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Scientific abstracts from the
6th International Barcode of Life
Conference
Résumés scientifiques du
6
e
congrès international
« Barcode of Life »
University of Guelph
Guelph, Ontario, Canada
18–21 August 2015
University of Guelph
Guelph (Ontario) Canada
Du 18 au 21 août 2015
Guest Editor
Sarah J. Adamowicz
Biodiversity Institute of Ontario &
Department of Integrative Biology
University of Guelph
Guelph, Ontario
Canada
Directrice scientifique invitée
Sarah J. Adamowicz
Biodiversity Institute of Ontario &
Department of Integrative Biology
University of Guelph
Guelph (Ontario)
Canada
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INTRODUCTION
International Barcode of Life: Evolution of a global research
community
Sarah J. Adamowicz
Abstract: The 6th International Barcode of Life Conference (Guelph, Canada, 18–21 August 2015), themed
Barcodes to Biomes, showcases the latest developments in DNA barcoding research and its diverse applica-
tions. The meeting also provides a venue for a global research community to share ideas and to initiate
collaborations. All plenary and contributed abstracts are being published as an open-access special issue of
Genome. Here, I use a comparison with the 3rd Conference (Mexico City, 2009) to highlight 10 recent and
emerging trends that are apparent among the contributed abstracts. One of the outstanding trends is the
rising proportion of abstracts that focus upon multiple socio-economically important applications of DNA
barcoding, including studies of agricultural pests, quarantine and invasive species, wildlife forensics, disease
vectors, biomonitoring of ecosystem health, and marketplace surveys evaluating the authenticity of seafood
products and medicinal plants. Other key movements include the use of barcoding and metabarcoding
approaches for dietary analyses—and for studies of food webs spanning three or more trophic levels—as well
as the spread of next-generation sequencing methods in multiple contexts. In combination with the rising
taxonomic and geographic scope of many barcoding iniatives, these developments suggest that several
important questions in biology are becoming tractable. “What is this specimen on an agricultural ship-
ment?”, “Who eats whom in this whole food web?”, and even “How many species are there?” are questions
that may be answered in time periods ranging from a few years to one or a few decades. The next phases of
DNA barcoding may expand yet further into prediction of community shifts with climate change and im-
proved management of biological resources.
Key words: DNA barcoding, conference, research trends, ecology, evolution, socio-economic applications, market
substitution, next-generation sequencing, plant barcoding, ethnobotany genomics, marker selection.
Résumé : Le 6e congrès international « Barcode of Life » (Guelph, Canada, 18 au 21 août 2015), dont le thème
est « Des codes a
`barres aux biomes », permet de présenter les plus récents développements en recherche sur
les codes a
`barres de l’ADN et leurs diverses applications. Ce congrès se veut également un lieu de rencontre
pour une communauté internationale de chercheurs afin d’échanger des idées et d’initier des collaborations.
Tous les résumés, tant des conférences plénières que des autres contributions, sont réunis pour publication
dans un numéro spécial de Génome a
`accès libre. Dans ce qui suit, l’auteure dresse une comparaison avec le
troisième congrès (Mexico, 2009) pour mettre en relief 10 tendances récentes et émergentes qui ressortent
parmi les résumés soumis. L’une des tendances qui ressort est la proportion croissante de résumés qui portent
sur des applications ayant des retombées socioéconomiques du codage a
`barres de l’ADN. Cela inclut des
études sur des ravageurs en agriculture, des espèces de quarantaine ou envahissantes, les causes de mortalité
de la faune, des vecteurs de maladies, la biosurveillance de la santé des écosystèmes ainsi que des analyses de
l’authenticité des produits de la mer ou des plantes médicinales qu’on retrouve sur le marché. D’autres
tendances clés incluent l’emploi du codage a
`barres et du méta-codage a
`barres dans le cadre d’analyses
nutritionnelles – dont des études de réseaux trophiques comprenant trois niveaux ou plus – ainsi que le
déploiement de méthodes de séquençage de nouvelle génération dans de nombreux contextes. Conjointe-
ment a
`la portée taxonomique et géographique croissante des initiatives de codage a
`barres, ces développe-
ments suggèrent qu’on peut maintenant s’attaquer a
`plusieurs questions importantes en biologie. « Quel est
ce spécimen de denrée agricole ? », « Qui mange qui dans ce réseau trophique ? », et même « Combien existe-il
d’espèces ? » sont des questions auxquelles il devient possible de répondre sur l’horizon de quelques années
a
`une ou plusieurs décennies. Les prochaines phases du codage a
`barres de l’ADN pourraient ouvrir de
Received 24 July 2015. Accepted 24 July 2015.
Corresponding Editor: Graham J. Scoles.
S.J. Adamowicz. Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone Road East,
Guelph, ON N1G 2W1, Canada.
Corresponding author: Sarah J. Adamowicz (e-mail: sadamowi@uoguelph.ca).
151
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nouveaux chantiers dans la prédiction de l’évolution des communautés biologiques liée aux changements
climatiques et dans la gestion des ressources biologiques. [Traduit par la Rédaction]
Mots-clés : codage a
`barres de l’ADN, congrès, tendances en recherche, écologie, évolution, applications so-
cioéconomiques, substitution de produits, séquençage de nouvelle génération, codage a
`barres chez les plantes,
ethnobotanique, génomique, sélection de marqueurs.
Introduction
The 6th International Barcode of Life Conference, held
18–21 August 2015 in Guelph, Ontario, Canada—themed
Barcodes to Biomes—highlights key recent expansions
to the DNA barcoding research program, including large-
scale investigations of entire assemblages of species. The
sum of the abstracts reveals that substantial work re-
mains to be done to understand the full extent of species
diversity and to build DNA barcode reference libraries to
enable specimen identifications to the species level. Nev-
ertheless, for focal ecosystems and taxonomic groups,
DNA barcode libraries are mature enough to move be-
yond library building, towards questions about trophic
interactions and the structure of food webs. Moreover,
key socio-economically important applications of DNA
barcoding are galloping forward, such as wildlife foren-
sics investigations and marketplace surveys of seafoods
and medicinal plants.
In this introductory article, I present a brief introduc-
tion to the history of DNA barcoding and comment upon
its international spread. I also review the most intriguing
trends that I have observed during the review of contrib-
uted abstracts. Being the largest of this biennual confer-
ence series to date, the 6th Conference received 500 oral
and poster presentations by presenting authors repre-
senting 56 nations. The special open-access volume of
conference abstracts to follow showcases a truly interna-
tional research movement reflective of the diverse indi-
vidual and national interests of the participants.
DNA sequences as barcodes: the origin of an idea
The ability to organize and recognize biological enti-
ties is essential for basic biological research and for di-
verse socio-economic applications in which humanity
and biodiversity intersect. The need to identify species is
perhaps particularly strong in the fields of systematics,
conservation, invasion biology, ecology, and evolution-
ary biology as well as for applications such as the foren-
sics of food, pest, and medicinal species. While estimates
of total global species diversity vary greatly (e.g., see
Mora et al. 2011 for a summary), there is a growing
community consensus that the number is sufficiently
vast—and contemporary extinction risk alarmingly
high—such that the incorporation of digital methods is
required to speed up the process of species discovery as
well as to store and retrieve information about species
collected at any life stage (e.g., Tautz et al. 2003;Janzen
et al. 2005;Packer et al. 2009;Padial et al. 2010).
One key form of digital information about biodiversity
is DNA itself, which stores vast biological information as
an information string consisting of four characters (A, C,
G, T). Genetic information in various forms has been
used for at least half a century for systematics research.
Early contributions in the 1970s and into the 1980s in-
cluded investigations of gross similarity and differences
in entire genomes through DNA–DNA hybridization ex-
periments, leading, for example, to an influential pro-
posal regarding the higher-level systematics of birds
(Sibley and Ahlquist 1990). Protein data, such as revealed
through allozyme analysis, also featured prominently in
earlier genetic studies. The invention of Sanger sequenc-
ing (Sanger et al. 1977) marked a critical point in the use
of genetic data for systematics, providing direct rather
than indirect evidence of the underlying DNA sequence
information.
Interestingly, the idea that DNA sequences can be used
as unique “barcodes” for identifying biological groups
has appeared at least three times independently in the
literature, i.e., without cross-citation. To my knowledge,
the first appearance of the term barcode—as used refer-
ring to genetic information rather than a commercial
product code—was by Arnot et al. (1993). They proposed
using hypervariable tandemly repeated sequences as bar-
codes for identifying strains of the parasite Plasmodium. Sec-
ond, Floyd et al. (2002) used the term barcodes in their
study proposing the use of nuclear small subunit ribo-
somal DNA (18S) sequences for defining Molecular Oper-
ational Taxonomic Units for a taxonomically difficult
group of animals, the nematodes. Third, the most recent
and most general independent introduction of the con-
cept was presented in two papers published by Hebert
and colleagues (Hebert et al. 2003a,2003b). These multi-
ple origins of the concept of codifying biological forms
through DNA, and the use of the barcode analogy, attest
to the utility inherent in the digital signal of DNA.
DNA barcoding: the spread of an idea (2003–2015)
The contributions by Hebert et al. (2003a,2003b) are
considered the most influential and are commonly cited
as representing the origin of the DNA barcoding ap-
proach and movement. What distinguished these contri-
butions from earlier treatments of DNA data as codes
in other systematics contexts (whether or not the term
barcodes was employed) was in providing a bold and
general proposal: that standardized DNA regions could
be used for identification of specimens to the species
level across all (or at least the vast majority) of animals.
Prior treatments tended to be more specific in their tax-
onomic scope. The more general proposal involved stan-
dardization of genetic regions to be sequenced, meaning
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that, in principle, any specimen of any life stage repre-
senting any taxon collected anywhere in the world could
be identified. Such a system would open new avenues for
research and for the detection of species at critical places
by non-taxonomic experts, such as at agricultural quar-
antine and international customs stations.
In their abstract entitled “Diffusing barcoding: the
global spread of a good idea”, Bubela et al. formally ana-
lyze publication patterns in the barcoding literature, in-
cluding all papers that cited four key papers. Bubela et al.
conclude “Barcoding is an exemplar of the rapid and
global spread of an innovation in the absence of formal
proprietization. Its diffusion is not only in volume but in
scope of applications. Institutional structures and opin-
ion leaders have been key drivers. Further diffusion is
likely with regulatory acceptance of the technology.”
(Unless otherwise indicated, all citations in this article
are to abstracts being published in this special volume.)
In this article, I delve into the contributed conference
abstracts in more detail to highlight emerging trends,
some of which are not yet apparent in the published
literature. The global spread of the idea of DNA barcod-
ing is clear, as evidenced by the diversity of nations rep-
resented among the authors and the varied study sites,
taxa, and research questions included in this conference
volume.
International Barcode of Life Conference series
A global research community was founded shortly af-
ter the publication of the first formal works proposing
DNA barcoding as a global, standardized initiative
(Hebert et al. 2003a,2003b). Beginning in 2005, the
International Barcode of Life Conference has been held
every other year. Prior meetings have been hosted by
cities spanning four continents: London, UK, in 2005;
Taipei, Taiwan, in 2007; Mexico City, Mexico, in 2009;
Adelaide, Australia, in 2011; and Kunming, China, in
2013.
As the 6th Conference will be hosted in Guelph,
Canada, the upcoming meeting represents a symbolic
homecoming for the DNA barcoding community. The
Biodiversity Institute of Ontario (BIO), University of
Guelph, is the home institution of Paul Hebert and col-
leagues. BIO is also home to the Secretariat of the Inter-
national Barcode of Life (iBOL) project (www.ibol.org)as
well as iBOL’s largest sequencing facility.
The International Barcode of Life Conferences have
grown in both participation and scientific scope over
time. For example, there were 215 attendees of the
1st Conference in London, while there were ⬃340 partic-
ipants at both the 2nd and 3rd Conferences. By the
4th Conference, in Adelaide, the number of conference del-
egates totaled 463, with 375 scientific presentations de-
livered. As of 22 July 2015, there are >500 registered
participants for the 6th Conference as well as 500 accepted
abstracts.
Research trends: Mexico City (2009) versus Guelph (2015)
As Guest Editor of this special volume, it was my priv-
ilege to review the contributed abstracts for publication.
During this process, I noticed several intriguing trends to
share with the broader community. I have elected to
partially quantify these emerging trends by comparing
the 6th Conference with the 3rd Conference. I selected
the Mexican conference for comparison because DNA
barcoding as a large-scale, standardized approach was
first proposed in 2003. Therefore, 2009 represents the
half-way point between 2003 and the present day, 2015.
Moreover, as both conferences were held in North Amer-
ica, inter-continental travel costs would be more similar
for these two conferences than for the other confer-
ences. I am assuming here that this would increase the
consistency of the pool of conference delegates at the
continental level, improving comparisons of conference
delegate composition across years. Nevertheless, I would
still expect that travel costs remain an important factor
influencing the representation of ongoing research
within these conference proceedings.
For each contributed, accepted abstract (both oral and
poster) for both conferences, I recorded key information
and “scored” the abstracts on several scales. First, I re-
corded the country of the presenting author. Second, I
assigned each abstract a score in terms of its taxonomic
scope: (1) 1–9 species, (2) 10–99 species, (3) 100–999 spe-
cies, (4) 1000–4999 species, and (5) 5000+ species. Third, I
scored each abstracts with regards to its geographic/
political scope: (1) local, (2) regional, (3) national, (4) conti-
nental, and (5) global. Fourth, I categorized all abstracts
in terms of their dominant theme: (1) barcode reference
library building, systematics, and (or) marker selection/
testing; (2) ecology (includes dietary analysis and com-
munity assembly); (3) evolutionary biology (includes
molecular evolution and phylogeography); (4) socio-
economic applications of DNA barcoding (e.g., agricul-
tural pests, disease vectors, medicinal species, wildlife
forensics, quarantine applications, invasive species de-
tection; this category included library building when the
socio-economic applications were stressed in the ab-
stract); and (5) methods development. Fifth, and lastly, I
recorded whether or not each abstract used next-
generation sequencing (NGS) methods.
Some abstracts did not receive a score in all categories.
When the above information was not explicitly pro-
vided, yet there was some reasonable indication of taxo-
nomic or geographic scope in the prose, I performed
some educated guessing. Therefore, the specific abstract
“scores” were somewhat subjective. Nevertheless, as I
conducted all of the scoring, the below comparison of
the 3rd and 6th Conferences will be at the least inter-
nally consistent and revealing of general trends. Further
details regarding the abstract scoring can be found in the
Adamowicz 153
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Supplementary data
1
. The 2015 numbers included here
represent all accepted abstracts as of 22 July 2015; due to
unforeseen circumstances, some of these abstracts may
not be presented at the 6th Conference. As well, I omit-
ted plenary abstracts, as those abstracts reflect in large
part the strategic vision for the conference that is crafted
by the individual organizing committees. By contrast,
contributed abstracts may be more comparable across
the conferences and more reflective of global research
activities in progress. Moreover, research by the plenary
speakers will be highlighted in a separate special issue of
full articles. While the Mexico City meeting featured
229 contributed presentations, the Guelph conference is
more than twice as large, with 470 contributed abstracts
(including both poster and oral presentations for both
meetings). Therefore, comparison in composition be-
tween the meetings is made in percentages.
Below, I highlight 10 of the most salient observations
and trends that I noted in the evolution of the Interna-
tional Barcode of Life community. For space consider-
ations, I emphasize trends apparent since the 2009
conference, acknowledging that many excellent ab-
stracts are not highlighted or cited below. I present
these observations as a commentary in the hope of pro-
moting reflection, discussion, collaboration, and research
planning.
1. Rise in socio-economic applications of DNA barcoding
The increased focus upon the socio-economic applica-
tions of DNA barcoding was one of the dominant trends
noted in the 2009 versus 2015 comparison. Contributed
abstracts for the 3rd Conference heavily featured library
building, genetic marker evaluation and comparisons,
and methods development, including primer design and
informatics tools for species-level identification. By con-
trast, in the 6th Conference, there is a distinct rise in the
number of abstracts that emphasize the socio-economic
applications of DNA barcoding (Fig. 1). For example,
there is a marked increase in both the number and di-
versity of marketplace surveys, with a particular empha-
sis upon herbal medicinal plants (Dhivya et al.; Ghorbani
et al.; Kumar et al.; Lekganyane et al.; Melo Palhares et al.;
Osathanunkul et al.; Ratsoma et al.; Schori et al.; Shiba
et al.) and fish products (Cawthorn et al.; Santos et al.;
Sarmiento Camacho and Valdéz-Moreno) but also includ-
ing spices (Saravanan et al.) and wild edible mushrooms (Xu
et al.). In 2015, the total quantity of studies on medicinal
plants—including reference library building, marker test-
ing, methods development for mixed-species products,
and marketplace studies—was striking.
The Fish Barcode of Life (FishBOL) initiative played a
prominent role in both conferences. While both confer-
ences include important studies that are building DNA
barcode reference libraries for fish from various geo-
graphic regions, some shifts in research focus relating to
fish and other commercially important aquatic taxa are
apparent. In particular, varied applications of DNA bar-
coding are apparent in 2015. These include developing
protocols for the barcoding of fish eggs and larvae
(Naaum et al.), using barcoding to investigate the larval
distributions of fish species (Bourque and Hanner; Cota-
Valentin et al.; Malca et al.; Steinke et al.) and also lob-
sters (Vasquez-Yeomans et al.), and unraveling trophic
interactions through dietary analysis in fish (Bartley
et al.; H. Liu et al.; Shortridge and Miner; Thielman et al.).
As well, several marketplace surveys are detailed in the
2015 conference proceedings (Cawthorn et al.; Santos
et al.; Sarmiento Camacho and Valdéz-Moreno), and the
contribution that barcoding can make to understanding
medicinal uses of fish is also highlighted under the term
ethnoichthyogenomics (Ravitchandirane and Thangaraj).
Studies on vectors of human and veterinary diseases;
agricultural pests, pathogens, and parasitoids (agricul-
tural biological control agents); and agricultural soil mi-
crobiota featured in both conferences, with an overall
increase towards the present in the frequency of studies
directed towards such socio-economically important
groups of organisms. As well, in 2009, there was a ten-
dency for abstracts to focus primarily on methodological
1
Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/gen-
2015-0094.
Fig. 1. Distribution of contributed presentations among
research themes in Mexico City (2009) versus Guelph
(2015). The number of abstracts in every category is higher
in Guelph, with a total of 470 contributed abstracts, in
comparison with Mexico, having a total of 229. The results
are therefore shown by percentage here to highlight
trends. Mexico more prominently featured DNA barcode
reference library building and systematics works, while in
Guelph there is a higher focus upon socio-economically
important applications of DNA barcoding as well as the
usage of barcoding for ecological and evolutionary
research.
0
10
20
30
40
50
60
70
Mexico City (2009)
Guelph (2015)
Percentage
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development and library building for these taxonomic
groups. In comparison, 2015 features more abstracts re-
porting studies conducted in real agricultural settings,
including cultivated fields (e.g., Bennur et al.; Gutiérrez
Gutiérrez et al.; Jindal et al.; Kamenova et al.; Shinde and
Khedkar; Wang et al.), greenhouses (Lenin et al.), fruit
tree orchards (Aslam et al.), and vineyards (Gutiérrez
Gutiérrez et al.). Such studies are uncovering new infor-
mation about species-level diversity and the specificity of
biological associations within agricultural systems. Pos-
sibly, in future conferences we may see yet a further
shift—going beyond elucidating the diversity inhabiting
agricultural systems towards directly applying barcod-
ing for agricultural management and for making pest
mitigation decisions.
Numerous studies also specifically highlight barcod-
ing progress, methods development, analysis of policy,
and (or) training programs relating to the detection
or understanding of quarantine and invasive species
(Bezeng et al.; Chain et al.; Deiner et al.; Frewin et al.;
Furlan et al.; Glover et al.; Gutiérrez Gutiérrez et al;
Hodgetts et al.; Jalali and Venkatesan; Kumar and
Smrithy; Layton et al.; Marinich et al.; Masson et al.;
Park et al.; M. Roy et al.; Salisbury et al.; Sambandan
et al.; R. Santos et al.; M.D. Santos et al.; Shimura and
Duthie; Sutou and Ito; Thomas et al.). Interest in the topic
of invasive and quarantine species spans many coun-
tries, focal taxa, and molecular approaches (DNA barcod-
ing, metabarcoding of mixed-species assemblages, and
eDNA detection using both qPCR and NGS methods).
Although the majority of all studies from both meet-
ings were contributed by university-based researchers,
2015 witnesses an increase in the number of abstracts
that were either written or coauthored by researchers
working within governmental agencies or other institu-
tions concerned with the regulation of biological mate-
rials, protection of biological resources, and protection
of environmental health. Using Canada as an example,
multiple contributions by Canadian teams highlight
barcode-related research within federal and provincial
governmental agencies, including both government-led
projects and strong academic-governmental collabora-
tions. These projects are diverse, including: library build-
ing and species delineation in aquatic (Castelin et al;
Young et al.) and terrestrial (Barrio et al.; Fernandez-
Triana et al.; Landry et al.; Solecki et al.) taxa, the trophic
ecology of fishes (Bartley et al.), the detection of threat-
ened (Boothroyd et al.; Currier et al.; Serrao et al.) and
invasive (Marinich et al.; Masson et al.; M. Roy et al.)
species, and a study revealing the composition of mixed-
species assemblages being used in freshwater ecotoxico-
logical assays (Capretta et al.).
Finally, there is a strong trend in using DNA barcoding
for wildlife forensics. Schindel and Trizna outline a ded-
icated six-country initiative to barcode endangered spe-
cies, particularly those regulated under the Convention
on International Trade in Endangered Species of Wild
Fauna and Flora (CITES), as well as related and “look-
alike” species. The strong data standards for this project
(Trizna and Schindel) are designed to enable barcodes to
be used in the prosecution of wildlife crimes to reduce
the trade of endangered species. As a participant in this
initiative, Mwale et al. report that all CITES-listed mam-
mal, bird, and reptile species of South Africa have been
barcoded. Shiba et al. expose the illegal presence of
CITES-listed species within a medicinal plant market in
South Africa. Other works relating to wildlife forensics
include the barcoding of captive animals at a zoo in India
(Kumar et al.) and the usage of barcodes to identify claw
samples originating from large cats in India (Hange and
Khedkar). Mendoza et al. focus upon increasing barcode
coverage for CITES-listed species within Colombia, with
particular emphasis upon a highly traded animal group,
the birds. Also, within India, Kalyankar et al. outline
investigations relating to the ornamental fish trade.
Beyond its conservation importance, wildlife forensics
also encompasses human safety-related investigations,
including understanding the identities of birds involved
in air strikes (Beentjes et al.). Arulandhu et al. outline a
strategy for identifying seized forensics samples, while
Topan provides an overview of the rising demand for
forensics-related services at the Biodiversity Institute of
Ontario.
2. International participation enhanced in 2015
The number of nations represented among presenting
authors of contributed presentations grew by 22% be-
tween Mexico City (44) and Guelph (54). These results
confirm that the Barcode of Life is indeed a highly inter-
national research discipline. Moreover, the distribution
of presentations among countries reflects broad partici-
pation among many nations, while several countries
emerge as being highly involved in DNA barcoding
(Fig. 2). Nations that are well represented (>2% of all con-
tributed abstracts) in both 2009 and 2015 are Canada,
India, the United States, Mexico, China, Brazil, the
United Kingdom, Germany, and New Zealand. As hosts of
the 2009 meeting, Mexico was prominently represented
at the 3rd Conference.
Several nations have become more heavily involved in
the 2015 conference. In particular, the rise in participa-
tion by India is striking; this increase in barcoding activ-
ities may be linked to the recent establishment of a
dedicated barcoding institute (see trend No. 4 below).
Interestingly, neither Malaysia nor Portugal are recorded
having any contributed abstracts in 2009, while both
reach >2% representation in 2015. An examination of
co-authorship patterns suggests that the rise of Malaysia
as a strongly represented nation is to a large degree due
to the establishment of a barcoding-intensive research
program at the University of Malaya. This group of re-
searchers is investigating the biodiversity of Southeast
Asia, including novel studies of wildlife harboured within
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large urban centres (Brandon-Mong et al.; Jisming-See
et al; Lee et al.; Lim and Wilson; Ng et al.; Sing et al.;
Wilson et al.). With multiple institutions involved from
from mainland Portugal and the Azores, Portugal exhib-
its particular strength in using DNA barcoding to explore
the diversity and evolution of marine life (Antunes
et al.; Costa et al.; Lobo et al.; Moura et al.; Oliveira et al.;
Paupério et al.; R. Santos et al.; Vieira et al.).
A comparison of 2009 versus 2015 also reveals an
increase in the number (but not the proportion) of
continental- and global-scale projects (Fig. 3). Several au-
thors specifically stress the importance of international
collaboration. For example, Lavinia et al. highlight the
role of international collaboration (among Argentina,
Bolivia, Brazil, and Mexico) in achieving a novel study of
evolutionary diversification in their abstract entitled
“From a local barcoding initiative to a continental-scale,
multi-institutional assessment of avian diversification in
the Neotropics”. Perez et al. describe a global-scale study
to elucidate terrestrial arthropod diversity, Schindel and
Trizna describe a six-country initiative to protect endan-
gered species, and Park et al. and Frewin et al. describe
continental or global efforts to generate species lists and
barcode libraries for pest and quarantine species. Such
initiatives are important for understanding and protect-
ing biodiversity, especially in the face of increasing
global trade and associated invasion risk (Shimura and
Duthie).
Interestingly, in 2015, there is an increasing propor-
tion of abstracts of narrow taxonomic focus (1–9 species)
and geographic scope (local or regional scale) (Fig. 3).
This may be reflective of the growing interest in DNA
barcoding; new researchers, institutions, and countries
are joining the 6th International Barcode of Life Confer-
ence. As well, a variety of the applied studies are highly
targeted; for example, several contributions relate to se-
lect taxonomic groups of agricultural pests collected
from fields in specific geographic regions. By contrast,
researchers from several “established” barcoding na-
tions are presenting very large-scale projects, represent-
ing a decade of barcoding-related research as well as
collaborations spanning multiple research teams.
3. Different countries/continents lead in specific
disciplines
While DNA barcoding as a whole is a highly interna-
tional endeavour, it is clear that institutional and na-
tional research priorities can vary. For example, India,
China, and South Africa have all emerged as leaders in
plant barcoding, and this includes the barcoding and
analysis of medicinal plant products. This result may
partially reflect institutional and (or) personal research
priorities; however, this finding also underscores inter-
national variability in the strength of association
between human communities and biodiversity. Partic-
ularly in rural communities in developing regions of
the world, plant-derived treatments are more common
than synthetic or industrially produced medicines,
and this trend is likely mirrored in research resources
being directed towards medicinal plants. Davies et al.
present a study entitled “Human population density in
Africa correlates with the evolutionary history of its
flora”, again highlighting the close association be-
tween humans and biodiversity.
As another example of regional leadership, South and
Central American researchers are global leaders in the
DNA barcoding of birds. Based upon patterns in abstract
coauthorships, this effort is to a large degree catalyzed
by Argentina, specifically the team at the Museo Argen-
tino de Ciencias Naturales “Bernardino Rivadavia”, but
this effort has now expanded far beyond Argentina’s bor-
ders and involves multiple institutions and research
teams. The building of more complete barcode reference
libraries enables not only wildlife forensics applications
Fig. 2. Distribution of countries that are represented by the presenting authors of contributed abstracts in Mexico City (2009)
versus Guelph (2015). The legend to the right of each pie chart can be associated with the figure by reading the legend from top to
bottom and comparing with the pie slices in a clockwise fashion, starting at the top. The 14 most represented countries from 2015
(>2% of contributed abstracts) are shown in both charts, with the countries placed in the same order to facilitate comparison.
Mexico City 2009 - Countries (by %)
Canada
India
USA
Mexico
China
Brazil
UK
Germany
New Zealand
France
Malaysia (0%)
Argenna
Portugal (0%)
South Africa
Guelph 2015 - Countries (by %)
Canada
India
USA
Mexico
China
Brazil
UK
Germany
New Zealand
France
Malaysia
Argenna
Portugal
South Africa
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but also excellence in fundamental scientific research.
This conference volume reports novel insights into the
systematics, phylogeography, species ages, diversification
patterns, molecular evolution, and barcodes of endan-
gered species within birds from the Neotropics, in-
cluding contributions from across South and Central
America (e.g., Barreira et al.; Bukowski et al.; Lavinia
et al.; Mendoza et al.; Salinas et al.).
4. New DNA barcoding centres rise to prominence
The 2015 abstracts volume also reveals emerging new
centres of DNA barcoding. I highlight here the research
scope and contributions of two key dedicated barcoding
centres, whose strongly prominent role in the confer-
ence proceedings has arisen since 2009.
The Paul Hebert Centre for DNA Barcoding and Biodi-
versity Studies (at the Dr. Babasaheb Ambedkar Marath-
wada University in Aurangabad, India) has emerged as a
major contributor to the 2015 conference proceedings,
which is especially noteworthy as its grand opening was
held only recently, in 2011. As authors from this institu-
tion submitted ⬃20 abstracts to the conference, this rep-
resents institutional participation second only to that of
the conference hosts, the University of Guelph. More-
over, a wide-ranging and diversified research program
on DNA barcoding and its socio-economic applications is
apparent, with conference contributions encompassing
the following: barcoding of mosquito disease vectors
(Ahirrao et al.); barcoding of both pest species (Shinde
and Khedkar) and parasitoids (Devi et al.) in agricultural
settings; reference library building, phylogenetic/
systematics works, and (or) barcoding methods develop-
ment for diverse taxa including aquatic plants (Kadam
et al.), freshwater and marine fishes (Khedkar;
Mohekar et al.; Rathod et al.), freshwater zooplankton
(Khobragade et al.), and corpse flies relevant for forensics
investigations (Zambare and Khedkar); conservation applica-
tions including a wildlife forensics investigation involv-
ing large cat species (Hange and Khedkar) and studies on
the trade of ornamental fishes (Kalyankar et al.); and a
marketplace survey of meats to evaluate law enforce-
ment (Naikwade et al.).
Another important continental centre is the African
Centre for DNA Barcoding (University of Johannesburg,
South Africa). With eight presented or coauthored ab-
stracts in 2015, compared to two from 2009, the Univer-
sity of Johannesburg team and collaborators are well
represented in the conference proceedings. Contributed
abstracts that include one or more coauthors from the
African Centre for DNA Barcoding are far-reaching and
build upon a solid foundation enabled by dedicated DNA
barcode library building. Specifically, this research
group and their collaborators have emerged as global
leaders in creating regional species-level plant phylog-
enies and using these to investigate the evolutionary
history and ecological mechanisms that underlie com-
munity assembly (Bello et al.; Davies et al.; Maurin et al.;
Yessoufou and van der Bank) as well as species invasion
success (Bezeng et al.). Members of this research group
have also investigated plant species identification suc-
cess and product authenticity from a traditional medici-
nal market (Lekganyane et al.; Ratsoma et al.; Shiba
et al.), revealing some trade in species with declining
populations as well as the illegal sale of CITES-listed
species.
The University of Waikato (New Zealand), with 10 con-
tributed abstracts, also features prominently among the
institutions participating in the 6th Conference, with
research focused upon the biodiversity of New Zealand
(Beet et al.; Doyle and Hogg; Doyle et al.; Mc Cormack
et al.; Podmore et al.; Riding et al.; West et al.; Woods
et al.) and Antarctica (Beet et al.; Collins and Hogg;
Collins et al.). Due to variability in the way institutions
were entered by authors into the abstract submission
form, the assessment in this section should be consid-
Fig. 3. A comparison of the scope of contributed abstracts in Mexico City (2009) versus Guelph (2015) using two metrics:
(A) taxonomic scope, and (B) geographic/political scope. For both metrics, the absolute number of abstracts was higher in every bin
in Guelph than in Mexico. The results are shown in percentages to demonstrate trends in the composition of abstracts.
0
5
10
15
20
25
30
35
40
45
Local Regional Naonal Connental Global
Mexico City (2009)
Guelph (2015)
0
5
10
15
20
25
30
35
40
45
0-9 species 10-99 species 100-999
species
1,000+
species
5,000+
species
Percentage
)B)A
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ered a qualitative rather than a comprehensive assess-
ment of institutional involvement. Nevertheless, the
trends reported here showcase several highly active re-
search programs and institutions.
5. Increase in the number of large projects
Both the number and proportion of taxonomically
broad projects, those including 1000+ species or even
5000+ species, has increased markedly between 2009 and
2015 (Fig. 3). This rise is not explained by an increase in
adoption of next-generation sequencing (NGS) methods.
Many studies that feature NGS were not included in the
“taxonomic scope” analysis, as the numbers of species or
MOTUs detected were not reported. Moreover, among
the nine contributed abstracts reporting work involving
5000+ species or Molecular Operational Taxonomic Units
(MOTUs) or 20 000+ specimens, all but one (Deiner and
Altermatt, a contribution on river biomonitoring) used
Sanger sequencing.
Among the contributed abstracts from 2015, these
large-scale projects include an overview of the Mexican
Barcode of Life (MexBOL) initiative (Eliás-Gutiérrez and
León-Règagnon; Martínez-Arce and Eliás-Gutiérrez), a
large and active national network that includes regional
barcoding facilities. The progress of another large na-
tional network, the Norwegian Barcode of Life (NorBOL),
which is tasked with advancing knowledge of Norwegian
and polar biodiversity, is also highlighted in a dedicated
presentation (Ekrem et al.). Although the Netherlands
were not represented among the presenting authors of
contributed abstracts in 2009, in 2015 a summary is pro-
vided regarding the substantial barcoding program at
the Naturalis Biodiversity Center, which is focused upon
the flora and fauna of the Netherlands (Beentjes et al.).
Interestingly, beyond these overviews of large institu-
tional programs and national networks, there are also
cases of specific research projects that are extensive in
their taxonomic scope. For example, Fernandez-Triana
et al. present a large study (10 000+ specimens) investi-
gating cryptic diversity and Holarctic distribution pat-
terns in a group of parasitoid wasps. Bukowski et al.
report progress in studying the diversity of terrestrial
arthropods as part of the Global Malaise Trap Program,
with a finding of >5000 MOTUs to date at a site in the
Atlantic Forest biome of Argentina. Perez et al. provide
an overview of the Global Malaise Trap Program, which
includes collaborators from 30 countries and which has
documented >65 000 MOTUs to date. Given these large
programs, the oft-posed but elusive question of “how
many species are there?” (e.g., Mora et al. 2011) may be
within reach within the coming few years, at least for
select taxonomic groups amendable to collection via
standardized methods.
6. Continued “hub” role for University of Guelph, Canada
About 18% (⬃84 of 470) of the contributed presenta-
tions include one or more coauthors from the University
of Guelph, particularly with representation from the Bio-
diversity Institute of Ontario (BIO), which span all five of
the research subject categories described above. This pre-
dominance in the scientific program in part reflects the
geographic reality that the host institution is expected to
contribute proportionally more abstracts due to lower
travel costs (Fig. 2). However, this also reflects the con-
tinued role of BIO as a hub for both research and for
high-throughput sequencing for the global barcoding
community. The Biodiversity Institute of Ontario (or Ca-
nadian Centre for DNA Barcoding, housed within) is ac-
knowledged for providing sequencing support within
approximately 25% of all publications relating to DNA
barcoding (Dirk Steinke, pers. comm., 17 July 2015).
BIO’s role as a high-throughput DNA barcoding centre
is enabling new, large-scale research projects that have
been developed since 2009. One such project being high-
lighted at this conference is the Global Malaise Trap Pro-
gram for elucidating terrestrial arthropod diversity,
which is featured in several abstracts led by researchers
from BIO (D’Souza; Perez et al.), Argentina (Bukowski
et al.), Bangladesh (Bhuiya and Mazumdar; Mazumdar
et al.), and the United States (Zlotnick et al.). The capac-
ity of BIO as biological repository—with a digitized,
highly barcoded specimen collection and an active
loan program—is also presented (Telfer et al.). BIO-
based researchers are also sharing new methodologi-
cal developments, including new NGS-based protocols
for barcoding type specimens (Prosser et al.), revealing
the composition of herbal medicines (Ivanova et al.), and
non-destructively sequencing DNA from bulk environ-
mental samples (Shokralla et al.).
University of Guelph researchers are also strongly in-
volved in building extensive plant (e.g., Kuzmina et al.;
Warne et al.) and animal (e.g., Blagoev et al.; Fernandez-
Triana et al.) barcode reference libraries; assessing the
uses and authenticity of medicinal plant species (e.g.,
Dhivya et al.; Sambandan et al.; Tahir et al.); studying
patterns of molecular evolution (e.g., Loeza-Quintana
and Adamowicz; Mitterboeck et al.; Young et al.) and
community assembly (e.g., Bringloe et al.; Martin et al.;
Pare and Smith; Smith et al.); developing approaches for
the biomonitoring of ecosystem health (e.g., Fahner
et al.; Gibson et al.); establishing methodological ap-
proaches (e.g., Naaum et al.), species checklists (Frewin
et al.), and best practices (Naaum et al.) relating to quar-
antine applications, product validation, and market-
place surveys; and using DNA barcoding to enhance
scientific education (Berzitis et al.).
7. Animal and fungal barcode markers: broad consensus
In the six years between 2003 (Hebert et al 2003a,
2003b) and 2009 (3rd Conference), the animal research
community widely adopted the standardized animal bar-
code marker, the 5=region of cytochrome c oxidase sub-
unit I (COI). This was despite the trend that earlier
barcoding-style works on species-level identification
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systems for animals (Bartlett and Davidson 1992;Parson
et al. 2000;Branicki et al. 2003), as well as phylogeography
studies of vertebrates, generally used a different mito-
chondrial gene, cytochrome b. This subsequent adoption
of COI for a broad DNA barcoding program in animals
may have been facilitated by the relatively similar pat-
tern of molecular evolution between the two mitochon-
drial protein-coding genes (e.g., see Lavinia et al.). The
greater conservatism of COI amino acid sequences may
assist with higher taxonomic placements (Hebert et al.
2003a). Phylogenetic signal of COI has previously been
explored in the literature (e.g., Wilson et al. 2011 and
other works), and the possibility of identifying speci-
mens to the family level based upon their DNA barcodes
is further developed at this conference (Kekkonen).
The barcoding community in general adopted the no-
tion of standardization as being important for creating
globally relevant DNA barcode reference libraries. In
both 2009 and 2015, most animal-focused contributions
used COI, either solely or in combination with other
markers. While a small number of contributions in 2009
solely explored “alternative” animal barcode markers,
the vast majority of animal contributions from 2015 use
COI. Some authors included the study of additional
genomic regions, often nuclear, to further their specific
study goals, such using nuclear DNA sequences to detect
hybridization (e.g., Aguilar-Velasco et al.). Terms such as
complementary rather than alternative, when referring
to markers other than COI, are more commonly em-
ployed in 2015 compared to 2009 among animal-related
abstracts.
While COI is broadly used as the barcode region for
most animal phyla, consideration of barcode markers is
apparently ongoing for nematodes. The nuclear small
subunit rRNA gene (18S) is typically used, sometimes in
combination with other markers, for studying free-living
nematodes in marine settings (Zhou et al.) as well as in
agricultural soils (Gutiérrez Gutiérrez et al.). Zhou et al.
contrast 18S and COI for marine nematodes and high-
light the trade-offs that may occur; they found lower
sequence variation (discrimination signal) in 18S but a
higher rate of sequence recovery compared to COI. Marker
evaluation is also ongoing in parasitic nematodes. COI,
ITS-1, and ITS-2 are used in a study of parasitic nematodes
infecting pelicans (Valles Vega et al.), while Velarde-
Aguilar and León-Règagnon evaluate the effectiveness of
the barcode region of COI for discriminating nematode
parasites of frogs.
Similar to the case in animals, there is broad consis-
tency in marker usage for fungi. Whether using Sanger
sequencing or NGS methods, most authors use one or
more ITS regions for fungi (Aslam et al.; Ivanova et al.;
Korpelainen and Pietiläinen; Mark et al.; Meyer et al.;
Porter et al.; S. Roy et al.; Xu et al.; Yu et al.), sometimes in
combination with other markers, especially nuclear
rRNA gene sequences. In light of the widespread usage of
ITS2 for fungal and plant barcoding, Ankenbrand et al.
present an extended informatics workbench for ITS2
sequences. However, Irinyi et al. report insufficient
species-level resolution of ITS within pathogenic fungi
and explore alternative markers. Although alternative or
complementary marker exploration is ongoing, ITS is
apparently now firmly established as the “core” marker
in use by the fungal barcoding community.
8. Plant barcode markers: community consensus remains
elusive
In contrast to the situation in animals, a consensus
regarding marker choice has not emerged in plants. In
2009, the CBOL Plant Working group published a dedi-
cated contribution in which they compared and tested
the merits of multiple candidate genetic regions. They
recommended that two chloroplast gene regions serve as
the “core” DNA markers for plant barcoding: rbcL and
matK. Given their study was published in 2009, it is not
surprising that at the 2009 conference, plant marker
choices were diffuse among studies. It is somewhat more
surprising that, six years later, this remains the case.
Many plant-related contributions in the present volume
employ or test a variety of different markers. Some stud-
ies do not even include the core markers as candidates.
Without marker standardization, it is difficult to achieve
a reference library that would enable identification any-
where in the world, such as at international customs
check-points.
Standardization also enables large-scale research into
entire biomes. In their contribution on the applications
of DNA barcoding for ecology, Yessoufou and van der
Bank point out: “Since the pair rbcLa and matK has been
accepted as the core DNA barcode for terrestrial plants,
many studies, perhaps surprisingly, continue to test the
discriminatory power of these markers in many lineages.
In Africa, and specifically in South Africa, we have moved
on with the application of the core barcode in phyloge-
netic ecological studies.” As highlighted above in the
section on the African Centre for DNA Barcoding, the
approach of this research group in using consistent
markers has yielded multiple intriguing findings. They
have used phylogenies to reveal mechanisms underlying
community assembly and to provide new knowledge in
invasion biology. As well, consistent marker usage en-
ables comprehensive regional libraries to be built, facil-
itating marketplace surveys of medicinal plants and
other biological products.
There are several potential reasons for the lack of con-
sensus. The primary reason may be that even when using
the recommended two-marker “core” plant barcode, spe-
cies discrimination rates for plants tend to be lower than
for animals. The CBOL Plant Working Group (2009) indi-
cated a species discrimination rate of 72% for the recom-
mended two-marker barcode among their test species,
with 100% success for genus-level assignments. Fazekas
et al. (2009) also reported species discrimination rates of
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⬃70% in plants, while species-level discrimination rates
for animal species in their review were much higher,
90%–98%. While lower rates of molecular evolution may
contribute, they attributed this difference primarily to
differences in hybridization rates between plants and
animals. Unfortunately, Fazekas et al. (2009) indicate this
issue cannot be readily addressed through adding a small
number of additional loci.
These typical plant “success” rates may not be high
enough for many purposes, such as distinguishing
closely related species that may have different medicinal
properties and in cases where geographic information
about the source location is not available (e.g., biological
forensics). The majority of plant authors either explicitly
evaluate and compare markers or comment upon the
species discrimination success rate. Upon seeking higher
resolution, some authors may forego global concerns
over marker standardization in favour of markers that
serve as taxon-specific “local optima” for species discrim-
ination. While many contributions in this conference
volume use rbcL, matK, or both, and demonstrate the
significant discriminatory signal in these markers, some
plant contributions did not employ either one of the core
barcode regions for plants.
A large number of plant contributions in this volume
have used a hybrid approach between standardization
and discrimination. These studies use the two “core”
plant barcode markers across the species in their study,
but also evaluated or added additional markers to in-
crease species-level discrimination in difficult groups
(e.g., Awad et al.; Gao et al.; Gawhari et al.; Kadam et al.;
Kress et al.; Kuzmina et al.; Malik and Babbar; Martínez
de la Vega et al.; Mitchell and Alemseged; S¸ahin et al.;
Schori et al.; Shaw et al.; Sheth and Thaker; Sheth et al.;
Shiba et al.; Silvis et al.; Vinitha et al.; Warne et al.; Zhou).
Fahner et al. also highlight the value of including multi-
ple markers in environmental assessments of below-
ground plant communities using NGS. These studies
could possibly serve as a model for future trends in plant
DNA barcoding, in which the core markers are always
included even when the specific study aims dictate addi-
tion of markers. One can hope that funding towards
plant barcoding projects can reflect the need for global
standardization of markers—for both scientific gain and
for enabling socio-economically important applications
of DNA barcoding—as well as the need for researchers to
achieve better species discrimination rates for plants for
their individual studies and specific applications.
9. Next-generation sequencing methods are becoming
widespread
In 2009, next-generation sequencing (NGS) methods
were used in just five presentations (2% of the 229
contributed abstracts). During the intervening years, re-
search that featured NGS methods were typically in-
cluded within dedicated oral presentation sessions. By
contrast, in 2015, NGS usage is widespread, being used in
19% (88 of 470) of all contributed abstracts by presenting
authors from 24 countries. Moreover, NGS methods are
widely distributed across various research themes. Top-
ics in which NGS usage is particularly prevalent include
dietary analysis of mammals, fish, and terrestrial arthro-
pods (see trend No. 10 below); simultaneous detection of
animal and microbe DNA in support of food authenticity
and safety analysis (Kaeppel et al.); restoration ecology
(Eaton et al.; McGee et al.); biodiversity assessment and
biomonitoring of entire communities for ecosystem
health (e.g., Bowser et al.; Gibson et al.; Ledger et al.; Lobo
et al.; Pawlowski et al.; Rougerie et al; Wright et al.); and
environmental DNA (eDNA) analysis, in which DNA is
sampled directly from the environment, such as from
sediments or water, rather than from specimens (e.g.,
Alsos et al.; Bista et al.; Deiner and Altermatt; Deiner
et al.; Fahner et al.; King et al.; Morey and Hanner; Pansu
et al.).
An interesting research avenue emerging at the 2015
conference is that several contributions use DNA for his-
torical reconstructions. Pansu et al. use sediments to re-
construct vegetation community shifts and to detect
changes in mammal species presence over 10 000 years,
with shifts detected that are corroborated with the
known history of human activities in the area. Similarly,
Alsos et al. demonstrate that eDNA in sediments can be
used to detect changes in vegetation communities, such
as planting of forests, over the past two centuries. Using
NGS barcoding of pollen from historical bee collections,
Gous et al. investigate shifts in plant usage by bees over a
93-year period. Finally, historic usage of different fish
species by humans is investigated through sequencing
mini-barcode regions from ancient fish remains from
archaeological sites (Puncher et al.; Royle et al.).
Several promising new technologies are also high-
lighted in 2015. Ramgren et al. and Boutain and Boutain
showcase diverse uses of the Oxford Nanopore Technol-
ogies’ MinION device, which is a sequencing instrument
“about the size of a smartphone” (Ramgren et al.). Sirianni
and Wangh also present an intriguing technology in
which species may be discriminated in a single closed-
tube reaction. Based upon such developments, the hoped-
for “hand-held field-friendly barcoder” (Janzen et al. 2005)
may be within reach, which would open remarkable op-
portunities for both research and applications of barcod-
ing by diverse user groups.
10. Species interactions are being studied across multiple
trophic levels
In Mexico, only one or possibly a small number of
studies investigated diet. Searches for the word “diet”
and for the phrase “gut content” in the titles/abstracts
spreadsheets yielded only a single match (barring a case
that mentioned that specific tissues were sampled for
earthworm barcoding to avoid amplifying gut contents).
The sole 2009 abstract on this topic presented a pub-
lished study on dietary analysis for eight sympatric
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species of bats (Clare et al. 2009). By contrast, in 2015,
there are enough dietary analyses among the contrib-
uted abstracts for a dedicated parallel presentation session
(plus there are posters and presentations in other sessions)
on the trophic interactions of mammals (Arrizabalaga-
Escudero et al.; Bennett et al; Jung; Kartzinel and Pringle;
Magalhaes de Oliveira et al.; Mallott et al.; Mata et al.; Mitch-
ell and Alemseged; Salinas-Ramos et al.). Moreover, there
are at least four abstracts reporting fish dietary analysis as
well (cited above).
The 2015 conference also witnesses the expansion of
dietary analysis beyond vertebrates, including dietary
analysis within marine copepods (Zhang et al.), squids
(Braid and Bolstad), and terrestrial arthropods. Kamenova
et al. use diet analysis to characterize the ecosystem ser-
vices (pest regulation) provided by carabid beetles in
managed wheat and oilseed rape fields, and Wang et al.
investigate the diet of generalist predators (spiders) in
rice paddies. Other studies investigate dietary breadth
in herbivorous terrestrial arthropods collected in
wild ecosystems (Burgess et al.; Garcia-Robledo et al.;
Kishimoto-Yamada and Ito; McClenaghan et al.), yielding
novel insights. For example, in their study investigating
the diets of chrysomelid beetles in Costa Rica, Garcia-
Robledo et al. found that barcoding “revealed several
cryptic insect herbivore species with narrow diets
and elevational distributions”. By contrast, Kishimoto-
Yamada and Ito emphasize the broad diets of several
species of chrysomelid beetles in a rainforest in Borneo
(e.g., “four species fed on several families of gymno-
sperms and (or) ferns together with multiple angiosperm
families”). While more research is needed to understand
broader patterns of specialization/generalization in in-
sect herbivores across taxonomic groups and sites, it is
clear that DNA barcoding and metabarcoding can play a
prominent role in uncovering feeding associations.
Species interaction studies have also expanded beyond
the (primarily) two trophic levels that are considered
during dietary analyses. For example, Roslin and Wirta
outline how they first constructed a comprehensive bar-
code library of terrestrial animals and vascular plants in
a focal High Arctic region of Greenland. They went on to
use this library to document predator–prey relationships
in animals, finding that the “the structure of the food
web proved extremely complex, showing dense linking
and no compartmentalization”. Beyond illuminating
new information about this Arctic food web, this study
showcases the value of DNA barcoding in food web ecol-
ogy. Another study on species interactions is explicitly
investigating interactions among three major taxo-
nomic groups representing multiple trophic levels
(Garcia-Robledo et al.; abstract entitled “Reconstructing
interactions among plants, insect herbivores, and pho-
retic mites using DNA barcodes: modeling coextinctions
under projected climate change”). This presentation un-
derscores the complex and novel ecological research
that can be facilitated by DNA barcoding. As a final
example, Fofanov et al. are exploring species interac-
tions across domains of life, studying bats, arthropods,
and guano-associated bacterial communities in subterra-
nean habitats.
Up to three interacting trophic levels are also being
investigated in the context of medically important stud-
ies. For example, Brugman et al. elucidate three interact-
ing levels: mosquito species identities (vectors), host
species identities (vertebrates which comprised the mos-
quito blood meals), and the presence/absence of the Myx-
oma virus (pathogen). Similarly, Lutomiah et al. analyze
the blood meal source and viral status associated with
mosquitoes collected during a Rift Valley Fever outbreak
in Kenya. To better understand the role of birds as reser-
voirs of three avian-transmitted viruses, Tseren-Ochir
et al. analyze bird fecal samples to identify the bird spe-
cies as well as screen for viral status. These studies dem-
onstrate that simultaneously considering the pathogen
and hosts is possible, and broader adoption of such
methods may provide substantial public health benefits
through monitoring and possibly mitigating vector and
pathogen distributions.
Concluding remarks
In just 12 years since it was formally proposed as a
standardized, large-scale endeavour, DNA barcoding has
matured into a broad-ranging international research
program. In August 2015, the 6th International Barcode
of Life Conference is hosted in Guelph, Canada, and is
currently set to include 470 contributed presentations by
presenting authors representing 54 nations. The breadth
of research topics ranges from reference barcode library
building for select taxa to food web studies spanning
three or more trophic levels. Barcoding-enabled research
is also providing new insights into the origins and distri-
bution of humans as well as our shared history and asso-
ciations with other species with whom we share the
planet (Davies et al.; Stoeckle and Thaler). Diverse socio-
economically important applications of DNA barcoding
are maturing, including multiple studies highlighting
quarantine/regulatory applications of DNA barcoding
and marketplace surveys of seafoods, medicinal plants,
and mushrooms.
Important questions for biology and for society are
becoming tractable. “What is this specimen on an agri-
cultural shipment?”, “Who eats whom in this whole food
web?”, and even “How many species are there?” are ques-
tions that can be answered in time periods ranging from
a few years to one or a few decades. The presentation by
Hebert outlines a vision for the “Planetary Biodiversity
Project”, a mega-science project that will run from 2020–
2040. Beyond elucidating the number and distributions
of species, the next phases of DNA barcoding may ex-
pand yet further into predicting community shifts with
climate change and improving the management of bio-
logical resources.
Adamowicz 161
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Research agendas enabled by DNA barcoding and meta-
barcoding are reflective of varied personal, institutional,
national, and international research interests relating to
biodiversity. Interestingly, standardization of genetic
markers is also permitting substantial creativity—with
scientifically novel projects showcased in these confer-
ence proceedings as well as socio-economically impor-
tant studies, such as those on medicinal species and
vectors. As attested by the diverse abstracts presented in
this special volume, DNA barcoding is enabling new re-
search avenues and new ways of accessing information
about biodiversity. This tool links a highly diverse com-
munity of researchers, and the 6th Conference repre-
sents an opportunity for inspiration, sharing of ideas,
discussion, and planning of the next exciting research
endeavours.
Acknowledgements
I sincerely thank Melania Cristescu and Graham Scoles
(Co-Editors of Genome) for the invitation to prepare this
contribution and for their vision regarding the value of
publishing this special open-access volume of confer-
ence abstracts. Thank you to Ryan Gregory for insightful
discussions as well as comments upon an earlier draft of
this manuscript. I also thank Robert Hanner and Paul
Hebert for helpful suggestions and discussions prior to
the preparation of this article. I gratefully acknowledge
the contributions of my colleagues co-serving with me
on the Conference Operating Committee (Mehrdad
Hajibabaei, Robert Hanner, Paul Hebert) as well as those
serving on the Local Support Committee (Suz Bateson,
Alex Borisenko, Jeremy deWaard, Meg Fritzsche, Sujeevan
Ratnasingham, Dirk Steinke, Brianne St. Jacques, Evgeny
Zakharov) and Scientific Organizing Committee (Inger
Greve Alsos, Filipe Costa, Torbjørn Ekrem, Manuel Elías
Gutiérrez, Winnie Hallwachs, Axel Hausmann, Paul Hebert,
Ian Hogg, Peter Hollingsworth, Dan Janzen, De-Zhu Li,
Scott Miller, Marko Mutanen, Cláudio Oliveira, Jan
Pawlowski, Rodolphe Rougerie, Elisabeth Stur, Nikolaus
Szucsich, Pablo Tubaro, Michelle van der Bank). All ab-
stracts were reviewed by 2–3 reviewers for acceptance to
the conference and for the organization of the parallel
sessions; thank you very much to the 60 reviewers/parallel
session organizers for this very valuable contribution to
the conference. Thank you to Meg Fritzsche for provid-
ing the contributed abstracts in spreadsheet format, en-
abling scoring and analysis.
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Contents / Contenu
DNA barcoding of Oeneis butterflies newly sampled in Mongolia Abe et al. 185
DNA barcoding and the origin of species Adamowicz 185
Species boundaries, mitochondrial introgression and nuclear mitochondrial
paralogs in the neotropical ant complex Ectatomma ruidum (Ectatomminae) Aguilar-Velasco et al. 185
Mosquito surveillance using DNA barcoding can help in vector-born
disease control Ahirrao et al. 185
DNA barcoding of small indigenous fish species from Tanguar Haor,
Bangladesh Ahmed et al. 186
DNA barcoding antlions (Myrmeleontidae: Neuroptera) of Pakistan Akhtar 186
Plant DNA in sediments: to which degree do they represent the flora? Alsos et al. 186
DNA barcoding for food safety and health consumer: the Italian experience Angelo et al. 186
Extending the ITS2-workbench with DNA barcoding capabilities Ankenbrand et al. 187
Comparison between morphological and DNA barcode-suggested species
boundaries among shallow-water amphipod fauna from the southern
European Atlantic coast
Antunes et al. 187
Integrative taxonomy reveals cryptic species and unusual speciation
pathway in flounders (Pleuronectiformes: Bothus) from the Brazilian coast Araújo Argolo et al. 187
Efficiency of DNA barcoding in the identification of flatfish
(Pleuronectiformes) from Brazilian coast Araújo Argolo et al. 188
What can DNA barcoding tell us about the dietary niche overlap of sibling
sympatric bat species? Arrizabalaga-Escudero et al. 188
DNA metabarcoding of endangered plant and animal species in seized
forensic samples Arulandhu et al. 188
DNA barcoding of plant pests clarifies cryptic species complexes Ashfaq 189
Application of DNA barcoding for phylogenetic identification of pathogenic
fungi associated with stem-end rot of mango in Pakistan Aslam et al. 189
A molecular phylogenetic study of pheretimoid species (Oligochaeta:
Megascolecidae) in Mindanao Island, Philippines Aspe et al. 189
Lessons from the first phase of the German Barcode of Life initiative
(2012–2015) Astrin et al. 190
Assessment of candidate DNA barcoding loci for the wheat and grass
family Poaceae in Egypt Awad et al. 190
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DNA barcoding, identification, and validation of the genus Phyllanthus in
Nigeria using rbcL and Atpf genetic markers and the taxonomic implication Awomukwu et al. 190
Myrmecophile communities of the African ant-plant Vachellia (Acacia)
drepanolobium Baker and Pierce 190
Molecular identification of mango hoppers infesting mango trees in Punjab
through DNA barcoding Banta et al. 190
The native bees of México and the DNA Barcode Of Life project Barajas and Packer 191
Is DNA barcode intraspecific variation associated with subspecies delimitation
and phenotypic variation? Barreira et al. 191
Is Nematocharax (Actinopterygii, Characiformes) a monotypic fish genus? Barreto et al. 191
New subspecies of an Arctic moth from SW Yukon: evolutionary and
ecological novelty Barrio et al. 192
Tiny, but complicated, mitochondrial genomes make barcoding challenging
in the parasitic phylum Apicomplexa Barta et al. 192
Food webs from fish guts: diet analysis using DNA barcoding increases
resolution and changes structure in Canadian boreal shield lake food webs Bartley et al. 192
Long-term monitoring of tropical arthropods and DNA barcoding Basset 193
DNA barcoding program at Naturalis Biodiversity Center, the Netherlands Beentjes et al. 193
All complete? Comparison of morphological and DNA-based biodiversity
assessments in one of the world’s best-studied stream ecosystems
(Breitenbach, Germany)
Beermann et al. 193
Assessing the distribution and genetic diversity of Antarctic springtails
(Collembola) Beet et al. 193
Assessing the diversity of New Zealand freshwater “EPT” macroinvertebrates Beet et al. 194
The effect of sample complexity and sequencing depth on DNA barcoding
of mixed-species pollen samples Bell et al. 194
DNA barcoding reveals microevolutionary signals in fire survival and
regeneration strategies in southern African Psoraleoid legumes Bello et al. 194
Barcoding as a tool to assess trophic impacts in an experimental
deforestation site in Borneo Bennett et al. 194
DNA barcoding of spidermites (Prostigmata: Tetranychidae) in vegetables
using COI and ITS2 markers Bennur et al. 195
Genomics applied to conservation and management of aquatic resources:
are the promises being filled? Bernatchez 195
Phylogenetic analysis, species identification and delimitation of
New Caledonian geckos and skinks using DNA barcoding Bernstein et al. 195
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The School Malaise Trap Program: the benefits of citizen science for
barcoding Berzitis et al. 196
Revisiting Darwin’s naturalization conundrum: explaining invasion success
of non-native trees and shrubs in southern Africa Bezeng et al. 196
DNA barcoding of dye-yielding plants from South India Bhattar et al. 196
Popularizing DNA barcoding in the identification of agricultural pests and
their natural enemies in Bangladesh Bhuiya and Mazumdar 196
Monitoring lake ecosystem health using metabarcoding of environmental
DNA: temporal persistence and ecological relevance Bista et al. 197
Building a DNA barcode reference library for Canadian spiders (Araneae) Blagoev et al. 197
Cryptic speciation among the spiders (Araneae) of North America: insights
from barcoding 2000 species Blagoev et al. 197
Metabarcoding of meiofauna and other eukaryotes in terrestrial and marine
ecosystems Blaxter and Jones 197
Environmental DNA detection and habitat occupancy of threatened spotted
gar (Lepisosteus oculatus)Boothroyd et al. 198
International Barcode Of Life: between the legal hammer and the policy
anvil Borisenko 198
Status report on barcode coverage for invasive alien species Borisenko et al. 198
DNA barcoding of larval fish and egg samples produced from the
driftnetting of two Lake Simcoe tributaries Bourque and Hanner 199
The long and the short of DNA barcodes: an approach using nanopore
sequencing Boutain and Boutain 199
A regional DNA barcode library for landscape-scale monitoring of
multi-taxa assemblages Bowser et al. 199
Ecology and systematics of New Zealand deep-sea squids Braid and Bolstad 199
DNA barcoding of coastal ichthyofauna from Bahia, northeastern Brazil,
South Atlantic: high efficiency for systematics and identification of cryptic
diversity
Brandão et al. 200
Optimizing metabarcoding of tropical Malaise trap samples and preliminary
results on seasonal changes in insect diversity in Penisular Malaysia Brandon-Mong et al. 200
The plastomes of mycoheterotrophic Ericaceae reveal extensive changes to
gene order and rare gene loss Braukmann and Stefanovic 200
Detecting signatures of competition from observational data: a novel
approach combining DNA barcoding, diversity partitioning, and
checkerboards at small spatial scales
Bringloe et al. 201
DNA metabarcoding of pollen: progress, technical limitations, and future
directions Brosi et al. 201
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Utility of DNA barcoding and other molecular methods for bloodmeal
analysis in mosquitoes (Diptera, Culicidae) of medical and (or) veterinary
importance in southern England, United Kingdom
Brugman et al. 201
Diffusing barcoding: the global spread of a good idea Bubela et al. 201
Evaluating the evolutionary and genetic relationships among the Andean
orchids of Ecuador Bucalo et al. 202
Using DNA barcodes to evaluate the level of endemism and isolation in
the avifauna of the Central Sierras in Córdoba, Argentina Bukowski et al. 202
First results of the Global Malaise Trap Program in Argentina: strikingly
high biodiversity in the southern extreme of the Atlantic Forest Bukowski et al. 202
DNA barcoding to determine the diets of Prairie grasshoppers Burgess et al. 202
Preliminary analysis of generic relationship of tribes Eucosmini and
Enarmoniini (Lepidoptera: Tortricidae) using DNA barcodes in Korea Byun et al. 203
Ecotoxicological applications of DNA barcoding to distinguish
laboratory-cultured organisms: a case study involving Hexagenia spp. Capretta et al. 203
Characterization of the COI gene in Carollia perspicilata (Chiroptera:
Phyllostomidae) from Amazonia Carvalho et al. 203
Use of integrative taxonomy and DNA barcoding for characterization of
ecological processes structuring marine benthic community assemblages in
British Columbia
Castelin et al. 204
Harnessing the power of DNA barcoding to detect and deter fish mislabeling
in South Africa Cawthorn et al. 204
DNA barcoding of mosquitoes (Culicidae) in the Yucatán Península Chable and Arce 204
Monitoring biodiversity for the early detection of aquatic invasive species
using metabarcoding applied across Canadian ports in the Pacific, Arctic,
Atlantic, and Great Lakes
Chain et al. 205
Assessing DNA barcodes as a diagnostic tool for North American reptiles
and amphibians in nature and natural history collections Chambers and Hebert 205
The complete chloroplast genomes of Aconitum: insight into taxonomy and
identification of complex plant lineages Chen et al. 205
Riverine barcoding: a proposed DNA barcoding study on the freshwater
fish species of Meenachil River of Kerala, India Cheriyan 205
Towards an universal genome-based DNA barcode – The PhyloAlps project Coissac et al. 206
Temperature-related activity of Gomphiocephalus hodgsoni (Collembola)
COI haplotypes in Taylor Valley, Antarctica: implications in a changing
climate
Collins and Hogg 206
An assessment of New Zealand rotifer diversity and global affinities using
COI barcodes Collins et al. 206
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The use of DNA barcodes in the identification of Biomphalaria species
(Mollusca: Planorbidae) for schistosomiasis control Cordeiro et al. 206
Genetic variation of endemic and endangered species of insular rodent
from Mexico Cortés-Calva et al. 207
Priming a DNA barcode library for marine Gastropoda of the continental
Portuguese coast and Azores Islands Costa et al. 207
Using different methods to access the difficult task to delimiting species in
a complex Neotropical hyperdiverse fish group Costa-Silva et al. 208
Advances in the identification of tuna larvae, Auxis, from western central
Atlantic using DNA barcoding Cota-Valentin et al. 208
From barcoding single individuals to metabarcoding biological communities:
towards understanding and managing invasive species Cristescu 208
Targeted detection of multiple species at risk (Unionidae) using environmental
DNA Currier et al. 208
Investigating terrestrial arthropod biodiversity in a tropical ecosystem using
barcode index numbers and phylogenetic community structure D’Souza 208
DNA barcoding of Javanese and Balinese freshwater fishes: molecular
insights into a poorly known ichthyofauna Darhuddin et al. 209
Human population density in Africa correlates with the evolutionary
history of its flora Davies et al. 209
Barcoding herpetological collections: discovering hidden biodiversity in a
hotspot Daza and Restrepo 209
Dissecting tropical earthworm biodiversity patterns in tropical rainforests
through the use of DNA barcoding Decaens et al. 210
The power and promise of environmental DNA for river biodiversity
monitoring Deiner and Altermatt 210
Using environmental DNA to track non-indigenous species in shipping
ports Deiner et al. 210
Species delimitation in the grasshopper genus Taeniopoda (Orthoptera:
Romaleidae) based on molecular and morphological evidence De Jesús-Bonilla and Riverón 210
DNA barcodes in resolving the taxonomic nomenclature of
Pseudoxytenanthera stocksii endemic to the Western Ghats, India Dhivya et al. 211
Authentication of Indian herbal products using DNA barcodes Dhivya et al. 211
DNA-based technologies for authentication of herbs and its admixtures –
a review Dhivya et al. 211
First DNA barcode reference library for the identification of South American
freshwater fish from Lower Paraná River Díaz et al. 212
DNA barcodes highlight unique research models in European butterflies Dinca et al. 212
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Phylogeography of the Indian Cobra (Naja naja) reveals genetically divergent
populations between the Indian subcontinent and Sri Lanka Dissanayake et al. 212
Barcoding Turkish geophythes: a new initiation for understanding biodiver-
sity and taxonomy Dönmez et al. 213
COI barcodes link population genetics with soil food web structure Donoso 213
Assessing the diversity of terrestrial invertebrates in the mangrove forests
of the Firth of Thames, New Zealand Doyle and Hogg 213
Finding whio: detection of blue duck using environmental DNA Doyle et al. 213
The effect of rainforest fragmentation on tropical mammals using leech
blood-meal analysis and DNA barcoding Drinkwater 213
Using metagenomics to show the efficacy of restoration in the New Jersey
Pine Barrens Eaton et al. 214
The Brazilian Barcode of Life (BrBOL) initiative and its potential to aid in
biodiversity conservation: an overview of the effort to catalogue mammals,
reptiles (including birds), and amphibians from a megadiverse region
Eizirik 214
The Norwegian Barcode of Life Network (NorBOL) Ekrem et al. 214
Testing primer bias and biomass – sequence relationships in metabarcod-
ing: implications for monitoring of freshwater invertebrate communities Elbrecht and Leese 215
PrimerMiner: An R package for the development of universal barcoding
primers and mini barcodes using partial COI sequences Elbrecht and Leese 215
Who is Moina micrura? An example of how barcodes can help to clarify
highly confused species Elena et al. 215
After 10 years of DNA barcoding in Mexico – where are we? Eliás-Gutiérrez and León-Règagnon 215
DNA barcoding of Skwala stoneflies from north-central British Columbia
reveals potential new species Erasmus et al. 216
COI-based identification of Orius species (Hemiptera: Anthocoridae)
from Iran Erfan et al. 216
Biodiversity assessment of plant communities from soil eDNA: impact of
marker selection on perceived community turnover Fahner et al. 216
The effect of anthropogenic disturbance on diversity and phylogenetic
structure of ants (Hymenoptera: Formicidae) Fairweather et al. 216
Barcodes, bugs, and bats Fenton 217
DNA barcoding of Holarctic Microgastrinae wasps (Hymenoptera): a major
step in the integrative taxonomy of these caterpillar parasitoids Fernandez-Triana et al. 217
Bats as drivers of bacterial biodiversity across multiple trophic levels of
subterranean biomes Fofanov et al. 217
Plant Pest Barcoding Campaign update Frewin et al. 217
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Application of DNA barcoding for the identification of intercepted pests Frewin et al. 218
A framework for estimating eDNA sensitivity Furlan et al. 218
Reconstructing interactions among plants, insect herbivores, and
phoretic mites using DNA barcodes: modeling coextinctions under
projected climate change
Garcia-Robledo et al. 218
Preliminary results from Malaise traps in southern Yucatan Peninsula,
Mexico Garibay 218
DNA barcoding for identification of Cephalotaxus and the discovery of
new species Gao et al. 219
Collection data of black flies, mosquitoes, and sand flies of Mexico for
further DNA barcode study Garza-Hernández et al. 219
Identifying Malva species in Libya through DNA barcodes techniques,
using four candidate DNA barcoding markers Gawhari et al. 219
Global perspectives on participating in the International Barcode of Life Project Geary and Bubela 219
The FREDIE project – different lessons from a large-scale DNA barcoding
campaign Geiger et al. 220
DNA barcoding in ethnobotany and ethnopharmacology: identifying
medicinal plants traded in local markets Ghorbani et al. 220
The good, the bad, and the ugly: DNA barcoding a nightmare taxon Gibbs 220
Biomonitoring boreal wetlands using environmental DNA barcoding and
high-throughput sequencing Gibson et al. 220
Evolution of ecological communities through the lens of an island
chronosequence Gillespie and Krehenwinkel 221
Metabarcoding for surveillance and monitoring: meeting policy objectives
in the real world Glover et al. 221
Barcodes, food webs, and insect community ecology Godfray 221
Integrating phylogenetic and functional diversity in REDD⫹projects:
a pilot in the Pacific rainforest in Colombia Gonzalez et al. 221
Barcoding as a useful tool for South American wild bee systematics Gonzalez-Vaquero et al. 222
DNA barcoding reveals a possible cryptic species complex of Mycalesis
mineus: a case study from Sri Lanka Goonesekera et al. 222
The application of next-generation sequencing barcoding in identifying
mixed-pollen samples from a historic bee collection Gous et al. 222
Integrative approach and molecular barcoding of dagger and needle
nematodes infesting grapevine soils in Portugal Gutiérrez Gutiérrez et al. 222
Potato cyst nematodes infesting potato fields in Ecuador: integrative
diagnosis and molecular phylogeny Gutiérrez Gutiérrez et al. 223
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Recovery of nucleic acids from microhymenopterans with four non-destructive
methodologies and considerations for museum slides preparations Guzmán-Larralde et al. 223
Evolving the concept, and use, of DNA barcode libraries Gwiazdowski and Hanner 223
DNA barcoding helps to fight against frauds – a case study of authentication
of deer products Ha and Lee 224
Environmental DNA barcoding: from the Arctic to the tropics – and
everywhere in between Hajibabaei 224
DNA barcoding of Philippine Helicarionidae (Mollusca: Gastropoda) Halili et al. 224
DNA barcode-based wildlife forensics for resolving the origin of claw
samples Hange and Khedkar 224
Barcoding the ants of Iguazú National Park, a biodiversity hotspot in
northeastern Argentina Hanisch et al. 224
Deep intraspecific barcode splits: cryptic species, Wolbachia or something
else? Hänninen and Mutanen 225
Balancing sensitivity and specificity in primer design for eDNA studies
using ePRIMER Harvey et al. 225
Calibrating the taxonomy of a megadiverse family on BOLD: 2700 geometrid
moth types barcoded (Geometridae, Lepidoptera) Hausmann et al. 225
Using DNA metabarcoding to investigate the medicinal properties of honey Hawkins et al. 225
From iBOL to planetary biodiversity Hebert 226
The San Diego Biodiversity Project Henter et al. 226
Diversity, species delimitation, and evolution of insect viruses Herniou et al. 226
Barcodes to investigate the plant visitor community: Brassicogethes spp. Hicks et al. 227
Arctic biodiversity – from barcodes to species interactions Hik 227
Impacts of anthropogenic disturbance on arthropod biodiversity and
community structure Ho and Smith 227
The place of barcoding in biodiversity informatics Hobern 227
The application of DNA barcoding for bio-security: a perspective from the UK Hodgetts et al. 228
Genomic analyses from highly degraded DNA Hofreiter et al. 228
International Society for the Barcode of Life Hollingsworth 228
Identifying road-killed animals in a Brazilian Biological Reserve crossed
by a highway using DNA barcodes Hollunder Klippel et al. 228
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DNA barcoding of 100 common medicinal plants in Malaysia for species
authentication Hong et al. 229
DNA barcoding Canadian scyphozoans Hotke 229
The phylogeography of the Hyalella azteca species complex in North
America Hrycyshyn and Witt 229
Recent trends in bio-diversity conservation Hummel 229
Comparative phylogeography of Javanese and Balinese freshwater fishes:
DNA barcodes shed light on Quaternary range expansion dynamic in a
biodiversity hotspot
Hutama et al. 230
Identification of six skate species by means of multiplex PNA fluorescence
melting curve analysis Hwang et al. 230
Subterranean pool party: determining the trophic links between subterranean
invertebrates in a groundwater system in Western Australia Hyde et al. 230
DNA barcode analysis of thrips (Thysanoptera) diversity in Pakistan
reveals cryptic species complexes Iftikhar and Ashfaq 230
Alternative barcodes for the identification of human and animal pathogenic
fungi Irinyi et al. 231
Pandora’s Box in a pill – unveiling the composition of herbal supplements Ivanova et al. 231
Quantification of diversity of agriculturally important insects through DNA
barcoding Jalali and Venkatesan 231
Combining citizen science and DNA-assisted species identification to
enable “a new kind of ecology” James 231
Barcoding from a tropical trench, looking upward and outward Janzen and Hallwachs 232
Genome size diversity and phylogenetic history in freshwater amphipod
(Hyalella spp.) species complexes Jeffery et al. 232
DNA barcoding of the tribe Archipini (Lepidoptera, Tortricidae, Tortricinae) in
Japan, with notes on the geographic variations of widely distributed species Jinbo and Ota 232
Cataloguing genetic variations in Sesamia inferens populations infesting
rice using DNA barcoding Jindal et al. 233
Monitoring of butterflies by schoolchildren to increase environmental
awareness and assess effects of climate change in peninsular Malaysia Jisming-See et al. 233
DNA barcoding contributes to the taxonomy of Afrotropical hover flies
(Insecta: Diptera: Syrphidae) Jordaens et al. 233
DNA barcoding identifies an introduced hover fly species (Diptera: Syrphidae:
Syrphinae) in the Afrotropics Jordaens et al. 234
DNA barcoding to monitor the biodiversity of marine mammals: species
identification along the French and Mauritanian Atlantic coasts, and diet
analysis
Jung 234
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DNA barcoding of aquatic plants may aid in understanding species
diversity and evolutionary relationships Kadam et al. 234
Parallel food authenticity and microbial composition analyses using DNA
barcoding and next-generation sequencing Kaeppel et al. 234
Use of BOLD systems in species conservation V. Kalyankar et al. 235
DNA barcoding may aid in conservation and fare trading of ornamental
fish in India A. Kalyankar et al. 235
High-throughput molecular approach for quantifying pest regulation
services and disservices provided by farmland insect communities Kamenova et al. 235
Barcoding of fresh water fishes from Pakistan Karim et al. 235
Genetic introgression between species: resolving challenges to Neo-Darwinism
and DNA barcoding Kartavtsev 236
DNA barcoding of green algae Caulerpa species (Caulerpales: Chlorophyta)
from Andaman Islands, India Karthick et al. 236
DNA metabarcoding illuminates dietary niche partitioning by large
mammalian herbivores in Africa Kartzinel and Pringle 236
The phylogenetic signal of DNA barcodes: insights on insect families Kekkonen 236
NGS pollen metabarcoding and automatic taxonomic classification Keller et al. 237
One fish many stories Khedkar 237
Freshwater zooplankton diversity and distribution pattern in the Godavari
River revealed by COI gene sequences Khobragade et al. 237
Application of environmental DNA methods for assessing biodiversity and
biomonitoring endangered species: a case study of Jefferson salamander
(Ambystoma jeffersonianum) in southern Ontario, Canada
King et al. 237
DNA barcoding plant–insect interactions in a tropical rainforest Kishimoto-Yamada and Ito 238
The global Trichoptera Barcode of Life campaign: implications and
applications Kjer and Zhou 238
Censusing marine life in the 21st Century Knowlton and Leray 238
Barcoding a biodiversity hotspot: Malaise-trapped insects of Southern
California Kohn et al. 238
Diversity of indoor fungi: what does it tell about the health of buildings? Korpelainen and Pietiläinen 239
Comparison of five extraction protocols and direct PCR for the recovery of
trace DNA in chironomid pupal exuviae Kranzfelder et al. 239
Tracking evolutionary diversity and phylogenetic structure across global
forest dynamics plots using plant DNA barcodes Kress et al. 239
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Development of a DNA barcode database of captive animals in
Thiruvananthapuram Zoo, Kerala, India U.S. Kumar et al. 239
Estimating the extent of adulteration in highly traded medicinal plants in
herbal raw drugs market in South India J.U.S. Kumar et al. 240
DNA barcoding of sea cucumbers (Echinodermata: Holothuroidea) of the
southwest coast of India Kumar and Deepa 240
DNA barcoding and invading suckermouth sailfin catfish Pterygoplichthys
(Siluriformes: Loricariidae) in India: issues with possible hybrids Kumar and Smrithy 240
What it takes to identify 5120 vascular plant species of Canada with DNA
barcodes Kuzmina et al. 240
Comparative phylogeography and population genetic structure of 10
widespread small vertebrate species in Morocco Lalis et al. 241
Barcoding Paralobesia cypripediana (Tortricidae): a stealthy micromoth
feeding on the threatened orchid Cypripedium reginae Landry et al. 241
Development of a molecular detection test, based on PCR technology, to
detect specific mushroom DNA in soil samples Laperriere et al. 241
Biodiversity discovery, documentation, and analysis in the digital era La Salle 241
From a local barcoding initiative to a continental-scale, multi-institutional
assessment of avian diversification in the Neotropics Lavinia et al. 242
Calibrating the molecular clock beyond cytochrome b: assessing the
evolutionary rate of COI in birds Lavinia et al. 242
Exploring diversity and distribution of a nudibranch genus (Chromodoris)
in the Indo-Pacific using DNA barcodes Layton et al. 242
DNA barcoding on the slime trail: the Canadian invasion of the terrestrial
gastropod Cepaea nemoralis (Stylommatophora: Helicidae) and new
perspectives for studying threatened species
Layton et al. 242
New methods give insight into DNA barcoding Canada’s freshwater
mussels (Bivalvia: Unionoida) Layton et al. 243
Utility of the ribosomal DNA ITS2 region for the identification of
Calliphoridae (Diptera: Calliphoridae) of forensic importance in Colombia Lea and Castro 243
A metabarcoding approach to measuring beta diversity: Costa Rican dry
forest arthropods and their associated bacteria Ledger et al. 243
Species delimitation of Eupithecia (Lepidoptera: Geometridae) using a
ddRAD-Seq approach Lee and Mutanen 244
Comparison of sampling methods including blowfly-derived mammal DNA
for tropical mammal diversity assessments Lee et al. 244
Muthi from the wild: a survey of bulbous and perennial herbs traded at the
Faraday Muthi Market in Johannesburg, South Africa, using DNA barcoding
as an identification tool
Lekganyane et al. 244
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Molecular approach in species determination of Tetranychus complex in
polyhouse cucumber Lenin et al. 244
Using mitochondrial genome targets within the phylum Apicomplexa:
divergent life cycle stages, cryptic species, and unexplored diversity in the
coccidia
Leveille et al. 245
Who laid the egg? Establishing the identity, distribution, and abundance of
fish eggs in the Mexican Caribbean with barcodes Leyva-Cruz et al. 245
High-accuracy de novo assembly and SNP detection of chloroplast genomes
for DNA-barcoding studies Li et al. 245
Application of Barcode of Life principles, to protect the international
cotton supply chain Liang et al. 245
Barcode UK – beyond the visible: a science–art collaboration Liggins and de Vere 246
Patterns of genetic diversification of bats in the Caribbean and their
relationship to other populations across the Neotropics Lim 246
Progress in DNA barcoding the bats of Peninsular Malaysia Lim and Wilson 246
Exploring genetic divergence in a species-rich insect genus using 2790
DNA barcodes Lin et al. 246
Testing DNA barcoding of the recently diverged species in the genus
Gentiana (Gentianaceae) J. Liu et al. 247
Dietary analysis of marine fishes: enhancing the detection of barcoded
reads for high-throughout sequencing in combination with blocking primers H. Liu et al. 247
Updates on the status of giant clams in the Philippines using mitochondrial
COI and 16S rRNA genes Lizano and Santos 247
DNA barcodes of polychaetes (Annelida: Polychaeta) from the southern
European Atlantic coast underscores the incipient state of the global
reference library for this taxon
Lobo et al. 247
Stepwise implementation of high-throughput sequencing metabarcoding to
estuarine macrobenthic communities Lobo et al. 248
Barcoding the Diplostomoidea (Platyhelminthes: Digenea) Locke and Marcogliese 248
An Arctic molecular clock? Genetic divergence in echinoderms across the
Bering Strait Loeza-Quintana and Adamowicz 248
Blood meal analysis and virus detection in blood-fed mosquitoes collected
during the 2006–2007 Rift Valley fever outbreak in Kenya Lutomiah et al. 249
NNEditor: neural N-label editor toward automated sequence finishing Ma et al. 249
Leaves of leaf-cutting bees: identity and diversity determined by DNA
barcoding MacIvor et al. 249
DNA barcodes of the genus Oxysarcodexia (Diptera: Sarcophagidae) from
south Brazil Madeira et al. 250
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DNA barcoding unravels the role of morphology and echolocation in
bat–insect relationships in Jamaica Magalhaes de Oliveira et al. 250
Environmental barcoding of aquatic invertebrates in Norway Majaneva et al. 250
Distribution and identification of larval Billfish (Istiophoridae) in the Gulf
of Mexico and Caribbean using DNA barcoding Malca et al. 250
DNA barcode reference library for Indian medicinal plants of high trade
volume Malik and Babbar 251
Integrating DNA barcode data with behavioral and ecological data in a
study of white-faced capuchin faunivory Mallott et al. 251
Sensitive detection of water soldier and other invasive aquatic plants using
environmental DNA Marinich et al. 251
Barcoding the Swiss lichens and associated fungal communities using
barcoded amplicon 454 pyrosequencing Mark et al. 252
Improving our understanding of metacommunity structure using DNA
barcoding Martin et al. 252
Mexican DNA barcoding laboratory (Chetumal Node): six years after Martínez-Arce and Eliás-Gutiérrez 252
Highlights and new discoveries in the Mexican cladocera Martínez-Caballero and Elías-Gutiérrez 252
DNA barcoding of Croton draco var. draco, an ethnomedicinal resource
for traditional indigenous doctors from Veracruz, México Martínez de la Vega et al. 253
Monitoring aquatic species’ invasions using environmental DNA Masson et al. 253
Diet analysis of European free-tailed bats Tadarida teniotis using
high-throughput sequencing Mata et al. 253
Savanna fire and the origins of the “underground forests” of Africa Maurin et al. 253
Biodiversity study of Bangladeshi parasitoid wasps (Insecta: Hymenoptera)
of Malaise trap collections using DNA barcoding techniques Mazumdar et al. 254
Determining grasshopper (Orthoptera: Acrididae) diet and niche overlap
using high-throughput sequencing and DNA barcodes recovered from gut
contents
McClenaghan et al. 254
DNA barcoding the Demospongiae (Porifera) from the Bay of Plenty, New
Zealand – connecting morphology with molecules Mc Cormack et al. 254
Effects of land management on soil biodiversity and nutrients using a DNA
metasystematic approach McGee et al. 254
Medicinal plants recommended by the World Health Organization: DNA
barcode identification associated with chemical analyses guarantees their
quality
Melo Palhares et al. 255
Recent adaptive origin of the cave fish Ancistrus cryptophthalmus Mendes et al. 255
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Cryptic diversity and geographic patterns revealed by building the DNA
barcode library of Colombian birds Mendoza et al. 255
Progress and prospects in using DNA barcoding to advance coevolutionary
biology Merilo et al. 255
Facing the challenge of growing numbers of fungal infections –
sequence-based fungal identification using the ISHAM-ITS reference
database
Meyer et al. 256
BOLD tools in support of STEM education Milton et al. 256
Dung, dorpers and diets: dietary analysis of sheep in rangelands through
DNA metabarcoding Mitchell and Alemseged 256
Testing for positive selection in mitochondrial and nuclear energy-related
genes in Pterygota (flying insects) Mitterboeck et al. 257
Molecular evolutionary rates in freshwater versus terrestrial insects Mitterboeck et al. 257
MiFish, a set of universal primers for metabarcoding environmental DNA
from fishes: detection of ⬎230 species from aquarium tanks and coral reefs
in the subtropical western North Pacific
Miya et al. 257
Validation of commercially important fish of India Mohekar et al. 258
Associating larvae and adults of high Andean aquatic insects: a preliminary
analysis using DNA barcoding Molina et al. 258
A look at the past to plan for the future – 10 years of fish barcodes in
Quintana Roo Moreno et al. 258
Identification of diverse fish species in a closed aquarium environment using
eDNA Morey and Hanner 258
Species identification in Malaise trap samples by DNA barcodes using
NGS – a “scoring matrix” of four amplicons Morinière et al. 259
Large-scale DNA barcoding of marine hydroids of the superfamily
Plumularioidea (Cnidaria: Hydrozoa) Moura et al. 259
DNA barcoding of vertebrate diversity in Qatar Mushtak et al. 259
Progress in the barcoding of illegally traded South African wildlife species
at the National Zoological Gardens of South Africa Mwale et al. 259
Real-time PCR for seafood authenticity: an extension of DNA barcoding Naaum et al. 260
Minimum information for conducting a DNA barcoding market survey Naaum et al. 260
DNA barcoding and real-time PCR for identification of entrained species Naaum et al. 260
DNA barcoding Congolese snakes Nagy et al. 260
Evaluation of recent enforcement to prohibit the slaughter of cow and its
progeny for beef in India using DNA barcoding Naikwade et al. 261
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Confirmation of genetic diversity in morphologically distinct accessions of
the Solanum nigrum L. complex using DNA barcodes Nalina et al. 261
The sustainable use of global biodiversity: scope and relevance of the
Nagoya Protocol and the Convention of Biological Diversity for Natural
History Collections and researchers
Neumann et al. 261
Placing the freshwater bivalves (Unionoida) of Peninsular Malaysia on the
bivalve tree of life Ng et al. 262
A great inventory of the small: combining BOLD datamining and focused
sampling hugely increases knowledge of taxonomy, biology, and distribution
of leafmining pygmy moths (Lepidoptera: Nepticulidae)
Nieukerken et al. 262
DNA barcodes highlight genetic diversity patterns in rodents from desert
and Andean areas of Argentina Ojeda et al. 262
Compilation and validation of a global DNA barcode reference library for
European marine fishes Oliveira et al. 262
Authenticating Thai herbal products, Boraphet: Tinospora crispa
(Menispermeae) by DNA barcoding coupled with high resolution melting
analysis
Osathanunkul et al. 263
Keeping the bees: identifying and protecting wild bees Packer 263
Ancient Lake Titicaca as an evolutionary arena for morphological
diversification in Hyalella amphipods Paez-Parent et al. 263
Dark taxa and hidden texts: obstacles to integrating barcodes and taxonomy Page 263
Environmental DNA metabarcoding to investigate historic changes in
biodiversity Pansu et al. 264
Contrasting soil biodiversity patterns along an altitudinal gradient Pansu et al. 264
By-passing the taxonomic impediment in Neotropical Collembola to
measure changes in diversity and phylogenetic structure Pare and Smith 264
A DNA barcode reference library for Asian quarantine pests Park et al. 264
BARCODING.MED: building a high taxonomic resolution database for the
Mediterranean basin Paupério et al. 265
Inferring biotic indices from metabarcoding data: promises and challenges Pawlowski et al. 265
Streamlining scholarly publication of Barcode of Life data Penev et al. 265
Amino acid variation and protein structure of COI barcodes – insights from
a Metazoa-wide sample Pentinsaari et al. 265
The Global Malaise Program: assessing global biodiversity using mass
sampling and DNA barcoding Perez et al. 266
Applying DNA barcoding to the manufacturing supply chain, to reduce
substitution and adulteration fraud Phelan and Jonker 266
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An exploration of sufficient sampling effort to describe intraspecific DNA
barcode haplotype diversity: examples from the ray-finned fishes (Chordata:
Actinopterygii)
Phillips et al. 266
What we need to know about biodiversity in order to conserve it Pimm 266
Using DNA barcoding (COI) to assess diversity of the New Zealand native
aphid genus Schizaphis (Hemiptera: Aphididae) Podmore et al. 266
Ribosomal DNA and plastid markers used to sample fungal and plant
communities from wetland soils reveal complementary biotas Porter et al. 267
Species tree inference from multilocus data sets Posada 267
Deep mitochondrial divergences within species are the rule rather than the
exception in western Palearctic leafcutter bees Praz 267
DNA barcodes from century-old type specimens using next-generation
sequencing Prosser et al. 267
Species identification of ancient tuna remains using a novel paleogenetic
protocol and barcoding techniques Puncher et al. 268
Mode and tempo of diversification of Hyalella (Crustacea: Amphipoda) in
ancient Lake Titicaca Pyle and Adamowicz 268
DNA barcoding and metabarcoding with the Oxford Nanopore MinION Ramgren et al. 268
Exploring the gut bacterial communities associated with larval Helicoverpa
armigera (Hübner) (Lepidopera: Noctuidae) based on next-generation
sequencing
Ranjith et al. 268
DNA barcodes for the fish of the second-largest river of India: the Godavari Rathod et al. 269
Informatics tools for the second decade of DNA barcoding Ratnasingham 269
A compendium of locally harvested trees traded as traditional medicine at
the Faraday Muthi Market in Johannesburg, South Africa Ratsoma et al. 269
Crabs, scallops, fish, and more: barcoding the marine fauna of the North
Sea Raupach et al. 270
DNA barcoding of Pteris species by psbA–trnH intergenic spacer:
taxonomically complex and polyploid ferns Ravi et al. 270
“Ethnoichthyogenomics”: identification, phylogeny and documentation of
ethnomedicinally important fishes using DNA barcodes Ravitchandirane and Thangaraj 270
DNA barcoding of rodent pests in South India Rengarajan and Archunan 271
DNA barcoding and cetacean species identification along the Mauritanian
coast, including the spectacular identification of a Balaenoptera omurai
specimen
Rey et al. 271
DNA barcoding reveals cryptic species and high genetic divergence in
pearl cichlid of Geophagus brasiliensis complex from northeastern Brazil Ribeiro de Souza et al. 271
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Pollen analysis: is metabarcoding the next generation? Richardson et al. 271
Assessing invertebrate dispersal among restored streams in the North Island
of New Zealand using DNA barcoding Riding et al. 272
Dissecting Arctic food webs by DNA barcodes Roslin and Wirta 272
DNA metabarcoding of saproxylic beetles – streamlining species identification
for large-scale forest biomonitoring Rougerie et al. 272
A tale of long tails: combining DNA barcoding and RAD sequencing to
investigate the diversification of comet and moon moths (Lepidoptera,
Saturniidae)
Rougerie et al. 272
Integrative taxonomy uncovers hidden diversity within three genera of
Canadian Osmiini (Hymenoptera: Megachilidae) Rowe et al. 273
Protection of Canadian biodiversity and trade through improved ability to
monitor invasive freshwater fish M. Roy et al. 273
Plant barcoding of a wildlife sanctuary across a wide climatic zone,
Uttaranchal, India S. Roy et al. 273
Using mini-barcodes to investigate the species composition of the Late Holocene
(3500 to 200 years BP) fishery at EeRb-144, British Columbia, Canada Royle et al. 273
Approaches for identification of Colchicum L. species in the flora of
Turkey by morphological parameters and DNA barcoding S¸ahin et al. 274
DNA metabarcoding of springtails (Collembola) Saitoh et al. 274
DNA barcoding to support biodiversity conservation, sustainable harvest-
ing, and trade in Peru Salinas et al. 274
Dietary overlap and seasonality in three species of mormoopid bats from a
tropical dry forest Salinas-Ramos et al. 275
The feasibility of detecting an Asian carp invasion using environmental
DNA and next-generation sequencing Salisbury et al. 275
Ethnobotany genomics – use of DNA barcoding to explore cryptic diver-
sity in medicinally important plants in the Indian subcontinent Sambandan et al. 275
Socio-economic impacts of DNA barcoding on Philippine fisheries and
aquatic resources M.D. Santos et al. 276
DNA barcoding of Philippine fish: first record of marine species in a
biodiversity hotspot M.D. Santos et al. 276
Genetic characterization of the red algae Asparagopsis armata and
Asparagopsis taxiformis (Bonnemaisoniaceae) from the Azores R. Santos et al. 276
DNA barcodes for authentication of commercially important Indian spices Saravanan et al. 276
What are you really eating in Mexico? A preliminary study of fish fillets Sarmiento Camacho and Valdéz-Moreno 277
The Muscidae of Canada: towards a complete DNA barcode reference library Savage et al. 277
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The Barcode of Wildlife Project, Part 1: a systemic barcoding initiative to
protect endangered species Schindel and Trizna 277
DNA barcoding medicinal plants from Pakistan Schori et al. 278
Using environmental DNA to detect endangered Redside Dace, Clinostomus
Elongatus
Serrao et al. 278
Phylogeography and conservation of endangered Redside Dace, Clinostomus
elongatus
Serrao et al. 278
The applications and limitations of DNA markers in authenticating herbal
materials Shaw et al. 278
Evaluation of four barcoding loci in the class Calyciflorae (phylum:
Polypetalae-dicotyledons) Sheth and Thaker 278
Molecular phylogenetic analysis of Cassia species using DNA barcoding Sheth and Thaker 279
DNA barcoding based phylogenetic assessment of some Vigna species
inferred from nrDNA internal transcribed spacer 2 (ITS2) sequences Sheth et al. 279
TCM-identifier: an integrated and user-friendly software package for
species identification of traditional Chinese herbal materials Shi et al. 279
Exposing the illegal trade in Encephalartos species at the Faraday Muthi
Market in South Africa using DNA barcoding Shiba et al. 279
An imperative action in invasives management – spreading rapid species
identification to developing countries Shimura and Duthie 280
DNA barcode-based true bugs (Heteroptera) surveillance for agriculture
crops from Maharashtra Shinde and Khedkar 280
Non-destructive DNA extraction approaches for massive parallel multiplex
sequencing for specimen identification and environmental DNA barcoding
using high-throughput sequencing platforms
Shokralla et al. 280
Gut content metabarcoding of Maumee River fishes: insights on fish diet
during early spring Shortridge and Miner 280
Model-based clustering techniques for analyzing RNA-seq data Silva et al. 281
Documenting the biodiversity of a local sandhill flora using DNA barcodes:
an investigation into the patterns of resolution in polytypic taxa Silvis et al. 281
Urban parks: refuges for tropical butterflies? Sing et al. 281
Virtual Barcoding: identification of every animal species in a single
closed-tube reaction Sirianni and Wangh 281
Elevation, crypsis, and phylogenetic community structure of Neotropical
arthropods Smith et al. 282
Changing oceans of biodiversity Snelgrove 282
Phylogeography of Diptera in northern North American glacial refugia Solecki et al. 282
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A special issue on DNA barcoding edited by the Belgian Network for
DNA Barcoding (BeBoL) Sonet et al. 283
Large-scale DNA barcoding of ants from Ecuador Sonet et al. 283
High-throughput sequencing of PCR amplicons: a test to barcode a bee
species complex (Hymenoptera: Apoidea: Halictidae) and survey
Wolbachia infections
Sonet et al. 283
Barcoding plant hotspots in Patagonian Monte Desert Souto et al. 283
Validation of NGS metabarcoding for detection and identification of
(freshwater) invertebrates Speksnijder et al. 284
Using next-generation sequencing to identify the botanic origin of pollen
collected from foraging honeybees Sprague et al. 284
Species admixtures in herbal trade: causes, consequences and mitigation Srirama et al. 284
Identification of species adulteration in medicinal plant raw drugs by DNA
barcoding Stalin et al. 285
Linking adults and immatures of South African marine fishes Steinke et al. 285
The International Union for Biological Sciences (IUBS): unified biology,
biodiversity, barcoding, and genomics Stenseth and Kevan 285
A recent evolutionary origin of most extant animal species? Mitogenome
and DNA barcode evidence from humans and other animals Stoeckle and Thaler 285
Future prospects and lessons learned from nine years of Chironomidae
(Diptera) DNA barcoding Stur and Ekrem 286
Threats to terrestrial vertebrates in Canada Stutchbury 286
DNA barcodes of Japanese Merodon hoverflies (Diptera: Syrphidae): high
morphological variation and low haplotype diversity of the invasive species Sutou and Ito 286
DNA barcoding the plants of Monte Desert, Argentina Tadey and Souto 286
Utility of DNA barcoding for the conservation and authentication of
medicinal plant species of economically poor areas of Pakistan to improve
the socio-economic condition of indigenous people
Tahir et al. 286
Using DNA barcoding to document interactions among bats, insects and
plants in the highly fragmented Atlantic forest of Brazil Teixeira et al. 287
BIO’s Bio-Inventory and Collections Unit (BIC): specimen vouchers and
barcodes create a unique natural history resource Telfer et al. 287
A comparison of traditional morphological and next-generation molecular
methods for the identification of benthic invertebrate and fish species in
central British Columbia streams
Thielman et al. 287
Towards a safe herbal medicine in Ghana, the role of DNA barcoding Thomas 288
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DNA barcoding of invasive species in relation to Canadian federal policy
and law Thomas et al. 288
New models, new data: can DNA barcoding help with the development of
general ecosystem models? Tittensor 288
The origin of the Sardinian Blue, Pseudophilotes barbagiae (Lepidoptera:
Lycaenidae): Out-of-Europe or Out-of-Africa? Todisco et al. 288
CSI: Guelph – forensic applications of DNA barcoding for wildlife
identification and food authentication Topan 289
The Barcode of Wildlife Project, part 2: data pipeline and GenBank
submission Trizna and Schindel 289
Integration of DNA barcoding for surveillance of avian influenza and
Newcastle disease in migratory birds Tseren-Ochir et al. 289
Molecular-phylogenetic reconstruction and taxonomic investigation of
eelpouts (Cottoidei: Zoarcales) based on two mitochondrial genes Turanov et al. 289
Plant DNA barcoding project in Kazakhstan Turuspekov and Abugalieva 290
DNA barcoding of selected Philippine pomacentrids Valeroso et al. 290
Pelecanus occidantalis infected by two related parasites species of
Contracaecum in Baja California peninsula: new records and ecological
perspectives
Valles Vega et al. 290
Insights from the Tree of Sex: why so many ways of doing it? Vamosi et al. 290
Advance in the identification of Palinuridae and Scyllaridae Phyllosomas
using DNA barcoding in front of the coast of the Mexican Caribbean Vásquez-Yeomans et al. 291
Utility of DNA barcodes for the identification of parasitic nematodes Velarde-Aguilar and León-Règagnon 291
Molecular identification of egg parasitoid, Trichogramma species of India
using COI and ITS-II regions and their phylogenetic relationships Venkatesan et al. 291
Investigating the floral preferences of pollinating insects using pollen DNA
metabarcoding Vere et al. 292
Identification of Belgian mosquito species (Diptera: Culicidae) by DNA
barcoding Versteirt et al. 292
Identifying forensic species of Diptera in southeast Brazil using DNA barcodes Viana Oliveira et al. 292
Phylogeographic structure of Dynamene edwardsi (Crustacea: Isopoda)
matches remarkably the sequential genesis of the Macaronesian islands Vieira et al. 293
Pattern of nucleotide variations in the standard DNA barcode loci in
different genera of Indian Zingiberaceae Vinitha et al. 293
Comprehensive phylogeographic assessments as a tool to understand and
protect biodiversity on islands Voda et al. 293
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Species from feces: reliably identifying global bat species with a DNA
mini-barcode assay Walker et al. 294
Using barcoding to analyse prey consumption by generalist predators in
rice ecosystems Wang et al. 294
DNA metabarcoding of marine hard-bottom communities using 18S and COI Wangensteen et al. 294
Barcoding the fishes of Australia – progress, uses, and lessons learnt Ward 294
DNA barcoding the plants of San Diego County, California: on the verge
of the first complete DNA barcode reference library for a globally
important regional flora
Warne et al. 295
A market survey of fish species substitutions in the United States to
advance seafood traceability requirements Warner et al. 295
Classifying DNA barcode multi-locus sequences with feature vectors and
supervised approaches Weitschek et al. 295
Barcoding forensic traces – practical challenges Wesselink and Kuiper 295
Barcoding New Zealand spiders West et al. 296
Urban biodiversity explored using intensive multi-year sampling of insects
in Los Angeles Wetzer and Brown 296
Diversity and human perceptions of bees in Southeast Asian megacities Wilson et al. 296
Testing the waters: using NGS to monitor zooplankton communities Woods et al. 297
Evaluation of the relation between phytochemical composition and genetic
diversity in tropical plant species using DNA barcodes Worthy et al. 297
Assessing benthic macroinvertebrate temporal turnover in a remote wetland
through environmental barcoding Wright et al. 297
Barcoding wild edible mushrooms in southwestern China Xu et al. 297
Plant diversity assessments in tropical forests of SE Asia Yahara 298
Utilizing DNA barcoding to conserve Canada’s endangered populations of
red mulberry (Morus rubra L., Moraceae) Yan et al. 298
DNA barcoding database for Chinese pharmacopoeia Yao et al. 298
Ecological applications of DNA barcoding: the African experiences Yessoufou and van der Bank 298
Patterns of protein evolution in cytochrome c oxidase subunit I (COI) from
the class Arachnida Young and Hebert 299
DNA barcoding in the marine realm: challenges, successes, and support for
DNA-based species delimitation among the Maxillopoda Young et al. 299
High-throughput monitoring of wild bee diversity and abundance via mi-
togenomics Yu et al. 299
Contents / Contenu 183
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Higher fungal diversity in dead wood reduces CO
2
emissions in a natural
forest Yu et al. 300
DNA barcoding of the Chinese fly Chrysomya megacephala and comparison
to its published data around the world Yue et al. 300
Identification of calliphorid flies in their different life stages using DNA
barcoding can improve the success of forensics investigations Zambare and Khedkar 300
Integration of DNA barcoding in a local diversity survey of Lepidoptera:
confronting morphospecies and DNA barcode OTUs of Arctiinae moths in
the Brazilian Atlantic forest
Zenker et al. 300
Molecular detection of in situ dietary composition of Calanus sinicus in
Taiwan Strait D. Zhang et al. 301
Detection of aquatic invasive species and biodiversity assessment in
Canadian ports G.K. Zhang et al. 301
Molecular and morphological evidence for underestimated biodiversity of
Clytia (Cnidaria: Hydrozoa) in the China Sea, with description of three
new species
Zheng et al. 301
DNA barcode of rare and endangered plants Zhou 301
The use of DNA barcoding in large-scale ‘omics projects Zhou and Kjer 302
Barcoding Chinese marine nematodes with mitochondrial cytochrome c
oxidase subunit I (COI) and small subunit 18S rDNA (18S) Zhou et al. 302
Use of DNA barcoding technology to appraise commercial Spatholobus
products Zhou et al. 302
The San Diego Barcode of Life: launching a regional DNA barcode
campaign in a globally important biodiversity hotspot Zlotnick 303
“Barcoding our Backyard” at ResMed, Inc.: 52-consecutive weeks Malaise
trap project at a corporate headquarters in a Global biodiversity hotspot Zlotnick et al. 303
184 Genome Vol. 58, 2015
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ABSTRACTS
DNA barcoding of Oeneis butterflies newly sampled in Mongolia
Takatoshi Abe,
1
Hidemi Watanabe,
1
Kanako O. Koyanagi,
1
and Botaro Inoue
2
1Hokkaido University, Excelsior Shinsapporo Hills, 105, Atsubetsuhigasi2-jyo2-tyome, 1-10, Atsubetsu-ku,
Sapporo-shi, Hokkai-do, Japan.
2Hokusei Gakuen Univ. High School, Japan.
Corresponding author: Takatoshi Abe (e-mail: t-abe@ist.hokudai.ac.jp).
Background: Butterflies of the genus Oeneis are distributed in the
arctic, subarctic, and alpine zones. Approximately 30 species consti-
tute the genus Oeneis, but classification criteria are controversial. The
Altai-Sayan is the ecoregion with the largest diversity of Oeneis species
in the world; it has been suggested that the genus Oeneis originated in
that ecoregion and migrated to the New World through the Bering
land bridge during the Last Glacial Maximum. Comparisons of the
barcode sequences of Oeneis species would be effective to decide their
speciation and origin. There are 1093 specimens of Oeneis with COI
barcode sequences registered in BOLD, but most of their collection
sites are concentrated in Canada, United States, and Russia. Results: We
collected 92 butterfly specimens around Lake Khövsgöl, Mongolia, lo-
cated in the Altai-Sayan ecoregion, from June 22 to June 29, 2014. Col-
lected specimens were identified based on their morphological
characters and DNA barcodes. As a result, 23 of 92 butterflies were clas-
sified as Oeneis species: O. jutta (n= 7), O. magna (n= 2), O. melissa (n= 7), O.
norna (n= 5), and O. urda (n= 2). This is the first report of O. melissa from this
site, and their COI barcode sequences coincided with barcode sequences
of O. melissa collected in Buryatia. O. jutta specimens were different genet-
ically from O. jutta collected in the Nearctic region. It is impossible to
distinguish among O. urda,O. nanna, and O. mongolica using COI barcode
sequences. Significance: These data show that O. melissa in Altai-Sayan
ecoregion are clustered independently on the phylogenic tree.
DNA barcoding and the origin of species
Sarah J. Adamowicz
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: sadamowi@uoguelph.ca.
Background: Since it was proposed in 2003, DNA barcoding has seen a
dramatic rise as the standard molecular-based method for specimen
identification for multicellular life. However, debate continues regarding
the best conceptual and analytical approaches for identifying unknown
specimens to the species level as well as for discovering new species. In
particular, criticisms have been aimed at the frequent lack of statistical
tests of explicit species hypotheses in the DNA barcoding literature, de-
spite diverse discussions of species boundaries. Meanwhile, large DNA
barcoding campaigns are generating continental-scale datasets of stan-
dardized DNA sequences for focal taxonomic groups that are nearly
comprehensive at the species level, which can be used to address
fundamental questions about the properties and origins of species.
Results: In this opinion piece, I will first briefly review the history of
application of species concepts in DNA barcoding studies. A gradual shift
from morphological concepts towards concordance and evolutionary
species concepts is apparent but not universal. Second, I will outline a
research program in which large-scale barcode datasets are applied, to-
gether with other data types, to address elemental questions about the
properties and evolutionary origins of species entities. For example, com-
parative barcode data from sexual versus asexual taxa can help us to
understand the strength of the role of reproductive isolation in the for-
mation of discrete biological entities. Significance: Critical examination
of barcode data will provide new insights into the nature of species.
Furthermore, improved knowledge of the evolutionary history of species
will contribute to selecting and operationalizing species concepts, which
is of substantial practical importance for choosing appropriate analysis
methods for specimen identification, species discovery, and species de-
limitation.
Species boundaries, mitochondrial introgression and nuclear
mitochondrial paralogs in the neotropical ant complex
Ectatomma ruidum (Ectatomminae)
R. Gabriela Aguilar-Velasco,
1
Chantal Poteaux-Leonard,
2
and Alejandro Zaldívar-Riverón
3
1Instituto de Biologia, UNAM, 3er circuito exterior s/n, Cd. Universitaria, Copilco, Coyoacán, CP 04510,
D. F., Mexico.
2LEEC, University of Paris XIII, Laboratoire d'Ethologie Expérimentale et Comparée, EA 4443,
Université Paris-Nord, UFR L.S.H.S., 99 avenue J.-B. Clément, 93430 Villetaneuse, France.
3Instituto de Biologia, UNAM, 3er Circuito exterior, Cd. Universitaria, Copilco, Coyoacan, D. F. Mexico.
Corresponding author: R. Gabriela Aguilar-Velasco (e-mail:
supernatural_fairy@hotmail.com).
Nuclear mitochondrial paralogs (numts) are non-functional frag-
ments of mtDNA integrated into the nuclear genome that can be
coamplified through amplification of conserved primers, and their
inclusion can overestimate the number of species in analyses based on
mtDNA sequence data. Since numts have relatively slow mutation
rates, they can pass undetected by conventional procedures such as
inspecting for internal stop codons, indels, or apparent polymor-
phism in chromatograms. Species boundaries based on the mt locus
therefore require a thorough assessment of numts, but especially in
insects, where this phenomenon is more frequent. Ectatomma ruidum
is a widely distributed Neotropical ant species that occurs from cen-
tral Mexico to northern Brazil. Due to its considerable external
morphological variation and close morphological similarity with other
related species, the species boundaries within E. ruidum need to be thor-
oughly investigated. Here we assessed the species boundries in this taxon
based on specimens collected from a number of localities along the Neo-
tropics. We examined external morphology and sequences belonging to
two mt (COI, cytochrome b) and one nuclear (H3) markers. Ancient and
recent mt paralogs were detected in several specimens, though pre-PCR
dilution of DNA template helped to recover most of the orthologs. There
was extensive variation in the barcoding locus among samples, which
precluded the use of BINs. Based on the congruence found between our
species delineation analyses and the morphospecies identified, we pro-
pose that E. ruidum is actually composed of at least three evolutionary
units, two of which have wide geographic distributions along the Neo-
tropics. The evidence obtained from the nuclear marker also suggests the
existence of hybrids between two of these species in southeast Mexico.
This study agrees with previous studies of other animal taxa, which have
revealed a complex evolutionary history and overlooked species diversity
along the Mexican Pacific coast.
Mosquito surveillance using DNA barcoding can help in
vector-born disease control
Kiran Ahirrao,
1
Rahul Jamdade,
2
and Gulab Khedkar
2
1Department of Zoology, Rani Laxmibai College Parola, Jalgaon District, Maharashtra, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Kiran Ahirrao (e-mail: drkiranahirrao@gmail.com).
Mosquitoes (Diptera: Culicidae) include some of the most important dis-
ease vectors. Morphological character-based identification of mosquito
species is difficult, particularly the identification of specimens collected
in the field as part of disease surveillance programs. To overcome the
challenge in taxonomic identification, we generated DNA barcodes of the
cytochrome c oxidase subunit I (COI) gene for the most common mos-
quito species present in Maharashtra state in India including the major
disease vectors. A total of 680 mosquito specimens were collected and
185
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assigned to 6 genera and 21 species and subspecies on the basis of mor-
phological characteristics. Individuals of the same species grouped
closely together in a neighbour-joining tree based on COI sequence sim-
ilarity, regardless of collection site. COI gene sequence divergence was
⬃28 times higher for species in the same genus than for members of the
same species. Divergence in over 98% of congeneric species ranged from
2.5% to 19.2%, whereas divergence in conspecific individuals ranged from
0% to 1.12%. Cryptic species may be common, and no pseudogenes were
evident.
DNA barcoding of small indigenous fish species from Tanguar
Haor, Bangladesh
Md. Sagir Ahmed,
1
Mohammad Mamun Chowdhury,
2
Monzillur Rahman,
3
and Luthfun Nahar
1
1University of Dhaka, Department of Zoology, Dhaka 1000, Bangladesh.
2University of Dhaka, Departmrnt of Fisheries, Dhaka 1000, Bangladesh.
3University of Dhaka, Department of Genetic Engineering and Boi-Technology, Dhaka 1000, Bangladesh.
Corresponding author: Md. Sagir Ahmed (e-mail: sagir@du.ac.bd).
Background: Bangladesh is very rich in its ichthyofaunal diversity
and ranked third largest in Asia after China and India, with ⬃260 species
of freshwater fish. Among those, about 143 species are considered as
small indigenous species (SIS) with high market demand and nutri-
tional value. The ichthyofaunal diversity of the country is under in-
creasing threat by overfishing, habitat destruction, and pollution.
Tanguar Haor is Bangladesh's most important freshwater wetland,
designated as an Ecologically Critical Area (ECA). It also is a protected
Ramsar site of international importance for the conservation and sus-
tainable utilization of wetlands. Results: An attempt has been made
for the genetic identification of SIS through DNA barcoding, a molec-
ular method that uses a short standardized DNA sequence as a species
identification tool. The standard 652 base-pair region of the mitochon-
drial cytochrome oxidase subunit I gene (COI) was sequenced in fresh-
water fish specimens from Taguar Haor. So far, we have sequenced
50 SIS species, and the high efficiency of species identification is demon-
strated in the present study. The average genetic distance was over
50-fold higher between species than within species, as Kimura two
parameter (K2P) genetic distances averaged 16.75% among congeners
and only 0.32% for intraspecific individuals. Significance: This DNA
barcode resource will enhance the capacity in many areas for fish
conservation biology, which can benefit from the improved knowl-
edge of genetic provenance. These include captive breeding and re-
stocking programs, life history studies, and ecological research into
the interactions between populations of native and exotic species.
DNA barcoding antlions (Myrmeleontidae: Neuroptera) of
Pakistan
Saleem Akhtar
Agriculture University, Peshawar, PIASA, NIGAB, National Agricultural Research Centre (NARC), Park
Road Chak Shahzad, Islamabad, Pakistan.
E-mail for correspondence: entomologist.nibge@gmail.com.
Background: Around 2000 species of the family Myrmeleontidae (ant-
lions) have been listed worldwide, but advances in global taxonomic
approaches and recent revisions have reduced the number of valid extant
species to 1500. Knowledge of species composition in different parts of
the world is also limited, and a significant number of species are syn-
onyms. Antlions are generally a complex group to identify, and taxo-
nomic expertise for their reliable identification is scarce. DNA barcoding
is a useful tool for species discrimination, and development of reference
DNA barcode libraries will enable future sequence-based identification of
specimens to the species level. Pakistan, with its diverse but poorly ex-
plored antlion fauna, is an excellent target for such a study. Results:
Antlions were collected from different areas of Pakistan and identified to
the species level using morphology. Of the 43 species we collected, 39
yielded successful barcode sequences from cytochrome c oxidase subunit
I (COI). Analysis of the barcode data revealed that most of the species did
not match closely (there was no match of >98% sequence similarity) with
any records presently on the Barcode of Life Data Systems (BOLD) or
GenBank, indicating either that the species are endemic to the region or
have been poorly represented in DNA barcoding surveys conducted else-
where. The intraspecific distances in all the species were lower than the
distances to their nearest-neighbour species. Assignment of the morpho-
logically identified species to unique Barcode Index Numbers supported
the capability of barcode data for discrimination among antlion species.
Significance: This study begins construction of the first DNA barcode
reference library for antlions of Pakistan. The availability of barcode data
will help to solve taxonomic confusion and avoid overestimation of spe-
cies numbers. The beneficial nature of antlions for biological control
highlights the importance of their correct identification.
Plant DNA in sediments: to which degree do they represent the
flora?
Inger Greve Alsos,
1
Eric Coissac,
2
Mary Edwards,
3
Marie Kristine Føreid,
1
Ludovic Gielly,
2
Per Sjögren,
1
Pierre Taberlet,
2
and Nigel Yoccoz
1
1UiT – The Arctic University of Norway, Tromsø, Norway.
2University Grenoble Alpes, LECA Grenoble, France.
3University of Southampton, Southampton, UK.
Corresponding author: Inger Greve Alsos (e-mail: inger.g.alsos@uit.no).
Background: Environmental DNA (eDNA) extracted from sediments
has promise as a new proxy in studies of modern biodiversity and palaeo-
biological reconstruction. However, little is known about how well this
method represents the flora. We used the g and h universal plant primers
for the short and variable P6 loop region of the chloroplast trnL (UAA)
intron to amplify DNA in lake sediment samples of different ages (mod-
ern, 100-200 years, and Holocene), as well as modern soil samples. We
compare this with modern vegetation and other proxies (pollen and
macrofossils) to evaluate the representation of different taxonomical
groups, the geographical area likely to be represented, and the tapho-
nomy of DNA. Results: Analyses of soil samples from the Arctic showed
that all species represented in the soil DNA grew within 3 m from the
sampling point, most of them within 0.5 m. Lake sediments represent the
flora of the catchment area, but a lower proportion of the flora were
represented in the DNA record compared to soil samples. DNA and pollen
of 100–200-year-old lake sediments from regions where forest was
planted in the mid-twentieth century showed that both proxies discern
major vegetation change at the time of plantation, with similar quanti-
tative changes. In a lake core from the Arctic dated to 8500-1200 cal. BP,
all except two genera identified as macrofossils were also identified with
DNA. Furthermore, DNA identified six additional taxa and more species
per sample. With one DNA extraction and one PCR per sample, we detect
most common species independent of sample type or age. Increasing the
number of extractions or PCR repeats increased the chances of detecting
rare species. Some taxonomic groups (e.g., Cyperaceae) were consistently
underestimated whereas other (e.g., water plants) were overrepresented
compared to the other proxies. Significance: The local flora was well
represented in the DNA of the sediments, and the method may have a
higher and (or) complementary taxonomic resolution than analyses of
pollen or macrofossils.
DNA barcoding for food safety and health consumer:
the Italian experience
Ferrari Angelo,
1
Vito Guendalina,
1
Goria Maria,
2
Acutis Pier Luigi,
2
and Razzuoli Elisabetta
1
1Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Laboratory of Food Control
(Genoa, Italy) piazza Borgo Pila 39/24 - 16129 Genoa, Italy.
2Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Laboratory of
Biotechnologies (Turin, Italy) via Bologna 148, 10154 Turin, Italy.
Corresponding author: Ferrari Angelo (e-mail: angelo.ferrari@izsto.it).
Background: The main type of fraud in the seafood market is the
substitution of a species with another of lower commercial value than
the declared one. Several measures, including consumer's informa-
tion, have been adopted by authorities to improve food safety levels.
186 Genome Vol. 58, 2015
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Furthermore, the analysis of DNA barcodes allows for efficiently coun-
teracting fish substitution fraud, especially concerning processed
products (e.g., slices, fillets). Since 2007 the Istituto Zooprofilattico
Sperimentale Piemonte, Liguria and Valle d'Aosta-Genoa's headquar-
ter belong to the FishBol. In collaboration with other institutions, the
IZS developed a project on traceability issues, supported by Italian
Ministry of Health. The objective was to obtain a standardized repro-
ducible method for routine analyses of seafood products. Results: The
validation method was performed on 30 different species from the
Ligurian Sea, already recorded in BOLD. A morphological investiga-
tion was also carried out. Results of both analyses types, molecular
and morphological, were consistent. The obtained sequences were
compared with those deposited in BOLD, and species names were
assigned when the similarity percentage was >98%; this was possible
for all analysed samples. The performance was evaluated: specificity =
100%; intra-laboratory reproducibility-index K= 1; sensitivity limit =
5 ng/L. We confirm the procedure's efficacy, although some problems
for closely related species were detected. Sequences of five Mediter-
ranean fish species have been deposited for the first time on BOLD:
Ophidion barbatum,Solea kleinii,Gymnammodytes cicerelus,Bothus podas
podas, and Scorpaena porcus.Significance: Our approach guarantees
product traceability and consumer health. Evidence of the impor-
tance of the correct knowledge of consumed fish species is repre-
sented by the “Oily Diarrhoea” case; this pathology can be induced by
inadequate practices of cooking and (or) preparation of Ruvettus pretiosus,
“confused” with other similar species. This case underlines the risks
for consumers resulting from intentional fraud or from unintentional
mislabeling.
Extending the ITS2-workbench with DNA barcoding capabilities
Markus J. Ankenbrand,
1
Alexander Keller,
1
Christian Koetschan,
2
Matthias Wolf,
2
Jörg Schultz,
2
and Frank Förster
2
1Department of Animal Ecology and Tropical Biology (Zoology III), University of Würzburg, Am
Hubland/Biozentrum, D-97074 Würzburg, Germany.
2Department of Bioinformatics, University of Würzburg, Am Hubland/Biozentrum, D-97074 Würzburg,
Germany.
Corresponding author: Markus J. Ankenbrand (e-mail: markus.ankenbrand@uni-
wuerzburg.de).
Background: The internal transcribed spacer 2 (ITS2) is a well-
established marker for DNA barcoding, especially for plants and
fungi. Yet so far, no specific reference database exists dedicated to this
purpose. Extracting ITS2 sequences for a taxonomic group from Gen-
Bank is tedious manual work, which is prone to errors due to misan-
notations. The same challenges also apply for phylogenetic analyses,
for which we have already developed the ITS2-workbench. It provides
structure-based annotations and quality control for sequences ex-
tracted from GenBank. Further, it offers tools for Hidden Markov
Models (HMM) annotation, secondary structure prediction, align-
ment, and tree calculation. However, it lacked the ability to taxo-
nomically classify barcode sequences with confidence estimations.
Results: Here, we present an extension of the ITS2-workbench for
the application in DNA barcoding. It is based on the underlying data
of 700 000 quality-controlled sequences from more than 175 000 species
(2015 update). This includes ⬃80 000 distinct species for plants and
fungi, each. Additionally, other eukaryotic groups that have been so
far less represented in barcoding studies are covered (e.g., the class
Bacillariophyceae). The workbench uses UTAX as the classifier, which
allows for barcoding of user-provided sequences and provides confi-
dence values for each taxonomic rank. For smaller data sets the results
can be immediately viewed on the website. For large data sets (e.g.,
next-generation sequencing amplicon data), the user is notified by
email as soon as the download of the classification results is ready.
Significance: The combination of a huge amount of quality-controlled
ITS2 sequences with the powerful classification algorithm UTAX facil-
itates the broad usage of ITS2 as a DNA barcode. The integration into
the ITS2-workbench provides the means to also use the rich phyloge-
netic methods and supplementary reference sequences for further
analyses. The ITS2-workbench is freely accessible online at http://
its2.bioapps.biozentrum.uni-wuerzburg.de.
Comparison between morphological and DNA
barcode-suggested species boundaries among shallow-water
amphipod fauna from the southern European Atlantic coast
Ilisa C. Antunes,
1
Maria S. Ferreira,
1
Jorge Lobo,
1,2
Marcos A.L. Teixeira,
1
Luisa M.S. Borges,
1,3
Ronaldo Sousa,
1,4
Pedro A. Gomes,
1
Maria Helena Costa,
2
Marina R. Cunha,
5
and Filipe O. Costa
1
1CBMA – Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
2MARE – Marine and Environmental Sciences Centre, New University of Lisbon, 2829-516 Monte de
Caparica, Portugal.
3Helmholtz-Zentrum Geesthacht, Centre for Material and Coastal Research, Max-Planck-Straße 1,
21502, Germany.
4CIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto,
Rua dos Bragas, 123, 4050-123, Porto, Portugal.
5CESAM – Centre for Environmental and Marine Studies, University of Aveiro, Campus de Santiago,
3810-193 Aveiro, Portugal.
Corresponding author: Ilisa C. Antunes (e-mail: ilisaantunes@gmail.com).
Background: Amphipods are one of the most diverse and widely
distributed groups of crustaceans, often constituting dominant or key
elements of marine and coastal communities. As with many other
marine invertebrate taxa, rigorous species identifications can be
rather challenging, and cryptic species reports have been increasing.
In this study we compared DNA barcode-suggested species boundaries
with morphology-based species identifications in the amphipod fauna
of the southern European Atlantic coast. Results: DNA sequences of
the cytochrome c oxidase subunit I barcode region (COI-5P) were ob-
tained from 197 specimens collected along the coast and estuaries of
continental Portugal, which were then assigned to 41 morphospecies
and 21 families. To this dataset we added publicly available COI-5P
sequences of the same species or genus, hence producing a final BOLD
dataset comprising 60 species and 273 sequences. Sequences from this
dataset were in turn assigned to 68 BINs (Barcode Index Numbers), of
which 38 were concordant (i.e., 1 BIN = 1 species), 11 were taxonomi-
cally discordant BINs, and 19 were singletons. A detailed examination
of the discordant BINs revealed that in most cases discordances likely
result from misidentifications. Rigorously identified specimens of
Corophium multisetosum (Stock, 1952) were split into two BINs, which
diverged 18%. Albeit fairly divergent, these BINs correspond to two
geographically close populations (≈115 km), lacking any apparent geo-
graphic barrier between them. Significance: Our results suggest that
specimens of C. multisetosum here analyzed comprise two overlooked
cryptic species. This morphospecies is locally abundant and frequently
dominant in the benthic communities of the investigated estuaries,
which makes these findings particularly relevant. For less-known fau-
nal groups as the amphipods, DNA barcodes offer great potential to
assist taxonomic revisions and inventories, species discovery, and rou-
tine species identifications.
Integrative taxonomy reveals cryptic species and unusual
speciation pathway in flounders (Pleuronectiformes: Bothus)
from the Brazilian coast
Leandro Araújo Argolo,
1
Silvia Britto Barreto,
1
Jamille de Araújo Bitencourt,
1
Robson Tamar Costa Ramos,
2
Iracilda Sampaio,
3
and Paulo Roberto Antunes de Mello Affonso
1
1Universidade Estadual do Sudoeste da Bahia, Departamento de Ciências Biológicas, Av. José Moreira
Sobrinho, s/n Jequiezinho, Jequié-BA 45206190, Brazil.
2Universidade Federal da Paraíba, Departamento de Sistemática e Ecologia, Cidade Universitária,
João Pessoa-PB 58059900, Brazil.
3Universidade Federal do Pará IECOS, Laboratório de Genética e Biologia Molecular, Alameda Leandro
Ribeiro s/n, Bairro Aldeia, Bragança-PA 68600000, Brazil.
Corresponding author: Paulo Roberto Antunes de Mello Affonso (e-mail:
paulomelloaffonso@yahoo.com.br).
Background: Flatfishes (order Pleuronectiformes) represent a tax-
onomically controversial group because of overlapping morpholog-
ical traits among species and the lack of uniformity in specialized
Abstracts 187
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literature, which justifies the necessity of integrative studies. In the
present work, we analysed flounder species (genus Bothus) from the
southwestern Atlantic Ocean using molecular markers (cyto-
chrome c oxidase subunit I – COI and 16S rRNA genes), traditional
morphology, and cytogenetics. Results: Most of samples were mor-
phologically identified as Bothus ocellatus while some individuals
from the coast of Bahia, northeastern Brazil, were clearly distin-
guished from congeneric Atlantic flounder species, probably corre-
sponding to a new species, here named Bothus sp. Moreover, while
B. ocellatus showed a karyotype with 2n= 32, as previously reported,
the karyotypic pattern of Bothus sp. was characterized by 2n= 44.
Molecular analyses using COI and 16S confirmed both the identifi-
cation of B. ocellatus and the accentuated genetic divergence of
Bothus sp. in relation to other species of Bothus from the South
Atlantic. Surprisingly, Bothus sp. was more closely related to B. pantherinus
from the Indo-Pacific. Indeed, the genetic distances between both
species were 2.3% for COI and 0.4% for 16S sequences, while mean
values of 19.6% (COI) and 9.1% (16S) were observed in relation to other
congeneric species. In addition, Bothus sp. and B. pantherinus clustered
together in neighbour-joining, maximum likelihood, and Bayesian
inference trees, with support values equal to or higher than 99%.
Significance: Bothus sp. corresponds to a new flounder species lacking
formal description. Thus, the richness of Bothus species along Brazilian
coast seems to be underrated and needs to be revisited. The unusual
genetic similarity between Bothus sp. and B. pantherinus, associated
with larval forms of flounder and biogeographic features, suggests a
ring speciation model with transoceanic dispersal via the Indian
Ocean and subequatorial currents in the South Atlantic.
Efficiency of DNA barcoding in the identification of flatfish
(Pleuronectiformes) from Brazilian coast
Leandro Araújo Argolo,
1
Jamille de Araújo Bitencourt,
1
Robson Tamar Costa Ramos,
2
Iracilda Sampaio,
3
and Paulo Roberto Antunes de Mello Affonso
1
1Universidade Estadual do Sudoeste da Bahia, Dep. Ciências Biológicas, Av. José Moreira Sobrinho,
s/n Jequiezinho, Jequié - BA 45206190, Brazil.
2Universidade Federal da Paraíba, Departamento de Sistemática e Ecologia, Cidade Universitária,
João Pessoa-PB 58059900, Brazil.
3Universidade Federal do Pará IECOS, Laboratório de Genética e Biologia Molecular, Alameda Leandro
Ribeiro s/n, Bairro Aldeia, Bragança-PA 68600000, Brazil.
Corresponding author: Paulo Roberto Antunes de Mello Affonso (e-mail:
paulomelloaffonso@yahoo.com.br).
Background: DNA barcoding is one of the most effective approaches to
describe biodiversity. The success of this molecular identification
method is partially related to an independent system of nomenclature,
named Barcode Index Number (BIN), in contrast to the Linnean system.
We performed a wide study of DNA barcoding of flatfish from the Brazil-
ian coast, South Atlantic, using cytochrome c oxidase subunit I (COI) and
16S rDNA sequences to test the efficiency of this methodology in identi-
fying species in this fish group. Results: A total of 16 species were iden-
tified among the Brazilian samples. A neighbour-joining tree including
collected specimens, public BINs (COI), and sequences from GenBank
(16S) from identified species and closely related groups yielded recipro-
cally monophyletic units for most taxa, with a 2% threshold serving to
discriminate species in the case of COI. Nonetheless, cases of a single
nominal species split into differentiated groups (up to 25.7% of genetic
distance in COI) or a single cluster composed of two taxonomic units
were observed. Moreover, the haplotype network revealed no genetic
structure among samples of the same species along the Brazilian coast in
contrast with the high genetic divergence observed between populations
from Brazil and the Caribbean. Significance: The remarkable intraspe-
cific divergence of Linnean taxa when some of collected specimens were
compared to flatfish sequences stored in BOLD and GenBank indicates
misidentification of species and (or) the presence of cryptic species. On
the other hand, the BIN system allows overcoming such identification
issues, making the use of BOLD an efficient strategy for taxonomic stud-
ies. Furthermore, the genetic divergence between samples from Brazil-
ian and Caribbean populations reinforces the isolation of both
biogeographic regions, possibly imposed by the Amazonas outflow.
Therefore, Atlantic flatfish require taxonomic revisions, and morpholog-
ical identification of controversial groups should be carefully performed
prior to uploading sequence data to online storage systems.
What can DNA barcoding tell us about the dietary niche
overlap of sibling sympatric bat species?
Aitor Arrizabalaga-Escudero,
1
Elizabeth L. Clare,
2
Antton Alberdi,
1
Egoitz Salsamendi,
1
Joxerra Aihartza,
1
Urtzi Goiti,
1
and Inazio Garin
1
1Department of Zoology and Animal Cell Biology, Faculty of Science and Technology, University of the
Basque Country, UPV/EHU Sarriena z/g, Leioa, E-48940, Spain.
2School of Biological and Chemical Sciences, Queen Mary University of London, Mile 9 End Road,
London E1 4NS, UK.
Corresponding author: Aitor Arrizabalaga-Escudero (e-mail:
arrizabalaga.aitor@gmail.com).
Background: According to the competitive exclusion principle,
species coexisting in the same community must differ in the way
they use spatial or food resources. When ecologically similar spe-
cies co-habit, resource partitioning is expected to occur, a mecha-
nism thought to enable the long-term coexistence of species. The
study of such a mechanism could be especially important when
investigating the foraging requirements of endangered species.
However, research on food requirements of endangered species has
been limited by the low taxonomic resolution of the employed
methodologies. DNA metabarcoding offers a solution to these lim-
itations, allowing species-level identification of consumed prey. In
this study, we analyse the dietary niche overlap between the sym-
patric sibling bat species Rhinolophus euryale and Rhinolophus mehelyi.
Previous studies suggested that segregation of foraging habitats is
the main mechanism allowing the coexistence of both species, but
prey identification was limited to ordinal level. We aimed to eluci-
date subtle but functionally relevant differences between the
consumed prey of these two bat species. Results: Using DNA me-
tabarcoding we identified 94 MOTUs from the guano of both spe-
cies, from which we were able to identify 65% in BOLD. Analysis of
niche breadth and overlap showed that the diet of both bats over-
laps to a high extent. Further analysis of the relationship between
bats' diet and their prey size and prey habitat preferences revealed
a weak association, which we would expect, given each species'
echo-morphology and foraging habitats. Significance: Our results
showed that both species ate virtually the same prey, suggesting
that, in line with previous studies for the same bat individuals, the
spatial segregation of the foraging grounds is the main mechanism
allowing coexistence between these sibling bats. Therefore, DNA
metabarcoding enabled us to gain a deeper understanding of
trophic interactions between bats and their insect prey.
DNA metabarcoding of endangered plant and animal species in
seized forensic samples
Alfred J. Arulandhu,
1
Martijn Staats,
1
Tamara Peelen,
2
and Esther Kok
1
1RIKILT Wageningen UR P.O. Box 230, 6700 AE Wageningen, Netherlands.
2Dutch Customs Laboratory Kingsfordweg 1, 1043 GN Amsterdam, Netherlands.
Corresponding author: Alfred J. Arulandhu (e-mail: alfred.arulandhu@wur.nl).
Background: Use of illegally traded endangered species (CITES) in prod-
ucts is a major issue throughout the world. In the EU, enforcement is
mainly focused at the borders, where illegally imported products con-
taining plants and (or) animals will be seized by Customs and CITES
authorities. To allow the Customs laboratories to effectively perform the
analysis of samples for the presence of endangered species, it is necessary
to develop improved methods that can broadly identify any species pres-
ent in samples such as in food supplements or traditional Chinese med-
icines (TCMs). As suspected samples may often contain highly degraded
DNA, it is a requirement that species can be identified on the basis of
short DNA sequences. DNA metabarcoding promises to overcome the
analysis problem of multiple ingredients, and it has been demonstrated
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to allow simultaneous identification of endangered species in highly
processed forensic samples including TCMs. Here, we propose a DNA
metabarcoding strategy for routine analysis of seized products that
makes use of an extended set of plant and animal barcode markers that
will improve resolution for identification (in case some markers fail) and
(or) improve quality assurance by verification of findings with more than
one barcode marker. Result: A panel of 12 full-length barcode and mini-
barcode markers has been identified that can efficiently be amplified
across a wide range of species using a single universal PCR protocol for all
primer sets. Since using a proper DNA isolation method is critical for
success, various DNA isolation kits and protocols have been evaluated on
various TCM samples. Optimized NGS protocols and a user-friendly data
analysis pipeline will be developed for robust and accurate identification
of (CITES) species in various experimental and real-life samples. Signifi-
cance: This study will significantly present the prospects and limitations
of DNA metabarcoding for endangered species identification in a routine
setting.
DNA barcoding of plant pests clarifies cryptic species complexes
Muhammad Ashfaq
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: mashfaq@uoguelph.ca.
Background: Although numerous insect species impact plants, some
are particularly important because of their broad distribution, invasive
nature, and capacity to transmit diseases. The identification of species in
four major groups of pest insects (aphids, scales, thrips, whiteflies) is a
challenge due to their small size and cryptic morphology. Analysis of
DNA barcode (COI-5A) sequences and the Barcode Index Number (BIN)
system has helped to discriminate closely related species in these groups.
Results: DNA barcoding was employed to identify species of whitefly
(Bemisia tabaci complex), thrips, aphids, and the mango scale insect and to
analyze their diversity in Pakistan. Barcode analysis of 593 whiteflies
revealed the presence of six BINs. Integration of these records with an-
other 173 from GenBank revealed 15 BINs in the B. tabaci complex. COI-3=
analysis from the corresponding B. tabaci lineages supported the BINs,
indicating that B. tabaci is a complex of multiple species. Barcode analysis
of 471 thrips revealed 56 species. Sequences for three major pest species
(Haplothrips reuteri,Thrips palmi,Thrips tabaci) and one predatory thrips
(Aeolothrips intermedius) showed deep intraspecific divergences, strongly
suggesting that each is a cryptic species complex. Likewise, the analysis
of 662 aphids revealed 42 species with a deep split in the barcodes of
Rhopalosiphum padi, leading specimens of this aphid species to be assigned
to two BINs. The mango scale, Drosicha mangiferae, has an uncertain status
in Pakistan because it has also been reported as D. stebbingi. Barcode
analysis of scale populations in Pakistan revealed the presence of a single
barcode lineage, indicating that the species are likely synonyms. Signif-
icance: DNA barcode analysis of four important pest groups was effective
in revealing cryptic species complexes and resolving species identities.
This barcode library will aid pest control by easing past problems in
identification.
Application of DNA barcoding for phylogenetic identification
of pathogenic fungi associated with stem-end rot of mango in
Pakistan
Sidra Aslam,
1
Aisha Tahir,
2
Abdul Rehman,
3
Khalil-ur-Rahman,
3
and Amer Jamil
1
1Molecular Biochemistry Laboratory, Department of Biochemistry, University of Agriculture,
Faisalabad, Pakistan.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
3University of Agriculture, Faisalabad, Pakistan.
Corresponding author: Aisha Tahir (e-mail: aishatahir85@gmail.com).
Background: DNA barcoding is a valuable tool for accurate species
identification. We employed it in this study to identify the causal
agents of stem-end rot disease of mango fruits, which is responsible
for huge economic losses every year in Pakistan. It is hypothesized that
along with Lasiodiplodia theobromae, other fungi such as Colletotrichum
gloeosporioides,Phomopsis mangiferae,Alternaria alternata, and Botrytis
cinerea are also involved in this disease. In order to evaluate this
hypothesis, diseased mango fruit were collected from different eco-
logical zones of Punjab Province of Pakistan and subjected to isola-
tion, purification, and molecular identification of fungal pathogens
present in rotten parts of the fruit. Results: Molecular identification
of the fungal species was performed using ITS and 18S rDNA gene as
DNA barcode markers. DNA barcodes delineated all the pathogenic
fungi species into clear clades corresponding to the morphological
classification. The study species C. gloeosporiodes,A. alternata,B. cinerea,
P. mangiferae,B. theobromae, and P. italicum are all belonging to the
division of Ascomycota based on traditional morphological classifica-
tion, and the current study also supported the classification by form-
ing each species into a single clear clade. Neighbour-joining and
maximum parsimony phylogenetic trees were constructed to visual-
ize the formation of species clades, and the results were the same in
both phylogenetic tress of neighbour-joining and maximum parsi-
mony and for both the genes ITS and 18S rDNA. Our study suggested
that both ITS and partial 18S rDNA are equally potential in distinguish-
ing the fungal species, which is greatly supported by inter- and
intraspecific genetic distances and also by the phylogenetic trees.
Significance: Correct identification of fungal pathogens using DNA
barcoding will provide a strong basis for understanding the lifecycle
of stem-end rot disease. Devising appropriate methods for treatment
is also dependent on the proper determination of causal agents. Re-
ducing the effects of stem-end rot of mango will resolve the socio-
economic problems of mango farmers, who are losing profit due to
low export value because of this disease.
A molecular phylogenetic study of pheretimoid species
(Oligochaeta: Megascolecidae) in Mindanao Island, Philippines
Nonillon Aspe,
1
Samuel James,
2
and Hiroshi Kajihara
1
1Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Japan.
2Department of Biology, University of Iowa, USA.
Hiroshi Kajihara, Department of Natural History Sciences, Graduate School of Science, Hokkaido
University, Japan.
Corresponding author: Nonillon Aspe (e-mail: nonillon_aspe@yahoo.com).
Background: Knowledge of the native earthworm fauna of the Phil-
ippines had been very limited until the last decade. With the recent
discovery of new species collected in various parts of the country, the
number of known species increased to around 200. Our study at-
tempts to infer the phylogenetic relationship among the members of
Pheretima s.lat. in Mindanao Island, which includes 18 morphospecies,
represented by Pheretima s.str. [including the two subgenera Pheretima
(13 spp.) and Parapheretima (2 spp.)] and the genus Amynthas (3 spp.); the
subgenus Pheretima includes five species in the P. urceolata group, six in
the P. sangirensis group, one monothecate, and one athecate mor-
phospecies. Four molecular markers were used: the mitochondrial
cytochrome c oxidase subunit I (COI) and 16S rRNA genes and the
nuclear 28S rRNA and protein-coding histone H3 genes. Results: Re-
sults of the maximum likelihood and Bayesian analyses of these data,
with Dichogaster sp. as the outgroup, show that (i) the P. sangirensis
group, defined by having a pair of spermathecae at intersegments 7/8,
is monophyletic, only if the monothecate and athecate species are
included, suggesting a fusion and loss of the spermathecae in each
lineage; (ii) the subgenus Parapheretima, defined by having secretory
diverticula on the copulatory bursae, is non-monophyletic, suggesting
that their respective secretory diverticula evolved independently;
(iii) the genus Amynthas is also non-monophyletic and is nested within
Pheretima s.str., suggesting that the evolution or loss of either the
nephridia on spermathecal ducts and (or) the secondary male pores
happened more than once; and (iv) the P. urceolata group, defined by
having a pair of spermathecae at intersegments 5/6, may or may not be
monophyletic. Significance: The addition of genetic markers with a
rate of divergence lower than COI and 16S but not lower than 28S and
H3 is necessary to obtain better resolution of the phylogeny of this
“problematic” group.
Abstracts 189
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Lessons from the first phase of the German Barcode of Life
initiative (2012–2015)
Jonas Astrin, Vera Fonseca, Matthias Geiger, Peter Grobe,
Björn Rulik, and Wolfgang Wägele
Zoological Research Museum Alexander Koenig, Adenauerallee 160, 53113 Bonn, Germany.
Corresponding author: Wolfgang Wägele (e-mail: w.waegele@zfmk.de).
GBOL (https://www.bolgermany.de/) is currently the largest barcoding
campaign in Europe, financed by the German Federal Ministry for
Education and Research. GBOL has five regional and thematic nodes
and is flanked by complementing projects (Barcoding Fauna Bavarica
(BFB), FREDIE for European freshwater fishes and some invertebrate
taxa, barcoding of the North Sea Fauna) and supported by more than
200 citizen scientists. The latter play a very important role, because
the taxonomic workforce and expertise in natural history museums
and universities is insufficient. We describe the efforts required to
motivate voluntary helpers and the importance of a national web
portal for communication in the native language, and the necessity
for a close cooperation with BOLD. To maximize synergies a strong
commitment to common data standards and repositories is needed, as
well as better trans-boundary cooperation between nations, which
both would accelerate database completion and increase cost-
efficiency. We present statistics for the work achieved in three years
(species numbers, taxonomic coverage, geographic coverage), some
problems that slow down the workflows, and observations on the
resolution of COI barcodes in different taxa. We discuss some unex-
pected discoveries (e.g., new species, new parasite-host relations).
Significance: This study shows that interdisciplinary knowledge of
biology, ecology, and barcode information—important tools in defi-
nitions of species—are required to describe new species.
Assessment of candidate DNA barcoding loci for the wheat
and grass family Poaceae in Egypt
Mohamed Awad,
1
Ragab M. Fahmy,
2
Kareem A. Mosa,
3
Mohamed Helmy,
4
and Fawzy A. El-feky
1
1Biotechnology Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt.
2Agricultural Research Center, Geza, Egypt.
3Department of Applied Biology College of Science, University of Sharjah, United Arab Emirates.
4The Donnelly Centre, University of Toronto, Toronto, Canada.
Corresponding author: Mohamed Awad (e-mail: m.awad@azhar.edu.eg).
DNA barcoding is widely used for biodiversity studies based on stan-
dard DNA regions. While many loci have been proposed as a DNA
barcodes in plants, standardizing regions as a DNA barcode poses a
challenge in many plant families. The Egyptian flora is rich with a
huge number of Poaceae plants that differ on their morphological
characteristics in advanced developmental stages, but which are very
difficult to discriminate in early developmental stages. This study
assessed the significance of using two DNA barcoding loci (Atpf and
rbcL) in the distinction between different Egyptian land races of
Triticum eastivum and Triticum turgidum subsp. durum using 11 different
landraces and 7 local varieties. Furthermore, the same two loci have
been tested for distinguishing between other Poaceae crops and herbs
including Avena fatua,Hordeum vulgare, and Hordeum apertum. Our re-
sults showed that Atpf and rbcL have a significant potential in distin-
guishing between the ambiguous Poaceae plants. We have also
attempted to pursue a bioinformatics approach by retrieving 10 chlo-
roplast genomes for different Triticum species and subspecies from
NCBI and using a python script to separate each gene in these ge-
nomes. CLC software has been used to perform multiple sequence
alignment for 80 chloroplast genes, and the intragenic regions be-
tween them, for in-silico analysis to determine the most useful DNA
barcode loci for Triticum species. Our results revealed the difficulty of
a single locus to distinguish between different Triticum species. How-
ever, the discrimination between Triticum species is possible by using
a combination of some gene loci including (Atpf, Atpf, and nadhA).
Interestingly, a combination of intragenic regions (trnF–trnT and
Atpf–trnQ) showed a very high resolution to distinguish the Triticum
species. We recommend further research on using intragenic regions
as standard DNA barcode loci in Poaceae.
DNA barcoding, identification, and validation of the genus
Phyllanthus in Nigeria using rbcL and Atpf genetic markers and
the taxonomic implication
Daniel Awomukwu,
1
Bio Louis Nyananyo,
2
Chiedozie Joel Uka,
3
Paula Spies,
4
and Bulelani Londoloza Sizani
4
1Department of Biological Sciences, Federal University Otuoke, Bayelsa State, Nigeria.
2Department of Plant Science and Biotechnology, University of Port Harcourt, Port Harcourt, Rivers
State, Nigeria.
3Department of Botany, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
4Department of Genetics, University of the Free State, Bloemfontein, Free State, South Africa.
Corresponding author: Daniel Awomukwu (e-mail: xdanny18@yahoo.com).
Background: DNA extraction, purification, PCR amplification, and
sequencing of five species of Phyllanthus in Nigeria—namely P. amarus
Schum and Thonn, P. urinaria Linn., P. odontadenius Mull-Arg.,
P. niruroides Mull-Arg., and P. muellerianus (O. Ktze) Excel, belonging
to the family of Phyllanthaceae—were carried out using plastid
ribulose-1,5 bisphosphate carboxylase large chain (rbcL) and mat-
urase K (Atpf) genetic markers to identify unknown Phyllanthus
species. The plastid region revealed that the Phyllanthus species
were able to be amplified optimally for sequencing. The results of
the nucleotide sequences were further compared using the Basic
Local Alignment Sequence Tool (BLAST) on GenBank and the Barcode
of Life Data System (BOLD) for validation. Results: Results revealed
that the closely related species, P. niruroides and P. odontadenius, had no
DNA record to separate them on either GenBank or BOLD using both
rbcL and Atpf gene regions, while P. amarus and P. urinaria were clearly
compatible with other works. P. muellerianus was only compatible with
other works using Atpf gene region but none for rbcL. BLAST valida-
tion also revealed that P. amarus is synonymous with P. niruri due to
overall similarity they share in both rbcL and Atpf gene regions, and
need to be subsumed. Results of the nucleotide sequences and frag-
ment analysis were published on BOLD for barcoding as a standard
coding marker translation matrix.
Myrmecophile communities of the African ant-plant Vachellia
(Acacia) drepanolobium
Christopher C.M. Baker and Naomi E. Pierce
Harvard University, Department of Organismic and Evolutionary Biology, 26 Oxford Street,
Cambridge, MA 02138, USA.
Corresponding author: Naomi E. Pierce (e-mail: npierce@oeb.harvard.edu).
Background: The African ant-plant Vachellia (Acacia) drepanolobium en-
gages in a classic protection mutualism with three species of ant that
occupy hollow swollen-thorn domatia on the host plant. Colonies of
these ant species are parasitized by a broad array of terrestrial arthro-
pods or “myrmecophiles” that inhabit the domatia alongside the ant
mutualists and often eat the ant brood. Results: Using COI barcodes,
we characterize the communities of myrmecophiles of the three dif-
ferent ant species. We examine how the abundance, diversity, and
specialization of these myrmecophile communities differ among the
different ant species. Significance: DNA barcoding has proven invalu-
able for differentiating difficult taxa, permitting rapid field sampling
by non-specialists, and matching up different life stages of myrmeco-
philic species.
Molecular identification of mango hoppers infesting mango
trees in Punjab through DNA barcoding
Geetika Banta, Vikas Jindal, and Manjinder Singh
Department of Entomology, Punjab Agricultural University, Ludhiana-141 004, India.
Corresponding author: Geetika Banta (e-mail: geetikabanta1@gmail.com).
Background: Mango hoppers are one of the most destructive pests of
all varieties of mango. The infestation results in retarded growth of
young trees, while older trees do not bear many fruits. Three species
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of mango hoppers, viz. Amritodus atkinsoni,Idioscopus clypealis, and
I. niveosparus, have been reported to infest mango in India; however,
no report is available on species prevailing in Punjab. This study
aimed to identify mango hoppers species occurring in Punjab using
DNA barcoding. The specimens were collected from the Patiala
(Nabha) and Gurdaspur districts of Punjab. The COI region was ampli-
fied using specific primers, cloned, and its nucleotide sequences were
determined through custom service of Xcelris Pvt Ltd., Ahmedabad.
Results: The obtained sequences were blasted in NCBI, and both pop-
ulations were identified as Amritodus atkinsoni due to 99% similarity of
COI gene sequence. The COI sequence of A. atkinsoni (2) from Lucknow
and Chidambaram, and sequences for Idioscopus niveosparus (2) and
I. clypealis (1), were retrieved from NCBI GenBank database. All the seven
sequences were aligned and processed to the 658-bp DNA barcode
region. A phylogenetic tree was generated using the neighbour-
joining method with 500 replicates for bootstrap using CLC software.
The Punjab population of A. atkinsoni was found to be genetically sim-
ilar to the Lucknow population, with 0.2% nucleotide difference, but it
was genetically distinct from the A. atkinsoni population of Chi-
dambaram. In comparison, the three populations of mango hopper,
Idioscopus spp. formed one separate cluster that differed by 18.0% and
16.98% from A. atkinsoni of Punjab and Tamil Nadu populations, respec-
tively. Significance: This study establishes the prevalence of Amritodus
atkinsoni in two districts of Punjab. However, as available reports es-
tablish existence of different species, numerous samples from differ-
ent regions of India need to be collected and characterized using DNA
barcoding to catalogue the diversity of the mango hopper.
The native bees of México and the DNA Barcode Of Life project
Ricardo Ayala Barajas
1
and Laurence Packer
2
1Estación de Biología Chamela, Instituto de Biologóa, UNAM, Antonio M. Cedeño 6, Jardines Vista
Hermosa, Colima, Colima, 28017, México.
2York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.
Corresponding author: Ricardo Ayala Barajas (e-mail: Ayala rayala@ib.unam.mx).
Background: Bees are considered some of the more important
pollinators of wild and cultivated plants; these provide an essential
ecosystem service that results in the out-crossing and sexual repro-
duction of many plants. The rich bee fauna of Mexico encompasses
6 families and 153 genera, with ⬃1839 currently recognized species.
The distribution of the richness between families is as follows:
Apidae 606 species, Andrenidae 535, Megachilidae 355, Halictidae
229, Colletidae 103, and Melittidae 11. Due to gaps in faunistic
studies, the bee fauna of Mexico is certainly much richer, perhaps
well in excess of 2000 species. The diversity of bees in Mexico
appears intermediate between that of the United States and coun-
tries of Central America. Despite their ecological and economic
importance, bees are facing threats from growing loss of habitats
principally in tropical areas. Thus, it is urgent to conduct more
taxonomic studies including the DNA Barcode of Life project,
which could help taxonomic studies by providing a practical
method for the identification of species for others with interest in
bees, or studies in environmental science, conservation, or pollina-
tion. We began our participation in the iBOL project in 2008, as part
of BEEBOL, which was promoted by Laurence Packer. At this point
there are about 3000 Mexican bee specimens in BOLD with about
1000 species. There are representatives of taxa with ecological im-
portance such as Centridini and Euglossini, as well as genera that
are important as pollinators for agriculture, such as Bombus,Osmia,
Xylocopini, and many Eucerini; as well as groups that are impor-
tant in meliponiculture (Meliponini). The following stage is the
accomplishment of new faunistic projects, to organize more spec-
imens of Mexican bees to be sequenced, in a strategy that includes
species of economic or biological importance. Also accomplished is
the completion of taxonomic revisions of some taxa of bees with
the aid of the DNA Barcode of Life, in addition to morphological
information.
Is DNA barcode intraspecific variation associated with
subspecies delimitation and phenotypic variation?
Ana S. Barreira, Natalia C. Garcia, and Pablo L. Tubaro
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” – CONICET, Avenida Ángel Gallardo
470, Buenos Aires, Argentina.
Corresponding author: Ana S. Barreira (e-mail: asbarreira@gmail.com).
Background: DNA barcodes are a powerful tool to discover unde-
tected genetic variation within species across large geographic areas.
Subspecies are defined on the basis of phenotypic differentiation
across the geographic distribution of the species. However, they are
not necessarily related to the occurrence of deep genetic differentia-
tion within species. Birds are an ideal model to assess the relationship
between previously described phenotypic variation and neutral ge-
netic markers, such as the mitochondrial COI gene, because of their
very well developed taxonomy. We selected 17 pairs of subspecies of
Neotropical passerines and took spectrophotometric measurements
of plumage colouration, given its frequent use in subspecies delimita-
tion. We used visual models to analyze colour measurements from an
avian perspective and assess quantitatively the level of variation in
plumage colour in relation to genetic differentiation between subspe-
cies. Results: Plumage colour differentiation between subspecies was
highly variable but not related to genetic variation. Some pairs of
subspecies showed small levels of genetic differentiation but large
levels of phenotypic variation and vice versa. In all cases, the levels of
plumage colour variation between subspecies was larger than the
colour differentiation threshold perceivable by birds. However, we
found large levels of variation in this character within subspecies.
Additionally, average genetic divergence in COI in 15 of the 17 pairs of
subspecies was larger than the average intraspecific genetic distance
found in Neotropical birds. Moreover, two of them had values that
were even larger than the average genetic distance between sister
species. Significance: Plumage colouration might be subject to selec-
tive pressures and therefore does not reflect the variation in neutral
markers such as DNA barcodes. Although subspecies delimitations
are not a direct measure of the genetic structure within species,
they are a good indicator of large levels of diversification that need
to be sampled in order for DNA barcodes to capture the entire
species variation.
Is Nematocharax (Actinopterygii, Characiformes) a monotypic
fish genus?
Silvia Britto Barreto, Lorena Andrade Nunes, André Teixeira da Silva,
Ricardo Jucá-Chagas, Débora Diniz,
and Paulo Roberto Antunes de Mello Affonso
Universidade Estadual do Sudoeste da Bahia, Dep. Ciências Biológicas, Av. José Moreira Sobrinho, s/n
Jequiezinho, Jequié – BA 45206190, Brazil.
Corresponding author: Silvia Britto Barreto (e-mail: silvinhabbarreto@hotmail.com).
Background: Since classic morphologic studies show intrinsic limita-
tions for species identification of some animal groups, the association of
traditional taxonomy with DNA barcodes and geometric morphometrics
has been useful to discriminate otherwise cryptic forms, providing more
precise estimates of biodiversity. Therefore, the goal of this study was to
access the diversity in Nematocharax, a controversial monotypic and sex-
ually dimorphic genus of Neotropical fish, based on sequencing of cyto-
chrome c oxidase subunit I (COI) and morphometric analyses of seven
populations of N. venustus from Contas, Almada, and the Jequitinhonha
River basins. Results: The intrapopulation divergence in COI sequences
ranged from 0% to 2.2%, with the highest value in samples from the
Gongogi River sub-basin. The interpopulation genetic divergence varied
from 0% to 7.5%, with the highest differentiation between populations
from the Upper Contas sub-basin and the Almada River basin. The
neighbour-joining tree resulted in four genetic groups with bootstrap
values equal to or higher than 99%, comprising 16 haplotypes. Morpho-
metric differences were also identified, mainly related to body height,
head length, head height, and eye diameter. Significance: Both genetic
and morphometric data revealed that specimens from the Upper Contas
sub-basin are highly divergent from other populations, thus suggesting a
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new species of Nematocharax established after the uprising of the Diaman-
tina Plateau in the state of Bahia, northeastern Brazil. Most likely, sexual
selection, restricted dispersal, and geographic isolation have been acting
as synergistic forces to the fast evolutionary split between populations
among and within hydrographic basins. These data challenge the current
view that Nematocharax is a monotypic genus inasmuch as unique evolu-
tionary units or cryptic species were identified. In addition, we recom-
mend that the coastal basins in northeastern Brazil, highly impacted by
human activities, should be prioritized in conservation plans.
New subspecies of an Arctic moth from SW Yukon:
evolutionary and ecological novelty
Isabel C. Barrio,
1
B.C. Schmidt,
2
S. Cannings,
3
and D.S. Hik
1
1Department of Biological Sciences, University of Alberta, Edmonton, AB T5N 0R5, Canada.
2Canada Canadian National Collection of Insects, Arachnids and Nematodes, Agriculture
and Agri-Food Canada, K.W. Neatby Bldg., 960 Carling Ave., Ottawa, ON K1A 0C6, Canada.
3Environment Canada, Canadian Wildlife Service, Whitehorse, YT Y1A 5X7, Canada.
Corresponding author: David Hik (e-mail: dhik@ualberta.ca).
Background: The Yukon is a refugium for species characteristic of
different biogeographic affinities. We describe a new subspecies of a
tussock moth previously considered a High Arctic endemic, the Arctic
moth Gynaephora groenlandica. Using morphological characters and
DNA sequences (the 658-bp “barcode” region of the cytochrome c
oxidase subunit I gene), we examined Gynaephora specimens from
two alpine populations in southwestern Yukon. We compared these
populations to Arctic populations of Gynaephora groenlandica from
Greenland and Nunavut. Barcode haplotypes were compared using
phylograms constructed by the neighbour-joining method imple-
mented in BOLD. We also investigated some unique ecological
characteristics of this new subspecies, including diet breadth and re-
sponses to experimental warming. Results: DNA barcode haplotypes
of the two Yukon populations differed from Arctic specimens by
0.46%–0.47% (Kimura 2-parameter distance model), while variation
within High Arctic specimens remained under 0.31%. Haplotypes from
the SW Yukon population consistently differed from DNA barcodes of
G. groenlandica of Arctic and Greenland populations in the BOLD data-
base. Given the differences in habitat, geography, wing phenotype,
and DNA barcode sequences, we recognized these alpine populations
as a distinct subspecies, Gynaephora groenlandica beringiana Schmidt
and Cannings. The DNA barcode sequences are available in GenBank
(accession numbers JN280825; JN280826). Feeding trials indicated
that Yukon alpine populations appear to have a broader diet spec-
trum than High Arctic populations. There is also evidence that cater-
pillar diets and physiology are influenced by experimental warming.
Significance: These southern, alpine populations of G. groenlandica
beringiana are of both biogeographic and ecological interest. Popula-
tions inhabiting arctic and alpine environments are subjected to dif-
ferent environmental pressures that may translate into localized
ecological, physiological, and behavioural adaptations. Comparisons
of arctic and alpine populations can help us to understand how spe-
cies may respond to recent and rapid environmental changes, partic-
ularly in regions of such biogeographic relevance as the SW Yukon.
Tiny, but complicated, mitochondrial genomes make
barcoding challenging in the parasitic phylum Apicomplexa
J.R. Barta, J.D. Ogedengbe, A. Leveille, M.A. Hafeez,
and M.E. Ogedengbe
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1,
Canada.
Corresponding author: J.R. Barta (e-mail: jbarta@uoguelph.ca).
Background: Major “groups” within the Apicomplexa have been recog-
nized for over a century: coccidia, piroplasms, plasmodia, haemogre-
garines, gregarines, and cryptosporidia. Molecular phylogenetics using
nuclear (nu) loci have inferred relationships among these groups but
relationships within each are largely unresolved. We showed the mito-
chondrial (mt) cytochrome c oxidase subunit I (COI) used as a “DNA
barcoding” locus to be far superior to many nuclear loci, especially nu
rDNA and ITS regions. Although complete mt sequences were obtained
from Plasmodium falciparum and Theileria parva over 20 years ago, the first
complete mt genome from a coccidium, Eimeria tenella, was sequenced in
2010. Our research explores the diversity of the mt genomes of taxonom-
ically and biologically diverse Apicomplexa and evaluates mt genetic loci
as potential DNA barcode targets. Results: In the short time since the
first coccidial mt genome was sequenced, we generated >28 additional
complete mt genomes, with representatives from seven coccidian gen-
era. Genome content and gene orders are largely conserved within major
apicomplexan groups but genome organization varied dramatically be-
tween major lineages. The mt COI locus was an excellent species-level
DNA barcode target for most coccidia. Combining mt genome sequences
(barcode data) with nu 18S rDNA sequences provided robust species de-
limitation as well as information on deeper evolutionary relationships.
Significance: DNA barcoding at the mt COI locus is an excellent species-
level delimiter for most coccidia and, with some procedural modifica-
tions, could provide reliable species identifications from mixed samples.
Multiplexing next-generation sequencing technologies could provide si-
multaneous enumeration of known species and identification of previ-
ously unknown Apicomplexa in a sample (with concurrent partial
molecular characterization) using the mt COI locus. For many vertebrate
hosts in which the diversity of apicomplexan parasites present may not
be known, this may be a highly productive area of investigation.
Food webs from fish guts: diet analysis using DNA barcoding
increases resolution and changes structure in Canadian boreal
shield lake food webs
Timothy J. Bartley,
1
Heather E. Braid,
2
Kevin S. McCann,
1
Nigel P. Lester,
3
Brian J. Shuter,
4
and Robert H. Hanner
1
1Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
2Institute for Applied Ecology, Auckland University of Technology, Private Bag 92006, Auckland, New
Zealand 1010.
3Science and Research Branch, Ontario Ministry of Natural Resources, Peterborough, ON K9J 7B8, Canada.
4Department of Ecology and Evolution, University of Toronto, Toronto, ON M5S 3G5, Canada.
Corresponding author: Timothy J. Bartley (e-mail: tbartley@uoguelph.ca).
Background: Food webs are important in understanding the struc-
ture, function, and behaviour of ecosystems, but methodological lim-
itations have historically restricted the resolution of food webs, which
impacts our understanding of food web structure. DNA barcoding has
proven useful in identifying feeding interactions; however, few stud-
ies have evaluated whether dietary data produced using barcoding
affect food web resolution or structure. We used predatory fish from
Canadian boreal shield lakes to evaluate barcoding as a tool to in-
crease dietary and food-web resolution and establish food-web struc-
ture. Results: Compared to traditional morphological approaches,
DNA barcoding yielded higher diversity and frequency of prey species
from stomach contents and significantly increased prey diversity for
individual predators and species, showing increased dietary resolu-
tion. Barcoding identified significantly more feeding links and prey
taxa, indicating increased food web resolution. Barcode-based food
webs had higher linkage density, higher proportions of possible feed-
ing links, increased intraguild predation (omnivory), and more pred-
ator species coupling habitats, demonstrating changes to food-web
structure. Significance: The application of DNA barcoding for diet
analysis has the potential to identify feeding interactions in an un-
precedented and highly detailed manner, improving food web resolu-
tion and changing our understanding of food web structure in ways
that have implications for their stability and dynamics. Highly re-
solved dietary data based on DNA barcodes have additional applica-
tions, such as inferring the behaviour of predators and observing
variability in food web structure. By harnessing the natural foraging
ability of predators to sample their environment, diet analysis based
on DNA barcodes could also help identify the presence of species of
interest, such as those that are invasive, rare, at risk, bioindicators,
economically important, or difficult to detect. DNA barcoding applied
to diet analysis has the potential to complement existing methods and
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improve our ability to establish structure, monitor species, and detect
change in ecosystems.
Long-term monitoring of tropical arthropods and DNA barcoding
Yves Basset
Smithsonian Tropical Research Institute, Apartado 0843-03092, Panama City, Panama.
E-mail for correspondence: bassety@si.edu.
Habitat degradation is currently the biggest threat to tropical in-
sects. However, the effects of climate change may soon be more
pervasive. Disentangling the short-term effects of local environ-
mental factors and seasonal variation from the long-term effects of
climate change on tropical communities can be challenging be-
cause of the diversity and complexity of these communities. Since
2009, the CTFS-ForestGEO Arthropod Initiative attempts to detect
long-term changes in the abundance and composition of focal assem-
blages of arthropods, driven primarily by climatic cycles and changes,
as opposed to short-term stochastic changes. The Initiative is cur-
rently active at nine tropical rainforest sites around the world. In this
context, DNA barcoding is used to refine delineation of insect mor-
phospecies and identifications of common, abundant species, likely to
be tractable in the long term. DNA barcoding also allows matching
casts of social insects and monitoring functional groups important to
the forest ecosystem but not well known taxonomically. Relying on
DNA barcoding, we provide examples of annual indices, recent popu-
lation dynamics, and change in species composition for a variety of
taxa during the past 6 years on Barro Colorado Island (BCI), Panama.
We also highlight the contribution of DNA barcoding to mining his-
torical data, with an example of an analysis of recent butterfly extinc-
tions on BCI. We believe that < 6% of the 601 species ever recorded on
BCI are locally extinct from the island and discuss species traits most
likely to influence the probability of extinction. We conclude that
with adequate protocols (including DNA barcoding) common species
of tropical insects can be precisely monitored in the long term. Be-
cause most insects have short generation times in the tropics, it may
therefore be possible to develop efficient warning systems that can
yield results within 5-10 years (equivalent to ⬃40–80 insect genera-
tions).
DNA barcoding program at Naturalis Biodiversity Center,
the Netherlands
Kevin Beentjes, Arjen Speksnijder, Berry van der Hoorn,
and Jan van Tol
Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA, Leiden, the Netherlands.
Corresponding author: Kevin Beentjes (e-mail: kevin.beentjes@naturalis.nl).
Background: The national DNA barcoding project funded by the
Dutch Ministry of Economic Affairs is nearing its final stages. The
DNA barcoding project at Naturalis focused on the Dutch flora and
fauna. Large projects involving bryophytes and vascular plants suc-
ceeded in covering most of the flora of the Netherlands, with a
combined total of over 6000 specimens barcoded. The Dutch fauna
mostly involved arthropod groups such as the Coleoptera, Diptera,
Hemiptera, and Hymenoptera, with over 10 000 specimens col-
lected by both employees and 200 citizen specialists, which were
subsequently barcoded. The Lepidoptera were represented by over
10 000 barcoded specimens as well, spread over several projects,
both Dutch and international. The Odonata collections of Naturalis
provided over 8000 sequenced specimens from all over the world.
Results: Whilst the original goal was to sequence 27 000 specimens,
the current total of sequenced samples has already exceeded 40 000.
With several additional projects processed in the high-throughput
facility, the Naturalis DNA bank has surpassed 60 000 DNA extracts.
All extracts are stored and are available for future research. With the
end of the project in sight, the contribution to BOLD by Naturalis is
growing. At least 27 000 successful sequences will be uploaded to
public databases. Voucher specimens will be accessible through the
Naturalis Bioportal (http://bioportal.naturalis.nl). Significance: The cur-
rent database is applied in identification requests relating to wildlife
forensics projects, bird strikes at civilian and air force airports, food
quality and food safety issues, and insurance claims. While there are
currently no plans for a continuation of the large-scale DNA barcod-
ing, there will be an additional focus on the barcoding of freshwater
organisms of the Water Framework Directive in the coming years,
with grants received from the Gieskes-Strijbis fund. In addition, vari-
ous projects have already been started using the DNA barcoding pipe-
line to solve different scientific questions within Naturalis.
All complete? Comparison of morphological and DNA-based
biodiversity assessments in one of the world's best-studied
stream ecosystems (Breitenbach, Germany)
Arne J. Beermann,
1
Volodymyr Pushkar,
1
Rüdiger Wagner,
2
and Florian Leese
1
1Ruhr University, Bochum Universitaetsstrasse 150, D-44801 Bochum, Germany.
2University of Kassel, Heinrich-Plett-Strasse 40, D–34132 Kassel, Germany.
Corresponding author: Arne J. Beermann (e-mail: arnebeermann@googlemail.com).
Background: DNA barcoding has often been demonstrated to be su-
perior to traditional biodiversity assessments. Yet, in most of these
comparisons, morphological species determinations have not been
performed by expert taxonomists or they have used immature life
stages, rendering the comparisons of resolution as inappropriate.
Here, we used species-level data obtained by expert taxonomists from
male adult insects and compared the resolution of DNA-based and
morphological determinations side-by-side. As a case study, we used
the Breitenbach (Hesse, Germany). With more than 50 years of re-
search and over 1800 reported metazoan species, the Breitenbach is
one of the ecologically and taxonomically best-studied freshwater eco-
systems. Results: We collected more than 11 000 adult insects with
emergence traps over one year. For more than 200 morphospecies, up
to 20 DNA barcodes per species were generated. The comparison of
DNA-based and morphological species assignments revealed a high
congruency between both approaches (⬃89% match). However, for
several important indicator taxa, DNA barcoding identified unrecog-
nized species diversity (9%) and cases of unnecessarily split species
(2%). In at least one case (Sericostoma sp.), DNA barcoding suggested the
presence of two species, whereas microsatellites revealed the presence of
a single species (populations in secondary contact). Significance: Mor-
phological identifications by expert taxonomists provide reliable and
high-resolution biodiversity information even without DNA barcod-
ing. However, in most cases (in particular ongoing biomonitoring
programs), species identification is performed not by taxonomic ex-
perts and is often done using immature life stages of freshwater in-
sects. This leads to a significant mismatch between the true and the
inferred stream biodiversity, which has potential negative conse-
quences for management. Therefore, as demonstrated in this study,
DNA barcoding provides a unique tool to quantify species diversity
independently of the taxonomic expertise and the life stages available
and thus significantly improves biodiversity assessments.
Assessing the distribution and genetic diversity of Antarctic
springtails (Collembola)
Clare Beet,
1
Ian Hogg,
1
Gemma Collins,
1
Don Cowan,
2
Byron Adams,
3
and Diana Wall
4
1University of Waikato, Hamilton, New Zealand.
2University of Pretoria, Pretoria, South Africa.
3Brigham Young University, Provo, Utah, USA.
4Colorado State University, Fort Collins, Colorado, USA.
Corresponding author: Clare Beet (e-mail: clarebeet17@gmail.com).
In order to predict future changes in biodiversity for Antarctic terres-
trial ecosystems, an accurate assessment of current levels of biodiver-
sity and an understanding of past responses is required. Here, we
sampled populations of three springtail (Collembola) species from
10 sites in the vicinity and to the north of the Mackay Glacier, which acts
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as a transitional zone between two biogeographic regions (northern
and southern Victoria Land). Of the year-round terrestrial animals in
Victoria Land, springtails have the narrowest environmental toler-
ances, and can therefore act as sensitive indicators of climate-driven
environmental change. Accordingly, we assessed levels of diversity
within and among populations of Gomphiocephalus hodgsoni,Cryptopygus
nivicolus, and Antarcticinella monoculata using the mitochondrial COI
(barcoding) gene. Results: In total, 105 springtails were successfully
sequenced, with highly divergent populations (cryptic species) found
for each of the three putative springtail species within the region.
Sampling also provided the first records of species from many of
the sites visited. New records for the springtail species Antarcticinella
monoculata and Cryptopygus nivicolus are of particular interest as they
may indicate changes in distribution from previous collections under-
taken in the early 1960s. Significance: This research will contribute to
our understanding of the evolutionary history of the region and pro-
vide an important baseline inventory against which to monitor future
changes as well as enable protection of biodiversity hotspots for Ant-
arctica's terrestrial biota.
Assessing the diversity of New Zealand freshwater “EPT”
macroinvertebrates
Clare Beet,
1
Ian Hogg,
1
Brian Smith,
2
Kristi Bennett,
1
and Gemma Collins
1
1University of Waikato, Hamilton, New Zealand.
2National Institute of Water and Atmospheric Research (NIWA), Hamilton, New Zealand.
Corresponding author: Clare Beet (e-mail: clarebeet17@gmail.com).
Background: The larval/nymphal stages of Ephemeroptera (mayflies),
Plecoptera (stoneflies), and Trichoptera (caddisflies; EPT taxa) are key
components of aquatic food webs and are frequently used as bioindi-
cators of water quality. Challenges in identifying larvae/nymphs to the
species level, however, are one factor limiting their more routine use.
New Zealand has over 50 mayfly species, 106 species of stonefly, and
244 species of caddisfly, most of which are endemic. The aim of this
study was to provide complete barcode coverage for the New Zealand
EPT taxa using curated collections of adult specimens available at the
Auckland and Canterbury museums. Results: Thus far, ⬃90% of
known caddisfly, 80% of mayfly, and 50% of stonefly species have been
barcoded as well as several undescribed species. Preliminary results
indicate average intraspecific divergences were generally <2% (range
0%–3.6%) for all taxa. In contrast, minimum interspecific distances
were generally >10% (range 6%–19%). COI sequences were congruent
with existing taxonomic literature and also highlighted some mis-
identifications within museum collections as well as groups in need of
further taxonomic review (e.g., Deleatidium spp., Hydrochorema spp.,
Pycnocentrodes spp.). Significance: This study has begun the assembly
of a complete reference library of the New Zealand EPT taxa. These
data enable rapid and accurate assessment of larval specimens and
additionally provide the opportunity to incorporate EPT taxa into
next-generation sequencing approaches to analyse entire aquatic in-
sect communities.
The effect of sample complexity and sequencing depth on DNA
barcoding of mixed-species pollen samples
Karen L. Bell,
1
Kevin S. Burgess,
2
Timothy D. Read,
3
and Berry J. Brosi
1
1Emory University, Department of Environmental Sciences, 400 Dowman Drive, Atlanta, GA 30322, USA.
2Columbus State University, Department of Biology, Columbus, GA 31907-5645, USA.
3Emory University, Department of Medicine, Division of Infectious Diseases and Department of Human
Genetics, 400 Dowman Drive, Atlanta, GA 30322, USA.
Corresponding author: Karen L. Bell (e-mail: karen.bell@emory.edu).
Background: Traditional microscopic identification of pollen is slow
and of limited taxonomic resolution. By contrast, DNA barcoding can
enhance both the speed and the accuracy of pollen identification.
Recent technological advances have enabled us to develop a method
for identifying pollen in mixed-species samples, through mixed-
amplicon sequencing of one of the standard DNA barcoding markers
(chloroplast rbcLa) on the Illumina MiSeq platform. Using pollen sam-
ples of known composition, we addressed the following questions:
(i) How many species can be detected within a sample? (ii) To what extent
is this affected by taxonomic relatedness? (iii) How rare can a species
be in a sample before it becomes undetectable? Results: First, we
show proof-of-concept for plastid-based DNA metabarcoding by dem-
onstrating that the plastid barcoding marker rbcL can be amplified
and sequenced from mixed-species pollen samples. We were able to
detect species in mixtures comprising up to nine species. We were also
able to distinguish closely related species and rare species in mixtures.
Subsampling of sequence reads enabled us to determine the sequenc-
ing depth required to detect all species present in mixtures of varying
complexities. Significance: We predict that DNA metabarcoding of
mixed-pollen samples will transform a wide range of fields, including
pollination biology, allergen monitoring, and forensics, by enhancing
both the speed and accuracy of species identification. Future advances
in this work, including standardization of the bioinformatics pipe-
line, development of methods to correct for bias in ptDNA copy num-
ber and amplification bias, and inclusion of multiple DNA barcoding
loci, would enable routine use of these methods in a range of fields.
DNA barcoding reveals microevolutionary signals in fire
survival and regeneration strategies in southern African
Psoraleoid legumes
Abubakar Bello,
1
Barnabas H. Daru,
2
Charles H. Stirton,
1
Samson B.M. Chimphango,
1
Michelle van der Bank,
3
Olivier Maurin,
3
and A. Muthama Muasya
1
1Bolus Herbarium, University of Cape Town, Private Bag X3, Rondebosch 7700, South Africa.
2Department of Plant Science, University of Pretoria, Private Bag X20, Hatfield 0028, Pretoria, South Africa.
3Department of Botany and Plant Biotechnology, University of Johannesburg, P.O. Box 524, Auckland
Park 2006, Johannesburg, South Africa.
Corresponding author: Abubakar Bello (e-mail: bllabu001@myuct.ac.za).
Background: Large-scale DNA barcoding provides a novel technique
for species identification and evaluation of relationships across vari-
ous levels (populations and species) and may reveal fundamental pro-
cesses in recently diverged species. Here, we analysed DNA sequence
variation in the recently diverged legumes from the Psoraleeae
(Fabaceae) occurring in the Cape Floristic Region (CFR) of southern
Africa to test the utility of DNA barcodes in species identification and
discrimination. We further explored the phylogenetic signal on fire
survival and regeneration strategies at species and generic levels.
Results: We showed that psoraleoid legumes of the CFR exhibit a bar-
coding gap and high K values, yielding Atpf as a better barcode than
rbcLa or combination of Atpf and rbcLa. We found a substantial match
between genetic and morphologically delimited genera and species,
supporting the discriminatory power of DNA barcoding. We also
found that different lineages showed a weak but significant phyloge-
netic conservatism in their affinity to fire as reseeders or resprouters,
with a stronger clustering of resprouters than would be expected by
chance. Significance: Our results showed that DNA barcoding may be
valuable in aiding species identification in recently diverged lineages
and for the detection of phylogenetic signal in some lineages from this
unique biodiversity hotspot. These novel microevolutionary patterns
might be acting continuously over time to produce multi-scale regu-
larities of biodiversity.
Barcoding as a tool to assess trophic impacts in an
experimental deforestation site in Borneo
David Bennett, Stephen Rossiter, and Elizabeth Clare
Queen Mary University of London, Mile End Rd., London, UK.
Corresponding author: David Bennett (e-mail: david.bennett@qmul.ac.uk).
Background: Tropical deforestation is causing widespread damage to
many biodiversity hotspots, with the need for timber and palm oil
causing significant fragmentation of Southeast Asia's remaining for-
est. The impact of this fragmentation on species interactions is poorly
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understood, and so it is important to assess any potential ecosystem-
wide changes that such habitat modification may cause. Through use
of 157-bp long “mini-barcodes” from the COI region, it is now possible
to identify the DNA in degraded material, such as faecal matter, with
high resolution. Combining this technique with next-generation se-
quencing, a suite of barcodes can be taken from a mixed slurry of
tissue. Using this combination of techniques, faecal matter and ecto-
parasites from 24 forest bat species from the SAFE project (Sustain-
ability of Altered Forest Ecosystems, based in Sabah, Malaysia) have
been collected along a gradient of habitat disturbance. Over the com-
ing 2 years the site is being subjected to experimental habitat frag-
mentation. During this period further samples will be collected over a
time-series, allowing for analysis of the temporal impacts on the stud-
ied plots of the deforestation of the surrounding matrix. Comple-
menting this approach, Malaise trap samples have been obtained at
each of the experimental sites for comparison with any potential
changes in bat diet. The experiment will identify any changes in eco-
system structure and stability across three trophic levels along a time-
series of habitat fragmentation. Results: Results will be presented
from analysis taking place prior to the conference, including tripartite
foodwebs of molecular operational taxonomic units (MOTUs) and
analysis of any potential trophic niche overlap between sympatric
species. Significance: Analysis of the impacts of habitat fragmenta-
tion on ecosystem structure are lacking, and the novel application of
DNA barcoding gives this study great potential to give new insights
into this phenomenon. This innovative study is also the first molecu-
lar investigation into the diet of bats on the island of Borneo.
DNA barcoding of spidermites (Prostigmata: Tetranychidae) in
vegetables using COI and ITS2 markers
Shruti Bennur,
1
P.S. Abida,
1
P.A. Valsala,
1
Deepu Mathew,
1
and Haseena Bhaskar
2
1Centre for Pant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, Kerala
Agricultural University, Thrissur, India.
2Department of Agricultural Entomology, College of Horticulture, Vellanikkara, Kerala Agricultural
University, Thrissur, India.
Corresponding author: Shruti Bennur (e-mail: shrubennur@gmail.com).
Background: Identification of spider mite species is an important
issue in pest control as members of this group are responsible for
significant crop losses. The use of DNA barcodes, short DNA sequences
from a standardized region of the genome, has recently been pro-
posed as a tool to facilitate species identification and discovery.
Results: DNA sequence data of economically important Tetranychidae
mites were studied: Tetranychus truncatus,T. macfarlanei, and T. okinawanus
(reported for the first time in India). These mites were collected from
different vegetable crops including cowpea, amaranthus, okra, doli-
chos bean, brinjal, cucumber, and ridge gourd from various locations
in Kerala, India. Spider mites were reared in the laboratory to obtain
an isoline from which DNA was isolated separately. Microscopic slides
were also prepared for morphological identification. The polymerase
chain reaction was used to amplify the second internal transcribed
spacer (ITS2) of the ribosomal DNA and the cytochrome c oxidase
subunit I (COI) locus of the mitochondrial genome. ITS2 and COI
yielded ⬃620- and 868-bp fragments, respectively. The fragments were
cloned and sequenced. Sequences were submitted to GenBank, and
sequences showed high similarity with other members of the above-
mentioned species within the database. The specimen data, specimen
images, sequence information, and trace files were further deposited
in the Barcode of Life Data Systems (BOLD). Genetic distances were
summarized, using the Kimura 2-parameter distance model. Intraspe-
cific COI sequence divergences (T. truncatus and T. okinawanus) were
0%–0.54%, while intraspecific divergences within this genus ranged
from 9.14% to 9.89%. ITS2 sequence divergence (T. truncatus,T. okinawanus,
T. macfarlanei) within species was 0%, but 7.7%–7.92% within the
genus. Significance: Our work has revealed that both ITS2 and COI
are effective markers to differentiate Tetranychus spp. DNA barcoding
helps in precise identification of species which can be further used
as a diagnostic tool for quarantine as well as in pest management
activities.
Genomics applied to conservation and management of aquatic
resources: are the promises being filled?
Louis Bernatchez
Université Laval, 1030, Ave de la Médecine, Pavillon Charles-Eugène Marchand, Quebec, Canada.
E-mail for correspondence: louis.bernatchez@bio.ulaval.ca.
Conservation genetics has been defined as the application of genetics to
preserve species as dynamic entities capable of coping with environmen-
tal change. Towards this end, molecular data can play two fundamental
roles. The first one, inventorial, pertains to documenting patterns and
has driven much of what we have accomplished until now. The second,
mechanistic, refers to deciphering evolutionary processes underlying
those patterns, and this is where most of us put big hopes in the use of
modern, high-throughput genomics methods. In particular, by allowing
to scaling up genome coverage for any non-model species, it is predicted
that genomics should refine our understanding of intra-specific variation
by improving estimates of population genetic and evolutionary parame-
ters. Secondly, this should also ease the identification of “genes that
counts” from an adaptive standpoint, and ultimately bring us closer to
the Holy Grail of conservation genetics, that is: Finding causal relation-
ships between genetic variation, phenotypes and the environment to
predict future dynamics of selectively important variation and potential
for adaptation to new conditions. In this presentation, I will present
several success stories to illustrate some of the progress that we have
made towards this end in the context of conservation and management
of aquatic species. I will also comment on some of the main challenges
that remain to be met, no matter the power and resolution of the current
genomics methods at hands.
Phylogenetic analysis, species identification and delimitation
of New Caledonian geckos and skinks using DNA barcoding
Justin Bernstein,
1
Aaron Bauer,
1
Todd Jackman,
1
and Yun-yu Wang
2
1Villanova University, Villanova, Pennsylvania, USA.
2Kunming Institute of Zoology, CAS Kunming, China 650223.
Corresponding author: Justin Bernstein (e-mail: jmbernst223@gmail.com).
Background: Due to its long isolation and subsequent in situ speciation,
New Caledonia has an extraordinary number of endemic animal and
plant species. Many species, including scincid and gekkotan lizards, are
still being discovered and are in danger of extinction due to anthropo-
genic activity. The COI gene is being used to barcode the most speciose
genera of endemic New Caledonian geckos and skinks. The utility of COI
for distinguishing undescribed species identified by other means and its
phylogenetic informativeness within the target genera will be assessed.
Results: The phylogenetic trees of COI sequence data from 143 Bavayia
(family Diplodactylidae) and 100 Caledoniscincus (family Scincidae) were
compared to trees based on the ND2 gene, which is generally believed to
provide a robust hypothesis of relationships at the intrageneric level in
most squamates. The Caledoniscincus COI tree grouped the 15 described
species of the genus together. The base of the tree was recovered with low
support. Several species groups came out as sister taxa to each other, with
the same topology as the ND2 tree. The Bavayia tree recovered 40 species
groups compared to 43 that were identified in the ND2 tree. The COI tree
groups all Bavayia species into the correct species groups. Significance:
The use of COI barcoding may prove beneficial for several research facil-
ities and conservation organizations. DNA barcoding would particularly
benefit conservation efforts in New Caledonia. New Caledonia has sev-
eral introduced fauna, one of which is feral cats that predate on these
lizard populations. Partial remains of lizards in feral cat scats are difficult
for non-specialists to identify. However, the tail tips and scales found in
cat scats can be used for DNA barcoding to allow for the accurate
identification of species and species groups of threatened reptiles in
New Caledonia.
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The School Malaise Trap Program: the benefits of citizen
science for barcoding
Emily Berzitis, Vanessa Breton, and Dirk Steinke
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada.
Corresponding author: Vanessa Breton (e-mail: bretonv@uoguelph.ca).
Background: With the primary goal of engaging students in current
research and fostering their interest in biodiversity and, more broadly,
the environment, the School Malaise Trap Program was developed by the
Biodiversity Institute of Ontario (BIO) in 2013. Since its inception, over
7000 elementary and secondary school students from nearly 200 schools
across Canada have participated in the program, deploying Malaise traps
in their schoolyards for a specific 2-week period in either the spring or
fall. By barcoding the specimens collected at each school, BIO is con-
tributing a wealth of new records to the Barcode of Life Data Systems
(BOLD) with the help of these young citizen scientists. Results: Over
the course of four offerings of the program, schools have collected
close to 200 000 specimens and more than 40 000 were then barcoded
at BIO. These specimens represented 4875 BINs, 733 of which were
new to BOLD. Only a small proportion of these species were collected
in both the spring and fall, demonstrating the importance, in terms of
library building, of running the program multiple times a year. Re-
sults also suggest that offering the program to schools located in
already heavily-sampled areas can still be scientifically (and education-
ally) valuable, with a school in Guelph, Ontario, collecting 12 species
that were new to BOLD in the fall of 2014. Significance: In addition to
encouraging environmental stewardship and introducing students of
all ages to DNA barcoding, the School Malaise Trap Program has dem-
onstrated its continued utility for library building, with great promise
for expansion outside of Canada.
Revisiting Darwin's naturalization conundrum: explaining
invasion success of non-native trees and shrubs in southern
Africa
Bezeng Simeon Bezeng,
1
T. Jonathan Davies,
2
Kowiyou Yessoufou,
3
Olivier Maurin,
1
and Michelle van der Bank
1
1African Centre for DNA Barcoding, University of Johannesburg, PO Box 524, APK Campus, 2006,
Johannesburg, South Africa.
2McGill University, 1205 Avenue Docteur Penfield, Montreal, Quebec, Canada.
3Department of Environmental Sciences, University of South Africa, Florida Campus, Florida 1710,
South Africa.
Corresponding author: Bezeng Simeon Bezeng (e-mail: bezengsimmy@gmail.com).
Background: Invasive species are detrimental ecologically and eco-
nomically. Their negative impacts in Africa are extensive and call for
a renewed commitment to better understand the correlates of inva-
sion success. In this study, we reconstructed the phylogeny of the
species pool using the core DNA barcoding regions (rbcLa and MatK).
Using this phylogeny, we explored several putative drivers of species
invasion among woody, non-native trees and shrubs. Results: We
found that non-native species generally have longer flowering dura-
tion compared to native species, are generally hermaphroditic, and
their dispersal is mostly abiotically mediated. We also revealed that
non-native species that have become invasive are less closely related
to native trees and shrubs than their non-invasive, non-native coun-
terparts. Non-natives that are more closely related to the native spe-
cies pool may be more likely to possess traits suited to the new
environment in which they find themselves, and thus have a greater
chance of establishment. However, successful invaders are less closely
related to the native pool, indicating evidence for competitive release
or support for the vacant niche theory. Significance: Non-native
species in southern Africa are characterized by a suite of traits,
including long flowering times, a hermaphroditic sexual system, and
abiotic dispersal, which may represent important adaptations promot-
ing establishment. We suggest that differences in the evolutionary
distances separating the native species pool from invasive and non-
invasive species might help resolve Darwin's Naturalization Conundrum.
DNA barcoding of dye-yielding plants from South India
Shikha Bhattar,
1
R. Siva,
1
S. Seethapathy,
2
U. Santhosh Kumar,
3
R. Uma Shaanker,
3
and G. Ravikanth
2
1School of Bioscience and Technology, VIT University, Vellore, Tamil Nadu, India.
2Ashoka Trust for Research in Ecology and the Environment, Royal Enclave, Srirampura, Jakkur Post,
Bangalore.
3Department of Crop Physiology and School of Ecology and Conservation, University of Agricultural
Sciences, GKVK, Bangalore-560065, India.
Corresponding author: G. Ravikanth (e-mail: gravikanth@atree.org).
Background: Plants are a rich source of dye and pigments. More than
2000 pigments have been identified in plants, of which about 150 are
commercially exploited. In India alone, over 450 plant species are known
to yield dyes. Interest in plant dyes has considerably increased during the
past few years because of the environmental safety concerns surround-
ing the production and use of synthetic dye. There are numerous reports
that some plant dyes also possess therapeutic properties. Due to their
non-toxic properties, natural dyes are extensively used in food, cosmetic,
and textile industries. The increasing market demand for natural dyes,
and the dwindling number of dye-yielding plants, has led to admixtures
of natural dyes with either toxic organic dyes or synthetic ones. These
admixtures could lead to adverse consequences on the health and safety
of consumers. A number of biochemical and molecular techniques have
been used to detect admixture in natural dyes. Results: In the present
study, we have developed DNA barcodes for a few economically impor-
tant dye-yielding plants to assess authenticity of natural dyes. Multiple
accessions of the dye-yielding plants were collected from different geo-
graphical locations in South India. For each species, a Biological Refer-
ence Material (BRM) was established by authenticating the plant material
by a taxonomist and depositing the same in reference herbaria. DNA
barcodes for the authenticated dye-yielding plants were developed using
both a nuclear region of the internal transcribed spacer (ITS) as well as a
chloroplast region (psbA–trnH). Significance: Barcoding of dye-yielding
plants will help in regulating the trade of endangered species and serve
as a deterrent to adulteration of dyes.
Popularizing DNA barcoding in the identification of
agricultural pests and their natural enemies in Bangladesh
Badrul Amin Bhuiya and Santosh Mazumdar
BRGB, Department of Zoology, University of Chittagong, Bangladesh.
Corresponding author: Badrul Amin Bhuiya (e-mail: badrulbhuiya@yahoo.com).
Background: In 2007, four university Professors began molecular taxon-
omy research in different universities within Bangladesh. To date, ⬃100
young scientists from five universities, 25 colleges, and four research
institutes have been trained in DNA barcoding at the DNA barcoding
laboratories of Chittagong University (CU), Chittagong Veterinary and
Animal Sciences University (CVASU), and Dhaka University (DU). Upon
return, many of the trainees focused on DNA barcoding research by
establishing new laboratories in their own institutions. In this contribu-
tion, we provide an overview of key barcoding activities within Bangla-
desh. Results: A total of 114 insect specimens have been barcoded by this
network of researchers, of which 24 sequences of Liriomyza sativae
Blanchard, a leafmining pest insect of vegetable crops, have been submit-
ted to NCBI GenBank and to BOLD by the first author. This was a new
species record for Bangladesh and was published in the online journal
DNA Barcodes in 2014. Two researchers from the DU Aquaculture & Fish-
eries Department barcoded 17 species of aquatic organisms and submit-
ted sequences to GenBank and BOLD. Additionally, one DU Zoology
Department member has barcoded nearly 80 species of freshwater fishes.
Recently, a Professor from Jahangirnagar University received funding
from the World Bank to research the diversity of insects through DNA
barcoding. Significance: The first author introduced DNA barcoding re-
search in Bangladesh in the year 2007. All individuals who received train-
ing became members of the Bangladesh Barcode of Life Forum
(BanglaBOL), founded and headed by the first author. Both the authors
are now involved in the Global Malaise Trap Program in Bangladesh, in
collaboration with the Biodiversity Institute of Ontario, for the study of
insect biodiversity of Bangladesh through next-generation sequencing.
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The few research examples highlighted here demonstrate an active and
growing DNA barcoding network in Bangladesh, which will further both
biodiversity knowledge and socio-economically important applications
of barcoding.
Monitoring lake ecosystem health using metabarcoding of
environmental DNA: temporal persistence and ecological
relevance
Iliana Bista,
1
Gary Carvalho,
1
Kerry Walsh,
2
Martin Christmas,
2
Mehrdad Hajibabaei,
3
Peter Kille,
4
Delphine Lallias,
1
and Simon Creer
1
1Bangor University, School of Biological Sciences, Molecular Ecology and Fisheries Genetics Laboratory,
Bangor, Gwynedd, LL57 2UW, UK.
2Environment Agency, Horizon House, Deanery Rd., Bristol, BS1 5AH, UK.
3University of Guelph, Biodiversity Institute of Ontario, Guelph, ON N1G 2W1, Canada.
4Cardiff University, School of Biosciences, Main Building, Museum Avenue, Cardiff, CF10 3AT, UK.
Corresponding author: Iliana Bista (e-mail: i.bista@bangor.ac.uk).
Background: The use of environmental DNA (eDNA) has been advocated
as a powerful tool for the identification of biodiversity and detection of
rare or invasive species in freshwater ecosystems. A related additional
application lies with monitoring ecosystem health or biomonitoring.
Traditionally, applications of biomonitoring have been based upon tax-
onomic identification of biota, such as macroinvertebrates. Shortcom-
ings of these approaches (low taxonomic resolution, need for high
taxonomic expertise, diverse life stages, etc.) could be addressed through
coupling eDNA with metabarcoding. Results: We are testing the utility
of eDNA for detecting chironomid midges (Diptera: Chironomidae) in
lentic ecosystems. Chironomids are a sentinel group for lake monitoring,
as they are highly diverse and can yield information on the status of the
ecosystems through species responses to environmental stressors. Water
and chironomid exuviae (CPET) community samples collected every 3
weeks, from an annual series of water samples in a natural lake ecosys-
tem in North Wales, were subjected to amplicon sequencing (MiSeq) of
the cytochrome c oxidase i (COI) gene. Parallel taxonomic identification
of community samples provides a real-time comparison of molecular
versus traditional approaches. Additionally, we are investigating the tem-
poral persistence of eDNA in the wild by recording taxon presence from
aqueous eDNA and the community samples. Significance: Findings of
this work will contribute towards establishing a framework for direct
application of eDNA as a tool for lake health assessment, ultimately
aiming to fulfil international directives such as the European Water
Framework Directive, with additional applications across aquatic sys-
tems.
Building a DNA barcode reference library for Canadian spiders
(Araneae)
Gergin A. Blagoev, Jeremy deWaard, and Paul D.N. Hebert
Biodiversity Institute of Ontario, University of Guelph, 579 Gordon Street, Guelph, ON, Canada.
Corresponding author: Gergin Blagoev (e-mail: gblagoev@uoguelph.ca).
Background: With 45 000 known species in 114 families, spiders are
one of the most diverse orders of arthropods. Similar to other arthro-
pod taxa, many spiders are difficult to identify morphologically.
Therefore, DNA barcode libraries can help to alleviate these identifi-
cation challenges. This study provides a DNA barcode reference li-
brary for 1103 (75%) of the 1460 species of spiders previously reported
from Canada, based upon the analysis of more than 45 000 specimens.
Results: The majority (96%) of the specimens analyzed were acquired
through sampling programs led by the Biodiversity Institute of On-
tario that employed diverse methods, including hand collecting, siev-
ing, sweep netting, and trapping (Malaise, pan, pitfall, sticky), and
that focused on National Park collections though the BIObus field
teams. The spider sequences were assigned to 1439 Barcode Index
Numbers (BINs), with 1216 of these BINs composed of specimens be-
longing to a single currently recognized species. There was a perfect
correspondence between BIN membership and a known species in
816 cases, while another 223 species were assigned to two or more
BINs. A small proportion of species (2%) was involved in BIN merges or
in a combination of merges and splits. Significance: Because 98% of
the species possessed a diagnostic array of sequences, the present DNA
barcode reference library will be highly effective for enabling barcode-
based identification of Canadian spiders. This study revealed that
nearest neighbour distances for Canadian spiders were 10 times
higher than maximum intraspecific values (averages of 7.85% versus
0.78%, respectively). These measures of variation presume that the
current taxonomic system is valid and that all specimens have been
correctly identified. The few species lacking barcode divergence need
to be investigated to validate their reproductive isolation by testing
for sequence divergence at nuclear loci, ideally at localities where they
are sympatric. The generation of a barcode reference library for Cana-
dian spiders revealed 17 morphological distinct undescribed species.
Also, 60 species were newly recorded for Canada, with first records
also presented for eight genera and one family.
Cryptic speciation among the spiders (Araneae) of North
America: insights from barcoding 2000 species
Gergin A. Blagoev, Jayme E. Sones, Jeremy R. deWaard,
and Paul D.N. Hebert
Biodiversity Institute of Ontario, University of Guelph, 579 Gordon Street, Guelph, ON, Canada.
Corresponding author: Gergin Blagoev (e-mail: gblagoev@uoguelph.ca).
Background: The existence of morphologically cryptic species is a
common phenomenon in diverse groups of organisms. Oftentimes,
genetically distinct lineages of organisms are classified as the same
species due to their similarity in morphology or lack of thorough
taxonomic studies. Once provisional cryptic species have been de-
tected through DNA barcode analysis, the poor resolution of morpho-
logical characteristics usually do not allow for the description of new
species. This study presents the first large-scale investigation of the
prevalence of cryptic species in spiders by characterizing barcode vari-
ability in 58 000 specimens representing nearly 2000 morphological
species from Canada and the United States, which represents half of
the recorded diversity from these countries and ⬃4.5% of the total
known global spider fauna. Results: Of the 967 species represented by
two or more specimens, ⬃90% exhibited low intraspecific variability
(typically <2.5% maximum divergence) as well as a one-to-one match
with genetic groupings termed BINs (Barcode Index Numbers). How-
ever, 350 were candidates for harbouring cryptic species. Maximum
sequence divergences between BINs assigned to a single valid species
name averaged 4.32%, but ranged from 1.24% to 12.11%. The majority of
these were split into two or just a few BINS, but three displayed sub-
stantial genetic diversity and were divided into 22, 16, and 11 BINs.
Deep genetic divergence was also detected within two charismatic
and widespread American species: the Spiny-backed orb-weaver
(Gasteracantha cancriformis) and the Orchard spider (Leucauge venusta).
Significance: The present study is the first to quantify the prevalence
of candidate cryptic species using such a large dataset. Although BIN
splits within morphological species could indicate evolutionarily sep-
arate but closely related species, the majority of cases require further
study to clarify the taxonomic status of the species involved. If most
BIN splits detected in this study reflect cryptic taxa, the true species
diversity of spiders in North America could be 20%–30% higher than
what is currently recognized.
Metabarcoding of meiofauna and other eukaryotes in
terrestrial and marine ecosystems
Mark Blaxter and Martin Jones
University of Edinburgh, Institute of Evolutionary Biology, Edinburgh EH9 3TF, UK.
Corresponding author: Mark Blaxter (e-mail: mark.blaxter@ed.ac.uk).
Background: The Linnaean project has been extraordinarily success-
ful: in only 250 years humanity has generated stable species descrip-
tions for over 2 million taxa, and these now underpin all of biology,
including evolution, ecology, conservation, and bioprospecting. This
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is not to say that there are no remaining problems: there is still lively
debate on what exactly a “species” is, and local disputes over whether
particular species are credible abound. However, a greater spectre
haunts the project. Estimates of total diversity suggest that the total
number of eukaryote species may exceed 100 million, and that
most of this majority of undescribed taxa are small or very small,
the meio- and micro-biota. Metabarcoding or metagenetics, using
high-throughput next-generation sequencing, offers a revelatory
method to survey, diagnose and count these unseen millions. Results:
We have been surveying terrestrial and marine ecosystems of meiobi-
ota (focusing on Metazoa) using specimen-independent, massively
parallel sequencing of the nuclear small subunit ribosomal RNA gene
from community DNA extractions. This reveals huge diversity, even in
compromised, urban ecosystems. Clustering of these sequences de-
fines molecular operational taxonomic units (MOTU), and these can be
used to define diversity and compare sites and samples. The MOTU can
be linked to known, Linnaean diversity by comparison to databases of
species-sequence links, and thus we can estimate their likely ecology
and relationships. Significance: This methodology may be the only
way to access the unseen majority of taxa, and indeed we identify
thousands of MOTU, some entirely unexpected, in each of our soil and
sediment samples. But how are we to treat the molecular operational
taxonomic units so defined? Is it time to move from Linnaean bi-
nomen into a new phase of anonymous taxa defined only by signature
barcode sequences?
Environmental DNA detection and habitat occupancy of
threatened spotted gar (Lepisosteus oculatus)
Maggie Boothroyd,
1,2
Nicholas E. Mandrak,
3,4
Michael Fox,
1
and Chris C. Wilson
2
1Environmental and Resource Studies Program and Department of Biology, Trent University,
Peterborough, ON, Canada.
2Aquatic Research and Monitoring Section, Ontario Ministry of Natural Resources and Forestry, 1600
West Bank Drive, Peterborough, ON K9J 7B8, Canada.
3Department of Fisheries and Oceans, Burlington, ON, Canada.
4Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto,
ON M1C 1A4, Canada.
Corresponding author: Maggie Boothroyd (e-mail: margaretbooth@trentu.ca).
Background: Access to DNA barcodes allows for rapid develop-
ment of species-specific markers. The environmental DNA (eDNA)
method is able to target DNA present within the environment as a
result of excretion, release of gametes, and shed epidermal cells.
Determining the occurrence and site occupancy of rare and endan-
gered species can be challenging, particularly in aquatic systems
where organisms are often underwater and hidden from view.
Barcode-based eDNA detection was used to assess habitat occu-
pancy of spotted gar (Lepisosteus oculatus), which is federally listed as
“Threatened” in Canada, with known occurrences limited to a
small number of locations in southern Ontario. Water samples
were collected in the spring at six locations based on distribution
information. Within these locations, 10-16 sites were sampled by
taking 3-5 replicate water samples. Additionally, sites were sam-
pled for spotted gar using hoop nets. Results: PCR assays were
developed targeting the cytochrome c oxidase subunit I gene (COI)
to detect spotted gar eDNA using both conventional and quantita-
tive PCR (qPCR). DNA from spotted gar was detected in all but one
previously recorded site, and the eDNA method was shown to be
more effective than traditional netting for detecting spotted gar
habitat use. The use of qPCR allowed for quantification of substan-
tial variation in detection strength (copy number) among replicate
eDNA samples, with implications for establishing sampling designs
for detection and surveillance. Significance: The use of eDNA for
detection and monitoring aquatic species shows great potential as
a non-invasive method for assessing species occurrences and habi-
tat occupancy, as well as for informing targeted sampling by con-
ventional capture methods.
International Barcode Of Life: between the legal hammer and
the policy anvil
Alex Borisenko
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Rd. East, Guelph, ON N1G 2W1,
Canada.
E-mail for correspondence: aborisen@uoguelph.ca.
Background: iBOL has globalized the effort of building an open
reference platform for DNA-based identification of living organisms.
This has allowed institutions in biodiverse but technologically
under-resourced nations to outsource the analytical component of
their research to core analytical facilities in countries possessing
sufficient technological capacity. The Nagoya Protocol (NP) under
the Convention on Biological Diversity sets out new guidelines for
international transfer of biological materials that may have a seri-
ous impact on the future of this collaboration. Results: It is critical
to ensure that ratification or accession to NP does not undermine
access of developing countries to genomic tools for assessing bio-
diversity or hamper the construction of global reference libraries.
First, it is vital to draw a clear distinction between basic DNA
barcoding analysis and genomic bioprospecting for commercial
purposes. This should be formalized by streamlining and standard-
izing the protocols for exchange of genetic resources across the
iBOL network; in particular, the development of a universal Mate-
rial Transfer Agreement template that would secure Prior Informed
Consent from the Provider and include clearly articulated Mutually
Agreed Terms of analyses. Such documents should address interna-
tional practices of genome data sharing (e.g., Fort Lauderdale Prin-
ciples) and be inclusive of iBOL partner countries that have not
ratified or acceded to NP. Next, it is important to alleviate discrep-
ancies between above protocols and changes to national legal
frameworks related to NP implementation. Significance: The iBOL
community is deeply familiar with real-world challenges of inter-
national collaboration in collecting, aggregating, and sharing bio-
diversity genomics information covered by NP. It should use its
unique position to provide a strong and consolidated voice on rel-
evant policy developments at both international and national lev-
els. Building a coordinated network-wide set of protocols for
specimen transfer will ensure that national sovereignty over ge-
netic resources is exercised without undermining basic biodiver-
sity research.
Status report on barcode coverage for invasive alien species
Alex Borisenko,
1
Junko Shimura,
2
and Robert Hanner
1
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East,Guelph, ON N1G 2W1,
Canada.
2Secretariat, Convention on Biological Diversity, 413 Rue St. Jacques, Suite 800, Montreal, QC H2Y 1N9,
Canada.
Corresponding author: Alex Borisenko (e-mail: aborisen@uoguelph.ca).
Background: Climate change and the globalization of commerce are
contributing to the spread of alien invasive species across both terres-
trial and aquatic biomes with sometimes devastating consequences
for native species. Their spread also poses significant socio-economic
challenges for humanity. The Global Invasive Alien Species Partner-
ship was developed to help identify and track invasive species in an
effort to aid their management. Molecular methods like DNA barcod-
ing provide a means of facilitating the rapid and accurate identifica-
tion of invasive species for all users, but the extent of their coverage in
barcode reference libraries is unknown. Here we perform a gap anal-
ysis of global invasive alien species against the Barcode of Life Data
System. Results: Barcode libraries are only partially complete for
many quarantine pests, vectors of zoonotic diseases, and other socio-
economically relevant taxa. There is at least some sequence data avail-
able to aid the identification of nearly all listed invasive alien species.
However, the number of barcode records for each species varies con-
siderably, as does the quality of the meta data associated with those
records. Significance: DNA barcoding is poised to support identifica-
tion efforts of invasive alien species and to facilitate biomonitoring
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programs using environmental or eDNA given a relatively modest
investment in validating and expanding the barcode libraries cur-
rently available.
DNA barcoding of larval fish and egg samples produced from
the driftnetting of two Lake Simcoe tributaries
Danielle Bourque and Robert Hanner
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Danielle Bourque (e-mail: dbourque@mail.uoguelph.ca).
Background: Larval driftnetting at four sites in two Lake Simcoe
tributaries was conducted in May 2014, generating 97 samples
(71 fish larvae, 26 fish eggs). All samples were photographed prior to
DNA extraction. Standard QIAGEN DNeasy Blood and Tissue Kit
extraction protocols were performed. The COI-5P region was ampli-
fied for all samples through polymerase chain reaction (PCR) using
C_FishF(R)1t1 or VF(R)1i_t1 primers. Sanger sequencing was per-
formed on the PCR products. Specimen data, images, trace files,
and sequences were uploaded to BOLD systems under the project
name [MNRFE]. Specimen identification was performed using
BOLD's Identification System (IDS). Results: In total, 74 sequences
between 420- and 602-bp in length were generated from Sanger
sequencing. Totally, 70 of these were identified to species with
100% sequence similarity using BOLD's IDS. The identification
breakdown is as follows: 39 specimens were Catostomus commersonii,
24 were Perca flavescens, 4 were Sander vitreus, 2 were Etheostoma
caeruleum, and 1 specimen was Rhinichthys obtusus. Of the original
71 larvae and 26 egg samples analyzed, 89% of the larval samples
obtained a species ID, while only 27% of the egg samples obtained a
species ID. Significance: In terms of obtaining a DNA sequence and
positive species identification, the low success rate of the egg sam-
ples compared to the larvae suggests that a different approach to
the DNA extraction of eggs should be considered for future DNA
barcoding studies. In our study, only half of an egg per egg sample
was used during extraction. Using whole eggs or an organic extrac-
tion method to remove inhibitors may improve both DNA extract
yield and DNA sequence yield.
The long and the short of DNA barcodes: an approach using
nanopore sequencing
Jeffrey R. Boutain
1,2
and Matthew R. Boutain
3
1Botanical Research Institute of Texas, 1700 University Drive, Fort Worth, TX 76107, USA.
2Department of Botany / Department of Tropical Plant and Soil Sciences, University of Hawai'i at
Maˉ noa, 3190 Maile Way, Room 101, Honolulu, HI 96822, USA.
3New Growth Botanical LLC, 691 Cloverlawn, Lincoln Park, MI 48146, USA.
Corresponding author: Jeffrey R. Boutain (e-mail: jeffrey.boutain@gmail.com).
Background: Nanopore sequencing emerges to classify the tree of
life using both genomes (long) and standard (short) DNA barcodes.
Results: A plant's maternal lineage was determined using phyloge-
netic analyses of DNA fragments generated by the MinION device.
In addition, the MinION device was used to sequence total genomic
DNA and PCR amplicons from fresh and historical specimens—
specifically hops, hemp, avocado, breadnut, and humans. Significance:
The entire chloroplast genome is an optimal DNA barcode for land
plants. The ability to sequence long and short DNA fragments is
useful to model DNA barcodes as well as to test the origins of plants
and animals that are common, rare, or endemic. With low costs for
high-throughput DNA sequencing, user-accommodating bioinfor-
matics, and mobile hardware, the model presented here shows how
to study long and short DNA barcodes with a nanopore sequencing
approach. Thus, nanopore sequencing will transform interdisci-
plinary research on historical and contemporary biological collec-
tions, particularly testing the evolutionary relationships and
species limits for cultivars and wild taxa with low genetic distance.
Updates on the long and the short of DNA barcodes can be found at
http://www.newgrowthbotanical.com/.
A regional DNA barcode library for landscape-scale monitoring
of multi-taxa assemblages
Matthew L. Bowser,
1
Derek S. Sikes,
2
John M. Morton,
1
and Sarah Meierotto
2
1U.S. Fish & Wildlife Service, Kenai National Wildlife Refuge, P.O. Box 2139, Soldotna, AK 99669, USA.
2University of Alaska Museum, 907 Yukon Dr., University of Alaska Fairbanks, AK 99775, USA.
Corresponding author: Matthew L. Bowser (e-mail: matt_bowser@fws.gov).
Background: Rapid climate change may result in ecological futures
with novel species assemblages, trophic mismatch, and mass extinc-
tion. Conventional inventory and monitoring approaches are gener-
ally not designed to address these changes, particularly at the
landscape scales needed to address climate change. On the 804 000 ha
Kenai National Wildlife Refuge in Alaska, we collaborated with the
USDA Forest Inventory and Analysis Program to inventory plants,
landbirds, and arthropods on a grid of 259 permanent plots system-
atically distributed at 5 km intervals in 2004–2008 (t1), yielding
1106 species: 80 birds, 256 invertebrates, 324 vascular plants, 297 lichens,
and 149 bryophytes. In preparation for t2 of a time series, we are
building a library of DNA barcode sequences to facilitate monitoring
of arthropods and other hyperdiverse groups (e.g., lichens) using next-
generation sequencing (NGS) methods. Initial library construction in-
cluded specimens from the grid (t1) and additional local sampling.
Regionally, we have been building a DNA barcode library from mate-
rial at the University of Alaska Museum. Real-world tests of the use-
fulness of the library for species identifications were performed using
Sanger sequencing and NGS methods. Results: Of the 8421 species in
the Draft Checklist of the Nonmarine Arthropods of Alaska, 4020 (47%)
are now represented on the Barcode of Life Data Systems (BOLD),
including 1138 species that we contributed. We obtained species iden-
tifications for 17% of 950 Sanger-sequenced unknowns. Three NGS
samples yielded 21–48 named taxa per sample, of which 9–17 were
species identifications. Significance: Our approach provides a statis-
tically robust spatial framework for landscape monitoring, allowing
us to make inferences at multiple spatial scales while ensuring the
capacity to detect ecological surprises that may result from a rapidly
warming climate. With continuing improvements in NGS technology
and dropping costs, we are setting the stage to deploy NGS methods
for rapid and efficient biomonitoring at the landscape scale in 2016.
Ecology and systematics of New Zealand deep-sea squids
Heather E. Braid and Kathrin S.R. Bolstad
Institute for Applied Ecology, Auckland University of Technology, Private Bag 92006, Auckland 1020,
New Zealand.
Corresponding author: Heather E. Braid (e-mail: heather.braid@gmail.com).
Background: New Zealand has one of the highest biodiversities
of squid and octopus species reported for any country to date. Al-
though squid are prey to a variety of predators, the dietary habits of
most squid species remain unknown. Squid use their sharp beaks to
masticate prey, which makes morphological identification difficult
(often impossible). The taxonomy of deep-sea squid species, in partic-
ular, remains unstable due to the delicate nature of the specimens,
which are often badly damaged during capture. Therefore, a genetic
analysis is most appropriate to assess the feeding habits, and an inte-
grative taxonomic approach is ideal for their systematics. Results:
The ecology and systematics of several families of deep-sea squids in
New Zealand waters have been analysed. Using a combination of tra-
ditional taxonomy with genetics (the DNA barcode region of COI and
16S rRNA), more than a dozen new species have been discovered.
Effort has been concentrated on families with high local diversity
(Onychoteuthidae), particularly unstable systematics (Mastigoteuthidae,
Chiroteuthidae, Octopoteuthidae), and high importance in the diets of
marine mammals (Histioteuthidae, Ommastrephidae). In addition to
clarifying the true local composition of these families, DNA barcoding
has also provided useful information on the dietary habits of large,
poorly known squid species (Architeuthis,Idioteuthis). Significance:
Taxonomy underpins all biological research. Accurate identifications
Abstracts 199
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are vital for species conservation and understanding ecological
relationships. Many of the locally occurring deep-sea species are com-
mercially valuable or play an important role in the diet of other
economically, ecologically, culturally, and inherently biologically
valuable animals including fish, marine mammals, and seabirds. An
accurate understanding of New Zealand squid biodiversity is essential
in monitoring and managing the local marine environment and re-
sources.
DNA barcoding of coastal ichthyofauna from Bahia,
northeastern Brazil, South Atlantic: high efficiency for
systematics and identification of cryptic diversity
José Henrique Souza Galdino Brandão,
1
Iracilda Sampaio,
2
Ana Maria Waldschmidt,
1
Jamille de Araújo Bitencourt,
1
and Paulo Roberto Antunes de Mello Affonso
1
1Universidade Estadual do Sudoeste da Bahia, Dep. Ciências Biológicas, Av. José Moreira Sobrinho, s/n
Jequiezinho, Jequié – BA 45206190, Brazil.
2Universidade Federal do Pará IECOS, Laboratório de Genética e Biologia Molecular, Alameda Leandro
Ribeiro s/n, Bairro Aldeia, Bragança-PA 68600000, Brazil.
Corresponding author: Paulo Roberto Antunes de Mello Affonso (e-mail:
paulomelloaffonso@yahoo.com.br).
Background: The utilization of cytochrome c oxidase subunit I
(COI) as a barcode system for species identification has been estab-
lished as a powerful approach to access marine biodiversity world-
wide. In Brazil, DNA barcoding was successful in identifying fish
species from the southeastern coast. On the other hand, similar
studies from the northeastern coast of Brazil (about 3000 km) are
absent. Therefore, we carried out an extensive analysis using DNA
barcoding for the coastal ichthyofauna from Bahia, the largest
state in northeastern Brazil. Results: We obtained a COI library of
470 specimens from 74 species (about six specimens per species),
45 genera, 29 families, and 11 orders. Collection data, voucher num-
bers, taxonomic identification, and COI sequences of all specimens
were uploaded to BOLD (project: BAHIA). All species were discrim-
inated by a neighbour-joining tree using the Kimura-2-parameter
distance model (bootstrap values = 99%–100%). The maximum in-
traspecific distance was 0.43% while the minimum interspecific
distance was 3.18%. The mean distances within species, genera, and
families were 0.15%, 12.5%, and 23.82%, respectively. The COI se-
quences were useful to reveal cryptic diversity within Eucinostomus
(mojarras). A total of 82 specimens of morphologically similar mo-
jarras, identifiable only to genus, were split into five species by
barcode analyses, with the highest intraspecific distance of 0.31%
and the lowest interspecific distance of 11.14%. Two of these species
seem to correspond to new species (Eucinostomus sp. 1 and sp. 2),
while E. jonesii was reported for the first time in a marine fish
inventory from northeastern Brazil. New barcode data were also
provided for Rypticus randalli,Halichoeres penrosei, and Thalassophryne
nattereri.Significance: The present results corroborate the effi-
ciency of DNA barcoding for the identification of marine fish from
northeastern Brazil, South Atlantic. Moreover, this method was
useful to evaluate the richness of the ichthyofauna of the Brazilian
coast.
Optimizing metabarcoding of tropical Malaise trap samples
and preliminary results on seasonal changes in insect diversity
in Penisular Malaysia
Guo-Jie Brandon-Mong,
1
Han-Ming Gan,
2
Kong-Wah Sing,
1
Ping-Shin Lee,
1
Phaik-Eem Lim,
3
and John-James Wilson
1
1Institute of Biological Sciences, Faculty of Science Building, University of Malaya, 50603 Kuala Lumpur,
Malaysia.
2School of Science, Monash University, Jalan Lagoon Selatan, Bandar Sunway, 46150 Petaling Jaya,
Selangor, Malaysia.
3Institute of Ocean and Earth Sciences (IOES), University of Malaya Institute of Ocean and Earth
Sciences (IOES), University of Malaya, 50603 Kuala Lumpur, Malaysia.
Corresponding author: Guo-Jie Brandon-Mong (e-mail: mong432@gmail.com).
Background: Global warming is considered a lower threat to insect
biodiversity in the tropics than in temperate regions because species
showing distinct seasonal patterns of activity are thought to be at
higher risk of climate-related extirpation. Tropical species are gener-
ally assumed to be aseasonal; however, seasonal patterns in insect
activity have rarely been examined in Southeast Asia. To determine
the extent to which insect species in Peninsular Malaysia exhibit sea-
sonal changes in activity, we have deployed (since October 2014) Mal-
aise traps at three stations and will use metabarcoding (bulk DNA
extraction, PCR, and high-throughput sequencing of COI) to charac-
terise the diversity within and between weekly samples. Before pro-
ceeding with the metabarcoding analysis, we first undertook a pilot
study to establish the best metabarcoding primers. Four published
and one newly designed primer set were tested across a diverse set
of 80 arthropod species, representing 11 orders, to establish optimal
protocols for Illumina-based metabarcoding of tropical Malaise trap
samples. Results: Two primer sets, those showing the highest ampli-
fication success with individual-specimen PCR (98%), were used for
bulk PCR and Illumina MiSeq sequencing. The sequencing outputs
were subjected to both manual and simple, automated metagenomics
quality control and filtering pipelines. We obtained acceptable detec-
tion rates after bulk PCR and high-throughput sequencing (80%–90%
of input species), but analyses were complicated by putative hetero-
plasmic sequences and contamination. The manual pipeline produced
similar or better outputs compared to the simple metagenomics pipe-
line (1.4 compared to 0.5 expected:unexpected Operational Taxo-
nomic Units). Significance: Our study suggests that metabarcoding is
slowly becoming as affordable, fast, and easy as conventional DNA
barcoding and that Malaise trap metabarcoding may soon fulfill its
potential, providing a thermometer for biodiversity.
The plastomes of mycoheterotrophic Ericaceae reveal extensive
changes to gene order and rare gene loss
Thomas Braukmann
1
and Sasa Stefanovic
2
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON NIG 2W1,
Canada.
2Department of Biology, University of Toronto Mississauga, 3359 Mississauga Rd. N, Mississauga, ON
L5L 1C6, Canada.
Corresponding author: Thomas Braukmann (e-mail: tbraukmann@gmail.com).
Background: About 1% of flowering plants have lost all or some of
their photosynthetic ability. These heterotrophic plants rely entirely
or partially on a host to supply water and nutrients. Heterotrophic
plants are divided into two distinct groups: haustorial parasites and
mycoheterotrophs. Haustorial parasitism has evolved at least 11 times
independently, and mycoheterotrophy has at least 10 independent
origins in angiosperms. Ericaceae, the heather family, is a large and
diverse group of plants with elaborate symbiotic relationships with
mycorrhizal fungi, including several mycoheterotrophic lineages.
Results: We investigated the evolution of mycoheterotrophy in Eri-
caceae, using select sampling across the trophic spectrum and a next-
generation sequencing approach, including multiple representatives
from the partially mycoheterotrophic pyroloids and fully mycohetero-
trophic monotropoids. Our results indicate that rearrangements of
the large single copy region of the plastome, as well as expansion
and contractions to the inverted repeat regions, are typical for
most members of Ericaceae. Associated with the extensive struc-
tural rearrangements of the plastome within the family, including
fully mycoheterotrophic species, is an increase in forward and pal-
indromic repeats. Additionally, most Ericaceae share the loss of the
plastid ycf1, ycf2, and clpP genes, which is rare even amongst fully
heterotrophic plant lineages. Significance: Consistent with other
heterotrophic lineages, hemi-mycoheterotrophic plants retain
most genes relating to photosynthesis but are polymorphic for the
presence of the plastid “ndh” genes, and the monotropoids exhibit
extreme reductions in plastome size and gene content. Surpris-
ingly, a number of different genes were found to be under positive
selection in both partially and fully mycoheterotrophic species. We
also discuss the limitations of plastid based markers and whole
plastome data for DNA barcoding.
200 Genome Vol. 58, 2015
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Detecting signatures of competition from observational data:
a novel approach combining DNA barcoding, diversity
partitioning, and checkerboards at small spatial scales
Trevor T. Bringloe,
1
Sarah J. Adamowicz,
1
Vivian F.I. Harvey,
1
John K. Jackson,
2
and Karl Cottenie
3
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Stroud Water Research Center, 970 Spencer Road, PA, USA.
3Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON, Canada.
Corresponding author: Sarah J. Adamowicz (e-mail: sadamowi@uoguelph.ca).
Background: Competitive interactions are assumed to play a role in
governing species distributions but are difficult to infer using obser-
vational data. In addition, morphological identification may overlook
species, obscuring patterns of competitive exclusion. To address these
limitations, we used a molecular (DNA barcoding) approach to species
delineation and sampled stream insect communities at small spatial
scales, where environmental filtering and dispersal limitations are
minimized. Thirty rocks from 15 riffles were sampled for stream in-
sects, in particular caddisfly (order Trichoptera), beetle (order Co-
leoptera), and black fly (family Simuliidae) larvae from filterer and
grazer guilds at White Clay Creek, Pennsylvania, USA (June 20, 2013).
After barcoding 1492 specimens, additive partitioning of species diver-
sity was used to establish at which spatial scale(s) variation in the
distribution of species occurs. The partition analysis, in turn, in-
formed the spatial scale at which checkerboard analyses tested for
signatures of species segregation (e.g., competitive exclusion among
specific taxa). Results: Only a small portion of the total species diver-
sity occurred at the smallest spatial level (mean = 30% on individual
rocks). Distributional variation in species was greatest at the kilome-
ter scale (where beta-diversity was 37% and 41% for filterers and graz-
ers, respectively). At the scale of White Clay Creek sampled (3 km
extent), species of filterers showed a strong tendency to segregate
(p< 0.001), while grazers showed random structure approaching ag-
gregation (p= 0.087). Significance: We introduced a novel combina-
tion of analyses—diversity partitioning followed by checkerboard
analyses—that facilitated linking patterns of segregation to competi-
tive interactions among species. We also emphasize the importance of
small spatial scales when inferring the importance of community
competition. This study further demonstrates the value of DNA bar-
coding in the rapid delineation of species units for ecological analyses.
DNA metabarcoding of pollen: progress, technical limitations,
and future directions
Berry Brosi, Karen Bell, Kevin Burgess, and Hsin-I Chu
Deptartment of Environmental Sciences, Emory University, 400 Dowman DR NE, Atlanta, GA 30322, USA.
Corresponding author: Berry Brosi: (e-mail: bbrosi@emory.edu).
Background: DNA metabarcoding of pollen has the potential to
transform a number of fields including plant–pollinator interactions,
aerobiological monitoring of pollen for allergies, forensic palynology,
and others. While proof-of-concept of DNA metabarcoding of pollen
has been shown by multiple research groups, several outstanding
questions remain. In this presentation we (i) synthesize results pre-
sented at this symposium; and (ii) outline results from work in our
group including shotgun sequencing, purification of non-pollen DNA
from samples, and other topics; and (iii) outline future research direc-
tions for DNA barcoding of pollen. Results: We present preliminary
results on shotgun sequencing, which holds promise for highly re-
solved genetic identifications of pollen, but remains highly limited
due to lack of reference samples. We also present a method to remove
non-pollen DNA from samples, while keeping pollen interior DNA intact.
We synthesize the findings presented in the symposium to give an over-
view of the field of DNA barcoding of pollen. Significance: DNA metabar-
coding of pollen is in its infancy but is rapidly evolving. Building an
understanding of the current state of the field, technical hurdles that
need to be overcome, and strategic directions for the future is critical for
realizing the transformative potential of this technology.
Utility of DNA barcoding and other molecular methods for
bloodmeal analysis in mosquitoes (Diptera, Culicidae) of
medical and (or) veterinary importance in southern England,
United Kingdom
Victor A. Brugman,
1
Luis M. Hernández-Triana,
2
Sean W.J. Prosser,
3
Chris Weland,
3
Dave Westcott,
2
Nicholas Johnson,
2
and Anthony Fooks
2
1The Pirbright Institute, Ash Road, Woking GU24 0NF, UK.
2Animal and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK.
3Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Luis M. Hernández-Triana (e-mail: lhernandt@gmail.com).
Background: Thirty-four species of mosquitoes are present in the UK,
of which nine have been implicated in West Nile virus (WNV) trans-
mission elsewhere. Enzootic arboviruses have not been recorded in
the UK, although serological studies in birds have reported virus-
specific neutralizing antibodies to arboviruses. The identification of
mosquito feeding preferences and species delineation is paramount to
the understanding of vector–host–pathogen interactions and would
help to predict transmission patterns. This, in turn, would assist in the
control of outbreaks. In this paper, we test the utility of DNA barcod-
ing for vertebrate species identification in blood-fed females follow-
ing previous methods. We also test bloodmeal DNA extractions to
molecularly delineate mosquito species and detect viruses associated
with mosquitoes. Results: Blood-fed females (n= 134) were collected
from sites in southern England and identified as seven morphospecies, of
which Anopheles maculipennis and Culex pipiens are species complexes. In
total, we positively identified the blood meal in 65 specimens (49%—
cow, human, dog, European rabbit, great tit, magpie, blackbird, and
barn swallow). Analysis of ITS2 and COI barcoding region sequences
in Anopheles maculipennis revealed the presence of two species,
An. atroparvus (33 specimens) and An. messae (7 specimens); one sample
was misidentified. Thirty three An. atroparvus had fed on European
rabbit, of which nine were positive for Myxoma virus. The results from
ITS2 and COI revealed that specimens identified as Cx. pipiens/torrentium
were Cx. pipiens pipiens.Significance: This study demonstrates the
application of DNA barcoding for the identification of vertebrate spe-
cies that mosquitoes have fed on. Moreover, the same sample can be
screened for the presence of pathogens, and there is sufficient inver-
tebrate DNA to perform ITS2 and COI DNA barcoding region PCRs to
molecularly identify the mosquito species.
Diffusing barcoding: the global spread of a good idea
Tania Bubela,
1
Janis Geary,
1
Mark Bieber,
1
Westerly Luth,
1
and David Hik
2
1School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
2Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Corresponding author: Janis Geary (e-mail: janis.geary@ualberta.ca).
Background: Twelve years since the inception of barcoding, it is
timely to examine the diffusion of this taxonomic tool. Our goal
was to apply theories on technological diffusion to barcoding. Theories
point to drivers of diffusion as (i) opinion leaders, (ii) early adopters,
(iii) enabling infrastructure, (iv) supportive institutional structures,
and (v) demonstrated utility. Results: We searched the Scopus data-
base on 23 April 2015 for all publications that cited four seminal
barcoding papers. We retrieved 2743 distinct publications that cited
these four seed articles and stored these in a MySQL database. We
disambiguated author names and geocoded individual author affilia-
tions to latitude and longitude. We used Sci2 (https://sci2.cns.iu.edu)
for co-author network analyses using these 10 375 unique authors and
their affiliations. Using Sci2 and enhanced network visualization capabil-
ities of Gephi (http://gephi.github.io/), we positioned the co-authorship
network on a global map. We used Gephi to calculate network statistics.
We characterized articles by taxonomic focus and theme (e.g., applied
biomonitoring, education, systematics, informatics). Barcoding research-
ers have spread from their base in North America to every populated
continent. There are strong linkages amongst researchers in developed
countries and increasing participation of researchers from emerging
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economies and developing countries. China has become a leader in plant
barcoding. While the taxonomic applications still dominate in the liter-
ature, barcoding applications have expanded over time. Significance:
Barcoding is an exemplar of the rapid and global spread of an innovation
in the absence of formal proprietization. Its diffusion is not only in vol-
ume but in scope of applications. Institutional structures and opinion
leaders have been key drivers. Further diffusion is likely with regulatory
acceptance of the technology.
Evaluating the evolutionary and genetic relationships among
the Andean orchids of Ecuador
Kylie Bucalo,
1
Jennifer M. Cruse-Sanders,
2
Alex Reynolds,
3
Alvaro Perez,
4
and Kevin S. Burgess
5
1Columbus State University, Atlanta Botanical Garden, 02 State Bridge Rd, Apt 8402, Duluth, GA, USA.
2Atlanta Botanical Garden, 1345 Piedmont Ave NE, Atlanta GA, USA.
3The Lovett School, 4075 Paces Ferry Road Northwest, Atlanta, GA 30327, USA.
4Pontifica Universidad Catolica del Ecuador Mariscal Sucre, Quito, Ecuador.
5Columbus State University, 4225 University Avenue, Columbus, GA 31907, USA.
Corresponding author: Kylie Bucalo (e-mail: kbucalo@atlantabotanicalgarden.org).
Background: With over 4000 orchid species, representing over
240 genera, Ecuador has the highest orchid species diversity in the world.
Orchids can be found in many regions of the country; however, one of the
largest concentrations can be found at high elevations, in the Andean
cloud forests of Ecuador. Present in these environments are orchid gen-
era that are considered to be complex, where traditional morphology-
based taxonomy has been challenged or has struggled to discriminate
between species well. As a phylogenetic tool DNA barcoding can solve
some of these issues using standardized loci to delimitate species, iden-
tify ambiguous taxa, further define genera, and explore evolutionary
relationships. The objective of this project is to develop a DNA barcode
library of ⬃200 native tropical orchid species, found at the Siempre
Verde Research Station in the Imbabura region of Ecuador. This barcode
library will then be used to achieve two research goals: (i) to assess the
efficacy of DNA barcodes to demarcate Andean orchid species, and (ii)to
evaluate the genetic and evolutionary relationships among the complex
genera found at the preserve. Results: To date over 100 species have been
collected that represent a broad taxonomic dispersion, as well as targeted
deep sampling of complex genera (Lepanthes,Pleurothallis,Epidendrum, and
Stelis). A further 20 species have been collected from herbarium samples,
representing a total of over 120 species collected and processed into
plates for sequencing. Significance: As many orchid taxa are under
threat due to deforestation, this project contributes to an immediate
conservation need to research, identify, and document the endemic flora
present in the Andean cloud forest habitat. Furthermore, this research
will facilitate future ecological research at the preserve and also increase
the relevancy of living collections and herbaria.
Using DNA barcodes to evaluate the level of endemism and isolation
in the avifauna of the Central Sierras in Córdoba, Argentina
Belén Bukowski, Pablo D. Lavinia, Natalia Trujillo-Arias,
Cecilia Kopuchian, Pablo L. Tubaro, and Dario A. Lijtmaer
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” – CONICET, Avenida Ángel Gallardo
470, Buenos Aires, Argentina.
Corresponding author: Dario A. Lijtmaer (e-mail: dariolijtmaer@gmail.com).
Background: The Andes mountains constitute one of the main fac-
tors promoting Neotropical diversification, but the role of other high-
land systems has been barely studied. The large, standardized barcode
library of the birds of the region provides a unique opportunity for
their analysis. In this context, we assessed the level of endemism
in the avifauna of the Central Sierras in Córdoba, Argentina, to evaluate
the effect of its geographic isolation from the Andes. There are 11 bird
species that include subspecies endemic to the area, but none of them
have been evaluated using molecular markers. Results: We studied 7
of these 11 species by obtaining the barcodes (and in some cases also
cytochrome b sequences) of specimens from the Central Sierras and
comparing them with those of other populations of these species in
Argentina. The analysis of genetic divergence and haplotype net-
works, supported when possible through AMOVAs, indicated that in
three of these species (Cinclodes atacamensis,Catamenia inornata, and
Phrygilus unicolor) the population in the Central Sierras is differenti-
ated from those of other regions of Argentina. In three of the remain-
ing species (Muscisaxicola rufivertex,Asthenes modesta, and Phrygilus
plebejus) there is a varying degree of differentiation, but results were
not conclusive. Finally, there is no differentiation in the case of
Sturnella loyca, the only species of this analysis that inhabits both high
and low areas and therefore has a continuous distribution between
the Central Sierras and the Andes. Significance: While further anal-
yses including more molecular markers, morphology, and vocaliza-
tions are needed, some avian species were shown to have populations
in the Central Sierras that are genetically differentiated, and therefore
likely evolutionarily isolated, from other populations of southern
South America. This clearly illustrates how the use of barcode se-
quences can constitute an initial step towards understanding evolu-
tionary patterns and the processes generating them.
First results of the Global Malaise Trap Program in Argentina:
strikingly high biodiversity in the southern extreme of the
Atlantic Forest
Belén Bukowski, Priscila E. Hanisch, Pablo L. Tubaro,
and Darío A. Lijtmaer
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” – CONICET, Avenida Ángel Gallardo
470, Buenos Aires, Argentina.
Corresponding author: Belén Bukowski (e-mail: belenbukowski@gmail.com).
Background: Environmental disturbance and global climate change are
modifying the species composition of terrestrial communities before we
get to know them. Notably, most species in terrestrial ecosystems are
arthropods, and therefore reliable tools for their collection and identifi-
cation are necessary. DNA barcoding can provide this. In this context, the
Global Malaise Program has been established a few years ago, and Argen-
tina is actively participating with traps placed in four different locations
throughout the country. In particular, the first Malaise trap was deployed
in Misiones province in the southern extreme of the Atlantic Forest,
generating the first results in South America. Results: Insects were col-
lected every week for 2 years. In total, 34 103 specimens were obtained
from the first 22 bottles sampled throughout the first year, which consti-
tutes the highest number of specimens collected by a Malaise trap in the
global project so far. A total of 30 771 barcode sequences were obtained,
and most of them received a BIN assignment. The order Diptera domi-
nated in specimen abundance (77%), followed by Hemiptera (8%), and
Lepidoptera (5%). For these 22 samples, 5646 unique BINs were generated,
and 92% of them were new to BOLD. The species accumulation curve
suggests that a large proportion of species have yet to be sampled, and
the Chao's estimate of species richness suggests that almost twice
as many BINs could be found by Malaise trapping in this location.
Significance: The results of this first trap allowed us to add many new
species into the growing library of DNA barcodes. It also contributed to
the knowledge of the insects of southern South America. The strikingly
high number of insects (and BINS/species) collected by this single trap
clearly indicates that even though northeastern Argentina harbors the
southern extreme of the Atlantic Forest, its biodiversity is remarkable.
DNA barcoding to determine the diets of Prairie grasshoppers
Kevin S. Burgess,
1
John A. Barone,
1
Scott Whitley,
1
and JoVonn Hill
2
1Department of Biology, Columbus State University, Columbus, GA, USA.
2Mississippi Entomological Museum, Mississippi State University, Starkville, MS, USA.
Corresponding author: Kevin S. Burgess (e-mail: burgess_kevin@columbusstate.edu).
Background: DNA barcoding is a molecular technique that allows for
the identification of taxa from small tissue samples. By barcoding the
plant material in feces, several studies have reported on the diets of
species of herbivorous mammals, as well as some tropical leaf beetles.
Here we describe the use of DNA barcoding to determine the diet of
grasshoppers from their feces (or frass). At three prairie sites in eastern
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Mississippi, grasshoppers were captured, identified, and held in test
tubes until they produced a frass pellet. They were then released, un-
harmed. Plant DNA was extracted from the frass, and the DNA was am-
plified through PCR using primers for the rbcL gene region found in the
chloroplast genome of plants. After the PCR product was sequenced, the
sequences were aligned and manually edited. Results: From 250 initial
samples, 63 yielded high quality, bi-directional sequences (with three
additional uni-directional sequences) from 18 species of grasshoppers. Of
these, 41 were homozygous, consisting of plant DNA from a single spe-
cies. These were compared to a pre-existing DNA barcode library for
prairie plants from Mississippi and Alabama. Fourteen samples were
identified to a single plant species with greater than 99.5% pairwise iden-
tity, while 20 were identified to genus. The remaining eight samples were
likely derived from plant species not in the barcode library. Twenty frass
samples appeared to contain plant DNA from more than one species.
Significance: By using a previously established barcode library for Prai-
rie plant species, our study demonstrates the utility of DNA barcoding to
non-destructively determine the diets of grasshoppers and provide in-
sight into the structure of community food webs.
Preliminary analysis of generic relationship of tribes
Eucosmini and Enarmoniini (Lepidoptera: Tortricidae) using
DNA barcodes in Korea
Bong-Kyu Byun,
1
Da-Som Kim,
1
and Jun Hyoung Jeon
2
1Department of Biological Sciences and Biotechnology, Hannam University, 461-6, Yuseong-gu, Daejeon
305-811, Republic of Korea.
2Microbiological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon
305-806, Republic of Korea.
Corresponding author: Bong-Kyu Byun (e-mail: bkbyun@hnu.kr).
Background: Tribe Eucosmini is one of the largest groups of Olethreu-
tinae in the family Tortricidae, comprising about 1000 species in the
world. In Korea, 78 species belonging to 23 genera have been recorded to
date. The tribe Enarmoniini was previously placed under Eucosmini, but
is now considered a small group that is a separate tribe. The taxonomic
position of the two tribes has been controverted among tortricid special-
ists. This study aimed to analyze and clarify the generic relationship of
the two tribes in Korea using their COI gene. We used all available spec-
imens collected from Korea, but, in some cases where material was not
available, the DNA barcode data were retrieved from BOLD. A total of 24
species, representing the main genera of the tribes found in Korea, were
selected and analyzed using the mtDNA barcode region. Data assembly
and tree building were conducted using MEGA 6.06 with maximum par-
simony. Results: Within the phylogenetic tree, the group Ancylis of the
tribe Enarmoniini clustered separately from the other genera. In the
Eucosmini clade, the genus Spilonota was monophyletic excluding Spi-
lonota lechriaspis. It is interesting that Rhyacionia pinivorana, which feeds on
Pinus, is phylogenetically close to Eucosma campoliliana, which feeds on
Compositae. Significance: Our analysis generally shows a similar trend
as seen with traditional classifications. Although the analysis was not a
complete one, it was useful to understand the taxonomic relationship
between the tribes. Further DNA barcode and morphological data will be
needed for complete reconstruction of the phylogeny for Eucosmini and
Enarmoniini tribes in Korea.
Ecotoxicological applications of DNA barcoding to distinguish
laboratory-cultured organisms: a case study involving
Hexagenia spp.
Gina Capretta,
1
Trudy Watson-Leung,
2
and Mehrdad Hajibabaei
1
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada.
2Laboratory Services Branch, Ontario Ministry of the Environment and Climate Change, 125 Resources
Road, Etobicoke, ON, Canada.
Corresponding author: Gina Capretta (e-mail: gcaprett@uoguelph.ca).
Background: Laboratory-based ecotoxicological bioassays aim to
evaluate the potential risk environmental toxicants pose to aquatic
organisms, including common bioindicator taxa such as the bur-
rowing mayfly (Hexagenia limbata and Hexagenia rigida). When col-
lected from the wild, eggs from these species are laboratory
cultured, resulting in a heterogeneous mixture of nymphs. H. limbata
and H rigida are also morphologically cryptic at the toxicity testing
stage (5 mg). Though differential abdominal pigmentation at the
bioaccumulation testing stage (20–30 mg) may help to morpholog-
ically distinguish each species, sorting individuals would require
much time and effort. As such, an unknown ratio of each species is
often used in ecotoxicological testing. Species-level responses to
environmental perturbations, however, are quite common. In an
effort to determine the ratio of H. rigida and H. limbata in a typical
ecotoxicological bioassay, a standard 96 h water-only toxicity test
with a water-only control, carrier-solution control, and three dif-
ferent concentrations of PCB-52 was performed in which 10 living
individuals were randomly selected from each test vessel upon test
completion and barcoded. Results: Across all five treatments, in-
cluding the controls, there was an uneven distribution of H. rigida
and H. limbata. The proportion of H. rigida across test vessels ranged
from 20% to 90%. Significance: Species identification via DNA bar-
coding is important for determining possible species-level effects
of environmental toxicant exposure across various levels of biolog-
ical organization. Standard ecotoxicological test species, such as
H. limbata and H. rigida, are often used as a mixture in conventional
bioassays. Application of DNA barcoding techniques to identify the
species used in these conventional bioassays will provide better
data with which to make recommendations to stakeholders.
Characterization of the COI gene in Carollia perspicilata
(Chiroptera: Phyllostomidae) from Amazonia
Tuane Carvalho,
1
Leonardo Trevelin,
2
and Juliana Cordeiro
1
1UFPel, Pelotas, Brazil.
2Museu Paraense Emílio Goeldi, Belém, Braazil.
Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br).
Background: The bats of the genus Carollia are among the most abun-
dant frugivorous species in the chiropteran communities along the
Neotropics. This genus is distributed from central Mexico to southern
Brazil and comprises small to medium-sized bats. Currently, it has
eight recognized species: C. benkeithi,C. brevicauda,C. castanea,C. manu,
C. monohernandezi,C. perspicillata,C. sowelli, and C. subrufa. Cytochrome
bgene analysis shows that the species of this genus can be phyloge-
netically divided in two clades, one with the small species (C. benkeithi
and C. castanea) and another group with the larger species (C. subrufa,
(C. sowelli,(C. brevicauda +C. perspicillata). In Brazil, there are only three
species formally registered (C. benkeithi,C. brevicauda, and C. perspicillata)
that occur in sympatry in some localities in the Amazon region.
Considering morphological taxonomy, C. brevicauda and C. perspicilata
are well distinguished. However, due to the use of continuous charac-
ters, accurate taxonomic identification can be problematic in some
cases. In this study, we analyzed COI polymorphism among different
populations of C. perspicilata from Amazonia, using this genetic
marker to help with species delimitation. Results: The COI sequences
showed nucleotide diversity for synonymous sites of 0.725%, with
30 polymorphic sites. It was possible to identify our 43 sequences as
C. perspicillata, comprising 22 haplotypes from six different localities.
In general, all genetic divergences were very low. The mean genetic
divergence among our sequences was 0.006. The mean intraspecific
genetic divergence for C. perspicilata sequences, considering the ones
available on BOLD and ours, was 0.009; and for C. brevicauda was 0.020.
The mean interspecific genetic divergence was 0.026. We identified
one very divergent sequence deposited in BOLD as C. brevicauda that is
also identified as Mormoops megalophylla in GenBank. Considering all
the 599 sequences here analyzed (those acquired from BOLD and ours),
it was not possible to detect a barcode gap between C. perspicillata
and C. brevicauda sequences using the ABGD method. In the network
analysis, considering only our 43 sequences, we could detect some
sequence clustering. Significance: In this study, we show that gene
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flow among very distant localities persists, even considering habitat
disturbance in regions where the samples were collected. Also, we
show the existence of great genetic similarity between C. brevicauda
and C. perspicillata.
Use of integrative taxonomy and DNA barcoding for
characterization of ecological processes structuring marine
benthic community assemblages in British Columbia
Magalie Castelin,
1
Niels Van Steenkiste,
1
Scott Gilmore,
1
Eric Pante,
2
Rick Harbo,
1
Geoff Lowe,
1
Bill Merilees,
1
Melissa Frey,
3
Thomas Therriault,
1
and Cathryn Abbott
1
1Fisheries and Oceans Canada Pacific Biological Station 3190 Hammond Bay Road Nanaimo, BC V9T
6N7, Canada.
2Environnement et Sociétés (LIENSs), UMR 7266 CNRS - Université de La Rochelle, 2 rue Olympe de
Gouges, 17042 La Rochelle, France.
3Royal BC Museum Royal BC Museum 675 Belleville Street, Victoria, BC V8W 9W2, Canada.
Corresponding author: Cathryn Abbott (e-mail: Cathryn.Abbott@dfo-mpo.gc.ca).
Background: Human-induced disturbances generate strong selection
pressures on the ecology and evolution of species, affecting commu-
nity dynamics, ecosystem functioning, and global processes. In the
marine environment, the taxonomy and distribution of most organ-
isms are poorly known. As ecologists strive to understand increasingly
complex processes, involving all species interacting within communi-
ties, reliable and accessible taxonomic tools to identify species in
broad community-level datasets are needed. This study aims to docu-
ment the species diversity of benthic marine communities in British
Columbia, Canada, and to establish a molecular inventory including
DNA barcodes to enable confident identification of species for ecolog-
ical research. Results: Intertidal and subtidal sites were sampled
across 700 km of BC coastlinem and 2000 specimens were collected.
For marine shelled gastropods and brachyuran crabs, we used a high-
throughput integrative taxonomic approach to generate fast and ac-
curate species hypotheses based on the phylogenetic species concept.
To date, 57 species have been delimited, including cryptic species that
can cause difficulty in ecological studies because of taxonomic uncer-
tainty. Rarefaction curves suggest 80%–100% of the actual diversity of
macro-species living in sampled habitats was collected. However, the
compilation of historic reports of species presence suggests that many
micro- and deep-sea species remain to be sampled in the North East
Pacific. Overall, the four habitat types analyzed contained distinct
species assemblages. Evidence of nonrandom co-occurrence patterns
indicative of ecological relationships or reflecting preference for
certain environmental conditions were observed in 35 species.
Significance: This is the first study to assess marine species richness
and ecological patterns in BC using a modernized approach to species
hypotheses including the incorporation of DNA barcode data. The
high-throughput taxonomic approach used should bring new oppor-
tunities for ecologists studying complex natural communities. The
molecular inventory of native macro-species generated provides base-
line data and will facilitate monitoring for invasive species in BC.
Harnessing the power of DNA barcoding to detect and deter
fish mislabeling in South Africa
Donna-Mareè Cawthorn,
1
Louwrens C. Hoffman,
1
Corli Witthuhn,
2
John Duncan,
3
Chris Kastern,
3
Junaid Francis,
3
and Harris Steinman
4
1Department of Animal Sciences, University of Stellenbosch, Private Bag X1, Matieland 7602, South
Africa.
2Office of the Dean: Faculty of Natural and Agricultural Sciences, Room 10, Biology Building, University
of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa.
3World Wildlife Fund South Africa - Southern African Sustainable Seafood Initiative (WWF-SASSI),
P.O. Box 23273, Claremont, 7735, South Africa.
4Food & Allergy Consulting & Testing Services, P.O. Box 565, Milnerton, 7435, South Africa.
Corresponding author: Donna-Mareè Cawthorn (e-mail: DonnaC@sun.ac.za).
Background: Seafood mislabeling has emerged as a globally perva-
sive problem, posing serious economic, health, and environmental
concerns. While DNA sequencing holds great promise for species au-
thentication monitoring, such methods must be backed up by robust
reference DNA sequence databases and statistically-sound sample col-
lection protocols to track changes in trading practices. This presenta-
tion will describe our 5-year research effort in South Africa to
systematically harness the power of DNA barcoding to detect, moni-
tor, and deter fish mislabeling in the country. Results: Based on rig-
orous surveys of fish species occurrence on the South African market,
reference specimens from 53 commonly available species were col-
lected and expertly identified. A comprehensive DNA sequence library
was thereafter established from the reference specimens based on
the sequencing of three mitochondrial gene regions (COI, 16S, and
12S rRNA genes). COI barcoding was capable of unequivocally identifying
the species origin of the vast majority (98%) of fish examined, whereas
discrimination of congeneric species was frequently problematic with
16S and 12S rRNA gene sequencing. Subsequent local market surveys
employing COI barcoding revealed that 53 of 248 (21%) collected fish
samples were incorrectly described in terms of species, including 43 of
140 (31%) from retailers and 10 of 108 (9%) from seafood wholesalers. A
recent follow-up study on fish mislabeling in the country demon-
strated some minor improvements, with 27 of 149 (18%) samples col-
lected from restaurants and retailers being potentially mislabeled.
Significance: DNA barcoding has been confirmed as an extremely
powerful method for the identification of fish species traded in South
Africa, the utility of which has been strengthened by the generation of
reference DNA sequence data for commonly marketed species. Such
an approach, when underpinned by structured and on-going seafood
testing protocols, can provide an effective and economical tool for
industry self-regulation, governmental monitoring, and prosecution
of illegal activities.
DNA barcoding of mosquitoes (Culicidae) in the Yucatán
Península
Rahuel Jeremías Chan Chable
1
and Arely Martínez Arce
2
1Instituto Tecnológico de Chetumal, Avenue Insurgentes No. 330, Col. David Gustavo Gtz. Chetumal,
Quintana Roo, Postal code 77013, Mexico.
2El Colegio de la Frontera Sur (ECOSUR), Unit Chetumal, Avenue Centenario Km. 5.5, Chetumal,
Quintana Roo, Postal code 77900, México.
Corresponding author: Rahuel Jeremías Chan Chable (e-mail:
rahuel_jere_1990@hotmail.com).
Background: There are over 3523 species of mosquitoes distributed
throughout the world; their identification is the first step in the pre-
vention and control of diseases that they transmit. During the last
10 years, “DNA barcoding” using the cytochrome c oxidase subunit I
(COI) gene as a molecular marker has achieved a better estimate of
biodiversity. In this study, the diversity of mosquitoes was evaluated
in four sampling sites in Quintana Roo State of Mexico. Morphological
and genetic analyses were used for identification. Results: Based on
morphology, 14 species were identified, and three could be deter-
mined to genus, giving a total of 17 morphotypes belonging to the
subfamily Culicinae. Four genera and eight species were new records
for the study area. Kimura 2-parameter (K2P) intraspecific distances
averaged 0.97%, while interspecific distances averaged 9.8%. Two
closely related species, Aedes Ae. (Ochlerotatus) angustivittatus and Ae.
(Ochlerotatus) trivittatus, showed just 0.89% sequence divergence be-
tween them. Furthermore, two individuals of Ae. (Ochlerotatus) angus-
tivittatus had an interspecific distance of more than 2% (2.21%). For
Haemagogus equinus, the intraspecific K2P distance was more than 2%
(2.63%), corresponding to two clades with different BINs. In Uranotaenia sp.,
two individuals had distances >2% (2.12%), but all individuals fell un-
der the same BIN. Finally, in Limatus durhamii, one individual showed
an intraspecific K2P distance of 3.43%. The total number of BINs as-
signed by BOLD systems was 19. The results suggest the need to add
more individuals to analyses and to review in detail the clades with
more than 2% of intraspecific K2P distances, which suggest the exis-
tence of cryptic genetic diversity or new species. Significance: In
total, 14 sequences were new to BOLD, and 15 sequences were new to
GenBank. The taxonomic and molecular database from this study will
be used as a reference for future studies on the identification of mos-
quitoes in the Quintana Roo State.
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Monitoring biodiversity for the early detection of aquatic
invasive species using metabarcoding applied across Canadian
ports in the Pacific, Arctic, Atlantic, and Great Lakes
Frédéric J.J. Chain,
1
Emily Brown,
1
Hugh MacIsaac,
2
and Melania E. Cristescu
1
1Department of Biology, McGill University, 1205 Docteur Penfield, Montréal, QC H3A 1B1, Canada.
2Great Lakes Institute for Environmental Research, University of Windsor, 401 Sunset Avenue, Windsor,
ON N9B 3P4, Canada.
Corresponding author: Frédéric J.J. Chain (e-mail: frederic.chain@mcgill.ca).
Background: Zooplankton community composition acts as a power-
ful bio-indicator of aquatic ecosystem health. Monitoring biodiversity
is especially important in the face of ongoing ecological and range
shifts driven by anthropogenic activities, which are accentuated in
ports where shipping promotes the introduction of non-native organ-
isms. Metabarcoding of environmental samples offers the opportu-
nity to rapidly characterize local biodiversity and potentially detect
the early stages of species invasions. Results: Mock zooplankton com-
munities were first used to assess the most effective bioinformatics
approach to identify species from taxonomically diverse metabarcod-
ing datasets. This approach was applied to characterize zooplankton
biodiversity from natural communities sampled from 16 major ports
spanning the three Canadian oceans and the Great Lakes basin. More
than 7 million sequenced reads were taxonomically classified into
over 400 families spanning 19 phyla. We detected the main taxonomic
groups previously identified in these waters as well as several non-
native species at low abundance, showcasing the breadth and sensi-
tivity of metabarcoding. Zooplankton communities were distinct
among ports from different coasts, and biodiversity profiles distin-
guished marine, estuarine, and freshwater samples. Temporal shifts
in community composition were also captured, in which the most
drastic turnover was observed in a sub-Arctic region with high beta
diversity (0.9) between samples from May and August. Significance:
We present an extensive survey of zooplankton biodiversity across
Canadian ports with heavy shipping traffic, setting a baseline to which
future surveys can be compared for monitoring changes in commu-
nity composition. Despite the need for more extensive reference da-
tabases, metabarcoding was shown to be effective in detecting both a
broad range of taxa and species that probably naturally occur locally
in low abundance. Our results provide a framework for the applica-
tion of metabarcoding to monitor biodiversity and for the early detec-
tion of invasive species in environmental samples.
Assessing DNA barcodes as a diagnostic tool for North
American reptiles and amphibians in nature and natural
history collections
E. Anne Chambers
1
and Paul D.N. Hebert
2
1Department of Integrative Biology, College of Natural Sciences, University of Texas, Austin, TX 78712,
USA.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: E. Anne Chambers (e-mail: eachambers@utexas.edu).
Background: High rates of loss and species discovery have lead to the
urgent need for more rapid assessments of species diversity and dis-
tribution in the herpetofauna, an approach now offered through DNA
barcoding. Prior DNA barcoding work on reptiles and amphibians has
revealed higher biodiversity counts than previously estimated due to
cases of cryptic and undiscovered species in both classes. Despite past
research, these taxa are very much in need of comprehensive species-
level coverage. Results: This study constructs a reference library of
DNA barcodes for North American reptiles and amphibians and as-
sesses their applicability as a technique for species delimitation. This
study also examines the correspondence of current species boundar-
ies with the BIN system. Barcodes were obtained from 732 specimens,
representing 282 species (44%) of the North American herpetofauna.
Mean intraspecific divergences were 1% and 3%, while average conge-
neric sequence divergences were 16% and 14% in amphibians and rep-
tiles, respectively. BIN assignments corresponded perfectly with
current species boundaries in 58% of these species. Barcode sharing
was observed in four genera of reptiles, while deep divergences (>2%)
were noted in 21% of the species. Using multiple primers and a refined
PCR regime, barcode fragments were recovered from 5 of 208 formalin-
fixed specimens, demonstrating that formalin collections can ex-
pand genetic databases. Significance: This is the first effort to
compile a reference library of DNA barcodes that provides species-
level identifications for reptiles and amphibians across a broad
geographic area. DNA barcodes from North American herpeto-
fauna were used to quickly and effectively flag errors in museum
collections, and cases of BIN splits and merges successfully identi-
fied taxa belonging to deeply diverged or hybridizing lineages. This
study also highlights the merit of further investigation into obtain-
ing genetic material from formalin-fixed tissue and the use of DNA
barcodes for biodiversity forensics.
The complete chloroplast genomes of Aconitum: insight into
taxonomy and identification of complex plant lineages
Xiaochen Chen,
1
Linchun Shi,
1
Ying Li,
1
Haibin Xu,
2
Jianping Han,
1
and Jingyuan Song
1
1Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical
College, No. 151 Malianwa North Road, Haidian District, Beijing 100193, P.R. China.
2Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16 Nanxiaojie,
Dongzhimennei Ave, Beijing, 100700, China.
Corresponding author: Linchun Shi (e-mail: lcshi@implad.ac.cn).
Background: Complete chloroplast genomes, regarded as super bar-
codes, are informative for studies of species identification and phyloge-
netic relationships, especially in complex plant lineages such as Aconitum.
We derived three complete chloroplast genomes belonging to the differ-
ent subgenera of the toxic Aconitum, aiming to discover new evidence for
taxonomic and identification studies. Results: Three complete chlo-
roplast genomes of Aconitum were derived using high-throughput se-
quencing platforms, and sequence divergence analysis was performed.
The three complete chloroplast genomes of Aconitum ranged from
155 769 to 157 327 bp in length and contain a total of 130 genes, consisting
of protein-coding genes, tRNA genes, and rRNA genes. The alignment of
the three chloroplast genomes resulted in a similarity of 97%. The six
highly conserved genes (cemA,psbB,psbC,rpoA,rpoC1, and rps4) amounted
to 8117 bp after alignment, from which 154 variable sites were detected
with a similarity of 98% and an average Kimura 2-parameter distance of
0.013. Significance: As cost is no longer the limitation to full-genome
sequencing, the establishment of a broad super-barcode database consist-
ing of chloroplast genomes has become practical. Compared with
current DNA barcodes, full chloroplast genomes represent a more
comprehensive approach to species taxonomy and identification of
the complex and toxic Aconitum.
Riverine barcoding: a proposed DNA barcoding study on the
freshwater fish species of Meenachil River of Kerala, India
Letha P. Cheriyan
Department of Zoology, Mar Thoma College, Tiruvalla-689103, Kerala, India.
E-mail for correspondence: lethapc@hotmail.com.
The knowledge on species inhabiting natural freshwater ecosystems
is a fundamental requirement for the management of any riverine
ecosystem. There are serious gaps in our knowledge on the biodiversity
of many of the Indian rivers due to the lack of robust documentation
efforts, also reflected in very few contributions to the international
literature on conservation and freshwater biology. Studies on the
freshwater fish species of Kerala, the southernmost state of India, are
gaining momentum, recently evident from the descriptions of around
15 new species from hitherto unknown freshwater habitats of Kerala
in a span of just 2 years. Documentation and bio banking of the still
unknown species before they turn extinct is an absolute priority for
the conservation of freshwater fish species of Kerala. The Meenachil
River originating in the Western Ghats is a degrading riverine ecosys-
tem in Central Kerala, home to a wide variety of fish with many
unknown possible endemic species. The proposed project of DNA bar-
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coding fish species may increase our understanding of the biodiversity
and the processes generating it and to produce information useful for
identifying management and conservation units. This also includes
possible identification of yet unknown (i.e., cryptic) species found in
Meenachil River. Resolving taxonomic ambiguities by the identifica-
tion of genetic structure of populations may also shed a light on the
phylogenetic (evolutionary) relationships between different fish taxa
and populations. In addition, DNA barcoding can be applied to illumi-
nate the taxonomic identity of fishes for future works. Apart of pro-
ducing results advancing our understanding of fundamental scientific
issues, studies on the diversity of fishes have the potential to pave a
road for the development of conservation strategies.
Towards an universal genome-based DNA barcode—The
PhyloAlps project
Eric Coissac,
1
Pierre Taberlet,
1
Cristina Roquet,
1
Marti Boleda,
1
Ludovic Gielly,
1
Adriana Alberti,
2
Patrick Wincker,
2
Inger Alsos,
3
Frédéric Boyer,
1
and Sébastien Lavergne
1
1LECA, CNRS/UJF Grenoble, France.
2Genoscope, CEA Evry, France.
3Tromsø University Museum Tromsø, Norway.
Corresponding author: Eric Coissac (e-mail: eric.coissac@inria.fr).
The DNA barcoding initiative, proposed in 2003, represented a big
step forward in standardized DNA-based species identification. It cor-
responds to the use of a single or few small portions of the genome
(= standard barcodes) as a discrete taxonomic character for identifying
unknown specimens by comparison with a reference database. The bar-
coding initiative was very successful and led to the collaboration of many
teams around the world, producing extensive reference databases. How-
ever, the standard barcodes were designed in the context of Sanger se-
quencing, and the recent development of next-generation sequencing
(NGS) allows further developments of the barcoding initiative. In light of
the PhyloAlps project aiming to sequence the whole alpine flora, we
suggest to complement the standard barcode strategy with an approach
taking advantage of the power of NGS. We propose to develop an ex-
tended barcode, simply composed of one or two gigabases of sequence
reads obtained using shotgun sequencing of genomic DNA. From this
extended barcode, we demonstrate for both an animal dataset and for a
plant dataset that it is possible to extract the initial standard barcodes,
the whole organelle genomes, the whole nuclear ribosomal tandem re-
peats, and a collection of sequence reads corresponding to single-copy
DNA regions. Such an extended barcode can be generated not only for
multicellular eukaryotes, but also for microorganisms via single-cell se-
quencing. As a consequence, the extended barcode has the potential of
being truly universal, encompassing organisms from the three domains
of life (eukaryotes, bacteria, archaea). After a four-year sampling effort,
the PhyloAlps 6000-sequence dataset will be produced by the end of 2015
at Genoscope in France. This tremendous dataset can be considered as a
large pilot experiment for this new DNA barcoding strategy.
Temperature-related activity of Gomphiocephalus hodgsoni
(Collembola) COI haplotypes in Taylor Valley, Antarctica:
implications in a changing climate
Gemma Collins
1
and Ian Hogg
2
1University of Waikato, 25 English Street, Saint Andrews, Hamilton, 3200, New Zealand.
2University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand.
Corresponding author: Gemma Collins (e-mail: gec9@students.waikato.ac.nz).
Background: In Southern Victoria Land (SVL), Western Antarctica,
Gomphiocephalus hodgsoni is the most abundant and widespread spring-
tail species. It is genetically diverse, and over 70 mitochondrial cyto-
chrome c oxidase subunit I (COI) haplotypes have been sequenced
to date. There is also considerable physiological variation among
G. hodgsoni individuals in their cold tolerance and metabolic activity.
Climate changes predicted for SVL are likely to result in glacial retreat
and increased connectivity among populations. Here, we assessed COI
haplotypes of G. hodgsoni relative to the environmental conditions
during which individuals were active. We sequenced 151 individuals
collected in pitfall traps from three sites within Taylor Valley in
January 2014. Results: We found 19 unique COI haplotypes that sepa-
rated into two distinct groups (1.6% divergence), with one haplotype
group comprising 80% of the sequenced population. Based upon sam-
pling every 2 h, air temperature was the strongest predictor of activity
between the two haplotype groups (R
2
= 0.56). Upon adding subsurface
soil temperature, relative humidity, and photosynthetically active ra-
diation, explanatory power increased to R
2
= 0.71. Significance: With
steadily increasing air temperatures predicted for much of Antarctica,
individuals with greater activity are likely to have greater dispersal
opportunities. Accordingly, some haplotypes may have a selective ad-
vantage, which could result in decreased genetic variability within
populations. We suggest that spatial and temporal monitoring of rel-
ative proportions of COI haplotypes may provide a subtle measure of
biological response to environmental changes within Antarctic terres-
trial ecosystems.
An assessment of New Zealand rotifer diversity and global
affinities using COI barcodes
Gemma Collins,
1
Ian Hogg,
1
Ian Duggan,
1
Clare Beet,
1
Jonathan Banks,
2
and Matthew Knox
3
1University of Waikato, Hamilton, New Zealand.
2Cawthron Institute, Private Bag 2, Nelson, 7042, New Zealand.
3Soil Biodiversity and Ecosystem Functioning Laboratory, Department of Biology, Colorado State
University, Fort Collins, CO 80523, USA.
Corresponding author: Gemma Collins (e-mail: gec9@students.waikato.ac.nz).
Background: Using traditional morphology-based taxonomy, only a
small proportion of rotifer species are classified as endemic to New
Zealand (<5%). This is surprising given the geographical isolation of
New Zealand and the high endemicity of other taxa. In order to assess
genetic diversity and endemicity of rotifers in New Zealand, we (i)
sequenced the cytochrome c oxidase subunit I (COI) gene region for
individuals of 45 currently recognised morpho-species collected from
freshwater lakes and ponds in New Zealand, and (ii) compared these
with available sequences on BOLD and GenBank from regions outside
of New Zealand. Results: We collected 177 individuals and found 73
unique sequences from the 45 morphologically recognised species. Of
the New Zealand morpho-species, 14 had high intraspecific COI se-
quence divergence (3.4%–39.0%). When compared with available se-
quences on GenBank and BOLD databases, we found that 18 species
had sequences that were highly divergent (>6.6%) from their conspe-
cifics elsewhere, including Lecane bulla (19.7% divergence) and Brachio-
nus calyciflorus (21.4% divergence). Significance: Based on our data, we
suggest that endemism among New Zealand rotifers may be higher
than originally suggested on the basis of morphological assessments.
However, of the 45 species we assessed, 27 had no data available on
GenBank or BOLD, highlighting the need for an enhanced interna-
tional effort in order to facilitate comparisons to assess the global
biodiversity of this phylum. From an applied perspective, these data
will be essential for detecting non-indigenous species’ incursions
globally, and as rotifers are often used as bioindicator species to mon-
itor water quality, the sequences can also be used to develop local,
DNA-based, water quality monitoring programs.
The use of DNA barcodes in the identification of Biomphalaria
species (Mollusca: Planorbidae) for schistosomiasis control
Juliana Cordeiro,
1
Patrícia Thyssen,
2
Mariana Xavier,
1
André Lopes,
1
and Demetrius Martins
1
1UFPel Pelotas, Brazil.
2Unicamp Campinas, Brazil.
Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br).
Background: Schistosomiasis is a chronic and acute parasitic disease
caused by bloodworms of the genus Schistosoma, one of the most com-
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mon parasitic diseases worldwide with a significant socio-economic
impact. This disease is prevalent in tropical and sub-tropical areas,
mainly in poor and rural areas that lack access to safe drinking
water and sanitation. In the Americas, the only human schistosome is
Schistosoma mansoni. Currently, in Brazil, schistosomiasis occurs en-
demically in nine states mainly from northeast Brazil, while south
Brazil is considered a low-transmission area. In this country there are
11 Biomphalaria snails species: three of them are natural intermediate
hosts of Schistosoma mansoni (B. glabrata,B. tenagophila tenagophila, and
B. straminea); three are potential hosts (B. amazonica,B. cousine, and
B. peregrina); and six non-hosts (B. intermedia,B. kuhniana,B. oligoza,
B. schrammi,B. occidentalis, and B. t. guaibensis). The taxonomic discrim-
ination of these species is based on morphological characters of the
reproductive system. The adult size, however, ranges from 8 to
40 mm, and these characters are highly variable. Another problem is
that there are two subspecies: one is a natural host, and the other is
non-host. In this study, we investigate the effectiveness of DNA bar-
coding to correctly identify these species, mainly the subspecies. We
analyzed 10 individuals collected from different places from south
Brazil, using DNA barcoding and morphological analyses. Results: It
was possible to discriminate the Biomphalaria species using the DNA
barcodes, with a barcode gap near 2.3% of genetic divergence. Also, we
identified a new occurence for one species in south Brazil. Besides
being possible to discriminate the species, we detected two different
possible taxonomic groups in the B. t. guaibensis sequences. Also, we
found a greater genetic similarity of B. t. guaibensis with B. occidentalis
than with its sister species, B. t. tenagophila.Significance: DNA barcod-
ing can be used as another tool to identify these species, overcoming
the taxonomic impediment. Also, using this approach, it was possible
to detect new species occurence, making it possible to help the health
system agencies to develop protocols for the disease control.
Genetic variation of endemic and endangered species of insular
rodent from Mexico
Patricia Cortés-Calva, Cristian Cornejo-Latorre,
and Sergio Ticul Álvarez-Castañeda
Centro de Investigaciones Biológicas del Noroeste SC, Instituto Politécnico Nacional 195, Mexico.
Corresponding author: Patricia Cortés-Calva (e-mail: pcortes04@cibnor.mx).
Background: The Baja California peninsula is the second longest and
one of the most geographically isolated peninsula on Earth. Its phys-
iography and the presence of many surrounding islands have facili-
tated studies of the underlying patterns and drivers of genetic
structuring for a wide spectrum of organisms. We analyzed a total of
20 species of the families Heteromyidae: Chaetodipus (n= 5), Dipodomys
(n= 1), and Cricetidae: Peromyscus (n= 12), Neotoma (n= 2), endemic to
the region, and occurring on 28 islands. Currently, all the insular
rodent populations are considered as endemic and threatened. The
islands of the surrounding area of the Baja California Peninsula were
isolated after the Last Glaciation Maximum (LGM, ⬃18 000 BP); under
that consideration we wanted to evaluate if haplotype and nucleotide
diversity has experienced a reduction in insular populations in rela-
tion to continental ones, considering the variation of adaptation be-
tween species of two different families. We evaluated sequence data
of the mitochondrial cytochrome c oxidase subunit I gene (COI) in
48 populations from 28 islands. Levels of genetic variation within
island populations for the different species were measured in terms of
the number of haplotypes and nucleotide diversity. Results: Our anal-
yses detected several islands populations with low haplotype and nu-
cleotide diversity in relation to the continental population. Reduction
in the genetic diversity was present in species of the two families and
four genera. The endemic species with continental population of the
Baja California Peninsula have two genetic diversity patterns. The first
one has a high genetic diversity in the southern part of the Peninsula,
and the second one a low genetic diversity in the northern region of
the Peninsula. The low genetic diversity has been considered as a
consequence of a recent range expansion of the species. The island
populations are within the second pattern, so the invasions of the
islands by endemic species were recent and after the LGM. The species
with a wide range in the continent are those that have the lowest
genetic diversity in haplotypes and nucleotide diversity, with no dif-
ferences independently if they are of the Cricetidae or Heteromyidae
families. The insular endemic species are in a range of genetic varia-
tion from H= 0.6923 ± 0.1154,
= 0.0012 ± 0.0011 in Peromyscus dickeyi to
H= 0.2476 ± 0.1307,
= 0.0008 ± 0.0008 in Peromyscus slevini.Significance:
Understanding the variation of the cytochrome c oxidase subunit I
gene (COI) in insular threatened rodent species endemic to the islands
of the Gulf of California is necessary to understand the current situa-
tion and future conservation strategies. Today, five insular endemic
species are recorded as having gone extinct from the islands surround-
ing Baja California, and many islands have exotic species that can
compete or predate over the endemic ones. This study will generate
information to understand the genetic status for future conservation
actions.
Priming a DNA barcode library for marine Gastropoda of the
continental Portuguese coast and Azores Islands
Filipe O. Costa,
1
L.M.S. Borges,
1,2
C. Hollatz,
1
J. Lobo,
1,3
A.M. Cunha,
1
A.P. Vilela,
1
G. Calado,
3,4,5
R. Coelho,
5
A.C. Costa,
6
M.S.G. Ferreira,
1
and M.H. Costa
3
1CMBA, Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
2Helmholtz-Zentrum Geesthacht, Centre for Material and Coastal Research, Max-Planck-Straße 1,
21502, Geesthacht, Germany.
3MARE, Marine and Environmental Sciences Centre, Faculty of Science and Technology, New University
of Lisbon, 2829-516 Monte de Caparica, Portugal.
4University Lusófona, Department of Life Science, School of Psychology and Life Sciences, University
Lusófona, Campo Grande 376, 1749-024 Lisbon, Portugal.
5IPM, Portuguese Institute of Malacology, Zoomarine, E.N. 125 km 65, Guia, 8201-864 Albufeira, Portugal.
6InBIO, Associate Laboratory CIBIO-Açores, Centre of Investigation of Biodiversity and Genetic
Resources, Biology Department, University of the Azores, 9501-801 Ponta Delgada, Azores, Portugal.
Corresponding author: Filipe O. Costa (e-mail: fcosta@bio.uminho.pt).
Background: The Gastropoda are among the most diverse taxonomic
groups of marine invertebrates and, together with other dominant
classes, such as Bivalvia, Malacostraca, and Polychaeta, constitute ma-
jor components of marine benthic communities. As part of a wider
effort to compile reference libraries for dominant benthic inverte-
brates of the NE Atlantic, we used DNA barcodes to investigate the
diversity of gastropods from continental Portugal and the Azores Is-
lands. Results: Sequences of the cytochrome c oxidase subunit I bar-
code region (COI-5P) were obtained for 105 specimens, assigned to
15 families, and 32 gastropod species. Among these, six were novel
barcodes. All sequences were DNA barcode compliant and were as-
signed to 35 barcode index numbers (BINs). Of the 35 BINs generated
from our sequences, 22 were concordant, 12 were discordant, and 1
was singleton. After reviewing the 12 discordant BINs, 7 were consid-
ered concordant, since the source of discordance was due to (i) cases of
species names that have not yet been updated in public repositories,
e.g., Osilinus lineatus that has not yet been updated to Phorcus lineatus;(ii)
misidentifications, e.g., one specimen identified morphologically as
Calliostoma virescens that is putatively Calliostoma zizyphinum; and (iii)
distinct species still identified as a single species, e.g., Patella ulyssipo-
nensis and Patella aspera identified interchangeably as P. aspera and P.
ulyssiponensis. To this dataset, we added publicly available COI-5P se-
quences of the same or taxonomically close species, to inspect for DNA
barcode discriminatory ability and data congruence. In total, we ex-
amined 183 specimens belonging to 51 species. Significance: The per-
centage of concordant BINs came up to over 80% after revision, which
shows a good reliability of our DNA barcode library. Nonetheless, the
amount of discordant data highlights the need for continuous revi-
sion and DNA barcode-based probation of the supposedly well-known
gastropod fauna from the European Atlantic coasts.
Abstracts 207
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Using different methods to access the difficult task to delimiting
species in a complex Neotropical hyperdiverse fish group
Guilherme J. da Costa-Silva,
1
Mónica S. Rodriguez,
2
Fábio F. Roxo,
1
Fausto Foresti,
1
and Claudio Oliveira
1
1Universidade Estadual Paulista, Departamento de Morfologia, Laboratório de Biologia e Genética de
Peixes, Botucatu, São Paulo State, Brazil.
2Universidade Federal de Viçosa, Rio Paranaíba, Minas Gerais State, Brazil.
Corresponding author: Claudio Oliveira (e-mail: claudio@ibb.unesp.br).
Background: The genus Rineloricaria is a group of Neotropical freshwater
fish with a long and problematic taxonomic history, attributed to a high
number of species and great similarity among them. For our work we
used taxonomic information and different approaches to a single locus
dataset to find species boundaries and to identify independent evolution-
ary units in an attempt to help with the problems associated with the
analysis of distinct sources of knowledge. Results: We analyzed 228 sam-
ples assembled in 53 distinct morphospecies. However, traditional anal-
yses, such as the barcode gap, failed to delineate them. In contrast, the
general mixed yule-coalescent (GMYC) analysis indicates the existence of
70 entities, confirming the identity of 43 of the 53 morphospecies, and
showing that some species are genetically indistinct, while others prob-
ably are species complexes. The geographic data helped to understand
deep genetic divergence among populations present in distinct river ba-
sins, and their ecological characterization suggested that species from
headwater drainages have greater genetic segregation between popula-
tions. Significance: The multiple approaches favor the recognition of
species boundaries and allow the conclusion that Rineloricaria probably
contains several undescribed species.
Advances in the identification of tuna larvae, Auxis, from
western central Atlantic using DNA barcoding
Yareli Cota-Valentin,
1
Lourdes Vásquez-Yeomans,
1
Selene Morales-Gutiérrez,
1
Arely Martínez-Arce,
1
and Estrella Malca
2
1El Colegio De La Frontera Sur, Unidad Chetumal Ave. Centenario Km 5.5 Col. Pacto Obrero
Campesino, Chetumal Q. Roo, Mexico.
2Cooperative Institute of Marine and Atmospheric Studies. University of Miami 4600 Rickenbacker
Causeway, Miami, FL 33149, USA.
Corresponding author: Yareli Cota-Valentin (e-mail: cotay0187@gmail.com).
Background: The bullet tuna Auxis rochei (Risso 1810) and the frigate tuna
Auxis thazard (Lacepède 1800) are widely distributed in tropical and temper-
ate seas. While these small tunas are economically and ecologically impor-
tant, little is known about their early life history. They inhabit the western
central Atlantic, and while the morphological identification of adults is rel-
atively easy, the identification of their larvae is difficult due to similarities
among the two species with regards to morphology and pigmentation pat-
terns. Currently, identification during the larval stage is only possible for
sizes greater than 5 mm. Major taxonomic difficulties arises when attempt-
ing to identify larvae less than 5 mm SL, because there are no distinctive
morphological and pigmentary characteristics between them. In this con-
text, we present preliminary results from DNA barcoding used to success-
fully identify Auxis larvae to species. The larvae were collected from several
oceanographic cruises in the Gulf of Mexico and along the Mesoamerican
Reef in 2006, 2007, and 2010. Results: Fourteen Auxis larvae were identified
to species: seven A. rochei (2.8–13 mm SL) and seven A. thazard (3.2–10 mm SL).
The smallest larvae (<4.5 mm SL) showed great similarity in pigmentation
patterns of the midbrain and hindbrain, gut, and cleithral symphysis. The
main difference between them was the pigmentation on the dorsal margin
in A. thazard (<3.5 mm SL). The samples analyzed to date with the COI showed
greater divergence than 2% (2.68%). Additional larval samples will be submit-
ted for DNA barcoding in order to establish a reliable pattern to facilitate the
morphological identification of larval Auxis.Significance: Combining DNA
barcoding with morphological identification techniques for the bullet and
frigate tuna can uncover spawning grounds and enhance our understanding
of the larval distribution particularly in the western central Atlantic.
From barcoding single individuals to metabarcoding biological
communities: towards understanding and managing invasive
species
Melania E. Cristescu
Department of Biology, McGill University, 1205 Docteur Penfield, Montréal, QC H3A 1B1, Canada.
E-mail for correspondence: melania.cristescu@mcgill.ca.
The continual destruction of natural barriers due to increased human
trade and travel generates massive biotic exchanges between previously
isolated regions and global biotic homogenization. Non-native species
dominate many ecosystems and often these introduced species display inva-
sive traits, altering species interactions within invaded communities. The
full extent of the on-going biotic exchange has been very difficult to assess
and its long-term impact notoriously difficult to predict. Genomic tools are
emerging as very efficient and cost-effective means for monitoring these
biotic exchanges and for understanding and managing invasive species.
The field of DNA-based species identification is rapidly integrating new
sequencing technologies, bioinformatics pipelines, computational infra-
structure, and experimental designs. The field is transitioning from bar-
coding single specimens to metabarcoding complex biotic communities.
This rapid revolution has multiple levels of challenges. However, many of
these challenges could be reconciled using a coordinated advancement
of DNA-based species identification that integrates taxonomic and bar-
coding information and facilitates access to taxonomic knowledge and
growing repository barcodes. Here I discuss the challenges that we face
when implementing genomics techniques to understand and manage inva-
sive species in the context of global environmental changes.
Targeted detection of multiple species at risk (Unionidae) using
environmental DNA
Charise Currier,
1
Todd Morris,
2
Chris Wilson,
3
and Joanna Freeland
1
1Trent University, 781 Water St., Peterborough, ON K9H 3N5, Canada.
2Fisheries and Oceans Canada, Great Lakes Laboratory for Fisheries and Aquatic Sciences.
3Ontario Ministry of Natural Resources and Forestry, LHS Building, Trent University, Peterborough, ON
K9J 7B8, Canada.
Corresponding author: Charise Currier (e-mail: charisecurrier@trentu.ca).
Background: Environmental DNA (eDNA) detection can be used to infer
species presence by using species-specific markers to screen DNA from
water samples. This technique is increasingly being used to track the
spread of invasive fish species but has largely not been applied to other
taxonomic groups. However, the low impact of eDNA sampling on
aquatic habitats makes the method ideal for sampling sensitive popula-
tions. Here we report on research that aims to quantify the opportunities
and limitations of eDNA for the detection of rare, endangered organisms.
Our study focuses on the detection of native freshwater mussel (Union-
idae) species at risk using species-specific qPCR markers. We will use a
multi-species approach to develop highly specific markers for each of the
five closely related species. Results: To date, species-specific qPCR
primer-probe sets have been developed, validated under controlled con-
ditions, and implemented across the species' ranges to map occurrences.
The eDNA-derived species occurrences will be compared with existing
distribution maps to evaluate the efficacy of the method. Significance:
eDNA shows promise as an effective tool for the targeted detection of
closely related at-risk invertebrates; however, a thorough knowledge of
the detection thresholds and limitations of the eDNA tools is crucial for
meaningful interpretation of results and the eventual use of eDNA for
management applications.
Investigating terrestrial arthropod biodiversity in a tropical
ecosystem using barcode index numbers and phylogenetic
community structure
Michelle D'Souza
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: mdsouza@uoguelph.ca.
Background: Current understanding of terrestrial arthropod biodi-
versity within the tropics is inadequate due to the inability to compre-
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hensively sample and identify specimens in these hyperdiverse
ecosystems. DNA barcoding is an excellent tool to overcome this bar-
rier. I analysed terrestrial arthropod biodiversity patterns at two sites
in Parque Nacional Cusuco, Honduras, by employing Malaise traps as
a standardized sampling technique and the Barcode Index Number
(BIN) as a proxy for species. BIN counts and phylogenetic community
structure were subsequently examined to determine diversity within
and between sites. Results: Malaise traps were deployed in two broa-
dleaf forest sites roughly 2 km apart with a 300 m elevational differ-
ence. A total of 1103 and 1804 BINs were collected at the two sites over
a 14-week period. Insecta dominated the arthropod communities at
both sites, comprising 95% of the BINs versus Arachnida (3%) and
Collembola (2%). The collection largely contained members of five
insect orders: Diptera (52%), Hymenoptera (17%), Coleoptera (11%), Lep-
idoptera (6%), and Hemiptera (4%). Despite the small distance between
the two sites and the analysis of more than 11 000 specimens, they
shared only 9% of their BINs. The low overlap was a consequence, at
least in part, of incomplete sampling as revealed by the BIN accumu-
lation curves and the high proportion of singletons (62%). Various
arthropod groups between the sites displayed more phylogenetic dis-
similarity than expected based on a null model, demonstrating that
variation between sites can be detected despite incomplete commu-
nity sampling. Significance: The examination of tropical biodiversity
patterns, despite the many challenges of comprehensive sampling,
can benefit from a phylogenetic perspective aided by null models and
DNA barcodes. As investigations into the phylogenetic community
structure of tropical arthropods are lacking, such studies can help
determine if species turnover is phylogenetically non-random and can
provide valuable insights into the various mechanistic theories of
community assembly.
DNA barcoding of Javanese and Balinese freshwater fishes:
molecular insights into a poorly known ichthyofauna
Hadi Darhuddin,
1
Aditya Hutama,
2
Frederic Busson,
3
Sopian Sauri,
1
Philippe Keith,
3
Robert Hanner,
4
Renny Hadiaty,
1
and Nicolas Hubert
5
1Indonesian Institute of Sciences, Research Centre for Biology, Gedung Widyasatwaloka, Jalan Raya
Bogor Km46, 16911 Cibinong, Indonesia.
2Department of Biology, Bogor Agricultural University, Faculty of Mathematics and Natural Sciences,
Kampus IPB Darmaga, 16680 Bogor, Indonesia.
3Muséum national d'Histoire naturelle, UMR 7208 (MNHN-CNRS-UPMC-IRD), DPMA CP 026, 43 rue
Cuvier, F-75231 Paris Cedex 05, France.
4Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
5Institut de Recherche pour le Développement, UMR 5554 (UM2-CNRS-IRD), ISEM Place Eugène
Bataillon, CC 065, F-34095 Montpellier cedex 05, France.
Corresponding author: Hadi Dahruddin (e-mail: hdahruddin@yahoo.com).
Background: To date, 224 fish species have been reported from the
Javanese and Balinese rivers, among which 38 are endemic to these is-
lands. While some emblematic islands of the Indonesian archipelago
have garnered considerable attention in the past due to the occurrence of
charismatic species (e.g., Borneo, Sumatra, Sulawesi), the Javanese and
Balinese biodiversity has been poorly explored so far, and freshwater
fishes are no exception to this. Java Island, with a human population that
has grown to more than 180 000 000 people sharing 128297 km
2
,isthe
most densely populated island of the Indonesian archipelago, and its
biodiversity is currently the most endangered of the country. The present
survey conducted between 2012 and 2014 aims to re-assess the Javanese
and Balinese ichthyodiversity based on systematic and standardized mo-
lecular screening. Results: Fishes were mainly collected using electro-
fishing, cast nets, and seine nets between November 2012 and December
2014 at nearly 70 sites distributed across rivers, lakes, ponds, estuaries,
and fish markets. More than 1000 specimens belonging to more than 160
nominal species have been sequenced and, together with collection data,
submitted to BOLD. Based on DNA barcode data, the number of mito-
chondrial lineages diverging by more than 2% is higher than the number
of nominal species, highlighting cryptic or undescribed diversity. In ad-
dition, three new species of amphidromous gobies (e.g., Sicyopus,Lentipes,
Stiphodon) are described and each is characterized by private mitochon-
drial lineages. Significance: The present study provides the first DNA
barcode reference library for the Javanese and Balinese ichthyofauna and
also represents the first large-scale DNA barcoding survey in Indonesia.
Given the accuracy of the identification based on DNA barcodes, this
preliminary library is expected to benefit a large community of users
from academic to managing authorities in Indonesia.
Human population density in Africa correlates with the
evolutionary history of its flora
Jonathan Davies,
1
Olivier Maurin,
2
Kowiyou Yessoufou,
3
Barnabas Daru,
4
Bezeng Simeon Bezeng,
2
Ledile Mankga,
2
Hanno Schaefer,
5
and Michelle van der Bank
2
1McGill University, Canada.
2University of Johannesburg, South Africa.
3University of South Africa, South Africa.
4University of Pretoria, South Africa.
5Technische Universität München, Germany.
Corresponding author: Jonathan Davies (e-mail: j.davies@mcgill.ca).
Background: There is growing evidence for a link between biodiver-
sity and ecosystem services. However, a correlation between human
population and species richness generates a conflict between human
population growth and conservation goals. Results: Using a DNA bar-
code phylogeny of southern African trees, we show that human pop-
ulation correlates not only with tree species richness, but also with
their phylogenetic diversity. Furthermore, we demonstrate that areas
rich in phylogenetic diversity support a greater diversity of natural
goods, indicating that the evolutionary processes responsible for gen-
erating variation among living organisms are also key to the provi-
sioning of ecosystem services. Significance: Our study suggests that
the distribution of humans in southern Africa may have been shaped
by the evolutionary history of its flora.
Barcoding herpetological collections: discovering hidden
biodiversity in a hotspot
Juan M. Daza and Adriana Restrepo
Biology Institute, Universidad de Antioquia Calle 67 Número 53 – 108, Colombia.
Corresponding author: Juan M. Daza (e-mail: jumadaza@gmail.com).
Background: High species diversity, the complexity of taxonomy in
poorly known groups, and the lack of adequate scientific staff hamper
curatorial work in natural history collections in tropical countries. As
a consequence, many specimens remain unassigned to a known
taxon. DNA barcoding will help to improve our understanding of
millions of specimens deposited in these repositories. The collection
at the Museo de Herpetología Universidad de Antioquia (MHUA) in
Colombia harbors more than 16 000 specimens of herpetofauna com-
ing mostly from the northwestern corner in South America. This re-
gion is similar in area to Costa Rica, but it has more species, and only
about 20% of the territory has been sampled in comparison to the
well-studied Central America's country. Here, we sequenced the COI
fragment along with other mitochondrial and nuclear markers. We
used monophyly, genetic distance, and the Automatic Barcode Gap
Discovery method to define evolutionary units. Results: We have se-
quenced more than 800 specimens of 217 nominal species. Prelimi-
nary analyses show hidden diversity in the amphibian genera
Pristimantis,Craugastor,Colostethus, and Bolitoglossa. High Andes assem-
blage barcodes highlight lineage divergence concordant with moun-
tain islands. Regarding reptiles, hidden diversity has been detected in
the lizard genera Cercosaura and Anolis. In contrast, barcodes have also
shown that highly polymorphic species are genetically indistinct, sug-
gesting a decoupling of phenotype and genotype in the Neotropical
herpetofauna. These results have fired up species descriptions, and six
new taxa are currently in the process of description. Significance:
Recent studies have demonstrated the outstanding underestimation
of Neotropical biodiversity when genetic data are analyzed. Thus, DNA
barcoding represents an opportunity to better quantify the biodiver-
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sity in tropical countries that in turn will benefit all the areas that
require information from natural history collections.
Dissecting tropical earthworm biodiversity patterns in tropical
rainforests through the use of DNA barcoding
Thibaud Decaens,
1
David Porco,
2
Samuel W. James,
3
George Brown,
4
Elodie Da Silva,
4
Lise Dupont,
5
Emmanuel Lapied,
6
Rodolphe Rougerie,
7
Pierre Taberlet,
8
and Virginie Roy
5
1CEFE UMR 5175 CNRS, University of Montpellier, 1919 Route de Mende, 34293 Montpellier Cedex 5, France.
2CEFE, CNRS, CEFE UMR 5175 CNRS, University of Montpellier, 1919 Route de Mende, 34293
Montpellier Cedex 5, France.
3University of Iowa, Department of Biology, University of Iowa, Iowa City, IA 52242, USA.
4EMBRAPA – Floresta, EMBRAPA Florestas, Colombo/PR, Brazil.
5University of Paris Est, UMR IEES Paris-DIIM, Université Paris-Est Créteil, 61 avenue du Général de
Gaulle, F-94010 Créteil Cedex, France.
6Bioforsk, Norwegian Institute for Agricultural and Environmental Research, Ås, Norway.
7MNHN, Muséum national d'Histoire naturelle, UMR 75205, 45 rue de Buffon, CP50, 75005 Paris, France.
8LECA, CNRS, UMR LECA, Grenoble, France.
Corresponding author: Thibaud Decaens (e-mail: thibaud.DECAENS@cefe.cnrs.fr).
Despite representing a key component of terrestrial biota, soil inverte-
brates in tropical rainforests have been poorly studied from both a taxo-
nomic and an ecological perspective. Earthworms in particular comprise
about 6000 described species, a number thought to represent less than
half of the actual biodiversity of this group. Earthworm taxonomy is
particularly weakly resolved in the tropics, resulting in difficulties for
species identifications, and a critical lack of ecological studies on earth-
worm communities. DNA barcoding has been proposed as a useful ap-
proach to counter this problem through allowing automated species
identifications or the use of molecular species proxy that are expected to
facilitate and increase the pace of ecological studies in poorly studied
tropical regions. During the past years, we conducted several projects in
South America and Central Africa with the aim of describing diversity
patterns of earthworm communities at different scales. In French Guy-
ana and Gabon, we collected thousands of earthworms in different local-
ities representing the main ecosystems of the region. Specimens were
DNA barcoded to delimit molecular operational taxonomic units (MO-
TUs), and we used MOTU number and composition to estimate species
richness and describe community structure in the different habitats/
localities. We found unexpectedly high levels of local species diversity
(e.g., 48 MOTUs found in the Nouragues site) and species turnover (

diversity ranging from 60% to 90% depending on geographical distance
between sites). The taxonomic spectrum of local communities suggested
the importance of long-term diversification processes and current eco-
logical factors for the structuring of local communities. In a few sites
MOTUs were described using a selection of relevant traits to explore
community functional structure. Combined with the use of community
phylogenetic tools, this approach was an interesting way to explore in
more detail the assembly rules that were responsible for the observed
community patterns.
The power and promise of environmental DNA for river
biodiversity monitoring
Kristy Deiner and Florian Altermatt
Eawag – Swiss Federal Institute of Aquatic Science and Technology, 133 U
¨berlandstrasse 133, 8600
Dübendorf, Switzerland.
Corresponding author: Kristy Deiner (e-mail: alpinedna@gmail.com).
Background: River channel restoration is used in water management to
restore biodiversity and ecosystem functioning. Recovery, however, is
often context dependent and intrinsically linked to whether or not spe-
cies occur locally and can recolonize a restored area. Determining the
local species pool from a river's catchment is therefore necessary in order
to predict potential recovery. In river systems, the power of using envi-
ronmental DNA (eDNA) to estimate the biodiversity of animals is just
starting to be explored. It is known that eDNA of individual species can be
transported downstream. Thus, water samples from rivers are likely to be
an integrated measure of aquatic biodiversity for a river's catchment.
Results: In this study we used next-generation sequencing of the animal
cytochrome c oxidase subunit I (COI) barcode from eDNA to describe
metazoan eukaryotes across 24 catchments in Switzerland. We found
over 10 000 molecular operational taxonomic units (MOTUs) across the
catchments, and these MOTUs can be assigned to nearly 20 animal phyla
(e.g., Annelida, Arthropoda, Chordata, Heterokontophyta, and Mollusca).
Additionally, terrestrial and riparian taxa, such as birds and spiders, were
detected. Together, these results validate that eDNA can be used to
biomonitor the animal kingdom living in or near freshwater resources
and provide evidence that it does so on a scale relevant to the catchment
of a watershed. Significance: By combining the COI barcode for the
animal kingdom and environmental DNA detection of species, we dem-
onstrate how to assess species pools in river catchments. A biodiversity
assessment tool such as this has the potential to inform restoration ef-
forts about recolonization potential and become a powerful tool for land-
scape biodiversity assessments.
Using environmental DNA to track non-indigenous species in
shipping ports
Kristy Deiner,
1
Nitesh Chawla,
1
Amanda Leister,
2
Thanuka Wickramarathne,
1
Erin Grey,
3
Jian Xu,
1
James Corbett,
4
Michael Pfrender,
1
Travis Warziniack,
2
and Yiyuan Li
1
1Notre Dame University, Notre Dame, IN 46556, USA.
2Colorado State University, 1200 Center Ave Mall, Fort Collins, CO 80523-1172, USA.
3Governors State University, 1 University Parkway, University Park, IL 60484-0975, USA.
4University of Delaware, 111 Robinson Hall, Newark, DE 19716, USA.
Corresponding author: Kristy Deiner (e-mail: alpinedna@gmail.com).
Background: The unintentional transport of invasive species through
the global shipping network causes substantial losses to social and eco-
nomic welfare—an economic externality that affects everyone. Address-
ing this global challenge requires a non-indigenous species (NIS) risk
assessment tool for ship-borne species. In this research, we develop and
employ novel methods in network analysis, data fusion, and environ-
mental DNA (eDNA) analysis of water from shipping ports to forecast NIS
risk under longer-term scenarios and integrate interdisciplinary models
of navigation infrastructure, global trade, and future climate change.
Results: We have developed a model that predicts six species flow clus-
ters based on trade and transportation data. A relatively few ports likely
contribute much of the potential inter-cluster species flow. For example,
Singapore alone contributes to 26% of total predicted inter-cluster flow
from (to) the Pacific cluster, which contains 818 ports. We are now in the
process of analyzing water from ports all over the world to assess the
similarity among species pools by using next-generation sequencing of
barcoding genes to describe port communities. Significance: Our empir-
ical assessments of taxonomic similarities among ports from eDNA will
be used to test model predictions of predicted port taxonomic similarity
based on shipping movement. Through iterative engagement with
decision-makers, our work will inform global and US policies and man-
agement practices that improve sustainability of coastal ecosystem ser-
vices.
Species delimitation in the grasshopper genus Taeniopoda
(Orthoptera: Romaleidae) based on molecular and
morphological evidence
Vladimir Salvador De Jesús-Bonilla and Alejandro Zaldívar Riverón
Instituto de Biologia, UNAM 3er. Circuito exterior s/n, Cd. Universitaria, Copiclo, Coyoacán, CP 04510,
D.F., Mexico.
Corresponding author: Vladimir Salvador De Jesús-Bonilla (e-mail: kmaxtli@gmail.com).
Taeniopoda is a small grasshopper genus distributed from southern
USA to Panama. Species of this genus are of economic importance
given they are pests in some of the regions they inhabit. These
grasshoppers are conspicuous, with large and striking coloration in
the hind wings. Twelve species are currently recognized, most of
which are distributed along the Mexican territory. However, the
systematics and taxonomy of Taeniopoda has been neglected for
nearly a hundred years, and the genus still has various taxonomic
problems. For instance, it has been proposed that some of the
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recognized species are actually chromatomorphs representing geo-
graphical variation, not valid species. In this study, we assessed the
species delineations of eight putative species of Taeniopoda based
on two mitochondrial (mt) markers (COI and cytb) and morpholog-
ical data (external morphology and male genitalia). Molecular data
was analyzed using genetic divergence thresholds of 1% and 2% for
COI and the GMYC model for the two concatenated mt markers. We
found evidence of introgression or incomplete lineage sorting in
two species (T. eques and T. tamaulipensis). An integrative taxonomic
approach helped to delimit a total of nine species out of the eight
initially recognized.
DNA barcodes in resolving the taxonomic nomenclature of
Pseudoxytenanthera stocksii endemic to the Western Ghats,
India
Shanmughanandhan Dhivya, Selvaraj Dhivya,
and Ramalingam Sathishkumar
Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
Corresponding author: Shanmughanandhan Dhivya (e-mail: dhivyas87@gmail.com).
Background: Bamboo is one of the most complicated taxonomic groups of
grasses belonging to the family Poaceae. In India, there are 128 bamboo
species distributed within 18 genera. Bamboo identification based on mor-
phological characters is confusing and is highly error prone during field
studies, collection, and classification. This has made identification of bam-
boo using molecular tools very desirable. In this study, we have used DNA
barcoding, a global molecular tool for species discrimination, to resolve the
taxonomical dispute present in the classification of bamboo belonging to
the genus Pseudoxytenanthera.Pseudoxytenanthera is considered to have enor-
mous economical value as they are used for making walking sticks, umbrella
handles, roofing for huts, tent poles, baskets, and javelin. Results: In the
present study, nuclear (ITS, ITS1, ITS2) and chloroplast (rbcL, psbA–trnH) bar-
code candidates were evaluated for their ability to identify the molecular
divergence within Bambusoideae species. Among the tested barcode candi-
dates, ITS was shown to be effective in species discrimination. Bayesian anal-
ysis of the barcode candidates ITS, rbcL, and psbA–trnH using MrBayes 3.04b
was carried out, which revealed the phylogenetic evolution of the species P.
stocksii. The molecular divergence calculated using pairwise distances re-
vealed the species P. stocksii and P. ritcheyi were closely related. Further, the
parsimony analysis of the ITS region performed using PAUP* confirmed that
the species P. stocksii cannot be placed under the genus Dendrocalamus. The
genes rbcL and psbA–trnH displayed poor resolution for species discrimina-
tion. The intergenic spacer psbA–trnH was found to be highly conserved with-
out any differences in the sequences for the bamboo species used in this
study. Significance: Most of the bamboos have very long vegetative periods
prior to flowering and the vegetative characters are variable at different
stages in the life cycle of the plants. This has resulted in much confusion in
the identification of bamboos, and many taxonomic uncertainties prevail as
most bamboo species have been described using vegetative characters alone.
DNA barcoding was proved to be a good complementary tool to taxonomy,
especially when diagnostic morphological characters are missing. The cur-
rent study proved the importance of DNA barcoding by evaluating the tax-
onomical lineage of Pseudoxytenanthera that differed from the morphology-
based classifications.
Authentication of Indian herbal products using DNA barcodes
S. Dhivya,
1
S.G. Newmaster,
2
S. Ragupathy,
2
M. Saravanan,
1
and R. Sathishkumar
1
1Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
2Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph,
ON N1G 2W1, Canada.
Corresponding author: R. Sathishkumar (e-mail: rsathish@buc.edu.in).
Background: Herbal products are plant-derived natural remedies
prone to frequent contamination and substitution by false or cheaper
alternatives. The deceptive exploitation of herbal remedies is expo-
nentially increasing due to illusive business practices, which has led
to the substantial health risks for consumers. The leading cause for
the unlawful practices is due to the lack of strict regulation of
guidelines for good collection practices (GCP's), good manufacturing
practices (GMP's), and market control laws, which are not being
strengthened to eliminate false and low-quality herbal products. In
this study we have applied DNA barcoding, a DNA-based method to
test the authenticity of commercially available herbal products from
India. We suggest the application of the technique by herbal indus-
tries for the enforcement of good quality control surveillance of
herbal products. Results: We have generated a Biological Reference
Material (BRM) herbal barcode library for 152 Indian herbal species
using barcode regions (ITS2 and rbcL). We tested the authenticity of
93 herbal products representing 50 herbal species. Of these, 46% were
determined to be authentic, and about 52% of the products contained
species of plants not listed on the labels. Nearly 23% of the samples
were contaminated with species from unrelated families, and 4% of
the products contained fillers. We found contamination of products
with legal substitutes of the drug that was scientifically supported. We
also found some inadvertent adulterant species that were never re-
corded before through scientific evidence. Our overall findings sug-
gest that no Indian herbal medicines contained products with pure/
single herbal species as mentioned on their labels. We assume that the
product is essentially mixed with one or few other herbs that can
alleviate the therapeutic activity of the main ingredients. Significance:
At present, the available quality control (QC) methods and stan-
dards of herbal preparations established with modern analytical
technologies are not very promising as the herbal preparations are
typically prone to batch-to-batch variability in composition, concen-
tration, contamination, and decisive adulteration. DNA barcoding has
proven to be robust for building DNA barcode reference libraries
and is reliable in identifying plant species from the processed plant
materials. Therefore, the technique can be applied in the field of
herbal pharmacovigilance to ensure the safety and efficacy of nat-
ural drugs.
DNA-based technologies for authentication of herbs and its
admixtures—a review
S. Dhivya,
1
S.G. Newmaster,
2
S. Ragupathy,
2
M. Saravanan,
1
and R. Sathishkumar
1
1Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
2Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph,
ON N1G 2W1, Canada.
Corresponding author: S. Dhivya (e-mail: dhivyas87@gmail.com).
Herbal or phyto-medicines are usually prone to frequent adulteration
or substitution with inferior or unintended herbs. In recent years,
increased globalization of trade, accompanied with financial gain, has
intensified the tendency for market substitution. Even though several
regulations and pharmacovigilance activities are followed to control
adulteration practices, there is a need for proper guiding material and
techniques for fast and easy determination of authenticity of crude
materials that will assure the quality of herbal products. The use of
DNA-based technology in herbal drug analysis is recently evolving as a
reliable alternative tool for the identification of herbs, and is consid-
ered to be a good choice, especially to identify powdered raw ma-
terials, where DNA is degraded or preserved poorly. Among
molecular techniques, DNA barcoding has been recognized as a
modern genomic tool that provides a robust, rapid, and cost-
effective approach for species identification. Standardization of
this technique would improve the ease of developing a diagnostic
tool for food authentication, as it is broadly applicable across a
wide range of taxa. Recently, few studies have documented the
potential scope and magnitude of market substitution for individ-
ual plant species such as teas, Cassia,Senna,Sida cordifolia,Serenoa
repens, natural health products (NHP), and other commercialized
medicinal plants using DNA barcodes. Even though utility of DNA
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barcoding for food regulation has been proved, one limitation of
the technique is that it can be used to identify single-ingredient
products. In such cases, advanced high-throughput sequencing
(next-generation sequencing) technologies are applied for validat-
ing the complex herbal mixtures containing more than a single
active ingredient species. Next-generation sequencing has many
advantages as it provides faster, better, and accurate genome-based
tests that can be used effectively for pharmacovigilance.
First DNA barcode reference library for the identification of
South American freshwater fish from Lower Paraná River
Juan Díaz,
1
Villanova Vanina,
2
Del Paso Felipe,
2
Brancolini Florencia,
3
Grimberg Alexis,
4
and Arranz Silvia E
1
1Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Universidad Nacional de Rosario
Ocampo y Emeralda sede CCT, Rosario, Argentina.
2Facultad de Ciencias Bioquímicas y Farmacéuticas, UNR Suipacha 531, Rosario, Argentina.
3Instituto de Limnología “Dr. Raúl A. Ringuelet” (ILPLA) CONICET, UNLP Boulevard 120 y 62, La Plata,
Argentina.
4Acuario “Río Paraná”, (FCByF y SECyT) Cordiviola y Paseo Ribereño, Rosario, Argentina.
Corresponding author: Juan Díaz (e-mail: diaz@ibr-conicet.gov.ar).
Background: The Lower Paraná River floodplain comprises one of the
largest and biodiverse wetland systems of South America. More than 164
freshwater fish species have been described in the lower Paraná River,
but most lack associated molecular information, and many lack barcode
reference sequences available through the international Barcode of Life
(iBOL.org) project. The present study aims to assemble a comprehensive
reference sequence library for fishes of the lower Paraná River and to
evaluate DNA barcode effectiveness for their identification. Results: Tax-
onomic identification of 309 fish specimens resulted in 75 species (70
genera, 31 families, and 10 orders) from the lower Paraná River. COI DNA
fragments were obtained for all specimens. The average K2P genetic
distance between specimens was 0.5% within species, 12.41% within gen-
era, and 20.15% within families. The average divergence within conspe-
cific specimens was 24-fold lower than the average found in congeneric
species, evidencing the existence of a “barcode gap” that allowed for
unambiguous discrimination of 97% of species. Interspecific genetic dis-
tances ranged from 4.06% to 23.03% (average of 14.45%), with the excep-
tion of two species of the genus Odontesthes (O. bonariensis and O. perugiae)
in which members showed a minimum genetic distance of 0%. In addi-
tion, four species showed deep intraspecific divergence (>2%): Hoplias
malabaricus (7.59%), Brycon orbignyanus (6.68%), Potamotrygon motoro (3.32%),
and Cnesterodon decemmaculatus (3.16%). Significance: A reference barcode
sequence library of fishes of the lower Paraná River is presented for the
first time and for future use in identification of these species and for use
in other applications. Five new records were generated and uploaded to
BOLD (Parastegophilus maculatus,Pseudohemiodon laticeps,Magalonema ar-
gentinum,Auchenipterus nigripinnis, and Xyliphius sp.). In addition, groups
that deserve further taxonomic study and one exotic species were iden-
tified.
DNA barcodes highlight unique research models in European
butterflies
Vlad Dinca,
1
Niclas Backström,
2
Leonardo Dapporto,
3
Magne Friberg,
2
Enrique García-Barros,
4
Paul D.N. Hebert,
1
Juan Hernández-Roldán,
4
Emily Hornett,
5
Vladimir Lukhtanov,
6
and František Marec
7
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, ON N1G 2W1, Canada.
2Uppsala University, Norbyvägen 18D, 75236 Uppsala, Sweden.
3Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, OX3
0BP, UK.
4Department of Biology, Universidad Autónoma de Madrid, Campus Cantoblanco 28049, Madrid, Spain.
5Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, UK.
6Department of Karyosystematics, Zoological Institute of Russian Academy of Sciences, Universitetskaya
nab. 1, 199034 St. Petersburg, Russia.
7Faculty of Science, University of South Bohemia, C
ˇeské Budeˇ jovice, 370 05, Czech Republic.
Corresponding author: Vlad Dinca (e-mail: vdinca@uoguelph.ca).
Background: There is a widely recognized need for more comprehen-
sive understanding of global biodiversity patterns. Such information
will not only provide insights into major scientific issues, such as
speciation mechanisms, but it will also add new rigor to conservation
programs (a critical need given the looming extinction crisis). We
exploit the power of DNA barcoding to explore biodiversity patterns
in European butterflies, one of the best-studied invertebrate groups in
the world. Ongoing research allowed the detection of unexpected
genetic patterns (exemplified here by Leptidea and Spialia), illustrating
the biological complexity that awaits discovery even in exhaustively
studied regions. Results: The intensively studied model species pair
Leptidea sinapis and Leptidea reali has recently been shown to actually
represent a triplet of species displaying genetic differences and repro-
ductive isolation due to female mate choice. Additional research has
shown that L. sinapis is currently the metazoan with the highest intras-
pecific chromosome number variability unrelated to polyploidy (2n=
56 to 2n= 110) and that its chromosomal races seem to follow a
longitudinally-oriented cline. This unique system prompted us to test
the role of chromosomal rearrangements in speciation and the con-
cept of clinal species by mating extreme chromosomal races based on
laboratory lines of L. sinapis. Within the genus Spialia, recent research
stimulated by DNA barcodes suggests the presence of a new cryptic
species confined to Iberia that has likely speciated through a shift in
larval host–plant. Complex patterns of Wolbachia infections have been
detected in both Leptidea and Spialia.Significance: Leptidea and Spialia
represent some of the most striking cases of cryptic species in Euro-
pean butterflies. These taxa exemplify the effort that lies ahead when
documenting biodiversity and show how patterns detected by DNA
barcodes can lead to the discovery of exciting systems that can often
act as models to improve our understanding of fundamental evolu-
tionary processes.
Phylogeography of the Indian Cobra (Naja naja) reveals
genetically divergent populations between the Indian
subcontinent and Sri Lanka
Duminda S.B. Dissanayake,
1
R.P.V. Jayantha Rajapakse,
1
K.B.A.T Bandara,
1
Kanishka D.B. Ukuwela,
2
S.A.M. Kularatne,
3
and J.G.S. Ranasinghe
3
1Veterinary Parasitology and Molecular Systematics Laboratory, Department of Veterinary
Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya,
Peradeniya, Sri Lanka.
2Department of Biological Sciences, Faculty of Applied Sciences, Rajarata University of Sri Lanka,
Mihintale, Sri Lanka.
3Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka.
Corresponding author: Duminda S.B. Dissanayake (e-mail: duminda.rusl@gmail.com).
Background: Sri Lanka and Western Ghats of India together are consid-
ered as a biodiversity hotspot of high conservation importance. Many
species and genera of fauna and flora are shared between these two
regions due to past geological connections between the two landmasses.
Sri Lanka has been connected to the mainland many times in the past,
the most recent case being in the Pleistocene ⬃10000 years ago when the
sea levels were low. It is assumed that these historical connections facil-
itated biotic exchanges between the two regions. Since the last connec-
tion, the fauna of Sri Lanka has been isolated from the mainland fauna,
and as a consequence co-distributed species are expected to show signif-
icant amounts of genetic divergence between the populations. We tested
this hypothesis by analyzing the mitochondrial cytocrome bgene in the
spectacled Cobra (Naja naja), a highly venomous snake commonly found
in Sri Lanka, India, Bangladesh, Pakistan, and southern Nepal. A 720-bp
region of the cytochrome b region was sequenced from samples collected
from different regions in Sri Lanka and was compared with sequences
downloaded from GenBank from specimens sampled from India and
Nepal. Results: Our maximum likelihood analysis of the cytochrome b
gene revealed two strongly supported (bootstrap support >70%) mono-
phyletic clades corresponding to Sri Lanka and the Indian subconti-
nent (India and Nepal). There was 4.5% genetic divergence between the
two clades, and the Sri Lankan clade was represented by four distinct
cytochrome b haplotypes. However, these haplotypes do not show any
geographically significant pattern. Significance: Our analyses of the
spectacled cobra cytochrome b genes indicate that the Sri Lankan
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congeners have considerably diverged genetically from the mainland
population due to isolation and that they show a distinct evolutionary
trajectory. This information would be useful for the molecular taxon-
omy of family Elapidae and also proteomics study to develop specific
antivenin for medical applications. The project is funded by the Min-
istry of Science and Technology of Sri Lanka (grant No. MRT/TRD/AGR/
3/1/7).
Barcoding Turkish geophythes: a new initiation for
understanding biodiversity and taxonomy
Ali A. Dönmez, Zübeyde Ug˘ urlu Aydın, and Aslı Dog˘ru Koca
Department of Biology, Faculty of Science, Hacettepe University, Beytepe, Ankara, Turkey.
Corresponding author: Ali A. Dönmez (e-mail: aliadonmez@gmail.com).
Background: Turkey is an important hotspot for plant diversity with
33% endemism and more than 600 geophyte species (35% endemism).
Due to floristic richness, the country attracts many botanists and plant
hunters from all over the world for describing new species, and the
country is still a target for the visitors. Moreover, descriptions are badly
needed for newly discovered taxa from some genera, such as Ophrys and
Crocus. Hence, for a better understanding of taxonomy and diversity of
these microspecies, a geophyte plant barcoding project was launched.
Plant DNA barcoding offers many opportunities compared to other mo-
lecular methods. Among the Turkish geophytes, 36 taxa belonging to 25
genera have been selected for evaluating potential barcode genes. Within
the scope of the ongoing project, a total of 108 samples have been exam-
ined for 11 potential barcoding loci. Results: The plant materials
have been stored in silica gel from both field locations and herbar-
ium samples. Specimen pictures have been taken, and observation
of populations is underway. DNA extraction is ongoing, but results
are not available yet. Significance: This project is an important initi-
ation for understanding geophyte diversity and taxonomy. The main
goal of this project is to correctly identify geophytes using a quick and
reliable method. Moreover, the barcoding results will contribute to
detection of illegal plant transportation at customs.
COI barcodes link population genetics with soil food web
structure
David Donoso
Museo de Colecciones Biológicas MUTPL, Universidad Técnica Particular de Loja San Cayetano Alto s/n
CP 1101608 Loja, Ecuador.
E-mail for correspondence: david.donosov@gmail.com.
Background: Ants are conspicuous members of tropical soil food webs,
where most plant detritus is recycled. The mechanisms that shape ant
community structure remain elusive. But species interactions (such as
competition) and environmental filters, expected to affect ant distribu-
tion, are mediated by ant traits. However, because ant traits determine
species attributes such as local abundance, dispersal ability, growth, and
generation time, the genetic variability within lineages should mirror
community composition. COI barcodes are standard pieces of mitochon-
drial DNA that provide us with both an identification tool and a measure
of genetic variability. Here, we explore how within-taxon haplotype di-
versity correlates with different ant traits and helps to explain commu-
nity composition. Results: For example, we expect more haplotype
variability among smaller ants with fast generation times. Increased vari-
ability is also expected among canopy ants with more profound ecologi-
cal barriers. On the contrary, we expect less variability among abundant
and widely distributed ants with less genetic isolation and more gene
flow. Similarly, long-legged ants with high dispersal ability (increased
migratory potential) are expected to show less genetic variability. Signif-
icance: We provide the first links between within-taxon barcode variabil-
ity and community structure.
Assessing the diversity of terrestrial invertebrates in the
mangrove forests of the Firth of Thames, New Zealand
Erin Doyle and Ian Hogg
University of Waikato, Hamilton, New Zealand.
Corresponding author: Erin Doyle (e-mail: edoyle@hotmail.co.nz).
Background: Mangroves provide habitat for a range of native species;
however, limited research has been done on the terrestrial inverte-
brate community within New Zealand's mangrove forests. This proj-
ect aims to address this gap through DNA barcoding of the fauna. We
also compared the mangrove arthropod communities to sequences of
individuals from other forest types and available on BOLD. Results:
Over one year, a total of 8254 individuals were collected, and 251 were
sequenced. COI sequences were congruent with morphospecies desig-
nations. The sequences formed 101 BINs, 39% of which contained spec-
imens from outside of New Zealand. Preliminary results show roughly
50% of species found at the Thames sites had not been previously
found in an inland habitat. Significance: This research project will
generate baseline data that may be used by future projects investigat-
ing topics such as species invasions, climate change, and mangrove
ecosystem function. By assessing the diversity of the invertebrates at
sites of varying age and health, it may also be possible to identify
indicator species useful to environmental monitoring programs. With
mangrove expansion and removal being a contentious issue in some
coastal communities, improving our understanding of the terrestrial
invertebrate communities in these habitats will facilitate appropriate
conservation strategies.
Finding whio: detection of blue duck using environmental DNA
Erin Doyle, Ian Hogg, and Jonathan Banks
University of Waikato, Hamilton, New Zealand.
Corresponding author: Erin Doyle (e-mail: edoyle@hotmail.co.nz).
Background: Population management efforts for New Zealand's en-
demic blue duck, or whio (Hymenolaimus malacorhynchos), are labour inten-
sive due to the difficulties in locating them in the high-elevation streams
they occupy in the wild, their camouflage, and their small population
sizes. The purpose of this research is to explore the potential for environ-
mental DNA (eDNA) analysis to be developed as a tool for detecting blue
ducks in rivers through water sampling. Results: Species-specific prim-
ers were designed to target a section of the mitochondrial control region.
Organic material was collected and concentrated from running water
within blue duck habitats using an in situ filtration system, and eDNA
was then extracted from the filter. Any blue duck DNA present in the
sample was selectively amplified using the species-specific primers,
allowing for blue ducks to be detected through sequencing of the
PCR product. Significance: This technology has the potential to be
further developed for use with other freshwater species, with ap-
plications in conservation pest management.
The effect of rainforest fragmentation on tropical mammals
using leech blood-meal analysis and DNA barcoding
Rosie Drinkwater
Queen Mary University London, Mile End Road, London, E1 4NS, UK.
Corresponding author: Rosie Drinkwater (e-mail: r.drinkwater@gmail.com).
Background: The island of Borneo is a biodiversity hotspot, supporting
high levels of biodiversity in tandem with high levels deforestation and
fragmentation. It is difficult to measure the effect large-scale fragmenta-
tion has on tropical mammals using common surveying methods, as they
can be time-consuming, expensive, and may be less accurate when sur-
veying rare and elusive species. DNA barcoding the blood meals of land
leeches provides a solution for indirectly identifying species, even from
partially digested DNA, by adapting methods for next-generation se-
quencing platforms. My aim is to investigate the effects of rainforest
fragmentation on mammal community composition by DNA blood-meal
analysis of land leeches (Haemadipsa sp.) in a large-scale biodiversity sur-
vey. The study site is the SAFE (Sustainability of Altered Forest Ecosys-
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tems) project in Sabah, Borneo, which is a long-term fragmentation
experiment that will leave forest fragments within an oil palm matrix
over several years. Results: Leeches have been sampled at all sites along
a fragmentation gradient, ranging from continuous logged forests to
different sized fragments and riparian reserves. It is expected that as
degradation increases, specialist species will be underrepresented. In ad-
dition to spatial differences in communities, temporal shifts and time
lags are expected as forest clearance and fragmentation increases in subse-
quent years. Significance: Previously, this method has been used to de-
tect rare and elusive species in Vietnam. Additionally, cryptic species
were also detected that could not have been found by non-molecular
methods. Borneo has high levels of endemism and rare species, but many
species remain listed as data deficient due, in part, to the difficulty of
direct sampling. This indirect barcoding approach will allow for rapid,
large-scale biodiversity surveys that will place important value on Bor-
neo's degraded forests and shape future policy protecting these forests
from further land conversion for agriculture.
Using metagenomics to show the efficacy of restoration in the
New Jersey Pine Barrens
William Eaton,
1
Shadi Shokralla,
2
and Mehrdad Hajibabaei
2
1Pace University, One Pace Plaza, New York, NY 10038, USA.
2Biodiversity Institute of Ontario, University of Guelph, 579 Gordon St., Guelph ON N1G 2W1, Canada.
Corresponding author: William Eaton (e-mail: weaton@pace.edu).
Background: The Franklin Parker Preserve within the Pine Barrens
region of New Jersey contains 5000 acres of wetlands habitat, includ-
ing old growth red maple (Acer rubrum) swamps, cranberry bogs, and
former cranberry bogs that have been restored with the goal of devel-
oping them into red maple forests. This provides excellent experimen-
tal conditions with which to test the efficacy of restoring cranberry
bogs into old growth red maple forests, using changes in soil bacterial,
fungal, and invertebrate communities and increases in efficiency of C
utilization as indicators of restoration success. Results: In this study,
it was shown that the organic C, C biomass, and C-use efficiency were
greater within old growth maple forest soils than in 8-year-old re-
stored red maple stands, which were greater than found in soil from
4-year-old red maple stands—the latter two stands being restored
from long-term cranberry bogs. A detailed metagenomic analysis of
eDNA extracted from these soils showed that these C-cycle trends
were clearly associated with increases in the relative numbers of DNA
sequences from copiotrophic bacterial groups (e.g., specific represen-
tative groups from the Acidobacteria, Actinomycetales, Bacteroidetes,
and Proteobacteria), complex C-decomposing fungal groups (e.g., spe-
cific representatives groups from the Sordiomycetes, Mortierellales,
and Thelephorales), and colembollan and formicid invertebrates. All
these groups are indicators of successionally more advanced soils and
are critical for soil C-cycle activities. Significance: The restoration
activities used at the Franklin Parker Reserve are enhancing critical
guilds of soil biota and increasing C-use efficiency in the soils of re-
stored habitats. The data from this study suggest the use of metag-
enomic analysis of eDNA from these soils can be used in the
development of a predictive model for soil recovery of these wetlands
following restoration. The Franklin Parker Reserve restoration strate-
gies should be modeled by others with regulatory control over similar
damaged wetlands.
The Brazilian Barcode of Life (BrBOL) initiative and its potential
to aid in biodiversity conservation: an overview of the effort to
catalogue mammals, reptiles (including birds), and amphibians
from a megadiverse region
Eduardo Eizirik
Department of Biodiversity and Ecology, School of Biosciences, PUCRS, Porto Alegre, RS 90619-900, Brazil.
E-mail for correspondence: eduardo.eizirik@pucrs.br.
Background: Biodiversity conservation efforts greatly depend on re-
liable knowledge of geographic patterns of species diversity, as it
allows the monitoring of anthropogenic shifts, the assessment of on-
going and future impacts, and the best possible allocation of re-
sources. Megadiverse regions of the world are especially challenging
in this regard, as their biodiversity may be extremely large, complex
and still poorly described. At the same time, they often suffer severe
anthropogenic threats as a consequence of rapid development and
various socio-economic issues. In this context, the DNA barcoding
initiative has a great potential to foster an acceleration of the reliable
cataloguing of taxa present throughout these regions, providing base-
line data for conservation decision making, allowing for more precise
and reproducible surveys in the context of impact assessment, and
being applied in the forensic identification of illegally traded fauna
and flora. Results and significance: In this talk, I will describe the
Brazilian Barcode of Life (BrBOL) initiative, and particularly our ongo-
ing effort to generate and analyze DNA barcodes for mammals, rep-
tiles (including birds), and amphibians from Brazil and adjacent
countries. The project congregates a consortium of 25 institutions,
comprising molecular genetics laboratories and zoological collec-
tions, including Brazil's largest natural history museums. We have so
far generated DNA barcodes for over 12 000 individuals, representing
⬃3000 species of all the included taxa. Ongoing analyses of these data
have revealed interesting patterns and demonstrated the usefulness
of DNA barcoding for species identification in these groups within a
megadiverse context. I will provide an overview of the current data set
and examples of ongoing or published analyses focusing on species
delimitation, assessment of geographic distribution, and application
of the database in forensic cases involving poached species.
The Norwegian Barcode of Life Network (NorBOL)
Torbjørn Ekrem,
1
Inger G. Alsos,
2
Arild Johnsen,
3
Endre Willassen,
4
Aina Maerk Aspaas,
1
Marie K. Foreid,
2
Katrine Kongshavn,
4
and Gunnhild Marthinsen
3
1NTNU University Museum, Department of Natural History, Norwegian University of Science and
Technology, NO-7491 Trondheim, Norway.
2Tromsø University Museum, NO-90370 Tromsø, Norway.
3University of Oslo, Natural History Museum, P.O. Box 1172 Blindern, NO-0318 Oslo, Norway.
4University Museum of Bergen, Natural History Collections, P.O. Box 7800, NO-5020 Bergen, Norway.
Corresponding author: Torbjørn Ekrem (e-mail: torbjorn.ekrem@ntnu.no).
Background: NorBOL (www.norbol.org) was formed in 2007 as a na-
tional network to (i) advance barcoding of Norwegian and Arctic bio-
diversity, (ii) raise funding, (iii) curate barcode reference material,
(iv) coordinate and initiate new barcoding projects, and (v) increase pub-
lic awareness of DNA barcoding and barcoding results in Norway.
NorBOL is a regional node within iBOL, with a particular responsibility
for Polar Regions. NorBOL is coordinated by the NTNU University Mu-
seum in Trondheim and connects 16 institutions, including all four
major natural history museums as well as all major research institutes
in Norway. Results: Despite strong support among research institu-
tions, substantial external funding was only first achieved in 2012
through a grant from the Norwegian Biodiversity Information Centre.
Further funding was obtained in 2014 from both the Research Council
of Norway and the Norwegian Biodiversity Information Centre. Since
then, barcoding progress of the Norwegian fauna, flora, and fungi has
increased, and the Barcode of Life Data Systems database currently
holds more than 42 000 published sequence records (27 000 DNA bar-
codes) of more than 7200 species from Norway. Significance: The goal
for NorBOL is to barcode 20 000 species by the end of 2018. NorBOL
currently targets barcoding of museum collections, lichens, fungi,
marine invertebrates, fish, and material from biosurveillance projects
supported by the Norwegian Taxonomy Initiative. We collaborate
with Swedish institutions on sampling of marine invertebrates, earth-
worms, and insects. An expansion of inter-Scandinavian barcoding
collaboration is in progress. As the standard barcode gene regions
have limited taxonomic resolution for the species level in many vas-
cular plant groups, we are engaged in testing if low-coverage shotgun
sequencing of herbarium material is useful to obtain the full plant
plastid genomes.
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Testing primer bias and biomass—sequence relationships in
metabarcoding: implications for monitoring of freshwater
invertebrate communities
Vasco Elbrecht and Florian Leese
University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany.
Corresponding author: Vasco Elbrecht (e-mail: vasco.elbrecht@rub.de).
Background: Metabarcoding combines DNA barcoding with next-
generation sequencing to reliably identify hundreds of specimens from
an environmental sample at once. However, detection rates for species-
rich stream invertebrate samples, as well as the capability to quantify
biomass or species abundances, have not been systematically tested. We
developed a cytochrome c oxidase subunit I (COI) metabarcoding proto-
col that uses the Illumina MiSeq platform and performed two controlled
experiments (with 10 replicates each) using stream invertebrate samples.
Results: In the first experiment we used 31 specimens of a single stonefly
species that differed by up to four orders of magnitude in biomass. We
found a clear biomass – sequence abundance relationship, but even the
smallest specimens were reliably detected. In the second experiment,
recovery of 52 different freshwater invertebrate taxa was tested using
similar amounts of biomass per specimen as template. With a single
universal primer pair we could recover 83% of the taxa. However, se-
quence abundance varied by four orders of magnitude between taxa.
Significance: Our experiments show that although biomass can be esti-
mated if only a single species is present in a sample, reliable species
biomass or abundance estimates from environmental samples are im-
possible due to primer bias. Thus, DNA-based ecosystem assessments
should rely on presence-absence rather than abundance data.
PrimerMiner: An R package for the development of universal
barcoding primers and mini barcodes using partial COI
sequences
Vasco Elbrecht and Florian Leese
Ruhr University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany.
Corresponding author: Vasco Elbrecht (e-mail: vasco.elbrecht@rub.de).
Background: DNA barcoding for species identification is increasingly
applied in ecological research and biodiversity monitoring. Conserved
“universal” primers are used to PCR-amplify a specific gene region of
the mitochondrial cytochrome c oxidase subunit I (COI) in animals.
Unfortunately, the universal primers do not amplify all taxa equally
well and may even fail. Therefore, many more group-specific, degen-
erate primers have been developed. While many COI barcode se-
quences are readily available in online databases such as the Barcode
of Life Data Systems (BOLD), sequence information about the primer-
binding region is often limited or even contains errors. Until now,
mitochondrial genomes have mostly been used to design improved
universal barcoding primers for animals. However, mitochondrial ge-
nomes are still not available for many groups, which limits the applica-
bility. Results: Here we developed improved degenerate COI primers for
freshwater invertebrates targeting the traditional “Folmer region”, using
a novel approach that also utilizes the full potential of partial COI se-
quences. COI sequences for important freshwater taxa were obtained
from online databases, clustered, and mapped against the COI consensus
from available mitochondrial genomes. Many partial COI sequences
overlapped with the Folmer region and could thus be used for designing
degenerate primers. The alignments created are also useful for the
development of mini-barcodes that lie within the Folmer region.
Significance: With this novel approach, we were able to design reliable
barcoding primers despite the few mitochondrial genomes available
for freshwater invertebrate taxa. Our approach of including partial
barcode sequences can be used to design and verify optimized degen-
erate primers for all taxonomic groups with unprecedented coverage.
An R package for downloading and processing sequences is available
on GitHub: https://github.com/VascoElbrecht/PrimerMiner.
Who is Moina micrura? An example of how barcodes can help to
clarify highly confused species
Lucia Montoliu Elena,
1
Manuel Eliás-Gutiérrez,
2
María Rosa Miracle Solé,
3
and Vladimír Koí
ˇnek
4
1Posgrado de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México and El Colegio
de la Frontera Sur - Chetumal Unit, Chetumal, México.
2El Colegio de la Frontera Sur - Chetumal Unit, Chetumal, México.
3Departamento de Microbiología y Ecología, Universitat de Valencia, Valencia, Spain.
4Department of Ecology, Charles University, Prague, Czech Republic.
Corresponding author: Lucia Montoliu Elena (e-mail: luciamontoliuelena@gmail.com).
Background: Moina micrura seems to be one of the most ubiquous clado-
cerans, with many studies on ecology, ecotoxicology, cultures, distribu-
tion, etc. It is really one of the most confused species of freshwater
cladocerans. As a result, all studies on this taxon cannot be compared or
repeated between different laboratories. With this study, based on mor-
phology, molecular data, and distribution we try to establish the identity
of the real Moina micrura.Results: Moina micrura was one common species
recorded in Albufera de Valencia (Spain). Detailed analyses and a compar-
ison with specimens from the type locality demonstrated the presence of
two taxa there, neither one of them belonging to this species. M. micrura
presents specific morphological traits at Limb II, the large lobe, the ar-
rangement of the seta and sensilla between the large lobe and the gna-
thobase, the ornamentation of the basal corner of the gnathobase, the
exopodite and the accessory setae of Limb V. All these traits are not
included at the original description, and they are of great taxonomic
importance to delineate species. Molecular data, namely COI and 12S
sequences, clearly allow us to differentiate it from its congeners. Signif-
icance: The significance of this study is to establish a baseline to identify
this species and discriminate it from all other similar taxa. This study
opens a new field to understand the taxonomy and general relationships
within the anomopods.
After 10 years of DNA barcoding in Mexico—where are we?
Manuel Eliás-Gutiérrez
1
and Virginia León-Règagnon
2
1El Colegio de la Frontera Sur, Av. Centenario Km 5.5, Chetumal, Mexico.
2Estacion de Biologia Chamela, Instituto de Biologia, UNAM, Mexico.
Corresponding author: Manuel Eliás-Gutiérrez (e-mail: melias@ecosur.mx).
Background: The 2 000 000 km
2
of territory occupied by the Mexican
Republic is the fourth most biodiverse country in the world. Regardless of
this outstanding “natural capital”, as the National Commission for
Knowledge and Use of Biodiversity (CONABIO) has called it, support for
research in this area has been quite limited by the federal agencies such
as the National Council of Science and Technology (CONACYT), com-
pared to other countries. Results: In spite of this resourcing challenge,
several strategies were developed by Mexican researchers when they
realized that DNA barcoding became an important tool to overcome the
taxonomic impediment: the Mexican Barcode Network (MEXBOL) was
established, including a National Laboratory focused on reducing the
costs of DNA analyses; alliances between interested institutions were
built; and main biorepositories got involved. The confidence of the aca-
demicians allowed this country to rank among the top 10 nations in
terms of DNA barcoding of the national biota, with results in almost all
important groups of animals, fungi, and plants. New species of fish,
echinoderms, crustaceans, insects, polychaetes, leeches, platyhelminths,
and acanthocephalans highlighted by the barcodes have been described.
New insights into the diversity of rotifers, molluscs, and the above-
mentioned groups have been discovered, with a much higher diversity
than expected. Diverse applications of barcoding, such as analyses of
seafood, exotic species, disease vectors, and a database of endangered
species to help in the control of trafficking, have been developed. With a
group of well-trained taxonomists, we are adopting new strategies, con-
sidering next-generation sequencing, and looking to integrative taxon-
omy in response to new questions. Finally, we started an educational
program with a mobile PCR laboratory in schools. Significance: In sum-
mary, Mexican researchers have developed creativity and efficiency in
order to use to maximal effect the limited support provided by national
science policies and funders. These advances are particularly important
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in the face of the urgent need for a better understanding of the amazing
and threatened biodiversity in this region of the world.
DNA barcoding of Skwala stoneflies from north-central British
Columbia reveals potential new species
Daniel Erasmus, Daemon Cline, and Ray Lyle
University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada.
Corresponding author: Daniel Erasmus (e-mail: daniel.erasmus@unbc.ca).
Background: DNA barcoding has become an effective tool to aid with
taxonomic identification and is well established for identification of
species within the order Plecoptera (stoneflies). Sampling of stonefly
nymphs from rivers in north-central British Columbia revealed 100%
sequence matches with species records from other locations in North
America, with the exception of the genus Skwala (family Perlodidae).
Results: Analyses of a 658-bp region of the COI gene indicated at least
a 1.7% difference between Skwala specimens collected from north-
central British Columbia and Skwala sequences derived from speci-
mens collected in Montana, Utah, and California. COI sequences from
north-central British Columbia also grouped separately from other
geographical regions in a neighbour-joining tree. Morphological
analyses of the abdominal tips of male and female adults indicate
differences between specimens collected from north-central British
Columbia and other recognised species (S. americana,S. curvata,
S. natorii,S. pusilla [S. brevis], and S. compacta [Arcynopteryx compacta]).
Significance: Collectively, our data suggest the existence of a previ-
ously undescribed species, or subspecies, in the genus Skwala.
COI-based identification of Orius species (Hemiptera:
Anthocoridae) from Iran
Delaram Erfan,
1
Alimorad Sarafrazi,
2
Ghadir Nouri Ghanbalani,
3
Hadi Ostovan,
4
and Mahmoud Shojaei
5
1Department of Entomology, Science and Research Branch, Islamic Azad University, Simoun Bulivar,
Hesarak st., Pounak sq., Tehran, Iran.
2Department of Insect Taxonomy Research, Iranian Research Institute of Plant Protection, Yaman st.,
Evin, Tehran 19395-1454, Iran.
3Department of Plant Protection, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil,
Iran.
4Science and Research Branch, Islamic Azad University, Fars, Iran.
5Department of Entomology, Science and Research Branch, Islamic Azad University, Simoun Bulivar,
Hesarak st., Pounak sq., Tehran, Iran.
Corresponding author: Delaram Erfan (e-mail: delaramerfan@yahoo.com).
Background: Species of the genus Orius (Hemiptera: Anthocoridae)
are important natural enemies of several key agricultural pests in
different climates in Iran. The efficiency and accuracy of DNA barcod-
ing for identification of the morphologically diverse Orius species were
tested using mitochondrial cytochrome c oxidase subunit I (COI) gene
sequences, in collaboration with the Canadian Center for DNA Barcod-
ing (CCDB). The present study analyzed DNA barcodes for 91 speci-
mens representing 12 species assigned to four subgenera. Results: The
average divergence values between species ranged from 0% to 18.3%.
There were two cases of species pairs (O. retamae–O. pallidicornis and O.
bulgaconus–O. minutus) exhibiting low divergence (down to 0% diver-
gence) that reflected cases of misidentification after rechecking the
specimens. Following correction of these cases, average interspecific
divergences were all above 7%. In terms of intraspecific divergences, a
range from 0% to 2.8% average within-species divergence was obtained
for 11 of the 12 species, but an abnormally large value was detected for
O. horvathi (15.7%), which is also explained by a case of misidentifica-
tion. The analysis of sequence variation in COI, which proved to be an
efficient method for species identification, revealed that all 12 species
had a unique suite of barcode sequences. Significance: This study
provides new knowledge about insect biodiversity in Iran. Moreover,
the patterns of sequence variability detected here indicate that future
barcode-based specimen identifications to the species level are likely
to be accurate for this genus. Therefore, our work provides reference
sequences that will enable rapid identification of Orius specimens,
which will be useful for future research into biological control in
agricultural systems.
Biodiversity assessment of plant communities from soil eDNA:
impact of marker selection on perceived community turnover
Nicole Fahner,
1
Donald J. Baird,
2
and Mehrdad Hajibabaei
1
1Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Environment Canada, Canadian Rivers Institute; Department of Biology, University of New Brunswick,
10 Bailey Drive, Fredericton, NB E3B 5A3, Canada.
Corresponding author: Nicole Fahner (e-mail: nfahner@uoguelph.ca).
Background: Soil captures a comprehensive picture of local plant
diversity because it contains seeds, pollen, active and dormant tissues,
as well as plant detritus. These sources of environmental DNA (eDNA)
can be extracted from soil samples and identified using DNA barcod-
ing in conjunction with next-generation sequencing (NGS) to assess
plant diversity. This approach differs from conventional aboveground
plant surveys, which are limited to a snapshot of concurrently grow-
ing species. Due to potential accumulation of plant tissues over time,
however, it is uncertain whether below-ground plant diversity is un-
changing year-to-year or if it exhibits interannual turnover. Addition-
ally, DNA markers commonly used for plant identification may differ
in their ability to detect interannual changes, and some may be less
suitable for DNA metabarcoding of eDNA due to trade-offs between
fragment size, taxonomic resolution, and degradation of eDNA. To
test this, soil cores were collected over three years from Wood Buffalo
National Park in Alberta through the Biomonitoring 2.0 project
(www.biomonitoring2.org), and four plant marker regions (rbcL, Atpf,
trnL intron P6 loop, and ITS2) were sequenced with the Illumina
MiSeq. Results: Below-ground plant diversity is temporally dynamic.
Interannual variability in richness and composition is consistent in
magnitude with previously observed above-ground variability at the
sites. The number of taxa observed and the temporal variability in
richness—but not composition—depend on DNA marker sequence
length and sequence diversity. Below-ground richness exceeds past
estimates of above-ground richness at the sites only when multiple
markers are combined, suggesting a multiple marker approach is
necessary. Significance: By demonstrating interannual variability in
below-ground plant diversity, this research alleviates concerns that
accumulations of so-called “zombie” DNA from long-dead organisms
will confound eDNA-based biodiversity assessments, thus helping to
validate the approach. As well, we show how DNA marker traits can
influence biodiversity assessments.
The effect of anthropogenic disturbance on diversity and
phylogenetic structure of ants (Hymenoptera: Formicidae)
Aaron Fairweather,
1
M. Alex Smith,
1
and Donald McAlpine
2
1University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
2New Brunswick Museum, 277 Douglas Ave., Saint John, NB E2K 1E5, Canada.
Corresponding author: Aaron Fairweather (e-mail: fairweaa@uoguelph.ca).
Background: Forests are threatened globally, and the loss of tree
cover continues within Canada. While there is empirical evidence
illustrating that forest disturbance reduces diversity in some taxa, our
understanding of how hyperdiverse invertebrate taxa are affected by
disturbance is limited. This is partly because the assessment of inver-
tebrate taxa diversity is hindered by “taxonomic impediments”. The
“taxonomic impediment” refers both to unappreciated morphologi-
cally cryptic diversity within named species and the paucity of avail-
able experts to describe and name individual specimens collected
from hyperdiverse taxa. Analysing diversity by utilizing DNA barcodes
in a shared public library allows for molecular discrimination be-
tween cryptic species. Integrating analyses of morphology and DNA
barcodes allows for a cohesive analysis of diversity and permits re-
search on the effects of disturbances on these groups. Parks and Pro-
tected Natural Areas (PNAs) are provincially protected nature reserves.
Many of these have a long history of forestry integrated into contem-
porary park management, thus creating a natural experiment to study
the impact of disturbance on invertebrate diversity. Results: Utilizing
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standardized collections and specimens identified by morphology and
DNA barcodes, our project will assess the diversity and phylogenetic
structure of ants within and around several parks and PNAs in New
Brunswick (NB) and Ontario (ON). If disturbed sites (i.e., forested) have
increased temperatures, food, and habitat resources compared to un-
disturbed sites, then we predict that communities from disturbed
sites will have greater diversity. Furthermore, we expect undisturbed
sites to be phylogenetically clustered since colder habitats create an
environmental filter, selecting ants with conserved traits for success
in that environment. Significance: Ants are valuable ecosystem engi-
neers, seed dispersers, and invertebrate and plant community regula-
tors. Measuring the diversity and phylogenetic structure of this group
will provide useful information regarding how anthropogenic distur-
bance affects forest communities.
Barcodes, bugs, and bats
Brock Fenton
Department of Biology, University of Western Ontario, London, Ontario, Canada.
E-mail for correspondence: bfenton@uwo.ca.
I will use two public programmes involving DNA barcode analysis
to illustrate the potential for increasing public awareness of sci-
ence. First is the Malaise programme operated by the Barcode of
Life endeavour at University of Guelph. Second is the urban bat
programme operating at High Park in Toronto. These two examples
illustrate the potential for engaging people, especially children, in
basic biodiversity.
DNA barcoding of Holarctic Microgastrinae wasps
(Hymenoptera): a major step in the integrative taxonomy of
these caterpillar parasitoids
Jose Fernandez-Triana,
1
Mark R. Shaw,
2
Sophie Cardinal,
3
and Peter G. Mason
3
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
2National Museums of Scotland, Chambers Street, Edinburgh EH1 1JF, United Kingdom.
3Canadian National Collection of Insects, Agriculture and Agri-Food Canada, 960 Carling Ave., Ottawa,
ON K1A 0C6, Canada.
Corresponding author: Jose Fernandez-Triana (e-mail: jftriana@uoguelph.ca).
Background: There is a considerable taxonomic impediment that
prevents further advances in the knowledge of microgastrine
wasps in the Holarctic region, which has resulted in species being
described twice (as different) on both sides of the Atlantic, holo-
types scattered across many collections, numerous morphologi-
cally cryptic species, and a lack of studies from a truly Holarctic
perspective. As a result, only 26% of the 1000+ species recorded
from the region are known from both Europe and North America,
while a significant number of undescribed or unreported species
remain, especially in the Nearctic and Eastern Palaearctic. Results:
Using an integrative taxonomic approach, DNA barcodes of 10 000+
samples from wasp specimens in European and Canadian collections
were analyzed together with morphological, biological (hosts), and
geographical data. These studies significantly increased the known
distribution of many species within the Holarctic, uncovered new
continental records (especially for North America), revealed a signifi-
cant number of cryptic and new species, and confirmed the establish-
ment of wasp species previously introduced for the biological control
of Lepidoptera pests. Significance: The new information available is
applicable to conservation efforts, biological control programs, biodi-
versity inventories, taxonomic revisions, and ecological studies on
this important group of parasitoid wasps. DNA barcoding arguably
provides the fastest and easiest way to organize and critically analyze
large datasets within taxonomically complex and hyperdiverse groups of
insects such as Microgastrinae.
Bats as drivers of bacterial biodiversity across multiple trophic
levels of subterranean biomes
Viacheslav Y. Fofanov,
1
Crystal M. Hepp,
2
Daniel E. Sanchez,
3
Colin J. Sobek,
3
Carol L. Chambers,
3
and Faith M. Walker
3
1Informatics and Computing Program, Northern Arizona University, 1297 S Knoles Drive, Flagstaff, AZ
86011, USA.
2Center for Microbial Genetics and Genomics, Northern Arizona University, 1297 S Knoles Drive,
Flagstaff, AZ 86011, USA.
3School of Forestry, Northern Arizona University, 200 East Pine Knoll Drive, Flagstaff, AZ 86011,
USA.
Corresponding author: Viacheslav Y. Fofanov (e-mail: viacheslav.fofanov@nau.edu).
Background: Subterranean ecosystems, such as caves and aban-
doned mines, are numerous (100 000+ exist in the western US
alone), yet understudied. In these environments, bats, through
their nutrient-rich guano, have long been known as a foundation
species, supporting a diverse food web including bacteria and ar-
thropods that use guano as a food source, as well as predators that
consume them. While bats are clearly important to this ecosystem,
the degree to which their presence, species composition, and gut
microbiota drive the diversity and stability of subterranean bacte-
rial communities, and what happens when bat assemblages are
significantly disrupted, is not known. Results: We have used a
variety of barcoding and shotgun metagenome sequencing tech-
niques to explore bacterial communities across multiple trophic
levels (bat guano, associated arthropods) in bat-occupied subterra-
nean sites (abandoned mines) within USA's southern Arizona re-
gion. Our results to date support the hypothesis that bats, and their
gut/guano microbiota, drive the bacterial biodiversity and compo-
sition of their immediate subterranean ecosystem. In particular,
we have observed bat guano pile microbiota affecting arthropod
gut bacterial composition, with detritivorous insect gut communi-
ties most closely mimicking the microbiota of guano from which it
was isolated. Shotgun metagenomic sequencing revealed a number
of bacterial taxa persisting across the guano pile and arthropod gut
niches. Finally, we observed bat species composition across different
sites closely correlating with guano pile microbiome compositions.
Significance: Exploration of subterranean biomes is particularly rel-
evant given that biodiversity within these ecosystems is threatened
from a variety of sources, including invasive species, human activities,
and disruptions to bat assemblages due to extinction, disease, habitat
loss, and climate change. Bats themselves are increasingly recognized
as an important reservoir for a number of human- and livestock-
affecting bacterial zoonotic diseases, further highlighting the need to
understand how their gut microbiota propagate through the environ-
ment.
Plant Pest Barcoding Campaign update
Andrew Frewin, Cynthia Scott-Dupree, and Robert Hanner
University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Andrew Frewin (e-mail: afrewin@uoguelph.ca).
Background: Plant Pest Barcoding (www.plantpestbarcoding.org) is a
campaign to DNA barcode agriculturally and environmentally important
arthropod pest species. Phytosanitary policy actions and on-farm pest
management decisions are both informed by the identification of pest
specimens. DNA barcoding can assist these identifications by overcom-
ing limitations of morphology-based methods such as the lack of taxo-
nomic resources, knowledge, and (or) expertise. However, for DNA
barcoding to address phytosanitary goals, barcode reference libraries
must contain representatives of known pest species. We compiled a
checklist of arthropod plant pests of global origin that includes species of
regulatory and (or) economic significance. Here, we present a gap analy-
sis of the DNA barcode library coverage for this checklist, contrast it with
one conducted two years previous, and discuss some of the current chal-
lenges facing the further implementation of DNA barcoding for phyto-
sanitary applications. Results: Altogether, 68% (638/943) of species on
our pest checklist are represented by specimens with associated barcode
sequences on BOLD; this represents an increase of 10% from two years
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prior. Over the last two years, 521 species had newly generated barcode
sequences, while 404 had no new additions, and 18 lost sequences, pre-
sumably due to curatorial processes. Although these additions resulted
in better barcode coverage across the checklist, only 41% (387/943) of
species are currently represented by 10 or more barcodes. Significance:
The potential phytosanitary applications of DNA barcoding are fre-
quently invoked to justify DNA barcode research. As such, it is important
that the wider barcode research community ensures that pests of varying
economic and regional importance are adequately represented in refer-
ence libraries. The current gap analysis suggests that library coverage for
many common pest species is progressing; however, gaps still exist. By
providing a checklist of pest species, Plant Pest Barcoding aims to address
practical library deficiencies, by highlighting gaps and guiding further
library development.
Application of DNA barcoding for the identification of
intercepted pests
Andrew Frewin, Cynthia Scott-Dupree, and Robert Hanner
University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Andrew Frewin (e-mail: afrewin@uoguelph.ca).
Background: International trade and travel is an important pathway for
the introduction of non-indigenous plant pests. Recognizing this, na-
tional phytosanitary organizations inspect baggage and cargo-containing
agricultural and plant-related commodities arriving at ports of entry for
plant pests. Data gained from these inspections, such as the abundance,
frequency, origin, and commodity-pest associations, are used to improve
inspection programs and to refine both pathway analyses and pest risk
assessments. Unfortunately, interception targets for many pest species
are the immature life stages, which are often more difficult to identify
compared to adult forms. In co-operation with the USDA-SEL, we exam-
ined the ability of current barcode reference libraries in Barcode of Life
Data Systems (BOLD) to assist in the identification of a set of larval Lepi-
doptera intercepted by USDA-PPQ at US points of entry, by comparing
morphological and barcode-based identification methods. Results:
There was no significant difference in the number of species-level iden-
tifications made using morphology or barcode-based methods. However,
morphology- and barcode-based identification combined significantly in-
creased the number of species-level identifications compared to either
method in isolation. Overall, sequence recovery rate was low at 84%, which
influenced method comparisons; and its implications are discussed.
Significance: This pilot project illustrates how DNA barcoding may en-
hance phytosanitary inspection programs. We also demonstrate the lim-
itation of current barcode reference libraries, which have relatively poor
representation for some taxa and regions. However, using the BINs
framework to assign unidentifiable specimens to MOTUs will help in
determining the frequency and commodity-association for intercepted
taxa and represents a transition from reactive to proactive management.
Finally, some unidentifiable larval specimens in this dataset were associ-
ated with unidentified adult specimens via BINs, which presents an in-
teresting opportunity for integrative taxonomy concerning taxa of
phytosanitary importance.
A framework for estimating eDNA sensitivity
Elise Furlan,
1
Dianne Gleeson,
1
Chris Hardy,
2
and Richard Duncan
1
1University of Canberra, IAE, Building 3, University Drive, Bruce, ACT 2617, Australia.
2CSIRO CSIRO, Land & Water Flagship, GPO Box 1700, Canberra, ACT 2601, Australia.
Corresponding author: Elise Furlan (e-mail: elise.furlan@canberra.edu.au).
Background: Environmental DNA (eDNA) is increasingly being used as a
survey tool to infer species distributions, yet the sensitivity of the tech-
nique has not been carefully evaluated. Imperfect sensitivity, or imper-
fect detection, is a feature of all survey methods and needs to be
accounted for in interpreting survey results. We will present a frame-
work to estimate the sensitivity of both the field and laboratory compo-
nents of an eDNA survey method and show how these can be combined
to estimate the overall sensitivity. We will apply this framework to
species-specific eDNA surveys to estimate the sensitivity—or probability
of detection—for three invasive aquatic species present in Australia:
Perca fluviatilis,Cyprinus carpio, and Misgurnus anguillicaudatus.Results: The
eDNA survey successfully detected all three invasive species in environ-
ments where they were known to be present, but detection sensitivities
differed greatly with season, with greater detection sensitivity typically
achieved in spring compared to autumn. These sensitivity estimates can
be used to explore different field survey designs to achieve the desired
detection sensitivity. This can be done by varying the number of water
samples and number of PCR replicates per sample. Significance: To
utilise eDNA as a management tool, the sensitivity of eDNA detection sur-
veys must be estimated and accounted for. The framework presented here
allows researchers to quantify the overall sensitivity of a particular eDNA
survey method and to optimise sampling regimes to improve detection sen-
sitivities. This has the potential to improve knowledge of species' distribu-
tions to assist management of both endangered and invasive taxa.
Reconstructing interactions among plants, insect herbivores,
and phoretic mites using DNA barcodes: modeling
coextinctions under projected climate change
Carlos Garcia-Robledo,
1
W. John Kress,
2
and Terry L. Erwin
3
1Institute of Ecology, Mexico, Carretera antigua a Coatepec 351, El Haya, 91070 Xalapa Enríquez, Ver.,
Mexico; and Department of Botany, National Museum of Natural History, Smithsonian Institution,
10th St. & Constitution Ave. NW, Washington, DC 20560, USA.
2Department of Botany, National Museum of Natural History, Smithsonian Institution, 10th St. &
Constitution Ave. NW, Washington, DC 20560, USA.
3Department of Entomology, National Museum of Natural History, Smithsonian Institution, 10th St. &
Constitution Ave. NW, Washington, DC 20560, USA.
Corresponding author: Carlos Garcia-Robledo (e-mail: carlos.garcia@inecol.mx).
Background: Coextinction, the loss of one species as a consequence of
the extinction of other species, is one of the main drivers of species loss
on earth. Unfortunately, we have limited knowledge of how coextinc-
tions propagate through trophic levels. One challenge to studying coex-
tinctions is the identification of interacting species. Using DNA barcodes,
we identified tri-trophic interactions along a tropical elevational gradient
between host plants, insect herbivores, and phoretic mites. The resulting
interaction networks were used to model cascades of extinctions for
organisms present at different elevations under projected global warm-
ing. Results: Along an elevational gradient in Costa Rica (60-2800
m.a.s.l.), we determined the elevational distributions and obtained DNA
barcodes (rbcL, ITS2) of plants from the order Zingiberales. These are the
hosts of rolled-leaf beetles (Cephaloleia, Chrysomelidae), a charismatic
group of insects that we are using to understand the complex process of
coextinctions. To reconstruct interactions between plants and herbi-
vores, we combined field records with novel molecular methods, identi-
fying host plants using plant DNA extracted from insect gut contents.
The DNA barcode COI revealed several cryptic insect herbivore species
with narrow diets and elevational distributions. We are currently obtain-
ing DNA barcodes (COI) from phoretic mites collected from beetles along
this elevational gradient. Coextinction analyses show that high-elevation
insect herbivores have a higher probability of extinction than insects in
the lowlands. Future analyses will determine if phoretic mites show sim-
ilar susceptibilities to extinction at different elevations. Significance: Fast
and accurate identifications of species and their trophic interactions us-
ing DNA barcodes are opening endless research avenues to understand
key issues in ecology, evolution, and conservation, such as the process of
coextinctions under projected global change.
Preliminary results from Malaise traps in southern Yucatan
Peninsula, Mexico
León Ibarra Garibay
Instituto Tecnológico de Chetumal, Av magisterio #20 col. Magisterial, Chetumal, Mexico.
E-mail for correspondence: leonibarragar@hotmail.com.
Background: In Mexico, specifically in the Yucatan Peninsula, there is
poor knowledge of insect diversity; only some groups have been in-
tensely studied. In BOLD, to date there are only four published projects
focused upon insects from the Yucatan Peninsula, three for families and
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one for a genus; highly diverse insect orders have yet to be studied. We
collected 12 763 specimens of the class Insecta using Malaise traps in
three different types of vegetation (a perturbed zone, a mature recovered
one, and a mangrove) during 159 days of sampling. Results: Our prelim-
inary results include the identification of 16 orders and 76 families of
insect. Nevertheless, the major orders Diptera, Hemiptera, and Lepidop-
tera have only 22 families identified, which is a small number in compar-
ison with the percentage of specimens in these orders (69.78%).
Specimens were distributed among the major orders as follows: Diptera
(57.06%), Hymenoptera (15.34%), Coleoptera (11.93%), Hemiptera (7.09%),
and Lepidoptera (5.63%). Preliminary results for 155 successfully se-
quenced specimens show 114 BINs, 61 of which were not previously reg-
istered in BOLD. Of the 114 Bin's, we identified 47 to the ordinal level, 53
to family, and 14 to generic level. The preliminary results show greater
diversity in the mangrove zone than in the other sites. Significance: This
is the first time this kind of sampling has been undertaken in this region.
We linked some immature stages with adults and some worker ants with
the reproductive stages of the same species. We also identified species
distribution patterns by comparing our data with published records in
BOLD. We found species that have not yet been sequenced in geograph-
ically close and well-studied regions such as Costa Rica. A library of insect
barcodes for this region is just in the starting phase.
DNA barcoding for identification of Cephalotaxus and the
discovery of new species
Lianming Gao, Dezhu Li, and Jie Liu
Kunming Institute of Botany, Chinese Academy of Sciences, No. 132 Lanhei Raod, Kunming, Yunnan, China.
Corresponding author: Lianming Gao (e-mail: gaolm@mail.kib.ac.cn).
The genus Cephalotaxus (Taxaceae) is comprised of eight species and two
varieties distributed in East Asia as understory trees in temperate mon-
tane forest. As there is a lack of clear-cut morphological differences
among species, taxonomy of this genus is difficult and controversial. In
this study, all species and varieties of Cephalotaxus were collected from the
distribution range, with 2–11 individuals per taxon, for species delimita-
tion using six candidate DNA barcodes (ITS, rbcL, Atpf, trnH–psbA,
trnL–F, and psbK–psbI). Among the six DNA barcodes, ITS showed the
highest species discrimination rate at 36.4%, followed by Atpf and trnL–F
(27.3%), while rbcL and trnH–psbA exhibited the lowest rate (9.1%), by
using tree-based (NJ) analysis. Combinations of all six DNA barcodes can
significantly improve the discriminatory power (63.6%) for Cephalotaxus
species identification. Based on DNA barcoding analysis, seven species,
including a new species, were identified in Cephalotaxus, which corre-
spond well with its distribution. Recent taxonomic revisions of
Cephalotaxus were not supported by our analysis. DNA barcoding is an
efficient tool for new species discovery and taxonomic revision.
Collection data of black flies, mosquitoes, and sand flies of
Mexico for further DNA barcode study
Javier A. Garza-Hernández,
1
Luis M. Hernández-Triana,
2
Aldo I. Ortega-Morales,
3
Erick de J. De Luna-Santillana,
1
and Mario A. Rodríguez-Pérez
1
1Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Tamaulipas, México.
2Animal and Plant Health Agency Woodham Lane, Addlestone, Surrey, KT15 3NB, UK.
3Departamento de Parasitología, Universidad Autónoma Agraria Antonio Narro Unidad Laguna
Torreón, Coahuila, México.
Corresponding author: Luis M. Hernández-Triana (e-mail: lhernand@uoguelph.ca).
Background: The dipterans, dubbed true flies, are one of the largest
insects orders, accounting for an estimated 120 000 species. Many species
of flies of this order are bloodsuckers and disease vectors of public health
concern. Accurate taxonomic identification of vectors is of paramount
importance for control programs and scientific research. For example,
the morphotaxonomy of black flies is extremely difficult because of phe-
notypic plasticity, genetic variability, cryptic diversity, and the presence
of different life-cycle stages and sexual dimorphism. Thus, the DNA bar-
code cytochrome c oxidase subunit I (COI) gene for the discovery of cryp-
tic diversity and species complexes in black flies is highly useful. Results:
The present study updates the collection data of 257 specimens of dipter-
ans, including important disease vectors such as black flies, mosquitoes,
and sand flies, collected from several localities in Mexico from 2012
through to 2015. In total 84 morphospecies were identified: two species
belong to Psychodidae (genus Lutzomyia), four species to Simuliidae
(genus Simulium), and 78 species to Culicidae (genera Aedeomyia,
Aedes,Anopheles,Coquillettidia,Culex,Culiseta,Deinocerites,Haemagogus,
Howardina,Limatus,Lutzia,Mansonia,Psorophora,Sabethes,Shannoniana,
Toxorhynchites,Trichoprosopon,Uranotaenia, and Wyeomyia). Significance:
All 257 specimens are currently processed using the standard COI bar-
code protocol for further analysis and study. Several vector species be-
longing to common genera, such as Aedes,Culex,Lutzomya, and Simulium,
have now been DNA barcorded which might facilitate their identi-
fication.
Identifying Malva species in Libya through DNA barcodes
techniques, using four candidate DNA barcoding markers
Ahmed Gawhari, Stephen Jury, and Alastair Culham
Centre for Plant Diversity and Systematics, Harborne Building, School of Biological Sciences, University
of Reading, Reading, RG6 6AS, UK.
Corresponding author: Ahmed Gawhari (e-mail: a.gawhari@pgr.reading.ac.uk).
The approach of DNA barcoding has been used to distinguish and iden-
tify the Malva species of Libya. This study has been conducted using both
official and novel DNA barcode regions tested on herbarium-derived
DNA samples. Twenty-three specimens representing eight Malva species
were collected from Libyan herbaria (Benghazi University, Omar
Mukhtar University, and Tripoli University) and University of Reading
herbarium (RNG). DNA fragments of regions rbcL, psbA–trnH, Atpf, and
ITS were used as DNA barcodes to test their ability to distinguish species
of Malva. Taxon DNA analysis and tree-based methods were used. The
average intra- and interspecific distances were calculated, and DNA bar-
coding gaps were used to investigate the molecular identification ability
of the chosen markers. The results showed that the best single barcode
region was psbA–trnH. It is considered as a good candidate for use as a
DNA barcode for identifying Malva, showing 100% identification effi-
ciency.
Global perspectives on participating in the International
Barcode of Life Project
Janis Geary and Tania Bubela
School of Public Health, University of Alberta, 3-300 ECHA, 11405 – 87 Ave., Edmonton, AB T6G 1C9, Canada.
Corresponding author: Janis Geary (e-mail: janis.geary@ualberta.ca).
Background: As an international effort to create a publicly accessible
database, the International Barcode of Life Project (iBOL) is vulnerable to
cultural differences and social dilemmas that may discourage individuals
from participating. We apply the Institutional Analysis and Development
(IAD) Framework, which is a tool used to study types of “commons”
(shared resources), to understand participation in iBOL. iBOL commons
include barcode databases and the repositories that store voucher speci-
mens. Collectively, the barcodes and specimens are genetic resources,
governed by national laws that implement the Nagoya Protocol to the
Convention on Biological Diversity (CBD). Concern over the use of genetic
resources differs between researchers in the Global North and the
resource-poor, but biodiversity-rich Global South. These cultural, soci-
etal, and legal differences can create divide in the global barcoding com-
munity over how the shared resources should be managed. The success
of iBOL is contingent on promoting use of the resource as well as re-
contribution of value-added data, while remaining sensitive to the divide
in interests of researchers. Accomplishing this balance requires a set of
rules that coordinates the behaviours of the different actors that com-
prise the international barcoding community. Results: We completed 44
semi-structured interviews with members of the iBOL community, fund-
ing agencies, and external stakeholders. Interviewees discussed many
topics including research collaborations, genetic resource collection,
data release, and knowledge about the CBD. Through applying the IAD
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Framework, we analysed these interviews within the broad context of
iBOL including print media representations, policies of iBOL and major
funding agencies, requirements of the CBD, and publication/data release
patterns of the barcoding community. Significance: Our research iden-
tifies cultural, societal, and legal differences between international par-
ticipants and stakeholders that are impacting participation in iBOL. We
suggest strategies to mitigate identified conflicts and will invite discus-
sion to further develop these strategies.
The FREDIE project—different lessons from a large-scale DNA
barcoding campaign
Matthias Geiger,
1
Fabian Herder,
1
and Jörg Freyhof
2
1Zoological Research Museum Alexander Koenig, Leibniz Institute for Animal Biodiversity, Foundation under
public law, Adenaueralle 160 / 53113 Bonn, Germany.
2German Centre for Integrative Biodiversity Research (iDiv), Deutscher Platz 5e / 04103 Leipzig, Germany.
Corresponding author: Matthias Geiger (e-mail: m.geiger@zfmk.de).
Background: In my talk I will first provide an overview of the aims
and structure of the FREDIE project and then focus on findings and
highlights of the freshwater fish diversity of the Western Palearctic.
Special emphasis will be on the Mediterranean biodiversity hotspot
as one of the most important temperate biodiversity areas, with a
remarkable diversity of freshwater fishes. Currently, 397 out of
526 species are recognized as endemic. In order to assess their diver-
sity, and to construct a molecular identification system that supports
conservation, we have built a DNA barcode library for 498 Mediterra-
nean freshwater fish species (98% extant species coverage) based on
3171 individuals. Results: Congruence of clusters with morphological
species ranged from 45% to 85% of the species and was highly depen-
dent on the method used to delineate clusters. Genetic discontinuities
suggest the existence of up to 64 possible new species or new records,
potentially increasing fish diversity by 12%. Shared haplotypes among
species highlight limits of a barcoding approach, although mis-
matches tend to occur in recently diverged (Pleistocene- or Holocene-
evolved) lineages. We found reduced reliability of DNA barcoding in a
highly biogeographically structured area, but much greater accuracy
when applied at the catchment scale. This scale effect can have impor-
tant implications for barcoding assessments, and our results suggest
that fairly simple identification pipelines can be successfully applied
for local freshwater fish monitoring. Inventorying and management
of large-scale diversity requires higher analytical and conceptual ef-
forts. Significance: The 98% extant species coverage of freshwater fish
species of the Mediterranean Biodiversity for barcode sequences rep-
resents a success story for biodiversity research in a geopolitically
complex region. The database will enable the recognition of conser-
vation units within the Hotspot and will strongly support the ongoing
exploration of global freshwater fish diversity.
DNA barcoding in ethnobotany and ethnopharmacology:
identifying medicinal plants traded in local markets
Abdolbaset Ghorbani,
1
Yousef Saeedi,
2
and Hugo J. de Boer
3
1Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D,
SE 75236 Uppsala, Sweden.
2Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical
Sciences, No. 10, Shams Alley, Valiassr Ave., Tehran, Iran.
3The Natural History Museum, University of Oslo, Oslo, Norway.
Corresponding author: Abdolbaset Ghorbani (e-mail: abdolbaset.ghorbani@ebc.uu.se).
Background: Local markets are important places for the trade of
medicinal plants harvested by rural villagers and provide rapid in-
sight into the medicinal plants growing in a region. However, accurate
taxonomic identification of medicinal plants from markets is chal-
lenging, as traded plant materials are sold in processed forms such as
dried roots and barks, powdered plant parts, and mixtures. Two meth-
ods of DNA barcode-based molecular identification are evaluated for
species-level identification performance: a purely objective sequence
matching method and an integrative approach that coalesces se-
quence matching with a priori and a posteriori data from other mark-
ers, morphology, ethnoclassification, and traditional knowledge.
Samples were purchased from seventeen herbal shops in Iran. Each
sample's vernacular name was recorded. Samples were identified
based on morphological characteristics and assigned to families,
genera, and species when possible. Putative scientific names were
extracted from the literature based on previously established corre-
spondence with vernacular names. DNA was extracted from 68 sam-
ples that could not be identified to species level using morphology,
and the nrITS and trnL markers were amplified and sequenced. The
resulting sequences were queried against the NCBI GenBank nucleo-
tide database and identified using both approaches. Results: Objec-
tive sequence matching yielded a 16.7% species-level identification
rate for trnL, a 33.3% rate for nrITS, and a 38.2% rate when both
markers were combined. The integrative approach resulted in a 75.0%
species-level identification rate for trnL, a 79.2% rate for nrITS, and a
80.9% rate when the markers were combined, and thus a 3.5-, 1.4-,
and 1.1-fold increase in species-level identification, respectively.
Significance: DNA barcoding is an effective method to identify traded
plant material that lacks morphological characteristics in processed
form, but requires integrative strategies to include data from multiple
markers, morphology, and traditional knowledge to optimize species-
level identification success.
The good, the bad, and the ugly: DNA barcoding a nightmare taxon
Jason Gibbs
Center for Integrated Plant Systems, Michigan State University, USA.
E-mail for correspondence: jgibbs@msu.edu.
Background: Lasioglossum (Hymenoptera: Halictidae) is the most
species-rich, behaviourally complex, and taxonomically challenging
genus of bees. Species from across North America have been se-
quenced as a component of multiple taxonomic monographs.
Results: DNA barcoding results support numerous cryptic species in
the genus Lasioglossum. In many cases DNA barcodes correspond well
with morphological and geographic data. In other cases, conflicts be-
tween DNA barcodes and other data are not easily resolved. Shared
haplotypes between and deep divergences within putative species can
be misleading if examined uncritically. Published data show that stan-
dard universal barcode primers are a poor choice for Lasioglossum
and lead to relatively frequent amplification of the endosymbiont
Wolbachia (Rickettsiales). Significance: DNA barcodes provide a useful
tool for challenging taxonomic groups, but data must be interpreted
with care. Future efforts to barcode bees should use bee-specific prim-
ers to increase successful amplification of barcode sequences.
Biomonitoring boreal wetlands using environmental DNA
barcoding and high-throughput sequencing
Joel F. Gibson,
1
Shadi Shokralla,
1
Colin Curry,
2
Ian King,
1
Donald J. Baird,
3
and Mehrdad Hajibabaei
1
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Canadian Rivers Institute, University of New Brunswick, Fredericton, New Brunswick, Canada.
3Environment Canada, Canadian Rivers Institute; Department of Biology, University of New Brunswick,
10 Bailey Drive, Fredericton, NB E3B 5A3, Canada.
Corresponding author: Joel F. Gibson (e-mail: jfgibson@uoguelph.ca).
Background: Advances in genomics and computational technologies
allow the efficient analysis of DNA barcode sequences from organisms
representing all trophic levels of a wetland ecosystem. High-throughput
sequencing (HTS) technology (e.g., Illumina MiSeq) can recover de-
tailed measures of the biodiversity of a wetland rapidly, accurately,
and at a much lower cost per sample compared to morphological
analysis. We have used environmental DNA barcoding to assess the
biodiversity contained within mixed environmental samples col-
lected in the boreal forest region of Canada. Each HTS sequencing run
generates over a million DNA sequences from the organisms con-
tained in each sample. DNA barcodes have been chosen to provide
taxonomic coverage for each kingdom of life, including bacteria,
plants, fungi, and animals. Bioinformatic processing of DNA sequence
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data recovers both taxonomic and phylogenetic diversity information
about the organisms whose DNA is present in the sample. Results:
Our processing of multiple, mixed environmental samples from two
river systems in Wood Buffalo National Park produced millions of
DNA barcode sequences. Comparison of these sequences to public
barcode libraries generated taxonomic lists for each sample up to and
including species-level identification. Advanced alpha and beta diver-
sity metrics were then generated based on these data. Significance:
All of the standard biomonitoring metrics employed by current prac-
titioners can be replicated using a high-throughput sequencing ap-
proach. The recovery of genus- and species-level data greatly increases
the resolution of the biodiversity assessment. By facilitating greater
biodiversity recovery in a shorter time, DNA-based massively-parallel
sequencing approaches will greatly multiply the force of human im-
pact on assessing, and improving, the status of pristine and impacted
wetlands.
Evolution of ecological communities through the lens of an
island chronosequence
Rosemary Gillespie and Henrik Krehenwinkel
University of California Berkeley, 130 Mulford Hall, Berkeley, CA, USA.
Corresponding author: Rosemary Gillespie (e-mail: gillespie@berkeley.edu).
Background: Understanding how ecological and evolutionary pro-
cesses synergistically determine biodiversity patterns remains a central
goal in biology. Oceanic islands provide simplified and discrete systems
for determining how biodiversity has developed within a discrete arena.
The Hawaiian Islands are particularly useful because they display an age
chronology that allows analysis of communities that are just starting to
establish, to more mature (≥5 mya). This chronology can be used as the
backdrop for a natural experiment to understand how early ecological
processes give way to longer-term processes of adaptation and species
diversification. In our work, we have been using the Hawaiian model
system to explore characteristics of arthropod communities over the
ecological/evolutionary continuum. Results: Using the space-for-time
chronosequence of Hawaii, we have incorporated a metabarcoding ap-
proach. Using next-generation sequencing, we generate a barcode data-
base of all arthropod taxa. Based on this reference collection, we
determine the identity of taxa within a given sample, as well as associ-
ated microbes, and identify host parasite and prey predator interactions.
The approach thus simultaneously provides information on the qualita-
tive and quantitative composition of the Hawaiian community. Finally,
the sequence data provides preliminary information on genetic relation-
ships between taxa across sites. These data are analyzed by comparing
arthropod communities at sites of different age to determine the pro-
cesses driving emergent patterns of island biodiversity. Specifically, we
compare the data to predictions of species abundances and food web
interactions generated from powerful theories based on the Maximum
Entropy Theory of Ecology (METE). Significance: Deviations of data from
theory, and how these deviations change across the island chronology,
can illuminate the mechanisms of species assembly and associated
changes in trophic interaction networks. Thus, our results allow us to
build a predictive understanding of the interplay between ecology and
evolution in dynamically shaping complex ecosystems.
Metabarcoding for surveillance and monitoring: meeting policy
objectives in the real world
Rachel Glover,
1
Mel Sapp,
1
Ian Adams,
1
Ummey Hany,
1
Erin Lewis,
1
Ian Brittain,
1
Kerry Walsh,
2
Martyn Kelly,
3
and Neil Boonham
1
1Fera, Sand Hutton, York, YO41 1LZ, UK.
2EA Environment Agency, Bristol, UK.
3Bowburn Consultancy, Bowburn, Durham DH6 5QB, UK.
Corresponding author: Rachel Glover (e-mail: Rachel.glover@fera.co.uk).
Background: Since the global economic downturn of the late
2000s, government laboratories have been trying to achieve their
monitoring activities with ever-dwindling budgets to achieve pol-
icy outcomes. In some cases, exploring novel approaches to surveil-
lance allows them to achieve more with less. Work at Fera has
focused on the development of metabarcoding approaches to iden-
tify species composition of organisms in water and in the air. Early
detection of airborne plant pathogens is essential to enable control
measures to be implemented. This is particularly challenging when
surveillance is for quarantine pathogens subject to control under
Council Directive 2000/29/EC where any one of a long list of patho-
gens need to be identified (https://secure.fera.defra.gov.uk/phiw/
riskRegister/). The community structure of freshwater benthic
diatoms is linked to water quality and is used to make assessments
as part of the EU Water Quality Framework. Identification work is
currently done using light microscopy to resolve species based on
the structure of the diatom “shell” or frustule. Results: We have
developed metabarcoding methods to allow monitoring of air-
borne fungal spores for quarantine/notifiable pathogens. We are
also assessing the suitability of using samples collected with air
samplers deployed for other purposes (e.g., pollen or air quality
networks), as this has the potential to save costs and improve effi-
ciency, potentially enabling profiling of samples to achieve multi-
ple outcomes from the same samples. In addition, we are
translating a metabarcode approach for diatom community profil-
ing into a high-throughput method to enable improvements in the
efficiency, speed, and cost of water quality assessments. Signifi-
cance: DNA-based methodologies have always had great promise
for delivering testing services. DNA barcoding in particular is a
generic technique for resolving taxa. When coupled with next-
generation sequencing, metabarcoding potentially offers efficient,
high-throughput species identification.
Barcodes, food webs, and insect community ecology
Charles Godfray
Oxford University, Department Zoology, South Parks Rd., Oxford, OX1 3PS, UK.
E-mail for correspondence: charles.godfray@zoo.ox.ac.uk.
Abstract: Insect ecologists construct food webs to understand how
communities are assembled and to generate hypotheses about species
interactions that can be tested by experimental manipulation. A par-
ticular premium is placed on quantitative food webs which are espe-
cially informative about community dynamics. There are, however,
formidable challenges to constructing quantitative food webs which I
shall describe in my talk. I will then explore how DNA barcodes and
related technologies might help overcome these issues, and what new
questions in community ecology they may allow us to address.
Integrating phylogenetic and functional diversity in REDD+
projects: a pilot in the Pacific rainforest in Colombia
Mailyn A. Gonzalez, Beatriz Salgado, Roy Gonzalez,
Ángela María Mendoza, Elkin Tenorio, Jhon Nieto, Sandra Medina,
and Hernando Garcia
Instituto Alexander von Humboldt, Calle 28A n 15-09, Colombia.
Corresponding author: Mailyn A. Gonzalez (e-mail: magonzalez@humboldt.org.co).
Background: The evolutionary diversity encompassed in a commu-
nity reflects the assembly rules that allow the coexistence of species
and is an indicator of the response capacity of ecosystems to change.
The Chocó biogeographic region on the Pacific coast of Colombia is
one of the world's biodiversity hotspots, with nearly 11 000 species of
vascular plants that remain poorly known. In this study, we assess the
taxonomic, functional, and phylogenetic diversity of trees in four 1 ha
plots from a REDD+ initiative. Results: We found 360 species of trees.
Phylogenetic diversity was measured based on four DNA barcode se-
quence regions (RbcLa, TrnH–psbA, trnL, and ITS) obtained for 120 spe-
cies and completed with 200 barcodes from GenBank. Taxonomic,
functional, and phylogenetic diversity patterns were decoupled,
pointing to areas of particular evolutionary history and functional
significance for conservation. Significance: The conservation value of
forests should consider not only the capacity to stock carbon but also
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all other dimensions of biodiversity. DNA barcoding constitutes an
efficient tool to assess the evolutionary history of communities and
understand the assembly rules that sustain biodiversity.
Barcoding as a useful tool for South American wild bee
systematics
Rocio Ana Gonzalez-Vaquero,
1
Arturo Roig Alsina,
1
and Laurence Packer
2
1Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” – CONICET, Avenida Ángel Gallardo
470, Buenos Aires, Argentina.
2York University, 4700 Keele St., Toronto, ON M3J 1P3, Canada.
Corresponding author: Rocio Ana Gonzalez-Vaquero (e-mail: rocioagv@yahoo.com).
Background: The bee genera Corynura and Halictillus (Hymenoptera:
Halictidae) contain species that are very abundant in Chile and Argen-
tinean Patagonia, and which are key elements in southern South
American ecosystems. For instance, C. chloris is among the major pol-
linators of crops such as raspberry, Chilean hazel, and buckwheat as
well as native wildflowers. These bee species are very difficult to iden-
tify due to close morphological similarity among species and extreme
sexual dimorphism. To assess this, we analyzed the barcodes of
15 species of Corynura, as part of a revision of the genus, as well as four
species of Halictillus.Results: We obtained 170 barcode-compliant se-
quences. Barcodes were useful to confirm gender associations and to
detect two cryptic species previously considered as one. The genera
showed a median interspecific distance (13.5%–14.3%), considerably
higher than among other closely related halictid genera. The se-
quence divergence between specimens of the same species was up to
0.9%, with a few exceptions. The barcodes of five specimens differed
from the rest by more than 2%, although these individuals were not
morphologically different from the others, nor collected in distant
areas. In contrast, C. patagonica showed a distance of 4.2% when any of
the specimens from Chile was compared to those from Argentina. The
only morphological difference found between the barcode clusters
was the colour of the metasomal terga of the female. BIN analysis was
useful to identify the species that showed high intraspecific variation.
Significance: Species delimitation and identification is particularly im-
portant in halictine bees, which are renowned for being morphologically
monotonous. The results suggest that Corynura and Halictillus species can
be identified through DNA barcodes, although some species showed a
high intraspecific variation which requires further study. We conclude
that C. patagonica is probably two cryptic species.
DNA barcoding reveals a possible cryptic species complex of
Mycalesis mineus: a case study from Sri Lanka
K.S. Goonesekera,
1
G. van der Poorten,
2
P.L.M. Lee,
3
and G.R. Ranawaka
1
1Department of Zoology, Open University of Sri Lanka, Nawala, Nugegoda, Sri Lanka.
2Ministry of Environment, Biodiversity Secretariat of Sri Lanka.
3School of Life and Environmental Sciences, Deakin University, Melbourne, Australia.
Corresponding author: Kshanika Goonesekera (e-mail: kshanika@outlook.com).
Background: The lepidopteran genus Mycalesis consists of over a
hundred species spread throughout the Oriental and Australasian
regions. Of these, M. perseus,M. patnia,M. mineus,M. subdita, and
M. rama are found in Sri Lanka, with the latter two being endemic to
the island. The Sri Lankan populations of M. perseus,M. mineus, and
M. patnia are closely related to those of the Oriental region but are
divergent enough to support subspecies status as M. perseus typhlus,
M. mineus polydecta, and M. patnia patnia, respectively. The aim of
this study was to investigate whether DNA barcoding can achieve
unambiguous species identification and delineation of Mycalesis
species in Sri Lanka. This group was selected for DNA barcoding
analysis because these species display close morphological similar-
ity with each other and also contain examples of high morpholog-
ical variation within species. Results: We analysed the genetic
divergence in the cytochrome c oxidase subunit I (COI) gene of
M. perseus,M. patnia,M. mineus, and M. subdita in Sri Lanka, supple-
mented with sequence data from GenBank. It was possible to un-
ambiguously distinguish M. perseus and M. patina from the M. mineus
and M. subdita cluster in neighbour-joining, maximum likelihood,
and Bayesian tree analyses. Sri Lankan M. mineus and M. subdita
appear relatively closely related, while the regional mineus group
formed a separate cluster from the Sri Lankan M. mineus with strong
bootstrap support (>90%). These clear barcode clusters may provide
evidence for a possible cryptic species complex within the cur-
rently recognised M. mineus.Significance: These barcode results
provide evidence for the presence of a genetically diverged
M. mineus population in Sri Lanka and highlight the necessity for
detailed morphological and ecological investigations to reveal any
overlooked species within the Mycalesis subspecies present in the
island.
The application of next-generation sequencing barcoding in
identifying mixed-pollen samples from a historic bee collection
Annemarie Gous,
1
Jurgens J. de Bruin,
1
Sandi Willows-Munro,
2
Connal Eardley,
3
and Dirk Swanevelder
1
1Biotechnology Platform, Agricultural Research Council, 1134 Park Street, Hatfield, Pretoria, South Africa.
2School of Life Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa Life Sciences
Campus, Carbus Road, Scottsville, Pietermaritzburg, South Africa.
3Plant Protection Research Institute, Agricultural Research Council, 1134 Park Street, Hatfield, Pretoria,
South Africa.
Corresponding author: Annemarie Gous (e-mail: gousa@arc.agric.za).
Background: Increasingly, studies are employing DNA barcoding and
next-generation sequencing (NGS) approaches to identify different
organisms in environmental samples. However, this approach in pol-
lination biology is still lacking, with available barcoding data only
assessing fresh pollen. NGS provides an easier, faster way to generate
large volumes of data on pollen sampled directly from bees, eliminat-
ing the need for separating the pollen grains by taxon prior to se-
quencing. In this study, DNA barcoding is combined with Illumina
NGS to provide taxonomic classification for pollen sampled from one
species of indigenous, solitary bee that was collected across South
Africa over a 93-year period. Three genomic regions were studied: the
internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the rbcL
gene. A bioinformatic workflow using custom-made databases for the
entire internal transcribed spacer region and rbcL was developed.
Results: In total, 22 mixed-origin pollen samples were assessed. Samples
represent eight decades, spanning over 90 years, of bee collection. Sam-
ples from as far back as 1910 were successfully sequenced and classified.
Species-level delimitation of pollen was possible for all genomic regions,
with higher confidence at family level. Significance: This is the first time
that samples from a historic insect collection have been used in studying
pollen origins using NGS and DNA barcoding techniques. This approach
provides a historical perspective on how floral choice in indigenous bees
changed over time and insights into the effects that land use and climate
change have on bee–plant interactions in regions with high levels of
oligolectic bees species.
Integrative approach and molecular barcoding of dagger
and needle nematodes infesting grapevine soils in Portugal
Carlos Gutiérrez Gutiérrez,
1
Maria Antonia Bravo,
2
Margarida Teixeira Santos,
2
Paulo Vieira,
1
and Manuel Mota
1
1NemaLab/ICAAM-Instituto de Ciências Agrárias e Ambientais Mediterrânicas; Departamento de
Biologia, Universidade de Évora Núcleo da Mitra, Ap. 94, 7006-554 Évora, Portugal.
2Instituto Nacional de Investigação Agrária e Veterinária I.P. Quinta do Marquês, Av. da República,
2780-159 Oeiras, Portugal.
Corresponding author: Carlos Gutiérrez Gutiérrez (e-mail: carlosg@uevora.pt).
Background: Dagger (Xiphinema spp.) and needle (Longidorus spp.,
Paralongidorus spp.) nematodes are two of the main groups of parasitic
nematodes in grapevines worldwide, causing severe damage to plants
by their direct feeding; in addition, some species may transmit plant
viruses. Some of these nematode species are included in the list of
quarantine organisms in many countries. Grapevine fanleaf virus
(GFLV) is specifically transmitted by Xiphinema index, and it is one of
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the most harmful viruses to vineyards wordwide. Despite their phyto-
pathological importance, this research area has been deserted for over
fifteen years in Portugal. In recent years, plant health of the Portu-
guese vineyards has declined, characterized mainly by reduced vigor
in plants. Thus, the main objective is to determine the ocurrence and
distribution of longidorids infesting vineyards from Portugal. Nema-
tode surveys have been conducted since 2015 on 30 commercial vine-
yards of the main Controlled Denomination of Origin (DOC) zones of
Portugal. Results: The use of an integrative strategy, based on the
combination of morphometric and morphological characterizations
with molecular analysis using barcode regions such as the ribosomal
DNA segments (rDNA) (D2–D3 regions of the 28S gene, ITS1 region, and
18S gene), has allowed the identification of collected longidorid spe-
cies, associated with severe infestations found in grapevine soils in the
studied DOC areas. We emphasize the successful identification and
detection of Xiphinema index due to its phytopathological importance.
The most important longidorid nematodes detected, in order of de-
creasing frequency of total soil infestation, were Xiphinema pachtaicum,
X. index,X. santos,Xiphinema sp., and X. italiae.Significance: Our study
highlights the validity of using an integrative approach based on the
combination of morphological data and molecular barcodes for the
correct and timely identification of this group of nematode species
characterized by high morphological similarity and phenotypic plas-
ticity. The high prevalence of X. index makes this species a severe
threat to grapevine production in Portugal.
Potato cyst nematodes infesting potato fields in Ecuador:
integrative diagnosis and molecular phylogeny
Carlos Gutiérrez Gutiérrez,
1
Pablo Llumiquinga,
2
Patricio Gallegos,
2
Katherine Orbe,
3
Néstor Castillo,
2
Jennifer Ulloa,
4
Carlos Guanotásig,
4
Wladimir Enriquez,
4
and Karina Proaño
4
1PROMETEO, INIAP - Instituto Nacional Autonómico de Investigaciones Agrarias, Estación
Experimental de Santa Catalina, Panamericana Sur s/n, Machachi, Ecuador; and NemaLab/ICAAM-
Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Biologia, Universidade
de Évora, Núcleo da Mitra, Ap. 94, 7006-554 Évora, Portugal.
2INIAP - Instituto Nacional Autonómico de Investigaciones Agrarias, Estación Experimental de Santa
Catalina, Departamento de Protección de Cultivos, Panamericana Sur s/n, Machachi, Ecuador.
3INIAP - Instituto Nacional Autonómico de Investigaciones Agrarias, Estación Experimental de Santa
Catalina, Departamento de Biotecnología, Panamericana Sur s/n, Machachi, Ecuador.
4Universidad de las Fuerzas Armadas, Departamento de Biotecnología Campus Central, Sangolquí,
Pichincha, Ecuador.
Corresponding author: Carlos Gutiérrez Gutiérrez (e-mail: carlosg@uevora.pt).
Background: Potato cyst nematodes (PCN), Globodera rostochiensis and
G. pallida, are serious pests of potatoes worldwide, with some species
included in the quarantine lists of many countries. Their accurate and
timely identification is a prerequisite in designing effective management
strategies. In Ecuador, potato is one of the main crops, and PCN represent
an important pest. This situation led to a national survey where a total of
85 soil samples were collected in 2013 from the major potato-growing
regions. The main objectives were to determine the prevalence of PCN
and to establish a method for the integrative diagnosis of species based
on the combination of morphological data with molecular analysis using
rRNA regions. PCR-based techniques for species-specific amplification of
the ITS region and for sequencing the D2–D3 expansion segments of the
28S rRNA gene were also developed. Results: Globodera pallida was the
only PCN species found in the potato fields, but it was widespread (55%
frequency of infestation). Moderate to high G. pallida population densities
were found. The results from the molecular methods were congruent
with the morphological data. Interspecific divergence of the D2–D3 re-
gion of PCN is low, and it is not possible to discriminate between G. pallida
and G. rostrochiensis based upon this molecular region, but it is useful for
the distinction of other species. Phylogenetic analysis yielded two major
well-defined and supported clades, where Ecuadorian sequences
grouped with sequences of G. pallida and G. rostochiensis populations
deposited in the GenBank database. Significance: The prevalence of
G. pallida makes it a severe threat to potato production in Ecuador. More-
over, management practices such as the short term of crop rotations and
the use of the “super-chola” susceptible cultivar could be worsening the
problem. A strategy based on the integration of morphological data and
molecular analysis is useful for identifying PCN species. In addition,
other molecular barcodes are currently being characterized (ITS-rRNA,
COI-mitDNA).
Recovery of nucleic acids from microhymenopterans with four
non-destructive methodologies and considerations for museum
slides preparations
Adriana Guzmán-Larralde,
1
Alba Suaste-Dzul,
2
Adrien Gallou,
2
and Alejandro González-Hernández
3
1UANL, Cerro Real 664, Valle Real, Saltillo, C.P. 21205 Coahuila, Mexico.
2CNRCB, Km 1.5 Carretera Tecoma-Estacion SSCC C.P. 28110, col Tepeyac, Tecoman, Colima, Mexico.
3UANL, Manuel L. Barragán, Ciudad Universitaria, San Nicolás de Los Garza, N.L., Mexico.
Corresponding author: Adriana Guzman-Larralde (e-mail: adri_guzman@yahoo.com).
Background: Microhymenopterans are used in biological control
programs of insect pests as an ecological alternative to chemical tox-
icants. These minute wasps require particular protocols and skills for
preparing the slides that allow a correct taxonomic identification.
DNA barcoding offers taxonomists the opportunity to improve the
identification of tiny hymenopterans. The molecular approach may
be of better use if, after DNA extraction, permanent slides of such
specimens are prepared for comparative analyses with taxonomic
keys. We evaluated four non-destructive methods for DNA extrac-
tion: (a) DNeasy Blood & Tissue Kit, (b) Protocol with CaCl
2
buffer,
(c) Method HotSHOT, and (d) Phire Tissue Direct PCR master mix. Using
PCR to amplify a ⬃680-bp sequence of the mitochondrial gene cyto-
chrome c oxidase subunit I, we demonstrated the efficiency of these
methods on insects maintained dry or in ethanol along a time course
of conservation (i.e., 23 years, 12 years, 3 years, 4 months, 1 day).
Results: Two techniques (a,b) yielded DNA extracts that were success-
fully PCR amplified for all samples, while technique (c) amplified the
last four samples in a faster, cheaper, and easier way than (a) and (b).
The last technique (d) amplified only the two more recent samples, but
it was the fastest one that did not produce non-specific PCR products,
as detected by observing multiple bands on an agarose gel. Moreover,
we adapted the traditional methodology of permanent slides prepara-
tion in Canada balsam for every technique after DNA extraction, in-
cluding the discoloration step. Significance: The results reported
here allow combining the utilization of classic and molecular biology
methodologies for taxonomic studies of microhymenopterans. The
most significant result was the recovery of DNA from 23-year-old in-
sects, allowing the description of additional biological traits for old
museum specimens.
Evolving the concept, and use, of DNA barcode libraries
Rodger Gwiazdowski
1
and Robert Hanner
2
1University of Massachusetts, Amherst ng, Amherst, MA 01003, USA.
2Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Robert Hanner (e-mail: rhanner@uoguelph.ca).
Background: DNA barcode library concepts and construction meth-
ods are diverse. As a result, they stand as important, but isolated
resources toward a coordinated mapping effort for a first draft of
eukaryotic life using DNA barcodes. In particular, annotating the bio-
logical and community relevance of DNA-barcoded specimens for
comparative identification in a reference library is a current frontier
for the use of DNA barcode data. Results & Significance: Global con-
sortium science methods have arisen as efficient ways to coordinate
annotation and knowledge development of large-scale genomic and
microbial data. These platforms stand as comparative templates to
advance eukaryotic biodiversity informatics via DNA barcodes. This
poster will present the structural elements of successful consortium
science methods toward promoting discussion of the complimentary
use of public, common-source DNA barcode libraries for identification
and discovery.
Abstracts 223
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DNA barcoding helps to fight against frauds—a case study of
authentication of deer products
Wai-yan Ha and Foo-wing Lee
7/F, Government Offices, 88 Chung Hau St., Homantin, Kowloon, Hong Kong SAR.
Corresponding author: Wai-yan Ha (e-mail: wyha@govtlab.gov.hk).
DNA-based methods for authenticity testing have been useful for identi-
fying the species origin of traded goods where the morphological char-
acteristics are frequently lost in the processed form. Deer are valuable
animals, as many of their body parts including velvet antler and tendons
are commonly consumed in tonics. There are dishonest traders tempted
to sell substitutes of deer products for increased profits. To provide sci-
entific evidence for identifying fraudulent material, an integrated DNA
protocol combining DNA barcode analysis and two deer-specific PCR sys-
tems was designed. The method was validated using different body parts
and processed products of deer, namely the velvet antler, ossified antler,
tendon, and also proprietary Chinese Medicines. The samples were first
tested with both deer-specific cytochrome b (cytb) and 16S ribosomal RNA
(16S) to authenticate the presence of deer substance. DNA barcoding of
cytochrome c oxidase I (COI) was then used to affirm the identity for
those samples that were found not to be deer. In summary, this method
provides a fast and accurate way to authenticate deer products.
Environmental DNA barcoding: from the Arctic to the tropics—
and everywhere in between
Mehrdad Hajibabaei
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: mhajibab@uoguelph.ca.
DNA barcoding has induced a paradigm shift in various branches of
biological sciences where accurate and timely specimen identification is
required. Minimalism and standardization—the main properties of DNA
barcoding—have elevated the utility of DNA barcoding from an artisanal
research tool to a global big data system for various research or societal
applications. Advances in high-throughput sequencing (HTS) and compu-
tational analysis have widened the scope of DNA barcoding studies. Here,
I will focus on the use of DNA barcode data in environmental biomoni-
toring programs aiming to assess ecosystem health. A fundamental ques-
tion in ecological analysis is: how to obtain consistently observed, high-
resolution biodiversity information. We have developed and used
environmental (meta) barcoding approaches using standard DNA bar-
code markers for animals, plants, fungi, and other eukaryotes, as well as
DNA markers for taxonomic and functional analysis of prokaryotes. So
far, environmental DNA (eDNA) data gathered from a wide range of
sites—from boreal to tropical forests and from various habitats—has
showcased the utility of this approach for ecological analyses. The avail-
ability of DNA barcode libraries allows for more robust taxonomic infer-
ence leading to richer biological information obtained from HTS data.
Importantly, our analyses show the utility of sequence-based operational
taxonomic units in deriving ecologically informative results even in the
absence of reference sequence libraries. Reduced cost and labour
achieved by the use of HTS, coupled with the biodiversity information
gained, will enable the development and implementation of ecological
monitoring programs with important scientific and societal implica-
tions.
DNA barcoding of Philippine Helicarionidae (Mollusca: Gastropoda)
Jordan Ferdin A. Halili,
1
Gizelle A. Batomalaque,
1
Ryan C. De Chavez,
2
Nengie Leander C. Figueras,
1
Benjamin O. Sosa, III,
1
and Ian Kendrich C. Fontanilla
1
1Institute of Biology, University of the Philippines, Diliman, Quezon City, the Philippines.
2Institute of Biological Sciences, University of the Philippines, Los Baños, the Philippines.
Corresponding author: Ian Kendrich C. Fontanilla (e-mail: ianfontanilla@hotmail.com).
Background: Helicarionidae is a very large and diverse family of pulmo-
nate land snails that includes about 150 genera and many hundred spe-
cies. In the Philippines, 8 out of the 80 endemic species of land snails
recorded belong to the family Helicarionidae. No molecular work has
been done regarding the phylogeny of Philippine helicarionids, and an
updated species list for this group is lacking. This study aimed to barcode
terrestrial snails belonging to the family Helicarionidae found in the
Philippines. Results: A total of nine species—namely Hemiglypta moussoni
(n= 2), Lepidotrichia luteofasciata (n= 1), Nipponochlamys semisericata (n= 1),
Ryssota uranus (n= 6), R. sagittifera batanica (n= 5), R. zeus (n= 6), and the
Philippine endemics Hemiglypta semperi (n= 3), R. otaheitana (n= 6), and R.
lamarckiana (n=4)—were barcoded using the standard marker cyto-
chrome c oxidase subunit I (COI) gene. A further two genera, Hemiglypta
sp. (n=3)andLepidotrichia sp. (n= 3), were also included. Helicarionid
individuals were obtained from various sites in the Philippines (Batanes,
Bicol, Laguna, Leyte, Marinduque, Mindoro, Romblon, and the Polillo
Group of Islands). The highest sequence divergence was found at the
family level (mean K2P distance=17.62%), and the least divergence oc-
curred at the species level (mean K2P distance=1.33%). Overlap was noted
between interspecific and intraspecific variation. A neighbour-joining
tree based on the Kimura-2-parameter model of DNA substitution re-
vealed most species to be monophyletic with the exception of R. otaheit-
ana; the species has been flagged for taxonomic re-evaluation.
Significance: This study provides the first molecular work on Helicari-
onidae in the Philippines, paving the way for further phylogenetic anal-
ysis of this economically important family of land snails.
DNA barcode-based wildlife forensics for resolving the origin of
claw samples
Rutuja Hange and Gulab Khedkar
Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Rautuja Hange (e-mail: raitujahange@gmail.com).
Background: Excessive wildlife hunting for commercial purposes can
have negative impacts on biodiversity and may result in species extinc-
tion. To ensure compliance with legal statutes, forensic identification
approaches relying on molecular markers may be used to identify the
species of origin of animal material including hairs, claw, blood, bone, or
meat. Using this approach, DNA sequences from the COI “barcoding”
gene have been used to identify material from a number of domesticated
animal species. However, many wild species of carnivores still present
great challenges in generating COI barcodes using standard “universal”
primer pairs. Results: In the work presented here, the mitochondrial COI
gene was successfully amplified using a novel primer cocktail, and the
products were sequenced to determine the species of twenty one un-
known samples of claw material collected as part of forensic wildlife case
investigations. Sixteen of the unknown samples were recognized to have
originated from either Panthera leo or P. pardus individuals. The remaining
five samples could be identified only to the family level due to the ab-
sence of reference animal sequences. Significance: This is the first report
on the use of COI sequences for the identification of P. pardus and P. leo
from claw samples as part of forensic investigations in India. The study
also highlights the need for adequate reference material to aid in the
resolution of suspected cases of illegal wildlife harvesting.
Barcoding the ants of Iguazú National Park, a biodiversity
hotspot in northeastern Argentina
Priscila E. Hanisch,
1
Pablo D. Lavinia,
2
Pablo L. Tubaro,
2
Andrew V. Suarez,
3
and Carolina I. Paris
4
1Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Av. Ángel Gallardo 470, Buenos
Aires, Argentina.
2Division Ornitologia, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Av. Ángel
Gallardo 470, Buenos Aires, Argentina.
3Department of Entomology, University of Illinois, Urbana, IL 61801, USA.
4Departamento Ecología, Genética y Evolución, Universidad de Buenos Aires, Ciudad Universitaria
Pabellón 2, Laboratory 28, Buenos Aires, Argentina.
Corresponding author: Priscila E. Hanisch (e-mail: phanisch@macn.gov.ar).
Background: Ants are a key component of terrestrial ecosystems be-
cause they provide several ecological services such as seed dispersal, or-
ganic matter decomposition, and soil nutrient cycling. Here, we assess,
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through the generation and analysis of DNA barcodes, the diversity of
ants of Iguazú National Park (INP) as part of a major project that aims to
barcode the ants of Argentina. The INP constitutes a biodiversity hotspot
located in northeastern Argentina with over 200 species of ants and
represents one of the biggest remnants of the Atlantic Forest, which is a
priority for conservation as it harbors 7% of world's diversity. Results: We
obtained 178 COI sequences from 332 individuals belonging to more than
100 species from over 30 genera. Unidentified species were not in-
cluded. The mean intraspecific sequence divergence was slightly over
0.70%, which was 23 times lower than the mean interspecific diver-
gence (16.6%). After removing a few species with strikingly deep intras-
pecific divergence, the mean intraspecific divergence fell to 0.30%.
There were more BINs (73) than species (67), with no BIN being shared
by different species. Seven species showed two clearly differentiated
barcode clusters, suggesting the possible presence of cryptic biodiver-
sity at INP. The existence of these divergent intraspecific lineages was
supported by high node support values in complementary phyloge-
netic analyses. Significance: Taxonomic keys for Neotropical ants are
mostly incomplete and based almost exclusively on workers, preclud-
ing the identification of males and queens of most species. Increasing
the reference barcode library for the ants of INP (and Argentina) will
certainly help to identify these castes and to register morphological
variability of Neotropical ants, which may eventually lead to the gen-
eration of new taxonomic keys. Lastly, our results support the biodi-
versity hotspot status of the Atlantic Forest and suggest that ant
diversity in INP is currently being underestimated.
Deep intraspecific barcode splits: cryptic species, Wolbachia or
something else?
Jonna Hänninen and Marko Mutanen
University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland.
Corresponding author: Marko Mutanen (e-mail: marko.mutanen@oulu.fi).
Background: Many studies of DNA barcodes have revealed extraordi-
nary variation in mitochondrial DNA in some species. Sometimes this
variation is highly clustered so that a species shows a deep intraspe-
cific split in mtDNA with no intermediates. While building a complete
reference library for ⬃2600 species of Finnish Lepidoptera, dozens of
such deep splits were detected. In order to investigate the presence of
cryptic species, we sequenced six taxonomically informative nuclear
genes (EF-1a,MDH,CAF,IDH,RpS5, and wingless) in 29 lepidopteran
species, each having a deep (>2%) sympatric intraspecific split in the
DNA barcode region. Results: The results suggest that some, but only
a minority of splits, are due to the presence of a previously undetected
morphologically cryptic species. Some cases may be attributed to his-
torical polymorphism. Several splits have likely resulted from intro-
gression, i.e., transfer of the mitochondrial genome from one species
to another. Wolbachia infection was detected in many cases, and some
splits may be explained by Wolbachia-driven spread of alien mitochon-
drial haplotypes in the population after an introgression event. Sig-
nificance: Overall, our results suggest that DNA barcodes have great
potential in revealing new species even among well-investigated
groups and areas. We also show that other biological processes may
result in mitochondrial polymorphisms that may occasionally impede
the usefulness of DNA barcodes in species identification.
Balancing sensitivity and specificity in primer design for eDNA
studies using ePRIMER
Daniel Harvey,
1,2
Sanjeena Subedi,
1
Robert Hanner,
2
and Sarah J. Adamowicz
2
1Department of Mathematics and Statistics, University of Guelph 50 Stone Road East, Guelph, ON N1G
2W1, Canada.
2Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Sarah J. Adamowicz (e-mail: sadamowi@uoguelph.ca).
Background: The detection of species-specific environmental DNA
(eDNA) via real-time polymerase chain reaction (qPCR) is emerging as
an important monitoring technique for rare and invasive species. To
be effective, a qPCR assay must be sensitive enough to detect the small
amounts of target DNA that may be present in the environment, but
specific enough to avoid detection of non-target DNA. We developed a
program (ePRIMER) that uses one or more target sequences and one or
more non-target sequences to produce a list of potential primers
ranked by relative efficiency and specificity, allowing the user to se-
lect a primer set with a desirable balance between the two. To illus-
trate the utility of ePRIMER, we designed and tested an eDNA
detection assay for the watersnake (Nerodia sipedon) in Ontario, with
the queensnake (Regina septemvittata) as a non-target species. Results:
Among 11 226 potential primer and probe sets produced by ePRIMER,
the most specific (with 11 base-pair mismatches between species) was
the 7572nd most efficient. Using tissue-derived watersnake DNA (with
initial quantities ranging from 7.1 × 10
−4
to 20 ng) and the most specific
primer and probe set, the efficiency of qPCR (as determined by 10-fold
serial dilution tests) was 68%. The assay did not amplify queensnake
DNA. Significance: Our study demonstrates that sensitivity and spec-
ificity may require a trade-off in primer design, and with ePRIMER we
provide a platform for assessment of this trade-off in the development
of qPCR assays for eDNA detection.
Calibrating the taxonomy of a megadiverse family on BOLD: 2700
geometrid moth types barcoded (Geometridae, Lepidoptera)
Axel Hausmann,
1
Scott E. Miller,
2
and Sean Prosser
3
1Bavarian State Collection of Zoology, Munich Muenchhausenstr. 21, 81247 Munich, Germany.
2Smithsonian Institution, P.O. Box 37012, MRC 105, Washington, DC 20013-7012, USA.
3Biodiversity Institute of Ontario, Canadian Centre for DNA Barcoding, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Axel Hausmann (e-mail: Axel.Hausmann@zsm.mwn.de).
Background: One of the major challenges in creating a global data-
base like BOLD is warranting the correct identification of the voucher
specimens. The strict BOLD policy to require images, indication of
specimen deposition, and accurate geo-referencing for all submitted
datasets is extremely helpful to control doubtful data and potential
misidentifications. Nevertheless, there are still many incomplete
identifications (to genus or subfamily level), interim names, or even
misidentifications on BOLD, mainly for species from tropical regions.
Unfortunately, experts are lacking for many problematic groups and
regions, and even when there are experts, they usually are not avail-
able for correcting the taxonomy of large amounts of data due to time
constraints. Results: The best way to reliably calibrate the system is to
barcode the original type specimens. In recent years, the challenge of
sequencing up to 250-year-old museum specimens has been overcome
by improved techniques and protocols developed by the Canadian
Centre for DNA Barcoding. These innovations allowed for the genera-
tion of barcode sequences for ⬃2700 geometrid type specimens,
which represent 2150 species corresponding to about 9% of the 23 000
described species worldwide. Significance: Here, we present case
studies to show the efficiency, reliability, and sustainability of this
approach as well as promising strategies to complete the calibration
of the reference library within a reasonable amount of time.
Using DNA metabarcoding to investigate the medicinal
properties of honey
Jennifer Hawkins,
1
Natasha de Vere,
2
Col R. Ford,
3
Matthew Hegarty,
4
and Les Baillie
5
1National Botanic Garden of Wales and School of Pharmacy and Pharmaceutical Sciences Llanarthne,
Carmarthenshire, SA32 8HG, UK and Cardiff University, Cardiff, UK.
2National Botanic Garden of Wales and Institute of Biological, Environmental and Rural Sciences,
Llanarthne, Carmarthenshire, SA32 8HG, UK and Aberystwyth University, Aberystwyth, UK.
3National Botanic Garden of Wales, Llanarthne, Carmarthenshire, SA32 8HG, UK.
4Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, UK.
5School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK.
Corresponding author: Jennifer Hawkins (e-mail: jhawkins24000@gmail.com).
Background: Honey possesses therapeutic properties that are the result
of a range of factors including high sugar content, low pH, hydrogen
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peroxide, and bee-derived peptides. Honey also contains antimicrobial
phytochemicals, which represent a rich source of leads for the develop-
ment of drugs for the treatment of microbial infections. Honey samples
donated by UK beekeepers (n= 217) and Manuka samples (n= 3) were
screened for the presence of novel antibacterial compounds by determin-
ing activity against methicillin-resistant Staphylococcus aureus (MRSA). The
majority (88%) of the honeys showed inhibitory activity; two of these
samples were found to contain potentially novel antibacterial com-
pounds, which were likely to be plant-derived. The pollen present in
honey represents a record of the plants that contributed to the making of
the honey and may be the source of specific antibacterial factors. For this
reason, pollen was extracted and characterised from honey samples us-
ing microscopic analysis and rbcL DNA metabarcoding (454 and Illu-
mina). Results: DNA metabarcoding provided superior discrimination
for some plant families and greater repeatability compared to micro-
scopic analysis. Key species identified in the antibacterial samples in-
cluded woodruff (Galium odoratum), bluebell (Hyacinthoides non-scripta),
and dandelion (Taraxacum officinale). Extracts from active honeys and
characterised plants demonstrated antibacterial activity against
MRSA, E. coli, and P. aeruginosa. Activity-guided characterisation using
a thin layer chromatography/mass spectrometry interface highlighted
the presence of some known and some novel compounds. Signifi-
cance: Understanding the floral composition of honey has a wide
variety of applications. DNA metabarcoding pollen extracted from
honey allows us to identify the plant species that may be contributing
to the antimicrobial effect of honey. The demonstration of antibacte-
rial activity may provide new lead compounds that could serve as
selective agents against MRSA and other antibiotic-resistant bacteria.
From iBOL to planetary biodiversity
Paul D.N. Hebert
Biodiversity Institute, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: phebert@uoguelph.ca.
Over the past decade, the DNA barcode research community has begun
to address the great uncertainties in biodiversity science. It has also
shown its capacity to lead a major research enterprise; the International
Barcode of Life (iBOL) project will achieve its primary goal of assembling
a DNA barcode reference library for 500 000 species by late 2015. This
work has confirmed the power of DNA-assisted taxonomy, and it has
helped to clarify and address the challenges in developing a DNA-based
identification system for all eukaryotes. The 1 500 000 barcodes now
available for 75 000 species of Canadian organisms provide a rather clear
preview of the task ahead. By exposing massive numbers of undescribed
taxa, the work in Canada has reinforced the need to automate taxonomic
placements by coupling DNA barcodes with expert systems. As we look to
the future, iBOL should be viewed as the foundational element for a
mega-science project that will deliver a comprehensive understanding of
global biodiversity by 2040. The next four years can be used to identify
and resolve the diverse barriers to activation of a $2B Planetary Biodiver-
sity Project (PBP). The PBP should focus on terrestrial and freshwater
species, as they are most at risk, but serious studies should also probe the
oceans. It will resolve the longstanding uncertainty surrounding the
number of species on our planet in a taxonomic heartbeat. Among its
many byproducts, the PBP will reveal species ages, their distributions,
and the factors modulating their diversification. However, these ad-
vances are only the first step in addressing the big challenge for our
discipline—developing the capacity to track the dynamics of all species
with precision.
The San Diego Biodiversity Project
Heather Henter, Madeline Butler, Stephanie Mel, and Joshua Kohn
University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093 0116, USA.
Corresponding author: Heather Henter (e-mail: hhenter@ucsd.edu).
Background: The San Diego Biodiversity Project is an effort to incor-
porate original research into the college biology curriculum through
the use of DNA barcoding. Undergraduate students at UC San Diego
are creating an inventory of invertebrate biodiversity at an ecological
reserve adjacent to campus. Ecology and molecular biology students
in large-enrollment laboratory classes collaborate to collect, docu-
ment, and generate DNA sequence data. Recently, we have scaled up
to include minority-serving community colleges in the region. To
make this possible, staff at the Biodiversity Institute of Ontario have
redesigned the BOLD Student Data Portal to cater to the university
classroom. Most importantly, the new University Student Data Portal
(uni.boldsystems.org) will facilitate sharing data across different class-
rooms and campuses. Results: In 2.5 years, over 1700 undergraduate
students have participated, successfully generating BOLD-compliant
consensus sequences for 67% of the 727 specimens that have been
collected. For certain taxa, students collect many species that are new
to BOLD; for example, 31% of the spider individuals are new additions
to the database. We have assessment data that documents student
perceptions of original research in the classroom and the effect of such
research on student self-efficacy and science identity. Significance: The
benefits of incorporating original research into the classroom are well
established in the science education literature. DNA barcoding, in
addition, helps students comprehend the biodiversity knowledge gap.
An undergraduate describes her reaction to the research this way, “I
think it did affect (me) because for whatever reason I assumed that all
the species in the near vicinity had already been discovered and se-
quenced and everything was all in place, like there were people doing
this. But then with the barcoding experiment you realize that there are a lot
more species out there that can be studied…”
Diversity, species delimitation, and evolution of insect viruses
Elisabeth Herniou,
1
Julien Thézé,
2
Jennifer Cory,
3
and Carlos Lopez Vaamonde
4
1CNRS - Insect Biology Research Institute UMR CNRS 7261, Université François Rableais de Tours,
France.
2Department of Zoology, University of Oxford, Oxford, UK.
3Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
4INRA UR633 Zoologie Forestière, Orléans, France.
Corresponding author: Elisabeth Herniou (e-mail: elisabeth.herniou@univ-tours.fr).
Background: Viruses can have major health, economic, and ecologi-
cal impacts, yet their diversity remains largely unexplored. To assess
viral diversity, though, one first needs to define what viral species are.
Viral taxonomy faces important challenges such as the propensity of
viruses to recombine and show fast evolutionary rates. Species delim-
itation criteria in viruses are usually based on genetic divergence and
host use. Here we use a phylogenetic clustering approach for species
delimitation. We then used a combination of phylogenetic and eco-
logical data to investigate the main ecological factors driving the di-
versification of baculoviruses (insect-specific large DNA viruses of the
family Baculoviridae) that mainly attack the insect order Lepidoptera.
Results: We generated a molecular phylogeny for ⬃500 viral isolates,
using four essential core genes (polh,lef-8,lef-9,pif-2). Among the bac-
ulovirus isolates, 88 have complete genomes and were used to recon-
struct a robust whole-genome backbone tree for the phylogeny. We
used GMYC and PTP methods to delimit without a priori 150 clusters,
including 106 new species. This approach recovered all known species
and allowed the unification in single taxa of isolates labeled under
different names. Comparative analyses revealed high levels of host
specificity and phylogenetic conservatism in Baculoviridae. Moreover,
the colonization of a new ecological niche (type of plant growth asso-
ciated with insect host) has in some cases led to baculovirus radiation.
Significance: The International Committee on Taxonomy of Viruses
recognizes currently 44 species in the Baculoviridae. Our study shows
that a set of four core genes could be used as DNA barcodes for the
family Baculoviridae from which we could identify 150 candidate spe-
cies. This study is the first to use a phylogenetic clustering approach,
inspired by DNA barcoding practices in Metazoa, for species delimita-
tion in viruses. Our results show that phylogenetic clustering could be
used to unify viral taxonomy.
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Barcodes to investigate the plant visitor community:
Brassicogethes spp.
Damien Hicks,
1
Graham Stone,
1
and Pierre Ouvrard
2
1School of Biological Sciences, University of Edinburgh, room 4.05 Ashworth Laboratories, Charlotte
Auerbach Road, Edinburgh, EH9 3FL.
2Earth and Life Institute, Université catholique de Louvain Bâtiment de Serres, Local B 331, Place Croix
du Sud 2 bte L7.05.14, B-1348 Louvain-la-Neuve, Belgium.
Corresponding author: Pierre Ouvrard (e-mail: pierre.ouvrard1@gmail.com).
Background: We explored structure within DNA barcode data col-
lected from pollen beetles, one of the commonest groups in a large
plant-pollinator project with sampling across the UK. We addressed
three questions: (1) Can we use barcodes to identify Brassicogethes pol-
len beetles? (2) Is the taxonomy of the genus supported by clustering
patterns in the barcode data? (3) Can barcodes provide within-species
indications of geographic origins? Results: 1. Our beetle specimens
were largely in a clade containing GenBank-identified specimens of
B. aeneus, with the second commonest species being B. viridescens. Other
individuals were placed in more divergent lineages that in some cases
could be matched with GenBank-identified specimens of Brassicogethes
species, but in other cases did not match closely enough (<95% BLAST
sequence similarity) to any identified specimen. 2. Individuals identi-
fied as species thought to be host plant-specific were found on a wide
range of plant species. Adults of B. aeneus and B. viridescens are associ-
ated with a range of plant taxa outside the Brassicaceae, to the extent
that we found no partitioning of Meligethes sensu lato sequences by
plant family using AMOVA. 3. The barcodes suggest a north–south
divide of B. aeneus in the UK, a pattern also observed with extended
sampling across Europe. Northern UK B. aeneus populations included
individuals from haplotype clades of northern Europe, whereas those
from the southern UK have haplotypes that cluster with central and
southern Europe. A similar pattern was observed for B. viridescens.
Significance: 1. There is apparently a good deal more species-level
diversity in this group than was either previously recognized or pre-
viously included in DNA barcoding surveys. 2. Barcode data combined
with host plant data provide an alternative, perhaps more reliable,
view of the taxonomy and ecology of this group. 3. An assessment of
spatial structure within MOTUs/species, and its implications for de-
limiting species with barcodes, would benefit from more extensive
sampling.
Arctic biodiversity—from barcodes to species interactions
David Hik
Biological Sciences, University of Alberta, Edmonton, AB T5N 0R5, Canada.
E-mail for correspondence: dhik@ualberta.ca.
The 2013 Arctic Biodiversity Assessment (ABA) has provided the most
comprehensive overview of the state of Arctic biodiversity. Arctic spe-
cies still have access to large areas of habitat that support diverse
ecological processes and interactions. However, rapid climate change,
industrial development, pollution, local disturbances and invasive
alien species are affecting the Arctic, and their impacts are increasing.
Warming temperatures and changes in precipitation are likely to give
rise to very different climatic conditions in the Arctic, seasonally and
across years. Indeed, the distribution of flora and fauna is shifting
northwards as the Arctic continues to warm. The ABA calls for im-
proved monitoring and research to survey, map, monitor and under-
stand Arctic biodiversity. This requires integrated and repeated data
collection following recommended standardized protocols and prior-
ities, and involving Arctic citizens in the survey and monitoring. Sup-
port for national and international coordinated efforts such as the
Circumpolar Biodiversity Monitoring Program (CBMP) and the Bar-
code of Life is essential to fill critical data gaps about Arctic terrestrial,
freshwater and marine species distributions, population abundances
and trends, and changes in the functioning and services of Arctic
ecosystems. Barcoding in particular has the potential to resolve local
species diversity and the potential for adaption to specific environ-
mental conditions. For example, the identification of a new subspe-
cies of Arctic moth (Gynaephora groenlandica beringiana) in the southern
Yukon, formerly known only from the high Arctic, has led to unex-
pected discoveries of facultative species interactions in these cold,
seasonal environments. These southern populations of moths are sub-
jected to different environmental pressures that can translate into
localized ecological, physiological and behavioural adaptations affect-
ing other tundra herbviores and plant communities. Species interac-
tions in Arctic food webs are not well documented, and understanding
the changing dynamics of these systems will depend on more exten-
sive documentation of species diversity.
Impacts of anthropogenic disturbance on arthropod
biodiversity and community structure
Chris Ho and M. Alex Smith
Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Chris Ho (e-mail: hoc@uoguelph.ca).
Background: Protected areas in Ontario preserve cultural and natural
features, maintain biodiversity, and provide economic opportunities
(such as forestry). Balancing conservation and economic usage ex-
poses parks to numerous kinds of disturbance. Arthropods are a major
component of forest biodiversity and are critical to nutrient cycling
and transfer. Certain arthropods, such as spiders, are sensitive to
changes in environmental conditions and could be used as ecological
indicators to monitor the effects of disturbance. However, assessment
of these indicators is hindered by the “taxonomic impediment” stem-
ming from both the difficulty in obtaining accurate species-level iden-
tifications and the lack of taxonomists able to identify these
organisms. We aimed to compare the differences in diversity and
community structure between sites with different disturbance histo-
ries in Algonquin Provincial Park. To alleviate the lack of taxonomic
expertise of hyper-diverse groups, we used Barcode Index Numbers
(BINs) derived from DNA barcodes as proxies for species. If niche avail-
ability influences arthropod communities and biodiversity in mixed-
boreal forests, and if forestry decreases niche availability, then we
expect to observe a decrease in diversity and a shift from arboreal to
litter-dwelling communities in harvested sites. Results: Preliminary
data shows that, contrary to our prediction, the phylogenetic struc-
ture of spider diversity in Algonquin Provincial Park displayed non-
significant clustering in uncut sites and overdispersion in cut sites.
Hunting spider diversity was greater in the cut sites (compared to
uncut), with no significant difference in species diversity between
treatments in weaver spiders. However, weaver spider species compo-
sition changed depending on the treatment, with 27 species unique to
the uncut treatment, 26 species unique to the cut sites, and 33 cosmo-
politan species. Significance: Arthropods play an important role in
regulating herbivore abundance and the rate of litter decay. Utilizing
DNA barcodes for phylogenetic analyses of diversity and community
structure, we can assess the impact of anthropogenic disturbance on
arthropod diversity in protected areas.
The place of barcoding in biodiversity informatics
Donald Hobern
Global Biodiversity Information Facility, Universitetsparken 15, 2100 København Ø, Denmark.
E-mail for correspondence: dhobern@gbif.org.
Background: Modern society and global science increasingly rely on
integration of massive quantities of data to answer questions and
guide planning. Many areas of human activity impact and are im-
pacted by biodiversity, yet it remains difficult to address these impacts
within models. A significant proportion of human knowledge of the
world's biota remains locked up in printed formats or distributed
among the world's natural history collections. Results: A range of
international biodiversity informatics organizations are working to
deliver digital representations of this historical knowledge and to
integrate it with contemporary data streams. The Global Biodiversity
Information Facility (GBIF, http://www.gbif.org/) focuses on standard-
ized evidence for the recorded occurrence of any species at any time
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and place. The Catalogue of Life (CoL, http://catalogueoflife.org/) seeks
to construct a comprehensive catalogue of described species and their
names. The Biodiversity Heritage Library (BHL, http://www.biodiversity
library.org/) is bringing the historical literature online. The Ency-
clopedia of Life (EOL, http://www.eol.org/) aims to integrate structured
data representing the properties and traits of species. However, these
activities remain only loosely linked with major biomolecular da-
tabasing activities. Significance: The barcoding community, and the
Barcode of Life Data Systems (BOLD) database in particular, forms a
bridge between the world of specimens and collections and the world
of sequences. Closer collaboration between BOLD and these other
biodiversity informatics activities offers the opportunity to fuse his-
torical knowledge with large-scale DNA-based biodiversity monitor-
ing to deliver the most accurate perspective possible on biodiversity
patterns and trends. Cooperation and planning now could deliver the
infrastructure to move from a legacy of primarily paper-based knowl-
edge to a stable digital knowledgebase which will be enhanced and
enriched by all new data, simultaneously facilitating future research
in taxonomy and biogeography and supporting the pressing need for
up-to-date knowledge to support conservation and wider sustainabil-
ity goals.
The application of DNA barcoding for bio-security:
a perspective from the UK
Jennifer Hodgetts,
1
Jayne Hall,
1
Joe Ostoja-Starzewski,
1
Dom Collins,
1
Chris Malumphy,
1
Sharon Reid,
1
Anastasia Korycinska,
2
Thomas Prior,
1
Rebecca Lawson,
1
and Neil Boonham
1
1Fera Science Ltd., Sand Hutton, York, YO41 1LZ, UK.
2Chief Plant Health Officer's Unit, Plant and Animal Health, Defra, Sand Hutton, York, YO41 1LZ, UK.
Corresponding author: Jennifer Hodgetts (e-mail: jennifer.hodgetts@fera.co.uk).
Background: With the continual increases in the importation of com-
modities into the UK from around the globe, the country is exposed to
the risk of introduction of non-native, quarantine pests and patho-
gens. Therefore, the ability to rapidly detect pests and pathogens in a
non-targeted approach has become increasingly important. To this
end, in parallel with specific detection tools such as real-time PCR and
LAMP applied in the laboratory and in the field, DNA barcoding has
been implemented for identification of unknown pathogens. Results:
DNA barcoding has been applied to a wide range of organismal
groups, in particular arthropod samples which are often intercepted
dead or at inappropriate life stages for morphological identification
without lengthy rearing processes. Numerous genera have been stud-
ied, leading to the first findings of quarantine species and numerous
new host records. Additionally, the method is deployed for confirma-
tion of morphological identifications, often to confirm that specimens
are not quarantine species. In the majority of instances, the standard
COI barcode is utilized; however, cases where this does not provide
the required level of discrimination have been identified, for example
within phytoseiid mites, where alternate barcode regions have been
assessed and deployed to allow sequence-based discrimination. Vari-
ous routes of entry have been demonstrated as potential risks, with
significant findings of pests and pathogens within imported wood
products, produce, and live plants for planting. Our approaches
have led to the detection of serious pests, including the nematode
Bursaphelenchus xylophilus and its vector Monochamus alternatus, nema-
todes such as Meloidogyne species, and fruit flies such as Drosophila
suzukii.Significance: DNA barcoding has been successfully used for
the identification of first interceptions of quarantine organisms
within imports to the UK. In addition, we have identified species of
possible concern on “alert lists”, and new host records have been
established. Barcoding is also being used in research and develop-
ment, combined with morphological expertise, to elucidate previ-
ously cryptic species; for example, we have developed barcode
markers for Leucinoides, a taxon for which effective barcode markers
were not previously available.
Genomic analyses from highly degraded DNA
Michael Hofreiter, Axel Barlow, Johanna Paijmans,
and Michael Westbury
Institute for Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476
Potsdam, Germany.
Corresponding author: Michael Hofreiter (e-mail: michi@palaeo.eu).
Next-generation sequencing allows obtaining exceptionally large DNA
sequence data sets. However, standard protocols—both in the wet labo-
ratory and with regard to bioinformatics approaches—are ill-suited to
deal with highly degraded DNA in fossil remains and many museum
specimens, especially from wet collections. I will show how careful adap-
tation of protocols opens up samples for genetic and genomic analyses
even with extremely degraded DNA, where average fragment lengths are
down to 40 bp or less, far below the length that allows amplification via
PCR. Selection of the right specimens and certain sample pre-treatments
as well as specifically tailored DNA extraction and NGS library-building
protocols allow full genome sequencing from samples exceeding 100 000
years in age, while DNA hybridization capture enables mitochondrial
genome typing even further back in time and also from samples where
high background microbial DNA levels prevent full genome sequencing
at justifiable prices. I will illustrate the power of both full genome se-
quencing and mitochondrial DNA typing using various examples from
fossil DNA studies.
International Society for the Barcode of Life
Peter Hollingsworth
Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, United Kingdom.
E-mail for correspondence: P.Hollingsworth@rbge.ac.uk.
Abstract: A preconference workshop on 17 August 2015 focused on
the establishment of an International Society for the Barcode of Life.
This workshop started by reviewing the Kunming Declaration, an-
nounced in 2013 at the 5th International Barcode of Life Conference in
China. The Kunming Declaration highlighted the importance of an
international society as a coordinating organization for DNA barcod-
ing. To take this forward, governance options for international soci-
eties, an update from national networks, and prospects for future
international conferences were discussed with a view to formalizing
the society at the close of the 6th International Barcode of Life Con-
ference. To achieve this, formal resolutions will be presented to the
conference plenary.
Identifying road-killed animals in a Brazilian Biological
Reserve crossed by a highway using DNA barcodes
Angélica Hollunder Klippel,
1
Gabrielle Ferreira Correia da Rocha,
1
Pablo Viana Oliveira,
1
Bárbara de Freitas Freire,
1
Marcel R. Moreno,
2
Alexandre Rosa dos Santos,
1
Aureo Banhos dos Santos,
1
and
Greiciane Gaburro Paneto
1
1Federal University of Espirito Santo, Alto Univeristário s/n Guararema, Alegre, ES, 29500-000, Brazil.
2Chico Mendes Institute of Biodiversity Conservation, Sooretama Biological Reserve, Highway BR-101,
km 101, Linhares, Espírito Santo, 29.900-970, Brazil.
Corresponding author: Greiciane Gaburro Paneto (e-mail: ggpaneto@gmail.com).
Background: Classical methods for species identification are based
on morphological characteristics and depend on the knowledge of
taxonomists who are experts in a particular group of organisms. New
methods, however, have been proposed to facilitate species identifi-
cation. One such method is DNA barcoding, which, in animals, uses a
specific region of mitochondrial DNA for specimen identification and
species discovery. This method may prove to be very useful in identi-
fying animals disfigured after road-kills. Roads are one of the main
causes of biodiversity loss in the world; that is why identification of
road-killed specimens is extremely important. Thus, the aim of this
study was to verify if DNA barcodes can identify road-killed samples
that cannot be determined using morphological identification. For
this, 222 vertebrate samples were collected along a stretch of BR-101
highway that crosses Sooretama Biological Reserve and Vale Natural
Reserve in Espirito Santo, Brazil. Mitochondrial COI sequences were
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generated and compared with the BOLD database; a species-level
match was defined as >99% sequence similarity to a single species
name in BOLD. Results: It was possible to identify 58% of samples,
totaling 57 different species, including three endangered species
(Pyrrhura cruentata,Chaetomys subspinosus, and Leopardus wiedii). The most
commonly identified animals were a bat (Molossus molossus), an opossum
(Didelphis aurita), and a frog (Trachycephalus mesophaeus). No reptile was
identified using the technique, probably due to lack of reference se-
quences in BOLD. Ambiguous identification was obtained from 3.6% of
samples, with BOLD reporting >99% similarity to more than one species.
Significance: Our data may contribute to a better understanding of the
impact of roads on biodiversity loss and may help to introduce DNA
barcoding techniques in road ecology research.
DNA barcoding of 100 common medicinal plants in Malaysia
for species authentication
Tnah Lee Hong, Lee Soon Leong, Tan Ai Lee, Lee Chai Ting,
Ng Kevin Kit Siong, Ng Chin Hong, Ling Sui Kiong,
and Nurul Farhanah Zakaria
Forest Research Institute Malaysia, 52109 Kepong, Selangor, Malaysia.
Corresponding author: Tnah Lee Hong (e-mail: leehong@frim.gov.my).
Background: Given the growing herbal market, the adulteration of
herbal products is rising as a global concern. Adulteration can occur
due to misidentification of herbs or intentional substitution with in-
expensive herbs or synthetic drugs, which may lead to public health
hazards and poor quality product formulations. As the fundamental
step to confirm the quality of an herbal product is through species
authentication, DNA barcoding offers a good alternative for detecting
plant-based adulterants in traded herbal medicinal materials. In the
present study, we aimed to establish a reference DNA barcoding data-
base for 100 common medicinal plants in Malaysia for species identi-
fication and authentication. Results: DNA barcodes were generated
using four plastid markers, rbcL, rpoC1, rpoB, and trnH–psbA. The
performance of all plastid markers in recovering species groups was
evaluated using similarity BLAST and phylogenetic trees. The rbcL,
rpoC1, and rpoB, which are more conserved, were chosen as core cod-
ing region to serve as a baseline for comparison. The analyses showed
that rbcL, rpoC1, and rpoB provided apparent resolution at the species
level and were able to separate the majority of the species. To improve
the identification to species, trnH–psbA appeared promising as a
second-tier locus. This particular region was able to increase the res-
olution of species identification significantly. As a whole, our analysis
indicates the potential of using a multigene, tiered approach for DNA
barcoding of common medicinal plants in Malaysia. Significance: A
methodology to identify and authenticate medicinal plant species is
very much in demand in the herbal industry. In Malaysia, the National
Pharmaceutical Control Bureau provides mostly chemical-based adul-
terant screening; therefore, DNA barcoding authentication would be a
good addition to complement the existing methods.
DNA barcoding Canadian scyphozoans
Kathryn Hotke
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
E-mail for correspondence: khotke@mail.uoguelph.ca.
Background: Members of the scyphozoan genus Cyanea are a conspic-
uous element of the marine zooplankton. Despite their large size,
broad distribution and abundance, the taxonomy of the genus is un-
certain. Many described taxa have been synonymized with C. capillata.
Along with weak morphological descriptions, the identification of
Cyanea specimens is often complicated by specimen fragility and
poorly preserved museum material. Molecular analyses have begun to
advance understanding of species boundaries in Cyanea, and also to
aid specimen identification. This study examines sequence variation
in the barcode region of COI to ascertain the number of Cyanea species
in Canadian marine waters. The extent of geographic variation in each
species was examined through comparison with lineages of the C.
capillata complex from other regions. Results: Sequence analysis re-
vealed five lineages of C. capillata s.l. with differing geographical dis-
tributions and showing from 5.9% to 18.0% divergence at COI.
Sequence variation was also examined in mitochondrial and nuclear
gene regions: 16S and internal transcribed spacer 1 and 2 (ITS1 and
ITS2). These regions revealed lower divergences (2.7%–10.8% for 16S,
1.3%–8.3% for ITS1, 2.2%–11.0% for ITS2), but associated with those at
COI, strongly suggesting that each clade represents a different species.
One lineage shows sequence congruence with C. capillata s.s., while
another lineage matches C. tzetlinii, recently described from the White
Sea. The other three species are likely to represent named taxa that
are currently viewed as synonyms of C. capillata.Significance: Interest
in Scyphozoa has grown due to increased occurrences of jellyfish
blooms causing adverse economic and ecological effects. This study
demonstrates the efficacy of using COI barcodes for scyphozoan spe-
cies identification, examination of phylogeographic patterns, as well
as to highlight cases of deep intra-specific divergence. This work ver-
ifies the need for greater effort in species documentation not only to
further understanding of marine biodiversity, but also to aid in con-
servation.
The phylogeography of the Hyalella azteca species complex in
North America
Matthew Hrycyshyn and Jonathan Witt
University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada.
Corresponding author: Matthew Hrycyshyn (e-mail: mjhrycys@uwaterloo.ca).
Background: Hyalella azteca (order: Amphipoda) is a morphologically
cryptic species complex of freshwater invertebrates. This group was once
thought to be a single species with a distribution that spans the North
American continent. Through the use of modern molecular techniques,
including sequencing of the COI gene, over eighty distinct species have
been identified. While most of these groups are endemic to the Great
Basin, several of them possess much larger distributions that include
previously glaciated habitats. This study is a comparative phylogeo-
graphic analysis of the major Hyalella lineages with a focus on glaciated
North America. The goal is to characterize present-day distributions and
patterns of molecular variation (using COI and ITS1 genes) in order to
delineate the effects of Pleistocene vicariance and post-glacial dispersal.
Results: Over 3000 Hyalella were collected from across North America,
and the distributions of nine major species were characterized. During
the Pleistocene glaciations, these major lineages persisted within refugia
south of the glacial limit or in ice-free areas of Beringia and subsequently
dispersed through meltwater lake systems. Several genetic patterns were
identified within these groups, including isolation by distance, demo-
graphic expansion, and long-distance colonization. These processes, in
conjunction with dispersal from multiple refugia, have resulted in elab-
orate spatial patterns. Notably, the Great Lakes region has become a
suture zone with habitats maintaining multiple Hyalella species. Signifi-
cance: Molecular studies remain a critical tool in the characterization of
species and the analysis of their demographic histories. The intricate
genetic and spatial patterns of the Hyalella azteca complex indicate an
elaborate history shaped by the Pleistocene glaciations. This study fur-
thers our understanding of the recolonization of glaciated regions by
freshwater invertebrates.
Recent trends in bio-diversity conservation
Monte Hummel
World Wildlife Fund Canada, Beeton, Ontario, Canada.
E-mail for correspondence: mhummel@WWFCanada.org.
I will outline two needed, but somewhat controversial, trends in
bio-diversity conservation: (i) Giving equal emphasis to protection,
management and restoration, not to just one or the other of these
approaches; and (ii) Moving our focus deeper and deeper, from saving
Abstracts 229
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species, to spaces, to underlying ecological processes—especially in
ways that demonstrate resilience to climate change. Human activities
are now affecting the very evolutionary potential of our planet.
Comparative phylogeography of Javanese and Balinese
freshwater fishes: DNA barcodes shed light on Quaternary
range expansion dynamic in a biodiversity hotspot
Aditya Hutama,
1
Hadi Darhuddin,
2
Frederic Busson,
3
Sopian Sauri,
2
Robert Hanner,
4
Philippe Keith,
3
Renny Hadiaty,
2
and Nicolas Hubert
5
1Bogor Agricultural University, Department of Biology, Faculty of Mathematics and Natural Sciences,
Kampus IPB Darmaga, 16680 Bogor, Indonesia.
2Indonesian Institute of Sciences, Research Centre for Biology, Gedung Widyasatwaloka, Jalan Raya
Bogor Km46, 16911 Cibinong, Indonesia.
3Muséum national d'Histoire naturelle, UMR 7208 (MNHN-CNRS-UPMC-IRD), DPMA CP 026, 43 rue
Cuvier, F-75231 Paris Cedex 05, France.
4Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
5Institut de Recherche pour le Développement, UMR 5554 (UM2-CNRS-IRD), ISEM Place Eugène
Bataillon, CC 065, F-34095 Montpellier cedex 05, France.
Corresponding author: Nicolas Hubert (e-mail: nicolas.hubert@ird.fr).
Background: Java and Bali islands belong to the Sundaland biodi-
versity hotspot, the most speciose hotspot of the Indonesian archi-
pelago. The eastern part of Java and Bali islands are located at the
margin of Sundaland, next to the Wallace line that marks the range
distribution boundaries of most of the primary freshwater fishes
from a continental origin (e.g., Cypriniformes, Siluriformes). Wide-
spread species of primary freshwater fishes are few in Java and Bali
but potentially informative on the timing and geography of the
colonization of this peripheral area of Sundaland. The biogeogra-
phy of the Sundaland ichthyodiversity is still poorly understood,
and the spatio-temporal dynamic of colonization of the peripheral
islands of Sundaland is still unknown. Results: Three species with
widespread distribution have been sampled across nearly 30 sites
throughout Java and Bali islands. A total of 100 specimens of Channa
gachua (Perciformes, Channidae), 123 specimens of Glyptothorax
platypogon (Siluriformes, Sisoridae), and 121 specimens of Puntius
binotatus (Cypriniformes, Cyprinidae) were sequenced and ana-
lyzed. For each species, mean intra-specific distances were high
ranging from 1.31% in P. binotatus to 3.18% in C. gachua, and seven to
eight allopatric lineages were observed with maximum pairwise
distance ranging from 5.3% to 6.1% among lineages. Patterns of
distribution of this cryptic diversity are congruent among species,
suggesting that a common history is at the origin of the distribu-
tion of the genetic diversity for the three species analyzed. Molec-
ular calibrations suggest that the initial diversification of those
mitochondrial lineages happened during the early Pleistocene.
Significance: The present study highlights that the evolutionary
history of the Javanese and Balinese freshwater fishes has been
influenced by the fragmentation of the landscapes during the Pleis-
tocene, likely as a result of the volcanic activity in the area during
the Pleistocene that promoted the rise of volcanic arches.
Identification of six skate species by means of multiplex PNA
fluorescence melting curve analysis
Chang-Nam Hwang,
1
Jae-Bum Cho,
2
Eun-Mi Hwang,
1
and Jung-Seon Lee
1
1National Fishery Products Quality Managment Service, Ministry of Oceans and Fisheries, 337,
Haeqang-ro, Yeongdo-gu, Busan, 606-080, Republic of Korea.
2National Fishery Products Quality Managment Service, Busan regional office, Ministry of Oceans and
Fisheries, 8,30beongil, Jongangdaero, Jung-gu, Busan, 600-016, Republic of Korea.
Corresponding author: Hwang ChangNam (e-mail: cnhwang7@korea.kr).
Background: The nucleotide sequences of the mitochondrial cyto-
chrome oxidase subunit 1 (COI) gene were analyzed for the identifica-
tion of six skate species and one sub species. Probe-based fluorescence
melting curve analysis (PNA FMCA) is a powerful tool for mutation
detection based on the melting temperature generated by thermal
denaturation of a probe-target hybrid. The PNA probes have several
advantageous features such as easy probe design and modification,
without the risk of false negatives. In this study we have developed a
molecular method based on real-time polymerase chain reaction (real-
time PCR) technology for the rapid identification of six Skate species.
Results: Six PNA probes were designed to identify Raja pulchra sub2,
Okamejei kenojei,Zearaja chilensis,Dipturus argentinensis,Raja binoculata,
and Zearaja maugeana. Six PNA probes in two reactions were designed
to cover the COI gene region. PNA FMCA with color multiplexing was
used to identify the skate species. The PNA FMCA system can distin-
guish target species from others in an efficient and high-throughput
manner and can be applied to species identification of skates. The
dual-labeled PNA probes offered the advantage of improved flexibility
in probe design, which would provide various applications for geno-
typing a wide range of spectra.
Subterranean pool party: determining the trophic links
between subterranean invertebrates in a groundwater system
in Western Australia
Josephine Hyde,
1
Steven Cooper,
2
and Andrew Austin
1
1Australian Centre for Evolutionary Biology and Biodiversity and School of Biological Sciences, The
University of Adelaide SA 5005, Australia.
2Evolutionary Biology Unit, South Australian Museum, North Terrace, Adelaide SA 5000, Australia.
Corresponding author: Josephine Hyde (e-mail: josephine.hyde@adelaide.edu.au).
Background: The Yilgarn Region of Western Australia comprises hun-
dreds of physically isolated calcretes, which resembles a subterranean
archipelago, and contains a rich diversity of subterranean invertebrates.
Each calcrete has a unique combination of subterranean aquatic species
(stygofauna), including diving beetles (Dytiscidae), crustaceans (Isopoda,
Amphipoda, Copepoda, Ostracoda, and Bathynellacea) and worms (Oli-
gochaeta). This project focuses on one calcrete (Sturt Meadows) within
the Yilgarn Region and aims to use metabarcoding analyses to identify
the food web of its subterranean ecosystem. The project also aims to
determine the primary source of energy for the ecosystem and discover if
the energy comes from external sources of carbon or whether it is pro-
duced directly via chemoautotrophic bacteria. Results: A reference data-
base of the stygofauna, surface flora, and microbes has been developed
using multiple genes including COI, 16S, and 12S (for stygofauna), and
rbcLa (for flora). Using laser dissection microscopy, the stomach contents
from individuals are being removed and analysed using metabarcoding
to determine prey items of species within the calcrete. Using this infor-
mation for multiple individuals and combining the data with stable iso-
tope analysis, insight can be provided into trophic interactions within
the ecosystem. Significance: This project will provide critical informa-
tion for the diversity of species present in a calcrete aquifer ecosystem,
and identify the structure of the food web and the source of energy that
is driving the system. Many of the calcretes are utilised for mineral pro-
cessing or provide groundwater for mines, pastoral (agricultural), and
domestic use. There is therefore an urgent need to develop a scientific
base to improve environmental assessment and monitoring of the im-
pacts of groundwater/ calcrete extraction on the fauna over the long
term.
DNA barcode analysis of thrips (Thysanoptera) diversity in
Pakistan reveals cryptic species complexes
Romana Iftikhar
1
and Muhammad Ashfaq
2
1National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang road, Faisalabad, Pakistan.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, ON NIG 2W1, Canada.
Corresponding author: Romana Iftikhar (e-mail: rmniftikhar299@gmail.com).
Background: Although thrips are globally important crop pests and
vectors of viral disease, species identifications are difficult because of
their small size and inconspicuous morphological differences. Se-
quence variation in the mitochondrial COI-5Agene region (DNA bar-
code) has proven effective for the identification of species of insect
pests. Results: We analyzed barcode sequence variation among
471 thrips from various plant hosts in north-central Pakistan. The
Barcode Index Number (BIN) system assigned these sequences to
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55 BINs, a number similar to the partitions recognized by Automatic
Barcode Gap Discovery. Sequence analysis revealed that intraspecific
distances ranged from 0.0% to 7.5%, while congeneric species showed
5.6% to 27% divergence. Neighbour-joining and Bayesian inference
trees supported the presence of 56 monophyletic lineages. Barcode
sequences for three major pest and vector species (Haplothrips reuteri,
Thrips palmi, and Thrips tabaci) and one predatory thrips (Aeolothrips
intermedius) showed deep intraspecific divergences, strongly suggest-
ing that each is a cryptic species complex. Significance: This study
compiles the first barcode reference library for the thrips of Pakistan
and examines global haplotype diversity in four important pest thrips.
Alternative barcodes for the identification of human
and animal pathogenic fungi
Laszlo Irinyi,
1
Aziza Khan,
1
William King,
1
Vincent Robert,
2
Benjamin Stielow,
2
Gianluigi Cardinali,
3
and Wieland Meyer
1
1Molecular Mycology Research Laboratory, CIDM, Sydney Medical School-Westmead Hospital, The
University of Sydney, Westmead Millennium Institute, Sydney, NSW, Australia.
2CBS-KNAW, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands.
3Department of Pharmaceutical Sciences-Universita
`degli Studi di Perugia, Perugia, Italy Via Borgo 20
Giugno, 74, 06123 Perugia, Italy.
Corresponding author: Laszlo Irinyi (e-mail: irinyil@yahoo.fr).
The generally agreed upon fungal barcode—the ITS region—proved to be
insufficient to discriminate between closely related/cryptic species
within human- and animal-pathogenic fungi. For these taxa, alternative
barcode loci needed to be identified to ensure the accurate and reliable
identification of species in a clinical setting. Potential alternative loci
with high levels of informativeness were identified by whole-genome
comparison of 25 fungal species. As a result, the three most-informative
regions: EF1, EF3, and 60S were selected as alternative barcode candi-
dates. Novel, allegedly universal primer pairs were designed from the
whole-genome data for these regions. As part of a pilot study, the three
new loci and a total set of nine primer pairs were tested on pathogenic
fungal species—including filamentous fungi and yeasts—to evaluate
their amplification efficiency under standardized laboratory conditions
and the universality of the primers across different taxa. The amplified
fragments were sequenced, and the average intraspecies variations were
defined. Barcoding gaps were calculated at the interspecies level to eval-
uate the resolution and discriminatory power of the novel loci. In patho-
genic fungi, the translation elongation factor 1␣gene (tef1
␣
) amplified
with the “Al33 primers set” proved to be the most promising candidate
for an alternative barcode in pathogenic fungi. The amplified region
showed less variability at the species level than the ITS region, and it
proved to yield a higher resolution at the interspecies level, thereby
enabling the separation of numerous closely related/cryptic species, such
as Cryptococcus. It also improved the size of the barcoding gap in other
taxa including the Debaryomycetaceae. As such, the tef1
␣
locus is a prom-
ising secondary barcode candidate for the identification of clinically rel-
evant species. However, further studies are necessary to test the
suitability of tef1
␣
as an alternative barcode and subsequently a new
public reference sequence repository needs to be created.
Pandora's Box in a pill—unveiling the composition of herbal
supplements
Natalia Ivanova, Maria Kuzmina, and Thomas Braukmann
Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Natalia Ivanova (e-mail: nivanova@uoguelph.ca).
Background: DNA-based testing has been gaining acceptance for a
wide range of food products and has potential for authentication of
herbal supplements. In our study we tested fifteen herbal supple-
ments corresponding to five medicinal plants, each from three differ-
ent manufacturers: Echinacea purpurea,Valeriana officinalis,Ginkgo
biloba,Hypericum perforatum, and Trigonella foenum-graecum. To maxi-
mize DNA recovery from processed plant materials, oil-free tablets or
gelatine capsules were chosen for testing. Listed ingredients consisted
of dried aerial parts, seeds or roots; plant extracts or extracts com-
bined with raw plant material (with or without fillers). Experimental
design included three modifications of DNA extraction, each tested
for three replicates and two lysate dilutions. To distinguish DNA deg-
radation from PCR inhibition, we utilized PCR amplification of target
DNA using primers targeting shorter regions in the presence of an
internal positive control. Results: Our results demonstrate reproduc-
ible detection of listed medicinal plant DNA in most supplements
represented by dry herb form. Multiple DNA sources rendered Sanger
sequencing results for rbcL and ITS2 regions non-interpretable, hence
indicating the strong need for next-generation sequencing (NGS). Be-
sides listed ingredients, we detected other plant DNA sources possibly
associated with harvesting, or introduced during the manufacturing
process, as airborne or equipment contaminants. The manufacturing
process of plant extracts does not take into account DNA preservation;
therefore, complementary analytical testing targeting bioactive com-
ponents may be required to verify false-negative results. NGS analysis
of the ITS2 region also revealed a diverse community of fungi, which
may be associated with raw plant material and (or) the fermentation
process utilized in production of plant extracts. Significance: We
have developed a robust NGS workflow for authentication of DNA
source in herbal supplements containing dry herb material, and some
plant extracts. This workflow can be utilized by manufacturers for
QA/QC of raw plant materials, production process, and final products.
Quantification of diversity of agriculturally important insects
through DNA barcoding
Sushil K. Jalali and T. Venkatesan
National Bureau of Agricultural Insect Resources, P.B. No. 2491, H.A. Farm Post, Hebbal, Bangalore--
560024, India.
Corresponding author: Sushil K. Jalali (e-mail: jalalisk1910@yahoo.co.in).
Background: India, occupying 2% of the global surface, is among the
top 10 nations in the world in terms of insect diversity, with about
7%–10% of the world insect fauna. About 65 000 species of insects have
been documented from India so far, and it is estimated that nearly
two-thirds of the Indian insect fauna remain to be described. The
combined specimen holdings of major insect repositories in India are
estimated to be fewer than 2 million. Results: In the past year, bar-
codes for agriculturally important insects from five orders have been
obtained including Lepidoptera (51.8%), Hymenoptera (33.9%), Diptera
(7.1%), Hemiptera (3.6%), and Coleoptera (3.6%). So far, barcodes for
256 insects have been obtained. Barcodes for geographical populations of
Leucinodes orbonalis (9), Plutella xylostella (13), and Culicoides innoxius (5)
have been obtained for analysis of genetic diversity based on the mi-
tochondrial COI gene. We have applied the DNA barcoding technique
to identify and confirm Acerophagus papayae, an important parasitoid
which showed spectacular success in controlling the invasive papaya
mealybug, Paracoccus marginatus, and saved millions of dollars' worth
of crop loss in India. In order to generate barcodes, we have opened
three projects in BOLD systems: Agriculturally Important Insects of
India (AGIMP), Veterinary Important Insects of India (VETIP), and Ants
of India (ANIND). To facilitate the barcode work on Indian insects, we
have developed a database “Insect Barcode Informatica (IBIn)” (URL:
http://www.cib.res.in/ibin/). Significance: This database provides bar-
code information on Indian and world insects, stores barcode images,
and has options for protein sequence and AT & GC content analysis.
Barcodes generated by users can also be registered in this database.
Combining citizen science and DNA-assisted species
identification to enable “a new kind of ecology”
Karen E. James
Mount Desert Island Biological Laboratory, P.O. Box 35, Salisbury Cove, ME, USA.
E-mail for correspondence: kjames@mdibl.org.
Climate change is shifting species' geographic ranges, patterns, abun-
dances, seasonal activities, and interactions with other species. Under-
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standing, predicting, and managing how species respond to climate
change and other environmental changes is a critical challenge in envi-
ronmental biology, which will require what Georgina Mace called “a new
kind of ecology…predicated on scaling up efforts, data sharing and
collaboration”. Collection of these kinds of data typically requires the
identification of biological specimens; yet, that task is difficult and labor-
intensive, and is compounded by a dearth of taxonomic expertise—the
“taxonomic impediment”. Citizen science is proposed—and has in some
cases been successfully implemented—as a mechanism for expanding
the scientific workforce to address difficult, large-scale, or computa-
tionally intensive ecological research questions. Unfortunately, train-
ing citizen scientists to accurately identify most organisms is, like the
task of identification itself, prohibitively time-consuming. Thus projects
that engage the public in identifying biological specimens, including
“bioblitzes”, still rely heavily on professional taxonomists to identify col-
lected specimens, and the taxonomic impediment remains in place. Auto-
mated identification tools that use morphology (e.g., Leafsnap), sound (e.g.,
Song Scope) and other characteristics, and online crowd-sourced photo iden-
tification services (e.g., iNaturalist, iSpot), are already being used to help
citizen scientists identify unknown specimens. DNA-assisted species identi-
fication (“DNA barcoding” and related techniques) can complement these
tools to empower researchers and citizen scientists alike to accurately
identify organisms and enable transformative increases in the scope and
scale at which ecology and biodiversity science are done. In this talk, I will
report on the progress of “BioTrails”, an NSF-funded collaboration of the
MDI Biological Laboratory, Acadia National Park, and the Schoodic Insti-
tute, to demonstrate the DNA-assisted citizen science concept and build
capacity for local, national, and international expansion.
Barcoding from a tropical trench, looking upward and outward
Daniel Janzen and Winnie Hallwachs
Department of Biology, 3740 Hamilton Walk, University of Pennsylvania, Philadelphia, PA 19104, USA.
Corresponding author: Daniel Janzen (e-mail: djanzen@sas.upenn.edu).
Thanks to massive DNA barcoding and analysis support from BIO,
iBOL, and BOLD, we are bumpily integrating CO1 barcoding with an
attempted full inventory of all the species of Lepidoptera, their cater-
pillars, and their parasitoid Hymenoptera and Diptera of a large
complex conserved tropical wildland in northwestern Costa Rica
(www.acguanacaste.ac.cr; www.gdfcf.org). Our barcoding adventure
and its integration with standard entomological practice has sug-
gested many rich possibilities and many stumbling blocks in the at-
tempt to connect centuries of what humanity knows and has tagged
with scientific and street names, to the unmistakeable and unavoid-
able future of machine+human identification and discovery of wild
biodiverse species, and the storage and retrieval of information about
them by the world team of 8 billion people who might have cause to
care at some point in their lives. Here we use a few of these new paths
and pitfalls as points of departure for anticipating what appears to be
coming down the road. It is conspicuous that there is much more
biodiversity in the wild than we had led ourselves to believe. Our
interspecies boundaries are not as genetight as we have wanted them
to be. The human enterprise is rapidly becoming uninterested in un-
derstanding all sorts of biodiversity. Museums and their specimens
are dynamic waystations on the path from wild populations to biodi-
versity awareness and subsequent attraction by a bioliterate human
populace, and they are costly and going extinct. DNA barcoding today,
and in whatever formats it evolves, is an essential tool in all these
dynamics. And the process and the philosophy extends far outside of
its birthplace as an academic research exercise here in Canada, and far
into the realms of creating a bioliterate humanity, ironically at the
same time that humanity is busily wiping the planet bare of the very
mold that created humanity and much of what we like (and fear)
about it. DNA barcoding just might rescue us from living in that white
box. Maybe. Literacy has been important to humanity. Bioliteracy
could be as well.
Genome size diversity and phylogenetic history in freshwater
amphipod (Hyalella spp.) species complexes
Nicholas Jeffery,
1
T. Ryan Gregory,
1
Sarah J. Adamowicz,
2
and Jonathan D.S. Witt
3
1Department of Integrative Biology, University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1,
Canada.
2Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
3Department of Biology, University of Waterloo, 200 University Ave W., Waterloo, Ontario, Canada.
Corresponding author: Nicholas Jeffery (e-mail: nick.w.jeffery3@gmail.com).
Background: Freshwater amphipods of the genus Hyalella show re-
markable genetic diversity in both North and South America. There
are an estimated 100 “species” in North America based on COI se-
quence variability when applying a 5% divergence threshold, and
more than 50 species within two subgenera in South America. Both of
these species groups show their highest diversity in different hot-
spots, namely the Great Basin of the western United States and Lake
Titicaca on the border of Peru and Bolivia. Here, we reinvestigate the
diversity of Hyalella using DNA barcoding (COI analysis) and phyloge-
netic reconstruction using 28S nuclear sequences, coupled with ge-
nome size estimation of representatives from each major lineage.
Genome size, defined as the total DNA content of the haploid chro-
mosome set, is known to vary ⬃700-fold within crustaceans overall.
Divergence in genome size can be associated with speciation, either
through gradual changes in genome size, particularly in allopatric
populations, or through rapid shifts via polyploidy, which can lead to
sympatric speciation. Results: We found that genome size varies
4-fold within both North and South American Hyalella. Upon phyloge-
netic mapping of genome sizes and seeking lineages with doubled
genome sizes, we find evidence for potential polyploidy in at least one
case among the North America lineages. Genome size also varied up to
4-fold in the South American species, suggesting polyploidy, but the
majority of lineages displayed more gradual genome size evolution.
Phylogenetic conservatism of genome size is apparent, with Pagel's
ranging from 0.68–0.94 and differing significantly from a phylogeny
with no signal. Significance: These results suggest changes in ge-
nome size may have played a role in sympatric and allopatric diver-
sification within Hyalella, while further study is needed using
karyotypes to determine the extent of polyploidy. This study also
reinforces the value of DNA barcoding as a useful tool to study cryptic
diversity while coupling it with research that aims to understand the
mechanisms behind the speciation process.
DNA barcoding of the tribe Archipini (Lepidoptera, Tortricidae,
Tortricinae) in Japan, with notes on the geographic variations
of widely distributed species
Utsugi Jinbo
1
and Aino T. Ota
2
1National Museum of Nature and Science, Amakubo 4-1-1 Tsukuba-shi, Ibaraki, Japan.
2Koganehara 1-19-1-505, Matsudo, Chiba, Japan.
Corresponding author: Utsugi Jinbo (e-mail: ujinbo@kahaku.go.jp).
Background: The growth of the DNA barcode library enables compre-
hensive comparisons of genetic variation. Such information is critical
for taxonomic revisions of widely distributed species. In particular,
the Barcode Index Number (BIN) is a good indicator to detect over-
looked geographic structures and unsolved taxonomic problems. Tor-
tricidae is one of the largest families of smaller-bodied moths. This
study is focused on the Japanese species of the tribe Archipini, belong-
ing to the subfamily Tortricinae. Eighty-five archipine species have
been known from Japan, including 21 species widely distributed from
Europe to Japan. Taxonomic issues remain, and thus an integrated
taxonomic study combining morphological and molecular evidence is
required. Results: To survey the geographic variation of widely dis-
tributed species and to build a DNA barcode library of Japanese Archi-
pini, DNA barcode sequences of the target group were obtained
through standard protocols. Together with morphological examina-
tion, barcodes were compared with those from other geographic pop-
ulations registered in BOLD. At present, 35 species of Japanese
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Archipini, including 12 widely distributed species recognized as
13 BINs, have been barcoded. While the European population of most
archipinine species is typically recognized as its own BIN, six BINs
were shared with European populations. Combined with morpholog-
ical data, this study reveals that one species (Pandemis monticolana)
from eastern Asia could be conspecific with other species (P. cerasana)
from the Eurasian continent. Significance: Japan consists of many
islands situated around the eastern boundary of the Palaearctic re-
gion, which is recognized as a biodiversity hotspot. Surveys on genetic
variation across taxonomic groups are indispensable to clarify the
importance and uniqueness of the fauna from this area. This study is
considered as the first step in using DNA barcode data for the taxo-
nomic study of insects widely distributed in the Palaearctic region
including Japan, enabling characterization of the Japanese fauna by
genetic comparisons across various groups. In addition, Tortricidae
contains many well-known serious agricultural pests. The obtained
barcode records are important as fundamental data to build a rapid
identification supporting system.
Cataloguing genetic variations in Sesamia inferens populations
infesting rice using DNA barcoding
Vikas Jindal, Amit Thakur, Geetika Banta, and Manjinder Singh
Department of Entomology, Punjab Agricultural University, Ludhiana-141 004, India.
Corresponding author: Vikas Jindal (e-mail: vikas_ento@pau.edu).
Background: The pink stem borer, Sesamia inferens (Walker), is an
important rice pest that is widely distributed in India and other coun-
tries. The complete mitochondrial genome of the pink stem borer has
been sequenced in China; however, few reports on the COI gene se-
quences are available in GenBank. This study reports on the genetic
variations in Sesamia inferens population from rice, collected from 18
different locations of Punjab, India. The DNA barcode region (COI) was
amplified using specific primers and the PCR product was cloned. The
nucleotide sequences were determined through custom service of
Xcelris Pvt Ltd., Ahmedabad. Results: The sequences obtained were
blasted in NCBI, and all the samples showed 99% similarity with
Sesamia inferens. Multiple alignment of all sequences revealed substi-
tutive variation at 22 different nucleotide positions that were distrib-
uted in the 658-bp sequence of COI gene obtained. A phylogenetic tree
was obtained using the maximum likelihood method in MEGA6,
which established a maximum genetic divergence of 1.57% among
Punjab populations. A low level of genetic difference was reported in
populations collected from the same district, viz. Faridkot, Bathinda,
Hoshiarpur, Barnala. Comparative analysis of the COI gene sequences
of the populations analyzed in this study with the sequences retrieved
for S. inferens from China and Korea established that the pink stem
borer populations from Punjab are genetically distinct from those of
Korea and China, with genetic variation ranging from 4.4% to 6.56%.
Significance: Genetic variations in Sesamia inferens populations in
Punjab, China, and Korea were reported in the present study. It estab-
lished that this pest occurs in nature as many distinct genotypes.
Therefore, there is need to continuously monitor the populations of
Sesamia inferens throughout the world so that appropriate pest man-
agement strategies are developed and employed for effective control
of this pest.
Monitoring of butterflies by schoolchildren to increase
environmental awareness and assess effects of climate change
in peninsular Malaysia
Shi-Wei Jisming-See, Kong-Wah Sing, and John-James Wilson
Institute of Biological Sciences, Faculty of Science, University of Malaya, Malaysia.
Corresponding author: Shi-Wei Jisming-See (e-mail: shiwei6337@gmail.com).
Background: Malaysia is a megadiverse country suffering from rapid
biodiversity loss, yet moves to address local conservation issues are
hindered by a lack of public awareness. The School Butterfly Project
began in September 2014 with the aims to increase awareness of bio-
diversity and to instill a sense of stewardship for local wildlife among
schoolchildren. Five schools in five different states of Peninsular
Malaysia were approached to participate in the project, with about
30 school children of each school, aged 9–12 years. A sixth group
included home-schooled children from the Kuala Lumpur area.
Results: During the first stage of the project, we visited each school
for an interactive training day. Following the training sessions, the
schoolchildren were provided with a sampling kit and tasked with
collecting butterfly samples in their schoolyards or local parks for
four times over the next 12 months. The first sampling day took
place in October 2014, and collectively the schoolchildren sampled
around 120 butterflies. Butterfly legs were sent to the Museum of
Zoology, University of Malaya for DNA barcoding, which revealed
that the sample included 40 butterfly species. Several dominant
species such as the Striped Wanderer, Appias olferna, and the Tawny
Coster, Acraea terpsicore, are relative newcomers to Peninsular Ma-
laysia. Significance: To the best of our knowledge, the School But-
terfly Project is the first project in Malaysia that introduces primary
schoolchildren to scientific research, and it has generated positive
interest from the local media. In the short term we look forward to
the next sampling days and to explore if, and how, butterfly diver-
sity changes throughout the year. In the long term we look forward
to exploring ways to expand the reach of the project across the
country and society.
DNA barcoding contributes to the taxonomy of Afrotropical
hover flies (Insecta: Diptera: Syrphidae)
Kurt Jordaens,
1,2
Georg Goergen,
3
Audrey Vokaer,
1
Nathalie Smitz,
1,4
Massimiliano Virgilio,
1
Gontran Sonet,
5
Thierry Backeljau,
2,5
and Marc De Meyer
1
1Royal Museum for Central Africa, Belgium.
2University of Antwerp, Belgium.
3International Institute of Tropical Agriculture.
4University of Liège, Belgium.
5Royal Belgian Institute of Natural Sciences, Belgium.
Corresponding author: Nathalie Smitz (e-mail: nathalie.smitz@africamuseum.be).
Background: The identification of Afrotropical hover flies is very
difficult because of limited recent taxonomic revisions and the lack of
comprehensive identification keys. In order to assist in their identifi-
cation and to improve the taxonomy of this group, we constructed a
reference dataset of 480 COI barcodes of 83 common nominal species
from Ghana, Togo, Benin, and Nigeria (West Africa), and evaluated its
identification accuracy with three methods (K2P distance-based,
neighbour-joining, and using SpeciesIdentifier). Results: Results of
the three methods were highly congruent and showed high identi-
fication success. Nine species pairs showed a low (< 3%) mean in-
terspecific K2P distance, with several cases of paraphyly and
misidentifications. A high (> 3%) mean intraspecific K2P distance
was observed in eight species, with several cases of para/polyphyly
that may indicate the occurrence of cryptic species. Optimal K2P
thresholds to differentiate intra- from interspecific K2P divergence
were highly different among the three subfamilies (Eristalinae:
3.5%; Syrphinae: 5.8%; Microdontinae: 0.7%–2%). Significance: In
addition to providing an alternative identification tool, our study
indicates that DNA barcoding improves the taxonomy of Afrotropi-
cal hover flies by highlighting groups of taxa that deserve further
taxonomic study, and by identifying the unknown sex of species
displaying sexual dimorphism. Expanding the reference database
by including less-common species with poor taxonomic placement
will prove challenging.
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DNA barcoding identifies an introduced hover fly species
(Diptera: Syrphidae: Syrphinae) in the Afrotropics
Kurt Jordaens,
1,2
Georg Goergen,
3
Ashley Kirk-Spriggs,
4
Audrey Vokaer,
1
Nathalie Smitz,
1,5
Thierry Backeljau,
2,6
and Marc De Meyer
1
1Royal Museum for Central Africa, Belgium.
2University of Antwerp, Belgium.
3International Institute of Tropical Agriculture.
4University of KwaZulu-Natal, South Africa.
5University of Liège, Belgium.
6Royal Belgian Institute of Natural Sciences, Belgium.
Corresponding author: Nathalie Smitz (e-mail: nathalie.smitz@africamuseum.be).
Background: Recently (2013–2014), several hover fly specimens from
two localities in Benin and Cameroon (West and Central Africa) were
caught that belonged to a species that we could not identify using
existing identification keys for Afrotropical Syrphidae. Results: Se-
quencing of the standard cytochrome c oxidase subunit I (COI) bar-
code region and a subsequent BLAST-IDS in the Barcode of Life Data
Systems (BOLD) revealed a 100% sequence similarity with Toxomerus
floralis from Suriname (South America). Species identification was sub-
sequently confirmed by morphology and sequencing of part of the
nuclear 18S rDNA gene. The species is widely spread in Benin, Nigeria,
and Cameroon; and eggs, larvae, and adults are abundant at several
localities. Yet, the full extent of its geographic distribution within
tropical Africa remains to be determined. Significance: This is only
the second known established introduction of a non-African hover fly
species in the Afrotropics. Interestingly, the larvae of the species are
pollinivorous, which is a rare feeding mode within the subfamily
Syrphinae. Moreover, it is the only Syrphinae species of which the
larvae feed on pollen from two plant species from different families
(Cyperaceae and Rubiaceae). This example illustrates how DNA bar-
coding may allow a fast and accurate identification of introduced
species.
DNA barcoding to monitor the biodiversity of marine
mammals: species identification along the French and
Mauritanian Atlantic coasts, and diet analysis
Jean-Luc Jung
Laboratory BioGeMME, University of Brest 6 avenue le Gorgeu, 29200 Brest, France.
E-mail for correspondence: jung@univ-brest.fr.
Background: Marine mammals are long-lived top predators relying
on lower trophic levels. As such, they act as sentinel species of the
health of marine and coastal ecosystems, especially when impacts of
anthropogenic origin are suspected. However, monitoring the pres-
ence and diversity of these mobile and elusive marine mammals is
challenging. For some years, we have applied DNA barcoding to the
study of marine mammal samples collected by stranding networks in
different areas presenting diverse levels of biodiversity and fieldwork
conditions. Results: Along the French Atlantic Coast, a National
Stranding Network has been taking samples since the end of the
1970s. We sequenced the 5=end of the COI gene of 15 different species
(12 cetaceans and three pinnipeds). We confirmed that field taxo-
nomic identifications were of a great accuracy and that undetermined
samples can be, as expected, identified using DNA barcodes. Along the
Mauritanian coast, surveys are organized in the frame of the program
“Biodiversité, Gaz, Pétrole” spearheaded by the Mauritanian Govern-
ment. We analysed more than 100 samples, taken from carcasses of
stranded animals. Some highly degraded carcasses led to technical
issues, but nevertheless we identified baleen and toothed whales, in-
cluding rare and unexpected species. We will also present results
focusing on two particular species, the harbour porpoise and the grey
seal, in terms of (i) phylogenetics and (ii) diet analysis, by barcoding
soft remains taken from the stomachs of predators. Significance:
The relevance and accuracy of the monitoring of marine mammal
strandings are strengthened by DNA barcoding at different levels: by
confirming the taxonomic identification, by identifying degraded car-
casses, by determining intraspecific variations, as well as for diet anal-
ysis. This is particularly applicable to remote areas where the rich
marine mammal fauna is poorly known and where anthropogenic
impacts are highly suspected, such as the Mauritanian coasts.
DNA barcoding of aquatic plants may aid in understanding
species diversity and evolutionary relationships
Ambadas Kadam,
1
Vikram Khilare,
2
and Gulab Khedkar
3
1Department of Botany, DSM College, Jintoor DSM College, Jintoor Dist. Parbahni, Maharashtra, India.
2Department of Botany, Vasantrao Naik College CIDCO, N4, Aurangabad 431004, India.
3Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Ambadas Kadam (e-mail: kadamambadas@rediffmail.com).
Aquatic vegetation is considered to be an integral part of an aquatic
ecosystem. It includes various species of flowering and nonflowering
plants, algae, and fungi. They play an important role in ecological pro-
cesses, and several species are known to inhabit such ecosystems. To
assess the extent of species diversity and genetic variability of such veg-
etation, and to evaluate species boundaries, we collected 450 samples of
aquatic vegetation from various aquatic habitats in Maharashtra State.
All collected plant species were well preserved and taxonomically anal-
ysed. A tissue sample was analysed for DNA barcoding using RBCL, ITS
region-2, and MatK to determine their robustness in species authentica-
tion in aquatic vegetation. Generated data is useful in the identification
of species of aquatic plants and in studying their intra- and interspecies
variability. Comparative analysis of various taxa and groups also focus on
an important aspect of co-evolution in these groups. The information
generated in this study may aid in controlling some weeds in irrigated
agricultural crops, where the source of irrigation is a large aquatic system
containing various vegetation.
Parallel food authenticity and microbial composition analyses
using DNA barcoding and next-generation sequencing
Christine Kaeppel,
1
Ilka Haase,
2
Elmar Schilling,
3
Christiana Cicicopol-Boicu,
3
Daniel Richter,
3
Lukas Windhager,
3
Katrin Juling,
2
Birgit Ottenwaelder,
3
and Rainer Schubbert
1
1Department of Applied Genomics, Eurofins Medigenomix GmbH, Eurofins Genomics GmbH Anzinger
Str. 7a, 85560 Ebersberg, Germany.
2Department of Applied Genomics Business Development, Eurofins Medigenomix GmbH, Eurofins
Genomics GmbH Anzinger Str. 7, 85560 Ebersberg, Germany.
3Department of Next Generation Sequencing, Eurofins Medigenomix GmbH, Eurofins Genomics GmbH
Anzinger Str. 7a, 85560 Ebersberg, Germany.
Corresponding author: Christine Kaeppel (e-mail: ChristineKaeppel@eurofins.com).
Background: Identifying animal and microbial species in food and
feed has become popular in recent years. Especially since the horse
meat scandal in 2013, authenticity tests and safety controls are neces-
sary. The current state of the art is species identification by Sanger
Sequencing of several DNA barcode regions. Nevertheless, Sanger Se-
quencing is limited by the overlay of several sequences in complex
samples. Furthermore, species-specific real-time PCRs allow determin-
ing only the ingredients that have been searched for and, thus, cannot
be applied to samples of unknown content. Next-generation sequenc-
ing (NGS) might be able to overcome these challenges and allows
analysis of animal and microbial composition in parallel. Results: We
have performed deep amplicon sequencing for mammals, birds,
fishes, and molluscs in mixed samples of known content using the
Illumina MiSeq technology. Real samples like fish meal were also
included in this trial. All NGS data were compared to the results of
species-specific real-time PCRs. Furthermore, we have analysed bacte-
rial composition of reference and real samples using the Illumina
MiSeq technology, combined with several bioinformatical pipelines.
Our results have shown that the Illumina MiSeq technology allows
detection of animal and microbial species in complex samples, while
the results are in accordance with real-time PCR results. The use of
different bioinformatical pipelines has demonstrated that the results'
quality is dependent on the PCR target used and database applied.
Furthermore, it has been shown that 16S copy number correction
should be applied when analyzing the bacterial content of a sample.
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Significance: Our experiments have shown that NGS is able to over-
come the drawbacks of Sanger Sequencing and real-time PCR in com-
plex animal and microbial samples. Constantly declining costs for
NGS and the ability to analyse animal and microbial components in
parallel make the method attractive for routine analyses.
Use of BOLD systems in species conservation
Vikas Kalyankar,
1
Shivaji Mane,
2
Sunil Ahirrao,
3
and Trupti Khedkar
4
1Department of Zoology, Toshniwal A.C.S.College, Sengaon, India.
2Krishi Vidnyan Kendra, Tondapur, Dist Hingoli, India.
3Department of Zoology, Shri Shivaji College, Parbhani, India.
4Department of Zoology, Nabira College, Katol, Nagpur, India.
Corresponding author: Vikas, Kalyankar (e-mail: vikasvb1@gmail.com).
Background: The identification of species by short DNA sequences
has been at the centre of DNA barcoding and is being adopted by all
fields of biology. Results: Herein we represent the role of BOLD sys-
tems for species conservation especially for the fish Anguilla bengalensis
bengalensis through uploading various data of biological and geograph-
ical importance with detailed photographs, strata of fish found, life
stages of individuals, latitude and longitude of the fish's occurrence,
etc. Significance: The documentation of fish life stage records main-
tained by the data base could add to the study of the status of popula-
tions. This can become a crucial part in conservation studies and
adding to the world's knowledge of living systems.
DNA barcoding may aid in conservation and fare trading of
ornamental fish in India
Amol Kalyankar,
1
Rahul Jamdade,
2
and Gulab Khedkar
2
1Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
2Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, India.
Corresponding author: Amol Kalyankar (e-mail: amol.hariom@gmail.com).
Background: In the wake of trade liberalization under WTO agree-
ment, international trading in agricultural products, foods, pharma-
ceuticals, and marine products has facilitated massive extraction of
natural resources and ultimately has created potential risks to biodi-
versity. Practical concerns to managing the ornamental fish species
under trade include the recognition of fish by their common names.
Interestingly, the rapid rate of hybridization and sometimes muta-
tions (natural or imposed) are creating variations among the same
species of fish, which are recognized by different names. Besides,
juvenile fish are often cumbersome to identify to the species level
even for experts. Therefore, the correct identification of ornamental
fish using morphological characters is often difficult and requires
expert help. Also, it is challenging to judge the origin of ornamentals,
such as wild, cultured, or exotic, etc., as over one billion individual
fish are traded annually through more than 148 countries. Results:
Our study covers a collection of 940 ornamental fishes collected from
aquarium retailers, wholesalers, and exporters from India. Moreover,
almost 673 wild ornamental fish specimens were collected from eight
major rivers in India. All collected specimens were barcoded and ana-
lysed to determine species boundaries, mean genetic differences, and
genetic distances to nearest neighbour species (barcode gap analysis).
Several cryptic species were recorded in this study. We detected some
issues with unexpectedly high intraspecific divergences, cases which
were resolved using different approaches. Overall, our paper demon-
strates high ambiguities in names currently used in fish trading, which
may help traders to escape regulation or prosecution. Significance:
This study highlights the misuse of common names in the trading
of banned fish, which may cause potential losses to biodiversity.
High-throughput molecular approach for quantifying pest
regulation services and disservices provided by farmland insect
communities
Stéfaniya Kamenova,
1
Vincent Bretagnolle,
2
and Manuel Plantegenest
3
1INRA UMR IGEPP Domaine de la Motte 35653 Le Rheu, France.
2CEBC Chizé CNRS UMR 7372 79360 Villiers-en-Bois, France.
3Agrocampus Ouest 65 Rue de Saint-Brieuc 35000 Rennes, France.
Corresponding author: Stéfaniya Kamenova (e-mail: stefaniya.kamenova@gmail.com).
Background: Maximizing farmland biodiversity has been widely pro-
moted through public policies as a sustainable alternative to chemical
inputs. The biodiversity of naturally occurring generalist predators,
such as spiders or ground-living beetles, has been positively associated
with pest control in intensively managed agro-ecosystems. However,
our ability to successfully predict ecological functions, such as pest
regulation by natural enemies, requires more direct and mechanistic
insight. Disentangling trophic interactions among service-providing
organisms in agricultural landscapes will afford a direct and holistic
view of the processes determining ecosystem services' achievement.
In order to elucidate factors affecting biological control of pests, we
built semi-quantitative trophic networks of a major group of insect
generalist predators (carabid beetles) by using a molecular metabar-
coding approach. Results: Gut contents from 500 individuals of 13
carabid species were collected in 6 intensively managed wheat and
oilseed rape fields and analyzed with a combination of plant and
animal universal primers. The most significant variation in carabids'
trophic choice was explained by the crop type and the field identity
(p< 0.01). This variation was mainly expained by differences in the
consumption of non-pest organisms, including other natural preda-
tors (i.e., disservice). Indeed, predation on non-pest prey was two times
higher in wheat crop compared to oilseed rape (p< 0.01). Finally, we
observed a significant effect of field identity on major properties of
the carabids' trophic network structure (nestedness, linkage density,
and connectance, p< 0.01). Significance: The physical mosaic of the
agricultural landscape plays a major role in the trophic behavior of
insect generalist predators, with significant consequences for the
success of biological control. This finding is important because of
the urgency to identify concrete actions for sustainable crop pro-
tection. It also emphasizes the value of DNA barcoding-based ap-
proaches for tackling questions of ecological, economic, and societal
relevance.
Barcoding of fresh water fishes from Pakistan
Asma Karim,
1
Asad Iqbal,
1
Rehan Akhtar,
1
Mohammad Rizwan,
1
Ali Amar,
2
Mohammad Usman Qamar,
2
and Shah Jahan
2
1Department of Zoology, Govt. College of Science, Wahdat Road, Lahore, Pakistan.
2University of Health Sciences, Lahore, Pakistan University of Health Sciences, Lahore, Pakistan.
Corresponding author: Asma Karim (e-mail: drasmakarim@gmail.com).
Background: DNA barcoding is a taxonomic method to identify spe-
cies. It uses a short genetic marker in an organism's mitochondrial
DNA for identification. It uses the sequence diversity of a 658-bp frag-
ment near the 5=end of the mitochondrial cytochrome c oxidase
subunit I (COI) gene for identification. When compared with morpho-
logical identification DNA barcoding is more accurate and reliable. It
is useful in both juvenile and adult stages of fishes. It also helps with
the identification of processed or damaged fish, which is impossible
by traditional methods. Results: A total of three COI barcodes were
recovered for three species from three genera of the family Cyprinidae
for the present study. Fish F1 and Fish R1 primers were used. The
species were Cyprinus carpio (1), Cirrhinus mrigala (1), and Ctenophyran-
godon idella (1). The average read length was 680 bp with 116 polymor-
phic sites, 109 singleton variable sites, and no parsimony-informative
sites. No insertions/deletions or pseudogenes or contaminant se-
quences (e.g., from bacteria) were observed, which supports the view
that all of the amplified sequences constituted functional mitochon-
drial COI sequences. Significance: From these findings it is concluded
that the gene sequence, COI, may serve as milestone for identification
Abstracts 235
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of related species at molecular level in Pakistan. In Pakistan the fresh
water fish fauna is represented by a minimum of 193 species identified
by traditional methods. These species belong to the class Actinoptery-
gii. Among the indigenous species of special importance, 43 species
have been identified as endemic to Pakistan and Kashmir.
Genetic introgression between species: resolving challenges to
Neo-Darwinism and DNA barcoding
Yuri Kartavtsev
A.V. Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of
Sciences, Vladivostok 690041; Far Eastern Federal University, Vladivostok 690095, Russia 17 Paltchevsy
St., Vladivostok 690041, Russia.
E-mail for correspondence: yuri.kartavtsev48@hotmail.com.
Background: The paper briefly summarises the notion of hybrids and
occurrence of hybridization events detected by molecular markers.
Evidence on possible impact of genetic introgression on species integ-
rity, evolutionary fate of species, including reticulation of phyloge-
netic lineages, and consistency with the main modern paradigm,
Neo-Darwinism, are considered. Three main points are discussed:
(i) What methods for hybrid detection and the estimation of genetic
introgression or gene flow are most appropriate? (ii) What facts ob-
tained on gene introgression by mtDNA and nDNA markers are
evidence for hybridization? (iii) Does the literature contain a corre-
spondence of data on molecular diversity in lineages with Biological
Species Concept (BSC) or is reticulation a major trend? Results: It
seems that claims on the disestablishment of the modern BSC para-
digm due to wide-scale gene introgression and phylogeny reticulation
are too premature, especially considering the long history of many
hybrid zones. Contrary to this, evidence summarized in the literature
shows that molecular genetic data are concordant with the BSC and
Neo-Darwinism in general. It is clear that introgression exists, al-
though even in a wide zone of Mytilus spp., for example, introgression
may be quite restricted or be asymmetric, resulting in at least the
“source” taxa remaining intact. Significance: If we accept that the
sexually reproducing species in marine and terrestrial realms are in-
trogressed, then we should recognize that the orthodox biological
species concept—in terms of complete absence of gene flow among
species—is inadequate in a sense that many zoological species are not
biological species yet. However, sooner or later zoological species def-
initely become biological species. Such a conclusion is supported by
genetic distance increasing with taxon rank, with lowest diversity at
intraspecies level and absence of interspecies reticulation for the
majority of gene trees. These outcomes have great impacts to iBOL -
FISH-BOL science policy and biological species identification in partic-
ular. This research is supported by RNF grant 14-50-00034.
DNA barcoding of green algae Caulerpa species (Caulerpales:
Chlorophyta) from Andaman Islands, India
P. Karthick, K.N. Murthy, C.H. Ramesh, S. Narayana,
and R. Mohanraju
Department of Ocean Studies and Marine Biology, Pondicherry University, Port Blair, Andaman
and Nicobar Islands, India. Pin: 744 112.
Corresponding author: P. Karthick (e-mail: karthickmicrobes@gmail.com).
A study was attempted to DNA barcode Caulerpa species (Caulerpales,
Chlorophyta) from the intertidal region of South and Little Andaman
Islands, India. Preliminary identification of five Caulerpa species was
carried out based on the morphological characters. For molecular
characterization two gene markers ITS2 and chloroplast tufA regions
were used to barcode five species of the genus Caulerpa. Among the
five species, four species (Caulerpa racemosa,C. serrulata,C. fergusonni,
and C. microphysa) were identified using the ITS2 gene and one species
C. mexicana var. pluriseriata was identified by the tufA gene. This study
is a first attempt on barcoding of seaweeds from the Andaman Islands.
DNA metabarcoding illuminates dietary niche partitioning by
large mammalian herbivores in Africa
Tyler Kartzinel and Robert Pringle
Princeton University, 106A Guyot Hall, Princeton, NJ 08544, USA.
Corresponding author: Tyler Kartzinel (e-mail: tylerk@princeton.edu).
Background: Ecological theory dictates that large mammalian herbi-
vores (LMH) must partition food resources to coexist. Traditional
frameworks categorize LMH along a spectrum of dietary niches from
grass-eating grazers to non-grass-eating browsers, yet it has never
been clear how finely LMH partition the enormous plant diversity
represented by these two broad plant types. Results: We are using a
DNA metabarcoding approach in conjunction with a local plant DNA
barcode library to compare the diets of a diverse assemblage of LMH
species (17 wild; 5 domestic) at Mpala Research Centre, Kenya. Using
independent carbon stable isotope measurements to evaluate the re-
liability of DNA metabarcoding for quantifying dietary grass:browse
ratios, we show that the diets of these species are shown to span a
continuum from almost-pure grazers to almost-pure browsers. More-
over, the composition of plant species within diets was similar within
LMH species and clearly divergent across species, irrespective of their
traditionally recognized feeding guild. Significance: These results
suggest that species-specific plant traits may be key to understanding
the dietary differences thought to shape the origin and maintenance
of LMH diversity. This hypothesis motivates ongoing experimental
research into the determinants of LMH dietary variation, as well as the
practical ways that rangelands can be managed to avoid diet-mediated
conflicts between livestock and wildlife.
The phylogenetic signal of DNA barcodes: insights on insect
families
Mari Kekkonen
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
E-mail for correspondence: kekkonen@uoguelph.ca.
Background: Although the identification of species has been the
main goal of DNA barcoding, the Barcode Index Number (BIN) system
together with other algorithmic approaches have enhanced the use of
barcodes for species discovery and delineation. These tools are ex-
tremely helpful for the evaluation of global patterns of species diver-
sity, but they can lack taxonomic context. Currently, many BINs (i.e.,
putative species), especially from poorly sampled taxonomic groups
and geographic regions, cannot be placed to a correct higher taxon
because there is no closely related species on BOLD. As much biolog-
ical insight can be gained from placement within the Linnean hierar-
chy, there is a need to see if barcodes contain enough phylogenetic
signal to permit such assignments. Results: Several efforts are under-
way to construct a functional identification system for higher taxo-
nomic levels based on the analysis of DNA barcode data. The present
study tests the cohesiveness of various insect families. Preliminary
results suggest that DNA barcodes possess enough phylogenetic signal
to enable family-level assignments, but there is much variation in the
difficulty of this task among and even within orders. The lepidopteran
superfamily Gelechioidea, a particularly challenging group, is consid-
ered as a case study. Significance: Family-level assignments for spe-
cies recognized through DNA barcoding have traditionally been based
on morphological characters. However, many studies employing next-
generation sequencing cannot use this approach because the link
between specimens and sequences is absent. Morphological determi-
nations can be difficult or impossible for many other reasons (e.g., no
taxonomic expert is available, the voucher is in poor condition, or
diagnostic characters for family identifications are homoplastic), leav-
ing DNA-based assignments as the only option. Due to the steeply
increasing number of studies where morphological approaches can-
not be employed, a reliable identification tool for the family level
would be a major aid to large-scale biodiversity assessments.
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NGS pollen metabarcoding and automatic taxonomic
classification
Alexander Keller, Wiebke Sickel, Markus Ankenbrand,
and Ingolf Steffan-Dewenter
Department of Animal Ecology and Tropical Biology, University of Wuerzburg Biocenter, Am Hubland,
97074 Wuerzburg, Germany.
Corresponding author: Alexander Keller (e-mail: a.keller@biozentrum.uni-wuerzburg.de).
Background: Identification of pollen plays an important role in ecology,
palaeo-climatology, honey quality control, and other areas. Expert
knowledge and reference collections are essential to identify pollen ori-
gin through light microscopy. Pollen identification through molecular
sequencing and DNA barcoding has been proposed as an alternative ap-
proach, but assessment of mixed-pollen samples originating from multi-
ple plant species is still a tedious and error-prone task. A new strategy to
tackle this is to use next-generation sequencing for assessment of com-
plete assemblages without manual separation. Especially, new sequenc-
ing devices allow high numbers of samples to be processed in parallel, to
the benefit of cost- and labour-efficiency. Results: We thus developed
molecular protocols to assess the biodiversity of pollen samples with ITS2
amplicons by different sequencing devices (454, MiSeq) and an automatic
classification pipeline designed for such high throughput. In general,
sequencing resulted in deeper assignments and more identified taxa
compared to light microscopy. Abundance estimations from sequencing
data were correlated with counted abundances through light micros-
copy. Simulation analyses of taxon specificity and sensitivity indicate
that 96% of taxa present in the database are correctly identifiable at the
genus level and 70% at the species level. Currently, the coverage of the
reference database is highly variable, exemplarily for US states ranging
between 78.2% and 88.4% (median: 85.3%) of known genera. Thus the
major limitation of the approach is low coverage for less well-sampled
bioregions. Significance: We successfully applied the method in a vari-
ety of ecological studies with nest provisions of social and solitary bees,
rigid corbicular pollen collections, pollinator surface swabs, and honey.
The obtained data was well suited to answer our specific hypotheses and
to identify diet and foraging patterns. Concluding, metabarcoding pres-
ents a useful workflow to assess plant origins of mixed-pollen samples in
ecology without requiring specialised palynological expert knowledge.
One fish many stories
Gulab Khedkar
Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
E-mail for correspondence: gdkhedkar@gmail.com.
Catfish aquaculture contributes to almost 17.5% of the overall freshwater
fish production. Production takes place mostly in tropical and subtropi-
cal areas of North America and Asia. Of the seven exploited catfish fam-
ilies, Ictaluridae and Clariidae represent over 95% of the entire
production. Clarias batrachus is one of the important clariid candidate
species for aquaculture. This fish is highly preferred by consumers but in
drastic decline in our natural resources over the last decade. Species
identification in the Asian region remains controversial, which is the
focus of this study, but can be resolved using DNA barcoding approaches.
This paper will focus on research strategy, important approaches, and
conservation opportunities for C. batrachus in India with a national per-
spective of where DNA barcoding can be substantially supported.
Freshwater zooplankton diversity and distribution pattern in
the Godavari River revealed by COI gene sequences
Kshama Khobragade,
1
Shil Abhyankar,
2
and Gulab Khedkar
2
1Department of Environmental Science, S.B. Science College, Aurangabad, Aurangabad, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Kshama Khobragade (e-mail: kshama_khobragade@yahoo.com).
Background: The recognition and discrimination of plankton species is
one of the foundations to freshwater biodiversity research and river mon-
itoring programs. Species identification is frequently a bottleneck in the
analytical chain from sampling to data analysis and subsequent environ-
mental status evaluation. Our study on zooplankton diversity data from
the Godavari River included three seasonal surveys assessed by COI gene
sequences and compared with identification based on morphology. Al-
though morphological characters can result in incorrect identification,
the success of COI amplification in plankton species has been quite poor,
hampering the contribution of DNA barcoding towards understanding
the biodiversity and distribution of these species. Results: Our study
explains the issues associated with DNA extraction, amplification, and
sequencing success, wherein small alterations in protocols can enhance
the results to a significant extent. DNA extraction protocols need to be
carefully selected, and usually commercially available kits were found to
be effective for PCR success. Generally, planktonic specimens are micro-
scopic, necessitating the use of the whole animal for DNA isolation, mak-
ing it difficult to conduct morphological species verification during
sequence analysis. Also, the time elapsed during specimen imaging in-
creases the chances of DNA degradation. Significance: Issues with cryp-
tic species and higher intraspecific distances also add to taxonomic
confusion, which suggest careful taxonomic evaluation and COI se-
quence studies are needed including the barcoding of multiple speci-
mens to ensure correct species assignments.
Application of environmental DNA methods for assessing
biodiversity and biomonitoring endangered species: a case
study of Jefferson salamander (Ambystoma jeffersonianum)in
southern Ontario, Canada
Ian King,
1
Steven Hill,
2
Jim Bogart,
3
Shadi Shokralla,
1
and Mehrdad Hajibabaei
1
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Dougan & Associates - Ecological Consulting and Design, Guelph, 77 Wyndham Street South, Guelph
ON N1E 5R3, Canada.
3Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Ian King (e-mail: kingi@uoguelph.ca).
Background: Use of environmental DNA (eDNA) has the potential to
greatly improve our knowledge of biodiversity and thereby help reduce
threats to it. Much of the impetus for the development of eDNA methods
has been the need to detect and monitor endangered organisms, so it is
crucial to expedite the standardization of eDNA monitoring protocols.
Collecting eDNA is a form of non-destructive sampling, which is espe-
cially important in situations where endangered or “at risk” species are
being monitored. One such case is the endangered Jefferson salamander
(Ambystoma jeffersonianum) and other congenerics in southern Ontario.
Results: We conducted a case study on using eDNA to improve monitor-
ing of salamanders in the genus Ambystoma. We collected water samples
from various ponds in southern Ontario where Jefferson salamanders
had been previously observed. Using tissue samples, multiple specimens
of A. jeffersonianum, two congenerics, and multiple polyploids that are
commonly found in the geographic range of A. jeffersonianum were bar-
coded using standard sequencing primers. The DNA barcodes were com-
piled into a database that served as an Ambystoma sequence library. We
designed three primer sets based on the sequences in this library. Envi-
ronmental DNA extracted from the pond water samples was sequenced
on an Illumina MiSeq platform using these primer sets. A comparison of
the eDNA sequences to the Ambystoma sequence library showed positive
matches, thereby confirming the successful detection of targeted sala-
manders from water samples via eDNA. Significance: Current monitor-
ing methods for salamanders rely on observation of individuals or egg
masses, and identification to species can be difficult. This problem is
compounded by the complex genetic system of Ambystoma, including the
presence of unisexual individuals. Based on our results, our method will
allow for more accurate detection and for a longer time window than is
currently possible.
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DNA barcoding plant–insect interactions in a tropical
rainforest
Keiko Kishimoto-Yamada and Motomi Ito
The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Corresponding author: Keiko Kishimoto-Yamada (e-mail: kky_kei@yahoo.co.jp).
Background: DNA barcoding of gut contents is an effective ap-
proach for constructing the food webs of terrestrial species living
in cryptic habitats such as the forest canopy. The general inacces-
sibility of the forest canopy makes it difficult to perform direct
observations of insect feeding, and thus few interactions between
plants and insects have been recorded in this habitat. We employed
DNA barcoding to examine the trophic associations of adult leaf-
chewing chrysomelid beetles (Coleoptera: Chrysomelidae: Gale-
rucinae), which are presumed to be canopy foragers, in a Bornean
rainforest. Adult chrysomelids were collected by light-trapping in the
canopy. Plant material ingested by the beetles was retrieved from
their bodies, and a portion of the chloroplast rbcL gene sequence was
then amplified from this material. The DNA template was also used
for barcoding chrysomelid beetles, and they were discriminated into
species. Results: The plants were identified to the family level using
an existing chloroplast DNA reference database. The study success-
fully identified the host plant families for 11 chrysomelid species,
indicating that five species fed on more than two families within the
angiosperms, and that four species fed on several families of gymno-
sperms and (or) ferns together with multiple angiosperm families.
Our findings suggest that generalist herbivores associated with eco-
logically and taxonomically distant plants constitute a part of the
plant–insect network of the Bornean rainforest. These observations
contrast with earlier estimates of extremely narrow host ranges
among tropical herbivores. Significance: Previous studies may have
underestimated the host ranges of insect herbivores and overlooked
the host generality of insect herbivores in tropical forests. Because our
DNA-based technique is less targeted, it reduces the possibility that
the trophic relationships of generalist herbivores will be overlooked.
Our study also highlights that DNA barcoding analyses revealed more
types of interaction between plants and chryosmelid species than
have previously been recorded by direct observations.
The global Trichoptera Barcode of Life campaign: implications
and applications
Karl Kjer
1
and Xin Zhou
2
1Rutgers University, Department of Ecology, Evolution, and Natural Resources, 14 College Farm Rd.,
Rutgers University, New Brunswick, NJ 08901, USA.
2China National GeneBank-Shenzhen & BGI-Shenzhen Shenzhen, Guangdong Province, 518083, China.
Corresponding author: Karl Kjer (e-mail: kjer@aesop.rutgers.edu).
We began working on the development of the Trichoptera barcode
database in 2007, as Xin Zhou joined the Biodiversity institute of
Ontario, as a postdoc. Kjer solicited specimen donations from
around the world, as Zhou worked with the Canadian team to
collect and process samples. The database has now passed a critical
tipping point, with over 4500 species and 55 000 specimens, and is
now useful in identifying species from around the world with high
efficiency. Several holes in the database still exist; mainly in Africa
and India, but North America, Europe, and Southeast Asia are very
well sampled. We will discuss examples of this utility in identifying
specimens from the Smithsonian collection, in developing a rea-
sonable phylogeny of Chimarra (Philopotamidae), and in identifying
specimens from the 1KITE initiative. We discuss several examples
in which the barcode database was used to associate Trichoptera
larvae from Churchill, Canada, and how we built an accelerated
regional database for Trichoptera in the Great Smoky Mountains
National Park. We also test how well the database identifies un-
known samples from Vietnam, the Philippians, and the Central
African Republic.
Censusing marine life in the 21st Century
Nancy Knowlton and Matthieu Leray
Smithsonian Institution, National Museum of Natural History, 10th & Constitution Avenue,
Washington, DC 20560, USA.
Corresponding author: Nancy Knowlton (e-mail: knowlton@si.edu).
Background: We still lack a well-constrained estimate for the di-
versity of marine life. The challenge stems from the fact that most
marine species are small, rare, and undescribed by science. How-
ever, high-throughput DNA sequencing used on standardized envi-
ronmental samples provides a cost-effective way to estimate the
number of species in a specific location and compare such esti-
mates with those made elsewhere. Results: A recent study of the
diversity of eukaryotic organisms on oyster reefs in Virginia and
Florida is an example of the power of this approach. Nine Autono-
mous Reef Monitoring Structures (ARMS) were deployed for 6
months in each location in three sets of three. Each ARMS consists
of a stack of ten 22.5 cm × 22.5 cm PVC plates (0.87 m
2
of surface
area) that are spaced to mimic the interstices of complex biogenic
environments. In total 983 056 sequences from the mitochondrial
cytochrome oxidase I gene were analyzed. They represented 2179
species belonging to a wide variety of major groups (e.g., 22 animal
phyla). More than two-thirds of these species were found in the
smallest size fraction analyzed (500-106 m). Remarkably, only 8.2%
of these could be matched to named species in any database, and
over 35% could not be confidently assigned to any group. These
figures reflect the fact that most marine life remains unrepresented
in genetic databases. Ongoing analyses include samples from shal-
low water reefs of the Red Sea, Panama, and Belize; mesophotic
reefs of Curacao; and acidified reefs of Papua New Guinea. Signif-
icance: Marine ecosystems are rapidly changing due to overfishing,
pollution, transport of non-native species, habitat destruction,
warming, and acidification. With the ability to obtain environmental
metabarcodes at relatively low cost, comprehensive analyses of hu-
man impacts on biodiversity are now possible.
Barcoding a biodiversity hotspot: Malaise-trapped insects of
Southern California
Joshua Kohn,
1
David O'Connor,
2
James Danoff-Burg,
3
Heather Henter,
1
and Bradley Zlotnick
4
1University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA.
2San Diego Zoo Institute for Conservation Research, 5600 San Pasqual Valley Road, Escondido, CA
92027, USA.
3New Knowledge Organization Ltd., 3630 Ocean Ranch Boulevard, Oceanside, CA 92056, USA.
4San Diego Barcode of Life, 3525 Del Mar Heights Road #139, San Diego, CA 92130, USA.
Corresponding author: Joshua Kohn (e-mail: jkohn@ucsd.edu).
Background: The California Floristic Province is recognized as a
global hotspot of endangered biodiversity owing to the high levels of
diversity and endemism present in various taxonomic groups (e.g.,
plants, vertebrates) and the high level of threat from anthropogenic
sources. Unfortunately, regional data on insect diversity that can be
readily compared with other regions is lacking. Results: We used a 60
km transect of six Malaise traps along the San Dieguito River Valley of
western San Diego County (California, USA) to begin a comprehensive
survey of insect diversity using DNA barcoding. Sequencing of 21 118
specimens revealed 2654 BINs (MOTUs) of which 1207 (45.5%) were
singletons. The large number of rare taxa precludes reliable estima-
tion of total species richness, but comparisons to studies of similar
sampling intensity suggest that the discovery rate of BINs in this re-
gion is not unusually high. This is surprising both because of the
region's high diversity in other taxonomic groups and also the sub-
stantial spatial -diversity evident in our data from different traps.
High -diversity should raise the discovery rate in comparison to stud-
ies where -diversity is low or only a single trap location was used to
sample a similar number of specimens. Significance: Either the in-
sect fauna of Southern California is not unusually diverse or total
diversity is so high, both here and elsewhere, that current levels of
Malaise trap sampling make broad-scale comparisons premature.
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Diversity of indoor fungi: what does it tell about the health of
buildings?
Helena Korpelainen and Maria Pietiläinen
Department of Agricultural Sciences, University of Helsinki, P.O. Box 27, 00014, Helsinki, Finland.
Corresponding author: Helena Korpelainen (e-mail: helena.korpelainen@helsinki.fi).
Background: Indoor human environments contain a variety of
microbes, some of which are detrimental to human health. Mould
in buildings is positively associated with several allergic and respi-
ratory effects, and certain moulds are toxigenic, meaning that they
can produce mycotoxins. Indoor fungi are identified traditionally
by culture-dependent methods, which inevitably have a low reso-
lution. In the present study, to increase accuracy in analyses and to
provide useful data for end-users, we conducted DNA metabarcod-
ing of the nuclear ITS2 region for indoor fungal samples. Results:
Both presumptive problem buildings and control buildings were
sampled multiple times, one of the buildings both before and after
a major renovation. Good-quality sequences were obtained, and the
results showed that DNA metabarcoding gives a high resolution in
fungal identification. The diversity and composition of fungal
classes, orders, families, genera, and species varied considerably
among samples; not only among samples from different buildings,
but also among samples from the same building. Also, temporal
variation was detected. Great variation among samples even within
the same building emphasizes the importance of multiple sam-
pling. We found that taxonomic diversity of fungi as such is not a
good indicator of indoor air quality—a diverse array of fungi occurs
even in a normal indoor environment. It is rather the presence of
certain fungal taxa and the relative proportion of fungi and plants (pri-
marily pollen) that are potential indicators of air quality. Significance:
The increased precision obtained through DNA metabarcoding provides
a potentially useful tool for analysing indoor mycoflora.
Comparison of five extraction protocols and direct PCR for the
recovery of trace DNA in chironomid pupal exuviae
Petra Kranzfelder,
1
Torbjørn Ekrem,
2
and Elisabeth Stur
2
1Department of Entomology, University of Minnesota, 1980 Folwell Avenue, 219 Hodson Hall, Saint
Paul, MN 55108, USA.
2NTNU University Museum, Department of Natural History, Norwegian University of Science and
Technology, NO-7491 Trondheim, Norway.
Corresponding author: Petra Kranzfelder (e-mail: kranz081@umn.edu).
Background: Efficient DNA extraction is critical to the success of
species identification using DNA barcoding and metabarcoding, espe-
cially when the total amount of extracted DNA is expected to be low.
Here, we compare the performance of five different DNA extraction
protocols and direct PCR in isolation of DNA from chironomid pupal
exuviae. Chironomidae (Insecta: Diptera) is a group of species-rich
aquatic macroinvertebrates widely distributed in freshwater environ-
ments and considered valuable bioindicators of water quality. A com-
monly used form of sampling chironomids involves collection of
pupal exuviae. Thus, using DNA barcoding to identify chironomid
pupal exuviae can be an important asset to understanding freshwater
ecology as well as advancing chironomid taxonomy through life stage
associations. Results: Genomic DNA was extracted from 61.2% of 570
sampled pupal exuviae. Several of the extracts were contaminated
with DNA from non-target organisms; only 13.7% of the sequences
produced from these extracts matched chironomid sequences. There
were significant differences in the extraction methods and direct PCR
with regards to cost, handling time, DNA quantity, PCR success, sequence
success, and the ability to sequence target taxa. The NucleoSpin Tissue
XS Kit, DNeasy Blood and Tissue kit, and QuickExtract DNA Extraction
Solution provided the best results in isolating DNA from single pupal
exuviae. Direct PCR and DTAB/CTAB methods gave poor results.
Significance: The observed differences in DNA extraction protocol
performance on trace DNA should be of interest to studies focusing on
noninvasive sampling in aquatic environments, such as environmen-
tal barcoding and metabarcoding.
Tracking evolutionary diversity and phylogenetic structure
across global forest dynamics plots using plant DNA barcodes
W. John Kress,
1
F. Andrew Jones,
2
Nathan G. Swenson,
3
and David L. Erickson
4
1Smithsonian Institution, Botany, MRC-166, National Museum of Natural History, Washington, DC
20013-7012, USA.
2Oregon State University, Department of Botany and Plant Pathology, Corvallis, Oregon, USA.
3University of Maryland, Department of Plant Biology, College Park, Maryland, USA.
4Food and Drug Administration, Beltsville, Maryland, USA.
Corresponding author: W. John Kress (e-mail: kressj@si.edu).
Forest dynamics plots span longitudes, latitudes, and habitat types
across the globe. These large plots, mostly located in primary and
secondary forested zones, provide natural laboratories for investiga-
tions of the ecological and evolutionary processes for species assembly
into communities. To understand how phylogenetic relationships
among species in a community influence these assembly processes a
mega-phylogeny of 1347 species of trees across 15 forest dynamics
plots in the Smithsonian ForestGEO network was constructed using
DNA barcode sequence data (rbcL, Atpf, and psbA–trnH). Three phylo-
genetic distance metrics that are commonly used to infer assembly
processes were estimated for each plot (phylogenetic distance [PD], mean
phylogenetic distance [MPD], and mean nearest taxon distance [MNTD]).
The partitioning of phylogenetic diversity among community plots was
quantified by comparing inter-community MPD and MNTD. Overall, evo-
lutionary relationships were highly resolved across the DNA barcode-
based mega-phylogeny, and phylogenetic resolution for each community
plot was improved when estimated within the context of the mega-
phylogeny. The comparisons among plots based on the mega-phylogeny
demonstrated that the communities in the ForestGEO plots in general
appear to be assemblages of more closely related species than expected
by chance. In addition, differentiation among communities is very low,
which suggests a more substantial floristic connection among commu-
nities than previously thought. The use of DNA barcodes for estimating
community phylogenies can also inform conservation priorities across
habitats and landscapes.
Development of a DNA barcode database of captive animals in
Thiruvananthapuram Zoo, Kerala, India
U. Suresh Kumar,
1
R.V. Ratheesh,
2
Jacob Alexander,
3
and E.V. Soniya
2
1Regional Facility for DNA Fingerprinting (RFDF), Rajiv Gandhi Centre for Biotechnology, Thycaud PO,
Thiruvananthapuram - 695014, Kerala, India.
2RFDF, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram - 695014, Kerala, India.
3Zoological Gardens, Department of Museum & Zoos, Thiruvananthapuram, Kerala, India.
Corresponding author: U. Suresh Kumar (e-mail: sureshkumar@rgcb.res.in).
Background: Animal poaching is one of the major threats to animals in
wild. It is imperative to punish the offenders to prevent illegal hunting.
Samples confiscated by forest officers in Kerala Forest Department are
forwarded to our laboratory for species-level identification, so as to en-
able them to charge the case and punish the offenders. DNA barcoding
helps to identify animals even from minute or cooked samples. The exact
identification of species from the Western Ghats region of Kerala, which
is one of the hottest among biodiversity hotspots, is often difficult or
impossible due to the lack of reference sequences in databases. This is
more prominent in the case of endemic species, where most of the times
reference data are not available. Results: We have collected blood/mus-
cle samples from 67 captive animals in Thiruvananthapuram Zoo, India.
Samples included endemic and threatened species present in the West-
ern Ghats and local as well as migratory birds. DNA was isolated from the
samples, and COI as well as cytochrome b genes were amplified and
sequenced using universal primers. The present study generated se-
quences of 16 mammal, 8 reptile, and 36 bird species, of which 9 species
were previously unrepresented in NCBI GenBank or BOLD. The newly
generated sequences have been deposited in BOLD under the project
WFDB. Significance: Species identification using DNA barcoding has
become an integral part of wildlife forensics, providing evidence to the
legal bodies to punish the offenders in poaching cases and thus aiding in
the conservation of endangered and endemic animals. An authentic DNA
barcode reference database is a pre-requisite for enabling the correct
Abstracts 239
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identification of species. The present study to develop a DNA barcode
reference database of the captive animals in the Thiruvananthapuram
Zoo, Kerala, India is an initiative in this regard, which will augment
future wildlife investigations.
Estimating the extent of adulteration in highly traded
medicinal plants in herbal raw drugs market in South India
J.U. Santhosh Kumar,
1
V. Krishna,
1
G.S. Seethapathy,
2
S. Ragupathy,
3
Steven G. Newmaster,
3
G. Ravikanth,
2
and R. Uma Shaanker
4
1Department of Post Graduate Studies and Research in Biotechnology, Kuvempu University, Shimoga-
577451, India.
2Ashoka Trust for Research in Ecology and the Environment, Royal Enclave, Srirampura, Jakkur Post,
Bangalore-560064, India.
3Centre for Biodiversity Genomics (CBG), Department of Integrative Biology, College of Biological
Science, University of Guelph, Guelph, ON N1G 2W1, Canada.
4Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore-560065, India.
Corresponding author: J.U. Santhosh Kumar (e-mail: santhu.ju@gmail.com).
Background: Medicinal plants are widely used in India, Africa, and
many other regions including China and South East Asia for treating
various disease and for improving general health and vigor. In recent
years, the global economy relating to international trade of herbal prod-
ucts and the alternative medicine market has been increasing at a rapid
rate of ⬃15% annually. However, with increasing international trade in
herbal medicinal products, there is also an increasing concern over the
safety and efficacy of these products. It is widely believed that there
might be wide spread adulteration and species admixtures in the herbal
trade due to lack of quality standards. A number of adverse consequences
of such species admixtures on the health and safety of consumers have
been reported. In this study, we assess the extent of species admixtures in
the raw herbal trade of three important medicinal climbers: Coscinium
fenestratum,Hemidesmus indicus, and Embelia ribes, in South India using the
nrDNA ITS and chloroplast psbA–trnH spacer regions. First, we developed
DNA barcodes for authentic species and then used these barcodes to
identify the ingredients in raw herbal products obtained from 15 markets
in southern India. Results: Our results indicate that there is widespread
adulteration of these medicinal climbers (Coscinium fenestratum and Embe-
liaribes) with other morphologically similar and co-occurring climbers
such as Berberis species, E. tsjeriam-cottam, and Maesa indica species. In the
case of Hemidesmus indicus, all of the market samples were sold as authen-
tic species. Significance: We discuss the results and the need for a robust
herbal drugs authentication system to regulate quality in the raw herbal
trade market.
DNA barcoding of sea cucumbers (Echinodermata:
Holothuroidea) of the southwest coast of India
A. Biju Kumar
1
and R. Deepa
2
1Department of Aquatic Biology and Fisheries, University of Kerala, Thiruvananthapuram, Kerala
695581, India.
2Department of Zoology, MSM College, Kayamkulam, Kerala, India.
Corresponding author: A. Biju Kumar (e-mail: abiju@rediffmail.com).
Background: One of the most valuable seafood items and organisms
harbouring a wide array of biologically active compounds, sea cucum-
bers, play critical roles in marine ecosystem functioning. The diversity of
holothurians is great in the Indo-Pacific area, and these organisms are
included in Schedule 1 of the Wildlife (Protection) Act of India, regulating
illegal capturing, trade, and export. However, there are only a few re-
cords of sea cucumbers from the southwest coast of India, and the com-
plexity in taxonomy is due to subtle morphological characteristics and
variations in the ossicles of the body wall. DNA barcoding is very signifi-
cant for the identification of holothurians at all developmental stages
and for controlling their illegal trade, both of which are pre-requisites for
conservation. We generated cytochrome c oxidase subunit I (COI) se-
quences for 15 species of holothurians collected from shallow coastal
waters of southwest coast of India. Results: COI unambiguously identi-
fied most of the commercially valuable holothurian species, and this
study generated barcodes of the remaining species for the first time,
providing a genetic barcode for the identification of known species from
India. The highest GC content in the sequences was obtained for H. hilla
(49.84%), and least was shown by Thyonina sp. (40.22%). Sequences of
Holothuria cinerascens,H. moebii,H. arenicola,H. leucospilota,H. hilla,H. fuscocinera,
H. imitans,Stichopus hermanni, and S. horrens showed 99% similarity with
the sequences of the same species available in GenBank. COI data were
generated for the first time for H. pardalis,H. difficilis,Phyllophorus cebuensis,
Trachasina crucifera,Thyonina articulata,Leptopentacta imbricata, and Aslia
forbesi.Significance: This study has shown that DNA barcoding can be used
for precise identification of holothurians and for further taxonomic studies;
the resulting database is also useful for conservation and management.
DNA barcoding and invading suckermouth sailfin catfish
Pterygoplichthys (Siluriformes: Loricariidae) in India: issues with
possible hybrids
A. Biju Kumar and R. Smrithy
Department of Aquatic Biology and Fisheries, University of Kerala, Thiruvananthapuram, Kerala
695581, India.
Corresponding author: A. Biju Kumar (e-mail: abiju@rediffmail.com).
Background: Non-native fishes have been known from freshwater eco-
systems of India since the 1950s. The recent invasions by a number of
suckermouth sailfin catfish species from the genus Pterygoplichthys in
India is a matter of concern, especially considering the fast pace of inva-
sion coupled with the ecological consequences to indigenous species.
Fishes of the genus Pterygoplichthys can be identified by key taxonomic
characters such as the presence of nine or more (usually 10) dorsal fin
rays, supraoccipital bone bordered posteriorly by one large plate, and the
absence of an elevated supraoccipital process. Native to the streams,
floodplain lakes, and marshes of South America, Pterygoplichthys spp.
have invaded inland water bodies of various countries around the world
through the uncontrolled pet trade, resulting in serious ecological and
economic consequences. Partial sequences of the mitochondrial genes
cytochrome c oxidase subunit I (COI), cytochrome b (cytb), and ribosomal
16S ribosomal RNA were prepared from tissue samples of invasive Ptery-
goplichthys spp. from various states of India. Results: Species-level match
queries for our Pterygoplichthys COI sequences in GenBank recorded 100%
genetic similarity with four species, namely P. ambrosettii,P. pardalis,P.
disjunctivus, and P. joselimaianus. Sequences of cytb were 99%–100% similar
to those of Hypostomus plecostomus,P. disjunctivus, and P. pardalis. Finally,
16S rRNA showed 98%–98% sequence similarity with P. ambrosettii and H.
cochliodon. The haplotypes demonstrated low genetic variation, and the over-
lapping morphological characters of the specimens examined indicated pos-
sible hybrids of Pterygoplichthys and that accidental introductions of the
hybrids into natural water bodies through the aquarium trade might have
brought about the sympatry. Significance: The present study discusses the
need for molecular markers to delineate hybrids and barcoding of topotypes
to establish the identity of invasive sailfin catfish in India.
What it takes to identify 5120 vascular plant species of Canada
with DNA barcodes
Maria Kuzmina,
1
Thomas Braukmann,
1
Anuar Rodrigues,
2
and Stephanie deWaard
1
1Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, 50 Stone Rd. E, Guelph, ON N1G
2W1, Canada.
2University of Toronto - Mississauga, 3359 Mississauga Rd, Mississauga, ON, Canada.
Corresponding author: Maria Kuzmina (e-mail: kuzminam@uoguelph.ca).
Background: The Biodiversity Institute of Ontario has joined with 10
Canadian herbaria to produce a complete DNA barcode library for the
vascular plants of Canada. Each of the 5117 species with non-hybrid origin
(VASCAN) is represented by at least one barcode. Results: From 2009-
2015, we sampled 1-3 specimens per species and generated a total of
10 642 DNA barcodes with 86% coverage for rbcL, 47% for Atpf, and 75%
for ITS2 (on average two loci per species). In order to assess the phylogeny-
based method that is most effective for DNA barcode identification of
Canadian vascular plant species, we compared the use of a mega phylog-
eny for all Canadian flora versus partial trees for the families (RAxML). In
addition, we explored several methods of sequence alignment (MUSCLE,
240 Genome Vol. 58, 2015
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MUFFT, transAlign, SATe) to ascertain their effect on identification when
using phylogeny. The advantages and limitations of using the marker
combination of rbcL+ITS2 versus rbcL+Atpf are also examined. In addi-
tion, species-rich genera, especially groups with poor species resolution,
were analyzed separately using all available sources of genetic informa-
tion (that are found on BOLD and from GenBank), resulting in a potential
recommendation for using additional supplementary markers for these
groups to improve species discrimination. Significance: This collabora-
tive effort with researchers and herbaria across Canada presents the
accumulation of the first DNA barcode library for an entire country's
vascular plant flora. It will serve as an important resource for a wide
range of applications that require species-level identifications, including
those in forensics, forestry, and conservation biology.
Comparative phylogeography and population genetic structure
of 10 widespread small vertebrate species in Morocco
Aude Lalis,
1
Violaine Nicolas,
1
Annemarie Ohler,
1
Aurélien Miralles,
2
Pierre André Crochet,
2
Raphael Leblois,
3
Soumia Fadh,
4
Ahmed El Hassani,
5
Touria Bennazou,
6
and Christiane Denys
1
1Muséum National d'Histoire Naturelle, UMR7205 ISYEB 55 rue Buffon, 75005 Paris, France.
2CEFE-CNRS Campus du CNRS, 1919 route du Mende, 34293 Montpellier, France.
3INRA - UMR1062 CBGP 34988 Montferrier-sur-Lez, France.
4Université Abdelmalek Essaadi Tétouan, Morocco.
5Institut Scientifique de Rabat, Avenue Ibn Battota, B.P. 703, Agdal 10106 Rabat, Morocco.
6Université Mohammed V-Agdal, Faculté des Sciences, Département de Biologie, 4 Avenue Ibn Battouta
B.P. 1014 RP, Rabat, Morocco.
Corresponding author: Aude Lalis (e-mail: lalis@mnhn.fr).
Background: Comparative phylogeography makes use of congruent
patterns of genetic variation among unrelated taxa with partially
overlapping geographical ranges to elucidate the influence of histor-
ical events on current patterns of biodiversity. Here we analyze spatial
divergence patterns of animal species and relate them to the evo-
lutionary history of fragmented areas among the most threatened
vegetation types in Morocco. We used comparative phylogeo-
graphic analyses to detect common geographic signals in taxa with
co-distributed ranges across Moroccan habitats and to elucidate the
influence of historical events on current patterns of biodiversity.
Results: We document spatial genetic divergence of four orders
(Rodentia, Soricomorpha, Anura, Squamata) including 10 species
(Rodentia: Meriones shawii,Apodemus sylvativus,Mus spretus,Gerbillus
campestris; Soricomorpha: Crocidura russula; Anura: Hyla meridionalis,
Bufo boulengeri,Pelophylax saharicus,Amietophrynus mauritanicus;
Squamata: Acanthodactylus erythrurus), and relate them to the evolu-
tionary history of the naturally or anthropogenically fragmented
areas in Morocco. Significance: The results will be discussed in the
context of biodiversity conservation. Coastal and central regions of
Morocco are characterized by a significant increase of urban areas
and tourism involving degradation of natural environments. Such
anthropogenic stresses and the increase of intensive agriculture in
these regions can cause significant damage to the biodiversity of
small vertebrates very subservient to the local environment.
Barcoding Paralobesia cypripediana (Tortricidae): a stealthy
micromoth feeding on the threatened orchid Cypripedium reginae
Jean-François Landry,
1
Marilyn Light,
2
and Michael MacConaill
2
1Agriculture & Agri-Food Canada, Canadian National Collection of Insects K.W. Neatby Building,
960 Carling Ave., Ottawa, ON K1A 0C6, Canada.
2Gatineau, Quebec, Canada.
Corresponding author: Jean-François Landry (e-mail: landryjf@agr.gc.ca).
Background: Paralobesia cypripediana is a rare tortricid whose larvae
feed on the seed pods of the Showy Ladyslipper, Cypripedium reginae,a
threatened North American orchid. Larvae appear to be strictly
monophagous on a single species of orchid, and thus the moth species
has few and highly localized populations where the orchid host is in
sufficient abundance. Results: DNA barcoding of all immature stages
(eggs, larvae, pupae) confirmed the presence of a single species on the
orchid host forming a compact BIN distinct from 14 other Paralobesia
BINs. Comparison with barcodes of other Paralobesia species showed that
occurrence of cypripediana has been overestimated and that many histor-
ical records and all barcode records of the species were based on misiden-
tifications of other Paralobesia species. Barcoding host-based specimens of
cypripediana had a cascading effect on rectifying the identification of
nearly all Paralobesia barcode records in BOLD. This included the correct
association of the grape berry moth, Paralobesia viteana, a pest of grapes
and vineyards. Adults of cypripediana were successfully reared after 5
years of trials. Evidence of past occurrences of cypripediana in areas where
the host plant no longer occurs was obtained from old herbarium plant
specimens. Significance: Barcoding can uncover evidence of misidenti-
fications among confusing or cryptic species, reveal unsuspected species,
help resolve taxonomic synonymies, and in absence of the morphologi-
cally diagnostic adult stage confirm range occurrences from immature
stages.
Development of a molecular detection test, based on PCR
technology, to detect specific mushroom DNA in soil samples
Genevieve Laperriere,
1
Mélodie B. Plourde,
2
and Hugo Germain
2
1Université du Québec a
`Trois-Rivières, département de biologie médicale, 3351 boulevard des Forges,
Trois-Rivières, Québec, Canada.
2Université du Québec a
`Trois-Rivières, département de chimie, biochimie et physique, 3351 boulevard
des Forges, Trois-Rivières, Québec, Canada.
Corresponding author: Genevieve Laperriere (e-mail: genevieve.laperriere@uqtr.ca).
Mushroom picking is a source of income, but it can be difficult and
unprofitable. Mushroom pickers have to possess knowledge about the
ecology, morphology, and life cycle of the mushrooms to assess where
and when it can be found. Likewise, fruiting in mushrooms appears at
specific times during their development when conditions are optimal.
In some cases, the mycelium can be abundant although no sporocarps
are observed; or the site may represent the appropriate ecological
niche, but the mycelium may be absent from the soil. Our research
project is to develop a molecular detection test, based on polymerase
chain reaction (PCR) technology, to detect mushroom DNA in soil
samples for four edible species that are of commercial interest: the
swollen-stalked cat (Catathelasma ventricosum), the chanterelle (Cantharellus
cibarius), the Canadian pine mushroom (Tricholoma magnivelare), and
the lobster mushroom (Hypomyces lactifluorum). DNA was extracted from
sporocarps, and the intergenic region and 5.6S were amplified with the
universal ITS1F-ITS4 primers. Several PCR products per species were se-
quenced at Genome Quebec, and sequences were aligned with additional
sequences gathered from GenBank. We designed specific primers for all
the species with multiplex PCR properties using a combination of soft-
ware (CLC MainWorkbench 7, PrimerPlex 2, and Primer3) and user input.
Primers were tested on different samples (plasmid and genomic DNA) to
confirm that they were species specific, they produced a band at the
appropriate molecular weight, that they could perform in multiplex con-
ditions, and to assess how many genome equivalents (dilution curve)
could be detected. We are currently doing validation of our test on DNA
extracts from soil samples. This molecular detection tool could confirm
the presence or the absence of the targeted mushroom, evaluate the
potential of sites presenting adequate ecological properties, and eventu-
ally help to develop the mushroom tourism and cultivation.
Biodiversity discovery, documentation, and analysis in the
digital era
John La Salle
Atlas of Living Australia, CSIRO National Collections and Facilities, GPO Box 1700, Canberra ACT 2601,
Australia.
E-mail for correspondence: john.lasalle@csiro.au.
The next generation of biodiversity analysis laboratories will need the
ability to keep pace with the impact humans are having on this planet.
This will require creating virtual environments where we can bring to-
gether various data types (genomic, phenomic, distribution) and com-
bine them with environmental information, phylogenetic hypotheses,
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and a suite of analysis tools. The Atlas of Living Australia is providing a
model of such an environment at a continental scale. This presentation
will provide an overview of some of the achievements and capabilities of
the Atlas, as well as exploring the concept of virtual museums and ways
of accelerating the capture of phenomics information in an attempt to
keep pace with the rest of the ‘omics explosion.
From a local barcoding initiative to a continental-scale, multi-
institutional assessment of avian diversification in the Neotropics
Pablo D. Lavinia,
1
Patricia Escalante,
2
Natalia C. Garcia,
1
Ana S. Barreira,
1
Natalia Trujillo-Arias,
1
Pablo L. Tubaro,
1
Kazuya Naoki,
3
Cristina Y. Miyaki,
4
Fabricio R. Santos,
5
and Dario A. Lijtmaer
1
1Division Ornitologia, Museo Argentino de Ciencias Naturales, Av. Ángel Gallardo 470, Buenos Aires,
Argentina.
2Instituto de Biología, Universidad Nacional Autónoma de México, Postal office box 70-153, 04510,
México DF, México.
3Instituto de Ecología, Universidad Mayor de San Andrés, Postal office box 6394, Correo Central, La
Paz, Bolivia.
4Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo,
Rua do Matão 277, 05508-090, São Paulo, Brazil.
5Departamento de Biologia Geral, Instituto Ciências Biológicas, Universidade Federal de Minas Gerais,
Av. Antônio Carlos 6627, 31270-901, Minas Gerais, Brazil.
Corresponding author: Pablo D. Lavinia (e-mail: pablodlo23@gmail.com).
Background: In 2005, the Museo Argentino de Ciencias Naturales joined
the All Birds Barcoding Initiative aiming to obtain the DNA barcodes of
the birds of Argentina. As the project expanded its frontiers to Bolivia, in
collaboration with local institutions, several cases of deep intraspecific
divergence were revealed. Among them, that of the Red-crowned Ant
Tanager (Habia rubica) stood out because of the strikingly high (around 7%)
COI distance found between the allopatric populations of the Atlantic
Forest in Argentina and the Yungas–Amazonia complex in Bolivia. Inde-
pendently, a similar pattern of east–west divergence was found in Mexico
and Middle America as part of the project to barcode the birds of Mexico
and Guatemala. All this eventually led to the joint effort of Argentinian,
Bolivian, Brazilian, and Mexican researchers to unveil the evolutionary
history of H. rubica at a continental scale. Results: We explored the phy-
logeographic patterns of intraspecific diversity within H. rubica through-
out its widespread but disjunct distribution from Mexico to Argentina.
We performed phylogenetic, phylogeographic and genetic population
analyses based on COI and other mitochondrial and nuclear genes, com-
plemented with the assessment of coloration and behavioural differen-
tiation. The evolutionary history of H. rubica through the last 5 million
years seems to have been shaped by the uplift of the Northern Andes, the
formation of the Isthmus of Panama, the establishment of the open
vegetation corridor, and Quaternary climatic fluctuations. This resulted
in levels of genetic, morphological, and behavioural divergence that jus-
tify considering at least three different species within this diversified
lineage. Significance: Our study clearly illustrates how local screening of
diversity through DNA barcodes can grow into large-scale, multi-
institutional collaborative projects able to provide meaningful insights
into the evolutionary history of certain regions and taxa of interest.
Calibrating the molecular clock beyond cytochrome b:
assessing the evolutionary rate of COI in birds
Pablo D. Lavinia,
1
Kevin C.R. Kerr,
2
Pablo L. Tubaro,
1
Paul D.N. Hebert,
3
and Dario A. Lijtmaer
1
1Division Ornitologia, Museo Argentino de Ciencias Naturales, Av. Ángel Gallardo 470, Buenos Aires,
Argentina.
2Toronto Zoo, Conservation, Education, and Wildlife Division, 361A Old Finch Avenue, Toronto,
ON M1B 5K7, Canada.
3Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Pablo D. Lavinia (e-mail: pablodlo23@gmail.com).
Background: Estimating the age of species or their component lineages
based on sequence data is crucial for many studies in avian evolutionary
biology. Although calibrations of the molecular clock in birds have been
performed almost exclusively using cytochrome b (cytb), they are com-
monly extrapolated to other mitochondrial genes. The existence of a
large, standardized cytochrome c oxidase subunit I (COI) library gener-
ated as a result of the DNA barcoding initiative provides the opportunity
to obtain a calibration for this mitochondrial gene in birds. Results: COI
evolves on average 14% slower than cytb, but considerable variation both
among and within avian orders was observed, precluding the use of this
value as a standard adjustment for the COI molecular clock for birds.
Distances for cytb are higher than those for COI for closely related species,
but the values become similar as the divergence between the species
increases. This pattern appears to result from the comparison of a gene
(COI) with a relatively constant rate and one (cytb) with a negatively time-
dependent rate, a difference that could be related to lower functional
constraints on a small number of sites in cytb that allow it to initially
accumulate mutations more rapidly than COI. Significance: Since most
species pairs do not fall into the “danger zone” of low divergences, both
COI or cytb could be used to estimate species ages with similar results in
most cases. However, when a sole calibration is used to assess the age of
different nodes in phylogenetic analyses COI could be more appropriate
than cytb because its evolutionary rate appears relatively more uniform.
Exploring diversity and distribution of a nudibranch genus
(Chromodoris) in the Indo-Pacific using DNA barcodes
Kara K.S. Layton,
1
Nerida G. Wilson,
2
and Jason Kennington
3
1University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
2Western Australian Museum, 49 Kew Street, Welshpool, WA 6106, Australia.
3Centre for Evolutionary Biology, University of Western Australia, 35 Stirling Highway, Crawley,
WA 6009, Australia.
Corresponding author: Kara K.S. Layton (e-mail: kara.layton@research.uwa.edu.au).
Background: Chromodoris is a genus of colourful sea slug that seques-
ters toxins from its prey and is widely distributed throughout the
Indo-Pacific. However, morphological similarities between congener-
ics, coupled with an incomplete knowledge of species ranges, make
species identification particularly difficult in this genus. The true di-
versity of Chromodoris in the Indo-Pacific is unknown, and this study
employs DNA barcoding to aid in species delimitation and the discov-
ery of hidden diversity. A portion of this project will focus on diversity
in the poorly-understood northwestern region of Western Australia,
a biodiversity hotspot known for intensive mining development.
Results: This work presents a biodiversity baseline for Chromodoris spe-
cies in the Indo-Pacific, with an emphasis on Western Australia, using
the barcode region of COI. A total of 181 individuals from 16 mor-
phospecies were sampled from wide geographic areas in the Indo-
Pacific, spanning entire species ranges, and providing crucial insight
into species-level relationships in this genus. We also use COI data to
explore phylogeographic patterns in two widespread species and
highlight an apparent rapid radiation of Chromodoris.Significance:
Documenting biodiversity is essential for identifying species in need
of conservation management, and is particularly important in areas
with high species diversity and endemism. This study fills in signifi-
cant sampling gaps in the Indian Ocean, which are often lacking for
“Indo-Pacific” taxa. We also advance the understanding of phyloge-
netic patterns in a recent radiation of nudibranchs and highlight
patterns of population structure in two widespread congenerics. Fu-
ture work will incorporate an exon-capture approach for resolving
species-level relationships.
DNA barcoding on the slime trail: the Canadian invasion of the
terrestrial gastropod Cepaea nemoralis (Stylommatophora:
Helicidae) and new perspectives for studying threatened
species
Kara K.S. Layton,
1
Jeremy R. deWaard,
1
Stephanie L. deWaard,
1
Monica R. Young,
1
Kareina D'Souza,
1
and Annegret Nicolai
2
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
2UMR 6553 EcoBio, Université Rennes 1, Campus Beaulieu, Bât. 14A, 35042 Rennes cedex, France.
Corresponding author: Annegret Nicolai (e-mail: annegret.nicolai@univ-rennes1.fr).
Background: The phylum Mollusca is a diverse animal group that is
poorly studied although many species are problematic in two opposite
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ways. Some species are successful colonizers that might become in-
vaders, whereas other species suffer from environmental changes and
might become endangered. We have begun the construction of a DNA
barcode reference library for Canada's native and introduced terres-
trial gastropods. Using the invasive snail Cepaea nemoralis, we also
tested the reliability of the non-invasive method of visceral swabs
from live specimens for obtaining barcodes. Results: Barcode records
for more than half of Canada's native and introduced terrestrial gas-
tropod species have been compiled. A new introduced slug species has
been discovered in New Brunswick: Arion flagellus, and the distribution
of some native gastropod has been revised (e.g., two rare slug species
of the genera Philomycus are sympatric on Pelee Island, ON). Thanks to
wide-ranging sampling of C. nemoralis, extending to both eastern and
western provinces in Canada as well as its native range in Europe, this
work has identified three separate introductions. Two of these intro-
ductions originate from widespread lineages in Europe, with a third
introduction from a rare lineage with a restricted range in Scotland
and Poland. Significance: A DNA barcode reference library for Cana-
dian terrestrial gastropods is an essential platform for future work,
particularly for the detection of cryptic ground-dwelling species from
soil samples using environmental DNA. Our study also highlights the
use of unconventional methods for DNA barcoding. The visceral swab
protocol for terrestrial gastropods has been optimized, allowing for
easier inter-continental shipping of DNA material. This method has
been successfully applied to identify unique C. nemoralis lineages in
Canada and to uncover three separate invasions from Europe. Further-
more, using such non-invasive protocols offer new possibilities of
studying gastropod distribution or the genetic structure of species at
risk. As such, this work has advanced our understanding of terrestrial
mollusc biodiversity in Canada.
New methods give insight into DNA barcoding Canada's
freshwater mussels (Bivalvia: Unionoida)
Kara K.S. Layton, Kareina D'Souza, Stephanie L. deWaard,
Monica R. Young, and Jeremy R. deWaard
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
Corresponding author: Kara K.S. Layton (e-mail: klayton@uoguelph.ca).
Background: Molluscs are the most endangered group of invertebrates
in Canada, and there is a need to document biodiversity in this phylum.
This group has seen substantial declines due to the presence of invasive
mussels in our waterways and because many species are sensitive to
environmental change. As such, we are constructing a DNA barcode ref-
erence library for Canada's freshwater mussels as well as testing the
utility of two non-invasive methods for obtaining barcodes: visceral
swabs from live specimens and periostracum scrapings from dry shells in
natural history collections. Results: Barcode records for more than half
of Canada's 54 freshwater mussel species have been compiled. We ob-
served 65% sequencing success with visceral swabs and 1% with periostra-
cum scrapings, suggesting additional work is needed to enhance success
in the latter. A preliminary experiment demonstrated a 1.4-fold increase
in sequencing success (92%) with swabs from store-bought blue mussels,
suggesting that in situ specimens face additional issues with PCR inhibi-
tion, likely caused by excessive polysaccharides. Low success from dry
museum samples is likely a result of DNA degradation and may be com-
bated through the use of internal primers. Significance: A DNA barcode
reference library for Canadian freshwater mussels is an essential plat-
form for future work, particularly for the detection of aquatic species
from water samples using environmental DNA. Our study highlights the
use of unconventional methods for DNA barcoding, having important
implications for the utilization of dry shell collections in natural history
archives. The optimization of field and laboratory protocols will increase
sequence acquisition from future collections as well as make monitoring
faster, easier, and more objective. In all, this work has advanced our
understanding of freshwater mussel diversity in Canada and presents
new methods for obtaining DNA barcodes from threatened populations.
Utility of the ribosomal DNA ITS2 region for the identification
of Calliphoridae (Diptera: Calliphoridae) of forensic
importance in Colombia
Edison Lea and Lyda Castro
Universidad del Magdalena, Colombia.
Corresponding author: Lyda R. Castro (e-mail: lydaraquelcastro@hotmail.com).
Background: Blowflies of the family Calliphoridae are recognized world-
wide for their importance in forensic entomology. However, identifica-
tion of these insects by morphological methods is often difficult,
particularly for poorly preserved specimens and larvae. The present
study evaluated the potential use of the ITS2 region of the nuclear ge-
nome for the identification of Calliphoridae species in Colombia. We
sequenced a total of 520 bp in 44 individuals representing 16 species and
calculated intraspecific and interspecific distance values using the K2P
model. Results: Intraspecific pairwise distance values ranged from 0% to
0.48%, while interspecific values varied between 4.5% and 55.4%, confirm-
ing the utility of the gene for specimen identification to the species level.
We used the NEBCutter application to develop an identification tool
using PCR-RFPLs, and we selected and tested four restriction enzymes
that successfully differentiated all the species. We also performed phylo-
genetic neighbour-joining and Bayesian phylogenetic analyses to evalu-
ate evolutionary relationships among Calliphoridae using the ITS2.
Significance: The obtained distance values using the ITS2 gene are
slightly higher than those obtained using the COI gene in the same spe-
cies, but confirmed the results found using the COI as a barcode. Our
results suggest that the ITS2 region is an effective and low-cost tool for
the identification of species of the family Calliphoridae in Colombia.
A metabarcoding approach to measuring beta diversity: Costa
Rican dry forest arthropods and their associated bacteria
Lisa Ledger, Shadi Shokralla, Joel F. Gibson, Rafal Dobosz,
and Mehrdad Hajibabaei
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Lisa Ledger (e-mail: lledger@uoguelph.ca).
Background: In a recent study, DNA metabarcoding was proven suc-
cessful at capturing the diversity of terrestrial arthropods and associated
bacteria within the contents of a single Malaise trap sample. Our research
evaluates whether DNA metabarcoding is an appropriate method for
measuring diversity in both terrestrial arthropods and their associated
bacterial groups using a study area of three plots of tropical dry forest
with differing land-use histories within a 2 square kilometer section of
the Area de Conservacion Guanacaste, Costa Rica. Environmental DNA
(eDNA) was obtained from nine Malaise traps and amplified using multi-
ple primer sets: three cytochrome c oxidase subunit I (COI) arthropod
minibarcode primers and two 16S primers targeting the V3, V4, and V6
variable regions. Following sequencing with an Illumina MiSeq, se-
quences were identified to species level using the Barcode of Life Data
Systems (BOLD) and GenBank databases for COI, and to genus level using
RDPipeline for 16S. Operational taxonomic units (OTUs) were assembled
for both COI and 16S sequences at 98% and 97% sequence similarity,
respectively. Statistical analysis of beta diversity using the VEGAN pack-
age for R was performed at various taxonomic levels and with OTUs for
both COI and 16S. Results: Significant arthropod and bacterial beta di-
versity is present for taxonomic data and OTUs. OTUs outperform taxo-
nomic identification at capturing the genetic diversity present within the
available sequence data (85% of good quality sequence vs. 19%) and in
correlating it to site differences. Of the COI OTU clusters, 62% could be
assigned to orders, yielding 19 orders inclusive of the seven identified
with species-level assignment. Significance: This research demonstrates
the viability of metabarcoding as a tool for the assessment of biodiversity
using eDNA. The use of OTUs in conjunction with species- or genus-level
identification captures available diversity where gaps in taxonomic clas-
sification are present.
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Species delimitation of Eupithecia (Lepidoptera: Geometridae)
using a ddRAD-Seq approach
Kyung Min Lee and Marko Mutanen
Insect Genomics Group, University of Oulu, FIN-90014, Oulu, Finland.
Corresponding author: Kyung Min Lee (e-mail: kyungmin.lee@oulu.fi).
Background: The order Lepidoptera (butterflies and moths) is one of the
most species-rich groups of organisms, with 500 000 species estimated
world-wide. Lepidoptera are also among the best-investigated insect
groups, and have the best coverage of DNA barcode data. It is widely
recognized, however, that DNA barcodes alone provide a narrow perspec-
tive for species delimitation in some groups. This study aims to address a
significant impact on the theoretical principles of taxonomic decisions,
especially species delimitation; with rapidly accumulating genomic data,
species delimitation no longer needs to be based on difficult to interpret
and measure morphological features and non-quantifiable criteria. Re-
sults: We used double digest restriction site associated DNA (ddRAD) tags
to contribute to delimitation of species within a diverse genus of Lepi-
doptera (Eupithecia) for which DNA barcodes provide an efficient tool for
species identification. We chose PstI and MseI to generate a large number
of markers from which genes and species will be constructed under a
multispecies coalescent framework. On the basis of two lanes of Illumina
sequencing, we delineated 36 species among 46 individuals identified by
morphological characters, running two RAD analysis pipelines (Stacks
and pyRAD) and estimating genetic distances using statistical methods.
Significance: Our study presents the first test of ddRAD-Seq based spe-
cies delimitation of any lepidopteran group. Results are expected to pro-
vide new insights on a standardized way to delimit insect species based
on genome-wide SNP data.
Comparison of sampling methods including blowfly-derived
mammal DNA for tropical mammal diversity assessments
Ping-Shin Lee,
1
Kong-Wah Sing,
1
Han-Ming Gan,
2
and John-James Wilson
1
1Museum of Zoology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603
Kuala Lumpur, Malaysia.
2School of Science, Monash University Malaysia, 46150 Selangor, Malaysia.
Corresponding author: Ping-Shin Lee (e-mail: leepingshin@gmail.com).
Most tropical mammal species are threatened or data-deficient. Tradi-
tional monitoring approaches impede data collection because they can
be laborious, expensive, and struggle to detect cryptic diversity. Monitor-
ing approaches using mammal DNA derived from invertebrates has re-
cently been suggested as cost- and time-effective alternatives. As a step
towards development of blowfly-derived DNA as an effective method for
mammal monitoring in tropical biodiversity hotspots (e.g., Peninsular
Malaysia), we (i) designed and tested primers that can selectively amplify
mammal cytochrome c oxidase subunit I (COI) DNA mini-barcodes in the
presence of high concentrations of blowfly DNA; (ii) determined the per-
sistence period of amplifiable mammal mtDNA in blowfly guts through a
laboratory feeding experiment; (iii) evaluated the effectiveness of
blowfly-derived mammal DNA in generating species inventories as com-
pared to other traditional approaches (i.e., cage trapping, mist netting,
hair trapping, and scat collection). A short (205 bp) mammal COI DNA
mini-barcode, suitable for high-throughput sequencing, could distin-
guish most mammal species (including separating dark taxa). The persis-
tence period of amplifiable mammal DNA in blowfly guts was in the
range of 24 h to 96 h post-feeding—this indicates the need for retrieving
flies within 24 h of capture to detect mammal mtDNA in sufficient quan-
tity and quality. Field surveys in Ulu Gombak Forest Reserve using differ-
ent sampling methods (November to December 2014) revealed that the
blowfly-derived DNA approach obtained the most diverse samples. This
includes a near-threatened species, Trachypithecus obscurus, the dusky leaf
monkey, which is a new record for the forest reserve.
Muthi from the wild: a survey of bulbous and perennial herbs
traded at the Faraday Muthi Market in Johannesburg, South
Africa, using DNA barcoding as an identification tool
Dorcas M. Lekganyane, Olivier Maurin, Herman van der Bank,
and Michelle van der Bank
African Centre for DNA Barcoding, University of Johannesburg, P.O. Box 524, Auckland Park, 2006,
South Africa.
Corresponding author: Dorcas M. Lekganyane (e-mail: didi.lekganyane@gmail.com).
Background: Approximately 80% of South Africans use traditional med-
icine (muthi) as an important component of primary health care, which
is traded at informal markets. Specialized gatherers harvest these plants
mostly from wild resources, which reach the market via middlemen.
Importantly, 86% of plants harvested will result in the death of the plant.
This results in noticeable levels of species depletion, whilst increasing the
incentive to adulterate products. Morphological similarities make iden-
tifying samples on a taxonomic level challenging. Furthermore, plants
are traded using local names, which do not always respond with scien-
tific names. Here we measure the efficacy of DNA barcoding to identify
bulbous and perennial herbs traded at one of the largest traditional me-
dicinal market in South Africa. A list of species traded, including their
conservation status, is compared against a known published checklist
(based on morphological identification) to note any decline or incline in
the number of species traded. Results: In total 61 plants were collected
and sequenced for the core barcoding regions; sequencing success was
higher for rbcLa (91%) than Atpf (72%). BLAST searches using rbcLa re-
sulted in high numbers of ambiguous identifications, whilst searches
using Atpf were less ambiguous, allowing the majority of samples to be
identified to genus level (95%). When combining rbcLa and Atpf most of
the samples could be identified to species level. Significance: Identifica-
tion success is generally high in our dataset, although closely related
species remain problematic to identify. Our survey shows a slight in-
crease in species traded at the market since 2003, and that the majority of
species traded are not currently endangered. However, 13% of species traded
are declining or near threatened in the wild. This poses a serious conserva-
tion issue, since if trade continues in an unsustainable way, these plants
could swiftly become critically endangered in the near future.
Molecular approach in species determination of Tetranychus
complex in polyhouse cucumber
Neena Lenin, Haseena Bhaskar, P.S. Abida,
and Maicykutty P. Mathew
Department of Agricultural Entomology, College of Horticulture, Kerala Agricultural University,
Thrissur, 680656, Kerala, India.
Corresponding author: Neena Lenin (e-mail: neenalenin@gmail.com).
Background: Polyhouse vegetable cultivation has been gaining mo-
mentum in Kerala, India in recent years. Among the vegetables grown
in polyhouses, salad cucumber is the most successful and widely cul-
tivated crop. The two-spotted spider mite, Tetranychus urticae, was con-
sidered as the most serious mite pest of cucumber. However, a recent
study revealed that more than one species of Tetranychus infests poly-
house cucumber in Kerala. The precise identification of the species
within Tetranychus is very difficult due to their high similarity and
limited number of informative morphological characters, which are
sex specific. Species are identified based on the male aedeagal charac-
ter, but field-collected samples mostly contained females due to the
female-biased sex ratio. Hence, a molecular approach was used as an
alternative tool for species determination. Results: Genomic DNA was
isolated, following a modified CTAB method. The sequences were PCR
amplified using primers for COI and ITS2 gene regions and sent to the
biological reagent company SciGenome, Kochi for sequencing. The
DNA sequences obtained were blasted against GenBank (NCBI) to
check the species identity in the database. Blasting revealed the pres-
ence of three species, namely Tetranychus urticae,T. truncatus, and
T. okinawanus. Later, the sequences along with digital specimen photo-
graphs were submitted to BOLD. Significance: The present study
helped in distinguishing the field-collected samples of T. urticae and
T. truncatus, where original taxonomic identification failed due to the
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absence of informative characters in female specimens. Also, the
study revealed the presence of a new species, T. okinawanus, from
polyhouse cucumber for the first time in India. This establishes the
significance of DNA barcoding as a diagnostic tool for specimen identifi-
cation and decision making in quarantine pest detection and control.
Using mitochondrial genome targets within the phylum
Apicomplexa: divergent life cycle stages, cryptic species,
and unexplored diversity in the coccidia
Alexandre N. Leveille, Mian A. Hafeez, and John R. Barta
Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road East,
Guelph, ON N1G 2W1 Canada.
Corresponding author: Alexandre N. Leveille (e-mail: aleveill@uoguelph.ca).
Background: Parasites in the protistan phylum Apicomplexa are
ubiquitous in metazoan hosts and cosmopolitan in distribution. The
complexity of their life cycles (encompassing both asexual and sexual
modes of replication), sometimes incorporating multiple hosts (e.g.,
as seen in malaria or toxoplasmosis) and multiple morphological
forms, make identification of these parasites challenging. Early at-
tempts to use sequence data to complement morphology-based para-
site identification and phylogenetic analyses focused on nuclear
ribosomal RNA genes (i.e., 18S rDNA); unfortunately, at least some
apicomplexan parasites possess 2-3 paralogous nuclear rDNA arrays
that can be highly divergent. Early work using mitochondrial (mt)
sequences focused on the use of cytochrome b (cytb) and, more re-
cently, cytochrome c oxidase subunit I (COI). More widespread use of
mt genetic targets was impeded by the huge variation in mt genome
structure within the phylum and the associated difficulty of generat-
ing useful DNA barcoding PCR primer sets. Results: PCR-based ampli-
fications of mt protein-coding regions and subsequent sequencing
(classical DNA barcoding) were developed for a number of groups of
apicomplexan parasites. Morphologically indistinguishable adele-
orinid parasites (Hepatozoon spp.) and eimeriid parasites (Isospora spp.)
could be differentiated readily using partial mt genome sequences.
Cryptic sympatric species could be detected in mixed samples, even in
the presence of a large surplus of host DNA. The generation of complete
(⬃6000 bp) mt genome sequences by amplifying overlapping PCR ampli-
cons confirmed that divergent mt genomes were present in both cases.
No evidence of paralogous mt gene copies was detected. Significance:
Widespread adoption of DNA barcoding for rapid identification and ge-
netic analyses of coccidian parasites may now be possible. Traditional
taxonomy and morphology will remain an essential source of well-
characterized specimens destined for barcoding. As more data become
available, barcoding will become increasingly useful as a diagnostic and
taxonomic tool; barcode generated sequences will help resolve evolu-
tionary relationships between morphologically similar parasites.
Who laid the egg? Establishing the identity, distribution, and
abundance of fish eggs in the Mexican Caribbean with barcodes
Elva Leyva-Cruz, Martha Valdez-Moreno,
and Lourdes Vasquez-Yeomans
El Colegio De La Frontera Sur, Unidad Chetumal Ave. Centenario Km 5.5 Col. Pacto Obrero Campesino, Chetumal
Q. Roo, Mexico.
Corresponding author: Elva María Leyva Cruz (e-mail: eleyva@ecosur.edu.mx).
Background: Along the Mesoamerican reef system there are many spawn-
ing and nursery places used by fishes of economic and ecological impor-
tance. It is almost impossible to identify the fish eggs due to the lack of
distinguishing markers. However, DNA barcodes have been effective in con-
necting early stages of fish development with adults. This study aimed to
recognize which species spawn in the southeast of the Yucatan Penin-
sula. Samples were collected 20–22 April 2011, during an oceanographic
survey supported by El Colegio de la Frontera Sur, National Oceanic and
Atmospheric Administration, and the University of Miami. In total, 17
sampling stations were reviewed. Results: A total of 1391 fish eggs were
sorted and differentiated into 94 morphotypes. Three hundred (at least
one from each morphotype) were photographed and described. The COI
gene was amplified and sequenced. We obtained 140 sequences that were
compared against the Barcode of Life Data Systems (BOLD) after which we
identified 42 taxa (32 with binomial name), 34 genera, and 23 families.
Among the game and commercially important species were Auxis thazard,
Caranx hippos,Coryphaena hippurus,Istiophorus platypterus,Kajikia albida,
Katsuwonus pelamis,Thunnus atlanticus, and Xiphias gladius. A station near
Banco Chinchorro had the highest abundance of eggs with 271, mainly
Katsuwonus pelamis. The most abundant species was Nesiarchus nasatus,
with 451 individuals. The species with the highest incidence were Diplospi-
nus multistriatus and Regalecus glesne, being present at eight stations. Signifi-
cance: Of the 32 species identified, 20 had not been described in their egg
stage. The first descriptions are presented in this work. The spawning season
and locations of 16 species are not known. Hence this work provides some
evidence of possible breeding areas. This pioneering research studies the fish
eggs for the area, and it is hoped that will help to establish better manage-
ment strategies for conservation purposes.
High-accuracy de novo assembly and SNP detection of chlo-
roplast genomes for DNA-barcoding studies
Qiushi Li,
1
Ying Li,
1
Haibin Xu,
1
Jingyuan Song,
1
and Shilin Chen
2
1Institute of Medicinal Plant Development (Peking Union Medical College & Chinese Academy of
Medical Sciences, China) No. 151 Malianwa North Road, Haidian District, Beijing, China.
2Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences No.16, Nanxiaojie of
Dongzhimen, Dongchen District, Beijing, China.
Corresponding author: Qiushi Li (e-mail: ashleyqsli@outlook.com).
Background: Chloroplast genome sequences are very important in
plant DNA barcoding studies. There many chloroplast regions used as
barcoding markers at higher taxonomic levels because of the relatively
low evolutionary rates. For distinguishing closely related plants at the
species or population level, whole chloroplast genome sequences with
sufficient variation information are expected to be “super barcodes”.
Traditionally, sequencing a chloroplast genome required tedious labour,
but next-generation sequencing (NGS) dramatically increased the acqui-
sition of complete chloroplast genomes. However, assembly of chlo-
roplast genomes using NGS usually requires a reference against which to
map contigs, and PCR is usually necessary to fill gaps. The sequencing
biases in NGS sequencing procedures also affect the estimation of single-
nucleotide polymorphism (SNP) variant frequency when developing the
chloroplast SNP markers. Results: Here we report a simple sequencing
and de novo assembly approach that yields complete high-quality chlo-
roplast genomes (without reference) using single-molecule, real-time
(SMRT) DNA sequencing technology. The 6% validation Sanger sequences
revealed 100% concordance with the assembled chloroplast genomes,
and the detected intraspecies SNPs at a minimum variant frequency of
15% were all confirmed. We have tested this method in some species of
both monocotyledons and dicotyledons. Significance: This full-chain,
PCR-free high-throughput approach eliminates the possible context-
specific biases in library construction and sequencing reaction. The se-
quence accuracy is significantly improved, and the reliable SNPs
detection is very sensitive compared with previous reports. We recom-
mend this approach for its powerful applicability to plant DNA barcoding
studies based on the sequences of chloroplast genomes.
Application of Barcode of Life principles, to protect the
international cotton supply chain
Ben Liang, Karim Berrada, Alex Tran, Yuhua Sun, Lucie Rows,
AnnaMarie Beckwith, Michael Hogan, and James Hayward
Applied DNA Sciences, 50 Health Sciences Blvd., Stony Brook, NY 11790, USA.
Corresponding author: Michael Hogan (e-mail: mike.hogan@adnas.com).
Background: The Barcode of Life exploits natural variation within
the chloroplast or mitochondrial genome, as a way to catalog species
diversity at a world-wide scale. At Applied DNA Sciences (ADNAS) we have
applied similar principles to protect and to manage the international
supply chain for natural product based commodities. Results: We de-
scribe here ADNAS technology in the cotton supply chain, from the gin
through processing into clothing. The study is based on a patented
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ADNAS technology, FiberTyping, which employs chloroplast genetic mark-
ers that can distinguish the highest quality of cotton Gossypium barbadense
(also known as Extra Long Staple, ELS) from the lower quality cotton
Gossypium hirsutum (also known as Upland). For this highly industrialized
analysis, it is necessary to simplify the chloroplast marker set employed
so that analysis can be done inexpensively upon thousands of samples
per day, and to focus on very small, highly repeated DNA sites so that
analysis can be performed on highly processed fabric where DNA has
become partially degraded due to cell death at harvest, and subsequently,
due to the chemical damage associated with processing, dying, and weav-
ing. Significance: We have demonstrated that cotton can be analyzed at
the gin, then as dyed yarn, and subsequently as woven fabric, to obtain
the ELS/Upland composition of the cotton at each step in the textile
supply chain, employing a straightforward extension of PCR. Based on
this pilot, we show that FiberTyping is now ready to be employed at an
international scale to protect the (high value) ELS supply chain from
accidental or criminal blending of (lower value) Upland cotton. More
broadly, we view cotton FiberTyping as a model for the use of DNA bar-
coding principles as a way to protect the supply chain associated with
other high-value commodities: wool, coffee, oils, and herbal products.
Barcode UK—beyond the visible: a science–art collaboration
Andrea Liggins
1
and Natasha de Vere
2
1University of Wales Trinity St. David, UK.
2National Botanic Garden of Wales and Aberystwyth University, UK.
Corresponding author: Natasha de Vere (e-mail: natasha.devere@gardenofwales.org.uk).
Background: Artists and scientists have been working, usually indepen-
dently, on a range of environmental research; however, art–science col-
laborations, particularly in the design and inception of research projects,
have been limited. Using as case studies two collaborative projects, this
research explored the insights, findings, and added value of art and sci-
ence collaborations. The art research in these projects investigated the
aesthetic representation of plants and landscape and how this affected
perceptions of DNA barcoding and of the perceived value of the plants
themselves. Results: The collaboration increased awareness and under-
standing of the Barcode Wales and Barcode UK projects, in a very large
sector of the arts and science communities, and the general public,
through exhibitions and journal articles. The research into aesthetics
and representation applied to the artwork (photography) confirmed that
alternative modes of composition can affect the way landscape, plants,
and the science are perceived. The work influenced the views of the
scientist and artist, and a further collaboration is planned focusing upon
an application of DNA metabarcoding pollen to investigate the floral
preferences of the honey bee. Significance: The Welsh and Chinese gov-
ernments supported a large-scale exhibition Barcode Wales—Beyond the
Visible at Nanshan Botanic Garden, Chongqing, which received over a
million visitors. Both the scientist and artist gave lectures and television
presentations to wide audiences, which included specialist researchers.
Exhibitions were also held in India, supported by the British Council, and
in Wales at the National Botanic Garden of Wales and the Eisteddfod
Science Pavilion. Following on from these projects, a network of artists,
scientists, and stakeholders is currently under development to provide a
hub that encourages collaboration on research into pollinators, which
will include the pollen DNA metabarcoding honey bee project.
Patterns of genetic diversification of bats in the Caribbean and
their relationship to other populations across the Neotropics
Burton Lim
Royal Ontario Museum, 100 Queen's Park, Toronto, Ontario, Canada.
E-mail for correspondence: burtonl@rom.on.ca.
Background: The majority of the terrestrial mammalian fauna in the
Caribbean are bats, and of these, more than half are endemic species.
However, this biogeographic region is a noticeable gap in world coverage
for this vertebrate group on the Barcode of Life reference database. Re-
sults: Recent biodiversity surveys in Jamaica, Dominican Republic, and
Bonaire have documented almost half of the bat species diversity known
from the Caribbean. Phylogenetic trees have low intraspecific but high
interspecific differentiation, which indicate that DNA barcoding is a re-
liable method for identifying species of bats. With a broader geographic
sampling across the Neotropics, there are also divergent lineages within
widely distributed taxa suggesting the presence of cryptic species. Sig-
nificance: The biodiversity of bats in the Caribbean is at least 25% under-
estimated. This indicates that more study is needed to better document
the ecological differences within and between insular and continental
populations that are associated with this high level of genetic variation.
In addition, the phylogeographic patterns of diversification warrant tax-
onomic revision and further scrutiny of the extent of morphological
evolution.
Progress in DNA barcoding the bats of Peninsular Malaysia
Voon-Ching Lim and John-James Wilson
Museum of Zoology and Ecology and Biodiversity Program, Institute of Biological Sciences, Faculty of
Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Corresponding author: Voon-Ching Lim (e-mail: voonchinglim@hotmail.com).
Background: Bats are ecologically and economically important as seed
dispersers, pollinators, and suppressors of arthropod populations. With
rapid urbanization, the roles of bats have shifted beyond ecological sig-
nificance; insectivorous bats could potentially combat urban diseases by
preying on disease-transmitting mosquitoes. Guano holds high nutri-
tional value for both cave ecosystems and the agriculture industry. Eco-
tourism promotes bat conservation while also boosting the national
economy and benefiting local communities socially and economically.
Bat monitoring can provide useful data for biodiversity assessment and
act as an indication of environmental contamination caused by human
activities. Despite these roles in ecosystems and as scientific models,
accurate species identification of bats remains challenging due to the
vast number of cryptic species. Results: There are at least 107 species of
bats in Peninsular Malaysia. At least 65 and 51 species have been recorded
in sympatry at the hotspots of Krau Wildlife Reserve and Ulu Gombak
Forest Reserve, respectively. We are updating the checklist of bat species
for the region and completing a DNA barcoding library for application in
ecological studies. This includes “dark” bat taxa, recognised species re-
corded under informal names (e.g., Hipposideros bicolor131, Cynopterus cf.
brachyotis Forest, and Chironax megacephalaGOMBAK). The species richness
of bats across Southeast Asia has been suggested to be underestimated by
at least 50%. Significance: Informal names for Malaysian bats (including
BINs) need not hinder progress in ecological research; they are essential
for providing precise understanding of species diversity and provide sta-
ble anchor points for biological data associated with individuals and
species.
Exploring genetic divergence in a species-rich insect genus
using 2790 DNA barcodes
Xiaolong Lin, Elisabeth Stur, and Torbjørn Ekrem
NTNU University Museum, Department of Natural History, Norwegian University of Science and
Technology, NO-7491 Trondheim, Norway.
Corresponding author: Xiaolong Lin (e-mail: xiaolong.lin@ntnu.no).
DNA barcoding using a fragment of the mitochondrial cytochrome c
oxidase subunit I gene (COI) has proven successful for species-level iden-
tification in many animal groups. However, most studies have been fo-
cused on relatively small datasets or on large datasets of taxonomically
high-ranked groups. We explore how well DNA barcodes delimit species
in the diverse chironomid genus Tanytarsus (Diptera: Chironomidae) by
using different analytical tools. Tanytarsus is the most species-rich genus
of tribe Tanytarsini (Diptera: Chironomidae) with more than 400 species
worldwide, some of which can be notoriously difficult to identify to
species level. Our dataset, based on sequences generated from own ma-
terial and publicly available data in BOLD, consist of 2790 DNA bar-
codes >500-bp long. A neighbour-joining tree of this dataset comprises
131 well-separated clusters representing 77 named, 15 unnamed, and 28
unidentified theoretical morphological species of Tanytarsus. For our geo-
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graphically widespread dataset, DNA barcodes unambiguously dis-
criminate 94.6% of the Tanytarsus species recognized through prior
morphological study. Deep intraspecific divergences exist in some spe-
cies complexes, and need further taxonomic studies using appropriate
nuclear markers as well as morphological and ecological data to be re-
solved. The DNA barcodes cluster into 120–242 molecular operational
taxonomic units (OTUs) depending on whether objective clustering, au-
tomatic barcode gap discovery (ABGD), subjective evaluation of the
neighbour-joining tree, or Barcode Index Numbers (BINs) are used. We
demonstrate that a 4%–5% threshold is appropriate to delineate species of
Tanytarsus non-biting midges.
Testing DNA barcoding of the recently diverged species in the
genus Gentiana (Gentianaceae)
Juan Liu, Hai-Fei Yan, and Xue-Jun Ge
South China Botanical Garden, #723, Xingke Road, Tianhe District, Guangzhou, Guangdong, China.
Corresponding author: Xue-Jun Ge (e-mail: xjge@scbg.ac.cn).
Background: DNA barcoding based on the sequence variation of short
and standard DNA regions has been used successfully in many ecological
applications. There are particular challenges in barcoding plant groups,
especially for differentiating recently diverged plant taxa. The genus Gen-
tiana—as the largest genus of the family Gentianaceae—is comprised of
⬃361 species, more than half of which are distributed in southwestern
China and its adjacent regions. The genus is recognized as one of the
taxonomically challenging plant groups in China due to recent radiative
speciation events. In this study, we surveyed two core plant barcode
markers (rbcL + Atpf), and the three most-promising complementary
barcode markers (trnH–psbA, ITS, and ITS2), from 30 Gentiana species
across six sections to estimate their discrimination efficiency as DNA
barcodes. Three methods—namely a genetic distance-based method, the
analysis of Best Close match, and a tree-based method—were employed
to evaluate five single markers and all their possible combinations. Re-
sults: The region of rbcL had the highest efficiency of PCR and sequenc-
ing success (100%), while ITS achieved the lowest performance on
sequence recoverability (68.35%). We also found that the presence of
indels and inversions in trnH–psbA led to difficulty in sequence align-
ment in the genus. In single-region analysis, ITS exhibited the highest
discriminatory power (70.37%–74.42%), and performed well in the
species-rich section Cruciata, while rbcL performed poorly. Of the com-
binations, Atpf + ITS provided the highest discrimination success
(71.43%–88.24%). Significance: Atpf + ITS was recommended as the DNA
barcoding region for the genus Gentiana. The utility of DNA barcoding in
Gentiana was also verified by authenticating medicinal plants “Qin-jiao”
(G. macrophylla,G. crassicaulis,G. straminea, and G. dahurica) correctly, allowing
these well-known Chinese traditional medicinal herbs to be used safely.
Dietary analysis of marine fishes: enhancing the detection of
barcoded reads for high-throughout sequencing in
combination with blocking primers
Huifen Liu, Zhenhao Liu, and Junbin Zhang
Shanghai Ocean University, No. 999, Hucheng Circle Road, Shanghai, China.
Corresponding author: Junbin Zhang (e-mail: jbzhang30@163.com).
Background: Fish play an integral role in complex marine food webs. A
greater understanding of the diets of marine fishes will greatly enhance
our understanding of the biology of individual species, as well as improv-
ing our ability to understand and model the function of marine ecosys-
tems. DNA barcoding, which allows the identification of individual
species by analysis of specific short DNA sequences, shows great promise
for species identification in a large variety of organisms from small
amounts of DNA. The robust combination of freely-available DNA bar-
code databases (such as the Barcode of Life Data Systems database, or
BOLD) and the robust data-generating power of high-throughout se-
quencing provides new opportunities for detailed dietary analyses. Re-
sults: In this study, we investigated the diet of four commercially
important fishes from the South China Sea: Acanthopagrus latus,Pampus
argenteus,Siganus argenteus, and Scomberomorus commerson. DNA was pre-
pared from the gut contents of each species, and two distinct fragments
of the mitochondrial cytochrome C oxidase subunit I (mtCOI) gene, a
small (130 bp) and a large (300 bp) amplicon, were amplified by PCR in the
presence of predator DNA-specific blocking primers. After the amplicons
from each species were tagged with a unique sequencing barcode, the
pooled DNA amplicons were sequenced using the Illumina MiSeq plat-
form, generating > 500 000 reads. Comparison of these data with mtCOI
sequences in BOLD and Genbank yielded 24 prey taxa for Acanthopagrus
latus, 15 prey taxa for Pampus argenteus, 46 prey taxa for Siganus argenteus,
and 56 prey taxa for Scomberomorus commerso, identified to the species
level. Significance: Our approach of combining Illumina MiSeq sequenc-
ing with blocking primers greatly enhances the speed and resolution of
dietary analysis of marine fishes and could provide new insights into
marine ecosystems.
Updates on the status of giant clams in the Philippines using
mitochondrial COI and 16S rRNA genes
Apollo Marco Lizano
1
and Mudjekeewis G. Santos
2
1University of the Philippines, Diliman, The Marine Science Institute Velasquez St., Quezon City 1101,
the Philippines.
2Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, 101 Mother
Ignacia St., Quezon City, the Philippines.
Corresponding author: Mudjekeewis G. Santos (e-mail: mudjiesantos@gmail.com).
Background: Six of the possible eight Philippine species of giant clam
(Hippopus hippopus,Tridacna gigas,T. crocea,T. squamosa,T. derasa, and Tri-
dacna sp.YCT-2005) under the Tridacnidae family were sequenced (COI
and 16S rRNA genes) for molecular-based species identification. We re-
ported the COI sequence of H. hippopus, which is now available online
through GenBank. We also reported the first sighting of Tridacna sp.
YCT-2005 in Philippine waters, a potentially new species that was
thought to be only in Taiwan. Phylogenetic trees of COI and 16S rRNA
gene sequences of giant clam samples from the Philippines were con-
structed using both neighbour-joining and maximum likelihood ap-
proaches. Results: Both trees showed similar topology in which Tridacna
and Hippopus formed two distinct clades. T. crocea,T. squamosa,T. maxima,
T. costata, and Tridacna sp. YCT-2005 showed a monophyletic grouping
under subgenus Chametrachea, confirming the recognized groupings of
giant clams based on morphology. On the other hand, restriction site
mapping based on the 16S rRNA gene showed a unique recognition site at
367–370 bp (5=AGCT3=) for T. maxima as opposed to Tridacna sp. YCT-2005.
AluI restriction endonuclease was identified as a candidate diagnostic
enzyme to differentiate between these species. Significance: This study
confirmed the identity of giant clams found in the Philippines using
molecular techniques. DNA barcoding can be a useful tool to identify
different species of giant clams in the Philippines, which is needed for
proper management and conservation of these endangered species.
DNA barcodes of polychaetes (Annelida: Polychaeta) from the
southern European Atlantic coast underscores the incipient
state of the global reference library for this taxon
Jorge Lobo,
1,5
Marcos A.L. Teixeira,
1
Luisa M.S. Borges,
2
Maria S.G. Ferreira,
1
Claudia Hollatz,
1
Pedro A. Gomes,
1
Ronaldo Sousa,
3
Ascensão Ravara,
4
Maria H. Costa,
5
and Filipe O. Costa
1
1CBMA – Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
2Helmholtz-Zentrum Geesthacht, Centre for Material and Coastal Research, Max-Planck-Straße 1,
21502, Germany.
3CIIMAR/CIMAR – Interdisciplinary Centre of Marine and Environmental Research, University of Porto,
Rua dos Bragas, 123, 4050-123, Porto, Portugal.
4CESAM – Centre for Environmental and Marine Studies University of de Aveiro, Campus de Santiago,
3810-193 Aveiro, Portugal.
5MARE – Marine and Environmental Sciences Centre New University of Lisbon, 2829-516 Monte de
Caparica, Portugal.
Corresponding author: Jorge Lobo (e-mail: j.arteaga@fct.unl.pt).
Background: The estuarine and coastal intertidal areas have a large
number of benthic invertebrates, where the annelid polychaetes are one
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of the most representative classes and, therefore, important indicators of
environmental quality in these ecosystems. Yet, these organisms have
been poorly studied, in comparison to other taxa of similar ecological
importance. Here we present a starting reference library of DNA bar-
codes for shallow water polychaetes of the southern European Atlantic
coast, focusing on specimens from Portugal. Results: Cytochrome cox-
idase subunit I DNA barcodes (COI-5P) from 164 specimens belonging to
51 morphospecies were analysed. Available published sequences from
same species, genus, or family were added for comparison. The final
dataset comprises 290 specimen records representing 79 morphospecies;
however, interestingly, 99 Barcode Index Numbers (BINs) were attrib-
uted. Only 47 BINs were considered concordant, where 1 morphospecies
equals 1 BIN, and approximately a third of all BINs were considered dis-
cordant. Some morphospecies were assigned to multiple BINs, among
which there were ecologically relevant species such as Hediste diversicolor
(O.F. Müller, 1776) and Owenia fusiformis delle Chiaje, 1844 (7 and 5 BINs,
respectively). Twenty-two BINs were singletons. Significance: The high
number of BINs compared to morphospecies found in this and other
studies suggests a considerable amount of hidden diversity in this group.
Reference libraries of DNA barcodes can be a key tool for a more exten-
sive and rigorous documentation of the diversity of polychaetes, where
numerous cryptic species have been found with the aid of molecular
approaches.
Stepwise implementation of high-throughput sequencing
metabarcoding to estuarine macrobenthic communities
Jorge Lobo,
1,3
Shadi Shokralla,
2
Maria H. Costa,
3
Mehrdad Hajibabaei,
2
and Filipe O. Costa
4
1CBMA – Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
2Biodiversity Institute of Ontario & Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
3MARE – Marine and Environmental Sciences Centre, New University of Lisbon. Campus de Caparica,
2829-516 Caparica, Portugal.
4CBMA – Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
Corresponding author: Jorge Lobo (e-mail: j.arteaga@fct.unl.pt).
Background: Benthic communities are key components of aquatic
ecosystem biomonitoring. However, taxonomic identification em-
ploying current methodologies is very time-consuming, technically
difficult, and frequently does not provide species-level data. Although
metabarcoding approaches have been tested and successfully imple-
mented on freshwater macrobenthos, no homologous approaches
have been proposed and tested for estuarine and marine macroben-
thic communities, which are particularly challenging due to their
much greater phylogenetic diversity. Results: To investigate the abil-
ity to successfully amplify a fragment of the DNA barcode region (658 bp
of the cytochrome c oxidase subunit I mtDNA gene—COI-5P) from
all species present in a whole-community DNA extract, we assembled
three phylogenetically diverse, simulated communities (SCs) with a
different number of species (9 to 36) and specimens per species (9 to
67). Four distinct primer combinations were used to amplify COI bar-
code fragments from DNA isolates of each of the SCs, and the respec-
tive PCR products were sequenced in an Illumina MiSeq platform.
Approximately 80% of the species present in the three SCs were recov-
ered after analyses of the MiSeq-generated sequences. Notably, this
recovery rate was attained using only two primer combinations. Sub-
sequently, we compared morphology and metabarcoding-based ap-
proaches to determine the species composition of macrobenthos from
four different collection sites of the Sado estuary, Portugal. In each
site, five sediment replicates were used separately for morphology-
and metabarcoding-based identifications. As expected, polychaete
annelids were the dominant component of the macrobenthic commu-
nity in this estuarine ecosystem. Significance: These promising
results indicate the viability of metabarcoding approaches for imple-
mentation in biodiversity assessments of estuarine macrobenthic
communities. Further developments are required including the com-
pletion of the DNA barcode reference library for marine and estuarine
macrobenthos and improvements in recovery rates.
Barcoding the Diplostomoidea (Platyhelminthes: Digenea)
Sean Locke
1
and David J. Marcogliese
2
1Department of Biology, University of Puerto Rico, Mayagüez, Puerto Rico, Box 9000, Mayagüez,
PR 00681-9000.
2Aquatic Biodiversity Section, Watershed Hydrology and Ecology Research Division, Water Science and
Technology Directorate, Science and Technology Branch, St. Lawrence Centre, Environment Canada,
105 McGill, 7th floor, Montreal, QC H2Y 2E7, Canada.
Corresponding author: Sean Locke (e-mail: sean.locke@upr.edu).
Background: Identification of digeneans (Platyhelminthes: Trema-
toda) to species is challenging. Morphological differences among con-
geners are often subtle in these small, soft-bodied organisms. Single
species may also display significant morphological variation in differ-
ent hosts. Digeneans typically infect three different hosts, in which
the developmental stages of the parasite are strongly dissimilar. Life
cycles can be elucidated with experimental infections, which may also
shed light on host-induced morphological variation, but this ap-
proach is impractical in biodiversity assessment. DNA barcodes pro-
vide an efficient way to link life cycle stages, identify specimens, and
discover potentially overlooked species. Herein, results will be presented
from barcodes obtained from digeneans in wildlife, with emphasis on
the Diplostomoidea. Parasites were collected from more than 120 birds
(40 species), 3000 fish (93 species), and 50 amphibians (7 species) from the
Americas (mainly), Europe, Iraq, and China. Results: In a surprising num-
ber of cases, barcodes from adult diplostomoids from definitive hosts
were not matched by those of sympatric larval parasites, even in in-
tensely sampled regions. In North America, barcodes connected larval
and adult parasites in 15 diplostomoid species, but because of the diffi-
culty of vouchering the taxonomically informative but small adults, the
number of positive identifications is smaller. Contrary to long-held
views, larval stages in most diplostomoid species are specific to a narrow
range of fish or frog hosts. COI distances are correlated with geo-
graphic distances, but patterns differ in two widely sampled genera.
Significance: In an intensely and widely sampled genus, Diplostomum,
sampling effort, but not spatial scale of sampling, has a significant but
non-problematic effect on metrics related to COI distance-based species
delineation. Species richness in Diplostomum is unevenly distributed
among larval infection sites, and an ecological mechanism is proposed to
explain the apparent difference in speciation rates.
An Arctic molecular clock? Genetic divergence in echinoderms
across the Bering Strait
Tzitziki Loeza-Quintana and Sarah J. Adamowicz
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Tzitziki Loeza-Quintana (e-mail: tloezaqu@uoguelph.ca).
Background: The molecular clock remains an invaluable tool yet a
controversial topic in evolutionary biology. The opening/reclosure of
the Bering Strait provides a calibration point to estimate molecular
divergence rates in northern marine taxa. Building upon a small num-
ber of prior studies, on polychaetes and starfish, we explore patterns
of molecular divergence in 16 sister pairs of echinoderms inhabit-
ing the Pacific versus Arctic–Atlantic coast of North America. Ge-
netic divergences (Kimura 2-parameter) were estimated between 16
trans-barrier sister pairs of echinoderms, using publicly available
cytochrome c oxidase subunit I (COI) sequences and with phylogenetic
relationships verified using whole-genus analysis when possible. To
be consistent with previous studies, we used a maximum time of
divergence of 3.5 million years (MY) to calibrate a molecular clock, as
this represents the approximate timing of the first major trans-Arctic
interchange. Results: K2P divergences between Arctic–Atlantic and
Pacific sister clades ranged from 0.4% to 15.45%. Assuming simultane-
ous isolation of all trans-Arctic pairs, these results would imply high
variation in evolutionary rates. Another possible scenario is that sister
clades migrated through the Bering Strait on separate occasions, which is
supported by clusters of divergence values within this large overall range.
In order to compare with prior polychaete research, we therefore used
the consistent cluster of points to calculate a preliminary average diver-
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gence rate of 2.8%/MY. Significance: Interestingly, our results contrast
with the apparently high rate of molecular evolution previously found in
polychaetes (2.8% divergence/MY in echinoderms vs. 4.4% in polychaetes).
Additionally, multiple pulses of trans-Bering migration are suggested for
Arctic invertebrates. Future work will include testing for rate variability,
increasing the sample size of pairs, and considering the full range of
possible trans-Bering migration times, with the goal of providing more
realistic ranges of potential rates for comparison with tropical rates cal-
ibrated using the Isthmus of Panama.
Blood meal analysis and virus detection in blood-fed
mosquitoes collected during the 2006–2007 Rift Valley fever
outbreak in Kenya
Joel Lutomiah,
1
David Omondi,
2
Daniel Masiga,
2
Collins Mutai,
1
Paul O. Mireji,
3
Juliette Ongus,
4
Ken J. Linthicum,
5
and Rosemary Sang
1
1Kenya Medical Research Institute (KEMRI), Nairobi, Kenya.
2International Centre of Insect Physiology and Ecology (ICIPE), Nairobi, Kenya.
3Egerton University, Department of Biochemistry and Molecular Biology, Njoro, Kenya.
4Jomo Kenyatta University of Agriculture and Technology, Ruiru, Kenya.
5USDA–ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, USA.
Corresponding author: David Omondi (e-mail: domondi@icipe.org).
Background: Rift Valley Fever (RVF) is a zoonosis of domestic rumi-
nants in Africa. Blood-fed mosquitoes collected during the 2006–2007
RVF outbreak in Kenya were analyzed to determine the virus infection
status and animal source of the blood meals. Blood meals from indi-
vidual mosquito abdomens were screened for viruses using Verocells
and RT-PCR. DNA was also extracted, and the cytochrome c oxidase
subunit I (COI) and cytochrome b (cytb) genes were amplified by PCR.
Purified amplicons were sequenced and queried in GenBank and the
Barcode of Life Data Systems (BOLD) to identify the putative blood
meal sources. Results: The predominant species in Garissa were Aedes
ochraceus (n= 561, 76%) and Ae. mcintoshi (n= 176, 24%), while Mansonia
uniformis (n= 24, 72.7%) predominated in Baringo. Ae. ochraceus fed on
goats (37.6%), cattle (16.4%), donkeys (10.7%), sheep (5.9%), and humans
(5.3%). Ae. mcintoshi fed on the same animals in almost equal propor-
tions. RVFV was isolated from Ae. ochraceus that had fed on sheep (n= 4),
goats (n= 3), human (n= 1), cattle (n= 1), and an unidentified host (n= 1),
with infection and dissemination rates of 1.8% (10/561) and 50% (5/10),
respectively; these values were 0.56% (1/176) and 100% (1/1), respec-
tively, in Ae. mcintoshi. In Baringo, Ma. uniformis fed on sheep (38%),
frogs (13%), duikers (8%), cattle (4%), goats (4%), and unidentified hosts
(29%), with infection and dissemination rates of 25% (6/24) and 83.3%
(5/6), respectively. Ndumu virus (NDUV) was also isolated from
Ae. ochraceus with infection and dissemination rates of 2.3% (13/561) and
76.9% (10/13), and 2.8% (5/176) and 80% (4/5) in Ae. mcintoshi, respectively.
Ten of the infected Ae. ochraceus had fed on goats, sheep (n= 1), and
unidentified hosts (n= 2), and Ae. mcintoshi on goats (n= 3), camel (n= 1),
and donkey (n= 1). Significance: This study has demonstrated that
RVFV and NDUV were concurrently circulating during the 2006–
2007 outbreak, and sheep and goats were the main amplifiers of these
viruses, respectively.
NNEditor: neural N-label editor toward automated sequence
finishing
Eddie Ma, Sujeevan Ratnasingham, and Stefan C. Kremer
University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Eddie Ma (e-mail: ema@uoguelph.ca).
Background: DNA barcodes, short segments of DNA used for identi-
fying species, have greatly expedited indexing life on the planet. Ap-
plication of DNA barcoding consists of DNA sequencing and
algorithms to assign species identifications based on DNA sequences.
This contrasts with the traditional method—differentiation by phys-
ical characteristics. DNA barcoding addresses a challenge termed “the
taxonomic impediment”—the limitation of experts to identify spe-
cies. However, DNA sequencing here has its own human challenges.
One limitation is sequence editing—correcting sequences using
instrument data. We address this challenge by applying Neural
Networks to automate editing. We present NNEditor, a tool that auto-
mates the most frequent human editing action, replacing ambiguous
N-labels with basecalls. Our basecalling approach is distinct from
other strategies. First, our solution is complementary to the instru-
ment basecaller, addressing only ambiguous bases. Second, our train-
ing strategy uses both human-edited sequences and complementary
basecalled sequences. Third, our tool automatically replaces N-labels,
only if it estimates a low probability of error for the new basecall.
Results: Our approach was applied to animal (COI) and plant (rbcL,
Atpf) barcodes with high success. Validation experiments resulted in
resolution of 80% of N-labels (0.78% error) from COI and 80% from Atpf
and rbcL (0.45% error). To test the generalizability, our system was also
validated on a range of non-protein-coding genes, replacing 58% of
N-labels (0.43% error). Significance: We sampled 450k tracefiles with
sequence length ≥500 from BOLD to estimate the impact of NNEditor.
Of these tracefiles, 76% are already barcode compliant, having <1%
N-labels. The application of NNEditor would result in 4.6% of sampled
tracefiles being made barcode compliant, and 20% improved beyond
the minimum barcode standard. Such improvements are critical for
diagnostic character analysis and species identity thresholds. In total,
a quarter of incoming tracefiles that could be improved using human
editing would be improved automatically using NNEditor.
Leaves of leaf-cutting bees: identity and diversity determined
by DNA barcoding
Scott J. MacIvor, Daniel de Ocampo, Baharak Salehi,
and Laurence Packer
Biology Department, York University, 4700 Keele St., Toronto, ON, Canada.
Corresponding author: Scott J. MacIvor (e-mail: jsmacivor@gmail.com).
Background: As their name suggests, leaf-cutting bees (most Megachile:
Megachilidae) use their mandibles to cut leaves from trees, shrubs,
wildflowers, and grasses. Each piece is carried back to the nest to
line and separate brood cells arranged in a linear series in a hollow
plant stem, decaying log, other tubular cavities or in the ground. Many
leaf pieces (>15) are required per brood cell. Determining host-leaf
preference is difficult, as it has depended on morphological identifi-
cation from leaf fragments from nests or from observation of known
bees cutting leaves from known plants. As such, data are poor on the
plant species used by leaf-cutting bees. This missing detail in inter-
preting the ‘needs of bees’ offers valuable information for conserva-
tion by accounting for necessary (and potentially limiting) nesting
materials. We use DNA barcoding and rbcL and ITS2 markers to
compare the identity and diversity of leaves used by three species of
above-ground nesting Megachile bees, one of which is economically
important with nesting material requirements generally unknown.
Results: To sample nests we used nestboxes set up throughout To-
ronto, Canada. Nests were opened, and one leaf piece from one cell per
nest of the native Megachile pugnata (N= 42 leaf pieces) and the intro-
duced M. rotundata (N= 51) and M. centuncularis (N= 59) were analyzed.
From 25 plant families and 40 genera identified, M. rotundata used 19
and 26, respectively, M. pugnata 7 and 10, and M. centuncularis 11 and 19.
Only three plant genera (Epilobium,Oenothera,Rosa) were collected by
all three species. Megachile pugnata (42.9% of the time) and M. centuncularis
(27.1%) visited Rosa most, while no plant genera were identified from
M. rotundata samples more than five times. Significance: In cities,
landscape design and gardening impact leaf types available for differ-
ent nesting bee species and these data can inform more complete
conservation planning that accounts for foraging and nesting mate-
rial requirements.
Abstracts 249
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DNA barcodes of the genus Oxysarcodexia (Diptera:
Sarcophagidae) from south Brazil
Taís Madeira,
1
Patrícia Thyssen,
2
Carina Souza,
2
and Juliana Cordeiro
1
1UFPel, Pelotas, Brazil.
2Unicampi Campinas, Brazil.
Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br).
Background: Necrophagous insects have been used for forensic pur-
poses, contributing to estimating the postmortem interval, which is
important for investigating crime scenes. Species of the Sarcophagi-
dae family (Diptera), in addition to Calliphoridae, are one of these
groups with forensically important species. This family shows a great
diversity of species in the Neotropical region. The genus Oxysarcodexia
is one of the most species-rich genera of Sarcophagidae with 81 known
species, most of them recorded from the Neotropical region, espe-
cially Brazil. This makes the adults of this genus very abundant and
often the most common flesh flies in field collections. Consequently,
due to the high species diversity of this genus and the great similarity
among the species, the discrimination of species is a complex task.
These species have a very high interspecific morphological similarity;
and the females lack morphological characters that allow a precise
diagnosis. In order to use these species as forensic tools, it is necessary
to be able to perform proper identification of males, females, and
larvae. Here, we analyzed DNA barcode sequence variability at each
collection site for 10 species from the genus Oxysarcodexia (O. admixta,
O. avuncula,O. bicolor,O. carvalhoi,O. culmiforceps,O. paulistanensis,
O. riograndensis,O. terminalis,O. thornax, and O. varia) distributed in Brazil.
The adult males and females were collected in São Paulo and Rio
Grande do Sul States; and only males could be morphologically iden-
tified. Results: DNA barcode sequences successfully discriminated all
10 species. Also, for some species, significant geographic population
structure was present, enabling some samples to be traced back to
their collection site. Significance: In this way, we show that DNA
barcodes could be used to help the taxonomy of Oxysarcodexia species,
especially to identify females and larvae. Furthermore, DNA barcod-
ing can be used as a forensic tool in this genus, helping to determine
the geographic origin of the larvae.
DNA barcoding unravels the role of morphology and
echolocation in bat–insect relationships in Jamaica
Hernani Fernandes Magalhaes de Oliveira,
1
Elizabeth Clare,
1
Stephen Rossiter,
1
Matthew Emrich,
2
Susan Koenig,
3
and Melville Brock Fenton
1
1Queen Mary University of London, 7 Cambay House, Harford Street, London, UK.
2Western University, London, Ontario, Canada.
3Windsor Research Centre, Trewlany, Jamaica.
Corresponding author: Hernani Fernandes Magalhaes de Oliveira (e-mail:
oliveira.hfm@gmail.com).
Background: Species interactions are the building blocks of ecosys-
tems, forming competitive, predatory, mutualistic, and parasitic rela-
tionships. Despite this, they are often difficult to observe and
untangle. Extreme generalist insectivores present a particular chal-
lenge as traditional analyses are based around morphological exami-
nation of faecal contents, providing only a general view of diet. DNA
barcoding solves this problem—identifying prey DNA in faeces with
species-level resolution. When coupled with next-generation sequenc-
ing it is possible to scale up the analysis from diagnosing single inter-
actions to studying the whole community of predators and prey. Bats
are an excellent model system for the study of dietary flexibility and
drivers of niche partitioning. Here we analyse the role of morphology
and echolocation behaviour in driving the dietary niche of seven sym-
patric insectivorous bats on the island of Jamaica, particularly their
relative niche size and degree of dietary overlap. Results: We ex-
tracted, sequenced, and identified the insect DNA obtained from the
faeces of several hundred individual bats using barcoding to gain
insights into their ecological roles. Species with longer, more narrow-
band echolocation calls and longer, narrower wings differed in diet
from the other species. Pteronotus parnellii—which uses high-duty-cycle
echolocation for detecting fluttering targets—was found to consume
Lepidoptera. Macrotus waterhousii uses low-intensity, broadband echo-
location calls and presumably uses prey-generated sounds to forage
and find its prey. Significance: Our study represents one of the first
whole community-level analyses of bat–insect interactions resolved to
species level. We show the role of morphology and echolocation be-
haviour in diet, and niche space. Our analysis provides a model for
how ecosystems and communities can be unravelled using powerful
sequencing approaches.
Environmental barcoding of aquatic invertebrates in Norway
Markus Majaneva,
1
Terje Bongard,
2
Ola H. Diserud,
2
Torbjørn Ekrem,
1
Vera G. Fonseca,
3
Mehrdad Hajibabaei,
4
Anders Hobaek,
5
Bernhard Misof,
3
and Elisabeth Stur
1
1NTNU University Museum, Department of Natural History, Norwegian University of Science and
Technology, NO-7491 Trondheim, Norway.
2Norwegian Institute of Nature Research, P.O. Box 5685 Sluppen, NO-7485 Trondheim, Norway.
3Center for Molecular Biodiversity, Zoological Research Museum Alexander Koenig, Adenauerallee 160,
53113 Bonn, Germany.
4Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
5Norwegian Institute of Water Research & Department of Biology, University of Bergen,
Thormøhlensgate 53 D, NO-5006 Bergen, Norway.
Corresponding author: Majaneva Markus (e-mail: markus.majaneva@gmail.com).
Background: The possibility to identify species in environmental sam-
ples using short, standardised DNA fragments and next-generation se-
quencing (NGS) techniques has the potential to revolutionize the way we
assess and monitor biodiversity in freshwater ecosystems. Results: In
this project, we will use an experimental setup to advance protocols for
environmental barcoding through analysis of recent and historical sam-
ples from the reference watershed Atna and Lake Jonsvatnet in central
Norway. Our aim is to develop general best-practice procedures to max-
imise the effectiveness and accuracy of species-level identification at
minimum cost using next-generation sequencing techniques. Signifi-
cance: Comparing results with traditional species determination, the
project will offer both a new practical tool and a scientific approach to
biodiversity monitoring. By doing so in an international framework and
including a postdoc position, we wish to promote knowledge transfer
and capacity building among the partnering institutions and ensure re-
cruitment of qualified personnel to this important area of nature re-
search and management. Through collaboration with the Norwegian
Environment Agency we will ensure that results are disseminated to
relevant user groups.
Distribution and identification of larval Billfish (Istiophoridae)
in the Gulf of Mexico and Caribbean using DNA barcoding
Estrella Malca,
1
Lourdes Vásquez-Yeomans,
2
Barbara A. Muhling,
3
John T. Lamkin,
4
Manuel Eliás-Gutiérrez,
2
and Trika L. Gerard
4
1Cooperative Institute of Marine and Atmospheric Studies, University of Miami/Rsmas, 4600
Rickenbacker Causeway, University Of Miami/Rsmas, Miami, Florida, USA.
2El Colegio De La Frontera Sur, Unidad Chetumal Ave. Centenario Km 5.5 Col. Pacto Obrero
Campesino, Chetumal Q. Roo, Mexico.
3Princeton University Program in Atmospheric and Oceanic Science, Forrestal Campus, Sayre Hall,
Princeton, NJ 08544, USA.
4National Oceanic Atmospheric Administration, Nmfs Sefsc. 75 Virginia Beach Drive, Miami, FL 33149,
USA.
Corresponding author: Lourdes Vásquez-Yeomans (e-mail: lvasquez@ecosur.mx).
Background: Billfishes (Istiophoridae) are a commercially prized
group of pelagic fishes that are globally distributed, including the
Atlantic Ocean. Larval billfish ecology is not well known, partially
because larvae are difficult to identify to species level, due to similar-
ities among species in larval morphology. To resolve this taxonomic
limitation, larval istiophorids were collected in the Gulf of Mexico and
the Caribbean Sea using neuston and MOCNESS nets during several
oceanographic cruises from April 2010 through September 2012. Lar-
vae were preserved in 95% ethanol, photographed, measured (SL), and
assigned a developmental stage (pre-flexion, flexion and post-flexion).
Tissues were barcoded using the COI gene, and the Barcode of Life
Data System was used to provide a species-level identification.
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Results: Preliminary barcoding results from 323 specimens indicated
that larval istophorids were predominantly Istiophorus platypterus
(65%), with Kajikia albidus (24%) and Makaira nigricans (10%) also present.
Although a wide range of sizes were collected (2.2–17.5 mm SL), the
majority (65%) of larval istiophorids were pre-flexion stage and were
likely less than one week old. Larvae were collected where surface
temperatures were between 25.2 and 31.6 °C, with most of them asso-
ciated with waters of 27–31 °C. Although all three species were found
each year, overlaying larval catch sites and surface temperature im-
ages showed that I. platypterus had a broad spatial distribution across
water masses, while K. albidus and M. nigricans were primarily collected
in the Caribbean Sea and in association with the Loop Current.
Significance: This is the first and most complete database that in-
cludes genetically verified specimens and the larval distribution of
three istiophorid species from a wide area of the western Central
Atlantic. Adequately identifying larval istiophorids is a first step to
improve our understanding of spawning times and areas, and deter-
mining larval distribution patterns, and will support overall billfish
conservation and management.
DNA barcode reference library for Indian medicinal plants of
high trade volume
Saloni Malik and S.B. Babbar
Department of Botany, University of Delhi, Delhi-110007, India.
Corresponding author: Saloni Malik (e-mail: malik.saloni10@gmail.com).
Background: Identification of herbal drugs, usually traded in frag-
mented form, is not possible by traditional morphology-based char-
acters, thus making it difficult to investigate the problems of
adulteration, substitution, and biopiracy. Of the estimated 17 500 spe-
cies of flowering plants existing in India, 6000–7000 are used as tradi-
tional medicines. Approximately 960 of these are traded, with 178
having high trade volume. DNA barcoding, a molecular method for
identification of unknown samples to the species level, could be an
effective tool for identifying plants in herbal medicines. Although the
Atpf and rbcL genes from the chloroplast genome and ITS/ITS2 (wher-
ever possible) from the nuclear genome have been suggested as the
barcode regions for plants, an in silico analysis was first conducted to
identify the effective loci for species-level identification of medicinal
plants. Results: ITS sequences representing 500 medicinal plant spe-
cies belonging to 442 genera and 117 families, and 278 Atpf and
302 rbcL sequences of these plants were downloaded from NCBI Gen-
Bank and individually checked for their uniqueness at the species
level using the BLAST method on NCBI and BOLD. About 96%, 91%, and
80% of ITS, Atpf, and rbcL sequences, respectively, matched with the
representative of its own species. ITS + Atpf used together provided
specific recognition tags for 99.6% of species. The addition of rbcL
raised this to 100%. Four potential DNA barcode markers (ITS, Atpf,
rbcL, and rpoC1) of 74 specimens representing 37 species of high trade
volume were amplified, sequenced, and analyzed. The sequencing
success for these loci was 77%, 92%, 84%, and 97%, respectively. Match
rates to the species level based on BLAST for ITS, Atpf, rbcL, and rpoC1
were 79%, 91%, 69%, and 47%, respectively, reflecting the current in-
complete state of barcode libraries for medicinal plant species. Sig-
nificance: Once further developed, the barcode reference library for
medicinal plants will be useful for checking the identity, purity, sub-
stitution, and adulteration of herbal samples.
Integrating DNA barcode data with behavioral and ecological
data in a study of white-faced capuchin faunivory
Elizabeth K. Mallott, Ripan S. Malhi, and Paul A. Garber
Department of Anthropology, University of Illinois at Urbana-Champaign 109A Davenport Hall,
607 S Mathews Ave, Urbana, IL 61801, USA.
Corresponding author: Elizabeth K. Mallott (e-mail: mallott2@illinois.edu).
Background: A number of changing social and ecological factors af-
fect which food resources primates choose to exploit. Previous field
studies of faunivorous primates have been limited by an inability to
accurately determine which animal prey are consumed, decreasing
the researchers' abilities to assess the influence of variation in animal
prey availability, age- and sex-based differences in prey choice, and
how prey choice relates to foraging microhabitats. Here, we use high-
throughput sequencing of invertebrate mtDNA from the feces of
white-faced capuchins (Cebus capucinus) to determine patterns of ani-
mal prey consumption over a yearlong study. From January 2013 to
January 2014, behavioural data was collected from a group of 22 white-
faced capuchins at La Suerte Biological Field Station, Costa Rica. In-
vertebrate availability was monitored using canopy traps and sweep
netting. Fecal samples were collected from adult female, adult male,
and juvenile white-faced capuchins (n= 235). A fragment (⬃300 bp) of
the COI mtDNA was amplified using universal invertebrate primers.
Samples were individually barcoded and sequenced on the Illumina
MiSeq platform. Sequences were clustered into operational taxo-
nomic units, which were compared to known COI sequences in Gen-
Bank and the Barcode of Life Data Systems. Results: Twenty new
orders of arthropods were identified in the white-faced capuchin diet
during the study period. Fluctuations in invertebrate availability influ-
enced observed foraging behavior, but did not influence the frequency of
consumption of specific orders of invertebrates. We observed few differ-
ences in invertebrate foraging behavior and diet of male and female
individuals; however, we found significant differences between adult
and juvenile foraging strategies and dietary choices. Significance: This
study greatly increases our knowledge of the diversity of invertebrates in
white-faced capuchin diets and gives us a more in-depth understanding
of both the influence of food availability and sex- and age-based differ-
ences on prey foraging strategies in primates.
Sensitive detection of water soldier and other invasive aquatic
plants using environmental DNA
Allison Marinich,
1
Michelle Scriver,
1
Chris Wilson,
2
and Joanna Freeland
1
1Trent University, 2140 East Bank Drive, Peterborough, Ontario, Canada.
2Ministry of Natural Resources and Forestry, 2140 East Bank Drive, Peterborough, Ontario, Canada.
Corresponding author: Allison Marinich (e-mail: almarinich@trentu.ca).
Background: Invasive species pose a substantial threat to global bio-
diversity, surpassed only by habitat loss. Water soldier (Stratiotes
aloides) is an invasive aquatic plant with a single wild population in
North America (Trent River, Ontario) that has the potential to spread
rapidly through dispersed vegetative fragments. Once established, wa-
ter soldier may become an extremely deleterious invasive species be-
cause it forms dense mats of floating vegetation that crowd out native
species, has the potential to alter water chemistry, and can hinder
aquatic recreational activities. Although it is now too late to prevent
its establishment, the distribution of water soldier remains limited,
and swift action following early detection via environmental DNA
(eDNA) markers could minimize its spread. Results: In this study, we
first screened three regions of chloroplast DNA (Atpf, rbcL, and trnH–
psbA) as potential eDNA markers using 10 different species of aquatic
plants, and found that Atpf was the region most likely to harbour
appropriate levels of intra- and interspecific variability appropriate
for designing species-specific markers. We designed markers for all 10
species and further tested the water soldier markers on eDNA ex-
tracted from water samples by using an experimental approach to
quantify the limits of detection based on plant biomass. We are now
using these markers to screen water samples from sites of known and
suspected water soldier occupancy in order to infer presence or ab-
sence. Significance: Although eDNA detection has some limitations,
our study has shown that eDNA markers have potentially important
applications for the early detection of invasive aquatic plants. This
method of detection can be used to complement physical search
methods, or it can be used independently when environmental barri-
ers inhibit physical monitoring. In conclusion, eDNA detection is a
promising tool that warrants further investigation.
Abstracts 251
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Barcoding the Swiss lichens and associated fungal
communities using barcoded amplicon 454 pyrosequencing
Kristiina Mark,
1
Carolina Cornejo,
2
Christine Keller,
2
and Christoph Scheidegger
2
1Institute of Botany and Ecology, University of Tartu, Lai Street 38, Tartu 51005, Estonia.
2Biodiversity and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland.
Corresponding author: Kristiina Mark (e-mail: kristiina.mark@ut.ee).
Background: As composite organisms, lichens (systematically lichen-
ized fungi) consist of a fungal partner (mycobiont) and one or more
photosynthetic partners (photobiont). Barcoding lichens—especially
crustose lichens—using Sanger sequencing can be problematic as sap-
rophytic, endophytic, and parasitic fungi live intimately admixed
with the lichen mycobiont, which results in low quality sequencing
reads. We sampled 100 epiphytic lichen species from the order Lecano-
rales (Ascomycota) from Switzerland to test barcoding of the myco-
biont using fungal-specific molecular markers in high-throughput
454 sequencing in a GS FLX System. Additionally, we aimed to gain
better insight into insufficiently explored epi- and endo-lichenic fun-
gal diversity. Results: Pyrosequencing of 100 lichens resulted in a total
of 128 449 reads. The full fungal ITS region (“barcode”) of the lichen
mycobiont was recovered for all sequenced species except one. In
26 samples, multiple ITS versions of expected mycobiont were de-
tected. Besides the target mycobiont, many other fungi were identi-
fied within our samples—on average 11 fungal lineages per lichen
sample. Many of these were identified as highly specific lichen-
associated (facultative parasites/lichenicolous) or plant-associated
(epi- or endo-phytes) fungi; others seem to be generalists with similar
fungal lineages shared in a variety of lichen samples, independent of
growth form (foliose/fruticose vs. crustose thallus), chemical compo-
sition, or substrate tree species. Significance: Our study shows the
potential of DNA barcoding using high-throughput 454 sequencing in
lichen identification. We demonstrate that GenBank can be used as a
reference database as the majority of lichen species were identified
correctly to species or at least genus level. The lichen-associated fun-
gal community proves to be much more diverse than previously sam-
pling suggested.
Improving our understanding of metacommunity structure
using DNA barcoding
Gillian K. Martin,
1
Sarah J. Adamowicz,
2
and Karl Cottenie
2
1Université du Québec a
`Montréal, 141 av. du Président-Kennedy, Montréal, QC H2X 1Y4, Canada.
2Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Gillian K. Martin (e-mail: gilliankmartin1@gmail.com).
Background: A longstanding area of debate in community ecology
has centered on niche theory versus neutral theory. While the debate
has resulted in observations supporting each theory, theoretical
frameworks have largely neglected the role of evolution. By incorpo-
rating genetic diversity data into community analyses it may be pos-
sible to determine under what conditions niche and neutral processes
play a greater role in shaping a community and whether the genetic
structure of a community impacts this. Barcode data will improve
species-level identifications and allow researchers to incorporate evo-
lutionary history into analyses. To test whether grouping species by
using coarse taxonomic resolution masks the relationship between
taxonomic composition and environmental variables, we character-
ized 21 stream invertebrate communities focusing on the five most
abundant aquatic insect orders: Coleoptera, Diptera, Ephemeroptera,
Plecoptera, and Trichoptera, plus the often-neglected class Oligochaeta.
Results: We found that the proportion of community composition
variability explained by the environment decreased or did not change
with increasing taxonomic resolution (family, genus, and DNA bar-
code cluster levels). This suggests ecological inter-changeability of
closely related taxa in this system, given the environmental variables
we measured. Significance: These findings suggest that ecological
filtering may play a greater role at a family level than a species level.
We will explore how estimates of variation in genetic diversity can
improve our understanding of the role of niche and neutral processes.
Determining the effect genetic diversity and species diversity have on
spatial dynamics will greatly improve our understanding of commu-
nity dynamics. In the face of great environmental change, due to
climate change and other anthropogenic factors, understanding why
species live in certain areas will help provide information about the
chance of local species extinctions, changes to species viability and
recovery, and predictions of how the communities will shift. Such
information will also help focus conservation efforts.
Mexican DNA barcoding laboratory (Chetumal Node): six years
after
Arely Martínez-Arce and Manuel Eliás-Gutiérrez
El Colegio de la Frontera Sur, Av. Centenario Km 5.5, Chetumal Quintana Roo, Mexico.
Corresponding author: Arely Martínez-Arce (e-mail: marce@ecosur.mx).
Background: The Barcode of Life Laboratory-Chetumal Node (BL-
Chetumal Node) belongs to the laboratory network of the Mexican Bar-
code of Life Network (MexBol), which is supported by the National
Council of Science and Technology (CONACYT). The creation of the labo-
ratory was to support the participation of researchers across Mexico in
the International Barcode of Life (iBOL) project. Results: From 2009 to
date, staff within the BL-Chetumal Node performed about 27 000 DNA
extractions and PCR amplifications in a wide range of taxa. For the DNA
sequencing, 20 615 PCR products were sent to the Canadian Centre for
DNA Barcoding (CCDB) as well as the Genome Sequencing Laboratory of
Biodiversity and Health, Institute of Biology in the Universidad Nacional
Autonoma de Mexico (UNAM). With results from this laboratory, it was
possible to describe a new species of cladoceran (Leberis chihuahuensis) and
a fish (Hypoplectrus ecosur). As well, many cryptic species have been high-
lighted, for example in the Lepidoptera (Adelpha,Asterocampa,Biblis, and
Hamadrya), copepods (Mastigodiaptomus albuquerquensis), and rotifers. For
the first time in Mexico, we identified the eggs and larvae of fishes to the
species level. This helped assess the ecological impact of some exotic
species, such as the identification of prey of the lionfish (Pterois volitans)in
the Caribbean and confirmation of the presence of an invasive frog,
Eleutherodactylus planirostris, in the Yucatan Peninsula. We also developed
new primers for difficult groups, such as freshwater microcrustaceans.
Significance: We consider that barcodes are a powerful tool to under-
stand this megadiverse region of the world, but continued and enhanced
efforts are required for the construction of the reference database, be-
cause most of Mexico's biodiversity still remains unknown.
Highlights and new discoveries in the Mexican cladocera
Ana L. Martínez-Caballero
1
and Manuel Elías-Gutiérrez
2
1Centro Tlaxcala de Biología de la Conducta, Carretera Tlaxcala-Puebla Km 1.5, C.P. 90062, Tlaxcala,
Tlax., México.
2El Colegio de la Frontera Sur (ECOSUR), Av. del Centenario Km. 5.5, Chetumal, Quintana Roo, México.
Corresponding author: Ana L. Martínez-Caballero (e-mail: almartinezc@hotmail.com).
Background: Mexico is one of the most-studied countries for its cladoceran
fauna using DNA barcodes; roughly 120 species have been barcoded to date.
Many new records and new species have been discovered in almost all
known genera, some of which have been described using integrative taxon-
omy, such as Leberis chihuahuensis and Scapholeberis duranguensis. This work was
undertaken in several water systems from the Central Plateau, which in-
cludes ancient crater lakes and temporary natural pools and is located 2000
m above sea level at its highest point. Results: We present new records and
new species of the Bunops genus, and several uncertain taxa belonging to the
genera Alona,Kurzia,Macrothrix,Picripleuroxus,Ilyocryptus,Pseudochydorus,
Moina, and Daphnia. The latter is difficult to identify using barcodes due to
possible hybridization between different species of the same genus; other
troublesome genera are Simocephalus and Pleuroxus.Significance: After 10
years of DNA barcode studies in Mexico, several new species have been
described using integrative taxonomy. The main challenge today is the lack
of types or topotypes to compare with our material. DNA barcodes are pro-
viding new insights on the freshwater zooplankton from this region; it is
paramount to promote these kinds of studies in other parts of the world.
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DNA barcoding of Croton draco var. draco, an ethnomedicinal
resource for traditional indigenous doctors from Veracruz,
México
Octavio Martínez de la Vega,
1
Ana Cristina Carrillo-Hernández,
2
Fernando Hernández-Godínez,
1
Corina Hayano-Kanashiro,
Corina Hayano-Kanashiro,
3
M. Humberto Reyes-Valdés,
4
and Feliza Ramón Farías
5
1Laboratorio de Biología Computacional LANGEBIO. Irapuato Gto. Mexico KM. 9.6 Libramiento Norte
Carretera Irapuato-Leon CP 36821 Irapuato-Guanajuato, Mexico.
2Facultad de Ciencias Biológicas y Agropecuarias. Universidad Veracruzana. Camino Peñuela -Amatlán
S/N, Peñuela MPIO. De Amatlán De Los Reyes. Apdo Postal 177, Córdoba, Veracruz, Mexico.
3Departamento de Investigaciones Científicas y Tecnológicas-Universidad de Sonora Luis Donaldo
Colosio S/N, Entre Sahuaripa Y Reforma. Col Centro. C.P. 83000. Hermosillo Sonora, Mexico.
4Universidad Autónoma Agraria Antonio Narro. Saltillo, Coah. Mexico. Calz. Antonio Narro 1923.
Buenavista. CP 25084, Saltillo, Coahuila, Mexico.
5Facultad de Ciencias Biológicas y Agropecuarias. Universidad Veracruzana. Córdoba, Veracruz.
Mexico Camino Peñuela -Amatlán S/N, Peñuela MPIO. De Amatlán De Los Reyes. Apdo Postal 177,
Mexico.
Corresponding author: Fernando Hernandez-Godinez (e-mail: fehernan@ira.cinvestav.mx).
Background: Croton draco var. draco Schltdl. & Cham. (sangregado) rep-
resents an important ethnomedicinal resource for traditional indige-
nous doctors (TID) in the centre of the Veracruz state (Mexico), who use it
as an injury healer, anti- inflammatory, antimicrobial, and antitumor
drug, among other applications. A comprehensive project is being car-
ried out on this species to generate basic information and validate the
medicinal properties that TID refer to this plant species. Phytochemical
studies revealed the presence of secondary metabolites produced by
C. draco and explain the diversity of uses in folk medicine. Considering the
biotechnological potential of this species, urgent steps are required to
disseminate its properties and facilitate the development and marketing
of herbal remedies. Results: We used three plastid markers: rbcL, Atpf,
trnH–F/psbA–R, and one nuclear marker: ITS, in order to identify Croton
draco var. draco. Two of them (rbcL, Atpf) were proposed by the Plant
Working Group (www.kew.org/barcoding). Leaf samples were used for
the analyses. The vouchers are deposited in the Herbarium “CORU” from
Universidad Veracruzana. Sequences for rbcL and Atpf were deposited in
the BOLD database. Sequences were BLASTed against public databases
and showed identity with the Croton genus. The four markers were com-
pared using many criteria. Significance: DNA barcoding will contribute
for an accurate authentication of this medicinal plant from adulterants.
It will also help in the protection of TID's knowledge through a patent (in
process) and the biotech products derived from this plant species.
Monitoring aquatic species' invasions using environmental DNA
Laurence Masson,
1
Chris C. Wilson,
2
and Michael G. Fox
1Trent University, 1600 West Bank Drive, Peterborough, ON K9J 7B8, Canada.
2ONNR, 1600 West Bank Drive, Peterborough, ON K9J 7B8, Canada.
Corresponding author: Laurence Masson (e-mail: laurencemasson2@trentu.ca).
Environmental DNA (eDNA) detection based on DNA barcoding is an
efficient method for detecting target species at low population densities,
where traditional census methods often fail. The sensitivity of the eDNA
method may improve conservation management strategies in response
to species invasions by enabling their detection at early invasion stages
before effective response options become limited. We tested the effi-
ciency of the eDNA method in tracking an invasive population of round
goby (Neogobius melanostomus) at an early stage of invasion. Paired testing
of both eDNA and a traditional survey method (angling) was used to
monitor the expansion of round goby along its invasive pathway in the
Trent–Severn Waterway in Central Ontario. The efficiency of the eDNA
method was also assessed by comparing the pattern of decrease in pop-
ulation density toward the expansion front as measured by angling, with
the strength of eDNA signal. We also tested the effects of distance from
the expansion front, habitat type, depth, and water flow on the eDNA
signal. Appropriate sampling and extraction methods were developed to
optimize final DNA yield and detection accuracy. The eDNA was shown to
detect the presence of round goby upstream from where the species was
detected with the traditional survey method. Our results indicate that a
well-designed monitoring protocol using eDNA would enhance our abil-
ity to detect a species invasion, which should increase the likelihood of
successful control.
Diet analysis of European free-tailed bats Tadarida teniotis using
high-throughput sequencing
Vanessa Mata, Francisco Amorim, Hugo Rebelo, and Pedro Beja
CIBIO/InBIO, R. Padre Armando Quintas, 4485-661 Vairao, Portugal.
Corresponding author: Hugo Rebelo (e-mail: hugo.rebelo@cibio.up.pt).
Background: Insectivorous bats are voracious predators that can ingest
as much as half of their own weight in insects per night. They provide
important ecosystem services by potentially controlling insect plagues,
thus greatly reducing the costs of pest control. European Free-tailed bats
are a common Mediterranean bat species with a large knowledge gap.
Traditional diet analysis of this species had an extremely low taxonomic
resolution at the family level. Recent DNA barcoding techniques capable
of generating large quantities of data have come to revolutionize diet
analysis, allowing a species-level identification of prey. Our study aimed
to further study the diet of this species using high-throughput sequenc-
ing and understand how season, bat gender, and bat age influence diet
composition. Results: We collected guano pellets of 143 individual bats
of five different roosts in the northwest of Portugal, from April to October
between 2012 and 2013. Using PCR to amplify a small fragment (157 bp) of
the insect's COI gene, we then used the BOLD database to identify the
prey present in the pellets. A total of 115 different prey species were
found, belonging to five insect orders and 17 families. The most common
and diverse prey belonged to the Lepidoptera family Noctuidae. Results
showed no marked temporal differences in diet composition, but re-
source partitioning was found between males and females, indepen-
dently of bat age, with females consuming larger prey and moth
species with migratory behavior. Significance: This study constitutes
a straightforward example on the use of the DNA barcoding analyses
in ecological studies and the results will also be the baseline for the
long-term diet monitoring of this bat population. Future fecal metag-
enomics studies will most likely provide unique information to detect
and understand the impacts of climate and land-use changes on the
trophic relationships of the ecosystems.
Savanna fire and the origins of the “underground forests” of
Africa
Olivier Maurin,
1
T. Jonathan Davies,
2
John E. Burrows,
3
Barnabas H. Daru,
4
Kowiyou Yessoufou,
5
A. Muthama Muasya,
6
Michelle van der Bank,
1
and William J. Bond
6
1African Centre for DNA Barcoding, University of Johannesburg, PO BOX 524, Auckland Park, 2006,
South Africa.
2Department of Biology, McGill University, 1205 Ave Docteur Penfield, Montreal, QC H3A 0G4, Canada.
3Buffelskloof Herbarium, P.O. Box 710, Lydenburg 1120, South Africa.
4Department of Plant Sciences, University of Pretoria, Private Bag X20, Hatfield 0028, Pretoria, South
Africa.
5Department of Environmental Sciences, University of South Africa, Florida Campus, Florida 1710,
Gauteng, South Africa.
6Department of Biological Sciences, University of Cape Town, Rondebosch 7701, Western Cape, South
Africa.
Corresponding author: Michelle van der Bank (e-mail: mvdbank@uj.ac.za).
Background: The evolution of fire adaptations in savanna plants is an
intriguing question in ecology and evolutionary biology. However,
deeper insight is hindered by the lack of comprehensive molecular phy-
logeny. Here, we explored the origin of mesic savannas in Africa using
geoxylic suffrutices (= geoxyles), White's underground forests of Africa,
as markers for fire-maintained ecosystems. First, we reconstructed a
time-calibrated phylogeny on the basis of DNA barcodes of southern
African trees using 1400 woody taxa representing 117 families and 562
genera of Gymnosperms and Angiosperms; a total of 53 geoxyle taxa,
representing 22 APG families, were included in the dataset. Then we used
this phylogeny to test if African geoxyles emerge at similar times to those
of the South American cerrado and used their phylogenetic position to
date the appearance of humid savannas. Results: Results suggest that
mesic savannas first appeared in the tropics with more recent speciation
Abstracts 253
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at lower latitudes in southern Africa. We found multiple independent
origins of the geoxyle life form mostly from the Pliocene (<5.3 Myr ago),
with the majority of divergences occurring within the last 2 million
years. Furthermore, geoxyles occur in regions characterized by higher
rainfall and greater fire frequency. Significance: This study provides the
first evidence for dates of emergence of higher rainfall savannas in Africa
and supports the role of fire in their origins. Furthermore, it illustrates
the value of phylogenies based on DNA barcode sequence data as an
alternative tool to explore origins of fire-adapted lineages and biomes.
Biodiversity study of Bangladeshi parasitoid wasps (Insecta:
Hymenoptera) of Malaise trap collections using DNA barcoding
techniques
Santosh Mazumdar,
1
Paul D.N. Hebert,
2
and Badrul Amin Bhuiya
3
1Bangladesh Council of Science and Industrial Research (BCSIR), Chittagong, Bangladesh.
2Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
3Department of Zoology, University of Chittagong, Bangladesh.
Corresponding authors: Badrul Amin Bhuiya (e-mail: badrulbhuiya@yahoo.com) and
Santosh Mazumdar (e-mail: mazumdarsantosh@gmail.com).
Parasitoid wasps are thought currently to comprise as much as one-
quarter of all named insects. They are recognized as a major biocontrol
agent for pest insects. In addition, basic taxonomic knowledge and the
ability to perform rapid and accurate species identifications are also
important for the sustainable development of natural resources and
ecosystem services. Nevertheless, only a few studies—most based on
morphological taxonomy—have been carried out in Bangladesh on para-
sitoids collected by Malaise traps. To integrate DNA barcoding with mor-
phological approaches, Malaise trap collections were made at two
locations in Bangladesh from March 2014–February 2015 following the
Standard Operating Protocol for the Global Malaise Trap Program. Spec-
imens were then barcoded at the Biodiversity Institute using Sanger se-
quencing to allow recovery of the voucher specimen responsible for
generating each sequence record. The analysis of 9079 specimens re-
vealed 1374 BINs (a proxy for species richness) of Bangladeshi insects in
the Malaise trap collections. Although taxonomic analysis is in progress,
these BINs include representatives of at least 71 families of Insecta with
strong representative from parasitic Hymenoptera.
Determining grasshopper (Orthoptera: Acrididae) diet and
niche overlap using high-throughput sequencing and DNA
barcodes recovered from gut contents
Beverly McClenaghan, Joel F. Gibson, Shadi Shokralla,
and Mehrdad Hajibabaei
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
Corresponding author: Joel F. Gibson (e-mail: jfgibson@uoguelph.ca).
Background: Species of grasshopper have been divided into three diet
classifications based on mandible morphology: specialists on forbs, spe-
cialists on grasses, and broad-scale generalists. Of southern Ontario spe-
cies, Melanoplus bivittatus and Dissosteira carolina are presumed to be broad-
scale generalists, Chortophaga viridifasciata is a specialist on grasses, and
Melanoplus femurrubrum is a specialist on forbs. These classifications, how-
ever, have not been verified in the wild. We selected 20 individuals of four
species upon which to utilize a high-throughput sequencing (HTS) ap-
proach to diet analysis. We recovered DNA from intact grasshopper guts
and amplified the rbcLa region of the chloroplast genome. We then se-
quenced these amplicons on an Illumina MiSeq platform and performed
bioinformatic processing and statistical analysis to determine the plant
diets for each species and the degree of interspecies dietary overlap.
Niche breadth and overlap measures reveal the degree to which each of
these species can be classified as specialists and generalists. Results: Gut
contents confirm both D. carolina and M. bivittatus as generalists and
C. viridifasciata as a specialist on grasses. For M. femurrubrum, a high niche
breadth was observed, and species of grasses were identified in the gut as
well as forbs. Niche overlap values did not follow predicted patterns;
however, the low values suggest low competition between these species.
Significance: Our research represents a major step forward in the use of
HTS for invertebrate ecological analysis. Family-level identifications of
the gut contents of different species living in the same habitat reveal the
presence of feeding niches and overlaps. DNA barcode data allow us to
test and refine previous anecdotal and morphological life history desig-
nations for important insect species.
DNA barcoding the Demospongiae (Porifera) from the Bay of
Plenty, New Zealand—connecting morphology with
molecules
Sam Mc Cormack,
1
Ian Hogg,
2
Chris Battershill,
3
and Phil Ross
3
1691 Pyes Pa Road, Tauranga, New Zealand.
2University of Waikato Te Whare Wananga o Waikato, Gate 1 Knighton Road, Private Bag 3105,
Hamilton 3240, New Zealand.
358 Cross Roads, Tauranga, New Zealand.
Corresponding author: Ian Hogg (e-mail: hogg@waikato.ac.nz).
Background: The phenotypic plasticity of marine sponges makes ac-
curate identification challenging. Morphologically based taxonomy
often fails to provide definitive evidence for differences among taxa.
Molecular approaches such as DNA barcoding have the potential to
assist with the identification of sponges. Here we evaluated sequence
diversity at the COI gene locus as a tool for resolving species of New
Zealand sponge. Results: A total of 95 species were examined. Of
these, we obtained sequences from 32 (success rate = 34%). In all cases,
species were successfully discriminated. Sequence divergence within
species was generally <1%, whereas divergences between species
were >9% in all cases. However, divergences for a single species of
Xestospongia were much higher (25%), and this could represent the
presence of an undescribed species. Significance: We conclude that
DNA barcoding is a useful tool for identifying New Zealand sponges,
particularly when used in combination with classical taxonomic tech-
niques.
Effects of land management on soil biodiversity and nutrients
using a DNA metasystematic approach
Katie McGee,
1
Mehrdad Hajibabaei,
1
Bill Eaton,
2
and Olivia Karas
3
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Department of Biology, Pace University, 1 Pace Plaza, New York, NY 10038, USA.
3Department of Biology, University of North Carolina at Chapel Hill, 174 Country Club Road, Chapel
Hill, NC 27599, USA.
Corresponding author: Katie McGee (e-mail: kmcgee@uoguelph.ca).
Background: The Northern Zone of Costa Rica has experienced
extraction-based land management practices for the past 40 years, result-
ing in a 70% reduction of lowland forests, which have likely impacted the
soil biota and carbon and nitrogen nutrient cycle activities. Development
of secondary forests has been used as a remediation strategy; however,
conservation managers are concerned that these secondary forests are
not regenerating at traditional rates. Therefore, it is critical to determine
how these practices impact the soil biotic ecosystems, yet too few studies
have examined this in the tropics. Thus, an eDNA metasystematic study
was conducted to assess the effects of land management on soil microbial
(16S and ITS gene regions) and arthropod (COI gene region) populations,
as well as carbon and nitrogen dynamics in Costa Rica. Soils were studied
from three unique habitats originally part of a large tract of upland
primary forest in the Northern Zone of Costa Rica, and that were man-
aged differently in the past 35 years (approximately): (i) unharvested pri-
mary forest; (ii) secondary forest cleared 32 years ago and allowed to
regenerate following clearing; (iii) secondary forest cleared 32 years ago
and used for pasture for ⬃10 years before allowing it to regenerate.
Results: The results of this study show statistically significant differences
between soil nutrient and biotic data among all three of the habitat soils
examined, as determined by ANOVA followed by post-hoc analyses. In
addition, the overall relatedness of the soil biotic and abiotic data across
the habitats was determined via ANOSIM using a Bray-Curtis index.
Significance: The data from this study suggest that two differently man-
aged secondary forests can affect the soil biotic and abiotic components,
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such that one regime is less conducive to efficient carbon utilization in
growth and biomass development, and thus carbon sequestration. These
metrics could prove useful as determinants of ecosystem condition, and
their utility in restoration ecology warrants further investigation.
Medicinal plants recommended by the World Health
Organization: DNA barcode identification associated with
chemical analyses guarantees their quality
Rafael Melo Palhares,
1
Marcela Gonçalves Drummond,
1
Bruno dos Santos,
2
Gustavo Pereira Cosenza,
3
Maria das Graças Lins Brandão,
3
and Guilherme Oliveira
4
1Myleus Biotechnology, Av. José Cândido da Silveira, n° 2100, sl. 11, Belo Horizonte, MG, Brazil.
2EMBRAPA Agroenergy, Parque Estação Biológica, Brasília, DF, 70770-901, Brazil.
3School of Pharmacy, UFMG Av. Presidente Antônio Carlos, 6627, UFMG, Belo Horizonte, MG,
31270-901, Brazil.
4Vale Institute of Technology, Rua Boventura da Silva 955, Belém, PA, Brazil.
Corresponding author: Guilherme Oliveira (e-mail: oliveirag@gmail.com).
Background: Medicinal plants are used throughout the world, and
the regulations defining their proper use, such as identification of the
correct species and verification of the presence, purity, and concen-
tration of the required chemical compounds, are widely recognized.
Medicinal plants are commercialized mostly as processed materials,
and these materials present a number of challenges for botanical
identification, leading to a high number of species substitutions in the
market. According to the World Health Organization (WHO), the use
of incorrect species is a threat to consumer safety. Results: We ob-
tained 257 samples from popular markets commercialized as eight
medicinal species that are of common use in the world (Hamamelis
virginiana,Matricaria recutita,Maytenus ilicifolia,Mikania glomerata,
Panax ginseng,Passiflora incarnata,Peumus boldus, and Valeriana officinalis).Using
DNA barcode identification, we found over 40% of samples contained
species substitutions. Most of these substitutions were with species
from another genus, or family, from the correct species. Some of the
samples were substituted with species with no previous knowledge of
medicinal use and, therefore, with no studies regarding their safety.
When applying the chemical analyses following procedures indicated
by official Pharmacopoeias and WHO monographs on medicinal
plants, we were able to detect additional problems. We found correct
species without the expected chemical compounds, or with those
compounds, but with lower concentrations than required. More trou-
blesome, we found species substitutions that contained the expected
chemical compounds. Significance: To our knowledge, this is the first
study that made a comparison between DNA barcode identification of
medicinal plants and the chemical analyses to assess the presence and
concentration of their required chemical compounds. This study
showed the complementarity of these tests, and has the potential to
improve the safety of this class of medicine. We urge governments to
recognize and demand the use of these methods from Pharmaceutical
industries.
Recent adaptive origin of the cave fish Ancistrus cryptophthalmus
Izabela Santos Mendes,
1
Alex Schomaker Bastos,
2
Francisco Prosdocimi,
2
Rodrigo Lopes Ferreira,
3
Paulo dos Santos Pompeu,
4
and Daniel Cardoso de Carvalho
1
1Pontifícia Universidade Católica de Minas Gerais, Conservation Genetics Laboratory, Av. Dom José
Gaspar, 500 - Coração Eucarístico - Belo Horizonte - MG, 30535-901.
2Universidade Federal do Rio de Janeiro, Genomics and Biodiversity Laboratory, Instituto de
Bioquímica Médica Leopoldo de Meis, Av. Pedro Calmon, 550 - Cidade Universitária, Rio de Janeiro -
RJ, 21941-901.
3Universidade Federal de Lavras, Subterranean Ecology Laboratory, Av. Doutor Sylvio Menicucci, 1001 -
Kennedy, Lavras - MG, 37200-000.
4Universidade Federal de Lavras, Fish Ecology Laboratory, Av. Doutor Sylvio Menicucci, 1001 - Kennedy,
Lavras - MG, 37200-000.
Corresponding author: Izabela Santos Mendes (e-mail:
izabelasantosmendes@hotmail.com).
Background: Cave fauna hosts a large diversity of species with evolu-
tionary adaptations to extreme conditions. Studies on these species
are important to understand evolutionary and adaptive processes re-
lated to cave environments with absence of light and restricted energy
resources. Ancistrus cryptophthalmus (Reis, 1987) is a small-sized lorica-
riid fish, found inside cave rivers located in the São Domingos' karst
system of the central-west region of Brazil. The fish possess tro-
glomorphic characteristics such as eye reduction and depigmenta-
tion compared with an undescribed surface species (Ancistrus sp.).
A. cryptophthalmus and the surface species were considered distinct species
due to morphological characteristics, but when using DNA barcoding
analysis no genetic divergences were found. In order to further inves-
tigate the taxonomic status of A. cryptophthalmus, we obtained the
mitogenome of one individual from a cave population and one
individual from the surface population of Ancistrus sp. using next-
generation sequencing (Illumina). We have also investigated the ge-
netic structure of two cave populations from distinct cave rivers and
compared them to one surface population using 570 bp of the hyper-
variable mitochondrial control region (CR). Results: We recovered
eight CR haplotypes and non-significant genetic structuring between
cave and surface populations. Mitogenome sequencing showed a high
genetic similarity between both morpho-species. Significance: We
show the first molecular evidence that the cave species A. cryptophthalmus
represents a recent adaptive origin, derived from the surface-dwelling
morpho-species Ancistrus sp.
Cryptic diversity and geographic patterns revealed by building
the DNA barcode library of Colombian birds
Ángela María Mendoza, Socorro Sierra, Fernando Forero,
María Fernanda Torres, Claudia Medina,
and Mailyn Adriana Gonzalez
Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, Calle 28A #15-09 Bogota,
Colombia.
Corresponding author: Ángela María Mendoza (e-mail: amendoza@humboldt.org.co).
Background: Colombia is the country with the largest number of
recognized bird species worldwide, most of them known to be geo-
graphically restricted to the Andean region. This study is the first
effort to build the DNA barcode library of bird species from Colombia.
We focus on species listed in the Convention on International Trade in
Endangered Species of Wild Fauna and Flora (CITES) appendices, with
the aim of contributing to global efforts to control illegal species
trading. Results: To date, we have obtained sequences from 287 sam-
ples corresponding to 156 species, representing 48% of the CITES bird
species registered from Colombia. The species analyzed are included
in nine families, with Trochilidae and Psittacidae being the most rep-
resented. We obtained for the first time barcode records for 41 hum-
mingbird, four parrot, two owl, and one hawk species; most of these
species have distributions that are restricted to Colombia or to the
northern Andes. Based on our results, we are able to recognize 85% of
the species using the K2P distance criterion. In general, haplotypes
from single species formed barcode groups that were clearly distin-
guishable from related species. However, seven species showed low
genetic differences, and 16 species showed profound genetic differences.
Significance: This study is the first attempt to build the reference library
of Colombian birds, one of the most trafficked groups in the world.
Futhermore, the divergent specimens found are located in different
mountain systems, which might suggest potential cryptic species.
Progress and prospects in using DNA barcoding to advance
coevolutionary biology
Mark T. Merilo,
1
Sarah J. Adamowicz,
1
and R. Ayesha Ali
2
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Department of Mathematics and Statistics, University of Guelph, 50 Stone Road East, Guelph, ON N1G
2W1, Canada.
Corresponding author: Mark T. Merilo (e-mail: mmerilo@uoguelph.ca).
Background: Symbiotic interactions between species are pervasive
across all types of ecosystems and range across parasitic, commensal,
and mutualistic relationships. Due to the prolific nature of these rela-
Abstracts 255
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tionships, and the impact they have on the evolution of biodiversity
and on ecosystem functioning, an understanding of coevolutionary
history is critical to understanding evolutionary trends. Additionally,
studying coevolution is practically important for understanding the
context and likelihood of host switches in agricultural pests and in
medical contexts. This paper will provide an overview of progress to
date as well as future opportunities for DNA barcoding campaigns
to contribute to our understanding of host-symbiont coevolution.
Results: To date, barcode-based research on symbionts has primarily
contributed towards our understanding of the diversity of species-like
entities in symbiotic groups in regions ranging from sub-Arctic Can-
ada to tropical ecosystems. Emerging methodologies yield unprece-
dented opportunities to gather host information through barcoding
and metabarcoding of gut contents, reducing the need for painstaking
rearing studies. Additionally, large datasets of standardized DNA se-
quences, ideally in combination with additional data from multi-gene
and genomics-based projects, will contribute to building species-level
phylogenies. These phylogenies, in combination with host association
data, will enable testing of large-scale, novel coevolutionary hypothe-
ses. We present results of such a comparison, using cophylogenies
collected from published literature. Evidence of significant differences
in the coevolutionary histories between ecto- and endo-symbionts was
found, with host switching more likely in ecto-symbionts. Significance:
The ever-expanding availability of barcode and host association data
provides opportunities for testing broad coevolutionary questions
and for research into comparative patterns of coevolution. Through
specimen barcodes and associated data, previously inaccessible lines
of research are now becoming possible. Do host switching rates and
levels of specialization/generalization differ across latitude zones?
What impact do various biological traits have on host-symbiont coevo-
lutionary history? What factors influence general coevolutionary
patterns?
Facing the challenge of growing numbers of fungal infections—
sequence-based fungal identification using the ISHAM-ITS
reference database
Wieland Meyer, Laszlo Irinyi, and The ISHAM Working Group for
Barcoding of Human and Animal Pathogenic Fungi
Molecular Mycology Research Laboratory, CIDM, Sydney Medical School-Westmead Hospital, The
University of Sydney, Westmead Millennium Institute, Sydney, NSW 2145, Australia.
Corresponding author: Wieland Meyer (e-mail: wieland.meyer@sydney.edu.au).
The numbers of human and animal fungal infections have increased
significantly in the last decade, causing serious public health burdens
and increased risk of biodiversity loss among animal species. Correct
and fast identification of the causative agents of mycoses is of great
importance to enable early diagnosis and targeted antifungal
therapy. DNA barcoding offers an accurate, fast, cost-effective,
culture-independent approach for species identification. The current
generally agreed upon fungal DNA barcode is the internal transcribed
spacer (ITS), but its widespread application in clinical settings has
been limited due to the lack of quality-controlled reference databases.
Recently, an international consortium of medical mycology laborato-
ries established the ISHAM-ITS database, the first fungal barcode
database for human and animal pathogenic fungi. The database
is available from http://www.isham.org/ or directly at http://its.
mycologylab.org/. It currently hosts 3200 complete ITS sequences cov-
ering 524 medically relevant fungal species, and it is open for further
submission. It is intended to cover all medically important fungal
species with a sufficient number of strains to represent the diversity of
a species. The gathered data have been used to evaluate the value of
the ITS region as a barcode at the intra- and interspecies levels. The
intraspecies variation of the ITS sequences currently included in the
database ranges from 0% to 2.25%, with 170 species having variation of
less than 1.5%. At the interspecies level, several taxa were identified
without a barcoding gap, and the ITS region was insufficient to dis-
criminate between closely related/cryptic species. Overall, clinically
important species show low intraspecies variability and a clear bar-
coding gap at interspecies level; consequently, ITS sequencing can be
used for the identification of most of them. However, for the species
with more than 1.5% diversity or taxa without a barcoding gap, alter-
native barcode loci need to be introduced to ensure reliable species
identification.
BOLD tools in support of STEM education
Megan A. Milton,
1
Paola Pierossi,
2
and Sujeevan Ratnasingham
1
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
2Alberta Biodiversity Monitoring Institute, University of Alberta, Edmonton, Alberta, Canada.
Corresponding author: Megan A. Milton (e-mail: mmilton@uoguelph.ca).
Background: Engaging students in STEM (Science, Technology, Engi-
neering and Mathematics) has become a major priority for educators.
As STEM-based careers emerge and define a global shift in innovation,
the need for hands-on STEM-oriented activities for educational pur-
poses becomes clear. DNA barcoding, through early studies, has been
shown to be an effective activity for STEM education. The application
of DNA barcoding combines knowledge from biodiversity, genomics,
and bioinformatics fields in a cost-effective way, enabling its use in
education. BOLD Systems now provides some of the infrastructure
necessary to make this technology accessible in supporting both
teacher-led and independent education. We discuss the tools devel-
oped by BOLD for this purpose and provide examples on their use.
Results: Many students and citizen scientists have expressed an inter-
est in contributing to the DNA barcoding initiative via the Barcode of
Life Data (BOLD) Systems, a dedicated platform developed for DNA
barcoding. However, this platform is targeted at professional scien-
tists and post-graduate students due to the complexity of interfaces
and assumed subject matter experience from users. This limitation is
addressed through two other systems, the BOLD Student Data Portal
(BOLD-SDP) and LifeScanner. BOLD-SDP was developed based on the
scientific barcoding workflow but with greatly simplified interfaces
targeting high-school and undergraduate classrooms. This portal sup-
ports teacher-led research projects involving the generation of DNA
barcodes. A key aspect of this system is that results of classroom
experiments lead to novel data being contributed to the BOLD data-
base and GenBank. SDP provides special tools for instructors to sup-
port the monitoring and publishing of student work. Since its
activation in 2013, BOLD-SDP has been used by over 200 instructors
and 4000 students from 10 countries. The LifeScanner mobile identi-
fication tool was developed in part to support independent barcode-
based research by users of any age. LifeScanner consists of mobile
software that provides a very simple interface to BOLD and specimen
collection kits that enable to acquisition of DNA barcodes and
barcode-based species identifications. Significance: By equipping stu-
dents and educators with technology and the support framework to
implement programs, BOLD exposes students to new technologies
that increase their understanding and interest in STEM careers.
Dung, dorpers and diets: dietary analysis of sheep in
rangelands through DNA metabarcoding
Andrew Mitchell
1
and Yohannes Alemseged
2
1Australian Museum, 6 College Street, Sydney NSW 2010, Australia.
2NSW Department of Primary Industries PMB 19, Trangie NSW 2823, Australia.
Corresponding author: Andrew Mitchell (e-mail: andrew.mitchell@austmus.gov.au).
About three-quarters of Australia's area is rangeland. It contains a
wealth of biodiversity including more than 1800 plant and 600 verte-
brate species. This land must be managed for a complex mix of inter-
ests, including agricultural production and conservation. The recent
introduction of a more drought-tolerant breed of sheep, the Dorper, to
western New South Wales (NSW) has raised environmental concerns
because of potential impacts on rangeland conditions. As part of a
larger project investigating best management practices for Dorpers
on NSW rangelands, we compared the diets of Dorpers, Merinos, and
goats using a DNA metabarcoding approach. We first produced a ref-
erence DNA barcode library for rbcL, Atpf, and ITS2 sequences for the
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20 most common species thought to comprise the bulk of sheep diets
in western NSW. Then we PCR amplified a 250-bp fragment of rbcL and
complete ITS2 (⬃260 bp) sequences from 24 dung samples, indexed
them using a Nextera kit and sequenced them in a single run on the
Illumina MiSeq platform. We obtained 1.1 million sequences, or a
mean of 46 000 sequences per sample. After quality controls, we had
around 20-100 unique haplotypes per sample. In total 90 “species”
were identified from all faecal samples but less than half of the refer-
ence library sequences were recovered from dung samples. Many se-
quences could not be exactly matched to one plant species because of
either insufficient resolving power (i.e., multiple species with exact
matches in BOLD, most rbcL sequences) or no exact match either in
BOLD or in the small reference library we generated (most ITS2 se-
quences). Family-level identification was possible for all sequences
recovered from all samples. At the family level, Dorper dung con-
tained seven plant families not found in Merino dung, and no families
were unique to Merino dung, confirming that Dorpers have broader
diets than merinos.
Testing for positive selection in mitochondrial and nuclear
energy-related genes in Pterygota (flying insects)
T. Fatima Mitterboeck,
1
Shanlin Liu,
2
Rui Zhang,
2
Wenhui Song,
2
Jinzhong Fu,
1
Sarah J. Adamowicz,
1
and Xin Zhou
2
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2China National GeneBank-Shenzhen & BGI-Shenzhen Shenzhen, Guangdong Province, China 518083.
Corresponding author: T. Fatima Mitterboeck (e-mail: tmitterb@uoguelph.ca).
Background: The gain and loss of flight in animals is associated with
increased rates of molecular evolution in mitochondrial genes, likely
due to positive or relaxed selection related to energy usage. Previous
observations have associated flight loss in insects with increased mo-
lecular rates in cytochrome c oxidase subunit I (COI). Recently, the
gain of flight in insects has been investigated for positive selection in
mitochondrial protein-coding genes. We expand on previous studies,
by using the newest transcriptome-based insect topology and more
complete sampling of apterygote hexapods, to explore positive selec-
tion in the lineage in which flight likely evolved. We include all mito-
chondrial protein-coding genes and over 900 nuclear protein-coding
genes for 27 hexapod species. Through inclusion of non-energy-
related genes, we can help to clarify whether elevated rates reflect
genome-wide patterns or are specifically linked to selection related to
energy demands. We furthermore specifically investigate positive
selection in mitochondrial and nuclear oxidative phosphorylation
(OXPHOS) genes, including COI, across all 32 hexapod orders. Results:
Although COI or other mitochondrial or nuclear OXPHOS genes them-
selves do not show a strong signal of positive selection associated with
the origin of Pterygota, categories of nuclear genes related to the
mitochondrion are found to be significantly often under positive se-
lection in the lineage where flight originated. Significance: This
study is the first exploration of positive selection in this particular
lineage using a phylogenetic approach and with the largest gene rep-
resentation to date for apterygote hexapods. A synthesis of evidence
across evolutionarily disparate instances of flight gain in animals reveals
parallel molecular evolutionary patterns in energy-related genes.
Molecular evolutionary rates in freshwater versus terrestrial
insects
T. Fatima Mitterboeck, Jinzhong Fu, and Sarah J. Adamowicz
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: T. Fatima Mitterboeck (e-mail: tmitterb@uoguelph.ca).
Background: Growing public databases of DNA sequence data pres-
ent new opportunities for broad-scale investigation of patterns of mo-
lecular evolution and macroevolution across life. Insects are a species-
rich group with diverse biological and ecological traits. Within this
group, the evolutionary shift between terrestrial and freshwater hab-
itats has occurred numerous times for both larval and adult life stages.
Using publicly available DNA sequence data, including cytochrome c
oxidase subunit I (COI), we investigated patterns of molecular evolu-
tionary rates, including both overall nucleotide substitution rates and
dN/dS ratios, associated with these habitat shifts within the insect
orders Coleoptera, Diptera, Lepidoptera, Hemiptera, Mecoptera,
Trichoptera, and Neuroptera. Results: Based upon a sample size of
over 40 phylogenetically independent habitat transitions in insects,
we did not detect any significant overall differences in the relative
pace of molecular evolution among paired lineages inhabiting these
broad habitat categories. Significance: Our results indicate that, on
average, the molecular clock ticks at a similar rate in related freshwa-
ter versus terrestrial insects. Further work will consider more specific
habitat categories that are more closely associated with differences in
habitat connectivity, which may influence molecular rates through an
effect mediated by effective population size.
MiFish, a set of universal primers for metabarcoding
environmental DNA from fishes: detection of >230 species from
aquarium tanks and coral reefs in the subtropical western
North Pacific
Masaki Miya,
1,8
Yukuto Sato,
2
Tsukasa Fukunaga,
3
Tetsuya Sado,
1
Keiichi Sato,
4
Toshifumi Minamoto,
5
Satoshi Yamamoto,
5
Hiroaki Yamanaka,
6
Hitoshi Araki,
7
Michio Kondoh,
6
and Wataru Iwasaki
8
1Natural History Museum and Institute, Chiba 260-8682, Japan.
2Tohoku University, Miyagi 980-8573, Japan.
3The University of Tokyo, Chiba 277-8568, Japan.
4Okinawa Churashima Research Center, Okinawa 905-0206, Japan.
5Kobe University, Hyogo 657-8501, Japan.
6Ryukoku University, Shiga 520-2194, Japan.
7Hokkaido University, Hokkaido 060-8589, Japan.
8The University of Tokyo, Tokyo 133-0032, Japan.
Corresponding author: Masaki Miya (e-mail: miya@chiba-muse.or.jp).
Background: DNA-based species identification is transitioning from
barcoding individuals to metabarcoding communities. We developed
a set of universal PCR primers (MiFish-U/E) for metabarcoding envi-
ronmental DNA (eDNA) from fishes. Results: The primer design was
based on the aligned whole-mitochondrial genome (mitogenome) se-
quences from 880 species, supplemented by partial mitogenome se-
quences from 160 elasmobranchs (sharks and rays). The primers target
a hypervariable region of the 12S rRNA gene (163–185 bp), which con-
tains sufficient information to identify fishes to taxonomic family,
genus, and species except for some closely related congeners. To test
the versatility of the primers across a diverse range of fishes, we sampled
eDNA from four tanks in the Okinawa Churaumi Aquarium with known
species compositions, prepared dual-indexed libraries, and performed
paired-end sequencing of the region using high-throughput, next-
generation sequencing (NGS) technologies. Out of the 180 marine fish
species contained in the four tanks with reference sequences in a custom
database, we detected 168 species (93.3%) distributed across 59 families
and 123 genera. These fishes are not only taxonomically diverse—includ-
ing sharks, rays, and higher teleosts—but are also greatly varied in their
ecology, including both pelagic and benthic species living in shallow
coastal to deep waters. We also sampled natural seawaters around coral
reefs near the aquarium and detected 93 fish species using this approach.
Of the 93 species, 64 were not detected in the four aquarium tanks,
rendering the total number of species detected as 232, belonging to 70
families and 152 genera. Significance: The metabarcoding approach pre-
sented here is non-invasive, more efficient, more cost-effective, and more
sensitive than the traditional survey methods. It has the potential to
serve as an alternative (or complementary) tool for biodiversity monitor-
ing that revolutionises natural resource management and ecological
studies of fish communities on larger spatial and temporal scales.
Abstracts 257
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Validation of commercially important fish of India
Ashok Mohekar,
1
Amol Kalyankar,
2
Dinesh Nalage,
2
and Gulab Khedkar
2
1Department of Zoology, SMD Mohekar College, Kalamb Dist. Osmanabad, Maharashtra-431004, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Ashok Mohekar (e-mail: smdmmkl@gmail.com).
DNA barcoding has emerged recently as a powerful tool for food authen-
tication and traceability studies. Several commercial market frauds have
been reported using this technique throughout the world. Steps involved
in DNA barcoding are relatively simple, cost effective, and less time con-
suming as compared to other analytical tools. Indian marine fish exports
for 2013–2014 were at an all-time high, with a growth of 5.96%. Validation
of species could further boost fish exports if steps are being followed to
ensure consumer confidence. Despite extensive taxonomic studies, the
identification of fishes can still be problematic for a variety of reason,
even for experts. DNA barcoding is proving to be a useful tool in this
context. However, its broad application is impeded by the need to con-
struct a comprehensive reference sequence library for all fish species. For
the effective utility of DNA barcoding as a tool in the Indian fish trade, we
have collected marine fish from five major coasts of India, and collected
over 1130 fish belonging to 197 species. DNA barcoding of the COI gene
was carried out for all samples. We represent almost one third of the
known fauna, and examine the utility of these data to address several key
taxonomic uncertainties pertaining to species in this region. This study is
an effort to create a valid DNA barcode library for marine fish under trade
in India. Collected fish were digitally photographed, and rigorous taxo-
nomic procedures were followed for fish identification. To cut down the
cost of analysis we have utilised our in-house nucleic acid purification
protocol, and PCR reactions were carried out in 12 L reaction volume.
This study constitutes a significant contribution to the universal barcode
reference sequence library for fishes, and establishes the utility of bar-
coding for regional species identification. As an independent valuation of
taxonomy, DNA barcodes provide robust support for most morphologi-
cally based taxon concepts, and also highlight key areas of taxonomic
uncertainty worthy of reappraisal.
Associating larvae and adults of high Andean aquatic insects: a
preliminary analysis using DNA barcoding
Carlos I. Molina,
1
François-Marie Gibon,
2
and Nina Rønsted
3
1Instituto de Ecología, University Campus, #23 Street, Cota Cota, Bolivia.
2Centre de Biologie pour la Gestion des Populations (UMR INRA / IRD / CIRAD / Montpellier Supagro)
Campus de Baillarguet, CS 30016, 34988 Montferrier sur Lez Cedex, France.
3Natural History Museum of Denmark, University of Copenhagen Sølvgade 83 Entrance S, Copenhagen
K, DK1307, Denmark.
Corresponding author: Carlos I. Molina (e-mail: camoar6088@gmail.com).
Background: Andean aquatic insect larvae are poorly known at the
species level, despite their importance in the understanding of fresh-
water fauna (biodiversity) and for water quality assessment through
biomonitoring. We analyzed the feasibility of using DNA barcoding as
a tool to achieve correct and rapid species-level associations between
different life stages of high Andean aquatic insects. We conducted a
short field survey in order to collect aquatic insect larvae of the orders
Trichoptera (caddisflies), Ephemeroptera (mayflies), and Plecoptera
(stoneflies) in four high Andean streams. We also collected adults by
carefully examining the stream banks from adjacent terrestrial envi-
ronments. We focused on sampling the high Andean environment
because stream water from glaciers is becoming scarce, which could
pose a risk of local disappearance of these aquatic species. Results:
Using cytochrome c oxidase subunit I (COI) gene sequences from 15
specimens, we found high interspecific divergences between speci-
mens of different families and genera (averaging 28%), whereas intras-
pecific genetic divergences were much lower (ranging from 0.2% to
2%). Using this barcoding approach, we associate larval instars with
their respective adults for one species of mayfly (Meridialaris tintin-
nabula), three species of stoneflies (Anacroneruria vagante,Claudioperla
tigrina, and C. ruhieri), and one species of caddisfly (Anomalocoesmoecus
cf. illiesi). Significance: We conclude that barcoding using COI gene
sequences is an effective tool to associate life stages for these aquatic
insect species in the High Andean region. Reference DNA barcode
libraries will enable research into biodiversity patterns of larval
aquatic insects in the High Andean region, with implications for eco-
logical understanding and biomonitoring applications.
A look at the past to plan for the future—10 years of fish
barcodes in Quintana Roo
Martha Valdez Moreno, Lourdes Vásquez-Yeomans,
José Angel Cohuo-Colli, and Selene Morales-Gutiérrez
El Colegio De La Frontera Sur, Unidad Chetumal Ave. Centenario Km 5.5 Col. Pacto Obrero Campesino,
Chetumal Q. Roo, Mexico.
Corresponding author: Martha Valdez Moreno (e-mail: mvaldez@ecosur.mx).
Background: The state of Quintana Roo is located in southeastern
Mexico, covering an area of 44 705 km
2
. It is bordered by Yucatán to
the northwest, Campeche to the west, to the south by Belize, and to
the north and east by the Caribbean Sea. The state has a privileged
geographic position because of its shoreline, which contains a major
part of the second-largest barrier reef in the world, the Mesoamerican
Reef. Also, four Marine National Parks and two Biosphere Reserves
(Banco Chinchorro and Sian Ka=an) are located there. Results: Fish
barcoding studies in this region began in 2005. After 10 years, 21
projects have been added to BOLD and about 3369 specimens se-
quenced, 3094 of them marine and 275 freshwater, including adults,
juveniles, larvae, and eggs. They represent 116 families, 261 genera,
and 397 species of marine fish; 3 families, 4 genera, and 5 species of
sharks; 6 families, 6 genera, and 5 species of rays; and 20 genera
comprising 27 species of freshwater fish. These results represent
about 17% of the Mexican ichthyofauna. As for territorial coverage,
almost 95% of the state has not been studied yet with this technique.
Significance: In the near future, we will continue to build the data-
base of barcodes especially for freshwater fishes, and we also plan to
develop new projects based on topics such as structural diversity,
stomach contents, food chains, detection of exotic species, ecosystem
health, all of them including information already generated, as well as
the use of new methodologies such as metagenomics and eDNA.
Identification of diverse fish species in a closed aquarium
environment using eDNA
Kevin Morey and Robert Hanner
University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Kevin Morey (e-mail: kmorey@mail.uoguelph.ca).
Background: Environmental DNA (eDNA) has been proposed as
having powerful utility for the detection of species in aquatic en-
vironments. Previously, eDNA detection has been tested in aquaria
containing few known species using ribosomal markers; however,
the cytochrome c oxidase subunit I (COI) DNA barcode region has
yet to have its efficacy tested in the large-scale identification of
fish species through environmental water samples. Water samples
were collected from a large marine tank at Ripley's Aquarium of
Canada containing 80 identified fish species. Species identification
efficiency was tested using a COI “minibarcode” region, the full COI
barcode region, and ribosomal gene markers using next-generation
sequencing (NGS) techniques. Results: We report on the utility and
accuracy of using the COI barcode region with NGS in the identifi-
cation of fish species in a closed aquarium environment using
eDNA. Significance: This study provides a useful proof-of-concept
test of the strength of eDNA for the identification of large numbers
of species simultaneously through the use of NGS techniques. Ad-
ditionally, this study provides a useful assessment of the COI DNA
barcode region against other markers previously used for identify-
ing fish species using eDNA.
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Species identification in Malaise trap samples by DNA barcodes
using NGS—a “scoring matrix” of four amplicons
Jérôme Morinière,
1
Bruno Cancian de Araujo,
1
Athena Wai Lam,
1
Axel Hausmann,
1
Stefan Schmidt,
1
Lars Hendrich,
1
Berthold Fartmann,
2
Samuel Arvidsson,
2
and Gerhard Haszprunar
1
1SNSB, Bavarian State Collection of Zoology, Münchhausenstrasse 21, 81247 München, Germany.
2LGC Genomics GmbH, Ostendstraße 25, TGS Haus 8, 12459 Berlin, Germany.
Corresponding author: Jérôme Morinière (e-mail: moriniere@zsm.mwn.de).
Twelve years after the start of DNA barcoding, ⬃380 000 animal barcode clusters have been uploaded
to BOLD. This reference library is particularly comprehensive in Central Europe which makes DNA
barcoding a suitable tool, for instance, to survey 90% of the biodiversity within forest ecosystems in
Germany.
Background: The German Barcode initiatives (BFB & GBOL) generated
a reference library of 16 000 German animal species which is now
ready for effective biodiversity assessment. We pre-sorted one sin-
gle Malaise trap sample (1 week) into 12 groups of arthropod orders
(Coleoptera, Hymenoptera, Lepidoptera, Diptera, Hemiptera, Ara-
neae, Collembola, Blattodea, Neuroptera & Mecoptera, Psocoptera,
Orthoptera, Plecoptera) and extracted each group separately. An
aliquot of each DNA extract was combined to simulate a non-sorted
sample. Each DNA extract was amplified with four different primer
sets targeting the COI-5' fragment and PCR products (150-400 bp)
and sequenced separately using an Illumina MiSeq, resulting in
1.5 million sequences and 5500 clusters (CD-HIT-EST, 98%). We used a
total of 100 000 DNA barcodes of reliably identified, central Euro-
pean Hymenoptera, Coleoptera, Diptera, and Lepidoptera down-
loaded from BOLD to establish a reference sequence database for a
local custom BLAST, which allowed us to identify Barcode Index
Numbers (BINs) for each cluster sequence obtained from the NGS
analyses. Results: We used BLAST results with a minimum 97%
sequence identity (in order to account for sequencing errors and
within-species variation), resulting in a total number of 553 BINs
(621 BINs including the taxa which are not Coleoptera, Hymenop-
tera, Lepidoptera, and Diptera). We established a “scoring matrix”,
which increases the plausibility of choice for candidate BINs within
the mixture of the four amplicons used (the score was ranging from
1360 max. and 70 min. points). A BIN was scored when it was iden-
tified in different amplicon analyses, whereas it was penalized if
this was not the case. Summarizing the preliminary results of the
different amplicons, 380 of the BINs were identified with a score ≥ 300
(173 BINs with a lower score were excluded), of which a total of 258
BINs (68%) was found in both the sorted samples as well as in the
combined fraction. Significance: The results of this study are prom-
ising for fast, efficient, and reliable analysis of next-generation data
from Malaise trap samples, avoiding time-consuming pre-sorting of
trap samples.
Large-scale DNA barcoding of marine hydroids of the
superfamily Plumularioidea (Cnidaria: Hydrozoa)
Carlos J. Moura,
1
Ricardo S. Santos,
1
Harilaos Lessios,
2
and Allen Collins
3
1University of the Azores - IMAR/MARE IMAR - Instituto do MAR, Centro do IMAR da Universidade dos
Açores Rua Prof. Doutor Frederico Machado, 4, 9901-862 Horta, Portugal; NMNH Smithsonian
Institution.
2Smithsonian Institution - STRI Smithsonian Tropical Research Institute ATT: Harilaos A. Lessios 9100
PANAMA CITY PL, Panama.
3Smithsonian Institution - NMNH Smithsonian Institution, National Museum of Natural History 10th
and Constitution Ave NW, Washington, DC 20560-0163, USA.
Corresponding author: Carlos J. Moura (e-mail: carlos.moura@mail.com).
Background: Hydroids are relatively simply marine invertebrates
and are widely spread across all oceans, both in shallow and deep
waters. The superfamily Plumularioidea is one of the most common
and species rich, comprising benthic colonies, mainly plumose, some-
times attaining considerable sizes. Many Plumularioidea are believed
to have large geographic distributions. However, few diagnostic mor-
phological characters are used to categorize its taxa, a consequence of
their structural simplicity, and the recent use of molecular characters
has uncovered cryptic taxa. Furthermore, the high levels of morpho-
logical plasticity that hydrozoans display often make it difficult to
differentiate intraspecific from interspecific variation. Results: We
generated DNA barcodes using mitochondrial 16S and COI markers
for about 800 different Plumularioidea specimens. This almost triples
the number of Genbank records presently available online. We fo-
cused sampling effort across the Northeast Pacific and throughout the
North Atlantic (in both shallow and deep waters), but we also gener-
ated molecular data for other marine areas worldwide. We summarize
the main findings of our integrative taxonomic approach to better
understand the systematics, phylogeography, and speciation patterns
in more than 150 species of Plumularioidea belonging to 19 nominal
genera. Significance: We uncovered significant new and cryptic di-
versity at the taxonomic level of species and genera, but also evidence
for few cases of synonymy and of species distributed widely in both
shallow and deep waters. Remarkably, few species indicate the main-
tenance of genetic connectivity across the Atlantic. Other species ap-
pear to have taken advantage of boat traffic to propagate to remote
locations. It is also noteworthy that most lineages of shallow-water
hydroids from the Azores arrived in the archipelago in distinct colo-
nizing episodes originating from the NE Atlantic, contradicting the
main influence of the Gulf Stream current into the area presently. We
will further assess the effectiveness and efficiency of 16S or COI for
DNA barcoding of hydroids.
DNA barcoding of vertebrate diversity in Qatar
Adham Mushtak,
1
Dhabiya Al-Kubaisi,
1
Wadha Almarri,
2
Ghizlane Bendriss,
1
Aurora M. Castilla,
3
and Kuei-Chiu Chen
1
1Weill Cornell Medical College in Qatar Eduction City, P.O. Box 24144, Doha, Qatar.
2Carnegie Mellon University in Qatar Eduction City, P.O. Box 24866, Doha, Qatar.
3Ministry of the Environment, Qatar Environment and Energy Research Institute, Doha, Qatar.
Corresponding author: Kuei-Chiu Chen (e-mail: kuc2005@qatar-med.cornell.edu).
Background: The recent worldwide endeavour of DNA barcoding is
slowly gaining momentum in the Middle East. The objective of this
study is to analyze the standard barcode sequence of all vertebrate
species in Qatar, with the initial student-centered research focusing
on lizard diversity. Lizard species were collected across Qatar, and
0.5 cm
3
of muscle tissue was obtained from each specimen for DNA
extraction. More than 19 primer pairs suggested in the literature and
additional primer pair combinations were used on nine species, and a
variety of PCR programs were employed in order to amplify the stan-
dard DNA barcode sequence. Results: Among the nine currently ana-
lyzed species, six species produced PCR products successfully, for the
most part from primer cocktails originally designed for mammals. In
some species, to eliminate the presence of multiple sequences in the
PCR products, the fragments were ligated to cloning vectors in order
to amplify one single sequence. Significance: Although it has been
more difficult to develop universal barcode primers for amphibians
and non-avian reptiles due to their deep phylogenetic divergence,
M13-tailed primer cocktails appear to be the most promising primers
tested in our study and will be used on the entire herpetofauna diver-
sity as well as other vertebrates in Qatar.
Progress in the barcoding of illegally traded South African
wildlife species at the National Zoological Gardens of South Africa
Monica Mwale, Desire L. Dalton, Marli Roelofse, Thando Radebe,
Kim Labuschagne, Antonie Kloppers, Takalani Musekwa,
Shivan Parusnath, and Antoinette Kotze
National Zoological Garderns of South Africa, 232 Boom Street, Pretoria 0001, South Africa.
Corresponding author: Monica Mwale (e-mail: monicam@nzg.ac.za).
Background: The Barcode of Wildlife Project (BWP) project has been
established at the National Zoological Gardens of South Africa (NZG)
to set up a DNA barcoding database for forensic analyses for South
African wildlife species (priority and look-alike) that are illegally
hunted and trafficked internationally. The aim of the project is to use
this mitochondrial DNA (cytochrome c oxidase subunit I) reference
database to begin using DNA barcodes to identify species from minute
samples in the investigation and prosecution of wildlife crimes. This is
Abstracts 259
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a national project managed by the South African National Biodiversity
Institute (SANBI), working with the South Africa Police Services foren-
sics unit and National Prosecuting Authority (NPA), and sponsored by
Google Global Impacts awards through the Smithsonian Institution.
Results: To date, 38 priority species have been identified as well as
98 look-alike species, that comprise all CITES-listed mammals, birds, and
reptiles that occur in South Africa. These species have been collected
from the wild, game reserves, zoos, and museum collections under
chain of custody, with five individuals representing each species. Ex-
amples of forensic cases that have been analysed using the BWP COI
databases will be discussed including other forensic analyses ap-
proaches that are done by the NZG. Significance: The significance of
the project will be to implement and demonstrate the use of DNA
barcoding in the prosecution of wildlife crime to support national
conservation efforts for critically endangered wildlife species of South
Africa.
Real-time PCR for seafood authenticity: an extension of DNA
barcoding
Amanda M. Naaum,
1
Rena Shimizu,
2
Neil Sharma,
2
and Robert Hanner
1
1University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1, Canada.
2Instant Labs, 800 W Baltimore St #407, Baltimore, MD 21201, USA.
Corresponding author: Amanda M. Naaum (e-mail: anaaum@uoguelph.ca).
Background: DNA barcoding is being used to catalogue biodiversity
and has been shown to be a useful and highly versatile tool for species
identification. For example, DNA barcoding has been used to document
significant levels of market fraud occurring in the seafood industry. How-
ever, this method is currently limited to use only in well-equipped labo-
ratories. The Barcode of Life Data System (BOLD) is an excellent source
of sequences for developing qPCR assays, which may be more suitable
in cases where rapid, portable testing is necessary, or where testing of
mixtures is required. Results: Expanding the use of BOLD beyond
DNA barcoding, qPCR assays based on DNA barcode sequences have
been developed and applied for species identification in seafood
applications including identification of blue crab, salmon, and red
snapper. Assays can be run on portable, simple instruments to
augment current authenticity testing by providing on-site testing.
Significance: Rapid, accurate, point-of-need testing for the seafood
industry is a key concern for authenticity and traceability of products.
However, the limitations of current DNA testing is a barrier to more
wide-spread uptake in the industry. Commercial availability real-time
PCR assays on portable instruments will allow businesses to imple-
ment more in-depth authenticity testing for key products on-site, and
provide a means to integrate DNA testing into current quality man-
agement practices.
Minimum information for conducting a DNA barcoding
market survey
Amanda M. Naaum,
1
Jason St. Jacques,
1
Kimberly Warner,
2
Linda Santschi,
3
Ralph Imondi,
3
and Robert Hanner
1
1University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1, Canada.
2Oceana, 1350 Connecticut Ave., NW, 5th Floor, Washington, DC 20036 USA.
3Coastal Marine Biolabs, 1559 Spinnaker Drive, Ventura, CA 93001, USA.
Corresponding author: Amanda M. Naaum (e-mail: anaaum@uoguelph.ca).
Background: DNA barcoding has been applied as a method to test
seafood authenticity in numerous market surveys. This trend is con-
tinuing to gain momentum as DNA barcoding is employed as a regu-
latory tool, by the media, and by students to test seafood products, in
addition to its use by scientific researchers to monitor seafood substi-
tution. However, as market surveys documenting mislabeling con-
tinue to be published by both the press and scientific journals, there is
a need for standardization in practices to aid in comparing and veri-
fying research. Results: Developed in collaboration with researchers
from academia, education, and NGOs, the best practices for conducting
and reporting DNA barcoding market studies for seafood identification
have been documented for use by citizen scientists, media outlets, and
academic researchers. Significance: This represents a means for stan-
dardizing future DNA barcoding market surveys to allow for better cohe-
sion between studies, and improve the chances for regulatory uptake of
this technology. These standards can also be used as a guideline for other
methods for conducting market surveys, or for market surveys employ-
ing DNA barcoding of other groups of organisms.
DNA barcoding and real-time PCR for identification of
entrained species
Amanda M. Naaum, Lauren Overdyk, Steve Crawford,
and Robert Hanner
University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1, Canada.
Corresponding author: Amanda M. Naaum (e-mail: anaaum@uoguelph.ca).
Background: Fish entrainment is a significant concern in power plants
that employ open water cooling systems. In order to assess environmen-
tal impacts, the species-level identification of entrained specimens is
necessary. However, entrained fish collected for identification are often
damaged or degraded to the point that they can be challenging to iden-
tify. Importantly, eggs and larvae can be very difficult to identify using
morphological means—even when fully intact—due to their small size
and the limited availability of diagnostic keys. Therefore, identification
of entrained material can be costly and time-consuming, as well as po-
tentially inaccurate or impossible if samples are small and badly dam-
aged. DNA-based analysis can be used to address these issues and
provides an opportunity for improving the unidentified fraction of en-
trained fish specimens. Results: A standardized protocol for collection,
preservation, and extraction of DNA from larval fish and eggs has been
developed. DNA barcoding was successful in improving species identifi-
cation from fish larvae and eggs. Since entrainment samples are often
mixed, real-time PCR assays for key species were developed to identify
target species from entrainment mixtures. Significance: DNA barcoding
can improve identification when added to existing methods for identify-
ing entrained specimens. Real-time PCR allows direct monitoring of spe-
cies of economic or environmental concern from mixed entrainment
samples. This method can be used to track invasive or endangered spe-
cies, and potentially quantitate their biomass as a percentage of en-
trained biological material. Both DNA barcoding and real-time PCR
provide means to improve the accuracy and reliability with which en-
trainment studies are carried out. This allows companies to adhere to
environmental legislation, and also provides better tools for biodiversity
assessment.
DNA barcoding Congolese snakes
Zoltan T. Nagy,
1
Stephanie Van Riet,
2
Gontran Sonet,
1
Eli B. Greenbaum,
3
Chifundera Kusamba,
4
Vaclav Gvozdik,
5
Steffi Matthyssen,
2
Akuboy Bodongola,
6
and Erik Verheyen
7
1OD Taxonomy and Phylogeny (JEMU), Royal Belgian Institute of Natural Sciences (RBINS)
29 Vautierstraat, B-1000 Brussels, Belgium.
2University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium.
3Department of Biological Sciences, University of Texas at El Paso 500 West University Avenue, El Paso,
TX 79968, USA.
4Laboratoire d'Herpétologie, Centre de Recherche en Sciences Naturelles Lwiro, The Democratic
Republic of the Congo.
5Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, 67502 Studenec, Czech
Republic.
6Faculty of Science, University of Kisangani Kisangani, The Democratic Republic of the Congo.
7Royal Belgian Institute of Natural Sciences, 29 Vautierstraat, B-1000 Brussels, Belgium.
Corresponding author: Zoltan T. Nagy (e-mail: zoltan-tamas.nagy@naturalsciences.be).
Background: Since 2008, we have conducted several field trips to the
Democratic Republic of the Congo, sampling both lowland (central, east-
ern, and western Congo Basin) and mid-altitude (Katanga Province) re-
gions. We surveyed a wide range of habitats including tropical
rainforests, inundated swamp forests, mangroves, savannah-shrub land
mosaics, agricultural fields, as well as grasslands, gallery forests, miombo
woodlands, springs, swamps, and streams. We conducted field searches
for various projects in order to evaluate the diversity of amphibians and
squamate reptiles of Central Africa; over 600 snake specimens (Squa-
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mata: Serpentes) were collected. In addition to voucher specimens, tissue
samples were taken for genetic analyses, and DNA barcoding was used to
assess intraspecific and intrageneric diversity and to test species bound-
aries—a complementary approach to morphological identification. Re-
sults: We compiled a data set of DNA barcodes including ⬃90 snake
species. Most species of colubrids, lamprophiids, elapids, and viperids are
represented by multiple specimens collected at distinct localities. Ac-
cording to our results, most snake species are easy to distinguish with
mitochondrial sequences, and intraspecific divergences appear modest.
In the case of some common and widespread species, however, unexpect-
edly high divergence was found. This observation could be a consequence
of spatial isolation (by distance, due to inappropriate habitats and (or)
topographic barriers), ancient polymorphism, or other, hitherto uniden-
tified reasons. Significance: This study is the first DNA-barcoding as-
sisted survey of snakes in Central Africa. It presents a reference data set
for roughly half of the snake fauna of the Democratic Republic of the
Congo. Further studies using an integrative approach may lead to alter-
ations of current classifications and challenge the biogeographic inter-
pretation of apparently widespread Afrotropical snake taxa.
Evaluation of recent enforcement to prohibit the slaughter of
cow and its progeny for beef in India using DNA barcoding
B.S. Naikwade,
1
Prakash Mahajan,
1
and G.D. Khedkar
2
1Aurangabad Municipal Corporation Zoo, Aurangabad, Maharashtra State, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. BAM University, Aurangabad,
Maharashtra State, India.
Corresponding author: B.S. Naikwade (e-mail: drbsnaikwade1@gmail.com).
Recent enforcement of law (4 March 2014) prohibiting the slaughter of
“cow” includes bull, bullock, ox, or calf for the purpose of beef produc-
tion in India. This law is enforced since Article 48 of the Indian constitu-
tion enjoins on the State to organise agriculture and animal husbandry
on modern and scientific lines and in particular to take steps for preserv-
ing and improving the breeds and prohibiting the slaughter of cow and
its progeny. In view of the consideration that the cow and its entire
progeny must be saved to provide milk, as well as manure, it becomes
imperative to impose a complete ban on cow slaughter. One month after
the law enforcement, we tried to evaluate the effectiveness of the proce-
dures involved the present law on ban of beef production. We surveyed
585 meat-selling shops in Aurangabad city of Maharashtra State in India,
of which 255 shops were found in beef retailing. Small meat samples
were collected from 219 shops and analysed using DNA barcoding and by
developing species-specific primers. Many shops claim the retailing of
buffalo meat. Overall, DNA barcoding could be one of the effective and
inexpensive tools in implementation of the law in reliable meat trading
in India.
Confirmation of genetic diversity in morphologically
distinct accessions of the Solanum nigrum L. complex using
DNA barcodes
L. Nalina, T. Nandhini, K. Rajamani,
1
P. Paramaguru,
1
S. Dhivya,
2
C. Jijo,
2
S. Ramachandran,
2
and R. Sathishkumar
2
1Tamil Nadu Agricultural University, Department of Medicinal & Aromatic Crops, HC & RI, TNAU,
Coimbatore- 641 003, Tamil Nadu, India.
2Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
Corresponding author: S. Dhivya (e-mail: dhivyas87@gmail.com).
Background: The correct identification and interrelationships among
the members of the Solanum nigrum L. complex have often been a puzzle
to taxonomists due to their intermediate forms and rapid natural hybrid-
ization between species. Significant morphological dissimilarity be-
tween some species due to spontaneous chromosome doubling and
external environmental influences cause difficulties during the identifi-
cation process and often lead to ambiguous interpretation. Recently,
DNA-based approaches have become increasingly useful for analysing
species diversity and for the rapid and accurate identification of species.
Therefore, in this study ITS was used as a barcode candidate to analyse
the 52 accessions of the S. nigrum L. complex collected from the regions of
southern India. Results: In this study, 13 morphological keys were used
to ascertain 52 accessions of S. nigrum species collected from different
geographical locations. Among these, 13 morphologically distinct acces-
sions, which could not be grouped under the species S. nigrum L. complex,
were subjected to DNA barcoding for identification. A barcode study
using ITS grouped them under three different species, viz. S. nigrum (3/13),
S. americanum (6/13), and S. villosum (4/13). Significance: Our study dis-
played that some species exhibit intraspecific polymorphism that distin-
guish them from closely related species, and the use of barcode candidate
ITS was proved to be the powerful tool for distinguishing the plant spe-
cies. This study provided a concrete result for proposing that a DNA-based
framework is essential for identification, documentation, and conserva-
tion of genetic diversity of the Solanum nigrum L. complex and its closely
related species. Though Solanum nigrum L. has several medicinal proper-
ties, there are scientific records suggesting toxicity effects on livestock
and humans. Hence it is desirable to authenticate morphologically sim-
ilar species using DNA barcoding.
The sustainable use of global biodiversity: scope and relevance
of the Nagoya Protocol and the Convention of Biological
Diversity for Natural History Collections and researchers
Dirk Neumann,
1
Cornelia Löhne,
2
Ana Casino,
3
Johan Bodegård,
4
Christopher H. C. Lyal,
5
Anne Nivart,
6
China Williams,
7
and Peter Giere
8
1Zoological State Collection Munich, Münchhausenstr. 21, DE 81247 Munich, Germany.
2Botanical Garden and Botanical Museum Berlin-Dahlem, Free University Berlin Königin-Luise-Str. 6-8,
14195 Berlin, Germany.
3Consortium of European Taxonomic Facilities (CETAF), c/o Ryal Belgian Institute of Natural Sciences,
rue Vautier, 29 1000, Brussels, Belgium.
4Swedish Museum of Natural History, Frescativägen 40, 104 05 Stockholm, Sweden.
5The Natural History Museum, Cromwell Road, London SW 7 5BD, UK.
6Muséum national d'Histoire naturelle, 57 rue Cuvier, 75 005 Paris, France.
7Royal Botanic Gardens, Kew, Surrey, TW9 3AB, UK.
8Museum für Naturkunde, Leibniz Institute for Research on Evolution and Biodiversity, Invalidenstr. 43,
10115 Berlin, Germany.
Corresponding author: Dirk Neumann (e-mail: dirk.neumann@zsm.mwn.de).
Background: With the Nagoya Protocol (NP) taking effect on 12 October
2014, the fair and equitable sharing of the benefits arising out of the
utilization of genetic resources is regulated under a strong global legal
framework. It obliges all parties of the Convention on Biological Diversity
(CBD) to access genetic resources outside national borders legally and to
ensure that utilisation inside their national borders is in compliance with
the international legal framework and its national implementation.
With the Nagoya Protocol in effect combined with implementation of
European Access and Benefit Sharing (ABS) legislation, illegal accession
and utilisation can be prosecuted. Results: This will affect traditional and
modern biodiversity research, natural history collections, and transfer of
objects and samples worldwide. As the NP regulates genetic resources
(GR) in general, it reaches beyond organismal research, including disci-
plines like climate science (GR in drill cores, water, or soil samples) or
archaeo sciences (archaeobotany, archaeozoology, and archaeology) ex-
ploring ancient DNA. To allow compliance with national and interna-
tional ABS laws, additional reporting and documentation is required to
demonstrate legal acquisition and utilisation of genetic resources ac-
cessed outside one's own national borders. This affects natural history
collections and collection management in signatory and non-signatory
countries (such as the USA), but also samples transferred for biocontrol or
for identification without NP-conform documentation. Significance:
The European legislation calls for voluntary measures to reach ABS com-
pliance, latest in October 2015. The Consortium of European Taxonomic
Facilities (CETAF), a major European network of taxonomic institutions,
set up a group to examine the legal issues. The set of guidance documents
developed so far include a common Code of Conduct and Best Practice for
taxonomic collections. Natural History Collections and researchers
worldwide are advised to revise processes and policies and to form net-
works of (taxonomic) institutions that have CBD-conform procedures in
place.
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Placing the freshwater bivalves (Unionoida) of Peninsular
Malaysia on the bivalve tree of life
Pei-Yin Ng,
1
Manuel Lopes-Lima,
2
Alexandra Zieritz,
3
Elsa Froufe,
2
Arthur Bogan,
4
Ronaldo Sousa,
5
and John-James Wilson
1
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur,
Malaysia.
2Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas,
n.289, 4050-123 Porto.
3University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Malaysia.
4North Carolina Museum of Natural Sciences.
5University of Minho, Department of Biology, Campus de Gualtar, 4710-057 Braga, Portugal.
Corresponding author: Pei-Yin Ng (e-mail: peiyin@siswa.um.edu.my).
Background: The unrevolved and conflicting accounts of the phylogeny
of the Unionoida (freshwater mussels) are attributable to an insufficient
amount of data (characters and species), a sampling bias towards North
American and European species, and neglect of tropical taxa. Almost one
quarter of unionoid species are taxonomically incertae sedis, and the
relationships among tropical lineages of freshwater mussels is consid-
ered one of the “Great Unanswered Questions in Malacology”. We aim to
start to fill this void by generating DNA barcodes (cytochrome c oxidase I
mtDNA sequences) for 12 unionoid species from 30 populations in Pen-
insular Malaysia. Results: The newly generated DNA barcodes enabled us
to integrate the freshwater mussel species from Peninsular Malaysia into
the global unionoid phylogeny. Significance: We now have an enhanced
understanding of the species boundaries, generic monophyly, and sub-
family level position of the the freshwater mussel species from Peninsu-
lar Malaysia. This will facilitate further studies of freshwater mussels in
this region and opens the potential for commercial applications such as
pearl culture, bioremediation, plankton bloom control, and freshwater
habitat restoration.
A great inventory of the small: combining BOLD datamining
and focused sampling hugely increases knowledge of
taxonomy, biology, and distribution of leafmining pygmy
moths (Lepidoptera: Nepticulidae)
Erik J. Nieukerken,
1
Camiel Doorenweerd,
1
Marko Mutanen,
2
Jean-François Landry,
3
Jeremy Miller,
1
and Jeremy R. deWaard
4
1Naturalis Biodiversity Center, P.O. Box 9517 2300 RA Leiden, the Netherlands.
2Zoological Museum, University of Oulu, P.O. Box 8000 FIN-90014 Oulu. Finland.
3Agriculture & Agri-Food Canada, Canadian National Collection of Insects K.W. Neatby Building, 960
Carling Ave., Ottawa, ON K1A 0C6, Canada.
4Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Erik J. Nieukerken (e-mail: nieukerken@naturalis.nl).
Background: The Nepticulidae form an early-radiating family of leaf-
mining moths with tight associations with their angiosperm hosts.
Around 850 named species in 10 genera form part of an unknown but
much larger global diversity. The group offers insights into evolution
between insects and host plants. Traditional methods to investigate their
diversity and host plant relations require considerable human effort, an
impediment further confounded by their extremely small body size (3-8
mm). DNA barcodes can speed up recognition of host plant relations,
distribution, and taxonomy. Results: We built a barcode dataset of leaf-
mining larvae and emerging adults from focused sampling in many parts
of the world, study of museum specimens, and barcoding initiatives such
as in Finland (www.finbol.org). We mined the Barcode of Life Datasys-
tems (BOLD) for all barcoded specimens labeled with the family name,
and additionally searched for closely-allied Barcode Index Numbers
(BINs) lacking family-level taxonomy. We found almost 5500 barcoded
specimens, representing 928 BINs and ⬃890 species, of which more than
55% are unnamed or unidentified. A large portion of the specimens
(around 2500) originated from the massive Malaise trapping program at
the Biodiversity Institute of Ontario. Most were initially identified only to
the ordinal or family level. We use our data for estimating the unknown
biodiversity of Nepticulidae, globally and per continent. For North Amer-
ica alone, our results suggest 40 unnamed species, confirm 12 species as
Holarctic (including several newly recorded), and link previously uniden-
tified host records to named adults. Significance: Barcoding mass-
trapped specimens showed an unprecedented increase in our knowledge
of distribution, host plant use, and invasiveness in an intractable and
otherwise poorly known group. Such records, however, acquire scientific
value only if validated by a solid barcode reference library that has been
vetted by taxonomic expertise. We advocate combining large-scale bio-
diversity inventories with DNA barcoding.
DNA barcodes highlight genetic diversity patterns in rodents
from desert and Andean areas of Argentina
Agustina A. Ojeda,
1
Alex Borisenko,
2
Natalia Ivanova,
2
Agustina Novillo,
1
Cecilia Lanzone,
3
Daniela Rodriguez,
1
Pablo Cuello,
1
Fernanda Cuevas,
1
Pablo Jayat,
4
and Ricardo A. Ojeda
1
1Instituto Argentino de Investigaciones de las Zonas Áridas (IADIZA-CCTMendoza- CONICET), Av. Ruiz
Leal s/n Parque Gral. San Martín. Ciudad. Mendoza. CP 5500 - CC 507, Argentina.
2Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East Guelph, ON N1G 2W1, Canada.
3Laboratorio de Genética Evolutiva, FCEQyN, IBS, UNaM-CONICET, Félix de Azara 1552, CPA
N3300LQF Posadas, Misiones, Argentina.
4Instituto de Ambiente de Montaña y Regiones Áridas (IAMRA) Univ. Nacional de Chilecito, 9 de Julio
22, 5360 Chilecito, La Rioja, Argentina; Instituto de Ecología Regional (IER) Universidad Nacional de
Tucuman, IER-LIEY, C.C. 34, 4107 Yerba Buena, Tucumán, Argentina.
Corresponding author: Agustina A. Ojeda (e-mail: agustinao@mendoza-conicet.gob.ar).
Background: Much of South America consists of diverse arid–semi-
arid regions characterized by high mammal endemism as a result of
interplay between place and lineage histories. Rodents are important
components in this scenario (with more than 2200 species represent-
ing this most diverse order among mammals). The high diversity and
complex evolutionary history has motivated researchers to study phy-
logenetic relationships and taxonomy, which have been under contin-
uous revision. Despite being an important component of local faunas,
rodents pose a major challenge for species-level diagnostics, due to
complex phylogeographic history and obscure morphological fea-
tures. We evaluated DNA barcodes as a tool for assessing the taxo-
nomic and genetic diversity in the two major lineages of rodents: the
“caviomorphs” and “sigmodontines”, from arid regions of Argentina.
We analyzed 310 COI barcodes representing 32 species from 56 local-
ities along the Argentinean Central Andes and lowland desert.
Results: The NJ, MP, and ML trees provided clear distance-based
separation between all currently recognized species in our dataset
with >95% bootstrap support. No cases of sequence overlap between
species were observed. Phyllotis xanthopygus showed divergences of up
to 13.0% between geographically separated lineages and strong corre-
spondence between genetic and geographic structure, suggesting
cryptic speciation. Akodon spegazzinii and Tympanoctomys barrerae con-
tained geographically separated clusters with up to 2.3% divergence.
Most species demonstrated low COI variation (mean P-distance 0%–
2.2%) and no geographic pattern. Mean nearest-neighbour distances
between congeneric species varied between 3.0% and 12.6%, except for
two closely related forms within Euneomys.Significance: The ob-
served DNA barcode divergence patterns provide insights into the
complex phylogeographic patterns and speciation scenarios in desert
Andean rodents and highlight areas requiring in-depth taxonomic
research, with potential implications in conservation and human
health.
Compilation and validation of a global DNA barcode reference
library for European marine fishes
Luís M. Oliveira,
1
Thomas Knebelsberg,
2
Monica Landi,
1
Pedro Soares,
1
Michael J. Raupach,
2
and Filipe O. Costa
1
1Centre of Molecular and Environmental Biology (CBMA), University of Minho, Campus de Gualtar,
4710-057 Braga, Portugal.
2German Center for Marine Biodiversity Research (DZMB), Suedstrand 44, 26382 Wilhelmshaven,
Germany.
Corresponding author: Luís M. Oliveira (e-mail: luismco12@gmail.com).
Background: Over the last few years, several comprehensive refer-
ence libraries of DNA barcodes for marine fishes of Europe have been
published with regional focus, but a global appraisal of the progress of
the compilation of a reference library for European marine ichthyo-
fauna is still missing. Here we assemble for the first time a large-scale
comprehensive reference library for this ichthyofauna, based on all
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publicly available DNA barcodes, with the aim to examine and anno-
tate consistency and reliability of records obtained independently
from multiple regions and studies. Results: We assembled a large
dataset comprising 4118 DNA barcodes from fish specimens collected
in Europe, representing 358 species, mined from 18 projects on BOLD
and a total of 13 research papers. A BIN (Barcode Index Number)
Discordance Report was generated for the BOLD dataset, showing
366 BINS (13 species were not available on BOLD) of which 213 were
concordant (1 BIN = 1 species), 141 discordant, and 12 singletons. Subse-
quent inspection of the BIN composition revealed potential artifacts
(i.e., synonyms, misidentifications, BIN=s incapability to resolve clus-
ter boundaries) resulting in 73% concordant species IDs, 5% with com-
paratively high intraspecific divergence (e.g., European seabass), and
8% of poorly represented species. Many economically important spe-
cies such as mackerel, salmonids, and various sharks are included in
the 14% of ambiguous species IDs. Significance: About 3/4 of the
European marine fishes species examined displayed DNA barcodes of
high taxonomic congruency, a proximate proportion to what has
been reported in sub-regional scope studies. The compilation of this
European-scale library unraveled pertinent cases of high within-
species divergence and taxonomic uncertainties that should be fur-
ther investigated. Despite the usefulness of this core reference library,
which includes most of the economically relevant species, the major-
ity of the European marine fish species is still missing. Therefore,
efforts to complete this library should continue.
Authenticating Thai herbal products, Boraphet: Tinospora crispa
(Menispermeae) by DNA barcoding coupled with high
resolution melting analysis
Maslin Osathanunkul,
1
Danupol Pintakum,
1
Sarawut Ounjai,
1
Panagiotis Madesis,
2
and Chatmongkon Suwannapoom
3
1Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
2Institute of Applied Biosciences, Centre for Research & Technology Hellas (CERTH), Thessaloniki, Greece.
3State Key Laboratory of Genetic Resources and Evolution State, and Yunnan Laboratory of Molecular
Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming
650223, China.
Corresponding author: Maslin Osathanunkul (e-mail: omaslin@gmail.com).
Background: Medicinal plants are used as a popular alternative to syn-
thetic drugs, both in developed and developing countries. The economic
importance of the herbal industry is increasing every year. As the herbal
industry grows, consumer safety is one issue that cannot be overlooked.
Herbal products in Thai local markets are commonly sold in processed
forms such as powders and capsules, making it almost impossible to
accurately identify the constituent species. Therefore, buying local
herbal products poses a high risk of acquiring counterfeited, substituted,
and (or) adulterated products. Due to these issues, a reliable method to
authenticate products is needed. Results: Here, DNA barcoding was used
in combination with high resolution melting analysis (Bar-HRM) to au-
thenticate Tinospora crispa species commonly used in Thailand. The ITS1
barcode was selected for use in primers design for HRM analysis to pro-
duce standard melting profiles of the selected species. Local products
made from T. crispa species were purchased from Thai markets and au-
thenticated by HRM analyses. Melting data from the HRM assay using the
designed primers showed that T. crispa could be distinguished from its
related species. The melting profiles of the ITS1 amplicons of each species
are clearly separated in all three replicates. The method was then applied
to authenticate local products in powdered form. HRM curves of all test
samples indicated that five of the tested products did not contain only
the indicated species, but other Tinospora species which have a high level
of morphological similarity. Significance: Incorrect information on
packaging and labels of the tested herbal products was the cause of the
results shown here. Morphological similarity among the species of inter-
est also hindered the collection process. The Bar-HRM method developed
here proved to be a cost-effective tool which can be used for rapid detec-
tion of adulteration and (or) substitution in herbal products.
Keeping the bees: identifying and protecting wild bees
Laurence Packer
York University, 4700 Keele Street, Toronto, Ontario, M3J 1P3, Canada.
E-mail for correspondence: xeromelissa@mail.com.
Abstract: Bees are almost constantly in the news, because they are re-
sponsible for some of the more nutritious and tasty components of our
diet and some species are in precipitous decline. Much of this news con-
cerns the domesticated western honey bee, which is only one of over
20 000 species worldwide and over 800 in Canada. In many agricultural
ecosystems, it is wild bees rather than domesticated ones that perform
most pollination, yet identifying these unsung heroines of the insect
world is problematic. I will provide examples of how DNA barcoding is
assisting us in providing identification tools for our bees.
Ancient Lake Titicaca as an evolutionary arena for
morphological diversification in Hyalella amphipods
Eric A. Paez-Parent,
1
Jonathan D.S. Witt,
2
and Sarah J. Adamowicz
3
1Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
2Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
3Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Eric A. Paez-Parent (e-mail: epaezpar@mail.uoguelph.ca).
Background: Ancient lakes are interesting systems for studying mor-
phological evolution as they harbour morphologically unique endemic
faunas, as exemplified by the diverse Hyalella amphipods of Lake Titicaca.
Phylogenetic evidence indicates multiple independent colonizations of
Lake Titicaca, providing a natural experiment for studying the conse-
quences of this major habitat transition. If long-term exposure to lake
conditions drives the sustained evolution of distinct morphologies in
Hyalella, then lineages that colonized earlier will be more morphologi-
cally divergent from their sister clades outside of the lake than lineages
that colonized later. Alternatively, rapid morphological evolution may
follow colonization due to exposure to new niches and predation. In this
study, we examine morphological and molecular (cytochrome c oxidase
subunit I) divergence between six Titicaca/non-Titicaca sister pairs of
Hyalella lineages to obtain insights into the pace of evolution of morpho-
logical novelty. Results: Based on multivariate statistical analysis of two
body size and 60 dorsal armature traits for 93 specimens, the Hyalella
genus is substantially more morphologically diverse within Lake Titicaca
than in surrounding environs. Additionally, there was significantly
higher prevalence of dorsal armature within Lake Titicaca. There was no
correlation, however, between morphological and molecular divergence
for paired Titicaca and non-Titicaca lineages, where molecular diver-
gence is assumed to approximate the relative time since lake coloniza-
tion among independent lineages. Two Titicaca clades exhibited
substantial morphological divergence from their sister lineages, despite
limited molecular divergence. Significance: Our study indicates that
independent Hyalella lineages have responded differently to Lake Titicaca
colonization, with two exhibiting rapid morphological evolution. Such
shifts may occur in response to new niche availability and predation
regimes and may be followed by a period of relative stasis. This study
provides new knowledge of the pattern and pace of morphological evo-
lution in ancient lakes and demonstrates the value of coupling morpho-
logical and barcode data in evolutionary study.
Dark taxa and hidden texts: obstacles to integrating barcodes
and taxonomy
Roderic Page
University of Glasgow, IBAHCM, MVLS, Glasgow G12 8QQ, UK.
E-mail for correspondence: Roderic.Page@glasgow.ac.uk.
Sequence databases are growing exponentially in size, whereas the rate
of description of new animal species has been roughly constant for de-
cades. This talk explores the implications of these two very different
trends, such as the growth in number of “dark taxa” (sequences not
associated with a named taxon). It discusses recent advances in making
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available and accessible the wealth of information in the primary taxo-
nomic literature, and the challenge of integrating this with the burgeon-
ing DNA barcode databases.
Environmental DNA metabarcoding to investigate historic
changes in biodiversity
Johan Pansu,
1
Charline Giguet-Covex,
1
Francesco Ficetola,
1
Ludovic Gielly,
1
Frederic Boyer,
1
Eric Coissac,
1
Isabelle Domaizon,
2
Lucie Zinger,
3
Jérôme Poulenard,
4
and Fabien Arnaud
4
1LECA, University Grenoble Alpes - CNRS LECA, BP 53 2233 Rue de la Piscine 38041 Grenoble Cedex 9.
2CARRTEL, INRA Thônon-Les-Bains INRA, 75 avenue de Corzent, 74200 Thonon-les-Bains, France.
3EDB, University Toulouse 3 Paul Sabatier - CNRS -ENFA Laboratoire Evolution & Diversité Biologique, Université
Toulouse III Paul Sabatier, Bâtiment 4R1 118, route de Narbonne, 31062 Toulouse cedex 9, France.
4EDYTEM, University Savoie Mont Blanc - CNRS - Ministère de la culture et de la communication
Université de Savoie, Laboratoire EDYTEM - UMR5204, Bâtiment « Pôle Montagne », Campus
scientifique Savoie Technolac, F-73376 Le Bourget du Lac cedex, France.
Corresponding author: Johan Pansu (e-mail: johan.pansu@gmail.com).
Background: Human activities have been modifying ecosystems for
thousands of years. Investigating biological community responses
to past anthropogenic disturbances is essential to understand and
anticipate long-term biodiversity changes. Over time, lake sediments accu-
mulate physical, chemical, and biological material—including DNA—
from the surrounding terrestrial environment through erosion and
sedimentation. Taking advantage of the promises of environmental DNA
metabarcoding to describe biodiversity, we investigated historic changes
in biodiversity in response to human-induced environmental modifica-
tions around a high-elevation lake over the last 10000 years. More specif-
ically, we (i) investigated the main past anthropogenic disturbances (i.e.,
pastoral activities) in this mountainous area by tracking mammal DNA in
the sediment records, (ii) reconstructed vegetation trajectories by target-
ing plant DNA, and (iii) evaluated the relative impact of these human
activities versus climate variations on plant biodiversity. Results: Our
high-quality data allowed reliable inference about presence/absence pat-
terns of numerous taxa along the chronological record of DNA in the
sediments. First, we detected the presence of livestock farming (cattle
and (or) sheep) associated with increased erosion during the Roman Pe-
riod and Middle Age. Second, we reconstructed plant community dynam-
ics from DNA. We particularly identified an abrupt replacement of
mountain forests and tall-herb communities by heathlands and grazed
lands ⬃4500 years ago. Plant community trajectories over the Holocene
were mostly related to soil evolution and pastoral activities, highlighting
the predominance of anthropogenic factors for their long-term evolu-
tion. All these results corroborated with the known anthropogenic his-
tory of the studied area. Significance: Sedimentary ancient DNA hence
provides reliable information on past biodiversity changes in relation to
human activities. This approach opens new avenues for research in pa-
leoecology. It will also enable the enhancement of forecasting models of
species distributions and biodiversity loss by giving access to biodiversity
records on temporal scales that were heretofore inaccessible.
Contrasting soil biodiversity patterns along an altitudinal gradient
Johan Pansu,
1
Lucie Zinger,
2
Loïc Chalmandrier,
1
Delphine Rioux,
1
Vincent Schilling,
2
Jérôme Chave,
2
Eric Coissac,
1
Wilfried Thuillier,
1
and Pierre Taberlet
1
1LECA, University Grenoble Alpes - CNRS Laboratoire d'Ecologie Alpine (LECA), BP 53, 2233 Rue de la
Piscine, 38041 Grenoble Cedex 9, France.
2EDB, University Toulouse III Paul Sabatier - CNRS - ENFA Laboratoire Evolution & Diversité Biologique
(EDB), Université Toulouse III Paul Sabatier, Bâtiment 4R1 118, route de Narbonne, 31062 Toulouse
cedex 9, France.
Corresponding author: Johan Pansu (e-mail: johan.pansu@gmail.com).
Background: In a global warming context, altitudinal diversity gradi-
ents for macro-organisms are subjects of intense research, but those of
soil biodiversity remain relatively poorly known. In this study, we at-
tempted to verify the generality of these patterns. We used environmen-
tal DNA metabarcoding to characterize diversity patterns for several soil
taxonomic groups (eukaryotes, fungi, bacteria, archaea, and plants)
along a 1000 m altitudinal gradient (from 1700 to 2700 m a.s.l.). Ten plots,
containing two 10m×10msub-plots, were placed every 100 m of altitude
change in a subalpine meadow located in the northern French Alps. In
each sub-plot, 22 soil cores were sampled along the diagonals and ana-
lyzed independently. Results: Our data allowed us to investigate varia-
tions of alpha- and beta-diversity of soil communities with elevation.
Responses of below-ground communities to elevation differed from
those usually reported for above-ground macro-organisms. Moreover,
altitudinal diversity patterns contrasted between the different studied
soil groups. Community data were also compared against environmental
variables. These results suggest that soil community composition arises
from different assembly rules. Significance: We show here that classical
altitudinal diversity patterns are not general to all organisms. This result
suggests that below-ground communities would not shift their distribu-
tion range in a similar way as reported for the above-ground biota. This
conclusion calls for a better characterization of the main drivers of soil
community assembly if we want to predict community responses to
global change.
By-passing the taxonomic impediment in Neotropical
Collembola to measure changes in diversity and
phylogenetic structure
Kate Pare and M. Alex Smith
Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1 Canada.
Corresponding author: Kate Pare (e-mail: kpare@uoguelph.ca).
Background: Taxonomic impediments can exist due to the presence of
cryptic lineages residing within named species and the lack of taxonomic
expertise available to recognise and formally assign names to these spe-
cies. Such overlooked diversity provides a challenge when studying bio-
geographical patterns in hyper-diverse groups or in diversity hotspots
such as the Neotropics. Using DNA barcodes as a proxy for species names
can remove the confounding factor of cryptic and unnamed diversity and
allow that diversity to be compared between multiple locations. Results:
Using DNA barcodes and rDNA sequences as proxies for species-level
identifications, we tested the relationship between phylogenetic diver-
sity and elevation as well as between phylogenetic community structure
and elevation. We predicted that Collembola diversity would increase
with elevation due to the increase in moisture. Furthermore, we ex-
pected phylogenetic structure to be more clustered at low elevations
compared to high elevations, due to dry environmental conditions select-
ing for specific lineages of Collembola able to survive such challenging
conditions. Significance: Collembola are small, abundant leaf-litter
hexapods, which are important prey items for arthropods and verte-
brates and aid in the decomposition of organic matter. Collembola are
also sensitive to desiccation, making them ideal organisms to study how
diversity changes along environmental gradients. However, because of
their small size and cryptic biology, there are challenges associated with
accurately estimating diversity within this group, necessitating the use of
techniques that analyse molecular estimates of diversity and phyloge-
netic structure.
A DNA barcode reference library for Asian quarantine pests
Doo-Sang Park,
1
Jun Hyung Jeon,
1
Bong-Kyu Byun,
2
Ki-Jeong Hong,
3
Seung Hwan Lee,
4
and Dong-Pyo Ryu
5
1Microbiological Resource Center, KRIBB 125 Gwahak-ro, Yuseong, Daejeon 305-806, Korea.
2Hannam University Daejeon, Korea.
3Soonchon National University Jeonnam, Korea.
4Seoul National University Seoul, Korea.
5Sanji University Gangwon, Korea.
Corresponding author: Jun Hyung Jeon (e-mail: dspark@kribb.re.kr).
Background: Increasing worldwide trade of plants enhances the risk of
spreading harmful organisms and may result in significant economic
damage. To construct an effective surveillance system against invasive
alien species, a standardized taxonomic framework between trading
countries is essentially required, and DNA barcoding is suggested as a
powerful tool for this purpose. Results: In this study, we developed a
DNA barcode reference library for quarantine pests, as well as their mor-
phologically similar and taxonomically related relatives, focused on four
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main categories of pests: Hemiptera, Micro-Lepidoptera, storage pests,
and ants. The 1500 sequences obtained represent 360 species that were
collected in South Korea, Southeast Asian countries, and some American
and Oceania countries. The library contains DNA barcode sequences and
relevant taxonomic and geographic data and images, which can be used
in plant quarantine applications. Significance: Building upon the previ-
ously assembled DNA barcode data for quarantine pests in Korea, we
expect to provide a comprehensive barcode registry for quarantine pests.
BARCODING.MED: building a high taxonomic resolution
database for the Mediterranean basin
Joana Paupério, José Manuel Grosso, Filipa Martins, Ana Filipa Filipe,
Andreia Penado, Vanessa Mata, Hugo Rebelo, Paulo Célio Alves,
and Pedro Beja
CIBIO/InBIO, R. Padre Armando Quintas, 4485-661 Vairao, Portugal.
Corresponding author: Hugo Rebelo (e-mail: hugo.rebelo@cibio.up.pt).
Background: Currently, the range of applications for DNA barcoding
in biodiversity research is very wide and has surpassed purely aca-
demic goals. The development of services for public and private insti-
tutions is now paramount to tackle several societal challenges in
diverse themes such as conservation biology, food security, and
forensics. Yet, the applicability of the DNA barcoding approach is
still undermined by the lack of taxonomic coverage in several
biodiversity-rich regions. In particular, the Mediterranean basin is an
underrepresented region in BOLD/GenBank, despite being a world biodi-
versity hotspot. Under the scope of the EDP Biodiversity Chair and an
ERA-Chair in Environmental Metagenomics (capacity building EU proj-
ects), we propose to develop a DNA barcoding database covering all ver-
tebrate and invertebrate taxa occurring in the Mediterranean basin.
Results: We are assembling a network of taxonomists and optimising
the analytical pipeline for barcoding a large number of voucher speci-
mens. Each sequenced specimen will be identified by expert taxono-
mists, uniquely labelled, and then stored in Natural History Museums,
CIBIO/InBIO, or private collections. Only taxonomically validated speci-
mens will be uploaded in Barcoding.med and internationally-free data-
bases like BOLD, constituting a source of development for services
relating to biodiversity research. The first phase of this project is focusing
on terrestrial and freshwater arthropods, particularly Plecoptera,
Ephemeroptera, Odonata, Trichoptera, Orthoptera, Coleoptera, and Hy-
menoptera. Significance: By providing cost-efficient approaches for bio-
diversity assessments and monitoring, we aim to promote the use of DNA
barcoding tools in public and private institutions, especially for provid-
ing services addressing current environmental legislation and interna-
tional obligations (e.g., water directive, detection of invasive species),
while also promoting research on ecosystem food webs. This initiative
will also be used to develop tailor-suited services to state and private
institutions, thus transferring skills and technology while also promot-
ing capacity building at a regional level.
Inferring biotic indices from metabarcoding data: promises
and challenges
Jan Pawlowski,
1
Franck Lejzerowicz,
1
Philippe Esling,
2
Laure Apothéloz-Perret-Gentil,
1
Joana Visco Amorim,
1
Loic Pillet,
1
Regis Vivien,
1
and Arielle Cordonier
3
1University of Geneva, Geneva, Switzerland.
2University of Paris, Paris, France.
3Water Ecology Service, Geneva, Switzerland.
Corresponding author: Jan Pawlowski (e-mail: jan.pawlowski@unige.ch).
Environmental diversity surveys are crucial for the bioassessment and
biomonitoring of anthropogenic impacts on aquatic ecosystems. Tradi-
tional monitoring is based on morphotaxonomic inventories of biologi-
cal communities, which are time-consuming, expensive, and require
excellent taxonomic expertise. High-throughput sequencing of environ-
mental DNA (metabarcoding) offers a powerful tool to describe biodiver-
sity. However, the capacity of the metabarcoding approach to meet the
quality standards of bioindication is a subject of controversy. Here, we
present the comparison of biotic indices inferred from morphotaxo-
nomic and molecular data for different groups of bioindicators (diatoms,
oligochaetes, meiofauna). We found that the molecular data faithfully
reflected the morphology-based indices and provide a similar assessment
of ecosystem status. We advocate that future biomonitoring should inte-
grate metabarcoding as a rapid and accurate tool for the evaluation of the
biological quality of aquatic ecosystems.
Streamlining scholarly publication of Barcode of Life data
Lyubomir Penev,
1
Stefan Schmidt,
2
Andrew Polaszek,
3
Teodor Georgiev,
1
Jose Fernandez-Triana,
4
Gunnar Brehm,
5
Pavel Stoev,
1
Axel Hausmann,
2
Sujeevan Ratnasingham,
6
and Paul D.N. Hebert
6
1Pensoft Publishers, Sofia, Bulgaria.
2Zoologische Staatssammlung, Munich, Germany.
3The Natural History Museum, London, UK.
4Canadian National Collection of Insects, Ottawa, Ont., Canada.
5Phyletisches Museum, Jena, Germany.
6Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
Corresponding author: Lyubomir Penev (e-mail: penev@pensoft.net).
Background: Biodiversity data are accumulating at an unprecedented
rate. However, their scholarly publication often happens at the expense
of tedious and time-consuming conversions that transform structured
data into non-machine-readable and non-reusable formats, such as PDF
and paper, thus adding to the ever-growing backlog of these types of data.
This is a deeply inefficient process that has to change. Publication of
biodiversity data needs to be fast, efficient, and structured. Results: The
Biodiversity Data Journal (BDJ) successfully tested several routes to export
specimen and other data from the Barcode of Life Data Systems (BOLD) to
BDJ's authoring tool via APIs and (or) Excel spreadsheets. The authoring
tool allows authors to further elaborate manuscripts online in collabora-
tion with co-authors and peers; for example, by providing specific epi-
thets, concise morphological descriptions and diagnoses, type specimen
data, and additional taxonomic remarks, to complete the formal publi-
cation in a way that meets the requirements of the codes of biological
nomenclature. The final version of the manuscript is submitted to BDJ
“at the click of a button” for further community peer-review, publica-
tion, and dissemination. On the very day of publication, BOLD can update
its original records either through automated metadata export or by a
harvesting mechanism. Occurrence records are automatically exported
and indexed in the Global Biodiversity Information Facility (GBIF). Data
paper manuscripts describing barcode data sets—also known as “bar-
code data release” papers—can also be extracted from the metadata de-
scriptors at BOLD and finalized in BDJ's authoring tool. Significance:
Data are imported into manuscripts and published in both human-
readable text and structured Darwin Core formats that can be down-
loaded by humans, or harvested by computers straight from the journal
article. This workflow saves a great deal of effort for all parties involved—
authors, editors, future users, and data aggregators.
Amino acid variation and protein structure of COI
barcodes—insights from a Metazoa-wide sample
Mikko Pentinsaari,
1
Heli Havukainen,
2
Marko Mutanen,
1
and Tomas Roslin
3
1Department of Genetics and Physiology, Pentti Kaiteran katu 1, 90014 University of Oulu, Finland.
2Department of Biosciences, Viikinkaari 1, 00140, University of Helsinki, Finland.
3Department of Agricultural Sciences, Latokartanonkaari 5, 00014 University of Helsinki, Finland.
Corresponding author: Mikko Pentinsaari (e-mail: mikko.pentinsaari@oulu.fi).
Background: Most studies published to date have treated the COI bar-
code simply as a conveniently readable identification tag. However, COI
plays a critical part in the cellular respiratory chain, and the barcode
fragment is located in the enzymatically active part of the COI protein.
Thus, changes in the amino acid sequence and protein structure may
affect energy metabolism. The massive number of sequences generated
by barcode initiatives around the globe allows us to explore the evolution
of this core metabolic enzyme in an unprecedented scale. We study ami-
no acid variation and evolution of COI protein structure in a crosscut of
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the entire Metazoa, and in denser samples of two megadiverse insect
orders, Coleoptera and Lepidoptera. Results: The COI barcode region
covers 219 amino acids: 23 were conserved across the entire Metazoa,
while 99 were highly variable. Based on 3D models of the protein, six of
these variable amino acids were located at atomic interaction distance
from the heme ligands, potentially affecting enzyme function. Deletions
were uncommon, and the vast majority of them were found in parasites
representing several different phyla. Unrelated parasite taxa also exhib-
ited some convergent amino acid changes at the heme-facing variable
sites. Coleoptera showed more amino acid variation than Lepidoptera (39
vs. 14 highly variable sites, respectively). Variation at sites potentially
affecting enzymatic activity was concentrated on a handful of herbivo-
rous and ancestrally fungivorous beetle lineages. Significance: Patterns
of COI barcode variation have rarely (if ever) been studied from the per-
spective of protein function and evolution. As expected, the protein
structure is generally conserved, but we managed to uncover some inter-
esting exceptions. Convergent evolution in parasites is particularly inter-
esting and possibly connected to similar hostile conditions faced by
endoparasites inside their hosts. The usefulness of large-scale barcode
libraries extends beyond identification and taxonomy.
The Global Malaise Program: assessing global biodiversity
using mass sampling and DNA barcoding
Kate H.J. Perez, Jayme E. Sones, Jeremy R. deWaard,
and Paul D.N. Hebert
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
Corresponding author: Kate H.J. Perez (e-mail: kperez@uoguelph.ca).
Background: The Global Malaise Program (GMP), a collaboration be-
tween the Biodiversity Institute of Ontario (BIO) and an ever growing
number of international contributors, represents a first step toward the
acquisition of detailed temporal and spatial information on terrestrial
arthropod communities across the globe. The standard methods of Mal-
aise trapping and DNA barcoding makes it possible to carry out large-
scale sampling programs and enables a time- and cost-efficient approach
for biodiversity assessments. Results: To date, 63 sites from 30 countries
have participated in GMP. From 2012 to 2014, Malaise traps were de-
ployed in ecosystems as diverse as Arctic tundra to tropical dry forest,
running anywhere from 4 to 62 weeks with samples collected weekly.
Over 638 000 specimens have been sorted from 36 sites (21 different
countries), and a total of 65 841 BINs were discovered. Identifications
were assigned by the BOLD-ID Engine where possible, allowing prelimi-
nary species inventories to be completed for each location and facilitat-
ing comparisons among them. As expected, species similarity was
negatively correlated to the distance between sites (p< 0.001) as well as
the latitudinal difference between sites (p< 0.001). However, there is no
evidence for higher species diversity in the tropics (i.e., the Latitudinal
Diversity Gradient; p> 0.10) although many confounding variables are at
play. Significance: Over the coming years, accumulated results from this
project will help establish comprehensive knowledge of species distribu-
tions and prevalence, which is critical for the accurate evaluation of
anthropogenic impacts. This project will also allow researchers to ex-
plore patterns of global biodiversity across latitudes, ecosystems, sea-
sons, and more. Furthermore, these established locations could act as
nodes for more intense, localized sampling efforts, paving the way for a
globally connected bio-monitoring program.
Applying DNA barcoding to the manufacturing supply chain,
to reduce substitution and adulteration fraud
Stephen Phelan
1
and David Jonker
2
1SAP, Holmes, New York, USA.
2SAP, Waterloo, Ont., Canada.
Corresponding author: Stephen Phelan (e-mail: stephen.phelan@sap.com).
DNA barcoding has become a mainstream tool in the food and health
products supply chains. It is particularly useful in distinguishing one
species from another, for example, tilapia from cod, or Coffea arabica
from Coffea canephora. Substitution of one species for another consti-
tutes not just fraud, but a consumer health hazard in many cases. The
integration of DNA testing into the business systems that monitor and
control the supply chains offers the possibility of reducing, or even
eliminating, this kind of substitution and adulteration, with major
benefits to manufacturers and consumers.
An exploration of sufficient sampling effort to describe
intraspecific DNA barcode haplotype diversity: examples from
the ray-finned fishes (Chordata: Actinopterygii)
Jarrett D. Phillips,
1
Rodger A. Gwiazdowski,
2
Daniel Ashlock,
3
and Robert Hanner
4
1Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
2Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
3Department of Mathematics and Statistics, University of Guelph, 50 Stone Road East, Guelph, ON, N1G
2W1, Canada.
4Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Jarrett D. Phillips (e-mail: jphill01@uoguelph.ca).
Background: Estimating appropriate sample sizes to measure species
abundance and richness is a fundamental problem for most biodiversity
research. In this study, we explore a method to measure sampling suffi-
ciency based on haplotype diversity in the ray-finned fishes (Animalia:
Chordata: Actinopterygii). To do this, we use linear regression and hy-
pothesis testing methods on haplotype accumulation curves from DNA
barcodes for 18 species of fishes, in the statistics platform R. We use a
simple mathematical model to estimate sampling sufficiency from a
sample-number based prediction of intraspecific haplotype diversity,
given an assumption of equal haplotype frequencies. Results: Our model
finds that haplotype diversity for most of the 18 fish species remains
largely unsampled, and this appears to be a result of small sample sizes.
Significance: Lastly, we discuss how our overly simple model may be a
useful starting point to develop future estimators for intraspecific sam-
pling sufficiency in studies using DNA barcodes.
What we need to know about biodiversity in order to
conserve it
Stuart Pimm
Duke University, Durham, NC, USA.
E-mail for correspondence: stuartpimm@me.com.
Currently, the salient feature about life on Earth is how fast we are
losing it. For birds, and a few well-known taxa, we know that extinc-
tions are running 10 000 times faster than the normal background
rate. We know where threatened species of these taxa live and we can
effect practical conservation efforts to prevent their extinction. Such
species represent perhaps 0.1% of all taxa; however, even that fraction
is uncertain. We do not need to know the names of all the species on
land and in the oceans. We do need to know which areas contain the
unique ones and ones in immediate danger to ensure conservation
efforts are broadly based taxonomically.
Using DNA barcoding (COI) to assess diversity of the
New Zealand native aphid genus Schizaphis (Hemiptera:
Aphididae)
Colleen Podmore,
1
Simon Bulman,
2
and Ian Hogg
1
1University of Waikato, Hillcrest Rd., Hamilton, New Zealand.
2Plant & Food, Lincoln, Christchurch, New Zealand.
Corresponding author: Colleen Podmore (e-mail: colleen.podmore@xtra.co.nz).
Background: We examined the diversity of COI in New Zealand
native aphids with a particular focus on the genus Schizaphis (syn.
Euschizaphis). Previously, the genus was thought to consist of two species
which are each host-specific to an endemic New Zealand plant (Aciphylla
and Dracophyllum). These unnamed native taxa are thought to be reason-
ably common but having a narrow distribution (Aciphylla-feeding) and
widespread but with low local abundance (Dracophyllum-feeding). There is
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also some uncertainty over the number of Schizaphis species present on
Dracophyllum.Results: Specimens from both host plants were collected
from 11 sites in the North (n= 3) and South (n= 8) Islands. A total of 29
individuals were sequenced for the COI gene marker. The dataset was
augmented by addition of sequences previously collected by researchers
at the New Zealand Institute for Plant and Food Research, Lincoln. Based
on maximum likelihood using the GTR+I model, two distinct well-
supported clades were clearly delineated according to the host plant.
However, we also found two distinct clusterings within the Aciphylla-
feeding group (South Island only) and five within the Dracophyllum-
feeding group (North and South Islands), suggesting seven potentially
cryptic species. This evidence indicates genetic differences between loca-
tions in the North and South Islands. Significance: Based on these data,
we suggest that the diversity of New Zealand native Schizaphis may be
underestimated. Ongoing work using the COI gene locus is likely to re-
veal additional diversity. The BOLD database will be particularly helpful
in examining global relationships within this taxon.
Ribosomal DNA and plastid markers used to sample fungal and
plant communities from wetland soils reveal complementary biotas
Teresita M. Porter,
1
Shadi Shokralla,
2
G. Brian Golding,
1
and Mehrdad Hajibabaei
2
1McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada.
2Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Teresita M. Porter (e-mail: terri@evol.mcmaster.ca).
Background: Although the use of metagenomic methods to sample
below-ground fungal communities is common, the use of similar meth-
ods to sample plants from their underground structures is not. In this
study we use the ribulose-bisphosphate carboxylase large subunit (rbcL)
plastid marker to sample the plant community as well as the internal
transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA (rDNA)
markers to sample the fungal community from two wetland sites.
Results: Observed community richness and composition varied by
marker. The two rDNA markers detected complementary sets of fungal
taxa. The taxonomic composition of fungi sampled across sites, but pro-
duced by the same primer, was more similar to each other than with
fungi sampled from the same site. By contrast, the taxonomic composi-
tion of the most frequently sampled rbcL OTUs was most similar within
sites. Significance: We suggest that future studies consider using multi-
ple markers—ideally generated from different primer sets—to detect a
more taxonomically diverse suite of taxa compared with what can be
detected by any single marker used alone. Conclusions drawn from the
presence of even the most frequently observed taxa should be made with
caution without corroborating lines of evidence.
Species tree inference from multilocus data sets
David Posada
Department of Biochemistry, Genetics and Immunology, University of Vigo, Spain.
E-mail for correspondence: dposada@uvigo.es.
The unprecedented amount of data resulting from next-generation se-
quencing has opened a new era in phylogenetics. However, although
large datasets should in theory increase phylogenetic resolution, multi-
locus data has also uncovered a great deal of phylogenetic incongruence
among different genomic regions. These findings have brought into the
limelight the distinction between gene trees and species trees. Indeed,
gene and species histories can be truly different due to the action of
different evolutionary process like incomplete lineage sorting, gene du-
plication and loss, and horizontal gene transfer. In this talk I will explain
some of the most important challenges we will have to face to recon-
struct species phylogenies from multilocus data sets. I will also describe a
new strategy for the phylogenetic analysis of multilocus data that we
have recently developed, which offers a very good compromise between
model complexity and computational feasibility.
Deep mitochondrial divergences within species are the rule
rather than the exception in western Palearctic leafcutter bees
Christophe Praz
University of Neuchatel, 2000 Neuchatel, Switzerland.
E-mail for correspondence: christophe.praz@unine.ch.
Background: DNA barcoding is used both for specimen identification
and as a tool for species delimitation. Molecular identification is prob-
lematic when distinct species share identical sequences, for example,
following mitochondrial introgression. Species delimitation is compli-
cated by deep within-species divergences that erode the barcoding gap.
Here I use the western Palaearctic bees of the large genus Megachile to
examine the performance of the mitochondrial barcode in molecular
identification and species delimitation. Results: Over 400 DNA barcodes
were generated for 70 of the ⬃100 described species in the western Pa-
laearctic. No evidence of mitochondrial introgression was revealed, even
among closely related species found in sympatry and infected with iden-
tical strains of Wolbachia, suggesting that this phenomenon does not pose
a major problem in this group of bees. However, in the majority of spe-
cies examined across their entire range, multiple, strongly divergent
mitochondrial haplotypes exist within single species. In most cases dif-
ferent haplotypes are found in different geographic regions, but in a few
cases widely different haplotypes are found in sympatry. In all cases, the
use of nuclear markers is necessary to determine species boundaries. The
fast-evolving marker ITS2 is particularly useful for species delimitation,
although it exhibited intragenomic diversity in most species examined.
Yet once clean sequences are obtained through cloning, the multiple
copies may highlight recent gene flow. Significance: Identification using
mitochondrial barcodes is not affected by these common deep within-
species divergences, provided that an accurate library is available across
the entire range of the species. However, these deep divergences greatly
limit the use of the mitochondrial barcode for species delimitation.
These results confirm that mitochondrial barcodes should not be used
alone to delimit species; even when nuclear markers are used, I recom-
mend detailed studies of the contact zone between genetically divergent
populations to determine species boundaries.
DNA barcodes from century-old type specimens using
next-generation sequencing
Sean Prosser,
1
Jeremy deWaard,
1
Scott Miller,
2
and Paul D.N. Hebert
1
1Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada.
2Smithsonian Institution, Washington, DC, USA.
Corresponding author: Sean Prosser (e-mail: sprosser@uoguelph.ca).
Background: Type specimens have high scientific importance because
they provide the only certain connection between the application of a
Linnean name and a physical specimen. Many other individuals may
have been identified as a particular species, but their linkage to the taxon
concept is inferential. Because type specimens are often more than a
century old and have experienced conditions unfavorable for DNA pres-
ervation, success in sequence recovery has been uncertain. The present
study addresses this challenge by employing next-generation sequencing
(NGS) to recover sequences for the barcode region of the cytochrome c
oxidase subunit I (COI) gene from small amounts of template DNA.
Results: DNA quality was first screened in more than 1800 type specimens
of Lepidoptera by attempting to recover 164- and 94-bp reads via Sanger
sequencing. This analysis permitted the assignment of each specimen to
one of three DNA quality categories—high (164-bp sequence), medium
(94-bp sequence), or low (no sequence). Ten century-old specimens from
each category were subsequently analyzed via a NGS protocol requiring
just 4 L of template DNA. Sequence information was recovered from all
specimens with average read lengths ranging from 458 to 610 bp for the
three DNA categories. Significance: With very little effort, 658-bp bar-
codes can be recovered from type specimens even in cases where DNA
degradation is too high for traditional Sanger sequencing. By sequencing
10 specimens in each NGS run, costs were similar to Sanger analysis.
Future increases in the number of specimens processed in each run
Abstracts 267
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promise substantial reductions in cost, making it possible to anticipate a
future where barcode sequences are available from most type specimens.
Species identification of ancient tuna remains using a novel
paleogenetic protocol and barcoding techniques
Gregory Neils Puncher,
1,2
Alessia Cariani,
1
Elisabetta Cilli,
3
Francesco Masari,
1
Arturo Morales,
4
Vedat Onar,
5
Nezir Yaşar Toker,
6
Thomas Moens,
2
and Fausto Tinti
1
1Department of Biological, Geological and Environmental Sciences / Laboratory of Genetics and
Genomics of Marine Resources and Environment (GenoDREAM), University of Bologna, Ravenna, Italy.
2Biology Department, Research Group Marine Biology, Ghent University, Ghent, Belgium.
3Laboratories of Physical Anthropology and Ancient DNA, Department of Cultural Heritage, University
of Bologna, Ravenna, Italy.
4Laboratory of Archeozoology, Department Biology, Autonomous University of Madrid, Madrid, Spain.
5Istanbul University, Faculty of Veterinary Medicine, Department of Anatomy, Osteoarchaeology
Research Centre Avcilar, Istanbul, Turkey.
6Istanbul University, Faculty of Veterinary Medicine, Department of Biochemistry Avcilar, Istanbul, Turkey.
Corresponding author: Gregory Neils Puncher (e-mail: greg@respectnature.com).
Background: Fishing has had a profound influence on the evolution of
Mediterranean civilizations over the past 20 000 years. Novel palaeoge-
netic tools can be used to identify ancient fish remains, allowing re-
searchers to better understand our all but forgotten relationship with the
sea. DNA was extracted from the remains of small fishes and large ma-
rine predators excavated from late iron age and ancient roman settle-
ments in coastal Iberia (4th–2nd century BCE; n= 23), Byzantine-era
Constantinople (4th–15th century CE; n= 6), and a 20th century collection
of vertebrae from the Ionian, Tyrrhenian, and Adriatic Seas (n= 208).
Mini-barcodes were amplified using newly developed primers targeting
diagnostic genes (cytochrome c oxidase subunit I, mitochondrial control
region, and internal transcribed spacer 1). Results: Samples were identi-
fied to genus level with a 78% success rate, while 70% of samples were
identified to species. BLAST, neighbour-joining trees, and a character-
based identification key were used to analyze DNA sequences, and the
latter was most effective due to the short length of DNA amplified (80-180
bp). Among the species identified were Atlantic bluefin tuna (Thunnus
thynnus), chub mackerel (Scomber colias), bonito (Sarda sarda), mackerel
(Scomber scombrus), bullet tuna (Auxis rochei), and swordfish (Xiphias gladius).
Significance: The techniques developed during this study have potential
for future use in both ancient and modern (traceability of cooked or
canned food products) forensic investigations. Used for archeological
purposes, our methods can help to identify damaged and incomplete
animal remains and provide information about historical human diets,
trade, species distribution and biodiversity.
Mode and tempo of diversification of Hyalella (Crustacea:
Amphipoda) in ancient Lake Titicaca
Michelle N. Pyle and Sarah J. Adamowicz
Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Michelle N. Pyle (e-mail: mpyle@mail.uoguelph.ca).
Background: Ancient lakes are renowned as sites of high biodiversity
and endemism. Evolutionary radiation within the genus Hyalella inhab-
iting ancient Lake Titicaca of Peru and Bolivia has resulted in an esti-
mated 100 endemic species. This study investigates two potentially
important mechanisms of diversification: geographic isolation of popu-
lations between the two sub-basins and population partitioning among
three depth zones. After barcoding 1273 specimens from the lake and
surrounding drainage basin, Barcode Index Numbers (BINs) were used to
group sequences into species-like entities for population genetic analysis.
Results: There was significant genetic partitioning among depth zones
within each sub-basin, with ⌽ST values within 3 BINs ranging from 0 to
0.211. The deep zone was consistently more different from the littoral
zone than was the middle zone across the two basins. By contrast, limited
genetic partitioning was observed between the sub-basins (⌽ST = 0.021–
0.051), although statistically significant structure was detected in the
deeper zones. The present-day distributions of BINs also suggest minimal
restrictions to dispersal between sub-basins, as Chao's Sørensen
Abundance-based similarity index (0.897) indicates that the majority of
BINs are shared between sub-basins. In sum, our results to date suggest
that divergence in different depth-linked habitats played a larger role in
diversification than geographic isolation; however, geographic isolation
could have played a greater role in the past and will be explored through
additional analyses. Investigation into the timescale of Hyalella diversifi-
cation using a molecular clock analysis revealed diversification coincided
with the formation of the lake ca. 2-3 MYA. Mapping depth occupancy
data onto a phylogenetic tree indicates that some lineages have subse-
quently diversified within depth zones. Significance: DNA barcoding
has permitted insight into this complex radiation, consisting of numer-
ous undescribed species, contributing to a comparative understanding of
the mechanisms underlying lineage proliferation within ancient lakes.
Depth-linked diversification is a recurring theme in several major an-
cient lakes radiations.
DNA barcoding and metabarcoding with the Oxford
Nanopore MinION
Anna C. Ramgren, Hannah S. Newhall, and Karen E. James
Mount Desert Island Biological Laboratory P.O. Box 35, Salisbury Cove, ME, USA.
Corresponding author: Karen E. James (e-mail: kjames@mdibl.org).
Background: Oxford Nanopore Technologies' MinION device is a minia-
turized platform for direct, electronic analysis of single molecules (DNA,
RNA, protein, etc.). The MinION is a portable, self-contained, real-time
instrument—about the size of a smartphone—that accepts reusable flow
cells and connects directly to a laptop or desktop computer via USB. The
device produces a “wiggle plot” which is uploaded to the cloud for real-
time base-calling. The MinION Access Programme (MAP) is a pre-
commercial beta-testing program in which several hundred participants
have been invited to apply the MinION to a range of biological questions
and research contexts. Results: As MAP participants, we used the Min-
ION to sequence PCR-amplified DNA from a broad range of individual
animal and plant specimens from Acadia National Park and from mito-
chondrial and chloroplast-enriched genomic DNA from mixed environ-
mental samples, including invasive green crab (Carcinus maenas) stomach
contents and marine sediments associated with disrupted, healthy, and
restored eelgrass (Zostera marina) habitat in Frenchman Bay, Maine. We
will report the results of our initial experiments including total basepairs,
read lengths, coverage of DNA barcode regions, raw wiggle plots, cloud-
based base-called sequences, and results from comparison of base-called
sequences with published animal and plant DNA barcode reference se-
quences using the Barcode of Life Data Systems (BOLD) identification
engine. Significance: From the moment DNA barcoding was proposed
more than a decade ago, members of the DNA barcoding community
have looked forward to a day when a handheld device might be used for
DNA-based identification of organisms in the field, in real time. The
MinION brings this once-distant possibility within reach and also repre-
sents a low-cost, next-generation platform for DNA metabarcoding.
Exploring the gut bacterial communities associated with larval
Helicoverpa armigera (Hübner) (Lepidopera: Noctuidae) based on
next-generation sequencing
M.T. Ranjith, Mani Chellappan, and E.R. Harish
Department of Agricultural Entomology, College of Horticulture, Vellanikkara, Kerala Agricultural
University, Kau (Po), Thrissur, Kerala 680656, India.
Corresponding author: M.T. Ranjith (e-mail: ranjith.mt16@gmail.com).
Background: Helicoverpa armigera (Lepidoptera: Noctuidae), a polypha-
gous agricultural pest of global importance, harbours diverse gut bacte-
rial communities. The gut bacteria community of H. armigera was
analysed previously by isolation and cultivation techniques and polym-
erized chain reaction (PCR) based cloning methods, resulting in identifi-
cation of few groups of bacteria. We explored the composition and
268 Genome Vol. 58, 2015
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diversity of gut bacteria in nine samples of H. armigera using Illu-
mina next-generation sequencing (NGS) of 16S ribosomal RNA am-
plicons. Results: Our data set consists of 864 813 high-quality,
paired-end sequences with average length of 150 bp. We found a
highly diverse bacterial community in the sample, containing
⬃2303 operational taxonomic units (OTUs) based on their sequence
similarity using the UCLUST program (similarity cutoff = 0.97). The
Illumina sequencing data were submitted to the Sequence Read
Archive (SRA) of GenBank as a file under accession number
SRR1914365. A total of 17 bacterial phyla, 34 classes, 84 orders, 173
families, 334 genera, and 707 species were identified by comparing
sequences against the Ribosomal Database Project (RDP) via the
metagenomics RAST (MG-RAST) server. Actinobacteria were the
most dominant group, followed by Proteobacteria, and Firmicutes.
Searches regarding the function of the different gut-inhabiting bacteria of
H. armigera revealed their role in nutrition, detoxification of lethal insecti-
cidal molecules, and defensive action against pathogens. Insecticidal toxin-
producing bacterial species were also found associated with H. armigera.
Significance: The present study paves the way to quantifying the total
bacteria community associated with the gut of H. armigera and under-
standing how it aids different physiological activities, which are impor-
tant factors that contribute to this species being a pest of global
importance. Further studies will focus on sequencing the whole gut met-
agenome of H. armigera, identifying the functional pathway of every rep-
resentative species, investigating the effect of removal of the gut
bacterial community on survival of the insect, and establishing whether
insecticidal toxin-producing bacterial species could play a major role as
biocontrol agent.
DNA barcodes for the fish of the second-largest river of India:
the Godavari
Sandeep Rathod,
1
Amol Kalyankar,
2
Anita Tiknaik,
2
Rahul Jamdade,
2
and Gulab Khdkar
2
1Department of Zoology, K.K.M.College, Manwath, Dist.Parbhani, Maharashtra State, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Gulab Khdkar (e-mail: gdkhedkar@gmail.com).
Background: Most of the Indian Rivers are heavily impacted by
human activities. For that reason there is great concern for the
conservation of aquatic life which is poorly understood. Many spe-
cies have yet to be described or to be discovered also because recent
ichthyological literature uncritically relies on earlier false data,
thereby propagating errors. It is estimated that 9% of Indian fresh-
water fish are endemic; however, this number may be even higher.
Results: Partial sequences (652 bp) of the mitochondrial gene COI
(cytochrome c oxidase subunit I) were used to barcode 622 individ-
uals, representing 12 orders, 41 families, 80 genera, and 103 species.
The mean conspecific, congeneric, and confamiliar genetic dis-
tances were 0.3%, 10.78%, and 16.26%, respectively. Molecular spe-
cies identification was in concordance with current taxonomical
classification in 92% of cases; based on the neighbour-joining trees,
in all but a few instances, members of a given genus clustered
within the same clade. A total of 12 cases of deep intraspecific
divergence (>3%) suggest the existence of cryptic species in the
Godavari River. Only 10% of the fishes recorded were recorded in
earlier studies based on morphology. On the other hand, 49 species
were newly recorded for the Godavari River. Various approaches
used in different studies led to the inflated and unrealistic number
of 220 fish species for the Godavari River, which indicates extensive
misidentification. We used this survey of genetic diversity as inde-
pendent calibration of current taxonomic resolution within the
Godavari fish fauna to reveal key areas of uncertainty whenever
discrepancies between genetic data and morphologically based tax-
onomy arise. Significance: This study can serve as a good example
demonstrating the need of complementary taxonomical proce-
dures to understand the diversity of life. Overall, the results
showed the usefulness of DNA barcodes for cataloguing the Goda-
vari river fish species and for identifying groups that deserve fur-
ther taxonomic attention.
Informatics tools for the second decade of DNA barcoding
Sujeevan Ratnasingham
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: sratnasi@uoguelph.ca.
The Barcode of Life Data System (BOLD) was launched in 2005 as a work-
bench and repository in support of a growing community of researchers
focused on building the DNA barcode library of all eukaryotic life. This
platform was highly successful during the first decade of DNA barcoding
with 4.2M+ barcodes representing 490K+ species hosted, and over 30K
species identifications provided per week. It is clear, however, that the
future informatics needs of the barcoding community will exceed the
capabilities of the current system. The overwhelming success of DNA
barcode studies across the taxonomic spectrum has resulted in the adop-
tion of this method in many life science fields, most notably in system-
atics, ecology, forensics, and conservation biology. With each field
finding novel uses, some extending and deviating from the original DNA
barcode concept, there are new and diverse requirements for informatics
tools. In recognition of this expanded landscape, the next generation of
informatics tools will need to employ new strategies, data standards, and
workflows. Important aspects of this future include an adoption of big-
data concepts and tools, democratization of DNA barcoding by improv-
ing access to this methodology, and a shift of focus from data collection
to knowledge generation. I present early solutions to these challenges,
including the latest version of BOLD (version 4), new tools, and future
plans to address the evolving informatics requirements in the second
decade of DNA barcoding.
A compendium of locally harvested trees traded as
traditional medicine at the Faraday Muthi Market in
Johannesburg, South Africa
Francinah Ratsoma, Olivier Maurin, Herman van der Bank,
and Michelle van der Bank
African Centre for DNA Barcoding, University of Johannesburg, PO BOX 524, Auckland Park, 2006,
South Africa.
Corresponding author: Francinah Ratsoma (e-mail: manchelaf@hotmail.com).
Background: Traditional medicine features in the lives of thousands of
people in South Africa every day, with current statistics estimating that
20 000 t of material, from more than 700 plant species, are traded annu-
ally. This intensive harvesting of wild medicinal plants represents a seri-
ous threat to biodiversity to such an extent that several traded plant
species are seldom found in unprotected areas. In South Africa, bark is
the most popular medicinal product harvested as medicine. On average
33% of the species sold at the Faraday Muthi Market are parts of trees (i.e.,
bark strips). Furthermore, the wide variety of products encountered at
local markets (protected flora, and more often their parts and derivatives)
makes it almost impossible to identify specimens to the taxonomic level
required to make critical law enforcement and conservation decisions,
which is a prerequisite for efficient control and seizure of illegally har-
vested forest products. In this study we used DNA barcoding as a tool to
identify forest products on the market that cannot be identified by tra-
ditional means. Here, we compared the unknown “bark” sequence from
the Faraday Muthi Market to a reference DNA barcode database for south-
ern African tree species. Results: Overall sequencing success was higher
for rbcLa (90%) than Atpf (81%) due to the fragmented nature of the ma-
terial. Of the market plants/bark identified, 55% are categorized as least
concern, 9% are vulnerable, 9% are declining, and 27% have not yet been
evaluated. Significance: The project produced an inventory of tree spe-
cies most affected by harvesting for traditional medicine for the manage-
ment of wild populations, and for Environmental Inspectors to monitor
the trade.
Abstracts 269
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Crabs, scallops, fish, and more: barcoding the marine fauna of
the North Sea
Michael Raupach,
1
Andrea Barco,
2
Jan Beermann,
3
Alexander Kieneke,
1
Silke Laakmann,
1
Inga Mohrbeck,
4
and Hermann Neumann
5
1DZMB/Senckenberg am Meer Suedstrand 44, 26382 Wilhelmshaven, Germany.
2GEOMAR - Helmholtz Centre for Ocean Research Duesternbrooker Weg 20, 24105 Kiel, Germany.
3Alfred Wegener Institute Helgoland Kurpromenade, 27498 Helgoland, Germany.
4Meeresforschung/Senckenberg am Meer Suedstrand 42, 26382 Wilhelmshaven, Germany.
5Thomas Knebelsberger, DZMB/Senckenberg am Meer Suedstrand 44, 26382 Wilhelmshaven, Germany.
Corresponding author: Michael Raupach (e-mail: mraupach@senckenberg.de).
Background: During the last years, the effectiveness of DNA barcoding for
animal species identification has been proven in many studies, analyzing
both vertebrate and invertebrate taxa. In terms of marine organisms, how-
ever, most barcoding studies typically focus on economically relevant spe-
cies, for example, fish, as well as on the documentation of hotspots of species
diversity, for example, tropical coral reefs or regions of the almost unex-
plored deep sea regions. In contrast to this, species diversity of “well-known”
habitats is nearly neglected. As part of our running project we started to
build up a comprehensive DNA barcode library for the metazoan taxa of the
North Sea, one of the most extensively studied ecosystems of the world. The
North Sea is characterized by a high amount of anthropogenic pressure such
as intensive fishing and ship traffic as well as offshore installations. Environ-
mental parameters (e.g., depth, sediment characteristics, temperature, and
salinity) of this semi-enclosed shelf sea follow a distinct pattern: high sea-
sonal fluctuations can be observed in southern areas, but low fluctuations
are given in the northern regions. This heterogeneity is also displayed in
macrobenthic community structures, with a lower number of species in the
shallow southern parts (i.e., the German Bight) and more species in the
central and northern North Sea. In addition to this, species with a typical
Mediterranean-Lusitanean distribution are also known to occur in parts of
the North Sea where oceanic influences prevail. Results: Our barcode library
includes a broad variety of taxa, including typical taxa of marine barcoding
studies, for example, fish or decapod crustaceans. Our on-growing library
also includes groups that are often ignored as cnidarians, parasitic crusta-
ceans, echinoderms, mollusks, pantopods, polychaets, and others. In total,
our library includes more than 4200 DNA barcodes of more than 600 species
at the moment. By using the Barcode of Life Data Systems (BOLD), unique
BINs were identified for more than 90% of the analyzed species. Signifi-
cance: Our data represent a first step towards the establishment of a com-
prehensive DNA barcode library of the Metazoa of the North Sea. Despite the
fact that various taxa are still missing or are currently underrepresented, our
results clearly underline the usefulness of DNA barcodes to discriminate the
vast majority of the analyzed species. It should be also kept in mind that the
benefits of DNA barcoding are not restricted to taxonomic or systematic
research only. The rise of modern high-throughput sequencing technologies
will change biomonitoring applications and surveys significantly in the com-
ing years. Following this, reference datasets such as ours will become essen-
tial for a correct identification of specimens sequenced as part of a
metabarcoding study. This is especially true for the North Sea, a marine
region that has been massively affected by cargo ship traffic, the exploitation
of oil and gas resources, offshore wind parks, and in particular extensive
long-term fisheries.
DNA barcoding of Pteris species by psbA–trnH intergenic spacer:
taxonomically complex and polyploid ferns
Baskaran Xavier Ravi,
1,2,3
Ramalingam Sathishkumar,
4
Selvaraj Dhivya,
4
Shanmughanandhan Dhivya,
4
and Shouzhou Zhang
1
1Shenzhen Key Laboratory of Southern Subtropical Plant Diversity, Fairylake Botanical Garden,
Shenzhen – 518004, P.R. China.
2Chinese Academy of Sciences, Shenzhen– 518004, P.R. China.
3Deptartment of Botany, St. Joseph's College, Tiruchirappalli, Tamil Nadu– 620 002, India.
4Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
Corresponding author: Shanmughanandhan Dhivya (e-mail: dhivyas87@gmail.com).
The fern genus Pteris is taxonomically complex to identify based on mor-
phological characters in wild ecosystems. Pteris species were proved to
exhibit bio-accumulation and antioxidant activities in previous studies.
The DNA was isolated from the plant samples using a modified CTAB
method (cetyl trimethyl ammonium bromide). PCR amplification of
the psbA–trnH intergenic spacer was carried out using following
gene-specific primers: F 5=-GTTATGCATGAACGTAAGCTC-3=;R5=-
CGCGCATGGTGGATTCAAATCC-3=in an Eppendorf thermal cycler (Ep-
pendorf, Germany). The PCR conditions were as follows: an initial
denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation
for 1 minute at 94°C, annealing for 1 minute at 55°C, and extension for
1 minute at 72°C; the final extension was at 72°C for 10 minutes. Then the
PCR product was separated in 1% agarose gel with ethidium bromide, and
the bands were visualized in a gel documentation unit (Alpha Digidoc,
USA). The evolutionary relationships of six Pteris species—namely
P. vittata L. (NCBI Accession No: JX987795), P. multiaurita Ag (JX987792),
P. confusa T.G.Walker (JX987791), P. otaria Bedd (JX987793), P. tripartita Sw.
(JX987794), and P. argyraea T.Moore (JX987790)—were studied using DNA
sequence data, and the sequences were submitted to NCBI. The sequence
alignments were carried out using Clustal X software for multiple se-
quence alignment, with a final alignment length of 402 bp. We found
variability in the sequence lengths among species: 355 bp (P. argyraea),
161 bp (P. confusa), 352 bp (P. multiaurita), 357 bp (P. otaria), and 300 bp
(P. vittata), suggesting that P. multiaurita was very closely related to P. otaria.
Furthermore, while comparing P. tripartita with other species of Pteris by
pairwise analysis, P. tripartita is the most similar to P. argyraea (9 bp ex-
hibited nucleotide differences between species), P. multiaurita (7 bp), and
P. otaria (8 bp), with 38%, 43%, and 37% gaps, respectively, between their
sequences, while the P. vittata (77 bp) and P. confusa (183 bp) showed more
genetic divergence along with 25% and 58% of gaps, respectively. Among
six Pteris species, significant distinct genetic variation was observed be-
tween P. tripartita and P. confusa. The evolutionary history was inferred by
the maximum parsimony method. The present study revealed that DNA
sequence data of the chloroplast psbA–trnH region was useful for resolv-
ing inter-specific relationships and performining identification among
six taxonomically complex Pteris species.
“Ethnoichthyogenomics”: identification, phylogeny and
documentation of ethnomedicinally important fishes using
DNA barcodes
Vaithilingam Ravitchandirane
1
and Muthusamy Thangaraj
2
1Department of Zoology, Kanchi Mamunivar Centre for Post Graduate Studies, Pondicherry-605008, India.
2CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai-608 502,
Tamilnadu, India.
Corresponding author: Vaithilingam Ravitchandirane (e-mail: vairavit@yahoo.co.in).
Background: Fishes play a vital role as traditional medicine in heal-
ing practices of many coastal nations. They are used in the treatment
of various ailments such as wound healing, joint pain (arthritis), and
asthma in Pondicherry and Tamil Nadu coastal villages, aside from
being a very important part of the cultural and religious life. We used
DNA barcoding as a tool for species identification to confirm authen-
ticity and unravel adulteration. Results: Ethnomedicinally important
fishes (6) have been collected based on traditional knowledge of prac-
titioners and local fishermen of the coastal villages of Pondicherry and
Tamil Nadu, India. Based on various classical morphometric charac-
ters, the fish were identified as Himantura imbricata,Naso annulatus,
Siganus javus,S. luridus,Trichiurus lepturus, and Leiognathus bindus. These
were authenticated by DNA barcoding the mitochondrial COI gene.
We also attempted genetic documentation and assessed the phyloge-
netic status of these fishes. Significance: The goal of the study was to
create Biological Reference Material (BRM) for ethnomedicinally im-
portant fishes from the east and west coast of India under the title
“ethnoichthyogenomics” in order to understand traditional use, ac-
tive compounds, and disease cure. Furthermore, this study explored
the biodiversity and evolutionary relationships using DNA sequences
to determine potential cryptic species, the distribution of rare spe-
cies, their ecological needs, including traditional ecological knowledge so
that management and conservation strategies can be implemented.
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DNA barcoding of rodent pests in South India
R.L. Rengarajan and G. Archunan
Center for Pheromone Technology, Department of Animal Science, Bharathidasan University,
Tiruchirappalli-620 024, India.
Corresponding author: R.L. Rengarajan (e-mail: srirenga21@gmail.com).
Background: Rodents constitute the largest and most successful group of
mammals worldwide. Their habitat, distribution, abundance, and economic
significance vary in different crops, seasons, and geographical regions across
India. Even though morphological observation is generally used to charac-
terize and identify rodent pest species, morphology-based identifications are
not always accurate. An alternative technique is to be developed for species
identification. DNA barcoding is a very effective and useful tool in species
identification even when classical approaches are not feasible. The present
investigation was undertaken to identify rodent species using 650 bp of
cytochrome C oxidase subunit I (COI). A total of 50 individuals from four
rodent pest species—namely Bandicota indica,Millardia meltada,Rattus rattus,
and Tatera indica, from six different places from South India—were taken for
individual species identification in the present study. Results: The present
study revealed that there was no intraspecific nucleotide variation within
Millardia meltada and Tatera indica among our barcoded specimens, and there
was low intraspecific variation within Bandicota indica and Rattus rattus at
maximum 0.3% and 0.5%, respectively. Among the four rodent pest species
included, the highest interspecific divergence was observed between Tatera
indica and Rattus rattus, at 22%, and the lowest between M. meltada and
B. indica,at4%.Significance: The present study concluded that the 650 bp of
the COI gene is valuable for accurate identification in rodent pest species.
The findings also support the use of COI-based DNA barcoding as a poten-
tially important tool for global pest species identification including rodent
pests.
DNA barcoding and cetacean species identification along the
Mauritanian coast, including the spectacular identification of a
Balaenoptera omurai specimen
Anaïs Rey,
1
Oumar Ba,
2
Wim C. Mullié,
3
Koen Van Waerebeek,
4
Jean-Luc Jung,
1
Moulaye Mohamed Wagne,
2
Abdellahi Samba Ould Bilal,
2
Zein el Abidine Ould Sidaty,
5
Marie Dominique Jezequel,
1
and Frédéric Marret
3
1Laboratoire BioGeMME, Université de Bretagne Occidentale, 6 avenue Le Gorgeu, 29200 Brest, France.
2Institut Mauritanien de Recherches Océanographiques et de Pêches (IMROP), BP 22, Nouadhibou, Mauritania.
3Programme Biodiversité Gaz Pétrole (BGP), GIZ BP 5217 Nouakchott, Mauritania.
4Conservation and Research of West African aquatic Mammals (COREWAM), Musée de la Mer de Gorée, Institut
Fondamental d'Afrique Noire (IFAN), Université Cheikh Anta Diop (UCAD), Dakar, Sénégal.
5Direction du Parc National du Diawling (PND), BP 3935, Nouakchott, Mauritania.
Corresponding author: Anaïs Rey (e-mail: anais.rey47@hotmail.fr).
Background: Mauritanian coastal waters are recognized as an ocean
province of high marine mammal diversity, hosting at least 26 cetacean
species, explainable by the presence of coastal upwelling associated with
the cool Canary Current. To enhance our knowledge of this key ecosys-
tem, highly impacted by human activities, and to detect possible tem-
poral trends, surveys of marine mammal strandings were conducted
every 3 months along 390 km of the Mauritanian coast. Combining
DNA analyses with morphological evidence allowed species identifi-
cation of even highly incomplete, damaged carcasses, and permitted
us to undertake population genetics studies for the harbour porpoise,
a vulnerable neritic odontocete. Results: More than 300 samples of
carcasses were collected from 2013 to 2015, including 33 non-identified.
The DNA barcoding approach, by using cox1 as a genetic marker, allowed
us to identify 30 specimens, including rare species, and led to the discov-
ery of a specimen of Balaenoptera omurai, 18 000 km away from its known
range, and a first record in the Atlantic Ocean. Among the 105 samples
collected of harbour porpoises, many were highly degraded so extracting
DNA remained a challenge. After experimental optimization, 91 individ-
uals (87%) were successfully sequenced for part of the mtDNA. Also, seven
microsatellites loci are being analyzed. For more than half of the sam-
ples, genetic sexing was achieved, revealing 31 males and 38 females.
Significance: Monitoring of cetacean strandings is essential to evaluate
anthropogenic impacts, especially where fisheries efforts and industrial
exploitation are high such as along the Mauritanian coast. Surveillance of
strandings linked to a barcoding approach is very useful and cost-
effective to assess long-term trends in cetacean species composition and
to detect unusual mortality events. The discovery of species formerly
unknown to occur, such as B. omurai, highlights the remarkable biodiver-
sity of the Canary Current ecosystem and re-emphasizes the importance
of systematic beach survey programme.
DNA barcoding reveals cryptic species and high genetic
divergence in pearl cichlid of Geophagus brasiliensis complex
from northeastern Brazil
Cássio Ribeiro de Souza,
1
Silvia Britto Barreto,
1
Paulo Roberto Antunes de Mello Affonso,
1
Iracilda Sampaio,
2
and Paulo Carneiro
1
1Universidade Estadual do Sudoeste da Bahia, Dep. Ciências Biológicas, Av. José Moreira Sobrinho, s/n
Jequiezinho, Jequié – BA 45206190, Brazil.
2Universidade Federal do Pará IECOS, Laboratório de Genética e Biologia Molecular, Alameda Leandro
Ribeiro s/n, Bairro Aldeia, Bragança – PA 68600000, Brazil.
Corresponding author: Silvia Britto Barreto (e-mail: silvinhabbarreto@hotmail.com).
Background: Pearl cichlid fish of the Geophagus brasiliensis complex
are widespread in Brazilian river basins, characterized by remarkable
morphological and color variation. Formerly considered a single spe-
cies, taxonomic studies have proposed that G. brasiliensis actually en-
compasses at least 12 taxa, even though the precise number of species
and their relationships remain unclear. In our study, we used DNA
barcoding to characterize genetic populations of pearl cichlids from
isolated coastal basins in northeastern Brazil throughout the north-
ern range of the G. brasiliensis complex. Results: A fragment of the
cytochrome c oxidase subunit I gene (COI) of 621 bp length and with 69
variable sites was amplified and sequenced bidirectionally from 93
specimens from 10 rivers along four coastal basins in the state of
Bahia, northeastern Brazil. Four genetic groups were observed in den-
drograms build using neighbour-joining, maximum likelihood, and
Bayesian inference, with high support values and mean K2P diver-
gence of 0.24% and 6.0% within and among populations, respectively.
Analysis of haplotype diversity revealed conspicuous genetic differen-
tiation of populations within the Contas River basin and suggests the
occurrence of headwater capture between nearby hydrographic sys-
tems. Significance: Our results indicated that each cluster represents
a unique species, with validation of G. itapicuruensis from the Itapicuru
River basin. Moreover, all evolutionary units herein identified are
genetically apart from Geophagus brasiliensis of other Brazilian basins.
Therefore, the taxonomic status of pearl cichlids in coastal basins of
northeastern Brazil needs to be revisited to provide a reliable scenario
of endemism rates and conservation issues of regional ichthyofauna,
characterized by insufficient knowledge.
Pollen analysis: is metabarcoding the next generation?
Rodney T. Richardson,
1
Chia-Hua Lin,
1
Juan O. Quijia Pillajo,
1
Natalia S. Riusech,
1
Karen Goodell,
2
and Reed M. Johnson
1
1The Ohio State University, Department of Entomology, 2021 Coffey Rd., Columbus, OH 43210, USA.
2The Ohio State University, Department of Evolution, Ecology and Organismal Biology, Founders Hall
2064, 1179 University Drive Newark, OH 43055, USA.
Corresponding author: Rodney T. Richardson (e-mail: richardson.827@osu.edu).
Background: Though useful in many areas of research, the difficulty
of microscopic palynology, or pollen analysis, has resulted in limited
implementation. Metabarcoding, a relatively novel approach, could
make palynology less onerous, but improved understanding of the
scope and quantitative capacity of various plant metabarcode regions
is needed to ensure that such applications are accurate and precise.
We applied metabarcoding, targeting the ITS2, Atpf, and rbcL loci, to
characterize six samples of pollen collected by honey bees, Apis
mellifera. In addition, samples were analyzed by microscopic palynology.
Results: We found significant rank-based associations between the
relative abundance of pollen types within our samples as inferred by
the two methods. Significance: Our findings suggest metabarcoding
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data from plastid loci, as opposed to ribosomal loci, are more reliable
for quantitative characterization of pollen assemblages. Additionally,
multi-locus metabarcoding of pollen may be more reliable quantita-
tively and qualitatively, underscoring the need for discovering novel
barcodes and barcode combinations optimized for molecular palynology.
Assessing invertebrate dispersal among restored streams in the
North Island of New Zealand using DNA barcoding
Morgan Riding,
1
Ian Hogg,
1
Brian Smith,
2
and Richard Storey
2
1University of Waikato, Gate 1 Knighton Road, Hamilton 3240, New Zealand.
2The National Institute of Water and Atmospheric Research (NIWA), Gate 10 Silverdale Road, Hillcrest
Hamilton, New Zealand.
Corresponding author: Morgan Riding (e-mail: morgan_riding@hotmail.com).
Background: Connectivity to source populations is critical for restor-
ing aquatic invertebrate communities. The Taranaki Region on the
West Coast of New Zealand consists of circular forested national park
(the quiescent stratovolcano, Mt Taranaki) surrounded by a ring-plain
of farmland. Restoration of farmland within the ring-plain has re-
sulted in a mosaic of newly forested patches that vary in temporal and
spatial scales. This has led to streams being fragmented, with varying
levels of connectivity to less-disturbed streams. Understanding rates
of inter-habitat dispersal is therefore essential in determining aquatic
invertebrate community recovery. The aim of the study was to deter-
mine the genetic connectivity of individuals among source popula-
tions and restored streams in the Taranaki Region. Five common
aquatic insect species were selected based on differences in flight capa-
bilities and longevity of adults: Archichauliodes diversus,Hydropsyche sp.,
Nesameletus sp., Coloburiscus humeralis, and Pycnocentrodes sp. A total of
10 sites within four streams around the mountain were used to exam-
ine connectivity within and among streams. We used sequencing of
the COI gene locus to measure genetic similarity among locations and
to determine potential geographic origins and dispersal pathways.
Results: Preliminary results indicate that highly mobile taxa such as
A. diversus share sequences between sites with little variation. Less
mobile species such as Hydopsyche sp. share sequences between the
closest sites; however, divergence increases with distance and pres-
ence of physical barriers (Mt Taranaki). This same general pattern is
also evident in Pynocentrodes sp., with the most remote location being
distinct from the other sites. Significance: These data will be used to
provide insight into the dispersal patterns of aquatic insects and to
assist with restoration efforts for stream ecosystems globally.
Dissecting Arctic food webs by DNA barcodes
Tomas Roslin and Helena Wirta
Department of Agricultural Sciences, P.O. Box 27 (Latokartanonkaari 5), FI-00014 University of
Helsinki, Finland.
Corresponding author: Tomas Roslin (e-mail: tomas.roslin@helsinki.fi).
Background: DNA barcodes offer efficient tools for describing both
the members of natural communities and the interactions between
them. In evidence of their utility, we have established a comprehen-
sive DNA barcode library for the terrestrial animals and vascular
plants of an intensively studied area of the High Arctic, the Zacken-
berg Valley of northeast Greenland. The resultant resource offers
species-level resolution in describing biotic interactions among com-
munity members. Over the last few years, we have used it to document
trophic links between predators and prey throughout this High Arctic
food web. Results: Overall, 425 terrestrial animal species and
160 vascular plant species were recorded by morphology-based tech-
niques. DNA barcodes based on COI offered high resolution in discrim-
inating among the local animal taxa, with rbcLa and ITS2 offering less
resolution among vascular plants. When DNA barcodes were applied
to resolve feeding associations among animals, the structure of the
food web proved extremely complex, showing dense linking and no
compartmentalization. Significance: Our description of the Zacken-
berg community comes with urgent implications both for our under-
standing of how arctic ecosystems are built and function, and of
how they respond to current climate change. To comprehend the
community-level consequences of rapid Arctic warming, we must em-
brace Arctic food web complexity, where impacts on single species
may reverberate widely across the community.
DNA metabarcoding of saproxylic beetles—streamlining species
identification for large-scale forest biomonitoring
Rodolphe Rougerie,
1
Mehrdad Hajibabaei,
2
Christophe Bouget,
3
Shadi Shokralla,
2
Joel F. Gibson,
2
and Carlos Lopez-Vaamonde
4
1Muséum national d’Histoire naturelle, UMR 75205, 45 rue de Buffon, CP50, 75005 Paris, France.
2Department of Integrative Biology and Biodiversity Institute of Ontario, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
3IRSTEA, Unité Ecosystèmes Forestiers, Nogent-sur-Vernisson, France.
4INRA, UR0633 Zoologie Forestiere, F-45075 Orleans, France.
Corresponding author: Carlos Lopez-Vaamonde (e-mail:
carlos.lopez-vaamonde@orleans.inra.fr).
Background: Forest ecosystems host most of the terrestrial biodiver-
sity on Earth. Climate change scenarios predict an increase in the
intensity and frequency of severe summer droughts, high tempera-
tures, and infestations of pathogens and insects, causing high mortal-
ity of some keystone tree species. These changes will affect forestry
policies and practices, strongly impacting biodiversity. Understand-
ing the responses of biodiversity to forest decline is therefore essential
to developing new climate-smart management options. Biomonitor-
ing of forest insects relies on techniques involving laborious and ex-
pensive sampling procedures. For instance, the study of indicators
such as saproxylic beetles is strongly impeded by their high abun-
dance and diversity, and by the deficit in taxonomists able to identify
them. Here, we propose and test the use of metabarcoding for bulk
samples of saproxylic beetles, in combination with the assembly of a
relevant barcode reference library, as a mean to streamline identifi-
cation. Results: Using a set of three primer pairs targeting short frag-
ments within the cytochrome c oxidase subunit I (COI) barcode, we
analyzed through metabarcoding a set of 32 bulk samples of saproxylic
beetles collected in France, containing hundreds of specimens that
were all initially counted and identified using morphology. To test the
efficiency of non-destructive analyses, we also sequenced libraries of
amplicons directly obtained from the ethanol used for preserving the
samples. Identifying the resulting reads with a newly assembled bar-
code library, we successfully recovered most species present in each of
the samples. Furthermore, our samples were selected to take into
account a variety of conditions and parameters possibly affecting the
results (species diversity, relative abundance and biomass, sampling
medium, and preservation method). Significance: The use of DNA
metabarcoding to monitor forest biodiversity can significantly im-
prove our capacity to measure, understand, and anticipate the impact
of global changes on forests, thus enhancing conservation strategies
and the sustainability of silvicultural practices.
A tale of long tails: combining DNA barcoding and RAD
sequencing to investigate the diversification of comet and
moon moths (Lepidoptera, Saturniidae)
Rodolphe Rougerie,
1
Astrid Cruaud,
2
and Jean-Yves Rasplus
2
1UMR 7205 ISYEB, Muséum national d’Histoire naturelle, 45, rue Buffon, CP50, 75005 Paris, France.
2INRA, UMR 1062 CBGP, Centre de Biologie et de Gestion des Populations, 755 avenue du campus
Agropolis CS 30016 34988 Montferrier-sur-Lez, France.
Corresponding author: Rodolphe Rougerie (e-mail: rodolphe.rougerie@mnhn.fr).
Background: RAD sequencing is becoming popular to infer phyloge-
netic relationships in organisms lacking genomic resources. Because
incomplete taxonomic coverage can confound macroevolutionary in-
ferences, taxon sampling must be representative and balanced. The
nearly complete DNA barcode libraries assembled in diverse insect
groups (e.g., Lepidoptera) provide refined accounts of species bound-
aries and greatly facilitate sampling design for phylogenetic studies
interested in relatively recent (up to 20–30 MY) diversification events.
Here, we use RAD-seq to infer phylogenetic relationships in comet and
moon moths (Saturniidae) and to investigate their diversification in
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space and time, also taking into account a set of adaptive traits (host-
plants, wing morphology). Results: Starting from a comprehensive
DNA barcode library, we selected 48 individuals representing 41 spe-
cies in the genera Actias,Argema, and Graellsia, plus 7 outgroups. We
generated a RAD library using genomic DNA extracted from individ-
uals representing contrasted preservation conditions and also using
DNA extracts previously obtained from specimen collections for DNA
barcoding. Whole Genome Amplification (WGA) was carried out when
initial DNA quantities were too small. The RAD library is used to
produce a robust and fully resolved phylogenetic hypothesis for these
moths, and their relationships are discussed in the light of species
distributions and life-history traits (e.g., pine-feeding species, length
of hindwing tails). Significance: Our results concur with those from a
growing number of recent studies demonstrating the power of RAD
sequencing to infer interspecific phylogenetic relationships with a
high degree of robustness. Here, our results allowed the investigation
of diversification history in an emblematic group of Lepidoptera. Our
approach also highlights the usefulness of DNA barcode libraries to
guide sample selection in groups incompletely resolved taxonomi-
cally. The potential use of DNA banks generated during the course of
DNA barcoding campaigns is discussed, as well as the recourse to WGA
when the quantity of DNA is limited.
Integrative taxonomy uncovers hidden diversity within three
genera of Canadian Osmiini (Hymenoptera: Megachilidae)
Genevieve Rowe,
1
Terry Griswold,
2
and Laurence Packer
1
1York University, 4700 Keele St., Toronto, ON M3J 1P3, Canada.
2USDA–ARS Bee Biology & Systematics Laboratory, 5310 Old Main Hill Logan, UT 84322-5310, USA.
Corresponding author: Genevieve Rowe (e-mail: genevieve.rowe@ymail.com).
Background: Osmiine bees are important both economically as man-
ageable bees and ecologically as major pollinators. They remain
poorly understood taxonomically. One aspect is the frequent division
of species into subspecies—a taxonomic level with an ambiguous
meaning. Here, the status of these entities is critically evaluated.
Results: Concurrent with previous and recent morphological assess-
ments, high levels of intraspecific divergence within several species
suggest the presence of species complexes within three genera of the
Osmiini—Atoposmia,Ashmeadiella, and Hoplitis. Deep divergences in
the mitochondrial cytochrome c oxidase subunit I (COI) sequences
and morphological assessments support the separation of several
polytypic species among these genera, in some cases elevating subspe-
cies to species status. Significance: One new species is described, and
changes in classification among the three genera are suggested.
Protection of Canadian biodiversity and trade through
improved ability to monitor invasive freshwater fish
Mélanie Roy,
1
Nellie Gagné,
1
Nicholas E. Mandrak,
2
and Charlotte Schoelinck
1
1Fisheries and Oceans Canada, 343, Avenue Université, Moncton, NB E1C 9B6, Canada.
2University of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada.
Corresponding author: Mélanie Roy (e-mail: melanie.roy@dfo-mpo.gc.ca).
Background: Although barcodes already exist for most Canadian
freshwater fish, there are many issues regarding taxonomic identifi-
cation. Development of a Canadian DNA database correctly con-
structed with independent markers and voucher specimens will
redress serious gaps relating to vouchering and data traceability of
existing barcodes. The DNA sequence database needs to have suffi-
cient depth and species coverage such that banned species can be
confidently discriminated from legitimate imported and native spe-
cies. A total of 13 species likely to be banned from importation into
Canada have been evaluated for DNA barcodes available and to iden-
tify gaps to address. Data generated in this project will enable confi-
dent identification of Canadian freshwater fish and potential
invaders, which is needed to develop monitoring tools from environ-
mental samples. Results: We obtained 906 samples representing 93%
of Canadian freshwater species. Each specimen was sequenced with
independent markers (COI, Rhodopsin), and a voucher has been sys-
tematically conserved at the Royal Ontario Museum to guarantee
traceability. Phylogenetic analyses showed good species delineation
except for lampreys and salmonids. Delineation issues within the ge-
nus Notropis have been found. In order to confidently distinguish
aquatic invasive species (AIS) from relatives, species included in the
genera containing the AIS were sequenced. Molecular identification
was especially challenging for the genera Carassius and Cyprinus where
hybridization is commonly detected. Specific qPCR assays and me-
tabarcode primers were developed for each potential AIS. Preliminary
results showed that NGS was able to detect Hypophthalmichthys molitrix
at a ratio of 0.01% in a mix of pooled DNA. This is now being
reproduced with eDNA from aquarium tanks. Significance: This
project generates the knowledge required to develop a DNA-based
tool for identifying taxa in live-food and aquarium-trade imports
that are banned by DFO based on their potential risk as invaders.
Plant barcoding of a wildlife sanctuary across a wide climatic
zone, Uttaranchal, India
Sribash Roy, Abhinandan Mani Tripathi, Gaurav Mishra, K.N. Nair,
T.S. Rana, and D.K. Upteri
CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow, India.
Corresponding author: Sribash Roy (e-mail: sribashroy@nbri.res.in).
Background: Earlier, we successfully worked on two plant DNA bar-
code projects, one genera specific and the other involving tree species
from a province. In recent efforts we are leading a consortium of six
Council of Scientific and Industrial Research institutes to barcode
plant species across India, where each institute is focusing on respec-
tive phytogeographic locations. As a part of this project, CSRI-NBRI is
involved in DNA barcoding of a wild life sanctuary, Govind Wildlife
Sanctuary, in the west Himalayan state of Uttarakhand. Results: We
have collected 238 angiosperms, 270 bryophytes, 255 pteridophytes,
265 lichens, and 154 algae species from the sanctuary, spanning a wide
climatic range, mainly due to steep altitudinal gradient (1300–6315 m a.s.l.).
So far we have analyzed 178 accessions of angiosperm plants from
this hot spot using the four standard plant barcode loci. MatK was not
used further after initial failure in PCR amplifications. PCR success
ranged from 88% to 95%, trnH–psbA being the lowest and rbcL being
the highest. ITS exhibited the lowest rate of sequencing success,
while trnH–psbA exhibited the highest. ITS and trnH–psbA exhibited
90% success in species identification. Among the non-flowering
plants, 75 accessions of lichen have been analyzed using ITS. PCR and
sequencing success was 75% and 80%, respectively. Contamination
with other fungus was a major problem faced during sequencing.
Barcoding of other lower group of plants from this region will be
carried out in the near future. Under the consortium, our main aim is
to develop a plant barcode data base of high-value medicinal plants.
Significance: Barcoding the flora, including a lower group of plant
species of a particular hot spot, spanning a wide range of climatic
zones, will certainly impact on conservation, policy making, and en-
vironmental protection by different government agencies. The study
will also help testing the efficacy in resolving species identification in
a specific geographic region.
Using mini-barcodes to investigate the species composition of
the Late Holocene (3500 to 200 years BP) fishery at EeRb-144,
British Columbia, Canada
Thomas C.A. Royle,
1
George P. Nicholas,
2
and Dongya Y. Yang
1
1Ancient DNA Laboratory, Department of Archaeology, Simon Fraser University, 8888 University Drive,
Burnaby, BC V5A 1S6, Canada.
2Department of Archaeology, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6,
Canada.
Corresponding author: Thomas C.A. Royle (e-mail: troyle@sfu.ca).
Background: EeRb-144 is a large Early (>7000 years BP) to Late
Holocene (3500 to 200 years BP) archaeological site located in the
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Interior Plateau region of southcentral British Columbia, Canada. A
multi-year excavation of the site recovered a large number of fish
remains (n= 1285) primarily associated with the Late Holocene
occupations of the site. Due to fragmentation and (or) a lack of
species-specific morphological features, most of these remains
(90.89%) could only be identified through morphological analysis to
the class level. As such, little is known about the species composi-
tion of EeRb-144's Late Holocene fishery. Consequently, this study
sought to assess the species composition of this fishery by using
mini-barcodes to identify a sample of Late Holocene fish remains
from the site. Results: In total, 29 of the 64 Late Holocene fish
remains from EeRb-144 selected for analysis yielded mini-barcodes
and were identified to the genus- or species-level. The majority of the
remains were identified as largescale sucker (Catostomus macrocheilus)
(41.38%) or northern pikeminnow (Ptychocheilus oregonensis) (27.59%).
This suggests the Late Holocene fishery at EeRb-144 was possibly
focused on harvesting largescale sucker and, to a lesser extent,
northern pikeminnow. A small proportion of the fish remains were
identified as Chinook salmon (Oncorhynchus tshawytscha) (3.45%),
longnose sucker (Catostomus catostomus) (3.45%), and peamouth
chub (Mylocheilus caurinus) (10.34%). This indicates EeRb-144's Late
Holocene fishery also harvested a variety of other species, albeit in
smaller quantities than largescale sucker and northern pikemin-
now. The remainder of the identified remains (13.79%) were as-
signed to the sucker genus (Catostomus sp.). Significance: This
study has provided detailed information about the species compo-
sition of the Late Holocene fishery represented at EeRb-144, and
perhaps elsewhere in the region at the time. More generally, this
study illustrates that mini-barcodes can be used to assign species
identifications to morphologically nondescript faunal remains re-
covered from archaeological sites.
Approaches for identification of Colchicum L. species in the
flora of Turkey by morphological parameters and DNA
barcoding
Ezgi Çabuk S¸ ahin,
1
Yıldız Aydın,
1
Erdal Kaya,
2
and Ahu Altınkut Uncuoğlu
1
1Marmara University, 34722, Istanbul, Turkey.
2Atatürk Horticultural Central Research Institute, 77102, Yalova, Turkey.
Corresponding author: Ezgi Çabuk S¸ ahin (e-mail: ezgicabuk@gmail.com).
The bulbous ornamental plant Colchicum L. belongs the family Colchi-
caceae, which has medicinal value due to the presence of colchicine.
In this study, we aimed to identify 49 Colchicum L. species (32 species
are endemic in Turkey), which contain 168 populations (16 popula-
tions are new candidate species), by using the multi-marker DNA bar-
coding method, proposed as a universal DNA-based tool for species
identification, and conventional morphological identification. For
identifying 168 Colchicum L. populations with DNA barcodes, rbcL,
Atpf, and trnH–psbA chloroplast genes were chosen, which were sug-
gested by The Consortium for the Barcode of Life Plant Working
Group. After amplifying these barcode genes from Colchicum DNA, PCR
products were sequenced, and SNP identification was conducted. Se-
quence data will be analyzed by using bioinformatic tools and biosta-
tistics programs such as phylogenetic trees, structure analysis, and
principal component analysis, to find DNA barcodes for identifying
species. Different morphological traits (flower size, petal size, shape of
petal surface, diameter of peduncle, anther length, filament length,
style length, tassellation) were used to perform the morphological
measurements of the Colchicum populations. The combination of
morphological and molecular data will shed light on identifying the
Colchicum gene pool at the species level.
DNA metabarcoding of springtails (Collembola)
Seikoh Saitoh,
1
Hiroaki Aoyama,
1
Saori Fujii,
2
Haruki Sunagawa,
3
Hideki Nagahama,
1
Masako Akutsu,
4
Naoya Shinzato,
1
Nobuhiro Kaneko,
2
and Taizo Nakamori
2
1Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan.
2Department of Environment and Natural Sciences, Graduate School of Environment and Information
Sciences, Yokohama National University Yokohama 240-8501, Japan.
3Okinawa Prefectural Agricultural Research Center, 820 Makabe, Itoman 901-0336, Japan.
4Department of Electrical Engineering and Computer Science, School of Industrial and Welfare
Engineering, Tokai University Toroku 9-1-1, Higashi-ku, Kumamoto 862-8652, Japan.
Corresponding author: Seikoh Saitoh (e-mail: h109296@comb.u-ryukyu.ac.jp).
Background: Springtails (Collembola) are a major group of soil mi-
croarthropods that mediate food webs during the decomposition pro-
cess. Collembola are conventionally identified to the species level by
microscopic examination of their morphological features, so commu-
nity assessment has been time-consuming until now. The application
of next-generation sequencing (NGS) technology to the community
assessment (DNA metabarcoding) of this group could be a solution.
Although several DNA metabarcoding methods for microscopic ani-
mals have been published to date, their use has been limited because
they tend to produce poor quantitative results, which is mainly due to
bias during PCR amplification. More quantitative identification meth-
ods are thus required. Results: We designed degenerate PCR primers
for the conserved regions in mitochondrial cytochrome c oxidase sub-
unit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on collem-
bolan mitogenomes that have already been sequenced. By examining
the amplification using all possible combinations, the best primer
pair was selected for each gene based on their ability to amplify the
gene, irrespective of species. The DNA extracted from simulated com-
munities was derived from seven collembolan species. The two gene
regions were then amplified using the selected primers, ligated with
adapters for the 454 technology (with multiplex identifier tags), and
sequenced. All the species were detected using the data from both
mtCOI and mt16S. After normalization, the sequence abundances for
each collembolan species showed linearity to the number of individ-
uals included in the community samples, although the sequence
abundances per individual varied depending on the species. The
mt16S data showed a little better linearity (R= 0.92–0.99) than the
mtCOI data (R= 0.91–0.99), which indicated that the mt16S data were
more quantitative. Significance: We present new methods for DNA
metabarcoding of Collembola using sequencing technology that
significantly improves quantification and community assessment
analysis.
DNA barcoding to support biodiversity conservation,
sustainable harvesting, and trade in Peru
Letty Salinas, Mónica Arakaki, Huber Trinidad, Marinoli Rivas,
Mirella Clavo, Víctor Pacheco, Diana Silva, Asunción Cano,
Rina Ramírez, and Betty Millán
UNMSM – Museo de Historia Natural, Av. Arenales 1256, Lima 14, Peru.
Corresponding author: Mónica Arakaki (e-mail: marakakim@unmsm.edu.pe).
Background: The Museum of Natural History (San Marcos University) is
the oldest biodiversity research institution in Peru and houses the largest
scientific collection of Peruvian flora and fauna. In 2014, under a Canada–
Peru Cooperation Agreement, the Museum welcomed a barcoding
initiative that helped to process a sample of our botanical and
zoological collections. In the process, we standardized protocols
and improved data collection and collection management in gen-
eral. These activities fell within the overall goal of strengthening
capacities for DNA barcoding in the country. Results: The USM Her-
barium and various departments in the Zoology Division sent a total of
2489 samples to the Biodiversity Institute of Ontario (BIO). The bird sam-
ples represent 60% of Peruvian bird species. Samples from the Depart-
ments of Entomology (95), Mammalogy (285), and the USM Herbarium
(190) were analyzed in order to validate their identities, and allow them to
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be used in phylogenetic reconstructions. Bird samples came from differ-
ent projects, a very important one of which was the characterization of
bird species associated with trade and increased industrial activity to
examine potential impacts on the biota. Significance: At the end of the
project, we expect to identify 20 species of birds that are important for
control of illegal trade (Business Calendar by the Ministries of Environ-
ment and Agriculture) and 26 species important in environmentally sus-
tainable agroecosystems (AGROKASA company). In the last decade, Peru
has experienced significant economic growth, mainly from mining, hy-
drocarbon, and hydroelectric industries. Six species collected from the
Camisea oil extraction project, 25 species from the Alto Chicama and
Conga mine, and six species from a hydroelectric project are scheduled to
be barcoded.
Dietary overlap and seasonality in three species of mormoopid
bats from a tropical dry forest
Valeria B. Salinas-Ramos,
1
L. Gerardo Herrera Montalvo,
2
Virginia León-Règagnon,
2
Aitor Arrizabalaga-Escudero,
3
and Elizabeth L. Clare
4
1Posgrado en Ciencias Biológicas Instituto de Biología, Universidad Nacional Autónoma de México Av.
Universidad 3000 D. F. 04510, México.
2Estación de Biología Chamela, Instituto de Biología, Universidad Nacional Autónoma de México A.P.
21, San Patricio Jalisco 48980, México.
3Department of Zoology and Animal Cell Biology, Faculty of Science and Technology, University of the
Basque Country UPV/EHU Sarriena z/g, Leioa E-48940, The Basque Country.
4School of Biological and Chemical Sciences, Queen Mary University of London, Mile end Road, London,
E1 4NS, UK.
Corresponding author: Valeria B. Salinas-Ramos (e-mail: airelav2@hotmail.com).
Background: Species' interactions are integral to understanding eco-
logical processes such as competition and niche partitioning. How-
ever, observing and describing these relationships is challenging,
particularly among generalist insectivores. Traditionally, morpholog-
ical classification of digested prey has been used to limited effect. DNA
barcoding provides a powerful solution to characterize these relation-
ships, providing species-level identifications even from degraded mate-
rial. When coupled with next-generation sequencing, these techniques
allow us to unravel the complexity of interactions between predators and
prey even in the most complex systems. Results: We applied DNA me-
tabarcoding to study three species of Pteronotus from a cave in Don
Panchito Island in Chamela, Jalisco, México. This area is characterized by
seasonal tropical dry forest. We investigate seasonal effects and the de-
gree of overlap and breadth of diet among bats that share roost and
foraging area. Lepidoptera and dipterans were the most consumed insect
orders, and there was a moderate level of dietary overlap between wet
and dry seasons. The highest degree of overlap was between P. parnellii
and P. personatus during the wet season. We found more overlap in insects
consumed between species in the same seasons than within any single
species across seasons. P. davyi and P. personatus increase their dietary
breadth during the dry season, and the diet of P. parnellii was broader and
had the highest effective number of species in all cases. Significance:
Our results suggest that the diet of these sympatric species is driven more
by prey availability than predator characteristic tracking seasonal re-
sources that vary throughout the year. This study provides one of the first
detailed views of tropical insectivores and their relationship with re-
sources.
The feasibility of detecting an Asian carp invasion using
environmental DNA and next-generation sequencing
Sarah Salisbury, Shadi Shokralla, and Mehrdad Hajibabaei
University of Guelph, 50 Stone Road East, Guelph, ON, Canada.
Corresponding author: Sarah Salisbury (e-mail: sarahsalisbury13@gmail.com).
Background: The use of environmental DNA (eDNA) has been pro-
posed as a potentially more sensitive method of invasive species de-
tection than traditional morphology-based methods. This barcoding-
based technique could allow for the identification of an invasive
species at a lower population density, thereby allowing earlier detec-
tion of an invasion and increasing the capacity to implement effective,
corrective measures. Asian carp are having devastating effects on
American waterways and threaten to invade the Great Lakes where, if
introduced, they are predicted to cause significant environmental and
economic damage. This study tested the usefulness of eDNA in the
detection of two Asian carp: the bighead carp, Hypophthalmichthys no-
bilis, and the grass carp, Ctenopharyngodonn idella. Muscle tissue of
each species was added to varying volumes of water sampled from
Lake Ontario and deionized water in order to mimic environmental
water samples that would be produced after an invasion of one or
both of these species. The DNA from the environmental samples
was extracted and amplified using three primer sets—cytochrome
c oxidase subunit I (COI), cytb, and D-loop—of varying specificity
and then sequenced using the Roche 454 Genome Sequencer.
Results: Successful amplification of Asian carp eDNA was observed
for all primer sets at varying concentrations of target Asian carp
eDNA and from a background of varying concentrations of non-
target eDNA. Significance: The successful detection of both target
Asian carp and other non-target species from lake water samples
using the COI primer set suggests the potential usefulness of this
universal primer in conjunction with next-generation sequencing
technologies for the detection of invasive species in combination
with biodiversity assessment.
Ethnobotany genomics—use of DNA barcoding to explore
cryptic diversity in medicinally important plants in the
Indian subcontinent
K. Sambandan,
1
S.G. Newmaster,
2
S. Ragupathy,
2
N. Dhatchanamoorthy,
1
R. Saravanan,
3
and R. Sathishkumar
3
1Department of Plant Science, Avvaiyar Government College for Women, Karaikal – 609 602, U.T of
Puducherry, India.
2Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph,
ON N1G 2W1, Canada.
3Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
Corresponding author: K. Sambandan (e-mail: sambandhan@gmail.com).
There is uncertainty in the number of species of Physalis L. occurring in
the Indian subcontinent, which may be due to (i) the introduction of
weeds and cultivated species, and (ii) cryptic diversity due to hybrid-
ization amongst closely related species. These factors may allow these
species to inhabit areas outside of their native range and habitats.
Cultivation and breeding may also influence the distribution and di-
versity of Physalis species in India. Physalis lagascae (Physalis lagascae
Roem. & Schult. = (P. minima auct non L.) is locally called ciyattini,
erumaittakkali, koticceni, koticcenittakkali, kutavayacceti; this is a
traditional medicinal plant used for treating chronic ailments such as
stomach ulcers and several forms of cancer. Another closely related
species, Physalis peruviana, is referred locally as cirutakkali, cirutak-
kali, itayakali, nattuttakkali, and it is known to inhabit the same areas
as P. lagascae. The two species are often confused due to their morpho-
logical similarity. However, P. peruviana has different medicinal prop-
erties from P. lagascae. Our research has shown that traditional healers
have often misidentified these species and their respective medicinal
properties. This is unfortunate because P. peruviana is invasive and
widely abundant, while P. lagascae is a local species with reduced pop-
ulations due to over exploitation for medicinal use and has even been
extirpated in some areas of India. Hence, the authentication of this
species is crucial for its conservation and for maintaining sufficient
populations for the herbal industry. Our research utilized rbcL and ITS
sequence data to clearly differentiate samples of Physalis peruviana and
Physalis lagasae. DNA barcoding using the tiered approach may provide
a useful authentication tool for differentiating these species, which
could be used to authenticate medicinal materials and support
conservation initiatives that support the preservation of small
populations.
Abstracts 275
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Socio-economic impacts of DNA barcoding on Philippine
fisheries and aquatic resources
Mudjekeewis D. Santos, Minerva H. Ventolero, Benedict Maralit,
Angellie Marie J.M. Asis, Rey C. Thomas, Altair Agmata,
Apollo Marco D. Lozano, Joanne Krisha M. Lacsamana,
June Feliciano F. Ordonez, and Roselyn Aguila
Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, 101 Mother
Ignacia St., Quezon City 1103, Philippines.
Corresponding author: Mudjekeewis D. Santos (e-mail: mudjiesantos@gmail.com).
The Philippines possess one of the largest fisheries in the world and is
now reported to be the center of marine biodiversity in the planet. Iron-
ically, however, basic taxonomic studies in the country are severely lack-
ing. Moreover, the Philippines is also considered as one of the hotspots in
terms of fisheries and marine conservation due to numerous threats. For
example, despite having a number of aquatic species being regulated in
the country, illegal trade still persists through the transport of live and
processed products. Fry or juvenile fisheries that potentially contribute
to growth overfishing are existent because they are an important source
of food and livelihood, especially by artisanal fishermen. Here, our stud-
ies revealed new records of a number of marine species. We reported the
presence of the recently resurrected beaked whale, Mesoplodon hotaula;a
recently resurrected giant clam, Tridacna noae; the reef manta ray, Manta
alfredi; and the mobula ray, Mobula japonica. We even discovered the pres-
ence of two possible invasive species, the tilapia flowerhorn Cichlasoma
urophthalmus and the black chin tilapia Sarotherodon melanotheron in Ma-
nila Bay. In terms of trade-related studies, we identified confiscated juve-
nile eels (elvers) at the Ninoy Aquino International Airport and
confiscated dried products of sharks and rays at the North Harbor in
Manila, and we showed mislabelling in various fishery products sold in
major supermarkets in Manila. Finally, we elucidated the following fry
(juvenile) fisheries for fisheries management: the siganid “padas” fishery
in Northern Luzon; the goby fry “ipon” and Anguilla eel “elvers” fisheries
in Aparri, Cagayan; the “dulong” fry fishery in Verde Island Passage,
Batangas; and the milkfish “bangus” fry fisheries and its bycatch. Taken
together, our studies highlighted DNA barcoding as a powerful tool in
addressing some of the major taxonomic and socio-economic issues in
Philippine fisheries management and marine biodiversity conservation.
DNA barcoding of Philippine fish: first record of marine species
in a biodiversity hotspot
Mudjekeewis D. Santos,
1
Minerva H. Ventolero,
1
Angellie Marie J.M. Asis,
1
Apollo Marco D. Lizano,
1
Joanne Krisha M. Lacsamana,
1
Billy Joel Catacutan,
1
John T. dela Pena,
1
June Feliciano F. Ordonez,
1
Jacqueline Pereda,
1
and Jom Acebes
2
1Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, 101 Mother
Ignacia St., Quezon City 1103, Philippines.
2Department of Biology, Ateneo de Manila University Katipunan, Quezon City, Philippines.
Corresponding author: Mudjekeewis D. Santos (e-mail: mudjiesantos@gmail.com).
The Philippines is home to the highest number of marine species per
square area in the world, gaining the distinction of being the center of
marine biodiversity in the world. Unfortunately, it is also considered as
one of the hotspots in terms of marine conservation due to numerous
threats to its biodiversity. This is compounded by the absence of a com-
prehensive taxonomic listing of marine species in the country. Here, we
present our recent results at the NFRDI-Genetic Fingerprinting Labora-
tory on the application of DNA barcoding to discover new records of
aquatic organisms in the Philippines. We established the first records of
rare and regulated species such as the recently resurrected beaked whale,
Mesoplodon hotaula stranded in southern Philippines; a recently resur-
rected giant clam, Tridacna noae; and the reef manta ray, Manta alfredi.We
established the first report in the country of two possible invasive spe-
cies, the tilapia flowerhorn Cichlasoma urophthalmus sampled from fish
ponds in Bulacan as well as the black chin tilapia Sarotherodon melan-
otheron in Manila Bay. We provide the first direct evidence of the presence
of mobula ray Mobula japonica sampled from an illegal shipment of dried
shark meat and bones. Finally, we also present recent discovery of new
fish species in the country by other collaborating authors. Our results
highlight the Philippines as truly a marine biodiversity haven because of
the still high rate of discovery of new species and that conserving it
should take paramount importance. It also highlights the important role
of DNA barcoding as a powerful tool in species discovery and conserva-
tion in a marine biodiversity hotspot.
Genetic characterization of the red algae Asparagopsis armata and
Asparagopsis taxiformis (Bonnemaisoniaceae) from the Azores
R. Santos,
1
A. Ferreira,
1
J. Micael,
1
M.C. Gil-Rodríguez,
2
M. Machín,
2
A.C. Costa,
1
D. Gabriel,
1
F.O. Costa,
3
and M.I. Parente
1
1INBIO/CIBIO Azores, Department of Biology, University of the Azores, Portugal.
2Department of Botany, Ecology and Plant Physiology, University of La Laguna, Canary Islands, Spain.
3CBMA – Centre of Molecular and Environmental Biology, Department of Biology, University of Minho,
Portugal.
Corresponding author: R. Santos (e-mail: rod_msantos@hotmail.com).
The Azores is situated in the North Atlantic Ocean (37–40N, 25–31W),
astride the Mid-Atlantic Ridge, and is strongly influenced by the sea-
surface pathway from the Gulf Stream. Nevertheless, the affinities of
the marine algal flora are to the continental coasts of Europe and
Africa, the Mediterranean Sea, and the other Macaronesian islands.
Azorean marine ecosystems are relevant because of their uniqueness,
geographic position, biogeographic mixed algal flora, and insularity—
all of which highlight their susceptibility to alien species introduc-
tion. Algae invasions in marine habitats represent a recognized
worldwide threat to the integrity of native communities, to econo-
mies, and even to human health. The genus Asparagopsis is known for
being invasive in several regions of the world. Until now, two species
of this genus have been reported to the Azores: Asparagopsis armata,
described from the western coast of Australia and displaying today a
worldwide distribution, although preferentially in cold temperate re-
gions; and Asparagospis taxiformis, originally described for the southern
hemisphere and widely distributed across tropical and temperate re-
gions. We investigated the phylogeography of these two species in the
Macaronesia, with focus in the Azores, to better understand biogeo-
graphic relationships within the North Atlantic. Populations of
A. armata and A. taxiformis from Azorean Islands, Madeira, Canary
Islands, and mainland Portugal were genetically scrutinized using two
molecular markers, the mitochondrial COI-5P barcode (cytochrome c
oxidase subunit I) and the nuclear ITS (ribosomal internal transcribed
spacer). Our preliminary results will be presented.
DNA barcodes for authentication of commercially important
Indian spices
M. Saravanan,
1
S.G. Newmaster,
2
S. Ragupathy,
2
S. Dhivya,
1
and R. Sathishkumar
1
1Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University,
Coimbatore – 641046, Tamil Nadu, India.
2Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO), University of Guelph, Guelph,
Ontario, Canada.
Corresponding author: M. Saravanan (e-mail: saravananmohan.bu@gmail.com).
Background: Spices are expensive aromatic and pungent food ingre-
dients that are added to food in several forms as whole, in ground
form, or as isolates from their extracts. An investigation on the bene-
ficial physiological consequences of spices is a thrust area for almost
three decades. India is one of the largest producers, consumers, and
exporters of a variety of spices. The spice trade has been rising globally
with increasing consumer demand for top quality, as it determines
the cost. There are several reports of illicit or fraudulent adulteration
of spices for commercial gains, and hence, a foolproof monitoring
system is mandatory. Three common examples of adulteration are
(i)Capsicum annumis is adulterated with Ziziphus nummularia fruit,
(ii)Brassica juncea is adulterated with Argemone mexicana seed, and (iii)Curcuma
longa is adulterated with the wild type Curcuma zedoaria. To date there
are several techniques reported for detecting the adulteration and
authentication of food material or its substances, one of which is DNA
barcoding. In this technique, universal genomic regions are used to
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resolve the species based on the DNA sequence variation between the
closely related species. Results: In this study, DNA barcoding analysis
of spices revealed non-Indian spices like P. racemosa,C. cartwrightianus,
L. sinense,E. phyllanthaceae, and O. syriacum in Indian market samples.
These adulterants are economically less valuable than what is used in
India traditionally. This may be intentional or unintentional trade
practice for commercial gains. Our study has concluded that ITS2 was
a better DNA barcode candidate for distinguishing different spice spe-
cies, as rbcLa identified only at the family/genus level. It is very clearly
demonstrated that DNA barcoding is a reliable and effective tech-
nique for the authentication of spices from the other closely related
species and its products. Significance: Molecular biology based DNA
barcoding studies have been very useful for the authentication of
socio-economically important spices. The spice industry should em-
brace the use of DNA barcoding for the authentication of spice prod-
ucts by testing its bulk materials, which would help in fair trade
practices. This would only amount to a minor cost to the spice indus-
tries that would result in high quality, authentic product. This is a
proof of concept study involving blind samples from Indian market
for which a Biological Reference Materials library was developed first
and used for comparison.
What are you really eating in Mexico? A preliminary study of
fish fillets
Stephanie Sarmiento Camacho
1
and Martha Valdéz-Moreno
2
1Escuela de Biología, Benemérita Universidad Autónoma de Puebla, Blvd. Valsequillo y Av. San Claudio,
Edificio 112-A, Ciudad, Universitaria, Col. Jardines de San Manuel zip 72570. Puebla Puebla, Mexico.
2El Colegio de la Frontera Sur, Unidad Chetumal, Av. Centenario km 5.5 Col. Pacto Obrero zip 77014,
Chetumal Quintana Roo, Mexico.
Corresponding author: Stephanie Sarmiento Camacho (e-mail: s.sar.cam@outlook.com).
Background: Substitution of highly valued species is a common prac-
tice today. But how do we know what we are really eating if we cannot
see the original product? DNA barcoding has been used to accurately
identify species of fish fillets. There have been studies on this topic but
not in Mexico. The main goal of our work was to identify fish fillets
sold by “La Viga”, the biggest seafood market in Mexico City, and other
markets on the Yucatan peninsula during 2010–2011. Results: We
sequenced 87 samples, comprising eight orders, 21 families, 25 genera,
and 28 species. The most commonly marketed sharks are Carcharhinus
falciformis,C. brevipinna,Isurus oxirinchus, and Galeocerdo cuvier. All of
them are sold under the common name “cazon”, which means “small
shark” without reference to any species. Nevertheless, all these spe-
cies have limited closed fishing seasons. Among the Actinopterygii,
Seriola dumerili,Scomberomorus cavalla,Bagre marinus,Sphyraena barracuda,
Mycteroperca microlepis, and Epinephelus morio are the most commercial-
ized. The latter falls under seasonal protection. We detected only
seven species with name substitutions on the markets. The most com-
mon freshwater species found was Pangasianodon hyphothalmus, which
is increasingly sold, but recently Mexican authorities reported the
arrival of some contaminated shipments. Significance: These results
will help to convince authorities that DNA barcoding is a reliable tool
to identify species and detect fish substitution when morphological
characterization is difficult or impossible. They can also be useful to
detect trade of protected species during closed seasons, which will
help to implement new strategies for the conservation of marine and
freshwater resources.
The Muscidae of Canada: towards a complete DNA barcode
reference library
Jade Savage,
1
Paul D.N. Hebert,
2
and Valérie Lévesque-Beaudin
2
1Bishop's University, 26 College Street, Sherbrooke, QC J1M 1Z7, Canada.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Jade Savage (e-mail: jsavage@ubishops.ca).
Background: The Muscidae (house-flies and relatives) is an ecologi-
cally diverse family of Diptera found in a wide range of terrestrial and
freshwater habitats. In Canada, the group is especially well repre-
sented in alpine and northern environments where they often ac-
count for more than 25% of all Diptera species. Muscids have recently
been the focus of various ecological and monitoring studies in alpine,
northern, and agricultural habitats; however, the large number of
species and the difficulties associated with morphology-based identi-
fication (especially of females) remain important challenges. Previous
work has demonstrated a high correspondence between morphology
and DNA barcodes for muscid flies. Using the largest data set available
to date, the present study aims at populating a complete reference library
of DNA barcodes (COI) for the Canadian muscid fauna, which is thought
to include ⬃500 species. Results: Our data set includes 22 386 sequences
representing 438 operational taxonomic units under the Barcoding
Index Number (BIN) system, half of which are already determined to
species. Based on morphological examination of representative spec-
imens, we have now completed a generic assignment for all BINs.
Preliminary results on species-level identification generally indicate
strong correspondence between BINs and morphology, with occa-
sional cases of over-splitting or over-lumping. We also report rare
cases where morphologically distinct taxa shared identical barcodes.
Significance: Our DNA barcode reference library for the Muscidae of
Canada will be relevant to the fields of agriculture, conservation, and
vector biology by (i) improving our ability to properly assess the status
of both invasive and native members of the Canadian insect fauna;
and (ii) facilitating future investigations into the community ecology,
biodiversity, and monitoring of insect assemblages, especially in
northern Canada.
The Barcode of Wildlife Project, Part 1: a systemic barcoding
initiative to protect endangered species
David E. Schindel and Michael G. Trizna
Consortium for the Barcode of Life, Smithsonian Institution, Washington, DC, USA.
Corresponding author: David E. Schindel (e-mail: schindeld@si.edu).
Background: The Consortium for the Barcode of Life (CBOL) was
granted a US$3 million Google Global Impact Award in December 2012
for a barcoding project with six developing “partner countries”. Mex-
ico, Kenya, South Africa, and Nigeria joined the project immediately,
and Nepal joined recently. A Latin American partner is still being
sought. The Barcode of Wildlife Project (BWP) is devoted to demon-
strating the impact of barcoding on the prosecution of poachers and
traffickers in endangered species. Each country selected 200 priority
endangered species that are protected under CITES and (or) their na-
tional laws. BWP has provided training, supplies, reagents, support for
fieldwork, technical, and other support to enable to begin contribut-
ing BARCODE records to GenBank for the priority endangered species
and related and look-alike species. The project had a roadmap, mile-
stones, and a timeline for library construction and development of
barcoding capabilities in enforcement agencies. SOPs for library con-
struction had to meet the needs of enforcement agencies. BWP was
committed to processing all samples in-country to avoid controversies
surrounding export of genetic resources. Results: CBOL had good
working relationships with barcode researchers in each country but
no links to enforcement agencies. Building strong working relations
between research and enforcement sectors has been a slow process
that began with a workshop on legal standards. After in-country train-
ing and regular support by videoconference, thousands of very high-
quality BARCODE records are being submitted to GenBank, and crime
scene evidence is being barcoded. Significance: Initiatives such as
BWP can convince government agencies to adopt barcoding as a reg-
ulatory system and support it on a long-term basis. The BARCODE data
standard will need to be adjusted to meet these requirements. As
regulations about access and benefit sharing are implemented, Gen-
Bank records will need to come increasingly from in-country barcod-
ing laboratories for which training and capacity-building will be
needed.
Abstracts 277
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DNA barcoding medicinal plants from Pakistan
Melanie Schori,
1
AlexaRae Kitko,
1
Kirk Emch,
1
Zabta K. Shinwari,
2
and Allan M. Showalter
1
1Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA.
2Department of Biotechnology, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
Corresponding author: Allan M. Showalter (e-mail: showalte@ohio.edu).
Background: In Pakistan, herbal products are a primary source of med-
icines, yet quality control for raw plant material sold to individuals or
companies is very limited. Large-scale cultivation of medicinal plants is
not prevalent, so the bulk of raw material is imported from other coun-
tries or collected from the wild. Roots, bark, twigs, leaves, flowers, and
seeds are sold under common names in local languages, so the potential
for misidentifications and mixed collections is high. Misidentifications
or adulteration of authenticated materials can lead to reduced effective-
ness of herbal products or accidental poisonings. Barcoding provides a
way to confirm the identification of raw plant material and establish a
level of quality assurance. Results: We have generated barcodes from 156
medicinal plant species, representing 49 different families, using the
rbcL, Atpf, and psbA–trnH spacer regions. Twenty-four market samples
(28% of barcoded market material) were documented as adulterated or
entirely misidentified, including common species like Melia azedarach,
Borago officinalis, and Althaea officinalis. Comparisons of medicinal plant
barcodes with those of sister species have indicated that the psbA–trnH
spacer is most useful for distinguishing species, followed by Atpf and
rbcL. Significance: Based upon scientific interactions and results of this
work, Qarshi Industries, a leading producer of natural products and
herbal pharmaceuticals in Pakistan, is enhancing its quality control by
setting up a DNA barcoding laboratory to confirm the identities of plants
used in their formulations and to detect potential adulteration.
Using environmental DNA to detect endangered Redside Dace,
Clinostomus Elongatus
Natasha Serrao,
1
Scott Reid,
2
and Chris Wilson
2
1Trent University, 2140 East Bank Drive, Peterborough, ON, Canada.
2Ontario Ministry of Natural Resources and Forestry, 2140 East Bank Drive, Peterborough, ON, Canada.
Corresponding author: Natasha Serrao (e-mail: natasha.rosemary.serrao@gmail.com).
Background: Effective management of endangered species requires sen-
sitive detection of their occurrences, which is often difficult for low-
abundance species. In aquatic environments, detection of rare species
can be further confounded by site accessibility, sampling gear, and cap-
ture efficiency, with direct observation being difficult at best. Redside Dace,
an endangered species native to southern Ontario, has been experiencing
population declines as a result of habitat fragmentation. Traditional
methods to detect their presence include seining and electrofishing;
however, species often go undetected even if present at the site. A novel
application of DNA barcoding has been to develop species-specific detec-
tion in aquatic habitats from environmental DNA (eDNA). The utility of
this methodology was tested at 29 sites where Redside Dace were histor-
ically known to be present. Results: The combined results indicate that
eDNA is a more sensitive method for species detection than conventional
methodology. The study determined that seasonality, temporal versus
spatial replicate sampling, and appropriate thresholds for classifying de-
tections versus non-detections are important for study design. Signifi-
cance: eDNA is a reliable method for species detection in freshwater
systems and can be used as an effective sampling technique for docu-
menting occurrences of aquatic endangered species.
Phylogeography and conservation of endangered Redside Dace,
Clinostomus elongatus
Natasha Serrao,
1
Scott Reid,
2
and Chris Wilson
2
1Trent University, 2140 East Bank Drive, Peterborough, ON, Canada.
2Ministry of Natural Resources and Forestry (MNRF), 2140 East Bank Drive, Peterborough, ON, Canada.
Corresponding author: Natasha Serrao (e-mail: natasha.rosemary.serrao@gmail.com).
Background: The rapid loss of biodiversity has provoked awareness
of the necessity of species conservation. Species at risk face immediate
danger and warrant fast attention if they are not to be lost. The Red-
side Dace, Clinostomus elongatus (Teleostei: Cyprinidae), is a small North
American minnow that is declining throughout its range as a result of
urban development. Fin clips and buccal swabs were obtained from
28 Redside Dace populations throughout Ontario and the United States
for sequence analysis. As Redside Dace exhibit very little variation in
COI, other mitochondrial genes (cytochrome b and ATPase 6+8) were
sequenced to investigate phylogeographic structuring among extant
populations. The populations were also genotyped using 10 microsat-
ellite loci to examine genetic diversity as well as contemporary spatial
structuring. Results: The combined sequencing and genotyping re-
sults indicate that Redside Dace persisted in multiple glacial refugia
during the Wisconsinan glaciation and exhibit significant geographic
structure, as well as retaining high genetic diversity within declining
populations. Significance: These results will be used to inform man-
agement decisions for Redside Dace recovery in Ontario and other
parts of the species range.
The applications and limitations of DNA markers in
authenticating herbal materials
Pang-Chui Shaw, Ka-Lok Wong, and Yat-Tung Lo
Institute of Chinese Medicine and School of Life Sciences, The Chinese University of Hong Kong, Shatin,
N.T., Hong Kong, China.
Corresponding author: Pang-Chui Shaw (e-mail: pcshaw@cuhk.edu.hk).
Background: DNA barcoding is now a widely used approach for the
identification of biological specimens to the level of species and discov-
ery of cryptic species. Through improvement on extraction protocols and
primer design, herbal species can now be identified in decoctions and
commercial products. Nevertheless, our studies have shown that DNA
markers have several limitations that influence the success rate and ac-
curacy of sequencing. Results: The first shortcoming is the inadequate
resolving power of DNA barcodes in some taxa. After analyzing seven
DNA loci, including rbcL, Atpf, trnH–psbA, rpl36–rps8, ITS, trnL–F, and 5S
rRNA, of the Gentiana taxa, only the latter two were able to discriminate
closely related species G. triflora,G. scabra, and G. manushruica. In the study
of Acontium, 19 taxa were clustered into 10 groups based on trnH–psbA,
while the two most commonly used medicinal species, A. carmchaeli and
A. kusnezoffii, could not be differentiated definitely. The second shortcom-
ing is the difficulty in amplifying DNA barcodes across a broad range of
taxa by universal primers. Alignment analysis revealed that there were
3–4 different nucleotides between the universal primer for (Atpf 3F Kim
f) and the sequences of 17 taxa in the genus Gentiana, and 4 out of 10
samples of Gentiana could be not amplified. The third issue is the ampli-
con size of DNA for highly degraded or processed samples. Only short
PCR products of about 100 bp were found in Panax ginseng decoction
boiled for 2 h. In such cases, a mini-barcode may be employed to enhance
the success rate. The fourth problem is amplification of the contaminant
species in old herbal materials. ITS was employed in a Chinese “cooling”
beverage study, in which among 145 sequences, 85.5% were derived from
intended species, while the rest were contaminated by fungal or other par-
asitic species. Significance: DNA barcoding is a powerful tool for authenti-
cation. For herbal materials, there are limitations in the applications due to
possessing, storage, and the existence of closely related materials.
Evaluation of four barcoding loci in the class Calyciflorae
(phylum: Polypetalae-dicotyledons)
Bhavisha P. Sheth and Vrinda S. Thaker
Centre for Advanced studies in Plant Biotechnology and Genetic Engineering (CPBGE), Department of
Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India.
Corresponding author: Bhavisha P. Sheth (e-mail: bhavisha.sheth@gmail.com).
Background: Molecular tools provide valuable data on diversity through
their ability to detect variation at the DNA level. The DNA barcoding
technique involves the generation of sequence datasets using wet labo-
ratory experimentation, followed by the analyses of the same using
bioinformatics and statistical tools. The present study is focused on the
analysis of the sequence data according to the taxonomic relationship.
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The nucleotide sequence dataset is composed of three barcoding loci
(four chloroplastic: rbcL, Atpf, trnH–psbA, as well as one nuclear: ITS).
The plants selected from the class Calyciflorae (phylum: Polypetalae- di-
cotyledons) were subjected to evolutionary analyses using various bioin-
formatics and statistical tools including MEGA 5.05, DnaSP v5.10, and
DAMBE v5.2.78. Results: The relatedness and distinction using the bioin-
formatics and statistical tools amongst the selected plants is discussed.
Significance: The work throws light on the comparative evaluation of
four barcoding loci at higher taxonomic levels.
Molecular phylogenetic analysis of Cassia species using
DNA barcoding
Bhavisha P. Sheth and Vrinda S. Thaker
Centre for Advanced studies in Plant Biotechnology and Genetic Engineering (CPBGE), Department of
Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India.
Corresponding author: Bhavisha P. Sheth (e-mail: bhavisha.sheth@gmail.com).
Background: Cassia is a genus of flowering plants in the pea family,
Fabaceae. Molecular identification tools provide valuable information
on genetic diversity through their ability to detect variation at the
DNA level. The present study comprises the phylogenetic analyses of
six species selected from the genus Cassia using DNA barcoding. The
nucleotide sequence dataset is composed of four barcoding loci (rbcL,
Atpf, trnH–psbA, and ITS2). The sequence data were subjected to evo-
lutionary analyses using various bioinformatics and statistical tools.
Results: The relatedness and distinction amongst the selected plants
is discussed. The bioinformatics analyses suggested that the trnH–
psbA and ITS2 are better than rbcL and Atpf to resolve the plants
at the species level. Significance: DNA barcoding and species-level
distinction of Cassia species might be useful for the appropriate
identification of these medicinally important plant species from their
adulterants.
DNA barcoding based phylogenetic assessment of some Vigna
species inferred from nrDNA internal transcribed spacer 2
(ITS2) sequences
Bhavisha P. Sheth,
1
Sumer Punia,
2
Meenakshi Dheer,
2
Purvi M. Rakhashiya,
1
Pooja P. Patel,
1
Vibhuti M. Jhala,
1
and Vrinda S. Thaker
1
1Centre for Advanced studies in Plant Biotechnology and Genetic Engineering (CPBGE), Department of
Biosciences, Saurashtra University, Rajkot 360005, Gujarat, India.
2Agricultural Research Station, Ummedganj Kota-324001, Rajasthan, India.
Corresponding author: Bhavisha P. Sheth (e-mail: bhavisha.sheth@gmail.com).
Background: The Vigna genus of the Fabaceae family consists of many
important food legumes. The nuclear internal transcribed spacer re-
gion (ITS1-ITS2) region has been useful for phylogenetic analyses at
lower taxonomic levels. In the present study, the molecular character-
ization between two Vigna species (Vigna radiata: 7 accessions and
Vigna mungo: 24 accessions) was inferred using the internal tran-
scribed spacer 2 (ITS2) region of the 18S–5.8S–26S ribosomal RNA gene
unit. Results: Sequence length in the accessions of Vigna species
ranged from 215 to 258 bp. The ITS2 sequences were very rich in G+C
content, ranging from 57.2% to 60.4% with an average of 58.8%. The
intraspecific Kimura-2 pairwise genetic distance (GD) was 0.0766 ±
0.0121 and 0.0456 ± 0.0068 for Vigna radiata and Vigna mungo, respec-
tively. The interspecific Kimura-2 pairwise genetic distance (GD) was
0.1062 ± 0.0229. The phylogenetic analysis was inferred using the
maximum parsimony (MP) and UPGMA methods using Cajanus cajan as
an outgroup. Both MP and UPGMA trees separated all 31 accessions of
Vigna into two distinct clusters of the two respective Vigna species.
Further, a total of 7 unique ITS2 haplotypes were obtained in Vigna
radiate, and 15 were obtained in Vigna mungo.Significance: ITS analy-
sis was useful in differentiating these two Vigna species at the generic
as well the infrageneric levels. The separation of all the accessions of
Vigna species in distinct clusters based on ITS2 sequence data was in
accordance with the morpho-taxonomic characteristics of the differ-
ent taxa. The study will be valuable for correct identification, differ-
entiation, and characterization of Vigna species and their genetic
resources.
TCM-identifier: an integrated and user-friendly software
package for species identification of traditional Chinese
herbal materials
Linchun Shi, Jingyuan Song, Hui Yao, Jianping Han, Xiaohui Pang,
and Shilin Chen
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, Beijing 100193, PR China.
Corresponding author: Linchun Shi (e-mail: lcshi@implad.ac.cn).
Background: The DNA barcoding system is established to identify
species of traditional Chinese herbal medicine stably and accurately.
The principles and methods of this system have been incorporated
into the third supplement of the Chinese Pharmacopoeia 2015 edition.
An integrated and user-friendly software package promotes the use of
DNA barcoding for species identification. Results: TCM-Identifier is
designed to meet all basic and advanced needs of herbal species iden-
tification by using the DNA barcoding method. It includes quality
control, sequence assembly, sequence annotation, and species identi-
fication. TCM-Identifier comprises a built-in reference database with
standard DNA barcodes for species listed in Chinese Pharmacopoeia.
This software accepts bidirectional trace files as input. First, TCM-
Identifier converts trace files to sequences with qualities and clips
low-quality bases from the ends of sequences. Short and low-quality
sequences are automatically removed after end clipping. Second, long
and high-quality sequences are assembled into contigs under assem-
bly criteria defined in TCM-Identifier. The forward and reverse prim-
ers can be trimmed from a specific contig when the trim-vector button
is clicked. Third, the contigs that meet the quality control criteria are
automatically annotated using the model-based annotation method of
Hidden–Markov. The annotated sequence is called the real DNA bar-
code. Finally, a blast method is used to search a DNA barcode from the
built-in database, and the corresponding species of this barcode can be
provided when the blast matches our criteria. Significance: DNA bar-
coding provides a rapid and effective tool for species identification of
traditional Chinese herbal medicine. TCM-Identifier, as an integrated
and user-friendly software package, can facilitate this process and has
been employed by national and local drug regulators. This software
also has been widely used in traditional herbal medicine enterprises,
especially those where a specific database is constructed.
Exposing the illegal trade in Encephalartos species at the
Faraday Muthi Market in South Africa using DNA barcoding
Sanele N.S. Shiba, Olivier Maurin, Herman van der Bank,
and Michelle van der Bank
African Centre for DNA Barcoding, University of Johannesburg, P.O. Box 524, Auckland Park, 2006,
South Africa.
Corresponding author: Sanele N.S. Shiba (e-mail: sanele.shiba@gmail.com).
Background: All Encephalartos species are listed in CITES Appendix I
and in the national Threatened or Protected Species regulations,
which prohibit trade in wild plants. Despite these regulations, illegal
bark harvesting for medicinal trade has increased in South Africa and
resulted in declines in cycad populations, and even complete loss of
sub-populations (e.g., type locality of Encephalartos natalensis). Encephalartos
species, locally known as isiGqiki-somkovu, are traded at traditional
medicinal markets in the form of “bark” strips and stem sections;
thus, determining the actual species traded presents a major chal-
lenge due to lack of characteristic plant parts. Here we present a case
study on the use of DNA barcoding to identify cycads sold at the
Faraday Muthi Market in South Africa. In total, 37 samples were col-
lected, and sequences for the two barcoding regions (rbcLa and Atpf)
and two additional genes (trnH–psbA and ITS) were generated. We
then utilized the large barcoding database for cycads available at the
University of Johannesburg to assign query samples to known species.
Three approaches were followed, viz. (i) tree-based method, (ii) BLAST
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algorithm, and (iii) character-based method (BRONX). Results: Market
species identified and their IUCN status were Encephalartos aemulans
(critically endangered), E. lebomboensis (endangered), E. natalensis (near
threatened), and E. senticosus (vulnerable). This is the first record of
E. aemulans being traded at the Faraday Muthi Market. This species is
critically endangered and known only from two populations in
KwaZulu-Natal. Furthermore, results indicate that the majority of
samples entering the Faraday Muthi Market are harvested in the
KwaZulu-Natal province of South Africa. Significance: Overall, we
determined that DNA barcoding is an ideal genetic-based tool for the
correct identification of fragmented cycad material that cannot be
identified by traditional means. Furthermore, results from this study
are crucial to make appropriate assessments and decisions on how to
manage these markets and for cycad conservation in general.
An imperative action in invasives management—spreading
rapid species identification to developing countries
Junko Shimura and David Duthie
Secretariat of the Convention on Biological Diversity, Montreal, Que., Canada.
Corresponding author: Junko Shimura (e-mail: junko.shimura@cbd.int).
Background: The Convention on Biological Diversity (CBD) is an in-
tegral policy framework for conservation and sustainable use of bio-
diversity, and sharing the benefits arising from utilization of genetic
resources with near-universal participation. Current human activities
are causing biodiversity loss at unprecedented speed and scale. Inva-
sive alien species are a threat to native species in all biogeographic
regions, with estimates of global costs rising to some billions of dol-
lars every year. According to the CBD Global Biodiversity Outlook 4th
edition, many countries have initiated measures to address invasive
alien species, although taking appropriate measures is challenging
unless alien species are identified rapidly and accurately. Results: The
Conference of the Parties to the Convention has requested the Secre-
tariat to facilitate capacity building on identification of invasive and
potentially invasive species. The Secretariat, in close partnership with
the International Barcode of Life and funding from the Government of
Japan, is providing training on rapid species identification for techni-
cal experts from 21 developing countries in 2015. Significance: These
trainees are expected to become in-country trainers spreading knowl-
edge and capacity for rapid species identification in various sectors
including trade, agriculture, and the environment. Although regula-
tion on hazards is fairly well developed in chemicals, pests, and com-
municable diseases management, a challenge still exists in addressing
growing numbers of alien species, for which invasion risk can vary in
different ecosystems. In this context, national or regional-level capac-
ity building is urgently needed for society to continue to develop and
secure ecosystem services for human well-being. In addition, forensic
DNA analysis for detection of environmental crimes, such as illegal
trade of protected species (e.g., CITES-listed species), enables legal
processes in biodiversity-rich countries. The high-throughput DNA
sequencing also opens possibility of rapid assessment of wildlife in
both aquatic and terrestrial environments, and mapping of phyloge-
netic diversity greatly helps setting conservation priority.
DNA barcode-based true bugs (Heteroptera) surveillance for
agriculture crops from Maharashtra
Anant Shinde
1
and Gulab Khedkar
2
1Department of Zoology, Yashwantrao Chavan Arts and Science Mahavidyalaya Mangrulpir, Dist.
Washim, Maharashtra, India.
2Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Anant Shinde (e-mail: anantshinde007@rediffmail.com).
Background: True bug species are an important component of insect
pest complexes of various commercially important crops in India. Effec-
tive control of such pests always requires accurate information on the
number of species involved within a particular crop system. Results: We
surveyed crop systems over 33 districts of Maharashtra and collected
763 bugs. Collected bugs were primarily identified based on morphological
characters, and 114 species were found. We next tested the effectiveness
of COI barcodes to identify these species and found that all of the species
had a unique assemblage of COI barcode sequences. A few exceptions
(⬃1%) were observed, where morphological and molecular identification
conflicted. Overall, the average interspecific genetic distance between
closely related species was much higher than the average intraspecific
genetic distance. Significance: DNA barcoding identified three putative
new species of true bug and revealed morphologically cryptic or very
recently diverged species. In sum, our results suggest that COI barcodes
can reveal new cryptic true bug species and are able to contribute the
exact identification of the true bugs.
Non-destructive DNA extraction approaches for massive
parallel multiplex sequencing for specimen identification and
environmental DNA barcoding using high-throughput
sequencing platforms
Shadi Shokralla,
1
Rachel Smith,
1
Joel F. Gibson,
1,2
Ian King,
1
Donald J. Baird,
3
Daniel Janzen,
4
Winnie Hallwachs,
4
and Mehrdad Hajibabaei
1
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Environment Canada, Canada Centre for Inland Waters, Burlington, Ontario, Canada.
3Environment Canada, Canadian Rivers Institute; Department of Biology, University of New Brunswick,
10 Bailey Drive, Fredericton, NB E3B 5A3, Canada.
4Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Corresponding author: Shadi Shokralla (e-mail: sshokral@uoguelph.ca).
Background: Advances in high-throughput sequencing (HTS) tech-
nologies allow the efficient analysis of DNA barcode sequences from
individual specimens and from bulk environmental DNA (eDNA) sam-
ples. Commonly used DNA extraction protocols involve homogeniz-
ing the samples, leading to loss of either a part or the entire
specimen's body structure. Here we introduce non-destructive and
inexpensive direct PCR protocols for barcoding of individual speci-
mens and a source of community DNA for environmental barcoding
using HTS without any physical disturbance of the specimen. We also
evaluate the amplification efficiency of a wide amplicon size range of
both mitochondrial and nuclear markers. Results: Our results indi-
cate the efficiency of a direct PCR approach in recovering sequence
data from hundreds of individual arthropod specimens from the Área
de Conservación Guanacaste (ACG), Costa Rica, as well as five bulk
benthos and five Malaise trap samples from Wood Buffalo National
Park, Canada. Our non-destructive protocols were effective in provid-
ing genomic template DNA for both mitochondrial and nuclear genes
for specimen-based or bulk HTS analysis of biodiversity in a wide
range of organisms. Significance: The non-destructive, direct PCR
approach presented here will avoid the labour-intensive DNA extrac-
tion procedure and its associated cost. Additionally, this approach will
allow for rapid scanning of the contents of bulk eDNA samples for
target taxa such as pathogens, vectors, and rare or endangered organ-
isms. With this method, the morphological features of the vouchered
specimens are well preserved for species identification, especially
when dealing with a large number of small and fragile specimens.
Gut content metabarcoding of Maumee River fishes: insights
on fish diet during early spring
Megan Shortridge
1
and Jeff Miner
2
1Bowling Green State University, 1833 Kettle Run Ct, Perrysburg, Ohio, USA.
2Bowling Green State University, Life Sciences, South College Drive, Bowling Green, Ohio, USA.
Corresponding author: Megan Shortridge (e-mail: mshortr@bgsu.edu).
Background: Early spring represents an interesting but poorly under-
stood period in the ecology of freshwater fishes. Many fishes enter the
Maumee River, an important tributary of Lake Erie, during spring to
spawn, and therefore, the potential for interspecific trophic interactions
to occur is high. As the timing of peak spawning activity is staggered
depending on the species, there is a high potential for incoming preda-
tors to consume eggs and larvae of earlier-spawning species. Early life
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stages of fishes digest quickly in the gut contents of predator fish, making
visual identification difficult. Therefore, predation on fish eggs and lar-
vae in this system is not well documented, and in order to characterize
the diet of fishes during this dynamic period, COI metabarcoding was
conducted on a small subsample of the homogenized gut contents of
192 bottom-feeding and pelagic fishes. A 313-bp region of COI was amplified
using primers from Leray et al. 2013 and sequenced using an Ion Torrent
PGM next-generation sequencer. Results: Results from the Maumee
River gut content metabarcoding study provided insight into fish diet
during this critical period and yielded possible instances of egg predation
by bottom-feeding fishes. Sequences matching with small-bodied forage
fish, chironomids, and insects were also detected in the diets of fishes.
Significance: Future research should attempt to reduce the degree of
predator swamping from the gut lining of predators species. This study
supports the use of gut content metabarcoding as an important tool to
investigate previously unknown trophic interactions between species.
Model-based clustering techniques for analyzing RNA-seq data
Anjali Silva, Gregory Downs, Yong-Mei Bi, Steven Rothstein,
and Sanjeena Subedi
University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Anjali Silva (e-mail: anjali@uoguelph.ca).
Background: Next-generation sequencing (NGS) enables rapid se-
quencing of genomes and has proven useful for understanding diver-
sity among organisms. One application of NGS is transcriptome
profiling or RNA sequencing (RNA-seq). RNA-seq provides counts of
transcripts, offering a method to quantify gene expression. Despite an
explosion of RNA-seq data, interpreting RNA-seq data in their biolog-
ical context remains a challenge. Clustering permits a systematic in-
vestigation of relationships between genes by identifying clusters of
genes that share similar expression patterns. In this study, a compar-
ative study of three model-based clustering techniques for RNA-seq
data was carried out. The methods included HTSCluster, MBClus-
ter.Seq, and Poisson.glm.mix, available on the Comprehensive R Ar-
chive Network (CRAN). Each clustering method was applied to an
RNA-seq dataset, and clusters of genes identified were analyzed for
biological significance. Results: Across all methods, the discrete na-
ture of read counts is accommodated by clustering via either or both
the Poisson or/and negative binomial distributions. For models using
the Poisson distribution, the Poisson mean is parameterized, while for
models using the negative binomial distribution, the variance is pa-
rameterized. Clustering followed by analysis identified clusters of
genes that show significance in the context of the experiment. Cross
tabulation of results identified similarities and differences in how the
genes were clustered by each method. Significance: A comparative
analysis of three clustering techniques for RNA-seq data is presented.
Clustering enables identification of networks of genes and could lead
to a better understanding of biological and regulatory pathways that
are active under certain conditions. Model-based clustering tech-
niques specific to RNA-seq data are still in their infancy. This is be-
cause only recently have expression profile studies shifted from the
use of microarray studies to RNA-seq. Further research is required to
improve clustering algorithms with respect to efficiency.
Documenting the biodiversity of a local sandhill flora using
DNA barcodes: an investigation into the patterns of resolution
in polytypic taxa
Scott D. Silvis,
1
Julie Ballenger,
1
Michele Elmore,
2
and Kevin Burgess
1
1Columbus State University, 4225 University Ave., Columbus, GA 31907, USA.
2The Nature Conservancy, 5884 1st Division Rd., Fort Benning, GA 31905, USA.
Corresponding author: Scott D. Silvis (e-mail: silvis_scott@columbusstate.edu).
Background: The Fall Line Sandhill region of the southeast United States
has been drastically altered from its original habitat, leading to the loss of
many species. DNA barcode libraries are fast becoming incorporated into
the restoration and conservation management plans of local floras, espe-
cially where taxonomic complexity can confound plant identification
based on morphology alone. This study outlines a preliminary evaluation
of a barcode library based on the four core barcoding gene regions (rbcL,
trnH–psbA, Atpf, and ITS). Specifically, we establish a barcode library for
the Sandhill Flora of Georgia, a fragile and threatened habitat of moder-
ate phylogenetic dispersion that holds a large number of rare and endan-
gered plant species. To assess DNA sequence variation for this barcode
region, DNA and Herbarium vouchers were collected across multiple
study sites in the Sandhill region of Georgia, USA. Results: Preliminary
analysis of the rbcL gene region shows that sequence recovery (91% of
specimens sequenced, 72% of total specimens screened) was relatively
high and comparable with studies of similar taxonomic complexity. Spe-
cies resolution (61%) was relatively low, but expected, for this flora of
moderate composition (29% polytypic genera). Significance: This study
underscores the potential utility of establishing a barcode library for the
Sandhill region of Georgia for future use by organizations like The Na-
ture Conservancy, Georgia Plant Conservation Alliance and Ft. Benning,
all of which are actively working to protect and improve the state of the
damaged and fragmented Sandhills wetlands. These barcode libraries
can assist with the confirmation of the genetic identity of specimens, the
detection of cryptic species, and the conservation and restoration of rare
and endangered taxa. Tools such as these are a necessity in a time where
taxonomy is, in many areas, being phased out of higher education, lead-
ing to a loss of trained taxonomists in the workforce.
Urban parks: refuges for tropical butterflies?
Kong-Wah Sing,
1
Wan Faridah Akmal Jusoh,
1
Nor Rasidah Hashim,
2
and John-James Wilson
1
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
2Faculty of Science, Technology, Engineering and Mathematics, International University of
Malaya-Wales, 50480 Kuala Lumpur, Malaysia.
Corresponding author: Kong-Wah Sing (e-mail: singkongwah@um.edu.my).
Rapid economic development has accelerated urbanisation and biodiver-
sity loss in Southeast Asia. Studies of urban ecology have suggested city
parks can be effective refuges for wildlife in temperate regions, but their
effectiveness as refuges for wildlife in tropical regions is unknown. We
examined the species diversity of butterflies in city parks in the Federal
Territory of Kuala Lumpur and investigated the relationships between
butterfly species richness and three park variables: (i) park age; (ii) park
size; and (iii) distance from the central business district. Standardised
butterfly sampling was conducted across different microhabitat types at
each park: (i) groves; (ii) hedges; (iii) flowerbeds; and (iv) unmanaged. We
collected 572 butterflies belonging to 60 species from five families. In this
study, 97% of species were considered common. Species richness was
positively correlated with park age and size and negatively correlated
with distance from the central business district; however, the correla-
tions were weak and not statistically significant (p> 0.05). The highest
richness (65% of observed species) was recorded in the unmanaged mi-
crohabitats. It is likely that both park planting scheme and the presence
of early-successional plants in unmanaged microhabitats contribute to
the stongest pattern observed, which was highest butterfly species rich-
ness in parks containing all the four microhabitat types. Whether a di-
verse planting scheme and increased size and number of unmanaged
areas in parks can improve the ability of parks to sustain populations of
rare butterflies in the face of other urban landscape parameters remains
to be seen.
Virtual Barcoding: identification of every animal species in a
single closed-tube reaction
Nicola Sirianni and Lawrence Wangh
Brandeis University, Department of Biology, 415 South St., Waltham, MA, USA.
Corresponding author: Lawrence Wangh (e-mail: wangh@brandeis.edu).
Background: Traditional DNA sequencing methods suggest that
the Folmer Region of the cytochrome c oxidase subunit I (COI) gene
Abstracts 281
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of each animal species on earth is unique. This is the basis of the
International Barcode of Life Initiative. Building on this, we have
described an alternative, rapid, easy-to-use, closed-tube method
called Virtual Barcoding that should also be able to distinguish all
animal species on earth without sequencing. Virtual Barcoding uti-
lizes linear-after-the-exponential PCR to generate single-stranded
DNA amplicons containing COI sequences. These targets are coated
at end-point with lights-on/lights-off probes in one or more fluores-
cent colours. The resulting fluorescent signatures can be character-
ized and catalogued automatically. Proof-of-principle experiments
using 10 probes to a portion of the Folmer Region in five species of
nematodes have been described. Results: A set of 15 universal
probes to the Folmer Region has now been designed. Because each
probe is mismatch tolerant and independently hybridizes over a
range of temperatures, this set can theoretically distinguish tens of
millions of sequence variants. The resulting fluorescent signatures
from nematodes to mammals and birds suggest major steps in COI
gene evolution. Significance: Virtual Barcoding is convenient,
highly reproducible, and inexpensive. It can be used for rapid char-
acterization of small multicellular and single-celled species that
are difficult to classify on the basis of morphology. Moreover, be-
cause one or both of the primers driving amplification can have
narrow specificity, the amplifiable sequences can be restricted to
species within a particular genus, populations within a species, or
mixtures of species. Virtual Barcoding is also amenable to use with
a handheld device for immediate detection and diagnosis of species
“in-the-field”. In addition, it will be possible to extend Virtual Bar-
coding to identification of plant species.
Elevation, crypsis, and phylogenetic community structure of
Neotropical arthropods
M. Alex Smith,
1
Megan McPhee,
1
Kate Pare,
1
Ellen Richard,
1
Connor P.K. Warne,
1
Winnie Hallwachs,
2
and Daniel H. Janzen
2
1Department of Integrative Biology, University of Guelph, 50 Stone Road East, University of Guelph,
Guelph, ON N1G 2W1, Canada.
2Department of Biology, University of Pennsylvania, 102 Leidy Laboratories, 433 S University Ave.,
Philadelphia, PA, USA.
Corresponding author: M. Alex Smith (e-mail: salex@uoguelph.ca).
Background: The diversity and phylogenetic community structure
of many organisms is negatively affected by factors that covary
with elevation. Many Neotropical species whose range is restricted
to tropical montane cloud forests are in danger of local or total
extinction due to warming and drying as air warmed by climate
change ascends these mountains. On the Pacific slope of the Cor-
dillera Guanacaste within Area de Conservación Guanacaste (ACG)
in northwestern Costa Rica, we used standardised collections and
DNA barcodes to quantify community size and diversity for some of
the most abundant leaf-litter fauna (ants, spiders, springtails, and
isopods). Results: The relationship between elevation and diversity
on each of three ACG volcanos is specific to the taxa in question. For
example, ants and spiders displayed a strong mid-elevation diver-
sity peak that was evident when diversity was measured through
molecular operational taxonomic units (MOTU) or by phylogenetic
diversity (PD) based on DNA barcodes or a multi-gene phylogeny.
For ants and spiders from the highest elevation cloud forest sites,
we found evidence of significant phylogenetic clustering, the ex-
pected result of filtering due to harsh environmental conditions.
Species turnover between sites on the same mountain and among
the three mountains was high. Significance: The narrow elevation
range of each species, coupled with the high diversity at each sam-
pling point, emphasizes that climate change will bring strong al-
terations in the location and composition of biodiversity on these
mountains. The structure and composition of the hyperdiverse
communities present at any one elevation is extremely vulnerable
to a changing climate.
Changing oceans of biodiversity
Paul Snelgrove
Ocean Sciences Centre Canada, Memorial University of Newfoundland, Newfoundland, Canada.
E-mail for correspondence: psnelgro@mun.ca.
Oceans encompass >95% of Earth's biosphere, and their massive
volume and remoteness ensure we will not fully quantify their
biodiversity in our lifetimes. Estimates of unknown marine biodi-
versity range from 33% to 90%+ of metazoans yet to be described.
Our knowledge of ocean life scales inversely with organism size;
new molecular tools suggest a possible billion unknown microbes,
and statistical models suggest thousands of undiscovered fishes.
Coral reefs, deep midwater environments, and the deep-sea floor
offer the greatest potential for species discovery, but even well-
known environments yield new species. Documented marine ex-
tinctions remain surprisingly rare, but local extirpations and
undocumented species loss raise concern about potential loss of
diversity and function and the need for conservation strategies to
mitigate loss. Importantly, new evidence of adaptability in some
species points to the importance of genetic diversity, and barcod-
ing and gene expression offer new opportunities to detect change.
Documentation of new environments and processes—even in the
last decade—point to untapped oceans of discovery and change.
Phylogeography of Diptera in northern North American
glacial refugia
Anna M. Solecki,
1
M. Alex Smith,
1
Jeffrey H. Skevington,
2
and Terry A. Wheeler
3
1Department of Integrative Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
2Agriculture and Agri-Food Canada, Canadian National Collection of Insects, Arachnids, and
Nematodes, 960 Carling Ave., Ottawa, ON K1A 0C6, Canada.
3Department of Natural Resource Sciences, McGill University, Macdonald Campus,
Ste-Anne-de-Bellevue, QC H9X 3V9, Canada.
Corresponding author: Anna M. Solecki (e-mail: asolecki@uoguelph.ca).
Background: Pleistocene glaciations are a significant abiotic driver of
current distributions of North American species and populations; con-
temporary species distributions are the result of dispersal from refu-
gia beyond ice margins. While most species survived glaciations south
of the ice sheets, many also survived in Beringia. Numerous recon-
structions show additional unglaciated areas in the Canadian Arctic
Archipelago, such as Banks Island; however, phylogeographic evi-
dence is divided on whether it was a refugium. In Canada, Diptera
(flies) are the most species-rich and diverse group of animals, but
studies on the impacts of glaciation on their distribution are lacking.
Our objective was to establish whether molecular patterns of diversity
in flies support a Banks Island refugium. To test this, we used pub-
lished and novel sequences (the DNA barcode region (cytochrome c
oxidase subunit I) and cytochrome b) from two Diptera species: Zaphne
barbiventris (Anthomyiidae) and Norrbomia fumipennis (Sphaeroceridae).
Results: Both species had high nucleotide diversity in the Yukon;
however, only Z. barbiventris also had high haplotype diversity there.
Neither species had high values of haplotype or nucleotide diversity in
Banks Island. Haplotype networks for both species had mostly sepa-
rate Yukon haplotypes and clusters, but few separate Banks Island
haplotypes. However, in the haplotype network of Z. barbiventris,
Banks Island haplotypes were shared with Cambridge Bay, which lies
on the adjacent Victoria Island. Molecular dating analysis suggested
that Yukon haplotypes of both species diverged from others early
during the Pleistocene glaciation. Significance: Patterns from both
species indicate that they survived Pleistocene glaciations in Beringia.
Although Banks Island did not have high haplotype diversity or many
unique haplotypes, a pattern often seen for refugia, current popula-
tions there could have arrived from another Arctic Archipelago refu-
gium. Although we did not find strong evidence for a Banks Island
refugium, we found evidence for a possible Arctic Archipelago
refugium.
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A special issue on DNA barcoding edited by the Belgian
Network for DNA Barcoding (BeBoL)
Gontran Sonet,
1
Zoltan T. Nagy,
1
Nathalie Smitz,
2,3
Massimiliano Virgilio,
3
Kurt Jordaens,
3,4
Thierry Backeljau,
1,4
Marc De Meyer,
3
and BeBoL Network
5
1OD Taxonomy and Phylogeny (JEMU), Royal Belgian Institute of Natural Sciences (RBINS),
29 Vautierstraat, B-1000 Brussels, Belgium.
2Conservation Genetics Unit, University of Liège, Belgium.
3Department of Biology (JEMU), Royal Museum for Central Africa (RMCA), 13 Leuvensesteenweg, B-3080
Tervuren, Belgium.
4Evolutionary Ecology Group, University of Antwerp, 13 Leuvensesteenweg, B-3080 Tervuren, Belgium.
5bebol.myspecies.info.
Corresponding author: Gontran Sonet (e-mail: gsonet@naturalsciences.be).
Background: The Belgian Network for DNA Barcoding (BeBoL) was
launched in 2011. It provides a multidisciplinary platform for 25 insti-
tutes active in DNA barcoding and molecular systematics. Between
2011 and 2013, collaborative projects of BeBoL resulted in about 45 ISI
publications on DNA barcoding, integrative taxonomy, phylogenetics,
phylogeography, identification of cryptic species, biosecurity, and
conservation genetics. BeBoL activities also included organization of
congresses (Third European Conference for the Barcode of Life in 2012,
DNA in Forensics 2014), symposia, workshops, and training activities.
More information about the Network is available on the BeBoL web-
site (http://bebol. myspecies. info). Results: In December 2013, the
Network edited a special issue of ZooKeys (issue 365) entitled “DNA
barcoding: a practical tool for fundamental and applied biodiversity
research”. This open-access publication (http://zookeys.pensoft.net/
browse_journal_issue_documents?issue_id=377) comprised 21 re-
search papers and included, among others, contributions of BeBoL
partners and participants of the ECBOL3 conference. Contributions
focused on many aspects of the DNA Barcode of Life initiative:
(i) technical challenges (DNA barcoding museum or processed biolog-
ical material, biobanking); (ii) testing the utility of COI and alternative
markers for species identifications of flatworms, molluscs, insects,
birds, and plants; (iii) validation of DNA barcoding identification pipe-
lines (for fish landings and forensic entomology); and (iv) applications
in biodiversity monitoring (marine mammals, forest resources).
Significance: As a supplement to large-scale DNA barcoding cam-
paigns, this collaborative initiative is providing methodological re-
sources together with reference DNA barcodes for specific purposes
(e.g., monitoring fish landings, forensics, medicinal plants, etc.).
Large-scale DNA barcoding of ants from Ecuador
Gontran Sonet,
1
Thibaut Delsinne,
2
David Donoso,
2
Adrian Troya,
3
Maurice Leponce,
4
Wouter Dekoninck,
5
Zoltan T. Nagy,
1
John Lattke,
6
and Thierry Backeljau
1,7
1OD Taxonomy and Phylogeny (JEMU), Royal Belgian Institute of Natural Sciences (RBINS),
29 Vautierstraat, B-1000 Brussels, Belgium.
2Museo de Colecciones Biológicas MUTPL, Departamento de Ciencias Naturales, Universidad Técnica
Particular de Loja, San Cayetano Alto, Calle París, Loja, Ecuador.
3Sección de Entomología, Instituto de Ciencias Biológicas, Escuela Politécnica Nacional Ladrón de
Guevara E11-253, EC170109, Quito, Ecuador.
4OD Natural Environment, Biodiversity Monitoring & Assessment (BIOMON), Royal Belgian Institute of
Natural Sciences (RBINS), 29 Vautierstraat, B-1000 Brussels, Belgium.
5Scientific Service Heritage, Royal Belgian Institute of Natural Sciences (RBINS), 29 Vautierstraat,
B-1000 Brussels, Belgium.
6Programa Biodiversidad, Bosques y Servicios Ecosistémicos, Universidad Nacional de Loja, Av. Pío
Jaramillo Alvarado y Reinaldo Espinosa, La Argelia, Ecuador.
7Evolutionary Ecology Group, University of Antwerp, 29 Vautierstraat, B-1000 Brussels, Belgium.
Corresponding author: Gontran Sonet (e-mail: gsonet@naturalsciences.be).
Background: Ecuador has a great diversity of habitat, rough topogra-
phy, and high species richness. Unfortunately, its ant fauna is poorly
understood. Some taxa are morphologically difficult to identify (caste
polymorphism, overlap between intraspecific variation and interspe-
cific differentiation, uniform worker morphology, lack of recent tax-
onomic revisions, etc.). To facilitate species identification, a joint
project of three Ecuadorian universities and two Belgian research
institutes was launched in 2015. It aims at creating a reference collec-
tion of DNA barcodes for every Ecuadorian ant species or morphospe-
cies. Results: A first focus of this project was on the genus Leptanilloides
(Formicidae: Dorylinae) from Central and northern South America
that is rarely collected due to its subterranean foraging habits. Seven
workers and one male were collected, and two putative unknown
species were recognized based on the worker morphology and COI
barcode sequences (p-distances ranging from 18.8% to 21.7%). These
also differed (18% to 21.8%) from the barcodes of the two Leptanilloides
species available in BOLD (6 specimens). Significance: Our results
show that DNA barcoding can distinguish among morphospecies of
ants of the genus Leptanilloides, and thus can complement morphology
for species identification. Unfortunately, Leptanilloides species are cur-
rently known from a limited number of workers and incomplete DNA
barcode libraries. Therefore, we cannot compare the new sequences
with those of all other Leptanilloides species. This case study illustrates
the importance of large-scale sampling in order to investigate a highly
diverse insect fauna, document intra- and interspecific variation, and
build a comprehensive reference library of DNA barcodes.
High-throughput sequencing of PCR amplicons: a test to
barcode a bee species complex (Hymenoptera: Apoidea:
Halictidae) and survey Wolbachia infections
Gontran Sonet,
1
Alain Pauly,
1
Nathalie Smitz,
2
Massimiliano Virgilio,
2
Zoltan T. Nagy,
1
Kurt Jordaens,
2,3
Sebastien Molle,
4
Thierry Backeljau,
1
and Marc De Meyer
2
1OD Taxonomy and Phylogeny (JEMU), Royal Belgian Institute of Natural Sciences (RBINS),
29 Vautierstraat, B-1000 Brussels, Belgium.
2Department of Biology (JEMU), Royal Museum for Central Africa (RMCA), 13 Leuvensesteenweg, B-3080
Tervuren, Belgium.
3Evolutionary Ecology Group, University of Antwerp, 13 Leuvensesteenweg, B-3080 Tervuren, Belgium.
4University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium.
Corresponding author: Gontran Sonet (e-mail: gsonet@naturalsciences.be).
Background: High-throughput sequencing of PCR amplicons, also
called targeted amplicon sequencing (TAS), combines the flexibility
of PCR amplification with next-generation sequencing (NGS) tech-
nologies. In comparison with Sanger sequencing, NGS potentially
improves the sequencing success rate and the detection of hetero-
plasmy, heterozygosity in nuclear markers, and endosymbionts.
Here, we applied TAS to simultaneously sequence the COI barcode
region, three nuclear markers (wingless, white gene, and HOG7036-
02), and a fragment of the Wolbachia surface protein (wsp) in 24 mu-
seum bee specimens of Halictus (Seladonia). This bee genus is frequently
infected by Wolbachia, and one of the species, Halictus smaragdulus
Vachal, 1895, is suspected to be a species complex on the basis of the
morphological variation in the male genitalia. Results obtained for
the DNA barcode fragment were compared to those obtained by
Sanger sequencing, using the same specimens and DNA extracts.
Results: Sequencing of COI was more successful with NGS (21/24 speci-
mens) than with Sanger sequencing (18/24 specimens). COI haplotypes
obtained from both approaches were identical and showed diver-
gences that were congruent with the male genitalia differentiation.
These results suggest that H. smaragdulus comprises more than one
species. No signs of heteroplasmy were observed. Nuclear markers
were successfully sequenced for 15-20 (62%–83%) of the specimens, and
Wolbachia was detected in ⬃50% of the individuals. Significance: By
sequencing standard DNA barcodes and specific DNA markers (includ-
ing DNA fragments from Wolbachia), we produced a dataset that allows
a better taxonomic interpretation of the species complex.
Barcoding plant hotspots in Patagonian Monte Desert
Cintia P. Souto, Paula Mathiasen, and Mariana Tadey
Laboratorio Ecotono Universidad Nacional del Comahue-CRUB, Quintral 1250 Bariloche (8400)
Río Negro, Argentina.
Corresponding author: Cintia P. Souto (e-mail: cintiap.souto@gmail.com).
Background: Under the current biodiversity crisis, an approach used to
prioritize areas for biological conservation is the identification of “biodi-
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versity hotspots,” which usually consist of large areas with exceptional
concentrations of endemic species that are under threat due to habitat
loss. In this study we used a method that integrates geographical infor-
mation with intraspecific genetic polymorphism of species within a hab-
itat type in order to identify genetic hotspots and suggest priority areas
for conservation. Genetic hotspots are areas where multiple species have
high genetic diversity and (or) contain unique genetic variants, and may
be used to set conservation priorities. We tested the hypothesis that
plants with similar ecological tolerances were similarly shaped by evolu-
tionary forces, resulting in genetic hotspots. Results: We mapped ge-
netic diversity and unique variants of DNA barcoded markers (rbcLa +
ITS2) for 75 plant species from Monte Desert, an area highly threatened
by anthropological activities. Spatial distribution patterns of genetic
polymorphisms differed among taxa according to their ecological tol-
erances. Several genetic hotspots were mapped, and we recommend
conservation actions for some of them. Significance: Biodiversity hot-
spots are defined at a coarse scale and are often based on richness,
endemism, threat, or a combination of these—but to date have not
incorporated genetic data. Preserving the evolutionary potential of
species is a major concern in conservation, as it may allow them to
respond to a changing climate and ensure long-term population per-
sistence. Mapping genetic hotspots can contribute to the design of
effective conservation actions by detecting local areas with high ge-
netic diversity, and the presence of unique gene variants—and thus
presumably high evolutionary potential. Existing spatially explicit ge-
netic data from the barcodes of multiple species can help to identify
biodiversity hotspots and guide conservation actions to establish
science-based protected areas that will preserve key habitats and species.
Validation of NGS metabarcoding for detection and
identification of (freshwater) invertebrates
Arjen Speksnijder, Kevin Beentjes, Elza Duijm, Youri Lammers,
and Berry van der Hoorn
Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA, Leiden, the Netherlands.
Corresponding author: Arjen Speksnijder (e-mail: arjen.speksnijder@naturalis.nl).
Background: The Waterscan project in the Netherlands aims to assess
freshwater macrofauna biodiversity by cytochrome c oxidase subunit I
(COI) metabarcoding of environmental DNA (eDNA) for water quality
analysis. The Dutch DNA barcoding database—which includes indicator
invertebrates described in the EU water framework directive—combined
with the extensive Barcode of Life Data Systems (BOLD) database pro-
vide the validated reference that is essential for reliable molecular
identification. We want to validate next-generation sequencing (NGS)
eDNA analysis with COI amplicons from defined complex macrofauna
samples compared to traditional morphological analysis. Therefore,
we have assessed different lengths for the COI barcode region, ranging
from 124 bp to the full 658 bp, on defined macrofauna samples. Fur-
thermore, we assessed different parameters and databases in the
bioinformatics pipeline. Results: Optimal results were obtained using
the full COI barcode, where we retrieved most species from defined
complex macrofauna samples. Using the full barcode region required
some additional work. Amplicons needed to be fragmented and la-
beled for IonTorrent analysis, but this led to a dataset that contained
95% of the expected species. It also included a variety of other species,
including endosymbionts and prey species present in stomach con-
tents. Commonly used universal primers for smaller regions caused
some taxa to be excluded due to primer-mismatch, resulting in detec-
tion of at most 85% of the expected species. Significance: The use of
blended macrofauna samples eliminates the need for cumbersome
morphological identification. In addition, this method increases the
number of species retrieved from samples, as it will also pick up
species that are only present as gut contents. The method is promising
for uniform evaluation of freshwater macrofauna in water quality
assessment and food web studies.
Using next-generation sequencing to identify the botanic origin
of pollen collected from foraging honeybees
Rowan Sprague,
1
Stephane Boyer,
2
and Stephen Wratten
1
1Bio-Protection Research Centre, P.O. Box 85084, Lincoln University, New Zealand.
2Unitec Institute of Technology, 39 Carrington Road, Mount Albert, Auckland 1025, New Zealand.
Corresponding author: Rowan Sprague (e-mail: rowan.sprague@lincolnuni.ac.nz).
Background: An estimated 70% of crops benefit from pollination,
and insects contribute the majority of pollination services. Honey-
bees (Apis mellifera) are the most-used insects for those services
because of their abundance, generalist floral preferences, and ease
of management and transportability. Increased dependence on
honeybees coupled with their recent population decline has led to
a proliferation of scientific research on them. An important aspect
of some of these studies is pollen identification, which is used to
determine from which plant species, genera, or families the bees
are foraging. This study discusses the relative usefulness of pollen
identification methods and suggests next-generation sequencing
(NGS) as an alternative method. Using pollen corbiculae collected
from honeybees, DNA was extracted, amplified with ITS 2 and ITS 5
primers, and sequenced using Roche 454. The samples were repli-
cated to test whether the same species of plants were identified
across each sample. Once the resulting sequences were sorted
and identified to species level, species presence and abundance
were compared between sample replicates to check for precision.
Results: The preliminary results showed that a total of approximately
29 species were present in the samples, with the majority consist-
ing of mainly radish (Raphanus sativus) and clover species (Trifolium
spp.). However, not the same species were found in the replicates of
the samples. Future analysis will reveal relative abundance of spe-
cies, making it possible to determine whether the species not pres-
ent in all replicates were rare. Significance: This approach will
quantify whether NGS can identify species presence and abundance
and thus determine whether NGS presents an efficient pollen iden-
tification method compared to existing methods. Success with this
method would enable pollination biologists to quantify honeybee
foraging preferences for flowering plant species. With a greater
understanding of honeybee foraging behavior, scientists using this
approach will be better equipped to design solutions to combat
honeybee population decline.
Species admixtures in herbal trade: causes, consequences
and mitigation
R. Srirama,
1
J.U. Santhosh Kumar,
2
G. Seethapathy,
1
S. Ragupathy,
3
Steven G. Newmaster,
3
K.N. Ganeshaiah,
4
R. Uma Shaanker,
2
and G. Ravikanth
1
1Ashoka Trust for Research in Ecology and the Environment, Royal Enclave, Srirampua, Jakkur Post,
Bangalore 560 064, India.
2School of Ecology and Conservation and Department of Crop Physiology, University of Agricultural
Sciences, GKVK, Bangalore 560 065, India.
3Centre for Biodiversity Genomics (CBG), College of Biological Sciences, Department of Integrative
Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
4School of Ecology and Conservation, University of Agricultural Sciences, GKVK, Bangalore 560
065, India.
Corresponding author: G. Ravikanth (e-mail: gravikanth@atree.org).
Background: The global economy of international trade of herbal prod-
ucts and the alternative medicine market have been increasing at a rapid
rate of ⬃15% annually. Most of these herbal products are sourced from
countries such as China and India. In India, about 8000 medicinal plant
species are either cultivated or collected from the wild; of these, ⬃880
medicinal plants are in active trade. However, with increasing interna-
tional trade in herbal medicinal products, there is also an increasing
concern about the widespread adulteration and species admixtures in
the raw herbal trade. The adverse consequences of such species admix-
tures on the health and safety of consumers are only recently beginning
to be recognized and documented. Results: We provide a comprehensive
review of the nature and magnitude of species admixtures in the raw
herbal trade and identify the underlying drivers that might lead to such
admixtures. We also discuss the possible biological and chemical equiv-
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alence of the species admixtures and substitutes and their consequences
to consumer health and safety. Significance: We propose a framework
for the development of an herbal trade authentication service that can
help regulate the herbal trade market.
Identification of species adulteration in medicinal plant raw
drugs by DNA barcoding
A. Nithaniyal Stalin, Sophie Lorraine Vassou, Poovitha Sundar,
Balaji Raju, and Parani Madasamy
SRM University Genomics Laboratory, Department of Genetic Engineering, School of Bioengineering,
SRM University, India.
Corresponding author: A. Nithaniyal Stalin (e-mail: nithaniyal88@gmail.com).
Background: Species adulteration in traded medicinal plant raw drugs
threatens the reliability and safety of complementary and alternative
medicine. Earlier studies have reported species adulteration in natural
health products, medicinal plants from southern Morocco, Chinese pat-
ent medicines, and North American herbal products. DNA barcoding
studies on medicinal plant raw drugs are very much limited in India. This
study reports a large-scale study on species adulteration in diverse me-
dicinal plant raw drugs. Results: We assembled a reference DNA barcode
library consisting of 1434 rbcL and Atpf barcodes from 521 medicinal
plant species in our study. Subsequently, we used this library to test for
species adulteration in 112 traded raw drugs in the form of dried roots,
rhizomes, leaves, seeds, fruits, whole plants, and powders that do not
posses intact morphological features for reliable identification. The rbcL
DNA barcodes from 90 raw drugs (80% approx.) showed matching with
expected species in the barcode library. The remaining 22 raw drugs (20%
approx.) had adulterated plant material, which showed 1.5%–10.2% ge-
netic divergence with expected species. All the adulterant species be-
longed to different genera, and 14 of them belonged to different family,
when compared with the species mentioned on the label. Species adul-
teration was more frequent with raw drugs in the form of dried roots,
powders, and whole plants than others. Eight adulterant species shared
some of the medicinal properties with expected species, and seven of
them had a completely different medicinal property or toxic property.
Significance: Morphological resemblance, co-occurrence, mislabeling,
confusing vernacular names, and unauthorized or fraudulent substitu-
tions may contribute to species adulteration. This pilot research will
enable more comprehensive surveys of species adulteration in the trade
of medicinal plant raw drugs. Our findings indicate that DNA barcoding
of the traded raw drugs will be useful to all the stakeholders: traders,
consumers, and regulatory agencies.
Linking adults and immatures of South African marine fishes
Dirk Steinke,
1
Allan Connell,
2
Tyler Zemlak,
1
and Paul D.N. Hebert
1
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road E, Guelph, ON N1G 2W1, Canada.
2South African Institute of Aquatic Biodiversity, Private Bag 1015, Grahamstown, South Africa 6140.
Corresponding author: Dirk Steinke (e-mail: dsteinke@uoguelph.ca).
Background: A comprehensive database of COI sequences, linked to
authoritatively identified voucher specimens for all fishes, promises a
significant advance for fisheries science. Aside from providing identifica-
tions for whole specimens, barcode analysis opens up new possibilities—
essentially barcodes can be used to gain identifications during any stage
of development or when examining fragmentary or processed remains.
The identification of immatures is one area of fisheries research that will
particularly benefit from a DNA barcoding system. Over 2500 immature
specimens were collected as part of a study that spans 10 years of collect-
ing early larvae of fishes spawning pelagic eggs on the inshore shelf,
within 5 km of the coast, along a short section of the KwaZulu-Natal
coastline, about 50 km south of Durban, South Africa. This study further
examined 3216 adult individuals, representing 915 fish species from
South African waters. Results: Using BOLD's library for COI sequences for
adults, 89% of all larval fishes were identified to species level. About 60%
of the unmatched sequences could be assigned to genus, family, or order
level. Only 4% of all sequences could not be assigned to any taxonomic
group. Among all immature individuals, there were representatives of
450 species. Accumulation curves based on inference of phylogenetic
diversity indicate near-completeness of the collecting efforts. The entire
set comprising both adult and larval fish represents about 43% of all fish
species known to occur in South African waters. In addition, it includes
189 species previously not recorded for this region. Significance: The early
life-history stages of fishes are poorly known, impeding acquisition of the
identifications needed to monitor larval recruitment and year-class strength.
In this study, we identified almost 90% of the fish larvae in our collections,
demonstrating both power and completeness of the DNA barcode reference
library of fishes that is available after 10 years of FishBOL.
The International Union for Biological Sciences (IUBS): unified
biology, biodiversity, barcoding, and genomics
Nils Christian Stenseth
1
and Peter G. Kevan
2
1Centre for Ecological and Evolutionary Synthesis, University of Oslo, Norway.
2School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Peter G. Kevan (e-mail: pkevan@uoguelph.ca).
The International Union for Biological Sciences (IUBS; established 1919
http://www.iubs.org) is the major organization representing unified
biology globally. It is now the lead organization within the biological
sciences cluster (comprising eight other international unions) of the
International Council of Science (ICSU) and, as such, has the role of
coordinating a world vision for biology in international sciences. The
membership of IUBS comprises 44 national and 80 international sci-
entific organizations. It is through national full memberships that
IUBS generates its funding for its meetings (the next, 32nd General
Assembly & Conference UBS 2015 – Frontiers in Unified Biology will be
in Berlin, 14–16 December 2015 (www.iubs2015.org)), operations, and
various programs. Of special relevance to Barcoding is that IUBS initi-
ated DIVERSITAS (running since 1991 www.diversitas-international.
org) in the late 1980s, has had long-term interests in genomics, and is
thoroughly interdisciplinary within biology. Through its approach to
unified biology, IUBS has promoted the importance of incorporating
concepts of biodiversity, genomics, evolution, and ecology into all
aspects of biology. IUBS welcomes the possibility that the International Bar-
code of Life has presented for future collaboration and membership.
A recent evolutionary origin of most extant animal species?
Mitogenome and DNA barcode evidence from humans and
other animals
Mark Stoeckle
1
and David Thaler
2
1The Rockefeller University, 1230 York Ave., New York, NY 10065, USA.
2University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
Corresponding author: Mark Stoeckle (e-mail: mark.stoeckle@rockefeller.edu).
Background: In most animals, mtDNA sequences cluster tightly
within species yet differ sharply between them. However, the under-
lying evolutionary mechanisms are unknown. Here we utilize human
data to evaluate alternative models—bottlenecks versus purifying se-
lection. Humans are an ideal test case because we are vastly better
studied than any other animal in terms of genetic diversity and evo-
lutionary history. Human mtDNA diversity is understood to reflect
our recent evolutionary origin and subsequent population expansion,
but it is not known whether humans are an exception among animals.
We analyzed 9413 human mitogenomes in public databases repre-
senting all major haplogroups and compared human mitogenome
variation to that of our closest living and extinct relatives (i.e., chim-
panzees and bonobos, and Neanderthals and Denosovians, respec-
tively) and to that other animals as recorded in mitogenome and COI
barcode libraries. Results: We found that human COI variation is
representative of the coding mitogenome as a whole and is typical of
that in large surveys of other animals, including birds (548 species),
fish (684 species), and moths (754 species). Unlike humans, chimpan-
zees and bonobos have distinct mitochondrial clusters associated with
subspecies or regional populations. Most (70%) synonymous codons in
the human mitogenome differed among one or more apparently
healthy individuals and variants were randomly distributed, findings
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inconsistent with a purifying selection model. Significance: Human
mtDNA diversity typifies that of the majority of other animals which
have single mtDNA clusters. Our results support similarly recent adap-
tive bottlenecks as underlying intraspecific mtDNA clustering in hu-
mans and most other animals. We propose that most extant animals,
like humans, are population outgrowths of recently evolved species.
Future prospects and lessons learned from nine years of
Chironomidae (Diptera) DNA barcoding
Elisabeth Stur and Torbjørn Ekrem
NTNU University Museum, Department of Natural History, Norwegian University of Science and
Technology, NO-7491 Trondheim, Norway.
Corresponding author: Torbjørn Ekrem (e-mail: torbjorn.ekrem@ntnu.no).
Background: The family Chironomidae has about 6200 species de-
scribed world-wide and is among the most widely distributed families
of Diptera. Chironomids are found in almost all kinds of terrestrial
and aquatic habitats but are particularly species rich and abundant in
freshwaters. Many species have specific habitat requirements, and
chironomids are therefore well suited for biological monitoring as
well as research projects focused on evolution and biogeography.
However, the full potential remains unleashed as many species are
difficult to identify, and a large number still is undescribed. DNA
barcoding of Chironomidae therefore represents a great tool for the
advanced use of non-biting midges in research and nature manage-
ment. Results: We summarize our work on DNA barcoding of Chi-
ronomidae over the last 9 years based on more than 6000 records of
900 named species (1267 BINs) in BOLD. Focus is on analytical success,
the importance of vouchers, association of life stages, detection of
cryptic diversity, and the lack of genetic variation. Significance: A
DNA barcode library provides a common base for species interpreta-
tion and paves the way for a broader use of Chironomidae in environ-
mental studies and biosystematics.
Threats to terrestrial vertebrates in Canada
Bridget Stutchbury
Department of Biology, York University, Toronto, Ont., Canada .
E-mail for correspondence: bstutch@yorku.ca.
Canada is home to over 150 terrestrial vertebrates that are nationally
designated as special concern, threatened or endangered; half these
are birds. Canada has an impressive biodiversity of birds, with some
450 species, but it is saddening to realize that over 15% are at risk of
extinction. Dozens of additional bird species are in steep decline. Most
of our birds are migratory and head south before winter, and so face
threats inside and outside of Canada. The good news for birds is that
they are intensively studied, the threats they face are well understood,
and they are highly valued by the public.
DNA barcodes of Japanese Merodon hoverflies (Diptera:
Syrphidae): high morphological variation and low haplotype
diversity of the invasive species
Mitsuaki Sutou and Motomi Ito
University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo, 153-8902 Japan.
Corresponding author: Mitsuaki Sutou (e-mail: mi.sutou@r8.dion.ne.jp).
Background: The genus Merodon includes ⬃160 species of medium-
sized hoverflies worldwide. Most are distributed in the Mediterranean
region, although several species occur in Central and East Asia. Japan
is home to a single native species of this genus, Merodon kawamurae,
distributed in central and western Japan. In addition, the European
species Merodon equestris (the large narcissus fly) invaded central and
eastern Japan in the second half of the 20th century. Studies of
M. equestris in the UK in the 20th century showed that this species has
remarkable colour polymorphism, and suggested that this species
mimics bumblebees. In this study, we compared the haplotype diver-
sity of these two species using DNA barcodes. Results: We sequenced
56 individuals of M. kawamurae collected from three sites in Japan
(Hyogo, Saitama, and Hachioji) and found that they possessed seven
haplotypes. High genetic divergence was also found between the
M. kawamurae populations of western (Hyogo) and central (Saitama
and Hachioji) Japan. We also sequenced 64 individuals of M. equestris
collected from five sites in Japan (Yokohama, Tokyo, Saitama, Ha-
chioji, and Sendai), and 63 of the 64 specimens had the same haplo-
type, while the specimen with the different haplotype differed from
the other 63 individuals by a single base. Significance: This study
shows that the haplotype diversity of the native species M. kawamurae
is higher than that of the invasive species M. equestris. This suggests
that the invasive species reduced its genetic variation during the in-
vasion. However, the specimens of M. equestris sequenced in this study
included several different colour types. By identifying the colour type
of each specimen, this study found that the major colour types of this
species share the same DNA barcode sequences.
DNA barcoding the plants of Monte Desert, Argentina
Mariana Tadey
1
and Cintia P. Souto
2
1CONICET-CRUB, Pasaje Gutiérrz 1125, Argentina.
2INIBIOMA-CONICET, Pasaje Gutiérrz 1125, Argentina.
Corresponding author: Cintia P. Souto (e-mail: cintiap.souto@gmail.com).
Background: Biodiversity conservation is a continuing challenge given
the increasing habitat loss due to human activities causing extintion,
local extinction, or reduction of species populations. Preserving species
evolutionary potential (i.e., genetic variability) is important because it
allows species to respond to changing environments, and therefore im-
prove persistence of populations. DNA barcoding to determine the vari-
ation in species attributable to evolutionary history and the assessments
of floristic surveys from geographic areas can enhance the development
of regional barcoding libraries, providing vital data for community phy-
logeny construction and studies in ecology and conservation biology.
Monte Desert occupies a vast area of Argentina and its being threatened
by human land use. This study aims to investigate the utility and species
resolution capability of DNA barcoding in the vegetation of the Monte
Desert. We used a phylogenetic method (neighbour-joining trees) of DNA
barcodes rbcLa and ITS2 to gain a better understanding of regional bar-
code variation. Results: We analysed 75 species from 400 samples col-
lected throughout Monte distribution. DNA barcodes were obtained for
190 specimens and 61 species. We obtained ITS2 sequences from 48 spec-
imens (27 species, 22 genera, 13 families) and rbcLa sequences from
76 specimens (36 species, 29 genera, 17 families). Barcode analysis showed
mean within-species divergences of 10.84% ± 0.74% and 10.32% ± 0.8%,
16.32% ± 0.64% and 10.32% ±0.6% within genus, and 24.99% ± 0.17% and
11.2% ± 0.2% within families for ITS2 and rbcLa, respectively. The mean
intra-specific divergence was 9.07% ± 1.12% and 1.66% ± 0.19%, and the
mean species divergence from the nearest neighbour was 15.15% ± 0.43%
and 1.93% ± 0.07% for ITS2 and rbcLa, respectively. Significance: These
results provide a better understanding of regional variation in barcode
sequences in plants of Monte Desert, Argentina. This study expanded the
global DNA barcode database for desert plants, and contributes to the
knowledge of the application of barcoding to biogeography and conser-
vation in plants.
Utility of DNA barcoding for the conservation and authentication of
medicinal plant species of economically poor areas of Pakistan to
improve the socio-economic condition of indigenous people
Aisha Tahir,
1
Steve G. Newmaster,
2
S. Ragupathy,
1
and Amer Jamil
3
1Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
2Department of Integrative Biology, Office 208, Centre for Biodiversity Genomics (CBG), University of
Guelph, Guelph, ON N1G 2W1, Canada.
3Deptartment of Biochemistry, Faculty of Sciences, University of Agriculture, Faisalabad-38040, Pakistan.
Corresponding author: Aisha Tahir (e-mail: atahir03@uoguelph.ca).
Background: Inhabitants of biodiversity-rich, but economically poor,
areas of Pakistan are using indigenous plants for treating ailments in
humans and animals, mainly because of unavailability of resources
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and basic medical facilities. Livelihood of many people in these areas
is dependent on the export of these medicinal species to herbal com-
panies. Traditional knowledge of the medicinal uses of plants pro-
vides new insights to the scientific community to explore the
medicinal ingredients of these plants; this is supported by DNA bar-
coding that provides rapid and accurate species identification, which
is a key to understanding the medicinal value of plants. The conser-
vation of medicinal plants has been neglected in many areas of Paki-
stan. This is the first attempt to assemble a reference DNA barcode
library for indigenous medicinal plants of Pakistan. Results: We sam-
pled a total of 250 plants representing 137 species of traditionally used
medicinal plants from different parts of the country. DNA barcoding
was completed using rbcL and ITS2 markers. rbcL correctly identified
88% of the species, while ITS2 identified 78% of the species, and most
taxa were identified at the family or genus level. This approach also
resolved morphological identification issues of almost 40 species.
Evaluation of the utility and species resolution power of two barcode
regions (rbcL and ITS2) included the phylogenetic (neighbour-joining
trees) and sequence similarity (all-to-all BLASTn searches) analyses.
Significance: This research expanded the reference library for medic-
inal plants of Pakistan, and provides a DNA barcoding tool for identi-
fying medicinal plants. We concluded that DNA barcoding using the
rbcL marker is an important tool for taxonomists for timely and cor-
rect species identifications to overcome the impediments of morpho-
logical identifications and identification of medicinal preparations.
Proper implementation of this tool to monitor overexploitation of
medicinal plants and adulteration of herbal products will improve the
quality of herbal medicines, and ultimately improve the rural econ-
omy and conservation of biodiversity in Pakistan.
Using DNA barcoding to document interactions among bats, insects
and plants in the highly fragmented Atlantic forest of Brazil
Tiago Souto Martins Teixeira, Stephen Rossiter, and Elizabeth Clare
Queen Mary University of London, Mile End Road, London, E1 4NS, UK.
Corresponding author: Tiago Souto Martins Teixeira (e-mail: t.s.m.teixeira@qmul.ac.uk).
Background: Habitat loss and fragmentation are serious threats to
biodiversity and ecosystem function, yet their impacts are not fully
understood. Observing ecological interactions is difficult. Bats are
highly diverse, consume wide-ranging resources, and perform numer-
ous ecosystem services. They are key species for forest maintenance
through predation, pollination, and seed dispersal. Traditionally, un-
derstanding these interactions required morphological examination
of gut content, faeces, and regurgitated pellets, with poor and taxo-
nomically biased resolution. DNA barcoding provides a meaningful
solution to this problem. We used high-throughput sequencing and
metabarcoding to document the interactions among bats and insects
and plants. Results: This study will be conducted in the Atlantic rain-
forest of SE Brazil. We are working in 16 forest fragments, sampling
each one for 12 nights over two field seasons. We expect to collect over
1500 faecal samples from bats, and conduct an inventory of insects
and plants to measure resource availability. Significance: The Atlan-
tic forest is of Brazil is one of world's top five hotspots for biodiversity
conservation, due to its rich biodiversity and high levels of endemism.
However, it has a history of large-scale clearance; it is now composed
of >200 000 fragments with 80% of these covering less than 50 ha. It is
necessary to understand the ecological and conservation value of
small fragments, and assess the relationship between community di-
versity and the nature of interactions between species in order to
comprehend the impact on the ecosystem services they provide. As a
result, the Atlantic forest can provide important insights into the
long-term impacts of fragmentation on biodiversity and ecosystem
function, which have important implications for understanding the
consequences of current clearance in Amazonia and Southeast Asia.
BIO's Bio-Inventory and Collections Unit (BIC): specimen
vouchers and barcodes create a unique natural history resource
Angela Telfer, Allison Brown, Paul D.N. Hebert,
and Jeremy R. deWaard
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada.
Corresponding author: Angela Telfer (e-mail: atelfer@uoguelph.ca).
Background: The Bio-Inventory and Collections Unit (BIC) of the Biodi-
versity Institute of Ontario (BIO) has committed substantial resources to
the collection of specimens from across Canada and the globe. This effort
has paid off, as exemplified by the over 95 000 BINs on the Barcode of Life
Data Systems (BOLD) that stem solely from BIC's collecting efforts. The
BIC's natural history collection contains over 2 million voucher speci-
mens; 80% have an associated cytochrome c oxidase subunit I (COI) bar-
code greater than 500 bp, and 20% have images. Exciting opportunities
for collaboration with other institutions are being pursued, first by the
publication of large datasets on the Global Biodiversity Information Fa-
cility (GBIF), and second by our expanding outgoing loan program. Re-
sults: As part of BIC's commitment to open-access specimen data, it has
contributed 320 000 specimen records to GBIF via Canadensys, which
accounts for one-third of arthropod records on the Canadensys reposi-
tory. Since 2012, BIC has loaned nearly 20 000 specimens to taxonomists
around the world. Some of these loans were initiated by BIC to obtain
identifications of unknown material, and other loans were initiated by
taxonomists interested in viewing recently collected specimens from
their area of interest. Case studies illustrate the ways in which BIC's
collection has contributed to work by taxonomists and biodiversity sci-
entists. Significance: BIC's ability to collect mass arthropod samples
each field season combined with high-throughput specimen processing
and DNA barcoding has created a constantly improving and evolving
natural history collection. The pairing of our specimens with DNA bar-
code sequences means we are not constrained by the availability of tax-
onomists to sort and identify incoming material. With increased online
visibility of the natural history collection, BIC is expanding its role as a
valuable resource for researchers worldwide.
A comparison of traditional morphological and next-generation
molecular methods for the identification of benthic invertebrate
and fish species in central British Columbia streams
Aynsley Thielman, Marla Schwarzfeld, Anne-Marie Flores,
Adam O'Dell, Jeanne Robert, Lisa Poirier, Brent Murray,
Daniel Erasumus, Mark Shrimpton, and Dezene Huber
University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada.
Corresponding author: Aynsley Thielman (e-mail: aynsley.thielman@unbc.ca).
Background: As part of the Biodiversity Monitoring and Assessment
Program (BMAP) at the University of Northern British Columbia (UNBC),
we aim to establish baseline biodiversity and to determine food web
dynamics in streams potentially associated with the construction of a
proposed natural gas pipeline in central British Columbia. In addition to
the use of traditional methods for specimen collection and morphologi-
cal identification, we are using DNA barcoding of individual specimens
and developing novel molecular techniques that use high-throughput
sequencing technology to aid in the identification of benthic inverte-
brate and fish species. Techniques employing next-generation sequenc-
ing currently in development include: fish diet (gut content) analysis,
environmental DNA (eDNA), and sampling preservative ethanol (from
Surber samples). Results: Throughout the 2013 and 2014 field seasons,
benthic invertebrates, fish, and eDNA samples were collected at multiple
sites from 14 permanent streams located in both coastal mountain and
interior plateau ecosystem units. Benthic invertebrate and fish identifi-
cation, and fish gut content analysis results to date, including compari-
son of morphological and molecular methods, will be presented.
Significance: Morphological identification and subsequent DNA barcod-
ing of benthic invertebrates have revealed important limitations in un-
derstanding taxonomic resolution using traditional methods, as well as
the immense potential for species discovery and expansion of known
distribution ranges in a very understudied region of Canada.
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Towards a safe herbal medicine in Ghana, the role of DNA barcoding
Gyimah Thomas
Ghana Wildlife Society, P.O. Box 13252, Accra, Ghana.
E-mail for correspondence: gyimthom@yahoo.com.
Background: Due to high poverty levels coupled with a high patient-to-
doctor ratio in Ghana, most people, especially the urban poor and rural
dwellers, resort to herbal medicines for their primary healthcare. There-
fore, there is an increasing demand for herbal medicine, leading to over-
harvesting of many important species. However, the safety and efficacy
of herbal medicine depends on the ability to correctly identify the plants
used in their preparation. This study sought to use DNA barcoding as an
identification tool in the authentication of herbal medicines and sustain-
able management of medicinal plants species in southern Ghana. Silica-
dried leaf samples from 57 different medicinal tree species covering
24 families were collected from three different conservation areas:
Ankasa, Bia, and Kakum. Sequences from the barcode region of rbcL were
generated for 119 samples, with a minimum sequence length of 431 bp
and a maximum of 579 bp. Results: A sequencing success rate of 94.6%
was achieved. Twenty out of the 57 medicinal plants already sequenced
were randomly selected and collected from farmlands within the
Akuapem North district of the Eastern region of Ghana and sequenced at
the same gene region. This was done to test the utility of the database
in identifying medicinal plants. Significance: Each of the se-
quences in the later-collected farm dataset matched closely with
their conspecific counterparts in our reference dataset, which sug-
gests DNA barcoding can be used in the identification of medicinal
plant species to ensure authenticity of herbal medicines in Ghana.
DNA barcoding of invasive species in relation to Canadian
federal policy and law
Vernon Thomas,
1
Robert Hanner,
2
and Alex Borisenko
2
1Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
Corresponding author: Vernon Thomas (e-mail: vthomas@uoguelph.ca).
Background: Invasive Alien Species (IAS) cause extreme economic and
environmental damage in Canada. Future invasions may happen via di-
verse pathways and ports of entry, calling for a robust nationwide system
for rapid detection and response. Numerous case studies demonstrate
the effectiveness of DNA barcoding as a tool for quarantine and environ-
mental detection of invasive organisms in both forensic and bulk sam-
ples; however, their scope has been limited. DNA barcoding should be
broadly deployed and integrated into a nation-wide biosurveillance ef-
fort with a conceptually novel operational framework, supported by
several important changes in legislation, policy, and governance. 1. DNA-
based identification should be incorporated into Canadian federal legis-
lation, as an accepted standard for validating the taxonomic identity of
invasive organisms and the basis of rapid response management, 2. It
should be broadly adopted by key relevant federal agencies, such as DFO,
CFIA, Transport Canada, Environment Canada, and Parks Canada as part
of their operating policies for monitoring and control of the spread of
invasive species. 3. The creation of a shared, federally accredited and
universally accessible DNA barcode reference dataset based on existing
data platforms (such as BOLD or CANADENSYS) should be authorized
under the respective articles of Canadian law for the above agencies. 4.
Legislative provisions/incentives need to be in place for the submission of
raw digital DNA-based information from environmental surveillance ac-
tivities (e.g., impact assessments) into a centralized data portal, to facili-
tate monitoring and detection of important invasive species. 5. Efforts
should be in place to garner support from provinces, territories, and
other jurisdictions in adopting similar legislations and facilitating data
sharing/exchange. 6. DNA barcoding should become part of the agenda
in trade negotiations between Canada and its key trading partners (e.g.,
those with signed FTA's), in an effort to harmonize regulatory frame-
works at an international level.
New models, new data: can DNA barcoding help with the
development of general ecosystem models?
Derek Tittensor
Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia, Canada.
E-mail for correspondence: derek.tittensor@unep-wcmc.org.
Background: The Madingley model, a General Ecosystem Model (GEM),
is an attempt to develop a tool analogous to the General Circulation
Models used by the earth science community for understanding the
mechanisms and consequences of climate change. It models the pro-
cesses that structure ecological assemblages on land and sea, and appears
particularly suitable for assessing the dynamic consequences of changes
including those due to human impacts. Results: GEMs integrate numer-
ous aspects of ecological thought and theory with the aim of modelling
entire ecosystems—including both discovered and undiscovered spe-
cies—at a global scale. Although this is a long-term effort and early mod-
els will be relatively crude, initial results are encouraging. However, their
development has led to the identification of large gaps in the types of
data required for constraining and parameterising them: gaps which
need to be filled in order to properly characterise the uncertainty associ-
ated with these models. Significance: Here I ask whether DNA barcoding
approaches, present and future, can be used as a prominent tool to
gather the types of data necessary to constrain such models, and appeal
for the development of creative approaches to acquire such data. In par-
ticular, most ecological data-gathering focuses on specific species rather
than cataloguing the components and fluxes of whole communities from
top to bottom. In contrast, GEMs such as Madingley may require a radi-
cally different approach, focussing on organismal functions, interac-
tions, the structure of the community and rates of flux through it, and it
may be that only data collection involving next-generation DNA sequenc-
ing will be able to provide the type and volume of data required.
The origin of the Sardinian Blue, Pseudophilotes barbagiae
(Lepidoptera: Lycaenidae): Out-of-Europe or Out-of-Africa?
Valentina Todisco,
1
Konrad Fiedler,
1
Elisabetta Puliga,
1
Vlad Dinca,
2,3
Raluca Voda,
3,4
and Andrea Grill
1
1Department of Botany and Biodiversity Research, Division of Tropical Ecology and Animal
Biodiversity, University of Vienna, Austria.
2Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
3Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Barcelona, Spain.
4Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Corresponding author: Valentina Todisco (e-mail: valentina.todisco@gmail.com).
Background: The island of Sardinia is characterized by an extraordinary
richness of endemic species, evolved as a result of its geographic position
and geological history. Here, we try to reconstruct the evolutionary his-
tory of the endemic Sardinian blue butterfly, P. barbagiae de Prins and van
der Poorten 1982, using the DNA barcoding region of the COI mitochon-
drial gene. The species is restricted to a few slopes in the mountainous
areas of the island, and has been described as distinct from the continen-
tal and Corsican P. baton by characteristics of the male genitalia and wing
markings. Results: We analyzed 11 individuals of P. barbagiae and com-
pared them with all European and north African congeneric species: a
group of 77 sequences including P. baton (Corsica, Italy, France, Spain), P.
vicrama (Romania, Turkey, Kazakhstan, Russia), and P. panoptes (Spain), as
well as several BINs containing 20 sequences of P. abencerragus (Spain,
Morocco, Tunisia, Portugal), 10 sequences of P. bavius (Romania, Russia),
and 4 sequences of P. fatma (Morocco). The Bayesan phylogenetic tree
showed that the group with P. baton,P. vicrama, and P. panoptes forms a
well-supported clade, but DNA barcodes do not distinguish these three
species. The largest interspecific distance (4.8%) was found between P.
fatma and the P. baton –P. vicrama –P. panoptes group, while the smallest
(1.2%) was between P.abencerragus and P. barbagiae. The latter also forms a
well-supported monophyletic group and has closer affinity to P. abencer-
ragus (1.2%) than to the P. baton –P. vicrama –P. panoptes group (1.8%).
Significance: Two hypotheses on a European versus an African origin of
P. barbagiae will be presented in the light of genetic and biogeographic
data. This study will contribute to the emerging picture of butterfly di-
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versification in the Palearctic region, and contribute to our understand-
ing of the evolution of endemism in the European butterfly fauna.
CSI: Guelph—forensic applications of DNA barcoding for
wildlife identification and food authentication
Janet Topan
Biodiversity Institute of Ontario, 50 Stone Road East, Guelph, ON N1G 2W1, Canada.
E-mail for correspondence: jtopan@uoguelph.ca.
Background: Ever since DNA fingerprinting had revolutionized crimi-
nal forensic science, interest in non-human DNA analysis has been grow-
ing. With endangered wildlife trade and adulteration of food products
being on the rise, DNA-based identification methods offer a solution that
empowers regulatory and enforcement agencies to prevent such illegal
activities. Forensic identification of wildlife and even more so verifica-
tion of food ingredients are often challenged by the lack of morphologi-
cal characters or access to taxonomic expertise in a timely fashion.
Forensic identification of food products—including meat, poultry, and
seafood—and the identification of wildlife samples thus require the use
of genetic species identification techniques such as DNA barcoding. Re-
sults: Since 2010, the Canadian Centre for DNA Barcoding (CCDB) core
laboratory has processed 74 cases, with over 2000 samples, for identifi-
cation of various animal and plants. Service was provided to a wide range
of clients including a number of government agencies (16), corporate
clients (30), universities (13), news media companies (5), private citizens
(3), and internal projects (7). Of the 2000 samples processed, 28% were
from seafood products; 16% were from other food items; 8.3% were for
wildlife identification; 13.2% were from cases involving insect identifica-
tion; 24.4% were from cases related to environmental sampling; 8.5%
were for plant identification; and 1.2% were related to health safety. For
these forensic cases, 92.6% of barcodes were identified to genus or spe-
cies, and 1.85% were identified to family. Significance: Detailed review of
a few of the most striking examples, spanning a wide range of sample
conditions and testing requirements, illustrates strong expertise of the
CCDB in forensic applications of DNA barcoding and confirms that DNA
barcoding provides a cost-efficient and effective solution for identifica-
tion of samples, with legal implications in wildlife conservation and
consumer health and safety.
The Barcode of Wildlife Project, part 2: data pipeline and
GenBank submission
Michael Trizna and David Schindel
Consortium for the Barcode of Life, Washington, DC, USA.
Corresponding author: Michael Trizna (e-mail: triznam@si.edu).
Background: The Barcode of Wildlife Project (BWP) is a multi-year proj-
ect with six developing partner countries that are preparing to use DNA
barcoding to prosecute wildlife crimes. This forensic use of barcodes
requires more complete documentation so CBOL required datafields in
addition to those required by the existing Barcode Data Standard. CBOL
worked with the BiSciCol project to construct a Field Information Man-
agement System (FIMS) that populated and validated high-quality speci-
men and collection metadata as soon as possible in the process. The FIMS
is able to serve specimen data to the Geneious Laboratory Information
Management System (LIMS), originally developed by the Moorea Biocode
barcoding project. The FIMS and LIMS feed data to a new timeline-based
dashboard to pinpoint bottlenecks in the workflow. Finally, CBOL
worked with Geneious developers to overhaul the GenBank Submission
Plug-in, which now meets all of the DNA barcode submission needs, as
well as the extended fields being used for the BWP Project. Training was
provided through an in-country workshop and frequent videoconferenc-
ing. Since the informatics pipeline was custom created for this project,
each step of the pipeline has been refined and reworked to address the
feedback and experience of the BWP users. Results: Three of the earliest
countries in the BWP have used the informatics pipeline to process over
a thousand high-quality BARCODE keyword records for protected plant
and animal species that will be the first BWP data release. The pipeline
software is open source and is available online along with documenta-
tion and training materials. Significance: This informatics pipeline pro-
vides the barcoding community with a system for assembling, validating,
and submitting GenBank records in compliance with the Barcode Data
Standard. The extended BWP Data Standard may meet the needs of other
regulatory applications of barcoding.
Integration of DNA barcoding for surveillance of avian
influenza and Newcastle disease in migratory birds
Erdene-Ochir Tseren-Ochir, Dong-Hun Lee, Jae-Keun Park,
Seong-Su Yuk, Jung-Hoon Kwon, Jin-Yong Noh,
and Chang-Seon Song
Avian Disease Laboratory, College of Veterinary Medicine, Konkuk University, Gwangjin-gu, Seoul,
Republic of Korea.
Corresponding author: Erdene-Ochir Tseren-Ochir (e-mail: erdeneochir88@gmail.com).
Background: Wild birds are recognized natural reservoirs and are po-
tentially responsible for the wide geographic distribution of various
pathogens, including the avian influenza virus (AI), Newcastle disease
virus (ND), and avian paramyxovirus Type 4 (APMV-4). Surveillance using
virus isolation from fresh fecal samples and bird species identification
have been key activities in providing information regarding the epidemi-
ology, geographic distribution, and host ecology of these diseases. How-
ever, bird species identification by observation is not always easy, and
therefore not always accurate. The DNA barcoding technique is a species
identification method that analyzes short mitochondrial DNA se-
quences, enabling accurate identification of the host bird species for AI,
ND, and APMV-4 viruses. DNA was extracted from fecal samples using a
DNeasy stool mini kit (Qiagen), and PCR was performed with novel prim-
ers (AvesF and AvesR) for cytochrome c oxidase subunit I (COI). DNA
sequences of PCR products were compared with the sequence database
offered by the Barcode of Life Data Systems (BOLD). Results: A total of 47
(AIV, n= 34, NDV n= 10, and APMV-4 n= 3) positive fecal samples were
isolated from nine (n= 9) different bird species, which were identified as:
mallard (Anas platyrhynchos,n= 21, 45%), Mandarin duck (Aix galericulata,
n= 11, 24%), spot-billed duck (Anas poecilorhyncha,n= 8, 17%), grey heron
(Ardea cinerea,n= 2, 4%), white-fronted goose (Anser albifrons,n= 1, 2%),
lesser white-fronted goose (Anser erythropus,n= 1, 2%), and bean goose
(Anser fabalis,n= 1, 2%). Significance: Identification of bird species by DNA
barcoding could be useful for large-scale avian disease surveillance. Im-
portantly, continued surveillance with DNA barcoding may significantly
improve our understanding of the role of migratory birds in the public
health and ecology of avian diseases.
Molecular-phylogenetic reconstruction and taxonomic
investigation of eelpouts (Cottoidei: Zoarcales) based on two
mitochondrial genes
S.V. Turanov,
1
Yu.Ph. Kartavtsev,
1
Y.-H. Lee,
2
V.V. Zemnukhov,
1
and A.A. Balanov
1
1A.V. Zhirmunsky Institute of Marine Biology of the Far Eastern Branch of the Russian Academy of
Sciences, Palchevskogo str. 17, Vladivostok, 690041, Russia.
2Marine Ecosystem Research Division, Korea Institute of Ocean Science & Technology, 787 Haean-ro,
Sangnok-gu, Ansan, Republic of Korea.
Corresponding author: S.V. Turanov (e-mail: sturcoal@mail.ru).
Background: The infraorder of the eelpouts (Cottoidei: Zoarcales) com-
prises about 300 species of coldwater fishes with their major diversity
concentrated in the Northern Pacific, but the distribution is extremely
wide and spreads also into the Atlantic ocean as well as into the southern
hemisphere. Molecular phylogenetics coupled with classic morphologi-
cal methods allowed a review of the group's taxonomic composition and
to confirm/refute the validity of large size families once they converge.
The application of DNA barcoding made it possible to discover cryptic
diversity and recently originated species in the group. Despite the grow-
ing abundance of new data on eelpout taxonomy and evolution, the
consideration of original and independent results is evidently required to
verify established knowledge. Results: Based on a concatenated matrix
of COI and cytb mitochondrial genes (2281 bp in total) for samples from 7
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families and 45 species of eelpouts and by using model and character-
based phylogenetics methods, we have obtained a phylogenetic scheme
that is generally consistent with previous results. Despite low resolution
of the original data matrix, we have demonstrated the isolation and
monophyly of the families Zoarcidae, Anarhichadidae Stichaeidae, Neo-
zoarcidae, and the recently revised Eulophiidae. The polyphyly amongst
some subfamilies within the Stichaeidae is confirmed, whereas Opistho-
centrinae and Pholidae seem to constitute a natural family-level taxon.
Significance: The study provides a new view on the problem of taxo-
nomic relationships in the complex and diverse eelpouts group, which
are not covered by the recently flourishing multi-locus phylogenies of
teleost fishes due to the lack of taxonomic representation. It becomes
clear that there is a need for the application of more unified and repre-
sentative mitochondrial phylogenomic approaches to resolve the issues
of evolution and taxonomy of eelpouts.
Plant DNA barcoding project in Kazakhstan
Yerlan Turuspekov and Saule Abugalieva
Institute of Plant Biology and Biotechnology, Timiryazev str 45, Kazakhstan.
Corresponding author: Yerlan Turuspekov (e-mail: yerlant@yahoo.com).
Kazakhstan is the ninth-largest country in the world by territory, and it is
home to more than 6000 plant species. In 2015, a new project was
launched with the main goal being to study genetic variation of endemic,
rare, and economically important plant species in National State Re-
serves and National Nature Parks of Kazakhstan. Genetic diversity of
plant populations will be studied by using different types of DNA mark-
ers, including universal markers of nuclear and chloroplast genomes.
The project combines the efforts of botanists and geneticists from Na-
tional State Reserves and National Nature Parks, National Universities,
Botanical Gardens, and two Biotechnology Research Institutes; more-
over, our team enthusiastically welcomes collaboration with foreign sci-
entists and organizations.
DNA barcoding of selected Philippine pomacentrids
Erika R. Valeroso,
1
Jonas P. Quilang,
1
and Ma. Josefa R. Pante
2
1Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, Philippines.
2Marine Science Institute, College of Science, University of the Philippines, Diliman, Quezon City,
Philippines.
Corresponding author: Ma. Josefa R. Pante (e-mail: drp.upmsi@gmail.com).
Background: Pomacentrids, more commonly known as damself-
ishes, are some of the most abundant reef fishes found throughout the
world's oceans. Out of the 387 species found worldwide, the Philip-
pines are home to 157 species. Members of the family exhibit a wide
range of colorful patterns and body shapes and hence are popular
aquarium fish. Visual identification of pomacentrids relies mainly on
distinction of color because in many instances the difference between
closely-related species is a matter of minor variances in a common
color pattern. Results: In this study, 60 individuals of 14 species of the
family Pomacentridae were barcoded using partial DNA sequences of
the mitochondrial cytochrome c oxidase subunit I (COI) gene. The
divergence of sequences within and between species was determined
using the Kimura-2-parameter (K2P) distance model, and a neighbour-
joining tree was generated with 1000 bootstrap replications with the
K2P model. All COI sequences obtained allowed discrimination of
species. The average genetic distances within species, genera, and
family were 0.20%, 8.90%, and 14.20%, respectively. Significance: Prior
to this study, DNA barcoding of Philippine pomacentrids has never
been done. Our results show that COI barcodes are truly effective for
the accurate identification of species in the family Pomacentridae.
This study and further barcoding studies on other Philippine poma-
centrids will become a useful tool as government agencies continue to
monitor the marine aquarium trade in the country.
Pelecanus occidantalis infected by two related parasites species
of Contracaecum in Baja California peninsula: new records and
ecological perspectives
Isabel Valles Vega, Sergio Hernandez Trujillo,
and Bárbara González Acosta
Centro de Interdisciplinario de Ciencias Marinas, Av. Instituto Politécnico Nacional s/n Col. Playa Palo
de Santa Rita Apdo. C P 23096, Mexico.
Corresponding author: Isabel Valles Vega (e-mail: iza_wega@hotmail.com).
Background: Anisakidae nematodes are parasites of marine mam-
mals and piscivorous birds and could produce health problems in
humans. Many of the species of this family have been sorted into
species complexes due to morphological similarities. Molecular tools
provided an efficient way to discriminate within species complexes,
and in this work we used two different molecular markers (ITS-1, ITS-2,
and COI) to identify Contracaecum multipapillatum and relate it with the
sibling species of its own complex (e.g., C. multipapillatum A, B, C, or D).
Our samples were obtained from brown pelicans (Pelecanus occidentalis),
both on the Gulf of California and the pacific coast of Baja California
Peninsula. Results: Sequence comparisons within the two markers sug-
gest the presence of two species due to the relatively high genetic differ-
ence computed with F84 similarities (ITS-1 = 13%; ITS-2 = 13%; COI = 3%). To
assign our sequences with some of the sibling species, we compared
them against GenBank data; only ITS-1 and ITS-2 sequences were avail-
able for C. multipapillatum. These comparisons grouped some of our se-
quences with C. multipapillatum, while other sequences were grouped
with C. bioccai.Significance: The presence of C. bioccai would be a new
record in our study area, and we suggest the possibility that the two
species have similar life cycles with an overlap in their definitive host and
co-occur in the same distribution because of pelican migrations.
Insights from the Tree of Sex: why so many ways of doing it?
Jana Vamosi,
1
Tia-Lynn Ashman,
2
Doris Bachtrog,
3
Heath Blackmon,
4
Emma E. Goldberg,
5
Matthew W. Hahn,
6
Mark Kirkpatrick,
7
Jun Kitano,
8
Judith E. Mank,
9
Itay Mayrose,
10
Ray Ming,
11
Sarah P. Otto,
12
Catherine L. Peichel,
13
Matthew W. Pennell,
14
Nicolas Perrin,
15
Laura Ross,
16
and Nicole Valenzuela
17
1University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada.
2Department Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
3Department of Integrative Biology, University of California, Berkeley, CA 94720, USA.
4Department of Biology, University of Texas, Arlington, TX 76019, USA.
5Department of Ecology, Evolution, and Behavior, University of Minnesota, Saint Paul, MN 55108, USA.
6Department of Biology and School of Informatics and Computing, Indiana University, Bloomington, IN
47405, USA.
7Department of Integrative Biology, University of Texas, Austin, TX 78712, USA.
8National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
9Department of Genetics, Evolution and Environment, University College London, London,
WC1E 6BT, UK.
10Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel-Aviv, 69978, Israel.
11Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA.
12Department of Zoology, University of British Columbia, Vancouver, BC V6J 3S7, Canada.
13Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
14Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA.
15Department of Ecology and Evolution, University of Lausanne, CH-1015 Lausanne, Switzerland.
16Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JT, UK.
17Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, IA 50011,
USA.
Corresponding author: Jana Vamosi (e-mail: jvamosi@ucalgary.ca).
Background: The vast majority of eukaryotic organisms reproduce
sexually, yet the nature of the sexual system and the mechanism of
sex determination often vary remarkably, even among closely related
species. Some species of animals and plants change sex across their
lifespan, some contain hermaphrodites as well as males and females,
some determine sex with highly differentiated chromosomes, while
others determine sex according to their environment. Testing evolu-
tionary hypotheses regarding the causes and consequences of this
diversity requires interspecific data placed in a phylogenetic context,
yet our understanding of the functional roles of species traits is often
undermined by a lack of knowledge of diversity in nature. Results:
With a new database developed to facilitate analysis of sexual systems
290 Genome Vol. 58, 2015
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and sex chromosomes across over 12 000 species, I summarize some of
the initial findings of the Tree of Sex Consortium (http://treeofsex.
org). One novel finding is that both the incidence among extant species
and the establishment rate of Y-autosome fusions is much higher than
for X-autosome, Z-autosome, or W-autosome fusions. Significance: Phy-
logenic reconstructions that include barcode data incorporate suffi-
cient taxon sampling to elucidate the role of sexual system diversity in
shaping (i) genome architecture in animals and (ii) speciation and
extinction rates in plants. By expanding our ability to observe empir-
ical patterns, we are often confronted with findings that overturn
current paradigms and thus must identify other combinations of evo-
lutionary forces that might account for the data.
Advance in the identification of Palinuridae and Scyllaridae
Phyllosomas using DNA barcoding in front of the coast of the
Mexican Caribbean
Lourdes Vásquez-Yeomans,
1
Ashanti Alejandra Canto-Garcìa,
1
and Jason S. Goldstein
2
1El Colegio De La Frontera Sur, Unidad Chetumal Ave. Centenario Km 5.5 Col. Pacto Obrero
Campesino, Chetumal Q. Roo, Mexico.
2University of New Hampshire, Department of Biological Sciences, Rudman Hall, 46 College Road,
Durham, NH 03824, USA.
Corresponding author: Lourdes Vásquez-Yeomans (e-mail: lvasquez@ecosur.mx).
Background: The Caribbean basin and adjacent Gulf of Mexico con-
tain three major groups of marine lobsters: Palinuridae, Scyllaridae,
and Sinaxidae. Spiny lobsters (Panulirus argus, Latreille 1804) are a
highly important fishery resource throughout the Caribbean basin
and in Quintana Roo. Although the early life history (larval and post-
larval phases) of this species has been well studied in two congeneric
species, Panulirus guttatus (Latreille 1804) and Panulirus laevicauda
(Latreille 1804), there is comparatively little information about larval
(phyllosoma) development. In the case of P. guttatus, it has been diffi-
cult to gather a valid and accurate description of their phyllosomal
development over its early stages due to taxonomic difficulties in
distinguishing their phyllosomas from other closely related species
(e.g., P. argus). A major distinguishing characteristic is the large size
that P. guttatus can obtain in later developmental stages. Phyllosoma
collections were obtained in March and April 2006 on an oceano-
graphic cruise (R/V Gordon Gunter), where samples were collected at
49 different stations in the northern part of the Mesoamerican Barrier
Reef System using a MOCNESS (1 m
2
diameter, 335 m mesh) system.
Results: We present preliminary results from DNA barcoding that
successfully identify P. guttatus from previously missing larval devel-
opmental stages (I–V). We obtained a total of 64 phyllosomas corre-
sponding to stages I–VII from P. guttatus, completing the phyllosomal
description for this species, and obtained 49 larvae of P. argus. Addi-
tionally, we obtained two phyllosomas from Scyllarus chacei and two
specimens from Scyllarides aequinoctialis, both slipper (Scyllaridae) lob-
sters. A total of 16 larvae (Scyllaridae) and one taxonomically un-
known phyllosoma were reported as Phyllosoma Q (Robertson, 1972).
Significance: Combining DNA barcoding with morphological identi-
fication techniques allows the possibility to decipher those stages of
other species of lobster phyllosomas as compared with the use of
previous traditional methods, in effect facilitating the identification
of missing stages and establishing a technique for taxonomic confir-
mation along with morphological identification.
Utility of DNA barcodes for the identification of
parasitic nematodes
María G. Velarde-Aguilar and Virginia León-Règagnon
Estación de Biología Chamela, Instituto de Biología, UNAM San Patricio, Jalisco, 48980, México.
Corresponding author: María G. Velarde-Aguilar (e-mail: velarde_guadalupe@hotmail.com).
Background: Identifying parasitic nematodes based on morphology
is often difficult and laborious, because clear diagnostic characters are
frequently absent or can be hard to obtain and interpret. In addition,
many diagnostic features are found exclusively in males, and pheno-
typic plasticity is frequent, which leads to the presence of cryptic
species. It is also common to find nematodes in larval stages, which
cannot be identified because of the lack of diagnostic features. Despite
the success of the Folmer region for identification of different animal
groups, its usefulness in parasitic nematodes has been scarcely tested.
Here, we try DNA barcodes to identify parasite nematodes from Mex-
ican anuran hosts that are semi-aquatic (Leptodactylus melanonotus),
terrestrial (Incilius marmoreus,Rhinella marina), and arboreal (Diaglena
spatulata,Smilisca baudinii). Results: A total of 64 individuals represent-
ing six genera from five families of common nematode parasites of
anurans were sequenced. All specimens were correctly assigned to
genus and species according to morphological characters. The intras-
pecific genetic divergence reached a maximum of 1.8%, but in most
cases it was less than 1%. Interspecific divergence varied from 8% to
10%. It was also possible to detect potential cryptic species in the genus
Aplectana. Larval stages could also be identified by comparison with
adult sequences from the same hosts. Significance: Helminth para-
sites are abundant in a wide range of marine and terrestrial hosts, are
highly diverse, and can be useful for monitoring environmental an-
thropogenic impacts and biodiversity studies. Molecular methods like
DNA barcoding offer an efficient alternative to the limitations of
morphological approaches in studying nematode biodiversity. DNA
barcoding also represents a tool for testing the real value of mor-
phological characters that traditionally have been considered diag-
nostic.
Molecular identification of egg parasitoid, Trichogramma
species of India using COI and ITS-II regions and their
phylogenetic relationships
T. Venkatesan, B. Reetha, S.K. Jalali, Y. Lalitha, C.R. Ballal,
Ravi P. More, and Abraham Verghese
National Bureau of Agricultural Insect Resources, P.B. No. 2491, H.A. Farm Post, Hebbal, Bangalore--
560024, India.
Corresponding author: T. Venkatesan (e-mail: tvenkat12@gmail.com).
Trichogrammatids are the most important egg parasitoids widely
used in different biocontrol programs worldwide. Since several spe-
cies, strains, and ecotypes are available, correct identification is the
first step for successful biological control programs. Morphological
identification remains difficult because of their minute size (<1 mm
long), is time consuming, and requires specialized skills, as species
exhibit subtle differences in male genitalia. However, male genitalia
may be similar in some species, and in some species males are not
available due to parthenogenetic reproduction. Therefore, species
identification must be rapid, simple, and widely applicable. Further,
many of the field-collected specimens are females, which are not iden-
tifiable using morphological keys. Therefore, to solve this difficulty,
we develop an identification method based on the COI and ITS-2 loci of
22 samples of nineteen species. Genomic DNA of this species was
isolated by using a DNA extraction kit (QIAGEN DNeasy blood and tissue kit
Cat. 69504, Germany). For COI gene amplification, the following primers
were used: Forward, 5=-GGTCAACAAATCATAAAGATATTGG-3=andReverse,
5=-TAAACTTCAGGGTGACCAAAAAATCA-3=. For ITS 2 amplification, the
following primers were used: Forward, 5=-TGTGAACTGCAGGACACATG-
3=, and Reverse, 5=- GTCTTGCCTGCTCTGAG-3=. Amplified PCR products
were sequenced, and a homology search was done using NCBI, BLAST.
The species identified based upon the COI and ITS-2 regions are
Trichogrammatoidea armigera,Tr. bactrae,Tr. robusta,Trichogramma achaeae,
T. pretiosum (France), T. pretiosum (Germany), T. chilonis,T. cacoeciae,
T. embryophagum,T. evanescens (Arrhenotokous), T. evanescens (Thelytok-
ous), T. semblidis,T. danausicida,T. cordubensis,T. japonicum,T. brassicae
(Italy), T. brassicae (Canada), Trichogramma mwanzai,T. chilotraeae,
T. dendrolimi,T. hebbalensis, and T. danaidiphaga. Along with available
homologous sequences in the GenBank database, a phylogenetic tree
was constructed using the maximum likelihood method based on the
predicted evolutionary model GTR+G (General Time Reversible) and
K2+G (Kimura 2 Parameters) for the COI (50 sequences) and ITS-2
(73 sequences) loci, respectively. We observed distinct phylogenetic
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clusters separated from each other by molecular distances. The results
confirm that our tested species can be identified in their respective
clades using COI and ITS2. We have also evaluated intra- and interspe-
cific evolutionary distances of both loci (COI & ITS-2), based on the mean
pairwise distance using the Kimura-2-parameter (K2P) distance model.
Variability and resolving power was observed in the case of both loci;
however, the ITS-2 locus has high discriminative capability based on
intra- and interspecies distances for identifying Trichogramma species
as compared to the COI locus.
Investigating the floral preferences of pollinating insects using
pollen DNA metabarcoding
Natasha de Vere,
1,4
Andrew Lucas,
1,2
Jenny Hawkins,
1,3
Col Ford,
1
Tegan Gilmore,
1
Abi Lowe,
1
Jake Moscrop,
1
Laura Jones,
1
John Warren,
1,4
and Matt Hegarty
1,4
1National Botanic Garden of Wales, UK.
2Swansea University, UK.
3Cardiff University, UK.
4Aberystwyth University, UK.
Corresponding author: Natasha de Vere (e-mail: natasha.devere@gardenofwales.org.uk).
Background: Insect pollination is a key regulating ecosystem service;
so there is considerable concern worldwide about declines in both
wild pollinators and honey bees due to habitat loss, agricultural in-
tensification, pests, disease, and climate change. Understanding the
reasons for declines and providing conservation guidance requires
detailed information on the habitat requirements and foraging pref-
erences of pollinating insects. Pollinator foraging can be tracked using
pollen DNA metabarcoding. Pollen is retrieved from the bodies of
insects, from pollen loads, or honey. DNA is extracted, amplified using
the rbcL and ITS2 DNA barcode markers, and sequenced using the
Illumina MiSeq platform. Key to the ability to identify unknown DNA
sequences is a comprehensive DNA barcode reference library. We
have DNA barcoded all of the native flowering plants of the UK along
with non-native, horticultural, and agricultural plants important for
pollinators. Results: We present three case studies. 1: Honey bee for-
aging has been assessed by characterising the floral composition of
honey collected throughout the UK. This provides a general overview
of commonly used plant species. 2: We extend this to investigate
honey bee floral preferences in a spatial and temporal context for
hives located within the National Botanic Garden of Wales. Plant spe-
cies composition of pollen loads and honey has been identified
throughout the year. We relate the plants used to those available for
foraging using flower maps of the entire botanic garden created from
ground and UAV surveys. 3: Hoverfly foraging has been investigated by
sampling a range of species from grassland communities to compare
floral constancy across individuals, species, and sites. Significance:
Pollen DNA metabarcoding provides a powerful method for tracking
pollinator foraging and can be used to assess floral preferences, hab-
itat requirements, and nutritional needs. This can be used to help
maintain resilient wild and agricultural ecosystems.
Identification of Belgian mosquito species (Diptera: Culicidae)
by DNA barcoding
V. Versteirt,
1,2
Z.T. Nagy,
3
P. Roelants,
1
L. Denis,
1
F.C. Breman,
4
N. Smitz,
4,5
D. Damiens,
6
W. Dekoninck,
3
T. Backeljau,
3,7
and M. Coosemans
1,7
1Institute of Tropical Medicine, Belgium.
2Avia-GIS, Belgium.
3Royal Belgian Institute of Natural Sciences, Belgium.
4Royal Museum for Central Africa, Belgium.
5University of Liège, Belgium.
6Universite Catholique de Louvain, Belgium.
7University of Antwerp, Belgium.
Corresponding author: N. Smitz (e-mail: nathalie.smitz@africamuseum.be).
Background: Since its introduction in 2003, DNA barcoding has
proven to be a promising method for the identification of many taxa,
including mosquitoes (Diptera: Culicidae). Many mosquito species are
potential vectors of pathogens, and correct identification in all life
stages is essential for effective mosquito monitoring and control. To
use DNA barcoding for species identification, a reliable and compre-
hensive reference database of verified DNA sequences is required.
Results: Genetic diversity of mosquitoes in Belgium was assessed us-
ing a 658-bp fragment of the mitochondrial cytochrome c oxidase
subunit I (COI) gene (N= 260), and a reference dataset was established
including specimens from the Aedes (147), Anopheles (30), Coquillettidia (15),
Culex (43) and Culiseta (25) genera. Most species appeared as well-
supported clusters. Intraspecific Kimura 2-parameter (K2P) distances
averaged 0.7%, and the maximum observed K2P distance was 6.2% for
Aedes koreicus. A small overlap between intra- and interspecific K2P
distances for congeneric sequences was observed. Overall, the identi-
fication success using best match and the best close match criteria
were high, above 98%. No clear genetic division was found between
the closely related species Aedes annulipes and Aedes cantans, which can
be confused using morphological identification only. The members of
the Anopheles maculipennis complex, that is Anopheles maculipennis s.s.
and An. messeae, were weakly supported as monophyletic taxa.
Significance: This study demonstrated that DNA barcoding offers a
reliable framework for mosquito species identification in Belgium,
with the exception of two pairs of closely related species.
Identifying forensic species of Diptera in southeast Brazil using
DNA barcodes
Pablo Viana Oliveira,
1
Natan Silva Matos,
1
Bárbara de Freitas Freire,
2
Janyra Oliveira-Costa,
3
Greiciane Gaburro Paneto,
12
and Francisco de Paula Careta1
4
1Federal University of Espirito Santo, Departamento de Farmácia e Nutrição, Alegre, ES, Brasil, Alto
Universitário s/n, Guararema, Alegre, ES, Brazil.
2Federal University of Espirito Santo, Programa de Pós-graduação em Biotecnologia, Vitória, ES, Brasil,
Alto Universitário s/n, Guararema, Alegre, ES, Brazil.
3Criminalistical Institute Carlos Éboli, Instituto de Criminalística Carlos Éboli, Departamento de Polícia
Técnico-Científica do Rio de Janeiro, RJ, Brasil, Rua Pedro I, 28, Centro, Rio de Janeiro, RJ, Brazil.
4Federal University of Espirito Santo, Programa de Pós-graduação em Ciências Veterinárias, Alegre, ES,
Brasil, Alto Universitário s/n, Guararema, Alegre, ES, Brazil.
Corresponding author: Francisco de Paula Careta (e-mail: franciscopcareta@gmail.com).
Background: Forensic entomology is the study of insects and other
arthropods to elucidate legal issues. Accurate insect specimen identi-
fication is usually a crucial first step in a forensic entomological anal-
ysis. It is traditionally done by morphological determination using
identification keys. However, due to problems identifying animal
specimens based on their morphology alone, new methods have been
developed, including species identification by DNA barcodes. The ob-
jective of this work was to identify forensically important species of
Diptera in southeast Brazil using DNA barcodes. For this, adult flies
were collected in Espirito Santo State, in the southeast region of Bra-
zil. After DNA extraction, a fragment of the cytochrome c oxidase
subunit I (COI) gene was PCR amplified and sequenced using a pair of
universal primers. All sequences were matched (>98%) to the BOLD
platform (Barcode of Life Data Systems) and alternatively to GenBank
using MegaBLAST. Results: In this study, 281 adult flies were col-
lected and identified morphologically according to characteristics
observed in an identification key. Of these, 36% were identified as Cal-
liphoridae, 34% as Muscidae, and 30% as Sarcophagidae. Approximately
10% of all collected samples were DNA barcoded. It was possible to iden-
tify, through the DNA barcode matches, Atherigona orientalis,Chrysomya
albiceps,Chrysomya megacephala,Musca domestica,Peckia chrysostoma, and
Oxysarcodexia thornax. However, barcode-based identifications to the spe-
cies level were achieved for only 35.7% of tested samples, which is prob-
ably due to the absence of the remaining species in these public
databases. Significance: Species detected in this study corroborate re-
sults from other authors who demonstrated the occurrence of these spe-
cies in the Atlantic Forest region of southeastern Brazil. Greater effort
should be directed towards creating publicly available reference libraries
of DNA barcodes for dipterans, which will allow the use of this identifi-
cation technique for routine forensic applications.
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Phylogeographic structure of Dynamene edwardsi (Crustacea:
Isopoda) matches remarkably the sequential genesis of the
Macaronesian islands
Pedro Emanuel Ferreira dos Reis Vieira,
1
Nuno Gomes,
2
David M. Holdich,
3
Henrique Queiroga,
1
and Filipe O. Costa
2
1Departamento de Biologia and CESAM - Centro de Estudos do Ambiente e do Mar, Universidade de
Aveiro, Campus Universitário de Santiago 3810-193 Aveiro, Portugal.
2CBMA - Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho,
Campus Gualtar 4710-057 Braga, Portugal.
3Aquatic Consultant, Keyworth, Nottinghamshire, England.
Corresponding author: Pedro Emanuel Ferreira dos Reis Vieira (e-mail:
pedroefrvieira@gmail.com).
Background: Although Dynamene is a species-poor genus of sphaero-
matid isopods, members of this genus are abundant in rocky shores of
the Northeast Atlantic, where they are represented by three species:
Dynamene bidentata,Dynamene edwardsi, and Dynamene magnitorata. Be-
cause isopods lack larval stages, and therefore have limited dispersal
ability, their populations are presumed more susceptible to isolation.
We examined DNA barcode diversity in this group, in rigorously iden-
tified specimens collected along European and Moroccan Atlantic
rocky shores, including the British Isles and Macaronesia. Results: As
expected, cytochrome c oxidase subunit I (COI-5P) DNA barcodes
clearly discriminated between the three monophyletic species clus-
ters (average distance of 24%), confirming their morphology-based
identifications. However, within D. edwardsi, four deeply divergent
lineages were present, displaying genetic distances between 15% and
22%, hence strongly suggesting the existence of a cryptic species com-
plex. Sequences of the 18S rRNA nuclear gene essentially confirmed
the complete sorting among the four lineages, although genetic dis-
tances were much lower. The most salient finding, however, was that
populations within a lineage were not grouped by geographic vicinity,
but instead by the known island emergence timing. For example,
populations as geographically close as Porto Santo and Madeira split
into two lineages displaying as much as 22% genetic distance. Upon
phylogenetic reconstruction, lineage branching depth matched
closely the sequential time of island emergence, with the Porto Santo/
continental Portugal branching most deeply, followed by Morocco/
Gran Canaria and finally Madeira/La Palma. Significance: DNA
barcodes unravelled a complex of three probable cryptic species
within D. edwardsi and a remarkable match between the sequential
genesis of the Macaronesian islands and the phylogeographic struc-
ture of this passively dispersed marine invertebrate. These findings
are highly significant for the investigation of comparative patterns of
evolution and speciation of marine invertebrates in Macaronesia and
contribute to the understanding of speciation processes in the marine
environment.
Pattern of nucleotide variations in the standard DNA
barcode loci in different genera of Indian Zingiberaceae
M.R. Vinitha,
1
U. Suresh Kumar,
1
M. Sabu,
2
and George Thomas
1
1Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Poojappura, Thiruvananthapuram, Kerala
695014, India.
2Department of Botany, University of Calicut, Calicut University P.O. 673 635, Kerala, India.
Corresponding author: M.R. Vinitha (e-mail: vinithamr@rgcb.res.in).
Background: A taxon-based sampling strategy, which evaluates
the potential of discriminating conspecific from congeneric indi-
viduals based on barcode sequences, has great applications for sam-
ple identification in Zingiberaceae in the pharmaceutical industry
since rhizomes, which harbour poor taxonomic descriptors, are the
medicinal source in many species. In view of the difficulty of iden-
tifying universal barcode loci for plants, attempts to sort barcode-
amenable and non-amenable genera in industrially important
families would help to design strategies for barcode applications
and to understand the evolutionary events that dampen barcode
success in a family. Results: We analyzed 262 accessions sampled
from 73 species belonging to 13 genera of family Zingiberaceae
represented in India using Atpf, rbcL, and ITS. Direct PCR sequenc-
ing retrieved good quality rbcL sequences in all accessions, Atpf
sequences in 249 accessions, and ITS sequence only in 120 acces-
sions. A total of 493 ITS sequences were retrieved additionally by
cloning the amplicons in 70 representative accessions. In phyloge-
netic analysis, chloroplast loci resolved all 13 genera (100%),
whereas ITS resolved only 11 genera (84%). At the species level,
resolution declined to 55.5% with respect to chloroplast loci and to
50.6% by ITS due to the occurrence of paraphyly in four genera:
Curcuma,Hedychium,Alpinia, and Amomum, rendering the barcode
applications in only 9 of the 13 genera examined. Further, the
analyses indicate that the dampening of barcode success observed
in some genera may be the result of recent genome duplication
events coupled with incomplete lineage sorting. Significance: The
study highlights the possible refinement in the identification of
taxonomically-recalcitrant rhizome materials in Zingiberaceae by a
barcode-based stratification approach from genera to species. Fur-
ther, the strategy for retrieving alternate barcode loci in Zingiber-
aceae should take into consideration the genomic disturbances
normally caused by genome duplication events in a taxa.
Comprehensive phylogeographic assessments as a tool to
understand and protect biodiversity on islands
Raluca Voda,
1
Leonardo Dapporto,
2
Vlad Dinca,
3
Tim Shreeve,
2
Mourad Khaldi,
4
Ghania Barech,
4
Khellaf Rebbas,
4
Paul Sammut,
Stefano Scalercio,
5
and Paul D.N. Hebert
3
1Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Maritim de la Barceloneta 37,
08003, Barcelona, Spain.
2Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, OX3
0BP, UK.
3Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada.
4Université Mohamed Boudiaf de M'sila, 28000 M'sila, Algeria.
5Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Unita
`di Ricerca per la
Selvicoltura in Ambiente Mediterraneo c.da Li Rocchi, I-87036 Rende (CS), Italy.
Corresponding author: Vlad Dinca (e-mail: vdinca@uoguelph.ca).
Background: Islands possess varied histories and geographical and
ecological settings, and they are exposed to different stochastic
events. Because of this complexity, the mechanisms determining and
maintaining species assemblages on islands are poorly understood,
and analyses of entire taxonomic groups of sufficiently large areas are
necessary to disentangle the array of factors that determine the com-
position and genetic attributes of island communities. We use com-
parative analyses linking community and phylogenetic approaches
for the butterfly fauna of the circum-Sicilian islands, a key intercon-
tinental region in the western Mediterranean, to understand the
factors that shaped the observed assemblages and to highlight pop-
ulations of exceptional conservation value. Results: Species richness
was mainly influenced by contemporary factors, but Pleistocene con-
nections also had a significant impact. A nested pattern was detected
when all species were considered, but this pattern disappeared when
they were divided into “widespread” and “uncommon” taxa. The fre-
quency of a species on the mainland was a weak predictor of its
frequency on islands, and most residuals appeared related to migra-
tory or sedentary behavior. DNA barcoding of more than 80% of the
butterfly species recorded in the region revealed that Europe and
north Africa formed two well-differentiated genetic groups and that
islands showed a high similarity with the mainland they were for-
merly connected to during Pleistocene low sea levels. Genetically di-
versified lineages were most frequent in “uncommon” species, while
“widespread” species were genetically homogeneous. Significance:
This study used an unprecedented integrative approach to examine
the biogeography of an entire superfamily in a complex biogeograph-
ical contact zone. For the first time, it was possible to recognize the
processes responsible for the observed species assemblages and to
highlight the uniqueness of each island community. These patterns
also revealed the value of assessing morphospecies coupled with in-
formation on intraspecific genetic diversity to obtain the information
needed for accurate conservation decisions.
Abstracts 293
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Species from feces: reliably identifying global bat species with a
DNA mini-barcode assay
Faith M. Walker,
1
Charles H.D. Williamson,
2
Colin J. Sobek,
1
Dan E. Sanchez,
1
and Carol L. Chambers
1
1Bat Ecology and Genetics Laboratory, School of Forestry and Center for Microbial Genetics and
Genomics, Northern Arizona University, Bldg. 56, 3rd floor, 1298 S Knoles Dr., Flagstaff, AZ 86011-4073,
USA.
2Center for Microbial Genetics and Genomics, Northern Arizona University, Bldg. 56, 3rd Floor, 1298 S
Knoles Dr., Flagstaff, AZ 86011-4073, USA.
Corresponding author: Colin J. Sobek (e-mail: Colin.Sobek@nau.edu).
Background: Bat guano is a relatively untapped reservoir of informa-
tion, with great utility as a DNA source because it is abundant in caves
and mines even when bats are not present, and it is stationary and
easy to collect. Three technologies have come of age that, together,
enable species identification from guano: reliable DNA typing from
feces, DNA barcoding (species-specific genetic identifiers), and bioinfor-
matic analysis. Taking advantage of these advances, we used 1.6 million
sequences to develop a DNA mini-barcode assay that targets a segment
of the mitochondrial cytochrome c oxidase subunit I gene, and that
we have found to be highly discriminatory among Chiroptera glob-
ally, readily accommodates fecal DNA, and selectively targets bat but
not prey DNA. Results: Our assay has high resolution (93%) for bar-
coded bat species; we have successfully validated it from the feces of
25 bat species (e.g., identification of Myotis septentrionalis,Eptesicus
fuscus,Corynorhinus townsendii) with aged fecal pellets (up to 3 months old)
and individual and pooled guano pellets, such that questions can
target individuals (using specific fecal pellets) or populations and com-
munities (long-term roost sites). Another benefit of our Species from
Feces tool is in confirming field identification, especially of morpho-
logically similar species. In several instances, our genetic approach
revealed misidentification of mist-netted species. We have developed
a searchable website (http://nau.edu/CEFNS/Forestry/Research/Bats/
Search-Tool/) that allows users to determine the discriminatory power
of our markers for bat species that interest them. Significance: Al-
though our Species from Feces tool has immediate application in the
US, where bats are under threat from White-Nose Syndrome, it is also
a potentially powerful application worldwide, for example, in deter-
mining the presence of bat species that are vulnerable or facing ex-
tinction.
Using barcoding to analyse prey consumption by generalist
predators in rice ecosystems
Xue-Qin Wang,
1
Guang-Hua Wang,
1
Zeng-Rong Zhu,
1
Hongye Li,
2
Kong Luen Heong,
1
and Jiaan Cheng
1
1Institute of Insect Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
2College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China.
Corresponding author: Xue-Qin Wang (e-mail: xqwang@zju.edu.cn).
Background: Food web studies can provide vital information for
ecologists to understand ecosystems. A detailed overview of species
interactions and dependencies in an ecosystem can help monitor
changes in functional relationships. The identification of ecosys-
tem players to the species level, especially arthropods collected
from fields, is a major task. DNA barcoding can potentially help in
the identification task and can be an important tool for rapid spec-
imen identification. In this study, arthropods (pests, predators,
parasitoids, neutral insects) were collected from rice paddies of
Zhejiang province, China. Standard DNA barcoding using the mi-
tochondrial cytochrome C oxidase subunit I gene (COI) was first
performed to establish a reference barcode library. We further
designed two pairs of primers for amplifying short sequences to
enable detection of partially digested prey species. We then used
Ion Torrent Amplicon sequencing to analyse the DNA extracted
from the whole body of four spider species. Using the DNA barcode
library, we determined the prey species composition in each spider
sample. We assume that DNA sequences besides those of the spi-
ders were those of the prey species. Results: We established a
reference barcode library including 356 records representing
140 species in 45 families. Based on the library, we found that the
average interspecies Kimura 2-parameter (K2P) genetic distance
was 0.304 (0.004–0.474) and intraspecies distance was 0.004 (0.000–
0.033). The average genetic distance was 75-fold higher between
species than within species. Using the two pairs of primers, we
could amplify 85.0% and 93.8% of the species represented in our COI
barcode vector library that was constructed for the evaluation. We
are now using the second pair for sequencing. Significance: This
paper describes the application of DNA barcoding to analyse food
web relationships among arthropods in agricultural fields, through
sampling and extracting the DNA within predator species.
DNA metabarcoding of marine hard-bottom communities
using 18S and COI
Owen S. Wangensteen,
1
Magdalena Guardiola,
1
Creu Palacín,
2
and Xavier Turon
1
1Center for Advanced Studies of Blanes (CEAB -CSIC), Carrer Accés Cala Sant Francesc, 14. 17300 Blanes
(Girona), Spain.
2University of Barcelona, Department of Animal Biology, Av. Diagonal, 643. 28028. Barcelona, Spain.
Corresponding author: Owen S. Wangensteen (e-mail: owenwangensteen@gmail.com).
Background: The use of DNA metabarcoding is progressively becom-
ing commonplace for characterizing eukaryotic diversity in relatively
homogeneous communities—such as soils or soft-bottom sediments—
but its implementation for characterizing macroscopically com-
plex marine hard-bottom communities has been little explored. We
applied a metabarcoding approach to assess the eukaryotic diversity
of two marine reserves: Islas Atlanticas (NE Atlantic) and Cabrera (W
Mediterranean). We studied three types of hard-bottom communities
in each location, including photophilous algal communities (with or
without invasive algae), sciaphilous communities, and maërl bottoms.
We separated each sample into three different size fractions that were
homogenized using a blender. We extracted total DNA from each
fraction and amplified two different metabarcoding markers, consist-
ing of fragments of the 18S rRNA and cytochrome c oxidase subunit I
(COI) genes. A Bayesian clustering algorithm was used to define mo-
lecular operational taxonomic units (MOTUs). Results: We proved
that DNA metabarcoding can be used successfully for simultaneously
detecting the presence and assessing the relative biomass of thou-
sands of MOTUs from complex hard-bottom communities. Compari-
son of 18S and COI in the same set of samples exposed the pros and
cons of each marker. The higher variability of COI allowed better
resolution, yielding a higher number of MOTUs than 18S. However,
taxonomic placement proved to be more difficult using COI, even
though our reference database for COI (based on the Barcode of Life
Data Systems - BOLD) included five times as many sequences as our
reference database for 18S. This highlights the need for improving
reference databases for accurate taxonomic assignment of metabarcoding-
derived sequences. Significance: We extended the application of the
metabarcoding approach to complex marine hard substrates, allow-
ing a deeper characterization of biodiversity than morphology or in-
dividual barcoding approaches. A significant improvement in marine
COI-barcoding databases is needed in order to get enhanced auto-
mated taxonomic inventories of marine communities.
Barcoding the fishes of Australia—progress, uses, and
lessons learnt
Robert D. Ward
CSIRO Oceans and Atmosphere Flagship, GPO Box 1538, Hobart, Tasmania 7001, Australia.
E-mail for correspondence: bob.ward@csiro.au.
Background: John White in 1790 was the first to describe fishes actu-
ally caught in Australian waters, and he described eight species. Now,
nearly 5000 species have been recorded from Australia, the vast ma-
jority marine, and the number is increasing monthly. DNA barcoding
this large fauna started in 2004, and remains far from complete.
Results: Currently around 10 000 specimens have been COI barcoded,
comprising about 2700 (⬃55%) of the 5000 species. Some 53% of the
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numerous bony fish species have been barcoded, and 80% of the much
less numerous elasmobranchs. Significance: In this talk I will sum-
marise where we are at and where the major gaps in our knowledge
are to be found. I will also summarise some of uses made of our
reference libraries in Australia (for example, in identifying shark fins,
in helping to describe foodwebs, in uncovering cryptic species, and in
identifying market products) and some of the lessons learnt (for ex-
ample, in the difficulties of making correct initial identifications).
DNA barcoding the plants of San Diego County, California: on
the verge of the first complete DNA barcode reference library
for a globally important regional flora
Connor P.K. Warne,
1
Stephanie L. deWaard,
1
Joshua R. Kohn,
2
Jon P. Rebman,
3
Maria L. Kuzmina,
1
and Bradley A. Zlotnick
4
1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1,
Canada.
2University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, USA.
3San Diego Natural History Museum, 1788 El Prado, San Diego, CA 92101, USA.
4San Diego Barcode of Life, San Diego, CA, USA.
Corresponding author: Connor P.K. Warne (e-mail: cwarne@uoguelph.ca).
Background: San Diego County is a hotspot of global biodiversity
within the similarly designated California Floristic Province. It holds
the greatest documented plant diversity of any county in the contig-
uous United States: more than 2600 taxa, approximately one-half as
many plant species as Canada. The vouchered Plant Atlas of the San
Diego Natural History Museum (SDNHM) previously consolidated
knowledge of species distributions, geo-referencing specimens of
each plant species within the County (http://www.sdplantatlas.org/).
We aimed to enhance the Plant Atlas by assembling DNA barcodes and
high-resolution images for all San Diego Country plant species, dem-
onstrating the feasibility of DNA barcoding specimens. Results: A
technician sampled 2619 specimens representing 2579 species in
17 days from the San Diego Synoptic Collection of Plants. Three loci
(rbcLa, ITS2, Atpf) were sequenced with 71.9%, 60.6%, and 52.0% spec-
imen and 71.8%, 60.4%, and 51.7% species success, respectively, reflect-
ing results consistent with previous barcoding projects on herbaria
specimens. One gene locus was recovered from a specimen that was
131 years old (rbcLa), and another that was 91 years old yielded se-
quences for all three loci. Cumulative barcode information was used
to construct a phylogeny for 2146 species of the plants of San Diego
County, supplemented by concurrently digitized images of each
taxon. Significance: We carried out the rapid assembly of a barcode
library for a globally important flora from a biodiversity hotspot,
adding value to the San Diego County Plant Atlas, and providing the
first genetic information for many of its taxa. SDNHM reference bar-
codes are being used for dietary analysis of regional herbivores and to
test phylogeny and community assembly of this diverse flora. This
success in DNA barcodes from the SDNHM herbaria has motivated
plans to barcode animal collections at the same museum to provide a
comprehensive San Diego Barcode of Life reference library.
A market survey of fish species substitutions in the United
States to advance seafood traceability requirements
Kimberly A. Warner,
1
Walker Timme,
2
Beth Lowell,
1
Michael Hirshfield,
1
and Robert Hanner
3
1Oceana, 1350 Connecticut Ave. NW, 5th Floor, Washington, DC 20036, USA.
2District of Columbia Public Schools, Washington, DC, USA.
3Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
Corresponding author: Kimberly A. Warner (e-mail: kwarner@oceana.org).
Background: Oceana, an international ocean conservation organi-
zation, launched a campaign in 2011 to expose the problem of
seafood fraud and to advocate for full supply chain traceability,
legal sourcing, and better consumer labeling for all seafood sold in
the United States. As part of this campaign, Oceana collected over
1200 seafood samples from 674 grocery stores and restaurants in
14 metropolitan areas in the US from 2010–2012 to determine if they
were properly labeled according to U.S. Food and Drug Administra-
tion guidelines. Most (95%) of the sample species identities were
determined by the Canadian Centre for DNA Barcoding and the
remainder by other genetic methods. Results: One third (410/1215)
of the samples were mislabeled, revealing a wide range of species
substitutions in 27 out of the 46 fish types collected. Species sub-
stitutions included those of lesser economic value and those posing
health and conservation risks. The fraction of fish mislabeled var-
ied according to the type of fish collected and type of retail outlet
where fish samples were purchased. Significance: Wide dissemi-
nation of survey findings helped raise awareness of the pervasive
problem of seafood fraud nationally and globally. Oceana's contin-
ued advocacy has contributed to advancing policy directives in the
US, including increased seafood documentation requirements,
traceability, and other measures.
Classifying DNA barcode multi-locus sequences with feature
vectors and supervised approaches
Emanuel Weitschek,
1
Giulia Fiscon,
2
Paola Bertolazzi,
1
and Giovanni Felici
1
1Institute of Systems Analysis and Computer Science – CNR, Via dei Taurini 19, Rome, Italy.
2Department of Computer, Control, and Management Engineering (DIAG), Sapienza University, Viale
Ariosto 25, Rome, Italy.
Corresponding author: Emanuel Weitschek (e-mail: emanuel@iasi.cnr.it).
Background: Due to the overwhelming increase in multi-locus DNA
barcode data provided by taxonomists and field scientists, sequence
analysis techniques have been widely developed to effectively com-
pare multi-locus sequences. On the one hand, traditional alignment-
based methods are time-consuming and cannot be used for the
analysis of non-alignable, multi-locus sequences. On the other hand,
alignment-free algorithms allow for the establishment of similarity
between biological sequences based on the counts of fixed-length sub-
strings (k-mers) and have proved successful in many applications,
including multi-locus DNA barcode analysis. Alignment-free algo-
rithms rely on counting and comparing the frequency of all the dis-
tinct k-mers that occur in the considered sequences. Results: Here,
we present LAF (Logic Alignment Free), a method that combines
alignment-free techniques and rule-based classifiers in order to assign
multi-locus DNA barcode sequences to their corresponding species.
LAF looks for a minimal subset of k-mers whose relative frequencies
are used to build the classification models as disjunctive-normal-form
logic formulas (“if-then rules”, e.g., “if the frequency of AACT>0.03,
then the species of the sequence is Mycena pura”). Significance: We
successfully applied LAF to the classification of DNA barcode se-
quences belonging to the plant and fungus kingdoms. In particular,
focusing our analysis on multi-locus barcode samples, we succeeded
in obtaining reliable classification performances at different taxo-
nomic levels by extracting a handful of rules.
Barcoding forensic traces—practical challenges
Monique Wesselink and Irene Kuiper
Netherlands Forensic Institute, Laan van Ypenburg 6, 2497 GB The Hague, the Netherlands.
Corresponding author: Monique Wesselink (e-mail: m.wesselink@nfi.minvenj.nl).
Background: Taxonomical identification of seized items is often
needed to determine whether a crime has been committed. When
seized items have been processed into art works, medicines, or other
objects, insufficient morphological identification characteristics are
often present; therefore, DNA-based techniques are increasingly be-
ing used. From a forensic point of view, the (local and international)
legislation determines the level of identification needed. In some
cases, only determination of the family or genus is necessary, but in
other cases, species, subspecies, or even population determination
may be required. Results: As legal definitions determine which level
of identification is needed, barcoding markers may be (i) applicable,
(ii) insufficient, or (iii) too informative. To benefit from barcoding
initiatives, most techniques for mammalian species identification are
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based on the mitochondrial marker COI. Many seized items can be
readily identified through use of (parts of) this marker, excluding all
but one species as the source of the DNA. Drawbacks include the
inability to detect hybrids due to the marker's maternal inheritance,
the fact that not all species are readily distinguished, and that subspe-
cies and population differentiation generally requires the use of other
techniques. Nuclear DNA markers, additional mitochondrial DNA
markers, and next-generation sequencing may be needed to overcome
these challenges. Significance: Although DNA-based techniques may
be the only methods to identify seized samples, the maternal inheri-
tance of mitochondrial DNA and inability to distinguish between spe-
cies and their hybrids has been pointed out as a potential pitfall in a
court of law. In our experience, some local and international flora and
fauna regulations are not at all hampered by nuances at the species/
hybrid level (e.g., when genera/families/orders are regulated), but in
other cases (e.g., distinction between species and their hybrids) mito-
chondrial DNA markers alone cannot be used to answer the relevant
forensic questions.
Barcoding New Zealand spiders
Annie West,
1
Nigel Binks,
1
Cor Vink,
2
Bryce McQuillan,
1
and Ian Hogg
1
1University of Waikato, School of Science, Private Bag 3105, Hamilton, New Zealand.
2Canterbury Museum, Christchurch, New Zealand.
Corresponding author: Ian Hogg (e-mail: hogg@waikato.ac.nz).
Background: New Zealand is home to an estimated 2000 species of spi-
der, although only 1134 species are presently described, and many can
only be assigned to the family level. Furthermore, morphological identi-
fication is complicated by sexual dimorphism as well as individual vari-
ation related to age and environmental factors. With 95% of species
thought to be endemic, accurate identification of New Zealand's spiders
will require the robust coverage of taxa envisaged as part of this research
project. Results: A total of 759 specimens have been processed to date. Of
these, 685 COI sequences were obtained covering roughly 130 known
species including 155 BINs. Several individuals could not be identified to
the species level, based on available taxonomic literature. In most cases,
within-species sequence divergence was <1%, whereas divergences
amongst known species were >2%. COI sequences also helped to resolve
confusion within the genera Badumna,Eriophora,Steatoda,Sidymella, and
Stiphidion, which all showed phenotypic plasticity. Several examples of
potential cryptic species were also highlighted. For example, sequence
divergences (>3%) for North and South Island specimens of Porrhothele
antipodiana suggested possible sibling species. Significance: These data
demonstrated the utility of COI sequences for the routine identification
of New Zealand spiders. Here, we have begun the assembly of a COI
library for New Zealand spiders, which will ultimately assist in the accu-
rate assessment of diversity within this taxon.
Urban biodiversity explored using intensive multi-year
sampling of insects in Los Angeles
Regina Wetzer and Brian Brown
Natural History Museum of Los Angeles, 900 Exposition Blvd., Los Angeles, CA 90007, USA.
Corresponding author: Regina Wetzer (e-mail: rwetzer@nhm.org).
Background: The Natural History Museum of Los Angeles County
(NHM) is engaged in a large-scale research study of urban biodiversity
in the Los Angeles area (part of the California Floristic Province, a
biodiversity hotspot). The Biodiversity Science: City and Nature (Bio-
SCAN) project is based on sampling insects weekly in an array of
30 localities for one year. Ultimately, the goal is to combine the biological
diversity data, physical measurements, and landscape parameters
into models that accurately predict the site-by-site diversity across
space and through time. Each site (mostly residents' backyards) has a
continuously operating insect trap (Malaise trap) with insects cap-
tured directly into 95% ethanol. Each trap is accompanied by a micro-
climate weather station and augmented with landscape information.
Sites range from downtown Los Angeles to less-urban areas near Grif-
fith Park. Results: Early results of the project have leveraged from the
enormous number of specimens in target taxa (specifically flies in the
family Phoridae). Detailed sorting of only a few months of samples has
already yielded dozens of completely new species in the single fly
genus Megaselia. Though intriguing, the results from morphological
work cannot possibly reflect the entire diversity of the insect samples
due to the impracticality of sorting and identifying the immense di-
versity in thousands of weekly samples. Significance: Whole-sample
molecular approaches are essential for addressing the extraordinary
diversity of small metazoan populations, enabling comparisons be-
tween samples across space and through time. Next-generation se-
quencing of entire unsorted samples, targeting barcode (or other)
genes, is the most promising route for characterizing the full multi-
taxon biodiversity in each sample. The significance of this approach is
particularly high in studies of urban biodiversity. In that domain,
mechanistic understandings of the effects of urbanization will un-
avoidably be based on quantitative approaches involving hyperdi-
verse small metazoans.
Diversity and human perceptions of bees in Southeast
Asian megacities
John-James Wilson,
1
Kong-Wah Sing,
1
Ping-Shin Lee,
1
Wen-Zhi Wang,
2
Zong-Xu Lee,
2
Xing Chen,
2
and Tao Wan
3
1Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia.
2Southern China DNA Barcoding Center, Kunming Institute of Zoology, Chinese Academy of Sciences,
Kunming, Yunnan 650223, China.
3Shenzhen Fairy Lake Botanical Garden, Chinese Academy of Sciences, Shenzhen, Guangdong 518004,
China.
Corresponding author: John-James Wilson (e-mail: wilso04@gmail.com).
Background: Rapid economic development has accelerated urbani-
sation and biodiversity loss in Southeast Asia. Yet, in urban areas, bees
retain important ecosystem services, e.g., pollination of garden plants
that can provide food for other animals. Furthermore, biodiversity in
urban ecosystems can enhance human well-being and is important
from a societal point of view—personal exposure to “nature” in every-
day life is a major determinant of sensitivity to environmental issues
and people's views on natural ecosystems. Without an understanding
of the diversity of bees in urbanisation hotspots we cannot predict
how future development will affect ecosystem functions provided by
bees. At the same time, basic information regarding perspectives of
Southeast Asia's urban community towards bees is essential for any
regional plan. Our first objective was to investigate the species rich-
ness and abundance of bees in four megacities in Southeast Asia—
Greater Bangkok, Kuala Lumpur (Klang Valley), Pearl River Delta
(Hong Kong/Shenzhen), Singapore (Singapore/Iskandar Malaysia)—
and their distribution from suburbs, through ornamental gardens, to
central business districts. Our second objective was to determine per-
spectives of the urban community regarding the presence of bees in
urban areas. Results: We conducted bee sampling and interviewed
locals at sites in the suburbs, ornamental gardens, and central busi-
ness districts. We used DNA barcoding to assess the species richness
and abundance at the sites. Preliminary analyses suggest species rich-
ness declined from the suburbs to the central business district; how-
ever, a few species were found in high abundance in central business
districts. Surprisingly, the presence of bees is largely unnoticed by the
human residents. Significance: This is the first regional study of bee
diversity in urban Southeast Asia and is unique in also examining
human perceptions. Education programs are necessary to challenge
both the generally negative perceptions towards bees and make urban
areas more bee friendly.
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Testing the waters: using NGS to monitor
zooplankton communities
Steve Woods,
1
Ian Hogg,
1
Ian Duggan,
1
Conrad Pilditch,
1
and Jonathan Banks
2
1University of Waikato, School of Science, Private Bag 3105, Hamilton, New Zealand.
2Cawthron Institute, 98 Halifax Street East, Nelson 7010, Private Bag 2, Nelson 7042, New Zealand.
Corresponding author: Steve Woods (e-mail: steve.woods00@gmail.com).
Background: A long-term study was conducted to assess the impacts
of common carp (Cyprinus carpio) on the zooplankton community. This
study showed wide variation in the zooplankton community through-
out the year. Our aim was to develop a more streamlined, DNA-based
approach to the routine monitoring of zooplankton communities and
to contrast these data with results from traditional morphological
assessments. Results: We obtained cytochrome c oxidase subunit I
(COI) and 28S sequences from a range of taxa. Unfortunately, the
diverse taxonomic range for zooplankton (rotifers to crustaceans)
meant that the current primers for the COI region were unable to
barcode all species successfully. As an interim measure, we shifted
focus to the D1 region of 28S, which had a much higher success rate.
An initial run using an Illumina MiSeq protocol found that in addition
to the zooplankton, phytoplankton were also being recovered. To
address this issue, capture probes (MYbaits) were used to select only
the zooplankton DNA in the sample. A further MiSeq run is currently
underway using the enriched sample material. Significance: Zoo-
plankton are currently used as indicators of lake trophic state. How-
ever, current techniques require taxonomists to sort each sample. A
sensitive genetic approach would speed up and potentially reduce
costs involved in monitoring. This approach could also be applied to
the detection of invasive species.
Evaluation of the relation between phytochemical
composition and genetic diversity in tropical plant species
using DNA barcodes
Samantha Jo Worthy,
1
Jennifer Cruse-Sanders,
2
Alex Reynolds,
3
Álvaro Pérez,
4
and Kevin S. Burgess
1
1Columbus State University, 4225 University Avenue, Columbus, GA 31907, USA.
2Atlanta Botanical Garden, 1345 Piedmont Avenue NE, Atlanta, GA 30309, USA.
3The Lovett School, 4075 Paces Ferry Rd NW, Atlanta, GA 30327, USA.
4Pontificia Universidad Católica del Ecuador, Mariscal Sucre, Quito, Ecuador.
Corresponding author: Samantha Jo Worthy (e-mail: worthy_samantha@columbusstate.edu).
Background: Amazonia is at the forefront of a conservation crisis
that includes the loss of tropical rainforest diversity, as well as a
cultural loss of indigenous knowledge, including the medicinal
uses of plants. While one of the fundamental goals of conservation
is to preserve genetic diversity within and among natural popula-
tions of potential socio-economic worth, very little is known about
the relation between genetic diversity and plants of medicinal
value. Medicinal plants typically have high levels of phytochemi-
cals that have evolved through a selective advantage to deter her-
bivores; the question remains as to whether selection for increased
phytochemicals has had a concomitant effect on the maintenance
of genetic diversity in tropical plant populations. Results: The goal
of this research was to investigate the relationship between the
genetic diversity of plant DNA barcodes and phytochemical com-
position in tropical plant taxa. To determine the relation between
genetic diversity and the presence/absence of phytochemicals previously
identified in Amazonian plant taxa, we evaluated the magnitude of rbcL
barcode diversity among 338 Amazonian tree sequences representing
47 families, 115 genera, and 195 species downloaded from GenBank. Specif-
ically, we evaluated the mean number of rbcL bp polymorphisms be-
tween plants of known medicinal value (high phytochemical content)
versus those of no known medicinal value. The average number of rbcL
bp polymorphisms was significantly higher (48.22) for medicinal plants
compared to non-medicinal plants (46.87). Significance: DNA barcoding
can be used as a potential predictive tool for the identification of tropical
plant species that contain medicinal phytochemicals. This research in-
creases the value of indigenous knowledge and aids in conservation ef-
forts to safeguard both cultural and biological diversity. The application
of these results to a long-term ethnobotanical research project that is
currently taking place at the Siempre Verde Preserve and Yasuní Re-
search Station, Ecuador will be discussed.
Assessing benthic macroinvertebrate temporal turnover in a
remote wetland through environmental barcoding
Michael Wright,
1
Donald J. Baird,
2
and Mehrdad Hajibabaei
1
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Environment Canada, Canadian Rivers Institute; Department of Biology, University of New Brunswick,
10 Bailey Drive, Fredericton, NB E3B 5A3, Canada.
Corresponding author: Michael Wright (e-mail: mwrigh06@uoguelph.ca).
Background: Benthic invertebrates are commonly used as biological
indicators of ecosystem change due to their sensitivity to environmen-
tal impacts and their ubiquity across aquatic systems. However, the
large numbers of individuals typically found within samples and the
large sample sizes within projects mean that a substantial amount of
time is required to sort and identify specimens. Environmental bar-
coding, or the next-generation sequencing of bulk environmental
samples for standard DNA markers (e.g., DNA barcodes), allows us to
rapidly identify a range of taxa with equal sequencing effort; however,
there are few examples of this technique highlighting temporal
changes in communities. The Peace-Athabasca Delta (PAD) is a dy-
namic, highly connected wetland system in northern Alberta that
hosts large numbers of migratory birds and some of the last free-
roaming wood buffalo. Because of its high diversity and international
importance, the PAD is an excellent study site to obtain baseline
measures of ecosystem change through environmental barcoding.
Results: As part of the Biomonitoring 2.0 project (www.biomonitoring2.
org), we sequenced two regions of the cytochrome c oxidase subunit I
(COI) gene from the bulk DNA extracts of 90 benthic samples col-
lected in June and August of 2012 and 2013, subsequently identify-
ing a range of invertebrate taxa including annelids, molluscs,
insects, and other arthropods. Through various measures of diver-
sity, our approach showed variation in communities at different
taxonomic levels from order to molecular operational taxonomic
units. Significance: Environmental barcoding is able to show tem-
poral shifts in benthic macroinvertebrate community assemblages,
which allows for the rapid assessment of wetlands without time
spent sorting and identifying specimens. When incorporated into a
biomonitoring program, this can allow for an increased focus on
sampling capacity. While there are still issues existing with refer-
ence database coverage, detecting abundance, and the destruction
of specimens during sample processing, environmental barcoding
has the potential to add information that traditional methods miss.
Barcoding wild edible mushrooms in southwestern China
Jianping Xu,
1
Ying Zhang,
2
Fei Mi,
2
Chunli Liu,
2
Yang Cao,
2
Xiaozhao Tang,
2
Xiaoxia He,
2
Pengfei Wang,
2
Dan Yang,
2
Jianyong Dong,
2
and Ke-Qin Zhang
2
1Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ont., Canada.
2Yunnan University, Kunming, Yunnan, China.
Corresponding author: Jianping Xu (e-mail: jpxu@mcmaster.ca).
Background: Wild edible mushrooms are important sources of nutri-
ents and income for many communities in both developed and devel-
oping countries. Southwestern China is a biodiversity hotspot and a
paradise for mushroom enthusiasts and connoisseurs. Wild mush-
rooms from that region are exported to many markets both within
and outside of China. However, the true biodiversity of wild edible
mushrooms from that region remains to be defined. Results: In this
study, we sampled the wild edible mushroom markets throughout
southwest China and obtained ITS sequences from 1908 samples rep-
resenting the major morphological types of wild edible mushrooms.
Our sequence analyses identified that these samples belonged to at
least 40 genera in 25 fungal families. Among the 1908 analyzed sam-
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ples, 952 (49.9%) were found to share >97% of ITS sequence identity to
the 68 closest-known species in databases; 179 (9.4%) shared 95%–
97% ITS sequence identity with the 25 closest-known species; 474 (24.8%)
shared 90%–95% ITS sequence identity with the 36 closest-known spe-
cies; and 303 (15.9%) shared <90% ITS sequence identity with the
35 closest-known species. Three genera—Lactarius,Ramaria, and
Russula—had the highest number of (putative) species in the wild
mushroom markets. Significance: Our results suggest a large cryptic
mushroom biodiversity in this region and that more than half of the
wild edible mushrooms in the markets belong to undescribed taxa.
Plant diversity assessments in tropical forests of SE Asia
Tetsukazu Yahara
Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
E-mail for correspondence: tet.yahara@gmail.com.
Tropical forests of SE Asia harbor extremely high and very unique
diversity of vascular plants. Unfortunately, this diversity has been
critically threatened by continuous forest loss since the 1950s, and we
urgently need to assess the status and trends of all the plant species in
tropical SE Asia and develop conservation plans for threatened spe-
cies. Responding to this need, in 2011, we started a project to visit
remaining forests in SE Asian countries and record all the vascular
plant species within a plot of 100m×5m.When we visited a moun-
tainous area, we placed some plots along elevational gradients. We
conducted this survey in ⬃100 plots. The specimens collected
amounted to ⬃20 000. For all of those specimens, we also collected
silica-gel dried samples for DNA barcoding. Many specimens we col-
lected are sterile, and thus identification is difficult. We overcame this
difficulty by determining sequences of rbcL and Atpf, with which we
could identify specimens usually to the level of genus. Then, we iden-
tified species using taxonomic literature and type specimens. This
identification work is still ongoing because it takes much time, espe-
cially in species-rich families. Our assessments revealed that (i) species
identification in forest plots with historical records are frequently
wrong, (ii) there remain many undescribed species within major tree
families (e.g., amounting to 30% in Lauraceae), and (iii) many species
are known only from a few localities and can be included in IUCN Red
List. Also, we found that some areas of Sumatra and southern Vietnam
harbor extremely high species richness comparable to northern Bor-
neo. So far, we have assessed as much as 10% of the total species in SE
Asia. A larger collaboration network is needed to record, assess, and
conserve all the vascular plant species in SE Asia.
Utilizing DNA barcoding to conserve Canada's endangered
populations of red mulberry (Morus rubra L., Moraceae)
Li-Jun Yan,
1
Scott Silvis,
2
Kevin S. Burgess,
2
Tammy Dobbie,
3
Lian-Ming Gao,
1
and De-Zhu Li
1
1Kunming Institute of Botany, CAS, 132# Lanhei Road, Heilongtan, Kunming 650201, Yunnan, China.
2Columbus State University, 4225 University Avenue, Columbus, GA, USA.
3Point Pelee National Park, 407 Monarch Lane, RR1, Leamington, ON N8H 3V4, Canada.
Corresponding author: Li-Jun Yan (e-mail: yanlijun@mail.kib.ac.cn).
Background: Red mulberry is a wind-pollinated tree species that is
distributed throughout eastern North America. In Canada, previous
studies show that hybridization with the introduced white mulberry
(M. alba L.) is leading to genetic assimilation of the remaining red
mulberry populations. Due to a lack of diagnostic morphological char-
acters, the confirmation of red, white, and hybrid mulberry remains
an elusive tool for the conservation management of this endangered
tree species. This is mostly due to costly, molecular-based tools that
are required to genetically identify red and white mulberry for pres-
ervation and removal, respectively. Results: In August of 2014, we
collected 61 putative red, white, and hybrid mulberry from two Na-
tional Park sites in southern Ontario, Canada: Middle Island and Point
Pelee. To circumvent costly and time-consuming sequence analysis to
evaluate the frequency of hybrids at each site, we developed species-
specific amplification (SS-PCR) of the nuclear ITS2 barcode region.
Using two primer pairs, we were able to obtain consistent SS-PCR
products that generated products of two sizes: 313 bp for M. rubra and
174 bp for M. alba. Hybrids yielded both products. Furthermore, we
found 17 samples (27.9%) were M. rubra, 21 (34.4%) were M. alba, and
23 (37.7%) were hybrids. To confirm these results as well as determine the
maternal parentage of hybrids, the rbcL barcode gene region was
sequenced for all samples. All species identifications were confirmed;
among the 23 samples identified as hybrids, 20 (87.0%) of them share
the red mulberry rbcL haplotype, while 3 (13%) share the rbcL haplo-
type with white mulberry. Significance: Collectively, our results not
only demonstrate an efficient use of DNA barcoding to identify the
red, white, and hybrid mulberries in Canada, but also confirm previ-
ous studies that suggest hybridization between red and white mul-
berry is bidirectional but asymmetric, and likely continues to be a
threat to remaining populations.
DNA barcoding database for Chinese pharmacopoeia
Hui Yao,
1
Jingyuan Song,
1
Jianping Han,
1
Linchun Shi,
1
Wei Sun,
2
and Shilin Chen
2
1Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, No. 151 Malianwa North Road, Haidian District, Beijing 100193, China.
2Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences No. 16, Nanxiao
Street, Dongzhimen, Dongcheng District, Beijing Municipality, China.
Corresponding author: Hui Yao (e-mail: scauyaoh@sina.com).
Background: In recent years, traditional identification methods (botan-
ical source and morphological identification, microscopic identification,
and physiochemical identification) have been unable to meet the de-
mands of rapid development in China's pharmaceutical industry. DNA
barcoding technology emerged in 2003 and revolutionized the available
identification methods. Our team proposed and verified that nuclear
ITS2 is suitable to identify traditional Chinese medicines (TCMs). A novel
identification system for Chinese herbal medicines in Chinese Pharma-
copoeia (ChP), which employs DNA barcodes, was set up and has been
adopted in the latest ChP (2015 edition). However, systematic research on
identification of TCMs in ChP by using DNA barcoding remains inade-
quate. Results: In this study, we established a DNA barcoding database
for medicinal species in ChP (http://www.tcmbarcode.cn/en/). One sample
of original species and three samples of medicinal materials were avail-
able for each Chinese materia medica. Most of the samples were collected
from China, whereas some of them were collected from other countries,
such as Japan, Korea, Canada, and the United States. To date, six different
modules have been implemented in the web server, namely Identifica-
tion, Protocol, Database, News, Documents, and Register. In the Identifi-
cation module, nr ITS2 was the core DNA barcode for identifying
medicinal plants, whereas the psbA–trnH region was a complementary
barcode. COI was used to identify medicinal animals. Significance: The
database provides standard sequences for each medicinal species in ChP,
thereby contributing great practical significance to clinical safety in uti-
lization of TCMs. Furthermore, this system can help prevent illegal drug
trade in circulation.
Ecological applications of DNA barcoding: the
African experiences
Kowiyou Yessoufou
1
and Michelle van der Bank
2
1University of South Africa, Chr de Wet avenue, 1710, Pretoria, South Africa.
2University of Johannesburg, University Road, 2006, Johannesburg, South Africa.
Corresponding author: Kowiyou Yessoufou (e-mail: kowiyouyessoufou1@gmail.com).
Background: Since the pair rbcLa and Atpf has been accepted as the core
DNA barcode for terrestrial plants, many studies, perhaps surprisingly,
continue to test the discriminatory power of these markers in many
lineages. In Africa, and specifically in South Africa, we have moved on
with the application of the core barcode in phylogenetic ecological stud-
ies. Using the core barcode to assemble the largest phylogeny of local,
regional, and continental floras in Africa, we explored important ques-
tions related to community ecology (biological invasions and community
responses to herbivory) and phylogenetic diversification (African cycads
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and “underground forests”). Results: We found that alien plants closely
related to native flora naturalize, but those distantly related become
invasive, thus resolving the Darwin's Naturalization Conundrum. We
further explored how native flora responds to disturbances driven by
large mammals. We found that native communities that are phylogeneti-
cally clustered tend to become overdispersed and vice-versa in response
to herbivory, indicating that community responses to disturbances are
contingent upon initial conditions. The evolutionary history of the Afri-
can native floras seems to be climatically mediated. For the African cy-
cads, we found a pattern of constant radiation with mass extinction in
addition to an explosive radiation in a Southern African clade that devel-
oped underground stems during the transition Pliocene–Pleistocene. We
then extended our interests to the diversification history of the entire
continental underground trees (beyond cycads). We found multiple inde-
pendent origins almost synchronous with the South American counter-
parts, and that the development of this life form may have been
mediated by the interactive effects of frequent fires and high precipita-
tion. Significance: Using the core DNA barcodes in phylogenetic ecolog-
ical studies, we shed light on contemporary and evolutionary events that
shape the present-day African flora, demonstrating the ecological signif-
icance of DNA barcoding beyond traditional species identification.
Patterns of protein evolution in cytochrome c oxidase subunit I
(COI) from the class Arachnida
Monica R. Young and Paul D.N. Hebert
Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON N1G2W1, Canada.
Corresponding author: Monica R. Young (e-mail: myoung02@uoguelph.ca).
Background: Broad patterns of mitochondrial evolution have seen little
investigation in Arachnida. Because sequence information is now avail-
able for the 648-bp barcode region of COI from nearly 400 000 animal
species, this gene segment can be used to probe patterns of mitochon-
drial evolution. The present study examines levels of amino acid substi-
tution and the frequency of indels in COI from 4177 species of arachnids,
including representatives from all 16 orders and 43% of its families (267/
625). Results: Divergences were examined at three taxonomic levels.
Order distances vary 4-fold (0.10–0.39), while the mean of the family
distances for each order ranges 5-fold (0.07–0.35). BIN distances show
great variation, ranging from 0.01 or less in 12 families to more than 0.25
in eight families. Groups whose COI genes have elevated levels of amino
acid substitution also regularly possess indels, and these rates are nega-
tively correlated with generation times. Patterns of amino acid substitu-
tion in COI are generally congruent with previously reported variation in
nucleotide substitution rates in arachnids, but provide some new in-
sights, such as clear rate acceleration in the Opiliones. Significance: By
revealing a strong association between elevated rates of nucleotide and
amino acid substitution, this study builds evidence for the selective im-
portance of the rate variation among arachnid lineages. Overall, this
study suggests that the mitochondrial genome of some arachnid groups
is dynamic with high rates of amino acid substitution and frequent in-
dels, while it is “locked down” in others. Dynamic genomes are most
prevalent in arachnids with short generation times, but the possible
impact of breeding system deserves investigation since many of the rap-
idly evolving lineages reproduce by haplodiploidy, a mode of reproduc-
tion absent in “locked down” taxa.
DNA barcoding in the marine realm: challenges, successes,
and support for DNA-based species delimitation
among the Maxillopoda
Robert G. Young,
1
Cathryn Abbott,
2
Thomas Therriault,
2
and Sarah J. Adamowicz
1
1Biodiversity Institute of Ontario & Department of Integrative Biology, University of Guelph, 50 Stone
Road East, Guelph, ON N1G 2W1, Canada.
2Department of Fisheries and Oceans, 3190 Hammond Bay Road, Nanaimo, BC V9T 6N7, Canada.
Corresponding author: R.G. Young (e-mail: ryoung04@uoguelph.ca).
Background: Biodiversity surveys in the marine realm increasingly rely
on the use of DNA barcoding and next-generation sequencing to describe
whole communities and to assess the likely number of species present.
Recent research has indicated the success of DNA barcoding for identify-
ing specimens belonging to a wide range of animal taxa, including plank-
tonic groups. The effectiveness and behaviour of methods for specimen
identification and species delineation remain to be examined on a large
scale for taxonomically diverse and widely distributed marine groups,
such as the crustacean class Maxillopoda. This study investigates whether
a biologically significant molecular threshold exists between intraspe-
cific and interspecific divergences in maxillopods for the barcode region
of the cytochrome c oxidase subunit I (COI) gene. Results: Using a data
set of all publically accessible maxillopod nucleotide sequences, we
determined the most general threshold value for the maxillopods that
separates apparent intraspecific from interspecific divergences, using
different analytical methods (e.g., ABGD, jMOTU, UPARSE, mothur). We
then compare the MOTUs generated using these threshold values and
analytical methods against morphological identifications and additional
methods (e.g., BINs, GMYC, PTP), using a new planktonic marine maxil-
lopod reference data set covering a wide geographic range from Canada's
three oceans. Our results indicate the presence of a biologically signifi-
cant COI threshold value of ⬃2.3% p-distance for maxillopods, which
typically yields molecular groupings that are largely concordant with
traditionally identified species. Significance: We provide evidence in
support of a general intra- and interspecific COI threshold value for the
marine maxillopods, which will help to facilitate rapid analysis of biodi-
versity data for biodiversity research and species discovery purposes. We
further highlight taxa where there was less concordance between mor-
phological and molecular delineations and recommend the need for con-
tinued biological research and the use of additional molecular markers
to determine the diversity, distributions, and niches of species within
these groups.
High-throughput monitoring of wild bee diversity and
abundance via mitogenomics
Douglas W. Yu,
1
Min Tang,
2
Chloe J. Hardman,
3
Yinqiu Ji,
1
Shanlin Liu,
2
Tim Nevard,
4
Simon G. Potts,
3
and Xin Zhou
2
1Kunming Institute of Zoology, 32 Jiaochang East Road, Kunming, Yunnan 650223, China.
2BGI-Shenzhen Shenzhen, Guangdong 518083, China.
3University of Reading, Reading RG66AR, UK.
4Charles Darwin University, Darwin NT0909, Australia.
Corresponding author: Douglas W. Yu (e-mail: dougwyu@gmail.com).
Background: Wild bees play a keystone role in the pollination of wild
plants and cultivated crops and thereby help to maintain biodiversity
and increase food production. Evidence of declines in wild pollinators
and insect-pollinated plants requires high-throughput methods to mon-
itor bee abundance and diversity, since the number of wild bee species
per country ranges from hundreds to thousands. Morphological identifi-
cation is inefficient because taxonomic expertise is scarce, and PCR-based
metabarcoding is error-prone. Results: We used a metagenomic ap-
proach to identify and quantify bee species from bulk samples. Reference
mitochondrial genomes from 48 bee species (9 genera) were assembled,
the total DNA of each of 10 bulk samples (containing a total of 204 bees
morphologically identified to 33 species) was shotgun-sequenced, and
the sequence reads were mapped to the reference mitogenomes. This
mitogenomic dataset showed high accuracy: successfully making 59
out of the 63 total species detections in the morphological dataset (i.e.,
four possible false negatives), plus making six detections not in the
morphological dataset (possible false positives). PCR analysis suggests
that these discrepancies were due to errors in the morphological iden-
tifications. The two datasets returned indistinguishable estimates of
community structure and extrapolated species diversity, and read
number per species was positively but weakly correlated with esti-
mated biomass per species (R
2
= 24.9%, p< 0.001). By contrast, meta-
barcoding exhibited greater error on all counts. Significance:
Mitogenomic resequencing of bulk samples is highly promising for
large-scale monitoring of wild bee communities and for assessing
conservation interventions and can easily be extended to other taxa.
Given their many practical and scientific advantages, mitogenomes
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deserve consideration as “super-barcodes” for economically and so-
cially important species.
Higher fungal diversity in dead wood reduces CO
2
emissions in
a natural forest
Douglas W. Yu,
1
Chunyan Yang,
1
Douglas A. Schaefer,
2
Viorel D. Popescu,
3
and Weijie Liu
2
1Kunming Institute of Zoology, 32 Jiaochang East Road, Kunming, Yunnan 650223, China.
2Xishuangbanna Tropical Botanical Garden, Menglun, Mengla, Yunnan 666303, China.
3Simon Fraser University, Burnaby, BC V5A 1S6, Canada.
Corresponding author: Douglas W. Yu (e-mail: dougwyu@gmail.com).
Background: Wood decomposition releases almost as much CO
2
as does
fossil-fuel combustion, so the factors regulating wood decomposition can
affect the global carbon cycle. Results: We used metabarcoding of the
ITS2 marker to estimate the fungal species diversities of naturally colo-
nized wood in subtropical China. Wood hosting more diverse fungal
communities emitted less CO
2
, with Shannon diversity explaining
26%–44% of emissions variation. Community analysis mostly supports
a “pure diversity” effect of fungi on decomposition rates. Our results
extend previous experiments on low-diversity, laboratory-inoculated
wood to a high-diversity, natural system, and we suggest that interfer-
ence competition is the underlying mechanism. Experiments are now
needed to determine the direction of causality. Significance: The
conservation of high levels of wood-decomposer fungal biodiversity
may preserve a globally important ecosystem service by reducing the
atmospheric contribution of CO
2
made by the world's stock of decom-
posing wood.
DNA barcoding of the Chinese fly Chrysomya megacephala and
comparison to its published data around the world
Qiaoyun Yue,
1
Charles C. Cook,
2
Deyi Qiu,
1
Jia Hu,
1
and Jian Chen
1
1Zhongshan Entry-Exit Inspection and Quaratine tecnical Center, 2, Zhongshan Six Road, Zhongshan
City, Guangdong, China.
2EMBL-European Bioinformatics Institute, Wellcome trust, Genome Campus, Hinxton, Cambridge, UK.
Corresponding author: Qiaoyun Yue (e-mail: 779721036@qq.com).
Background: The blowfly Chrysomya megacephala (Fabricius), or ori-
ental latrine fly, is the most common human-associated fly in
South, Southeast, and Eastern Asia. C. megacephala larvae develop in
feces and decomposing flesh and, though native to Eurasia, have
spread through human activity around the world. C. megacephala
larvae are used in wound therapy and for forensic entomology.
Dried larvae are also used as animal feed and in traditional Chinese
medicine, while adults may vector bacterial pathogens. We used
cytochrome c oxidase subunit I (COI) barcodes to survey variation
in C. megacephala related blowflies, and other flies in China, and to
assess the utility of barcodes to identify the morphologically cryp-
tic larvae of these species. Results: We collected C. megaphala, its
congener C. pinguis, and 12 other species from 42 locations in China
as well as in Manila and Lima. In addition to the two chyrosomids,
these included flies from five other species in the family Calliphori-
dae, three from the Muscidae, and three from the Sarcophagidae.
We successfully sequenced the 658-bp COI barcoding region from
645 individuals, including 208 C. megacephala and 36 C. pinguis.
Kimura-2-parameter distance plots distinguish C. megacephala from
C. pinguis and all other fly species. Additionally, there is no indica-
tion of geographic variation in C. megacephala within China. Signif-
icance: The larvae of C. megacaphala are important in forensic death
investigations, in wound therapy, and in traditional Chinese med-
icine, yet can be difficult to identify. Our work shows that the
standard COI barcode can quickly distinguish C. megacepahala from
other flies in China, including C. pinguis, its closest congener. This
confirms previous work on this species from other locations. We
are currently comparing our sequences from Chinese flies with all
other reported C. megacephala barcodes (152 sequences from BOLD)
to assess worldwide geographical variation and will report these
results as well.
Identification of calliphorid flies in their different life stages using
DNA barcoding can improve the success of forensics investigations
Sureshchandra Zambare and Gulab Khedkar
Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar
Marathwada University, Aurangabad, India.
Corresponding author: Sureshchandra Zambare (e-mail: sureshchandraz@yahoo.co.in).
Background: Calliphorid and sarcophagid flies are the first to attack
corpses and lay their eggs in the openings within corpses. Such flies can
be used as evidence in forensic investigations. Despite their substantial
forensic potential, their use has been limited because morphological
species identification, at any life stage, is greatly puzzling. Results: Our
study investigated whether DNA could be extracted and cytochrome c
oxidase subunit I (COI) barcode sequences obtained for molecular iden-
tification of each immature life stage of the forensically important Indian
flesh flies belonging to the families Calliphoridae and Sarcophagidae.
Genomic DNA extracts were prepared from all larval instars and puparia.
Amplification of the barcoding region was successful from all extracts,
but puparia amplicons were weak. All sequences were identified with
99.98% confidence using the Barcode of Life Data Systems (BOLD) data-
base. In some cases, PCR inhibition is correlated with the presence of a
corpse. All together, we barcoded 35 flies in their different life stages. The
species we recorded include Chrysomya megacephala,C. rafigaices,C. suffranea,
Sarcophaga dux,S. peregrena, and S.S. ruficornis.Significance: These results
can improve the investigation success in forensic cases using evidence
from immature flies.
Integration of DNA barcoding in a local diversity survey of
Lepidoptera: confronting morphospecies and DNA barcode
OTUs of Arctiinae moths in the Brazilian Atlantic forest
Mauricio Moraes Zenker,
1
Rodolphe Rougerie,
2
José Augusto Teston,
3
Michel Laguerre,
2
Marcio R. Pie,
3
and André Victor Lucci
4
1Universidade Estadual de Campinas, Campinas-SP, Brazil.
2Muséum national d'Histoire naturelle, Paris, France.
3Universidade Federal do Oeste do Pará, Santarém-PA, Brazil.
4Freitas Universidade Estadual de Campinas, Campinas-SP, Brazil.
Corresponding author: Mauricio Moraes Zenker (e-mail: maurizenker@gmail.com).
Background: Surveys of insect diversity, especially in the tropics, are
strongly impeded by the difficulty in obtaining accurate species delimi-
tation and identification. DNA barcoding proved an efficient method to
enhance species diversity assessment in Lepidoptera, permitting reliable
species identification and frequently unveiling overlooked or cryptic di-
versity. In this study, 14 026 individuals of Tiger-Moths (Erebidae: Arctii-
nae) were sampled over 2 years along an elevational gradient in a large
fragment of the endangered Atlantic Forest in southern Brazil. Results:
After an initial sorting of 294 species based on external morphology and
color patterns, we successfully sequenced DNA barcodes for 1100 speci-
mens representing 291 of these species. Operational taxonomic units
(OTUs) were defined using a 2% sequence divergence threshold and com-
pared to morphospecies defined from our initial morphological assess-
ment (IMA). Because species delimitation based on wing patterns is
known to potentially overlook closely resembling “cryptic” species, we
also carried out a morphological reassessment (MRA) of species diversity
based on the comparison of male genital structure to further investigate
cases where IMA species and DNA barcode OTUs were incongruent. From
a total of 291 IMA species, 201 matched their respective OTUs. Morphol-
ogy could not be reassessed in three species, and the species delimita-
tions changed in the remaining 87 IMA species, suggesting that as much
as 30% of the diversity might be overlooked when using superficial mor-
phological comparison. Interestingly, a close examination of the male
genital morphology revealed that only 14 IMA species did not match
barcode OTUs. All the results reported here, including the several cases of
cryptic species and sexual dimorphism found among the 87 IMA species
that did not match barcode OTUs, are available on BOLD under the name
of Lepidoptera of Serra do Mar Project. Significance: In addition to being
useful for further taxonomic studies, the data available in the Lepidop-
tera of Serra do Mar Project allow automated species identifications of a
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considerable part of the arctiines in the Atlantic Forest and thus is a
valuable tool for conservation purposes.
Molecular detection of in situ dietary composition of Calanus
sinicus in Taiwan Strait
Dangni Zhang,
1
Lianming Zheng,
2
Weiwei Li,
1
Hua Lin,
1
Wenqing Cao,
3
and Lvping Fang
1
1College of Ocean and Earth Sciences, 320, Zhou Long Quan Building, Xiang'an Campus, Xiamen
University, Xiamen 361102, P.R. China.
2College of Ocean and Earth Sciences, 313, Zhou Long Quan Building, Xiang'an Campus, Xiamen
University, Xiamen 361102, P.R. China.
3College of Ocean and Earth Sciences, 318, Zhou Long Quan Building, Xiang'an Campus, Xiamen
University, Xiamen 361102, P.R. China.
Corresponding author: Lvping Fang (e-mail: zhlm@xmu.edu.cn).
Background: Planktonic copepods represent a critical link between
primary producers and higher trophic level consumers in marine food
webs. However, in situ diet status of copepods remains largely unre-
vealed due to the lack of effective approaches. With the development
of rapid and reliable species-detection methods based on different
DNA markers, detailed prey composition and quantification of cope-
pods are becoming accessible. Results: In this study, in situ dietary
composition of Calanus sinicus (Brodsky, 1962) was analyzed using a
DNA and PCR-based method from two sampling stations, A1 (in-shore)
and B9 (off-shore) in the Taiwan Strait. In the laboratory, 95% ethanol-
preserved individuals were carefully washed, had their appendages
removed, and were checked under a fluorescence microscope to ex-
clude the influences of attaching organisms; then genomic DNA were
extracted using the CTAB protocol. Finally, two loci of chloroplast
ribulose-bisphosphate carboxylase gene (rbcL ID and IA/B) and one
partial nuclear 18S rDNA gene were PCR amplified, cloned, and se-
quenced. A total of 50 individuals of C. sinicus from A1 station and
10 from B9 were analyzed. For station A1, 26 OTUs from clone libraries
(282 sequences) were detected, clustering into 11 plankton groups;
phytoplankton was the dominant group, comprising 73.1% of the total
OTUs and 53.5% of the total sequences. Diatoms and chlorophytes
were the most abundant groups among phytoplankton, accounted for
⬃34.6% and ⬃11.5% of the total OTUs, respectively. The detected dia-
tom sequences were most closely related to the genus Thalassiosira. For
station B9, 30 OTUs were detected, clustering into 10 plankton groups.
Phytoplankton was the dominant group as well, comprising 60.0% of
the total OTUs and 44.4% of the total sequences. Diatoms and chlo-
rophytes were also the most abundant groups among phytoplankton,
except representing a lower coverage of 16.7% for diatoms and a
higher coverage of 13.3% for the chlorophytes. The results revealed
that dietary composition of C. sinicus in these two locations varied
significantly, with only 9 OTUs shared by both stations, corresponding
to 19.1% of the total OTUs. Besides the formerly recognized groups
with traditional analyses, such as phytoplankton and ciliates, our
study unveiled several novel dietary compositions, like Hydromedu-
sae (3 sequences of 1 OTUs, 2.1%) and Thalia (1 sequences of 1 OTUs,
2.1%), suggesting a possible predation on eggs or larvae of these groups
by C. sinicus.Significance: Our results supported DNA-based molecu-
lar detection as an effective approach in copepods trophic ecology
studies, and it has laid the foundation for its further applications in
analyzing matter cycling and energy flow in marine ecosystem.
Detection of aquatic invasive species and biodiversity
assessment in Canadian ports
Guang K. Zhang,
1
Emily Brown,
1
Frédéric J.J. Chain,
1
Cathryn Abbott,
2
and Melania E. Cristescu
1
1Department of Biology, McGill University, 1205 Docteur Penfield, Montréal, QC H3A 1B1, Canada.
2Fisheries and Oceans Canada, 3190 Hammond Bay Rd, Nanaimo, British Columbia, Canada.
Corresponding author: Guang K. Zhang (e-mail: guang.zhang@mail.mcgill.ca).
Background: Aquatic invasive species (AIS) negatively impact
economies and ecosystem functions, making their early detection
of great importance for conservation and environmental sustain-
ability. Metabarcoding can be used as a rapid method for monitor-
ing biodiversity, but species-level identification and quantification
are limited by finding a suitable metabarcode, lack of high-quality
taxonomic reference libraries, and complex bioinformatic analy-
ses. The mitochondrial cytochrome c oxidase subunit I (COI) gene
has high interspecific genetic variation and comprehensive refer-
ence databases, but designing primers that successfully amplify
diverse taxa remains a challenge. We address this issue by testing
and developing a set of group-specific COI primer pairs that will be
used to identify zooplankton species present in major Canadian
ports. These primers will first be tested on mock zooplankton com-
munities for which it will also be possible to examine the extent to
which species abundance is correlated to the number of sequence
reads. Results: The amplification success of 11 previously published
COI primer pairs were compared with the aim to cover most major
taxonomic groups of zooplankton (Copepoda, Branchiopoda, Deca-
poda, Mollusca, Tunicata, Rotifera, and Cnidaria). A set of COI
primer pairs was selected to perform separate PCR reactions on
targeted groups, and then pooled for next-generation sequencing.
Our preliminary results based on mock communities indicated that
raw reads show strong correlation with the number of individuals
(R
2
= 0.7647). Significance: The method that we developed has the
potential to eliminate a major limitation in the use of COI primers
for biodiversity assessment and for effective early detection of aquatic
invasive species. This project could contribute to global sampling net-
works that aim to generate catalogs of aquatic invertebrates. More-
over, the biodiversity estimates revealed by metabarcoding of natural
port communities can enable broader research on invasion biology,
trophic interactions, and evolution.
Molecular and morphological evidence for underestimated
biodiversity of Clytia (Cnidaria: Hydrozoa) in the China Sea,
with description of three new species
Lianming Zheng, Weiwei Li, Hua Lin, Lvping Fang, and Wenqing Cao
College of Ocean and Earth Sciences, Xiamen University, Zhou Long Quan Building, Xiang'an Campus,
Xiamen University, Xiamen 361102, China.
Corresponding author: Wenqing Cao (e-mail: zhlm@xmu.edu.cn).
Background: The genus Clytia is distributed worldwide, but most
accepted species in this genus have been examined either only at the
hydroid or medusa stage. The challenge in identifying Clytia species
reflects their complex life cycles and phenotypic plasticity. Results: In
this study, molecular and morphological investigations of Clytia spec-
imens from the coastal waters of China revealed three as yet unre-
ported species, designated Clytia xiamenensis sp. nov., Clytia gulangensisi
sp. nov., and Clytia tankahkeei sp. nov., that were considered as conspe-
cific to two nearly cosmopolitian species, Clytia hemisphaerica and
C. gracilis. DNA barcoding based on partial mitochondrial cytochrome
c oxidase subunit I (COI) and large subunit ribosomal RNA (16S) gene
sequences confirmed the highly distinct lineage of these three species.
These results were corroborated by the detailed observations of their
mature medusae and colonies, which showed that these three spe-
cies were morphologically distinct from other species of Clytia.
Significance: Thus, based on our findings, the nearly cosmopolitan
distribution attributed to some species of Clytia might rather be due to
the misidentification, and life cycle and DNA-based studies should be
a standard approach in future biodiversity investigations of Clytia spe-
cies in order to establish the systematic validity of all species within
the genus Clytia.
DNA barcode of rare and endangered plants
Shiliang Zhou
State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of
Sciences, Beijing 100093, China.
E-mail for correspondence: slzhou@ibcas.ac.cn.
China is rich in plant species diversity—according to the Flora of
China, there are 31 362 vascular plant species in 3328 genera belong-
ing to 312 families (sensu Engler). Among them, 809 species are con-
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sidered endangered in the wild. Some species are living fossils from
the Carboniferous (e.g., Ginkgo biloba) or Cretaceous (Metasequoia
glyptostroboides), and some species are threatened due to historical
demand for timber (e.g., Dalbergia tonkinensis for 600 years) or recent
construction of hydroelectric power stations (e.g., Adiantum reniforme
var. sinense). Some species are critically endangered (e.g., Carpinus
putoensis, one individual; Abies beshanzuensis, three individuals; and Ostrya
rehderiana, five individuals), yet illegal logging or trade is still happen-
ing. The project “DNA Barcode of Rare and Endangered Plants (BREP)
in China” aims to (i) create a web-based platform for governmental
officers to help determine whether material in question is from rare
or endangered species; (ii) quantify the genetic diversity of the rare
and endangered species for conservation purposes. Thus we employ
four universal barcode markers (ITS, Atpf, rbcL, and ycf1b) and some
taxon-specific supplementary barcodes (atpB-rbcL, rps4, trnH–psbA,
trnL–F, and trnS–trnG). DNA material is mostly from the national her-
barium (PE). DNA templates were first reconstructed by primer-free
PCR. All primers were modified by adding a universal tail, which is
used subsequently to label samples. Multiple PCRs were performed,
and the DNA-labeled PCR products were mixed and sequenced on an
Illumina MiSeq platform. The MiSeq reads were sorted into barcodes
according to primers and into individuals according to DNA labels.
Alleles were verified by referring to known sequences or using phylo-
genetic methods. By following the above pipeline we were able to
complete the project in half a year. The platform is now available
(http://www.brep.ac.cn), and the genetic diversity of rare and endan-
gered species is being evaluated.
The use of DNA barcoding in large-scale ‘omics projects
Xin Zhou
1
and Karl Kjer
2
1BGI-Shenzhen, Bldg 11, Beishan Rd., Beishan Industrial Zone, Yantian Dist., Shenzhen, Guangdong
Province, China.
2Department of Entomology, Rutgers University, New Jersey, USA.
Corresponding author: Xin Zhou (e-mail: xinzhou@genomics.cn).
Abstract: DNA barcodes have been adopted in routine applications in
a wide range of research fields (e.g., species identification, DNA tax-
onomy, life-stage association, phylogenetics, diet analysis, biodiver-
sity survey, biomonitoring). As a critical linkage that bridges
taxonomy, biology, and genomics of a particular taxon, DNA barcodes
have also demonstrated significant contribution to large-scale ‘omics
studies. Genome sequencing typically would start with a sufficient
amount of tissues—sometimes from varied resources and times—
creating an unpredictable chance for errors in mixing cross-taxon
tissues. On the other hand, RNA-seq of non-model organisms requires
field samples collected directly into liquid nitrogen or RNAlater, pro-
viding a huge challenge for taxonomic identification of sequenced
taxa. This presentation will demonstrate the usefulness of DNA bar-
coding in the pipeline of ‘omics studies suffering from taxonomic or
QA/QC issues.
Barcoding Chinese marine nematodes with mitochondrial
cytochrome c oxidase subunit I (COI) and small subunit
18S rDNA (18S)
Hong Zhou, Qing Yu, Yating Wang, and Zhinan Zhang
Ocean University of China, 5 Yushan Road, 266003, Qingdao, China.
Corresponding author: Hong Zhou (e-mail: hzhou@ouc.edu.cn).
Background: The nuclear small subunit 18S rDNA gene (18S) is the
most widely used molecular marker to estimate the diversity in bio-
diversity surveys of meiofauna and nematodes. The cytochrome c ox-
idase subunit I (COI) mtDNA has been used as a standard barcoding
marker for animals. Different efficiency using these two markers for
barcoding marine nematodes has been noticed and has led to the
inconsistent pace between the rapid increase of environmental
metabarcode sequences of 18S and the slow growth of COI barcode
sequences in BOLD (Barcode of Life Data Systems). There is recom-
mendation against the use of 18S as a marker for biodiversity surveys
due to its diversity underestimation, and there are several attempts to
improve the success rate of COI by designing specific primers, but few
of these works were based on marine nematodes. Results: We com-
pared the efficiency of using partial sequences of 18S (608 bp) and COI
(658 bp) as molecular markers for barcoding, using 192 specimens of
marine nematodes sampled from intertidal and subtidal habitats in
Chinese coastal waters, and evaluated the estimation of species rich-
ness against morphospecies. Our results show that 18S reduced se-
quence divergence by a factor of 0.25 relative to COI, but increased
success rates by a factor of 4.0 (66%/16%) and 2.8 (85%/30%) for speci-
mens and species, respectively. 18S recovered 100% of morphospecies,
whereas COI increased species richness by a factor of 0.02 due to poten-
tial cryptic species. Significance: Our results suggested that even
though 18S has the potential to underestimate cryptic diversity of
meiofauna, its sequencing efficiency makes it a useful complemen-
tary marker to COI and it can be used in conjunction in marine nem-
atode barcoding and biodiversity survey projects. But before large-
scale application of the 18S marker, its underestimation for cryptic
diversity need to be thoroughly evaluated and the sequence segment
needs to be standardized.
Use of DNA barcoding technology to appraise commercial
Spatholobus products
Hong Zhou,
1
Pei Yang,
1
Shuang-jiao Ma,
1
Jing-yuan Song,
1
Shi-lin Chen,
2
Bei-bei Chen,
3
Zheng-zhou Han,
3
and Hui Yao
1
1Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union
Medical College, No. 151 Malianwa North Road, Haidian District, Beijing 100193, China.
2Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, No. 16, Nanxiao
Street, Dongzhimen, Dongcheng District, Beijing Municipality, PR China.
3Research Center, China Resources of Sanjiu Medical & Pharmaceutical Co., Ltd., Guanqing Road,
Guanlan High-tech Industrial Park, Longhua District, Shenzhen, Guangdong, China.
Corresponding author: Hong Zhou (e-mail: zhouhong1013@126.com).
Background: Spatholobi Caulis (Ji Xueteng) exhibits blood-activating
and stasis-dissolving properties (“Huoxuehuayu” in Chinese medi-
cine). Therefore, this medicinal plant is regarded as a panacea for
blood stasis and an effective medicine for gynecological diseases.
Given that Spatholobus suberectus Dunn leaks reddish-brown juices
when cut, it has been named “Xueteng”. Moreover, Sargentodoxae
Caulis (Da Xueteng), Kadsurae Caulis (Dian Jixueteng), and other
stems with that same feature can also be called “Xueteng”. The phe-
nomenon of homonyms in traditional Chinese medicine may lead to
misuse because of different sources and varied efficacy. At present,
various commercial Spatholobus products exist, including tablets, gran-
ules, syrups, and capsules. To guarantee clinical medication safety and
better protect the legal rights and interests of consumers, we should
clarify the species composition of these commercial Spatholobus prod-
ucts, which are difficult to identify using morphological characters. In
this study, we use DNA barcoding as an authentication method.
Results: Up to 115 samples were collected from different habitats in
China, which contained S. suberectus and its closely related species.
ITS2 and psbA–trnH sequences were obtained in accordance with the
DNA barcoding standard operating procedure. The maximum intras-
pecific genetic distances of Spatholobi Caulis in the ITS2 and psbA–
trnH regions were 0.02 and 0.014, respectively. These distances were
smaller than those of the interspecific ones. Furthermore, Spatholobi
Caulis formed into a single branch in the NJ tree and can be distin-
guished from other species efficiently. The reference database was
established from these samples, and its stability and reliability
have already been tested. Significance: We will use the existing
database to investigate the materia medica of “Xueteng” and the
commercial Spatholobus products, as well as to test the recovery of
standard DNA barcodes for Spatholobi Caulis from a large array of
commercial Spatholobus products to ensure accurate ingredient la-
bels for consumers.
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The San Diego Barcode of Life: launching a regional DNA
barcode campaign in a globally important biodiversity
hotspot
Bradley Zlotnick
San Diego Barcode of Life, 3525 Del Mar Heights Road Suite #139, San Diego, CA, USA.
E-mail for correspondence: sdbarcodeoflife@gmail.com.
Background: Inspired by Dan Janzen's DNA barcoding public lectures
(2007 and 2012), and visionary presentations by BIO/BOLD researchers
(2013), the San Diego Barcode of Life (SDBOL) aspires to be a preemi-
nent node and example for exploration, dissemination, and imple-
mentation of biodiversity DNA barcoding and its infrastructure.
SDBOL leads and partners in San Diego's innovative and entrepreneur-
ial scientific, corporate, civic, philanthropic, and academic communi-
ties to advance mutual goals in biodiversity understanding. Results:
Since mid-2013, SDBOL contributed to the assembly of nearly 38 000 barcode
sequences to BOLD: 26 000 specimens from a 38-mile land conser-
vancy Malaise trap transect; 7000 from the first 52-consecutive
week corporate Malaise trap project; 2600 from museum herbaria;
and thousands from collaborating pilot sites and institutions. To date,
SDBOL has negotiated over $1.7M in cash and in-kind investment value
to iBOL, not including voluntary professional time and expense. SD-
BOL established unprecedented collaboration among the University of
California, San Diego (UCSD), San Diego Zoo Global, and local land
trusts including San Dieguito River Valley Conservancy. SDBOL initi-
ated a multi-year in-kind contribution of reagents and consumables to
BIO, and secured iBOL's first “altruistic” corporate sponsor, ResMed,
Inc. It facilitated UCSD's and BOLD's NSF-funded collegiate Student
Data Portal. SDBOL continues to champion world-class interactive,
artistic depictions of DNA barcoding with Seattle artist Joseph Ros-
sano, bringing the public to bioliteracy and highlighting iBOL innova-
tions. Throughout, SDBOL has sustained close relations with the
Consul of Canada in San Diego and the Trade Commissioner for Life
Sciences, matching and leveraging their strategic seed funding by
more than an order of magnitude in executing SDBOL projects.
Significance: The San Diego Barcode of Life is achieving critical mass
as a unique regionally-led initiative evolving from and underpinning
iBOL, BIO, and BOLD in their global missions.
“Barcoding our Backyard” at ResMed, Inc.: 52-consecutive
weeks Malaise trap project at a corporate headquarters in a
Global biodiversity hotspot
Bradley Zlotnick,
1
Joshua Kohn,
2
David Dannecker,
2
and Valérie Lévesque-Beaudin
3
1San Diego Barcode of Life, 3525 Del Mar Heights Road Suite #139, San Diego, CA, USA.
2University of California, San Diego, CA, USA.
3Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
Corresponding author: Bradley Zlotnick (e-mail: sdbarcodeoflife@gmail.com).
Background: In February 2014, ResMed, Inc., a leading San Diego medi-
cal sciences company, engaged the San Diego Barcode of Life (SDBOL) and
became the first corporate partner to join iBOL in DNA barcoding its
community's biodiversity. ResMed committed to an initial 52-consecutive
week collection, instituting the Global Malaise Program (GMP) protocol
at their nine-acre headquarters site (half of which is an open-space sculp-
ture garden) in central urban/industrial San Diego. ResMed barcoding
aimed to document flying insect diversity in its urban setting, advance
barcode indexing generally, model corporate community involvement in
science for employees and the public, and inspire expansion of Malaise
trapping in the region. Managed by University of California, San Diego,
faculty and students from their San Diego Biodiversity Project, the trap
supplements ongoing SDBOL GMP and related activities. Results: Thirty-
nine weeks of consecutive sampling acquired 8103 specimens (range,
43-403 individuals/week). 86% of specimens were successfully barcoded,
with 631 putative species in at least 15 orders, generating 129 unique BINs
for BOLD. Species accumulation curves were similar to less urban sites,
suggesting ⬃1303 species will potentially surface with continued sam-
pling. Employee engagement began with facilities staff, adding an
employee vegetable garden group, and developing a corporate
communications video of the ResMed iBOL project for an all-employee
assembly and global website in April 2015. The ResMed project's progress
stimulated further investment in SDBOL and iBOL: ResMed sponsored
the first complete DNA barcoding of a globally important regional
flora—the San Diego Plant Atlas—at the San Diego Natural History Mu-
seum. A regional technology and life sciences consortium is considering
expansion of a Corporate Malaise Program with SDBOL. Significance:
ResMed's substantial participation with SDBOL marks notable iBOL firsts:
52-consecutive week Malaise dataset; corporate Malaise site; corporate
participant in SDBOL, GMP, iBOL. With their desire to model corporate
engagement, ResMed is stimulating SDBOL growth and inspiring other
iBOL regions.
Abstracts 303
Published by NRC Research Press
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