Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 164 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 165 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 166 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 167 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 168 Genome Vol. 58, 2015 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 biodiversity 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 metabarcoding: 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 169 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 170 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 171 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 172 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 173 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 174 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 175 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 176 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 177 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 178 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 179 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 Şahin et al. 274 DNA metabarcoding of springtails (Collembola) Saitoh et al. 274 DNA barcoding to support biodiversity conservation, sustainable harvesting, 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 diversity 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 180 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 181 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 182 Genome Vol. 58, 2015 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Contents / Contenu 183 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 mitogenomics Yu et al. 299 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 184 Genome Vol. 58, 2015 Higher fungal diversity in dead wood reduces CO2 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 Published by NRC Research Press 185 ABSTRACTS DNA barcoding of Oeneis butterflies newly sampled in Mongolia Takatoshi Abe,1 Hidemi Watanabe,1 Kanako O. Koyanagi,1 and Botaro Inoue2 is of substantial practical importance for choosing appropriate analysis methods for specimen identification, species discovery, and species delimitation. 1Hokkaido Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 constitute 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, located in the Altai-Sayan ecoregion, from June 22 to June 29, 2014. Collected specimens were identified based on their morphological characters and DNA barcodes. As a result, 23 of 92 butterflies were classified 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 genetically 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, despite diverse discussions of species boundaries. Meanwhile, large DNA barcoding campaigns are generating continental-scale datasets of standardized 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, together with other data types, to address elemental questions about the properties and evolutionary origins of species entities. For example, comparative barcode data from sexual versus asexual taxa can help us to understand the strength of the role of reproductive isolation in the formation 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 Genome 58: 185–303 (2015) dx.doi.org/10.1139/gen-2015-0087 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-Riverón3 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 fragments 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 polymorphism 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 central 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 thoroughly investigated. Here we assessed the species boundries in this taxon based on specimens collected from a number of localities along the Neotropics. 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 propose that E. ruidum is actually composed of at least three evolutionary units, two of which have wide geographic distributions along the Neotropics. 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 Khedkar2 1Department of Zoology, Rani Laxmibai College Parola, Jalgaon District, Maharashtra, India. Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, India. Corresponding author: Kiran Ahirrao (e-mail: drkiranahirrao@gmail.com). 2Paul Mosquitoes (Diptera: Culicidae) include some of the most important disease 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 mosquito species present in Maharashtra state in India including the major disease vectors. A total of 680 mosquito specimens were collected and Published by NRC Research Press 186 assigned to 6 genera and 21 species and subspecies on the basis of morphological characteristics. Individuals of the same species grouped closely together in a neighbour-joining tree based on COI sequence similarity, 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Md. Sagir Ahmed,1 Mohammad Mamun Chowdhury,2 Monzillur Rahman,3 and Luthfun Nahar1 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 nutritional value. The ichthyofaunal diversity of the country is under increasing 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 sustainable utilization of wetlands. Results: An attempt has been made for the genetic identification of SIS through DNA barcoding, a molecular method that uses a short standardized DNA sequence as a species identification tool. The standard 652 base-pair region of the mitochondrial cytochrome oxidase subunit I gene (COI) was sequenced in freshwater fish specimens from Taguar Haor. So far, we have sequenced 50 SIS species, and the high efficiency of species identification is demonstrated 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 knowledge of genetic provenance. These include captive breeding and restocking 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 (antlions) 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 synonyms. Antlions are generally a complex group to identify, and taxonomic 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 explored 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 Genome Vol. 58, 2015 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 elsewhere. The intraspecific distances in all the species were lower than the distances to their nearest-neighbour species. Assignment of the morphologically 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 species 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 Yoccoz1 1UiT – The Arctic University of Norway, Tromsø, Norway. Grenoble Alpes, LECA Grenoble, France. of Southampton, Southampton, UK. Corresponding author: Inger Greve Alsos (e-mail: inger.g.alsos@uit.no). 2University 3University Background: Environmental DNA (eDNA) extracted from sediments has promise as a new proxy in studies of modern biodiversity and palaeobiological 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 (modern, 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 taphonomy 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 quantitative 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 Elisabetta1 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 information, have been adopted by authorities to improve food safety levels. Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts Furthermore, the analysis of DNA barcodes allows for efficiently counteracting 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 headquarter 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 reproducible 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 investigation 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 Mediterranean 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 importance of the correct knowledge of consumed fish species is represented 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 Förster2 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@uniwuerzburg.de). Background: The internal transcribed spacer 2 (ITS2) is a wellestablished 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 GenBank is tedious manual work, which is prone to errors due to misannotations. 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 extracted from GenBank. Further, it offers tools for Hidden Markov Models (HMM) annotation, secondary structure prediction, alignment, and tree calculation. However, it lacked the ability to taxonomically 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 confidence 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 facilitates the broad usage of ITS2 as a DNA barcode. The integration into the ITS2-workbench provides the means to also use the rich phylogenetic methods and supplementary reference sequences for further 187 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. Costa1 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 obtained 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 taxonomically 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 geographic 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 faunal groups as the amphipods, DNA barcodes offer great potential to assist taxonomic revisions and inventories, species discovery, and routine 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 Affonso1 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 taxonomically controversial group because of overlapping morphological traits among species and the lack of uniformity in specialized Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 188 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 (cytochrome c oxidase subunit I – COI and 16S rRNA genes), traditional morphology, and cytogenetics. Results: Most of samples were morphologically identified as Bothus ocellatus while some individuals from the coast of Bahia, northeastern Brazil, were clearly distinguished from congeneric Atlantic flounder species, probably corresponding 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 identification 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 Affonso1 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 Brazilian coast, South Atlantic, using cytochrome c oxidase subunit I (COI) and 16S rDNA sequences to test the efficiency of this methodology in identifying species in this fish group. Results: A total of 16 species were identified 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 reciprocally 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 intraspecific 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 studies. Furthermore, the genetic divergence between samples from Brazilian and Caribbean populations reinforces the isolation of both Genome Vol. 58, 2015 biogeographic regions, possibly imposed by the Amazonas outflow. Therefore, Atlantic flatfish require taxonomic revisions, and morphological 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 Garin1 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 species co-habit, resource partitioning is expected to occur, a mechanism 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 limitations, allowing species-level identification of consumed prey. In this study, we analyse the dietary niche overlap between the sympatric 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 elucidate subtle but functionally relevant differences between the consumed prey of these two bat species. Results: Using DNA metabarcoding we identified 94 MOTUs from the guano of both species, from which we were able to identify 65% in BOLD. Analysis of niche breadth and overlap showed that the diet of both bats overlaps 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 Kok1 1RIKILT Wageningen UR P.O. Box 230, 6700 AE Wageningen, Netherlands. Customs Laboratory Kingsfordweg 1, 1043 GN Amsterdam, Netherlands. Corresponding author: Alfred J. Arulandhu (e-mail: alfred.arulandhu@wur.nl). 2Dutch Background: Use of illegally traded endangered species (CITES) in products is a major issue throughout the world. In the EU, enforcement is mainly focused at the borders, where illegally imported products containing 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 present in samples such as in food supplements or traditional Chinese medicines (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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 minibarcode 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. Significance: 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 another 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. Significance: 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 Jamil1 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 189 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 ecological zones of Punjab Province of Pakistan and subjected to isolation, 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 classification, and the current study also supported the classification by forming each species into a single clear clade. Neighbour-joining and maximum parsimony phylogenetic trees were constructed to visualize the formation of species clades, and the results were the same in both phylogenetic tress of neighbour-joining and maximum parsimony and for both the genes ITS and 18S rDNA. Our study suggested that both ITS and partial 18S rDNA are equally potential in distinguishing 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. Reducing the effects of stem-end rot of mango will resolve the socioeconomic 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 Kajihara1 1Department of Natural History Sciences, Graduate School of Science, Hokkaido University, Japan. 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). 2Department Background: Knowledge of the native earthworm fauna of the Philippines 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 attempts 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 morphospecies. 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: Results 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. Published by NRC Research Press 190 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 costefficiency. 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 unexpected 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 definitions 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-feky1 1Biotechnology Department, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt. Research Center, Geza, Egypt. 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). Genome Vol. 58, 2015 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 Sizani4 1Department of Biological Sciences, Federal University Otuoke, Bayelsa State, Nigeria. 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). 2Department 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 maturase 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 validation 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 fragment analysis were published on BOLD for barcoding as a standard coding marker translation matrix. 2Agricultural 3Department DNA barcoding is widely used for biodiversity studies based on standard 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 results showed that Atpf and rbcL have a significant potential in distinguishing between the ambiguous Poaceae plants. We have also attempted to pursue a bioinformatics approach by retrieving 10 chloroplast genomes for different Triticum species and subspecies from NCBI and using a python script to separate each gene in these genomes. CLC software has been used to perform multiple sequence alignment for 80 chloroplast genes, and the intragenic regions between 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. However, 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 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 engages 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 arthropods 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 different 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 invaluable for differentiating difficult taxa, permitting rapid field sampling by non-specialists, and matching up different life stages of myrmecophilic 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 amplified 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 populations 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 neighbourjoining method with 500 replicates for bootstrap using CLC software. The Punjab population of A. atkinsoni was found to be genetically similar to the Lucknow population, with 0.2% nucleotide difference, but it was genetically distinct from the A. atkinsoni population of Chidambaram. 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, respectively. Significance: This study establishes the prevalence of Amritodus atkinsoni in two districts of Punjab. However, as available reports establish existence of different species, numerous samples from different 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 Barajas1 and Laurence Packer2 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 reproduction 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 countries 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 pollination. 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 importance 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 important in meliponiculture (Meliponini). The following stage is the accomplishment of new faunistic projects, to organize more specimens 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. 191 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 undetected 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 differentiation within species. Birds are an ideal model to assess the relationship between previously described phenotypic variation and neutral genetic 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 delimitation. 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 subspecies. 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 selective 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 limitations 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 sexually dimorphic genus of Neotropical fish, based on sequencing of cytochrome 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. Morphometric 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 Published by NRC Research Press 192 new species of Nematocharax established after the uprising of the Diamantina 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 evolutionary units or cryptic species were identified. In addition, we recommend 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. Hik1 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 1Department of Biological Sciences, University of Alberta, Edmonton, AB T5N 0R5, Canada. 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). 2Canada 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 implemented in BOLD. We also investigated some unique ecological characteristics of this new subspecies, including diet breadth and responses 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 database. 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 spectrum than High Arctic populations. There is also evidence that caterpillar diets and physiology are influenced by experimental warming. Significance: These southern, alpine populations of G. groenlandica beringiana are of both biogeographic and ecological interest. Populations inhabiting arctic and alpine environments are subjected to different environmental pressures that may translate into localized ecological, physiological, and behavioural adaptations. Comparisons of arctic and alpine populations can help us to understand how species may respond to recent and rapid environmental changes, particularly 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 recognized for over a century: coccidia, piroplasms, plasmodia, haemogregarines, 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 mitochondrial (mt) cytochrome c oxidase subunit I (COI) used as a “DNA Genome Vol. 58, 2015 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 taxonomically 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 genera. Genome content and gene orders are largely conserved within major apicomplexan groups but genome organization varied dramatically between 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 delimitation as well as information on deeper evolutionary relationships. Significance: DNA barcoding at the mt COI locus is an excellent specieslevel delimiter for most coccidia and, with some procedural modifications, could provide reliable species identifications from mixed samples. Multiplexing next-generation sequencing technologies could provide simultaneous enumeration of known species and identification of previously 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. Hanner1 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 structure, function, and behaviour of ecosystems, but methodological limitations 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 studies 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 increase dietary and food-web resolution and establish food-web structure. 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 resolution. 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 feeding links, increased intraguild predation (omnivory), and more predator 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 unprecedented and highly detailed manner, improving food web resolution and changing our understanding of food web structure in ways that have implications for their stability and dynamics. Highly resolved dietary data based on DNA barcodes have additional applications, 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 Published by NRC Research Press Abstracts improve our ability to establish structure, monitor species, and detect change in ecosystems. Long-term monitoring of tropical arthropods and DNA barcoding Yves Basset Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 insects. However, the effects of climate change may soon be more pervasive. Disentangling the short-term effects of local environmental factors and seasonal variation from the long-term effects of climate change on tropical communities can be challenging because 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 assemblages of arthropods, driven primarily by climatic cycles and changes, as opposed to short-term stochastic changes. The Initiative is currently active at nine tropical rainforest sites around the world. In this context, DNA barcoding is used to refine delineation of insect morphospecies 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 population 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 historical data, with an example of an analysis of recent butterfly extinctions 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. Because 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 generations). 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 succeeded 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 collected 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 193 Naturalis Bioportal (http://bioportal.naturalis.nl). Significance: The current 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 barcoding, 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, various projects have already been started using the DNA barcoding pipeline 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 Leese1 1Ruhr University, Bochum Universitaetsstrasse 150, D-44801 Bochum, Germany. of Kassel, Heinrich-Plett-Strasse 40, D–34132 Kassel, Germany. Corresponding author: Arne J. Beermann (e-mail: arnebeermann@googlemail.com). 2University Background: DNA barcoding has often been demonstrated to be superior 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 research and over 1800 reported metazoan species, the Breitenbach is one of the ecologically and taxonomically best-studied freshwater ecosystems. 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 unrecognized 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: Morphological identifications by expert taxonomists provide reliable and high-resolution biodiversity information even without DNA barcoding. However, in most cases (in particular ongoing biomonitoring programs), species identification is performed not by taxonomic experts and is often done using immature life stages of freshwater insects. This leads to a significant mismatch between the true and the inferred stream biodiversity, which has potential negative consequences 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 Wall4 1University of Waikato, Hamilton, New Zealand. of Pretoria, Pretoria, South Africa. Young University, Provo, Utah, USA. 4Colorado State University, Fort Collins, Colorado, USA. Corresponding author: Clare Beet (e-mail: clarebeet17@gmail.com). 2University 3Brigham In order to predict future changes in biodiversity for Antarctic terrestrial ecosystems, an accurate assessment of current levels of biodiversity 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 194 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 tolerances, 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 undertaken in the early 1960s. Significance: This research will contribute to our understanding of the evolutionary history of the region and provide an important baseline inventory against which to monitor future changes as well as enable protection of biodiversity hotspots for Antarctica's terrestrial biota. Genome Vol. 58, 2015 amplicon sequencing of one of the standard DNA barcoding markers (chloroplast rbcLa) on the Illumina MiSeq platform. Using pollen samples 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 demonstrating 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 sequencing 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 pipeline, development of methods to correct for bias in ptDNA copy number and amplification bias, and inclusion of multiple DNA barcoding loci, would enable routine use of these methods in a range of fields. Assessing the diversity of New Zealand freshwater “EPT” macroinvertebrates Clare Beet,1 Ian Hogg,1 Brian Smith,2 Kristi Bennett,1 and Gemma Collins1 1University of Waikato, Hamilton, New Zealand. Institute of Water and Atmospheric Research (NIWA), Hamilton, New Zealand. Corresponding author: Clare Beet (e-mail: clarebeet17@gmail.com). 2National 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 bioindicators 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 misidentifications 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 insect 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. Brosi1 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- 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 Muasya1 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 various levels (populations and species) and may reveal fundamental processes 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 barcoding 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 phylogenetic 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 regularities 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 forest. The impact of this fragmentation on species interactions is poorly Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts understood, and so it is important to assess any potential ecosystemwide 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 sequencing, a suite of barcodes can be taken from a mixed slurry of tissue. Using this combination of techniques, faecal matter and ectoparasites from 24 forest bat species from the SAFE project (Sustainability of Altered Forest Ecosystems, based in Sabah, Malaysia) have been collected along a gradient of habitat disturbance. Over the coming 2 years the site is being subjected to experimental habitat fragmentation. During this period further samples will be collected over a time-series, allowing for analysis of the temporal impacts on the studied plots of the deforestation of the surrounding matrix. Complementing 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 ecosystem structure and stability across three trophic levels along a timeseries 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 fragmentation 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 molecular 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 Bhaskar2 1Centre 195 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 environmental 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 parameters. 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 relationships 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. 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). Phylogenetic analysis, species identification and delimitation of New Caledonian geckos and skinks using DNA barcoding 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 proposed 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, dolichos 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 abovementioned 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. Intraspecific 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 1Villanova Justin Bernstein,1 Aaron Bauer,1 Todd Jackman,1 and Yun-yu Wang2 University, Villanova, Pennsylvania, USA. Institute of Zoology, CAS Kunming, China 650223. Corresponding author: Justin Bernstein (e-mail: jmbernst223@gmail.com). 2Kunming 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 anthropogenic 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 facilities and conservation organizations. DNA barcoding would particularly benefit conservation efforts in New Caledonia. New Caledonia has several 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. Published by NRC Research Press 196 The School Malaise Trap Program: the benefits of citizen science for barcoding Emily Berzitis, Vanessa Breton, and Dirk Steinke Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 contributing 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. Results also suggest that offering the program to schools located in already heavily-sampled areas can still be scientifically (and educationally) 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 demonstrated 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 Bank1 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 economically. Their negative impacts in Africa are extensive and call for a renewed commitment to better understand the correlates of invasion 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 duration 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 counterparts. Non-natives that are more closely related to the native species 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 promoting establishment. We suggest that differences in the evolutionary distances separating the native species pool from invasive and noninvasive species might help resolve Darwin's Naturalization Conundrum. Genome Vol. 58, 2015 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. Ravikanth2 1School of Bioscience and Technology, VIT University, Vellore, Tamil Nadu, India. 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). 2Ashoka 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 surrounding 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 important dye-yielding plants to assess authenticity of natural dyes. Multiple accessions of the dye-yielding plants were collected from different geographical locations in South India. For each species, a Biological Reference 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 taxonomy 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 contribution, we provide an overview of key barcoding activities within Bangladesh. 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 submitted 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 & Fisheries Department barcoded 17 species of aquatic organisms and submitted 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 research in Bangladesh in the year 2007. All individuals who received training 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. Published by NRC Research Press Abstracts 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 Creer1 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 taxonomic identification of biota, such as macroinvertebrates. Shortcomings 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 ecosystem 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 temporal 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 systems. 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 arthropod taxa, many spiders are difficult to identify morphologically. Therefore, DNA barcode libraries can help to alleviate these identification challenges. This study provides a DNA barcode reference library 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 Ontario that employed diverse methods, including hand collecting, sieving, 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 belonging 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 197 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 barcodebased 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 Canadian 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 detected through DNA barcode analysis, the poor resolution of morphological 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 variability 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). However, 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 substantial 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 separate 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 successful: in only 250 years humanity has generated stable species descriptions for over 2 million taxa, and these now underpin all of biology, including evolution, ecology, conservation, and bioprospecting. This Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 198 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 meiobiota (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 defines 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 binomen 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. Wilson2 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 development 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 endangered 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 occupancy 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 sampled 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 quantitative 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 substantial 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 habitat occupancy, as well as for informing targeted sampling by conventional capture methods. Genome Vol. 58, 2015 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 serious 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 biodiversity 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 standardizing the protocols for exchange of genetic resources across the iBOL network; in particular, the development of a universal Material 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 international practices of genome data sharing (e.g., Fort Lauderdale Principles) and be inclusive of iBOL partner countries that have not ratified or acceded to NP. Next, it is important to alleviate discrepancies 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 international collaboration in collecting, aggregating, and sharing biodiversity genomics information covered by NP. It should use its unique position to provide a strong and consolidated voice on relevant policy developments at both international and national levels. Building a coordinated network-wide set of protocols for specimen transfer will ensure that national sovereignty over genetic resources is exercised without undermining basic biodiversity research. Status report on barcode coverage for invasive alien species Alex Borisenko,1 Junko Shimura,2 and Robert Hanner1 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 terrestrial 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 Partnership was developed to help identify and track invasive species in an effort to aid their management. Molecular methods like DNA barcoding provide a means of facilitating the rapid and accurate identification of invasive species for all users, but the extent of their coverage in barcode reference libraries is unknown. Here we perform a gap analysis 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 socioeconomically relevant taxa. There is at least some sequence data available to aid the identification of nearly all listed invasive alien species. However, the number of barcode records for each species varies considerably, as does the quality of the meta data associated with those records. Significance: DNA barcoding is poised to support identification efforts of invasive alien species and to facilitate biomonitoring Published by NRC Research Press Abstracts programs using environmental or eDNA given a relatively modest investment in validating and expanding the barcode libraries currently available. 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 Meierotto2 DNA barcoding of larval fish and egg samples produced from the driftnetting of two Lake Simcoe tributaries 1U.S. Danielle Bourque and Robert Hanner Background: Rapid climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Conventional inventory and monitoring approaches are generally 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 systematically 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 nextgeneration sequencing (NGS) methods. Initial library construction included specimens from the grid (t1) and additional local sampling. Regionally, we have been building a DNA barcode library from material at the University of Alaska Museum. Real-world tests of the usefulness 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 identifications 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 statistically 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. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 199 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 amplified for all samples through polymerase chain reaction (PCR) using C_FishF(R)1t1 or VF(R)1i_t1 primers. Sanger sequencing was performed 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 samples 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 extraction 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. Boutain1,2 and Matthew R. Boutain3 Fish & Wildlife Service, Kenai National Wildlife Refuge, P.O. Box 2139, Soldotna, AK 99669, USA. of Alaska Museum, 907 Yukon Dr., University of Alaska Fairbanks, AK 99775, USA. Corresponding author: Matthew L. Bowser (e-mail: matt_bowser@fws.gov). 2University Ecology and systematics of New Zealand deep-sea squids Heather E. Braid and Kathrin S.R. Bolstad 2Department 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: 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 phylogenetic 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 bioinformatics, 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 interdisciplinary research on historical and contemporary biological collections, 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/. Background: New Zealand has one of the highest biodiversities of squid and octopus species reported for any country to date. Although 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 particular, 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 integrative 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 traditional 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 1Botanical Research Institute of Texas, 1700 University Drive, Fort Worth, TX 76107, USA. of Botany / Department of Tropical Plant and Soil Sciences, University of Hawai'i at Maˉnoa, 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). Published by NRC Research Press 200 are vital for species conservation and understanding ecological relationships. Many of the locally occurring deep-sea species are commercially 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 resources. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Affonso1 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 established as a powerful approach to access marine biodiversity worldwide. 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 numbers, taxonomic identification, and COI sequences of all specimens were uploaded to BOLD (project: BAHIA). All species were discriminated by a neighbour-joining tree using the Kimura-2-parameter distance model (bootstrap values = 99%–100%). The maximum intraspecific 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 sequences were useful to reveal cryptic diversity within Eucinostomus (mojarras). A total of 82 specimens of morphologically similar mojarras, 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 efficiency 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 Wilson1 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 Genome Vol. 58, 2015 showing distinct seasonal patterns of activity are thought to be at higher risk of climate-related extirpation. Tropical species are generally 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 seasonal changes in activity, we have deployed (since October 2014) Malaise traps at three stations and will use metabarcoding (bulk DNA extraction, PCR, and high-throughput sequencing of COI) to characterise the diversity within and between weekly samples. Before proceeding 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 amplification 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 detection rates after bulk PCR and high-throughput sequencing (80%–90% of input species), but analyses were complicated by putative heteroplasmic sequences and contamination. The manual pipeline produced similar or better outputs compared to the simple metagenomics pipeline (1.4 compared to 0.5 expected:unexpected Operational Taxonomic 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 Braukmann1 and Sasa Stefanovic2 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 Ericaceae, using select sampling across the trophic spectrum and a nextgeneration sequencing approach, including multiple representatives from the partially mycoheterotrophic pyroloids and fully mycoheterotrophic 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 structural rearrangements of the plastome within the family, including fully mycoheterotrophic species, is an increase in forward and palindromic 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. Surprisingly, 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. Published by NRC Research Press Abstracts 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 Cottenie3 1Biodiversity Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 observational 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 insects, in particular caddisfly (order Trichoptera), beetle (order Coleoptera), 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 diversity was used to establish at which spatial scale(s) variation in the distribution of species occurs. The partition analysis, in turn, informed 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 diversity occurred at the smallest spatial level (mean = 30% on individual rocks). Distributional variation in species was greatest at the kilometer scale (where beta-diversity was 37% and 41% for filterers and grazers, 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 aggregation (p = 0.087). Significance: We introduced a novel combination of analyses—diversity partitioning followed by checkerboard analyses—that facilitated linking patterns of segregation to competitive 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 barcoding 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 presented 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 directions for DNA barcoding of pollen. Results: We present preliminary results on shotgun sequencing, which holds promise for highly resolved 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 overview of the field of DNA barcoding of pollen. Significance: DNA metabarcoding 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. 201 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 Fooks2 1The Pirbright Institute, Ash Road, Woking GU24 0NF, UK. and Plant Health Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, UK. 2Animal 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) transmission elsewhere. Enzootic arboviruses have not been recorded in the UK, although serological studies in birds have reported virusspecific 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 barcoding for vertebrate species identification in blood-fed females following 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 species that mosquitoes have fed on. Moreover, the same sample can be screened for the presence of pathogens, and there is sufficient invertebrate 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 Hik2 1School of Public Health, University of Alberta, Edmonton, Alberta, Canada. of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada. Corresponding author: Janis Geary (e-mail: janis.geary@ualberta.ca). 2Department 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 database 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 affiliations 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 capabilities 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 researchers 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 Published by NRC Research Press 202 economies and developing countries. China has become a leader in plant barcoding. While the taxonomic applications still dominate in the literature, 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 volume 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Kylie Bucalo,1 Jennifer M. Cruse-Sanders,2 Alex Reynolds,3 Alvaro Perez,4 and Kevin S. Burgess5 1Columbus State University, Atlanta Botanical Garden, 02 State Bridge Rd, Apt 8402, Duluth, GA, USA. 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). 2Atlanta 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 genera that are considered to be complex, where traditional morphologybased 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, identify 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 factors promoting Neotropical diversification, but the role of other highland 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 Genome Vol. 58, 2015 Argentina. The analysis of genetic divergence and haplotype networks, 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 differentiated from those of other regions of Argentina. In three of the remaining 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 analyses including more molecular markers, morphology, and vocalizations 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 sequences can constitute an initial step towards understanding evolutionary 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 identification are necessary. DNA barcoding can provide this. In this context, the Global Malaise Program has been established a few years ago, and Argentina 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 collected every week for 2 years. In total, 34 103 specimens were obtained from the first 22 bottles sampled throughout the first year, which constitutes 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 dominated 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 Hill2 1Department of Biology, Columbus State University, Columbus, GA, USA. Entomological Museum, Mississippi State University, Starkville, MS, USA. Corresponding author: Kevin S. Burgess (e-mail: burgess_kevin@columbusstate.edu). 2Mississippi 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts Mississippi, grasshoppers were captured, identified, and held in test tubes until they produced a frass pellet. They were then released, unharmed. Plant DNA was extracted from the frass, and the DNA was amplified 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 species. 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 identity, 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 Prairie plant species, our study demonstrates the utility of DNA barcoding to non-destructively determine the diets of grasshoppers and provide insight 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 Jeon2 203 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 morphologically 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 different 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, including 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 barcoding is important for determining possible species-level effects of environmental toxicant exposure across various levels of biological 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. 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 Olethreutinae 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 specialists. This study aimed to analyze and clarify the generic relationship of the two tribes in Korea using their COI gene. We used all available specimens 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 parsimony. 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 Spilonota 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 Hajibabaei1 1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, Canada. 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). 2Laboratory Background: Laboratory-based ecotoxicological bioassays aim to evaluate the potential risk environmental toxicants pose to aquatic organisms, including common bioindicator taxa such as the burrowing mayfly (Hexagenia limbata and Hexagenia rigida). When col- Characterization of the COI gene in Carollia perspicilata (Chiroptera: Phyllostomidae) from Amazonia Tuane Carvalho,1 Leonardo Trevelin,2 and Juliana Cordeiro1 1UFPel, Pelotas, Brazil. Paraense Emílio Goeldi, Belém, Braazil. Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br). 2Museu Background: The bats of the genus Carollia are among the most abundant 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 b gene analysis shows that the species of this genus can be phylogenetically 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 characters, 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 Published by NRC Research Press 204 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 Abbott1 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 community dynamics, ecosystem functioning, and global processes. In the marine environment, the taxonomy and distribution of most organisms are poorly known. As ecologists strive to understand increasingly complex processes, involving all species interacting within communities, reliable and accessible taxonomic tools to identify species in broad community-level datasets are needed. This study aims to document 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 ecological 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 highthroughput integrative taxonomic approach to generate fast and accurate 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 uncertainty. 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 opportunities for ecologists studying complex natural communities. The molecular inventory of native macro-species generated provides baseline 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 Steinman4 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 pervasive problem, posing serious economic, health, and environmental concerns. While DNA sequencing holds great promise for species authentication monitoring, such methods must be backed up by robust reference DNA sequence databases and statistically-sound sample col- Genome Vol. 58, 2015 lection protocols to track changes in trading practices. This presentation will describe our 5-year research effort in South Africa to systematically harness the power of DNA barcoding to detect, monitor, and deter fish mislabeling in the country. Results: Based on rigorous surveys of fish species occurrence on the South African market, reference specimens from 53 commonly available species were collected 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 demonstrated some minor improvements, with 27 of 149 (18%) samples collected 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 Chable1 and Arely Martínez Arce2 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 prevention 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 determined 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 between them. Furthermore, two individuals of Ae. (Ochlerotatus) angustivittatus 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 under the same BIN. Finally, in Limatus durhamii, one individual showed an intraspecific K2P distance of 3.43%. The total number of BINs assigned 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 existence 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 mosquitoes in the Quintana Roo State. Published by NRC Research Press Abstracts 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. Cristescu1 1Department of Biology, McGill University, 1205 Docteur Penfield, Montréal, QC H3A 1B1, Canada. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Great Background: Zooplankton community composition acts as a powerful 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 organisms. Metabarcoding of environmental samples offers the opportunity to rapidly characterize local biodiversity and potentially detect the early stages of species invasions. Results: Mock zooplankton communities were first used to assess the most effective bioinformatics approach to identify species from taxonomically diverse metabarcoding 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 nonnative species at low abundance, showcasing the breadth and sensitivity of metabarcoding. Zooplankton communities were distinct among ports from different coasts, and biodiversity profiles distinguished 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 community composition. Despite the need for more extensive reference databases, 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 application of metabarcoding to monitor biodiversity and for the early detection 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 205 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 formalinfixed specimens, demonstrating that formalin collections can expand genetic databases. Significance: This is the first effort to compile a reference library of DNA barcodes that provides specieslevel identifications for reptiles and amphibians across a broad geographic area. DNA barcodes from North American herpetofauna were used to quickly and effectively flag errors in museum collections, and cases of BIN splits and merges successfully identified taxa belonging to deeply diverged or hybridizing lineages. This study also highlights the merit of further investigation into obtaining 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 Song1 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 barcodes, are informative for studies of species identification and phylogenetic relationships, especially in complex plant lineages such as Aconitum. We derived three complete chloroplast genomes belonging to the different subgenera of the toxic Aconitum, aiming to discover new evidence for taxonomic and identification studies. Results: Three complete chloroplast genomes of Aconitum were derived using high-throughput sequencing 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 consisting 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. E. Anne Chambers1 and Paul D.N. Hebert2 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 distribution 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 specieslevel coverage. Results: This study constructs a reference library of DNA barcodes for North American reptiles and amphibians and assesses their applicability as a technique for species delimitation. This study also examines the correspondence of current species boundaries 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 congeneric sequence divergences were 16% and 14% in amphibians and reptiles, respectively. BIN assignments corresponded perfectly with current species boundaries in 58% of these species. Barcode sharing 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 ecosystem in Central Kerala, home to a wide variety of fish with many unknown possible endemic species. The proposed project of DNA barPublished by NRC Research Press 206 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 identification 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 illuminate the taxonomic identity of fishes for future works. Apart of producing 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 Lavergne1 1LECA, CNRS/UJF Grenoble, France. CEA Evry, France. 3Tromsø University Museum Tromsø, Norway. Corresponding author: Eric Coissac (e-mail: eric.coissac@inria.fr). 2Genoscope, The DNA barcoding initiative, proposed in 2003, represented a big step forward in standardized DNA-based species identification. It corresponds 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 barcoding initiative was very successful and led to the collaboration of many teams around the world, producing extensive reference databases. However, the standard barcodes were designed in the context of Sanger sequencing, 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 extended 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 repeats, 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 sequencing. 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 Collins1 and Ian Hogg2 1University of Waikato, 25 English Street, Saint Andrews, Hamilton, 3200, New Zealand. of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand. Corresponding author: Gemma Collins (e-mail: gec9@students.waikato.ac.nz). Genome Vol. 58, 2015 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 separated into two distinct groups (1.6% divergence), with one haplotype group comprising 80% of the sequenced population. Based upon sampling every 2 h, air temperature was the strongest predictor of activity between the two haplotype groups (R2 = 0.56). Upon adding subsurface soil temperature, relative humidity, and photosynthetically active radiation, explanatory power increased to R2 = 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 advantage, which could result in decreased genetic variability within populations. We suggest that spatial and temporal monitoring of relative proportions of COI haplotypes may provide a subtle measure of biological response to environmental changes within Antarctic terrestrial 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 Knox3 1University of Waikato, Hamilton, New Zealand. 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). 2Cawthron 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 sequence divergence (3.4%–39.0%). When compared with available sequences on GenBank and BOLD databases, we found that 18 species had sequences that were highly divergent (>6.6%) from their conspecifics elsewhere, including Lecane bulla (19.7% divergence) and Brachionus 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 international 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 monitor water quality, the sequences can also be used to develop local, DNA-based, water quality monitoring programs. 2University Background: In Southern Victoria Land (SVL), Western Antarctica, Gomphiocephalus hodgsoni is the most abundant and widespread springtail species. It is genetically diverse, and over 70 mitochondrial cytochrome 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 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 Martins1 1UFPel Pelotas, Brazil. Campinas, Brazil. Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br). 2Unicamp Background: Schistosomiasis is a chronic and acute parasitic disease caused by bloodworms of the genus Schistosoma, one of the most comPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 endemically 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 discrimination 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 barcoding 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 barcoding 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 physiography and the presence of many surrounding islands have facilitated 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 relation to continental ones, considering the variation of adaptation between 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 analyses detected several islands populations with low haplotype and nucleotide 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 207 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 differences independently if they are of the Cricetidae or Heteromyidae families. The insular endemic species are in a range of genetic variation 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 situation and future conservation strategies. Today, five insular endemic species are recorded as having gone extinct from the islands surrounding 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. Costa3 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 major components of marine benthic communities. As part of a wider effort to compile reference libraries for dominant benthic invertebrates of the NE Atlantic, we used DNA barcodes to investigate the diversity of gastropods from continental Portugal and the Azores Islands. Results: Sequences of the cytochrome c oxidase subunit I barcode 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 assigned 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 considered 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 ulyssiponensis and Patella aspera identified interchangeably as P. aspera and P. ulyssiponensis. To this dataset, we added publicly available COI-5P sequences of the same or taxonomically close species, to inspect for DNA barcode discriminatory ability and data congruence. In total, we examined 183 specimens belonging to 51 species. Significance: The percentage 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 revision and DNA barcode-based probation of the supposedly well-known gastropod fauna from the European Atlantic coasts. Published by NRC Research Press 208 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 Oliveira1 1Universidade Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 evolutionary units in an attempt to help with the problems associated with the analysis of distinct sources of knowledge. Results: We analyzed 228 samples assembled in 53 distinct morphospecies. However, traditional analyses, 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 probably are species complexes. The geographic data helped to understand deep genetic divergence among populations present in distinct river basins, and their ecological characterization suggested that species from headwater drainages have greater genetic segregation between populations. 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 Malca2 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 temperate seas. While these small tunas are economically and ecologically important, little is known about their early life history. They inhabit the western central Atlantic, and while the morphological identification of adults is relatively easy, the identification of their larvae is difficult due to similarities among the two species with regards to morphology and pigmentation patterns. Currently, identification during the larval stage is only possible for sizes greater than 5 mm. Major taxonomic difficulties arises when attempting to identify larvae less than 5 mm SL, because there are no distinctive morphological and pigmentary characteristics between them. In this context, we present preliminary results from DNA barcoding used to successfully 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 submitted 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. Genome Vol. 58, 2015 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 invasive 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 infrastructure, and experimental designs. The field is transitioning from barcoding 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 barcoding 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 invasive 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 Freeland1 1Trent University, 781 Water St., Peterborough, ON K9H 3N5, Canada. 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). 2Fisheries 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 populations. 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 (Unionidae) 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 conditions, 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 biodiversity within the tropics is inadequate due to the inability to comprePublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts hensively sample and identify specimens in these hyperdiverse ecosystems. DNA barcoding is an excellent tool to overcome this barrier. 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 broadleaf forest sites roughly 2 km apart with a 300 m elevational difference. 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%), Lepidoptera (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 accumulation curves and the high proportion of singletons (62%). Various arthropod groups between the sites displayed more phylogenetic dissimilarity than expected based on a null model, demonstrating that variation between sites can be detected despite incomplete community 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 209 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 Bank2 1McGill University, Canada. 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). 2University Background: There is growing evidence for a link between biodiversity 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 barcode phylogeny of southern African trees, we show that human population 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 generating variation among living organisms are also key to the provisioning 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. Hadi Darhuddin,1 Aditya Hutama,2 Frederic Busson,3 Sopian Sauri,1 Philippe Keith,3 Robert Hanner,4 Renny Hadiaty,1 and Nicolas Hubert5 Barcoding herpetological collections: discovering hidden biodiversity in a hotspot 1Indonesian Biology Institute, Universidad de Antioquia Calle 67 Número 53 – 108, Colombia. Corresponding author: Juan M. Daza (e-mail: jumadaza@gmail.com). 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 islands. 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 128 297 km2, is the 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 molecular screening. Results: Fishes were mainly collected using electrofishing, 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 mitochondrial lineages diverging by more than 2% is higher than the number of nominal species, highlighting cryptic or undescribed diversity. In addition, three new species of amphidromous gobies (e.g., Sicyopus, Lentipes, Stiphodon) are described and each is characterized by private mitochon- Juan M. Daza and Adriana Restrepo 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 coming mostly from the northwestern corner in South America. This region 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 sequenced more than 800 specimens of 217 nominal species. Preliminary analyses show hidden diversity in the amphibian genera Pristimantis, Craugastor, Colostethus, and Bolitoglossa. High Andes assemblage barcodes highlight lineage divergence concordant with mountain 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, suggesting 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 biodiverPublished by NRC Research Press 210 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 Roy5 1CEFE UMR 5175 CNRS, University of Montpellier, 1919 Route de Mende, 34293 Montpellier Cedex 5, France. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 invertebrates in tropical rainforests have been poorly studied from both a taxonomic 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 earthworm communities. DNA barcoding has been proposed as a useful approach 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 Guyana and Gabon, we collected thousands of earthworms in different localities representing the main ecosystems of the region. Specimens were DNA barcoded to delimit molecular operational taxonomic units (MOTUs), 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 ecological 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 Überlandstrasse 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 species 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 environmental 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 Genome Vol. 58, 2015 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 demonstrate how to assess species pools in river catchments. A biodiversity assessment tool such as this has the potential to inform restoration efforts about recolonization potential and become a powerful tool for landscape 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 Li1 1Notre Dame University, Notre Dame, IN 46556, USA. 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). 2Colorado Background: The unintentional transport of invasive species through the global shipping network causes substantial losses to social and economic welfare—an economic externality that affects everyone. Addressing 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 environmental 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 clusters 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 empirical 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 management practices that improve sustainability of coastal ecosystem services. 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 Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. recognized species are actually chromatomorphs representing geographical 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 morphological 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 morphological characters is confusing and is highly error prone during field studies, collection, and classification. This has made identification of bamboo 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 enormous 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) barcode candidates were evaluated for their ability to identify the molecular divergence within Bambusoideae species. Among the tested barcode candidates, ITS was shown to be effective in species discrimination. Bayesian analysis 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 revealed 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 discrimination. The intergenic spacer psbA–trnH was found to be highly conserved without 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 current study proved the importance of DNA barcoding by evaluating the taxonomical lineage of Pseudoxytenanthera that differed from the morphologybased classifications. Authentication of Indian herbal products using DNA barcodes S. Dhivya,1 S.G. Newmaster,2 S. Ragupathy,2 M. Saravanan,1 and R. Sathishkumar1 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- 211 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 industries 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 recorded before through scientific evidence. Our overall findings suggest 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 standards 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, concentration, 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 natural 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. Sathishkumar1 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 considered to be a good choice, especially to identify powdered raw materials, 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 costeffective 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 individual 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 Published by NRC Research Press 212 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 validating 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 E1 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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: Taxonomic 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 genera, and 20.15% within families. The average divergence within conspecific 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 distances ranged from 4.06% to 23.03% (average of 14.45%), with the exception of two species of the genus Odontesthes (O. bonariensis and O. perugiae) in which members showed a minimum genetic distance of 0%. In addition, 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 argentinum, Auchenipterus nigripinnis, and Xyliphius sp.). In addition, groups that deserve further taxonomic study and one exotic species were identified. 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 Marec7 1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, ON N1G 2W1, Canada. 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, České Budějovice, 370 05, Czech Republic. Corresponding author: Vlad Dinca (e-mail: vdinca@uoguelph.ca). 2Uppsala Background: There is a widely recognized need for more comprehensive understanding of global biodiversity patterns. Such information Genome Vol. 58, 2015 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 reproductive isolation due to female mate choice. Additional research has shown that L. sinapis is currently the metazoan with the highest intraspecific 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 concept 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 European 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 evolutionary 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. Ranasinghe3 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 considered 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 facilitated biotic exchanges between the two regions. Since the last connection, the fauna of Sri Lanka has been isolated from the mainland fauna, and as a consequence co-distributed species are expected to show significant amounts of genetic divergence between the populations. We tested this hypothesis by analyzing the mitochondrial cytocrome b gene 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%) monophyletic clades corresponding to Sri Lanka and the Indian subcontinent (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 Published by NRC Research Press Abstracts 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 taxonomy of family Elapidae and also proteomics study to develop specific antivenin for medical applications. The project is funded by the Ministry 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 Uğurlu Aydın, and Aslı Doğru Koca Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 molecular 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 examined for 11 potential barcoding loci. Results: The plant materials have been stored in silica gel from both field locations and herbarium 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 initiation 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 distribution, 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 mitochondrial DNA that provide us with both an identification tool and a measure of genetic variability. Here, we explore how within-taxon haplotype diversity correlates with different ant traits and helps to explain community composition. Results: For example, we expect more haplotype variability among smaller ants with fast generation times. Increased variability is also expected among canopy ants with more profound ecological 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. Significance: We provide the first links between within-taxon barcode variability and community structure. 213 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 invertebrate community within New Zealand's mangrove forests. This project 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 designations. The sequences formed 101 BINs, 39% of which contained specimens 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 investigating 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 endemic blue duck, or whio (Hymenolaimus malacorhynchos), are labour intensive 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 environmental DNA (eDNA) analysis to be developed as a tool for detecting blue ducks in rivers through water sampling. Results: Species-specific primers 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 applications 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 fragmentation has on tropical mammals using common surveying methods, as they can be time-consuming, expensive, and may be less accurate when surveying 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 sequencing 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 survey. The study site is the SAFE (Sustainability of Altered Forest EcosysPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 214 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 addition to spatial differences in communities, temporal shifts and time lags are expected as forest clearance and fragmentation increases in subsequent years. Significance: Previously, this method has been used to detect 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 Borneo'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 Hajibabaei2 1Pace University, One Pace Plaza, New York, NY 10038, USA. Institute of Ontario, University of Guelph, 579 Gordon St., Guelph ON N1G 2W1, Canada. Corresponding author: William Eaton (e-mail: weaton@pace.edu). 2Biodiversity Background: The Franklin Parker Preserve within the Pine Barrens region of New Jersey contains 5000 acres of wetlands habitat, including old growth red maple (Acer rubrum) swamps, cranberry bogs, and former cranberry bogs that have been restored with the goal of developing them into red maple forests. This provides excellent experimental 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 restored 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 representative groups from the Acidobacteria, Actinomycetales, Bacteroidetes, and Proteobacteria), complex C-decomposing fungal groups (e.g., specific 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 restored habitats. The data from this study suggest the use of metagenomic 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 strategies 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 reliable knowledge of geographic patterns of species diversity, as it Genome Vol. 58, 2015 allows the monitoring of anthropogenic shifts, the assessment of ongoing and future impacts, and the best possible allocation of resources. 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 baseline 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 ongoing effort to generate and analyze DNA barcodes for mammals, reptiles (including birds), and amphibians from Brazil and adjacent countries. The project congregates a consortium of 25 institutions, comprising molecular genetics laboratories and zoological collections, 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 Marthinsen3 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 national network to (i) advance barcoding of Norwegian and Arctic biodiversity, (ii) raise funding, (iii) curate barcode reference material, (iv) coordinate and initiate new barcoding projects, and (v) increase public 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 Museum 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 institutions, 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 barcodes) 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, earthworms, 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 vascular plant groups, we are engaged in testing if low-coverage shotgun sequencing of herbarium material is useful to obtain the full plant plastid genomes. Published by NRC Research Press Abstracts Testing primer bias and biomass—sequence relationships in metabarcoding: implications for monitoring of freshwater invertebrate communities Vasco Elbrecht and Florian Leese Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany. Corresponding author: Vasco Elbrecht (e-mail: vasco.elbrecht@rub.de). Background: Metabarcoding combines DNA barcoding with nextgeneration sequencing to reliably identify hundreds of specimens from an environmental sample at once. However, detection rates for speciesrich 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 protocol 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, sequence abundance varied by four orders of magnitude between taxa. Significance: Our experiments show that although biomass can be estimated if only a single species is present in a sample, reliable species biomass or abundance estimates from environmental samples are impossible 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, degenerate primers have been developed. While many COI barcode sequences are readily available in online databases such as the Barcode of Life Data Systems (BOLD), sequence information about the primerbinding 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 genomes are still not available for many groups, which limits the applicability. 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 sequences. 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 degenerate 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. 215 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 Koí̌nek4 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 cladocerans, with many studies on ecology, ecotoxicology, cultures, distribution, 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 morphology, 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 comparison 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 arrangement of the seta and sensilla between the large lobe and the gnathobase, 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. Significance: 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-Gutiérrez1 and Virginia León-Règagnon2 1El Colegio de la Frontera Sur, Av. Centenario Km 5.5, Chetumal, Mexico. de Biologia Chamela, Instituto de Biologia, UNAM, Mexico. Corresponding author: Manuel Eliás-Gutiérrez (e-mail: melias@ecosur.mx). 2Estacion Background: The 2 000 000 km2 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), compared 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 academicians 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 abovementioned 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, considering next-generation sequencing, and looking to integrative taxonomy in response to new questions. Finally, we started an educational program with a mobile PCR laboratory in schools. Significance: In summary, 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 Published by NRC Research Press 216 in the face of the urgent need for a better understanding of the amazing and threatened biodiversity in this region of the world. Genome Vol. 58, 2015 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 Hajibabaei1 DNA barcoding of Skwala stoneflies from north-central British Columbia reveals potential new species Daniel Erasmus, Daemon Cline, and Ray Lyle Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 northcentral British Columbia and Skwala sequences derived from specimens 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 previously 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 Shojaei5 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 barcoding 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 Barcoding (CCDB). The present study analyzed DNA barcodes for 91 specimens 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% divergence) 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 misidentification. 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. 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 barcoding 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 growing species. Due to potential accumulation of plant tissues over time, however, it is uncertain whether below-ground plant diversity is unchanging year-to-year or if it exhibits interannual turnover. Additionally, 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 McAlpine2 1University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada. Brunswick Museum, 277 Douglas Ave., Saint John, NB E2K 1E5, Canada. Corresponding author: Aaron Fairweather (e-mail: fairweaa@uoguelph.ca). 2New 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 invertebrate taxa diversity is hindered by “taxonomic impediments”. The “taxonomic impediment” refers both to unappreciated morphologically cryptic diversity within named species and the paucity of available 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 between cryptic species. Integrating analyses of morphology and DNA barcodes allows for a cohesive analysis of diversity and permits research on the effects of disturbances on these groups. Parks and Protected Natural Areas (PNAs) are provincially protected nature reserves. Many of these have a long history of forestry integrated into contemporary park management, thus creating a natural experiment to study the impact of disturbance on invertebrate diversity. Results: Utilizing Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 undisturbed 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 engineers, seed dispersers, and invertebrate and plant community regulators. Measuring the diversity and phylogenetic structure of this group will provide useful information regarding how anthropogenic disturbance 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 science. 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. Mason3 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, holotypes scattered across many collections, numerous morphologically 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 significant number of cryptic and new species, and confirmed the establishment 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, biodiversity 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. 217 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. Walker3 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 abandoned 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 arthropods 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 bacterial 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 techniques to explore bacterial communities across multiple trophic levels (bat guano, associated arthropods) in bat-occupied subterranean sites (abandoned mines) within USA's southern Arizona region. Our results to date support the hypothesis that bats, and their gut/guano microbiota, drive the bacterial biodiversity and composition of their immediate subterranean ecosystem. In particular, we have observed bat guano pile microbiota affecting arthropod gut bacterial composition, with detritivorous insect gut communities 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 relevant 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 livestockaffecting bacterial zoonotic diseases, further highlighting the need to understand how their gut microbiota propagate through the environment. 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 overcoming limitations of morphology-based methods such as the lack of taxonomic 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 analysis of the DNA barcode library coverage for this checklist, contrast it with one conducted two years previous, and discuss some of the current challenges facing the further implementation of DNA barcoding for phytosanitary 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 Published by NRC Research Press 218 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. prior. Over the last two years, 521 species had newly generated barcode sequences, while 404 had no new additions, and 18 lost sequences, presumably 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 frequently 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 reference 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. Genome Vol. 58, 2015 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 environments 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 surveys 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 sensitivities. This has the potential to improve knowledge of species' distributions to assist management of both endangered and invasive taxa. 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, national 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 examined the ability of current barcode reference libraries in Barcode of Life Data Systems (BOLD) to assist in the identification of a set of larval Lepidoptera 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 identifications made using morphology or barcode-based methods. However, morphology- and barcode-based identification combined significantly increased 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 enhance phytosanitary inspection programs. We also demonstrate the limitation 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 associated with unidentified adult specimens via BINs, which presents an interesting 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 Duncan1 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. Erwin3 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 coextinctions propagate through trophic levels. One challenge to studying coextinctions 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 warming. 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 herbivores, we combined field records with novel molecular methods, identifying 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 obtaining 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 similar susceptibilities to extinction at different elevations. Significance: Fast and accurate identifications of species and their trophic interactions using 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. 1University of Canberra, IAE, Building 3, University Drive, Bruce, ACT 2617, Australia. CSIRO, Land & Water Flagship, GPO Box 1700, Canberra, ACT 2601, Australia. Corresponding author: Elise Furlan (e-mail: elise.furlan@canberra.edu.au). 2CSIRO Background: Environmental DNA (eDNA) is increasingly being used as a survey tool to infer species distributions, yet the sensitivity of the technique has not been carefully evaluated. Imperfect sensitivity, or imperfect detection, is a feature of all survey methods and needs to be accounted for in interpreting survey results. We will present a framework to estimate the sensitivity of both the field and laboratory components of an eDNA survey method and show how these can be combined to estimate the overall sensitivity. We will apply this framework to 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 intensely studied. In BOLD, to date there are only four published projects focused upon insects from the Yucatan Peninsula, three for families and Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 preliminary results include the identification of 16 orders and 76 families of insect. Nevertheless, the major orders Diptera, Hemiptera, and Lepidoptera have only 22 families identified, which is a small number in comparison 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 sequenced specimens show 114 BINs, 61 of which were not previously registered 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 geographically 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 montane 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 delimitation 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 correspond 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-Pérez1 1Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa, Tamaulipas, México. 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). 2Animal 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 phenotypic plasticity, genetic variability, cryptic diversity, and the presence of different life-cycle stages and sexual dimorphism. Thus, the DNA barcode cytochrome c oxidase subunit I (COI) gene for the discovery of cryptic diversity and species complexes in black flies is highly useful. Results: 219 The present study updates the collection data of 257 specimens of dipterans, 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 barcode protocol for further analysis and study. Several vector species belonging to common genera, such as Aedes, Culex, Lutzomya, and Simulium, have now been DNA barcorded which might facilitate their identification. 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 identify 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 barcoding 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 efficiency. 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 specimens. 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, societal, and legal differences can create divide in the global barcoding community over how the shared resources should be managed. The success of iBOL is contingent on promoting use of the resource as well as recontribution 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 comprise the international barcoding community. Results: We completed 44 semi-structured interviews with members of the iBOL community, funding 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 Published by NRC Research Press 220 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 identifies cultural, societal, and legal differences between international participants and stakeholders that are impacting participation in iBOL. We suggest strategies to mitigate identified conflicts and will invite discussion 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 Freyhof2 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 diversity, and to construct a molecular identification system that supports conservation, we have built a DNA barcode library for 498 Mediterranean 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 dependent 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 mismatches tend to occur in recently diverged (Pleistocene- or Holoceneevolved) 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 important 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 efforts. Significance: The 98% extant species coverage of freshwater fish species of the Mediterranean Biodiversity for barcode sequences represents a success story for biodiversity research in a geopolitically complex region. The database will enable the recognition of conservation 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 Boer3 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 insight into the medicinal plants growing in a region. However, accurate taxonomic identification of medicinal plants from markets is challenging, as traded plant materials are sold in processed forms such as dried roots and barks, powdered plant parts, and mixtures. Two methods 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 sequence matching with a priori and a posteriori data from other markers, morphology, ethnoclassification, and traditional knowledge. Genome Vol. 58, 2015 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 correspondence with vernacular names. DNA was extracted from 68 samples 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 nucleotide database and identified using both approaches. Results: Objective 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 specieslevel 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 sequenced 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 between 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 standard 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 primers 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 Hajibabaei1 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 detailed 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 collected in the boreal forest region of Canada. Each HTS sequencing run generates over a million DNA sequences from the organisms contained 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 diversity metrics were then generated based on these data. Significance: All of the standard biomonitoring metrics employed by current practitioners can be replicated using a high-throughput sequencing approach. 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 impact 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 processes 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 approach. Using next-generation sequencing, we generate a barcode database of all arthropod taxa. Based on this reference collection, we determine the identity of taxa within a given sample, as well as associated microbes, and identify host parasite and prey predator interactions. The approach thus simultaneously provides information on the qualitative and quantitative composition of the Hawaiian community. Finally, the sequence data provides preliminary information on genetic relationships between taxa across sites. These data are analyzed by comparing arthropod communities at sites of different age to determine the processes 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 Boonham1 1Fera, Sand Hutton, York, YO41 1LZ, UK. Environment Agency, Bristol, UK. 3Bowburn Consultancy, Bowburn, Durham DH6 5QB, UK. Corresponding author: Rachel Glover (e-mail: Rachel.glover@fera.co.uk). 2EA 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 policy outcomes. In some cases, exploring novel approaches to surveil- 221 lance allows them to achieve more with less. Work at Fera has focused on the development of metabarcoding approaches to identify 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 pathogens 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 airborne 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 efficiency, potentially enabling profiling of samples to achieve multiple outcomes from the same samples. In addition, we are translating a metabarcode approach for diatom community profiling into a high-throughput method to enable improvements in the efficiency, speed, and cost of water quality assessments. Significance: 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 nextgeneration 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 particular premium is placed on quantitative food webs which are especially 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 community 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 sequence regions (RbcLa, TrnH–psbA, trnL, and ITS) obtained for 120 species 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 Published by NRC Research Press 222 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 Packer2 1Museo Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Argentinean Patagonia, and which are key elements in southern South American ecosystems. For instance, C. chloris is among the major pollinators of crops such as raspberry, Chilean hazel, and buckwheat as well as native wildflowers. These bee species are very difficult to identify 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 sequences. 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 sequence 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 important 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. Ranawaka1 1Department of Zoology, Open University of Sri Lanka, Nawala, Nugegoda, Sri Lanka. of Environment, Biodiversity Secretariat of Sri Lanka. of Life and Environmental Sciences, Deakin University, Melbourne, Australia. Corresponding author: Kshanika Goonesekera (e-mail: kshanika@outlook.com). 2Ministry 3School 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 similarity with each other and also contain examples of high morphological 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- Genome Vol. 58, 2015 mented with sequence data from GenBank. It was possible to unambiguously 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 currently 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 Swanevelder1 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 pollination 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, eliminating the need for separating the pollen grains by taxon prior to sequencing. 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. Samples 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 Mota1 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts the most harmful viruses to vineyards wordwide. Despite their phytopathological importance, this research area has been deserted for over fifteen years in Portugal. In recent years, plant health of the Portuguese 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. Nematode surveys have been conducted since 2015 on 30 commercial vineyards 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 species, 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 decreasing 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 plasticity. 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 Proaño4 1PROMETEO, INIAP - Instituto Nacional Autonómico de Investigaciones Agrarias, Estación Experimental de Santa Catalina, Panamericana Sur s/n, Machachi, Ecuador; and NemaLab/ICAAMInstituto 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 region 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. Moreover, 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 223 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-Hernández3 1UANL, Cerro Real 664, Valle Real, Saltillo, C.P. 21205 Coahuila, Mexico. 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). 2CNRCB, Background: Microhymenopterans are used in biological control programs of insect pests as an ecological alternative to chemical toxicants. 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 extraction: (a) DNeasy Blood & Tissue Kit, (b) Protocol with CaCl2 buffer, (c) Method HotSHOT, and (d) Phire Tissue Direct PCR master mix. Using PCR to amplify a ⬃680-bp sequence of the mitochondrial gene cytochrome 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 successfully 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 preparation in Canada balsam for every technique after DNA extraction, including 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 insects, allowing the description of additional biological traits for old museum specimens. Evolving the concept, and use, of DNA barcode libraries Rodger Gwiazdowski1 and Robert Hanner2 1University of Massachusetts, Amherst ng, Amherst, MA 01003, USA. 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). 2Biodiversity Background: DNA barcode library concepts and construction methods 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 biological 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 consortium 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. Published by NRC Research Press 224 DNA barcoding helps to fight against frauds—a case study of authentication of deer products Wai-yan Ha and Foo-wing Lee Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 identifying the species origin of traded goods where the morphological characteristics 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 scientific evidence for identifying fraudulent material, an integrated DNA protocol combining DNA barcode analysis and two deer-specific PCR systems 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 computational analysis have widened the scope of DNA barcoding studies. Here, I will focus on the use of DNA barcode data in environmental biomonitoring programs aiming to assess ecosystem health. A fundamental question in ecological analysis is: how to obtain consistently observed, highresolution biodiversity information. We have developed and used environmental (meta) barcoding approaches using standard DNA barcode 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 availability of DNA barcode libraries allows for more robust taxonomic inference 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 implications. DNA barcoding of Philippine Helicarionidae (Mollusca: Gastropoda) Genome Vol. 58, 2015 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 cytochrome c oxidase subunit I (COI) gene. A further two genera, Hemiglypta sp. (n = 3) and Lepidotrichia 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 occurred 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 revealed most species to be monophyletic with the exception of R. otaheitana; the species has been flagged for taxonomic re-evaluation. Significance: This study provides the first molecular work on Helicarionidae in the Philippines, paving the way for further phylogenetic analysis 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 extinction. 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 unknown 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 absence 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. Paris4 1Museo of Biology, University of the Philippines, Diliman, Quezon City, the Philippines. of Biological Sciences, University of the Philippines, Los Baños, the Philippines. Corresponding author: Ian Kendrich C. Fontanilla (e-mail: ianfontanilla@hotmail.com). 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: Helicarionidae is a very large and diverse family of pulmonate land snails that includes about 150 genera and many hundred species. In the Philippines, 8 out of the 80 endemic species of land snails Background: Ants are a key component of terrestrial ecosystems because they provide several ecological services such as seed dispersal, organic matter decomposition, and soil nutrient cycling. Here, we assess, 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. Fontanilla1 1Institute 2Institute Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 included. The mean intraspecific sequence divergence was slightly over 0.70%, which was 23 times lower than the mean interspecific divergence (16.6%). After removing a few species with strikingly deep intraspecific 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 biodiversity at INP. The existence of these divergent intraspecific lineages was supported by high node support values in complementary phylogenetic analyses. Significance: Taxonomic keys for Neotropical ants are mostly incomplete and based almost exclusively on workers, precluding 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 generation of new taxonomic keys. Lastly, our results support the biodiversity hotspot status of the Atlantic Forest and suggest that ant diversity in INP is currently being underestimated. 225 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 select a primer set with a desirable balance between the two. To illustrate 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 specificity 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 Prosser3 1Bavarian 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 extraordinary variation in mitochondrial DNA in some species. Sometimes this variation is highly clustered so that a species shows a deep intraspecific 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 historical polymorphism. Several splits have likely resulted from introgression, 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 mitochondrial haplotypes in the population after an introgression event. Significance: 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 State Collection of Zoology, Munich Muenchhausenstr. 21, 81247 Munich, Germany. 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). 2Smithsonian Background: One of the major challenges in creating a global database 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 available 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 generation 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 Baillie5 1National 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). 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: The detection of species-specific environmental DNA (eDNA) via real-time polymerase chain reaction (qPCR) is emerging as Background: Honey possesses therapeutic properties that are the result of a range of factors including high sugar content, low pH, hydrogen Daniel Harvey,1,2 Sanjeena Subedi,1 Robert Hanner,2 and Sarah J. Adamowicz2 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 226 peroxide, and bee-derived peptides. Honey also contains antimicrobial phytochemicals, which represent a rich source of leads for the development 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 determining 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 compounds, 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 using microscopic analysis and rbcL DNA metabarcoding (454 and Illumina). Results: DNA metabarcoding provided superior discrimination for some plant families and greater repeatability compared to microscopic analysis. Key species identified in the antibacterial samples included 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. Significance: 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 antibacterial activity may provide new lead compounds that could serve as selective agents against MRSA and other antibiotic-resistant bacteria. Genome Vol. 58, 2015 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, document, 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 classrooms 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 sequenced 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 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 Biodiversity 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 advances 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 incorporate original research into the college biology curriculum through Elisabeth Herniou,1 Julien Thézé,2 Jennifer Cory,3 and Carlos Lopez Vaamonde4 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 ecological 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 delimitation 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 ecological data to investigate the main ecological factors driving the diversification 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 baculovirus isolates, 88 have complete genomes and were used to reconstruct 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 associated 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 species. This study is the first to use a phylogenetic clustering approach, inspired by DNA barcoding practices in Metazoa, for species delimitation in viruses. Our results show that phylogenetic clustering could be used to unify viral taxonomy. Published by NRC Research Press Abstracts Barcodes to investigate the plant visitor community: Brassicogethes spp. Damien Hicks,1 Graham Stone,1 and Pierre Ouvrard2 1School Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 collected 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 pollen 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 identified 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 associated 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 previously 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 delimiting 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 species 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 improved monitoring and research to survey, map, monitor and understand Arctic biodiversity. This requires integrated and repeated data collection following recommended standardized protocols and priorities, and involving Arctic citizens in the survey and monitoring. Support for national and international coordinated efforts such as the Circumpolar Biodiversity Monitoring Program (CBMP) and the Barcode 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 environmental conditions. For example, the identification of a new subspecies of Arctic moth (Gynaephora groenlandica beringiana) in the southern 227 Yukon, formerly known only from the high Arctic, has led to unexpected discoveries of facultative species interactions in these cold, seasonal environments. These southern populations of moths are subjected to different environmental pressures that can translate into localized ecological, physiological and behavioural adaptations affecting other tundra herbviores and plant communities. Species interactions in Arctic food webs are not well documented, and understanding the changing dynamics of these systems will depend on more extensive 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 exposes 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” stemming from both the difficulty in obtaining accurate species-level identifications 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 histories 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 availability influences arthropod communities and biodiversity in mixedboreal 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 structure of spider diversity in Algonquin Provincial Park displayed nonsignificant 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 composition changed depending on the treatment, with 27 species unique to the uncut treatment, 26 species unique to the cut sites, and 33 cosmopolitan 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 impacted 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 standardized evidence for the recorded occurrence of any species at any time Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 228 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 Encyclopedia 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 databasing 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 historical knowledge with large-scale DNA-based biodiversity monitoring 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 knowledge 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 sustainability 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 Boonham1 1Fera Science Ltd., Sand Hutton, York, YO41 1LZ, UK. 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). 2Chief Background: With the continual increases in the importation of commodities into the UK from around the globe, the country is exposed to the risk of introduction of non-native, quarantine pests and pathogens. 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 studied, leading to the first findings of quarantine species and numerous new host records. Additionally, the method is deployed for confirmation 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. Various 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, nematodes 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 development, combined with morphological expertise, to elucidate previously cryptic species; for example, we have developed barcode markers for Leucinoides, a taxon for which effective barcode markers were not previously available. Genome Vol. 58, 2015 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 laboratory 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 adaptation 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 sequencing 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, announced 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 barcoding. To take this forward, governance options for international societies, 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 Conference. 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 Paneto1 1Federal University of Espirito Santo, Alto Univeristário s/n Guararema, Alegre, ES, 29500-000, Brazil. 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). 2Chico 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 identification. 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 identifying 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 Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 sequences 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 inexpensive 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 database for 100 common medicinal plants in Malaysia for species identification 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 coding 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 resolution 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 adulterant 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 conspicuous element of the marine zooplankton. Despite their large size, broad distribution and abundance, the taxonomy of the genus is uncertain. 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 229 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 revealed five lineages of C. capillata s.l. with differing geographical distributions 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 species identification, examination of phylogeographic patterns, as well as to highlight cases of deep intra-specific divergence. This work verifies the need for greater effort in species documentation not only to further understanding of marine biodiversity, but also to aid in conservation. 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 phylogeographic 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, demographic expansion, and long-distance colonization. These processes, in conjunction with dispersal from multiple refugia, have resulted in elaborate spatial patterns. Notably, the Great Lakes region has become a suture zone with habitats maintaining multiple Hyalella species. Significance: 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 furthers 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 Published by NRC Research Press 230 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 Hubert5 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 biodiversity hotspot, the most speciose hotspot of the Indonesian archipelago. 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). Widespread 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 biogeography 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 analyzed. 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 distribution of the genetic diversity for the three species analyzed. Molecular 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 Pleistocene, 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 Lee1 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 cytochrome oxidase subunit 1 (COI) gene were analyzed for the identification 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 Genome Vol. 58, 2015 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 (realtime 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 distinguish 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 genotyping 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 Austin1 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 hundreds 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 (Oligochaeta). 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 produced directly via chemoautotrophic bacteria. Results: A reference database 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 information for multiple individuals and combining the data with stable isotope analysis, insight can be provided into trophic interactions within the ecosystem. Significance: This project will provide critical information 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 processing 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 impacts 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 Iftikhar1 and Muhammad Ashfaq2 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. Sequence variation in the mitochondrial COI-5A gene region (DNA barcode) 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 Published by NRC Research Press Abstracts 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 suggesting 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. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Meyer1 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-Università 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 candidates. 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 evaluate the resolution and discriminatory power of the novel loci. In pathogenic 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 promising secondary barcode candidate for the identification of clinically relevant 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 supplements corresponding to five medicinal plants, each from three different manufacturers: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum, and Trigonella foenum-graecum. To maximize DNA recovery from processed plant materials, oil-free tablets or gelatine capsules were chosen for testing. Listed ingredients consisted 231 of dried aerial parts, seeds or roots; plant extracts or extracts combined 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 degradation 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 reproducible 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). Besides 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 components 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, barcodes 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 mitochondrial 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 barcode 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, abundances, seasonal activities, and interactions with other species. UnderPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 232 standing, predicting, and managing how species respond to climate change and other environmental changes is a critical challenge in environmental 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 laborintensive, 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 computationally intensive ecological research questions. Unfortunately, training 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 collected specimens, and the taxonomic impediment remains in place. Automated identification tools that use morphology (e.g., Leafsnap), sound (e.g., Song Scope) and other characteristics, and online crowd-sourced photo identification services (e.g., iNaturalist, iSpot), are already being used to help citizen scientists identify unknown specimens. DNA-assisted species identification (“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 Institute, 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 caterpillars, 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 suggested many rich possibilities and many stumbling blocks in the attempt to connect centuries of what humanity knows and has tagged with scientific and street names, to the unmistakeable and unavoidable 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 understanding all sorts of biodiversity. Museums and their specimens are dynamic waystations on the path from wild populations to biodiversity 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 Vol. 58, 2015 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. Witt3 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 remarkable genetic diversity in both North and South America. There are an estimated 100 “species” in North America based on COI sequence 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 hotspots, 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 phylogenetic reconstruction using 28S nuclear sequences, coupled with genome size estimation of representatives from each major lineage. Genome size, defined as the total DNA content of the haploid chromosome 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 phylogenetic 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 genome size may have played a role in sympatric and allopatric diversification 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 Jinbo1 and Aino T. Ota2 1National Museum of Nature and Science, Amakubo 4-1-1 Tsukuba-shi, Ibaraki, Japan. 1-19-1-505, Matsudo, Chiba, Japan. Corresponding author: Utsugi Jinbo (e-mail: ujinbo@kahaku.go.jp). 2Koganehara Background: The growth of the DNA barcode library enables comprehensive 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 overlooked geographic structures and unsolved taxonomic problems. Tortricidae is one of the largest families of smaller-bodied moths. This study is focused on the Japanese species of the tribe Archipini, belonging 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 distributed species and to build a DNA barcode library of Japanese Archipini, DNA barcode sequences of the target group were obtained through standard protocols. Together with morphological examination, barcodes were compared with those from other geographic populations registered in BOLD. At present, 35 species of Japanese Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 morphological 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 region, 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 taxonomic 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 countries. The complete mitochondrial genome of the pink stem borer has been sequenced in China; however, few reports on the COI gene sequences 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 substitutive variation at 22 different nucleotide positions that were distributed 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 established 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 management 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 233 hindered by a lack of public awareness. The School Butterfly Project began in September 2014 with the aims to increase awareness of biodiversity 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 Malaysia. Significance: To the best of our knowledge, the School Butterfly 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 diversity 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 Meyer1 1Royal Museum for Central Africa, Belgium. 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). 2University 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 identification 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 identification success. Nine species pairs showed a low (< 3%) mean interspecific 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 Afrotropical 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. Published by NRC Research Press 234 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 Meyer1 1Royal Museum for Central Africa, Belgium. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2University 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: Sequencing of the standard cytochrome c oxidase subunit I (COI) barcode 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 subsequently 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 barcoding 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 presence 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 taxonomic 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 Government. 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, including 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 carcasses, by determining intraspecific variations, as well as for diet anal- Genome Vol. 58, 2015 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 Khedkar3 1Department of Botany, DSM College, Jintoor DSM College, Jintoor Dist. Parbahni, Maharashtra, India. of Botany, Vasantrao Naik College CIDCO, N4, Aurangabad 431004, India. Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, India. Corresponding author: Ambadas Kadam (e-mail: kadamambadas@rediffmail.com). 2Department 3Paul 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 processes, and several species are known to inhabit such ecosystems. To assess the extent of species diversity and genetic variability of such vegetation, 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 analysed. A tissue sample was analysed for DNA barcoding using RBCL, ITS region-2, and MatK to determine their robustness in species authentication 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 Schubbert1 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 necessary. The current state of the art is species identification by Sanger Sequencing of several DNA barcode regions. Nevertheless, Sanger Sequencing is limited by the overlay of several sequences in complex samples. Furthermore, species-specific real-time PCRs allow determining only the ingredients that have been searched for and, thus, cannot be applied to samples of unknown content. Next-generation sequencing (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 bacterial 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. Published by NRC Research Press Abstracts Significance: Our experiments have shown that NGS is able to overcome the drawbacks of Sanger Sequencing and real-time PCR in complex 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. 235 High-throughput molecular approach for quantifying pest regulation services and disservices provided by farmland insect communities Stéfaniya Kamenova,1 Vincent Bretagnolle,2 and Manuel Plantegenest3 1INRA UMR IGEPP Domaine de la Motte 35653 Le Rheu, France. Chizé CNRS UMR 7372 79360 Villiers-en-Bois, France. Ouest 65 Rue de Saint-Brieuc 35000 Rennes, France. Corresponding author: Stéfaniya Kamenova (e-mail: stefaniya.kamenova@gmail.com). 2CEBC Use of BOLD systems in species conservation Vikas Kalyankar,1 Shivaji Mane,2 Sunil Ahirrao,3 and Trupti Khedkar4 1Department of Zoology, Toshniwal A.C.S.College, Sengaon, India. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Krishi 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 systems for species conservation especially for the fish Anguilla bengalensis bengalensis through uploading various data of biological and geographical 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 maintained by the data base could add to the study of the status of populations. 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 Khedkar2 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 agreement, international trading in agricultural products, foods, pharmaceuticals, and marine products has facilitated massive extraction of natural resources and ultimately has created potential risks to biodiversity. 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 mutations (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 analysed 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 demonstrates 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. 3Agrocampus Background: Maximizing farmland biodiversity has been widely promoted 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 metabarcoding 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 predators (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 protection. It also emphasizes the value of DNA barcoding-based approaches 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 Jahan2 1Department of Zoology, Govt. College of Science, Wahdat Road, Lahore, Pakistan. of Health Sciences, Lahore, Pakistan University of Health Sciences, Lahore, Pakistan. Corresponding author: Asma Karim (e-mail: drasmakarim@gmail.com). 2University Background: DNA barcoding is a taxonomic method to identify species. It uses a short genetic marker in an organism's mitochondrial DNA for identification. It uses the sequence diversity of a 658-bp fragment near the 5= end of the mitochondrial cytochrome c oxidase subunit I (COI) gene for identification. When compared with morphological 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 Ctenophyrangodon idella (1). The average read length was 680 bp with 116 polymorphic sites, 109 singleton variable sites, and no parsimony-informative sites. No insertions/deletions or pseudogenes or contaminant sequences (e.g., from bacteria) were observed, which supports the view that all of the amplified sequences constituted functional mitochondrial COI sequences. Significance: From these findings it is concluded that the gene sequence, COI, may serve as milestone for identification Published by NRC Research Press 236 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 Actinopterygii. 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 integrity, evolutionary fate of species, including reticulation of phylogenetic 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 obtained on gene introgression by mtDNA and nDNA markers are evidence for hybridization? (iii) Does the literature contain a correspondence 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 paradigm 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, although 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 introgressed, 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 definitely 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 particular. 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. Genome Vol. 58, 2015 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 herbivores (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 reliability 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. Moreover, 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 extremely helpful for the evaluation of global patterns of species diversity, 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 biological insight can be gained from placement within the Linnean hierarchy, there is a need to see if barcodes contain enough phylogenetic signal to permit such assignments. Results: Several efforts are underway to construct a functional identification system for higher taxonomic 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 considered as a case study. Significance: Family-level assignments for species recognized through DNA barcoding have traditionally been based on morphological characters. However, many studies employing nextgeneration sequencing cannot use this approach because the link between specimens and sequences is absent. Morphological determinations 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), leaving DNA-based assignments as the only option. Due to the steeply increasing number of studies where morphological approaches cannot be employed, a reliable identification tool for the family level would be a major aid to large-scale biodiversity assessments. Published by NRC Research Press Abstracts NGS pollen metabarcoding and automatic taxonomic classification Alexander Keller, Wiebke Sickel, Markus Ankenbrand, and Ingolf Steffan-Dewenter Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but assessment of mixed-pollen samples originating from multiple 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 complete assemblages without manual separation. Especially, new sequencing 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 microscopy. 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 variety 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 presents 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 subtropical areas of North America and Asia. Of the seven exploited catfish families, 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 perspective 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 Khedkar2 1Department of Environmental Science, S.B. Science College, Aurangabad, Aurangabad, India. 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). 2Paul Background: The recognition and discrimination of plankton species is one of the foundations to freshwater biodiversity research and river mon- 237 itoring programs. Species identification is frequently a bottleneck in the analytical chain from sampling to data analysis and subsequent environmental 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. Although 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 microscopic, necessitating the use of the whole animal for DNA isolation, making it difficult to conduct morphological species verification during sequence analysis. Also, the time elapsed during specimen imaging increases the chances of DNA degradation. Significance: Issues with cryptic species and higher intraspecific distances also add to taxonomic confusion, which suggest careful taxonomic evaluation and COI sequence studies are needed including the barcoding of multiple specimens 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 Hajibabaei1 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 especially 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 monitoring 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 barcoded using standard sequencing primers. The DNA barcodes were compiled into a database that served as an Ambystoma sequence library. We designed three primer sets based on the sequences in this library. Environmental 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 salamanders from water samples via eDNA. Significance: Current monitoring 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. Published by NRC Research Press 238 DNA barcoding plant–insect interactions in a tropical rainforest Censusing marine life in the 21st Century Keiko Kishimoto-Yamada and Motomi Ito Smithsonian Institution, National Museum of Natural History, 10th & Constitution Avenue, Washington, DC 20560, USA. Corresponding author: Nancy Knowlton (e-mail: knowlton@si.edu). 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Genome Vol. 58, 2015 Background: DNA barcoding of gut contents is an effective approach for constructing the food webs of terrestrial species living in cryptic habitats such as the forest canopy. The general inaccessibility 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 leafchewing chrysomelid beetles (Coleoptera: Chrysomelidae: Galerucinae), 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 successfully 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 gymnosperms and (or) ferns together with multiple angiosperm families. Our findings suggest that generalist herbivores associated with ecologically 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. Nancy Knowlton and Matthieu Leray Background: We still lack a well-constrained estimate for the diversity of marine life. The challenge stems from the fact that most marine species are small, rare, and undescribed by science. However, high-throughput DNA sequencing used on standardized environmental samples provides a cost-effective way to estimate the number of species in a specific location and compare such estimates 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 Autonomous 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 m2 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 shallow water reefs of the Red Sea, Panama, and Belize; mesophotic reefs of Curacao; and acidified reefs of Papua New Guinea. Significance: 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 human 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 Zlotnick4 1University 1Rutgers of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA. 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). 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 reasonable 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 unknown samples from Vietnam, the Philippians, and the Central African Republic. 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 estimation of total species richness, but comparisons to studies of similar sampling intensity suggest that the discovery rate of BINs in this region is not unusually high. This is surprising both because of the region's high diversity in other taxonomic groups and also the substantial spatial ␤-diversity evident in our data from different traps. High ␤-diversity should raise the discovery rate in comparison to studies where ␤-diversity is low or only a single trap location was used to sample a similar number of specimens. Significance: Either the insect 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. The global Trichoptera Barcode of Life campaign: implications and applications Karl Kjer1 and Xin Zhou2 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). 2San Published by NRC Research Press Abstracts Diversity of indoor fungi: what does it tell about the health of buildings? Tracking evolutionary diversity and phylogenetic structure across global forest dynamics plots using plant DNA barcodes Helena Korpelainen and Maria Pietiläinen W. John Kress,1 F. Andrew Jones,2 Nathan G. Swenson,3 and David L. Erickson4 Department of Agricultural Sciences, University of Helsinki, P.O. Box 27, 00014, Helsinki, Finland. Corresponding author: Helena Korpelainen (e-mail: helena.korpelainen@helsinki.fi). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 239 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 respiratory 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 resolution. In the present study, to increase accuracy in analyses and to provide useful data for end-users, we conducted DNA metabarcoding 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 sampling. 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 (primarily 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 Stur2 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, especially 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 environments and considered valuable bioindicators of water quality. A commonly 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 environmental barcoding and metabarcoding. 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 investigations 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 phylogenetic 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, evolutionary relationships were highly resolved across the DNA barcodebased mega-phylogeny, and phylogenetic resolution for each community plot was improved when estimated within the context of the megaphylogeny. 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 communities 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. Soniya2 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 enable 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/muscle samples from 67 captive animals in Thiruvananthapuram Zoo, India. Samples included endemic and threatened species present in the Western 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 sequences 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 Published by NRC Research Press 240 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 Shaanker4 Genome Vol. 58, 2015 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. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 1Department of Post Graduate Studies and Research in Biotechnology, Kuvempu University, Shimoga577451, 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 products 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 Embeliaribes) 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 authentic 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 Kumar1 and R. Deepa2 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 ecosystems 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 invasion 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 Pterygoplichthys 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 overlapping morphological characters of the specimens examined indicated possible 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. 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 cucumbers, 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 records of sea cucumbers from the southwest coast of India, and the complexity in taxonomy is due to subtle morphological characteristics and variations in the ossicles of the body wall. DNA barcoding is very significant 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) sequences for 15 species of holothurians collected from shallow coastal waters of southwest coast of India. Results: COI unambiguously identified 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 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 deWaard1 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 20092015, 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 phylogenybased method that is most effective for DNA barcode identification of Canadian vascular plant species, we compared the use of a mega phylogeny for all Canadian flora versus partial trees for the families (RAxML). In addition, we explored several methods of sequence alignment (MUSCLE, Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 addition, species-rich genera, especially groups with poor species resolution, were analyzed separately using all available sources of genetic information (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 collaborative 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 241 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 historical records and all barcode records of the species were based on misidentifications 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 misidentifications among confusing or cryptic species, reveal unsuspected species, help resolve taxonomic synonymies, and in absence of the morphologically diagnostic adult stage confirm range occurrences from immature stages. 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 Denys1 Development of a molecular detection test, based on PCR technology, to detect specific mushroom DNA in soil samples 1Muséum 1Université Background: Comparative phylogeography makes use of congruent patterns of genetic variation among unrelated taxa with partially overlapping geographical ranges to elucidate the influence of historical events on current patterns of biodiversity. Here we analyze spatial divergence patterns of animal species and relate them to the evolutionary history of fragmented areas among the most threatened vegetation types in Morocco. We used comparative phylogeographic 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 evolutionary 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. 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 sequenced 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 software (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 conditions, 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 eventually help to develop the mushroom tourism and cultivation. 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). Barcoding Paralobesia cypripediana (Tortricidae): a stealthy micromoth feeding on the threatened orchid Cypripedium reginae Jean-François Landry,1 Marilyn Light,2 and Michael MacConaill2 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 Genevieve Laperriere,1 Mélodie B. Plourde,2 and Hugo Germain2 du Québec à Trois-Rivières, département de biologie médicale, 3351 boulevard des Forges, Trois-Rivières, Québec, Canada. 2Université du Québec à 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). 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 together various data types (genomic, phenomic, distribution) and combine them with environmental information, phylogenetic hypotheses, Published by NRC Research Press 242 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, multiinstitutional assessment of avian diversification in the Neotropics Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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. Lijtmaer1 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. Independently, 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 phylogeographic patterns of intraspecific diversity within H. rubica throughout 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, complemented with the assessment of coloration and behavioural differentiation. 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 justify 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, multiinstitutional 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. Lijtmaer1 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 commonly extrapolated to other mitochondrial genes. The existence of a Genome Vol. 58, 2015 large, standardized cytochrome c oxidase subunit I (COI) library generated 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 timedependent 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 Kennington3 1University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Australian Museum, 49 Kew Street, Welshpool, WA 6106, Australia. 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). 2Western 3Centre Background: Chromodoris is a genus of colourful sea slug that sequesters toxins from its prey and is widely distributed throughout the Indo-Pacific. However, morphological similarities between congenerics, coupled with an incomplete knowledge of species ranges, make species identification particularly difficult in this genus. The true diversity of Chromodoris in the Indo-Pacific is unknown, and this study employs DNA barcoding to aid in species delimitation and the discovery 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 species in the Indo-Pacific, with an emphasis on Western Australia, using the barcode region of COI. A total of 181 individuals from 16 morphospecies were sampled from wide geographic areas in the IndoPacific, 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 significant sampling gaps in the Indian Ocean, which are often lacking for “Indo-Pacific” taxa. We also advance the understanding of phylogenetic patterns in a recent radiation of nudibranchs and highlight patterns of population structure in two widespread congenerics. Future 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 Nicolai2 1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada. 6553 EcoBio, Université Rennes 1, Campus Beaulieu, Bât. 14A, 35042 Rennes cedex, France. Corresponding author: Annegret Nicolai (e-mail: annegret.nicolai@univ-rennes1.fr). 2UMR Background: The phylum Mollusca is a diverse animal group that is poorly studied although many species are problematic in two opposite Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts ways. Some species are successful colonizers that might become invaders, 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 terrestrial 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 gastropod 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 introductions 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 Canadian 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. Furthermore, 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 reference 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 observed 65% sequencing success with visceral swabs and 1% with periostracum 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 inhibition, likely caused by excessive polysaccharides. Low success from dry museum samples is likely a result of DNA degradation and may be combated through the use of internal primers. Significance: A DNA barcode reference library for Canadian freshwater mussels is an essential platform 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. 243 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 worldwide for their importance in forensic entomology. However, identification 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 genome 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%, confirming 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 phylogenetic neighbour-joining and Bayesian phylogenetic analyses to evaluate 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 species, 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 successful 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 multiple 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, sequences 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 package for R was performed at various taxonomic levels and with OTUs for both COI and 16S. Results: Significant arthropod and bacterial beta diversity is present for taxonomic data and OTUs. OTUs outperform taxonomic 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 classification are present. Published by NRC Research Press 244 Species delimitation of Eupithecia (Lepidoptera: Geometridae) using a ddRAD-Seq approach Kyung Min Lee and Marko Mutanen Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 perspective 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. Results: We used double digest restriction site associated DNA (ddRAD) tags to contribute to delimitation of species within a diverse genus of Lepidoptera (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 species delimitation of any lepidopteran group. Results are expected to provide 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 Wilson1 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. Traditional monitoring approaches impede data collection because they can be laborious, expensive, and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates has recently 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 persistence 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 compared 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 distinguish most mammal species (including separating dark taxa). The persistence 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 quantity and quality. Field surveys in Ulu Gombak Forest Reserve using different 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. Genome Vol. 58, 2015 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 medicine (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 identifying samples on a taxonomic level challenging. Furthermore, plants are traded using local names, which do not always respond with scientific names. Here we measure the efficacy of DNA barcoding to identify bulbous and perennial herbs traded at one of the largest traditional medicinal 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 resulted 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: Identification success is generally high in our dataset, although closely related species remain problematic to identify. Our survey shows a slight increase 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 conservation 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 momentum in Kerala, India in recent years. Among the vegetables grown in polyhouses, salad cucumber is the most successful and widely cultivated crop. The two-spotted spider mite, Tetranychus urticae, was considered as the most serious mite pest of cucumber. However, a recent study revealed that more than one species of Tetranychus infests polyhouse 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 character, 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 presence of three species, namely Tetranychus urticae, T. truncatus, and T. okinawanus. Later, the sequences along with digital specimen photographs 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 Published by NRC Research Press Abstracts 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 identification 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 attempts to use sequence data to complement morphology-based parasite 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 recently, 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 generating useful DNA barcoding PCR primer sets. Results: PCR-based amplifications of mt protein-coding regions and subsequent sequencing (classical DNA barcoding) were developed for a number of groups of apicomplexan parasites. Morphologically indistinguishable adeleorinid 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 amplicons 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 genetic analyses of coccidian parasites may now be possible. Traditional taxonomy and morphology will remain an essential source of wellcharacterized 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 evolutionary 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 spawning and nursery places used by fishes of economic and ecological importance. It is almost impossible to identify the fish eggs due to the lack of distinguishing markers. However, DNA barcodes have been effective in connecting early stages of fish development with adults. This study aimed to recognize which species spawn in the southeast of the Yucatan Peninsula. 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 245 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 Diplospinus multistriatus and Regalecus glesne, being present at eight stations. Significance: 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 management strategies for conservation purposes. High-accuracy de novo assembly and SNP detection of chloroplast genomes for DNA-barcoding studies Qiushi Li,1 Ying Li,1 Haibin Xu,1 Jingyuan Song,1 and Shilin Chen2 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 acquisition of complete chloroplast genomes. However, assembly of chloroplast 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 singlenucleotide 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 chloroplast 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 contextspecific biases in library construction and sequencing reaction. The sequence accuracy is significantly improved, and the reliable SNPs detection is very sensitive compared with previous reports. We recommend 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 describe here ADNAS technology in the cotton supply chain, from the gin through processing into clothing. The study is based on a patented Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 246 ADNAS technology, FiberTyping, which employs chloroplast genetic markers 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 weaving. 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 barcoding 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 Liggins1 and Natasha de Vere2 1University of Wales Trinity St. David, UK. Botanic Garden of Wales and Aberystwyth University, UK. Corresponding author: Natasha de Vere (e-mail: natasha.devere@gardenofwales.org.uk). 2National Background: Artists and scientists have been working, usually independently, on a range of environmental research; however, art–science collaborations, 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 science 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 understanding 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 governments 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. Results: Recent biodiversity surveys in Jamaica, Dominican Republic, and Genome Vol. 58, 2015 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 reliable 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. Significance: The biodiversity of bats in the Caribbean is at least 25% underestimated. 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 taxonomic 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 significance; insectivorous bats could potentially combat urban diseases by preying on disease-transmitting mosquitoes. Guano holds high nutritional value for both cave ecosystems and the agriculture industry. Ecotourism 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 recorded 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 stable 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 identification in many animal groups. However, most studies have been focused 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 material and publicly available data in BOLD, consist of 2790 DNA barcodes >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 geoPublished by NRC Research Press Abstracts graphically widespread dataset, DNA barcodes unambiguously discriminate 94.6% of the Tanytarsus species recognized through prior morphological study. Deep intraspecific divergences exist in some species complexes, and need further taxonomic studies using appropriate nuclear markers as well as morphological and ecological data to be resolved. The DNA barcodes cluster into 120–242 molecular operational taxonomic units (OTUs) depending on whether objective clustering, automatic 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. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Testing DNA barcoding of the recently diverged species in the genus Gentiana (Gentianaceae) 247 argenteus, Siganus argenteus, and Scomberomorus commerson. DNA was prepared 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 platform, 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 sequencing with blocking primers greatly enhances the speed and resolution of dietary analysis of marine fishes and could provide new insights into marine ecosystems. 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 Gentiana—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. Results: The region of rbcL had the highest efficiency of PCR and sequencing 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 alignment 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 combinations, 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 improving our ability to understand and model the function of marine ecosystems. 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 barcode databases (such as the Barcode of Life Data Systems database, or BOLD) and the robust data-generating power of high-throughout sequencing provides new opportunities for detailed dietary analyses. Results: In this study, we investigated the diet of four commercially important fishes from the South China Sea: Acanthopagrus latus, Pampus Updates on the status of giant clams in the Philippines using mitochondrial COI and 16S rRNA genes Apollo Marco Lizano1 and Mudjekeewis G. Santos2 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 Tridacna sp.YCT-2005) under the Tridacnidae family were sequenced (COI and 16S rRNA genes) for molecular-based species identification. We reported 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 constructed using both neighbour-joining and maximum likelihood approaches. 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. Costa1 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 248 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 barcodes for shallow water polychaetes of the southern European Atlantic coast, focusing on specimens from Portugal. Results: Cytochrome c oxidase 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 attributed. Only 47 BINs were considered concordant, where 1 morphospecies equals 1 BIN, and approximately a third of all BINs were considered discordant. 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 extensive 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. Costa4 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 employing current methodologies is very time-consuming, technically difficult, and frequently does not provide species-level data. Although metabarcoding approaches have been tested and successfully implemented on freshwater macrobenthos, no homologous approaches have been proposed and tested for estuarine and marine macrobenthic communities, which are particularly challenging due to their much greater phylogenetic diversity. Results: To investigate the ability 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 barcode fragments from DNA isolates of each of the SCs, and the respective PCR products were sequenced in an Illumina MiSeq platform. Approximately 80% of the species present in the three SCs were recovered after analyses of the MiSeq-generated sequences. Notably, this recovery rate was attained using only two primer combinations. Subsequently, we compared morphology and metabarcoding-based approaches 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 morphologyand metabarcoding-based identifications. As expected, polychaete annelids were the dominant component of the macrobenthic community in this estuarine ecosystem. Significance: These promising results indicate the viability of metabarcoding approaches for implementation in biodiversity assessments of estuarine macrobenthic communities. Further developments are required including the completion of the DNA barcode reference library for marine and estuarine macrobenthos and improvements in recovery rates. Genome Vol. 58, 2015 Barcoding the Diplostomoidea (Platyhelminthes: Digenea) Sean Locke1 and David J. Marcogliese2 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: Trematoda) to species is challenging. Morphological differences among congeners are often subtle in these small, soft-bodied organisms. Single species may also display significant morphological variation in different 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 approach is impractical in biodiversity assessment. DNA barcodes provide 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 number of cases, barcodes from adult diplostomoids from definitive hosts were not matched by those of sympatric larval parasites, even in intensely sampled regions. In North America, barcodes connected larval and adult parasites in 15 diplostomoid species, but because of the difficulty 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 geographic 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 number of prior studies, on polychaetes and starfish, we explore patterns of molecular divergence in 16 sister pairs of echinoderms inhabiting the Pacific versus Arctic–Atlantic coast of North America. Genetic 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 simultaneous 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 diverPublished by NRC Research Press Abstracts 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 calibrated using the Isthmus of Panama. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Sang1 1Kenya Medical Research Institute (KEMRI), Nairobi, Kenya. Centre of Insect Physiology and Ecology (ICIPE), Nairobi, Kenya. 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). 2International 3Egerton Background: Rift Valley Fever (RVF) is a zoonosis of domestic ruminants 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 individual 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 proportions. 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), respectively, 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 identifying species, have greatly expedited indexing life on the planet. Application 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 physical characteristics. DNA barcoding addresses a challenge termed “the 249 taxonomic impediment”—the limitation of experts to identify species. 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 automates 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 instrument basecaller, addressing only ambiguous bases. Second, our training 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 identification 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 interpreting the ‘needs of bees’ offers valuable information for conservation 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 Toronto, Canada. Nests were opened, and one leaf piece from one cell per nest of the native Megachile pugnata (N = 42 leaf pieces) and the introduced 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 different nesting bee species and these data can inform more complete conservation planning that accounts for foraging and nesting material requirements. Published by NRC Research Press 250 DNA barcodes of the genus Oxysarcodexia (Diptera: Sarcophagidae) from south Brazil Taís Madeira,1 Patrícia Thyssen,2 Carina Souza,2 and Juliana Cordeiro1 1UFPel, Pelotas, Brazil. Campinas, Brazil. Corresponding author: Juliana Cordeiro (e-mail: juliana.cordeiro@ufpel.edu.br). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Unicampi Background: Necrophagous insects have been used for forensic purposes, contributing to estimating the postmortem interval, which is important for investigating crime scenes. Species of the Sarcophagidae 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, especially 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 identified. 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 barcoding 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 Fenton1 1Queen Mary University of London, 7 Cambay House, Harford Street, London, UK. University, London, Ontario, Canada. 3Windsor Research Centre, Trewlany, Jamaica. Corresponding author: Hernani Fernandes Magalhaes de Oliveira (e-mail: oliveira.hfm@gmail.com). 2Western Background: Species interactions are the building blocks of ecosystems, forming competitive, predatory, mutualistic, and parasitic relationships. Despite this, they are often difficult to observe and untangle. Extreme generalist insectivores present a particular challenge as traditional analyses are based around morphological examination 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 sequencing it is possible to scale up the analysis from diagnosing single interactions 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 sympatric insectivorous bats on the island of Jamaica, particularly their relative niche size and degree of dietary overlap. Results: We extracted, 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 narrowband echolocation calls and longer, narrower wings differed in diet Genome Vol. 58, 2015 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 echolocation 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 behaviour 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 Stur1 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 samples using short, standardised DNA fragments and next-generation sequencing (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 samples from the reference watershed Atna and Lake Jonsvatnet in central Norway. Our aim is to develop general best-practice procedures to maximise the effectiveness and accuracy of species-level identification at minimum cost using next-generation sequencing techniques. Significance: 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 recruitment of qualified personnel to this important area of nature research 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. Gerard4 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 similarities 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. Larvae 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. Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 associated with waters of 27–31 °C. Although all three species were found each year, overlaying larval catch sites and surface temperature images 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 includes 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 determining 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 fragmented form, is not possible by traditional morphology-based characters, thus making it difficult to investigate the problems of adulteration, substitution, and biopiracy. Of the estimated 17 500 species of flowering plants existing in India, 6000–7000 are used as traditional 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 (wherever 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 species belonging to 442 genera and 117 families, and 278 Atpf and 302 rbcL sequences of these plants were downloaded from NCBI GenBank 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 incomplete state of barcode libraries for medicinal plant species. Significance: Once further developed, the barcode reference library for medicinal plants will be useful for checking the identity, purity, substitution, 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 affect which food resources primates choose to exploit. Previous field 251 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 highthroughput sequencing of invertebrate mtDNA from the feces of white-faced capuchins (Cebus capucinus) to determine patterns of animal prey consumption over a yearlong study. From January 2013 to January 2014, behavioural data was collected from a group of 22 whitefaced capuchins at La Suerte Biological Field Station, Costa Rica. Invertebrate 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 taxonomic units, which were compared to known COI sequences in GenBank 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 influenced observed foraging behavior, but did not influence the frequency of consumption of specific orders of invertebrates. We observed few differences 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 differences 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 Freeland1 1Trent University, 2140 East Bank Drive, Peterborough, Ontario, Canada. of Natural Resources and Forestry, 2140 East Bank Drive, Peterborough, Ontario, Canada. Corresponding author: Allison Marinich (e-mail: almarinich@trentu.ca). 2Ministry Background: Invasive species pose a substantial threat to global biodiversity, 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, water soldier may become an extremely deleterious invasive species because 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 extracted 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 absence. 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 barriers inhibit physical monitoring. In conclusion, eDNA detection is a promising tool that warrants further investigation. Published by NRC Research Press 252 Barcoding the Swiss lichens and associated fungal communities using barcoded amplicon 454 pyrosequencing Kristiina Mark,1 Carolina Cornejo,2 Christine Keller,2 and Christoph Scheidegger2 1Institute of Botany and Ecology, University of Tartu, Lai Street 38, Tartu 51005, Estonia. and Conservation Biology, Swiss Federal Research Institute WSL, Switzerland. Corresponding author: Kristiina Mark (e-mail: kristiina.mark@ut.ee). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Biodiversity Background: As composite organisms, lichens (systematically lichenized 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 saprophytic, 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 Lecanorales (Ascomycota) from Switzerland to test barcoding of the mycobiont 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 fungal 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 detected. Besides the target mycobiont, many other fungi were identified within our samples—on average 11 fungal lineages per lichen sample. Many of these were identified as highly specific lichenassociated (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 composition, 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 fungal community proves to be much more diverse than previously sampling suggested. Improving our understanding of metacommunity structure using DNA barcoding Gillian K. Martin,1 Sarah J. Adamowicz,2 and Karl Cottenie2 1Université du Québec à Montréal, 141 av. du Président-Kennedy, Montréal, QC H2X 1Y4, Canada. 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). Genome Vol. 58, 2015 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 community 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 (BLChetumal Node) belongs to the laboratory network of the Mexican Barcode of Life Network (MexBol), which is supported by the National Council of Science and Technology (CONACYT). The creation of the laboratory 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 highlighted, 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 understand this megadiverse region of the world, but continued and enhanced efforts are required for the construction of the reference database, because most of Mexico's biodiversity still remains unknown. 2Biodiversity 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 incorporating genetic diversity data into community analyses it may be possible 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 evolutionary history into analyses. To test whether grouping species by using coarse taxonomic resolution masks the relationship between taxonomic composition and environmental variables, we characterized 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 barcode 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 Highlights and new discoveries in the Mexican cladocera Ana L. Martínez-Caballero1 and Manuel Elías-Gutiérrez2 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 taxonomy, such as Leberis chihuahuensis and Scapholeberis duranguensis. This work was undertaken in several water systems from the Central Plateau, which includes 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 providing new insights on the freshwater zooplankton from this region; it is paramount to promote these kinds of studies in other parts of the world. Published by NRC Research Press Abstracts 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 Farías5 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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) represents an important ethnomedicinal resource for traditional indigenous 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 carried 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 compared 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. 253 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 sequencing 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 resource partitioning was found between males and females, independently 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 metagenomics 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 Laurence Masson,1 Chris C. Wilson,2 and Michael G. Fox 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. Bond6 1Trent 1African Monitoring aquatic species' invasions using environmental DNA University, 1600 West Bank Drive, Peterborough, ON K9J 7B8, Canada. 1600 West Bank Drive, Peterborough, ON K9J 7B8, Canada. Corresponding author: Laurence Masson (e-mail: laurencemasson2@trentu.ca). 2ONNR, 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 efficiency 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 population 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- 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 phylogeny. 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 Published by NRC Research Press 254 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Santosh Mazumdar,1 Paul D.N. Hebert,2 and Badrul Amin Bhuiya3 1Bangladesh Council of Science and Industrial Research (BCSIR), Chittagong, Bangladesh. 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). 2Biodiversity Parasitoid wasps are thought currently to comprise as much as onequarter 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 parasitoids collected by Malaise traps. To integrate DNA barcoding with morphological 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. Specimens were then barcoded at the Biodiversity Institute using Sanger sequencing to allow recovery of the voucher specimen responsible for generating each sequence record. The analysis of 9079 specimens revealed 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. Genome Vol. 58, 2015 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 designations 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 Ross3 1691 Pyes Pa Road, Tauranga, New Zealand. 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). 2University Background: The phenotypic plasticity of marine sponges makes accurate 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 techniques. 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 Karas3 1Biodiversity 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, specialists on grasses, and broad-scale generalists. Of southern Ontario species, Melanoplus bivittatus and Dissosteira carolina are presumed to be broadscale generalists, Chortophaga viridifasciata is a specialist on grasses, and Melanoplus femurrubrum is a specialist on forbs. These classifications, however, have not been verified in the wild. We selected 20 individuals of four species upon which to utilize a high-throughput sequencing (HTS) approach to diet analysis. We recovered DNA from intact grasshopper guts and amplified the rbcLa region of the chloroplast genome. We then sequenced 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. 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, resulting 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 managed differently in the past 35 years (approximately): (i) unharvested primary 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 managed secondary forests can affect the soil biotic and abiotic components, Published by NRC Research Press Abstracts 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 Oliveira4 1Myleus Biotechnology, Av. José Cândido da Silveira, n° 2100, sl. 11, Belo Horizonte, MG, Brazil. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2EMBRAPA 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 concentration 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 obtained 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 troublesome, 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 Carvalho1 255 lated to cave environments with absence of light and restricted energy resources. Ancistrus cryptophthalmus (Reis, 1987) is a small-sized loricariid fish, found inside cave rivers located in the São Domingos' karst system of the central-west region of Brazil. The fish possess troglomorphic characteristics such as eye reduction and depigmentation 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 investigate 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 nextgeneration sequencing (Illumina). We have also investigated the genetic structure of two cave populations from distinct cave rivers and compared them to one surface population using 570 bp of the hypervariable 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 geographically 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 samples 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 represented. We obtained for the first time barcode records for 41 hummingbird, 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 distinguishable 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. 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 evolutionary adaptations to extreme conditions. Studies on these species are important to understand evolutionary and adaptive processes re- Progress and prospects in using DNA barcoding to advance coevolutionary biology Mark T. Merilo,1 Sarah J. Adamowicz,1 and R. Ayesha Ali2 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 relaPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 256 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 Canada to tropical ecosystems. Emerging methodologies yield unprecedented 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 sequences, 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 hypotheses. 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 coevolutionary 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 laboratories 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 covering 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 discriminate between closely related/cryptic species. Overall, clinically important species show low intraspecies variability and a clear bar- Genome Vol. 58, 2015 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, alternative 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 Ratnasingham1 1Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada. Biodiversity Monitoring Institute, University of Alberta, Edmonton, Alberta, Canada. Corresponding author: Megan A. Milton (e-mail: mmilton@uoguelph.ca). 2Alberta Background: Engaging students in STEM (Science, Technology, Engineering 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 purposes 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 developed by BOLD for this purpose and provide examples on their use. Results: Many students and citizen scientists have expressed an interest 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 scientists 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 supports 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 database and GenBank. SDP provides special tools for instructors to support 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 identification tool was developed in part to support independent barcodebased 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 students 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 Mitchell1 and Yohannes Alemseged2 1Australian Museum, 6 College Street, Sydney NSW 2010, Australia. Department of Primary Industries PMB 19, Trangie NSW 2823, Australia. Corresponding author: Andrew Mitchell (e-mail: andrew.mitchell@austmus.gov.au). 2NSW About three-quarters of Australia's area is rangeland. It contains a wealth of biodiversity including more than 1800 plant and 600 vertebrate species. This land must be managed for a complex mix of interests, 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 reference DNA barcode library for rbcL, Atpf, and ITS2 sequences for the Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 257 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 reference library sequences were recovered from dung samples. Many sequences 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 sequences). Family-level identification was possible for all sequences recovered from all samples. At the family level, Dorper dung contained seven plant families not found in Merino dung, and no families were unique to Merino dung, confirming that Dorpers have broader diets than merinos. rich group with diverse biological and ecological traits. Within this group, the evolutionary shift between terrestrial and freshwater habitats 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 evolutionary 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 freshwater 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. Testing for positive selection in mitochondrial and nuclear energy-related genes in Pterygota (flying insects) 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 T. Fatima Mitterboeck,1 Shanlin Liu,2 Rui Zhang,2 Wenhui Song,2 Jinzhong Fu,1 Sarah J. Adamowicz,1 and Xin Zhou2 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 molecular 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 selection in the lineage in which flight likely evolved. We include all mitochondrial protein-coding genes and over 900 nuclear protein-coding genes for 27 hexapod species. Through inclusion of non-energyrelated 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 themselves 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 selection 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 representation 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 present new opportunities for broad-scale investigation of patterns of molecular evolution and macroevolution across life. Insects are a species- 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 Iwasaki8 1Natural History Museum and Institute, Chiba 260-8682, Japan. 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). 2Tohoku 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 environmental DNA (eDNA) from fishes. Results: The primer design was based on the aligned whole-mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163–185 bp), which contains 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, nextgeneration 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—including 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 presented 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 monitoring that revolutionises natural resource management and ecological studies of fish communities on larger spatial and temporal scales. Published by NRC Research Press 258 Validation of commercially important fish of India Ashok Mohekar,1 Amol Kalyankar,2 Dinesh Nalage,2 and Gulab Khedkar2 1Department of Zoology, SMD Mohekar College, Kalamb Dist. Osmanabad, Maharashtra-431004, India. Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, India. Corresponding author: Ashok Mohekar (e-mail: smdmmkl@gmail.com). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Paul DNA barcoding has emerged recently as a powerful tool for food authentication 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 consuming 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 construct 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 taxonomic 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 barcoding for regional species identification. As an independent valuation of taxonomy, DNA barcodes provide robust support for most morphologically 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 Rønsted3 1Instituto de Ecología, University Campus, #23 Street, Cota Cota, Bolivia. 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). 2Centre Background: Andean aquatic insect larvae are poorly known at the species level, despite their importance in the understanding of freshwater 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 environments. 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 specimens of different families and genera (averaging 28%), whereas intraspecific 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 tintinnabula), three species of stoneflies (Anacroneruria vagante, Claudioperla Genome Vol. 58, 2015 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 ecological 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 km2. 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 sequenced, 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 database 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 environments. 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 identification 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. Additionally, this study provides a useful assessment of the COI DNA barcode region against other markers previously used for identifying fish species using eDNA. Published by NRC Research Press Abstracts 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 Haszprunar1 1SNSB, Bavarian State Collection of Zoology, Münchhausenstrasse 21, 81247 München, Germany. 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. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2LGC 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 single Malaise trap sample (1 week) into 12 groups of arthropod orders (Coleoptera, Hymenoptera, Lepidoptera, Diptera, Hemiptera, Araneae, 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 European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded 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, Hymenoptera, 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 identified 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 promising 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 Collins3 259 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 focused sampling effort across the Northeast Pacific and throughout the North Atlantic (in both shallow and deep waters), but we also generated 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 diversity 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 maintenance of genetic connectivity across the Atlantic. Other species appear 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 colonizing 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 Chen1 1Weill Cornell Medical College in Qatar Eduction City, P.O. Box 24144, Doha, Qatar. 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). 2Carnegie 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 cm3 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 standard DNA barcode sequence. Results: Among the nine currently analyzed 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 diversity as well as other vertebrates in Qatar. 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, sometimes attaining considerable sizes. Many Plumularioidea are believed to have large geographic distributions. However, few diagnostic morphological 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 morphological plasticity that hydrozoans display often make it difficult to 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 260 a national project managed by the South African National Biodiversity Institute (SANBI), working with the South Africa Police Services forensics 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. Examples of forensic cases that have been analysed using the BWP COI databases will be discussed including other forensic analyses approaches 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 Hanner1 1University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1, Canada. Labs, 800 W Baltimore St #407, Baltimore, MD 21201, USA. Corresponding author: Amanda M. Naaum (e-mail: anaaum@uoguelph.ca). 2Instant 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. However, this method is currently limited to use only in well-equipped laboratories. 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 implement more in-depth authenticity testing for key products on-site, and provide a means to integrate DNA testing into current quality management 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 Hanner1 1University of Guelph, 50 Stone Road E., Guelph, ON N1G 2W1, Canada. 1350 Connecticut Ave., NW, 5th Floor, Washington, DC 20036 USA. Marine Biolabs, 1559 Spinnaker Drive, Ventura, CA 93001, USA. Corresponding author: Amanda M. Naaum (e-mail: anaaum@uoguelph.ca). 2Oceana, 3Coastal Background: DNA barcoding has been applied as a method to test seafood authenticity in numerous market surveys. This trend is continuing to gain momentum as DNA barcoding is employed as a regulatory tool, by the media, and by students to test seafood products, in addition to its use by scientific researchers to monitor seafood substitution. However, as market surveys documenting mislabeling continue to be published by both the press and scientific journals, there is a need for standardization in practices to aid in comparing and verifying 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 Genome Vol. 58, 2015 have been documented for use by citizen scientists, media outlets, and academic researchers. Significance: This represents a means for standardizing future DNA barcoding market surveys to allow for better cohesion 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 employing 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 environmental 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 identify. 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 potentially inaccurate or impossible if samples are small and badly damaged. DNA-based analysis can be used to address these issues and provides an opportunity for improving the unidentified fraction of entrained 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 identification 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 identifying entrained specimens. Real-time PCR allows direct monitoring of species of economic or environmental concern from mixed entrainment samples. This method can be used to track invasive or endangered species, and potentially quantitate their biomass as a percentage of entrained biological material. Both DNA barcoding and real-time PCR provide means to improve the accuracy and reliability with which entrainment 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 Verheyen7 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, eastern, and western Congo Basin) and mid-altitude (Katanga Province) regions. 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 (SquaPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 boundaries—a complementary approach to morphological identification. Results: 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. According 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, unexpectedly 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 unidentified reasons. Significance: This study is the first DNA-barcoding assisted 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 alterations of current classifications and challenge the biogeographic interpretation 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. Khedkar2 1Aurangabad Municipal Corporation Zoo, Aurangabad, Maharashtra State, India. 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). 2Paul Recent enforcement of law (4 March 2014) prohibiting the slaughter of “cow” includes bull, bullock, ox, or calf for the purpose of beef production in India. This law is enforced since Article 48 of the Indian constitution enjoins on the State to organise agriculture and animal husbandry on modern and scientific lines and in particular to take steps for preserving 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 procedures 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. Sathishkumar2 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 hybridization between species. Significant morphological dissimilarity between some species due to spontaneous chromosome doubling and external environmental influences cause difficulties during the identification 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 261 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 accessions, 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 displayed that some species exhibit intraspecific polymorphism that distinguish them from closely related species, and the use of barcode candidate ITS was proved to be the powerful tool for distinguishing the plant species. This study provided a concrete result for proposing that a DNA-based framework is essential for identification, documentation, and conservation of genetic diversity of the Solanum nigrum L. complex and its closely related species. Though Solanum nigrum L. has several medicinal properties, there are scientific records suggesting toxicity effects on livestock and humans. Hence it is desirable to authenticate morphologically similar 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 Giere8 1Zoological State Collection Munich, Münchhausenstr. 21, DE 81247 Munich, Germany. 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). 2Botanical 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 disciplines like climate science (GR in drill cores, water, or soil samples) or archaeo sciences (archaeobotany, archaeozoology, and archaeology) exploring ancient DNA. To allow compliance with national and international ABS laws, additional reporting and documentation is required to demonstrate legal acquisition and utilisation of genetic resources accessed 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 compliance, 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 networks of (taxonomic) institutions that have CBD-conform procedures in place. Published by NRC Research Press 262 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 Wilson1 1Institute Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 considered 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 Peninsular 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 subfamily level position of the the freshwater mussel species from Peninsular 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. deWaard4 1Naturalis Biodiversity Center, P.O. Box 9517 2300 RA Leiden, the Netherlands. 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). 2Zoological Background: The Nepticulidae form an early-radiating family of leafmining 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 leafmining 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 Datasystems (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 America alone, our results suggest 40 unnamed species, confirm 12 species as Holarctic (including several newly recorded), and link previously unidentified host records to named adults. Significance: Barcoding masstrapped specimens showed an unprecedented increase in our knowledge Genome Vol. 58, 2015 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 biodiversity 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. Ojeda1 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–semiarid 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 representing this most diverse order among mammals). The high diversity and complex evolutionary history has motivated researchers to study phylogenetic relationships and taxonomy, which have been under continuous 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 features. We evaluated DNA barcodes as a tool for assessing the taxonomic 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 localities 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 correspondence between genetic and geographic structure, suggesting cryptic speciation. Akodon spegazzinii and Tympanoctomys barrerae contained 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 observed 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. Costa1 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 reference 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 ichthyofauna is still missing. Here we assemble for the first time a large-scale comprehensive reference library for this ichthyofauna, based on all Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 263 publicly available DNA barcodes, with the aim to examine and annotate 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. Subsequent inspection of the BIN composition revealed potential artifacts (i.e., synonyms, misidentifications, BIN=s incapability to resolve cluster boundaries) resulting in 73% concordant species IDs, 5% with comparatively high intraspecific divergence (e.g., European seabass), and 8% of poorly represented species. Many economically important species 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 withinspecies divergence and taxonomic uncertainties that should be further investigated. Despite the usefulness of this core reference library, which includes most of the economically relevant species, the majority 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 Suwannapoom3 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 synthetic 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 authenticate Tinospora crispa species commonly used in Thailand. The ITS1 barcode was selected for use in primers design for HRM analysis to produce standard melting profiles of the selected species. Local products made from T. crispa species were purchased from Thai markets and authenticated 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 interest also hindered the collection process. The Bar-HRM method developed here proved to be a cost-effective tool which can be used for rapid detection 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 responsible for some of the more nutritious and tasty components of our diet and some species are in precipitous decline. Much of this news concerns 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. Adamowicz3 1Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada. 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). 2Department Background: Ancient lakes are interesting systems for studying morphological 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 consequences 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 morphologically 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 morphological 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 divergence is assumed to approximate the relative time since lake colonization 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 evolution in ancient lakes and demonstrates the value of coupling morphological 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 decades. 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 Published by NRC Research Press 264 Genome Vol. 58, 2015 available and accessible the wealth of information in the primary taxonomic literature, and the challenge of integrating this with the burgeoning 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 Arnaud4 1LECA, University Grenoble Alpes - CNRS LECA, BP 53 2233 Rue de la Piscine 38041 Grenoble Cedex 9. INRA Thônon-Les-Bains INRA, 75 avenue de Corzent, 74200 Thonon-les-Bains, France. 2CARRTEL, Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 accumulate 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 modifications around a high-elevation lake over the last 10000 years. More specifically, 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 targeting 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 patterns 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 Period and Middle Age. Second, we reconstructed plant community dynamics 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 evolution. All these results corroborated with the known anthropogenic history 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 paleoecology. 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 Taberlet1 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 gradients for macro-organisms are subjects of intense research, but those of soil biodiversity remain relatively poorly known. In this study, we attempted to verify the generality of these patterns. We used environmental 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 10 m × 10 m sub-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 analyzed independently. Results: Our data allowed us to investigate variations 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 distribution 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 species. Such overlooked diversity provides a challenge when studying biogeographical 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 diversity 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 expected phylogenetic structure to be more clustered at low elevations compared to high elevations, due to dry environmental conditions selecting 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 vertebrates 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 phylogenetic 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 Ryu5 1Microbiological Resource Center, KRIBB 125 Gwahak-ro, Yuseong, Daejeon 305-806, Korea. 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). 2Hannam 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 morphologically similar and taxonomically related relatives, focused on four Published by NRC Research Press Abstracts 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 previously assembled DNA barcode data for quarantine pests in Korea, we expect to provide a comprehensive barcode registry for quarantine pests. 265 present the comparison of biotic indices inferred from morphotaxonomic 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 integrate 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 BARCODING.MED: building a high taxonomic resolution database for the Mediterranean basin Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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). 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. Hebert6 1Pensoft Publishers, Sofia, Bulgaria. Staatssammlung, Munich, Germany. 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). 2Zoologische 3The Background: Currently, the range of applications for DNA barcoding in biodiversity research is very wide and has surpassed purely academic goals. The development of services for public and private institutions 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 biodiversity hotspot. Under the scope of the EDP Biodiversity Chair and an ERA-Chair in Environmental Metagenomics (capacity building EU projects), we propose to develop a DNA barcoding database covering all vertebrate 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 specimens. Each sequenced specimen will be identified by expert taxonomists, uniquely labelled, and then stored in Natural History Museums, CIBIO/InBIO, or private collections. Only taxonomically validated specimens will be uploaded in Barcoding.med and internationally-free databases 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 Hymenoptera. Significance: By providing cost-efficient approaches for biodiversity assessments and monitoring, we aim to promote the use of DNA barcoding tools in public and private institutions, especially for providing services addressing current environmental legislation and international 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 promoting capacity building at a regional level. 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 collaboration with co-authors and peers; for example, by providing specific epithets, concise morphological descriptions and diagnoses, type specimen data, and additional taxonomic remarks, to complete the formal publication 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, publication, 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 “barcode data release” papers—can also be extracted from the metadata descriptors at BOLD and finalized in BDJ's authoring tool. Significance: Data are imported into manuscripts and published in both humanreadable text and structured Darwin Core formats that can be downloaded 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. Inferring biotic indices from metabarcoding data: promises and challenges Amino acid variation and protein structure of COI barcodes—insights from a Metazoa-wide sample 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 Cordonier3 Mikko Pentinsaari,1 Heli Havukainen,2 Marko Mutanen,1 and Tomas Roslin3 1University 2Department Environmental diversity surveys are crucial for the bioassessment and biomonitoring of anthropogenic impacts on aquatic ecosystems. Traditional monitoring is based on morphotaxonomic inventories of biological communities, which are time-consuming, expensive, and require excellent taxonomic expertise. High-throughput sequencing of environmental DNA (metabarcoding) offers a powerful tool to describe biodiversity. However, the capacity of the metabarcoding approach to meet the quality standards of bioindication is a subject of controversy. Here, we Background: Most studies published to date have treated the COI barcode 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 amino acid variation and evolution of COI protein structure in a crosscut of of Geneva, Geneva, Switzerland. 2University of Paris, Paris, France. 3Water Ecology Service, Geneva, Switzerland. Corresponding author: Jan Pawlowski (e-mail: jan.pawlowski@unige.ch). 1Department of Genetics and Physiology, Pentti Kaiteran katu 1, 90014 University of Oulu, Finland. of Biosciences, Viikinkaari 1, 00140, University of Helsinki, Finland. of Agricultural Sciences, Latokartanonkaari 5, 00014 University of Helsinki, Finland. Corresponding author: Mikko Pentinsaari (e-mail: mikko.pentinsaari@oulu.fi). 3Department Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 266 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 exhibited 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 herbivorous and ancestrally fungivorous beetle lineages. Significance: Patterns of COI barcode variation have rarely (if ever) been studied from the perspective of protein function and evolution. As expected, the protein structure is generally conserved, but we managed to uncover some interesting exceptions. Convergent evolution in parasites is particularly interesting 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 between 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 Malaise trapping and DNA barcoding makes it possible to carry out largescale 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 deployed 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 preliminary species inventories to be completed for each location and facilitating 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 distributions and prevalence, which is critical for the accurate evaluation of anthropogenic impacts. This project will also allow researchers to explore patterns of global biodiversity across latitudes, ecosystems, seasons, 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. Genome Vol. 58, 2015 from Coffea canephora. Substitution of one species for another constitutes 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 Hanner4 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 sufficiency based on haplotype diversity in the ray-finned fishes (Animalia: Chordata: Actinopterygii). To do this, we use linear regression and hypothesis 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 sampling 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 extinctions 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 Hogg1 Applying DNA barcoding to the manufacturing supply chain, to reduce substitution and adulteration fraud Stephen Phelan1 and David Jonker2 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 1University of Waikato, Hillcrest Rd., Hamilton, New Zealand. & Food, Lincoln, Christchurch, New Zealand. Corresponding author: Colleen Podmore (e-mail: colleen.podmore@xtra.co.nz). 2Plant 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 reasonably common but having a narrow distribution (Aciphylla-feeding) and widespread but with low local abundance (Dracophyllum-feeding). There is Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 wellsupported clades were clearly delineated according to the host plant. However, we also found two distinct clusterings within the Aciphyllafeeding group (South Island only) and five within the Dracophyllumfeeding group (North and South Islands), suggesting seven potentially cryptic species. This evidence indicates genetic differences between locations 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 reveal 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 Hajibabaei2 1McMaster University, 1280 Main Street West, Hamilton, ON L8S 4L8, Canada. 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). 2Biodiversity Background: Although the use of metagenomic methods to sample below-ground fungal communities is common, the use of similar methods 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 produced by the same primer, was more similar to each other than with fungi sampled from the same site. By contrast, the taxonomic composition of the most frequently sampled rbcL OTUs was most similar within sites. Significance: We suggest that future studies consider using multiple 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 sequencing has opened a new era in phylogenetics. However, although large datasets should in theory increase phylogenetic resolution, multilocus 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 duplication and loss, and horizontal gene transfer. In this talk I will explain some of the most important challenges we will have to face to reconstruct 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. 267 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 problematic when distinct species share identical sequences, for example, following mitochondrial introgression. Species delimitation is complicated 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 Palaearctic. No evidence of mitochondrial introgression was revealed, even among closely related species found in sympatry and infected with identical strains of Wolbachia, suggesting that this phenomenon does not pose a major problem in this group of bees. However, in the majority of species examined across their entire range, multiple, strongly divergent mitochondrial haplotypes exist within single species. In most cases different 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 withinspecies 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 recommend 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. Hebert1 1Biodiversity Institute of Ontario, University of Guelph, Guelph, ON, Canada. Institution, Washington, DC, USA. Corresponding author: Sean Prosser (e-mail: sprosser@uoguelph.ca). 2Smithsonian 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 preservation, 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 barcodes 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 Published by NRC Research Press 268 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 Tinti1 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 palaeogenetic tools can be used to identify ancient fish remains, allowing researchers to better understand our all but forgotten relationship with the sea. DNA was extracted from the remains of small fishes and large marine predators excavated from late iron age and ancient roman settlements 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 identified to genus level with a 78% success rate, while 70% of samples were identified to species. BLAST, neighbour-joining trees, and a characterbased 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 inhabiting ancient Lake Titicaca of Peru and Bolivia has resulted in an estimated 100 endemic species. This study investigates two potentially important mechanisms of diversification: geographic isolation of populations 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 Genome Vol. 58, 2015 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 diversification 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 subsequently diversified within depth zones. Significance: DNA barcoding has permitted insight into this complex radiation, consisting of numerous 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 ancient 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 miniaturized 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 realtime base-calling. The MinION Access Programme (MAP) is a precommercial 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 MinION to sequence PCR-amplified DNA from a broad range of individual animal and plant specimens from Acadia National Park and from mitochondrial and chloroplast-enriched genomic DNA from mixed environmental 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, cloudbased base-called sequences, and results from comparison of base-called sequences with published animal and plant DNA barcode reference sequences 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 represents 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 polyphagous agricultural pest of global importance, harbours diverse gut bacterial communities. The gut bacteria community of H. armigera was analysed previously by isolation and cultivation techniques and polymerized chain reaction (PCR) based cloning methods, resulting in identification of few groups of bacteria. We explored the composition and Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts diversity of gut bacteria in nine samples of H. armigera using Illumina next-generation sequencing (NGS) of 16S ribosomal RNA amplicons. 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 insecticidal molecules, and defensive action against pathogens. Insecticidal toxinproducing 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 understanding how it aids different physiological activities, which are important factors that contribute to this species being a pest of global importance. Further studies will focus on sequencing the whole gut metagenome of H. armigera, identifying the functional pathway of every representative 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 Khdkar2 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 species 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 freshwater 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 individuals, representing 12 orders, 41 families, 80 genera, and 103 species. The mean conspecific, congeneric, and confamiliar genetic distances were 0.3%, 10.78%, and 16.26%, respectively. Molecular species 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 independent calibration of current taxonomic resolution within the Godavari fish fauna to reveal key areas of uncertainty whenever discrepancies between genetic data and morphologically based taxonomy arise. Significance: This study can serve as a good example demonstrating the need of complementary taxonomical procedures to understand the diversity of life. Overall, the results 269 showed the usefulness of DNA barcodes for cataloguing the Godavari river fish species and for identifying groups that deserve further 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 workbench 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 adoption of this method in many life science fields, most notably in systematics, 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 bigdata concepts and tools, democratization of DNA barcoding by improving 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 annually. This intensive harvesting of wild medicinal plants represents a serious 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 harvested forest products. In this study we used DNA barcoding as a tool to identify forest products on the market that cannot be identified by traditional means. Here, we compared the unknown “bark” sequence from the Faraday Muthi Market to a reference DNA barcode database for southern African tree species. Results: Overall sequencing success was higher for rbcLa (90%) than Atpf (81%) due to the fragmented nature of the material. 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 species most affected by harvesting for traditional medicine for the management of wild populations, and for Environmental Inspectors to monitor the trade. Published by NRC Research Press 270 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 Neumann5 1DZMB/Senckenberg am Meer Suedstrand 44, 26382 Wilhelmshaven, Germany. - Helmholtz Centre for Ocean Research Duesternbrooker Weg 20, 24105 Kiel, Germany. 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). 2GEOMAR Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 3Alfred 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, however, most barcoding studies typically focus on economically relevant species, for example, fish, as well as on the documentation of hotspots of species diversity, for example, tropical coral reefs or regions of the almost unexplored 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. Environmental parameters (e.g., depth, sediment characteristics, temperature, and salinity) of this semi-enclosed shelf sea follow a distinct pattern: high seasonal 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 crustaceans, 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. Significance: Our data represent a first step towards the establishment of a comprehensive 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 coming years. Following this, reference datasets such as ours will become essential 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 Zhang1 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 morphological characters in wild ecosystems. Pteris species were proved to Genome Vol. 58, 2015 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=; R 5=CGCGCATGGTGGATTCAAATCC-3= in an Eppendorf thermal cycler (Eppendorf, 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 sequence 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 exhibited 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 between 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 resolving 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 Ravitchandirane1 and Muthusamy Thangaraj2 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 healing 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 authenticity and unravel adulteration. Results: Ethnomedicinally important fishes (6) have been collected based on traditional knowledge of practitioners and local fishermen of the coastal villages of Pondicherry and Tamil Nadu, India. Based on various classical morphometric characters, 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 phylogenetic status of these fishes. Significance: The goal of the study was to create Biological Reference Material (BRM) for ethnomedicinally important fishes from the east and west coast of India under the title “ethnoichthyogenomics” in order to understand traditional use, active 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 species, their ecological needs, including traditional ecological knowledge so that management and conservation strategies can be implemented. Published by NRC Research Press Abstracts 271 DNA barcoding of rodent pests in South India R.L. Rengarajan and G. Archunan Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 characterize 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, at 4%. 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 potentially 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 Marret3 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 ecosystem, highly impacted by human activities, and to detect possible temporal 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 identification 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 discovery 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 individuals (87%) were successfully sequenced for part of the mtDNA. Also, seven microsatellites loci are being analyzed. For more than half of the samples, 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 costeffective 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 biodiversity 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 Carneiro1 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 species, taxonomic studies have proposed that G. brasiliensis actually encompasses 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 northern 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 dendrograms build using neighbour-joining, maximum likelihood, and Bayesian inference, with high support values and mean K2P divergence of 0.24% and 6.0% within and among populations, respectively. Analysis of haplotype diversity revealed conspicuous genetic differentiation of populations within the Contas River basin and suggests the occurrence of headwater capture between nearby hydrographic systems. 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. Johnson1 1The Ohio State University, Department of Entomology, 2021 Coffey Rd., Columbus, OH 43210, USA. 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). 2The 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 Published by NRC Research Press 272 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 quantitatively 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 how they respond to current climate change. To comprehend the community-level consequences of rapid Arctic warming, we must embrace 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 Morgan Riding,1 Ian Hogg,1 Brian Smith,2 and Richard Storey2 Rodolphe Rougerie,1 Mehrdad Hajibabaei,2 Christophe Bouget,3 Shadi Shokralla,2 Joel F. Gibson,2 and Carlos Lopez-Vaamonde4 1University 1Muséum 2The 2Department of Waikato, Gate 1 Knighton Road, Hamilton 3240, New Zealand. 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). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Genome Vol. 58, 2015 Background: Connectivity to source populations is critical for restoring 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 resulted 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 determine the genetic connectivity of individuals among source populations and restored streams in the Taranaki Region. Five common aquatic insect species were selected based on differences in flight capabilities 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 examine 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 presence 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 comprehensive DNA barcode library for the terrestrial animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley of northeast Greenland. The resultant resource offers species-level resolution in describing biotic interactions among community 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 techniques. DNA barcodes based on COI offered high resolution in discriminating 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 Zackenberg community comes with urgent implications both for our understanding of how arctic ecosystems are built and function, and of national d’Histoire naturelle, UMR 75205, 45 rue de Buffon, CP50, 75005 Paris, France. 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 biodiversity on Earth. Climate change scenarios predict an increase in the intensity and frequency of severe summer droughts, high temperatures, and infestations of pathogens and insects, causing high mortality of some keystone tree species. These changes will affect forestry policies and practices, strongly impacting biodiversity. Understanding the responses of biodiversity to forest decline is therefore essential to developing new climate-smart management options. Biomonitoring of forest insects relies on techniques involving laborious and expensive sampling procedures. For instance, the study of indicators such as saproxylic beetles is strongly impeded by their high abundance 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 identification. Results: Using a set of three primer pairs targeting short fragments 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 barcode 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 improve 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 Rasplus2 1UMR 7205 ISYEB, Muséum national d’Histoire naturelle, 45, rue Buffon, CP50, 75005 Paris, France. 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). 2INRA, Background: RAD sequencing is becoming popular to infer phylogenetic relationships in organisms lacking genomic resources. Because incomplete taxonomic coverage can confound macroevolutionary inferences, 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 boundaries 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts space and time, also taking into account a set of adaptive traits (hostplants, wing morphology). Results: Starting from a comprehensive DNA barcode library, we selected 48 individuals representing 41 species in the genera Actias, Argema, and Graellsia, plus 7 outgroups. We generated a RAD library using genomic DNA extracted from individuals 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 taxonomically. 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 Packer1 1York University, 4700 Keele St., Toronto, ON M3J 1P3, Canada. Bee Biology & Systematics Laboratory, 5310 Old Main Hill Logan, UT 84322-5310, USA. Corresponding author: Genevieve Rowe (e-mail: genevieve.rowe@ymail.com). 2USDA–ARS Background: Osmiine bees are important both economically as manageable 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 assessments, 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 subspecies 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 Schoelinck1 1Fisheries and Oceans Canada, 343, Avenue Université, Moncton, NB E1C 9B6, Canada. of Toronto Scarborough, 1265 Military Trail, Toronto, ON M1C 1A4, Canada. Corresponding author: Mélanie Roy (e-mail: melanie.roy@dfo-mpo.gc.ca). 2University Background: Although barcodes already exist for most Canadian freshwater fish, there are many issues regarding taxonomic identification. Development of a Canadian DNA database correctly constructed 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 sufficient depth and species coverage such that banned species can be confidently discriminated from legitimate imported and native species. A total of 13 species likely to be banned from importation into Canada have been evaluated for DNA barcodes available and to identify gaps to address. Data generated in this project will enable confident identification of Canadian freshwater fish and potential invaders, which is needed to develop monitoring tools from environmental samples. Results: We obtained 906 samples representing 93% of Canadian freshwater species. Each specimen was sequenced with 273 independent markers (COI, Rhodopsin), and a voucher has been systematically conserved at the Royal Ontario Museum to guarantee traceability. Phylogenetic analyses showed good species delineation except for lampreys and salmonids. Delineation issues within the genus 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 metabarcode 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 barcode 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 respective 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 environmental 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. Yang1 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 274 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 composition 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 pikeminnow. The remainder of the identified remains (13.79%) were assigned to the sucker genus (Catostomus sp.). Significance: This study has provided detailed information about the species composition 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 recovered from archaeological sites. Approaches for identification of Colchicum L. species in the flora of Turkey by morphological parameters and DNA barcoding Ezgi Çabuk Şahin,1 Yıldız Aydın,1 Erdal Kaya,2 and Ahu Altınkut Uncuoğlu1 1Marmara University, 34722, Istanbul, Turkey. Horticultural Central Research Institute, 77102, Yalova, Turkey. Corresponding author: Ezgi Çabuk Şahin (e-mail: ezgicabuk@gmail.com). 2Atatürk The bulbous ornamental plant Colchicum L. belongs the family Colchicaceae, 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 populations are new candidate species), by using the multi-marker DNA barcoding 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 suggested 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. Sequence data will be analyzed by using bioinformatic tools and biostatistics 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. Genome Vol. 58, 2015 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 Nakamori2 1Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara 903-0213, Japan. 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). 2Department Background: Springtails (Collembola) are a major group of soil microarthropods that mediate food webs during the decomposition process. Collembola are conventionally identified to the species level by microscopic examination of their morphological features, so community 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 animals 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 methods are thus required. Results: We designed degenerate PCR primers for the conserved regions in mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on collembolan 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 communities 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 individuals 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 general. These activities fell within the overall goal of strengthening capacities for DNA barcoding in the country. Results: The USM Herbarium and various departments in the Zoology Division sent a total of 2489 samples to the Biodiversity Institute of Ontario (BIO). The bird samples represent 60% of Peruvian bird species. Samples from the Departments of Entomology (95), Mammalogy (285), and the USM Herbarium (190) were analyzed in order to validate their identities, and allow them to Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. be used in phylogenetic reconstructions. Bird samples came from different 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 Environment and Agriculture) and 26 species important in environmentally sustainable agroecosystems (AGROKASA company). In the last decade, Peru has experienced significant economic growth, mainly from mining, hydrocarbon, 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. Clare4 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 ecological processes such as competition and niche partitioning. However, observing and describing these relationships is challenging, particularly among generalist insectivores. Traditionally, morphological classification of digested prey has been used to limited effect. DNA barcoding provides a powerful solution to characterize these relationships, providing species-level identifications even from degraded material. 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 metabarcoding 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 degree 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 resources that vary throughout the year. This study provides one of the first detailed views of tropical insectivores and their relationship with resources. 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 proposed as a potentially more sensitive method of invasive species detection than traditional morphology-based methods. This barcodingbased technique could allow for the identification of an invasive species at a lower population density, thereby allowing earlier detec- 275 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 nobilis, 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 nontarget 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. Sathishkumar3 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 hybridization 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 diversity 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, cirutakkali, itayakali, nattuttakkali, and it is known to inhabit the same areas as P. lagascae. The two species are often confused due to their morphological similarity. However, P. peruviana has different medicinal properties 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 populations 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. Published by NRC Research Press 276 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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. Ironically, however, basic taxonomic studies in the country are severely lacking. 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 studies 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 presence of two possible invasive species, the tilapia flowerhorn Cichlasoma urophthalmus and the black chin tilapia Sarotherodon melanotheron in Manila Bay. In terms of trade-related studies, we identified confiscated juvenile 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 Acebes2 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 comprehensive taxonomic listing of marine species in the country. Here, we present our recent results at the NFRDI-Genetic Fingerprinting Laboratory 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 resurrected giant clam, Tridacna noae; and the reef manta ray, Manta alfredi. We established the first report in the country of two possible invasive species, the tilapia flowerhorn Cichlasoma urophthalmus sampled from fish ponds in Bulacan as well as the black chin tilapia Sarotherodon melanotheron 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 Genome Vol. 58, 2015 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 conservation 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. Parente1 1INBIO/CIBIO Azores, Department of Biology, University of the Azores, Portugal. of Botany, Ecology and Plant Physiology, University of La Laguna, Canary Islands, Spain. – Centre of Molecular and Environmental Biology, Department of Biology, University of Minho, Portugal. Corresponding author: R. Santos (e-mail: rod_msantos@hotmail.com). 2Department 3CBMA The Azores is situated in the North Atlantic Ocean (37–40N, 25–31W), astride the Mid-Atlantic Ridge, and is strongly influenced by the seasurface 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 introduction. Algae invasions in marine habitats represent a recognized worldwide threat to the integrity of native communities, to economies, 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 regions; and Asparagospis taxiformis, originally described for the southern hemisphere and widely distributed across tropical and temperate regions. We investigated the phylogeography of these two species in the Macaronesia, with focus in the Azores, to better understand biogeographic 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. Sathishkumar1 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 ingredients that are added to food in several forms as whole, in ground form, or as isolates from their extracts. An investigation on the beneficial 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 species, as rbcLa identified only at the family/genus level. It is very clearly demonstrated that DNA barcoding is a reliable and effective technique 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 embrace the use of DNA barcoding for the authentication of spice products by testing its bulk materials, which would help in fair trade practices. This would only amount to a minor cost to the spice industries 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 Camacho1 and Martha Valdéz-Moreno2 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 practice 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 species have limited closed fishing seasons. Among the Actinopterygii, Seriola dumerili, Scomberomorus cavalla, Bagre marinus, Sphyraena barracuda, Mycteroperca microlepis, and Epinephelus morio are the most commercialized. The latter falls under seasonal protection. We detected only seven species with name substitutions on the markets. The most common 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-Beaudin2 1Bishop's University, 26 College Street, Sherbrooke, QC J1M 1Z7, Canada. Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada. Corresponding author: Jade Savage (e-mail: jsavage@ubishops.ca). 2Biodiversity Background: The Muscidae (house-flies and relatives) is an ecologically diverse family of Diptera found in a wide range of terrestrial and 277 freshwater habitats. In Canada, the group is especially well represented in alpine and northern environments where they often account 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 identification (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 specimens, 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 occasional 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”. Mexico, 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 demonstrating 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 national laws. BWP has provided training, supplies, reagents, support for fieldwork, technical, and other support to enable to begin contributing BARCODE records to GenBank for the priority endangered species and related and look-alike species. The project had a roadmap, milestones, and a timeline for library construction and development of barcoding capabilities in enforcement agencies. SOPs for library construction 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 training and regular support by videoconference, thousands of very highquality 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 regulatory 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, GenBank records will need to come increasingly from in-country barcoding laboratories for which training and capacity-building will be needed. Published by NRC Research Press 278 DNA barcoding medicinal plants from Pakistan Melanie Schori,1 AlexaRae Kitko,1 Kirk Emch,1 Zabta K. Shinwari,2 and Allan M. Showalter1 1Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA. of Biotechnology, Quaid-i-Azam University, Islamabad, 45320, Pakistan. Corresponding author: Allan M. Showalter (e-mail: showalte@ohio.edu). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Department Background: In Pakistan, herbal products are a primary source of medicines, 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 countries 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 effectiveness 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 Wilson2 1Trent University, 2140 East Bank Drive, Peterborough, ON, Canada. Ministry of Natural Resources and Forestry, 2140 East Bank Drive, Peterborough, ON, Canada. Corresponding author: Natasha Serrao (e-mail: natasha.rosemary.serrao@gmail.com). 2Ontario Background: Effective management of endangered species requires sensitive detection of their occurrences, which is often difficult for lowabundance species. In aquatic environments, detection of rare species can be further confounded by site accessibility, sampling gear, and capture 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 detection in aquatic habitats from environmental DNA (eDNA). The utility of this methodology was tested at 29 sites where Redside Dace were historically 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 detections versus non-detections are important for study design. Significance: eDNA is a reliable method for species detection in freshwater systems and can be used as an effective sampling technique for documenting occurrences of aquatic endangered species. Genome Vol. 58, 2015 danger and warrant fast attention if they are not to be lost. The Redside 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 microsatellite loci to examine genetic diversity as well as contemporary spatial structuring. Results: The combined sequencing and genotyping results 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 management 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 discovery 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 accuracy 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 shortcoming 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 amplicon 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 parasitic species. Significance: DNA barcoding is a powerful tool for authentication. 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 Phylogeography and conservation of endangered Redside Dace, Clinostomus elongatus 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). Natasha Serrao,1 Scott Reid,2 and Chris Wilson2 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 laboratory 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. 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 Published by NRC Research Press Abstracts 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- dicotyledons) were subjected to evolutionary analyses using various bioinformatics and statistical tools including MEGA 5.05, DnaSP v5.10, and DAMBE v5.2.78. Results: The relatedness and distinction using the bioinformatics 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 evolutionary 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. Thaker1 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 region (ITS1-ITS2) region has been useful for phylogenetic analyses at lower taxonomic levels. In the present study, the molecular characterization between two Vigna species (Vigna radiata: 7 accessions and Vigna mungo: 24 accessions) was inferred using the internal transcribed 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, respectively. 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 analysis 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 different taxa. The study will be valuable for correct identification, differ- 279 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 identification by using the DNA barcoding method. It includes quality control, sequence assembly, sequence annotation, and species identification. 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, TCMIdentifier 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 assembly criteria defined in TCM-Identifier. The forward and reverse primers 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 barcode. 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 barcoding 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 challenge 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 collected, 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 Published by NRC Research Press 280 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 integral policy framework for conservation and sustainable use of biodiversity, 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. Invasive alien species are a threat to native species in all biogeographic regions, with estimates of global costs rising to some billions of dollars 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 Secretariat 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 technical experts from 21 developing countries in 2015. Significance: These trainees are expected to become in-country trainers spreading knowledge and capacity for rapid species identification in various sectors including trade, agriculture, and the environment. Although regulation on hazards is fairly well developed in chemicals, pests, and communicable 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 capacity 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 phylogenetic diversity greatly helps setting conservation priority. DNA barcode-based true bugs (Heteroptera) surveillance for agriculture crops from Maharashtra Anant Shinde1 and Gulab Khedkar2 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. Effective 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 Genome Vol. 58, 2015 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 Hajibabaei1 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) technologies allow the efficient analysis of DNA barcode sequences from individual specimens and from bulk environmental DNA (eDNA) samples. Commonly used DNA extraction protocols involve homogenizing 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 specimens 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 indicate 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 providing 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 extraction 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 organisms. 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 Shortridge1 and Jeff Miner2 1Bowling Green State University, 1833 Kettle Run Ct, Perrysburg, Ohio, USA. Green State University, Life Sciences, South College Drive, Bowling Green, Ohio, USA. Corresponding author: Megan Shortridge (e-mail: mshortr@bgsu.edu). 2Bowling Background: Early spring represents an interesting but poorly understood 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 predators to consume eggs and larvae of earlier-spawning species. Early life Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts stages of fishes digest quickly in the gut contents of predator fish, making visual identification difficult. Therefore, predation on fish eggs and larvae 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 sequencing of genomes and has proven useful for understanding diversity 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 biological context remains a challenge. Clustering permits a systematic investigation of relationships between genes by identifying clusters of genes that share similar expression patterns. In this study, a comparative study of three model-based clustering techniques for RNA-seq data was carried out. The methods included HTSCluster, MBCluster.Seq, and Poisson.glm.mix, available on the Comprehensive R Archive 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 nature 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 parameterized. 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 techniques specific to RNA-seq data are still in their infancy. This is because 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 Burgess1 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- 281 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 moderate phylogenetic dispersion that holds a large number of rare and endangered 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. Species 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 Nature 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, leading 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 Wilson1 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 biodiversity 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 correlations were weak and not statistically significant (p > 0.05). The highest richness (65% of observed species) was recorded in the unmanaged microhabitats. 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 richness in parks containing all the four microhabitat types. Whether a diverse 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 282 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 utilizes 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 fluorescent colours. The resulting fluorescent signatures can be characterized 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 characterization of small multicellular and single-celled species that are difficult to classify on the basis of morphology. Moreover, because 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 Barcoding to identification of plant species. Genome Vol. 58, 2015 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 biodiversity 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 wellknown environments yield new species. Documented marine extinctions 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 barcoding 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. Wheeler3 1Department 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. Janzen2 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 Cordillera 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 diversity 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 expected 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 sampling point, emphasizes that climate change will bring strong alterations 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. 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; contemporary species distributions are the result of dispersal from refugia beyond ice margins. While most species survived glaciations south of the ice sheets, many also survived in Beringia. Numerous reconstructions show additional unglaciated areas in the Canadian Arctic Archipelago, such as Banks Island; however, phylogeographic evidence 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 published 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 separate 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 populations there could have arrived from another Arctic Archipelago refugium. Although we did not find strong evidence for a Banks Island refugium, we found evidence for a possible Arctic Archipelago refugium. Published by NRC Research Press Abstracts 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 Network5 1OD Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 institutes 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 website (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 research 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 biological 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 pipelines (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 campaigns, this collaborative initiative is providing methodological resources 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 Backeljau1,7 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 topography, 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 interspecific differentiation, uniform worker morphology, lack of recent taxonomic 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 collection of DNA barcodes for every Ecuadorian ant species or morphospecies. Results: A first focus of this project was on the genus Leptanilloides 283 (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 currently 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 Meyer2 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) technologies. In comparison with Sanger sequencing, NGS potentially improves the sequencing success rate and the detection of heteroplasmy, heterozygosity in nuclear markers, and endosymbionts. Here, we applied TAS to simultaneously sequence the COI barcode region, three nuclear markers (wingless, white gene, and HOG703602), and a fragment of the Wolbachia surface protein (wsp) in 24 museum 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 specimens) than with Sanger sequencing (18/24 specimens). COI haplotypes obtained from both approaches were identical and showed divergences 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 (including 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 “biodiPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 284 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 information with intraspecific genetic polymorphism of species within a habitat 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 evolutionary forces, resulting in genetic hotspots. Results: We mapped genetic 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 tolerances. Several genetic hotspots were mapped, and we recommend conservation actions for some of them. Significance: Biodiversity hotspots 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 persistence. Mapping genetic hotspots can contribute to the design of effective conservation actions by detecting local areas with high genetic diversity, and the presence of unique gene variants—and thus presumably high evolutionary potential. Existing spatially explicit genetic 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 provide 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. Furthermore, 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 labeled 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 contents. Commonly used universal primers for smaller regions caused some taxa to be excluded due to primer-mismatch, resulting in detection 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. Genome Vol. 58, 2015 Using next-generation sequencing to identify the botanic origin of pollen collected from foraging honeybees Rowan Sprague,1 Stephane Boyer,2 and Stephen Wratten1 1Bio-Protection Research Centre, P.O. Box 85084, Lincoln University, New Zealand. Institute of Technology, 39 Carrington Road, Mount Albert, Auckland 1025, New Zealand. Corresponding author: Rowan Sprague (e-mail: rowan.sprague@lincolnuni.ac.nz). 2Unitec Background: An estimated 70% of crops benefit from pollination, and insects contribute the majority of pollination services. Honeybees (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 replicated 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 consisting 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 species, making it possible to determine whether the species not present 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 identification 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. Ravikanth1 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 products 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 international 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 admixtures 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 equivPublished by NRC Research Press Abstracts 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 patent 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 medicinal 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% genetic divergence with expected species. All the adulterant species belonged to different genera, and 14 of them belonged to different family, when compared with the species mentioned on the label. Species adulteration 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 substitutions 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. 285 450 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 Stenseth1 and Peter G. Kevan2 1Centre for Ecological and Evolutionary Synthesis, University of Oslo, Norway. of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada. Corresponding author: Peter G. Kevan (e-mail: pkevan@uoguelph.ca). 2School 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 scientific 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 initiated 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 Barcode 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 Stoeckle1 and David Thaler2 1The Linking adults and immatures of South African marine fishes Dirk Steinke,1 Allan Connell,2 Tyler Zemlak,1 and Paul D.N. Hebert1 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 identifications 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 collecting 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 Rockefeller University, 1230 York Ave., New York, NY 10065, USA. of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland. Corresponding author: Mark Stoeckle (e-mail: mark.stoeckle@rockefeller.edu). 2University Background: In most animals, mtDNA sequences cluster tightly within species yet differ sharply between them. However, the underlying evolutionary mechanisms are unknown. Here we utilize human data to evaluate alternative models—bottlenecks versus purifying selection. Humans are an ideal test case because we are vastly better studied than any other animal in terms of genetic diversity and evolutionary 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 representing all major haplogroups and compared human mitogenome variation to that of our closest living and extinct relatives (i.e., chimpanzees and bonobos, and Neanderthals and Denosovians, respectively) 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, chimpanzees 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 Published by NRC Research Press 286 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 adaptive bottlenecks as underlying intraspecific mtDNA clustering in humans 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 described 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 management. Results: We summarize our work on DNA barcoding of Chironomidae 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 interpretation and paves the way for a broader use of Chironomidae in environmental 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 mediumsized 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 diversity of these two species using DNA barcodes. Results: We sequenced Genome Vol. 58, 2015 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, Hachioji, and Sendai), and 63 of the 64 specimens had the same haplotype, 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 invasion. 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 Tadey1 and Cintia P. Souto2 1CONICET-CRUB, Pasaje Gutiérrz 1125, Argentina. Pasaje Gutiérrz 1125, Argentina. Corresponding author: Cintia P. Souto (e-mail: cintiap.souto@gmail.com). 2INIBIOMA-CONICET, 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 improve persistence of populations. DNA barcoding to determine the variation 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 phylogeny 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 barcode variation. Results: We analysed 75 species from 400 samples collected throughout Monte distribution. DNA barcodes were obtained for 190 specimens and 61 species. We obtained ITS2 sequences from 48 specimens (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 conservation 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 Jamil3 1Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada. 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). 2Department 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts and basic medical facilities. Livelihood of many people in these areas is dependent on the export of these medicinal species to herbal companies. Traditional knowledge of the medicinal uses of plants provides new insights to the scientific community to explore the medicinal ingredients of these plants; this is supported by DNA barcoding that provides rapid and accurate species identification, which is a key to understanding the medicinal value of plants. The conservation of medicinal plants has been neglected in many areas of Pakistan. This is the first attempt to assemble a reference DNA barcode library for indigenous medicinal plants of Pakistan. Results: We sampled 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 medicinal plants of Pakistan, and provides a DNA barcoding tool for identifying medicinal plants. We concluded that DNA barcoding using the rbcL marker is an important tool for taxonomists for timely and correct species identifications to overcome the impediments of morphological 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 economy 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 287 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 Biodiversity 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 specimens; 80% have an associated cytochrome c oxidase subunit I (COI) barcode 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 Facility (GBIF), and second by our expanding outgoing loan program. Results: 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 repository. 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 scientists. 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 barcode sequences means we are not constrained by the availability of taxonomists 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. 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 numerous ecosystem services. They are key species for forest maintenance through predation, pollination, and seed dispersal. Traditionally, understanding these interactions required morphological examination of gut content, faeces, and regurgitated pellets, with poor and taxonomically 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 rainforest 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 Atlantic 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 diversity 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. 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 morphological 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 invertebrate and fish species. Techniques employing next-generation sequencing 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 identification, and fish gut content analysis results to date, including comparison of morphological and molecular methods, will be presented. Significance: Morphological identification and subsequent DNA barcoding of benthic invertebrates have revealed important limitations in understanding 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. Published by NRC Research Press 288 Towards a safe herbal medicine in Ghana, the role of DNA barcoding Gyimah Thomas Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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-todoctor ratio in Ghana, most people, especially the urban poor and rural dwellers, resort to herbal medicines for their primary healthcare. Therefore, there is an increasing demand for herbal medicine, leading to overharvesting 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 sustainable management of medicinal plants species in southern Ghana. Silicadried 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 sequences in the later-collected farm dataset matched closely with their conspecific counterparts in our reference dataset, which suggests 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 Borisenko2 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 diverse 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 environmental detection of invasive organisms in both forensic and bulk samples; however, their scope has been limited. DNA barcoding should be broadly deployed and integrated into a nation-wide biosurveillance effort with a conceptually novel operational framework, supported by several important changes in legislation, policy, and governance. 1. DNAbased identification should be incorporated into Canadian federal legislation, 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 activities (e.g., impact assessments) into a centralized data portal, to facilitate 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 frameworks at an international level. Genome Vol. 58, 2015 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 processes 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 numerous aspects of ecological thought and theory with the aim of modelling entire ecosystems—including both discovered and undiscovered species—at a global scale. Although this is a long-term effort and early models 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 associated 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 particular, 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 radically different approach, focussing on organismal functions, interactions, the structure of the community and rates of flux through it, and it may be that only data collection involving next-generation DNA sequencing 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 Grill1 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 history of the endemic Sardinian blue butterfly, P. barbagiae de Prins and van der Poorten 1982, using the DNA barcoding region of the COI mitochondrial gene. The species is restricted to a few slopes in the mountainous areas of the island, and has been described as distinct from the continental and Corsican P. baton by characteristics of the male genitalia and wing markings. Results: We analyzed 11 individuals of P. barbagiae and compared 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. abencerragus (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 diPublished by NRC Research Press Abstracts versification in the Palearctic region, and contribute to our understanding of the evolution of endemism in the European butterfly fauna. CSI: Guelph—forensic applications of DNA barcoding for wildlife identification and food authentication 289 software is open source and is available online along with documentation and training materials. Significance: This informatics pipeline provides 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. Janet Topan Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 criminal forensic science, interest in non-human DNA analysis has been growing. 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 verification of food ingredients are often challenged by the lack of morphological 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. Results: Since 2010, the Canadian Centre for DNA Barcoding (CCDB) core laboratory has processed 74 cases, with over 2000 samples, for identification 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 identification; 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 species, 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 identification 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 project 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 Management System (FIMS) that populated and validated high-quality specimen 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 videoconferencing. 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 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 potentially 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 epidemiology, geographic distribution, and host ecology of these diseases. However, 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 sequences, 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 primers (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. Importantly, 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. Balanov1 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) comprises 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 morphological 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 growing 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 290 families and 45 species of eelpouts and by using model and characterbased 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, Neozoarcidae, and the recently revised Eulophiidae. The polyphyly amongst some subfamilies within the Stichaeidae is confirmed, whereas Opisthocentrinae and Pholidae seem to constitute a natural family-level taxon. Significance: The study provides a new view on the problem of taxonomic 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 representative 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 Reserves and National Nature Parks of Kazakhstan. Genetic diversity of plant populations will be studied by using different types of DNA markers, including universal markers of nuclear and chloroplast genomes. The project combines the efforts of botanists and geneticists from National State Reserves and National Nature Parks, National Universities, Botanical Gardens, and two Biotechnology Research Institutes; moreover, our team enthusiastically welcomes collaboration with foreign scientists and organizations. DNA barcoding of selected Philippine pomacentrids Erika R. Valeroso,1 Jonas P. Quilang,1 and Ma. Josefa R. Pante2 Genome Vol. 58, 2015 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 mammals 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 suggest the presence of two species due to the relatively high genetic difference 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 available for C. multipapillatum. These comparisons grouped some of our sequences 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 Valenzuela17 1University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada. 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). 1Institute 2Department Background: Pomacentrids, more commonly known as damselfishes, are some of the most abundant reef fishes found throughout the world's oceans. Out of the 387 species found worldwide, the Philippines 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 neighbourjoining 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 pomacentrids will become a useful tool as government agencies continue to monitor the marine aquarium trade in the country. 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 evolutionary 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 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). Published by NRC Research Press Abstracts Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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: Phylogenic reconstructions that include barcode data incorporate sufficient 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 empirical patterns, we are often confronted with findings that overturn current paradigms and thus must identify other combinations of evolutionary 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. Goldstein2 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 contain 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 postlarval 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 difficult 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 oceanographic 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 m2 diameter, 335 ␮m mesh) system. Results: We present preliminary results from DNA barcoding that successfully identify P. guttatus from previously missing larval developmental stages (I–V). We obtained a total of 64 phyllosomas corresponding to stages I–VII from P. guttatus, completing the phyllosomal description for this species, and obtained 49 larvae of P. argus. Additionally, we obtained two phyllosomas from Scyllarus chacei and two specimens from Scyllarides aequinoctialis, both slipper (Scyllaridae) lobsters. A total of 16 larvae (Scyllaridae) and one taxonomically unknown phyllosoma were reported as Phyllosoma Q (Robertson, 1972). Significance: Combining DNA barcoding with morphological identification 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 confirmation 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- 291 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 Mexican 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 representing 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 intraspecific 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 parasites are abundant in a wide range of marine and terrestrial hosts, are highly diverse, and can be useful for monitoring environmental anthropogenic 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 morphological characters that traditionally have been considered diagnostic. 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 species, 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 identifiable 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= and Reverse, 5=-TAAACTTCAGGGTGACCAAAAAATCA-3=. For ITS 2 amplification, the following primers were used: Forward, 5=-TGTGAACTGCAGGACACATG3=, 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 (Thelytokous), 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 Published by NRC Research Press 292 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 interspecific 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. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 Hegarty1,4 1National Botanic Garden of Wales, UK. University, UK. University, UK. 4Aberystwyth University, UK. Corresponding author: Natasha de Vere (e-mail: natasha.devere@gardenofwales.org.uk). 2Swansea 3Cardiff 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 intensification, pests, disease, and climate change. Understanding the reasons for declines and providing conservation guidance requires detailed information on the habitat requirements and foraging preferences 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 foraging 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 species 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, habitat 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. Coosemans1,7 1Institute of Tropical Medicine, Belgium. Belgium. 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). 2Avia-GIS, 3Royal 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 Genome Vol. 58, 2015 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 comprehensive reference database of verified DNA sequences is required. Results: Genetic diversity of mosquitoes in Belgium was assessed using 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 wellsupported 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 identification 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 Careta14 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 identification is usually a crucial first step in a forensic entomological analysis. 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 objective 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 Brazil. 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 collected and identified morphologically according to characteristics observed in an identification key. Of these, 36% were identified as Calliphoridae, 34% as Muscidae, and 30% as Sarcophagidae. Approximately 10% of all collected samples were DNA barcoded. It was possible to identify, through the DNA barcode matches, Atherigona orientalis, Chrysomya albiceps, Chrysomya megacephala, Musca domestica, Peckia chrysostoma, and Oxysarcodexia thornax. However, barcode-based identifications to the species level were achieved for only 35.7% of tested samples, which is probably due to the absence of the remaining species in these public databases. Significance: Species detected in this study corroborate results from other authors who demonstrated the occurrence of these species 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 identification technique for routine forensic applications. Published by NRC Research Press Abstracts 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. Costa2 1Departamento Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 sphaeromatid 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. Because 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 identified 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 clusters (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 complex. Sequences of the 18S rRNA nuclear gene essentially confirmed the complete sorting among the four lineages, although genetic distances 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 structure 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 Thomas1 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 individuals based on barcode sequences, has great applications for sample 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 identifying universal barcode loci for plants, attempts to sort barcodeamenable 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 sequencing retrieved good quality rbcL sequences in all accessions, Atpf 293 sequences in 249 accessions, and ITS sequence only in 120 accessions. A total of 493 ITS sequences were retrieved additionally by cloning the amplicons in 70 representative accessions. In phylogenetic 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. Further, the strategy for retrieving alternate barcode loci in Zingiberaceae 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. Hebert3 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, Unità 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 composition and genetic attributes of island communities. We use comparative analyses linking community and phylogenetic approaches for the butterfly fauna of the circum-Sicilian islands, a key intercontinental region in the western Mediterranean, to understand the factors that shaped the observed assemblages and to highlight populations of exceptional conservation value. Results: Species richness was mainly influenced by contemporary factors, but Pleistocene connections 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 frequency of a species on the mainland was a weak predictor of its frequency on islands, and most residuals appeared related to migratory 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 formerly connected to during Pleistocene low sea levels. Genetically diversified 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 biogeographical 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 information on intraspecific genetic diversity to obtain the information needed for accurate conservation decisions. Published by NRC Research Press 294 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. Chambers1 1Bat Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 information, 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 bioinformatic 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 globally, readily accommodates fecal DNA, and selectively targets bat but not prey DNA. Results: Our assay has high resolution (93%) for barcoded 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 communities (long-term roost sites). Another benefit of our Species from Feces tool is in confirming field identification, especially of morphologically 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: Although 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 determining the presence of bat species that are vulnerable or facing extinction. 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 Cheng1 1Institute of Insect Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China. of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang, 310058, China. Corresponding author: Xue-Qin Wang (e-mail: xqwang@zju.edu.cn). 2College 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 ecosystem 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 specimen identification. In this study, arthropods (pests, predators, parasitoids, neutral insects) were collected from rice paddies of Zhejiang province, China. Standard DNA barcoding using the mitochondrial 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 spiders were those of the prey species. Results: We established a reference barcode library including 356 records representing Genome Vol. 58, 2015 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 Turon1 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 becoming commonplace for characterizing eukaryotic diversity in relatively homogeneous communities—such as soils or soft-bottom sediments— but its implementation for characterizing macroscopically complex 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, consisting of fragments of the 18S rRNA and cytochrome c oxidase subunit I (COI) genes. A Bayesian clustering algorithm was used to define molecular 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 thousands of MOTUs from complex hard-bottom communities. Comparison 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 metabarcodingderived sequences. Significance: We extended the application of the metabarcoding approach to complex marine hard substrates, allowing a deeper characterization of biodiversity than morphology or individual barcoding approaches. A significant improvement in marine COI-barcoding databases is needed in order to get enhanced automated 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 actually caught in Australian waters, and he described eight species. Now, nearly 5000 species have been recorded from Australia, the vast majority 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 Published by NRC Research Press Abstracts numerous bony fish species have been barcoded, and 80% of the much less numerous elasmobranchs. Significance: In this talk I will summarise 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 example, 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Connor P.K. Warne,1 Stephanie L. deWaard,1 Joshua R. Kohn,2 Jon P. Rebman,3 Maria L. Kuzmina,1 and Bradley A. Zlotnick4 295 were properly labeled according to U.S. Food and Drug Administration 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 substitutions included those of lesser economic value and those posing health and conservation risks. The fraction of fish mislabeled varied according to the type of fish collected and type of retail outlet where fish samples were purchased. Significance: Wide dissemination of survey findings helped raise awareness of the pervasive problem of seafood fraud nationally and globally. Oceana's continued advocacy has contributed to advancing policy directives in the US, including increased seafood documentation requirements, traceability, and other measures. 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 contiguous 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, demonstrating 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% specimen and 71.8%, 60.4%, and 51.7% species success, respectively, reflecting 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 sequences 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 barcodes 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. Classifying DNA barcode multi-locus sequences with feature vectors and supervised approaches Emanuel Weitschek,1 Giulia Fiscon,2 Paola Bertolazzi,1 and Giovanni Felici1 1Institute of Systems Analysis and Computer Science – CNR, Via dei Taurini 19, Rome, Italy. of Computer, Control, and Management Engineering (DIAG), Sapienza University, Viale Ariosto 25, Rome, Italy. Corresponding author: Emanuel Weitschek (e-mail: emanuel@iasi.cnr.it). 2Department 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 compare multi-locus sequences. On the one hand, traditional alignmentbased 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 substrings (k-mers) and have proved successful in many applications, including multi-locus DNA barcode analysis. Alignment-free algorithms rely on counting and comparing the frequency of all the distinct 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 sequences 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 taxonomic levels by extracting a handful of rules. Barcoding forensic traces—practical challenges 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 Hanner3 1Oceana, 1350 Connecticut Ave. NW, 5th Floor, Washington, DC 20036, USA. of Columbia Public Schools, Washington, DC, USA. 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). 2District 3Biodiversity Background: Oceana, an international ocean conservation organization, 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 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 being 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 Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 296 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 subspecies 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 inheritance of mitochondrial DNA and inability to distinguish between species 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) mitochondrial 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 Hogg1 1University of Waikato, School of Science, Private Bag 3105, Hamilton, New Zealand. Museum, Christchurch, New Zealand. Corresponding author: Ian Hogg (e-mail: hogg@waikato.ac.nz). 2Canterbury Background: New Zealand is home to an estimated 2000 species of spider, although only 1134 species are presently described, and many can only be assigned to the family level. Furthermore, morphological identification is complicated by sexual dimorphism as well as individual variation 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 accurate 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 (BioSCAN) 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- Genome Vol. 58, 2015 tured directly into 95% ethanol. Each trap is accompanied by a microclimate weather station and augmented with landscape information. Sites range from downtown Los Angeles to less-urban areas near Griffith 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 diversity in thousands of weekly samples. Significance: Whole-sample molecular approaches are essential for addressing the extraordinary diversity of small metazoan populations, enabling comparisons between samples across space and through time. Next-generation sequencing of entire unsorted samples, targeting barcode (or other) genes, is the most promising route for characterizing the full multitaxon 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 unavoidably be based on quantitative approaches involving hyperdiverse 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 Wan3 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 urbanisation 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 everyday 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 richness 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 perspectives 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 business districts. We used DNA barcoding to assess the species richness and abundance at the sites. Preliminary analyses suggest species richness declined from the suburbs to the central business district; however, 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. Published by NRC Research Press Abstracts Testing the waters: using NGS to monitor zooplankton communities Steve Woods,1 Ian Hogg,1 Ian Duggan,1 Conrad Pilditch,1 and Jonathan Banks2 297 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 Research Station, Ecuador will be discussed. 1University of Waikato, School of Science, Private Bag 3105, Hamilton, New Zealand. Institute, 98 Halifax Street East, Nelson 7010, Private Bag 2, Nelson 7042, New Zealand. Corresponding author: Steve Woods (e-mail: steve.woods00@gmail.com). Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 2Cawthron 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 throughout 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: Zooplankton are currently used as indicators of lake trophic state. However, 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. Burgess1 1Columbus State University, 4225 University Avenue, Columbus, GA 31907, USA. Botanical Garden, 1345 Piedmont Avenue NE, Atlanta, GA 30309, USA. 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). 2Atlanta 3The 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 populations 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 phytochemicals that have evolved through a selective advantage to deter herbivores; 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 composition 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. Specifically, we evaluated the mean number of rbcL bp polymorphisms between 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 increases the value of indigenous knowledge and aids in conservation ef- Assessing benthic macroinvertebrate temporal turnover in a remote wetland through environmental barcoding Michael Wright,1 Donald J. Baird,2 and Mehrdad Hajibabaei1 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 environmental 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 barcoding, 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 dynamic, highly connected wetland system in northern Alberta that hosts large numbers of migratory birds and some of the last freeroaming 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 collected in June and August of 2012 and 2013, subsequently identifying a range of invertebrate taxa including annelids, molluscs, insects, and other arthropods. Through various measures of diversity, our approach showed variation in communities at different taxonomic levels from order to molecular operational taxonomic units. Significance: Environmental barcoding is able to show temporal 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 reference 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 Zhang2 1Department of Biology, McMaster University, 1280 Main St. West, Hamilton, Ont., Canada. University, Kunming, Yunnan, China. Corresponding author: Jianping Xu (e-mail: jpxu@mcmaster.ca). 2Yunnan Background: Wild edible mushrooms are important sources of nutrients and income for many communities in both developed and developing countries. Southwestern China is a biodiversity hotspot and a paradise for mushroom enthusiasts and connoisseurs. Wild mushrooms 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 representing 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 samPublished by NRC Research Press 298 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 species; 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 Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 species. 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 100 m × 5 m. When we visited a mountainous 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 collected 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 identified species using taxonomic literature and type specimens. This identification work is still ongoing because it takes much time, especially 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 Borneo. 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 Li1 1Kunming Institute of Botany, CAS, 132# Lanhei Road, Heilongtan, Kunming 650201, Yunnan, China. State University, 4225 University Avenue, Columbus, GA, USA. Pelee National Park, 407 Monarch Lane, RR1, Leamington, ON N8H 3V4, Canada. Corresponding author: Li-Jun Yan (e-mail: yanlijun@mail.kib.ac.cn). 2Columbus Genome Vol. 58, 2015 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 haplotype 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 previous studies that suggest hybridization between red and white mulberry 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 Chen2 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 (botanical source and morphological identification, microscopic identification, and physiochemical identification) have been unable to meet the demands 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 Pharmacopoeia (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 inadequate. 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 available 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 Identification, Protocol, Database, News, Documents, and Register. In the Identification 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 utilization of TCMs. Furthermore, this system can help prevent illegal drug trade in circulation. 3Point 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 characters, 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 preservation and removal, respectively. Results: In August of 2014, we collected 61 putative red, white, and hybrid mulberry from two National 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 speciesspecific amplification (SS-PCR) of the nuclear ITS2 barcode region. Ecological applications of DNA barcoding: the African experiences Kowiyou Yessoufou1 and Michelle van der Bank2 1University of South Africa, Chr de Wet avenue, 1710, Pretoria, South Africa. of Johannesburg, University Road, 2006, Johannesburg, South Africa. Corresponding author: Kowiyou Yessoufou (e-mail: kowiyouyessoufou1@gmail.com). 2University 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 studies. Using the core barcode to assemble the largest phylogeny of local, regional, and continental floras in Africa, we explored important questions related to community ecology (biological invasions and community responses to herbivory) and phylogenetic diversification (African cycads Published by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. Abstracts 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 phylogenetically 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 African native floras seems to be climatically mediated. For the African cycads, we found a pattern of constant radiation with mass extinction in addition to an explosive radiation in a Southern African clade that developed 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 independent origins almost synchronous with the South American counterparts, and that the development of this life form may have been mediated by the interactive effects of frequent fires and high precipitation. Significance: Using the core DNA barcodes in phylogenetic ecological studies, we shed light on contemporary and evolutionary events that shape the present-day African flora, demonstrating the ecological significance 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 available for the 648-bp barcode region of COI from nearly 400 000 animal species, this gene segment can be used to probe patterns of mitochondrial evolution. The present study examines levels of amino acid substitution 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 negatively correlated with generation times. Patterns of amino acid substitution in COI are generally congruent with previously reported variation in nucleotide substitution rates in arachnids, but provide some new insights, 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 importance 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 indels, 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 rapidly evolving lineages reproduce by haplodiploidy, a mode of reproduction 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. Adamowicz1 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 299 whole communities and to assess the likely number of species present. Recent research has indicated the success of DNA barcoding for identifying specimens belonging to a wide range of animal taxa, including planktonic 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 intraspecific 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 maxillopod reference data set covering a wide geographic range from Canada's three oceans. Our results indicate the presence of a biologically significant 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 biodiversity data for biodiversity research and species discovery purposes. We further highlight taxa where there was less concordance between morphological and molecular delineations and recommend the need for continued 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 Zhou2 1Kunming Institute of Zoology, 32 Jiaochang East Road, Kunming, Yunnan 650223, China. 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). 2BGI-Shenzhen 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 monitor bee abundance and diversity, since the number of wild bee species per country ranges from hundreds to thousands. Morphological identification is inefficient because taxonomic expertise is scarce, and PCR-based metabarcoding is error-prone. Results: We used a metagenomic approach 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 identifications. The two datasets returned indistinguishable estimates of community structure and extrapolated species diversity, and read number per species was positively but weakly correlated with estimated biomass per species (R2 = 24.9%, p < 0.001). By contrast, metabarcoding 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 Published by NRC Research Press 300 deserve consideration as “super-barcodes” for economically and socially important species. Genome Vol. 58, 2015 Identification of calliphorid flies in their different life stages using DNA barcoding can improve the success of forensics investigations Sureshchandra Zambare and Gulab Khedkar Higher fungal diversity in dead wood reduces CO2 emissions in a natural forest 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). Douglas W. Yu,1 Chunyan Yang,1 Douglas A. Schaefer,2 Viorel D. Popescu,3 and Weijie Liu2 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 identification 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) database. 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. 1Kunming Institute of Zoology, 32 Jiaochang East Road, Kunming, Yunnan 650223, China. Tropical Botanical Garden, Menglun, Mengla, Yunnan 666303, China. Fraser University, Burnaby, BC V5A 1S6, Canada. Corresponding author: Douglas W. Yu (e-mail: dougwyu@gmail.com). 2Xishuangbanna Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 3Simon Background: Wood decomposition releases almost as much CO2 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 colonized wood in subtropical China. Wood hosting more diverse fungal communities emitted less CO2, 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 interference 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 CO2 made by the world's stock of decomposing 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 Chen1 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 oriental 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 cryptic 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 Calliphoridae, 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 indication of geographic variation in C. megacephala within China. Significance: The larvae of C. megacaphala are important in forensic death investigations, in wound therapy, and in traditional Chinese medicine, 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. 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 Lucci4 1Universidade Estadual de Campinas, Campinas-SP, Brazil. 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). 2Muséum Background: Surveys of insect diversity, especially in the tropics, are strongly impeded by the difficulty in obtaining accurate species delimitation 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 diversity. In this study, 14 026 individuals of Tiger-Moths (Erebidae: Arctiinae) 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 specimens representing 291 of these species. Operational taxonomic units (OTUs) were defined using a 2% sequence divergence threshold and compared to morphospecies defined from our initial morphological assessment (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. Morphology could not be reassessed in three species, and the species delimitations changed in the remaining 87 IMA species, suggesting that as much as 30% of the diversity might be overlooked when using superficial morphological 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 Lepidoptera of Serra do Mar Project allow automated species identifications of a Published by NRC Research Press Abstracts 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 Fang1 1College Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 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 unrevealed 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 copepods 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% ethanolpreserved individuals were carefully washed, had their appendages removed, and were checked under a fluorescence microscope to exclude 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 sequenced. 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 diatom 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 chlorophytes 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 Hydromedusae (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 molecular 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. Cristescu1 1Department of Biology, McGill University, 1205 Docteur Penfield, Montréal, QC H3A 1B1, Canada. and Oceans Canada, 3190 Hammond Bay Rd, Nanaimo, British Columbia, Canada. Corresponding author: Guang K. Zhang (e-mail: guang.zhang@mail.mcgill.ca). 2Fisheries Background: Aquatic invasive species (AIS) negatively impact economies and ecosystem functions, making their early detection of great importance for conservation and environmental sustainability. Metabarcoding can be used as a rapid method for monitor- 301 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 analyses. The mitochondrial cytochrome c oxidase subunit I (COI) gene has high interspecific genetic variation and comprehensive reference 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 communities 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, Decapoda, 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 (R2 = 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 networks that aim to generate catalogs of aquatic invertebrates. Moreover, 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 specimens from the coastal waters of China revealed three as yet unreported species, designated Clytia xiamenensis sp. nov., Clytia gulangensisi sp. nov., and Clytia tankahkeei sp. nov., that were considered as conspecific 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 species 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 species 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 belonging to 312 families (sensu Engler). Among them, 809 species are conPublished by NRC Research Press Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 302 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 happening. 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 herbarium (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 phylogenetic 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 endangered species is being evaluated. The use of DNA barcoding in large-scale ‘omics projects Xin Zhou1 and Karl Kjer2 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 taxonomy, life-stage association, phylogenetics, diet analysis, biodiversity 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, providing a huge challenge for taxonomic identification of sequenced taxa. This presentation will demonstrate the usefulness of DNA barcoding 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 biodiversity surveys of meiofauna and nematodes. The cytochrome c oxidase 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 Genome Vol. 58, 2015 sequences in BOLD (Barcode of Life Data Systems). There is recommendation 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 compared 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 richness against morphospecies. Our results show that 18S reduced sequence 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 specimens and species, respectively. 18S recovered 100% of morphospecies, whereas COI increased species richness by a factor of 0.02 due to potential 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 complementary marker to COI and it can be used in conjunction in marine nematode barcoding and biodiversity survey projects. But before largescale 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 Yao1 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 medicine). 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 phenomenon 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, granules, 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 products, 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 intraspecific 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 distinguished 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 labels for consumers. Published by NRC Research Press Abstracts The San Diego Barcode of Life: launching a regional DNA barcode campaign in a globally important biodiversity hotspot “Barcoding our Backyard” at ResMed, Inc.: 52-consecutive weeks Malaise trap project at a corporate headquarters in a Global biodiversity hotspot Bradley Zlotnick Bradley Zlotnick,1 Joshua Kohn,2 David Dannecker,2 and Valérie Lévesque-Beaudin3 San Diego Barcode of Life, 3525 Del Mar Heights Road Suite #139, San Diego, CA, USA. E-mail for correspondence: sdbarcodeoflife@gmail.com. Genome Downloaded from www.nrcresearchpress.com by 175.157.185.230 on 08/18/15 For personal use only. 303 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 preeminent node and example for exploration, dissemination, and implementation of biodiversity DNA barcoding and its infrastructure. SDBOL leads and partners in San Diego's innovative and entrepreneurial scientific, corporate, civic, philanthropic, and academic communities 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 conservancy 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. SDBOL 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 initiated 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 Rossano, bringing the public to bioliteracy and highlighting iBOL innovations. 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. 1San Diego Barcode of Life, 3525 Del Mar Heights Road Suite #139, San Diego, CA, USA. of California, San Diego, CA, USA. Institute of Ontario, University of Guelph, Guelph, Ontario, Canada. Corresponding author: Bradley Zlotnick (e-mail: sdbarcodeoflife@gmail.com). 2University 3Biodiversity Background: In February 2014, ResMed, Inc., a leading San Diego medical 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 sculpture 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: Thirtynine 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 sampling. 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 Museum. 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. Published by NRC Research Press