Complete issue - IMA Fungus

The Global 

Mycological Journal 

Volume 3 · No. 2 · December 2012 

NEWS · REPORTS · AWARDS AND PERSONALIA · RESEARCH NEWS 

BOOK NEWS · forthcoming MEETINGS · ARTICLES

Colofon 

IMA Fungus 

Compiled by the International 

Mycological Association for the 

world’s mycologists. 

Scope: All aspects of pure and 

applied mycological research and 

news. 

Aims: To be the flagship journal 

of the International Mycological 

Association. IMA FUNGUS is 

an international, peer-reviewed, 

open-access, full colour, fast-track 

journal. 

Frequency: Published twice per year 

(June and December). Articles are 

published online with final pagination 

as soon as they have been 

accepted and edited. 

ISSN 

E-ISSN 

2210-6340 (print) 

2210-6359 (online) 

Websites: www. imafungus.org 

www.ima-mycology.org 

E-mail: d.hawksworth@nhm.ac.uk 

Volume 3 · No. 2 · December 2012 

Cover: Leucoagaricus variicolor, 

a new species from Aragón, 

northern Spain, which forms 

basidiomes in the winter. See pp. 

127–133 of this issue. Photo taken 

in Spain by Guillermo Muñoz 

González. 

EDITORIAL BOARD 

Editor-in-Chief 

Prof. dr D.L. Hawksworth CBE, Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense 

de Madrid, Plaza Ramón y Cajal, 28040 Madrid, Spain; and Department of Life Sciences, The Natural History 

Museum, Cromwell Road, London SW7 5BD, UK; E-mail: d.hawksworth@nhm.ac.uk 

Managing Editor 

Prof. dr P.W. Crous, CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands; 

E-mail: p.crous@cbs.knaw.nl 

Layout Editors 

M.J. van den Hoeven-Verweij & M. Vermaas, CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD 

Utrecht, The Netherlands; E-mail: m.verweij@cbs.knaw.nl 

Associate Editors 

Dr T.V. Andrianova, M.G. Kholodny Institute of Botany, Tereshchenkivska Street 2, Kiev, MSP-1, 01601, Ukraine; 

E-mail: tand@darwin.relc.com 

Prof. dr D. Begerow, Lehrstuhl für Evolution und Biodiversität der Pflanzen, Ruhr-Universität Bochum, Universitätsstr. 

150, Gebäude ND 03/174, 44780, Bochum, Germany; E-mail: dominik.begerow@rub.de 

Dr S. Cantrell, Department of Plant Pathology and Crop Physiology, Louisiana State University, Agricultural Centre, 455 Life 

Sciences Bldg., Baton Rouge, LA 70803, USA; E-mail: scantrel@suagm.edu 

Prof. dr D. Carter, Discipline of Microbiology, School of Molecular Biosciences, Building G08, University of Sydney, 

NSW 2006, Australia; E-mail: d.carter@mmb.usyd.edu.au 

Prof. dr J. Dianese, Departamento de Fitopatologia, Universidade de Brasília, 70910-900 Brasília, D.F., Brasil; E-mail: 

jcarmine@unb.br 

Dr P.S. Dyer, School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 

2RD, UK; E-mail: paul.dyer@nottingham.ac.uk 

Dr M. Gryzenhout, Dept. of Plant Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South 

Africa; E-mail: Gryzenhoutm@ufs.ac.za 

Prof. dr L. Guzman-Davalos, Instituto de Botánica, Departamento de Botánica y Zoología, Universidad de Guadalajara, 

A.P. 1-139 Zapopan, 45101, México; E-mail: lguzman@cucba.udg.mx 

Dr K. Hansen, Kryptogambotanik Naturhistoriska Riksmuseet, Box 50007, 104 05 Stockholm, Sweden; E-mail: karen. 

hansen@nrm.se 

Prof. dr K.D. Hyde, School of Science, Mae Fah Luang University, Tasud, Chiang Rai, Thailand; E-mail: kdhyde3@ 

gmail.com 

Prof. dr L. Lange, Vice Dean, The Faculties of Engineering, Science and Medicine, Aalborg University; Director of 

Campus, Copenhagen Institute of Technology (CIT), Lautrupvang 15, DK-2750 Ballerup, Denmark; E-mail: lla@ 

adm.aau.dk 

Prof. dr L. Manoch, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, 

Thailand; E-mail: agrlkm@ku.ac.th 

Prof. dr W. Meyer, Molecular Mycology Research Laboratory, CIDM, ICPMR, Level 3, Room 3114A, Westmead 

Hospital, Darcy Road, Westmead, NSW, 2145, Australia; E-mail: w.meyer@usyd.edu.au 

Dr D. Minter, CABI Bioservices, Bakeham Lane, Egham, Surrey, TW20 9TY, UK; E-mail: d.minter@cabi.org 

Dr L. Norvell, Pacific Northwest Mycology Service, LLC, 6720 NW Skyline Boulevard, Portland, Oregon 97229-1309, 

USA; E-mail: llnorvell@pnw-ms.com 

Dr G. Okada, Microbe Division / Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, 

Wako, Saitama 351-0198, Japan; E-mail: okada@jcm.riken.jp 

Prof. dr N. Read, Fungal Cell Biology Group, Institute of Cell and Molecular Biology, Rutherford Building, University 

of Edinburgh, Edinburgh EH9 3JH, UK; E-mail: nick@fungalcell.org 

Prof. dr K.A. Seifert, Research Scientist / Biodiversity (Mycology and Botany), Agriculture & Agri-Food Canada, K.W. 

Neatby Bldg, 960 Carling Avenue, Ottawa, ON, K1A OC6, Canada; E-mail: seifertk@agr.gc.ca 

Prof. dr J.W. Taylor, Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, 

CA 94720, USA; E-mail: jtaylor@berkeley.edu 

Prof. dr M.J. Wingfield, Forestry and Agricultural Research Institute (FABI), University of Pretoria, Pretoria 0002, 

South Africa; E-mail: mike.wingfield@fabi.up.ac.za 

Prof. dr W.-Y. Zhuang, Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy 

of Sciences, Beijing 100080, China; E-mail: zhuangwy@sun.im.ac.cn 

 

ima fUNGUS

Organizing mycology 

Questions, and quizzical or glazed expressions, are not unfamiliar amongst audiences of mycologists when the acronyms or 

names of committees and organizations start to be banded about as if everyone should instinctively know what they were 

and what they did. In order to alleviate this situation, Andrew Miller (Secretary, International Commission on the Taxonomy 

of Fungi, ICTF) has produced a helpful diagram for the ICTF webpage (www.fungaltaxonomy.org/affiliations) 

which is reproduced here. 

EDITORIAL 

The overarching body concerned 

with the promotion of science 

internationally is ICSU: the International 

Council of Science, established in 

1931, and named the International Council 

of Scientific Unions until the name was 

changed in 1998. The mission of ICSU is 

to strengthen international science for the 

benefit of society, through the mobilization 

of knowledge and resources. ICSU is 

supported financially by national scientific 

members, that is national academies, 

research councils, or equivalents, currently 

representing 140 countries. In addition 

it has 31 scientific unions as members, 

of which two are of major importance to 

mycologists: (1) the International Union 

of Biological Sciences (IUBS) formed long 

before ICSU itself in 1919 and now with 40 

national and 80 scientific members; and (2) 

the International Union of Microbiological 

Societies (IUMS) which was formed in 

1980 after separation from IUBS, to which 

microbiological organizations had previously 

adhered. 

The IMA is a scientific member of 

IUBS, as is the International Association 

for Lichenology (IAL), International 

Society for Mushroom Science (ISMS), 

and the International Society for Plant 

Pathology (ISPP). IUBS includes all 

organizations involved with the regulation 

of the naming of eukaryotes, including 

those responsible for the six-yearly 

International Botanical Congresses, 

the first of which was held in 1865, 

and at which the International Code 

of Nomenclature for algae, fungi, and 

plants (ICN) is revised. That Congress 

appoints the Nomenclature Committee 

for Fungi (NCF) to rule and advise 

on all aspects of fungal nomenclature. 

IUMS has three divisions, one of which 

is the Division of Mycology, and that has 

several Commissions devoted to topics 

in mycology, as indicated in the diagram, 

and to which the International Society for 

Human and Animal Mycology (ISHAM) 

adheres. 

It is also possible to have inter-union 

bodies, and the ICTF is one of these. The 

ICTF complements the NCF in dealing 

with taxonomic rather than nomenclatural 

issues, for example in relation to the 

development of good practice and the 

establishment of Subcommissions focussing 

on different groups of fungi, but is currently 

working closely with the NCF on the 

development of lists of fungal names to 

be accorded special protection or to be 

suppressed. A second example of an interunion 

body of interest to mycologists is the 

World Federation for Culture Collections 

(WFCC). 

For further information on the history 

and roles of all the bodies mentioned here, 

and full lists of the different societies and 

other organizations that adhere to them, 

explore the pertinent web pages, which may 

prove a fascinating insight into the world of 

bioorganization. 

The issue of having mycology 

represented within two ICSU unions may 

seem strange, and indeed this has been a 

matter of some discussion at various times 

in the past (Simmons 2010). However, 

while this situation may not be optimal, and 

surely merits revisiting at some future date, 

it currently creates few practical problems 

and the IMA and IUMS now endeavour to 

operate synergistically wherever appropriate 

for the good of mycology. 

Simmons EG (2010) The International Mycological 

Association: its history in brief with summaries 

of its International Mycological Congresses and 

diverse international relationships. IMA Fungus 

1: 18–100. 

David L. Hawksworth 

Editor-in-Chief, IMA Fungus 

(d.hawksworth@nhm.ac.uk) 

BREAKING NEWS 

Official nomenclatural 

repositories for fungal 

names announced. 

See p. (44)–(45) 

volume 3 · no. 2 

(43)

News 

MycoBank, Index Fungorum, and Fungal Names 

recommended as official nomenclatural repositories 

for 2013 

The Nomenclature Committee for Fungi, 

which voted to support multiple official 

repositories over a single repository during 

a recent ballot, has accepted three starting 

1 January 2013: Fungal Names, Index 

Fungorum, and MycoBank. During 

November, repository representatives signed 

a Memorandum of Cooperation that will 

continue until the 2017 International 

Botanical Congress. The 2014 International 

Mycological Congress must ratify the 

NCF recommendation after reviewing the 

effectiveness of this arrangement. 

Beginning 1 January 2013, a prerequisite 

for valid publication of a fungal name is the 

citation in the protologue of an identifier 

issued by a recognized repository (Art. 

42.1, International Code of Nomenclature 

for algae, fungi, and plants [Melbourne 

Code], McNeill et al. 2012). Article 42.3 has 

empowered the Nomenclature Committee 

for Fungi (NCF), a body appointed by the 

International Botanical Congress, with the 

ability to appoint and recognize one or 

more repositories subject to later ratification 

by an International Mycological Congress. 

This action would appear to be a simple 

task that would have been decided several 

months ago. However, as with all things 

nomenclatural, the ‘Realpolitik’ behind this 

task was far more complex. 

Attendees of the 2011 International 

Botanical Congress (Melbourne) regarded 

MycoBank , 

the most frequently used online registry 

established in 2005, as prominent enough 

to serve as a cited example of a potential 

repository in the new Code (Art. 42. 1 

Ex. 1). However, two other repositories 

had been developed in anticipation of 

the need for official repositories. These 

were Index Fungorum and Fungal Names < 

http://fungalinfo.im.ac.cn/fungalname/ 

fungalname.html>. Currently, MycoBank 

is owned by the International Mycological 

Association and is run off servers in Belgium 

and The Netherlands. Index Fungorum, 

which began functioning as a repository in 

2009, was run by a partnership that changed 

during 2012, initially comprising three 

partners — CABI, UK , CBS-KNAW Fungal Biodiversity 

Centre, The Netherlands , and Landcare Research, New 

Zealand — but by mid 2012 consisting 

of two, CABI and Landcare Research; by 

November 2012, following the transfer of 

Index Fungorum curator, Paul Kirk, from 

CABI (on whose servers IF resided) to the 

Royal Botanic Gardens Kew on 1 November 

2012, the IF partnership consisted of a 

single partner, Landcare Research, with 

servers in New Zealand. Fungal Names is an 

initiative of the Institute of Microbiology, 

Chinese Academy of Sciences (IM-CAS), 

with servers in Beijing. 

As noted here previously (Norvell & 

Redhead 2012), the differing views in the 

mycological community became readily 

apparent at this year’s (April 12–13) 

Amsterdam CBS symposium: One Fungus 

= Which Name? 

and the April 15 th meetings of the 

International Mycological Association 

Executive Committee and the International 

Commission on the Taxonomy of Fungi 

in Utrecht. On May 14 th , IMA president 

John Taylor wrote to the NCF urging 

that MycoBank be selected as the “central 

registry” while acknowledging that other 

repositories (Index Fungorum, Fungal 

Names) might be recognized. Further 

discussions on registries were held during 

the 16 July 2012 nomenclatural session at 

the 2012 Mycological Society of America 

annual meeting (Yale University, New 

Haven CT). 

Major decisions on registries were 

delayed pending a meeting of representatives 

of the three repositories (Paul Kirk, Vincent 

Robert, and Yi-Jian Yao) at the “New Era 

of Fungal Nomenclature” symposium in 

Beijing (9–10August 2012), . Notably, Kirk 

and Yao are also members of the NCF. 

Following negotiations between these 

representatives (Fig. 1), NCF Chairman 

Scott Redhead reported an agreement by 

the parties to work towards a Memorandum 

of Cooperation (MOC), noting that letters 

of institutional support might be needed 

to help the NCF decide which repositories 

could be recommended. 

On 16 August 2012, the Chinese 

Academy of Science provided a letter of 

support for Fungal Names, and Robert 

initiated a draft MOC starting with 

MycoBank and IMA. All documents were 

circulated within the NCF (10 October 

2012) and among the three repositories. 

(44) 

 

ima fUNGUS

NEWS 

Geoffrey C. Ainsworth (1905–1998), whose 

vision and actions led to the formation of the 

IMA in 1971, proposed, along with Raffaele 

Ciferri (1897–1964) that newly published 

fungus names should be registered 58 years 

before this was to become a reality (Ainsworth 

& Ciferri, Taxon 4: 3–6. 1955). 

Deliberations were delayed during transfer 

of Index Fungorum to Landcare Research 

while still curated by Kirk, but on 19 

November the NCF began voting on 

repositories. On 27–29 November, the 

MOC among CBS (Pedro Crous) and 

the IMA ( John Taylor) for MycoBank, 

Landcare Research (Richard Gordon) 

for Index Fungorum, and Institute of 

Microbiology, CAS (Li Huang) for Fungal 

Names, was signed. The NCF voting period 

closed officially on 3 December, after 14 

(out of 17) NCF members had sent in their 

ballots; no further votes were received after 

that date. 

NCF voting protocols dictate at least 

60 % of the total membership (here 11 of 

17) must agree in order to reach consensus, 

and therefore the actual percentages for 

the received votes are higher than the rules 

require. There were 13 items on the ballot, 

of which 11 received 65 % consensus. The 

following principal issues were resolved by 

ballot: 

A majority of 65 % did not favour 

recognizing only one repository, while 

71 % favoured more than one repository, 

recommending the following three: Fungal 

Names (~71 %), Index Fungorum (~71 %), 

and MycoBank (~82 %). A 71 % majority 

felt that the NCF should require responsible 

repository representatives to sign an 

MOC agreeing to cooperate as requested 

by the NCF. Another ~71 % felt that 

synchronizing data-sharing in the minimal 

fields among multiple registries is essential, 

and ~65 % recommended that the NCF 

should require shared unique identifier 

numbers among all participating registries. 

While ~71 % agreed that all registration 

numbers be prefixed with the same 

identifying acronym, there was no consensus 

on which unique prefix to use, an item now 

under discussion. In view of the fact that we 

have less than a month before registration is 

required, ~65 % recommended that during 

2013 the NCF recognize the current prefix 

for the three repositories (i.e. FN, IF, MB). 

~76 % agreed that only the ‘minimal’ 

requirements (Art. 42.2) are required for an 

official repository [these include scientific 

name, rank (Art. 37.1); basionym with 

citation (Art. 41.5); validating description/ 

diagnosis (Latin or English) of new taxon 

names (Art. 39.2); place of effective 

publication of name (Art. 32.1); holotype 

[or equivalent when new] (Art. 40.1) 

including holotype specimen identifier 

number or other identifying data for species 

and subspecific taxon names and type taxon 

name and authorship [or identifier] for 

supra-specific taxon names; and location of 

holotype [herbarium, institute, collection] 

(Art. 40.7)]. However, the single ‘no’ vote 

was accompanied by concern over current 

irregularities in existing databases, raising a 

legitimate question that is now being further 

discussed within committee. 

The Committee, which continues to 

discuss how best to implement registration, 

will review and evaluate the effectiveness of 

the arrangement after one year, thereafter 

assembling a full report for the IMC10 in 

Bangkok in 2014. It will also determine 

whether any repositories are not functioning 

as expected and should be removed, or 

whether additional repositories are to be 

considered. The current MOC among the 

Chinese Academy of Sciences, CBS, IMA, 

and Landcare Research, runs until August 

2017, coinciding with the next International 

Botanical Congress, at which time it 

automatically expires unless renewed. 

McNeill J, Barrie FR. Buck WR, Demoulin V, 

Greuter W, Hawksworth DL, Herendeen PS, 

Knapp S, Marhold K, Prado J, Prud’homme 

van Reine WF, Smith GE, Wiersema JH, 

Turland NJ (eds) (2012) International Code 

of Nomenclature for algae, fungi, and plants 

(Melbourne Code) adopted by the Eighteenth 

International Botanical Congress Melbourne, 

Australia, July 2011. [Regnum Vegetabile no. 

154.] A.R.G. Ganter Verlag, Ruggell. 

Norvell LL, Redhead SA (2012) Registries of names 

and the Code. IMA Fungus 3: (2). 

Scott A. Redhead 1 and Lorelei L. Norvell 2 

1 

Chair, Nomenclature Committee for 

Fungi, National Mycological Herbarium, 

Science and Technology Branch, 

Agriculture and Agri-Food Canada, 960 

Carling Avenue, Ottawa, ON, Canada K1A 

0C6; scott.redhead@agr.gc.ca 

2 

Secretary, Nomenclature Committee for 

Fungi, Pacific Northwest Mycology Service, 

Portland, Oregon 97229-1309, USA; 

llnorvell@pnw-ms.com 


(45)

News 

Violins and mushrooms 

superior tonal qualities, were constructed 

from Norway spruce that had grown mostly 

during the Maunder Minimum (1645–1715), 

a period of reduced solar activity when 

relatively low temperatures caused trees to lay 

down wood with narrow annual rings and 

rendering the wood softer. He then carried 

out experiments on the resonance resulting 

from woods infected with various fungi, and 

reported on his results using sycamore (Acer 

pseudoplatanus) and Norway spruce (Picea 

abies) and two fungi, Physisporinus vitreus 

and Xylaria longipes (Schwarze et al. 2008). 

He went on to have violins constructed 

from infected and untreated wood by master 

violin makers. The fungi in the wood were 

first killed to ensure decay did not continue. 

This was followed by a blind trial in which 

a UK violinist, Matthew Trusler, played the 

new violins and one that had been made by 

Stradivarius in 1711. Experts considered 

that the new infected wood violin was the 

Stradivarius, but you can check for yourself in 

audio-files available through The Economist 

website (see above). Schwarze had solved a 

problem that had defeated instrument makers 

for three centuries. It would be interesting to 

know if the wood Stradivarius had used was 

also infected by wood-decay fungi, or if the 

results he achieved were due to climate alone 

as assumed. 

Schwarze FWMR, Spycher M, Fink S (2008) 

Superior wood for violins – wood decay fungi 

as a substitute for cold climate. New Phytologist 

179: 1095–1104. 

On 22 September 2012 The Economist has 

a striking headline “Violins constructed 

from infected wood sound like those of 

Stradivari” (http://www.economist.com/ 

node/21563276). The investigations of 

mycologist Francis W. M. R. Schwarze, 

based in the Swiss Federal Laboratories 

for Materials Science and Technology (St 

Gallen, Switzerland) had caught the public 

eye. Schwarze had noticed that sound travels 

faster through healthy wood than through 

wood softened by fungal attack. He discovered 

that the violins produced by Antonio 

Stradivarius during the late 17 th and early 18 th 

centuries, which are recognized as having 

Physisporinus vitreus. Photo: Francis Schwarze. 

Establishing authenticity in newly generated ITS 

sequences 

The issue of reliability in the scientific names appended to 

sequences in public databases such as GenBank is a matter of major 

concern, especially as these may be used uncritically in barcoding, 

environmental diversity assessments, and even phylogenetic studies. 

This thorny topic is addressed in a most thoughtful and constructive 

article by Nilsson et al. (2012) who not only pin-point the key 

targets required for confidence, but present guidance on how 

those targets may be realized in a particular case. The five targets 

recognized and guidelines presented are: 

· Establish that the sequence come from the intended gene or 

marker. 

Guideline 1: It is simple to check that all query sequences 

represent the ITS region. 

· Establish that all sequences are given the correct (5’ to 3’) 

orientation. 

Guideline 2: A single alignment step can assess the orientation 

of the query sequence. 

· Establish that there are no (bad cases of ) chimeras in the dataset. 

Guideline 3: PCR chimeras tend to lack full counterparts in the 

sequence databases and are therefore usually easy to spot through 

BLAST. 

· Establish that there are no other major technical errors in the 

sequences. 

Guideline 4: Sequences can be broken in other, puzzling ways; 

BLAST again, will tell. 

· Establish that any taxonomic annotations given to the sequences 

make sense. 

Guideline 5: Taxonomic annotations should be verified before 

the sequences are used. 

In each case, there are detailed practical step-wise accounts of what 

can be done, and attention is drawn to the issue of what can be done 

over erroneously labeled sequences. The article has been prepared 

(46) 

 

ima fUNGUS

y a team of particularly experienced 

molecular mycologists, primarily concerned 

with basidiomycetes, but merits the close 

attention of mycologists involved in 

sequencing studies of any kinds of fungi, 

and whether for applied, systematic, or 

ecological purposes. 

Nilsson RH, Tedersoo L, Abarenkov K, Ryberg 

M, Kristiansson E, Hartmann M, Schoch 

CL, Nylander JAA, Bergsten J, Porter TM, 

Jumpponen A, Vaishampayan P, Ovaskainen 

P, Hallenberg N, Bengttsson-Palme J, Eriksson 

KM, Larson K-H, Larsson E, Kõljalg U (2012) 

Five simple guidelines for establishing basic 

authenticity and reliability of newly generated 

fungal ITS sequences. MycoKeys 4: 37–63. 

NEWS 

Possible mutagen effects on genetic stability of 

fungi in living collections 

1 

The issue of long-term genetic stability 

of fungal strains preserved in biological 

resource centres with collections of cultures 

has been a topic of concern for at least half a 

century, especially with respect to the loss of 

ability to form particular extrolites or loss of 

pathogenicity. The advent of cryopreservation 

techniques has made a major contribution 

to alleviating this problem, but, nevertheless, 

Paterson & Lima (2012) point out that 

there are grounds for continuing vigilance 

and a need for awareness of possible 

biochemical mutagens. Three sources 

of possible biochemical mutagens when 

isolating material from the environment 

are recognized: (1) mutagenic antibiotics 

included in media; (2) microbial mixtures 

may include some taxa able to produced 

mutagens; and (3) mutagens formed by the 

target fungus in culture. The types of damage 

to DNA are wide-ranging, although some 

effects appear to be epigenetic and not to 

involve the fungal DNA, and around 90 

fungi producing mutagenic mycotoxins are 

now known – including some agarics. It 

is also noted that changes can conceivably 

arising during subculturing and preservation 

procedures. Amongst various suggestions 

made to alleviate the problem, is growing 

the fungi for different time periods and on 

different media prior to preservation. This is 

clearly a topic meriting further investigation, 

and perhaps particularly critical strains, such 

as name-bearing types or patent strains, 

should be routinely preserved in or on a range 

of media for long-term storage. 

Paterson RRM, Lima N (2012) Biochemical 

mutagens affect the preservation of fungi and 

biodiversity estimation. Applied Microbiology 

and Biotechnology: DOI:10.1007/s00253- 

0124554-6. 

Representation of kinds of damage to DNA that 

may be caused by mutagenic extrolites. Reproduced 

from Paterson & Lima (2012). 

CBS Fungal Biodiversity Calendar: Battle of the pixels 

CBS is initiating a new (12 month) calendar 

series, which will focus on the beauty of 

fungal biodiversity. 

The first calendar is scheduled for 2014, 

and will subsequently appear annually. 

To this end we invite all those making 

photographs or micrographs to submit 

their most beautiful fungal illustrations. 

Photographs of fungi cultivated in the 

laboratory, or observed in nature will be 

considered. Illustrations should be identified 

by the species name, and preferably also 

have a DNA barcode. Images should be in 

landscape layout, at least 300 dpi (3600 ˣ 

2400 px) and in full colour. 

If the image is selected, the mycologist 

who took the actual photograph and 

submitted it for publication will receive 

three copies of the calendar, and a choice 

of any CBS publication. All submissions 

will subsequently also be added to 

MycoBank. 

The publication of the 2014 calendar 

is scheduled for April 2013 and the 

submissions for the 2014 calendar are 

welcome until 15 February 2013. 

Submissions can either be sent to 

p.crous@cbs.knaw.nl or r.samson@cbs. 

knaw.nl; for larger files we recommend using 

www.wetransfer.com. 


(47)

REPORTS 

The Mycological Society of Japan and the 

Melbourne Code 

The Mycological Society of Japan (MSJ) 

held two meetings in 2012, related to the 

Melbourne Code, the International Code 

of Nomenclature for algae, fungi and plants 

(ICN). The Code took effect from 30 July 

2011, with some provisions operative from 

either 1 January 2012 or 1 January 2013. 

These meetings were: (1) the “Symposium 

on Nomenclatural Change” held during 

the 56 th annual meeting of the MSJ on 26 

May in Gifu, Japan; and (2) the “Forum on 

the Future of Microbial Databases” held 

on 28 May in Tokyo, and organized by the 

MSJ in collaboration with the Federation 

of Microbiological Societies of Japan and 

a number of other academic societies or 

associations. The programmes and speakers 

are shown in the accompanying two boxes. 

Symposium on Nomenclatural Change 

Mycologists have it easy. Paul M. Kirk, CABI Bioservices, UK. 

The name that can be named is not the everlasting name - the new rules for the nomenclature of Asco- and Basidiomycota and their 

implications. Roland Kirschner, National Central University, Taiwan & Walter Gams, formerly CBS-KNAW Fungal Diversity 

Centre, The Netherlands. 

One Fungus Which Name: report of the Amsterdam symposium (12-13 April 2012). Robert A. Samson, CBS-KNAW Fungal 

Diversity Centre, The Netherlands. 

Impact of the current change of botanical nomenclature at the Melbourne Conference and a practical consideration on its application, 

especially related to alteration of the Article 59. Takayuki Aoki, National Institute of Agrobiological Sciences, Japan. 

Forum on the Future of Microbial Databases 

Fungal diversity and systematics projects derived from the Tree of Life. Kentaro Hosaka, National Museum of Nature and Science, 

Japan. 

Transition of the use of microbial genome information and future perspective. Natsuko Ichikawa, National Institute of Technology and 

Evaluation, Japan. 

Moving from a web of information to a web of data. Paul M. Kirk, CABI Bioservices, UK. 

WDCM databases and biological databases in China. Juncai Ma, Institute of Microbiology, Chinese Academy of Sciences, China. 

MycoBank an on-line database as a service to the mycological and scientific society. Robert A. Samson, CBS-KNAW Fungal Diversity 

Centre, The Netherlands. 

MicrobeDB in National Bioscience Database Center project. Hideaki Sugawara, National Institute of Genetics, Japan. 

Requirements for industrial microbial genome database. Tatsunari Nishi, Genaris, Inc., Japan. 

As a leverage for research in industry; microbiological database and stock cultures. Hideharu Anazawa, Japan Bioindustry Association, 

Japan. 

Together with the two meetings above, we 

report on the actions taken by the MSJ in 

relation to the latest nomenclatural changes 

(Hawksworth 2011, 2012, Knapp et al. 

2011, McNeill et al. 2011, 2012). These are 

presented here as they may be found of value 

by other mycological societies considering 

actions they should take to comply with the 

new Code. 

(1) Actions in publication of the 

MSJ official journals 

The MSJ publishes two journals, Mycoscience 

(in English) and the Japanese Journal of 

Mycology (in Japanese). Important actions 

were taken by the Editorial Board for the 

effective publication of fungal names in 

Mycoscience, but none are proposed for the 

Japanese Journal of Mycology as it is not 

expected to have nomenclatural novelties 

(though this cannot be ruled out). However, 

use of earlier sexual (teleomorphic) and 

asexual (anamorphic) names in the Japanese 

Journal of Mycology is also expected because 

the corresponding names are now treated 

as synonyms, irrespective of the morphs 

represented. 

For Mycoscience, changes to 

requirements for effective publication relate 

to: (1) online publication, i.e. electronic 

distribution of articles in PDF format via 

the worldwide web (Arts 29–31, Recs 

29A, 30A, and 31A); (2) deposition of key 

nomenclatural information in a recognized 

repository (Art. 37); and (3) acceptance of 

a Latin or English diagnosis or description 

(Art. 36). 

Online publication: For this to be 

considered effective, Art. 29 requires 

electronic distribution of papers in 

Portable Document Format (PDF) with 

an International Standard Serial Number 

(ISSN). Rec. 29A.1 recommends that the 

PDF complies with the PDF/A archival 

standard (ISO 19005). The PDF format 

for papers in Mycoscience volume 53 is 

available at the website (http://www. 

springer.com/life+sciences/microbiology/ 

journal/10267; until 31 December 2012), 

and those of volume 54 at a new site 

(http://www.sciencedirect.com/science/ 

journal/13403540; from 20 July 2012); 

these meet the PDF/A archival standard 

(48) ima fUNGUS

(ISO 19005). Currently, the ISSN number 

does not appear in the PDF and the Board 

has requested the publisher (Elsevier Japan), 

to prominently show this on each PDF. 

Parallel to the PDF version, a Hypertext 

Markup Language (HTML) version is also 

available at the same site which has the 

online-publication date. Mycoscience is being 

published by Elsevier Japan from volume 54 

(2013). All papers published in the printed 

version of volume 53 (2012) by Springer 

Japan were already becoming available on 

the MSJ/Springer Japan site from the end of 

May 2012. 

Art. 29.4 does not permit any alteration 

in the content of a particular electronic 

publication after it is released, which is 

the date of effective publication. To meet 

this requirement, Mycoscience is to publish 

papers in the electronic version with the 

final volume number and pagination so that 

the electronically published PDF and the 

hard-copy version are exactly the same; i.e. 

adopting “Article Based Publication”. 

Rec. 29A.2 recommends the deposition 

of the online published materials in 

multiple trusted online digital repositories. 

Both Springer and Elsevier, as most major 

publishers, participate in CLOCKSS 

(Controlled Lot of Copies Save Stuff ), a 

trusted community-governed archive, in 

which Mycoscience is now placed. 

Art. 30.2 requires evidence associated 

with or within the publication that the 

publisher considers a particular version 

final; and Rec. 30A.1 recommends a clear 

indication that an electronically published 

version is final. “Article Based Publication” 

fulfills this requirement. 

Deposition of key nomenclatural 

information: Under Art. 37, the deposition 

of key nomenclatural information in a 

recognized online repository becomes 

mandatory for valid publication of all new 

scientific names of fungi. The requirement 

to cite the protologue of an identifier 

issued from the repository where the 

nomenclatural information has been 

deposited becomes effective on 1 January 

2013. The following repositories are 

currently operating, although at the time 

of writing none has been “recognized” by 

the Nomenclature Committee for Fungi 

(NCF) appointed by the Melbourne 

Congress: MycoBank (MB; http:// 

www.mycobank.org), Index Fungorum 

(http://www.indexfungorum.org/names/ 

IndexFungorumRegister.htm), and Fungal 

Name (http://www.fungalinfo.net/ 

fungalname/fungalname.html) (Norvell 

& Redhead 2012). The latter is built in 

Chinese, and the former two were created 

originally in English, but are now translated 

into several languages. The deposition of 

key nomenclatural information in MB and 

citation of that number is already required 

for papers in Mycoscience. This practice will 

continue, and the MSJ supports a Japanese 

translation of the interface. 

Acceptance of either a Latin or an 

English diagnosis or description: An 

amended Art. 36 permitted the use of 

either English or Latin for the diagnosis or 

description on all new scientific names from 

1 January 2012. Mycoscience recommends 

that authors write both an English diagnosis 

and a detailed English description for new 

fungal taxa, although the use of a Latin 

diagnosis is not rejected. 

If a Latin or English diagnosis is 

provided, the description could then 

continue to be in any language of the 

author’s choice. However, Mycoscience 

requires authors to use only English 

except for Latin diagnoses and citations 

of original writings (in quotation marks). 

In the Japanese Journal of Mycology, a 

taxonomic novelty with a Latin or English 

diagnosis and then a Japanese description 

would be possible, but that practice is not 

recommended to authors. 

Practices at the MSJ Editorial Office: 

All submitted taxonomic papers are 

checked against the major changes made in 

the new Code. In particular, the amended 

Art. 59 does not allow the proposal of 

two or more names simultaneously for a 

single taxon. Since 1 May 2012, taxonomic 

papers proposing two or more names for 

different morphs of a new fungus have 

been sent back to authors, notifying them 

of the changes in the Code. In such cases, 

however, the Editorial Office neither judges 

nor suggests which of two or more names 

is correct or appropriate for a fungus under 

consideration. 

A notice of the major changes in the 

new ICN is provided for authors at the 

following site: http://www.elsevier.com/ 

framework_products/promis_misc/myc_ 

Fungal_Nomenclature.pdf. 

(2) Recent meetings in Japan with 

a focus on the Code 

Based on the symposium held at the 

56 th MSJ meeting, together with the 

publication of Okada (2011), the MSJ 

has endeavoured to distribute the most 

recent information related to the current 

Paul Kirk at MSJ. 

rule changes, especially on Art. 59, with 

members who may work with pleomorphic 

fungi. At the symposium, the now unified 

fungal nomenclature system was explained 

by Paul Kirk and Roland Kirschner, 

respectively, from their own standpoints. 

Nomenclatural discussion from the One 

Fungus = Which Name? (1F=WN) 

symposium in Amsterdam was also reported 

on by Rob Samson, together with the 

proposals of the International Commission 

on Penicillium and Aspergillus (ICPA). 

Possible practical examples of procedures 

towards unification of sexual and asexual 

states were provided by Takayuki Aoki, 

with examples from Fusarium and related 

ascomycetes. In moving to the unification 

of names of pleomorphic fungi, priority of 

generic names and species epithets should 

be considered independently. When an 

asexually typified (anamorphic) generic 

name has priority by date over a sexually 

typified (teleomorphic) one, and the 

sexually typified epithet has priority by date 

over an asexually typified one, or vice versa, 

a recombination of the priorable specific 

epithet to the priorable generic name will 

be necessary. This process could result in 

many unfamiliar recombinations of generic 

and specific names. In order to minimize 

disruption, a democratic process is being 

initiated, in which active participation by 

all mycologists, whether users or generators 

of names, is encouraged. In due course, 

it is envisaged that lists of protected and 

suppressed names will be internationally 

REPORTS 


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REPORTS 

agreed. In the interim, enquiries should 

be made to the NCF or the International 

Commission on the Taxonomy of Fungi 

(ICTF) on doubtful or ambiguous cases and 

processes being put in place. The symposium 

participants saw this as a complicated 

situation which would be laborious and 

time-consuming, and a lively discussion 

on how to reach a unified nomenclature 

followed (see the below box). 

At the Forum on the Future of 

Microbial Databases, Kentaro Hosaka 

overviewed projects since Deep Hypha, and 

mentioned some DNA barcode projects in 

which he was involved. Natsuko Ichikawa 

introduced a new function of genome 

database created by NITE; information 

on secondary metabolite genes was being 

accumulated, which will be attractive to 

many working in applied areas. Paul Kirk 

stressed the importance of developing a 

Global Names Architecture in order to 

assemble all the mycological data in the 

world. Juncai Ma announced the transfer 

of WDCM/WFCC to Beijing. He intends 

to develop a user friendly catalogue of 

fungi and microbes that member biological 

resource centers can distribute, so that onestop 

ordering of strains will be possible. 

Rob Samson introduced a variety of useful 

functions being implemented in MycoBank, 

and Hideaki Sugawara a new project, 

MicrobialDB.Jp, for the virtual integration 

of microbial databases in Japan. A physical 

integration of heterogeneous databases 

is a considerable challenge, but Semantic 

Web Technologies will facilitate virtual 

integration and will be one of the future 

directions for numerous kinds of databases. 

Major questions or comments on the procedures in the new Code from the two meetings 

For the registration of new names, three different databases are expected to be recognized: Index Fungorum, MycoBank, and Fungal 

Name. Although each database is different in structure, the registration of names and their data release are planned to be 

synchronized in a minute (Norvell & Redhead 2012). 

If an author describes a morph of a new taxon, a separate name of another morph of the same taxon cannot be adopted in that 

publication. Some saw this as a problem, but there was no obstacle to the use of informal names (e.g. acremonium-morph). 

The 1F=1N process has just begun implementation, and will take a considerable time to work through all fungal organisms affected by 

1F=WN. Anybody can participate in the particular working groups or committees now being established, or propose to start a 

new group. 

A user, a geneticist who does not know much about taxonomy or nomenclature, wondered why the dual naming system had been 

abandoned. 

Many scientists confuse taxonomic changes and nomenclatural changes. Nomenclatural change is one based on the requirements of the 

rules in the Code. It is, however, a major challenge to avoid the misapplication of names and to reach taxonomic consensus. All 

taxonomists need to cooperate closely in this. 

Microbial genome datasets will be accelerated to increase; probably more than 1,000 per year. If metagenome data is counted, an 

enormous increase in the information available is expected. 

(3) Expectations of concerned 

mycologists in Japan 

As Rob Samson stated at the symposium, 

the 1F=1N process has just started and 

requires a great deal of collaborative work. 

The MSJ therefore encourages concerned 

mycologists in Japan to participate in 

this important mission. Anyone who is 

interested in a particular taxon is requested 

to contact Keith Seifert (Chair, ICTF) 

to either join an existing working group 

or committee, or initiate a new group 

(see the ICTF website; http://www. 

fungaltaxonomy.org/subcommissions). 

Hawksworth DL (2011) A new dawn for the 

naming of fungi: impacts of decisions made 

in Melbourne in July 2011 on the future 

publication and regulation of fungal names. 

MycoKeys 1: 7–20; IMA Fungus 2: 155–162. 

Hawksworth DL (2012) Managing and coping 

with names of pleomorphic fungi in a period of 

transition. IMA Fungus 3: 15–24. 

Knapp S, McNeil, J, Turland NJ (2011) Changes to 

publication requirements made at the XVIII 

International Botanical Congress in Melbourne 

— what does e-publication mean for you? 

Taxon 60: 1498–1501. 

McNeill J, Barrie FR. Buck WR, Demoulin V, 

Greuter W, Hawksworth DL, Herendeen PS, 

Knapp S, Marhold K, Prado J, Prud’homme 

van Reine WF, Smith GF, Wiersema JH, 

Turland NJ (eds) (2012) International Code 

of Nomenclature for algae, fungi, and plants 

(Melbourne Code) adopted by the Eighteenth 

International Botanical Congress Melbourne, 

Australia, July 2011. [Regnum Vegetabile no. 

154.] Ruggell: A.R.G. Ganter Verlag. 

McNeill J, Turland NJ, Monro A, Lepschi BJ (2011) 

XVIII International Botanical Congress: 

preliminary mail vote and report of Congress 

action on nomenclature proposals. Taxon 60: 

1507–1520. 

Norvell LL, Redhead SA (2012) Registries of names 

and the new Code. IMA Fungus 3: (2). 

Okada G (2011) Unified nomenclature for 

anamorphic fungi or fungi with a pleomorphic 

life cycle adopted at the 18 th International 

Botanical Congress (IBC2011, Melbourne). 

Japanese Journal of Mycology 52: 82–97. [in 

Japanese] 

Toru Okuda, Yoshitaka Ono, Takayuki 

Aoki, and Gen Okada 

(torula@lab.tamagawa.ac.jp) 

(50) ima fUNGUS

COST Action FA1103: European scientists 

investigating endophytic microrganisms and fungi 

As announced in IMA Fungus 3 (1): (7) 

( June 2012), this European Cooperation 

in Science and Technology (COST) 

programme aims to promote research into 

the exploitation of endophytic fungi and 

bacteria in biotechnology and agriculture. 

In large funding schemes related to “White 

Biotechnology” and “Bioeconomy”, these 

organisms are now being exploited not 

only as biocontrol agents, but as producers 

of fine chemicals, industrial enzymes, and 

even biofuel from plant waste (Stadler 

& Schulz 2009). However, there are still 

bottlenecks limiting the full exploitation of 

their potential, and insufficient knowledge 

of their ecology. 

COST Action FA1103, “Endophytes 

in Biotechnology and Agriculture” 1 , 

is now getting underway. The Action 

has so far been joined by 20 countries, 

and around 150 scientists from over 50 

institutions are actively involved. Most 

have already contributed to the scientific 

programme, and the number of interested 

scientists is steadily increasing. The ratios 

of bacteriologists vs. mycologists, and 

applied vs. basic scientists, involved are 

about balanced. Indeed, several scientists 

and companies involved are dealing with 

both bacteria and fungi, and numerous 

active European research groups in basic 

and applied mycology and microbiology are 

represented. 

The Action is divided into four 

thematic working groups (WG), which 

do, however, closely interact with one 

another: WG1 (Ecology of endophytes), 

WG2 (Identification of new competent 

endophytes), WG3 (Development of 

new microbial inocula), and WG4 (New 

industrial products in life sciences). One 

important goal will be to bring expertise in, 

for example, molecular ecology, taxonomy, 

and other fields of basic research, together 

with applied aspects, such as bioprospecting 

and biocontrol. 

Even though the COST Action cannot 

provide direct funding for joint research 

activities, several joint projects, based on 

1 

For more information see: www.endophytes.eu 

(Action website), and http://www.cost.eu/domains_ 

actions/fa/Actions/FA1103?parties (corresponding 

COST website). 

Participants in the Reims COST workshop (March 2012). 

Plants are associated with micro-and nano-organisms: endophytic bacteria and fungi, which live interand 

intra-cellularly in plants without inducing pathogenic symptoms, while interacting with the host 

biochemically and genetically. Endophytic microorganisms may function as plant growth and defense 

promoters by synthesising phytohormones, producing biosurfactants, enzymes or precursors for 

secondary plant metabolites, fixing atmospheric nitrogen and CO 2 , or controlling plant diseases, as 

well as providing a source for new bioactive natural products with utility in pharmaceutical, 

agrochemical and other LifeScience applications. The use of these endophytic microorganisms to 

control plant-pathogenic bacteria and fungi is receiving increased attention as a sustainable alternative 

to synthetic pesticides and antibiotics. Furthermore, endophytes may be adapted to the presence and 

metabolism of complex organic molecules and therefore can show useful biodegradation properties. In 

Participants order to reduce in the inputs San Michele of pesticides dell’Adige and COST fertilizers workshop and add (November value to 2012). eco-friendly agriculture in Europe, 

it will be important to develop inocula of biofertilizers, stress protection and biocontrol agents. But 

there are currently bottlenecks limiting the development of endophytes for use in biotechnology and 

synergies agriculture. and institutional budgets of the 

participating institutes and companies, have 

already been initiated. The meetings of the 

Action provide a fruitful atmosphere for 

discussions about future international grant 

applications on interdisciplinary themes 

that could eventually result in successful 

applications for calls by the European 

Commission. 

Two well-attended workshops have 

already been held; in Reims (France) in 

March 2012) and San Michele dell’ Adige 

near Trento (Italy) in November 2012. 

Members of the Action presented their 

scientific results in symposia and poster 

sessions at these workshops. International 

experts were invited to deliver keynoe 

lectures, for example Linda Johnson (New 

Zealand) and T. S. Suryanarayanan (India) 

addressed the Trento meeting. 

It is envisaged that members of the 

Action will co-organise some symposia 

at the conference “Endophytes for plant 

protection: the state of the art” in Berlin 

in May 2013. The German Society for 

Plant Protection and Plant Health (DPG) 

is to sponsor this meeting, which is also 

being promoted by IUBS (International 

Union of Biological Sciences). The topics 

the Working Group sessions will cover 

include one on the construction and design 

of a European database on endophytes. 

Furthermore, training schools are also 

planned, for instance on analyses of natural 

products and statistical computing and 

graphics. A special issue of Fungal Diversity, 

covering the mycological parts of the 

Action is planned for publication during 

2013. Complementary publications are also 

planned by participating bacteriologists, 

and participation in several important 

European and international conferences 

will follow. Outreach activities include an 

interview recently reported in International 

Innovation Reports. 

 

Aside from networking, the Action 

particularly supports early stage researchers 

(ESR). Further, the programme also 

provides for “Short Term Scientific 

Missions” (STMS), during which ESR 

and other scientists will receive funding 

from COST to visit different European 

laboratories for up to three months for 

training in complementary disciplines or to 

conduct joint research; 8-10 such postings 

can be funded each year. The Action can 

also provide travel grants to enable highly 

qualified ESR to attend international 

scientific meetings. 

An example of the kind of results to 

be expected from the joint investigations 

of bioprospectors and biodiversity experts 

is that of Bills et al. (2012). That study 

emphasised that culturing of apparently new 

phylogenetic lineages will be imperative not 

only to make them available for sustainable 

biotechnological exploitation, but also 

to elucidate life-cycles and ecological 

To increase understanding about these hidden associations between plants, bacteria and fungi, and to 

identify bottlenecks in the development and implementation of technologies using endophytes, a 

network of scientists was recently formed. The present COST Action FA1103: ‘Endophytes in 

biotechnology and agriculture’ will operate all over Europe during the next four years and will provide a 

forum for the identification of bottlenecks limiting the use of endophytes in biotechnology and 

agriculture and ultimately provide solutions for the economically and ecologically compatible 

exploitation of these organisms within Europe and beyond. 

Four working group will be held during the meeting as follow: 

WG1. Ecology of endophytes 

WG2. Identification of new competent endophytes 

WG3. Development of new microbial inocula 

WG4. New industrial products in life sciences 

Enjoy your meeting and welcome in Reims, 

REPORTS 


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REPORTS 

interactions. In this respect, pure in silico 

mycology and microbial ecology, merely 

relying on PCR-based methods, should be 

discouraged. 

In summary, this new COST Action 

provides a forum for the identification of 

bottlenecks limiting the use of endophytes 

in biotechnology and agriculture. It is 

anticipated that it will ultimately provide 

solutions for economically and ecologically 

compatible exploitation of these organisms 

within Europe and beyond. Non-European 

participants and European researchers 

from non-COST countries are invited 

to participate in the Action, but will 

not be eligible for direct reimbursement 

from the Action budget. I trust that this 

report will alert mycologists to these new 

opportunities to fund networking activities 

and international collaborations. After all, 

the scope of the COST Actions may also be 

attractive for other interdisciplinary research 

fields involving mycological expertise. 

Indeed some actions targeting plant 

pathogenic fungi and chemical biology 

approaches are already under way. The next 

deadline for proposals will be in March 

2013. 

Bills GF, González-Menéndez V, Martín J, 

Platas G, Fournier J, Peršoh D, Stadler 

M (2012) Hypoxylon pulicicidum sp. nov. 

(Ascomycota, Xylariales), a pantropical 

insecticide-producing endophyte. PLoS 

ONE 7(10): e46687; DOI:10.1371/ 

journal.pone.0046687. 

Stadler M, Schulz B (2009) High energy 

biofuel from endophytic fungi? Trends in 

Plant Science 14: 353-355 

Marc Stadler 

(marc.stadler@helmholtz--hzi.de) 

(52) ima fUNGUS

AWARDS 

Ana Crespo 

Ana M. Crespo de Las Casas, who was 

honoured with the Acharius Medal of the 

International Association for Lichenology 

(IAL) at their congress in Bangkok in 

January, was received as a full member into 

the prestigious Real Academia de Ciencas 

Exactas, Fisicas y Naturales of Spain at a 

ceremony in the Academy’s rooms on 28 

November 2012. This was a most formal 

occasion at which she had to read the full 

text of a specially prepared dissertation. 

This was a wide-ranging work entitled “El 

discurrir de una Ciencia amable y la vigencis 

de sus objectivos: de Linneo al código 

de barras de AND se pasa por Darwin”, 

published in book-form and distributed 

to those present. Ana is currently head of 

the Departamento de Biología Vegetal 

II in the Facultad de Farmacia of the 

Universidad Complutense 

de Madrid, and renowned 

in particular for her 

pioneering work on the 

molecular phylogenetics 

and systematics of the 

parmelioid lichens 

initiated in 1995–96 

when she was a visiting 

scientist at the former 

International Mycological 

Institute in Egham 

(UK). The Academy was 

established in 1847 by 

royal decree, and Ana 

appears to be the first 

in the field of wholeorganism 

mycology ever to be admitted, as 

well as one of the few women. Many of her 

family, friends, former and current graduate 

students, and also academic colleagues 

attended the two-hour ceremony, and IMA 

Fungus also adds its congratulations to Ana 

on this well-deserved further recognition of 

her achievements. 

AWARDS AND PERSONALIA 

Emil M. Mrak International Award 

José Carmine Dianese, Emeritus Professor in 

the University of Brasilia and a member of 

the IMA Executive Committee, is to receive 

the 2013 Emil M. Mrak International 

Award. The award honours a graduate of 

the University of California at Davis “who 

is distinguished in his or her career or in 

service outside the United States.” José was 

the first Brazilian to gain a PhD in plant 

pathology at Davis in 1970. For more than 

40 years he has been training mycologists 

and plant pathologists in Brazil, 15 of whom 

went on to become university professors 

themselves, as well as painstakingly 

documenting the hitherto unexplored 

microfungal biota of the cerrado. The Award 

was established in 1988, and he appears to 

be the first systematic mycologist to have 

been so honoured. The IMA wishes to add 

its congratulations to those he will already 

have received. 

IN MEMORIAM 

Gouri Rani Ghosh (1924–2012) 

Gouri Rani Ghosh died in Pondichery, 

Tamil Nadu, on 8 January 2012 at the age 

of 88. She was born on 6 June 1924 in 

Orissa, and was the first female graduate of 

the Department of Botany at Ravenshaw 

College in Orissa. By the late 1940s she was 

writing student science text books in the 

Oriya language. She secured a Fulbright 

Fellowship in 1952 and completed a PhD 

in mycology at the University of Illinois in 

1954. Back in Orissa, she started to work on 

myxomycetes, and also on Gymnoascaceae 

in collaboration with Harold H. Kuehn and 

G. F. Orr, on which she became a leading 

authority. Her research on these fungi 

continued into the 1980s, securing grants, 

and training four PhD students of her own; 

there were also further collaborations with 

mycologists overseas, notably Allan Piers in 

the USDA. Her investigations were both 

ontogenetic and systematic. Amongst the 

new fungi in the family she named, are 

the genera Gymnoascoideus and Orromyces 

which remain in use today, and species of 


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AWARDS AND PERSONALIA 

various genera in the family. She was also 

one of the founder members of the Indian 

Mycological Society. 

Lennart Holm, a leading authority on 

pyrenomycete taxonomy for over sixty 

years, and a nomenclatural sage, passed 

away in Uppsala on 28 July 2012. Born in 

Kirity Roy kindly provided notes on Gouri’s life and 

achievements, as well a the portrait included here. 

Carl Lennart Holm (1921–2012) 

Umeå on 29 April 1921, he was aged 91. 

Lennart was nevertheless busy working 

almost to the end on a fascicle of Fungi 

Exsiccati Suecici, bringing the total number 

of specimens distributed in the series to a 

staggering 3800. The exsiccate had been 

initiated in 1934 by Lennart’s mentor, 

Johan A. F. Nannfeldt (1904–1985), 

and Lennart assumed that mantle from 

Nannfeldt. Lennart’s most influential work 

was perhaps his PhD on Pleosporaceae 

(1957), which laid the foundations of a 

new taxonomy for the family, including 

revised generic concepts and clarifications 

of the application of many species names 

through careful typifications. He went 

on to publish revisions of various genera, 

especially of pyrenomycetes in the Nordic 

countries, and also developed interests in 

rusts. In addition he started to reveal the 

previously hardly recognized diversity 

of microfungi associated with particular 

Scandinavian plants, such as those on Dryas, 

Equisetum, Juniperus, Rubus chamaemorus, 

and ferns – often in conjunction with his 

wife Kerstin. Lennart devoted much time 

to clarifying the nomenclatural status of 

generic names, and served for many years 

on the Nomenclature Committee for Fungi 

established by successive International 

Botanical Congresses, including the role 

of Chair. He was also responsible for the 

recommendation to use the colon (:) 

notation to indicate the sanctioned status 

of names in author citations introduced 

at the Sydney Congress in 1981. Lennart 

was always generous with his time, and 

with Kerstin always enjoyed attending field 

meetings, and also entertaining mycologists 

at their home near Uppsala. 

Dorothy Jean Stamps (1927–2012) 

Jean Stamps, as she was always known, was 

born in West Bromwich on 10 February 

1927, and died in London on 3 October 

2012 aged 85 years. She graduated in 

plant biology from the University of 

Birmingham in 1948, continuing there 

to complete a PhD on variability in 

Phytophthora cactorum in 1951. Jean 

was immediately recruited by the then 

Commonwealth Mycological Institute in 

Kew as subeditor of the Review of Applied 

Mycology. However, under the tutelage 

of Grace M. Waterhouse (1906–1996) 

she became increasingly involved in the 

identification of Phytophthora and Pythium 

species, becoming a world authority on 

Phytophthora. She was always pleased to 

put her immense knowledge at the disposal 

of others, distilling that on Phytophthora 

into the renowned “tabular keys”; first 

with F J Newhook and Grace (Mycological 

Papers 143, 1978), and then with a new 

edition in 1990 (Mycological Papers 

162, 1990) in which she was assisted by 

Geoffrey S. Hall. Those keys, which placed 

the species into six categories, remain a 

standard work and continue to be regularly 

cited. In addition she prepared numerous 

authoritative accounts of selected species 

for the Institute’s Descriptions of Pathogenic 

Fungi and Bacteria. Jean was modest 

and unassuming, but on courses and at 

conferences she would amaze with her 

expertise. From 1967 until her retirement 

in 1987, Jean had the responsibility 

for what became the Review of Plant 

Pathology, overseeing its migration onto 

a computerized production system and 

also to CAB International headquarters 

in Wallingford, Oxfordshire. Jean, an 

accomplished musician, also served as 

Librarian of the British Mycological 

Society for many years, receiving the 

Society’s Benefactors’ Medal in 1986. She 

also has the distinction of serving under 

six of the seven Directors the Institute had 

since its establishment in 1920 until it 

ceased in 1998. 

The portrait presented here was kindly supplied by 

Gerald Crowther. 

(54) ima fUNGUS

Coal Measure formation and lignin-degrading fungi 

The first speculation of which I am aware 

that the formation of the Carboniferous 

Coal Measures could be linked to the 

evolution of fungi able to decompose 

lignin was that of Corner (1964: 112), 

who in discussing the origins of fungi 

commented: “to judge from the great 

accumulation of plant debris which makes 

the Coal measures, either they were not 

then established or they were unable to cope 

with the chemistry of those plants”. When 

I read this as an undergraduate it made a 

huge impression on me as to the importance 

of fungi in shaping the world we know 

today, and I have alluded to this from time 

to time in lectures and publications. Now, 

comparative genomics have shown Corner 

was, as in so many aspects of mycology, 

spot-on. 

Floudas et al. (2012) analysed 31 

fungal genomes, 12 of which were 

generated for their study, to ascertain when 

lignin decomposition had arisen within 

Agaricomycotina. They used a 26-gene data 

set and conducted molecular clock analyses. 

David S. Hibbett (Clark University, 

Worcester, MA) co-ordinated this massive 

team-effort, which achieved more than any 

lab could have contemplated doing alone. 

The results were striking, and suggest that 

both brown-rot and ectomycorrhizal fungi 

evolved from white-rot ancestors, with the 

origin of lignin-decomposing brown-rot 

fungi revealed as coinciding with a sharp 

decrease in the rate of organic carbon 

burial at the end of the Carboniferous 

period, at around 290 Myr ago. Their study 

also places the split between ascomycetes 

and basidiomycetes at 662 Myr, and 

diversification of the ascomycetes from the 

Cambrian period, 518 Myr ago. 

Had lignin-decomposing fungi been 

around earlier, with no Coal Measures 

could there ever have been an Industrial 

Revolution in the 18 th century and 

subsequent exponential development of 

the iron and steel industries that laid the 

foundations of the modern world? A worthy 

topic perhaps for an exam question to raise 

awareness of the relevance of fungi in the 

shaping of the world we live in today. 

Corner EJH (1964) The Life of Plants. London: 

Weidenfeld and Nicolson. 

Floudas D, Binder M, Riley R, Barry K, 

Blanchette RA, et al. [and 66 others] (2012) 

The Palaeozoic origin of enzymatic lignin 

decomposition reconstructed from 31 fungal 

genomes. Science 336: 1715–1719. 

B 

RESEARCH NEWS 

COPCI 

Agaricomycetidae 1,2,2,2 

1 

(149) 1,2,2,2 

LACBI 1 Agaricales 

Key to symbols: 

1,2,2,2 

SCHCO 0 

Ecological modes in 

0,0,0,0 

Agaricomycotina: 

CONPU 0 

(196, 2 

White rot 

6,2,3,7 

Boletales 

SERLA 0 

Brown rot 

* 6,3,4,6 

Mycorrhizal 

Character coding: 

HETAN 8 

Russulales Mn-binding site 

Soil saprotroph 

STEHI 6 

Typical (3 aa) 

Mycoparasite / 

1,1,1,2 

Animal Pathogen 

Atypical (2 aa) 

7,3,6,16 

POSPL 1 

B 

Absent (0-1 aa) 

POD copy numbers: 

* 

1,1,1,2 

WOLCO 

12 Extant 

8,3,6,11 

1 

Trp171 

7,2,3,7 Estimated 

FOMPI 

Present 

1 

13,7,8,13 

Polyporales 

Absent 

Contractions in PODs (p

RESEARCH News 

Slime mould navigation 

Slime moulds, traditionally and still recorded 

and studied by mycologists despite their 

classification outside the kingdom Fungi, 

Foraging Physarum polycephalum in the laboratory. 

Photo: Steven L. Stephenson. 

continue to amaze. Plasmodia migrating 

across dead wood are delightful to observe, 

not least when they are of brightly coloured 

species. But what controls the mycelium-like 

strands the colonies often express? Previous 

studies have shown abilities to negotiate 

complex mazes and discover the shortest 

paths, but the mechanisms have remained 

obscure. Just what happens in the case of 

Physarum polycephalum has now been 

investigated in the laboratory by Reid et al. 

(2012). Plasmodia were first presented with 

a choice between agar with extracellular 

slime from the mould and also blank agar in 

a Y-shaped maze, with a food source at the 

end of each arm. They had a most dramatic 

result; 39 of 40 plasmodia chose the blank 

arm. On blank agar with a U-shaped rather 

than a Y-shaped configuration, 96 % reached 

a glucose goal in 120 h, while on slime-coated 

agar only 33 % achieved that in the same time 

The authors concluded that the plasmodium 

was foraging, avoiding areas previously visited 

(i.e. those with slime formed by previous 

trips) in favour of ones that did not appear to 

have previously been explored, and thus were 

more likely to have untapped food resources. 

Complex navigational behaviour does not, 

therefore, necessarily depend on an organism 

having an internal memory, but can result 

from an externalized spatial memory based 

on signals left from previous roaming. 

Reid CR, Latty T, Dussutour A, Beekman M 

(2012) Slime mold uses an externalized 

spatial “memorary” to navigate in complex 

environments. Proceedings of the National 

Academy of Sciences, USA 109: 17490–17494. 

Stratified algal and cyanobacterial lichens from the 

Lower Devonian 

The search for the origins of lichenization as 

a biological strategy within fungi has taken 

a dramatic advance. Honegger et al. (2013) 

have discovered not loose associations 

between fungi and photosynthetic algae 

or cyanobacteria, but layered (“stratified”) 

thalli strongly reminiscent of extant 

foliose lichens. These fossils come from 

the Lower Devonian in the borderland 

between England and Wales, which the 

second author, eminent palaeobotanist 

Dianne Edwards, has been investigating 

for several decades. Two new fossils 

reported on here are in deposits 415 Myr 

Chlorolichenomycites salopensis. A. Entire fragment 

seen from the upper surface. B. Detail as marked 

in “A” seen from the lower surface, and showing 

hyphae of the alga and algal layer connected 

to the peripheral fungal cortex. C. Detail of a 

partly factored hyphae, the arrows pointing to a 

tangentially fractured septum and to a septum 

within the hypha. D. Thallus cross-section, with a 

cortex, algal layer, and medulla; the white arrows 

point to presumed green algal cells with framboidal 

pyrite contents, and black arrows to ones with lost 

contents. E. Fungal hyphae in contact with remains 

of globose algal cells, the right one having retained 

its delicate wall. Scanning electron micrographs by 

Rosmarie Honegger. 

(56) ima fUNGUS

old, and are interpreted as representing 

stratified thalli, one with a cyanobacterial 

partner (Cyanolichenomycites devonicus) 

and one with a green algal partner 

(Chlorolichenomycites salopensis). The 

structures were compared with modern 

freshly collected lichens which had been 

“charcoalified” to facilitate comparison 

with the Lowe Devonian specimens. The 

results are remarkable and leave no doubt 

that complex stratified lichen thalli similar 

to that seen in extant Lecanoromycetes had 

already evolved by this early date. These 

predate the earliest previous reports of 

fossil stratified lichens from the Triassic 

by some 195 Myr. The paper is also of 

value in including a critical assessment of 

previously discovered fossils that have been 

interpreted as lichens, including citations of 

several papers scarcely known outside the 

palaeobotanical community. 

Of further interest is that 

while no ascomata were found, the 

Cyanolichenomycites had what is clearly a 

pycnidium, within which young conidia and 

conidiophores were visualized by superbly 

skilled scanning electron microscopy. 

This is an extraordinarily meticulously 

executed and elegant study, and I 

understand that there will be future papers 

documenting other fascinating fungal 

fossils from these ancient deposits. Such 

fossils have major implications for the 

calibration of molecular clocks and the 

dating of divergence points in phylogenetic 

trees. In this case the authors are confident 

their two fossils belong to Pezizomycotina, 

but, perhaps over-cautiously, prefer not 

to refer them to a class in the absence of 

any sexual reproductive structures despite 

the obvious structural similarity to extant 

Lecanoromycetes. However, I do feel that 

possible classification now needs to be 

considered in future attempts to reconstruct 

and date the origins of that class, and of 

lichenization itself, even in the absence of 

ascomata. Structurally differentiated lichen 

thalli had clearly started to develop well 

before the Lower Devonian to enable such 

complex fossil to have been around by that 

time. 

Honegger R, Edwards D, Axe L (2013) The earliest 

records of internally strafified cyanobacterial 

and algal lichens from the Lower Devonian of 

the Welsh borderland. New Phytologist 197: 

264–275; DOI 10:1111/nph.12009. 

RESEARCH NEWS 

Trichoderma trichothecenes in biocontrol and 

plant defence gene induction 

Molecular tools are increasingly enabling us 

to understand something of the complexity 

of interactions between different fungi 

and plants. Some Trichoderma species 

produce trichothecenes, most importantly 

trichodermin and harzianum A (HA), 

but the genes encoding these have a 

different genomic organization from 

that seen in trichothecene producing 

gene clusters of Fusarium species. There 

have been some previous studies on the 

effects of trichodermin produced by T. 

brevicompactum on plants, but the role 

of harzianum A had remained obscure. 

Now, the pertinent genes in a transformed 

strain of T. arundinaceum, labelled tri4 

and involved in HA biosynthesis, were 

silenced, enabling Malmierca et al. (2012) 

to explore its effects and possible relevance 

to the use of the fungus in biocontrol. They 

demonstrated that disruption of this gene 

led to reduced antifungal activity against 

both Botrytis cinerea and Rhizoctonia solani, 

and further to a reduced ability to induce 

the expression of plant defence related genes 

in tomato plants compared to the wildtype 

Trichoderma strain. Their experiments 

lead to the conclusion that harzianum A 

has a role in sensitizing the tomato plants 

to attack by other fungi, as well as in its 

antifungal mycoparasitic activity. They also 

found that the plant pathogenic fungi and 

the tomato plants had a role in regulating 

the expression of the tri genes in T. 

arundinaceum. 

Schematic representation of the network of interactions established among Trichoderma arundinaceum 

(Ta37), Botrytis cinerea, and tomato plants deduced from the present work. Arrows indicate response 

stimulation or gene upregulation, and blunt-ended lines indicate gene repression or growth inhibition. 

Red, blue, and green lines indicate interactions mediated by B. cinerea, tomato plant, and the Trichoderma, 

respectively. a, sensitizing effect of Trichoderma-pretreated tomato plants mediated by the trichothecene 

harzianum A (HA); b, coupled action of HA and extracellular hydrolytic enzymes to inhibit B. cinerea growth; 

c, other metabolites produced by T. arundinaceum that, in addition to HA, would also affect its interaction 

with plants and with its fungal targets. Reproduced from Malmierca et al. (2012). 


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RESEARCH News 

This appears to be the first report of 

an interaction between trichothecenes and 

plant defence responses, indicating that 

these compounds are involved in a complex 

network of interactions in which each 

partner regulates the other. The complexity 

of this particular situation is indicated in the 

accompanying figure, but it seems probable 

that extrolites from other fungi may also 

have similar roles in biocontrol scenarios. 

Malmierca MG, Cardoza RE, Alexander NH, 

McCormick SP, Hermosa R, Monte E, 

Gutiérrez SW (2012) Involvement of 

Trichoderma trichothecenes in the biocontrol 

activity and induction of plant defense-related 

genes. Applied and Environmental Microbiology 

78: 4856–4868. 

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ima fUNGUS

Marine Fungi and Fungal-Like Organisms. Edited by E. B. Gareth Jones and Ka-Lai 

Pang. 2012. ISBN 978-3-11-026398-5. Pp. xvi + 532. Göttingen: Walter de Gruyter. 

Price: 139.95 €. 

There have been several books devoted to 

marine fungi over recent decades, such as 

those edited by Hyde & Pointing (2000) 

and Hyde (2002), can there be room for 

another? Definitely yes, as this new book is 

not an identification manual, but provides 

an authoritative overview of the phylogeny, 

biodiversity, and applications of marine 

fungi. As noted in the Introduction, there 

was no comprehensive analysis of the 

group as a whole previously available. In 

order to achieve this goal, the editors have 

marshalled 44 contributors, drawn from 17 

countries, to prepare 24 chapters designed 

to cover all aspects of the field. 

The book starts with a masterly 

overview of marine fungi and fungal-like 

organisms by the editors, covering their 

classification, and numbers; around 530 

species are known, most described in the 

1980s and 1990s, but the actual number 

is estimated here at 12,060 species. 

Molecular phylogenetics has provided a new 

understanding of the diversity of fungal 

groups, and the first two sections of the 

book are devoted to phylogeny. There are 

chapters on ascomycetes (including lichenforming 

representatives), basidiomycetes, 

conidial fungi, yeasts, and zoosporic fungi, 

with overviews of the orders, families, and 

sometimes genera and species represented. 

These are followed by a series on fungal-like 

organisms, including the recently recognized 

Cryptomycota as well as Hyphochytriomycota, 

Oomycota, Perkinsozoa, Labyrinthulomycota, 

and Phytomyxea. The phylogenetic 

sections are up-to-date, and in addition 

to information on the characters of the 

organisms, data on their ecology, life-cycles, 

and products is also often provided. 

I was especially pleased to see the 

chapters on biodiversity, which consider 

the fungi on mangroves (625 species, of 

which 287 occur on submerged mangrove 

substrata), a palm (Nypa fruticans; with 

135 taxa of which just 97 are described), 

those that are endophytes (tabulated by 

host family) or otherwise associated with 

marine plants and animals, marine algae, 

occur in salt marshes (with lists for Juncus 

roemerianus, Phragmites australis, and 

Spartina species; 332 species in total of 

which 89 % are exclusively associated with 

one host), are associated with sponges, or 

detected in deep-sea habitats by culture or 

molecular methods. 

The last group of chapters on 

applications has contributions which cover 

natural products (with structural formulae), 

enzymes (with tabulations of species that 

produce them), and the decomposition of 

materials. There is also a pragmatic chapter 

devoted to the culture and long-term 

preservation of marine fungi which includes 

details of commended methodologies. 

The volume concludes with an epilogue 

by the editors, stressing the importance 

of marine fungi both ecologically and 

industrially, as sources of novel bioactive 

compounds, but also as agents of diseases in 

their hosts. Aspects meriting more attention 

are highlighted, but the 15 laboratories 

tabulated as currently studying the diversity 

and ecology of marine fungi are all located 

in tropical countries. Overall, this is a 

masterly overview of the subject, which will 

be a key reference for decades to come, but 

what else would one expect with the doyen 

and master of marine fungi, who has been 

devoted to expanding our knowledge of 

these fungi for over 50 years, as an editor? 

Hyde KD (ed.) (2002) Fungi in Marine 

Environments. [Fungal Diversity Research 

Series no. 7.] Hong Kong: Fungal Diversity 

Press. 

Hyde KD, Pointing SB (eds) (2000) Marine 

Mycology: a practical approach. Hong Kong: 

Fungal Diversity Press. 

BOOK NEWS 

Neurospora: genomics and molecular biology. Edited by Durgados P. Kasbekar and 

Kevin McCluskey. 2013. ISBN 978-1-908230-12-6. Pp. x + 294. Caister, Norfolk: Caister 

Academic Press. Price: £ 159 or US$ 319.00. 

Ever since Beadle & Tatum (1941) 

established Neurospora crassa as a model 

system for the elucidation of genetics, it 

has been the focus of elegant in-depth 

research into fungal genetics. The ability to 

grow quickly and for strains to mate readily 

commended this fungus to geneticists, and 

this fungus has continued to have a pivotal 

role in the bioinformatics and genomic era. 

This new wide-ranging work “aims to distil 

the most important findings and provide 

snapshots of the current research landscape” 

(p. ix), and does that by bringing together 

leading researchers on different aspects of 

the genetics of this fascinating fungus. 

For those unfamiliar with Neurospora, 

“Tony” Griffiths first provides an overview 

of the methodology in making crosses, 

mutants, and heterokaryons, with the 

procedures illustrated by clear line-drawings. 

This is followed by 14 chapters which 

cover a staggering array of topics. These 

include: non-self recognition systems 

(i.e. incompatability); the control and 

mathematical modelling of branching 

patterns; glycosyl hydrolases, and the 

numerous genes involved; quantitative trait 

locus mapping; recombination processes 

and mechanisms, including chromosomal 

markers; chromosome segment duplications, 

repeat-induced point mutations and meiotic 

silencing; mutagen response and repair; 

regulation of gene transcription by light, 

which involves a blue light photoreceptor; 

regulation and physiological role of protein 

kinase pathways; the heterotrimeric G 

protein signalling pathway, responding 

to environmental factors and affecting 

conidiation; calcium signalling, which 


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BOOK NEWS 

involves 48 signalling proteins; 

carotenoid biosynthesis and its regulation; 

and the circadian system and the series of 

processes involved. 

A concluding chapter looks at what 

is being achieved through whole-genome 

sequencing. The whole haploid genome 

is about 43 Mb and contains less than 

10,000 genes, and several other species 

of the genus in addition to N. crassa have 

now been sequenced. The numerous 

carefully characterised strains maintained 

at the Fungal Genetics Stock Center are 

proving to be of especial value generating 

new information and reinforcing earlier 

discoveries with cutting-edge techniques; 

an example is the paper in the last issue 

of this journal, which evidently came out 

after the book went to press, and reports on 

mitochondrial genome variation in some of 

the classic strains (McCluskey 2012). 

While very much a state-of-the-art 

review of Neurospora genetics, the depth 

of understanding achieved and complexity 

revealed can only be marvelled at. This 

synthesis will undoubtedly also be of value 

to those working in different model genetic 

fungal systems, notably Aspergillus nidulans 

and Coprinopsis cinerea, as it will facilitate 

comparisons with them – something hardly 

addressed in the present volume, but perhaps 

of interest to a wider range of mycologists, 

and a topic for a different book. 

Beadle GW, Tatum EL (1941) Genetic control 

and biochemical reactions in Neurospora. 

Proceedings of the National Academy of Sciences, 

USA 27: 499–506. 

McCluskey K (2012) Variation in mitochondrial 

genome primary sequence among wholegenome-sequenced 

strains of Neurospora crassa. 

IMA Fungus 3: 93–98. 

Fungal Secondary Metabolites: methods and protocols. Edited by Nancy P. Keller and 

Geoffrey Turner. 2012. ISBN 978-1-62703-121-9. Pp. xii + 288. New York: Humana 

Press. [Methods in Molecular Biology no. 944.] Price: 101.60 €. 

This is very much a how-to-do book, 

prepared by two particularly distinguished 

mycologists, and designed for those wishing 

to investigate the chemical possibilities 

of filamentous fungi. It has become 

increasingly clear that the compounds a 

fungus actually expresses are only a fraction 

of those that it has the genetic systems to 

produce. In order to reveal the full spectrum 

of what a species is capable of, it is necessary 

to develop ways of encouraging silenced 

genes to be expressed, i.e. upregulated. As 

the potential of a fungus will necessarily 

be included in the genome, the book starts 

with chapters on library preparation and 

data analysis packages for rapid genome 

sequencing, and the bioinformatic 

approaches and software available for the 

detection of pertinent gene clusters. Steps 

to be commended are detailed and practical 

procedures illustrated, accompanied by 

discussions of the strength and weaknesses 

of different packages. 

The selection of media and growth 

conditions has long been recognized as 

critical for the induction of particular 

chemical products. Fifteen solid media to 

try are detailed by Frisvad, as agar plugs can 

easily be analyzed, but he recognizes that 

while generalizations can be made, optimal 

conditions for a particular fungus will 

depend on its ecological and physiological 

requirements. Multi-well plates in largely 

automated systems have proved especially 

valuable for high throughput screening in 

major laboratories, and the contribution 

on this by Tormo et al. is so well-illustrated 

by photographs that many mycologists 

will be fascinated to see these procedures 

in operation. As solid-state fermentations 

have been found to generally exhibit 

more complex metabolite profiles, Merck 

Research Laboratories (Rahway, NJ) 

exploited this in the FERMEX programme 

in the 1980s and 1990s; Bills et al. not only 

describe the method, but tabulate significant 

discoveries made from it, including 

antifungal compounds and HIV-1 enzyme 

inhibitors. 

Twelve chapters concern methodologies 

applied to Aspergillus species, although 

in many cases they could be utilized also 

in other fungi. These consider: methods 

for the upregulation of normally silent 

metabolite producing gene clusters A. 

nidulans; non-ribosomal peptide synthetase 

products in A. fischeri (under the name 

Neosartorya fischeriana); targeted gene 

deletions and promoter replacement to 

awaken gene clusters in A. nidulans; a 

site-directed mutagenesis method for the 

rapid construction of plasmid vectors 

in A. nidulans; the use of plasmids with 

different selection markers to transform 

A. oryzae so that it can express up to three 

genes simultaneously; the identification 

of novel regulators in A. nidulans 

through multi-copy genetic screening; 

the identification of protein complexes 

through tandem affinity purification, 

demonstrated in A. nidulans; comparative 

metabolomics based on differential 

analysis by two-dimensional NMRspectroscopy 

and liquid chromatography/ 

mass spectrometry to pursue orphan gene 

clusters in A. fumigatus; in vivo proteinprotein 

interactions in A. nidulans conidia; 

chromatin immunoprecipitation analysis 

to map interactions between proteins and 

(60) ima fUNGUS

a particular genome locus, using antibodies 

from A. nidulans and Neuropsora crassa; 

purification of the aflatoxin-storing 

vesicle-vacuole fraction from A. parasiticus 

protoplasts; and isolation of surfacegrown 

mycelium from A. nidulans after 

confrontation with Drosophila melanogaster 

for analysis of gene expression. 

Other topics covered concern: clavinetype 

ergot alkaloids; the analysis of volatiles 

using solid-phase microextraction-gas 

chromatography/mass spectrophotometry; 

targeted proteomics for metabolite 

pathway optimization; and a hollow fibre 

assay for the discovery of novel anticancer 

compounds using mice. 

In all the contributions, the protocols to 

be adopted are presented in recipe-format 

numbered steps, and the illustrations are 

especially helpful, and in many cases in 

colour. The editors and publishers must be 

congratulated in the extent to which they 

have managed to marshal their contributors 

into such a common and lucid style. The 

focus is, however, very much on the exciting 

and promising new technologies, of product 

discovery and gene expression, rather than 

chemical detection and characterization. 

This is consequently not a volume for 

mycologists wishing to learn of advances in 

microchemical detection, and for that it will 

be necessary to look elsewhere. 

The title is also available as an e-book at 

the slightly lower price of 89.99 €, for those 

that prefer to read on screen or have run out 

of shelf space. 

BOOK NEWS 

Glomeromycota. By Janusz Błaszkowski. 2012. ISBN 978-83-89648-82-2. Pp. 303, illustr. 

Kraków: W. Szafer Institute of Botany. Price: 65 zł. 

There have been enormous advances in our 

understanding of Glomeromycota in the 

last few years as the results of molecular 

systematics have been incorporated into 

revised classifications. However, an account 

of the currently recognized genera and 

species, will detailed descriptions and 

illustrations, has previously been lacking. 

This work is based on the author’s personal 

study, and he indicates he collected most of 

the species and grew them in trap and singlespecies 

cultures. Others were obtained as 

microscopic preparations received on loan 

from other institutions. In total, 137 species 

are accepted and described and illustrated 

in detail, including one new species and one 

new combination. Full information is also 

included on the names and their synonyms, 

the plants the species are associated with, 

their phylogenetic position, distribution, 

and habitat, followed by details of specimens 

examined and often lengthy notes. 

Dichotomous keys are provided, and the 

coloured photomicrographs of the spores, 

with details of the wall layers meticulously 

labelled are superb. This work will therefore 

greatly facilitate the identification of 

the known species of this ecologically 

and economically important group of 

fungi without the need for molecular 

sequence data. Further, in addition to a 

concise synopsis of previous studies on 

glomeromycetes since their discovery by 

Polish mycologist Franciszek Kamieński in 

1881, Błaszkowski also provides a practical 

guide to the collection, isolation of spores, 

establishment of pot cultures, preparation 

of diagnostic slides, and visualizing these 

fungi in roots. The whole work is beautifully 

presented, large (A4)-format, and the 

author’s passion for these fungi is evident 

throughout. He should be extremely proud 

of this work. All those concerned with the 

identification of arbuscular mycorrhizal 

fungi by microscopic methods will find this 

an enormous asset to have on their shelves. 

It will certainly help me personally, as I 

encounter glomeromycete spores regularly 

in palynological preparations I examine in 

connection with forensic cases. 

However, it must be noted that there are 

some differences in the system adopted here 

from that of Oehl et al. (2011). The main 

reason for this is undoubtedly a consequence 

of Błaszkowski’s book being in production 

for a considerable time. Indeed, the most 

recent paper I could find cited was from 

2010. Only the single class Glomeromycetes 

is accepted in the phylum here, and neither 

Archaeosporomycetes nor Paraglomeromycetes. 

The same orders are nevertheless recognized, 

apart from Gigasporales, which is included as 

a family in Diversisporales here. Differences 

in classification do not of course devalue the 

importance of the keys, detailed descriptions 

and illustrations; although taxonomic 

systems and names may change, the 

characters of the fungi do not. That is what 

makes monographs like this of enduring 

value. 

Oehl F, Sieverding E, Palenzuela J, Ineichen K, Alves 

de Silva G (2011) Advances in Glomeromycota 

taxonomy and classification. IMA Fungus 2: 

191–199. 

Mushrooms: the natural and human world of British fungi. By Peter Marren. 2012. 

ISBN 978-0-9564902-3-0. Pp. 272, illustr. Gillingham, UK: British Wildlife Publishing. 

[British Wildlife Collection no. 1.] Price: £ 24.95. 

This was a real pleasure to read. Peter is 

not a professional mycologist, but began 

his interest in fungi while still at school, 

before coming under the spell of John 

Webster at the University of Exeter, and 

later, after moving into nature conservation, 

Roy Watling and later Malcolm Storey and 

Ted Green. However, Peter has become 

an accomplished author, with 20 titles on 

different aspects of natural history already 

to his credit. Further, he authored a column 

in British Wildlife since 1990, and also 

contributed many pieces on macrofungi. 

This skill results in a style of writing that 

is sure to appeal to the public at large, and 

the book is packed with references to the 

most recently reported discoveries, and 

many personal experiences. The topics 


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BOOK NEWS 

are also wide-ranging, with a strong 

emphasis on conservation aspects. While 

not an identification manual, the chapter 

“Mushrooms on parade” (a super title!) 

categorises the macrofungi into groups to 

which he allocates common names, such 

as oysterlings, cockleshells, redleafs, and 

stagshorns; I can see some of these catching 

on amongst the UK´s numerous naturalist 

mycologists. Amongst the other catchy 

chapter titles are “What mushroom is that?, 

“In our midst: our fungal neighbours”, 

“The good, the bad and the crazy”, and 

“Picking for the pot”. Personally, I might 

have devoted more text to mycorrhizal 

associations, responses to pollutants, and 

distribution patterns, but what to include in 

such a work is necessarily eclectic. The whole 

is superbly laid out and illustrated by high 

quality colour photographs, some on almost 

every page and in some cases particularly 

dramatic photographs are spread over two. It 

was also pleasing to see that the first title in 

this new book series was devoted to fungi; if 

subsequent titles can aspire to the standard 

Peter sets here, the long-established and 

prestigious New Naturalist series, for which 

Peter has written, may find a challenger 

has been born. As made explicit in the title 

and subtitle, this is almost exclusively on 

mushrooms, although there are occasional 

exceptions, and focussed on the British 

Isles. Regionally orientated works are not 

normally covered in IMA Fungus, but I 

decided to feature this book here as I am 

confident that it can have a role in increasing 

the awareness of larger fungi, their roles, and 

uses, amongst a wide range of naturalists in 

the English-speaking world. 

Hungry Planet: stories of plant diseases. By Gail L. Schumann and Cleora J. D’Arcy. 

2012. ISBN 978-0-89054-399-3. Pp. ix +294. St Paul, MN: APS (American Phytopathological 

society) Press. Price: US$ 65.00. 

This book has been prepared by two 

experienced university teachers of plant 

pathology in the USA, who also previously 

co-authored the established and muchused 

textbook, Essential Plant Pathology 

(Schumman & D’Arcy 2009). The present 

title, however, is introduced as being an 

update to Plant Diseases: their biology and 

social impact by the first author (Schumann 

1991) and directed to a broader audience. 

The aim is to heighten general awareness 

of the vulnerability of plants used as food 

through stories of plant diseases and their 

impacts, and the need to balance safety with 

the cost of producing food, fibre, and fuel. 

The emphasis is on diseases caused by fungi, 

but bacteria, nematodes, viruses and viroids 

are also treated. 

There are chapters introducing readers 

to the range of fungi and fungus-like 

organisms, the requirements for healthy 

plant growth, and the basics of genetics 

and genetic engineering. The examples of 

diseases featured are much as expected, 

starting with the Irish potato famine, and 

also focussing on the serious problems 

that have impacted coffee and rubber 

production, the ravages of wheat stem 

rust, southern corn blight, white pine 

blister rust, Dutch elm disease, chestnut 

blight, and others. Effects on people are 

also reviewed, encompassing, for example, 

ergotism, mycotoxins, and edible corn smut. 

In relation to disease management, the 

topics addressed include epidemiology and 

control through the use of pesticides, soil 

fumigation, crop protection, integrated pest 

management, quarantine, and regulations. 

A particular feature to help the nonscientist 

is the provision of boxed “Science 

Sidebars” on a diverse range of topics, 

amongst which are: ascospore formation, 

-mycetes versus –mycota, the Ames test, 

regulation of genetic engineering, DAS- 

ELISA, mistletoe rituals, new names for elms, 

and endophytes. There is also a particularly 

full, and perhaps a little overfull, glossary. 

However, there are no literature references or 

even suggestions for further reading to guide 

the more inquisitive reader. While there are 

numerous photographs, all are half-tones and 

their reproduction is rather poor, though I 

am sure many of the originals were superb in 

colour. This is particularly unfortunate in a 

book aimed at a general audience. 

The concluding chapter, with the same 

title as the book, is something of a call 

to arms. It draws attention to issues that 

will affect the ability of the Earth to feed 

the exponentially rising human load of 

the planet. These include population size 

(surely the key!), reduction in the areas 

devoted to arable crops, air pollution, water 

resources, soil fertility, and climate change. 

The authors “are optimistic that educated 

citizens can make a difference” (p. 266). 

That might become true in some of the 

more developed OECD countries, but I am 

personally, sadly, pessimistic at the global 

scale. This book may help a little, but at 

such a price and without more dramatic 

and coloured illustrations I wonder if those 

the authors laudibly wish to address would 

select it from a bookstore shelf. Perhaps the 

authors should consider preparing a much 

smaller and eye-catching book and securing 

funding to make it available in the countries 

that need it at no or nominal cost? 

Schumann GL (1991) Plant Diseases: their biology 

and social impact. St Paul, MN: APS Press. 

Schumann GL, D’Arcy CJ (2009) Essential Plant 

Pathology. 2 nd edn. St Paul, MN: APS Press. 

(62) ima fUNGUS

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Contact: Congress Care, P.O. Box 440, 5201 AK ’s-Hertogenbosch, The Netherlands; info@congresscare.com 

 

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9 th International Conference on Cryptococcus and Cryptococcosis 

15–19 May 2014 

Royal Tropical Institute, Amsterdam, The Netherlands 

 


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(64) ima fUNGUS

doi:10.5598/imafungus.2012.03.02.01 

IMA Fungus · volume 3 · no 2: 103–108 

Development of merosporangia in Linderina pennispora (Kickxellales, 

Kickxellaceae) 

Mohamed E. Zain 1,2 , Steve T. Moss 3† , and Hussein H. El-Sheikh 2 

1 

Center of Excellence in Biotechnology Research, King Saud University, Riyadh, Saudi Arabia; corresponding author e-mail: Mohamed E. Zain, 

mzain@ksu.edu.sa 

2 

Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt 

3 

[ † Deceased 2001] School of Biological Sciences, University of Portsmouth, King Henry Building, King Henry 1 Street. Portsmouth PO1 2DY, UK 

ARTICLE 

Abstract: The vegetative and sporulating structures of Linderina pennispora are described using scanning and 

transmission electron microscopy. The vegetative hyphae and sporangiophores were regularly septate, possessed 

a two-layered wall, and coated with rod-shaped, 0.2–0.7 µm long, 0.15–0.25 µm wide ornamentations. The 

sporangiophore was erect, cylindrical, and narrower (4–8 µm) than the vegetative mycelium (8–12 µm). The mature 

sporocladium was ovoid to dome-shaped, sessile, non-septate, 18–24 µm diam, possessed a two-layered wall, 

and coated with rod-shaped ornamentations. Mature pseudophialides were ellipsoid, 2.0–2.5 µm wide, 4–7 µm 

long, possessed a two-layered wall, and formed in a series of concentric groups radiating from the “apex” of the 

sporocladium. The pseudophialides had a round, ca. 1.5 µm diam, base with a narrower, 0.7–0.8 µm diam lobed, 

cylindrical neck structure in the distal region which extended to the pseudophialide neck. The merosporangia were 

obovate, 3–4 µm wide near the base, and narrowed distally to 2.0–2.5 µm wide, 18–23 µm long, possessed a threelayered 

wall, with regular surface annulation with interconnecting ridges, but lacked rod-shaped ornamentations. The 

merosporangia contained a single, obovate, 2.3–2.5 µm diam merosporangiospore, with a ca. 1 µm diam papilla-like 

base, that possessed a four-layered wall. Detached merosporangia had a single, acicular, unbranched, 3–5 µm long, 

ca. 0.1 µm diam “appendage” that was attached to the merosporangiospore inner cell wall layer and passed through 

the septum plug to the pseudophialide. 

Key words: 

Kickxellomycotina 

ontogeny 

ultrastructure 

zygomycetes 

Article info: Submitted: 16 January 2012; Accepted: 11 July 2012; Published: 20 August 2012. 

INTRODUCTION 

The order Kickxellales was established by Kreisel 

(1969) to accommodate the families Kickxellaceae and 

Dimargaritaceae. Benjamin (1979) suggested the separation 

of the Dimargaritaceae and the Kickxellaceae at the ordinal 

level, within the Zygomycetes. He established the order 

Dimargaritales for the Dimargaritaceae, and retained the 

order Kickxellales for the family Kickxellaceae (Benjamin 

1979). Although the Kickxellales had been classified 

traditionally within the class Zygomycetes, recently it was 

segregated from other orders of this class to establish the 

subphylum Kickxellomycotina with Harpellales, Asellariales, 

and Dimargaritales (Hibbettt et al. 2007, Kurihara et al. 2008). 

Kickxellales are phylogenetically closest to the 

harpellalean genus Orphella (White 2006). Most species of 

Kickxellaceae are saprobess and are commonly isolated 

from soil, dung, humus, dead insects, or other organic debris. 

However, Martensella pectinata and M. corticii are obligate 

mycoparasites (Kurihuara et al. 2008). The classification of 

Kickellaceae and its genera has undergone several changes 

(Moss & Young 1978, Young 1985, Benny 1995, O’Donnell et 

al. 1998, Hibbett et al. 2007). The family currently contains 12 

genera (Kirk et al. 2008, Benny 2012). 

Young (1974) described a labyrinthiform organelle within 

the pseudophialides of Kickxella alabastrina, and a similar 

structure was demonstrated by Benny & Aldrich (1975) 

within the pseudophialides of Linderina pennispora. They 

coined the term “abscission vacuole” for that structure, and 

believed that this was responsible for the detachment of the 

merosporangia from their pseudophialides. Moss & Young 

(1978) demonstrated that Kickxellaceae (Zygomycetes) 

were are closely related to Harpellales and Asellarials 

(Trichomycetes) on the basis of the septa; they consist of 

a cross-wall with a central pore occluded by a biumbonate 

plug, the form of the asexual reproductive apparatus, and 

the similar wall structure. The labyrinthiform organelle 

in Kickxellaceae was speculated to be analogous to the 

trichospore appendage of Harpellales (Moss & Young 1978, 

Young 1985). This contribution describes the ontogeny of the 

sporulating structures of Linderina pennispora. 

© 2012 International Mycological Association 

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volume 3 · no. 2 103

Zain, Moss & El-Sheikh 

ARTICLE 

MATERIALS AND METHODS 

Isolate 

The isolate used in this study was Linderina pennispora 

(IMI 174729) provided as a culture from CABI Bioscience 

(Egham, UK). Malt extract agar (20 g Difco malt extract, 20 

g dextrose, 1 g peptone, 20 g agar, 1 L distilled water) was 

used for experimental studies and maintenance of a stock 

culture during the investigation. 

Scanning Electron Microscopy 

Colonized agar squares of 6–8 mm with sporulating material 

were fixed in 2 % (w/v) aqueous osmium tetroxide (OsO4) 

at 4 °C for 12 h in the dark, and then washed in distilled 

water. Fixed and washed material was dehydrated through 

a graded (10 % steps) ethanol series from 10–90 %, and 

finally absolute ethanol. The absolute alcohol was replaced 

with acetone via a stepwise series (ethanol: acetone 3:1, 1:1, 

1:3), and then finally maintained in water-free acetone for 

1 h with three changes. Specimens dehydrated to acetone 

were critical-point-dried using a Polaron E3000 apparatus 

with liquefied carbon dioxide as the drying agent. Using a 

stereomicroscope, the critical-point dried specimens were 

orientated and then attached to 2.5 cm diam aluminium stubs 

with carbon adhesive, and allowed to dry in a desiccator for 

at least 12 h. Specimens were coated with gold-palladium 

(60 : 40; ca. 50 nm thickness) using a Polaron diode sputtering 

system (E5000). Coated specimens were then examined 

using a JEOL T20 scanning electron microscope at 20 KV. 

Transmission Electron Microscopy 

Fungal material (ca. 2 mm 3 ) was fixed in 1 % (w/v) aqueous 

potassium permanganate for 5 min at 20 ± 2 °C. Fixed 

material was then washed in distilled water for 15 min. Some 

fungal materials were fixed in 4 % (v/v) glutaraldehyde in 0.1 

M sodium cacodylate buffer, two changes each of 15 min, and 

then post-fixed in 2 % (w/v) osmium tetroxide in 0.1 M sodium 

cacodylate buffer, 2 changes each of 15 min. Fixed material 

was dehydrated through a graded ethanol series following 

the procedure described for SEM. Dehydrated specimens 

were embedded in an epoxy resin. For material fixed with 

potassium permanganate, agar 100 resin, 31 mL, DDSA 

(hardener) (dodecenyl succinic anhydride), 50 mL and DMP- 

30 (accelerator; 2.4 mL) were used. For material fixed with 

glutaraldehyde-osmium tetroxide, mixture A [agar 100 resin (62 

mL) and DDSA (100 mL)], mixture B [agar 100 resin (100 mL) 

and NMA (89 mL)], and BDMA (benzyl-dimethyl amine) were 

mixed in the ration (3:7:0.15). Dehydrated specimens were 

infiltrated with the resin through a graded series (Resin : acetone 

(1:3), (1:1), (3:1) for 24 h with rotation at room temperature for 

each grade). 

The resin was polymerised at 60 °C for 72 h and then 

allowed to cool to room temperature in a desiccator for 

24 h. Flat embedded material was examined with a light 

microscope and different stages of fungal development 

identified. Selected specimens were cut from the flat blocks, 

glued to resin stubs in the desired orientation, and placed 

in the oven at 60 °C for 24 h to allow polymerization of the 

glue. Using a stereomicroscope, the mounted blocks were 

trimmed with razor blades to give a trapezoid-shaped block 

face, less than 1 mm in width and height with, when possible, 

most of the block faces comprising the embedded fungal 

material. Surface resin was trimmed off the block face using 

a Cambridge Instruments Huxley MK2 ultramicrotome and 

glass knives. 

Ultrathin sections were cut with a LKB MRIII ultratome and 

the sections floated onto water. The knife had a clearance 

angle of 4° and the block a cutting speed of 1 mm/s; for 

routine work, silver-grey or gold (30–60 nm) sections were 

cut. Sections were then flattened with chloroform vapour and, 

using an eye-lash, sections were manipulated to the centre of 

the boat prior to picking up on hexagonal copper mesh (3.05 

mm pores), coated with Parlodion (2 % Parlodion in amyl 

acetate) grids. Sections were double stained in a carbon 

dioxide-free atmosphere with lead citrate (Pb 3 

[C 6 

H 5 

O7] 2 

) 

followed by uranyl acetate. The lead citrate solution was 

centrifuged for 15 min to remove any precipitate and then 

single drops of the supernatant were transferred onto dental 

wax. A single grid was floated on to each drop of the stain, 

with sections facing the stain, for 15 min. Stained sections on 

grids were washed with 0.02 N NaOH followed by distilled 

water and then stained with uranyl acetate for 30 min in the 

dark. Stained sections were examined with a JEOL 100S 

transmission electron microscope at 80 kV. 

RESULTS 

Linderina pennispora grows on malt extract agar (MEA), with 

sporulation occurring at 20 °C within 6–8 d of inoculation. 

The axenic cultures were yellow, and having two types of 

hyphae occurred; submerged and aerial. Sporangiophores 

were erect, “zigzag”-shaped, and produced from submerged 

hyphae. The asexual apparatus was composed of a 

non-septate, dome-shaped sporocladium that produced 

numerous pseudophialides. Each pseudophialide produced 

a single merosporangium (Fig. 1F). 

Sporocladia 

The mature sporocladium was ovoid to dome-shaped, 

sessile, non-septate, 18–24 µm diam, and coated with rodshaped 

ornamentation similar in shape but fewer in density 

per unit area than ones coating the sporangiophore (Fig. 

1B). Five to eight sporocladia were arranged on alternate 

sides of the sporangiophore. The sporocladium initials were 

initially apical (Fig. 1A), but sympodial branching of the 

sporangiophore beneath of the sporocladium later displaced 

the mature sporocladium laterally. The sporocladium initials 

were spherical, and coated with rod-shaped ornamentation. 

Each sporocladium produced several pseudophialide initials. 

The wall was two-layered, similar in nature to the 

sporangiophore cell wall, with an outer electron-dense and an 

inner less electron-dense layer. The wall of the sporocladium 

was irregularly undulate, which may represent the early 

stages of pseudophialide formation (Fig. 1C). 

Pseudophialides 

Pseudophialides were initially spherical, but with maturity 

became ellipsoid, non-septate, 2.0–2.5 µm wide, 4–7 µm 

long, and possessed a two-layered wall. They formed in 

104 

ima fUNGUS

Merosporangia of Linderina pennispora 

ARTICLE 

Fig. 1. Scanning electron (A, B, D, F–I) and transmission micrographs (C, E, J, K) of sporulating structures of Linderina pennispora. A. Immature, 

globose sporocladia formed apically on the sporangiophore (Sp). Bars = 5 µm. B. Pseudophialide initials formed on sporocladia (S). Bars = 5 µm. 

C. Longitudinal sections of a sporocladium (S) with pseudophialides initials. Bar = 1 µm. D. Ellipsoid pseudophialides (Ps) with merosporangium 

initials. S = Sporocladium, Sp = Sorangiophores. Bar = 5 µm. E. Longitudinal sections through the distal region of the pseudophialide (Ps), 

the septum between a pseudophialide neck (PsN) and merosporangium (M). The pore in the septal cross wall (CW) contains a biconvex 

septal-plug (P), and the base of the merosporangiospore (Ms) is constricted towards the septum. P = septal-plug. Bar = 0.25 µm (c). F. Mature 

sporocladium (S), pseudophialides (Ps) and merosporangia (M). Bar = 5 µm. G. Obovate merosporangia (M) on pseudophialides (Ps). Note 

the regular annulations on the merosporangia surface, and the septum on the pseudophialide neck (N). Bar = 5 µm. H. Merosporangium and 

merosporangiospore cell wall. Three wall-layered of merosporangium (MW) and four-layered wall of the merosporangiospore (MsW). Bar = 

0.1 µm. I. Released merosporangia (M) with a single, basally-attached “appendage”. Bar = 2 µm. J. Ellipsoid pseudophialides (Ps) coated 

with rod-shape ornamentations. Bar = 2 µm. K. Base of merosporangiospore (MB) with appendage (A) attached to the inner layer of the 

merosporangiospore cell wall and passing through the septum pore to the pseudophialide neck. Bar = 0.1 µm. 

a series of concentric groups radiating from the “apex” 

of the sporocladium. Pseudophialides at the “apex” of 

the sporocladium form first, and those at the periphery, 

last (Fig. 1D). The pseudophialides were produced 

holoblastically from the sporocladium. Only the peripheral 

pseudophialides possessed surface ornamentation and 

each arose approximately perpendicularly to the surface of 

the sporocladium. The distal region of the pseudophialides 

comprised a 1–1.5 µm diam neck region which lacked surface 

ornamentation (Fig. 1J). The necks were formed at the apex 

of the pseudophialides on those at the centre of the cluster, 

but subterminally and towards the inner pseudophailides 

on those at the periphery. Each pseudophialide produced a 

single merosporangium. 

An different structure occurred in the distal region and 

extended to the pseudophialide neck (Fig. 1I). Here the 

pseudophialide had a round, ca. 1.5 µm diam base and 

a narrower, 0.7–0.8 µm diam, lobed, cylindrical neck. 



ARTICLE 

The structural cell membrane was contiguous with the 

membrane of the septum, between the pseudophialide 

and merosporangium cross-walls. Here the pseudophialide 

necks were cylindrical, 1.2–1.4 µm long, ca. 1–1.1 µm 

diam, with a septum delimiting the merosporangium 

(Fig. 1E). Pseudophialides from which merosporangia 

had been released had a flared-shape structure from the 

merosporangium cell wall attached to the pseudophialide 

neck; the septum and septal plug, and structure within the 

pseudophialide, was shrivelled (Fig. 1J). 

Merosporangia and merosporangiospores 

Immature merosporangia were obovoid, whereas mature 

ones were obovate. Merosporangia matured first on those 

pseudophialides towards the “apex” of the sporocladium with 

those on the peripheral pseudophialides the last to mature. 

The merosporangia of L. pennispora were obovate, 3–4 µm 

wide near the base, narrowed distally to 2.0–2.5 µm wide, 

18–23 µm long, and possessed regular surface annulation 

with interconnecting ridges, but lacked any rod-shaped 

ornamentation (Fig. 1G). 

The merosporangia were produced terminally or 

subterminally and singly on the pseudophialides. A septum 

formed at the apex of the pseudophialide neck delimited the 

merosporangium from the pseudophialide. Merosporangia 

had a three-layered wall continuous with the pseudophialide 

wall. The merosporangium wall comprised an outer, 150– 

200 nm thick, electron-opaque layer; a middle, 200–250 

nm thick, electron-opaque layer; and an inner, 40–50 nm 

thick, electron-dense layer (Fig. 1H). Each merosporangium 

contained a single merosporangiospore. 

The merosporangiospore was obovate, 2.3–2.5 µm diam, 

with a ca. 1 µm diam papilla-like base. The merosporangiospore 

had a four-layered wall: an outer, 2–5 nm thick, electrondense 

layer; adpressed to the outer layer, a thick, 5–10 

nm, electron-dense layer; and an innermost fourth, 90– 

100 nm thick, amorphous, electron-transparent layer. 

The merosporangiospore wall was contiguous with the 

merosporangium wall at the distal region, and separated by 

an electron-opaque layer at the base of the merosporangium. 

Detachment of the merosporangia from their pseudophialides 

occurred at the base of the merosporangium. 

Detached merosporangia possessed a single, 3–5 µm 

long, “appendage” that was attached to the base of the 

merosporangium (Fig. 1I). The appendage was acicular, 

unbranched, ca. 0.1 µm diam and attached to the 

merosporangiospore inner cell wall layer and passed through 

the septum plug to the pseudophialide. Pseudophialides from 

which merosporangia had been released possessed a flared 

collar-like distal region with the septum, which delimited the 

pseudophialide from the merosporangium, retained at the 

base of the collar (Fig. 1J). 

The detachment of the merosporangium from the 

pseudophialide occurred by rupture of the merosporangium 

wall near the base, when the merosporangiospore wall 

becomes coated with the electron-opaque layer (Fig. 1K). 

When the merosporangium has been detached, a part 

of the merosporangium wall remained attached to the 

pseudophialide neck, as well as the septum between the 

pseudophialide and merosporangium. The cytoplasmic 

layer between the merosporangiospore base and the 

merosporangium wall appeared as a spherical-shape 

structure attached to the septal-plug. 

DISCUSSION 

Aerial hyphae of Linderina pennispora were investigated 

previously at the ultrastructure level (Young 1969, 1970b, 

Benny & Aldrich 1975). The hyphae had a two-layered wall 

which comprised an outer amorphous layer and an inner 

fibrillar layer (Young 1969, 1970b, Benny & Aldrich 1975). 

Numerous spines were described as attached to the outer 

layer of the hyphal wall (Young 1970b). Benny & Aldrich 

stated that the spines were attached to the inner layer of the 

wall and appeared to be covered by material from the outer 

wall layer (Benny & Aldrich 1975). The results presented here 

show that the surface ornamentations of the aerial hyphae 

of Linderina pennispora appears rod-shaped rather than 

spine-like. The ornamentation is attached to the outer layer 

of the hyphal wall. It is fibrillar, electron-dense, and seem 

to be derived from the same material as the outer layer. 

There was no definite description for the sporangiophore in 

all the previously published studies on the morphology of 

the species. This study revealed that the sporangiophore 

of L. pennispora arose as a lateral branch of the vegetative 

hyphae. The sporangiophores are narrower in diameter 

than the vegetative hyphae, and the ontogeny of the 

sporangiophore and its sympodial growth are described here 

for the first time, and explain the diagnostic “zigzag” form of 

the sporangiophore. 

Benjamin (1966) described the sporocladia of Kickxellaceae 

species as the most highly developed sporiferous 

branchlets in Zygomycetes. The present study provides details 

of the sporocladia and their ontogeny. The sporocladium 

initials are produced terminally by the sporangiophore, and, 

when the sporangiophore resumes its growth, the sporocladia 

are displaced laterally. The terminal sporocladium is displaced 

after the formation of the merosporangia, particularly at 

the late stages of merosporangia development. Benny & 

Aldrich (1975) observed the surface ornamentations of 

pseudophialides of L. pennispora and stated that they were 

coated with fewer rod-shape surface ornamentations than 

the sporocladium, and comprised a structure they termed 

an “abscission vacuole”. However, Young (1974) described 

a similar structure in the pseudophialides of Kickxella 

alabastrina, and then termed the structure a “labyrinthiform 

organelle” based on its morphology. A similar structure was 

also demonstrated in the pseudophialides of Dipsacomyces 

acuminosporus and Martensiomyces pterosporus (Young 

1968). Benny & Aldrich (1975) suggested that this structure 

was related to the abscission and dispersal mechanism of 

the wet-spored species of Kickxellaceae. They believed that 

this structure was produced from the septum delimiting the 

merosporangium (Benny & Aldrich 1975). 

Our electron microscopic studies of the pseudophialides 

of Linderina pennispora show, for the first time, a concentric 

arrangement of the pseudophialides on the sporocladium, 

and that only the peripheral pseudophialides are coated 

with a rod-shaped ornamentation. Ultra-thin sections 

106 

ima fUNGUS

Merosporangia of Linderina pennispora 

showed this structure was in the distal, neck region of the 

pseudophialides. The structure has a round base located 

in the distal part of the pseudophialide, with a cylindrical 

neck occupying the whole of the remaining pseudophialide 

neck. The structure is covered with a membrane-like layer 

contiguous to both the septum cross walls and the inner 

wall of the neck. The electron microscopy results of the 

merosporangia ontogeny and its detachment confirms 

that this structure has no role in its release, in addition this 

confirmed that the merosporangiospore appendage contains 

the appendage. Consequently, in future this structure would 

be better termed “appendage sac” rather than abscission 

vacuole or labyrinthiform organelle. 

The ultrastructure of the merosporangia of Linderina 

pennispora has been the subject of many previous studies 

(Young 1968, 1970a, 1971, Benny & Aldrich 1975, Moss & 

Young 1978, McKeown et al. 1996). However, no obvious 

differentiation was made between the merosporangium and 

the merosporangiospore since some of these studies used 

the term “spore” (Young 1968, 1970a, 1971) without any 

reference to the merosporangium or merosporangiospore. 

Differentiation between merosporangium and 

merosporangiospore has been made in the present study. 

The merosporangium prior to release is characterised by 

a surface ornamentations comprising annular rings with 

interconnecting ridges. The merosporangiospore is included 

within the merosporangium, and has a papilla-like base lies 

above the septum delimiting the merosporangium from the 

pseudophialide. 

The detached spore of L. pennispora was found to be 

the merosporangiospore covered with the merosporangium 

wall, except at the base where that remained attached to 

the pseudophialide neck. On the other hand, the surface 

ornamentation that characterises the morphological maturity 

of the merosporangium is caused by the formation of a dentatelike 

surface ornamentation by the merosporangiospore wall. 

Young (1971) and Benny & Aldrich (1975) described this 

dentate-like ornamentation as spines regularly arranged on 

the surface of the merosporangiospore, which they believed 

to be the liberated spore. This situation is now established, 

and in addition to the new interpreation of the liberated spore 

of L. pennispora, explains the results of McKeown et al. 

(1996) who described two different regions of microfibrills in 

the arrangement of the merosporangium wall of this fungus. 

It is conceivable that the surface ornamentations are involved 

in the merosporangium detachment by pushing out the cell 

wall. 

The two-layered nature of the wall of the aerial 

hyphae of L. pennispora was confirmed. However, the 

results of the transmission electron microscopy revealed 

that the merosporangium had a three-layered wall: an 

outer, an electron-dense, and a thinner layer. The wall at 

the merosporangium base was similar and continuous 

with the two-layered wall of the pseudophialides that 

comprised an outer, electron-dense, thinner layer, and an 

inner, electron-opaque, thicker layer. The rupture of the 

merosporangium wall appears to occur at the point where 

the two-layered pseudophialide wall is contiguous with the 

three-layered merosporangium wall. On the other hand, 

the merosporangiospore possessed a four-layered wall: 

an outer, 2–5 nm thick, electron-dense layer; adpressed 

to the outer layer a thick, 5–10 nm, electron-dense layer; 

and an innermost fourth, 90–100 nm thick, amorphous, 

electron-transparent layer. Young (1970a) described the 

merosporangiospore wall of Linderina pennispora as an 

outer and inner complex, but it is possible that he actually 

described the wall of the detached merosporangiospore 

within the merosporangium wall. 

Our results reveal that the merosporangiospore of L. 

pennispora possess an “appendage”. This is the first such 

observation not only for the species, but also within the 

family. The formation of the merosporangiospore appendage 

proceeds at a late stage of merosporangia development, 

almost prior to merosporangium detachment. The appendage 

is attached to the collar-like base, particularly to the inner 

layer of the merosporangiospore. It is ca. 3–5 µm long and 

formed inside the “appendage sac” in the pseudophialides. 

The function of this appendage is unknown and necessitates 

more work in order to be resolved. 

The septum that comprises a cross-wall, a central pore 

occluded by a biumbonate plug, the coemansioid form of the 

asexual reproductive apparatus, the similar wall structure, 

and the serological affinity indicate that Kickxellaceae are 

closely related to Harpellales and Asellariales (Moss & 

Young 1978). The demonstration of the merosporangiospore 

“appendage” strongly supports this hypothesis. 

AcknowledgEment 

This project was supported by King Saud University, Deanship of 

Scientific Research, College of Science, Research Center. 

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ARTICLE 

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the Kickxellales (Zygomycetes, Zygomycotina). Mycologia 70: 

944–963. 

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108 

ima fUNGUS

doi:10.5598/imafungus.2012.03.02.02 


Homortomyces gen. nov., a new dothidealean pycnidial fungus from the 

Cradle of Humankind 

Pedro W. Crous 1 , Johannes Z. Groenewald 1 , Lorenzo Lombard 1 and Michael J. Wingfield 3 

1 

CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; corresponding author e-mail: p.crous@cbs.knaw.nl 

2 

Department of Microbiology and Plant Pathology, DST/NRF Centre of Excellence in Tree Health Biotechnology, Forestry and Agricultural 

Biotechnology Institute (FABI), University of Pretoria, Private Bag X20 Hatfield, Pretoria 0028, Pretoria, South Africa 

3 

Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria 0002, South Africa 

ARTICLE 

Abstract: Homortomyces is introduced as a new coelomycetous genus associated with leaf spots on Combretum 

erythrophyllum trees growing near and around the Sterkfontein caves, Maropeng, South Africa. Based on its 

transversely septate, brown conidia, the presence of paraphyses, and percurrent proliferation of the conidiogenous 

cells, the genus resembles Stilbospora (Melanoconidaceae, Diaporthales). It is distinct in having pycnidial condiomata, 

conidia lacking mucoid sheaths, and becoming muriform when mature. Its morphology and phylogenetic placement 

based on analyses of sequence data for the large subunit nuclear ribosomal RNA gene (LSU, 28S) as well as the ITS 

and 5.8S rRNA gene of the nrDNA operon, show that Homortomyces represents a novel genus in Dothideomycetes, 

although its familial relationships remain unresolved. 

Key words: 

coelomycetes 

Combretum 

Dothideomycetes 

ITS 

LSU 

Stilbospora 

systematics 

Article info: Submitted: 1 September 2012; Accepted: 10 October 2012; Published: 5 November 2012. 

Introduction 

The Sterkfontein caves at Maropeng (meaning “returning 

to the place of origin” in the southern African language, 

Setswana) form part of the Cradle of Humankind, a World 

Heritage Site close to Johannesburg, Gauteng Province, 

South Africa. The site is well known for the 2.3-million yearold 

fossil Australopithecus africanus, named “Mrs. Ples”, 

which was found there in 1947 by Robert Broom and John 

T. Robinson (Fleminger 2008). Although much attention has 

been devoted to fossils buried in the area, little is known 

of the fungi on the surrounding vegetation. The area is 

characterised by Rocky Highveld Grassland that harbours 

a diversity of plants and animals. During a recent visit to 

Maropeng, it was noted that Combretum erythrophyllum 

(River bushwillow; Combretaceae) trees suffered from a 

serious leaf spot disease, which appears to eventually kill the 

young shoots and lead to the development of prominent stem 

cankers. A Stilbospora-like coelomycete was consistently 

found sporulating on the leaf and shoot lesions. 

The genus Stilbospora is based on S. macrosperma, 

a coelomycetous fungus that occurs on dead branches of 

Carpinus betulus in Europe. Stilbospora macrosperma has 

been linked to the sexual morph Prosthecium ellipsoporum 

(Melanoconidaceae, Diaporthales) based on culture studies, 

and supported by DNA sequence data (Voglmayr & Jaklitsch 

2008). Stilbospora is characterised by acervular conidiomata 

that give rise to brown, transversely distoseptate conidia 

with mucilaginous sheaths, formed on hyaline, percurrently 

proliferating conidiogenous cells, intermingled with septate 

and hyaline paraphyses (Sutton 1980). The genus includes 

more than 80 names representing many disjunct taxa, and 

is in urgent need of taxonomic revision. The aim of this study 

was to isolate and characterise the fungus associated with 

the leaf spots on Combretum erythrophyllum, and to compare 

this taxon to species in Stilbospora. 

Materials and methods 

Isolates 

Single conidial colonies established from sporulating 

conidiomata were grown in Petri dishes containing 2 % malt 

extract agar (MEA; Crous et al. 2009b) as described earlier 

(Crous et al. 1991). Colonies were subcultured onto potatodextrose 

agar (PDA), oatmeal agar (OA), MEA (Crous et al. 

2009b), and pine needle agar (PNA) (Smith et al. 1996), and 

incubated at 25 °C under continuous near-ultraviolet light to 

promote sporulation. Reference strains were deposited at 

the CBS-KNAW Fungal Biodiversity Centre in Utrecht, The 

Netherlands (CBS), and taxonomic novelties were deposited 

in MycoBank (Crous et al. 2004). 

DNA isolation, amplification and analyses 

Genomic DNA was extracted from fungal colonies growing 

on MEA using the UltraClean TM Microbial DNA Isolation Kit 

(MoBio Laboratories, Inc., Solana Beach, CA, USA) following 

the manufacturer’s protocols. Part of the nuclear rDNA 



Attribution: 







Crous et al. 

ARTICLE 

Table 1. Collection details and GenBank accession numbers of isolates for which novel sequences were generated in this study. 

Species Strain no. 1 Substrate Country Collector GenBank accession no. 2 

ITS LSU 

Homortomyces combreti CBS 132554; CPC 19800 Combretum erythrophyllum, leaves South Africa: Maropeng P.W. Crous & M.J. 

Wingfield 

CBS 132555; CPC 19808 Combretum erythrophyllum, leaves South Africa: Maropeng P.W. Crous & M.J. 

Wingfield 

JX517280 — 

JX517281 JX517291 

Sclerostagonospora sp. CBS 118142; CMW 18281 Elegia equisetacea, dead culm South Africa: Kirstenbosch S. Lee DQ286766 / 

JX517282 

DQ286770 

CBS 118146; CMW 17948 Cannomois virgata, dead culm South Africa: Jonkershoek S. Lee DQ286765 DQ286769 

CBS 118152; CMW 18025 Thamnochortus spicigerus, dead culm South Africa: Kirstenbosch S. Lee JX517283 JX517292 

CBS 118224; CMW 18063 Ischyrolepis subverticellata, dead culm South Africa: Kirstenbosch S. Lee JX517284 JX517293 

Stilbospora macrosperma (syn. Prosthecium 

ellipsosporum) 

CBS 121692 Carpinus betulus, dead corticated twig Austria: Niederösterreich H. Voglmayr JX517285 JX517294 

CBS 121693 Carpinus betulus, dead corticated twig Austria: Niederösterreich H. Voglmayr JX517286 JX517295 

CBS 121694 Carpinus betulus, dead corticated twig Austria: Oberösterreich H. Voglmayr JX517287 JX517296 

CBS 121695 Carpinus betulus, dead corticated twig The Netherlands: Utrecht H. Voglmayr JX517288 JX517297 

CBS 121882 Carpinus betulus, dead corticated twig Austria: Niederösterreich, Wassergspreng 

CBS 121883 Carpinus betulus, dead corticated twig Austria: Oberösterreich, Leithenbachtal 

H. Voglmayr JX517289 JX517298 

H. Voglmayr JX517290 JX517299 

1 CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CMW: Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; 

CPC: Culture collection of P.W. Crous, housed at CBS. 

2 ITS: Internal transcribed spacers 1 and 2 together with 5.8S nrDNA; LSU: 28S nrDNA; TEF: partial translation elongation factor 1-alpha. 

110 ima fUNGUS

Homortomyces gen. nov. (Dothideomycetes) 

operon spanning the 3’ end of the 18S rRNA gene, both 

internal transcribed spacer regions, the 5.8S rRNA gene, 

and the 5’ end of the 28S rRNA gene (ITS) was amplified 

using the primers V9G (de Hoog & Gerrits van den Ende 

1998) and LR5 (Vilgalys & Hester 1990). The primers ITS4 

(White et al. 1990) and LSU1Fd (Crous et al. 2009a) were 

used as internal sequence primers to provide sequences 

of high quality over the entire length of the amplicon. The 

LSU sequence alignment of Voglmayr & Jaklitsch (2008) was 

downloaded from TreeBASE (matrix M3536; www.treebase. 

org/treebase/index.html) and modified with additional 

sequences from NCBI’s GenBank nucleotide database. The 

sequence alignment and subsequent phylogenetic analyses 

were carried out using methods described by Lombard et al. 

(2011); gaps were treated as “fifth state” data. Sequences 

derived in this study were lodged in GenBank (Table 1), 

the alignment in TreeBASE (www.treebase.org/treebase/ 

index.html), and taxonomic novelties in MycoBank (www. 

MycoBank.org; Crous et al. 2004). 

Morphology 

Descriptions were based on slide preparations mounted 

in clear lactic acid from colonies sporulating on PNA. 

Observations were made with a Zeiss V20 Discovery stereomicroscope, 

and with a Zeiss Axio Imager 2 light microscope 

using differential interference contrast (DIC) illumination and 

an AxioCam MRc5 camera and software. Colony characters 

and pigment production were noted after 1 mo of growth on 

MEA, PDA and OA (Crous et al. 2009b) incubated at 25 ºC. 

Colony colours (surface and reverse) were established using 

the colour charts of Rayner (1970). 

calculated (Fig. 1). 

Neighbour-joining analyses using three substitution 

models on the same LSU sequence alignment yielded trees 

with identical topologies and differed mainly with regard to the 

arrangement of the clades representing Umbilicariales and 

Teloschistales compared to that obtained from the Bayesian 

analysis (Fig. 1). 

Parsimony analysis of the LSU alignment yielded 88 

equally most parsimonious trees (data not shown; TL = 795 

steps; CI = 0.540; RI = 0.885; RC = 0.478). Similar to the 

tree generated by MrBayes, the clades representing the 

Umbilicariales and Teloschistales were differently ordered 

in the parsimony phylogeny compared to the neighbourjoining 

and Bayesian analyses. Also, the Stilbospora-like 

strain isolated in this study moved to a basal position in 

Botryosphaeriales as sister to Phyllosticta in the parsimony 

analyses (data not shown). However, its position in 

Botryosphaeriales was not supported in the bootstrap 

analysis (data not shown). 

A megablast search of the ITS sequence failed to reveal 

any high similarity hits in the general nucleotide database 

of GenBank. Highest levels of similarity were observed with 

Bagnisiella examinans (GenBank EU167562; Identities 

= 522/628 (83 %), Gaps = 54/628 (9 %)), Botryosphaeria 

dothidea (GenBank DQ008327; “Identities” = 497/600 (83 

%), Gaps = 58/600 (10 %)) and Sclerotinia homoeocarpa 

(GenBank GU002301; “Identities” = 515/622 (83 %), Gaps 

= 58/622 (9 %)). The Stilbospora-like strain isolated in this 

study is described in a new genus below. 

Taxonomy 

ARTICLE 

RESULTS 

Phylogenentic comparisons 

Amplicons of approximately 1 700 bases were obtained for 

the ITS region, including the first approximately 900 bp of 

LSU, for the isolates listed in Table 1. The LSU sequences 

were used to obtain additional sequences from GenBank, 

which were added to an alignment modified from that of 

Voglmayr & Jaklitsch (2008). The manually adjusted LSU 

alignment contained 46 sequences (including the outgroup 

sequence) and 850 characters including alignment gaps 

(available in TreeBASE) were used in the phylogenetic 

analysis; 253 of these were parsimony-informative, 36 

were variable and parsimony-uninformative, and 561 were 

constant. The ITS sequences were used in a blast search of 

the GenBank nucleotide database in an attempt to identify 

the species. 

A Bayesian analysis was conducted on the aligned 

LSU sequences using a general time-reversible (GTR) 

substitution model with inverse gamma rates and dirichlet 

base frequencies. The Markov Chain Monte Carlo (MCMC) 

analyses of two sets of 4 chains started from a random tree 

topology and lasted 506 000 generations, after which the 

split frequency reached less than 0.01. Trees were saved 

each 1 000 generations, resulting in 1 012 saved trees. 

Burn-in was set at 25 %, leaving 760 trees from which the 

consensus tree and posterior probabilities (PP’s) were 

Homortomyces Crous & M.J. Wingf., gen. nov. 

MycoBank MB801349 

Etymology: Homortomyces, derived from “homo” (human 

being), “orto or origo” (origin) and “-myces” (fungus). 

Hormotomyces resembles Stilbospora (Melanoconidaceae, 

Diaporthales), but is distinguished from that genus by having 

pycnidial condiomata, and conidia characterised by muriform 

septa (in exceptional cases), and lacking mucoid sheaths. 

Description: Foliicolous, associated with leaf spots. 

Conidiomata pycnidial, black, globose, with central ostiole; 

wall consisting of 4–7 layers of brown textura angularis. 

Conidiophores reduced to conidiogenous cells or one 

supporting cell, hyaline, cylindrical, with 1–4 inconspicuous 

percurrent proliferations at apex. Paraphyses intermingled 

among conidiogenous cells, extending above conidia, hyaline, 

smooth, cylindrical, flexuous, apex obtuse, sparingly septate. 

Conidia brown, ellipsoid to subcylindrical, verruculose, 

transversely euseptate, septa with visible central pore, 

becoming muriformly septate in older cultures, apex obtuse, 

base truncate with visible scar, basal or displaced towards 

the side. 

Type species: Homortomyces combreti Crous & M.J. Wingf. 

2012. 


111

Crous et al. 

ARTICLE 

Magnaporthe grisea AB026819 

1 Diaporthe acaciigena JF951160 

Diaporthe eres AF408350 

“Prosthecium” galeatum EU039988 

1 

1 

“Prosthecium” pyriforme EU039987 

1 “Prosthecium” acerophilum EU039993 

1 “Prosthecium” acerinum EU039996 

1 “Prosthecium” opalus EU039997 

Stilbospora macrosperma EU039986 

Diaporthales 

Stilbospora macrosperma CBS 121692 


1 Stilbospora macrosperma CBS 121694 




0.96 Umbilicaria decussata EF489960 

0.89 Umbilicaria dendrophora HM161600 

1 Caloplaca scopularis JQ301559 

Caloplaca marina JQ301557 

Homortomyces combreti CPC 19808 

Umbilicariales 

Teloschistales 

incertae sedis 

1 Hysteropatella clavispora AY541493 

Glonium chambianum GQ221883 

0.92 0.98 

Gloniopsis praelonga FJ161195 

Hysteriales 

0.01 

1 Psiloglonium araucanum FJ161190 

0.86 

1 

Curreya proteae EU552117 

0.95 

1 Misturatosphaeria tennesseensis GU385207 

1 Sclerostagonospora sp. CBS 118224 

Sclerostagonospora sp. CBS 118152 

1 

Neosetophoma samarorum GQ387579 

Phaeosphaeriopsis musae DQ885894 

Botryosphaeria melanops DQ377856 

1 Saccharata proteae EU552145 

1 Saccharata intermedia GU229889 

1 Phyllosticta vaccinii FJ588245 

Phyllosticta concentrica DQ377878 

Neofusicoccum mediterraneum FJ755232 

0.84 

Neoscytalidium dimidiatum DQ377924 

1 

Botryosphaeria mamane DQ377855 

Dothiorella sarmentorum DQ377860 

Botryosphaeriales 

0.87 

1 Neofusicoccum ribis DQ246263 

Neofusicoccum arbuti DQ377919 

Tiarosporella tritici DQ377941 

Lasiodiplodia venezuelensis DQ377904 

1 

Lasiodiplodia pseudotheobromae FN645639 

1 

Diplodia porosum DQ377895 

Diplodia pinea DQ377893 

Pleosporales 

Fig. 1. Bayesian consensus phylogeny obtained from the analysis of the LSU sequence alignment. The scale bar represents the average 

number of substitutions per site, and posterior probability values are shown at the nodes. The novel species treated in this study is shown in red 

and novel sequences in bold. Orders are indicated in the coloured blocks. Branches also present in the strict consensus tree of the parsimony 

analysis are thickened and the tree was rooted on a sequence of Magnaporthe grisea (GenBank accession no. AB026819). 

112 ima fUNGUS


ARTICLE 

Fig. 2. Homortomyces combreti (CPC 19800). A. Rocky Highveld Grassland at Sterkfontein Caves, Maropeng. B–D. Prominent leaf spots on 

Combretum erythrophyllum, with black pycnidia. E. Sporulating pycnidial conidiomata on MEA. F. Paraphyses. G–J. Conidiogenous cells giving 

rise to conidia. K–P. Distoseptate conidia, showing septal pores, transverse septa, and flattened, eccentric, basal conidial hila. Scale bars = 10 

µm. 

Homortomyces combreti Crous & M.J. Wingf., sp. 

nov. 


(Fig. 2) 

Etymology: After the genus Combretum on which the fungus 

was first found. 

Type: South Africa: Gauteng, Maropeng, Sterkfontein 

Caves, The Cradle of Humankind, on leaves of Combretum 

erythrophyllum (River bushwillow; Combretaceae), 4 July 

2011, P.W. Crous & M.J. Wingfield (CBS H-21049 holotype; 

cultures ex-type CPC 19800 = CBS 132554, 19801, 19808 = 

CBS 132555, CPC 19809). 

Description: Leaf spots amphigenous, circular to subcircular, 

medium brown with dark brown margin, 2–7 mm diam. On 

MEA: Conidiomata pycnidial, amphigenous on leaves, 

black, globose, up to 500 µm diam with central ostiole; 

wall consisting of 4–7 layers of brown textura angularis. 

Conidiophores reduced to conidiogenous cells or one 

supporting cell, hyaline, cylindrical, 20–60 × 3–5 µm, with 


113

Crous et al. 

ARTICLE 

1–4 inconspicuous percurrent proliferations at their apex. 

Paraphyses intermingled among conidiogenous cells, 

extending above the conidia, to 100 µm long, 2–4 µm diam, 

hyaline, smooth, cylindrical, flexuous, sparingly (1–3)-septate 

with obtuse apex; in old paraphyses the apical cell becoming 

swollen and clavate, with walls becoming thickened. Conidia 

(27–)32–38(–40) × (11–)13–16(–18) µm, brown, ellipsoid 

to subcylindrical, verruculose, 3(–4)-euseptate, septa with 

visible central pore, becoming muriformly septate in older 

cultures, apex obtuse, base truncate with visible scar, basal 

or displaced towards the side, 3–3.5 µm diam. 

Cultural characteristics: Colonies on MEA on 25 ºC spreading, 

erumpent with sparse aerial mycelium and lobate, feathery 

margins, reaching 35 mm diam after 1 mo. Surface umber to 

chestnut; reverse chestnut, outer margin ochraceous. 

Discussion 

In a recent phylogenetic study, the type species of the genus 

Stilbospora, S. macrosperma was linked to a Prosthecium 

sexual state, P. ellipsosporum (Voglmayr & Jaklitsch 2006). 

Stilbospora macrosperma Pers. 1794 is the type species of 

Stilbospora Pers. 1794, while P. ellipsosporum Fresen. 1852 

is the type species of Prosthecium Fresen. 1852. In moving 

to a single nomenclature (Hawksworth et al. 2011, Wingfield 

et al. 2012), it would be prudent to retain Stilbospora over 

Prosthecium, as the former genus includes a greater number 

of taxa, is the older genus (thus having priority), and is the 

more commonly used name by plant pathologists. Other than 

confirming this link, Voglmayr & Jaklitsch (2006) described 

several other Prosthecium-like species, which also had 

Stegonsporium Corda 1827 conidial morphs. Although 

Stegonsporium resembles Stilbospora, it differs from that 

genus in that conidia have longitudinal septa. Furthermore, 

taxa with Stegonsporium morphs cluster adjacent to 

Stilbospora s.str. (Voglmayr & Jaklitsch 2006), and represent 

a different morphological and phylogenetic entity, to which 

the name Stegonsporium applies. Prosthecium, however, 

is a later synonym of Stilbospora (Melanconidaceae, 

Diaporthales) in this taxonomy. 

Homortomyces closely resembles Stilbospora in 

morphology, but can be distinguished by the pycnidial 

conidiomata with a central ostiole, whereas Stilbospora has 

acervulate conidiomata. Conidia of Homortomyces also 

lack mucoid sheaths, and are transversely distoseptate, 

becoming muriformly septate in older cultures. Other genera 

with rather similar conidia to consider include Endocoryneum, 

Hendersoniopsis, Angiopomopsis, and Ceratopycnis, but none 

of these genera have paraphyses (Sutton 1980), and thus are 

easily distinguished morphologically from Homortomyces. 

Based on our parsimony analysis, Homortomyces 

resides in Botryosphaeriales (Dothideomycetes), in which it 

appears to represent a family basal to Botryosphaeriaceae 

(results not shown). The Botryosphaeriaceae includes more 

than 17 genera that have Botryosphaeria-like ascomata 

(Crous et al. 2006, Damm et al. 2007 Phillips et al. 2008, 

Rojas et al. 2008), and are commonly associated with stem 

cankers and leaf spots of woody hosts (Slippers & Wingfield 

2007). Several conidial genera in Botryosphaeriales have 

pycnidial conidiomata with paraphyses and conidiogenous 

cells with percurrent proliferation. However, the description 

of Homortomyces as a coelomycetous genus characterised 

by distoseptate conidia does not fully fit the morphological 

concept for this order. Both the distance and Bayesian 

analyses place Homortomyces in the backbone of the 

phylogenetic tree of Dothideomycetes (e.g. Fig. 1) and, 

pending collection of additional species of this genus or more 

closely allied genera, it is best treated as incertae sedis rather 

than referred to an any existing or a new family. 

Homortomyces combreti is the only fungus closely 

associated with a destructive leaf and shoot disease of C. 

erythrophyllum, and it is most likely the causal agent of this 

disease, though this has not yet been proven experimentally. 

Given the damage caused to these trees, it will be important 

to establish its pathogenicity and then to consider strategies 

to manage the disease, which is damaging large numbers 

of amenity trees. Although the primary infections occur on 

young leaves and shoots, the infections subsequently appear 

on larger branches and main stems, resulting in obvious stem 

cankers. 

Acknowledgements 

We thank the technical staff, Arien van Iperen (cultures), Marjan 

Vermaas (photographic plate), and Mieke Starink-Willemse 

(DNA isolation, amplification and sequencing) for their invaluable 

assistance. 

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doi:10.5598/imafungus.2012.03.02.03 


A new Leucoagaricus species of section Piloselli (Agaricales, Agaricaceae) 

from Spain 

Guillermo Muñoz 1 , Agustín Caballero 2 , Marco Contu 3 , and Alfredo Vizzini 4 

1 

Avda. Valvanera 32, 5.º dcha. 26500 Calahorra, La Rioja, Spain 

2 

C/ Andalucía 3, 4.º dcha. 26500 Calahorra, La Rioja, Spain 

3 

Via Marmilla, 12. 07026, Olbia. Italy 

4 

Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Viale P.A. Mattioli 25, I-10125 Torino, Italy; corresponding author 

e-mail: alfredo.vizzini@unito.it 

ARTICLE 

Abstract: The new species Leucoagaricus variicolor is described from a public park in Zaragoza, Spain, based on 

both morphological and molecular characters. Illustrations of fresh basidiomata in situ and of the main macro- and 

micromorphological features are added. Leucoagaricus variicolor belongs to section Piloselli and is compared with 

similar taxa. 

Key words: 

Agaricomycetes 

Basidiomycota 

nrITS rDNA 

taxonomy 

Article info: Submitted: 25 July 2012; Accepted: 14 October 2012; Published: 5 November 2012. 

INTRODUCTION 

During a survey of macrofungi conducted by the first author in 

a public park in Zaragoza (Parque José Antonio Labordeta), 

four collections of a remarkable species of Leucoagaricus 

were recorded on clayey soil near a Pinus halepensis 

plantation. The park covers an area of 409 000 sq. m to the 

south of the city; it is regarded as an important green space 

because of its botanical biodiversity. The species has been 

collected from an area of 50 sq. m. 

The collections fit morphologically into Leucoagaricus sect. 

Piloselli, a section within the Leucoagaricus/Leucocoprinus 

clade (Agaricaceae) that encompasses species whose 

basidiomata usually stain orange-red when bruised and turn 

green with ammonia (Singer 1973, 1986, Vellinga 2010). 

In this section, species identification depends particularly 

on morphological characters such as pileus colour, colour 

reactions of the basidiome surface when bruised or exposed 

to ammonia, the structure of the pileipellis, and the shape 

of the cheilocystidia and spores (Bon 1993, Vellinga 2010). 

Species of the morphologically similar sect. Rubrotincti differ 

mainly in the immutable context (not changing colour when 

bruised) and the absence of a green reaction with ammonia 

on the basidiome surface (Singer 1948, 1986, Bon 1993, 

Vellinga 2001). 

Our taxon is distinguished from all other species in sect. 

Piloselli by the cream-ochre pileus which becomes entirely 

pink-rose in herbarium specimens, abundant velar remnants 

on the pileus surface, mainly pyriform to sphaeropedunculate 

cheilocystidia, and subglobose to broadly ellipsoid spores. 

An exhaustive search of the literature, including monographic 

treatments and papers by Bon (1981, 1993), Candusso 

& Lanzoni (1990), Contu (1990), Bon & Caballero (1997), 

Caballero (1997), Gennari & Migliozzi (1999), Migliozzi & 

Resta (2001), Migliozzi et al. (2001), Vellinga (2001, 2006, 

2010), and Vellinga et al. (2010), confirmed the unique nature 

of this species: its characteristics do not match any published 

species. In addition, an ITS sequence analysis supported this 

statement. Therefore, a detailed description and illustrations of 

this previously undescribed Leucoagaricus are presented here. 


Morphology 

All the studied collections were photographed in situ, using 

a Nikon D50 digital camera, with a tripod, and in natural 

light. Macromorphological features are described from fresh 

specimens. The microscopic structures were observed in 

both fresh and dried material, using several mountants 

and stains: water, 2 % KOH, ammoniacal Congo red, 

Brilliant Cresyl blue, and Melzer’s reagent. Dried fragments 

were rehydrated in 2 % KOH. All microscopic measurements 

were carried out with a ×1000 oil immersion objective. In 

the description below, spore measurements are based on 

120 elements in ammoniacal Congo red randomly selected 

from four collections. Only mature, normally developed and 

non-aberrant spores from spore prints were measured. 

Dimensions of the spores are given as follows: (minimum 

value–) 1st decile – average value – 9th decile (–maximum 

value). The width of basidia was measured at the widest part, 

and the length was measured from the apex (sterigmata 



Attribution: 







Muñoz et al. 

ARTICLE 

100 

Leucoagaricus flammeotinctoides GU136173 

100 

78 

Leucoagaricus flammeotinctoides GQ258476 

Leucoagaricus flammeotinctoides AY243620 

98 

100 Leucoagaricus pyrrhophaeus GU136199 

Leucoagaricus pyrrhophaeus GQ258473 

100 

75 

Leucoagaricus pyrrhulus GQ258474 

Leucoagaricus pyrrhulus GU136201 

100 

Leucoagaricus erythrophaeus GQ258468 

97 Leucoagaricus erythrophaeus GQ258470 

Lepiota roseifolia GQ203805 

Leucoagaricus decipiens GQ203803 

100 Leucoagaricus pardalotus GU136202 

Leucoagaricus pardalotus GQ258479 

100 Lepiota decorata GU136197 

88 

Lepiota decorata AY243645 

68 

Leucoagaricus ionidicolor AY176415 

Leucoagaricus marriagei GQ329049 

100 Leucoagaricus sp. Weber 6019 AY243633 

100 

Leucoagaricus brunnescens GQ203804 

89 

100 Leucoagaricus georginae AY176413 

Leucoagaricus georginae GU136198 

Leucoagaricus jubilaei AY243635 

98 Leucoagaricus badhamii GQ329056 

78 

Leucoagaricus sp. ecv2484 GU136182 

100 Leucoagaricus dyscritus GU136181 

Leucoagaricus dyscritus GU136180 

100 Leucoagaricus hesperius GU139789 

Leucoagaricus hesperius GU139788 

Lepiota fuliginescens GU136183 

98 


52 Lepiota fuliginescens GU136189 

Lepiota flammeotincta GU136170 

100 Leucoagaricus sp. Vellinga 2746 AY176440 

95 

Lepiota flammeotincta GU136167 

54 Lepiota fuliginescens GU136187 

Leucoagaricus sp. Vellinga 2619 AY243637 


Leucoagaricus sp. Huijser s.n. AY243643 

Leucoagaricus adelphicus GQ258478 

100 

68 

Leucoagaricus adelphicus AY243622 Piloselli 

Leucoagaricus adelphicus AY243624 

100 Leucoagaricus pilatianus GQ329040 

Leucoagaricus pilatianus GQ329057 

85 

93 Leucoagaricus cupresseus GQ258477 

95 Leucoagaricus cupresseus GU136194 

87 Leucoagaricus cupresseus AY243629 

Leucoagaricus cupresseus AY243627 

Leucoagaricus cupresseus AY243632 

Leucoagaricus variicolor coll. GM-2485 JX880032 

100 Leucoagaricus variicolor coll. AH 40328 (holotype) JX880030 



89 Leucoagaricus rubrotinctus JN944081 

58 Leucoagaricus rubrotinctus FJ481050 

100 

Leucoagaricus rubrotinctus JN944082 

98 

Leucoagaricus sublittoralis AY176442 Rubrotincti 

100 Leucoagaricus littoralis GQ329041 

Leucoagaricus wychanskyi AF482874 

Cystolepiota seminuda AY176350 

0.06 

Fig. 1. Maximum Likelihood phylogram obtained from the ITS (ITS1-5.8S-ITS2) sequence alignment of Leucoagaricus spp. Cystolepiota 

seminuda was used as outgroup taxon. MLB values over 50 % are given above branches. Newly sequenced collections are in bold. 

excluded) to the basal septum. Microscopic pictures were 

taken on a Moticam 2500 digital camera connected to a Motic 

BA300 microscope. Colour notations for the macroscopic 

descriptions are from Munsell (1994), hereafter shortened as 

Mu. Herbarium acronyms follow Index Herbariorum, except 

for GM and AC that refer to the personal herbaria of Guillermo 

Muñoz and Agustín Caballero. The type collection is housed 

at AH. The name and description of the new species are 

deposited in MycoBank (Crous et al. 2004). 

DNA extraction, PCR amplification, and DNA 

sequencing 

Genomic DNA was isolated from 1 mg of a dried herbarium 

specimen from four collections (AH-40328, GM-2454, GM- 

2485, and GM-2486), using the DNeasy Plant Mini Kit 

(Qiagen, Milan) according to the manufacturer’s instructions. 

Universal primers ITS1F/ITS4 were used for the ITS region 

amplification (White et al. 1990, Gardes & Bruns 1993). 

Amplification reactions were performed in a PE9700 thermal 

cycler (Perkin-Elmer, Applied Biosystems) following Vizzini et 

al. (2011). The PCR products were purified with the AMPure 

XP kit (Beckman) and sequenced by MACROGEN (Seoul, 

Republic of Korea). The sequences were submitted to 

GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and their 

accession numbers are reported in Fig. 1. 

Sequence alignment and phylogenetic 

analysis 

The sequences obtained in this study were checked and 

assembled using Geneious v. 5.3 (Drummond et al. 2010) 

and compared to those available in the GenBank database 

by using the Blastn algorithm. Based on the Blastn results, 

sequences were selected according to the outcomes of 

recent phylogenetic studies on Leucoagaricus (Vellinga 

2010, Vellinga et al. 2010, 2011). Alignments were generated 

using MUSCLE (Edgar 2004) with default conditions for gap 

openings and gap extension penalties. The alignment was 

then imported into MEGA v. 5.0 (Tamura et al. 2011) for manual 

adjustment. The phylogenetic analysis was performed using 

the Maximum Likelihood (ML) approach. Following Vellinga 

(2010) and Vellinga et al. (2010), a Cystolepiota seminuda 

sequence (AY176350) was used as outgroup. ML estimation 

118 ima fUNGUS

A new Leucoagaricus species from Spain 

ARTICLE 

Fig. 2. Leucoagaricus variicolor. Macroscopic characters. A–C. Fresh basidiomata in situ. D. Herbarium specimens. A, D from AH 40328 

(holotypus); B from GM-2454; C from GM-2485. Bars = A–B = 50 mm; C–D = 20 mm. 

was performed through RAxML v. 7.0.4 (Stamatakis 2006) 

with 1000 bootstrap replicates (Felsenstein 1985) using the 

GTRGAMMA algorithm to perform a tree inference and search 

for a good topology. Support values from bootstrapping runs 

(MLB) were mapped on the globally best tree using the “-f a” 

option of RAxML and “-x 12345” as a random seed to invoke 

the novel rapid bootstrapping algorithm. Only MLB over 50 % 

are reported in the resulting tree (Fig. 1). 

RESULTS 

Phylogenetic analysis 

The amplification of the ITS regions was successful for the 

four specimens, yielding a PCR product of about 700 bp. The 

ITS data matrix comprises a total of 59 sequences (including 

55 from GenBank). In the obtained ML phylogram (Fig. 1), our 

four sequences of the new Leucoagaricus clustered together 

and are distinct and basal to all the existing sequences of 

previously sequenced species of section Piloselli. 

Taxonomy 

Leucoagaricus variicolor G. Muñoz, A. Caball., Contu 

& Vizzini, sp. nov. 


(Figs 2–4) 

Etymology: The specific epithet “variicolor” refers to the highly 

variable colours of the pileus surface depending on fresh or 

dry conditions. 

Diagnosis: Differs from every other described species of 

Leucoagaricus sect. Piloselli in having a cream to pink to eggyellow, 

or ochre pileus in fresh basidiomata that becomes 

dark rose in the herbarium, the presence of universal veil 

remnants on the pileus surface and stipe base, subglobose 

to broadly ellipsoid spores, pyriform to spheropedunculate 

cheilocystidia, and a unique ITS sequence. 

Type: Spain: Aragón: Zaragoza, “José Antonio Labordeta” 

(antea: “Parque Grande”) park, UTM 30TXM7511, N 41.634 W 

0.894, alt. 225 m, on argillose-sandy soil, in Pinus halepensis 

litter, 3 Dec. 2011, G. Muñoz (AH 40328 – holotype; GM- 

2453 and AC-4896 – isotypes). 

Description: Pileus 40–100 mm wide, when young 

hemispherical-convex to hemispherical, expanding to 

subplane, finally plane, without an umbo, with an entire, 

slightly exceeding margin, involute or incurved in young 

stages; pileus surface dry, almost smooth to finely feltedfibrillose, 

variable in colour, starting from whitish or pale 

cream or egg-yellow to ochre (Mu 2.5Y 8/1-2; Mu 5Y 8/1 

“White”; Mu 2.5Y 8/2-4 “Pale yellow”) to orange-pink (Mu 5YR 

6/6-8 “Yellowish red”; Mu 2.5YR 5/6-8 “Red”); in adult stages 


119

Muñoz et al. 

ARTICLE 

Fig. 3. Leucoagaricus variicolor. Microscopic characters. A–B. Elements of the pileipellis. C–D. Spores (in ammoniacal Congo red). E–F. 

Cheilocystidia (in ammoniacal Congo red). A, C, E from AH 40328 (holotype); B, D, F from GM-2485. Bars: A–B, E–F = 20 µm; C–D = 10 µm. 

these tinges are mixed, in dried herbarium material the main 

tinge is pink-rose (Mu 10R 5/3-4; 6/3-4) (Fig. 2D); surface 

not reddening but tardily darkening on handling, covered, 

mainly toward the disc, but often up to the antimarginal zone, 

with abundant, fibrillose to submembranaceous white velar 

patches, as remnants of the universal veil. Lamellae not 

reaching the stipe, attached to a pseudocollarium, crowded, 

to 6 mm broad, with (0–)1–3 lamellulae, white to cream or 

slightly beige, darker towards the margin, not reddening when 

bruised, with an even or slightly flocculose, concolorous edge. 

Stipe 40–100 × 12–18 mm, stout, solid, cylindrical, in most 

specimens with a napiform to submarginate bulb and then 

up to 23 mm wide; surface white, shiny, pruinose-floccose 

at the apex, at times slightly browning on handling or due to 

the environmental conditions, in young stages with minute 

universal veil remnants (as coarse flecks) towards the base. 

120 ima fUNGUS


ARTICLE 

Fig. 4. Leucoagaricus variicolor. 

Line drawings of microscopic 

characters (from AH 40328, 

holotype). A. Spores. B. Basidia. 

C. Cheilocystidia. D. Pileipellis. 

Bars: A–C = 20 µm; D = 100 µm. 

Annulus thin, simple, membranous, persistent, not movable, 

usually descending (rarely ascending), entirely white or 

ochre towards margin. Context fleshy, white, unchanging 

or slightly turning ochre-pink towards the base of the stipe. 

Smell and taste not distinctive, fungoid, pleasant. Edibility 

unknown. Spore-print: white. Chemical reactions: surface of 

the pileus, stipe, annulus, and lamellae green in ammonia. 

Spores (5.6–)6.0–6.8–7.6 (–8.2) × (4.5–)4.8–5.2–5.6(–5.7) 

µm, Q = (1.1–)1.2–1.3–1.4 (–1.6) (n = 120), subglobose to 

broadly ellipsoid or slightly ovoid, smooth, without a germpore, 

dextrinoid, metachromatic in Cresyl Blue (Figs 3C, D, 

4A). Basidia 20–30 × 7–9 µm four-spored, clavate, without 

basal clamp connection (Fig. 4B). Lamella edge sterile. 

Cheilocystidia abundant, 20–45 × 10–16 µm, pedicellate, 

pyriform to sphaeropedunculate, rarely with a very short 

mucro, nearly hyaline or with light brown contents (diluted 

and homogeneous cytoplamatic pigment) (Figs 3E, F, 4C). 

Pleurocystidia absent. Pileipellis a trichoderm consisting of 

erect, long cylindrical to fusiform, not gelatinized elements, 

occasionally septate, 150–350(–400) × 8–16 µm, without 

a subtending (basal) hymeniform layer (Figs 3A, B, 4D); 

pigment brownish, usually parietal, smooth, but sometimes 

also intracellular in some terminal elements. Velar patches 

of the pileus surface composed of hyaline, tightly interwoven, 

cylindrical, 3–7 µm wide hyphae. Clamp-connections absent. 

Habitat and distribution: Terrestrial, on clayey soil, in the litter 

of a Pinus halepensis plantation, in a park with considerable 

public pressure. Basidiomes produced in winter (December). 

Known only from the province of Aragón, Spain, at this time. 

Additional collections examined: Spain: Aragón: Zaragoza, “José 

Antonio Labordeta” (antea: “Parque Grande”) park, N 41.634 W 

0.894, alt. 225 m, near Pinus halepensis, on argillose-sandy soil, 


121

Muñoz et al. 

ARTICLE 

basidiomata nearly covered by the substrate, 3 Dec. 2011, G. Muñoz 

(GM-2454); ibid., in closeby neighbourhoods, on soil, 10 Dec. 2011, 

G. Muñoz (GM-2485, GM-2486). 

DISCUSSION 

According to morphological data and phylogenetic analyses 

of ITS sequences (Fig. 1), the collections studied merit 

recognition as an independent species within Leucoagaricus 

sect. Piloselli. No similar species could be found in the literature 

since all the previously described species are distinguished 

by different tinges in the pileus, the absence of pyriform to 

spheropedunculate cheilocystidia, or more elongated and 

differently shaped spores (Candusso & Lanzoni 1990, Bon 

1993, Caballero 1997, Gennari & Migliozzi 1999, Migliozzi & 

Resta 2001, Migliozzi et al. 2001, Vellinga 2001, 2006, 2010). 

Among the macromorphologically most similar species, 

Lepiota decorata (Leucoagaricus idae-fragum fide Vellinga 

2006), known from the western parts of North America 

(California and Oregon) and the western parts of France 

(Atlantic coast), differs in the rose-vinaceous purple, raspberry 

deep pink overall colours from the first, narrowly clavate, 

cylindrical, to slightly utriform cheilocystidia, and ellipsoid to 

amygdaliform spores (Guinberteau et al. 1998, Vellinga 2006). 

Leucoagaricus cupresseus, known from under Cupressaceae 

in California, and the Atlantic and Mediterranean coasts 

of France, differs in the ellipsoid to oblong, amygdaliform 

spores with a faint papilla, and variably sized and shaped 

cheilocystidia (clavate, fusiform-clavate, lageniform-utriform, 

to cylindrical) (Sundberg 1976, Boisselet & Guinberteau 

2001, Vellinga 2010). Finally, L. pseudopilatianus and its 

varieties, which occur in Spain and Italy, is distinguished 

by the red-brownish pileus, amygdaliform spores with an 

indistinct apical papilla, broader clavate cheilocystidia with 

evident brownish contents, a pileipellis with a subhymeniform 

basal layer, terminal elements of the pileipellis with rounded 

(not attenuated) tips, and basidiomes turning black on drying 

(Migliozzi & Resta 2001, Migliozzi et al. 2001); according to 

Vellinga (2010), that species could prove to be identical to L. 

cupresseus. 

ACKNOWLEDGEMENTS 

Fernando Esteve-Raventós (Madrid) is thanked for facilitating the 

deposit of the holotype in the collections of the Universidad de Alcalá 

(AH) as well as for his advice. Our most sincere thanks are also due to 

Else C. Vellinga (Berkeley, CA) for her opinion on this species, Enrico 

Ercole (Turin) for technical support, and Peter Lee Heesacker (Olbia, 

Italy) and Caroline Hobart (Sheffield) for improving the English text. 

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124 ima fUNGUS

doi:10.5598/imafungus.2012.03.02.04 


Ascus apical apparatus and ascospore characters in Xylariaceae 

Nuttika Suwannasai 1 , Margaret A. Whalley 2 , Anthony J.S. Whalley 2 , Surang Thienhirun 3 , and Prakitsin Sihanonth 2 

1 

Department of Biology (Microbiology), Faculty of Science, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok, 10110, Thailand; 

corresponding author e-mail: snuttika@hotmail.com 

2 

Department of Microbiology, Faculty of Science Chulalongkorn University, Bangkok, Thailand 

3 

Forest Products Research Division Royal Forest Department, Chatuchak, Bangkok, 10900, Thailand 

ARTICLE 

Abstract: Members of Xylariaceae (Ascomycota) are recognized and classified mainly on the morphological features 

of their sexual state. In a number of genera high morphological variation of stromatal characters has made confident 

recognition of generic and specific boundaries difficult. There are, however, a range of microscopical characteristics 

which can in most cases make distinctions, especially at generic level, even in the absence of molecular data. These 

include details of the apical apparatus in the ascus (e.g. disc-shaped, inverted hat-shaped, rhomboid, composed 

of rings, amyloid, non-amyloid); position and length of the germ slit; and presence and type of ascospore wall 

ornamentation as seen by scanning electron microscopy (SEM). Unfortunately many of the classical studies on 

xylariaceous genera omitted these features and were undertaken long before the development of scanning electron 

microscopy. More recent studies have, however, demonstrated their value as diagnostic characters in the family. 

Camillea is for example, instantly recognizable by its rhomboid or diamond shaped apical apparatus, and the 

distinctive inverted hat or urniform type is usually prominent in Xylaria, Rosellinia, Kretzschmaria, and Nemania. At 

least six categories of apical apparatus based on shape and size can be recognized. Ascospore ornamentation as 

seen by SEM has been exceptionally useful and provided the basis for separating Camillea from Biscogniauxia and 

other xylariaceous genera. 

Key words: 

Ascomycota 

ascospores 

iodine reaction 

scanning electron 

microscopy 

systematics 

Xylariales 

Article info: Submitted: 5 July 2012; Accepted: 11 October 2012; Published: 7 November 2012. 

INTRODUCTION 

Xylariaceae is one of the best-known and widely distributed 

families of Ascomycota. The majority of the species are 

wood inhabitants, and are particularly well represented in the 

tropics. Ju & Rogers (1996) recognized 38 genera, Whalley 

(1996) 40, and the number has grown to at least 76 (Lumbsch 

& Huhndorf 2010), although the total varies according to 

individual opinion and the status of several genera in the 

family awaits confirmation. The separation of genera and 

subsequent identification of taxa has been problematic 

mainly as a result of diversity of form and variation in many 

morphological characteristics (Whalley 1996, Rogers 2000). 

Genera within Xylariaceae were traditionally recognized on 

the basis of stromal form, stromal colour, and ascospore 

shape and dimensions (Fig. 1). As a consequence other 

important taxonomic features were neglected (Rogers 1979, 

Whalley 1996). Details of the ascus, including the apical 

apparatus, and ascospore topography were not considered. 

The subsequent advent of scanning electron microscopy 

(SEM) has demonstrated the value of spore ornamentation 

and details of stromatal surfaces (Læssøe et al. 1989, Whalley 

1996). In this paper we assess the importance of these 

characteristics based on our experience and extrapolations 

from recent publications. 

METHODS 

Squash preparations of asci and ascospores mounted in 

water, Melzer’s iodine reagent, and lactophenol cotton blue 

were microscopically examined by bright field microscopy 

and differential interference contrast (DIC) light microscopy 

with an Olympus BH2 research microscope using x10, 

x40 and x60 dry objectives. Images were captured by 

Camera (INFINITY 1) and were analyzed by Infinity Analyze 

software provided with measurement functions and image 

enhancement options. For examination by SEM, small 

sections of dried stromata were mounted using Electrodag 

high conductivity paint (Acheson Colloids Company) on a 

1cm diam aluminium stub. Additionally perithecial contents 

were Åspread on the surface of stubs. The specimens were 

sputter-coated with a film of gold approximately 500 Å thick in 

an Emitech K550X coating unit. The coated specimens were 

then loaded into a FEI (Quanta 200) ESEM (Environmental 

Scanning Electron Microscopy, 2008) and examined over a 

range of magnifications at an accelerating voltage of 5kV. 

Images for all methods were obtained using an image capture 

system (Oxford Instruments, INCA system, Oxford, UK). 



Attribution: 







Suwannasai et al. 

ARTICLE 

Fig. 1. Stromatal characteristics of some xylariaceous fungi. A. Daldinia eschscholzii (SUT 039) B. Biscogniauxia capnodes (SUT 212) C. 

Hypoxylon monticulosum (SUT 189). D. Rhopalostroma lekae (PK 148). Kretzschmaria clavus (PK 270). F. Annulohypoxylon bovei var. 

microspora (SUT 025). G. Rosellinia procera (SUT113). H. Astrocystis mirabilis (SUT 051). I. Xylaria sp.(PK 017). J. X. cubensis (PK 108). K. 

X. magnoliae var. microspora (PH 072). L. X. allantoidea (PK 088). Bars A–B, I–L = 1 cm; C, F–H = 5 mm; D–E = 2 mm. 

* Collection abbreviations: AJSW = Liverpool John Moores 

University, UK; SUT = Suranaree University of Technology Nakhon 

Ratchasima, Thailand; ST Royal Forest Department, Bangkok, 

Thailand; SWU Srinakharinwirot University, Bangkok, Thailand – 

incorporating collections from national parks and forests of Thailand 

H (Khao Kra Yang Plantation, Phitsanulok Province), PK (Phu Kheio 

Wildlife Sanctuary, Chaiyaphum Province), and PH (Phu Hin Rong 

Kla National Park, Phitsanulok Province). 

RESULTS AND DISCUSSION 

In most of the currently recognized genera of Xylariaceae 

the asci contain eight spores. Exceptions include Wawelia, 

with 4-spored asci (Minter & Webster 1983, Lundqvist 1992) 

and Thuemenella with 6-spored asci (Samuels & Rossman 

1992). In general, the xylariaceous ascus is cylindrical and 

possesses a stipe. In Biscogniauxia the stipe is frequently 

short in relation to the spore-containing part of the ascus, 

126 ima fUNGUS


ARTICLE 

Fig. 2. Asci and different types of apical apparatus. A. Hypoxylon fuscum with disc-like apical apparatus stained in Melzer’s reagent (AJSW 

078*). B. Camillea selangorensis ascus (IMI – isotype). C. Kretzschmaria clavus ascus with apical apparatus stained in Melzer’s reagent (PK 

270). D. Nemania bipapillata ascus with stipe (AJSW 693). E. K. clavus showing distinctive urniform apical apparatus stained dark blue in 

Melzer’s reagent (PK 270). F. C. fusiformis with rhomboid apical apparatus stained in Melzer’s reagent (MAW S21, IMI) G. Hypoxylon lividicolor 

ascus with long stipe (ST 1047 RFD). H. Xylaria aristata ascus with apical apparatus arrowed (ST 1411 RFD). Bars A–B, F–H = 10 µm; C–D = 

25 µm; E = 5 µm. 


127


ARTICLE 

whilst in Xylaria and Kretzschmaria the stipes are usually 

long. Hypoxylon begae, H. haematostroma and H. polyporum 

are notable within the genus for their very long stipes which 

appear to have diagnostic value (Ju & Rogers 1996). The 

apical tip of the ascus is usually rounded and encloses an 

apical apparatus which is mostly amyloid, staining blue 

in Melzer’s iodine reagent. There are a number of taxa in 

which no apical apparatus can be seen by light microscopy 

although the possibility of some remnant structures cannot 

be excluded as such taxa have not yet been studied by 

transmission electron microscopy. The shape and size of 

the apical apparatus is one of the more important taxonomic 

features exhibited in Xylariaceae (Fig. 2). The general 

appearance of the apical apparatus has been successfully 

applied in taxonomic studies of the family (e.g. Munk 1957, 

Carroll 1963, 1964, Martin 1967, 1968a, b, 1969a, b, Krug 

& Cain 1974a, b, Francis 1975, Rogers 1979, Læssøe et 

al. 1989, van der Gucht 1995, Ju & Rogers 1996, Whalley 

1996). Unfortunately, a number of important taxonomic 

studies in the family have not considered this feature. On the 

basis of shape and size, at least five types of amyloid apical 

apparatus can be recognized plus a category in which there 

is no visible apparatus: 

1) Stacks of small rings, as in Hypocopra and Poronia 

(Krug & Cain 1974b, Jong & Rogers 1969). 

2) Discoid or triangular, as in most species of Hypoxylon 

s. str. and Daldinia (Ju & Rogers 1996, Ju et al. 

1997). 

3) Broad band to discoid, as in Biscogniauxia (Ju et al. 

1998). 

4) Rhomboid to diamond-shaped in Camillea (Læssøe 

et al. 1989). 

5) Inverted hat or urniform, as in Xylaria, Rosellinia, 

Kretzschmaria and Nemania (Petrini & Muller 1986, 

Whalley 1996, Rogers 2000). 

6) No visible apical apparatus under the light microscope 

as in Rhopalostroma and most species of Ascotricha 

(Whalley & Thienhirun 1996, Hawksworth 1971) 

In most species the apical apparatus stains blue, usually 

dark blue, or occasionally reddish brown (dextrinoid) in 

Melzer’s iodine reagent. The significance of the iodine 

reaction in the apical apparatus, including Xylariaceae has 

been discussed by Eriksson (1966), Kohn & Korf (1975), and 

Nannfeldt (1976). It has been shown that pre-treatment with 

potassium hydroxide (KOH) can induce a positive reaction in 

a previously iodine negative species (Nannfeldt 1976). Baral 

(1987) has questioned the effectiveness of Melzer’s reagent 

demonstrating that Lugol’s solution is superior in the detection 

of amyloidity in ascomycetes. Species of Xylariaceae can, 

however, be grouped according to the response of their 

apical apparatus to Melzer’s reagent as: 

7) Apical apparatus consistently iodine positive (blue). 

8) Apical apparatus varying in its reaction to iodine, i.e. 

some collections give a positive amyloid reaction 

whilst other collections of the same species do not, 

as in Hypoxylon cohaerens and Nemania serpens 

(Pouzar 1985a, b, Petrini & Rogers 1986). 

9) Apical apparatus consistently iodine-negative, as in 

Hypoxylon intermedium and H. cercidicola (Pouzar 

1972, Ju & Rogers 1996). 

The iodine positive nature of the apical apparatus is 

considered, however, to be a cardinal character of the 

Xylariaceae in spite of the presence of certain iodine negative 

taxa in what are undoubted taxa of the Xylariaceae (Rogers 

1979, 1994, 2000). 

The structure of the apical apparatus appears to be 

relatively simple when studied by transmission electron 

microscopy (Greenhalgh & Evans 1967, Beckett & Crawford 

1973, Griffiths 1973). Chadefaud (1942, 1973) proposed 

a much more complex structure on the basis of light 

microscopicy, but many of his studies were carried out on 

old material with degenerating asci which might also be the 

case here. Regardless of structure or reaction to iodine, the 

function of the apical apparatus is not clear. Greenhalgh & 

Evans (1967) and Beckett & Crawford (1973) considered 

the apical apparatus to act as a sphincter through which the 

ascospores pass. Martin (1967a), however, was of the opinion 

that the ascospores bypass the apical apparatus during 

discharge and that the function of the apical apparatus was 

therefore unclear. Rogers (1979) suggested that the apical 

apparatus served as a strengthening device in the ascus and 

that it becomes everted, pushed to one side, or blown off 

by the ascospores once sufficient pressure has developed 

in the ascus. Certainly, the dimensions and shapes of many 

ascospores are not suited for passage through the central 

channel in the apical apparatus and the suggestion of Rogers 

(1979) is currently the most plausible. In a study of Barron’s 

strain of Nemania serpens which unusually produces mature 

stromata in culture, Kenerley & Rogers (1976) demonstrated 

that the ascospores were passively discharged under wet 

conditions, but forcibly discharged under dry conditions. 

The ascospores of most xylariaceous fungi are described 

as more or less bean-shaped (phaseoliform), single-celled, 

smooth walled, light to dark brown, and with a conspicuous 

germ slit usually running the full length of the spore (Rogers 

1979). In reality, there is considerable variation on this basic 

theme (Fig. 2). In most species the ascopores are uniseriate 

in their arrangement in the ascus, but variation occurs in 

relation to their shape. The basic shape is ellipsoid, but this 

can become subglobose, oblong, fusiform, inequilaterally 

ellipsoid, navicular or broadly crescent-shaped. The ends 

can be narrowly or broadly rounded, attenuated, or apiculate. 

In Biscogniauxia species, which possess appendages, the 

loss of an appendage results in a truncate end (Whalley et 

al. 1990). In Hypoxylon s. str. and Daldinia the spores are 

usually inequilaterally ellipsoid, in Biscogniauxia they are 

more frequently subglobose, in Xylaria they are often broadly 

crescent-shaped, and in Rosellinia many are characterized 

by long attenuated ends (Petrini 1992). Most xylariaceous 

spores are brown, but range from light to medium or dark 

brown, sometimes appearing almost black. In Camillea, 

however, the spores are pale yellow or almost colourless, 

and almost all of them lack germ slits or pores, except for C. 

labiatrima which have a distinct slit (Rogers et al. 2002), and 

are ornamented. Their very pale colour, lack of a germ slit 

and presence of spore wall ornamentation, as observed by 

scanning electron microscopy, drew attention to the incorrect 

placement of many applanate species in Hypoxylon, which 

were subsequently transferred to Camillea (Rogers 1977, 

Læssøe et al. 1989). Thus, the genus Camillea is partially 

128 ima fUNGUS


ARTICLE 

Fig. 3. Variation in ascospore shape and germ slits. A. Rosellinia bunodes with elongated ascospore ends (AJSW 937). B. Entoleuca mammata 

with broadly rounded ascospores (AJSW 803). C. Biscogniauxia nummularia with broadly rounded ascospores. (AJSW 236). D. Hypoxylon 

comedens with straight germ slits 2/3 length of the spore (ST 1142 RFD). E. Xylaria longipes with spiral germ slit. (AJSW 576). F. H. monticulosum 

with spiral germ slits (SUT 189). G. Rhopalostroma kanyae with germ slit on the dorsal side of the ascospore (IMI 368200 – isotype). H. 

Biscogniauxia anceps showing bicelled spores and germ slit (AJSW 1009) I. H. fuscum showing dehiscent perispore following treatment with 10 

% KOH (AJSW 078). J. Kretzschmaria clavus with straight germ slit almost full length of the ascospore (PK 270). A–B, D–G scanning electron 

micrographs. C, H–J bright field light microscopy. Bars A–B, D, G–H = 10 µm; C, E = 2 µm; F = 1 µm; I–J = 15 µm. 


129


ARTICLE 

Fig. 4. Ascospore ornamentation. A. Camillea fusiformis longitudinal reticulate. (MAW S21, IMI). B. C. tinctor poroid (SUT 260). C. C. fusiformis 

details of reticulate ornamentation (MAW S21, IMI).D. C. selangorensis verrucose (IMI). E. Nemania chestersii longitudinal ribbed (AJSW433). 

F. C. selangorensis faint ornamentation by light microscopy (IMI). G. Daldinia eschscholzii transverse (SUT 039). H. C. cyclops poroid 

(MAW S18) A–E, G–H scanning electron micrographs. F, bright field light microscopy. Bars A–B, F = 10 µm; C–D, G–H = 1 µm; E = 2 µm. 

130 ima fUNGUS


circumscribed on the basis of ascospore wall ornamentation 

which may be poroid, reticulate, or ribbed (Camillea subgen. 

Camillea), or echinulate to verrucose (Camillea subgen. 

Jongiella) (Læssøe et al. 1989, Rogers et al. 1991, Whalley 

1995, 1996, Whalley et al. 1996, 1999). 

Most xylariaceous ascospores are smooth walled, but 

ornamentation occurs spasmodically throughout the family 

(Figs 3–4). Thus, Stromatoneurospora possesses striate 

ascospores (Jong & Davis 1973), and some species of 

Hypoxylon s. str. have ascospores with faint transverse 

striations perpendicular to the long axis of the spore (Rogers 

& Candoussau 1982, Rogers 1985, van der Gucht & van 

der Veken 1992, Ju & Rogers 1996). Van der Gucht (1993) 

and Stadler et al. (2002) emphasized the significance of 

transverse striations of the ascospores in certain species of 

Daldinia. A single species of Biscogniauxia, B. reticulospora, 

exhibits reticulately ornamented ascospores (Ju et al. 1998), 

and the genera Helicogermslita and Spirodecospora were 

erected mainly on the presence of a spiral ornamentation on 

the ascospores (Hawksworth & Lodha 1983, Lu et al. 1998). 

In their revision of Hypoxylon, Ju & Rogers (1996) placed 

considerable importance on ascospore ornamentation, 

noting that it can be found on the perispore, epispore, 

and/or beneath the epispore. Perispore ornamentation 

is evident in those taxa where perispores dehisce in 10 

% potassium hydroxide. The ornamentation falls into two 

major patterns, which Ju & Rogers (1996) used as one of 

the three major characters to delimit the two sections of 

Hypoxylon. Transversely orientated, coil-like ornamentation 

can be found in sect. Hypoxylon, whereas a thickening of 

the perispore situated towards one end is almost universal in 

sect. Annulata (Ju & Roger 1996). It was also recognized that 

the conspicuousness of the coil-like ornamentation in sect. 

Hypoxylon is an important character at species level. This 

feature is useful in the separation of closely related taxa such 

as H. anthochroum, H. duranii, H. fendleri, and H. retpela (Ju 

& Rogers 1996). Epispore ornamentation appears to be rare 

in Hypoxylon, but shallow pits can be found in H. rubellum 

(Rogers et al. 1987), striations in H. californicum (Ju & Rogers 

1996), and pleated folds in H. rectangulosporum (Rogers 

et al. 1992) and H. thouarsianum (Miller 1961). Transverse 

striations are also apparent in some Daldinia species (van 

der Gucht 1993, Stadler et al. 2002). Stadler et al. (2002) 

examined representative specimens of Daldinia species 

with the SEM and found that ornamentation of their outer 

spore layers were species-consistent. They reported them 

as having either smooth or transversely striated ascospores, 

with the striated spores always ellipsoid-equilateral to 

ellipsoid-inequilateral with narrowly rounded ends. Smooth 

ascospores were broadly ellipsoid to cylindrical. Daldinia 

concentrica was found to have very faint ornamentation, but 

this was only visible at ×1000 in an SEM. Ju et al. (1997) 

had previously found that ascospores of some species of 

Daldinia undergo perispore dehiscence in 10 % potassium 

hydroxide and have ornamentation similar to that exhibited 

by members of Hypoxylon sect. Hypoxylon. In H. fragiforme a 

shedding or eclosion, likened to the hatching of insect pupae, 

of the perispore in response to specific chemical stimuli 

has been interpreted as part of an intricate fungus-host 

recognition system (Chapela et al. 1990, 1991). Whether 

this phenomenon occurs in other Hypoxylon species or 

indeed in other xylariaceous taxa has not been tested. In the 

coprophilous genera Poronia, Podosordaria, and Hypocopra, 

the ascospores are usually surrounded by thick gelatinous 

sheaths which are assumed to facilitate the spores adhering 

to plant materials, mainly leaf lamina (Rogers 1979). 

Details of the asci and ascospores, in conjunction with 

features of any asexual stages (Ju & Rogers 1996), have 

proved to be valuable in making identifications, and they also 

provide insights into species groups and generic separations. 

However, knowledge on the distribution and patterns of 

extrolite chemicals in Xylariaceae and application of DNA 

technology has been pivotal in resolving boundary issues 

(Whalley & Edwards 1995, Stadler & Hellwig 2005, Triebel 

et al. 2005). 

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from Thailand. Mycological Research 100: 866–868. 

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doi:10.5598/imafungus.2012.03.02.05 


A new species of the lenticel fungal genus Claviradulomyces (Ostropales) 

from the Brazilian Atlantic forest tree Xylopia sericea (Annonaceae) 

Robert W. Barreto 1 , Peter R. Johnston 2 , Pedro W. Crous 3 , and Harry C. Evans 1, 4 

1 

Departamento de Fitopatologia, Universidade Federal de Viçosa, 36750 Viçosa, Minas Gerais, Brazil; corresponding author email: rbarreto@ 

ufv.br 

2 

Landcare Research, Private Bag 92170, Auckland 1142, New Zealand 

3 

CBS-KNAW Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD, Utrecht, The Netherlands 

4 

CAB International, E-UK Centre, Egham, Surrey TW20 9TY, UK 

ARTICLE 

Abstract: Claviradulomyces xylopiae sp. nov. is introduced for a fungus occurring in association with abnormal 

(enlarged, spongy) lenticels of Xylopia sericea (Annonaceae), a common tree of the Atlantic forest and Cerrado 

ecosystems in Brazil. This is the second species described in the genus and, although it is morphologically distinct 

from the type species, C. dabeicola from West Africa, it possesses the same characteristics. Apothecial ascomata 

have periphysoids and paraphyses that are inflated apically (clavate), and ornamented with denticles (raduliform). 

Furthermore, similar to the type species, it also has long-cylindric or acerose, aseptate ascospores and conidia. An 

additional asexual morph was produced in culture and is described. Molecular studies of C. dabeicola and the new 

species confirmed a placement in Ostropales, although a relationship to Odontotremataceae was not supported. 

Both species were consistently in association with abnormal lenticular development on their woody hosts. It remains 

to be ascertained, however, if these are the causal agents of the bark disorders, or, simply, opportunistic colonisers. 

The finding of the second species in the genus Claviradulomyces on a plant from a distantly related family to that 

of the host of C. dabeicola (Erythroxylaceae) for the genus on a different continent suggests that fungi in this genus 

may be common on lenticels of other woody plants, and could even have a pantropical distribution. It is possible 

that fungi in the genus have remained unreported until now because lenticels have remained neglected as a habitat 

surveyed by mycologists. 

Key words: 

Ascomycota 

mycobiota 

Ostropales 

phylogeny 

plant disease 

taxonomy 

Article info: Submitted: 25 September 2012; Accepted: 3 November 2012; Published: 15 November 2012. 

Introduction 

Xylopia sericea (Annonaceae) is a fast-growing native tree of 

the Brazilian Atlantic forest and Cerrado ecosystems (Lorenzi 

1992), known locally as pimenteiro. During a collection of 

entomopathogenic fungi associated with armoured scale 

insects in the canopy of X. sericea (Fig. 1A), a high proportion 

of the pruned branches showed abnormal development 

of lenticels: the latter appearing as prominent eruptions 

along the branches (Fig. 1B–D). A discomycete fungus was 

observed consistently colonising the lenticular tissues, which 

was identified provisionally as similar to Claviradulomyces 

dabeicola (Evans et al. 2010). On closer inspection, 

morphological differences were found that distinguished 

the fungus on X. sericea from C. dabeicola. The fungus 

was isolated into pure culture, from the sexual morph and 

from the purported asexual morph, allowing for molecular 

data to be generated to confirm the sexual relationship. A 

detailed description of the new species and a discussion 

of Claviradulomyces phylogeny and its placement within 

Ostropales are presented. 

Materials and methods 

Isolates and morphology 

Stems bearing pronouncedly developed lenticels were 

collected from the canopy of Xylopia sericea with the aid of 

a pruning pole (Fig. 1) from two sites in the municipality of 

Viçosa (state of Minas Gerais, Brazil): at the edge of a well 

preserved stretch of Atlantic rainforest,and a roadside stand 

adjacent to farmland. Samples were air-dried and specimens 

were deposited in the collections of the Universidade Federal 

de Viçosa (VIC) and of the CBS-KNAW Fungal Biodiversity 

Centre Herbarium (CBS). 

Isolations were performed either by transferring 

sporocarps to sterile distilled water agar (DWA), breaking 

them open with fine forceps, and streaking the spores across 

the agar surface to await germination, when germinating 

spores (ascospores, conidia) were selected with the aid of 

a sterile fine-pointed needle under a stereo-microscope with 

transmitted light and placed on potato carrot agar (PCA), 

or by direct transfer of ascomata or pycnidia onto plates 



Attribution: 







Barreto et al. 

ARTICLE 

Fig. 1. A. Collecting on Xylopia sericea at type locality of Claviradulomyces xylopiae – margin of Atlantic forest, Mata do Seu Nico, Fazenda 

Bonsucesso, Viçosa, state of Minas Gerais Brazil. B–D. Close-up of bark of X. sericea colonised by C. xylopiae showing hypertrophied lenticels 

bearing bearing apothecial ascomata, showing fully opened habit (C–D) after 24 h in a humid chamber. Bars: C = 0.5 cm; D = 1 cm. 

containing vegetable broth agar (VBA), as described in 

Pereira et al. (2003). Representative cultures were deposited 

in the CBS-KNAW Fungal Biodiversity culture collection. 

Colony characters were noted on malt extract agar (MEA) 

and PCA, either in the dark or with a 12 h light/ 12 h dark 

regime, at 25 ºC. Colony colour was assessed according to 

Rayner (1970). Morphological observations were made in 

lactic acid or lacto-fuchsin from hand sections of sporocarps 

or those teased from the lenticels and macerated. 

DNA isolation, amplification and analyses 

Genomic DNA was isolated from fungal mycelium grown 

on MEA, using the UltraCleanTM Microbial DNA Isolation 

Kit (MoBio Laboratories, Solana Beach, CA) according to 

the manufacturer’s protocols. The primers V9G (de Hoog 

& Gerrits van den Ende 1998) and LR5 (Vilgalys & Hester 

1990) were used to amplify part of the nuclear rDNA operon 

spanning the 3’ end of the 18S rRNA (SSU), ITS1, 5.8S rRNA 

gene, ITS2 and the first 900 bases at the 5’ end of the 28S 

rRNA (LSU) genes. The primers ITS4 (White et al. 1990) and 

LSU1Fd (Crous et al. 2009a) were used as internal sequence 

primers to ensure good quality sequences over the entire 

length of the amplicon. The PCR conditions followed the 

methods of Crous et al. (2006, 2009b). 

Sequences were compared with those from 

Claviradulomyces dabeicola (Evans et al. 2010) and from the 

taxa treated by Baloch et al. (2010). LSU and mtSSU sequences 

from the two Claviradulomyces spp. were concatenated and 

incorporated into the alignments of Baloch et al. (2010) using 

Geneious (Drummond et al. 2011). Data were analysed with 

Bayesian phylogenetic methods using MrBayes v. 3.1.2 

(Huelsenbeck & Ronquist 2001; Ronquist & Huelsenbeck 2003), 

with gaps treated as missing data, applying the GTR+I+G model 

for both genes, the models selected using the AIC method in 

MrModelTest v. 2.3 (Nylander 2004). The data set was run 

with two chains for 10 M generations, and trees sampled every 

1000 generations. Convergence of all parameters was checked 

using the internal diagnostics of the standard deviation of split 

frequencies and performance scale reduction factors (PSRF), 

and then externally with Tracer v. 1.5 (Rambaut & Drummond 

2007). On this basis, the first 25 % of generations were discarded 

as burnin. Bayesian posterior probabilities were obtained from 

50 % majority rule consensus trees. 

RESULTS 

Taxonomy 

Claviradulomyces xylopiae R.W. Barreto, H.C. 

Evans, P.R. Johnst., sp. nov. 


(Figs 1–5) 

Etymology: derived from Xylopia, the generic name of the 

host plant. 

136 ima fUNGUS

Claviradulomyces xylopiae sp. nov. from Xylopia sericea 

ARTICLE 

Fig. 2. Claviradulomyces xylopiae asexual morph (VIC 31417 mounted in lactofuchsin). A. Pycnidium with long rostrate ostiole. B, C. Conidia 

[note subtle heel at base of conidium in C (arrowed)]. D. Group of immature conidia attached to conidiogenous cells. Bars: A = 50 µm; B = 15 

µm; C = 5 µm; D = 10 µm, 

Fig. 3. Claviradulomyces xylopiae sexual morph (VIC 31417 mounted in lactic acid-cotton blue). A. Cross section of fully opened, apothecial 

ascoma (note group of denticulate periphysoids at the margins of apothecium). B. Close-up of periphysoids. C. Hymenium with parallel asci 

and paraphyses. D. Muricate and denticulate paraphyses extending above the top of asci. Bars: A = 15 µm; B = 10 µm; C = 10 µm; D = 15 µm. 


137


ARTICLE 

Fig. 4. Claviradulomyces xylopiae asci and ascospores (VIC 31417 mounted in lactofuchsin). A. Mature asci containing parallel to somewhat 

spirally arranged ascospores. B. Single vermiform ascospore within ascus. C, D. Ascospores. Bars: A = 40 µm; B = 35 µm; C = 10 µm; D = 10 µm. 

Diagnosis: Similar to Clavariadulomyces dabeicola, from 

which it is distinguished by the longer periphysoids (16–39 

µm, shorter asci (35–50 µm), a longer ostiolar neck in the 

asexual state, shorter conidia (14–34.5 µm) with a discrete 

heel region, and molecular sequence data. 

Type: Brazil: Minas Gerais: Viçosa, Piúna, on living branches 

of Xylopia sericea (Annonaceae), 11 June 2010, R.W. Barreto 

(VIC31417 — holotype; CBS 133260 and CBS 133261 – exholotype 

cultures). 

Paratype: Brazil: Minas Gerais: Viçosa, “Mata do Seu Nico”, 

Fazenda Bonsucesso, on living branches of Xylopia sericea, 

20 May 2010, H.C. Evans & R.W. Barreto (VIC 31416). 

Description: Internal mycelium intra- and intercellular, 1–2 

µm diam, septate, branched, hyaline. Ascomata erumpent 

from spongy tissues of lenticels on bark of living branches; 

apothecial when mature and turgid, but perithecium-like and 

opening by a large, round pore when dry; sessile, urceolate, 

0.12–0.25 mm diam, wall black, extending above the surface 

of the hymenium, partially covering the hymenium when dry, 

opening pore lined with a whitish fringe of periphysoids. In 

vertical section, lower part of ascomatal wall often ill-formed 

and restricted to a loose hyphal layer from which asci and 

paraphyses arise, 5–15 µm thick, composed of tangled 

hyphae 1–2 µm diam; upper part of wall dark brown to 37 

µm thick, narrowing and becoming paler towards the base 

and there 10–12 µm wide. Periphysoids lining the upper 

wall above the level of the hymenium, cylindrical or clubshaped, 

sinuose or curved, 16–39 µm long and 3–4 µm 

wide along the axis, often slightly swollen in the upper 

part up to 6 µm, wall hyaline along most of the length, 

bearing abundant blunt denticles, arising from short brown 

smooth basal stalks. Paraphyses 1–2 µm wide, to 60 

µm long, apex swollen and bulbous, to 5–7 µm wide and 

bearing abundant blunt denticles, imparting a mace-like 

or muricate appearance, extending beyond the asci but 

usually prostrate over the hymenial surface. Asci parallel, 

clavate with a broadly rounded to somewhat flattened apex, 

becoming ellipsoidal when free of the hymenium, without a 

basal stalk, 35–50 × 5–10 µm, apex non-amyloid, 8-spored. 

Ascospores in single fascicles extending to the base of the 

ascus, parallel to spirally or partly spirally arranged in the 

upper half, cylindrical to vermiform, attenuating towards 

the sub-acute ends, straight to slightly curved or sigmoid, 

sometimes strongly curved at the apices, (21–) 28–43 × 2–3 

µm, aseptate, hyaline, smooth, strongly guttulate. Asexual 

morph: formed separately or in combination with the sexual 

morph in the same lenticel. Conidiomata pycnidial, semiimmersed, 

globose, 76–137 µm diam; thin walled, walls 4–15 

µm thick, with long cylindrical ostiolate necks, 110–360 × 

24–46 µm, composed of parallel hyphae 1–3 µm wide, often 

reduced to a narrower cylinder of bristle-like hyphal tips at 

the apex, 15–21 ×15 µm. Conidiophores usually reduced to 

conidiogenous cells, occasionally consisting of a small stalk 

138 ima fUNGUS


ARTICLE 

Fig. 5. Claviradulomyces xylopiae in culture . A. Colony formed on PCA under 12 h daily light regime. B. Colony formed on MEA under 12 h 

daily light regime. C. Close-up of margin of colony formed on PCA (Note groups of black pycnidia, isolated and in groups). D. Close-up of colony 

formed on MEA (Note mucilaginous mass of conidia oozing from black spermogonia). Bars = 2 mm. 

of one or two cells. Conidiogenous cells lining the pycnidial 

wall, holoblastic, seemingly monoblastic, subcylindrical, 

lageniform or oblong, straight or curved, solitary, occasionally 

branched, 4.5–12.5 × 1.5–2 µm, hyaline, smooth. Conidia 

probably mucilaginous, acerose to narrowly cymbiform, 

mostly straight or slightly curved or sigmoid, attenuated 

towards a basal subtle heel continuing as a short cylindrical 

penducle and ending in a rounded base, aseptate, guttulate, 

hyaline, smooth, 14–34.5 × 2–3.5 µm. 

Cultures: Slow growing, to 76 mm diam after 23 d, either 

totally immersed or flat to slightly raised centrally, with 

radiating grooves of compressed medium, immersed at 

periphery; felt-like or entirely slimy centrally, comprising a 

dense, rosy vinaceous mat of dark brown monilioid hyphae 

within a pale hyphal matrix; embedded, black setose pycnidia 

densely formed on PCA in light, producing a white-creamy 

ooze of cylindrical to oval hyaline conidia (2−4 × 1.5−2 µm) 

and oblong hyaline spermatia (1.5 × 1 µm); pycnidia fewer 

and sterile in the dark. 

Phylogenetic analysis 

The sequences generated from both collections were 

identical (GenBank accession numbers ITS JX843524; LSU 

JX843525; SSU JX843526). The two Claviradulomyces spp. 

formed a strongly supported clade within the Ostropales, but 

the family level relationship within the order was not resolved 

(Fig. 6). 

Discussion 

Claviradulomyces xylopiae represents a novel species on an 

indigenous Brazilian plant distantly related to Erythroxylum 

mannii, the host of the type species, C. dabeicola, in West 

Africa (Evans et al. 2010). This suggests that fungi in this 

genus could have a pantropical distribution, perhaps as 

endophytic colonisers of tropical woody plants, with the 

ability to sporulate on the lenticular tissues of living plants. 

This somewhat cryptic niche has not traditionally been 

explored by mycologists, and this could explain the absence 

of previous records of this genus. However, it remains to be 

proven whether or not these fungi are benign endophytes, or 

acting as systemic pathogens that promote abnormal lenticel 

growth to facilitate sporulation, or opportunistic invaders of 

trees with bark disorders. 

Claviradulomyces xylopiae has considerable similarity 

to C. dabeicola,and has the same muricate periphysoids, 

which characterise the genus (Evans et al. 2010). It can, 

however, be separated from the type species by the longer 

periphysoids, 16–39 µm compared to 6.5–14 µm, and shorter 

asci, 35–50 µm compared with 60–75 µm. Nevertheless, the 

most significant morphological divergences are to be found in 

the asexual state of the two species. The ostiolar neck in the 

new species is long, sometimes reaching up to three times 

the length of the pycnidial body, whereas in C. dabeicola the 

neck is reduced to a short protrusion. Conidia in C. xylopiae 

are also shorter, 14–34.5 µm in length, than in C. dabeicola 


139


ARTICLE 

Fig. 6. Bayesian analysis (50 % majority rule consensus tree) of nucLSU and mtSSU sequences. Bayesian posterior probabilities are shown 

where above 90 %. Sequences for all taxa except Claviradulomyces dabeicola (GenBank records records GQ337897, GQ337900) and C. 

xylopiae (GenBank LSU JX843525; SSU JX843526) are from Baloch et al. (2010). The clade labels follow Baloch et al. (2010). 

where they measure 33–42 µm, and also have a discrete 

heel region not seen in C. dabeicola. 

Evans et al. (2010) referred Claviradulomyces to 

Odontotremataceae based on morphology. At that time, no 

DNA sequences of Odontotrematcaeae had been published. 

A subsequent comprehensive phylogenetic study of the 

Ostropales (Baloch et al. 2010) allowed a re-evaluation 

of the phylogenetic position of Claviradulomyces, which 

we now regard as “incertae sedis” within Ostropales. The 

morphological similarity with the Odontotremataceae is not 

reflected in the phylogenetic position of these purported 

woody plant endophytes. 

140 ima fUNGUS



We thank the Freitas family of the Fazenda Bonsucesso for allowing 

access to their forest reserve. P.R.J. was supported by the Landcare 

Research Systematics Portfolio, with Core funding from the Science 

and Innovation Group of the New Zealand Ministry of Business, 

Innovation and Employment. H.C.E. and R.W.B. acknowledge 

financial support from the Conselho Nacional do Desenvolvimento 

Cientifico e Tecnológico (CNPq) and the Coordenação de 

Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 

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doi:10.5598/imafungus.2012.03.02.06 


Shivasia gen. nov. for the Australasian smut Ustilago solida that historically 

shifted through five different genera 

Matthias Lutz 1 , Kálmán Vánky 2 , and Marcin Piątek 3 

1 

Evolutionäre Ökologie der Pflanzen, Institut für Evolution und Ökologie, University of Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, 

Germany; corresponding author e-mail: matthias.lutz@uni-tuebingen.de 

2 

Herbarium Ustilaginales Vánky (H.U.V.), Gabriel-Biel-Straße 5, D-72076 Tübingen, Germany 

3 

Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Kraków, Poland 

ARTICLE 

Abstract: The generic position of the enigmatic smut fungus Ustilago solida is evaluated applying molecular 

phylogenetic analyses using ITS and LSU rDNA sequences as well as light and scanning electron microscopical 

investigations of several collections of this species. Ustilago solida has previously been included in five different 

genera (Ustilago, Urocystis, Sorosporium, Cintractia, and Tolyposporium), however, molecular analyses 

revealed that this smut does not belong to any of these genera and represents a distinct ustilaginalean 

lineage. The closest known phylogenetic relative of Ustilago solida is Heterotolyposporium lepidospermatis, 

the type species of the monotypic genus Heterotolyposporium. Both smuts differ considerably in both LSU 

sequences and in several morphological traits, such as the structure of sori and the characteristics of spore 

balls. Accordingly, the new genus Shivasia is described to accommodate Ustilago solida. This smut infects 

different Schoenus species (Cyperaceae) in Australia and New Zealand. The description of Shivasia increases 

the number of endemic smut genera in Australasia to ten. Compared to all other continents the number of 

endemic smut genera is exceptionally high, which may point at fast evolving characters and/or may be caused 

by the regional history, including the long-term geographic isolation of Australasia. 

Key words: 

Basidiomycota 

Biogeography 

Molecular phylogenetics 

Plant pathogens 

Schoenus 

Ustilaginales 

Article info: Submitted: 22 August 2012; Accepted: 5 November 2012; Published: 15 November 2012. 

INTRODUCTION 

The order Ustilaginales, currently classified within the 

subclass Ustilaginomycotina (Bauer et al. 2006), contains 

the largest number of smut fungi, both in terms of the number 

of genera and species, among all ustilaginomycotinous 

orders. Forty-five genera are currently included in the 

Ustilaginales (Begerow et al. 2007 – excluding Pseudozyma, 

Vánky 2008a – excluding Thecaphora; Vánky 2011, Vánky 

& Lutz 2011), but this number is likely to increase since at 

least some of these genera are undoubtedly polyphyletic 

(Stoll et al. 2005), and several smut species with unusual 

characteristics could still be wrongly classified. All members 

of Ustilaginales share a similar ultrastructure (Bauer et al. 

1997), which accordingly is useless for generic classification. 

However, molecular phylogenetic analyses proved to be 

useful in the Ustilaginales. Indeed, molecular phylogenetic 

analysis has been crucial in the generic placement of several 

ustilaginalean species with ambiguous morphological 

characteristics (Piepenbring et al. 1999, Cunnington et 

al. 2005, Vánky et al. 2006, Bauer et al. 2007, González 

et al. 2007) and has also helped to elucidate the generic 

placement for several smuts from other orders (Vánky et 

al. 1998, Bauer et al. 1999, 2001a, 2005, Castlebury et al. 

2005, Bauer et al. 2007, 2008, Chandra & Huff 2008, Vánky 

et al. 2008a, b, Lutz et al. 2012). 

The generic classification of an enigmatic smut growing 

in the ovaries of several Schoenus species in Australasia, 

which had been described under the name Ustilago solida by 

Berkeley (in Hooker 1859), troubled several generations of 

smut researchers. This smut was originally described from the 

ovaries of “Chaetophora imberbis”, now Schoenus apogon, 

collected in Tasmania. Berkeley remarked that “This species 

connects Ustilago and Sporisporium [sic!]”. Consecutively, 

it was moved to Urocystis (Fischer von Waldheim 1877), 

Sorosporium (McAlpine 1910), and Cintractia (Piepenbring 

2000), in each of these genera constituting a discordant 

element according to the current generic concepts (Vánky 

2002). Recently, Vánky (2009) transferred Ustilago solida 

to Tolyposporium. Indeed, the spore balls of Ustilago solida 

show some resemblance to spore balls of Tolyposporium 

junci (type species), T. isolepidis, T. neillii, and T. piluliforme, 

which form a monophyletic group according to phylogenetic 

analyses presented by Lutz (in Vánky 2008b). This taxonomic 

decision could suggest that Ustilago solida reached the 

appropriate genus. However, since molecular data were 

still lacking for this species, it could not be excluded that the 

phenotypic similarity of spore balls may have resulted from 



Attribution: 







Lutz, Vánky & Piątek 

ARTICLE 

Table 1. List of examined specimens of Shivasia solida. 

Host Locality, date, collectors GenBank acc. no. Reference specimens 

Schoenus apogon AU: Tasmania, Penquite, 21 Dec. 1845, R.C. Gunn – holotype K(M) 171338, isotype 

DAR 59818 

Schoenus apogon AU: Tasmania, Hobart, 4 Nov. 1894, L. Rodway – H.U.V. 17613 

Schoenus apogon AU: Victoria, Port Campbell Natl Park, 30 Oct. 1966, 

G. Beaton 114 

Schoenus apogon NZ: Auckland, Waikumete Cemetry, 1 Sep. 1976, 

S. Bowman & W.S.M. Versluys 

Schoenus apogon NZ: Auckland, Waikumete Cemetry, 26 Oct. 1989, 

E.H.C. McKenzie 

Schoenus apogon AU: Tasmania, 170 km NE of Hobart, 8 Mar. 1996, 

C. Vánky & K. Vánky 

Schoenus latelaminatus AU: Victoria, between Moora Channel and Mairstrack, 

18 Jan. 1969, A.C. Beauglehole 30303 

– H.U.V. 17483 

– H.U.V. 16467 

LSU: JF966729 H.U.V. 15059, H.U.V. 15060, 

KRAM F-49115 

ITS: JF966731, 

LSU: JF966730 

– H.U.V. 20072 

Schoenus maschalinus NZ: Wellington, Upper Hutt, 13 Nov. 1952, A.J. Healy – H.U.V. 16477 

Schoenus nitens var. NZ: Wanganui, Himatangi, 29 Jan. 1932, H.H. Allan – H.U.V. 18757 

concinnus 

Schoenus pauciflorus NZ: Canterbury, near Cass, Kettlehole Bog, 1 Feb. – H.U.V. 16755 

1990, K. Vánky 

Schoenus tesquorum AU: New South Wales, Sydney, Enfield Sate Park, 

Drevers Road, 28 Nov. 1996, J. Dickins 

– H.U.V. 20073 

epitype H.U.V. 17649, isoepitype 

TUB 20001 

morphological convergence, which is quite often observed in 

different smut fungi. 

The present work aims to clarify the generic position of 

Ustilago solida applying molecular phylogenetic analyses 

using rDNA sequences as well as light and scanning electron 

microscopical investigation of several collections of this 

fungus. 


Specimen sampling and documentation 

The specimens examined in this study are listed in Table 

1. The voucher specimens have been deposited in DAR, 

K, KRAM F, and H.U.V. The latter abbreviation refers 

to the personal collection of Kálmán Vánky, “Herbarium 

Ustilaginales Vánky” currently held at his home (Gabriel- 

Biel-Straßr 5, D-72076 Tübingen, Germany). Nomenclatural 

novelties were registered in MycoBank (www.MycoBank. 

org, Crous et al. 2004). The genetype concept follows the 

proposal of Chakrabarty (2010). 

Morphological examination 

Sorus structure, spore ball development, mature spore balls 

and spore characteristics were studied using dried herbarium 

specimens. For soral studies, young sori from herbarium 

specimens were rehydrated by briefly boiling in distilled 

water, and fixed with 2 % glutaraldehyde in 0.1 M sodium 

cacodylate buffer (pH 7.2) at room temperature. Following six 

transfers in 0.1 M sodium cacodylate buffer, samples were 

postfixed in 1 % osmium tetraoxide in the same buffer for 1 

h in the dark, washed in distilled water, and stained in 1 % 

aqueous uranyl acetate for 1 h in the dark. After five washes 

in distilled water, samples were dehydrated in acetone, using 

10 min changes at 25 %, 50 %, 70 %, 95 %, and three times 

in 100 % acetone. Samples were embedded in Spurr’s plastic 

and sectioned with a diamond knife. Semi-thin sections were 

transferred to a microscope slide, stained with new fuchsin 

and crystal violet, mounted in Entellan under a cover slip, and 

studied by light microscopy (LM) at various magnifications. 

For LM, spore balls and spores were dispersed in a 

droplet of lactophenol on a microscope slide, covered with 

a cover slip, gently heated to boiling point to rehydrate the 

spores and to eliminate air bubbles, and examined at 1000× 

magnification. For examination of spore ball development, 

sori were boiled in a mixture of lactophenol with cotton blue 

and distilled water, and hand sectioned with a razor blade 

under a stereomicroscope. Pieces of host tissues from 

the basal part of the sori and very young spore balls were 

transferred into a droplet of lactophenol with cotton blue and 

covered with a cover slip. Gentle pressure was applied until 

the host tissue became flat. Air bubbles were eliminated by 

gently heating to boiling point. 

For scanning electron microscopy (SEM), spore balls 

and spores were mounted on carbon tabs and fixed to an 

aluminium stub with double-sided transparent tape. The stubs 

were sputter-coated with carbon using a Cressington sputtercoater 

and viewed under a Hitachi S-4700 scanning electron 

microscope, with a working distance of ca. 11 mm. SEM 

micrographs were taken in the Laboratory of Field Emission 

Scanning Electron Microscopy and Microanalysis at the 

Institute of Geological Sciences of Jagiellonian University, 

Kraków (Poland). 

DNA extraction, PCR, and sequencing 

Genomic DNA was isolated directly from the herbarium 

specimens. For methods of isolation and crushing of fungal 

material, DNA extraction, amplification, purification of PCR 

products, sequencing, and processing of the raw data see Lutz 

et al. (2004). ITS 1 and ITS 2 regions of the rDNA including the 

5.8S rDNA (ITS, about 780 bp) were amplified using the primer 

pair M-ITS1 (Stoll et al. 2003) and ITS4 (White et al. 1990). 

144 ima fUNGUS

Shivasia, a new genus for Ustilago solida 

100/96 

100/100 

97/76 

100/70 

100/81 

61/67 

58/64 

100/94 

64/99 

100/98 

100/ 

100 

100 

/93 

69/76 

Franzpetrakia microstegii GU139170 

Sporisorium sorghi AF009872 

Anomalomyces panici DQ459347 

Melanopsichium pennsylvanicum AY740093 

Ustilago hordei AY740122 

Leucocintractia scleriae AJ236154 

Ustanciosporium standleyanum DQ846888 

Tranzscheliella hypodytes DQ875373 

Macalpinomyces eriachnes AY740090 

Moesziomyces bullatus DQ875365 

Eriomoeszia eriocauli AY740094 

Farysia chardoniana AF009859 

Stegocintractia luzulae AJ236148 

Schizonella melanogramma AF009870 

Shivasia solida (syn.: Ustilago solida) JF966730 

Shivasia solida (syn.: Ustilago solida) JF966729 

Heterotolyposporium lepidospermatis DQ875362 

Tolyposporium piluliforme AF009871 

Tolyposporium isolepidis EU246949 

Tolyposporium junci AF009876 

Tolyposporium neillii EU246952 

Anthracoidea caricis AY563589 

Portalia uljanishcheviana EF118824 

Dermatosorus cyperi AJ236157 

Cintractia axicola DQ631906 

Trichocintractia utriculicola AF009877 

Parvulago marina DQ185437 

Moreaua fimbristylidis DQ875367 

Pericladium grewiae DQ875370 

64/- 

84/61 

97/100 

60/- 

100/100 

88/62 

100/69 

91/- 

92/- 

98/63 

79/- 

86/- 

100/98 

79/62 

100/95 

60/- 

100/78 

Restiosporium meneyae DQ875371 

Websdanea lyginiae AJ236159 

Melanotaenium endogenum DQ789979 

Melanoxa oxalidis EF635908 

Melanustilospora ari EF517924 

92/- Ustacystis waldsteiniae AF009880 

Vankya ornithogali EF517926 

Urocystis ficariae EF517939 

Flamingomyces ruppiae DQ185436 

Mundkurella kalopanacis AF009869 

Antherospora vaillantii EF653980 

Floromyces anemarrhenae EU221284 

Mycosyrinx cissi DQ875368 

Doassansiopsis deformans AF009849 

Thecaphora seminis-convolvuli AF009874 

Thecaphora saponariae EF200047 

Tilletia caries AY819007 

Exobasidium vaccinii FJ644526 

Entyloma microsporum DQ185435 

71/53 

100/100 

0.05 substitutions/site 

Ustilaginaceae 

Melanotaeniaceae 

Urocystidaceae 

Floromycetaceae 

Mycosyringaceae 

Doassansiopsidaceae 

Glomosporiaceae 

Ustilaginales 

Urocystidales 

ARTICLE 

Fig. 1. Bayesian inference of phylogenetic relationships within the sampled Ustilaginomycetes: Markov chain Monte Carlo analysis of an alignment 

of LSU sequences using the GTR+I+G model of DNA substitution with gamma distributed substitution rates and an estimated proportion of 

invariant sites, random starting trees and default starting parameters of the DNA substitution model. A 50 % majority-rule consensus tree is shown 

computed from 45 000 trees that were sampled after the process had reached stationarity. The topology was rooted with the exobasidiomycetous 

species Entyloma microsporum, Exobasidium vaccinii, and Tilletia caries. Numbers on branches before slashes are estimates for a posteriori 

probabilities, numbers on branches after slashes are ML bootstrap support values. Branch lengths were averaged over the sampled trees. They 

are scaled in terms of expected numbers of nucleotide substitutions per site. The taxonomic concept used here follows Bauer et al. (2001b). 

The 5’-end of the nuclear large subunit ribosomal DNA (LSU, 

about 650 bp) was amplified using the primer pair NL1 and 

NL4 (O´Donnell 1993). PCR primers were also used for cycle 

sequencing. For amplification the annealing temperature was 

adjusted to 45 °C. DNA sequences determined for this study 

were deposited in GenBank. GenBank accession numbers are 

given in Table 1 and Fig. 1. 

Phylogenetic analyses 

The Ustilago solida specimens examined in this study are 

listed in Table 1. For molecular phylogenetic analyses the 

following sequences from GenBank were additionally used 

(Begerow et al. 1997, Piepenbring et al. 1999, Castlebury et 

al. 2005, Hendrichs et al. 2005, Stoll et al. 2005, Matheny et 

al. 2006, Vánky et al. 2006, Bauer et al. 2007, Begerow et 


145


ARTICLE 

al. 2007, González et al. 2007, Vánky & Lutz 2007, Bauer et 

al. 2008, Vánky & Lutz, in Vánky 2008b, Vánky et al. 2008b, 

Kottke et al. 2010, Lutz et al. 2012): Anomalomyces panici 

DQ459347, Antherospora vaillantii EF653980, Anthracoidea 

caricis AY563589, Cintractia axicola DQ631906, 

Dermatosorus cyperi AJ236157, Doassansiopsis deformans 

AF009849, Entyloma microsporum DQ185435, Eriomoeszia 

eriocauli AY740094, Exobasidium vaccinii FJ644526, Farysia 

chardoniana AF009859, Flamingomyces ruppiae DQ185436, 

Floromyces anemarrhenae EU221284, Franzpetrakia 

microstegii GU139170, Heterotolyposporium lepidospermatis 

DQ875362, Leucocintractia scleriae AJ236154, 

Macalpinomyces eriachnes AY740090, Melanopsichium 

pennsylvanicum AY740093, Melanotaenium endogenum 

DQ789979, Melanoxa oxalidis EF635908, Melanustilospora 

ari EF517924, Moesziomyces bullatus DQ875365, Moreaua 

fimbristylidis DQ875367, Mundkurella kalopanacis AF009869, 

Mycosyrinx cissi DQ875368, Parvulago marina DQ185437, 

Pericladium grewiae DQ875370, Portalia uljanishcheviana 

EF118824, Restiosporium meneyae DQ875371, Schizonella 

melanogramma AF009870, Sporisorium sorghi AF009872, 

Stegocintractia luzulae AJ236148, Thecaphora saponariae 

EF200047, T. seminis-convolvuli AF009874, Tilletia caries 

AY819007, Tolyposporium isolepidis EU246949, T. junci 

AF009876, T. neillii EU246952, T. piluliforme AF009871, 

Tranzscheliella hypodytes DQ875373, Trichocintractia 

utriculicola AF009877, Urocystis ficariae EF517939, 

Ustacystis waldsteiniae AF009880, Ustanciosporium 

standleyanum DQ846888, Ustilago hordei AY740122, Vankya 

ornithogali EF517926, and Websdanea lyginiae AJ236159. 

To elucidate the phylogenetic position of the Ustilago 

solida specimens their LSU sequences were analysed within 

a dataset covering all urocystidalean and ustilaginalean 

genera of which sequences were available in GenBank. If 

present in GenBank the respective type species were used. 

Additionally, all Tolyposporium LSU sequences available in 

GenBank and the LSU sequence of Thecaphora saponariae 

(type species of Sorosporium) were added. 

Sequence alignment was obtained using MAFFT 6.853 

(Katoh et al. 2002, 2005, Katoh & Toh 2008) using the L-INS-i 

option. To obtain reproducible results, manipulation of the 

alignment by hand as well as manual exclusion of ambiguous 

sites were avoided as suggested by Giribet & Wheeler 

(1999) and Gatesy et al. (1993), respectively. Instead, highly 

divergent portions of the alignment were omitted using 

GBlocks 0.91b (Castresana 2000) with the following options. 

‘Minimum Number of Sequences for a Conserved Position’: 

25, ‘Minimum Number of Sequences for a Flank Position’: 25, 

‘Maximum Number of Contiguous Non-conserved Positions’: 

8, ‘Minimum Length of a Block’: 5, and ‘Allowed Gap Positions’ 

to ‘With half’. 

The resulting alignment (new number of positions: 586; 

21 % of the original 2790 positions; number of variable sites: 

312) was used for phylogenetic analyses using a Bayesian 

Approach and Maximum Likelihood (ML). A Bayesian 

approach using a Markov chain Monte Carlo (MCMC) 

technique was used as implemented in the computer program 

MrBayes 3.1.2 (Huelsenbeck & Ronquist 2001, Ronquist & 

Huelsenbeck 2003). Four incrementally heated simultaneous 

Markov chains were run over 5 M generations using the 

general time reversible model of DNA substitution with gamma 

distributed substitution rates and an estimated proportion of 

invariant sites, random starting trees and default starting 

parameters of the DNA substitution model as recommended 

by Huelsenbeck & Rannala (2004). Trees were sampled 

every 100 th generation resulting in an overall sampling of 

50 001 trees. From these, the first 5 001 trees were discarded 

(burnin = 5 001). The trees sampled after the process had 

reached stationarity (45 000 trees) were used to compute a 

50 % majority rule consensus tree to obtain estimates for the 

a posteriori probabilities of groups of species. This Bayesian 

approach of phylogenetic analysis was repeated five times to 

test the independence of the results from topological priors 

(Huelsenbeck et al. 2002). ML analysis (Felsenstein 1981) 

was conducted with the RAxML 7.2.8 software (Stamatakis 

2006), using raxmlGUI (Silvestro & Michalak 2012), invoking 

the GTRCAT and the rapid bootstrap option (Stamatakis et al. 

2008) with 1000 replicates. 

Trees were rooted with the exobasidiomycetous species 

Entyloma microsporum, Exobasidium vaccinii, and Tilletia 

caries. 

RESULTS 

Morphology 

The morphological characteristics of Ustilago solida are 

included in the species description and depicted in illustrations 

(Figs 2–4). 


The LSU sequences of the two Ustilago solida specimens 

analysed were identical. Compared to the LSU of 

Heterotolyposporium lepidospermatis DQ875362 they 

differed in 51 positions (9.75 %) in 36 different sections. 

BLAST searches (Altschul et al. 1997) for the ITS 

sequence of Ustilago solida H.U.V. 17649 revealed closest 

similarity to Tolyposporium isolepidis EU246950 and T. neillii 

EU246951. However, ITS sampling of ustilaginaceous species 

in GenBank is limited (e.g., there is no Heterotolyposporium 

lepidospermatis ITS sequence available) and sequences 

differ to a great extent (e.g., compared to the ITS of 

Tolyposporium isolepidis and T. neillii the ITS sequence of 

Ustilago solida differed in 211 positions (27.51 %) in 123 

different sections). Thus, molecular phylogenetic analyses of 

the available ITS sequences, similar to the LSU analyses, 

supported the ustilaginaceous affiliation of Ustilago solida but 

did not resolve the placement within the Ustilaginaceae (data 

not shown). 

The different runs of the Bayesian phylogenetic analyses 

that were performed and the ML analyses yielded consistent 

topologies. To illustrate the results, the consensus tree of one 

run of the Bayesian phylogenetic analyses is presented (Fig. 

1). Estimates for a posteriori probabilities are indicated on 

branches before slashes, numbers on branches after slashes 

are ML bootstrap support values. 

In all analyses the two Ustilago solida specimens 

formed a cluster within the Ustilaginaceae sensu Bauer et 

al. (2001b), which was revealed in a group consisting of 

Heterotolyposporium lepidospermatis, and the cluster of 

146 ima fUNGUS


ARTICLE 

Fig. 2. Shivasia solida on 

Schoenus apogon. A. Holotype 

of Ustilago solida [K(M) 171338]. 

B. The habit of infected plants 

and two enlarged sori (H.U.V. 

15059). Bars: A = 1 cm, B = 1 cm 

and 3 mm, respectively. 

Farysia chardoniana, Schizonella melanogramma, and 

Stegocintractia luzulae. A closer relation of the Ustilago solida 

specimens to any of the genera Cintractia, Sorosporium (here: 

Thecaphora saponariae, the type species of Sorosporium), 

Tolyposporium, Urocystis or Ustilago was not revealed by 

any of the phylogenetic analyses. 

Taxonomy 

Molecular phylogenetic analyses, sorus, spore ball and 

spore morphology, as well as the type of spore germination, 

supplemented by host plant taxonomy give evidence that 

a new genus should be erected to accommodate Ustilago 

solida. 

Shivasia Vánky, M. Lutz & M. Piątek, gen. nov. 


Etymology: This genus is named in the honour of Roger 

Graham Shivas, a multi-talented Australian mycologist, 

interested in plant parasitic microfungi, author of two books 

and over 130 scientific papers, in which over 160 new species 

and several new genera are described. Roger is a sharpeyed, 

ardent collector who collected not only in Australia but 

also in Bolivia, Burma, China, India, Indonesia, Malaysia, 

New Zealand, Norway, Papua New Guinea, Philippines, 

South Africa, Thailand, and Vietnam. 

Type species: Shivasia solida (Berk.) Vánky, M. Lutz & M. 

Piątek 2012. 

Sori in the flowers of Schoenus (Cyperaceae) forming 

hard, globoid, black bodies composed of spore balls and 

spores on the surface of innermost floral organs developed 

in sporogenous hyphae within U-shaped pockets, at first 

covered by a fungal peridium. Spore balls few to manyspored, 

composed of spores only, enclosed by a subhyaline 

mucilaginous layer. Spores pigmented (brown). Sterile cells 

absent. 

Shivasia solida (Berk.) Vánky, M. Lutz & M. Piątek, 

comb. nov. 


(Figs 2–4) 

Basionym: Ustilago solida Berk., in Hooker, Flora Tasmaniae 

2: 270 (1859). 

Synonyms: Urocystis solida (Berk.) A.A. Fisch. Waldh., 

Aperçu Syst. Ustil.: 38 (1877). 

Sorosporium solidum (Berk.) McAlpine, Smuts of Australia: 

185 (1910). 

Cintractia solida (Berk.) M. Piepenbr., Nova Hedwigia 70: 310 

(2000). 

Tolyposporium solidum (Berk.) Vánky, Mycotaxon 110: 320 

(2009). 

Type: [Australia: Tasmania]: “Herb. Berk. 4751, 1879. 

Ustilago solida, Berk. on Chaetospora imberbis Penquite 

21/12/45 h°581.” [original label of holotype specimen, Fig. 

2a] (K(M) 171338 – holotype; DAR 59818 -- microscope slide 

ex-holotype). – Australia: Tasmania: 170 km NE of Hobart, 

on Schoenus apogon, 8 March 1996, C. Vánky & K. Vánky 

(H.U.V. 17649 – epitype designated here; TUB 20001 – 

isoepitype). 


147


ARTICLE 

Fig. 3. Shivasia solida on Schoenus apogon. A. Embedded, stained, semi-thin section of a sorus (H.U.V. 17649), sh = sporogenous hyphae, sp 

= spore balls, p = peridium. B. Fungal cells of the peridium covering the sori, formed of thick-walled, sterile hyphae (H.U.V. 15059). C. Spore ball 

formation in sporogenous fungal layer on the surface of innermost floral organs, in U-shaped pockets, hand sectioned, stained with cotton blue 

in lactophenol (H.U.V. 15059). D–E. Young spores and spore balls covered by fungal cells of the young peridium, embedded in plastic, sectioned 

and stained with new fuchsin and cristal violet (H.U.V. 15059). F. Spore balls in different developmental stages, hand sectioned, stained with 

cotton blue in lactophenol (H.U.V. 15059). G–H. Spore germination in water, at room temperature, in 3–5 days (H.U.V. 15059). Bars: A = 100 

µm, B–H = 10 µm. 

148 ima fUNGUS


ARTICLE 

Fig. 4. Shivasia solida on Schoenus apogon (KRAM F-49115). A–C. Spore balls and spores seen in LM, note mucilaginous layer (subhyaline 

caps) around spores marked by arrows. D–E. Spore balls and spores seen in SEM, note that spores are enclosed by remnants of mucilaginous 

layer that form (pseudo-)ornamentation, while the spore surface is verruculose as marked by arrow. Bars = 10 µm. 

Sori (Figs 2–3) in all flowers of an inflorescence, comprising 

the innermost floral organs, visible between the glumes as 

black, globose to ovoid bodies, 1–2 mm diam, rarely also on 

the stems, then fusiform, at first covered by a thick, whitish 

brown fungal peridium of thick-walled, sterile hyphae that 

early flakes away exposing the compact mass of spore balls 

with spores, powdery on the surface. Spore balls (Fig. 4) 

usually irregular or globoid to ellipsoidal, composed of 2–15 

spores, loose but rather permanent, 25–55(–70) × 20–40 

µm, reddish brown, enclosed by subhyaline mucilaginous 

layer. Spores (Fig. 4) subglobose, ovoid, elongate or 

irregular, flattened on one or two sides, 15–20 × 12–16 µm, 

yellowish to pale reddish brown; wall uneven, 0.5–1.5 µm 

thick, smooth to rough, in SEM finely, densely, irregularly 

verruculose and covered by remnants of the mucilaginous 

layer which form irregularly warty (pseudo-)ornamentation. 

Spore balls and spores produced on the surface of host 

tissues in hyaline, sporogenous fungal layer within radially 

arranged, U-shaped pockets (Fig. 3C–F). Spore germination 

(Fig. 3G–H; on water, at room temperature, in 3–5 d) results 

in long, aseptate basidia on which apically elongated, 

cylindrical basidiospores are produced that germinate by 

filaments. 

The ITS/LSU epigenetype sequences are deposited in 

GenBank with the accession numbers JF966731/JF966730, 

respectively. 

Hosts: On different Schoenus species (Cyperaceae): 

S. apogon, S. calyptratus, S. carsei, S. cruentus, S. 

latelaminatus, S. maschalinus, S. nanus, S. nitens var. 

concinnus, S. pauciflorus, S. tesquorum, and Schoenus sp. 

(Table 1, Vánky & McKenzie 2002, Vánky & Shivas 2008). 

Distribution: The genus and species are restricted to southeastern 

Australasia: south-east Australia, including Tasmania, 

and north-west New Zealand (Fig. 5, based on the specimens 

included in Table 1 as well as in Vánky & McKenzie 2002, and 

Vánky & Shivas 2008). 

DISCUSSION 

In the present study molecular phylogenetic analyses and 

morphological data were used to clarify the systematic 

position of Ustilago solida. The molecular analyses revealed 

that this smut does not belong to any genus in which it 


149


ARTICLE 

Fig. 5. Global distribution of Shivasia solida. 

was included during the last 150 years. The morphological 

characteristics also contradict the placement of this species 

in Ustilago, Urocystis, Sorosporium as well as in Cintractia. 

In the past the generic concept of Ustilago was very broad 

(Zundel 1953), but now this genus is restricted to smuts 

infecting poacean hosts, producing sori in vegetative or 

generative host plant organs, and having sori composed only 

of single spores, never forming spore balls (Vánky 2002). The 

species included in the genus Urocystis infect a wide range 

of host species from both mono- and dicotyledonous families, 

and are characterised by the production of spores in spore 

balls, which are usually enclosed by a complete or incomplete 

layer of sterile cells (Vánky 2002). The genus Sorosporium 

included in the past many, often unrelated smuts having 

spore balls (Zundel 1953), but the generic type, Sorosporium 

saponariae, has features of the genus Thecaphora with which 

it was merged based on both morphological and molecular 

phylogenetic analyses (Vánky 1998a, b, Vánky & Lutz 

2007, Vánky et al. 2008a). Thecaphora (incl. Sorosporium) 

is restricted to smut fungi parasitic on dicotyledonous host 

plants, having sori with a granular-powdery spore ball mass 

that is yellowish, pale brown or dark reddish-brown, but never 

black or almost black (Vánky 2002, Vánky et al. 2008a). 

Cintractia species infect cyperaceous host plants forming 

single spores only (Vánky 2002, Piątek & Vánky 2007). The 

placement of Ustilago solida in Cintractia was in any case 

considered as provisional (Piepenbring 2000). 

The most surprising result of the molecular analyses is that 

Ustilago solida is also not a member of the genus Tolyposporium, 

with which it shares many morphological traits, including the 

formation of sori in the floral organs and stems, sporulation 

within U-shaped pockets formed on the sterile stroma, and 

the development of spore balls (Piepenbring 2000, Vánky 

2002). However, there are morphological differences between 

Ustilago solida and Tolyposporium that support the affiliation 

to different phylogenetic lineages. Ustilago solida differs from 

the type species of Tolyposporium (i.e. T. junci) in spore wall 

ornamentation (rough or finely verruculose vs. irregularly, 

coarsely warty) and most notably in that spore balls of U. solida 

are enclosed by a mucilaginous layer that in light microscope 

is seen as subhyaline cap on the outer sides of spores, and in 

scanning electron micrographs as an irregularly warty (pseudo-) 

ornamentation. A mucilaginous layer around the spore balls is 

absent in Tolyposporium. This is in line with the observation 

of Piepenbring (2000) who used these two characteristics to 

differentiate Tolyposporium from Cintractia solida. It is worth 

noting that these two diagnostic features of Tolyposporium are 

easily applied to that three remaining species currently classified 

in this genus (T. isolepidis, T. neillii, and T. piluliforme). 

The molecular analyses revealed that the closest 

phylogenetic relative of Ustilago solida may be 

Heterotolyposporium lepidospermatis, the type of the 

monotypic genus Heterotolyposporium. However, these two 

smuts differ considerably in LSU base sequences as well as 

in several morphological characteristics. In particular, the 

structure of the sori could hardly be considered congeneric. 

Heterotolyposporium lepidospermatis develops sori in the 

inflorescence of Lepidosperma ensiforme, with powdery 

spore masses replacing all central floral organs. A peridium 

is lacking and the sori are covered only by the outermost 3–4 

glumes (Vánky 1997, 2002). This contrasts with the sori of 

Ustilago solida that are coated, at least in young stages, by 

a peridium, and the innermost floral organs are covered by 

a sterile stroma with U-shaped pockets within which spore 

balls are produced. The characteristics of the spore balls 

that differentiate Ustilago solida and Tolyposporium also 

distinguish Ustilago solida and Heterotolyposporium. The 

presence of two kinds of spores, originally used as the main 

diagnostic feature of Heterotolyposporium (Vánky 1997), is 

systematically irrelevant at the genus level since this character 

150 ima fUNGUS


Table 2. Endemic smut genera in particular continents. 

Continent 

Africa 

Australasia 

Asia 

Europe 

North America 

South America 

Endemic smut genera 

Eriosporium, Geminago, Talbotiomyces 

Anomalomyces, Centrolepidosporium, Farysporium, Fulvisporium, Heterotolyposporium, Macalpinomyces, Pseudotracya, 

Restiosporium, Shivasia, Websdanea 

Ahmadiago, Floromyces, Franzpetrakia, Georgefischeria, Liroa, Phragmotaenium, Zundeliomyces 

Doassinga, Flamingomyces, Melanustilospora, Parvulago 

Clintamra, Exoteliospora, Planetella, Salmacisia, Tilletiaria 

Kuntzeomyces, Oberwinkleria, Uleiella 

ARTICLE 

evolved convergently in Tolyposporium piluliforme, which is 

only distantly related to Heterotolyposporium (Piepenbring et 

al. 1999, Vánky & Lutz, in Vánky 2008b). 

Both genetic and morphological features reveal Ustilago 

solida as a unique smut that should be accommodated in 

a distinct genus, described here as Shivasia. This is in line 

with the current generic concept in smut fungi where distinct 

phylogenetic lineages distinguishable by morphological and/ 

or ecological characters are referred to distinct genera (e.g., 

Piepenbring et al. 1999, Vánky et al. 2006, 2008b, Lutz et al. 

2012). The close phylogenetic relation of Heterotolyposporium 

and Shivasia is reflected in the close phylogenetic relation 

of their host genera. Both Lepidosperma and Schoenus are 

placed in the same tribe Schoeneae within the Cyperaceae 

(Verboom 2006, Simpson et al. 2007, Muasya et al. 2009). 

Thus co-speciation of parasites and hosts may have played a 

role in the evolution of these two smuts. 

In addition to resolving the phylogenetic and systematic 

placement of Ustilago solida, this study reports an emerging 

number of smut genera currently known exclusively from 

Australasia. Based on the present knowledge, and considering 

the relatively uniform and stabilized generic concept in smut 

fungi, they may be treated as endemic to this ecozone. The 

high rate of endemic smuts in Australia has been discussed 

at length by Shivas & Vánky (2003) and less extensively also 

by Vánky & Shivas (2008). Amongst the 309 species reported 

from the continent (Vánky & Shivas 2008, Barrett et al. 2009, 

McTaggart & Shivas 2009a, b, Shivas & McTaggart 2009, 

Vánky 2009, Shivas et al. 2010, Piątek & Shivas 2011, Shivas 

et al. 2011, Crous et al. 2012) about half are endemic. Six 

genera were considered to be endemic to Australia by Vánky & 

Shivas (2008), namely Anomalomyces, Centrolepidosporium, 

Farysporium, Fulvisporium, Pseudotracya, and Websdanea, 

but in fact Farysporium should be deleted as a strictly 

Australian endemic since it also occurs in New Zealand. 

This removal is balanced by adding Heterotolyposporium 

because, in the present circumscription, that genus has 

only one species, H. lepidospermatis, known from only one 

locality in Tasmania. Four additional genera are endemic to 

Australasia, being present in both Australia and New Zealand 

(Farysporium, Restiosporium, and Shivasia) or in Australia 

and Papua New Guinea (Macalpinomyces). Until recently 

Macalpinomyces included many unrelated smuts from 

different parts of the globe, but recent molecular analyses 

(Stoll et al. 2005) narrowed this genus to the type species M. 

eriachnes that occurs exclusively in Australasia. Thus, ten 

endemic genera occur in the Australasian ecozone and this 

number is not comparable with any other continent since all 

of them have lower numbers of endemic smut genera (Table 

2). The high number of unique smut genera may point to 

rapidly evolving characters and/or may result from the regional 

history, including the long geographic isolation of Australasia 

which started with the break-up of Gondwana and the initial 

separation of Australia and Tasmantia (incl. New Zealand) 

terranes from Antarctic terranes about 96 Myr and 84 Myr ago, 

respectively (McLoughlin 2001). 

Shivasia solida has been reported on ten different 

Schoenus species. Recent molecular analyses of different 

smut genera (Lutz et al. 2005, Carris et al. 2007, Vánky & 

Lutz 2007, Bauer et al. 2008, Lutz et al. 2008, Kemler et al. 

2009, Piątek et al. 2011, Lutz et al. 2012, Piątek et al. 2012, 

Savchenko et al. 2012) revealed that such polyphagous 

species are usually complexes of morphologically similar 

cryptic species that are often restricted to single host plant 

species. However, in the present study sequences were 

obtained only from specimens on Schoenus apogon, while 

repeated attempts to obtain sequences from specimens 

on two other hosts (Schoenus pauciflorus, H.U.V. 16755, 

and Schoenus tesquorum, H.U.V. 20073) failed. Thus, the 

question whether the collections on other Schoenus species 

than Schoenus apogon are genetically identical or cryptic 

species exist within what we now consider as one species 

is left open for future studies. To stabilise the taxonomy of 

Shivasia solida and to make future genetic comparisons 

possible an epitype is selected here based on the sequenced 

specimen collected on the same host (Schoenus apogon) 

in the same geographical area (Tasmania) as the holotype. 

This is in concordance with recent recommendations for 

epitypifications (Hyde & Zhang 2008). 

The genus Schoenus shows greatest species diversity 

in Australasia. This is reflected by the inhabiting smut fungi 

– amongst seven smuts known on this genus worldwide, 

five occur in Australasia (Vánky & Websdane 1995, Vánky 

2009). The two extralimital species are Anthracoidea andina 

in southern South America (Argentina: Tierra del Fuego) and 

Moreaua kochiana in Central Europe (Italy and Switzerland). 

The Schoenus smuts belong to three genera, Anthracoidea, 

Moreaua and Shivasia, that are not closely related, suggesting 

that at least three independent colonisation events took place 

onto this host plant genus in the course of evolution. 


We thank Michael Weiß, Sigisfredo Garnica, and Robert Bauer 

(Tübingen) for providing facilities for molecular analyses, Michael 


151


ARTICLE 

Weiß for critically reading the manuscript, Christine Vánky (Tübingen) 

for technical assistance with the illustrations, Jolanta Piątek (Kraków) 

for preparing the world map, Magda Wagner-Eha (Tübingen) for 

technical assistance with the semi-thin sections, Anna Łatkiewicz 

(Kraków) for help with the SEM photomirographs, and the Curators 

of DAR and K (M) for the loan of specimens. 

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154 ima fUNGUS

doi:10.5598/imafungus.2012.03.02.07 


Addressing the conundrum of unavailable name-bearing types 

David L. Hawksworth 

Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal, 28040 Madrid, Spain; 

and Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK; e-mail: d.hawksworth@nhm.ac.uk 

ARTICLE 

Abstract: Access to name-bearing type material can be a particular frustration for those mycologists in the tropics, or 

working outside established institutions, where the specimens are known to exist but cannot be examined. They can 

be inaccessible because of loans policies and the inability of mycologists to make personal visits. Each case has to be 

considered separately, but a pragmatic nine-point approach is presented which may provide some guidance as to what 

can be done in such instances. A postscript draws attention to 12 points to consider when designated or handling namebearing 

types. 

Key words: 

collections 

epitype 

fungi 

holotype 

lichens 

nomenclature 

proxy type 

typification 

Article info: 29 October 2012; Accepted: 11 November 2012; Published: 19 November 2012. 

BACKGROUND 

Many fungi have yet to be collected and named, and it 

appears that the number of undescribed species is at least 

1.4 million and probably as many as 3 million (Hawksworth 

2012a). It is anticipated that many of these species are to 

be found in the tropics, and this poses particular constraints 

to their formal description. Until the 1990s, tackling this task 

was generally by opportunist, short-stay visits to the tropics 

by European and North American mycologists; something 

that can be likened to “smash-and-grab” raids. The material 

is often retained in the collector’s institution though, where 

possible, some mycologists have split, and diligently 

repatriated, at least some of the specimens. As there were 

few centres anywhere in the tropics where fungal material 

could be deposited and safeguarded for examination by future 

generations of mycologists in the 18 th and 19 th centuries, 

this situation was unavoidable in those times. In the last 

few decades in particular, the situation has changed. There 

has been a remarkable expansion in systematic mycology 

in universities, research institutions, and museums located 

in some tropical regions, especially in parts of Asia, South 

America, and southern Africa. 

THE PROBLEM 

In endeavouring to check if a previously named fungus is 

the same as one recently collected, or when undertaking 

revisionary work or preparing monographs, it is often 

necessary to consult material at institutions in Europe and/or 

North America. This is particularly so in the case of the namebearing 

type material where original descriptions, especially 

from the 19 th century, are meagre and lack information on 

characters essential for interpretation today; and they may 

not be accompanied by photomicrographs or line drawings. 

Personal visits to the holding institutions are ideal, but may 

be prohibitively expensive for those lacking secure funding. At 

the same time, collection curators are increasingly reluctant 

to dispatch material around the world. This is understandable 

as there are instances where irreplaceable types have been 

lost or damaged in the postal systems, or even destroyed at 

points of entry by customs officials. Also, the problems of loss 

or damage in transit are not confined to tropical countries; for 

example, I know of cases where type material, dispatched to 

the UK from institutions in Poland and Russia, failed to arrive 

at all. 

In the case of microfungi in particular, there is often an 

additional problem of few or even single sporocarps being 

present on a specimen. There are concerns at their being 

destroyed in examination, with no permanent preparations 

having been made, or being used in abortive attempts to 

extract DNA. Some institutions have developed a policy of 

sending only a portion of the material at one time, with the 

remainder sent only when the first part has been returned. 

Further, to minimise destructive sampling, whenever slides 

had been prepared, these were often also included and 

loaned with the type material to preclude the necessity for 

more preparations. The splitting of samples and slides was 

a practice adopted at the former International Mycological 

Institute (Kew and Egham, UK) in the 1980s and 1990s. 

That Institute was anxious to promote the study of tropical 

fungi in universities and other institutions in tropical regions. 

Many European and North American institutions, however, 



Attribution: 







Hawksworth 

ARTICLE 

have policies of only lending material to other established 

institutions, and not to individuals or institutions lacking 

collections or curators. Others, especially ones with 

collections dating from the early 19 th century and before, will 

no longer lend material under any circumstances, generally 

as a result of unfortunate experiences in the past. However, 

in some cases, where the lending of type material is not 

allowed, curators of collections have been pleased to supply 

photographs, and prepare microscopic preparations instead 

which may be sufficient in some cases. An increasing number 

are actively preparing digital images of the specimens they 

hold, and making the images available through the worldwide 

web. However, although digital macroscopic images can be 

excellent for studying vascular plants, they are of limited 

value for studying most fungi. 

There is also a category of institutions that are willing, in 

principle, to lend material, but have neither the appropriately 

experienced staff to look out, pack, and dispatch material, nor 

the funds to cover the costs of secure postal services. 

The loan issue is particularly acute and frustrating where 

it is known that the desired name-bearing type material is in 

existence in a collection, but which cannot be examined for 

any of the reasons summarized above. What is a mycologist 

waiting to complete a publication to do? This is a conundrum 

that has the potential to shackle, frustrate, and delay progress 

in systematic studies in regions with the highest proportions 

of yet undescribed fungi – and where many of those able to 

undertake that work are now located. 

This is not a new situation, indeed the mycologist and 

botanical polymath Corner (1946) observed that “there is 

no reason why research should be held up because the 

[mycologist] is unable to consult earlier investigations”. He 

advised “young [mycologists] brazenly to face the situation 

and to ignore, of necessity, what they cannot possibly obtain, 

through distant libraries”. He went on to remark that following 

the destruction of so many libraries and collections in World 

War II, “Few will be able to consult the early periodicals, the 

early books, and the type specimens”. His suggestion was to 

produce encyclopaedic works and, in effect, treat those as 

new starting points for future work. I suspect he would have 

been a strong supporter of the changes enacted in July 2011 

to establish protected lists of names of fungi (Hawksworth 

2012b, McNeill et al. 2012). He would also be pleased to see 

that increasing amounts of early mycological literature are 

becoming available free of charge through the Biodiversity 

Heritage Library (BHL; http://www.biodiversitylibrary.org/ ) and 

CyberLiber (http://www.cybertruffle.org.uk/cyberliber/ ) initiatives. 

The issue of access to name-bearing types unfortunately 

remains a constraint almost seven decades on. 

A PRAGMATIC APPROACH 

A pragmatic approach has to be adopted to alleviate this 

particular constraint on systematic mycology, especially in the 

tropics. Each case must be considered individually, and no 

generalization can be made, but some guidelines may prove 

helpful to mycologists when confronted with the frustrating 

situation of not being able to examine name-bearing type 

material which is known still to exist in some collection. 

(1) Request either a member of staff in the collection (or the 

institution in which it is housed), or another mycologist living 

near the holding institution, or a visiting mycologist, to take 

high-power digital images, make measurements or notes, or 

prepare microscopic slides that can be sent on loan. 

(2) See if any duplicate material (isotypes) of the desired 

name-bearing type is available in collections of other 

institutions known to house material of the author or the 

collector, as these might be willing lend material. Information 

on where material of deceased authors of fungal names 

is held is included in Hawksworth (1974) and Taxonomic 

Literature (TL-2; Stafleu & Cowan 1976–2009). 

(3) In some cases, there will be evidence in the published 

literature that later mycologists have examined a specimen, 

and these may have provided a detailed description and/or 

illustrations. In such instances the type collection may be 

cited but with “n.v.” (non vide; i.e. not seen), added after the 

collection acronym to show it was not examined. This is a 

common practice where a taxon is well established, and the 

circumscription is not controversial. 

(4) Request photocopies of the labels to verify the status 

of the located specimens to confirm that they qualify as 

holotypes, or be potential material for lectotypification. 

The label should give locality and date of collection, or 

other indications on the packets, such as “Orig. mat.”, 

“Sp. nov.”, or “Typus”, in the author’s handwriting. If there 

is no such evidence, it is possible that material which 

had been previously considered to be the name-bearing 

type, proves not to be when the provenance is studied 

more critically. For example, a specimen with no date, 

even though made by the describing author and from the 

original locality, may have been collected after the date 

of effective publication of the name. This would mean 

that there was no obstacle to designating some other 

collection that was available for study as a neotype. A 

neotype does not have to be of material ever seen by 

the original author but, ideally, should be from the same 

geographical area of collection and, where appropriate, 

from the same host or substrate. 

(5) In cases, where it is really necessary to clarify an 

ambiguous situation and fix unequivocally the application 

of a name, and where a holotype/lectotype/neotype 1 exists, 

but cannot be studied, an interpretative type, termed an 

“epitype” could perhaps be justified. This would be a broad 

interpretation of the phrase “cannot be critically identified for 

purposes of the precise application of the name of a taxon” 

(McNeill et al. 2012: Art 9.8). 

That epitype would stand unless, and until, it was “shown 

that an epitype and the type it supports differ taxonomically” 

1 

As an epitype is an interpretive type linked to the name-bearing type, 

if there are syntypes that are not accessible it would be necessary 

first to select one of those as a lectotype and to base the epitype on 

that. 

156 ima fUNGUS

Unavailable name-bearing types 

(Art. 9.20 2 ). When designating an epitype, the extant namebearing 

type which it acts as an interpretative type for has 

to be stated. This is, however, a somewhat controversial 

interpretation of the Code, where the material might be 

identifiable were it studied. Consequently, such a step should 

not be undertaken without the most careful consideration, and 

this issue will be explored further in a separate paper currently 

being prepared by Kevin D. Hyde and colleagues. However, 

this may be the most appropriate solution, and justifiable, 

where cryptic species (ones that are morphologically 

indistinguishable) are involved, and where DNA sequence 

data are not available for the extant (but unexamined) namebearing 

type, but are for the proposed epitype. 

(which cannot be studied), a lectotype which is available 

for examination may be selected from among elements 

associated with either the original protologue or the 

sanctioning treatment (Art. 9.10). This is only likely to 

be a potential solution in a small number of cases as 

all specimens cited in sanctioning works may also be 

unavailable for examination. However, if one or more 

illustrations are cited in the sanctioning work they may be 

available for designation as lectotypes in which case the 

procedure noted in (8) above could be considered. 

POSTSCRIPT 

ARTICLE 

(6) For names that are NOT in current use, the name can 

simply be listed as of uncertain application, and not be 

adopted – but this procedure should not be followed for 

names in use today as that could lead to new undesirable 

names shaving to be adopted. Now that mechanisms for 

the protection of fungal names through the adoption of lists 

of Accepted Names (i.e. protected names) and Rejected 

Names (i.e. suppressed names) have been incorporated 

into the Code (Arts 14.13, 56.3), the importance of fixing 

the application of all proposed names is reduced. While 

it is desirable that all names are typified and discussed in 

systematic work, older names subsequently discovered to 

pre-date ones in use may be listed either as synonyms in an 

Accepted list, or alternatively added to a Rejected list. 

(7) For names in current use, where it is not clear whether 

any name-bearing type material definitely exists, for example 

because of uncertainty of the provenance of a specimen 

previously considered as the type, or not being able to check 

the describing author’s collections, designate a particular, 

freshly studied specimen and use a phrase such as 

“representative specimen” or “proxy type”; this is a practice 

sometimes used in zoology. “Pragmatype” and “protype”, as 

used in zoology, are both better avoided as their meaning is 

closer to that of epitype (Hawksworth 2010). A proxy type, 

although an unofficial designation, would remain available for 

selection in the future as a neotype if it later became clear that 

no holotype or original material eligible for lectotypification 

was extant. 

(8) Where there is no holotype, a lectotype has not previously 

been designated, and the name was introduced prior to 1 

January 2007, in some cases an illustration may be available 

for use (Art. 40.4). This situation arises where the original 

material consists of both one or more candidate specimens 

that cannot be studied and also an illustration, there is no 

obstacle under the Code to selecting the illustration as 

lectotype, and not the specimen. The illustrations can be 

unpublished, or published either before or with the validating 

diagnosis (Art. 9.3). It would then be possible to designate 

another specimen or metabolically inactive culture as epitype. 

Bearing in mind the current problems over access to namebearing 

types addressed here, mycologists can take some 

action to preclude, or at least minimise any future difficulties 

when designating a holotype, or any other official category 

of type. 

(1) Deposit the name-bearing type (“holotype”) in a public 

collection in the country of origin where there is a fungal 

curator. Many types of fungi are deposited in collections 

located in countries other than that in which they were 

collected; this was the case for 41 % of the name-bearing 

types of fungi described in the period 1991–94 (Hawksworth 

& Kirk 1995). This situation should not be exacerbated 

where alternative public collections exist in the countries of 

origin. 

(2) Deposit duplicates (“isotypes”) of the name-bearing type 

in one or more different public collections located in other 

countries where the specimens are sufficiently large to enable 

them to be subdivided. Where material cannot be split, where 

possible deposit duplicates of other collections cited in the 

original publication (“paratypes”) instead. 

(3) Where a name-bearing type (“holotype”) is a permanently 

preserved and metabolically inactive culture, it is prudent to 

deposit subcultures prepared from that (“ex-type” cultures) in 

at least three service collections of fungal cultures from which 

they can be obtained. 

(4) Provide as detailed and comprehensive description 

as possible of the fungus and accompany it with 

photomicrographs and line drawings, and note the advice in 

Seifert & Rossman (2010). 

(5) Along with the designated type material, deposit 

permanent microscopic slide preparations which show 

essential features. 

(6) Where DNA sequence data have been obtained, deposit 

them in GenBank, or a similar public repository. 

(9) Where a name is sanctioned for use by either Fries 

or Persoon (Art. 13.1(d)), no holotype of the namebringing 

epithet exists, and a lectotype has not previously 

been designated from amongst existing original material 

2 

The Article (Art.) numbers of the Code used in this contribution are 

those of the Melbourne Code (McNeill et al. 2012), some of which 

differ from those allocated to the same points in previous editions. 


157

Hawksworth 

ARTICLE 

(7) When designating a lectotype, neotype, or epitype of 

a name, remember that this, as other nomenclatural acts, 

has to be published; an annotation on a label attached to a 

specimen does not constitute effective typification. 

(8) Mention any designations of a lectotype, neotype, or 

epitype in the abstract of the paper in which these are 

published. Such later typifications are otherwise easily 

overlooked by other mycologists. Also, record such published 

typifications in a nomenclatural database if possible. I 

understand that this facility will be available in MycoBank 

shortly, and in the future I personally would wish to see this 

become mandatory for the recognition of later typifications 

under the Code. 

(9) In view of the scant and fragile nature of many older 

type specimens, it is recommended that they never be 

consulted unless absolutely necessary. Examination of types 

is essential in the course of revisionary or monographic 

studies, or to confirm differences from a newly discovered 

taxon, but inappropriate in the case of routine identifications 

– except where ex-type cultures are available. Remember 

that holotypes in particular are irreplaceable and so always 

merit treatment with respect. Mycologists today sometimes 

need to consult specimens collected in the 18 th and even the 

17 th centuries; unnecessary handling and slide-making may 

jeopardize the value of the specimens to future generations. 

(10) Any microscopic preparations from specimens should 

only be made when essential, and the slides made from the 

material should be permanently preserved along with the 

type material from which they were derived. 

(11) Destructive sampling of dried specimens for DNA 

extraction should only be undertaken with the prior permission 

of the curator concerned. This is not, however, a problem 

where ex-type cultures are available. 

(12) Any type material received on loan should always be 

packed carefully and returned using secure delivery services 

– and within the specified period of the loan. 

DISCLAIMER 

The recommendations in this contribution are based on my 

personal opinions and experience, good taxonomic practice, 

and questions which I have been asked by other mycologists, 

particularly ones based in the tropics. The recommendations 

do not necessarily reflect the views of the Nomenclature 

Committee for Fungi (NCF), the International Commission 

on the Taxonomy of Fungi (ICTF), or the International 

Mycological Association (IMA). Further, the options presented 

are not claimed to be exhaustive and other possibilities may 

be appropriate in some instances. In each case mycologists 

should consult the Code (McNeill et al. 2012) to ensure their 

actions are in accordance with its provisions. As the Code 

is now such a complex and even forbidding document, a 

valuable guide to it and its operation has recently been 

prepared by Turland (2013); that work merits a place on the 

shelves of all systematic mycologists. 


I am indebted to Kevin D. Hyde for stimulating me to prepare this 

note, which was written while in receipt of funding from the Spanish 

Ministerio de Ciencia e Innovación project CGL2011-25003. 

REFERENCES 

Corner EJH (1946) Suggestions for botanical progress. New 

Phytologist 45: 185–192. 

Hawksworth DL (1974) Mycologist’s Handbook: an introduction to 

the principles of taxonomy and nomenclature in the fungi and 

lichens. Kew: Commonwealth Mycological Institute. 

Hawksworth DL (2010) Terms used in Bionomenclature: the naming 

of organisms (and plant communities). Copenhagen: Global 

Environment Facility. 

Hawksworth DL (2012a) Global species numbers of fungi: are tropical 

studies and molecular approaches contributing to a more robust 

estimate? Biodiversity and Conservation 21: 2425–2433. 

Hawksworth DL (2012b) Managing and coping with names of 

pleomorphic fungi in a period of transition. Mycosphere 3: 143– 

155; IMA Fungus 3: 15–24. 

Hawksworth DL, Kirk PM (1995) Passing round the standards. 

Nature 378: 341. 

McNeill J, Barrie FR. Buck WR, Demoulin V, Greuter W, Hawksworth 

DL, Herendeen PS, Knapp S, Marhold K, Prado J, Prud’homme 

van Reine WF, Smith GE, Wiersema JH, Turland NJ (eds) (2012) 

International Code of Nomenclature for algae, fungi, and plants 

(Melbourne Code) adopted by the Eighteenth International 

Botanical Congress, Melbourne, Australia, July 2011. [Regnum 

Vegetabile no. 154.] Ruggell: ARG Ganter Verlag. 

Seifert KA, Rossman AY (2010) How to describe a new fungal 

species. IMA Fungus 1: 109–116. 

Stafleu FA, Cowan ST (1976–2009) Taxonomic Literature: a 

selective guide to botanical publications and collections with 

dates, commentaries and types. 2 nd edn. 7 vols + Supplementum 

(8 vols). [Regnum Vegetabile, nos. 94, 98, 105, 110, 112, 115, 

116, 125, 130, 132, 134, 135, 137, 149, and 150 .] Utrecht: Bohn, 

Scheltema & Holkema. 

Turland NJ (2013) The Code Decoded: a user’s guide to the 

International Code of Nomenclature for algae, fungi, and plants. 

[Regnum Vegetabile, in press.] Ruggell: ARG Gantner Verlag. 

158 ima fUNGUS

doi:10.5598/imafungus.2012.03.02.08 


Two novel species of Aspergillus section Nigri from indoor air 

Željko Jurjević 1 , Stephen W. Peterson 2 , Gaetano Stea 3 , Michele Solfrizzo 3 , János Varga 4 , Vit Hubka 5 , and Giancarlo Perrone 3 

1 

EMSL Analytical, Inc., 200 Route 130 North, Cinnaminson, New Jersey 08077 USA; corresponding author e-mail: zjurjevic@emsl.com 

2 

Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research 

Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 USA 

3 

Institute of Sciences of Food Production, CNR, Via Amendola 122/O, 70126 Bari, Italy 

4 

Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary 

5 

Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01, Praha 2, Czech Republic 

ARTICLE 

Abstract: Aspergillus floridensis and A. trinidadensis spp. nov. are described as novel uniseriate species 

of Aspergillus section Nigri isolated from air samples. To describe the species we used phenotypes from 

7-d Czapek yeast extract agar culture (CYA), creatine agar culture (CREA) and malt extract agar culture 

(MEA), with support by molecular analysis of the β-tubulin, calmodulin, RNA polymerase II (RPB2), and 

translation elongation factor-alpha (TEF) gene amplified and sequenced from 56 air isolates and one 

isolate from almonds belonging to Aspergillus section Nigri. Aspergillus floridensis is closely related to A. 

aculeatus, and A. trinidadensis is closely related to A. aculeatinus. Aspergillus brunneoviolaceus (syn. A. 

fijiensis) and A. uvarum are reported for the first time from the USA and from the indoor air environment. 

The newly described species do not produce ochratoxin A. 

Key words: 

Aspergillus brunneoviolaceus 

Aspergillus floridensis 

Aspergillus trinidadensis 

Aspergillus uvarum 

Aspergillus violaceofuscus 

black aspergilli 

environment 

phylogeny 

ochratoxin A 

Article info: Submitted: 21 August 2012; Accepted: 26 November 2012; Published: 30 November 2012. 

INTRODUCTION 

Aspergillus section Nigri (Gams et al. 1985), commonly known 

as the black aspergilli, contains many common species in the 

environment (Klich 2009), and some have been implicated in 

human and animal diseases (de Hoog et al. 2000, Abarca et 

al. 2004, Klich 2009). They have a worldwide distribution and 

occur on a large variety of substrates including soil, grains, 

dairy and forage products, various fruit, vegetables, beans 

and nuts, cotton textiles and fabrics, and meat products 

(Raper & Fennell 1965, Pitt & Hocking 2007, 2009). Black 

aspergilli are used in the fermentation industry to produce 

various enzymes and organic acids (Raper & Fennell 1965, 

Varga et al. 2000). Some black aspergilli produce ochratoxin 

A (Abarca et al. 1994, 2003, 2004, Wicklow et al. 1996, Varga 

et al. 2000, Cabanes et al. 2002, Sage et al. 2004, Samson 

et al. 2004). 

Although black aspergilli occur in clinical samples, they are 

much less frequent than A. fumigatus, A. terreus, or A. flavus. 

Aspergillus species are widely documented as causative 

pathogens in invasive and non-invasive infections as well as 

in allergic reactions especially Types III and IV (Richardson 

2005). Indeed, the allergic forms of the disease appear to 

be “almost exclusively caused by Aspergillus species” 

(Moss 2002, Knutsen 2011). Some strains of black aspergilli 

are often misidentified as A. niger due to the difficulties of 

identifying the species in this group (Samson et al. 2007). 

Perrone et al. (2012a, b) recognized two new species of 

black aspergilli that may be involved in human disease from 

Sri Lanka: A. brunneoviolaceus (= A. fijiensis) in pulmonary 

aspergillosis, and A. aculeatinus in human dacryocystitis. 

We collected 56 isolates of black uniseriate Aspergillus 

species from air (52 homes and four outside samples) 

from 17 states of the USA, Bermuda, Martinique, Trinidad 

&Tobago, and one from almonds in the Czech Republic. 

Using molecular data and macro- and micro-morphological 

observations, we discovered and describe here two new 

species related to A. aculeatus and A. aculeatinus. 


Fungal isolates 

The provenance of fungal isolates examined is detailed in 

Table 1. 

Culture methods 

Observations were made on Czapek yeast extract agar 

(CYA), CYA with 20 % sucrose (CY20S), malt extract agar 

(MEA), oatmeal agar (OA), and creatine agar (CREA), (Pitt 

1980, Samson et al. 2004) cultures incubated at 25 °C for 

7 d in darkness, and CYA cultures incubated at 5 °C, 35 °C 

and 37 °C for 7 d. The cultures were grown on one plate 

as a three-point inoculation and on another plate as a single 



Attribution: 







Jurjević et al. 

ARTICLE 

Table 1. Provenance of fungal isolates characterized in this study. 

*ITEM number Provenance 

Aspergillus aculeatus 

14807 USA: Georgia, iso. ex indoor air sample, 2010, Ž. Jurjević. 

Aspergillus brunneoviolaceus (syn. A. fijiensis) 

14784 USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević. 



14790 Bermuda: isol. ex indoor air sample, 2010, Ž. Jurjević. 


14794 USA: Texas, isol. ex indoor air sample, 2010, Ž. Jurjević. 




14802 USA: Arizona, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14804 Trinidad and Tobago: Tunapuna, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14806 USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević. 




14825 USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević. 

14831 USA: Alabama, isol. ex outside air sample, 2011, Ž. Jurjević. 


Aspergillus floridensis sp. nov. 

14783 T USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević, ex-type. 

** (=NRRL 62478 T ) 

***CCF 4046 

Czech Republic: Brno, isol. ex almonds in shells imported from USA, 2010, V. Ostrý. 

CCF 4236 

Martinique: Fort de France, isol. ex outside air sample, 2004, N. Desbois. 

****CRI 323-04 

Thailand: Phi Phi Islands, isol. ex Xestospongia testudinaria, 2006, T.S. Bay. 

*****IFM 55703 

Japan: isol. ex soil from grapery, 2007, K. Yokoyama. 

Aspergillus violaceofuscus (syn. A. japonicus) 


14788 USA: Kentucky, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14789 USA: New Jersey, isol. ex indoor air sample, 2010, Ž. Jurjević. 


14793 USA: Louisiana, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14797 USA: New York, isol. ex indoor air sample, 2010, Ž. Jurjević. 




14803 USA: Georgia, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14805 USA: Louisiana, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14808 USA: South Carolina, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14810 USA: Tennessee, isol. ex indoor air sample, 2010, Ž. Jurjević. 

14811 USA: Hawaii, isol. ex indoor air sample, 2010, Ž. Jurjević. 



14815 USA: Delaware, isol. ex indoor air sample, 2011, Ž. Jurjević. 

14816 USA: Maryland, isol. ex indoor air sample, 2011, Ž. Jurjević. 



160 ima fUNGUS

Novel species of Aspergillus sect. Nigri 

Table 1. (Continued) 

*ITEM number Provenance 





14828 USA: Georgia, isol. ex indoor air sample, 2011, Ž. Jurjević. 


14834 USA: New Jersey, isol. ex indoor air sample, 2011, Ž. Jurjević. 

14835 USA: North Carolina, isol. ex indoor air sample, 2011, Ž. Jurjević. 

14836 USA: New York, isol. ex indoor air sample, 2011, Ž. Jurjević. 


Aspergillus trinidadensis sp. nov. 

14821 T 

(=NRRL 62479 T ) 

14829 

(=NRRL 62480) 


Trinidad and Tobago: Tunapuna, isol. ex indoor air sample, 2011, Ž. Jurjević, ex-type. 

USA: California, isol. ex indoor air sample, 2011, Ž. Jurjević. 



14833 USA: New Jersey, isol. ex outside air sample, 2011, Ž. Jurjević. 

*ITEM, Agri-Food Toxigenic Fungi Culture Collection, Institute of Sciences of Food Production, Bari, Italy; **NRRL (Northern Regional Research 

Laboratory), the National Center For Agricultural Utilization Research, USA; ***CCF (Culture Collection of Fungi), Department of Botany, Faculty 

of Science, Charles University, Prague, Czech Republic; ****CRI (Chulabhorn Research Institute), Vibhavadi-Rangsit Road, Laksi, Bangkok, 

Thailand; *****IFM (Medical Mycology Research Center) Chiba University, Chiba, Japan. 

ARTICLE 

T 

= ex-type strain 

center-point inoculation on each medium in 9 cm diam Petri 

dishes. Colony diameters and appearance were recorded and 

photographs were made from 7 d culture plates incubated at 

25 °C. 

Microscopy 

Microscopic examination was performed by gently pressing 

a ca. 20 × 5 mm piece of transparent tape onto a colony, 

rinsing the tape with one or two drops of 70 % ethanol and 

mounting the tape in lactic acid with fuchsin dye. Additional 

microscopic samples were made by teasing apart a small 

amount of mycelium in a drop of water containing 0.5 % 

Tween 20. A Leica DM 2500 microscope with bright field, 

phase contrast and DIC optics was used to view the slides. 

A Spot camera with Spot imaging software was mounted 

on the microscope and used for photomicrography. A Nikon 

digital SLR camera with a D70 lens was used for colony 

photography. Photographs were resized and fitted into plates 

using Microsoft PowerPoint 2010. 

Ochratoxin A (OTA) assay 

Aspergillus strains were grown in duplicate in 100-ml stationary 

liquid cultures (5 cm diam) of Czapek w/20 % Sucrose Broth 

(200 g l –1 Sucrose, 1 g l –1 K 2 

HPO 4 

, 3 g l –1 NaNO 3 

, 0.5 g l –1 

KCl, 0.05 g MgSO 4·7•H 2 

O, 0.01 g l –1 FeSO 4 

7•H 2 

O) (Health 

Link ® , Jacksonville, FL) and Yeast Extract Broth {2 g l –1 Yeast 

extract, 15 g l –1 Sucrose, 0.5 g l –1 MgSO 4·7•H 2 

O, 1 ml l –1 trace 

metal solution (1 g l –1 ZnSO 4·7•H 2 

O, 0.5 g l –1 CuSO 4 

5•H 2 

O)} 

(Health Link ® , Jacksonville, FL) in 250 ml flasks for 7 d at 

25 °C± 0.2 °C in the dark. Each sample was inoculated with 

10 6 spores counted by hemocytometer, previously grown on 

MEA. 

Sample preparation (extraction and cleanup) 

100 ml of liquid culture was homogenized (Waring ® , USA) 

for 2 min. Two millilitre (ml) aliquots were diluted with 2 ml 

of acetonitrile/water (50/50, v/v) containing 0.5 % acetic 

acid, vortex mixed for 30 sec and then filtered through 

Acrodisc syringe filters with 0.45 µm PTFE membrane (Pall 

Corporation, http://www.pall.com/main/home.page) before 

LC/MS analysis. 

Standard 

Ochratoxin A standard was purchased from Sigma (http://www. 

sigmaaldrich.com/united-states.html) and stored at -20 °C. 

LC/MS equipment and parameters 

Analyses were performed on an Agilent 6330 series ion trap 

LC/MS system (http://www.home.agilent.com/), equipped 

with an ESI interface and an 1100 series LC system 

comprising a quaternary pump and an auto-sampler, from 

Agilent Technologies. 

The analytical column was an Allure Bi-Phenyl column 

30 mm x 2.1 mm with 5 µm particle sizes (http://www.restek. 

com/). The column oven was set at 40 °C. The flow rate of the 

mobile phase was 250 µl min l –1 and the injection volume was 

10 µ l –1 . The column effluent was directly transferred into the 

ESI interface, without splitting. 


161


Table 2. GenBank accession numbers of reference and ex-type strains. Sequence IDs in red are the sequences deposited for this manuscript. 

ARTICLE 

Species Source * RPB2 TEF CaM BenA 

Aspergillus acidus ITEM 4507 = CBS 564.65 EF661052 FN665410 AM419749 AY585533 

Aspergillus aculeatus ITEM 7046 T = CBS 172.66 T EF661046 HE984381 AJ964877 AY585540 

Aspergillus ‘aculeatus’ ITEM 4760 = CBS 620.78=NRRL 2053 EF661044 HE984382 EF661145 EU982087 

Aspergillus ‘aculeatus’ ITEM 15927 = NRRL 359 EF661043 HE984383 EF661146 EF661106 

Aspergillus aculeatinus CBS 121060 T = IBT 29077 T HF559233 HF559230 EU159241 EU159220 

Aspergillus aculeatinus ITEM 13553 HE984359 HE984385 HE984422 HE984407 

Aspergillus awamori ITEM 4509 T = CBS 557.65 T HE984360 FN665395 AJ964874 AY820001 

Aspergillus brasiliensis ITEM 7048 = CBS 101740 T EF661063 FN665411 AM295175 AY820006 

Aspergillus brunneoviolaceus ITEM 7047 T = CBS 621.78 T EF661045 HE984384 EF661147 EF661105 

Aspergillus carbonarius ITEM 4503 T = CBS 556.65 T EF661068 FN665412 AJ964873 AY585532 

Aspergillus costaricaensis ITEM 7555 T = CBS 115574 T HE984361 FN665409 EU163268 AY820014 

Aspergillus ellipticus ITEM 4505 T = CBS 482.65 T EF661051 HE984386 AM117809 FJ629279 

Aspergillus fijiensis ITEM 7037 T = CBS 119.49 T HE984362 HE984387 HE818081 HE818086 

Aspergillus helicothrix ITEM 4499 = CBS 677.79 HE984363 HE984389 AM117810 FJ629279 

Aspergillus heteromorphus ITEM 7045 = CBS 117.55 - HE984388 AM421461 FJ629284 

Aspergillus homomorphus ITEM 7556 T = CBS 101889 T HE984365 HE984390 AM887865 AY820016 

Aspergillus ibericus ITEM 4776 T = IMI 391429 T EF661065 HE984391 AJ971805 AM419748 

Aspergillus indologenus ITEM 7038 = CBS 114.80 HE984366 HE984392 AM419750 AY585539 

Aspergillus japonicus ITEM 7034 T = CBS 114.51 T EF661047 HE984393 AJ964875 HE577804 

Aspergillus japonicus ITEM 15926 = NRRL 35494 EU021639 HE984394 EU021690 EU021665 

Aspergillus lacticoffeatus ITEM 7559 T = CBS 101883 T HE984367 FN665406 EU163270 AY819998 

Aspergillus niger ITEM 4501 T = CBS 554.65 T XM_001395124 FN665404 AY585536 AJ964872 

Aspergillus pulverulentus ITEM 4510 T = CBS 558.65 T HE984368 HE984395 HE984423 HE984408 

Aspergillus saccharolyticus ITEM 16159 T = CBS 127449 HF559235 HF559232 HM853554 HM853553 

Aspergillus sclerotioniger ITEM 7560 T = CBS 115572 T HE984369 HE984396 EU163271 AY819996 

Aspergillus tubingensis ITEM 7040 T = CBS 134.48 T EF661055 FN665407 AJ964876 AY820007 

Aspergillus uvarum ITEM 4834 T = IMI 388523 T HE984370 HE984397 AM745755 AM457751 

Aspergillus vadensis ITEM 7561 T = CBS 113.365 T HE984371 FN665408 EU163269 AY585531 

Aspergillus violaceofuscus ITEM 16177 T = CBS 102.23 T HF559234 HF559231 FJ491698 HE577805 

Eluent A was 95 % water: 5 % acetonitrile, and eluent 

B was 95 % acetonitrile: 5 % water, both containing 0.5 % 

acetic acid. Gradient elution was performed starting with 

100 % eluent A, the proportion of eluent B was linearly 

increased to 100 % over a period of 5 min and then kept 

constant for 5 min. The column was re-equilibrated with 100 

% eluent A for 5 min. For LC/MS analyses, the ESI interface 

was used in positive ion mode, with parameters set at: Dry 

TEMP 350 °C; NEBULIZER 40 psi, nitrogen, DRY GAS 10 

l min –1 , Capillary voltage -3500 V. The mass spectrometer 

operated in MRM (multiple reaction monitoring) mode, by 

monitoring three transitions (1 quantifier, 2 qualifiers) for each 

compound, with a dwell time of 200 ms. Quantification of 

ochratoxin A was performed by measuring peak areas in the 

MRM chromatogram, and comparing them with the relevant 

calibration curve. 

Tuning experiments were performed by direct infusion 

at a flow rate of 0.6 ml h –1 of 1µg l –1 standard solutions in 

acetonitrile/water (50/50, v/v) containing 0.5 % acetic acid. 

The infusion was performed by using a model KDS100CE 

infusion pump (KDS Scientific Holliston, MA). 

Interface parameters were: Dry temp 350 °C; 

nebulizer 10 psi nitrogen, DRY GAS 5L/min, Capillary 

voltage -3500 V, spacer was removed for flow infusion. 

Fungal cultures, DNA extraction and DNA 

sequencing 

Monoconidial isolates of each fungal strain were deposited at 

the ITEM Collection (CNR-ISPA, Bari, Italy) and received an 

ITEM accession number (Table 1). Supplemental information 

about the isolates can be recovered from the ITEM electronic 

catalogue (http: www.ispa.cnr.it/Collection). 

For mycelium production, a suspension of spores from 

each fungal strain was grown in Wickerham’s medium 

(glucose 40 g, peptone 5 g, yeast extract 3 g, malt extract 

3 g and distilled water to 1 l). Mycelia were filtered and 

lyophilized for total DNA isolation. The fungal DNA was 

extracted with mechanical grinding using 5 mm iron beads in 

a Mixer Mill MM 400 (http://www.retsch.com/), and a “Wizard ® 

Magnetic DNA Purification System for Food” kit (Promega, 

http://www.promega.com/), starting from 10 mg of lyophilized 

mycelium. The quality of genomic DNA was determined by 

electrophoresis and it was quantified using a ND-1000 (Nano 

Drop) spectrophotometer. 

162 ima fUNGUS


Table 3. GenBank accession numbers of Aspergillus strains isolated from air. 

Species Source RPB2 TEF CaM BenA 

Aspergillus aculeatus ITEM 14807 HE984372 HE984398 HE984424 HE984409 

Aspergillus brunneoviolaceus ITEM 14784 HE984374 HE984400 HE984426 HE984411 

Aspergillus brunneoviolaceus ITEM 14785 HE984375 HE984427 

Aspergillus brunneoviolaceus ITEM 14795 HE984428 

ARTICLE 



Aspergillus floridensis sp. nov. ITEM 14783 T =NRRL 62478 T HE984376 HE984403 HE984429 HE984412 

Aspergillus violaceofuscus ITEM 14787 HE984377 HE984404 HE984430 HE984413 

Aspergillus violaceofuscus ITEM 14788 HE984431 

Aspergillus violaceofuscus ITEM 14789 HE984432 HE9844174 



Aspergillus violaceofuscus ITEM 14805 HE984378 HE984416 





Aspergillus trinidadensis sp.nov. ITEM 14821 T =NRRL 62479 T HE984379 HE984406 HE984434 HE984420 

Aspergillus trinidadensis ITEM 14829 =NRRL 62480 HE984373 HE984399 HE984425 HE984410 

Aspergillus uvarum ITEM 14819 HE984380 HE984435 HE984421 

Aspergillus uvarum ITEM 14826 HE984364 HE984437 

Aspergillus uvarum ITEM 14833 HE984436 

* The sequences were deposited only for the strains that differ in their sequences from the sequence of the type strain for a specific locus/gene. 

Beta-tubulin (BenA, ca. 400 nt) was amplified using 

BT2a and BT2b primers and PCR conditions described by 

Glass & Donaldson (1995), calmodulin (CaM, ca. 650 nt) 

was amplified using CL1 and CL2A primers (O’Donnell et al. 

2000), translation elongation factor-1 alpha (TEF-1α, ca. 700 

nt) was amplified using A-EF_F/A-EF_R primers (Perrone 

et al. 2011) and RNA polymerase II (RPB2, ca. 950 nt) was 

amplified using primers 5F and 7CR (Liu et al. 1999). After 

amplification, the products were purified with the enzymatic 

mixture EXO/SAP (Exonuclease I, Escherichia coli / Shrimp 

Alkaline Phosphatase; Fermentas International, http://www. 

fermentas.com/en/home). 

Bidirectional sequencing was performed for all loci and 

isolates. Sequence reactions were performed with the Big 

Dye Terminator Cycle Sequencing Ready Reaction Kit for 

both strands, purified by gel filtration through Sephadex G-50 

(Amersham Pharmacia Biotech) and analyzed on the “ABI 

PRISM 3730 Genetic Analyzer” (Applied Biosystems, http:// 

www.appliedbiosystems.com/absite/us/en/home.html). 

The preliminary alignments of sequences from each 

of the four loci was performed using the software package 

BioNumerics 5.1 from Applied Maths (http://www.appliedmaths.com/bionumerics/bionumerics.htm) 

with manual 

adjustments where judged necessary. 

Sequence Data Analysis 

DNA sequences were aligned using the Clustal W algorithm 

(Thompson et al. 1994) in MEGA version 5 (Tamura et al. 

2011). Sequences were deposited in GenBank (Tables 2 & 

3). Each locus was aligned separately and then concatenated 

in a super-gene alignment used to generate the phylogenetic 

tree. Phylogenetic analysis was performed in MEGA5 

using both Neighbor-Joining (NJ) (Saitou & Nei 1987) and 

Maximum Likelihood (ML) methods and the Tamura-Nei 

model (Tamura & Nei 1993). Evolutionary distances for NJ 

were computed using the Tamura-Nei method of the package 

and are in units of number of base substitutions per site. All 

positions containing gaps and missing data were eliminated 

from the dataset (Complete deletion option). Bootstrap 

values (Felsenstein 1985, 1995) were calculated from 1000 

replications of the bootstrap procedure using programs within 

MEGA5. 

The evolutionary history was inferred by using the 

Maximum Likelihood method based on the Tamura-Nei 

model implemented in MEGA5. The percentage of trees 

in which the associated taxa clustered together is shown 

next to the branches. Initial tree(s) for the heuristic search 

were obtained automatically as follows. When the number 

of common sites was < 100 or less than one fourth of the 

total number of sites, the maximum parsimony method was 


163


ARTICLE 

Table 4. Provenance of Aspergillus section Nigri isolates used as reference strains 

Species Source * Provenance 

Aspergillus acidus 

Aspergillus aculeatus 

ITEM 4507 T = IMI 104688 T = CBS 564.65 T 

= NRRL 4796 T JAPAN: unknown, R. Nakazawa. 


= NRRL 5094 T USA: isol. ex tropical soil, 1962, K. B. Raper. 

Aspergillus ‘aculeatus’ ITEM 4760 = CBS 620.78 = NRRL 2053 

= IMI 358696 

New Guinea: isol. ex canvas tent, 1946, received from D. L. 

White 

Aspergillus ‘aculeatus’ ITEM 15927 = NRRL 359 Thom and Raper 1945 received it from Dr. A. F. Blakeslee. 

Aspergillus brunneoviolaceus 

ITEM 7047 T = CBS 621.78 T = IMI 312981 T 

= NRRL 4912 T BRAZIL: culture contaminant, A. C. Batista and H. Maia. 

Aspergillus aculeatinus ITEM 16172 T = CBS 121060 T = IBT 29077 T THAILAND: Chumporn Prov.: isol. ex arabica coffee, P. Noonim. 

Aspergillus aculeatinus ITEM 13553 SRI LANKA: isol. ex human dacryocystitis. 

Aspergillus awamori 

Aspergillus brasiliensis 

Aspergillus carbonarius 

ITEM 4509 T = CBS 557.65 T = NRRL 4948 T Unknown - Raper and Fennel 1965 received it from the Instituto 

= IMI 211394 T Ozwaldo Cruz 

ITEM 7048 = IMI 381727 T = CBS 101740 T 

= NRRL 26652 T BRAZIL: Pedreira: isol. ex soil, J. H. Croft. 


= NRRL 369 T Unknown: paper, A. F. Blakeslee. 

Aspergillus costaricaensis ITEM 7555 T = CBS 115574 T COSTA RICA: Taboga island: isol. ex soil, 2000, M. Christensen. 

Aspergillus ellipticus 


= NRRL 5120 T COSTA RICA: isol. ex soil, 1962, K. J. Kwon. 

Aspergillus fijensis ITEM 7037 T = CBS 119.49 T INDONESIA: Palembang: Lactuca sativa, 1949. 

Aspergillus heteromorphus 

ITEM 7045 T = CBS 117.55 T = IMI 172288 T 

= NRRL 4747 T BRAZIL: Recife: culture contaminant, A. C. Batista. 

Aspergillus homomorphus ITEM 7556 T = CBS 101889 T ISRAEL: isol. ex soil 2 km away from Dead Sea. 

Aspergillus ibericus 

ITEM 4776 T = CBS 121594 T = IMI 391429 T 

= NRRL 35644 T PORTUGAL : Iberian Peninsula: isol. ex grapes, 2001, R. Serra. 

Aspergillus indologenus ITEM 7038 T = CBS 114.80 T = IBT 3679 T INDIA: isol. ex soil. 

Aspergillus japonicus ITEM 7034 T = CBS 114.51 T Unknown, K. Saito. 

Aspergillus japonicus ITEM 15926 = NRRL 35494 Unknown. 

Aspergillus lacticoffeatus ITEM 7559 T = CBS 101883 T INDONESIA: South Sumatra: isol. ex coffee bean, J. M. Frank. 

Aspergillus niger 

Aspergillus pulverulentus 

ITEM 4501 T = IMI 050566 T = CBS 554.65 T USA: Connecticut: tannin-gallic acid fermentation, 1913, A. 

= NRRL 326 T Hollander. 

ITEM 4510 T = CBS 558.65 T = NRRL 4851 T AUSTRALIA: Victoria: isol. ex Phaseolus vulgaris, ~1907, D. 

= IMI 211396 T McAlpine. 

Aspergillus sclerotioniger ITEM 7560 T = CBS 115572 T = IBT 22905 T INDIA: Karnataka: isol. ex green arabica coffee J. M. Frank. 

Aspergillus tubingensis ITEM 7040 T = CBS 134.48 T = NRRL 4875 T Unknown: 1948, deposited by R. Mosseray. 


ITEM 4834 T = IMI 388523 T = CBS 127591 T 

= IBT 26606 T ITALY: Brindisi: isol. ex grapes, 2001, P. Battilani. 

Aspergillus uvarum ITEM 4685 = IMI 387209 PORTUGAL: Régua, Douro Region: isol. ex grapes. 

Aspergillus uvarum ITEM 4962 = IMI 3888715 SPAIN: isol. ex grapes. 

Aspergillus uvarum ITEM 4997 = IMI 388670 ISRAEL: Lichron: isol. ex grapes. 

Aspergillus uvarum ITEM 5020 = IMI 388660 ITALY: Brindisi, Apulia: isol. ex grapes. 

Aspergillus uvarum ITEM 5321 = IMI 389195 FRANCE: Narbonne, Languedoc: isol. ex grapes. 

Aspergillus uvarum ITEM 5350 = IMI 389166 ISRAEL: Pdaya: isol. ex grapes. 

Aspergillus vandensis ITEM 7561 T = IMI 313493 T = CBS 113365 T EGYPT: air, A. H. Moubasher. 

Aspergillus violaceofuscus ITEM 16159 T = CBS 102.23 T FRANCE: Strassbourg: received by D Borrel, 1923 

Aspergillus saccharolyticus ITEM 16177 T = CBS 127449 T = IBT 28509 T DENMARK: Gentofte: ex under a toilet seat made of treated oak 

wood, P. J. Teller. 

* 

CBS, Centraalbureau voor Scimmelcultures, Utrecht, The Netherlands; IMI, CABI Bioscience Genetic Resource Collection, Egham, United 

Kingdom; ITEM, Agri-Food Toxigenic Fungi Culture Collection, Institute of Sciences of Food Production, Bari, Italy; NRRL (Northern Regional 

Research Laboratory), the National Center For Agricultural Utilization Research, USA. 

T 

= ex-type strain 

164 ima fUNGUS


Table 5. Sequence characteristics and phylogenetic information for RPB2, TEF, CaM, BenA and combined MLS. 

Locus Region Sites Net Sites % 

GC 

No. of 

variable 

sites 

No. of 

informative 

sites 

No. of 

mutations 

(Eta) 

Nucleotide 

diversity 

BenA 1-369 369 326 58 180 149 229 0.101 

CaM 370-937 568 496 53 269 224 349 0.103 

RPB2 938-1922 985 878 52 370 271 442 0.063 

TEF 1923-2552 630 629 57 93 55 105 0.023 

MLS 1-2560 2552 2329 55 912 699 1125 0.067 

ARTICLE 

used; otherwise the BIONJ method with MCL distance matrix 

was used. A discrete Gamma distribution was used to model 

evolutionary rate differences among sites (five categories; 

+G, parameter = 0.2036). All positions containing gaps and 

missing data were eliminated. There were 2329 positions 

in the final dataset. The tree is drawn to scale, with branch 

lengths measured in the number of substitutions per site. 

A Markov Chain Monte Carlo (MCMC) algorithm was used 

to generate phylogenetic trees with Bayesian probabilities 

using MrBayes v3. 2 (Huelsenbeck & Ronquist 2001, 

Ronquist & Huelsenbeck 2003) for the combined sequences 

datasets. The analysis was run in duplicate with four MCMC 

chains and setting random trees for 10 7 generations sampled 

every 100 generations. A total of 15 738 trees were read 

in the two runs, 7869 for each, and the first 1967 trees (25 

%) were discarded in each run as the burn-in phase of the 

analysis and posterior probabilities were determined from the 

remaining trees (5902 in each run). 

Maximum Parsimony analysis (MP) was performed for 

all data sets using the heuristic search option and Close- 

Neighbor-Interchange algorithm (with search level 1 in which 

the initial trees were obtained with the random addition of 

sequences). To assess the robustness of the topology, 1000 

bootstrap replicates were run. The tree is drawn to scale, 

with branch lengths calculated using the average pathway 

method and are in units of the number of changes over the 

whole sequence (Nei & Kumar 2000). The analysis involved 

data from 86 isolates and all positions containing gaps and 

missing data were eliminated. There were a total of 2329 

positions in the final dataset. Evolutionary analyses were 

conducted in MEGA5 (Tamura et al. 2011). 

RESULTS 

Phylogenic analysis of sequence data 

The multilocus analysis was performed on 56 isolates collected 

from air (52 homes and 4 outside samples) from 17 states of 

the United States, Bermuda, Martinique, Trinidad and Tobago, 

and one isolated from almonds in the Czech Republic (Table 

1), along with 28 reference and ex-type strains from Aspergillus 

section Nigri (Table 4). The ex-type strain of Aspergillus flavus 

(ITEM 7526) was used as outgroup. The percentage of variable 

sites and parsimony informative sites for each locus differ, the 

benA sequences have the highest percentage of variable and 

parsimony informative sites, the CaM sequences have the 

highest nucleotide diversity, TEF sequences have the lowest 

variability and RPB2 has lower sequence diversity than CaM 

and benA but the highest number of informative sites (Table 5). 

After a preliminary analysis using MEGA5 Neighbour-Joining, 

the best substitution model among the evolutionary models 

in MEGA5 was calculated. The best model was Tamura-Nei 

with Gamma distribution (TN93 + G). Evolutionary history was 

inferred using the Neighbor-Joining method. The tree with the 

highest log likelihood is shown (Fig 1). Bootstrap proportions 

are shown next to the branches. The tree is drawn to scale, 

with branch lengths reflecting evolutionary distance computed 

using the Maximum Composite Likelihood method as number 

of base substitutions per site (MEGA5). The rate variation 

among sites was modeled with a gamma distribution (shape 

parameter = 0.3). Phylogenetic analysis was conducted first 

on the four single locus alignments and subsequently the 

combined alignment of the four loci. The single locus and 

four locus combined data trees contained the same topology 

fulfilling the requirements of genealogical concordance 

phylogenetic species recognition (GCPSR, Taylor et al. 2000). 

Of the 56 strains collected from air, 30 strains were A. 

violaceofuscus (syn. A. japonicus), 18 A. brunneoviolaceus 

(syn. A. fijiensis), three A. uvarum, one (ITEM 14807) was 

A. aculeatus, two (ITEM 14821 and 14829) were grouped 

(high bootstrap) in a distinct cluster from A. aculeatinus, 

and three (ITEM 14783, CCF 4046 and CCF 4236) were 

phylogenetically isolated (with strong statistical support) 

from A. aculeatus, and are described as two new species 

here. In addition, two strains previously characterized by 

CaM analysis CRI 323-04 (sequence accession number 

FJ525444, Ingavat et al 2009) and IFM 55703 (sequences 

from Tetsuhiro Matsuzawa, Chiba University, Japan) 

resulted to have an homology > 99.5% with ITEM 14783. 

They also showed a different phylogenetic position from the 

uniseriate species decribed as A. indologenus (CBS 114.80), 

A. brunneoviolaceus (ITEM 7047) and from two atypical 

Aspergillus “aculeatus” (ITEM 4760 and ITEM 15927) strains, 

not yet well-defined and characterized as belonging in any of 

the known uniseriate species. 

Bayesian inference analysis of the multilocus (benA, 

CaM, TEF-1α, RPB2) data set produced a phylogenetic 

tree (log likelihood 14835.94) with high PP values for 

the same monophyletic group obtained with Maximum 

Likelihood analysis, the atypical A. aculeatus isolate 

ITEM 4760 clustered together with ITEM 14873, while 

the two other atypical A. aculeatus isolates were placed 

in the A. brunneoviolaceus clade. The results obtained 

by the Maximum Parsimony analysis are represented 

by one of the 80 equally most parsimonious trees (Fig. 

2). The consistency index is (0.491302), the retention 


165


ARTICLE 

99 

99 

77 

99 

98 

97 

93 

ITEM 14828 

ITEM 14836 

ITEM 14818 

ITEM 14817 

ITEM 14814 

ITEM 14808 

ITEM 14797 

ITEM 14789 

ITEM 14792 

ITEM 14835 

ITEM 14793 

ITEM 14822 

ITEM 14801 

ITEM 16159 A. violaceofuscus T 

ITEM 14805 

ITEM 14837 

ITEM 14798 

ITEM 14815 

ITEM 14824 

ITEM 14803 

ITEM 14810 

ITEM 14800 

ITEM 14788 

ITEM 14787 

ITEM 7034 A. japonicus T 

ITEM 14811 

ITEM 14813 

ITEM 14816 

ITEM 14823 

ITEM 14830 

ITEM 14834 

ITEM 14827 

ITEM 15926 (NRRL35494) 

ITEM 7038 A. indologenus T 

ITEM 14826 

ITEM 14819 

ITEM 4834 A. uvarum T 

ITEM 14833 

ITEM 7046 A. aculeatus T 

ITEM 14807 

ITEM 14783 A. floridensis sp nov. 

CCF 4046 

CCF 4236 

ITEM 4760 A. aculeatus atypic (CBS 620.78) 

97 ITEM 16172 A. aculeatinus T (CBS 121060) 

ITEM 13553 A. aculeatinus 

92 

ITEM 14821 A. trinidadensis sp. nov. 

93 ITEM 14829 

95 

78 

99 

99 

99 

95 

99 

ITEM 15297 A. aculeatus atypic (NRRL 359) 

ITEM 7047 A. brunneoviolaceus T (CBS 621.78) 

ITEM 14784 

76 

ITEM 14825 

73 

ITEM 14802 

ITEM 14795 

ITEM 14832 

ITEM 14804 

ITEM 7037 A. fijiensis T 

ITEM 14785 

ITEM 14786 

ITEM 14790 

ITEM 14791 

72 ITEM 14794 

ITEM 14796 

99 

70 

ITEM 14799 

ITEM 14806 

ITEM 14809 

ITEM 14812 

ITEM 14820 

ITEM 14831 

99 

99 

97 

86 

98 

92 

ITEM 7526 A. flavus T 

ITEM 7556 A. homomorphus T 

ITEM 16177 A. saccharolyticus T 

ITEM 4503 A. carbonarius T 

ITEM 7560 A. sclerotioniger T 

ITEM 4776 A. ibericus T 

ITEM 4505 A. ellipticus T 

ITEM 7048 A. brasiliensis T 

ITEM 4501 A. niger T 

ITEM 7559 A. lacticoffeatus T 

ITEM 4509 A. awamori T 

ITEM 4510 A. pulverulentus T 

ITEM 7040 A. tubingensis T 

ITEM 4507 A. acidus T 

ITEM 7555 A. costaricaensis T 

ITEM 7561 A. vadensis T 

ITEM 7045 A. heteromorphus T 

0.02 

Fig. 1. Phylogenetic trees produced from the combined sequence data of four loci (CaM, benA, RPB2 and TEF) of 57 strains of uniseriate black 

Aspergillus, 28 reference strains of species belonging to Aspergillus section Nigri, and A. flavus (ITEM 7526) as outgroup. Numbers above 

branches are bootstrap values. Only values above 70 % are indicated. The evolutionary history was inferred using the Neighbour-Joining method 

computed with the Maximum Likelihood Evolutionary method. 

166 ima fUNGUS


99/1.0 

100/1.0 

-/0.97 

97/1.0 

ITEM 7526 A. flavus T 

100/1.0 

84/1.0 

97/1.0 

99/1.0 

82/0.99 

100/1.0 

ITEM 16159 A. violaceofuscus T 

ITEM 14805 

ITEM 14837 

ITEM 14813 

ITEM 14811 

ITEM 14810 

ITEM 14798 

ITEM 14815 

ITEM 14816 

ITEM 14830 

ITEM 14824 

ITEM 14823 

ITEM 14788 

ITEM 14801 

ITEM 14834 

ITEM 14822 

ITEM 14793 

ITEM 14835 

ITEM 14818 

ITEM 14792 

ITEM 14814 

ITEM 14817 

ITEM 14808 

ITEM 14797 

ITEM 14828 

ITEM 14789 

ITEM 14836 

ITEM 14803 

ITEM 7034 A. japonicus T 

ITEM 14800 

ITEM 14787 

ITEM 14827 

ITEM 15926 (NRRL35494) 

ITEM 7038 A. indologenus T 

ITEM 14826 

ITEM 4834 A. uvarum T 

ITEM 14819 

ITEM 14833 

ITEM 7046 A. aculeatus T 

ITEM 14807 

ITEM 14783 A. floridensis sp. nov. 

96/1.0 

CCF 4046 

CCF 4236 

ITEM 4760 A. aculeats atypic (CBS 62078) 

ITEM 16172 A. aculeatius T (CBS 121060) 

ITEM 13553 A. aculeatinus 

ITEM 14821 A. trinidadensis sp. nov . 

ITEM 14829 

ITEM 15297 A. aculeatus atypic (NRRL 359) 

ITEM 7047 A. brunneoviolaceus T (CBS 621.78) 

ITEM 14802 

96/1.0 

-/0.91 

-/0.97 79/0.97ITEM 14825 

98/1.0 

76/1.0 

-/0.99 

84/0.89 

99/1.0 

97/1.0 100/1.0 

98/1.0 

92/1.0 

84/1.0 

80/0.91 

-/0.97 

-/0.81 

-/0.95 

-/0.82 

-/0.81 

ITEM 14784 

ITEM 14832 

ITEM 14794 

ITEM 14799 

ITEM 14812 

ITEM 14804 

ITEM 14820 

ITEM 14795 

ITEM 14831 

ITEM 14786 

ITEM 14785 

ITEM 7037 A. fijiensis T 

ITEM 14790 

ITEM 14809 

ITEM 14791 

ITEM 14796 

ITEM 14806 

ITEM 7556 A. homomorphus T 

ITEM 16177 A. saccharolyticus T 

ITEM 4505 A. ellipticus T 

ITEM 7045 A. heteromorphus T 

81/1.0 

100/1.0 

-/- 

-/- 

-/- 

100/1.0 

100/1.0 

ITEM 4776 A. ibericus T 

ITEM 7560 A. sclerotioniger T 

ITEM 4503 A. carbonarius T 

ITEM 4501 A. niger T 

ITEM 7559 A. lacticoffeatus T 

ITEM 4509 A. awamori T 

ITEM 7048 A. brasiliensis T 

84/1.0 

99/1.0 

-/0.80 

83/0.99 

ITEM 4510 A. pulverulentus T 

ITEM 7040 A. tubingensis T 

ITEM 4507 A. acidus T 

ITEM 7555 A. costaricaensis T 

ITEM 7561 A. vadensis T 

ARTICLE 

50 

Fig. 2. Maximum parsimony phylogram derived from the combined sequence data of four loci (CaM, benA, RPB2 and TEF) of 57 strains of 

uniseriate black Aspergillus, 28 reference strains of Aspergillus section Nigri, and A. flavus (ITEM 7526) as outgroup. Numbers at nodes are 

bootstrap values/Bayesian posterior probabilities. A dash indicates the support for the branch was < 70 % BP or < 0.80 PP. 

index is (0.863662), and the composite index is 0.466789 

(0.424319) for all sites and parsimony-informative sites (in 

parentheses). The MP phylogenetic analysis agreed with 

the evolutionary results obtained by the ML and Bayesian 

analysis. A. violaceofuscus, A. brunneoviolaceus and A. 

uvarum are the principal black aspergilli “uniseriate” species 

collected in these indoor air samples with the identification 

of two possible new species (ITEM 14821 and 14783). The 

topology of MP, Bayesian phylogenetic trees is concordant, 

and the two trees are represented together in Fig. 2. The 

phylogenetic tree obtained by ML analysis has also the same 

topology of the other two phylogenetic analyses with some 

minor exception regarding the clades of A. ellipticus and 

A. heteromorphus (Fig. 1). All three phylogenetic analyses 

performed give evidence with high bootstrap that the two 

new species belong to different monophyletic groups (Figs 

1–2), and that the atypical strain (ITEM 15297) belongs to 

the A. brunneoviolaceus clade, and that ITEM 4760 needs 

further characterization as belonging alone in a clade close 

to the new species A. floridensis but with no high supported 

bootstrap (Figs 1–2). 

TAXONOMY 

Previously described species 

Aspergillus brunneoviolaceus Batista & Maia, Anais 

Soc. Biol. Pernambuco 13: 91 (1955). 

Synonym: Aspergillus fijiensis Varga et al., Stud. Mycol. 

69: 9 (2011). 

(Fig. 3a–f) 


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ARTICLE 

A 

D 

B 

C 

D 

E 

F 

Fig. 3. Aspergillus brunneoviolaceus (syn. A. fijiensis; ITEM 7037), culture plates are 9 cm diam, colonies grown at 25 °C for 7 d. A. CYA 

colonies. B. MEA colonies. C. CREA colonies. D–E. Stipes smooth or with a limited surface granulation just below the vesicle, globose to 

ellipsoidal vesicle, and conidia. F. Globose to ellipsoidal, conidia, with echinulate surface. Bars = 10 µm. 

Type: (CBS 621.78 T =NRRL 4912 T ). 

Description: Colony diameters after 7 d incubation at 25 °C on 

CYA (Fig. 3a) > 85 mm (50–75 mm 5 d), MEA (Fig. 3b) 45– 

75(< 85) mm, CY20S 50–65 mm, OA 55–70 mm, CREA (Fig. 

3c) displayed poor sporulation but commonly good to very 

good acid production, conidial heads on CYA brown to dark 

brown near black, commonly abundant, velutinous to slightly 

floccose, white to buff mycelium, commonly moderate radial 

sulcation, exudate clear to brown, sparse to abundant, soluble 

pigment not seen, occasionally present and brown at 37 °C, 

if present sclerotia subglobose to elongate 250–800 µm long, 

buff to orange-brown, reverse buff to yellow. On MEA conidial 

heads are brown, sclerotia absent, mycelium white, reverse 

uncolored to yellowish-gray. Incubation for 7 d on CYA at 5 °C 

produced no growth or germination of conidia. Incubation for 7 

d on CYA at 35 °C and 37 °C produced growth of 35–63 mm, 

and (12–)17–26 mm diam, respectively. 

Stipes (Fig. 3d–e) smooth or with a limited surface 

granulation just below the vesicle, hyaline or pigmented just 

below the vesicle, (75–)200–800(–1600) × (8–)10–15(–21) 

µm, isolate ITEM 7037 has longer stipes (400–)800–2000 

(–3400) × (8–)10–15(–18) µm than other A. brunneoviolaceus 

isolates, vesicles globose to elipsoidal, (30–)35–70(–90) µm 

diam, conidial heads uniseriate, phialides (6–)7–9(–10) × 3.5– 

4.5(–5) µm covering entire vesicle, conidia (Fig. 3e) globose 

to ellipsoidal, 3.5–4.5(–6) × 3.5–4.5(–5) µm, occasionally 

subglobose to angular 2.5–3.5 µm, brown near black, with 

coarsely roughened to echinulate surface. 

Aspergillus uvarum G. Perrone et al., Int. J. Syst. 

Evol.Microbiol. 58: 1036 (2008). 

MycoBank MB510962. 

(Fig. 4a–f). 

Type: Italy: Apulia, Brindisi, isol. ex grapes (ITEM 4834 T ; 

= IMI 388523 T ). 

Description: Colony diameters after 7 d incubation at 

25 °C on CYA (Fig. 4a) > 85 mm (47–88 mm at 5 d), MEA 

(Fig. 4b) > 85 mm (58–84 mm at 5 d), CY20S 55–65 mm, 

OA (Fig. 4c) 60-70 mm, CREA produced good growth, acid 

production ranged from good to very poor to none depending 

on the isolates, conidial heads on CYA brown to dark brown 

near black, sporulating abundantly, granular, mycelium 

white to buff, moderate to deep radial sulcation, exudate 

168 ima fUNGUS


ARTICLE 

A 

B 

C 

D 

E 

F 

Fig. 4. Aspergillus uvarum (ITEM 4834 T ), culture plates are 9 cm diam, colonies grown at 25 °C for 7 d. A. CYA colonies. B. MEA colonies. C. OA 

colony. D–E. Smooth stipes, globose to ellipsoidal vesicle, and conidia. F. Globose to ellipsoidal, conidia, with echinulate surface. Bars = 10 µm. 

clear to brown, soluble pigments when present pale-yellow, 

brown at 35 °C, at 37 °C rarely present, brown, sclerotia 

when present abundant in the center of the colony, globose 

to elongate, buff to brown, reverse wrinkled, white to dull 

brown occasionally pink-orange. On MEA conidial heads 

are brown to dark brown, sporulating abundantly, mycelium 

white and commonly inconspicuous, reverse yellowishgrayish-green. 

Incubation for 7 d on CYA at 5 °C produced no 

growth or germination of conidia. Incubation for 7 d on CYA 

at 35 °C and 37 °C produced growth of 18–27(–46) mm, and 

(germinate)3–15(–21) mm diam, respectively. 

Stipes (Fig. 4d–e) smooth, hyaline, becoming brown with 

age (250–)600–1800(–3600) × (8–)10–18(–24) µm, vesicles 

globose to ellipsoidal, (30–)45–100(–121) µm diam, conidial 

heads uniseriate, phialides 7–10(–12) × (3–)3.5–4.5(–7) µm 

covering entire vesicle, conidia (Fig. 4f) globose to ellipsoidal, 

(4–)4.5–7(–9) × 3.5–7 µm, with echinulate surface. 

New species 

Aspergillus floridensis Ž. Jurjević, G. Perrone & 

S. W. Peterson, sp. nov. 


(Fig. 5a–g) 

Etymology: Isolated in Florida. 

Type: USA: Florida: isol. ex air sample, August 2010, Ž. 

Jurjević (BPI 883907 – holotype; from dried colonies of ITEM 

14783 (=NRRL 62478) grown 7 d at 25 °C on CYA and MEA) 

. 

Diagnosis: Stipes uniseriate, mycelium white to yellow on 

MEA, vesicles globose to subglobose occasionally ellipsoidal 

(14–)35–65(–105) µm diam, conidia globose to ellipsoidal, 

4–5(–6) × 3.5–5.5 µm, with echinulate surface, incubation at 

37 °C produced growth of 18–24 mm diam. 

Description: Colony diameters after 7 d incubation at 25 °C 

on CYA (Fig. 5a) 80–85 (> 85) mm, MEA (Fig. 5c) 50–55 (> 

85) mm, CY20S 32–53 mm, OA 55–60 mm, CREA (Fig. 5d) 

displayed poor sporulation but good acid production, conidial 

heads on CYA dark brown to black, abundantly produced, 

globose to subglobose at first and later radiate, developing into 

columns, mycelium white to buff-yellow, velutinous, moderate 

radial sulcation, exudate clear to brown, sparse to abundant, 

soluble pigment not seen, occasionally producing buff-yellowish 

sclerotia (Fig. 5b) subglobose to elongate (200–)400–700(– 

1100) µm long, reverse brownish-yellow to yellow-brown. On 

MEA conidial heads are brown, sclerotia absent, mycelium 


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ARTICLE 

A 

B 

C 

D 

D E F 

E 

F 

G 

Fig. 5. Aspergillus floridensis (ITEM 14783 T ) culture plates are 9 cm diam, colonies grown at 25 °C for 7 d. A. CYA colonies. B. CYA colony, buffyellowish 

sclerotia, subglobose to elongate (200–)400–700(–1100) µm long, clear to brown exudates. C. MEA colonies. D. CREA colony. E–F. 

Smooth stipes, globose to ellipsoidal vesicle, and conidia. G. Globose to ellipsoidal, conidia, with echinulate surface. Bars = 10 µm. 

white to yellow, reverse gray to grayish-yellow. Incubation for 7 

d on CYA at 5 °C produced no growth or germination of conidia. 

Incubation for 7 d on CYA at 35 °C and 37 °C produced growth 

of 40–50 mm and 18–24 mm diam, respectively. 

Stipes smooth, hyaline (50–)200–650(–950) × (8–)10– 

15(–21) µm, vesicles (Fig. 5e–f) globose to subglobose 

occasionally ellipsoidal (14–)35–65(–105) µm diam, conidial 

heads uniseriate, phialides (6–)7–9(–11) × 3.5–4.5(–5) µm 

covering entire vesicle, conidia (Fig. 5g) globose to ellipsoidal, 

4–5(–6) × 3.5–5.5 µm, with echinulate surface. No ochratoxin 

A produced. 

Aspergillus trinidadensis Ž. Jurjević, G. Perrone & 

S. W. Peterson, sp. nov. 


(Fig. 6a–g) 

Etymology: Isolated in Trinidad. 

Type: Trinidad & Tobago:Tunapuna, isol. ex air sample, July 

2011, Ž. Jurjević (BPI 883908 – holotype; from dried colonies 

of ITEM 14821 T (=NRRL 62479 T ) grown 7 d at 25 °C on CYA 

and MEA). 

Diagnosis: Stipes uniseriate, mycelium white to orangishyellow 

on CYA, vesicles globose to subglobose occasionally 

ellipsoidal (10–)30–70(–100) µm diam, conidia large 4–7(–8) 

× 3.5–7 µm, if borne from monophialides up to 13 × 10 µm, 

with finely spiny to echinulate surface, and range from no 

growth to 7 mm diam growth at 37 °C. 

Description: Colony diameters after 7 d incubation at 25 °C 

on CYA (Fig. 6a) 65–78 mm, MEA (Fig. 6b) 57 to > 85 mm, 

CY20S 50–55 mm, OA 55–60 mm, CREA (Fig. 6c) showed 

poor sporulation and no acid production, conidial heads on 

CYA brown to dark brown, globose to subglobose initially, 

later radiate, then developing into columns, sporulating 

well, mycelium white to yellowish creamy or orangishyellow 

toward the center of the colony, white at margins, 

floccose, moderate to deep radial sulcation, exudate clear 

to brownish, soluble pigments and sclerotia not seen, 

occasionally globose to elongate chlamydospores present, 

reverse brown to brownish-yellow. On MEA conidial heads 

brown to dark brown, sporulating well centrally, mycelium 

white to buff-yellowish-orange, reverse buff. Incubation 

for 7 d on CYA at 5 °C produced no growth or germination 

of conidia. Incubation for 7 d on CYA at 35 °C and 37 °C 

170 ima fUNGUS


ARTICLE 

A 

B 

C 

F 

F 

D 

E 

G 

Fig. 6. Aspergillus trinidadensis (ITEM 14821 T ) culture plates are 9 cm diam, colonies grown at 25 °C for 7 d. A. CYA colonies. B. MEA colonies. 

C. CREA colonies. D–E. Smooth stipes, globose to subglobose vesicle, and conidia. F. Globose to ellipsoidal, conidia, with echinulate surface. 

G. Monophialides and conidia. Bars = 10 µm. 

produced growth 4–21 mm and from no growth to 7 mm 

diam, respectively. 

Stipes (Fig. 6d–e) smooth or occasionally with a limited 

surface granulation just below the vesicle, hyaline or 

occasionally pigmented just below the vesicle, long if from 

substrate, short with small vesicles if borne from aerial 

hyphae (50–)150–800(–1800) × (5–)8–14(18) µm, vesicles 

globose to subglobose occasionally ellipsoidal (10–)30–70(– 

100) µm diam, conidial heads uniseriate, phialides (5-)7–9(– 

12) × (3–)3.5–4.5(–6) µm commonly covering entire vesicle, 

occasionally producing monophialides (Fig. 6g) 3–42 × 3.5–8 

µm, conidia (Fig. 6f) globose to ellipsoidal, rarely pyriform, 

4–7(–8) × 3.5–7 µm, if borne from monophialides up to 13 × 

10 µm, with finely spiny to echinulate surface. No ochratoxin 

A produced. 

DISCUSSION 

In our studies of the indoor environment the dominant species 

of uniseriate Aspergillus section Nigri were A. violaceofuscus 

(syn. A. japonicus) (30 of 55 isolates) and A.brunneoviolaceus 

(syn. A. fijiensis) (18 of 55 isolates). Aspergillus violaceofuscus 

was isolated from 15 states in the USA, mainly from Southern 

and Mid-Atlantic states (Table 1). Aspergillus violaceofuscus 

(syn. A. japonicas) and A. aculeatus have previously only 

been found in the tropics (Nielsen et al. 2009). Nine isolates 

(ITEM 14800, 14803, 14805, 14810, 14827, 14828, 14830, 

14834, and14837) of the 30 A. violaceofuscus isolates 

produced sclerotia on CYA or OA, buff to yellowish-orange 

or orange-brown, subspherical to elongate, 300–1000 µm 

long. Also, three isolates (ITEM 14794, ITEM 14799, and 

ITEM 14802) of A. brunneoviolaceus produced abundant 

buff to orange-brown sclerotia, 250–800 µm long. None of 

the three sclerotium producing A. brunneoviolaceus isolates 

produced ochratoxin A. Aspergillus brunneoviolaceus 

isolates commonly have good to very good acid production. 

However, two isolates ITEM 14806 and ITEM 14831, did not 

show acid reactions on CREA agar, nor were they sulcate 

on CYA. Aspergillus brunneoviolaceus (syn. A. fijiensis) was 

previously isolated from soil, Fiji (CBS 313.89), Lactuca 

sativa, Indonesia (CBS 119.49) (Varga et al. 2011), guano, 

Peru (IHEM 18675), corneal scraping keratitis, India (IHEM 

22812), droppings of Coenobita sp., Bahamas (IHEM 4062) 

(Hendricks at al. 2011), and industrial material, China (CCF 

108) (Hubka & Kolarik 2012). This is the first report of A. 


171


ARTICLE 

brunneoviolaceus isolated from the indoor air environment 

and the first reported isolation in the United States. We found 

only one isolate of A. aculeatus and three isolates of A. 

uvarum (Table 1). A. uvarum was previously known only from 

grapes in the Mediterranean basin (Perrone et al. 2008). This 

is the first time that A. uvarum was isolated from the indoor air 

environment and its first isolation in the USA. 

A. brunneoviolaceus, A. uvarum, and A. violaceofuscus 

are the uniseriate black aspergilli occurring in the indoor 

environment in the USA. The A. brunneoviolaceus clade 

(Fig. 1) showed the presence of two statistically supported 

subgroups, one included 15 strains and the ex-type strain 

of A. fijiensis ITEM 7037, while the other included 3 strain 

(ITEM 14802, 14825, and 14784). Two strains previously 

characterized as atypical A. aculeatus (ITEM 7047 – the extype 

strain of A. brunneoviolaceus, and NRRL 359) belong 

to the same subclade as A. brunneoviolaceus with high 

bootstrap in all the three phylogenetic analysis conducted 

(Figs 1–2). These findings confirm the data of Hubka & 

Kolarik (2012) that suggest treating A. fijiensis as a synonym 

of A. brunneoviolaceus because they are indistinguishable by 

multilocus sequence analysis and belong in the same highly 

supported clade. Then, as A. brunneoviolaceus has been 

previously described at species level, we suggest treating A. 

fijiensis as a synonym of it, in agreement with findings of Hubka 

& Kolarik (2012). The same should be done for A. japonicus 

and A. violaceofuscus, previously proposed as separate taxa 

(Varga et al. 2011), as our phylogenetic results do not support 

this separation and suggest they should be treated as the 

same taxon; i.e. A. japonicus should be treated as a synonym 

of A. violaceofuscus which was described earlier. 

In the case of the atypical A. aculeatus isolate ITEM 4760, 

although the molecular difference suggests the possible 

recognition of further new species, there is no unique 

topology among the four single locus trees. Two loci indicate 

it belongs to A. brunneoviolaceus and the other two loci form 

a clade with the A. floridensis (data not shown). When the 

combined multilocus alignment was conducted, ML, MP, and 

PP criteria put it close to A. floridensis (Figs 1–2), but not with 

a high bootstrap/PP value. 

Phenotypically, the atypical A. aculeatus (ITEM 4760) 

grows slower on CY20S (30 mm diam) and CYA (70-78 mm 

diam) after 7 d at 25 °C than A. brunneoviolaceus isolates 

that grow on CYA < 85 mm (50–75 mm diam 5 d), and CY20S 

50–65 mm. ITEM 4760 also has slower growth on CYA when 

compared with Aspergillus floridensis that grows on CYA 80– 

85(> 85) mm diam after 7 d at 25 °C. 

The phylogenetic analysis evidenced both in single locus 

and in a multilocus analysis showed that the two strains 

ITEM 14821 and 14829 of A. trinidadensis belong to the A. 

aculeatinus clade, a black Aspergillus species known only 

from Thai coffee beans (Noonim et al. 2008). Finally, the newly 

described A. floridensis was highly supported in both the MP, 

ML, and Bayesian analysis (Figs 1–2), and in particular the 

five strains (Table 1) isolated from different world geographic 

area belonging in the same group by phylogenetic calmodulin 

analysis (data not shown). 


We thank Filomena Epifani (ISPA-CNR) for his valuable technical 

help in growing, DNA extraction, and sequencing of the fungal 

strains. Frank Robinson (Paul VI High School, Haddonfield, NJ) 

kindly advised us on Latin usage. J. Varga was partly supported 

by OTKA grant no. K 84077. Mention of a trade name, proprietary 

product, or specific equipment does not constitute a guarantee or 

warranty by the United States Department of Agriculture and does 

not imply its approval to the exclusion of other products that may be 

suitable. USDA is an equal opportunity provider and employer. 

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doi:10.5598/imafungus.2012.03.02.09 


Clarifications needed concerning the new Article 59 dealing with 

pleomorphic fungi 

Walter Gams 1 , Hans-Otto Baral 2 , Walter M. Jaklitsch 3 , Roland Kirschner 4 , and Marc Stadler 5 

1 

Formerly Centraalbureau voor Schimmelcultures, Utrecht; Molenweg 15, 3743CK Baarn, The Netherlands; corresponding author e-mail: 

walter.gams@online.nl 

2 

Blaihofstraße 42, D-72074 Tübingen 9, Germany 

3 

Faculty Centre of Biodiversity, University of Vienna, Rennweg 14, A-1030 Vienna, Austria 

4 

Department of Life Sciences, National Central University, No. 300, Jhongda Rd., Jhongli City, Taoyuan County 32001, Taiwan (R.O.C.) 

5 

Department Microbial Drugs, Helmholtz Centre for Infection Research, Bldg. B, Room 175a, Inhoffenstraße 8, D-38124 Braunschweig, Germany 

ARTICLE 

Abstract: The new rules formulated in Article 59 of the International Code of Nomenclature for algae, fungi, 

and plants (ICN) will cause numerous, often undesirable, name changes, when only phylogenetically defined 

clades are named. Our task is to name fungal taxa and not just clades. Two suggestions are made here 

that may help to alleviate some disadvantages of the new system. (1) Officially an epithet coined in a listdemoted 

genus that is older than the oldest one available in the list-accepted genus would have to be 

recombined in the accepted genus. We recommend that individual authors and committees establishing 

lists of protected names should generally not recombine older epithets from a demoted genus into the 

accepted genus, when another one from pre-2013 is available in that genus. (2) Because the concepts of 

correlated teleomorph and anamorph genera are often incongruent, enforced congruence leads to a loss 

of information. Retaining the most suitable generic name is imperative, even when this is subordinated to 

another, list-accepted, generic name. Some kind of cryptic dual generic nomenclature is bound to persist. 

We therefore strongly recommend the retention of binomials in genera where they are most informative. With 

these recommendations, the upheaval of fungal nomenclature ensuing from the loss of the former Art. 59 can 

be reduced to an unavoidable minimum. 

Key words: 

anamorph 

Kew rule 

list-demoted generic name 

nomenclature 

teleomorph 

Article info: Submitted: 19 November 2012; Accepted: 26 November 2012; Published: 30 November 2012. 

INTRODUCTION 

The new ruling and abandonment of the former Article 59 of 

the International Code of Botanical Nomenclature (ICBN) not 

only has abandoned the intricacies of dual nomenclature for 

pleomorphic fungi but also sacrificed the formerly recognized 

precedence of teleomorph-typified names over those of the 

associated anamorphs (McNeill et al. 2012). This precedence 

was not an expression of sexism, but it simply recognized that 

with the description of a teleomorph–anamorph association 

the knowledge of a fungus was more complete and more 

thorough than without it. It is not a matter of chance that the 

suprageneric classification is and remains generally based 

on teleomorph names. According to the new rules, many 

teleomorph-generic names will have to be replaced by older 

anamorph-generic names in cases where each morph of a 

fungus can unequivocally be tied to a particular taxon. 

Hawksworth (2012) analyzed the consequences of the 

new rules in coping with the names involved in a period of 

transition. He did, however, not question the rigid priority of 

all kinds of names and analyze and propose a solution for the 

two problems addressed here. The examples below are given 

not to criticize the respective authors, who tried to find the best 

solution for a difficult nomenclature. For example, when an 

author did not give preference to the older anamorph-generic 

name against the corresponding teleomorph name, he/she still 

followed the new Code correctly which states (Art. 14.13 ICN): 

“…lists of names may be submitted to the General Committee, 

which will refer them to the Nomenclature Committee for Fungi 

(see Div. III) for examination by subcommittees established by 

that Committee in consultation with the General Committee 

and appropriate international bodies. Accepted names on 

these lists, which become Appendices of the Code once 

reviewed and approved by the Nomenclature Committee for 

Fungi and the General Committee, are to be listed with their 

types together with those competing synonyms (including 

sanctioned names) against which they are treated as 

conserved (see also Art. 56.3).” These lists do thus not dictate 

that a particular taxonomy has to be adopted; that choice 

remains a matter of judgement; the list indicates only the 



Attribution: 







Gams et al. 

ARTICLE 

choice of names in whatever taxonomy an author wishes to 

adopt. We maintain that it is important that the list entries not 

be made before a careful analysis and a decision taken by 

the competent committees. We are aware that difficulties may 

have arisen from misleading formulations in the Code and thus 

indicate the need for clarification. 

Gams et al. (2012) pointed out the desirability of calling 

the preferred/listed names “prioritized” to distinguish the 

situation from that of conservation. The opposite would have 

been a “suppressed” name (which in certain situations can 

still be used). This terminology is not sufficiently clear and 

may cause misunderstandings. Therefore we speak now of 

list-accepted and list-demoted names, in order to distinguish 

the situation from that of conservation/rejection. 

CLARIFICATIONS REQUIRED 

I. The new rules would imply that an older 

epithet coined in a list-demoted genus has to be 

recombined in the list-accepted genus 

This would entail a very high number of undesirable name 

changes in some genera. 

Examples 

(1) If the genus name Trichoderma 1794 is listed with 

preference over Hypocrea 1825, the older epithet of 

Hypocrea schweinitzii (Fr. 1828) Sacc. 1883 would have to be 

recombined in Trichoderma, displacing the established name 

Trichoderma citrinoviride Bissett 1984. This is fortunately 

not done by Samuels et al. (2012). Conversely, according to 

this suggestion, the now established name Hypocrea citrina 

(Overton et al. 2006) would have to be called Trichoderma 

lacteum Bissett 1992, a so far hardly used name that may 

also not be desirable, because Hypocrea lactea is now 

regarded as a synonym of H. citrina. Thus a critical judgement 

is required when establishing a list of accepted names. 

(2) Hirooka et al. (2011), in a paper written just before the 

new rules were set, retained Nectria canadensis Ellis & 

Everh. 1884, although the anamorph name Tubercularia 

grayana (Sacc. & Ellis 1882) Seifert 1985 is older. Similarly: 

Nectria pseudotrichia Berk. & M.A. Curtis 1853 is predated by 

the anamorph Tubercularia lateritia (Berk. 1843) Seifert 1985 

but not replaced nomenclaturally. Note that the generic name 

Nectria (Fr. 1825) Fr. 1849 also is younger than Tubercularia 

Tode 1790, but obviously deserves preference. 

(3) Orbilia brochopaga (Drechsler 1937) comb. nov. would 

have to be introduced to replace Orbilia orientalis (Raitv. 

1991) Baral 1999, simply because of the available older 

anamorph epithet of Drechslerella brochopaga (Drechsler 

1937) M. Scholler et al. 1999. More about this question below. 

Comments 

The new wording of Art. 59 may be misleading in this respect. 

Its explicit statement that names introduced before 2013 

separately for teleomorphs and associated anamorphs are 

not automatically each other’s (legitimate or illegitimate) 

synonyms as they are based on different types permits 

retention of either name. Braun (2012) rightly emphasized 

that “names published prior to 1 January 2013 for the same 

taxon, but based on different morphs, are neither considered 

to be alternative names according to Art 34.2 nor superfluous 

names according to Art. 52.1, i.e. they are legitimate (if not 

illegitimate due to other reasons). Such synonyms are valid 

names, and valid names remain available for use.” 

Therefore individual authors and committees 

establishing lists of protected names should generally 

not recombine older epithets from a list-demoted genus 

into the list-accepted genus, when another one from pre- 

2013 is already available in that genus. This is in line with 

the botanical ‘Kew Rule’, adopted in the first volumes of Index 

Kewensis but never in the Code, which says: “Under this rule, 

priority within a genus was reckoned from the date when a 

specific epithet was first associated with that generic name. 

Older epithets, previously associated with species placed in 

other genera, were ignored” (Stevens 1991). 

II. Presently the concepts of correlated teleomorph 

and anamorph genera are often incongruent, while 

both of them are meaningful. Enforcing congruence 

leads to unnatural and unworkable Procrustean 1 

beds and loss of information 

In such cases, retaining the most suitable generic name is 

imperative, even when this is subordinated to another listaccepted 

generic name. Many ‘orphan’ species (Hawksworth 

2012) remain anyhow, which cannot yet be properly classified. 

Examples 

(1) Crous et al. (2009) found Mycosphaerella sensu stricto to 

phylogenetically coincide with species having anamorphs in 

Ramularia, and gave preference to binomials in that genus, 

but the same author (Crous 2009) happily continued to use 

the generic name Mycosphaerella for the hundreds of species 

that are not yet phylogenetically reorganized. 

(2) Scopulariopsis Bainier 1907 is predated by the associated 

teleomorph-generic name Microascus Zukal 1885, but older 

than Kernia Nieuwl. 1916, which also has Scopulariopsis 

anamorphs. Merging these genera into one would be 

confusing and undesirable. 

(3) In the monophyletic genus Hypocrea, a name to be 

subordinated under the older anamorph name Trichoderma, 

as accepted by a majority of members of the International 

Subcommission on Trichoderma and Hypocrea (ISTH), 

certain species lack an anamorph or have anamorphs quite 

different from Trichoderma. Would it not be the best solution 

to simply retain these in Hypocrea? 

(4) In the example of Orbilia, discussed under “I” above, 

it would be the simplest solution to retain for the species in 

question the anamorph name Drechslerella brochopaga, 

because the generic name Drechslerella, like Arthrobotrys 

1 

Procrustes, in Greek mythology, a son of Poseidon who placed his 

guests on an iron bed, stretching them or cutting off their legs, so as 

to force them to fit the size of the bed. 

176 

 

ima fUNGUS

Clarifications needed in naming pleomorphic fungi 

and other anamorph-generic names for nematode-trapping 

species, conveys phylogenetic and ecological information that 

would be lost by merging all species in Orbilia. Unpublished 

morphological and phylogenetic data on a vast number of 

species of Orbiliaceae indicate that the nematode-trapping 

fungi form a comparatively young clade out of many further 

taxonomic groups that comprise very numerous species. These 

remaining groups are rather well-defined by teleomorphic 

features and possess various other, non-nematophagous 

anamorphs. When treating the nematode-trapping group as 

three or four different genera, the remaining groups would then 

need to be handled similarly. The associated anamorphs are 

only diagnostic for some of these genera in regard to conidial 

morphology, and trapping organs are unknown in all of them. 

Hence, a classification according to teleomorph and DNA 

characteristics may be the preferable option. 

Classifying the nematode-trappers in the genera 

Arthrobotrys, Drechslerella, Dactylellina and Gamsylella, as 

proposed by Scholler et al. (1999), may be the beginning of 

a generic inflation. Such a procedure could eventually lead 

to the erection of numerous genera within the large genus 

Orbilia as presently circumscribed. As a further complication, 

trapping organs are also known in Lecophagus and 

Hyalorbilia, two quite basal genera of the Orbiliomycetes with 

no genetic connection to the nematode-trapping taxa. 

(5) Cordyceps militaris (L. 1753) Link 1833 is the oldest and 

indispensable name of a well-known fungus, in contrast to 

its still not definitely named and less known Lecanicillium 

anamorph. It would, however, be totally irresponsible to 

combine all species of the paraphyletic genus Lecanicillium 

into Cordyceps. 

Comment 

Some kind of cryptic dual generic nomenclature is therefore 

bound to persist. For binomials of species it will be easier to 

choose the most plausible unique name. Many systematists 

seem to forget that our task is to name fungal taxa, and not 

just clades. We therefore strongly recommend to retain 

binomials in genera where they are most informative. 

When following these recommendations, the upheaval of 

fungal nomenclature ensuing from abandoning the old Art. 59 

can be reduced to an unavoidable minimum. 

References 

Braun U (2012) The impacts of the discontinuation of dual 

nomenclature of pleomorphic fungi: the trivial facts, problems, 

and strategies. IMA Fungus 3: 81–86. 

Crous PW (2009) Taxonomy and phylogeny of the genus 

Mycosphaerella and its anamorphs. Fungal Diversity 38: 1–24. 

Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, Hunter 

GC, Burgess TI, Andjic V, Barber PA, Groenewald JZ (2009) 

Unravelling Mycosphaerella: do you believe in genera? 

Persoonia 23: 99–118. 

Gams W, Humber RA, Jaklitsch WM, Kirschner R, Stadler M (2012) 

Minimizing the chaos following the loss of Article 59: suggestion 

sfor a discussion. Mycotaxon 119: 495–507. 

Hawksworth DL (2012) Managing and coping with names of 

pleomorphic fungi in a period of transition. Mycosphere 1: 143– 

155. Doi 10.5943/mycosphere/3/2/4; IMA Fungus 3: 15–24. 

Hirooka Y, Rossman AY, Samuels GJ, Lechat C, Chaverri P 

(2012) A monograph of Allantonectria, Nectria and Pleonectria 

(Nectriaceae, Hypocreales, Ascomycota) and their pycnidial, 

sporodochial, and synnematous anamorphs. Studies in Mycology 

71: 1–210. 

McNeill J, Barrie FR. Buck WR, Demoulin V, Greuter W, Hawksworth 

DL, Herendeen PS, Knapp S, Marhold K, Prado J, Prud’homme 

van Reine WF, Smith GE, Wiersema JH, Turland NJ (eds) (2012) 

International Code of Nomenclature for algae, fungi, and plants 

(Melbourne Code) adopted by the Eighteenth International 

Botanical Congress Melbourne, Australia, July 2011. [Regnum 

Vegetabile no. 154.] Ruggell: A.R.G. Gantner Verlag. 

Overton BE, Stewart EL, Geiser DM, Jaklitsch WM (2006) 

Systematics of Hypocrea citrina and related taxa. Studies in 

Mycology 56: 1–40. 

Samuels GJ, Ismaiel A, Mulaw TB, Szakacs G, Druzhinina IS, 

Kubicek CP, Jaklitsch WM (2012) The Longibrachiatum clade of 

Trichoderma: a revision with new species. Fungal Diversity 55: 

77–108. 

Scholler M, Hagedorn G, Rubner A (1999) A reevaluation of predatory 

orbiliaceous fungi. II. A new generic concept. Sydowia 51: 89– 

113. 

Stevens PF (1991) George Bentham and the Kew Rule. In: Improving 

the Stability of Names: needs and options (Hawksworth DL, 

ed.): 157–168. [Regnum Vegetabile no. 123.] Königstein: Koeltz 

Scientific Books. [Also available from the International Plant 

Names Index website, http://ipni.org/understand_the_data.html.] 

ARTICLE 

CONCLUSION 

At the moment we can only offer guidelines for taxonomic 

revisions and the work of committees involved in establishing 

lists of names to be protected. It is hoped that such 

mechanisms of fine-tuning will eventually also find their way 

into subsequent editions of the Code. 


We are grateful to John McNeill for making parts of the new Code 

available before its publication, and to him and David L. Hawksworth 

for constructive comments on the present text. 


177

ARTICLE 

 

ima fUNGUS

doi:10.5598/imafungus.2012.03.02.10 


The treasure trove of yeast genera and species described by Johannes van 

der Walt (1925–2011) 

Maudy Th. Smith and Marizeth Groenewald* 

CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; corresponding author’s e-mail: m.groenewald@cbs. 

knaw.nl 

ARTICLE 

Abstract: Yeast taxonomy and systematics have in recent years been dealt with intensively primarily by a small group of 

individual researchers with particular expertise. Amongst these was Johannes P. van der Walt, who had a major role in 

shaping our current understanding of yeast biodiversity and taxonomy. Van der Walt based his taxonomic studies not only 

on available cultures, but also by going into the field to isolate yeasts from various substrates. This pioneering work led to 

the discovery of many new genera and species, which were deposited in the Centraalbureau voor Schimmelcultures (CBS) 

collections for future studies in taxonomy, genomics, and industrial uses. These treasures collected during more than 60 

years provide an outstanding legacy to the yeast community and will continue to exist in his absence. This contribution 

provides a comprehensive overview of the current nomenclatural and taxonomic status of the yeast genera and species 

introduced by van der Walt during his career. 

Key words: 

South Africa 

biodiversity 

taxonomy 

Article info: Submitted: 24 October 2012; Accepted: 30 November 2012; Published: 3 December 2012. 

INTRODUCTION 

Johannes van der Walt passed away after a short illness on 

13 November 2011. He will be remembered as a person very 

much interested in the biodiversity of yeasts, a passion which 

is apparent from the many yeast strains representing novel 

taxa that he isolated from various, mainly South African, 

sources. 

The first yeast species that was isolated in South Africa 

was from an infected human nail and was described as 

Hanseniaspora guilliermondii by Adrianus Pijper (Pijper 

1928), a pathologist practicing in Pretoria. The type strain of 

this species was deposited by Pijper in the yeast collection 

of the Centraalbureau voor Schimmelcultures (CBS), at 

that time located in Delft. The yeast collection had been 

transferred from Baarn to Delft after the appointment of Albert 

Jan Kluyver as Professor of Microbiology of the Technical 

University in Delft in 1921 (Samson et al. 2004), and came 

back together with the CBS filamentous fungal collection in 

Utrecht in 2000. 

As a result of Pijper’s mediation, Johannes van der Walt 

started to study for his PhD in Delft under the guidance of 

Kluyver in 1949, obtaining his degree in 1952 for a thesis 

entitled “On the yeast Candida pulcherrima and its pigment 

pulcherrimine” (van der Walt 1952). It was also in Delft that 

van der Walt was instructed in the use of specific enrichment 

techniques for the isolation of soil-borne microorganisms. 

After his return to South Africa in 1952, van der Walt started 

to search for novel yeast species. Applying a wide range 

of enrichment methods, van der Walt and his collaborators 

spent almost 50 years hunting intermittently for new taxa 

associated not only with natural sources such as uncultivated 

grassland soils, arboricolous beetle infestations and other 

similar niches, but also manufactured products such as wine 

and beer. This broad-based survey led to the discovery of 

many novel sexual and asexual ascomycetous taxa and some 

of heterobasidiomycetous affinity. Some of these species are 

still only known from South African isolates. 

Although originally trained in chemistry, van der Walt 

developed a great interest in the systematics, ecology, and 

genetics of yeasts. His interest in yeast systematics was a 

consequence having the CBS yeast collection close to his 

work-place in Delft, facilitating his study of these organisms. 

From that time, van der Walt maintained strong connections 

with the CBS, consulting their yeast taxonomists on taxonomic 

problems, and by depositing 492 strains in the collection. 

These strains formed the basis for 20 new genera and 109 

new species. Because of his broad knowledge of enrichment 

techniques, but also of yeast systematics, van der Walt was 

invited to contribute several chapters to the second and third 

editions of The Yeasts: a taxonomic study (Lodder 1970, 

Kreger-van Rij 1984). 

Van der Walt’s broad knowledge of yeasts and his 

discovery of previously unrecognized genera and species 

was much respected by other yeast taxonomists, who named 

four genera and four species in his honour: Vanderwaltia 

(Novak & Zsolt 1961; now included in Hanseniaspora), 

Waltomyces (Yamada & Nakase 1985; now included in 



Attribution: 







Smith & Groenewald 

ARTICLE 

Fig. 1. Asci with ascospores of Kluyveromyces polysporus 

(Vanderwaltozyma polyspora) After Barnett et al. (2000). A. YM agar, 

16 d. B. McClary acetate agar, 2 weeks. Bar = 5 µm. 

genera and species. At that time, these features were 

considered as important for generic assignment and species 

distinction. One relevant practical contribution for species 

characterization was the introduction of the Diazonium Blue 

B (DBB) test by van der Walt & Hopsu-Havu (1976). In cases 

where the sexual cycle of a yeast was unknown, this DBB test 

could be used by yeast taxonomists to determine whether 

the yeast had an ascomycetous or basidiomycetous affinity. 

Basidiomycetous yeasts gave a dark red colour reaction 

when the DBB solution was applied, while this reaction was 

absent in ascomycetous yeasts. 

Since the 1970s, following the trends set for bacterial 

taxonomy, molecular criteria such as mol% G+C and DNA- 

DNA hetero-duplex formations, and later gene sequencing, 

were introduced for the yeasts. Today, the introduction 

of novel species is predominantly based on molecular 

information obtained by sequencing one or several genes. 

This evolution in yeast taxonomy can be reconstructed from 

the five monographs on yeasts that have been published 

over the years (Lodder & Kreger-van Rij 1952, Lodder 1970, 

Kreger-van Rij 1984, Kurtzman & Fell 1998, Kurtzman et al. 

2011). 

YEAST GENERA 

Fig. 2. Three famous yeast taxonomists (left to right): Herman J. 

Phaff, Nico van Uden, and Johannes P. van der Walt. Photograph 

taken in 1987 at the international symposium “The expanding realm 

of yeast-like fungi”, Amersfoort, The Netherlands, 

Lipomyces), Waltiozyma (Muller & Kock 1986; now included 

in Wickerhamomyces), Vanderwaltozyma by Kurtzman 

(2003), Torulopsis vanderwaltii (Vidal-Leira 1966; reclassified 

by Yarrow & Meyer 1978, as Candida vanderwaltii), 

Kluyveromyces waltii (Kodama 1974; reclassified by Kurtzman 

2003, as Lachancea waltii), Myxozyma vanderwaltii (Spaaij 

et al. 1993), and Bullera waltii (Sugita et al. 1999; reclassified 

by Wang & Bai 2008, as Derxomyces waltii). 

In the early years of taxonomy, a group of scientist that 

included Johannes van der Walt, Nico van Uden and Herman 

J. Phaff (Fig. 1), as well as Lynford J. Wickerham, made a 

huge contribution by using phenotypic characteristics of 

morphology and physiology for the description of novel 

Between 1964 and 1995, twenty novel yeast genera were 

introduced by van der Walt (Table 1). The first of these genera 

was Dekkera. Species of this genus are known as spoilage 

organisms of soft drinks and alcoholic beverages (Dequin 

et al. 2003, Dufour et al. 2003, Stratford & James 2003). 

Besides Dekkera, seven more genera were introduced by 

van der Walt as single author. Of the remaining genera, eight 

were published in collaboration with researchers at CBS and 

four with other authors. 

As a consequence of the application of DNA sequence 

comparisons, eight of these genera were not accepted in the 

most recent edition of The Yeasts (Kurtzman et al. 2011), 

but were reduced to synonymy (Table 1). The generic name 

Debaryozyma (van der Walt & Johannsen 1978) was not 

accepted because the proposal of Lodder & Kreger-van Rij 

(1978) to conserve the name Debaryomyces was approved 

(Greuter et al. 1988) The monospecific genus Wingea is not 

now retained because the type species of this genus was 

phylogenetically shown to belong in Debaryomyces (Suzuki 

et al. 2012). Further, since the ex-type culture Aessosporon 

was found to mate with strains of Sporidiobolus salmonicolor 

(Sampaio 2011, unpubl.), this generic name can be 

considered a synonym of the earlier Sporidiobolus. The status 

of the genus Entelexis is uncertain; Lachance et al. (2011) 

commented on this in a discussion of Candida magnolia 

(previouslyTorulopsis magnoliae), since that species was 

indicated as the type species of Entelexis by van der Walt & 

Johannsen (1973). 

YEAST SPECIES 

Van der Walt was (co-)responsible for the introduction of 109 

novel yeast species during the period 1956 to 1999 (Table 2). 

180 ima fUNGUS

Yeasts described by J P van der Walt 

Table 1. Genera introduced by van der Walt and co-authors. 

Year Genus Author(s) Present generic status 1 (year of description of the genus) 

1964 Dekkera Van der Walt Recognized 

1970 Aessosporon Van der Walt Not recognized (ex-type culture mates with Sporidiobolus salmonicolor) 

1971 Kluyveromyces Van der Walt Recognized 

Lodderomyces Van der Walt Recognized 

Cyniclomyces Van der Walt & D.B. Scott Recognized 

Wingea Van der Walt Not recognized (type species belongs to the genus Debaryomyces) 

1972 Ambrosiozyma Van der Walt Recognized 

1973 Wickerhamiella Van der Walt Recognized 

Entelexis Van der Walt & Johannsen Not recognized (status of the genus uncertain) 

1976 Hyphopichia Arx & van der Walt Recognized 

Stephanoascus M.T. Sm., Van der Walt & Johannsen = Trichomonascus (1947) 

1978 Pachytichospora Van der Walt = Kazachstania (1971) 

Debaryozyma Van der Walt & Johannsen Not recognized (the genus name Debaryomyces is conserved) 

1980 Yarrowia Van der Walt & Arx Recognized 

1981 Myxozyma Van der Walt, Weijman & Arx Recognized 

Arxiozyma Van der Walt & Yarrow = Kazachstania (1971) 

ARTICLE 

1987 Zygozyma Van der Walt & Arx = Lipomyces (1952) 

1990 Arxula Van der Walt, M.T. Sm. & Y. Yamada = Blastobotrys (1967) 

1995 Babjevia Van der Walt & M.T. Sm. = Dipodascopsis (1978) 

Smithiozyma Kock, Van der Walt & Y. Yamada = Lipomyces (1952) 

1 

Present status in Kurtzman et al. (2011) 

Of the taxa compiled in Table 2, 30 species were described 

by van der Walt alone, 15 in collaboration with co-authors 

at the CBS, and the remaining species with mycologists in 

other countries. Most of the type strains of these species are 

isolates from South African sources, and only 20 are from 

elsewhere. Thirty types were isolated from soil in different 

localities of South Africa; eight came from vegetable material; 

35 from insect-related sources such as frass, tunnels or 

insect guts; ten are from processed food products such as 

beer, wine, and buttermilk; and three are from lichens. 

One of the highlights of his career was the isolation of 

a strain that produced asci with more ascospores than the 

normal 1–4 ascospores which he described as Kluyveromyces 

multisporus (now Vanderwaltozyma polyspora; Fig. 2). 

One of his new species, Saccharomyces inusitatus, is now 

considered to have a hybrid genome on the basis of DNA/ 

DNA reassociation experiments by A. Vaughan and A. Martini 

(Kurtzman et al. 2011) with high levels of similarity to both S. 

bayanus (94 %) and S. pastorianus (91 %). 

Van der Walt introduced 16 new combinations of species 

of which the basionyms were described previously by other 

yeast taxonomists. As these species are not seen as species 

first introduced by van der Walt we have not included them in 

Table 2. These species names, introduced by van der Walt on 

basis of basionyms of other yeast taxonomist and presently 

recognized, are listed below: 

Ambrosiozyma monospora (Saito) Van der Walt 1972 

Ambrosiozyma platypodis (J.M. Baker & Kreger) Van der 

Walt 1972 

Cyniclomyces guttulatus (C.P. Robin) Van der Walt & D.B. 

Scott 1971 

Hyphopichia burtonii (Boidin et al.) Arx & Van der Walt 1976 

Kluyveromyces aestuarii (Fell) Van der Walt 1971 

Kluyveromyces dobzhanskii (Shehataet al.) Van der Walt 

1971 

Kluyveromyces lactis (Dombrowski) Van der Walt 1986 

Kluyveromyces marxianus (E.C. Hansen) Van der Walt 1971 

Kluyveromyces wickerhamii (Phaff et al.) Van der Walt 1971 

Lodderomyces elongisporus (Recca & Mrak) Van der Walt 

1971 

Myxozyma melibiosi (Shifrine & Phaff) Van der Walt et al. 

1981 

Myxozyma mucilagina (Phaff et al.) Van der Walt et. al. 1981 

Saccharomycopsis vini (Kreger-van Rij) Van der Walt & D.B. 

Scott 1971 

Torulaspora globosa (Klöcker) Van der Walt & Johannsen 

1975 

Torulaspora microellipsodes (Osterwalder) Van der Walt & E. 

Johannsen 1975 

Yarrowia lipolytica (Wickerham et al.) Van der Walt & Arx 

1980 

CONCLUSIONS 

Most of the species described early in his career by van 

der Walt were based on phenotypic features, and, as with 

genera, molecular data have led to the revision of the status 

of species described in the “pre-molecular era”. This is 


181


ARTICLE 

Table 2. Species introduced by van der Walt and co-authors. 

Year Species name Authors Type strains of South 

African source 

Type strains from other source Present status of the type strain 1 

1956 Kluyveromyces africanus Van der Walt Soil = Kazachstania africana 

Saccharomyces transvaalensis Van der Walt Soil = Kazachstania transvaalensis 

Saccharomyces delphensis Van der Walt & Tscheuschner Dried figs = Nakaseomyces delphensis 

Saccharomyces capensis Van der Walt & Tscheuschner Soil = Saccharomyces cerevisiae 

Pichia vanriji (= P. vanrijiae) Van der Walt & Tscheuschner Soil = Schwanniomyces vanrijiae var. Vanrijiae 

Saccharomyces pretoriensis Van der Walt & Tscheuschner Soil = Torulaspora pretoriensis 

Kluyveromyces polysporus Van der Walt Soil = Vanderwaltozyma polyspora 

1957 Hanseniaspora vineae Van der Walt & Tscheuschner Soil Recognized 

Saccharomyces telluris Van der Walt Soil = Kazachstania telluris 

Hansenula beijerinckii Van der Walt Soil = Lindnera saturnus 

Saccharomyces lodderae Van der Walt & Tscheuschner Soil = Kazachstania lodderae 

Pichia terricola Van der Walt Soil Recognized 

Pichia pijperi Van der Walt & Tscheuschner Buttermilk = Wickerhamomyces pijperi 

Candida natalensis Van der Walt & Tscheuschner Soil Recognized 

1959 Endomycopsis wickerhamii Van der Walt Insect frass = Barnettozyma wickerhamii 

Pichia robertsii (= P.robertsiae) Van der Walt Insect = Debaryomyces robertsiae 

Endomyces reessii Van der Walt water-rotted Hibiscus cannabis, Indonesia = Galactomyces reessii 

1960 Torulopsis domercqii 

(=T. domerqiae) 

Van der Walt & Kerken Wine vat = Wickerhamiella domerqiae 

1961 Brettanomyces custersianus Van der Walt Brewery Recognized 

Torulopsis vanzylii Van der Walt & Kerken Equipment of wine making = C. norvegica 

Candida ingens Van der Walt & Kerken Wine cellar = Saprochaete ingens 

Torulopsis cantarellii Van der Walt & Kerken Industrial grape must = Trigonopsis cantarellii 

Torulopsis capsuligena Van der Walt & Kerken Wine cellar = Filobasidium capsuligenum 

1962 Schwanniomyces persoonii Van der Walt Soil = S. occidentalis var. persoonii 

1963 Saccharomyces vanudenii Van der Walt & E.E. Nel Soil = Kluyveromyces lactis var. drosophilarum 

Fabospora phaffii Van der Walt Winery equipment = Tetrapisispora phaffii 

1964 Dekkera bruxellensis Van der Walt From Belgian stout, Belgium Recognized 

Dekkera intermedia Van der Walt Tea-beer = Dekkera bruxellensis 

1965 Saccharomyces vafer Van der Walt Unknown = Torulspora delbrueckii 

Saccharomyces inconspicuus Van der Walt Grapes, France = Torulspora delbrueckii 

182 ima fUNGUS






Saccharomyces inusitatus Van der Walt Beer Possible hybrid between S. pastorianus and 

S. bayanus 1 

= Saccharomyces bayanus2 

1966 Kluyveromyces cicerisporus Van der Walt, E.E. Nel & Kerken Unknown = K. marxianus 

Kluyveromyces wikenii Van der Walt, E.E. Nel & Kerken Bantu beer = K. marxianus 

Pichia acaciae Van der Walt Insect frass = Millerozyma acaciae 

1968 Candida edax Van der Walt Insect tunnels = Sugiyamaella smithiae 

Torulopsis humilis E.E. Nel & Van der Walt Bantu beer = C. humilis 

1970 Saccharomyces amurcae Van der Walt “Alpechin”, Malaga, Spain = Lachancea fermentati 

Saccharomyces saitoanus Van der Walt Sour milk, Japan = Torulspora delbrueckii 

Hansenula philodendri Van der Walt & D.B. Scott Insect frass = Ogataea philodendri 

Hansenula sydowiorum D.B. Scott & Van der Walt Insect frass = Wickerhamomyces sydowiorum 

Syringospora stellatoidea Van der Walt Sputum = C. albicans 

Syringospora claussenii Van der Walt Unknown = C. albicans 

Aessosporon salmonicolor Van der Walt Carious dentine of man Synonym of Sporidiobolus salmonicolor 

Bullera dendrophila Van der Walt & D.B. Scott Insect frass Recognized 

Sterigmatomyces polyborus D.B. Scott & Van der Walt Insect tunnels = Fellomyces polyborus 

Trichosporon melibiosaceum D.B. Scott & Van der Walt Insect frass = C. fennica 

1971 Pichia ambrosiae Van der Walt & D.B. Scott Insect tunnels = Ambrosiozyma ambroasiae 

Pichia cicatricosa D.B. Scott & Van der Walt Insect tunnels = Ambrosiozyma cicatricosa 

Saccharomycopsis synnaedendra D.B. Scott & Van der Walt Insect tunnels Recognized 

Hansenula dryadoides D.B. Scott & Van der Walt Insect tunnels = Starmera dryadoides 

Torulopsis dendrica Van der Walt, Klift & D.B. Scott Insect frass = C. dendrica 

Candida silvanorum Van der Walt, Klift & D.B. Scott Insect frass Recognized 

Candida dendronema Van der Walt, Klift & D.B. Scott Insect frass Recognized 

Candida entomophila D.B. Scott, Van der Walt & Klift Insect tunnels Recognized 

Torulopsis insectalens D.B. Scott, Van der Walt & Klift Insect tunnels = C. insectalens 

Torulopsis nemodendra Van der Walt, Klift & D.B. Scott Insect tunnels = C. nemodendra 

Torulopsis silvatica Van der Walt, Klift & D.B. Scott Insect tunnels = C. silvatica 

Candida hylophila Van der Walt, Klift & D.B. Scott Insect tunnels = Rhodotorula hylophila 

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Torulopsis philyla Van der Walt, Klift & D.B. Scott Insect tunnels = Rhodotorula philyla 

1972 Ambrosiozyma philentoma Van der Walt, D.B. Scott & Klift Insect tunnels Recognized 

Pichia melissophila Van der Walt & Klift Gut honey bee = Priceomyces melissophilus 

Candida nitrativorans Van der Walt, D.B. Scott & Klift Insect tunnels = Wickerhamomyces sydowiorum 

Candida entomaea Van der Walt, D.B. Scott & Klift Insect tunnels = Yamadazyma mexicana 

Candida insectamans D.B. Scott, Van der Walt & Klift Insect frass Recognized 

Candida insectorum D.B. Scott, Van der Walt & Klift Insect frass Recognized 

Candida silvicultrix Van der Walt, D.B. Scott & Klift Insect frass Recognized 

Candida amylolenta Van der Walt, D.B. Scott & Klift Insect frass = Cryptococcus amylolentus 

1973 Wickerhamiella domercqiae Van der Walt Wine vat Recognized 

Candida homilentoma Van der Walt & Nakase Insect frass Recognized 

Candida naeodendra Van der Walt, Johannsen & Nakase Insect frass Recognized 

Entelexis magnoliae Van der Walt & Johannsen Flower = C. magnoliae 

Aessosporon dendrophilum Van der Walt Frass of larvae in galleries 

of Dichrostachys cinerea 

= Bullera dendrophila 

1975 Hansenula lynferdii Van der Walt & Johannsen Soil = Wickerhamomyces lynferdii 

Pichia philogaea Van der Walt & Johannsen Soil = Yamadazyma philogaea 

Trichosporon terrestre Van der Walt & Johannsen Soil = Blastobotrys terrestris 

1976 Stephanoascus ciferrii M.T. Sm., Van der Walt & Johannsen Mating type a from soil = Trichomonascus ciferrii 

1978 Pachytichospora transvaalensis Van der Walt Soil = Kazachstania transvaalensis 

Torulopsis azyma Van der Walt, Johannsen & Yarrow Lichen = C. azyma 

Torulopsis geochares Van der Walt, Johannsen & Yarrow Soil = C. geochares 

Candida fermenticarens Van der Walt Lichen Recognized 

1980 Debaryozyma yamadae Van der Walt & Johannsen Soil = Schwanniomyces yamadae 

1982 Hansenula euphorbiaphila Van der Walt Flower = Cyberlindnera euphorbiiphila 

Pichia meyerae Van der Walt Flower = Cyberlindnera meyerae 

Pichia kodamae Van der Walt & Yarrow Insect infestations = Ogataea kodamae 

1983 Pichia euphorbiae Van der Walt & Opperman Flower = Cyberlindnera euphorbiae 

= Vanderwaltozyma yarrowii 

1986 Kluyveromyces yarrowii Van der Walt, Johannsen, Opperman 

& Halland 

Stable mutant of crossing auxothrophic 

subcultures of CBS 2684 and CBS 6070, 

both isolated from tanning liquors of bark 

tree, France 

184 ima fUNGUS






Sporobolomyces kluyveri-nielii Van der Walt Leaf Recognized 

1987 Zygozyma oligophaga Van der Walt & Arx Insect frass = Lipomyces oligophaga 

Candida lyxosophila Van der Walt, N.P. Ferreira & Steyn Soil Recognized 

Myxozyma geophila Van der Walt, Y. Yamada & Nakase Soil Recognized 

Myxozyma lipomycoides Van der Walt, Y. Yamada & Nakase Lichen Recognized 

Sterigmatomyces wingfieldii Van der Walt, Y. Yamada & N.P. 

Ferreira 

Insect frass = Cryptococcus amylolentus 

1988 Sporobolomyces phyllomatis Van der Walt & Y. Yamada Leaf Recognized 

1989 Debaryomyces udenii Van der Walt, M.T. Sm. & Y. Yamada Soil, Ontario, Canada Recognized 

Zygozyma arxii Van der Walt, M.T. Sm. & Y. Yamada Soil = Lipomyces arxii 

Lipomyces japonicus Van der Walt, M.T. Sm., Y. Yamada & 

Nakase 

Garden soil, Japan Recognized 

Zygozyma suomiensis M.T. Sm., Van der Walt & Y. Yamada Skin lesion of a cow, Finland = Lipomyces suomiensis 

Myxozyma kluyveri Van der Walt, Spencer-Martins & Y. 

Yamada 

Soil Recognized 

Sporobolomyces phylladus Van der Walt & Y. Yamada Leaf = Bensingtonia phyllada 

1990 Zygozyma smithiae Van der Walt, Wingfield & Y. Yamada Insect frass = Lipomyces smithiae 

Myxozyma udenii Spaaij, Weber, Oberwinkler & van der 

Walt 

Soil around Magnifera indica, Florida, USA Recognized 

1992 Kluyveromyces picaeae Weber, Spaaij & Van der Walt Rhizosphere of Picea abies, Germany = Kazachstania picaeae 

1997 Lipomyces spencer-martinsiae (Van der Walt & M.T. Sm.) van der 

Walt & M.T. Sm. 

Soil, Nigeria Recognized 

1998 Myxozyma neglecta Spaaij, Van der Walt & Weber-Spaaij Cactus Recognized 

1999 Lipomyces doorenjongii Van der Walt & M.T. Sm. Soil Recognized 

Lipomyces kockii M.T. Sm. & Van der Walt Soil Recognized 

Lipomyces mesembrius Van der Walt & M.T. Sm. Soil Recognized 

Lipomyces yamadae Van der Walt & M.T. Sm. Soil Recognized 

Lipomyces yarrowii M.T. Sm. & Van der Walt Soil, Mauritius Recognized 

1 

Present status in Kurtzman et al. (2011) 

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185


ARTICLE 

evident by comparing the initial status of the species with that 

in the present classification. From Table 2, it can be seen 

that 20 species were placed in synonymy with existing taxa, 

while 54 species were reassigned to different genera and 

are still recognized as well defined species. However, even 

after the addition of DNA sequence data, 34 species have 

retained their original status and stand as tribute to a great 

yeast taxonomist. 

Even after his official retirement, van der Walt did not 

lose his passion for isolating interesting yeasts. For example, 

in 2010, over 20 years later, in collaboration with Teresa 

Coutinho, mating types of the presumed asexual species 

Candida deformans were isolated from lichens and soil 

(Groenewald & Smith, unpubl.). The last manuscript that 

he was actively involved with, resolving species within the 

Geotrichum/Galactomyces group (Groenewald et al. 2012), 

was possible because South African strains he isolated in 

2009 had been sent to CBS. 

The yeast community is indebted to van der Walt for his 

contribution to the yeast biodiversity and taxonomy over 63 

years. It is also likely that further novel taxa remain to be 

discovered among the strains that he has deposited over the 

years, supporting the quotation of Pliny (23–79 AD) “Ex Africa 

semper aliquid novi” 1 , a quotation that Johannes van der Walt 

was fond of citing. 

On a personal note, one of us, M. T. S., who collaborated 

with van der Walt for many years adds: “Those who may 

have had the privilege to meet Johannes van der Walt or to 

collaborate with him, as I have, will definitely remember him 

not only from his taxonomic work, but will also remember him 

as an amiable person full with stories to tell while enjoying a 

fine dinner with a good glass of wine.” 

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ima fUNGUS

doi:10.5598/imafungus.2012.03.02.11 


Westerdykella reniformis sp. nov., producing the antibiotic metabolites 

melinacidin IV and chetracin B 

Ghada A. Ebead 1 , David P. Overy 2,3 , Fabrice Berrué 2,3 , and Russell G. Kerr 1,2,3 

1 

Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Ave., Charlottetown, PEI, 

Canada, C1A 4P3; corresponding author e-mail: rkerr@upei.ca 

2 

Department of Chemistry, University of Prince Edward Island, 550 University Ave., Charlottetown, PEI, Canada, C1A 4P3 

3 

Nautilus Biosciences Canada, Duffy Research Center (NRC-INH), 550 University Ave., Charlottetown, PEI, Canada, C1A 4P3 

ARTICLE 

Abstract: Westerdykella reniformis Ebead & Overy sp. nov. is described based on morphology and phylogenetic 

analyses using ITS, nLSU rDNA, and β-tubulin gene sequences. Westerdykella reniformis is characterized by 

the production of cleistothecioid ascomata, containing small globose to subglobose asci with 32, aseptate, dark 

colored, pronouncedly reniform ascospores having a concave central groove. The isolate was obtained from 

a red alga (Polysiphonia sp.) collected from the tidal zone in Canada at low tide. Organic extracts enriched 

in extrolites, obtained from fermentation on a rice-based media, inhibited the growth of methicillin-resistant 

Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), S. warneri, and Proteus 

vulgaris. Presented here is the identification of the compounds responsible for the observed antimicrobial activity, 

the taxonomic description of W. reniformis, and a dichotomous key to the known species of Westerdykella based 

on macro- and micromorphological characters. 

Key words: 

antimicrobial screening 

Ascomycota 

ITS phylogeny 

multigene phylogeny 

Sporormiaceae 

Article info: Submitted: 15 September 2012; Accepted: 5 December 2012; Published: 11 December 2012. 

INTRODUCTION 

While screening organic solvent extracts of isolates of 

algicolous fungi obtained from Prince Edward Island 

(Canada) for antimicrobial activity, we found several strains 

with unique ITS rDNA gene sequences and associated 

extracts having antibiotic activity. Of particular interest was 

an isolate that was phylogenetically related to the genus 

Westerdykella within the family Sporormiaceae. Taxa 

in Sporormiaceae occur worldwide, especially on dung, 

but also as endophytes and as soil saprobes. The family 

currently comprises seven genera representing around 

100 species: Chaetopreussia, Pleophragmia, Preussia, 

Sporormia, Sporormiella, Spororminula, and Westerdykella 

(Kruys et al. 2006, Lumbsch & Huhndorf 2007, Kruys & 

Wedin 2009). 

The genus Westerdykella, first described by Stolk in 1955, 

was named after Johanna Westerdijk, the founding director 

of what is now the KNAW-CBS Fungal Biodiversity Centre 

in Utrecht, The Netherlands (Stolk 1955). Westerdykella 

species occur worldwide on a variety of substrates including 

soil, mud, dung, and plant material (Clum 1955, Ito & Nakagiri 

1995, Stolk 1955, Cain 1961, Rai & Tewari 1962, Malloch 

& Cain 1972). Kruys & Wedin (2009) retypified the genus, 

and distinguished it from other genera in the family by the 

production of cleistothecioid ascomata containing small asci 

(< 50 µm tall) with a short or almost absent stipe, encasing 

one-celled ascospores without germ slits. 

Species delineation within the genus historically has been 

based primarily on asci and ascospore shape. Originating 

with the description of the ex-type strain, W. ornata 

(Stolk 1955), to date nine species have been described 

withinWesterdykella: W. ornata, W. angulata, W. aurantiaca, 

W. cylindrica, W. dispersa, W. globosa, W. multispora, W. 

nigra, and W. purpurea. Over time and through various 

taxonomic revisions, several species of the genera Preussia, 

Pycnidiophora, and Eremodothis have been reclassified in 

Westerdykella. Pycnidiophora multispora was the first taxon 

to be transferred into the genus by Cejp & Milko (1964). 

Subsequently, Arx (1973) reclassified Preussia cylindrica 

in the genus due to the production of cylindrical, larger 

ascospores and the presentation of an asexual Phomalike 

state, and also P. nigra due to the production of short 

cylindrical asci ellipsoidal ascospores, and the absence of 

a conidial state. Subsequently, Preussia purpurea was also 

transferred to the genus by Arx (1975) due to the production 

of an orange pigment in culture, non-ostiolate ascomata 

with often a central columnar body and ascospores without 

germ pores. Ito & Nakagiri (1995) added P. globosa on the 

basis of the production of asci containing 32 ascospores, 

each having a single semicircular spiral ridge on the 

spore surface, and so conforming to the generic concept 



Attribution: 







Ebead et al. 

ARTICLE 

of Westerdykella as described by Stolk (1955). From a 

multilocus phylogenetic study based of ITS and nLSU rDNA, 

mtSSU and β-tubilin gene sequences, Kruys & Wedin (2009) 

reclassified Pycnidiophora dispersa and P. aurantiaca in the 

genus. Furthermore, Eremodothis angulata was found to be 

phylogenetically related to Westerdykella, despite producing 

eight pyramidal star-shaped ascospores per ascus compared 

to the typical 32 ascospores of other Westerdykella species, 

and was therefore also reclassified within it. 

In this study, a unique Westerdykella isolate from 

algae collected in the littoral zone was evaluated for 

morphological similarity to other taxa of the genus and 

for phylogenetic relatedness within Sporormiaceae. Multigene 

phylogenies were constructed using data sets of 

ITS and nLSU rDNA and β-tubulin gene sequences. Two 

extrolites were responsible for the observed antibiotic 

activity and these metabolites were isolated and identified 

from organic extracts of rice-based medium fermentations 

of the isolate. Presented here is the taxonomic description 

of the novel Westerdykella species, W. reniformis sp. nov., 

a dichotomous key to the known species of the genus based 

on macro- and micromorphological characteristics, and 

the isolation and identification of the antibiotic secondary 

metabolites melinacidin IV and chetracin B, production 

which is particular feature of W. reniformis. 


Sample collection 

Algal material (Polysiphonia sp.) was collected by G.A.E. 

from the shoreline at Point Prim, Prince Edward Island 

(46’04”N, 62’59”W) at low tide on 4 June 2009. Immediately 

after removal from the sea, the algal sample was deposited 

in a sterile plastic bag and seawater was added. The sample 

was kept cold until arrival at the laboratory where it was 

maintained at 4 °C until the following day. Before being 

processed, the sample was shaken three times with sterile 

seawater in order to wash the surface free of any adhering 

particulate material. 

Sample plating and fungal isolation 

The sample was homogenized in sterile seawater and 

the resulting homogenate was plated on a 9 cm Petri dish 

containing YM media (Yeast extract Malt agar; 2 g yeast 

extract, 10 g malt extract, 10 g glucose, 20 g agar, 50 mg 

chloramphenicol, 18 g Instant Ocean in 1 L Millipore H 2 

O) 

and inspected daily for fungal growth. The plates were 

incubated at 22 °C for 5 d and then examined under the 

dissecting microscope. Emerging fungal colonies were 

transferred via a flame-sterilized needle to another Petri dish 

containing YM. After obtaining a pure isolate, seed inoculum 

was prepared by excising cubes (1–3 mm 3 ) from an actively 

growing culture into 15 mL of yeast extract-maltose medium 

(10 g peptone, 40 g maltose, 10 g yeast extract, 18 g Instant 

Ocean and 1 g agar in 1 L Millipore H 2 

O) in a 50 mL test tube 

and incubated at 22 °C, 200 rpm for 5 d, after which 500 µL of 

mycelial suspension was removed for DNA extraction and the 

remainder reserved to inoculate fermentations. 

Culture characteristics and morphology 

For morphological and molecular comparisons, six 

Westerdykella isolates were obtained from CBS: W. cylindrica 

CBS 454.72, W. dispersa CBS 297.56, W. multispora CBS 

391.51, W. nigra CBS 416.72, W. ornata CBS 379.55, and W. 

rapa-nuiensis ined. CBS 604.97. For macro-morphological 

comparisons, fungi were grown on OA (Oatmeal Agar; 30 g 

oatmeal, 15 g agar in 1 L Millipore H 2 

O), Mannitol Soya agar 

(20 g mannitol, 20 g soya flour, 20 g agar in 1L Millipore H 2 

O) 

and Rice agar (75 g brown rice, 20 g agar in 1 L Millipore 

H 2 

O) at 22 °C and their growth rates were measured and 

colonies were evaluated after 7 and 14 d of incubation. 

Colour descriptions were qualified using Kornerup & 

Wanscher (1978). Measurements were repeated twice. For 

micro-morphological measurements and photographs, fungal 

structures from 26 d-old cultures were mounted on glass 

slides in lactic acid; photographs were taken while viewing 

using either bright field or phase contrast microscopy. For 

measurements, a Leica DME light microscope with phase 

contrast optics accompanied by a Leica EC3 camera (Leica 

Microsystems, Switzerland), was used at 100× magnification 

and a total of 25 ascospores and 25 asci were measured 

from crushed mounts and the dimension range (minimum and 

maximum) and average were determined (measurements 

were adjusted to the nearest 0.5 microns to avoid false 

impression of accuracy). Bright-field photomicrographs were 

obtained with a Carl Zeiss microscope, Axio Imager A1m 

model with a HRc Axiocam digital camera and AxioVision v. 

3.1 software (Carl Zeiss, Heerbrugg, Switzerland). 

Salt tolerance testing 

Isolate RKGE 35 was point-inoculated onto OA media and OA 

media with artificial seawater (+ASW; 18 g L -1 Instant Ocean) 

and plates were incubated at 22 °C. Radial growth rates and 

colony features were noted after 7 d and 14 d of incubation. 

Fermentation and extraction 

Strains were fermented on a rice-based medium (10 g brown 

rice; 50 mL YNB (6.7 g YNB + 5 g sucrose in 1 L Millipore 

H 2 

O)) in 250 ml Erlenmeyer flasks. The brown rice medium 

was autoclaved twice for 20 min at 121 °C, first with only 

brown rice, which was allowed to cool before YNB was 

added and the mixture was autoclaved again. Flasks were 

inoculated with 1.5 mL of seed inoculum. An uninoculated 

control flask was used to inspect medium purity and to be 

used as a negative control for antimicrobial screening. All 

experiments were incubated under stationary conditions at 

22 °C for 21 d. 

After 21 d of incubation, fermentations were extracted by 

first disrupting the fungal colony using a sterile spatula and 

adding 30 mL EtOAc:MeOH (1:1), followed by shaking at 50 

rpm for 1 h at room temperature. Organic extracts were then 

vacuum-filtered through Whatman #3 filter paper and dried 

using a GeneVac vacuum evaporating system (model: EZ-2 

MK2) prior to fractionation. Extracts were fractionated on 

Thermo HyperSep C-18 Sep Pack columns (500 mg C-18, 

6 mL column volume) using a vacuum manifold by eluting 

with 14 mL of each of the following solvent combinations: 

8:2 H 2 

O:MeOH (fraction 1), 1:1 H 2 

O:MeOH (fraction 2), 

2:8 H 2 

O:MeOH (fraction 3), EtOH (fraction 4), and 1:1 

190 ima fUNGUS

Antibiotic producing Westerdykella reniformis sp. nov. 

MeOH:DCM (fraction 5). The eluent representing fractions 

2–5 were retained and using a GeneVac (model: EZ-2 MK2) 

evaporating system, weighed and submitted for antimicrobial 

testing, and analyzed by LC/HRMS using a Kinetex 1.7 µm 

C18 UPLC column (Phenomenex, 50 × 2.1 mm) and Accela 

Thermo equipment coupled with MS-ELSD-UV detection 

(Orbitrap Excactive mass spectrometer fitted with an ESI 

source, PDA, and LT-ELSD Sedex 80 (Sedere)). 

Additional fermentation of strain RKGE 35 was carried 

out in 10 Erlenmeyer flasks and following the same growth 

conditions and extraction protocol as described above in 

order to obtain sufficient material to determine the structural 

identity of the secondary metabolites responsible for the 

observed antimicrobial activity. The resulting EtOAc:MeOH 

extract was partitioned between EtOAc:H 2 

O (1:1) and 

the organic layer (EtOAc) was dried under vacuum. The 

resulting gum was resuspended in a biphasic solvent mixture 

of Hexane:MeOH:H 2 

O (6:7:2) and the H 2 

O:MeOH layer 

after evaporation was subjected to a flash chromatography 

using bulk C-18 to yield to five fractions: 9:1 H 2 

O:MeOH, 

1:1 H 2 

O:MeOH, 2:8 H 2 

O:MeOH, EtOH, acetone, and 1:1 

MeOH:DCM. The 2:8 H 2 

O:MeOH fraction was further 

fractionated on normal phase silica by using automated 

medium pressure chromatography system (Combiflash 

Rf200 (Teledyne Isco)) to yield to 15 fractions. Fractions 

3–5 were purified by semi-preparative normal phase HPLC 

(Phenomenex Luna Silica, 250 × 10 mm, 5 μm) with isocratic 

conditions using 15% CHCl 3 

:MeOH (9:1) in 85 % CHCl 3 

and a 

flow rate of 2.5 ml min -1 to afford melanicidin IV and chetracin 

B. 

Antimicrobial bioassay 

All microbroth antibiotic susceptibility testing was carried 

out in 96-well plates in accordance with Clinical Laboratory 

Standards Institute testing standards (Ferraro, 2003) using 

the following pathogens: methicillin-resistant Staphylococcus 

aureus ATCC 33591 (MRSA), S. warneri ATCC 17917, 

vancomycin-resistant Enterococcus faecium EF379 (VRE), 

Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris 

ATCC 12454, and Candida albicans ATCC 14035. Extract 

fractions and pure compounds were tested in triplicate 

against each organism. Extract fractions were resuspended 

in sterile 20 % DMSO and assayed at 250 µg/mL with a 

final well volume concentration of 2 % DMSO while pure 

compounds were serially diluted to generate a range of 

twelve concentrations (128 µg mL -1 to 0.0625 µg mL -1 ) in a 

final well volume concentration of 2% DMSO. Each plate 

contained eight uninoculated positive controls (media + 

20 % DMSO), eight untreated negative controls (Media 

+ 20 % DMSO + organism), and one column containing a 

concentration range of a control antibiotic (vancomycin for 

MRSA, and S. warneri, rifampicin for VRE, gentamycin for 

P. aeruginosa, ciprofloxacin for P. vulgaris, or nystatin for C. 

albicans). The optical density of the plate was recorded using 

a BioTek Synergy HT plate reader at 600 nm at time zero and 

then again after incubation of the plates for 22 h at 37 °C. 

After subtracting the time zero OD600 from the final reading, 

the percentages of microorganism survival relative to vehicle 

control wells were calculated. 

DNA extraction and PCR amplification 

Genomic DNA was obtained from all strains using the fast 

DNA extraction kit (FASTDNA SPIN KIT FOR SOIL®, MP 

Biomedicals) according to the manufacturer’s protocols. 

Double-stranded copies of the ITS and nLSU rRNA gene 

and the β-tubulin gene were obtained by polymerase chain 

reaction (PCR) amplifications using 50 µL of reaction mixture 

consisting of 25 µL of Econo Taq ® PLUS GREEN 2× Master 

Mix (Lucigen), 17 µL of sterile ddH 2 

O, 2 µL of each primer 

and 4 µL of genomic DNA. Reactions were run in a Biometra 

thermocycler using the following settings for ITS amplicon 

generation: an initial denaturation step at 96 °C for 3 min, 

35 cycles consisting of denaturation at 96 °C for 45 s, primer 

annealing at 54.5 °C for 45 s and extension at 72 °C for 1 

min. The PCR was completed with a final extension step of 

10 min at 72 °C. Amplification protocols were similar for both 

β-tubulin and nLSU rDNA genes with the exception of the 

employed annealing temperatures: 58 °C for β-tubulin and for 

50 °C nLSU. Primers used for the ITS rDNA gene were ITS- 

1 and ITS-4 (White et al. 1990), for the β-tubulin gene were 

BT1819R and BT2916 (Miller & Hundorf 2005); for the nLSU 

rDNA gene were LROR and LR7 (Vilglys & Hester 1990, 

Rehner & Samuels 1994). PCR amplicons were checked for 

correct length and concentration by electrophoresis in 1 % 

agarose gel in 1× TAE buffer (Tris Base 2.42 g, glacial acetic 

acid 0.572 lL, 0.5 M EDTA 1 mL; add ddH 2 

O to 500 mL. 

DNA sequencing and sequence alignment 

The ITS, nLSU, and β-tubulin amplicons were sent to a 

commercial sequencing facility (Eurofins MWG Biotech) and 

sequenced on a 3730xl DNA Analyzer coupled with BigDye 

Terminator v. 3.1 Cycle Sequencing reagents, Applied 

Biosystems (ABI). The generated sequences were compared 

with other fungal DNA sequences from NCBI’s GenBank 

sequence database using a Blastn search algorithm. 

Phylogenetic analysis of the ITS rDNA gene were performed 

using the software Molecular Evolutionary Genetics Analysis 

v. 5 (MEGA5) (Tamura et al. 2011). Sequence data generated 

in this study were aligned with additional sequences of 

representative Westerdykella spp. as well as several 

isolates belonging to Sporormiaceae and other Pleosporales 

available in GenBank (Table 1). In total, 34 sequences were 

aligned using the ClustalW algorithm, with a DNA Gap Open 

Penalty = 15.0, DNA Gap Extension Penalty = 6.66 and a 

delay divergent cutoff of 30 %. For result optimization, the 

alignments were refined by manual correction when needed. 

The evolutionary history was inferred using the neighborjoining 

method employing the maximum composite likelihood 

model using pairwise deletion and the clade stability was 

evaluated using the bootstrap method (n = 2000 bootstrap 

replications). Novel sequences were accessioned in 

GenBank under accession numbers JX235699–JX235707. 

A multigene phylogeny was constructed using 23 isolates 

(Table 1). Relevant sequence data were downloaded from 

GenBank and used to construct aligned and trimmed ITS, nLSU 

and β-tubulin data matrices in MEGA5. A Bayesian analysis 

was performed using MrBayes 3.2 (Ronquist et al. 2012) with 

the following settings: nst = 6, therefore using GTR (General 

Time Reversible) model; rates = invgama, setting acrosssite 

rate variation for gamma distribution with a proportion of 

ARTICLE 


191

Ebead et al. 

ARTICLE 

Table 1. Sequences included in this study, newly generated sequences are highlighted in bold. 

Species Isolate Origin GenBank accession no. 

ITS 28S β-tubulin 

Herpotrichia juniper CBS 468.64 Switzerland, Pinus mugo GQ203759 DQ384093 GQ203681 

Pleospora herbarum ATCC 11681 USA, onion leaf AF229479 AF382386 AY749032 

Preussia australis Lundqvist 20884-a France, rabbit dung GQ203773 GQ203732 GQ203695 

P. funiculata Huhndorf 2577 USA, porcupine dung GQ203762 GQ203722 GQ203685 

P. isomera CBS 388.78 Venezuela, cow dung GQ203763 GQ203723 GQ203686 

P. lignicola CBS 363.69 Netherlands, rabbit dung GQ203783 DQ384098 GQ203703 

P. tenerifae CBS 354.86 Tenerife, rabbit dung GQ203794 GQ203752 GQ203713 

P. terricola CBS 317.65 Honduras, Musa sapientum GQ203765 GQ203725 GQ203688 

P. terricola CBS 527.84 Tanzania, elephant dung GQ203764 GQ203724 GQ203687 

P. typharum CBS 107.69 Japan, deer dung GQ203766 GQ203726 GQ203689 

P. vulgaris Strid 18884 Sweden, hare dung GQ203767 GQ203727 GQ203690 

Sporormia fimetaria Lundqvist 2302-c Sweden, cow dung GQ203768 GQ203728 GQ203691 

Sporomiella affinis Lundqvist 17739-j Denmark, rabbit dung GQ203770 – – 

S. dakotensis Thulin 2570-g Ethiopia, cow dung GQ203776 – – 

S. heptamera Lundqvist 3090-b Sweden, horse dung GQ203778 – – 

S. irregularis Lundqvist 16568-f Hungary, cow dung GQ203780 GQ203739 GQ203700 

S. leporina Lundqvist 19873-a Sweden, hare dung GQ203781 – – 

S. pulchella Richardson MJR67/01 USA, dung GQ203789 – – 

S. vexans UME23 Sweden, moose dung GQ203793 – – 

Trematosphaeria heterospora CBS 644.86 Switzerland, Iris sp. GQ203795 AY016369 GQ203714 

Westerdykella angulata IMI 090323 India, rice-field soil GQ203758 GQ203720 GQ203680 

W. angulata CBS 610.74 India, rice-field soil GQ203757 – – 

W. aurantiaca IMI 086825 India, mud AY943057 – – 

W. aurantiaca FNBR-03 India, soil JN118571 – – 

W. cylindrica ATCC 24077 = CBS 454.72 Kenya, cow dung AY943056 AY004343 JX235707 

W. dispersa CBS 297.56 Virginia, damp seedlings GQ203797.1 GQ203753.1 GQ203716.1 

W. dispersa CBS 156.67 Nigeria, soil DQ468016 – – 

W. dispersa CBS 508.75 Armenia, salt-marsh soil GQ203798 – – 

W. dispersa CBS 712.71 The Netherlands, greenhouse DQ468031 – – 

soil 

W. globosa IFO 32588 India, soil AY943046 – – 

W. multispora CBS 383.69 France, saline soil GQ203799 GQ203754 GQ203717 

W. multispora CBS 391.51 Japan AY943048 – – 

W. nigra CBS 416.72 Pakistan, soil GQ203800 GQ203755 GQ203718 

W. nigra ATCC 12756 AY943049 – – 

W. ornata CBS 379.55 Mozambique, mangrove mud GQ203801 AY853401 GQ203719 

W. purpurea CBS 297.75 Togo, sandy soil AY943050 – – 

W. purpurea HN6-5B China, mangrove FJ624258 – – 

W. rapa-nuiensis ined. CBS 604.97 Chile, soil JX235699 JX235703 JX235705 

W. reniformis RKGE35 = DAOM 242243 Canada, red algae JX235700 JX235704 JX235706 

Verruculina enalia CBS 304.66 Liberia, drift wood GQ203796 AY016363 GQ203715 

invariant sites; MCMC heated chain set with nchains = 4 and 

temp = 0.2, ngen = 500 000, samplefreq = 100, sumt burnin = 

1250; the analysis was continued for 500 000 generations in 

order obtain an average standard deviation of split frequencies 

below 0.01. The first 25 % of sampled trees were discarded 

as burn-in. Resulting trees were viewed in FigTree v. 1.3.1. 

Sequence alignments and trees presented were deposited in 

TreeBASE (accession number 13676). 

RESULTS 

Sequencing analysis 

In order to verify the taxonomic placement of isolate 

RKGE 35, gDNA was extracted and amplified by PCR 

for different genes resulting in sequence lengths of 472, 

1295 and 935 nucleotides for the ITS rDNA, nLSU rDNA, 

and β-tubulin genes respectively. The Blastn search for 

192 ima fUNGUS


Westerdykella dispersa CBS 297.56 


Westerdykella multispora CBS 391.51 

65 


98 

Westerdykella rapa-nuiensis ined. CBS 604.97 

74 Westerdykella dispersa CBS 508.75 

99 Westerdykella aurantiaca FNBR-03 

Westerdykella aurantiaca IMI 086825 


100 Westerdykella nigra CBS 416.72 

Westerdykella nigra ATCC 12756 

100 Westerdykella angulata IMI 090323 

Westerdykella angulata CBS 610.74 

98 Westerdykella purpurea CBS 297.75 

81 

Westerdykella purpurea HN6-5B 

Westerdykella globosa IFO 32588 

99 

Westerdykella cylindrica CBS 454.72 

51 Westerdykella reniformis RKGE35=DAOM 242243 

Westerdykella ornata CBS 379.55 

Sporormia fimetaria Lundqvist2302-c 

Sporormiella affinis Lundqvist17739-j 

92 Sporormiella heptamera Lundqvist3090b 

99 

Sporormiella vexans UME23 

Sporormiella leporina Lundqvist19873-a 

Sporormiella irregularis Lundqvist 16568-f 

Preussia isomera CBS 388.78 

Preussia tenerifae CBS 354.86 

78 

Sporormiella dakotensis Thulin 2570-g 

Sporormiella pulchella Richardson MJR93/01 

Preussia australis Lundqvist 20884-a 

Preussia lignicola CBS 363.69 

Preussia vulgaris Strid 18884 

100 

Preussia funiculata Huhndorf 2577 

76 

Preussia typharum CBS 107.69 

Trematosphaeria heterospora CBS 644.86 

Herpotrichia juniperi CBS 468.64 

Pleospora herbarum ATCC 11681 

Verruculina enalia CBS 304.66 

ARTICLE 

59 

54 

59 

58 

96 

0.02 

Fig. 1. Bootstrap consensus tree inferred from 2000 replicates using the neighbor-joining method based on ITS rDNA sequences. The percentage 

of replicate trees (> 50 %) in which the associated taxa clustered together in the bootstrap tests of 2000 replicates are shown next to the 

branches. Evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number of base 

substitutions per site. The tree was rooted with Verruculina enalia (CBS 304.66). 

the sequences showed that isolate RKGE 35 is classified 

within the genus Westerdykella. For the ITS region, the 

closest sequence matches with 95 % maximum identity 

and complete coverage were to those of W. ornata CBS 

379.55 (AY943045.1; matching 455/477 bases with 7 

gaps), W. dispersa CBS 297.56 (AY943055.1; matching 

454/477 bases with 6 gaps), and W. aurantiaca IMI 08625 

(AY943048.1; matching 452/477 bases with 6 gaps). For 

the amplified nLSU region, the closest sequence matches 

with complete coverage were to W. angulata IMI 090323 

(GQ203720.1; matching 1281/1296 bases with 1 gap) with 

99 % maximum identity followed by W. cylindrica ATCC 

24077 (NG027595.1; matching 1266/1269 bases with 1 

gap) with 98 % maximum identity. For the β-tubulin gene, the 

closest sequence matches with complete coverage were to 

W. dispersa CBS 297.56 (GQ203716.1; matching 876/941 

bases with 6 gaps) with 93 % maximum identity and W. 

ornata CBS 379.55 (GQ203719.1; matching 869/945 bases 

with 10 gaps) with 92 % maximum identity as well as with 

W. angulata IMI090323 (GQ203680.1; matching 368/926 

bases with 6 gaps) with 94 % maximum identity and only 

98 % coverage. 


The ITS rDNA gene was analysed to determine the relative 

evolutionary history of isolate RKGE 35 with multiple isolates 

of other representative Westerdykella spp. The evolutionary 

history was inferred by the bootstrap consensus tree (Fig. 

1) constructed using the neighbour-joining method and 2000 

bootstrap replicates. The analysis involved 39 sequences 


193

Ebead et al. 

Herpotrichia juniperi CBS 468.64 

ARTICLE 

Westerdykella angulata IMI 090323 

64 

100 Westerdykella dispersa CBS 297.56 

100 Westerdykella rapa-nuiensis ined. CBS 604.97 

72 


98 

Westerdykella ornata CBS 379.55 

100 

Westerdykella reniformis RKGE35=DAOM 242243 

100 

Westerdykella cylindrica CBS 454.72 

Westerdykella nigra CBS 416.72 

Sporormia fimetaria Lundqvist 2302-c 

70 Preussia funiculata Huhndorf 2577 

100 

Preussia typharum CBS 107.69 

Preussia vulgaris Strid 18884 

Preussia australis Lundqvist 20884 

Preussia lignicola CBS 363.69 

Preussia isomera CBS 388.78 

100 Preussia terricola CBS 527.84 

Preussia terricola CBS 317.65 

Preussia tenerifae CBS 354.86 

Sporormiella irregularis Lundqvist 16568-f 

Trematosphaeria heterospora CBS 644.86 

Pleospora herbarum CBS 191.86 

Verruculina enalia CBS 304.66 

100 

52 

89 

86 

99 

85 

89 

99 

63 

64 

0.05 

Fig. 2. Consensus tree inferred from a Bayesian analysis of ITS and nLSU rDNA and β-tubulin gene sequences. Bayesian posterior probabilities 

are given as % values at the nodes. The tree was rooted with Verruculina enalia (CBS 304.66). 

and included 409 nucleotide positions in the final dataset 

with an overall mean distance calculated as 0.137 with a 

standard error of 0.013. The genus Westerdykella formed 

a well-supported monophyletic clade distinct from other 

members of Sporormiaceae. Within the Westerdykella clade, 

sequences from individual isolates formed distinct species 

groups with high bootstrap support; however, evolutionary 

relatedness of species within the genus was difficult to infer 

due to separation with low associated bootstrap values. 

Isolate RKGE 35 formed a sister clade to that of W. cylindrica, 

represented by isolate CBS 454.72 (= ATCC 24077). Isolate 

CBS 604.97, representing the unpublished species W. rapanuiensis 

ined., clustered together within the W. dispersa 

clade along with the W. multispora isolate CBS 391.51. 

Evolutionary history within the genus Westerdykella was 

also inferred by a multigene Bayesian analysis involving 

sequences of the ITS and nLSU rDNA and β-tubulin genes 

from 23 strains. The aligned dataset consisted of 417 

nucleotides from the ITS rDNA, 872 nucleotides from the 

nLSU rDNA and 511 nucleotides from the β-tubulin gene 

sequences. Convergence was assumed as an average 

standard deviation of split frequencies of 0.007432 was 

achieved following 500 000 generations. From the generated 

phylogenetic tree (Fig. 2), representative isolate sequences of 

Westerdykella species once again clustered together forming 

a distinct clade with 100 % Bayesian posterior probability 

support. Isolate RKGE 35 clustered within the Westerdykella 

clade, forming its own discrete lineage with 100 % posterior 

probability support. Isolate CBS 604.97 representing the not 

yet formally named W. rapa-nuiensis clustered together and 

most proximal to W. dispera (CBS 297.56). 

Antimicrobial metabolite identification 

Two successive orthogonal fractionations (reverse phase 

then normal phase) of the MeOH:H 2 

O extract obtained after 

liquid-liquid partitions yielded three fractions (3–5) exhibiting 

strong antimicrobial activities. Chemical profiling by LC- 

HRMS coupled to a universal detector (ELSD) suggested 

that two major compounds were responsible for the observed 

biological activities (Fig. 3). The interpretation of the HRMS 

data indicated the molecular formulae C 30 

H 28 

N 6 

O 8 

S 4 

(m/z 

729.09076 [M+H] + , Δ -2.2 ppm) and C 30 

H 28 

N 6 

O 8 

S 5 

(m/z 

761.06274 [M+H] + , Δ -2.3 ppm) respectively and was in 

agreement with the observed isotopic pattern and the 

presence of sulfur atoms. After searches in databases 

Antibases and SciFinder, the two prominent components 

with antimicrobial properties were identified as the known 

metabolites melinacidin IV and chetracin B (Fig. 4) which 

belong to the important class of biologically active metabolites: 

epipolythiodioxopiperazines (ETPs) (Argoudelis & Mizsak 

1977, Li et al. 2012). This conclusion was further confirmed 

by 1 H NMR analysis after the purification of both metabolites 

by normal phase HPLC. 

194 ima fUNGUS


3.56 

milliVolts 

300 

250 

200 

150 

3.17 

NL: 

3.35E2 

ELSD 

ARTICLE 

100 

0.48 

Relative Abundance 


50 

100 

80 

60 

40 

20 

0 

100 

80 

60 

40 

* 

3.24 

3.40 

3.60 

* 

3.66 



100 

50 

[M+H] + 

729.09076 

[M+Na] + 

0 

720 730 740 750 760 

m/z 

100 

50 

melinacidin IV 

751.07135 

[M+H] + 

761.06274 

[M+Na] + 

chetracin B 

783.04559 

0 

200 300 400 

nm 

20 

0 

0 

750 760 770 780 790 200 300 400 

m/z 

nm 

0 

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 

Time (min) 

Relative Absorbance 

Relative Absorbance 

100 

50 

100 

50 

NL: 

4.54E5 

m/z= 729.0900- 

729.0950 

NL: 

1.02E5 

m/z= 761.0600- 

761.0650 

Fig. 3. ELSD (top) and single ion monitoring LCMS traces (middle, bottom) of fraction 3 (2:8 H 2 

O:MeOH) generated from rice fermentations of 

W. reniformis. Due to differences in tubing length, there is a 4 sec delay between the ELSD and MS detectors. High resolution mass spectra and 

UV absorbance traces (200–400 nm) are provided confirming the production of melinacidin IV and chetracin B. *Denotes possible artifacts due 

to reverse phase chromatography or presence of analogs. 

Bioactivity 

Microbroth dilution antibiotic susceptibility was determined at 

a concentration of 250 µg mL -1 for all of the fractions generated 

from rice fermentation extracts of the representative 

Westerdykella strains. Antibiotic activity was observed for 

all of the strains tested and the more potent antimicrobial 

response was observed in fraction 3 (2:8 H 2 

O:MeOH) 

(summarized in Table 2). It is notable that none of these 

fractions inhibited the growth of Pseudomonas aeruginosa 

and the yeast Candida albicans at a concentration of 250 

µg mL -1 . Gram positive antibiotic activity against methicillinresistant 

Staphylococcus aureus (MRSA) and S. warneri was 

observed for all of the strains tested whereas activity against 

vancomycin-resistant Enterococcus faecium (VRE) and the 

Gram negative bacterium Proteus vulgaris was exhibited only 

for the strain RKGE 35. Strain RKGE 35 exhibited a distinct 

antibiotic phenotype relative to the other strains tested. 

This observation was further confirmed by the comparison 

of the LC-HRMS data. Indeed, melinacidin derivatives were 

exclusively detected for the isolate RKGE 35 and constituted 

the main components of fraction 3 (2:8 H 2 

O:MeOH). LC- 

HRMS analysis of the extract fractions of the remaining 

Westerdykella strains examined confirmed the absence of 

melinacidin IV and chetracin B from both fraction 3 and fraction 

4. Rather, the metabolite profiles of these other strains for 

fraction 3 and fraction 4 were dominated by the presence of 

fatty acids characterized by the molecular formulae C 16 

H 32 

O 2 

, 

C 18 

H 34 

O 2 

, and C 18 

H 32 

O 2 

and oxidized fatty acids characterized 

by the molecular formulae C 18 

H 34 

O 3 

and C 18 

H 32 

O 3. 

Further 

purification and identification of the metabolites responsible 

for the antibacterial effect observed from the remaining 

Westerdykella strains has not been followed up here as it 

beyond the intended scope of this manuscript. Additional 

antimicrobial testing was carried out on purified melinacidin A 

and chetracin B to determine minimal inhibitory concentration 

(MIC) and half maximal inhibitory concentration (IC 50 

) values 


195

Ebead et al. 

O 

O 

ARTICLE 

HO 

HO 

O 

O 

dykellic acid 

HO 

HO 

O 

O 

gelastatin B 

O 

N 

S 2 

N 

H 

N 

O 

H 

N 

O 

N 

S 2 

N 

H 

N 

O 

H 

N 

HO 

O 

gelastatin A 

O O 

HO 

OH 

HO 

OH 

O 

N 

S 2 

N 

O 

O 

N 

S 3 

N 

O 

O 

O 

melinacidin IV 

OH 

chetracin B 

OH 

O 

lanomycin 

NH 2 

HO 

O 

HO 

O 

O 

O 

O 

HO 

O 

O 

O 

HO 

O 

O 

O 

O 

O 

H 

N 

glucolanomycin 

O 

OH 

OH 

HO 

auranticin A 

O 

auranticin B 

OH 

OH 

Fig. 4. Chemical structures of biologically active secondary metabolites produced by Westerdykella species. 

Table 2. Observed biological activity, presented as a percentage of inhibition, of fraction 3 (2:8 H 2 

O:MeOH) generated from organic extracts of 

rice fermentations of various Westerdykella spp. against various pathogens tested at 250 µg mL -1 in a microbroth dilution assay (results of values 

less than 50 % were not included). 

Species Strain MRSA VRE P. aeruginosa P. vulgaris S. warneri C. albicans 

W. reniformis RKGE35 = DAOM 242243 100 100 – 99 100 – 

W. ornata CBS 379.55 87 – – – 74 – 

W. nigra CBS 416.72 93 – – – 84 – 

W. multispora CBS 383.69 87 – – – 73 – 

W. cylindrica CBS 454.72 74 – – – – – 

W. dispersa CBS 297.56 97 – – – 86 – 

W. rapa-nueinsis ined. CBS 604.97 93 – – – 82 – 

Table 3. Biological activity of melinacidin IV and chetracin B against the drug resistant Gram-positive bacteria, methicillin-resistant Staphylococcus 

aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) and the Gram-negative bacterium Proteus vulgaris along with antibiotics 

tested as a positive control. All assays were run in triplicate, averaged and activity values are expressed in µM. If an assay was not performed, 

table entry was left blank. MIC: minimal inhibitory concentration. IC 50 

: half maximal inhibitory concentration. 

MRSA VRE P. vulgaris 

Compound MIC IC 50 

MIC IC 50 

MIC IC 50 

melinacidin IV 0.7 0.1 22 6 175.8 71.4 

chetracin B 0.7 0.2 10.5 1.1 168.4 76.3 

vancomycin 1.4 0.6 – – – – 

rifampicin – – 2.4 1 – – 

ciprofloxacin – – – – 0.024 0.012 

196 ima fUNGUS


of the compounds against MRSA, VRE and P. vulgaris 

(summarized in Table 3). Both melinacidin IV and chetracin 

B were slightly more efficacious than vancomycin against 

MRSA and were considerably less efficacious than rifampicin 

and ciprofloxacin against VRE and P. vulgaris respectively. 

Taxonomy 

The Westerdykella isolate RKGE 35 was clearly distinguished 

from other Westerdykella species studied based on DNA 

sequence comparisons of three gene regions, and growth 

inhibition to both Gram positive and Gram negative bacteria 

due to the production of melinacidin IV and chetracin B. 

Additional differences in both macro- and micro-morphological 

characters were also observed from those of the closest 

phylogenetically related species, confirming this isolate as 

representing a new taxon: 

Westerdykella reniformis G.A. Ebead & D.P. Overy, 

sp. nov. 


(Fig. 5) 

Etymology: The species name reflects the pronounced 

reniform (kidney shape) of the ascospores. 

Diagnosis: Colonies appressed, velvety, faint brown on 

oatmeal agar; attaining 16 mm diam after 7 d at 22 °C; 

producing distinct, glabrous, brownish black cleistothecia 

after 26 d; containing globose to subglobose, rarely ovoid 

asci (12–18 × 11–17 µm); each bearing 32, black, glabrous, 

reniform ascospores with a distinct central grove (2–4 × 4–6 

µm); pycnidal stage unknown. 

Type: Canada: Prince Edward Island: Point Prim (46’04”N, 

62’59”W), from Polysiphonia sp. collected from littoral tidal 

zone at low tide, 4 June 2009, G.A. Ebead, (DAOM 242243 – 

holotype; culture ex-type RKGE35). 

Description: Cleistothecia discrete, occurring in the upper layer 

of the culture medium, normally forming underneath a dense 

mat of hyphae exuding clear exudates, globose, glabrous 

and brownish black to black. Ascus initials somewhat clavate, 

asci later becoming globose to subglobose, occasionally 

ovoid when mature, measuring 12–18 (av. 14.7) × 10.5 – 16.5 

(av. 12.8) µm, containing 32 ascospores. Ascospores 2.5–4 

(av. 2.9) × 4 – 6 (av. 4.9) µm, black, glabrous, pronouncedly 

reniform in shape, having a central groove on the concave 

side, no oil droplets or germ-slits observed, germinating 

readily in 24 h at 22 °C. No conidial stage observed. 

Colony morphology: Colonies on oatmeal agar slow growing, 

attaining 16 mm diam in 7 d, and 40 mm diam in 14 d at 22 

°C. Mycelial development appressed, velvety, with no or little 

aerial hyphae. The colonies are faint brown in colour (6E4), the 

reverse brown (6E4) to dark grey-black in parts according to 

age of the colony. In older cultures (26 d), forming white mycelial 

aggregates covering brownish black cleistothecia, overall 

texture of the culture varies with density of the cleistothecia 

and the extent of hyphal overgrowth. Colonies on oatmeal agar 

(with artificial seawater) attaining 20 mm diam in 7 d and 40 

mm diam in 14 d at 22 °C, colony and reverse faint brown 

(6E4) and mycelia appressed to the agar surface, cleistothecia 

absent at 26 d, appearing later after 32 d. Colonies on mannitol 

soya agar slow growing, attaining 12 mm diam in 7 d and 36 

mm diam in 14 d at 22 °C, mycelia appressed and velvety, 

greyish brown (6D4), later becoming darker (6E4) with age, 

aerial mycelia present. Concentric circles of black cleistothecia 

apparent upon review of the colony reverse after 26 d. Colonies 

on rice agar, reaching 15 mm diam after 7 d and 44 mm diam 

after 14 d at 22 °C, dense, floccose aerial mycelia apparent, 

reverse progressing from a lighter to darker brown with age 

(6E4–6F4), cleistothecia absent after 26 d. 

ARTICLE 

Key to the known species of Westerdykella 

As previously evaluated by Kruys & Wedin (2009), the morphological characteristics of ascus shape and dimensions, along with 

ascospore shape, dimensions and ornamentation were found to be diagnostic in distinguishing species within the genus. The 

following dichotomous key was produced to facilitate the morphological identification of Westerdykella species. 

1 Ascospores ornamented; asci 32-spored .......................................................................................................................... 2 

Ascospores not ornamented; asci 8- or 32-spored............................................................................................................ 3 

2 (1) Ascospores globose with semicircular ridge; asci subglobose-ovoid ..................................................................... globosa 

Ascospores globose with 4–5 spiral bands, asci subglobose-elliptical .................................................................... ornata 

3(1) Ascospores reniform, cylindrical, or subglobose; asci 32-spored ..................................................................................... 4 

Ascospores angular with rounded ends, asci globose, 8-spored .......................................................................... angulata 

4(3) Ascospores reniform ......................................................................................................................................................... 5 

Ascospores subglobose or cylindrical ............................................................................................................................... 6 

5 (4) Asci globose; pycnidial state present; conidia globose to pyriform ....................................................................... dispersa 

Ascospores with a pronounced central groove; asci globose to subglobose, sometimes ovoid; 

pycnidial state absent ..................................................................................................................................... reniformis 


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6 (4) Asci clavate to cylindrical-clavate ...................................................................................................................................... 7 

Asci globose to ellipsoidal ................................................................................................................................................. 8 

7 (6) Ascospores ovoid to cylindrical; asci cylindrical-clavate to ovoid; pycnidial state present; 

conidia ovoid to ellipsoidal ............................................................................................................................... cylindrica 

Ascospores ellipsoid, rarely with one mid septum; asci distinctly clavate; pycnidial state absent .............................. nigra 

8 (6) Pycnidial state absent ....................................................................................................................................................... 9 

Pycnidial state present; conidia oblong; ascospores ovoid to cylindrical;asci ellipsoidal to pyriform ................ aurantiaca 

9 (8) Colonies violet to purple; cleistothecia 120–208 µm diam; ascospores ellipsoid; asci globose to subglobose ....... purpurea 

Colonies ochraceous to salmon; cleistothecia 150–600 µm diam; ascospores cylindrical with rounded ends; 

asci globose to subglobose ........................................................................................................................... multispora 

DISCUSSION 

The new taxon, Westerdykella reniformis, conforms to 

the classical morphological characterization of the genus 

Westerdykella, including the production of cleistothecioid 

ascomata containing small asci with an almost absent ascus 

stipe, and each ascus containing 32, 1-celled, dark-coloured 

ascospores lacking germ slits. Phylogenetic analyses using 

ITS and combined ITS and nLSU rDNA and β-tublin genes 

confirmed the placement in Westerdykella. 

Morphologically, W. reniformis is differentiated from both 

W. ornata and W. globosa on ascospore characters, as both 

those species produce globose, ornate ascospores (Stolk 

1955, Ito & Nakagiri 1995) while W. reniformis produces 

reniform ascospores lacking ornamentation. Westerdykella 

reniformis is also easily distinguished from W. cylindrica and 

W. nigra as both species produce clavate asci (Cain 1961, 

Malloch & Cain 1972), while the asci of W. reniformis are 

globose to subglobose. Both the asci and ascospores of W. 

reniformis are morphologically most similar to those of W. 

dispersa, W. multispora, and W. purpurea in both shape and 

dimension ranges (Cain 1961); however, phylogenetically W. 

reniformis is distinct from W. dispersa, W. multispora, and 

W. purpurea in both the ITS and the multigene analyses. 

Additionally, W. dispersa produces a pycnidial asexual 

morph (Clum 1955), which is absent in W. reniformis. CBS 

604.97, previously classified and deposited but yet not validly 

published under the name W. rapa-nueinsis, also has reniform 

ascospores; however based on phylogenetic analyses of all 

three genes sequenced and compared, W. rapa-nueinsis 

was phylogenetically distinct from W. reniformis and most 

similar to W. dispersa. Moreover, CBS 604.97 presented 

a pycnidial asexual morph comparable to W. dispersa. 

Based on micromorphological observations and the ITS and 

multigene phylogenetic comparisons, CBS 604.97 should be 

considered as a strain of W. dispersa. CBS 391.51, identified 

as W. multispora, was also found to cluster with W. dispersa 

in both our ITS phlyogenetic analysis as well as a previous 

ITS-nLSU phylogeny (Kruys & Wedin, 2009), suggesting 

that the strain has been misidentified. In order to confirm this 

synonmy, a morphological comparison of this strain to that of 

the ex-type strain of W. dispersa is warranted. 

Westerdykella species have been isolated from a wide 

variety of environmental substrates, including soil/sediment, 

and dung and plant debris. Our isolate was obtained from 

algal debris collected from the littoral zone at low tide. 

Growth measurement with W. reniformis on OA media 

varying in salt concentrations demonstrated that this fungus 

is capable of growing and sporulating in both a saline and 

non-saline environment. The ability of this fungus to grow and 

sporulate in the absence of salt suggests that this fungus is 

not obligate marine (as defined by Kohlmeyer & Kohlmeyer 

1979); an obligate marine fungus must be able to grow 

and sporulate exclusively in a marine or estuarine habitat. 

Although Westerdykella species are commonly isolated 

from terrestrial environments, they have also been isolated 

from both aquatic, estuarine and marine environments. 

In particular, W. aurantiaca and W. multispora have been 

isolated from mangrove sediments (Lee & Baker 1973, Poch 

& Gloer 1991) while W. dispersa has been isolated from a 

saline lake in Egypt (El-Sharouny et al. 2009). Westerdykella 

dispersa and W. multispora have also been isolated from low 

salinity and fresh water environments, both from sediment 

samples from lakes (Mishra 1995), and river delta flood plains 

(Bettucci et al. 2002) and estuaries (da Silva et al. 2003). 

Furthermore, they occurred as endophytes within the leaves 

of the freshwater lake reed Phragmites australis (Angelini 

et al. 2012). Therefore species of the genus Westerdykella 

appear to be widespread and most likely play a saprobic role 

in the decomposition of plant organic material within these 

ecosystems. 

Several research groups have previously examined 

Westerdykella isolates for the production of bioactive 

compounds (Fig. 4). Dykellic acid is an apoptosis inhibitor, 

isolated from W. multispora with indications as a therapeutic 

in a range of apotopsis-mediated diseases, such as hepatitis, 

neurodegeneration, and stroke (Lee et al. 1999a, 2003). 

Dykellic acid inhibited Ca 2+ influx, Ca 2+ -activated DNA 

endonuclease activity and suppressed caspase-3 protease 

activation preventing the cell from entering the execution of 

apoptosis (Lee et al. 2003). The gelastatins (A and B) are 

stereoisomers structurally related to dykellic acid that were 

isolated from the same strain of W. multispora (Lee et al. 

1997). A mixture of the isomers was found to selectively inhibit 

198 ima fUNGUS


ARTICLE 

Fig. 5. Macro and micro-morphology of Westerdykella reniformis (RKGE 35 = DAOM 242243). A. Colony grown on oatmeal agar (left) and 

oatmeal agar with sea salts (right) at 14d. B–C. Ascospores. D–F. Asci. 

the metalloproteinase gelatinase MMP-2 (involved in the 

cleavage of type IV collagen), demonstrating reversible and 

competitive inhibition of the enzyme; the mixture has therefore 

been proposed as lead compounds for the development as 

an antimetastatic agent (Lee et al. 1999b). Lanomycin and 

glucolanomycin, were two antifungal metabolites isolated 

from liquid fermentations of W. dispersa inhibiting growth of 

various dermatophytes and some species of Candida, but 


199

Ebead et al. 

ARTICLE 

were inactive against C. albicans, Aspergillus flavus and 

Gram-positve and Gram-negative bacteria (O’Sullivan et al. 

1992). Antifungal activity of lanomycin and glucolanomycin 

was attributed to the inhibition of lanosterol 14α-demethylase, 

suggesting a similar mode of action to the azole and bistriazole 

class of antifungal agents (O’Sullivan et al. 1992). 

From this survey, culture extracts generated from each 

of the Westerdykella species strains tested, demonstrated 

an antibiotic effect. Antibiotic activity was first associated 

with the genus Westerdykella from a mangrove isolate of 

W. aurantiaca. The depsidones, auranticins A and B, were 

isolated and demonstrated to have activity against both the 

Gram-positive bacteria Staphylococcus aureus and Bacillus 

subtilis, with auranticin A being more potent in antibiotic 

activity compared to auranticin B using a disc diffusion assay 

(Poch & Gloer 1991). In our survey, antibiosis, measured 

as growth inhibition, was assayed against several Grampositive 

and Gram-negative bacteria, including the drug 

resistant pathogens MRSA and VRE, and the pathogenic 

yeast, C. albicans. All of the Westerdykella isolates tested 

inhibited growth of the Gram-positive bacteria MRSA and S. 

warneri; however activity against VRE and Proteus vulgaris 

was unique to W. reniformis. LC-HRMS analysis confirmed 

the absence of auranticin A and B in each of the fraction 3’s 

obtained from W. cylindrica, W. dispersa, W. multispora, W. 

nigra, W. ornata, and W. reniformis. The observed biological 

activity of fraction 3 derived from culture extracts from W. 

reniformis was attributed to the production of melinacidin 

IV and chetracin B, which was found to be exclusive within 

the genus to W. reniformis. Melinacidin derivatives have 

been reported previously from a variety of different fungi: 

Acrostalagmus luteoalbus (syn. A. cinnabarinus; Argoudelis 

& Mizsak 1977), Chaetomium nigricolor (syn. C. abuense; 

Saito et al. 1985), Cladobotryum sp. (Feng et al. 2003), 

and Oidiodendron truncatum (Li et al. 2012); indicating that 

melinacidin production is not uncommon, nor limited to a 

particular taxonomic order. Both melinacidin IV and chetracin 

B are epipolythiodioxopiperazines, an important class of 

biologically active metabolites which possess a wide variety 

of biological activities, including antiproliferative, cytotoxic, 

immunomodulatory, antiviral, and antimicrobial activities (Li 

et al. 2012). We have reported potent antibiotic activity of 

melinacidin IV against the drug resistant bacteria methicillinresistant 

S. aureus and vancomycin-resistant Enterococcus 

faecium for the first time. 


We gratefully acknowledge financial support from the Natural 

Sciences and Engineering Council of Canada (NSERC), Canada 

Research Chair Program, University of Prince Edward Island, Atlantic 

Canada Opportunities Agency (funding from the AIF program), and 

Jeanne and Jean-Louis Lévesque Foundation. G.A.E. was supported 

by a scholarship from the Egyptian Cultural and Educational Mission 

Sector, Ministry of Scientific Research, Egypt. We also acknowledge 

experimental assistance from Martin Lanteigne who carried out all 

antimicrobial assays. 

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Coal Measure formation and lignin-degrading fungi – Slime mould navigation – Stratified algal and cyanobacterial 

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Articles 

“Development of merosporangia in Linderina pennispora (Kickxellales, Kickxellaceae)” by Mohamed E. Zain, Steve T. 103 

Moss, and Hussein H. El-Sheikh 

“Homortomyces gen. nov., a new dothidealean pycnidial fungus from the Cradle of Humankind” by Pedro W. Crous, 109 

Johannes Z. Groenewald, Lorenzo Lombard and Michael J. Wingfield 

“A new Leucoagaricus species of section Piloselli (Agaricales, Agaricaceae) from Spain” by Guillermo Muñoz, Agustín 117 

Caballero, Marco Contu, and Alfredo Vizzini 

“Ascus apical apparatus and ascospore characters in Xylariaceae” by Nuttika Suwannasai, Margaret A. Whalley, 

125 

Anthony J.S. Whalley, Surang Thienhirun, and Prakitsin Sihanonth 

“A new species of the lenticel fungal genus Claviradulomyces (Ostropales) from the Brazilian Atlantic forest tree Xylopia 

sericea (Annonaceae)” by Robert W. Barreto, Peter R. Johnston, Pedro W. Crous, and Harry C. Evans 

135 

“Shivasia gen. nov. for the Australasian smut Ustilago solida that historically shifted through five different genera” by 143 

Matthias Lutz, Kálmán Vánky, and Marcin Piątek 

“Addressing the conundrum of unavailable name-bearing types” by David L. Hawksworth 155 

“Two novel species of Aspergillus section Nigri from indoor air” by Željko Jurjević, Stephen W. Peterson, Gaetano 159 

Stea, Michele Solfrizzo, János Varga, Vit Hubka, and Giancarlo Perrone 

“Clarifications needed concerning the new Article 59 dealing with pleomorphic fungi” by Walter Gams, Hans-Otto 175 

Baral, Walter M. Jaklitsch, Roland Kirschner, and Marc Stadler 

“The treasure trove of yeast genera and species described by Johannes van der Walt (1925–2011)” by Maudy Th. 

179 

Smith and Marizeth Groenewald 

“Westerdykella reniformis sp. nov., producing the antibiotic metabolites melinacidin IV and chetracin B” by Ghada A. 189 

Ebead, David P. Overy, Fabrice Berrué, and Russell G. Kerr

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