Volume 3 No. 2 December 2012

Transcription

1 The Global Mycological Journal Volume 3 No. 2 December 2012 NEWS REPORTS AWARDS AND PERSONALIA RESEARCH NEWS BOOK NEWS forthcoming MEETINGS S

2 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 (print) (online) Websites: www. imafungus.org 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 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, Madrid, Spain; and Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK; 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; 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; 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; 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; 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; scantrel@suagm.edu Prof. dr D. Carter, Discipline of Microbiology, School of Molecular Biosciences, Building G08, University of Sydney, NSW 2006, Australia; d.carter@mmb.usyd.edu.au Prof. dr J. Dianese, Departamento de Fitopatologia, Universidade de Brasília, Brasília, D.F., Brasil; jcarmine@unb.br Dr P.S. Dyer, School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 2RD, UK; 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; Gryzenhoutm@ufs.ac.za Prof. dr L. Guzman-Davalos, Instituto de Botánica, Departamento de Botánica y Zoología, Universidad de Guadalajara, A.P Zapopan, 45101, México; lguzman@cucba.udg.mx Dr K. Hansen, Kryptogambotanik Naturhistoriska Riksmuseet, Box 50007, Stockholm, Sweden; karen. hansen@nrm.se Prof. dr K.D. Hyde, School of Science, Mae Fah Luang University, Tasud, Chiang Rai, Thailand; 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; lla@ adm.aau.dk Prof. dr L. Manoch, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand; 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; w.meyer@usyd.edu.au Dr D. Minter, CABI Bioservices, Bakeham Lane, Egham, Surrey, TW20 9TY, UK; d.minter@cabi.org Dr L. Norvell, Pacific Northwest Mycology Service, LLC, 6720 NW Skyline Boulevard, Portland, Oregon , USA; llnorvell@pnw-ms.com Dr G. Okada, Microbe Division / Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama , Japan; 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; 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; 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; jtaylor@berkeley.edu Prof. dr M.J. Wingfield, Forestry and Agricultural Research Institute (FABI), University of Pretoria, Pretoria 0002, South Africa; mike.wingfield@fabi.up.ac.za Prof. dr W.-Y. Zhuang, Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China; zhuangwy@sun.im.ac.cn ima fungus

3 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 ( 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 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: 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)

4 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 Ex. 1). However, two other repositories had been developed in anticipation of the need for official repositories. These were Index Fungorum < indexfungorum.org> and Fungal Names < 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 < (left to right) Paul Kirk, Yi-Jian Yao, and Vincent Robert shake hands upon reaching agreement on a sketched out data sharing arrangement between MycoBank, Index Fungorum and Fungal Names in Beijing, August 10, Photo: Scott A Redhead. org>, CBS-KNAW Fungal Biodiversity Centre, The Netherlands < cbs.knaw.nl>, and Landcare Research, New Zealand < co.nz/home> 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? < nl/news/newsdetails.aspx?rec=70> 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), < mycolab.org.cn/templates/t_second_en/ index.aspx?nodeid=248>. 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

5 NEWS Geoffrey C. Ainsworth ( ), whose vision and actions led to the formation of the IMA in 1971, proposed, along with Raffaele Ciferri ( ) that newly published fungus names should be registered 58 years before this was to become a reality (Ainsworth & Ciferri, Taxon 4: ). 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 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 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 [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 , USA; llnorvell@pnw-ms.com volume 3 no. 2 (45)

6 News Violins and mushrooms superior tonal qualities, were constructed from Norway spruce that had grown mostly during the Maunder Minimum ( ), 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 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: On 22 September 2012 The Economist has a striking headline Violins constructed from infected wood sound like those of Stradivari ( node/ ). 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

7 by 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: NEWS Possible mutagen effects on genetic stability of fungi in living collections 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: /s 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 Submissions can either be sent to p.crous@cbs.knaw.nl or r.samson@cbs. knaw.nl; for larger files we recommend using volume 3 no. 2 (47)

8 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 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 ( springer.com/life+sciences/microbiology/ journal/10267; until 31 December 2012), and those of volume 54 at a new site ( journal/ ; from 20 July 2012); these meet the PDF/A archival standard (48) ima fungus

9 (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 Art 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 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 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; Index Fungorum ( IndexFungorumRegister.htm), and Fungal Name ( 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 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: 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 volume 3 no. 2 (49)

10 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; 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: Hawksworth DL (2012) Managing and coping with names of pleomorphic fungi in a period of transition. IMA Fungus 3: 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: 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 [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: 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: [in Japanese] Toru Okuda, Yoshitaka Ono, Takayuki Aoki, and Gen Okada (torula@lab.tamagawa.ac.jp) (50) ima fungus

11 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: (Action website), and actions/fa/actions/fa1103?parties (corresponding COST website). Participants in the Reims COST workshop (March 2012). Participants in the San Michele dell Adige COST workshop (November 2012). synergies 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 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 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 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 REPORTS volume 3 no. 2 (51)

12 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 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: / journal.pone Stadler M, Schulz B (2009) High energy biofuel from endophytic fungi? Trends in Plant Science 14: Marc Stadler (marc.stadler@helmholtz--hzi.de) (52) ima fungus

13 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 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 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 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 ( ) 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 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 volume 3 no. 2 (53)

14 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 Born in Kirity Roy kindly provided notes on Gouri s life and achievements, as well a the portrait included here. Carl Lennart Holm ( ) 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 The exsiccate had been initiated in 1934 by Lennart s mentor, Johan A. F. Nannfeldt ( ), 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 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 ( ) 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 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 ( ) 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 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 The portrait presented here was kindly supplied by Gerald Crowther. (54) ima fungus

15 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: 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 White rot 6,2,3,7 Boletales SERLA 0 Brown rot * 6,3,4,6 Mycorrhizal C HETAN 8 Russulales M Soil saprotroph STEHI 6 Mycoparasite / 1,1,1,2 Animal Pathogen 7,3,6,16 POSPL 1 B POD copy numbers: * 1,1,1,2 WOLCO 12 Extant 8,3,6,11 1 T 7,2,3,7 Estimated FOMPI 1 13,7,8,13 Polyporales Contractions in PODs (p<0.01) 7,3,4,15 DICSQ 12 Expansions in PODs (p<0.01) B* TRAVE 26 (290) Mean node ages (mya) 8,3,6,13 PHACH 16 7,3,4,11 (237) B GLOTR 0 Gloeophyllales Agaricomycetes 7,2,3,7 * PUNST 11 Corticiales (290) A 7,2,2,4 FOMME 17 Hymenochaetales 2,1,1,1 (350) AURDE 19 Auriculariales Agaricomycotina 1,1,1,1 (429) DACSP 0 Dacrymycetes 0,0,0,0 CRYNE 0 1,1,1,1 Tremellomycetes (153) TREME 0 (491) Basidiomycota MALGL 1,1,1,1 0,0,0,0 0 (521) Ustilaginomycotina (273) USTMA 0 Dikarya (662) 900 A 1,--,--,0 --,--,--,0 (812) Cryogenian 800 1,1,1,2 (770) Ascomycota 700 Ediac. 600 * 1,1,1,1 Cam. Ord. Sil. Dev ,0,0,0 (518) (382) 1,1,1, ,1,1,1 (344) * Carb. Per. Tri. Jur ,1,1,1 200 Cret. 100 Cen. 0 MELLA SPORO CRYPA TRIRE ASPNI STANO PICST PHYBL BATDE Pucciniomycotina Pezizomycotina B (3 Saccharomycotina Mucoromycotina Chytridiomycota (631, 7 RESEARCH NEWS David S. Hibbett kindly supplied the figure reproduced here. Chronogram produced with BEAST from a 26-gene data set. Light blue bars are 95 % highest posterior density intervals for node ages; mean ages of selected nodes (millions of years) are in parentheses. Blue and red branches indicate significant expansion and contraction, respectively. The node A is the ancestor of Agaricomycetes, and an asterisk (*) indicates nodes that did not receive maximal support. See Floudas et al. (2012) for more detailed explanation of figures and species names, from whom this figure has been adapted. volume 3 no. 2 (55)

16 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: 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

17 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: ; DOI 10:1111/nph 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). volume 3 no. 2 (57)

18 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: (58) ima fungus

19 Marine Fungi and Fungal-Like Organisms. Edited by E. B. Gareth Jones and Ka-Lai Pang ISBN Pp. xvi Göttingen: Walter de Gruyter. Price: 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 ISBN Pp. x Caister, Norfolk: Caister Academic Press. Price: 159 or US$ 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 volume 3 no. 2 (59)

20 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: McCluskey K (2012) Variation in mitochondrial genome primary sequence among wholegenome-sequenced strains of Neurospora crassa. IMA Fungus 3: Fungal Secondary Metabolites: methods and protocols. Edited by Nancy P. Keller and Geoffrey Turner ISBN Pp. xii New York: Humana Press. [Methods in Molecular Biology no. 944.] Price: 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

21 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 ISBN 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 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: Mushrooms: the natural and human world of British fungi. By Peter Marren ISBN Pp. 272, illustr. Gillingham, UK: British Wildlife Publishing. [British Wildlife Collection no. 1.] Price: 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 volume 3 no. 2 (61)

22 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 ISBN Pp. ix St Paul, MN: APS (American Phytopathological society) Press. Price: US$ 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

23 International and regional meetings which are entirely mycological or have a major mycological component One Fungus = Which Gene(s)? April 2013 Royal Dutch Academy of Arts and Sciences, Amsterdam, The Netherlands. Contact: Pedro Crous; p.crous@cbs.knaw.nl <cbs.knaw.nl/> 23 rd European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) April 2013 Berlin, Germany Contact: ECCMID 2013, c/o Congrex Switzerland Ltd, Peter Merian-Strasse 80, 4002 Basel, Switzerland; basel@congrex.com <congrex.com> FEMS 2013: 5 th Congress of European Microbiologists July 2013 Congress Center Leipzig, Messe-Allee 1, Leipzig, Germany Contact: Kenes International, 1-3, rue de Chantepoulet, P.O. Box 1726, CH-1211 Geneva 1, Switzerland; fems@kenes.com < FORTHCOMING MEETINGS 3 rd Annual World Congress of Microbes 2013 (WCM-2013): Non-Virus Microbes August 2013 Chongquing, China Contact: BIT Congress Inc., Chongquing, China; chinajob.com < Asian Mycological Congress and 13th International Marine and Freshwater Mycology Symposium August 2013 Beijing International Convention Center, Beijing, China Contact: Na Jiang; AMC2013@163.com <amc2013.com> 19 th Australasian Plant Pathology Congress: Protecting our Cops and Native Flora August 2013 University of Auckland, Auckland, New Zealand Contact: events@plantandfood.co.nz < Bio-security, Food Safety and Plant Pathology: The Role of Plant Pathology in a Globalized Economy 10 th International Congress of Plant Pathology August 2013 Beijing International Convention Center, Beijing, China <bicc.com.cn> and <icppb2013.org/file/> 6 th Trends in Medical Mycology (TIMM) October 2013 Tivoli Hotel and Conference Center, Copenhagen, Denmark Contact: Congress Care, P.O. Box 440, 5201 AK s-hertogenbosch, The Netherlands; info@congresscare.com <congresscare.com> th International Conference on Cryptococcus and Cryptococcosis May 2014 Royal Tropical Institute, Amsterdam, The Netherlands < volume 3 no. 2 (63)

24 FORTHCOMING MEETINGS IUMS XIV International Congress of Mycology [with the Congresses of Bacteriology and Applied Microbiology, and also Virology] 27 July 1 August 2014 Montreal, Canada Contact: iums3014@nrc-cnrc.gc.ca < 10 th International Mycological Congress (IMC10) 3 8 August 2014 Queen Sirikit National Convention Center, Bangkok, Thailand Contact: Lekha Manoch; agrlkm@ku.ac.th VIII Latin-American Congress of Mycology (VIII CLAM) Colombia: the Crossroads of Latin-America s Fungal Diversity 4 7 November 2014 Centro de Convenciones Plaza Mayor, Medellín, Colombia Contact: Aida Vasco; almycol@gmail.com NOTICES IMA Fungus is compiled by David L. Hawksworth (Facultad de Farmacia, Universidad Complutense de Madrid) on behalf of the Executive Committee of the International Mycological Association. All unsigned items in the journal are by, and may be attributed to, him. Items for consideration for inclusion in all sections of the journal should be submitted to him at d.hawksworth@nhm.ac.uk. Books for possible coverage in the Book News section should be mailed to: IMA Fungus, Milford House, The Mead, Ashtead, Surrey KT21 2LZ, UK. (64) ima fungus

25 doi: /imafungus IMA Fungus volume 3 no 2: 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 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 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, µm long, 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, µm diam, possessed a two-layered wall, and coated with rod-shaped ornamentations. Mature pseudophialides were ellipsoid, 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, 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 µm wide, µm long, possessed a threelayered wall, with regular surface annulation with interconnecting ridges, but lacked rod-shaped ornamentations. The merosporangia contained a single, obovate, 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 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 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

26 Zain, Moss & El-Sheikh MATERIALS AND METHODS Isolate The isolate used in this study was Linderina pennispora (IMI ) 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 %, 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, 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, µm wide, 4 7 µm long, and possessed a two-layered wall. They formed in 104 ima fungus

27 Merosporangia of Linderina pennispora 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 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, µm diam, lobed, cylindrical neck. volume 3 no

28 Zain, Moss & El-Sheikh 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, µm long, ca 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 µm wide, 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, nm thick, electron-opaque layer; a middle, nm thick, electron-opaque layer; and an inner, nm thick, electron-dense layer (Fig. 1H). Each merosporangium contained a single merosporangiospore. The merosporangiospore was obovate, 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, 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

29 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, 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. REFERENCES Benjamin RK (1966) The merosporangium. Mycologia 58: Benjamin RK (1979) Zygomycetes and their spores. In Kendrick B (ed.), The Whole Fungus 2: Ottawa: National Museums of Canada. Benny GL (1995) Classical morphology in zygomycete taxonomy. Canadian Journal of Botany 73: Benny GL (2012) Current systematics of Zygomycota with a brief reiew of their biology. In: Misra JK, Tewari JP, Deshmukh SK (eds), Systematics and Evolution of Fungi: Enfield, NH: Science Publishers. Benny GL, Aldrich HC (1975) Ultrastructural observations on septal and merosporangial ontogeny in Linderina penispora (Kickxellales, Zygomycetes). Canadian Journal of Botany 53: Benny GL, Humber RA, Morton JB (2001). Zygomycota: Zygomycetes. In: McLaughlin DJ, McLaughlin EG, Lemke PA (eds). The Mycota, Vol 7. Systematics and Evolution, A: Berlin: Springer-Verlag. Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, et al. (2007) A higher-level phylogenetic classification of the fungi. Mycological Research 111: Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Ainsworth & Bisby s Dictionary of the Fungi. 10 th edn. Wallingford: CAB International. volume 3 no

30 Zain, Moss & El-Sheikh Kreisel (1969) Grundzuge eines naturlichen Systems der Pilze. J. Cramer, Lehre. McKeown TA, Moss ST, Jones EBG (1996) Atomic force and electron microscopy of sporangial wall microfibrils in Linderina pennispora. Mycological Research 100: Moss ST, Young TWK (1978) Phyletic considerations of the Harpellales and Asellariales (Trichomycetes, Zygomycotina) and the Kickxellales (Zygomycetes, Zygomycotina). Mycologia 70: O Donnell K, Cigelnik E, Benny G (1998) Phylogenetic relationship among the Harpellales and Kickxellales. Mycologia 90: Raper KB, Fennell DI (1952) Two noteworthy fungi from Liberian soil. American Journal of Botany 39: White MM, James TY, O Donnell K, Cafaro MJ, Tanabe Y, Sugiyama J (2006) Phylogeny of the Zygomycota based on nuclear ribosomal sequence data. Mycologia 98: Young TWK (1968) Electron microscopic study of asexual spores in Kickxellaceae. New Phytologist 67: Young TWK (1969) Ultrastructure of aerial hyphae in Linderina pennispora. Annals of Botany 33: Young TWK (1970a) Ultrastructure of the spore wall of Linderina. Transactions of the British Mycological Society 54: Young TWK (1970b) Arrangement of the microfibrils in walls of aerial hyphae of Linderina. Transactions of the British Mycological Society 55: Young TWK (1971) Ultrastructure of the wall of the germinating sporangiospore of Linderina pennispora (Mucorales). Annals of Botany 35: Young TWK (1974) Ultrastructure of the sporangiospore of Kickxella alabastrina (Mucorales). Annals of Botany 38: Young TWK (1985) Ultrastructure of mucoralean sporangiospores. Botanical Journal of the Linnean Society 91: ima fungus

31 doi: /imafungus IMA Fungus volume 3 no 2: 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 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 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

32 Crous et al. 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 ; CPC Combretum erythrophyllum, leaves South Africa: Maropeng P.W. Crous & M.J. Wingfield CBS ; CPC Combretum erythrophyllum, leaves South Africa: Maropeng P.W. Crous & M.J. Wingfield JX JX JX Sclerostagonospora sp. CBS ; CMW Elegia equisetacea, dead culm South Africa: Kirstenbosch S. Lee DQ / JX DQ CBS ; CMW Cannomois virgata, dead culm South Africa: Jonkershoek S. Lee DQ DQ CBS ; CMW Thamnochortus spicigerus, dead culm South Africa: Kirstenbosch S. Lee JX JX CBS ; CMW Ischyrolepis subverticellata, dead culm South Africa: Kirstenbosch S. Lee JX JX Stilbospora macrosperma (syn. Prosthecium ellipsosporum) CBS Carpinus betulus, dead corticated twig Austria: Niederösterreich H. Voglmayr JX JX CBS Carpinus betulus, dead corticated twig Austria: Niederösterreich H. Voglmayr JX JX CBS Carpinus betulus, dead corticated twig Austria: Oberösterreich H. Voglmayr JX JX CBS Carpinus betulus, dead corticated twig The Netherlands: Utrecht H. Voglmayr JX JX CBS Carpinus betulus, dead corticated twig Austria: Niederösterreich, Wassergspreng CBS Carpinus betulus, dead corticated twig Austria: Oberösterreich, Leithenbachtal H. Voglmayr JX JX H. Voglmayr JX JX 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

33 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; 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 ( 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 RESULTS Phylogenentic comparisons Amplicons of approximately 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 generations, after which the split frequency reached less than Trees were saved each generations, resulting in 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 MB 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 volume 3 no

34 Crous et al. Magnaporthe grisea AB Diaporthe acaciigena JF Diaporthe eres AF Prosthecium galeatum EU Prosthecium pyriforme EU Prosthecium acerophilum EU Prosthecium acerinum EU Prosthecium opalus EU Stilbospora macrosperma EU Diaporthales Stilbospora macrosperma CBS Stilbospora macrosperma CBS Stilbospora macrosperma CBS Stilbospora macrosperma CBS Stilbospora macrosperma CBS Stilbospora macrosperma CBS Umbilicaria decussata EF Umbilicaria dendrophora HM Caloplaca scopularis JQ Caloplaca marina JQ Homortomyces combreti CPC Umbilicariales Teloschistales incertae sedis 1 Hysteropatella clavispora AY Glonium chambianum GQ Gloniopsis praelonga FJ Hysteriales Psiloglonium araucanum FJ Curreya proteae EU Misturatosphaeria tennesseensis GU Sclerostagonospora sp. CBS Sclerostagonospora sp. CBS Neosetophoma samarorum GQ Phaeosphaeriopsis musae DQ Botryosphaeria melanops DQ Saccharata proteae EU Saccharata intermedia GU Phyllosticta vaccinii FJ Phyllosticta concentrica DQ Neofusicoccum mediterraneum FJ Neoscytalidium dimidiatum DQ Botryosphaeria mamane DQ Dothiorella sarmentorum DQ Botryosphaeriales Neofusicoccum ribis DQ Neofusicoccum arbuti DQ Tiarosporella tritici DQ Lasiodiplodia venezuelensis DQ Lasiodiplodia pseudotheobromae FN Diplodia porosum DQ Diplodia pinea DQ 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

35 Homortomyces gen. nov. (Dothideomycetes) 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. MycoBank MB (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 holotype; cultures ex-type CPC = CBS , 19801, = CBS , 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, µm, with volume 3 no

36 Crous et al. 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, 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 is the type species of Stilbospora Pers. 1794, while P. ellipsosporum Fresen is the type species of Prosthecium Fresen 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 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. References Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) MycoBank: an online initiative to launch mycology into the 21st century. Studies in Mycology 50: Crous PW, Schoch CL, Hyde KD, Wood AR, Gueidan C, et al. (2009a) Phylogenetic lineages in the Capnodiales. Studies in Mycology 64: Crous PW, Slippers B, Wingfield MJ, Rheeder J, Marasas WFO, et al. (2006) Phylogenetic lineages in the Botryosphaeriaceae. Studies in Mycology 55: Crous PW, Verkley GJM, Groenewald JZ, Samson RA (eds) (2009b) Fungal Biodiversity. [CBS Laboratory Manual Series 1.] Utrecht: Centraalbureau voor Schimmelcultures. Crous PW, Wingfield MJ, Park RF (1991) Mycosphaerella nubilosa a synonym of M. molleriana. Mycological Research 95: Damm U, Fourie PH, Crous PW (2007) Aplosporella prunicola, a novel species of anamorphic Botryosphaeriaceae. Fungal Diversity 27: Fleminger D (2008) World Heritage Sites of South Africa: the cradle of humankind. Johannesburg: 30 Degrees South Publishers. Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, et al. (2011) The Amsterdam Declaration on Fungal Nomenclature. IMA Fungus 2: ; Mycotaxon 116: Hoog GS de, Gerrits van den Ende AHG (1998) Molecular diagnostics of clinical strains of filamentous basidiomycetes. Mycoses 41: ima fungus

37 Homortomyces gen. nov. (Dothideomycetes) Lombard L, Polizzi G, Guarnaccia V, Vitale A, Crous PW (2011) Calonectria spp. causing leaf spot, crown and root rot of ornamental plants in Tunisia. Persoonia 27: Phillips AJL, Alves A, Pennycook SR, Johnston PR, Ramaley A, Akulov A, Crous PW (2008) Resolving the phylogenetic and taxonomic status of dark-spored teleomorph genera in the Botryosphaeriaceae. Persoonia 21: Rayner RW (1970) A Mycological Colour Chart. Kew: Commonwealth Mycological Institute. Rojas EI, Herre EA, Mejía LC, Chavarri P, Samuels GJ (2008) Endomelanconium endophyticum, a new Botryosphaeria leaf endophyte from Panama. Mycologia 100: Slippers B, Wingfield MJ (2007) The Botryosphaeriaceae as endophytes and latent pathogens of trees: identification, ecology and potential impact. Fungal Biology Reviews 21: Smith H, Wingfield MJ, Crous PW, Coutinho TA (1996) Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62: Sutton BC (1980) The Coelomycetes: fungi imperfecti with pycnidia, acervuli, and stromata. Kew: Commonwealth Mycological Institute. Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: Voglmayr H, Jaklitsch WM (2008) Prosthecium species with Stegonsporium anamorphs on Acer. Mycological Research 112: White TJ, Bruns T, Lee J, Taylor SB (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: a guide to methods and applications: San Diego: Academic Press. Wingfield MJ, de Beer ZW, Slippers B, Wingfield BD, Groenewald JZ, et al. (2012) One fungus, one name promotes progressive plant pathology. Molecular Plant Pathology 13: volume 3 no

38 ima fungus

39 doi: /imafungus IMA Fungus volume 3 no 2: 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 Calahorra, La Rioja, Spain 2 C/ Andalucía 3, 4.º dcha Calahorra, La Rioja, Spain 3 Via Marmilla, , Olbia. Italy 4 Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Viale P.A. Mattioli 25, I Torino, Italy; corresponding author alfredo.vizzini@unito.it 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 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 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. MATERIALS AND METHODS 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

40 Muñoz et al. 100 Leucoagaricus flammeotinctoides GU Leucoagaricus flammeotinctoides GQ Leucoagaricus flammeotinctoides AY Leucoagaricus pyrrhophaeus GU Leucoagaricus pyrrhophaeus GQ Leucoagaricus pyrrhulus GQ Leucoagaricus pyrrhulus GU Leucoagaricus erythrophaeus GQ Leucoagaricus erythrophaeus GQ Lepiota roseifolia GQ Leucoagaricus decipiens GQ Leucoagaricus pardalotus GU Leucoagaricus pardalotus GQ Lepiota decorata GU Lepiota decorata AY Leucoagaricus ionidicolor AY Leucoagaricus marriagei GQ Leucoagaricus sp. Weber 6019 AY Leucoagaricus brunnescens GQ Leucoagaricus georginae AY Leucoagaricus georginae GU Leucoagaricus jubilaei AY Leucoagaricus badhamii GQ Leucoagaricus sp. ecv2484 GU Leucoagaricus dyscritus GU Leucoagaricus dyscritus GU Leucoagaricus hesperius GU Leucoagaricus hesperius GU Lepiota fuliginescens GU Lepiota fuliginescens GU Lepiota fuliginescens GU Lepiota flammeotincta GU Leucoagaricus sp. Vellinga 2746 AY Lepiota flammeotincta GU Lepiota fuliginescens GU Leucoagaricus sp. Vellinga 2619 AY Lepiota fuliginescens GU Leucoagaricus sp. Huijser s.n. AY Leucoagaricus adelphicus GQ Leucoagaricus adelphicus AY Piloselli Leucoagaricus adelphicus AY Leucoagaricus pilatianus GQ Leucoagaricus pilatianus GQ Leucoagaricus cupresseus GQ Leucoagaricus cupresseus GU Leucoagaricus cupresseus AY Leucoagaricus cupresseus AY Leucoagaricus cupresseus AY Leucoagaricus variicolor coll. GM-2485 JX Leucoagaricus variicolor coll. AH (holotype) JX Leucoagaricus variicolor coll. GM-2486 JX Leucoagaricus variicolor coll. GM-2454 JX Leucoagaricus rubrotinctus JN Leucoagaricus rubrotinctus FJ Leucoagaricus rubrotinctus JN Leucoagaricus sublittoralis AY Rubrotincti 100 Leucoagaricus littoralis GQ Leucoagaricus wychanskyi AF Cystolepiota seminuda AY 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 ( 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

41 A new Leucoagaricus species from Spain Fig. 2. Leucoagaricus variicolor. Macroscopic characters. A C. Fresh basidiomata in situ. D. Herbarium specimens. A, D from AH (holotypus); B from GM-2454; C from GM Bars = A B = 50 mm; C D = 20 mm. was performed through RAxML v (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 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. MycoBank MB (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 W 0.894, alt. 225 m, on argillose-sandy soil, in Pinus halepensis litter, 3 Dec. 2011, G. Muñoz (AH holotype; GM and AC-4896 isotypes). Description: Pileus 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 volume 3 no

42 Muñoz et al. 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 (holotype); B, D, F from GM 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 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

43 A new Leucoagaricus species from Spain 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 ) ( 8.2) (4.5 ) ( 5.7) µm, Q = (1.1 ) ( 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 µm four-spored, clavate, without basal clamp connection (Fig. 4B). Lamella edge sterile. Cheilocystidia abundant, 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, ( 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 W 0.894, alt. 225 m, near Pinus halepensis, on argillose-sandy soil, volume 3 no

44 Muñoz et al. 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. REFERENCES Boisselet P, Guinberteau J (2001) Leucoagaricus cupresseus (Burlingham) Boisselet & Guinberteau comb. nov., une lépiote cupressicole d origine américaine récoltée en France. Bulletin de la Féderation des Assocations mycologiques méditerranéennes, n.s. 19: Bon M (1981) Clé monographique des Lépiotes d Europe. Documents Mycologiques 11 (43): Bon M (1993) Flore Mycologique d Europe 3: Les Lépiotes. Lepiotaceae Roze. Documents mycologiques, Mémoire hors série 3: Bon M, Caballero A (1997) Le genre Leucoagaricus dans La Rioja (Espagne). Documents Mycologiques 27 (106): Caballero A (1997) Flora Micológica de La Rioja, 1: Lepiotaceae. CD- ROM. Calahorra-La Rioja: A Caballero. Candusso M, Lanzoni G (1990) Lepiota s.l. [Fungi Europaei vol. 4.] Saronno: Giovanna Biella. Contu M (1990) Nuovi taxa di Agaricales (Basidiomycetes) dalla Sardegna. Boletim da Sociedade Broteriana 63(2): Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) MycoBank: an online initiative to launch mycology into the 21st century. Studies in Mycology 50: Drummond AJ, Ashton B, Cheung M, Heled J, Kearse M, Moir R, Stones-Havas S, Thierer T, Wilson A (2010) Geneious. Version 5.3. < Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Molecular Ecology 2: Gennari A, Migliozzi V 1999 ( 1998 ) Una nuova entità della sezione Piloselli: Leucoagaricus aurantiovergens sp. nov. Rivista di Micologia 41: Guinberteau J, Boisselet P, Dupuy G (1998) Leucoagaricus idaefragum, sp. nov., un nouveau Leucoagaricus des dunes atlantiques françaises de coloration rose framboise. Bulletin trimestriel de la Societé mycologique de France 114(3): Migliozzi V, Resta G (2001) Note sulla sottosezione Pilatiani del genere Leucoagaricus. Due nuove varietà: Leucoagaricus pseudopilatianus var. rugosoreticulatus e Leucoagaricus pseudopilatianus var. roseodiffractus. Micologia e Vegetazione Mediterranea 15: Migliozzi V, Rocabruna A, Tabarés M (2001) Leucoagaricus pseudopilatianus: una nueva especie de la sección Piloselli. Revista Catalana de Micologia 23: Munsell C (1994) Munsell Soil Color Charts. New Windsor, NY: Kollmorgen Instruments. Singer R (1948) Diagnoses fungorum novorum Agaricalium. Sydowia 2: Singer R (1973) Diagnoses fungorum novorum Agaricalium III. Beihefte zur Sydowia 7: Singer R (1986) The Agaricales in Modern Taxonomy. 4th edn. Königstein: Koeltz Scientific Books. Stamatakis A (2006) RAxML-VI-HPC: Maximum Likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: Sundberg WJ (1976) Lepiota sensu lato in California. II. Type studies of Lepiota cupressea and Lepiota marginata. Mycotaxon 3: Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony methods. Molecular Biology and Evolution 28: ima fungus

45 A new Leucoagaricus species from Spain Vellinga EC (2001) Leucoagaricus (Locq. ex) Sing. In: Flora Agaricina Neerlandica (ME Noordeloos, TW Kuyper & EC Vellinga EC, eds) 5: Lisse: AA Balkema Publishers. Vellinga EC (2006) Lepiotaceous fungi in California, U.S.A. 3. Pink and lilac species in Leucoagaricus sect. Piloselli. Mycotaxon 98: Vellinga EC (2010) Lepiotaceous fungi in California, U.S.A. Leucoagaricus sect. Piloselli. Mycotaxon 112: Vellinga E, Contu M, Vizzini A (2010) Leucoagaricus decipiens and La. erythrophaeus, a new species pair in sect. Piloselli. Mycologia 102: Vellinga E, Sysouphanthong P, Hyde KD (2011) The family Agaricaceae: phylogenies and two new white-spored genera. Mycologia 103: Vizzini A, Contu M, Musumeci E, Ercole E (2011) A new taxon in the Infundibulicybe gibba complex (Basidiomycota, Agaricales, Tricholomataceae) from Sardinia (Italy). Mycologia 103: White TJ, Bruns TD, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: method and applications (MA Innis, DH Gelfand, JJ Snisky & TJ White, eds): San Diego: Academic Press. volume 3 no

46 Muñoz et al. 124 ima fungus

47 doi: /imafungus IMA Fungus volume 3 no 2: 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 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 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 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) International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

48 Suwannasai et al. 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

49 Ascus apical apparatus and ascospore characters in Xylariaceae 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. volume 3 no

50 Suwannasai et al. 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

51 Ascus apical apparatus and ascospore characters in Xylariaceae 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 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. volume 3 no

52 Suwannasai et al. 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

53 Ascus apical apparatus and ascospore characters in Xylariaceae 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). REFERENCES Baral H-O (1987) Lugol s solution/iki versus Melzer s reagent: hemiamyloidity, a universal feature of the ascus wall. Mycotaxon 29: Beckett A, Crawford RM (1973) The development and fine structure of the ascus apex and its role during spore discharge in Xylaria longipes. New Phytologist 72: Carroll G (1963) Studies on the flora of Thailand 24. Pyrenomycetes. Dansk Botansk Arkiv 23: Carroll G (1964) Pyrenomycetes, mainly Xylariaceae, from some South Pacific Islands. Botanisk Tidskrift 59: Chadefaud M (1942) Structure et anatomie comparee de 1 appareil apical des asques chez divers Discomycètes et Hypocopra, sa Pyrenomycètes. Revue Mycologie 7: Chadefaud M (1973) Les asques et la systematique des Ascomycètes. Bulletin de la Société Mycologique de France 89: Chapela IH, Petrini O, Hagmann L (1991) Monolignol glucosides as specific recognition messengers in fungus/plant symbioses. Physiological and Molecular Plant Pathology 39: Chapela IH, Petrini O, Petrini LE (1990) Unusual ascospore germination In Hypoxylon fragiforme first steps in the establishment of an endophytic symbiosis. Canadian Journal of Botany 68: Eriksson OE (1966) On Anthostomella Sacc., Entosordaria (Sacc.) Höhn. and some related genera (Pyrenomycetes). Svensk Botanisk Tidskrift 60: Francis SM (1975) Anthostomella Sacc. (Part I). Mycological Papers 139: Greenhalgh GN, Evans LV (1967) The structure of the ascus apex in Hypoxylon fragiforme with reference to ascospore release in this and related species. Transactions of the British Mycological Society 50: Griffiths HB (1973) Fine structure of seven unitunicate pyrenomycete asci. Transactions of the British Mycological Society 60: Hawksworth DL (1971) A revision of the genus Ascotricha Berk. Mycological Papers 126: Hawksworth DL, Lodha BC (1983) Helicogermslita: a new stromatic xylariaceous genus with a spiral germ slit from India. Transactions of the British Mycological Society 81: Jong SC, Davis EE (1973) Stromatic Neurosporas. Mycologia 65: volume 3 no

54 Suwannasai et al. Jong SC, Rogers JD (1969) Poronia oedipus in culture. Mycologia 60: Ju Y-M, Rogers JD (1996) A Revision of the Genus Hypoxylon. St Paul, MN: American Phytopathological Society Press. Ju Y-M, Rogers JD, San Martin F (1997) A revision of the genus Daldinia. Mycotaxon 61: Ju Y-M, Rogers JD, San Martin F, Granmo, A (1998) The genus Biscogniauxia. Mycotaxon 67: Kenerley CM, Rogers JD (1976) On Hypoxylon serpens in culture. Mycologia 68: Kohn LM, Korf RP (1975) Variation in ascomycete iodine reactions: KOH pretreatment explored. Mycotaxon 3: Krug JC, Cain RF (1974a) A preliminary treatment of the genus Podosordaria. Canadian Journal of Botany 52: Krug JC, Cain RF (l974b) New species of Hypocopra. Canadian Journal of Botany 52: Læssøe T, Rogers JD, Whalley AJS (1989) Camillea, Jongiella and light- spored species of Hypoxylon. Mycological Research 93: Lumbsch HT, Huhndorf SM (2010) Outline of Ascomycota Fieldiana, Life and Earth Sciences 14: Lu BS, Hyde KD, Ho WH (1998) Spirodecospora gen. nov. (Xylariaceae, Ascomycotina), from bamboo in Hong Kong. Fungal Diversity 1: Lundqvist N (1992) Wawelia effusa Lundqvist spec. nov. (Xylariaceae). Persoonia 14: Martin P (1967) Studies in the Xylariaceae: I. New and old concepts. Journal of South African Botany 33: Martin P (1968a) Studies in the Xylariaceae III. South African and foreign species of Hypoxylon section Entoleuca. South African Journal of Botany 34: Martin P (l968b) Studies in the Xylariaceae IV. Hypoxylon, sections Papillata and Annulata. South African Journal of Botany 34: Martin P (1969a) Studies in the Xylariaceae V. Euhypoxylon. Journal of South African Botany 35: Martin P (l969b) Studies in the Xylariaceae VI. Daldinia, Numulariola and their allies. Journal of South African Botany 35: Miller JH (1961) A Monograph of the World Species of Hypoxylon. Athens, GA: University of Georgia Press. Minter DW, Webster J (1983) Wawelia octospora sp. nov., a xerophilous and coprophilous member of the Xylariaceae. Transactions of the British Mycological Society 80: Munk A (1957) Danish pyrenomycetes. Dansk Botansk Arkiv 17: Nannfeldt JA (1976) Iodine reactions in ascus plugs and their taxonomic significance. Transactions of the British Mycological Society 67: Petrini LE (1992) Rosellinia species of the temperate zones. Sydowia 44: Petrini LE, Müller E (1986) Haupt- und Nebenfruchtformen europäischer Hypoxylon-Arten (Xylariaceae, Sphaeriales) und velwandter Pilze. Mycologia Helvetica 1: Petrini LE, Rogers JD (1986) A summary of the Hypoxylon serpens complex. Mycotaxon 26: Pouzar Z (1972) Hypoxylon fraxinophylum spec. nov. and H. moravicium spec. nov., two interesting species found on Fraxinus angustifolia. Česká Mykologie 26: Pouzar Z (1985a) Reassessment of Hypoxylon serpens-complex 1. Česká Mykologie 39: Pouzar Z (1985b) Reassessment of Hypoxylon serpens-complex II. Česká Mykologie 39: Rogers JD (1977) Surface features of the light colored ascospores of some applanate Hypoxylon species. Canadian Journal of Botany 55: Rogers JD (1979) The Xylariaceae: systematic, biological and evolutionary aspects. Mycologia 71: Rogers JD (1985) Hypoxylon duranii sp. nov. and the anamorphs of H. caries, H. papiflatum, and Rosellinia subicutata. Mycotaxon 23: Rogers JD (1994) Problem genera and family interfaces in the eupyrenomycetes In: Ascomycete Systematics: problems and perspectives in the nineties (Hawksworth DL, ed.): New York: Plenum Press. Rogers JD (2000) Thoughts and musing on tropical Xylariaceae. Mycological Research 104: Rogers JD, Callan BE, Samuels GJ (1987) The Xylariaceae of the rain forests of North Sulawesi (Indonesia). Mycotaxon 29: Rogers JD, Candoussau F (1982) Hypoxylon gillesii, a new species with ornamented ascospores from Madagascar. Mycotaxon 15: Rogers JD, Læssøe T, Lodge DJ (1991) Camillea: new combinations and a new species. Mycologia. 83: Rogers JD, Martin FS, Ju YM (2002) Three new taxa of Camillea from Costa Rica. Sydowia 54: Rogers JD, Ju Y-M, Hemmes DE (1992) Hypoxylon rectangulosporum sp. nov., Xylaria psidii sp. nov., and comments on taxa of Podosordaria and Stromatoneurospora. Mycologia 84: Samuels GJ, Rossman AY (1992) Thuemenella and Sarawakus. Mycologia 84: Stadler M, Baumgartner M, Ide K, Popp A, Wollweber H (2002) Importance of ascospore ornamentation in the taxonomy of Daldinia. Mycological Progress 1: Stadler M, Hellwig V (2005) Chemotaxonomy of the Xylariaceae and remarkable bioactive compounds from the Xylariales and their associated asexual stages. Recent Research Developments in Phytochemistry 9: Triebel D, Persoh D, Wollweber H, Stadler M (2005) Phylogenetic relationships among Daldinia, Entonaema, and Hypoxylon as inferred from nr DNA analyses of Xylariales. Nova Hedwigia 80: van der Gucht K, van der Veken P (1992) Contributions towards a revision of the genus Hypoxylon s. str. (Xylariaceae, Ascomycetes) from Papua New Guinea. Mycotaxon 44: van der Gucht K (1993) Spore ornamentation makes a nice difference: Daldinia eschscholzii and D. concentrica. In: Aspects of Tropical Mycology Isaac S, Frankland JC, Watling R, Whalley AJS, eds): Cambridge, UK: Cambridge University Press. van der Gucht K (1995) Illustrations and descriptions of xylariaceous fungi collected in Papua New Guinea. Bulletin de la Jardin Botanique National de Bélgique 64: Whalley AJS (1996) The xylariaceous way of life. Mycological Research 100: Whalley AJS, Edwards RL (1995) Secondary metabolites and systematic arrangements in the Xylariaceae. Canadian Journal of Botany 73 (Suppl 1): S802 S810. Whalley AJS, Læssøe T, Kile GA (1990) A new species of 132 ima fungus

55 Ascus apical apparatus and ascospore characters in Xylariaceae Biscogniauxia with appendaged ascospores from Tasmania. Mycological Research 94: Whalley AJS, Thienhirun S (1996) Rhopalostroma kanyae sp. nov. from Thailand. Mycological Research 100: Whalley MA (1995) Camillea fusiformis sp. nov. from Ecuador. Sydowia 47: Whalley MA (1996) Distinctive features of Camillea (Xylariaceae) from Cuyabeno as revealed by scanning electron microscopy. Mycologist 10: Whalley MA, Whalley AJS, Jones EBG (1996) Camillea selangorensis sp. nov. from Malaysia. Sydowia 48: l. Whalley MA, Whalley AJS, Thienhirun S, Sihanonth P (1999) The genus Camillea in southeast Asia. Kew Bulletin 54: volume 3 no

56 Suwannasai et al. 134 ima fungus

57 doi: /imafungus IMA Fungus volume 3 no 2: 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, Viçosa, Minas Gerais, Brazil; corresponding author 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 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

58 Barreto et al. 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 (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. MycoBank MB (Figs 1 5) Etymology: derived from Xylopia, the generic name of the host plant. 136 ima fungus

59 Claviradulomyces xylopiae sp. nov. from Xylopia sericea Fig. 2. Claviradulomyces xylopiae asexual morph (VIC 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 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. volume 3 no

60 Barreto et al. Fig. 4. Claviradulomyces xylopiae asci and ascospores (VIC 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 ( 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 and CBS 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, 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 µm wide. Periphysoids lining the upper wall above the level of the hymenium, cylindrical or clubshaped, sinuose or curved, 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, 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 ) 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, µm diam; thin walled, walls 4 15 µm thick, with long cylindrical ostiolate necks, µm, composed of parallel hyphae 1 3 µm wide, often reduced to a narrower cylinder of bristle-like hyphal tips at the apex, µm. Conidiophores usually reduced to conidiogenous cells, occasionally consisting of a small stalk 138 ima fungus

61 Claviradulomyces xylopiae sp. nov. from Xylopia sericea 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, 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, 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 ( 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, µm compared to µm, and shorter asci, µm compared with 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, µm in length, than in C. dabeicola volume 3 no

62 Barreto et al. 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 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

63 Claviradulomyces xylopiae sp. nov. from Xylopia sericea Acknowledgements 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). REFERENCES Baloch E, Lucking R, Lumbsch HT, Wedin M (2010) Major clades and phylogenetic relationships between lichenized and non-lichenized lineages in Ostropales (Ascomycota: Lecanoromycetes). Taxon 59: Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, et al. (2011) Geneious. Version 5.4, Evans HC, Johnston PR, Park D, Barreto RW, Soares DR (2010) Claviradulomyces, a new genus of Odontotremataceae from West African rainforest. Fungal Biology 114: Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by Maximum Likelihood. Systematic Biology 52: Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17: org/ /bioinformatics/ Johnston PR, Park D (2005) Chlorociboria (Fungi, Helotiales) in New Zealand. New Zealand Journal of Botany 43: Lorenzi H (1992) Árvores Brasileiras. Nova Odessa, São Paulo: Editora Plantarum. Miadlikowska J, Kauff F, Hofstetter V, Fraker E, Grube M, et al. (2006) New insights into classification and evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from phylogenetic analyses of three ribosomal RNA- and two proteincoding genes. Mycologia 98: Nylander JAA (2004) MrModeltest v2. Program distributed by the author. Uppsala: Evolutionary Biology Centre, Uppsala University. Pereira JM, Barreto RW, Ellison CA, Maffia LA (2003) Corynespora cassiicola f. sp lantanae: a potential biocontrol agent from Brazil for Lantana camara. Biological Control 26: Rambaut A, Drummond AJ (2007) Tracer. Version bio.ed.ac.uk/tracer. Rayner RW (1970) A Mycological Colour Chart. Kew: Commonwealth Mycological Institute. Ronquist F, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates. Thompson JD, Higgins DJ, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22: Wedin M, Döring H, Könberg K, Gilenstam G (2005) Generic delimitation in the family Stictidaceae (Ostropales, Ascomycota): the Stictis-Conotrema problem. Lichenologist 37: White TJ, Bruns T, Lee J, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: a guide to methods and applications: San Diego: Academic Press. Zoller S, Scheidegger C, Sperisen C (1999) PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31: volume 3 no

64 Barreto et al. 142 ima fungus

65 doi: /imafungus IMA Fungus volume 3 no 2: 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 Tübingen, Germany; corresponding author matthias.lutz@uni-tuebingen.de 2 Herbarium Ustilaginales Vánky (H.U.V.), Gabriel-Biel-Straße 5, D Tübingen, Germany 3 Department of Mycology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL Kraków, Poland 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 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

66 Lutz, Vánky & Piątek 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) , isotype DAR Schoenus apogon AU: Tasmania, Hobart, 4 Nov. 1894, L. Rodway H.U.V 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 H.U.V H.U.V LSU: JF H.U.V , H.U.V , KRAM F ITS: JF966731, LSU: JF H.U.V Schoenus maschalinus NZ: Wellington, Upper Hutt, 13 Nov. 1952, A.J. Healy H.U.V Schoenus nitens var. NZ: Wanganui, Himatangi, 29 Jan. 1932, H.H. Allan H.U.V concinnus Schoenus pauciflorus NZ: Canterbury, near Cass, Kettlehole Bog, 1 Feb. H.U.V , K. Vánky Schoenus tesquorum AU: New South Wales, Sydney, Enfield Sate Park, Drevers Road, 28 Nov. 1996, J. Dickins H.U.V epitype H.U.V , isoepitype TUB 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. MATERIALS AND METHODS 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 Tübingen, Germany). Nomenclatural novelties were registered in 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

67 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/ /93 69/76 Franzpetrakia microstegii GU Sporisorium sorghi AF Anomalomyces panici DQ Melanopsichium pennsylvanicum AY Ustilago hordei AY Leucocintractia scleriae AJ Ustanciosporium standleyanum DQ Tranzscheliella hypodytes DQ Macalpinomyces eriachnes AY Moesziomyces bullatus DQ Eriomoeszia eriocauli AY Farysia chardoniana AF Stegocintractia luzulae AJ Schizonella melanogramma AF Shivasia solida (syn.: Ustilago solida) JF Shivasia solida (syn.: Ustilago solida) JF Heterotolyposporium lepidospermatis DQ Tolyposporium piluliforme AF Tolyposporium isolepidis EU Tolyposporium junci AF Tolyposporium neillii EU Anthracoidea caricis AY Portalia uljanishcheviana EF Dermatosorus cyperi AJ Cintractia axicola DQ Trichocintractia utriculicola AF Parvulago marina DQ Moreaua fimbristylidis DQ Pericladium grewiae DQ /- 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 DQ Websdanea lyginiae AJ Melanotaenium endogenum DQ Melanoxa oxalidis EF Melanustilospora ari EF /- Ustacystis waldsteiniae AF Vankya ornithogali EF Urocystis ficariae EF Flamingomyces ruppiae DQ Mundkurella kalopanacis AF Antherospora vaillantii EF Floromyces anemarrhenae EU Mycosyrinx cissi DQ Doassansiopsis deformans AF Thecaphora seminis-convolvuli AF Thecaphora saponariae EF Tilletia caries AY Exobasidium vaccinii FJ Entyloma microsporum DQ /53 100/ substitutions/site Ustilaginaceae Melanotaeniaceae Urocystidaceae Floromycetaceae Mycosyringaceae Doassansiopsidaceae Glomosporiaceae Ustilaginales Urocystidales 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 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 volume 3 no

68 Lutz, Vánky & Piątek 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 AJ 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 (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 (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 trees. From these, the first trees were discarded (burnin = 5 001). The trees sampled after the process had reached stationarity ( 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 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). Phylogenetic analyses The LSU sequences of the two Ustilago solida specimens analysed were identical. Compared to the LSU of Heterotolyposporium lepidospermatis DQ 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 revealed closest similarity to Tolyposporium isolepidis EU and T. neillii EU 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

69 Shivasia, a new genus for Ustilago solida Fig. 2. Shivasia solida on Schoenus apogon. A. Holotype of Ustilago solida [K(M) ]. B. The habit of infected plants and two enlarged sori (H.U.V ). 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. MycoBank MB 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 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. MycoBank MB (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, Ustilago solida, Berk. on Chaetospora imberbis Penquite 21/12/45 h 581. [original label of holotype specimen, Fig. 2a] (K(M) holotype; DAR microscope slide ex-holotype). Australia: Tasmania: 170 km NE of Hobart, on Schoenus apogon, 8 March 1996, C. Vánky & K. Vánky (H.U.V epitype designated here; TUB isoepitype). volume 3 no

70 Lutz, Vánky & Piątek Fig. 3. Shivasia solida on Schoenus apogon. A. Embedded, stained, semi-thin section of a sorus (H.U.V ), 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 ). 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 ). 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 ). F. Spore balls in different developmental stages, hand sectioned, stained with cotton blue in lactophenol (H.U.V ). G H. Spore germination in water, at room temperature, in 3 5 days (H.U.V ). Bars: A = 100 µm, B H = 10 µm. 148 ima fungus

71 Shivasia, a new genus for Ustilago solida 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) µm, reddish brown, enclosed by subhyaline mucilaginous layer. Spores (Fig. 4) subglobose, ovoid, elongate or irregular, flattened on one or two sides, µm, yellowish to pale reddish brown; wall uneven, 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 volume 3 no

72 Lutz, Vánky & Piątek 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

73 Shivasia, a new genus for Ustilago solida 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 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 , and Schoenus tesquorum, H.U.V ) 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. ACKNOWLEDGEMENTS We thank Michael Weiß, Sigisfredo Garnica, and Robert Bauer (Tübingen) for providing facilities for molecular analyses, Michael volume 3 no

74 Lutz, Vánky & Piątek 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. REFERENCES Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: Barrett MD, Barrett RL, Shivas RG, McTaggart AR (2009) Fungal Planet no. 33. Tilletia micrairae. Persoonia 22: Bauer R, Begerow D, Nagler A, Oberwinkler F (2001a) The Georgefischeriales: a phylogenetic hypothesis. Mycological Research 105: Bauer R, Begerow D, Oberwinkler F, Piepenbring M, Berbee ML (2001b) Ustilaginomycetes. In: The Mycota. Vol. VII, Part B. Systematics and Evolution (DJ McLaughlin, EG McLaughlin, PA Lemke, eds): Berlin: Springer-Verlag. Bauer R, Begerow D, Sampaio JP, Oberwinkler F (2006) The simpleseptate basidiomycetes. Mycological Progress 5: Bauer R, Begerow D, Vánky K, Oberwinkler F (1999) Ustilaginomycetes on Selaginella. Mycologia 91: Bauer R, Lutz M, Begerow D, Piątek M, Vánky K, Bacigalova K, Oberwinkler F (2008) Anther smut fungi on monocots. Mycological Research 112: Bauer R, Lutz M, Oberwinkler F (2005) Gjaerumia, a new genus in the Georgefischeriales (Ustilaginomycetes). Mycological Research 109: Bauer R, Lutz M, Piątek M, Vánky K, Oberwinkler F (2007) Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina). Mycological Research 111: Bauer R, Oberwinkler F, Vánky K (1997) Ultrastructural markers and systematics in smut fungi and allied taxa. Canadian Journal of Botany 75: Begerow D, Bauer R, Oberwinkler F (1997) Phylogenetic studies on nuclear LSU rdna sequences of smut fungi and related taxa. Canadian Journal of Botany 75: Begerow D, Stoll M, Bauer R (2007( 2006 ). A phylogenetic hypothesis of Ustilaginomycotina based on multiple gene analyses and morphological data. Mycologia 98: Carris LM, Castlebury LA, Huang GM, Alderman SC, Luo JF, Bao XD (2007) Tilletia vankyi, a new species of reticulate-spored bunt fungus with non-conjugating basidiospores infecting species of Festuca and Lolium. Mycological Research 111: Castlebury LA, Carris LM, Vánky K (2005) Phylogenetic analysis of Tilletia and allied genera in order Tilletiales (Ustilaginomycetes; Exobasidiomycetidae) based on large subunit nuclear rdna sequences. Mycologia 97: Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Molecular Biology and Evolution 17: Chakrabarty P (2010) Genetypes: a concept to help integrate molecular phylogenetics and taxonomy. Zootaxa 2632: Chandra A, Huff DR (2008) Salmacisia, a new genus of Tilletiales: reclassification of Tilletia buchloëana causing induced hermaphroditism in buffalograss. Mycologia 100: Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) MycoBank: an online initiative to launch mycology into the 21 st century. Studies in Mycology 50: Crous PW, Summerell BA, Shivas RG, Burgess TI, Decock CA, Dreyer LL, Granke LL, Guest DI, Hardy GESJ, Hausbeck MK, Hüberli D, Jung T, Koukol O, Lennox CL, Liew ECY, Lombard L, McTaggart AR, Pryke JS, Roets F, Saude C, Shuttleworth LA, Stukely MJC, Vánky K, Webster BJ, Windstam ST, Groenewald JZ (2012) Fungal Planet description sheets: Persoonia 28: Cunnington JH, Vánky K, Shivas RG (2005) Lundquistia is a synonym of Sporisorium (Ustilaginomycetes). Mycologia Balcanica 2: Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 17: Fischer von Waldheim AA (1877) Aperçu systématique des Ustilaginées, leurs plantes nourricières et la localisation de leurs spores. Paris: Lahure. Gatesy J, DeSalle R, Wheeler W (1993) Alignment-ambiguous nucleotide sites and the exclusion of systematic data. Molecular Phylogenetics and Evolution 2: Giribet G, Wheeler WC (1999) On gaps. Molecular Phylogenetics and Evolution 13: González V, Vánky K, Platas G, Lutz M (2007) Portalia gen. nov. (Ustilaginomycotina). Fungal Diversity 27: Hendrichs M, Begerow D, Bauer R, Oberwinkler F (2005) The genus Anthracoidea (Basidiomycota, Ustilaginales): a molecular phylogenetic approach using LSU rdna sequences. Mycological Research 109: Hooker JD ( ) The botany of the Antarctic Voyage. Vol. 3. Flora Tasmaniae. Vol. 2. London: Reeve. Huelsenbeck JP, Larget B, Miller RE, Ronquist F (2002) Potential applications and pitfalls of Bayesian inference of phylogeny. Systematic Biology 51: Huelsenbeck JP, Rannala B (2004) Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology 53: Huelsenbeck JP, Ronquist F (2001) MRBAYES: bayesian inference of phylogenetic trees. Bioinformatics Applications Note 17: Hyde KD, Zhang Y (2008) Epitypification: should we epitypify? Journal of Zhejiang University, Science B 9: Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research 33: Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30: Katoh K, Toh H (2008) Recent developments in the MAFFT multiple sequence alignment program (outlines version 6). Briefings in Bioinformatics 9: Kemler M, Lutz M, Göker M, Oberwinkler F, Begerow D (2009) Hidden diversity in the non-caryophyllaceous plant-parasitic members of Microbotryum (Pucciniomycotina: Microbotryales). Systematics and Biodiversity 7: Kottke I, Suarez JP, Herrera P, Cruz D, Bauer R, Haug I, Garnica S (2010) Atractiellomycetes belonging to the rust lineage 152 ima fungus

75 Shivasia, a new genus for Ustilago solida (Pucciniomycotina) form mycorrhizae with terrestrial and epiphytic neotropical orchids. Proceedings of the Royal Society of London, B: Biological Sciences 277: Lutz M, Bauer R, Begerow D, Oberwinkler F, Triebel D (2004) Tuberculina, rust relatives attack rusts. Mycologia 96: Lutz M, Göker M, Piątek M, Kemler M, Begerow D, Oberwinkler F (2005) Anther smuts of Caryophyllaceae: molecular characters indicate host-dependent species delimitation. Mycological Progress 4: Lutz M, Piątek M, Kemler M, Chlebicki A, Oberwinkler F (2008) Anther smuts of Caryophyllaceae: molecular analyses reveal further new species. Mycological Research 112: Lutz M, Vánky K, Bauer R (2012) Melanoxa, a new genus in the Urocystidales (Ustilaginomycotina). Mycological Progress 11: Matheny PB, Gossmann JA, Zalar P, Arun Kumar TK, Hibbett DS (2006) Resolving the phylogenetic position of the Wallemiomycetes: an enigmatic major lineage of Basidiomycota. Canadian Journal of Botany 84: McAlpine D (1910) The Smuts of Australia: their structure, life history, treatment, and classification. Melbourne: J. Kemp. McLoughlin S (2001) The breakup history of Gondwana and its impact on pre-cenozoic floristic provincialism. Australian Journal of Botany 49: McTaggart AR, Shivas RG (2009a) Fungal Planet No. 36. Tilletia challinorae. Persoonia 23: McTaggart AR, Shivas RG (2009b) Fungal Planet No. 37. Macalpinomyces mackinlayi. Persoonia 23: Muasya AM, Simpson DA, Verboom GA, Goetghebeur P, Naczi RFC, Chase MW, Smets E (2009) Phylogeny of Cyperaceae based on DNA sequence data: current progress and future prospects. Botanical Review 75: O Donnell KL (1993) Fusarium and its near relatives. In: The Fungal Holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematics (DR Reynolds, JW Taylor, eds): Wallingford: CAB International. Piątek M, Lutz M, Ronikier A, Kemler M, Świderska-Burek U (2012) Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System. Fungal Biology 116: Piątek M, Lutz M, Smith PA, Chater AO (2011) A new species of Antherospora supports the systematic placement of its host plant. IMA Fungus 2: Piątek M, Shivas RG (2011) Sporisorium warambiense sp. nov., a fourth smut fungus on Xerochloa in Australia. Mycological Progress 10: Piątek M, Vánky K (2007) Cintractia bulbostylidicola sp. nov. (Ustilaginomycotina) from North America. Nova Hedwigia 85: Piepenbring M (2000) The species of Cintractia s. l. (Ustilaginales, Basidiomycota). Nova Hedwigia 70: Piepenbring M, Begerow D, Oberwinkler F (1999) Molecular sequence data assess the value of morphological characteristics for a phylogenetic classification of species of Cintractia. Mycologia 91: Ronquist FR, Huelsenbeck JP (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: Savchenko KG, Lutz M, Piątek M, Heluta VP, Nevo E (2012) Anthracoidea caricis-meadii is a new North American smut fungus on Carex sect. Paniceae. Mycologia: DOI: / Shivas RG, Barrett MD, Barrett RL, Vánky K (2011) Two new species of Moreaua (Ustilaginomycetes), on Actinoschoenus and Chrysitrix, from Western Australia. Mycologia Balcanica 8: Shivas RG, McTaggart AR, Ryley MJ, Scharaschkin T, Gambley CF (2010) First report of the smut fungus Ustanciosporium appendiculatum in Australia. Australasian Plant Disease Notes 5: Shivas RG, McTaggart AR (2009) Three new species of Tilletia on native grasses from northern Australia. Australasian Plant Pathology 38: Shivas RG, Vánky K (2003) Biodiversity of Australian smut fungi. Fungal Diversity 13: Silvestro D, Michalak I (2012) raxmlgui: a graphical front-end for RAxML. Organisms Diversity and Evolution 12: Simpson DA, Muasya AM, Alves MV, Bruhl JJ, Dhooge S, Chase MW, Furness CA, Ghamkhar K, Goetghebeur P, Hodkinson TR, Marchant AD, Reznicek AA, Nieuwborg R, Roalson EH, Smets E, Starr JR, Thomas WW, Wilson KL, Zhang XF (2007) Phylogeny of Cyperaceae based on DNA sequence data a new rbcl analysis. Aliso 23: Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology 57: Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: Stoll M, Begerow D, Oberwinkler F (2005) Molecular phylogeny of Ustilago, Sporisorium, and related taxa based on combined analyses of rdna sequences. Mycological Research 109: Stoll M, Piepenbring M, Begerow D, Oberwinkler F (2003) Molecular phylogeny of Ustilago and Sporisorium species (Basidiomycota, Ustilaginales), based on internal transcribed spacer (ITS) sequences. Canadian Journal of Botany 81: Vánky K (1997) Heterotolyposporium, a new genus of Ustilaginales. Mycotaxon 63: Vánky K (1998a) Proposal to conserve the generic name Thecaphora Fingerhuth against Sorosporium Rudolphi (Fungi, Ustilaginales). Taxon 47: Vánky K (1998b) Taxonomical studies on Ustilaginales XVIII. Mycotaxon 69: Vánky K (2002) Illustrated Genera of Smut Fungi, 2nd edn. St Paul, MN: American Phytopathological Society Press. Vánky K (2008a) Smut fungi (Basidiomycota p.p., Ascomycota p.p.) of the world: novelties, selected examples, trends. Acta Microbiologica et Immunologica Hungarica 55: Vánky K (2008b) Taxonomic studies on Ustilaginomycetes 28. Mycotaxon 106: Vánky K (2009) Taxonomic studies on Ustilaginomycetes 29. Mycotaxon 110: Vánky K (2011) Bambusiomyces, a new genus of smut fungi (Ustilaginomycetes). Mycologia Balcanica 8: Vánky K, Bauer R, Begerow D (1998) Doassinga, a new genus of Doassansiales. Mycologia 90: Vánky K, Lutz M, Bauer R (2008a) About the genus Thecaphora (Glomosporiaceae) and its new synonyms. Mycological Progress 7: volume 3 no

76 Lutz, Vánky & Piątek Vánky K, Lutz M, Bauer R (2008b) Floromyces, a new genus of Ustilaginomycotina. Mycotaxon 104: Vánky K, Lutz M, Shivas RG (2006) Anomalomyces panici, new genus and species of Ustilaginomycetes from Australia. Mycologia Balcanica 3: Vánky K, Lutz M (2007) Revision of some Thecaphora species (Ustilaginomycotina) on Caryophyllaceae. Mycological Research 111: Vánky K, Lutz M (2011) Tubisorus, a new genus of smut fungi (Ustilaginomycetes) for Sorosporium pachycarpum. Mycologia Balcanica 8: Vánky K, McKenzie EHC (2002) Smut Fungi of New Zealand. [Fungi of New Zealand. Vol. 2.]: Fungal Diversity Press. Vánky K, Shivas RG (2008) Fungi of Australia: the smut fungi. Melbourne: CSIRO Publishing. Vánky K, Websdane K (1995) Ustilaginales of Schoenus (Cyperaceae). Mycotaxon 56: Verboom GA (2006) A phylogeny of the schoenoid sedges (Cyperaceae: Schoeneae) based on plastid DNA sequences, with special reference to the genera found in Africa. Molecular Phylogenetics and Evolution 38: White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods and applications (MA Innis, DH Gelfand, JJ Sninsky, TJ White, eds): San Diego: Academic Press. Zundel GL (1953) The Ustilaginales of the world. Pennsylvania State College School of Agriculture Department of Botany Contribution 176: xi ima fungus

77 doi: /imafungus IMA Fungus volume 3 no 2: 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, Madrid, Spain; and Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK; d.hawksworth@nhm.ac.uk 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 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, 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

78 Hawksworth 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; ) and 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 ). (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

79 Unavailable name-bearing types (Art ). 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 (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 (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. volume 3 no

80 Hawksworth (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. ACKNOWLEDGEMENTS 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 CGL REFERENCES Corner EJH (1946) Suggestions for botanical progress. New Phytologist 45: 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: Hawksworth DL (2012b) Managing and coping with names of pleomorphic fungi in a period of transition. Mycosphere 3: ; IMA Fungus 3: 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 [Regnum Vegetabile no. 154.] Ruggell: ARG Ganter Verlag. Seifert KA, Rossman AY (2010) How to describe a new fungal species. IMA Fungus 1: Stafleu FA, Cowan ST ( ) 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

81 doi: /imafungus IMA Fungus volume 3 no 2: 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 USA; corresponding author 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 USA 3 Institute of Sciences of Food Production, CNR, Via Amendola 122/O, 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, , Praha 2, Czech Republic 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 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. MATERIALS AND METHODS 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. 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82 Jurjević et al. Table 1. Provenance of fungal isolates characterized in this study. *ITEM number Provenance Aspergillus aculeatus USA: Georgia, iso. ex indoor air sample, 2010, Ž. Jurjević. Aspergillus brunneoviolaceus (syn. A. fijiensis) USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević Bermuda: isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Texas, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Arizona, isol. ex indoor air sample, 2010, Ž. Jurjević Trinidad and Tobago: Tunapuna, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Alabama, isol. ex outside air sample, 2011, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević. Aspergillus floridensis sp. nov T USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević, ex-type. ** (=NRRL 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 Thailand: Phi Phi Islands, isol. ex Xestospongia testudinaria, 2006, T.S. Bay. *****IFM Japan: isol. ex soil from grapery, 2007, K. Yokoyama. Aspergillus violaceofuscus (syn. A. japonicus) USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Kentucky, isol. ex indoor air sample, 2010, Ž. Jurjević USA: New Jersey, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Louisiana, isol. ex indoor air sample, 2010, Ž. Jurjević USA: New York, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Georgia, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Louisiana, isol. ex indoor air sample, 2010, Ž. Jurjević USA: South Carolina, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Tennessee, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Hawaii, isol. ex indoor air sample, 2010, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Delaware, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Maryland, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Alabama, isol. ex indoor air sample, 2011, Ž. Jurjević. 160 ima fungus

83 Novel species of Aspergillus sect. Nigri Table 1. (Continued) *ITEM number Provenance USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Georgia, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Texas, isol. ex indoor air sample, 2011, Ž. Jurjević USA: New Jersey, isol. ex indoor air sample, 2011, Ž. Jurjević USA: North Carolina, isol. ex indoor air sample, 2011, Ž. Jurjević USA: New York, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Texas, isol. ex indoor air sample, 2011, Ž. Jurjević. Aspergillus trinidadensis sp. nov T (=NRRL T ) (=NRRL 62480) Aspergillus uvarum Trinidad and Tobago: Tunapuna, isol. ex indoor air sample, 2011, Ž. Jurjević, ex-type. USA: California, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Florida, isol. ex indoor air sample, 2011, Ž. Jurjević USA: Missouri, isol. ex indoor air sample, 2011, Ž. Jurjević 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. 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 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 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, before LC/MS analysis. Standard Ochratoxin A standard was purchased from Sigma ( 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 ( 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 ( 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. volume 3 no

84 Jurjević et al. Table 2. GenBank accession numbers of reference and ex-type strains. Sequence IDs in red are the sequences deposited for this manuscript. Species Source * RPB2 TEF CaM BenA Aspergillus acidus ITEM 4507 = CBS EF FN AM AY Aspergillus aculeatus ITEM 7046 T = CBS T EF HE AJ AY Aspergillus aculeatus ITEM 4760 = CBS =NRRL 2053 EF HE EF EU Aspergillus aculeatus ITEM = NRRL 359 EF HE EF EF Aspergillus aculeatinus CBS T = IBT T HF HF EU EU Aspergillus aculeatinus ITEM HE HE HE HE Aspergillus awamori ITEM 4509 T = CBS T HE FN AJ AY Aspergillus brasiliensis ITEM 7048 = CBS T EF FN AM AY Aspergillus brunneoviolaceus ITEM 7047 T = CBS T EF HE EF EF Aspergillus carbonarius ITEM 4503 T = CBS T EF FN AJ AY Aspergillus costaricaensis ITEM 7555 T = CBS T HE FN EU AY Aspergillus ellipticus ITEM 4505 T = CBS T EF HE AM FJ Aspergillus fijiensis ITEM 7037 T = CBS T HE HE HE HE Aspergillus helicothrix ITEM 4499 = CBS HE HE AM FJ Aspergillus heteromorphus ITEM 7045 = CBS HE AM FJ Aspergillus homomorphus ITEM 7556 T = CBS T HE HE AM AY Aspergillus ibericus ITEM 4776 T = IMI T EF HE AJ AM Aspergillus indologenus ITEM 7038 = CBS HE HE AM AY Aspergillus japonicus ITEM 7034 T = CBS T EF HE AJ HE Aspergillus japonicus ITEM = NRRL EU HE EU EU Aspergillus lacticoffeatus ITEM 7559 T = CBS T HE FN EU AY Aspergillus niger ITEM 4501 T = CBS T XM_ FN AY AJ Aspergillus pulverulentus ITEM 4510 T = CBS T HE HE HE HE Aspergillus saccharolyticus ITEM T = CBS HF HF HM HM Aspergillus sclerotioniger ITEM 7560 T = CBS T HE HE EU AY Aspergillus tubingensis ITEM 7040 T = CBS T EF FN AJ AY Aspergillus uvarum ITEM 4834 T = IMI T HE HE AM AM Aspergillus vadensis ITEM 7561 T = CBS T HE FN EU AY Aspergillus violaceofuscus ITEM T = CBS T HF HF FJ HE 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 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 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: 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 ( and a Wizard Magnetic DNA Purification System for Food kit (Promega, 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

85 Novel species of Aspergillus sect. Nigri Table 3. GenBank accession numbers of Aspergillus strains isolated from air. Species Source RPB2 TEF CaM BenA Aspergillus aculeatus ITEM HE HE HE HE Aspergillus brunneoviolaceus ITEM HE HE HE HE Aspergillus brunneoviolaceus ITEM HE HE Aspergillus brunneoviolaceus ITEM HE Aspergillus brunneoviolaceus ITEM HE Aspergillus brunneoviolaceus ITEM HE Aspergillus floridensis sp. nov. ITEM T =NRRL T HE HE HE HE Aspergillus violaceofuscus ITEM HE HE HE HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE Aspergillus violaceofuscus ITEM HE Aspergillus trinidadensis sp.nov. ITEM T =NRRL T HE HE HE HE Aspergillus trinidadensis ITEM =NRRL HE HE HE HE Aspergillus uvarum ITEM HE HE HE Aspergillus uvarum ITEM HE HE Aspergillus uvarum ITEM HE * 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, 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, The preliminary alignments of sequences from each of the four loci was performed using the software package BioNumerics 5.1 from Applied Maths ( 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 volume 3 no

86 Jurjević et al. Table 4. Provenance of Aspergillus section Nigri isolates used as reference strains Species Source * Provenance Aspergillus acidus Aspergillus aculeatus ITEM 4507 T = IMI T = CBS T = NRRL 4796 T JAPAN: unknown, R. Nakazawa. ITEM 7046 T = IMI T = CBS T = NRRL 5094 T USA: isol. ex tropical soil, 1962, K. B. Raper. Aspergillus aculeatus ITEM 4760 = CBS = NRRL 2053 = IMI New Guinea: isol. ex canvas tent, 1946, received from D. L. White Aspergillus aculeatus ITEM = NRRL 359 Thom and Raper 1945 received it from Dr. A. F. Blakeslee. Aspergillus brunneoviolaceus ITEM 7047 T = CBS T = IMI T = NRRL 4912 T BRAZIL: culture contaminant, A. C. Batista and H. Maia. Aspergillus aculeatinus ITEM T = CBS T = IBT T THAILAND: Chumporn Prov.: isol. ex arabica coffee, P. Noonim. Aspergillus aculeatinus ITEM SRI LANKA: isol. ex human dacryocystitis. Aspergillus awamori Aspergillus brasiliensis Aspergillus carbonarius ITEM 4509 T = CBS T = NRRL 4948 T Unknown - Raper and Fennel 1965 received it from the Instituto = IMI T Ozwaldo Cruz ITEM 7048 = IMI T = CBS T = NRRL T BRAZIL: Pedreira: isol. ex soil, J. H. Croft. ITEM 4503 T = IMI T = CBS T = NRRL 369 T Unknown: paper, A. F. Blakeslee. Aspergillus costaricaensis ITEM 7555 T = CBS T COSTA RICA: Taboga island: isol. ex soil, 2000, M. Christensen. Aspergillus ellipticus ITEM 4505 T = IMI T = CBS T = NRRL 5120 T COSTA RICA: isol. ex soil, 1962, K. J. Kwon. Aspergillus fijensis ITEM 7037 T = CBS T INDONESIA: Palembang: Lactuca sativa, Aspergillus heteromorphus ITEM 7045 T = CBS T = IMI T = NRRL 4747 T BRAZIL: Recife: culture contaminant, A. C. Batista. Aspergillus homomorphus ITEM 7556 T = CBS T ISRAEL: isol. ex soil 2 km away from Dead Sea. Aspergillus ibericus ITEM 4776 T = CBS T = IMI T = NRRL T PORTUGAL : Iberian Peninsula: isol. ex grapes, 2001, R. Serra. Aspergillus indologenus ITEM 7038 T = CBS T = IBT 3679 T INDIA: isol. ex soil. Aspergillus japonicus ITEM 7034 T = CBS T Unknown, K. Saito. Aspergillus japonicus ITEM = NRRL Unknown. Aspergillus lacticoffeatus ITEM 7559 T = CBS T INDONESIA: South Sumatra: isol. ex coffee bean, J. M. Frank. Aspergillus niger Aspergillus pulverulentus ITEM 4501 T = IMI T = CBS T USA: Connecticut: tannin-gallic acid fermentation, 1913, A. = NRRL 326 T Hollander. ITEM 4510 T = CBS T = NRRL 4851 T AUSTRALIA: Victoria: isol. ex Phaseolus vulgaris, ~1907, D. = IMI T McAlpine. Aspergillus sclerotioniger ITEM 7560 T = CBS T = IBT T INDIA: Karnataka: isol. ex green arabica coffee J. M. Frank. Aspergillus tubingensis ITEM 7040 T = CBS T = NRRL 4875 T Unknown: 1948, deposited by R. Mosseray. Aspergillus uvarum ITEM 4834 T = IMI T = CBS T = IBT T ITALY: Brindisi: isol. ex grapes, 2001, P. Battilani. Aspergillus uvarum ITEM 4685 = IMI PORTUGAL: Régua, Douro Region: isol. ex grapes. Aspergillus uvarum ITEM 4962 = IMI SPAIN: isol. ex grapes. Aspergillus uvarum ITEM 4997 = IMI ISRAEL: Lichron: isol. ex grapes. Aspergillus uvarum ITEM 5020 = IMI ITALY: Brindisi, Apulia: isol. ex grapes. Aspergillus uvarum ITEM 5321 = IMI FRANCE: Narbonne, Languedoc: isol. ex grapes. Aspergillus uvarum ITEM 5350 = IMI ISRAEL: Pdaya: isol. ex grapes. Aspergillus vandensis ITEM 7561 T = IMI T = CBS T EGYPT: air, A. H. Moubasher. Aspergillus violaceofuscus ITEM T = CBS T FRANCE: Strassbourg: received by D Borrel, 1923 Aspergillus saccharolyticus ITEM T = CBS T = IBT 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

87 Novel species of Aspergillus sect. Nigri 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 CaM RPB TEF MLS 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 = ). 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 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 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 (sequence accession number FJ525444, Ingavat et al 2009) and IFM (sequences from Tetsuhiro Matsuzawa, Chiba University, Japan) resulted to have an homology > 99.5% with ITEM They also showed a different phylogenetic position from the uniseriate species decribed as A. indologenus (CBS ), 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 ) 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 ( ), the retention volume 3 no

88 Jurjević et al ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM A. violaceofuscus T ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM 7034 A. japonicus T ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM (NRRL35494) ITEM 7038 A. indologenus T ITEM ITEM ITEM 4834 A. uvarum T ITEM ITEM 7046 A. aculeatus T ITEM ITEM A. floridensis sp nov. CCF 4046 CCF 4236 ITEM 4760 A. aculeatus atypic (CBS ) 97 ITEM A. aculeatinus T (CBS ) ITEM A. aculeatinus 92 ITEM A. trinidadensis sp. nov. 93 ITEM ITEM A. aculeatus atypic (NRRL 359) ITEM 7047 A. brunneoviolaceus T (CBS ) ITEM ITEM ITEM ITEM ITEM ITEM ITEM 7037 A. fijiensis T ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM 7526 A. flavus T ITEM 7556 A. homomorphus T ITEM 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

89 Novel species of Aspergillus sect. Nigri 99/ /1.0 -/ /1.0 ITEM 7526 A. flavus T 100/1.0 84/1.0 97/1.0 99/1.0 82/ /1.0 ITEM A. violaceofuscus T ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM 7034 A. japonicus T ITEM ITEM ITEM ITEM (NRRL35494) ITEM 7038 A. indologenus T ITEM ITEM 4834 A. uvarum T ITEM ITEM ITEM 7046 A. aculeatus T ITEM ITEM A. floridensis sp. nov. 96/1.0 CCF 4046 CCF 4236 ITEM 4760 A. aculeats atypic (CBS 62078) ITEM A. aculeatius T (CBS ) ITEM A. aculeatinus ITEM A. trinidadensis sp. nov. ITEM ITEM A. aculeatus atypic (NRRL 359) ITEM 7047 A. brunneoviolaceus T (CBS ) ITEM /1.0 -/0.91 -/ /0.97ITEM /1.0 76/1.0 -/ / /1.0 97/ /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 ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM ITEM 7037 A. fijiensis T ITEM ITEM ITEM ITEM ITEM ITEM 7556 A. homomorphus T ITEM A. saccharolyticus T ITEM 4505 A. ellipticus T ITEM 7045 A. heteromorphus T 81/ /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 -/ /0.99 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 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 ( ), and the composite index is ( ) 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 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) volume 3 no

90 Jurjević et al. 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 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 mm, OA 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 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 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 ) ( 1600) (8 )10 15( 21) µm, isolate ITEM 7037 has longer stipes (400 ) ( 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) ( 5) µm covering entire vesicle, conidia (Fig. 3e) globose to ellipsoidal, ( 6) ( 5) µm, occasionally subglobose to angular 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 MB (Fig. 4a f). Type: Italy: Apulia, Brindisi, isol. ex grapes (ITEM 4834 T ; = IMI 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 mm, OA (Fig. 4c) 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

91 Novel species of Aspergillus sect. Nigri 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 ) ( 3600) (8 )10 18( 24) µm, vesicles globose to ellipsoidal, (30 )45 100( 121) µm diam, conidial heads uniseriate, phialides 7 10( 12) (3 ) ( 7) µm covering entire vesicle, conidia (Fig. 4f) globose to ellipsoidal, (4 )4.5 7( 9) µm, with echinulate surface. New species Aspergillus floridensis Ž. Jurjević, G. Perrone & S. W. Peterson, sp. nov. MycoBank MB (Fig. 5a g) Etymology: Isolated in Florida. Type: USA: Florida: isol. ex air sample, August 2010, Ž. Jurjević (BPI holotype; from dried colonies of ITEM (=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) µm, with echinulate surface, incubation at 37 C produced growth of mm diam. Description: Colony diameters after 7 d incubation at 25 C on CYA (Fig. 5a) (> 85) mm, MEA (Fig. 5c) (> 85) mm, CY20S mm, OA 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 ) ( 1100) µm long, reverse brownish-yellow to yellow-brown. On MEA conidial heads are brown, sclerotia absent, mycelium volume 3 no

92 Jurjević et al. A B C D D E F E F G Fig. 5. Aspergillus floridensis (ITEM 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 ) ( 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 mm and mm diam, respectively. Stipes smooth, hyaline (50 ) ( 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) ( 5) µm covering entire vesicle, conidia (Fig. 5g) globose to ellipsoidal, 4 5( 6) µm, with echinulate surface. No ochratoxin A produced. Aspergillus trinidadensis Ž. Jurjević, G. Perrone & S. W. Peterson, sp. nov. MycoBank MB (Fig. 6a g) Etymology: Isolated in Trinidad. Type: Trinidad & Tobago:Tunapuna, isol. ex air sample, July 2011, Ž. Jurjević (BPI holotype; from dried colonies of ITEM T (=NRRL 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) µm, if borne from monophialides up to 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) mm, MEA (Fig. 6b) 57 to > 85 mm, CY20S mm, OA 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

93 Novel species of Aspergillus sect. Nigri A B C F F D E G Fig. 6. Aspergillus trinidadensis (ITEM 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 ) ( 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 ) ( 6) µm commonly covering entire vesicle, occasionally producing monophialides (Fig. 6g) µm, conidia (Fig. 6f) globose to ellipsoidal, rarely pyriform, 4 7( 8) µm, if borne from monophialides up to 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, µm long. Also, three isolates (ITEM 14794, ITEM 14799, and ITEM 14802) of A. brunneoviolaceus produced abundant buff to orange-brown sclerotia, 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 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 ), Lactuca sativa, Indonesia (CBS ) (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. volume 3 no

94 Jurjević et al. 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 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 and 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). ACKNOWLEDGEMENTS 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 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. REFERENCES Abarca ML, Accensi F, Bragulat MR, Castella G, Cabanes FJ (2003) Aspergillus carbonarius as the main source of ochratoxin A contamination in dried wine fruits from the Spanish market. Journal of Food Protection 66: Abarca ML, Accensi F, Cano J, Cabanes FJ (2004) Taxonomy and significance of black aspergilli. Antonie van Leeuwenhoek 86: Abarca ML, Bragulat MR, Castella G, Cabanes FJ (1994) Ochratoxin A production by strains of Aspergillus niger var. niger. Applied and Environmental Microbiology 60: Cabanes FJ, Accensi F, Bragulat MR, Abarca ML, Castella G, Minguez S, Pons A (2002) What is the source of ochratoxin A in wine? International Journal of Food Microbiology 79: de Hoog GS, Guarro J, Figueras MJ, Gené J (2000) Atlas of Clinical Fungi. Baarn: Centraalbureau voor Schimmelcultures. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: Gams W, Christensen M, Onions AHS, Pitt JI, Samson RA (1985) Infrageneric taxa of Aspergillus. In: Advances in Penicillium and Aspergillus Systematics (RA Samson & JI Pitt, eds): New York: Plenum Press. Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61: Hendrickx M, Beguin H, Detandt M (2012) Genetic re-identification and antifungal susceptibility testing of Aspergillus section Nigri strains of the BCCM IHEM collection. Mycoses 55: Hubka V, Kolarik M (2012) β-tubulin paralogue tubc is frequently misidentified as the bena gene in Aspergillus section Nigri taxonomy: primer specificity testing and taxonomic consequences Persoonia 29: Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: Ingavat N, Dobereiner J, Wiyakrutta S, Mahidol C, Ruchirawat S, Kittakoop P (2009) Aspergillusol A, an alpha-glucosidase inhibitor from the marine-derived fungus Aspergillus aculeatus. Journal of Natural Products 72: Klich MA (2009) Health effects of Aspergillus in food and air. Toxicology and Industrial Health 25: Knutsen AP (2011) Immunopathology and immunogenetics of allergic bronchopulmonary aspergillosis. Journal of Allergy: doi: /2011/ Liu YL, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. 172 ima fungus

95 Novel species of Aspergillus sect. Nigri Molecular Biology and Evolution 16: Moss RB (2002) Allergic bronchopulmonary aspergillosis. Clinical Reviews in Allergy and Immunology 23: Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. New York: Oxford University Press. Nielsen KF, Mogensen JM, Maria Johansen, Larsen TO, Frisvad JC (2009) Review of secondary metabolites and mycotoxins from the Aspergillus niger group. Analytical and Bioanalytical Chemistry 395: Noonim P, Mahakarnchanakul W, Varga J, Frisvad JC, Samson RA (2008) Two novel species of Aspergillus section Nigri from Thai coffee beans. International Journal of Systematic and Evolutionary Microbiology 58: O Donnell K, Nirenberg HI, Aoki T, Cigelnik E (2000) A multigene phylogeny of the Gibberella fujikuroi species complex: detection of additional phylogenetically distinct species. Mycoscience 41: Perrone G, Varga J, Susca A, Frisvad JC, Stea G, et al. (2008) Aspergillus uvarum sp. nov., an uniseriate black Aspergillus species isolated from grapes in Europe. International Journal of Systematic and Evolutionary Microbiology 58: Perrone G, Stea G, Epifani F, Varga J, Frisvad JC, Samson RA (2011) Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biology 115: Perrone G, Epifani F, Rambukwelle K, Parahitiyawa N, Wijedasa H, Arseculeratne SN (2012a) Aspergillus aculeatinus n. sp. in chronic human dacryocystitis; the first report. Journal of Infectious Disease and Antimicrobial Agents 29: Perrone G, Stea G, Kulathunga CN, Wijedasa H, Arseculeratne SN (2012b) Aspergillus fijiensis isolated from broncial washings in a case of bronchiectasis with invasive aspergillosis; the first report of probable pathogenicity. Journal of Infectious Disease and Antimicrobial Agents (in press). Pitt JI (1980) [ 1979 ] The Genus Penicillium and its teleomorph states Eupenicillium and Talaromyces. London: Academic Press. Pitt JI, Hocking AD (2007) Fungi and Food Spoilage. 2 rd edn. London: Blackie. Pitt JI, Hocking AD (2009) Fungi and Food Spoilage. 3 rd edn. London: Springer. Raper KB, Fennell DI (1965) The Genus Aspergillus. Baltimore, MD: Williams & Wilkins. Richardson MD (2005) Aspergillosis. In: Topley & Wilson s Medical Mycology. (WG Merz, RJ Hay, eds): th edn. London: Hodder Arnold. Ronquist F, Huelsenbeck JP (2003) MrBayes3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: Sage L, Garon D, Seigle-Murandi F (2004) Fungal microflora and ochratoxin A risk in French wineyards. Journal of Agricultural and Food Chemistry 52: Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: Samson RA, Hoekstra ES, Frisvad JC (2004) Introduction to Foodand Airborne Fungi. 7th edn. Utrecht: Centraalbureau voor Schimmelcultures. Samson RA, Noonim P, Meijer M, Houbraken J, Frisvad JC, Varga J (2007) Diagnostic tools to identify black Aspergilli. Studies in Mycology 59: Tamura K, Nei M (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10: Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony methods. Molecular Biology and Evolution 28: Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22: Varga J, Kevei F, Hamari Z, Tóth B, Téren J, Croft JH, Kozakiewicz Z (2000) Genotypic and phenotypic variability among black aspergilli. In: Integration of Modern Taxonomic Methods for Penicillium and Aspergillus Classification (RA Samson & JI Pitt, eds): Amsterdam: Harwood Academic Publishers. Varga J, Frisvad JC, Kocsubé S, Brankovics B, Tóth B, Szigeti G, Samson RA (2011) New and revisited species in Aspergillus section Nigri. Studies in Mycology 69: Wicklow DT, Dowd PF, Alftafta AA, Gloer JB (1996) Ochratoxin A: an antiinsectan metabolite from the sclerotia of Aspergillus carbonarius NRRL 369. Canadian Journal of Microbiology 42: volume 3 no

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97 doi: /imafungus IMA Fungus volume 3 no 2: 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 walter.gams@online.nl 2 Blaihofstraße 42, D 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 Braunschweig, Germany 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 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). 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98 Gams et al. 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 would have to be recombined in Trichoderma, displacing the established name Trichoderma citrinoviride Bissett 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 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 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 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

99 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: Crous PW (2009) Taxonomy and phylogeny of the genus Mycosphaerella and its anamorphs. Fungal Diversity 38: 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: 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: Hawksworth DL (2012) Managing and coping with names of pleomorphic fungi in a period of transition. Mycosphere 1: Doi /mycosphere/3/2/4; IMA Fungus 3: 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: 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 [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: 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: Scholler M, Hagedorn G, Rubner A (1999) A reevaluation of predatory orbiliaceous fungi. II. A new generic concept. Sydowia 51: Stevens PF (1991) George Bentham and the Kew Rule. In: Improving the Stability of Names: needs and options (Hawksworth DL, ed.): [Regnum Vegetabile no. 123.] Königstein: Koeltz Scientific Books. [Also available from the International Plant Names Index website, 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. Acknowledgements 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. volume 3 no

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101 doi: /imafungus IMA Fungus volume 3 no 2: The treasure trove of yeast genera and species described by Johannes van der Walt ( ) Maudy Th. Smith and Marizeth Groenewald* CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands; corresponding author s m.groenewald@cbs. knaw.nl 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 INTRODUCTION Johannes van der Walt passed away after a short illness on 13 November 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author s moral rights. volume 3 no

102 Smith & Groenewald 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

103 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) 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 Myxozyma mucilagina (Phaff et al.) Van der Walt et. al 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 volume 3 no

104 Smith & Groenewald 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 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

105 Yeasts described by J P van der Walt Table 2. (Continued) Year Species name Authors Type strains of South African source Type strains from other source Present status of the type strain 1 Saccharomyces inusitatus Van der Walt Beer Possible hybrid between S. pastorianus and S. bayanus 1 = Saccharomyces bayanus 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 volume 3 no

106 Smith & Groenewald Table 2. (Continued) Year Species name Authors Type strains of South African source Type strains from other source Present status of the type strain 1 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 = Bullera dendrophila of Dichrostachys cinerea 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

107 Yeasts described by J P van der Walt Table 2. (Continued) Year Species name Authors Type strains of South African source Type strains from other source Present status of the type strain 1 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) volume 3 no

108 Smith & Groenewald 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. REFERENCES Barnett JA, Payne RW, Yarrow D (2000) Yeasts: characteristics and identification. 3 rd edn. Cambridge, UK: Cambridge University Press. Boekhout T, Kurtzman CP, O Donell K, Smith MTh (1994) Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences. International Journal of Systematics and Bacteriology 44: Dequin S, Salmon J-M, Nguyen H-V, Blondin B (2003) Wine yeasts. Yeasts in Food: beneficial and detrimental aspects (Boekhout T, Robert V, eds): Hamburg: Behr s Verlag. Dufour J-P, Verstrepen K, Derdelinckx G (2003) Brewing yeasts. In: Yeasts in Food: beneficial and detrimental aspects (Boekhout T, Robert V, eds): Hamburg: Behr s Verlag. Greuter W, Burdet H, Chaloner WG, Demoulin V, Grolle R, Hawksworth DL, Nicolson DH, Silva PC, Stafleu FA, Voss EG, McNeill J (eds) (1988) International Code of Botanical Nomenclature adopted by the Fourteenth International Botanical Congress, Berlin, July August [Regnum Vegetabile vol. 118.]. Königstein: Koeltz Scientific Books. There is always something new in Africa. Groenewald M, Coutinho T, Smith MTh, van der Walt J (2012) Reclassification and mating type behavior of Geotrichum bryndzae, Geotrichum phurueaensis, Geotrichum silvicola and Geotrichum vulgare. International Journal of Systematic and Evolutionary Microbiology: DOI: /ijs Kodama K (1974) Ascosporogenous yeasts isolated from tree exudates in Japan. Annual Microbiology Milano 24: Kreger-van Rij NJW (1984) The Yeasts: a taxonomic study. 3 rd edn. Amsterdam: Elsevier. Kurtzman CP (2003) Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora. FEMS Yeast Research 4: Kurtzman CP, Fell JW (1998) The Yeasts: a taxonomic study. 4 th edn. Amsterdam: Elsevier. Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts: a taxonomic study. 5 th edn. Amsterdam: Elsevier. Kurtzman CP, Albertyn J, Basehoar-Powers E (2007) Multigene phylogenetic analysis of the Lipomycetaceae and proposed transfer of Zygozyma species to Lipomyces and Babjevia anomala to Dipodascopsis. FEMS Yeast Research 7: Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008) Phylogenetic relationships among species of Pichia, Issatchenkia and Williopsis determined from multigene phylogenetic analysis, and the proposal of Barnettozyma gen. nov., Lindnera gen. nov. and Wickerhamomyces gen. nov. FEMS Yeast Research 8: Lachance M-A, Boekhout T, Scorzetti G, Fell JW, Kurtzman CP (2011) Candida Berkhout. In: The Yeasts: a taxonomic study (Kurtzman CP, Fell JW, Boekhout T, eds): th edn. Amsterdam: Elsevier. Lodder J (1970) The Yeasts: a taxonomic study. 2 nd edn. Amsterdam: North-Holland Publishing. Lodder J, Kreger-van Rij NJW (1952) The Yeasts: a taxonomic study. Amsterdam: North-Holland Publishing. Lodder J, Kreger-van Rij NJW (1978) Proposal (446) for the conservation of the generic name Debaryomyces Lodder et Kreger-van Rij against Debaryomyces Klöcker. Taxon 27: Muller HB, Kock JLF (1986) Waltiozyma gen. nov. (Saccharomycetaceae), a new genus of the Endomycetales. South African Journal of Science 82: Novák EK, Zsolt J (1961) A new system proposed for yeasts. Acta Botanica Academiae Scientiarum Hungaricae 7: Pijper A (1928) Een nieuwe Hanseniaspora. Verhandelingen Koninklijke Nederlandse Akademie van Wetenschappen Afdeling Natuurkunde 37: Samson RA, Aa H van der, de Hoog GS (2004) Centraalbureau voor Schimmelcultures: hunderd years microbial resource centre. Studies in Mycology 50: 1 8. Sampaio JP (2011) Sporidiobolus Nyland. In: The Yeasts: a taxonomic study (Kurtzman CP, Fell JW, Boekhout T, eds): th edn. Amsterdam: Elsevier. Spaaij F, Weber G, Smith MTh (1993) Myxozyma vanderwaltii sp. nov. (Candidaceae), a new yeast species isolated from a flower of Protea repens (L.) L. Antonie van Leeuwenhoek 63: Stratford M, James SA (2003) Non-alcoholic beverages and yeasts. In: Yeasts in Food: beneficial and detrimental aspects (Boekhout T, Robert V, eds): Hamburg: Behr s Verlag. 186 ima fungus

109 Yeasts described by J P van der Walt Sugita T, Cañete-Gibas CF, Takashima M, Nakase T (1999) Three new species of Bullera isolated from leaves in the Ogasawara Islands. Mycoscience 40: Suzuki M, Prasad GS, Kurtzman CP (2011) Debaryomyces Lodder & Kreger-van Rij. In: The Yeasts: a taxonomic study (Kurtzman CP, Fell JW, Boekhout T, eds): th edn. Amsterdam Elsevier. van der Walt JP (1952) On the yeast Candida pulcherrima and its pigment. PhD thesis, Technological University Delft. van der Walt JP, Hopsu-Havu VK (1976) A colour reaction for the differentiation of ascomycetous and hemibasidiomycetous yeasts. Antonie van Leeuwenhoek 42: van der Walt JP, Johannsen E (1973) The perfect state of Torulopsis magnoliae. Antonie van Leeuwenhoek 39: Vidal-Leiria M (1966) Torulopsis vanderwaltii sp. nov. Antonie van Leeuwenhoek 32: Wang Q-M, Bai F-Y (2008) Molecular phylogeny of basidiomycetous yeasts in the Cryptococcus luteolus lineage (Tremellales) based on nuclear rrna and mitochondrial cytochrome b gene sequence analyses: proposal of Derxomyces gen. nov. and Hannaella gen. nov., and description of eight novel Derxomyces species. FEMS Yeast Research 8: Yamada Y, Nakase T (1985) Waltomyces, a new ascosporogenous yeast genus for the Q10-equipped, slimeproducing organisms whose asexual reproduction is by multilateral budding and whose ascospores have smooth surfaces. Journal of General and Applied Microbiology. 31: Yarrow D, Meyer SA (1978) Proposal for amendment of the diagnosis of the genus Candida Berkhout nom. cons. International Journal of Systematic and Bacteriology. 28: volume 3 no

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111 doi: /imafungus IMA Fungus volume 3 no 2: 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 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 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 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 2012 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. 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112 Ebead et al. 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. MATERIALS AND METHODS Sample collection Algal material (Polysiphonia sp.) was collected by G.A.E. from the shoreline at Point Prim, Prince Edward Island (46 04 N, W) at low tide on 4 June 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 , W. dispersa CBS , W. multispora CBS , W. nigra CBS , W. ornata CBS , and W. rapa-nuiensis ined. CBS 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

113 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, 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, 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 (MRSA), S. warneri ATCC 17917, vancomycin-resistant Enterococcus faecium EF379 (VRE), Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and Candida albicans ATCC 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 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 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 JX JX 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 volume 3 no

114 Ebead et al. 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 Switzerland, Pinus mugo GQ DQ GQ Pleospora herbarum ATCC USA, onion leaf AF AF AY Preussia australis Lundqvist a France, rabbit dung GQ GQ GQ P. funiculata Huhndorf 2577 USA, porcupine dung GQ GQ GQ P. isomera CBS Venezuela, cow dung GQ GQ GQ P. lignicola CBS Netherlands, rabbit dung GQ DQ GQ P. tenerifae CBS Tenerife, rabbit dung GQ GQ GQ P. terricola CBS Honduras, Musa sapientum GQ GQ GQ P. terricola CBS Tanzania, elephant dung GQ GQ GQ P. typharum CBS Japan, deer dung GQ GQ GQ P. vulgaris Strid Sweden, hare dung GQ GQ GQ Sporormia fimetaria Lundqvist 2302-c Sweden, cow dung GQ GQ GQ Sporomiella affinis Lundqvist j Denmark, rabbit dung GQ S. dakotensis Thulin 2570-g Ethiopia, cow dung GQ S. heptamera Lundqvist 3090-b Sweden, horse dung GQ S. irregularis Lundqvist f Hungary, cow dung GQ GQ GQ S. leporina Lundqvist a Sweden, hare dung GQ S. pulchella Richardson MJR67/01 USA, dung GQ S. vexans UME23 Sweden, moose dung GQ Trematosphaeria heterospora CBS Switzerland, Iris sp. GQ AY GQ Westerdykella angulata IMI India, rice-field soil GQ GQ GQ W. angulata CBS India, rice-field soil GQ W. aurantiaca IMI India, mud AY W. aurantiaca FNBR-03 India, soil JN W. cylindrica ATCC = CBS Kenya, cow dung AY AY JX W. dispersa CBS Virginia, damp seedlings GQ GQ GQ W. dispersa CBS Nigeria, soil DQ W. dispersa CBS Armenia, salt-marsh soil GQ W. dispersa CBS The Netherlands, greenhouse DQ soil W. globosa IFO India, soil AY W. multispora CBS France, saline soil GQ GQ GQ W. multispora CBS Japan AY W. nigra CBS Pakistan, soil GQ GQ GQ W. nigra ATCC AY W. ornata CBS Mozambique, mangrove mud GQ AY GQ W. purpurea CBS Togo, sandy soil AY W. purpurea HN6-5B China, mangrove FJ W. rapa-nuiensis ined. CBS Chile, soil JX JX JX W. reniformis RKGE35 = DAOM Canada, red algae JX JX JX Verruculina enalia CBS Liberia, drift wood GQ AY GQ invariant sites; MCMC heated chain set with nchains = 4 and temp = 0.2, ngen = , samplefreq = 100, sumt burnin = 1250; the analysis was continued for generations in order obtain an average standard deviation of split frequencies below The first 25 % of sampled trees were discarded as burn-in. Resulting trees were viewed in FigTree v 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

115 Antibiotic producing Westerdykella reniformis sp. nov. Westerdykella dispersa CBS Westerdykella dispersa CBS Westerdykella multispora CBS Westerdykella dispersa CBS Westerdykella rapa-nuiensis ined. CBS Westerdykella dispersa CBS Westerdykella aurantiaca FNBR-03 Westerdykella aurantiaca IMI Westerdykella multispora CBS Westerdykella nigra CBS Westerdykella nigra ATCC Westerdykella angulata IMI Westerdykella angulata CBS Westerdykella purpurea CBS Westerdykella purpurea HN6-5B Westerdykella globosa IFO Westerdykella cylindrica CBS Westerdykella reniformis RKGE35=DAOM Westerdykella ornata CBS Sporormia fimetaria Lundqvist2302-c Sporormiella affinis Lundqvist17739-j 92 Sporormiella heptamera Lundqvist3090b 99 Sporormiella vexans UME23 Sporormiella leporina Lundqvist19873-a Sporormiella irregularis Lundqvist f Preussia isomera CBS Preussia tenerifae CBS Sporormiella dakotensis Thulin 2570-g Sporormiella pulchella Richardson MJR93/01 Preussia australis Lundqvist a Preussia lignicola CBS Preussia vulgaris Strid Preussia funiculata Huhndorf Preussia typharum CBS Trematosphaeria heterospora CBS Herpotrichia juniperi CBS Pleospora herbarum ATCC Verruculina enalia CBS 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 ). 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 (AY ; matching 455/477 bases with 7 gaps), W. dispersa CBS (AY ; matching 454/477 bases with 6 gaps), and W. aurantiaca IMI (AY ; matching 452/477 bases with 6 gaps). For the amplified nlsu region, the closest sequence matches with complete coverage were to W. angulata IMI (GQ ; matching 1281/1296 bases with 1 gap) with 99 % maximum identity followed by W. cylindrica ATCC (NG ; 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 (GQ ; matching 876/941 bases with 6 gaps) with 93 % maximum identity and W. ornata CBS (GQ ; matching 869/945 bases with 10 gaps) with 92 % maximum identity as well as with W. angulata IMI (GQ ; matching 368/926 bases with 6 gaps) with 94 % maximum identity and only 98 % coverage. Phylogenetic analyses 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 volume 3 no

116 Ebead et al. Herpotrichia juniperi CBS Westerdykella angulata IMI Westerdykella dispersa CBS Westerdykella rapa-nuiensis ined. CBS Westerdykella multispora CBS Westerdykella ornata CBS Westerdykella reniformis RKGE35=DAOM Westerdykella cylindrica CBS Westerdykella nigra CBS Sporormia fimetaria Lundqvist 2302-c 70 Preussia funiculata Huhndorf Preussia typharum CBS Preussia vulgaris Strid Preussia australis Lundqvist Preussia lignicola CBS Preussia isomera CBS Preussia terricola CBS Preussia terricola CBS Preussia tenerifae CBS Sporormiella irregularis Lundqvist f Trematosphaeria heterospora CBS Pleospora herbarum CBS Verruculina enalia CBS 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 ). and included 409 nucleotide positions in the final dataset with an overall mean distance calculated as with a standard error of 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 (= ATCC 24077). Isolate CBS , representing the unpublished species W. rapanuiensis ined., clustered together within the W. dispersa clade along with the W. multispora isolate CBS 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 was achieved following 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 representing the not yet formally named W. rapa-nuiensis clustered together and most proximal to W. dispera (CBS ). 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 [M+H] +, Δ -2.2 ppm) and C 30 H 28 N 6 O 8 S 5 (m/z [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

117 Antibiotic producing Westerdykella reniformis sp. nov millivolts NL: 3.35E2 ELSD Relative Abundance Relative Abundance * * 3.66 Relative Abundance Relative Abundance [M+H] melinacidin IV [M+Na] m/z [M+H] chetracin B [M+Na] nm m/z nm Time (min) Relative Absorbance Relative Absorbance NL: 4.54E5 m/z= NL: 1.02E5 m/z= 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 ( 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 volume 3 no

118 Ebead et al. O O HO HO O O dykellic acid HO HO O O gelastatin B O N S 2 N O H N O N S 2 N O H N HO O gelastatin A O O HO OH HO OH N H O N S 2 N O N H 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 W. ornata CBS W. nigra CBS W. multispora CBS W. cylindrica CBS W. dispersa CBS W. rapa-nueinsis ined. CBS 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 chetracin B vancomycin rifampicin ciprofloxacin ima fungus

119 Antibiotic producing Westerdykella reniformis sp. nov. 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. MycoBank MB (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 ( µm); each bearing 32, black, glabrous, reniform ascospores with a distinct central grove ( µm); pycnidal stage unknown. Type: Canada: Prince Edward Island: Point Prim (46 04 N, W), from Polysiphonia sp. collected from littoral tidal zone at low tide, 4 June 2009, G.A. Ebead, (DAOM 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 (av. 14.7) (av. 12.8) µm, containing 32 ascospores. Ascospores (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. 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 (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 (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 volume 3 no

120 Ebead et al. 6 (4) Asci clavate to cylindrical-clavate... 7 Asci globose to ellipsoidal (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 µm diam; ascospores ellipsoid; asci globose to subglobose... purpurea Colonies ochraceous to salmon; cleistothecia 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 , 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 presented a pycnidial asexual morph comparable to W. dispersa. Based on micromorphological observations and the ITS and multigene phylogenetic comparisons, CBS should be considered as a strain of W. dispersa. CBS , 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

121 Antibiotic producing Westerdykella reniformis sp. nov. Fig. 5. Macro and micro-morphology of Westerdykella reniformis (RKGE 35 = DAOM ). 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 volume 3 no