From the Cover: Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi (original) (raw)

• Journal List
• Proc Natl Acad Sci U S A
• v.109(16); 2012 Apr 17
• PMC3341068

Proc Natl Acad Sci U S A. 2012 Apr 17; 109(16): 6241–6246.

From the Cover

Conrad L. Schoch,a,1 Keith A. Seifert,b,1 Sabine Huhndorf,c Vincent Robert,d John L. Spouge,a C. André Levesque,b Wen Chen,b and Fungal Barcoding Consortiuma,2

aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20892;

bBiodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada, K1A 0C6;

cDepartment of Botany, The Field Museum, Chicago, IL, 60605; and

dCentraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS-KNAW), 3508 AD, Utrecht, The Netherlands

Conrad L. Schoch

aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20892;

Keith A. Seifert

bBiodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada, K1A 0C6;

Sabine Huhndorf

cDepartment of Botany, The Field Museum, Chicago, IL, 60605; and

Vincent Robert

dCentraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS-KNAW), 3508 AD, Utrecht, The Netherlands

John L. Spouge

aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20892;

C. André Levesque

bBiodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada, K1A 0C6;

Wen Chen

bBiodiversity (Mycology and Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada, K1A 0C6;

Fungal Barcoding Consortium

aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, 20892;

Fungal Barcoding Consortium Author List, Elena Bolchacova,5 Kerstin Voigt,6 Pedro W. Crous,4 Andrew N. Miller,7 Michael J. Wingfield,8 M. Catherine Aime,9 Kwang-Deuk An,10 Feng-Yan Bai,11 Robert W. Barreto,12 Dominik Begerow,13 Marie-Josée Bergeron,14 Meredith Blackwell,15 Teun Boekhout,4,16 Mesfin Bogale,17 Nattawut Boonyuen,18 Ana R. Burgaz,19 Bart Buyck,20 Lei Cai,11 Qing Cai,21 G. Cardinali,22 Priscila Chaverri,23 Brian J. Coppins,24 Ana Crespo,25 Paloma Cubas,25 Craig Cummings,5 Ulrike Damm,4 Z. Wilhelm de Beer,8 G. Sybren de Hoog,4 Ruth Del-Prado,25 Bryn Dentinger,26,57 Javier Diéguez-Uribeondo,27 Pradeep K. Divakar,25 Brian Douglas,28 Margarita Dueñas,27 Tuan A. Duong,29 Ursula Eberhardt,4 Joan E. Edwards,28 Mostafa S. Elshahed,30 Katerina Fliegerova,31 Manohar Furtado,5 Miguel A. García,27 Zai-Wei Ge,22 Gareth W. Griffith,28 K. Griffiths,32 Johannes Z. Groenewald,4 Marizeth Groenewald,4 Martin Grube,33 Marieka Gryzenhout,34 Liang-Dong Guo,11 Ferry Hagen,4 Sarah Hambleton,2 Richard C. Hamelin,14 Karen Hansen,35 Paul Harrold,24 Gregory Heller,9 Cesar Herrera,23 Kazuyuki Hirayama,36 Yuuri Hirooka,23 Hsiao-Man Ho,37 Kerstin Hoffmann,6 Valérie Hofstetter,38 Filip Högnabba,39 Peter M. Hollingsworth,24 Seung-Beom Hong,40 Kentaro Hosaka,41 Jos Houbraken,4 Karen Hughes,42 Seppo Huhtinen,43 Kevin D. Hyde,44,45 Timothy James,46 Eric M. Johnson,71 Joan E. Johnson,28 Peter R. Johnston,47 E.B. Gareth Jones,18 Laura J. Kelly,24,26 Paul M. Kirk,48 Dániel G. Knapp,49 Urmas Kõljalg,50 Gábor M. Kovács,49 Cletus P. Kurtzman,51 Sara Landvik,52 Steven D. Leavitt,3 Audra S. Liggenstoffer,30 Kare Liimatainen,53 Lorenzo Lombard,4 J. Jennifer Luangsa-ard,18 H. Thorsten Lumbsch,3 Harinad Maganti,54 Sajeewa S. N. Maharachchikumbura,44 María P. Martin,27 Tom W. May,32 Alistair R. McTaggart,9 Andrew S. Methven,55 Wieland Meyer,56 Jean-Marc Moncalvo,57 Suchada Mongkolsamrit,18 László G. Nagy,58 R. Henrik Nilsson,59 Tuula Niskanen,53 Ildikó Nyilasi,58 Gen Okada,10 Izumi Okane,60 Ibai Olariaga,35 Jürgen Otte,61 Tamás Papp,58 Duckchul Park,47 Tamás Petkovits,58 Raquel Pino-Bodas,27 William Quaedvlieg,4 Huzefa A. Raja,62 Dirk Redecker,63 Tara L. Rintoul,2 Constantino Ruibal,25 Jullie M. Sarmiento-Ramírez,27 Imke Schmitt,61,64 Arthur Schüßler,65 Carol Shearer,66 Kozue Sotome,67 Franck O.P. Stefani,32 Soili Stenroos,39 Benjamin Stielow,4 Herbert Stockinger,63 Satinee Suetrong,18 Sung-Oui Suh,68 Gi-Ho Sung,69 Motofumi Suzuki,10 Kazuaki Tanaka,36 Leho Tedersoo,70 M. Teresa Telleria,26 Eric Tretter,71 Wendy A. Untereiner,17 Hector Urbina,15 Csaba Vágvölgyi,58 Agathe Vialle,14 Thuy Duong Vu,4 Grit Walther,4 Qi-Ming Wang,11 Yan Wang,71 Bevan S. Weir,47 Michael Weiß,72 Merlin M. White,71 Jianping Xu,54 Rebecca Yahr,24 Zhu L. Yang,21 Andrey Yurkov,13 Juan-Carlos Zamora,27 Ning Zhang,73 Wen-Ying Zhuang,11 and David Schindel74

3Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

5Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 9440

6Jena Microbial Resource Collection, Institute of Microbiology, University of Jena & Leibniz Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11 a, 07745 Jena, Germany

7University of Illinois, Illinois Natural History Survey, 1816 South Oak Street, Champaign, IL 61820

8Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0001, South Africa

9Louisiana State University Agricultural Center, Department of Plant Pathology and Crop Physiology, Baton Rouge, LA 70803

10Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

12Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil

13Ruhr-Universität Bochum, Geobotanik ND03/174, Universitätstr. 150, 44801 Bochum, Germany

14Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 du PEPS, PO Box 10380, Stn. Sainte-Foy, Québec G1V 4C, Canada, 7

15Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803

16Department of Internal Medicine and Infectious Diseases, University Medical Center, Utrecht, The Netherlands

17Department of Biology, Brandon University, Brandon, Manitoba R7A 6A9, Canada

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

19Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense de Madrid, Madrid 28040, Spain

20Muséum National d’Histoire Naturelle, Dépt. Systématique et Evolution CP39, UMR7205, 12 Rue Buffon, F-75005 Paris, France

21Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China

22Dipartimento Biologia Applicata- Microbiologia, Università degli Studi di Perugia, Perugia, Italy

23University of Maryland, Department of Plant Sciences and Landscape Architecture, 2112 Plant Sciences Building, College Park, MD 20742

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

26Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

28Institute of Biological, Environmental and Rural Sciences, Prifysgol Aberystwyth, Aberystwyth, Ceredigion Wales SY23 3DD, UK

29Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0001, South Africa

30Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74074

31Institute of Animal Physiology and Genetics, Czech Academy of Sciences, v.v.i., Prague, Czech Republic

32Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VIC 3141, Australia

33Institut für Pflanzenwissenschaften, Karl-Franzens-Universität Graz, Holteigasse 6, 8010 Graz, Austria

34Department of Plant Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa

35Department of Cryptogamic Botany, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05, Stockholm, Sweden

36Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

37Department of Science Education, National Taipei University of Education, Taipei 10642, Taiwan

38Agroscope Changins-Wädenswil Research Station ACW, Département de recherche en Protection des végétaux grandes cultures et vigne/Viticulture et oenologie, CP 1012, CH-1260 Nyon, Switzerland

39Botanical Museum, Finnish Museum of Natural History, FI-00014 University of Helsinki, Finland

40Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA, Suwon, 441-707, Korea.

41National Museum of Nature and Science, Tsukuba, Japan

42Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37920

43Herbarium, University of Turku, FI-20014 University of Turku, Finland

44Mae Fah Luang University, 57100 Chiang Rai, Thailand

45Botany and Microbiology Department, College of Science, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia

46Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109

47Landcare Research, Private Bag 92170, Auckland 1142, New Zealand

48CABI-UK, Bakeham Lane, Egham, Surrey TW20 9TY, UK

49Eötvös Loránd University, Institute of Biology, Department of Plant Anatomy, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary

50Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai St, Tartu 51005, Estonia

51National Center for Agricultural Utilization Research, ARS, USDA, 1815 N. University St, Peoria, IL

52Fungal Screening, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark

53University of Helsinki, Plant Biology, Department of Biosciences, P.O. Box 65, 00014, Finland

54Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

55Deppartment of Biology, Eastern Illinois University, Charleston, IL 61920

56Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney Western Clinical School at Westmead Hospital, Westmead, Australia

57Department of Natural History, Royal Ontario Museum, and Department of Ecology and Evolutionary Biology, University of Toronto, 100 Queen's Park, Toronto, Ontario, M5S 2C6, Canada

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

59Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, Gothenburg, Sweden

60NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Kisarazu, Chiba 292-0818, Japan

61Biodiversity and Climate Research Center (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt, (Main), Germany

62The University of North Carolina at Greensboro, Department of Chemistry and Biochemistry, 457 Sullivan Science Building, P.O. Box 26170, Greensboro, NC 27402-6170

63Université de Bourgogne/INRA, UMR1347 Agroécologie, BP 86510, F-21000 Dijon, France

64Goethe Universität Frankfurt, Department of Biological Sciences, Siesmayerstr. 70, 60323 Frankfurt, (Main), Germany

65Genetics, Department Biology, Biocenter of Ludwig-Maximilians-University Munich, Grosshaderner Strasse 2-4, 82152 Martinsried, Germany

66University of Illinois, Department of Plant Biology, 505 South Goodwin Avenue, Urbana, IL 61801

67Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University 4-101 Koyama-Minami, Tottori 680-8553, Japan

68Mycology and Botany Program, American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110

69Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Department Administration, Suwon, 404-707, Korea

70Natural History Museum of Tartu University, 46 Vanemuise Street, 51014 Tartu, Estonia

71Boise State University, Department of Biological Sciences, 1910 University Dr., Boise, ID 83725

72Institute of Evolution and Ecology, University of Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

73Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901

74CBOL, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, MRC-105, Washington, DC 20013-7012

Elena Bolchacova

5Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 9440

Kerstin Voigt

6Jena Microbial Resource Collection, Institute of Microbiology, University of Jena & Leibniz Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11 a, 07745 Jena, Germany

Pedro W. Crous

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Andrew N. Miller

7University of Illinois, Illinois Natural History Survey, 1816 South Oak Street, Champaign, IL 61820

Michael J. Wingfield

8Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0001, South Africa

M. Catherine Aime

9Louisiana State University Agricultural Center, Department of Plant Pathology and Crop Physiology, Baton Rouge, LA 70803

Kwang-Deuk An

10Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan

Feng-Yan Bai

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

Robert W. Barreto

12Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-000 Viçosa, Minas Gerais, Brazil

Dominik Begerow

13Ruhr-Universität Bochum, Geobotanik ND03/174, Universitätstr. 150, 44801 Bochum, Germany

Marie-Josée Bergeron

14Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 du PEPS, PO Box 10380, Stn. Sainte-Foy, Québec G1V 4C, Canada, 7

Meredith Blackwell

15Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803

Teun Boekhout

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

16Department of Internal Medicine and Infectious Diseases, University Medical Center, Utrecht, The Netherlands

Mesfin Bogale

17Department of Biology, Brandon University, Brandon, Manitoba R7A 6A9, Canada

Nattawut Boonyuen

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

Ana R. Burgaz

19Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense de Madrid, Madrid 28040, Spain

Bart Buyck

20Muséum National d’Histoire Naturelle, Dépt. Systématique et Evolution CP39, UMR7205, 12 Rue Buffon, F-75005 Paris, France

Lei Cai

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

Qing Cai

21Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China

G. Cardinali

22Dipartimento Biologia Applicata- Microbiologia, Università degli Studi di Perugia, Perugia, Italy

Priscila Chaverri

23University of Maryland, Department of Plant Sciences and Landscape Architecture, 2112 Plant Sciences Building, College Park, MD 20742

Brian J. Coppins

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

Ana Crespo

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

Paloma Cubas

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

Craig Cummings

5Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 9440

Ulrike Damm

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Z. Wilhelm de Beer

8Department of Microbiology and Plant Pathology, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0001, South Africa

G. Sybren de Hoog

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Ruth Del-Prado

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

Bryn Dentinger

26Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

57Department of Natural History, Royal Ontario Museum, and Department of Ecology and Evolutionary Biology, University of Toronto, 100 Queen's Park, Toronto, Ontario, M5S 2C6, Canada

Javier Diéguez-Uribeondo

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Pradeep K. Divakar

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

Brian Douglas

28Institute of Biological, Environmental and Rural Sciences, Prifysgol Aberystwyth, Aberystwyth, Ceredigion Wales SY23 3DD, UK

Margarita Dueñas

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Tuan A. Duong

29Department of Genetics, Forestry & Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 0001, South Africa

Ursula Eberhardt

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Joan E. Edwards

28Institute of Biological, Environmental and Rural Sciences, Prifysgol Aberystwyth, Aberystwyth, Ceredigion Wales SY23 3DD, UK

Mostafa S. Elshahed

30Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74074

Katerina Fliegerova

31Institute of Animal Physiology and Genetics, Czech Academy of Sciences, v.v.i., Prague, Czech Republic

Manohar Furtado

5Life Technologies, 850 Lincoln Centre Drive, Foster City, CA 9440

Miguel A. García

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Zai-Wei Ge

22Dipartimento Biologia Applicata- Microbiologia, Università degli Studi di Perugia, Perugia, Italy

Gareth W. Griffith

28Institute of Biological, Environmental and Rural Sciences, Prifysgol Aberystwyth, Aberystwyth, Ceredigion Wales SY23 3DD, UK

K. Griffiths

32Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VIC 3141, Australia

Johannes Z. Groenewald

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Marizeth Groenewald

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Martin Grube

33Institut für Pflanzenwissenschaften, Karl-Franzens-Universität Graz, Holteigasse 6, 8010 Graz, Austria

Marieka Gryzenhout

34Department of Plant Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa

Liang-Dong Guo

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

Ferry Hagen

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Richard C. Hamelin

14Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 du PEPS, PO Box 10380, Stn. Sainte-Foy, Québec G1V 4C, Canada, 7

Karen Hansen

35Department of Cryptogamic Botany, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05, Stockholm, Sweden

Paul Harrold

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

Gregory Heller

9Louisiana State University Agricultural Center, Department of Plant Pathology and Crop Physiology, Baton Rouge, LA 70803

Cesar Herrera

23University of Maryland, Department of Plant Sciences and Landscape Architecture, 2112 Plant Sciences Building, College Park, MD 20742

Kazuyuki Hirayama

36Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

Yuuri Hirooka

23University of Maryland, Department of Plant Sciences and Landscape Architecture, 2112 Plant Sciences Building, College Park, MD 20742

Hsiao-Man Ho

37Department of Science Education, National Taipei University of Education, Taipei 10642, Taiwan

Kerstin Hoffmann

6Jena Microbial Resource Collection, Institute of Microbiology, University of Jena & Leibniz Institute for Natural Product Research and Infection Biology, Beutenbergstrasse 11 a, 07745 Jena, Germany

Valérie Hofstetter

38Agroscope Changins-Wädenswil Research Station ACW, Département de recherche en Protection des végétaux grandes cultures et vigne/Viticulture et oenologie, CP 1012, CH-1260 Nyon, Switzerland

Filip Högnabba

39Botanical Museum, Finnish Museum of Natural History, FI-00014 University of Helsinki, Finland

Peter M. Hollingsworth

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

Seung-Beom Hong

40Korean Agricultural Culture Collection, National Academy of Agricultural Science, RDA, Suwon, 441-707, Korea.

Kentaro Hosaka

41National Museum of Nature and Science, Tsukuba, Japan

Jos Houbraken

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Karen Hughes

42Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN 37920

Seppo Huhtinen

43Herbarium, University of Turku, FI-20014 University of Turku, Finland

Kevin D. Hyde

44Mae Fah Luang University, 57100 Chiang Rai, Thailand

45Botany and Microbiology Department, College of Science, King Saud University, P.O. Box: 2455, Riyadh 1145, Saudi Arabia

Timothy James

46Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109

Eric M. Johnson

71Boise State University, Department of Biological Sciences, 1910 University Dr., Boise, ID 83725

Joan E. Johnson

28Institute of Biological, Environmental and Rural Sciences, Prifysgol Aberystwyth, Aberystwyth, Ceredigion Wales SY23 3DD, UK

Peter R. Johnston

47Landcare Research, Private Bag 92170, Auckland 1142, New Zealand

E.B. Gareth Jones

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

Laura J. Kelly

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

26Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

Paul M. Kirk

48CABI-UK, Bakeham Lane, Egham, Surrey TW20 9TY, UK

Dániel G. Knapp

49Eötvös Loránd University, Institute of Biology, Department of Plant Anatomy, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary

Urmas Kõljalg

50Institute of Ecology and Earth Sciences, University of Tartu, 40 Lai St, Tartu 51005, Estonia

Gábor M. Kovács

49Eötvös Loránd University, Institute of Biology, Department of Plant Anatomy, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary

Cletus P. Kurtzman

51National Center for Agricultural Utilization Research, ARS, USDA, 1815 N. University St, Peoria, IL

Sara Landvik

52Fungal Screening, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark

Steven D. Leavitt

3Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605

Audra S. Liggenstoffer

30Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74074

Kare Liimatainen

53University of Helsinki, Plant Biology, Department of Biosciences, P.O. Box 65, 00014, Finland

Lorenzo Lombard

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

J. Jennifer Luangsa-ard

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

H. Thorsten Lumbsch

3Department of Botany, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605

Harinad Maganti

54Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

Sajeewa S. N. Maharachchikumbura

44Mae Fah Luang University, 57100 Chiang Rai, Thailand

María P. Martin

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Tom W. May

32Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VIC 3141, Australia

Alistair R. McTaggart

9Louisiana State University Agricultural Center, Department of Plant Pathology and Crop Physiology, Baton Rouge, LA 70803

Andrew S. Methven

55Deppartment of Biology, Eastern Illinois University, Charleston, IL 61920

Wieland Meyer

56Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, University of Sydney Western Clinical School at Westmead Hospital, Westmead, Australia

Jean-Marc Moncalvo

57Department of Natural History, Royal Ontario Museum, and Department of Ecology and Evolutionary Biology, University of Toronto, 100 Queen's Park, Toronto, Ontario, M5S 2C6, Canada

Suchada Mongkolsamrit

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

László G. Nagy

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

R. Henrik Nilsson

59Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, Gothenburg, Sweden

Tuula Niskanen

53University of Helsinki, Plant Biology, Department of Biosciences, P.O. Box 65, 00014, Finland

Ildikó Nyilasi

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

Gen Okada

10Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan

Izumi Okane

60NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Kisarazu, Chiba 292-0818, Japan

Ibai Olariaga

35Department of Cryptogamic Botany, Swedish Museum of Natural History, P.O. Box 50007, SE-104 05, Stockholm, Sweden

Jürgen Otte

61Biodiversity and Climate Research Center (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt, (Main), Germany

Tamás Papp

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

Duckchul Park

47Landcare Research, Private Bag 92170, Auckland 1142, New Zealand

Tamás Petkovits

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

Raquel Pino-Bodas

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

William Quaedvlieg

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Huzefa A. Raja

62The University of North Carolina at Greensboro, Department of Chemistry and Biochemistry, 457 Sullivan Science Building, P.O. Box 26170, Greensboro, NC 27402-6170

Dirk Redecker

63Université de Bourgogne/INRA, UMR1347 Agroécologie, BP 86510, F-21000 Dijon, France

Constantino Ruibal

25Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain

Jullie M. Sarmiento-Ramírez

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Imke Schmitt

61Biodiversity and Climate Research Center (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt, (Main), Germany

64Goethe Universität Frankfurt, Department of Biological Sciences, Siesmayerstr. 70, 60323 Frankfurt, (Main), Germany

Arthur Schüßler

65Genetics, Department Biology, Biocenter of Ludwig-Maximilians-University Munich, Grosshaderner Strasse 2-4, 82152 Martinsried, Germany

Carol Shearer

66University of Illinois, Department of Plant Biology, 505 South Goodwin Avenue, Urbana, IL 61801

Kozue Sotome

67Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University 4-101 Koyama-Minami, Tottori 680-8553, Japan

Franck O.P. Stefani

32Royal Botanic Gardens Melbourne, Birdwood Avenue, South Yarra, VIC 3141, Australia

Soili Stenroos

39Botanical Museum, Finnish Museum of Natural History, FI-00014 University of Helsinki, Finland

Benjamin Stielow

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Herbert Stockinger

63Université de Bourgogne/INRA, UMR1347 Agroécologie, BP 86510, F-21000 Dijon, France

Satinee Suetrong

18National Center for Genetic Engineering and Biotechnology (BIOTEC),113 Paholyothin Road, Pathum Thani 12120, Thailand

Sung-Oui Suh

68Mycology and Botany Program, American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110

Gi-Ho Sung

69Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Department Administration, Suwon, 404-707, Korea

Motofumi Suzuki

10Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351-0198, Japan

Kazuaki Tanaka

36Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan

Leho Tedersoo

70Natural History Museum of Tartu University, 46 Vanemuise Street, 51014 Tartu, Estonia

M. Teresa Telleria

26Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK

Eric Tretter

71Boise State University, Department of Biological Sciences, 1910 University Dr., Boise, ID 83725

Wendy A. Untereiner

17Department of Biology, Brandon University, Brandon, Manitoba R7A 6A9, Canada

Hector Urbina

15Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803

Csaba Vágvölgyi

58University of Szeged, Faculty of Science and Informatics, Department of Microbiology, Közép fasor 52, Szeged, H-6726, Hungary

Agathe Vialle

14Laurentian Forestry Centre, Canadian Forest Service, Natural Resources Canada, 1055 du PEPS, PO Box 10380, Stn. Sainte-Foy, Québec G1V 4C, Canada, 7

Thuy Duong Vu

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Grit Walther

4Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, The Netherlands

Qi-Ming Wang

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

Yan Wang

71Boise State University, Department of Biological Sciences, 1910 University Dr., Boise, ID 83725

Bevan S. Weir

47Landcare Research, Private Bag 92170, Auckland 1142, New Zealand

Michael Weiß

72Institute of Evolution and Ecology, University of Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany

Merlin M. White

71Boise State University, Department of Biological Sciences, 1910 University Dr., Boise, ID 83725

Jianping Xu

54Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada

Rebecca Yahr

24Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, UK

Zhu L. Yang

21Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China

Andrey Yurkov

13Ruhr-Universität Bochum, Geobotanik ND03/174, Universitätstr. 150, 44801 Bochum, Germany

Juan-Carlos Zamora

27Real Jardín Botánico, RJB-CSIC, Plaza de Murillo 2, 28014 Madrid, Spain

Ning Zhang

73Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901

Wen-Ying Zhuang

11State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Bei-Chen-Xi Road, Chaoyang District, Beijing, 100101, China

David Schindel

74CBOL, National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, MRC-105, Washington, DC 20013-7012

Edited* by Daniel H. Janzen, University of Pennsylvania, Philadelphia, PA, and approved February 24, 2012 (received for review October 18, 2011)

Author contributions: C.L.S. and K.A.S. designed research; K.A.S., V.R., E.B., K.V., P.W.C., A.N.M., M.J.W., M.C.A., K.-D.A., F.-Y.B., R.W.B., D.B., M.-J.B., M. Blackwell, T.B., M. Bogale, N.B., A.R.B., B.B., L.C., Q.C., G.C., P. Chaverri, B.J.C., A.C., P. Cubas, C.C., U.D., Z.W.d.B., G.S.d.H., R.D.-P., B. Dentinger, J.D-U., P.K.D., B. Douglas, M.D., T.A.D., U.E., J.E.E., M.S.E., K.F., M.F., M.A.G., Z.-W.G., G.W.G., K.G., J.Z.G., M. Groenewald, M. Grube, M. Gryzenhout, L.-D.G., F. Hagen, S. Hambleton, R.C.H., K. Hansen, P.H., G.H., C.H., K. Hirayama, Y.H., H.-M.H., K. Hoffmann, V. Hofstetter, F. Högnabba, P.M.H., S.-B.H., K. Hosaka, J.H., K. Hughes, Huhtinen, K.D.H., T.J., E.M.J., J.E.J., P.R.J., E.B.G.J., L.J.K., P.M.K., D.G.K., U.K., G.M.K., C.P.K., S.L., S.D.L., A.S.L., K.L., L.L., J.J.L., H.T.L., H.M., S.S.N.M., M.P.M., T.W.M., A.R.M., A.S.M., W.M., J.-M.M., S.M., L.G.N., R.H.N., T.N., I.N., G.O., I. Okane, I. Olariaga, J.O., T. Papp, D.P., T. Petkovits, R.P.-B., W.Q., H.A.R., D.R., T.L.R., C.R., J.M.S.-R., I.S., A.S., C.S., K.S., F.O.P.S., S. Stenroos, B.S., H.S., S. Suetrong, S.-O.S., G.-H.S., M.S., K.T., L.T., M.T.T., E.T., W.A.U., H.U., C.V., A.V., T.D.V., G.W., Q.M.W., Y.W., B.S.W., M.W., M.M.W., J.X., R.Y., Z.-L.Y., A.Y., J.-C.Z., N.Z., W.-Y.Z., and D.S performed research; V.R., J.L.S., C.A.L., and W.C. analyzed data; C.L.S., K.A.S., and S.H. wrote the paper.

Freely available online through the PNAS open access option.

Supplementary Materials

Supporting Information

GUID: 03826FFD-C27A-4D99-96D0-BCB2E2A5B52B

GUID: 930BB42A-EB22-447A-B1DF-9740B33AAC24

GUID: 0025544C-5E7F-4076-B758-7A9CE19EF51E

GUID: 5FCAB5AC-A818-43CE-A2D8-4A287F7D4019

Abstract

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Keywords: DNA barcoding, fungal biodiversity

The absence of a universally accepted DNA barcode for Fungi, the second most speciose eukaryotic kingdom (1, 2), is a serious limitation for multitaxon ecological and biodiversity studies. DNA barcoding uses standardized 500- to 800-bp sequences to identify species of all eukaryotic kingdoms using primers that are applicable for the broadest possible taxonomic group. Reference barcodes must be derived from expertly identified vouchers deposited in biological collections with online metadata and validated by available online sequence chromatograms. Interspecific variation should exceed intraspecific variation (the barcode gap), and barcoding is optimal when a sequence is constant and unique to one species (3, 4). Ideally, the barcode locus would be the same for all kingdoms. A region of the mitochondrial gene encoding the cytochrome c oxidase subunit 1 (CO1) is the barcode for animals (3, 4) and the default marker adopted by the Consortium for the Barcode of Life for all groups of organisms, including fungi (5). In Oomycota, part of the kingdom Stramenopila historically studied by mycologists, the de facto barcode internal transcribed spacer (ITS) region is suitable for identification, but the default CO1 marker is more reliable in a few clades of closely related species (6). In plants, CO1 has limited value for differentiating species, and a two-marker system of chloroplast genes was adopted (7, 8) based on portions of the ribulose 1-5-biphosphate carboxylase/oxygenase large subunit gene and a maturase-encoding gene from the intron of the _trn_K gene. This system sets a precedent for reconsidering CO1 as the default fungal barcode.

CO1 functions reasonably well as a barcode in some fungal genera, such as Penicillium, with reliable primers and adequate species resolution (67% in this young lineage) (9); however, results in the few other groups examined experimentally are inconsistent, and cloning is often required (10). The degenerate primers applicable to many Ascomycota (11) are difficult to assess, because amplification failures may not reflect priming mismatches. Extreme length variation occurs because of multiple introns (9, 12–14), which are not consistently present in a species. Multiple copies of different lengths and variable sequences occur, with identical sequences sometimes shared by several species (11). Some fungal clades, such as Neocallimastigomycota (an early diverging lineage of obligately anaerobic, zoosporic gut fungi), lack mitochondria (15). Finally, because most fungi are microscopic and inconspicuous and many are unculturable, robust, universal primers must be available to detect a truly representative profile. This availability seems impossible with CO1.

The nuclear rRNA cistron has been used for fungal diagnostics and phylogenetics for more than 20 y (16), and its components are most frequently discussed as alternatives to CO1 (13, 17). The eukaryotic rRNA cistron consists of the 18S, 5.8S, and 28S rRNA genes transcribed as a unit by RNA polymerase I. Posttranscriptional processes split the cistron, removing two internal transcribed spacers. These two spacers, including the 5.8S gene, are usually referred to as the ITS region. The 18S nuclear ribosomal small subunit rRNA gene (SSU) is commonly used in phylogenetics, and although its homolog (16S) is often used as a species diagnostic for bacteria (18), it has fewer hypervariable domains in fungi. The 28S nuclear ribosomal large subunit rRNA gene (LSU) sometimes discriminates species on its own or combined with ITS. For yeasts, the D1/D2 region of LSU was adopted for characterizing species long before the concept of DNA barcoding was promoted (19–21).

Currently, ∼172,000 full-length fungal ITS sequences are deposited in GenBank, and 56% are associated with a Latin binominal, representing ∼15,500 species and 2,500 genera, derived from ∼11,500 scientific studies in ∼500 journals. An important fraction of the sequences lacking binominals is from environmental samples (22, 23). In a smaller number of environmental studies, ITS has been used combined with LSU (24, 25). ITS is also used in some fungi for providing an indication of delimitation by a measure of the genetic distances (26). However, phylogenetic approaches are also being used to identify taxonomic units in environmental sampling of fungi (27) and are often more effective in comparison (28).

Protein-coding genes are widely used in mycology for phylogenetic analyses or species identification. For Ascomycota (including mold genera such as Aspergillus), they are generally superior to rRNA genes for resolving relationships at various taxonomic levels (29). Specialized identification databases use several markers [e.g., translation elongation factor 1-α for _Fusarium_ (30) and β-tubulin for Penicillium (31)], but there is little standardization. Available primers for such markers usually amplify a narrow taxonomic range. Among protein-coding genes, the largest subunit of RNA polymerase II (RPB1) may have potential as a fungal barcode; it is ubiquitous and single copy, and it has a slow rate of sequence divergence (32). Its phylogenetic use was shown in studies of Basidiomycota, zygomycota, Microsporidia (33–36), and some protists (37). RPB1 primers were developed for the Assembling the Fungal Tree of Life (AFToL) project, and the locus is included in the subsequent AFToL2 (38). However, its use as a barcode remains untested.

This paper stems from a multilaboratory, multinational initiative to formalize a standard DNA barcode for kingdom Fungi (excluding nonfungal organisms traditionally treated as fungi). We compared barcoding performance of three nuclear ribosomal regions (ITS, LSU, and SSU) and one region from a representative protein-coding gene, RPB1, based on probability of correct identification (PCI) and barcode gap analysis using newly generated sequences for representatives of the 17 major fungal lineages (Fig. 1). Contributors used standard primers and protocols developed by AFToL and submitted sequences to a customized database for analysis. Some also contributed sequences from regions of two additional optional genes, namely the second largest subunit of RNA polymerase II (RPB2; also an AFToL marker) (39) and a gene encoding a minichromosome maintenance protein (_MCM_7), which were chosen based on their usefulness in phylogenetic studies and ease of amplification across Ascomycota (40–42).

Dendrogram of 17 fungal lineages sampled in this study showing consensus relationships and sampling. Relationships with high levels of uncertainty are indicated by stippled lines. Lineages are labeled and listed together with the approximate number of currently described species. The currently accepted node for delineating Fungi is indicated by F. The phyla Ascomycota and Basidiomycota are indicated by A and B, respectively. Gray bars to the left indicate numbers of strains in the barcode database, with the longest bar equal to 1,176 strains. Black bars indicate the proportions selected for a PCI analysis. The four datasets analyzed for PCI are numbered 1–4: 1, Pezizomycotina; 2, Saccharomycotina; 3, Basidiomycota; 4, early diverging lineages. Pie charts indicate the proportion of success from attempts to amplify the four-marker regions in the following order: ITS, LSU, SSU, and RPB1. Black, successful PCRs and sequences; gray, uncertain cases where no report was given; white, unsuccessful PCR.

Results

We compared the barcoding performance of four markers using newly generated sequences from 742 strains or specimens, with two additional protein-coding markers analyzed for a smaller subset of about 200 fungi. Our taxon sampling was comprehensive and covered the main fungal lineages, with heavier sampling in the most speciose clades. Comparisons of PCI for all combinations of ITS, LSU, SSU, and RPB1 for all Fungi are shown in Figs. S1 and S2. We attempted to include Glomeromycota in the four-marker comparison, but RPB1 could only be amplified for some species of Glomeraceae. A simplified analysis of ITS vs. LSU in Glomeromycota (Fig. S3) indicated high levels of intraspecific variation in this group. We were unable to include Neocallimastigomycota because of the absence of sufficient sequence data spanning the full length of the ribosomal cistron. We omitted the Cryptomycota and Microsporidia clades; arguments for and against their inclusion within Fungi continue (43, 44), although they presently are classified within the kingdom. For practical reasons, we had to assume that species concepts used by the taxonomists in the consortium were accurate and consistent, relying on the current circumscription of each species as assessed by the participants’ expertise. Genealogical concordance phylogenetic species recognition is commonly applied in mycology (45).

PCR Success.

The survey (Fig. S4) showed that PCR amplifications of ribosomal RNA genes were more reliable across the Fungi than the protein-coding markers (Fig. 1). As expected, the success varied by taxonomic group [e.g., ITS PCR amplification success ranged from 100% (Saccharomycotina) to 65% (early diverging lineages)]. Ranges for the other ribosomal markers were similar. In comparison, success for RPB1 varied from 80% (Saccharomycotina) to 14% (basal lineages). About 80% of respondents reported no problems with PCR amplification of ITS, 90% scored it as easy to obtain a high-quality PCR product, and 80% reported no significant sequencing concerns. In comparison, >70% reported PCR amplification problems for RPB1; 40–50% reported primer failure as the biggest problem.

Species Identification.

We performed several analyses to allow direct comparison of the barcoding use of the four main markers under consideration (i.e., ITS, LSU, SSU, and RPB1) (Figs. 2 and ​3). To assess the PCI, data were divided into subsets according to taxonomic affinity. The combined four-marker PCI comparisons (Fig. 2) included 742 samples, with 142 species represented by more than one sample and 84 species represented with one sample. With all taxa considered, the PCI of ITS (0.73) was marginally lower than RPB1 (0.76). RPB1 consistently yielded high levels of species discrimination in all of the fungal groups except the early diverging lineages, which is comparable with multigene combinations (Fig. 2). Within Dikarya, ITS had the most resolving power for species discrimination in Basidiomycota (0.77 vs. 0.67 for RPB1). For Pezizomycotina, the PCI of RPB1 (0.80) outperformed ITS (0.71). ITS had lower discriminatory power than SSU and LSU in early diverging lineages, but margins of error were high. LSU had variable levels of PCI (0.66–0.75) among all groups but was generally lower than RPB1 or ITS (Fig. 2). In Saccharomycotina, LSU had the lowest PCI (0.67), but all four markers performed similarly. SSU was consistently the worst performing marker, with the lowest species discrimination in Pezizomycotina (Fig. 2) and Basidiomycota (Fig. 2). In the early diverging lineages (Fig. 2), SSU had a better PCI, on par with LSU and better than both ITS and RPB1.

Barcode gap probability of identification for the four-marker datasets of ITS, LSU, SSU, and RPB1. The plots show the combinations of barcode markers investigated on the y axis. I, ITS; L, LSU; S, SSU; R, RPB1. The x axis shows the barcode gap PCI estimate for Ascomycota, Pezizomycotina (142 species), Basidiomycota (43 species), Ascomycota, Saccharomycotina (13 species), early diverging lineages (8 species), and combined groups (206 species). The error bars indicate 95% confidence intervals for the PCI estimate.

Barcode gap analyses using distance histograms for each marker. Histograms display intraspecific variation in light gray and interspecific variation in dark gray. Inserts summarize distance data.

In the multigene combinations, the most effective two genes in the combined analysis were either ITS and RPB1 or LSU and RPB1, both yielding a PCI of 0.78. This finding represented an increase of 0.02 from the highest-ranked single gene. The highest-ranked three- and four-gene combinations gave comparable increases.

Two supplementary three-marker comparisons expanded diversity for some major clades underrepresented in the four-gene analysis. For lichen-forming fungi, SSU was often absent, because the protocols favored amplicons from the photobiont rather than the fungus. Eliminating the requirement for SSU allowed more intensive sampling, yielding 683 sequences that included 179 species represented by more than one sample and 117 species represented by one sample (Fig. S5_A_). There was no apparent difference in ranking of the four candidate barcodes for the Pezizomycotina compared with the four-gene comparison in this analysis. Similarly, early diverging lineages yielded only 43 RPB1 sequences, and a comparison of ribosomal markers (ITS, SSU, and LSU) allowed inclusion of a larger set of 152 samples, with 34 species represented by more than one sample and 50 species by one sample. In this dataset, all sequences were unique to their species (Fig. S5_B_), and there was again no difference from the original four-gene comparison.

The barcode gap analyses (Fig. 3) largely confirmed the trends seen in the PCI analysis. The clearest indication of a barcode gap is seen for RPB1 followed by ITS. LSU and SSU performed poorly, each lacking a significant barcode gap.

To test whether other single-copy protein-coding markers might have a similar barcoding performance to RPB1, RPB2 and MCM7 sequences were tested for a subset of taxa. Neither yielded data from the early diverging lineages, but a combination of remaining groups yielded 207 strains, including 55 species with more than one sample and 23 species with one sample (Fig. S6). For both markers, all sequences were unique to their species. The two supplementary genes had a similar barcoding performance to RPB1, with RPB2 yielding slightly superior results followed by RPB1 and MCM7.

Discussion

Overall, ribosomal markers had fewer problems with PCR amplification than protein-coding markers (Fig. 1 and Fig. S4). Based on overall performance in species discrimination, SSU had almost no barcode gap (46) and the worst combined PCI, and it can be eliminated as a candidate locus (Figs. 2 and ​3). LSU, a favored phylogenetic marker among many mycologists, had virtually no amplification, sequencing, alignment, or editing problems, and the barcode gap was superior to the SSU. However, across the fungal kingdom, ITS was generally superior to LSU in species discrimination and had a more clearly defined barcode gap (Fig. 3). The overall probability of correct species identification using ITS is comparable with the success reported for the two-marker plant barcode system (0.73 vs. 0.70) (7). Higher species identification success can be expected in the major macrofungal groups in Basidiomycota (0.79), and slightly lower success can be expected in the economically important microfungal groups in filamentous Ascomycota (0.75). ITS performed as a close second to the most heavily sampled of our protein-coding markers, RPB1. However, the much higher PCR amplification success rate for ITS is a critical difference in its performance as a barcode (Fig. 1). ITS primers used in this study were applied to a range of fungal lineages, and several primers function as almost universal primers. However, all primer sets have a range of biases, and an appropriate solution will be to use more than one primer combination (47).

Taking all these arguments into account, we propose ITS as the standard barcode for fungi. The proposal will satisfy most fungal biologists but not all. Given the fungal kingdom's age and genetic diversity, it is unlikely that a single-marker barcode system will be capable of identifying every specimen or culture to species level. Furthermore, the limitations of ITS sequences for identifying species in some groups and the failure of the universal ITS primers to work in a minority of other groups will have to be carefully documented (14, 43, 48). ITS sequences shared among different species have already been documented in species-rich Pezizomycotina genera with shorter amplicons, such as the economically important genera Cladosporium (49), Penicillium (50), and Fusarium (51). In Aspergillus, ITS sequences are identical in several complexes of critical mycotoxigenic, industrial, and medically important species, and additional markers are necessary (52). Although the ITS region is a potentially effective DNA barcode in several lichenized lineages (53), genetic drift may prevent lineage sorting of ancestral polymorphisms in some slowly evolving groups.

Intragenomic variation, such as the existence of multiple paralogous or nonorthologous copies within single fruiting bodies of basidiomycetes (54, 55) and ascomycetes (56) or within axenic cultures (51), may lead to higher estimates of infraspecific variability (57, 58) or generation of barcodes that act only as representative sequences of multiple variable repeats (59, 60). Highly variable lengths and high evolutionary rates for the nuclear ribosomal cistron in species of Cantharellus, Tulasnella (Cantherellales, Basidiomycota) (61–63), and some lichens (53) may provide challenges for sequencing and analysis. The upper range of this ITS region variation is likely found in the Glomeromycota, with up to 20% divergence within a single multinucleate spore (64, 65).

We acknowledge that species delimitations vary from one fungal group to another and are often influenced by scarcity of sampling and lack of detailed biological knowledge (43, 45). This influence is reflected when ITS distances are compared between phyla, subphyla, and species (Figs. S7 and S8). In an expanded dataset of ITS sequences from our fungal DNA barcoding database, the highest variation was most often found in the early diverging lineages. This finding confirms the fact that fungal diversity remains poorly sampled with DNA sequences for these lineages (43, 48). It is, therefore, very likely that high divergence reflects the presence of multiple cryptic species, indicating important focal points for additional study. Despite these challenges, ITS combines the highest resolving power for discriminating closely related species with a high PCR and sequencing success rate across a broad range of Fungi.

In addition to Fungi, ITS may also be applicable as a barcode for other organisms. Its use has already been shown in Chlorophyta and plants (66, 67) as well as in Oomycota (6). The possibility of multikingdom analyses of complex ecosystems like soil using the species-informative, stable, high copy number ITS mirrors the original vision of DNA barcoding, and it already seems feasible, for example, to amplify Fungi and other eukaryotes from soil (23).

Protein-coding genes are popular phylogenetic markers in mycology, and they are used as de facto barcodes of limited taxonomic scope in several groups of fungi. We chose RPB1 as a representative marker to include in our broad comparisons, with RPB2 and MCM7 analyzed for a smaller sampling. In general, such protein markers had more species resolving power, but PCR and sequencing failures eliminate them as potential universal barcodes for the broad phylogenetic scope of the kingdom Fungi. Reliable kingdom-wide PCR amplification needs to be tested for other widely used protein-coding markers, such as translation elongation factor 1-α, β-tubulin, or actin.

The possibility of a two-marker barcoding system for fungi, as adopted for plants, is often discussed among mycologists, particularly researchers working on ascomycetous yeasts (19–21) and Glomeromycota (68) who prefer a system combining ITS and LSU. Data from this study (Fig. S5) indicate that ITS and LSU perform very similarly as barcodes and that differences in these sequences correlate well with current species concepts. Combinations of both ITS and LSU sequences are also applied in environmental sampling (69), where tandem amplification can allow simultaneous species identification with ITS and phylogenetic analysis with LSU. Our analyses with two-, three-, or four-marker barcode systems (Fig. 2) reveal only a modest increase in the PCI over a single-marker ITS barcode. The need for a second marker depends on the intended purpose of an investigation (i.e., whether a broad and general survey is intended or whether particular critical species are being monitored). If these taxa are taxa with low ITS interspecific variability, secondary markers must be used to accurately report genetic diversity (70). Genome mining efforts have identified a few single-copy genes that might be amenable for broad-range priming, and these efforts should continue (71, 72).

Although the genome diversity of fungal species is studied with increasing intensity, the vast majority of fungal species remains unknown. The recent discovery of a ubiquitous fungal class from soil (73) and a diverse early diverging phylum, Cryptomycota, tied to Rozella (74–76) from riverine and marine sites illuminates this fact. More than 90% of Fungi may be awaiting discovery, posing a tremendous pressure to increase the pace of fungal species discovery (1, 2). In addition to this, the Melbourne Botanical Congress has recently approved large-scale changes to the process of naming fungi (77), and sequence data from type specimens will increasingly be essential to the stability of fungal nomenclature. Continuing discovery of novel biodiversity while classifying knowledge already available will demand well-coordinated initiatives, and DNA barcoding has a crucial role to play.

Materials and Methods

DNA Isolation, Amplification, and Sequencing.

DNA was isolated and purified from cultures or specimens using the methods routinely used by the participating laboratories. Similarly, PCR protocols (Table S1) and thermocyclers varied from laboratory to laboratory. PCR primers were those primers from the AFToL project (Table S1). Several samples were sent by contributors for PCR amplification and sequencing at LifeTech. For PCR at LifeTech, 1–2 μL fungal DNA were amplified in a final volume of 30 μL with 15 μL AmpliTaq Gold 360 Mastermix, PCR primers, and water. All forward primers contained the M13F-20 sequencing primer, and reverse primers included the M13R-27 sequencing primer. PCR products (3 μL) were enzymatically cleaned before cycle sequencing with 1 μL ExoSap-IT and 1 μL Tris EDTA and incubated at 37 °C for 20 min followed by 80 °C for 15 min. Cycle sequencing reactions contained 5 μL cleaned PCR product, 2 μL BigDye Terminator v3.1 Ready Reaction Mix, 1 μL 5× Sequencing Buffer, 1.6 pmol M13F or M13R sequencing primer, and water in a final volume of 10 μL. The standard cycle sequencing protocol was 27 cycles of 10 s at 96 °C, 5 s at 50 °C, 4 min at 60 °C, and hold at 4 °C. Sequencing cleaning was performed with the BigDye XTerminator Purification Kit as recommended by the manufacturer for 10-μL volumes. Sequencing reactions were analyzed on a 3730xl Genetic Analyzer.

Sampling.

Closely related but separately named asexual and sexual species were coded with one genus name and then divided into subsets to allow taxonomically targeted assessment of markers for each major clade (Fig. 1). From the barcoding database of 2,920 samples, we selected a subset of 742 strains with sequences for all four markers (ITS, LSU, SSU, and RPB1). This subset was divided into four taxonomically delimited datasets: 416 strains in Pezizomycotina (filamentous ascomycetes), 81 strains in Saccharomycotina (ascomycete yeasts), 202 strains in Basidiomycota, and 43 strains from the combined polyphyletic early diverging lineages. Two additional analyses were performed for samples with three markers to enhance evaluation of certain undersampled lineages: the first analysis for 683 strains of Pezizomycotina with ITS, LSU, and RPB1 sequences and the second analysis for 152 representatives of basal lineages with ITS, LSU, and SSU sequences. Finally, a six-marker comparison was made for a selection of 207 strains of Pezizomycotina, Basidiomycota, and Saccharomycotina, with the first four markers supplemented with the two optional markers, MCM7 and RPB2. The species and strains used in the analysis are shown in Dataset S1.

PCR success.

Participants recorded their experience on the success of PCR amplification and sequencing for the genes and taxa that they contributed to this study. They also documented specific problems with PCR, quality of PCR amplification, primer problems (PCR and sequencing), and whether cloning was required. The genes were ranked for their ability to discriminate species and their overall taxonomic and phylogenetic use in specialized taxonomic groups. Comments were parsed to identify taxon-specific problems and are summarized in Fig. S4.

Data Analyses.

Database.

A query-based BioloMICS database (78) was established for 2,920 strains (1,022 species including subspecies) provided by >70 members of the consortium (www.fungalbarcoding.org). The complete database sets consist of 213 different genera and 915 unique species; there was an average of four species per genus and three strains per species. Approximately one-third (1,029) of the strains were scored as sibling species of other species in the sample, with 156 unique sibling species groups. All data are based on deposited voucher specimens or cultures identified by taxonomic specialists. The database allowed pairwise sequence alignments or polyphasic identifications using one or any combination of the six genes used in this study. The taxon sampling covered 15 of 17 major lineages attributed to the Fungi (Fig. 1) that were weighted to species-rich higher taxa such as the Pezizomycotina (the largest group of Ascomycota) and the Agaricomycotina (mushrooms and other macrobasidiomycetes).

PCI.

For each dataset, we calculated the barcode gap PCI. All alignments used the BLAST default DNA scoring system (79, 80). Two kinds of sequence alignment were calculated between every sample pair, namely (i) a global alignment using the Needleman–Wunsch algorithm, which aligns the entire sequence length with penalties for gaps at the alignment ends (81), and (ii) a semiglobal alignment using a variant Needleman–Wunsch algorithm that includes both ends of one sequence and finds the alignment with the highest score without penalizing end gaps in the other sequence. The latter algorithm does the same for the other sequence, returning the alignment with the higher of the two scores. Thus, the global alignment matches the whole length of two sequences, and the semiglobal alignment matches one sequence to a subset of the other and then vice versa. Semiglobal alignment checks whether disparate sequence lengths degrade species identification; if they do not, global and semiglobal alignment should result in similar identifications. For the two types of alignment, the _p_-distance (the proportion of aligned nucleotide pairs consisting of differing nucleotides) was calculated. The sequence diameter of a species is defined as the greatest _p_-distance between any two samples from within a species. Based on the sequence diameter, correct identification of a species occurs if, for every sample in the species, no sample from another species lies within the sequence diameter. The corresponding barcode gap PCI is the fraction of species correctly identified (7). The Wilson score interval yielded 95% confidence intervals for each PCI estimate (82). PCI was also calculated for all possible combinations of two, three, or four genes to evaluate the potential payoff of a multigene barcoding system.

Sequence divergence and DNA gap analyses.

Using the same dataset as for the PCI analysis, a DNA barcode gap analysis was performed using matrix algebra and Statistic Analysis Software (SAS Institute) as described previously (6) except that the lower triangular uncorrected distance matrix was calculated using mothur (83). The results are indicated in Fig. 3. Additional comparisons were done and are described in Figs. S2, S3, and S7–S9.

Supplementary Material

Acknowledgments

We thank David L. Hawksworth, Martin Bidartondo, and numerous other colleagues for critical comments. This work was organized under the Fungal Working Group of the Consortium for the Barcode of Life (CBOL), which provided support from its funding from the Alfred P. Sloan Foundation. Support was also provided by the Intramural Research Program of the National Library of Medicine at the National Institutes of Health, Life Technologies Corporation, and the individual funders to authors who provided sequences for our analysis. Publication charges were provided by the International Barcode of Life Network from Genome Canada through the Ontario Genomics Institute.

Footnotes

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