Fungi in the Built EnvironmentJason StajichUniversity of California, Riverside

http://fungidb.org http://lab.stajich.org@hyphaltip @fungalgenomes @fungidb

Coprinopsis cinerea Ellison & Stajich

Puccinia graminis J. F. Hennen

Cryptococcus neoformans X. Lin

Laccaria bicolor Martin et al.

Aspergillus niger. N Read

Xanthoria elegans. Botany POtD

Neurospora crassa. Hickey & Reed

Glomus sp. Univ Sydney

Phycomyces blakesleansus T. Ootaki

Rhizopus stolonifera.Amanita phalloides. M WoodUstilago maydis Kai Hirdes

Batrachochytrium dendrobatidis J. Longcore

Rozella allomycis. James et al

Blastocladiella simplex Stajich & Taylor

Fungal diversity of forms, functions, ecosystems

1500 1000 500 0

Rozella

Microsporidia

Entomophthoromycotina

Chytridiomycota

Agaricomycotina

Choanozoa

Amoebozoa

Ustilaginomycotina

Metazoa

Glomeromycota

Pezizomycotina

Pucciniomycotina

Plantae

Mucoromycotina

Zoopagomycotina

Kickxellomycotina

Saccharomycotina

Blastocladiomycota

Taphrinomycotina

Loss of flagellum

Mitotic sporangiato mitotic conidia

Regular septa

Meiotic sporangia to external meiospores

Multicellular with differentiated tissues

Millions of years

Basidiomycota

Ascomycota

Fungi

Stajich et al. Current Biol 2009

Fungi interact with many organisms

Endophytes

10.3389/fpls.2011.00100

doi: 10.3389/fpls.2011.00100

Mycorrhiza

Betsy Arnold

F. Martindoi: 10.1016/j.pbi.2009.05.007,

Organisms interacting with Fungi - fungi as the host

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 4063–4075 Vol. 76, No. 120099-2240/10/$12.00 doi:10.1128/AEM.02928-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Diverse Bacteria Inhabit Living Hyphae of PhylogeneticallyDiverse Fungal Endophytes!†

Michele T. Hoffman and A. Elizabeth Arnold*Division of Plant Pathology and Microbiology, School of Plant Sciences, 1140 E. South Campus Drive,

University of Arizona, Tucson, Arizona 85721

Received 3 December 2009/Accepted 20 April 2010

Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, theinterplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recentlybacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at leastone species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylo-genetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including repre-sentatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photo-synthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteriausing microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophyticPezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regionssurveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies wereincongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a smallnumber of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequentloss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineagesof endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endo-phytes representing four of the most species-rich classes of fungi, and highlights for the first time theirdiversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants inwhich these endophyte-bacterium symbiota occur.

Traits related to the establishment and outcome of plant-fungus symbioses can reflect not only abiotic conditions andthe unique interactions of particular fungal and plant geno-types (49, 50, 56, 59, 62, 67) but also additional microbes thatinteract intimately with fungal mycelia (4, 12, 42). For example,mycorrhizosphere-associated actinomycetes release volatilecompounds that influence spore germination in the arbuscularmycorrhizal (AM) fungus Gigaspora margarita (Glomeromy-cota) (14). Levy et al. (34) describe Burkholderia spp. thatcolonize spores and hyphae of the AM fungus Gigaspora de-cipiens and are associated with decreased spore germination.Diverse “helper” bacteria have been implicated in promotinghyphal growth and the establishment of ectomycorrhizal sym-bioses (23, 26, 57, 70). Minerdi et al. (43) found that a consor-tium of ectosymbiotic bacteria limited the ability of the patho-gen Fusarium oxysporum to infect and cause vascular wilts inlettuce, with virulence restored to the pathogen when ectosym-bionts were removed.

In addition to interacting with environmental and ectosym-biotic bacteria, some plant-associated fungi harbor bacteriawithin their hyphae (first noted as “bacteria-like organisms” ofunknown function) (38). These bacteria, best known from liv-

ing hyphae of several species of the Glomeromycota andMucoromycotina, can alter fungal interactions with host plantsin diverse ways (see references 12, 31, and 51). For example,the vertically transmitted bacterium “Candidatus Glomeri-bacter gigasporarum” colonizes spores and hyphae of the AMfungus Gigaspora gigasporarum (9, 10). Removal of the bacte-rial partner from the fungal spores suppresses fungal growthand development, altering the morphology of the fungal cellwall, vacuoles, and lipid bodies (37). In turn, the discovery ofphosphate-solubilizing bacteria within Glomus mossae spores(44), coupled with the recovery of a P-transporter operon inBurkholderia sp. from Gigaspora margarita (54), suggests acompetitive role in phosphate acquisition and transport bythese bacteria within the AM symbiosis. Within the Mucoro-mycotina, Partida-Martinez and Hertweck (51) reported that asoilborne plant pathogen, Rhizopus microsporus, harbors en-dosymbiotic Burkholderia that produces a phytotoxin (rhi-zoxin) responsible for the pathogenicity of the fungus.

These examples, coupled with the discovery of bacteriawithin hyphae of the ectomycorrhizal Dikarya (Tuber borchii;Ascomycota; Laccaria bicolor and Piriformospora indica;Basidiomycota) (5–8, 58), suggest that the capacity to harborendohyphal bacteria is widespread among fungi. To date, how-ever, endocellular bacteria have been recovered only fromfungi that occur in the soil and rhizosphere (12, 31). Here wereport for the first time that phylogenetically diverse bacteriaoccur within living hyphae of foliar endophytic fungi, includingmembers of four classes of filamentous Ascomycota. We usea combination of light and fluorescence microscopy to visu-alize bacterial infections within living hyphae of represen-

* Corresponding author. Mailing address: Division of Plant Pathol-ogy and Microbiology, School of Plant Sciences, University of Arizona,1140 E. South Campus Drive, Tucson, AZ 85721. Phone: (520) 621-7212. Fax: (520) 621-9290. E-mail: arnold@ag.arizona.edu.

† Supplemental material for this article may be found at http://aem.asm.org/.

! Published ahead of print on 30 April 2010.

4063

on September 18, 2012 by UNIV O

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.org/Downloaded from

Domestication: Ant farmed fungi

Plant + Fungus + Mycovirusto the Midwest Regional Center of Excellence forBiodefense and Emerging Infectious Disease Research(MRCE) and by NIH grant AI53298. The DDRCC issupported by NIH grant DK52574. W.W.L. was supportedby the Clinical/Translational Fellowship Program of theMRCE, the W.M. Keck Foundation, and the NIH NationalResearch Service Award (NRSA) F32 AI069688-01. P.A.P.

was supported by the NIH Institutional NRSA T32GM07067 to the Washington University School ofMedicine.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/315/5811/509/DC1Materials and Methods

SOM TextFigs. S1 to S4Tables S1 and S2References

6 November 2006; accepted 14 December 200610.1126/science.1137195

A Virus in a Fungus in a Plant:Three-Way Symbiosis Required forThermal ToleranceLuis M. Márquez,1 Regina S. Redman,2,3 Russell J. Rodriguez,2,4 Marilyn J. Roossinck1*

A mutualistic association between a fungal endophyte and a tropical panic grass allows bothorganisms to grow at high soil temperatures. We characterized a virus from this fungus that isinvolved in the mutualistic interaction. Fungal isolates cured of the virus are unable to conferheat tolerance, but heat tolerance is restored after the virus is reintroduced. The virus-infectedfungus confers heat tolerance not only to its native monocot host but also to a eudicot host,which suggests that the underlying mechanism involves pathways conserved between these twogroups of plants.

Endophytic fungi commonly grow withinplant tissues and can be mutualistic insome cases, as they allow plant adaptation

to extreme environments (1). A plant-fungalsymbiosis between a tropical panic grass fromgeothermal soils, Dichanthelium lanuginosum,and the fungus Curvularia protuberata allowsboth organisms to grow at high soil temperaturesin Yellowstone National Park (YNP) (2). Fieldand laboratory experiments have shown thatwhen root zones are heated up to 65°C, non-symbiotic plants either become shriveled andchlorotic or simply die, whereas symbiotic plantstolerate and survive the heat regime. Whengrown separately, neither the fungus nor the plantalone is able to grow at temperatures above 38°C,but symbiotically, they are able to tolerate ele-vated temperatures. In the absence of heat stress,symbiotic plants have enhanced growth ratecompared with nonsymbiotic plants and alsoshow significant drought tolerance (3).

Fungal viruses or mycoviruses can modulateplant-fungal symbioses. The best known exam-ple of this is the hypovirus that attenuates thevirulence (hypovirulence) of the chestnut blightfungus,Cryphonectria parasitica (4). Virus regu-lation of hypovirulence has been demonstratedexperimentally in several other pathogenic fungi(5–8). However, the effect of mycoviruses onmutualistic fungal endophytes is unknown. Thereis only one report of a mycovirus from the well-

knownmutualistic endophyte, Epichloë festucae,but no phenotype has been associated with thisvirus (9).

Fungal virus genomes are commonly com-posed of double-stranded RNA (dsRNA) (10).Large molecules of dsRNA do not normallyoccur in fungal cells and, therefore, their presenceis a sign of a viral infection (9). Using a protocolfor nucleic acid extraction with enrichment fordsRNA (11), we detected the presence of a virusin C. protuberata. The dsRNA banding patternconsists of two segments of about 2.2 and 1.8 kb.A smaller segment, less than 1 kb in length, wasvariable in presence and size in the isolatesanalyzed and, later, was confirmed to be a sub-genomic element, most likely a defective RNA(fig. S1 and Fig. 1, A and B). Using taggedrandom hexamer primers, we transcribed thevirus with reverse transcriptase (RT), followed byamplification and cloning. Sequence analysisrevealed that each of the two RNA segmentscontains two open reading frames (ORFs) (fig.

S2). The 2.2-kb fragment (RNA 1) is involved invirus replication, as both of its ORFs are similarto viral replicases. The first, ORF1a, has 29%amino acid sequence identity with a putativeRNA-dependent RNA polymerase (RdRp) fromthe rabbit hemorrhagic disease virus. The aminoacid sequence of the second, ORF1b, has 33%identity with the RdRp of a virus of the fungalpathogen Discula destructiva. These two ORFsoverlap and could be expressed as a singleprotein by frameshifting, a common expressionstrategy of viral replicases. The two ORFs ofRNA 2 have no similarity to any protein withknown function. As in most dsRNA mycovi-ruses, the 5! ends (21 bp) of both RNAs areconserved. Virus particles purified from C.protuberata are similar to those of other fungalviruses: spherical and ~27 nm in diameter (fig.S3). This virus is transmitted vertically in theconidiospores. We propose naming this virusCurvularia thermal tolerance virus (CThTV) toreflect its host of origin and its phenotype.

The ability of the fungus to confer heattolerance to its host plant is related to thepresence of CThTV. Wild-type isolates of C.protuberata contained the virus in high titers, asevidenced by their high concentration of dsRNA(~2 mg/g of lyophilized mycelium). However, anisolate obtained from sectoring (change inmorphology) of a wild-type colony contained avery low titer of the virus, as indicated by a lowconcentration of dsRNA (~0.02 mg/g of lyophi-lized mycelium). These two isolates were iden-tical by simple sequence repeat (SSR) analysiswith two single-primer polymerase chain reac-tion (PCR) reactions and by sequence analysis ofthe rDNA ITS1-5.8S-ITS2 region (figs. S4 andS5). Desiccation and freezing-thawing cycles areknown to disrupt virus particles (12); thus, my-celium of the isolate obtained by sectoring was

1Plant Biology Division, Samuel Roberts Noble Foundation,Post Office Box 2180, Ardmore, OK 73402, USA. 2Depart-ment of Botany, University of Washington, Seattle, WA98195, USA. 3Department of Microbiology, Montana StateUniversity, Bozeman, MT 59717, USA. 4U.S. Geological Sur-vey, Seattle, WA 98115, USA.

*To whom correspondence should be addressed. E-mail:mroossinck@noble.org

Fig. 1. Presence or absence ofCThTV in different strains of C.protuberata, detected by ethid-ium bromide staining (A),Northern blot using RNA 1 (B)and RNA 2 (C) transcripts ofthe virus as probes, and RT-PCR using primers specific fora section of the RNA 2 (D). Theisolate of the fungus obtainedby sectoring was made virus-free (VF) by freezing-thawing.The virus was reintroduced intothe virus-free isolate through hyphal anastomosis (An) with the wild type (Wt). The wild-type isolate ofthe fungus sometimes contains a subgenomic fragment of the virus that hybridizes to the RNA 1 probe(arrow).

www.sciencemag.org SCIENCE VOL 315 26 JANUARY 2007 513

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DOI: 10.1126/science.1136237

Estimates of the number of species of Fungi

426

American Journal of Botany 98(3): 426–438. 2011.

American Journal of Botany 98(3): 426–438, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America

What are Fungi? — Fungal biologists debated for more than 200 years about which organisms should be counted as Fungi. In less than 5 years, DNA sequencing provided a multitude of new characters for analysis and identifi ed about 10 phyla as members of the monophyletic kingdom Fungi ( Fig. 1 ). Mycolo-gists benefi ted from early developments applied directly to fungi. The “ universal primers, ” so popular in the early 1990s for the polymerase chain reaction (PCR), actually were de-signed for fungi ( Innis et al., 1990 ; White et al., 1990 ). Use of the PCR was a monumental advance for those who studied min-ute, often unculturable, organisms. Problems of too few mor-phological characters (e.g., yeasts), noncorresponding characters among taxa (e.g., asexual and sexual states), and convergent morphologies (e.g., long-necked perithecia producing sticky ascospores selected for insect dispersal) were suddenly over-come. Rather than producing totally new hypotheses of rela-tionships, however, it is interesting to note that many of the new fi ndings supported previous, competing hypotheses that had been based on morphological evidence ( Alexopoulos et al., 1996 ; Stajich et al., 2009 ). Sequences and phylogenetic analy-ses were used not only to hypothesize relationships, but also to identify taxa rapidly ( Kurtzman and Robnett, 1998 ; Brock et al., 2009 ; Begerow et al., 2010 ).

Most fungi lack fl agella and have fi lamentous bodies with distinctive cell wall carbohydrates and haploid thalli as a result

of zygotic meiosis. They interact with all major groups of or-ganisms. By their descent from an ancestor shared with animals about a billion years ago plus or minus 500 million years ( Berbee and Taylor, 2010 ), the Fungi constitute a major eukary-otic lineage equal in numbers to animals and exceeding plants ( Figs. 2 – 10 ). The group includes molds, yeasts, mushrooms, polypores, plant parasitic rusts and smuts, and Penicillium chrysogenum , Neurospora crassa , Saccharomyces cerevisiae , and Schizosaccharomyces pombe , the important model organ-isms studied by Nobel laureates.

Phylogenetic studies provided evidence that nucleriid pro-tists are the sister group of Fungi ( Medina et al., 2003 ), nonpho-tosynthetic heterokont fl agellates are placed among brown algae and other stramenopiles, and slime mold groups are ex-cluded from Fungi ( Alexopoulos et al., 1996 ). Current phyloge-netic evidence suggests that the fl agellum may have been lost several times among the early-diverging fungi and that there is more diversity among early diverging zoosporic and zygosporic lineages than previously realized ( Bowman et al., 1992 ; Blackwell et al., 2006 ; Hibbett et al., 2007 ; Stajich et al., 2009 ).

Sequences of one or several genes are no longer evidence enough in phylogenetic research. A much-cited example of the kind of problem that may occur when single genes with differ-ent rates of change are used in analyses involves Microsporidia. These organisms were misinterpreted as early-diverging eu-karyotes in the tree of life based on their apparent reduced mor-phology ( Cavalier-Smith, 1983 ). Subsequently, phylogenetic analyses using small subunit ribosomal RNA genes wrongly supported a microsporidian divergence before the origin of mi-tochondria in eukaryotic organisms ( Vossbrinck et al., 1987 ). More recent morphological and physiological studies have not upheld this placement, and analyses of additional sequences, including those of protein-coding genes, support the view that these obligate intracellular parasites of insect and vertebrate

1 Manuscript received 10 August 2010; revision accepted 19 January 2011. The author thanks N. H. Nguyen, H. Raja, and J. A. Robertson for

permission to use their photographs, two anonymous reviewers who helped to improve the manuscript, and David Hibbett, who graciously provided an unpublished manuscript. She acknowledges funding from NSF DEB-0417180 and NSF-0639214.

2 Author for correspondence (e-mail: mblackwell@lsu.edu)

doi:10.3732/ajb.1000298

THE FUNGI: 1, 2, 3 … 5.1 MILLION SPECIES? 1

Meredith Blackwell 2

Department of Biological Sciences; Louisiana State University; Baton Rouge, Louisiana 70803 USA

• Premise of the study: Fungi are major decomposers in certain ecosystems and essential associates of many organisms. They provide enzymes and drugs and serve as experimental organisms. In 1991, a landmark paper estimated that there are 1.5 million fungi on the Earth. Because only 70 000 fungi had been described at that time, the estimate has been the impetus to search for previously unknown fungi. Fungal habitats include soil, water, and organisms that may harbor large numbers of understudied fungi, estimated to outnumber plants by at least 6 to 1. More recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist.

• Methods: Technological advances make it possible to apply molecular methods to develop a stable classifi cation and to dis-cover and identify fungal taxa.

• Key results: Molecular methods have dramatically increased our knowledge of Fungi in less than 20 years, revealing a mono-phyletic kingdom and increased diversity among early-diverging lineages. Mycologists are making signifi cant advances in species discovery, but many fungi remain to be discovered.

• Conclusions: Fungi are essential to the survival of many groups of organisms with which they form associations. They also attract attention as predators of invertebrate animals, pathogens of potatoes and rice and humans and bats, killers of frogs and crayfi sh, producers of secondary metabolites to lower cholesterol, and subjects of prize-winning research. Molecular tools in use and under development can be used to discover the world ’ s unknown fungi in less than 1000 years predicted at current new species acquisition rates.

Key words: biodiversity; fungal habitats; fungal phylogeny; fungi; molecular methods; numbers of fungi.

DOI:10.3732/ajb.1000298

Mycol. Res. 9S (6): 641--655 (1991) Printed in Great Britain 641

Presidential address 1990

The fungal dimension of biodiversity: magnitude, significance,and conservation

D. L. HAWKSWORTH

International Mycological Institute, Kew, Surrey TW9 3AF, UK

Fungi, members of the kingdoms Chromista, Fungi S.str. and Protozoa studied by mycologists, have received scant consideration indiscussions on biodiversity. The number of known species is about 69000, but that in the world is conservatively estimated at1'5 million; six-times higher than hitherto suggested. The new world estimate is primarily based on vascular plant:fungus ratios indifferent regions. It is considered conservative as: (1) it is based on the lower estimates of world vascular plants; (2) no separateprovision is made for the vast numbers of insects now suggested to exist; (3) ratios are based on areas still not fully knownmycologically; and (4) no allowance is made for higher ratios in tropical and polar regions. Evidence that numerous new speciesremain to be found is presented. This realization has major implications for systematic manpower, resources, and classification. Fungihave and continue to playa vital role in the evolution of terrestrial life (especially through mutualisms), ecosystem function and themaintenance of biodiversity, human progress, and the operation of Gaia. Conservation in situ and ex situ are complementary, and thesignificance of culture collections is stressed. International collaboration is required to develop a world inventory, quantify functionalroles, and for effective conservation.

'Biodiversity', the extent of biological variation on Earth, hascome to the fore as a key issue in science and politics for the1990s. First used as 'BioDiversity' in the title of a scientificmeeting in Washington, D.C. in 1986 (Wilson, 1988: p. v), ithas been rapidly adopted as a contraction of 'biotic diversity'and 'biological diversity'. Interest has been inflamed byconcern over the conservation of genetic resources, destrudionof forests, extinction of species, and the effects of globalwarming. A plethora of texts and reports has resulted; someof the more significant since 1985 are Norton (1986a), Soule(1986), U.S. Congress Office of Technology Assessment(1987), Wolf (1987), Cronk (1988), Lugo (1988), Wilson(1988), Knutson & Stoner (1989), U.s. National Science Board(1989), di Castri & Younes (1990), Keystone Center (1990),

McNeely et al. (1990), and u.s. Board on Agriculture(1991).

While many of the principles and discussions of broaderissues raised in these works are relevant to mycology, mostlack any substantive content on fungi, or indeed in many caseson any micro-organisms. Exceptions with sections on at leastsome micro-organism aspects are: U.s. Congress Office ofTechnology Assessment (1987), Knutson & Stoner (1989),U.S. National Science Board (1989), di Castri & Younes (1990),and U.S. Board on Agriculture (1991).

The aim of this address is to broaden the biodiversitydebate by focusing on its fungal dimension; the magnitude ofthe task and its implications for systematics; the significanceof fungi in evolution, ecosystem function, human progress,and to Gaia; and the conservation of fungi. Biodiversity canbe explored at a variety of levels: in terms of ecosystems,

41

species, or populations. Knowledge of all of these is pertinentto a thorough appreciation of the fungal dimension, but hereI will centre on species biodiversity; that is basal to discussionsat other levels.

DAVID L. HAWKSWORTHPresident, British Mycological Society, 1990

MYC 95

1.5 Million based on fungus to plant ratio of 6:1

Don’t forget the endophytes...and the soil...

Upwards of 6M species - Lee Taylor (pers comm)“Thus, the Fungi is likely equaled only by the Insecta with respect to eukaryote species richness.”

Fungal genome sequencing

http://www.diark.org/diark/statistics

400+ genomes of Fungi

http://1000.fungalgenomes.org

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Blue = completed or in progress, Red= proposed for Tier One sampling,Green = remaining unsampled families Numbers or Percent of Families in each clade and their current or proposed genome sampling

Addressing the phylogenetic diversity: 1000 Fungal genomes project

FungiDBStrategy queries

Data-miningGenome Browser

Gene function curation tool

Functional Genomics

Data

Phylogenomic profiles

Annotation & Function

Synteny

• Eurotiomycetes; Ascomycota◦ Aspergillus clavatus◦ Aspergillus flavus◦ Aspergillus fumigatus strain Af293◦ Aspergillus nidulans strain A4◦ Aspergillus niger◦ Aspergillus terreus◦ Coccidoidies immitis strain RS◦ Coccidoidies immitis strain H538.4

• Sordariomycetes; Ascomyocta◦ Fusarium oxysporum f. sp. lycopersici◦ Fusarium graminearum◦ Gibberella moniliformis (Fusarium verticillioides)◦ Magnaporthea oryzae◦ Neurospora crassa strain OR74A◦ Neurospora tetrasperma◦ Neurospora discreta◦ Sordaria macrospora

• Taphrinomycotina; Ascomyocta◦ Schizosaccharomyces pombe

• Saccharomycotina; Ascomyocta◦ Candida albicans◦ Saccharomyces cerevisiae

• Basidiomycota◦ Cryptococcus neoformans var. grubii) strain H99◦ Cryptococcus neoformans var. neoformans) strain JEC21◦ Cryptococcus neoformans var. neoformans) strain B3501◦ Cryptococcus gattii strain WM276◦ Cryptococcus gattii strain R265◦ Tremella mesenterica

• Oomycetes; Stramenopiles◦ Phytophthora capsici◦ Phytophthora infestans◦ Phytophthora ramorum◦ Phytophthora sojae◦ Pythium ultimatum◦ Hyaloperonospora arabidopsidis

FungiDB 2.0

• 31 genomes - 25 Fungi and 6 Oomycetes

• RNA-Seq for 6 species, Microarray for 2 sp, KEGG, EC, and GO annotations for several species

• Ortholog tables with OrthoMCL

• Gene function predictions from InterPro

Combining queries

• Results from one query combined with a second one.

• Can be intersection, union, or left or right overlaps

Microbial Ecology of Indoor Fungi

• Sloan Foundation initiative to provide a data coordination center for indoor microbiome data

• In collaboration with Rob Knight (QIIME), Mitch Sogin (VAMPS), Folker Meyer (MG-RAST)

• Fungi - names and taxonomy in flux

• Marker Genes and data collection approaches

• A sample indoor environment dataset analysis

One fungus, one name

Fungal Taxonomy and naming undergoing a revolutionARTIC

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V O L U M E 2 · N O . 1 105

© 2011 International Mycological Association

You are free to share - to copy, distribute and transmit the work, under the following conditions:Attribution:� � <RX�PXVW�DWWULEXWH�WKH�ZRUN�LQ�WKH�PDQQHU�VSHFL¿HG�E\�WKH�DXWKRU�RU�OLFHQVRU��EXW�QRW�LQ�DQ\�ZD\�WKDW�VXJJHVWV�WKDW�WKH\�HQGRUVH�\RX�RU�\RXU�XVH�RI�WKH�ZRUN�� Non-commercial:�� <RX�PD\�QRW�XVH�WKLV�ZRUN�IRU�FRPPHUFLDO�SXUSRVHV��No derivative works:� <RX�PD\�QRW�DOWHU��WUDQVIRUP��RU�EXLOG�XSRQ�WKLV�ZRUN��For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. 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.

GRL���������LPDIXQJXV�������������� IMA FuNgus · voluMe 2 · No 1: 105–112

The Amsterdam Declaration on Fungal Nomenclature

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Abstract: The Amsterdam Declaration on Fungal Nomenclature was agreed at DQ� LQWHUQDWLRQDO� V\PSRVLXP�FRQYHQHG� LQ�$PVWHUGDP�RQ���±���$SULO� �����XQGHU� WKH�DXVSLFHV�RI�WKH�,QWHUQDWLRQDO�&RPPLVVLRQ�RQ�WKH�7D[RQRP\�RI�)XQJL��,&7)���7KH�SXUSRVH�of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data DUH� URXWLQHO\� DYDLODEOH�� 7KH� LVVXH� LV� XUJHQW� DV�P\FRORJLVWV� FXUUHQWO\� IROORZ� GLIIHUHQW�SUDFWLFHV��DQG�QR�FRQVHQVXV�ZDV�DFKLHYHG�E\�D�6SHFLDO�&RPPLWWHH�DSSRLQWHG�LQ������E\� WKH� ,QWHUQDWLRQDO� %RWDQLFDO� &RQJUHVV� WR� DGYLVH� RQ� WKH� SUREOHP��The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become HQGDQJHUHG��7KDW�LV��PHDQLQJ�WKDW�SULRULW\�VKRXOG�EH�JLYHQ�WR�WKH�ILUVW�GHVFULEHG�QDPH��H[FHSW�ZKHUH�WKDW�LV�D�\RXQJHU�QDPH�LQ�JHQHUDO�XVH�ZKHQ�WKH�ILUVW�DXWKRU�WR�VHOHFW�D�name of a pleomorphic monophyletic genus is to be followed, and suggests controversial FDVHV� DUH� UHIHUUHG� WR� D� ERG\�� VXFK� DV� WKH� ,&7)��ZKLFK�ZLOO� UHSRUW� WR� WKH�&RPPLWWHH�for Fungi��,I�DSSURSULDWH��WKH�,&7)�FRXOG�EH�PDQGDWHG�WR�SURPRWH�WKH�LPSOHPHQWDWLRQ�RI� WKH�'HFODUDWLRQ�� ,Q�DGGLWLRQ��EXW�QRW� IRUPLQJ�SDUW�RI� WKH�'HFODUDWLRQ��DUH�UHSRUWV�RI�discussions held during the symposium on the governance of the nomenclature of fungi, DQG�WKH�QDPLQJ�RI�IXQJL�NQRZQ�RQO\�IURP�DQ�HQYLURQPHQWDO�QXFOHLF�DFLG�VHTXHQFH�LQ�SDUWLFXODU��3RVVLEOH�DPHQGPHQWV�WR�WKH�Draft BioCode��������WR�DOORZ�IRU�WKH�QHHGV�RI�P\FRORJLVWV�DUH�VXJJHVWHG�IRU�IXUWKHU�FRQVLGHUDWLRQ��DQG�D�SRVVLEOH�H[DPSOH�RI�KRZ�D�IXQJXV�RQO\�NQRZQ�IURP�WKH�HQYLURQPHQW�PLJKW�EH�GHVFULEHG�LV�SUHVHQWHG��

Key words: Anamorph$UWLFOH���%LR&RGH&DQGLGDWH�VSHFLHV(QYLURQPHQWDO�VHTXHQFHV,QWHUQDWLRQDO�&RGH�RI�%RWDQLFDO�1RPHQFODWXUH0\FR&RGHPleomorphic fungiTeleomorph

Article info:�6XEPLWWHG�����0D\�������$FFHSWHG�����0D\�������3XEOLVKHG����-XQH������

1'HSDUWDPHQWR�GH�%LRORJtD�9HJHWDO�,,��)DFXOWDG�GH�)DUPDFLD��8QLYHUVLGDG�&RPSOXWHQVH�GH�0DGULG��3OD]D�5DPyQ�\�&DMDO��(�������0DGULG��6SDLQ��DQG�'HSDUWPHQW�RI�%RWDQ\��1DWXUDO�+LVWRU\�0XVHXP��&URPZHOO�5RDG��/RQGRQ�6:���%'��8.��FRUUHVSRQGLQJ�DXWKRU�H�PDLO��G�KDZNVZRUWK#QKP�DF�XN���&%6�.1$:�)XQJDO�%LRGLYHUVLW\�&HQWUH��8SSVDODODDQ���������&7�8WUHFKW��7KH�1HWKHUODQGV��S�FURXV#FEV�NQDZ�QO��3National Mycological +HUEDULXP��$JULFXOWXUH�DQG�$JUL�)RRG�&DQDGD��1HDWE\�%XLOGLQJ������&DUOLQJ�$YHQXH��2WWDZD��2QWDULR�.�$��&���&DQDGD���+HUEDULXP��8QLYHUVLW\�RI�&DOLIRUQLD�%HUNHOH\�������9DOOH\�/LIH�6FLHQFHV�%XLOGLQJ�������%HUNHOH\��&$�������������86$���Department of Plant and Microbial Biology, University RI�&DOLIRUQLD��%HUNHOH\��&$�������������86$��6)RUHVWU\�DQG�$JULFXOWXUDO�%LRWHFKQRORJ\�,QVWLWXWH��)$%,���8QLYHUVLW\�RI�3UHWRULD��3ULYDWH�EDJ�;����+DW¿HOG�������3UHWRULD�������6RXWK�$IULFD��7Department of Biology, Basic and Industrial Microbiology Section, Faculty of Science, Ege University,

http://www.biology.duke.edu/fungi/mycolab/primers.htm

Barcoding consortium has chosen ITS as primary markerNuclear ribosomal internal transcribed spacer (ITS)region as a universal DNA barcode marker for FungiConrad L. Schocha,1, Keith A. Seifertb,1, Sabine Huhndorfc, Vincent Robertd, John L. Spougea, C. André Levesqueb,Wen Chenb, and Fungal Barcoding Consortiuma,2

aNational Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20892; bBiodiversity (Mycologyand Microbiology), Agriculture and Agri-Food Canada, Ottawa, ON, Canada K1A 0C6; cDepartment of Botany, The Field Museum, Chicago, IL 60605; anddCentraalbureau voor Schimmelcultures Fungal Biodiversity Centre (CBS-KNAW), 3508 AD, Utrecht, The Netherlands

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

Six DNA regions were evaluated as potential DNA barcodes forFungi, the second largest kingdom of eukaryotic life, by a multina-tional, multilaboratory consortium. The region of the mitochondrialcytochrome c oxidase subunit 1 used as the animal barcode wasexcluded as a potential marker, because it is difficult to amplify infungi, often includes large introns, and can be insufficiently vari-able. Three subunits from the nuclear ribosomal RNA cistron werecompared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largestsubunit of RNA polymerase II, and minichromosome maintenanceprotein). Although the protein-coding gene regions often hada higher percent of correct identification compared with ribosomalmarkers, low PCR amplification and sequencing success eliminatedthem as candidates for a universal fungal barcode. Among theregions of the ribosomal cistron, the internal transcribed spacer(ITS) region has the highest probability of successful identificationfor the broadest range of fungi, with the most clearly defined bar-code gap between inter- and intraspecific variation. The nuclearribosomal large subunit, a popular phylogenetic marker in certaingroups, had superior species resolution in some taxonomic groups,such as the early diverging lineages and the ascomycete yeasts, butwas otherwise slightly inferior to the ITS. The nuclear ribosomalsmall subunit has poor species-level resolution in fungi. ITS will beformally proposed for adoption as the primary fungal barcodemarker to the Consortium for the Barcode of Life, with the possibil-ity that supplementary barcodes may be developed for particularnarrowly circumscribed taxonomic groups.

DNA barcoding | fungal biodiversity

The absence of a universally accepted DNA barcode for Fungi,the second most speciose eukaryotic kingdom (1, 2), is a seri-

ous limitation for multitaxon ecological and biodiversity studies.DNA barcoding uses standardized 500- to 800-bp sequences toidentify species of all eukaryotic kingdoms using primers that areapplicable for the broadest possible taxonomic group. Referencebarcodes must be derived from expertly identified vouchers de-posited in biological collections with online metadata and vali-dated by available online sequence chromatograms. Interspecificvariation should exceed intraspecific variation (the barcode gap),and barcoding is optimal when a sequence is constant and uniqueto one species (3, 4). Ideally, the barcode locus would be the samefor all kingdoms. A region of the mitochondrial gene encoding thecytochrome c oxidase subunit 1 (CO1) is the barcode for animals(3, 4) and the default marker adopted by the Consortium for theBarcode of Life for all groups of organisms, including fungi (5). InOomycota, part of the kingdom Stramenopila historically studiedby mycologists, the de facto barcode internal transcribed spacer(ITS) region is suitable for identification, but the default CO1marker 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 trnK gene. This system sets a precedent forreconsidering CO1 as the default fungal barcode.CO1 functions reasonably well as a barcode in some fungal

genera, such as Penicillium, with reliable primers and adequatespecies resolution (67% in this young lineage) (9); however,results in the few other groups examined experimentally are in-consistent, and cloning is often required (10). The degenerateprimers applicable to many Ascomycota (11) are difficult to as-sess, because amplification failures may not reflect primingmismatches. Extreme length variation occurs because of multipleintrons (9, 12–14), which are not consistently present in a species.Multiple copies of different lengths and variable sequences oc-cur, with identical sequences sometimes shared by several species(11). Some fungal clades, such as Neocallimastigomycota (anearly diverging lineage of obligately anaerobic, zoosporic gutfungi), lack mitochondria (15). Finally, because most fungi aremicroscopic and inconspicuous and many are unculturable, ro-bust, universal primers must be available to detect a truly rep-resentative profile. This availability seems impossible with CO1.The nuclear rRNA cistron has been used for fungal dia-

gnostics and phylogenetics for more than 20 y (16), and itscomponents 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 in-ternal transcribed spacers. These two spacers, including the 5.8Sgene, are usually referred to as the ITS region. The 18S nuclearribosomal small subunit rRNA gene (SSU) is commonly used inphylogenetics, and although its homolog (16S) is often used asa species diagnostic for bacteria (18), it has fewer hypervariable

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., andW.C. analyzed data; C.L.S.,K.A.S., and S.H. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in GenBank.Sequences are listed in Dataset S1.1To whom correspondence may be addressed. E-mail: schoch2@ncbi.nlm.nih.gov or Keith.Seifert@AGR.GC.CA.

2A complete list of the Fungal Barcoding Consortium can be found in the SI Appendix.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1117018109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1117018109 PNAS Early Edition | 1 of 6

MICRO

BIOLO

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http://fungalbarcoding.org

One published indoor microbiome

• Amend et al PNAS 2010 “Indoor fungal composition is geographically patterned and more diverse in temperate zones than in the tropics.”

• 72 samples of fungi from 6 continents. Sampled ITS2 region and the D1-D2 region of LSU with 454-FLX

• Main finding of increasing species diversity with increasing latitude

Fig 1. Amend et al 2010

PCA  of  normalized  counts  –  Painted  by  rRNA  type

ITS 28S

MG-­‐RAST  tools

PCA  of  normalized  counts  –  Painted  by  sampled  country

MG-­‐RAST  tools

PCA  of  normalized  counts  –  Painted  by  sampled  elevaCon

MG-­‐RAST  tools

Dilution to Extinction (d2e)

‘High throughput’ isolation from global dust samplesSarea resinae

Cryptocoryneum rilstonei

From barcodes to organisms

Keith Seifert

Summary

• New tool development for interacting with genome and metagenome data for Fungi

• FungiDB is a resource for genome investigations and repeatable queries and workflows

• Development of a centralized resource for ITS sequences will enable better analysis of amplicon metagenomics of Fungi

Acknowledgements

Stajich  lab  @UCR  labPeng  LiuBrad  CavinderSofia  RobbSteven  AhrendtDivya  Sain  Yizhou  WangYi  Zhou

FungiDB  ProgrammersDaniel  BorcherdingRaghu  RamamurthyEdward  LiawGreg  Gu

UndergraduatesJessica  De  AndaSapphire  EarLorena  RiveraCarlos  RojasErum  KhanRamy  WissaAnnie  Nguyen

Marine  Biological  Lab  -­‐  VAMPSMitch  SoginSue  HuseAnna  Shipunova

Univ  of  Colorado  at  Boulder  -­‐  QIIMERob  KnightScoW  BatesGail  AckermanJesse  Stombaugh

Argonne  NaConal  Lab  -­‐  MG-­‐RASTFolker  MeyerDaniel  BraithwaiteTravis  HarrisonKevin  KeeganAndreas  Wilke

EuPathDB @UPenn & UGADavid Roos, Jessica Kissinger, Chris StoeckertSteve Fischer - John BrestelliBrian Brunk - Debbie PinneyWei Li - Sufen Hu

Anthony AmendKeith Seifert