Towards better tools for fungal environmental metagenomics

Jason StajichPlant Pathology & Microbiology http://lab.stajich.org

http://fungalgenomes.orghttp://fungidb.org

twitter: hyphaltip, stajichlab, fungalgenomes

AcknowledgementsPeng  LiuBrad  CavinderSofia  RobbJinfeng  ChenAnastasia  Gio@

Steven  AhrendtDivya  Sain  Yizhou  WangYi  Zhou

Raghu  RamamurthyEdward  LiawGreg  GuDaniel  Borcherding

Sapphire  EarErum  Khan  Lorena  RiveraCarlos  RojasMegna  TiwariJessica  De  AndaAnnie  Nguyen  Ramy  Wissa

Univ  of  Colorado,  BoulderRob  KnightDaniel  McDonald

Noah  FiererSco0  BatesJon  Leff

Marine  Biological  LaboratoryMitch  SoginSue  Huse

Argonne  Na@onal  LabFolker  Meyer

Henrik  NilssonKeith  Seifert

IIGB Computational Core

Molecular Ecology of microbes

• What microbes live where?

• Using molecular techniques improve upon culture based methods reducing bias in just fast-growing and or culturable organisms.

• Many efforts to examine Bacteria and Archaeal diversity with sequencing developed important standards - e.g. Human Microbiome Project.

• Efforts towards improving methods of studying of fungi in the environment

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.

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F CALIFORNIA RIVERSIDE

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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

How many species of Fungi are there?

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.”

Microbial Ecology is not just outside

• Most humans spend majority of lives indoors

• What are the organisms that live in the built environment?

• Are there beneficial organisms that influence overlal composition of communities?

• How does the composition change when environmental conditions change (moisture, temperature, food sources)

Microbial ecology

Bik et al., 2012

Microbial Ecology in simple terms

• Collecting what’s there (sampling and PCR amplifying) [LAB]

• Put labels on things by matching to knowns (BLAST or other approach to see what matches in a database) [COMPUTER]

• See what is different (compare communities) [COMPUTER]

http://xkcd.com/1133/

Sampling and amplifying

• Total DNA extracted from a sample - soil, plant tissue, swab

• PCR with primers designed to amplify a conserved locus

• Sequencing with Sanger sequencing -> Next Generation Sequencing

Metagenomics - Amplicon

• Amplify a targeted locus for sequencing.

• Works best if there are universal primers which can amplify from all the species of interest

• For Bacteria most successful locus has been Ribosomal Small Subunit gene (16S)

• Primers that work well to amplify most groups of Bacteria and Archea

• Other loci are useful markers for sometimes better species resolution (phylogenetics) or community functional diversity by targeting a protein coding gene

Universal primersDevelopment one of first primer sets and

amplified regions of rRNA small subunit gene

Woese, Pace

Barcoding for multiplexing samples

http://www.hmpdacc.org/doc/HMP_MDG_454_16S_Protocol.pdf

Fungal Markers for molecular ecology• Needs to be universally amplifying across all groups

• Ribosomal rRNA (

• Small Subunit and Large Subunit genes

• Internal Transcribed Spacer 1 and 2

• Protein coding genes

• EF1alpha, RPB1, RPB2 (Fungal Tree of Life project)

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

White, Bruns, Lee, Taylor

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

White, Bruns, Lee, Taylor

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

Illumina HiSeq

320k curated sequences

2-3 Billion sequences per run (10-14 days)

Roche-4541M sequences per run

Illumina MiSeq3-5 M reads (1 day)

IonTorrent4-8 M reads (2hrs)

There’s a data storm coming

Fungal-specific Challenges

• Alignment of ITS

• Establishment of a reference tree

• Unalignable sequence into tree with LSU

• Naming and Curation of datasets

The problem with Alignment of ITS

ITS1 5.8S

ITS is most useful as a barcode sequenceNuclear 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

GY

Solutions

• ITS is hard to align across diverse taxa, but LSU is not.

• Marker with both sequences would be useful for both phylogenetic placement and barcoding.

• ITS + LSU amplicon proposed - primer testing with Illumina is under testing - a bit too large by current chemistry but could work in the near future

5.8S LSU

Putting a name on it

• Most sequences will not have identified names

• Grouping all observed sequences together to define OTU clusters even if no name can be assigned

• Curated ITS databases - UNITE project

• ~300,000 sequences in UNITE, ~200,000 which are full length (SSU + ITS + LSU)

• 50% are identified to a species level (18,000 distinct latin binomials)

UNITE project for

http://unite.ut.eeH. Nilsson

Soil Clone Group 1 - highly abundant, uncultured organism

Porter et al. 2008

Soil Clone Group 1 - highly abundant, uncultured organism

Porter et al. 2008

What’s in a name? Would a mold by any other name smell as sweet?

• “One fungus, one name” is eliminating dual nomeclature (naming of sexual and asexual forms separately)

• How to name species from molecular data alone?

• Name by close relatives on the tree?

• Use marker loci that contain both ITS and LSU to better place sequence in tree.

• Proposal to name species in Botanical code directly from sequence

• Good old fashioned microbiology

HIbbett and Taylor 2013

incomplete taxon sampling in reference sequence databases.

The perceived incompatibility of the code with sequence-based taxonomy is a consequence of the requirement for type specimens. However, the code places no restrictions on the form of type specimens, which need not be complete or representa-tive; all that is required of a type specimen is that it should be a physical specimen. In principle, an aliquot of DNA extracted from an environmental sample, or a por-tion of the substrate from which the DNA was isolated, can serve as a legitimate type specimen. To prove this point, Kirk et al.20 recently described a new species of rumen chytrid, Piromyces cryptodigmaticus, based on sequence data, and typified it with a sample from the fermenter from which the DNA was extracted. The new taxon name was validly published, even though the fungus was never directly observed. In the future, if purely sequence-based taxonomy is incorporated into the code, it may be pos-sible to forego the deposition of physical type materials altogether. In the meantime,

the publication of P. cryptodigmaticus pro-vides a model for environmental molecular biologists who would like to formalize their discoveries through code-compliant taxonomic names.

Errors and incomplete taxonomic sampling in sequence databases, such as GenBank, present a psychological barrier to naming environmental sequences; if an environmental sequence has no match in GenBank, it could still represent a described but unsequenced species. Faced with such uncertainty, fungal taxonomists might be reluctant to describe new species based on environmental sequences. They should not be; current estimates of the actual diversity in the kingdom Fungi range from as few as 500,000 species to millions of species2, suggesting that most unmatched environmental sequences probably do rep-resent new species5. Even if some environ-mental species prove to be redundant, taxonomists are accustomed to resolving synonymy based on the principle of priority. Finally, the solution to the GenBank prob-lem is conceptually straightforward — that

is, generate well-documented reference sequences21 — and is already being pur-sued through the fungal bar-coding initia-tive22 and the creation of custom-curated databases of well-documented reference sequences, such as the RefSeq collection within GenBank, and the UNITE database for mycorrhizal fungi23.

Lessons from prokaryotic taxonomyMany of the taxonomic challenges faced by mycologists parallel those faced by researchers studying prokaryotes, but the nomenclatural practices adopted by the two groups are often divergent. For exam-ple, the expanded power of the GC to rule on the legitimacy of choices among exist-ing names under the forthcoming ICN might worry some mycologists, who could fear a loss of taxonomic freedom, but the new system for fungi might seem familiar to prokaryote taxonomists, who have long used a Judicial Commission to accept or reject newly proposed names24,25. Another key difference between the nomenclatu-ral codes for prokaryotes26 and fungi3 is

Nature Reviews | Microbiology

Uncultured fungus clone unisequences#37-3808_2763 ITS2, PS

Uncultured Agaricomycotina clone 6_g19 18S rRNA gene

Uncultured fungus clone MOTU_4043_GVUGB5B04JK5N2 18S rRNA gene, PS, ITS2

Uncultured fungus clone MOTU_1778_GVUGB5B04IF01X 18S rRNA gene, PSUncultured fungus clone LT5P_EUKA_P5H04 18S rRNA gene, 18S–25/28S rRNA gene

Uncultured fungus clone F66N0BQ02H1NX5 18S rRNA geneUncultured fungus clone MOTU_43

Uncultured fungus clone unisequences#69-3466_2373 ITS2, PS Uncultured fungus clone MOTU_4349_GOKCVYYY06GR7WA 18S rRNA gene, PS, ITS2

Uncultured fungus clone unisequences #65-3574_00447, ITS2, PSTrichosporonales sp. LM559 18S rRNA gene

Uncultured Tremellales clone LTSP_EUKAUncultured fungus clone unisequence

Fungi 3 leavesUncultured fungus clone unisequence#65-3936_0554 ITS2, PS

Uncultured basidiomycete ITSUncultured fungus clone MOTU_601_GOK

Uncultured Tremellales clone LTSP_EUKA_P4L03 18S rRNA gene, PS, ITSUncultured fungus clone MOTU_141_GOKCVYYY06G5FYL 18S rRNA gene, PS, ITS

Fibulobasidium murrhardtense strain CB59109 18S rRNA gene Uncultured fungus clone MOTU_2930_GOKCVYYY06G7201 18S rRNA gene, PS, ITS

Uncultured fungus clone MOTU_2993_GOKCVYYY06HH12J 18S rRNA gene, PS, ITSUncultured fungus clone MOTU_1888_GVUGV5B04JJTLJ 18S rRNA gene

Uncultured fungus clone MOTU_3006_GVUGV5B04JIHT 18S rRNA geneUncultured fungus clone MOTU_2635_GVUGVSB04J56R4 18S rRNA gene, PS, ITS

gi|22497358|gb|FJ761130.1| uncultured fungus clone singleton_70-3063_2201 18S rRNA gene

Uncultured fungus clone singleton_70-3063_2201 18S rRNA gene, PS, ITSUncultured fungus clone OTU_403_GW5CJXV07IOX5A 18S rRNA gene

Uncultured fungus clone U_QM_090130_240_B_plate1a12.b1 18S rRNA gene, PS, ITS1

Uncultured fungus clone OTU_1445_1GW5CJXV07HXDTO 18S rRNA geneUncultured fungus clone U_QM_090130_127_1A_plate1g12.b1 18S rRNA gene, PS, ITS1

Uncultured fungus clone MOTU_3163_GYUGV5B0412KQP 18S rRNA gene, PS, ITS1Uncultured fungus clone MOTU_533_GOKCVYYY06GU3JA18S rRNA gene, PS, ITS1

Uncultured Tremellales clone 5_D20 18S rRNA, ITS1, 5.8S rRNA gene, ITS1Uncultured Rhodotorula IT51, 5.8S rRNA, ITS2 and partial 28S rRNA, clone MNIB2FAST_K1

Uncultured fungus clone MOTU_3797_GOKCVYYY06HBZ1X 18S rRNA gene, PS, ITS2

Uncultured fungus clone MOTU_2412

Figure 2 | Unnamed diversity. A demonstration of the problem posed by unnamed fungi that are known only from environmental DNA sequences. When a new environmental sequence (the bottom-most opera-tional taxonomic unit, gi|22497358; blue box) was used in a BLAST search of the GenBank database and the result displayed using the BLAST

distance tree tool, only two of the 35 most closely related sequences were from cultured organisms (green boxes), and only one was named (Fibulobasidium murrhardtense). Without names, the information content of this tree leaves much to be desired. ITS, internal transcribed spacer; PS, partial sequence.

PERSPECT IVES

NATURE REVIEWS | MICROBIOLOGY VOLUME 11 | FEBRUARY 2013 | 131

© 2013 Macmillan Publishers Limited. All rights reserved

Dilution to Extinction (d2e)

‘High throughput’ isolation from global dust samplesSarea resinae

Cryptocoryneum rilstonei

From barcodes to organisms - low throughput but effective

Keith Seifert

Community comparisons

• Pie charts of taxonomic differences varied across treatments

• 16S Community composition varies with smoking and COPD status

Erb-Downward et al 2011.

Comparing communities

• Taxonomic diversity varies across ant worker type and time of year

Bik et al., 2012

Workflows for data processing

Tools - QIIME: Quantitative Insight Into Molecular Ecology

• For amplicon based datasets (16s, 18s, ITS)

• Alpha diversity - phylogenetic diversity, Chao, number of observed species

• Generate species diversity plots to assess community diversity

• Beta diversity - Unifrac distance, Bray-Curtis, Jaccard

• Need reference phylogenetic tree to compute these, unavailable

• Support for shotgun metagenomics

Approaches to clustering sequences

• De novo clustering

• Requires all-vs-all searches, very expensive

• Known Knows - “Closed reference”

• Match sequences to a database of representative known sequences

• Fast, but throw out unknowns

• Known Knowns and Known Unknowns - “Open reference”

• Match to known set and de novo cluster the remainder

QIIME on fungal data

• New (Dec 2012) Fungal ITS reference database from UNITE incorporated as QIIME resource

• Can use it to match against known set (closed-reference) or match and cluster unknowns (open reference)

• One dataset of Indoor dust samples from Kerry Kinney (UT Austin) group

• A second indoor sampled (Amend et al)

QIIME taxonomic distribution for samplesGreg Gu

A previously published indoor mycobiome

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

• Sequencing dust from houses and office buildings

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

• A primary finding was increasing species diversity with increasing latitude

Fig 1. Amend et al 2010

MG-RAST with Fungal Data

MG-RAST summary statistics

Hits summarized by different taxonomic levels

Rarefaction curve (1 sample)

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  eleva@on

MG-‐RAST  tools

Metagenomics - shotgun approach

• For non-amplicon based studies of community composition

• Will be the future approaches for community studies with the increased sequencing depth

• Metatranscriptomics for studying what is expressed

• Support in QIIME and MG-RAST for the studies, but limited by the diversity of genome/protein sequences which can be matched.

Increasing fungal genome diversity

http://1000.fungalgenomes.org

Fungal genome sequencing

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

400+ genomes of Fungi

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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

Community can propose new genomes

Summary

• Fungal microbial ecology is embracing highthroughput sequencing technologies for community studies

• Limitations due to lack of curated sequences and the properties of the marker loci used

• Building new databases and tools to help with the analyses will improve utility

• Improvements in sequencing chemistry (read length x depth) make this a moving target for establishing the best practices

• Deeper studies will improve our understanding of the fungal diversity and role of fungi in different ecosystems - 1000 genomes project can help provide anchor representatives of this diversity.