Ascomycetes associated with ectomycorrhizas:molecular diversity and ecology with particularreference to the Helotialesemi_2020 3166..3178
Leho Tedersoo,1,2* Kadri Pärtel,1 Teele Jairus,1,2
Genevieve Gates,3 Kadri Põldmaa1,2 andHeidi Tamm1
1Department of Botany, Institute of Ecology and EarthSciences, University of Tartu, 40 Lai Street, 51005Tartu, Estonia.2Natural History Museum of Tartu University, 46Vanemuise Street, 51005 Tartu, Estonia.3Schools of Agricultural Science and Plant Science,University of Tasmania, Hobart, Tasmania 7001,Australia.
Summary
Mycorrhizosphere microbes enhance functioning ofthe plant–soil interface, but little is known of theirecology. This study aims to characterize the asco-mycete communities associated with ectomycorrhi-zas in two Tasmanian wet sclerophyll forests. Wehypothesize that both the phyto- and mycobiont,mantle type, soil microbiotope and geographical dis-tance affect the diversity and occurrence of the asso-ciated ascomycetes. Using the culture-independentrDNA sequence analysis, we demonstrate a highdiversity of these fungi on different hosts andhabitats. Plant host has the strongest effect on theoccurrence of the dominant species and communitycomposition of ectomycorrhiza-associated fungi.Root endophytes, soil saprobes, myco-, phyto- andentomopathogens contribute to the ectomycorrhiza-associated ascomycete community. Taxonomicallythese Ascomycota mostly belong to the orders Helo-tiales, Hypocreales, Chaetothyriales and Sordariales.Members of Helotiales from both Tasmania and theNorthern Hemisphere are phylogenetically closelyrelated to root endophytes and ericoid mycorrhizalfungi, suggesting their strong ecological and evolu-tionary links. Ectomycorrhizal mycobionts from Aus-tralia and the Northern Hemisphere are taxonomicallyunrelated to each other and phylogenetically distant
to other helotialean root-associated fungi, indicatingindependent evolution. The ubiquity and diversity ofthe secondary root-associated fungi should be con-sidered in studies of mycorrhizal communities toavoid overestimating the richness of true symbionts.
Introduction
Endophytic and mycorrhizosphere microbes, especiallyBacteria, Archaea and microfungi, synthesize plantgrowth regulators and vitamins facilitating the develop-ment and functioning of the mycorrhizal system in soil(Schulz et al., 2006). These root-associated microbessuch as mycorrhiza helper bacteria and the nitrogen-fixing actinobacteria and rhizobia differ substantially intheir function and ecology, including host preference pat-terns (Benson and Clawson, 2000; Sprent and James,2007; Burke et al., 2008). Of microfungi, foliar endo-phytes may considerably vary according to the special-ization to different host species and even organs(Neubert et al., 2006; Arnold, 2007; Higgins et al., 2007).On the contrary, facultative root-associating fungi suchas endophytes (e.g. the Phialocephala–Acephala andMeliniomyces–Rhizoscyphus complexes) form mostlynon-specific associations with many plant hosts (Vrålstadet al., 2002; Chambers et al., 2008), although host pref-erence may occur on the cryptic species level (Grüniget al., 2008).
Despite numerous studies on isolation and morphol-ogical identification of ascomycetous microfungi fromectomycorrhizal (EcM) root tips, their specificity for hostplants, fungi and substrate types remains unknown(Melin, 1923; Fontana and Luppi, 1966; Summerbell,1989; Girlanda and Luppi-Mosca, 1995). Molecular toolshave only recently been used to characterize and distin-guish the secondarily associated microfungi from EcMfungi in situ. These microfungi were identified from EcMroot tips by either cutting additional bands from the gel(Rosling et al., 2003; Tedersoo et al., 2006), using specificprimers (Urban et al., 2008) or cloning (Morris et al.,2008a,b; 2009; Wright et al., 2009). Cloning from DNAextracts comprising pooled individual EcM root tipsreveals many ascomycete taxa of uncertain ecologicalrole (Bergemann and Garbelotto, 2006; Smith et al.,
Received 9 April, 2009; accepted 22 June, 2009. *For correspon-dence. E-mail [email protected]; Tel. (+372) 7376222; Fax(+372) 7376222.
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2007). Many of these are endophytic or rhizoplane colo-nists that are accidentally reported as forming mycorrhi-zas both in research publications and InternationalSequence Database (INSD) entries (Grünig et al., 2008).Such uncertain reports are especially common in ericoidmycorrhizas (ErM) and EcM for which universal fungal-specific primers are routinely used for the identification ofmycobionts.
Helotiales (Ascomycota) comprises the largest numberof undescribed root-associated fungi in addition toapproximately 2000 described species with contrastinglifestyles (Wang et al., 2006). Various subgroups ofHelotiales such as the Phialocephala–Acephala andRhizoscyphus–Meliniomyces complexes and Lachnumspp. are identified from EcM and arbutoid mycorrhiza inforest trees and subshrubs of the Northern Hemisphere(Vrålstad et al., 2002; Rosling et al., 2003; Tedersoo et al.,2003; 2007; 2008a; Bergemann and Garbelotto, 2006)and often erroneously reported as truly mycorrhizal.Indeed, the ecologically heterogeneous Rhizoscyphus–Meliniomyces and Phialocephala–Acephala complexesboth include distinct EcM-forming species nested withinnumerous pathogenic, ErM and root endophytic taxa(Vrålstad et al., 2002; Hambleton and Sigler, 2005;Münzenberger et al., 2009).
In previous EcM fungal community studies in Tasmania,we encountered frequent secondary colonization of EcMroot tips by Ascomycota besides the predominatelybasidiomycetous EcM fungi (Tedersoo et al., 2008b;2009). The present study was undertaken to identify anddistinguish these EcM-associated Ascomycota (EAA)from the true EcM-forming mycobionts using a culturing-independent approach. Utilizing the DNA extracts fromsingle EcM root tips with preidentified plant and EcMfungal hosts and developing several ascomycete-specificprimers, we hypothesized that these EAA have prefer-ence for either host tree, host fungus lineage, EcM mantletype, soil microbiotope, plot and site. Because of thedominance of Helotiales in this and previous studiesinvolving root-associated fungi, we addressed the phylo-genetic relations of helotialean EcM, ericoid mycorrhizal,endophytic and EAA isolates using the rDNA 28Ssequence data.
Results
Identification and distribution of EAA
Application of the newly designed taxon-specific primers(Fig. 1; Appendix 1) allowed us to specifically amplifyAscomycota from EcM root tips. Based on the rDNA ITSsequence analysis, 251 individuals of EAA were identifiedfrom 226 out of 675 (33.5%) analysed root tips (148individuals from the Mt. Field site and 103 from the Warrasite). 88.9% of the EcM root tips yielded a single ampliconof EAA. Based on the 99% ITS barcoding threshold, EAAwere assigned to 105 species, including 69 (65.7%)singletons and 15 (14.3%) doubletons (Appendix 2).
At both sites, species of EcM fungi and EAA wereaccumulating at similar rates with increasing samplingeffort (Fig. 2). The species accumulation curves hadstrongly overlapping confidence intervals (not shown)suggesting no substantial difference in EAA diversityamong mantle types, sites, plots, microsites or plant andfungal hosts. Similarly, there were no statistically signifi-cant differences in the relative frequency of colonization ofEAA among these habitats.
Trends in the distribution of eight most frequent EAAspecies were statistically analysed at both two sites(Table 1). Five of these species differed significantlyaccording to the site. Only Lecanicillium flavidum (syn.Verticillium fungicola var. flavidum) displayed a statisti-cally significant preference for EcM fungal lineage (Fish-er’s exact test: d.f. = 4; P = 0.002). This species occurredmore frequently on root tips colonized by members ofthe/cortinarius lineage, compared with the other four mostcommon EcM lineages. Due to elevated abundanceon the/cortinarius EcM, L. flavidum was more commonon plectenchymatous mantles than expected (d.f. = 1;P = 0.004). In contrast, Helotiales sp016 colonized exclu-sively mycorrhizas with pseudoparenchymatous mantles(d.f. = 1; P = 0.001).
Among the seven most common EAA species at Mt.Field, host trees and plots affected the distribution of sixand two species respectively (Table 1). Putative rootendophytes, root parasites and mycoparasites includedspecies with significant host plant preference. Forexample, Helotiales sp008 (putative endophyte, d.f. = 2;
Fig. 1. Map of primers used for amplification of the ITS and 28S rDNA. Newly designed primers are given in bold.
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P = 0.001) and L. flavidum (mycoparasite; d.f. = 2;P = 0.043) occurred significantly more frequently on EcMof Eucalyptus regnans, while Hypocreales sp086 (rootparasite Neonectria cf. radicicola; d.f. = 2; P = 0.018)
preferred Pomaderris apetala compared with the othertwo hosts (Nothofagus cunninghamii is the third host). Atthe Warra site, only L. flavidum was significantly morecommon in the forest floor soil compared with decayedwood (d.f. = 1; P = 0.027).
At Mt. Field and Warra, respectively, the multivariatemodel explained 19.3% and 17.6% of the total variation inthe distribution of EAA. At both sites, the fungal lineageand mantle anatomy explained < 2% of the total variationthat remained non-significant. At Mt. Field, host plant andplot contributed 7.2% (SS = 141.2; P = 0.001) and 3.0%(SS = 59.0; P = 0.003) respectively. At Warra, substratetype contributed 3.5% to the total variation (SS = 34.7;P = 0.008).
Phylogenetic affinities of Tasmanian EAA
Based on blastN matches, the Tasmanian EAA belongedto 12 orders of Pezizomycotina. Helotiales, Hypocreales,Chaetothyriales and Sordariales comprised 54, 21, 9 and8 species respectively. Many of the hypocrealean taxawere assigned to parasitic lifestyle, including six putativemycoparasites (L. flavidum, Hypomyces spp.), four rootparasites (e.g. Neonectria radiciicola, Cylindrocarpon sp.)and two insect parasites (Cordyceps spp.). Sordarialesand Chaetothyriales, respectively, comprised mostlysaprotrophic and putatively endophytic members.
Helotiales comprised most of the dominant species thatwere assigned to the endophytic lifestyle based on ITSmatches (Appendix 2) and phylogenetic analysis (Fig. 3).The 30 species of Tasmanian EAA with available 28Ssequence formed 10 distinct, more or less supported lin-eages, including both monospecific branches and aggre-gates comprising up to 17 species (the Hyphodiscuscomplex) (Fig. 3). Helotialean EAA and root endophytesfrom Tasmania and the Northern Hemisphere were
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Fig. 2. Species accumulation curve of ectomycorrhizal (opensymbols) and ectomycorrhiza-associated ascomycetes (closedsymbols) at the Mt. Field (triangles) and Warra (circles) sites. Forclarity, the overlapping confidence intervals are not shown.
Table 1. Statistical significance of host plant, microbiotope, site and mantle type on the occurrence of eight most common species ofectomycorrhiza-associated ascomycetes. Significant P-values are indicated in bold.
Species Putative ecology
P-values of a Fisher’s exact test
Mt. Field Warra Both sitesa
Host plant(d.f. = 2)
Plot(d.f. = 2)
Micro-biotope(d.f. = 1)
Mantle type(d.f. = 1)
Site(d.f. = 1)
Helotiales sp007 Endophyte 0.018 0.091 na 0.401 < 0.001Helotiales sp008 Endophyte 0.001 0.007 na 0.241 0.342Helotiales sp013 Endophyte 0.554 0.193 na 0.540 0.004Helotiales sp016 Endophyte < 0.001 0.012 na 0.001 < 0.001Helotiales sp029 Endophyte na na 0.059 0.733 < 0.001Helotiales sp038 Endophyte 0.007 0.556 0.077 0.782 0.010Hypocreales sp086 Root parasite 0.018 0.279 na 0.542 0.635Lecanicillium flavidum Mycoparasite 0.043 0.125 0.027 0.003 0.854
a. Based on G-tests.Molecular identification of these species is shown in Appendix 2.na, not applied because of insufficient replication.
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Fig. 3. Maximum-likelihood phylogram of Tasmanian ectomycorrhiza-associated ascomycetes (in bold) among identified taxa and otherplant-associated fungi within Helotiales. Fast bootstrap values > 70 and Bayesian posterior probabilities >95% are indicated below and abovethe branches respectively. Asterisks denote confirmed and putative EcM isolates, although it is possible that other EcM Helotiales exist.
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phylogenetically closely related to ErM and saprobic iso-lates in the Hyphodiscus, Cryptosporiopsis–Neofabraeaand Rhizoscyphus–Meliniomyces complexes (Fig. 3,Appendix 2). Similarly, the majority of EAA sequencesfrom the Pinaceae and Fagaceae hosts in the NorthernHemisphere usually clustered with the well-recognizedendophytic and/or ErM lineages, whereas a few groupsformed monotypic lineages with yet unknown ecology(e.g. isolates AY394891, DQ273463, DQ273467 andEU563495).
The Tasmanian helotialean EcM species that have beenconfirmed by mycorrhiza anatomy and consistent molecu-lar identification (cf. Tedersoo et al., 2008b, 2009; L. Ted-ersoo, unpublished) were clustered in four distinctlineages (Fig. 3). These Tasmanian EcM lineages wereclearly distinguished from EAA, endophytes, ErM isolatesand the two EcM lineages distributed in the NorthernHemisphere (/meliniomyces and an unnamed lineage)based on both ITS (Appendix 2) and 28S (Fig. 3)sequence data.
Discussion
Diversity of EAA
Ascomycota comprises common and taxonomicallydiverse secondary colonists on EcM root tips. Our resultscorroborate previous reports of high local diversity of rootand foliar endophytes in various ecosystems (Vandenk-oornhuyse et al., 2002; Sieber and Grünig, 2006; Arnold,2007; Higgins et al., 2007). We detected no statisticaldifference in the diversity of EAA among the host plantsand fungi, microbiotope, plot and site, indicating thatnone of these substantially affect the naturally high EAAdiversity.
Among the factors investigated, host plant had thestrongest effect on the frequency of individual EAAspecies. This agrees with studies on foliar endophytes(Arnold and Lutzoni, 2007), but contrasts with previousresearch on root endophytes that detected no significantdifferentiation according to the host plant (Narisawa et al.,2002; Sieber and Grünig, 2006; Chambers et al., 2008;but see Grünig et al., 2008). However, methodologicaldifferences such as choice of a barcoding threshold andinclusion of the culturing step may account for the discrep-ancies. We speculate that host generalists, e.g. mostmembers of the Trichoderma, Phialocephala–Acephalaand Rhizoscyphus–Meliniomyces complexes, may befavoured by culturing due to their relatively rapid mycelialgrowth and non-specialized ecology. Similarly, lower DNAbarcoding thresholds may result in lumping of closelyrelated taxa that are often ecologically differentiated(Summerell and Leslie, 2004; Sharon et al., 2006; Grüniget al., 2008). Host preference among EAA (this study),
foliar endophytes (Arnold, 2007), EcM symbionts (Molinaet al., 1992; Tedersoo et al., 2008b) and arbuscularmycorrhizal symbionts (Vandenkoornhuyse et al., 2003)suggest that plant diversity, through the niche comple-mentarity effect, may promote the diversity of both com-mensal and mutualistic fungi above and below ground.The relatively stronger effect of plant host compared withplot, site and microbiotope effects on EAA species andcommunities suggests that interspecific differences inphytochemistry play a more important role in structuringthe distribution of EAA compared with qualitative differ-ences in the soil matrix and geographical distance.
Based on INSD search and phylogenetic analysis, mostof the EAA represented root endophytes, with a minorityhaving strongest affinities to plant parasites, mycopara-sites, insect parasites and soil saprobes. While the lattermay be rhizoplane fungi, root endophytes and plant para-sites are the expected root colonists (Vandenkoornhuyseet al., 2002; Neubert et al., 2006). Their ubiquitous asso-ciation with ectomycorrhizas and the negligible effect offungal host indicates that the fungal mantle is not neces-sarily an effective barrier to the colonization of endophyticand potentially parasitic fungi. Moreover, endophytic fungioften proliferate in the fungal mantle, developing hyphaewith hyaline or melanized cell walls (L. Tedersoo, pers.obs.).
Our results suggest that EcM root tips provide a habitatfor mycoparasitic fungi that normally infect fruit-bodiesabove ground. This study confirms previous reports onidentification of the Lecanicillium fungicola complex fromEcM root tips and associated soil (Summerbell, 1989) andsuggests that some mycoparasites (such as L. flavidum)may be relatively frequent in ectomycorrhizas. Theobserved preference of L. flavidum for cortinarius EcM hasnot been detected in case of the more general fungalfruit-body parasitism by L. flavidum or the closely relatedL. fungicola (Zare and Gams, 2008; K. Põldmaa, pers.obs.). The rarity of other mycoparasites among the EAAmay be ascribed to their host specificity (Põldmaa, 2000) orscarcity of their below ground associations. However,given the strongly seasonal production of suitable fruit-bodies, it is not surprising to find mycoparasites on EcMthat are active throughout the year. The ecology of EAA onEcM root tips, particularly the potential to spread along withthe EcM hyphae and rhizomorphs that give rise to fruit-body primordia, warrants further investigation. Similarly,Arnold (2008) noted the presence of certain entomopatho-gens among foliar endophytes and suggested that their lifecycle may include both insect and plant hosts.
In the perspective of EcM communities, the presence ofEAA hamper molecular identification and interpretation ofresults. Even small supplement of EAA DNA may becoamplified, resulting in double signal in sequence chro-matograms or, worse, the preferential amplification of
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EAA (Rosling et al., 2003). To be able to separate thetargeted EcM fungi from these ‘contaminant’ secondarycolonizers, both morphological and molecular aspects ofEcM fungal communities need to be addressed. This cau-tions against pooling large amounts of roots prior to DNAextraction that disables post hoc morphological confirma-tion of the molecularly identified fungi (Bergemannand Garbelotto, 2006). We predict that ongoing high-throughput sequencing studies addressing mutualisticEcM fungi and soil eukaryotes will create thousands ofsequences from putative root endophytes, whose identityand actual ecology can easily be misinterpreted (Nilssonet al., 2009).
Phylogenetic affinities of EAA
Most of the EAA in the two Tasmanian sites belong to theHelotiales. This agrees well with previous sporadic reportsof EAA in EcM fungal communities (Bergemann and Gar-belotto, 2006; Smith et al., 2007; Morris et al., 2008a,b;2009; Urban et al., 2008; Wright et al., 2009) as well asthe more focused studies on root endophytes (Vandenk-oornhuyse et al., 2002; Vrålstad et al., 2002) and ErMfungi (Allen et al., 2003; Bergero et al., 2003) in theNorthern Hemisphere. Similarly, the Cladophialophora–Exophiala–Capronia group of Chaetothyriales is acommon facultative root endophytic taxon, althoughmembers of this group have been found from a variety ofsubstrates (Narisawa et al., 2002; 2007). The Hypocre-ales and Sordariales, represented here by parasitic orsaprobic members, are detected as infrequent root colo-nizers in studies on root endophytic fungi. These twoorders, as well as Dothideomycetes, dominate amongfoliar endophytes in angiosperms (Arnold, 2007; Higginset al., 2007).
We paid particular attention to phylotype distributionwithin Helotiales, because this was the most importantorder in terms of EAA frequency and diversity, andalso comprised EcM-forming fungi. The Tasmanian EAAspecies formed several mono- or multispecific lineagesthroughout the Helotiales, particularly clustering withthe Hyphodiscus, Oidiodendron and Cryptosporiopsis–Neofabraea complexes from the Northern Hemisphere.Except for the Cryptosporiopsis–Neofabraea clade, thesetaxa are not included in the 18S + 28S rDNA phylogeniesof Wang and colleagues (2006) and therefore, their phy-logenetic position within the Helotiales remains obscure.
Both the phylogenetic analysis and INSD searchessuggest substantial taxonomic overlap among Tasmanianand boreal EAA, root endophytes and ErM fungi. Previousstudies have suggested that several ErM fungi and rootendophytes may be conspecific based on identical culturemorphology (McNabb, 1961), synthesis trials (Bergeroet al., 2000) and molecular identification (Bergero et al.,
2000; Piercey et al., 2002). Moreover, many of these fungiare common saprobes in soil and peat (Piercey et al.,2002). Enzymatic tests have revealed relatively highlevels of cellulolytic activities in ErM and endophytic fungicompared with EcM symbionts (Mandyam and Jump-ponen, 2005). These elevated enzymatic activities maycontribute to the improved nutrition of ericoid plants inhighly organic, nutrient-poor soils (Read et al., 2004).Among hyperdiverse soil saprobes, members of the Cha-etothyriales and Helotiales in particular display frequentendophytic colonization. We suggest that Ericalesevolved capacities to host these endophytes in individualroot cells and stimulated the formation of coils forimproved nutrient exchange, thus giving rise to the ericoidmycorrhiza. ErM fungi and root endophytes largelyoverlap in many groups within Helotiales (Bergero et al.,2000; Chambers et al., 2008), Chaetothyriales (Usuki andNarisawa, 2005) and Sebacinales (Selosse et al., 2007).
Species of confirmed helotialean EcM fungi from Aus-tralia and Europe formed four and two distinct lineagesrespectively. There were no close relationships betweenEcM Helotiales from Australia and the Northern Hemi-sphere, suggesting that in both regions, EcM lifestylemay have evolved multiple times independently in thistaxon. Except for the /meliniomyces and /acephalamacrosclerotiorum lineages (Hambleton and Sigler, 2005;Münzenberger et al., 2009), EcM fungal taxa have noclear closely related ErM or root endophytic sister groups,suggesting different origin of EcM and other rootbiotrophic lifestyles. Therefore, taxonomic breadth of thepostulated common guild between EcM and ErM myco-bionts (Vrålstad et al., 2002; Bougoure et al., 2007) aswell as their evolutionary and ecological differencesrequire further clarification.
In conclusion, ascomycetes associated with ectomyc-orrhizas are highly diverse and comprise root endophytes,saprotrophs, myco-, phyto- and entomopathogens. Thedistribution of these microfungi is influenced by plant hostrather than EcM fungi, substrate type or geographicalvariables. Within Helotiales, EAA and the putative ecto-mycorrhizal symbionts are distantly related and probablyevolved multiple times independently in the Northernand Southern Hemisphere. In studies of mycorrhizalcommunities, the ubiquity and diversity of secondaryroot-associated fungi should be considered to avoidoverestimating the diversity of the true mycorrhizalsymbionts.
Experimental procedures
Sample preparation
Root sampling was performed in two Tasmanian wet sclero-phyll forest sites, Mt. Field (42°41′-S, 146°42′-E) and Warra(43°04′-S; 146°40′-E) as described in detail in Tedersoo and
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colleagues (2008b; 2009). Briefly, EcM root tips of matureN. cunninghamii (Hook.) Oerst., E. regnans F. Muell. andP. apetala Labill. were sampled in forest floor soil from 45 soilcores (15 cm¥ 15 cm to 5 cm depth) in three 1 ha plots at Mt.Field. At Warra, root tips of only N. cunninghamii weresampled from 42 cores in decayed wood and 22 cores inforest floor soil. EcM root tips were sorted into morphotypesand anatomotypes using a stereomicroscope. Particular carewas taken to characterize and record the anatomy of EcMformed by Ascomycota (L. Tedersoo, unpublished). SingleEcM root tips, one to four from each anatomotype per soilcore, were carefully cleaned from the adhering soil and debrisand subjected to DNA extraction, PCR amplification andsequencing as described in Tedersoo and colleagues(2008b). The EcM symbionts were identified based on bar-coding of the rDNA Internal Transcribed Spacer (ITS) region.Roots from different host trees were initially distinguishedbased on morphological characters such as colour, branchingand thickness, and confirmed based on the length polymor-phism of the plastid trnL region (Tedersoo et al., 2008b).
From the DNA extracts of single root tips that were suc-cessfully ascribed to EcM plant and fungal species (Tedersooet al., 2008b; 2009), we targeted the associated Ascomycotausing the combination of a fungal-specific primer ITSO-FT(5′-acttggtcatttagaggaagt-3′) and the Ascomycota-specificLA-W (5′-cttttcatctttcgatcactc-3′) (Fig. 1). The Ascomycotawere specifically addressed, because a vast majority of soilfungi and endophytes belong to this phylum (Vandenkoorn-huyse et al., 2002; O’Brien et al., 2005; Arnold, 2007). We areaware that certain Basidiomycota such as Ceratobasidiumand Cryptococcus, and Zygomycota may also contribute tothe root endophytic fungal community especially in theculture-based studies, but form a minor component(Summerbell, 1989; Hoff et al., 2004; Neubert et al., 2006).Because most EcM root tips were associated with a single orno species of EAA (based on preliminary PCR surveys), onlydirect sequencing of the PCR products was performed,neglecting the cloning step. When several fungi were presenton root tips as revealed from double DNA bands on 1%agarose gels, the DNA fragments were cut from the geland re-amplified using the internal primers ITS5 (5′-ggaagtaaaagtcgtaacaagg-3′) or ITS1 (5′-tccgtaggtgaacctgcgg-3′)and ITS4 (5′-tcctccgcttattgatatgc-3′). Because the resultsrevealed dominance of Helotiales and Sordariomycetes, wefurther designed taxon-specific reverse primers, ITS4-Sord(5′-cccgttccagggaatct-3′), LR6-Sord (5′-gtttgagaatggatgaaggc-3′) and LR6-LS (5′-aaaatggcccactagtgttg-3′) in the 28SrDNA to specifically target these taxa. To address phyloge-netic relationships among the species of Helotiales, weamplified the 28S rDNA using a fungal specific primerLR0R (5′-acccgctgaacttaagc-3′) in combination with eitherof the newly developed Pezizomycotina-specific primerLR3-Asc (5′-cacytactcaaatccwagcg-3′) or Leotio- andSordariomycetes-specific LR6-LS (Fig. 1; Appendix 1). TheITS region of EAA was sequenced using the primers ITS4,ITS1, ITS5 and/or LF340 (5′-tacttgtkcgctatcgg-3′); 28S rDNAwas sequenced using the primers ctb6 (5′-gcatatcaataagcggagg-3′), TW13 (5′-ggtccgtgtttcaagacg-3′) and/or LR5(5′-tcctgagggaaacttcg-3′). Sequences were further trimmed,assembled and edited using Sequencher 4.7 software(Genecodes Corp., Ann Arbor, MI, USA). Based on the clus-
tering of ITS sequences belonging to the Helotiales in thepresent study and in previous research (Hambleton andSigler, 2005; Grünig et al., 2009), 99.0% was selected as auniversal barcoding threshold to distinguish between putativespecies. All unique ITS and partial 28S rDNA sequences aredeposited both in INSD (Accession Numbers FN298677–FN298803) and UNITE (UDB004100–UDB004230) publicdatabases.
Thirty 28S rDNA sequences (typically 600–900 bp span-ning divergent domains D1–D2 or D1–D3) of helotialean EAAfrom Tasmania were automatically aligned with publishedendophyte, ErM, EcM and preidentified fruit-body sequences(retrieved from INSD and UNITE public databases) usingMAFFT 5.861 (Katoh et al., 2005). Obvious alignment errorswere checked and edited manually. The final data set com-prised 167 taxa and 1335 characters. Using the onlineversion of RAxML 7.0.4 (Stamatakis et al., 2008), a maximumlikelihood phylogram with 100 fast bootstrap replicates wasconstructed following the GTR + G + P base substitutionmodel. Posterior probabilities were estimated with MrBayes3.1.2 (Ronquist and Huelsenbeck, 2003) using the samemodel (parameters: lset nst = 6, rates = invgamma). Two par-allel MCMC analyses were performed, both initiated withrandom starting trees and run for 10 000 000 generations.Every 100th generation was sampled. The first 10 000 treeswere discarded as burn-in. Posterior probabilities werecalculated from the remaining 90 000 trees sampled from9 000 000 generations.
Statistical analyses
G-tests were applied to detect statistically significant differ-ences in the occurrence of EAA among host fungal lineages,host plant species, mantle types, sites, plots and micro-biotopes. The effects of host fungus, mantle type and site onthe frequency of the eight most common EAA were furtherstudied based on the pooled sites and occurrence of EAAindividuals in each class, using Fisher’s exact tests. Morespecifically, the effects of plant host and plot were addressedat the Mt. Field site and microbiotope at the Warra site,following the same procedure. To study the differences inaccumulating species richness among each of the factors, wecalculated individual-based rarefaction curves with 95% con-fidence intervals using a computer program EstimateS 8.0(Colwell, 2006). Accumulating species richness of EAA wascompared with that of EcM fungal species identified from thesame EcM root tips.
Using a computer program DISTLM forward 1.3 (McArdleand Anderson, 2001), we studied the effect of fungal hostlineage, microsite and mantle anatomy on EAA communitycomposition of N. cunninghamii EcM at the Warra site. Inanother analysis, we addressed the effects of fungal hostlineage, plant host species, plot and mantle anatomy on EAAcommunity structure at the Mt. Field site. In both multivariateanalyses, occurrence of an EAA formed a sampling unit (i.e.individual). Singletons were removed from the analyses.Thus, the Mt. Field and Warra data sets comprised 22 and 17species as well as 112 and 65 individuals respectively. EcMfungal lineages with at least three occurrences were includedas dummy variables. The occurrence of individuals was
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standardized by sums of variables (EAA species). Chi-squaredistance was used as a distance metric with 999 permuta-tions. Significance level a = 0.05 was used in all statisticalanalyses.
Acknowledgements
We thank G. Kantvilas, D. Ratkowsky, N. Ruut and D.Puskaric for support in Tasmania; H.-O. Baral for voucherspecimens; D. Ratkowsky for helpful suggestions on anearlier draft of the manuscript. This study was funded byEstonian Science Foundation Grants no. 6606, 6939, 7434and JD92, Doctoral School of Environmental Sciences, Krist-jan Jaak scholarship and FIBIR/rloomtipp. G.G. is supportedby Forestry Tasmania, Holsworth Wildlife Research Endow-ment Fund, CRC for Forestry and Bushfire.
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Appendix 1
Characterization of the primers (in bold) designed for the identification of Ascomycota in this study. Multiple sequence alignmentswith target and non-target taxa are indicated.
ITS4-Sord (calculated TM = 56°C); specific to SordariomycetesPrimer 5′-CCCGTTCCAGGGAACTC-3′Sordariomycetes (45) *****************Ascomycota other (101) **Y******A**R***TBasidiomycota (95) *********A*AR***TPlants (6) ***C********G***TLA-W (calculated TM = 58°C); specific to AscomycotaPrimer 5′-CTTTTCATCTTTCGATCACTC-3′Ascomycota (incl. SCGI*) (98) *********************
Pachyella (2) ****************T****Heterobasidiomycetes, Sebacinales (92) *************CC****GG
Cantharellus cibarius (1) *************CC*TG*GGSistotrema confluens (1) *************CC**G*GGTulasnella (2) ******C******CC****GG
Plants (6) *************CC**G*GGLR3-Asc (calculated TM = 57°C); specific to PezizomycotinaPrimer 5′-CACYTACTCAAATCCAAGCG-3′Ascomycota [75; incl. Pezizales (26)] ***Y****************
Pezizales (18) ***Y***********T**A*Orbiliales (1) *T*A************TC**SCGI* (2) *T*T******C****TTCG*
Basidiomycota (62) ***TAC*K*NG*WS*G**RYPinaceae (2) ***GC****G**C**T**GCAngiosperms (4) ***W*****G*****T**TCNOTE: This primer is not recommended for further useLR6-Sord (calculated TM = 58°C); specific to SordariomycetesPrimer 5′-GTTTGAGAATGGATGAAGGC-3′Sordariomycetes (40) ********************Ascomycota (other) (60) **********A*G*T****WBasidiomycota (70) **********A*G*T****WPlants (6) **********A*G*CG***GLR6-LS (calculated TM = 58°C); specific to Leotio- and Sordariomycetes.Primer 5′-AAAATGGCCCACTAGTGTTG-3′Sordariomycetes, Leotiomycetes (60) ********************Ascomycota (other) (40) ****************AAC*Basidiomycota (70) ***************A**CTPlants (6) *************T*G*G**LR6-Asc (calculated TM = 58°C); specific to Ascomycota; not experimentally testedPrimer component #1 5′-AAAATGGCCCACTAGTAACG-3′Primer component #2 +AAAATGGCCCACTAGTGTTGSordariomycetes, Leotiomycetes (60) ****************GTT*Ascomycota (other) (40) ****************AAC*Basidiomycota (70) ***************A**CTPlants (6) *************T*G*G**
*CSGI, Soil Clone Group I of the basal Ascomycota (cf. Porter et al., 2008).
Ectomycorrhiza-associated ascomycetes 3175
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Ap
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L354
8U
DB
0041
31E
ndop
hyte
plan
tlit
ter
asso
ciat
edfu
ngus
DQ
9147
30S
piro
spha
era
beve
rwijk
iana
EF
0292
0195
.088
.41
00
01
Hel
otia
les
sp03
5L3
287
UD
B00
4133
End
ophy
teC
allu
naro
ot-a
ssoc
iate
dfu
ngus
DQ
3091
34S
cler
opez
icul
aal
nico
laA
F14
1168
.199
.489
.90
02
00
Hel
otia
les
sp03
6L3
583f
UD
B00
4134
End
ophy
teR
hodo
dend
ron
root
-ass
ocia
ted
fung
usA
Y69
9684
Hum
icol
asp
.D
Q06
9025
97.7
93.7
01
03
0
Hel
otia
les
sp03
7L3
705
UD
B00
4136
End
ophy
teLe
ptod
ontid
ium
elat
ius
AY
1292
8592
.90
01
10
Hel
otia
les
sp03
8L3
739
UD
B00
4138
End
ophy
teE
pacr
idro
ot-a
ssoc
iate
dfu
ngus
AY
2791
89Le
ptod
ontid
ium
elat
ius
AY
1292
8597
.691
.80
61
141
Hel
otia
les
sp04
0L3
388
UD
B00
4141
End
ophy
teO
idio
dend
ron
chla
myd
ospo
ricum
AF
0627
8994
.81
00
00
3176 L. Tedersoo et al.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 3166–3178
Hel
otia
les
sp04
1L3
590
UD
B00
4142
End
ophy
teE
pacr
isro
ot-a
ssoc
iate
dfu
ngus
AY
2682
08P
ezic
ula
cinn
amom
eaA
F14
1186
97.7
97.1
00
12
0
Hel
otia
les
sp04
3L3
129b
fU
DB
0041
43E
ndop
hyte
Cad
opho
rasp
.AY
3715
1387
.41
00
00
Cap
nodi
ales
sp04
6L3
390
UD
B00
4144
Pla
ntpa
rasi
teM
ycos
phae
rella
cann
abis
AY
1525
49.1
96.8
10
00
0C
haet
othy
riale
ssp
047
L320
0U
DB
0041
45E
ndop
hyte
Cla
doph
ialo
phor
ach
aeto
spira
EU
0354
04.1
95.2
00
20
0C
haet
othy
riale
ssp
048
L301
7bU
DB
0041
47E
ndop
hyte
Cla
doph
ialo
phor
ach
aeto
spira
EU
0354
04.1
91.3
00
00
0P
eziz
ales
sp04
9L3
240
UD
B00
4149
Sap
robe
Bet
ula
EcM
root
-ass
ocia
ted
fung
usA
B21
8200
94.2
01
10
0S
orda
riale
ssp
050
L334
1U
DB
0041
50S
apro
beC
haet
omiu
msp
AM
2624
01.1
83.9
00
10
0H
elot
iale
ssp
051
L335
5U
DB
0041
51S
apro
beLe
otia
lubr
ica
AY
1445
4399
.80
02
00
Con
ioch
aeta
les
sp05
2L3
702
UD
B00
4153
Sap
robe
Lecy
thop
hora
sp.A
Y21
9880
88.1
00
00
2E
urot
iale
ssp
053
L307
3U
DB
0041
54S
apro
beP
enic
illiu
mth
omii
DQ
1328
26.1
99.8
10
00
1E
urot
iale
ssp
054
L333
4U
DB
0041
55S
apro
beP
enic
illiu
mro
seop
urpu
reum
AF
0334
15.1
98.4
00
20
0H
ypoc
real
essp
055
L305
0U
DB
0041
56S
apro
beP
hial
emon
ium
aff.
dim
orph
ospo
rum
AY
1883
7181
.10
01
00
Hyp
ocre
ales
sp05
6L3
161e
UD
B00
4157
Myc
opar
asite
Hyp
ocre
opsi
ssp
EU
0731
98.1
88.4
00
10
0S
orda
riale
ssp
057
L318
7U
DB
0041
58C
opro
phile
Pod
ospo
radi
dym
aA
Y99
9127
80.9
00
10
0H
ypoc
real
essp
058
L353
7U
DB
0041
59In
sect
para
site
Cor
dyce
psin
egoe
nsis
AB
0273
68.1
82.7
00
00
1S
orda
riale
ssp
059
L354
8U
DB
0041
60S
apro
beS
oilf
ungu
sE
U55
4812
91.8
00
00
1C
apno
dial
essp
062
L336
9bU
DB
0041
61S
apro
beC
lado
spor
ium
ossi
frag
iEF
6793
8210
0.0
10
03
1H
elot
iale
ssp
063
L366
4aU
DB
0041
62E
ndop
hyte
Scl
erop
ezic
ula
alni
cola
AF
1411
6991
.00
00
20
Hyp
ocre
ales
sp06
4L3
388
UD
B00
4163
Sap
robe
Clo
nost
achy
sca
ndel
abru
mA
F21
0668
95.6
10
10
0S
orda
riale
ssp
066
L301
8U
DB
0041
65E
ndop
hyte
Lith
ocar
pus
root
-ass
ocia
ted
fung
usD
Q27
3344
Aqu
atic
ola
hong
kong
ens
AF
1771
5689
.871
.10
02
00
Cha
etot
hyria
les
sp06
7L3
598m
UD
B00
4166
End
ophy
teC
lado
phia
loph
ora
min
utis
sim
aE
F01
6385
92.0
00
02
0C
haet
othy
riale
ssp
068
L360
9bU
DB
0041
67S
apro
beC
haet
osph
aeria
talb
otii
DQ
9146
6698
.00
00
20
Hel
otia
les
sp07
0L3
637m
UD
B00
4168
End
ophy
teLe
ptod
ontid
ium
sp.
DQ
0690
3395
.60
00
20
Hel
otia
les
sp07
1L3
609s
UD
B00
4169
End
ophy
teR
oesl
eria
subt
erra
nea
EF
0603
09.1
85.3
00
01
0C
haet
othy
riale
ssp
073
L372
7U
DB
0041
70S
apro
beD
acty
laria
appe
ndic
ulat
aA
Y26
5339
92.0
00
01
0E
urot
iale
ssp
074
L370
5bU
DB
0041
71S
apro
beP
enic
illiu
min
dicu
mA
Y74
2699
83.6
00
01
0C
haet
othy
riale
ssp
075
L366
9U
DB
0041
72S
apro
beC
haet
osph
aeria
verm
icul
ario
ides
AF
1785
5093
.30
00
10
Hel
otia
les
sp07
6L3
574S
UD
B00
4173
End
ophy
teC
allu
naro
ot-a
ssoc
iate
dfu
ngus
DQ
3091
26C
lado
phia
loph
ora
min
utis
sim
aE
F01
6385
80.7
83.5
00
01
0
Cha
etot
hyria
les
sp07
7L3
640b
UD
B00
4174
End
ophy
teE
xoph
iala
xeno
biot
ica
EF
0254
0810
0.0
00
01
0S
orda
riale
ssp
078
L364
8aU
DB
0041
75S
apro
beM
yrm
ecrid
ium
schu
lzer
iEU
0417
7588
.00
00
10
Art
honi
ales
sp07
9L3
744
UD
B00
4176
Sap
robe
Ope
grap
hava
riaA
F13
8838
82.4
00
01
0H
ypoc
real
essp
081
L367
3cU
DB
0041
77S
apro
beTr
icho
derm
avi
ride
X93
979
99.6
00
03
0H
ypoc
real
essp
082
L334
5U
DB
0041
78P
lant
para
site
Cyl
indr
ocar
pon
sp.
DQ
9146
7098
.40
01
00
Hyp
ocre
ales
sp08
3L3
350
UD
B00
4179
Unk
now
nV
ertic
illiu
msp
.D
Q91
4739
97.6
00
10
0H
ypoc
real
essp
084
L302
9U
DB
0041
80M
ycop
aras
iteLe
cani
cilli
umsp
.AB
3603
6898
.60
01
00
Ple
ospo
rale
ssp
085
L372
5U
DB
0041
81S
apro
beE
pico
ccum
nigr
umE
U52
9998
97.1
00
01
0H
ypoc
real
essp
086
L335
2U
DB
0041
82P
lant
para
site
Neo
nect
riara
dici
cola
DQ
1328
4699
.80
06
30
Hyp
ocre
ales
sp08
7L3
093a
UD
B00
4183
Inse
ctpa
rasi
teC
ordy
ceps
bass
iana
DQ
6798
9799
.20
10
00
Hyp
ocre
ales
sp08
8L3
070a
UD
B00
4184
Sap
robe
Myr
othe
cium
verr
ucar
iaA
J301
999
88.6
10
00
0H
elot
iale
ssp
089
L357
7U
DB
0041
85E
ndop
hyte
Cal
luna
root
-ass
ocia
ted
fung
usD
Q30
9162
Pse
udae
gerit
avi
ridis
EF
0292
3598
.390
.90
00
10
Hyp
ocre
ales
sp09
0L3
184p
UD
B00
4186
Pla
ntpa
rasi
teN
eone
ctria
radi
cico
laA
J875
331
85.0
00
10
0H
ypoc
real
essp
091
L332
9U
DB
0041
87S
apro
beC
lono
stac
hys
cand
elab
rum
AF
2106
6876
.70
01
00
Hel
otia
les
sp09
2L3
364
UD
B00
4188
End
ophy
teS
cler
opez
icul
aal
nico
laA
F14
1168
91.4
10
00
0H
elot
iale
ssp
093
L359
5U
DB
0041
89E
ndop
hyte
Hum
icol
asp
.D
Q06
9025
90.8
00
01
0H
ypoc
real
essp
094
L309
0eU
DB
0041
90M
ycop
aras
iteM
ycog
one
pern
icio
saE
U38
0317
88.6
02
00
0H
elot
iale
ssp
096
L358
3sU
DB
0041
92E
ndop
hyte
Lept
odon
tidiu
mel
atiu
sA
Y78
1230
87.4
00
01
0S
orda
riale
ssp
097
L304
2aU
DB
0041
93S
apro
beC
epha
loth
eca
sulfu
rea
AB
2781
9479
.30
01
00
Ectomycorrhiza-associated ascomycetes 3177
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 3166–3178
Ap
pen
dix
2co
nt.
Spe
cies
Isol
ate
UN
ITE
acce
ssio
nP
utat
ive
lifes
tyle
Bes
tfu
ll-le
ngth
ITS
sequ
ence
mat
chR
elat
ive
freq
uenc
yof
indi
vidu
als
Bes
tm
atch
ing
isol
ate(
s)%
ID
Mt.
Fie
ldW
arra
Euc
alyp
tus
regn
ans
(n=
49)
Not
hofa
gus
cunn
ingh
amii
(n=
25)
Pom
ader
risap
etal
a(n
=74
)
For
est
floor
soil
(n=
80)
Dea
dw
ood
(n=
23)
Con
ioch
aeta
les
sp09
8L3
050a
UD
B00
4194
Sap
robe
Leaf
litte
ras
soci
ated
fung
usE
F15
9553
Con
ioch
aeta
sp.A
M26
2406
98.5
90.7
00
10
0
Hel
otia
les
sp09
9L3
585
UD
B00
4195
End
ophy
teS
oilf
ungu
sE
F43
4152
Lept
odon
tidiu
mel
atiu
sA
Y80
5569
94.2
94.2
00
02
0
Pez
izal
essp
100
L311
9eU
DB
0041
96S
apro
beS
trum
ella
cory
neoi
dea
AF
4850
6993
.21
00
00
Hyp
ocre
ales
sp10
1L3
176a
UD
B00
4197
Unk
now
nV
ertic
illiu
mle
ptob
actr
umA
B21
4657
92.0
00
10
0H
elot
iale
ssp
102
L320
8eU
DB
0041
98E
ndop
hyte
Phi
alop
hora
sp.
DQ
0690
4698
.50
10
00
Leca
nora
les
sp10
3L3
306
UD
B00
4199
Sap
robe
Ste
reoc
aulo
nal
pinu
mD
Q21
9308
81.1
00
10
0H
elot
iale
ssp
104
L332
9U
DB
0042
00E
ndop
hyte
Epa
cris
root
-ass
ocia
ted
fung
usA
Y62
7817
Oid
iode
ndro
nm
aius
AY
6243
0895
.594
.10
01
00
Hel
otia
les
sp10
9L3
382
UD
B00
4201
End
ophy
teO
idio
dend
ron
pilic
ola
AF
0627
8795
.81
00
00
Cha
etot
hyria
les
sp11
0L3
101e
UD
B00
4202
Sap
robe
Cha
etos
phae
riaac
utat
aA
F17
8553
85.6
01
00
0H
elot
iale
ssp
111
L359
1U
DB
0042
03E
ndop
hyte
Lept
odon
tidiu
mel
atiu
sA
Y78
1230
92.4
00
02
1H
elot
iale
ssp
112
L353
9U
DB
0042
04E
ndop
hyte
Lept
odon
tidiu
mel
atiu
sA
Y78
1230
90.7
00
00
1X
ylar
iale
ssp
113
L358
9U
DB
0042
05S
apro
beM
icro
doch
ium
niva
leA
B27
2124
99.4
00
01
0H
elot
iale
ssp
114
L359
8SU
DB
0042
06E
ndop
hyte
Leoh
umic
ola
verr
ucos
aA
Y70
6325
89.9
00
01
0H
elot
iale
ssp
115
L363
5U
DB
0042
07E
ndop
hyte
Cad
opho
rafin
land
ica
AY
2490
7495
.30
00
10
Hel
otia
les
sp11
7L3
678f
UD
B00
4208
End
ophy
teP
yren
opez
iza
gent
iana
eU
DB
0030
7590
.50
00
10
Sor
daria
les
sp11
8L3
685
UD
B00
4209
Sap
robe
Glo
eotin
iate
mul
enta
DQ
2356
9798
.50
00
10
Sor
daria
les
sp11
9L3
691f
UD
B00
4210
Sap
robe
Fim
etar
iella
rabe
nhor
stii
AM
9217
1781
.90
00
10
Hel
otia
les
sp12
0L3
716
UD
B00
4211
End
ophy
teLe
ptod
ontid
ium
elat
ius
AY
1292
8581
.10
00
10
Hel
otia
les
sp12
1L3
710
UD
B00
4212
End
ophy
teE
pacr
isro
ot-a
ssoc
iate
dfu
ngus
AY
6691
37Le
ptod
ontid
ium
elat
ius
AY
7812
3097
.192
.90
00
10
Cap
nodi
ales
sp12
2L3
261
UD
B00
4213
Sap
robe
Cla
dosp
oriu
mcl
ados
porio
ides
AF
1777
3690
.40
11
00
Hyp
ocre
ales
sp12
5L3
675
UD
B00
4216
Sap
robe
Tric
hode
rma
aspe
rellu
mD
Q09
3705
99.6
00
01
0H
elot
iale
ssp
126
L359
8fU
DB
0042
17E
ndop
hyte
Unc
ultu
red
soil
fung
usE
F43
4152
Lept
odon
tidiu
mel
atiu
sA
Y80
5569
95.2
95.2
00
01
0
Hel
otia
les
sp12
7L3
636
UD
B00
4218
End
ophy
teLa
nzia
huan
gsha
nica
DQ
9864
8480
.80
00
10
Hel
otia
les
sp12
8L3
615S
UD
B00
4219
End
ophy
teO
idio
dend
ron
pilic
ola
AF
0627
8796
.10
00
10
Hel
otia
les
sp13
0L2
620L
UD
B00
4240
End
ophy
teB
otry
otin
iafu
ckel
iana
AY
5446
51*
99.0
00
00
1H
ypoc
real
essp
131
L327
8aU
DB
0042
21M
ycop
aras
iteH
ypom
yces
aura
ntiu
sA
B37
4290
74.9
01
00
0H
ypoc
real
essp
132
L320
7nU
DB
0042
22M
ycop
aras
iteH
ypom
yces
chlo
rinig
enus
AF
0548
6681
.10
10
00
Hyp
ocre
ales
sp13
3L3
195s
UD
B00
4223
Sap
robe
Soi
lfun
gus
AF
5048
33P
aeci
lom
yces
carn
eus
AB
2583
6999
.889
.90
01
00
Leca
nici
llium
flavi
dum
L370
9U
DB
0042
24M
ycop
aras
iteLe
cani
cilli
umfla
vidu
mE
F64
1878
99.8
83
39
0
*Bas
edon
28S
rDN
Am
atch
.
3178 L. Tedersoo et al.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 3166–3178