1

Fems Microbiology Ecology A chimer December 2014, Volume 90 Issue 3 Pages 908-921 r http://dx.doi.org/10.1111/1574-6941.12447 http://archimer.ifremer.fr http://archimer.ifremer.fr/doc/00247/35810/ © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

In-depth analyses of deep subsurface sediments using 454-pyrosequencing reveals a reservoir of buried fungal communities at record-breaking depths

Rédou Vanessa 1, * , Ciobanu Maria Cristina 2, 3, 4, Pachiadaki Maria G. 5, Edgcomb Virginia 5, Alain Karine 2, 3, 4, Barbier Georges 1, Burgaud Gaëtan 1

1 Univ Brest, Lab Univ Biodiversite & Ecol Microbienne, EA3882, F-29280 Plouzane, France. 2 UBO, LMEE, UEB, IUEM,UMR 6197, Plouzane, France. 3 CNRS, UMR 6197, IUEM, LMEE, Plouzane, France. 4 IFREMER, UMR 6197, LMEE, Plouzane, France. 5 Woods Hole Oceanog Inst, Dept Geol & Geophys, Woods Hole, MA 02543 USA.

Corresponding author : Vanessa Rédou, email address : [email protected]

Abstract :

The deep subseafloor, extending from a few centimeters below the sediment surface to several hundred meters into sedimentary deposits, constitutes the deep biosphere and harbors an unexpected microbial diversity. Several studies have described the occurrence, turnover, activity and function of subseafloor prokaryotes; however, subsurface eukaryotic communities still remain largely underexplored. Ribosomal RNA surveys of superficial and near-surface marine sediments have revealed an unexpected diversity of active eukaryotic communities, but knowledge of the diversity of deep subseafloor microeukaryotes is still scarce. Here, we investigated the vertical distribution of DNA and RNA fungal signatures within subseafloor sediments of the Canterbury basin (New Zealand) by 454 pyrotag sequencing of fungal genetic markers. Different shifts between the fungal classes of Tremellomycetes, Sordariomycetes, Eurotiomycetes, Saccharomycetes, Wallemiomycetes, Dothideomycetes, Exobasidiomycetes and Microbotryomycetes were observed. These data provide direct evidence that fungal communities occur at record depths in deep sediments of the Canterbury basin and extend the depth limit of fungal presence and activity, respectively 1740 and 346 mbsf. As most of the fungal sequences retrieved have a cosmopolitan distribution, it indicates that fungi are able to adapt to the deep subseafloor conditions at record-depth and must play important ecological roles in biogeochemical cycles.

Keywords : 454 pyrotag, Canterbury basin, deep subseafloor, fungi, microeukaryotes

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. 2

Introduction

Exploration of the marine subsurface was initiated in the 1930s (ZoBell & Anderson, 1936) and since that time it has been demonstrated that abundant and diverse microbial communities inhabit this ecosystem with the potential to impact large-scale biogeochemical cycles (Fredrickson & Balkwill, 2006; Edwards et al., 2012; Anderson et al., 2013). The deep subseafloor biosphere hosts large numbers of living microbial cells, estimated at 2.9 × 1029 cells, as revealed by recent cell counts (Kallmeyer et al., 2012) and thus represents a significant biome structured by several spatial, physical and energetic constraints (e.g. confinement, pressure, temperature, refractory organic matter, etc.). Studies of subsurface microbial communities (Parkes et al., 2000; Fry et al., 2008; Orcutt et al., 2011) and metabolisms (D'Hondt et al., 2002, 2004; Biddle et al., 2006, 2011) have provided a foundation for understanding the ecological roles of subsurface microorganisms. Prokaryotes remain the common targets of investigations that aim to study subsurface microbial diversity, and debate continues whether Archaea or Bacteria predominate (Schippers et al., 2005; Biddle et al., 2006; Briggs et al., 2012).

Microeukaryotes remain understudied although their presence in marine extreme environments is increasingly documented. Culture-based and culture-independent methods have demonstrated their occurrence in hydrothermal vents (Edgcomb et al., 2002; Burgaud et al., 2009, 2010; Le Calvez et al., 2009), anoxic environments (Stoeck & Epstein, 2003; Takishita et al., 2005; Jebaraj et al., 2010), deep hypersaline anoxic basins (Alexander et al., 2009; Stock et al., 2012), cold seeps (Takishita et al., 2007; Nagano et al., 2010; Nagahama et al., 2011) and associated with sunken wood (Barghoorn & Linder, 1944; Dupont et al.,

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. Accepted Article thein deep biosphere. (after component microbial sequences resp 1740346 mbsf detected at and richnessspecies isextremely with only low, 18OTUsdetected. onBased rDNA andrRNA subseafloor, deep althoughrecord-depth the in at signaturesappear that showfungal we Here totheprerequisite understandingof the ecological offungiroles in the deep biosphere. operational fungal taxonomicdifferent units (OTUs) atthe level, present species a and DNA,RNA and re fungal ITS1 targetin used a 454-pyrosequencingapproach natureinsights and onthe extentof subsurface further provide Canterburybasin sediment to cores from communities microeukaryotic indepths the subseafloor. Inourstudy, we conducted an in-depth investigationof biosphere, and revealed theoccurrence of deep the of limits hypothesescore the regarding sediment wastest investigated to That the Canterbury core from sediment (1922m) record-depth usinglife a of microbial the boundaries recently extended conditions conditions thus and may be capable of colonizing thedeep subseafloor. Ciobanu surface-dwellingterrestrial and fungimaybe of capable biosphere adaptation todeep This article This protectedis byAllcopyright. rights reserved. (Richards environments terrestrial in widespread typically are significanthave roles ecological deepin the biosphere. Fungi in revealed sediments marine be diverse(Edgcomb 2011; Orsi etal subsurface marine at depths 5 from (Edgcomb belowseafloorto 159 meters (mbsf) definitelyhave microeukaryoticdemonstrated (Ciobanu al et microeukaryotic life enoughsupport appear large to microorganisms.multicellular However,deepmicrons systemsseveral cavities of with spatialconstraints that viewpoint wereobstacleprevailing growth to an the of cellsorlarger subseafloorthe delayed hasbeen deep by microeukaryotes inthe search for The 2009). ., 2013a, 2013b). Among microeukaryotes, 2013a,Among communities fungal to., microeukaryotes, 2013b). appear not et al basin (New Zealand). Zealand). (New basin ., 2011, Xu Bacteria gions in order to analyze the presence and activity ofthe and et al et Archaea ., 2014) but consistently dominate, and thus, may consistently dominate, andthus, ., 2014)but Bacteria g eukaryotic smallg eukaryotic subunit (18S) ribosomal presence, activity thein andmetabolisms fungal reservoirs in the deep deep fungalreservoirsbiosphere. inthe We ) with potentially important ecological roles ecological potentially important ) with ectively, fungi may be viewed as a third viewed asa ectively, be may fungi , Archaea and et al. Eukarya ., 2014).., Recent studies , 2012), indicating, that at these record these at et alet . (2014) et al ., Accepted Article 1990) and fungal primers ITS1 (G Captair cabinet; samplesto lowbiomass(PCR sediment dedicated PCRcabinet exclusively outin a were manipulationsavoidcontamination, carried all to 1).In order 9samples along performed (Table collected core were the from DNA extractions following the manufacturer’s instructions. Ten instructions. manufacturer’s the following This article This protectedis byAllcopyright. rights reserved. (White ITS1F/ITS2 516R and prim followedPCR amplifications with fusion 42F(López-García Euk primers eukaryotic universal PCR were using amplifications performed the DNA 25µL For each extract, 4 independent described in Ciobanu are sampling of time the parameters at and environmental core lithology The periods. Eocene depth,sedimenthadandthe coreHolocenewater spanning adepthof 1927.5mbsf, tolatethe U1352Site (44° sediment corewasdrilled at (RV Leg 317Expedition IODPduring New Zealand, of South Island Materials and Methods volumes containing 1×Taq DNA polymerase buffer with MgCl with buffer DNAvolumes containing 1×Taq polymerase MgCl treatment. treatment. exogenous amplifiable DNA during or thedifferentcDNA occurred stages of sample cDNA extraction) was forprepared each work toensure that with nocontamination stage, inincluded each set ofPCRreactions. Inaddition, a negative control (e.g., DNA negative or (reac controls Negative (Thermo-Scientific). of Concentration DNAextracted was meas fi subsequentAtprecipitation stages.the in Ambion/Applied Biosystems) were added the to be retrieved, with the the retrieved, with FastDNA be 5x gextracted 0.5-1 from samples(-8 frozen 2. Sediment samples were collected from the Canterbury basin, on the eastern margin of the ontheeastern of margin Canterburythe fromwere collected samples basin, Sediment 1.

2 , 240 Nucleic acidNucleic extractions, PCR amplifications and 454 sequencing Site description and sediment sampling and sediment sampling description Site μ M dNTP, 0.4 et al ., (2014). μ M of M of each primer, 1 5×volume of GC-rich buffer, 1 unit of et al TM ardes &Bruns, 1993)/ITS2ardes (White ., 1990). All PCR reactions were performed in 25 µL 25µL werein performed AllPCRreactions 1990). ., Spin Kit for Soil (#6560-200, MP Biomedicals SoilKit (#6560-200, for Spin ured with aNanoDropSpectrophotometer 1000 nal step, DNA was eluted in a 50 µL volume. nal step, DNA inwas eluted a50 µL 56’26.62’’S; 172°1’36.30’’E) (Fig. 1) in 344m 1)in (Fig. 172°1’36.30’’E) 56’26.62’’S; 0°C), where no fluorescent couldmicrospheres no fluorescent where 0°C), tion without mixture DNA or cDNA) were er sets Euk 82F (Dawson & Pace, 2002) / Euk 2002)/ Pace, 82F(Dawson & setsEuk er microliters of linearmicroliters acrylamide (5mg.ml protein lysisprotein buffer in orderto favor DNA et al ., 2003)/Euk 516R(Amann ., 2003)/Euk 2 (2mM), 1 mM of additional additional of 1 mM (2mM), ® ). A ). Resolution JOIDES Bio, DNA wasErlab). et al ., 1990). Nested et al ® -1 ), ), ., , Accepted Article PowerSoil and 9318 g 346mbsf samples for of uncontaminated frozen samples (-80°C) RNA with the 2xand around sample 12 mbsf g for 4 2 x from extracted was 1).RNA (Table 931 mbsf and RNA extractions wereperformed using 3 samples along collected the core, including 12,346, previously (Ciobanu 18S PCRpyrosequencing. sequencing and detail programfor strategy describedin were werepooledproducts two by two,so as second PCR amplification wasperformed in 95°C, 1min at 56°C min 72°C and1.5 at anda final extension step of at 72°C.7 min The denaturation byregion step started at initial 95°C 5 followed by for min, 20 of cycles 1 minat This article This protectedis byAllcopyright. rights reserved. DNAFastStart and1 Taq polymerase MgCl 2,5 of mM each primer, of 0.2µM dNTPs,0.4 volumes containingGoTaq Buffer, mM 1X reaction the eukaryoticAllPCR 18SrRNA.in25µL were(~380 bp)of reactions performed (Cole primers gesmall subunitribosomalRNA of amplification Technologies). Removal contaminatingof DNA was byconfirmed theabsence of visible (Life kit DNA-free using Turbo were the treated RNAextracts contamination, DNA potential any Toremove instructions. manufacturer’s according wasused Technologies) the (Life kit instructions. manufacturer’s Topurify the ResearchLP, Texas)and pyrosequencing was performedon a GSFLX+ (Roche).platform sequencing Barcodes instructions. byligation(Molecular bythe added companywere GelZymoclean DNA RecoveryKit (Zymo Research) according to the manufacturer’s usingthe purified 72°C. were at Amplicons of 7min step extension final 72°C anda 50 sat at95°C,35 s 45s by 35cycles 48°C step ofdenaturation 95°C 4min,followed at for at and TAReukFWD1 and TAReukREV3 (Stoeck TAReukREV3 (Stoeck TAReukFWD1 and according(Quiagen) Kitmanufacturer’s The the primers universal instructions. Transcription extracted RNA was immediately transcribed reverse to usingcDNA theQuantiTect 2 , 1 U of GoTaq polymerase and 1 µL of cDNA. ThePCRby assay initialcDNA. started of GoTaq 1UofandµL polymerase ,

® Total RNA Isolation Kit (#12866-25, MO BIO Laboratories), following the following Laboratories), MO BIO Kit (#12866-25, RNAIsolation Total et al ., 2009)40cycles ofRNAafter PCRastemplate.Total usingthe extracts et al ., 2014). μ L of DNA template. The first PCR assay for The assay for ITS1 PCR L of DNAfirst template. RNA extracts, the MEGAclearthe RNA purification RNA extracts, et alet the same conditions using 10cycles.PCR ., 2010) were usedregion., V4 toamplify2010) were the to have two independent replicates two independent for to have nes using bacterial hypervariable V4 region Accepted Article database for for fungi (Koljalg database sequencesidentified and were similar to18S processing data raw sfffrom files to the dereplication Chimericstep. generated from UPARSE pipeline were processed and analyzed using QIIME pipelineusing QIIME were processed analyzed and pipeline UPARSE from generated weighted UniFrac& (Lozupone metrics Knight,2005). 1963).Weaver, diversity Beta samples of were assessed patterns using unweightedand the1 (Chao,1984) Simpsonand andShannon diversity Chaoestimator Alpha wasassessedrichnessby QIIME. the calculating in command determined using were thecore_diversity_analyses.py beta metrics Both and diversity alpha th with phylogenetic analysis wasperformed contained andpositionsgreater that alignment parameters using filtered default was alignment using (DeSantis PYNAST with theassign_taxonomy.py Sequence command. within QIIME. assign_taxonomy.py command usingperformed Silva_111the database and in analyzed using the QIIME (Caporaso usingpipeline the analyzed (Edgar (Quastetal Silva_111 sequencingretained. Chimeric were duplicates both in OnlyOTUs command. cluster_otus present threshold usingthe 97% identity sorted by decreasing abundancewith thesortbysize OTUs delineatedcommand. were at a derep_fulllengththe command with the sizeout optionsetat64. Dereplicated reads were (Edgar, 2013).Theremoval sequences duplicated of was usingperformed v7 USEARCH and UPARSE function and the (Edgar, 2010) usingv7 performed USEARCH was clustering 200bp.The at set trimming was after length readretained removed. A minimum were method. error filtering which Sequences a for format. A quality filtering was performed with USEARCH using themaximum expected This article This protectedis byAllcopyright. rights reserved.

3. DNA- and RNA-based 18S data processing. dataprocessing. 18S DNA- andRNA-based

DNA-based ITS1 data processing. Quality control, control, Quality clustering assignment 454pyrotags andtaxonomic of et al et ., 2011).The generatedOTU from table theUPARSE was pipeline processed and ., 2013) as thereferencedatabase., 2013)as eukaryotes UCHIME for within et al et et al,. removed using UNITE (Abarenkov UNITE removed using ., 2006) and classified using the Silva_111 rep set. The classified ., 2006)and repset. Silva_111 using the 2013), within UCHIME (Edgar within UCHIME(Edgar 2013), Data processing used for the ITS1 marker was marker ITS1 processingData the for used e make_phylogeny.py command within QIIME. QIIME. e make_phylogeny.py within command ll base pairs had a Phred quality score under 15 score a Phredquality base had pairs ll diversity indices (Simpson,diversityindices 1949;Shannon& sequences were identified andremoved using . The 10% most variable positions within the within positions variable 10%most The . than 80% gapsthan re were 80% The taxonomic mapping file was generated was taxonomicmappingfile The cluding only withthesequences eukaryotic The raw sff files were converted to FASTQ toFASTQ converted were files sff raw The et al representatives of each OTU were aligned each OTU were of representatives ., 2010).Taxonomicwas assignment et al et al ., 2010) as a referenceas a 2010) ., moved. Aeukaryotic ., 2011). OTU ., table 2011). Accepted Article observed OTUs indicating a complete coverage of the fungal DNA thefungal TheShannon of coverage diversity. observed complete OTUsindicating a sequences recovered. Chao richness estimators (T total of 54100% represent to 18Sdataset DNA-based Fungi 2. inthis inTable summarized number cleaned fungal reads, the of 454 reads, theand numberof OTUsfungal are and weregrouped into 13 fungal OTUs. For sample, the number each fungal total of 454 sequences the were 1740 mbsf.After sequences) quality48% of analyzed (14,036 control, 28,868 sequences were 454 bygenerated pyr the Results XLSTAT(Addinsoft, USA, York). New environmental parametersand sample. for each analyses performedwere statistical usingAll PCAand Depth). Ethane an Methane, Porosity, Carbon, Inorganic Carbon, Organic (OTUs, Depth, parameters different of the contribution samplesMFA themapping allows sediment of on a2-dimensional plane showing the This article This protectedis byAllcopyright. rights reserved. environmental parameters acquired during IODP Expedition 317 (Fulthorpe toprocessed elucidate relationships between fungal community structure and some selected Analyses Factorial Multiple (MFA) andPr 2005). Knight,& UniFrac samples wereassessedmetrics(Lozupone weighted using and unweighted 1949; (Simpson, diversity Shannon& Weaver, indices 1963). ofBeta diversity patterns Chaocalculating therichness 1984)andtheSimpson 1(Chao, estimator andShannon core_diversity_analyses.py in the command assign_taxonomy.py Both command. alpha and beta assign_taxonomy.py withthe Taxonomic was file performed mapping command. (Caporaso DNA-based eukaryotic gene dataset based on V1-V3 region of 18S rRNA gene of 18S on V1-V3 based dataset DNA-based eukaryotic gene region 1. 4.

Fungal diversity Statistical analyses Statistical

et al ., 2010). with usingTaxonomic assignment was performed UNITE., 2010). database incipal Componentincipal Analyses (PCA) were QIIME. Alpha diversity was assessed bydiversityassessed was Alpha QIIME. alyses OTUs based ondifferent obtained are osequencing for the 9 depths from 346to from the9depths osequencing for able 3)areable consistent numbers with the of diversity metrics werediversity determined using metrics et al . A . total of ., 2011). Accepted Article complexity of of the complexity diversity withcomplexversus notcomplex samples, detailed below.as Fig. Italsoclearly 2A, 3A). left the (Fig. highlighting on the high-depth/low-porosity and plot the rightof the on porosity samples low-depth/high with structuring parameters gravity of MFA variables. thedifferent cl of 2A,3A representations (Fig. Fig. superimposed emerging from thedataset. Differences between samples were deduced based on the factors. of Usingit was MFA, toge possible a toolfor convenient represents comparing se Analysis (MFA) Co withaPrincipal coupled theof thestructure fungalcommu in Variations weakly diversified along the core. suggesting, as DNA-based 18Sdataset, that thefor fungal using communities also are ITS1 DNA diversity. The Shannon diversity everyfor index computed depthranged 0 from to 1.86 fungal coveragethe complete of a indicating observed of OTUs, numbers the consistent with resulting of number OTUsaresummarized in Table 2. Chao richness estimatorsare reads andthe quality of after retained number fungal the obtained, 454 reads control, This article This protectedis byAllcopyright. rights reserved. 18 OTUs grouped into were singletons of removal qualityAfter 59% control, ITS1sequences were of (10,421analyzed sequences), and after sequences were generated by 454 pyrosequencin groupedinto 185 OTUs, amongthem only 1 fungalobserved OTUs at12 and 346mbsf. sequencesquality 39%of mbsf. After control, analyzedwere (63,564sequences) andwere R generatedsequencesfor were species diversityof fungal communities along the core. 3) 2.11 (Table suggesting 0to alow every from for computed diversity depthranged index 2. DNA-based eukaryotic basedgene dataset onITS1 fungal region 18SrRNA. of region onV4 based dataset gene eukaryotic RNA-based

Distribution patterns of patterns fungal Distribution communities NA-based 454 pyrotags 3depths,i.e. for veral samples characterized by subset samples the same veral early depthandporositypositioned as strong t the t an of common structureoverall picture ) on which samples) on are represented as centers nities were determined by Multiple Factorial Factorial nities weredetermined by Multiple g for 5 samples from 346 mbsf to 1711 mbsf. 1711 to 346 mbsf from samples 5 gfor mponent Analysismponent (Fig. 2,Fig.MFA 3). . For each sample, the total number of number total the sample, each For . A of 17,672total A total of 164,743of A total 12, 346 and 931346 12,and Accepted Article the different depths clearly indicate contrasted fungal communities along the sediment sediment different along depths fungalcore. clearly the the communities indicate contrasted 1577 mbsf and 1711 mbsf samples. As 18Sfor the data, directions differentOTU vector at 583 mbsf 931 and mbsf samples, with long length OTU vectors, were complexmore than concentration. guilliermondii indicating that that indicating withmethane. Clustercorrelated II appeared negatively with carbon, organic correlated In III, depth. cluster the shallowest from only OTUs contained III cluster while depth, horizon sediment deeper the OTUs from of sp., Exophiala dermatitidis Exophiala Malassezia pachydermatis, Meyerozyma guilliermondii, Pleurostomophora richardsiae, globisporum,Filobasidium complex anddiverse.visualization3 PCA (I) ofclusters: allowed depths th that different confirm directions at contrasted at the different depths. Indeed, th analyzed. Using the OTU vector, MFA also cl demonstrates sedimentthatlayers diversityfirst and the OTU vectors fungal is higher in the to compared complex samples representing with 346 ornotcomplex. complex vectors, samples, longmbsf and583 OTU length 1367 samples. mbsf MFA allowed samplesdifferentiation sediment based of on diversity, samples were1740 mbsf depth 634mbsf to583mbsf, and relativelyorganic-rich compared samplesorganic mbsf and 1740carbon. It that 931for clearly indicates 346mbsf, mbsf and 583 mbsf, 634mbsf and 1367mbsfsamples inversely with346 correlated mbsf, 931 mbsf This article This protectedis byAllcopyright. rights reserved. values, DNA-based 18Sdataset were used to detail communities distribution. ustoMFA distribution allowed infer general 1740 mbsfand931 for mbsf samples caorganic Cryptococcus curvatus DNA-based ITS1 dataset. i.e. 1367 mbsf, 634 mbsf and 1740 mbsf samples for inorganic carbon, 346 mbsf, 346 carbon, inorganic mbsf samples for 1740 and mbsf mbsf, 634 1367 wereOTUs sedimentcarbon sampleslow in with onlyorganic found T . mucoides and (III) . Therepresentation. MFA to allowed cluster samples with close and

, Cryptococcus surugaensis M. Cyberlindnera jadinii. Leptosphaerulina chartarum, Fusarium solani,Trichoderma Consistent 18S mbsf, the indicated MFA with data, that 346

pachydermatis Cryptococcus curvatus patterns. PrincipalThen, Component Analyses e lengths differentOTUvector butalsothe e first sediment layers analyzed were more were analyzed layers sediment first e rbon of some and differentiation samples, , other sediment samples with short lengthshort sediment samples with other early indicated earlythat diversity indicated washighly E . The first cluster wascomposed mainly The cluster first dermatitidis ; (II) , Trichosporon mucoides, appeared significantly P.

richardsiae Wallemia muriae, Wallemia and e.g. i.e. M.

Accepted Article This article This protectedis byAllcopyright. rights reserved. PCA clearly differentiated 3 clusters: (i) Exobasidiomycetes Pleurostomophora richardsiae, respectively. Tremellomycetes kratochvilovae, pattern, with noapparentpattern,the with ofdepths. one specificity for Saccharomycetes of Signatures respectively. Basidiomycota were detected in ourDNA-based with 63% of libraries, compared against SILVA_111, UNITE and GenBank databases. To evaluatethe taxonomic composition of each sample, therepresentative reads were 931 mbsf sample OTU and 2 withthe 1711 mbsf sample(Table 4). the with shared sample sample. OTU3 583mbsf 931mbsf The OTU3 with the sample and 11. In ITS1dataset, the 346 mbsf the sample shared OTU OTU 3and 6 the 583mbsfwith shared between samples, observed inthesame OTUOTU 9, 3,OTU 6, OTU andOTU OTU4). 10, 13(Table Amaximum 4OTUs of are eukaryotic primer OTUs with obtained the13 Of ethane.methane and cluster III, some of which affiliated to the to whichaffiliated of some III, cluster the of Members matter. organic with correlated inversely were PCA the of right the III on and wereatmost OTUsthe only found agive and of Trichosporon pseudolongus, Cyberlindnera jadinii, Galactomyces candidum, leucadendriBatcheloromyces 3.

DNA-based eukaryoticDNA-based 18Sdataset composition Taxonomic Sordariomycetes sp., and . The reads The . were classified 7 into classes. Signatures of

were the abundantmost recovered, with 38%and 35% the of reads, Rhodotorula Rhodotorula were represented less with 15%, Meyerozyma guilliermondii , , sp., Elmerina caryae

Eurothiomycetes, Chaetothyriales Tremella moriformis . Within kingdom,. onlythe fungal the Within andTremellomycetes Crypococcus sample type, OTU 4, OTU 5, OTU 8 and OTU and OTU 8 OTUsample 5, OTU type,4, n depth. Many OTUsbelongingII to clusters n depth. , sp., s, 6 appear to be depth dependent, OTU2,depth dependent, 6appeartobe s,

Rhinocladiella . Those3clusters depth-specific, appeared . Dothideomycetes Cryptococcus saitoi, ExophialaPenicillium spinifera, 6%, the 3%,2%and1%of reads, , Dothideomycetes, Sordariomycetes genus, appearedcorrelated with Leucosporidiella muscorum Leucosporidiella

Leptosphaerulina chartarum sp., and (iii) (iii) sp., and Ascomycota had a broaddistribution , Wallemiomycetes Saccharomycetes

Rhodosporidium and37% of Cryptococcus Dikarya ; (ii) and sp., sp., and , Accepted Article Wallemia muriae pachydermatis and detected in the931 while mbsfsample, Signatures of samples shallowest 634mbsf. the up to dermatitidis, Trichoderma Exophiala chartarumLeptosphaerulina eukaryotic reads).eukaryotic 346 mbsf. No fungal signatures were detected in the 931 mbsfsample (outof 17,397 passed quality observed control) 18,897 12 (outof reads)at at eukaryotic mbsf and 1 read 27,988eukaryoticreads thatof decreased (out 11 with depth, of reads reads with Occurrence was basidiomyceteand this assignedto the yeast approach, using RNA-based an kingdom, only 1fungal OTU wasdetected the in samples analyzed at12, 931 346, and mbsf This article This protectedis byAllcopyright. rights reserved. sequences, while affiliated to the species appear tobe at the depths unique different analyzed. taxonomic level aspecies mbsf. OTUswith as 1740 with affiliatingSequences and Wallemiomycetes 18S dataset, were were absent18S dataset, in Sordariomycetes depth. with noapparent pattern, specificityone distribution for 1% reads, ofthe respectively. The of Signatures with ITS1 primers. mbsf, a result consistent with the DNA-based 18S DNA-based with the mbsf, aresult data. OTUsaspeciesconsistent taxonomic and level Microbotryomycetes, Eurotiomycetes atrepresented the class level with 57% Eurotiomycetes Cryptococcus surugaensis V4 region of RNA-based regionthe V4 eukaryoticdataset 18S rRNA DNA-based fungal ITS1 dataset. dataset. DNA-based fungal ITS1 was only at931to1689mbsf. detected and Ascomycota wereonlyfound inthe deepest 4) layer (Fig. Dikarya and Eurotiomycetes had only been detected in the first samples detected onlyhad inthe583mbsf. from been 346 to first Exobasidiomycetes, . Dominance. ofBasidiomycota Wallemiomycetes Wallemiomycetes the ITS dataset. Incontrast, ITS dataset. the were only 346 found in the mbsf sample. communities were less represented with 39% of thesequences.with of represented less 39% were communities were detected samples inall analyzed. Saccharomycetes Tremellomycetes sp. and Pleurostomophora were detected richardsiae in and have been detected in the upperhave samples beendetected inthe downto582 and 26% of the reads,of the respectively. 26% and Meyerozyma guilliermondii Sequences recovered in the ITS1 dataset were ITS1 dataset inthe recovered Sequences Dothideomycetes2% and represented 10%,4%, which previously were in theDNA-based found are seen exclusively seen are from sample deepest inour signment are presentedsignment are and andSaccharomycetes Microbotryomycetes Trichosporon mucoides Tremellomycetes was observed the with 61% of Filobasidium globisporum Filobasidium . , Fusarium and solani Cryptococcus curvatus Cyberlindnera jadinii, . Within thefungal Within . displayed a broad in 4.in FungalTable Sordariomycetes, Dothideomycetes, Dothideomycetes, were only werefound were were mostthe Malassezia Malassezia Malassezia were only were and .

Accepted Article immediately frozen at–80°C onshoremolecularanalyses. for Potential of thecontamination underwithin interior, subsampled sterileconditions, core thesefrom the and subsamples were contamination from seawater and drilling fluids. Onboard, whole drilling fluids. seawaterOnboard, and from contamination were subsequently usingfluorescence investigated microscopy to determine the potential for cellsmicrobial thatwere incorporated dril into R/V onboard the contamination of samples was investigated. tests Different during drilling performed were formicrobiological potential the Expedition, the IODP317 erroneousresults. During to lead risks,sinceregarding the contamination presen studiesAccurate of the deepstrict biosphererequire quality thecontrols ofsamples analyzed Archive under the study accession numberPRJEB6764. Nucleotide European tothe wasdataset submitted entire The subseafloor. colonizing the different insights taxa the fungal dataprovide into sets These 12 mbsf. at sampled layer genes rRNA alongthis transect of analysis twodepths complementaryand one shallowfrom In addition, inaseparate1740 mbsf. study that depths subsurface from sediments incollected the Canterbury basinspanning 346 to mbsf IT rRNAand eukaryotic of analysis DNA-based theCa subsurfaceof sediments deep in occurring study of the of distribution to aim vertical The this was fungal communities investigate Discussion appearto deepest dominatethe layers analyzed (Fig. 4). from thefrom 931sample, mbsf while moriformis This article This protectedis byAllcopyright. rights reserved. Chaetothyriales Penicillium of Signatures 4. Table in given are assignment mbsf. dominated the upper horizons down to 582 mbsf. Meyerozyma guilliermondii 1.

Sequences affiliating with Controlling contamination contamination Controlling , Cryptococcus saitoi sp., Joides Resolution Elmerina caryae , Pleurostomophora richardsiae and Leucosporidiella muscorumLeucosporidiella Cryptococcus pseudolongus in order to quantify fluorescent microbeads mimicking quantify fluorescent to order in were only recovered recovered only Rhodosporidium kratochvilovae were , Rhinocladiella l fluids during drilling. The exteriors of l fluidsduring Theexteriorsof cores drilling. we include here, weinclude weapplied an RNA-based ce of exogenous cells or nucleic mayacids nterbury basin. Toward that aim, we used a aim,we that Toward nterbury basin. Trichosporon S1 signatures in samples from 9 different different 9 samplesfrom S1 in signatures sp., sp., Leptosphaerulina chartarum Batcheloromyces leucadendri Batcheloromyces sp. was sp. detected downto 931 , and ‒ Rhodotorula round sediment cores weresediment cores round and Cyberlindnera jadinii Exophiala spinifera spinifera Exophiala sp., Tremella , , Accepted Article with with highcorrelated porosity and high carbon Sequencesorganic concentration. affiliating were only detected at583 mbsf. These genera, associated the with lower seemdepths, to be Leptosphaerulina Leptosphaerulina, fungal forms in deep-sea environments (Takishita al., et consistent withprevious where studies basidi to OTUsbelongingof numbers high However, the to of 0.64 primers and eukaryotic universal ofAscomycotaratio suggestingabundance orreally absence low samples. ofThe in earlythese lineages diverging datasets,fungal ITS1 onlythe 18Sand Among DNA-based the conservative pictureofsubsurface communities. remove contaminant libraryanyin orderto contaminant andtopresent potential a stringent possibleas (2 independent pyrosequenc to bioma sterilematerialsdedicated low (i) of wereprecautions takenduring withthe laboratory use inthe analysis contamination toavoid This article This protectedis byAllcopyright. rights reserved. of sample the very5–11core isg low andwas atcellsinterior estimated 1.99 g cm properties, the ITS2 marker is less variable in length compared with ITS1 and is also well many share groupstaxonomic ITS2ITS1 and specificfungi. Also,if of of recovery not sincewere surprising, biases of primer but only inthe deeper layers withthe marker.fungal These differences between data sets ITS1 marker. fungal the signatures taxon this with sedimentary onlylayers of were presentinthe upper signaturesset of and organismthis pr were on observation of on observation beads fluorescent and average of densities 1.85 g cm Cryptococcus, Meyerozyma, Exophial 2011). Signatures of many common genera were detected between the two datasets; 2. Meyerozyma

Comparison of DNA-basedthe V1-V3SSU ITS1forand markers understanding fungal community diversity community fungal –3 insedimentary rocks U1352at site (Ciobanu Cryptococcus

which showed the same pattern which core. the showed alongthe distribution was found only at346mbsf. Similarly, guillermondii to Basidiomycota wasdetected all along withthe thecore 18Smarker,eukaryotic formed the most the abundant most OTU 18S inthe eukaryotic formed data was quite with of different, 1.7 the totalreads using a, Trichosporon, Pleurostomophoraa, Trichosporon, esent throughoutthe sediment core. By contrast, s are well known and each primer set favors primer known andeach well s are omycete yeasts were found to bethe dominant ss samples, (ii) data processing strategy as as strategy processing data (ii) ss samples, tal readsusingtal the fungal specific primers. ing replicates) and (iii) the analysis of a Basidiomycota 2006; Bass Pleurostomophora et al et in the ITSdataset are et al., Dikarya Dikarya ., 2014). Inaddition, -1 –3 of sediment, ofsediment, based 2007; Singh 2007; insediments and and were detected, Exophiala et al et and .,

Accepted Article tolerate hasbeen it Indeed, shown the membranecomposition able that to fungi their are tochange colonize habitats. to deep-sea fungi suggesting able that fungi, are toknown terrestrial close including including (Gadanho &Sampaio, 2006) and subsurface sedime already shown theability marine to have tocolonize includingenvironments waters acidic 2013a). sediments down 48to mbsf in an RNA-based study PeruMargin sediments of (Orsi methane Knoll Kuroshima from seep libraries genus and this was detected subsurfacein al., (2006)identified diversity in study microeukaryotic another of Cryptococcus. affiliated with OTUsinourdataset mostabundant the the among genera sediat each detected uniqueOTUswere since is quitelowin samples.these Fungal communities different tobeappear differentdepths at 454approach tag-encoded pyrosequencingUsing the This article This protectedis byAllcopyright. rights reserved. sediment tosedimentary 931which occursfrom rock, transition around mbsf,diversity 583 thanindeeper samples. the mbsf 346 and samplesfrom After was greater inthe Although fungal communities exhibited quite low phylogenetic overall diversity, diversity (Palmero pathogens andmycotoxinplant producers.It al., 2013a). sediments in marine previously detected approaches thus appearedmore efficient for capturingbroader apictureof fungal diversity. databases in represented (Lindahl biosphere. deep the in role ecological an have might and depths sediment greater at suggests(Ramaiah, knownin fungi 2006).This that 3. 4.

Fungal communities in deep sediments arenotdiverse and areclose to terrestrial Distribution patterns patterns Distribution taxa in situ in Cyberlindnera et al., Meyerozyma,Rhodosporidium Malassezia, Fusarium conditions such conditions such pressure highhydrostatic as (Simonato a 2009) and itmay also be opportunistic pathogenan of deep-sea animals Dikarya - present down oursamplesto 346mbsfin - is a includesgenus that Cryptococcus curvatus yeasts instudy were our 1711mbsf.found downto These yeasts were .

Interestingly, these deep sediment fungithese are phylogenetically deep Interestingly, et al et

(Kutty & Philip (Kutty & ., 2013). Multiple-primer and multiple-marker Multiple-primer ., 2013). as eukaryote thedominant ingene 18SrRNA deep-sea methane-rich sediments. Takishita ment depth. Our study identified 22different Our depth. ment identified study has been reported has beenin reported river and seawater terrestrial environments persist mayalso terrestrial , we discovered diversity, we that thefungal , nts (Orsi et al., 2013a). Other yeasts,Other 2013a). al., et nts (Orsi Trichosporon , 2008; Edgcomb and This is consistent with consistent is This et al., Filobasidium et al., 2006).Oneof 2011;Orsi et al were were et et ., Accepted Article (Nagano& Nagahama, 2012).Although deep-sea water columns sedimentdeep-sea samples (Amend,and 2014). Gao et al. (2008)revealed a previously andthat thought, indicating samples, like thistaxon marine that analysis skin diseases,frequentlyis also of by DNA-based recovered thisyeast agents of class yeast ubiquitous exclusively the with with caution. Fungal both from OTUsrevealed DNA- and RNA-based affiliated methods be interpreted OTUs must fungal recovery particular different, wereof in analyses variations addition andto our study, given and primers al (Edgcomb sediments toPeruMargin Canterbury contrast in the basinin oractive diverse ourDNA-basedOTUs in 18Sdataset. Asa firs OTUwas in detected 1fungal Surprisingly only microeukaryotic communities inmarine sediments. concentration – structuring as parameters andmethaneorganic –here specificmatter withcomplementary parameters environmental Fungalthat isunlikely. observation communities contamination appearthusdepth- mostly thesupportsresources but differentalso depths, communities fungal accordingat available to thedifferent depths OTUs of suggestsfungal between aspatialpoor overlap differentiationof methanogenic/ethanogenic prokaryotes in deep marine sediments (Hinrichs sediments marine deep prokaryotes in methanogenic/ethanogenic Cryptococcus subseafloor has Aalso been revealeddeep with cycle. methane ethane correlation with environmentsrich (Takishita methane- deep-sea marine in thatbasidiomycetes appearsuch dominant idea the supports and This article This protectedis byAllcopyright. rights reserved. withrepresentatives correlation of the a18Smarkers. Also,distribution patternpositive was observed with ITS1and This oligotrophicthat mayconditions. matteran adaptation organic to indicate with correlated supports correlationwithdepth organic and matter withsomecommunities the negatively materialthan isavailablein rocky subsurfa appears todecrease. li Fungal communities ., 2011;Orsi 5.

Exobasidiomycetes Many fungal OTUs may representdormant taxa and might someindicate interactionsbetween this fungus and et al ., 2013a, 2013b). Since the the RNA-based analysesSince ., 2013a, 2013b). anunanticipated were are related theto genus Malassezia 2006) and that they might be indirectly involved inthe mightinvolved beindirectly et al.,2006)andthatthey Malassezia. Malassezia. may becommon environmentsin marine that include Cryptococcus Malassezia ce horizons. Indeed, PCA of OTUdistribution Indeed, horizons. PCAof ce kely depend on a greater amount of organic organic kelya greater amountof dependon protocols used for the RNA- and DNA-basedand RNA- the protocols used for likely to influence the distribution ofthe the distribution likely toinfluence t conclusion, fungi donot appear be veryto ly wider range than niches of a occupies vs.our RNA-based 13fungal 18Sdataset, The most ubiquitous species within the most ubiquitous The species within species well as knownare causative the genus and methane has been revealedmethanehas been genus and Malassezia in deep-sea in environments et al., 2006). The 2006). et Accepted Article This article This protectedis byAllcopyright. rights reserved. sediment (Edgcomb greater depths at matter suggests that they RNA-based recoveryclone libraries also may in survive onburiedorganic suggesting deep-sea alsobeopportunisticof they pathogens Their that mammals. may Malassezia of diversity high of GDR Ecchis and the MaCuMBA Europ theMaCuMBA GDREcchis of and research, and highertheFrench education of ministry support of with the financial conducted ies scientific andshipboard part crew co-chiefs, of IODP 317. Expedition This study was the thank We IODP. support wereprovided by and expedition facilities shipboard Samples, Acknowledgments to colonize and have an ecological role in the deep subseafloor. ar indicating thatterrestrial environments, fungi in knowntobewidespread basin are inthe Canterbury subseafloor revealed fungi deep The ecosystem. inthis role ecological animportant have communities fungal that hypothesis inthisobtained work have beenpreviously thesequencesfungal some of that fact The 346 mbsf. active fungi downto of evidence to arecorddepthof 1740 using mbsf DNA-based pyrotag sequencing. RNA provided subseafloor.the fungal unexpected diversity down indeep communities Wefoundan fungal this specific ourknowledge, tothe of workisthefirst bestof description dedicated To the Conclusion fungalseem taxa colonize thedeep subsurface. Canterburyin thisfungi basinsubsurface sediments appear slightlyand active fewpersistent could V4 primers bethe complimentaryless targets.Themajorityto fungal subsurfaceof diversity data setssuggests RNA-based significantlyin the recovery taxonomic that lower of Although ourRNA-andDNA-based 18Sdatasets are not strictly thecomparable, the ecological significanceof the approa culture-independent and culture-dependent deep-seadeep-sea obtainedfromwaterand been sediments.employing Futurestudies ubiquitous presencethe of Malassezia lineages associated sponges andinvertebrates, withmarine Malassezia phylotypes deep-seaenvironments,have in no cultures group inmarineenvironment. et al., ean program (FP7, Grant Agreement 311975). Agreement (FP7,Grant ean program reported from marine ecosystems marine ecosystems from supports thereported e highlyadaptable organisms,potentially able 2011). Whereas 2011). molecularstudies suggest ches shouldrevealuseful oninformation Accepted Article metagenomes. comparison 1320): Basin (IODPsite Trinity Biddle JF, White JR, TeskeHouse APCH( & subsurface ecosystems offPeru. JF, Biddle LeverLipp JS, MA, oceans. N, Brown A, Howe D, Bass 1 Barghoorn ES & Linder DH (1944) Marine fungi: their taxonomy and biology. taxonomy and fungi: their Marine DH (1944) Linder ES & Barghoorn 75:649-675. impact on microbial community dynamics in the deepsubsurface.community the dynamics in onmicrobial impact Anderson RE, Brazelton WJ Baross JA& deepviriosphere: (2013)The assessing theviral hyper-diverse. PLoSPathogens todeep-sea Amend Adandruff ve(2014) From nts: Malassezia-like Fungi ecologicallyare analyzingmicrobial mixed populations. oligonucleotideCombination of 16SrRNA-targeted probeswith flow cytometry for Amann RI, Binder BJ, OlsonRJ, Chis 11:360-381. Microbial eukaryotesMicrobial hypersalinin the T(2009) &Stoeck MM Yakimov A, HW,J, Bunge Behnke StockA,Breiner E, Alexander This article This protectedis byAllcopyright. rights reserved. identification - of fungi recent updates and future perspectives. AbarenkovK, R, Nilsson KH, Larsson References and WHOI’s Ocean Life Institute to VE supported the RNA-based work. G.UngerFundsthe Foundation, from Vetlesen :395-467. Proc R Soc B Proc ISME J

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126:11-25.

69:2657-2663. 10:165-169. Microb

Accepted Article T.sp: Malassezia pachydermatis, Fusarium solani, Cryptococcus surugaensis, This article This protectedis byAllcopyright. rights reserved. 2. Figure 1. Figure GeolBull Pet Assoc sediments. marine bacteria in of distribution Vertical QA (1936) Anderson CE & ZoBell Tremella moriformis, moriformis, Tremella Rhodosporidium kratochvilovae, Rhi.sp: chartarum,Leptosphaerulina Figure 3. Figure (a) and ITS1 region (b). depthsSample are given in table 1. 4 Figure environmental parameters. C.c: OTUsof sediment and samples, ribution Organic P: Porosity (b) Dist Oc: carbon, environmental different parameters. D:Depth, samples) (sediment individuals representation based on454-pyrosequencingand 18Sreads parameters. environmental (a) of Superimposed guilliermondii, guilliermondii, caryae, Elmerina environmental parameters. B.l: parameters. environmental OTUsof sediment and samples, ribution Organic P: Porosity (b) Dist Oc: carbon, environmental different parameters. D:Depth, samples) (sediment individuals representation anden reads 454-pyrosequencing ITS of based on C.p: Cryptococcuspseudolongus, Trichoderma :Distribution of fungalOTUs at the species based level on DNA-based V1-V3 SSU Sampling site. Sampling Multiple Factorial Analysis Factorial (MFA) a Multiple Multiple Factorial Analysis Factorial (MFA) a Multiple P.r: L.c: sp., W.m:

20 Pleurostomophora richardsiae, Pleurostomophora E.s: T.sp: Leptosphaerulinachartarum, :258-269. Exophiala spinifera, Exophiala Trichosporon P.r: E.d: Wallemia muriae. Pleurostomophora richardsiae, Exophiala dermatitidis,F.g: Exophiala Batcheloromyces leucadendri, Cryptococcus curvatus, C.j: L.m: C.s: Leucosporidiella muscorum, Cryptococcussaitoi, Rhinocladiella Rhinocladiella sp.

Sample depthsSample are given in table 1

E:M: Ethane, Ic:Inorganic Methane, carbon, E:M: Ethane, Ic:Inorganic Methane, carbon, Sample depthsSample are given in table 1. with the contribution OTUsandthe withthecontribution of with the contribution OTUsandthe withthecontribution of nd Principal Component Analysisnd Principal (PCA) nd Principal Component Analysisnd Principal (PCA) vironmental parameters. (a) Superimposed (a) parameters. vironmental G.c: M.g: Galactomyces candidum, sp Meyerozyma guilliermondii, guilliermondii, Meyerozyma

., ., P.sp: Filobasidium globisporum, C.sp: Rho.sp: Cyberlindnera jadinii, T.m: C.j: Penicillium Penicillium Chaetothyriales Chaetothyriales Trichosporon mucoides, Cyberlindnera jadinii, Rhodotorula M.g: Meyerozyma sp., R.k: sp., sp., T.m: sp., E.c: sp., M.p: C.s: L.c: F.s: Am

Accepted Article eukaryotic V1-V3 primers (a) and fungal ITS1 primers(b). ITS1 fungal primers (a) and V1-V3 eukaryotic

This article This protectedis byAllcopyright. rights reserved. Table 4 Table 3. Table 2. Table 1. Retained of fungal of fungal Number Number Number of reads quality Sample name Sample OTUs reads

128R 125R 122R 110R 87R 34R 73X 68X 42X 2H . Diversity indices for the 18S surveyscalculatedbased on the fungal OTUs. Number of sequences and number of OTUs in the DNA-based datasets. List of studied samples and depths.

List ofoperationalunits fungal taxonomic indeep-seafound sediment using 6425 2491 18S ITS 18S ITS 18S 18S ITS 18S ITS 18S ITS 18S 4 bf 583mbsf 346 mbsf 7 2366 4625 3 Core depthbelow the seafloor (m) 4747 5868 7 1740 1711 1690 1577 1367 931 634 583 346 12 3789 1448 10 mbsf mbsf 2973 7646 634 2 td efre Target marker performed Study DNA / cDNA 18S 18S rDNA /ITS DNA /cDNA DNA / cDNA 18S 18S rDNA /ITS DNA /cDNA 2260 4521 931 mbsf 4 cDNA DNA DNA DNA DNA DNA DNA 18S 18S rDNA /ITS DNA DNA 1485 983 6 mbsf mbsf 2318 1367 919 1 mbsf mbsf 3568 4377 1577 18S rDNA 18S rDNA 18S rDNA 18S 18S rDNA 18S rDNA 18S 1 ITS ITS mbsf mbsf 1809 2866 1690 3 mbsf mbsf 2056 3399 1711

2 mbsf mbsf 1240 2659 1740 3 Accepted Article

This article This protectedis by Allcopyright. rights reserved. (Shannon) (Shannon) (a) OTU_13 346 OTU_12 346, 1690 OTU_11 346, 583 OTU_10 1740 (Simpson) (Simpson) OTU Id OTU_6 346 OTU_6 OTU_2 931 OTU_2 OTU_1 Evenness OTU_9 583 OTU_9 346,583, 634 OTU_8 346, OTU_7 1740 346, 583, 1740 OTU_5 OTU_4 583 OTU_3 Diversity Richness Richness (Chao1) (Chao1)

Sample depth Sample 583, 634, 931, 346, 583, 931, 0.73 0.73 2.11 18S ITS 18S ITS 18S 18S ITS 18S ITS 18S ITS 18SITS 18S 18SITS 18S ITS ITS 18S 18S 18S ITS 4 bf 583mbsf 346 mbsf 1367, 1690 7 (mbsf) 1690 0.42 0.42 1.04 4 0.51 0.51 1.21 10 0.47 1.71 Cryptococcus curvatus Exophiala dermatitidisExophiala Cyberlindnera jadinii 11 Pleurostomophora Leptosphaerulina Wallemia muriae muriae Wallemia Trichoderma Fusarium solani pachydermatis guilliermondii Cryptococcus Trichosporon Most similar Filobasidium Meyerozyma Meyerozyma globisporum surugaensis richardsiae Malassezia mbsf mbsf chartarum 0.16 0.43 sequence mucoides mucoides 634 2 0.12 0.36 931 mbsf 4 sp. 0.64 1.86

6 5,00E- 2,00E- 2,00E- 1,00E- 4,00E- 3,00E- 2,00E- 3,00E- 2,00E- 7,00E- 3,00E- 2,00E- value mbsf mbsf 1367 118 147 176 124 129 174 131 174 131 176 130 131 . 6/6(9) AB100440.1 0.0 360/361(99%) E 0 0 1 339/339(100%) AB491674.1339/339(100%) AY741381.1246/246(100%) AY729812.1338/338(100%) mbsf mbsf 1577 237/238(99%) AB473810.1 237/238(99%) DQ457640.1 327/345(95%) X79317.1 257/258(99%) EF550447.1 338/339(99%) AB075546.1 261/262(99%) HM216185.1 338/339(99%) AB032626.1 261/262(99%) AB001763.2 259/260(99%) JQ698913.1 261/262(99%) 0 0 1 Identity Identity mbsf mbsf 1690 0.54 0.54 1.28 3 mbsf mbsf 1711 0.02 0.07 number of the most the of number GenBank accession 2 similar sequence mbsf mbsf 1740 0.60 1.44 3 Accepted Article This article This protectedis by Allcopyright. rights reserved. (b)

OTU_18 583 OTU_17 1711 OTU_15 583 OTU_14 583 OTU_13 583 OTU_12 931 OTU_16 931 OTU_11 931 OTU_10 583 OTU Id OTU_2 583, OTU_2 1711 1577 OTU_1 OTU_8 346 OTU_8 931 OTU_7 346, OTU_6 931 OTU_5 583 583 OTU_4 346,583, 931 OTU_3 OTU_9 583 OTU_9

Sample depth Sample (mbsf) Cyberlindnera jadinii Tremella moriformis moriformis Tremella Cryptococcus saitoi Exophiala spiniferaExophiala Chaetothyriales Pleurostomophora Rhinocladiella Leptosphaerulina Batcheloromyces Trichosporon Leucosporidiella Elmerina caryae caryae Elmerina Rhodosporidium Rhodotorula Rhodotorula Penicillium kratochvilovae guilliermondii Galactomyces pseudolongus Cryptococcus Most similar Meyerozyma Meyerozyma leucadendri richardsiae chartarum muscorum candidum candidum sequence sp. sp. sp. sp. sp.

1,00E- 6,00E- 1,00E- 3,00E- 9,00E- 4,00E- 3,00E- 7,00E- 4,00E- 3,00E- 3,00E- 3,00E- 3,00E- 4,00E- 7,00E- 2,00E- 9,00E- 3,00E- value 136 133 100 104 92 44 98 55 46 73 60 59 24 35 40 86 86 50 E 188/188(100%) JN662395.1188/188(100%) KC119207.1 266/266(100%) JX188127.1262/262(100%) KF751183.1 101/101(100%) AB051048.1121/121(100%) 128/128(100%) KF811431.1 KF811431.1 128/128(100%) KC464351.1 177/177(100%) JX966556.1111/111(100%) 204/205(99%) JX270559.1 204/205(99%) FJ865435.1 201/202(99%) 122/131(93%) JF499832.1 JF499832.1 122/131(93%) JN974290.1 161/164(98%) AF042426.1 132/133(99%) JQ764655.1 125/152(82%) HQ634635.1 153/183(84%) AM901696.1 106/112(95%) FR717869.1 187/192(97%) 214/216(99%) AM231400.1 214/216(99%) Identity Identity number of the most the of number GenBank accession similar sequence Accepted Article

This article This protectedis by Allcopyright. rights reserved. OTU_15 583 OTU_12 931 OTU_10 583 OTU Id Sample depth Sample (mbsf) Rhinocladiella similis similis Rhinocladiella Rhodotorula minuta Most similar minioluteum minioluteum Penicillium Penicillium sequence 8,00E- 2,00E- 7,00E- value 62 100% SH210380.06FU 47 97% SH227573.06FU 89 100% SH225847.06FU E Identity Identity number of the most the of number UNITE accession similar sequence

Accepted Article This article This protectedis by Allcopyright. rights reserved.

Accepted Article This article This protectedis by Allcopyright. rights reserved.

Accepted Article This article This protectedis by Allcopyright. rights reserved.