Posted on Authorea 14 Apr 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161839272.27561225/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. hr il:Mtbroigbae ii eeto fEDF Reynolds of K. detection Nicole limit biases Metabarcoding samples environmental title: detec- from Short limit fungi that diverging biases early methodological of unknown: tion and underrepresented, Understudied, these missed Title: of be role may the community understand EDF to the needed context. are of ecological techniques much broader modified suggest a and in results critical approaches taxa this Our metabarcoding To of “standard” advantages Dikarya. sequences. PCR with the than unidentified misidentified exacerbated illustrate EDF and or and variation for identified reconstruction greater length investigating phylogenetic was for performed fragment effect we tool Target this amplicons but EDF. LSU assignment assignments, of and taxonomy taxonomic identification two improve downstream fungi The influenced taxonomy of software. two and AMPtk communities compared biases We with amplification different sequence compatible MiSeq. and strikingly database Illumina amplify reference with recovered to LSU samples methods example. LR22F) environmental an an 146 primer created as of new and set Zoopagomycota, with methods a lineage, paired assignment methodological and EDF LR3 and a community one and choice mock comprise on LROR/LR3, marker Zoopagomycota EDF (ITS1F/ITS2, focused DNA a We sets that how primer samples. suggests investigate environmental three fungal we studies evaluated from investigate Here multiple We EDF to from sampling. of used community recovery evidence impact in However, been diversity biases (EDF). also unknown Basidiomycota) fungi has + of diverging (Ascomycota rDNA portion Dikarya early (LSU) target large to to subunit the designed attention large is mostly little rDNA The were (ITS) with primers spacer metabarcoding problems. LSU transcribed known available internal but has The communities but communities. fungal barcode understanding fungal for accepted tool important an is Metabarcoding Abstract 2021 14, April 3 2 1 Reynolds Nicole from fungi diverging early samples environmental of detection limit that methodological biases unknown: and underrepresented, Understudied, Abstract nicolereynolds1@ufl.edu author: *Corresponding 92521 California Riverside, California–Riverside, of 3 2 1 nvriyo aionaRiverside California of University College Mary and Williams Florida of University ilasadMr olg,Dprmn fBooy ilasug igna23185 Virginia Williamsburg, Biology, of Department 32611 College, Florida Mary Gainesville, and Pathology, Williams Plant of Department Florida, of University eateto ln ahlg irbooyadIsiuefrItgaieGnm ilg,University Biology, Genome Integrative for Institute and Microbiology & Pathology Plant of Department 1 1* ihleJusino Michelle , ihleA Jusino A. Michelle , 2 ao Stajich Jason , 2 ao .Stajich E. Jason , 1 3 n ate Smith Matthew and , 3 ate .Smith E. Matthew , 1 1 Posted on Authorea 14 Apr 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161839272.27561225/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. S a ugse oprombte hnISa acd o D u ogetrPRscesada and success PCR greater to 2012). al., due et EDF (Schoch and for SSU evaluated than barcode been variation) a intraspecific have and as ITS inter- ITS and between than difference LSU, 2015; (i.e. al., better SSU, gap et perform the barcode (Hart which larger to used for suggested commonly performed was AMF is been markers LSU the copies two not early are rDNA have of Among exception more metabarcoding combination templates. for some An with mixed markers groups. individuals of from to many and comparisons sequencing direct for 2019), individual lead copy and lineages, single al., can amplification rDNA (EDF) a variation et PCR fungal wide diverging from during rDNA (Lofgren found derived dominate Interspecific mode also OTUs potentially samples. trophic study multiple could environmental with recent of in uncorrelated formation A among unpredictable was divergence the 1996). 6% variation that al., is large to Fungi such up et and ITS kingdom making found (SSU) (Lloyd-MacGilp the study across al., small spore One (AMF) variation et the 2012). single number al., Li fungi to Thi´ery a et 2010; 2018; mycorrhizal compared from al., al., variation arbuscular et sequences et intra-spore (Egan among (Bellemain and subunits example, groups intraspecific rDNA (LSU) For some high variation exclude has 2016). number which and Lindahl, copy the hypervariable biases use rDNA & primer to interspecific Tedersoo inability and 2011), therefore 2020; are 2011), al., (and et There Banik, discrimination Gazis & interspecific groups. (e.g. between of (Lindner determinations) taxonomic length lack Engelbrektson species-level 2020; ITS 2007), all al., Vivanco, for (Casta˜no in for et fragments) variability & marker shorter barcode Manter high al., for 2010; suitable including et sequencing al., lineages, Yahr a and et fungal PCR 2006; not favored some al., is in in et (resulting ITS ITS assignments groups (Nilsson the sequencing taxonomic caution with from use, with of results issues widespread interpreted that known representation its be mean poor Despite must problems These (OTUs) including 2018; However,2016). units al., 2012). problems, et taxonomic al., 2019). (Abarenkov are et operational al., annotation Nilsson there of taxonomic et 2019; incorrect databases Nilsson al., and et these 2020; sequences, Hofstetter quality al., of low the et groups, usage decades, (Abarenkov taxonomic two (UNITE widespread 1996; some approximately databases updated the reference for Bruns, and ITS barcode with & the curated a and even consistently (Gardes as developed used are well studies are been INSD) sequencing extensively data has and amplicon environmental used region processing ITS been in for the has tools particularly Because operon bioinformatic fungi, rDNA 2012). al., the for et of barcode Schoch region investigating DNA (ITS) for spacer the tool powerful transcribed as a internal is The technologies ecology. high-throughput fungal using communities fungal of Metabarcoding Zoopagomycota community, Introduction Mock context. Metabarcoding, ecological LSU, broader ITS, a MiSeq, in Illumina taxa these suggest of and results approaches role Words metabarcoding the Our Key “standard” understand the with sequences. to illustrate misidentified needed unidentified or and are missed and techniques be reconstruction identified may modified investigating phylogenetic community EDF for performed the Dikarya. tool of we than much critical EDF amplicons amplification for this reference LSU greater PCR of was LSU advantages of strikingly exacerbated effect variation an recovered this identification but length improve methods created assignments, fragment To assignment taxonomic and Target downstream taxonomy EDF. influenced methods two and and assignment biases The fungi taxonomy of samples software. primer two communities environmental new AMPtk 146 different compared with of with We set paired compatible a LR3 and as database community and MiSeq. Zoopagomycota, mock LROR/LR3, lineage, Zoopagomycota Illumina (ITS1F/ITS2, a EDF biases with sequence sets one methodological and unknown primer on amplify and three of focused to choice We evaluated LR22F) portion marker fungi We samples. large DNA diverging environmental example. a how from early an investigate comprise EDF mostly to we EDF of were Here attention recovery that has impact primers little suggests sampling. rDNA studies community metabarcoding with (LSU) multiple in LSU Basidiomycota) subunit diversity from available + large evidence but The (Ascomycota However, communities Dikarya problems. (EDF). fungal target known investigate has to spacer but to transcribed designed barcode internal used The fungal been accepted communities. also fungal the understanding is for rDNA tool (ITS) important an is Metabarcoding 2 pke l,21) diinly the Additionally, 2014). al., et Opik ¨ Posted on Authorea 14 Apr 2021 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.161839272.27561225/v1 | This a preprint and has not been peer reviewed. Data may be preliminary. u a.Fnly eue usto S Tsgnrtdfo ahpie arta eeietfidas identified were that pair primer and each from GenBank generated from 2014) OTUs sequences al., LSU additional manually et of with (Cole a subset RDP along search a the databases to used combining reference 2010) we by 2013) Finally, (Edgar, database Na¨ıve lab. UTAX al., Bayesian LSU RDP our and et new the 2007) the (Quast assignment: SILVA created al., taxonomy we and We OTU et primer communities, database. LSU fungal designed (Wang reference profiling for newly classifier) for curated the methods marker RDP the two with sole of (hereafter paired of a regions as Classifier LR3 efficacy different LSU and two the used (LROR/LR3 to compared have LSU region studies also marker for few ITS1 relatively pairs Because the primer LR22F). arthropod compared two we 2017; obligate using samples. communities, al., rDNA, are environmental fungal LSU et many they detect from Lazarus to because absent 2016; order Entomophthoromycotina or al., In include rare fun- et be not Benny Zoopagomycota to did for 1961; likely We sampled and 1959, distant 2019). heavily pathogens 1958, geographically al., been two (Benjamin have et (FL), methods locations Reynolds Florida culturing Both selective and soils. using (CA) and gi California climate in divergent microinvertebrate sites with and multiple states Orphellales), from (Harpellales, samples water (Dimargaritales, and (Zoopagales) (Asel- soil sediments collected species We freshwater gut-dwelling (Kickxellales). collection. Zoopagales), (Dimargaritales) saprotrophs arthropod Kickxellales, mycoparasites putative culture commensalistic and includes Ascomycota, (and large Orphellales), (Zoopa- and and Harpellales, Mucoromycota a Mucoromycota subphylum rotifers) lariales, the on have diverse nematodes, parasitic most we in amoebae, are the which (e.g. fungi that is microinvertebrates for host Kickxellomycotina of and The attack parasites gales). sites primarily are Kickxellomycotina study or and that Ascomycota) Zoopagomycotina our mycoparasites sometimes the from are of isolated species members regularly includes Zoopagomycotina work This been and three samples. have ha- culture, the length soil that of isolate, that sequence from taxa Out to on isolated tool. ITS difficult investigative sequence focused been are large important we reference ve they particularly is Zoopagomycotina) 4) a limited and al., there and sampling Kickxellomycotina, are available (Entomophthoromycotina, environmental et 2019), subphyla making are there Benny al., lab, data et 2) 1958; the Reynolds sequence in metabarcoding, Benjamin 2017; with which (e.g. with al., for et studies detected because: species (Lazarus culture-based readily biases variation among during not methodological 3) evaluating soil are available, for in they data 2016; case found yet test al., routinely and a et 2019). are as 2016) al., Benny Zoopagomycota fungi et in of 1958; these Reynolds found species Benjamin, 1) 2017; or some al., (e.g. absent on et are dung focused (Lazarus We species studies and some Zoopagomycota metabarcoding litter, that findings, most indicate leaf in these or methods abundance soils, Despite 2016) enrichment low 1955). in al., Specialized Duddington, parasites widespread et animals. 1938; fungal Spatafora and the includes Drechsler, small and – diverse is of diverse Mucoromycota ecologically are EDF parasites is + of taxa phylum as (Dikarya This groups well fungi 2021). as al., understudied terrestrial et (mycoparasites) other this most (Li indicate Mucoromycota all the the studies to Among to phylogenomic especially sister sister but OTUs. EDF 2016), unresolved either al., the to still is et is to belong (Grossart lineage Zoopagomycota assigned taxa of fungi” placement matter reliably undescribed The “dark can be of Zoopagomycota. these methods Powell, about can Metabarcoding portion & 2020). more Letcher taxonomy large al., learning 2015; et if a for Walsh James, 2017; tool that & al., essential et suggest Lazarus an Torres-Cruz provide sequence 2020; 2017; data Many 2017, Available no 2014, al. lab. or al. 2021). et the (Tedersoo limited et al. lineages in have Corsaro et manipulate and 2016; lineages symbionts, and Malar al. diverging obligate collect et 2019; et Early are to Benny Ihrmark techniques, 2015). challenging (e.g. standard (e.g. al., available generally using Dikarya data are et culturable to Tedersoo and not relative fungi 2015; are despite studied sequences al., EDF EDF, least target et few the divergent Stielow a among highly 2017; or are have al., none For P´erez-Izquierdo often et contain metabarcoding. 2012; fungi often for al., these performance primers that primer fungal validate fact “universal” to the generally develop used also communities to are chytrids mock EDF attempts as studies. example, during such sequencing EDF environmental underrepresented c 3 Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ape eecletdfo h Senyws”st nClfri eas hr a osadn water (protists, standing Microinvertebrates no rocks. was between there depression because wet California Vacuum water a No removed. in CTAB. from were with site (6 collected filled leaves wash” paper were tubes of “Sweeney filter mL samples clumps 50 the through mud sterile and water from although in rocks, the placed collected immediately sticks, filter were were to as samples papers used floating such filter water or were the debris the B¨uchner bottom filtration, funnel Large into the After sterile jar water. on a the present the dipping and debris collected by (CTAB) of filtration and was jars buffer sediment top Mason soil lysis some sterilized the Bromide of included mL Ammonium on and mL samples 950 Trimethyl Water water in 15-25 Cetyl embankment. the collected 2x approximately the to were sterile sample from closest samples with Water each at was filled mL. 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ITS1F/ITS4 BLAST 1994; not pair by Vilgalys (data verified primer and reconstruction were and (Hopple sequences LSU LROR/LR5 Reference community for pair ITS. final 1990) from primer and The sequences our Hester using Sanger markers. amplification in reference members and different generated variation reduced community also across length We in mock 1). known biases (TABLE resulted with fungi individual potential Zoopagomycota taxa taxa these of additional isolates these examine and 30 contained in further taxa those to length (Benjamin, fungi included community ITS1 axenically We mock the grown increased 1). both FIG were that from (SUPP single and from indicated DNA sequencing were an saprotrophic DNA on DNA study primers contained are contained soil ITS1F/ITS2 (2019) Kickxellales also also of with al. of test cultures and et preliminary Species these A methods Davis organisms. 1958). from the displacement host from multiple extracts their DNAs by DNA and genomic amplified the Similarly, Accordingly, genomes DNA. cell 2016b). host of al., of quantity University et unknown the (Benny Dimargaritales, from of fungi grown Species host 1). cultures (TABLE collaborators from from obtained received or Piptocephalis, was and collection Kickxellales, members culture Dimargaritales, Benny community Gerald of mock Florida species from of DNA DNA genomic of Zoopagales. aliquots equilibrated combining community by mock created the OTUs. in EDF fungi other Zoopagomycota taxonomy and 3) Zoopagomycota and of Methods reconstruction, identification outperform phylogenetic and the would and Materials in pair clustering differ bias Zoopagomyco- primer OTU strongly sequencing LR22F/LR3 of of would and the terms absence methods in 2) amplification assignment the pair species, length to primer fragment Zoopagomycota LROR/LR3 contributing inhibiting of 1) the length) factors detection that: fragment the hypothesized both and We decrease Are choice would studies? 3) marker metabarcoding as and rare in (such truly Zoopagomycota?, ta community Zoopagomycota biases af- of Are mock methodological pipeline 1) detection Zoopagomycota questions: Are metabarcoding a the following 2) the the created environment?, how address We the investigate to data. identify aimed to in as We ITS samples specifically. well with group environmental as that pipeline possible the fected our not alongside by evaluated is made was assignments that taxonomic that step am- the Analyzing a validate reconstructions. to errors, phylogenetic us putative in allowed them way included this and in Fungi plicons kingdom to only or Zoopagomycota and Syncephalis – ecletdevrnetlsmlsfo v ie nC n w nF (SUPP FL in two and CA in sites five from samples environmental collected We r asoilmcprstsadwr rw nda utrswt their with cultures dual in grown were and mycoparasites haustorial are – okcmuiycmrsdo opgmct ug was fungi Zoopagomycota of comprised community mock A 4 Piptocephalis and Syncephalis μ pce mixed species oesize). pore m Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hncendwt h eetaSz N la n ocnrtrkt(yo rie A S) l samples All and USA). intensity CA, band Irvine, similar (Zymo, on kit based Concentrator three and of Clean GoTaq groups DNA protocol: into Select-a-Size program following pooled the thermocycling the were with the cleaned products with and then PCR step reaction Indexed the PCR minutes. for 10 separate used for XT a 94 was Nextera at in USA) the cycles WI, USA) nine using Madison, included added CA, (Promega, were Diego, mix indexing Indices San master gels. the amplification Green (Illumina, agarose initial to the 1.5% v2 prior both on kit combined from verified were index Amplicons were for reactions. reaction replicates all attachment as the -0.2 in index same and included of and were the replicates down controls was separate step PCR LR22F Negative three a reaction. for in was program amplified temperature were thermocycling annealing samples The the temperature. that step annealing except amplification LROR best 72 initial the of the determine step for with to program elongation sequencing thermocycling final and MA, the a Waltham, that Amplification and except Scientific, modifications. 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No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eepromdfrec rmrstsprtl n h crswr xrce sdtfaeojcswhich objects dataframe as extracted were ordinations scores NMDS the OTUs. and EDF the separately of set ordina- subsets These primer on 2016). each and (Wickham, community were for ggplot2 fungal ordinations using performed entire (NMDS) plotted the 2019) were scaling and for al., community vegan multidimensional visualized et in visualize Non-metric were (Oksanen function To 2003). tions package metaMDS al., vegan fungi. and the et the all format with in (Koleff performed presence/absence to function method into betadiver compared “w” were converted the the and comparisons was with using Community EDF table calculated 2020). 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OTUs and OTU the remaining output the Any create from the To sets. sets, deleted in primer direct were data OTUs enabled three filtering both LSU UNOISE3 of the after using for number both controls with Additionally, community. negative 99% For the generated mock and datasets 2016). doubled the between DADA2 98, al., of comparisons recovery because (97, et improve DADA2 cutoffs significantly (Callahan of not different DADA2 did instead at against used data UNOISE3 was matched the comparing UNOISE3 best processing and output community for after sets) mock thresholds which composition primer a clustering evaluating community The by bp, and available. determined true 8) 200 for was were set the threshold AMPtk datasets AMPtk under training in LR22F clustering with (RDP sequences and installed outdated compatible a LROR was was ITS1 database the database and database LSU have LSU LSU bp comprehensive custom built-in species other 225 A the fungal no LSU. because LSU of than below) some The ITS length ITS1F (see Because sets. assignment for minimum The taxonomy used primer method. a method. was across with UNOISE3 length hybrid comparisons minimum the processed the shorter for were using via classifier sets 98% identification RDP primer of taxonomic the built-in 150 both the with for of and from taxonomy used length algorithm, data assigned minimum 2010) was 600 (Edgar a also that AMPtk UNOISE3 only data, were found the in ITS1F are OTUs and using the database 97% cutoffs Reads For was length ITS AMPtk. formation. threshold different contig UNITE clustering bp in several consistent the 600 processing tested standard used, for The We was downstream results a cutoff. clustering. bp best for bp, length were during the reads the used 600 errors returned above of was at bp expected are filtering truncation set two they chimera the if was of reference-based to truncated maximum length and refers set, maximum a length 2018). was the primer, al., maximum 0.5% each filtering, et of (Palmer in quality bleed 1.4.1 allowed and index v were demultiplexing pipeline mismatches AMPtk during are the nucleotide allowed data with two Raw processed to were Biology. bp data Up Genome 300 sequencing Center Integrative All Interdisciplinary MiSeq PRJNA660245). for – UF Illumina (BioProject Bioinformatics Institute the Archive the either Riverside Read with at Sequence UC USA) sequenced NCBI’s the CA, at then or available Diego, and San Research (Beckman and dimers (Illumina, Biotechnology kit chemistry equilibrated, primer XP for V3 AMPure remove USA), Agencourt using to MA, the protocol Waltham, with USA) PE purified (Invitrogen, CA, further Fluorometer were Brea, Libraries 4.0 Coulter, library. Qubit final the a for using combi > 6 0 eune faias ug,pat,adprotists and plants, fungi, animals, of sequences 200 B i dis- sim Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. fLO n R2 Tst o-uglgop hnteUA ehd(725%RPv 38.8-33.2% vs followed Ascomycota RDP to taxonomy (47.2-54% assigned were method of datasets proportion UTAX three greater methods all the a from LSU) reads than assigned taxonomy fungal (for groups but of two UTAX Fungi majority non-fungal The the followed to and to C). 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PERMANOVA within of samples results the ANOVA ficant of dispersion to B matrices sensitive the dissimilarity are of the the 2001) tests of composition, Anderson, PERMANOVA effect - community (PERMANOVA the variance EDF compare of and To analysis B multivariate fungal coordinates. permutational on the with of type analyzed were modification sample without and plot location single sampling a onto overlayed then were i itnemtie a odce ots o infiatdffrne ewe ruso neet Signi- interest. of groups between differences significant for test to conducted was matrices distance sim i itnemtie Adro ta. 06 sn h dns ucini h ea akg.Because package. vegan the in function adonis2 the using 2006) al., et (Anderson matrices distance sim B i uptidct htdseso ewe rusmycnon eut from results confound may groups between dispersion that indicate output sim 7 – h T1 aae nldd16smls(105 samples 146 included dataset ITS1F The B i tp cnri”,adboth and “centroid”), = (type sim B sim Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. sFnio h usto Tsietfida D.TeISFpie e eoee ,4 uglOU,1,757 OTUs, fungal 6,140 recovered identified set OTUs primer of ITS1F subset The the EDF. as on identified based OTUs are of analyses subset Community the or – Fungi analyses as et statistical (Dutton and content plots G:C Ordination high as fungi such these amplification, 3) reduce and/or community, 1993). that the al., features in sequence present uncharacterized fragments that have DNA may found host we shorter from 1) by competition well: amplification fungi sequence possible un- or as multiple were amplify are and identified members There not , sets. mock accurately did primer Zoopagales were taxa three isolates the all these mock but by sets, reasons Many undetected RDP primer remaining 1). all isolates with (TABLE several Across classifier with RDP unclassified database. derrepresented RDP by LSU remained RDP+SILVA the identified the or to were mycoparasites and Metazoa database members UTAX the community LSU as RDP+SILVA of mock classified the fungi the against were host of UTAX Mucoromycota few of putative that Comparison to shows mock. assigned all the less OTUs almost one in recovered recovered identified included sets dataset also LR22F primer sets the LSU that primer assignment Both except taxonomy LSU community 1). UTAX mock (TABLE the the dataset of using members ITS1F datasets the the LSU the than The community. with database RDP+SILVA RDP. mock accurate identi- and by more the method Fungi was from recovery kingdom classifier OTU community to RDP Kickxellales Mock only one The classified only and added. were had OTUs originally in OTUs dataset fungi were 21 present ITS1F in host remaining isolates the Kickxellales DNA Mucoromycota Similarly, to 21 putative genomic sequenced included. assigned though 5 communities mixed actually OTUs even fied were mock the 12 OTUs taxa returned the from nine Zoopagales also in amplified only three dataset whereas returned been hybrid community have not ITS1F mock The could were the isolates. OTUs OTUs they mock non-fungal These and non-axenic and controls primers. the Dikarya negative LSU These the the Haptista. in with or found Metazoa, OTU not Basidiomycota, The were as identity). identified 78.08% were coverage, community 72% to matches and recurvatus, identity BLAST myces identified 91.5% had method coverage, each as assignment (73% but identified taxonomy (SAR), member hybrid community Stramenopiles AMPtk the mock as The in OTUs 1). Kickxellales ITS1F (TABLE of two assignment inflation taxonomic the mirrors OTU This the communities sets. mock primer below). was Kickxellales of LSU (see to Chytridiomycota Comparison dataset assigned the datasets. but OTUs ITS1F of LSU 36 the each ITS1F, the had in for of dataset for community fewer either ITS1F mock EDF the or than Likewise, five among OTUs 3). to Amoebozoa). of (FIG taxon compared markers number (kingdom dominant LSU the Physariida both the times in for several was dominant molds had Rozellomycota fungal- slime dataset from example, and ITS1F OTUs For SAR) LR22F more the recovered and (kingdom cases, also LROR Oomycetes other datasets The In class LR22F 6). and in LROR FIG that organisms The SUPP groups 3). like 3; (FIG and OTU-rich (FIG ITS1F Cladochytriales, Zoopagales, than less sets Chytridiales, 6; Neocallimastigales, Microsporidia primer Calcarisporiellales, among Monoblepharidales, FIG three Blastocladiales, Gromochytriales, were (SUPP the from Entomophthorales, samples sets OTUs Endogonales, of and EDF two primer more sets or between recovered one primer datasets differences only all by greatest across recovered dominant The were and were LROR. 3). fewer Dikarya for 23.8%, TABLE recovered 24.1% of with classifier SUPP and orders LROR RDP LR22F, several by The for Fungi, respectively. 18.3% followed ITS1F Within ITS1F, methods, 1.9%, the UTAX for Finally, are with and 0% 5). variables hybrid with OTUs FIG these 548 OTUs the unidentified (SUPP that LR22F unidentified using datasets of bp, indicate 35.5% LSU 534 number percentage with the (ITS1F read lowest LR22F for sets versus the not primer length but EDF had all OTU ITS1F, to dataset for of assigned for similar were plots related was that negatively Correlation length sequences significantly bp). OTU of 546 maximum proportion LROR The the bp, 4). in differed 2, datasets (FIGS three groups the but Basidiomycota by Stylopage iagrscristilligena Dimargaris pce akpr fteIS rmn ie(e lgmn lsa tp:/s.oc3h) 2) https://osf.io/cz3mh/), at files alignment (see site priming ITS2 the of part lack species h oto hsmcprst.Fv te Tsfudi h T1 opgmct mock Zoopagomycota ITS1F the in found OTUs other Five mycoparasite. this of host the – npcino h T1 okcmuiysossm ieyerr in errors likely some shows community mock ITS1F the of Inspection a LS ac f10 oeaead9.%iett to identity 97.4% and coverage 100% of match BLAST a had 8 cuoaedichotoma Acaulopage Acaulopage Coemansia Zoaae) a (Zoopagales), , .tetraceros A. T.Both OTU. Cokero- Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nyOU65 L2F i o lc nacaemthn t aooi lsicto rmtetaxonomy the from 2019). classification al., taxonomic its et matching (Wang DISCUSSION clade studies a other and in by Dimargaritales place found not the as did with pipeline. Kickxellomycotina (LR22F) 2019), 6654 within al., OTU nested Only et than (Davis rather studies subphyla subphyla two two other the phylogeny, to Zoopagomycota-only contrary the Ramicandelaber polyphyletic, In Glomeromycetes OTU. as The LR22F recovered LROR. one the from are and in none OTUs but clades LROR OTUs and four LR22F Aphelidiomycetes, En- contained contained had Archaeorhizomycetes, within Sordariomycetes Orbiliomycetes Chytridiomycetes placed The and primer the the were 1, Zoopagomycetes. both in dogonomycetes in OTUs and placed from LROR placed Umbelopsidomycetes, OTUs Only OTUs Eurotiomyctes, OTUs many orders. fungal Basidiomycota, contrast, The fungal Most In other Orbiliomycetes. Orbiliomycetes. range. to (except and to matches 75-78% matches matches Chytridiomycetes the the matches BLAST the all with in close but in clade had scores Chytridiomycetes, larger resolved identity the a were had within and above) sets nested two Stramenopiles, coverage was mentioned in to 20% sequences clade resolved one matches only were protist protist had the protists but LR22F the to that LROR, The matches to OTUs for Rhizaria. BLAST identity three scores had to 100% with that identity OTUs and one BLAST LROR coverage clades, The higher LROR 81% different LR22F. than had of for fewer identity maximum OTUs had 98.86% a LROR dataset and OTUs, LR22F these LR22F the of the but majority 5), than TABLE The (SUPP 6). sequences protist near (FIG and nodes to phylogeny ( whereas the matches support unsupported bp in BLAST mostly used 186-230 bootstrap had were OTUs phylogeny and higher LSU the LSU LR22F had the of the for tips lists nodes bp 5 sites, the Backbone TABLE OTU. 216-239 ambiguous SUPP each of of and for phylogeny length results exclusion the BLAST final After shows a 7 taxa. had Figure 436 OTUs LROR. for included and characters that 1,104 alignment 7). communities contained sequence FIG EDF alignment the (SUPP of to plots separation added NMDS and were OTUs the 4) LSU in TABLE test Tukey’s observable of (SUPP reconstruction 5). also primer Phylogenetic (FIG are Sweeney by markers site LSU and from and both mud fungi and for type all and diversity mud, sample EDF and soil by water, Conversely, highest EDF three many the 5). for among all ITS1F, (FIG varied ITS1F had For across soil results Desert for differed. than Mojave types higher the diversity patterns was sample in diversity EDF but sites all greater EDF sets for However, 8). had primer richness FIG set. all samples (SUPP invertebrate across OTU primer diversity samples highest LSU richness highest invertebrate either the fungal the for overall than had lower among that was generally had showed richness samples Jacinto Fungal type soil primers. San sample but sets, and and primer Canyon site, value three set, Evey a than all primer with less across each LR22F, and similar between vs. was LR22F richness ITS1F vs. fungal fungal LROR for of and was Plots LROR statistic vs. R EDF, Mantel ITS1F highest fungal and The 0.71. for fungi comparisons. (TABLEof 0.50 in both EDF datasets than communities all for three less for the significant on was between 0.60 all (R) impact correlated were correlation significantly stronger The were sets a communities 3). primer recovered had between the site comparisons that indicating test whereas Mantel FL, clusters A). (EDF). the from site 4 sample of communities among (R CA (FIG results communities size ANOVA fungal datasets EDF, effect fungal The and LSU the CA. the the but EDF on the For 2), type on in D). (TABLE sample sets overlapped effect 4B, and primer communities (FIG site the those of of overlapped but effects each sites significant Desert dataset, FL Mojave found ITS1F given the partitioning two analyses from the PERMANOVA are additional the separately separately in with clustered whereas set distinct separated sites were CA, Jacinto primer were San FL each in and and for the Canyon sites CA Evey plots on Sweeney The from (individual based site. communities 4) sets sampling fungal EDF). on (FIG primer However, (447 based between plots 7). fungi recovered FIG NMDS 3,126 community SUPP recovered the fungal in LR22F in the and samples in EDF), of differences (369 grouping observable fungi no 2,095 recovered were LROR There EDF, were which of etdwti h opgmctn FG7.Adtoal,teHrellsaesse othe to sister are Harpellales the Additionally, 7). (FIG Zoopagomycotina the within nested B i arcsfrC eealsgicn xetfrISFst fni and (fungi) site ITS1F for except significant all were CA for matrices sim > 0.Bt rmrst eoee oohltccae fOU that OTUs of clades monophyletic recovered sets primer Both 70). – h 0LO n 0L2FOU dnie nya “Fungi” as only identified OTUs LR22F 50 and LROR 50 The 9 2 a ml.Gnrly apetp a greater a had type sample Generally, small. was ) Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. u ovral ehd fsml olcin C mlfiain eunig n iifrais However, bioinformatics. and challenging sequencing, from are amplification, data studies PCR different collection, using sample from results of datasets our methods metabarcoding variable between to to al., Comparisons due able et – not Heeger comparison was 2021; be Primer al., technology will et PacBio to (Furneaux issues by made assignment assignment offered taxonomic are reference-based taxonomic once and efforts 2018). of at that reads intensive pitfalls long markers clear the Until the rDNA overcome is example, marker. entirely multiple For curation it bioinformatics. metabarcoding of and databases, and sequencing rDNA additions sequencing within simultaneous further an in gaps improvements 2020), done as of taxonomic al., been irrespective ITS et has the problematic much with Hanafy fill Although 2019; par and 2019b). al., on al., order, curate et et small LSU Vu (Davis relatively (e.g. bring data a databases sequence can within LSU any Even lack fungal 2020). genera improve 2017, 22 to al., of et out Tedersoo 13 including 2015; EDF, Zoopagales, represent James, sequences unidentified & many (Lazarus that identification suggest Zoopagomycota alleviate data available completely and placement cannot substantial phylogenetic is methods unidentifiedsequences the reference-based reflect other and not to the did BLAST (L matches that that or problems matches best indicating best 2011) had OTU, or identification OTUs the 5) al., OTU unidentified TABLE of SUPP LSU et our (e.g. improve of GenBank (Huson to many in potential OTUs. However, MEGAN sequences the 2012). of by have Golding, classification 2008), followed & the al., (Porter BLAST on et have (Munch like Package databases Assignment region approaches, reference Statistical this assignment that for effect taxonomic primers profound Other because the data- demonstrate LSU reference results for robust the These especially a reinforces sequences, Therefore, also eukaryotic fungal-specific. 7). phylogeny less of (FIG often reduced diversity pipeline are a our our by include in made should OTUs classifications compared base Zoopagomycota taxonomic sequences of se- non-fungal the versus non-fungal placement of fungal additional accuracy supported determining with method. The of database RDP+SILVA taxonomy RDP. accuracy our hybrid the to populating the improved that using GenBank however, from members note, quences mock to the important of is 27% It recovered com- with RDP+SILVA community ITS1F 1) our mock contrast, (TABLE However, the databases In classifier OTUs. RDP+SILVA. in reference RDP LSU isolates of the the Zoopagomycota scores in to of taxonomy representation pared identification on correlation taxonomic improved impact significant substantially that greater no database implies a was LSU has This a there length 5). for was OTU ITS1F, FIG there than for relationship (SUPP rather while number datasets However, positive read refe- non-Dikarya. LSU a and length longer the shorter length than but for had the databases OTU 9), score of the between representation identity FIG in relationship greater negative species taxonomic (SUPP the Dikarya reflects and sets from likely sequences length LSU primer mi- rence for OTU LSU relationship indicate fungal negative the community The of for mock ITS1F. plots relationships the Correlation negative from 1). significant OTUs (TABLE ITS1F ITS1F Zoopagales) of than community (e.g. (e.g. assignment the sequences taxonomy recapitulated reference the closely of more sidentification in datasets Errors LSU ITS 1). both the (TABLE analyses, Because community databases. mock LSU the versus In OTUs. ITS Bellemain ITS1F the (i.e. – for of Dikarya specificity assignment completeness for primer taxonomic the biases accurate in RDP and known differences the 2015) has to by al., have but et due databases non-fungal greatest Tedersoo fungal-specific expected as 2010; the more was identified al., had is result et be combination dataset This to even primer ITS1F Fungi. likely was ITS1F/ITS2 The as the more This (33-38%). identified considera- were database sets. OTUs RDP+SILVA found OTUs primer of our We LSU number all to detection. where for compared EDF level (47-54%) methods on and Kingdom classifier taxonomy impact length the between profound fragment identifications at a target taxonomic true have particular, in choi- each In differences methodological by can communities. ble more Dikarya method impacted than assignment is sampling communities taxonomy EDF metabarcoding of during Recovery ces – OTUs of assignment Taxonomic cige l,22) h iprt ewe ae uglseisadunmdenvironmental unnamed and species fungal named between disparity The 2020). al., et ucking ¨ > ,0,0 eeec eune oprdto compared sequences reference 1,000,000 > 2 niomna ape upr h ra atr ffna community fungal of pattern broad the support samples environmental 127 Dimargaris ≤ 0 fmc ebr dnie ihRPvru 3 with 63% versus RDP with identified members mock of 10% 10 n loalc frfrnesqecsfrsm taxa some for sequences reference of lack a also and ) < 0,0 o S aaae,w xetdmore expected we databases, LSU for 200,000 Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. o eoee rmmxdsmls hswstu ept digDAfo hs pce ttiethe twice at community mock species in those reiterated from were was DNA bp) pattern initial adding This ([?]400 our despite ITS1 1). In true FIG longest 2018). was (SUPP the al., DNA This with “background” et species the Palmer samples. Zoopagomycota of 2019; mixed concentration set, al., from primer et metabarcoding recovered ITS1F the Jusino not fragments affects the 2020; shorter strongly when with al., PCR fragments) (Casta˜no during et experiments LSU occurs amplified putatively and bias preferentially strongest ITS1 the Zoopagomycotaare both ITS1F, For of (for steps. different example length at process the region target EDF: to kingdom that of beyond Orbiliomycetes)evidence detection identified to impacting be matches biases could BLAST Methodological example, taxonomy. database high For the the had 2013). of in al., (and accuracy sequences et the Orbiliomycetes reference increase (Glass the corresponding investigation well within the as further that placed sequences need indicate were the fungal that to that EDF errors of correspond OTUs divergent taxonomic utility that potential the or the clades identifying artifacts illustrate BLAST in for sequence results placed their OTUs as were These of LSU well majority resolved of class pipeline. UTAX/RDP+SILVA OTUs the reconstruction fungal the LR22F and phylogenetic the and from 7), 6). LROR matched output (FIG both (FIG that Conversely, assignment tree OTUs OTUs. clade taxonomy Zoopagomycota LROR LROR a 50 smaller shorter the in the than of placed in 15 resolution were only higher OTUs to compared with LR22F where reliable match, placed analysis 50 less phylogenetic generally be the our may were influenced of OTUs length Twenty-two OTUs LROR sequence al., LR22F that of et found identification longer Porras-Alfaro also taxonomic We 2012; the the OTUs. longer al., result, LR22F Furthermore, et a longer 2019). the (Liu As al., than 2012). methods et Golding, bioinformatic Truong & using 2011; Porter identified 2014; al., more accurately during et reads: problems more (Bartram reduce unpaired are sequences assignment over sequences longer taxonomy advantages and several and errors, reads, have clustering sequence forward reduce reads OTU the sequences paired to overlapping However, restricted utilized, entirely are loss. almost data consistently data fragments therefore were significant target are LROR dataset data in The resulting LR22F to resulting 1). The the chemistry. closer (FIG were MiSeq sets from estimates commonly Illumina primer results reads richness were LROR of Similar members recovered and reverse community ITS1F 8). which and mock the the primer, forward FIG differences both for LR22R to the SUPP significant contrary related that 4; contigs, found into the found TABLE analyses paired We for (SUPP diversity LROR. (2016) LROR fungal dataset than al. with LR22F ITS and et Zoopagomycota found The and Mueller LROR not pipeline. broadly, by than bioinformatics fungi LR22F found OTUs the amplify for total in to groups and ability performance between their their fungal compare for more as pairs had well primer as LSU specifically, test fungi to aimed accuracy. further analyses. for These dataset, We phylogenetic evaluated our 2016). in with be Rozellomycota tested easily et Vˇetrovsk´y (e.g. al., Taylor markers cannot be refining between 2018; 2015; et 3) cannot representation and al., in FIG al., OTUs et differences errors ITS Palmer dramatic et with of methodological 2011; al., groups Nguyen assignments al., Thus, detecting (Casta˜no et et taxonomic 2019; for Furthermore, studies (Caporaso al., 2016). thresholds communities ITS metabarcoding clustering al., et mock in as of such of Jusino communities number parameters importance 2017; mock bioinformatic the the al., in of underscore taxa et inflation results methodological various Filippis Artificial and/or for variation) De factors. number shown OTUs. 2020; threshold) copy 12 been recovered rDNA identity but has intraspecific clustering taxa (e.g. OTUs nine OTU of biological OTUs added numbers of inappropriate of originally greater result number we (e.g. a the community; reported mock inflated be (2018) than marker the could al., detected in ITS1F This et were Kickxellales the Benucci OTUs Conversely, the and similar ITS. LSU to were than (2019) recovered assigned more there LROR Shaw which However, datasets with and for 6). LR22F OTUs Nelson orders FIG Chytridiomycota and NMDS Similarly, chytrid (SUPP Our 3). LROR, some lineages al., 2019). (FIG ITS1F, like OTU-rich et al., ITS1F groups, the Johansen same et rarer 2014; Xue the that al., among 2019; mostly demonstrate et discrepancies al., detected 7) Brown et and 2014; Skelton FIG found communities al., 2019; and SUPP fungal et Shaw, 3) 4; Bonito & (TABLE 2019; Nelson (FIG tests al., 2019; plots Mantel et al., si > 0 p(AL ) elbyn h eunebelength sequenceable the beyond well 1), (TABLE bp 700 11 – efudadditional found We Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hr ogrfamnsaddi nqa rprin osotrfamnswr eeeyunderrepresented communities severely mock were in fragments shorter pattern and/or to a lengths proportions such fragment unequal found target rooted in variable (2020) are added highly al. biases fragments with Casta˜no et These longer organisms others. representation. of where of group sequence absence any reference symbiotic and affect outcomes, poor groups (with can metabarcoding some and heterogeneity on of methodology impact sample representation in synergistic inflated environmental a artificially have and to may currently biases leading abundance) lower than methodological having distributed likely an of widely Zoopagomycota be effects more and could and combined 3) reports (FIG diverse The of soil more dearth from be the detected may 7). but be arthropods clade (FIG can et rare, with recognized Kickxellales the Kurihara be that association 1943; indicate to demonstrate Linder in analyses assumed results 1948; phylogenetic are or be Our Deardon, species to fungi undersampling. & assumed most of other (Jackson Furthermore, are artifact culture on samples, fungi in 2008). growing Kickxellales growth mud 2001, species Although fastidious or al., isolated exhibit some unclear. be water species of remain can many from and modes reports and axenically OTUs trophic are grow their Harpellales Kickxellales there but column of saprotrophic, detect water dung, Species the or to OTUs. in soil chytrid unable transmission by from through one were dominated hosts least mostly we between at were pass However, contained which to the Florida) thought Benny than from are 1986). example, less fungi (mostly (Lichtwardt For Harpellales samples still of soil techniques. is spores 520 one culturing detected the recovered of only specialized OTUs 46% we analyses using of but that phylogenetic species, sites found number and these (2016) set, the al., from primer of Nonetheless, number recovered et each chance small by isolates 7). our types A of (FIG sample increase number 3). other identification thereby (FIG from their dominated and detected Mucoromycota generally supported were and fungi were OTUs Blastocladiomycota samples these well? 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(Drechsler Chytridiomycota soil potential that Baermann material by concentrate the using species plant by decaying to in Zoopagales them and Similarly, attempted hosts dung. detecting moss We like attack from soil? example, substrates isolated the actively wet For frequently in 1955). they from are persist isolated do straightforward. also spores been or parasites, less they have their soil, Zoopagomycota coprophilic, do metabarcoding from from long among mainly for detection isolated How dynamics species types their be host/parasite sample these of can the probability and Are species these mycoparasitic about the locations of the lowering known of distribution although time, is choice the through Little result, the variable a making As highly sample. interactions. and single abundance host/parasite patchy the any with on likely linked dependent is is sample and given fungi organisms any in host symbiotic abundance The their their considered. that of be means also fungi must Zoopagomycota Zoopagomycota of of nature biology the for bioinformatics, among and length sequencing range community,Beyond fragment the shortest mock ITS1F. the Likewise, our the for Although ITS1F. In and bp for 1,100 ITS1F. longest 247 have bp 2001). for versus the that Lichtwardt, 508 than between Harpellales versus & lower some length LR22F (Gottlieb is in for fragment variation ITS1 as in the such for difference ITS1, Zoopagomycota, extreme bp the than among have 500 longer variable occurs is only Harpellales variation is ITS2 length but of LR22F cases Similar ITS2 some Species 2001). in for Lichtwardt, & and bp results. (Gottlieb among region bp ITS1F ITS2 variation 250–1,000 the the found of in range also in ITS1 have overrepresentation an we with their variation and to 2019) contributed the al., likely However, et Reynolds 1). 2017; (TABLE al., ITS1 an et example, have For bias (Lazarus examined This we ITS1 and taxa. species have Basidiomycota, Zoopagomycota may detected. between most Ascomycota, Cantharellales not hand, of (e.g. were other exceptions species the bp) notable ( Many ([?]400 few regions a ITS1 biased. ITS1 with longest taxonomically short the also have with is Mucoromycota species fragments target short where towards 1) (TABLE results Coemansia Piptocephalis Syncephalis sltshdsotrIS hnmn te okmmes which members, mock other many than ITS1 shorter had isolates < 0-0 p Bleane l,21;Bklc il,2013), Mills, & Bokulich 2010; al., et (Bellemain bp) 200-300 and T rmtosmlsuigteLO rmrst Likewise, set. primer LROR the using samples two from OTU Syncephalis 12 pce aeIS htrne 0 0 bp 800 – 300 ranges that ITS1 have species Piptocephalis > ,0 p–Fiemne l,19) On 1994). al., et Feibelman – bp 1,000 Syncephalis > 0 pwt infiatvariation significant with bp 300 pce a 5b o LR22F for bp 85 was species pce a ny4 bp 45 only was species Coemansia Syncephalis species Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. mn,A . efr,K . asn . rn,T .(00.Ido uglcmoiini geographically tropics. is America the composition of in fungal States than Indoor United (2010). zones the D. temperate T. of in Bruns, Sciences diverse & R., O. more Samson, Ovaskainen, and A., patterned & K. transcribed Seifert, N., internal S., fungal Abrego, A. of Amend, T., placement Huotari, taxonomic M., probabilistic P. for sequences. Kirk, tool spacer web-based H., a R. Protax-fungi: Nilsson, (2018). P., Somervuo, Kessy, Abarenkov, Fungi for P dataset T., TE+INSD Piirmann, A., the Zirk, for K, Abarenkov, PRJNA660245 the BioProject and at analyses, data. (SRA) supplemental archive data References the Illumina read for for 1. Table sequence PRJNA428770 in used available NCBI’s and are dataset scripts on paper main available The this R in (https://osf.io/cz3mh/). are reported OSF AMPtk, sequences sequences from Sanger raw in download the for for processing numbers available accession data are GenBank file raw FASTA database for manuscript LSU RDP+SILVA the used wrote notebooks and NKR bioinformatics Jupyter co-authors. MAJ. all the from from conceived input Statement help MAJ included Availability troubleshooting and and Data MES with NKR California. by from MAJ. data edited samples of thoroughly the collect was assistance analyzed and which reserves the NKR to with and access samples work analyses arrange collected lab NKR helped research. JES performed the for and NKR funding Florida provided JES and and California MES research. from the planned MES and NKR Desert Contributions Station Mountains Author Biological Ordway-Swisher Granite System Florida Florida. Sweeney of County, Benny Reserve University Putnam and Natural the Gerald in 10.21973/N3KQ0T) at California (https://ordway-swisher.ufl.edu/) from and of (doi: 10.21973/N3S942) input University culture (doi: Reserve the Center the and Mountains Research with at guidance helped Jacinto performed and San the collection was culture rDNA James appreciate sampling zygomycete Nematology reference environmental the also generate and to The We to access Mycology work. community. provided used (USDA who mock samples Florida) Davis DNA of the J. (University Zoopagales in William sharing Florida. to for and and of 1441715 Lab) University Michigan) sequences Biology the DEB of at and MES; (University Sciences Diversity Agricultural Genetic to James award and Tim 1441677 under Food USDA-NIFA thank for (DEB the Pathology, Institute We Foundation Plant the of Science and Department the FLA-PLP-005289 National by number provided the was by funding Additional supported JES). was 2016). work al., et This (Grossart fungi” help matter are can “dark approaches methods of Acknowledgements novel sampling roles popula- al., environmental but ecological et host Mamanova improved and mysteries on (e.g. diversity approaches, these capture effect the culturing hybrid of illuminate their on-array targeted some PCR-free and with as unravel webs Combined such help 2010). food biases, to methodological microbial overcome potential in questions to the unanswered needed roles Many has their studies. indi- Metabarcoding as metabarcoding involved Rozellomycota), such in tions. similarly Blastocladiomycota, misidentified remain, severely are or (e.g. Zoopagomycota sequences EDF missed data reference often about of sequence likewise of lineages reference (Tedersoo are lack Other lack they method and samples. cating and bias loading still mixed length associations sample was from PCR symbiotic by Zoopagomycota bias both in influenced of length that be detection a demonstrate also the discrepancies, results can impact severe Our that less 2017b). output effect al., produced greater an et 57% sequencing 2020), to II (up al., overrepresented (Casta˜no RS et highly detected PacBio be Although could fragments input). shorter than while results MiSeq Illumina in e Phytologist New NT omnt.https://doi.org/https://doi.org/10.15156/BIO/786372 Community. 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Data may be preliminary. ahdtsti itdaoetebxsadtenme fttl(..fna n o-ugl Tsfudafter found OTUs non-fungal) and fungal for (i.e. returned total below. reads of given number total is the of control and contigs quality number form boxes and The to the merged filtering LR22F/LR3). above were listed and that is LROR/LR3 reads reverse dataset (ITS1F/ITS2, and each set forward doi: of primer (https://osf.io/cz3mh/); percentage each average OSF the from 2020; of fungi: M.E.; plots in Violin Smith, 1. diversity J.E., Figure Stajich, hidden M.A., of Jusino, discovery N.K., up Reynolds, Scaling [dataset] (2016). M. T. B. approaches. 371 Dentinger, fungal Q., barcoding of & Shen, of analyses L., Q., on Impacts Huang, C. databases W., reference Schoch, Ran, and R., markers B., Yahr, genetic Zhang, ITS M., and Zhao, LSU Q., of Wang, Effect C., communities. (2019). Penton, (Eds.), M. Ryan J. White X., Tiedje, Pu, J. & Y., T. Hao, C., & Xue, Sninsky, J. (2016). J. Gelfand, ps://ggplot2.tidyverse.org H. 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(pp. 1,7–8 https://doi.org/10.1007/s00374-018-1331-4 79–88. (1), species. 21 ple n niomna Microbiology Environmental and Applied , nPress In ora fBacteriology of Journal tde nMycology in Studies . BioRxiv pigrVra.htt- Springer-Verlag. . 000.3179.htt- 2020.07.03.187096. , , , 172 154 oeua Ecology Molecular 8,4238–4246. (8), 3–5.htt- 135–154. , Mycologia , 73 PCR (16), , , Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ycpai cornu Syncephalis moniliformis Piptocephalis itcpai cylindrospora Piptocephalis xerosporica Dimargaris interrupta Coemansia omni erecta Coemansia pseudoplumigaleata Syncephalis californica Syncephalis microcephala Piptocephalis cruciata Piptocephalis thaxteri Coemansia aciculifera Coemansia parentheses). parentheses) (in ITS1F) (outside (for taxonomy method numberIsolate classifier hybrid the RDP AMPtk list the the columns or by primer LSU) detected (for the fragment database member and LSU community target RDP+SILVA LSU), the mock the (ITS, versus each numbers including + com- accession of taxonomy sets, GTR GenBank OTUs OTU primer the (bp), of community LR22F/LR3 pairs using mock and base 8 of in LROR/LR3, results v length ITS1F/ITS2, and RAxML the members shown. in across community are include parisons mock performed [?]70 and Zoopagomycota were supports bolded Analyses Branch 1. are Table replicates. numbers classified. the bootstrap Zoopagomycota was and 1,000 as OTU included and identified were each model OTUs datasets which GAMMA study. LR22F to this and re- order LROR from the including the isolates OTUs of sequences sequenced all each LSU newly of from Zoopagomycota list species and of a GenBank reconstruction has from phylogenetic 5 ferences TABLE likelihood matches. SUPP Maximum sequences BLAST clades. 7. protist their protists” diverging to Figure to with early matches match along indicate GenBank “BLAST phylogeny Asterisks had grey the if that red. in dark in colored OTUs in The performed included are of IDs not. fungi were comprised OTU do of are Analyses they the Classes clades of if with replicates. bolded. these grey each bootstrap are that shaded 1,000 from indicates or numbers and “Fungi” OTUs shading model the as LSU GAMMA only unknown and + include identified GTR included they the OTUs were using 50 8 datasets study. v LR22F RAxML this and from from references LROR isolates including the sequenced sequences LSU newly fungal and of (colors) reconstruction site GenBank phylogenetic by likelihood set Maximum primer 6. each Figure for fungi diverging (shapes). early type for dataset. measures sample each diversity and for alpha listed of and Comparison are locations 5. values sampling Figure Stress different diverging set. represent California early colors primer all Point and indicate for sites. LR22F/LR3) shapes fungi sampling LROR/LR3, point environmental all (ITS1F/ITS2, (FL) for sets Florida for plots and primer type (CA) the ordination sample by (NMDS) by recovered scaling communities fungi fungal multi-dimensional diverging early Non-metric of 4. phylum Figure a each is taxonomy. to Glomeromycotina ITS assigned set. that the reads primer Note with of each comparison dataset. abundance each for Relative the separately in 3. indicate categorized OTUs Percentages Figure separately. is D) data but (B, LROR Mucoromycota fungal the the resulting and of bp, within C) graphs 283 subphylum each additional (A, of to contains length non-fungal whiskers a assigned 4 and of with FIG reads height reads proportion SUPP of forward plot line. of proportion Box comprised flat the D). entirely a represents (C, almost in width was classifier hybrid set plot RDP the primer box the LROR number using the The and phylum read group. whereas B) fungal range, (A, and and length method length Kingdom OTU LSU) by represent OTU (for assignment UTAX in taxonomic or LR22F/LR3) to ITS1F) according (for LROR/LR3, OTU (ITS1F/ITS2, each variation to set assigned Primer 2. Figure + M 312319 IMI RLA54 (61) A-5447 NRRL M 214463 IMI CC34489 BCRC B 826.97 CBS RL2694 NRRL A23985 RL3178 NRRL RL13723 NRRL B 418.77 CBS S 2659 RSA S71 22 407 335 2 5 8 G660 G6630()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (0) 0 MG764623 MG764680, 783 455 427 728 216 5 3 6 G660 G6690()1()1()0()0(1) 0 (1) 0 (1) 1 (1) 1 (0) 0 MG764609 MG764650, 764 437 287 457 1 363 433 412 1 6 8 1 1 1 1 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) 1 (1) 1 (1) 1 (0) 0 MG764611 MG764652, 773 689 446 365 778 527 412 bp length ITS1F/ITS2 + ˆ + ˆ + + ˆ ++ § 0 1 T035 Y0830()0()0()0()0(0) 0 (1) 2 (0) 0 (1) 2 (0) 0 (1) 3 (0) 0 (1) 3 (0) 0 (0) 0 KY001803 KT601335, MG775651 MG764647, 813 780 501 468 4 6 N464 N8920()0()0()0()0(2) 0 (2) 0 (2) 0 (2) 0 (4) 0 JN982932 JN942674, 568 261 241 361 503 0 1 Y067 Y0740()0()0()0()0(0) 0 (0) 0 (0) 0 (0) 0 (0) 0 KY001764 KY001697, 817 503 R2/R eghb RRL3lnt pGnakAcsin# Accession GenBank bp length LROR/LR3 bp length LR22F/LR3 + + + ˆ 592 687 845 700 + + + ˆ Y075 Y0760()0()0()0()0(0) 0 (0) 0 (0) 0 (0) 0 (0) 0 KY001776 KY001705, Y903 Q7710()0()0()0()0(0) 0 (0) 0 (0) 0 (0) 0 (1) 0 DQ273791 AY997043, ¶ 0 2 2 1 (1) 0 (1) 0 (2) 0 (2) 0 (0) 0 0 0 0 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 T1 TsLO TsLO TsL2FOU R2 OTUs LR22F OTUs LR22F OTUs LROR OTUs LROR OTUs ITS1F Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ug Site fungi FL ITS1F Sample x Site fungi CA ITS1F Sample fungi CA ITS1F Site fungi CA ITS1F type PERMANOVA sample by with type, (˜300) and sample (ITS1F/ITS2, short and FL) Typically set site Florida, between primer interaction and each the ( as p-values CA by well significant recovered as (California, indicating water), communities asterisks states and the (EDF) soil, within mud, of fungal invertebrates, sites analyses (including diverging by ANOVA early LR22F/LR3) whether and LROR/LR3, and fungi PERMANOVA all of for Results study. 2. this Table sequences, for rDNA of sequenced available exception newly with the isolates species with indicate only numbers species, the same with on the Lengths based of are sites. isolate sequencing, different priming amplification, a more after on or recovered one were missing that sequence identifications OTU processing. to bioinformatic and refer “isolates” remaining The + “Fungi” or fungi) “Unclassified” Dikarya protists, (animals, Non-zygomycetes Kickxellales Unidentified ( Hosts Zoopage odontospora Cochlonema Acaulopage Zoopage acanthospora Acaulopage pectospora Zoophagus hadra Stylopage coronantispora Pinnaticoemansia yatsukahoi Myconymphaea scoparia Mycoemilia obconica Syncephalis digitata Syncephalis graefenhanii Piptocephalis Coemansia Coemansia tetraceros Acaulopage parasiticus Tieghemiomyces depressa Syncephalis Isolate slt ae nbl niaeseisfrwihgnmcDA eecmie omk h okcommunity. mock the make to combined were DNAs genomic which for species indicate bold in names Isolate + oeoye,Cnigael,Riou,Umbelopsis Rhizopus, Cunninghamella, Cokeromyces, p Zo2 sp. C3 sp. p S 2604 RSA sp. 1933 RSA sp. p Ap sp. B35 .77005 .59000*yes 0.0001* yes 0.0001* 4.5509 no 0.0001* 3.0065 0.0001* 0.0855 4.5242 0.19658 8.8969 1.3797 0.08068 9.149 0.21154 3.755 1 9.845 11 R squares Sum 3 4 freedom of Degrees 51(K12) S521 BC100468 NBRC 27(K10) S227 16(4) S116 B39 T2 E1 Ac1 S99022101 BC100467 NBRC S 861 RSA ++ egh with Lengths B 131509 CBS < 0.05). ˆ niaeta h ubri netmt u otereference the to due estimate an is number the that indicate 23 2 + niaeta h eghi netmt based estimate an is length the that indicate .bacillispora D. iagrsxerosporica Dimargaris ) au value P value F nnw 6 1 0 0 0 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) (0) 0 0 (0) 0 (0) (0) 0 0 (0) 0 (0) (0) 0 0 (0) 0 811 794 677 461 461 354 Unknown 326 Unknown nnw 8 2 0 1 1 1 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) (1) 0 0 (1) 0 (1) 0 (1) (1) 0 0 (0) 0 (1) 0 (0) (1) 0 0 (1) 0 (1) 0 (1) 0 (0) 0 (1) 0 KY001806 KY001695, 721 746 808 389 683 416 733 370 Unknown 458 354 Unknown 416 Unknown Unknown 270 281 271 2 5 0 U166 Y0780()0()0()0()0(0) 0 (0) 0 (0) 0 (0) 0 (0) 0 KY001788 KU317676, 808 698 (˜300) short Typically 458 370 544 246 483 Unknown 227 321 280 186 219 Unknown bp length ITS1F/ITS2 B . ˆ ˆ + + i uptwr significant were output sim ¶ oddGnakaccession GenBank Bolded o hc estimates which for ++ § 9 3 Y063 Y0760()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (1) 0 KY001766 KY001683, 833 491 1 8 0 0 0 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 789 745 416 421 522 R2/R eghb RRL3lnt pGnakAcsin# Accession GenBank bp length LROR/LR3 bp length LR22F/LR3 significant? ANOVA ˆ + B sim 698 848 ˆ ˆ + ˆ ˆ X153 X2090()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (1) 0 JX128019 JX312513, A B8000()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (0) 0 AB288000 NA, A B8990()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (0) 0 AB287999 NA, A B8980()0()0()0()0(1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (0) 0 (1) 0 AB287998 NA, (0) 0 KF848916 NA, ¶ 7 0 0 0 (0) 8 (0) 0 (5) 5 (0) 8 (0) 0 (5) 5 (0) 4 (0) 1 (5) 5 (0) 4 (0) 1 (5) 5 (1) 0 (1) 0 (7) 0 (7) 0 (4) 5 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (1) 0 (0) 0 OTUs LR22F (0) 0 OTUs LR22F OTUs LROR OTUs LROR OTUs ITS1F 1()2 0 1()1 0 (1) 9 (0) 11 (0) 11 (0) 21 (0) 21 Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. D Sample EDF FL LROR Site EDF FL LROR Sample x Site EDF CA LROR Sample EDF CA LROR Site EDF CA LROR Sample x Site fungi FL LROR Sample fungi FL LROR Site fungi FL LROR Sample x Site fungi CA LROR Sample fungi CA LROR Site fungi CA LROR Sample x Site EDF FL ITS1F Sample EDF FL ITS1F Site EDF FL ITS1F Sample x Site EDF CA ITS1F Sample EDF CA ITS1F Site EDF CA ITS1F Sample x Site fungi FL ITS1F Sample fungi FL ITS1F PERMANOVA .730338579 .01 yes no 0.0001* 0.0001* yes 5.7291 yes 0.0001* 6.4159 0.0001* 0.34388 0.0001* 2.0418 0.12837 4.3787 no 0.0001* 3.4713 5.4378 0.15779 no 1.2958 0.09228 0.0001* 2.6082 0.1528 0.0001* 8.899 3 yes 5.205 4.399 0.15512 0.0001* 1 yes 8.618 4.4197 0.0001* 0.26163 11 2.1557 2.8612 0.0001* 0.08762 3 yes 5.6033 3.6358 4 no 0.0007* 0.19185 7.6923 1.2176 0.0001* 3 0.10247 0.0001* 2.0617 9.542 0.18756 no 3 4.1235 yes 0.0001* 5.096 1 4.7734 0.1205 0.0001* 9.328 0.241 11 0.0001* 2.7287 yes 0.093 1.9184 3.8844 3 0.0001* 3.8367 7.4519 0.19306 4 0.0001* 1.4805 0.07495 3 2.4872 0.19172 9.746 3.7382 3 3.784 0.14018 1 9.678 0.2107 11 2.2622 3 3.4 4 R 3 squares Sum 3 freedom of Degrees 24 2 au value P value F significant? ANOVA B sim Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. RRv R2 ug .43006 .44001 .610.0771 0.0667 0.0740 0.0698 0.0543 0.0601 0.0641 0.0545 0.0637 0.0598 0.0487 0.0518 0.0481 0.0539 0.0532 0.0483 0.0408 0.0424 0.0373 0.0436 0.0389 0.0356 0.0318 0.006* 0.006* 0.0345 0.006* 0.4483 0.006* 0.006* 0.7101 fungi LR22F vs LROR 0.4716 fungi Significance LR22F 0.006* vs ITS1F 0.5849 statistic fungi LROR R 0.5603 vs ITS1F EDF LR22F vs 0.5397 LROR EDF LR22 vs ITS1F EDF LROR vs ITS1F set primer Spear- each using of Comparison OTUs matrices (EDF) ( p-values distance fungal significant the diverging indicate between early asterisks analyses and correlation, correlation fungal rank the man’s for for LR22F/LR3) results LROR/LR3, test (ITS1F/ITS2, Mantel 3. Table Sample x Site EDF FL LR22F Sample EDF FL LR22F Site EDF FL LR22F Sample x Site EDF CA LR22F Sample EDF CA LR22F Site EDF CA LR22F Sample x Site fungi FL LR22F Sample fungi FL LR22F Site fungi FL LR22F Sample x Site fungi CA LR22F Sample fungi CA LR22F Site fungi CA LR22F Sample x Site EDF FL LROR PERMANOVA .140327606 .01 no 0.0001* no 0.0001* 0.0001* 3.3381 yes 6.0965 0.0001* yes 0.16583 6.0836 0.0001* 0.30287 0.0001* 2.1516 0.10074 2.2544 3.5568 yes 0.0001* no 4.1174 0.16648 5.7059 1.3695 0.07506 0.0001* 0.0001* 3 2.7758 0.16055 9.68 3 yes 4.364 4.9772 0.14753 5.1877 0.0001* 1 yes 9.335 0.0001* 0.26452 11 2.1189 0.0919 3.2395 0.0001* 3 5.6324 3.7993 4 0.0001* 0.20461 9.1561 1.32 3 0.09702 2.7925 9.274 0.2103 3 4.397 1 0.16761 9.531 11 1.692 3 R 4 squares Sum 3 freedom of Degrees + 25 0 unie9%qatl 75 unie9%quantile 99% quantile 97.5% quantile 95% quantile 90% 2 < 0.05). au value P value F significant? ANOVA B sim Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. + -ausajse o utpecmaiosuigteHl ehd(om 1979). (Holm, method Holm the using comparisons multiple for adjusted P-values C A OTU Length bp OTU Length bp 200 200 300 400 500 300 400 500 Average % merged reads 30 60 90 0 17.3% 0% ITS1F 7,609 OTUs 14,233,141 reads Total 33.2% 38.8% 54%

LR22F ITS1F LR22F 47.2% LROR Kingdom RDP Kingdom UTAX/hybrid Viridiplantae Rhodophyta Rhodophyta Fungi Excavata Eukaryota Fungi Filasterea Eukaryota Filasterea Haptista Hacrobia Metazoa Metazoa SAR SAR Choanozoa Viridiplantae Amoebozoa Apusozoa Amoebozoa 10,028 OTUs 7,403,607 reads Total 26 200 300 400 500 200 300 400 500 D B 80.7% 100% ITS1F 27.7% 28.5% 31.2% LR22F 5,786 OTUs 9,135,843 reads Total 36.2% LROR LROR Fungal Phylum RDP Fungal Phylum UTAX/hybrid Not assigned Blastocladiomycota Basidiomycota Mucoromycota Glomeromycotina Chytridiomycota Rozellomycota Entorrhizomycota Not assigned Blastocladiomycota Basidiomycota Mucoromycota Microsporidia Zoopagomycota Zoopagomycota Ascomycota Glomeromycotina Chytridiomycota Ascomycota Aphelidiomycota Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Phylum C A Rozellomycota Mucoromycota Microsporidia Blastocladiomycota Glomeromycotina Chytridiomycota Zoopagomycota NMDS2 NMDS2 Aphelidiomycota -0.25 −0.4 −0.2 0.00 0.25 0.50 0.0 0.2 0.4 −0.40.2 0.0 0.2 Florida early diverging fungi California early diverging fungi

Relative Abundance 1000000 1500000 2000000 500000 -0.25 0.00 0.25 0 Invertebrate NMDS1 NMDS1 Mud ITS1F Soil 0.4 0.6 Water B D 1000000 2000000 3000000

-0.25 NMDS2

NMDS2 0.00 0.25 0.50 -0.50 -0.25 0.00 0.25 0 -0.50 -0.25 0.00 Invertebrate Mud -0.50 -0.25 0.00 California allfungi Florida allfungi 27 LR22F NMDS1 Soil NMDS1 Water 0.25 1000000 2000000 2500000 0.25 0 Invertebrate Mud Location Primer Fungi stress: 0.06929218 EDF stress:0.07588488 Fungi stress: 0.1281375 EDF stress:0.1502559 Fungi stress: 0.1429781 EDF stress:0.1556327 Fungi stress: 0.1168332 EDF stress:0.1367924 Fungi stress: 0.08640903 EDF stress:0.05850112 Fungi stress: 0.09405482 EDF stress:0.07262848 Location Primer Sweeney 2 Sweeney 1 Sweeney Wash San Jacinto Evey Canyon LROR LR22F ITS1F LROR Goose Lake Blue Pond LROR LR22F ITS1F Soil Water Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary.

Alpha Diversity Measure 100 150 10 20 30 40 50 20 40 60 0 0 0 LROR ITS1F LR22F Observed Observed Observed 100 150 10 20 30 40 20 40 60 50 0 0 0 Chao1 Chao1 Chao1 Samples 0 1 2 3 0 1 2 3 4 0 1 2 3 Shannon Shannon Shannon 28 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 Simpson Simpson Simpson Substrate Location Water Soil Mud Invertebrate Sweeney 2,CA Sweeney 1,CA Sweeney wash,CA San Jacinto, CA Goose Lake,FL Evey Canyon, CA Blue Pond, FL

Posted on Authorea 14 Apr 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161839272.27561225/v1 — This a preprint and has not been peer reviewed. Data may be preliminary.

ATCC16579 minor var. Dentiscutata heterogama DQ27379213188 haptosporus Basidiobolus

Scutellospora heterogama AFTOL ID138 Rhizophagus intraradices FJ23557113086 Dentiscutata heterogama INVAM FL225

Jimgerdemannia flammicorona KPMNC0024738 Paraglomus occultumAFTOL ID844

Acaulospora colliculosa GU3263391

Archaeospora trappei S003841656 Paraglomus occultum S003824525

MH8737831 macrocystopsis Mortierella

Geosiphon pyriformis AM1839202 Rhizophagus irregularis DAOM229456

Gigaspora margarita S004127012

AF0098521

Diversispora celataAM7134192 casparyana Entorrhiza EF3924221

Diversispora celata pHS006 01 meristosporus Basidiobolus

S000622265 brassicae Olpidium

CBS130.62

Sphaerocreas pubescens LC4311101 microsporus Basidiobolus

MH8685881

Glomus etunicatum S003857707 parvispora Mortierella

Gigaspora margarita BEG152

DQ2737941 verticillata Mortierella

CBS432.76 cola fi Glomus intraradices MUCL 43194 tur Mortierella

Rhizophagus fasciculatus BEG53

S003872849 jenkinii Mortierella

CBS157.71 kuhlmanii Mortierella

KP4130811

Jimgerdemannia lactiflua LC4311011 tenuis Entorrhiza

EF3924281 serpentis Schizangiella

KC0183281 Endogone pisiformis KPMNC0024229 alpina Mortierella EF3924191 ranarum Basidiobolus

NRRLA5450 lepidula Piptocephalis

CBS418.77

Protomycocladus faisalabadensisUmbelopsis JN2065581 ramanniana S000622244 microcephala Piptocephalis RSA1564 tieghemiana Piptocephalis

IMI210884 pseudocephala Piptocephalis

RSA2468

Umbelopsis gibberispora CBS109328 lepidula Piptocephalis D98122701 Umbelopsis longicollis S003857285 freseniana Piptocephalis RSA2551

Umbelopsis autotrophicaEndogone CBS310.93 incrassata LC1073621 lepidula Piptocephalis

Brazil2

Glomus sp MUCL 43206 Diversispora sp EE1 platyclados Piptocephalis sp. NRRLA10083 sp.

Cunninghamella bertholletiae JA6068584484035 Piptocephalis

BCRC34206 Apophysomyces variabilis JN9806991 formosana Piptocephalis Fennellomyces heterothallicusSaksenaea trapezispora JN2065391 LT6074071

LROR OTU2626

Thamnostylum lucknowense JN2065461 OTU1679 LROR LROR OTU1205 LROR OTU3048 LROR

LROR OTU2016 LROR LROR OTU3050 LROR LROR OTU2835 LROR

LROR OTU3137 LROR

CBS826.97 cruciata Piptocephalis

LROR OTU5731 LROR LROR OTU3431 LROR LROR OTU876 OTU1183 LROR

Mycocladus corymbiferus FJ3453501 LR22F OTU8379 OTU875 LROR

LROR OTU2661 LROR

LROR OTU733 LROR

NRRLA12035 indica Piptocephalis NRRL66062 brijmohanii Piptocephalis

LROR OTU507 LROR

LROR OTU5816 Horse Idaho Horse Umbelopsis ovata CBS499.82 tieghemiana Piptocephalis

BCRC34516 indica Piptocephalis

sp. Sky0202 sp.

LR22F OTU1998 Piptocephalis

RSA1275

LR22F OTU2454 indica Piptocephalis

BCRC34715 mbriata fi

Circinella angarensis JN2065511 Piptocephalis

LR22F OTU336

sp. CBS945.95 sp.

Gongronella butleri JN2066071 Piptocephalis

NRRLA21622 brijmohanii Piptocephalis RSA1396 unispora Piptocephalis

RSA1397

SporodiniellaSyzygites umbellata megalocarpusAbsidia FLAS-F-62758 panacisoli JQ0432301 NG0639481 trachypus Piptocephalis

sp. BennyS2-5 sp.

BackusellaAbsidia variabiliscalifornica KC0126581 EU7363011 LROR OTU1539 Piptocephalis

RSA525 cylindrospora Piptocephalis

RSA737 trachypus Piptocephalis RSA2659

Rhizopus delemar MH8666671 cylindrospora Piptocephalis

RSA786 cylindrospora Piptocephalis

CBS794.97

PilobolusRhizopus crystallinus lyococcus JX6445171 KJ4085621 debaryana Piptocephalis

CBS260.77

ChoanephoraCokeromyces infundibulifera recurvatus JN2065141 NG0588131 debaryana Piptocephalis

sp. S114 sp. Piptocephalis

BLAST match to protists to match BLAST

NRRLA10082 Glomeromycetes xenophila Piptocephalis S98092309 xenophila Piptocephalis

Uncultured soil fungus clone FunCON4 02H FunCON4 clone fungus soil Uncultured NRRL13723

Anthracocystis andrewmitchelliiBenjaminiellaMycotypha youngii JQ9953701 indica MH8738481 NG0641351 moniliformis Piptocephalis

MucorEllisomyces racemosus anomalus JA6068602263946Pilaira moreaui NG0673651 JX6445141 M

Macalpinomyces muelleri KX6869491 Endogonomycetes 1 B o

sp. HTS P14 HTS sp. Piptocephalis

Endogonomycetes 2 a

r

Geminibasidium hirsutum S003870098 s t

sp. NRRL26521 sp. Piptocephalis

i

i d

Paratritirachium cylindroconium CBS838.71 Umbelopsidomycetes e CBS100869 curvata Piptocephalis

i

o

r

CBS100814 graefenhanii Piptocephalis

b

e CBS927.95 indica Piptocephalis BCRC34436 graefenhanii Piptocephalis

o

Symg0108

Meredithblackwellia eburnea NG0583331 curvata Piptocephalis l

l l

o

o

sp. FD1 sp. Piptocephalis

m CBS950.95 mbriata fi

Rhodotorula mucilaginosa KY1091371 Piptocephalis

SymmetrosporaRhodotorula chungnamensispseudomarina KJ7012141 AY9539491 Mucoromycetes m

LR22F OTU2179 y

c

y

e

Phragmidium tuberculatum KJ8419191 c B39

LROR OTU1847 pectospora Zoophagus

Cystofilobasidium infirmominiatum CBS323 t

LROR OTU3006 e

B38 pectospora Zoophagus e

Maravalia cryptostegiae KT1994011 s

LROR OTU3054 t

e

Ac1

* acanthospora Acaulopage

Ac3 acanthospora Acaulopage B35 hadra Stylopage

Naganishia vishniacii KY1086251 s B36 hadra Stylopage

*

T2 CerinomycesTremella crustulinus globispora S000451434 CBS6972 tetraceros Acaulopage Geminibasidiomycetes * Zoopagomycetes

*

Zi2

Geastrum smithii KF9885751 insidians Zoophagus sp. C3 sp. * Zoopage

sp. Ap sp. Acaulopage

E1

* odontospora Cochlonema NRRLA13833 plumigaleata cf.

LROR OTU853 Syncephalis Pucciniomycotina *

sp. Zo2 sp. Zoopage

NRRLA-23985

Sebacina incrustansLROR FJ6445132 OTU2235 californica Syncephalis LROR OTU2199 LROR

BCRC34472

Craterocolla cerasiLROR AY5055421 OTU4134 pyriformis Syncephalis

Tulasnella eremophila KJ7011891 OTU3765 LROR

Sistotrema albopallescensLROR KHL11070 OTU1531

NRRL6268

Tremellomycetes cornu Syncephalis

DTSTM Sistotrema confluens AY5867121 intermedia Syncephalis STDTFEBa

BLAST match to protists depressa Syncephalis S114

Lactifluus medusae KR3641981 depressa Syncephalis

SCANSAa

Neolentinus cyathiformisRussula KM4114771 solaris 559 * sphaerica Syncephalis NRRL6269

MarchandiomycesAbortiporus marsonii biennis NG0594341 FD319 aurantia Syncephalis NRRL6286 intermedia Syncephalis

S439-M11 Tomentella pulvinulata BAFC52370 parvula Syncephalis AFTOLID 142 AFTOLID elegans Rhopalomyces AFTOLID 136 AFTOLID

Abundisporus mollissimusTomentella NG0570151 botryoides bacillispora Dimargaris

NRRLA-16106 cornuta Dispira NRRL2924 Ganoderma enigmaticumThelephora NG0581561 sp. IR 2013 parasiticus Tieghemiomyces

Trechisporasp. MPM 2013 LROR OTU2720 LROR

BCRC34191

PsathyrellaTrechispora candolleana nivea AFTOL AY5867201 ID1507 linderi Coemansia sp. RSA2424 sp. Coemansia

sp. RSA1694 sp.

Crucibulum laeve TUB011564 Coemansia

AB287993.1

Cyathus striatus AF3362471 aciculifera Coemansia

BCRC34190

Boletellus deceptivus KP3276191 A furcata Coemansia NRRL5531

gar furcata Coemansia

Xerocomus badius Xb2 icomycetes

sp. S171 sp.

Marasmiellus synodicus AY6394351 Coemansia

0.3

avoconia fl Amanita

Marasmius anomalus EF1600861 FJ8900411

Amanita muscaria Amanita FJ8900381

texasorora sp. Amanita

MN614386 MN614386 RSA2049 Coemansia sp.

Bolbitius titubans Bolbitius JX9683691 JX9683691 RSA1939 Coemansia sp. 29

avostellifera fl Clavaria BRACR16695 BRACR16695 RSA1358 Coemansia sp.

Camarophyllopsis schulzeri Camarophyllopsis

AM9464151 AM9464151 NRRL3115 Coemansia spiralis

*

Hebeloma mesophaeum Hebeloma IB2005309 IB2005309 RSA720 Coemansia sp.

Hymenogaster vulgaris Hymenogaster AF3362581 AF3362581 RSA522 Coemansia sp.

Hebeloma sacchariolens Hebeloma

BP B B BP Kick IMI279145 Coemansia erecta Hebeloma sacchariolens Hebeloma

KT5915611 KT5915611 xellom AF031068.1 Spirodactylon aureum

Entoloma vinaceum Entoloma

TB8870 TB8870 ycetes NRRL1564 Coemansia reversa

Tricholoma frondosae Tricholoma AM9464721 AM9464721 BCRC34628 Coemansia asiatica

Archaeorhizomycetes Tricholoma_apium EL37 99 EL37 NRRL2693 Kickxella alabastrina

Sphagnurus paluster Sphagnurus AY2073041 AY2073041 NBRC100470 Pinnaticoemansia coronantispora

* Calocybe gambosa Calocybe AM9464141 AM9464141 NBRC100467 Myconymphaea yatsukahoi

Schizosaccharomyces octosporus Schizosaccharomyces NG0661861 NG0661861 AF031066.1 Martensiomyces pterosporus

Protomyces inouyei Protomyces NG0424061 NG0424061 NRRL2925 Dipsacomyces acuminosporus

Taphrina deformans Taphrina

DQ4709731 DQ4709731 H NRRL3781 Linderina pennispora

* Neolecta vitellina Neolecta

DQ4709851 DQ4709851 ar BCRC 31802 Linderina macrospora

nlayi fi Archaeorhizomyces

JF8360221 JF8360221 pel OR-14-W25A Orphella pseudohiemalis

OTU1667 LR22F OTU1667 lom NBRC100468 Mycoemilia scoparia Archaeorhizomyces borealis Archaeorhizomyces

FG15P2b FG15P2b E y Spiromyces aspiralis

Pichia fermentans Pichia

KY1088121 KY1088121 nt ce NRRL3067 Spiromyces minutus Saccharomyces cerevisiae Saccharomyces

LC0943861 LC0943861 om tes CA-18-W17 Bojamyces sp. Scheffersomyces stipitis Scheffersomyces NG0426371 NG0426371 OR-11-W8 Pteromaktron sp.

Orbiliomycetes o Wilcoxina mikolae Wilcoxina

WS36 WS36 p NF-15-5A Harpella melusinae

Urnula hiemalis Urnula

JX6698671 JX6698671 * ht S003865602 Genistelloides hibernus na aurantiopsis na fi Wol

JN0120181 JN0120181 ho KF8489091 Smittium culicis Tuber spinoreticulatum Tuber

NG0599191 NG0599191 r NG0600761 Ramicandelaber taiwanensis

Tuber verrucosivolvum Tuber

NG0597301 NG0597301 om ARSEF6175 Ramicandelaber longisporus Orbilia auricolor Orbilia

AY2611231 AY2611231 Chytridiomycetes y EF3923891 Furia americana

Arthrobotrys elegans Arthrobotrys

FJ1768641 FJ1768641 BLAST match to protists ce EF3923841 Pandora delphacis Monacrosporium shuzhengense Monacrosporium S003827888 S003827888 JN9391821Erynia radicans

Sordariomycetes te Dactylellina robusta Dactylellina

S003827891 S003827891 s EF3924001 Zoophthora phalloides OTU3604 LR22F OTU3604 EF3923771 Massospora cicadina

OTU3586 LR22F OTU3586 GQ2858741 Entomophthora planchoniana

*

OTU4841 LROR OTU4841 EF3923951 Entomophaga maimaiga

OTU3547 LROR OTU3547 KC1210581 Conidiobolus paulus

OTU4126 LR22F OTU4126 JX9466931 Conidiobolus undulatus

*

OTU3368 LR22F OTU3368 JQ0047921 Conidiobolus coronatus

Eurotiomycetes

OTU2374 LR22F OTU2374 Chytridiomycetes EF3924011 Batkoa major

Brachyphoris brevistipitata Brachyphoris HQ1107021 HQ1107021 JX2425911 Batkoa gigantea

OTU2802 LR22F OTU2802 OTU413 LROR Aphelidiomycetes

OTU3390 LR22F OTU3390 OTU373 LR22F

Microdochiella fusarioidea Microdochiella

S004606307 S004606307 OTU1708 LR22F

OTU2208 LROR OTU2208 OTU3056 LR22F

OTU1260 LROR OTU1260 OTU2378 LR22F

OTU3126 LROR OTU3126 OTU889 LR22F

Vermispora spermatophaga Vermispora

S003854464 S003854464 OTU3084 LR22F

OTU3248 LR22F OTU3248 OTU1843 LR22F

OTU2952 LR22F OTU2952 OTU773 LR22F

OTU2685 LR22F OTU2685 OTU2253 LR22F

OTU2653 LR22F OTU2653 OTU2070 LR22F

OTU3214 LR22F OTU3214 OTU1177 LR22F

Tetracladium globosum Tetracladium

HAILUO215 HAILUO215 OTU761 LR22F

cariarum fi Botryotinia

NG0673701 NG0673701 OTU1711 LR22F

Bifusella camelliae Bifusella

KF7974501 KF7974501 OTU3519 LR22F

Xylaria grammica Xylaria

JQ8626081 JQ8626081 OTU3241 LR22F

Apiospora setosa Apiospora

AY3462591 AY3462591 OTU3095 LR22F

OTU2799 LR22F OTU2799 OTU2434 LR22F

Cercophora atropurpurea Cercophora

SMH2961 SMH2961 OTU1714 LR22F

Apiosphaeria guaranitica Apiosphaeria

MG5733361 MG5733361 OTU3001 LR22F

Clonostachys pityrodes Clonostachys

GJS9526 GJS9526 OTU3201 LR22F

Fusarium solani Fusarium

FJ34535213830 FJ34535213830 NG0672751 Spizellomyces acuminatus

Trichoderma harzianum Trichoderma

MF13025 MF13025 S004058249 Fimicolochytrium jonesii

Trichoderma peltatum Trichoderma

CBS127115 CBS127115 DQ27382613589 Triparticalcar arcticum

Toninia sedifolia Toninia

AY3008681 AY3008681 OTU2307 LR22F

Chaenotheca phaeocephala Chaenotheca

KF1579841 KF1579841 OTU1822 LR22F

Chaenotheca trichialis Chaenotheca

JX0000851 JX0000851 OTU5379 LR22F

Hypotrachyna caraccensis Hypotrachyna

DQ9123361 DQ9123361 OTU4046 LR22F

Xylographa opegraphella Xylographa

KJ4623661 KJ4623661 OTU3880 LR22F

Nannizzia corniculata Nannizzia

KT1552281 KT1552281 OTU3437 LR22F

Gymnascella littoralis Gymnascella

NG0578101 NG0578101 OTU4294 LR22F

Aspergillus citocrescens Aspergillus

CCF4001 CCF4001 OTU1878 LR22F

Eurotium amstelodami Eurotium

JF9220301 JF9220301 OTU3817 LR22F

Elaphomyces granulatus Elaphomyces

KT2322171 KT2322171 OTU8142 LR22F

OTU1188 LROR OTU1188 DQ27378513155 Dendrochytrium crassum

lcdu arboreum Placidium

EF6437651 EF6437651 MH9339701 Rhopalophlyctis sarcoptoides

Clavascidium umbrinum Clavascidium

EF6437491 EF6437491 JN9409991 Allochytridium luteum

a perforans a fi Knu

CBS885.95 CBS885.95 EU8285091 Nowakowskiella sp.

Cladophialophora boppii Cladophialophora

CBS126.86 CBS126.86 OTU1976 LROR

Cladophialophora subtilis Cladophialophora

CBS122642 CBS122642 NG0602511 Kappamyces laurelensis

Trinosporium guianense Trinosporium

NG0426821 NG0426821 NG0275661 Boothiomyces macroporosum

Xylona heveae Xylona NG0661601 NG0661601 DQ2737841 Entophlyctis helioformis

Symbiotaphrina buchneri Symbiotaphrina

FJ1768871 FJ1768871 NG0276191 Batrachochytrium dendrobatidis

KF8162291 Abrothallus parmeliarum Abrothallus KF8162291 OTU1509 LROR

Aliquandostipite khaoyaiensis Aliquandostipite

EF1756491 EF1756491 OTU1790 LROR

sp. Coreomyces

KY3505001 KY3505001 OTU1317 LROR

agellata fl Laboulbenia

KY3505381 KY3505381 OTU4779 LROR

a helicomyces a fi Tubeu

DQ7676541 DQ7676541 FJ8041531 Mesochytrium penetrans

Acidiella americana Acidiella

LT6272411 LT6272411 MH6264961 Zygorhizidium affluens

a cylindrica a fi Chlamydotubeu

NG0662301 NG0662301 NG0602501 Lobulomyces angularis

Fusicladium proteae Fusicladium

JN7125511 JN7125511 OTU3554 LR22F

Diplodia seriata Diplodia

KX4642811 KX4642811 OTU4179 LROR

Arthonia dispersa Arthonia

AY5713811 AY5713811 KJ6681211 Monoblepharis hypogyna

Lecanactis submollis Lecanactis

NG0602571 NG0602571 KJ6680981 Monoblepharis polymorpha

Mycosphaerella pneumatophorae Mycosphaerella

FJ1768561 FJ1768561 DQ27380413972 Oedogoniomyces sp.

Taeniolella pyrenulae Taeniolella

KX2449761 KX2449761 OTU2728 LROR

Cenococcum geophilum Cenococcum

1-1-5 1-1-5 OTU1859 LROR

Aquastroma magniostiolata Aquastroma

AB8075101 AB8075101 OTU5666 LROR

Epicoccum viticis Epicoccum

KY7422721 OTU1150 LROR

macrospora var. medicaginicola Ascochyta

CBS112.53 CBS112.53 OTU3750 LROR

Didymella vitalbina Didymella

FJ5156331 FJ5156331 DQ2738221 Neocallimastix sp.

Neocucurbitaria cava Neocucurbitaria CBS115979 CBS115979 AJ8644751 Orpinomyces sp.

Pyrenochaeta lycopersici Pyrenochaeta CBS26759 CBS26759 JX9672741 Amoeboaphelidium sp.

Pleospora typhicola Pleospora MUT4379 MUT4379 KY2496411 Aphelidium desmodesmi

Pyrenophora phaeocomes Pyrenophora AFTOLID 283 283 AFTOLID JX50729824085774 Amoeboaphelidium protococcarum

MFLUCC17 0783 Alternaria hampshirensis Alternaria 0783 MFLUCC17 OTU4118 LROR

Curvularia eragrostidis Curvularia KP6987271 KP6987271 OTU1935 LROR

AFTOLID 271Cochliobolus sativus AFTOLID OTU2011 LROR OTU1412 LROR