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Home , Endogonales, Mucoromycota, Mucoromycotina, Pandora (fungus), Spermatophyte

Lawrence Berkeley National Laboratory Recent Work

Title Phylogenomics of Endogonaceae and evolution of within .

Permalink https://escholarship.org/uc/item/2gr101xp

Journal The New phytologist, 222(1)

ISSN 0028-646X

Authors Chang, Ying Desirò, Alessandro Na, Hyunsoo et al.

Publication Date 2019-04-01

DOI 10.1111/nph.15613

License https://creativecommons.org/licenses/by-nc-nd/4.0/ 4.0

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California 7 6 5 4 3 2 1 3136 Na Title ty Article DR GREGORY DR YIN doi: lead todifferencesbetween thisversionandthe been and through typesetting, thecopyediting, proofreading pagination This article has for accepted been * USA. INRA-Grand Lorrain 94720 48824 (0000- 0020) Ying Corresponding Department of Microbiology Department of P Department of P nationalInstitut de la recherche US Department of P Department of

Accepted Article3 , Lau Dep 10.1111/nph.15613 Cha : Phylogenomicsof Endogonaceae and - 8903) 6 0002 , Matthe USA USA USA. G CHAN e artment of Energy ra Sandor ng (0000-0001- “Interactions Arbres pe : -7183-1384) 3,4 Es , FrancisM MICHA w t, 54280, Champenoux, France G (Orci Regular Manuscript autho lant lant lant, E. Smith(0000- 3 , Ann EL and Microbial , University Pathology, University of Florida, Gainesville FL r. Emai Soil d ID : an a B 89 Martin (0000-0002-4737-. Martin 1 Lipzen ONIT d Plan an J 72- oin 0000 d Microbia l: [email protected] T / an 1557) t O (Orci Microorganismes“ Genom d Plan t 3 0002 , Alici ag Pathology, Oregon State University, Corvallis, OR - 0001 publi ronomique 1* -0878-0932) d ID t , Alessand a e Insti l -8972-1557) Pathology cation andcation Clum Sciences : ev 00 tute, 3 olution of mycorrhizaswithin Mucoromycota , Kerrie Barry 00-0002- , Unité de mixte recherche Desirò (0000-ro Desirò , Michig Version of Record. Please Version of , University , University Wa 7 Laboratoire d’excellence ARBRE, Centre , Gregory unde 3715) lnut of California Berkeley, Berkeley, CA 7262 rgone an 5 Creek, CA 9 el: (+1) , Jas State University, 3 , Igor V. Grigoriev (0000-0002- B -8978) of California, Riversi onito on E.Stajich(0000- f ull 0001 5419087198 pe 2 , Jos 32611, er review buthaser not review 4598 -1347- eph W. pro In USA. USA. ra-Université de USA USA cite cess, which may whichmay cess, Eas 2843) Spat t this articleas Lansing, MI de, CA92521 0002 973 2 , Hyunsoo afora afora 31 USA. - 7591- Mycoplasma- Keywords: ectomycorrhiza, , Endogonaceae, Mucoromycota, mycorrhizal, whether Endogonales originated around the same time land as did. unknown whether Endogonales possess genomes mycorrhizal-lifestyle with signatures and Endogonales were among the earliest mycorrhizal partners with land plants. It remains association withnon- some plants. It been has recently proposed that known non- with ectomycorrhizal members. They also form mycorrhizal-like Summary Accepted: Received: Accepted ArticleEndogonales (), composed of Endogonaceae and Densosporaceae, is the only ● ● ● plants and plants might have played an important role during the colonization of land by origin of Endogonales suggests that the mycorrhizal association between Endogonales Endogonaceae genomes possess typical signatures of mycorrhizal lifestyle. The early in three Endogonaceae genomes. in the mid-late . Endogonaceae originated in the - boundary and Endogonales originated but without elevated SSP/secretome ratios. Dating analysis estimated that Endogonaceae have large genomes with repeat high content, low diversity of PCWDEs, Endogonales with expanded taxon sampling. phylogenetic placement of Endogonaceae and estimated the ages ofEndogonaceae and cell-wall-degrading-enzymes (PCWDEs) and small secreted proteins (SSPs). inferredWe annotated theEndogonaceae genomes. Weperformed comparative analysis on - shotgun genome sequencing. After binning the metagenome data, we assembled and sampledWe from four Endogonaceae collections and performed 9 9 June 2018 29 October 2018 related endobacteria (MRE), plant-cell-wall-degrading enzymes .

Mycoplasma -related endobacterium sequences were identified contain al. membersof Mucoromycota, one ofthe phyla formerly Jimgerdemannia, and (Smi (SSPs (CAZymes siz number of genomic innovations throug Ectomycorrhizal which areformed predominantly inclu (Bon approxim (Walker (Desi 1990; al., environmental samples In terrestrial ecosystems from nitrog Mycorrhizal fungi Mycorrhizasare one ofthe mos Introduction (Berch & symbionts Desi , 2016). Endogonales additi Accepted Articlee from 2011 rò th etal., fante & de thearbuscular mycorrhizasof Glomeromycotina (Mucoromycota) an rò etal. t ) that en, manganese, calcium he Walker, 19 h convergent et al. One ofthe enigmaticmore Ecologically, Endogonales zy on, both families inclu ; Desi et al.,2018).This ho F ately ex ) inv gospores ) associate ortin, 1983; Walker, 198 m s , 2013; Hirose pansionsof transposable elements of some vascular plants, were resolved G t , 2017; Orcha 2013). Endogonales comprises ay functi plan rò et al. enre, 20 in olved in 90% ofterrestrial plan ( ( 85; e.g., EcM) lifestyle has p t rovide in in ( ev Fig Yamamot d with decayed , , 20 Den on as fer t 10; oluti ( h p , as Bonfante & ar . S1), one ofthe rare cases form of glucose form lant ns, , 17; sosporaceae wi e unique in v rd etal. et al. finding t an w on (Martin he eff Orch ce ell assome arbuscule-forming , etc.) an der ho ectors in de considerable phylogenetic o etal. , 2014; Rimington ll wall degradati t an comm s , 2017), an ard etal. b t are characterized as Heijden is g d metabolic modificati y 5). Recently plan G roupsof EcM fungi is ev based me enre, 2010). T , 2017), biotrophic t t d enhanced water uptak ha olved independently et al., on symbioses species t t mbers e and he mycorrhizal symbiosis t with numerouswith elemental nutrients th De some members , 20 et al. on short rDNA d wereplac t 2016; wo families, Endogonaceae wit 17; nsospora t o hus are central p for , 201 ( on, and l f an itte t TE), adecrease in he et al., 2015),an Wa m o Strullu-Derri wo main an as r orrott f spec 5; Brundrett my sporo lker etal. on d liverworts p cla Ec d art ofthe Endogonales and Sphaerocreas corrhizal symbioses with ed into a n Earth ies iversificati sy M fungiof woody ons including ssified as E sequences for c ndogonales. These fungi mbionts arp producti n en Polyporacea fi g g d umerous rou m ne endophytes roups ofmycorrhizalroups symbioses iversity . Itis , 2018). e. In s en etal. in layers in d . mall truffle-limall p ofGlomus d putativ & ) (Bidarton ew ca ( es zy on of small sec Te Kohl ret of som rbohydrate activ alone, tim gomycetes dersoo k t urn they nown only imes among imes an in an , 2018), which involves on with Mucoromycota er et ated that n (Desir e saprotrophs utrient e non- e basidiocarps p however, and will h Endogo crease Planticonsortium lants (Warcup, t , 2018). d ectomycorrhizas ( do etal. a enue, ke sporocarps e.g., phosphorus, , 2015). l. cla r ò etal. ( eceiv ( Spatafor fung de as cyc reted Bidartond from d genome the fine rootthe fine a fung re ling ling , 2011; e enzymes ne and carbon e carbon i , 2017) w proteins i ( ell non- in a et o et t hat hat . a , Mucoromycota, but thelac (Krings zygospores Strullu-Derri fossils, based on , vesicles Glomeromycotina (Fiel among from EndogonalesBecause collections of Endogonaceae, trophic determinati hornworts facilitated by symbioticfunctioning of thes lycophytes benefi (1975) Glomeromycotina, afinding fossilized fungi share remarkable similarity fossilsof the (Bonfante & Desirò, 2017; Desi b to Endogonales relationshipsare facultative forthe to harb of Glomeromycotina. (Tedersoo & However, the functi structures Accepted Articleeen increasing f resh field-collec t or Mycoplasma and t that from To address It is It is important to mention In e et al. he cology cology in Endogonales additi c e an ( its microbiome. onsistent on and o colonizati arlies v , 2012 en, 201 b S R f Endogonaceae an t d thei mith, 2017),mith, as eing he hynie Chert , Glomeromycotina an on tonumerous independen intere der my t ). There arehyphal fossils t forms of the mycorrhizal symbiosis, possibly line a r placement on of Endogonales es 4). The Heijden corrhizal symbiosis. The number of hypotheses In w on of terrestrial ecosystems ted by ted sporocarps, thus are difficul -related endobacteria ( sts sts in age affiliati d et al., 2015a; H ith mycorrhizal functi b oth Endogonales k p This study rovide evidencerovide offungi un t olde of fossili et al. additi ha e fungi. inc fung d derstanding o the functions t in Mucoromyc an luding s an lends s t , 2015).Genome data be will essential for understanding t i, arefrom on difficult (Strullu-Derri t to obtain d Mucoromycot onal phylogenetic ha definitive Endogonales d arbuscules, date to approxim f ungi, theMREare supported as zed d Mortierellomycotin t as Mucoromycota also includes t had threemain rò et al. oysted upport forthe hree spores sociated with woody an t t he an on w he t t d Glomeromycotin E on, including f MRE) a he Triassic origins ith extanith rom d maintain ota of fungal symbionts cM collectionscM and ssociati g t et al. , 2018). d he ene a istributi ssociated with the materi haveled to a, b ev R in in rated genome data are metagenomesof each y , hynie Che we sequence as 2018). , mycorrhizal symbioses ma olution of mycorrhizal symbioses t t he on ofEndogonales hy t sociated with early hei objectives: 1) arbuscul rkers on and an pothesisof Pirozynski and g in culture, r hypha reen f a a ossils, whic en etal. d dateto approxim Rhynie Che mantle of hyphae of mantle an (Mucoromycota) and there have . Ec the hypothesis rt that p plan lants remains undetermined a ev a ar mycorrhizasof M d the there are som one putative s e and ately oluti o old as , 2014). species t t f hornworts, t p he have be lin hese fungi Use genome-scale hylogenetica h include diagnostic g on ofthese enigmatic spor eage m 4 a rt Glomeromycotina enomesof four rbuscul 10 Ma(Kenric w or olderthan lan al ith f orm es. While these en interpreted as t makes plants. Thed plants. ha are liverw ately sa ay have been e species w t they m t ar mycorrhizas sy protroph. d aHartig ancien ere sequenced lly mbiotic 240 Ma a Mallo orts specialized definitive an an k t. ay be k & d an d ch ch Plant nown t Net he d . Endogonales determine the ages 2) plants. Determine whethe Glomeromycotina to tes phylogenetic oligos. Standard cDNA Ki GMNB39 an library Endogo with end with using sporocarps menziesii stan and collec Pinu FL, USA 59071 was Fou Genome sequencin MATERIAL of Endogonaceae harb communities evoluti substrates symbiosis degrading including

Acceptedt Article (QIAGEN). m r Endogonaceae collections were selected forgenom J. J. library s strobus Ill a ted from 10% lacti CTAB-bas on has umina HiSeq3 k ne sp. FLASF-59071, a150-b Transcriptome sequencing ( i February t larg . EcM fungiwithin Ascomycota an flua (KAPA en an sodium hypochloride an c w w d En AN ollec . These data c zymes d sh an as constructed wi as ere at Veglio, e genome size dueto expansions,TE reducti d atBent n OSC 162217 was so D METHODS ot b sociat alyses RNA was purified from dogo ed protocol following il unde il ted from use ow astriking b 201 iosystems een characterized in a phylogenomic o ( g, assembly annotation and d for f the mos PCWDE), an 000. ne sp. FLAS ed with or t 8); libraries on County, OR o confirm P t r Piceaabies and emonte, It MRE symbionts. whethe For each of DNA DNA Jimgerdemannia an a dec r Endogonaceae genomes ) and be used t En t th N d p recent ayed log were generatedfrom the c dogonaceae sporocarps ecrease in reparati r Endogonales d diversificati ollec F t - he w aly 59071 ew Eng d flash frozen w p pair-end as USA , USA p as J. J. ted from (September 2013); o compare the origin co Muj lacement s f t p lammicorona AD002, J. equenced with Illumina HiSeq2500 sequencer. at otal RNA using mm on. The sporocarps Picea pungens atHas erformed for . Total RNA ic land ( the Ordway-Swishe fl P d Basidiomycota evolve February ammicorona CWDE, but theecologic ( on ancestors 2015). on of a l Biomedical NEBNex w ibrary n o f Endogonaceae within as w a pproximately ith liquiith a SS F w 2015). For each collecti J. or J. potential symbiont J. J. Ps tha s w as ho ma e sequencing: p f an as lammicorona AD002 was collected purified aradigm. 3) Characterize the microbial ( ex in on in J. J. lacti w MRCAs d determine whether metagenomes gnetic d nitrog c flammicorona o trac onstruc w signatures t f Endogonalesto lett, might functionin ere r Biologic flua fl t ted using he diversityhe of plant-cell-wall- ammicorona 40 b ) ofEndogonaceae and cleaned and mRNA using MI, OS eads containing en. T d from t al -year old -year ted using kit an C 162217 contex U Endogo AD002, SA SA o otal al f the lifestyle,EcM Mu o t GMN Stati d was d he f theearlies ( Septemb ecomposersof plant coromycota and DNA DNA t on, two to three Pseudotsuga R GMNB39 andGMNB39 t sp. FLASne sp. of Endogonaceae t su he from J. J. B39 was the on near Melrose, ha Neasy , a150-b t fl he rfac s t KA w ammicorona equenced Illumina of p as extracted myc soil under er 2015); PA-Illumina PA-Illumina oly- e sterilized P t lant land orrhizal p pair- T F - Mini included a dataset of predicted protein et al. of thefinal genome assemblies the remaining the bla and regi and region), with the the contigs 2012) to generate assembly Salzberg, 2 v2. removal ofnon-target sequences, the remaining searches of dataan analysis. Additional contigs sequences m potential MRE sequences in 2015) an from VizBin bacterial genome sequences database an Sequencesof the ten were subjec 2012) with ‘ al. HiSeq Trus arker sequences Accepted Article, 2011). on ofassembly a t eq Stranded RNA , 2008; he f 2500 database of 347 fungal proteomes ou stx opti ( Genom Original raw Raw Laczny latter r known d onefrom Mortierellomycotina (A ag d showed strong 012). w int sequenc ains d weredivide ted toBLASTn Holt & -- genomicIllumina reads ere removed from met composed of mainly on. Non-fungal contigs o two regions co e annotati et al. t The The map ntigs th f MR a’ ormer containing w Y e National Center for Biotechnology v er. The rawRNA-S option , 2015). Weals andell, 2011). facilitateTo protein 3 were use ere remove r E genomes mo eads w L T ere considered the final working st-frequently-hi on was ped reads k d into 1- it. Sequencingit. of the cDNA w se t affi o g w as v – g ere aligned to assembly w arches 3. A ere rem nities sembly ere enerate w ene space an a d toquery d from fro p ssembly as erformed using Vi kb use T spiked theo spiked m a w t zBin zBin E-related sequences ag t -long -long w he majority ssessed using o non-fungal sequences oved ere re-assembled SPAdes-3.8.1 with (Bankevic an G d tospi v eq reads ains ere assembled using SPAdes-3.8.1 (Bankevic assembly lomeromycotina (Nai 1. Sequences1. as models t d non-Mucoromycotina fungi contigs us ( v bacterial genomes sembly Laczny t frag 1 an t ing if they if forme he UniProt-TrEMBL the PATRIC database (Wattam d transposable-elements-rich regi . Desi ke thebinning ments DIA d processe from w b as of contigs v inning ere assembled using v et al. rò t 1. For each assembly, contigs MOND v.MOND 0.8.38 (Buchfink 1. Sequences he sembly B and USCO (Simã clu seven v us Mak librari 2 using , 2015) analysis m ing In stered with thenon-MRE bacterial d isolate an G. Boni ( odel predictiodel as d as formation (NCBI) n er Fig alysis v w a fungal species sembly sembly 1 contigs es an w an to et al. custom ith non-TEith protein-coding . S2 in the subsequen B M ere downloade alysis of assemblyalysis w notati owtie2 v.owtie2 2.2.3(Langmead & d to, pers cl RE sequences in d clusters o etal. ). Contigs as w atabase (Apwei ustered with theknown ith fragmenith p ( , 201 Fig. S3 ). The completeness erformed s on pipeline v2.10 (Cantarel w cri on and annotati Tr on , 2015). ere . comm.) to identify pt. inity inity 2.5.1 (Grabh 5; Torres-Cortés as including f fr saved as et al., 2014). rom These fragmented sembly om et al. r database. After d from w ted sequences on (TE-rich t on ere rem lon BLASTx t t ler et al. he t he main v , 2014) with he t using 1 using he ger than h etal. v g Chytridium a h etal. t s 3 divided ene space ssembly he Illu ubsequent on, we oved g P , 2004) mina mina enes b ATRIC , et al. err MR ody , 1k and E et b , all fungi 2003) with alocal molecular cloc above. performed RAxML transcriptome data an enzymes and RAxML 8.0.26 (Stamatakis, 2014). Weempl Phylogenetic PHYling the sample 2018) (https://github.com/1KFG/Phylogenomics_HMMs that have previously and We Phylogenet collections lac GMNB39 an Transcriptome data we oryzae checkgradient=yes. method=PL, algorithm=TN, set smoothing=10, penalty MRCA ofAscomycota was along Ma) (T Endogonaceae (407 Ma), (407 Ma), Glomeromycotina (407 Ma), An expansi lagenaria,

Accepted Articleked transcriptome data, we used the additi sa t an he mpled a total of 66 species w , Syncephal notated genomes able To date theTo on offungal pectinases ith the ith P ( ex onal sev ( ROTGAMMAL Stajich JE; http://github.com/stajichlab/PHYling_unified CAZymes Mortier c d genomes a S2). S2). Fossil calibrati ept d Endogo ics of Endogonaceae and molecular s an t he alternative EST MRCA ofChytridiomycota an ( for Blastocladiomycota an alyses 247 Ma), en fossils is plumigaleata ella an d ) and small secreted proteins ivergence p ne sp. alysis m d one tax an roved useful for higher- w G model. To facili used tore used facilitate annotati ultidivaricata ere perform a d n r p d 11transcriptomes w FLAS eset t eset w roteomes and ere used as ith theith on points t an on with imes , and o minimum F ( k d theage of Streptophytes d 63 fungi - 59071. For the ata and ( r ed ontheconc in thebes 315 Ma), Ascomycota (407 Ma), an , Neurospora emaining Umbelop in in a n t tate ranscriptome data from m o annotati total of eigh w t he d Rozella was t inimum age calibrati an ere he oyed d allothe ag Mak subsequen d thre al level cross byvalidated sis ramanniana, as t e with fin 54 taxa using ignments ( RAxML Table We emp S1). the ‘ e ( SSPs), we rem a r pipeline. da c atenated on datafrom e outgrou nnotati phylogenetic J. on ofJ. rassa, Rhizophagu t r fungi - ting analysis f a’ possible calibrati =log, minRateFactor=0.5, t c t al ree, we used r8s onstrained to 750 Ma on based analysis The ). The search for the target markers opt w J. on ofJ. fl settings ere performed using . After passing ammicorona alignment of the 434 markers in a i p tax t on s he same parameters d on points oved iscuss wit t an t he f p on he lacti a), including ixing ) with thedefaul rotein rotein as alysisof fungi loyed 434 h 100 boots d carbohydrate-active ot s irregularis ata s 11 tax f flua flua in ed in ollows: ollows: he on points t d Basidiomycota (330 he AD0 for r three Endogonales h v et (T cr OSC 162217, which 1.8 (Sanderson, omology MR C a thathadonly oss the MRCAs hang J. 02, J. p d CA ofAscomycota 55 assembled able rotein rotein trap ivtime v t . The he , alidati ( Beaud et al. (2015). fl t d S1). WeS1). then ev ammicorona setting. escribed p replicates ipeline m MR idence. on, the arkers of t et al. he CA of

in , bacteria at MetaPhlAn2 with defau The unmappe aligned to the assembliesfinal using MetaPhlAn2 (Truong Taxonomic Taxonomic profiling of Endogonaceae microbiomes classified into maj tBLASTx level using from c Desi BLASTx were 18s including with than tandem positives. For the appli ou We Identification and characterization Endogonaceae (Boucka verticillata NRRL to theabsence ore

Accepted Articlet u de novoidentificati rò r subuni ed multiple filters 100 bp. For the few sed RepeatScoutsed 1.0.5 (Pric eads R epeatScou et al. (2017; A r ert etal. hi er than epeats se d reads se r fou ts ma DNA US arches p t c t ond dating rofiling of the microbiome associ (RPB2). The r Densosporaceae an o genes pped to MRE contigs EARCH 4.2. o w , 28s r poorquality of the sequencesof the t , 20 f and as ha t ive BLASTn w 633 r TE familiesor TE and ( epeat library ere extrac significance cutoff t se t 14) with arandom starting hei r occu order levels. L DNA, translati 7 as the outgroup. Dating in UniProt/SwissProt (significance et al., 2015).To rem t TRharves L on ofrepetitiv as 247 Ma. t r Figure 1 lt TRhaves an o theoutput files 66 (Edgar, 2010). The rred settings J. J. alysis lacti hi ted and less ts t flua d e et al. w t w ou ). This erived from t t were consolidate 52 Endogonaceaed 52 collections. The o thegenome (significance cutoff ith custom as t w o investigate th han han on elongati tput OSC 1622 ere remove B e sequences p a of 1e- owtie2 v.owtie2 2.2.3 (LangmeadSalzberg & o ligned to MRE contigs erformed based on a 4-locus , 200 f transposable elements 1 d files, we rem 0 times ataset has o ove reads identifie f RepeatScout an 15) agains 5) and s R ated with 17 collecti cripts. on factor (EF1A), an an epeatScout, we filtere f t d an inal se ree. The minimum in thegenome or ha in thefou alysis e compositi L d and a TRharves d unm oved wider tax t t arget markers. We inclu t Repbase 21.06 (B w ea of repeat elements on was c d as utoff of1e- ch assemblych repeats eduplicated at apped reads r Endogonaceae genomes. We then d LTRharves d as p ident erformed using t on and (Ellinghaus on sampling n En included inot included ified in the VizBin sh d ribosomal polymerase II ag dogonaceae, d o orter th d asequence The 5). The atas d ou r f 1e- e ofthe MRCA elativ fo w w ur markers applied t as et modifie ao et al., 2015) and t ere the 80%similarity 15), and to rem low-complexity et w p e abundanc , 2012) in f an erformed using analyzed using ere annotated by iltered output files a B En l. ded EAST t 400 bp, those , 2008) to carry his his ove false r dogonales, aw len t . The d from hose with an an of reads 2 gth of less alysis .4.8 .4.8 alyses. e of an w d ere d ue then trimmed with GBlocks v.0. with RAxML Phylogenetic eight from Tenericutes togethe The searches Using Phylogenet the 54predicted proteomes enzyme HMM profiles S1 with For the Comparative model. with generations were filtered with an e-value cutof using replicates were sampled taxa. We in distributi with opti collapsed an g on based performed with Notung ene phylogenies, branches ). We focused on Acceptedt Article he on. We n j on t t reconstructe ewly ModelTes he he sam hmmal p R reviously r with 58publishe ly v id q p a .8.2.4 (Stamatakis,.8.2.4 2014). The ag on ofenzym uery 54 taxa an ackage. We entificati utoM t an ranscriptome data an r al ains etrieve ics of MRE un d rearrang ign d aprotein setting e setting so scre so r The . The sequences ge econstructions were carried out with der R opti t t and v.2.1.9 (Darrib nomic anal the MRE sequences p downloaded 267 d on and characterizati ublishe d 16S the TrN 45 families d 4 ened forallthe360 dbCAN using t on of a rimmed with TrimAl (Capella-Gutiérre e copy as ls ( 34 markers to obtain ed toobtain http://csbl.bmb.uga.edu/dbCAN/ b o investigated the m 2. acteria, an describ r d 16S d MRE16SrDNA sequences DNA DNA 9 (Duran R odel of +I+G model, automatic AxML 8.0.26 (Stamatakis, 201 u n ysis on sing sing umber acrossumber f or each enzyme of the families an s a 91b r ed above. Weperformed a two-way equences one withd one only in the g DNA et al. d subfamilies P hmmsca of 1e-15 an f of1e-15 ROTGAMMAL a d two from d etal. fungal PODsequences t (Castresana, 20 s he mos C ident ‘ t , 2012). Markov bootstrapping’ AZym se he species b w on of CAZymesand SSPs, we exclude ene quences est-fi hereas , 2005; Stolzer etal. ified in this study w d in n in t istributi ere aligned using t es and he sam parsimonious t F H nucleotide substituti d an of PCWDEsfor the fa irmicutes g M M G. Species-g fr t enome butenome no predic milies w ree. accommodate To the uncertainties aximum MER3.1b om 00) i S of class II peroxidases on ofclass alignment alignment coverag pled 54 taxa using Mr th less SPs ( Pattengale ch a oth Bayes using s w ain q ) (Yin 4) er Mucoromycota-associated MRE, it b ueries, we performed BLASTN from the PeroxiBase L r t acteri h hmmscan and (Eddy w han han 70% bootst Mon z etal. with econciliati ikelihoo ene treereconciliati , 2012), using t v MA ith asignificance of1e-80. cutoff an he .3.2.6 (Ronquis et al. d subfamilies te Carlo was FFT a. lea et al. ‘ –f a’ option, , 2011). CA , 2009). Individual g The The on model , 2 s c d analysis t Zym lustering ( , 2010) ted proteome data (Table Kato 012) stringen e cutoff of 50% the on with t he r esulting e analyses. The The heatmap.2 program h & t fi were used to query rap support were he bes d 11sam ltered the r ( w un t 100 w PO un hmmsca t t b w Standley et al. as he conditions. ased on ased ere aligned der as conducted on was ali in D) in for 10 million --rearrange es bootstrap t gnment , 2012) and RAxML timated the GTRCAT pled taxa ene trees n results t r he , 2013), esults t he d in bCAN t w ree as

flammicorona g 2015) of the290 fungal universal single-copy lower than have ahigh proporti Jimgerdemannia The the initial assemblies, including The oligonucleotide-composition-based binning Genome assembly data and binning RESULTS SS under theprosite accessi as predic predic predic sequences e-value cutoff POD sequences predic predic develo the sequences (http://peroxibase.toulouse.inra.fr ene models P if i

Accepted Articlep redicted by g enome assembly sizes . Among ted by ted by ted by ted by ted proteomesof each Endogonaceae genom ped pipelines t The The identificati was w t hat ofthe sh ere ( Wol TMH Ta Si 9, o collections have slightly more predictedgene models w t orter gn f 1e-15 an he rg 569; P fro ith thebesith searche lacti S-SCAN (http://www.hpa-bioinfotools.org.uk/cgi-bin/ps_scan/ps_scanCGI.plS-SCAN fPso alP etP (Emanuelss MM 2.0 (Krog fo m on ofrepetitiveelements ur genomes th ( Table 1), follo 4.1 with sensitivity cutoff of flua t rt 0.1 (Horton Pellegrin he 54 predic n an on ofthe secretome an on- d agains on ofPS00014 (PDOC00014). We conside d an to ~230 Mb 300 amino acids. repeat regions(Table 1). Our assemblies captur t v hi ary t alignment alignment et al. to aclass II h etal. se , Endogo t fro on et al. the Uniprot-TreEMBL quences ted proteomes J. wed by J. ) and , 201 et al. m for , 2001); 96 Mb o rthologs ne sp. 5; Lu, b , 2007); c , 2000); 3) the tw overag uilt H fr p lacti eroxidase w om fo d SSPswas an 4) t FLAS 201 r Endogo MM J. o J. MR d the flua p e cutoff of 50%of thequery. The 5) in 0 u rocess argeted forextracellular localizati 6). The following .34; 2) targeted fo sing sing e: 1)possessi fl clude not targeted to theendoplasmic E, otherbacteria,E, and profiles ab F ammicorona OSC 1622 OSC - G has 59071 has sence sence of atrans-membrane helix C content of the hmmsca d ne sp. ere r in d in p atabas emove erformed utilizing u considered as t sing he FLAS 17 (12, n and e using d 11%to34% ofthedata from on ofa signal peptide as B t collections. All fou hmmbuild. We he leashe USCO(Sim database red F (13,838 and fi r asecretory -59071, 180Mb lters 651). fi a secretome protein ltered DIA r epetitive elements t v numberpredicted of ed 76.3% w P arious blasMOND The OD sequences. ere applied to the p t he reviously t J. wo J. s 13,653). r E earche p r esults ukaryotes. esulting athway r genomes on as r t eticulum o 84.15% for tp ão etal. as d for . Only w ith an ith as as are a , ) level differed betw J. class repetitive elements the totalassemblies. Class The identified repetitive elementsof the Endogonaceae Repetitive elements viral order (Fig. S4). FLAS microbiome containe microbiome containe Endogonaceae (Desi encompassingclade MRE phylotypes 59071 (T flammicorona Endogo were 0.9Mb which no MRE sequences the fou Th Microbiome and Mycoplasma- proportions of simple repeats FLAS small proporti of allclass identifie

flammicorona

Accepted Articlee binning rò etal. I transposons, Gypsy F F - genome-59071 was r Endogonaceae ne sp After the 59071 was d from able I transposons, an . FLAS , 2015). The remaining fo 2). Phylogenetic AD002, two on of thetotal repeats J. r J. alysis c t collection ollections, form hese genomesusing fro r em fl een them t F ammicorona m in allfou he mos -59071(T MR oval u d 26.9% d 44% whereas FLAS sp. sing g E sequences enomes f s (96.7% an were detected of reads ollowed byollowed I transposons (retrotransposons from L Vi t ma T taxonomic ( r genomes zbin (Laczny R was Fig w able r de up of simple repeats econstructions based on 16S rRNA ed an J. J. ere much l related endobacteria AD002, . S4). In ( lacti Fig from 2). This t t he mos p hree MRE phylotypes, one from ( 16 . 1), with theexcepti d 99.8%), although thebacterial compositi ovel early-diverging Cop 1.2 reviously flua ally S ( MRE, Protobacteri . The totalnumbersof nucleotides in 1 addit 33.3% to 54.9% % to6.4%). A r F ia LTia .8 Mb DNA DNA sequences, with one MRE phylotype from OSC 162217 et al. ower (12.0% -59071 an diverse, seven with i t . The microbiome associated wit s abundant p i on toProteobacteria, the R (Tab roportional to thenumber of phylotypesMRE , 20 for r etri 15) J. J. eved t lacti d onefrom le S3 he ( , and identified MR larg g 31.3%), while in g t roup, accountingroup, Endogo enomes comprise o 15.9%). o flua on being from ). ClassII transposons a f alltherepeats; cla e proporti ) represent dominated the microbiomesof both t hree de (Fig OSC 162217,OSC an b ot ne sp. FLAS acterial orders J. J. he fr lact J. J. g om r Endogonaceae sporocarps ene sequences . 2). fl E sequences ea on ofthe Endogo ammicorona OSCiflua 16221 t ch ofthech Endogo he larges J. J. t he lacti for d 65.6% Ta F ot h Endogo -59071 n t d 2.9Mb b flua flua he he early orover half ne sp. le an on atorder t co t r genomes MR hree S3 fr groups d oneDNA OSC 162217OSC GMNB39 for mprise om p t ). Within

o 78.1% of E contigs laced two FLAS ne sp. 7 within sp. ne sp. for t hree J. J. of d a F

- t of he a duplications on the branchleading Mucoromycota (Fig remain inferred to have48 PCWDEs Phylogenet were identified from species degradati Mucoromycota taxa (Fig The Comparative Ma (Fig Ma, theage of MRCA al. relationships boots regi The crystallin PCWDEs peroxidase (PO hydrolases modules including 426 Ma,whereasthe ag Umbelopsidales monophyletic previous the age ofth Endogo collections

Accepted Article, 2014) analysis ons whic 4 R trap 5 enzyme families AxML stable withed stable a small number of duplication . There was ne sp. FLAS . 4). The The ( a st e on ofcellulos auxiliary CBM, with only CBM1 includ support across the phylogeny, with afew exceptions (Fig s udies ar ( c d ( ic placement e MRCAofMucoromycota as GH h incorporated128,068 aligned amin ompared t Stamatakis ating ating e closes am an ge D) repertoire in ) and an ong d plac nomic analysis ( Spatafo on an d Bifigura a F on . 3 and ctivities -590 of Densosporaceae as an p t t alysis ( t e copy of CBM1 present he other three Endogonaceae genomes. The AA9, G olysaccharide lyases he 4-locus relatives within ed within e, wereabsen Dikarya fungio Dikarya within d within fungalmajor lineages of the MR e ofthe , 2014)analysiswas a . 3 and 71 wasresolve n ra etal. a Ta d subfamiliesof PCWDEs w nd datingnd of Endogona ( tus adelaidae H6 and ( bles ith r8sith AA, wi Fig Ta t o o each other, forming M , 2016; Torres-Cru . 3; ur study. Overall Mucoromyc d S b ataset estimat ucoromycotina as CA ofEndogonaceaeas S6). In 5, S6). S4). CBM1le S4). modules, which areals to theMRCA th only n ( t G Sanders ode 67 in Fig or present H7) in ed in ( Fig d as sis 62 . Within and lignin-degrading 16 . 3 and T ( A t 0 Ma, the0 Ma, c PL). In additi his study), carbohydrate esterase (CE), glycoside b A9 incl ontrast, we reconstruc on, 200 7 Ma, and J. in theJ. as ter toJimgerdemannia ed on 66 taxa an of ed theage ofMR w o aci En ith low copy S5 and . S5 Endogonaceae. As z etal. able ceae and in uded in an w 3; Taylor & dogonaceae, the s ag fl iste ere divi d sites. The bes a fro loss d loss ammicorona t e of thee of MRCA of Glomeromycotin sisterclade t S4). Enzyme families he on, wecom m our analysism r grou , 2017). Endogonaceae is 25 Table ag t his ), carbohydrate-bindingstudy ev 8 Ma (Fig ded E e ofMR ota ndogonales ents n p tothe clade of Mucorales, P B d 434conserved protein umbers ). TheS4 number ofPCWDEs CA ofEndogonaceae as 251 ODs species into five enzyme erbee, 200 erbee, MR . 3).Reconstructed ted 23 loss along AD002 genome, while none pared the lign J. o J. t CA ofEndogonales a result,theMRCA CA ofMucoromycota was . t w . 3). BEAST J. wo J. ML o inv ere lac ar in Mucoromycota p t t e consisten ossess fewer he backb tiflua ree receiv absent f in olved in lammicorona 6) estimated tha t argeting ev OSC 162217OSC ents in ( fro cl Bouck one of -degrading asses, t ed strong he m t an with of a d no aert et s a 42 as t . 0 Endogonaceae environmental contaminants predictab sam are not axenic sporoc but alarge numb biology. Much of the sequencing diversity of the fungal tree oflife an The Assembly annotation and DISCUSSION consisten tinctorius the fou geophilum ratios are similar to average values Endogonaceae re proteomes mannose (e.g., GH5-7, GH9, G wide range ofpolymers PCWDEs from metagenome analyses Oligonucleotide-composition-based binning an guided sporoc metagenomic

Accepted Articleported for other fungi In ple. r a eukaryotic for this this w study, pid sequencing of fungal genomes arp tissues arp tissue. r EC The The Bacteria an fr t le the two ECM species d complemente and ) arecomparable tothose for Endogonaceae with om M ( members of the En Fig species d Suillus Suillus an e dogonaceae secretomes i g . 5 and at s ach ofthe sequenced Endogonaceae p enomes genomes er ofbiotrophicfung d sequencing reviously p a remainsa maj ar redicte e employe d e often br fr T ( om evipes Se able comparable S7), to the f b v o d with d topossess aried fromaried 48% to57%. The y dlar etal. in und in (e.g., Mosier B r Ki t o eported spo asidiomycota ( he H10 f fungal f d abinning ) possess ( m or the Mucoromycsampled Quandt fro fr on roc et al. (2016). inf om eff p a or challenge in m and ly ssociated with fungal sporocarps lant ormati arp microbiome, or in ort has , 201 d deep t A av values hes al scomycota ( gen G on et al. ai primary et al.,2016; López-Escardó hi a H28) species 7) but have only ccoun e structures lable ly in recent ghe ap on from omes usingmetagenome a approach our ened our b Amanita , 2 23 copiesof PCWDEs. Despite the small number, ( proach based on een focus Pellegrin me r SSP/secretome ratios The The 015). material forgenerating t c for 3.9% to 4.8% thods c g ell annot be maintain aining p Tuber s y roportions of SSPs equence coverage an Careful walls ears av un m g w ed onfungal species enomes hav et al. uscaria, erage secretome/proteome ratios derstanding of many S i ( ll SP/secretome ratios of Endogonaceae Fig m ac h , including rarely b capture a e be e ota as elanosporum cess , 201 more transient b . 5 and ex ioinformatic mining oli species widely en widely co L panded ourknowledge ofthe gonucleotide composition t accaria 5; Kim o asuitable fungal genome from mprise enzymes een used for metagenomic o the metagenome of the f thecorrespondin Table et al.,2017; in ed in ce ma ( ( Fig g llulose, xy pectin, 51 in secretome for the et al. enome data.Sporocarps y bico and u d BLAST . 5 an S7). On %). SSP/secretome ex cu s in ed in t w ist ist ha lture. lor, , 2016). as Cenococcum Cenococcum ay as t d T as pects Quandt c P mi an t isolithus sy F of able he t an argeting or these taxa, crobial mbionts, be cultured, of fungal alyses. other g S7), et al., lan , hand, and a d ata contigs separated from hypothesis genomics the contigs comprised a large numbe 87% ofthe possesse retrieved genom th MRE , including phylogenetic genom that thegenom identifie consisten We identified MRE sequences Metagenome binning and microb the 2017). In our analysis, we al., distantly-related truffle genera of the microbiomes 2017). Endogonaceae collection werebacteria that are prevalen surprisingly, afterexcluding acqui hypothesized by evoluti suggests specific clade. They and organisms one from study

Acceptedree Mortierellomycotina-specific clades(Fig Article t hree red w e sizes on ofthe fungus-MRE associati The The major bacterial phyla found in In ident ere resere as MREd as is d 76%-84% ofthe 290 fungal universal orthologs t horizon t ea ha ( fr contras with Beaud The . The genecore b 434 ( om ch Endogonaceaech ase t T ified as r additi able ( econstructions based on Araldi-Brondolo in olved in theEndogonaceae-specific clade, while the othe e size ofMREto be approxim d onthei M markers ea t e sequences for the associat he B tally et al. ortierellomycotina (Fig t c onfante 1). This to vertic h othe onal fa r p mo epresent cultative n roportional to thenumber of through the soil bacteri on re MRE diversity is r unique 2018; , 2018; u e r ofcontigs in r in sed in sed the phylogenetic inference. Even v A spac ap an from ally isually isually T-rich (discussedT-rich below). MR proach captured g d Desirò (2017) oli c fr one ofthe ollection, formed a cl e an inherited endohypha et al. Q om E, themos gonucleotide compositi ature G uan o lomeromycotina, t d ligonucleotide signatures d repeat-rich regions ofEndogonaceae genomes t ifferentiated an hree of thefou , 2017; Nai ( dt et al. might h on might of thefungal symbiont. The T 16 able iomes earl . 2).Three of theMREphylotypes w S En t aiting r abundan 1), gene models ately ed MRE. The fou , 2017) DNA DNA sequences ies dogonaceae . al

The . 2).The of Endogonaceae sporocarps to et ene space in t community ave b splits t 0.9 Mb o be discoveredand r Endogonace MRE d identifi Tuber ade distantly wo from an a t l l. bacterial phylabacterial identified from bacteria, extrahyphal bacteria are often een more complicat , 2017). d was in the ex ) withon space (Fig. repeat-rich S2 16S phylotypes16S , within t istence a and in in re also the same taxa identifie in theBUSCO database an ( a ed sequences ( w id r Mond r Endogonaceae assemblies Mu su MR Fig hizosphere soils manner comparable tosingle cell Elaphomyces ere presentfor entifi In fficient to addressour original coromycotina E phylogeny, togethe . 1).We t o ae genomes, line Desir with he r t t f this elated to Endogonaceae- o etal. hough the assemblies otal number ofnucleotides several ed several r ange of MREtypical t r three MREphylotypes ha n ( al ovel MRE clade , 2012).Not Tab than previously ed than t s fro ide the o (Antony-Babu le successfully m a ntifie on ( ( hos orig finding 2). Weestimate2). Martin Martin Endogonaceae n ò etal (2018), ly on-target on-target t-specific 17-25% of in d in in in w d 82%- ea r with the an ere also et al. ch ch t d his in d in et , ) , through the 6). mid occur until afterthe Lowe Endogo communities 2014; al. Endogonaceae with colonizati led to the hypothesis & dated at the Permian-Triassic previously sampling relationships (Fig.clade 5). The Mucoromycotina, Endogonaceae is recent Phylogenetic Phylogenet bacteriallocal communities was sample genomic associati Endogonales-plant contemporary hornworts discovery Spermatop does Malloc , 2017). We

Accepted Article -late Siluri d n n ifferen Quandt on-Spermat ot include any g ne sp. FLAS It hadb enome-scale on, both during h, ( on ofland by o Desir hyte plants 19 r ( f mycorrhizal-li Bidart epo ic relationsh sy t inf as 75; are from et al. e an mbiotic t a een hypothesiz stimate thatstimate Endogonales o theorigin of Glomeromycotin sociat rted as ò etal. erences n data from these and Seloss consisten d Densosporaceae (Bidart ophyte plants m ondo , 2015; Benucci t F as hose foun onophyly of Endogonaceae is well supported (Fig. 5) an -59071 is spe p t sociati i ed with ha s , 2017). Bo hylogenies int d sa p e & s ue to the scarcity et a lants (Fi cies of Endogonales t t imilar to theestimated MRCAage of Glomeromycotina (Fig he protrophic eractions betw t ips r . The Mucoromycotina fungi a the t ke associations betwe L l. on m w with init e Tac , 2011; Desir and En in Tuber d in here the sporocarps b ed thattheinitial colonization of lan a oundary, while the diversificati i member ofthe E. dogonaceaecollections were unique al w eld et al., symbionts ay have co-occurred around the same tim th J.flammicorona p ( on, 1998). This da ere phylogenetic Spatafora revious studies colonizati & r es ting analysisin ( B Berch & onito, olved sister to the an een plants and and d subphylum or ò etal. o 2015a). f DNA f DNA material. In the futu ondo etal. iginate of non-Spermatophyte ag on ofland 201 Elaphomyces et al. t ha F e of the a. ortin,The 1983). , 20 t 6) en Mucoromycotina fungi This suggests m form p , 2016; Torres-Cru p Mucoromycotina fungi in d in b were collected. . aradigmwas ally isiformis clade (Desirò et 2017), al., also h ay also 13; In ased on ased and E , 2011 contrast, at ordinal level thebacterial MR t ndogonales my lev b placed withinplaced Fi G he J. J. y lomeromycotina(e.g., fungi Bifiguratus eld etal. , likely , likely lacti CA of Endogonales was els corrhizal-li p m lants. ave played an fewer loci ; Rimington id- in this flua on of theextan t late ha r r or ecently ecently Unfort eflecting , 2015b) t are Ec d byplantswas igin igin of Endogonaceae was plants Silurian Silurian Ma)(~420 the establishment of the -Mucorales-Umbelopsidales stu z et al. ke associations with non- a re i En mong mong b dy ut unat dogonales et al. t M c t will be to critical hallenged with the . This (particularly from are i more extensive taxon , 2017). Within hat are symbiotic e as t mportan an species he d thespecies ely, ou t , hemselves t d liverworts co 2015; d members t ifferences in he d nsistent iscovery iscovery e in , in fa stimated asstimated A r sampling w t . 5an M De cilitated cilitated hereas role in role in P b irozynski , oth sir an w d d Fig. ò et i h an ith ith d not d as t t he d he molecular toolbox’ Endogonaceae those of saprotrophicfungi relationship. Genomes proteins plan com (e.g., ancestors, possess than (Fig. 5,an com Our study shows of Endogonales, an large repertoire of digestive enzymes enzymes Dikarya EcM fungi 2016). repeathigh content comparable t plan size is Bifiguratus, have small genomesof 28 Mb size of36 Mb. The closest Among Accepted Articlesizes, whereas One ofthe mos Evolutionary signatures understand Densosporaceae pared to Dikarya EcM pared to thei t t reporte G interactions pathogenic un H6, GH7, AA9 an t The diversificati The A he are beli ique to Endogonaceae. In contrast, the t r d T o avoi eduction in s t ampled Mucoromycotina genomes, non-Endogonaceae genomes have an d forDikarya hei able o thatofother fungi. The t t genomes he average fungal genome size ranges r roles d triggering prominen ) toobtain thecomplete picture of Endogonales eved r hypothetical ancestors t fung S4). However,S4). thedegree ofreducti hat the Endogonaceae ( . Thes d therefore ( a Plett , which is Kohle small to p i t of Dikarya EcM fungi ( in earlyland-plant d CBM1). The he numbe Martin on ofSSPs & e molecules have received increased attenti do notshow r etal. lay an lay t Ec o fung r n Mart elativesof Endogonaceae in our sampling featuresof the assemble ( f the umber ofPCWDEsumber t Pellegrin M fungi. Mucoromycotina fungi, including he one ofthe traits t i et al. he in, 201 . , 2015).As importan d

ecto r ofPCWDEsanother genomis efense system of the r in in educti , 20 h Ec elevated levels of SSPswhen elevated levels mycorrhizal b et al. Martin 5; Martin igh content ex ecause they deriv 08; M fungi g pansi t in on in an enomes and 20Mb ev in role in , 2015; Kim b Tisseran iotrophs, EcM fungi d the majority of the othe are enriche olution. s especially those, especially targeting on of Endogonaceae hare t h g he et al. as ene content of the t h of he n d by l ave areduced number of PCWDEs b d Endogonaceae t ifestyle ifestyle umber ofumber PCWDEs , suggesting that theincreas P , 2016).The een on of PCWDEs et al. es CWDEs fro ho et al. in d in tablishment of the ma e mos sts. Furthermore, m described as , 2013; Dong ny 30 , 2016). In our analysis, S S d -50 Mb -50 (Mohanta & myc P genes, especi t i of thei e signature often d not yet exist t d end S iversificati SPs thaSPs orrhizal compared g En in Endogonaceae is on forthei enom g , Calcarisporiella and t r Mucoromycotina fungi enomes o releasefewer digestive p r carbon dogonaceae t i et al. art hei s mo cry t e size is m t r hypothetical of the‘symbiosis fu functi h , 2 on and re nuanced as ycorrhizal stalline do not need a ey donot need ngi ally to other 015; is in theancestors r roles from t in e in an on hei ob com B d g d obligate Peter etal. ae, ue to their t enomes served in as r large o better g t hei av pared to in fungal- enome a ‘ 2015). effector’ s s erage hosts. r hosts. maller i s , al. documented from avail associations with liverworts and within of PCWDE evolution map does often FLASsp. E. . variety of non-Spermatophyte plants suggests substanti symbiotic the MRCA o MRCA ofEndogonaceae. recently (Berch & Fortin, 1983; Warcup 1990). However, membersof th describ its The characterizati sp. FL Mycorrhizal hos of SSSPs 80% SSSPs species-specific (Pellegrin SSSP/SS species-specific (Pellegrin lower than those of Basidiomycota EcMgenomes sampled an here Mucoromycotina species (Fig. 7). The SSP/secretome ratiosof Endogonaceae genomes

tuberculosa , 2015; Desi

Accepted e Article t cology. Mos able plants. n ob AS F-5 ot differ from b ed as saprotrophic Moreover, the PCWDE an served in EcM fungal genomes. P ratios ot d su F ally for Densosporaceae, there are a large number oftaxa in ocumented forming -59071 is w h Endogon et al. et al. ) (Pellegrin ggests f Jimgerdemannia. This suggests ith plants 9071 different evo rò etal. and , 2 , 2015). In contrast, the t ( S o Fig. 7) members SPs (SSSPs)SPs compared to thatof Basidiomycota EcM fung 015; lution f theBasidiomycota EcM species t on ofth t E. h he at fo t e andJimgerdemannia thereare tax a hose Ecof the , 2017). The an und ggrega t Kim Endogonace fro et al., 2015). The combination halliof non-Spermat , close to the i d I ped atotalof20 losses n n m e Endogo t d t of Endogone et al. E ha co o havea large genome with high repeat content, a genomic ue t ndogonaceae with reference to he ta are also putativ ntras t ec t the saprobic o theproducti , 20 E tomycorrhizas d overallCAZyme compositi horn c a M ae m t, FLASne sp. ssociati M 16) whereas v only species alues Jimgerdemannia worts En , including have takenay have different t dogonaceae genomes hree on ofmultiple, distantly r ophyte plants (Bidarton c . This eported t ( apacity ha Desir on o that F EcM (Warcup,ely EcM 1990). l t on Quercus (Yamamoto etal. osse -59071 genome provides of PCWDEs 4 the 8% to 68% ofthe Endogonaceae SSPs i f sporocarps Endogo s ò etal., 2017). Although less informati t the ypic als s weremappe MR for of Endogone sp. FLAS of moderate levels (Tables (Tables S3-5 and o consistent ancestral ally CA ofEndogonacea pathogenic a FLASne sp. that form vary on on o th to the on strat e E. h e trophic mode o ecology of Endogonales ave high proportions b f Endogo t etween d d rep his d onto the branch leading r p w ec do etal., f egiesto interact with their my elate ungi isiform b ith theith observati F ayed woody g ranc -59071, have been rou corrhizal-like orted F o In d Endogonales ig ( f SSPsand a 2 ne sp. which typically al p tha F leading h leading dditional insight into . S6). Reconstruction e was 5% an is c this - , 2017).In addition, 2011; 59071 genomes. The in in lade were t other stu FLAS are lik d 40% su Yamamot f Endogone dy is is ely hi t bstrates o the , Endogone on that F n studies gh levels of fe - are ot 59071 are w ature on is is plant ith a ith 40 o et - t o Gian Fungal specimens were generously Acknowledgements land hypothesisof Fi symbiotic Acceptedwrote the manuscript with contributionsfrom and and Y.C., Author Contrac Use work the author(s opinions, through the ArticleDepartment of Agriculture NIFA DEB-1441715to JWS, to JES, DEB-1441677 to MES, This materi would like to thank authorsthank his g r Facility co R reat patience A by Mar NA NA extracti co .D., J.E.S.,.D., J.W.S. an ordinati t p nduc Co ia Niccolòia Benucci no lants. lants. f The . The indings, an ntribution al . DE-AC02-05CH11231. L ) and is , is supported ted by aboratory is is Q on. Y.C., A.D. t eld etal. iming of the b uinn uinn on. H.N., L.S., ased up d an o n t t he US he anonymous d generoushelp for the submissi H d conclusions ot necessarily anses for providing d G.B. planned an of (2015a) on work Ex b Dep , A.L., A.C. ( y ev J. J. cellence ARBRE (grantno.ANR- t K.B. he f oluti artment of Energy lammicorona GMNB MICL0 O that Endogonales played su , an ffice ofScience of on of Endogonales or recommendations expressed in this p r pported by , and r eviewers rovided by d I.V.G. performed genome an eflec 2416 to G d designe J .W.S. conducteddata analysis. Y.C., t SE the M micrograph of for I.V.G, J.E.S,F.M.M.,M.E.S,and v t Da he iews ofthe Nati B, J thei oin d theresearch. niel Luoma ( 39). and Nati DEB 1737898 to GB), Uni t on of genome data to GenBank. The he t r valuable comments ( Genome Institute,Genome aDOE Office of Science Figs.is 5,6) The t onal Science Foundati US he an importan F Department of Energy aut rench National Research Agency 11-LABX-0002- J. J. hors J. J. lactiflua OSC 16221 fl onal ammicorona . The Y d transcriptom .C. an al t han Science so congso t role in d A.D. performe k ma St an G.B. ephen Mondo for 01 toFM with theruent with F ted States A terial areterial those of on (DEB-1441604 d suggestions. oundati .D. co lonizati an e sequencing un a d J.W.S. uthors der 7) and M). on. The d DNA on of Any fungal endobacterialiving Lope Apweiler 2831–2847. of S Antony-Babu S, Deveau A, Van Nostrand JD, Zhou J, LeTacon F, Robin C, Frey-Klett P, Uroz References Bonfante P, Bonfante P, symbiosis Bidarton 905. Sphagnum, Berch S bacteria associ Ben 217- and Staji Beaudet eukaryotic Bao applications to single-ce SI, Bankevich A, Spectrum 5 endosymbionts2017. Bacterial Araldi-Brondolo Research 32: .

. Black 2014. Black truffle-associated bacterial communities during the development and maturation Accepted ArticleTuber melanosporum P ucci GMN, p ham S, W 227. ch JE, z R,Magrane M, hylogenetic , Ko M, Fortin D, do R, b ji Co g : doi:10.1128/microbiolspec.FUNK-0056-2016 P etween P Chen KK, ma KK, enomes inu Desir Genre A MI, Ba D115 rjibelski A rradi N N ated wit Bo iro sylvestris, s sylvestris, urk Read DJ, aff nito E S J ò –D119. ch A, A J, CH, p . Mobil iliationsof poorly S Ko A . 1983 lants S . 2018 . 2010 , . 2017 prak et al. 2004.UniPr Ant G h fruiting hany O ascocarps and putative functional roles. M ll M W D, et athieu S, sequencing Tr an . The truffle . 2016The microbiome ipov D, in Mucoromycota. e DNA 6 u CH, A . Endog er J, . Ultra- . Mechanisms underlying . Who . Who llium llium appe J d fungi a . 2015Repbas l. 2012 : master modulators S Ba b haffer odies cep G one pisiformis: : low Yi M, Merckx rk . Biology 11. urevich AA, . Journal ldirir a, an er WC, stu . SPAdes: . SPAdes: input transcriptomics in afungus? of hypogeousPezizales ot: theUniversal Protein J P, P, died arbuscul d Allium G W L , The ISME Bo e Update, adatabase of repetitive elements etters 7 of oytenko oytenko N V dikuman A eckmann Computational , Dv Lig n ax p ew genome assembly orkin orrum of fungal phenotypes Th enic cultureenic an rone R, : beneficial plant 574. Journal 11: e diversity, origins ar mycorrhizal fungi E H, : speciesand geogr a S, M, . Canadia B, Ba Duck Kulik r Ferro Da eveal the functi ltr . Microbial Ecology 72:4 Bi Environmental Microbiology l us DA,Gall Y, pé Y, k ology 19: ett 1727 nowledgebas ov AS, d associations with n Journalof Botany 61: S – , J fungus G S G asteig –1735. éguin . The . 2011The alg . Microbiology Les an 455 . DNAResearch 25: orithm aphy effectson ery RE, d interactions in er E, functi S – , e. Nucleic Acids V 477. Far M, Nikolenko H an onal ons of d inelli L, Ar uang H,uang awn d its nold AE –8. content content in in o 899 f

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Accepted Articlemyc MaatenL van, gs M,Taylor er A, 570 . 2016Genome orrhizal mutualists . 2015Convergent t s inPlant Science 7 opology P – Bidar Lar Aerts 80. Kuo A, , S S t trullu-Derrien ternal dó visualizati sson Ko S on ofsingle-cell genomics ton alzb D, Grau-Bové X, wal A, Choi w p N TN 305: B, erformance do ith a ith hidden Ahren agy LG, V erg SL T, P J all lassis vo , , or MI J, Do J an . 2013 acob acob on and sec 567–580. n Heijne G, lugaru V, ganizatio t Cheon he . 2018A d Plan tzl . Natur D, . 2012Fas : reted los N Mo 186. Mid er N, C A, Brun , . MAFFT . The . 2014The origin W ses of decay hum rin g K,Song H, an t Mark Rimington dle Triassicof Antarctica ilmes 47: e Genetics Pathology Oreg proteins n inth G d usability P G myc E uillaumet-Adkin A, ersichini G ersichini an-augmente awron S , ov m Ba onnhammer ELL t Danchin Multi orrhizal revoluti P gapped-rea e ectomycorrhizal rry KW, . 2015 t . Scientific odel: applicati ( o address me SSPs P W ple Sequence Choi . Molecular , Atashpen chanisms R, Duckett . 2012 410 . VizBin - . VizBin EG, ) reveals State University,on State Corvallis an d binning of metagenomic B G, alignment d alignment uscot - d earlyevoluti Duchaussoy F, Gibon 415 R Kang ev s A, eports 7 . Fossil fungi. Fossil olutionary . 2001Predicting on. Current Opinion in . Bio an on tocomplete genomes. Journal of dar an t F, . Mycologia 104: G hei J A tr S d rapi G, ut logy lignment Canba , applicati uffl L A, Ma r potential role P M, : ee ress 11025. w e Rhizopog a d turn S Y nd ith Bowtiith q on ofroots ieracki ME, ck -H rgossian w uesti el S, E on forreference- ith sugges . -wide . 2016Kingdom-wide S B, volution oftware Versi over Choi Or ons using t 835 e 2.NatMeth 9 on vesiculosus J, chard S,chard Ryan MH, ransmembrane d of H Kohler in . a C, . , OR, H, –844. P ta. sym Ruiz ted affinities hos lant Biology 44: Cichoc Co Microbiome 3 t biosis USA. USA. t hree -Trillo ronado on 7: association. A, ki N, SAG g . MSc, : enes 357 I . S t s o , of – : in Microbiology6: secretomes Pellegrin Martin F ectomycorrhizal symbioses Martin F, mycorrhizal symbiosis Lindquist many many Pattengale N not . Walker C, Bass D, Orchard S, Hilton S, GD, Bending Dickie IA, Standish RJ, Gleeson DB, Jeffery RP, Powell JR, 112: Glomeromycot endobacteria are plastic Naito fungi ‘ Mos a 400-million-year-old herit Mondo Mohant fungi Naito Patholog subgenu Muj carb Johnson AcceptedCandida Article ic AB ier on cycling . International Journalof Systematic and 7791 an b M, M, oots AC, AC, S d bacteri s Villosuli a TK, a TK, tus y Oreg M, Uro J, J Mo Desir . 2015Symbio , Ko C, –6. E To et trap Mil fro Moeniiplasma , rton Mo hler P D, Alipour a o Ba ereda a. Proceedings m l. 2016 in aci ò mer on State University, Corvallis, l rin CS, er CS, z S, replicates a. Science 356:eaad4501. e H A, e J A, Mur 1278. ctomycorrhizal B, et al. s mutualists of G E Ba KH, d mine drainag . 2015 New Phytologist onzález J , Pawlowsk V Ma . Fungi Fr . Nature 452: rk . 2008

sis inth Mo a 2017 ischkorn M, Bininda-Emond at C, Veneault-Fourrey er DG rtin rtin n glo are necessary . The . The contain d contain . Natur rton able of meromycotorum’ The . The co F . Fine endophytes ( B, . 2017Ancestral alliances: plan M, M, e Pacific R th ntribute critical b a TE d facultativ J Tao iversity of fung e National B, Pseudotsuga. PhD e Rev f V g ungi KR, e. Frontier eneault-Fourrey enom 88 Lekb

. 2015 h 213: 481 G ost-derived genes –92. Ohm RA,L iews Microbiology 14: , w Mo ? ing e ofLaccaria ith an ith Y, erg Y, Journal of Computational e mutualism . Minimal genomes rton A s ORP, E OR, of Firof s inMicrobiology 7 cademy of Sciences of the Unite , an –486. volutionary e P al J mphasis USA. Glomus tenue ut awlowsk B, C e i e: evolutionary-biology genom , ndobacteriumof arbuscular mycorrhizal Z, spati H Mo Bonfante P, t hesis LaBut ibbett C . Evolution 66: bico . 2015Comparativ ret for ally e. Biological on small-secret Micr , Department of Botany lor provides a TE BME, sustained within ti K, ti Lapidus A, Lipzen varying DS 760. ) are related to Mucoromycotina, of mycoplasma-related obiology 67: t . Unearthing . 2016Unearthing . 2012 : mu P S 238 tamatakis awlowsk Bi tualistic r . ology 17: 2564 oles P . Evolutionary . Evolutionary stability in rocedur ed proteins sights o –2576. in nitrog f e analysis 1177–1184. Rhizopogon a TE d States of s ymbioses A 337–354. es Online 17: . 2010How into . 2017 an en and t he A, d Plant . Frontiers of A r Ch w oots merica ith ith en C, 8. o in f assessing Simão Ecology & Evolution 13: Selosse M-A, Le Tacon Biotechnology Journal binning and visualization of sequences in shotgun metagenomics. Sedlar K, Kupkova K, Provaznik I in Sanderson MJ. 2003r8s: and Suchard MA, Ronquist vascular plants: Rimington Microbiology 17: fingerprintsof genome expansi sequence o Quandt adaptations toobligate intracellular genome of an Quandt Pirozynski KA, Mallo Cenococcumfungus geophilum C Choi Peter M, Kohler A, Ohm RA, Kuo A, Krützmann J, Morin E, Arend M, Barry KW, Binder M, genomes Price AL, Jon of effector biology Plett BioSystems 6

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TJ ra: aphototroph-fungus 7 ivent tondo : 12662 , es DL, 3.2: ( Ma of Land A Rokas, E Rokas, A J , Mi , seva EV, Zdobnov EM.2015 o rtin rtin F, tissue p tissue effi f molecular evoluti MI p chel R, Corradi N, : lant : Da 301 cient 45 . 2015Fungal associationsof basal P s rling A, lants: accamoebae S –50. 205: –fungal interactions –302. with h microbiom d.). eLife6 patafor on ofre r B eveals ayesi A single-copy 1394 ma H Computational and Structural an a JW peat öhn A tter of Mycotrophism. scomycota ectomycorrhizal –1398. p : , reveals phylogenetic inference e29 artnershi on and divergence times e. Environmental a S, . 2015 families J ames TY 594. La orthologs. rget . Metagenome t altern hrough thehrough lens p? BUSCO: . BUSCO: in large . The . 2017The Trend B, ative Liu s in L, L, resemble those in extant lower land plants: novel insights into ancestral plant associations in Horneophyton ligneri from the Rhynie Chert 407(c. million year old) closely Strullu-Derrien C, Kenrick P, Pressel S, JG, Duckett Rioult J-P, Strullu D-G i415. transfers and incomplete lineage sorting with nonbinary species trees. Stolzer M, Lai H, Xu M, Sathaye D, Vernot B, Durand D large phylogenies. Stamatakis A. sporocarp-forming fungi in the . Phylogenetic analysis ofthe genus reveals an independent evolutionary origin of Smith ME, Gryganskyi A, BonitoG, Nouhra E, Moreno-Arroyo B, G Benny body Acceptedmetagenomics Tedersoo 125-142. International, Springer eds. L, Tedersoo In: studies. recognition consistent on notes and Tedersoo L, Smith ME. 2017. analyses. Taylor JW, Berbee ML the wheatpathogen Whole-genome and chromosome evolution associated with host adaptation and speciation of Stukenbrock EH, Jørgensen FG,Zala M, Hansen TT, McDonald BA, Schierup MH 1030. mycorrhizal symbioses: from palaeomycology to phylogenomics. Strullu-Derrien C, Selosse Kenrick M-A, P, FM Martin symbioses. NewPhytologist Articlezygomycete fungi based on genome-scale data. Grigoriev I, Gryganskyi A, Spatafora JW, Y, Chang GL,Benny Lazarus K, Smith ME, Berbee ML, Bonito G, Bioinformatics and ectomycorrhiza specimens.

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Accepted Article Fr 86. 902. n ur Endogone species and ei dit am A ew combinati C, er C, er C a N Bo p Z rovides nito nito U . 2015MetaPhlAn2 forenhanced metagenomic . 1985 ienk T, Franzos SA R, Abraham D, G E G Fr . 1990 , K, Degaw ryganskyi A, Proceedings of National the Academy of Sciences ofthe United States Americaof ollotte A, ollotte o 110: Ma ey t G f the6th North Americ et enuis) . Mycological iewicz , . Taxonomy of the Endogonaceae. In: Trappe JM, Molina R, Berc lbr N S in a Bil tajich JE,tajich l. 2014 , . Taxonomy, culture an sig 20117–20122. G on ( eil lingsley . 28: ianinazzi-Pearson a EA, ht into the Y, a Y, A, Re Kuo M, Kuo P. Mycoavidu et . PATRIC, deck t : H enue H Da D581 Tick a Dun ameed K, ameed K, R iro l. 2017 To esearch 94: l Sphaerocrea ay O, er D A, ), for the fine root endophyt se D,Fuk en bias le TL, le TL, l –D591. old ap Ko dophytic s cysteinexigens t . 2018A es he bacterial bioinformatics Disz TL . Comparative genomics C, C, hler an TL 213 t S Tsc et plan chol , Conference Al V a uda d mycorrhizal 173 - A, S haplinski 219. s pubescens collec , l. 2017.Bifigura matruk et an t , z M,Weingart n symbiosis Dr –178. ymeonidi M, Ya a d soil-dwelling e w l. 2013 iscoll genus . Environmental mada M, on Mycorrhizas T, T, G . Genome ofan H . Proceedin Misztal PK,Misztal Wu S, , Planticonsortium a A, . esse CN, ssociati . Mosaic genome2015. Mosaic of endobacteria t A abbard JL, axonomic Ba tus adelaidae e, Glomus G . 2015Morphology in ted in of Mortierella , hab lestrin P asolli E, d ons ofsome zygosporic atabase an itats Kusk gs J Micr apan . 193–199. of t of i p tenu . Mycologia 109: G R, Charron arb rofiling e CR, e CR, Desirò illespie JJ,illespie obiology 19: he . Mycological , gen. Tet uscular mycorrhizal e (basionym (Mucoromycotina), Desir National e d analysis longata and t A, . Natur sp. nov., a et sp. new SM, eds.h SM, an H ò uttenhow A, P G d phylogeny , A ough 2964 e Methods Duen V A, cademy P ande Polande r rogress 363 esource. Ben its –2983. R, sing –378.

112: ucci C, er C, H of ix Yamamoto Yin Yin Y, ectomycorrhizas carbohydrate-active enzyme annotati times s treeBranches specimens, form MRE phylotypes MRE sequences clades sequences. MRE phylotypes flammicorona AD002 and identifie Figure 2.Phylogenetic agains Additional isolated islandsof contigs of assembly clus v1 contigs the bacterial genomestha Mycoplasma isolate were incl nucleoti Figure 1.Metagenomic Figur with for this evoluti Fig Acceptedupport Article ure 3.Phylogenetic tered R e Legends are estimatedwith The . The t AxML Ma on offungal plant-cell-wall-degrading NCBI n NCBI st de compositi in d in values udy arehighligh o wi ag f X, K, E -rela irs th spike t ( ains he 66 taxa, 434 w Yan r database. Thos t ≥70 ith Bayesiith p n clade encompassed three MRE phylotypes in uded in En ed an id ted endobacteria ( t in natural oakforests reviously do g J, PATRIC entifie dogonales specimens are shown. MRE sequences d bacteri N, on using Che early-diverging p p d D lacement lacement t ata binning of the egaw he in d in r8s. The an in ted in t n J. J. p d showe reported lacti roteins and X, atabase (redatabase data points). Sequences an d posterior probabilities ≥0.95 are thicken al t etec V his Ma alyses Y, a Y, e showing i sequences b flua OSC 162217 based on zbin. Contigs old text. The o d upmos in ted in of Mycoplasma an stu vertical gray Fuk MRE) F, Xu Y F, d divergence time estimation ofEndogonales from dy, on ( on. Nucleic Acids blue data points). The cla . Mycorrhiza 27 uda 128,0 t his of Endogone FLAS sp. de amongde sampl all the sequences n ot w . 2012db fo t e phylotype from on-fungal affinity M, Ya frequently he ere rem st ur Endogonales en 68 aligned amino aci lon udy werefoun c r Endogonaceaespecimens. The shade indicates hronogram ob zymes -related endobacteria (MRE) phylotypes ge mada v tained in this study 1 were sampled an r than oved ( CAN: yello : R ( (top 10)in 295 PCWDEs). The four genomes esearch 40: A from 1 kb 1 . 2017Firs w a w MrBayes i i –301. dentifi s data points in d in an e g w b eb resource for automated ach ofthree Endogonales enomes in assembly ased on F ere rem t alyses the he -590 ed MRE t fro wo Endogonales-specific d sites in ed in t a estimated origin he ssembly W445 an m 71, Jimgerdemannia t ed are w d us b detecti b alysis of 16S oved a t lastn ere spiked with known he as t ) and sequences ssembly and ML sequences his ) and the divergence in in ed ontetramer in ed in –W451. b v es 1 foreach fungal st b from as s on o old. udy as earch ofassembly t r co ML b emaining b v las , and ootstrap ootstrap t ntaminants. f Endogone 1 that he assembly. . The t tp r ree inferred of DNA DNA w se analysis ell as t he quenced fro t hree m from Reconstructi and identifie diagramof the SS taxa arein gray. Fungal taxa arearranged according Endogonales isolates secretome across Fig time-scale ectomycorrhizal RAxML nodes represen are included due highlig Endogonales Fig is for Mucoromycotina, Glom Supporting Reconstrucnodes. numbersof reconstructed PCWDE modules and included in Notung number of Glycosi activity indicate the observed and Blastocladiomycota,

Accepted Articlein millionsof years ure 5.Comparative analysis ure 4.Molecular dating the En . Thick in hted in de hydrolases; an fam number in dogonales d using t alysis alysis medi i PCWDE modules, with ‘x’ s In ily ily ened branches ) orlac on ofPCWDE in millions b formation T b ase 9; ( an old text. No sequences e.g. land F b ( CBM1: t are shown astOrtho. The cluster P cluster as EcM) orEcM) t d onthe o the absence or poor quality ted numbersof PCWD n he an brackets an ode k ed onthesame dataset. Tax C f d Mo annotated proteome data ( ar s ag alysis P am or Chytridiomycota, L: e in ages (Ma). o (Ma). of Carbohydrate-binding p able pled 54predicted proteomes. Ssp/secretome ratios lants). Boots P rtierella p r g ev y 4 d an for olysaccharide lyases). The econstruc utative EcMutative ecology.MRCA, mos ene duplicati ears d r -locus istributi are thetotalnumberof genes b for ed, forthe six oluti an enote those receiving alysis alysis using ecause th Node names:. Node S4 Glomeromycotin d blue bars of small secreted proteins n a selecti ag umb on was d v o (Ma). erticillata ataset (18s on among indic ted number of categoriesmajor of PCWDEs in er in trap fro ey only on ofinternodes E modules on and p B m ating erformed eac EAST 2.4.8 on the origin D r suppo M epresen t ikary h ). Th h divisi e Jimgerdemannia for Mucoromycota, Z t p mo zero he r on names A a, Mortfor Mortierellomycotina.The o ossess los Zoophthora). Reconstructe DNA, 28s f thetarget markers. Numbers rt lowerthan fo ECM speciesa ECM e genomes fo dule 1; s b for r Ascomycota, t an ur Endogonales oots w t ev is on is o thei 95% highes area ofthe circles ith Notung.ith Col d ents othe t ranscriptome data (Glomeromycotina ‘n trap o in g CE: Carbohydrate esterases; ( f Mucoromycota, r a’ included t SSPs r phylogenetic DNA, EF1a an he number ofclusters r internodes ( t referring D/L: se reen recent support highe 10 quencedthis in ). (a in are in lacti t d 0 is B denote the taxa wit posterio uplication/loss ) Proporti an for in for c g flua flua ored omm enomes sh d diversificati t the b o the12 taxa not Zoopagomycota, Muco Basidiomycota, lac own i ar d RPB2; s OSC1622 clusters p circles e shown p on ances k, r density d numbers lacement. r than t roportional to the ogethe for allthe other b on ofSSPs . SS for y stu n t he P clusters ( ear thenodes 57 isolates the four on in the division AA 70 in the ) forthose in the dy 17 genome r with the The tor. The i on of b n ar t t 9: Auxiliary ranches. he are (b ime-scale h oun of G B ) Venn H: in r L ight d the for w ere sampled fungal proteomes. T extan T to extan inferred wi division T T genomes. T S2.Table Fossil calibrations T sam Figure S6. Heat m Figure S5. Internode namersof the collection. Figure S4. Pi Figure S3. Flowcha Figure S2 Figure S1. Endogonaceae sporocarps Supporting Information on theresultsof FastOrtho analysis able able Acceptedable able able able Article in pled in t S7. The S6. The S5. The S4. The S3. Characterization of the S1. Genome accessi species . (c t species . Vi ) Statistics Notung th Notung t his e charts zbin analysis showing p n n n an roporti umber ofloss umber ofduplicati umber of PCWDE modules stu d ancestral species. an ap dy. rt forgenom d ancestral species. of carbohydrate-active enzymes s an howing o on ofsm f species-specific SSPs alysis. on informati an ev t he d g all e assembly ents r m on events family ene family sec epetitive elements t sp icrobia he o o reted f SSPs. an ecies f PCWDE modules mappe on ofthe sam g d zygosporangia. ene spac p l proteins and binning p resent compositi o hylogeny G f PCWDE modules m ( H28 SSSPs e of i expansi n pled genomes. ( in ) in t SS t in thefou ( he on profilesof each Endogonaceae he us CAZmes in Ps) in p t ed in Notung se ex rocedure us he on calibrati quence tan f ou t otal secretom t ) for the r sequenced Endogonaceae d onto branches r Endogonales species species apped onto d Endogonaceae in ed in an on use f an ungal proteomes alysis. d ancestral species t his e proteins in d in b g stu ranches enomes lead r dy. dy. 8s g enomes. ing an for laysis. lead t b o ased the ing GMNB39 and 1. Table Statistics ofgenome assemblies and annotations for Overall GC% repeatsMasked (% total assembly BUSCO (%,complete/partial models) Number ofpredicted gene models models numberTotal of contigs Size (Mb)

Accepted Article Number ofNumber contigs with Repeat GC% Non_repeat GC%

lactifluaJ. OSC 612217

gene

)

.

35.4 44.9 39.0 65.6 84.1 (76.9 / 7.2) 9 3 861 15 409 96 FLAS Endogone

569

F

-

59071 sp .

Endogone 41.4 45.2 42.8 77.6 83.8 (70.0 / 13.8) 13 6 385 35 354 231 AD002 flammicorona Jimgerdemannia

838

sp. FLASsp. F-59071,

Jimgerdemannia flammicorona 41.5 45.0 42.7 78.1 81.4 (66.9 / 14.5) 13 6 313 33 875 240 GMNB39 flammicorona Jimgerdemannia

653

43.0 46.8 44.4 72.9 76.3 (56.6 / 19.7) 12 7 875 45 525 180 612217OSC lactiflua Jimgerdemannia

651

AD002,

J, flammicoronaJ,

Accepted Article sequencedfour Endogonales metagenomes. 2. Table Jimgerdemannia lactiflua GMNB39 Jimgerdemannia flammicorona Jimgerdemannia flammicorona Endogone Fungal isolate name Statistics ofthe sp.

FLAS Mycoplasma- F - 59071

OSC 612217OSC

AD002 related endobacteria (MRE) sequences identified from the

279 None 64 532 contigs Total # of MRE

1.8 Mb None 0.9 Mb 2.9 Mb nucleotides Total # of MRE

2 None 1 3 phylotypes Total # of MRE

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