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New Phytologist Letters (Field et al, 2014) also form associations with vascular plants. To Fungal associations of basal test this possibility, we carried out molecular and microscopical vascular plants: reopening a analyses of the fungal associations of all the major lineages of lycopods and ferns. closed book? Materials and Methods Sampling sites were globally distributed (Supporting Information Table S1). At least one mature plant colony was collected from each Introduction site. Plants were processed for cytological and molecular analyses The widely held hypothesis that Glomeromycota fungi alone within 1 wk of collection by removing and cleaning roots with formed the ancestral land plant—fungus symbiosis (Pirozynski & forceps and sterile water. Roots were prepared for scanning and Da!pi, 1989; Selosse & Le Tacon, 1998; Wang & Qiu, 2006; transmission electron microscopy as previously described (Pressel Parniske, 2008) has recently been challenged by new lines of et al, 2010; Desire end, 2013). Extraction and sequencing of evidence from molecular, cytological, functional and palaeonto- genomic fungal DNA were performed using the method of logical studies. First, liverworts of the earliest divergent Glade, the Bidartondo et al (2011). In brief, the universal fungal 18S primer Haplomitriopsida, form a mutualistic mycorrhiza-like relation- combination NS1 (White et 4, 1990) and EF3 (Smit et at, 1999) ship, whereby there is reciprocal exchange of plant carbon (C) for was used to amplify DNA which was cloned (TOPO TA; fungal nitrogen (N) and phosphorus (P), with members of the Invitrogen) and sequenced using an Applied Biosystems Genetic Mucoromycotina (Bidartondo et al, 2011; Field et 2014), a Analyser 3730 (Waltham, MA, USA). Between four and eight fungal lineage considered basal or sister to the Glomeromycota clones were sequenced for each sample and identified using NCBI (James etal., 2006; Lin nal, 2014). Second, other basal plants, BLAST (Altschul et al, 1997). Sequence editing and assembly were including complex and simple thalloid liverworts and homworts, performed in Geneious v5.6 (http://www.geneious.com). The enter into associations with both Mucoromycotina and Glomer- alignment algorithms ofMUSCLE were used within MEGA v5.1 omycota fungi, sometimes simultaneously (Bidartondo et at, (Tamura et al, 2011), with reference sequences from GenBank. 2011; Desire, eta!, 2013). Third, dual partnerships involving Using UCHIME (Edgar et al, 2011) within MOTHUR (http:// fungi with affinities to Glomeromycota and Mucoromycotina have www.mothur.org), confirmed sequences were not chimeric. Evo- been reported in fossils of early vascular plants from the Devonian lutionary models were tested in MEGA. Bayesian inference was (Strullu-Derrien et al, 2014). carried out using MrBayes (Huelsenbeck & Ronquist, 2001) and Turning to the fungal associations ofthe extant representatives of FigTree v1.4 (http://tree.bio.ed.ac.uk) for visualization and edit- the early diverging vascular plant lineages, the glomeromycete ing. Representative DNA sequences have been deposited in identity of fungi in ferns (Monilophyta) has never been questioned GenBank (KJ952212—KJ952241). —a consensus borne out by cytology and limited DNA sequencing data (Wang & Qiu, 2006; Ogura-Tsujita etal., 2013). By contrast, Results the unusual cytology of fungal colonization in lycopods (Lycopodiophyra), highly reminiscent of the cytology reported in the Molecular and cytological analyses showed that both Mucoromy- Haplomitriopsida genus Treubia (Duckett et al, 2006), suggested cotina and Glomeromycota fungi associate with lycopods and ferns unique fungal partnerships or ilycopodioid mycothallus interac- (Figs 1, 2). We examined samples from 20 lycopod and 18 fern tions' (Duckett & Ligrone, 1992; Schmid & Oberwinlder, 1993) species, and detected fungi in seven and 13 species, respectively until a molecular study detected Glomeromycota in this group (Table S1). Glomeromycota fungi were present in three lycopod (Winther & Friedman, 2008), thus 'laying to rest over a century of species while Mucoromycotina were found in four. Fungal speculations and uncertainty' surrounding their identity (Leake colonization was detected in only 17 of the 101 lycopod samples et al, 2008). However, Windier & Friedman's study, and a more analysed. Diverse Mucoromycotina fungi colonized lycopods, recent investigation proposing a basidiomycete as the main sometimes occurring within the same species, and even the same symbiont in a member of the Lycopodiaceae (Horn eta!, 2013; plant, and six new Mucoromycotina clades were discovered (Fig. but see rebuttal in Strullu-Derrien eta& 2014 criticizing their S2). Colonization rates in ferns were higher (33 out of 58 samples) limited molecular and microscopical data), used methods that do and showed specificity to Glomeromycota (Fig. SI). Ferns not detect Mucoromycotina fungi. Therefore, it remains to be exclusively contained members of the order Glomerales, with the determined whether members of the Mucoromycotina related to exception of one Ophioglossum (Diversisporales), one Psi/arum the fungi known to enter into mutualism with basal liverworts (Archaeosporales), one Tmetipteris (Archaeosporales), and three 1394 New Phytologist (2015) 205: 1394-1398 g) 2014 The Audio's wmv.newphytologisteom New !biologist cD 2014 New Phrologist Trust EFTA01620819 Ncv. Phytulo,•kt Letters Forum 1395 Sens osaccharomyces pombe X51866 I C r nus ccmatus A Y665772 1 sith 8oltychium duntlii 8536 or Gloms erancum HM153420 1 d ,_ with Selagoaria sedaginoides 8530 Uri , r Fonnerforma caledcnium 1r17635.3 with Anogranyna leptophyle WR856-8 ea olEr with Glecheno mcrophyffe 8963-E t with Ophrogbssum yuktatum 8949.0 Gorrus .-acro:arpu- FR750376 I oat .it_ with Lycopodum cemuum 8964 2-8 WO Tmesiptens Warden 8369 eloaAwoJawoo with Ancgramma loptophyNa WR864-A Rr zepriagus .r!fariK:ces Ai30189 I o si V watt Setagmet.tu kraussona WR9I 1-C with Prune purpurascens A139-1 with Lycoredium comuum WR90743 I r with Nephrolopo hrsutute AU98b-4 L— with Xrphoptens ascensonensis Alan 3 inn with Salagmelfe kraussrana WR911, A with PsAatum radon A11.2 o 11 with Oplacglossum ythOatum 8956-D I Dwersopora (Ivaco FP686936 1 on I with Anogramnie hyptophytia WR865-8 with Pbsena porpurascens (gamotophyte) A181-2 Archaeospcea serenely, FR773150 1 ayth Psibium nut:turn .412-3 Paraglomus cauttum 4J276081 3 r -- Sphaorccreas pubescent 48755407 I—with Treutaa pygmaea 8356 0414230.1 r with Lycopodrella mwntiala Th6-2 o9 i_ ,,,,, Rhoeoceros carohnsanus 8445 3 KC708396 1 with Anogramme itiptophylle WR86543 - o ' I I f —with Lycopodmille inundate ThEF4 with Phaeoceros carohnianus 8873 4 KC708428 1 euRopAumioDniry with Lyoopodiette inundate 77•86-4 0 .yeth Nothott3f0S vineenhanus 8843 6 KC 708395 I cycepodreile inundate Th8b-3 L WW1 LyttpotheAla inundate 8528 with Anogramma leplophyab JD92 Endogone podorrms 10322628 Endogone intrastate 90444 r Endorne fectifius MA59900-A I Endogone /Num.:corona $414206 r vath Lyvopodium ennotinurn WR266-C Froth Lycoporilum fastiglatum WR148 843 I- YAM PRasomEetatos coataceos 8797 4 KC708415 I Mortrerela mutdevancata AF157144 004 Fig. 1 Representative fungal associates of basal vascular plants in a Bayesian full 18SnrDNA analysis. Both lycopods and ferns harbourdivene Mucoromycotina and Glomeromycota fungi. Reference sequences from GenBank are highlightedin grey.Analysiswasperformed using an HKY85 model (nit = 2)and invgamma rates. Four heated chains were run simultaneously with a chain length of 1.1 x 106. Anogramme (Mucoromycotina and Diversisporales) specimens; host cytoplasm packed with mitochondria (Fig. 2a). Fungal Anogramme was the only fern genus harbouring Mucoromycotina associations in both the roots and gametophytes of Anima fungi. All samples analysed were sporophytes, with the exception of (Fig. 2c—g) comprise structures typical of Glomeromycota coloni- one fern gametophyte (Presents sp.), which contained Gigaspora- zation, including arbuscules, large vesicles and hyphal coils, which ceae fungi. This investigation added two new samples to the still are intimately associated with the plant cell wall. limited database of Endogone fruiting body DNA sequences (including the first E. incresram) and supported the placement of Discussion Spharrortrar pubes-nu (Hirose etal., 2014) in Mucoromycotina Group L (ram Desire et al, 2013). This study demonstrates for the first time that the extant The cytology of fern—fungal associations hitherto undescribed is representatives of the earliest diverging clades of vascular plants, illustrated in Fig. 2. In Anogramma colonized by Mucoromycotina lycopods and ferns, form associations with both Mucoromycotina (Fig. 22,6), the exclusively intracellular fungus produces large and Glomeromycota fungi. Lycopod sporophytes rely on a variety hyphae, finer short-lived coils and vesicles (Fig. 2b). Fungal ofstrategies, entering into partnership with either Glomeromycota structures are surrounded by host plasma membrane and healthy or Mucoromycotina, both or often neither. By contrast, all the ferns SD 2014 The Audio's New Phoyfoght (2015) 205: 1394-1358 New PhytologistO 2014 New Phytologist Titorc www.newphytologistcom EFTA01620820  New 1396 Forum Liners Phytologist Fig. 2 Fungal colonization in ferns. (a, b) Transmission electron micrographs of Anogramma leptophyna colonized by Mucoromycotina fungi. Fungal colonization is largely confined to a zone where the tubers join the main root system and the lipid-filled tubers, as in hornworts and liverworts, are fungus-free. (a) Early stage in fungal colonization showing living farrowed) and collapsed (9 hyphae surrounded by healthy host cytoplasm packed with mitochondria (M). (b) Later stage of colonization showing a large hypha, clusters of collapsed short-lived hyphae and a vesicle farrowed). (c-g) Scanning electron micrographs of Ptisana purpurascern colonized by Glomeromycota fungi. (c) Fungal structures (indicated by arrows) in root inner cortex cells packed with amyloplasts. (d) Large vesicle and fine hyphal coil. (e) Hyphae tightly appressed to the inner walls of colonized cells (indicated by arrows). (f) Arbuscules. (g) Fungal entry is via the root hairs (indicated by arrows). Bars: (O 50 pm; (d-g) 20 pm. sampled associated exclusively with Glomeromycota, with the (Berbee & Taylor, 2007; Krings ttat, 2007a,b), formed associa- exception of the derived genUS Anogramma where dual partnerships tions with Mucoromycotina fungi (Pressel etal., 2010; Strullu- were detected. Our discovery finally provides an explanation for the Derrien et al, 2014). Nonetheless, where the fungi are exclusively unusual colonization patterns reported before in some lycopods intracellular (e.g. Anogramma), it is impossible to ascertain from (Duckett & Ligrone, 1992; Schmid & Obenvinkler, 1993), cytology alone to which fungal group they belong, as both consisting of an intracellular phase and extensive fungal prolifer- Glomeromycota and Mucoromycotina produce vesicles and ation in gametophytic mucilage-filled intercellular spaces, as also hyphal coils. The short-lived fungal swellings or lumps typical of reported in other Mucoromycotina-associated groups: the Haplo- Mucoromycotina colonization in the Haplomitriopsida (Carafa miotriopsida liverwort genus Treubia (Duckett a al., 2006), several et al, 2003; Duckett et al, 2006) are unique to this group, the only hornwort genera (Desire eta!, 2013), and the Devonian fossil land plant lineage to date known to associate exclusively with plant Horneoplryton hstneri (Strullu-Derrien etnl., 2014). We Mucoromycotina fungi (Field cal, 2014). Arbuscules, the hypothesize that the associations between Mucoromycotina fungi signature of Glomeromycota colonization in angiosperms, are and vascular plants are mutualistic. Beyond microscopy, our main produced in some lycopod and fern—Glomeromycota associations line ofevidence is the recent demonstration ofmutualism between (e.g. Ptisana, Angioptens, °mune& — Ogura-Tsujita et A, 2013) Haplomitriopsida liverworts and Mucoromycotina fungi (Field but are lacking in others (see Strullu-Derrien cal, 2014 and a al., 2014) closely related to those now detected in vascular plants. references therein), as is also often the case in liverworts and Our observations demonstrate that intercellular fungal prolif- hornworts. eration is a signature of Mucoromycotina colonization, and lend The presence ofGlomeromycota and Mucoromycotina fungi in further support to the hypothesis that the early Devonian vascular lycopods and the predominance of Glomeromycota in the later plant Nothia, which also harboured inter- and intracellular fungi diverging ferns fit the phylogenetic distribution of these fungi in New Ph)tologht (2015) 205: 1394-1398 X) 2014 Tht Authors www.newphytologist.com New Phywlogist €.> 2014 New Phytologist Trust EFTA01620821 New Phytologist Lams Forum 1397 other 'lower land plant groups. As such, dual partnerships are the 3 Department of Life Sciences, Plants Division, Natural History norm in basal thalloid liverworts, while more derived clades have, Museum, Cromwell Road, London, SW7 5BD, UK like ferns, the specificity to Glomeromycota typical oflater vascular ("Author for correspondence: tel +44 (0)20 8332 5379; plant lineages (Smith & Read, 2008). Together with the occurrence email [email protected]) ofmultiple fungal associations in Devonian plants (Strullu-Derrien mat, 2014), this lends further weight to the notion of shifting References symbiotic encounters between early land colonists and soil- dwelling fungi before the Glomeromycota became dominant. Altschul SF. Madden TL.SchafferAA, ZhangJH, Zhang Z, Miller W,Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database The presence of Mucoromycotina in Anogramma may be a recent search programs. 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A light and electron-microscope study of the fungal vascular plants. as well as fossil plants (Strullu-Derrien et 2014), endophytes in the sporophyte and gametophyte of Lyeoporlisent ant:tune with observations on the gametophyte—sporophyce junction. CanadianJournal of indicate that associations between Mucoromycotina and land Botany70: 58-72. plants are extremely ancient. Edgar RC. Haas BJ.Clemente JC, Quince C.Knight R. 2011. UCHIME improves During this investigation, we examined sporophytes only and it sensitivity and speed of chimera detection. Bioinformatia 27: 2194-2200. would be desirable now to study the cryptic nonphotosynthetic Field KJ. Rimington WEL Bidartondo MI. Munson KE, Seeding DJ, Cameron gametophytes ofa range oflycopods and ferns, which are expected DD. Duckett JG, Leaks JR.PresselS.2014. First evidence ofmuntalism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi to be more heavily and consistently colonized by fungi (Read eta!, and its response to simulated Palaeozoic changes in atmospheric CO2. New 2000; Ogura-Tsujita et al, 2013). Nevertheless, our discovery that Plrologin 205: 743-756. lycopods enter into partnerships with both Mucoromycotina and Goebel K. 1905. aganografrby ofplant, Pan IL TranslatedI. B. Balfour. Oxford. Glomeromycota fungi opens a new chapter in understanding the UK: Clarendon Press. origins and evolution of fungal symbioses in vascular plants. Hirose D. Degawa Y, Yamamoto K, Yamada A. 2014. Sphaentereas pubnewu is a member of the Mucoromycotina closely related to fungi associated withliverworts Functional studies into the nature of these associations, like those and hornworts. *continue55: 221-226. conducted by Field etal. (2014) on Haplomitriopsida—Mucoro- Horn K. Franke T. Untersehet M, Schnieder M. Deaden.L 2013. Morphological mycotina symbioses, are now needed. and molecular analyses of fungal endophytes ofachlorophyllous gametophytes of Diphasiastrumalpinum (Lycopodiaceae). AnwrintnJournalofBotany WO: 2158- 2174. Acknowledgements HuelsenbeckJP.Ronquist F. 2001. MRBAYES: Bayesian inference ofphylogenetic trees. Bioinfonnatin 17:754-755. M.i.B. and S.P. thank NERC for grants NEJI027193/1 and NE/ James TY. Kauff F. Schoch CL Matheny PB. Hofiteuer V. Cox CJ, Celio G, I025360/1. J.G.D. thanks the Leverhulme Trust for an Emeritus Gueidan C, Faker E, Miadlikowska J et al. 2006. Reconstructing the early Fellowship. A Darwin Initiative Grant enabled S.P. and J.G.D. to evolution of Fungi using a six-gene phylogeny. Nature 443: 818-822. collect fungal samples from Ascension Island. We thank Jim Krthp M. Taylor Tit Han H. Kerp H. Dottier N. Herman EJ. 2007a. An alternative mode of early land plant colonization by putative endomycorrhial Trappe (Oregon State University) and Maria Martin (Royal fungi. Plant Signaling & Behavior 2: 125-126. Botanic Garden ofMadrid) for fungal fruiting bodies, and Tatiana Krinp M. Taylor TN. Han H. Keep H. Dottier N. Hermon EJ. 20076. Fungal Solovieva (supported by the Society for Biology and Imperial endophytes in a 400-million-yr-old land plant: infection pathways. spatial College Undergraduate Research Opportunities Programme) for distribution, and host responses. New Apologist 174:648-657. Leak. JFt. Cameron DD, Beetling DJ. 2008. Fungal fidelity in the analysing Ascension Island samples. Our thanks go to the Editor myco-Imerotroph-to-autotroph life cycle of Lycopodiaceae a case of parental and three anonymous referees for their comments. nurture? New Phytologist 177: 572-576. Lin K. Umpens E, Zhang ZH, Nam S, Saunders DGO, Mu DS. Pang EL Cao William R. Rimington11213*, Silvia Breese"), Jeffrey G. Duckett3 HF, Ora HH, Lin T et AL 2014. Single nucleus gnome sequencing reveals high and Martin I. Bidartondola similarity among nuclei of an endomyconhiral fungus. PLoS Gerwtia 10: e1004078. Ogura-Tsujita Y. Sake& A. Ebihara A. Yukawa T. lmaichi R. 2013. 'Department of Life Sciences, Imperial College London, London, Arboscular mycorrhiza formation in cordate gametophytes of two ferns. SW7 2AZ, UK; Angiopteris lygoaSifolio and Onnumia japonica. Journal of Plant Research 126: 2Jodrell Laboratory. Royal Botanic Gardens, ICew,TW9 3DS,UK; 41-50. 8i 2014 The Authors Nam Apologist (2015) 205: 1394-1398 New Phytologist €3 2014 New Phytologist Trust www.newphytologist.com EFTA01620822  New 1398 Forum Letters Phytologist Pamiske NI. 2008. Arbuscular mycorthira: the mother ofplant root endosymbioses. of myconhiras in the colonisation of land by plants. New Phytologist 186: Nature Perinea Mierobielagy6: 763-775. 514-525. Pircaynski KA. Dalpi Y. 1989. 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Myer:whin-like interaction between the achlorophyllous gametophyte of Lyropodiron clamant L. and its lunged Supporting Information endophyte studied by light and eleaton-microscopy. New Phyrolegist 124: 69-81. Additional supporting information may be found in the online Sdosse MA. Le Taeon F. 1998. The land flora: a phototroph-fungus partnership? version of this article. Trends in Ecology and Errohetion 13: 15-20. Smit E. Leeflang P. GlandorfB. van Ebas JD, Weman K. 1999. Analysis of fungal Fig. Si Glomeromycota associates of basal vascular plants in a diversity in the wheat rhisosphete by sequencing of cloned PC:R-amplificd genes Bayesian full 18S nrDNA analysis. encoding 185 rRNA and temperature gradient gel electrophoresis. Applied and Enriremmental slcicrobielogy65: 2614-2621. Smith SE. Read DJ. 2008. Myron-hindgm/Pink Cambridge. UK: Academic Press. Fig. S2 Mucoromycotina associates of basal vascular plants in a StrulltrDerrien C. Kenrick P. Pressel S, Duckett JG, Ftioult J-P.Strunu D.C. Bayesian full I 85 nrDNA analysis. 2014. Fungal associations in Harneophyran hymn from the Rhynie Chest (e. 407 million year old) closely resemble those in extant lower land plants: novel insights into ancestral plant—fungus symbioses. New Phyrologit 203: 964-979. Table SI Lycopod, fern and fungal fruiting body samples analysed Tamun K, Peterson D. Peterson N. &ether G, Nei M. Kumar S. 2011. MEGAS: with their origin and fungi detected Molecular evolutionary genetics analysis using maximum likelihood. evolutionary distance, and maximum parsimony methods. Molecular Biology and Please note: Wiley Blackwell are not responsible for the content or Eve anion 28: 2731-2739. functionality of any supporting information supplied by the Walker C 1985. Enekoneletaillua forming ectomycorrhizas with Phew canton& Transaction, *film British AfyrologiralSeeiety84: 353-355. authors. Any queries (other than missing material) should be Wang B, Qiu YL 2006. Phylogenetic distribution and evolution of mycorthirm in directed to the New Phytologist Central Office. land plants. Myeerrhiza 16: 299-363. Wang B, Yeun LH. Xue JY. Liu Y, Ane JM. Qiu YL 2010. Presence of three Key words: ferns. Glomeromycota. lycopods. Mucoromycotina. myconhin. mymrrhizal genes in the common ancestor of land plants suggests a key role • About New Phytologist lib CI • NewPhytologist is an electronic (online-only) journal owned by the New Phytologist Trust, a not-for-profit organization dedicated. to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews. • Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication 'as ready' via Early View- our average time to decision Is <26 days. 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