Portal de Periódicos da CAPES

Fungal associations of basal vascular plants: reopening a closed book?

2014; Wiley; Volume: 205; Issue: 4 Linguagem: Inglês

10.1111/nph.13221

1469-8137

William R. Rimington, Silvia Pressel, Jeffrey G. Duckett, Martin I. Bidartondo,

Plant and Fungal Species Descriptions

New PhytologistVolume 205, Issue 4 p. 1394-1398 LettersFree Access Fungal associations of basal vascular plants: reopening a closed book? William R. Rimington, Corresponding Author William R. Rimington Department of Life Sciences, Imperial College London, London, SW7 2AZ UK Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS UK Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UK(Author for correspondence: tel +44 (0)20 8332 5379; email [email protected])Search for more papers by this authorSilvia Pressel, Silvia Pressel Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UKSearch for more papers by this authorJeffrey G. Duckett, Jeffrey G. Duckett Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UKSearch for more papers by this authorMartin I. Bidartondo, Martin I. Bidartondo Department of Life Sciences, Imperial College London, London, SW7 2AZ UK Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS UKSearch for more papers by this author William R. Rimington, Corresponding Author William R. Rimington Department of Life Sciences, Imperial College London, London, SW7 2AZ UK Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS UK Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UK(Author for correspondence: tel +44 (0)20 8332 5379; email [email protected])Search for more papers by this authorSilvia Pressel, Silvia Pressel Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UKSearch for more papers by this authorJeffrey G. Duckett, Jeffrey G. Duckett Department of Life Sciences, Plants Division, Natural History Museum, Cromwell Road, London, SW7 5BD UKSearch for more papers by this authorMartin I. Bidartondo, Martin I. Bidartondo Department of Life Sciences, Imperial College London, London, SW7 2AZ UK Jodrell Laboratory, Royal Botanic Gardens, Kew, TW9 3DS UKSearch for more papers by this author First published: 23 December 2014 https://doi.org/10.1111/nph.13221Citations: 63AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Introduction The widely held hypothesis that Glomeromycota fungi alone formed the ancestral land plant–fungus symbiosis (Pirozynski & Dalpé, 1989; Selosse & Le Tacon, 1998; Wang & Qiu, 2006; Parniske, 2008) has recently been challenged by new lines of evidence from molecular, cytological, functional and palaeontological studies. First, liverworts of the earliest divergent clade, the Haplomitriopsida, form a mutualistic mycorrhiza-like relationship, whereby there is reciprocal exchange of plant carbon (C) for fungal nitrogen (N) and phosphorus (P), with members of the Mucoromycotina (Bidartondo et al., 2011; Field et al., 2014), a fungal lineage considered basal or sister to the Glomeromycota (James et al., 2006; Lin et al., 2014). Second, other basal plants, including complex and simple thalloid liverworts and hornworts, enter into associations with both Mucoromycotina and Glomeromycota fungi, sometimes simultaneously (Bidartondo et al., 2011; Desirò et al., 2013). Third, dual partnerships involving fungi with affinities to Glomeromycota and Mucoromycotina have been reported in fossils of early vascular plants from the Devonian (Strullu-Derrien et al., 2014). Turning to the fungal associations of the extant representatives of the early diverging vascular plant lineages, the glomeromycete identity of fungi in ferns (Monilophyta) has never been questioned – a consensus borne out by cytology and limited DNA sequencing data (Wang & Qiu, 2006; Ogura-Tsujita et al., 2013). By contrast, the unusual cytology of fungal colonization in lycopods (Lycopodiophyta), highly reminiscent of the cytology reported in the Haplomitriopsida genus Treubia (Duckett et al., 2006), suggested unique fungal partnerships or 'lycopodioid mycothallus interactions' (Duckett & Ligrone, 1992; Schmid & Oberwinkler, 1993) until a molecular study detected Glomeromycota in this group (Winther & Friedman, 2008), thus 'laying to rest over a century of speculations and uncertainty' surrounding their identity (Leake et al., 2008). However, Winther & Friedman's study, and a more recent investigation proposing a basidiomycete as the main symbiont in a member of the Lycopodiaceae (Horn et al., 2013; but see rebuttal in Strullu-Derrien et al., 2014 criticizing their limited molecular and microscopical data), used methods that do not detect Mucoromycotina fungi. Therefore, it remains to be determined whether members of the Mucoromycotina related to the fungi known to enter into mutualism with basal liverworts (Field et al., 2014) also form associations with vascular plants. To test this possibility, we carried out molecular and microscopical analyses of the fungal associations of all the major lineages of lycopods and ferns. Materials and Methods Sampling sites were globally distributed (Supporting Information Table S1). At least one mature plant colony was collected from each site. Plants were processed for cytological and molecular analyses within 1 wk of collection by removing and cleaning roots with forceps and sterile water. Roots were prepared for scanning and transmission electron microscopy as previously described (Pressel et al., 2010; Desirò et al., 2013). Extraction and sequencing of genomic fungal DNA were performed using the method of Bidartondo et al. (2011). In brief, the universal fungal 18S primer combination NS1 (White et al., 1990) and EF3 (Smit et al., 1999) was used to amplify DNA which was cloned (TOPO TA; Invitrogen) and sequenced using an Applied Biosystems Genetic Analyser 3730 (Waltham, MA, USA). Between four and eight clones were sequenced for each sample and identified using NCBI BLAST (Altschul et al., 1997). Sequence editing and assembly were performed in Geneious v5.6 (http://www.geneious.com). The alignment algorithms of MUSCLE were used within MEGA v5.1 (Tamura et al., 2011), with reference sequences from GenBank. Using UCHIME (Edgar et al., 2011) within MOTHUR (http://www.mothur.org), confirmed sequences were not chimeric. Evolutionary models were tested in MEGA. Bayesian inference was carried out using MrBayes (Huelsenbeck & Ronquist, 2001) and FigTree v1.4 (http://tree.bio.ed.ac.uk) for visualization and editing. Representative DNA sequences have been deposited in GenBank (KJ952212–KJ952241). Results Molecular and cytological analyses showed that both Mucoromycotina and Glomeromycota fungi associate with lycopods and ferns (Figs 1, 2). We examined samples from 20 lycopod and 18 fern species, and detected fungi in seven and 13 species, respectively (Table S1). Glomeromycota fungi were present in three lycopod species while Mucoromycotina were found in four. Fungal colonization was detected in only 17 of the 101 lycopod samples analysed. Diverse Mucoromycotina fungi colonized lycopods, sometimes occurring within the same species, and even the same plant, and six new Mucoromycotina clades were discovered (Fig. S2). Colonization rates in ferns were higher (33 out of 58 samples) and showed specificity to Glomeromycota (Fig. S1). Ferns exclusively contained members of the order Glomerales, with the exception of one Ophioglossum (Diversisporales), one Psilotum (Archaeosporales), one Tmesipteris (Archaeosporales), and three Anogramma (Mucoromycotina and Diversisporales) specimens; Anogramma was the only fern genus harbouring Mucoromycotina fungi. All samples analysed were sporophytes, with the exception of one fern gametophyte (Ptisana sp.), which contained Gigasporaceae fungi. This investigation added two new samples to the still limited database of Endogone fruiting body DNA sequences (including the first E. incrassata) and supported the placement of Sphaerocreas pubescens (Hirose et al., 2014) in Mucoromycotina Group L (sensu Desirò et al., 2013). Figure 1Open in figure viewerPowerPoint Representative fungal associates of basal vascular plants in a Bayesian full 18S nrDNA analysis. Both lycopods and ferns harbour diverse Mucoromycotina and Glomeromycota fungi. Reference sequences from GenBank are highlighted in grey. Analysis was performed using an HKY85 model (nst = 2) and invgamma rates. Four heated chains were run simultaneously with a chain length of 1.1 × 106. Figure 2Open in figure viewerPowerPoint Fungal colonization in ferns. (a, b) Transmission electron micrographs of Anogramma leptophylla 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 (arrowed) and collapsed (*) 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 (arrowed). (c–g) Scanning electron micrographs of Ptisana purpurascens 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: (a, b) 2 μm; (c) 50 μm; (d–g) 20 μm The cytology of fern–fungal associations hitherto undescribed is illustrated in Fig. 2. In Anogramma colonized by Mucoromycotina (Fig. 2a,b), the exclusively intracellular fungus produces large hyphae, finer short-lived coils and vesicles (Fig. 2b). Fungal structures are surrounded by host plasma membrane and healthy host cytoplasm packed with mitochondria (Fig. 2a). Fungal associations in both the roots and gametophytes of Ptisana (Fig. 2c–g) comprise structures typical of Glomeromycota colonization, including arbuscules, large vesicles and hyphal coils, which are intimately associated with the plant cell wall. Discussion This study demonstrates for the first time that the extant representatives of the earliest diverging clades of vascular plants, lycopods and ferns, form associations with both Mucoromycotina and Glomeromycota fungi. Lycopod sporophytes rely on a variety of strategies, entering into partnership with either Glomeromycota or Mucoromycotina, both or often neither. By contrast, all the ferns sampled associated exclusively with Glomeromycota, with the exception of the derived genus Anogramma where dual partnerships were detected. Our discovery finally provides an explanation for the unusual colonization patterns reported before in some lycopods (Duckett & Ligrone, 1992; Schmid & Oberwinkler, 1993), consisting of an intracellular phase and extensive fungal proliferation in gametophytic mucilage-filled intercellular spaces, as also reported in other Mucoromycotina-associated groups: the Haplomiotriopsida liverwort genus Treubia (Duckett et al., 2006), several hornwort genera (Desirò et al., 2013), and the Devonian fossil plant Horneophyton ligneri (Strullu-Derrien et al., 2014). We hypothesize that the associations between Mucoromycotina fungi and vascular plants are mutualistic. Beyond microscopy, our main line of evidence is the recent demonstration of mutualism between Haplomitriopsida liverworts and Mucoromycotina fungi (Field et al., 2014) closely related to those now detected in vascular plants. Our observations demonstrate that intercellular fungal proliferation is a signature of Mucoromycotina colonization, and lend further support to the hypothesis that the early Devonian vascular plant Nothia, which also harboured inter- and intracellular fungi (Berbee & Taylor, 2007; Krings et al., 2007a,b), formed associations with Mucoromycotina fungi (Pressel et al., 2010; Strullu-Derrien et al., 2014). Nonetheless, where the fungi are exclusively intracellular (e.g. Anogramma), it is impossible to ascertain from cytology alone to which fungal group they belong, as both Glomeromycota and Mucoromycotina produce vesicles and hyphal coils. The short-lived fungal swellings or lumps typical of Mucoromycotina colonization in the Haplomitriopsida (Carafa et al., 2003; Duckett et al., 2006) are unique to this group, the only land plant lineage to date known to associate exclusively with Mucoromycotina fungi (Field et al., 2014). Arbuscules, the signature of Glomeromycota colonization in angiosperms, are produced in some lycopod and fern–Glomeromycota associations (e.g. Ptisana, Angiopteris, Osmunda – Ogura-Tsujita et al., 2013) but are lacking in others (see Strullu-Derrien et al., 2014 and references therein), as is also often the case in liverworts and hornworts. The presence of Glomeromycota and Mucoromycotina fungi in lycopods and the predominance of Glomeromycota in the later diverging ferns fit the phylogenetic distribution of these fungi in other 'lower' land plant groups. As such, dual partnerships are the norm in basal thalloid liverworts, while more derived clades have, like ferns, the specificity to Glomeromycota typical of later vascular plant lineages (Smith & Read, 2008). Together with the occurrence of multiple fungal associations in Devonian plants (Strullu-Derrien et al., 2014), this lends further weight to the notion of shifting symbiotic encounters between early land colonists and soil-dwelling fungi before the Glomeromycota became dominant. The presence of Mucoromycotina in Anogramma may be a recent reacquisition, on a par with Endogone forming ectomycorrhizas with pines (Walker, 1985), and probably relates to its unique life cycle among ferns – comprising short-lived sporophytes and aestivating tubers (Goebel, 1905). It is also possible that associations with Mucoromycotina in lycopods and other plants represent recent acquisitions. However, this seems unlikely, given that the genes required for mycorrhiza formation in angiosperms are highly conserved across major plant lineages and that mycorrhizal genes from Mucoromycotina-associated Haplomitriopsida liverworts recovered the Glomeromycota mycorrhizal phenotype in a transformed mutant of the angiosperm Medicago truncatula (Wang et al., 2010). These findings, coupled with the occurrence of Mucoromycotina in extant basal groups of both nonvascular and vascular plants, as well as fossil plants (Strullu-Derrien et al., 2014), indicate that associations between Mucoromycotina and land plants are extremely ancient. During this investigation, we examined sporophytes only and it would be desirable now to study the cryptic nonphotosynthetic gametophytes of a range of lycopods and ferns, which are expected to be more heavily and consistently colonized by fungi (Read et al., 2000; Ogura-Tsujita et al., 2013). Nevertheless, our discovery that lycopods enter into partnerships with both Mucoromycotina and Glomeromycota fungi opens a new chapter in understanding the origins and evolution of fungal symbioses in vascular plants. Functional studies into the nature of these associations, like those conducted by Field et al. (2014) on Haplomitriopsida–Mucoromycotina symbioses, are now needed. Acknowledgements M.I.B. and S.P. thank NERC for grants NE/I027193/1 and NE/I025360/1. J.G.D. thanks the Leverhulme Trust for an Emeritus Fellowship. A Darwin Initiative Grant enabled S.P. and J.G.D. to collect fungal samples from Ascension Island. We thank Jim Trappe (Oregon State University) and María Martín (Royal Botanic Garden of Madrid) for fungal fruiting bodies, and Tatiana Solovieva (supported by the Society for Biology and Imperial College Undergraduate Research Opportunities Programme) for analysing Ascension Island samples. Our thanks go to the Editor and three anonymous referees for their comments. Supporting Information Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Filename Description nph13221-sup-0001-Supinfo.pdfapplication/PDF, 615.3 KB Fig. S1 Glomeromycota associates of basal vascular plants in a Bayesian full 18S nrDNA analysis.Fig. S2 Mucoromycotina associates of basal vascular plants in a Bayesian full 18S nrDNA analysis.Table S1 Lycopod, fern and fungal fruiting body samples analysed with their origin and fungi detected Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. References Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389– 3402. Berbee ML, Taylor JW. 2007. Rhynie chert: a window into a lost world of complex plant–fungus interactions. New Phytologist 174: 475– 479. Bidartondo MI, Read DJ, Trappe JM, Merckx V, Ligrone R, Duckett JG. 2011. The dawn of symbiosis between plants and fungi. Biology Letters 7: 574– 577. Carafa A, Duckett JG, Ligrone R. 2003. Subterranean gametophytic axes in the primitive liverwort Haplomitrium harbour a unique type of endophytic association with aseptate fungi. New Phytologist 160: 185– 197. Desirò A, Duckett JG, Pressel S, Villarreal JC, Bidartondo MI. 2013. Fungal symbioses in hornworts: a chequered history. Proceedings of the Royal Society Series B–Biological Sciences 280: 20130207. Duckett JG, Carafa A, Ligrone R. 2006. A highly differentiated glomeromycotean association with the mucilage-secreting, primitive antipodean liverwort Treubia (Treubiaceae): clues to the origins of mycorrhizas. American Journal of Botany 93: 797– 813. Duckett JG, Ligrone R. 1992. A light and electron-microscope study of the fungal endophytes in the sporophyte and gametophyte of Lycopodium cernuum with observations on the gametophyte–sporophyte junction. Canadian Journal of Botany 70: 58– 72. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27: 2194– 2200. Field KJ, Rimington WR, Bidartondo MI, Allinson KE, Beerling DJ, Cameron DD, Duckett JG, Leake JR, Pressel S. 2014. First evidence of mutualism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2. New Phytologist 205: 743– 756. Goebel K. 1905. Organography of plants Part II. Translated I. B. Balfour. Oxford, UK: Clarendon Press. Hirose D, Degawa Y, Yamamoto K, Yamada A. 2014. Sphaerocreas pubescens is a member of the Mucoromycotina closely related to fungi associated with liverworts and hornworts. Mycoscience 55: 221– 226. Horn K, Franke T, Unterseher M, Schnittler M, Beenken L. 2013. Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes of Diphasiastrum alpinum (Lycopodiaceae). American Journal of Botany 100: 2158– 2174. Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754– 755. James TY, Kauff F, Schoch CL, Matheny PB, Hofstetter V, Cox CJ, Celio G, Gueidan C, Fraker E, Miadlikowska J et al. 2006. Reconstructing the early evolution of Fungi using a six-gene phylogeny. Nature 443: 818– 822. Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ. 2007a. An alternative mode of early land plant colonization by putative endomycorrhizal fungi. Plant Signaling & Behavior 2: 125– 126. Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ. 2007b. Fungal endophytes in a 400-million-yr-old land plant: infection pathways, spatial distribution, and host responses. New Phytologist 174: 648– 657. Leake JR, Cameron DD, Beerling DJ. 2008. Fungal fidelity in the myco-heterotroph-to-autotroph life cycle of Lycopodiaceae: a case of parental nurture? New Phytologist 177: 572– 576. Lin K, Limpens E, Zhang ZH, Ivanov S, Saunders DGO, Mu DS, Pang EL, Cao HF, Cha HH, Lin T et al. 2014. Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus. PLoS Genetics 10: e1004078. Ogura-Tsujita Y, Sakoda A, Ebihara A, Yukawa T, Imaichi R. 2013. Arbuscular mycorrhiza formation in cordate gametophytes of two ferns, Angiopteris lygodiifolia and Osmunda japonica. Journal of Plant Research 126: 41– 50. Parniske M. 2008. Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology 6: 763– 775. Pirozynski KA, Dalpé Y. 1989. Geological history of the Glomaceae with particular reference to mycorrhizal symbiosis. Symbiosis 7: 1– 36. Pressel S, Bidartondo MI, Ligrone R, Duckett JG. 2010. Fungal symbioses in bryophytes: new insights in the Twenty First Century. Phytotaxa 9: 238– 253. Read DJ, Duckett JG, Francis R, Ligrone R, Russell A. 2000. Symbiotic fungal associations in 'lower' land plants. Philosophical Transactions of the Royal Society of London. Series B–Biological Sciences 355: 815– 830. Schmid E, Oberwinkler F. 1993. Mycorrhiza-like interaction between the achlorophyllous gametophyte of Lycopodium clavatum L. and its fungal endophyte studied by light and electron-microscopy. New Phytologist 124: 69– 81. Selosse MA, Le Tacon F. 1998. The land flora: a phototroph-fungus partnership? Trends in Ecology and Evolution 13: 15– 20. Smit E, Leeflang P, Glandorf B, van Elsas JD, Wernars K. 1999. Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis. Applied and Environmental Microbiology 65: 2614– 2621. Smith SE, Read DJ. 2008. Mycorrhizal symbiosis. Cambridge, UK: Academic Press. Strullu-Derrien C, Kenrick P, Pressel S, Duckett JG, Rioult J-P, Strullu D-G. 2014. Fungal associations in Horneophyton ligneri from the Rhynie Chert (c. 407 million year old) closely resemble those in extant lower land plants: novel insights into ancestral plant–fungus symbioses. New Phytologist 203: 964– 979. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731– 2739. Walker C. 1985. Endogone lactiflua forming ectomycorrhizas with Pinus contorta. Transactions of the British Mycological Society 84: 353– 355. Wang B, Qiu YL. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza 16: 299– 363. Wang B, Yeun LH, Xue JY, Liu Y, Ane JM, Qiu YL. 2010. Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizas in the colonization of land by plants. New Phytologist 186: 514– 525. White T, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M Innis, D Gelfand, J Sninsky, T White, eds. PCR protocols: a guide to methods and applications. Orlando, FL, USA: Academic Press, 315– 322. Winther JL, Friedman WE. 2008. Arbuscular mycorrhizal associations in Lycopodiaceae. New Phytologist 177: 790– 801. Citing Literature Volume205, Issue4Special Issue: Ecology and evolution of mycorrhizasMarch 2015Pages 1394-1398 This article also appears in:Ecology and evolution of mycorrhizas FiguresReferencesRelatedInformation