pp 1–15 | Cite as

Exploring the natural microbiome of the model liverwort: fungal endophyte diversity in Marchantia polymorpha L

  • Jessica NelsonEmail author
  • A. Jonathan Shaw


Within their tissues, plants host diverse communities of fungi, termed fungal endophytes. These fungi can affect plant growth, competitiveness, and resistance to stressors, thereby influencing plant community structure. Research characterizing fungal endophyte communities has so far mostly focused on seed plants, but information on the endophytes of other plant lineages is needed to understand how plant microbiomes impact whole ecosystems and how major changes through land plant evolution have affected plant-microbe relationships. In this study, we assess the fungal endophyte community of the model liverwort Marchantia polymorpha L. by both culturing and Illumina amplicon sequencing methods. We detect a very diverse fungal community that is distinct between M. polymorpha patches and only shares a few core fungi between populations across the United States. We also show low overlap in taxa detected by the different methods. This study helps build a foundation for using M. polymorpha and other Marchantia species as models for the ecology and dynamics of bryophyte microbiomes.


Bryophyte Liverwort Fungal endophytes Marchantia polymorpha Plant microbiota 



The authors thank the following for assistance with field work logistics and facilities: G. Hermann and M. Metz (Lewis & Clark College); S. LaGreca and K. Hodge (Cornell University); K. McFarland (University of Tennessee); D. Allard and M. Tierney (University of Vermont); P. Ball (Oregon State University, Cascades); D. Taylor (Daniel Boone National Forest); B. Overton (Lock Haven University); A. DeMarais, M. Morrison, and B. Kirkpatrick (University of Puget Sound); C. Crisafulli (Pacific Northwest Research Station); K. Golinski (Smithsonian Institution); S. Heiney (North Carolina Botanical Garden). We thank R. Vilgalys, F. Lutzoni, F. Dietrich, and P. Manos for their advice on manuscript development. We also thank our undergraduate assistants: K. Atherton, C. Chen, R. Hinson, and S. Ou. Funding for this work was provided by grant no. DEB-1501826, U.S. National Science Foundation, an Anderson & Crum grant from the American Bryological and Lichenological Society, and a Grant in Aid of Research from the Duke University Biology Department.


  1. Abarenkov K, Henrik Nilsson R, Larsson KH, Alexander IJ, Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, Pennanen T, Sen R, Taylor AFS, Tedersoo L, Ursing BM, Vrålstad T, Liimatainen K, Peintner U, Kõljalg U (2010) The UNITE database for molecular identification of fungi–recent updates and future perspectives. New Phytol 186:281–285CrossRefPubMedGoogle Scholar
  2. Afkhami ME, Strauss SY (2016) Native fungal endophytes suppress an exotic dominant and increase plant diversity over small and large spatial scales. Ecology 97:1159–1169CrossRefPubMedGoogle Scholar
  3. Aguilar-Trigueros CA, Rillig MC (2016) Effect of different root endophytic fungi on plant community structure in experimental microcosms. Ecol Evol 6:8149–8158CrossRefPubMedGoogle Scholar
  4. Akita M, Lehtonen MT, Koponen H, Marttinen EM, Valkonen JPT (2011) Infection of the Sunagoke moss panels with fungal pathogens hampers sustainable greening in urban environments. Sci Total Environ 409(17):3166–3173CrossRefPubMedGoogle Scholar
  5. Alcaraz LD, Peimbert M, Barajas HR, Dorantes-Acosta AE, Bowman JL, Arteaga-Vázquez MA (2018) Marchantia liverworts as a proxy to plants’ basal microbiomes. Sci Rep 8:12712CrossRefPubMedGoogle Scholar
  6. Arnold AE (2002) Neotropical fungal endophytes: diversity and ecology. Dissertation, The University of ArizonaGoogle Scholar
  7. Arnold AE (2007) Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biol Rev 21:51–66CrossRefGoogle Scholar
  8. Arnold AE, Maynard Z, Gilbert GS (2001) Fungal endophytes in dicotyledonous neotropical trees: patterns of abundance and diversity. Mycol Res 105:1502–1507CrossRefGoogle Scholar
  9. Berg G, Rybakova D, Grube M, Köberl M (2015) The plant microbiome explored: implications for experimental botany. J Exp Bot 67:995–1002CrossRefPubMedGoogle Scholar
  10. Bischler-Causse H, Boisselier-Dubayle M (1991) Lectotypification of Marchantia polymorpha L. J Bryol 16:361–365CrossRefGoogle Scholar
  11. Boisselier-Dubayle MC, Jubier MF, Lejeune B, Bischler H (1995) Genetic variability in the three subspecies of Marchantia polymorpha (Hepaticae): isozymes, RFLP and RAPD markers. Taxon 44(3):363CrossRefGoogle Scholar
  12. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120Google Scholar
  13. Bowman JL, Araki T, Kohchi T (2016) Marchantia: past, present and future. Plant Cell Physiol 57:205–209CrossRefPubMedGoogle Scholar
  14. Bowman JL et al (2017) Insights into land plant evolution garnered from the Marchantia polymorpha genome. Cell 171:287–304CrossRefPubMedGoogle Scholar
  15. Bradbury S (2006) Response of the post-fire bryophyte community to salvage logging in boreal mixedwood forests of northeastern Alberta, Canada. For Ecol Manag 234:313–322CrossRefGoogle Scholar
  16. Bresinsky A, Schotz A (2006) Behaviour in cultures and habitat requirements of species within the genera Loreleia and Rickenella (Agaricales). Acta Mycol 41:189–208Google Scholar
  17. Bunyard BA, Nicholson MS, Royse DJ (1994) A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86:762–772CrossRefGoogle Scholar
  18. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336CrossRefPubMedGoogle Scholar
  19. Carbone I et al (2016) T-BAS: tree-based alignment selector toolkit for phylogenetic-based placement, alignment downloads and metadata visualization: an example with the Pezizomycotina tree of life. Bioinformatics 33:1160–1168Google Scholar
  20. Chen KH, Liao HL, Arnold AE, Bonito G, Lutzoni F (2018) RNA-based analyses reveal fungal communities structured by a senescence gradient in the moss Dicranum scoparium and the presence of putative multi-trophic fungi. New Phytol 218:1597–1611CrossRefPubMedGoogle Scholar
  21. Cordier T, Robin C, Capdevielle X, Fabreguettes O, Desprez-Loustau ML, Vacher C (2012) The composition of phyllosphere fungal assemblages of European beech (Fagus sylvatica) varies significantly along an elevation gradient. New Phytol 196:510–519CrossRefPubMedGoogle Scholar
  22. Davey ML, Currah RS (2006) Interactions between mosses (Bryophyta) and fungi. Can J Bot 84(10):1509–1519CrossRefGoogle Scholar
  23. Davey ML, Currah RS (2009) Atradidymella muscivora gen. Et sp. nov.(Pleosporales) and its anamorph Phoma muscivora sp. nov.: a new pleomorphic pathogen of boreal bryophytes. Am J Bot 96:1281–1288CrossRefPubMedGoogle Scholar
  24. Davey ML, Tsuneda A, Currah RS (2009) Pathogenesis of bryophyte hosts by the ascomycete. Atradidymella muscivora. Am J Bot 96:1274–1280CrossRefPubMedGoogle Scholar
  25. Davey ML, Heegaard E, Halvorsen R, Ohlson M, Kauserud H (2012) Seasonal trends in the biomass and structure of bryophyte-associated fungal communities explored by 454 pyrosequencing. New Phytol 195:844–856CrossRefPubMedGoogle Scholar
  26. Davey ML, Heegaard E, Halvorsen R, Kauserud H, Ohlson M (2013) Amplicon-pyrosequencing-based detection of compositional shifts in bryophyte-associated fungal communities along an elevation gradient. Mol Ecol 22:368–383CrossRefPubMedGoogle Scholar
  27. Davey ML, Kauserud H, Ohlson M (2014) Forestry impacts on the hidden fungal biodiversity associated with bryophytes. FEMS Microbiol Ecol 90:313–325CrossRefPubMedGoogle Scholar
  28. Davey ML, Skogen MJ, Heegaard E, Halvorsen R, Kauserud H, Ohlson M (2017) Host and tissue variations overshadow the response of boreal moss-associated fungal communities to increased nitrogen load. Mol Ecol 26:571–588CrossRefPubMedGoogle Scholar
  29. Davis EC, Shaw AJ (2008) Biogeographic and phylogenetic patterns in diversity of liverwort-associated endophytes. Am J Bot 95:914–924CrossRefPubMedGoogle Scholar
  30. Davis EC, Franklin JB, Shaw AJ, Vilgalys R (2003) Endophytic Xylaria (Xylariaceae) among liverworts and angiosperms: phylogenetics, distribution, and symbiosis. Am J Bot 90:1661–1667CrossRefPubMedGoogle Scholar
  31. Delaux P-M, Nanda AK, Mathé C, Sejalon-Delmas N, Dunand C (2012) Molecular and biochemical aspects of plant terrestrialization. Perspect Plant Ecol Evol Syst 14:49–59CrossRefGoogle Scholar
  32. Desirò A, Duckett JG, Pressel S, Villarreal JC, Bidartondo MI (2013) Fungal symbioses in hornworts: a chequered history. Proc R Soc Lond B Biol Sci 280:20130207CrossRefGoogle Scholar
  33. Döbbeler P (1997) Biodiversity of bryophilous ascomycetes. Biodivers Conserv 6:721–738CrossRefGoogle Scholar
  34. Egertová Z, Eckstein J, Vega M (2015) Lamprospora tuberculata, Octospora ithacaensis, O. orthotrichi and O. affinis–four bryoparasitic ascomycetes new to the Czech Republic. Czech Mycol 67Google Scholar
  35. Field KJ, Cameron DD, Leake JR, Tille S, Bidartondo MI, Beerling DJ (2012) Contrasting arbuscular mycorrhizal responses of vascular and non-vascular plants to a simulated. Palaeozoic CO2 decline. Nat Commun 3:835CrossRefPubMedGoogle Scholar
  36. Field KJ, Rimington WR, Bidartondo MI, Allinson KE, Beerling DJ, Cameron DD, Duckett JG, Leake JR, Pressel S (2016) Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline. ISME J 10:1514–1526CrossRefPubMedGoogle Scholar
  37. Garcia G, Van Vooren N (2005) Un discomycète inoperculé plutôt discret, Pezoloma ciliifera, et remarques sur le genre Pezoloma. Publications de la Société Linnéenne de Lyon 74:115–130Google Scholar
  38. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefPubMedGoogle Scholar
  39. Glime JM (2013a) Protozoa diversity. In: Bryophyte ecology volume 2: byological interactionGoogle Scholar
  40. Glime JM (2013b) Invertebrates: nematodes. In: Bryophyte ecology volume 2: bryological interactionGoogle Scholar
  41. Glime JM (2017) The fauna: a place to call home. In: Bryophyte ecology volume 2: bryological interactionGoogle Scholar
  42. Glynou K, Ali T, Buch AK, Haghi Kia S, Ploch S, Xia X, Çelik A, Thines M, Maciá-Vicente JG (2016) The local environment determines the assembly of root endophytic fungi at a continental scale. Environ Microbiol 18:2418–2434CrossRefPubMedGoogle Scholar
  43. Graff PW (1936) Invasion by Marchantia polymorpha following forest fires. Bull Torrey Bot Club 63:67–74CrossRefGoogle Scholar
  44. Guminska B, Mierzenska M (1992) Gerronema marchantiae Sing et Clem-a fungus associating with Marchantia polymorpha L and Nostoc sp Zeszyty Naukowe Uniwersytetu Jagiellońskiego Prace Botaniczne 24:171–177Google Scholar
  45. Hamayun M, Khan SA, Khan AL, Rehman G, Sohn EY, Shah AA, Kim SK, Joo GJ, Lee IJ (2009) Phoma herbarum as a new gibberellin-producing and plant growth-promoting fungus. J Microbiol Biotechnol 19:1244–1249PubMedGoogle Scholar
  46. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M (2004) Trichoderma species—opportunistic, avirulent plant symbionts. Nat Rev Microbiol 2:43–56CrossRefPubMedGoogle Scholar
  47. Heckman DS, Geiser DM, Eidell BR, Stauffer RL, Kardos NL, Hedges SB (2001) Molecular evidence for the early colonization of land by fungi and plants. Science 293:1129–1133CrossRefPubMedGoogle Scholar
  48. Hipol R, Tamang SMA, Gargabite BF, Broñola Hipol R (2015) Diversity of fungal endophytes isolated from Marchantia polymorpha populations from Baguio City, Philippines bulletin of environment, Pharmacology and Life Sciences 4:87–91Google Scholar
  49. Hoshina R, Kusuoka Y (2016) DNA analysis of algal endosymbionts of ciliates reveals the state of algal integration and the surprising specificity of the symbiosis. Protist 167:174–184CrossRefPubMedGoogle Scholar
  50. Humphreys CP, Franks PJ, Rees M, Bidartondo MI, Leake JR, Beerling DJ (2010) Mutualistic mycorrhiza-like symbiosis in the most ancient group of land plants. Nat Commun 1:103CrossRefPubMedGoogle Scholar
  51. Innis MA, Gelfand DH, Sninsky JJ, White TJ (2012) PCR protocols: a guide to methods and applications. Academic Press, LondonGoogle Scholar
  52. Junker C, Draeger S, Schulz B (2012) A fine line–endophytes or pathogens in Arabidopsis thaliana. Fungal Ecol 5:657–662CrossRefGoogle Scholar
  53. Kivlin SN, Lynn JS, Kazenel MR, Beals KK, Rudgers JA (2017) Biogeography of plant-associated fungal symbionts in mountain ecosystems: a meta-analysis. Divers Distrib 23:1067–1077CrossRefGoogle Scholar
  54. Knack J et al (2015) Microbiomes of streptophyte algae and bryophytes suggest that a functional suite of microbiota fostered plant colonization of land. Int J Plant Sci 176:405–420CrossRefGoogle Scholar
  55. Kowal J, Pressel S, Duckett JG, Bidartondo MI (2016) Liverworts to the rescue: an investigation of their efficacy as mycorrhizal inoculum for vascular plants. Funct Ecol 30:1014–1023CrossRefGoogle Scholar
  56. Kowal J, Pressel S, Duckett JG, Bidartondo MI, Field KJ (2018) From rhizoids to roots? Experimental evidence of mutualism between liverworts and ascomycete fungi. Ann Bot 121:221–227CrossRefPubMedGoogle Scholar
  57. Ligrone R, Carafa A, Lumini E, Bianciotto V, Bonfante P, Duckett JG (2007) Glomeromycotean associations in liverworts: a molecular, cellular, and taxonomic analysis. Am J Bot 94:1756–1777CrossRefPubMedGoogle Scholar
  58. Lundberg DS, Yourstone S, Mieczkowski P, Jones CD, Dangl JL (2013) Practical innovations for high-throughput amplicon sequencing. Nat Methods 10:999–1002CrossRefPubMedGoogle Scholar
  59. Marks RA, Smith JJ, Cronk Q, McLetchie DN (2018) Variation in the bacteriome of the tropical liverwort, Marchantia inflexa, between the sexes and across habitats. Symbiosis 75:93–101Google Scholar
  60. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12CrossRefGoogle Scholar
  61. Nelson JM (2017) Diversity and effects of the fungal endophytes of the liverwort Marchantia polymorpha. Dissertation. Duke UniversityGoogle Scholar
  62. Nelson JM, Hauser DA, Hinson R, Shaw AJ (2018) A novel experimental system using the liverwort Marchantia polymorpha and its fungal endophytes reveals diverse and context-dependent effects. New Phytol 218:1217–1232CrossRefPubMedGoogle Scholar
  63. Nguyen NH, Smith D, Peay K, Kennedy P (2015) Parsing ecological signal from noise in next generation amplicon sequencing. New Phytol 205:1389–1393CrossRefPubMedGoogle Scholar
  64. Oksanen, J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, O’Hara RB, Simpson GL, Solymos P, Stevens MH (2017) vegan: Community Ecology Package. R package version 2.4–3Google Scholar
  65. Peay KG, Kennedy PG, Talbot JM (2016) Dimensions of biodiversity in the earth mycobiome. Nat Rev Microbiol 14:434–447CrossRefPubMedGoogle Scholar
  66. Peršoh D (2015) Plant-associated fungal communities in the light of meta’omics. Fungal Divers 75:1–25CrossRefGoogle Scholar
  67. Ploch S, Rose LE, Bass D, Bonkowski M (2016) High diversity revealed in leaf-associated protists (Rhizaria: Cercozoa) of Brassicaceae. J Eukaryot Microbiol 63:635–641CrossRefPubMedGoogle Scholar
  68. Porras-Alfaro A, Bayman P (2011) Hidden fungi, emergent properties: endophytes and microbiomes. Annu Rev Phytopathol 49:291–315CrossRefPubMedGoogle Scholar
  69. Pressel S, Bidartondo MI, Ligrone R, Duckett JG (2010) Fungal symbioses in bryophytes: new insights in the twenty first century. Phytotaxa 9:238–253CrossRefGoogle Scholar
  70. Radhakrishnan G (2017) Tracing the evolution of the arbuscular mycorrhizal symbiosis in the plant lineage. Doctoral dissertation, University of East AngliaGoogle Scholar
  71. Reese WD (1981) " Chlorochytrium," a green alga endophytic in Musci. Bryologist 84:75–78CrossRefGoogle Scholar
  72. Reese WD (1992) More mosses with Chlorochytrium1. J Phycol 28:707–707CrossRefGoogle Scholar
  73. Rognes T, Flouri T, Nichols B, Quince C, Mahé F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584CrossRefPubMedGoogle Scholar
  74. Ross A, Yasutake W, Leek S (1975) Phoma herbarum, a fungal plant saprophyte, as a fish pathogen. J Fish Res Board Can 32:1648–1652CrossRefGoogle Scholar
  75. Rudgers JA, Koslow JM, Clay K (2004) Endophytic fungi alter relationships between diversity and ecosystem properties. Ecol Lett 7:42–51CrossRefGoogle Scholar
  76. Sapp M, Ploch S, Fiore-Donno AM, Bonkowski M, Rose LE (2018) Protists are an integral part of the Arabidopsis thaliana microbiome. Environ Microbiol 20:30–43CrossRefPubMedGoogle Scholar
  77. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefPubMedGoogle Scholar
  78. Schulz B, Boyle C (2005) The endophytic continuum. Mycol Res 109:661–686CrossRefPubMedGoogle Scholar
  79. Shaw J, Renzaglia K (2004) Phylogeny and diversification of bryophytes. Am J Bot 91:1557–1581CrossRefPubMedGoogle Scholar
  80. Spatafora JW, Chang Y, Benny GL, Lazarus K, Smith ME, Berbee ML, Bonito G, Corradi N, Grigoriev I, Gryganskyi A, James TY, O’Donnell K, Roberson RW, Taylor TN, Uehling J, Vilgalys R, White MM, Stajich JE (2016) A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia 108:1028–1046CrossRefPubMedGoogle Scholar
  81. Stenroos S, Laukka T, Huhtinen S, Döbbeler P, Myllys L, Syrjänen K, Hyvönen J (2010) Multiple origins of symbioses between ascomycetes and bryophytes suggested by a five-gene phylogeny. Cladistics 26:281–300CrossRefGoogle Scholar
  82. Stone JK, Bacon CW, White J (2000) An overview of endophytic microbes: endophytism defined. Microbial Endophytes 3:29–33Google Scholar
  83. Taylor TN, Krings M (2005) Fossil microorganisms and land plants: associations and interactions. Symbiosis 40:119–135Google Scholar
  84. Team RC (2018) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  85. Trémouillaux-Guiller J, Huss VA (2007) A cryptic intracellular green alga in Ginkgo biloba: ribosomal DNA markers reveal worldwide distribution. Planta 226:553–557CrossRefPubMedGoogle Scholar
  86. U’Ren JM, Lutzoni F, Miadlikowska J, Laetsch AD, Arnold AE (2012) Host and geographic structure of endophytic and endolichenic fungi at a continental scale. Am J Bot 99:898–914CrossRefPubMedGoogle Scholar
  87. Venables WN, Ripley BD (2002) Random and mixed effects. In: Modern applied statistics with S Springer, pp 271–300Google Scholar
  88. Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246CrossRefPubMedGoogle Scholar
  89. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267CrossRefPubMedGoogle Scholar
  90. Wickham H (2009) ggplot2: elegant graphics for data analysis. Springer-Verlag, New YorkGoogle Scholar
  91. Wickham H (2011) The split-apply-combine strategy for data analysis. J Stat Softw 40:1–29Google Scholar
  92. Wu L, Han T, Li W, Jia M, Xue L, Rahman K, Qin L (2013) Geographic and tissue influences on endophytic fungal communities of Taxus chinensis var. mairei in China. Curr Microbiol 66:40–48CrossRefPubMedGoogle Scholar
  93. Yuan Z-L, Chen Y-C, Yang Y (2009) Diverse non-mycorrhizal fungal endophytes inhabiting an epiphytic, medicinal orchid (Dendrobium nobile): estimation and characterization. World J Microbiol Biotechnol 25:295CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of BiologyDuke UniversityDurhamUSA
  2. 2.Boyce Thompson InstituteIthacaUSA

Personalised recommendations