Abstract
Mixotrophic plants obtain carbon by their own photosynthetic activity and from their root-associated mycorrhizal fungi. Mixotrophy is deemed a pre-adaptation for evolution of mycoheterotrophic nutrition, where plants fully depend on fungi and lose their photosynthetic activity. The aim of this study was to clarify mycorrhizal dependency and heterotrophy level in various phenotypes of mixotrophic Pyrola japonica (Ericaceae), encompassing green individuals, rare achlorophyllous variants (albinos) and a form with minute leaves, P. japonica f. subaphylla. These three phenotypes were collected in two Japanese forests. Phylogenetic analysis of both plants and mycorrhizal fungi was conducted based on DNA barcoding. Enrichment in 13C among organs (leaves, stems and roots) of the phenotypes with reference plants and fungal fruitbodies were compared by measuring stable carbon isotopic ratio. All plants were placed in the same clade, with f. subaphylla as a separate subclade. Leaf 13C abundances of albinos were congruent with a fully mycoheterotrophic nutrition, suggesting that green P. japonica leaves are 36.8% heterotrophic, while rhizomes are 74.0% heterotrophic. There were no significant differences in δ13C values among organs in both albino P. japonica and P. japonica f. subaphylla, suggesting full and high mycoheterotrophic nutrition, respectively. Among 55 molecular operational taxonomic units (OTUs) detected as symbionts, the genus Russula was the most abundant in each phenotype and its dominance was significantly higher in albino P. japonica and P. japonica f. subaphylla. Russula spp. detected in P. japonica f. subaphylla showed higher dissimilarity with other phenotypes. These results suggest that P. japonica sensu lato is prone to evolve mycoheterotrophic variants, in a process that changes its mycorrhizal preferences, especially towards the genus Russula for which this species has a marked preference.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abadie J-C, Püttsepp Ü, Gebauer G et al (2006) Cephalanthera longifolia (Neottieae, Orchidaceae) is mixotrophic: a comparative study between green and nonphotosynthetic individuals. Can J Bot 84:1462–1477. https://doi.org/10.1139/b06-101
Altschul SF, Madden TL, Schaffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Bidartondo MI, Bruns TD (2001) Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographical structure. Mol Ecol 10:2285–2295. https://doi.org/10.1046/j.1365-294X.2001.01358.x
Bidartondo MI, Burghardt B, Gebauer G et al (2004) Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proc R Soc B Biol Sci 271:1799–1806. https://doi.org/10.1098/rspb.2004.2807
Bidartondo MI, Read DJ (2008) Fungal specificity bottlenecks during orchid germination and development. Mol Ecol 17:3707–3716. https://doi.org/10.1111/j.1365-294X.2008.03848.x
Braukmann T, Stefanović S (2012) Plastid genome evolution in mycoheterotrophic Ericaceae. Plant Mol Biol 79:5–20. https://doi.org/10.1007/s11103-012-9884-3
Cameron DD, Preiss K, Gebauer G, Read DJ (2009) The chlorophyll-containing orchid Corallorhiza trifida derives little carbon through photosynthesis. New Phytol 183:358–364. https://doi.org/10.1111/j.1469-8137.2009.02853.x
CBOL Plant Working Group (2009) A DNA barcode for land plants. Proc Natl Acad Sci 106:12794–12797
Cullings KW, Szaro TM, Bruns TD (1996) Evolution of extreme specialization within a lineage of ectomycorrhizal epiparasites. Nature 379:63–66
De Cáceres M, Legendre P (2009) Associations between species and groups of sites: indices and statistical inference. Ecology 90:3566–3574. https://doi.org/10.1890/08-1823.1
Eriksson O, Kainulainen K (2011) The evolutionary ecology of dust seeds. Perspect Plant Ecol Evol Syst 13:73–87. https://doi.org/10.1016/j.ppees.2011.02.002
Farquhar GD, O’Leary MH, Berry JA (1982) On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves. Aust J Plant Physiol 9:121–137. https://doi.org/10.1071/PP9820121
Figura T, Tylová E, Šoch J et al (2019) In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development. Ann Bot 123:625–639
Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes- application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x
Gebauer G, Meyer M (2003) 15N and 13C natural abundance of autotrophic and myco-heterotrophic orchids provides insight into nitrogen and carbon gain from fungal association. New Phytol 160:209–223. https://doi.org/10.1046/j.1469-8137.2003.00872.x
Gebauer G, Schulze E-D (1991) Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge, NE Bavaria. Oecologia 87:198–207
Girlanda M, Selosse MA, Cafasso D et al (2006) Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae. Mol Ecol 15:491–504. https://doi.org/10.1111/j.1365-294X.2005.02770.x
Gomes SIF, Aguirre-Gutierrez J, Bidartondo MI, Merckx VSFT (2017) Arbuscular mycorrhizal interactions of mycoheterotrophic Thismia are more specialized than in autotrophic plants. New Phytol 213:1418–1427. https://doi.org/10.1111/nph.14249
Gomes SIF, van Bodegom PM, Merckx VSFT, Soudzilovskaia NA (2019) Global distribution patterns of mycoheterotrophy. Glob Ecol Biogeogr 28:1133–1145. https://doi.org/10.1111/geb.12920
Gonneau C, Jersáková J, de Tredern E et al (2014) Photosynthesis in perennial mixotrophic Epipactis spp. (Orchidaceae) contributes more to shoot and fruit biomass than to hypogeous survival. J Ecol 102:1183–1194. https://doi.org/10.1111/1365-2745.12274
Hashimoto Y, Fukukawa S, Kunishi A et al (2012) Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids. New Phytol 195:620–630. https://doi.org/10.1111/j.1469-8137.2012.04174.x
Hynson NA, Bruns TD (2009) Evidence of a myco-heterotroph in the plant family Ericaceae that lacks mycorrhizal specificity. Proc R Soc B Biol Sci 276:4053–4059. https://doi.org/10.1098/rspb.2009.1190
Hynson NA, Preiss K, Gebauer G, Bruns TD (2009) Isotopic evidence of full and partial myco-heterotrophy in the plant tribe Pyroleae (Ericaceae). New Phytol 182:719–726. https://doi.org/10.1111/j.1469-8137.2009.02781.x
Hynson NA, Mambelli S, Amend AS, Dawson TE (2012) Measuring carbon gains from fungal networks in understory plants from the tribe Pyroleae (Ericaceae): a field manipulation and stable isotope approach. Oecologia 169:307–317. https://doi.org/10.1007/s00442-011-2198-3
Hynson NA, Madsen TP, Selosse MA et al (2013a) The physiological ecology of mycoheterotrophy. In: Merckx VSFT (ed) Mycoheterotrophy: the biology of plants living on fungi. Springer-Verlag, New York, pp 297–342
Hynson NA, Weiß M, Preiss K et al (2013b) Fungal host specificity is not a bottleneck for the germination of Pyroleae species (Ericaceae) in a Bavarian forest. Mol Ecol 22:1473–1481. https://doi.org/10.1111/mec.12180
Jacquemyn H, Merckx VSFT (2019) Mycorrhizal symbioses and the evolution of trophic modes in plants. J Ecol 107:1567–1581. https://doi.org/10.1111/1365-2745.13165
Johansson VA, Müller G, Eriksson O (2014) Dust seed production and dispersal in Swedish Pyroleae species. Nord J Bot 32:209–214. https://doi.org/10.1111/j.1756-1051.2013.00307.x
Johansson VA, Mikusinska A, Ekblad A, Eriksson O (2015) Partial mycoheterotrophy in Pyroleae: nitrogen and carbon stable isotope signatures during development from seedling to adult. Oecologia 177:203–211. https://doi.org/10.1007/s00442-014-3137-x
Johansson VA, Bahram M, Tedersoo L et al (2017) Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development. Mol Ecol 26:2591–2604. https://doi.org/10.1111/mec.14050
Jolles DD, Wolfe AD (2012) Genetic differentiation and crypsis among members of the myco-heterotrophic Pyrola picta species complex (pyroleae: Monotropoideae: Ericaceae). Syst Bot 37:468–477. https://doi.org/10.1600/036364412X635511
Julou T, Burghardt B, Gebauer G et al (2005) Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol 166:639–653. https://doi.org/10.1111/j.1469-8137.2005.01364.x
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. https://doi.org/10.1093/molbev/mst010
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. https://doi.org/10.1093/molbev/msw054
Lallemand F, Gaudeul M, Lambourdière J et al (2016) The elusive predisposition to mycoheterotrophy in Ericaceae. New Phytol 212:314–319. https://doi.org/10.1111/nph.14092
Lallemand F, Puttsepp Ü, Lang M et al (2017) Mixotrophy in Pyroleae (Ericaceae) from Estonian boreal forests does not vary with light or tissue age. Ann Bot 120:361–371. https://doi.org/10.1093/aob/mcx054
Lallemand F, Figura T, Damesin C et al (2019) Mixotrophic orchids do not use photosynthates for perennial underground organs. New Phytol 221:12–17. https://doi.org/10.1111/nph.15443
Leake J (1994) Tansley Review No. 69—the biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol 127:171–216
Leake J, Johnson D, Donnelly D et al (2004) Networks of power and influence: the role of mycorrhizal mycelium in controlling plant communities and agroecosystem functioning. Can J Bot 82:1016–1045. https://doi.org/10.1139/b04-060
Liu ZW, Zhou J, Liu E, De, Peng H (2010) A molecular phylogeny and a new classification of Pyrola (Pyroleae, Ericaceae). Taxon 59:1690–1700. https://doi.org/10.2307/41059866
Liu ZW, Jolles DD, Zhou J et al (2014) Multiple origins of circumboreal taxa in Pyrola (Ericaceae), a group with a Tertiary relict distribution. Ann Bot 114:1701–1709. https://doi.org/10.1093/aob/mcu198
Matsuda Y, Okochi S, Katayama T et al (2011) Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan. Mycorrhiza 21:569–576. https://doi.org/10.1007/s00572-011-0365-3
Matsuda Y, Shimizu S, Mori M et al (2012) Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments. Am J Bot 99:1177–1188. https://doi.org/10.3732/ajb.1100546
May M, Jąkalski M, Novotná A et al (2020) Three-year pot culture of Epipactis helleborine reveals autotrophic survival, without mycorrhizal networks, in a mixotrophic species. Mycorrhiza 30:51–61. https://doi.org/10.1007/s00572-020-00932-4
Merckx VSFT (2013) Mycohetrotrophy: an introduction. In: Merckx V (ed) Mycoheterotrophy: the biology of plants living on fungi. Springer-Verlag, New York, pp 1–17
Merckx VSFT, Freudenstein JV, Kissling J et al (2013) Taxonomy and classification. In: Merckx V (ed) Mycoheterotrophy: the biology of plants living on fungi. Springer-Verlag, New York, pp 19–101
Ogura-Tsujita Y, Yokoyama J, Miyoshi K, Yukawa T (2012) Shifts in mycorrhizal fungi during the evolution of autotrophy to mycoheterotrophy in Cymbidium (Orchidaceae). Am J Bot 99:1158–1176. https://doi.org/10.3732/ajb.1100464
Oksanen J, Blanchet FG, Friendly M et al (2018) vegan: community ecology package. R package version 2.5-2
Preiss K, Adam IKU, Gebauer G (2010) Irradiance governs exploitation of fungi: fine-tuning of carbon gain by two partially myco-heterotrophic orchids. Proc R Soc B Biol Sci 277:1333–1336. https://doi.org/10.1098/rspb.2009.1966
R Core Team (2020) R: a language and environment for statistical computing, Vienna, Austria. https://www.R-project.org/. Accessed 17 Oct 2020
Roy M, Gonneau C, Rocheteau A et al (2013) Why do mixotrophic plants stay green? A comparison between green and achlorophyllous orchid individuals in situ. Ecol Monogr 83:95–117. https://doi.org/10.1890/11-2120.1
Sakamoto Y, Ogura-Tsujita Y, Ito K et al (2016) The tiny-leaved orchid Cephalanthera subaphylla obtains most of its carbon via mycoheterotrophy. J Plant Res 129:1013–1020. https://doi.org/10.1007/s10265-016-0856-6
Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. https://doi.org/10.1128/AEM.01541-09
Schneider G, Chicken E, Becvarik R (2020) NSM3: functions and datasets to accompany hollander, wolfe, and chicken-nonparametric statistical methods, 3rd edn. R package version 1.14. https://CRAN.R-project.org/package=NSM3. Accessed 17 Oct 2020
Selosse MA, Weiß M, Jany JL, Tillier A (2002) Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) L.C.M. Rich. and neighbouring tree ectomycorrhizae. Mol Ecol 11:1831–1844. https://doi.org/10.1046/j.1365-294X.2002.01553.x
Selosse MA, Faccio A, Scappaticci G, Bonfante P (2004) Chlorophyllous and achlorophyllous specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal septomycetes, including truffles. Microb Ecol 47:416–426. https://doi.org/10.1007/s00248-003-2034-3
Selosse MA, Martos F (2014) Do chlorophyllous orchids heterotrophically use mycorrhizal fungal carbon? Trends Plant Sci 19:683–685. https://doi.org/10.1016/j.tplants.2014.09.005
Selosse MA, Roy M (2009) Green plants that feed on fungi: facts and questions about mixotrophy. Trends Plant Sci 14:64–70. https://doi.org/10.1016/j.tplants.2008.11.004
Selosse MA, Charpin M, Not F (2017) Mixotrophy everywhere on land and in water: the grand écart hypothesis. Ecol Lett 20:246–263. https://doi.org/10.1111/ele.12714
Shefferson RP, Roy M, Püttsepp Ü, Selosse MA (2016) Demographic shifts related to mycoheterotrophy and their fitness impacts in two cephalanthera species. Ecology 97:1452–1462. https://doi.org/10.1890/15-1336.1
Shutoh K, Kaneko S, Kurosawa T, Pyroleae, Ericaceae (2017) Taxonomy and distribution of Pyrola subaphylla Maxim. Acta Phytotaxon Geobot 68:181–192. https://doi.org/10.18942/apg.201707
Shutoh K, Suetsugu K, Kaneko S, Kurosawa T (2018) Comparative morphological analysis of two parallel mycoheterotrophic transitions reveals divergent and convergent traits in the genus Pyrola (Pyroleae, Ericaceae). J Plant Res 131:589–597. https://doi.org/10.1007/s10265-018-1040-y
Shutoh K, Tajima Y, Matsubayashi J et al (2020) Evidence for newly discovered albino mutants in a pyroloid: implication for the nutritional mode in the genus Pyrola. Am J Bot 107:650–657
Suetsugu K, Yamato M, Miura C et al (2017) Comparison of green and albino individuals of the partially mycoheterotrophic orchid Epipactis helleborine on molecular identities of mycorrhizal fungi, nutritional modes and gene expression in mycorrhizal roots. Mol Ecol 26:1652–1669. https://doi.org/10.1111/mec.14021
Suetsugu K, Yamato M, Matsubayashi J, Tayasu I (2019) Comparative study of nutritional mode and mycorrhizal fungi in green and albino variants of Goodyera velutina, an orchid mainly utilizing saprotrophic rhizoctonia. Mol Ecol 28:4290–4299. https://doi.org/10.1111/mec.15213
Suetsugu K, Matsubayashi J, Tayasu I (2020) Some mycoheterotrophic orchids depend on carbon from dead wood: novel evidence from a radiocarbon approach. New Phytol 227:1519–1529. https://doi.org/10.1111/nph.16409
Takahashi H (1991) Distribution of Japanese Pyrola and Orthilia, and a distribution pattern with a gap in the central to southern Tohoku district. Acta Phytotaxon Geobot 42:23–43. https://doi.org/10.18942/bunruichiri.KJ00001078702
Tedersoo L, Suvi T, Larsson E, Kõljalg U (2006) Diversity and community structure of ectomycorrhizal fungi in a wooded meadow. Mycol Res 110:734–748. https://doi.org/10.1016/j.mycres.2006.04.007
Tedersoo L, Pellet P, Kõljalg U, Selosse MA (2007) Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae. Oecologia 151:206–217. https://doi.org/10.1007/s00442-006-0581-2
Tĕšitel J, Těšitelová T, Minasiewicz J, Selosse M-A (2018) Mixotrophy in land plants: why to stay green? Trends Plant Sci 23:656–659. https://doi.org/10.1016/j.tplants.2018.05.010
Toftegaard T, Iason GR, Alexander IJ et al (2010) The threatened plant intermediate wintergreen (Pyrola media) associates with a wide range of biotrophic fungi in native Scottish pine woods. Biodivers Conserv 19:3963–3971. https://doi.org/10.1007/s10531-010-9940-8
Trudell SA, Rygiewicz PT, Edmonds RL (2003) Nitrogen and carbon stable isotope abundances support the myco-heterotrophic nature and host-specificity of certain achlorophyllous plants. New Phytol 160:391–401. https://doi.org/10.1046/j.1469-8137.2003.00876.x
Uesugi T, Nakano M, Selosse M-A et al (2016) Pyrola japonica, a partially mycoheterotrophic Ericaceae, has mycorrhizal preference for russulacean fungi in central Japan. Mycorrhiza 26:819–829. https://doi.org/10.1007/s00572-016-0715-2
Vincenot L, Tedersoo L, Richard F et al (2008) Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests. Mycorrhiza 19:15–25. https://doi.org/10.1007/s00572-008-0199-9
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (eds) PCR Protocols. Academic Press, New York, pp 315–322
Zimmer K, Hynson NA, Gebauer G et al (2007) Wide geographical and ecological distribution of nitrogen and carbon gains from fungi in pyroloids and monotropoids (Ericaceae) and in orchids. New Phytol 175:166–175. https://doi.org/10.1111/j.1469-8137.2007.02065.x
Zimmer K, Meyer C, Gebauer G (2008) The ectomycorrhizal specialist orchid Corallorhiza trifida is a partial myco-heterotroph. New Phytol 178:395–400. https://doi.org/10.1111/j.1469-8137.2007.02362.x
Acknowledgements
We thank Drs. T. Fukiharu and T. Furuki for the information on plant habitats, the staffs of the cities of Tomakomai and Sanmu for access to the study sites and David Marsh for English corrections. The authors also appreciate that anonymous reviewers improved the quality of this manuscript. The study was partly supported by KAKENHI (25,304,026) to YM.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Matsuda, Y., Yamaguchi, Y., Matsuo, N. et al. Communities of mycorrhizal fungi in different trophic types of Asiatic Pyrola japonica sensu lato (Ericaceae). J Plant Res 133, 841–853 (2020). https://doi.org/10.1007/s10265-020-01233-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10265-020-01233-9