Abstract
Although the absence of normal leaves is often considered a sign of full heterotrophy, some plants remain at least partially autotrophic despite their leafless habit. Leafless orchids with green stems and capsules probably represent a late evolutionary stage toward full mycoheterotrophy and serve as valuable models for understanding the pathways leading to this nutritional strategy. In this study, based on molecular barcoding and isotopic analysis, we explored the physiological ecology of the leafless orchid Eulophia zollingeri, which displays green coloration, particularly during its fruiting phase. Although previous studies had shown that E. zollingeri, in its adult stage, is associated with Psathyrellaceae fungi and exhibits high 13C isotope signatures similar to fully mycoheterotrophic orchids, it remained uncertain whether this symbiotic relationship is consistent throughout the orchid’s entire life cycle and whether the orchid relies exclusively on mycoheterotrophy for its nutrition during the fruiting season. Our study has demonstrated that E. zollingeri maintains a specialized symbiotic relationship with Psathyrellaceae fungi throughout all life stages. However, isotopic analysis and chlorophyll data have shown that the orchid also engages in photosynthesis to meet its carbon needs, particularly during the fruiting stage. This research constitutes the first discovery of partial mycoheterotrophy in leafless orchids associated with saprotrophic non-rhizoctonia fungi.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The sequence data have been deposited in the Sequence Read Archive of the DNA Data Bank of Japan (accession no. DRA017590). Additional supporting information is available online in the Supporting Information section at the end of the article.
References
Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389
Arditti J, Ghani AKA (2000) Numerical and physical properties of orchid seeds and their biological implications. New Phytol 145:367–421
Bellino A, Alfani A, Selosse MA et al (2014) Nutritional regulation in mixotrophic plants: new insights from Limodorum abortivum. Oecologia 175:875–885. https://doi.org/10.1007/s00442-014-2940-8
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 Lond B 271:1799–1806. https://doi.org/10.1098/rspb.2004.2807
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
Dearnaley JDW, Martos F, Selosse MA (2013) Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects. In: Hock B (ed) Fungal associations. Springer, Berlin, Germany, pp 207–230
DeNiro MJ, Epstein S (1976) You are what you eat (plus a few ‰): the carbon isotope cycle in food chains. Geological Society of America Abstracts with Programs 8:834–835
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
Gebauer G, Dietrich P (1993) Nitrogen isotope ratios in different compartments of a mixed stand of spruce, larch and beech trees and of understorey vegetation including fungi. Isot Environ Health Stud 29:35–44
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, Preiss K, Gebauer AC (2016) Partial mycoheterotrophy is more widespread among orchids than previously assumed. New Phytol 211:11–15. https://doi.org/10.1111/nph.13865
Gebauer G, Schulze ED (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 (2005) 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
Gleixner G, Danier HJ, Werner RA, Schmidt HL (1993) Correlations between the 13C content of primary and secondary plant products in different cell compartments and that in decomposing basidiomycetes. Plant Physiol 102:1287–1290
Gomes SIF, Giesemann P, Klink S et al (2023) Stable isotope natural abundances of fungal hyphae extracted from the roots of arbuscular mycorrhizal mycoheterotrophs and rhizoctonia-associated orchids. New Phytol 239:1166–1172. https://doi.org/10.1111/nph.18990
Hynson NA, Madsen TP, Selosse MA et al (2013) The physiological ecology of mycoheterotrophy. In: Merckx VSFT (ed) Mycoheterotrophy: The biology of plants living on fungi. Springer, Berlin, Germany, pp 297–342
Hynson NA, Schiebold JMI, Gebauer G (2016) Plant family identity distinguishes patterns of carbon and nitrogen stable isotope abundance and nitrogen concentration in mycoheterotrophic plants associated with ectomycorrhizal fungi. Ann Bot 118:467–479. https://doi.org/10.1093/aob/mcw119
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, 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
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
Kim Y-K, Jo S, Cheon S-H et al (2020) Plastome evolution and phylogeny of Orchidaceae, with 24 new sequences. Front Plant Sci 11:22. https://doi.org/10.3389/fpls.2020.00022
Kobayashi K, Suetsugu K, Wada H (2021) The leafless orchid Cymbidium macrorhizon performs photosynthesis in the pericarp during the fruiting season. Plant Cell Physiol 62:472–481. https://doi.org/10.1093/pcp/pcab006
Kumar S, Stecher G, Li M et al (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https://doi.org/10.1093/molbev/msy096
Leake JR (1994) The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol 127:171–216
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence - a practical guide. JExpBot 51:659–668
Mayor JR, Schuur EA, Henkel TW (2009) Elucidating the nutritional dynamics of fungi using stable isotopes. Ecol Lett 12:171–183
McCutchan JH Jr, Lewis WM Jr, Kendall C, McGrath CC (2003) Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur. Oikos 102:378–390. https://doi.org/10.1034/j.1600-0706.2003.12098.x
Merckx V, Freudenstein JV (2010) Evolution of mycoheterotrophy in plants: a phylogenetic perspective. New Phytol 185:605–609. https://doi.org/10.1111/j.1469-8137.2009.03155.x
Merckx VS, Freudenstein JV, Kissling J et al (2013) Taxonomy and classification. In: Merckx VSFT (ed) Mycoheterotrophy. Springer, Berlin, Germany, pp 19–101
Minagawa M, Wada E (1984) Stepwise enrichment of 15N along food chains: further evidence and the relation between δ15N and animal age. GeochimCosmochimActa 48:1135–1140. https://doi.org/10.1016/0016-7037(84)90204-7
Motomura H, Selosse M-A, Martos F et al (2010) Mycoheterotrophy evolved from mixotrophic ancestors: evidence in Cymbidium (Orchidaceae). Ann Bot 106:573–581. https://doi.org/10.1093/aob/mcq156
Nilsson RH, Larsson K-H, Taylor AFS et al (2019) The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res 47:D259–D264
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
Ogura-Tsujita Y, Yukawa T (2008) High mycorrhizal specificity in a widespread mycoheterotrophic plant, Eulophia zollingeri (Orchidaceae). AmJBot 95:93–97
Ogura-Tsujita Y, Yukawa T, Kinoshita A (2021) Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi. J Plant Res 134:19–41. https://doi.org/10.1007/s10265-020-01244-6
Op De Beeck M, Lievens B, Busschaert P et al (2014) Comparison and validation of some ITS primer pairs useful for fungal metabarcoding studies. PLoS ONE 9:e97629
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BBA - Bioenergetics 975:384–394
Rasmussen HN, Rasmussen FN (2014) Seedling mycorrhiza: a discussion of origin and evolution in Orchidaceae. BotJLinnSoc 175:313–327
Ritchie GA (2006) Chlorophyll fluorescence: what is it and what do the numbers mean? USDA Forest Service Proceedings RMRS 43:34–43
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
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
Schiebold JMI, Bidartondo MI, Karasch P et al (2017) You are what you get from your fungi: nitrogen stable isotope patterns in Epipactis species. AnnBot 119:1085–1095. https://doi.org/10.1093/aob/mcw265
Schweiger JMI, Bidartondo MI, Gebauer G (2018) Stable isotope signatures of underground seedlings reveal the organic matter gained by adult orchids from mycorrhizal fungi. Funct Ecol 32:870–881
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
Selosse M-A, 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
Stöckel M, Meyer C, Gebauer G (2011) The degree of mycoheterotrophic carbon gain in green, variegated and vegetative albino individuals of Cephalanthera damasonium is related to leaf chlorophyll concentrations. New Phytol 189:790–796. https://doi.org/10.1111/j.1469-8137.2010.03510.x
Suetsugu K, Haraguchi TF, Okada H, Tayasu I (2021a) Stigmatodactylus sikokianus (Orchidaceae) mainly acquires carbon from decaying litter through association with a specific clade of Serendipitaceae. New Phytol 231:1670–1675. https://doi.org/10.1111/nph.17523
Suetsugu K, Haraguchi TF, Tanabe AS, Tayasu I (2021b) Specialized mycorrhizal association between a partially mycoheterotrophic orchid Oreorchis indica and a Tomentella taxon. Mycorrhiza 31:243–250
Suetsugu K, Haraguchi TF, Tayasu I (2022) Novel mycorrhizal cheating in a green orchid: Cremastra appendiculata depends on carbon from deadwood through fungal associations. New Phytol 235:333–343. https://doi.org/10.1111/nph.17313
Suetsugu K, Matsubayashi J (2021a) Evidence for mycorrhizal cheating in Apostasia nipponica, an early-diverging member of the Orchidaceae. New Phytol 229:2302–2310
Suetsugu K, Matsubayashi J (2021b) Subterranean morphology modulates the degree of mycoheterotrophy in a green orchid Calypso bulbosa exploiting wood-decaying fungi. Funct Ecol 35:2305–2315. https://doi.org/10.1111/1365-2435.13864
Suetsugu K, Matsubayashi J (2022) Foliar chlorophyll concentration modulates the degree of fungal exploitation in a rhizoctonia-associated orchid. J Exp Bot 73:4204–4213
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
Suetsugu K, Ohta T, Tayasu I (2018) Partial mycoheterotrophy in the leafless orchid Cymbidium macrorhizon. Am J Bot 105:1595–1600
Suetsugu K, Yamato M, Matsubayashi J, Tayasu I (2021c) Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile. Mycorrhiza 31:301–312. https://doi.org/10.1007/s00572-021-01032-7
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
Syed F, Grunenwald H, Caruccio N (2009) Optimized library preparation method for next-generation sequencing. NatMethod 6:1–2
Tanabe AS, Toju H (2013) Two new computational methods for universal DNA barcoding: a benchmark using barcode sequences of bacteria, archaea, animals, fungi, and land plants. PLoS ONE 8:e76910. https://doi.org/10.1371/journal.pone.0076910
Tayasu I, Hirasawa R, Ogawa NO et al (2011) New organic reference materials for carbon- and nitrogen-stable isotope ratio measurements provided by Center for Ecological Research, Kyoto University, and Institute of Biogeosciences, Japan Agency for Marine-Earth Science and Technology. Limnology 12:261–266. https://doi.org/10.1007/s10201-011-0345-5
Taylor DL, McCormick MK (2008) Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas. New Phytol 177:1020–1033. https://doi.org/10.1111/j.1469-8137.2007.02320.x
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
Tsukaya H (2018) How have leaves of mycoheterotrophic plants evolved–from the view point of a developmental biologist. New Phytol 217:1401–1406
Waud M, Busschaert P, Ruyters S et al (2014) Impact of primer choice on characterization of orchid mycorrhizal communities using 454 pyrosequencing. Mol Ecol Resour 14:679–699. https://doi.org/10.1111/1755-0998.12229
Yagame T, Lallemand F, Selosse M-A et al (2021) Mycobiont diversity and first evidence of mixotrophy associated with Psathyrellaceae fungi in the chlorophyllous orchid Cremastra variabilis. J Plant Res 134:1213–1224
Zahn FE, Lee Y-I, Gebauer G (2022) Fungal association and root morphology shift stepwise during ontogenesis of orchid Cremastra appendiculata towards autotrophic nutrition. AoB Plants 14:1–10
Zahn FE, Söll E, Chapin TK et al (2023) Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons. New Phytol 239:1449–1463. https://doi.org/10.1111/nph.18991
Zanden MJ, Rasmussen JB (2001) Variation in δ15N and δ13C trophic fractionation: implications for aquatic food web studies. LimnolOceanogr 46:2061–2066. https://doi.org/10.4319/lo.2001.46.8.2061
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
The authors thank Nobuyuki Inoue and Tadashi Minamitani for their invaluable support during the field studies. We also thank Hidehito Okada, Kazuma Takizawa, and Takako Shizuka for their technical assistance.
Funding
This study was financially supported by the PRESTO (JPMJPR21D6, KS) from the Japan Science and Technology Agency and the JSPS KAKENHI (16H02524, IT and 17H05016, KS) and a Joint Research Grant for the Environmental Isotope Study of Research Institute for Humanity and Nature.
Author information
Authors and Affiliations
Contributions
KS planned and designed the research, conducted field and laboratory experiments, carried out analyses, and wrote the initial draft. TO conducted isotopic experiments, performed analyses, and contributed to manuscript revisions. IT supervised the isotopic experiments conducted by TO and revised the manuscript. All authors have approved the final version.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Suetsugu, K., Ohta, T. & Tayasu, I. Partial mycoheterotrophy in the leafless orchid Eulophia zollingeri specialized on wood-decaying fungi. Mycorrhiza 34, 33–44 (2024). https://doi.org/10.1007/s00572-024-01136-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00572-024-01136-w