Skip to main content
Log in

Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile

  • Original Article
  • Published:
Mycorrhiza Aims and scope Submit manuscript

Abstract

Most green orchids form mycorrhizal associations with rhizoctonia fungi, a polyphyletic group including Serendipitaceae, Ceratobasidiaceae, and Tulasnellaceae. Although accumulating evidence indicated that partial mycoheterotrophy occurs in such so-called rhizoctonia-associated orchids, it remains unclear how much nutrition rhizoctonia-associated orchids obtain via mycoheterotrophic relationships. We investigated the physiological ecology of green and albino individuals of a rhizoctonia-associated orchid Cypripedium debile, by using molecular barcoding of the mycobionts and stable isotope (13C and 15 N) analysis. Molecular barcoding of the mycobionts indicated that the green and albino individuals harbored Tulasnella spp., which formed a clade with the previously reported C. debile mycobionts. In addition, stable isotope analysis showed that both phenotypes were significantly enriched in 13C but not in 15 N. Therefore, green and albino individuals were recognized as partial and full mycoheterotrophs, respectively. The green variants were estimated to obtain 42.5 ± 8.2% of their C from fungal sources, using the 13C enrichment factor of albino individuals as a mycoheterotrophic endpoint. The proportion of fungal-derived C in green C. debile was higher than that reported in other rhizoctonia-associated orchids. The high fungal dependence may facilitate the emergence of albino mutants. Our study provides the first evidence of partial mycoheterotrophy in the subfamily Cypripedioideae. Partial mycoheterotrophy may be more general than previously recognized in the family Orchidaceae.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abadie JC, Püttsepp Ü, Gebauer G, Faccio A, Bonfante P, Selosse MA (2006) Cephalanthera longifolia (Neottieae, Orchidaceae) is mixotrophic: A comparative study between green and nonphotosynthetic individuals. Can J Bot 84:1462–1477

    Article  CAS  Google Scholar 

  • Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bellino A, Alfani A, Selosse MA, Guerrieri R, Borghetti M, Baldantoni D (2014) Nutritional regulation in mixotrophic plants: new insights from Limodorum abortivum. Oecologia 175:875–885

    Article  PubMed  Google Scholar 

  • Bidartondo MI (2005) The evolutionary ecology of myco-heterotrophy. New Phytol 167:335–352

    Article  PubMed  Google Scholar 

  • Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004) Changing partners in the dark: isotopic and molecular evidence of ectomycorrhizal liaisons between forest orchids and trees. Proc Roy Soc B 271:1799–1806

    Article  CAS  Google Scholar 

  • Bidartondo MI, Bruns TD, Weiss M, Sergio C, Read DJ (2003) Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort. Proc Roy Soc B 270:835–842

    Article  Google Scholar 

  • Bougoure J, Ludwig M, Brundrett M, Grierson P (2009) Identity and specificity of the fungi forming mycorrhizas with the rare mycoheterotrophic orchid Rhizanthella gardneri. Mycol Res 113:1097–1106

    Article  CAS  PubMed  Google Scholar 

  • Cribb P (1997) The genus Cypripedium. Timber Press, Oregon

    Google Scholar 

  • Dearnaley JDW, Martos F, Selosse MA (2012) Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects. In: Hock B (ed) The Mycota IX: fungal associations, 2nd edn. Springer, Berlin, pp 207–230

    Chapter  Google Scholar 

  • Fochi V, Chitarra W, Kohler A, Voyron S, Singan VR, Lindquist EA, Barry KW, Girlanda M, Grigoriev IV, Martin F, Balestrini R, Perotto S (2017) Fungal and plant gene expression in the Tulasnella calosporaSerapias vomeracea symbiosis provides clues about nitrogen pathways in orchid mycorrhizas. New Phytol 213:365–379

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Gebauer G, Preiss K, Gebauer AC (2016) Partial mycoheterotrophy is more widespread among orchids than previously assumed. New Phytol 211:11–15

    Article  PubMed  Google Scholar 

  • Gonneau C, Jersáková J, de Tredern E, Till-Bottraud I, Saarinen K, Sauve M, Roy M, Hájek T, Selosse MA (2014) Photosynthesis in perennial mixotrophic Epipactis spp. (Orchidaceae) contributes more to shoot and fruit biomass than to hypogeous survival. J Ecol 102:1183–1194

    Article  CAS  Google Scholar 

  • Halbwachs H, Dentinger BT, Detheridge AP, Karasch P, Griffith GW (2013) Hyphae of waxcap fungi colonise plant roots. Fungal Ecol 6:487–492

    Article  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hynson NA, Preiss K, Gebauer G (2009) Is it better to give than to receive? A stable isotope perspective on orchid-fungal carbon transport in green orchid species Goodyera repens and Goodyera oblongifolia. New Phytol 182:8–11

    Article  CAS  PubMed  Google Scholar 

  • Hynson NA, Madsen TP, Selosse MA, Adam IKU, Ogura-Tsujita Y, Roy M, Gebauer G (2013) The physiological ecology of mycoheterotrophy. In: Merckx V (ed) Mycoheterotrophy: the biology of plants living on fungi. Springer, New York, pp 297–342

    Chapter  Google Scholar 

  • Jacquemyn H, Merckx VS (2019) Mycorrhizal symbioses and the evolution of trophic modes in plants. J Ecol 107:1567–1581

    Article  Google Scholar 

  • Jacquemyn H, Waud M, Brys R, Lallemand F, Courty PE, Robionek A, Selosse MA (2017) Mycorrhizal associations and trophic modes in coexisting orchids: an ecological continuum between auto-and mixotrophy. Front Plant Sci 8:1497

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Julou T, Burghardt B, Gebauer G, Berveiller D, Damesin C, Selosse MA (2005) Mixotrophy in orchids: insights from a comparative study of green individuals and nonphotosynthetic individuals of Cephalanthera damasonium. New Phytol 166:639–653

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lallemand F, Robionek A, Courty P, Selosse M (2018) The 13C content of the orchid Epipactis palustris (L.) Crantz responds to light as in autotrophic plants. Bot Lett 165:1–9

    Google Scholar 

  • Leake JR (1994) The biology of myco-heterotrophic ('saprophytic’) plants. New Phytol 127:171–216

    Article  PubMed  Google Scholar 

  • Lee YI, Yang CK, Gebauer G (2015) The importance of associations with saprotrophic non-Rhizoctonia fungi among fully mycoheterotrophic orchids is currently under-estimated: Novel evidence from sub-tropical Asia. Ann Bot 116:423–435

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liebel HT, Bidartondo MI, Gebauer G (2015) Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance? Ann Bot 115:251–261

    Article  CAS  PubMed  Google Scholar 

  • Liebel HT, Bidartondo MI, Preiss K, Segreto R, Stöckel M, Rodda M, Gebauer G (2010) C and N stable isotope signatures reveal constraints to nutritional modes in orchids from the Mediterranean and Macaronesia. Am J Bot 97:903–912

    Article  CAS  PubMed  Google Scholar 

  • Martos F, Dulormne M, Pailler T, Bonfante P, Faccio A, Fournel J, Dubois MP, Selosse MA (2009) Independent recruitment of saprotrophic fungi as mycorrhizal partners by tropical achlorophyllous orchids. New Phytol 184:668–681

    Article  CAS  PubMed  Google Scholar 

  • Matsuda Y, Yamaguchi Y, Matsuo N, Uesugi T, Ito J, Yagame T, Figura T, Selosse MA, Hashimoto Y (2020) Communities of mycorrhizal fungi in different trophic types of Asiatic Pyrola japonica sensu lato (Ericaceae). J Plant Res 133:841–853

    Article  CAS  PubMed  Google Scholar 

  • Maxwell K, Johnson GN (2000) Chlorophyll fluorescence - A practical guide. J Exp Bot 51:659–668

    Article  CAS  PubMed  Google Scholar 

  • Mayor JR, Schuur EAG, Henkel TW (2009) Elucidating the nutritional dynamics of fungi using stable isotopes. Ecol Lett 12:171–183

    Article  PubMed  Google Scholar 

  • Merckx V (2013) Mycoheterotrophy: the biology of plants living on fungi. Springer, New York

    Book  Google Scholar 

  • Monje OA, Bugbee B (1992) Inherent limitations of nondestructive chlorophyll meters: a comparison of two types of meters. HortScience 27:69–71

    Article  CAS  PubMed  Google Scholar 

  • Nurfadilah S, Swarts ND, Dixon KW, Lambers H, Merritt DJ (2013) Variation in nutrient-acquisition patterns by mycorrhizal fungi of rare and common orchids explains diversification in a global biodiversity hotspot. Ann Bot 111:1233–1241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ogura-Tsujita Y, Gebauer G, Hashimoto T, Umata H, Yukawa T (2009) Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena. Proc Roy Soc B 276:761–767

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Preiss K, Gebauer G (2008) A methodological approach to improve estimates of nutrient gains by partially myco-heterotrophic plants. Isotopes Environ Health Stud 44:393–401

    Article  CAS  PubMed  Google Scholar 

  • Riess K, Oberwinkler F, Bauer R, Garnica S (2013) High genetic diversity at the regional scale and possible speciation in Sebacina epigaea and S. incrustans. BMC Evol Biol 13:102

  • 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

    Article  Google Scholar 

  • Selosse MA, Martos F (2014) Do chlorophyllous orchids heterotrophically use mycorrhizal fungal carbon? Trends Plant Sci 19:683–685

    Article  CAS  PubMed  Google Scholar 

  • Selosse MA, Roy M (2009) Green plants that feed on fungi: facts and questions about mixotrophy. Trends Plant Sci 14:64–70

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Selosse MA, Weiss M, Jany JL, Tillier A (2002) Communities and populations of sebacinoid basidiomycetes associated with the achlorophyllous orchid Neottia nidus-avis (L.) LCM Rich. and neighbouring tree ectomycorrhizae. Mol Ecol 11:1831–1844

    Article  CAS  PubMed  Google Scholar 

  • Shefferson RP, Taylor DL, Weiss M, Garnica S, McCormick MK, Adams S, Gray HM, McFarland JW, Kull T, Tali K, Yukawa T, Kawahara T, Miyoshi K, Lee YI (2007) The evolutionary history of mycorrhizal specificity among lady’s slipper orchids. Evolution 61:1380–1390

    Article  PubMed  Google Scholar 

  • Shefferson RP, Bunch W, Cowden CC, Lee Y, Kartzinel TR, Yukawa T, Downing J, Jiang H (2019) Does evolutionary history determine specificity in broad ecological interactions? J Ecol 107:1582–1593

    Article  Google Scholar 

  • Shutoh K, Tajima Y, Matsubayashi J, Tayasu I, Kato S, Shiga T, Suetsugu K (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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Stöckel M, Tešitelová T, Jersáková J, Bidartondo MI, Gebauer G (2014) Carbon and nitrogen gain during the growth of orchid seedlings in nature. New Phytol 202:606–615

    Article  PubMed  CAS  Google Scholar 

  • Suetsugu K, Matsubayashi J (2021) Evidence for mycorrhizal cheating in Apostasia nipponica, an early-diverging member of the Orchidaceae. New Phytol 229:2302–2310

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Suetsugu K, Ohta T, Tayasu I (2018) Partial mycoheterotrophy in the leafless orchid Cymbidium macrorhizon. Am J Bot 105:1595–1600

    Article  CAS  PubMed  Google Scholar 

  • Suetsugu K, Haraguchi TF, Tanabe AS, Tayasu I (2021) Specialized mycorrhizal association between a partially mycoheterotrophic orchid Oreorchis indica and a Tomentella taxon. Mycorrhiza 31:243–250

    Article  CAS  PubMed  Google Scholar 

  • Suetsugu K, Yamato M, Matsubayashi J, Tayasu I (2019) Comparative study of nutritional mode and mycorrhizal fungi in green and albino individuals of Goodyera velutina, an orchid mainly utilizing saprotrophic rhizoctonia. Mol Ecol 28:4290–4299

    Article  PubMed  Google Scholar 

  • Suetsugu K, Yamato M, Miura C, Yamaguchi K, Takahashi K, Ida Y, Shigenobu S, Kaminaka H (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

    Article  CAS  PubMed  Google Scholar 

  • Tayasu I, Hirasawa R, Ogawa NO, Ohkouchi N, Yamada K (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

    Article  CAS  Google Scholar 

  • Taylor DL, McCormick MK (2008) Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorrhizas. New Phytol 177:1020–1033

    Article  CAS  PubMed  Google Scholar 

  • Tedersoo L, May TW, Smith ME (2010) Ectomycorrhizal lifestyle in fungi: Global diversity, distribution, and evolution of phylogenetic lineages. Mycorrhiza 20:217–263

    Article  PubMed  Google Scholar 

  • Tedersoo L, Bahram M, Ryberg M, Otsing E, Kõljalg U, Abarenkov K (2014) Global biogeography of the ectomycorrhizal/sebacina lineage (Fungi, Sebacinales) as revealed from comparative phylogenetic analyses. Mol Ecol 23:4168–4183

    Article  PubMed  Google Scholar 

  • Veldre V, Abarenkov K, Bahram M, Martos F, Selosse MA, Tamm H, Kõljalg U, Tedersoo L (2013) Evolution of nutritional modes of Ceratobasidiaceae (Cantharellales, Basidiomycota) as revealed from publicly available ITS sequences. Fungal Ecol 6:256–268

    Article  Google Scholar 

  • Weiß M, Waller F, Zuccaro A, Selosse MA (2016) Sebacinales–one thousand and one interactions with land plants. New Phytol 211:20–40

    Article  PubMed  Google Scholar 

  • Yagame T, Yamato M, Suzuki A, Iwase K (2008) Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid Chamaegastrodia sikokiana. Mycorrhiza 18:97–101

    Article  PubMed  Google Scholar 

  • Yagame T, Ogura-Tsujita Y, Kinoshita A, Iwase K, Yukawa T (2016) Fungal partner shifts during the evolution of mycoheterotrophy in Neottia. Am J Bot 103:1630–1641

    Article  PubMed  Google Scholar 

  • Yagame T, Orihara T, Selosse MA, Yamato M, Iwase K (2012) Mixotrophy of Platanthera minor, an orchid associated with ectomycorrhiza-forming Ceratobasidiaceae fungi. New Phytol 193:178–187

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Marc-Andre Selosse and anonymous reviewers for their constructive comments on the manuscript. We thank Mr. Masayuki Sato for the help with field study. We also thank Dr. Chikage Yoshimizu for the assistance with the stable isotope abundance measurements.

Funding

This work was financially supported by the JSPS KAKENHI (Grant Numbers 17H05016 (KS) and 16H02524 (IT)).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kenji Suetsugu.

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.

Supplementary file1 (XLSX 13 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Suetsugu, K., Yamato, M., Matsubayashi, J. et al. Partial and full mycoheterotrophy in green and albino phenotypes of the slipper orchid Cypripedium debile. Mycorrhiza 31, 301–312 (2021). https://doi.org/10.1007/s00572-021-01032-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00572-021-01032-7

Keywords

Navigation