, Volume 29, Issue 2, pp 159–166 | Cite as

A leafless epiphytic orchid, Taeniophyllum glandulosum Blume (Orchidaceae), is specifically associated with the Ceratobasidiaceae family of basidiomycetous fungi

  • Kento Rammitsu
  • Takahiro Yagame
  • Yumi Yamashita
  • Tomohisa Yukawa
  • Shiro Isshiki
  • Yuki Ogura-TsujitaEmail author
Original Article


Leafless epiphytes in the Orchidaceae undergo a morphological metamorphosis in which the root has chloroplast-containing cortical cells and is the sole photosynthetic organ for carbon gain. All orchids are entirely dependent on mycorrhizal fungi for their carbon supply during seed germination, and this mycorrhizal association generally persists in adult plants. However, our knowledge of the mycorrhizal association of leafless epiphytic orchids remains limited, and the contribution of the mycorrhizal association to nutrient acquisition in these orchid species is largely unknown. In this study, the mycorrhizal fungi of a leafless epiphytic orchid, Taeniophyllum glandulosum, were identified molecularly using 68 mature plants and 17 seedlings. In total, 187 fungal internal transcribed spacer sequences were obtained, of which 99% were identified as Ceratobasidiaceae. These sequences were classified into five operational taxonomic units (OTUs) based on 97% sequence similarity. The most frequent sequence was OTU1, which accounted for 91% of all Ceratobasidiaceae sequences, although other phylogenetically distinct Ceratobasidiaceae fungi were detected. These results show that T. glandulosum is specifically associated with a particular group of Ceratobasidiaceae. All mycorrhizal fungi found in T. glandulosum seedlings belonged to OTU1, which was also found in adult plants on the same host tree. The mycorrhizal fungi from 13 host tree species were compared, and T. glandulosum was preferentially associated with OTU1 on 11 tree species. In conclusion, T. glandulosum is specifically associated with Ceratobasidiaceae fungi and this specific association remains throughout the orchid life cycle and is found on divergent host tree species.


Ceratobasidiaceae Epiphytic orchid Leaflessness Orchid mycorrhizal fungi In situ seed bating 



The authors thank Y. Endo, M. Gotoh, K. Higaki, S. Mori, T. Shimizu, M. Takashima, A. Takizawa, and K. Tanaka for collecting samples and K. Ureshino for valuable advices. The DNA sequencing analysis were made using a Genetic Analyzer spectrometer at Analytical Research Center for Experimental Sciences, Saga University.


This work was supported by JSPS KAKENHI Grant Numbers 17K07536 and 18H02500.


  1. Batty AL, Dixon KW, Brundrett M, Sivasithamparam K (2001) Constraints to symbiotic germination of terrestrial orchid seed in a Mediterranean bushland. New Phytol 152:511–520CrossRefGoogle Scholar
  2. Burgeff H (1936) Samenkeimung der orchideen. Gustav Fischer. In: JenaGoogle Scholar
  3. Carlsward BS, Whitten WM, Williams NH, Bytebier B (2006) Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness. Am J Bot 93:770–786CrossRefGoogle Scholar
  4. Christenhusz MJM, Byng JW (2016) The number of known plants species in the world and its annual increase. Phytotaxa 261:201–217CrossRefGoogle Scholar
  5. Cruz-Higareda JB, Luna-Rosales BS, Barba-Álvarez A (2015) A novel seed baiting technique for the epiphytic orchid Rhynchostele cervantesii, a means to acquire mycorrhizal fungi from protocorms. Lankesteriana 15:67–76CrossRefGoogle Scholar
  6. Dearnaley JDW, Martos F, Selosse MA (2012) Orchid mycorrhizas: molecular ecology, physiology, evolution and conservation aspects. In: Hock B (ed) Fungal associations, 2nd edn. Springer, Berlin, pp 207–230CrossRefGoogle Scholar
  7. Felsenstein D, Carney WP, Iacoviello VR, Hirsch MS (1985) Phenotypic properties of atypical lymphocytes in cytomegalovirus-induced mononucleosis. J Infect Dis 152:198–203CrossRefGoogle Scholar
  8. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118CrossRefGoogle Scholar
  9. Gónzalez D, Rodriguez-Carres M, Boekhout T, Stalpers J, Kuramae EE, Nakatani AK, Vilgalys R, Cubeta MA (2016) Phylogenetic relationships of Rhizoctonia fungi within the Cantharellales. Fungal Biol 120:603–619CrossRefGoogle Scholar
  10. Graham RR, Dearnaley JDW (2012) The rare Australian epiphytic orchid Sarcochilus weinthalii associates with a single species of Ceratobasidium. Fungal Divers 54:31–37CrossRefGoogle Scholar
  11. Herrera H, Valadares R, Contreras D, Bashan Y, Arriagada C (2017) Mycorrhizal compatibility and symbiotic seed germination of orchids from the coastal range and Andes in south Central Chile. Mycorrhiza 27:175–188CrossRefGoogle Scholar
  12. Hoang NH, Kane ME, Radcliffe EN, Zettler LW, Richardson LW (2017) Comparative seed germination and seedling development of the ghost orchid, Dendrophylax lindenii (Orchidaceae), and molecular identification of its mycorrhizal fungus from South Florida. Ann Bot 119:379–393CrossRefGoogle Scholar
  13. Irawati I (2009) Self and cross inoculation of Papilionanthe hookeriana and Taeniophyllum obtusum orchid mycorrhiza. Bull Kebun Raya 12:11–18Google Scholar
  14. Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Gafur A, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53:396–401CrossRefGoogle Scholar
  15. Jacquemyn H, Merckx V, Brys R, Tyteca D, Cammue BPA, Honnay O, Lievens B (2011) Analysis of network architecture reveals phylogenetic constraints on mycorrhizal specificity in the genus Orchis (Orchidaceae). New Phytol 192:518–528CrossRefGoogle Scholar
  16. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  17. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  18. Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse MA (2012) The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids. Mol Ecol 21:5098–5109CrossRefGoogle Scholar
  19. Mújica EB, Mably JJ, Skarha SM, Corey LL, Richardson LW, Danaher M, González EH, Zettler LW (2018) A comparison of ghost orchid (Dendrophylax lindenii) habitats in Florida and Cuba, with particular reference to seedling recruitment and mycorrhizal fungi. Bot J Linn Soc 186:572–586CrossRefGoogle Scholar
  20. Mutsuura O, Ito I, Nakahira R (1962) Studies on the germination and the development of seedlings of Taeniophyllum aphyllum (Makino) Makino. Sci Rep Kyoto Pref Univ 3:189–194Google Scholar
  21. Nurfadilah S, Yulia ND, Ariyanti EE (2016) Morphology, anatomy, and mycorrhizal fungi colonization in roots of epiphytic orchids of Sempu Island, East Java, Indonesia. Biodiversitas 17:592–603CrossRefGoogle Scholar
  22. Ogura-Tsujita Y, Yukawa T (2008) High mycorrhizal specificity in a widespread mycoheterotrophic plant, Eulophia zollingeri (Orchidaceae). Am J Bot 95:93–97CrossRefGoogle Scholar
  23. 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 99:1158–1176CrossRefGoogle Scholar
  24. Otero JT, Ackerman JD, Bayman P (2002) Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids. Am J Bot 89:1852–1858CrossRefGoogle Scholar
  25. Otero JT, Flanagan NS, Herre EA, Ackerman JD, Bayman P (2007) Widespread mycorrhizal specificity correlates to mycorrhizal function in the neotropical, epiphytic orchid Ionopsis utricularioides (Orchidaceae). Am J Bot 94:1944–1950CrossRefGoogle Scholar
  26. Rasmussen HN (2002) Recent developments in the study of orchid mycorrhiza. Plant Soil 244:149–163CrossRefGoogle Scholar
  27. Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids. Am J Bot 80:1374–1378CrossRefGoogle Scholar
  28. Shefferson RP, Cowden CC, McCormick MK, Yukawa T, Ogura-Tsujita Y, Hashimoto T (2010) Evolution of host breadth in broad interactions: mycorrhizal specificity in east Asian and north American rattlesnake plantains (Goodyera spp.) and their fungal hosts. Mol Ecol 19:3008–3017CrossRefGoogle Scholar
  29. Sneh B, Jabaji-Hare S, Neate S, Dijst G (1996) Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control. Kluwer, DordrechtGoogle Scholar
  30. Suarez JP, Weiss M, Abele A, Garnica S, Oberwinkler F, Kottke I (2006) Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest. Mycol Res 110:1257–1270CrossRefGoogle Scholar
  31. Taylor DL, McCormick MK (2008) Internal transcribed spacer primers and sequences for improved characterization of basidiomycetous orchid mycorhizas. New Phytol 177:1020–1033CrossRefGoogle Scholar
  32. Tedersoo L, Bahram M, Jairus T, Bechem E, Chinoya S, Mpumba R, Leal M, Randrianjohany E, Razafimandimbison S, Sadam A, Naadel T, Kõljalg U (2011) Spatial structure and the effects of host and soil environments on communities of ectomycorrhizal fungi in wooded savannas and rain forests of continental Africa and Madagascar. Mol Ecol 20:3071–3080CrossRefGoogle Scholar
  33. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar
  34. Valadares RB, Pereira MC, Otero JT, Cardoso EJ (2012) Narrow fungal mycorrhizal diversity in a population of the orchid Coppensia doniana. Biotropica 44:114–122CrossRefGoogle Scholar
  35. Wang H, Fang H, Wang Y, Duan L, Guo S (2011) In situ seed baiting techniques in Dendrobium officinale Kimuraet Migo and Dendrobium nobile Lindl.: the endangered Chinese endemic Dendrobium (Orchidaceae). World J Microbiol Biotechnol 27:2051–2059CrossRefGoogle Scholar
  36. Warcup JH, Talbot PHB (1967) Perfect states of Rhizoctonias associated with orchids. New Phytol 66:631–641CrossRefGoogle Scholar
  37. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322Google Scholar
  38. Winter K, Medina E, Garcia V, Mayoral ML, Muniz R (1985) Crassulacean acid metabolism in roots of a leafless orchid, Campylocentrum tyrridion Garay & Dunsterv. J Plant Physiol 118:73–78CrossRefGoogle Scholar
  39. Xing X, Gai X, Liu Q, Hart MM, Guo S (2015) Mycorrhizal fungal diversity and community composition in a lithophytic and epiphytic orchid. Mycorrhiza 25:289–296CrossRefGoogle Scholar
  40. Yamato M, Yagame T, Suzuki A, Iwase K (2005) Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae). Mycoscience 46:73–77CrossRefGoogle Scholar
  41. Yukawa T (2015) The handbook of Japanese wild orchids, vol 1. Bunichi sogo shuppan, Tokyo (in Japanese)Google Scholar
  42. Yukawa T, Ogura-Tsujita Y, Shefferson RP, Yokoyama J (2009) Mycorrhizal diversity in Apostasia (Orchidaceae) indicates the origin and evolution of orchid mycorrhiza. Am J Bot 96:1997–2009CrossRefGoogle Scholar
  43. Zi XM, Sheng CL, Goodale UM, Shao SC, Gao JY (2014) In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid, Dendrobium aphyllum (Orchidaceae). Mycorrhiza 24:487–499CrossRefGoogle Scholar
  44. Zotz G (2013) The systematic distribution of vascular epiphytes – a critical update. Bot J Linn Soc 171:453–481CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Faculty of AgricultureSaga UniversitySagaJapan
  2. 2.United Graduate School of Agricultural SciencesKagoshima UniversityKagoshimaJapan
  3. 3.Mizuho Municipal MuseumTokyoJapan
  4. 4.National Museum of Nature and ScienceIbarakiJapan

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