REGULAR PAPER The rare terrestrial orchid Nervilia nipponica consistently associates with a single group of novel mycobionts Naofumi Nomura • Yuki Ogura-Tsujita • Stephan W. Gale • Ayako Maeda • Hidetaka Umata • Kentaro Hosaka • Tomohisa Yukawa Received: 12 April 2012 / Accepted: 15 January 2013 / Published online: 6 April 2013 Ó The Botanical Society of Japan and Springer Japan 2013 Abstract Nervilia nipponica is a tuberous terrestrial orchid that has a highly restricted distribution within common secondary evergreen forest communities in cen- tral and western Japan. Such a limited occurrence could be attributable to a requirement for a specific mycorrhizal fungus. As part of a broader examination of this hypothe- sis, we sought to elucidate the mycorrhizal associations of N. nipponica. Seventy-five samples of mycorrhizae from forty individuals were collected at ten populations throughout the orchid’s range in Japan. The identity of mycorrhizal fungi was investigated by sequencing two genetic markers (nrDNA ITS and nrDNA 28S LSU) and their relationships were assessed via phylogenetic analyses. The most frequently encountered mycorrhizal fungi con- sisted of four closely related Agaricomycetes that infected an average of 78.7 % of individuals per population. All four formed a discrete, monophyletic clade with low sequence homology to other fungi registered in GenBank, indicating that they belong to a novel, unnamed family. Two additional fungal groups, belonging to Ceratobasidi- aceae and ‘‘Group B’’Sebacinales, were found in 22.0 and 21.5 % of individuals per population, respectively. The orchid probably uses these two groups opportunistically, because they were found at lower densities and always in combination with the unidentified Agaricomycete. These findings suggest that a group of novel Agaricomycete fungi constitutes the dominant mycobiont of N. nipponica. Keywords Agaricomycetes Á Mycorrhizae Á Rarity Á Symbiosis Á Terrestrial orchid Introduction With an estimated 26,000 species (Govaerts and Carr 2011), the Orchidaceae is one of the largest families of flowering plants. Its members are found in many distinct habitats and they encompass great diversity in life history and growth form. Many species exhibit a high degree of specialisation to biotic and abiotic habitat factors, and this is thought to both constrain the ecological niche they may successfully occupy and place them at risk of extirpation through extraneous and anthropogenic threats (Cribb et al. 2003). For example, of the ca. 300 orchid species native to Japan, 200 are listed as threatened or endangered (Ministry of the Environment 2007). Rarity among orchid species has been linked to two intrinsic (natural) factors that are crucial to the completion of their life cycle. The first is a mycorrhizal association with a fungal endophyte for seed germination and N. Nomura and Y. Ogura-Tsujita contributed equally. N. Nomura (&) Á H. Umata Á T. Yukawa Tsukuba Botanical Garden, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba 305-0005, Japan e-mail: nomura.naofumi@gmail.com Y. Ogura-Tsujita Botanical Gardens, Tohoku University, Kawauchi 12-1, Aobaku, Sendai 980-0862, Japan S. W. Gale Kadoorie Farm and Botanic Garden, Lam Kam Road, Tai Po, New Territories, Hong Kong A. Maeda Kochi Prefectural Makino Botanical Garden, Godaisan 4200-6, Kochi 781-8125, Japan K. Hosaka Department of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba 305-0005, Japan 123 J Plant Res (2013) 126:613–623 DOI 10.1007/s10265-013-0552-8