Epipactis helleborine shows strong mycorrhizal preference towards ectomycorrhizal fungi with contrasting geographic distributions in Japan

Epipactis helleborine (L.) Crantz, one of the most widespread orchid species, occurs in a broad range of habitats. This orchid is fully myco-heterotrophic in the germination stage and partially myco-heterotrophic in the adult stage, suggesting that a mycorrhizal partner is one of the key factors that determines whether E. helleborine successfully colonizes a specific environment. We focused on the coastal habitat of Japanese E. helleborine and surveyed the mycorrhizal fungi from geographically different coastal populations that grow in Japanese black pine (Pinus thunbergii Parl.) forests of coastal sand dunes. Mycorrhizal fungi and plant haplotypes were then compared with those from inland populations. Molecular phylogenetic analysis of large subunit rRNA sequences of fungi from its roots revealed that E. helleborine is mainly associated with several ectomycorrhizal taxa of the Pezizales, such as Wilcoxina, Tuber, and Hydnotrya. All individuals from coastal dunes were exclusively associated with a pezizalean fungus, Wilcoxina, which is ectomycorrhizal with pine trees growing on coastal dunes. Wilcoxina was not detected in inland forests. Coastal populations were indistinguishable from inland populations based on plant trnL intron haplotypes. Our results indicate that mycorrhizal association with geographically restricted pezizalean ectomycorrhizal fungi is a key control upon this orchid species' distribution across widely different forest habitats.