Isotopic and molecular evidence for contrasting fungal associations in two Epipogium orchids in cool-temperate Japan.

Abstract

Fully mycoheterotrophic orchids depend on fungus-derived carbon. Associations with wood- and litter-decaying saprotrophic (SAP) fungi have been reported mainly in fully mycoheterotrophic orchids from warm, humid forests, whereas ectomycorrhizal (ECM) fungi commonly support fully mycoheterotrophic orchids in boreal and temperate forests. For example, the subtropical and tropical Epipogium roseum associates with wood-decaying Psathyrellaceae fungi, whereas the cool-temperate E. aphyllum associates with ECM fungi, including Inocybe. Field observations suggest possible SAP associations in E. japonicum in cool-temperate to subalpine forests in Japan, based on its frequent occurrence near decaying logs and coarse woody debris. Here, we characterized the fungal associates and carbon sources of E. japonicum from moist ravines and compared them with those of nearby E. aphyllum from ECM-dominated coniferous forests. We combined high-throughput ITS2 metabarcoding with δ¹³C and δ¹⁵N measurements from plants collected on Mount Kitadake. Sequencing showed that mycorrhizal rhizome tissues of E. japonicum were dominated by Psathyrellaceae, particularly Coprinopsis, whereas those of E. aphyllum were dominated by Inocybe. Epipogium japonicum showed stronger ¹³C enrichment relative to co-occurring autotrophic plants than did E. aphyllum, consistent with carbon acquisition through deadwood-associated SAP fungi. These results indicate that two fully mycoheterotrophic congeners exploit contrasting fungal guilds within the same cool-temperate mountain landscape.