Divergent mycorrhizal strategies and nutritional modes in the East Asian orchid genus Ephippianthus

Orchids provide an ideal system for examining the evolution of heterotrophy because all species depend on fungal carbon during germination, whereas adult nutritional modes range from autotrophy to full mycoheterotrophy. The subtribe Calypsoinae illustrates this diversity through repeated shifts in fungal partners and nutritional strategies. Recent phylogenomic analyses place the Dactylostalix-Ephippianthus clade as sister to the Corallorhiza clade, which includes several heterotrophic lineages, raising questions about adult nutrition in Ephippianthus. Here, we combined fungal metabarcoding with δ¹³C and δ¹⁵N analyses to investigate the physiological ecology of the two known Ephippianthus species, which are restricted to Japan and the adjacent Russian Far East. We found that Ephippianthus schmidtii was primarily associated with Tulasnellaceae (Cantharellales). Its δ¹³C and δ¹⁵N values showed no significant positive enrichment relative to co-occurring autotrophic plants, providing no evidence for substantial fungal nutrient gain based on these isotope markers. By contrast, Ephippianthus sawadanus was mainly associated with saprotrophic Cyphellaceae (Agaricales), representing, to our knowledge, the first documented case in which a member of this family was detected as the dominant mycorrhizal associate of a green orchid. Moreover, E. sawadanus exhibited elevated δ¹³C and δ¹⁵N values relative to autotrophic plants, consistent with substantial fungal contributions to its carbon and nitrogen budgets. Together, these findings reveal divergent mycorrhizal strategies and nutritional modes in the East Asian orchid genus Ephippianthus.