Stable isotope signatures confirm carbon and nitrogen gain through ectomycorrhizas in the ghost orchid Epipogium aphyllum Swartz

Epipogium aphyllum is a rare Eurasian achlorophyllous forest orchid known to associate with fungi that form ectomycorrhizas, while closely related orchids of warm humid climates depend on wood- or litter-decomposer fungi. We conducted 13C and 15N stable isotope natural abundance analyses to identify the organic nutrient source of E. aphyllum from Central Norway. These data for orchid shoot tissues, in comparison to accompanying autotrophic plants, document C and N flow from ectomycorrhizal fungi to the orchid. DNA data from fungal pelotons in the orchid root cortex confirm the presence of Inocybe and Hebeloma, which are both fungi that form ectomycorrhizas. The enrichment factors for 13C and 15N of E. aphyllum are used to calculate a new overall average enrichment factor for mycoheterotrophic plants living in association with ectomycorrhizal fungi (epsilon 13C +/- 1 SD of 7.2 +/- 1.6 parts per thousand and epsilon 15N +/- 1 SD of 12.8 +/- 3.9 parts per thousand). These can be used to estimate the fungal contribution to organic nutrient uptake by partially mycoheterotrophic plants where fully mycoheterotrophic plants are lacking. N concentrations in orchid tissue were unusually high and significantly higher than in accompanying autotrophic leaf samples. This may be caused by N gain of E. aphyllum from obligate ectomycorrhizal fungi. We show that E. aphyllum is an epiparasitic mycoheterotrophic orchid that depends on ectomycorrhizal Inocybe and Hebeloma to obtain C and N through a tripartite system linking mycoheterotrophic plants through fungi with forest trees.