Integrating stable isotope and fungal association evidence for nutrient acquisition in the rare orchid, Risleya atropurpurea.

Risleya atropurpurea, a fully mycoheterotrophic species in the subtribe Calypsoinae, is a rare Himalayan orchid whose fungal associations and nutritional strategy remain poorly understood. In this study, we combined high-throughput ITS amplicon sequencing and stable isotope natural abundance analyses to investigate the mycorrhizal associations and nutrient acquisition strategy of R. atropurpurea in a natural habitat in southwestern China. Fungal community profiling revealed the dominance of a single operational taxonomic unit affiliated with Basidiodendron caesiocinereum (Auriculariales). Stable isotope analyses showed that R. atropurpurea was significantly enriched in both ¹³C and ¹⁵N relative to neighboring autotrophic reference plants, consistent with isotope patterns characteristic of fully mycoheterotrophic orchids associated with saprotrophic wood-decaying fungi. Together, these findings indicate that Basidiodendron is likely to contribute to carbon and nitrogen acquisition in R. atropurpurea and suggest that Auriculariales-associated saprotrophic fungi may represent an under-recognized nutritional partner in fully mycoheterotrophic orchids. Our study expands the known diversity of fungal partners involved in orchid mycoheterotrophy and provides new insights into the ecological evolution of fungal-dependent nutrition in orchids.