Partial mycoheterotrophy in Apostasia wallichii, an early-diverging Asian tropical orchid

All orchids exhibit mycoheterotrophy during their early development stages, which predisposes certain species to retain this nutritional mode into adulthood. Consequently, many orchids adopt partial mycoheterotrophy, a mixotrophic strategy combining carbon acquisition through both autotrophy and mycoheterotrophy. However, whether this strategy represents an ancestral trait remains contested. This study examines the fungal symbionts and nutritional strategies of the early-diverging orchid Apostasia wallichii and a sympatric, photosynthetic orchid, Cystorchis variegata, in tropical Asia (Sabah, Malaysian Borneo). Specifically, we explored their potential nutritional modes and mycobionts by analysing δ13C and δ15N isotopic profiles and employing high-throughput DNA sequencing. Community profiling via metabarcoding revealed that the A. wallichii individuals investigated were predominantly associated with putatively saprotrophic Botryobasidium fungi, while C. variegata was simultaneously associated with non-ectomycorrhizal rhizoctonias, saprotrophic non-rhizoctonia fungi, and ectomycorrhizal fungi. Additionally, stable isotope analysis showed that both A. wallichii and C. variegata were significantly enriched in 13C and 15N compared to co-occurring autotrophic plants, indicating partial mycoheterotrophy. Our findings, particularly the discovery of partial mycoheterotrophy associated with non-ectomycorrhizal fungi in A. wallichii, suggest that partial mycoheterotrophy in green orchids may be more widespread than previously believed and could represent an ancestral trait intrinsic to orchids.