Diverse mycorrhizal associations and nutrition in Didymoplexis orchids

Fully mycoheterotrophic (FMH) orchids rely entirely on mycorrhizal fungi for carbon and nutrients, with tropical Asian FMH orchids typically associating with saprotrophic fungi, though some known relationships also with ectomycorrhizal fungi, leaving much to learn about their fungal partners. Didymoplexis belongs to tribe Gastrodieae, which represents one of the largest fully mycoheterotrophic orchid lineages. Although mycorrhizal associations of its sister genus Gastrodia have been relatively well-studied, those of Didymoplexis remain largely unexplored. Here, we used molecular barcoding to analyze fungal associations and stable isotope analysis to elucidate the nutritional strategies of Didymoplexis micradenia, Didymoplexis pallens, and Didymoplexis siamensis in subtropical and tropical forests across Taiwan. In Didymoplexis pallens and Didymoplexis micradenia, most fungal partners were litter-decaying fungi (Mycena, Clitocybula, Marasmius, Gymnopus) with smaller contributions from ectomycorrhizal and rhizoctonia fungi. In Didymoplexis siamensis, ectomycorrhizal fungi dominated, particularly Sebacinales, however, with additional associations with wood-decaying Delicatula. The pattern of carbon and nitrogen isotope enrichments found for the three Didymoplexis species was in the typical range known for fully mycoheterotrophic orchids associated with litter- or wood-decaying fungi. ¹⁵N enrichments of all investigated Didymoplexis species distinguished from fully mycoheterotrophic orchids associated with ectomycorrhizal fungi. Despite its ectomycorrhizal association, Didymoplexis siamensis was weakly enriched in ¹⁵N and more enriched in ¹³C than found for exclusively ectomycorrhizal fully mycoheterotrophic orchids. Thus, Didymoplexis siamensis covered its carbon and nitrogen demand obviously through the additional association with wood-decaying Delicatula. These findings enhance our understanding of the diverse fungal associations and physiological ecology of Didymoplexis species in subtropical and tropical ecosystems.