Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionary early diverging orchid subfamily Apostasioideae

Abstract Background and Aims The earliest diverging orchid lineage Apostasioideae consists only of two genera: Apostasia and Neuwiedia. Previous report of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. Methods We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundance (δ13C, δ15N, δ2H, δ18O) of different developmental stages to autotrophic reference plants. Key Results Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition. Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. Conclusions The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows for various habitat options in the earliest diverging orchid lineage Apostasioideae. While A. nipponica relies on the heterotrophic C gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic C gain as adult allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.