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Background and Aims The earliest-diverging orchid lineage, Apostasioideae, consists only of two genera: Apostasia and Neuwiedia. Previous reports 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 abundances (δ 13C, δ 15N, δ 2H, δ 18O) of different developmental stages with those of autotrophic reference plants. Key Results Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and the 13C signature was characteristic of their fully mycoheterotrophic nutrition. Seedlings and adults were 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 various habitat options in the earliest-diverging orchid lineage, Apostasioideae. While A. nipponica relies on the heterotrophic carbon gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic carbon gain as an adult, allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur. [ABSTRACT FROM AUTHOR]

Although most orchid species produce minute, light seeds with a one-layer thin seed coat, some in the subfamily Vanilloideae show seed coat with sclerotization and wing development. We investigated the processes of their development by analyzing the anatomy of ovules of two species in the Vanilloideae, Cyrtosia septentrionalis (Rchb. f.) Garay and Erythrorchis altissima (Blume) Blume, that produce seeds with, respectively, rudimentary and well-developed wings. In both species, lignified dark material accumulated in the cells of the outermost layer of the outer integument, forming a sclerotic seed coat. Accumulation started from the cell walls of the outer periclinal surface. C. septentrionalis formed a large embryo and a rudimentary wing and E. altissima formed a small embryo and a well-developed wing. Fully developed seeds of C. septentrionalis and E. altissima were dispersed by animal and wing, respectively. In C. septentrionalis, vascular-bundle-like cells extended from the tip of the funiculus to the chalaza. In E. altissima, they branched into two parts: one reached the chalaza and the other reached a group of cells adjacent to the embryo sac in the micropylar region. The group of cells structurally and functionally resembled hypostase, although hypostase is reported to be present in the chalazal region. A helical vessel was found in one ovule in E. altissima. These observations indicate that the specialized cells represent degenerated vascular bundle cells. [ABSTRACT FROM AUTHOR]