In situ seed baiting and DNA metabarcoding support developmental continuity of a Coprinellus association in the predominantly leafless orchid Cremastra aphylla.

Although orchids depend on mycorrhizal fungi for germination, their early-stage fungal partners may be replaced during subsequent development. This possibility is particularly relevant in Calypsoinae, in which many green species shift from juvenile associations with wood-decaying fungi to adult associations with rhizoctonia fungi. To determine whether a comparable shift occurs in the predominantly leafless orchid Cremastra aphylla, we combined in situ seed packet burial with ITS2 metabarcoding in a single population. We investigated fungal associations among small protocorms, large protocorms, and adult coralloid rhizomes, characterized germination microsites, and compared these fungal associations with published fungal community data for the leafy sister species C. appendiculata. All developmental categories of C. aphylla were overwhelmingly dominated by the same operational taxonomic unit assigned to Coprinellus (Psathyrellaceae). These results are consistent with retention of a Coprinellus association across the categories examined, contrasting with the pronounced ontogenetic shift from Psathyrellaceae to Tulasnellaceae and Ceratobasidiaceae reported in C. appendiculata. Germination occurred in five seed packets at three burial points, all of which were located near adult plants and contained deadwood, whereas most packets buried under apparently similar conditions showed no germination. These findings indicate that suitable germination microsites are highly localized and that spatially restricted recruitment may contribute to the rarity and patchy distribution of C. aphylla.