Species‑specific responses of orchid mycorrhizal associations to geographic and edaphic gradients

Orchid mycorrhizal fungi (OMF) are essential for orchid germination and ecological resilience, yet the factors shaping OMF diversity and distribution across species and geographic regions remain poorly understood. We investigated OMF communities of four European orchid species with varying degrees of fungal specificity: Dactylorhiza sambucina, Anacamptis morio, Platanthera bifolia, and Orchis mascula, along a latitudinal gradient from Italy, across the Czech Republic, to Sweden.We integrated culture-dependent isolation and ITS metabarcoding to identify OMF community composition across geographical and environmental gradients. Coupled with detailed soil analyses, these approaches revealed 57 OMF OTUs. Orchids associated with Tulasnellaceae except P. bifolia, which associated mainly with Ceratobasidiaceae. Orchid species identity determined OMF community composition, whereas geographical and soil variables varied according to species. OMF richness and diversity did not vary according to latitude; however, some OTUs were shared across species and countries, with orchid fungal specificity decreasing with latitude. Soil calcium was a strong driver of the OMF community structure identified by metabarcoding. Overall, metabarcoding revealed orchid species-specific responses of OMF taxa to soil properties. The low regional variation in OMF diversity suggests a high degree of spatial consistency in orchid-OMF associations, despite pronounced differences in host specificity and species-specific responses to soil conditions.