Generalist core orchid mycorrhizal fungi structure symbiotic associations across multiple orchid species in the Ryukyu archipelago, an island biodiversity hotspot.

Plant-fungal symbioses influence plant community structure and dynamics, and orchids provide a model system for examining these interactions due to their strict dependence on orchid mycorrhizal fungi (OMF) throughout the life cycle. However, the organization of OMF communities across multiple orchid hosts in island ecosystems, and the identity of fungal lineages functioning as core symbionts, remain insufficiently resolved. We characterised OMF associated with 28 orchid species across the Ryukyu Archipelago, Japan using a two-step approach: preliminary Sanger sequencing followed by high-throughput sequencing (HTS) with multiple primer sets to improve taxonomic and phylogenetic coverage. HTS detected 57 OMF operational taxonomic units (OTUs), dominated by Tulasnellaceae, and recovered all Sanger-detected OTUs while capturing additional diversity. Six OTUs exhibited exceptionally broad host ranges, each occurring in more than ten orchid species, forming an ecologically influential generalist group that provides the basis for the core OMF concept proposed in this study. All orchids showed broad and flexible fungal associations with low specificity. Population-level patterns revealed both conserved and site-specific partners, reflecting the coexistence of apparent and true generalism among Ryukyu orchids. Primer choice significantly influenced the recovery of Tulasnellaceae clades, demonstrating that single universal primers underestimate lineage-level diversity. Together, these results indicate that orchid communities in the Ryukyus are structured around a core set of ecologically versatile generalist fungi that function as key symbionts across diverse hosts and habitats. These lineages represent promising candidates for multispecies symbiotic propagation and conservation initiatives, although their functional roles require validation through symbiotic culture assays.