Endophytic fungal community structure and diversity in paphiopedilum barbigerum across different habitats

Objective This study aimed to investigate the response patterns of root-associated fungal communities in the rare and endangered species Paphiopedilum barbigerum across different habitat types, and to identify key taxonomic groups that differentiate natural habitats from reintroduction sites, thereby providing a theoretical basis for the species artificial propagation and field reintroduction.Methods Using PacBio high-throughput sequencing, we sequenced the ITS region of 21 root samples of P. barbigerum collected from seven sites representing three habitat categories: natural habitats within a nature reserve (SL, DE, YL, KL), natural habitats outside the reserve (FQ, KY), and a field reintroduction site (HG). Alpha diversity, beta diversity, LEfSe analysis, and co-occurrence network analysis were employed to characterize root-associated fungal community structure, diversity, and habitat-driven assembly patterns.Results A total of 792,178 clean reads were obtained and classified into 8 phyla, 37 classes, 134 orders, 352 families, 1,012 genera, and 2,311 species. The communities were dominated by Ascomycota (52.90%–91.41%) and Basidiomycota (7.79%–54.33%). Key findings include: (1) Fungal species richness was significantly higher in natural habitats within the reserve than at the reintroduction site and natural habitats outside the reserve, with specific enrichment of desiccation-tolerant taxa such as Chaetothyriales and Exophiala (relative abundance 12.58%–33.53%). (2) The fungal community structure at the reintroduction site converged with that of natural habitats outside the reserve. Core OTUs included Epulorhiza sp., Cladosporium sp., and Lachancea thermotolerans, all of which exhibited significantly higher relative abundances in these two habitat types; notably, Epulorhiza sp. reached a relative abundance of 44.79% at the reintroduction site, suggesting a key role in host growth and development.Conclusion Habitat heterogeneity is associated with adaptive restructuring of the root-associated fungal community by selecting for distinct functional guilds: desiccation-tolerant taxa are enriched in natural habitats within the reserve, whereas growth-promoting taxa are enriched in disturbed habitats. We recommend prioritizing the conservation of natural habitats within the reserve to preserve high fungal diversity and stress-tolerant guilds, and emphasize the targeted utilization and monitoring of core growth-promoting fungi such as Epulorhiza during reintroduction programs.