Impact of plant sex on endophytic microbial diversity and community assembly in dioecious species.

Dioecious plants show marked sexual dimorphism in morphology, physiology, defense, and life-history allocation, yet differences in endophytic microbiomes remain understudied. We compared endophytic bacterial and fungal communities between male and female individuals of four dioecious species-Eucommia ulmoides, Taxus wallichiana var. mairei, Ginkgo biloba, and Morus alba-by high-throughput sequencing of bark (E. ulmoides and T. wallichiana var. mairei) and leaves (G. biloba and M. alba). Sex significantly influenced the assembly of bacterial and fungal communities in all species. Bacterial alpha diversity (Shannon index) was significantly higher in males than females for E. ulmoides, T. wallichiana var. mairei, and M. alba. The bacterial phylum Spirochaetota was only detected in male samples of E. ulmoides, G. biloba, and M. alba, while the fungal genera Albophoma and Striaticonidium were detected only in female samples across all four species. Acidobacteria were significantly enriched in males of E. ulmoides and T. wallichiana var. mairei, whereas the fungal genus Sporidesmium was more abundant in females of T. wallichiana var. mairei and M. alba. Despite these sex-driven differences, dominant endophytic composition was similar between sexes: Actinobacteriota and Proteobacteria were the most prevalent bacterial phyla, whereas Ascomycota and Basidiomycota were the most prevalent fungal phyla across all samples. Co-occurrence analysis indicated stronger links between Actinobacteriota or Proteobacteria and other phyla than among remaining phyla. These results enhance understanding of sex-associated endophytic communities in dioecious plants and may inform studies on plant sex differentiation and potential plant-microbiome associations.IMPORTANCEPlant sex influences physiology, defense, and resource allocation, but its effects on internal microbial communities remain poorly understood. We compared endophytic bacteria and fungi in male and female Eucommia ulmoides, Taxus wallichiana var. mairei, Ginkgo biloba, and Morus alba using high-throughput sequencing and found consistent sex-associated differences in microbial diversity and specific taxa (e.g., Spirochaetota enriched in males; Albophoma and Striaticonidium enriched in females). These results indicate that sex is a reproducible driver of endophyte community assembly across diverse hosts. Recognizing sex-specific microbiomes has practical implications for conservation, forest and crop health management, and targeted bioprospecting, and underscores the need to include host sex as a key factor in ecological, functional, and evolutionary studies of plant-microbe interactions.