Abstract
Species identification and phylogenetic relationship clarification are fundamental goals in species delimitation. However, these tasks pose challenges when based on morphologies, geographic distribution, and genomic data. Previously, two species of the fern genus Bolbitis, B. × multipinna and B. longiaurita were described based on morphological traits; they are phylogenetically intertwined with B. sinensis and fail to form monophyletic groups. To address the unclear phylogenetic relationships within the B. sinensis species complex, RAD sequencing was performed on 65 individuals from five populations. Our integrated analysis of phylogenetic trees, neighbor nets, and genetic structures indicate that the B. sinensis species complex should not be considered as separate species. Moreover, our findings reveal differences in the degree of genetic differentiation among the five populations, ranging from low to moderate, which might be influenced by geographical distance and gene flow. The Fst values also confirmed that genetic differentiation intensifies with increasing geographic distance. Collectively, this study clarifies the complex phylogenetic relationships within the B. sinensis species complex, elucidates the genetic diversity and differentiation across the studied populations, and offers valuable genetic insights that contribute to the broader study of evolutionary relationships and population genetics within the Bolbitis species.