Objective Whitmania pigra is the dominant leech species used in traditional Chinese medicine. This study aims to evaluate the genetic diversity among five wild W. pigra populations .

Methods Based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) and cytochrome b (Cyt b) gene sequences, we assessed genetic diversity in 146 wild W. pigra specimens from five populations: Daxing of Beijing (BJ), Cangzhou of Hebei (HB), Nanjing of Jiangsu (JS), Dezhou of Shandong (SD), and Baodi of Tianjin (TJ).

Results A+T content greatly exceeded G+C content in both genes, indicating a pronounced AT bias. The COⅠand Cyt b fragments contained 78 and 120 variable sites that defined 39 and 51 haplotypes, respectively. Genetic diversity analyses revealed that the BJ population had the highest mean haplotype diversity (Hd, 0.937 9 and 0.970 1 for COⅠ and Cyt b, respectively). The SD population exhibited the highest mean number of pairwise nucleotide differences (k, 18.653 8 and 23.264 5) and the highest nucleotide diversity (π, 0.026 7 and 0.022 9). In contrast, the TJ population consistently showed the lowest values for all indices: mean haplotype diversity (0.685 1 and 0.694 3), mean number of pairwise nucleotide differences (1.514 9 and 2.308 1), and nucleotide diversity (0.002 2 and 0.002 3). Pairwise F-statistics (F_st), genetic distance, and analysis of molecular variance (AMOVA) all indicated that most variation resides within rather than among populations. Phylogenetic trees constructed from COⅠand Cyt b haplotypes were congruent, displaying an intermingled distribution without clear geographic structuring. Neutrality tests suggested that the combined populations have remained demographically stable without significant expansion.

Conclusion Overall, BJ and SD harbour abundant genetic diversity, reflecting healthy wild resources, whereas the depauperate TJ population warrants strengthened protection and management to ensure sustainable utilization of W. pigra germplasm. The results of the study provide baseline data for the conservation and utilization of germplasm resources of W. pigra.