Population Genetics of the Medicinal Leech Whitmania pigra Based on Mitochondrial Genes

Background: Leeches possess significant medicinal value as antithrombotic agents, and elucidating their population genetic background is of great importance for the conservation of medicinal germplasm resources and the development of pharmaceutical applications. Methods: In this study, mitochondrial genes (COI, Cytb, and ND1) were used as molecular markers to systematically investigate the population genetic characteristics of wild Whitmania pigra from 13 regions across China. A total of 137 individuals were sequenced, yielding a concatenated alignment of 3554 bp with 441 variable sites. Haplotype networks and phylogenetic trees were constructed based on the concatenated sequences, alongside BAPS clustering, neutrality tests, and Bayesian Skyline Plot (BSP) analyses. Results: Genetic diversity analyses revealed that the species-level concatenated dataset and most populations exhibited high haplotype diversity (Hd > 0.5) and low nucleotide diversity (Pi < 0.02), with JXJA as a notable exception (concatenated Hd = 0.200). Neutrality tests (Fu’s Fs = −6.002, p = 0.001) supported a scenario of historical bottlenecks followed by rapid demographic expansion, whereas the reduced diversity in JXJA may reflect a local bottleneck or founder effect. Haplotype networks, phylogenetic trees, and BAPS clustering consistently divided the 13 populations into three significantly differentiated groups: northeastern (HLJ), southern, and northern-central. The HLJ population exhibited extremely high genetic differentiation (FST = 0.902–0.977), suggesting it may represent a highly differentiated mitochondrial lineage and a candidate Evolutionarily Significant Unit (ESU). The BSP analysis reconstructed four phases of demographic history, broadly corresponding to climatic fluctuations since the Late Pleistocene. Conclusions: Overall, this study reveals high haplotype diversity and low nucleotide diversity in W. pigra, with JXJA representing a distinct local bottleneck case. The populations are strongly structured into three lineages, with the HLJ population representing a highly differentiated mitochondrial lineage and a candidate ESU. Demographic dynamics appear broadly associated with Late Pleistocene climatic fluctuations, though the timing remains uncertain due to the substitution rate. These findings provide a basis for W. pigra conservation, and future studies incorporating nuclear markers are warranted.