Phylogenetic characterization and relationship of leaf-nosed bats (Family: Hipposideridae) from Tirunelveli District, Southern Tamil Nadu, India

Resolving evolutionary relationships among closely related microchiropteran species remains challenging due to overlapping morphological characteristics. The present study investigated genetic diversity and phylogenetic relationships among three Hipposideros species, namely H. bicolor, H. speoris, and H. ater, using RAPD-PCR and mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. RAPD-PCR analysis was performed using six random primers (OPA-01 to OPA-06), of which OPA-06 generated distinct and reproducible species-specific banding profiles. Species-specific amplicons of approximately 750 bp, 1500 bp, and 1250 bp were obtained for H. bicolor, H. speoris, and H. ater, respectively, demonstrating the potential of RAPD markers for species discrimination. COI sequences generated a 575 bp aligned fragment without insertions, deletions, or stop codons. The dataset comprised 244 conserved sites, 163 variable sites, 114 parsimony-informative sites, and 54 singleton sites. The average nucleotide composition was A (24.9%), T (31.9%), G (18.7%), and C (24.4%), with transitions occurring more frequently than transversions. Pairwise genetic distances ranged from 0.000 to 0.240, with an average interspecific divergence of 0.193. Phylogenetic relationships were reconstructed using Maximum Likelihood, Neighbor-Joining, and UPGMA methods based on the Kimura two-parameter model. All three approaches consistently resolved H. speoris and H. bicolor as a closely related species pair, while H. ater formed another strongly supported species group. Overall, the results demonstrate that RAPD-PCR and COI DNA barcoding provide complementary molecular approaches for species identification, assessment of genetic divergence, and reconstruction of phylogenetic relationships among Hipposideros bats. The findings further support the utility of mitochondrial COI sequences as reliable molecular markers for resolving species-level relationships within the family Hipposideridae.