Comparative Analysis of Mitochondrial Genomes and Phylogeny of Barbastelle Bats Across China

ABSTRACT Barbastelle bats are notable for their nearly square outer ears that converge at the forehead. Knowledge of their mitochondrial genome is limited. This study sequenced the complete mitochondrial genomes of Barbastella beijingensis and B. darjelingensis , the latter being reported for the first time. Subsequently, we performed a comprehensive comparative analysis with the published mitochondrial genome of B. capsica . The mitochondrial genomes measured 16,667 bp and 16,434 bp, respectively, and included 13 protein‐coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and one D‐loop. Their gene order matched other bat species in the Vespertilionidae and Phyllostomidae families. All three species showed low K a / K s ratios in the PCGs, indicating strong purifying selection, especially in cytochrome oxidase respiratory chain complex ( COX1 , COX2 , and COX3 ), while ATP8 faced relatively relaxed selective pressure. In all three species, tRNAs showed typical cloverleaf structures, except for trnS1 , which lacked a D arm. The D‐loop region is divided into ETAS, CD, and CSB domains. Both ETAS domains have ETAS1 and ETAS2 motifs, and the CD domain contains conserved motifs box B–F. While CSB1–CSB3 were conserved in B. capsica , only CSB1 was found in B. beijingensis and B. darjelingensis. Phylogenetic analysis of the 13 PCGs strongly confirmed the three barbastelle species as distinct within Barbastella , closely related to genera Plecotus and Corynorhinus in the Plecotini tribe. This study enhances the molecular data available for the genus Barbastella and offers essential mitochondrial genomic evidence to resolve persistent taxonomic disputes within the Vespertilionidae family.