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

## TL;DR

This study sequenced and compared the mitochondrial genomes of two barbastelle bat species from China, revealing insights into their genetic structure and evolutionary relationships.

## Contribution

The study provides the first complete mitochondrial genome of B. darjelingensis and offers new phylogenetic evidence for resolving taxonomic disputes in the genus Barbastella.

## Key findings

- The mitochondrial genomes of B. beijingensis and B. darjelingensis were sequenced and found to have typical gene structures with 13 protein-coding genes and 22 tRNA genes.
- Phylogenetic analysis confirmed the distinctness of the three barbastelle species and their close relationship to Plecotus and Corynorhinus.
- The D-loop region showed conserved motifs in B. capsica but not in B. beijingensis and B. darjelingensis.

## 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.

Barbastelle bats are characterized by their distinct approximately square‐shaped outer ears, which converge at the forehead. In China, three species have been documented, and we present two mitochondrial genomes for
Barbastella beijingensis
and B. darjelingensis. Every mitochondrial genome is composed of 13 protein‐coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and one D‐loop. Phylogenetic analysis based on the 13 PCGs strongly supported
B. beijingensis
and B. darjelingensis as distinct species within the genus Barbastella, which exhibits relatively close phylogenetic relationships with the genera Plecotus and Corynorhinus within the family Vespertilionidae.