Gene-Specific Substitution Rates for the Vespertilionidae (Chiroptera: Mammalia) Mitochondrial Genome, with the Description of Three new Mitogenomes.

Molecular data are irreplaceable resources for reconstructing the tree of life. Gene-specific substitution rates are essential for estimating divergence times in the absence of fossil calibration or converting coalescent units into absolute time in phylogeographic approaches, among other uses. However, substitution rate estimates are often derived from limited genomic loci, narrow taxonomic comparisons, and model organisms, hindering their applicability to understudied taxa. Among mammals, bats (Order Chiroptera)-despite their ecological diversity and evolutionary significance-remain underrepresented in substitution rate studies, particularly within the family Vespertilionidae, the third largest mammal family. Here, we investigate mitochondrial genome (mitogenome) evolutionary rates in this group, while also describing the first complete mitogenomes of three Neoeptesicus species: N. brasiliensis, N. diminutus, and N. furinalis. Using fossil-calibrated Bayesian phylogenetic analyses, we estimated that protein-coding genes evolve at rates between 0.0055-0.0089 substitutions per site per million years (subs/site/Ma), while ribosomal RNA genes evolve at rates between 0.0035-0.0049 subs/site/Ma. Notably, the ND4, ND4L, and ND5 genes exhibited the highest rates, whereas non-coding regions showed the lowest, suggesting that gene-specific evolutionary constraints influence these rates. These findings provide the first comprehensive substitution rate framework for Vespertilionidae mitogenomes, addressing a critical gap in genomic resources for this taxonomically complex group. By integrating novel mitogenomic data with rigorous rate estimation, this study advances our capacity to resolve evolutionary patterns in bats, offering a benchmark for future phylogenetic and phylogeographic studies in non-model mammals.