Phylogenetic Analysis of the Brazilian Free-tailed Bat (Tadarida brasiliensis) in Chihuahua Using Partial Sequencing of the Mitochondrial Control Region (D-loop)

Bats are among the most diverse mammalian groups and provide essential ecological services, yet the mitochondrial diversity and matrilineal connectivity of Mexican populations remain poorly characterized. Tadarida brasiliensis is a mobile, colonial species whose phylogeographic structure in northern Mexico remains incompletely resolved. This study evaluated the phylogenetic placement and mitochondrial variability of T. brasiliensis from the Bustillos mine in Chihuahua using partial mitochondrial control region (D-loop) sequences within a broader comparative dataset.

Forty-eight adult bats of both sexes, including non-pregnant and non-lactating females, were captured with mist nets from June to October 2025 (permission SPARN/DGVS/11298). Individuals were handled with biosafety protection, morphologically identified using specialized taxonomic keys, and sampled through 2-mm wing biopsies preserved in absolute ethanol. DNA was extracted using standard procedures, and an approximately 650-bp D-loop fragment was amplified by PCR and sequenced by the Sanger method. Chihuahua sequences were analyzed with reference D-loop sequences. After quality control and alignment trimming, the population dataset comprised 239 sequences and 458 bp. Genetic diversity, haplotype networks, maximum-likelihood phylogeny, AMOVA/ΦST, Mantel tests, and demographic statistics were estimated.

The comparative dataset contained 208 haplotypes and 224 segregating sites, with high haplotype diversity (Hd = 0.9986) and nucleotide diversity (π = 0.0643). Among Chihuahua samples, 19 haplotypes were detected, 17 of which were not shared with the reference dataset and are therefore considered putatively novel. AMOVA revealed strong mitochondrial structure among countries (ΦST = 0.8190), regions (ΦST = 0.7580), and localities (ΦST = 0.7591; all p < 0.001). Mantel analyses supported isolation by distance. Overall, despite the migratory ecology of T. brasiliensis, D-loop variation retained a strong spatial signal, reflecting historical matrilineal structure rather than contemporary movement alone. These results establish a useful reference for northern Mexican colonies and support future integrative analyses using nuclear markers, morphology, and broader temporal sampling in Mexico