In the species-rich family Phyllostomidae, the genus Macrotus (‘big eared’ bats) contains only two species; Macrotus waterhousii, distributed in western, central, and southern Mexico, Guatemala and some Caribbean Islands, and Macrotus californicus, distributed in the southwestern USA, and in the Baja California peninsula and the state of Sonora in Mexico. In this study, we sequenced and assembled the mitochondrial genome of Macrotus waterhousii and characterized in detail this genome and that of the congeneric M. californicus. Then, we examined the phylogenetic position of Macrotus in the family Phyllostomidae based on protein coding genes (PCGs). The AT-rich mitochondrial genomes of M. waterhousii and M. californicus are 16,792 and 16,691 bp long, respectively, and each encode 13 PCGs, 22 tRNA genes, 2 rRNA genes, and a putative non-coding control region 1,336 and 1,232 bp long, respectively. Mitochondrial synteny in Macrotus is identical to that reported before for all other cofamilial species. In the two studied species, all tRNAs exhibit a ‘typical’ cloverleaf secondary structure with the exception of trnS1, which lacks the D arm. A selective pressure analysis demonstrated that all PCGs are under purifying selection. The CR of the two species feature three domains previously reported in other mammals, including bats: extended terminal associated sequences (ETAS), central (CD), and conserved sequence block (CSB). A phylogenetic analysis based on the 13 mitochondrial PCGs demonstrated that Macrotus is monophyletic and the subfamily Macrotinae is a sister group of all remaining phyllostomids in our analysis, except Micronycterinae. The assembly and detailed analysis of these mitochondrial genomes represents a step further to continue improving the understanding of phylogenetic relationships within the species-rich family Phyllostomidae.
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