Single nucleotide polymorphisms (SNPs) provide unprecedented resolution of species boundaries, phylogenetic relationships, and genetic diversity in the mastiff bats (Molossus).

Mammals are one of the better known groups of animals, and in the Neotropics bats typically comprise about half of the mammalian species diversity. But, well resolved species-level phylogenies are still lacking for most taxa of bats. One broadly distributed genus is the mastiff bats, Molossus. Species within this genus are morphologically very similar, which results in a confusing and unstable taxonomy. In addition, low levels of genetic divergence among some clades make resolution of phylogenetic relationships difficult. Most authors recognize Molossus as being monophyletic, however, phylogenetic relationships within the genus remain poorly understood based on traditional Sanger sequencing of individual genes. We propose a more comprehensive framework based on large-scale genomic data derived from Next Generation Sequencing techniques to better understand evolutionary relationships within a group of closely related species with a rich taxonomic history. In this study, we utilized the NGS method of Genotype by Sequencing (GBS) to test the monophyly of the genus, understand evolutionary relationships within Molossus and investigate the genetic integrity of currently recognized species. Given that both de novo and reference genome pipelines are often used in the assembly of Single Nucleotide Polymorphism data from GBS, and that several tree inference methodologies have been proposed for SNP data, we test whether different alignments and phylogenetic approaches produce similar results. We also examined how the process of SNP identification and mapping can affect the consistency of the analyses. Our data provide the first high resolution phylogeny for the genus Molossus, bringing new insights into recognition of species boundaries and relationships among taxa. This study clarifies the taxonomy of Molossus and elevates the number of species in the genus from 11 to 14. We suggest the revalidation of the names M. nigricans, and M. fluminensis, which were synonymized under the name M. rufus; and M. bondae, previously synonymized under the name M. currentium. Different alignments and phylogenetic inferences produce consistent results, supporting use of SNP approach in addressing evolutionary questions on a macroevolutionary scale where the genetic distance among clades is low.