Caracterização genética das unidades taxonômicas do gênero Bokermannohyla (Anura: Hylidae) do Parque Estadual do Ibitipoca, Minas Gerais

Abstract

The taxonomic and phylogenetic characterization of amphibian species of the genus Bokermannohyla has been historically challenging, largely due to the absence of diagnostic morphological characters, the occurrence of cryptic species and hybridization events. In this scenario, species delimitation is highly dependent on molecular data, making the use of reliable sets of molecular markers a promising alternative given the high cost of whole-genome sequencing. The Ibitipoca State Park (PEIB), located in the municipality of Lima Duarte, Minas Gerais state, within the Serra da Mantiqueira mountain range, harbors species of this genus with historical and conservation relevance, including taxa described from the protected area itself. However, current knowledge regarding taxonomic boundaries and the possible occurrence of hybridization remains limited. This study aims to characterize the genetic structure and delimit the taxonomic units of Bokermannohyla present in PEIB, to investigate the possible occurrence of hybrids, and to place these taxa within a phylogeny of the genus, while evaluating the efficiency of the FrogCap protocol for Neotropical anurans. We sequenced 56 individuals of the genus using the FrogCap molecular marker set. We analyzed genetic structure based on Single Nucleotide Polymorphisms (SNPs) using the softwares Structure and PopCluster. We performed phylogenetic reconstructions using nuclear and mitochondrial genes, combining sequences obtained in this study with those from previous studies deposited in GenBank, employing Bayesian Inference in MrBayes and Maximum Likelihood in IQ-TREE. The FrogCap protocol demonstrated high efficiency, capturing approximately 82% of the targeted markers and providing robust data for downstream molecular analyses. Genetic structure analyses revealed three well-defined clusters (K=3), corresponding to Bokermannohyla feioi, Bokermannohyla ibitipoca and Bokermannohyla vulcaniae (considered endangered by the IUCN), with no evidence of mixed ancestry. This suggests the absence of hybridization among the species, despite indications of reproductive interference between B. feioi and B. ibitipoca in the locality. Phylogenetic analyses were congruent with these delimitations and confirmed the occurrence of B. vulcaniae in the PEIB, revising the historical identification as B. circumdata in this locality. However, we found a clear phylogenetic separation between the populations from the municipalities of Poços de Caldas and Ouro Branco (Minas Gerais state), suggesting that a simple geographic range expansion of B. vulcaniae should be handled with caution when reassessing the conservation status of the species. Our results reinforce the importance of integrative taxonomy and the use of multiple methodological approaches to resolve taxonomic problems, and provide relevant information for the conservation of Bokermannohyla species in the PEIB.