Mitochondrial 16S rDNA alignment used to perform phylogenetic analysis of Bokermannohyla (Amphibia: Anura)

Mitochondrial 16S rDNA alignment used to test the monophyly of <em>Bokermannohyla nanuzae</em>, as well as the presence of candidate candidate species. Based on <em>16S</em> phylogenetic analysis, we recovered <em>B. nanuzae</em> as paraphyletic, with <em>B. sagarana</em> nested within it, and have shown that there are at least two species hidden under the name <em>B. nanuzae</em>. We recovered two main groups, with the geographic distribution generally corresponding to the Cerrado (North and Central Espinhaço) and Atlantic Forest boundaries (Quadrilátero Ferrífero, Southern Mantiqueira, Serra Negra, and Alto do Cariri). We revalidate <em>Bokermannohyla feioi</em> (Napoli &amp; Caramaschi, 2004; type locality Ibitipoca State Park, district of Conceição do Ibitipoca, municipality of Lima Duarte, state of Minas Gerais, Brazil) and apply this name to the populations from the Quadrilátero, Southern Mantiqueira, Serra Negra, and Alto do Cariri. <br> We extracted genomic DNA using the DNeasy Blood &amp; Tissue kit (Qiagen), following the protocol described by the manufacturer for vertebrate samples. We amplified the <em>16S</em> rDNA by Polymerase Chain Reaction (PCR) and sequenced with primers 16Sar-L and 16Sbr-H as described in Palumbi et al., (1991). For PCRs, we used the following conditions, using approximately 10-50 ng genomic DNA: 2.5 μL of 10× reaction buffer (100 mM Tris-HCl, pH 8.3, and 500 mM KCl), 2.4 μL of 50 mM MgCl2, 2 μL of the dNTP mix (2 mM each), 0.5 μL of each primer at 10 μM, 0.5 U of Taq polymerase (Thermo Fisher) and Milli-Q water qsp 25 μL. The PCR was performed on a Veriti 96-Well Thermal Cycler (Thermo Fisher), with the following conditions: initial denaturation at 94 °C for 2 min, followed by 35 cycles of amplification at 45 s at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, with a final extension step at 72 °C for 7 min.