Sampling and gDNA Extraction

A total of 144 individuals were sampled from 17 localities, comprising eight specimens of A. alalia and 136 of A. mantiqueira (Fig. 1 and Table 1).Three individuals corresponding to A. catarina and two samples ofA. dalmeidai were also collected from the same localities to be used as outgroups in some analyses. The localities (municipalities) were defined as populations for downstream analyses. Ten localities were sampled for A. mantiqueira, representing most of its known distribution. For A. alalia , however, fresh samples were obtained from two localities throughout its distribution.
Total genomic DNA (gDNA) was purified using the CTAB protocol (Doyle & Doyle, 1987), modified after Silva-Brandão et al. (2018). Total gDNA was eluted with 50 μL of AE buffer, quantified using a Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA), and stored at -20 ºC. Gel electrophoreses were performed with 1% agarose gel in 50 mM Tris-acetate (TAE buffer), using 5 μL of each sample to check extraction quality. The ratios of the wavelengths 280/260 and 260/230 were estimated with a NanoDrop UV spectrophotometer (ThermoFisher Scientific).