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).