Obtaining sequences from the COI barcode region
The barcode region (Hebert et al., 2003), which is the 5′ end of the
mitochondrial DNA (mtDNA) gene cytochrome oxidase subunit I (COI, ca.
658 bp), is widely used in the delimitation of butterfly species
(Silva-Brandão et al., 2009). Hence, to assess the species limit betweenA. alalia and A. mantiqueira with this traditional
molecular marker, 59 barcode sequences were investigated, including all
samples available on GenBank (Benson et al., 2005) and BOLD
(Ratnasingham & Hebert, 2007). The barcode region was amplified
from 39 samples, which were also evaluated with GBS libraries (Table 1).
In total, these specimens represent 13 sampling localities (collection
data and GenBank accession codes are shown in Table 1). The barcode
region was amplified using the primers LCO (F) and NANCY (R) (Caterino
& Sperling, 1999; Folmer et al., 1994), following the procedures
described in Magaldi et al. (2021). Purified PCR products were sequenced
using both forward and reverse primers in a 3730xl DNA Analyzer
(ThermoFisher Scientific).