DNA extraction and genotyping

DNA was extracted from a small posterior part of the abdomen at the “Laboratorio de Ecología Molecular y Conservación" (Laboratory of Molecular Ecology and Conservation) at the “El Colegio de la Frontera Sur” research center in Chetumal, Mexico. Each DNA sample was rehydrated in ultra-pure water for one hour before extraction, which was performed using a Wizard Genomic DNA Purification Kit (Promega) following the manufacturer’s instructions. Afterwards, DNA products were stored at -20°C until amplification. Concentration of genomic DNA was determined with the Qubit®2.0 fluorometer (Invitrogen), and quality was tested using agarose gel (1% with TAE buffer 1X; Promega) and the post-gel staining method using GelRed™ (Quimica Valaner).

Mitochondrial DNA sequences were obtained at the Butterfly Diversity and Evolution Lab (Institut de Biologia Evolutiva, Barcelona, Spain). GenBank accession numbers are provided in S1 Table. They consisted of the COI gene that was performed on a subset of data, which consisted of 83 individuals. The target region was amplified using primers with demonstrated efficacy in butterfly studies [17, 32–33], amplifying a fragment of ~657 pb: LepR1 (5´-TAAACTTCTGGATGTCCAAAAAATCA-3’) and LepF1 (5’-ATTCAACCAATCATAAAGATATTGG-3’). The PCR mix consisted of 14.4 μl ddH₂O, 5 μl 5X Green Buffer, 0.5 μl dNTPs mix, 0.1 μl Taq Polymerase 5 U/μl (Promega), 2 μl MgCl₂, 2 μl of template DNA, and 0.5 μl of each primer (10 μM). Amplifications were carried out under specific conditions: initial denaturation at 92°C for 60 s, 35 cycles of denaturation at 92°C for 15 s, primer annealing temperature at 49°C for 45 s, an extension at 62°C for 150 s, and a final extension at 62°C for 7 min. Amplification products were visualized by electrophoresis using 3 μl of PCR products on a 1% agarose gel with SBYR-safe staining, at 80 V for 30 min. PCR products were sent for purification and sequencing to Macrogen Inc. (Seoul, Korea).

Two different nuclear genes were used: the ribosomal protein subunit 5 (RpS5), a relatively fast-evolving gene [34], and the wingless gene (Wg), involved in wing pattern formation (eyespot center formation) [35]. RpS5 sequence was performed on a subset of data that consisted of 74 individuals. The target region was amplified using the hybrid primers with demonstrated efficacy in butterfly studies [36–37], amplifying a fragment of ~613 pb [38]: HybRpS5deg (5'-TAATACGACTCACTATAGGGATGGCNGARGARAAYTGGAAYGA-3'; forward) and HybRpS5deg (5'-ATTAACCCTCACTAAAGCGGGTTRGAYTTRGCAACACG-3'; reverse). A subset of data, consisting of 77 individuals, resulted in good sequences for Wg using primers with demonstrated efficacy in butterfly studies [39–40], amplifying a fragment of ~400 pb.: LepWg1 (5'-GARTGYAARTGYCAYGGYATGTCTGG-3'; forward) and LepWg2 (5'-ACTICGCARCACCARTGGAATGTRCA-3'; reverse).

The PCR mix consisted of 14.15 μl (RpS5) and 14.4 μl (Wg) ddH₂O, 5 μl 5X Green Buffer, 0.5 μl dNTPs mix, 0.15 μl (RpS5) and 0.1 μl (Wg) Taq Polymerase 5 U/μl (Promega), 2.2 μl (RpS5) and 2 μl (Wg) of MgCl₂, 2 μl of template DNA, and 0.5 μl of each primer (10 μM). Amplifications were carried out under specific conditions: initial denaturation at 95°C for 6 min(RpS5) and 3 min (Wg), 40 cycles of denaturation at 95°C for 30 s, primer annealing temperature at 51°C for 30 s (RpS5) and 60 s (Wg), an extension at 72°C for 90 s, and a final extension at 72°C for 10 min (RpS5) and 6 min (Wg). The protocol for visualizing the PCR products was identical to mitochondrial DNA. The PCR products were sent for purification and sequencing to Macrogen Inc. (Seoul, Korea). GenBank accession numbers are presented in S1 Table.

We tested a total of 24 different ISSR markers. The selection of markers for this study was based on the identification of a particular band profile to facilitate the identification of each morphospecies, in addition to presenting good resolution and a high number of bands. Two ISSR markers were retained for our study: (AG)₈Y and (GA)₈C (Table 2).

Percentage of guanine and cytosine content (%GC), melting temperature (Tm), annealing temperature (Ta), total number of bands per primer over all localities (N bands), size range of the DNA fragments for each primer (size). The designation Y (C or T) was used for degenerated sites.

PCR amplifications were performed in 15 μl reaction volume containing ~20 ng of template DNA, 1.5 μl 5X Green Buffer (Promega), 200 μM dNTP (dNTP mix; Promega), 3 mM MgCl₂ (Promega), 1 μM of primer (Integrated DNA Technologies), and 1.25 U GoTaq Flexi DNA Polymerase (Promega); finally, the volume was adjusted with ultrapure water. Amplifications were conducted in a T100 Thermal Cycler (Bio-Rad™): initial denaturation step at 94°C for 4 min, 39 cycles of denaturation at 94°C for 45 s, annealing temperature (Ta) 54°C or 56°C depending on the ISSR primer (Table 2), extension temperature at 72°C for 2 min, and a final extension at 72°C for 10 min. DNA banding patterns were visualized by electrophoresis, performed with 3 μl of amplified products on a 2% agarose gel using 1X TAE buffer and post-staining with GelRed™ (Biotium), at 110 V for 2 h. A 100 bp DNA Ladder (Promega) was used to estimate amplification product lengths. Fragment (band) patterns were visualized and digitized using an imaging system (PhotoDoc-it, UVP®).