Deleterious load and fixed mutations
We used the G. alexis annotations to create a SNPeff database that we used to annotate our callings generated with angsd and predict their putative effect (12). Additionally, we took a more in-depth view to genes which could alter the coloration patterns in the individuals (19–23). First we located those genes in our annotation with BLAST and their homologs in other butterfly species (58), setting an E-value lower than 0.001 and an Identity value above 60%. Then, the coordinates were called using GATK v3.7 UnifiedGenotyper. Variants were filtered for a indels and minimum Genotype Quality of 30 using vcftools (45). Variants were kept regardless of their coverage. A variant was considered as fixed in a specie if it is covered in at least 2 individuals of each specie, do not present heterozygous genotypes, and when one of the species present all their genotypes calls as homozygous for the derived allele while in the other are homozygous of the ancestral allele. Additionally, we checked the coverage in the after mention genes using bedtools (41) (Fig. S9). The results were displayed using ggplot2 (59).