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).