3.4. Outlier identification
We explored potential selective pressures driving population genomic differentiation by identification of outlier SNPs. Intersection of outputs from two independent methods (PCAdapt and Bayescan) provided a total of 18 candidate outliers for the COMBINED dataset, 222 for the GBS and 190 for the RADSeq dataset (n total =446, q<0.01 andF ST>0.8, see Table S4 for a complete list).
Pattern of distribution of outliers per chromosome was highly concordant among the three datasets, with most outliers falling within the sexual chromosome Z (>16% for all datasets), followed by a large representation on chromosome 2 (>9%) and 15 (12 and 8%, respectively) (Figure 7A). Outliers in chromosomes 3 and 4 were detected only by RADSeq.
About 60% of the outliers fell within protein-coding genes (44% for the COMBINED) corresponding to a total of 182 proteins (Figure 7A, pie charts). Only a minor proportion fell within CDS (n=14 outliers, corresponding to 11 annotated proteins). Five of these SNPs caused a nonsynonymous substitution (ALT) with respect to the reference genome (REF) and were detected in the glypican-3, B-cell lymphoma 6 protein, the cytochrome P450 2C19-like, the pore complex Nup214 genes and an uncharacterized protein (see details in Table S4).
Functional enrichment analysis of annotated proteins with outliers indicated a comparable profile for GBS and RADSeq, showing a significant overrepresentation of binding proteins and catalytic activity (MF category), and metabolic process, growth, and cellular homeostasis (BP category) (BH, p<0.001, Figure S3).
Of the overall 182 proteins with outliers, a small subset (n=13) was consistently retrieved by more than one dataset and most (n=9) were target of multiple outliers per dataset (up to seven) (Figure 7B). Three of these proteins were ion channels: the calcium channels inositol 1,4,5-trisphosphate receptor type 2 and the voltage-dependent L-type calcium channel subunit alpha-1C-like, and the potassium voltage-gated channel subfamily KQT member 1-like. Two proteins presented an outlier position identified by more than one dataset: the cullin-1 protein (SNP Position POS:937545) detected by all three datasets, and the voltage-dependent L-type calcium channel subunit alpha-1C-like (POS:55226525) detected by both GBS and COMBINED (Table S4).