2.5. Population genetic differentiation and clustering
For differentiation and structural analyses, loci under Linkage Disequilibrium (LD) were removed using plink v2.00a3.3 (Purcell et al., 2007) (–indep-pairwise: 50 [’kb’] 5 0.20) (Table 2). Pairwise Fst values (Weir & Cockerham, 1984) were estimated with the functionstamppFst from ‘StAMPP’ R package (Pembleton et al., 2013), and significant differences for each population pair were evaluated with confidence limits at 95% after 1000 bootstrap iterations. Heatmaps based on Fst were created using pheatmap in R (Raivo Kolde, 2019).
Isolation by distance (IBD) was evaluated by correlating the Fst genetic distances with linear geographic distances among populations, using a Mantel test (Mantel, 1967) with function mantel.randtest in the ‘ade4’ package in R (Dray and Dufour, 2007) with 1000 permutations. The test was performed for Menorca and Mallorca/Cabrera archipelago separately to control for distance effect between major archipelagos.
To reconstruct evolutionary relationships among island populations, we first used a Discriminant Analysis of Principal Components (DAPC), integrated in the ‘Adegenet’ R package (Jombart, 2008). In this multivariate statistical approach, sample genetic variance is partitioned into groups to maximize discrimination, without prior knowledge on genetic structure. The number of meaningful PCs retained was chosen by a cross-validation with the function xvalDapc(Jombart & Collins, 2015) with default parameters (1000 replicates).
Population structure, including contemporary and historic admixture among populations and their relationships, were further investigated using ADMIXTURE v1.3.0 (Alexander et al., 2009), setting the numbers of co-ancestry clusters (K ) from 2 to 16 (maximum number of populations). Cross-validation was performed with the -cv option (10-fold) to retain the best value of K clusters (lowest cv error). Membership probabilities were calculated according to the retained discriminant functions and specimens showing above 90% of assignment probability to an island different from their source were considered as recently translocated, or potential mislabeled and removed (none detected).