2.4 | Genetic and Statistical Analysis
Fixation index coefficient (FIS ) was calculated based on the variance found in the allelic frequencies (Weir & Cockerham, 1984) and the intra-population genetic diversity (expected and observed heterozygosities) was estimated under the Hardy-Weinberg equilibrium. Both analyses were performed using the ‘het’ option in VCFtools implemented in r2vcftools (Danecek et al. 2011 (https://github.com/rojaff/LanGen_pipeline). FSTcalculations between populations were performed using the dartR package v.183 in R software (R Core, Jombart, 2008; Jombart & Ahmed, 2011; Team, 2019). Genetic distance was estimated according to Nei (1972), where a constant and equal mutation rate is considered for all loci with equal population sizes for all generations and a mutation-drift balance, using the adegenet package in R (Jombart, 2008; Jombart & Ahmed, 2011). From the genetic distance analysis, cluster analyses were performed based on the UPGMA method using the poppr package in R (Kamvar et al., 2014-2015). To validate the groups, a 1,000-repetition bootstrap analysis was applied using the aboot function of the poppr package. Supported nodes were those with values ​​≥ 70% (Hillis & Bull, 1993). Additionally, the geographical distance (in meters) between pairs of populations was calculated considering the Earth’s curvature (assuming the spherical model) from the georeferences of each population using the geosphere package in R (R Core, Hijmans, 2019; Team, 2019). Once the distance matrices were determined, a Mantel test based on Spearman’s correlation with 9,999 permutations was performed using the vegan package (Oksanen et al., 2019). A Principal Coordinate Analysis (PCO) based on Nei’s distance (1972) was conducted using the pcoa command of the ape package (Paradis & Schliep, 2019).