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).