Computational protocol: Molecular Analyses Reveal Unexpected Genetic Structure in Iberian Ibex Populations

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Protocol publication

[…] Expected (HE) and observed (HO) heterozygosity, and deviations from the Hardy-Weinberg equilibrium (HWE) were calculated using Genepop (v.3.4; []). Fisher’s exact tests and sequential Bonferroni corrections were used to evaluate deviations from HWE. Linkage disequilibrium (LD) between pairs of microsatellite loci within populations was estimated with Arlequin 3.11 [].Genetic divergence in the three Iberian ibex populations was assessed using pairwise FST based on allele frequencies as implemented in Arlequin 3.11.We estimated standardized allelic richness in FSTAT version 2–9.3. [] to account for the substantially different sample sizes of the studied Iberian ibex populations.The relationships between the C. pyrenaica populations were assessed using a dendrogram calculated using Nei's standard genetic distance based on allele frequencies [] constructed with the POPGENE software package []. Additionally, we used two further methods, namely:The clustering program INSTRUCT v1.1 []: population structure was inferred for K (number of clusters) = 1 to 8, with 10 chains for each K. The Gelman–Rubin statistic (Rc) was used to check for convergence []. The optimal K was inferred using the deviance information criterion (DIC: []).The DAPC [] software implemented in the adegenet R package [] to identify the number of clusters. The maximum number of clusters was set at eight. The number of clusters was determined using the Bayesian Information Criteria (BIC) following the k-means algorithm.In recently bottlenecked populations, observed heterozygosity is higher than expected given the number of alleles in the population if the population is at mutation-drift equilibrium []. This heterozygosity excess was used to test for the genetic signature of bottlenecks in Iberian ibex as implemented in Bottleneck (v.1.2.02; []). We used 10,000 iterations for each of the three tests (sign test, standardized differences test and Wilcoxon sign-rank test) per population. We used the intermediate two-phase mutation model (TPM) [], which has been shown to deliver the most realistic results for the typical mutational events of microsatellite loci []. The heterozygosity excess tested by means of BOTTLENECK refers to “the fact that heterozygosity computed from a sample of genes is larger than the heterozygosity expected from the number of alleles found in the sample if the population were at mutation-drift equilibrium (HEQ)” []. All input file preparations were made using Convert (v. 1.31; []). […]

Pipeline specifications

Software tools Genepop, Arlequin, POPGENE, adegenet
Application Population genetic analysis
Organisms Capra ibex, Capra pyrenaica