Computational protocol: Catchment-Scale Conservation Units Identified for the Threatened Yarra Pygmy Perch (Nannoperca obscura) in Highly Modified River Systems

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Protocol publication

[…] Fisher's exact test of linkage disequilibrium and tests for departures from Hardy-Weinberg equilibrium (HWE) were conducted using GENEPOP 4.1.4 , and GenoDive 2.0 , respectively. Significance levels were Bonferroni-corrected to avoid type I errors associated with multiple tests . For each site, the number of alleles (NA), expected (HE) and observed (HO) heterozygosity, and inbreeding coefficient (FIS) were calculated in GenoDive 2.0 . Allelic richness (AR) was estimated using the rarefaction procedure in HP-RARE , and the percentage of polymorphic loci was calculated in GenAlEx 6.5 . [...] Population genetic structure was assessed at multiple spatial scales, both across the entire distribution and within each of the proposed ESUs, using a combination of several frequency-based and genotype-based statistical methods. Pairwise FST and RST tests were performed in Arlequin to evaluate between-site differentiation. Given the potential for temporal variation in population structure, an assessment of pairwise FST and RST was also performed between years at 12 sites where samples were collected on multiple occasions. In order to determine if either FST or RST was more appropriate for this study, the relative contribution of genetic drift and mutation to population differentiation was assessed . SPAGeDi 1.3 was used to permutate global allele sizes for each locus and to compare observed RST with permutated RST (pRST) values. Arlequin was used to perform an analysis of molecular variance (AMOVA) with 1000 permutations based on FST . Hierarchical structure was assessed using AMOVA among the major genetic lineages, among sites within lineages, and among individuals within sites. Separate AMOVAs were also performed for each primary lineage.A Bayesian clustering analysis of individual genotypes using STRUCTURE 2.3.4 was initially performed using all samples to identify primary population structure across the entire distribution, before repeating the analysis within each of the primary clusters to assess hierarchical population structure at smaller spatial scales . Twenty independent runs for each K value (1–27) were completed to ensure reproducibility , using a burn in of 100 000 followed by 1 million Monte-Carlo Markov chain (MCMC) iterations. We used the admixture model, with independent allele frequencies among populations and no prior information on sampling location. The most likely K value was inferred using the Evanno et al. method implemented in STRUCTURE HARVESTER . Results of the 20 replications were then combined using the software CLUMPP 1.1.2 , and visualised using Distruct 1.1 . A different analytical approach based on assignment of individual genotypes was performed using GeneClass2 . This was conducted using the Bayesian approach of Rannala and Mountain to calculate the probability that each individual originates from its sampling locality or from other sites.Principal coordinates analysis (PCA) was also employed to allow visual examination of the genetic affinities of individuals across the entire distribution and to clusters identified within each lineage. Pairwise genetic distances between individuals were first calculated before those results were subjected to PCA analysis. Both procedures were completed in GenAlEx 6.5 . […]

Pipeline specifications

Software tools Genepop, Genodive, GenAlEx, Arlequin, SPAGeDi, Structure Harvester, CLUMPP, DISTRUCT, GeneClass
Applications Phylogenetics, Population genetic analysis
Organisms Homo sapiens, Danio rerio, Nannoperca obscura
Diseases Genetic Diseases, Inborn