Computational protocol: The evolutionary history of sharp- and blunt-snouted lenok (Brachymystax lenok (Pallas, 1773)) and its implications for the paleo-hydrological history of Siberia

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Protocol publication

[…] All sequences were easily aligned by eye including an alignment incorporating one sequence from South Korea (GenBank accession no. AF125519), and 14 from China []. Quantitative assessment was limited to the sequences produced in our laboratory.Sequences were imported into PAUP*4.0b10 [] for phylogenetic analyses, pairwise sequence divergence (uncorrected p distances) and the number of transitions and transversions. Saturation in ND1 was assessed by plotting the number of transitions and transversions against uncorrected p distances for each codon position. A chi-square (χ2) test was used to evaluated base composition homogeneity in the ND1 gene for each codon position.To evaluate relationships among closely related haplotypes, unrooted networks were constructed with a 95% parsimony criterion using TCS ver 1.13 []. Between-group variation was calculated using net nucleotide divergence (Da) in MEGA version 2.1 []. Haplotype or clade divergence was also calculated using Da distances between groups using the Kimura two-parameter model. Uncorrected p distances were used for divergence estimates between in- and outgroup taxa.Maximum parsimony (MP), Maximum-Likelihood (ML) and Neighbor-Joining (NJ) were used for phylogenetic reconstruction. Modeltest 3.0 [] was used to choose models of nucleotide evolution. To estimate the most likely topology for ML and MP methodologies, heuristic searches (10 replicates) started with stepwise addition trees, with each replicate beginning with a random order of sequences. Bootstrap analysis was used to estimate node support with 10000 (NJ and MP) or 1000 replicates (ML). Full heuristic search algorithms were applied for the MP and the "fast" stepwise addition method for the ML analysis. [...] Genetic variation among and within major basins was evaluated with an analysis of molecular variance (AMOVA) using Arlequin 3.1 []. The AMOVA structure was defined by major ocean basins (Arctic/Pacific), then by large river basins or regions (Ob, Enisei, Lena including Indigirka and Kolyma, Amur, Islands, Primor'e, and Uda and Tugur) (see Table ). A more detailed overview of within and among basin differentiation is provided with a table of average (and corrected) pairwise differences also done with Arlequin. [...] To assess introgression between sharp- and blunt-snouted lenoks, we applied bi-parentally inherited microsatellites to three sets of sympatric populations from the Amur basin: the Anui (n = 56), Khor (n = 48), and Sukpai (n = 26) rivers.Four tri-nucleotide and three tetra-nucleotide microsatellite loci [], and four unpublished di-nucleotide microsatellite loci were analyzed (see Supplementary Materials). All forward primers were fluorescently labeled and PCR and genotyping were performed as described in [], (see Additional File ). Exact probability tests for deviations from Hardy-Weinberg equilibrium (HWE) across populations (within loci) and loci (within populations), exact tests for deviations from genotypic linkage equilibrium (LE) across populations, and tests for genic differentiation among populations were performed with Genepop 3.2a []. Corrections for multiple significance tests were performed using a sequential Bonferroni-type correction [].To estimate the proportion of each individual's genome originating in each parental species and the patterns of intraspecific variability among sharp- and blunt snouted lenoks we used the Structure []. This software implements a Bayesian model-based clustering algorithm that reveals population structure in a data set by resolving clusters of individuals that minimize Hardy-Weinberg and linkage disequilibrium. The parameter settings included the assumption of admixture and a correlated allele frequencies model. In exploratory runs we did not provide the software with prior information regarding the origin of individuals, but in final runs, and when analyzing the three sympatric populations separately, individuals from the two forms were assumed to represent pure blunt- or sharp-snouted lenoks, and used as a proxy for determining the degree of admixture of the individuals within each sympatric population. Structure was run for 100,000 steps, of which the first 10,000 where discarded as burn-in and we conducted five independent replicates of the MCMC for each value of k. We also performed analyses using the independent allele frequencies model to test for robustness of our conclusions to the violation of prior assumptions because of recent suggestions that the choice of the model might strongly influence the outcome of the clustering algorithm [,]. […]

Pipeline specifications

Software tools PAUP*, MEGA, ModelTest-NG, Arlequin, Genepop
Applications Phylogenetics, Population genetic analysis
Organisms Brachymystax lenok, Hemisus marmoratus