Computational protocol: Mitochondrial DNA reveals secondary contact in Japanese harbour seals, the southernmost population in the western Pacific

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Protocol publication

[…] Genomic DNA was extracted from samples using the standard phenol-chloroform method []. A total segment of the mtDNA control region was amplified using a polymerase chain reaction (PCR) with primers PvsF (5’-GTACTCATACCCATTGCCAGC-3’) and PvsR (5’-GCGCGGAGGCTTGCATGTAT-3’) designed for this study. PCRs were conducted in a 25 μl reaction volume containing 1.0 μl of DNA template, 2.5 μl 10X buffer, 2.0 μl dNTP (0.2 mM), 0.1 μl taq polymerase (5U/ μl), 1.25 μl (1 mM) of each primer, and 16.9 μl Mili-Q water. MtDNA amplification consisted of an initial denaturation step for 5 min at 94°C, 30 cycles of 94°C for 1 min, 63°C for 1 min, 72°C for 1 min 30 s, and a final extension at 72°C for 5 min. PCR products were checked on agarose gel by electrophoresis and sequenced using a BigDye terminator cycle sequencing kit v3.1 (Applied Biosystems). The same forward primer and an additional reverse primer PvsFR (5’-GTAACGTAACTATGTCCCGC-3’) was used for DNA sequencing, and sequences were read in both directions. Sequence editing and running CLUSTALW for alignment were implemented in MEGA version 6 [].A sequence of 454 base pairs (bp) was used for analysis to examine the phylogeny of Japanese harbour seals. Only the data of Stanley et al. [] (GenBank accession numbers U36342–U36371) was included for the analysis since they have the longest sequence deposited in the GenBank database. [...] For the phylogenetic tree, the most appropriate model of substitution was determined using the Baysian Information Criterion (BIC) in MEGA6 [], and the K2+G+I model was used for the maximum-likelihood (ML) tree. A tree based on the neighbour-joining (NJ) method using same substitution model (K2+G+I) was also created in MEGA6 to validate the phylogenetic tree [].To visualise patterns of geographical distribution and haplotype relationships, the median-joining network (MJ Network) was generated using Network [] with default parameters (epsilon = 0, weight = 10).The results for the phylogenetic tree and network were combined to examine groupings of Japanese haplotypes. The proportions of haplotypes belonging to the different groups were then compared between the four districts to investigate trends.Mismatch distribution analysis, which compares the distribution of the observed numbers of pairwise differences among all haplotypes in a sample, was also conducted using Arlequin version [] to investigate past demographic fluctuations. The goodness of fit between the expected and observed values was tested using the sum of squared deviation (SSD) and Harpending’s raggedness index (Hrag). […]

Pipeline specifications

Software tools Clustal W, MEGA, Arlequin
Applications Phylogenetics, Population genetic analysis
Organisms Phoca vitulina