Computational protocol: Incorporating deep and shallow components of genetic structure into the management of Alaskan red king crab

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Protocol publication

[…] The mitochondrial DNA cytochrome oxidase subunit I (COI) universal primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′; ) were used to amplify a 665-bp fragment (‘barcode’ segment). PCR mixtures consisted of a 50-μL mixture of 2.0 μL template DNA in 1× Colorless GoTaq Flexi Buffer, 2.5 mm MgCl2, 1 mm dNTPs (ABI), 1 μm of forward and reverse primers, and 5 U GoTaq Flexi DNA polymerase (Promega Inc., Madison, WI, USA). PCR amplifications were conducted in ABI 9700 thermocyclers with an initial denaturation of 1 min at 95°C, 37 cycles of 40 s at 95°C, 40 s at primer annealing temperature 41°C, and 1 min at 72°C; the final cycle was at 4°C for 5 min. Cycles were made at a ramp speed of 1°C/s. The PCR amplifications were sequenced in the forward and reverse directions by the High-Throughput Genomics Unit (Seattle, WA, USA). Sequences were aligned with a published sequence for red king crab (GenBank accession no. AB211303) with the muscle algorithm (mega 5, ) and adjusted by eye. Polymorphic sites were confirmed with reverse sequences. GenBank accession nos. (JF738153–JF738249) also include sequences shared with the study of . [...] Single nucleotide polymorphism genotypes for the 15 loci were tested for fit of genotypic frequencies to Hardy–Weinberg expectations (HWE) in each sample with exact probability tests of 20 batches of 10 000 permutations and a burn-in of 10 000 permutations (genepop 4, ). Probabilities over loci per sample, over loci, and over samples were determined with Fisher’s method. Sample heterozygosities were estimated with genepop. Linkage disequilibrium between loci in each sample and overall was tested with genepop with 100 batches of 5000 iterations and with a burn-in of 10 000 iterations. Analysis of molecular variation (amova) in arlequin 3.11 () was used to explore geographic and temporal structures in both SNP allele frequencies and mtDNA haplotypes. These amovas were based on FST estimated from allele (SNPs) or haplotype (mtDNA) frequencies. Additional amovas of mtDNA haplotypes were based on an FST analog statistic (ΦST) that accounts for both haplotype-frequency differences between samples and sequence divergences between haplotypes. We used the substitution model in the amovas. The probability an F statistic was larger than 0.0 was estimated with 50 000 permutations of individuals among samples.Statistical power to detect significant differences with 15 SNP loci and 17 samples was estimated with powsim 4.0 (), using Ne = 2000 and 1000 replicate runs. The incorporation of divergences between haplotypes in tests of differentiation with amova and estimates of ΦST adds additional power to tests of geographic structure. Geographic groupings of samples were visualized with principal coordinates analysis (PCoA) computed with NTSYS (Exeter Software; Exeter, New York, NY, USA). Haplotype richness was estimated with HP-rare (, ) with a minimal sample size of 29 crabs. Mitochondrial DNA nucleotide diversity (Θπ) and haplotype diversity (h) and their standard deviations were estimated with arlequin. A 95% plausible parsimony network of mtDNA haplotypes in 1278 crabs was made with TCS 1.21 (). This network was used as a template to construct networks for the three regional groups identified with amova.Historical demographies were examined in two ways. First, we tested for population and spatial range expansions with mismatch distributions between haplotypes using arlequin. Both recent demographic and spatial expansions are expected to produce a unimodal mismatch distribution from the accumulation of mutations (). With time, the mode of the distribution increases and gives way to a multimodal mismatch distribution (). The time since the population expansion can be estimated with τ = 2ut, where τ is estimated from the distribution, u is the whole-sequence mutation rate and t is time since expansion. The use of mismatch distributions is discussed further in . Tajima’s DT was estimated from the distributions of haplotype frequencies in a sample and used to test for neutrality (). Significance was determined with 10 000 randomizations in arlequin. Population expansions can lead to excesses of low-frequency haplotypes before populations reach drift–mutation equilibria.Historical demographies of the three major groups were examined with coalescence theory and Bayesian skyline plots (BSPs) with beast 1.6.1 () with MCMC runs of up to 400 million steps that yielded effective sample sizes (ESS) of at least 200. Effective population size (Ne) can be estimated from the results with Neμg, where μ is the mutation rate and g is generation time (about 5 years for red king crab). However, estimates of μ are problematic, as no temporally anchored phylogenetic estimates are available for Paralithodes. Even so, phylogenetic estimates appear to greatly underestimate contemporary mutation rates and do not appear to be appropriate for BSP calibrations (). To help interpret the results of the BSPs, we simulated samples (n = 200) of sequences (bp = 665) with Mesquite 2.74 (). The scaling factor (roughly mutation rate) and base effective population size were varied until the simulated sequences had similar haplotype and nucleotide diversities (within ± SE) as those in the three major groups of red king crab. We used a demographic model based on Antarctic ice core paleoclimatic temperatures over the last four glacial cycles reaching to 450 kyr (). We reasoned that abundances of red king crabs were proportional to the amount of shallow-water habitat, which was related exponentially to a linear increment in warming following the LGM. BSPs were constructed from the sequences with beast and with appropriate substitution models, as determined with jModelTest (). This empirical approach to calibrating the molecular clock is similar to that described by Crandall et al. (2012), who used the post-Pleistocene expansion onto the Sunda Shelf to date a genetic signature of population growth in three invertebrates. […]

Pipeline specifications

Software tools MUSCLE, MEGA, Genepop, Arlequin, BEAST, Mesquite, jModelTest
Applications Phylogenetics, Population genetic analysis, Nucleotide sequence alignment
Organisms Paralithodes camtschaticus