Computational protocol: Mechanisms of peripheral phylogeographic divergence in the indo-Pacific: lessons from the spiny lobster Panulirus homarus

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Protocol publication

[…] Aligned and edited 618 bp sequence fragments of mtCR (using Geneious vR9 []), were used for further analysis. A median-joining haplotype network [] was constructed using mtCR and COI sequences in Network with star contraction []. After preliminary analysis, samples from adjacent locations with small sample size that were not significantly different genetically, and did not cross proposed biogeographic barriers, were pooled together for further regional analysis, but also compared with analyses from unpooled samples (Table ). Gene (haplotype) and nucleotide (л) diversities, Tajima’s D [] as well as Fu’s F [] tests and analysis of molecular variance (AMOVA) were calculated using Arlequin version 3.5 []. The most likely clusters of genetically similar individuals were estimated from mtCR data using a Bayesian approach in the program Baps v.6 with varying numbers of populations (k) [].Microsatellite alleles were sized in Geneious using the microsatellite plugin version 1.4.0 []. Of the 14 loci initially genotyped, 9 were retained for further population analysis following quality control analysis, taking regard of reliability, interpretability, and fit of genotype distributions to null expectations. Measures of variability for each locus analysed are reported in Additional file : Table A1.2. Analysis of Molecular Variance (AMOVA), and pairwise FST comparisons between populations and regions were carried out with Arlequin 3.5 [], Genepop [] and GenAlEx []. Principal coordinate analyses (PCoA) of pairwise genetic distances were undertaken in GenAlEx to display the genetic relationships among sampled populations. Genalex was used to estimate allele frequency, effective number of alleles, observed and expected heterozygosity and fixation index. Allelic richness and gene diversity was analysed in Fstat 2.9.3 []. The number of private alleles in each region were adjusted for sample size with a rarefaction approach implemented in Adze []. Spatial genetic discontinuities were determined using the Bayesian clustering algorithm Structure [] either using or excluding a priori locations and groupings (see Additional file for detailed methods). The Benjamini and Hochberg [] method was used to adjust the family-wide false discovery rate in microsatellite and mtCR sequence data. Additional analysis parameters are provided in Additional file .The ratio of male to female gene flow (mm/mf) between the two lineages was estimated from the differences between mtDNA ФST and nDNA FST, assuming neutral divergence ([] eq. 7c).The time to most recent common ancestor (TMRCA), was calculated from the COI data using the Bayesian MCMC approach implemented in Beast 2.4.2 []. To enable as direct comparisons as possible with divergence dates calculated in Iacchei et al., [], the same COI calculation parameters were used, including the 1.39% per lineage divergence rate in a strict clock model. Unfortunately, there are no confirmed divergence times of closely related species on which to base a confident prior. Thus, although there may remain some imprecision in the dates calculated, we can be confident that they are directly comparable with the estimated dates presented in the related studies we discuss. Specific parameters and further details are again provided in Additional file .Gene flow between the P. h. rubellus and P. h. homarus lineages were estimated in the isolation-with-migration model using IMa2 [] on the mitochondrial and microsatellite data. Estimates were made independently from the mtDNA and nDNA data sets, as they differed substantially, using search parameters detailed in Additional file . […]

Pipeline specifications

Software tools Geneious, Arlequin, BAPS, Genepop, GenAlEx, BEAST
Applications Phylogenetics, Population genetic analysis
Organisms Panulirus homarus