Computational protocol: Integrative Biology of Idas iwaotakii (Habe, 1958), a ‘Model Species’ Associated with Sunken Organic Substrates

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Protocol publication

[…] Sequences were edited using Sequencher 4.1.4 and aligned using the Clustal W module implemented in Mega 4.0 . Sequences were deposited both in BOLD and GenBank (COI: KC861674-KC861725; 28S: KC861726-KC861749 and KC904226-KC904234).For the COI dataset, for each locality, the number of haplotypes (Hap), the number of segregating sites (Seg), haplotype (Hd) and nucleotide (Pi) diversity were determined using Arlequin . Phylogenetic relationships among haplotypes were constructed using the Median-Joining algorithm and maximum parsimony post-processing calculation implemented in Network ( was estimated among sampling locations using the formula of Weir and Cockerham (1984) to assess population genetic structure. These calculations were based both on haplotype frequencies and on their pairwise nucleotide differences. A distribution of FST values under the null hypothesis of spatial genetic homogeneity was drawn from 10,000 datasets simulated by permutations of specimens between south-western localities. Genetic structure was also analysed with an exact test of differentiation using a Markov chain process with a burnin of 1000 steps. An Analysis of Molecular Variance (AMOVA; ) was performed among populations of the south-western part of the sampling region (Papua New Guinea, the Solomon Islands, Vanuatu and New Caledonia). An AMOVA was also assessed in order to evaluate hypothesised patterns of bathymetric genetic structure by using two depth categories (sampling600 m). All tests were performed in Arlequin .Tajima’s D and Fu’s FS statistics were calculated to trace demographic events. Their statistical significance at alpha = 5% was estimated with a permutation test (10,000 replicates). Tests were performed for south-western localities, and for groups of localities among which no structure was detected. The parameters of a demographic expansion model, including the expansion factor (τ), the mutation parameter (θ = 2 µN, where µ is the mutation rate and N is the effective population size) initial (θ0) and actual (θ1) of the south-western populations were estimated using Arlequin. These estimators were used to plot the distribution of the number of pairwise differences between mitochondrial sequences (‘mismatch distributions’) based on Slatkin and Hudson (1991) for south-western Pacific populations. Under the coalescent model modified from Hudson (1990) 1,000 replicates were carried out to calculate parameters, to define 99% confidence intervals, and to calculate the sum of squared deviations (SSD) of these parameters between the observed and estimated mismatch distribution. [...] The sequences obtained for the bacterial 16S fragment were aligned using the Clustal W module implemented by Mega 4.0 . Sequences with ≥99% identity were considered as a single phylotype and a single representative clone from each phylotype was chosen for phylogenetic analysis. Sequences were deposited on GenBank (KC904235-KC904238). Sequences were compared with the NCBI ( database using BLAST . The most similar BLAST sequences were included in phylogenetic analysis. Phylogenetic reconstructions were performed using maximum likelihood (ML) and Bayesian (BA) analyses.The most appropriate evolutionary model was estimated using jModelTest 0.1.1. The GTR+I+Γ model was chosen based on AIC. The ML analysis was performed using the software Treefinder and node support was assessed with 1,000 bootstrap replicates . BA was performed using MrBayes v3.1.2 . Two independent analyses were run in parallel. Each analysis comprised four Markov chains and each chain was run over 8 million generations with a sampling frequency of one tree every hundred generations and a burnin period of 40,000 generations. Convergence between the two analyses was assessed using likelihood curves, standard deviation of split frequencies and potential scale reduction factor . […]

Pipeline specifications

Software tools Sequencher, Clustal W, MEGA, Arlequin, jModelTest, MrBayes
Applications Phylogenetics, Population genetic analysis
Organisms Bacteria, Drosophila melanogaster, Escherichia coli