Computational protocol: Phylogeography of the Sponge Suberites diversicolor in Indonesia: Insights into the Evolution of Marine Lake Populations

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Protocol publication

[…] The poriferan origin of the obtained sequences was verified through BLAST searches against Genbank (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were handled in SEQUENCHER 4.10.1 (Gene Codes Corporation) and aligned with CLUSTALW and MUSCLE as implemented in DAMBE and SEAview v 4.3.0 . Alignment was conducted under default settings and optimized by eye. Alignments were collapsed to contain only unique sequence types in DAMBE. Haplo-/genotypes and nucleotide diversity as well as Tajima's D neutrality test were calculated per population with Arlequin v. 3.11 .Phylogeographic analyses were carried out for COI and ITS. We used ITS outgroup sequences obtained from Genbank from the family Halichondriidae (), as the available sequences for ITS of other Suberitidae were more distant than those from Halichondriidae. Several studies have shown that the families Suberitidae and Halichondriidae are sister groups , , . To be consistent we also used species of the family Halichondriidae for the outgroup of the COI phylogram. The relatively best-fit DNA substitution model was selected by the Akaike Information Criterion deployed in jMODELTEST v. 0.1.1 and this model (COI: HKY and ITS: GTR+G+I) was used for subsequent Bayesian and maximum likelihood phylogeny inferences. Phylogenetic reconstructions were performed under Bayesian inference criteria implemented in MrBayes v. 3.1.2. . Each analysis consisted of two independent runs of four Metropolis-coupled Markov-chains, sampled at every 1,000th generation at the default temperature (0.2). Analyses were terminated after the chains converged significantly as indicated by an average standard deviation of split frequencies <0.001. Convergence was also checked in Tracer v. 1.5.0 . For comparison, maximum likelihood bootstrap analyses were conducted using MEGA v. 5.01 using a heuristic search with 1,000 bootstrap replicates. The Bayesian and maximum likelihood phylograms were combined and visualized using TreeGraph 2 . Within group p-distance (uncorrected), as well as net nucleotide divergence between groups were calculated in MEGA. A Kruskall-Wallis test was performed to test whether color or substrate preference significantly differed between lineages. To test for spatial structuring of samples we performed an analysis of molecular variance (AMOVA) and calculated pairwise Φst values between separate populations using Arlequin 3.5.1.2 . Significance of pairwise Φst values (based on p-distances) was determined by 10,000 permutations and exact tests of population differentiation in Arlequin. […]

Pipeline specifications

Software tools Sequencher, Clustal W, DAMBE, SeaView, Arlequin, jModelTest, MrBayes, MEGA-V, TreeGraph, MEGA
Applications Phylogenetics, Population genetic analysis, GWAS
Organisms Hemisus marmoratus