Computational protocol: Phylogenetic and Morphologic Analyses of a Coastal Fish Reveals a Marine Biogeographic Break of Terrestrial Origin in the Southern Caribbean

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[…] Taxonomic sampling within the genus Cathorops was designed following the phylogenetic hypotheses of Betancur-R. et al. and Betancur-R . In addition to the C. mapale group (C. mapale and Cathorops sp.), the ingroup included the closely related C. fuerthii group (C. fuerthii, C. aff. fuerthii, and C. manglarensis; from the Eastern Pacific) and C. cf. higuchii (from Nicaraguan Caribbean). We used C. spixii, C. agassizii, and C. hypophthalmus as outgroups. Sample size within the C. mapale group consisted of 17 individuals from each lineage collected at 10 locations along its distributional range, with a focus on neighboring localities from either side of the Parque Nacional Natural Tayrona (PNNT) in Santa Marta, Colombia (; ), which represents the distributional breakpoint between the lineages (see below). This sample size represents individuals collected during multiple field trips to Venezuela and Colombia from 2003 to 2008 by RBR and AAP. Institutional abbreviations are as listed at ASIH website (2010), with the addition of stri-x: tissue collection, Smithsonian Tropical Research Institute. SL is standard length. Two letter country codes follow ISO-3166.Targeted mitochondrial regions included partial cytochrome b (cyt b) and the complete ATP synthase subunits 8 and 6 (ATPase 8/6) protein-coding genes. Nucleic acid extractions, PCR conditions, utilized primers, and sequence alignment procedures are as described in Betancur-R. et al. . The software DnaSP v. 5 was used to estimate haplotype diversity as well as levels of sequence polymorphism. Corrected genetic distances were calculated in PAUP* v.4.0b10 . [...] Phylogenetic reconstructions were performed under maximum likelihood (ML), Bayesian inference (BI), and maximum parsimony (MP) criteria. For ML and BI, the number of model parameters was estimated using the Akaike information criterion (AIC) in ModelTest v. 3.7 . The ML analyses were performed in Garli v. 0.96 with ten runs from random-starting seeds to ensure convergence of likelihood scores. Model parameters were estimated simultaneously (i.e., unfixed) and remaining settings left at default values. The ML nodal support was assessed using the fast bootstrapping algorithm via automatic estimation of runs in RAxML as implemented in the CIPRES portal v.1.15 (2010) BI analyses were performed in MrBayes v.3.1.2 via Markov chain Monte Carlo (MCMC) iterations. The MCMC searches were conducted in triplicate using four chains. Each search was run for 4.0×106 generations, with tree sampling every 100 generations. Ten percent of the initial trees sampled in each MCMC run were discarded as burn-in. To confirm that post-burn-in trees represent the actual MCMC posterior distribution, marginal parameters (i.e., the MrBayes log file) were analyzed using the Effective Sample Size (ESS) statistic in the program Tracer . ESS values greater than 200 were obtained for all parameters, suggesting that the MCMC searches were run for a sufficient duration to accurately represent the posterior distribution . The post-burn-in samples of the three independent runs were combined in order to estimate marginal probabilities of summary parameters, consensus phylograms, and posterior probabilities of nodes. The MP reconstructions were conducted in PAUP* via heuristic searches with random addition of sequences (10000 replicates) and the tree-bisection-reconnection algorithm. [...] Molecular clock analyses were performed to infer the divergence time for the Cathorops sp./C. mapale stem node (: †2). Two different methods were conducted to assess rate heterogeneity among sequences: relative rate tests (RRT) based on likelihood, as implemented in the software r8s v.1.71 , , and likelihood ratio tests (LRT) as implemented in PAUP*. Both tests failed to reject the null hypothesis of clock-like behavior (see ); thus, divergence times were estimated under the assumption of a molecular clock via the likelihood-based Langley-Fitch (LF) method in r8s . For clock calibration, the final rise of the Panama isthmus (3.1–2.8 mya ) was utilized as the hypothetical vicariant event leading to the divergence of the Cathorops mapale (Southern Caribbean) and C. fuerthii (Eastern Pacific; node †1, ) groups from a common ancestor. The mitochondrial distances calculated from protein-coding sequences between the two groups (2.2–2.8% ) are similar to those reported for other transisthmian fish pairs assumed to have diverged during the final rise of the isthmus , . Both maximum and minimum age constraints (3.1 and 2.8 my, respectively) were applied to node †1 (see also ). […]

Pipeline specifications

Software tools DnaSP, ModelTest-NG, GARLI, RAxML, CIPRES Science Gateway, MrBayes, r8s
Application Phylogenetics
Organisms Danio rerio, Drosophila melanogaster