Computational protocol: Molecular Phylogeny and Biogeography of the Amphidromous Fish Genus Dormitator Gill 1861 (Teleostei: Eleotridae)

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Protocol publication

[…] Whole genomic DNA was extracted from all of the tissue samples using a standard proteinase K and phenol-chloroform protocol [] or Qiagen DNeasy Tissue Kits (Qiagen, Inc., Valencia, CA, USA). The mitochondrial Cytochrome b gene (Cytb) was amplified by polymerase chain reaction (PCR) using the primers GluDG [] and H16460 []. Additionally, a subset of samples representative of the genetic variation found in the Cytb gene tree were sequenced using the primers RhF193 and RhR1039 [] and the primers BactFor and BactRev [] for the nuclear Rhodopsin (Rh) and Beta actin (β-actin) genes, respectively. PCRs were carried out in 25 μL volume reactions containing the following: 10X reaction buffer, 0.5 μM each primer, 0.2 mM dNTP, 2 mM MgCl2 and 1U of Taq DNA polymerase (Invitrogen). Thermocycling conditions consisted in an initial denaturation step at 94°C (2 min) followed by different time and temperature cycles depending on the gene: 35 cycles of denaturation at 94°C (45 s), annealing at 46°C (1 min) and extension at 72°C (90 s) for Cytb; 5 cycles of denaturation at 94°C (30 s), annealing at 50°C (45 s) and extension at 72°C (45 s), followed by 35 cycles of denaturation at 94°C (30 s), annealing at 54°C (45 s) and extension at 72°C (45 s) for Rh; 35 cycles of denaturation at 94°C (30 s), annealing at 55°C (40 s), extension at 72°C (90 s) for β-actin and a final extension at 72°C (5 min) in all cases. All gene fragments were sequenced in both directions using the same PCR primers. Sequencing was performed by MACROGEN Inc. (Korea) sequencing service, High-Throughput Genomics Unit sequencing service (USA) and the Smithsonian Tropical Research Institute sequencing facility (Panama). Chromatograms were visually examined and then edited and assembled using Bioedit 7.2.5 []. DNA sequences are available in the GenBank database under the following accession numbers: KU764787-KU765046 for Cytb, KU765047-KU765129 for Rh and KU958382-KU958464 for β-actin. [...] The evolutionary substitution models that best fit our data were determined for each gene using jModeltest2 [] and the Akaike information criterion (AIC, ). Once best-fit models were determined, they were used in all of the subsequent analyses. Phylogenetic hypotheses were independently inferred for each molecular marker, nuclear data set (Rh + β-actin) and the complete concatenated data set (Cytb + Rh + β-actin). Concatenated data sets only included individuals for which all genes were successfully sequenced. Maximum likelihood (ML) trees were generated using RAxMLBlackBox []. Genes were considered as individual partitions and treated independently with respect to evolutionary models and the optimization of branch lengths. Node support was assessed using 100 bootstrap ML replicates. Bayesian inference (BI) analyses were performed using MrBayes v.3.2.2 [] via the CIPRES portal []. Each gene was considered as a distinct partition with unlinked maximum likelihood models. Two simultaneous Markov chain Monte Carlo (MCMC) searches were completed with four chains for 1 x 107 generations, and trees were sampled every 1000 generations with the first 25% of the trees discarded as burn-in. Convergence between runs was assessed by monitoring the standard deviation of split frequencies with MrBayes v.3.2.2 and by using the effective sampling size (ESS) criterion in Tracer v.1.6 [].Mean uncorrected genetic p-distances (Dp) and their associated standard errors (S.E., 1000 bootstrap replicates) were calculated for the three genes independently and for the complete concatenated data set (Cytb + Rh + β-actin) within and between clades using MEGA v.6 []. [...] The time to the most recent common ancestor (TMRCA) and confidence intervals (95% highest posterior density: HPD) were estimated for each clade in the Cytb data set using a relaxed molecular clock with an uncorrelated lognormal distribution of rates in BEAST v.1.8.0 []. A Yule speciation model was assumed as a tree prior (i.e., a constant rate of speciation per lineage []). Two independent analyses were performed by running the MCMC for 5 x 107 generations, with trees sampled every 5000 generations. The runs were examined for convergence and adequate ESS using Tracer v.1.6 and combined using LogCombiner v.1.8.0 with a burn-in fraction of 10%. The final consensus tree was produced using TreeAnnotator v.1.8.0.To calibrate the molecular clock, we used previously published sequence and fossil record data of Gobiiformes (). Because the Yule speciation model assumes that each tip of the tree represents one species, we selected one sequence from each of the main lineages of Dormitator here generated and included 37 sequences from 32 genera of Gobiiformes and two genera of Kurtiformes () to infer a molecular phylogeny anchored by four calibration points derived from six fossil species of Gobiiformes, with mean ages ranging from 12.5 to 52 million years ago (Mya) (). To account for uncertainties in fossil dates or conflicts between fossils and molecules [], fossil data points were included as soft calibration points using lognormal prior distributions in the stem nodes of interest.Also, we estimated divergence times among Dormitator clades using the multispecies coalescent method *BEAST implemented in BEAST v.1.8.0. This method estimates a species tree while taking into account variation among gene trees []. We ran *BEAST using all sequences from all individuals and assigning them to five species, corresponding to the monophyletic clades obtained in the previous gene-tree phylogenetic hypothesis.We used a relaxed lognormal molecular clock and a Birth-Death speciation model for each gene tree. We calibrated the molecular clock, incorporating as normal prior the substitution rate of 5.49 x 10−2 substitutions/site/million years (S.D. = 2.94 x 10−2) estimated for the Cytb gene in the fossil calibrated phylogeny of Gobiiformes described above, and estimated the substitution rate of Rh and β-actin genes relative to Cytb. We performed two independent MCMC runs, each for 5 x 107 generations, sampling every 5000 iterations. Each run was checked for convergence and adequate ESS sampling in Tracer v.1.8, combined using LogCombiner v.1.8.0 and summarized with TreeAnnotator v.1.8.0. All BEAST v.1.8.0 analyses were run in the CIPRES portal. […]

Pipeline specifications

Software tools BioEdit, jModelTest, MrBayes, CIPRES Science Gateway, MEGA-V, BEAST
Applications Phylogenetics, WGS analysis, GWAS
Organisms Danio rerio