Computational protocol: Using Different Methods to Access the Difficult Task of Delimiting Species in a Complex Neotropical Hyperdiverse Group

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[…] Total genomic DNA was isolated from fins or muscle tissues of each specimen with a DNeasy Tissue Kit (Qiagen), according to the manufacturer’s instructions. Amplifications were performed in a total volume of 12.5 μl, with 1.25 μl of 10X buffer (10 mM Tris-HCl+15 mM MgCl2), 0.5 μl dNTPs (200 nM of each), 0.5 μl each 5 mM primer (FishF1, FishR1 or FishF2, FishR2 described in [], 0.25 U Platinum Taq Polymerase (Invitrogen), 1 μl template DNA (12 ng), and 8.7 μl ddH2O. The PCR reactions consisted of 30–40 cycles for 30 s at 95°C, 15–30 s at 48–54°C (according to each species), and 45 s at 72°C. All PCR products were first visually identified on a 1% agarose gel and then purified using ExoSap-IT (USB Corporation) following the instructions of the manufacturer. The purified PCR products were sequenced using a Big Dye Terminator v 3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems), purified again by ethanol precipitation and loaded onto an automatic sequencer 3130-Genetic Analyzer (Applied Biosystems). [...] Consensus sequences from forward and reverse strands were obtained using Geneious Pro 5.4.2 []. To avoid analyzing sequences of nuclear mitochondrial pseudogenes (numts) we followedthe recommendations of Song et al. [] and only the sequences that have gone through all quality steps were used to the analysis on present study. Alignments were generated using Muscle [] under default parameters. After alignments, the matrix was checked by eye for any obvious misalignments and to detect potential cases of sequencing errors, and the presence of stop codons was checked using Geneious.Nucleotide variation, substitution patterns and genetic distances were examined using the BOLD system tools. To evaluate the occurrence of substitution saturation, we estimated the Iss index in DAMBE 5.2.31 [], as described by Xia et al. [] and Xia and Lemey [], and the rate of transitions/transversions was also evaluated with the software DAMBE 5.2.31. The best nucleotide evolution models for the COI gene were evaluated using Modeltest 3.06 [] under the information-theoretical measure of Akaike Information Criterion (AICc). [...] The lognormal relaxed molecular clock tree was estimated using BEAST v.1.6.2 [] because the GMYC requires an ultrametric tree. The nucleotide evolutionary model used to estimate the ultrametric tree was the GTR model with a Gamma distribution (estimated by the program Modeltest 3.06). Briefly, we used Bayesian inference of phylogeny with a relaxed lognormal clock and birth-death speciation process rate on an arbitrary timescale. A random tree was used as a starting tree for the Markov chain Monte Carlo searches. Eight chains were run simultaneously for 10,000,000 generations, and a tree was sampled every 100th generation. The above analysis was performed twice. The distribution of log-likelihood scores was examined to determine the stationary phase for each search and to decide whether extra runs were required to achieve convergence using the program Tracer 1.6 []. All sampled topologies beneath the asymptote (2,500,000 generations) were discarded as part of a burn-in procedure, and the remaining trees were used to construct a 50% majority-rule consensus tree in TreeAnnotator v1.6.2.We performed the GMYC analysis with R 3.0.0 [] with a single threshold method with the Species Limits by Threshold Statistics (“splits”) package ( on standard parameters. […]

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