Computational protocol: Comparative cytogenetic analysis of some species of the Dendropsophus microcephalus group (Anura, Hylidae) in the light of phylogenetic inferences

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[…] Genomic DNA was extracted from liver or muscle tissue stored at −70°C in the tissue bank of the Department of Structural and Functional Biology-UNICAMP, Campinas, SP, Brazil, using the TNES method. Tissue samples were immersed in TNES buffer solution (50 mM Tris pH 7.5, 400 mM NaCl, 20 mM EDTA, 0.5% SDS). The solution was subsequently supplemented with proteinase K (to a final concentration of 100 μg/mL), and the samples were incubated for 5 hours at 55°C. Then, 1/3 volume of NaCl 5M was added, and the samples were centrifuged. DNA was precipitated from the supernatant with isopropyl alcohol, washed with ethanol (70%), resuspended in TE (10 mM Tris–HCl, 1 mM EDTA pH 8.0) and stored at −20°C.The mitochondrial 12S ribosomal gene was partially amplified using the primers MVZ 59(L) and MVZ 50(H) []. The PCR-amplified products were purified with the GFX PCR and Gel Band DNA Purification Kits (GE Healthcare, England) and directly used as templates for sequencing in an automatic ABI/Prism DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator Kit (Applied Biosystems, Foster City, CA, USA), as recommended by the manufacturer. DNA sequences were bi-directionally sequenced and edited using Bioedit version 7.0.1 ( [...] Fragments of approximately 810 bps of the 12S ribosomal genes from 80 specimens of Dendropsophus were sequenced as described above, and a data matrix consisting of 133 OTUs, including five sequences from outgroup species and a total of 37 species of Dendropsophus, was constructed. The GenBank accession numbers for all of the sequences used are presented in Table . Because parsimony [] and likelihood [,] criteria have been employed for the phylogenetic studies of Dendropsophus, we conducted both types of analyses. When using parsimony criterion, phylogenetic relationships were inferred (i) from analyses under dynamic homology, as implemented in the software POY v. [], or (ii) from aligned sequences using the software TNT v.1.1 []. A Bayesian analysis was implemented in the software MrBayes v.3.1.2 [] using the model GTR + I + G, inferred with the software MrModeltest v.2.3 []. For the analyses using TNT and MrBayes, the sequences were first aligned with Clustal W [], and a matrix was generated with 852 characters.The phylogenetic searches performed with POY included tree building (of Wagner trees), tree bisection–reconnection (TBR) swapping, perturbation using a parsimony ratchet and tree fusing. The analyses were run with a maximum execution time of 48 h and an opening indel cost of 3, indel extension cost of 1 and nucleotide substitution cost of 1 using the command “transform (tcm:(1, 1), gap_opening:2)”. To obtain an implied alignment from the POY analysis, the characters were transformed into static characters, and the generated matrix was exported using the command “phastwinclad.” The exported matrix was loaded with TNT v.1.1 to calculate the bootstrap support based on 1,000 pseudoreplicates.For the phylogenetic analysis using TNT software, the most parsimonious trees were inferred through heuristic searches performed using the command xmult, which combined sectorial searches, the ratchet, tree drifting and tree fusing. Gaps were considered to be missing data. The bootstrap values of the branches inferred in this analysis were calculated with 1000 pseudoreplicates.For the Bayesian inferences, two simultaneous analyses were run, each with four chains (three heated and one cold). In each analysis, 2,980,000 generations were run and one tree was sampled every 100 generations. A consensus topology and the posterior probability for each node were produced after discarding the first 25% of the trees generated. The ASDSF (Average Standard Deviation of Split Frequencies) value was below 0.01, and the PSRF (Potential Scale Reduction Factor) values were approximately 1.000. […]

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