Computational protocol: Two New Species of Liuixalus (Rhacophoridae, Anura): Evidence from Morphological and Molecular Analyses

Similar protocols

Protocol publication

[…] Taxonomic sampling included 34 sequences. For our sequencing, genomic DNA was extracted from either muscle, or live tissues using the standard phenol-chloroform extract protocol []. Primer sequences of Wilkinson et al. [–] were used to amplify and sequence a fragment that included 1467 aligned nucleotide positions encompassing from 12S to 16S ribosome RNA (rRNA) of the mtDNA genome. Double-stranded polymerase chain reaction (PCR) amplification was carried out using the following parameter: 94°C initial hot start (3 min), then 30 cycles of 94°C denaturation (1 min), 52°C annealing (1 min), and 72°C extension (2 min). Final extension at 72°C was conducted 10 min. PCR products were directly sequenced with an ABI 3730 automated DNA sequencer and in both directions. The resulting sequences were submitted to Blast searching [] in GenBank to ensure the required sequence had been sequenced. The outgroup taxa (and GenBank Accession Nos.) included Buergeria japonica (DQ283055), Kurixalus hainanus (DQ283054), Rhacophorus moltrechti (DQ283080), Polypedates megacephalus (DQ283073), Chiromantics hansenae (AB813161), Buergeria oxycephala (AB813156), Rhacophorus rhodopus (AB813151), Philautus jinxiuensis (EU215525), and Buergeria buergeri (AB127977).Alignments first used CLUSTALX 1.81 [–] with default parameter followed by visual confirmation and manual adjustments. Nucleotide sites with ambiguous alignments were removed from analyses. Gaps were analyzed as missing data. Our de novo were trimmed to 16s rRNA only to match data downloaded from NCBI. The aligned sequences were analyzed using Bayesian inference (BI) in MrBayes 3.12 []. Markov Chain Monte Carlo (MCMC) generations used three million iterations and we sampled every 1000th step. The first 25% of the samples were discarded as conservative burn-in. The remaining samples were used to generate a majority-rule consensus tree (). […]

Pipeline specifications

Software tools MUSCLE, Clustal W, MrBayes
Application Nucleotide sequence alignment
Organisms Labiobarbus ocellatus, Lepus hainanus