Computational protocol: The Complete Mitochondrial Genome of Coptotermes ‘suzhouensis’ (syn. Coptotermes formosanus) (Isoptera: Rhinotermitidae) and Molecular Phylogeny Analysis

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[…] Whole genomic DNA was extracted from the heads of 20 soldiers using One-tube General Sample DNAup Kit (Sangon Biotech, Shanghai, China) for polymerase chain reaction (PCR), following manufacturer’s instructions. Mitochondrial genome sequences of the termites related to C. suzhouensis were downloaded from the NCBI database. ClustalX ver1.8 was used for alignment, and Premier Primer 5 software was applied to design five pairs of primers from conserved regions (). Fragments were amplified by long distance PCR using Takara LA Taq (Takara Bio, Japan) with the following cycling conditions: an initial denaturation for 3 min at 94°C, followed by 35 cycles of denaturation at 94°C (30 s), annealing at 52−58°C (30 s; ), elongation at 72°C (10 min); and a final extension period 72°C (10 min). PCR products were checked using 1% agarose gels and were sequenced at MAP Biotech Company (Shanghai, China) with ABI3730 Genetic Analyzer. [...] Sequences were assembled and aligned with complete mitogenomes from C. formosanus in Sequencher 4.1.4. The location, size, and coding direction of each gene, including 13 protein coding genes (PCGs), 22 tRNA genes, and two rRNA genes were determined with DOGMA (). Secondary structures of tRNA were predicted using MITOS () and tRNAscan-SE Search Server v.2.0 online (). Amino acid composition and coding region for each PCG was identified with ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Nucleotide composition statistics (excluding stop codons) and relative synonymous codon usage (RSCU) of 13 PCGs were calculated with MEGA 5.1 (). Composition skew analysis was calculated according to formulas AT skew = [A−T]/[A+T] and GC skew = [G−C]/[G+C], respectively (). [...] Along with the C. suzhouensis mitochondrial genome, 41 termite mitogenomes and two outgroup species (Shelfordella lateralis and Periplaneta australasiae: Blattodea) were used in phylogenetic analysis. Sampling and sequence availability used in this study are summarized in . Concatenated amino acid sequences from the 13 PCGs was used in phylogenetic analysis, with maximum likelihood (RAxML7.03; ), maximum parsimony (PAUP 4.0b10; ), and Bayesian inference methods (Mr Bayes v.3.1.2) (). Modeltest ver3.06 () was used to infer best-fitting model for Bayesian inference (BI) and maximum likelihood (ML) analysis. The GTR + I + G model was selected based on the Akaike information criterion (). Bayesian inference was performed with the following settings: four MCMC chains (one cold chain and three hot chains) for 10,000,000 generations until the average standard deviation of split frequencies reached a value less than 0.01. Bayesian posterior probabilities (BPP) were calculated from the sample points after the MCMC algorithm had started to converge. In ML and MP analyses, a heuristic search with 100 random addition replicates was applied. BPP values were mapped onto the tree, and nodal support for ML&MP was assessed using nonparametric bootstrapping () in PAUP for the MP analyses (MPBP) and RAxML () for ML (MLBP) using 1,000 pseudoreplicates each. […]

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