1 (Applied Biosystems). Sequences were obtained using an automatic DNA sequencer (3730 DNA analyzer, ABI) and were
deposited small molecule library screening in the Wolbachia MLST and GenBank databases with alleles and accession numbers, respectively (Table 1). Samples from each of the 14 termite colonies were amplified using the MLST genes, in order to isolate at least two sequences from the same gene per colony in different individuals. Wolbachia gene sequences from all the colonies were identical within each nest, confirming that the descendants inherited the same Wolbachia strain from the infected queen. Estimates of genetic diversity (Pi), variable sites (VI) and the ratio of synonymous substitutions per synonymous site over nonsynonymous substitutions per nonsynonymous site (Ka/Ks) were performed using DNAsp version 4.10.2 (Rozas
& Rozas, 1999). Recombination analyses were carried out on single and concatenated gene alignments using the MaxChi method implemented in rdp3 program (Martin et al., 2005). Parameters were set as follows: triplets were scanned using different values of the fraction Alectinib of variable sites per window, a Bonferroni correction was applied and 1000 permutations were generated. The highest acceptable P value cut-off was set to 0.05. The Wolbachia gene sequences and termite 16S rRNA gene sequences generated in this study were aligned with homologous sequences deposited in Wolbachia MLST and GenBank database using clustalx (version 2.0.9) (Larkin et al., 2007). All sequences were manually edited using mega4 (Tamura et al., 2007). Unrooted phylogenetic trees were constructed using Bayesian inference and the neighbor-joining method for each dataset. For the Bayesian inference of phylogeny, the program mrbayes 3.1.2 (Huelsenbeck & Ronquist, 2001) was used. The analysis for each gene consisted of 3 000 000 generations
with sampling every 100 generations. The first 12 000 trees (40%) were discarded as burnin. Before carrying out the probabilistic phylogenetic analyses, appropriate models of sequence evolution for each dataset were chosen via the Akaike Information Criterion (AIC) using program mrmodeltest2.2 (Nylander, 2002). The selected model of nucleotide substitution was ‘GTR+G’ for all genes. The final alignments consisted of 435 bp for ftsZ, 2079 bp for concatenated MLST gene sequences, 803 bp for the Wolbachia 16S rRNA and 398 bp for insect 16S the rRNA gene fragments. Only the strains with at least four complete allele sequences out of five MLST alleles were selected to construct the phylogenetic tree for the concatenated dataset. The strains TO, TL, TER30, T2, TERMITE3 and TLR were therefore excluded from the concatenated analysis. Three independent runs were performed for each dataset. In phylogenetic trees, the levels of confidence for each node are shown in the form of Bayesian posterior probabilities (BPP). BPP below 0.50 are not shown. STs, allele number and accession number are shown after each species name in parenthesis.