The scale bar represents substitutions per site. NJ trees were constructed using mega 4.1 with 1000 bootstrap replications and Kimura 2-parameter as
a model selleck screening library of nucleotide substitution. All the termites from 14 populations representing two genera, and two families (seven Odontotermes horni, five Odontotermes spp. and two C. heimi colonies), screened initially for the presence of Wolbachia, using PCR assays with the wsp gene were found to be positive. At least one MLST gene was sequenced for all the 14 colonies (Table 1). Repeated failures to PCR amplify Wolbachia genes (MLST and 16S rRNA gene) resulted in incomplete profiles. Sequence typing was performed using the Wolbachia MLST database (http://pubmlst.org/Wolbachia/), which resulted in the assignment of full sequence type (ST) to four strains (Table 1). All of the alleles and allelic profiles characterized for the termite strains were new to the MLST database, except for B supergroup C. heimi Wolbachia (Table 1). Alleles were shared among some of the strains, showing their relatedness, but at the same time showing a distinct difference for other strains. The divergence among the strains accounted for 591 variable learn more sites (VI) out of 2079 sites (28.43%), with a concatenated alignment of all five Wolbachia MLST genes (Table 2). The gene hcpA showed the highest
nucleotide divergence with 154 variable sites out of 444 (34.38%), followed by fbpA with 128 variable sites out of 429 (29.83%) (Table 2). The average Ka/Ks per gene was found to be ≪1 (the average Ka/Ks across genes is 0.0933), which indicated strain evolution mainly by synonymous substitutions. This is compatible with a scenario of a strong purifying selection. Recombination
events were not indicated in either single gene or concatenated MLST dataset alignments (P<0.05) by MaxChi analyses. Phylogenetic reconstructions for all genes by both Bayesian inference and neighbor-joining methods showed similar results, and therefore only Bayesian phylogenetic trees were included. Phylogenetic reconstructions based on the concatenated alignment Bay 11-7085 of hcpA, gatB, coxA, ftsZ and fbpA genes indicated a strong clustering of termite Wolbachia from this study with Wolbachia from bed bug Cimex lectularis and form a sister clade within F supergroup with different species of scorpion genus Opistophthalmus (Fig. 1). Two major clusters were observed for termite Wolbachia in the present study (MCT, W5, G29 and RA) (T1, T3, T21 and THYD) (Fig. 1). On the basis of single-gene analyses, all the strains consistently belonged to the same supergroup. All Odontotermes spp. harbored F supergroup Wolbachia, with the exception of a population from O. horni (T2). Two C. heimi colonies harbored two different supergroup Wolbachia. One of these (TERMITE3) belonged to supergroup F, whereas the other (TLR) belonged to supergroup B (Table 1, Fig. 2).