Posterior probability (PP) values were subsequently calculated. Stabilization of model parameters (burn-in) occurred around 2 400 000 and 800 000 generations for 16S rRNA and surface-encoding genes, respectively. Every 100th tree after stabilization (burn-in) INCB024360 was sampled to calculate a 50% majority-rule

consensus tree. All trees were constructed using the program figtree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). dnasp (Librado & Rozas, 2009) was used to calculate synonymous (dS) and nonsynonymous (dN) rates and two common measures of nucleotide variation, π and θW, for determining ompA intraspecies variation within Glossina. Neutrality tests were also performed in dnasp. The McDonald–Krietman test and neutrality index

(NI) were calculated by comparing the ratio of dS to dN mutations within either individual Glossina species for ompA, or among Glossina isolates for ompC, and an E. coli outgroup. The outgroup was composed of ecologically diverse E. coli representatives NC_000913, Osimertinib clinical trial NC_008253, and NC_002655. These adaptive evolution tests have been shown to be most powerful when taxa are closely related (Clark et al., 2003). We chose E. coli as our representative outgroup because it is a close relative of Sodalis, and has a wide representation of publicly available genome strains. The

nucleotide sequences determined in this study have been deposited in the NCBI GenBank database under accession numbers HM626140–HM626149 and HQ914651-HQ914697. To examine the evolutionary relationships of the newly identified Sodalis-like symbionts, we constructed phylogenetic trees based on 16S rRNA gene sequences. Bayesian analysis supports the monophyly of Gammaproteobacteria symbionts Adenosine isolated from diverse insect orders (i.e. Diptera, Coleoptera, Hemiptera, and Phthiraptera) (Fig. 1). In general, there is a tight clustering of symbionts with respective insect host Order. Our Bayesian analysis also suggests the closer relationship of hippoboscid symbionts to weevil and pigeon louse symbionts, rather than to Sodalis, despite a common ancestry of their respective hosts within the Hippoboscoidea (Petersen et al., 2007), thus further substantiating a previous hypothesis of independent symbiont acquisition events by these hosts (Novakova & Hyspa, 2007). However, there is only moderate Bayesian support for this relationship (PP=77, data not shown) that is further decreased (PP=51) when symbionts of the recently reported chestnut weevil Curculio sikkimensis (Toju et al., 2010) and the stinkbug Cantao ocellatus (Kaiwa et al., 2010) are included in the analyses.