The same procedure was performed on the ATP synthase subunit alpha (AtpA) reference sequences that were collected for the species in the OMPLA protein list by searching the protein NCBI database (See Appendix 1 for the Protein IDs used). The consensus tree of AtpA and OMPLA sequences were generated from the 1000 PhyML bootstrap

trees using Phylip’s Consense package [54]. Results were visualized as circular trees using FigTree http://tree.bio.ed.ac.uk/software/figtree/. Detection of adaptive molecular evolution of pldA sequences To study evolutionary divergence among the pldA sequences, the mean numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site were estimated using the Nei and Gojobori method [63] in SWAAP [57]. The Ks value is the mean number of synonymous (silent) substitutions per site, while Ka represents the mean number of nonsynonymous substitutions mTOR inhibitor per site (a change of amino acid is observed). The MEGA5 [52] codon-based Z-test for purifying selection was used to estimate the probability of rejecting strict neutrality (null hypothesis where Ka equals Ks) in favor of the alternate hypothesis Ka < Ks. The PAML program [64] estimates the nonsynonymous/synonymous ratio, omega (ω), using maximum likelihood codon substitution

models. In this study, four different models (M1, M2, M7, and M8) were used to estimate ω as described by Yang et al.[65]. These models are nested KU55933 cost pairs in

which one (M1 and M7) does not allow for positive selection, while the other (M2 and M8) includes an additional parameter to detect positively selected sites. The neutral model M1 assumes two classes of proteins, highly conserved codons (ω = 0) and neutral codons (ω = 1), and is nested within the M2 model, which has a third category for positive selection (ω > 1). The two most advanced models, M7 and M8, use a discrete ß distribution; M8 has an extra class of codons that allows positive Tenofovir molecular weight detection (ω > 1) and simplifies to M7. The two pairs of nested models (M1 vs. M2 and M7 vs. M8) were compared using the likelihood ratio test (LRT) statistic, where 2ΔlnL equals 2*(lnL1 – lnL0). The CP-868596 clinical trial lnL1-value is the log-likelihood for the more advanced model and lnL0 is the log-likelihood for the simpler model. The 2ΔlnL value follows a χ2 distribution, where the degree of freedom is the difference in the number of parameters used in the two models. The identification of positive selected sites implemented in PAML uses Bayes empirical Bayes where the posterior probabilities of each codon was calculated from the site class of the M2 and M8 models; sampling errors have been accounted for through Bayesian prior [66, 67]. A pldA tree generated in PhyML using the K80 model (the best fit as determined in MEGA5) was used in the PAML analysis. PAML also calculated possible transition (ts) to transversion (tv) bias (κ = ts/tv).