Wang et al. BMC Genomics 2012, 13:529 http://www.biomedcentral.com/1471-2164/13/529 Table 5 Sequence divergence between MEAM1 and Asia II 3 % Differences % CpG % GC Loci mean SE Compared kb ts/tve 5' UTRsa All No CpG CpG CDSb All No CpG CpG nd sitesc All No CpG CpG 4d sitesd All No CpG CpG 3' UTRs All No CpG CpG 7.33 37.36 210 7.13 43.44 2966 6.27 44.17 2966 12.07 37.64 2966 4.68 35.74 337 3.35 0.21 3.05 0.21 8.28 1.03 1.73 0.02 1.29 0.02 7.77 0.12 0.47 0.02 0.42 0.02 1.27 0.07 5.30 0.07 3.43 0.06 20.11 0.36 2.91 0.16 2.52 0.15 12.42 0.97 22.11 20.49 1.62 1434.41 1332.11 102.30 840.49 787.78 52.71 200.59 176.37 24.22 46.95 44.75 2.20 1.47 1.34 2.38 2.94 2.53 4.17 1.43 1.29 2.41 2.18 1.55 3.52 2.01 1.68 4.48 aUTRs: untranslated regions. bCDS: coding sequence. cnd sites: non-degenerative sites. d4d sites: fourfold-degenerate sites where no changes cause any amino acid replacement. ets/tv: ration of transitions (ts) over transversions (tv). divergence at 4d sites (5.3%) is 11.3 times of that at the nd sites. This is due to the fact that any nucleotide sub- stitutions at an nd site will produce an amino acid change; therefore nd sites evolve under extensive func- tional constraints. To understand the mechanism of gene evolution in the B. tabaci complex, we also com- pared the ratio of transition (ts) and transversion (tv) of the three species [35]. Overall, the transitional differ- ences are more frequent than transversional differences in 5’UTRs (1.47), CDS (2.94) and 3’UTRs (2.01) (Table 5). Interestingly, for all regions, the transition-transversion ratio is higher in the CpG positions than the non-CpG positions (Table 5). This is consistent with the sugges- tion that, in the CpG sites, the predominant type of mutations is cytosine deamination, which results in tran- sitional differences [36]. The sequence divergence between Asia II 3 and MED For Asia II 3 and MED, the average differences between the 5’UTRs, CDS and the 3’UTRs are 3.57%, 1.84% and 3.13%, respectively (Additional file 6). The sequence divergences are almost the same between Asia II 3 and Page 7 of 13 MEAM1 and nearly twice of the divergence between MEAM1 and MED which is 1.66%, 0.83% and 1.43% (Figure 5) [30]. Likewise, in the 4d and nd regions, the same phenomenon was observed (Figure 5 and Add- itional file 6). It confirmed the previous analysis that the phylogenetic distance between MEAM1 and MED is shorter than that of the invasive whiteflies to the native Asia II 3 (Figure 4) [2,16]. These results indicate that despite high-sequence identity in their coding sequences, the Asia II 3, MEAM1 and MED B. tabaci species have diverged substantially between their transcriptomes. The levels of sequence divergence provide further supports to the previous proposition that Asia II 3, MEAM1 and MED are different species [25,26,37]. Analysis of synonymous and non-synonymous sites The nonsynonymous (Ka) and synonymous (Ks) substi- tution rates have been widely used to measure the inten- sity of gene evolution. To identify genes undergoing purifying or positive selections, we estimated Ka and Ks rates of orthologous gene pairs. Among the 2,966 pairs of CDS between Asia II 3 and MEAM1, both a Ka and a Ks rate could be calculated for 1,373 orthologs (Add- itional file 7). The 1,373 sequence pairs had mean values of Ka, Ks and Ka/Ks of 0.0094, 0.0623 and 0.198. These ratios are similar to the average Ka/Ks ratio of MEAM1 & MED (0.225), Ka/Ks ratio of rat & mouse (0.19) and human & chimpanzee (0.22) [30,38,39]. The distribution of the Ka/Ks ratio showed that the majority of genes (98.2%, 1348/1373) have Ka/Ks ratios less than 1, indi- cating the strong purifying selection for these genes. In addition, Fisher's exact test reports that nearly 56% of the genes are statistically significant (P<0.01) (Additional MEAM1/MED Asia II 3/MEAM1 Asia II 3/MED 6 5 s4 e c rne3 e f f id2 % 1 0 5' UTRs CDS nd sites 4d sites 3' UTRs Figure 5 Summary of the sequence divergence between Asia II 3 & MEAM1, Asia II 3 & MED and MEAM1 & MED. Average divergences of the orthologous gene pairs are given for the 5’UTR, CDS, fourfold degenerate sites (4d), nondegenerate sites (nd) and 3’UTRs, respectively. The divergence between Asia II 3 & MEAM1 and Asia II3 & MED are nearly twice of that between MEAM1 & MED. CDS: coding sequence; UTRs: untranslated regions.