Esteban and Hutchinson BMC Genomics 2011, 12:261 http://www.biomedcentral.com/1471-2164/12/261 by testing if the data fits a model (which does not allow for sites under diversifying selection) better than an alternative model, which does allow for sites under diversi- fying selection. Two model comparisons were applied: M1a vs. M2a, M7 vs. M8, where M1a and M7 are models and M2a and M8 are alternate models. A log like- lihood ratio test was then applied to determine whether allowing for a site class with ω>1 results in a significantly better fit of the model to the data. Evidence for diversifying selection was considered to be strong where both comparisons were found to be sig- nificant at p < 0.05. 25 genes were identified by both models as having an amino acid site class experiencing diversifying selection (null models rejected, p < 0.05) (Table 1). With model M8 (a less conservative model) an additional 54 genes were identified as having sites experiencing diversifying selection (null model rejected, p < 0.05), for a total of 79 genes under diversifying selection (Additional file 4). All significant genes under model M2a were also significant under model M8, except one (Unknown YMTV120.5L). Alternative models M2a and M8 allow for the exis- tence of a codon site class under diversifying selection. The proportion of sites that fall into this site class pro- vides insight into the selective pressure experienced by the gene. For each gene, the proportion of sites that are under diversifying selection was determined (Additional file 5). Among the genes for which the diversifying selection models showed a significantly better fit of the data, the proportion of sites under diversifying selection ranged from 0.1% to 14.6% (model M2a) and 0.1% to 29.5% (model M8). A gene does not necessarily need a large proportion of sites under diversifying selection to fit the alternative model better. In many genes, a large proportion of amino acids may be virtually invariable due to structural or functional constraints, while diversi- fying selection occurs at only a few key sites. For exam- ple, the F6L homolog (unknown function) has a small proportion of sites under diversifying selection (2.9%), while the mitochondrial associated apoptosis inhibitor has a large proportion of sites under diversifying selec- tion (14.0%) and for both the model was rejected. Using the CPXV-BR genome as a reference for gene positions within the genome, we noted a distinct pattern of genes with a high proportion of sites under diversify- ing selection being located closer to the termini of the genome, as well as a tendency for significant genes (model M2a) to be located in the terminal regions (Fig- ure 1). A centrally located core set of 90 genes is pre- sent in all chordopoxvirues [2]. A chi square test revealed that genes experiencing diversifying selection identified by model M2a were significantly more likely to be found among the 85 terminally located less con- served genes (c2 = 12.56, df = 1, p < 0.05). The 79 Page 3 of 12 genes identified by model M8 however were proportio- nately distributed between the core and less conserved genes (c2 = 0.05, df = 1, p > 0.05). The non-synonymous/synonymous rate ratio (ω = dN/ dS ) is an important indicator of selective pressure at the protein level. The dN/dS ratio of a gene is calculated as the average of the ω of each codon in the gene. A higher dN/dS ratio suggests a greater role for diversifying selec- tion in the molecular evolution of the gene. Again we note that genes with high dN/dS ratios are located in the termini of the genome (Figure 2). Some significant genes have an average dN/dS ratio less than 1, indicating that diversifying selection has occurred in these genes, but not sufficiently to cause the average to be greater than 1. These genes may have only a small proportion of amino acids under diversifying selection or a larger proportion that experiences weaker diversifying selection. The dN/dS ratio of the site class under diversifying selection (ω2) was also determined (Additional file 5). This value is a measurement of the intensity of diversify- ing selection within that site class. For example, Sema- phorin (CPXV-BR-182) experiences very strong diversifying selection (ω2 = 10.417) in only a small pro- portion of total sites (2.7%). The F7L homolog (CPXV- BR-055, unknown function) also has a site class experien- cing diversifying selection (ω2 = 8.343), however this slightly weaker selection is spread over a greater propor- tion of sites (12.9%). For this reason, diversifying selec- tion cannot be identified solely on the basis of quantity of sites experiencing diversifying pressure along the gene, but also the strength of the pressure acting on these genes. A few genes have ω2 > 1 but the data do not fit the diversifying selection model significantly better than the model, indicating that a site class under diversi- fying selection likely does not exist for these genes. Function and localization of genes under diversifying selection The 79 genes identified were broadly categorized by function and localization (Table 1, Additional file 4 and Figure 3) based on annotation in the Viral Bioinfor- matics Resource Center curated genes database [13] and several reports on poxvirus genomes [1,14,15]. The lar- gest category is that of genes of unknown function (20 genes), but many host response modifiers or host-range genes (16 genes) were also found. Enzymes and struc- tural components were also identified. Classified by localization, 36 of the proteins under diversifying selec- tion have been found to be virion associated (Figure 3B and 3C). Localization in the virion was determined from two proteomics studies [16,17] and previous literature [1]. All of the proteins of unknown function, and most (81%) of the host response modifiers are not found in the virion. 18 out of 25 (72%) of the enzymes involved