course, it is possible that the value of 0.80 may represent a signal for purifying selection averaged together with one for positive selection. In our analyses, we have tried to account for this in so far as current models permit, by using models that allow for both site- and lineage-specific evolution simultaneously. For the Mcts family, lineage-specific models were not a sig- nificantly better fit to the data than site-specific models. Although the genes are still resolved into distinct lineages by the phyloge- netic reconstruction, there was no evidence to support adaptive evolution of the Mcts2 gene. The best model was determined to be Model 3 with two discrete ω values (K = 2). Overall this model predicts purifying selective pressure on the Mcts family with 89% of the codons estimated to have ω = 0.01, and 11% of the codons with ω = 0.27. All Mcts gene family members are under purify- ing selection. There is a strong evolutionary pressure on all these gene family members to retain the same amino acid sequence. The Nap1l family shows similarity, in terms of selective pres- sures, to the Mcts family, although purifying selection is generally less strict in this case. Like the Mcts family, the lineage-specific models of evolution do not fit the data significantly better than the site-specific models. Again this indicates that differences in codon evolution between the Nap1l5 retrogene lineage and the putative parent genes Nap1l3 and Nap1l2 were not detected by codon evolution analysis. The best site-specific model is Model 3 (K = 2) with 50% of the codons in the Nap1l family under strin- gent purifying selection with ω = 0.06, and the other 50% under slightly less-stringent purifying selection with ω = 0.38. Taking the codon evolution results alone, the Nap1l family would seem to have evolved in a similar way to the Mcts family. However, the MSA for the Nap1l family shows major changes in the open reading frames of the various family members as the family grew due to multiple transposition events. (See Supplementary File 6 for alignments). These changes could very well have profoundly affected protein function, although further examinations of the Nap1l protein structure and function are needed to verify this. Interestingly, the U2af1-rs gene family does show evidence of positive selection (ω = 3.74). Model 3 (K = 3) is the best site-specific model. This model detects positive selection across 10% of sites, but these may only be positively selected in a par- ticular lineage, and a site-specific model cannot address this, also site-specific models in general do not fit these data well. Using lineage-specific models, we have investigated this possibility and have found that Model B fits the data significantly better (P < 0.05). According to this model, 74% of the sites are evolving under purifying selection with ω = 0.06, and 18% of the sites have ω = 0.79, regardless of the lineage. We found that 6% of sites are predicted to be under purifying selection in the parent gene U2af1-rs2 lineages (ω = 0.06), whereas these exact sites are under positive selection in the retrogene U2af1-rs1 lineage (ω = 3.74). A further 1% of sites were evolving neutrally (ω = I M P R I N T E D R E T RO G E N E E VO L U T I O N 0.78) in the parent gene U2af1-rs2 lineages, but showed positive selection in the retrogene U2af1-rs1 lineages (ω = 3.74). In sum- mary, after the retroposition event that created ancestral U2af1-rs1 from its parent gene, certain amino acids in the U2af1-rs1 protein have been under evolutionary pressure to change (i.e., adaptive Darwinian selection), whereas the corresponding codons in the parent lineage are either under purifying selection or are evolv- ing neutrally. This is suggestive of neofunctionalization unique to the retrogene U2af1-rs1 lineage following the retroposition event. These pressures are absent from its parent gene lineage. There were a total of 15 positively selected amino acid changes in the U2af1-rs1 retrogene lineage compared (Table 3). Very similar results were obtained when the pruned species phylogeny is used (Supplementary File 9). As the retrogene and parent proteins are dissimilar at their extreme C terminus, it is not surprising that examination of the U2af1-rs family protein align- ment (Supplementary File 6) revealed that the last four positively selected residues (numbers 480, 485, 491, and 493 in the MSA) are in a poorly aligned region, are likely to be false positives and were subsequently disregarded. Apart from this region, all posi- tively selected residues fall into regions of the alignment with high conservation between the different proteins, indicating a func- tional importance for these regions. We have examined the sites under positive selection for the U2af1 protein using both gene and species phylogenies. The sites estimated using both topologies were similar, see Supplementary File 9. We examined the sites from the gene phylogeny in further detail at the 3D structure level. THREE-DIMENSIONAL STRUCTURE OF U2af1-rs PROTEINS The 3-D structure of the U2af1-rs proteins was investigated to see what effects the positively selected sites might have on the overall fold stability of the protein. We carried out disorder prediction for all the U2af1-rs proteins to identify areas of the proteins that are predicted to be disordered, and areas that might have secondary and tertiary ordered structure. Predictions consistently showed high levels of disorder at the beginning and at the end of the pro- tein, with an ordered area toward the centre (disorder probability of >0.5 is considered disordered). Figure 4A shows the predicted level of disorder across the protein for mouse U2af1-rs1. The pos- itively selected amino acid changes are shown as triangles, and are observed to cluster particularly in the disordered regions. The same pattern is seen in all the U2af1-rs proteins (data not shown). Within the nondisordered central region of the U2af1-rs pro- teins, a region homologous to the human U2af35 RNA binding domain was found. The crystal structure of the human U2af35 domain has been solved (Kielkopf et al. 2001). This structure was used as a template to model the structures of the homologous domains in mouse U2af1-rs1 and U2af1-rs2. EVOLUTION MAY 2011 1 4 2 1