RU T H B . M c C O L E E T A L . MULTIPLE SEQUENCE ALIGNMENT (MSA) AND PHYLOGENETIC RECONSTRUCTION Protein-coding sequences were translated using in-house software and aligned using ClustalW (Thompson et al. 1994). Alignments were inspected using Se-Al (Rambaut 1996) and JalView 12.2.0 (Clamp et al. 2004). Phylogenetic reconstruction was carried out using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). The model for amino acid substitution to be used, JTT + G for all MSAs, was determined using ModelGenerator 0.85 (Jones et al. 1992). SHIMODAIRA–HASEGAWA (SH) TEST As the gene phylogenies did not map precisely onto the pruned species phylogeny for each MSA, we performed the SH test to determine if these phylogenies were significantly different. The pruned species phylogeny was generated by simply removing taxa from the canonical species phylogeny as resolved by Murphy et al. (2001). This comparison was carried out using the SH test (Shimodaira and Hasegawa 2001) implemented in the TreePuzzle 5.2 (Schmidt et al. 2002). The results are given in Supplementary File 3. There is no significant difference between the topologies for the Inpp5f_v2-Vam21, Nap1l, and U2af1-rs families. In the Mcts family, the gene tree was a better fit to the data and so this was used for all further analysis. MODELS OF CODON EVOLUTION We estimated ω across the four alignments individually using both site-specific and lineage-specific models implemented in PAML version 4.2a, these models are described in Yang ( 1997), Yang and Nielsen (2002), and updated in Zhang et al. ( 2005). ω is an estimate of the ratio of nonsynonymous to synonymous substitutions at each site in the MSA, normalized by the number of possible substitutions of each type. ω provides an estimate at each codon of the type of selection pressure (positive, neutral or purifying) that has occurred during evolution. Nine different models of codon evolution were tested, along with two “null” models that are essential for statistical validation. For full descriptions of all models used in this study and parame- ters therein please see (Yang 1997; Yang and Nielsen 2002; Zhang et al. 2005). A brief description is given in Supplementary File 4. For each gene family, the model that fit the data best following statistical tests was chosen. In some cases, this was a site-specific model that provided estimates on evolutionary rates in specific regions of the protein. In other cases, a lineage-specific model was chosen. When a lineage-specific model fits the data best, this indicates asymmetry in evolutionary rates between the phyloge- netic branches in question, or between parent gene and retrogene lineages as is the case in this study. In all four gene families, either Model 3 (K = 2) or Model B was the best fit to the data. Model 3 (K = 2) is a site-specific 1 4 1 6 EVOLUTION MAY 2011 model, where each site is only allowed one of two values of ω. No constraint is placed on the value of ω, which can be larger than 1, so positively selected sites are allowed. If Model 3 (K = 2) is the best fit to the data, there is no evidence that the foreground (retrogene lineage) has evolved differently from the background (parent gene lineage). Model B is the lineage-specific extension of Model 3 (K = 2). Sites are allowed to have different values of ω simultaneously, so the foreground lineage can be shown to have evolved differently from the background. Four possible values of ω are allowed, which can be greater than 1 (positive selection). A summary of all models tested is in Supplementary File 4. PROTEIN THREE-DIMENSIONAL (3D) STRUCTURE MODELLING The modeled structures of the U2af1-rs proteins were obtained by homology modeling from the crystal structure of the U2AF35 central domain (chain A, pdb code = 1JMT) (Kielkopf et al. 2001). The sequence alignment used to build the models was gener- ated with the program PRALINE with the homology-extended alignment strategy (Simossis et al. 2005). Three-dimensional models were generated using the MODELLER package (Marti- Renom et al. 2000). The selected model was chosen on the basis of the MODELLER objective function’s score. Images were pro- duced with visual molecular dynamics (VMD) 1.8.5 software (Humphrey et al. 1996). The VSL2 package was used for disorder prediction (Peng et al. 2006). The software provides a disorder probability for each residue. To achieve the most accurate results, we have used VSL2 with four features sets; amino acid composition, two indepen- dent secondary structure predictions, and PSI-BLAST profiles as described in Peng et al. (2006). Results IMPRINTED RETROGENES HAVE BEEN ACCURATELY DATED We have previously shown estimates of the ages of the four retro- gene insertions in question (Wood et al. 2007). We have been able to refine these estimations for Inpp5f_v2, Nap1l5, and U2af1- rs1 orthologs (Fig. 1). Inpp5f_v2 orthologs were known not to be present in the opossum (Wood et al. 2007), but we have found orthologs in the elephant and armadillo (Supplementary File 5). Retroposition of Inpp5f_v2 must have occurred after the Marsupalia/Placentalia split, but before the split of the Xenarthra and Afrotheria. The retroposition event that formed Nap1l5 occurred af- ter Xenarthra and Afrotheria clades diverged, but before the Laurasiatheria/Euarchontoglires divergence. We were unable to