Position in retrogene protein U2af1-rs1 (M. musculus) Position in parent protein U2af1-rs2 (M. musculus) 38 46 62 154 206 313 355 361 384 385 388 475 480 486 488 RU T H B . M c C O L E E T A L . Table 3. Positions of positively selected codons in the U2af1-rs1 retrogene lineage. Position in alignment1 Amino acid in retrogene lineage Amino acid in parent lineage 38 46 63 154 206 313 355 361 384 385 388 4803 485 491 493 M A L E V V P S Y(mouse) H(rat) H S E G H T L L E G I M D F R R P S R R R P value2 0.659 0.57 0.996 0.576 0.745 0.678 0.997 0.501 0.875 0.965 0.528 0.993 0.593 0.942 0.802 33 41 55 142 192 300 342 348 371 372 373 415 420 426 428 1The position differs from alignment to protein as the alignment ﬁle contains sequence gaps. 2Our conﬁdence in each of these sites being positively selected is calculated using the posterior probability and summarized in the P values shown. P values vary from 0.00 (no evidence for belonging in the positively selected category) to 1.00 (100% conﬁdence of belonging in the positively selected category). 3Dark gray area refers to residues deemed to be false positives due to poor alignment of the U2af1-rs sequences. Only one of the positively selected amino acid changes was found to fall within the ordered region of U2af35 homology, this was the codon at position 206 in the MSA, corresponding to isoleucine in the all parent gene sequences in all species (residue position 206 in the mouse U2AF1-RS2), and a valine residue in all the retrogene sequences in all species (position 192 in mouse U2AF1-RS1). We analyzed the difference between the two sets of models and we focused on the immediate neighboring residues of the two mutations (Figure 4B and C). Many iterations of the modeling procedure are depicted; hence each residue has multiple representations of its position. Any atom within 6 Angstroms of the residue of interest is colored. In the U2AF1-RS1 structure, two residues (atoms belonging to Phenylalanine 238 and Pheny- lalanine 279, both magenta) are on average closer to the positively selected valine residue, compared to the isoleucine in the U2AF1- RS2 structure. The more bulky isoleucine residue of U2AF1-RS2 induces a larger perturbation in neighboring residues, pushing them away. Discussion DISPARATE MODES OF EVOLUTION FOR DIFFERENT IMPRINTED RETROGENE FAMILIES Although studies of large gene cohorts (Cusack and Wolfe 2007) can be informative on the general trends in evolutionary rates 1 4 2 2 EVOLUTION MAY 2011 of parent genes and retrogenes, analysis of individual retrogene- parent pairs, as in this study, can reveal much heterogeneity in evolutionary rate among retrogenes. Indeed, the four retrogenes examined here have many features in common, other than their origins as retroposition products, such as their imprinted regula- tion and X-chromosome derivation. However, each gene family examined showed very different evolutionary trajectories. The Inpp5f_v2 retrogene is evolving under a more relaxed selective constraint than its parent gene Vma21. The Nap1l gene family has evolved under a strict regime, with a high constraint on codon evolution. However, major deletions to the Napl15 open reading frame may have impacted on this protein’s function. In the case of the Mcts gene family, selective pressure analyses results show that all gene sequences from all lineages (both parent and retrogene), are under purifying selection suggestive of evolution- ary pressure to maintain the same protein function in the parent and retrogene. The U2af1-rs1 retrogene has been under positive Darwinian selection, in contrast to its parent gene, which has been under a mixture of purifying and neutral evolutionary pressures. NONUNIFORM EVOLUTIONARY INNOVATION ALONG THE U2af1-rs1 PROTEIN Regions of the U2af1-rs genes are homologous to the U2af35 RNA-binding domain. After the emergence of the ances- tral U2af1-rs1 retrogene, one residue in the homologous