RU T H B . M c C O L E E T A L . Figure 2. Expression of the retrogene and parent gene transcripts in multiple tissues and developmental stages. Expression was assayed by reverse-transcriptase PCR with Actin as a control for each different tissue sample (bottom row). Tissues where only one member of a gene family is expressed are highlighted with black squares. PHYLOGENY RECONSTRUCTION SUPPORTS THE SEPARATION OF RETROGENES AND PARENT GENES INTO DISTINCT LINEAGES For every gene family, the parent genes and retrogenes were separated into distinct lineages with high posterior probabilities (Fig. 3). The Nap1l family phylogeny is in agreement with previ- ously published data (Wood et al. 2007), with Nap1l2 predicted to be the closest relative to Nap1l5. Within each lineage (parent gene or retrogene) the correct species phylogeny is not always preserved. It is not unusual for a gene tree to be discordant with the species tree, and these have previously been used for codon evolution analysis (Ward et al. 2002 Spady et al. 2005; Sassi et al. 2007; Chapman et al. 2008). Indeed, there may be systematic reasons for gene tree–species tree discordance. Other factors apart from the ancestry of the gene se- quences in question, can have an impact on topology such as the presence of strong negative or positive selection (Massey et al. 2008). For each of the four datasets, we have also constructed a pruned canonical species phylogeny. These species phylogenies were compared to the gene phylogenies using a statistical frame- work implemented in the SH test (Shimodaira and Hasegawa 2001). The results of the comparisons are given in Supplementary File 3. 1 4 1 8 EVOLUTION MAY 2011 In summary, the gene and species phylogenies did not vary significantly, for all gene families but Mcts. Following SH test analysis of the Mcts gene tree and species tree, the gene tree was a statistically better fit to the data, and so all further analyses for Mcts were carried out using the gene tree. For the remaining three gene families, Nap1l, U2af1-rs, and Inpp5f_v2-Vma21, we have applied the species phylogenies to the codon evolution anal- yses. The results from the codon evolution analyses did not differ significantly based on the gene or species phylogeny, see Supple- mentary File 7. This is consistent with the results from the SH test where there was no significant difference between the gene and species trees for Nap1l, U2afs, and Inpp5f. The results described below for codon evolution analyses are from the inferred gene family phylogenies for all genes. EACH GENE FAMILY HAS EVOLVED DIFFERENTLY We investigated the evolution of the coding DNA sequence of each gene family. For lineage-specific models, the imprinted ret- rogene lineages were each in turn designated as foreground, as shown by the positions of the stars in Figure 3. Table 2 shows the models determined to fit the data best following LRT analysis for each gene. See Supplementary File 8 for full codon evolution results for each gene family from the gene