RU T H B . M c C O L E E T A L . Model1 Inpp5f_v2-Vma21 family Site specific model: Model 3: Discrete(K=2) Lineage specific model: Model B Mcts family Site specific model: Model 3: Discrete(K=2) Nap1l family Site specific model: Model 3: Discrete(K=2) U2af1-rs family Site specific model: Model 3: Discrete(K=3) Lineage specific model: Model B Table 2. Summary of codon evolution model that ﬁts each gene family best following likelihood ratio test analysis. Estimates of parameters Chi-squared test result Positively selected sites2 p0=0.49, p1=0.51 ω0=0.02, ω1=0.19 p0=0.23, p1=0.22, p2=0.28, p3=0.27 Parent Lineages (Background): ω0=0.01, ω1=0.19, ω2=0.15, ω3=0.19 Retrogene Lineages (Foreground): ω0=0.01, ω1=0.19, ω2=0.80, ω3=0.80 p0=0.89, p1=0.11 ω0=0.01, ω1=0.27 p0=0.50, p1=0.50 ω0=0.06, ω1=0.38 p0=0.58, p1=0.31, p2=0.10 ω0=0.03, ω1=0.24, ω2=1.30 p0=0.74, p1=0.18, p2=0.06, p3=0.01 Parent gene lineages (Background): ω0=0.06, ω1=0.79, ω2=0.06, ω3=0.78 Retrogene lineages (Foreground): ω0=0.06, ω1=0.79, ω2=3.74, ω3=3.74 M0 v M3k2 2(2039.75–2024.97)=29.56∗ Critical value≥5.99 M3k2 v Model B 2(2024.97–2020.32)=9.3∗ Critical value≥5.99 M0 v M3k2 2(2554.31–2528.13)=52.36∗ Critical value≥5.99 M0 v M3k2 2(12832.55– 12667.28)=330.54∗ Critical value≥5.99 M3k2 v M3k3 1(7302.16–7293.06)=9.1∗ Critical value≥1.00 M3k2 v Model B 2(7302.16–7284.63)=107.06∗ Critical value≥5.99 No positive selection No positive selection No positive selection No positive selection 36 sites, p.p.>0.5 15 sites, p.p.>0.95 4 sites, p.p>0.99 Foreground: 10 sites, p.p.>0.5 2 sites, p.p.>0.95 3 sites, p.p.>0.99 1Model 3 categorizes each site in the alignment into either two (K=2) or three (K=3) categories of ω, the values for ω are estimated based on the data. The proportion of sites with these ω values is given as “p” with the corresponding subscript for the ω value. Model B allows a speciﬁc branch of the phylogenetic tree to be marked as foreground and categorizes the sites into four proportions, p0,1,2, and 3, with four different values of ω estimated for the foreground and background independently. 2Where models predicted categories of sites with ω>1, indicating positive selection, the estimated numbers of sites with posterior probabilities >0.5, >0.95, and >0.99 of belonging to this category are listed. Codons are estimated as belonging to the category of positively selected using Naı¨ve Empirical Bayes analysis only if Bayes Empirical Bayes is not available. tree analyses. What follows is a brief synopsis of these results on a gene-by-gene basis. For the Inpp5f_v2-Vma21 gene family, the best site-specific model was Model 3 (K = 2) with all codons in the MSA under purifying selection (ω < 0.5). The lineage-specific model that fits the data significantly better (P < 0.05) than this is Model B, which allows differences in the retrogene (foreground) compared with the parent gene (background) lineages. Model B indicates that 23% of the sites have a predicted ω of 0.01 (purifying selection), 22% have ω predicted at 0.19 (slightly more relaxed purifying selection). This means that approximately 45% of the alignment is under purifying selection, regardless of lineage. A further 28% of the sites have ω predicted at 0.15 in the parent gene lineages and 0.80 in the retrogene lineage, and the remaining 27% have ω at 0.19 in the parent gene and 0.80 in the retrogene lineages. So, at 55% of the sites, the Vma21 parent lineage is under purifying selection, whereas the Inpp5f_v2 lineage is evolving neutrally (ω close to 1) at these sites. This means that natural selection is acting to preserve the protein sequence in the parent gene lineage, but 55% of the retrogene lineage codons are unconstrained. Of 1 4 2 0 EVOLUTION MAY 2011