Wang et al. Genetics Selection Evolution 2010, 42:24 http://www.gsejournal.org/content/42/1/24 Page 4 of 6 Figure 3 Percentage of tissues with duplicates co-expressed. Me- dians for tandem duplicates and retrogenes in mouse genomes are 8.5 and 1.8, respectively (p = 0.002, one-tailed rank sum test); medians for the both of duplicates in human genomes are 7.9 and 2.0, respectively (p = 0.036) Nonetheless, the dN/dS test is directed to single site substitutions, which is not suitable for the case of whole gene substitutions such as the addition of duplicates. Lynch [32] has presented a new strategy for this issue by examining the role of effective population size. Briefly, if the new duplicates are nearly neutral and fixed by genetic drift, a small population size is favourable for their reten- tion. On the contrary, if the new duplicates are advanta- geous and fixed by positive selection, the opposite should be true. In fact, Lynch has suggested that the long-term increase of duplicates from prokaryotes to eukaryotes is initially a neutral process in response to the reduction of population size [32]. However, Shiu et al. [33] have argued that positive selection also plays an important role at least in mammalian genomes because there are more duplicates retained in the mouse lineage (larger popula- tion size) than in the human lineage (smaller population size), which cannot be explained by the difference in their duplication rate. Furthermore, since the generation time in mice is shorter than in humans, there will be more gen- erations that are subject to selective pressures for mice and consequently, more duplicates retained in the mouse genome. In our dataset, there are both more tandem duplicates and more retrogenes in the mouse genome. To Figure 4 Role of positive selection. (A) Comparison of dN/dS ratios for tandem duplicates and retrogenes (log-scale). The non-synony- mous substitutions in most duplicates are under selective constraints (dN/dS < 1); there is little difference in the dN/dS ratios between both types of duplicates (human: p = 0.607, mouse: p= 0.257, t-test) (B) Dis- tribution of the duplicates before and after the split of human and mouse lineages (≈80 million years ago [33]). dS/2 was used to estimate the duplication age, which can be translated to the absolute time scale by using 2.5e-3 substitutions per site per million years [9]; there are sig- nificantly more tandem duplicates and retrogenes arising in the mouse-specific lineage (p < 1e-4 for both types, chi-square test) test if the excessive duplicates are really created in the mouse lineage, we grouped the age of the duplicates (inferred from dS) according to the divergence time between the two species (Figure 4B). The result shows that, while the duplicates generated prior to the split of the two genomes are more or less the same, there are more duplicates arising in the mouse-specific lineage (p < 1e-4 for both types, chi-square test). Based on the same assumption with Shiu et al. [33], this result implies that positive selection plays essential roles in the retention of both types of duplicates. Discussion Dosage effect is more prevalent in tandem duplicates Of the two key dimensions to determine the retention mechanisms, we have found that the extent of functional divergence is distinct for tandem duplicates and retro- genes, whereas the underlying evolutionary forces are the same. As tandem duplicates are generated at the DNA level and easily influenced by gene conversion, they are more likely to be maintained (Figure 2 and 3). Two main