Wang et al. Genetics Selection Evolution 2010, 42:24 http://www.gsejournal.org/content/42/1/24 Page 3 of 6 Figure 1 Gene duplicability. Distribution of the duplicates among small (≤5 members) and large (>5 members) families; tandem dupli- cates are more likely to be enriched in large families than the retro- genes (p < 0.01 for both genomes, chi-square test) Sequence similarity Similarity of coding sequences has been widely used to indicate whether the new duplicates undergo gene con- servation or functional divergence. While some reports have suggested that the duplicates really undergo sequence divergence when they are newly produced [9,26], other reports have found that they still remain more conserved than singletons [27]. However, taking all duplicates as a whole will neglect some specific factors that belong to different molecular bases. For example, the effect of gene conversion, which keeps duplicates appear- ing similar through local DNA recombination [28], may have greater influence on tandem duplicates than retro- genes. The higher duplicability of tandem duplicates may also leave more recent and less divergent gene pairs. To test the hypothesis, we compared the amino acid identity between both types of duplicates (Figure 2). The result shows that, the sequence identity of tandem duplicates is significantly higher than that of retrogenes (human: p = 0.021, mouse: p = 0.034, rank sum test). In agreement with Jun et al. [7], this result implies that tandem dupli- cates tend to be more conserved, whereas retrogenes tend to be more divergent. Expression similarity In addition to the coding sequences, the evolution of reg- ulatory elements is also important to determine the fate of duplicates. In fact, the differentiation of regulatory motifs can increase the expression specificity of the duplicates among various tissues and developmental stages, which is perhaps the most common form of sub- functionalization [29]. Previous reports have found that a rapid expression divergence exists between duplicates [30], and that the expression diversity is also increased compared to singletons [31]. However, as tandem dupli- cations directly occur at the DNA level, it is more likely that the new duplicates preserve their original regulatory motifs and expression patterns. In contrast, as retrogenes Figure 2 Percentage of amino acid identity. Medians for tandem duplicates and retrogenes in mouse genomes are 74 and 68.75, re- spectively (p = 0.034, one-tailed rank sum test); medians for both types of duplicates in human genomes are 63 and 55.75, respectively (p = 0.021) are randomly inserted into the genome via mRNAs, they are more likely to acquire distinct regulatory motifs and expression patterns. To test this hypothesis, we compared the expression similarity for tandem duplicates and retro- genes by using microarray data across diverse tissues (Figure 3). Although a lot of duplicates have been quite differentiated for both types, the expression similarity between tandem duplicates is still significantly higher than that between retrogenes (human: p = 0.036, mouse: p = 0.002, rank sum test). Therefore, the gene expression profiles also support the difference in functional diver- gence for both types of duplications. Role of positive selection As mentioned in the section Background, the retention mechanisms are determined by both functional diver- gence and evolutionary forces. To compare the evolution- ary forces for both types of duplicates, we first performed the traditional dN/dS analysis (Figure 4A). The result shows little difference in the dN/dS ratios between tan- dem duplicates and retrogenes (human: p = 0.607, mouse: p = 0.257, t-test). In addition, the non-synonymous sub- stitutions in most duplicates are under selective con- straints (dN/dS < 1).