As paleopolyploid genomes evolve, the expression profiles of retained gene pairs are expected to diverge. To examine this divergence process on a large scale in a vertebrate system, we compare Xenopus laevis, which has retained Ϸ40% of loci in duplicate after a recent whole-genome duplication (WGD), with its unduplicated relative Silurana (Xenopus) tropicalis. This comparison of ingroup pairs to an outgroup allows the direction of change in expression profiles to be inferred for a set of 1,300 X. laevis pairs, relative to their single orthologs in S. tropicalis, across 11 tissues. We identify 68 pairs in which X. laevis is inferred to have undergone a significant reduction of expression in at least two tissues since WGD. Of these pairs, one-third show evidence of subfunctionalization, with decreases in the expression levels of different gene copies in two different tissues. Surprisingly, we find that genes with slow rates of evolution are particularly prone to subfunctionalization, even when the tendency for highly expressed genes to evolve slowly is controlled for. We interpret this result to be an effect of allopolyploidization. We then compare the outcomes of this WGD with an independent one that happened in the teleost fish lineage. We find that if a gene pair was retained in duplicate in X. laevis, the orthologous pair is more likely to have been retained in duplicate in zebrafish, suggesting that similar factors, among them subfunctionalization, determined which gene pairs survived in duplicate after the two WGDs.rate of evolution ͉ Silurana tropicalis ͉ whole-genome duplication P olyploidy, also termed whole-genome duplication (WGD) is a frequent phenomenon in eukaryotes (1). A WGD is followed by extensive and rapid genome restructuring involving many gene losses, so that only one of the two gene copies remains in most genomes that underwent ancient polyploidization [for example, fish and yeast (2, 3)]. Alterations in function are expected among genes retained in duplicate. In some cases, one copy may acquire a new function (neofunctionalization), while the other keeps the ancestral function. The models of Lynch and Force (4, 5) also propose the existence of subfunctionalization, in which each copy retains a subset of the functions of the ancestral gene. Sub and neofunctionalization models make different predictions about the rate and symmetry of sequence evolution in the duplicates.Asymmetry in evolutionary rates between the protein sequences of the two copies is often interpreted as a footprint of neofunctionalization, especially if it is associated with evidence of positive selection in the accelerated copy (6). Several studies of paleopolyploid genomes have shown that rate asymmetry between the two copies can be widespread. For example, asymmetry was seen in 6% of retained gene pairs in Xenopus laevis and in 25-36% of pairs in teleost fishes (6-8). Relatively few examples of subfunctionalization of duplicated genes have been demonstrated so far, the best-known being those of fish mitf (9), sox9 (10), ...