There is no significantly higher dN than dS in the pairwise comparisons of the sequences from clade III, suggesting that no positive selection acted on IFITM5 (Fig. 6A) . Further site-specific analyses using PAML (Table 3 ) and HyPhy (data not shown) confirmed no positive selection acting on IFITM5 genes. There are three pairs of IFITM10 genes (in fugu: IFITM10-like3 vs. IFITM10-like2, and IFITM10-like1 vs. IFITM10-like2; in medaka: IFITM10-like1 vs. IFITM10-like2) with dN/dS.1 (Fig. 6A) , indicating positive selection acting on aquatic IFITM10 gene. Because gene expansion of IFITM10 was observed in frog (Fig. 2C) , we analyzed the five IFITM10 related genes (including one IFITM10 and four IFITM10-like genes) using the site-specific model. A strong signal of positive selection was detected. Eight sites were identified as under positive selection at the level of posterior probability.0.8 (Table 3) . We further constructed the phylogeny of frog IFITM10-related genes and counted the numbers of non-synonymous (n) and synonymous (s) substitutions on each branch (Fig. S6) . The gn/gs ratio (1.75) is significantly smaller than the N/S ratio (2.96) (P = 0.0112, chi-square test), showing no positive selection. However, a very strong signal of positive selection (v = 999, P = 0.0077, chi-square test) was Within clade I, 12 of 5996 pairwise comparisons exhibit significantly higher dN than dS (Fig. 6B and Table S3 ), indicating the presence of positive selection. Among 12 pairwise comparisons with significantly higher dN than dS, eight occur between the primate sequences (Fig. 5C) , three between the rodent sequences (Fig. 5D) , and one between cow sequences. Further site-specific analysis detected two positively selected sites (PSS) in the primate IR-IFITM genes and one in the rodent IR-IFITM genes ( Table 3) . No PSS was detected in the primate IFITM1 and IFITM2&3 subgroups. Because positive selection is generally associated with the occurrence of gene duplication [35] , and a large number of gene duplications were observed in the marmoset IR-IFITM genes, we also performed site-specific analysis on marmoset IR-IFITM genes. As expected, one significant PSS was detected in marmoset IR-IFITM genes.