E2F4/5 subgroup evolved slower than E2F1/2/3 subgroup. (A) Distance between Human E2F proteins and predicted ancestral E2F sequence. The pairwise distances between human E2Fs and predicted ancestral E2F sequence, E2F-Mb(gi|167523471). (B) The group average pairwise distance of E2F4/5 subgroup and E2F1/2/3 subgroup in invertebrates. Based on the sequence data from Ta, Nv, Ci. Sp and Bf, the average distance of E2F4/5 subgroup is smaller than E2F1/2/3 subgroup (P < 0.001, with Pair Student's T Test), standard deviation values are showed as error bars. (C) Distance between human E2F proteins and other vertebrate orthologues. Pair wise distances between human E2F proteins and their orthologues from Gg, Xt. Dr, Tn were computed respectively. (D) The group average distances of E2F4, E2F1, and E2F3 in vertebrates. Based on protein sequence data from 8 organisms (Hs, Mm, Cf, Bt, Rn, Gg, Xt, Tn), the group average distance of E2F4 is smaller than E2F1 and E2F3 (E2F1:E2F4, P < 0.001; E2F3:E2F4, P < 0.001). (E) Ka/Ks values for E2F genes based on codon sequences from human and mouse. (F) The group average Ka/ks of E2F1, E2F2, E2F3, and E2F4 in mammals. Based on Ka/ks values for protein-coding DNA sequences from four mammals (Hs, Cf, Bt, Mm) in each subgroup, The group average Ka/ks value of E2F4 subgroup is smaller than E2F1 subgroup, E2F2 subgroup and E2F3 subgroup (For E2F1:E2F4, P < 0.001; E2F2:E2F4, P < 0.001; E2F3:E2F4, P < 0.05.). Detail information could be found in additional file 5 and additional file 6. Abbreviations: Hs, H. sapiens; Mm, M. musculus; Cf, C. familiaris; Bt, B. Taurus; Gg, G. gallus; Xt, X. tropicalis; Dr, D. rerio; Tn, T. nigroviridis; Ci, C. intestinalis; Sp, S. purpuratus; Bf, B. floridae, Ta, T. adhaerens.