Accelerated Evolution of Limb-Related Gene Hoxd11 in the Common Ancestor of Cetaceans and Ruminants (Cetruminantia)

Abstract

Reduced numbers of carpal and tarsal bones (wrist and ankle joints) are extensively observed in the clade of Cetacea and Ruminantia (Cetruminantia). Homebox D11 (Hoxd11) is one of the important genes required for limb development in mammals. Mutations in Hoxd11 can lead to defects in particular bones of limbs, including carpus and tarsus. To test whether evolutionary changes in Hoxd11 underlie the loss of these bones in Cetruminantia, we sequenced and analyzed Hoxd11 coding sequences and compared them with other 5' HoxA and HoxD genes in a taxonomic coverage of Cetacea, Ruminantia and other mammalian relatives. Statistical tests on the Hoxd11 sequences found an accelerated evolution in the common ancestor of cetaceans and ruminants, which coincided with the reduction of carpal and tarsal bones in this clade. Five amino acid substitutions (G222S, G227A, G229S, A240T and G261V) and one amino acid deletion (G254Del) occurred in this lineage. In contrast, other 5' HoxA and HoxD genes do not show this same evolutionary pattern, but instead display a highly conserved pattern of evolution in this lineage. Accelerated evolution of Hoxd11, but not other 5' HoxA and HoxD genes, is probably related to the reduction of the carpal and tarsal bones in Cetruminantia. Moreover, we found two amino acid substitutions (G110S and D223N) in Hoxd11 that are unique to the lineage of Cetacea, which coincided with hindlimb loss in the common ancestor of cetaceans. Our results give molecular evidence of Hoxd11 adaptive evolution in cetaceans and ruminants, which could be correlated with limb morphological adaptation.

Keywords: 5′Hox genes; Hoxd11; Limb; carpal; mutation; tarsal.

Figure 1

Evolution of carpal and tarsal bone numbers in mammals. (A) Carpus. (B) Tarsus. The numbers of carpal and tarsal bones in extant species are shown to the left side of the names of each species. Numbers for the ancestors are shown on the branches of the species tree. The lineage leading to Cetruminantia is shown in orange and the lineage leading to Cetacea is in pink. Clades for cetaceans and ruminants are indicated in blue and green, respectively.

Figure 2

Molecular evolution of Hoxd11 in mammalian ancestors. Branch lengths are proportional to nucleotide substitutions per codon. The ω values, numbers of nonsynonymous and synonymous substitutions inferred from the free-ratio model are shown on the species tree (the three numbers on the branches). The five amino acid substitutions on the lineage leading to Cetruminantia (branch “C”), shown in orange, and the two amino acid substitutions on the lineage leading to Cetacea, shown in pink, were mapped onto the branches. The clades for cetaceans and ruminants are indicated in blue and green respectively. Abbreviations for taxonomy are “Pe” (Perissodactyla), “Ca” (Carnivora), “Ch” (Chiroptera), “La” (Lagomorpha) and “Af” (Afrosoricida).

Figure 3

Alignment of mammalian Hoxd11 protein sequences. Partial amino acid sequences from 53 mammalian species are aligned. The five amino acid substitutions and one amino acid deletion that occurred in the ancestor of Cetruminantia are highlighted by the red arrows. The two amino acid substitutions that occurred in the ancestor of Cetacea are highlighted by the blue arrows. The gray box in the schematic of Hoxd11 is homeodomain.

Figure 4

Evolution of the six amino acids in Hoxd11 proteins with mutations that occurred on the Cetruminantia branch. Evolutionary changes in the 53 mammals at the sites of the amino acid substitutions or deletion G222S, G227A, G229S, A240T, G261V and G254Del are shown in A, B, C, D, E and F, respectively.

Figure 5

Evolution of the two amino acids that are substituted on the branch leading to Cetacea in the Hoxd11 protein. Evolutionary changes in the 53 mammals at the sites of the amino acid substitutions G110S and D223N are shown in A and B, respectively.

Figure 6

Sliding window analysis of Hoxd11. ω values were calculated in a sliding window across the Hoxd11 sequence. The orange line represents ω values within cetaceans, the black line is ruminants, and the dotted gray line is other mammals. The homeobox is shown in the gray rectangle.