Pseudogenization of the tooth gene enamelysin (MMP20) in the common ancestor of extant baleen whales

Abstract

Whales in the suborder Mysticeti are filter feeders that use baleen to sift zooplankton and small fish from ocean waters. Adult mysticetes lack teeth, although tooth buds are present in foetal stages. Cladistic analyses suggest that functional teeth were lost in the common ancestor of crown-group Mysticeti. DNA sequences for the tooth-specific genes, ameloblastin (AMBN), enamelin (ENAM) and amelogenin (AMEL), have frameshift mutations and/or stop codons in this taxon, but none of these molecular cavities are shared by all extant mysticetes. Here, we provide the first evidence for pseudogenization of a tooth gene, enamelysin (MMP20), in the common ancestor of living baleen whales. Specifically, pseudogenization resulted from the insertion of a CHR-2 SINE retroposon in exon 2 of MMP20. Genomic and palaeontological data now provide congruent support for the loss of enamel-capped teeth on the common ancestral branch of crown-group mysticetes. The new data for MMP20 also document a polymorphic stop codon in exon 2 of the pygmy sperm whale (Kogia breviceps), which has enamel-less teeth. These results, in conjunction with the evidence for pseudogenization of MMP20 in Hoffmann's two-toed sloth (Choloepus hoffmanni), another enamel-less species, support the hypothesis that the only unique, non-overlapping function of the MMP20 gene is in enamel formation.

Figure 1.

Alignment of CHR-2 SINE sequences for representatives of the FL subfamily (Balaenoptera bonaerensis Minke14), MDI subfamily (B. bonaerensis Minke12), MDII subfamily (Physeter macrocephalus Macco13), DT subfamily (Megaptera novaeangliae Hump14), CD subfamily (B. brydei BRY28, M. novaeangliae Hump20, B. bonaerensis Sei23, B. brydei IWA31, Caperea marginata MMP20) and CDO subfamily (Pontoporia blainvillei Isi38). Diagnostic features of different subfamilies are highlighted with coloured boxes as follows: green, MDI subfamily deletion; yellow, MDII subfamily deletion; blue, central deletion of DT, CD and CDO subfamilies; red, examples of diagnostic substitutions that are shared by members of the CD and CDO subfamilies; purple, CDO subfamily deletion. Poly-AT regions of SINEs are not shown.

Figure 2.

A phylogenetic hypothesis for living and extinct taxa that summarizes the evolution of teeth, enamel and enamel-specific genes within Cetacea. Cetaceans in the tree are toothless as adults (white circles), have enamel-less teeth (grey circles) or have enamel-capped teeth (black circles). Circles at internal nodes of the tree show parsimony reconstructions of these three states and indicate the loss of teeth within Mysticeti (baleen whales) and the loss of enamel in Kogia (pygmy and dwarf sperm whales). Frameshift mutations (red bars) and nonsense substitutions (red hexagons) in four enamel genes (AMEL, A; AMBN, B; ENAM, E; MMP20, M) are mapped onto the tree (deltran parsimony optimization). The MMP20 SINE insertion in the common ancestor of extant baleen whales is indicated with a red arrow, and may have occurred on the branch before or after the indicated node. Phylogenetic relationships and divergence times among extant lineages (grey branches) are according to McGowen et al. [38]; the placements of extinct lineages (dotted lines) are as in Bianucci & Landini [47] for the physeteroid, Zygophyseter and as in Fitzgerald [9] for stem mysticetes (Eomysticetus, Aetiocetus, Mammalodon, Janjucetus) and the archaeocete outgroup, Basilosaurus.

Figure 3.

One of the two ML phylograms for MMP20 exon 2 protein-coding sequences (−ln L = 1027.63005). The second tree (not shown) includes a short branch (1.94 × 10⁻⁷ substitutions per site) that groups Hippopotamus with Cetacea to the exclusion of other cetartiodactyls. Branches coloured red indicate evolutionary lineages that lack enamel-capped teeth according to parsimony reconstructions. Bootstrap scores ≥70% are shown. Branch lengths are proportional to the amount of change in nucleotide substitutions per site. Scale bar, 0.01 substitutions per site.

Figure 4.

Maximum-likelihood phylogram for mysticete CHR-2 SINE sequences (−ln L = 1068.74802). The clade of SINE sequences from the MMP20 locus is coloured black, and groupings that are congruent with the supermatrix topology of McGowen et al. [38] are marked by grey circles at nodes. The CHR-2 SINE tree was rooted with Megaptera novaeangliae Hump14, which belongs to the DT subfamily. Bootstrap scores greater than 70% are shown. Branch lengths are proportional to the amount of change in nucleotide substitutions per site. Mysticete genera are abbreviated as follows: Balaenoptera, B.; Balaena, Ba.; Caperea, C.; Eschrichtius, Es.; Eubalaena, Eu.; Megaptera, M. Scale bar, 0.01 substitutions per site.