The Evolution of Unusually Small Amelogenin Genes in Cetaceans; Pseudogenization, X-Y Gene Conversion, and Feeding Strategy

Abstract

Among extant cetaceans, mysticetes are filter feeders that do not possess teeth and use their baleen for feeding, while most odontocetes are considered suction feeders, which capture prey by suction without biting or chewing with teeth. In the present study, we address the functionality of amelogenin (AMEL) genes in cetaceans. AMEL encodes a protein that is specifically involved in dental enamel formation and is located on the sex chromosomes in eutherians. The X-copy AMELX is functional in enamel-bearing eutherians, whereas the Y-copy AMELY appears to have undergone decay and was completely lost in some species. Consistent with these premises, we detected various deleterious mutations and/or non-canonical splice junctions in AMELX of mysticetes and four suction feeding odontocetes, Delphinapterus leucas, Monodon monoceros, Kogia breviceps, and Physeter macrocephalus, and in AMELY of mysticetes and odontocetes. Regardless of the functionality, both AMELX and AMELY are equally and unusually small in cetaceans, and even their functional AMELX genes presumably encode a degenerate core region, which is thought to be essential for enamel matrix assembly and enamel crystal growth. Furthermore, our results suggest that the most recent common ancestors of extant cetaceans had functional AMELX and AMELY, both of which are similar to AMELX of Platanista minor. Similar small AMELX and AMELY in archaic cetaceans can be explained by gene conversion between AMELX and AMELY. We speculate that common ancestors of modern cetaceans employed a degenerate AMELX, transferred from a decaying AMELY by gene conversion, at an early stage of their transition to suction feeders.

Keywords: Amelogenin; Cetaceans; Dental enamel; Gene conversion; Mammalian sex chromosomes; Tooth development.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1

Phylogeny of modern Cetartiodactyla. A scale for the divergence times is shown at the bottom (Mya, million years ago). Cetaceans are thought to be adapted to life in fully aquatic environments ~ 40 Mya (dark background). The phylogeny and divergence times are based on previous studies (Montgelard et al. 1997; Benton and Donoghue 2007; Gatesy 2009; McGowen et al. 2009; Gatesy et al. 2013). The divergence time of N. a. asiaeorientalis and N. phocaenoides is not to scale (shown with dashed branches). D, P, and M represent Delphinidae, Phocoenidae, and Monodontidae, respectively. For the Tursiops, Sousa, and Kogia genera, T. truncatus, S. sinensis, and K. sima are shown. See Table S1 for common names

Fig. 2

Characteristics of the core region encoded by AMELX and AMELY. The X axis represents the size (the number of amino acid residues; AA in a), the average disorder prediction (PONDR) score (b), the number of amino acid residues predicted to adopt disordered conformations (AA in c), and the disorder index (DI in d). The Y axis shows the percentage of AMELX genes (%AMELX) in cetaceans (a) or odontocetes (b–d; see the text) and in artiodactyls (a–d) above the X axis, and the percentage of AMELY genes (%AMELY) in cetaceans (a) or odontocetes (b–d) and in artiodactyls (a–d) below the X axis. The percentage of AMELX and AMELY genes in cetaceans (or odontocetes) and 10 different families of artiodactyls are shown by bars in different colors, and the ranges of these characteristics in cetaceans (odontocetes) and artiodactyls are shown by dotted brackets. The largest value of each characteristic in cetaceans (odontocetes) and the smallest value of each characteristic in artiodactyls are indicated. The number of samples used to calculate these characteristics is also shown (e.g., N = 53 for artiodactyl AMELX genes). See Table S2 for the raw data and the correspondence between colors of the bars and names of the families

Fig. 3

Gene conversion of the LTR sequence from AMELY to AMELX (a) and phylogenetic trees of the nucleotide sequences of the LTR region (b), the LTRup region (c), and exon 6 (d) of AMEL genes. (a) An LTR sequence was inserted in intron 5 of AMELY in a common ancestor of Ruminantia, Hippopotamidae, and Cetacea, and this LTR was presumably transferred from AMELY to AMELX by gene conversion in a common ancestor of odontocetes. (b–d) Nodes supported by 95% or higher bootstrap values are shown with a closed circle and 75–95% with an open circle. A scale is shown at the bottom of each tree. (c) The node for the two clusters (1) one composed of Mysticeti, Hippopotamidae, and Ruminantia AMELX sequences and (2) the other composed of Mysticeti, Hippopotamidae, and Ruminantia AMELY sequences and Odontoceti AMELX and AMELY sequences, is supported by a bootstrap value of 57%. (d) DPX represents the cluster consisting of Delphinidae, Phocoenidae, M. monoceros, D. leucas, L. vexillifer, and P. blainvillei exon 6X sequences, while DPY represents the cluster comprising Delphinidae and Phocoenidae exon 6Y sequences. The LTRdown (see Fig. 4) sequences are too small and were not used in our phylogenetic analysis

Fig. 4

Nucleotides potentially underwent gene conversion in cetaceans. The four regions used in this analysis (LTRup, LTR, LTRdown, and exon 6) and their lengths (nucleotides) are illustrated on the top. Each column below shows the position (number) within the region and the nucleotides (A, G, C, or T; a hyphen represents a gap in the sequence alignment) at the position in various cetacean and artiodactyl AMELX and AMELY genes. Substitutions possibly involved in gene conversion are highlighted in yellow. Deletions extending positions 183–184 and 216–245 in the LTR region (highlighted in sky blue) were considered to be two discrete sites, because each site probably underwent a single gene conversion event. The timing of each putative gene conversion event (Timing) can be assigned to three most likely periods: (1) in common ancestors of odontocetes and mysticetes (2) in the odontocete lineage, and (3) in the mysticete lineage, or a combination of two events at different timings (2/3 or 1/2). For each position in exon 6, whether the putative gene conversion is associated with a synonymous substitution (+) or a non-synonymous substitution (−) is shown in the “AA change” row. Complete multiple nucleotide sequence alignments are provided in Fig. S1 for exon 6 and Fig. S6 for the LTRup, LTR, and LTRdown regions. Substitutions from A or T to G or C, and those from G or C to A or T are shown by “X” in the “A/T to G/C” and “G/C to A/T” rows, respectively, while substitutions between G and C and between A and T are shown with “X” in the “Btw G&C or A&T” row