Pseudogenized Amelogenin Reveals Early Tooth Loss in True Toads (Anura: Bufonidae)

Abstract

Extant anurans (frogs and toads) exhibit reduced dentition, ranging from a lack of mandibular teeth to complete edentulation, as observed in the true toads of the family Bufonidae. The evolutionary time line of these reductions remains vague due to a poor fossil record. Previous studies have demonstrated an association between the lack of teeth in edentulous vertebrates and the pseudogenization of the major tooth enamel gene amelogenin (AMEL) through accumulation of deleterious mutations and the disruption of its coding sequence. In this study, we have harnessed the pseudogenization of AMEL as a molecular dating tool to correlate loss of dentition with genomic mutation patterns during the rise of the family Bufonidae. Specifically, we have utilized AMEL pseudogenes in three members of the family as a tool to estimate the putative date of edentulation in true toads. Comparison of AMEL sequences from Rhinella marina, Bufo gargarizans and Bufo bufo, with nine extant, dentulous frogs, revealed mutations confirming AMEL inactivation in Bufonidae. AMEL pseudogenes in modern bufonids also exhibited remarkably high 86-93% sequence identity among each other, with only a slight increase in substitution rate and relaxation of selective pressure, in comparison with functional copies in other anurans. Moreover, using selection intensity estimates and synonymous substitution rates, analysis of functional and pseudogenized AMEL resulted in an estimated inactivation window of 46-60 million years ago in the lineage leading to modern true toads, a time line that coincides with the rise of the family Bufonidae.

Fig. 1

MicroCT scan of a Lithobates [Rana] pipiens skull showing maxillary teeth (red triangles) and edentulous mandible, while Rhinella marina exhibits complete edentulation. “Frog” is representative of nonbufonid, dentulous anurans, while “Toad” is representative of Bufonidae

Fig. 2

Conserved AMEL structure in representative tetrapod lineages. Exons are depicted as boxes and are drawn to scale; introns are lines and are not to scale. Coding regions are in full color, while non-coding regions are at 50% opacity. Represented species are Human (Homo sapiens; NCBI NM_182680.1), Mouse (Mus musculus; NCBI NM_009666.4), Bovine (Bos taurus; NCBI NM_001014984), Lizard (Anolis carolinensis; NCBI KP792754), Alligator (Alligator mississippiensis; NCBI XM_014607781.2), and Frog (Xenopus tropicalis; NCBI NM_001113681.1)

Fig. 3

Nucleotide alignment of exons 2–6. Bufonid AMEL pseudogenes share deleterious mutations not found in dentulous anurans or salamander. Red box denotes loss of start codon (“ACC” in R.m. and “GCC” in B.b. and B.g.), green boxes denote frameshift deletions, blue boxes denote frameshift insertions, red text indicates premature stop codons, and purple text indicates donor and acceptor splice site mutations. Species include three members of Bufonidae (Rhinella marina, Bufo bufo and Bufo gargarizans), four nonbufonid frogs (Ranoidea chloris, Lithobates [Rana] pipiens, Xenopus tropicalis, and Scaphiopus multiplicatus), and one salamander (Ambystoma mexicanum)

Fig. 4

Gene phylogeny and selection intensity follows evolutionary patterns of respective lineages. (A) Bayesian phylogram generated from codon-specific alignment of AMEL. Branch labels represent Bayesian posterior probabilities (BPP) where 0.50 ≤ BPP ≤ 0.99, while unlabeled branches indicate BPP =100. Multifurcations indicate branches with BPP <0.5. Scale bar indicates the number of substitutions per site. Asterisks indicate species where the gene tree and species tree disagree. (B) Evolutionary chronogram with species relationships and divergence date estimates from Feng et al. (2017). Branches are labeled with dN and dS values, estimated in CODEML under a free-ratio model (M1) with CodonFreq = 2. The resulting ω values are included in parentheses in bold. Photo is representative of a R. marina specimen

Fig. 5

Substitution rate increase in the stem lineage to modern Bufonidae. Substitution rates were estimated under a local clock model for Toads (members of Bufonidae) (0.23 ± 0.04), the stem bufonid branch (1.22 ± 0.15), and Frogs (nonbufonid, dentulous anurans) (0.13 ± 0.01)