Rampant tooth loss across 200 million years of frog evolution

Teeth are present in most clades of vertebrates but have been lost completely several times in actinopterygian fishes and amniotes. Using phenotypic data collected from over 500 genera via micro-computed tomography, we provide the first rigorous assessment of the evolutionary history of dentition across all major lineages of amphibians. We demonstrate that dentition is invariably present in caecilians and salamanders, but teeth have been lost completely more than 20 times in frogs, a much higher occurrence of edentulism than in any other vertebrate group. The repeated loss of teeth in anurans is associated with a specialized diet of small invertebrate prey as well as shortening of the lower jaw, but it is not correlated with a reduction in body size. Frogs provide an unparalleled opportunity for investigating the molecular and developmental mechanisms of convergent tooth loss on a large phylogenetic scale.


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The evolution of teeth is considered a key innovation that promoted the radiation of jawed 31 vertebrates, facilitating the transition from a passive to active predatory lifestyle (Gans and 32 Northcutt 1983). Teeth are complex mineralized tissues that originated in stem gnathostomes 33 more than 400 million years ago (Rücklin et al. 2012) and have been broadly maintained across 34 living chondrichthyans, actinopterygians, and sarcopterygians due to the critical role these 35 structures play in the acquisition and processing of food. The shape, size, location, and number 36 of teeth differ widely across vertebrates, especially in response to broad variation in food type.

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Although dentition is generally conserved across vertebrates, teeth have been lost completely

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Teeth are likely lost following the evolution of a secondary feeding tool that improves the 41 efficiency of food intake (e.g., beak, baleen, specialized tongue), leading to relaxed functional 67 odontogenesis occurs ontogenetically late in frogs (during or after metamorphosis) compared to 68 other vertebrates.

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Using the most recent species-rich phylogeny of extant amphibian species (Jetz and

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Pyron 2018) and our extensive taxonomic sampling via high-resolution X-ray micro-computed 71 tomography of over 500 of the 561 currently recognized amphibian genera (AmphibiaWeb 72 2021), we 1) evaluated the phylogenetic distribution of teeth and reconstructed the evolutionary 73 history of dentition across all major lineages of amphibians and 2) tested whether dietary 74 specialization, relative jaw length, and body size are correlated with the loss of teeth in frogs.

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Our results demonstrate that the presence and location of teeth are highly conserved in 76 salamanders and caecilians, but labile in frogs. We found that teeth have been repeatedly lost in 77 frogs and at a much higher frequency than in any other vertebrate group. The evolution of 78 edentulism in anurans is correlated with a microphagous diet and shortening of the lower jaw 79 but not with a reduction in body size over evolutionary time. Six reversals, from edentulous to 80 toothed jaws, were inferred in frogs.

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We recorded the presence or absence of teeth on each dentigerous bone of the lower jaw, 85 upper jaw, and palate for 524 amphibian species ( Fig. 1; Dataset S1). Taxa were coded as 86 "toothed" if teeth were observed on any cranial element and "edentulous" if teeth were entirely 87 absent. Our survey of amphibian dentition across the majority of extant genera confirmed that 88 all salamanders and caecilians retain teeth, while 134 of the 429 frog species examined are 89 entirely edentulous (Dataset S1). All anuran species lack dentary teeth with the exception of

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The loss of teeth may be associated with the evolution of a secondary feeding apparatus

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We identified a phylogenetic correlation between the evolution of edentulism and a 239 microphagous diet, and these two traits co-occur in more than 50 genera belonging to 14 240 families (Dataset S2 ; Fig. 3). The majority of these species specialize on eating ants and 241 termites, despite that these insects have many defense behaviors (biting, stinging, chemical

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The complete loss of teeth in frogs is associated with the shortening of the lower jaw 255 (Fig. 4), a skeletal trait that is known to occur in species that eat smaller prey (Emerson 1985,           Physalaemus, Pipa, Telmatobius, Uperodon, Uperoleia) with two sampled lineages that 381 represent known dental variation within these genera (Dataset S1). All scans were run using a 382 240kv x-ray tube containing a diamond-tungsten target, with the voltage, current, and detector 383 capture time adjusted for each scan to maximize absorption range for each specimen. Raw x-384 ray data were processed using GE's proprietary datos|x software version 2.3 to produce a 385 series of tomogram images and volumes, with final voxel resolutions ranging from 1 to 147 μm.

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The resulting microCT volume files were imported into VG StudioMax version 3.2.4 (Volume

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Graphics, Heidelberg, Germany), the skull and skeleton were isolated using VG StudioMax's 388 suite of segmentation tools, and then exported as high-fidelity mesh files. We deposited image 389 stacks (TIFF) and 3D mesh files of the skull and skeleton for each specimen in MorphoSource 390 (see Dataset S1 for DOIs).

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Survey of amphibian dentition variation and ancestral state reconstructions.

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We recorded the presence or absence of teeth on each dentigerous bone of the lower jaw, 393 upper jaw, and palate for 524 amphibian species ( Fig. 1; Dataset S1). We conducted ancestral

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(lower jaw is longer than the head) and a value less than one indicates an anteriorly shifted jaw 426 joint (lower jaw is shorter than the head).

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We used phylogenetic comparative methods to test for evolutionary correlations among 428 dentition, diet, and body size in frogs. We compiled diet records for 267 taxa, representing 258