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doi:10.1111/evo.14379
Re-evaluating the morphological evidence
for the re-evolution of lost mandibular
teethinfrogs
Daniel J. Paluh,1,2 ,3 Wesley A. Dillard,2Edward L. Stanley,1Gareth J. Fraser,2and David C. Blackburn1
1Department of Natural History, Florida Museum of Natural History, University of Florida, Gainesville, Florida 32611
2Department of Biology, University of Florida, Gainesville, Florida 32611
3E-mail: dpaluh@u.edu
Received July 16, 2021
Accepted October 5, 2021
Dollo’s law of irreversibility states that once a complex structure is lost, it cannot be regained in the same form. Several putative ex-
ceptions to Dollo’s law have been identied using phylogenetic comparative methods, but the anatomy and development of these
traits are often poorly understood. Gastrotheca guentheri is renowned as the only frog with teeth on the lower jaw. Mandibular
teeth were lost in the ancestor of frogs more than 200 million years ago and subsequently regained in G. guentheri. Little is known
about the teeth in this species despite being a frequent example of trait “re-evolution,” leaving open the possibility that it may
have mandibular pseudoteeth. We assessed the dental anatomy of G. guentheri using micro-computed tomography and histology
and conrmed the longstanding assumption that true mandibular teeth are present. Remarkably, the mandibular teeth of G. guen-
theri are nearly identical in gross morphology and development to upper jaw teeth in closely related species. The developmental
genetics of tooth formation are unknown in this possibly extinct species. Our results suggest that an ancestral odontogenic path-
way has been conserved but suppressed in the lower jaw since the origin of frogs, providing a possible mechanism underlying the
re-evolution of lost mandibular teeth.
KEY WORDS: Anura, dentition, Dollo’s law, Hemiphractidae, trait reversal.
Dollo’s law of irreversibility proposes that complex structures
that are lost over evolutionary time cannot be regained in the same
form (Gould 1970). Recent studies exploring trait evolution in a
phylogenetic context, however, suggest that many complex traits
have been lost and then regained, including wings in stick in-
sects (Whiting et al. 2003), limbs in lizards (Brandley et al. 2008),
aquatic larvae in salamanders (Chippindale et al. 2004), and teeth
on the lower jaw in frogs (Wiens et al. 2011). In most cases, the
evidence for these putative exceptions to Dollo’s law derives from
reconstructing trait evolution on phylogenies. Further evaluating
the plausibility of these exceptions requires a more thorough un-
derstanding of the anatomy, development, and genetics of these
traits (Kerney et al. 2011; Sadier et al. 2021).
Teeth originated in stem gnathostomes more than 400 mil-
lion years ago (Rücklin et al. 2012) and have been broadly
maintained across vertebrates. Within modern amphibians
(Lissamphibia), all salamanders and caecilians maintain teeth on
the upper and lowers jaws, but nearly all frogs lack dentition on
the lower jaw (i.e., mandible) and variably possess teeth on the
upper jaw and palate (Paluh et al. 2021). Of the more than 7000
species of frogs, only one species, the marsupial frog Gastrotheca
guentheri (Boulenger 1882; Hemiphractidae), possesses teeth on
the dentary bone of the lower jaw. Boulenger (1882) recognized
the distinctiveness of these mandibular teeth when describing
G. guentheri and thus placed the species in a monotypic fam-
ily (Amphignathodontidae) and genus (Amphignathodon; from
the Greek: amphí—on both sides, gnathos—jaw, and odon—
tooth). Noble (1922) illustrated the toothed mandible of Amphig-
nathodon, described the mandibular teeth as being similar to the
maxillary teeth, and noted that the mandibular teeth contained
a pulp cavity, dentin, and enamel, but presented no histological
evidence to support this. He further recognized that the species
1
© 2021 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.
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was “simply a specialized Gastrotheca,” which was later con-
firmed with immunological and molecular data (Duellman et al.
1988; Wiens et al. 2007). Laurent (1983) suggested that G. guen-
theri re-evolved mandibular teeth due to the lifting of a “very
old inhibition,” such as a suppressor regulatory gene that disap-
peared, and that the developmental and genetic basis for teeth on
the lower jaw had not been lost in the ancestors of these frogs.
Recently, Wiens (2011) used ancestral state reconstruction meth-
ods to demonstrate that mandibular teeth were lost in the ancestor
of crown-group frogs more than 200 million years ago and sub-
sequently regained in G. guentheri during the Miocene.
The “re-evolution” of mandibular teeth in frogs may be un-
likely given the exceptionally long period of time between their
loss and subsequent re-acquisition (>200 million years). Only
two brief anatomical descriptions (Boulenger 1882; Noble 1922),
one illustration (Noble 1922), and one photograph (Berkovitz and
Shellis 2016) of the teeth of G. guentheri have been published.
There remains a need to verify that the mandibular dentition of
G. guentheri is composed of true teeth. Alternatively, these struc-
tures may be bony odontoid serrations (i.e., pseudoteeth) as seen
in some other anurans, such as the mandibular odontoids found
in Cornufer guentheri and species of Hemiphractus (Shaw 1989;
Fabrezi and Emerson 2003; Paluh et al. 2021), which would
indicate that they do not represent a “re-evolved” trait. If true
mandibular teeth are present, they may be degraded or simplified,
such as through the loss of enamel or a bicuspid shape, due to de-
terioration of the odontogenic pathway. If the mandibular teeth
of G. guentheri are identical to those on the upper jaw, these re-
sults may suggest the ancestral tooth development program has
been highly conserved, but long suppressed, on the lower jaw
of frogs. Gastrotheca guentheri was always a rare species in the
cloud forests of Colombia and Ecuador, and a living specimen has
not been observed since 1996 (De la Riva et al. 2020). Museum
specimens are thus our only resource to investigate this putative
violation of Dollo’s law. Here, we re-evaluate the dental anatomy
of G. guentheri using both high-resolution micro-computed to-
mography scanning and histology.
Methods
SAMPLING
Females in the genus Gastrotheca brood developing embryos in
a pouch on their back (Duellman 2015). Some species, includ-
ing G. guentheri, have direct development (i.e., no free-living
and feeding larval stage; Gomez-Mestre et al. 2012) and young
emerge from the pouch as miniature versions of the adult. We
examined six postnatal specimens of G. guentheri that vary in
ontogenetic stage: a neonate froglet (KU:KUH:200260, 17.1 mm
snout-vent length [SVL]), two juveniles (KU:KUH:178464, 30.9
mm SVL; KU:KUH:221634, 47.6 mm SVL), and three adults
(KU:KUH:195628, 62.4 mm SVL; KU:KUH:164226, 71.5 mm
SVL; KU:KUH:221635, 78.9 mm SVL). The largest reported
body size for the species is 82.00 mm SVL (Duellman 2015).
One adult (KU:KUH:221635) was a brooding female with late-
stage embryos in its dorsal pouch, which allowed us to assess em-
bryological anatomy of individuals with an approximate SVL of
13 mm. We compared the jaw and tooth morphology of G. guen-
theri to 19 additional specimens of other Gastrotheca (Table S1;
four juveniles and 15 adults), representing 14 species and each of
the eight recognized subgenera (Duellman 2015; Duellman and
Cannatella 2018; but see Echevarría et al. 2021). Specimens for
this study are from the herpetological collections of the Univer-
sity of Kansas Biodiversity Institute (KU) and Florida Museum
of Natural History (UF).
MicroCT
To examine the dental ontogeny of G. guentheri, we generated
high-resolution X-ray computed tomography (microCT) scans of
the entire body, as well as for just the head at higher resolution,
for each specimen using a GE v|tome|x M 240 at the University of
Florida’s Nanoscale Research Facility. A-180 kV X-ray tube and
diamond-tungsten target were used for all scans. We adjusted the
voltage (between 70 and 90 kV) and current (between 100 and
200 µA) to maximize absorption range for each specimen, with
final voxel resolutions ranging from 7 to 46 µm. The raw X-ray
data were processed using the GE datos|x software to produce to-
mogram and volume files. The volume files were imported into
VG StudioMax (Volume Graphics, version 3.4), and the skull and
lower jaw were segmented using the region-growing and smooth-
ing tools. Linear measurements of the skull, jaws, and teeth were
recorded using the polyline length tool in VG StudioMax. One
late-stage embryo was removed from the pouch of the brood-
ing female (KU:KUH:221635) and scanned at 4.9 µm to iden-
tify the state of developing teeth. We additionally CT-scanned
a dissected segment of the upper jaw (maxilla and premaxilla)
and lower jaw (dentary) of one juvenile specimen of G. guen-
theri (KU:KUH:221634) at 1.6 µm to examine the jaw and dental
anatomy at high resolution prior to histological sectioning. Tooth
counts (number of attached functional teeth and number of to-
tal tooth loci) were recorded for each paired dentigerous element
(left and right maxilla, premaxilla, vomer, dentary) in the series
of G. guentheri and in the additional 19 specimens of other Gas-
trotheca species for the right side of the skull only. All resulting
CT data are available via MorphoSource (see Table S1).
HISTOLOGY
We dissected upper and lower jaw tissue from a juvenile speci-
men of G. guentheri (KU:KUH:221634) to evaluate the cellular
structure and tissue composition of the upper jaw, lower jaw, and
corresponding dentition. For comparison, we sampled two other
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species of Gastrotheca (Gastrotheca peruana, UF:Herp:65788;
Gastrotheca riobambae, UF:Herp:98222) and a representative
from the Hylidae (Osteopilus septentrionalis, UF:Herp:171513),
a family that is closely related to the Hemiphractidae (Feng et al.
2017; Streicher et al. 2018). We removed one late-stage embryo
from the dorsal pouch of a brooding female (KU:KUH: 221635);
there is no staging table for direct-developing hemiphractid
frogs, but the staging table developed for Eleutherodactylus
by Townsend and Stewart (1985) suggests that this embryo is
at a stage just before hatching (Townsend-Stewart [TS] 14 or
15). The embryo and the dissected jaw tissues from G. guen-
theri and the comparative species were decalcified in EDTA for
48hand4−6 days, respectively, and processed for paraffin sec-
tioning using a standard protocol. The embedded paraffin blocks
were sectioned to 8−11 µm on a Leica RM2145 microtome and
stained using a standard Hematoxylin and Eosin (H&E) Alcian
blue protocol (Johanson et al. 2019). Slides were imaged on
a Leica DM2500 LED compound microscope. We additionally
stained the G. guentheri jaw sections using a fluorescent DAPI
(diamidino-2-phenylindole) stain to visualize cell nuclei and as-
sess gross cell morphology.
Results
DENTAL ONTOGENY IN G. guentheri
Pedicellate, bicuspid teeth are present on the maxilla, premax-
illa, vomer, and dentary in all six postnasal G. guentheri speci-
mens examined (Fig. 1). Tooth size and shape vary little across
the four dentigerous elements within each specimen, indicating a
homodont condition. Pedicel height (range: 0.3−1.2 mm), pedi-
cel width (range: 0.1−0.4 mm), and crown height (range: 0.1−
0.4 mm) increase with body size (Table S2; Fig. S1). Tooth num-
ber varies considerably across the four dentigerous elements and
through ontogeny (Fig. 1; Table S2). The vomer has the fewest
functional teeth (range: 0−5) and tooth loci (range: 3−7) and
the maxilla has the most (functional teeth range: 11−40; tooth
loci range: 28−53). The smallest postnatal individual examined
has seven to 10 functional teeth and 21 tooth loci on each den-
tary, whereas the three adults have 27−30 functional teeth and
40−49 tooth loci on each dentary. The relative increase in tooth
loci and functional teeth is similar on the maxilla and dentary
through ontogeny (Fig. S2). The dentition in G. guentheri first
develops as an alternating series in which each functional tooth
on the premaxilla, maxilla, dentary, and vomer is separated by
a locus undergoing replacement (either empty, undergoing re-
sorption, or developing the next generation tooth; Fig. 1). In the
three adult specimens examined, this alternating sequence is bro-
ken on the maxilla and dentary where several functional teeth
are adjacent to one another (a similar pattern has been shown
in the closely related Hemiphractus; Shaw 1989). On all ele-
ments, the teeth are replaced using a one-for-one tooth replace-
ment system, which is typical of amphibians (Davit-Béal et al.
2007). The late-stage embryos within the pouch of the brood-
ing female (KU:KUH:221635) do not have observable teeth, al-
though many skeletal elements are undergoing ossification, in-
cluding those of the axial skeleton (vertebral column, urostyle),
appendicular skeleton (ilium, femur, tibiofibula, humerus), and
skull (basicranium, maxilla, premaxilla). The lower jaw is not
observable in the microCT data and thus presumably not yet os-
sified.
Gastrotheca TOOTH COUNT VARIATION
The number of functional teeth and tooth loci generally increases
with body size across all species of Gastrotheca examined and
for all four dentigerous elements (Fig. 2; Table S1). The numbers
of functional teeth and tooth loci on the premaxilla, maxilla, and
vomer in the G. guentheri series are within the range observed
across other species of Gastrotheca.However,G. guentheri often
has fewer teeth and tooth loci than congeners at a similar body
size. For example, an adult Gastrotheca weinlandii, which is the
sister taxon to G. guentheri (Wiens et al. 2011), has 15 more tooth
loci on the right maxilla and two more tooth loci on the right
premaxilla and right vomer than a similarly sized G. guentheri.
Dentary teeth are present only in G. guentheri.
HISTOLOGY
True teeth were confirmed present in the upper and lower jaws
of G. guentheri through standard histology stained with H&E
and Alcian blue (Figs. 3 and 4). A successional dental lamina
is present on both the upper and lower jaws of G. guentheri,
suggesting a similar method of tooth addition and replacement.
The dental lamina is a band of dental epithelium continuous with
the oral epithelium. It invaginates into the underlying dental mes-
enchyme and continuously develops new teeth in polyphyodont
vertebrates (Fraser et al. 2020). This essential tissue for tooth re-
placement appears to play an important and conserved role in
both tooth development and replacement in the dentary teeth of
G. guentheri. Replacement teeth at various stages of development
(from tooth buds to ankylosed functional teeth) are in close asso-
ciation with the dental lamina and are composed of a pulp cavity,
odontoblasts, dentin, ameloblasts, enamel organ, and a thin layer
of enamel. Similar dental tissues were observed in the replace-
ment teeth on the upper jaw in Osteopilus septentrionalis, G. pe-
ruana,andG. riobambae (Fig. 4). In the four species examined,
the tissue composition and progression of tooth replacement on
the upper jaw resembles previously published dental descriptions
in toothed frogs (Rana pipiens, Gillette 1955; Hyla cinerea,Goin
and Hester 1961; Xenopus laevis, Shaw 1979; Hemiphractus pro-
boscideus, Shaw 1989). The dental lamina of the upper jaw ex-
tends on the lingual side of each functional tooth, from which the
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Figure 1. Dental ontogeny of Gastrotheca guentheri. (A) 17.1 mm snout-vent length (SVL) neonate, KU:KUH:200260; (B) 30.9 mm SVL
juvenile, KU:KUH:178464; (C) 47.6 mm SVL juvenile, KU:KUH:221634; (D) 62.4 mm SVL adult, KU:KUH:195628; (E) 71.5 mm SVL adult,
KU:KUH:164226; and (F) 78.9 mm SVL adult, KU:KUH:221635. Skulls in lateral view: dentigerous cranial elements are colored. Isolated
premaxilla (yellow), maxilla (green), dentary (blue), and vomer (purple) in lingual views. Scale bars =1 mm.
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Figure 2. Relationship between tooth loci number and log transformed skull length on the (A) maxilla, (B) premaxilla, (C) vomer, and
(D) dentary across the Gastrotheca guentheri ontogenetic series (black points) and other Gastrotheca species (gray points). Tooth loci
counts were recorded from the right side of each specimen. Specimen point labels are provided in Figure S3 and corresponding data are
provided in Table S1.
next tooth will develop. The dental lamina of the lower jaw in G.
guentheri also extends on the lingual side of each functional tooth
and is similar to the lower jaw dental morphology seen in sala-
manders (Davit-Béal et al. 2007). There is no evidence of a dental
lamina or dentition on the lower jaw in Osteopilus,G. peruana,
or G. riobambae (Fig. 4).
Although no teeth are visible in the microCT reconstruc-
tion of the late-stage G. guentheri embryo, histological sections
confirm that odontogenesis has initiated on both the upper and
lower jaws with the formation of the primary dental lamina that
gives rise to the first-generation teeth (Fig. 5). The dental lam-
ina on the upper jaw is proliferating into a cup shape at its ex-
tremity and is therefore at a more advanced developmental stage
than the dental lamina of the lower jaw. Due to tissue shrink-
age that likely resulted from poor initial preservation of the de-
veloping embryos in the dorsal pouch, the oral epithelium and
dental lamina have detached from the underlying mesenchyme
(see Fig. 5).
Discussion
Perhaps our most remarkable finding is that the teeth on the lower
jaw in G. guentheri are nearly identical in gross morphology, his-
tology, and development to the teeth on the upper jaw and palate.
Our microCT and histological results confirm the longstanding
assumption that G. guentheri has true teeth on the lower jaw. Bi-
cuspid, pedicellate teeth are present on the upper jaw (premaxilla
and maxilla), palate (vomer), and lower jaw (dentary) through-
out postnatal ontogeny, and the tissue composition of the denti-
tion, from dental lamina to erupted teeth, is similar on both jaws.
Tooth size and shape are similar across the tooth-bearing bones
within each G. guentheri specimen, but tooth size increases with
body size. The numbers of functional teeth and tooth loci increase
through ontogeny on all four dentigerous elements. This is gener-
ally consistent with the limited data for dental ontogeny available
from other anuran species (Smirnov and Vasil’eva 1995; Davit-
Béal et al. 2007). Gastrotheca guentheri has relatively fewer teeth
and tooth loci on the premaxilla, maxilla, and vomer than sev-
eral other species of Gastrotheca that attain a similar body size
(Fig. 2). One explanation for this pattern is that a consequence
of re-evolving mandibular teeth might be a reduction in tooth
number on other dentigerous elements due to the energetic costs
of continuous dental development and replacement on an addi-
tional skeletal element. Alternatively, tooth development in G.
guentheri may be delayed (see below), and this slowed devel-
opment could be responsible for the reduced tooth count. Other
likely consequences of re-evolving mandibular teeth warrant fur-
ther investigation, such as modifications to the blood supply and
innervation of the lower jaw and modifications to tongue mor-
phology.
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Figure 3. High-resolution dental anatomy of Gastrotheca guentheri (KU:KUH:221634) from the upper jaw (A–C) and lower jaw (D–F and
G–I) based on microCT volume segmentation (A and D), microCT tomograms (B and E), DAPI stained sections (C and F), and H&E-stained
sections (G–I). Abbreviations: ab, ameloblasts; as, angulosplenial; c, crown; d, dentary dt, dentin, e, enamel, ft, functional tooth; ob,
odontoblasts; oe, oral epithelium; pc, pulp cavity; pd, pedicel; pm, premaxilla; rt, replacement tooth; sdl, successional dental lamina; tb,
tooth bud. Scale bars =100 µm.
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Figure 4. Jaw anatomy of Osteopilus septentrionalis (microCT of UF:Herp:63656, histology of UF:Herp:171513), Gastrotheca peruana
(microCT of UF:Herp:65783, histology of UF:Herp:65788), G. riobambae (both UF:Herp:98222), and G. guentheri (both KU:KUH:221634)
based on microCT volume segmentation (center; green =maxilla, blue =dentary; anterior is to the left) and H&E-stained sections of the
upper law (left) and lower jaw (right). Upper jaw teeth are present in all species and mandibular teeth are present only in G. guentheri.
Abbreviations: ab, ameloblasts; as, angulosplenial; d, dentary dt, dentin, e, enamel; ob, odontoblasts; oe, oral epithelium; m, maxilla; pc,
pulp cavity; rt, replacement tooth; sdl, successional dental lamina.
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Figure 5. Anatomy of a late-stage Gastrotheca guentheri embryo (A) removed from the pouch of a brooding female (KU:KUH:221635)
examined using histology (B) and microCT (C). (D) Inset of mouth opening outlined in panel B. No teeth are visible in the microCT re-
construction of the embryo, but histological sections show the initiation of tooth development on the upper and lower jaws with the
formation of the primary dental lamina. Abbreviations: oe, oral epithelium; pdl, primary dental lamina.
The initiation of odontogenesis typically occurs during early
larval or embryonic development (Lainoff et al. 2015), includ-
ing in salamanders and caecilians (Davit-Béal et al. 2007). In
frogs that have a biphasic life history, the onset of tooth devel-
opment is delayed and typically occurs during metamorphosis
(Gosner Stages [GS] 40−46; Smirnov and Vasil’eva 1995, Davit-
Béal et al. 2007) or during the mid-larval phase in Xenopus laevis
(Niewkoop-Faber Stage 53−55/GS 31−35; Shaw 1979, Davit-
Béal et al. 2007). Tooth formation likely occurs ontogenetically
late in frogs because the anuran mouth undergoes dramatic re-
structuring during metamorphosis (McDiarmid and Altig 1999),
transitioning from an herbivorous tadpole with a keratinized beak
and pseudoteeth (i.e., keratodonts or labial teeth) to an insectiv-
orous frog with true teeth. Tooth development has not been di-
rectly studied in any frogs that have direct development, but the
evolution of this life history mode may provide an opportunity to
repattern the jaw and alter the timing of tooth germ initiation.
No erupted teeth are present in hatchlings of Eleutherodacty-
lus jasperi (Eleutherodactylidae; Wake 1978) or Pseudophilautus
silus (Rhacophoridae; Kerney et al. 2007), although tooth buds
are present and undergoing mineralization on the upper jaw of E.
jasperi up to 4 days prehatching (Wake 1978). Maxillary teeth
are visible in Haddadus binotatus (Craugastoridae) in the final
stage (TS 15) prior to hatching (Vera Candioti et al. 2020), but
it is difficult to interpret from the images of cleared-and-stained
specimens whether these teeth are erupted and functional. Cumu-
latively, these observations suggest that odontogenesis does not
shift to earlier embryonic stages in direct-developing frogs. The
embryo of G. guentheri that we examined is likely near the final
stage of development prior to hatching (TS 14 or 15) because of
the presence of fully developed limbs, toepads, and dorsal pig-
mentation. If true, tooth development may be further delayed in
G. guentheri in comparison to what little is known in other direct-
developing lineages. The earliest stages of odontogenesis (for-
mation of the primary dental lamina; no mineralization present)
may begin shortly before emerging from the maternal pouch. The
stage of tooth development between the upper and lower jaws is
slightly offset with the dental lamina of the upper jaw being more
advanced, forming into a cup shape at its extremity (Davit-Béal
et al. 2007).
Little is known of the ecology of G. guentheri. The stomach
contents of one specimen (KU:KUH:164226) revealed that this
species is capable of consuming large prey relative to its body
size, suggesting that mandibular dentition may play some role in
prey capture (Paluh et al. 2019). The species has also been re-
ported to consume small vertebrates, including frogs and lizards
(Arteaga et al. 2013; De la Riva et al. 2020). The shape, hyper-
ossification, and articulation of the skull in G. guentheri are simi-
lar to other carnivorous frog species that specialize on eating large
vertebrate prey, such as Ceratophrys,Pyxicephalus,Cornufer
guentheri,andHemiphractus (Paluh et al. 2020). These frogs are
typically sit-and-wait predators that independently evolved bony
odontoid fangs and serrations on the lower jaw that are thought
to improve prey capture but are not true teeth (Fabrezi and Emer-
son 2003). Wiens (2011) speculated that the repeated evolution of
odontoid fangs in these anurans suggests that selection can favor
tooth-like structures on the mandible, but true teeth have not re-
evolved in any of these taxa due to an unspecified developmental
constraint. The longstanding question of how G. guentheri re-
gained mandibular dentition after more than 200 million years
of absence in frogs and their nearest relatives (Boulenger 1882;
Noble 1922; Laurent 1983; Wiens et al. 2011) remains chal-
lenging to investigate using developmental genetic techniques
due to the lack of living specimens and tissue resources for this
species.
Recent work has documented that teeth have been com-
pletely lost more than 20 times in frogs, which is a higher oc-
currence of edentulism than in any other major vertebrate lineage
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(Paluh et al. 2021). The analyses of Paluh et al. (2021) also in-
dicated as many as six reversals from a complete edentulous to
a toothed state and at least one reversal from toothless vomers to
toothed vomers in Hylambates (formerly Phlyctimantis)leonardi
(Hyperoliidae). Five of the six inferred reversals from toothless to
toothed jaws occurred in the Microhylidae. These results suggest
that the regain of mandibular teeth in G. guentheri is only one of
several dental reversals in frogs, although still the only reversal
documented on the dentary. The remaining inferred reversals re-
quire further anatomical and developmental investigation to con-
firm that these all represent true teeth and not, for example, small
bony serrations that lack the tissue composition of true dentition
(Shaw et al. 1989).
Several studies have used comparative methods to identify
traits that seem to reject Dollo’s law (Whiting et al. 2003; Bran-
dley et al. 2008; Wiens et al. 2011), but few of these phyloge-
netic patterns have been further scrutinized using integrative ap-
proaches to assess the developmental and genetic mechanisms
responsible for the evolutionary regain of these traits (Collin and
Miglietta 2008). The lack of developmental studies has hindered
the interpretation of “re-evolved” traits, especially because many
of these structures may only be absent in adult life stages (Sadier
et al. 2021). If lost structures are developmentally transient, their
gene regulatory networks and developmental pathways are likely
retained and provide a mechanism for recovering lost phenotypes
(Collin and Miglietta 2008; Kerney et al. 2011; Smith-Paredes
et al. 2021). Similarly, Wake et al. (2011) hypothesized that the
re-evolution of serially repeated structures in different positions
within an organism may be more likely to occur through the
retention of ancestral developmental pathways. We hypothesize
that a suppressed tooth developmental program may be main-
tained in the lower jaw of anurans and is disrupted by a con-
served mechanism that originated in the ancestor of all frogs.
Triadobatrachus—the oldest known “stem frog” that lived during
the Early Triassic—already lacked teeth on the lower jaw (Ascar-
runz et al. 2016), and the absence of mandibular dentition is con-
sidered a synapomorphy of Salientia (Milner 1988). Presumably
this suppression was somehow removed to generate true teeth on
the lower jaw in G. guentheri. If true, signaling molecules and
transient rudimentary structures, such as early thickenings of the
oral epithelium that typically gives rise to tooth buds, might be
seen before the abortion of tooth development in the mandible of
frogs.
The loss of mandibular teeth in anurans may be due to the
loss of a single signal that orchestrates odontogenesis and thus
arrests tooth formation early in development. This would then be
comparable to the loss of odontogenic Bmp4 expression in birds
(Chen et al. 2000) or termination of Msx2 expression in turtles
(Tokita et al. 2013). Alternatively, Wiens (2011) suggested that
because most frog species maintain teeth on the premaxilla, max-
illa, and vomer, G. guentheri may have re-evolved mandibular
teeth by using the developmental machinery used in the forma-
tion of upper jaw teeth. If true, the entire odontogenic pathway
may have decayed in the lower jaw of all other frogs and thus
there may be no early signs of tooth development. Regional tooth
loss among different regions of the skull has repeatedly occurred
in vertebrates, including in fishes (Aigler et al. 2014), amphibians
(Paluh et al. 2021), squamates (Voris 1966), and birds (Brockle-
hurst and Field 2021). The gene regulatory network that controls
tooth initiation, development, and differentiation is likely modu-
lar (Stock 2001; Sadier et al. 2020), and this organization may
be responsible for shaping this pattern of heterogeneous tooth
loss across decoupled jaw regions. Odontogenesis is poorly un-
derstood in amphibians, especially when compared to our un-
derstanding of tooth development in fishes and amniotes (Fraser
et al. 2004; Tucker and Sharpe 2004; Thiery et al. 2017). It is not
yet known if all the genes critical for tooth formation in fishes
and amniotes are also expressed during the morphogenesis of
teeth in amphibians (but see Soukup et al. 2021). Investigating
the developmental genetics of tooth formation in the jaws of frogs
may provide insights into whether a transient tooth signaling pro-
gram is present in the lower jaw, providing the possible mech-
anism underlying the re-evolution of lost mandibular teeth in
G. guentheri.
ACKNOWLEDGMENTS
This research was supported by the National Science Foundation Grad-
uate Research Fellowship to D.J.P. under Grants DGE-1315138 and
DGE-1842473. The authors thank R. Brown and L. Welton (University
of Kansas Biodiversity Institute) for access to specimens. The authors
thank L. Echevarria for providing access to the Gastrotheca pulchra scan.
CT scanning was performed as part of the openVertebrate (oVert) The-
matic Collections Network (National Science Foundation DBI-1701714,
to DCB). The authors also thank three anonymous reviewers for helpful
comments that improved an earlier version of this manuscript.
AUTHOR CONTRIBUTIONS
DJP curated the data, performed visualization, and wrote the original
draft. DJP, GJF, and DCB conceptualized the idea of the study. DJP and
DCB acquired funding. GJF provided resources. All authors performed
investigation and reviewed and edited the manuscript.
DATA ARCHIVING
Computed tomography data have been deposited in MorphoSource (see
Table S1). All measurement and tooth count data are available in Tables
S1 and S2.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Associate Editor: T. Kohlsdorf
Handling Editor: A. G. McAdam
Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article.
Figure S1. Ontogenetic variation in pedicel height (left), crown height (center), and pedicel width (right) on the maxilla (row A), premaxilla (row B),
vomer (row C), and dentary (row D) in Gastrotheca guentheri.
Figure S2. Increase in tooth loci and functional tooth number on the maxilla and dentary (right and left side of each specimen) in Gastrotheca guentheri
through ontogeny.
Figure S3. Relationship between tooth loci number and log transformed skull length (Fig. 2) with specimen point labels. Corresponding data are provided
in Table S1.
Tab l e S1. Species and specimens examined in this study with associated measurement and tooth count (functional teeth and total tooth loci) data.
Tab l e S2. Linear measurement (in mm) and tooth count (functional teeth and total tooth loci) data for Gastrotheca guentheri specimens examined.
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