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Dias et al. Zoological Letters (2024) 10:17
https://doi.org/10.1186/s40851-024-00241-0 Zoological Letters
†Pedro Henrique dos Santos Dias and Jackson R. Phillips contributed
equally to this work.
*Correspondence:
Pedro Henrique dos Santos Dias
pedrodiasherpeto@gmail.com
Philippe J. R. Kok
philippe.kok@biol.uni.lodz.pl; pjrkok@gmail.com
1Leibniz Institut zur Analyse des Bioaliationersitätswandels,
Zoologisches Museum Hamburg, Zentrum für Taxonomie und
Morphologie, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany
2Utah State University, 5305 Old Main Hill, Logan, Utah 84322, USA
3CONICET - Agencia INTA General Acha, Avellaneda 530 (8200), General
Acha, La Pampa, Argentina
4Coastal Plains Institute and Land Conservancy, 1313 Milton Street,
Tallahassee, Florida 32303, USA
5Department of Ecology and Vertebrate Zoology, Faculty of Biology and
Environmental Protection, University of Łódź, 12/16 Banacha Str.,
Łódź 90-237, Poland
6Life Sciences, The Natural History Museum, Cromwell Road,
London SW7 5BD, UK
7Department of Biological Science, Florida State University, Tallahassee,
Florida 32303, USA
Abstract
Tadpoles serve as crucial evidence for testing systematic and taxonomic hypotheses. Suctorial tadpoles collected in
Guyana were initially assigned to Rhaebo nasicus through molecular phylogeny. Subsequent analysis of larval and
adult morphological traits revealed synapomorphies within the clade encompassing R. nasicus and R. ceratophrys,
prompting the recognition of a new genus described herein as Adhaerobufo. The new genus is distinguished
from other bufonids by specic phenotypic traits including an enlarged, suctorial oral disc with distinct papillae
arrangements, and the presence of certain muscles and narial vacuities at the larval stage. However, only a few
adult external characteristics (e.g., enlarged eyelids, infraocular cream spot), seem to be reliably discriminative from
related genera. This study underscores the signicance of larval morphology in anuran systematics and oers new
insights into the evolution of suctorial and gastromyzophorous larvae within bufonids.
Keywords Evolution, Larval traits, Musculoskeletal system, Pantepui, Suctoriality, Systematics, Taxonomy
The remarkable larval morphology of Rhaebo
nasicus (Werner, 1903) (Amphibia: Anura:
Bufonidae) with the erection of a new
bufonid genus and insights into the evolution
of suctorial tadpoles
Pedro Henrique dos SantosDias1*†, Jackson R.Phillips2†, Martín O.Pereyra3, D. BruceMeans4,7, AlexanderHaas1 and
Philippe J. R.Kok5,6*
Page 2 of 26Dias et al. Zoological Letters (2024) 10:17
Introduction
While adult traits have dominated the eld of anuran
systematics, biologists have long recognized the poten-
tial of larval morphology in better understanding evo-
lutionary relationships. e earliest instance of a larval
trait being used in this way can be traced to the late 19th
century, when the French zoologist Fernand Lataste [1]
proposed a new classication of frogs based on the posi-
tion of spiracles. A few years earlier, Pizarro [2] had pro-
posed the erection of the genus Batrachychthis for the
bizarre tadpoles of Pseudis. During the following century,
the impact of larval morphology on the systematics and
taxonomy of anurans was further explored, especially by
Noble, who published a series of papers [3–8] advocating
the use of larval characters and natural history informa-
tion in the classication of amphibians. Later, Orton [9]
published a seminal paper in which she proposed that
four major groups of frogs could be recognized based on
larval characters (see also [10]).
Although some authors have argued against the usage
of larval characters in taxonomic and systematic studies
(e.g., [11, 12]), tadpoles are largely recognized as a source
of useful evidence for such studies (e.g., [13–20]). For
instance, Haas [21] used larval morphology to propose
a new anuran phylogeny that anticipated several phylo-
genetic trends that have since been supported by the fol-
lowing generation of large-scale molecular studies (e.g.,
[22–23]).
e past two decades have witnessed constant growth
in studies on tadpoles and the exploration of larval char-
acters. Grosjean et al. [24] set a benchmark of the impor-
tance of larval characters in systematics, describing a
new species based on its tadpole — Clinotarsus penelope
(Ranidae). Several bizarre and previously unknown lar-
val phenotypes have been described (e.g., [25–32]), and
many new characters and synapomorphies for dierent
groups have been proposed (e.g., [33–47]). In the present
paper, we discuss the impact of larval morphology on the
systematics and taxonomy of a clade of toads of the fam-
ily Bufonidae.
e true toads, bufonids, are one of the most diverse
and speciose anuran clades, with a nearly cosmopolitan
distribution (found on all continents except Australia and
Antarctica [48]). Currently, the 655 recognized species
are allocated in 54 genera [48]. Bufonid diversity is also
reected in their numerous reproductive strategies and
developmental modes (e.g., [49–58]). Bufonid tadpoles
are also quite diverse, and while many genera have con-
served a lentic-benthic larval phenotype (e.g., [59]), there
is signicant variation in ecology and morphology within
the family, including inter alia suctorial (sucker mouth)
and gastromyzophorous (belly sucker) forms, which rep-
resent adaptations to life in fast-owing waters (e.g., [33,
60, 61]), phytotelma dwellers with endotrophic nutrition
(e.g., [62–64]), open-water species with large, vascular
crests [65], semiterrestrial tadpoles that live on wet rocks
(e.g., [66]), and direct developers that retain larval traits
(e.g., [55]). However, the tadpoles of many bufonid spe-
cies remain unknown, and while some have assumed that
their larval morphology will likely prove to be a typical
benthic, lentic form, tadpoles continue to surprise us.
e Pantepui biogeographical region is located in
northeastern South America, in the western Gui-
ana Shield highlands, and is famed for its iconic table
mountains of Proterozoic sandstone (locally known as
“tepuis”). Tepuis are remnants of an enormous landmass
(called the Roraima Supergroup or Mataui Formation)
resulting from the sedimentation and subsequent uplifts
of sandstones produced by the erosion of ancient Gond-
wanan highlands [67–69]. Over the last two decades
a substantial number of new endemic amphibian spe-
cies (e.g., [70–86], to only cite a few) and even endemic
genera and families [87–89] have been described from
the region, highlighting the importance of this often
neglected biome in the evolution of Neotropical amphib-
ians (see also [90, 91]).
During multiple expeditions in the Eastern Pantepui
uplands and highlands of Guyana, DBM and PJRK
observed and collected series of brightly colored tad-
poles in fast-owing mountain streams. Until recently,
these larvae were assumed to be Atelopus cf. hoogmoedi
based on overall external characteristics and microhabi-
tat (fast-owing mountain streams). However, a closer
examination of the suctorial apparatus and recent molec-
ular phylogenetic analyses indicated that these larvae do
not belong to the genus Atelopus and should instead be
assigned to Rhaebo nasicus. As the tadpole of R. nasicus
is undescribed, we re-examined the larvae of these Pan-
tepui “Atelopus” in detail. Our new ndings strongly
impact the understanding of the taxonomy of these toads
and the evolution of bufonid tadpoles more generally.
Materials and methods
Sample determination, molecular data collection and
analyses
Species assignment
Adults were assigned to Rhaebo nasicus based on external
morphology characters, such as the eyelid projection. In
Guyana, R. nasicus is the only species known to present
this character-state. Additional to the phylogenetic place-
ment, the tadpoles were assigned to the family Bufonidae
based on the presence of larval synapomorphies of the
family: anterolateral process of crista parotica absent,
m. diaphragmatopraecordialis absent, lateral bers of
m. subarcualis rectus II–IV invading branchial septum,
larval lungs rudimentary, and a single pair of infralabial
papillae [21, 33]. In Guyana, there are four genera of
bufonids: Atelopus, Oreophrynella, Rhaebo, and Rhinella
Page 3 of 26Dias et al. Zoological Letters (2024) 10:17
[48]. All known tadpoles of Atelopus present a belly
sucker [32], and Oreophrynella exhibits endotrophic
development [53, 55, 92]. Tadpoles of Rhaebo guttatus,
Rhinella marina, and R. merianae have been described
[93]. us, these tadpoles could only be assigned to R.
nasicus, R. beebei, R. martyi, or R. nattereri (the three lat-
ter being absent from our collection localities).
Tissue sampling, DNA extraction, amplication and
sequencing
Genomic DNA was isolated from a small piece of the tail
of a preserved tadpole (whole larva xed in 99% ethanol
in the eld) from Mount Wokomung, Guyana (CPI10704;
05˚00’08”N, 59˚52’47”W at 1,573m elevation) and from
liver tissues of two adult Rhaebo nasicus (tissues xed
in 99% ethanol in the eld) from two localities in Guy-
ana: Kaieteur National Park (IRSNB14518 [PK1348];
05˚08’N, 59˚25’W at ca. 540m elevation), and the slopes
of Maringma-tepui (PK1895; 05˚12’28”N, 60˚33’60”W at
1,060m elevation).
Tissue samples were digested overnight at 56°C in a
solution of 5 µL of proteinase K and 100 µL of lysis buf-
fer (100 mM NaCL, 100 mM Tris, 25 mM EDTA, 0.5%
SDS). DNA extraction was performed using Sera-Mag™
SpeedBeads™ (ermo Fisher Scientic) at a concen-
tration of ca. 1.7 × (105µl of digested tissue to 180 µL
of beads) and eluted into 200µl of 10 mM Tris buer.
Using polymerase chain reaction (PCR; for primers and
PCR conditions see [94]), we amplied a fragment of the
barcoding 16S ribosomal RNA gene (16S; 507 base pairs
[bp]). PCR amplications were conrmed on a 1% aga-
rose gel, and negative controls were run on all ampli-
cations to exclude contamination. PCR products were
puried, and Sanger sequenced (along both strands using
the same primers used for PCR) at the Natural History
Museum’s (NHM, London, UK) sequencing facility.
Chromatograms were assembled and edited in Codon-
Code Aligner 10.0.2 (Codon Code Cooperation, Ded-
ham, USA). Novel sequences have been catalogued in
GenBank (PQ200682–PQ200684). e newly generated
sequences were uploaded onto BLAST NCBI (https://
blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the most
similar sequences on GenBank.
Sequences editing and alignment settings
Based on the results of the BLAST analysis and guided
primarily by the phylogenetic frameworks established by
[95–97], we designed a sampling strategy to determine
the placement of the sequenced specimens and eluci-
date their evolutionary relationships. Accordingly, our
phylogenetic analyses focused on a mitochondrial frag-
ment comprising the 12S RNA, tRNA valine, and 16S
RNA genes (12s-trna-val-16s), complemented by three
nuclear loci: a fragment of the C-X-C motif chemokine
receptor 4 gene (cxcr4), a fragment of the proopiomelano-
cortin gene (pomc), and a fragment of the recombination
activating 1 gene (rag1) for 82 bufonid specimens and
12 outgroups. Sequences were aligned using MAFFT v7
online software [98–99] with the strategy E-INS-i (for the
12s-trna-val-16s fragment) and L-INS-i (for remaining
fragments). Subsequently, the individual alignments were
concatenated using SequenceMatrix v1.8 [100], result-
ing in a nal alignment of 4,668bp. e brachycephaloid
Ischnocnema guentheri was used as the outgroup for tree
rooting. Details regarding specimens, locality data, and
GenBank accession numbers for the sequences used in
our analyses are provided in Appendix MS1.
Maximum parsimony phylogenetic analysis
Phylogenetic analysis under Maximum Parsimony (MP)
was performed in TNT version 1.6 [101, 102] using
“New Technology” searches and treating gaps as a fth
state. e analysis utilized a combination of secto-
rial searches, ratchet, and tree-fusing techniques [103,
104] until the consensus tree was stabilized 10 times
(see [103]). e parameters set of the search were:
xmult = replications 10 ratchet 5 drift 5 fuse 5 consense
10. e support for each clade was evaluated by esti-
mating two types of resampling support-measures for
the datasets: (1) parsimony jackknife absolute frequen-
cies (JAF; [105]) and (2) parsimony jackknife frequency
dierences (JGC; [106]). Jackknife supports were esti-
mated performing 1000 replicates using “New Technol-
ogy” searches with the following settings: xmult = hit 2
replications 12 xss fuse 3.
Maximum likelihood phylogenetic analysis
For Maximum Likelihood (ML) analysis, we initially
determined the best partition scheme and correspond-
ing models of nucleotide evolution using ModelFinder
[107], as implemented in IQ-TREE 2.2.0 [108] with the
command TESTNEWMERGEONLY. Coding genes
were partitioned by codon position, while mitochondrial
sequences (non-coding) were considered as a single par-
tition. Dened initial partitions are detailed in Appendix
MS2.
Subsequently, we searched for the best ML tree in
IQ-TREE 2.2.0 with the partition scheme and models of
nucleotide evolution selected by ModelFinder. We per-
formed 10 independent searches with dierent values
of perturbation parameter (-pers option) and the tree
with the highest likelihood was selected as the optimal
tree. For searches we consider edge linked-propor-
tional partition model but separate substitution mod-
els and rate evolution between partitions (-spp option).
e maximum-likelihood tree was conducted with
1000 ultrafast bootstrap (UFBoot) replicates (-B 1000
option; [109]).
Page 4 of 26Dias et al. Zoological Letters (2024) 10:17
Genetic distances
Uncorrected pairwise distances (UPDs) were calculated
in PAUP* [110] for a dataset of the 16S gene (507bp,
aligned in MAFFT under the
G-INS-i strategy) and containing only sequences of
species of Rhaebo (see Appendix MS3).
Larval morphology
Larval morphology description is based on four tadpoles
in developmental stages 25–38 (sensu [111]): three tad-
poles (stages 25–26) housed in the National Museum of
Natural History, Smithsonian Institution (USNM 592409-
11) and one individual (CPI10704) at stage 38 (whose
skeleton remains preserved [CPI10704]). All these larvae
were originally collected as a single lot (CPI10704) in the
Kamana Creek on Mount Kopinang of the Wokomung
Massif in Guyana (site MK4; 05˚00′08′′N, 59˚52′47′′W
at 1,573m elevation). Terminology for external morphol-
ogy characters follows [112, 113]. For the study of inter-
nal morphology, one tadpole in stage 38 (CPI10704) was
submitted to the clearing and double staining protocol of
[114]; the process was stopped after the alcian blue step,
and the specimen was manually dissected for inspection
of larval muscles. After photographic documentation
of muscle characters, the palatoquadrate and the hyo-
branchial skeleton were gently disarticulated; upper and
lower jaws were separated and the buccopharyngeal cav-
ity exposed for study of its morphology. After recording
characters from muscles and buccopharyngeal cavity, we
concluded the clearing process for the study of the larval
cranium and hyobranchial morphology. Terminology for
the musculoskeletal system follows [47]; buccopharyn-
geal cavity follows Wassersug [19, 115].
Additionally, one tadpole in Gosner stage 25 (USNM
592409) was stained with phosphotungstic acid [116] and
subjected to high-resolution micro-computed tomogra-
phy (µCT). e tadpole was µCT-scanned using a Nikon
X TH 225 ST 2x µCT scanner. Volumetric reconstruction
was performed in Nikon CT agent and post-processed in
VG Studio Max. Finally, we also examined other tadpoles
of dierent bufonid species (See Appendix MS4).
Adult morphology
We investigated the adult osteology of one individual of
Rhaebo nasicus housed at the Royal Belgian Institute of
Natural Sciences (IRSNB14518) using µCT scans. e
individual was µCT-scanned using a YXLON FF20 CT.
We also µCT-scanned two adult R. ceratophrys (UTA-
A4061, UTA-A4062) and two adult R. haematiticus
(UTA-A57567, UTA-A57572) housed in the herpetologi-
cal collection of the University of Texas, Arlington, using
a Nikon X TH 225 ST 2x µCT scanner. Some additional
species were studied for osteology in (1) cleared and
double stained specimens prepared following the tech-
niques of Wassersug [117] and (2) reconstruction from
µ-CT scans (see Appendix MS4).
Evolution of suctoriality
We performed a parsimony optimization of tadpoles’
general ecomorphological types in the bufonid tree
of life. e evolution of ecomorphological types was
assessed using ancestral character state reconstruction
as modeled on Fitch’s [118] optimization on the Portik
et al.’s [97] topology using TNT [101, 102]. Ecomorpho-
logical information was taken from Vera Candioti et al.
[57].
Results
Phylogenetic analyses and genetic distances
A summary tree of Rhaebo and other bufonids is shown
in Figs.1 and 2 (for complete topologies, see MS5 and
MS6). e topologies inferred by the MP and ML anal-
yses consistently recover our new sequences within a
highly supported clade along with Rhaebo ceratophrys
and R. nasicus (JAF and JGC = 100%; UFBoot = 100%).
e new sequences PQ200683 [IRSNB14518 (PK1348)]
and PQ200684 (PK1895) were similar to the only
available sequence of Rhaebo nasicus in GenBank
(DQ158477 = ROM20650 [erroneously reported as
ROM20560], from Tukeit in Kaieteur National Park, Guy-
ana) with a genetic distance ranging from 0.21 to 0.43%.
On the other hand, the sequence PQ200682(CPI10704)
was recovered as the sister lineage of that clade showing a
genetic distance ranging from 4.84 to 5.78%. Rhaebo cer-
atophrys is, in turn, sister to the clade composed by the
three new sequences and R. nasicus ROM20650. In the
MP analysis (Fig.1), the clade R. ceratophrys + R. nasicus
collapses in a polytomy with (1) a moderately well sup-
ported clade (JAF = 93%, JG C = 90%) composed of the
remaining included species of Rhaebo, (2) the highly
supported Peltophryne (JAF and JGC = 100%) and (3) a
moderately well supported clade (JAF = 92%, JGC = 89%)
composed of the “New World” Anaxyrus, Incilius and
Rhinella, and all the sampled “Old World” bufonids. In
the ML analysis (Fig.2), the internal topology of the clade
R. ceratophrys + R. nasicus is mostly identical to the MP
analysis, nevertheless, the relations of this clade with
other bufonids are less conicting. e clade R. ceratoph-
rys + R. nasicus is recovered as sister of the remaining
Rhaebo with high support (UFBoot = 98%), and Rhaebo
is sister to Peltophryne with low support (UFBoot = 55%).
Finally, Rhaebo + Peltophryne are sister to a well-sup-
ported clade (UFBoot = 100%) composed of the “New
World” Anaxyrus, Incilius, and Rhinella, and all the sam-
pled “Old World” bufonids.
Page 5 of 26Dias et al. Zoological Letters (2024) 10:17
Larval morphology
External morphology (Figs.3, 4 and 5)
Body compressed (Fig.3A), elliptical in dorsal (Fig.3B)
and lateral views. Snout rounded in dorsal view, sloped in
lateral view. Nostrils positioned dorsofrontally, elliptical,
with a medial eshy projection, anterolaterally directed.
Eyes dorsal, laterally directed. Nasolacrimal duct visible
(Fig. 3B). Spiracle sinistral, lateral, short, directed pos-
teroventrally; centripetal wall presents as slight ridge.
Digestive tract coiled; switchback point laterally dislo-
cated from the center of abdominal region. Vent tube
medial, directed posteroventrally, short, distal portion
free from ventral n. Tail higher than body; tail muscle
almost reaching tail tip; tail tip rounded. Dorsal and ven-
tral ns convex, about the same height; higher portions
between the middle and posterior thirds of the tail. Dor-
sal n originating on the tail. Lateral line system barely
visible in preserved material. Oral disc (Fig.4) enlarged,
positioned and directed ventrally, laterally emarginate; a
single, continuous row of conical, marginal papillae; no
gaps in marginal papillation; submarginal papillae pres-
ent, in all extension of the lower lip and laterally in the
upper lip, with multiple parallel rows. Labial tooth row
formula (LTRF) 2/3; A1 and A2 length subequal; P2 and
P3 length subequal, slightly longer than P1. Jaw sheaths
present, serrate, keratinized; upper jaw sheath arch-
shaped (slightly less keratinized medially in the photo-
graphed specimen); lower jaw sheath V-shaped.
Fig. 1 Summary tree of the maximum parsimony analysis depicting the relationships of Rhaebo and other Bufonidae. This tree represents the stabilized
strict consensus derived from three most parsimonious trees (of length 15,782 steps). Values at nodes are parsimony jackknife frequencies (absolute/fre-
quency dierences). The numbers between parentheses following the names of genera denote the total condensed terminals at that tip. The complete
MP strict consensus tree is shown in MS5
Page 6 of 26Dias et al. Zoological Letters (2024) 10:17
Color in life
In life (Fig.5), the overall coloration is yellow-gold dor-
sally and ventrally but is divided into ve yellow-gold
bands by four transverse dark bands of approximately
the same width. e tadpole snout is yellow-gold from
the tip to the eye, then the narrowest yellow-gold band
encircles the midbody with an overwash of dark pigment.
e posterior one-third of the tadpole body is densely
black set o by the rst of three yellow bands on the tail,
the tip of which is the last yellow-gold band. e oral disc
is translucent. Ventral views reveal a fading of the dark
banding pattern along the body, with translucent skin
oering glimpses of internal organs. Upon preservation,
the vibrant hues subside, and the yellow-gold bands take
on a cream-colored appearance separated by dark brown
bands with scattered light brown blotches (Fig.2).
Buccopharyngeal cavity (Fig.6)
Buccal roof (Fig.6A) triangular. Prenarial arena (Fig.6C)
rectangular, with a triangular protuberance. Internal
nares elliptical (Fig.6C), transversally oriented; posterior
valve free, with small, triangular projections in the ante-
rior wall. Vacuities present, circumscribed by margins
of inner nares. Postnarial arena diamond-shaped, two
conical, short postnarial papillae. Lateral ridge papillae
short, trifurcated. Median ridge low, triangular, with a
medial notch at its apex. Buccal roof arena poorly delim-
ited, dened by a single pair of conical papillae each side.
Glandular zone poorly dened. Dorsal velum medi-
ally continuous, devoid of papillae or projections, arch
shaped.
Buccal oor (Fig. 6B) triangular. Single pair of at,
wide, branched, infralabial papillae; small papilla-like
structures after mouth opening (Fig. 6D). Lingual bud
well developed, rounded; lingual papillae absent. Buccal
oor arena bell-shaped; 7–8 papillae each side. Buccal
oor arena lacking pustulations. Prepocket papillae and
pustulation absent. Buccal pockets deep, wide, oblique
slit shaped. Ventral velum present; spicular support
conspicuous; medial notch absent; secretory pits poorly
developed; secretory ridges present. Branchial basket tri-
angular, short, poorly developed, wider than long.
Larval cranium (Fig.7)
Neurocranium longer than wide; greatest width at the
subocular bar level (Fig. 7A–B). Suprarostral cartilage
(Fig.7C) formed by the suprarostral alae and suprarostral
corpora; both corpora are medially fused and connected
to the proximal region of the triangular alae. An adro-
stral tissue mass is present close to the posterior process
Fig. 2 Summary tree of the maximum likelihood analysis depicting the relationships of Rhaebo and other Bufonidae. Values at nodes are bootstrap
values. The numbers between parentheses following the names of genera denote the total condensed terminals at that tip. The complete ML tree is
shown in MS6
Page 7 of 26Dias et al. Zoological Letters (2024) 10:17
of the alae (Fig.7C); under dissection, it did not appear to
be chondried, but histological analysis should be done
to conrm. Ethmoidal region short; trabecular horns
long, diverging in a “V” pattern; trabecular horns greatly
expanded anteriorly. Basicranial fenestra weakly chondri-
ed, partially occluded by a thin membrane. Taenia tecti
medialis and transversalis present and conuent (Fig.7A),
dividing the frontoparietal fontanelle in three. Orbital
cartilage low. Otic capsules robust, rhomboidal in dorsal
view, representing ca. 1/4 of chondrocranium length; syn-
otic tectum connects the two capsules. Palatoquadrate,
thin in lateral view, attached to neurocranium through a
wide anterior quadratocranial commissure and an almost
perpendicular ascending process. Articular process wide.
Fig. 4 The oral disc of “Rhaebo” nasicus (CPI10704) tadpole at stage 38 in natural, preserved coloration (A) and stained with methylene blue to highlight
anatomical features (B). Scale bars = 1.0mm. Photos by Pedro H. Dias
Fig. 3 The tadpole of “Rhaebo” nasicus (CPI10704) at stage 38 in lateral (A), dorsal (B), and ventral (C) views. Scale bar = 1.0mm. Photos by Pedro H. Dias
Page 8 of 26Dias et al. Zoological Letters (2024) 10:17
Muscular process triangular, well-developed, and curved
dorsomedially. Connection between the tip of the muscu-
lar process and the neurocranium through a chondried
quadrato-orbtial commissure. Palatoquadrate C-shaped,
clearly concave; posterior curvature of palatoquadrate
reaching the level of the otic capsules.
In the lower jaw (Fig. 7D), Meckel’s cartilage sigmoid,
transversely oriented, almost perpendicular to the chondro-
cranium longitudinal axis. Infrarostral cartilages rectangular
in frontal view, curved, joined at the symphysis (Fig.7D).
Ceratohyals (Fig.7E) long, at, and subtriangular; ante-
rior margin with well-developed anterior and anterolat-
eral processes; posterior processes triangular and long.
Ceratohyals conuently joined by a chondried pars
reuniens. Basibranchial rectangular, with rounded uro-
branchial process present. Basihyal absent. Hypobran-
chial plates long, triangular. Branchial basket with four
curved ceratobranchials bearing lateral projections.
Ceratobranchial I with a triangular anterior branchial
process, continuous with the hypobranchial plate. Cera-
tobranchials II and III joined by the proximal commis-
sure. Four long, curved spicules projecting dorsally from
the ceratobranchials. Ceratobranchials distally joined by
terminal commissures.
Muscles (Figs.8, 9 and 10)
We identied 32 muscles (Table 1); most of Rhaebo
nasicus muscles followed general patterns of origin and
insertion of other bufonids and other anurans (Figs.8, 9
and 10). Interestingly, the lateral bers of the subarcualis
rectus II–IV invade the interbranchial septum IV (Fig.9)
and the presence of the rectus abdominis anterior.
Visceral components
Digestive tract short; coiled gut with switchback point
sinistral. Liver enlarged, occupying a signicant portion
of the abdominal cavity. Lungs short, inated, pigmented.
Adult morphology
e adult morphology of both Rhaebo nasicus and R.
ceratophrys has been widely reviewed in the literature,
including aspects of their osteology (e.g., [96, 119–121]).
e most obvious shared character between R. nasicus
and R. ceratophrys is the presence of an enlarged eyelid
in both species (although more distinctly projecting in R.
ceratophrys). An infraocular cream spot is also evident in
adult specimens of both species. Additionally, both spe-
cies share a narrow sphenethmoid (see below).
Pramuk dened the “Bufo guttatus group” (= Rhaebo)
as presenting two unique, unreversed synapomorphies:
the sphenethmoid in ventral view is distinctively broad,
and the posterior process of the prootic is prominent and
notched ([121]:434). Pramuk did not consider B. nasicus
to be part of that clade and stressed that B. nasicus and
the B. guttatus group share the presence of a well-devel-
oped omosternum and an elongated transverse process of
Fig. 5 Living tadpole of “Rhaebo” nasicus in right lateral (A), dorsal (B), and ventral (C) views. Photos by D. Bruce Means
Page 9 of 26Dias et al. Zoological Letters (2024) 10:17
vertebra VI ([121]:434). However, most of the osteologi-
cal characters for B. nasicus were missing in her analysis.
Ron et al. ([95]:354) proposed a redenition for the
states “narrow” and “distinctively broad” for the sphen-
ethmoid condition of Pramuk ([121]: ch35), considering
the species of Rhaebo to have a “wide condition” due to
the lateral edges of the sphenethmoid being in contact
with the frontoparietals. In species where the frontopa-
rietals do not extend to the anterior portion of the orbit
(e.g., Peltophryne), a more accurate denition of the
“wide” condition of the sphenethmoid could be as fol-
lows: the sphenethmoid reaches the margin of the orbit
immediately posterior to the palatines. Both “R.” cera-
tophrys and “R.” nasicus have a narrow condition of the
sphenethmoid (i.e, the sphenethmoid does not reach the
margin of the orbit immediately posterior to the pala-
tines), dierentiating them from other Rhaebo (Fig.11).
e narrow condition of the sphenethmoid is also
observed in bufonids closely related to “R.” ceratophrys
and “R.” nasicus, such as Amazophrynella, Nannophryne
and Peltophryne, which suggests the wide sphenethmoid
to be a synapomorphy of Rhaebo sensu stricto.
Regarding the second synapomorphy of Rhaebo pro-
posed by Pramuk ([121]; i.e, posterior process of the
prootic prominent and notched), Ron et al. [95] pointed
out a perceived error in the identication of the ante-
rior prootic processes (sic) by Pramuk [121], stating that
they were, in fact, the occipital condyles, which are part
of the exoccipital rather than the prootic. However, both
structures are clearly illustrated and identied in Fig.5A
of Pramuk’s work [121], suggesting a possible misunder-
standing of these structures by Ron et al. [95], so we fol-
low Pramuk [121]. In this regard, “R.” ceratophrys and
“R.” nasicus have prominent and notched posterior pro-
cess of the prootic as other species of Rhaebo, but also
seen in some species of Peltophryne ([121]: Fig.2).
Fig. 6 The buccopharyngeal cavity of “Rhaebo” nasicus (CPI10704) tadpole at stage 38. Buccal roof (A) and oor (B) morphologies, with details of the pre-
and postnarial arenas (C) and of the infralabial and lingual (D) regions. BFA, buccal oor arena; BFAP, buccal oor arena papillae; BRAP, buccal roof arena
papillae; DV, dorsal velum; ILP, infralabial papillae; IN, internal nares; LR, lateral ridge; MR, median ridge; NV, narial vacuities; PNP, postnarial arena papillae;
TA, tongue anlage; TP, triangular projection; UJ, upper jaw sheath; VV, ventral velum. Scale bars = 1.0mm. Photos by Pedro H. Dias
Page 10 of 26Dias et al. Zoological Letters (2024) 10:17
Comments on the taxonomic and systematic history of
“Rhaebo” ceratophrys and “Rhaebo” nasicus
Rhaebo ceratophrys was rst described in 1882 by Bou-
lenger ([122] as Bufo ceratophrys) based on a juvenile
specimen from Ecuador (BMNH 1880.12.5.151). e spe-
cies was characterized by a unique feature, a long eyelid
projection. Since then, the species has been transferred to
several dierent species groups within the former genus
Bufo. For instance, Gallardo [123] allocated it in the B.
marinus group, whereas Cei [124], Hoogmoed [120] and
Pramuk [121] considered the species as belonging to the
B. typhonius/margaritifer group.
e taxonomic history of Rhaebo nasicus has also
been convoluted. Werner [125] found a specimen of
an unknown toad (IRSNB1015, formerly IRSNB4792),
which he named Bufo nasicus. Later, studies of the con-
tents of its digestive tract suggested a South American
origin (Smith and Laurent 1950), and eventually Hoog-
moed [120] accessed additional specimens from Guyana
and Venezuela. He compared these individuals with the
redescription and illustrations made by Smith and Lau-
rent [126], identifying them as the Bufo nasicus of Wer-
ner [125]. Hoogmoed [120] redescribed the species and
suggested it to be restricted to the Guiana Shield. Hoog-
moed [120] noted the presence of an enlarged eyelid in
B. nasicus and argued that the shared presence of such
an eyelid in B. nasicus and in B. ceratophrys, as well
as similar color patterns indicate a close relationship
between the two species. Hoogmoed [120] also noted
that B. ceratophrys was much smaller than B. nasicus — a
Fig. 7 The larval cranium of “Rhaebo” nasicus (CPI10704) tadpole at stage 38. Dorsal (A), ventral(B) views, details of the suprarostral (C) and Meckel’s
cartilage (D), and hyobranchial apparatus (E). ALPH, antelateral process hyalis; AP, articular process; APH, anterior process hyalis; AT, adrostral tissue; CB,
constrictor branchialis; CH, ceratohyal; HP, hypobranchial plate; HQP, hyoquadrate process; IR, infrarostral cartilage; JF, jugular foramen; LP, lateral process;
MC, Meckel`s cartilage; MP, muscular process; OC, otic capsule; PCM, proximal commissure; PP, posterior process; PU, process urobranchialis; QOC, quadro-
orbital commissure; SA, suprarostral ala; SB, subocular bar; SC, suprarostral copora; SP, spicule; TH, trabecular horns; TS, tectum synoticum; TTM, taenia tecti
medialis; TTT, taenia tecti transversalis. Scale bars = 1.0mm. Photos by: Pedro H. Dias
Page 11 of 26Dias et al. Zoological Letters (2024) 10:17
Fig. 9 The larval muscles of “Rhaebo” nasicus (CPI10704) tadpole at stage 38 in ventral view. CB, constrictor branchialis; HA, hyoangularis; IH, interhyoideus;
IM, intermandibularis; OH, orbitohyoideus; QA, quadrato-angularis; RA, rectus abdominis; RC, rectus cervicis; SAR I, subarcualis rectus I; SAR II–IV, subarcua-
lis rectus II–IV; SO, subarcualis obliquus. Scale bars = 1.0mm. Photos by Pedro H. Dias
Fig. 8 The larval muscles of “Rhaebo” nasicus (CPI10704) tadpole at stage 38 in ventral view (A); detail of the tendon of the m. rectus abdominis anterior
(B). HA, hyoangularis; IH, interhyoideus; IHP, interhyoideus posterior; IM, intermandibularis; OH, orbitohyoideus; RA, rectus abdominis; RAA, rectus abdomi-
nis anterior. Scale bars = 1.0mm. Photos by: Pedro H. Dias
Page 12 of 26Dias et al. Zoological Letters (2024) 10:17
misinterpretation repeated by others (e.g., [127]), since
the holotype of B. ceratophrys is a juvenile.
Pramuk [121] performed an extensive phylogenetic
analysis of Bufonidae, using both morphology and
molecular data. She recovered Bufo nasicus as sister to
other species of the Bufo guttatus group of Blair [128]
in all analyses (morphological, mitochondrial genes,
nuclear genes, and combined analyses). She also exam-
ined specimens of B. ceratophrys (her appendix 1; p.443),
but the species does not appear in any of her phyloge-
netic hypotheses. Concurrently, Frost et al. [23] resur-
rected the genus Rhaebo to accommodate species of the
Bufo guttatus group of Blair [128]. ey also transferred
Bufo ceratophrys and Bufo nasicus to the genus Rhinella.
Finally, Frost [23], considering the evidence of Pramuk
[121], transferred Rhinella nasica to Rhaebo, which
remains the current taxonomy.
Fenolio et al. [119] recognized that the diagnosis of
Rhaebo ceratophrys (as Rhinella ceratophrys) proposed
by Hoogmoed [120] needed to be revised in the light of
new collections. ey performed a detailed morphologi-
cal study and provided a new diagnosis for the species,
recognizing it as having: (1) triangular projecting dermal
aps on the eyelids, (2) projecting dermal aps at the cor-
ners of mouth, and (3) a larger adult size ([119]:10).
Ron et al. [95] studied the poorly known genus Andi-
nophryne (now included in Rhaebo). ey performed
separate phylogenetic analyses for mitochondrial and
Fig. 10 The larval muscles of “Rhaebo” nasicus (CPI10704) tadpole at stage 38 in dorsal (A-C) and lateral (D-E) views. LMA, levator mandibulae articularis;
LMEP, levator mandibulae externus profundus; LMES, levator mandibulae externus super cialis; LMI, levator mandibulae internus; LMLS, levator mandibu-
lae longus supercialis; OH, orbitohyoideus; SA, suspensorioangularis; SH, suspensoriohyoideus. Scale bars = 1.0mm. Photos by: Pedro H. Dias
Page 13 of 26Dias et al. Zoological Letters (2024) 10:17
Table 1 Muscles origin and insertion in the larva of “Rhaebo” nasicus
Muscle Origin Insertion Comments
Mandibular group,n. trigeminus (c.n. V) innerved
Levator mandibulae longus supercialis External posterior margin of subocular
bar
Dorsomedial Meckel’s cartilage Via long tendon
Levator mandibulae longus profundus External margin (curvature) of subocu-
lar bar
External margin of suprarostral ala Via a long tendon
Levator mandibulae longus internus Ventral otic capsule and processus
ascendens
Lateral Meckel`s cartilage Via a long tendon
Levator mandibulae externus supercialis Inner muscular process (superior) Adrostral tissue mass
Levator mandibulae externus profundus Inner muscular process (medial) Distal suprarostral ala Share a tendon
with LMLP
Levator mandibulae articularis Inner muscular process (inferior) Dorsal Meckel’s cartilage
Levator mandibulae lateralis Articular process Adrostral tissue mass
Submentalis (intermandibularis anterior) - -
Intermandibularis Median aponeurosis Ventromedial Meckel’s cartilage
Mandibulolabialis Ventromedial Meckel’s cartilage Lower lip
Mandibulolabialis superior - -
Hyoid group, n. facialis (c.n. VII)
Hyoangularis Dorsal ceratohyal Retroarticular process of Meckel’s
cartilage
Quadratoangularis Ventral palatoquadrate Retroarticular process of Meckel’s
cartilage
Suspensorioangularis Ventral palatoquadrate Retroarticular process of Meckel’s
cartilage
Orbitohyoideus Muscular process Lateral edge of ceratohyal
Suspensoriohyoideus Posterior descending margin of muscu-
lar process and subocular bar
Lateral process of ceratohyal
Interhyoideus Median aponeurosis Ventral ceratohyal
Branchial group, n. Glossopharyngeus (c.n. IX) and vagus (c.n. X)
Levator arcuum branchialium I Lateral subocular bar Ceratobranchial I
Levator arcuum branchialium II Lateral otic capsule Ceratobranchial II
Levator arcuum branchialium III Lateral otic capsule Ceratobranchial III
Levator arcuum branchialium IV Lateroventral otic capsule Ceratobranchial IV
Tympanopharyngeus Lateroventral otic capsule Ceratobranchial IV
Constrictor branchialis I - -
Constrictor branchialis II Branchial process II Terminal commissure I
Constrictor branchialis III Branchial process II Terminal commissure II
Constrictor branchialis IV Ceratobranchial III Terminal commissure II I
Subarcualis rectus I Posterior lateral base of ceratohyal Branchial processes II and III, and
ceratobranchial I
Subarcualis rectus II-IV Ceratobranchial IV Ceratobranchial II Lateral bers invad-
ing the interbran-
chial septum IV
Subarcualis obliquus II Urobranchial process Ceratobranchials II Single slip
Diaphragmatobranchialis Peritoneum (diaphragm) Distal Ceratobranchial III
Spinal group, spinal nerve innervation
Geniohyoideus Hypobranchial plate Infrarostral cartilage At the level of CB III
Rectus abdominis Peritoneum (diaphragm) Pelvic girdle Six open myomers
Rectus abdominis anterior Peritoneum (diaphragm) Ventral palatoquadrate Very short bers;
via a long tendon
Rectus cervicis Peritoneum (diaphragm) Branchial process III
Page 14 of 26Dias et al. Zoological Letters (2024) 10:17
nuclear data, placing Andinophryne olallai and A. colo-
mai within Rhaebo —Rhaebo nasicus was sister to all
other Rhaebo + Andinophryne. Rhinella ceratophrys was
not included in that study. According to the authors,
they preferred to synonymize Andinophryne with Rhaebo
rather than erecting a new genus for Rhaebo nasicus, as
their study did not include all Rhaebo. Other large-scale
studies (e.g., [129–130]) have also recovered Rhaebo
nasicus as sister to all other Rhaebo (Fig.12).
Pereyra et al. [96] studied the evolution and system-
atics of Rhinella with a large and dense taxon sam-
pling (including an extensive outgroup sampling). ey
included representatives of Rhinella ceratophrys in a phy-
logenetic analysis for the rst time. In their total evidence
analysis under Maximum Parsimony ([96]: Fig.10), they
recovered Rhaebo nasicus as sister to Rhinella ceratoph-
rys and the rest of Rhaebo as sister to Rhaebo nasicus
and “Rhinella” ceratophrys + other bufonids, render-
ing both Rhinella and Rhaebo non-monophyletic. e
authors transferred R. ceratophrys to Rhaebo, despite the
paraphyly of Rhaebo, arguing that their analysis was not
designed to rigorously test the monophyly of Rhaebo.
Recently, Portik et al. [97] published a large study on
the phylogeny of anurans. ey included seven of the
14 valid species assigned to Rhaebo. In their topology,
Rhaebo nasicus and Rhaebo ceratophrys are sister taxa
and together form the sister group to all other Rhaebo.
In summary, neither Rhaebo nasicus or R. ceratophrys
have ever been recovered as nested within other Rhaebo
species in any phylogenetic hypothesis (Fig.12) and the
most inclusive analysis of Bufonidae strongly supports
the clade formed by Rhaebo nasicus and R. ceratophrys
as the sister clade to all other Rhaebo. e larval mor-
phology of other Rhaebo species is a generalized, ben-
thic type (e.g., [131–132]), while the larval morphology
of R. nasicus (and likely R. ceratophrys) is a specialized
torrential form (see above). erefore, combining larval
and adult morphological synapomorphies for the clade
of R. nasicus and R. ceratophrys (e.g., [96, 119, 120]; this
study), along with phylogenetic evidence supporting
their monophyly([96, 97]; this study), we propose that
this clade should be recognized as new genus, which is
named hereafter.
Taxonomic account
Adhaerobufo gen. nov.
ZooBank registration urn: lsid: zoobank.org: act:
C757A1FA-A343-4134-8371-6A42797F162A
Type species Bufo nasicus (Werner, 1903 [125]) comb.
nov.
Immediately more inclusive taxon Bufonidae Gray,
1825 [134].
Fig. 11 Variation in the sphenethmoid morphology in Amazophrynella, Nannophryne, Peltophryne, and Rhaebo sensu lato (sl). Dierent cranial bones are
colored as follows for reference: blue (maxilla and premaxilla), dark grey (vomers), light grey (parasphenoid), pink (palatines), and yellow (sphenethmoid).
Figures of Peltophryne guentheri and Rhaebo colomai were redrawn and slightly modied from [95] and [121]
Page 15 of 26Dias et al. Zoological Letters (2024) 10:17
Content Adhaerobufo ceratophrys (Boulenger, 1882
[122]) comb. nov, and Adhaerobufo nasicus (Werner, 1903
[125]) comb. nov.
Etymology Adhaerobufo gen. nov. (gender masculine)
is derived from the Latin adhaerens, meaning adherent
and the Latin būfo, meaning toad. e name refers to the
unique suctorial morphology of their tadpoles.
Denition and diagnosis: Adhaerobufogen. nov. can be
dierentiated from all other Bufonidae by the combina-
tion of the following characters: (1) tadpole with enlarged,
suctorial, oral disc; (2) tadpole oral disc with a complete
row of marginal papillae; (3) tadpole oral disc with mul-
tiple rows of submarginal papillae on the lower lip and
by a single row of marginal papillae on the upper lip; (4)
tadpole oral disc with an uninterrupted second anterior
row of keratodonts; (5) presence of the m. interhyoideus
posterior at larval stage; (6) presence of the m. rectus
abdominis anterior at larval stage; (7) presence of narial
vacuities in the buccopharyngeal cavity at larval stage;
(8) projecting, enlarged eyelid in adults; (9) presence of
an infraocular cream spot in adults, (10) sphenethmoid
relatively narrow, overlapping only the medial ends of the
palatines; and (11) posterior process of the prootic promi-
nent and notched.
Fig. 12 Summarized relationships of “Rhaebo” nasicus (and R. ceratophrys when included) according to the several published phylogenetic hypotheses
for Bufonidae: Pramuk ([121]: Fig.1; morphological data alone, MP tree); Pramuk et al. ([133]: Fig.1; molecular data alone, Bayesian analysis tree); Ron et al.
([95]: Fig.1; molecular data alone, ML tree); Pereyra et al. ([96]: Fig.10; phenotypic + molecular data, MP tree), and Portik et al. ([97]: Fig.57; molecular data
alone, ML tree)
Page 16 of 26Dias et al. Zoological Letters (2024) 10:17
Comment e genetic diversity observed in Adhaero-
bufogen. nov. strongly suggests the occurrence of at least
one additional species within the genus (see Appendix
MS3). Given the imprecise type locality of A. nasicus
and the high genetic divergence observed between the
sequences of the tadpole and adult specimens from sev-
eral dierent localities, there is likely a hidden diversity in
the genus, with more species to be described.
Further comparisons with other genera
Larval characters
Adhaerobufo gen. nov. presents several of the bufonid
larval synapomorphies, such as the absence of the m.
diaphragmatopraecordialis, the lateral bers of m. subar-
cualis rectus II-IV invading branchial septum IV, the lar-
val lungs being rudimentary or absent, and presence of
single pairs of infralabial papillae. Nevertheless, it lacks
other bufonid synapomorphies, such as the oral disc with
a wide ventral gap in marginal papillae and the absence of
the m. interhyoideus posterior.
e complete row of marginal papillae dierentiates
Adhaerobufogen. nov. from all other bufonids, includ-
ing other members of Rhaebo. e enlarged, suctorial
disc dierentiates Adhaerobufogen. nov. from all other
bufonids except Ansonia, Blaira, Phrynoidis, and Wer-
neria. e lack of a belly sucker dierentiates it from
Adenomus, Atelopus, Bufo (part; Bufo aspinus), Rhinella
(part; Rhinella veraguensis group), and Sabahphrynus.
e uninterrupted second anterior row of keratodonts
dierentiates Adhaerobufo gen. nov. from most gen-
era, except Amazophrynella, Phrynoidis, and Werneria
(although some few species within some genera, such
as, Adenomus, Ansonia, Atelopus, Bufo, Bufotes, Capen-
sibufo, Ingerophrynus, Melanophryniscus, Rhinella, and
Sclerophrys, have been reported lacking the interrup-
tion). e multiple rows of submarginal papillae in the
lower lip and a single row of marginal papillae in the
upper lip dierentiate Adhaerobufo gen. nov. from all
genera but Werneria. e presence of the m. interhyoi-
deus posterior dierentiates it from all other bufonids
except Amazophrynella. Finally, the presence of narial
vacuities in the buccopharyngeal cavity dierentiates
Adhaerobufo gen. nov. from all other bufonids except
Ansonia, Atelopus, Incilius (part; Incilius coniferus),
Schismaderma, and Werneria.
Several Rhaebo species have their tadpoles described,
including R. glaberrimus, R. guttatus, R. haematiticus,
and R. caeruleostictus (e.g., [131, 132, 135]). None of
these species has a suctorial form, and most typify the
benthic, lentic type common across bufonids. Several
other members of the genus have no published data on
tadpole morphology, and to our knowledge no collec-
tions have been made. Previous attempts to collect the
tadpole of Rhaebo olallai have been unsuccessful, and
while recently metamorphized froglets were found along-
side a fast-owing mountain stream in the Ecuadorian
Andes, no tadpoles were found within the stream ( [136],
Trageser S., pers comm).
Finally, we would like to stress that a phenotypically
similar tadpole from Amazonia, which shares all exter-
nal morphology characters with A. nasicus, including the
color pattern, the enlarged oral disc, and the complete
row of marginal papillae, is awaiting formal description
(T. Grant and T. Pezzuti, pers comm). Individuals of that
species in late Gosner developmental stage [111] present
a dorsal color pattern very similar to that of adults of A.
ceratophrys (thus contra juveniles of Rhaebo [95]). Like-
wise, juveniles of A. nasicus have the same color pattern
as adults, both in life and in preservative.
Adult characters
As noted by Hoogmoed [120], Adhaerobufo nasicus and
A. ceratophrys share a projecting ap above the eyelid.
is character is especially pronounced in A. ceratophrys,
where it is enlarged to form a spiny projection above the
eye. e lateral surfaces of head and body (including the
ventral portion of the parotoid macroglands) in Adhaero-
bufogen. nov. are dark, similar to some species of Rhaebo
(e.g., R. blombergi, R. guttatus, R. haematiticus). Nev-
ertheless, both species have a well-dened infraocular
cream spot. e combination of dark pattern contrast-
ing with an infraocular cream spot is a putative synapo-
morphy of Adhaerobufo gen.nov., as it does not occur
in other related genera of Bufonidae. Adhaerobufogen.
nov. shares several characters previously associated with
Rhaebo, including an elongate transverse process of ver-
tebra VI, well-developed omosternum, and large and
notched posterior processes of the prootic ([95]: Fig. 6
for R. blombergi and R. colomai, the authors pers. obs.).
Adhaerobufogen. nov. diers from Rhaebo in having a
distinctly narrow sphenethmoid.
Distribution (Fig.13)
Northwestern Guyana and eastern Venezuela (Adhaero-
bufo nasicus) and upper Amazon Basin in western Brazil,
southeastern Colombia, eastern Ecuador, northeastern
Peru, and southern Venezuela (A. ceratophrys).
Natural history
Tadpoles of Adhaerobufo nasicus were scraped by aquar-
ium-mesh dip-net from the sides of large, submerged
boulders of Roraima Supergroup sandstones in the bed
of Kamana Creek, upstream within 100 m of Kamana
Waterfall (Fig.14A), draining Mt. Kopinang, one of the
peaks of the Wokomung Massif, Potaro-Siparuni Dis-
trict, Guyana. Tadpoles were observed clinging by their
mouthparts to the vertical sides of big boulders on 7
December 2006 (DBM-3372); 18 July 2007 (observed
Page 17 of 26Dias et al. Zoological Letters (2024) 10:17
when water was shallow on 18 July but not collected on
19 July due to torrential ow overnight); and 25 June
2012 (CPI10704). Figure12B is a view of an unnamed
stream on the slopes of Maringma-tepui on 22 Novem-
ber 2007 where Adhaerobufo tadpoles were also observed
(same ecological data as above). Figure14C–D are of an
amplexing pair of A. nasicus in situ (Wokomung Massif)
when rst discovered on 20 July 2012 (14C), and shortly
thereafter when placed on a leaf for photography (14D).
Amplexus is inguinal, and couples have been observed
in shallow waters, on the side of rivers. Tadpoles and
adults were observed in similar microhabitats at the base
and on the slopes of Maringma-tepui in western Guyana
in November 2007 (e.g., Fig.14B), and in the La Escal-
era region of Venezuela in November 2010. Adult indi-
viduals were collected/observed all year long in Kaieteur
National Park (west-central Guyana), although tadpoles
were not found at that location. In Kaieteur National
Park adults were often found relatively far away from any
fast-owing streams suggesting either periodical migra-
tion to suitable breeding sites, or plasticity in egg deposi-
tion site. Since we never collected any A. nasicus tadpole
in non-owing waterbodies, we favor the rst hypothesis.
Discussion
Larval morphology, systematics, and taxonomy
e impact of larval morphology on the systemat-
ics of bufonids has been widely discussed recently [33,
63]. Larval synapomorphies of Bufonidae are: (1) oral
disc with wide ventral gap in marginal papillae; (2)
anterolateral process of crista parotica absent; (3) m.
diaphragmatopraecordialis absent; (4) lateral bers of m.
subarcualis rectus II–IV invading branchial septum IV;
(5) larval lungs rudimentary or absent; (6) the m. interhy-
oideus posterior absent; and (7) a single pair of infralabial
papillae [21, 33, 47, 63]. Additionally, several synapomor-
phies have been reported for less inclusive clades (e.g.,
[33, 60, 63, 137, 138]).
Adhaerobufo nasicus shares several of these synapo-
morphies, but reverted some states; for instance, it is
characterized by the complete row of marginal papillae
and by presenting the m. interhyoideus superior. Addi-
tionally, other autapomorphic traits are present in the
larvae of Adhaerobufo gen. nov., such as (1) the enlarged,
suctorial, oral disc; (2) multiple rows of submarginal
papillae in the lower lip and by a single row of marginal
papillae in the upper lip; and (3) the presence of narial
vacuities in the buccopharyngeal cavity. e combination
of traits supports Adhaerobufo gen. nov. in Bufonidae
but also distinguishes it from all other bufonids.
Adhaerobufo gen. nov. has been consistently recov-
ered as either sister taxon of Rhaebo (e.g., [96, 97, 121].,;
this work, Fig.2) or closely allied to this genus ( [96]; this
work Fig. Figure1) and the morphology of their larvae is
unique—especially in comparison with “typical” Rhaebo
larvae (Fig. 15)—including several apomorphic trans-
formations, supporting our proposition of a new genus.
Furthermore, additional characters from adult mor-
phology and osteology also underscore the distinctive-
ness of this taxon. e genus Rhaebo has relatively few
potential synapomorphies, and the widened shape of the
sphenethmoid has been used previously as an important
Fig. 13 Geographical distribution of Adhaerobufogen. nov. in northwestern Guyana, eastern Venezuela and upper Amazon Basin. Inset map of South
America, highlighting the geographical area occupied by the genus (A). Known distribution of A. ceratophrys and A. nasicus (B). Examples of macrohabi-
tats in which the new genus is present; Kaieteur Falls in Guyana (C), uplands and highlands of western Guyana (D), and lowlands, Amazon Forest, Icá River,
Brazil (E). Shape les of the geographical distribution were downloaded from the IUCN website. Adult and tadpole are from A. nasicus. Photos by: Philippe
Kok (C and D) and Pedro H. Dias (E)
Page 18 of 26Dias et al. Zoological Letters (2024) 10:17
generic trait. We nd that Adhaerobufogen. nov. lacks
this character, therefore, the inclusion of A. nasicus and
A. ceratophrys in Rhaebo would potentially destabilize
its taxonomy. Additionally, by recognizing Adhaerobufo
gen. nov.as a new genus, Andinophryne can be revali-
dated without aecting the monophyly of Rhaebo. We
refrained from making this change, as we have not per-
sonally examined specimens of R. olallai or R. colomai.
Furthermore, Ron et al. [95] suggested some phenotypic
characters, including a widened sphenethmoid, to sup-
port Andinophryne as part of Rhaebo.
Ron et al. [95] also mentioned that the coloration pat-
tern of juveniles, described as “dorsal coloration con-
sisting of a dark background with contrasting thin clear
stripes or dots”, could be a synapomorphy of Rhaebo. e
fact that (1) tadpoles closely related to A. ceratophrys
(see above) in late developmental stage already present
the adult dorsal color pattern; and (2) that juveniles of A.
nasicus have the same color pattern as the adults suggests
that the synapomorphy proposed by Ron et al. [95] sup-
ports the monophyly of Rhaebo, including Andinophryne.
Nevertheless, just as in Rhaebo sl, Peltophryne juveniles
change their color pattern (e.g., [139, 140], which could
aect the optimization of that character. us, we rec-
ommend caution when considering this potential syn-
apomorphy. e present results reinforce the potential of
larval morphology in the elds of systematics, taxonomy,
and evolution. Tadpoles are highly variable regarding
Fig. 14 Kamana Creek, upstream within 100m of Kamana Waterfall, draining Mt. Kopinang low waters where tadpoles of Adhaerobufo were collected
(A) and an unnamed stream on the slopes of Maringma-tepui where tadpoles were also observed (B). Amplexing couple of A. nasicus (C and D). Photos
by D. Bruce Means (A, C, D) and Philippe J. R. Kok (B)
Page 19 of 26Dias et al. Zoological Letters (2024) 10:17
their morphology (e.g., 25, 27, 28, 35, 43, 138, 141, 142),
ecology and behavior (e.g., [143, 144]), among others.
Such variation makes tadpoles a powerful source of evi-
dence to test hypotheses of evolutionary relationships
among frogs. Recently, several studies have approached
larval morphology in a phylogenetic context (e.g., [35,
45]), resulting in the identication of novel synapomor-
phies and strengthening the support of clades.
It is also evident that the exploration of larval mor-
phology in previously unstudied groups has widened our
perception of larval diversity. In the past 20 years, aston-
ishing novel phenotypes have been reported (e.g., [27, 28,
141]), but many of these new characters have never been
included in any phylogenetic analysis. We strongly advo-
cate for the usage of larval morphology in further studies
about the evolution and diversication of anurans.
Finally, we believe that the taxonomy of anurans (and
of other organisms with complex life cycles) could greatly
benet from the usage of non-adult semaphoronts. His-
torically, anuran taxonomists have concentrated their
eorts in metamorphosed adult (mainly males), ignoring
larval individuals. When such dogma is broken, taxono-
mists can better delimit, recognize, and describe species,
and other supraspecic clades. For example, Grosjean et
al. [24] were able to describe Clinotarsus penelope (Rani-
dae), referring a tadpole as the holotype. Our study fol-
lows the same path, and larval characters were pivotal for
the proposition of Adhaerobufogen. nov. – named after
larval characteristics.
The evolution of suctoriality in bufonid tadpoles
Recently, Dias and Anganoy-Criollo [33] discussed the
convergent evolution of suctorial and gastromyzopho-
rous ecomorphologies across anurans. ey stressed that
the presence of enlarged oral disc and/or of a belly sucker
were dierent strategies shaped by natural selection in
tadpoles occupying fast-owing waters. ese strate-
gies have evolved independently multiple times across
13 families of anurans. ese authors, however, also dis-
cussed the dierences among these larvae, suggesting
that the real diversity of suctorial forms is unknown.
Fig. 15 Phenotypic dierences between Adhaerobufo nasicus (CPI10704) (A) and Rhaebo larvae; R. caeruleostictus (KU112307) (B) and R. haematiticus
(KU68327) (C). Note the striking dierences in body shape, mouthparts, and coloration. Scale bars = 1.0mm. Photos by: Pedro H. Dias (A) and Jackson
Phillips (B and C)
Page 20 of 26Dias et al. Zoological Letters (2024) 10:17
Suctorial and gastromyzophorous larvae evolved inde-
pendently at least 10 times in Bufonidae (Fig.16). Gas-
tromyzophorous tadpoles have been reported in all
known Atelopus larvae, in three species of the Rhinella
veraguensis group (R. chrysophora, R. quechua, and R.
veraguensis), in Sabahphrynus maculatus, in Adeno-
mus kandianus, and in Bufo aspinius [33, 60, 145–147],
whereas suctorial tadpoles have evolved in Adhaerobufo
gen. nov., Ansonia, Blaira, Phrynoidis, Bufo pageoti, Bufo
torrenticola, Bufo tuberospinus, and Werneria ([66, 148–
151]; the present study).
As stressed by Dias and Anganoy-Criollo [33], suc-
torial larvae of bufonids share many traits, but also dif-
fer widely. Suctorial larvae share a series of convergent
traits, such as the presence of a developed element in
the prenarial arena and of narial vacuities [33], a widen-
ing of the palatoquadrate, enlarged and short cornua tra-
beculae, robust lower jaw, upper jaw with fused elements
and with a well-developed processus posterior dorsalis,
adrostral elements often present, reduction of elements
of the branchial basket, modications in the insertion of
the abdominal muscles, presence of a rectus abdominis
superior, suspensorio-angularis with a sub- or postor-
bital origin, and well developed axial muscles ( [21, 47,
60, 152]; PHD, the authors, pers. obs.). Each independent
instance of bufonid suctoriality is also unique. e most
obvious dierence among many is the presence of a belly
sucker in gastromyzophorous species, as opposed to an
enlarged oral disc, but there are other variable states.
For instance, jaw sheaths are interrupted in Ansonia,
Fig. 16 Gastromyzophorous and suctorial larvae evolved independently at least 10 times within bufonids, revealed by the phylogenetic hypothesis of
Portik et al. [95] showing the genera in which these tadpoles have evolved. The dots next to the genera indicate derived conditions within them. Photos
by: Pedro H. Dias and Jackson Phillips
Page 21 of 26Dias et al. Zoological Letters (2024) 10:17
but continuous in other taxa. Other variable characters
are the presence and distribution of submarginal papil-
lae, tail tip morphology, and body color pattern. Despite
the great potential of this system in the study of novelty
and ecomorphological evolution, the signicance of such
variation remains largely unexplored.
Adhaerobufogen. nov. represents an interesting case,
given that the adult form is rather unspectacular, being
confused with many unrelated bufonid sub-clades over
time. It is remarkable that the adult form appears to be so
unaected by radical evolutionary changes to the larva.
We interpret this as further evidence of the decoupling
power of metamorphosis, whereby evolutionary changes
in the larval form can operate semi-independently of the
adult phenotype, despite sharing a genome and being
part of the same developmental sequence [153–154].
From a macroevolutionary perspective, it is interesting
to note that the evolution of suctoriality may be par-
ticularly common in bufonids. Vera Candioti et al. [57]
demonstrated that suctorial forms are the exclusive lar-
val form of four anuran families, (Ascaphidae, Conraui-
dae, Heleophrynidae, and Nasikabatrachidae), and that
while suctoriality has evolved in several other families,
it is a relatively rare phenomenon in anurans (number of
suctorial species/number of species). Future studies that
identify the features that make suctoriality a more com-
mon evolutionary outcome in some lineages (including
bufonids) than others could provide insight into not only
adaptive ecomorphological evolution, but also non-adap-
tive factors that limit such evolutionary changes.
Conclusion
We describe the tadpole of “Rhaebo” nasicus and pres-
ent evidence supporting the erection of a new genus,
Adhaerobufogen. nov., to recognize the evolutionary dis-
tinctiveness of this group of South American toads. e
tadpole of Adhaerobufo nasicus is a brightly colored, suc-
torial form adapted to living in fast-owing streams. e
oral morphology of that tadpole is unique among bufo-
nids, with a complete row of marginal papillae that dier-
entiates it from all other tadpoles known from the family
Bufonidae. Suctorial larvae have evolved independently
at least 10 times in bufonids; in each case, a combination
of convergent and unique traits can be observed. Our
ndings echo the importance of tadpoles in systematic
and taxonomic studies.
Fig. 17 Torrential environments that were colonized by suctorial/gastromyzophorous larvae of bufonids. Adult of Atelopus sp. in Tacarcuna, Colombia (A);
fast owing waters occupied by Atelopus elegans at Isla Gorgona, Colombia (B); larvae of Ansonia guibei attached to rocks of fast owing streams in Borneo
(C and D). Photos by Marco A. Rada (A), David Velázquez (B), and Alexander Haas (C and D)
Page 22 of 26Dias et al. Zoological Letters (2024) 10:17
Abbreviations
ALPH Antelateral process hyalis
AP Articular process
APH Anterior process hyalis
AT Adrostral tissue
BFA Buccal oor arena
BFAP Buccal oor arena papillae
BRAP Buccal roof arena papillae
CB Constrictor branchialis
CH Ceratohyal
DV Dorsal velum
HA Hyoangularis
HP Hypobranchial plate
HQP Hyoquadrate process
IH Interhyoideus
IHP Interhyoideus posterior
ILP Infralabial papillae
IM Intermandibularis
IN Internal nares
IR Infrarostral cartilage
JAF Jackknife absolute frequencies
JF Jugular foramen
JGC Parsimony jackknife frequency dierences
LMA Levator mandibulae articularis
LMEP Levator mandibulae externus profundus
LMES Levator mandibulae externus supercialis
LMI Levator mandibulae internus
LMLS Levator mandibulae longus supercialis
LP Lateral process
LR Lateral ridge
LTRF Labial tooth row formula
MC Meckel’s cartilage
µCT High-Resolution micro-computed tomography
ML Maximum likelihood
MP (in larval cranium) Maximum parsimony
MP (in phylogenetics) Muscular process
MR Median ridge
NV Narial vacuities
OH Orbitohyoideus
OC Otic capsule
PCM Proximal commissure
PNP Postnarial arena papillae
PP Posterior process
PU Process urobranchialis
QA Quadrato-angularis
QOC Quadro-Orbital commissure
RA Rectus abdominis
RAA Rectus abdominis anterior
RC Rectus cervicis
SA Suprarostral ala
SAR I Subarcualis rectus I
SAR II–IV Subarcualis rectus II–IV
SB Subocular bar
SC Suprarostral copora
SH Suspensoriohyoideus
SO Subarcualis obliquus
SP Spicule
TA Tongue anlage
TH Trabecular horns
TP Triangular projection
TS Tectum synoticum
TTM Taenia tecti medialis
TTT Taenia tecti transversalis
UFBoot Ultrafast Bootstrap Approximation
UJ Upper Jaw Sheath
VV Ventral Velum
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s40851-024-00241-0.
Supplementary Material 1: Appendix MS1 take ESM 1
Supplementary Material 2: Appendix MS2 take ESM 2
Supplementary Material 3: Appendix MS3 take ESM 3
Supplementary Material 4: Appendix MS4 take ESM 4
Supplementary Material 5: Figure MS5 take ESM 5
Supplementary Material 6: Figure MS6 take ESM 6
Acknowledgements
Pedro H. Dias thanks the Marie Skłodowska-Curie Actions (MSCA-IF-2020,
MEGAN/101030742). We thank the Smithsonian Institution and curatorial sta
for providing specimens for examination and scanning. The work of Jackson R.
Phillips was supported by start-up funds awarded to Molly C. Womack by Utah
State University. Martín O. Pereyra acknowledges the support from Agencia
Nacional de Promoción Cientíca y Tecnológica (ANPCyT PICTs 2019 − 346,
2019–2519, 2019–2557) and Consejo Nacional de Investigaciones Cientícas y
Técnicas (CONICET PIP 11220200102800CO). Fieldwork of D. Bruce Means was
supported by a National Geographic Society Expeditions Council Grant and
Guyana EPA permission to conduct Biodiversity Research (Reference Numbers
1007003 BR 0098; 011206 BR 065; 120707 BR 075; 061212c BR 020) and to
export specimens for scientic research (Reference Numbers 030310 SP 001;
151206 SP 030; 230707 SP 008; 072412 SP 054). The work of Philippe J. R. Kok
was supported by a Marie Skłodowska-Curie Actions grant (MSCA-IF-2020,
HOSTILE/101022238). We thank Marco Rada and David Velázquez for kindly
providing photos of the environment occupied by Atelopus species. Olivier
Pauwels kindly provided access to the individual housed at the Royal
Belgian Institute of Natural Sciences. Agustín Elias-Costa contributed to the
improvement of Fig.11. We also thank Kevin de Queiroz, Esther Langan, and
the Smithsonian Institution, Eric Smith, Greg Pandalis, and the University of
Texas Arlington ARDRC, as well as Rafe Brown, Ana Motta, and the Kansas
University Biodiversity Institute and Natural History Museum for providing
specimens and permission for CT scanning. David Blackburn, contributors,
and the sta of MorphoSource (Duke Library Digital Repository) made µCT
images of relevant species available. Daniel Paluh shared µCT images of
Amazophrynella. Finally, we thank three anonymous reviewers for comments
that improved the quality and clarity of our manuscript.
Author contributions
Pedro H. Dias and Jackson R. Phillips conceived and designed the study.
Philippe J. R. Kok and D. Bruce Means acquired samples. Pedro H. Dias, Philippe
J. R. Kok and Martín O. Pereyra acquired funding. Pedro H. Dias, Jackson R.
Phillips, Martín O. Pereyra and Philippe J. R. Kok performed the analyses and
analyzed the data. Pedro H. Dias and Jackson R. Phillips wrote the original draft
with inputs by all authors. Pedro H. Dias, Jackson R. Phillips, Martín O. Pereyra,
and Philippe J. R. Kok prepared/contributed to all gures.
Funding
This work was supported by two Marie Skłodowska-Curie Actions (MSCA)
grants (MEGAN/101030742 to Pedro H. Dias and HOSTILE/101022238 to
Philippe J. R. Kok). Martín O. Pereyra is supported by the Agencia Nacional de
Promoción Cientíca y Tecnológica (ANPCyT PICTs 2019 − 346, 2019–2519,
2019–2557) and the Consejo Nacional de Investigaciones Cientícas y
Técnicas (CONICET PIP 11220200102800CO). Fieldwork of D. Bruce Means was
supported by a National Geographic Society Expeditions Council grant.
Data availability
Further information and requests for additional resources should be directed
to and will be fullled by the corresponding authors. This article has been
registered in ZooBank with the life science identier urn: lsid: zoobank.org:
pub: 95161DF6-0C46-4CC1-9BD1-045545B57C57.
Declarations
Ethical approval
Not applicable.
Page 23 of 26Dias et al. Zoological Letters (2024) 10:17
Consent for publication
Not applicable.
Competing interests
Not applicable.
Received: 8 April 2024 / Accepted: 13 August 2024
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