First description of buccopharyngeal anatomy in Pelodryadinae larvae: Morphological comparison and systematic implications (Anura: Hylidae: Pelodryadinae: Litoria rubella and Ranoidea caerulea)

Pelodryadinae, the Australian tree frogs, is a monophyletic group endemic to the Australo‐Papuan region. Although we have a relatively good knowledge about tadpoles' phenotypic diversity in terms of external morphology, information about internal anatomy is rare for the subfamily; for instance, their buccopharyngeal cavity is completely unknown. Herein I describe for the first time the buccopharyngeal anatomy of two pelodryadins: Litoria rubella and Ranoidea caerulea. I compare my results with available evidence from Phyllomedusidae, that is, the sister clade to Pelodryadinae, and briefly comment on buccopharyngeal cavity within Hylidae. Both species can be readily distinguished based on lateral ridge, postnarial, buccal roof arena, infralabial papillae, and lingual papillae. Variation between the two species may suggest a large diversity within Pelodryadinae. Pelodryadinae and Phyllomedusinae present similar buccopharyngeal morphologies, although Agalychnis callidryas has a unique morphology and putative apomorphic transformations can be observed in Pithecopus + Phyllomedusa, Ranoidea, and Phasmahyla.

lacking for many species (Altig et al., 2021).On a broad scale, most taxa are well represented, but Allophrynidae and Melanobatrachinae are the only suprageneric taxa for which data on tadpoles (or the absence of it) and development is missing (Altig, 2018).Also, at the generic level, or considering just a particular phenotypic system, there are many gaps in our knowledge.
Australian anurofauna seems particularly poorly studied.
Although there was a massive effort of some researchers to understand the depth of external morphology variation (e.g., Anstis, 2002Anstis, , 2010Anstis, , 2017)), data on internal morphology of Australian tadpoles is virtually inexistent.To date, only cranial morphology of few pelodryadin species (Haas, 2003;Haas & Richards, 1998)
Pelodryadinae, the Australian tree frogs, is a monophyletic group endemic to the Australo-Papuan region, but introduced to other regions including New Zealand, that currently comprises 234 species (Frost, 2023).This clade has been consistently considered sister to the Neotropical subfamily Phyllomedusinae (e.g., Faivovich et al., 2005Faivovich et al., , 2010;;Frost et al., 2006;Jetz & Pyron, 2018), but taxonomy and internal relationships are far from being stable (Faivovich et al., 2005(Faivovich et al., , 2010)).Although the sister relationship of Pelodryadinae and Phyllomedusinae appears to be well supported by molecular and phenotypic evidence (e.g., Elias-Costa et al., 2021;Faivovich et al., 2010), the poor taxon sampling and the lack of data of pelodryadins precludes the optimization and the understanding of the evolution of many phenotypic characters.
Tadpoles of Pelodryadinae are phenotypically and ecologically diverse, including neustonic and suctorial larvae (Anstis, 2002(Anstis, , 2017;;Sherratt et al., 2017), that differ significantly both in external and in cranial morphology (Haas & Richards, 1998), however phenotypic diversity within pelodryadins is highly underestimated.In this paper, I intend to contribute to the knowledge of larval diversity within Pelodryadinae by describing the buccopharyngeal morphology for representatives of the subfamily.Moreover, I compare my results with available evidence from Phyllomedusinae and discuss the evolution of this system within hylids.

| MATERIALS AND METHODS
All specimens used in this study are deposited at the herpetological collection of the Leibniz Institut zur Analyse des Biodiversitätswandels, Hamburg, Germany.Litoria rubella (Gray, 1842) tadpoles came from Townsville, Queensland, Australia and were identified by direct comparison with the description; according to Anstis (2017), in the region Queensland, the species that could be misled as L. rubella are Litoria dentata (Keferstein, 1868), Litoria electrica (Ingram & Corben, 1990), and Litoria verreauxi (Duméril, 1853).Nevertheless, at L. rubella is not sympatric with any of them in Queensland (Cutajar et al., 2022).Tadpoles of Ranoidea caerulea (White, 1790) came from a captivity breed colony.Three individuals of L. rubella (ZMH12193) and two of R. caerulea (ZMH12040) in Gosner's (1960) stages ranging from 32 to 36 were manually dissected according to Wassersug (1976) to expose the buccopharyngeal cavity and, after inspection under a stereoscopic microscope with the aid of methylene blue staining, one individual pers species submitted to a protocol for scanning electron microscopy.Buccopharyngeal terminology follows Wassersug (1976Wassersug ( , 1980)).

| Comparison between species
Both species can be readily distinguished regarding their buccopharyngeal cavity.L. rubella has a simple, conical lateral ridge papilla (trifurcated in R. caerulea), single pair of postnarial papilla (two pairs in  have dorso-lateral compressed bodies, with wide snouts, a ventral, enlarged oral disc fully papillated, strong tail muscles, slow-flowing, nektonic species, such as Litoria adelaidensis, have globular bodies, high tail fins, and flagellated tail tip (Anstis, 2017).Some species, such as Ranoidea subglandulosa, are benthic, and possess a bizarre oral disc, that lacks labial teeth and jaw sheaths, but has a series of pointy, long, papillae scattered within its entire surface (Anstis, 2017).One might expect that such variation in external morphology would also be reflected in internal anatomy.The results of this paper support this idea and stress the need for further studies within the subfamily; the fact that several differences could be observed in the studied species suggests that analysis of other taxa will uncover novel buccopharyngeal morphologies.
Pelodryadinae and Phyllomedusinae larvae seem to have similar buccopharyngeal morphology; in general, members of these two clades have two pairs of infralabial papillae and one pair of lingual papillae, like in many hylids (e.g., Oliveira et al., 2017;Pezzuti et al., 2015;Wassersug, 1980), differing slightly in the number of buccal roof and A. callidryas is quite divergent of the "phyllomedusid pattern," bearing multiple infralabial papillae, and presenting a pair of keratinized spurs in the lower jaw (Wassersug, 1980).The funnelmouthed tadpoles of Phasmahyla also present several derived, unique character states, such as the v-shaped crest in the prenarial arena and the obtuse postnarial papillae (Dias et al., 2018).This suggests that we are far from knowing the real phenotypic diversity of larvae in that clade.
Although most clades have representatives with described buccopharyngeal cavity, we are far from understanding the real phenotypic diversity of hylids.Although most clades have been studied, the number of taxa studied per major group is still reduced; for instance, regarding Dendropsophini, of the 108 recognized species (Frost, 2023), the buccopharyngeal cavity is known to only 13 species (Candioti, 2007;Echeverría, 1997;Kaplan & Ruiz, 1997;Dias et al., 2019Dias et al., , 2023;;Wassersug, 1980), less than 10% of the clade's diversity.
We are still being surprised with the description of novel character states as new hylid species are studied (e.g., Dias & Pie, 2021) and more efforts should be concentrated in uncovering a hidden information that can improve our understanding of evolution, ecology, systematics, and conservation of these charismatic frogs.

| CONCLUSIONS
Tadpole biology is an exciting field with prospect for improvement in the future.In the last 20 years we were surprised with the description of several novel, and sometimes bizarre, phenotypes (e.g., Haas et al., 2006Haas et al., , 2014;;Rowley et al., 2012;Dias, 2020;Dias et al., 2021;Vera Candioti et al., 2017) as previous unstudied taxa are investigated.Pelodryadinae tadpoles seem to hide uncovered phenotypic variation.The fact that the two species studied in this paper greatly differed in their morphologies suggests that future research may discover novel phenotypes and will help to understand the diversification of the group.
3.2.2| Buccal roofBuccal roof (Figure3b) triangular, longer than wide.Prenarial arena long and wide, with a transverse, crenulate crest.Internal nares elliptical, arranged perpendicular to the anteroposterior axis; prenarial papillae absent; small, conical papilla projecting from the inner anterior margin; inner surface of internal nares covered with ciliated epithelium.Postnarial arena triangular, bearing two pairs of conical postnarial papillae; single pair of triangular papillae anteriorly to median ridge.Median ridge triangular, tall, with irregular margin.Lateral ridge papillae present, flap-like, trifurcated.Buccal roof arena delimited by 5−6, short, conical papillae each side.Glandular zone not evident.Dorsal velum with smooth margin, interrupted medially, lacking papillae.

F
I G U R E 3 Ranoidea caerulea (ZMH12040), scanning electron micrographs of the buccopharyngeal cavity of a tadpole at stage 32.Buccal floor (a) and buccal roof (b).Scale bars = 200 µm.BFA, buccal floor arena; BFAP, buccal floor arena papillae; BP, buccal pocket; BRA, buccal roof; BRAP, buccal roof arena papillae; DV, dorsal velum; IL, infralabial papillae; IN, internal nares; LP, lingual papillae; LRP, lateral ridge papillae; MR, median ridge; PNP, postnarial papillae; VV, ventral velum.F I G U R E 4 Ranoidea caerulea (ZMH12040), scanning electron micrographs of the buccopharyngeal cavity of a tadpole at stage 32.Detail of the infralabial and lingual papillae (a), and of the ventral velum and glottis (b).IL, infralabial papillae; LP, lingual papillae.floor papillae.The reduction of infralabial papillae in Litoria and in Pithecopus + Phyllomedusa are independent and could represent synapomorphies in these groups, although more data is necessary to test this hypothesis.The presence of four lingual papillae in Ranoidea is also an apomorphic condition; within Pelodryadinae and Phyllomedusinae, four lingual papillae have only been described in Phasmahyla (Dias et al., 2018), and it could represent independent, synapomorphic transformation in both clades.Nevertheless, additional data is necessary to test these hypotheses.