Larval anatomy of Dendropsophus decipiens (A. Lutz 1925) (Anura: Hylidae: Dendropsophini) with considerations to larvae of this genus

The Dendropsophus decipiens clade comprises four species: D. berthalutzae, D. decipiens, D. haddadi, and D. oliveirai. Tadpoles of these species were described, but data on their internal morphology are lacking. We provide the first description of the buccopharyngeal anatomy, chondrocranial morphology, and cranial, hyoid and hyobranchial musculature of the tadpole of D. decipiens. Larvae of D. decipiens are characterized by the absence of lingual papillae, presence of fan-like papilla on the buccal floor, presence of a single-element suprarostral cartilage, presence of a small triangular process at the basis of the processus muscularis, m. levator mandibulae lateralis inserted on the nasal sac, and m. subarcualis rectus II-IV with a single, continuous slip. Tadpoles are likely macrophagous, although not as specialized as those of other species of the genus, suggesting some degree of diversification on the feeding habits within Dendropsophus.

The external larval morphology in the Dendropsophus decipiens clade has been described for D. berthalutzae [18], D. decipiens [16], D. haddadi [19,20], and D. oliverai [16], but no aspect of their internal anatomy has been analyzed so far. We describe the buccopharyngeal anatomy, chondrocranial morphology, and cranial, hyoid and hyobranchial musculature of the tadpole of D. decipiens. We also provide comments on the larval internal morphology of Dendropsophus based on our observations complemented with data from the literature.

Materials and methods
Tadpoles were collected (ICMBio/RAN permit # 13256-1) in a dam of Rio Borboleta (225 9'19"S, 44˚06'13"W) at Reserva Rio das Pedras, Mangaratiba, Rio de Janeiro, Brazil. Specimens were euthanized by topical application of 20% benzocaine anesthetic mixed with water, preserved in 5% formaldehyde and deposited in the Coleção de Anfíbios do Laboratório de Biossistemática de Anfíbios da Universidade Federal do Estado do Rio de Janeiro (Lot UNI-RIO 3635). Some tadpoles were raised in the laboratory to corroborate species identification. Developmental stages were determined according to Gosner [21]. Two individuals (stages 34 and 35) were dissected according to Wassersug [22] to expose the buccopharyngeal cavity. One individual (stage 34) was submitted to the protocol of Alcalde and Blotto [23] for scanning electron microscopy (SEM). Descriptive terminology follows Wassersug [22,24]. For observations of the chondrocranium and cranial muscles, six individuals (stages 30-36) were treated following the protocol of Dingerkus and Uhler [25] for clearing and staining; we interrupted the procedure just after the staining with alcian blue for two individuals (stage 36) which were then dissected manually for the study of larval muscles. After observations and illustrations, we finished the clearing protocol. Terminology for the chondrocranium and muscles follows Haas [26][27][28]. The character-states discussed through the text were optimized on the phylogenetic hypothesis proposed by Duellman et al. [1] with the software TNT v1.5 [29].

External morphology
The tadpole of Dendropsophus decipiens (stage 37) was described by Pugliese et al. [16]. It is characterized by having a triangular body, high tail fins with brown stripes, and a reduced oral disc with blunt marginal papillae and absence of labial teeth.
Ceratohyals long, flat, and subtriangular ( Fig 2C); their anterior margin bearing two triangular, well-developed processes: processus anterior hyalis and processus anterolateralis hyalis. Posterior processes triangular, tall. Ceratohyals are joined by a pars reuniens, which is chondrified. Basibranchial rectangular, bearing a rounded processus urobranchialis. Basihyal present, slim, cylindrical. Planum hypobranchiale long, triangular, contacting each other along their anterior half. Branchial basket has four curved ceratobranchials with numerous lateral projections. Ceratobranchial I continuous with the planum hypobranchiale; dorsally, it bears a triangular processus anterior branchialis. Ceratobranchials II and III also fuse to the planum hypobranchiale plate and bear round branchial processes. Ceratobranchial IV is shorter, wider, and fused to the planum hypobranchiale. Four long, curved spicules develop dorsally; the third and fourth spicules are connected to the planum hypobranchiale by a thin cartilaginous bridge. Ceratobranchials are distally joined by terminal commissures.

Cranial, hyoid and hyobranchial musculature
We found a total of 31 muscles in larvae of Dendropsophus decipiens (origins and insertions in Table 1; Figs 3 and 4). Interhyoideus posterior and diaphragmatopraecordialis absent. Subarcualis obliquus present in two slips, inserting on the processus branchialis II and III. Subarcualis rectus II-IV continuous, inserting on the ceratobranchial I. Mandibulolabialis with single slip, corresponding to mandibulolabialis inferior. Intermandibularis arch-shaped. Larval levator mandibulae externus with two slips, superficialis and profundus. Levator mandibulae lateralis inserting in the nasal sac. Ramus mandibularis (cranial nerve V 3 ) runs dorsally to longus and externus groups.

Larval internal morphology
Dendropsophus has been repeatedly found as sister taxon of Xenohyla (always well supported) in molecular phylogenetic analyses for Hylidae (e.g. [1,3,6,8]). However, the phylogenetic relationships of the tribe Dendropsophini (Dendropsophus + Xenohyla; sensu [2]) within Hylidae remain controversial. It is recovered as sister taxon of Sphaenorhynchini, closely related to Pseudini and Scinaxini [3] or alternatively, as sister taxon of Pseudini, distantly related to Scinaxini and Sphaenorhynchini (e.g. [1,8] [24,[30][31][32]. Similarly, descriptions of cranial muscles and the chondrocranium morphology are only available for D. ebraccatus, D. microcephalus, and D. nanus [28,[32][33][34]. Dendropsophus ebraccatus was included in the phylogenetic analyses of Haas [28]. Aside from those studies, there is some scattered information available as the relative size of the buccal depressors muscles of D. microcephalus and D. phlebodes  The controversial phylogenetic position of the tribe Dendropsophini (e.g. [1,3]) within Hylidae, added to the poor knowledge about on internal larval anatomy in Dendropsophus prevents us to assess the polarity of most character-states and, therefore, to enhance broad discussions about larval character evolution in Dendropsophus; however, there are some interesting exceptions that deserve comments.
Several species of Dendropsophus have a reduced number of elements in the buccopharyngeal cavity [24,[30][31][32]. For example, the median ridge is missing in D. ebraccatus and D. nanus [24,32], and a lower number of papillae in both buccal floor and roof and the absence of lingual papillae were also reported for larvae of Dendropsophus (e.g. [31,32]). Faivovich et al. [3] suggested the absence of lingual papillae as a putative synapomorphy for the clade composed of Dendropsophus, Lysapsus, Pseudis, Scarthyla, Scinax, and Sphaenorhynchus (the former Dendropsophini tribe sensu [3]), with reversion in Lysapsus and Pseudis. Our observations showed that lingual papillae are also present at least in some species of Sphaenorhynchus (S. dorisae, S. lacteus, S. prasinus; P.H.S. Dias and K. Araujo-Vieira personal observations). This characterstate is unknown for Xenohyla. The absence of lingual papillae optimizes ambiguously in the phylogenetic hypothesis of Duellman et al. [1] due to the unknown character-state of Xenohyla; it is a synapomorphy of Dendropsophus or of a more inclusive clade, the tribe Dendropsophini (Dendropsophus + Xenohyla sensu [2]).
Dendropsophus decipiens (D. microcephalus group) has a fan-like papilla on the buccal floor, adjacent to the buccal pockets. This character was first described by Kaplan and Ruíz-Carranza [31] for D. garagoensis, D. padreluna, D. virolinensis (D. garagoensis group), and D. stingi  putative synapomorphy of the genus Dendropsophus; however, the taxonomic distribution of this character-state within Dendropsophus remains poorly known.
A putative synapomorphy of the Dendropsophus garagoensis group is the presence of two pairs of infralabial papillae, which were reported for D. garagoensis and D. padreluna [31]with independent evolution in D. decipiens [this work]. Other species of Dendropsophus that have been studied present a single pair of infralabial papillae (e.g. D. minutus, D. nanus, D. phlebodes; [24,30,32]).
Larvae of Dendropsophus decipiens differ from those of its related species D. microcephalus and D. nanus (characters in parenthesis) in having generalized processus muscularis (broad processus muscularis), marginal projections on arcus subocularis of palatoquadrate (smooth margin), thin ceratohyals (stout and thick ceratohyals), regular branchial basket (reduced branchial basked), and presence of spicules on the ceratobranchialia (absence of spicules) [32-34]. However, D. decipiens share with those species short and narrow cornu trabeculae, the presence of a small triangular process at the basis of the processus muscularis, and a single-element suprarostral cartilage. The latter could be a synapomorphy of Dendropsophus (with one reversion to a separated element suprarostral cartilage in D. ebraccatus within the D. leucophyllatus group; [28]) or of the D. microcephalus group. In larvae of Lysapsus, Pseudis, and some Scinax, the suprarostral corpora are fused distally, but with a medial indentation that allow the identification of both elements; several other hylids have completely separated suprarostral cartilage (e.g. [32,33,[40][41][42][43]). This character-state is still unknown for Scarthyla, Sphaenorhynchus, and Xenohyla.
It is worth to note that a single-element suprarostral cartilage is not very common in anuran larvae. Besides some Dendropsophus, such morphological condition have been reported in some microhylids [32], Occidozyga baluensis (Dicroglossidae) [44], and Ceratophrys and Lepidobatrachus (Ceratophryidae) [32,[45][46][47]. It is interesting to point that, regarding Ceratophryidae, Chacophrys pierotti possesses the suprarostral alae and corpora completely fused (with a small fenestra), but both corpora have a medial indentation [48]. Lavilla and Fabrezi [49] suggested that the complete fused suprarostral could be a synapomorphy of Ceratophrys + Lepidobatrachus. Given the phylogenetic relationships within Ceratophryidae [50]-Chacophrys and Lepidobatrachus are sister taxa, and both sister to Ceratophrys-this is not possible. However, the complete fusion between the alae and corpora could be a synapomorphy for the family. While microhylids are filter-feeders, Ceratophrys, Lepidobatrachus, and Occidozyga are macrophagous larvae. Further information are still needed to understand the relationship between macrophagy and the fusion of the elements of the suprarostral cartilage.
Larvae of Dendropsophus, Lysapsus, and Pseudis that have been studied share the m. levator mandibulae lateralis inserted on the nasal sac [32,41] [this work]-although Haas [28] mentioned that it inserted on the processus posterior dorsalis of the suprarostral or in the rostral tissue in D. ebraccatus [28: ch. 57.0]. In larvae of Scinax, the m. levator mandibulae lateralis inserts on the pars alaris of the suprarostral cartilage [32,34,42]. The taxonomic distribution of these character-states on the phylogenetic hypothesis of Duellman et al. [1] suggests that the m. levator mandibulae lateralis inserted on the nasal sac could be a synapomorphy of the Dendropsophini + Pseudini clade (sensu [2]) or arose twice independently in Dendropsophus and the Lysapsus + Pseudis clade. Other character-state, the absence of the superior slip of the m. mandibulolabialis also optimize ambiguously as a synapomorphy of the Dendropsophini + Pseudini clade or of Dendropsophus and the Lysapsus + Pseudis clade (with some instances of reversion within Pseudis; [28,41]). Larvae of Scinax have the superior slip of the m. mandibulolabialis [42]. The optimizations of both character-states mentioned above are dependent on the conditions, still unknown, present in larvae of Scarthyla, Sphaenorhynchus, and Xenohyla.

Feeding habits
Tadpoles can be distinguished regarding their deeding habits accordingly with the size of their prey (macrophagous or microphagous) and the nature of the feeding items (herbivore, carnivore, or omnivore). Larvae of Dendropsophus have been described as macrophagous feeders (e.g. [51]), or macrophagous herbivores according to Wassersug [24]. However, other reports suggest that tadpoles of Dendropsophus could be omnivorous. For example, Vera Candioti [32] described the presence of entire oligochaetes in the digestive system of D. nanus. Ruas et al.
[52] observed tadpoles of D. novaisi chasing and preying on tadpoles of Rhinella crucifer. Also, larvae of D. minutus were observed attacking and preying on living tadpoles of Physalaemus sp.-although it was considered a facultative behavior, given that D. minutus feeds commercial fish food and plant residues in captivity [53].
Most of the macrophagous tadpoles (both herbivorous or carnivores) have terminal mouth, reduced branchial basket, well-developed orbitohyoideus, reduced labial tooth rows, reduced or lost elements of the buccopharynx, massive jaw sheaths, fused elements of suprarostral cartilages, and short intestines [32,35-36,44-47,54] [P.H.S. Dias personal observation]. Almost all these character-states are present, with different combinations, in larvae of Dendropsophus. Wassersug [24] pointed out several buccopharyngeal characters associated with macrophagy in larvae of Dendropsophus, and also reported that D. phlebodes "presented the most extreme reduction in structures associated with fine suspended matters".
We observed that larvae of Dendropsophus decipiens have some character associated with a macrophagy as a terminal mouth lacking labial teeth, reduction in the elements of the buccopharyngeal cavity, such as papillation and secretory tissue, and single-element suprarostral cartilage. Nevertheless, tadpoles of D. decipiens differ from those of D. microcephalus and D. nanus, which have marked reduction of branchial baskets (well-formed and bearing spicules in D. decipiens) and massive orbitohyoideus [32] that is correlated with powerful suction capacity [35]. This combination of characters suggests that larvae of D. decipiens feed on large elements and have reduced filter capacity, although they are not as specialized for predation (carnivory/omnivory) as the tadpoles of D. microcephalus and D. nanus. Morphological and behavioral observations for more species, as well as the usage of techniques such as stable isotope (see [55]) are necessary for a fully appreciation of the evolution of feeding habits in Dendropsophus.
Our results suggest that larval internal morphology could provide interesting insights into the evolution and diversification of Dendropsophus. However, additional studies on other species of Dendropsophus and its related taxa (e.g. Scarthyla, Sphaenorhynchus, and Xenohyla) are required to corroborate the different putative synapomorphies mentioned in this work. Further, the feeding habits of tadpoles of the genus are, putatively, more diverse than previously observed, with variable degrees of specialization for a macrophagous diet.