Occurrence of a Calcified Pseudodontoid in Troglodytes (Anura: ) Author(s): Antonio Sebben, Natan M. Maciel, Leandro A. Campos, Marcelo N. C. Kokubum, and Hélio Ricardo da Silva Source: Journal of Herpetology, 41(2):337-340. Published By: The Society for the Study of and Reptiles https://doi.org/10.1670/0022-1511(2007)41[337:OOACPI]2.0.CO;2 URL: http://www.bioone.org/doi/ full/10.1670/0022-1511%282007%2941%5B337%3AOOACPI%5D2.0.CO %3B2

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Journal of Herpetology, Vol. 41, No. 2, pp. 337–340, 2007 Copyright 2007 Society for the Study of Amphibians and Reptiles

Occurrence of a Calcified Pseudodontoid in Leptodactylus troglodytes (Anura: Leptodactylidae)

1,2 1,3 1,3 4 ANTONIO SEBBEN, NATAN M. MACIEL, LEANDRO A. CAMPOS, MARCELO N. C. KOKUBUM, AND 5 HE´ LIO RICARDO DA SILVA

1Laborato´rio de Anatomia Comparativa de Vertebrados, Departamento de Cieˆncias Fisiolo´gicas, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, ICC-Sul, Mo´dulo 5, Campus Darcy Ribeiro, 70910-900, Brası´lia, Distrito Federal, Brazil 3Programa de Po´s-graduac¸a˜o em Biologia , Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Campus Darcy Ribeiro, 70910-900, Brası´lia, Distrito Federal, Brazil 4Programa de Po´s-graduac¸a˜o em Ecologia, Instituto de Cieˆncias Biolo´gicas, Universidade de Brası´lia, Campus Darcy Ribeiro, 70910-900, Brası´lia, Distrito Federal, Brazil 5Departamento de Biologia Animal, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro, 23851-970, Serope´dica, Rio de Janeiro, Brazil

ABSTRACT.—A calcified pseudodontoid in the mandibular symphysis of Leptodactylus troglodytes was described from fresh, macerated, and cleared/double-stained preparations. Histology confirmed that the pseudodontoid was mineralized and composed of fibrocartilage. This structure was found in all specimens of both sexes, suggesting a role in either prey capture or defense rather than in courtship or combat. The structure in L. troglodytes differed morphologically and histologically from the pseudodontoid observed in other Leptodactylus and in other neobatrachian . Our sampling indicates that the presence and morphology of the pseudodontoid is synapomorphic to a clade recently named Tinctanura. However, a broader sample is still necessary to strengthen this assessment.

Fanglike structures of bone or cartilage are found on October 2004. These specimens were euthanized by the maxillary arch or mandible of a number of injection of 1.5% lidocaine into the central nervous anurans. Fabrezi and Emerson (2003) reviewed the system, fixed in 10% formalin, and then analyzed diverse morphologies, sizes, and locations of these morphologically and histologically. We also examined structures in various lineages. These structures, which several preserved specimens of L. troglodytes (see are different from true teeth in tissue composition Appendix 1). We examined approximately the same (Peyer, 1968), may appear as outgrowths on the number of males and females (no sexual dimorphism neopalatine, vomer, or bones of the mandible (Trueb, was observed; see Results). Specimens of 22 other 1973). These structures may appear superficially so species of Leptodactylus, and seven other neobatra- similar to true teeth that some authors have confused chian species, were also examined and are listed in the the two structures (Miranda-Ribeiro, 1926). Appendix 1. Odontoids are present in certain hylids, ranids, To study the morphology of the pseudodontoid, we leptodactylids, and even in such toothless taxa as the made fresh preparations (Sebben, in press), macera- bufonids and pipids (Trueb, 1973). Fabrezi and tions, and cleared/double-stained whole preparations Emerson (2003) actually described two kinds of tooth- (Taylor and van Dyke, 1985). Specimens of L. like structures. Bony projections from dermal bones troglodytes were submitted to histological (N 5 1), within the mouth cavity are called odontoids. Pseudo- cleared-and-stained (N 5 2), and macerated prepara- dontoids, however, are composed of unmineralized tions (N 5 1). Additionally we examined macerated connective tissues and are located in the mandibular specimens of Leptodactylus furnarius (N 5 1) and symphysis in some species of Ranidae, Bufonidae, and Leptodactylus pustulatus (N 5 1); cleared-and-stained Leptodactylidae (Fabrezi and Emerson, 2003). specimens of L. furnarius (N 5 2), L. pustulatus (N 5 2), Herein, we describe a toothlike structure present in and Leptodactylus ocellatus (N 5 1); and histological the mandibular symphysis of Leptodactylus troglodytes, preparations of Leptodactylus ocellatus (N 5 1). The a small leptodactylid found in northeastern other specimens (Leptodactylus and other neobatra- Brazil. The presence of this projection was first chian) were examined by direct observation of the recorded by Lutz (1926) in the description of this species. This structure has not been studied closely; mandibular symphysis under stereomicroscope. This thus, we present a more morphological description; was made to observe the presence/absence of the we also compare the pseudodontoid of L. troglodytes to pseudodontoid among a variety of species (see that of 22 congeneric and seven outgroup species. Appendix 1). Histological preparations were used to elucidate MATERIALS AND METHODS tissue composition of the pseudodontoid of L. Adult specimens of L. troglodytes were collected in troglodytes. The mandibles were decalcified with nitric the Cocos municipality, State of Bahia, Brazil, in acid (5%), submitted to routine histological tech- niques, and stained with Mallory’s Trichome and hematoxylin-eosin. Observations and measurements 2 Corresponding Author. E-mail address: sebben@ were made with the aid of stereomicroscopes (Zeiss unb.br SV 11 and Zeiss Axioskop 2) equipped with an MC80 338 SHORTER COMMUNICATIONS

camera (and micrometric eyepiece) or a Sony Cyber- shot digital camera DSC2W7.

RESULTS All specimens of both sexes of L. troglodytes show a large, calcified, toothlike structure in the middle of the mandibular symphysis (Figs. 1, 2B, C). This structure, which appears to result from superficial calcification of the tissue that lies on the superior surface of the symphyseal cartilage, stained with alizarin red in adult specimens (N 5 3) but not in a juvenile specimen (N 5 1). The pseudodontoid from one adult specimen (SVL 5 48.4 mm; Fig. 2C) measured 1.20 mm high by 0.35 mm wide at thickest point. The whole length of the structure is calcified. There was no notable variation in the size and shape of the pseudodontoid between males and females. We also observed that there is a medial notch in the lingual shelf of the partes palatinae of the premaxillae FIG. 1. The calcified pseudodontoid of Leptodacty- in L. troglodytes (Fig. 2A); the pseudodontoid fits in lus troglodytes (macerated). (A) Frontal view; (B) this space when the frog’s mouth is closed. Lateral view. The pseudodontoid of L. troglodytes is composed of chondrocytes that lie in the lacunae among the collagen fibers, as is characteristic of a fibrocartilage tissue (Fig. 2B). However, these fibers are distributed

FIG. 2. (A) The notch between the palatine process of the premaxillae where the pseudodontoid fits (cleared/ double stained preparation). (B) The calcified pseudodontoid stained by Mallory’s Trichome showing the fibrocartilage tissue composition of this structure. (C) and (D) Cleared/double-stained preparation showing the pseudodontoid of Leptodactylus troglodytes and Leptodactylus pustulatus, respectively. SHORTER COMMUNICATIONS 339 in different directions in the tissue. The pseudodon- present study). The presence/absence and condition toid is basally fixed to the middle region of the of this structure may be a phylogenetically informa- mandibular symphysis by abundant dense connective tive character. The presence of a pseudodontoid in all tissue. The pseudodontoid of L. ocellatus is composed taxa we examined suggests that it could be a synapo- mainly of collagen fibers without chondrocytes, morphy for the clade Tinctanura (sensu Frost et al., characterizing a dense connective tissue (data not 2006). In the literature, we found references to shown). structures in ranines that resemble pseudodontoids In the cleared-and-stained Leptodactylus specimens (original references unseen, cited by Fabrezi and that we examined, as well as in Physalaemus cuvieri Emerson, 2003). However, a much more extensive (N 5 1) and Chaunus granulosus (N 5 1), a pseudo- sampling of taxa for the presence/absence and dontoid similar to that shown in Fig. 2D is present calcification of pseudodontoids is needed to assess in the same position. The other specimens examined the real value of this trait. An investigation is now by direct observation of the mandibular symphysis underway to survey the phylogenetic distribution of also show a structure similar to the pseudodontoid this structure. (data not shown). We can distinguish L. troglodytes from all other species, because it is the only species Acknowledgments.—The collections in Cocos munic- whose pseudodontoid is stained by alizarin red ipality were made under permits from the Centro de (Fig. 2C). Conservac¸a˜o e Manejo de Re´pteis e Anfı´bios/Instituto The mandibulary arch is formed by the mentomeck- Brasileiro do Meio Ambiente e dos Recursos Naturais elian, dentary, and angulosplenial. The former is Renova´veis (RAN/IBAMA) 012/03 and process fused with the dentary by its lateral surface. The 02010.006106/02201. We thank the administration of anterior and posterior ends of mentomeckelian are Fazenda Trijunc¸a˜o in Cocos, State of Bahia, for field projected toward the premaxillary bones, forming assistance. A. D. Santos helped with the histological a concavity in its middle region (Figs. 1B, 2C). This preparations. C. Griso´lia granted permission to use character was also observed in L. pustulatus (Fig. 2D). the steromicroscope located in Laborato´rio de Gene´t- By comparison, in L. laticeps (Ponssa, 2006), only the ica, Universidade de Brası´lia. G. R. Colli, L. B. anterior end of mentomeckelian is projected toward Nascimento, J. P. Pombal Jr., and H. E. D. Zaher gave the premaxillary bones. The dentary lies on the us access to the collections in their care. We thank external surface of the mandibular arch. Leptodactylus Conselho Nacional de Desenvolvimento Cientı´fico e troglodytes possesses odontoids on the dorsal edge of Tecnolo´gico (CNPq) for fellowships to NMM and the angulosplenial and on the anterior region of the LAC and Coordenac¸a˜o de Aperfeic¸oamento de Pes- dentary (Fig. 1A, 1B). These features were only soal de Nı´vel Superior (CAPES) for fellowship to observed in L. troglodytes among the species analyzed MNCK. We are grateful to P. Andreadis, R. W. Heyer, (Appendix 1). We observed a hypertrophied coronoid and one anonymous reviewer for significant improve- process on the angulosplenial of both L. troglodytes ments and suggestions in the manuscript. and L. ocellatus. This attribute has been previously described in Leptodactylus laticeps (sensu Ponssa, 2006); it is less well developed in L. laticeps and L. ocellatus LITERATURE CITED than in L. troglodytes. FABREZI, M., AND S. B. EMERSON. 2003. Parallelism and convergence in anuran fangs. Journal of Zoology DISCUSSION 260:41–51. The calcified pseudodontoid of L. troglodytes is FROST, D., T. GRANT,J.FAIVOVICH,R.BAIN,A.HAAS,C.B. unique among the anuran species that we studied. HADDAD,R.O.DE SA´ ,A.CHANNING,M.WILKINSON, The fibrocartilaginous composition of this structure S. C. DONNELLAN,C.J.RAXWORTHY,J.A.CAMPBELL,B. confers rigidity and resistance, suggesting that the L. BLOTTO,P.MOLER,R.C.DREWES,R.A.NUSSBAUM, pseudodontoid has a role in biting. Although other J. D. LYNCH,D.M.GREEN, AND W. C. WHEELER. 2006. Leptodactylus possess pseudodontoids (Fabrezi and The tree of life. Bulletin of the Emerson, 2003; present study), in all of them, the American Museum of Natural History 297:1–370. pseudodontoids are composed of dense connective LUTZ, A. 1926. Observac¸o˜es sobre batra´chios brasi- tissue. leiros. Parte 1: o geˆnero Leptodactylus Fitzinger. The function of the calcified pseudodontoid is Memo´rias do Instituto Oswaldo Cruz 19:149–150. unknown. It may serve to prevent prey escape or to MIRANDA-RIBEIRO, A. 1926. Notas para servirem ao subdue prey by causing wounds. Alternatively, the estudo dos Gymnobatrachios (Anura) Brasileiros. structure may be used as a weapon against predators Arquivos do Museu Nacional 27:1–227. or conspecific competitors. Fabrezi and Emerson PEYER, B. 1968. Comparative Odontology. University (2003) emphasized a positive correlation between fang of Chicago Press, Chicago. size of male anurans and intensity of sexual selection. PONSSA, M. L. 2006. On the osteology of a distinctive We observed the pseudodontoids of male and female specie of the genus Leptodactylus: Leptodactylus L. troglodytes to be similar in size and shape. In laticeps (Boulenger, 1917) (Anura; Leptodactyli- addition, we did not find any wounds or scars dae). Zootaxa 1188:23–36. indicative of aggressive interactions. Hence, our SEBBEN, A. In press. Microdissecc¸a˜o fisiolo´gica a results suggest that the calcified pseudodontoid of L. fresco:umanovavisa˜ o sobre anatomia de troglodytes is not under sexual selection. anfı´bios e re´pteis. In L. B. Nascimento and M. E. Pseudodontoids (calcified or otherwise) has now Oliveira (eds.) Herpetologia no Brasil II, Pontifı´cia been identified in approximately 60 species of anurans Universidade Cato´lica de Minas Gerais, Belo from multiple families (Fabrezi and Emerson, 2003; Horizonte, Brazil. 340 SHORTER COMMUNICATIONS

TAYLOR, W. R., AND G. C. VAN DYKE. 1985. Revised Leptodactylus fuscus (N 5 1), , ASUnB procedures for staining and clearing small fishes 2040. and other vertebrates for bone and cartilage study. Leptodactylus knudseni (N 5 2), Rondoˆnia, CHUNB Cybium 9:107–119. 22863, 22865. TRUEB, L. 1973. Bones, and evolution. In J. L. Vial Leptodactylus labyrinthicus (N 5 3), Distrito Federal, (ed.), Evolutionary Biology of the Anurans: Con- ASUnB 32; Bahia, ASUnB 1460; Mato Grosso, ASUnB temporary Research on Major Problems, pp. 65– 2051. 132. University of Missouri Press, Columbia. Leptodactylus leptodactyloides (N 5 1), Rondoˆnia, CHUNB 29296. Accepted: 12 January 2007. Leptodactylus mystaceus (N 5 3), Amapa´, CHUNB 1002, 1003; Para´, CHUNB 34305. Leptodactylus mystacinus (N 5 3), Goia´s, ASUnB 191, APPENDIX 1 2243, 2364. The specimens examined were deposited in the Leptodactylus natalensis (N 5 3), Pernambuco, following collections: Colec¸a˜o Antonio Sebben—Uni- CHUNB 29043–29045. versidade de Brası´lia (ASUnB); Colec¸a˜o Herpetolo´gica Leptodactylus ocellatus (N 5 3), Bahia, ASUnB 1014; da Universidade de Brası´lia (CHUNB); Museu de Sa˜o Paulo, ASUnB 1015, 1714; Distrito Federal, ASUnB Biodiversidade do , Universidade Federal de 2474. Uberlaˆndia (AAG2UFU); Museu de Cieˆncias Natur- Leptodactylus pentadactylus (N 5 2), Amapa´, CHUNB ais da Pontifı´cia Universidade de Cato´lica de Minas 1000, 1001. Gerais (MCNAM); and Museu de Zoologia da Uni- Leptodactylus petersi (N 5 1), Brazil, CHUNB 45784. versidade de Sa˜o Paulo (MZUSP). Leptodactylus podicipinus (N 5 2), Mato Grosso, The following specimens (N 5 62) of Leptodactylus ASUnB 984, 2332. troglodytes, all from Brazil, were examined. States are Leptodactylus pustulatus (N 5 4), Goia´s, ASUnB 1162, listed, followed by collection/identification numbers. 2408, 2409, 2473. Bahia: CHUNB 39064–39078, 42547, 15876, MZUSP Leptodactylus rhodomystax (N 5 2), Para´, CHUNB 2 106433A–106442A, AAG UFU 3576 (specimen sub- 40128, 44910. mitted to histological procedure), ASUnB, 2404–2405 Leptodactylus spixii (N 5 2), Pernambuco, CHUNB (cleared-and-stained specimens), 2,406 (macerated 29029; Minas Gerais, CHUNB 36467. specimen). Goia´s: CHUNB 35409235423, ASUnB Leptodactylus stenodema (N 5 1), Amapa´, CHUNB 2239. 1035. Minas Gerais: CHUNB 38807, 38809, 34018234022, Leptodactylus syphax (N 5 1), Mato Grosso, ASUnB MCNAM 211, 3313, 2948. Paraı´ba: CHUNB 29058. 2042. Rio Grande do Norte: CHUNB 14003, 30578. 5 : CHUNB 37706, 41976. Leptodactylus (Lithodytes) lineatus (N 3), Amazo- The following specimens of other species were nas, ASUnB 1300; Mato Grosso, CHUNB 2137, 46716. 5 examined. For each species, sample size is in Outgroup (N 15 specimens from other families). parentheses, followed by state of Brazil, and specimen Chaunus granulosus (formerly Bufo; N 5 1), Tocan- identification numbers. Some specimens of Leptodac- tins, ASUnB 2412. tylus bufonius are from Argentina. Ceratophrys cornuta (N 5 5), Rondoˆnia, CHUNB Other Leptodactylus (N 5 44 specimens of 22 22649–22653. species). Crossodactylus gaudichaudii (N 5 5), Rio de Janeiro, Leptodactylus andreae (formerly Adenomera; N 5 2), MCNAM 5109–5113. Amazonas, ASUnB 1399, 1865. Gastrotheca sp. (N 5 1), Rio de Janeiro, ASUnB 2414. Leptodactylus martinezi (formerly Adenomera; N 5 1), Hypsiboas faber (formerly Hyla; N 5 1), Minas Gerais, Goia´s, ASUnB 1526. ASUnB 684. Leptodactylus bufonius (N 5 4), Mato Grosso, MNRJ Odontophrynus salvatori (N 5 1), Distrito Federal, 3298; Argentina, MNRJ 3458, 3472, 3459. ASUnB 2413. Leptodactylus chaquensis (N 5 1), Mato Grosso, Eupemphix nattereri (formerly Physalaemus; N 5 1), ASUnB 2045. Goia´s, ASUnB 2411. Leptodactylus furnarius (N 5 4), Distrito Federal, Physalaemus cuvieri (N 5 1), Distrito Federal, ASUnB ASUnB 2410, 2470, 2471, 2358. 2472.