The Tadpole of Physalaemus moreirae (Anura: Leiuperidae) Author(s) :Diogo B. Provete, Michel V. Garey, Natacha Y. N. Dias, and Denise de C. Rossa-Feres Source: Herpetologica, 67(3):258-270. 2011. Published By: The Herpetologists' League DOI: 10.1655/HERPETOLOGICA-D-11-00004.1 URL: http://www.bioone.org/doi/full/10.1655/HERPETOLOGICAD-11-00004.1 BioOne (www.bioone.org) is a a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Herpetologica, 67(3), 2011, 258–270 E 2011 by The Herpetologists’ League, Inc. THE TADPOLE OF PHYSALAEMUS MOREIRAE (ANURA: LEIUPERIDAE) DIOGO B. PROVETE1,2,3,5, MICHEL V. GAREY1,2, NATACHA Y. N. DIAS1,4, DENISE DE C. ROSSA-FERES2 1 AND Pós-Graduação em Biologia Animal, Departamento de Zoologia e Botânica, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, campus São José do Rio Preto, São Paulo, Brasil, 15054-000 2 Departamento de Zoologia e Botânica, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista, campus São José do Rio Preto, São Paulo, Brasil, 15054-000 ABSTRACT: We describe the external morphology of the tadpole of the frog Physalaemus moreirae. We also provide a review of internal oral features, chondrocrania, and a characterization of the genus based on larval traits. The internal oral features and the external morphology of P. moreirae are most similar to P. jordanensis, a species allocated to the P. gracilis group. None of the larval traits supports the current intrageneric arrangement of species, because several features are shared by species belonging to different species groups. We argue for the inclusion of larval traits in future phylogenetic studies in order to understand the evolution of larval characters. Key words: Atlantic Rain Forest; Chondrocranium; Internal oral features; Larval morphology; Physalaemus signifer group THE GENUS Physalaemus comprises 42 species distributed throughout northern and central Argentina, eastern Bolivia, Paraguay, Uruguay, Brazil, the Guianas, the lowlands of southern Venezuela, the llanos of southeastern Colombia, and western Ecuador (Frost, 2011). Seven phenetic species groups have been recognized within the genus: the P. albifrons, P. cuvieri, P. deimaticus, P. gracilis, P. henselli, P. olfersii, and P. signifer groups (Nascimento et al., 2005). According to these authors, P. moreirae belongs to the P. signifer species group, along with more 12 species: P. angrensis, P. atlanticus, P. bokermanni, P. caete, P. camacan, P. combrei, P. irroratus, P. maculiventris, P. nanus, P. obtectus, P. signifer, and P. spiniger. Each species occurs in a narrow geographic range (Heyer and Wolf, 1989) along the Atlantic Rain Forest in eastern Brazil, from the state of Alagoas through Rio Grande do Sul (Nascimento et al., 2005). 3 PRESENT ADDRESS: Laboratório de Ecologia de Insetos, Departamento de Ecologia, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, Goiás, Brasil, 74001-970 4 PRESENT ADDRESS: Pós-graduação em Zoologia, Museu Nacional, Departamento de Zoologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil 5 CORRESPONDENCE: e-mail, dbprovete@gmail.com Chondrocranial and internal oral features of the larvae of frogs in the family Leiuperidae have been little explored, and only a few detailed descriptions are available (e.g., Alcalde et al., 2006; Vera Candioti, 2007). Additionally, no larval characters have been included so far in phylogenetic studies of the family, despite their utility in reconstructing anuran phylogenies (Haas, 2003). Physalaemus moreirae is endemic of the Atlantic forest, and is known from some localities in the Serra do Mar range in the state of São Paulo, including Santos, Guarujá, Salesópolis, Bertioga, and Santo André at elevations up to 1200 m. This species is currently considered as a ‘‘Data Deficient’’ species because of the lack of information on its extent of occurrence, status, and ecological requirements (Nascimento and Verdade, 2004). The only data available about the species are its advertisement call and male reproductive behavior (Giaretta et al., 2009; Heyer, 1985; Heyer et al., 1990). Our aim in the present article is to provide information about the tadpole of P. moreirae, including external morphology, internal oral features and chondrocranium, along with an extensive review of the information available for these characteristics in tadpoles of the genus Physalaemus. Previous attempts to synthesize the morphological descriptions of Physalaemus 258 September 2011] HERPETOLOGICA 259 FIG. 1.—Tadpole of Physalaemus moreirae at Gosner Stage 37, (A) lateral view, (B) dorsal view, scale bar 5 5 mm; (C) oral apparatus, scale bar 5 1 mm. 260 HERPETOLOGICA [Vol. 67, No. 3 TABLE 1.—Tadpole measurements in millimeters (mean 6 standard deviation) taken from Physalaemus moreirae tadpoles Gosner Stages 29–39. Abbreviations: total length (TOL), body length (BL), tail length (TL), body height (BH), tail height (TH), dorsal membrane height (DMH), ventral membrane height (VMH), tail musculature width (TMW), eye diameter (ED), interorbital distance (IOD), naris diameter (ND), internarial distance (IND), oral disc diameter (OD), upper jaw sheath width (UJSW), upper jaw sheath height (UJSH), eye–snout distance (ESD), naris–snout distance (NSD), body width (BW), tail musculature height (TMH). Stage (n) 29 (3) 30 (1) 31 (6) 33 (6) 34 (1) 35 (4) 37 (6) 38 (1) 18.41 6.83 11.59 4.44 3.49 1.27 1.11 1.42 0.69 1.27 0.29 0.83 2.06 0.92 0.14 1.47 0.64 5.34 1.32 larvae were made by Wassersug and Heyer (1988) and Alcalde et al. (2006), but these authors included some species that now are placed in the related genera Eupemphix and Engystomops. Despite such efforts, the diagnosis of the genus remains fully based on adult traits (Nascimento et al., 2005). We hope that our article, together with the previous ones, contributes to an integrative taxonomy (sensu Dayrat, 2005) of the genus that will help clarify the relationships within Physalaemus (see Funk et al., 2008, for a recent discussion). We fed the larvae with commercial fish food and kept them in a mixture of pond and tap water at room temperature and daylight regime. Larval stages follow Gosner (1960). For the morphometric characterization, we took 20 measurements based on Altig and McDiarmid (1999a) in 35 tadpoles, ranging from Stage 29 to 39: total length (TOL), body length (BL), tail length (TL), body height (BH), tail height (TH), dorsal membrane height (DMH), ventral membrane height (VMH), tail musculature width (TMW), eye diameter (ED), naris diameter (ND), oral disc diameter (OD), upper jaw sheath width (UJSW), upper jaw sheath height (UJSH), eye–snout distance (ESD), naris–snout distance (NSD), body width (BW), tail musculature height (TMH). The interorbital distance (IOD) and internarial distance (IND) were taken between the internal margins of eye and external naris, respectively. For the description of internal oral features, we dissected one tadpole at Stage 36 and another at Stage 37. We recorded all measurements, proportions, and coloration under a stereomicroscope. In addition, the Stage 37 larva was studied with the use of a Zeiss LEO MATERIALS AND METHODS The field work was carried out in the Parque das Neblinas, Bertioga, State of São Paulo, southeastern Brazil (23u44937.80S, 46u08957.50W; datum 5 SAD69), monthly between March 2006 and March 2007. We collected the tadpoles with a dip net that had 3-mm mesh, they were anesthetized with a solution of 5% benzocaine in the field, and later fixed and preserved with 10% formalin. To confirm the identification, we collected an egg clutch from an amplectant pair and reared tadpoles in plastic vials until metamorphosis. 20.56 7.02 13.34 4.01 4.32 1.51 1.47 1.24 0.69 1.42 0.27 0.93 2.13 0.96 0.16 1.86 0.86 5.87 1.43 36 (7) TOL 17.46 6 0.96 17.46 17.96 6 0.36 18.99 6 0.37 BL 6.51 6 0.18 6.35 6.64 6 0.08 6.77 6 0.16 TL 11.48 6 0.61 11.11 11.32 6 0.3 12.13 6 0.28 BH 3.33 6 0.09 3.81 3.46 6 0.08 3.84 6 0.1 TH 3.6 6 0.1 3.65 3.36 6 0.12 3.7 6 0.15 DMH 1.22 6 0.05 1.27 1.22 6 0.11 1.32 6 0.07 VMH 1.16 6 0.05 1.27 1.14 6 0.1 1.16 6 0.05 TMW 1.0 6 0.04 1.13 1.03 6 0.05 1.17 6 0.03 ED 0.59 6 0.03 0.59 0.59 6 0.01 0.63 6 0.03 IOD 1.22 6 1.13 1.27 1.29 6 0.04 1.35 6 0.07 ND 0.28 6 0.01 0.25 0.24 6 0.01 0.26 6 0.01 IND 0.8 6 0.03 0.74 0.86 6 0.01 0.88 6 0.05 OD 1.98 6 0.03 1.86 1.97 6 0.06 2.03 6 0.08 UJSW 0.84 6 0 0.8 0.88 6 0.03 0.95 6 0.04 UJSH 0.17 6 0.01 0.14 0.15 6 0.01 0.15 6 0.01 ESD 1.36 6 0.17 1.37 1.63 6 0.05 1.53 6 0.07 NSD 0.54 6 0.1 0.54 0.79 6 0.04 0.66 6 0.07 BW 4.79 6 0.12 5.1 4.87 6 0.12 5.37 6 0.13 TMH 1.18 6 0.03 1.13 1.22 6 0.05 1.31 6 0.02 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 0.33 23.27 6 0.62 21.69 6 1.48 23.49 0.19 7.96 6 0.19 7.65 6 0.67 6.46 0.28 15.31 6 0.49 14.05 6 0.95 16.03 0.12 4.36 6 0.11 4.39 6 0.55 4.44 0.12 4.9 6 0.11 4.50 6 0.49 4.6 0.1 1.7 6 0.08 1.56 6 0.11 1.27 0.08 1.57 6 0.07 1.43 6 0.11 1.43 0.01 1.48 6 0.07 1.36 6 0.09 1.72 0.02 0.64 6 0.04 0.71 6 0.04 0.64 0.09 1.6 6 0.08 1.50 6 0.07 1.62 0.01 0.28 6 0.01 0.26 6 0.01 0.29 0.02 1.01 6 0.03 0.99 6 0.05 1.03 0.06 2.3 6 0.08 2.29 6 0.11 2.25 0.01 1.05 6 0.03 1.02 6 0.03 0.98 0.01 0.17 6 0.01 0.17 6 0.01 0.12 0.04 1.92 6 0.04 1.96 6 0.06 1.81 0.01 0.87 6 0.02 0.91 6 0.03 0.74 0.16 6.35 6 0.24 6.25 6 0.32 6.62 0.04 1.62 6 0.07 1.59 6 0.05 1.86 September 2011] HERPETOLOGICA 261 FIG. 2.—Internal oral features of the tadpole of Physalaemus moreirae at Gosner Stage 37. (A) Buccal floor, inset: detail of infralabial papillae; (B) buccal roof. Scale bar 5 300 mm. 450 VPi Scanning Electron Microscope following Conte et al. (2007); the terminology for those results follows Wassersug (1976). For chondrocranium descriptions, we cleared and stained two Stage 36–37 larvae with the technique of Taylor and Van Dyke (1985); chondrocranial terms follow Cannatella (1999) and Haas (2003). All specimens used in descriptions are housed at the tadpole collection of the Departamento de Zoologia e Botânica, Universidade Estadual Paulista, campus São José do Rio Preto, São Paulo (DZSJRP) (see Appendix 1). RESULTS External Morphology Physalaemus moreirae has Orton Type IV, exotrophic, lenthic, and benthic (Altig and McDiarmid, 1999b) tadpoles (Fig. 1A,B; Table 1). At Stage 36 the body is ovoid in dorsal view (BL/BW 5 1.5) and depressed in lateral view (BL/BH 5 2), with the broadest part at the level of the spiracle. The snout is rounded in lateral and dorsal views. The eyes are very small, dorsally positioned, dorsolaterally directed. The nares are rounded, dorsally positioned, and located on a depression of the body wall with a continual marginal rim; they project upward and are closer to the eye than to the snout. The spiracle is short and sinistral, directed posterodorsally, with the inner wall present as a slight ridge, located right below the median line; the spiracle aperture is oval. The vent tube is short, dextral, with the right wall displaced dorsally and laterally. The tail is long, with the tail musculature not reaching the end of the rounded tail (Fig. 1A). The dorsal fin originates on the body, and is slightly higher than the ventral fin, with maximum height at the middle third. The oral apparatus is anteroventral and laterally emarginated (Fig. 1C). The marginal papillae are long, fingerlike, arranged in a single row laterally, and alternated ventrally. The lateral papillae are pigmented and curved toward the oral opening. There are about 16 papillae per millimeter. The rostral gap comprises approximately 2/3 of the P-1 row. Submarginal papillae are present ventrally (range 2–9 papillae, n 5 10) and are usually taller than marginal papillae. The labial teeth are strongly keratinized and arranged in a single row. There are about 54 labial teeth per millimeter (estimated on P-1), curved toward the oral opening. Labial Teeth Row Formula 2(2)/3(1), A1 and A2 equal in length; P1 equal in length to P3, P2 larger than P1 and P3. The A-2 gap equivalent to 13–21 labial teeth, whereas the P-1 gap corresponds to 2–4 labial teeth. Both jaw sheaths are strongly keratinized and finely serrated; the serrations are small, triangular-shaped, and oriented downwards. There are about 45 serrations per millimeter. The upper jaw sheath is Mshaped. The lower jaw is V-shaped (Table 1). 262 HERPETOLOGICA [Vol. 67, No. 3 FIG. 3.—Chondrocranium and mandibular arch of a Gosner Stage 37 tadpole of Physalaemus moreirae (A) dorsal and (B) ventral views of the neurocranium and mandibular arch, (C) frontal view of the cartilago labialis superior, and (D) ventral view of the hyobranchial apparatus. Abbreviations—for (A) and (B), ca, capsula auditiva; ci, cartilago infralabiale; cm, cartilago meckeli; cq, commissura quadrato-orbitalis; ct, cornua trabeculae; fc, foramen craneopalatinum; nc, notochordal canal; par, processus articularis; pas, processus ascendens; pm, processus muscularis quadrati; pra, processus retroarticularis; sf, fenestra subocularis; tm, taenia tecti marginalis; ts, tectum synoticum; for (C), pa, pars September 2011] HERPETOLOGICA Coloration We do not have data on the color in life. In formalin, the dorsum is light brown and the venter is cream. The body wall is transparent. The tail is cream-colored, with small, sparse melanophores, and dark rectangles on the dorsal portion. The fins are transparent, with a few melanophores. The iris is black. Internal Oral Features Buccal floor.—The buccal floor is triangular, and about 10% wider than it is long. The oral opening occupies about 30% of the buccal floor width (BFW; Fig. 2A). There are two pairs of infralabial papillae; the posteromedial pair is composed of a large multiple branching papilla, with two pustulations at its base; the other anteromedial pair is composed of fingerlike papillae (Fig. 2A, inset). There are four tall, conical, and transversally arranged lingual papillae; the medial one is taller than the lateral ones. There are one or two small prepocket papillae. The buccal pockets are oriented at 45u, about two times wider than high, perforated. The buccal floor arena (BFA) is triangular, densely pigmented, and bordered by 10–16 conical papillae arranged in a U-shaped pattern, projecting towards the BFA, with the larger ones located anteriorly. There are about 30 pustulations on the posterior portion of BFA. The free velar surface is about 20% of the BFW, scattered with melanophores; spicular support conspicuous, spicules about 20% of the BFW. The posterior margin of the free velar surface is slightly curved downward. The peaks over the filter cavities are equivalent to about 5% of the BFW, with no projections on the midline. The median notch occupies about 9% of the BFW. Secretory pits are conspicuous. The lung buds are small and not inflated. Ventral pharynx.—The branchial baskets are triangular and slightly wider than long (width 45% of the BFW). The filter cavities are angled 45u from the midline. The ceratobranchiales 3 bears 10 filter rows, cb 4 bears 4 filter rows. The glottis is very small and fully occluded, two times wider than long, lips 263 broad. The width of esophageal funnel is approximately 20% of the BFW. Buccal roof.—The buccal roof (Fig. 2B) is oval and 1.5 times longer than it is wide. The prenarial arena is square-shaped with four pustulations arranged in an arch. The internal nares are elliptical, their length is about 38% of the buccal roof width (BRW), and positioned 20% of the distance from front of the mouth to the esophagus. The internarial distance is 25% of the BRW. The median ridge is positioned about 40% of the distance from front of the mouth to the esophagus. The anterior wall of the internal nares is tall and thin, with 4–5 small papillae concentrated on the median portion; the posterior wall is smooth, about 30% longer than high. the narial valve is distinct but short. Eight conical, serrated papillae on the postnarial arena form an arch; the four smallest ones are medially located, whereas the four largest ones are laterally located. The median ridge is triangular with serrated margins, its length about 8% of the BRW. There are two fingerlike and bifurcated lateral-ridge papillae; they bear pustulations along their margins and their length is approximately 20% of the BRW. The buccal roof arena (BRA) is like an elongated rectangle, about 7% wider then high; it has four conical papillae on each side, the largest of which are medially located, with none bifurcated. There are also 35 pustulations distributed homogeneously along the BFA. The glandular zone is densely pigmented, with distinct secretory pits. The dorsal velum lacks papillation and does not extend to the midline. Chondrocranium The neurocranium is as long as it is wide and is depressed (height/width ratio 5 0.4), with its greatest width at the posterior part of the arcus subocularis (Fig. 3A,B). The maximum width of neurocranium is 4 mm, and the maximum length is 4.4 mm. The U-shaped pars corporis of the cartilago labialis superior connects dorsally to the pars alaris by a thin proximal connection. The broad partes alares have rounded ventral margins and bear a r alaris; pc, pars corporis; ppd, processus posterior dorsalis; for (d), ca, condylus articularis; cb, ceratobranchiales; cp + pu, copula posterior and processus urobranchialis; h, hypobranchiales; pab, processus anterior branchialis; pal, processus anterolateralis hyalis; pp, processus posterior dorsalis; pr, pars reuniens; s, spicula; tc, terminal commissura. 264 HERPETOLOGICA pointed processus posterior dorsalis (Fig. 3C). The cornua trabeculae are enlarged anteriorly, with truncated and flat tips; they comprise about 22% of the total neurocranium length. The thin taenia tecti marginalis surrounds the fenestra frontoparietalis and communicates with the ethmoid plate anteriorly. Although the foramina carotica primaria are well defined at the studied stages, the foramina craneopalatina are poorly defined. At the posterior end of the neurocranium lies the tectum synoticum, which is the only tectal structure present at the studied stages. The oval-shaped capsula auditiva represents about 30% of the neurocranium length. The larval crista parotica is not well defined. The operculum is not chondrified at the developmental stages we studied. The notochordal canal comprises about 29% of the total neurocranium length. The palatoquadrate bears a tall and thin processus muscularis quadrati, a wide comissura quadrato-cranialis superior, and a small and pointed processus quadrato-ethmoidalis, along with a processus articularis quadrati. The larval processus oticus and processus pseudopterygoideus are absent. The processus ascendens of the palatoquadrate meets the pila antotica behind the oculomotor foramen (intermediate union, Sokol, 1981). The posterior margin forms an edge in dorsal view, but it is not deeply concave. The lower jaw is composed of a pair of L-shaped cartilago meckeli, with three processes each, and a paired cartilagines infrarostrales (Fig. 3B). The basihyal is absent. Both ceratohyalia articulate medially via a narrow, trapezoid pars reuniens. The copula posterior bears a tiny processus urobranchialis. All ceratohyalia processes are well developed. The condylus articularis is robust and bears a rounded dorsal protuberance. The small and pointed processus anterolateralis hyalis is curved to the midline. The processus anterior hyalis is wider and taller than the processus lateralis hyalis, but the processus posterior hyalis is the widest of them. The planum hypobranchiale shows a V-shaped posterior margin. The triangular-shaped processus anterior branchialis is present at ceratobranchial I. The ceratobranchialia are joined distally by a terminal commissura. Spiculae I– III are present and associated with the ceratobranchialia I–III (Fig. 3D). [Vol. 67, No. 3 Natural History Tadpoles of P. moreirae were usually found sheltering within the muddy bottom of one temporary woodland swamp (80 m in diameter, 40 cm in depth) from October to December (the beginning of the rainy season), with the peak of abundance in November. The swamp was formed by two rivulets and held water for 9 mo of the year; there was little herbaceous vegetation on its margin. Other tadpoles that cooccurred with P. moreirae were Aplastodiscus leucopygius, Bokermannohyla hylax, Hypsiboas pardalis, Scinax hayii, S. hiemalis, Physalaemus olfersii, and Crossodactylus dispar. Some predatory insects that co-occurred with tadpoles of P. moreirae were adults of Belostoma sp. (Heteroptera: Belostomatidae), Buenoa sp. (Heteroptera: Notonectidae), Lancetes sp. (Coleoptera: Dytiscidae), and larvae of Coryphaeschna sp. and Aeshna sp. (Odonata: Aeshnidae). DISCUSSION Currently, 26 species of the genus Physalaemus have known tadpoles. However, little information is available on internal oral features and chondrocranial morphology. Despite this lack of information, we discuss the variation of some larval characters within the genus in order to identify apomorphic traits that distinguish P. moreirae larvae from other larvae. Some external morphological characters of larvae are somewhat plastic and are shared by most species of Physalaemus (Altig and McDiarmid, 1999b). However, other character states vary within the genus (Table 2). Most of the species (69%) have rounded nares, 19% have elliptical nares, and 12% have reniform nares. The spiracle is oriented posterodorsally in the majority of species (69%), but it may be oriented posteriorly (19%), postero-ventrally (4%), or dorsally (8%). The vent tube is dextrally oriented in 54% of the species, with the remaining having a medial vent tube. The oral apparatus is anteroventral in 50% of the known tadpoles of Physalaemus and ventral in the other 50% of species. Larvae from most of the species (74%) bear a single row of (simple or alternated) marginal papillae, but variations such as a double row of marginal papillae are found in 19% of the species, including P. moreirae. Only 7% of the species have a single Species Group SO VTO OAP MP GMP SP LTRF DFO Reference A A A A A P A 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) ABT BTJ ABT BTJ BTJ BTJ BTJ ? 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) 2(2)/2(1) 2(2)/2(1) 2(2)/2(1) 2(2)/3(1) 2(2)/3(1) 2(2)/2(1) 2(2)/3(1) 2(2)/2 2(2)/3(1) 2(2)/3(1) 2/3(1) 2(2)/3(1) 2(2)/3(1-2) 2(2)/3(1) 2(2)/3(1) 2(2)/3(1) BTJ BTJ BTJ BTJ BTJ ABT ABT BTJ ABT ABT BTJ BTJ BTJ BTJ BTJ BTJ BTJ BTJ BTJ BTJ Haddad and Sazima (2004) Cardoso and Haddad (1985) Pombal and Madureira (1997) Pimenta et al. (2005) Bokermann (1963) Present study Weber and Carvalho-e-Silva (2001) Haddad and Pombal (1998) Langone (1989) Gomes et al. (2010) Both et al. (2006) Borteiro and Kolenc (2007) Nomura et al. (2003) Perotti and Cespedez (1999) Alcade et al. (2006) Kolenc et al. (2006) Borteiro and Kolenc (2007) Kehr et al. (2004) Rossa-Feres and Jim (1993) Vieira and Arzabe (2008) Perotti (1997) Heyer et al. (1990) Baêta et al. (2007a) Nascimento et al. (2001) Pimenta and Cruz (2004) Baêta et al. (2007b) Weber et al. (2005) P. P. P. P. P. P. P. atlanticus bokermanni caetae camacan maculiventris moreirae signifer P. P. P. P. P. P. P. signifer signifer signifer signifer signifer signifer signifer F ? R E R R R PD ? PD PD P PD P M* ? M M* D D M* V V V V V A A SLDV SA D D S SA SA D D D D D D D P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. spiniger gracilis jordanensis lisei biligonigerus marmoratus santafecinus fernandezae henselii riograndensis albonotatus centralis cicada cuqui cuvieri erythros rupestris aguirrei maximus soaresi P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. signifer gracilis gracilis gracilis albifrons albifrons albifrons henselii henselii henselii cuvieri cuvieri cuvieri cuvieri cuvieri deimaticus deimaticus olfersii olfersii olfersii R E R R R R E E R R R R R F R R R F E R P PD PD PD PD PD PD PD P PD PD PV PD PD P PD ? PD PD PD M D* D* D D* D* D M M M D M* M* D D D M M D* D* V A A A A V A A A A A V A V V V V A A V SLDV D S S SA SA S D S S S S S S S SA S SA S S D D D D D DVS D DV DV DVS DVLS DVS DV DVS DVS D D D DL D P A A A P P A A A A A A ? P A A P A A HERPETOLOGICA NS September 2011] TABLE 2.—External morphological traits used to distinguish tadpoles from the seven phenetic species groups of Physalaemus (Nascimento et al., 2005). Abbreviations: narial shape (NS): elliptical (E), reniform (F), and rounded (R); spiracle orientation (SO): posteroventrally (PV), posterodorsally (PD), and posterior (P); vent tube orientation (VTO): medial (M) and dextral (D); oral apparatus position (OAP): anteroventral (A) and ventral (V); marginal papillae (MP): single (S), single alternated (SA), double (D), or single laterally and double ventrally (SLDV); presence of gap on marginal papillae (GMP): one dorsal (D), one dorsal and one ventral (DV), one dorsal and two laterally (DL), one dorsal, one ventral, and two ventrolaterally (DVS), or one dorsal, one ventral, two laterally and two ventrolaterally (DVLS); submarginal papillae (SP): present (P) or absent (A); labial tooth row formula (LTRF); dorsal fin origin (DFO): anterior to body–tail junction (ABT) or at body–tail junction (BTJ). * Vent tube attached to the ventral fin. ? Data not available from original descriptions. 265 266 HERPETOLOGICA [Vol. 67, No. 3 TABLE 3.—Main internal oral features of tadpoles in the genus Physalaemus. Abbreviations: BFA 5 buccal floor arena; BRA 5 buccal roof arena. See text for explanations of the characters. BFA Prenarial arena Infralabial papillae (number) Lingual papillae (number) P. moreirae P. jordanensis 4 4 4 4 12/14 12/30–37 4 6–10 + + P. marmoratus 4 2 8–12/30–40 ? P. santafecinus 4 1 15/30–35 4–6/6–10 pustulations pustulations present P. fernandezae P. lisei P. cuqui 2 6 1 5 3 12/.60 8/? P. biligonigerus 4 P. cuvieri 4 ? Papillae/ Pustulations Secretory pits ? Features Pustulations Square-shaped With one serrated ridge anteriorly Two semicircular arches of pustulations Bilobated ridge 4 3 Two trifid papillae No papillae 3 ? ? ? ? ? 12 ? ? ? ? ? ? 1 10–12/20 ? ? ? 3 8–10/14–33 2–8/2–14 pustulations 2–6/8–14 pustulations 5–7 ? Two ridges ? Without papillae ? Oval ? P. centralis 4 4 12–16/7–10 P. albonotatus 4 4 10/30 ? Prepocket papillae 2 ? 1–4 Data not available from original descriptions. row of marginal papillae laterally and a double row ventrally. The pattern of gaps on the marginal papillae is complex and varies among species: A single dorsal gap in the marginal papillae is found in approximately 63% of the species, whereas 18% of the species have a single dorsal gap, one ventral gap, and two ventrolateral gaps, and 11% of the species bear one dorsal and one ventral gap. Physalaemus fernandezae (4%) larvae have one dorsal gap and two lateral gaps, P. albonotatus (4%) larvae have one dorsal, one ventral, two lateral, and two ventrolateral gaps. Submarginal papillae are absent in 76% of the species. The LTRF in the majority of species of Physalaemus is 2(2)/ 3(1); however, P. biligonigerus, P. marmoratus, P. santafecinus, P. riograndensis, P. centralis, P. cuvieri, and P. rupestris have a different LTRF. The dorsal fin originates anterior to body–tail junction in 22% of the species and at body–tail junction in 78% of the species. Finally, with respect to external morphological characters, the tadpole of P. moreirae can be diagnosed by the following combination of traits (Table 2): (1) nares rounded, (2) spiracle oriented posterodorsally, (3) vent tube orientation dextral, (4) oral apparatus anteroventral, (5) marginal papillae in a single row, alternated ventrally, (6) marginal papillae gap only present dorsally, (7) submarginal papillae present, (8) labial tooth row formula 2(2)/3(1), (9) dorsal fin originating at body–tail junction. The internal oral features of P. moreirae are very similar to those of P. jordanensis (Table 3). The most common number of infralabial papillae found in Physalaemus larvae is four. Wassersug and Heyer (1988) argued that variations in the number of infralabial papillae should be considered significant because four was the most common number of them in the 54 species of ‘‘leptodactyloids’’ they analyzed. In this context, the presence of two infralabial papillae in P. fernandezae (Alcalde et al., 2006) and six in P. lisei (Both et al., 2006) is thought to be taxonomically significant and useful in species recognition. In addition, independent from the number of infralabial papillae, there is always a pair that is larger and multiple-branching (conical and serrated in P. centralis and P. cuvieri). Another extremely variable trait of Physalaemus larvae is the number of lingual papillae. The most common numbers are four (P. moreirae, P. jordanensis, and P. albonotatus; this study; Gomes et al. 2010; Miranda and Ferreira, 2009), three (P. cuqui, P. centralis, P. September 2011] HERPETOLOGICA 267 TABLE 3.—Extended. Postnarial arena Median ridge (shape/ pustulations) Papillae/ pustulations BRA Lateral ridge papillae Papillae/ pustulations Dorsal velum Secretory pits Continuous across midline Papilate medial edge Reference 4/? 7/2 Triangular/serrated Trapezoid 6 4 8/40 8/44 + + 2 2 + + This study Gomes et al. (2010) small/? Semicircular/pustulated 2 + 2 2 Nomura et al. (2003) 4/? Trapezoid 2 6–8/ 50–60 10–12/ numerous + 2 2 5/1 Semicircular/serrated Trapezoidal/pustulated 2 4 ? ? ? ? ? ? ? 8/.60 8/? ? ? ? ? ? Triangular/pustulated 2 8/17 + 2 1–2/1–4 Semicircular/pustulated 4 + 2 2/2 Semicircular/pustulated 4 + 2 2/? Triangular 4 4–12/ 30–35 6–10/ 20–30 8/40 Perotti and Céspedez (1999); Vera Candioti (2007) Alcalde et al. (2006) Both et al. (2006) Fabrezi and Vera (1997) Perotti and Cespedez (1999) Miranda and Ferreira (2009) Miranda and Ferreira (2009) Miranda and Ferreira (2009) ? ? /3 ? cuvieri; Miranda and Ferreira, 2009; Perotti, 1997), and one (P. santafecinus, P. fernandezae, P. biligonigerus). A remarkable exception is the larvae of P. lisei, with five lingual papillae, which is very uncommon in other larvae (Wassersug and Heyer, 1988). The most common number of BFA papillae found among Physalaemus larvae is approximately 10. It is common to find bifid papillae laying on the BFA, as in P. moreirae, P. marmoratus, P. santafecinus and P. cuvieri. The prepocket papillae of P. moreirae are similar to those of P. marmoratus, P. cuvieri and P. centralis. Some characters of the buccal roof also vary. Physalaemus marmoratus, P. biligonigerus, and P. santafecinus share a bilobated ridge located anteriorly to the prenarial arena (Alcalde et al., 2006; Nomura et al., 2003). This character apparently supports the inclusion of these species in the P. albifrons species group, but whether or not it is a synapomorphy should be evaluated in other species of the genus. Furthermore, this area of the buccal cavity of Physalaemus tadpoles is extremely variable, and the condition found in P. moreirae (with four pustulations) is similar to that found in P. cuvieri and P. jordanensis. 2 ? ? ? ? ? All Physalaemus tadpoles with described internal oral features bear pustulations on the anterior wall of the elliptical internal nares, and have distinct narial valves. The postnarial arena is very similar in P. centralis and P. cuvieri larvae, bearing two conical serrated papillae (Miranda and Ferreira, 2009), whereas P. moreirae and P. santafecinus larvae have four papillae. The median ridge is a lunate or trapezoid flap with a pustulated free margin in the majority of larvae, but it is triangle-shaped in P. moreirae and P. biligonigerus (Perotti and Cespedez, 1999; this study). All known larvae of Physalaemus have two lateral ridge papillae, usually serrated, and two postnarial papillae. The number of BRA papillae varies from approximately eight in the majority of species, to four in P. cuvieri (Miranda and Ferreira, 2009) and 10–12 in P. santafecinus (Vera Candioti, 2007). The dorsal velum is papillated only in P. jordanensis and P. centralis. In summary, we were able to recognize some internal oral features that are shared by the most Physalaemus larvae: (1) a median ridge that is lunate or trapezoid, (2) four infralabial papillae, (3) approximately 10 BFA papillae, (4) two lateral ridge papillae and two 268 HERPETOLOGICA postnarial papillae, (5) approximately eight BRA papillae, (6) the velum lacking papillae and interrupted at the midline (Table 3). The chondrocranium of P. moreirae is very similar to that described in other species of the genus (Alcalde et al., 2006; Fabrezi and Vera, 1997; Prado, 2006; Vera Candioti, 2007). In all reported larvae of the genus, the cartilago labialis superior bears a V- or Ushaped corpus, which is dorsally fused to the alae. Additionally, the processus anterioris alaris varies in length among species of Physalaemus. The cornua trabeculae are uniformly wide and sometimes anteriorly enlarged. In relation to the palatoquadrate, it is possible to recognize two groups that share common characters: (1) processus pseudopterygoideus present, palatoquadrate with a foramen on the posterior region (P. cuvieri, P. centralis, and P. marmoratus; Prado, 2006); (2) processus pseudopterygoideus absent (P. moreirae, P. santafecinus, P. fernandezae; this study; Vera Candioti, 2007). The basihyal is absent in all larvae of Physalaemus. The processus anterolateralis of the ceratohyal is shorter than the processus anterior, which is usually larger in all species. The basibranchial bears a short processus urobranchialis in all species. The processus anterior branchialis is present in all species. There is little variation in the external morphological characters of Physalaemus tadpoles, probably because most larvae are of the generalized pond-type (Orton, 1953) and are found in almost the same set of environments, mainly temporary and permanent ponds in forested areas (e.g., Haddad and Sazima, 2004; Pimenta et al., 2005; Pombal and Madureira, 1997; Weber and Carvalho-eSilva, 2001) and open areas (e.g., Baêta et al., 2007a; Heyer et al., 1990; Perotti, 1997; Vasconcelos and Rossa-Feres, 2008). Some authors (e.g., Borteiro and Kolenc, 2007; Kolenc et al., 2006) have argued that larval external characters do not support the current arrangement of phenetic species groups (Nascimento et al., 2005) within the genus Physalaemus. Except for the tadpoles of P. albifrons group, which can be characterized by the labial tooth row formula [2(2)/2(1)] and by some internal oral features (e.g., a bilobated ridge anteriorly located to the prenarial [Vol. 67, No. 3 arena), our results for external morphology, internal oral features, and the chondrocranium support that statement. Larval characters have been used in several attempts to classify anuran species (e.g., Orton, 1953; Sokol, 1975; Starrett, 1973). In addition, larval characters have proven to be useful in recent phylogenetic analyses at the ordinal level (Haas, 2003). Therefore, our results may be useful in future phylogenetic studies of the genus Physalaemus, and may help to identify generic and intrageneric synapomorphies. Acknowledgments.—We thank the IBAMA-RAN for the collecting permits (093/96). We are also grateful to F. Nomura for his help in the fieldwork. V. H. M. Prado critically reviewed the manuscript. F. B. R. Gomes made the line drawings of the tadpole. For their help with the SEM analysis we thank Professor Dr. L. Madi-Ravazzi, L. R. Falleiros and the Centro de Microscopia e Microanálise of the IBILCE-UNESP. L. F. Belussi and R. Soleman helped with pictures of tadpole chondrocranium. This work was supported by a fellowship to NYDN (proc. 06/54308-2) from FAPESP and also by a grant (01/ 13341-3, Biota Program). DBP and MVG were assisted by fellowships from FAPESP during the final preparation of this manuscript (proc. 08/55744-6 and 08/50575-1, respectively). The work of DCRF is supported by CNPq (proc. 302838/2007-4 and 563075/2010-4) and FAPESP (proc. 10/52321-7) grants. LITERATURE CITED ALCALDE, L., G. S. NATALE, AND R. CAJADE. 2006. 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CARVALHO-E-SILVA. 2001. Descrição da larva de Physalaemus signifer (Girard, 1853) (Amphibia, Anura, Leptodactylidae) e informações sobre a reprodução e a distribuição geográfica. Boletim do Museu Nacional 462:1–6. WEBER, L. N., S. P. CARVALHO-E-SILVA, AND L. P. GONZAGA. 2005. The tadpole of Physalaemus soaresi Izecksohn, 1965 (Anura: Leptodactylidae), with comments on taxonomy, reproductive behavior, and vocalizations. Zootaxa 1072:35–42. .Accepted: 24 May 2011 .Associate Editor: Richard Lehtinen APPENDIX I Specimens Examined DZSJRP 1187.2, Brazil: São Paulo, Bertioga, Parque das Neblinas, cols. N.Y.N. Dias, F. Nomura, 20/X/2006; DZSJRP 1200.6, Brazil: São Paulo, Bertioga, Parque das Neblinas, cols. N.Y.N. Dias, F. Nomura, 18/XI/2006; DZSJRP 1208.9, Brazil: São Paulo, Bertioga, Parque das Neblinas, cols. N.Y.N. Dias, F. Nomura, 09/XII/2006.