article a survey of the internal oral features and external morphology of physalaemus larvae (anura,...

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ZOOTAXAISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2012 · Magnolia Press

Zootaxa 3200: 1–26 (2012) www.mapress.com/zootaxa/ Article

A survey of the internal oral features and external morphology of Physalaemus larvae (Anura, Leptodactylidae)

JOICE RUGGERI¹ & LUIZ NORBERTO WEBER²¹Departamento de Zoologia, Instituto de Biologia, Universidade Federal da Bahia, Campus Universitário, Rua Barão de Jeremoabo s/n, Ondina, 40170-115 Salvador, Bahia, Brasil. E-mail: [email protected] de Biologia, Universidade Federal do Maranhão, Campus Universitário do Bacanga, Av. dos Portugueses, s/n, 65080-040, São Luis, Maranhão, Brasil. E-mail: [email protected]

Abstract

There are 45 species currently described for the genus Physalaemus that are allocated into seven morphological groups and 29 of them have their tadpoles described, of which 12 have information on their internal oral anatomy. In order to help resolving taxonomic and systematic problems, tadpoles from this genus had their external and internal oral morphologies studied, described and compared. During this study, it was noticed that different terms are sometimes used to refer to the same character and a standardized nomenclature is suggested. This study is divided into two parts. First, we describe the oral cavity anatomy of the tadpoles of Physalaemus aguirrei, P. atlanticus, P. camacan, P. cicada, P. gracilis, P. irroratus, P. maximus, P. rupestris, P. signifer and P. soaresi. Second, we present a comparison among tadpoles of these ten species plus the tadpoles of P. albifrons, P. angrensis, P. centralis, P. crombiei, P. cuvieri, P. henselii, P. jordanensis, P. marmora-tus, P. moreirae and P. spiniger. This study adds information on tadpoles of the genus Physalaemus that corroborates some of the species groups proposed for adult specimens, but also shows that although they have a typical pond-living external morphology, being very similar to one another, the oral disc and the internal oral morphologies present many features with interspecific variation that may be used to identify the specimens.

Key words: systematics, larval anatomy, external anatomy, internal oral anatomy

Introduction

The Neotropical genus Physalaemus Fitzinger belongs to the subfamily Leiuperinae according to Pyron and Wiens (2011), and comprises small-sized frogs widely distributed from northern to central Argentina, eastern Bolivia, Par-aguay, Uruguay, Brazil, the Guianas, lowlands of southern Venezuela, southeastern Colombia, and western Ecua-dor (Frost, 2011). Currently, there are 45 recognized species in the genus allocated into seven morphological groups based on characteristics of the adults (Nascimento et al., 2005), 29 with the tadpole described.

These larvae are typical pond-living tadpoles that are assumed to occur in the bottom of these lentic water bod-ies (Rossa-Feres et al., 2004; Vera Candioti, 2007) and are very similar to one another regarding to their external morphology (Vieira & Arzabe, 2008).

For 13 of the 30 tadpoles described in the genus, there is information upon the internal oral morphology: P. santafecinus and P. biligonigerus (Perroti & Céspedez, 1999); P. riograndensis (Sandoval, 2002); P. lisei (Both et al., 2006) P. marmoratus (Nomura et al, 2003); P. fernandezae (Alcade et al., 2006), P. albonotatus, P. centralis and P. cuvieri (Miranda & Ferreira, 2009), P. jordanensis (Gomes et al., 2010), P. albifrons (Oliveira et al., 2010), P. moreirae (Provete et al., 2011), and P. angrensis (Ruggeri et al., 2011). According to Wassersug (1976, 1980), the oral cavity of the tadpoles is characterized by having many papillae whose arrangement, length, and number differ among genera and species and contain important information that may contribute on the understanding of the systematic of anuran groups (Spirandeli-Cruz, 1991; d’Heursel & de Sá, 1999).

In order to provide new features that allow the differentiation of tadpoles of this genus, herein we analyze 20 species from all seven morphological groups and we compare the external morphology, the oral morphology and the internal oral cavity morphology of these tadpoles, suggesting a standardized nomenclature by listing the most relevant characters that should be considered in descriptions.

Accepted by M. Vences: 12 Jan. 2012; published: 21 Feb. 2012 1

mailto:joice.ruggeri@gmai

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Material and methods

We studied specimens housed at the zoological collections of: CFBH (Universidade Estadual Paulista—Rio Claro, São Paulo), DZSJRP (Universidade Estadual Paulista—São José do Rio Preto, São Paulo), MNRJ (Museu Nacio-nal, Rio de Janeiro), ZUFRJ (Universidade Federal do Rio de Janeiro, Rio de Janeiro), and UFBA (Universidade Federal da Bahia, Bahia).

The following species are analyzed in this study: Physalaemus albifrons (Spix) and P. marmoratus (Reinhardt and Lütken), from the P. albifrons group; P. centralis Bokermann, P. cicada Bokermann, and P. cuvieri Fitzinger, from the P. cuvieri group; P. rupestris Caramaschi, Carcerelli, and Feio, from the P. deimaticus group; P. gracilis (Boulenger) and P. jordanensis Bokermann, from the P. gracilis group; P. henseli (Peters), from the P. henseli group; P. aguirrei Bokermann, P. maximus Feio, Pombal and Caramaschi, and P. soaresi Izecksohn, from the P. olf-ersii group; and P. angrensis Weber, Gonzaga, and Carvalho-e-Silva, P. atlanticus Haddad and Sazima, P. camacan Pimenta, Cruz and Silvano, P. crombiei Heyer and Wolf, P. irroratus Cruz, Nascimento and Feio, P. moreirae (Miranda-Ribeiro), P. signifer (Girard) and P. spiniger (Miranda-Ribeiro), from the P. signifer group.

To confirm species identity, we used the following methods: tadpoles from the original descriptions; tadpolesobtained from foam nests, raised in captivity for species identification; and those specimens previously identified were compared to original descriptions. Additional information on the examined material is presented in Appendix I.

Tadpole developmental stages were determined according to Gosner (1960) and all them analyzed are at stages 33–38 (Table 1). Measurements in millimeter of the total length (TL), body length (BL), body maximum height (BH), tail length (Tail L), tail maximum height (Tail H), tail musculature maximum height (HTM), interorbital dis-tance (IOD), eye diameter (ED), nostril diameter (ND), nostril-snout distance (NSD), eye-snout distance (ESD), snout-spiracle distance (SSD), oral disc width (ODW), buccal roof height (BRH), buccal roof width (BRW), inter-narial distance (IND), median ridge height (MRH), median ridge width (MRW), buccal floor height (BFH) and buccal floor width (BFW) were obtained by using an ocular micrometer in a stereo microscope (Olympus SZ40).

For studying the internal oral morphology, one or two tadpoles of each species were dissected under the stereo microscope and the oral morphological features were stained with 3% Methylene Blue solution. Procedure follows Wassersug (1976) and Spirandeli-Cruz (1991). Dissections were made carefully inserting the micro dissecting scis-sors into the left corner of the mouth between the upper and lower beaks and cutting back to the posterodorsal cor-ner of the pharynx. This cut separated Meckel’s cartilage from the palatoquadrate bar and that from the ceratohyal. The same procedure was made in the right side. The mouth could then be opened and oral surface exposed. A lon-gitudinal cut was made along the side of the mouth, freeing the roof from the floor.

The study of these tadpoles is divided into two different parts. First we present the description following the terminology proposed by Wassersug (1976) of the oral cavity morphology for the tadpoles of P. aguirrei, P. atlanti-cus, P. camacan, P. cicada, P. gracilis, P. irroratus, P. maximus, P. rupestris, P. signifer, and P. soaresi.

In the second part, we provide comparisons of the external and internal morphologies of the tadpoles studied, except for the internal oral anatomy of P. henseli and P. spiniger that were not studied herein.

The external morphology of the tadpoles analyzed in this study has been previously described but in order to compare it to our data, it was necessary to standardize the nomenclature. For this purpose, we used the key pro-vided by Rossa-Feres & Nomura (2006), with modifications.

We built a matrix with 32 characters and 75 states of characters (see Appendix II), and performed a clustering analysis, the UPGMA (unweighted pair group method with arithmetic mean) (Sneath & Sokal, 1973) based on the Gower distance, in order to simplify comparisons among species. The analysis was conducted using the software PAST version 2.04 (Hammer et al., 2001).

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Results

Oral cavity features—Table 2.

TABLE 2. Mean and Standard Deviation of the internal oral features of the tadpoles of Physalaemus aguirrei (N =1), P. atlan-ticus (N =2), P. camacan (N =2), P. cicada (N =2), P. gracilis (N =2), P. irroratus (N =2), P. maximus (N =2), P. rupestris (N =2), P. signifer (N =2) and P. soaresi (N =1), Stage 33–38 (Gosner, 1960) in millimeter (mm). BRH = Buccal Roof Height; BRW = Buccal Roof Width; IND = Internarial Distance Median Ridge; MRH = Median Ridge Height; MRW = Median Ridge Width; BFH = Buccal Floor Height; BFW = Buccal Floor Width.

Physalaemus aguirrei Bokermann

Ventral aspect (Fig. 1A): At Stage 37, buccal floor triangular. Presence of two pairs of infralabial papillae. The anterior pair (hidden under the posterior pair) is thin with flattened apex, much smaller than the posterior and directed upward. The posterior pair is large, long and curved upward, easily discernible from the anterior pair. Tongue anlage elliptical, with four long and tapered lingual papillae in a transverse row. Buccal pockets horizon-tally oriented. In the buccal floor arena (BFA) there are three papillae on each side of the arena, pointing to the cen-ter; the first and last papillae are finger-like and the one in the middle is chela-shaped; and about ten pustulations dispersed on it. Ventral velum round shaped with small projections along its external margin. Dorsal aspect (Fig. 1B): Buccal roof somewhat triangular. Prenarial arena longer than wide, without papillae or pustulations on it. Elliptical choanae, large, oriented 45° from transverse plane. Internarial distance 5% of the roof width. In the post-narial arena, presence of one pair of finger-like papillae pointing to the center. The median ridge is trapezoid, twice wider than long, with few projections in the superior margin. Presence of a pair of chela-shaped lateral ridge papil-lae. In the buccal roof arena (BRA), about twenty pustulations dispersed, and four finger-like papillae on each side directed to the center of BFA. Dorsal velum with no projections.

Physalaemus atlanticus Haddad & Pombal

Ventral aspect (Fig. 1C): At Stage 37, buccal floor triangular. Presence of two pairs of infralabial papillae, both are large in the basis and flattened in the extremity with irregular margins and apices, but the posterior pair is larger and much discernible (the anterior pair is hidden under the posterior pair). Tongue anlage elliptical, with four tapered lingual papillae in a transverse row, directed upwards; the two in the middle are longer than the ones in the extremities. Buccal pockets horizontally oriented. In the BFA, presence of one bifid, long and tapered papilla on each side and another five long and finger-like papillae on each side, pointing to the center of the arena and few pustulations dispersed. Ventral velum round shaped with small projections. Dorsal aspect (Fig. 1D): Buccal roof somewhat triangular. Prenarial arena longer than wide; there are some short, conical papillae distributed in a trans-verse row. Elliptical choanae, large, oriented 45° from transverse plane. Internarial distance 8% of the roof width. In the postnarial arena, presence of one pair of long papillae, large in the basis and tapered in the end, directed to

Species BHR BRW IND MRH MRW BFH BFW

P. aguirrei 4.0 3.3 0.2 0.4 0.2 2.6 2.6

P. atlanticus 3.8 (±0.2) 2. 9 (±0.2) 0.2 0.5 0.2 2.4 (±0.1) 2.9 (±0.1)

P. camacan 3.3 3.3 0.2 0.3 0.2 2.3 2,8 (±0.2)

P. cicada 3.0 2.6 (±0.4) 0.2 0.5 (±0.1) 0.2 2.0 2.31

P. gracilis 3.5 (±0.2) 3.6 0.3 (±0.1) 0.5 (±0.1) 0.2 (±0.1) 3.0 3.3

P. irroratus 3.1 (±0.2) 2.5 (±0.2) 0.2 0.4 0.2 (±0.1) 2.2 (±0.1) 2.6

P. maximus 4.3 3.8 (±0.2) 0.3 0.7 0.3 (±0.1) 3.3 3.8 (±0.2)

P. rupestris 3.6 3.1 (±0.2) 0.3 (±0.1) 0.6 (±0.1) 0.3 (±0.1) 2.9 (±0.1) 3.6

P. signifer 3.6 (±0.5) 3.0 0.2 0.4 (±0.1) 0.2 2.3 2.3

P. soaresi 3.3 3.0 0.2 0.5 0.1 2.5 2.6



FIGURE 1. (A) Floor and (B) roof of Physalaemus aguirrei; (C) Floor and (D) roof of P. atlanticus; (E) Floor and (F) roof of P. camacan. Stage 37. Scale = 1mm.



the center; and a pair of short and flattened papillae. Presence of a pair of bifid lateral ridge papillae, large in the basis, with tapered branches. The median ridge in semicircle is about five times wider than long, with few projec-tions in the superior margin. In the BRA about thirty pustulations dispersed in the arena; it is limited by five papil-lae on each side, directed to the center of BFA; the first papilla on each side is slender but bifid and the others are finger-like papillae. Dorsal velum with no projections.

Physalaemus camacan Pimenta, Cruz & Silvano

Ventral aspect (Fig. 1E): At Stage 37, buccal floor slightly triangular. Presence of two pairs of infralabial papillae. The posterior pair is large and has irregular margins and apex, while the anterior pair is short and tapered, with irregular margins. The posterior pair is easily discernible from the anterior pair and it hides the anterior pair. Tongue anlage oval with four long, conical lingual papillae in a transverse row, with smooth surfaces. Small buccal pockets horizontally oriented. In the BFA, presence of five–six papillae on each side; the second papillae are bifid and the others are finger-like papillae, all point to the center of the arena, except for the last papillae that are directed upward. There are few pustulations dispersed on the arena. Ventral velum round with some projections in the margin. Dorsal aspect (Fig. 1F): Buccal roof slightly triangular. Prenarial arena wider than longer, without papillae or pustulations on it. Choanae elliptical, oriented 45° from transverse plane. Internarial distance 5% the buccal roof length. In the postnarial arena, presence of a pair of tapered papillae, irregular and large in the basis, directed to the center. One chela-shaped lateral ridge papilla at each side. The median ridge is in semicircle, longer than wide, with some irregularities in the margin. The BRA has about 15 pustulations dispersed, limited by four thin and tapered papillae on each side, directed to the center of the arena. Dorsal velum with irregular margin.

Physalaemus cicada Bokermann

Ventral aspect (Fig. 2A): At Stage 36, buccal floor triangular. Presence of two pairs of infralabial papillae. The pos-terior pair is large in the basis, tapered in the apex, and with irregular margins, while the anterior pair (hidden under the posterior pair) is small, thin and tapered, and is directed upward. The posterior pair is easily discernible from the anterior. Tongue anlage elliptical with four small and tapered lingual papillae in a transverse row, directed upward. Buccal pockets horizontally oriented. In the BFA presence of four–five finger-like papillae on each side, pointing to the center of the arena and about 20 pustulations dispersed on it. Ventral velum round shaped with small projections. Dorsal aspect (Fig. 2B): Buccal roof triangular. Prenarial arena longer than wide. Some short, conical papillae are distributed in a transverse row in the prenarial arena. Elliptical choanae, large, oriented 45° from trans-verse plane. Internarial distance 7% of the roof width. In the postnarial arena, presence of one large finger-like papilla on each side. The median ridge is trapezoid, about three times wider than long, with irregular superior mar-gin. Presence of a pair of lateral ridge papillae, large and chela-shaped. In the BRA about 40 pustulations dispersed on the arena, limited by three to five finger-like papillae on each side, directed to the center of the arena. Dorsal velum with projections.

Physalaemus gracilis (Boulenger)

Ventral aspect (Fig. 2C): At Stage 37, buccal floor triangular. Presence of two pairs of infralabial papillae. The anterior pair is more centrally positioned, is smaller, tapered and directed upward, while the posterior pair is large, bifid, with irregular apices. The posterior pair is much discernible from the anterior pair. Tongue anlage oval with four tapered lingual papillae in a transverse row, directed upward. Buccal pockets horizontally oriented. In the BFA presence of five to seven papillae on each side. Some papillae are large and chela-shaped and some are finger-like; there are about 30 pustulations dispersed in the arena. Ventral velum round shaped. Dorsal aspect (Fig. 2D): Buccal roof triangular. Prenarial arena longer than wide, with three short, flattened papillae on it. Elliptical choanae, large, oriented 45° from transverse plane, serrated in the superior margin. Internarial distance 8% of the roof width. In the postnarial arena, presence of two papillae on each side, one shorter thin and flattened papilla and another large



FIGURE 2. (A) Floor and (B) roof of P. cicada; (C) Floor and (D) roof of P. gracilis; (E) Floor and (F) roof of P. irroratus. Stage 36–37. Scale = 1mm.



finger-like papilla that points to the center. The median ridge is in semicircle, postulated in the superior margin. Presence of a pair of large chela-shaped lateral ridge papillae, with irregular margins. In the BRA five–six papillae on each side; there are three short, thin finger-like lateral roof papillae. Presence of 30 pustulations dispersed on the arena. Dorsal velum with no projections.

Physalaemus irroratus Cruz, Nascimento & Feio

Ventral aspect (Fig. 2E): At Stage 36, buccal floor triangular. Presence of two pairs of infralabial papillae. The pos-terior pair is large in the basis, tapered in the apex, with irregular margin. The anterior pair is tapered, directed upward and hidden under the posterior pair. The posterior pair is easily discernible from the anterior. Tongue anlage elliptical with three small and tapered lingual papillae in a transverse row, directed upwards. Buccal pockets horizontally oriented. In the BFA about ten papillae on each side, pointing to the center of the arena, thin and tapered, except for the second papillae, which are chela-shaped. There are few pustulations dispersed on the arena. Ventral velum with small projections. Dorsal aspect (Fig. 2F): Buccal roof somewhat triangular. Prenarial arena longer than wide, with presence of some short, conical papillae arranged in semicircle on it. Elliptical choanae, large, oriented 45° from transverse plane. Internarial distance 7% of the roof width. In the postnarial arena, pres-ence of one finger-like papilla on each side, large in the basis and serrated in the superior margin, directed upward. The median ridge is in semicircle, twice wider than long, with small projections along its superior margin. Presence of a pair of large chela-shaped lateral ridge papillae. In the BRA about 30 pustulations dispersed on it, and four long and tapered papillae arranged parallel to each other on each side, all directed to the center of BFA. Dorsal velum with no projections.

Physalaemus maximus Feio, Pombal & Caramaschi

Ventral aspect (Fig. 3A): At Stage 36, buccal floor somewhat triangular. Presence of one short and tapered infrala-bial papilla on each side, directed upward. Tongue anlage elliptical with four tapered lingual papillae in a trans-verse row. Buccal pockets horizontally oriented. In the BFA about 20 pustulations and five to seven long, tapered papillae on each side, some pointing to the center and some directed upward, except for the first on each side that may be larger and bifid. Ventral velum with no projections. Dorsal aspect (Fig. 3B): Buccal roof somewhat trian-gular. Prenarial arena longer than wide, with about some short, conical papillae on it. Elliptical choanae, large, ori-ented 45° from transverse plane. Internarial distance 8% of the roof width. In the postnarial arena, presence of two large papillae on each side. The posterior pair is larger and has irregular margins and apices; the anterior pair is large in the basis, becoming tapered. The posterior pair is much discernible from the anterior. The median ridge is trapezoid, twice wider than long, with some projections along its margin. Presence of a large and bifid lateral ridge papilla on each side. In the BRA about 40 pustulations dispersed and four–five finger-like papillae on each side directed to the center of BFA. The last papilla on each side may be bifid. Dorsal velum with no projections.

Physalaemus rupestris Caramaschi, Carcerelli & Feio

Ventral aspect (Fig. 3C): At Stage 36, buccal floor triangular. Presence of one pair of infralabial papillae, large in the basis and tapered, directed upward. Tongue anlage elliptical with four long and tapered lingual papillae in a transverse row, directed upwards; the papillae found in the extremities are smaller and flattened. Buccal pockets horizontally oriented. Presence of few pustulations dispersed on the BFA. There are four long, finger-like papillae on each side, pointing to the center of the arena, and about ten papillae closer to the velum, directed upwards. Ven-tral velum with small projections. Dorsal aspect (Fig. 3D): Buccal roof somewhat triangular. Prenarial arena longer than wide, with two short, flattened papillae. Elliptical choanae, large, oriented 45° from transverse plane. Internar-ial distance 9% of the roof width. In the postnarial arena, presence of two papillae on each side. The posterior pair is a large finger-like papilla with irregular margin that points to the center and the anterior pair is a smaller and tapered. The posterior pair is discernible from the anterior. There is one lateral ridge papilla on each side, large in



FIGURE 3. (A) Floor and (B) roof of P. maximus; (C) Floor and (D) roof of P. rupestris; (E) Floor and (F) roof of P. signifer. Stage 36–37. Scale = 1mm.



the basis and chela-shaped. The median ridge is triangular, about two times wider than long. In the BRA 30 pustu-lations in the arena and about six finger-like papillae on each side, arranged parallel to each other and directed to the center of BRA. Dorsal velum with no projections.

Physalaemus signifer (Girard)

Ventral aspect (Fig. 3E): At Stage 37, buccal floor triangular, as long as wide. Presence of one pair of large infrala-bial papillae, with irregular margins. Tongue anlage elliptical with two small and conical lingual papillae in a trans-verse row. Absence of prepocket papillae. Buccal pockets horizontally oriented. In the BFA about 20 pustulations dispersed and five papillae on each side, pointing to the center of the arena. The first papilla on each side may be chela-shaped and the others are shorter, slender and tapered. Ventral velum with small projections. Dorsal aspect(Fig. 3F): Buccal roof somewhat triangular. Prenarial arena longer than wide, with no pustulations or papillae on it. Elliptical choana, large, oriented 45° from transverse plane. Internarial distance 7% of the roof width. In the post-narial arena, presence of two pairs of papillae. One pair is large and bifid and the other pair is finger-like with irreg-ular margins, and points to the center. Presence of one large, bifid lateral ridge papillae on each side. The median ridge is in semicircle, about two times wider than long. In the BRA 20 pustulations dispersed on the arena and about one-two slender and tapered papillae on each side directed to the center of BFA. Dorsal velum with no pro-jections.

Physalaemus soaresi Izeckson

Ventral aspect (Fig. 4A): At Stage 33, buccal floor triangular. Presence of two pairs of infralabial papilla. The pos-terior pair is a large finger-like papillae; the anterior pair is short and tapered, directed upward. Tongue anlage oval with three long and tapered lingual papillae in a transverse row, directed upwards. Buccal pockets horizontally ori-ented. In the BFA there are few pustulation dispersed and about two–three papillae on each side, parallel to each other. The sencond papillae are chela-shaped and the others are finger-like and long. Ventral velum with small pro-jections. Dorsal aspect (Fig. 4B): Buccal roof somewhat triangular. Prenarial arena longer than wide, with three short, flattened papillae on the midline. Elliptical choanae, large, oriented 45° from transverse plane. Internarial distance 6% of the roof width. In the postnarial arena, presence of one long finger-like papilla on each side, directed upward. The median ridge is trapezoid, four times wider than long, with some projections in the superior margin. Presence of a pair of lateral ridge papillae, large and bifid in the basis with irregular margins. In the BRA there are few pustulations dispersed and four long and tapered papillae on each side arranged parallel to each other and directed to the center of BFA. Dorsal velum with no projections.

FIGURE 4. (A) Floor and (B) roof of P. soaresi. Stage 33. Scale = 1mm.


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Morphological characters

The morphological characters of the tadpoles of Physalaemus species used in this study is presented and described below. All tadpoles are at Stages 33–38.

I. EXTERNAL MORPHOLOGY1. Body shape in lateral view (LV): The body of the tadpoles studied varied from elliptical (0) to globular/

depressed (1) in lateral view (Fig. 5A,B). The elliptical body found in the literature can be synonym of ovoid, because the tip of the snout as well as the terminal body is rounded, while the globular/depressed body consists of a triangular and long body. In dorsal and ventral views, all tadpoles have an ovoid body.

2. Shape of the snout in lateral view (SSL): The snout may vary interspecific regarding to its shape. Some tad-poles have an ovoid (0) snout and some have it rounded (1) in lateral view (Fig. 5C,D). The difference is subtle, but the ovoid snout is a little less curved than the round snout.

3. Shape of the snout in dorsal view (SSD): In dorsal view, the snout of tadpoles could be ovoid (0) or rounded (1) (Fig. 5E,F).

4. Shape of nostrils (NS): The nostrils vary in shape; it can be round (0) or ovoid (1) (Fig. 5G,H). 5. Size of nostrils (N): The nostrils also vary in length; it can be large (0) or small (1) (Fig. 5G,H). 6. Spiracle position (SpP): The spiracle is sinistral and located at the middle third of the body in all species stud-

ied, but it can be lateral (0), or ventrolateral (1) (Fig.6A,B). It is ventrolateral when the spiracle originates in the vent region.

7. Spiracle (Sp): Regarding to the length of the spiracle, two states of character occur within species; it can be short (0), or medium sized (1)

8. Vent tube (VT): The vent tube can be attached to the ventral fin in the right side (0) or in the left side (1). 9. Fins (TF): The dorsal fin can be slightly taller than the ventral fin (0) or it may be about twice taller than the

ventral fin (1) (Fig. 6C,D).10. Tail (TS): The tail muscle may be straight (0) or arched and the arched tail range from much arched (1) to little

arched (2).11. Tail musculature (TM): The musculature of the tail has three states of character: it can be developed (0), slen-

der (1) or even moderately developed (2).

II. ORAL DISC12. Position (POD): The oral disc varies interspecifically and is an important feature for the species identification

(Fig. 6E,F). In the tadpoles of Physalaemus, the oral disc can be ventral (0) or anteroventral (1).13. Sheath (Sh): The jaw sheaths are weakly keratinized (0) or strongly keratinized (1). 14. Row of marginal papillae (RP): The oral disc may be surrounded by a single row (0) of marginal papillae; or by

a double row (1) (Fig. 7A,B). 15. Ventral gaps in the marginal papillae - total (MP): In Physalaemus species this character occurred in four dif-

ferent states: absent (0), one mental gap and no ventrolateral gaps (1), two ventrolateral gaps and no mental gap (2), one mental gap and two ventrolateral gaps (3) (Fig. 7A,B,C,D).

16. Tooth row formula (TR): It was found three different tooth row formulas (Fig. 7E,F,G) in the tadpoles: [2(2)/2] (0); [2(2)/3(1)] (1); [2(2)/3(1-2)] (2); [2(2)/2(1)] (3).

17. Length of posterior teeth rows (PTR): There are three states of character observed regarding to the length of the posterior tooth rows: they can be all equal in size (0); the last row can be little smaller than the others (1); or the last row can be twice (or more) smaller than the other rows (2).

III. BUCCAL FLOOR18. Number of infralabial papillae (IP): Tadpoles of Physalaemus may have one pair of infralabial papillae (0) or

two pairs (1). 19. Shape of infralabial papillae (SIP): This character describes the most visible pair (in case of two pairs), and it

may be slender (0) or large (1). The posterior pair, when present, is always larger and easily discernible from the anterior pair.

20. Shape of the anterior pair of infralabial papillae (IPP): Absence of the anterior pair (0). When present it may be thin (1) or large (2).



FIGURE 5. (A) P. albifrons: elliptical body in lateral view, and (B) P. cuvieri: globular/depressed body in lateral body. (C) P. aguirrei: snout ovoid in lateral view, (D) P. atlanticus: snout round in lateral view; (E) P. aguirrei: snout ovoid in dorsal view, and (F) P. atlanticus: snout round in dorsal view. (G) P. gracilis: nostrils large and oval, and (H) P. henselii: nostrils small and round.



FIGURE 6. Spiracle (A) P. aguirrei: lateral and (B) P. centralis: ventrolateral. (C) P. atlanticus: dorsal fin slightly taller than ventral fin, and (D) P. centralis: dorsal fin twice taller than ventral fin and. (E) P. camacan: ventral oral disc, and (F) P. jordan-ensis: anteroventral oral disc.

21. Number of lingual papillae (LP): This trait occurred in three different states (Fig. 8A,B,C) may be present in one pair (0); in odd numbers (1); or in two pairs (2).

22. Number of buccal floor papillae (BFP): The buccal floor arena is limited by papillae on both sides. The number of these papillae may vary interspecifically. In the tadpoles analyzed, they were found in three states: 4–8 (0); 10–14 (1); 16–20 (2).

23. Number of pustulations at buccal floor arena (BFPu): The buccal floor arena presents pustulations dispersed on it. Pustulations on the arena varied from few, when it was ≤ 10 (0) to moderate, 11–29 (1) and dense ≥ 30 (2).



FIGURE 7. (A) P. albifrons: single row of marginal papillae; a mental gap (*) and two ventrolateral gaps (**); (B) P. aguirrei: double row of marginal papillae; no mental gap and two small ventrolateral gaps; (C) P. cicada: single row of marginal papil-lae; a mental gap and no ventrolateral gaps; and (D) P. signifer; single row of marginal papillae; no mental or ventralateral gaps. (E) P. crombiei: tooth row formula 2(2)/3(1), where P-3 = P-2 = P-1; (F) P. albifrons: tooth row formula 2(2)/3(1,2), where P-3 < P-2 < P-1; and (G) P. centralis: tooth row formula 2(2)/2, where P-2 = P-1. Scale = 1 mm.



FIGURE 8. (A) P. crombiei: two lingual papillae; (B) P. albifrons; three lingual papillae; and (C) P. cicada: four lingual papil-lae. (D) P. marmoratus: median ridge semicircular; (E) P. rupestris: median ridge triangular; and (F) P. maximus: median ridge trapezoid. Scale = 1 mm.



IV. BUCCAL ROOF24. Projections at prenarial arena (PreNar): Pustulations or papillae on the prenarial arena can be absence (0) or

present (1). 25. Number of papillae in the postnarial arena (PosNarP): The papillae on the arena were found in one pair (0), two

pairs (1) or more than two pairs (2). 26. Shape of papillae in the postnarial arena (SPosNarP): When there is one pair, papillae may be thin (0) or large

(1); when there are more than one pair, some papillae are large and tapered and some are short and conical (2)27. Shape of median ridge (MR): It was found three states of character: median ridge semicircular (0), median

ridge trapezoid (1) and median ridge triangular (2) (Fig. 8D,E,F)28. Lateral median ridge papillae (MRP): The lateral ridge papillae may be chela–shaped (0) or bifid in the

extremity (1). 29. Lateral roof papillae (LRP): Lateral roof papillae may absent (0) or present (1).30. Buccal roof papillae (BRP): The buccal floor arena is also limited by papillae on both sides that may vary

interspecifically. In tadpoles analyzed, the character “number of papillae” was found in two states: 4–10 (0) and 12–18 (1).

31. Number of pustulations at buccal floor arena (BRPu): The buccal floor arena is also filled with pustulations that vary from few, ≤ 10 (0), to moderate, 11–29 (1), and dense ≥ 30 (2).

32. Margin of velum dorsal (VD): absence of projections in the margin (0); presence of projections in the margin (1).

Comparison among species

By analyzing only the external features from tadpoles (Fig. 9), Physalaemus jordanensis and P. signifer are the most similar species, differing only as the jaw sheaths that are strongly keratinized in the first, and as the teeth rows, that are all equal in sizes in P. signifer. This group is similar to the group formed by P. marmoratus and P. maximus and they all share large nostrils, round snout, a laterally positioned spiracle, a vent tube attached in the left side, arched tail with a slender muscle. The oral disc is anteroventral bordered by a single row of papillae.

The tadpoles of P. cicada and P. irroratus form a group that shares the shape of the body and snout and the size of nostrils, the left vent tube, the dorsal fin about twice taller than the ventral fin, the medium sized spiracle, the anteroventral oral disc, tooth row formula 2(2)/3(1) and a single row of marginal papillae.

Tadpoles of P. moreirae have a quite different external morphology, sharing two features with the group formed by tadpoles of P. cicada, P. irroratus, P. jordanensis, P. marmoratus, P. maximus and P. signifer: anteroven-trally positioned oral disc bordered by a single row of marginal papillae.

Tadpoles of P. angrensis, P. atlanticus, P. crombiei and P. soaresi form a group divided into P. angrensis and P. atlanticus, and P. crombiei and P. soaresi. They all share round snout and round nostrils, the vent tube attached in the right side, a ventrally positioned oral disc with strongly keratinized jaw sheaths, and the tooth row formula 2(2)/3(1). The tadpole of P. albifrons shares with this group the position of the vent tube, the body shapes, and the posi-tion of the oral disc.

Another group formed in this analysis unites P. camacan, P. centralis, P. gracilis, P. henselii, P. rupestris and P. spiniger, but they only share the vent tube attached in the left side and the round snout in dorsal view. On the other hand, P. centralis and P. spiniger differ only as the lateral medium sized spiracle in P. camacan, a much arched tail and slender muscle in P. spiniger, and the size of the last teeth row in the oral disc, that is much small than the other rows in P. camacan. The tadpoles of P. gracilis and P. rupestris are the most similar to each other in the group, dif-fering only as the oral disc: jaw sheaths strongly keratinized in P. gracilis, and tooth row formula 2(2)/3(1,2) and teeth rows equally sized in P. rupestris.

Tadpoles of P. aguirrei and P. cuvieri are similar to each other but the most different group formed. They only differ from each other as the shape of nostrils (ovoid in the first), position of the vent tube (attached at the left size of the body in P. aguirrei), the arched tail in the last,

By considering only morphological features from the internal oral cavity (Fig. 10), P. jordanensis presents the most different tadpole. On the other hand, the most similar species are P. angrensis and P. signifer, differing just as two traits from the buccal roof: presence of two large papillae on the postnarial arena in the first and presence of four papillae in the latter, one large and one small at each side of the BRA.



Physalaemus angrensis, P. maximus, P. rupestris and P. signifer have two infralabial papillae, a chela-shaped lateral median ridge on each side, absence of lateral roof papillae, a great amount of pustulations dispersed on the buccal roof arena and the absence of projections in the dorsal velum.

Tadpoles of P. albifrons, P. atlanticus, P. crombiei and P. gracilis share four infralabial papillae, one pair ante-rior and one pair posterior, the posterior pair is large in them all, presence of papillae or pustulations on the prenar-ial arena, four papillae on the postnarial arena where two are larger and the presence of 4–8 papillae on the buccal roof arena.

Physalaemus centralis and P. soaresi share some traits of the buccal floor, as the number and shape of infrala-bial papillae (four thin and tapered papillae) and the number of papillae on the buccal floor (10–14), besides some features in the buccal roof, as the presence of papillae or pustulations on the prenarial arena, presence of two thin papillae on the postnarial arena, a trapezoid shaped median ridge, absence of lateral roof papillae, presence of 4–10 papillae on the buccal roof arena and absence of projections in the dorsal velum.

Tadpoles of P. aguirrei, P. camacan, P. cicada, P. cuvieri, P. irroratus and P. moreirae have a large pair of infralabial papillae, tapered papillae on the postnarial arena, absence of lateral roof papillae, chela-shaped lateral ridge papillae and 4–10 buccal roof papillae. Inside this group, P. cicada and P. cuvieri share the presence of struc-tures (papillae or pustulations) on the prenarial arena, one pair of papillae on the postnarial arena, trapezoid median ridge, a great amount of pustulations dispersed on the buccal roof arena and presence of few projections in the dor-sal velum.

When considering all morphological features (Fig. 11), the tadpoles of P. henselii and P. spiniger, were removed from the analysis.

The results show that P. crombiei and P. gracilis, are the most similar tadpoles, sharing 26 traits: four infrala-bial papillae, one large and one slender on each side, 10–14 papillae on the buccal floor arena, papillae or pustula-tions are present on the prenarial arena, four small and tapered papillae on the postnarial arena, a semicircle median ridge with a chela-shaped lateral ridge papilla on each side, 4–10 papillae on the buccal roof arena, which is full of pustulations, and without projections in the ventral velum; regarding to the oral disc, they share all features: ventral oral disc, strongly keratinized jaw sheaths, marginal papillae in a single row, tooth row formula 2(2)/3(1) where P–3 is smaller than P–1 and P–2, no mental gap present; both have elliptical body in lateral view and rounded snout in dorsal view, the nostrils are large and round, the spiracles are ventrolateral and short, and tail are very arched with the muscle developed. They differ as the amount of pustulations on the buccal floor arena and as the shape of lat-eral ridge papillae on the buccal roof, besides the shape of tail and the snout profile, and the position of vent tube. They are inserted in the group formed by P. centralis, P. crombiei, P. gracilis, P. maximus, P. moreirae, and P. rup-estris. They all have a little arched tail, a single row of marginal papillae in the oral disc, presence of postulations (or small papillae) on the prenarial arena, and chela–shaped lateral ridge papillae.

Another group was formed by the tadpoles of P. angrensis, P. irroratus, P. signifer, and P. soaresi, sharing eight traits. They have round snout and slender tail muscle, a single row of marginal papillae and tooth row formula 2(2)/3(1), absence of lateral roof papillae, buccal roof arena limited by 4–10 papillae, and absence of projections in the margin of the dorsal velum.

Physalaemus aguirrei, P. camacan, P. cicada, P. cuvieri, and P. jordanensis formed a group that shares a later-ally positioned spiracle, tooth row formula 2(2)/3(1), a pair of thin papillae on the postnarial arena, chela–shaped lateral ridge papillae, and absence of lateral roof papillae.

In the group formed by P. albifrons, P. atlanticus and P. marmoratus, the first two species are most similar to one another; they differ as the oral disc and shape of nostrils (that is ovoid in P. albifrons), and a pair of pair of thin infralabial papillae (large in P. atlanticus, two pais of lingual papillae in P. atlanticus, buccal floor arena limited by 4–8 and few pustulations on the buccal roof arena in P. albifrons, and absence of projections on the dorsal velum in P. atlanticus. The tadpole of P. marmoratus share with these larvae, the globular/depressed body, round snout, large nostrils, lateral and medium sized spiracle and slender tail; in the buccal floor, they share two pairs of large infrala-bial papillae and few pustulations on the arena; in the buccal roof, they share the presence of pustulations or small papillae on the prenarial arena, the semicircular median ridge with bifid lateral ridge paipillae, absence of lateral ridge papillae, and 4–10 papillae at each side, on the buccal roof arena.



Discussion

External morphology. The tadpoles studied are between stages 33–38 and showed little interspecific variation regarding to the external morphology.

There are subtle differences regarding to the body and the snout shapes. The tadpoles of P. aguirrei have an oval snout like the tadpole of P. henselii, which is different from the rounded snout of P. atlanticus and P. camacan. The tadpoles of P. irroratus have the shortest snout among the specimens analyzed and it is also round in both lat-eral and dorsal views.

The spiracle of P. henselii is the shortest among the studied tadpoles; it is originally described (Kolenc et al., 2006) as laterally positioned, but it was noticed herein that it originates above the median line of the body and can-not be seen in dorsal view, so it is in fact ventrolateral. The spiracle is also ventrolateral in the tadpoles of P. spini-ger although Haddad & Pombal (1998) did not mention it, different from P. signifer, which spiracle is laterally positioned.

The tail also shows some interspecific differences; the dorsal fin is always taller than the ventral fin but in the tadpoles of P. cicada, P. crombiei, P. moreirae, P. signifer, and P. soaresi, the dorsal fin is about twice taller than the ventral fin.

The oral disc presents many features that vary interspecific among tadpoles of the genus Physalaemus. The position of the oral disc in the tadpoles analyzed was ventral in the tadpoles of P. albifrons, P. angrensis, P. atlanti-cus, P. camacan, P. crombiei, P. cuvieri, P. gracilis, P. irroratus, and P. spiniger, and anteroventral in the tadpoles of P. aguirrei, P. centralis, P. cicada, P. henselii, P. jordanensis, P. marmoratus, P. maximus, P. moreirae, P. rupestris, P. signifer, and P. soaresi. The lower lip may have a mental gap and/or ventrolateral gaps, or the papillary may be continuous. Tadpoles of P. aguirrei, P. albifrons, P. centralis, P. cicada, P. cuvieri, P. henselii, and P. marmoratushave gaps in the lower lip (Pimenta & Cruz, 2004; Oliveira et al., 2010; Rossa-Feres & Jim, 1993; Vieira & Arzabe, 2008; Heyer, 1990; Kolenc et al., 2006; Nomura et al., 2003).

It seems that the number of marginal papillae in the lower lip may vary. Nomura et al. (2003) described for P. marmoratus (as P. fuscomaculatus) the presence of “a single row (23.7%), alternate row (65.8%) or mixed of both (10.5%)”, although all specimens analyzed herein presented only the second type with two ventrolateral gaps. Sim-ilar occurs to P. atlanticus; its oral disc is bordered by “one or two rows of small papillae, interrupted along a large area on the anterior labium” according to Haddad and Sazima (2004), but this variation could not be observed in this study, and in all specimens the lower lip was bordered by a double row of marginal papillae. Langone (1989) noticed two rows of marginal papillae in the lower lip of P. gracilis, but it seems to be in fact a single row of alter-nate papillae projected anteriorly and posteriorly, emulating a double row.

A character that seems to vary intraspecific, although it was not observed herein, is the tooth row formula. For P. cuvieri it is described two different tooth row formula; for the specimens analyzed by Bokermann (1962), it is 2/2(1) and for the specimens analyzed by Heyer et al. (1990) and the present study, it is 2(2)/3(1). The formula 2(2)/3(1) is the most common for species of Physalaemus, only P. albifrons, P. rupestris, and P. centralis present varia-tions, which is (2(2)/3(1-2) for the first two species and 2(2)/2 for the later species.

This study shows that tadpoles of Physalaemus herein analyzed are very similar to each other and the oral disc appeared to present important traits that allow the species differentiation, especially regarding to the presence of mental and/or ventrolateral gaps in the lower lip.

Internal oral morphology. The buccal pocket and the glandular zone could not be seen in all specimens stud-ied probably because of dissection mistakes (Spirandeli-Cruz, 1991) and were not considered for comparison in this study. The same happens to the shape of the buccal roof as well as the buccal floor because as stated by Was-sersug and Heyer (1988), the shape is easily affected by the plane of dissection.

On the other hand, the oral cavity varies substantially among species of Physalaemus and there are many important features that could be used on their differentiation, like the lateral roof papillae (absent or present), the shape of the median ridge, and the number of lingual papillae.

The four species in the Physalaemus albifrons group have their tadpole described. Physalaemus albifrons, P. biligonigerus, P. marmoratus, and P. santafecinus have four infralabial papillae in the buccal floor, and the median ridge is in semicircle (Perroti & Céspedez, 1999; Sandoval & Alvarez, 2001; Nomura et al., 2003; Oliveira et al., 2010). The number of lingual papillae varied in the group: P. albifrons have three tapered lingual papillae (Oliveira et al., 2010), P. marmoratus have two (Nomura et al., 2003) and P. santafecinus have one (Perroti & Céspedez, 1999).



The tadpoles of P. albonotatus, P. centralis, P. cuvieri, and P. cicada belong to the P. cuvieri group (Nasci-mento et al. 2005). Miranda and Ferreira (2009) described the presence of four infralabial papillae in the buccal floor of P. albonotatus, P. centralis and P. cuvieri, which are also present in the tadpole of P. cicada. According to original descriptions (Miranda & Ferreira, 2009), P. albonotatus and P. cuvieri have two papillae on the postnarial arena as well as P. cicada, differing from P. centralis that have six.

In the Physalaemus deimaticus group, only P. rupestris has the tadpole described (Nascimento et al., 2001)and this study provided the description of the internal oral anatomy. This tadpole shares some traits with tadpoles of the P. henselii group, which comprises two species with tadpoles described: P. fernandezae (Alcade et al., 2006) and P. riograndensis (Sandoval, 2002). Physalaemus riograndensis have a triangular median ridge, similar to P. rupestris, and differing from P. fernandezae that have a semicircular median ridge. The BFA of P. rupestris and P. fernandezae are limited by six papillae on each side, differing from P. riograndensis that have only four papillae (Sandoval, 2002; Alcade et al., 2006). Different from P. rupestris, P. fernandezae and P. riograndensis have about four papillae on each side of the BRA (five–six in P. rupestris) and one lingual papilla on the tongue anlage (four in P. rupestris).

In the P. olfersii group, the tadpoles of P. aguirrei, P. maximus and P. soaresi have a trapezoid median ridge. The tadpoles of P. aguirrei and P. soaresi have four infralabial papillae, differing from P. maximus that have two. On the other hand, P. aguirrei and P. maximus present four labial papillae while P. soaresi have three. The prenarial arena of P. maximus and P. soaresi are filled with pustulations, differing from P. aguirrei, that have neither pustula-tions nor papillae on the prenarial arena.

Physalaemus lisei (Both et al., 2006), P. jordanensis (Gomes et al., 2010) and P. gracilis belong to the P. gra-cilis group. The tadpoles of P. jordanensis have six infralabial papillae on each side and P. lisei have three multiple-brancnhing papillae on each side, differing from P. gracilis that have two papillae on each side. Physalaemus lisei have five lingual papillae (Both et al., 2006), differing from P. jordanensis and P. gracilis. Like the tadpoles in the P. olfersii group, the median ridge is trapezoid in P. jordanensis and in P. lisei; but it is in semicircle in the tadpoles of P. gracilis. Both P. gracilis and P. jordanensis have lots of pustulations on the the buccal floor and buccal roof arenas, but they differ as the number of papillae on those arenas. Physalaemus lisei have four papillae on each side of both BRA and BFA (Both et al., 2006), differing from P. gracilis that have four–five papillae on each side of theBRA and five-seven on each side of the BFA, and from P. jordanensis that have eight papillae on each side of the BRA and six on each side of the BFA. The tadpole of P. gracilis has few lateral roof papillae close to the ventral velum like P. albonotatus, differing from other tadpoles from its morphological group.

The tadpoles of P. angrensis, P. atlanticus, P. camacan, P. crombiei, P. irroratus, and P. signifer, belong to theP. signifer group and share the semicircular median ridge, differing from P. moreirae, which has a triangular median ridge (Provete et al., 2011), similar to that found in P. rupestris. Physalaemus angrensis and P. signifer have a pair of large infralabial papillae, while the other tadpoles have four large tadpoles except for the P. atlanti-cus, and P. moreirae, which two papillae are large and two are slender. The lingual papillae can vary among spe-cies: P. irroratus have three small and tapered papillae on the tongue anlage; P. angrensis, P. crombiei and P. signifer have two lingual papillae, in P. angrensis and P. crombiei they are tapered, in P. signifer they are flattened; P. atlanticus, P. camacan and P. moreirae have four tapered lingual papillae. In the postnarial arena, in the baccal roof, the tadpoles may have two or four papillae; P. angrensis, P. camacan, and P. irroratus, have two papillae that differ as the shape, which are larger in P. camacan and P. irroratus; and P. atlanticus, P. crombiei, P. moreirae, andP. signifer have two pairs of papillae, one much larger and discernible than the other, which is slender.

The presence of prepocket papillae has been described for the tadpoles of P. centralis, P. cuvieri, P. jordanen-sis, P. lisei, and P. moreirae (Miranda & Ferreira, 2009; Gomes et al., 2010; Both et al., 2006; Provete et al., 2011) but in the present study those papillae were not observed. However, according to Wassersug and Heyer (1988), the actual number of prepocket papilla is difficult to assess because of the continuation of the prepocket papillary field with the BFA and do not show any meaningful patterns of variation.

Miranda and Ferreira (2009) inferred that the presence of three–four labial papillae should be a diagnostic character for the P. cuvieri group, once tadpoles from other species group with the oral anatomy available, have one or two labial papillae. This study does not corroborate such inference due to some tadpoles in the P. signifer group also presenting three or four lingual papillae.



FIGURE 9. Cluster analysis resulted from external morphological features of 20 species of Physalaemus. Abbreviations stand for: P. ag = Physalaemus aguirrei; P. al = P. albifrons; P. an = P. angrensis; P. at = P. atlanticus; P. ca = P. camacan; P. ce = P. centralis; P. ci = P. cicada; P. cr = P. crombiei; P. cu = P. cuvieri; P. gr = P. gracilis; P. he = P. henselii; P. ir = P. irroratus; P. jo = P. jordanensis; P. ma = P. marmoratus; P. mx = P. maximus; P. mo = P. moreirae; P. ru = P. rupestris; P. si = P. signifer; P. so= P. soaresi; P. sp = P. spiniger.



FIGURE 10.Cluster analysis resulted from internal oral features of 18 species of Physalaemus. Abbreviations stand for: P. ag= Physalaemus aguirrei; P. al = P. albifrons; P. an = P. angrensis; P. at = P. atlanticus; P. ca = P. camacan; P. ce = P. centralis; P. ci = P. cicada; P. cr = P. crombiei; P. cu = P. cuvieri; P. gr = P. gracilis; P. ir = P. irroratus; P. jo = P. jordanensis; P. ma = P. marmoratus; P. mx = P. maximus; P. mo = P. moreirae; P. ru = P. rupestris; P. si = P. signifer; P. so = P. soaresi.



FIGURE 11. Cluster analysis resulted from morphological features of 18 species of Physalaemus. Abbreviations stand for: P. ag = Physalaemus aguirrei; P. al = P. albifrons; P. an = P. angrensis; P. at = P. atlanticus; P. ca = P. camacan; P. ce = P. centra-lis; P. ci = P. cicada; P. cr = P. crombiei; P. cu = P. cuvieri; P. gr = P. gracilis; P. ir = P. irroratus; P. jo = P. jordanensis; P. ma = P. marmoratus; P. mx = P. maximus; P. mo = P. moreirae; P. ru = P. rupestris; P. si = P. signifer; P. so = P. soaresi.



Acknowledgments

We thank Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB) for financial support and the curators from Universidade Federal da Bahia (UFBA), Universidade Federal do Rio de Janeiro (UFRJ), Universidade Estadual Paulista (UNESP) and Museu Nacional (MNRJ), for the loan of specimens and loggistic support. We also thank Ana Carolina Guedes for the drawings of the internal oral morphology and Lucas Menezes for the drawings of the external morphology.

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APPENDIX I. Material examined.

From Museu Nacional, Rio de Janeiro:Physalaemus aguirrei (MNRJ 30591), from Parque Nacional Descobrimento, Prado, Bahia—specimens used in original

description; P. angrensis (MNRJ 35071), from Ariró, Agra dos Reis, Rio de Janeiro—specimens obtained from foam nests and raised in

captivity; at least one specimen reared until metamorphorsis for species identification;P. camacan (MNRJ 33342), from Reserva Biológica de UMA, UMA, Bahia—specimens used in original description;P. crombiei (MNRJ 54313), from Reserva Biológica de Duas Bocas, Cariacica, Espírito Santo—specimens reared in captivity

from a couple in amplex; P. irroratus (MNRJ 41460), from Fazenda Duas Barras, Santa Maria do Salto, Minas Gerias—specimens used in original

description; P. maximus (MNRJ 46717), from Santa Rita de Ouro Preto, Ouro Preto, Minas Gerais—specimens used in original description;P. rupestris (MNRJ 25479), from Parque Nacional de Ibitipoca, Lima Duarte, Minas Gerais—specimens used in original

description;

From Universidade Estadual Paulista, campus Rio Claro:P. henselii (CFBH 13175), from São Sepé, Rio Grande do Sul—specimens compared to original description;P. spiniger (CFBH 9066), from Ilha do Cardoso, São Paulo—specimens compared to original description.

From Universidade Estadual Paulista, campus São José do Rio Preto:P. centralis (DZSJRP L411.3), from Santa Fé do Sul, São Paulo—specimens compared to original description; P. cuvieri (DZSJRP L1331.2), from Icém, São Paulo—specimens compared to orginal descriptions; P. gracilis (DZSJRP L956.7), from São José dos Pinhais, Paraná—specimens compared to orginal description; P. jordanensis (DZSJRP L1359.1), from Campos do Jordão, São Paulo—specimens used in original description; P. marmoratus (DZSJRP L117.1), from Nova Itapirema, Nova Aliança, São Paulo—specimens used in original description; P. moreirae (DZSJRP L1200.6), from Parque das Neblinas, Bertioga, São Paulo—specimens used in original description;

From Universidade Federal da Bahia:P. albifrons (UFBA 010375), from Brotas de Macaúbas, Chapada Diamantina, Bahia—specimens used in original description;P. cicada (UFBA 1001-1002), from Serra do Ramalho, Bahia—specimens compared to orginal description;

Universidade Federal do Rio de Janeiro:From P. atlanticus (ZUFRJ 7382), from Picinguaba, Ubatuba, São Paulo—specimens reared in captivity, at least one specimen

reared until metamorphosis; P. signifer (ZUFRJ 5121), from Parque Natural Municipal da Taquara, Duque de Caxias, Rio de Janeiro—specimens obtained

from foam nests and raised in captivity; at least one specimen reared until metamorphorsis for species identification; P. soaresi (ZUFRJ 7436), from Floresta Nacional Mário Xavier, Seropédica, Rio de Janeiro—specimens used in original

description.


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