JOURNAL OF MORPHOLOGY 238:287–305 (1998)

Chondrocranial Morphology of Larvae (: Leptodactylinae): Its Utility in Phylogenetic Reconstruction

PETER M. LARSON AND RAFAEL O. DE SA´ * Department of Biology, University of Richmond, Richmond, Virginia 23173

ABSTRACT Chondrocranial morphology of leptodactylid is scarcely known and has not been completely described for any species of Leptodacty- lus. We describe the diversity of chondrocranial morphology in the genus Leptodactylus based on the analysis of 22 species, representing the four species groups: the fuscus Group, ocellatus Group, melanonotus Group, and pentadactylus Group. Furthermore, 26 characters are identified and used in a phylogenetic analysis. The phylogenetic analysis using Physalaemus, Cros- sodactylus, and Hylodes as outgroups suggests two monophyletic clades within Leptodactylus: the melanonotus-ocellatus clade and the pentadactylus- fuscus clade. However, it does not support the monophyly of the species groups as currently recognized and it suggests a paraphyletic Leptodactylus. Enforc- ing the monophyly of the ingroup, i.e., Leptodactylus, results in the same major two clades of Leptodactylus. Leptodactylus riveroi, a taxon previously unassigned to any species group, appears most closely related to the melano- notus-ocellatus clade based on chondrocranial characteristics. J. Morphol. 238:287–305, 1998. ௠ 1998 Wiley-Liss, Inc. KEY WORDS: Chondrocrania; anatomy; larvae; Leptodactylus; variation; phylogeny

Most studies of cranial morphology in an- resents the other end of the phylogenetic urans have focused on variation and charac- spectrum. Furthermore, it is important if we teristics of the ossified adult skull. Whereas are going to use chondrocranial characters characters derived from the study of adult in phylogenetic studies. This study is the osteology have played an important role in first to examine phylogenetic variation in anuran systematics, few authors have stud- the chondrocrania of 22 closely related spe- ied characters of the larval chondrocranium cies. in an evolutionary context. In part, this can Leptodactylus currently consists of ap- be attributed to a lack of available compara- proximately 61 recognized species, which are tive baseline data needed to understand clustered into four species groups: the fuscus character diversity and chondrocranial evo- Group, the ocellatus Group, the melanono- lution (de Sa´ and Trueb, ’91). Recently, a tus Group, and the pentadactylus Group number of papers have contributed to ex- (Heyer, ’69). The current clustering of spe- pand our understanding of tadpole chondro- cies of Leptodactylus into these four ‘‘species groups’’ points to the presence of morphologi- crania (e.g., Wiens, ’89; Lavilla and Fabrezi, cal variation in the genus; at least enough ’92; Lavilla and De la Riva, ’93; Pu´ gener and variation to recognize ‘‘species groups’’ within Maglia, ’97; Wild, ’97). A few studies have the genus. Consequently, Leptodactylus is used chondrocranial characters in a phyloge- an ideal candidate to examine phylogenetic netic context (Sokol ’77, ’81; de Sa´ and Trueb, ’91; Haas, ’95, ’96, ’97); however, those pa- pers compared chondrocrania among dis- tantly related taxa. No study has attempted Contract grant sponsor: National Science Foundation; Con- a comparative morphological analysis of a tract grant number: BIR-9510228 (to R.D.S.). large number of congeneric species. Under- *Correspondence to: Rafael de Sa´, Department of Biology, The University of Richmond, Richmond, VA 23173. standing the variation of chondrocranial E-mail: [email protected] characters among closely related species rep- Received 1 January 1998; Accepted 14 August 1998

௠ 1998 WILEY-LISS, INC. 288 P.M. LARSON AND R.O. DE SA´ variation of chondrocranial morphology them as L. melanonotus CR (Costa Rica sam- among closely related species and its pos- ple) and L. melanonotus B (Belize sample) sible correlation to currently recognized spe- and describe them separately. Specimens are cies groups. deposited in the United States National Mu- Whereas there is some available informa- seum of Natural History (USNM), Smithso- tion on adult osteology (Lynch, ’71, ’73), there nian Institution. Specimens examined, develop- is no complete description of the chondrocra- mental stages available, collection data, and nium of any species of Leptodactylus. Brief catalogue numbers are listed in Appendix A. references to individual chondrocranial All specimens were wild-caught, preserved structures are available for Leptodactylus in 4% formalin, and staged using the table of chaquensis (Sokol, ’81), L. pentadactylus, and Gosner (’60). Tadpoles were cleared and L. ocellatus (Haas, ’95). Analysis of chondro- double-stained for bone and cartilage by us- cranial variation within Leptodactylus may ing a modified version of the technique of provide additional characters to support or Dingerkus and Uhler (’77). Chondrocranial refute the monophyly of the species groups; terminology follows de Sa´ (’88), de Sa´ and furthermore, it may help to resolve relation- Trueb (’91), and Haas (’95). Illustrations of ships among the species groups. Addition- chondrocrania and branchial baskets were ally, such analysis may help to determine made using a Wild MC3 stereomicroscope whether chondrocranial anatomy provides with a camera-lucida attachment. Speci- useful diagnostic characters to differentiate mens were illustrated at comparable devel- closely related species. opmental stages (stages 30–37) whenever Herein, we analyze chondrocranial mor- possible; however, all available stages were phology for approximately one third of the examined for chondrocranial descriptions. currently recognized species of Leptodacty- Phylogenetic analysis was done using lus. The chondrocrania of 22 species within PAUP program version 3.1 (Swofford, ’93). the genus Leptodactylus are analyzed with Multistate characters were run unordered the following goals: 1) to describe and ana- to avoid a priori assumptions of the evolu- lyze chondrocranial anatomy in the genus tionary path of the transformation series. Leptodactylus; 2) to identify characters that Out-group comparison was used to polarize may help to further define the species groups all characters. Relationships within the Lep- and their evolutionary relationships; and 3) todactylidae, and within the leptodactylid to contribute baseline chondrocranial data subfamilies, are unresolved (Maxon and that can be used for comparative purposes Heyer, ’88; Ford and Cannatella, ’93; Ruvin- within the Leptodactylidae and Neobatra- sky and Maxson, ’96). Lynch (’71) considered chia as a whole. both the Hylodinae and the Leptodactylinae to be derived from the Telmatobinae. Herein, MATERIALS AND METHODS we take a conservative approach and use Chondrocranial descriptions are based on representatives from these two subfamilies a total of 151 tadpoles and juveniles repre- as outgroups. Outgroups included are Physa- senting 22 species and four species groups laemus pustulosus (Leptodactylinae), Hy- within the genus Leptodactylus. Species in- lodes nasus, and Crossodactylus gaudichau- cluded in this study are Leptodactylus albila- dii (Hylodinae). Hylodes and Crossodactylus bris, L. bufonius, L. fuscus, L. gracilis, L. must be considered more distantly related labialis, L. labrosus, L. latinasus, L. longiros- outgroups to Leptodactylus, whereas Physa- tris, and L. mystacinus of the fuscus Group; laemus represents a more closely related L. chaquensis, L. insularum, and L. ocella- outgroup. tus of the ocellatus Group; L. melanonotus, L. petersii, L. podicipinus, and L. validus of RESULTS the melanonotus Group; and L. knudseni, L. The chondrocranial description presented labyrinthicus, L. pentadactylus, and L. rhod- here is representative for all species; indi- omystax of the pentadactylus Group. In addi- vidual divergences from the general descrip- tion, L. silvanimbus, which has recently been tion, as well as ontogenetic changes in chon- associated with the melanonotus Group drocranial morphology, are noted when (Heyer et al., ’96), and one unassigned spe- applicable. If information for a given species cies, L. riveroi, were examined. Specimens of is not presented, it is because the character L. melanonotus that come from localities in state for that species could not be deter- Belize and Costa Rica show clear differences mined based on the limited specimens avail- in chondrocranial morphology. We refer to able. LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 289 The typical chondrocranium of Leptodacty- chondrocranial width is approximately equal lus is slightly longer than it is wide, until to the length. Chondrocranial height is about about stage 41 when major metamorphic 20–25% of its total length and approxi- rearrangements of the chondrocranium be- mately 30% of its total width. gin. The chondrocranial width is approxi- mately 85–90% of the total length (Fig. 1); Neurocranium exceptions are L. bufonius, in which the Ethmoidal region chondrocranium is slightly narrower (about The paired suprarostral cartilages sup- 82% of chondrocranial length) and L. knud- port the upper horny break; each supraros- seni, L. insularum, and L. validus, in which tral consists of a central corpus and a lateral

Fig. 1. Chondrocranium of Leptodactylus melanono- foramen opticum; fov, fenestra ovalis; fp, foramen prooti- tus B larva (melanonotus Group), stage 33 (USNM cum; ft, foramen trochleare; hp, hypobranchial plate; i, #519940). (A) dorsal, (B) ventral, and (C) lateral views; infrarostral cartilage; lcp, larval crista parotica; mc, (D) ventral view of hyobranchial apparatus. cb I-IV, Meckel’s cartilage; oc, otic capsule; pah, processus ante- ceratobranchials I-IV; cbp, closed processus branchialis; rior hyalis; pal, processus anterolateralis hyalis; pph, ch, ceratohyal; cop, copula posterior; cot, commisura processus posterior hyalis; pas, processus ascendens; terminalis; cq, commisura quadratoorbitalis; cqa, com- paq, pars articularis quadrati; pm, processus muscula- missura quadratocranialis anterior; ct, cornua trabecu- ris quadrati; pq, palatoquadrate; pqe, processus quadra- lae; fah, facies articularis hyalis; fb, fenestra basicrania- toethmoidalis; pr, pars reuniens; pu, processus urobran- lis; fcp, foramen caroticum primarium; fcrp, foramen chialis; s, spicule; sa, suprarostral ala; sc, suprarostral craniopalatinum; fj, foramen jugulare; foc, foramen ocu- corpus; tn, tectum nasi; ts, tectum synoticum; ttm, tae- lomotorium; fpi, foramen perilymphaticum inferior; fo, nia tecti marginalis. Bar ϭ 1.0 mm. 290 P.M. LARSON AND R.O. DE SA´ ala (Fig. 2). The central corpora lie between ovoid hole. The suprarostrals of all species of the cornua trabeculae at the anterior end of the ocellatus and melanonotus Groups exam- the chondrocranium. The two corpora are ined, L. riveroi, and L. silvanimbus have fused ventromedially, forming a continuous, slender corpora (Fig. 2c,d). In these taxa, the horizontal bar of cartilage between the cor- corpora are fused ventromedially by a rela- pora. In all species, the corpora are continu- tively long, thin connection and the space ous dorsolaterally with the alae through a between the corpora acquires a wider U-to relatively narrow strip of cartilage. The lat- V-shaped appearance. No ventral connec- eral alae are flat, wide sheets of cartilage tion or lateral projections between the cor- that are rounded ventrally; each ala curves pora and alae are present in any members of posteriorly from its point of fusion with the the ocellatus and melanonotus Groups, L. corpora. A small, rounded enlargement near riveroi, or L. silvanimbus. the dorsomedial margin of the ala serves as In most Leptodactylus species, the cornua the point of articulation of the cornua tra- trabeculae represent about 20% of the total beculae to the suprarostral. The posterodor- chondrocranial length. However, in mem- sal margin of each ala possesses a well devel- bers of the pentadactylus Group (except L. oped processus posterior dorsalis. An rhodomystax) they are shorter, representing adrostral tissue mass is present near the tip between 10–15% of the chondrocranial of the processus posterior dorsalis. length (Figs. 3c, 4h,i). The cornua trabecu- The suprarostrals of members of the fus- lae diverge anteriorly from the planum tra- cus Group and Leptodactylus rhodomystax beculare anticum and are expanded distally have wide and, in frontal view, U-shaped to varying degrees. Their anterior margins corpora (Fig. 2a). In L. rhodomystax and L. are uneven in most species and flex ven- mystacinus the ventral margins of the cor- trally to articulate with the suprarostral pora have lateral projections facing similar alae. The widths of the cornua trabeculae of projections from the lateral alae; however, L. rhodomystax (Fig. 4j) are similar to other these are not fused with each other (Fig. 2a). species in the pentadactylus Group and they In L. albilabris, L. bufonius, and L. longiros- tris these projections are fused, forming a also bear a relatively straight anterior mar- narrow, poorly chondrified ventral connec- gin. However, they resemble the cornua tra- tion between the corpora and the alae. The beculae in some members of the fuscus ventromedial connection between the cor- Group, particularly L. albilabris, L. latina- pora of L. pentadactylus, L. knudseni, and L. sus, L. longirostris, and L. fuscus, in terms of labyrinthicus is very thin (Fig. 2b). Further- the depth and narrowness of their diver- more, in these species the medial separation gence and in having a slightly outwardly between the corpora is narrow, forming a rounded lateral margin. The cornua trabecu- deep U, and there is an extensive ventral lae are of variable width in the fuscus Group, connection between the corpora and the alae. with those of L. bufonius (Fig. 4b) being As a result, in these taxa each suprarostral slightly narrower among the species exam- is a single element bearing a small round or ined. The cornua trabeculae of all species of the ocellatus and melanonotus Groups exam- ined, L. riveroi, and L. silvanimbus are nar- rower proximally and only slightly wider distally (Figs. 1A, 3b, 4k–r). In addition, in the ocellatus Group, L. melanonotus CR, and L. silvanimbus the V-shaped area between the cornua trabeculae is slightly wider than in other species. Leptodactylus labyrinthi- cus, L. knudseni, and L. pentadactylus have shorter and wider cornua trabeculae than any other species examined. Their anterior margins are almost straight and the cornua trabeculae are of almost uniform width throughout their length, except for L. knud- Fig. 2. Suprarostral cartilages. a: Leptodactylus mys- seni (Fig. 4h), in which they are slightly tacinus, stage 36 (USNM #519948). b: L. pentadactylus, wider distally. The divergence of the cornua stage 29 (USNM #519997). c: L. ocellatus, stage 36 (USNM #519962). d: L. melanonotus B, stage 33 (USNM trabeculae in these taxa forms a sharp and #519940). Bar ϭ 1.0 mm. distinct V shape. LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 291

Fig. 3. Dorsal, ventral and lateral views of chondrocrania and ventral view of hyobranchial apparatus. a: Leptodactylus latinasus larva ( fuscus Group), stage 35 (USNM #520298). b: L. ocellatus larva (ocellatus Group), stage 37 (USNM #519962). c: L. labyrinthicus larva ( penta- dactylus Group), stage 36 (USNM #520296). Bar ϭ 1.0 mm. 292 ..LRO N ..D SA DE R.O. AND LARSON P.M. ´

Fig. 4. Dorsal view of chondrocranium. a: , stage 32 33 (USNM #520304). m: L. validus, stage 37 (USNM #520313). n: L. melanonotus (USNM #520280). b: L. bufonius, stage 35 (USNM #520286). c: L. gracilis, stage 35 CR, stage 35 (USNM #520283). o: L. chaquensis, stage 37 (USNM #519881). p: L. (USNM #519898). d: L. labialis, stage 37 (USNM #519928). e: L. labrosus, stage 31 insularum, stage 36 (USNM #520291). q: L. riveroi, stage 27 (USNM #520308). r: (USNM #520295). f: L. longirostris, stage 33 (USNM #520299). g: L. mystacinus, L. silvanimbus, stage 31 (USNM #520311). a–g: are species in the fuscus Group, stage 37 (USNM #519952). h: L. knudseni, stage 36 (USNM #520293). i: L. h–j are species in the pentadactylus Group, k–m are species in the melanonotus pentadactylus, stage 29 late/30 early (USNM #519995). j: L. rhodomystax, stage 27 Group, and n–p are species in the ocellatus Group. Bar ϭ 1.0 mm. (USNM #520306). k: L. petersii, stage 36 (USNM #520301). l: L. podicipinus, stage LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 293 The ligamentum quadratoethmoidale ex- early stages. This connection forms the dor- tends between the commissura quadratocra- sal margin of the foramen prooticum, a large, nialis anterior and the lateral margin of the elongate foramen located between the ante- cornua trabeculae near their point of diver- rior edge of the otic capsule and the poste- gence. This ligament attaches to a variably rior margin of the orbital cartilage (Fig. 1C). developed ventrolateral process on the cor- The prootic foramen is somewhat variable in nua trabeculae, the processus lateralis tra- size. Two other large foramina are visible in beculae. The processus lateralis trabeculae the posteroventral portion of the orbital car- is poorly developed in Leptodactylus bufo- tilage. The slightly larger anterior foramen nius, L. fuscus, L. gracilis, L. knudseni, L. is the foramen opticum, whereas the smaller melanonotus CR, L. chaquensis, L. riveroi, posterior foramen is the foramen oculomoto- and L. silvanimbus. However, in L. albila- rium. Additionally, a small trochlear fora- bris, L. labialis, L. mystacinus, L. labyrinthi- men is located anterodorsal to the optic fora- cus, L. rhodomystax, L. ocellatus, L. melano- men; this foramen is variably visible. notus B, and L. validus it is a better The frontoparietal fontanelle is large and developed and blunt process. The processus ovoid in shape. It is bordered laterally by the lateralis trabeculae is not distinct in L. labro- taenia tecti marginales, posteriorly by the sus, L. latinasus, L. longirostris, L. pentadac- tectum synoticum, and anteriorly by the pla- tylus, L. insularum, L. petersii, and L. podi- num ethmoidale. In some species the fronto- cipinus. parietal fontanelle further divides during The posterior confluence of the cornua tra- development into an anterior frontal and beculae is continuous with the planum tra- two posterior parietal fenestrae (Fig. 4a,d, beculare anticum, which, in turn, continues j,m,o). This subdivision occurs by the medial with the planum ethmoidale that forms the growth of the taenia tecti transversales from anterior wall of the braincase. In the region the taenia tecti marginales and the elonga- just posterior to this confluence, the planum tion of the taenia tecti medialis anteriorly trabeculare anticum is slightly longer and from the medial portion of the tectum synoti- narrower in Leptodactylus riveroi, L. silvan- cum. The subdivision of the frontoparietal imbus, and species of the ocellatus and mela- fenestra begins at stage 34 in Leptodactylus nonotus Groups. In most specimens illus- melanonotus B; however, it already has be- trated (Figs. 1A, 3, 4), the tectum nasi has gun by stage 27 in L. rhodomystax, stage 31 started to form on each side as a rounded in L. silvanimbus, and stage 32 in L. albila- projection located dorsal to the commissura bris. Among the available specimens of L. quadratocranialis anterior (as early as stage chaquensis, L. labialis, and L. riveroi, fron- 27 in L. riveroi and L. rhodomystax). tal and parietal fenestrae are present by stage 37 and by stage 38 in L. podicipinus. Orbitotemporal region Leptodactylus mystacinus is unique among The central area of the cranial floor is examined species in having the posterior closed by a thin cartilage, the planum inter- half of the frontoparietal fenestra covered by trabeculare, which eventually occludes the a continuous sheet of cartilage as early as fenestra basicranialis and contributes to the stage 35 (Fig. 4g). Formation of parietal cranial floor during development. The pla- fenestrae was not observed in this species num intertrabeculare forms from extensions among the specimens examined. of the trabeculae cranii laterally and the planum trabeculare anticum anteriorly. The Otooccipital region cranial floor has two sets of foramina. The The otic capsules are ovoid and represent anterior, smaller pair are the foramina cra- about 30% of the total chondrocranial length. niopalatina, while the larger, posterior pair In dorsal view, the otic capsule bears a later- correspond to the foramina carotica prima- ally projecting and wide larval crista par- ria (Fig. 1A,B). In early stages, the foramina otica in most species. Anteriorly, the crista craniopalatina are particularly difficult to parotica bears a well-developed processus identify because of the large fenestra basicra- anterolateralis; posteriorly, it is enlarged nialis. forming a distinct, triangular processus pos- The lateral walls of the braincase are terolateralis. The processus anterolateralis formed by the orbital cartilages. The orbital is stout and triangular in all members of the cartilages attach posterodorsally to the otic pentadactylus Group, Leptodactylus silvan- capsules, although this connection may not imbus, L. albilabris, and L. labrosus; long be completely developed or clearly visible in and finger-like in all other members of the 294 P.M. LARSON AND R.O. DE SA´ fuscus Group; stout and rectangular in L. nonotus CR (Fig. 4b,n), where the palato- melanonotus CR, L. ocellatus, and L. chaque- quadrate appears relatively flat in dorsal nsis; a small rounded to triangular bump in view. L. insularum, L. melanonotus B, and L. podi- Posteriorly, the palatoquadrate attaches cipinus; and poorly developed and difficult to to the orbital cartilage via the processus identify in L. petersii, L. validus, and L. ascendens, which is a narrow, rod-like carti- riveroi. The processus anterolateralis ap- lage connecting the posteromedial margin of proaches a knob-like projection of the pos- the palatoquadrate to the region of the pila terolateral curvature of the palatoquadrate antotica of the orbital cartilage (Figs. 1C, 3, in L. chaquensis, L. ocellatus, L. melanono- 6). The processus ascendens attaches just tus CR, and L. melanonotus B. However, posterior to the oculomotor foramen (see Fig. while they are nearly in contact in L. ocella- 3a,c), a condition defined as ‘‘intermediate’’ tus and L. melanonotus CR, fusion of these by Sokol (’81), in the fuscus Group, pentadac- structures does not occur. The processus pos- tylus Group, L. ocellatus, and L. chaquensis. terolateralis is reduced in L. melanonotus B, In the melanonotus Group, L. insularum, L. L. podicipinus, and L. riveroi and seems to riveroi, and L. silvanimbus the processus be absent in L. petersii and L. validus. A ascendens attaches below the posteroven- large fenestra ovalis is visible ventrolater- tral margin (see Fig. 1C) of the oculomotor ally on the otic capsule, directly below the foramen in a typical ‘‘low’’ condition (Sokol, crista parotica. ’81). The arcus occipitalis extends ventrally The processus ascendens is almost perpen- from the posteromedial margin of the otic dicular to the main axis of the chondrocra- capsule, fusing with the planum basale and nium in the fuscus Group, its posterior mar- giving rise to the occipital condyles. The gin forming an angle of about 85–90° with planum basale and the arcus occipitalis form the main axis of the body. This angle is about the ventral and medial margins of the fora- 75–80° in the pentadactylus Group, Lepto- men jugulare. The foramen perilymphati- dactylus chaquensis, and L. ocellatus, around cum inferior is found lateral to the foramen jugulare in the ventromedial margin of the 72° in L. melanonotus CR, and about 70° in otic capsule (visible ventrally, Fig. 5). L. melanonotus B and L. silvanimbus. In other species the angle is more acute, being Visceral components about 65° in remaining species of the melano- Palatoquadrate notus Group and L. insularum, and about The palatoquadrate of Leptodactylus is 60° in L. riveroi. narrow anteriorly and it expands continu- Anteriorly, the palatoquadrate connects to ously posteriorly, becoming outwardly the braincase via a wide commissura quadra- rounded in all species except L. bufonius tocranialis anterior. The commissura quadra- (Fig. 4b), in which it is not expanded posteri- tocranialis anterior extends between the orly. In most species of the fuscus Group, the palatoquadrate, at a level just posterior to posterior curvature of the palatoquadrate is the pars articularis quadrati, and the floor of at the level of the point of attachment of the the neurocranium. The anterior margin of processus ascendens to the orbital cartilage. the commissura quadratocranialis anterior In L. albilabris, L. labrosus, L. melanonotus bears an overall triangular processus CR, all species of the pentadactylus Group, quadratoethmoidalis, which serves as the and most species of the ocellatus Group, the point of attachment for the ligamentum posterior curvature extends slightly past this quadratoethmoidale. In addition, a triangu- level. In L. melanonotus B and L. silvanim- lar-shaped processus pseudopterygoideus is bus the posterior curvature extends beyond present on the posterior margin of the com- the anterior margin of the otic capsule; how- missura quadratocranialis anterior in Lepto- ever, in all other members of the melanono- dactylus fuscus and L. mystacinus (Fig. 4g). tus Group, L. insularum, and L. riveroi, this The latter process was not clearly identifi- curvature extends even farther posteriorly able in other Leptodactylus. A rod-like com- to about one-third of the way back on the otic missura quadratoorbitalis is present; it is capsule (Fig. 4k–m,p,q). The lateral and pos- formed by the fusion of the processus antor- terior margins of the palatoquadrate also bitalis with the medial tip of the processus curve slightly dorsally, giving the palato- muscularis quadrati. quadrate a basket-like shape in dorsal view The palatoquadrate bears two distinct an- in all species except L. bufonius and L. mela- terior processes, the processus muscularis LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 295 quadrati and the pars articularis quadrati. portion of Meckel’s cartilage expands in the The processus muscularis quadrati is broad, vertical plane to form a dorsal processus flat, and extends dorsally from the lateral dorsomedialis and a ventral processus ven- margin of the palatoquadrate at a level just tromedialis. The posterodorsal portion of the posterior to the pars articularis quadrati. infrarostral cartilage articulates with Meck- The processus muscularis quadrati is wide el’s cartilage in the region between these two in the melanonotus Group, ocellatus Group, processes. Leptodactylus silvanimbus, and L. riveroi, to The infrarostral cartilages provide sup- the degree that, in lateral view, it is almost port for the lower horny beak. Overall, the impossible to distinguish the pars articu- infrarostrals are wedge-shaped and their laris quadrati (Fig. 1C). In other Leptodacty- dorsolateral half is rounded, slightly thicker, lus examined, the processus muscularis qua- and wider; they connect ventromedially, drati is narrower and the pars articularis forming a V-shaped structure in anterior quadrati is clearly distinguishable in lateral view. The anterior margins of the infraros- view (Fig. 3a,c). The dorsal edge of the proces- trals are outwardly curved laterally and sus muscularis quadrati inclines medially notched medially in dorsal view. Further- and attaches to the commissura quadratocra- more, they are notched on their posterior nialis anterior via the commissura quadrato- margin. The posterior notch is deep in Lepto- orbitalis. Immediately below the processus dactylus validus (Fig. 5m). In the pentadac- muscularis and ventrally on the palatoquad- tylus Group, except L. rhodomystax (which rate, there is a notch, the facies articularis resembles all other species), the infraros- hyalis, that serves as the point of articula- trals are unique in being overall rectangu- tion of the ceratohyal to the palatoquadrate. lar, stout, wide throughout their length, and The facies articularis hyalis is partly formed outwardly rounded over their entire ante- by the hyoquadrate process, a rounded, ven- rior margin (Figs. 3c, 5h,i). The infrarostrals tral enlargement of the palatoquadrate. In of L. longirostris are more slender than in lateral view, the hyoquadrate process is small other species (Fig. 5f). and triangular in most species; however, in Hyobranchial apparatus members of the melanonotus Group (except Leptodactylus melanonotus CR), L. riveroi, The ceratohyalia (Figs. 1D, 3, 7) are wide, and L. insularum it is enlarged and rounded flat cartilages medially; laterally, they are in lateral view. The pars articularis quadrati twisted dorsally to articulate with the pala- corresponds to the short, wide segment of toquadrate at the facies articularis hyalis. the palatoquadrate that extends anterior to The anterior margin of each ceratohyal bears the level of the processus muscularis. The two anterior processes, the processus ante- pars articularis quadrati articulates almost rior hyalis and the processus anterolateralis over its entire anterior width with Meckel’s hyalis. Both processes are overall triangular cartilage. in shape, with the processus anterior hyalis being larger and the processus anterolatera- Meckel’s and infrarostral cartilages lis hyalis inclining slightly medially. The Meckel’s cartilages, together with the in- processus anterior hyalis varies somewhat frarostral cartilages, form the mandible in in shape, being pointed in the fuscus Group Leptodactylus. Meckel’s cartilage is stout, and Leptodactylus rhodomystax (Figs. 3, 7), oriented almost perpendicular to the main wide and blunt in L. knudseni, L. labyrinthi- body axis, and is sigmoid-shaped, with its cus, and L. pentadactylus (Figs. 3c, 7h, i), anteromedial margin being concave and its and variably pointed to slightly rounded in anterolateral margin convex (Fig. 1B). Later- the melanonotus Group, ocellatus Group, L. ally, Meckel’s cartilage articulates broadly riveroi, and L. silvanimbus (Figs. 1D, 3, 7). with the pars articularis quadrati via the The processus anterolateralis hyalis is vis- processus retroarticularis. This process ible only as a small bump in L. ocellatus and curves ventrally beneath the pars articu- L. melanonotus CR (Figs. 3b, 7n). The cerato- laris. Medially, Meckel’s cartilage articu- hyal also possesses a large and well-devel- lates with the pars articularis quadrati oped processus posterior hyalis. Medially, through an enlarged, knob-like area of the the ceratohyalia are connected by a poorly ventromedial margin of Meckel’s cartilage. chondrified pars reuniens. The pars reu- While it is clearly present in all species, this niens is attached posteriorly to a well- knob tends to be more visible in some species developed copula posterior, which bears a than in others. In lateral view, the medial large processus urobranchialis. The copula 296 ..LRO N ..D SA DE R.O. AND LARSON P.M. ´

Fig. 5. Ventral view of Leptodactylus chondrocrania. Species as in Figure 3. Bar ϭ 1.0 mm. LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 297

Fig. 6. Lateral view of Leptodactylus chondrocrania. Species as in Figure 3. Bar ϭ 1.0 mm. posterior connects posteriorly to the hypo- L. insularum each plate bears a small, finger- branchial plates. like projection on its anterior margin lateral The hypobranchial plates are wide, flat to its point of attachment to the copula poste- sheets of cartilage that serve as the point of rior (Fig. 7p). attachment for the ceratobranchials. In most The branchial baskets consist of four cera- species, the plates are separated medially tobranchials that are distally continuous via along most of their lengths and are only commissurae terminales. Proximally, cerato- distinctly fused posteriorly. However, in Lep- branchial I attaches to the hypobranchial todactylus bufonius, L. fuscus, and L. graci- plate by a wide strip of cartilage; on its lis the hypobranchial plates are medially anterior margin it bears a distinct, widely fused throughout their length. Similarly, in triangular to hook-like processus anterior L. labrosus, L. latinasus, L. petersii, and L. branchialis that slants medially. Ceratobran- validus, the plates are medially connected chials II and IV are also fused to the hypo- throughout most of their length, except for branchial plate, the former via a narrow bar the area immediately posterior to the copula of cartilage and the latter via a wider carti- posterior. Posteriorly, the hypobranchial laginous connection. Ceratobranchial III ap- plates diverge, leaving an inverted U-shaped pears to be only very narrowly attached to separation between their posterior edges. In the hypobranchial plate. Ceratobranchials I, 298 P.M. LARSON AND R.O. DE SA´

Fig. 7. Ventral view of hyobranchial apparatus of Leptodactylus. Species as in Figure 3, except that (i) L. pentadactylus corresponds to USNM #519992, stage 31. Bar ϭ 1.0 mm. LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 299 II, and III bear a dorsally projecting spicu- TABLE 1. List of 26 chondrocranial characters lum near their point of attachment to the used in the present study hypobranchial plate. In all members of the a. ventromedial fusion of suprarostral corpus: (0) not ocellatus and melanonotus Groups exam- fused, (1) fused. ined, Leptodactylus riveroi, and L. silvanim- b. dorsal fusion of suprarostral corpus and ala: (0) not fused, (1) fused. bus, ceratobranchials II and III are con- c. ventrolateral projections of suprarostral corpus and nected proximally by a closed processus ala: (0) absent, (1) present, (2) fused. branchialis (see Fig. 1D). In all examined d. contact between the suprarostral corpora: (0) con- members of the fuscus Group and pentadac- tact nearly along their length, (1) narrowly sepa- rated, (2) widely separated. tylus Group, the processus branchialis is e. chondrocranial width relative to chondrocranial visible as two opposing, ventrally projecting length: (0) 80–85%, (1) 85–90%, (2) equal. cartilages extending from ceratobranchials f. length of cornua trabeculae relative to chondrocra- II and III (see Fig. 3a,c). Although they ap- nial length: (0) 25%, (1) 20%, (2) 10–15%. g. planum trabeculare anticum: (0) wide, (1) narrow. pear to be connected by more lightly stained h. frontoparietal fenestra: (0) open, (1) posterior half ligaments in some cases, they never fuse via closed. a cartilaginous connection throughout devel- i. otic capsules: (0) Ͻ than 30% of chondrocranial opment in these species. length, (1) 30% of chondrocranial length. j. processus anterolateralis of crista parotica: (0) The present study identified 26 variable small and triangular, (1) large and triangular, (2) chondrocranial characters in Leptodactylus. long finger-like projection, (3) large and rectan- Description of characters is provided in Table gular, (4) absent. 1; character states for each taxa included in k. projection of posterolateral curvature of palato- quadrate: (0) absent, (1) present. this analysis are given in Table 2. l. processus posterolateralis of crista parotica: (0) dis- tinct, (1) reduced. DISCUSSION m. posterolateral extension of the palatoquadrate: (0) Chondrocranial morphology does not reach the level of attachment of the pro- cessus ascendens to the braincase, (1) reaches the Analysis of chondrocranial morphology in level of attachment of the processus ascendens to the genus Leptodactylus provided a number the braincase, (2) extends beyond the level of of interesting findings. Chondrocrania of the attachment of the processus ascendens to the brain- 22 species included in this study share a case, (3) past anterior margin of otic capsules, (4) 1 reaches ⁄3 the length of the otic capsule. number of features. Overall, with the excep- n. attachment of the processus ascendens: (0) low, (1) tion of three species, the chondrocranium in intermediate. Leptodactylus tends to be slightly longer than o. angle of processus ascendens: (0) Ͻ70°, (1) 70–80°, wide. All members of the genus Leptodacty- (2) 80–90°. p. processus pseudopterygoideus: (0) absent, (1) pre- lus studied are characterized by the follow- sent. ing set of characters: 1) quadripartite supra- q. pars articularis quadrati: (0) distinct from pro- rostral cartilage with corpora fused to each cessus muscularis, (1) indistinct from the processus other and the alae; 2) processus posterior muscularis. r. processus muscularis: (0) large, (1) reduced. dorsalis present on suprarostral alae; 3) lar- s. commissura quadratoorbitalis: (0) absent, (1) pre- val crista parotica present; 4) larval otic sent. process absent; 5) commissura quadratoor- t. Meckel’s cartilage: (0) short and stout, (1) long and bitalis present; 6) processus quadratoethmoi- narrow. u. infrarostral cartilages: (0) large and notched anteri- dalis present; 7) processus dorsomedialis, orly, (1) small and notched anteriorly, (2) small, processus ventromedialis, and processus ret- thick, and rounded anteriorly, (3) small, slender, roarticularis of Meckel’s cartilage present; and notched anteriorly. 8) proximal ends of all ceratobranchials fused v. processus anterolateralis hyalis: (0) absent, (1) pre- sent. to hypobranchial plate; 9) spicules present w. anterior process of hypobranchial plate: (0) absent, on ceratobranchials I, II, and III; 10) proces- (1) present. sus anterior branchialis present; 11) palato- x. processus branchialis: (0) open, (1) closed. quadrate wide and outwardly rounded; and y. dorsal curving of the posterior margin of the palato- quadrate: (0) present, (1) absent. 12) otic capsules ovoid and representing ap- z. hyoquadrate process: (0) small and triangular, (1) proximately 30% of the length of the chondro- large and rounded. cranium. Also, a number of variable characters were identified. They include morphology of the the processus ascendens; and the presence suprarostral and infrarostral cartilages; size, or absence of a fused processus branchialis. shape, and degree of divergence of the cor- These characters are discussed below as ex- nua trabeculae; shape of the palatoquad- amples of the extent of variation in chondro- rate; orientation and point of attachment of cranial morphology in Leptodactylus. 300 P.M. LARSON AND R.O. DE SA´ TABLE 2. DATA matrix showing character states components of the suprarostrals (Fig. 2b). As of 26 variable chondrocranial characters found a result, these species have a small, overall in Leptodactylus and outgroup taxa circular hole perforating their supraros- Character states trals. Species abcdefghijklmnopqrstuvwxyz The overall shape of the infrarostrals is H. nasus 00000000000000000000000000 less variable than that of the suprarostrals. C. gaudichaudii 00000000040000000100010000 Overall, the infrarostrals are wide and rect- P. pustulosus 11021100110110200011110010 angular in Leptodactylus. However, the infra- L. melanonotus CR 11021110131020101011110110 L. chaquensis 11021110131021101011110100 rostrals of L. longirostris (Fig. 5f) are slightly L. insularum 11022110100040001011111101 more slender and in L. validus (Fig. 5m) L. ocellatus 11021110131021101011110100 their posterior indentation is more pro- L. melanonotus B 11021110101130101011110101 nounced than in most Leptodactylus species. L. petersii 11021110140140001011110101 L. podicipinus 11021110100140001011110101 Furthermore, in L. knudseni, L. labyrinthi- L. validus 11022110140140001011110101 cus, and L. pentadactylus the infrarostrals L. knudseni 11212200110021100011210000 differ in being very stout, wide, and out- L. labyrinthicus 11211200110021100011210000 wardly rounded on their anterior margin L. pentadactylus 11211200110021100011210000 L. rhodomystax 11111100110021100011110000 (Figs. 3c, 5h,i). The more robust infraros- L. albilabris 11211100110021200011110000 trals, along with the extensive distal fusion L. bufonius 11210100120001200011110010 of the suprarostral elements, may relate to L. fuscus 11011100120011210011110000 L. gracilis 11011100120011200011110000 the carnivorous diet previously reported for L. labialis 11011100120011200011110000 these tadpoles (Heyer, ’79). L. labrosus 11011100110021200011110000 Another character that has also been pre- L. latinasus 11011100120011200011110000 viously considered a modification correlated L. longirostris 11211100120011200011310000 L. mystacinus 11111101120011210011110000 with a carnivorous diet is the presence of L. riveroi 11021110140140001011110101 short cornua trabeculae (de Sa´, ’94). Lepto- L. silvanimbus 11021110130030101011110100 dactylus knudseni, L. labyrinthicus, and L. pentadactylus have cornua trabeculae that are uniquely short and uniformly wide Specimens of Leptodactylus melanonotus throughout their length (Figs. 3c, 4h,i). The from Belize (B) and Costa Rica (CR) show cornua trabeculae are narrow in the melano- clear differences in chondrocranial struc- notus and ocellatus Groups, as well as in L. tures. These include the processes of the silvanimbus and L. riveroi (Figs. 1A, 3b, crista parotica of the otic capsule, the poste- 4k–r). Additionally, the divergence between rior curvature of the palatoquadrate, and the cornua trabeculae is wider and more the dorsolateral margin of the palatoquad- triangular in shape in members of the ocella- rate. These differences could represent intra- tus Group, L. melanonotus CR, and L. silvan- specific variation or could suggest that one imbus. In L. rhodomystax (Fig. 4j) and spe- of these samples represents an undescribed cies in the fuscus Group (Figs. 3a, 4a–g) the taxon. No information has been reported on cornua trabeculae are wider and their diver- the amount of geographic variation in chon- gence is less marked. drocranial characteristics. However, samples The palatoquadrate of Leptodactylus pos- of L. validus and L. mystacinus used herein sesses a wide and rounded posterior curva- are also from two disjunct localities each ture; however, the posterior extent of this and show no significant variation. curvature varies within the genus. Further- Species of Leptodactylus possess a quadri- more, this character is highly variable among partite cartilago suprarostralis with a proces- species within the traditionally recognized sus posterior dorsalis extending from the ‘‘species groups.’’ posterior margin of the alae (Fig. 2a–d). Sokol (’81) defined three possible condi- However, the characteristics of the central tions of attachment: low, intermediate, and corpus and its attachment to the alae are high—between the processus ascendens and variable among Leptodactylus. Haas (’95) the braincase, although Haas (’95) indicated discussed different states of distal connec- that varying degrees of these conditions are tion between the pars corporis and pars likely to be found. Overall, two conditions alaris in dendrobatid larvae. Among species were present in Leptodactylus. In the penta- examined, L. knudseni, L. labyrinthicus, and dactylus and fuscus Groups, L. chaquensis, L. pentadactylus are unique in having an and L. ocellatus the processus ascendens extensive distal connection between these attaches behind and at the same level as the LEPTODACTYLUS CHONDROCRANIAL MORPHOLOGY 301 foramen oculomotorium, corresponding to beculare anticum posterior to the confluence Sokol’s (’81) ‘‘intermediate’’ condition (e.g., of the cornua trabeculae, and a pars articu- Fig. 3a,c). In the melanonotus Group, L. laris quadrati that is indistinct from the insularum, L. silvanimbus, and L. riveroi, processus muscularis in lateral view. The the processus ascendens attaches below (e.g., fuscus-pentadactylus clade is supported by Fig. 1C) the posteroventral margin of the having an open processus branchialis, pars foramen oculomotorium (Sokol’s ‘‘low’’ condi- articularis quadrati distinct from the proces- tion). The angle between the posterior mar- sus muscularis in lateral view, wide and gin of the processus ascendens and the main narrowly separated suprarostral corpora, in- axis of the body is also variable. The most termediate attachment of the processus as- acute angles are present in the melanonotus cendens to the braincase, and a wide pla- Group, L. insularum, L. silvanimbus, and L. num trabeculare anticum posterior to the riveroi (Figs. 1B, 5k–m,p–r). confluence of the cornua trabeculae. The processus branchialis of the visceral In these trees (Fig. 9a,b), Leptodactylus skeleton possess two distinct states: closed melanonotus CR, L. chaquensis, and L. ocel- (a distinct cartilaginous bridge between cera- latus grouped together. However, L. insula- tobranchials II and III) or open (no cartilagi- rum clustered with most species examined nous bridge between ceratobranchials II and of the melanonotus Group plus L. riveroi. III). Among the species examined, this char- Leptodactylus riveroi has not been previ- acter points to a major evolutionary di- ously associated with any of the species chotomy within Leptodactylus. Species tradi- groups. The present analyses suggest a closer tionally placed in the pentadactylus and relationship of this taxon to the melanonotus- fuscus Groups have an open processus bran- ocellatus clade, particularly to L. petersii chialis (see Fig. 3a,c), whereas all other spe- and L. validus (Fig. 9a). Leptodactylus insu- cies of Leptodactylus possess a closed proces- larum (traditionally placed in the ocellatus sus branchialis (e.g., Fig. 1D). Group) and L. riveroi share the following Phylogenetic analysis characteristics with the melanonotus Group: The 26 chondrocranial characters identi- posterior curvature of the palatoquadrate fied here (Table 1) were used in phylogenetic reaching well beyond the anterior margin of analyses enforcing and not enforcing a mono- the otic capsule, an acute angle between the phyletic Leptodactylus, with Physalaemus posterior margin of the processus ascendens pustulosus (Leptodactylidae: Leptodactyli- and the braincase, and a ‘‘low’’ attachment of nae), Hylodes nasus, and Crossodactylus gau- the processus ascendens to the braincase. dichaudii (Leptodactylidae: Hylodinae) as Based on external larval morphology (Mc- outgroups. Cranie et al., ’86) and call characteristics The analysis that did not enforce a mono- (Heyer et al., ’96), Leptodactylus silvanim- phyletic Leptodactylus resulted in 17 equally bus has been associated with the melanono- parsimonious trees, (51 steps long, C.I. 0.69) tus Group. In the present analyses, L. with Leptodactylus being paraphyletic rela- silvanimbus always clustered with the ocel- tive to Physalaemus. The consensus tree latus-melanonotus clade. Surprisingly, the (50% majority-rule) for these trees is given two samples of L. melanonotus did not group in Figure 9a. A second analysis, with a mono- together. In the analyses, L. melanonotus B phyletic constrained Leptodactylus, resulted grouped with other species of the traditional in 66 equally parsimonious trees, (52 steps melanonotus Group, whereas L. melanono- long, C.I. 0.67); the consensus tree (50% tus CR represents the sister group to the majority-rule) for these trees is given in Fig- ocellatus-chaquensis clade. ure 9b. The four species of the traditional penta- The present analyses identify two major dactylus Group, Leptodactylus knudseni, L. clades within Leptodactylus, the melanono- labyrinthicus, L. pentadactylus, and L. rhod- tus-ocellatus clade and the pentadactylus- omystax, clustered together, with a closer fuscus clade. The ocellatus-melanonotus relationship among the former three taxa. clade is supported by the following synapo- The strong relationship among these three morphies: presence of a closed processus taxa is supported by the unique characteris- branchialis, narrow suprarostral corpora tics of their suprarostrals, infrarostrals, and that are widely separated and not attached cornua trabeculae. Their association with L. distally to the alae, narrow and widely diver- rhodomystax is based on characteristics of gent cornua trabeculae, narrow planum tra- the palatoquadrate and crista parotica. 302 ..LRO N ..D SA DE R.O. AND LARSON P.M. ´

Fig. 8. Dorsal (a) and (e), ventral (b) and (f) and lateral (c) and (g) views of chondrocrania, and ventral (d) and (h) view of hyobranchial apparatus of a–d, Hylodes nasus, stage 25 and e-h, Crossodactylus gaudichaudii, stage 31. Bar ϭ 1.0 mm. Fig. 9. 50% majority-rule consensus trees. a: Analysis of data without topologi- chaudii as outgroups. Numbers above branches correspond to bootstrap values cal constraints. b: Analysis with a monophyletic constrained Leptodactylus. Both (values below 50% are not shown), letters and numbers below branches correspond analyses used Physalaemus pustulosus, Hylodes nasus, and Crossodactylus gaudi- to character states. 304 P.M. LARSON AND R.O. DE SA´ Among the traditionally recognized species embryos and larvae with notes on identification. Her- groups, the pentadactylus Group would rep- petologica 16:183–190. Haas, A. (1995) Cranial features of Dendrobatid larvae resent a paraphyletic assemblage because L. (Amphibia: Anura: Dendrobatidae). J. Morphol. 224: albilabris and L. labrosus (Fig. 9a,b) appear 241–264. more closely related to species in the penta- Haas, A. (1996) Das larvale cranium von Gastrotheca dactylus Group than to any other Leptodac- riobambae und seine metamorphose (Amphibia, Anura, Hylidae). Verh. Naturwiss. Ver. Hamburg 36:33–162. tylus examined. Haas, A. (1997) The larval hyobranchial apparatus of All members of the traditional fuscus discoglossoid frogs: its structure and bearing on the Group clustered together in the present systematics of the Anura (Amphibia: Anura). J. Zool. analyses, except for Leptodactylus albilabris Syst. Evol. Research 35:179–197. and L. labrosus. Within this strict fuscus Heyer, W.R. (1969) The adaptive ecology of the species groups of the genus Leptodactylus (Amphibia, Lepto- clade, L. mystacinus-L. fuscus and L. bufo- dactylidae). Evolution 23:421–428. nius-L. longirostris appear as sister taxa, Heyer, W.R. (1979) Systematics of the pentadactylus whereas other relationships are unresolved. species group of the genus Leptodactylus (Am- Overall, relationships among species of phibia: Leptodactylidae). Smith. Cont. Zool. 301:1–43. Heyer, W.R., R. de Sa´, J.R. McCranie, and L.D. Wilson Leptodactylus are strikingly similar (Fig. (1996) Leptodactylus silvanimbus (Amphibia: Anura: 9a,b) in both analyses (i.e., when Leptodacty- Leptodactylidae): Natural history notes, advertise- lus is constrained or not constrained to be ment call, and relationships. Herp. Nat. Hist. 4:169– monophyletic). However, the analysis that 174. does not constrain a monophyletic ingroup Lavilla, E.O., and I. De la Riva (1993) La larva de Telmatobius bolivianus (Anura, Leptodactylidae). results in a paraphyletic Leptodactylus by Alytes 11:37–46. placing Physalaemus as the sister group to Lavilla, E.O., and M. Fabrezi (1992) Anatomia craneal the fuscus-pentadactylus clade (Fig. 9a). De- de larvas de Lepidobatrachus llanensis y Ceratophrys spite the present results, Physalaemus and cranwelli (Anura: Leptodactylidae). Acta Zool. Lill. XLII 1:5–11. Leptodactylus are supported as distinct gen- Lynch, J.D. (1971) Evolutionary relationships, osteol- era by characteristics derived from the exter- ogy, and zoogeography of leptodactyloid frogs. Univ. nal morphology of adults and larvae (Lynch, Kansas Mus. Nat. Hist., Misc. Publ. 53:1–238. ’71). Consequently, herein we do not favor a Lynch, J.D. (1973) The transition from archaic to ad- vanced frogs. pp. 133–182. In: J.L. Vial (ed): Evolution- systematic re-arrangement until chondrocra- ary Biology of the Anurans. Contemporary Research nial data for more species of both genera Problems. Columbia: University of Missouri Press, become available and these data are ana- 470 pp. lyzed together with data from adult and Maxson, L., and W.R. Heyer (1988) Molecular systemat- ics of the frog genus Leptodactylus (Amphibia: Lepto- larval external morphology. dactylidae). Fieldiana: Zoology 41:1–13. McCranie, J.R., L.D. Wilson, and K.L. Williams (1986) ACKNOWLEDGMENTS The tadpole of Leptodactylus silvanimbus, with com- We thank the United States National Mu- ments on the relationships of the species. J. Herpetol. seum of Natural History, Smithsonian Insti- 20:560–562. Pu´ gener, L.A., and A.M. Maglia (1997) Osteology and tution, for the loan of specimens. skeletal development of Discoglossus sardus (Anura: Discoglossidae). J. Morphol. 232:233–267. LITERATURE CITED Ruvinsky, I., and L.R. Maxson (1996) Phylogenetic rela- de Sa´, R.O. (1988) Chondrocranium and ossification tionships among bufonoid frogs (Anura: Neobatra- sequence of Hyla lanciformis. J. Morphol. 195:345– chia) inferred from mitochondrial DNA sequences. 355. Mol. Phylogenet. Evol. 5:533–547. de Sa´, R.O. (1994) Chondrocranial structure and adapta- Sokol, O.M. 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APPENDIX A. Specimens examined and collection data.1 Number of Gosner Species specimens stages Locality USNM number L. albilabris 3 32, 34, 37 Tortola, Virgin Islands 520280-282 L. bufonius 3 33, 35, 37 Salta, Argentina 520285-287 L. chaquensis 3 37, 38, 40 Salta, Argentina 519879-881 L. fuscus 3 28, 34, 36 Roraima, Brazil 520288-290 L. gracilis 39 29–42 Dept. Canelones, Uruguay 519881-920 L. insularum 1 36 Isla San Andres 520291 L. knudseni 2 34, 36 Manaus, Amazonas, Brazil 529292-293 L. labialis 10 37–44 Cayo District, Belize 519928-937 L. labrosus 2 31, 36 Pichincha, Ecuador 520294-295 L. labyrinthicus 1 36 Amambay, Paraguay 520296 L. latinasus 2 35, 37 Salta, Argentina 520297-298 L. longirostris 2 33, 34 Para, Brazil 520299-300 L. melanonotus B 4 31–34 Cayo District, Belize 519938-941 L. melanonotus CR 2 35, 38 Guanacaste, Costa Rica 520283-284 L. mystacinus 4 31, 33, 35, 40 Montevideo, Uruguay 519942-945 L. mystacinus 9 35–38, 45 Misiones, Argentina 519946-954 L. ocellatus 18 40–46, 7 juv. Dept. Canelones, Uruguay 519882-920 L. ocellatus 16 31, 34–39 San Jose, Uruguay 519955-970 L. pentadactylus 11 27–31, 33, 35–36, 39 Puntarenas, Costa Rica 519989-999 L. petersii 1 36 Madre de Dios, Peru 520301-302 L. podicipinus 2 33, 38 Alto Parana, Paraguay 520303-304 L. rhodomystax 2 27, 31 Manaus, Amazonas, Brazil 520305-306 L. riveroi 2 27, 37 Manaus, Amazonas, Brazil 520307-308 L. silvanimbus 3 27, 31, 35 Ocotepeque, Honduras 520309-312 L. validus 1 37 St. Paul, Tobago 520313 L. validus 2 31, 35 St. George, St. Vincent 520314-315

1USNM United States National Museum of Natural History, Smithsonian Institution.