A Phylogenetic Analysis of Vanzolinius Heyer, 1974 (Amphibia, Anura, Leptodactylidae): Taxonomic and Life History Implications '

Home , Adenomera hylaedactyla, Leptodactylinae, Leptodactylus chaquensis

Arquivos do Museu National, Rio de Janeiro, v.63, n.4, p.707-726, out./dez.2005 ISSN 0365-4508

A PHYLOGENETIC ANALYSIS OF VANZOLINIUS HEYER, 1974 (AMPHIBIA, ANURA, LEPTODACTYLIDAE): TAXONOMIC AND LIFE HISTORY IMPLICATIONS '

(With 1 figure)

RAFAEL O. DE SA2 W. RONALD HEYER3 ARLEY CAMARGO4

ABSTRACT: The validity of the monotypic leptodactylid frog genus Vanzolinius Heyer, 1974 has been questioned recently. We explore the relationships of Vanzolinius discodactylus within the cluster of closely related genera Adenomera, Leptodactylus, and Lithodytes with both morphological and molecular data sets. Morphological and combined morphological and molecular data were analyzed using maximum parsimony; molecular data sets were analyzed with maximum likelihood methods. The resultant relationships are unambiguous in Vanzolinius being imbedded within Leptodactylus. In order to maintain Leptodactylus as a monophyletic genus, Vanzolinius is placed in the synonymy of Leptodactylus Fitzinger, 1826. The implications of relationships analyzed in this study are discussed in terms of both nomenclature and life-history evolution. Key words: Leptodactylus. Vanzolinius. Phylogenetic relationships. Life history evolution. RESUMO: Analise filogenetica de Vanzolinius Heyer, 1974 (Amphibia, Anura, Leptodactylidae): implicacoes taxonomicas e sobre a historia de vida. A validade do genero monotipico de leptodactilideo Vanzolinius Heyer, 1974, tern sido questionada recentemente. Neste estudo exploramos as relacoes de Vanzolinius discodactylus dentro do agrupamento de generos proximamente relacionados Adenomera, Leptodactylus e Lithodytes por meio de dados morfologicos e moleculares. Dados morfologicos e dados morfologicos e moleculares combinados foram analizados por parcimonia maxima, dados moleculares foram analisados por maxima verossimilhanca. As relacoes resultantes sao inequivocas em Vanzolinius ter que ser incluido em Leptodactylus. Para manter Leptodactylus como um genero monofiletico, Vanzolinius Heyer 1974, e colocado na sinonimia de Leptodactylus Fitzinger, 1826. As implicacoes dos relacionamentos analisados neste estudo sao discutidas em termos de nomenclatura e evolucao dos modos reprodutivos. Palavras-chave: Leptodactylus. Vanzolinius. Relacoes filogeneticas. Evolucao da historia de vida.

INTRODUCTION (HEYER, 1974b). The most recent morphological analysis indicated that Vanzolinius shared distinctive The frog genera Adenomera Fitzinger, 1867, characteristics with Leptodactylus diedrus (HEYER, Lithodytes Fitzinger, 1843, and Vanzolinius Heyer, 1998). Previous analyses of relationships agreed that 1974 have, atone time or another, been included in within the subfamily Leptodactylinae the genera the genus Leptodactylus. BOULENGER (1883) Adenomera, Leptodactylus, Lithodytes, and described the currently recognized monotypic Vanzolinius formed a monophyletic clade and that Vanzolinius as, Leptodactylus discodactylus. HEYER the genus Physalaemus Fitzinger, 1826, was more (1970) associated this taxon with the Leptodactylus distantly related to this clade (HEYER, 1974a, 1975; melanonotus species group. Later, HEYER (1974a) LYNCH, 1971). placed the taxon within Lithodytes commenting on It is necessary to establish convincingly whether its possible distinctiveness and subsequently created the genus Leptodactylus as currently understood the genus Vanzolinius to accommodate this species is monophyletic, if we wish to understand the

' Submitted on February 28, 2005. Accepted on June 17, 2005. 2 University of Richmond, Department of Biology, Richmond, VA 23173, USA. E-mail: [email protected]. 3 National Museum of Natural History, Amphibians & Reptiles, MRC 162. PO Box 37012, Smithsonian Institution, Washington, DC 20013-7012, USA. 4 Universidad de la Republica, Facultad de Ciencias, Seccion Zoologia Vertebrados. 4225 Igua, Montevideo 11400, Uruguay. 708 ROSA, W.R.HEYER & A.CAMARGO evolution of life history variation in Leptodactylus. following exceptions. We had no tissue samples for In this paper, we are particularly interested in Adenomera marmorata and Physalaemus determining the phylogenetic relationships of pustulosus, two of the taxa used in HEYER (1998), Vanzolinius. Preliminary findings on relationships so we used morphological data for Adenomera of previously proposed monophyletic clades in the hylaedactyla and Physalaemus gracilis, for which Leptodactylus cluster (Adenomera, Leptodactylus, we do have molecular data. Data taken for A. Lithodytes, Vanzolinius) are also presented in this hylaedactyla and P. gracilis were taken from HEYER paper, and we discuss the implications of our (1974a), HEYER, DE SA & MULLER (2005), USNM results for understanding aspects of life history 292477 (cleared-and-stained A, hylaedactyla) and evolution in this cluster. RdS 511 (larval P. gracilis from Uruguay, Canelones, Balneario Atlantida, Rafael de Sa field number). MATERIAL AND METHODS These two species have a few states that differ from their congeners, and require recoding of states and/ Taxon sampling - Species groups within or redefinition of states as follows. Leptodactylus were previously recognized on the Character 7, toe webbing. Physalaemus pustulosus basis of morphological and life history characters was coded as having a unique state in the data set (HEYER, 1969). We included samples from each of of HEYER (1998), toes with weak basal fringes and the four species groups to sample the morphological webbing. Physalaemus gracilis has toes without web diversity within Leptodactylus. Leptodactylus riveroi or fringes, a condition found in other taxa in the Heyer & Pyburn, 1983, a species of uncertain data set. The new definitions are: State 0 - toes species group affinity, and L. silvanimbus McCranie without web or fringes; State 1 - toes with fringes et al., 1980, a species recently suggested as basal extending length of toes except for tips; State 2 - within the genus (HEYER, DE SA & MULLER, females with weakly developed lateral toe fringes 2005), were also included. Physalaemus has been and males either with ridges or weakly developed shown to function well as an outgroup for fringes. The state ordering is 0-1-2. Leptodactylus using both morphological and Character 15, depressor mandibulae muscles. The molecular data (HEYER, 1998; HEYER, DE SA & depressor mandibulae may have one to three slips MULLER, 2005); herein Physalaemus gracilis of origin, from the dorsal fascia (df), the zygomatic (Boulenger, 1883) was the outgroup taxon. and/or otic ramus of the squamosal (sq), and the The taxa analyzed in this study are: Leptodactylus tympanic annulus (at) (following the terminology bufonius Boulenger, 1894, L. fuscus (Schneider, defined by STARRETT, 1968). Lower case indicates 1799) [fuscus species group); L. leptodactyloides small slips of the muscle, upper case indicates large (Andersson, 1945), L, melanonotus (Hallowell, slips. Physalaemus pustulosus has the dfSQat 1861) (melanonotus species group); L. chaquensis condition, whereas P. gracilis has DFSQat. The Cei, 1950, L. insularum Barbour, 1906 (ocellatus DFSQat condition is state 0 in our data matrix. species group); L. pentadactylus (Laurenti, 1768) (pentadactylus species group); L, diedrus Heyer, Character 18, anterior petrohyoideus muscle. 1994, L, riveroi, L. silvanimbus (Leptodactylus of Adenomera hylaedactyla has a state not found in unclear species group affinity); Adenomera the data set of HEYER (1998). The new definitions hylaedactyla (Cope, 1868), Lithodytes lineatus are: State 0 - the anterior petrohyoideus muscle (Schneider, 1799), Vanzolinius discodactylus inserts entirely on the edge of the hyoid (Boulenger, 1883); and Physalaemus gracilis (as apparatus; State 1 - the muscle inserts on the the outgroup). For both the morphological and edge of the hyoid and on the ventral body of the molecular data, the data for L. pentadactylus are hyoid in part; State 2 - the muscle inserts entirely from Middle American specimens. See Tissue on the ventral surface of the hyoid body. The state Voucher Specimens section at the end of this ordering is 0-1-2. paper for specimen data used for molecular Character 24, sartorius muscle. The condition in analyses. Museum abbreviations follow LEVITON P. gracilis does not differ from some other taxa in etal. (1985). the data set, in contrast to the condition found in Morphological data set - The morphological matrix P. pustulosus. The new definitions are: State 0 - is provided in Appendix 1. The character state muscle moderate; State 1 - intermediate condition descriptions and ordering information are the same between States 0 and 2; State 2 - muscle broad. as those published in HEYER (1998) with the The state ordering is 0-1-2.

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Character 32, sacral diapophyses. Physalaemus data sets (i.e., morphology, 12S, and 16S data sets) gracilis does not differ in this character from other as well as combined analyses (i.e., 12S+16S matrix, taxa. Thus characters 32-37 in our data set equal morphology+12S+16S matrix). In combined characters 33-38 in the HEYER (1998) data set. analyses gaps were alternatively considered as Molecular methodology - DNA extraction followed missing or as 5th characters; we also evaluated HILLIS et al. (1996). Two segments of the the effect of the substitution bias in the analysis of mitochondrial genome were amplified using the the combined data matrix using MP by down- polymerase chain reaction (PCR). A segment of the weighting transitions to transversions 5:1. 12S r RNA of ~ 900 nucleotides and a segment of the 16s r RNA of ~ 700 nucleotides were amplified. RESULTS Double-stranded (DS) PCR amplifications were performed in a final volume of 50|al containing 0.4jjl There is modest variation in the 12S, 16S, and of each primer, 1.0^1 of each dNTP, 3.0|al of 25mM 12S+16S data sets (Tabs. 1-3). The maximum MgCl, and 1.25 units of Taq (Thermus aquaticus) DNA sequence divergences between pairs of taxa are 21% polymerase; the reaction was overlaid with 50(il of for the 128 data, 16% for the 168 data, and 18% mineral oil. PCR conditions were as follows: 94°C for the 128+168 data. for 60s, 57°C for 60s, and 72°C for 60s, with 25 The results of all cladistic analyses are almost cycles for the 12S amplification and 30 cycles for identical; consequently we present the maximum the 16S amplification. Amplified product was parsimony combined data set results and point out purified using Wizard® PCR Preps Kit (Promega). where the analyses differ (Fig. 1). The parsimony Of the purified DS fragment, 0.5jjl were mixed with analysis of the combined data matrix results in a 1.5(il of a single IRD-labeled primer, 7.2jul of single tree (length= 1430, consistency index=0.56) in Sequencing Buffer, l.Ojul of Sequitherm Excel•II which Vanzolinius exhibits a sister taxa relationship (Epicentre Technologies Co.) DNA polymerase, and with L, diedrus. This relationship is also recovered in 6.8(il of dH20. Subsequently, 4.0pl of this mix was the analyses of the combined molecular data added to each of 4 tubes containing 2jjl of each partitions as well as in all analyses of the 12S data nucleotide respectively. PCR conditions were as partition. The analyses of the 16S data partition follows (30 cycles): 92°C for 30s, 55°C for 30s, and position Vanzolinius in the following clade (L, diedrus 70°C for 30s. SB amplified and IR labeled fragments (L. leptodactyloides+Vanzolinius)). The distance data were sequenced in a LI-COR 4200 IR DNA Sequencer matrices show that the close relationship of L, diedrus on 6% acrylamide gels. A total of 839 128 and 648 with Vanzolinius is unambiguous in the 12S data 16S nucleotide positions were aligned unambigously (Tab. 1), but not at all clear in the 16S data, where L, using Clustal X and positions of ambiguous diedrus and Vanzolinius have lower sequence distance alignments were not used in the phylogenetic values with L, silvanimbus and several members of analyses. GenBank accession numbers for the the L, fuscus, L, melanonotus, and L. ocellatus group sequence data are AY943217—242. The alignment members than with each other (Tab.2). The matrix is provided in Appendix 2. morphological data set demonstrates strong support Phylogenetic Analysis - Maximum Parsimony (MP) for a L. diedrus-V. discodactylus sister species analysis using PAUP* 4.0 (SWOFFORD, 2002) was relationship with 100% bootstrap support. used for both the morphological data set and the combined morphological and molecular data set. DISCUSSION Molecular data sets were analyzed with maximum likelihood (ML) in PAUP* under the GTR+I+G model Phylogenetic conclusions - The following recommended by both the Hierarchical Likelihood conclusions are supported by the analyses Ratio Test and the Akaike Information Criterion used performed on our data. by Modeltest 3.04 (POSADA & CRANDALL, 1998). First, Vanzolinius always clusters within We obtained a total of 37 morphological characters Leptodactylus. The data are very clear and convincing and i486 base pairs (bp) for each taxon (839 bp for this conclusion. There are two nomenclatural corresponding to the 12S rDNA gene and 647 bp options to resolve the phylogenetic conclusion that to the 16S rDNA gene). Sequences were aligned Vanzolinius is imbedded within Leptodactylus: using Clustal X (THOMPSON, HIGGINS & GIBSON, Vanzolinius could be synonymized with 1994). We ran individual analyses for each of the Leptodactylus; or one or more clades within

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Leptodactylus could be raised to generic status. Current (unpublished) data are inconclusive regarding the phylogenetic relationships among Leptodactylus species, and rule out elevating certain clades within Leptodactylus to generic status at this time. However, we think there are compelling 3 arguments for placing Vanzolinius in the synonymy > f- of Leptodactylus. The previous actions on generic 6 placement of the species discodactylus were all based ^ o on morphological and karyotype data. The strongest support for generic recognition of Vanzolinius as a d d genus distinct from Leptodactylus involved two oo o CN morphological features of the toes: the toe tips of V. O o u oo discodactylus are expanded into small disks with odd longitudinal grooves on the dorsal surface and the CO co lO f- terminal phalanges are T-shaped (HEYER, 1974b). d d d d With the discovery of Leptodactylus diedrus, the >-i io <*• CN <*• morphological distinctiveness between Leptodactylus CO CO t- oo IO and Vanzolinius was bridged to a large extent O O O O O (HEYER, 1998). Thus, the morphological data used u G t> lO O CO 00 CN to define Vanzolinius as a genus distinct from u a* H co io t- io O i o ^ ^ ^ ^ ^ Leptodactylus are seriously compromised by d d d d d d inclusion of the data for L. diedrus and the molecular a h » n o H t- * oo oo H ^H io oo io data strongly support synonymizing Vanzolinius with l O ^ ^ ^ ^ ^ •a 0 Leptodactylus. Consequently, we hereby synonymize 3 d d d d d d d the genus Vanzolinius Heyer, 1974 with the genus lOCNt-t-cQWW^ Leptodactylus Fitzinger, 1826. otcooct + ô •a u dddddddd Second, the genera Adenomera and Lithodytes may •a share a sister-group relationship and our data [•-cQcQr-ooair-coco a CTiCTi^^^CNWt-W provide support that both are evolutionarily distinct ddddddddd from Leptodactylus (including Vanzolinius). •3 t-m^t-t-iooococooO'-* Third, the previously recognized "traditional" a t-OiOiOO'-HCOlOcQW species groups may not all be monophyletic, o dddddddddd although the two members of the L. fuscus group lOlOKttlHtlHNiol^ form a well-supported clade in this study. ^cot-aiooo^^-t-t-co ddddddddddd Fourth, a sister-group relationship between L. u discodactylus and L. diedrus, previously suggested t-^-lOaicOWcQOcQt-COCN 6 COÎ-O'-HCOCOÎ-OOOiO'-H by HEYER (1998), is reasonably well supported by o G dddddddddddd the morphological and combined molecular data sets. u oocoaicooocO'-Ht-î-cot-t-io Finally, Leptodactylus riveroi, a taxon of uncertain ^"CNCOCOCNCNCOCO^-^t-OOO ^H^H^H^H^H^H^H^H^H^H^HCN^ relationships, exhibits suggestive affinities to the ddddddddddddd L. melanonotus species group. CD Life history implications - All members of the & 13 subfamily Leptodactylinae (except Limnomedusa), 3 a CO o S CM c c e place their eggs in foam nests (LANGONE, 1995). s CD o •a •a c 3 c CD 3 c 5 a Within the Leptodactylus cluster, however, there is 5 a « £ 1 variation regarding where the foam nests are a <2, s a, I 42 deposited and considerable variation occurs in c« -J 1-1 1-1 -J 1-1 1-1 1-1 -4 -1 -4 ^ < i-i o •-I CN CO other life history aspects. Two examples illustrate si- IO kO f- how an understanding of phylogenetic relationships in this group is critical to deciphering life history evolution in the genus Leptodactylus.

Arq. Mus. Nac, Rio de Janeiro, v.63, n.4, p.707-726, out./dez.2005 A PHYLOGENETIC ANALYSIS OF VANZOLINIUS: TAXONOMIC AND LIFE HISTORY IMPLICATIONS 711

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First, two clades (Adenomera and the L. fuscus Second, there is considerable variation in female species group) within Leptodactylinae share the attendance of foam nests and larvae, whether same pattern of males constructing a terrestrial attending females communicate with their larvae, subsurface chamber, attracting females to the and how females communicate with their larvae chamber acoustically, and depositing the foam nest (VAZ-FERREIRA & GEHRAU, 1975; WELLS & in the chamber where at least embryonic and early BARD, 1988). As far as is known, parental care larval development take place (see KOKUBUM & does not occur in any species of the L. fuscus group. GIARETTA, 2005 and references cited therein). Our Our preliminary data indicate that intensive taxon data indicate that this complex life history pattern sampling with additional data is required to resolve was independently derived in both clades and is relationships among the Leptodactylus species that not the result of shared ancestral adaptations. Also, demonstrate female attendance and at least some members of the L. pentadactylus communication with their offspring in order to group use pre-existing terrestrial burrows in which understand the evolution of parental care in they deposit their foam nest (see GIBSON & BULEY, Leptodactylus. 2004 and references cited therein). Additional taxon More intensive taxon sampling and the sequencing sampling is required to determine whether this of nuclear and more slowly evolving genes should pattern served as a precursor to the actual provide a well-supported phylogeny for construction of terrestrial incubating chambers in Leptodactylus at the species level that will allow a the L. fuscus group. Our preliminary data suggest better understanding of the evolution of life history support for this scenario. variation in the Leptodactylus cluster.

L. diedrus 94 69 95 Vanzolinius 59 ,L. riveroi L. silvanimbus 78 L. melanonotus 75 . L. chaquensis 77 97 75 . L. leptodactyloides L. insularum 100 L. bufonius 99 55 100 L. fuscus 75 L. pentadactylus . Adenomera 69 95 Lithodytes Physalaemus

Fig.l- Maximum Parsimony Tree of combined (morphological and molecular) data sets. Gaps were considered as a fifth character. Numbers above branches correspond to bootstrap support in parsimony analysis; numbers below branches are bootstrap support values from Maximum Likelihood analysis of the combined molecular data set.

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TISSUE VOUCHER SPECIMENS (Universidade Federal de Sergipe, Brazil), Andrew Chek (Organization for Tropical Studies, USA), Adenomera hylaedactyla - BRAZIL: PARA: Alter do Reginald B. Cocroft (University of Missouri, USA), Chao (MZUSP 70958) Ronald I. Crombie (California Academy of Sciences, Leptodactylus bufonius - ARGENTINA: SALTA: 54 USA), Miriam M. Heyer (Smithsonian Institution, km NE of Joaquin V. Gonzalez on provincial route USA), Roberto Ibanez D. (Circulo Herpetologico de 41 (USNM field number 175816, deposited in FML). Panama), Esteban O. Lavilla (Institute Miguel Lillo, Argentina), James R. McCranie (Miami, USA), Roy Leptodactylus chaquensis- ARGENTINA: TUCUMAN: W. McDiarmid (Biological Resources Division, United ca 40 km SE San Miguel de Tucuman at km post States Geological Survey, USA), Alejandro Olmos 1253 on International Route 9 (USNM 319708). (Montevideo, Uruguay), Miguel T. Rodrigues Leptodactylus diedrus - VENEZUELA: AMAZONAS: (Universidade de Sao Paulo, Brazil), Larry David Rio Negro, near Neblina base camp on left bank of Wilson (Miami-Dade College,USA), and Addison Rio Baria (= Rio Mawarinuma) (USNM 30715). Wynn (Smithsonian Institution, USA). Leptodactylus discodactylus - ECUADOR (QCAZ 16788). LITERATURE CITED Leptodactylus fuscus - BRAZIL: RORAIMA: BOULENGER, G.A., 1883. On a collection of frogs from Caracarana, near Normandia (MZUSP 67073). Yurimaguas, Huallaga River, northern Peru. Proceedings of the Zoological Society of London, Leptodactylus insularum - PANAMA: PANAMA: Rio London, 1883:635-638. Indio, camino hacia Las Minas (CH 4956). GIBSON, R.C. & BULEY, K.R., 2004. Maternal care and Leptodactylus leptodactyloides - BRAZIL: PARA: obligatory oophagy in Leptodactylus fallax: a new reproductive mode in frogs. Copeia, Lawrence, Serra de Kokoinhokren (MZUSP 70969). 2004(1):128-135. Leptodactylus melanonotus - BELIZE: CAYO: HEYER, W.R., 1969. The adaptive ecology of the between San Jacinto and Spanish Lookout road on species groups of the frog genus Leptodactylus Webster Highway, Caesar's Hotel (USNM 535964). (Amphibia, Leptodactylidae). Evolution, Lawrence, 23(3):421-428. Leptodactylus ocellatus - BRAZIL: SANTA HEYER, W.R., 1970. Studies on the frogs of the genus CATARINA: Campeche (MZUSP 68993). Leptodactylus (Amphibia: Leptodactylidae). VI. Bio system at ics of the melanonotus group. Leptodactylus "pentadactylus" - PANAMA: BOCAS Contributions in Science, Los Angeles County DELTORO: Isla Popa (USNM 347153). Museum Los Angeles, 253:1-46 Leptodactylus riveroi - VENEZUELA: AMAZONAS: HEYER, W.R., 1974a. Relationships of the marmoratus Rio Negro, Neblina base camp on left bank of Rio species group (Amphibia, Leptodactylidae) within the Baria (= Rio Mawarinuma) (USNM 562029). subfamily Leptodactylinae. Contributions in Science, Los Angeles County Museum Los Angeles, 191:1-48. Leptodactylus silvanimbus - HONDURAS: HEYER, W.R., 1974b. Vanzolinius, a new genus proposed OCOTEPEQUE; Belen Gualcho (USNM 348631). for Leptodactylus discodactylus (Amphibia, Leptodactylidae). Proceedings of the Biological Lithodytes lineatus - BRAZIL: MATO GROSSO: Society of Washington, Washington, 87(11):81-90. Apiacas (MZUSP 80874). HEYER, W.R., 1975. A preliminary analysis of the intergeneric relationships of the frog family Physalaemus gracilis - URUGUAY: SALTO: Leptodactylidae. Smithsonian Contributions to Espinillar (RdS 788 field number). Zoology, Washington, 199:1-55. HEYER, W.R., 1998. The relationships of Leptodactylus ACKNOWLEDGMENTS diedrus (Anura, Leptodactylidae). Alytes, Paris, 16(l-2):l-24. This work was supported through National Science HEYER, W.R.; DE SA, R.O. & MULLER, S., 2005. On Foundation Awards #9815787 and 0342918 to ROS the enigmatic distribution of the Honduran endemic Leptodactylus silvanimbus (Amphibia: Anura: and WRH, and the Neotropical Lowlands Research Leptodactylidae). In: DONNELLY, M.A.; CROTHER, Program, Smithsonian Institution (Richard P. Vari, B.I.; GUYER, C.; WAKE, M.H. & WHITE, M.E. (Eds.) Principal Investigator). We thank the following Ecology and evolution in the tropics: a historical colleagues that kindly provided tissue specimens perspective. Chicago: University of Chicago Press, and/or helped in collecting: Celso Morato de Carvalho p.81-101.

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HILLIS, D.M.; MABLE, B.K.; LARSON, A.; DAVIS, S.K. the model of DNA substitutions. Bioinformatics, & ZIMMER, E.A., 1996. Nucleic acids IV: Sequencing Oxford, 14:817-818 and cloning. In: HILLIS, D.M.; MORITZ, C. & MABLE, STARRETT, PH., 1968. The phylogenetic significance B.K. (Eds.) Molecular systematics. Second edition. of the jaw musculature in anuran amphibians. Ann Sunderland, Massachusetts: Sinauer Associates, Inc. Arbor, Michigan. 179p. Dissertation (Ph.D. in p.321-381. Zoology), University of Michigan. KOKUBUM, M.N.C. & GIARETTA, A.A., 2005. SWOFFORD, D.L., 2002. PAUP*. Phylogenetic Analysis Reproductive ecology and behaviour of a species of Using Parsimony (*and other methods). Version Adenomera (Anura, Leptodactylinae) with endotrophic 4.0bl0. Sunderland, Massachusetts: Sinauer tadpoles: systematic implications. Journal of Natural Associates. History, London, 39(20):1745-1758. THOMPSON, J.D.; HIGGINS, D.G. & GIBSON, T.J., LANGONE, J.A., 1995 ("1994"). Ranas y sapos del 1994. CLUSTAL W: improving the sensitivity of Uruguay (reconocimientos y aspectos biologicos). progressive multiple sequence alignment through Museo Damaso Antonio Larranaga, Serie de sequence weighting, position specific gap penalties Divulgacidn, Montevideo, 5:1-123. and weight matrix choice. Nucleic Acids Research, LEVITON, A.E.; GIBBS JR., R.H.; HEAL, E. & Oxford, 22:4673-4680. DAWSON, C.E., 1985. Standards in herpetology VAZ-FERREIRA, R. & GEHRAU, A., 1975. and ichthyology: Part I. Standard symbolic codes Comportamiento epimeletico de la rana comun, for institutional resource collections in Leptodactylus ocellatus (L.) (Amphibia, herpetology and ichthyology. Copeia, Lawrence, Leptodactylidae) I. Atencion de la cria y actividades 1985(3):802-832. alimentarias y agresivas relacionadas. Physis, LYNCH, J.D., 1971. Evolutionary relationships, Buenos Aires, Seccion B, 34(88):1-14. osteology, and zoogeography of leptodactyloid frogs. WELLS, K.D. & BARD, K.M., 1988. Parental behavior of University of Kansas Museum of Natural History, an aquatic-breeding tropical frog, Leptodactylus Miscellaneous Publication, Lawrence, 53:1-238. bolivianus. Journal of Herpetology, Athens, POSADA, D. &CRANDALL, K.A. 1998. Model-test: testing 22(3):361-364.

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