Open Access Open Access  Restricted Access Subscription or Fee Access

The cane or marine toad, Rhinella marina (Anura, Bufonidae): two genetically and morphologically distinct species



Rhinella marina is a Neotropical toad that has been introduced widely worldwide. Its toxic effects to frog-eating predators threaten the native and domestic fauna of some regions where it has been introduced. Despite previous studies suggesting two genetically distinct cryptic species within R. marina, one east and one west of the Andes, its taxonomic status remained unresolved due to the absence of morphological complementary evidence. For the first time, data from two mitochondrial genes (ND3 and CR) and 23 morphometric landmarks are combined to evaluate the taxonomic status of this species. Our results support the hypothesis of two separate evolutionary lineages within R. marina and demonstrate that these lineages have significantly diverged in skull shape. We identified two distinct morphotypes, one eastern and one Andean western, with no overlapping morphospaces. The geographic pattern of genetic variation was consistent with a stable structured population with no evidence of recent demographic or geographic expansions. The concordance between the observed geographic patterns in morphometric and genic traits calls for the recognition of two species under R. marina name.


biological control, mitochondrial DNA, morphometric geometry, phylogeny, taxonomy, Venezuela, Amphibia

Full Text:



Adams, D.C., Rohlf, F.J. & Slice, D.E. (2004) Geometric morphometrics: Ten years of progress following the ‘revolution’. Italian Journal of Zoology, 71, 5–16.

Adams, D.C., Rohlf, F.J. & Slice, D.E. (2013) A field comes of age: geometric morphometrics in the 21st century. Hystrix, the Italian Journal of Mammalogy, 24, 7–14.

Alonso, R., Crawford, A.J. & Bermingham, E. (2012) Molecular phylogeny of an endemic radiation of Cuban toads (Bufonidae: Peltophryne) based on mitochondrial and nuclear genes. Journal of Biogeography, 39, 434–451.

Avise, J.C., Shapira, J., Daniel, S.W., Aquadro, C.F. & Lansman, R.A. (1983) Mitochondrial DNA differentiation during the speciation process in Peromyscus. Molecular Biology and Evolution, 1, 38–56.

Bandelt, H.J., Forster, P. & Röhl, A. (1999) Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution, 16, 37–48.

Bickford, D., Lohman, D.J., Sodhi, N.S., Ng, P.K., Meier, R., Winker, K., Ingram, K.K. & Das, I. (2007) Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution, 22, 148–155.

Birchard, S.J. (2002) Manual Clínico de Procedimientos en Pequeñas Especies. Mc Graw-Hill Interamericana de Editores S.A. de C.V., Mexico, D.F., 1960 pp.

Bookstein, F.L. (1989) Principal warps: thin-plate splines and the decomposition of deformations. IEEE Transactions on Pattern Analysis and Machine Intelligence, 11, 567–585.

Bookstein, F.L. (1991) Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, Cambridge, 435 pp.

Borkin, L.Y., Litvinchuk, S.N., Rozanov, Y.M. & Skorinov, D.V. (2004) On cryptic species (from the example of amphibians). Zoologichesky Zhurnal, 83, 936–960.

Duellman, W.E. & Trueb, L. (1994) Biology of Amphibians. The John Hopkins University Press, Baltimore, 670 pp.

Easteal, S. (1981) The history of introductions of Bufo marinus (Amphibia: Anura): a natural experiment in evolution. Biological Journal of the Linnean Society, 16, 93–113.

Easteal, S. (1985) The ecological genetics of introduced populations of the giant toad Bufo marinus. II. Efective population size. Genetics, 110, 107–122.

Fouquet, A., Recoder, R., Teixeira, M., Cassimiro, J., Amaro, R.C., Camacho, A., Damasceno, R., Carnaval, A.C., Moritz, C. & Rodrigues, M.T. (2012) Molecular phylogeny and morphometric analyses reveal deep divergence between Amazonia and Atlantic Forest species of Dendrophryniscus. Molecular Phylogenetics and Evolution, 62, 826–838.

Fouquet, A., Vences, M., Salducci, M.D., Meyer, A., Marty, C., Blanc, M. & Gilles, A. (2007) Revealing cryptic diversity using molecular phylogenetics and phylogeography in frogs of the Scinax ruber and Rhinella margaritifera species groups. Molecular Phylogenetics and Evolution, 43, 567–582.

Frost, D.R. (2015) Amphibian Species of the World: an Online Reference. Version 6.0. American Museum of Natural History. Available from: (accessed 7 December 2015)

Fu, Y.X. (1997) Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics, 147, 915–925.

Global Invasive Species Database (2005) Rhinella marina (=Bufo marinus). Available from: (accessed 8 May 2015)

Goebel, A.M., Donnelly, J.M. & Atz, M.E. (1999) PCR primers and amplification methods for 125 ribosomal DNA, the control region, cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully. Molecular Phylogenetics and Evolution, 11, 163–199.

Graybeal, A. (1997) Phylogenetic relationships of bufonid frogs and tests of alternate macroevolutionary hypotheses characterizing their radiation. Zoological Journal of the Linnean Society, 119, 297–338.

Jaeger, J.R., Riddle, B.R. & Bradford, D.F. (2005) Cryptic Neogene vicariance and Quaternary dispersal of the red-spotted toad (Bufo punctatus): insights on the evolution of North American warm desert biotas. Molecular Ecology, 14, 3033–3048.

Junca, F.A., Rohr, D.L., Lourenco-de-Moraes, R., Santos, F.J.M., Protazio, A.S., Merces, E.A. & Sole, M. (2012) Advertisement call of species of the genus Frostius Cannatella 1986 (Anura: Bufonidae). Acta Herpetologica, 7, 189–201.

Kaliontzopoulou, A. (2011) Geometric morphometrics in herpetology: modern tools for enhancing the study of morphological variation in amphibians and reptiles. Basic and Applied Herpetology, 25, 5–32.

Kendall, D.G. (1977) The diffusion of shape. Advances in Applied Probability, 9, 428–430.

Klingenberg, C.P. (2011) MorphoJ: an integrated software package for geometric morphometrics. Molecular Ecology Resources, 11, 353–357.

Kwet, A., Di-Bernardo, M. & Maneyro, R. (2006) First record of Chaunus achavali (Anura, Bufonidae) from Rio Grande do Sul, Brazil, with a key for the identification of the species in the Chaunus marinus group. Iheringia. Série Zoologia, 96, 479–485.

Lever, C. (2001) The Cane Toad. The History and Ecology of a Succesful Colonist. Westbury Academic and Scientific Publishing, West Yorkshire, 230 pp.

Librado, P. & Rozas, J. (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25, 1451–1452.

Muller, L. & Hellmich, W. (1936) Wissenschaftliche Ergebnisse der Deutschen Gran Chaco-Expedition. Amphibien und Reptilien. I. In: Amphibia, Chelonia, Loricata. Strecker und Schröder Verlag, Stuttgart, 120 pp.

Posada, D. (2008) jModelTest: Phylogenetic Model Averaging. Molecular Biology and Evolution, 25, 1253–1256.

Pramuk, J.B. (2006) Phylogeny of South American Bufo (Anura : Bufonidae) inferred from combined evidence. Zoological Journal of the Linnean Society, 146, 407–452.

Rohlf, F.J. (2008) tpsRelwarp v1.46. Ecology and Evolution. State University of New York at Stony Brook, Stony Brook.

Rohlf, F.J. & Marcus, L.F. (1993) A revolution in morphometrics. Trends in Ecology & Evolution, 8, 129–132.

Sambrook, J. & Russel, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2344 pp.

Sequeira, F., Sodré, D., Ferrand, N., Bernardi, J.A., Sampaio, I., Schneider, H. & Vallinoto, M. (2011) Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers. BMC Evolutionary Biology, 11, 1–15.

Shine, R. (2010) The ecological impact of invasive cane toads (Bufo marinus) in Australia. Quarterly Review of Biology, 85, 253–291.

Slade, R.W. & Moritz, C. (1998) Phylogeography of Bufo marinus from its natural and introduced ranges. Proceedings of the National Academy of Sciences, USA, 265, 769–777.

Stuart, B.L., Inger, R.F. & Voris, H.K. (2006) High level of cryptic species diversity revealed by sympatric lineages of Southeast Asian forest frogs. Biology Letters, 2, 470–474.

Swofford, D.L. & Olsen, G.J. (1990) Phylogeny Reconstruction. In: Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts, pp. 411–501.

Tajima, F. (1989) Statistical-method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123, 585–595.

Vallinoto, M., Sequeira, F., Sodré, D., Bernardi, J.A.R., Sampaio, I. & Schneider, H. (2010) Phylogeny and biogeography of the Rhinella marina species complex (Amphibia, Bufonidae) revisited: implications for Neotropical diversification hypotheses. Zoologica Scripta, 39, 128–140.

Zelditch, M.L., Swiderski, D.L., Sheets, H.D. & Fink, W.L. (2004) Geometric Morphometrics for Biologists: A Primer. Elsevier Academic Press, London, 416 pp.



  • There are currently no refbacks.

ISSN 1175-5326 (Print Edition) & ISSN 1175-5334 (Online Edition)
Published by Magnolia Press, Auckland, New Zealand