DNA barcoding in Neotropical tadpoles: evaluation of 16S rRNA gene for the identification of anuran larvae from northeastern Brazil


  • Marcos J. Matias Dubeux
  • Filipe A. Cavalcanti do Nascimento
  • Larissa L. Correia
  • Tamí Mott

Palabras clave:

Amphibia, Mitochondrial Gene, Genetic Divergence, Species Diversity


The challenge in studying Neotropical tadpoles is identifying species using only their external morphology. However, the DNA barcode protocol is often implemented to help elucidate taxonomic issues. In fact, the identification of frogs through their unknown tadpoles has already been achieved accurately using this protocol. Despite the successful application of this tool, the efficiency of the 16S rRNA gene as a DNA barcode for Neotropical tadpoles has not been fully assessed. Herein we evaluate the efficacy of the 16S rRNA gene for identifying tadpoles from northeastern Brazil. Samples of 100 tadpole specimens from 12 locations were analyzed. The DNA sequences were individually submitted to a BLAST search and were then aligned with a matrix containing available sequences in the GenBank based on the anurans known to occur in the study area. The 16S rRNA fragment successfully identified the analyzed anuran species. Based on DNA barcoding, 8% of the tadpoles morphologically identified at the species level were incorrect. When an incongruence between morphological and molecular identifications was detected, the morphology of the target morphotype was reexamined, and previously neglected morphological characteristics were identified. DNA barcoding using the 16S rRNA gene facilitated the assessment of tadpole richness in northeastern Brazil. This DNA protocol can be used as a starting point for detecting high levels of genetic divergence, highlighting potential taxa that should be studied from phylogenetic and taxonomic perspectives.


Almeida, J.P.F.; Nascimento, F.A.C.; Torquato, S.; Lisboa, B.S.; Tiburcio, I.C.S.; Palmeira, C.N.S.; Lima, M.G. & Mott, T. 2016. Amphibians of Alagoas State, northeastern Brazil. Herpetology Notes 9: 123-140.

Altig, R. & McDiarmid, R.W. 1999. Diversity: familial and generic characterizations. In: Tadpoles: the biology of anuran larvae. Edited by R.W. Mcdiarmid, R. Altig. University of Chicago Press, Chicago. pp. 295-337.

Altig, R.; McDiarmid, R.W. & Dias, P.H.S. 2021. Bibliography for the identification, morphology and development of amphibian gametes and larvae. https://www.researchgate. net/publication/359195811_Bibliography_for_the_identification_morphology_and_development_of_amphibian_gametes_and_larvae. Accessed on 07 February 2022.

Andrade, F.S.; Haga, I.A.; Ferreira, J.S.; Recco-Pimentel, S.M.; Toledo, L.F. & Bruschi, D.P. 2020. A new cryptic species of Pithecopus (Anura, Phyllomedusidae) in north-eastern Brazil. European Journal of Taxonomy 723, 108-134.

Asfora, P.H. & Pontes, A.R.M. 2009. The small mammals of the highly impacted North-eastern Atlantic Forest of Brazil, Pernambuco Endemism Center. Biota Neotropica 9: 31-35.

Brandão, D.; Bastos, R.; De Souza, M.; Vieira, C.; Bini, L.; Oliveira, L. & Diniz-Filho, J.A. 2004. Spatial patterns in species richness and priority areas for conservation of anurans in the Cerrado region, Central Brazil. Amphibia-Reptilia 25: 63-75.

Bruford, M.W.; Hanotte, O.; Brookfield, J.F.Y. & Burke, T. 1992. Single-locus and multilocus DNA fingerprint. In: Hoelzel AR (Ed.) Molecular Genetic Analysis of Populations: A Practical Approach. IRL Press, Oxford: pp. 225-270.

Carcerelli, L.C. & Caramaschi, U. 1993. Ocorrência do gênero Crossodactylus Duméril & Bibron, 1841 no nordeste brasileiro, com descrição de duas espécies novas (Amphibia, Anura, Leptodactylidae). Revista Brasileira de Biologia 52: 415-422.

Crawford, A.J.; Lips, K.R. & Bermingham, E. 2010. Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama. Proceedings of the National Academy of Sciences 107: 13777-13782.

Dubeux, M.J.M.; Gonçalves, U.; Nascimento, F.A.C. & Mott, T. 2020a. Anuran amphibians of a protected area in the northern Atlantic Forest with comments on topotypic and endangered populations. Herpetology Notes 13: 61-74.

Dubeux, M.J.M.; Nascimento, F.A.C.; Lima, L.R.; Magalhães, F.D.M.; Silva, I.R.S.; Gonçalves, U.; Almeida, J.P.F.; Correia, L.L.; Garda, A.A.; Mesquita, D.O.; Rossa-Feres, D.D.C. & Mott, T. 2020b. Morphological characterization and taxonomic key of tadpoles (Amphibia: Anura) from the northern region of the Atlantic Forest. Biota Neotropica 20: 1-24.

Dubeux, M.J.M.; Silva, T.; Mott, T. & Nascimento, F.A.C. 2020c. Redescription of the tadpole of Leptodactylus natalensis Lutz (Anura: Leptodactylidae), an inhabitant of the Brazilian Atlantic Forest. Zootaxa 4732: 346-350.

Dubeux, M.J.M.; Silva, G.R.S.; Nascimento, F.A.C.; Gonçalves, U. & Mott, T. 2019. Síntese histórica e avanços no conhecimento de girinos (Amphibia: Anura) no estado de Alagoas, nordeste do Brasil. Revista Nordestina de Zoologia 12: 18-52.

Fávero, E.R.; Veiga-Menoncello, A.C.; Rossa-Feres, D.C.; Strüssmann, C.; Giaretta, A.A.; Andrade, G.D.; Colombo, P. & Recco-Pimentel, S.M. 2011. Intrageneric karyotypic variation in Pseudopaludicola (Anura: Leiuperidae) and its taxonomic relatedness. Zoological Studies 50(6): 826-836.

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

Forti, L.R.; Silva, T.R.Á. & Toledo, L.F. 2017. The acoustic repertoire of the Atlantic Forest Rocket Frog and its consequences for taxonomy and conservation (Allobates, Aromobatidae). ZooKeys (692): 141-153.

Fouquet, A.; Gilles, A.; Vences, M.; Marty, C.; Blanc, M. & Gemmell, N.J. 2007. Underestimation of species richness in Neotropical frogs revealed by mtDNA analyses. PLoS one 2: e1109.

França, R.C.; Morais, M.; França, F.G.; Rödder, D. & Solé, M. 2020. Snakes of the Pernambuco Endemism Center, Brazil: diversity, natural history and conservation. ZooKeys 1002: 115-158.

Frost, D.R. 2022. Amphibian Species of the World: Online Reference. Version 6.0. http://research.amnh.org/herpetology/amphibia/index.php. Accessed on 07 February 2022.

Gehara, M.; Crawford, A.J.; Orrico, V.D.; Rodríguez, A.; Lötters, S.; Fouquet, A.; Barrientos, L.S.; Brusquetti, F.; de la Riva, I.; Ernst, R.; Gagliardi-Urrutia, G.; Glaw, F.; Guayasamin, J.M.; Hölting, M.; Jansen, M.; Kok, P.J.R.; Kwet, A.; Lingnau, R.; Lyra, M.; Moravec, J.; Pombal Jr., J.P.; Rojas-Runjaic, F.J.M.; Schulze, A.; Señaris, J.C.; Solé, M.; Rodrigues, M.T.; Twomey, E.; Haddad, C.F.B.; Vences, M. & Köhler, J. 2014. High levels of diversity uncovered in a widespread nominal taxon: continental phylogeography of the Neotropical tree frog Dendropsophus minutus. PloS one 9: e103958.

Gomes, M.D.R.; Alves, A.C.R. & Peixoto, O.L. 2014. O girino de Scinax nebulosus (Amphibia, Anura, Hylidae). Iheringia. Série Zoologia 104(2): 184-188.

Grosjean, S.; Ohler, A.; Chuaynkern, Y.; Cruaud, C. & Hassanin, A. 2015. Improving biodiversity assessment of anuran amphibians using DNA barcoding of tadpoles. Case studies from Southeast Asia. Comptes Rendus Biologies 338(5): 351-361.

Haas, A. 2003. Phylogeny of frogs as inferred from primarily larval characters (Amphibia: Anura). Cladistics 19: 23-89.

Hall, T. 2011. BioEdit: an important software for molecular biology. GERF Bull Biosci 2: 60-61.

Hebert, P.D.N. & Gregory, T.R. 2005. The promise of DNA barcoding for taxonomy. Systematic Biology 54: 852-859.

Hebert, P.D.N.; Cywinska, A.; Ball, S.L. & Waard, J.R. 2003. Biological identification through DNA barcodes. Proceedings of the Royal Society B 270: 313-321.

IUCN. 2022. The IUCN Red List of Threatened Species: Online Reference. Version 2020-1. http://www.iucnredlist.org. Accessed on 07 February 2022.

Jordani, M.X.; Mouquet, N.; Casatti, L.; Menin, M.; Rossa‐Feres, D.C. & Albert, C.H. 2019. Intraspecific and interspecific trait variability in tadpole meta‐communities from the Brazilian Atlantic rainforest. Ecology and Evolution 9(7): 4025-4037.

Katoh, K. & Standley, D.M. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30(4): 772- 780.

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120.

Koroiva, R.; Rodrigues, L.R.R. & Santana, D.J. 2020. DNA barcoding for identification of anuran species in the central region of South America. PeerJ 8: e10189.

Kumar, S.; Stecher, G.; Li, M.; Knyaz, C. & Tamura, K. 2018. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549.

Larson, P.M. 2005. Ontogeny, phylogeny, and morphology in anuran larvae: morphometric analysis of cranial development and evolution in Rana tadpoles (Anura: Ranidae). Journal of Morphology 264: 34-52.

Larsson, A. 2014. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30(22): 3276-3278.

Lima, L.R.; Dubeux, M.J.M.; Nascimento, F.A.C.; Bruschi, D.P. & Mott, T. 2019. Uncovering Neotropical treefrog diversity: integrative taxonomy reveal paraphyly in Boana atlantica (Amphibia, Anura, Hylidae). Amphibia-Reptilia 40: 511-521.

Lisboa, B.; Santos, W.F.S.; Torquato, S.; Guarnieri, M.C. & Mott, T. 2019. A new state record of the glassfrog Vitreorana baliomma (Anura: Centrolenidae), with notes on its reproductive biology. Herpetology Notes 12: 957-960.

Lourenço, L.B.; Targueta, C.P.; Baldo, D.; Nascimento, J.; Garcia, P.C.; Andrade, G.V.; Haddad, C.F.B. & Recco-Pimentel, S.M. 2015. Phylogeny of frogs from the genus Physalaemus (Anura, Leptodactylidae) inferred from mitochondrial and nuclear gene sequences. Molecular Phylogenetics and Evolution 92: 204-216.

Lyra, M.L.; Haddad, C.F.B. & Azeredo‐Espin, A.M.L. 2017. Meeting the challenge of DNA barcoding Neotropical amphibians: polymerase chain reaction optimization and new COI primers. Molecular Ecology Resources 17(5): 966-980.

Lyra, M.L.; Lourenço, A.C.C.; Pinheiro, P.D.; Pezzuti, T.L.; Baêta, D.; Barlow, A.; Hofreiter, M.; Pombal, J.P.; Haddad, C.F.B. & Faivovich, J. 2020. High-throughput DNA sequencing of museum specimens sheds light on the long-missing species of the Bokermannohyla claresignata group (Anura: Hylidae: Cophomantini). Zoological Journal of the Linnean Society 190(4): 1235-1255.

Magalhães, F.M.; Dantas, A.K.B.P.; Brito, M.R.M.; Medeiros, P.H.S.; Oliveira, A.F.; Pereira, T.C.S.O.; Queiroz, M.H.C.; Santana, D.J.; Silva, W.P. & Garda, A.A. 2013. Anurans from an Atlantic Forest-Caatinga ecotone in Rio Grande do Norte State, Brazil. Herpetology Notes 6: 1-10.

Malkmus, R. & Kosuch, J. 2000. Beschreibung einer neuen Ansonia-Larve (Ansonia guibei) von Borneo. Salamandra- Bonn 36(2): 121-124.

Moravec, J.; Lehr, E.; Cusi, J.C.; Córdova, J.H. & Gvoždík, V. 2014. A new species of the Rhinella margaritifera species group (Anura, Bufonidae) from the montane forest of the Selva Central, Peru. ZooKeys 2014: 35-56.

Oliveira, R.F.D.; Magalhães, F.M.; Teixeira, B.F.D.V.; Moura, G.J.B.D.; Porto, C.R.; Guimarães, F.P.B.B.; Giaretta, A.A. & Tinôco, M.S. 2021. A new species of the Dendropsophus decipiens Group (Anura: Hylidae) from Northeastern Brazil. Plos one (7): e0248112.

Palumbi, S.; Martin, A.; Romano, S.; McMilan, W.O.; Stice, L. & Grabowski, G. 2002. The Simple Fool’s Guide to PCR. University of Hawaii.

Perl, R.G.B.; Nagy, Z.T.; Sonet, G.; Glaw, F.; Wollenberg, K.C. & Vences, M. 2014. DNA barcoding Madagascar’s amphibian fauna. Amphibia-Reptilia 35: 197-206.

Provete, D.B.; Garey, M.V.; Silva, F. & Jordani, M.X. 2012. Knowledge gaps and bibliographical revision about descriptions of free-swimming anuran larvae from Brazil. North-Western Journal of Zoology 8(2): 283-286.

Ranvestel, A.W.; Lips, K.R.; Pringle, C.M.; Whiles, M.R. & Bixby, R.J. 2004. Neotropical tadpoles influence stream benthos: evidence for the ecological consequences of decline in amphibian populations. Freshwater Biology 49: 274-285.

Ribeiro, M.C.; Metzger, J.P.; Martensen, A.C.; Ponzoni, F.J. & Hirota, M.M. 2009. The Brazilian Atlantic Forest: How much is left, and how is the remaining forest distributed? Implications for conservation. Biological Conservation 142: 1141-1153.

Roberto, I.J.; Araujo-Vieira, K.; Carvalho-e-Silva, S.P. & Ávila, R.W. 2017. A new species of Sphaenorhynchus (Anura: Hylidae) from northeastern Brazil. Herpetologica 73: 148- 161.

Rossa-Feres, D.C. & Nomura, F. 2006. Characterization and taxonomic key for tadpoles (Amphibia: Anura) from the northwestern region of São Paulo State, Brazil. Biota Neotropica 6: 1-26.

Rossa-Feres, D.D.C.; Jim, J. & Fonseca, M.G. 2004. Diets of tadpoles from a temporary pond in southeastern Brazil (Amphibia, Anura). Revista Brasileira de Zoologia 21: 745-754.

Rossa-Feres, D.D.C.; Venesky, M.D.; Nomura, F.; Eterovick, P.C.; Vera Candioti, M.F.; Menin, M.; Juncá, F.A.; Schiesari, L.C.; Haddad, C.F.B.; Garey, M.V.; Anjos, L.A. & Wassersug, R. 2015. Taking tadpole biology into the 21st century: a consensus paper from the First Tadpoles International Workshop. Herpetologia Brasileira 4(2): 48-59.

Sambrook, J.; Fritsch, E.F. & Maniatis, T. 1989. Molecular cloning: a laboratory manual (No. Ed. 2). Cold Spring Harbor Laboratory Press.

Schulze, A; Jansen, M & Köhler, G. 2015. Tadpole diversity of Bolivia's lowland anuran communities: molecular identification, morphological characterisation, and ecological assignment. Zootaxa 4016(1): 1-111.

Segalla, M.V.; Berneck, B.; Canedo, C.; Caramaschi, U.; Cruz, C.A.G.; Garcia, P.C.A.; Grant, T.; Haddad, C.F.B.; Lourenço, A.C.C.; Mângia, S.; Mott, T.; Nascimento, L.B.; Toledo, L.F.; Werneck, F.P. & Langone, J.A. 2021. List of Brazilian Amphibians. Herpetologia Brasileira 10(1): 121-216.

Silva, F.R. 2010. Evaluation of survey methods for sampling anuran species richness in the neotropics. South American Journal of Herpetology 5: 212-220

Silva, L.A.; Magalhaes, F.M.; Thomassen, H.; Leite, F.S.; Garda, A.A.; Brandao, R.A.; Haddad, C.F.B.; Giaretta A.A. & Carvalho, T.R. 2020. Unraveling the species diversity and relationships in the Leptodactylus mystaceus complex (Anura: Leptodactylidae), with the description of three new Brazilian species. Zootaxa 4779: 151-189.

Thomas, M.; Raharivololoniaina, L.; Glaw, F.; Vences, M. & Vieite, D.R. 2005. Montane tadpoles in Madagascar: molecular identification and description of the larval stages of Mantidactylus elegans, Mantidactylus madecassus, and Boophis laurenti from the Andringitra Massif. Copeia 2005: 174-183.

Vacher, J.P.; Chave, J.; Ficetola, F.G.; Sommeria‐Klein, G.; Tao, S.; Thébaud, C.; Blanc, M.; Camacho, A.; Cassimiro, J.; Colston, T.J.; Dewynter, M.; Ernst, R.; Gaucher, P.; Gomes, J.O.; Jairam, R.; Kok, P.J.R.; Lima, J.D.; Martinez, Q.; Marty, C.; Noonan, B.P.; Nunes, P.M.S.; Ouboter, P.; Recoder, R.; Rodrigues, M.T.; Snyder, A.; Marques-Souza, S. & Fouquet, A. 2020. Large‐scale DNA‐based survey of frogs in Amazonia suggests a vast underestimation of species richness and endemism. Journal of Biogeography 47: 1781-1791.

Vancine, M.H.; Duarte, K.D.S.; Souza, Y.S.; Giovanelli, J.G.R.; Martins‐Sobrinho, P.M.; López, A.; Bovo, R.P.; Maffei, F.; Lion, M.B.; Júnior, J.W.R.; Brassaloti, R.; Costa, C.O.R.; Sawakuchi, H.O.; Forti, L.R.; Cacciali, P.; Bertoluci, J.; Haddad, C.F.B. & Ribeiro, M.C. 2018. ATLANTIC AMPHIBIANS: a data set of amphibian communities from the Atlantic Forests of South America. Ecology 99: 1692-1692.

Vences, M.; Thomas, M.; Bonett, R.M. & Vieites, D.R. 2005a. Deciphering amphibian diversity through DNA barcoding: chances and challenges. Philosophical Transactions of the Royal Society B: Biological Sciences 360: 1859-1868.

Vences, M.; Thomas, M.; Van der Meijden, A.; Chiari, Y. & Vieites, D.R. 2005b. Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology 2: 1-12.

Verdade, V.K. & Rodrigues, M.T. 2007. Taxonomic review of Allobates (Anura, Aromobatidae) from the Atlantic Forest, Brazil. Journal of Herpetology 41(4): 566-580.

Vieites, D.R.; Wollenberg, K.C.; Andreone, F.; Köhler, J.; Glaw, F. & Vences, M. 2009. Vast underestimation of Madagascar's biodiversity evidenced by an integrative amphibian inventory. Proceedings of the National Academy of Sciences USA 106: 8267-8272.




Cómo citar

Dubeux, M. J. M. ., Cavalcanti do Nascimento, F. A. ., Correia, L. L., & Mott, T. . (2022). DNA barcoding in Neotropical tadpoles: evaluation of 16S rRNA gene for the identification of anuran larvae from northeastern Brazil. Cuadernos De Herpetología, 36(2). Recuperado a partir de http://ojs.aha.org.ar/index.php/CdH/article/view/31