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Published: 2017-09-18

Phylogenetic and divergence time analysis of the Chelonoidis chilensis complex (Testudines: Testudinidae)

Laboratorio de Ecología Molecular, Centro Austral de Investigaciones Científicas (CADIC-CONICET). Bernardo A. Houssay 200 (9410) Ushuaia, Tierra del Fuego, Argentina. Instituto de Ciencias Polares, Ambiente y Recursos Naturales, ICPA-UNTDF, Avenida Los Ñires 2382 (9410), Ushuaia, Tierra del Fuego, Argentina.
Instituto de Diversidad y Evolución Austral (IDEAus-CONICET). Blvd. Alte. Brown 2915, U9120ACF Puerto Madryn, Chubut, Argentina.
Laboratorio de Ecología Molecular, Centro Austral de Investigaciones Científicas (CADIC-CONICET). Bernardo A. Houssay 200 (9410) Ushuaia, Tierra del Fuego, Argentina. Instituto de Ciencias Polares, Ambiente y Recursos Naturales, ICPA-UNTDF, Avenida Los Ñires 2382 (9410), Ushuaia, Tierra del Fuego, Argentina.
Laboratorio de Citogenética y Mutagénesis, Instituto Multidisciplinario de Biología Celular (IMBICE, CICPBA-UNLP-CONICET La Plata) y Facultad de Ciencias Naturales y Museo, UNLP, Avenida 122 y 60, 1900 La Plata, Buenos Aires, Argentina.
Laboratorio de Genética Molecular Poblacional, Instituto Multidisciplinario de Biología Celular (IMBICE, CICPBA-UNLP-CONICET La Plata) y Facultad de Ciencias Naturales y Museo, UNLP, Avenida 122 y 60, 1900 La Plata, Buenos Aires, Argentina.
Reptilia tortoises Testudines mitochondrial DNA haplotypes phylogeny molecular clock


We present a phylogenetic and divergence time analysis of the Chelonoidis chilensis complex (the southernmost tortoises of South America) within crown Testudinidae. We compiled a dataset of 1118 bp cytochrome b (cyt b) sequences derived from 111 individuals sampled across the known geographic range of the species, and performed a phylogenetic analysis employing Maximum Parsimony, Maximum Likelihood and Bayesian Inference methods. The resulting trees showed similar topologies and support values. The C. chilensis complex was always recovered as a monophyletic group composed by two major clades (i.e. haplogroups). The biogeographic distribution of one of these clades overlaps with the Dry Chaco eco–region, while the biogeographic distribution of the other overlaps with the Monte eco–region. In order to date the origin and diversification time of these two clades, we employed a previously published two-step molecular clock method. In the first step we dated the time of origin of C. chilensis as a clade within the Testudinidae family using new and previously published sequences, extinct testudinid taxa for age calibration and the Fossilized Birth-Death (FBD) model. In the second step we dated the divergence between the haplogroups of C. chilensis based on the time of origin estimated in the first step and a coalescent evolution model. Our results suggest that divergence between Dry Chaco and Monte tortoises may have occurred about 2.47 million of years ago. We interpret these results in the light of the environmental and geological changes that occurred during the late Pliocene to Middle Pleistocene of South America.



  1. Albino, A.M. (1996) Snakes from the Miocene of Patagonia (Argentina) Part II. The Colubroidea. Neues Jahrbuch für Geologie und Paläontologie—Abhandlungen, 200, 353–360.

    Albino, A.M. (2011) Evolution of squamata reptiles in Patagonia based on the fossil record. Biological Journal of the Linnean Society, 103, 441–457.

    Albino, A.M., Brizuela, S. & Montalvo, C.I. (2006) New Tupinambis remains from the Late Miocene of Argentina and a review of the South American Miocene Teiids. Journal of Herpetology, 40, 206–213.

    Andrews, C.W. & Beadnell, H.J.L. (1903) A preliminary notice of a land tortoise from the upper Eocene of the Fayum, Egypt. Survey Department Ed. National Printing Department, Cairo, 10 pp.

    Andrews, C.W. (1904) I.—Note on the Gigantic Land Tortoise (Testudo ammon, Andrews), from the Upper Eocene of Egypt. Geological Magazine (Decade V), 1 (11), 527–530.

    Attum, O., Baha el Din, S., Carranza, S., Earley, R., Arnold, E.N. & Kingsbury, B. (2007) An evaluation of the taxonomic validity of Testudo werneri. Amphibia-Reptilia, 28 (3), 393–401.

    Auffenberg, W. (1971) A new fossil tortoise, with remarks on the origin of South American Testudinines. Copeia, 1971 (1), 106–117.--

    Barbarán, F.R. (2003) Factibilidad de Caza de Subsistencia, Comercial y Deportiva en el Chaco Semiárido de la Provincia de Salta, Argentina. Available from: (accessed 15 November 2016)

    Bouckaert, R.R., Heled, J., Kuehnert, D., Vaughan, T.G., Wu, C.-H., Xie, D., Suchard, M.A., Rambaut, A. & Drummond, A.J. (2014) BEAST 2: A software platform for Bayesian evolutionary analysis. PLoS Computational Biology, 10 (4), e1003537.

    Brizuela, S. (2010) Los lagartos continentales fósiles de la Argentina (excepto Iguania). Tesis doctoral N°1057, Universidad Nacional de La Plata, La Plata, 408 pp.

    Burkart, R., Bárbaro, N.O., Sánchez, R.O. & Gómez, D.A. (1999) Ecorregiones de la Argentina. Administración de Parques Nacionales y Secretaría de Recursos Naturales y Desarrollo Sustentable. Argentina: Buenos Aires. Available from: (accessed 15 November 2016)

    Buskirk, J.R. (1993) Distribution, status and biology of the tortoise, Geochelone chilensis, in Río Negro Provence, Argentina. Studies on Neotropical Fauna Environments, 28 (4), 233–249.

    Cabrera, M.R. (1998) Las tortugas continentales de Sudamérica austral. BR copias, Córdoba, 108 pp.

    Caccone, A., Gibbs, J.P., Ketmaier, V., Suatoni, E. & Powell, J.R. (1999) Origin and evolutionary relationships of giant Galápagos tortoises. Proceedings of the National Academy of Sciences, 96 (23), 13223–13228.

    Cei, J.M. (1986) Reptiles del centro, centro-oeste y sur de la Argentina. Herpetofauna de las zonas áridas y semiáridas. Museo Regionale di Scienze Naturali Torino, 4, 1–527.

    Cei, J.M. (1993) Reptiles del Noroeste, Nordeste y Este de la Argentina. Herpetofauna de las selvas subtropicales, Puna y Pampas. Museo Regionale di Scienze Naturali Torino, Monografía XIV, 1–949.

    Corsini, J.A., Böhme, M. & Joyce, W.G. (2014) Reappraisal of Testudo antiqua (Testudines, Testudinidae) from the Miocene of Hohenhöwen, Germany. Journal of Paleontology, 88 (5), 948–966.

    De la Fuente, M.S. (1994) Descripción de nuevos especímenes y relaciones filogenéticas de Chelonoidis gringorum (Chelonii: Testudinidae) del Mioceno temprano de Patagonia central. Available from: (accessed 21 June 2017)

    Drummond, A.J. & Rambaut, A. (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7 (1), 214.

    Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29 (8), 1969–1973.

    Duchêne, S., Lanfear, R. & Ho, S.Y. (2014) The impact of calibration and clock-model choice on molecular estimates of divergence times. Molecular Phylogenetics and Evolution, 78, 277–289.

    Endicott, P., Ho, S.Y.W., Metspalu, M. & Stringer, C. (2009) Evaluating the mitochondrial timescale of human evolution. Trends in Ecology & Evolution, 24 (9), 515–521.

    Fernández, M.S. (1988) Las Testudinidae (Reptilia: Chelonii) argentinas: Osteología, sistemática y distribución geográfica. Tesis doctoral Nº 505, Universidad Nacional de La Plata, La Plata, 300 pp.

    Freiberg, M.A. (1973) Dos nuevas tortugas terrestres de Argentina. Boletín de la Sociedad de Biología de Concepción, 46, 81–93.

    Freiberg, M.A. (1979) El mundo de las tortugas. 2º Ed. Ed. Albatros, Buenos Aires, 154 pp.

    Fritz, U., Ayaz, D., Buschbom, J., Kami, H.G., Mazanaeva, L.F., Aloufi, A.A., Auer, M., Rifai, L., Šilić, T. & Hundsdörfer, A.K. (2008) Go east: phylogeographies of Mauremys caspica and M. Rivulata - Discordance of morphology, mitochondrial and nuclear genomic markers and rare hybridization. Journal of Evolutionary Biology, 21 (2), 527–540.

    Fritz, U., Auer, M., Bertolero, A., Cheylan, M., Fattizzo, T., Hundsdörfer, A.K., Sampayo, M.M., Pretus, J.L., široký, P. & Wink, M. (2006) A rangewide phylogeography of Hermann´s tortoise, Testudo hermanni (Reptilia: Testudines: Testudinidae): implications for taxonomy. Zoologica Scripta, 35 (5), 531–543.

    Fritz, U., Hundsdörfer, A.K., Široký, P., Auer, M., Kami, H., Lehmann, J., Mazanaeva, L.F., Türkozan, O. & Wink, M. (2007) Phenotypic plasticity leads to incongruence between morphology-based taxonomy and genetic differentiation in western Palaearctic tortoises (Testudo graeca complex; Testudines, Testudinidae) Amphibia- Reptilia, 28 (1), 97–121.

    Fritz, U., Široký, P., Kami, H. & Wink, M. (2005) Environmentally caused dwarfism or a valid species—Is Testudo weissingeri Bour, 1996 a distinct evolutionary lineage? New evidence from mitochondrial and nuclear genomic markers. Molecular Phylogenetics and Evoluttion, 37 (2), 389–401.

    Fritz, U., Alcalde, L., Vargas-Ramírez, M., Goode, E., Fabius-Turoblin, D.U. & Praschag, P. (2012) Northern genetic richness and southern purity, but just one species in the Chelonoidis chilensis complex. Zoologica Scripta, 41 (3), 220–232.

    Fritz, U. & Bininda-Emonds, O.R. (2007) When genes meet nomenclature: tortoise phylogeny and the shifting generic concepts of Testudo and Geochelone. Zoology, 110 (4), 298–307.

    Fritz, U., Harris, D.J., Fahd, S., Rouag, R., Gracía Martínez, E., Giménez Casalduero, A., Široký, P., Kalboussi, M., Jdeidi, T.B. & Hundsdörfer, A.K. (2009) Mitochondrial phylogeography of Testudo graeca in the Western Mediterranean: Old complex divergence in North Africa and recent arrival in Europe. Amphibia-Reptilia, 30 (1), 63–80.

    Fusco, G. & Minelli, A. (2010) Phenotypic plasticity in development and evolution: Facts and concepts. Philosophical Transactions of the Royal Society B, 365 (1540), 547–556.

    Gavryushkina, A., Welch, D., Stadler, T. & Drummond, A.J. (2014) Bayesian inference of sampled ancestor trees for epidemiology and fossil calibration. PLoS Computational Biology, 10 (12), e1003919.

    Gemmell, N.J. & Akiyama, S. (1996) An efficient method for extraction of DNA from vertebrate tissues. Technical Tips, 12 (9), 338–339.

    Gernhard, T. (2008) The conditioned reconstructed process. Journal of Theoretical Biology, 253 (4), 769–778.

    Goloboff, P.A., Mattoni, C.I. & Quinteros, A.S. (2008a) TNT, a free program for phylogenetic analysis. Cladistics, 24 (5), 774–786.

    Goloboff, P., Farris, J. & Nixon, K., (2008b) TNT: tree search using new technology. Vers. 1.1 (Willy Hennig Society Edition) Program and documentation. Available from: (accessed 11 August 2017)

    González-Acuña, D., Beldoménico, P.M., Venzal, J.M., Fabry, M., Keirans, J.E. & Guglielmone, A.A. (2005) Reptile trade and the risk of exotic tick introductions into southern South American countries. Experimental and Applied Acarology, 35 (4), 335–339.

    González-Porter, G.P., Hailer, F., Flores-Villela, O., García-Anleu, R. & Maldonado Higgins, J.E. (2011) Patterns of genetic diversity in the critically endangered Central American river turtle: human influence since the Mayan age?. Conservation Genetics, 12 (5), 1229–1242.

    Guglielmone, A.A., Luciani, C.A. & Magnold, A.J. (2001) Aspects of the ecology of Amblyomma argentinae Neumann, 1904 [=Amblyomma testudinis (Connil 1877)] (Acari: Ixodidae) Systematic and Applied Acarology Special Publications, 8, 1–12.

    Hay, O.P. (1904) On some fossil turtles belonging to the Marsh collection in Yale University Museum. American Journal of Science, 106, 261–276.

    Heath, T.A., Huelsenbeck, J.P. & Stadler, T. (2014) The fossilized birth-death process for coherent calibration of divergence-time estimates. Proceedings of the National Academy of Sciences, 111 (29), 2957–2966.

    Holroyd, P.A. & Parham, J.F. (2003) The antiquity of African tortoises. Journal of

    Vertebrate Paleontology, 23 (3), 688–690.

    Hutchison, J. H. (1996) Testudines, In: Prothero, D.R. & Emry, R.J. (Eds.), The Terrestrial Eocene-Oligocene Transition in North America. Cambridge University Press, Cambridge, pp. 337–353.

    Joshi, B.D. (2012) Sequence Divergence and Phylogenetic Status of Four Species of Testudinidae Family. Available from: (accessed 15 November 2016)

    Joyce, W.G., Parham, J.F., Lyson, T.R., Warnock, R.C. & Donoghue, P.C. (2013) A divergence dating analysis of turtles using fossil calibrations: an example of best practices. Journal of Paleontology, 87 (4), 612–634.

    Kehlmaier, C., Barlow, A., Hastings, A.K., Vamberger, M., Paijmans, J.L., Steadman, D., Albury, N., Franz, R., Hofreiter, M. & Fritz, U. (2017) Tropical ancient DNA reveals relationships of the extinct Bahamian giant tortoise Chelonoidis alburyorum. Proceedings of the Royal Society B, 284 (1846), 20162235.

    Kim, K.Y., Nam, Y.K. & Bang, I.C. (2012) Phylogeny and divergence time estimation of Coreoleuciscus splendidus populations (Teleostei: Cypriniformes) endemic to Korea based on complete mitogenome sequences. Genes & Genomics, 34 (2), 149–156.

    Kingman, J.F.C. (1982) The coalescent. Stochastic Processes and their Applications, 13 (3), 235–248.

    Lanfear, R., Calcott, B., Ho, S.Y.W. & Guindon, S. (2012) PartitionFinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses. Molecular Biology and Evolution, 29 (6), 1695–1701.

    Lapparent de Broin, F. (2001) The European turtle fauna from the Triassic to the

    Present. Dumerilia, 4, 155–217.

    Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. & Higgins, D.G. (2007) Clustal W and Clustal X version 2.0. Bioinformatics, 23 (21), 2947–2948.

    Le, M., Raxworthy, C.J., McCord, W.P. & Mertz, L. (2006) A molecular phylogeny of tortoises (Testudines: Testudinidae) based on mitochondrial and nuclear genes. Molecular Phylogenetics and Evolution, 40 (2), 517–531.

    Librado, P. & Rozas, J. (2009) DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25 (11), 1451–1452.

    Lourenço, J.M., Claude, J., Galtier, N. & Chiari, Y. (2012) Dating cryptodiran nodes: origin and diversification of the turtle superfamily Testudinoidea. Molecular Phylogenetics and Evolution, 62 (1), 496–507.

    Martínez, P.A., Boeris, J.M., Sánchez, J., Bolzán, A.D. & Ledesma, M.A. (2009) Karyotypic characterization of Trachemys dorbigni (Testudines: Emydidae) and Chelonoidis donosobarrosi (Testudines: Testudinidae), two species of Cryptodiran turtles from Argentina. Genetica, 137, 277–283.

    Matschiner, M., Musilová, Z., Barth, J.M., Starostová, Z., Salzburger, W., Steel, M. & Bouckaert, R. (2016) Bayesian node dating based on probabilities of fossil sampling supports trans-Atlantic dispersal of cichlid fishes. bioRxiv, 2016, 038455.

    McCartney, M.A. & Barreto, F.S. (2010) A mitochondrial DNA analysis of the species status of the endemic Waccamaw darter, Etheostoma perlongum. Copeia, 2010 (1), 103–113.

    Meylan, P. & Auffenberg, W. (1986) New land tortoises (Testudines: Testudinidae) from the Miocene of Africa. Zoological Journal of the Linnean Society, 86 (3), 279–307.

    Montalvo, C.I. & Verzi, D.H. (2004) El registro de roedores Octodontoidea (Caviomorpha) de la formación Cerro Azul (Mioceno Tardío, La Pampa, Argentina): sistemática, biocronología, evolución y paleoclimas. Ameghiniana, 41 (Suplemento), 56R.

    Morrone, J.J. (2006) Biogeographic areas and transition zones of Latin America and the Caribbean Islands based on panbiogeographic and cladistic analyses of the entomofauna. Annual Review of Entomology, 51, 467–494.

    Noss, A.J., Montaño, R.R., Soria, F., Deem, S.L., Fiorello, C.V. & Fitzgerald L.A. (2013) Chelonoidis carbonarius (Testudines: Testudinidae) Activity Patterns and Burrow use in the Bolivian Chaco. South American Journal of Herpetology, 8 (1), 19–28.

    Palumbi, S.R., Martin, A.P., Romano, S., McMillan, W.O., Stice, L. & Grabowski, G. (1991) The simple fools guide to PCR, version 2.0. Available from: (accessed 15 November 2016)

    Pascual, R. & Odreman Rivas, O. (1973) Las unidades estratigráficas del Terciario portadoras de mamíferos. Su distribución y sus relaciones con los acontecimientos diastróficos. Actas del Quinto Congreso Geológico Argentino, 3, 293–338

    Pascual, R., Ortiz Jaureguizar, E. & Prado, J.L. (1996) Land mammals: paradigm of Cenozoic South American geobiotic evolution. In: Arratia, G. (Ed.), Müncher Geowissenchaftliche Abhandlungen (A), 39, 265–319.

    Posada, D. (2008) jModelTest: phylogenetic model averaging. Molecular Biology and Evolution, 25 (7), 1253–1256.

    Prado, W.S., Waller, T., Albareda, D.A., Cabrera, M.R., Etchepare, E., Giraudo, A., González Carman, V., Prosdocimi, L. & Richard, E. (2012) Categorización del estado de conservación de las Tortugas de la República Argentina. Available from: (accessed 15 November 2016)

    Quattrocchio, M., Deschamps, C., Martínez, S., Grill, S. & Zavala, C. (1988) Caracterización paleontológica y paleoambiental de sedimentos cuaternarios, arroyo Napostá Grande, provincia de Buenos Aires. Actas de las 2° Jornadas Geológicas Bonaerenses, Bahía Blanca, 1988, 37–46.

    Rambaut, A. & Drummond, A.J. (2009) TRACER: MCMC Trace Analysis Tool Version 1. 5.0. University of Oxford, Oxford.

    Red List, IUCN (2015) (accessed 11 August 2017)

    Reynoso, V.H. & Montellano-Ballesteros, M. (2004) A new giant turtle of the genus Gopherus (Chelonia: Testudinidae) from the Pleistocene of Tamaulipas, México, and a review of the phylogeny and biogeography of gopher tortoises. Journal of Vertebrate Paleontology, 24 (4), 822–837.

    Richard, E. (1999) Tortugas de las regiones áridas de Argentina. Nº10. L.O.L.A., Buenos Aires, 200 pp., 8 pls.

    Rohland, N. & Hofreiter, M. (2007a) Ancient DNA extraction from bones and teeth. Nature Protocols, 2 (7), 1756–1762.

    Rohland, N. & Hofreiter, M. (2007b) Comparison and optimization of ancient DNA extraction. Available from: (accessed 15 November 2016)

    Sánchez, J., Alcalde, L. & Bolzán, A.D. (2015) First evidence of chromosomal variation within Chelonoidis chilensis (Testudines: Testudinidae). Available from: (accessed 15 November 2016)

    Schulte, J.A., Macey, J.R., Espinoza, R.E. & Larson, A. (2000) Phylogenetic relationships in the iguanid lizard genus Liolaemus: multiple origins of viviparous reproduction and evidence for recurring Andean vicariance and dispersal. Biological Journal of the Linnean Society, 69, 75–102. 1999.034

    Scillato-Yané, G.J., Carlini, A.A. & Vizcaíno, S.F. (1993) Los Xenarthra (Mammalia Edentata) del Mioceno medio de Argentina. Ameghiniana, 30 (3), 352.

    Silvestro, D. & Michalak, I. (2012) raxmlGUI: a graphical front-end for RAxML. Organisms Diversity and Evolution, 12 (4), 335–337.

    Simpson, G.G. (1942) A Miocene tortoise from Patagonia. Available from: (accessed 15 November 2016)

    Široký, P. & Fritz, U. (2007) Is Testudo werneri a distinct species? Biologia, 62 (2), 228–231.

    Spinks, P.Q., Shaffer, H.B., Iverson, J.B. & McCord, W.P. (2004) Phylogenetic hypotheses for the turtle family Geoemydidae. Molecular Phylogenetics and Evolution, 32 (1), 164–182.

    Stamatakis, A. (2006) RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22 (21), 2688–2690.

    Turtle Taxonomy Working Group (2014) Turtles of the World. Annotated Checklist of Taxonomy, Synonymy, Distribution with Maps, and Conservation Status. Conservation biology of freshwater turtles and tortoises: a compilation project of the IUCN/SSC Tortoise and Freshwater Turtle Specialist Group. Chelonian Research Monographs, 5, 000–329.

    Vaidya, G., Lohman, D.J. & Meier, R. (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene data sets with character set and codon information. Cladistics, 27 (2), 171–180.

    Vargas-Ramírez, M., Maran, J. & Fritz, U. (2010) Red- and yellow-footed tortoises, Chelonoidis carbonarius and C. denticulatus (Reptilia: Testudines: Testudinidae), in South American savannahs and forests: do their phylogeographies reflect distinct habitats? Organisms Diversity & Evolution, 10 (2), 161–172.

    Vivo M. & Carmignotto, A.P. (2004) Holocene vegetation change and the mammal faunas of South America and Africa. Journal of Biogeography, 31, 943–957.

    Ware, J.L., Simaika, M.P. & Samways, M.J. (2009) Biogeography and divergence time estimation of the relict Cape dragonfly genus Syncordulia: global significance and implications for conservation. Zootaxa, 2216 (2216), 22–36.

    Wermuth, H. & Mertens, R. (1977) Testudines, Crocodylia, Rhynchocephalia. Das Tierreich, Liefer 100, 1–173. [Ed. De Gruyter]

    Zacarías, G.G., de la Fuente, M.S., Fernández, M.S. & Zurita, A.E. (2013) Nueva especie de tortuga terrestre gigante del género Chelonoidis Fitzinger, 1835 (Cryptodira: Testudinidae), del miembro inferior de la formación Toropí/Yupoí (Pleistoceno Tardío/Lujanense), Bella Vista, Corrientes, Argentina. Ameghiniana, 50 (3), 298–318.

    Zachos, J., Pagani, M., Sloan, L., Thomas, E. & Billups, K. (2001) Trends, rhythms, and aberrations in global climate 65 Ma to Present. Science, 292, 686–693.