Abstract
Wild populations of many species are declining as a result of habitat destruction and climate change but also through the over-collection for wild meat and the pet trade. With a long history of trade around the Mediterranean, populations of the spur-thighed tortoise (Testudo graeca graeca) have become highly disturbed. In this study we utilise a molecular approach to investigate the diversity, population admixture and structure of T. g. graeca populations in northern Africa and southern Spain, as well as to obtain an insight into the origin of newly established populations in the south of Europe. We infer this from the sequencing of two partial regions of the mitochondria (12s rRNA + cyt b) and genotyping at 16 microsatellite markers in 448 tortoises. Our results are consistent with the hypothesis that Spanish populations were founded from North Africa, the consequence of multiple introductions or exchanges in genetic material as a result of trans-oceanic dispersal. Despite the trade of individuals between both sides of the Gibraltar Strait, our analysis of population structure showed clear differences between both the African and European populations, suggesting an incipient evolutionary lineage in southeast of Spain. As such, these populations possess unique genetic identities and should be treated as different management units. Surprisingly, the genetic data identified a great deal of diversity contained within pet (captive) stock and also allowed us to infer hybrids among individuals with another species of terrestrial tortoise from northern Spain (T. hermanni hermanni). Additionally, our results provide insight into the local movement and trade of individuals that has occurred around the Mediterranean basin (between northern Africa and southern Spain) and as such provides guidance for the effective management of T. g. graeca captive stock and the illegal trafficking.
Similar content being viewed by others
References
Alvarez Y, Mateo JA, Andreu AC, Diaz-Paniagua C, Diez A, Bautista JM (2000) Mitochondrial DNA haplotyping of Testudo graeca on both continental sides of the straits of Gibraltar. J Hered 91:39–41
Andreu AC, López-Jurado LF (1998) Los reptiles ibéricos: género Testudo. In: Ramos MA et al (eds) Fauna Ibérica. Reptiles. Museo Nacional de Ciencias Naturales, CSIC, Madrid
Asian Turtle Trade (2000) Proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in Asia. In: van Dijk PP, Stuart BL, Rhodin AGJ (eds) Chelonian Research Monographs No. 2
Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press, Cambridge
Avise JC, Bowen BW, Lamb T, Meylan AB, Bermingham E (1992) Mitochondrial DNA evolution at a turtles’s pace: evidence for low genetic variability and reduced microevolutionary rate in the Testudines. Mol Biol Evol 9:457–473
Bailón S (2001) Revisión de la asignación a Testudo cf. graeca del yacimiento del Pleistoceno superior de Cueva Horá (Darro, España). Rev Esp Herpetol 15:61–65
Bandelt HJ (1999) Combination of data in phylogenetic analysis. Plant Syst Evol 9:355–361
Benson RH, Rakic-El Beid K, Bonaduce G (1991) An important current reversal, influx in the Rifian corridor, Morroco, at the Tortonian-Messinian boundary: the end of the Tethys ocean. Paleoceanography 6:164–172
Bowcock AM, Ruiz-Linares A, Tomfohrde J, Minch E, Kidd JR, Cavalli-Sforza LL (1994) High resolution of human evolutionary trees with polymorphic microsatellite. Nature 368:455–457
Busack SD (1986) Biogeographic analysis of the herpetofauna separated by the formation of the Strait of Gibraltar. Natl Geogr Res 2:339–348
Chkhikvadze VM (1983) Iskopaemye cherepakhi Kavkaza i Severnogo Prichernomorya. Metsniereba, Tbilisi
Chkhikvadze VM (1989) Neogenovye cherepakhi SSSR. Metsniereba, Tbilisi
Ciofi C, Milinkovitch MC, Gibbs JP, Caccoce A, Powell JR (2002) Microsatellite analysis of genetic divergence among populations of Giant Galápagos Tortoises. Mol Ecol 11:2265–2283
Connor M (1989) Molecular biology and the turtle: the desert tortoise and its relatives. Tortuga Gaz 25:10–11
Cornuet JM, Luikart G (1996) Description and power analysis of two test for detecting recent population bottleneck from allele frequency data. Genetics 1444:2001–2014
Crandall KA, Bininda-Edmonds O, Mace G, Wayne RK (2000) Considering evolutionary processes in conservation biology: an alternative to “evolutionary significants units”. Trends Ecol Evol 15:290–295
Danilov IG (2005) Die fossilen Schildkröten Europas. In: Fritz U (ed) Handbuch der Reptilien und Amphibien Europas. Band 3/IIIB: Schildkröten (Testudines) II. Aula-Verlag, Wiebelsheim
Dupre A (2002) Situation de Testudo graeca au Maghreb. Chelonii 3:300–301
Edwards T, Goldberg CS, Kaplan ME, Schwalbe CR, Swann DE (2003) PCR primers for microsatellite loci in the desert tortoise (Gopherus agassizii, Testudinidae). Mol Ecol Notes 3:589–591
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software structure: a simulation study. Mol Ecol 14:2611–2620
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Ferrández M (2003) Illegal trade of Greek tortoise (Testudo graeca) coming from Algeria admitted in the Wildlife Preservation Centre Santa Faz Alicante (Spain). Presented at the second international congress on chelonian conservation, Dakar, Senegal, 18–22 June 2003
Forlani A, Crestanello B, Mantovani S, Livoreil B, Zane L, Bertorelle G, Congiu L (2005) Identification and characterization of microsatellite markers in Hermann’s tortoise (Testudo hermanni, Testudinidae). Mol Ecol Notes 5:228–230
Fritz U, Hundsdörfer AK, Široký P, Auer M, Kami H, Lehmann J, Mazanaeva LF, Türkozan O, Wink M (2007) Phenotipic plasticity leads to incongruence between morphology-based taxonomy and the genenetic differentiation in western Paleartic tortoises (Testudo graeca complex; Testudines, Testudinidae). Amphib-Reptil 28:97–121
Fritz U, Harris DJ, Fahd S, Rouag R, Graciá E, Giménez A, Siroky P, Kalboussi M, Jdeidi TB, Hundsdörfer AK (2009) Mitochondrial phylogeography of Testudo graeca in the Western Mediterranean: old complex divergence in North Africa and recent arrival in Europe. Amphib-Reptil 30:63–80
González J (1993) Rèunion Island. Still a land tortoises. Chelonian Conserv Biol 1:51–52
Goudet J (1995) FSTAT version 1.2: a computer program to calculate F-statistics. J Hered 86:485–486
Harpending HC (1994) Signature of ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Hum Biol 66:591–600
Huelsenbeck JP, Ronquist F (2001) MRBAYES: bayesian inference of phylogenetic trees. Bioinformatics 17:754–755
Kabisch K (2001) Bastardisierung von Testudo horsfieldii (GRAY, 1844) und Testudo graeca ibera (PALLAS, 1814). Sauria 23:7–11
King TL, Julian SE (2004) Conservation of microsatellite DNA flanking sequence across 13 Emyidid genera assayed with novel bog turtle (Glytemys muhlenbergii) loci. Conserv Genet 5:719–725
Kirsche W (1984) Bastardierung von Testudo horsfieldii (GRAY, 1844) und Testudo h. hermanni (GMELIN). Amphib-Reptil 5:311–322
Klemens MW, Thorbjarnarson JB (1995) Reptiles as a food resource. Biodivers Conserv 4:218–298
Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX (1989) Dynamics of mitochondrial DNA evolution in animals: Amplification and sequencing with conserved primers. Proc Natl Acad Sci USA 86:6196–6200
Kumar S, Tamura K, Jakobsen IB, Nei M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics 5:150–163
Lambert MRK (1979) Trade and the mediterranean tortoises. Oryx 15:81–82
Lapparent de Broin F (2000) African chelonians from the Jurassic to the present: a preliminary catalog of the African fossil chelonians. Palaeontol Afr 36:43–82
Manel S, Berthier P, Luikart G (2002) Detecting poaching: identifying the origin of individuals using Bayesian assigment tests and multi-locus genotypes. Conserv Biol 16:650–659
Minch E, Ruiz-Linares A, Goldstein D, Feldman M, Cavalli-Sforza LL (1997) Microsat v.1.5d: a computer program for calculating various statistics on microsatellite allele data
Moon JC, McCoy ED, Mushinsky HR, KarlMultiple SA (2006) Multiple paternity and breeding system in the gopher tortoise, Gopherus polyphemus. J Hered 97:150–157
Nei M (1987) Molecular evolutionary genetics. Columbia University Press, New York
Oosterhout CV, Hutchinson WF, Wills DPM, Shipley P (2004) Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538
Pamilo P, Nei M (1988) Relationships between gene trees and species trees. Mol Biol Evol 5:568–583
Parham JF, Macey JR, Papenfuss TJ, Feldman CR, Türkozan O, Polymeni R, Boore J (2006) The phylogeny of Mediterranean tortoises and their close relatives based on complete mitochondrial genome sequences from museum specimens: with coments on mitochondrial genomic features, phylogenetic, and paleogeography. Mol Phylogenet Evol 38:50–64
Pariset L, Cappuccio I, Ajmone Marsan P, Dunner S, Luikart G, Obexer-Ruff G, Peter C, Marletta D, Pilla F, Valentini A (2006) Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds. J Chromatogr B 833:117–120
Pérez I, Gimenez A, Sanchez-Zapata JA, Anadón JD, Martínez M, Esteve MA (2004) Non-commercial of spur-thighed tortoises (Testudo graeca graeca): a cultutal problem in Southeast Spain. Biol Conserv 118:175–181
Pleguezuelos JM, Márquez R, Lizana M (eds) (2002) Atlas y libro rojo de los anfibios y reptiles de España. Dirección General de la Conservación de la Naturaleza-Asociación Herpetológica Española, 2nd edn. Spain, Madrid
Posada D, Buckley T (2004) Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests. Soc Syst Biol 53:793–808
Posada D, Crandall K (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 1:817–818
Posada D, Crandall K (2001) Performance of methods for detecting recombination from DNA sequences: computer simulations. Proc Natl Acad Sci USA 98:13757–13762
Pritchard JK, Stephens M, Donelly P (2000) Inference of population structure from multilocus genotypes data. Genetics 155:945–949
Ramos-Onsins S, Rozas J (2002) Statistical properties of new neutrality tests against population growth. Mol Biol Evol 19:2092–2100
Raymond M, Rousset F (1995) GENEPOP: population genetics software for exact tests and ecumenicism. J Hered 86:248–249
Reed J, Tollit D, Thompson P, Amos W (1997) Molecular scatology: the use of molecular genetic analysis to assign species, sex and individual identity to seal faeces. Mol Ecol 6:225–234
Rogers AR, Harpending H (1992) Population growth makes waves in the distribution of pairwise genetic differences. Mol Biol Evol 9:552–569
Roques S, Díaz-Paniagua C, Andreu AC (2004) Microsatellite markers reveal multiple paternity and sperm storage in the Mediterrranean spur-thighed tortoise, Testudo graeca. Can J Zool 82:153–159
Rozas J, Sanchez-Delbarrio JC, Messenger X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and others methods. Bioinformatics 19:2496–2497
Saitou N, Nei M (1987) The Neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Schwartz T, Osentoski M, Lamb T, Karl A (2003) Microsatellite loci for the North American tortoises (genus Gopherus) and their applicability to other turtle species. Mol Ecol Notes 3:283–286
Slatkin M, Hudson RR (1991) Pairwise comparisons of mitochondrial DNA sequences in stable and exponentially growing populations. Genetics 129:555–562
Swofford DL (2002) PAUP* Phylogenetic analysis using parsomony (*and Other Methods), Version 4. Sinauer Associates, Sunderland, MA
Takahata N (1987) On the overdispersed molecular clock. Genetics 116:169–179
Thorbjarnarson JB, Lagueux CJ, Bolze D, Klemens MW, Meylan AB (2000) Human use of turtles: a worldwide perspective. In: Klemens MW (ed) Turtle conservation. Smithsonian Institution Press, Washington, pp 33–88
Turtle Conservation Fundation (2002) A global action plan for conservation of tortoises and freshwater turtles. Strategy and funding prospectus 2002–2007. Washington, DC: Conservation International and Chelonian Research Foundation, 30 pp
van der Kuyl AC, Ballasina D, Dekker J, Maas J, Willensem RE, Goudsmit J (2002) Phylogenetic relationships among the species of the Genus Testudo (Testudines: Testudinidae) inferred from mitochondrial 12s rRNA gene sequences. Mol Phylogenet Evol 22:174–183
van der Kuyl AC, Ballasina D, Zorgdrager F (2005) Mitochondrial haplotype diversity in the tortoises species Testudo graeca from North Africa and the middle East. BMC Evol Biol 5:29–37
Waples RS (2006) A bias correction for estimates of effective population size based on linkage disequilibrium at unlinked gene loci. Conserv Genet 7:167–184
Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370
Woodbury AM (1952) Hybrids of Gopherus berlandieri and G. agassizii. Herpetologica 8:33–36
Zhang D, Hewitt G (2003) Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects. Mol Ecol 12:563–584
Znari M, Germano DJ, Mace JC (2005) Growth and population structure of the Moorish Tortoise (Testudo graeca graeca) in Westcentral Morocco: possible effects of over-collecting for the tourist trade. J Arid Environ 62:55–74
Acknowledgments
The authors gratefully acknowledge to Fundación Global Nature (Murcia Region, Spain); the Wildlife Rehabilitation Center “El Valle” (Murcia Region, Spain); Marcos Fernández (Wildlife Rehabilitation Center Santa Faz, Alicante, Spain) and Albert Martinez-Silvestre (CRARC, Barcelona, Spain). Thanks also to Peter Spencer for his revisions of the manuscript and referees for their comments and suggestions that helped to improve the previous manuscript. The financial support was provided by both Fundación Séneca, Spain (Proyect 00655/PI/04) and by MEC, Spain (Grant AP-2004-4048).
Author information
Authors and Affiliations
Corresponding author
Appendix
Appendix
See Appendix Table 3.
Rights and permissions
About this article
Cite this article
Salinas, M., Altet, L., Clavel, C. et al. Genetic assessment, illegal trafficking and management of the Mediterranean spur-thighed tortoise in Southern Spain and Northern Africa. Conserv Genet 12, 1–13 (2011). https://doi.org/10.1007/s10592-009-9982-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10592-009-9982-1