The Genetic Structure of the Loggerhead Sea Turtle (Caretta
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Conserv Genet DOI 10.1007/s10592-006-9224-8 ORIGINAL PAPER The genetic structure of the loggerhead sea turtle (Caretta caretta) in the Mediterranean as revealed by nuclear and mitochondrial DNA and its conservation implications C. Carreras Æ M. Pascual Æ L. Cardona Æ A. Aguilar Æ D. Margaritoulis Æ A. Rees Æ O. Turkozan Æ Y. Levy Æ A. Gasith Æ M. Aureggi Æ M. Khalil Received: 16 March 2006 / Accepted: 18 September 2006 Ó Springer Science+Business Media B.V. 2006 Abstract The population genetic structure of the small populations of Lebanon and Israel. This popu- loggerhead sea turtle (Caretta caretta) nesting in the lation structure indicates that assessing population eastern Mediterranean was assessed by sequencing a relevance only on the basis of genetic variability and fragment of the control region of the mitochondrial size would be misleading, as some populations not DNA (n = 190) and seven microsatellites (n = 112). fulfilling those requirements may play a relevant role in The two types of markers revealed genetic structuring genetic exchange and hence contribute to the overall (mtDNA: cst = 0.212, P < 0.001; nDNA Fst = 0.006, genetic variability. P < 0.001), thus indicating that both females and males are philopatric and that gene flow between populations Keywords Caretta caretta Á Mitochondrial DNA Á is restricted. Mitochondrial DNA data indicate that the Microsatellite DNA Á Mediterranean Á female populations nesting on the islands of Crete and Genetic structure Cyprus have suffered a recent bottleneck or coloniza- tion event. However, no bottleneck or founder effect was revealed by nuclear markers, thus indicating male- Introduction mediated gene flow from other populations that would increase nuclear genetic variability. Crete, and to a The circumtropical loggerhead sea turtle (Caretta lower extent Cyprus, are thought to play a central role caretta) is the most common sea turtle breeding in the in such male-mediated gene flow that may reduce the Mediterranean sea (Broderick et al. 2002). It is esti- negative effect of genetic drift or inbreeding on the mated that about 5,000 nests are laid every year in the Mediterranean (Margaritoulis et al. 2003), much less than the large rookeries of the Atlantic (about 80,000 C. Carreras Á L. Cardona Á A. Aguilar Department of Animal Biology, Faculty of Biology, University of Barcelona, Avda. Diagonal 645, O. Turkozan E-08028 Barcelona, Spain University of Adnan Menderes, 09010 Aydin, Turkey C. Carreras (&) Y. Levy Estacio´ n Biolo´ gica de Don˜ ana-CSIC-Apdo., The Israel Sea Turtle Rescue Centre, Nature Parks 1056-E-41013 Sevilla, Spain Authority, Mevoot Yam, Mikhmoret 40297, Israel e-mail: [email protected] Y. Levy Á A. Gasith M. Pascual Department of Zoology, Faculty of Life Sciences, Department of Genetics, Faculty of Biology, Tel-Aviv University, Tel-Aviv, Israel University of Barcelona, Avda, Diagonal 645, E-08028 Barcelona, Spain M. Aureggi Naucrates, Via Corbetta, 11, 22063 Cantu` , CO, Italy D. Margaritoulis Á A. Rees ARCHELON, The Sea Turtle Protection Society of Greece, M. Khalil Solo´ mou 57, GR-104 32 Athens, Greece MEDASSET, P.O. Box 19, Tyre, Lebanon 123 Conserv Genet nests/year; Ehrhart et al. 2003) or the Indian Ocean loci (Karl et al. 1992), then as randomly amplified (about 20,000–40,000 females/year that represents polymorphic DNA (RAPDs) (Schroth et al. 1996) and about 70,000–140,000 nests/year; Baldwin et al. 2003). more recently as microsatellites (e.g. Bowen et al. 2005; Most rookeries are located in the eastern Mediterranean FitzSimmons et al. 1997b), biparentally inherited basin (Margaritoulis et al. 2003) although sporadic markers that, combined with mtDNA, provide a fur- nesting has been reported from the western basin ther insight into the structure of marine turtle popu- (Llorente et al. 1992; Tomas et al. 2002; Delauguerre and lations, as mtDNA ignores male-mediated gene flow. Cesarini 2004). This combined approach has revealed that that the The species is listed as ‘‘endangered’’ globally by the Atlantic populations of the loggerhead sea turtle in the IUCN (IUCN 2006) although Groombridge (1990) northern hemisphere are highly structured with considered the Mediterranean populations as ‘‘criti- mtDNA, hence reflecting the high philopatry of the cally endangered’’. The Mediterranean population has females, but no structure exists for the nDNA due to declined due to incidental catch by fishing activity, egg male-mediated gene flow. As a result, male-mediated harvest and tourism development (Margaritoulis et al. gene flow would prevent genetic isolation in Atlantic 2003). Moreover, genetic studies on Mediterranean loggerhead sea turtles despite female philopatry loggerhead sea turtles (Bowen et al. 1993; Encalada (Bowen et al. 2005). Similar results were reported by et al. 1998) provided more arguments for considering FitzSimmons et al. (1997b) for Australian green turtles. the Mediterranean a relevant area for conservation of Available information suggests that this may not be the loggerhead sea turtle as it is for green sea turtle, a likely scenario in the Mediterranean, as regional Chelonia mydas (IUCN 2006). genetic structuring has been observed not only for Population genetic studies with marine turtles first mitochondrial DNA markers (Laurent et al. 1993, used the mitochondrial DNA (mtDNA) using restric- 1998; Schroth et al. 1996; Encalada et al. 1998), but also tion fragment length polymorphisms (RFLPs) (e.g. in nuclear DNA (Schroth et al. 1996). Unfortunately, Bowen et al. 1992, 1993) and then focused on the con- sample effort has been uneven and some areas have trol region of the mtDNA by sequencing (e.g. Bowen been poorly sampled or not sampled at all. Without et al. 1998, 2005; Encalada et al. 1996, 1998; Laurent more detailed information on the genetic structure of et al. 1998). The mtDNA is maternally inherited and the populations nesting in the eastern Mediterranean, high levels of genetic structuring were found all around identifying management units is difficult and may lead the world either by RFLPs or by sequencing. These to inappropriate management decisions. The absence high levels of genetic structuring indicated that the of internal structuring would indicate that the Medi- females of marine turtles were highly philopatric to the terranean rookeries may be treated as a single man- beaches were they were born (Meylan et al 1990). agement unit and hence, the loss of small local nesting Studies on the Mediterranean loggerhead sea turtle sites would not hinder the conservation of the overall populations (Bowen et al. 1993; Encalada et al. 1998) genetic variability. Otherwise, if internal structuring indicated that these rookeries became isolated from the exists, several management units would be defined and Atlantic populations at the beginning of the Holocene, should be preserved to guarantee healthy populations. and should be considered an independent management The purposes of this paper are (1) to assess the unit. Genetic isolation from the Atlantic populations is population structure of Mediterranean loggerhead likely to further increase the vulnerability of the Med- populations, including gene flow and possible bottle- iterranean population, not only because the strength of necks among the nesting sites, using both nuclear stochastic phenomena increases, but also because of the (nDNA) and mitochondrial (mtDNA) markers and (2) potential reinforcement of inbreeding depression and identify those populations that need to be protected in loss of diversity through genetic drift. Furthermore, the order to preserve the genetic diversity of the Medi- existence of internal genetic structuring has been pro- terranean rookeries. posed to increase the probability of local extinctions in animal populations (Frankham et al. 2002). Although this has not been proved for marine turtles, the exis- Methods tence of several different management units within the Mediterranean, some of them of extremely small size Collection and DNA extraction (Margaritoulis et al. 2003; Khalil et al. in press), would make extinction risk even higher. During the nesting seasons of 2003 and 2004, hatchlings Nuclear markers (nDNA) were incorporated to were sampled from 112 independent nests found in marine studies first as RFLPs of anonymous nuclear seven different nesting areas in the Mediterranean 123 Conserv Genet (Fig. 1). Individuals were collected from Zakynthos, 1998) were also included in the present work for Lakonikos and Crete in Greece; Fethiye, in west Tur- statistical analysis (Table 1). In order to establish key; northern Cyprus; El Mansouri in Lebanon; and haplotype relationships, a haplotype network was sites scattered along the Israeli coastline (Table 1). To constructed using package TCS v1.02 (Clement et al. avoid pseudoreplication (e.g. sampling hatchlings from 2000), which implements the statistical parsimony (SP) two nests of the same female), sampled females were described in Templeton et al. (1992). tagged with external flipper tags or subcutaneous PIT Genetic differentiation between different samples tags. When this was unfeasible, samples were collected from the same location was assessed with the Chi- only from clutches laid within a 15-day window, as fe- square test (Cuadras 1983) as implemented in the males rarely nest at intervals shorter than this period. program CHIRXC (Zaykin and Pudovkin 1993). When These procedures are expected to ensure independency statistical differences were not found between pub- of the collected samples. Furthermore, when dead lished and present data of the same nesting area, the embryo/hatchlings from a nest were found, only one two sets were considered to be subsamples of the same was collected and analyzed. From each individual, population. Both groups of studies were cautious in muscle or skin samples were stored in 95% ethanol. order to avoid pseudoreplication but, because inde- DNA was extracted using the QIAamp extraction kit pendence of data across different sampling datasets (QIAGENÒ) following the manufacturer’s instructions. cannot feasibly be totally ensured, the largest sample was considered for each nesting area, as detailed in Mitochondrial DNA analysis Table 1.