caraccio2003malasia

Caraccio M. N., A. Formia, M. Hernandez, A. Fallabrino and M. Bruford. (In press).Preliminary Mixed Stock Analysis of Juvenile Green Turtles in Uruguay using Mitochondrial Dna Sequences. Proceedings of the 23° International Symposium on Sea Turtle Biology and Conservation, Kuala Lumpur, Malasia. U.S. Dep. Commer. NOAA Tech. Mem (2003). PRELIMINARY MIXED STOCK ANALYSIS OF JUVENILE GREEN TURTLES IN URUGUAY USING MITOCHONDRIAL DNA SEQUENCES Maria Noel Caraccio 1,3 , Angela Formia 2, Martin Hernandez1,3, Alejandro Fallabrino1 and Michael W Bruford2 1C.I.D., Proyecto Karumbé, Tortugas Marinas del Uruguay , J. Paullier 1198, Montevideo, Uruguay, [email protected] 2School of Biosciences, Cardiff University, Cardiff CF10 3TL, United Kingdom 3Sección Genética Evolutiva, Facultad de Ciencias. Iguá 4225, Montevideo, Uruguay INTRODUCTION The green turtle, Chelonia mydas, similarly to other sea turtle species, has a life cycle that is hard to study directly due to the difficulty of tracking migratory movement among feeding and nesting areas, and to the long maturation times. After reaching the sea, the hatchlings are not seen again until they enter neritic foraging grounds as juveniles. These areas often contain mixed stocks (individuals belonging to more than one nesting population) and are located many kilometers away from the nesting grounds. Studies carried out in nesting areas indicate that adult females return to nest at their natal beach. Most of the knowledge concerning sea turtle distribution is obtained from tagging experiments. While nesting females are easily accessible on the beach, feeding grounds require greater efforts, including catching individuals at sea with tangle nets. For some years, Karumbé Project has been monitoring the feeding grounds of juvenile green turtles along the Uruguayan coast, and performing activities such as capturing and tagging turtles through netting, and locating strandings or incidental fishery catches. Recently, tissue sampling has been carried out in order to determine the genetic characteristics of green turtles inhabiting Uruguayan waters. The control region of mitochondrial DNA (mtDNA) is a molecular tag commonly used in phylogeographic studies of this species in the Atlantic, contributing to reveal their molecular evolution, population structure, reproductive behavior and migratory patterns (Bowen et al. 1992, Allard et al. 1994, Lahanas et al. 1994, Encalada et al. 1996, Lahanas et al. 1998). Mitochondrial DNA evidence can also be used to define management units for conservation (Moritz 1994). In this work we present the preliminary results of analysis carried out in the Biodiversity and Environmental Process Research Group at Cardiff University (UK) using the control region of mtDNA of juvenile green turtles found along the Uruguayan coast. MATERIALS AND METHODS Tissue samples from twenty juvenile green turtles were collected from stranded individuals, from turtles that were captured by netting efforts, and from turtles incidentally caught by artisanal fisheries. The size of the turtles ranged between 32 and 52cm of curved carapace length notch to tip (CCLn-t) (Bolten 1999). The samples were preserved in absolute ethanol. Extraction of the mtDNA was perfomed using a modification of the protocol by Allen et al. (1998). A fragment of 486 bp from the control region was amplified with the primers HDCM1 and LDCM1 (Allard et al. 1994), and HDCM1.1 and LDCM1.1 for degraded DNA (Formia 2002). PCR products were visualised on 1.5% agarose gels and were later sequenced using automated sequencing protocols with an ABI3100 Sequencer. The sequences obtained were aligned using SEQUENCHER software (Gene Codes Corporation) and analysed using ARLEQUIN (Schneider et al. 2000). Also, mixed stock analyses were carried out with BAYES (Pella & Masuda 2001), to estimate the proportion of nesting populations that contributed to our juvenile stock. Caraccio M. N., A. Formia, M. Hernandez, A. Fallabrino and M. Bruford. (In press). Preliminary Mixed Stock Analysis of Juvenile Green Turtles in Uruguay using Mitochondrial Dna Sequences. Proceedings of the 23° International Symposium on Sea Turtle Biology and Conservation, Kuala Lumpur, Malasia. U.S. Dep. Commer. NOAA Tech. Mem (2003). RESULTS Five polymorphic sites were found in the twenty samples, defining six haplotypes already described for nesting beaches of the Atlantic Ocean: CM5 (previously detected in Mexico, Aves Island, Surinam and Sao Tome), CM6 (also found in Bioko, Ascension Island,Sao Tome, Suriname), CM8 (Bioko, Ascension Island, Guinea Bissau, Sao Tome, Principe and Atol das Rocas), CM9 (Ascension Island and Atol das Rocas), CM10 (Ascension Island) and CM24 (Ascension Island) (Lahanas et al. 1994, Encalada et al. 1996, Lahanas et al. 1998, Formia 2002). Eleven of the twenty individuals exhibited the CM8 haplotype, five individuals had CM5, and CM6, CM9, CM10 and CM24 haplotypes were each represented once. Haplotypic diversity was found to be 0.6579 +/0.0957, comparable to values for other mixed stocks (Formia 2002) and indicating relatively high genetic variation. The results of mixed stock analyses (using the nine baseline rookeries listed above as potential contributors, Figure 1) showed that the nesting population which contributed most to the feeding ground of Uruguay was the Ascension Island rookery with a mean of 62% followed by, Matapica (Surinam) with 14% and Aves Island (Venezuela) with 12% (see Table 1). The lower confidence limit for Ascension Island (above zero) confirmed that it was definitely present among the Uruguayan green turtle stock. DISCUSSION When analyzing these preliminary results, we have to be aware of the number of samples used and the incomplete descriptions available for several of the potentially contributing rookeries. Results clearly showed the necessity for further studies based on greater sample sizes. Despite this fact, the sequences obtained from these juvenile greens provided enough information to presume that the coast of Uruguay is an area hosting green turtles from several nesting beaches in the Atlantic Ocean. The presence of 6 different haplotypes in only 20 samples, showed this recruitment zone for juvenile greens to be genetically diverse. The results obtained using BAYES suggested that Ascension Island is likely to be the greatest contributor to the Uruguayan genetic stock (a mean of Caraccio M. N., A. Formia, M. Hernandez, A. Fallabrino and M. Bruford. (In press). Preliminary Mixed Stock Analysis of Juvenile Green Turtles in Uruguay using Mitochondrial Dna Sequences. Proceedings of the 23° International Symposium on Sea Turtle Biology and Conservation, Kuala Lumpur, Malasia. U.S. Dep. Commer. NOAA Tech. Mem (2003). 62%). However, we cannot exclude other areas, particularly the large rookery of Trindade, for which genetic data is not yet available. A comprehensive study and increased sample sizes, could elucidate some unresolved questions about origin, distribution, dispersal movements and migratory behavior of these green turtle populations. We hope that a comprehensive genetic survey will aid in developing appropriate management strategies for the endangered green turtles in the region. ACKNOWLEDGEMENTS We are grateful to the British Embassy in Uruguay for financial support of the research at Cardiff University. Thank to British Petrole um Conservation Programme, National Fish and Wildlife Foundation for supporting Karumbe Project. We would also like to thank Karumbe's friends for collecting the samples and the David and Lucille Packard Foundation for supporting our participation at the Symposium. Scientific and collection permit : Nº 270/01 (Dirección General de Recursos Naturales Renovables). 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