MTN 66:1-3 Forensic Applications of Mitochondrial DNA Markers: Origin of a Confiscated Green Turtle

seaturtle.org : MTN : ARCHIVES : INDEX Marine Turtle Newsletter 66:1-3, 1994

Forensic Applications of Mitochondrial DNA Markers: Origin of a Confiscated Green Turtle

S. E. Encalada1, S. A. Eckert2, and B. W. Bowen3 1Department of Wildlife and Range Sciences, University of Florida, Gainesville, Florida 32611 and Archie Carr Center for Sea Turtle Research, 223 Bartram Hall, University of Florida, Gainesville, Florida 32611 2Hubbs-Sea World Research Institute, 2595 Ingraham Street, San Diego, California 92109 3BEECS Genetic Analysis Core, P. O. Box 110699, Univ. Florida, Gainesville, Florida 32611 USA On 28 September 1993, a 35.6 cm green sea turtle (Chelonia mydas) was left at the Steinhart Aquarium (San Francisco, California) by a private citizen with the explanation that the turtle had been purchased in Abidjan, Ivory Coast, West Africa by a crew member of a cargo ship which had recently docked in San Francisco. Apparently the intention had been to consume the turtle, but the crew member had decided otherwise during the voyage. The turtle was in a poor state of health when left at the aquarium and was retained for rehabilitation. After the turtle was restored to health, Steinhart Aquarium requested guidance from the National Marine Fisheries Service (NMFS) and one of the authors (Eckert) on how and where the turtle should be released. Because of the circumstances surrounding acquisition, there was some concern that the turtle may not have originated in Africa, but rather had been taken from the Pacific. NMFS wished to accurately determine the region of origin before a repatriation plan was formulated. In practical terms, the question at hand was whether to release the turtle in the Atlantic or the Pacific Ocean. Green turtle nesting populations typically contain distinctive mtDNA haplotypes (Bowen et al., 1992; Allard et al., 1994) which can be used to indicate the location of origin for sea turtles on distant feeding grounds (Norman et al., 1994; Broderick et al., 1994). The BEECS Genetic Analysis Core maintains an inventory of sea turtle DNA, including specimens from 16 Chelonia mydas rookeries in the Atlantic, Indian, and Pacific Oceans. This data bank provided the basis for the forensic application presented here. A blood sample (<1 ml) was taken from the confiscated turtle following the protocol of Owens and Ruiz (1980). Blood was stored in a lysis buffer [100 mM Tris-HCI, pH 8; 100 mM EDTA, pH 8, 10 mM NaCI; 1.0% sodium dodecyl sulfate] in a blood:buffer ratio of approximately 1:10, as recommended by White and Densmore (1992). Our experience indicates that blood collected for
http://www.seaturtle.org/mtn/archives/mtn66/mtn66p1.shtml (1 of 3)09/02/2005 09:56:27 a.m.

MTN 66:1-3 Forensic Applications of Mitochondrial DNA Markers: Origin of a Confiscated Green Turtle

DNA analysis can be preserved in lysis buffer for extended periods (>1 year) without refrigeration. This is a convenient approach for biologists who wish to stockpile material for genetic analysis. Lysis buffer is inexpensive to prepare and the ingredients can be found in any basic biochemical or genetic lab. Whole genomic DNA was isolated from blood samples with a phenol/chloroform protocol (Hillis et al., 1990). To generate a DNA sequence from the confiscated turtle, biotinylated versions of primers described by Allard et al. (1994) were used to amplify mtDNA control region sequence with standard polymerase chain reaction (PCR) methodology (Innes et al., 1990). PCR products were purified with streptavidin coated magnetic particles. Single stranded sequencing reactions were conducted with fluorescently labelled primers in a robotic work station (Applied Biosystems model 800), and the labelled extension products were analyzed with an automated sequencer (Applied Biosystems model 373A) in the DNA Sequencing Core at University of Florida. In this case, a 373 base fragment of the mtDNA control region was generated and compared to known sequences from Atlantic and Pacific nesting colonies (Allard et al., 1994; Encalada, unpublished data). The mtDNA sequence from this turtle exactly matched a genotype observed in nesting colonies in Brazil, Ascension Island (on the mid-Atlantic ridge), and Guinea Bissau (West Africa). While we cannot determine which specific nesting colony this animal came from, we can say with a high degree of certainty that the confiscated turtle is derived from the South or East Atlantic. Previous research has revealed a marked genetic difference between Atlantic and Pacific green turtles (Bowen et al., 1989), and available evidence indicates that populations in these two oceans probably diverged on the order of 1.5-3 million years ago (Bowen et al., 1992). In light of this evolutionary separation, it would be poor management practice to release the confiscated turtle in the Pacific Ocean. If the turtle mated with Pacific conspecifics, subsequent introgression could potentially disrupt coadapted gene complexes which have evolved in isolation over millions of years. The possible consequences of outbreeding include introduction of maladaptive geno-types, reduced survivorship of progeny, and increased susceptibility to disease. Release of an Atlantic turtle into a Pacific gene pool could also obscure the evolutionary history of Pacific populations beyond recovery. In addition to these genetic problems, release of the captured animal in the Pacific could introduce foreign (endemic Atlantic) parasites or microfauna into the Pacific Ocean. The analysis of mtDNA markers indicate that this turtle should be released in the southern or eastern tropical Atlantic Ocean, or maintained in captivity for educational purposes. Green turtles are known to migrate extensively and to have a very precise sense of location, such that this animal could in principle return to resident feeding grounds from any release location in the tropical Atlantic. A release in the Atlantic Ocean at least provides the animal with a chance to return to resident foraging areas. NMFS is to be commended for considering the genetic and life history consequences of releasing animals outside their natural range. This case history illustrates the potential applications of mtDNA markers for forensic inquiry. The

http://www.seaturtle.org/mtn/archives/mtn66/mtn66p1.shtml (2 of 3)09/02/2005 09:56:27 a.m.

MTN 66:1-3 Forensic Applications of Mitochondrial DNA Markers: Origin of a Confiscated Green Turtle

PCR-based approach used here can be applied to small tissue samples, partially degraded material, or dry (taxidermic) specimens. Genetic markers have powerful applications in identification of endangered species material and in determining the region of origin for endangered species products. Based on results of this investigation, arrangements are underway to ensure that the turtle is returned to the Atlantic. For logistical support we thank the staff of the Steinhart Aquarium, as well as E. Almira, K. Bjorndal, A. Bolten, T. Boomershine, J. Cordaro, R. Ferl, A. Garcia, P. Lahanas, M. Miyamoto, and the crew of the DNA Sequencing Core at University of Florida. This research program is supported by the Interdisciplinary Center for Biotechnology Research (University of Florida), the U. S. Fish and Wildlife Service, and the U. S. National Science Foundation. Allard, M. W., M. M. Miyamoto, K. A. Bjorndal, A. B. Bolten and B. W. Bowen. 1994. Support for natal homing in green turtles from mitochondrial DNA sequences. Copeia 1994:34-41. Bowen, B. W., A. B. Meylan and J. C. Avise. 1989. An odyssey of the green sea turtle, Chelonia mydas: Ascension Island revisited. Proc. Natl. Acad. Sci. USA 86:573-576. Bowen, B. W., A. B. Meylan, J. P. Ross, C. J. Limpus, G. H. Balazs and J. C. Avise. 1992. Global population structure and natural history of the green turtle (Chelonia mydas) in terms of matriarchal phylogeny. Evolution 46:865-881. Hillis, D. M., A. Larson, S. K. Davis and E. A. Zimmer. 1990. Nucleic Acids III: Sequencing, p.318370. In: D. M. Hillis and C. Mortiz (Editors), Molecular Systematics. Sinauer Associates, Sunderland, Massachusetts, USA. Innis, M. A., D. H. Gelfand, J. J. Sninsky and T. J. White. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, California, USA. Norman, J. A., C. Moritz and C. J. Limpus. 1994. Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles. Molecular Ecology (in press). Owens, D. W. and G. W. Ruiz. 1980. New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica 36(1):17-20. White, P. S. and L. D. Densmore. 1992. Mitochondrial DNA isolation, p.29-58. In: A. R. Hoezel (Editor), Molecular Genetic Analysis of Populations: A Practical Approach. IRL Press, Oxford University Press, New York, USA.

http://www.seaturtle.org/mtn/archives/mtn66/mtn66p1.shtml (3 of 3)09/02/2005 09:56:27 a.m.