GEOGRAPHIC ORIGINS OF ILLEGALLY HARVESTED HAWKSBILL SEA TURTLE PRODUCTS By Elizabeth Freese Shattuck A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Forensic Science 2011 ABSTRACT GEOGRAPHIC ORIGINS OF ILLEGALLY HARVESTED HAWKSBILL SEA TURTLE PRODUCTS By Elizabeth Freese Shattuck The hawksbill sea turtle (Eretmochelys imbricata) is one of seven sea turtle species, all of which are protected by international law. One of the distinguishing characteristics of the hawksbill is its carapace (shell), which is covered with colorful and ornate keratinous overlapping plates called scutes. These scutes are the source of ―tortoiseshell‖ products, which are widely, though illegally, available in many countries. The hawksbill has a circumglobal distribution, making coordinated and focused conservation efforts aimed at preventing poaching of the species difficult. In this research, a procedure was developed to extract and amplify mitochondrial DNA from tortoiseshell items, in an effort to determine where these sea turtles are being poached, thus establishing tortoiseshell products as a novel source of genetic material. DNA from 56 confiscated tortoiseshell items donated by the United States Fish and Wildlife Service were analyzed. An average of 506 base pairs of mitochondrial DNA was sequenced. Sixteen haplotypes were indentified including 10 that were unique. All but one item (94%) corresponded to genetic stocks in the Indo-Pacific Ocean region, two were plastic tortoiseshell forgeries, and the remaining item was Atlantic in origin. These results indicate that poaching is more common in the Indo-Pacific Ocean and the multiple Indo-Pacific haplotypes imply that poaching is occurring at more than one location in this region. Based on these results, conservation efforts should be focused on the Indo-Pacific region. This research will allow for specific legislation, effective enforcement and the inclusion of evidence during prosecution, all addressing the need to reduce the poaching of hawksbills that supply the tortoiseshell trade. AKNOWDLEGMENTS First I would like to thank Dr. David Foran for his assistance throughout the project. I would also like to thank Dr. Meredith Gore, and experts from the World Wildlife Fund and TRAFFIC, Crawford Allan and Adrian Reuter, for help in forming the premise of my research. I would like to thank Dr. Kim Scribner for his conservation genetics guidance. This research would also not have been possible without tortoiseshell items and assistance from Laura Abraczinskas, the collections manager at the Michigan State University Museum, and Doni Sprague, United States Fish and Wildlife repository specialist. Finally, I would like to thank my family and friends who have surrounded me with encouragement and support over the past two years. iii TABLE OF CONTENTS List of Tables .............................................................................................................................. v List of Figures ............................................................................................................................ vi Introduction ................................................................................................................................ 1 Hawksbill status and biology................................................................................................ 1 The tortoiseshell trade .......................................................................................................... 4 DNA and wildlife conservation ............................................................................................ 7 mtDNA and hawksbill conservation ..................................................................................... 9 Goals of this study ............................................................................................................. 11 Materials and methods .............................................................................................................. 12 Samples and sample preparation ........................................................................................ 12 DNA extraction .................................................................................................................. 13 DNA amplification............................................................................................................. 14 mtDNA sequencing ............................................................................................................ 18 Sequence analysis .............................................................................................................. 18 Results ...................................................................................................................................... 20 Sample preparation and DNA extraction ............................................................................ 20 mtDNA amplification ........................................................................................................ 20 mtDNA sequencing of tortoiseshell items .......................................................................... 28 mtDNA haplotypes ............................................................................................................ 30 Haplotype analysis ............................................................................................................. 33 Discussion ................................................................................................................................. 39 Recommendations ..................................................................................................................... 50 Appendix .................................................................................................................................. 51 References ................................................................................................................................ 57 iv LIST OF TABLES TABLE 1: Primer summary ...................................................................................................... 17 TABLE 2: Summary of amplification and sequencing success .................................................. 22 TABLE 3: Confiscated tortoiseshell item sequence lengths ....................................................... 28 TABLE 4: Confiscated tortoiseshell item haplotypes ................................................................ 31 TABLE 5: Polymorphisms of unique sequences ........................................................................ 33 TABLE 6: Description of samples ............................................................................................ 52 TABLE 7: Reference sequence accession numbers.................................................................... 54 v LIST OF FIGURES FIGURE 1: Hawksbill sea turtle .................................................................................................. 1 FIGURE 2: Geographic locations of nesting sites ........................................................................ 3 FIGURE 3: Tortoiseshell items ................................................................................................... 4 FIGURE 4: Map of vertebrate mtDNA ........................................................................................ 8 FIGURE 5: Confiscated tortoiseshell item ................................................................................. 12 FIGURE 6: Primer map............................................................................................................. 15 FIGURE 7: Universal sea turtle primer amplification ................................................................ 23 FIGURE 8: EI1 – EI4 primer amplification ............................................................................... 24 FIGURE 9: EI5 – EI8 primer amplification ............................................................................... 25 FIGURE 10: Annealing temperature optimization. .................................................................... 26 FIGURE 11: Confiscated tortoiseshell item amplification. ........................................................ 27 FIGURE 12: Unique sequence phylogenetic comparison........................................................... 34 vi INTRODUCTION Hawksbill status and biology The hawksbill sea turtle (Eretmochelys imbricata; Figure 1) is one of seven sea turtle species. While all are listed as endangered or threatened, each species is unique, requiring individualized conservation considerations. The hawksbill sea turtle is protected under the Endangered Species Act, is listed under the Convention on International Trade in Endangered Species (CITES) in Appendix I, and is cited as critically endangered on the 2000 International Union for Conservation of Nature (IUCN) red list (Mortimer and Donnelly 2008). The species has been exploited for its ornate shell for centuries, making it difficult to assess the extent of the population decline that the hawksbill population has experienced, since historical baseline data are unknown (Mortimer and Donnelly 2008). The goal of this study was to determine the geographic locations of where the exploitation of hawksbills, specifically for their shell, is occurring. Figure 1. Hawksbill sea turtle (E. imbricata) (http://www.fws.gov). For interpretation of the references to color in this and all other figures, the reader