A Review of Current Wildlife Species Genetic Research: Identification of a priority list of wildlife species in trade, where DNA research would assist law enforcement Revised Final Report February 22, 2002 (Revised Mar 2004) Report No 3 LGC/LS/2004/001 A Review of Current Wildlife Species Genetic Research: Identification of a priority list of wildlife species in trade, where DNA research would assist law enforcement Revised Final Report Report No 3 Contact Point: Carole Foy Tel: 020 8943 7335. Prepared by: LGC: Carole Foy Lydia Ballam TRAFFIC: Crawford Allan Angela Barden Approved by: Alison Woolford ________________________________ Date: 22nd February 2002 (Revised Mar 2004) ________________________________ The work described in this report was supported under contract with DEFRA LGC/LS/2004/001 © LGC (Teddington) Limited 2004 Contents 1. Executive Summary 1 2. Project Aims 2 3. Background 2 4. Approach 5 4.1 Development of Priority Species Selection Criteria 5 4.1.1 The Primary Filter Process and Selection Criteria 5 4.1.2 Development of a DNA search strategy 8 4.2 Development of Analytical Database System 11 4.3 The Ranking, Scoring and Weighting Systems 11 4.3.1 DNA Ranking Strategy 11 5. Output 17 6. Recommendations 20 7. Conclusion 21 8. Acknowledgements 22 9. Appendices 24 9.1 Appendix 1 : Alternative animal ranking and prioritisation strategy 24 9.2 Appendix 2 : Species Database Construction and Use 26 9.3 Appendix 3 : Individuals/organisations contacted 37 9.4 Appendix 4 : Individuals/organisations offering assistance 37 9.5 Appendix 5 : DNA References 37 9.6 Appendix 6 : Summary of animal DNA information 65 9.7 Appendix 7 : Summary of plant DNA information 73 9.8 Appendix 8 : Wildlife trade regulation in the european union 75 9.9 Appendix 9 : Definitons for the Red List categories 77 Review of Current Wildlife Species Genetic Research - i - Final Report 1. Executive Summary DNA testing is becoming a standard tool to assist law enforcement through provision of robust evidence, and it is being applied more widely for wildlife trade law enforcement cases. There are limitations on its use for cases involving particular species, when DNA tests are not developed for that target species. Extensive research is ongoing to develop species-specific tests but often this may be duplicating the effort of others or may be misplaced. A need was recognised to develop a system to focus research where it was most needed, to support enforcement cases. Usually the need for a test arises from an enforcment case but development takes time, which is often too long for the requirements of a particular case. Development of a system to prioritise where research on DNA should be focussed, to meet the needs before they arise was an obvious step. Enforcement resources are highly restricted and it was determined that efforts should focus on those species most threatened with extinction and protected in law, as these would most likely be the species for which resources could be allocated. DNA tests are able to provide assistance in enforcement cases in many instances and there is a need to prioritise those species where such tests would have the greatest impact. Once prioritised, there is a further need to determine the current availability, or otherwise, of DNA markers such that future research to develop markers can be focussed effectively. Under support from the Department for the Environment, Food and Rural Affairs (DEFRA), LGC and TRAFFIC International have identified a priority list of endangered species in trade where DNA information is currently lacking but, if available, could help in wildlife trade law enforcement cases. In producing this list two additional resources have been developed: • an analytical system that can be revised or adapted to generate priority lists in future if conditions change or different scenarios are applied; • a catalogue of details of the main papers, researchers and laboratories involved in genetic studies on endangered wildlife The development and implementation of the system and the results are outlined and discussed in this report. The work focussed on developing a filter and ranking mechanism within a database structure, which scored species based on a variety of criteria such as extent of illegal trade, DNA research undertaken, breeding capacity, legal controls and degree of threat. From a first cut of over 5000 species in the database, the system was filtered down to a working data set of nearly 350 species, from which a priority list of 100 species has been produced. Whilst the system has been developed to take the most significant factors into consideration and has incorporated a great deal of research and analysis, its value is as the best guide available and it should not be seen as providing a definitive answer. Review of Current Wildlife Species Genetic Research - 1 - Final Report 2. Project Aims The overall aims of the project were directed by DEFRA in their original tender document, as follows: • Identify a priority list of 75-100 endangered species in trade where DNA-based research to support enforcement activity is likely to deliver the most significant benefits; • Assess the current availability, or otherwise, of DNA markers for these species by reviewing current literature and research effort; • Identify individuals and institutions with expertise in the development and/or utilisation of these DNA techniques; Although these were the main aims, the project did expand somewhat with the need identified by the project executants to ensure that an analytical system be developed, which would allow for future updating with changes in DNA research, legislation and enforcement needs. The additional aim of the project was therefore: • To develop an analytical process that lends itself to future revisions, should the need arise to produce a new priority list as a result of changes that would significantly impact the existing list. 3. Background There is no doubt that the existence of many species is being threatened by illegal trade in them, either as living animals or plants or their parts and derivatives. National and international laws have been established to regulate and prohibit trade in wildlife in an attempt to ensure that trade in some species is sustainable and in others is banned to limit further risk of extinction. Enforcing these laws can be complicated and many fraudulent methods have been developed to avoid detection by those who would wish to contravene the laws. Increasingly sophisticated criminal methods are employed in illegal wildlife trade and enforcers have responded by relying on advanced technologies to counter the trade, particularly DNA testing. Research to develop DNA tests, which can assist in efforts to protect endangered species by supporting enforcement activities, is increasingly being carried out in many centres around the world. DNA can assist enforcement bodies in determining whether shipments being inspected or cases investigated involve infraction of wildlife trade laws and can provide evidence for prosecution in a variety of ways. It can provide a means of species identification to establish whether a plant or animal (including parts and derivatives) is an endangered species that is protected in law. This is useful, as many species will not be traded as a more easily recognisable whole animal or plant. For example, a plant could be traded as a seed or powdered leaves in medicines, while animals could be traded as lumps of bush meat, pelts or ground up bones to be used in herbal medicines and remedies. Alternatively, DNA testing provides a means to discriminate between individuals, where population and/or familial-specific markers can identify whether an animal has been bred in captivity or taken from the wild. DNA markers can also be used to link an individual specimen to a suspected criminal believed to be involved in the poaching or smuggling of that specimen. DNA can also provide geographical and population information as markers can discriminate at the sub species / geographical population level e.g. South China Tiger or a Sumatran Tiger. Review of Current Wildlife Species Genetic Research - 2 - Final Report DNA is the analyte of choice for species identification for a variety of reasons. It is a very robust molecule, DNA can be highly degraded e.g. cooked (as in the case of bushmeat) or very old (as in the case of ivory) and a positive identification can still be obtained. The DNA tests developed are also extremely sensitive with only minute quantities of DNA needed to gain a positive result. Sensitivity and assay robustness is further enhanced through the use of markers derived from mitochondrial DNA, as this molecule is present in many copies within each cell. This is especially useful in mixed herbal medicines where the sample may only make up a small percentage of the overall mixture. DNA analysis will also have few collection issues because DNA is in all biological cells and can therefore be obtained from the smallest of samples from any part of the animal or plant. As DNA research is at the cutting edge of molecular biology, research is on going with new and improved techniques constantly being developed to aid detection. DNA markers available include: Microsatellite markers • Microsatellites are short polymorphic repeat sequences. Microsatellite markers can identify specific species, familial relationships, whether an animal is captive bred or wild and what geographical location or population it came from. Microsatellite markers have been developed across taxonomically similar species so sets of primers for one species may amplify a related species or closely related taxa. Mitochondrial markers • Mitochondrial or chloroplast markers are increasingly being used to identify species and sub species. Mitochondrial DNA is chosen over nuclear DNA for the development of DNA markers, as it is more prone to genetic drift, meaning that a greater proportion of variation is distributed between populations.