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Understanding illegal trade in pangolins through forensics: applications in law enforcement
C H A P T E R
20 Understanding illegal trade in pangolins through forensics: applications in law enforcement Antoinette Kotze1,2, Rob Ogden3,4, Philippe Gaubert5,6, Nick Ahlers7, Gary Ades8, Helen C. Nash9,10 and Desire Lee Dalton1,11 1
National Zoological Garden, South African National Biodiversity Institute, Pretoria, South Africa Genetics Department, University of the Free Sate, Bloemfontein, South Africa 3TRACE Wildlife Forensics Network, Edinburgh, United Kingdom 4Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, United Kingdom 5Laboratoire E´volution & Diversite´ Biologique (EDB), Universite´ de Toulouse Midi-Pyre´ne´es, CNRS, IRD, UPS, Toulouse, France 6 CIIMAR, University of Porto, Matosinhos, Portugal 7TRAFFIC, N IUCN, Hatfield Gables, Pretoria, South Africa 8Fauna Conservation Department, Kadoorie Farm & Botanic Garden, Hong Kong SAR, P.R. China 9Department of Biological Sciences, National University of Singapore, Singapore 10IUCN SSC Pangolin Specialist Group, N Zoological Society of London, Regent’s Park, London, United Kingdom 11University of Venda, Thohoyandou, South Africa 2
O U T L I N E Introduction
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Past, present and future methods Species identification Geographic origin Individual identification Kinship investigations Age determination Gender determination
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Pangolins DOI: https://doi.org/10.1016/B978-0-12-815507-3.00020-4
Coordinating and managing wildlife forensics at global and local scales
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Research and method development
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Developing pangolin forensic capacity
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Conclusion
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© 2020 Elsevier Inc. All rights reserved.
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Introduction The global illegal trade in pangolins and their body parts has seemingly increased in both Africa and Asia in recent decades, to supply a growing demand, primarily for use as traditional medicines and wild meat (Chapters 14 16; Heinrich et al., 2016; Ingram et al., 2018). Analyses of pangolin seizures reveal an apparent, stark increase in the number of pangolin scales originating in Africa that are supplying traditional medicine markets in Asia, while scales and whole and eviscerated bodies are more frequently traded within Asia (Challender and Waterman, 2017; Heinrich et al., 2017). Knowledge of illegal trade dynamics is important for informing law enforcement priorities and individual investigations, and desk-based studies of pangolin trafficking can both inform and are informed by scientific analysis. Seized pangolins and their body parts cannot usually be identified as anything more specific than “pangolin” by enforcement officers. This may be sufficient for prosecution in cases of international illegal trade where all pangolin species are protected (though see Chapter 17), but it limits investigations into range states and prevents the collection of potentially important information regarding the sources of seized pangolins. Conversely, from a forensic perspective, traditional trade studies guide the scientists’ understanding of the species, transport routes and sample types to be encountered in pangolin trade control and allow forensic scientists to prepare for the types of investigative questions and evidential material they may face. Collaboration between the scientific community and trade monitoring bodies, including integration of their respective datasets, therefore represents an important aspect of illegal pangolin trade investigations. Wildlife forensic analyses may be applied to investigations of poaching, illegal trade,
verification of species that are captive-bred versus wild caught, and identification of protected species in traditional medicine trade. Different methods to support law enforcement have been developed to determine (1) species identification, (2) geographic origin, (3) individual identification, (4) kinship, (5) age, and (6) gender. This chapter reviews these methods, their potential relevance and application to pangolin trafficking, and how local and global efforts to apply them should be coordinated in order for them to be effective tools in combating pangolin trafficking.
Past, present and future methods Species identification Morphological methods such as osteology or microscopy can be used to identify species. These methods require specialist knowledge of comparative anatomy at macroscopic and microscopic scales (Bell, 2011). A study on the radiological anatomy and scales of Temminck’s pangolin (Smutsia temminckii) indicated that patterns of scales on the body are associated with the underlying skeletal structure and thus may be used to identify the different pangolin species (Steyn, 2016). However, it is not always possible to identify different species of pangolin from scales alone as scales from different parts of the body differ in shape and size, and can vary with age and environmental variables. Microscopic analysis of animal material can be used to exclude possible pangolin species but it is generally not used for identification purposes due to overlapping ranges of measurements between species and due to limited access to reference data (Hillier and Bell, 2007). Molecular genetic approaches to pangolin species identification rely on DNA barcoding using mitochondrial DNA (mtDNA) markers
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or species specific microsatellite markers. Targeted mitochondrial gene regions include Cytochrome c Oxidase I (COI), Cytochrome b (Cytb), control region (CR), 12 S and 16 S ribosomal RNA, which have all been validated in previous studies for forensic application (Balitzki-Korte et al., 2005; Dawnay et al., 2007; Gaubert et al., 2015). Using these approaches, a region of the mitochondrial genome is sequenced and compared with sequences in public online databases such as GenBank, the European Molecular Biology Laboratory (EMBL), the Barcode of Life Database (BOLD; Ratnasingham and Hebert, 2007), and/or expert-curated online identification tools such as ForCyt (Ahlers et al., 2017) and DNAbushmeat (Gaubert et al., 2015). Critical to successful analysis of any species identification in a forensic case is the construction of a DNA reference library. A case study demonstrating the utility of DNA barcoding in pangolins is provided in Box 20.1. The ForCyt initiative differs from other publicly accessible databases in that it is designed for wildlife forensic casework and only contains quality assured sequence data generated from validated voucher specimens. At a broader level, collaboration with and critical input from relevant police services, prosecuting authorities and environmental enforcement agencies are essential to ensuring the impact of such forensic techniques. There is a need to raise awareness of DNA barcoding within law enforcement and prosecutor communities globally to promote the technology, which is currently being incorporated into actual investigations and court cases. Standard Operating Procedures (SOPs) and guideline documents are required, and staff involved in the collecting and analysis of samples need adequate training to ensure that the protocols are followed, reference libraries used and any subsequent investigations are legally defensible.
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Geographic origin Geographic source may be required for criminal investigations involving either live pangolins for potential repatriation, or dead animals or their parts and derivatives for understanding poaching hotspots and trade routes. There are two primary analytical approaches used for the geographic origin assignment of seized wildlife samples in trade: stable isotope analysis and molecular genetic (DNA) analysis. The geographic origin of biological materials, sometimes referred to as biogeolocation, can be determined using stable light isotopes (Hobson and Wassenaar, 2018; Oulhote et al., 2011), in a process which utilizes isotopic maps (or isoscapes) to assign a sample to its likely origin based on its isotopic profile. Measurements of stable isotopes have been used to trace origins of a range of wildlife products including African elephant ivory (van der Merwe et al., 1990; Vogel et al., 1990; Ziegler et al., 2016) and are widely used in analysis of traded foodstuffs. The technique is based on differences in elemental stable isotope ratios among geographic regions and may relate to underlying environmental differences (e.g., hydrogen, oxygen isotope ratios), vegetation type (e.g., nitrogen isotope ratios) or underlying geology (e.g., strontium isotope ratios). For stable isotope analysis to be useful in geographic origin assignment, it is usually necessary to generate a combined elemental profile for a sample and assign it to an isoscape map of multiple elements. One limitation to the approach is that despite having environmental isoscape maps available for many geographic regions, the way in which isotopes are incorporated into biological material may vary among species and among tissue types, so that separate isoscapes often need to be generated for each type of sample being used in the analysis. This limitation, combined with difficulties in accessing
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BOX 20.1
Case Study: Forensic application of DNA barcoding for identification of illegally traded African pangolin scales (Mwale et al., 2017). Between 2014 and 2015, 3.3 tonnes of pangolin scales were confiscated by the CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) Management Authority in Hong Kong SAR. It was suspected that the consignment originated from Africa and a representative subsample of ten bags, each representing a different consignment with a scale net weight of 27 kg was sent to the forensic laboratory of the National Zoological Garden in South Africa for analysis (Fig. 20.1A B). The contents were visually sorted into distinct scale types and provisionally assigned to a species by a taxonomic expert based on shape, coloration and morphology. Five samples per scale morph type were
voucher reference specimens of three African pangolin species, Temminck’s pangolin, blackbellied pangolin (Phataginus tetradactyla) and white-bellied pangolin (P. tricuspis), were analyzed. References were supplemented with sequences of the giant pangolin (S. gigantea), Sunda pangolin (Manis javanica) and Chinese pangolin (M. pentadactyla) retrieved from GenBank. The results indicated that the samples came from both African and Asian pangolins, and that confiscated scales may represent multiple pangolin species in one bag. This has laid the foundation for accurate identification of pangolin species in further forensic cases in South Africa.
selected from each of the ten bags for molecular characterization. In addition to the samples, 15
FIGURE 20.1 (A) Extraction of pangolin scale tissue and its DNA for forensic analysis. (B) Pangolin scales seized from illegal wildlife trade.
appropriate laboratory equipment and validating test methods means that isotopic profiling has seen limited use in forensic investigations (Meier-Augenstein et al., 2013) and although may have good potential
for pangolin origin analysis, remains unproven to date. Molecular genetic methods that rely on the analysis of mitochondrial DNA variation and nuclear DNA variation (microsatellite markers
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or single nucleotide polymorphisms [SNPs]) to assign individuals to a particular population provide an alternative option (Ogden and Linacre, 2015). These methodologies utilize genetic differentiation that has occurred as a result of gradual genetic separation between isolated populations in different geographic regions. For mitochondrial DNA, populations may show fixed discrete genetic differences, whereas for nuclear DNA, differences in the frequencies of genetic markers can be used to characterize this genetic structure. Accurate identification relies on the development of large genetic databases that are representative of the candidate source populations across the species’ geographic distribution (Ogden et al., 2009; Wasser et al., 2015). Bayesian methods developed by Pritchard et al. (2000) and Falush et al. (2003) can be used for assignment of individuals to clusters. For example, the geographic origin of 46 rescued chimpanzees has been conducted using mtDNA sequences and microsatellite genotypes (Ghobrial et al., 2010). Specifically for pangolins, the examination of a series of mtDNA and nuclear genes have allowed for the identification of six geographic lineages within the white-bellied pangolin (Gaubert et al., 2016), a pattern that could help track the global trade of the species at a sub-regional scale.
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moved in separate shipments (Wasser et al., 2015). Such applications are not typically essential for securing a conviction and are usually only practicable in cases where the number of individuals trafficked is relatively low (tens to hundreds). For pangolins, often traded in many thousands of individuals (or hundreds of thousands of scales), the evidential or intelligence value of individual identification relative to the potential cost of the work is much reduced. Apart from the occasional presence of specific morphological features on live pangolins (e.g., easily identifiable scales or scale patterns), it is difficult to distinguish between individual animals. While photographic libraries of confiscated animals (live or dead) may prove useful reference materials, particularly to support evidential control processes, there is currently little immediate requirement or capacity for morphological identification of pangolins at an individual level. The construction of DNA profiles can enable individualization, just as in humans. Individual DNA profiles may be utilized to regulate legal trade of species that are subject to quotas, or that can be used to determine the captive origin of an animal (e.g., where databases exist for captive stock). However, there are currently no techniques in place to use individual DNA profiling of pangolins for trade monitoring or forensic investigation, nor is there a requirement to do so.
Individual identification Individual identification of animals or plants in wildlife forensics may be very useful when trying to link samples from different crime scenes or points along the supply chain. This is most common where the numbers of traded individuals are relatively low and the value of the information is considered high. For example, the use of individual DNA profiling to link horns back to individual rhinoceros carcasses through the RHODIS database (Harper et al., 2018), or linking pairs of elephant tusks being
Kinship investigations Establishing levels of relatedness between animals in forensic investigations is generally employed in order to differentiate between captive-bred and wild caught animals (Ogden et al., 2009) and can currently only be achieved with DNA-based methods. The patterns of inheritance from parent to offspring allow DNA profiles to be used to verify family relationships. Parentage is refuted if all or part of
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the profile observed in an offspring individual is not present in its claimed parents. In addition to a DNA profiling system, kinship investigations usually rely on breeding records that allow parent-offspring trios to be identified for testing. A requirement for such breeding records should be included in breeding license regulations if any DNA testing system is to be effectively employed for enforcement. With the potential advent of pangolin farms (see Chapter 32), such a regulatory framework may be necessary to prevent the laundering of wild animals through captive breeding centers.
Age determination In some wildlife crime investigations, it may be necessary to determine whether an individual was living at a certain point in time. For example, rhino horn or ivory collected prior to 1947 pre-dates laws prohibiting trade. The primary method of ageing recent biological material is a form of radio carbon dating, known as bomb-testing. During the early part of the 1950s, atmospheric nuclear weapons testing became common. This resulted in an artificial increase in the amounts of different carbon isotopes, particularly carbon 14 (δ14C), which had doubled in abundance by 1965. As such, biological samples that pre-date this period will be expected to have a lower ratio of δ14C than more modern specimens. Determining the actual age of dead individuals may be achieved through analysis of morphological features, such as growth rings in otoliths in fish (Campana, 2001), and tooth cementum annulation (Wittwer-Backofen et al., 2004) in mammals, but for application to time-bound legislation these would need to be accompanied by reliable estimates of date of death. Determining the age of live animals largely relies upon external features which change predictably over time in a discriminate
fashion, for example, pigmentation patterns on the ventral side of humpback whale (Megaptera novaeangliae) flukes; or artificial markings, such as unique numbered bands attached to the legs of birds (Sherley et al., 2014). Research on genetic age determination has also made significant progress, primarily in humans and suggests promise for forensic application (Jarman et al., 2015). However at present, investigations concerning the illegal trade in pangolins do not require knowledge of individual age or period of life; but this situation could change, for example if legal movement of pre-CITES convention pangolins were to create a loophole for the illegal trade in contemporary specimens, such as pangolin scales. Such horizon scanning would be required to allow time for the forensic community to develop and validate ageing methods for pangolins.
Gender determination Gender determination via morphological and molecular genetic methods are available for many whole specimens or body parts of species in trade and may occasionally have application to law enforcement. For pangolins, sexing via direct observation of the genital region may be possible depending on species (e.g., easier in Temminck’s pangolin to determine gender due to penis size) and if the full carcass is available for dead animals. Molecular gender determination can be conducted if only parts of the animal are available or when sex-specific characters are either absent or difficult to observe. Methods of DNA gender determination in pangolins are not currently available, but their development should be readily achievable if required. However, as with ageing, there is no immediate requirement for this type of information in support of law enforcement investigations.
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Coordinating and managing wildlife forensics at global and local scales The global capacity to undertake wildlife forensic analysis and the number of casework investigations performed are increasing year on year. Across Africa and Southeast Asia in particular, a significant increase in wildlife forensic capacity-building activities occurred in the decade from 2009 to 2019, resulting in the routine use of wildlife forensic evidence to support prosecutions in many countries. This process of international development is far from complete, with laboratories in different countries operating at different levels and offering a range of techniques related to variation in both national need and technical capability. The international nature of the illegal pangolin trade, both with respect to the number of range states across Africa and Asia and the subsequent movement of pangolins through non-range states, means that coordination of forensic activities also needs to operate at an international scale. There are two key aspects that should be considered in this respect: the availability and harmonization of pangolin identification techniques among countries; and the ability to implement these methods with sufficient forensic rigor to secure prosecutions and prevent the emergence of weak links in forensic capacity along trade routes.
Research and method development Despite the high level of global attention given to pangolins due to their illegal trade, the group as a whole has remained remarkably under-studied. Although the species-level taxonomy of the African and Southeast Asian taxa has been described, relatively few phylogenetic studies on populations were conducted prior to 2010. This lack of data has prevented
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the rapid development of forensic genetic tools, requiring the implementation of biological baseline research before species identification and geographic origin tests could be designed to support pangolin trade investigations. Since 2010, several international groups have focused on the application of research on pangolin biology, ecology and evolutionary history, towards production of a number of validated traceability tools and forensic identification techniques. Work at the University of Toulouse, France, by Phillippe Gaubert and colleagues, has focused on the evolutionary history of pangolins (Gaubert et al., 2018) and population genetics of African pangolins (Gaubert et al., 2016), distinguishing pangolins from different geographic regions based on genetic variation, and thus laying the foundations for traceability of seized pangolins to areas within broad species distributions. A complimentary approach is being employed by the South African National Biodiversity Institute (SANBI) research group at the National Zoological Garden, under Antoinette Kotze, where the wildlife forensics laboratory has extensive experience in performing a combination of species identification (Dalton and Kotze, 2011) and origin assignment (Mwale et al., 2017) to help investigate the illegal pangolin trade across southern Africa. In East Asia, the University of Hong Kong’s School of Biological Sciences recently established a forensic laboratory to support wildlife crime enforcement work including a focus on pangolin analysis. The Conservation Genetics laboratory at Kadoorie Farm & Botanic Garden (KFBG) is also providing an analytical function and has carried out preliminary investigations on pangolin scales seized in Hong Kong Special Administrative Region (hereafter “Hong Kong”), and donated to the center for scientific use (Zhang et al., 2015). KFBG has more recently focused on the illegal trade in African pangolins. Scales originating from
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BOX 20.2
Case Study: Illegal trade in Sunda pangolins across insular Southeast Asia. Sunda pangolins across insular Southeast Asia have been heavily poached for the illegal wildlife trade. For example in April 2015, five tons of frozen pangolins, 77 kg of pangolin scales, and 96 live pangolins were seized from a single haul in Medan, Indonesia (Nijman et al., 2016). Due to the large, transnational range of the species, even when it is possible via morphological features or other methods to identify a pangolin to species level, it is difficult to determine the national or local geographic origins of a seizure, unless informants have provided further details (although these can often be unreliable). The application of advanced genetic techniques was trialed across insular Southeast Asia to help provide information about the origin of 97 Sunda pangolins within several seizures. It included the use of geolocated reference samples from across the
Africa seized in Hong Kong since 2009 are being analyzed in combination with statistical modeling to characterize the illegal trade and identify priority areas for targeted policy and enforcement efforts. In Southeast Asia, research projects in Indonesia, Malaysia and Singapore have led to the development of novel methods for pangolin identification. The collaborative work on Sunda pangolin geolocation in Singapore (see Box 20.2) has demonstrated the feasibility of applying research and development to pangolin trade investigations in this arena, and the partnership approach employed across multiple government, academic and non-government organizations offers a powerful model for
region to help assign the seized individuals to their likely geographic origin. The DNA analysis revealed three previously unrecognized genetic lineages of Sunda pangolins, from Borneo, Java and Singapore/Sumatra. For the seizure samples, it was possible to conclude that most of the pangolins had been captured from Borneo and exported to Java (Nash et al., 2018). The project was a collaboration between The Indonesian Institute of Sciences (LIPI), the IUCN SSC Pangolin Specialist Group, the National University of Singapore (NUS), the University of Malaya (UM), the Universiti Malaysia Terengganu (UMT), and Wildlife Reserves Singapore (WRS), supported by funding from the Southeast Asian Regional Centre for Tropical Biology (SEAMEO BIOTROP DIPA) and other partners.
broader work within the region. At the same time, the Malaysian National Wildlife Forensics Laboratory at Perhilitan, Kuala Lumpur, has embarked on genomic analysis of pangolin species from around the region, resulting in novel insights into both the taxonomy of the Southeast Asian pangolins and the potential to generate DNA-based traceability tools for Malaysia and beyond.
Developing pangolin forensic capacity The transfer and application of such research initiatives into the international wildlife forensic community represents the next significant step
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Conclusion
in the use of forensic science in investigations into illegal pangolin trade. For the results of pangolin sample analysis to be used successfully in law enforcement, it is necessary to raise awareness and build expertise from crime scene to courtroom. The use of biological material as forensic evidence requires seizures to be made following robust protocols that maintain both the evidential and biological integrity of the pangolin products confiscated. Laboratories analyzing such evidential samples need to operate under strict quality management systems using test methods previously validated to generate robust, reproducible, accurate results. Lastly, forensic reports must be generated and communicated to the legal profession, such that the prosecution, defence and judiciary alike are capable of evaluating and accepting the evidence placed before them. These issues are not unique to pangolins and the international wildlife forensic community works to ensure that national enforcement agencies have access to forensic services that offer internationally recognized tests delivered within a common forensic framework. The Society for Wildlife Forensic Science (SWFS) sets international standards for the discipline. Long-term capacity building programmes supported by The United States Agency for International Development (USAID), the U.S State Department and the European Commission are all contributing to the dissemination and harmonization of best practice in this field, largely implemented through the work of technical specialist organizations such as TRACE Wildlife Forensics Network and the Netherlands Forensic Institute (NFI). A number of initiatives specifically relating to pangolin forensics are being developed within these over-arching programs, including field officer training in the identification of pangolin parts to support seizures, protocols for the sampling and collection of pangolin evidence for forensic analysis, laboratory training in DNA
recovery from pangolin scales, and the production of guidelines for the coordination of pangolin seizure analysis among countries and continents (Fig. 20.2).
Conclusion The development and application of wildlife forensic analysis is driven by the needs of the enforcement community. Enforcement agencies may be looking for intelligence level data that inform investigations concerning trade patterns and likely shipment routes, or they may need forensic evidence concerning the identity of a sample for use in legal proceedings. These requirements must guide the scientific community in how it can help to support the ongoing fight against the illegal pangolin trade. Within the field of wildlife forensics, only a subset of possible methods and applications are currently applicable to pangolin law enforcement; these focus primarily on species identification and geographic origin assignment. Species identification of whole specimens can be achieved using expert morphological examination, or more routine DNA sequencing analysis. Approaches for determining geographic origin are available for certain species in specific regions and work is underway to extend these capabilities to enable all pangolins to be traced back to their origin to at least some level of geographic resolution. As with other species subject to wildlife forensic investigation, the initial development of analytical methods and their subsequent validation and application to legal casework crosses the boundary between academic research and forensic casework laboratory environments, requiring informed coordination between these two scientific communities. As the need for analytical data relating to the illegal trade in pangolins increases, the
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FIGURE 20.2 Proposed guidelines for wildlife forensic investigations involving pangolin confiscations. Excerpts from UNODC, 2014.
response from the scientific community is leading to increases in the availability of both pangolin-specific techniques and laboratories capable of implementing them for forensic application.
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Zhang, H., Miller, M.P., Yang, F., Chan, H.K., Gaubert, P., Ades, G., et al., 2015. Molecular tracing of confiscated pangolin scales for conservation and illegal trade monitoring in Southeast Asia. Glob. Ecol. Conserv. 4, 414 422. Ziegler, S., Merker, S., Streit, B., Boner, M., Jacob, D.E., 2016. Towards understanding isotope variability in elephant ivory to establish isotopic profiling and sourcearea determination. Biol. Conserv. 197, 154 163.
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20 Understanding illegal trade in pangolins through forensics: applications in law enforcement Antoinette Kotze1,2, Rob Ogden3,4, Philippe Gaubert5,6, Nick Ahlers7, Gary Ades8, Helen C. Nash9,10 and Desire Lee Dalton1,11 1
National Zoological Garden, South African National Biodiversity Institute, Pretoria, South Africa Genetics Department, University of the Free Sate, Bloemfontein, South Africa 3TRACE Wildlife Forensics Network, Edinburgh, United Kingdom 4Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, United Kingdom 5Laboratoire E´volution & Diversite´ Biologique (EDB), Universite´ de Toulouse Midi-Pyre´ne´es, CNRS, IRD, UPS, Toulouse, France 6 CIIMAR, University of Porto, Matosinhos, Portugal 7TRAFFIC, N IUCN, Hatfield Gables, Pretoria, South Africa 8Fauna Conservation Department, Kadoorie Farm & Botanic Garden, Hong Kong SAR, P.R. China 9Department of Biological Sciences, National University of Singapore, Singapore 10IUCN SSC Pangolin Specialist Group, N Zoological Society of London, Regent’s Park, London, United Kingdom 11University of Venda, Thohoyandou, South Africa 2
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Past, present and future methods Species identification Geographic origin Individual identification Kinship investigations Age determination Gender determination
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Introduction The global illegal trade in pangolins and their body parts has seemingly increased in both Africa and Asia in recent decades, to supply a growing demand, primarily for use as traditional medicines and wild meat (Chapters 14 16; Heinrich et al., 2016; Ingram et al., 2018). Analyses of pangolin seizures reveal an apparent, stark increase in the number of pangolin scales originating in Africa that are supplying traditional medicine markets in Asia, while scales and whole and eviscerated bodies are more frequently traded within Asia (Challender and Waterman, 2017; Heinrich et al., 2017). Knowledge of illegal trade dynamics is important for informing law enforcement priorities and individual investigations, and desk-based studies of pangolin trafficking can both inform and are informed by scientific analysis. Seized pangolins and their body parts cannot usually be identified as anything more specific than “pangolin” by enforcement officers. This may be sufficient for prosecution in cases of international illegal trade where all pangolin species are protected (though see Chapter 17), but it limits investigations into range states and prevents the collection of potentially important information regarding the sources of seized pangolins. Conversely, from a forensic perspective, traditional trade studies guide the scientists’ understanding of the species, transport routes and sample types to be encountered in pangolin trade control and allow forensic scientists to prepare for the types of investigative questions and evidential material they may face. Collaboration between the scientific community and trade monitoring bodies, including integration of their respective datasets, therefore represents an important aspect of illegal pangolin trade investigations. Wildlife forensic analyses may be applied to investigations of poaching, illegal trade,
verification of species that are captive-bred versus wild caught, and identification of protected species in traditional medicine trade. Different methods to support law enforcement have been developed to determine (1) species identification, (2) geographic origin, (3) individual identification, (4) kinship, (5) age, and (6) gender. This chapter reviews these methods, their potential relevance and application to pangolin trafficking, and how local and global efforts to apply them should be coordinated in order for them to be effective tools in combating pangolin trafficking.
Past, present and future methods Species identification Morphological methods such as osteology or microscopy can be used to identify species. These methods require specialist knowledge of comparative anatomy at macroscopic and microscopic scales (Bell, 2011). A study on the radiological anatomy and scales of Temminck’s pangolin (Smutsia temminckii) indicated that patterns of scales on the body are associated with the underlying skeletal structure and thus may be used to identify the different pangolin species (Steyn, 2016). However, it is not always possible to identify different species of pangolin from scales alone as scales from different parts of the body differ in shape and size, and can vary with age and environmental variables. Microscopic analysis of animal material can be used to exclude possible pangolin species but it is generally not used for identification purposes due to overlapping ranges of measurements between species and due to limited access to reference data (Hillier and Bell, 2007). Molecular genetic approaches to pangolin species identification rely on DNA barcoding using mitochondrial DNA (mtDNA) markers
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or species specific microsatellite markers. Targeted mitochondrial gene regions include Cytochrome c Oxidase I (COI), Cytochrome b (Cytb), control region (CR), 12 S and 16 S ribosomal RNA, which have all been validated in previous studies for forensic application (Balitzki-Korte et al., 2005; Dawnay et al., 2007; Gaubert et al., 2015). Using these approaches, a region of the mitochondrial genome is sequenced and compared with sequences in public online databases such as GenBank, the European Molecular Biology Laboratory (EMBL), the Barcode of Life Database (BOLD; Ratnasingham and Hebert, 2007), and/or expert-curated online identification tools such as ForCyt (Ahlers et al., 2017) and DNAbushmeat (Gaubert et al., 2015). Critical to successful analysis of any species identification in a forensic case is the construction of a DNA reference library. A case study demonstrating the utility of DNA barcoding in pangolins is provided in Box 20.1. The ForCyt initiative differs from other publicly accessible databases in that it is designed for wildlife forensic casework and only contains quality assured sequence data generated from validated voucher specimens. At a broader level, collaboration with and critical input from relevant police services, prosecuting authorities and environmental enforcement agencies are essential to ensuring the impact of such forensic techniques. There is a need to raise awareness of DNA barcoding within law enforcement and prosecutor communities globally to promote the technology, which is currently being incorporated into actual investigations and court cases. Standard Operating Procedures (SOPs) and guideline documents are required, and staff involved in the collecting and analysis of samples need adequate training to ensure that the protocols are followed, reference libraries used and any subsequent investigations are legally defensible.
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Geographic origin Geographic source may be required for criminal investigations involving either live pangolins for potential repatriation, or dead animals or their parts and derivatives for understanding poaching hotspots and trade routes. There are two primary analytical approaches used for the geographic origin assignment of seized wildlife samples in trade: stable isotope analysis and molecular genetic (DNA) analysis. The geographic origin of biological materials, sometimes referred to as biogeolocation, can be determined using stable light isotopes (Hobson and Wassenaar, 2018; Oulhote et al., 2011), in a process which utilizes isotopic maps (or isoscapes) to assign a sample to its likely origin based on its isotopic profile. Measurements of stable isotopes have been used to trace origins of a range of wildlife products including African elephant ivory (van der Merwe et al., 1990; Vogel et al., 1990; Ziegler et al., 2016) and are widely used in analysis of traded foodstuffs. The technique is based on differences in elemental stable isotope ratios among geographic regions and may relate to underlying environmental differences (e.g., hydrogen, oxygen isotope ratios), vegetation type (e.g., nitrogen isotope ratios) or underlying geology (e.g., strontium isotope ratios). For stable isotope analysis to be useful in geographic origin assignment, it is usually necessary to generate a combined elemental profile for a sample and assign it to an isoscape map of multiple elements. One limitation to the approach is that despite having environmental isoscape maps available for many geographic regions, the way in which isotopes are incorporated into biological material may vary among species and among tissue types, so that separate isoscapes often need to be generated for each type of sample being used in the analysis. This limitation, combined with difficulties in accessing
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BOX 20.1
Case Study: Forensic application of DNA barcoding for identification of illegally traded African pangolin scales (Mwale et al., 2017). Between 2014 and 2015, 3.3 tonnes of pangolin scales were confiscated by the CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) Management Authority in Hong Kong SAR. It was suspected that the consignment originated from Africa and a representative subsample of ten bags, each representing a different consignment with a scale net weight of 27 kg was sent to the forensic laboratory of the National Zoological Garden in South Africa for analysis (Fig. 20.1A B). The contents were visually sorted into distinct scale types and provisionally assigned to a species by a taxonomic expert based on shape, coloration and morphology. Five samples per scale morph type were
voucher reference specimens of three African pangolin species, Temminck’s pangolin, blackbellied pangolin (Phataginus tetradactyla) and white-bellied pangolin (P. tricuspis), were analyzed. References were supplemented with sequences of the giant pangolin (S. gigantea), Sunda pangolin (Manis javanica) and Chinese pangolin (M. pentadactyla) retrieved from GenBank. The results indicated that the samples came from both African and Asian pangolins, and that confiscated scales may represent multiple pangolin species in one bag. This has laid the foundation for accurate identification of pangolin species in further forensic cases in South Africa.
selected from each of the ten bags for molecular characterization. In addition to the samples, 15
FIGURE 20.1 (A) Extraction of pangolin scale tissue and its DNA for forensic analysis. (B) Pangolin scales seized from illegal wildlife trade.
appropriate laboratory equipment and validating test methods means that isotopic profiling has seen limited use in forensic investigations (Meier-Augenstein et al., 2013) and although may have good potential
for pangolin origin analysis, remains unproven to date. Molecular genetic methods that rely on the analysis of mitochondrial DNA variation and nuclear DNA variation (microsatellite markers
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or single nucleotide polymorphisms [SNPs]) to assign individuals to a particular population provide an alternative option (Ogden and Linacre, 2015). These methodologies utilize genetic differentiation that has occurred as a result of gradual genetic separation between isolated populations in different geographic regions. For mitochondrial DNA, populations may show fixed discrete genetic differences, whereas for nuclear DNA, differences in the frequencies of genetic markers can be used to characterize this genetic structure. Accurate identification relies on the development of large genetic databases that are representative of the candidate source populations across the species’ geographic distribution (Ogden et al., 2009; Wasser et al., 2015). Bayesian methods developed by Pritchard et al. (2000) and Falush et al. (2003) can be used for assignment of individuals to clusters. For example, the geographic origin of 46 rescued chimpanzees has been conducted using mtDNA sequences and microsatellite genotypes (Ghobrial et al., 2010). Specifically for pangolins, the examination of a series of mtDNA and nuclear genes have allowed for the identification of six geographic lineages within the white-bellied pangolin (Gaubert et al., 2016), a pattern that could help track the global trade of the species at a sub-regional scale.
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moved in separate shipments (Wasser et al., 2015). Such applications are not typically essential for securing a conviction and are usually only practicable in cases where the number of individuals trafficked is relatively low (tens to hundreds). For pangolins, often traded in many thousands of individuals (or hundreds of thousands of scales), the evidential or intelligence value of individual identification relative to the potential cost of the work is much reduced. Apart from the occasional presence of specific morphological features on live pangolins (e.g., easily identifiable scales or scale patterns), it is difficult to distinguish between individual animals. While photographic libraries of confiscated animals (live or dead) may prove useful reference materials, particularly to support evidential control processes, there is currently little immediate requirement or capacity for morphological identification of pangolins at an individual level. The construction of DNA profiles can enable individualization, just as in humans. Individual DNA profiles may be utilized to regulate legal trade of species that are subject to quotas, or that can be used to determine the captive origin of an animal (e.g., where databases exist for captive stock). However, there are currently no techniques in place to use individual DNA profiling of pangolins for trade monitoring or forensic investigation, nor is there a requirement to do so.
Individual identification Individual identification of animals or plants in wildlife forensics may be very useful when trying to link samples from different crime scenes or points along the supply chain. This is most common where the numbers of traded individuals are relatively low and the value of the information is considered high. For example, the use of individual DNA profiling to link horns back to individual rhinoceros carcasses through the RHODIS database (Harper et al., 2018), or linking pairs of elephant tusks being
Kinship investigations Establishing levels of relatedness between animals in forensic investigations is generally employed in order to differentiate between captive-bred and wild caught animals (Ogden et al., 2009) and can currently only be achieved with DNA-based methods. The patterns of inheritance from parent to offspring allow DNA profiles to be used to verify family relationships. Parentage is refuted if all or part of
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the profile observed in an offspring individual is not present in its claimed parents. In addition to a DNA profiling system, kinship investigations usually rely on breeding records that allow parent-offspring trios to be identified for testing. A requirement for such breeding records should be included in breeding license regulations if any DNA testing system is to be effectively employed for enforcement. With the potential advent of pangolin farms (see Chapter 32), such a regulatory framework may be necessary to prevent the laundering of wild animals through captive breeding centers.
Age determination In some wildlife crime investigations, it may be necessary to determine whether an individual was living at a certain point in time. For example, rhino horn or ivory collected prior to 1947 pre-dates laws prohibiting trade. The primary method of ageing recent biological material is a form of radio carbon dating, known as bomb-testing. During the early part of the 1950s, atmospheric nuclear weapons testing became common. This resulted in an artificial increase in the amounts of different carbon isotopes, particularly carbon 14 (δ14C), which had doubled in abundance by 1965. As such, biological samples that pre-date this period will be expected to have a lower ratio of δ14C than more modern specimens. Determining the actual age of dead individuals may be achieved through analysis of morphological features, such as growth rings in otoliths in fish (Campana, 2001), and tooth cementum annulation (Wittwer-Backofen et al., 2004) in mammals, but for application to time-bound legislation these would need to be accompanied by reliable estimates of date of death. Determining the age of live animals largely relies upon external features which change predictably over time in a discriminate
fashion, for example, pigmentation patterns on the ventral side of humpback whale (Megaptera novaeangliae) flukes; or artificial markings, such as unique numbered bands attached to the legs of birds (Sherley et al., 2014). Research on genetic age determination has also made significant progress, primarily in humans and suggests promise for forensic application (Jarman et al., 2015). However at present, investigations concerning the illegal trade in pangolins do not require knowledge of individual age or period of life; but this situation could change, for example if legal movement of pre-CITES convention pangolins were to create a loophole for the illegal trade in contemporary specimens, such as pangolin scales. Such horizon scanning would be required to allow time for the forensic community to develop and validate ageing methods for pangolins.
Gender determination Gender determination via morphological and molecular genetic methods are available for many whole specimens or body parts of species in trade and may occasionally have application to law enforcement. For pangolins, sexing via direct observation of the genital region may be possible depending on species (e.g., easier in Temminck’s pangolin to determine gender due to penis size) and if the full carcass is available for dead animals. Molecular gender determination can be conducted if only parts of the animal are available or when sex-specific characters are either absent or difficult to observe. Methods of DNA gender determination in pangolins are not currently available, but their development should be readily achievable if required. However, as with ageing, there is no immediate requirement for this type of information in support of law enforcement investigations.
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Coordinating and managing wildlife forensics at global and local scales The global capacity to undertake wildlife forensic analysis and the number of casework investigations performed are increasing year on year. Across Africa and Southeast Asia in particular, a significant increase in wildlife forensic capacity-building activities occurred in the decade from 2009 to 2019, resulting in the routine use of wildlife forensic evidence to support prosecutions in many countries. This process of international development is far from complete, with laboratories in different countries operating at different levels and offering a range of techniques related to variation in both national need and technical capability. The international nature of the illegal pangolin trade, both with respect to the number of range states across Africa and Asia and the subsequent movement of pangolins through non-range states, means that coordination of forensic activities also needs to operate at an international scale. There are two key aspects that should be considered in this respect: the availability and harmonization of pangolin identification techniques among countries; and the ability to implement these methods with sufficient forensic rigor to secure prosecutions and prevent the emergence of weak links in forensic capacity along trade routes.
Research and method development Despite the high level of global attention given to pangolins due to their illegal trade, the group as a whole has remained remarkably under-studied. Although the species-level taxonomy of the African and Southeast Asian taxa has been described, relatively few phylogenetic studies on populations were conducted prior to 2010. This lack of data has prevented
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the rapid development of forensic genetic tools, requiring the implementation of biological baseline research before species identification and geographic origin tests could be designed to support pangolin trade investigations. Since 2010, several international groups have focused on the application of research on pangolin biology, ecology and evolutionary history, towards production of a number of validated traceability tools and forensic identification techniques. Work at the University of Toulouse, France, by Phillippe Gaubert and colleagues, has focused on the evolutionary history of pangolins (Gaubert et al., 2018) and population genetics of African pangolins (Gaubert et al., 2016), distinguishing pangolins from different geographic regions based on genetic variation, and thus laying the foundations for traceability of seized pangolins to areas within broad species distributions. A complimentary approach is being employed by the South African National Biodiversity Institute (SANBI) research group at the National Zoological Garden, under Antoinette Kotze, where the wildlife forensics laboratory has extensive experience in performing a combination of species identification (Dalton and Kotze, 2011) and origin assignment (Mwale et al., 2017) to help investigate the illegal pangolin trade across southern Africa. In East Asia, the University of Hong Kong’s School of Biological Sciences recently established a forensic laboratory to support wildlife crime enforcement work including a focus on pangolin analysis. The Conservation Genetics laboratory at Kadoorie Farm & Botanic Garden (KFBG) is also providing an analytical function and has carried out preliminary investigations on pangolin scales seized in Hong Kong Special Administrative Region (hereafter “Hong Kong”), and donated to the center for scientific use (Zhang et al., 2015). KFBG has more recently focused on the illegal trade in African pangolins. Scales originating from
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BOX 20.2
Case Study: Illegal trade in Sunda pangolins across insular Southeast Asia. Sunda pangolins across insular Southeast Asia have been heavily poached for the illegal wildlife trade. For example in April 2015, five tons of frozen pangolins, 77 kg of pangolin scales, and 96 live pangolins were seized from a single haul in Medan, Indonesia (Nijman et al., 2016). Due to the large, transnational range of the species, even when it is possible via morphological features or other methods to identify a pangolin to species level, it is difficult to determine the national or local geographic origins of a seizure, unless informants have provided further details (although these can often be unreliable). The application of advanced genetic techniques was trialed across insular Southeast Asia to help provide information about the origin of 97 Sunda pangolins within several seizures. It included the use of geolocated reference samples from across the
Africa seized in Hong Kong since 2009 are being analyzed in combination with statistical modeling to characterize the illegal trade and identify priority areas for targeted policy and enforcement efforts. In Southeast Asia, research projects in Indonesia, Malaysia and Singapore have led to the development of novel methods for pangolin identification. The collaborative work on Sunda pangolin geolocation in Singapore (see Box 20.2) has demonstrated the feasibility of applying research and development to pangolin trade investigations in this arena, and the partnership approach employed across multiple government, academic and non-government organizations offers a powerful model for
region to help assign the seized individuals to their likely geographic origin. The DNA analysis revealed three previously unrecognized genetic lineages of Sunda pangolins, from Borneo, Java and Singapore/Sumatra. For the seizure samples, it was possible to conclude that most of the pangolins had been captured from Borneo and exported to Java (Nash et al., 2018). The project was a collaboration between The Indonesian Institute of Sciences (LIPI), the IUCN SSC Pangolin Specialist Group, the National University of Singapore (NUS), the University of Malaya (UM), the Universiti Malaysia Terengganu (UMT), and Wildlife Reserves Singapore (WRS), supported by funding from the Southeast Asian Regional Centre for Tropical Biology (SEAMEO BIOTROP DIPA) and other partners.
broader work within the region. At the same time, the Malaysian National Wildlife Forensics Laboratory at Perhilitan, Kuala Lumpur, has embarked on genomic analysis of pangolin species from around the region, resulting in novel insights into both the taxonomy of the Southeast Asian pangolins and the potential to generate DNA-based traceability tools for Malaysia and beyond.
Developing pangolin forensic capacity The transfer and application of such research initiatives into the international wildlife forensic community represents the next significant step
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in the use of forensic science in investigations into illegal pangolin trade. For the results of pangolin sample analysis to be used successfully in law enforcement, it is necessary to raise awareness and build expertise from crime scene to courtroom. The use of biological material as forensic evidence requires seizures to be made following robust protocols that maintain both the evidential and biological integrity of the pangolin products confiscated. Laboratories analyzing such evidential samples need to operate under strict quality management systems using test methods previously validated to generate robust, reproducible, accurate results. Lastly, forensic reports must be generated and communicated to the legal profession, such that the prosecution, defence and judiciary alike are capable of evaluating and accepting the evidence placed before them. These issues are not unique to pangolins and the international wildlife forensic community works to ensure that national enforcement agencies have access to forensic services that offer internationally recognized tests delivered within a common forensic framework. The Society for Wildlife Forensic Science (SWFS) sets international standards for the discipline. Long-term capacity building programmes supported by The United States Agency for International Development (USAID), the U.S State Department and the European Commission are all contributing to the dissemination and harmonization of best practice in this field, largely implemented through the work of technical specialist organizations such as TRACE Wildlife Forensics Network and the Netherlands Forensic Institute (NFI). A number of initiatives specifically relating to pangolin forensics are being developed within these over-arching programs, including field officer training in the identification of pangolin parts to support seizures, protocols for the sampling and collection of pangolin evidence for forensic analysis, laboratory training in DNA
recovery from pangolin scales, and the production of guidelines for the coordination of pangolin seizure analysis among countries and continents (Fig. 20.2).
Conclusion The development and application of wildlife forensic analysis is driven by the needs of the enforcement community. Enforcement agencies may be looking for intelligence level data that inform investigations concerning trade patterns and likely shipment routes, or they may need forensic evidence concerning the identity of a sample for use in legal proceedings. These requirements must guide the scientific community in how it can help to support the ongoing fight against the illegal pangolin trade. Within the field of wildlife forensics, only a subset of possible methods and applications are currently applicable to pangolin law enforcement; these focus primarily on species identification and geographic origin assignment. Species identification of whole specimens can be achieved using expert morphological examination, or more routine DNA sequencing analysis. Approaches for determining geographic origin are available for certain species in specific regions and work is underway to extend these capabilities to enable all pangolins to be traced back to their origin to at least some level of geographic resolution. As with other species subject to wildlife forensic investigation, the initial development of analytical methods and their subsequent validation and application to legal casework crosses the boundary between academic research and forensic casework laboratory environments, requiring informed coordination between these two scientific communities. As the need for analytical data relating to the illegal trade in pangolins increases, the
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FIGURE 20.2 Proposed guidelines for wildlife forensic investigations involving pangolin confiscations. Excerpts from UNODC, 2014.
response from the scientific community is leading to increases in the availability of both pangolin-specific techniques and laboratories capable of implementing them for forensic application.
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