Forensic Science International: Genetics 10 (2014) 1–11

Contents lists available at ScienceDirect

Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig

Review

Current and future directions of DNA in wildlife forensic science Rebecca N. Johnson a,*, Linzi Wilson-Wilde b, Adrian Linacre c a

Australian Museum Research Institute, Australian Centre for Wildlife Genomics, Science and Learning Division, Australian Museum, Sydney, Australia Australia New Zealand Policing Advisory Agency – National Institute of Forensic Science, Melbourne, Australia c School of Biological Sciences, Flinders University, Bedford Park, Adelaide, Australia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 June 2013 Received in revised form 13 December 2013 Accepted 18 December 2013

Wildlife forensic science may not have attained the profile of human identification, yet the scale of criminal activity related to wildlife is extensive by any measure. Service delivery in the arena of wildlife forensic science is often ad hoc, unco-ordinated and unregulated, yet many of those currently dedicated to wildlife conservation and the protection of endangered species are striving to ensure that the highest standards are met. The genetic markers and software used to evaluate data in wildlife forensic science are more varied than those in human forensic identification and are rarely standardised between species. The time and resources required to characterise and validate each genetic maker is considerable and in some cases prohibitive. Further, issues are regularly encountered in the construction of allelic databases and allelic ladders; essential in human identification studies, but also applicable to wildlife criminal investigations. Accreditation and certification are essential in human identification and are currently being strived for in the forensic wildlife community. Examples are provided as to how best practice can be demonstrated in all areas of wildlife crime analysis and ensure that this field of forensic science gains and maintains the respect it deserves. This review is aimed at those conducting human identification to illustrate how research concepts in wildlife forensic science can be used in the criminal justice system, as well as describing the real importance of this type of forensic analysis. Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: CITES DNA Mitochondrial DNA STR Wildlife

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Wildlife forensic science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legislation and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . International trade and legislation . . . . . . . . . . . . . . . . . . . . . . 2.1. 2.2. National regulation and legislation . . . . . . . . . . . . . . . . . . . . . DNA-based tests in wildlife forensic science . . . . . . . . . . . . . . . . . . . 3.1. STR loci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allelic ladders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Allele databases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Mitochondrial DNA typing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Data analysis – species identification . . . . . . . . . . . . . . . . . . . 3.5. 3.6. Data analysis – individualisation and population assignment New DNA-based technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Accreditation and certification in wildlife forensic science . . . . . . . . Accreditation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. 5.2. Certification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: +61 2 9320 6297; fax: +61 2 9320 6015. E-mail address: [email protected] (R.N. Johnson). 1872-4973/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsigen.2013.12.007

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1. Wildlife forensic science Wildlife forensic science is a wide-ranging discipline compared to human identification and takes many guises depending on the nature of the allegation. A key difference is that in alleged crimes against wildlife there can often be no ‘victim’ to provide information regarding the investigation. Additionally, the list of species encountered in wildlife forensic science is extensive in contrast to the single species analysed in human identification. Wildlife forensic science is no longer the realm of the ‘wellmeaning conservation geneticist’. Despite being a relatively new sub-discipline of forensic science, analyses of evidence handled in wildlife crime cases may be presented in court in just the same way as more traditional forensic evidence; it therefore should be treated as such. From a quality control and quality assurance perspective, a complicating factor is that most accredited forensic laboratories and associated scientists do not handle non-human samples. This is due to the particular complexities of wildlife crime analysis techniques requiring a completely different expertise and skill set to those possessed by scientists in traditional forensic laboratories [1]. The scope of sample types encountered in wildlife forensic science is vast, an analysis can be requested for whole animals (live or dead), skins or skeletons of vertebrate species, exoskeletons and shells of invertebrate species (such as butterflies, rhinoceros beetles and mollusc shells) and animal body parts (intact or processed, such as internal organs, whole feet/legs/wings/heads/ fins, furs, feathers, scales, teeth, beaks, claws, muscle fillets, powdered shells/skeletons/skins and blood samples); see for instance [2–11] and examples in Fig. 1. Additionally, some cases can involve trace DNA [12–16] or mixtures of genetic material [17]. The most frequently requested analyses are for species identification, individualisation or assignment of unidentified sample/s to a particular individual. For both species identification

and individualisation analyses it is not uncommon for a scientist to encounter a species that has not previously been worked on in a forensic context. This requires the scientist to conduct extensive research to identify previous studies that have described the taxonomy of the species or any known congeners (closely related species), and then choose markers that are appropriate for both the forensic question and animal group. When conducting a species identification test, morphological or molecular techniques could be used depending on the state of the specimen being analysed. For molecular techniques, scientists may choose specific markers designed for species identification of mammals [12,18–22] which could be different from those used in reptiles [13], amphibians [23,24], fish [25,26] or birds [3,27]. Marker choice is vital and must take into account the known evolutionary relationships of the group, for example did they diverge relatively recently from a common ancestor? If so, a faster evolving gene would be recommended for species identification. Similarly for alleged wildlife crime cases requiring individualisation, DNA is typically used. If markers do not exist for the species in question they must be developed and validated. Further, the scientist should take into account any known characteristics of the population such as high levels of relatedness and local genetic endemism [28,29]. A set of 13 recommendations by an ISFG Commission on the use of non-human DNA samples in the criminal justice system has previously been proposed [30]. More recently in the USA the Scientific Working group on Wildlife Forensic Science (SWGWILD) have developed a comprehensive set of Standards and Guidelines (accessed from www.wildlifeforensicscience.org/swgwild/). Together, these documents illustrate the desire within the wildlife forensic science community to implement best practice by incorporating these recommendations, standards and guidelines. It is essential that the discipline of wildlife forensic science remains in step with the broader forensic community ensuring the highest

Fig. 1. (a) Seized bird egg and embryo from the order Psittaciformes requiring DNA analysis for definitive species identification. (b) Rhinoceros horn (family Rhinocerotidae) requiring DNA analysis for definitive species identification.

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standards are in place. This review is aimed at readers of FSI Genetics familiar with human identification methods to illustrate the need for good quality wildlife DNA testing and how this relatively new discipline is meeting the challenges of attaining the same standards accepted in human identification. 2. Legislation and regulation 2.1. International trade and legislation The global trade in wildlife is regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Countries may make formal declarations to be bound to the CITES Convention, however this is voluntary and countries may withdraw at any time. There are currently 178 countries who are signatories to the CITES Convention (see www.cites.org). CITES provides a mechanism to regulate trade and minimise the impact on threatened or endangered species. It operates by listing species on one of the three CITES Appendices according to the level of protection the particular species requires. Appendix I lists species whose trade is prohibited (with exceptions for non-commercial purposes such as research) and includes species threatened with extinction. Appendix II lists species whose trade is regulated so that they do not become endangered. Appendix III lists species (or sometimes even specific populations) requested by a particular country that may be threatened in that country but not in another (Chapter 1 in [31]). Once a country becomes a Party to CITES it is legally bound to the Convention for the period it remains a signatory (as Parties can withdraw) and is required to implement a licencing system through the national adoption of domestic legislation. Approximately 5000 species of animals and 29,000 species of plants are listed on one of the three CITES Appendices. The identity of a species involved in compliance or regulation is paramount as the prosecution of offenders relies on the ability of investigators to prove the wildlife crime evidence relates to a species listed within a CITES Appendix. Species may be listed as individual species, as groups, sub-species or a geographical population (such as when a country lists a species population on Appendix III). In some cases it can be difficult to identify a particular species, as genetically the concept of a species is not always clearly defined and legal and scientific definitions are not always aligned. Species delimitation can also be complicated due to hybridisation, introgression, homoplasy and cryptic species [32]. At the international level there are many groups operating in both government and non-government arenas to combat wildlife crime. This includes Interpol, the world’s largest international police organisation, based in Lyon, France. Interpol has 190 member countries and in 1992 formed the Environmental Crime Committee, under which sits the Wildlife Crime Working Group and the Pollution Crime Working Group. The Wildlife Crime Working Group meets every two years to develop and facilitate projects to combat the poaching, trafficking, or possession of legally protected flora and fauna. In 2009 Interpol launched the Environmental Crime Program in response to increasing links between environmental crime and organised crime. In March 2012 the Environmental Crime Committee was re-structured into the Environmental Compliance and Enforcement Committee (ECEC), which now includes the Fisheries Crime Working Group. In addition to the Interpol initiatives the International Consortium on Combating Wildlife Crime (ICCWC) was created in November 2010. The Consortium consists of a partnership between CITES, Interpol, the United Nations Office of Drugs and Crime (UNODC), the World Bank and Customs. The aim of the group is to provide co-ordinated support for national wildlife crime enforcement agencies. Assistance is focused in the areas of

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education and training, capacity building, information exchange, skills co-ordination, provision of relevant tools and generally promoting the issue of wildlife crime. 2.2. National regulation and legislation At the national level each member country to CITES must enact federal or national legislation to facilitate the requirements as detailed in the CITES convention. For example in the USA the Endangered Species Act 1973, the Marine Mammal Protection Act 1972 and the Lacey Act 1900 and local state laws and regulation all assist in the legislation and regulation of wildlife crime. The main agencies working in the US are the Environment Protection Agency, the National Parks Service and the US Fish and Wildlife Service (USFWS). The USFWS operates the world’s only dedicated forensic facility for wildlife crime; established in 1979 in Ashland, Oregon. In Europe, the European Union (EU) is not technically a signature to the CITES Convention (although member EU countries are) but would be admitted as a member as an economic integration organisation under the Gaborone Amendment to the Convention, once the amendment is ratified. Currently the EU establishes regulations that apply to the member countries and also EU Directives. An example being Regulation 338/97/EC provides for the implementation of CITES and Directive 92/43/ EEC on Fauna-Flora-Habitat and Directive 2009/147/EC on Birds. In Australia the Environment Protection and Biodiversity Conservation Act 1999 enacts the CITES Convention. The Act is regulated by the Department of Sustainability, Environment, Water, Populations and Communities (SEWPaC). SEWPaC also works with the Australian Customs Service and the Australian Quarantine and Inspection Service for border control and the Environment Departments at the State and Territory level. SEWPaC also supports the Australasian Environmental Law Enforcement and Regulators Network (AELERT). Effective control to combat and minimise wildlife crime requires a co-ordinated network of police and enforcement agencies, customs, quarantine, border control agencies and nongovernment organisations (e.g. World Wide Fund for Nature (WWF)). Unfortunately this rarely exists. In addition, environment enforcement agencies often operate differently to crime investigation agencies (i.e. police) in that they do not have access to the same level of infrastructure and communication channels, including forensic analytical services. Often environment officers rely on ‘traditional’ policing techniques such as surveillance, which are resource intensive and time consuming. Any evidence in cases of wildlife trade must also meet legal evidentiary requirements. Local legislation applicable in these cases can include anything from statutes (both state and national) to protect endangered species, to enforce local quarantine restrictions, legislation to prevent cruelty to animals, and laws that dictate various fishing and hunting practices. The transnational nature of wildlife crime can be challenging to prosecutors, particularly the trade in endangered species as it frequently crosses international borders meaning inter-country co-operation is desirable [33]. DNA-based testing is frequently used in the testing of samples suspected to be on the CITES list of protected species. To date the most common application is in determining species identification, but increasingly DNA is being applied to analyse geographic location of origin or claims that a listed species has been captive bred as opposed to taken from a wild population. 3. DNA-based tests in wildlife forensic science The types of DNA markers used in wildlife forensic science are more varied than those familiar to the forensic human identification

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community due to the complexities of species identification. Microsatellite markers (or Short Tandem Repeat – STR loci) and increasingly Single Nucleotide Polymorphisms (or SNPs) are used in putative individualisation, pedigree assignment and also the assignment of an unknown sample to a population. Mitochondrial DNA (mtDNA) is most commonly used in species identification but can be used for putative individualisation where analysis of the female lineage is sufficient. 3.1. STR loci Use of DNA for individual identification in wildlife forensic applications is becoming increasingly frequent. This can involve investigation to determine if two samples are from the same individual, to determine the pedigree of a particular individual or for questions requiring assignment to population of origin. Human identification for forensic purposes is currently based predominantly on STRs [34,35]. Polymorphic STR loci have been identified and characterised on autosomal chromosomes with an initial use of four loci in 1994 [36] evolving to 24 loci in 2013 with the introduction of GlobalFilerTM from Life Technologies. In parallel, STR loci on both the Y and X chromosomes now complement these autosomal loci allowing a large suite of polymorphic STR loci to be employed in a criminal investigation or for civil purposes such as paternity testing [37–41]. These loci have been developed for forensic purposes with the aim of determining if there is a statistical link between an unknown biological sample and a reference DNA profile. In this regard STR loci are highly effective with the combination of 21 loci having powers of identification in the order of 1018 [42]. The identification of such loci was only possible due to an increased knowledge of the human genome sequence [43], which provided the ability to map each locus to its specific location on each chromosome. Each locus should be tested for genetic linkage, polymorphic content and inheritance in a standard Mendelian manner [44]. Standard human identification will use one of a battery of commercially available kits where the primer sets have been premixed at appropriate concentrations to allow even amplification across all the loci. Ideally, the same extensive characterisation of STR loci is required if they are to be used in a forensic wildlife investigation. This can lead to a significant amount of investment if a separate set of STR loci is needed for every species encountered in wildlife forensic science. Further, there is a requisite level of knowledge required for development of markers in species that can range widely from a beetle to a snake, an exotic bird to a wombat, or an oyster to mahogany. There is only a limited number of species for which adequate STR loci have been characterised; and this has not been predominantly for forensic purposes but for commercial or conservation genetic reasons [29,45–50]. If markers need to be developed and/or validated the resource implications can be high, particularly in terms of time and funding. This can be a delicate balance as there are often limited resources for enforcement agencies to spend on development and validation. Human identification settled on tetra-repeats from the outset and through extensive research on the human genome ensured that the loci are either on different chromosomes or are not genetically linked. The most common STR loci to be isolated are dinucleotide repeats where there are issues with increased stutter [51]. The International Society for Animal Genetics still advocates the use of di-nucleotide repeat STRs and these loci are commonly used for domesticated species [52,53]. Ensuring that the STR loci used in wildlife investigations behave as if independent, with sufficient studies to ensure equilibrium, are required; which again requires time and resources that are not commonly available to a forensic wildlife laboratory. Simple tests, however, for linkage

disequilibrium should be routine for any set of loci to be used in a wildlife forensic context [54]. 3.2. Allelic ladders Comparison of alleles to an allelic ladder is routine in human identification with all commercially available STR kits having a ladder supplied. The companies that supply these products have taken the time to ensure that the ladder is as comprehensive as possible. The construction of an allelic ladder is complex but does allow for laboratory-to-laboratory comparison and accurate allele designations making all results from all machines and laboratories directly comparable where it is used. This process is a recommendation of the ISFG Commission on non-human DNA testing [30], yet not accepted by all in the area of wildlife forensic science due to the obvious difficulty in this work [55]. Whether a complete and comprehensive allelic ladder or representative alleles, where available, are used as a separation control, unambiguous allele designation is required to allow inter-laboratory sharing of data to occur. 3.3. Allele databases Allied to STR testing is the requirement for an allele frequency database used to calculate statistical weightings of match results. Sampling 250 humans from different local populations is not a difficult task, providing ethical approval is obtained, and is standard practice prior to the use of any STR locus. Allele frequencies of numerous human populations are published in every edition of FSI Genetics as a Letter to the Editor and represent a constantly enriched and updated aspect of the scientific literature for the forensic DNA community. For non-human forensic DNA testing, the development of an allele frequency database may be possible in the case of captive and commercial species, or if the species is abundant. It is more often the case that forensic investigations are needed as the species is endangered and protected. Obtaining samples from such wild species can be problematic, particularly so for rare and endangered species where the numbers in any one population may be limited. Additionally, obtaining samples for a research type project can be difficult if the species is subject to restrictions via legislation and/or CITES permits. Recent instances of such validated databases include the European brown bear [51] and wolves [2,50] but these are few in comparison to the large number of species encountered in wildlife forensic science. It is normal practice to collect samples from individuals not known to be directly genetically related and then account for shared alleles as identical by descent with the use of a kinship factor. In many human populations this kinship factor (via an estimate of FST or theta (u)) is commonly between 0.01 and 0.03 for small populations [44]. Rare and endangered species are very likely to be highly inbred with a large amount of homozygosity and deviation from Hardy–Weinberg Equilibrium. Further, there may be significant population sub-structuring in some species for which difference in allele frequency may be significant. Any population sub-structuring should also be accounted for using an estimate such as FST. The use of a relatively high u value, such as assuming a highly structured population where all members of the local population are second cousins, is probably conservative and an overestimate of the actual genotype frequency [56] but should be considered on a case-by-case basis. In all such calculations, a u value needs to be used and the value must be justified and ideally conservative. It has been suggested that a realistic u value in wildlife populations is 0.05 (Chapter 12 in [10]) also suggested as suitable by Menotti–Raymond for cats [21].

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Allele frequency databases are likely to be based on a limited subset of the population and therefore many rare alleles may not be included in the database in the absence of extensive sampling. A minimum allele frequency needs to be applied on a regular basis. A figure of 5/2N, where N is a reflection of the size of the database is used in human populations (see Chapter 5 of [35] and Chapter 5 of [11]) where there is a large enough population sample size and known karyotype; although for smaller populations a figure of 2/8N can be applied (Chapter 5 of [11]). The temporary addition of alleles to the database, adding 2 if a heterozygote or 4 if a homozygote [56], is logical compared to the arbitrary assignment of a minimal frequency, but both methods have advantages and limitations when dealing with alleles that are rare or absent due to problems with sampling. 3.4. Mitochondrial DNA typing MtDNA typing is more commonly used in forensic wildlife applications for three key reasons. The first is that mitochondrial loci are regularly used in molecular taxonomy and phylogenetics for the purpose of species assignment and therefore there is already a wealth of data available in the scientific literature. The second is that universal primers exist that can be applied to almost any unknown sample and generate a result. Thirdly many of the samples encountered in wildlife forensic science are highly degraded and therefore nuclear markers have a low amplification success rate compared to mtDNA which is present in up to 1000fold higher quantities than the nuclear genome depending on the tissue type [57]. The most commonly used mitochondrial loci in molecular taxonomy and phylogenetics are the cytochrome b (cyt b) gene [3,6,7,11,13,18,58–63] and the cytochrome oxidase 1 (COI) gene [11,64–72]. While other loci on the mitochondrial genome have been used, such as the two ribosomal RNA genes [73,74], the control region (or D-loop) [75], and subunits of mitochondrial encoded NADH dehydrogenase [76]. However there has been a lack of standardisation in determining the preferred locus for use in species identification. Historically cyt b was used for most animal species testing [4–6,12,18,23,58], however, more recently COI has been promoted by the Barcode for Life Consortium [65,77]. This lack of standardisation may seem odd to those not familiar with species testing but equally it would be odd if only one locus was suitable for all species, from insects to birds, grasses and deciduous trees. Instead, standardisation within a taxonomic class should be supported by the science and the availability of data. The availability of data for the locus chosen for a species test should be a guide along with the polymorphic nature of the sequences used. Ideally there should be reliable sequence data for the species under consideration along with species known to be closely related genetically. It should be noted that green plants use two loci on the chloroplast genome for species assignment and therefore a different choice of loci for species testing from either cyt b or COI. Information on species testing in plants can be found in various articles [78–81]. Within the mitochondrial gene loci there are signature DNA bases or sometimes ‘fixed differences’ for particular species. These bases can only be identified by aligning large amounts of sequence data for each locus to detect interspecies variation and measure any amount of variation within a species (also known as intraspecies variation). By designing primers to these single nucleotide polymorphisms and using standard methods in forensic science such as SNaPSHOT1 it is possible to develop and validate a method suitable for the criminal justice system [82]. A complete specificity test is required to ensure that the Single Nucleotide Polymorphism (SNP) test provides unambiguous species identification. Unlike other methods of species identification, described in

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Section 3.5, SNP testing provides essentially a yes/no result and hence caution is required in using such a result unless there is an extensive specificity study to support the conclusion. MtDNA sequencing is used in human identification where the DNA is highly degraded and the opportunity for nuclear DNA testing is limited [83–87]. Forensic laboratories that conduct mtDNA testing for human identification purposes tend to be highly specialised as the risk of contamination from the operator or other contemporary extraneous sources is extremely high [88]. Many smaller operational laboratories may not have the space and resources to develop a mtDNA laboratory. In Australia for instance, human mtDNA testing for court purposes is only conducted in two laboratories; yet it is a standard tool in wildlife forensic DNA testing to determine species. Only a few wildlife DNA laboratories have the facilities to perform this type of testing to the same standards expected in human forensic identification. It should be noted that a benefit of working on non-human mtDNA is that contamination from the operator is more easily detected than for human identification. However, care should still be taken to avoid cross-contamination between different species being worked on in the same laboratory when broad-spectrum DNA markers such as COI are being analysed. DNA sequences from mitochondrial loci such as cyt b or COI generated from samples submitted for a forensic investigation need to be compared to a reference DNA sequence. One of the key recommendations of the ISFG Commission [30] is that this reference sample should come from a voucher specimen (i.e. a specimen that has been lodged in a museum, herbaria or other designated ‘collecting institution’); yet very few laboratories have access to such samples as these are only available from certain museums, zoos or botanical gardens and these collections are almost never comprehensive, potentially leaving gaps in the reference data. Instead there is a reliance on DNA databases such as GenBank (or BOLD for COI data, some from voucher specimens). For many in the wildlife arena GenBank has been for many years a tremendous benefit as it is an extensive repository of sequence data. A downside that needs to be appreciated is that GenBank has limited regulation and there are erroneous sequences on the database [89–91] meaning that any analyst using GenBank derived data must have the appropriate expertise and experience to rigorously quality control the data. Analysis of results obtained via mtDNA typing methodology is analogous to the sequence comparison of HVI or HVII for human identification, except instead of a revised Cambridge Reference Sequence, sequences from reference sources are used (as discussed above from either a voucher specimen, or if not available, GenBank or BOLD). Reporting of results between an unknown sample and a reference sequence tends to be reported as either a statistically supported match, i.e. the unknown sample is from the matching species, or a definitive exclusion, i.e. the unknown sample is not from the species of the voucher sample. In human identification, studies and publications have addressed the issue of a mismatch of 1, 2 or more bases when comparing sequences from the hypervariable loci between 2 samples; with recommendations on the reporting in such circumstances [92–94]. These recommendations are based on the knowledge of mutation rates at these sequence sites after conducting studies of large amounts of population data. The same in-depth studies are rare in wildlife forensic science as they would require large numbers of DNA samples from known mothers and their offspring. While a 100% match with 400 bp of cyt b gives extreme confidence in the match for most species, more frequently there is less than a 100% match between sequences. Further, the difference in using a 400 bp sequence of cyt b compared to a 648 bp sequence of COI on the results of a species identification test is difficult to apply as a fixed ‘rule’. This is due in part to the diversity of species

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Fig. 2. An example alignment of COI mtDNA data (607 bp gene region) in the Molecular Evolutionary Genetics Analysis (MEGA) program across divergent taxa of birds and mammals.

that may be subject to analysis and the lack of intra- and interspecies boundaries for many closely related species. Frequently, the DNA will be degraded such that only a fragment of the sequence of these loci is available, hence there may only be a match of 99 bp out of 100 bp for either locus and it requires an experienced wildlife forensic scientist to ascertain whether there can still be a high level of confidence in the match given the limited sequence information. For mammals there have been some indepth studies into intraspecies and interspecies variation at commonly used loci [95] however for many other taxonomic groups this is rarely available. 3.5. Data analysis – species identification Allied to new methodologies in DNA sequencing is the concordant development in software coupled to improvements in computing technologies to analyse the data. Advances in analytical processes and the reduced costs of Sanger sequencing have allowed much greater data than ever to be obtained. However, the collection of data is only of value if it can be interpreted, evaluated and supported conclusions made. Sequence matching, as outlined above, is the first step in species identification. To include the necessary statistical robustness for identification to some taxonomic level (i.e. species, Genus, Family) a phylogenetic tree is frequently constructed. This is where some level of zoological or botanical knowledge or reference to botanical or zoological literature is required. The phylogenetic tree should contain reference sequences from the most appropriate species (or genera) available and its branches should also include a measure of statistical support that is known to be taxonomically robust for the species in question. Known and unknown sequences from the genetic marker analysed must be confirmed and aligned. Molecular Evolutionary Genetics Analysis (MEGA) [96] and Clustal are standard free

software for the alignment of DNA sequences and are frequently used in forensic analysis (an example alignment for partial COI gene region across diverse taxa can be seen in Fig. 2). Sequencher and Geneious are commercially available software that perform sequence alignments. These software programs allow the rapid alignment of unknown DNA sequences to known sequences allowing a confident assignment of an unknown sample to a particular species. Once sequences have been confirmed and aligned, and any missing data accounted for, phylogenetic trees should be constructed. This will allow for placement of the unknown sample onto the tree so that the clustering on the tree of the unknown sample can be determined. The nodal length and bootstrap support will indicate the relationships and strength of the population group and the unknown sample. Software algorithms used in phylogenetics include: Maximum Likelihood [97], Maximum Parsimony [98], Neighbor-Joining (NJ) [99], and Bayesian [100], using various programs such as MrBayes [101]. An issue familiar to those who generate phylogenetic trees using these programs is that a different tree can be generated from the same data using different programs, particularly if there is variability in the data. For example, if one laboratory uses Maximum Likelihood (ML) and another Maximum Parsimony (MP) then the results may be different due to different underlying assumptions in these algorithms, this is especially so if the data are weak or do not contain an appropriate level of genetic variation. Currently there is little international standardisation as to which program to use; this has potential problems when presented in the criminal justice system. Choosing the appropriate reference sequences from the correct population and also the appropriate marker for that population is extremely important to ensure that the relationships of the population are being measured and not the evolution of the gene/marker. Where possible, trees should also be rooted with an out-group. Regardless of phylogenetic method used

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Fig. 4. Species identification (highlighted) of an unknown sample using appropriate congeneric and family level mammalian reference sequences. The tree depicted was calculated using the Maximum likelihood algorithm and 500 bootstrap replicates. The same tree topology was obtained using both Maximum Parsimony and Neighbor-Joining methods.

Fig. 3. Comparison of tree topology using the three most commonly used treebuilding algorithms using the aligned data set in Fig. 2 showing robust taxonomic groupings. (a) Neighbor-Joining tree, (b) Maximum Likelihood tree, and (c) Maximum Parsimony tree.

however, providing there is robust support for nodes which are correctly interpreted in the context of appropriately chosen reference sequences by the analyst, it should not lead to an incorrect identification. Fig. 3 depicts trees built using three different algorithms from the dataset presented as an alignment in Fig. 2 and demonstrates poor tree resolution when extremely divergent taxa are used. The two robust groupings – Family Bovidae and Order Primate are highlighted. Fig. 4 shows a more focused group of reference sequences and includes placement of an ‘unknown’ sequence within the tree allowing for species identification. Which tree is used is dependent on the theoretical approach taken as each of the methods operates in different ways. The NJ tree uses a distance method that finds the shortest distance between taxa by converting the sequences to pairwise distances. The method is simple and therefore fast, however, it has been largely superseded by other more advanced techniques as it is affected by variations in rates of evolution. The strength of the nodes on the tree can be displayed as bootstrapped values. The MP and ML methods are discrete methods that look at the sequences themselves. MP looks for the ‘least steps’ to explain the differences between the sequences being compared. It is still a relatively simple method and quick on small data sets. MP does not perform well when there are variations in the evolutionary change along the branches of the tree as MP tends to prefer longer tree branches. ML looks for the tree that is most likely (likelihood function) to explain the observed data. However, it can be time intensive and large data sets may require an online bioinformatics platform for analysis; further if a chosen evolution model for nucleotide substitution is inappropriate it can lead to inconsistent results. The strength of the nodes on the MP and ML trees are also displayed as bootstrapped values. Bayesian inference using Markov Chain Monte Carlo has become more popular in recent years. The trees can then be summarised in a number of ways such as the majority rule consensus tree. The strength of the nodes on the trees are displayed as probabilities for the clades being true given the priors. As with ML the results of the Bayesian inference can be inconsistent if the incorrect model of evolution is chosen, however the Bayesian method offers a robust model of phylogenetic reconstruction with a decrease in the computation time required compared to ML. 3.6. Data analysis – individualisation and population assignment As has already been discussed above, a significant time and resource investment is typically involved in developing and

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validating markers suitable for individualization for the purposes of wildlife forensic analysis. The validation and analysis process should involve assessment of the number of alleles for each locus, the observed and expected heterozygosity for each marker, along with the probability of identity (i.e. the probability that two animals share a common genotype by chance), often called the Population Match Probability, for each species to maximise the exclusion power for each suite of markers. Further, for pedigree analyses, when at least one parent is known the parentage exclusion probability can be calculated. Programs commonly used for these purposes include CERVUS and GenAlEx [102,103]. Broadly, individualisation data can interpreted in the following ways:  Exclusion = non match  Inclusion = match; but requiring a statistical estimate to assess likelihood  Inconclusive = no decision can be made Population assignment is possible if there is enough resolution in the data to provide statistical power to associate a particular sample with a specific population from a known location, however if it is a species subject to a lot of movement (human mediated or otherwise) inconclusive results for population assignment may be obtained. Population assignment is typically done using software such as STRUCTURE [104] where a DNA sequence can be compared to data sets from known populations (Chapter 5 in [11]). The use of STRUCTURE has been shown in the tracking of wolf [2,105] and bear populations [51,106]. Population assignment using STR data has been applied to investigations in the movement of ivory [8,107–109]. Further examples include the population assignment of commercially caught fish and shark species [110]. Population assignment to a high degree of confidence is possible if there is extensive sampling in the populations beforehand and if there are distinct sub-populations developing. As noted, all the above methods and software were developed for other purposes and then adopted for forensic science applications. Validation of any new process is a key aspect when transferring to the criminal justice system. Experiments designed to address sensitivity, specificity, stability, reproducibility, robustness, reliability, and false positives and negatives are familiar to the forensic science community. These same processes are less familiar those to conducting scientific research outside of the forensic community. Implementation and adherence to quality assurance and quality management systems offer a framework within which potentially complicating variables can be detected, controlled and then can be mitigated.

4. New DNA-based technologies Decoding complete genomes was an extremely lengthy and laborious process using Sanger sequencing [43]. The advent of mass parallel sequencing, also termed Next Generation Sequencing (NGS) or high-throughput sequencing, has revolutionised this process such that whole genomes can be sequenced within a few weeks and at a fraction of the cost [111]. Comparison of whole sequence data currently provides limited evidential value as closely related species will share much of their DNA. The process of mass parallel sequencing allows for the identification of repetitive DNA sequences and has led to significant reduction in time for the identification of new STRs and other highly informative markers such as SNP [112–117]. These newly detected STR loci can later be characterised for their polymorphic content, number of alleles, heterozygosity, linkage, and other relevant forensic parameters that must be considered, such as likelihood of size homoplasy,

possibility of null (or no amplification) alleles and whether the marker is under selection or neutral. NGS has recently been cleverly applied in a Research & Development capacity to investigate the content (often of mixed species of origin) of some traditional medicines [17]. Once properly validated, this could become an important tool in investigating species content and even quantifying the content of such samples. Additionally, recent improvements to new synthetic DNA polymerases allow significantly longer amplifications of sections of DNA combined with increased enzyme accuracy. Amplification of over 10 kb is now possible. As a result, instead of using 20 primer pairs to amplify a whole mammalian mitochondrial genome it now only requires three primer pairs (personal observation based on [118]). Further, complete mitochondrial sequences can now be rapidly obtained by mass parallel sequencing in one reaction. The concept of choosing a specific locus to sequence on the mitochondrial genome becomes less of an issue if data from longer sequence regions can be rapidly obtained on an individual basis. This is only an option if the DNA is not degraded, or if smaller sections of fragmented DNA can be associated together to create a contiguous sequence. 5. Accreditation and certification in wildlife forensic science 5.1. Accreditation Recent years have seen a significant increase in the number of forensic science laboratories being accredited to the international standard ISO 17025 globally; this standard is focused mainly on testing and calibration with a strong emphasis on quality and documentation. The standard is not directly aimed at forensic science testing but can be adapted to provide an overall quality system for a laboratory and is appropriate for application in wildlife forensic science. The process of accreditation is expensive as nationally recognised companies must be engaged to facilitate and award accreditation status. The process can be onerous to go thorough but ultimately demonstrates to any outside agency that the laboratory processes meet the recognised international standard. The purpose of accreditation to a standard is to ensure the results of any testing by the laboratory are robust, repeatable and reliable. In order to achieve this ISO 17025 sets out a number of criteria that must be met by a laboratory. This includes documented procedures, validated/verified methods, appropriate and documented training for all laboratory personnel and regular checks of the work conducted by personnel involved in testing and the testing system. The development and maintenance of this quality process is time consuming and resource intensive and may not be possible for small laboratories, particularly in wildlife forensic science where laboratories may be housed in museums or universities. Additionally, accreditation may require extensive quality processes as a new analysis test may be required for each species and tests are often developed for each case, leading to an almost exponential documentation and testing requirement. Some European laboratories have chosen to implement accreditation to another standard; ISO 17020. However this standard is more applicable to crime scene investigations which rely more on recording and not laboratory based analysis techniques. More recently there has been a move to develop forensic specific standards, with Standards Australia developing a framework of four core forensic standards [119]. There is a possibility that these may be developed as international ISO standards, however again these may also be resource intensive to implement and therefore be prohibitive to smaller laboratories. Other agencies include universities where there may be academic staff with knowledge of population genetics or

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phylogenetics who may provide expert opinion and be familiar with the tests to be performed. To fulfil the same requirements as an accredited laboratory, the academic will need to adhere to previously written SOPs, take part in proficiency tests, and have their casefile reviewed along with any statements. Non-dedicated forensic science laboratories can play a role in wildlife forensic science provided that the working practices are recognised as meeting international standards and that all who work within the laboratory undertake and document the quality assurance process recognised by the rest of the forensic science community; see reviews on this topic [1,120]. Museum’s, herbaria and other collecting institutions have a particularly important role to play in wildlife forensic science through their voucher specimens and the potential for creation of additional gene databases (i.e. even though BOLD contains a suite of vouchered data it is only for the COI gene in animals) populated with sequences derived from appropriately identified voucher specimens. Working with local enforcement agencies it may be possible to develop a list of priority groups of species that are regularly encountered in an enforcement context. Using this list data from voucher specimens suitable for species identification, along with STR and/or SNP markers for individualisation, could be developed for use by analysts accredited to work with wildlife forensic samples. The combination of laboratory accreditation with the voucher specimens of a museum or herbaria can create a strong local hub for wildlife forensic analyses. In 2009 at the 23rd Congress of the International Society for Forensic Genetics in Buenos Aires, interested parties in the field of forensic genetics met and instigated a commission to recommend standards of best practice in the use of non-human DNA in forensic science. The culmination of this commission was the publication of 13 recommendations ranging from the use of voucher specimens to the production of a statement [30]. The commission was comprised of members of the ISFG Board plus three others with an extensive research record in this field. These recommendations were a development on recommendations contained in a previous publication [121]. 5.2. Certification The National Academy of Science (NAS) Report on forensic practice in the US criticised many areas of forensic science as lacking in scientific foundation [122]. In many instances human DNA identification was labelled as the ‘gold standard’. Some aspects of wildlife forensic science practice were open to criticism in the report. Significant recommendations of the NAS report included a recommendation for all practitioners to gain certification and all forensic laboratories to obtain accreditation. Also in 2009, a number of scientists with an interest in wildlife forensic science formed the Society for Wildlife Forensic Science (SWFS). The whole field of wildlife forensic science encompasses not only DNA, but chemical profiling and morphology, hence while there is an overlap between the ISFG and SWFS there are also members of SWFS (for example chemists or morphologists) that are not naturally members of the ISFG. One of the aims of SWFS is to promote ‘‘professional competence, uniform qualifications, certification and ethical behaviour among nonhuman/wildlife forensic scientists’’ [33]. To promote this aim and also to address some of the criticisms raised by the review of forensic practice in the US [122], the Scientific Working Group for Wildlife Forensics (SWGWILD) was formed in December 2010. Standards and guidelines developed by SWGWILD would be passed to members of SWFS for ratification. The first meeting was held in Ashland Oregon in 2010, and the second held in Jackson Wyoming in 2012 where SWFS members adopted and ratified the first set of Standards and Guidelines (www.wildlifeforensicscience.org/swgwild/). These

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guidelines and standards provide a comprehensive framework on how to attain and adhere to recognised best practice; particularly for small laboratories that are unable to attain ISO 17025 accreditation. Measuring professional competence in a forensic laboratory can be by a number of means. This includes successful completion of proficiency tests, which in the field of human identification includes samples to be tested by well-established STR markers. The field of wildlife forensic science is complicated greatly by the vast number of species that could form part of a proficiency test. It would seem to have limited relevance if a wildlife forensic scientist took part in the proficiency tests designed for human identification, yet this is perhaps of greater relevance than taking part in a proficiency test for a mammalian expert if the tests provided are for a fish species. If a scientist working on reptilian species in particular wishes to undergo proficiency testing then the cost of another laboratory putting such a test together is prohibitive at present. The only non-human proficiency tests are prepared and organised by members of the US Fish and Wildlife Laboratory in Ashland Oregon, hence the forensic wildlife community are reliant on this laboratory for all such tests. Certification of an expert is fraught with difficulty and can be open to criticism unless there is a rigorous and relevant assessment process. Qualifications by themselves indicate knowledge of the subject matter at the time of taking any test but are not always relevant to wildlife forensic science. Publication of research articles or attendance at conferences indicates continuing professional development but not necessarily a reason to certify someone. Examination of the casefile, as now adopted by SWGWILD as a key part of the certification process, captures aspects of the standards and guidelines as well as all those conducting casework. This process of certification is similar to the process developed by the now defunct Certification of Registered Forensic Practitioners in the UK which was, for its time, as rigorous a method of checking for competence as was possible. Any process, such as case file review, with a real and robust assessment process against published guidelines meets many of the criteria of certification. Certification, by whatever robust means attained, provides credibility to an expert witness and should be welcomed. 6. Future developments The immediate aim of the forensic wildlife community is to ensure that all its members can demonstrate that they work to a professional standard. Wildlife crime shows no sign of decreasing, rather the reverse is true, and hence there is more reason than ever to have a forensic process in place that meets international standards. Those involved in DNA typing for wildlife purposes need to be seen to be on a par with those conducting human DNA identification. The requirement to determine sample provenance via tracking populations and tracing samples by genetic testing is rare in human identification but is commonplace in wildlife genetics. The methods and samples used, along with the software applied, need to be demonstrably robust, reproducible and reliable and validated if used for court purposes rather than only as an investigative tool. By applying the principles outlined above we believe it will significantly advance the reproducibility and reliability of wildlife forensic analysis using DNA. In this way the courts can have confidence in the results and the expert opinion being provided. References [1] R. Ogden, N. Dawnay, R. McEwing, Wildlife DNA forensics—bridging the gap between conservation genetics and law enforcement, Endang. Species Res. 9 (2009) 179–195, http://dx.doi.org/10.3354/esr00144.

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