Species identification in wildlife crime investigation using diprotodontia

Abstract

This research investigated the issues surrounding species identification in a forensic wildlife crime context using Diprotodontia as a model group. Wildlife crime covers a broad range of offences where there is a deliberate and purposeful illegal activity involving animals and plants for which purposeful gain is the principle motive. Worldwide it is thought to cost between US$10 and US$20 billion dollars annually. Native Australian marsupials such as sugar gliders and wallabies are thought to be targeted for their unique appeal and are currently sold overseas as pets. Numerous marsupials are also the subject of regulated harvesting and international trade, some of which are listed on the Convention on International Trade in Endangered Species of Fauna and Flora Appendices. The application of molecular DNA techniques and population genetics theory, in the context of a broader understanding of genetic variation within and among taxa, can provide the basis for determining the provenance of animals or their parts that have been seized as a result of wildlife crime investigations or regulation of legal trade. Current human and non-human analysis systems and equipment in forensic laboratories were reviewed. This information was used to inform what changes in equipment and training are required to implement methods for species identification and the investigation of wildlife crime. Current expertise and methods used for species identification were assessed. It was found that the Ouchterlony antigen/antibody method was being used under the (now confirmed) mistaken belief that it was a definitive or confirmatory test, as opposed to an indicative or opinion based test, as it should correctly be used. Since the Ouchterlony test is limited, an alternative method for species identification is recommended. Informative nuclear markers have been shown many times to be useful in phylogenetic studies by resolving relationships between taxa. A survey of a broad range of nuclear markers for their phylogenetic utility was conducted in Diprotodonts. One hundred and two sets of primers for nuclear markers obtained from the literature were evaluated for phylogenetic performance using a representative phylogeny of Diprotodonts. Eighteen nuclear markers were identified as having potential for further work. Of these,nine were optimised and analysed using parsimony,likelihood and Bayesian approaches. Four new nuclear markers were developed to assist future genetic studies. A robust phylogeny must underpin any species identification test. A phylogeny containing a large number of taxa generated from nuclear and mitochondrial data was constructed. Mitochondrial markers COI and ND2 were combined with nuclear markers ApoB, IRBP and GAPD to amplify target sequence from 27 genera of Diprotodontia. Two suborders were resolved, Vombatiformes and Phalangeriformes. Phalangeriformes was subsequently split into two clades. The first clade contained the Macropodiformes and Burramyidae. The second clade contained Petauridae, grouping with Phalangeroidae. Of the markers tested, ApoB and ND2 provided the greatest number of diagnostic characters. ND2,owing to its presence on the mitochondria and therefore its ease of amplification in difficult samples, is recommended for use as a phylogenetics species identification tool to complement the COI barcoding marker for Diprotodonts. The use of the Cytochrome Oxidase I (COI) barcoding marker has been suggested as an alternative species identification test but would require a dedicated laboratory space and additional equipment and training. Whilst the technique is very similar to current methods and it would therefore require minimal training to conduct the analysis, the interpretation of the results would require significant training. The COI barcoding marker was assessed in detail using Diprotodontia as a model group and compared to two other forensically relevant mitochondrial DNA markers, Cytochrome b and ND2. The ND2 marker provided the most informative results for the Diprotodontia. However, the COI marker offers an international database of relevant sequences for comparison and as such might still provide the best solution for forensic laboratories. An additional recommendation arising from this work is that other supplementary markers, such as ND2,for Diprotodonts also be implemented as an adjunct to the COI barcoding marker. Degraded and difficult samples are often encountered in wildlife crime investigations. Shortened amplicons and primer modification techniques for improving the amplification efficiency of these types of samples were investigated. The COI 5’ 150bp shortened amplicon segment identified by the Barcode of Life Initiative for species identification was targeted in a range of Diprotodont samples. Shortened amplicons were found to be effective in increasing amplification efficiency. However, consistent amplification was difficult when targeting within a gene across multiple species, owing to the lack of conserved sites for primer binding. The COI shortened amplicon was found to be insufficient for species identification within the diprotodonts, although this segment may be useful in other species. Primer modification techniques focused on the use of Locked Nucleic Acids (LNAs) for improving the amplification efficiency of degraded samples. LNAs were spiked into primers for the entire COI barcoding marker and the primers producing the COI 5’ shortened amplicons. The addition of LNAs to the primers improved amplification efficiency by up to an order of magnitude. The design of the primers however was found to be the critical factor in amplification success of primers, even with LNA additions. Use of M13 primer additions was not recommended as these appear to interfere with the function of the LNA spiked primers. The results of this research highlight the difficulties associated with species identification and the broad level of underpinning information for species delimitation required to interpret the results. The final recommendation must therefore be that if DNA-based species identification is introduced into Government forensic laboratories, it be implemented in a ‘centres of specialisation’ approach where one laboratory becomes the expert service provider for all others. This specialised laboratory could act as a repository for forensically relevant samples, the analysis laboratory for samples of interest for crime investigations and provide a role for coordination of appropriate research and method development.

Date of Award	2010
Original language	English
Supervisor	Stephen Sarre (Supervisor), Arthur GEORGES (Supervisor) & James Robertson (Supervisor)