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Crime in Australia: International Connections Australian Institute of Criminology International Conference Hilton on the Park, Melbourne, Australia 29-30 November 2004 CONFERENCE PAPER: PHYLOGENETIC DNA PROFILING A Tool for the Investigation of Poaching Paul Roffey, Pam Provan, Michelle Duffy, Aisuo Wang, Chris Blanchard and Lyndall Angel School of Biomedical Sciences, Charles Sturt University (New South Wales) http://www.aic.gov.au/conferences/2004/index.html PHYLOGENETIC DNA PROFILING - A TOOL FOR THE INVESTIGATION OF POACHING 1 Paul Roffey, 2Pam Provan, 3Michelle Duffy, 4 Aisuo Wang, 5Chris Blanchard and 6Lyndall Angel 1 Lecturer, School of Biomedical Sciences, Charles Sturt University Honours Graduate, School of Biomedical Sciences, Charles Sturt University 3 Honours Graduate, School of Biomedical Sciences, Charles Sturt University 4 PhD Candidate, School of Biomedical Sciences, Charles Sturt University 5 Senior Lecturer, School of Wine and Food Science, Charles Sturt University 6 Head of School of Biomedical Sciences, Charles Sturt University 2 1 Abstract Poaching of abalone is a multi-million dollar crime and single-handedly is the greatest threat to the viability of the Australian and New Zealand abalone industries. Contained within the genome of each organism is a full history of its ancestry, documented as a series of accumulated mutations. The rate at which mutations accumulate in any particular gene sequence directly correlates with its importance in life functions where sequences involved with critical life functions accumulate mutations less rapidly. Hence mutation provides a window through which evolutionary relationships can be examined. Traditionally this procedure, known as phylogenetic DNA profiling, has been used to investigate the taxonomy of life forms and to study the origin and development of species. However, it has powerful potential as a forensic tool to assist in the investigation of poaching by being able to trace ancestry, and hence geographical origin, of poached specimens. In this paper we will show the forensic application of phylogenetic DNA profiling to the investigation of poaching using abalone as a model. Introduction Phylogeny is the term given to the field of science that focuses on the assessment of evolutionary relationships between organisms. Analyses of this type can be conducted across taxa from individuals to kingdoms. The analysis relies on the comparison of heritable traits. Pre-“DNA,” morphological, biochemical and immunological markers were used to determine relationships, however with the development of sophisticated molecular genetic technology emphasis has shifted towards the utilisation of genetic markers. Ultimately DNA is a more accurate measure of change since changes in morphology, biochemistry and immunology all result from changes at the genetic level. DNA essentially functions as an evolutionary clock, where the changes that occur over time are recorded as inherited mutations. The rate of mutation is constant, however the inheritance of mutations is dependant upon the function of the mutated DNA sequence; sequences that are involved in critical life processes accumulate mutations at a slower rate than those with less important function. In many ways the science behind phylogenetics is akin to the basic principles of photography. To be successful it must be focussed at a particular evolutionary depth. This is achieved through the careful selection of sequences that will provide the exposure, depth of field and resolution that is sought. The sequence chosen must be present in all the organisms to be examined (exposure). The more important the sequence, the more likely it will be maintained in a line of descent (depth of field). However, the more important the sequence the less likely the variation and therefore ability to distinguish (resolution). This process is less complicated than it sounds, for in many cases the examination of a single gene can provide resolution across large evolutionary scales. This is because different regions within genes are under different selective pressures. This in turn manifests as variations in sequence flexibility. In this paper we present phylogenetic analyses that were primarily targeted to Australian and New Zealand abalone species using a number of genomic and mitochondrial gene markers. The application of this technology to the abalone industry and the investigation of poaching is discussed. 2 Materials and Methods Abalone Species: Australian species examined were Haliotis rubra, H.conicopora, H.roei, H.laevigata and H.scalaris. The New Zealand species examined were H.iris, H.australis and H.virginea. Also examined was H.asinina which represents a species that inhabits diverse global geographical locations. Genomic DNA Extraction: Whole cell DNA was extracted from specimens using either the Chelex extraction method described by Walsh, et al, (1991) or salt purified (Aljanabi & Martinez, 1997) followed by standard phenol chloroform extraction (Sambrook, et al, 1989). Gene Amplification: Where possible, sequences were amplified using a standardised PCR procedure. Reactions were performed in a total volume of 25 µl using 1-10 ng template DNA. The final concentration of the components in reaction mix was as follows; 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.25 µM each primer, 200 µM dNTP’s, 1 unit Taq polymerase (Life Technologies, Australia). After an initial denaturation at 94°C for 2 min, the amplifications were performed using a “touchdown” amplification procedure consisting of 10 cycles of denaturation at 94°C for 45 sec, annealing for 10 sec at 62°C (decreasing 0.5°C per cycle) and extension at 72°C for 30 sec, then 35 cycles of denaturation at 94°C for 45 sec, annealing for 10 sec at 55-57°C and extension at 72°C for 30 sec. A final extension was performed for 10 mins at 72°C. Positive and negative controls were included in each reaction batch to verify the specificity and efficiency of amplification and to monitor for contamination. Genetic Loci: Sequences within the following genetic loci were targeted for amplification; 28s rRNA (genomic), Cytochrome Oxidase 1 (CO1, mitochondrial), 16s rRNA (mitochondrial), 12s rRNA (mitochondrial), Cytochrome Oxidase 2 (CO2, mitochondrial) and NAD Dehydrogenase 2 (ND2, mitochondrial). The 28s rRNA gene is a highly conserved genetic sequence whereas the mitochondrial loci represent more variable sequences. Of the mitochondrial sequences cytochrome oxidase 1 is the gene with greatest conservation. Electrophoretic Techniques: Preliminary analysis of amplification products was performed following separation in a 2% agarose gel. The size of fragments was estimated by comparison with known molecular weight markers (100 bp ladder, Progen) using the method of Priefer (1984) and the quantity of specifically amplified product estimated by comparison of fluorescence with bands of known concentration in the marker. DNA Sequencing, Sequence Analysis and Phylogenetic Studies: DNA sequencing was performed on 20-100 pg PCR amplified product using the DyeNamic fluorescent dye terminator sequencing kit (Amersham Australia) and analysed using an ABI Prism® 377 DNA Sequencer (Applied Biosystems). Sequences were compared to databases using the FastaA algorithm within the Australia Nucleic Acid Genome Information Service (ANGIS; http://www.angis.org.au), aligned using ClustalW and phylogenetic reconstructions performed using MEGA v2.1 (Kumar, et al, 2001) using the neighbour-joining (NJ) method. Results Phylogenetic analysis of the highly conserved 28s rRNA gene sequence. The H.australis 28s rRNA sequence was compared with sequences within databases containing DNA sequences of known origin. This analysis identified close similarities with over a thousand known ribosomal RNA sequences, and amongst those with the highest similarity were 18 3 gastropods each with a sequence similarity over 91%. By comparison, primates demonstrated a 8182% sequence identity when compared to H.australis and a 96-99% identity when compared to one another. Phylogenetic analyses clustered the gastropods into two major subgroups that were linked at a more distant level (Figure 1). This arrangement is consistent with the current classification of the Vetigastropoda and Ceanogastropoda respectively (both subgroups of the class Gastropoda). By comparison the primates grouped separately and demonstrated a very distant link with the gastropods. Haliotis (abalone) correctly grouped within the Vetigastropoda; this is a well documented and recognised evolutionary relationship. Phylogenetic analysis of mitochondrial gene sequences. The CO1 sequences were retrieved from all species tested. When compared with DNA databases CO1 sequences from a number of other abalone species were identified, including representatives from North America, and South Africa. Phylogenetic reconstruction using all CO1 sequences clearly separated the abalone species according to geographical location (Figure 2). Phylogenetic reconstructions for the 16s rRNA gene, 12s rRNA gene, ND2 and CO2 showed similar geographical separations. Discussion The abalone industry is a lucrative trade. Australia and New Zealand are amongst the few countries in the world that still have thriving abalone industries based on natural stock. Not surprisingly these countries are also prime targets for poaching. The policing of poaching is a difficult task which largely relies on either catching the poachers “red-handed” or detecting the sale of illegal harvests. In either case subsequent prosecution and conviction can often hinge on the ability of fisheries scientists to identify the abalone species and/or subspecies type and therefore the likely country of origin. Genetic tests that could identify abalone at the species and sub-species level will greatly improve the detection and prosecution of those involved in the illegal trade and/or poaching of abalone. The use of phylogeny in forensic application is not new. Indeed the standard DNA tests for human discrimination are classic examples of phylogenetic tests. Hence the application of this technology to poaching does not represent a completely new facet to forensic science. The challenge in application to non-human samples is to find the suitable sequences that are sufficiently different to be useful for phylogenetic studies but sufficiently similar to be used across the desired range of target species. Genes such as the 28s rRNA gene are very highly conserved hence have application at higher taxonomic levels. Indeed when applied to the gastropods it successfully resolved the order Vetigastrpoda from the remainder of the gastropods. This follows conventional taxonomic theory and reinforces a number of other genetic studies (eg Tillier, et al, 1992; Tillier, et al, 1994; McArthur & Koop, 1999; Colgan, et al, 2000; Ponder & Lindberg, 1996; Ponder & Lindberg, 1997; Salvini-Plawen, 1980; Salvini-Plawen & Haszprunar, 1987). It is clear that application of this gene to forensic samples would be useful to confirm abalone identification at the genus, family and order levels. Species identification requires genetic sequences with greater variation. The mtDNA genes examined to date appear suited for this level of resolution. In forensic application mtDNA markers have three great advantages over genomic DNA markers. First, mtDNA genes code for essential life processes hence remain conserved over many generations. Second, each cell has thousands of identical copies hence tests targeted to mtDNA provide a significant improvement in sensitivity and 4 robustness over the single copy genomic markers. And third, mtDNA shows maternal rather than Mendelian inheritance. This means the reshuffling of chromosomes that takes place during sexual reproduction does not influence the inheritance of mtDNA. MtDNA remains essentially over many generations hence is a more stable marker for family lines and populations. The mtDNA genes examined to date clearly demonstrate their ability to provide useful information about gross geographical location. In theory mtDNA should be able to provide a much higher level of geographical resolution. The level of precision will be determined by the variability of the fragment chosen where fine detail mapping will require genes with even greater variation. If genetic sequences can provide high geographical resolution (eg within a 100 kilometre radius) then the genetic data may be used in conjunction with vessel movements to substantially improve the usefulness of forensic evidence in poaching investigations. Studies are currently in progress to find suitable sequences. Acknowledgements The authors would like to thank Dr Graeme Bremner (New Zealand Fisheries) for specimens and for advice on the usefulness of tests to fisheries investigations. This research was supported by funds from the Charles Sturt University, the Australian Research Council and New Zealand Fisheries. References Aljanabi, S.M. & Martinez, I. 1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research, 25: 4692-4693. Colgan, D.J., Ponder, W.F. & Eggler, P.E. 2000. Gastropod evolutionary rates and phylogenetic relationships assessed using partial 28s rDNA and histone H3 sequences. Zoologica Scripta, 29: 29-63. Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. 2001. Mega2: Molecular Evolutionary Genetics Analysis software. Arizona State University, Tempe, Arizona, USA. McArthur, A.G. & Koop, B.F. 1999. Partial 28S rDNA sequences and the antiquity of hydrothermal vent endemic gastropods. Molecular Phylogenetics & Evolution, 13: 255-274. Ponder, W.F. & Lindberg, D.R. 1996. Gastropod phylogeny - challenges for the 90s. In: Origin and evolutionary radiation of the Mollusca (J. Taylor, ed.), 135-154. Oxford University Press, London. Ponder, W.F. & Lindberg, D.R. 1997. Towards a phylogeny of gastropod molluscs: analysis using morphological characters. Zoological Journal of the Linnean Society, 119: 83-265. Priefer, U. 1984. Characterization of plasmid DNA by agarose gel electrophoresis. In: Advanced molecular genetics (A. Puhler & K.N. Timmis, eds), 26-37. Springer-Verlag, Berlin. Salvini-Plawen, L. 1980. A reconsideration of systematics in the Mollusca (phylogeny and higher classification). Malacologia, 19: 249-278. Salvini-Plawen, L.V. & Haszprunar, G. 1987. The Vetigastropoda and the systematics of streptoneurous Gastropoda (Mollusca). Journal of Zoology, London, 211: 747-770. 5 Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Phenol extraction of DNA samples. In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Tillier, S., Masselot, M., Guerdoux, J. & Tillier, A. 1994. Monophyly of major gastropod taxa tested from partial 28s rRNA sequences, with emphasis on Euthyneura and hot-vent limpets Peltospiroidea. Nautilus, Supplement 2: 122-140. Tillier, S., Masselot, M., Philippe, H. & Tillier, A. 1992. Phylogenie molecularie des Gastropoda (Mollusca) fondee sur le sequencage partiel de l’ARN ribosomique 28s. Comptes Rendus Academie de Sciences (Paris), 134: 79-85. Walsh, P.S., Metzger, D.A. & Higuchi, R. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 10: 506-513. 6 Figure 1 –Phylogram generated from the comparison of 28s rRNA sequences. The family designations for each gastropod species are shown in parentheses. Major clades are highlighted. 7 Figure 2 - Phylograms generated from alignment of CO1 sequences. Geographical locations are indicated. 8 Figure 3 - Phylograms generated from alignment of 16s rRNA, 12s rRNA, ND2 and CO2. Geographical locations are indicated. Unnamed branches represent non-Haliotis species. 16s rRNA 12s rRNA ND2 CO2 9