The Development of Forensic DNA Techniques to Assist with Criminal Investigations involving the Australasian Carpet Python (Morelia spilota)

Author: Sherryn Ciavaglia

Ciavaglia, Sherryn, 2019 The Development of Forensic DNA Techniques to Assist with Criminal Investigations involving the Australasian Carpet Python (Morelia spilota), Flinders University, College of Science and Engineering

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The analysis of non-human DNA to provide evidence in criminal cases involving wildlife species is a relatively young area of forensic science. Forensic genetic tests have been developed for only a small handful of the plethora of species persecuted and exploited, despite their statutory protection. Poor resourcing is ever an issue for wildlife crime, which most often falls below crimes against humans in the financial priorities of governments. Forensic DNA profiling techniques are resource intensive to develop, so the wildlife forensic scientist is continually working to develop and validate new techniques and expand their capacity for forensic testing between casework.

Furthermore, wildlife species bring additional challenges not met by forensic scientists specialising in human DNA. Species identification is peculiar to wildlife forensic science and the elevated genetic structure and fragmented populations and low sample sizes encountered when validating individualisation tests for wildlife species provide difficulties of interpretation and application unique to this field. This PhD thesis has progressed the field of wildlife forensic science by providing novel phylogenetic and population genetic analyses of Australasian python species commonly encountered in wildlife crime, and in particular one species commonly encountered by Australian enforcement officials, the carpet python, Morelia spilota. Forensic tests for both species identification and individualisation of carpet pythons are developed and validated for direct application to casework involving this species.

This thesis explores the arrangement and sequence level variability of mitochondrial genomes in a wide range of snake species to identify a locus with characteristics desirable for species identification of individuals belonging to Pythonidae. Species identification by DNA requires priming sites located in DNA regions highly conserved across snakes taxa to facilitate wide amplification utility. The amplified region ideally represents a ‘Goldilocks’ sequence, containing a fine balance of sequence variability between species and conservation within a species. Such a locus is identified to separate all eleven species within the genus Morelia, excluding M. bredli. However, mitochondrial and nuclear DNA analyses within this thesis suggest that the species differentiation of M. spilota and M. bredli is not reflected at a genetic level. In the context of reporting species identification, any match to either of these species should be reported as M. spilota or M. bredli. A more conservative approach would be to report to genus rather than species, however the former approach is far more useful and this decision falls to the comfort of the reporting officer. Furthermore, this project has identified a small target region for species identification of Morelia, suitable for application to degraded forensic remains. A developmental validation of the resulting assay is provided to facilitate direct application to criminal casework involving python species. Mock case samples demonstrate the utility of this test on unknown samples suspected to be carpet pythons.

The primary focus of this thesis is the development and validation of a novel STR test to individualise carpet pythons. This DNA test aids investigations involving matching of evidentiary items, parentage testing and ultimately also repatriation of a poached python to the wild through identification of the most likely population of origin, which also facilitates concentration of enforcement efforts on identified poaching ‘hotspots’. New STR markers are identified with characteristics desirable for forensic application. A set of 24 tetra- and penta-nucleotide markers are optimised into three 11-plex multiplex assays with inbuilt mechanisms to maintain high quality assurance. Allelic ladders accompany the three assays for robust single base-pair genotyping and inter-laboratory utility. Sequence level characteristics of the STR loci are explored and certain features are identified that can provide information subsequent to genotyping by size migration, where case scenarios involve M. bredli or M. spilota imbricata. The applicability of these STR markers to other Australasian python species of forensic significance is also explored.

A DNA reference database of carpet pythons representative of their native range is presented to facilitate the calculation of statistical conclusions. However, the complete database violates assumptions made when calculating the forensic statistics ultimately reported in casework. Population genetic analyses are conducted to identify more appropriate sub-populations with which to calculate forensic statistics. The dataset offers a number of limitations that confound straight-forward reporting of the forensic statistics, caused by small sample sizes, sparse sampling across large geographic areas and high genetic structuring within sub-populations. Approaches to application involving varying degrees of conservativism are presented, but the best approach to apply will need to be assessed on a case by case basis, depending on the scenario and population involved.

The overall reference DNA dataset is divided into six sub-populations that are more appropriate than the total database for calculating forensic statistics. Samples unsuitable for inclusion in the reference database (for example, missing data at >2 loci) are used to test the effectiveness of geographic assignment to the six sub-populations and the resulting population specific statistical calculations. The resulting statistics offer strong discrimination power in all sub-populations, even when samples in poor condition yield DNA profiles of around half a dozen loci. Geographic assignment to the six populations is also effective even when less than half the total loci have been obtained. Only one interface between two populations showed limitations for assignment to sub-population. A limited developmental validation of the STR assays lays the foundation for application in criminal casework involving the Australasian carpet python.

Keywords: PhD, wildlife, forensic science, carpet python, morelia spilota, snake, forensic, DNA, species identification.

Subject: Forensic & Analytical Chemistry thesis

Thesis type: Doctor of Philosophy
Completed: 2019
School: College of Science and Engineering
Supervisor: Prof. Adrian Linacre