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ABSTRACT The exotic pet trade is a major pathway for the introduction, establishment, and spread of novel invasive alien species. Reptiles are common in the exotic pet trade and are prominent invasive alien vertebrate species that have dire impacts if allowed to establish. The North American corn snake (Pantherophis guttatus) is particularly common in the international pet trade and has been identified as a vertebrate pest priority species in Australia due to widespread climate suitability and prevalence in pre‐ and post‐border seizure records. Consequently, rapid, and presumptive post‐border biosecurity detection is essential to prevent its establishment and spread. Loop‐mediated isothermal amplification (LAMP) is an emerging biosecurity tool that has shown promise for rapid detection of several high‐risk species. We developed two LAMP assays for the detection of P. guttatus, validated against: synthetic DNA; DNA extracted from snap‐frozen tissue, and shed skins; and then compared their performance for the detection of trace DNA collected from swabs of glass tanks post reptile presence. Our results include laboratory optimization and assessment of two mobile devices for in‐field integration (Franklin Real‐Time PCR Thermocycler, Biomeme, USA, and Genie III, Optigene, UK). The results indicate that LAMP is a viable biosecurity tool, with DNA detection possible for a range of sample types in a total of c.30 min, when including a rapid extraction step (8 min). Herein, we provide tools for rapid, presumptive detection of the North American corn snake from trace DNA samples in Australian biosecurity and wildlife compliance settings. The international exotic pet trade is a major pathway for the introduction, establishment, and spread of novel invasive alien species. The North American corn snake (Pantherophis guttatus) is particularly common in the international pet trade and has been identified as a vertebrate pest priority species in Australia due to widespread climate suitability and prevalence in pre‐ and post‐border seizure records. We developed two LAMP assays for the detection of P. guttatus, to provide tools suited to biosecurity detection for a species of highest vertebrate pest priority in Australia.

Massively parallel sequencing following hybridisation enrichment provides new opportunities to obtain genetic data for various types of forensic testing and has proven successful on modern as well as degraded and ancient DNA. A customisable forensic intelligence panel that targeted 124 SNP markers (67 ancestry informative markers, 23 phenotype markers from the HIrisplex panel, and 35 Y-chromosome SNPs) was used to examine biogeographic ancestry, phenotype and sex and Y-lineage in samples from different ethnic populations of Pakistan including Pothwari, Gilgit, Baloach, Pathan, Kashmiri and Siraiki. Targeted sequencing and computational data analysis pipeline allowed filtering of variants across the targeted loci. Study samples showed an admixture between East Asian and European ancestry. Eye colour was predicted accurately based on the highest p-value giving overall prediction accuracy of 92.8%. Predictions were consistent with reported hair colour for all samples, using the combined highest p-value approach and step-wise model incorporating probability thresholds for light or dark shade. Y-SNPs were successfully recovered only from male samples which indicates the ability of this method to identify biological sex and allow inference of Y-haplogroup. Our results demonstrate practicality of using hybridisation enrichment and MPS to aid in human intelligence gathering and will open many insights into forensic research in South Asia.

Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Furthermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps.

The human colonization of Remote Oceania remains one of the great feats of exploration in history, proceeding east from Asia across the vast expanse of the Pacific Ocean. Human commensal and domesticated species were widely transported as part of this diaspora, possibly as far as South America. We sequenced mitochondrial control region DNA from 122 modern and 22 ancient chicken specimens from Polynesia and Island Southeast Asia and used these together with Bayesian modeling methods to examine the human dispersal of chickens across this area. We show that specific techniques are essential to remove contaminating modern DNA from experiments, which appear to have impacted previous studies of Pacific chickens. In contrast to previous reports, we find that all ancient specimens and a high proportion of the modern chickens possess a group of unique, closely related haplotypes found only in the Pacific. This group of haplotypes appears to represent the authentic founding mitochondrial DNA chicken lineages transported across the Pacific, and allows the early dispersal of chickens across Micronesia and Polynesia to be modeled. Importantly, chickens carrying this genetic signature persist on several Pacific islands at high frequencies, suggesting that the original Polynesian chicken lineages may still survive. No early South American chicken samples have been detected with the diagnostic Polynesian mtDNA haplotypes, arguing against reports that chickens provide evidence of Polynesian contact with pre-European South America. Two modern specimens from the Philippines carry haplotypes similar to the ancient Pacific samples, providing clues about a potential homeland for the Polynesian chicken.

Falsified medicines are a major threat to global health. Antimalarial drugs have been particularly targeted by criminals. As DNA analysis has revolutionized forensic criminology, we hypothesized that these techniques could also be used to investigate the origins of falsified medicines. Medicines may contain diverse adventitious biological contamination, and the sealed nature of blister-packages may capture and preserve genetic signals from the manufacturing processes allowing identification of production source(s). We conducted a blinded pilot study to determine if such environmental DNA (eDNA) could be detected in eleven samples of falsified and genuine artesunate antimalarial tablets, collected in SE Asia, which could be indicative of origin. Massively Parallel Sequencing (MPS) was used to characterize microbial and eukaryote diversity. Two mitochondrial DNA analysis approaches were explored to detect the presence of human DNA. Trace eDNA from these low biomass samples demonstrated sample specific signals using two target markers. Significant differences in bacterial and eukaryote DNA community structures were observed between genuine and falsified tablets and between different packaging types of falsified artesunate. Human DNA, which was indicative of likely east Asian ancestry, was found in falsified tablets. This pilot study of the ‘pharmabiome’ shows the potential of environmental DNA as a powerful forensic tool to assist with the identification of the environments, and hence location and timing, of the source and manufacture of falsified medicines, establish links between seizures and complement existing tools to build a more complete picture of criminal trade routes. The finding of human DNA in tablets raises important ethical issues that need to be addressed.

Drivers and dynamics of initial human migrations across individual islands and archipelagos are poorly understood, hampering assessments of subsequent modification of island biodiversity. We developed and tested a new statistical-simulation approach for reconstructing the pattern and pace of human migration across islands at high spatiotemporal resolutions. Using Polynesian colonisation of New Zealand as an example, we show that process-explicit models, informed by archaeological records and spatiotemporal reconstructions of past climates and environments, can provide new and important insights into the patterns and mechanisms of arrival and establishment of people on islands. We find that colonisation of New Zealand required there to have been a single founding population of approximately 500 people, arriving between 1233 and 1257 AD, settling multiple areas, and expanding rapidly over both North and South Islands. These verified spatiotemporal reconstructions of colonisation dynamics provide new opportunities to explore more extensively the potential ecological impacts of human colonisation on New Zealand’s native biota and ecosystems.

Causes of late Quaternary extinctions of large mammals (\"megafauna\") continue to be debated, especially for continental losses, because spatial and temporal patterns of extinction are poorly known. Accurate latest appearance dates (LADs) for such taxa are critical for interpreting the process of extinction. The extinction of woolly mammoth and horse in northwestern North America is currently placed at 15,000-13,000 calendar years before present (yr BP), based on LADs from dating surveys of macrofossils (bones and teeth). Advantages of using macrofossils to estimate when a species became extinct are offset, however, by the improbability of finding and dating the remains of the last-surviving members of populations that were restricted in numbers or confined to refugia. Here we report an alternative approach to detect 'ghost ranges' of dwindling populations, based on recovery of ancient DNA from perennially frozen and securely dated sediments (sedaDNA). In such contexts, sedaDNA can reveal the molecular presence of species that appear absent in the macrofossil record. We show that woolly mammoth and horse persisted in interior Alaska until at least 10,500 yr BP, several thousands of years later than indicated from macrofossil surveys. These results contradict claims that Holocene survival of mammoths in Beringia was restricted to ecologically isolated high-latitude islands. More importantly, our finding that mammoth and horse overlapped with humans for several millennia in the region where people initially entered the Americas challenges theories that megafaunal extinction occurred within centuries of human arrival or were due to an extraterrestrial impact in the late Pleistocene.

Environmental biosecurity risks associated with the transnational wildlife trade include the loss of biodiversity, threats to public health, and the proliferation of invasive alien species. To assist enforcement agencies in identifying species either intentionally (trafficked) or unintentionally (stowaway) entrained in the trade-chain pathway, rapid forensic techniques are needed to enable their detection from DNA samples when physical identification is not possible. Loop Mediated Isothermal Amplification (LAMP) is an emerging technique, with recent applications in biosecurity and forensic sciences, which has potential to function as a field-based detection tool. Here we provide an overview of current research that applies LAMP to environmental biosecurity, including identification of ornamental wildlife parts, consumer products, and invasive species monitoring and biosecurity detection. We discuss the current scope of LAMP as applied to various wildlife trade scenarios and biosecurity checkpoint monitoring, highlight the specificity, sensitivity, and robustness for these applications, and review the potential utility of LAMP for rapid field-based detection at biosecurity checkpoints. Based on our assessment of the literature we recommend broader interest, research uptake, and investment in LAMP as an appropriate field-based species detection method for a wide range of environmental biosecurity scenarios.

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make \"phylogenetically diverse\" probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.

Conservation genetics has informed threatened species management for several decades. With the advent of advanced DNA sequencing technologies in recent years, it is now possible to monitor and manage threatened populations with even greater precision. Climate change presents a number of threats and challenges, but new genomics data and analytical approaches provide opportunities to identify critical evolutionary processes of relevance to genetic management under climate change. Here, we discuss the applications of such approaches for threatened species management in Australia in the context of climate change, identifying methods of facilitating viability and resilience in the face of extreme environmental stress. Using genomic approaches, conservation management practices such as translocation, targeted gene flow, and gene-editing can now be performed with the express intention of facilitating adaptation to current and projected climate change scenarios in vulnerable species, thus reducing extinction risk and ensuring the protection of our unique biodiversity for future generations. We discuss the current barriers to implementing conservation genomic projects and the efforts being made to overcome them, including communication between researchers and managers to improve the relevance and applicability of genomic studies. We present novel approaches for facilitating adaptive capacity and accelerating natural selection in species to encourage resilience in the face of climate change.