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The extinction of the woolly rhinoceros (Coelodonta antiquitatis) at the onset of the Holocene remains an enigma, with conflicting evidence regarding its cause and spatiotemporal dynamics. This partly reflects challenges in determining demographic responses of late Quaternary megafauna to climatic and anthropogenic causal drivers with available genetic and paleontological techniques. Here, we show that elucidating mechanisms of ancient extinctions can benefit from a detailed understanding of fine-scale metapopulation dynamics, operating over many millennia. Using an abundant fossil record, ancient DNA, and high-resolution simulation models, we untangle the ecological mechanisms and causal drivers that are likely to have been integral in the decline and later extinction of the woolly rhinoceros. Our 52,000-y reconstruction of distribution-wide metapopulation dynamics supports a pathway to extinction that began long before the Holocene, when the combination of cooling temperatures and low but sustained hunting by humans trapped woolly rhinoceroses in suboptimal habitats along the southern edge of their range. Modeling indicates that this ecological trap intensified after the end of the last ice age, preventing colonization of newly formed suitable habitats, weakening stabilizing metapopulation processes, triggering the extinction of the woolly rhinoceros in the early Holocene. Our findings suggest that fragmentation and resultant metapopulation dynamics should be explicitly considered in explanations of late Quaternary megafauna extinctions, sending a clarion call to the fragility of the remaining large-bodied grazers restricted to disjunct fragments of poor-quality habitat due to anthropogenic environmental change. [ABSTRACT FROM AUTHOR]

Context. Wildlife trade is a prominent pathway for invasive species introductions into novel environments. Deliberate or accidental release of exotic pets can result in the establishment of alien populations, with damaging impacts for native species and environmental assets. This process is well documented for reptiles globally and is of considerable biosecurity concern in Australia. Boa constrictor is one species at high risk of establishment in Australia, and has insufficient biosecurity detection and post-border control capacity. Aims. We aimed to develop rapid DNA-based presumptive testing capacity for detecting B. constrictor, with appropriate sensitivity and specificity to operate in a trace DNA biosecurity context. Methods. Loop Mediated Isothermal Amplification (LAMP) is an emerging biosecurity tool that provides highly specific, sensitive, low-resource methods for detection of trace DNA in the absence of physical evidence. We developed colourimetric and fluorescent LAMP assays targeting the mitochondrial DNA control region of B. constrictor. We tested and validated these assays against synthetic DNA fragments, as well as DNA extracted from: (1) vouchered museum B. constrictor tissue; (2) shed B. constrictor skin samples; (3) a range of non-target species to test specificity; and (4) trace DNA recovered from glass tanks post B. constrictor presence. Key results. We successfully detected synthetic target DNA down to 1 fg and genomic B. constrictor DNA from tissue and shed skins down to <10 pg in under 30 minutes with our fluorescence-based LAMP assay. Additionally, we were able to detect B. constrictor trace DNA following 24 h of presence utilising a traditional laboratory-based DNA extraction method (approximately 180 min) and a rapid lysis step (approximately 8 min). Conclusions. Both colourimetric and fluorescent assays show promise for the specific detection of B.constrictor in biosecurity contexts, including post-border enforcement and compliance checksin the domestic illicit wildlife trade. Implications. Our findings greatly strengthen the ongoing development of biosecurity tools for trace DNA detection of commonly traded and trafficked species (i.e. reptiles) in wildlife enforcement contexts, advancing both preparedness and surveillance. [ABSTRACT FROM AUTHOR]

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence‐related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real‐time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an "inhibition study" and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in‐house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000‐fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR‐based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin. [ABSTRACT FROM AUTHOR]

Reintroduction programs can face issues maintaining genetic diversity due to founder effects, and subsequent bottlenecks related to mortality and reproductive skews in the first generations after release. We assessed genetic diversity and undertook a pedigree analysis of 12 founders and 23 first-generation western quolls (Dasyurus geoffroii) at a reintroduced population at Arid Recovery, South Australia, in 2018. Genetic pedigrees showed that five of the eight females and three of the four males produced offspring. We also identified multiple paternity in this species. However, skewed paternity was evident with one male siring 65% of the sampled offspring. The reason for the paternity skew is unclear. The most successful male was smaller in body mass but had the largest home range compared to the other males, was released 4 days prior to two of the other males and spent more time inside the reserve. Failure of 33% of founders to breed in the first year combined with the strong paternity skew indicate that genetic drift and inbreeding pose a risk to the long-term success of this reintroduction. Genetic management, including the release of additional males, has already been undertaken, but may be required longer-term. Future quoll reintroductions should test if releasing all males simultaneously reduces paternity skew, and paternity should be measured through several generations to determine if paternity skew is a reintroduction protocol issue or one that is common in small populations more generally. In 2018 12 adult western quolls were reintroduced to a fenced reserve with 30 offspring produced in the first year. We used DNA to assess pedigrees and found that 65% of offspring were fathered by one male. We tried to work out why he was the most successful and include some notes for future quoll reintroductions. [ABSTRACT FROM AUTHOR]