52,000 years of woolly rhinoceros population dynamics reveal extinction mechanisms.

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.

Metal-DNA interactions: Exploring the impact of metal ions on key stages of forensic DNA analysis.

Forensic DNA analysis continues to be hampered by the complex interactions between metals and DNA. Metal ions may cause direct DNA damage, inhibit DNA extraction and polymerase chain reaction (PCR) amplification or both. This study evaluated the impact of metal ions on DNA extraction, quantitation, and short tandem repeat profiling using cell-free and cellular (saliva) DNA. Of the 11 metals assessed, brass exhibited the strongest PCR inhibitory effects, for both custom and Quantifiler Trio quantitation assays. Metal ion inhibition varied across the two quantitative PCR assays and the amount of DNA template used. The Quantifiler Trio internal PCR control (IPC) only revealed evidence of PCR inhibition at higher metal ion concentrations, limiting the applicability of IPC as an indicator of the presence of metal inhibitor in a sample. Notably, ferrous ions were found to significantly decrease the extraction efficiency of the DNA-IQ DNA extraction system. The amount of DNA degradation and inhibition in saliva samples caused by metal ions increased with a dilution of the sample, suggesting that the saliva matrix provides protection from metal ion effects.

Process-explicit models reveal pathway to extinction for woolly mammoth using pattern-oriented validation.

Pathways to extinction start long before the death of the last individual. However, causes of early stage population declines and the susceptibility of small residual populations to extirpation are typically studied in isolation. Using validated process-explicit models, we disentangle the ecological mechanisms and threats that were integral in the initial decline and later extinction of the woolly mammoth. We show that reconciling ancient DNA data on woolly mammoth population decline with fossil evidence of location and timing of extinction requires process-explicit models with specific demographic and niche constraints, and a constrained synergy of climatic change and human impacts. Validated models needed humans to hasten climate-driven population declines by many millennia, and to allow woolly mammoths to persist in mainland Arctic refugia until the mid-Holocene. Our results show that the role of humans in the extinction dynamics of woolly mammoth began well before the Holocene, exerting lasting effects on the spatial pattern and timing of its range-wide extinction.

Historical museum samples enable the examination of divergent and parallel evolution during invasion.

During the Anthropocene, Earth has experienced unprecedented habitat loss, native species decline and global climate change. Concurrently, greater globalization is facilitating species movement, increasing the likelihood of alien species establishment and propagation. There is a great need to understand what influences a species' ability to persist or perish within a new or changing environment. Examining genes that may be associated with a species' invasion success or persistence informs invasive species management, assists with native species preservation and sheds light on important evolutionary mechanisms that occur in novel environments. This approach can be aided by coupling spatial and temporal investigations of evolutionary processes. Here we use the common starling, Sturnus vulgaris, to identify parallel and divergent evolutionary change between contemporary native and invasive range samples and their common ancestral population. To do this, we use reduced-representation sequencing of native samples collected recently in northwestern Europe and invasive samples from Australia, together with museum specimens sampled in the UK during the mid-19th century. We found evidence of parallel selection on both continents, possibly resulting from common global selective forces such as exposure to pollutants. We also identified divergent selection in these populations, which might be related to adaptive changes in response to the novel environment encountered in the introduced Australian range. Interestingly, signatures of selection are equally as common within both invasive and native range contemporary samples. Our results demonstrate the value of including historical samples in genetic studies of invasion and highlight the ongoing and occasionally parallel role of adaptation in both native and invasive ranges.

The development of a tool to predict temperature-exposure of incinerated teeth using colourimetric and hydroxyapatite crystal size data.

This study presents a novel tool to predict temperature-exposure of incinerated pig teeth as a proxy for understanding impacts of fire on human teeth. Previous studies on the estimation of temperature-exposure of skeletal elements have been limited to that of heat-exposed bone. This predictive tool was developed using a multinomial regression model of colourimetric and hydroxyapatite crystal size variables using data obtained from unheated pig teeth and teeth incinerated at 300 °C, 600 °C, 800 °C and 1000 °C. An additional variable based on the observed appearance of the tooth was included in the tool. This enables the tooth to be classified as definitely burnt (600 °C-1000 °C) or uncertain (27 °C/300 °C). As a result, the model predicting the temperature-exposure of the incinerated teeth had an accuracy of 95%. This tool is a holistic, robust and reliable approach to estimate temperature of heat-exposed pig teeth, with high accuracy, and may act as a valuable proxy to estimate heat exposure for human teeth in forensic casework.

Evaluation of the efficiency of Isohelix™ and Rayon swabs for recovery of DNA from metal surfaces.

PURPOSE: We investigated the recovery and extraction efficiency of DNA from three metal surfaces (brass, copper, steel) relevant to forensic casework, and plastic (control) using two different swabbing systems; Rayon and Isohelix™ swabs, with sterile water and isopropyl alcohol respectively, as the wetting solutions. METHODS: Twenty nanograms of human genomic DNA were applied directly to Isohelix™ and Rayon swabs; and to the metal and plastic substrates. All substrates were left to dry for 24 h, followed by single wet swabbing and extraction with the DNA IQ™ System. DNA extracts were quantified using real time quantitative PCR assays with SYBR green chemistry. RESULTS: DNA was extracted from directly seeded Isohelix™ swabs with a high efficiency of 98%, indicating effective DNA-release from the swab into the extraction buffer. In contrast, only 58% of input DNA was recovered from seeded Rayon swabs, indicating higher DNA retention by these swabs. Isohelix™ swabs recovered 32 - 53% of DNA from metal surfaces, whilst the Rayon swabs recovered 11-29%. DNA recovery was lowest from copper and highest from brass. Interestingly, Rayon swabs appeared to collect more DNA from the plastic surface than Isohelix™ swabs, however, due to the lower release of DNA from Rayon swabs they returned less DNA overall following extraction than Isohelix™ swabs. CONCLUSION: These results demonstrate that DNA samples deposited on metal surfaces can be more efficiently recovered using Isohelix™ swabs wetted with isopropyl alcohol than Rayon swabs wetted with sterile water, although recovery is affected by the substrate type.

Comparison of Isohelix™ and Rayon swabbing systems for touch DNA recovery from metal surfaces.

A previous study evaluating two swabbing systems found that DNA was best recovered from sterile metal substrates using an Isohelix™ swab wetted with isopropyl alcohol rather than a Rayon swab with water as the wetting agent. We tested the same swabbing systems on metal (aluminum, brass, and stainless steel) and plastic substrates in a regularly touched environment to simulate the non-deliberate transfer of touch evidence likely seen in a casework scenario, to ascertain the performance of these swabs in an uncontrolled situation. Higher amounts of touch DNA were recovered with Isohelix™ swabs (0.5 - 3.3 ng) compared to Rayon swabs (0.13 - 1.2 ng). The Isohelix™ swabbing system was found to significantly recover more touch DNA (p = 0.04) from the metal substrates than the Rayon swabbing system, consistent with the findings of our previous work. The results contribute to our understanding of the impact of sample collection techniques on touch DNA recovery from problematic metal surfaces and suggest that supplemental cleaning of substrates as a precautionary step against the spread of infections may affect touch DNA persistence and the recovery efficiency of swabs.

Fifty thousand years of Arctic vegetation and megafaunal diet.

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25-15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

High-quality fossil dates support a synchronous, Late Holocene extinction of devils and thylacines in mainland Australia.

The last large marsupial carnivores-the Tasmanian devil (Sarcophilis harrisii) and thylacine (Thylacinus cynocephalus)-went extinct on mainland Australia during the mid-Holocene. Based on the youngest fossil dates (approx. 3500 years before present, BP), these extinctions are often considered synchronous and driven by a common cause. However, many published devil dates have recently been rejected as unreliable, shifting the youngest mainland fossil age to 25 500 years BP and challenging the synchronous-extinction hypothesis. Here we provide 24 and 20 new ages for devils and thylacines, respectively, and collate existing, reliable radiocarbon dates by quality-filtering available records. We use this new dataset to estimate an extinction time for both species by applying the Gaussian-resampled, inverse-weighted McInerney (GRIWM) method. Our new data and analysis definitively support the synchronous-extinction hypothesis, estimating that the mainland devil and thylacine extinctions occurred between 3179 and 3227 years BP.

Genetic diversity and drivers of dwarfism in extinct island emu populations.

Australia's iconic emu (Dromaius novaehollandiae novaehollandiae) is the only living representative of its genus, but fossil evidence and reports from early European explorers suggest that three island forms (at least two of which were dwarfs) became extinct during the nineteenth century. While one of these-the King Island emu-has been found to be conspecific with Australian mainland emus, little is known about how the other two forms-Kangaroo Island and Tasmanian emus-relate to the others, or even the size of Tasmanian emus. We present a comprehensive genetic and morphological analysis of Dromaius diversity, including data from one of the few definitively genuine Tasmanian emu specimens known. Our genetic analyses suggest that all the island populations represent sub-populations of mainland Dnovaehollandiae Further, the size of island emus and those on the mainland appears to scale linearly with island size but not time since isolation, suggesting that island size-and presumably concomitant limitations on resource availability-may be a more important driver of dwarfism in island emus, though its precise contribution to emu dwarfism remains to be confirmed.

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality.

Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.

Using ancient DNA to study the origins and dispersal of ancestral Polynesian chickens across the Pacific.

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.

Ancient mitochondrial DNA reveals convergent evolution of giant short-faced bears (Tremarctinae) in North and South America.

The Tremarctinae are a subfamily of bears endemic to the New World, including two of the largest terrestrial mammalian carnivores that have ever lived: the giant, short-faced bears Arctodus simus from North America and Arctotherium angustidens from South America (greater than or equal to 1000 kg). Arctotherium angustidens became extinct during the Early Pleistocene, whereas Arctodus simus went extinct at the very end of the Pleistocene. The only living tremarctine is the spectacled bear (Tremarctos ornatus), a largely herbivorous bear that is today only found in South America. The relationships among the spectacled bears (Tremarctos), South American short-faced bears (Arctotherium) and North American short-faced bears (Arctodus) remain uncertain. In this study, we sequenced a mitochondrial genome from an Arctotherium femur preserved in a Chilean cave. Our molecular phylogenetic analyses revealed that the South American short-faced bears were more closely related to the extant South American spectacled bear than to the North American short-faced bears. This result suggests striking convergent evolution of giant forms in the two groups of short-faced bears (Arctodus and Arctotherium), potentially as an adaptation to dominate competition for megafaunal carcasses.

Molecular phylogeny, biogeography, and habitat preference evolution of marsupials.

Marsupials exhibit great diversity in ecology and morphology. However, compared with their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs.

PURPOSE: The primary method for analysis of low template DNA (LTDNA) is known as the low copy number (LCN) method involving an increased number of PCR cycles (typically 34). In common with other LTDNA methods, LCN profiles are characterized by allelic imbalance, drop in, and drop out that require complicated interpretation rules. They often require replicate PCR reactions to generate a "consensus" profile in a specialized facility. An ideal method for analysis of LTDNA should enhance profiling outcomes without elevated error rates and be performed using standard facilities, with minimum additional cost. METHODS: In this study, we present a comparison of four method variations for the amplification of STRs from LTDNA with a widely used, commercially available kit (AmpFℓSTR(®) Profiler Plus(®)): the standard method, the standard method with a post-PCR clean up, the LCN method, and a reduced reaction volume with increased Taq DNA polymerase concentration. RESULTS: Using telogen hairs-a common source of LTDNA-and matched reference DNA, the LCN method produced the highest number of concordant and non-concordant (i.e., dropped-in) alleles. In comparison, the reduced reaction volume with increased Taq polymerase yielded more full and concordant DNA profiles (all alleles combined) and less off-ladder alleles from a broad range of input DNA. In addition, this method resulted in less non-concordant alleles than LCN and no more than for standard PCR, which suggests that it may be preferred over increased PCR cycles for LTDNA analysis, either with or without consensus profiling and statistical modelling. CONCLUSIONS: Overall, this study highlights the importance and benefit of optimizing PCR conditions and developing improved laboratory methods to amplify and analyze LTDNA.

Extensive population decline in the Tasmanian devil predates European settlement and devil facial tumour disease.

The Tasmanian devil (Sarcophilus harrisii) was widespread in Australia during the Late Pleistocene but is now endemic to the island of Tasmania. Low genetic diversity combined with the spread of devil facial tumour disease have raised concerns for the species' long-term survival. Here, we investigate the origin of low genetic diversity by inferring the species' demographic history using temporal sampling with summary statistics, full-likelihood and approximate Bayesian computation methods. Our results show extensive population declines across Tasmania correlating with environmental changes around the last glacial maximum and following unstable climate related to increased 'El Niño-Southern Oscillation' activity.

Evaluation of carrier RNA and low volume demineralization for recovery of nuclear DNA from human teeth.

PURPOSE: Teeth and bones are frequently used in the genetic analysis of degraded and ancient human and animal remains. Standard extraction methods, including most commercially available systems, may not yield sufficient DNA to enable successful genetic analysis. Addition of a carrier molecule and demineralization (via EDTA) can increase yields from samples containing limited amounts of DNA. However the benefits of carrier molecules have not been demonstrated for bones and teeth and demineralization introduces large reagent volumes that are difficult to integrate into commercial DNA extraction systems. METHODS: We compared nuclear DNA yields recovered from small samples of partially decomposed human teeth using a commercial silica-based DNA extraction system with and without the addition of carrier RNA and/or a low-volume demineralization step. RESULTS: DNA yield was significantly improved with demineralization, but there was no significant effect of carrier RNA. The DNA content of a sample did not influence the significance of the effect of demineralization. CONCLUSION: Using a simple low-volume (1 mL) demineralization step, prior to DNA extraction with the QIAmp DNA Investigator kit (Qiagen), as little as 50 mg of tooth powder can yield more than 500 ng of nuclear DNA.

Reconstructing colonization dynamics to establish how human activities transformed island biodiversity.

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.

Molecular patterns of introgression in a classic hybrid zone between the Australian tree frogs, Litoria ewingii and L. paraewingi: evidence of a tension zone.

Hybrid zones provide a rare opportunity to explore the processes involved in reproductive isolation and speciation. The southern hybrid zone between the southeastern Australian tree frogs Litoria ewingii and L. paraewingi has been comprehensively studied over the last 40 years, primarily using reproductive compatibility experiments and male advertisement calls. We used mitochondrial DNA (mtDNA) and eight nuclear microsatellite markers to characterize this hybrid zone along a historically studied transect and to test various dispersal-dependent and dispersal-independent hybrid zone models. The species are genetically distinct and the level of hybridization within the contact zone is low, with the majority of admixed individuals representing later-generation hybrids. Based on previous experimental genetic compatibility studies, we predicted that hybrids with L. paraewingi mtDNA would be more frequent than hybrids with L. ewingii mtDNA. Surprisingly, a greater proportion of the identified hybrids had L. ewingii mtDNA. Geographical cline analyses showed a sharp transition in allele frequencies across the transect, and both the mtDNA and microsatellite data showed concordant cline centres, but were best supported by a model that allowed width to vary. Overall, the L. ewingii-L. paraewingi hybrid zone is best characterized as a tension zone, due to the narrow cline width, concordant genetic clines and low levels of hybridization.

Low major histocompatibility complex diversity in the Tasmanian devil predates European settlement and may explain susceptibility to disease epidemics.

The Tasmanian devil (Sarcophilus harrisii) is at risk of extinction owing to the emergence of a contagious cancer known as devil facial tumour disease (DFTD). The emergence and spread of DFTD has been linked to low genetic diversity in the major histocompatibility complex (MHC). We examined MHC diversity in historical and ancient devils to determine whether loss of diversity is recent or predates European settlement in Australia. Our results reveal no additional diversity in historical Tasmanian samples. Mainland devils had common modern variants plus six new variants that are highly similar to existing alleles. We conclude that low MHC diversity has been a feature of devil populations since at least the Mid-Holocene and could explain their tumultuous history of population crashes.