Species identification of Australian marsupials using collagen fingerprinting.

The study of faunal remains from archaeological sites is often complicated by the presence of large numbers of highly fragmented, morphologically unidentifiable bones. In Australia, this is the combined result of harsh preservation conditions and frequent scavenging by marsupial carnivores. The collagen fingerprinting method known as zooarchaeology by mass spectrometry (ZooMS) offers a means to address these challenges and improve identification rates of fragmented bones. Here, we present novel ZooMS peptide markers for 24 extant marsupial and monotreme species that allow for genus-level distinctions between these species. We demonstrate the utility of these new peptide markers by using them to taxonomically identify bone fragments from a nineteenth-century colonial-era pearlshell fishery at Bandicoot Bay, Barrow Island. The suite of peptide biomarkers presented in this study, which focus on a range of ecologically and culturally important species, have the potential to significantly amplify the zooarchaeological and paleontological record of Australia.

Micro Methods for Megafauna: Novel Approaches to Late Quaternary Extinctions and Their Contributions to Faunal Conservation in the Anthropocene.

Drivers of Late Quaternary megafaunal extinctions are relevant to modern conservation policy in a world of growing human population density, climate change, and faunal decline. Traditional debates tend toward global solutions, blaming either dramatic climate change or dispersals of Homo sapiens to new regions. Inherent limitations to archaeological and paleontological data sets often require reliance on scant, poorly resolved lines of evidence. However, recent developments in scientific technologies allow for more local, context-specific approaches. In the present article, we highlight how developments in five such methodologies (radiocarbon approaches, stable isotope analysis, ancient DNA, ancient proteomics, microscopy) have helped drive detailed analysis of specific megafaunal species, their particular ecological settings, and responses to new competitors or predators, climate change, and other external phenomena. The detailed case studies of faunal community composition, extinction chronologies, and demographic trends enabled by these methods examine megafaunal extinctions at scales appropriate for practical understanding of threats against particular species in their habitats today.

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.

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial.

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000-4000 years in association with a dramatic population decline. In addition, we obtained near-complete 11-loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of 'new' microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current-day conservation strategies.

Scrapheap challenge: a novel bulk-bone metabarcoding method to investigate ancient DNA in faunal assemblages.

Highly fragmented and morphologically indistinct fossil bone is common in archaeological and paleontological deposits but unfortunately it is of little use in compiling faunal assemblages. The development of a cost-effective methodology to taxonomically identify bulk bone is therefore a key challenge. Here, an ancient DNA methodology using high-throughput sequencing is developed to survey and analyse thousands of archaeological bones from southwest Australia. Fossils were collectively ground together depending on which of fifteen stratigraphical layers they were excavated from. By generating fifteen synthetic blends of bulk bone powder, each corresponding to a chronologically distinct layer, samples could be collectively analysed in an efficient manner. A diverse range of taxa, including endemic, extirpated and hitherto unrecorded taxa, dating back to c.46,000 years BP was characterized. The method is a novel, cost-effective use for unidentifiable bone fragments and a powerful molecular tool for surveying fossils that otherwise end up on the taxonomic "scrapheap".

An Aboriginal Australian genome reveals separate human dispersals into Asia.

We present an Aboriginal Australian genomic sequence obtained from a 100-year-old lock of hair donated by an Aboriginal man from southern Western Australia in the early 20th century. We detect no evidence of European admixture and estimate contamination levels to be below 0.5%. We show that Aboriginal Australians are descendants of an early human dispersal into eastern Asia, possibly 62,000 to 75,000 years ago. This dispersal is separate from the one that gave rise to modern Asians 25,000 to 38,000 years ago. We also find evidence of gene flow between populations of the two dispersal waves prior to the divergence of Native Americans from modern Asian ancestors. Our findings support the hypothesis that present-day Aboriginal Australians descend from the earliest humans to occupy Australia, likely representing one of the oldest continuous populations outside Africa.

Fossil avian eggshell preserves ancient DNA.

Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes.

Prolonged coexistence of humans and megafauna in Pleistocene Australia.

Recent claims for continent wide disappearance of megafauna at 46.5 thousand calendar years ago (ka) in Australia have been used to support a "blitzkrieg" model, which explains extinctions as the result of rapid overkill by human colonizers. A number of key sites with megafauna remains that significantly postdate 46.5 ka have been excluded from consideration because of questions regarding their stratigraphic integrity. Of these sites, Cuddie Springs is the only locality in Australia where megafauna and cultural remains are found together in sequential stratigraphic horizons, dated from 36-30 ka. Verifying the stratigraphic associations found here would effectively refute the rapid-overkill model and necessitate reconsideration of the regional impacts of global climatic change on megafauna and humans in the lead up to the last glacial maximum. Here, we present geochemical evidence that demonstrates the coexistence of humans and now-extinct megafaunal species on the Australian continent for a minimum of 15 ka.

The size of the largest marsupial and why it matters.

We show that at 2786 kg, the largest known marsupial, Diprotodon optatum, was much larger than has previously been suggested. Our results contradict the conclusion that the maximum attainable body mass of an Australian marsupial has been constrained by low productivity.