Using ancient DNA to investigate extinction, extirpation and past biodiversity of Australian macropods

The field of ancient DNA (aDNA) involves the isolation and retrieval of trace amounts of degraded DNA from a variety of substrates including fossils, sediments and historical material. The fragmentary nature of aDNA necessitates the use of methods with the ability to capture and amplify short segments of DNA. Collectively aDNA studies have made significant and unique contributions to a wide field of research including conservation, population genetics, taxonomy and phylogeny. The primary aim of this thesis research is to explore the utility of aDNA techniques to study extirpation, extinction and past biodiversity of Australian macropods. Using a combination of historical, Holocene and Pleistocene aged fossils, this research will attempt to investigate what ancient mitochondrial DNA (mtDNA) can add to our knowledge of Australia’s macropods. Traditional aDNA techniques have largely been used to isolate mtDNA from single fossil samples - an example of this approach is shown in Chapter Two where a wellpreserved wallaby fossil bone from Depuch Island (Western Australia) was studied. The ancient mtDNA (cytochrome b and control region) data produced strong phylogenetic signal and shows that the Depuch Island rock-wallaby specimen is most similar to the mainland Petrogale lateralis lateralis. This finding has conservation implications for ongoing rehabilitation and translocation efforts in the Pilbara region. Chapter Three of this thesis also uses mitochondrial aDNA techniques, to explore questions regarding interrelationships and former distribution of a macropod species complex; Bettongia spp. Cytochrome b and control region data retrieved from 88 historical samples, along with ~214 already sequenced samples, place the most recent common ancestor of the brush-tailed bettongs at c. 2.5 Myr. Ancient mtDNA is suggestive of connectivity between what are now highly fragmented populations, a result that has implications for how critically endangered brush-tailed bettongs should be managed. Ancient DNA analyses and DNA sequencing technology have evolved over recent years and during the course of this study. Therefore in keeping up with the latest high-throughput sequencing (HTS) technology, aDNA analyses in ~70 bones and 20 sediment samples excavated from a Late Pleistocene–Holocene cave deposit on Kangaroo Island, South Australia was undertaken. Samples were selected from 15 stratigraphic layers, ranging in age from >20 ka to ~6.8 ka. The successful retrieval of bona fide aDNA sequences, back to at least 20 ka, demonstrates excellent longterm DNA preservation at the site. All unidentified bones that were screened revealed a number of taxa from the assemblage including, Macropus, Onychogalea, Potorous, Bettongia, Dasyurus, Rattus and Notechis. The results from this study add significant value to the late Pleistocene-mid-Holocene paleontological record, detailing the past diversity of flora and fauna on Kangaroo Island. Lastly, Chapter 5 introduces the latest molecular techniques in capturing and enriching highly fragmented aDNA bone from four sites across Australia. Ancient DNA extractions techniques, targeting ultra-short DNA fragments, were employed in an attempt to obtain Pleistocene-aged material. The warm conditions, a factor common in Australian caves, are not conductive to long-term DNA preservation at many sites. Shotgun sequencing was only successful on six bone samples (including one incisor) from a total of 25 samples that were screened. Three samples were successfully captured and enriched for endogenous DNA; one bettong sample generated 89.6% of a mtDNA genome with 5.4X coverage. Overall, the decay rate of DNA and preservation across all four sites was high, and extremely degraded, with an average fragment length between 47 bp and 57 bp. These data demonstrate that recovery of Pleistocene-aged aDNA from warm climate sites across Australia will remain a challenge and that better ways to screen and predict DNA survival are needed. This thesis presents a combination of work from multiple sites across Australia using a range of aDNA techniques and sequencing technologies that have evolved over the tenure of this thesis. Collectively, this body of work has demonstrated the value of integrating aDNA data into modern-day conservation decision-making and has contributed to a wider understanding of Australian macropods both past and present