Charlotte Oskam

Eggshell is frequently encountered within palaeontological and archaeological deposits and due to various chemical and physical properties, it is highly valued as a substrate for geochronology, palaeodietary and palaeoecological applications. Despite the wellrecognised preservation of biomolecules in fossil eggshell, the substrate had never been evaluated for ancient DNA (aDNA) preservation. Herein, this thesis describes the first exploration involving the recovery and characterisation of aDNA from fossil eggshell. The primary aim of this thesis research is to explore eggshell aDNA and how genetic data, when combined with stable isotope profiles, provides new insights into hunting practices, zooarchaeology, palaeodiets, palaeobiology and extinction processes. The visualisation of fluorescently-stained DNA in fossil eggshell by confocal microscopy revealed that genetic material was present in the eggshell of the extinct New Zealand moa (Aves: Dinornithiformes) and Madagascan elephant bird (Aves: Aepyornithiformes). The immediate task was to determine how to go about characterising the aDNA encapsulated in this previously unrecognised substrate. Quantitative PCR assays were employed to thoroughly evaluate different methods to maximise the recovery and amplification of aDNA from powdered eggshell. This study resulted in the successful characterisation of both mitochondrial and nuclear DNA isolated from 19,000 year-old emu eggshell and the first ever DNA sequence from the elephant bird, Aepyornis. All data pointed toward fossil eggshell as a reliable source of relatively pure aDNA. This work culminated in a formal description of the methodology that should be employed to maximise recovery of aDNA from eggshell. Next, this thesis investigates whether eggshell aDNA could be applied to ‘first-contact’ archaeological sites in New Zealand, to gain new insights into the Moa-hunter period of prehistory. The abundance of moa eggshell within the ‘first-contact’ Polynesian archaeological sites is well recognised from previous excavations. With nine species of moa contained within the New Zealand archipelago however, the scientific value of these remains has been limited by the lack of definitive species assignments. Genetic species assignments of moa eggshell combined with morphological identification of bone (literature and museum catalogued specimens) has enabled the most comprehensive audit of zooarchaeology assemblages from several significant 13th-15th century AD deposits. In total, mitochondrial DNA (mtDNA) was amplified from 251 of 323 (78 %) eggshell fragments and 22 of 27 (88 %) bone samples, and the analyses revealed the presence of four moa species: Anomalopteryx didiformis; Dinornis robustus; Emeus crassus; and Euryapteryx curtus. The mtDNA, together with highly polymorphic microsatellite markers, were used in combination to individualise eggshell fragments. These data revealed over 50 individual eggs from Wairau Bar – a number that likely represents a considerable proportion of the total reproductive output of moa in the area. This emphasises that human predation of all life stages of moa was intense. In addition, an excess of male bones at the Wairau Bar site (determined by molecular sexing) suggests that males might have been preferential targets, perhaps while incubating eggs. Lastly, moa eggshell genetics and stable isotope profiles were combined to examine the palaeodiets, palaeobiology, palaeoecology and the human mediated extinction of the moa. Organic (δ13Corg and δ15N) and carbonate (δ13Ccarb and δ18O) stable isotope profiles were analysed from 247 genetically identified eggshell fragments. The combination of ancient DNA and stable isotope profiles revealed moa consumed a diet consisting exclusively of C3 vegetation and occupied the varied landscape that surrounded the archaeological deposits. Given that moa were foraging in such diverse landscapes suggests that the first colonisers hunted these moa away from the homogenous landscapes immediately surrounding these archaeological sites and thus engaged in ‘central point foraging’. Discriminant functional analyses indicate moa eggshells can be assigned with good precision to their parent species based purely on their stable isotope profiles. In conclusion, this thesis research has revealed a ‘new’ aDNA substrate that has a number of useful applications in archaeology, palaeogenomics and palaeoecology. The research on New Zealand moa eggshell demonstrated, first-hand, how this substrate can provide valuable insights into the biology, ecology and extinction of a megafaunal species. It is hoped the research will have a lasting influence on eggshell discovered across the globe.