Collagen fingerprinting and sequence analysis provides a molecular phylogeny of extinct Australian megafauna

During the Late Pleistocene, Sahul-the former land mass of Australia, Tasmania and New Guinea-faced one of the greatest waves of megafaunal extinctions on the planet, for reasons that remain highly debated. Yet how some of these extinct species relate to each other also remains unclear, with poor DNA preservation causing challenges for reconstructing phylogenies of extinct taxa using biomolecular data. Here, we use ZooMS collagen peptide mass fingerprinting to screen 51 marsupial bones from Tasmania, ranging in age from late Holocene to over 100 000 years old, to locate specimens of extinct megafauna with the best potential for peptide sequence analysis. We then carried out phylogenetic analyses of collagen peptide sequences, providing the first biomolecular evidence for the relationships of the extinct marsupial genera <i>Zygomaturus</i>, <i>Palorchestes</i> and <i>Thylacoleo</i>. Most notably, our collagen data raise the possibility that the koala (<i>Phascolarctos cinereus</i>) may be the closest living relative of <i>Thylacoleo carnifex</i>, the so-called 'marsupial lion'. Furthermore, by yielding biomolecular data from specimens that far pre-date human arrival, our study demonstrates that ZooMS can be an important tool for establishing higher-resolution extinction chronologies for extinct megafauna from Sahul, which may help to more conclusively establish the cause of their extinction.