Each recovered sequence showed some signs of molecular damage in the form of DNA fragmentation and type 2 miscoding lesions to a lesser extent, indicating authentic ancient DNA. DNA was extracted in a dedicated ancient DNA laboratory and a control region and COI amplicon were independently replicated for each of two specimens at a separate ancient DNA facility. The independent replication showed identical sequences, thereby ruling out laboratory contamination from PCR products. However there is the unlikely possibility that all four King Island Emu specimens were contaminated by modern Emu specimens beforehand, although the overlapping multiplex approach and observed molecular damage make this scenario extremely unlikely. The same loci were recovered from an additional eighteen modern Emu blood samples from Emu farms in Medina, Western Australia and Palmerston North, New Zealand, these farmed emu represent varying origins from the wild population of modern Emu. The recovered King Island Emu MC1R fragments were identical to those of modern Emu and interestingly did not display a SNP most commonly associated with melanism in birds. This does not necessarily indicate that the modern Emu and the supposedly quite black King Island Emu shared the same plumage colour Other Azlocillin sodium salt genetic or non-genetic factors might be responsible for the reported difference in plumage colour. However, the fact that this likely cause of darker plumage coloration in birds is not detected in the King Island Emu sequences brings into question the validity of this taxonomic trait. The control and COI regions recovered for both taxa show very little diversity, only seven and six sites respectively are polymorphic in alignments including the modern Emu mitochondrial genome reference sequence. The sequences show no individual sites that fully discriminate both taxa,Labetalol hydrochloride the King Island Emu sequences group phylogenetically with three modern Emu that share several segregating sites when compared to other modern Emu. In order to confirm its authenticity the haplotype for modern Emu specimen AU01 has been replicated using several independent amplifications, including long range PCR to avoid nuclear copies and contamination. Although the King Island Emu display unique haplotypes for both the control and the COI regions, they fall within the diversity of modern Emu for both regions. This, in combination with the low control region and COI diversity, suggests that future studies may identify King Island Emu specific haplotypes in modern Emu. Hence this study would suggest that research aiming to distinguish both taxa using DNA should not be limited to the control or COI regions. Perhaps more highly variable nuclear sequences, like those often used in population studies, may be better able to distinguish these taxa. The sequence data recovered from both mitochondrial DNA regions indicate that the modern and the King Island Emu are very closely related. The control and COI regions of the King Island Emu fall within the diversity of modern Emu, showing the latter is a paraphyletic taxon. The low diversity in the sequences recovered for both taxa however indicates that incomplete lineage sorting is a likely cause for this pattern, in particular the processes involved in divergence of peripheral isolates as a result of founder effects. Both taxa show a very close paraphyletic relationship, the maximum distance between any King Island and modern Emu control and COI region haplotype is 0.46 and 0.13%, respectively.