Significance This report is the first publication, to our knowledge, to report the complete mitochondrial genome of an ancient Aboriginal Australian. In addition, it also provides important evidence about the reliability of the only previous publication of this kind. The paper attained international significance, although its conclusions have remained controversial. Using second generation DNA sequencing methods, we provide strong evidence that the DNA sequences reported by Adcock et al. were, indeed, contamination. Our manuscript is also important, because the research was planned and conducted and is published with the support of the Barkindji, Ngiyampaa, and Muthi Muthi indigenous groups.

Abstract The publication in 2001 by Adcock et al. [Adcock GJ, et al. (2001) Proc Natl Acad Sci USA 98(2):537–542] in PNAS reported the recovery of short mtDNA sequences from ancient Australians, including the 42,000-y-old Mungo Man [Willandra Lakes Hominid (WLH3)]. This landmark study in human ancient DNA suggested that an early modern human mitochondrial lineage emerged in Asia and that the theory of modern human origins could no longer be considered solely through the lens of the “Out of Africa” model. To evaluate these claims, we used second generation DNA sequencing and capture methods as well as PCR-based and single-primer extension (SPEX) approaches to reexamine the same four Willandra Lakes and Kow Swamp 8 (KS8) remains studied in the work by Adcock et al. Two of the remains sampled contained no identifiable human DNA (WLH15 and WLH55), whereas the Mungo Man (WLH3) sample contained no Aboriginal Australian DNA. KS8 reveals human mitochondrial sequences that differ from the previously inferred sequence. Instead, we recover a total of five modern European contaminants from Mungo Man (WLH3). We show that the remaining sample (WLH4) contains ∼1.4% human DNA, from which we assembled two complete mitochondrial genomes. One of these was a previously unidentified Aboriginal Australian haplotype belonging to haplogroup S2 that we sequenced to a high coverage. The other was a contaminating modern European mitochondrial haplotype. Although none of the sequences that we recovered matched those reported by Adcock et al., except a contaminant, these findings show the feasibility of obtaining important information from ancient Aboriginal Australian remains.

Since the publication in 1863 of Thomas Henry Huxley’s Man’s Place in Nature, there has been considerable interest and debate regarding the origins of the first Australians. It was evolutionary biologist Ernst Haeckel who initially argued that humans originated in South Asia, a theory that enjoyed considerable support for the first half of the 20th century. The Asian origin theory was provided with a “missing link” after the discoveries by Eugene Dubois of the Java Man fossils in Trinil, Indonesia in the 1890s. Although there is once again increasing acceptance that Asia played a significant role in hominin evolution, particularly after the discoveries of new hominin species at Dmanisi and Flores (1), few researchers have suggested that Asia has played a key role in modern human origins. Some paleoanthropologists still argue that Australian origins are closely linked with archaic hominins from Indonesia, such as the Ngandong series of Homo erectus (2). The results of the work by Adcock et al. (3) have also been argued to support a significant Asian contribution to modern human origins. Several recent studies that focus on genomic data, however, suggest that archaic hominins, such as the Denisovans and Neanderthals, only made a small genetic contribution to Aboriginal Australians (4, 5).

Dating methods have established the presence of Aboriginal Australians on the Australian continent at least at 47,000 y B.P. (6). The existence of morphologically gracile and robust forms of Aboriginal Australian people from the Pleistocene subfossil record suggested to some (7) a complex history of the settlement of the Australian continent. For many years, the robust Pleistocene Australian morphology was used to argue for a close phylogenetic relationship to archaic humans from Java, Indonesia, providing evidence for the regional evolution of modern humans in Southeast Asia. Although the basic nature and usefulness of the “robusticity” debate has been questioned (8), it has been, in the past, a significant component of many theories of human evolution in the region.

In recent decades, the debate regarding the origins of the first Australians has largely focused on the mtDNA diversity of contemporary Aboriginal Australians. Generally, this debate has centered on whether this diversity was the result of a single migration or multiple migrations into Australia. In the context of these discussions, the publication by Adcock et al. (3) of mtDNA sequences from ancient robust and gracile forms originating from two sites, Kow Swamp and the Willandra Lakes (Fig. 1), stood as an important test of Thorne’s Dihybrid model for Aboriginal Australian origins. Adcock et al. (3) made four landmark claims. First, they had recovered authentic mitochondrial sequences from 10 sets of ancient Aboriginal Australian remains. Second, most of these recovered ancient sequences fell within the diversity of contemporary human sequences, whereas Mungo Man [Willandra Lakes Hominid (WLH3)] and Kow Swamp 8 (KS8) yielded unique mtDNA sequences. Importantly, these two sequences fell outside the range of contemporary human variation and clustered with a nuclear DNA insert. Moreover, the divergence between the mitochondrial and nuclear inserts predated the divergence of all contemporary modern humans. The implications of these claims were profound, because they suggested that multiple waves of migration to Australia were, in fact, possible, involving population replacement or selective sweeps to explain modern genetic and morphological diversity. Third, to explain the disconnect between the morphological and genetic data, Adcock et al. (3) invoked selective sweeps. Fourth, Adcock et al. (3) suggested that a reinterpretation of the “Out of Africa” theory was required and questioned the use of contemporary mitochondrial data in support of a single origin of modern humans from Africa. Given the obvious importance of these claims, the work by Adcock et al. (3) deserves serious reevaluation using more recent advances in DNA methods and powerful analytical approaches.

Fig. 1. The locations of the Willandra Lakes and Kow Swamp where the remains for this study originated. (A) Map of Australia showing the locations of Lake Mungo (B on map) and Kow Swamp (C on map). (B) Map of the Willandra Lakes detailing where each of the remains were excavated. (C) Map of Kow Swamp detailing where the remains were excavated.

The majority of ancient mitochondrial sequences reported by Adcock et al. (3), including those from individuals with both “robust” and “gracile” morphologies, were shown to fall into a single clade that included sequences of living Aboriginal Australians. These mtDNA sequences failed to phylogenetically differentiate individuals with robust or gracile morphologies (3). Two of the sampled individuals, Mungo Man (WLH3) [Lake Mungo 3 (LM3) in the study by Adcock et al. (3)] and KS8, were identified by Adcock et al. (3) as lying outside the range of modern Aboriginal Australian mitochondrial variation. The latter observation was argued to support the highly debated multiregional hypothesis (9, 10).

These controversial findings were further challenged by researchers who questioned the authenticity of the sequences reported (11⇓–13), highlighted problems with the phylogenetic approach (11⇓⇓⇓–15), and expressed concern about the validity (11, 12, 14, 15) of the conclusions by Adcock et al. (3) . It is worth noting that Adcock et al. (16, 17) rebutted some of these criticisms, defending their conclusions about modern human origins, particularly with regard to the phylogenetic placement of Mungo Man (WLH3).

To assess the results obtained by Adcock et al. (3), we were given consent from the Willandra Lakes World Heritage Area Aboriginal Elders Committee (comprising the Barkindji, Ngiyampaa, and Muthi Muthi elders) to resample material from this important fossil series. In addition, we obtained access to several samples and extracts from the original research by Adcock et al. (3), including those from KS8.

These samples are either Late Pleistocene or Holocene in age (Table 1). WLH4 was excavated by Wilfred Shawcross in 1974 but does not have an absolute date. WLH4 is likely to be Holocene in age, because the skeletal remains are not heavily mineralized. The teeth from WLH4 exhibit a pattern of occlusal wear typical of Aboriginal hunter-gatherer populations and includes no evidence of dental caries. Interestingly, they also exhibit a pattern of interproximal tooth wear bilaterally at the second mandibular molars, indicating that some task activity that incorporated repeatedly dragging fibrous material between these teeth had occurred (18). This wear identifies WLH4 as an individual from a traditional hunter-gatherer population and combined with the lack of mineralization in the bone and its position in the stratigraphic sequence at Lake Mungo, indicates that the remains are estimated to be late Holocene in age (∼3,000–500 y B.P.).

Table 1. Details of archaeological material examined in this study

The investigated remains are shown in Fig. 2. To avoid and monitor previously hypothesized contamination issues and PCR artifacts, we used a range of methods that were independently performed at different locations. We focused our next generation sequencing efforts on all four Willandra Lakes individuals and our PCR and single-primer extension (SPEX) cloning/sequencing efforts on the KS8 and Mungo Man (WLH3) studied by Adcock et al. (3).

Fig. 2. Details of five sets of skeletal remains of individuals from the Willandra Lakes that were investigated in this study. These are, from left to right: WLH3, WLH4, WLH15, WLH55 and KS8. Gray indicates skeletal elements that have survived for each individual.

Conclusion The Aboriginal Australian individuals referred to as WLH3 and KS8 have played a critical role in the interpretation of modern human evolution outside of Africa. The fact that we could not replicate the original results by Adcock et al. (3) is perhaps not surprising given that they were obtained by PCR-based methods applied to samples recovered from Holocene and Pleistocene deposits in semiarid and temperate Australia. The methods used here present advantages over PCR, in that they are able to target very degraded templates and improve the ability to distinguish authentic sequences from contaminants (19, 28, 29, 33, 34). These approaches have allowed us to clarify the results for the four Willandra Lakes individuals analyzed by Adcock et al. (3); only WLH3 (Mungo Man) and WLH4 contained verifiable human DNA, whereas nothing could be retrieved from extracts of WLH15 and WLH55. However, in principle, we cannot exclude the possibility that these specimens contain endogenous human DNA, which may be differentially preserved within the same individual. It has been shown that some regions of the skeleton, such as the petrous bone, are better suited than other regions for preserving ancient DNA (35). The recovery of an authentic Aboriginal Australian haplotype (S2) from WLH4 together with a European contaminating sequence (V3c) show the advantages of the non–PCR-based approach used here. We did not observe any amplified product for the extraction and library build blanks, suggesting that no contamination occurred during the laboratory procedures. For WLH3, however, we found a total of five European mtDNA contaminants, presumably derived from peopling handling the remains. Our study also suggests that the original 2001 extracts contained modern contaminants, although the haplogroup K sequence found in WLH3 was likely introduced during the extraction process. The SPEX assay, which has the ability to amplify shorter molecules, supports the next generation sequencing results using the new extractions. A similar conclusion can be drawn for KS8, because we were unable to amplify any diagnostic human mtDNA from either the SPEX assay or the next generation sequencing of enriched secondary libraries. The abundance of contamination that can be identified from these remains highlights the necessity for careful precautions and specially constructed laboratory environments now typical of ancient DNA facilities. With ancient DNA studies focusing on increasingly older and more marginal samples, it is important to reduce direct contact with the remains to an absolute minimum. As a further control against contamination within the laboratory, it may also prove beneficial to sequence the genomes of all of those who handled the remains. This procedure would have the advantage of providing data on other loci that may be similar to the individual being studied (e.g., nuclear inserts of the mtDNA in this case). In relation to the four landmark claims made by Adcock et al. (3), we were unable to verify that the original study recovered any authentic Aboriginal Australian mitochondrial sequences from the four Willandra Lakes samples that they studied (claim 1). The contamination observed for WLH3 and the absence of human DNA for KS8 suggest that the validity of the originally reported sequences should be reappraised in light of the technological advances available to this study. These observations together with the uncertainties of the phylogenetic resolution obtained using HVR1 sequences provide no support for the historical existence of mitochondrial lineages in Australia that fall outside contemporary human variation (claim 2). As a consequence, there is no need to explain the presence of robust and gracile morphologies by invoking population replacement or selective sweeps (claim 3). We suggest that all of the sequences reported by Adcock et al. (3) were either modern contamination or PCR artifacts. As a result, it seems that contemporary mitochondrial data are consistent with the Out of Africa theory (claim 4). Of the four Willandra Lakes samples, we show that WLH4 does contain authentic Aboriginal Australian DNA sequences, and we report the complete mitochondrial sequence from this individual. This report is the first example, to our knowledge, of DNA being recovered from ancient remains in an Australian archaeological context. The recovery of a complete mitochondrial genome to a high level of coverage of one Willandra Lake sample suggests that future research into First Australian material has the potential to lead to the recovery of complete nuclear genomes from these iconic remains.

Materials and Methods Sample Collection. In partnership with the Barkindji, Ngiyampaa, and Muthi Muthi tribal elders, we sampled material from four sets of remains of First Australians. Originally, these remains derived from the Willandra Lakes region (a copy of the approval is supplied in SI Appendix) and were held in storage at the Australian National University. Subsamples were taken from Mungo Man (WLH3), WLH4, WLH15, and WLH55. The following procedure was conducted in a fume hood that had previously not been used for any human-based research. We treated the surface of bones with concentrated sodium hypochlorite to remove any contaminating surface DNA, and ∼200 mg bone shavings were then removed from the treated surface using a sterile Dremel on low speed. This method is minimally invasive and has been generally successful at removing contaminating human DNA, thereby allowing the recovery of authentic endogenous ancient DNA. All pre-PCR procedures were carried out in the dedicated ancient DNA laboratory at Griffith University or a similar laboratory located at Oxford University (Ancient Biomolecules Centre). DNA was extracted from ∼50 mg bone powder and eluted in 50 μL water using a protocol that retains short DNA fragments and removes inhibitory substances (32). Extraction blanks were included throughout all procedures. Illumina DNA Libraries were built according to the methods described by Meyer and Kircher (36) and Rasmussen et al. (4). Using the NEBNext DNA Library Prep Master Mix Set for 454 (NEB), 21.25 μL DNA extract was subjected to one-quarter volume end repair reaction. After a MinElute (Qiagen) purification with 10× binding buffer PN (Qiagen) or PB (Qiagen), the resulting solution was subjected to quick ligation in a one-half volume reaction with blunt end adapters. After an additional MinElute purification, the DNA was subjected to a one-half volume fill-in reaction. The libraries were amplified to levels required for sequencing with one of three protocols (SI Appendix, Table S5), and if necessary, a secondary PCR was used to increase quantities. For all three protocols, the primary 100-μL library PCR was done according to the manufacturer’s instructions using the InPE1.0 and indexing primers. The primary PCR was subsequently cleaned using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. The secondary PCR used 5 μL purified primary PCR and primers IS5 and IS6. The volume of the secondary PCR was 33 μL for the AccuPrime Pfx SuperMix (Life Technologies) protocol. Cycling was as follows for both PCR stages: 95 °C for 10 min, cycling (95 °C for 15 s, 60 °C for 30 s, and 68 °C for 30 s), and 10 °C indefinitely. The volume of the secondary PCR was 50 μL for the KAPA HiFi HotStart Uracil+ ReadyMix (Kapa Biosystems) protocol, and cycling was as follows for both PCR stages: 98 °C for 2 min, cycling (98 °C for 30 s, 60 °C for 15 s, and 72 °C for 15 s), 72 °C for 1 min, and 10 °C indefinitely. The volume of the secondary PCR was 50 μL for the Platinum Taq DNA Polymerase High Fidelity (Life Technologies) protocol, and cycling was as follows for both PCR stages: 94 °C for 1 min, cycling (94 °C for 15 s, 60 °C for 30 s, and 68 °C for 30 s], and 10 °C indefinitely. DNA Enrichment Using Whole-Genome Capture and Bioinformatics. Details of DNA capture methods, second generation sequencing, and bioinformatic processing of sequence reads together with mitochondrial sequence analyses and phylogenetics can be found in SI Appendix, SI Methods. In addition, details of the SPEX method and related PCR methods can also be found in SI Appendix.

Acknowledgments This research was conducted in partnership with the Barkindji, Ngiyampaa, and Muthi Muthi elders. We thank these people for their permission to study the Lake Mungo remains and their advice and guidance. We thank Manaasa Raghavan and Morten Rasmussen for help with Illumina library construction methods, Hannes Schroeder and Morten Allentoft for help with the DNA capture methods, Greg Baillie for advice regarding DNA extraction methods, and Gregory Adcock for providing the original DNA extracts for WLH3 and KS8 as well as undigested homogenized bone powder from WLH3. We also thank Christina Strobl (Innsbruck Medical University) for second generation DNA sequencing and Paul Brotherton (Department of Zoology, Oxford University) for assistance with SPEX assays. We thank the anonymous reviewers for helpful suggestions and comments. We also thank The Danish National High-Throughput DNA Sequencing Centre for sequencing the samples. We thank the Australian Research Council for support through Discovery and Linkage Grant Projects DP140101405, DP110102635, and LP120200144. J.L.W. thanks Griffith University for a PhD scholarship and the Environmental Futures Research Institute for support. P.E. was funded by the Wenner–Gren Foundation.

Footnotes Author contributions: M.C.W., C.D.M., E.W., and D.M.L. designed research; T.H.H., S.S., J.L.W., P.E., L.H., and W.P. performed research; T.H.H., S.S., P.E., and L.H. contributed new reagents/analytic tools; S.S., J.L.W., C.D.M., and D.M.L. analyzed data; and T.H.H., S.S., J.L.W., P.E., M.C.W., C.D.M., E.W., and D.M.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequence (WLH4, haplogroup S2) reported in this paper has been deposited in the GenBank database (accession no. KU659023).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1521066113/-/DCSupplemental.