Ancient DNA Chronology within Sediment Deposits: Are Paleobiological Reconstructions Possible and Is DNA Leaching a Factor? James Haile,* Richard Holdaway, Karen Oliver,* Michael Bunce,à M. Thomas P. Gilbert,§ Rasmus Nielsen,§ Kasper Munch,§ Simon Y. W. Ho,* Beth Shapiro,* and Eske Willerslev*§ *Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, South Parks Road, Oxford, United Kingdom; Palaecol Research Ltd, Christchurch, New Zealand; àDepartment of Biological Sciences and Biotechnology, Murdoch University, Perth, Australia; and §Centre for Ancient Genetics, Niels Bohr Institute & Biological Institute, University of Copenhagen, Copenhagen, Denmark In recent years, several studies have reported the successful extraction of ancient DNA (aDNA) from both frozen and nonfrozen sediments (even in the absence of macrofossils) in order to obtain genetic ‘‘profiles’’ from past environments. One of the hazards associated with this approach, particularly in nonfrozen environments, is the potential for vertical migration of aDNA across strata. To assess the extent of this problem, we extracted aDNA from sediments up to 3300 years old at 2 cave sites in the North Island of New Zealand. These sites are ideal for this purpose as the presence or absence of DNA from nonindigenous fauna (such as sheep) in sediments deposited prior to European settlement can serve as an indicator of DNA movement. Additionally, these strata are well defined and dated. DNA from sheep was found in strata that also contained moa DNA, indicating that genetic material had migrated downwards. Quantitative polymerase chain reaction analyses demonstrated that the amount of sheep DNA decreased as the age of sediments increased. Our results suggest that sedimentary aDNA is unlikely to be deposited from wind-borne DNA and that physical remains of organisms or their ejecta need to have been incorporated in the sediments for their DNA to be detected. Our study indicates that DNA from sediments can still offer a rich source of information on past environments, provided that the risk from vertical migration can be controlled for. Introduction aging, but the potential for DNA being leached in nonfro- Ancient DNA (aDNA) from diverse mammals and zen conditions remains to be examined (Pa¨a¨bo et al. 2004). plants has been obtained directly from minor amounts of This is especially important because temperate and desert permafrost (permanently frozen) sediments many thou- cave sites are major sources of aDNA used in reconstructing sands of years old (Willerslev et al. 2003; Lydolph et al. past environments (Poinar et al. 1996,1998; Hofreiter et al. 2005). Likewise, under nonfrozen conditions, trace 2000, 2003; Willerslev et al. 2003). DNA leaching would amounts of sediment have yielded aDNA sequences of di- significantly complicate, or even invalidate, the interpreta- verse vertebrate and plant species, even in the absence of tion of results in some contexts (Poinar et al. 1996, 1998; macrofossils (Hofreiter et al. 2003; Willerslev et al. Hofreiter et al. 2000). If the fidelity of DNA sequences can 2003). The immediate sources of this DNA is unclear. A be established from strata (of defined age), however, then possible source of plant DNA in sedimentary deposits is this technology will enable paleofaunal reconstructions fine rootlets (Willerslev et al. 2003). A variety of sources spanning thousands of years (Willerslev and Cooper 2005). have been suggested for animal DNA, including dung, Apart from leaching, other potential sources of bias in taxon urine, skin, hair, and keratin (Lydolph et al. 2005). Also representation include taxon-dependent factors such as uncertain is whether the DNA is extracellular and bound body size and the likely presence of remains, such as hair, to clay minerals or if cellular DNA is released during the feathers, eggs, and dung. extraction procedure (Ogram et al. 1988). In this study, we used sediments from 2 cave sites in To date, most aDNA sedimentary analyses have exam- New Zealand to gain insights to the origin of sedimentary ined soil profiles from permafrost regions (Hansen et al. aDNA and to test for DNA leaching in nonfrozen sedi- 2001; Willerslev et al. 2003; Willerslev, Hansen, Poinar ments. New Zealand has an environment ideal for investi- 2004; Willerslev, Hansen, Rønn, et al. 2004; Lydolph gating possible DNA leaching because the preservation of et al. 2005; Mitchell et al. 2005). In this environment, 2 bones and other materials is excellent. Most importantly, sources of evidence suggest that DNA leaching and rede- New Zealand had a limited range of large vertebrates, position are not significant problems: firstly, changes ob- mostly birds, prior to the arrival of humans; terrestrial mam- served in floral and faunal communities through time mals were entirely absent, with the exception of 3 bat spe- agree broadly with those predicted by macrofossil records cies. This distinctive faunal composition allows leaching to (Willerslev et al. 2003; Lydolph et al. 2005); secondly, the be readily identified, because the presence of nonindige- recovered DNA fragments were damaged in clear age- nous mammal species in presettlement strata is necessarily dependent patterns, despite discontinuous sediment chro- the result of downward movement of DNA in the sediments. nology and the presence of free water (Willerslev, Hansen, For example, sheep (Ovis aries), whose numbers currently Poinar 2004; Hansen et al. 2006). These results are encour- exceed 40 million, were introduced to New Zealand by European settlers only from the 1830s; they did not reach the study area until approximately 1870. It is also well es- Key words: aDNA, sediment, environmental reconstructions, extinc- tablished that the large, indigenous, herbivorous ratite tions, leaching. birds known as moa (Aves; Dinornithiformes) became E-mail: [email protected]. extinct by approximately 550 years ago (Holdaway and Mol. Biol. Evol. 24(4):982–989. 2007 doi:10.1093/molbev/msm016 Jacomb 2000), and so the presence of moa DNA in Euro- Advance Access publication January 25, 2007 pean layers would imply upward movement of sedimentary Ó The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: [email protected] aDNA Chronology within non-cryosediment Deposits 983 FIG. 1.—Map of New Zealand, showing location of Hukanui sites: (A) General view of rock pile containing Hukanui Pool site, (B) General view of rock pile containing Hukanui #7a site. DNA or of the sediment constituents that retained the DNA. By contrast, the Waimihia tephra (Froggatt and Lowe These and other distinct changes in the biota provide an op- 1990) was deposited as a ‘‘rain’’ of pumice lapilli approx- portunity to assess the extent of DNA leaching within non- imately 1–2 mm in diameter, which stripped leaves from permafrost sediments. trees but did not necessarily kill them. The tephra formed a layer approximately 100 mm thick (Froggatt and Lowe 1990) in the Hukanui Pool site, which was more open, but did not enter Hukanui #7a in any significant amounts. Materials and Methods The sediments in Hukanui Pool are more or less Sediment samples were taken from freshly excavated horizontal over most of the floor, but the layers slope sections in 2 dry caves located on the southern face of the downwards toward the western wall. In Hukanui #7a, Hukanui range, North Island, New Zealand (fig. 1): Huka- sediments were incorporated both as horizontal fill amongst nui Pool and Hukanui #7a (fig. 2) are approximately 300 m and above boulder debris and as a small fan in the north- apart and at 860 and 800 m above sea level, respectively western corner of the excavation, where the aDNA samples (Holdaway and Beavan-Athfield 1999). The sites are be- were collected. The fan had been formed from sediments neath large erratic limestone blocks and both contain sed- entering through a small opening from an upper chamber iment layers ranging in age from .3000 years old to the (Holdaway et al. 2002). present. Importantly, the Hukanui locality itself is well Additionally, the sharp, sand-sized particles of fossil suited for investigating paleogenetic reconstructions be- barnacle shells and other marine organisms (derived from cause it contains 2 layers of volcanic ejecta from massive the enclosing limestone rocks) that compose much of the eruptions from the Lake Taupo volcano (fig. 1); the Taupo cave sediments contain variable amounts of clay. The clay ignimbrite deposited at AD 232 6 15 (Sparks et al. 1995) binds the sediments, making them firm and cohesive and and the Waimihia tephra (deposited at 3280 6 20 yr 14C lending stability to vertical sections, so that macroscopic years before present (Froggatt and Lowe 1990). particles cannot migrate through or between the layers. The Taupo eruption emplaced the Taupo ignimbrite Any DNA found out of stratigraphic context must therefore from a pyroclastic flow, which traveled the 80 km to the have migrated as free molecules or bound to microscopic sites at high speed and at a relatively high temperature, particles. Significantly, there are no rooted plants or soil ho- destroying all vegetation and fauna in its path. It disturbed rizon development in the sediments of either site, and so the sediment surfaces and entrained bones and sediment they are not soils. particles in the sites and now forms a tephra layer up to The sediments were moist as a result of condensation 600 mm thick, with charcoal, sediment, and (rare) bone in a humid environment, but there is no fall of water onto inclusions. The Taupo ignimbrite has the consistency of the surface nor flow across it. These are rock shelters, not bulk cement and can also support vertical sections; all dis- true water-carved caves, and there is no free water flowing turbances in the layers are easily visible. in the sites (Holdaway and Beavan-Athfield 1999). Both 984 Haile et al.