MURDOCH RESEARCH REPOSITORY
This is the author’s final version of the work, as accepted for publication
following peer review but without the publisher’s layout or pagination.
The definitive version is available at
http://dx.doi.org/10.1126/science.1141758
Willerslev, E., Cappellini, E., Boomsma, W., Nielsen, R.,
Hebsgaard, M. B., Brand, T. B., Hofreiter, M., Bunce, M., Poinar,
H. N., Dahl-Jensen, D., Johnsen, S., Steffensen, J. P., Bennike,
O., Schwenninger, J.-L., Nathan, R., Armitage, S., de Hoog, C.-J.,
Alfimov, V., Christl, M., Beer, J., Muscheler, R., Barker, J., Sharp,
M., Penkman, K. E. H., Haile, J., Taberlet, P., Gilbert, M. T. P.,
Casoli, A., Campani, E. and Collins, M. J. (2007) Ancient
biomolecules from deep ice cores reveal a forested southern
Greenland. Science, 317 (5834). pp. 111-114.
http://researchrepository.murdoch.edu.au/5130/
Copyright: © 2007 American Association for the Advancement of Science
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Ancient Biomolecules from Deep Ice Cores Reveal a Forested
Southern Greenland
Eske Willerslev
1,*
, Enrico Cappellini
2
, Wouter Boomsma
3
, Rasmus Nielsen
4
, Martin B.
Hebsgaard
1
, Tina B. Brand
1
, Michael Hofreiter
5
, Michael Bunce
6,7
, Hendrik N. Poinar
7
,
Dorthe Dahl-Jensen
8
, Sigfus Johnsen
8
, Jørgen Peder Steffensen
8
, Ole Bennike
9
, Jean-Luc
Schwenninger
10
, Roger Nathan
10
, Simon Armitage
11
, Cees-Jan de Hoog
12
, Vasily
Alfimov
13
, Marcus Christl
13
, Juerg Beer
14
, Raimund Muscheler
15
, Joel Barker
16
, Martin
Sharp
16
, Kirsty E.H. Penkman
2
, James Haile
17
, Pierre Taberlet
18
, M. Thomas P. Gilbert
1
,
Antonella Casoli
19
, Elisa Campani
19
, and Matthew J. Collins
2
1
Centre for Ancient Genetics, University of Copenhagen, Denmark
2
BioArch, Departments of Biology and
Archaeology, University of York, UK
3
Bioinformatics Centre, University of Copenhagen, Denmark
4
Centre
for Comparative Genomics, University of Copenhagen, Denmark
5
Max Planck Institute for Evolutionary
Anthropology, Germany
6
Murdoch University Ancient DNA research laboratory, Murdoch University,
Australia
7
McMaster Ancient DNA Center, McMaster University, Canada
8
Ice and Climate, University of
Copenhagen, Denmark
9
Geological Survey of Denmark and Greenland, Denmark
10
Research Laboratory
for Archaeology and the History of Art, University of Oxford, UK
11
Department of Geography, Royal
Holloway, University of London, UK
12
Department of Earth Sciences, University of Oxford, UK
13
PSI/ETH
Laboratory for Ion Beam Physics, Institute for Particle Physics, ETH Zurich, Switzerland
14
EAWAG,
Switzerland
15
GeoBiosphere Science Center, Lund University, Sweden
16
Department of Earth and
Atmospheric Sciences, University of Alberta, Canada
17
Ancient Biomolecules Centre, Oxford University,
UK
18
Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, BP 53, 38041 Grenoble
Cedex 9, France
19
Dipartimento di Chimica Generale e Inorganica, Università di Parma, Italy
Abstract
One of the major difficulties in paleontology is the acquisition of fossil data from the 10% of Earth’s
terrestrial surface that is covered by thick glaciers and ice sheets. Here we reveal that DNA and amino
acids from buried organisms can be recovered from the basal sections of deep ice cores and allow
reconstructions of past flora and fauna. We show that high altitude southern Greenland, currently
lying below more than two kilometers of ice, was once inhabited by a diverse array of conifer trees
and insects that may date back more than 450 thousand years. The results provide the first direct
evidence in support of a forested southern Greenland and suggest that many deep ice cores may
contain genetic records of paleoenvironments in their basal sections.
The environmental histories of high latitude regions such as Greenland and Antarctica are
poorly understood because much of the fossil evidence is hidden below kilometer thick ice
sheets (1-3). Here, we test the idea that the basal sections of deep ice cores can act as archives
for ancient biomolecules and show that these molecules can be used to reconstruct significant
parts of the past plant and animal life in currently ice covered areas.
The samples studied come from the basal impurity rich (silty) ice sections of the 2km long Dye
3 core from south-central Greenland (4), the 3km long GRIP core from the summit of the
*
Author of correspondence: ewillerslev@bi.ku.dk
UKPMC Funders Group
Author Manuscript
Science. Author manuscript; available in PMC 2009 June 11.
Published in final edited form as:
Science. 2007 July 6; 317(5834): 111–114. doi:10.1126/science.1141758.
UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscript
Greenland ice sheet (5), and the Late Holocene John Evans Glacier on Ellesmere Island,
Nunavut, northern Canada (Fig. 1A,B). The latter sample was included as a control to test for
potential exotic DNA because the glacier has recently overridden a land surface with a known
vegetation cover (6). As an additional test for long-distance atmospheric dispersal of DNA, we
included five control samples of debris-free Holocene and Pleistocene ice taken just above the
basal silty samples from the Dye 3 and GRIP ice cores (Fig. 1B). Finally, our analyses included
sediment samples from the Kap København Formation from the northernmost part of
Greenland, dated to 2.4 million years before present (Ma BP) (1,2).
The silty ice yielded only few pollen grains and no macrofossils (7). However, the Dye 3 and
John Evans Glacier silty ice samples showed low levels of amino acid racemization (Fig. 1A,
insert), indicating good organic matter preservation (8). Therefore, following previous success
with permafrost and cave sediments (9-11), we attempted to amplify ancient DNA from the
ice. This was done following strict criteria to secure authenticity (12-14), including covering
the surface of the frozen cores with plasmid DNA to control for potential contamination that
may have entered the interior of the samples through cracks or during the sampling procedure
(7). PCR products of the plasmid DNA were obtained only from extracts of the outer ice
scrapings but not from the interior, confirming that sample contamination had not penetrated
the cores.
We could reproducibly PCR amplify short amplicons (59-120 base-pairs, bp) of the chloroplast
DNA (cpDNA) rbcL gene and trnL intron from about 50g of the interior ice melts from the
Dye 3 and the John Evans Glacier silty samples. From Dye 3, we also obtained 97bp amplicons
of invertebrate cytochrome oxidase subunit I (COI) mitochondrial DNA (mtDNA). Attempts
to reproducibly amplify DNA from the GRIP silty ice and from the Kap København Formation
sediments were not successful. These results are consistent with the amino acid racemization
data demonstrating superior preservation of biomolecules in the Dye 3 and John Evans silty
samples, which is likely due to these samples being colder (Dye 3) or younger (John Evans)
than the GRIP sample (Fig. 1A, insert). We also failed to amplify DNA from the five control
samples of Holocene and Pleistocene ice taken just above the silty samples from the Dye 3 and
GRIP ice cores (volumes 100g to 4kg, Fig. 1B, (7)). None of the samples studied yielded
putative sequences of vertebrate mtDNA.
A previous study has shown that simple comparisons of short DNA sequences to GenBank
sequences using BLAST Search make misidentification likely (15). Therefore, we assigned
the sequences obtained to the taxonomic levels of order, family, or genus using a new rigorous
statistical approach (7). In brief, this Bayesian method calculates the probability that each
sequence belongs to a particular clade by considering its position in a phylogenetic tree based
on similar GenBank sequences. In the calculation of these probabilities, uncertainties regarding
phylogeny, models of evolution and missing data are taken into account. Sequences with >90%
posterior probability of membership to a taxonomic group were assigned to that group.
Additionally, a given plant taxon was only considered genuine if at least two sequences
assigned to that taxon were found to be 100% identical and reproducibly obtained in separate
analyses (for Dye 3, by independent laboratories and for the John Evans Glacier control sample
within laboratory). This strict criterion of authenticity obviously dismisses many putative taxa
that are present at low abundance or have heterogeneous distributions, as is typical of
environmental samples (16), but efficiently minimizes the influence of possible low-level
contamination and misidentifications due to DNA damage (17).
Approximately 31% of the sequences from the John Evans Glacier silty sample were assigned
to plant taxa passing the authentication and identification criteria. These belong to the Rosales
(an order of flowering plants, including nine families such as the rose family Rosaceae), the
Salicaceae (willow) family, and the genus Saxifraga (Table 1). This result is consistent with
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the John Evans Glacier forming no more than a few thousand years ago in a high Arctic
environment (18), characterized by low plant diversity and sparse vegetation cover similar to
that currently surrounding the glacier which consists mainly of Arctic willow (family
Salicaceae), purple saxifrage (genus Saxifraga), Dryas (order Rosales), and Arctic poppy
(19). Thus, by confirming the expected result, the John Evans Glacier study can be regarded
as a positive control showing that DNA data from silty ice reliably record the local ecology.
In contrast to the John Evans Glacier silty sample, the 45% of the Dye 3 DNA sequences that
could be assigned to taxa reveal a community very different from that of Greenland today. The
taxa identified include trees such as alder (genus Alnus), spruce (genus Picea), pine (genus
Pinus), and members of the yew family (Taxaceae) (Table 1). Their presence indicates a
northern boreal forest ecosystem rather than today’s Arctic environment. The other groups
identified, including Asteraceae, Fabaceae, and Poaceae, are dominated by herbaceous plants
and are represented by many species found in northern regions at present (Table 1). The
presence of these herb-dominated families suggests an open forest, allowing heliophytes to
thrive. Additionally, we recorded taxa that are common in the Arctic and/or Boreal regions but
lacked 100% sequence identity between independent laboratories. These are yarrow
(Achillea), birch (Betula), chickweed (Cerastium), fescue (Festuca), rush (Luzula), plantain
(Plantago), bluegrass (Poa), saxifrage (Saxifraga), snowberry (Symphoricarpos), and aspen
(Populus). Although not independently authenticated at the sequence level the presence of
these taxa adds further support to the conclusion of a northern boreal forest ecosystem at Dye
3.
To date, the youngest well-dated fossil evidence of native forest in Greenland is from
macrofossils in the deposits of the Kap København Formation from the northernmost part of
Greenland and dates back to around 2.4Ma (1,2). Other less well-dated traces of forests in
Greenland include wood at two other late Cenozoic sites in northern Greenland (20), pollen
spectra of unknown age in marl concretions found in a late glacial moraine, and wood and
spruce seeds in eastern Greenland (21). Dye 3, almost exactly 2000km to the southwest of the
Kap København Formation (Fig. 1A), therefore provides the first direct evidence of a forested
southern-central Greenland.
The invertebrate sequences obtained from the Dye 3 silty ice are related to beetles (Coleoptera),
flies (Diptera), spiders (Arachnida), brushfoots (Nymphalidae), and butterflies and moths
(Lepidoptera) (probability supports between 50% and 90%). However, only sequences of the
latter two are supported by more than 90% significance (Table 1). Thus, although detailed
identifications of the COI sequences are in general not strongly supported, the results show
that DNA from a variety of invertebrates can be obtained from sediments even in the absence
of macrofossils as was previously shown for plants, mammals, and birds (9-11).
Several observations suggest that the DNA sequences we obtained from the Dye 3 ice are of
local origin and not due to long-distance dispersal. The reproducible retrieval of diverse DNA
from the silty basal ice but not from similar or larger volumes of the overlying “clean” ice
largely precludes long-distance atmospheric dispersal of microfossils as a source of the DNA.
Although pollen grains are found in the Greenland ice sheet, including the Dye 3 silty ice (7),
the concentrations are in general too low (0.3-15 grains/liter (22), Bourgeois pers. comm.) for
them to be present in the sample volumes studied. Furthermore, long-term survival of DNA in
pollen has proved fairly poor (23) and the vast majority of angiosperm pollen does not contain
cpDNA (24). These factors effectively exclude pollen as the general source of the silty ice plant
DNA. Moreover, the Dye 3 silty ice appears to have originated as solid precipitation without
going through stages of superimposed ice and most likely formed by mixing in the absence of
free water (25), effectively excluding subsurface transportation. As explained in (26) the ice
is believed to be predominantly of local origin having been shielded from participating in the
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large-scale glacier-flow by a bedrock trough, in agreement with the solid ice-mixing hypothesis
(25). Thus, being of local origin, the DNA sequences from the Dye 3 silty ice must derive from
the plants and animals that inhabited this region the last time it was ice free, as possible older
DNA records from previous ice-free periods will vanish with the establishment of a new
ecosystem, or at least be out-competed during PCR by DNA from the most recent record.
Interestingly, the plant taxa suggest that this period had average July temperatures that
exceeded 10°C and winter temperatures not colder than −17°C, which are the limits for northern
boreal forest and Taxus, respectively (1). Allowing for full recovery of the isostatic depression
that is produced by two-kilometers of ice, Dye 3 would have been about a thousand meters
above sea level. In combination, these factors suggest that a high altitude boreal forest at Dye
3 may date back to a period considerably warmer than present.
There are no established methods for dating basal ice, and it remains uncertain whether the
overlying clean ice of Dye 3 is temporally contiguous with the lower silty section. Therefore,
in order to obtain a tentative age estimate for the Dye 3 silty ice and its forest community, we
applied a series of dating techniques;
10
Be/
36
Cl isotope ratios, single grain luminescence
measurements, amino acid racemization coupled with modeling of the basal ice temperature
histories of GRIP and Dye 3, and maximum likelihood estimates for the branch length of the
invertebrate COI sequences (7). All four dating methods suggest that the Dye 3 silty ice and
its forest community predate the Last Interglacial (LIG, ~130-116Ka) (Fig 2), which contrasts
with the results of recent models suggesting that Dye 3 was ice-free during this period (27,
28). Indeed, all four dating methods give overlapping dates for the silty ice between 450Ka
and 800Ka (Fig. 2), exceeding the current record of long-term DNA survival from Siberian
permafrost of 300-400Ka (9). However, due to the many assumptions and uncertainties
connected with the interpretation of the age estimates (7), we cannot rule out the possibility of
a LIG age for the Dye 3 basal ice.
In conclusion, our results reveal that ancient biomolecules from basal ice offer a novel means
for environmental reconstruction from ice covered areas and can yield new insights into the
climate and the ecology of communities from the distant past. As many deep ice cores exist
from both hemispheres and further drillings are planned, this new approach may be used on a
larger scale. Excitingly, basal ice at even lower temperatures than Dye 3 may contain an archive
of genetic data of even greater antiquity.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank S. Funder, P. Hartvig, J. Bourgeois, O. Seberg, J. J. Böcher, K. Høegh, J. W. Leverenz, and S. Y. W. Ho
for helpful discussions and R. Bailey, N. Belshaw, N. Charnley, C. Doherty and D. Peat for technical assistance and
advice. EW, TB, MBH were supported by the Carlsberg Foundation, DK, and the National Science Foundation. EW
and KP were both supported by Wellcome Trust Bioarchaeology Fellowships. NERC supported KP and MC. EC was
in receipt of a Marie Curie Intra European Fellowship, grant number 501340. EW and MC acknowledge support from
Europe (MEST-CT-2004-007909). MB and HNP were supported by NSERC grant #299103-2004 and McMaster
University. MS and JB were supported by NSERC and the Polar Continental Shelf Project. MH was supported by the
Max Planck Society.
References and Notes
1. Bennike O. Meddelelser om Grønland, Geoscience 1990;23:85.
2. Funder S, et al. Bulletin of the Geological Society of Denmark 2001;48:117.
3. Francis JE, Hill RS. Palaios 1996;11:389.
4. Dansgaard W, et al. Science 1982;218:1273. [PubMed: 17770148]
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