Ancient DNA reveals lack of postglacial habitat
tracking in the arctic fox
Love Dale ´n*†‡, Veronica Nystro ¨m†, Cristina Valdiosera*, Mietje Germonpre ´§, Mikhail Sablin¶, Elaine Turner?,
Anders Angerbjo ¨rn†, Juan Luis Arsuaga*, and Anders Go ¨therstro ¨m*,**
*Centro UCM-ISCIII de Evolucio ´n y Comportamiento Humanos, C/ Sinesio Delgado 4, Pabello ´n 14, 28029 Madrid, Spain;†Department of Zoology, Stockholm
University, S-106 91 Stockholm, Sweden;§Department of Palaeontology, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels,
Belgium;¶Zoological Institute RAS, Universitetskaya nab.1, St. Petersburg 199034, Russia;?Ro ¨misch-Germanisches Zentralmuseum Mainz, Forschungsbereich
Altsteinzeit, Schloss Monrepos, 56567 Neuwied-Segendorf, Mainz, Germany; and **Department of Evolutionary Biology, Evolutionary Biology
Centre, Uppsala University, S-752 36 Uppsala, Sweden
Contributed by Juan Luis Arsuaga, February 22, 2007 (sent for review January 16, 2007)
How species respond to an increased availability of habitat, for
example at the end of the last glaciation, has been well established.
In contrast, little is known about the opposite process, when the
amount of habitat decreases. The hypothesis of habitat tracking
predicts that species should be able to track both increases and
decreases in habitat availability. The alternative hypothesis is that
populations outside refugia become extinct during periods of unsuit-
able climate. To test these hypotheses, we used ancient DNA tech-
through an expansion/contraction cycle. The results show that the
arctic fox in midlatitude Europe became extinct at the end of the
Instead, a high genetic similarity between the extant populations in
Scandinavia and Siberia suggests an eastern origin for the Scandina-
new insights into how species respond to climate change, since they
suggest that populations are unable to track decreases in habitat
avaliability. This implies that arctic species may be particularly vul-
nerable to increases in global temperatures.
climate change ? evolutionary stasis ? extinction ? phylogeography ?
forced temperate species in North America and Europe to
endure repeated isolations in southern refugia, the warmer
interglacials have allowed them to expand northwards to recol-
onize previously glaciated regions (2). Genetic analysis has in
recent years been used to study the patterns of postglacial
recolonization after the last glaciation, where the genetic com-
position of recently recolonized regions is compared with that of
refugial populations (2, 3).
However, this approach is of limited value if one or several
glacial populations have gone extinct during the Holocene.
Moreover, although phylogeographic inference based on mod-
ern samples can be used to investigate the process of recoloni-
zation, it cannot be used to directly study the process by which
species contract. Current knowledge about this process is there-
fore limited, despite its importance for evolutionary theory.
Darwin suggested that when species ‘‘moved first southward and
afterward backwards to the north, in unison with the changing
climate, they will not have been exposed during their long
migration to any great diversity of temperature [..]. Hence, in
accordance with the principles inculcated in this volume, these
forms will not have been liable to much modification’’ (4). The
idea that species are able to increase their distribution during
periods of suitable climate and, in a similar way, literally contract
as the hypothesis of habitat tracking (5), which was proposed as
an explanation for the high degree of evolutionary stasis ob-
served in the fossil record (6). The alternative to habitat tracking
during the contraction phase would be that populations inhab-
he glacial cycles have influenced the distribution of organ-
isms worldwide (1). Whereas cold periods have generally
iting areas outside refugia become extinct when climate deteri-
orates (3, 7).
One possible approach to test these two hypotheses is to use
ancient DNA technology to investigate if populations that
inhabited geographic regions outside refugia during the expan-
sion phase have contributed to the genetic composition of the
refugial populations. For temperate species, this would require
DNA to be recovered from samples predating the last intergla-
cial, which would be problematic since samples of that age are on
the limits of ancient DNA recovery. Arctic species, on the other
hand, expand during glacials and contract during interglacials (8,
9). Testing the hypothesis of habitat tracking on an arctic species
would thus be possible by comparing the genetic variation of
southern Late Pleistocene populations with that of contempo-
rary northern populations.
We have analyzed genetic variation in the arctic fox (Alopex
lagopus), which had a large distribution during the last glacial
maximum (LGM) and inhabited large parts of central and
northeastern Eurasia (10–15). Today, the arctic fox is restricted
to the tundra regions in the northern hemisphere, including
regions that were glaciated during the LGM, such as Scandinavia
(16). There are three possible hypotheses for the origin of the
Scandinavian arctic fox: (i) Scandinavia was colonized from
the south by foxes tracking the retreating ice edge at the end of
the LGM, (ii) Scandinavia was colonized by foxes expanding
from the ice-free regions in the east (e.g., Beringia) after the end
of the LGM, and (iii) the arctic fox survived the LGM in a local
Scandinavian refugium (17). These three scenarios are all plau-
sible because Scandinavia has been colonized from both the
south and east by other species (3), and LGM survival in a local
refugium has been suggested for the Norwegian lemming (Lem-
mus lemmus) (18).
We addressed these hypotheses by comparing mitochondrial
DNA (mtDNA) sequences retrieved from Late Pleistocene
arctic foxes in midlatitude Europe with those from extant
Siberian and Scandinavian arctic foxes. Furthermore, to avoid
confounding effects of a recent bottleneck in Scandinavia caused
by human overexploitation 100 years ago, which may have
altered the population’s genetic composition, we also included
42 prebottleneck samples from Scandinavia (19). For hypotheses
J.L.A., and A.G. performed research; L.D. analyzed data; and L.D., A.A., J.L.A., and A.G.
wrote the paper.
The authors declare no conflict of interest.
Abbreviations: AMOVA, analysis of molecular variance; LGM, last glacial maximum.
Data deposition: The DNA sequences reported in this paper have been deposited in the
GenBank database (accession nos. EF095220–EF095229).
‡To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or love.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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(i) and (ii), we would expect to find Scandinavian mitochondrial
DNA haplotypes that are identical to, or recently derived from,
the haplotypes in the source population (2, 3), whereas hypoth-
esis (iii) predicts that several unique haplotypes should have
evolved in Scandinavia (18).
Results and Discussion
Late Pleistocene fossils were sampled from four locations in
midlatitude Europe [Fig. 1; and see supporting information (SI)
Table 1]. We used ancient DNA technology to retrieve 292 bp of
the mitochondrial control region from seven specimens, and
103–209 bp from an additional three specimens. Although
shorter, the latter sequences were unique compared with all
other sequences, and their position could therefore be inferred
in a minimum spanning network.
population, compared with 10 haplotypes in the contemporary
Siberian population. In Scandinavia, 7 haplotypes were observed
not been observed elsewhere in the world (9), despite extensive
in Scandinavia were shared with Siberia, whereas only 1 was shared
with the Pleistocene European population, and that haplotype was
found also in Siberia (Fig. 1). The 7th haplotype in Scandinavia,
although not shared with Siberia or Pleistocene Europe, is not
unique and exists also in Greenland, Alaska, and Svalbard (9). In
an analysis of molecular variance (AMOVA), the most probable
geographic structure was with Pleistocene Europe in one group
versus Siberia and both Scandinavian samples in the other group
(?CT ? 0.18, P ? 0.001, all other ?CT values were ?0.06 and
Both founder effects during recolonization and demographic
bottlenecks have been theorized to cause a loss of genetic
haplotypes seem to have been lost as a consequence of the
recolonization process, whereas human overexploitation 100
of mtDNA haplotypes during the postglacial recolonization is
intriguing because the arctic fox is known for its capacity for
long-distance dispersal. Such species are expected to display
leptokurtic expansions (i.e., long-distance dispersers setting up
colonies ahead of the main advance), which models have shown
should lead to particularly high losses of genetic variation (2, 21).
We suggest that the reason for the disparity between the models
and our data are that, although leptokurtic dispersal may initially
lead to lower genetic variation, the high gene flow inherent in
this type of species will, over time, reverse this effect and allow
a higher level of variation to be maintained.
The haplotype diversity was higher in Pleistocene Europe than
in Siberia (t33? 7.74, P ? 0.001), which, in turn, had higher
haplotype diversity than both pre- and postbottleneck Scandi-
navia (t50? 7.99, P ? 0.001 and t28? 5.94, P ? 0.01; see SI Table
2). The high haplotype diversity in the Pleistocene European
population indicates a large effective population size. This is in
agreement with the high frequency of arctic fox remains found
mutational step. Haplotypes missing in a particular population are shown with small unfilled circles, whereas overall missing haplotypes are shown with black
dots. Gray areas represent the current distribution of arctic fox habitat, whereas gray circles indicate sample sites for Late Pleistocene samples.
Minimum spanning networks for the sampled populations. The size of each haplotype illustrates its relative frequency. Each branch represents one
Dale ´n et al.
April 17, 2007 ?
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in palaeontological sites across Europe (10–15). As observed in
contemporary populations around the arctic (9), there was no
mtDNA phylogeographical structure within Pleistocene Europe,
suggesting gene flow or a recent shared history within Europe
(SI Fig. 2 and SI Table 1). This is congruent with the lack of
osteometrical differences between the fossil Russian and West
European foxes (11). However, the Pleistocene European foxes
had smaller metatarsal and metacarpal bones, implying a smaller
paw size, compared with contemporary Siberian foxes (11, 12),
which is consistent with the genetic differentiation between
Siberia and Pleistocene Europe found in this study.
The genetic similarity between Scandinavia and Siberia, and
the absence of unique haplotypes in Scandinavia, suggest that
Scandinavia was recolonized from Beringia in northeastern
Siberia after the retreat of the Scandinavian ice sheet (Fig. 1).
Consequently, the arctic foxes in midlatitude Europe became
extinct during the contraction phase and their genes did not
contribute to the makeup of present-day populations. This
suggests that the arctic fox was unable to track the changing
environment as the climate shifted, and there was thus no
support for the hypothesis of habitat tracking (4, 5). Such
inability to track decreases in habitat availability may be due
to, for example, behavioral constraints or that the habitat shifts
faster than the species is capable of dispersing. However, the
arctic fox is a highly mobile species (9), suggesting that lack of
habitat tracking may be a general pattern among species. This
implies that the explanation for evolutionary stasis (22) during
the Quaternary may be found inside, rather than outside,
The results in this study have far-reaching implications for our
understanding of how species respond to climate change, be-
cause they provide empirical evidence for how populations
behave when the distribution of their habitats shift. The habitat-
tracking hypothesis (4, 5) assumes similar processes during both
the expansion and contraction phase. However, as opposed to
when species expand in range to occupy newly available habitats,
a decrease in habitat availability may cause local extinction of
populations. This confirms the significance of climate change as
an agent for extinction (23), and demonstrates the importance of
refugia for the long-term persistence of species. Our results also
highlight an important difference between arctic and temperate
species in the light of current climate change. If lack of habitat
tracking is a general phenomenon during range contraction,
arctic species may be unable to track the shifting habitat as the
temperature increases. This may result in losses of genetic
variation as local populations become extinct.
Materials and Methods
Sampling. Late Pleistocene samples for DNA analysis were col-
lected from Belgium (n ? 17), Germany (n ? 9), and southwestern
Russia (n ? 17; SI Table 1). Approximately 50 mg of bone powder
was sampled by using a Multitool drill. DNA sequences from the
extant populations in Scandinavia (n ? 20) and Siberia (n ? 25) as
well as the prebottleneck population in Scandinavia (n ? 42) were
gathered from published data sets (9, 19).
DNA Analysis. DNA was extracted with a collagenase and
phosphate buffer-extraction protocol (24). After this, the
mtDNA control region was lifted from the extract as described
(24) by using biotinylated probes (primers Pex1R, Pex2F,
Pex2R, and Pex3F) (19). Amplifications were done by using
three overlapping primer pairs (19), each pair amplifying ?150
bp. PCRs were set up in 25-?l volumes containing 0.2 ?M
solutions of each primer, 1? PCR-buffer (Qiagen, Valencia,
CA), 2.5 mM MgCl2(Qiagen), 0.4 mM dNTPs, 3 units Hotstar
Taq (Qiagen), and 9 ?l of DNA extract. The PCR profile was
10-min denaturation at 95°C, followed by 55 cycles of 30-s
denaturation at 94°C, 30-s annealing at 50°C, 30 s of extension
at 72°C, and a final 7-min extension step at 72°C. All PCR
products were cloned by using JM109-competent cells and
pGEM-T Vector System II cloning kit (both from Promega,
Madison, WI), and resulting products were cleaned with
ExoSAP-IT (USB, Cleveland, OH). Sequencing reactions were
performed by using the DYEnamic cycle sequencing kit
(Amersham Biosciences, Piscataway, NJ) and were analyzed
on a MegaBACE 1000 (Amersham Biosciences).
Precautions and Authentication. Standard precautions for ancient
DNA work were taken (25). Extractions were done in an
ancient DNA laboratory (Madrid, Spain), physically isolated
from the post-PCR laboratory, where no work with modern fox
samples had taken place. All equipment and working surfaces
were sterilized by using HCl, sodium hypochlorite, or UV-
light. To monitor contamination, and to avoid a bias caused by
the carrier effect (26), samples from Late Pleistocene horses
(Equus caballus), to which the arctic fox primers should not
anneal, were used as negative controls (one horse sample for
every two arctic fox samples). Ancient DNA can contain
damages that cause misincorporated bases during PCR (27).
To identify such misincorporated bases, all amplicons were
cloned, and an average of 10.5 (SE ? 0.9) clones per sample
were sequenced. A subset of five samples were also sent to the
Archaeological Research Laboratory in Stockholm, Sweden,
for independent replication, by using the methods described
above. None of the negative controls produced arctic fox DNA.
However, one sample gave a DNA sequence identical to red
fox (Vulpes vulpes). This sample was one of the Russian canine
teeth, which are difficult to morphologically identify down to
species level. Contamination from dog (Canis familiaris) was
observed in two samples (once during initial work and once
during replication). During replication, a PCR product con-
tamination was observed for one of the fragments. Results for
this fragment were therefore excluded from the replication.
However, one of the other fragments was successfully repli-
cated for one sample. This sequence, which has not been
observed in any other arctic fox, was identical to the one
Data Analysis. Clone sequences were aligned by using Sequencher
a minimum spanning network were computed in Arlequin ver. 3.01
(28), by using previously described parameters (9). To investigate
geographical structuring of genetic variation, we used an AMOVA
different hierarchical groupings: [Pleistocene Europe vs. Siberia
and both Scandinavian samples], [Siberia vs. Pleistocene Europe
and both Scandinavian samples], [Siberia and prebottleneck Scan-
dinavia vs. Pleistocene Europe and postbottleneck Scandinavia],
and prebottleneck Scandinavia]. The most probable geographic
structure was assumed to be represented by the groupings that
maximized values of ?CT(30). Although cloning of PCR products
allowed us to identify several misincorporated bases, it is possible
that some damage remained because of a few starting templates in
the PCR. Because the extraction method we used lifts out all
template copies from the extract, we could not perform multiple
amplifications from the same extract. As an additional precaution
(31), we therefore treated C/G to A/T nucleotide substitutions (27)
that were observed only in one single sample (compared with both
modern and ancient samples) as unknown characters in the phy-
logenetic analysis. It should be noted that such characters do not
have any phylogenetic signal. This approach did therefore not
change the tree topology but reduced branch length in some cases.
Diversity analyses were done with and without these characters
being treated as unknown.
www.pnas.org?cgi?doi?10.1073?pnas.0701341104 Dale ´n et al.
We thank M. Street, H. H. Wegner, A. von Berg and N. Abramson for Download full-text
assistance with samples, and K. Lide ´n for providing laboratory space.
We also thank I. Barnes, J. R. Stewart, M. T. P. Gilbert, R. Quam,
P. Hersteinsson, and M. Street for comments on the manuscript. The
study was financed by the Swedish Research Council, EU-Life to
SEFALO? and the Ministerio de Ciencia y Tecnologı ´a in Spain.
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