Ancient DNA and the Population Genetics of Cave Bears (Ursus spelaeus) Through Space and Time

Ludovic Orlando*, Dominique Bonjean{dagger}, Herve Bocherens{ddagger}, Aurelie Thenot*, Alain Argant§, Marcel Otte|| and Catherine Hänni*,3

*CNRS UMR 5534, Centre de Genetique Moleculaire et Cellulaire, Universite Claude Bernard Lyon 1, Villeurbanne, France;
{dagger}Archeologie Andennaise, Sclayn, Belgium;
{ddagger}CNRS UMR 5554, Institut des Sciences de l'Evolution, Universite Montpellier 2, Montpellier, France;
§CNRS UMR 6636 ESEP GIRRPA, Institut Dolomieu, Grenoble, France;
||Universite de Liege, Service de Prehistoire, Liege, Belgium


    Abstract
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
The cave bear spread from Western Europe to the Near East during the Riss glaciation (250 KYA) before becoming extinct approximately 12 KYA. During that period, the climatic conditions were highly dynamic, oscillating between glacial and temperate episodes. Such events have constrained the geographic repartition of species, the movements of populations and shaped their genetic diversity. We retrieved and analyzed ancient DNA from 21 samples from five European caves ranging from 40 to 130 KYA. Combined with available data, our data set accounts for a total of 41 sequences of cave bear, coming from 18 European caves. We distinguish four haplogroups at the level of the mitochondrial DNA control region. The large population size of cave bear could account for the maintenance of such polymorphism. Extensive gene flow seems to have connected European populations because two haplogroups cover wide geographic areas. Furthermore, the extensive sampling of the deposits of the Scladina cave located in Belgium allowed us to correlate changes in climatic conditions with the intrapopulational genetic diversity over 90 KY.


    Introduction
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
The retrieval of DNA preserved in fossil remains has now become a valuable tool to understand the phylogenetic relationships that link extinct to extant species (reviewed in Hofreiter et al. 2001bCitation ). Given that, according to environmental conditions and time elapsed since burial, DNA decay results in genomic fragments no longer than a few hundred base pairs (bp), ancient DNA studies have mostly concerned short sequences. One approach to overcome this problem was to amplify numerous overlapping fragments to obtain complete gene or even whole mitochondrial genome sequences (Noro et al. 1998Citation ; Cooper et al. 2001Citation ; Loreille et al. 2001Citation ). But studying short sequences on large numbers of samples has also opened the field of ancient DNA to the level of population genetics (Loreille et al. 2001Citation ; Vila et al. 2001Citation ), providing an elegant means of detecting historical bottlenecks (Weber et al. 2000Citation ), to estimate the genetic diversity between-individual (Krings et al. 2000Citation ; Ovchinnikov et al. 2000Citation ), to follow Pleistocene migration routes (Leonard, Wayne, and Cooper 2000Citation ; Barnes et al. 2002Citation ), or conversely to show evidence of long-term genetic isolation of a given population (Hadly et al. 1998Citation ).

The cave bear (Ursus spelaeus) is the macro fauna species that gave rise to the largest amount of fossils at the Pleistocene period in caves. Cave bears have inhabited Europe and the Near East since the Riss glacial period (250,000 years ago, 250 KYA; Mazza and Rustioni 1994Citation ). Despite clear synapomorphies, such as reduction of forelimb length, a domed forehead and teeth adapted toward vegetarianism, the great level of morphological polymorphism of the cave bears has for a long time blurred their phylogenetic position among the Ursidae family (Kurten 1976Citation ; Stuart 1991Citation ). Molecular phylogenetic analysis of ancient mtDNA control region (CR) fragments recently clarified the situation and showed that brown and cave bears separated from a common ancestor 1.2–1.6 MYA (Hänni et al. 1994Citation ; Loreille et al. 2001Citation ).

Numerous local forms of cave bear have been described, which suggested to paleontologists that limited migration movements occurred between populations across Europe; but to date it has never been possible to directly evaluate the relationships that connected them (Kurten 1968Citation ; Pacher 2000Citation ). The DNA analysis of contemporary cave bear samples excavated from different sites in Europe appears to be the only tool able to shed light on the relationships that have linked cave bear populations. Here, we report 21 new mtDNA CR sequences of cave bear samples excavated from five caves located from France to Slovakia, and compare them to already published ones in order to map the genetic relationships between European cave bear populations.

The cave bear became extinct at the end of the late glacial period (12 KYA, Guérin and Patou-Mathis 1996Citation , p. 172). Climatic changes might have shaped the genetic diversity profiles of cave bears to eventually lead to their extinction. To check if the climatic changes had an effect on the genetic diversity of cave bears, we focused on an extensive analysis of partial mtDNA CR sequences of 20 samples excavated from different layers of one cave, the Scladina cave. This Belgian cave preserved a stratigraphic record of fossils from 130 to 40 KY before present (BP), a period during which the European climate changed drastically. In Scladina cave, oscillations between glacial and more temperate episodes have been demonstrated by microfaunal, palinological, and isotopic data. Thus, the Scladina cave provides an interesting opportunity to correlate the extent of genetic diversity sampled in each layer to the climatic variations through time.


    Material and Methods
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
Extracted Samples
We extracted 29 cave bear samples that consisted of either phalanx or tooth root remains. Eight of them never yielded any DNA, despite numerous PCR attempts (table 1 Go ). The 21 samples that gave positive results come from five European caves (table 1 ): Scladina in Belgium (Namur, 17 samples denoted SC), Gigny, Azé, and Prélétang in France (Burgundy, Saone et Loire, and Isère, respectively, three samples), and Mokrav in Slovakia (Moravia, one sample). The sample from Azé, whose age has been estimated to be older than 80 KY, deserves special attention. Contrary to all the other samples recognized as typical U. spelaeus, it was defined as a specimen of U. spelaeus deningeroides because it presents a third premolar and weak frontal bumps like U. deningeri, but second premolar characteristics of U. spelaeus (Argant 1989Citation ).


View this table:
[in this window]
[in a new window]
 
Table 1 List of the Cave Bear Sequences Data Set

 

View this table:
[in this window]
[in a new window]
 
Table 1 Continued

 
DNA Extraction
Extraction procedures have been conducted in a laboratory devoted to ancient DNA analyses with facilities for analyzing ancient DNA (Hänni et al. 1994Citation ; Loreille et al. 2001Citation ). After an overnight decalcification and protein digestion at 55°C with agitation (EDTA 0.5 M, pH = 8.5, proteinase K 1–2 mg/ml, N-lauryl Sarcosyl 0.5%), the pellets were eliminated by 10 min of centrifugation (1,200 rpm). The supernatants (5–10 ml) were further extracted three times using phenol-chloroform-isoamylic alcohol (25:24:1). Subsequently, the aqueous phase was concentrated in 80–100 µl of sterile distilled water with Centricon-30 columns (Amicon®) according to the instructions of the manufacturer. The Gigny 18.9.F3 and JAL108 extracts yielded great amounts of PCR inhibitors so they were further purified with isopropanol according to the protocol developed by Hänni et al. (1995)Citation . An extraction blank and different species samples (Ursus arctos, Coelodonta antiquitatis, Cervus elaphus... unpublished data) were coextracted with the cave bear samples during each extraction session to trace potential cross-contamination between samples. No more than three ancient samples were coextracted at the same time.

DNA Amplification
The pre-PCR mix was prepared in a DNA-free room in the ancient DNA laboratory. Each PCR reaction was carried out in a total volume of 100 µl. PCR conditions were as follows: 10 units of Taq Gold polymerase (Perkin-Elmer®), 2 mM MgCl2, 1 mg/ml BSA, 250 µM of each dNTP, and 300 ng of each primer. One to five microliters of ancient DNA extract was added. Fifty cycles of amplification were conducted (92°C, 1 min—50 to 54°C, 45 s—72°C, 45 s) on a Master Gradient apparatus (Ependorf®). Primers designed to amplify only short overlapping fragments in mtDNA CR (92–206 bp, fig. 1 ) were described elsewhere (Loreille et al. 2001Citation ). Three independent blanks were carried out for each set of PCR experiment as reported in Loreille et al. 2001Citation .



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.—Position of the amplified fragments on the mtDNA CR. Primer names and fragment lengths are indicated. The sequences of each primer are described in Loreille et al. (2001)Citation

 
Cloning and Sequencing
The products of ancient DNA amplification generally contain a large number of mutations generated by Taq polymerase errors during the elongation of a degraded and chemically modified template (Handt et al. 1996Citation ). Therefore, PCR products were subcloned into bacterial vectors using the Topo TA cloning kit (Invitrogen®) according to the manufacturer's instructions. Plasmids were purified using the QIAprep spin miniprep kit (QIAGEN®), and the sequences of both-strands were obtained on a Megabace1000 automatic capillary sequencer (Amersham®). The authentic sequence was deduced from the "consensus" between several amplification products of several clones (table 1 ).

Sequence Analyses
Our 21 sequences were compared with the published sequences. In earlier studies (Hänni et al. 1994Citation ; Loreille et al. 2001Citation ), we published 10 cave bear sequences from five caves located in Belgium (Scladina, Namur), Spain (Cova-Linarès, Galicia), and France (Mialet, Gard; La Balme à Collomb and Prélétang, Isère). For three of the 10 latter samples (SC11700, SC15700, and TAB2), the length of the mtDNA CR analyzed has been extended here (table 1 ). Other authors published 13 mtDNA CR sequences: one of a 35-KY-old sample from Chiemsee (Austria; Kühn, Schröder, and Rottman 2001Citation ) and 12 sequences of 26.5 to over 50-KY-old samples from nine European caves (Hofreiter et al. 2001aCitation ). In this study, the sampling is completed at the geographic scale with 18 mtDNA CR sequences coming from four European caves (Scladina in Belgium; Azé and Gigny in France; Mokrav in Slovakia). Thus, our data set includes 41 sequences of cave bear coming from 18 European caves (table 1 ). Sequences were aligned manually using the Seaview software (Galtier, Gouy, and Gautier 1996Citation ). All the phylogenies were computed with the Phylo_win program (Galtier, Gouy, and Gautier 1996Citation ). To ensure that the results were not depending on the methods used, distance and parsimony algorithms were tested. One thousand bootstrap replicates were performed. Jukes & Cantor, Kimura 2, or Tajima & Nei corrections were applied to pairwise distance matrices and yielded the same topologies. Molecular dating was based on the brown bear standard of 7.03% substitutions per 850 KY (Taberlet and Bouvet 1994Citation ). The date of the cave bear radiation was estimated by the time of the last common ancestor of all sequences related to the cave bear monophyletic group adding 12 KY, which is the date this species became extinct according to paleontological record.


    Results
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
Amplification Success
From the 29 extracted samples, eight (27%) did not yield any DNA though extensive amplifications were attempted (table 1 ). Five fragments whose length varies from 92 to 206 bp were targeted by PCR (fig. 1 ). The majority of the samples yielded amplification products when the shortest fragments were targeted (5/7 = 71% and 18/29 = 62%, respectively, for the 92- and 127-bp fragments, table 1 ). In contrast, only 10/27 (37%) and 9/26 (31%) yielded the two largest fragments (193- and 206-bp, respectively), suggesting that the DNA template was very fragmented, as expected with authentic ancient DNA. No cross-contamination appears to have occurred during our experiments because (1) all PCR showed all blanks as negative (three per experiment), (2) 27% of our samples did not yield any DNA, (3) large fragments amplified on some samples were never retrieved on other samples, and (4) amplification of cave bear DNA never succeeded on samples of other species coextracted (data not shown).

DNA Modifications in Ancient Extracts
Modifications such as hydrolysis and oxidation that affect the DNA of an organism after its death may lead Taq polymerase to incorporate incorrect nucleotides during the elongation step (Hofreiter et al. 2001aCitation ). Such damage-induced errors may blur the genetic message preserved in fossils by adding artifactual polymorphism (Cooper and Poinar 2000Citation ). Repeated amplification of the same fragment and the sequencing of several clones from each amplification product is the only known strategy that allows such errors to be detected. In this study, some PCR fragments have been independently amplified, and all the PCR products have been cloned (table 1 ). The extensive overlap of the fragments amplified with primers H3–H1 and DF311–DR500, DF311–DR500, and H16143–H16299, respectively, enabled us to identify the base present at specific polymorphic sites several times. The authentic sequence of each sample has been deduced from the consensus between the different clones of all its PCR products. In summary, 167 clones from 59 independent PCR products were analyzed.

When comparing the different clones of the same extract with the consensus sequence, two major types of substitutions appear. In the first case, all the clones of a given PCR product carry a substitution never observed in the clones of the other amplifications of the same fragment. Such a substitution may be due to a damage-induced error occurring at the very first cycle of the amplification. This type of substitution occurs in 29 cases among which transitions, and more specially GC->AT substitutions, are more frequently recovered. In the second case, some substitutions appear in some but not all the clones of the same PCR product, which may be due to damage-induced errors introduced during the latter cycles of the amplification. This type accounts for the majority of the substitutions observed among clones (188/217, 87%; see table 2 ). Once again, transitions are dominant (145/188, 77%), GC->AT transitions being the most frequent (78/145, 54%, table 2 ). AT->GC transitions are also retrieved with high frequency (67/188, 35%) as noted in other studies (Hofreiter et al. 2001aCitation ). Overall, the observed pattern of damage-induced errors is consistent with the pattern generally described in ancient sequences, which argues in favor of the authenticity of the sequences retrieved.


View this table:
[in this window]
[in a new window]
 
Table 2 Pattern of the PCR Damage-induced Errors

 
Sequence Characteristics
The mean base composition of the brown bear (U. arctos), the closest extant species from the cave bear, is 24.2% A, 25.0% C, 17.1% G, and 33.7% T for the part of the mtDNA CR under study (calculated from the sequences of Taberlet and Bouvet 1994Citation ). The mean base composition of our samples is quite similar (26.3% A, 20.4% C, 13.4% G, and 39.9% T) and even more closely related to the mean base composition of the cave bear sequences already published (26.1% A, 23.0% C, 14.4% G, and 36.5% T, calculated from the cave bear sequences reported in Hänni et al. 1994Citation ; Hofreiter et al. 2001aCitation ; Kühn, Schröder, and Rottman 2001Citation ; Loreille et al. 2001Citation ). Most of the substitutions among cave bear sequences are transitions, which is consistent with strong transitional bias of mitochondrial DNA. Furthermore, substitutions occur most often in sites either polymorphic between the sequences of cave bear already published or known to be polymorphic in brown and black bears. Finally, gaps are only observed in a central T-rich region that we have already shown to be partly deleted in brown bears (T5–6C2T3AT6–11, fig. 2 and Loreille et al. 2001Citation ).



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 2.—Alignment of the 21 new cave bear sequences. A brown bear sequence is used as a reference (denoted PYR, Accession number: X75878). A part of the tRNAPro gene is presented between positions 1 and 32. The mtDNA CR starts at the 33rd site. For comparison, at the top of the alignment, three sequences reported by three independent laboratories have been aligned (denoted #, Loreille et al. 2001Citation ; Kühn, Schröder, and Rottman 2001Citation ; Hofreiter et al. 2001aCitation ). Dots indicate identity to the reference sequence. The T-rich region (T5–6C2T3AT6–11) is indicated. After their divergence with cave bears, brown bears have lost part of this region (denoted {Delta}, Loreille et al. 2001Citation ). The polymorphic sites used to define haplogroups are given in bold and indicated by the $ symbol. The transition observed at site 27 in the tRNAPro region is indicated by §. The position of each primer, their name, and orientation are reported. The different combination of sites taken into account in the phylogenetic analyses are shown between asterisks (213, 179, 145, 127, and 92 sites)

 
Cave Bear Phylogeny
Distance and parsimony methods were used for the data set of cave bear sequences completed with the sequences of the American black bear and the brown bear reported in Taberlet and Bouvet (1994)Citation . According to the PCR fragments retrieved, not all samples have the same sequence length (table 1 ); groups of 23, 26, 26, and 24 samples share 213, 179, 145, and 127 bp, respectively (fig. 2 ). Altogether, all the sequences of our data set share 79 sites, between positions 188 and 266 of the alignment (fig. 2 ). Therefore, phylogenetic reconstructions were conducted with either 213, 179, 145, 127, or 79 sites to avoid loss of information and to construct trees with sites shared by all sequences. In all cases, all U. spelaeus sequences cluster together in a well-supported monophyletic group (bootstrap values 68%–99%, fig. 3 and table 3 ). Likewise, brown bear monophyly receives high bootstrap support (table 3 ). Because branches leading on the one hand to the sequence of the Eastern lineage of brown bears and on the other hand to the Geissenklösterle, Nixloch, Potocka, Vindija, and SC15700 sequences, are longer than any other branches, the topology built may have been biased by mutation rate acceleration. Nevertheless, excluding these sequences neither affects the tree topology nor the bootstrap supports of the main branches, suggesting that a long-branch artifact does not account for the resulting trees. Assuming a molecular clock rate of 7.03% difference each 850 KY for the mtDNA CR (Taberlet and Bouvet 1994Citation ), cave bears expanded 400–680 KY BP depending on the fragments analyzed. This estimate is in good agreement with paleontological data suggesting that cave bear radiation occurred during the Mindel Glaciations (350–650 KY BP, Kurten 1968Citation ; Guérin and Patou-Mathis 1996Citation ).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3.—Molecular phylogeny of cave bears based on 179 mtDNA CR sites. The sites used in the analysis correspond to the region between primers H3–H1 and H16143–H2, respectively. The Kimura 2 parameters correction for substitution bias was applied to the pairwise distance matrix. Thousand bootstrap replicates were performed; only bootstrap values greater than 60% are reported. Bootstrap supports to cave and brown bear monophyly, and cave bear haplogroups, are reported. The sequences reported in this study are reported with gray characters. Letters stand for the four U. spelaeus haplogroups. The accession numbers of the cave bear sequences are reported in table 1 ; for the black and brown bear sequences: U. americanus (X75863), DAL (X75868), NOR (X75871), CAN (X75865), GRE (X75870), BUL (X75864), ABR (X75862), PYR (X75878), TIB1 (AB010727), JAP1 (AB010727), JAP2 (AB010726), RUS (X75875), and RO1 (X75872)

 

View this table:
[in this window]
[in a new window]
 
Table 3 Bootstrap Values Supporting the Main Bear Clades According to the Phylogenetic Method Used

 
Definition of Cave Bear Mitochondrial Haplogroups
Comparison of alignments of the mtDNA CR sequences of cave bears shows they can be clustered in four groups of samples that share the same pattern of substitutions at specific sites (fig. 4 ). The sequences of U. spelaeus deningeroides (Azé), which shows morphological characters of both U. deningeri and U. spelaeus (Argant 1989Citation ), cluster in haplogroup A with the sequences of SC11600, SC12500, SC13800, SC85F16 (Belgium), TAB2, Grotte Merveilleuse (France), and Conturines (Italy). Although the Conturines and the Grotte Merveilleuse samples disrupt the monophyly of the haplogroup A (fig. 4B ), they were clustered into this haplogroup because (1) only one C -> T substitution at positions 218 and 226, respectively, relate them to haplogroup A when (2) between 4–8 substitutions are needed to relate them to the other haplogroups (fig. 4A ). From the 14 sites that define haplogroup A, only two differ from brown bears, which suggests haplogroup A as the most ancestral haplogroup. Most of the samples (22/41) belong to haplogroup B (fig. 4 ). Inside this haplogroup, the samples Gailenreuth and TAB15 show a T -> C substitution at position 218. With two substitutions, A -> G and T -> C at positions 168 and 237, the sample Hohlefels appear as the most divergent inside the haplogroup B. Three samples cluster inside haplogroup C (Chiemsee, Ramesch1, and Ramesch2). Haplogroup D includes the Belgian sample SC15700, the two Croatian (Vindija1, Vindija2), two German (Geissenkloestere1, Geissenkloestere2), one Slovenian (Potocka), and one Austrian sample (Nixloch). Haplogroup D members are separated by five substitutions from the brown bears (positions 50, 53, 204, 226, and 237); between 7–11 substitutions distinguish them from the other cave bear haplogroups (fig. 4A ), suggesting it represents the most divergent cave bear lineage sequenced to date. Sample SC15700, which shows T 205, T 226, and C 237, respectively, never observed in other haplogroups, can be attributed without doubt to the haplogroup D, although being distant by two substitutions from the true combination of sites of this haplogroup (fig. 4A , C -> T and G -> A at positions 204 and 227).



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 4.—The 4 mtDNA control region haplogroups of cave bears. A, Definition of haplogroups A, B, C, and D. For each cave bear sequence, the base at specific polymorphic sites is reported. The position of the site is given in accordance to the alignment presented in figure 2 . Dots indicate identity to the closest ursine extant species (U. arctos). The sites used for definition of each haplogroup are given in bold. B, Unrooted phylogenetic tree of the 41 cave bear samples. The distance reported between each couple of samples corresponds to the percentage of observed divergence on the 79 common sites between position 188 and 266 in the alignment presented in figure 2

 
Interestingly, two of the haplogroups defined above are supported by significant phylogenetic bootstrap values (fig. 3 ). Even when taking into account only the 79 sites in common for all the sequences of our data set, haplogroup C is continuously supported by bootstrap values of 87%–93% according to the phylogenetic method used. Likewise, haplogroup D is supported by bootstrap values of 77%–80%.

Geographic Genetic Diversity
Cave bears show less genetic diversity than brown bears at the level of the mtDNA CR locus (4.4% for cave bears, < 7.5% for brown bears, Taberlet and Bouvet 1994Citation ). But four distinct haplogroups can be recognized. The geographic distribution of haplogroup A extends in the Western European region, in France (Azé, Grotte Merveilleuse, Prélétang), Italy (Conturines), and Belgium (Scladina). Members of haplogroup B spread over a very large geographic area because it includes samples from Spain (Cova-Linarès), France (Balme à Collomb, Gigny, Mialet), Germany (Gailenreuth, Hohlefels), Belgium (Scladina), and Slovakia (Mokrav). In Scladina cave, members of haplogroup A and B coexist in layers 1A and four (40–130 KYA). In France, contemporary caves located in close proximity also possess members of those two haplogroups (40 KYA in Prélétang and 25–35 KYA in La Balme à Collomb). Thus, in the France and Belgium area at least, haplogroup A and B coexisted in sympatry (fig. 5 ). Given the small number of samples (three in Austria) defining the haplogroup C, its geographic repartition cannot be estimated precisely. The last haplogroup, haplogroup D, was widespread in Eastern and Northern Europe (Croatia, Slovenia, Germany, and Belgium). In Scladina, members of haplogroups B and D are found in layer five (90–130 KY). Both haplogroups B and D spread in Germany (Gailenreuth, Hohlefels; Geissenklösterle1, Geissenklösterle2). Thus, those two haplogroups may have coexisted in a sympatric context in Belgium and Germany (fig. 5 ).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 5.—Geographic distribution of the four cave bear haplogroups. A dashed line reports the location of the Alps

 
Climate and Genetic Diversity
The Scladina cave has preserved a sedimental and paleontological record from 150 to 30 KY BP, thus offering a unique opportunity to trace back how the genetic diversity evolved inside a local population during the last Quaternary climatic oscillations. Grouping the samples relative to the layer from which they have been excavated would give insight into the intrapopulational genetic diversity at the time of each deposit, using pairwise-observed divergence. A large number of bones of cave bears were sampled in four layers of the Scladina cave (1A, 1B, 4A, and 5), ranging from 40–45 KY to 90–130 KY. mtDNA CR sequences were obtained in 20/25 samples (80%). SC5300 is the unique sample of the layer 1B that yielded a sequence. Because the genetic variability of a layer cannot be estimated with one sample, it was excluded from the analysis. The 19 remaining samples consist of 1, 5, and 13 members of haplogroups D, A, and B, respectively. If the sequences of the three haplogroups are considered altogether, the genetic variability of a layer would principally reflect the presence or the absence of one haplogroup inside. We considered the genetic variability of a layer as better described by the observed divergence inside a haplogroup. With 13 samples distributed in layers 1A, 4, and 5, haplogroup B was selected to obtain statistically reliable estimates. During the cold episodes of the layers 1A and 5, the intrapopulational genetic diversity is estimated to be 1.1 ± 1.0% and 0.6 ± 0.6%, respectively (fig. 6 , right and left). The diversity reaches 3.3 ± 1.2% during the time of the warmer deposit of layer 4A (fig. 6 , center). Thus, in the layer 4A, i.e., during more temperate conditions than in the layer 5 deposit, the intrapopulational genetic diversity increases, whereas it decreases during the cold periods of the layer 1A deposits (Wilcoxon rank test, P value < 1.3%).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.—mtDNA control region diversity through Scladina layers. Only sequences of the haplogroup B have been taken into account. Individual numbers refer to the number of samples used to estimate mean and standard deviation on the 79 sites in common with mtDNA CR sequences. Relative temperature gives an indication of local climatic conditions deduced from microfauna correlations, isotopic, and palinological data. Circles and bars indicate the mean and the standard deviation values for each layer, respectively; crosses indicate the exact values of the pairwise-observed divergence.

 

    Discussion
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
Authenticity of the Sequences
We present data suggesting that during the extraction and the amplification steps, no contamination occurred between our samples. The DNA extracted from the bones appears highly fragmented because the efficiency of PCR increases when the length of the fragments targeted decreases. The pattern of substitutions observed among clones suggests deoxy-cytosine deamination as the major base modifications, a lesion generally described in fossil material (Hoss et al. 1996Citation ; Hofreiter et al. 2001aCitation ). Both base composition and the pattern of substitutions observed among our sequences are consistent with what is observed in the mtDNA CR of extant Ursids. All our sequences form a monophyletic group, both with the 10 ancient cave bear sequences we had already published (Loreille et al. 2001Citation ) and the 13 reported by two other laboratories (Kühn, Schröder, and Rottman 2001Citation ; Hofreiter et al. 2001aCitation ). Taken together, these arguments plead in favor that the sequences we describe are authentic ancient sequences.

Origin and Maintenance of Mitochondrial Haplogroups
In this study, we obtained 18 new CR sequences from four caves located from France to Slovakia; moreover, the length of the sequences published for three samples has been increased. Added to 20 other sequences retrieved West to East from France to Slovenia, and North to South from Germany to Croatia (Hänni et al. 1994Citation ; Hofreiter et al. 2001aCitation ; Kühn et al. 2001Citation ; Loreille et al. 2001Citation ), our data set allows us to faithfully evaluate the genetic diversity of cave bear populations. Both phylogenetic clustering and the sharing of specific substitutions allowed us to use 12–14 polymorphic sites to designate four mitochondrial haplogroups. The haplogroup A exhibits only two transitions when compared with the sequence of the closest extant species, U. arctos (fig. 4 ). It could represent a very ancient haplogroup. Interestingly, one sample of this haplogroup (Azé) was described as U. spelaeus deningeroides because it conserved some archaic characters of the ancestor of U. spelaeus (U. deningeri), such as the presence of the third premolar and a weak development of frontal bumps (Argant 1989Citation ). From all the sequences analyzed the haplotype Grotte Merveilleuse, related to haplogroup A, is closer to the haplogroup D (four transitions) than to the remaining haplogroups or U. arctos sequences (fig. 4 ). Haplogroup D could thus have emerged from the haplogroup A. Haplogroups B and C are equally distant from brown bear sequences and other cave bear haplogroups.

In Scladina cave, each layer yielded members of haplogroup A, which suggests the maintenance of this haplogroup between 40 to 130 KY BP. Similarly, haplogroup B members are recovered from layers 1A and 4, two deposits separated by 40 KY (fig. 4 ). In France, haplogroup A is tracked from 35 KYA in the Prélétang cave up to over 80 KYA in the Azé cave. Large effective sizes of cave bear populations (Ne), as suggested by the numerous remains excavated through Europe, has probably contributed to maintain these polymorphisms for such long periods of time because under neutralism the mean time of an allelic fixation equals 4Ne (Kimura 1989Citation ). Furthermore, because (1) the pairwise mismatch distribution between all the cave bear sequences is trimodal and (2) the null substitution class is not overrepresented, the effective size of populations would have been rather large and stable (fig. 7 , Excoffier and Schneider 1999Citation ).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.—Pairwise observed substitutions between cave bear samples. The frequency of the number of substitutions between each couple of cave bear sequences or between couples inside each haplogroup has been deduced from pairwise substitutions among 79 common sites. Because haplogroup C consists of only three samples, the distribution of pairwise substitutions has not been reported. The number of sequences taken into account are indicated in parentheses

 
High Gene Flow Between Populations
Because of the existence of numerous local forms and subspecies, such as U. spelaeus crimaeus, paleontologists have always considered cave bear populations as poorly connected by migration (Kurten 1968Citation ; Guerin and Patou-Mathis 1996Citation ). At the genetic level, the situation appears to be completely the opposite. Each haplogroup (except haplogroup C represented to date by only three samples) spread through a wide geographic area. Members of the haplogroup B were retrieved throughout Western and Northern Europe, in Spain, France, Belgium, Germany, and Slovakia. Similarly, the geographic distribution of the haplogroup D stretches on the Eastern and Northern side of Europe, in Croatia, Slovenia, Germany, and Belgium. Haplogroups B and D overlap partially in France and Belgium at the same period. Thus, although separated by hundreds or even thousands of kilometers, some caves share the same haplogroups; in Scladina cave for example, we found members of both the haplogroup B also present in Spain (Cova-Linarès) and in Slovakia (Mokrav), and members of the haplogroup C, which extends to Croatia. Such data are a clear indication of extensive gene flow between cave bear populations.

Interestingly, according to phylogeographic studies of many European species, the Alps often appear as a barrier to migration because they were covered with ice during glacial periods (Taberlet and Bouvet 1994Citation ; Hewitt 2000Citation ). With a living area that overlaps the territory of the Alpine glacier, cave bears thus appear as suitable models to evaluate if the alpine topographic barrier has opposed gene flow (Stuart 1991Citation ). The sample Conturines excavated on the Italian side of the Alps shows strong proximity with other samples from haplogroup A from France (0.4% from TAB2 and 0.7% from Grotte Merveilleuse). Therefore, the geographic distribution of the haplogroup A appears to overlap the Alpine boundary. But because this assumption is based on only one sample, a more exhaustive sampling of Southern Italian caves would be necessary to check in more detail whether the Alps have influenced cave bear migration.

Climate Incidence on Genetic Diversity
At the level of the mtDNA CR (haplogroup B), we found that the genetic diversity in Scladina reached 3.3% at the time of the 4A deposit (80 KYA). On the contrary, it remained much weaker during the 5 (0.6%, 90–130 KYA) and 1A episodes (1.1%, 40–45 KYA, fig. 6 ). Palinological data, datation and magnetic susceptibility studies, and the presence or absence of animals associated with glacial conditions, all suggest that (1) cold conditions affected the layer 5, (2) temperate conditions prevailed at the time of the deposition of the layer 4A (either the 5c or the 5a oxygen isotopic stage), and (3) harder glacial conditions dominated during the deposition of layer 1A (for details, see Appendix A).

Bears from the layer 4A revealed a significant maximum of mean genetic diversity (fig. 4 ). On the contrary, the genetic variability sampled in the other layers, 1A and 5, remained much smaller. Thus, intrapopulational genetic diversity decrease clearly correlates with glaciation intensity: the colder the climate, the smaller the genetic diversity. Regional mixing during warmer periods and geographic isolation imposed during glacial intervals might have patterned the genetic diversity observed. According to this model, the return of colder conditions would have pushed back the invading populations which came into contact at the time of the relatively warm layer 4A. Under this assumption, the layer 4 would carry the traces of these divergent newcomers with samples showing divergent haplotypes inside the haplogroup B, which are not observed in the other layers. Interestingly, inside the layer 4A, the sequence of the SC11800 sample, related to the haplogroup B, exhibits two transitions never recovered in the 19 other samples analyzed from the cave (T->C and A->G at positions 197 and 220, fig. 1 ). Alternatively, local genetic drift might have shaped the genetic variations. Consistent with that model, Lopez Gonzalez and Grandal d'Anglade (2000)Citation reported that the mortality in the Eiros (Spain) cave during a cold phase (24 KYA) has been biased toward juveniles, although older individuals were present in the neighboring Cova-Linarès cave, which experienced a relative warmer phase (35 KYA). But the strong correlation found between the intrapopulational genetic variability and the climatic conditions should be interpreted as a preliminary approach. Layers 4A and 5 need further sampling because only 3 and 4 sequences account for the global diversity estimations of these layers. Additionally, low levels of genetic diversity found in the layers 1A and 5 could simply be explained by the sampling of maternally linked relatives. Such bias could also explain why inside haplogroup A the four samples excavated from the layer 4A exhibit the same sequences.

Bearing in mind that cave bears disappeared at the end of the last glacial maximum (12 KYA), it is interesting to note that when the cave bear started to become extinct, the climatic conditions were cold and a significant loss of genetic diversity is observed. Ancient DNA appears a valuable tool to provide a temporal dimension to the study of genetic variation. Hadly et al. (1998)Citation reported the genetic variation of an isolated pocket gopher population (Thomomys talpoides) during the past 2.4 KY. Here, it has been possible to genetically track one cave bear population over 90 KY. In future, it will be important to develop such studies to be able to evaluate how species might adapt to climatic changes.


    Appendix A
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
Until recently, in either microfaunal, macrofaunal, or palinological analyses, layer C5 in Scladina cave was thought to be related to the temperately cold isotopic interstadium 5c. The layer 5 yielded signs of a Neanderthal occupation and thermo luminescence conducted on a burnt-Mousterian silex from the layer 5 delivered an age 20 KY younger than the isotopic interstadium 5c (130 KYA, Bonjean 1998Citation ). Magnetic susceptibility studies have solved this discrepancy, linking the layer 5 to the cold crisis of the previous isotopic interstadium (5d).

Though arboreal pollen is absent at the time of the 5 deposition, hazel pollen dominates in the layer 4A. Palinological correlation suggests that the layer 4A corresponds to the temperate oxygen isotopic stage 5a (Bastin 1992Citation , the interglacial episode Saint-Germain II, around 80 KYA). On the other hand, autochthonous microfauna retrieval, macrofauna predominance of forested species with confirmation of a closed-forest habitat by carbon stable isotopes (Bocherens, Billiou, and Mariotti 1999Citation ), magnetic susceptibility and the thermoluminescence dating of a top-layer calcite to 100 KY BP (Huxtable and Aitken 1992Citation ), all these data relate the layer 4A to another temperate episode, the oxygen isotopic stage 5c.

In the Scladina cave, the middle Paleolithic layer 1A is the second layer which has a relatively rich Mousterian industry; consistent radiocarbon dates of around 40 KY BP were obtained from associated animal fossils and were further confirmed by thermoluminescence analysis on stalagmitic calcite (Debenham 1998Citation ). Layer 1A clearly shows an unambiguous decrease of arboreal pollens and a prevalence of snow sp. voles and collared sp. lemmings in microfauna fossils, indicating that at the time of the deposit pleniglacial conditions prevailed (Cordy 1998Citation ). Such a palaeoenvironmental interpretation is confirmed by the analysis of carbon and nitrogen isotopic compositions of bone collagen of large mammals (Bocherens et al. 1997Citation ).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 
We thank M. Patou-Mathis for the generous gift of Scladina samples, and J. A. Leonard for the generous gift of JAL104, JAL108, and JAL113 samples (F: AM11100, F: AM96635, F: AM21949) from the American Museum of Natural History, L. Granger and C. Lerondel for helpful technical assistance in sequencing, F. Sauvage, P. Taberlet, and L. Excoffier for fruitful discussions, and F. Depaulis, M. Schubert, S. Hugues, E. Derrington, M. Robinson-Réchavi, and V. Laudet for critical reading of the manuscript. We particularly acknowledge both reviewers for their constructive comments. Finally, we thank V. Laudet for availability of the PCR and DNA extraction facilities in ENS de Lyon. This work has been supported by CNRS (APN), MENRT (ACI) and Université Claude Bernard (BQR). L.O. receives a fellowship from the ENS de Lyon.


    Footnotes
 
Manolo Gouy, Reviewing Editor

Abbreviations: CR, mtDNA control region; BP, before present; KYA, thousand years ago; KY, thousand years. Back

Keywords: Ursus spelaeus ancient DNA population genetics climatic change Back

Address for correspondence and reprints: Catherine Hänni, CNRS UMR 5534, Centre de Genetique Moleculaire et Cellulaire, Universite Claude Bernard Lyon 1, 16 Rue Raphael Dubois, 69622 Villeurbanne Cedex, France. hanni{at}univ-lyon1.fr Back


    References
 TOP
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 Appendix A
 Acknowledgements
 References
 

    Argant A., 1989 Carnivores quaternaires de Bourgogne Diplôme de doctorat presente devant l'Universite Claude Bernard, Lyon 1

    Barnes I., P. Matheus, B. Shapiro, D. Jensen, A. Cooper, 2002 Dynamics of Pleistocene population extinctions in Beringian brown bears Science 295:2267-2270[Abstract/Free Full Text]

    Bastin B., 1992 Analyses pollinique des sediments detritiques, des coprolites et des concretions stalagmitiques du site prehistorique de la grotte Scladina (Province de Namur, Belgique) In M. Otte, M. Patou-Mathis, and D. Bonjean, eds. La grotte Scladina, Vol. 1. Le contexte

    Bocherens H., D. Billiou, A. Mariotti, 1999 Palaeoenvironmental and palaeodietary implications of isotopic biogeochemistry of last interglacial Neanderthal and mammal bones in Scladina cave (Belgium) J. Arc. Sci 26:599-607[ISI]

    Bocherens H., D. Billiou, M. Patou-Mathis, D. Bonjean, M. Otte, A. Mariotti, 1997 Isotopic biogeochemistry (13C, 15N) of fossil mammal collagen from Scladina cave (Sclayn, Belgium) Quat. Res 48:370-380[ISI]

    Bonjean D., 1998 Chronologie a la grotte de Sclayn Pp. 45–57 in M. Otte, M. Patou-Mathis, and D. Bonjean, eds. La grotte Scladina, Vol. 2. L'archeologie. Belgium: Eraul 79.

    Cooper A., C. Lalueza-Fox, S. Anderson, A. Rambaut, J. Austin, R. Ward, 2001 Complete mitochondrial genome sequences of two extinct moas clarify ratite evolution Nature 409:704-707[ISI][Medline]

    Cooper A., H. J. H. J. Poinar, 2000 Ancient DNA: do it right or not at all Science 289:1139.[ISI][Medline]

    Cordy J. M., 1998 Bio- et chronostratigraphie des depôts quaternaires de la grotte Scladina (province de Namur, Belgique) a partir des micromammifères Pp. 79–125 in M. Otte, M. Patou-Mathis, and D. Bonjean, eds. La grotte Scladina, Vol. 1. Le contexte. Belgium: Eraul 27.

    Debenham N. C., 1998 Thermoluminescence dating of stalagmitic calcite from La grotte Scladina at Sclayn (Namur) Pp. 39–43 in M. Otte, M. Patou-Mathis, and D. Bonjean, eds. La grotte Scladina, Vol. 2. L'archeologie. Belgium: Eraul 79.

    Excoffier L., S. Schneider, 1999 Why hunter-gatherer populations do not show signs of Pleistocene demographic expansions Proc. Natl. Acad. Sci. USA 96:10597-10602[Abstract/Free Full Text]

    Galtier N., M. Gouy, C. Gautier, 1996 SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny Comput. Appl. Biosci 12:543-548[Abstract]

    Guerin C., M. Patou-Mathis, 1996 Les grands Mammifères plio-pleistocènes d'europe Masson, Paris.

    Hadly E. A., M. H. Kohn, J. A. Leonard, R. K. Wayne, 1998 A genetic record of population isolation in pocket gophers during Holocene climatic change Proc. Natl. Acad. Sci. USA 95:6893-6896[Abstract/Free Full Text]

    Handt O., M. Krings, R. H. Ward, S. Pääbo, 1996 The retrieval of ancient human DNA sequences Am. J. Hum. Genet 2:368-376

    Hänni C., T. Brousseau, V. Laudet, D. Stehelin, 1995 Isopropanol precipitation removes PCR inhibitors from ancient bone extracts Nucleic Acids Res 23:881.[ISI][Medline]

    Hänni C., V. Laudet, D. Stehelin, P. Taberlet, 1994 Tracking the origins of the cave bear (Ursus spelaeus) by mitochondrial DNA sequencing Proc. Natl. Acad. Sci USA 91:12336-12340[Abstract/Free Full Text]

    Hewitt G., 2000 The genetic legacy of the Quaternary ice ages Nature 405:907-913[ISI][Medline]

    Hofreiter M., V. Jaenicke, D. Serre, A. von Haeseler, S. Pääbo, 2001a. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA Nucleic Acids Res 29:4793-4799[Abstract/Free Full Text]

    Hofreiter M., D. Serre, H. N. Poinar, M. Kuch, S. Pääbo, 2001b Ancient DNA Nat. Rev. Genetics 2:353-359[ISI][Medline]

    Hoss M., P. Jaruga, T. H. Zastawny, M. Dizdaroglu, S. Paabo, 1996 DNA damage and DNA sequence retrieval from ancient tissues Nucleic Acids Res 24:1304-1307[Abstract/Free Full Text]

    Huxtable J., M. J. Aitken, 1992 Thermoluminescence dating of a burned flint and stalagmitic calcite Pp. 175–178 in M. Otte, M. Patou-Mathis, and D. Bonjean, eds. La grotte Scladina, Vol. 1. Le contexte. Belgium: Eraul 27.

    Kimura M., 1989 The neutral theory of molecular evolution and the world view of the neutralists Genome 31:24-31[ISI][Medline]

    Krings M., C. Capelli, F. Tschentscher, H. Geisert, S. Meyer, A. von Haeseler, K. Grosschmidt, G. Possnert, M. Paunovic, S. Pääbo, 2000 A view of Neandertal genetic diversity Nat. Genetics 26:144-146[ISI][Medline]

    Kühn R., W. Schröder, O. Rottman, 2001 Sequencing mtDNA of the cave bear (Ursus spelaeus) from the Bavarian Alps is feasible by nested and touchdown PCR Acta Theorol 46:61-68

    Kurten B., 1968 Pleistocene mammals of Europe Weindenfeld & Nicholson, London

    ———. 1976 The cave bear story Columbia University Press, New York

    Leonard J. A., R. K. Wayne, A. C. Cooper, 2000 Population genetics of ice age brown bears Proc. Natl. Acad. Sci. USA 97:11651-11654

    Lopez Gonzalez F., A. Grandal D'Anglade, 2000 A paleobiological approach to the cave bears from Linarès and Eiros (Galicia, Spain) Pp. 28–30 Sixth International Cave Bear Symposium, 27–30 September, 2000

    Loreille O., L. Orlando, M. Patou-Mathis, M. Philippe, P. Taberlet, C. Hänni, 2001 Ancient DNA analysis reveals divergence of the cave bear, Ursus spelaeus, and brown bear, Ursus arctos, lineages Curr. Biol 11:200-203[ISI][Medline]

    Mazza P., M. Rustioni, 1994 On the phylogeny of Eurasian bears Paleontograph. Abt. A 230:1-38.

    Noro M., R. Masuda, I. A. Dubrovo, M. C. Yoshida, M. Kato, 1998 Molecular phylogenetic inference of the woolly mammoth, Mammuthus primigenius, based on complete sequences of mitochondrial cytochrome b and 12S ribosomal RNA genes J. Mol. Evol 46:314-26[ISI][Medline]

    Ovchinnikov I. V., A. Götherström, G. P. Romanova, V. M. Kharitonov, K. Liden, W. Goodwin, 2000 Molecular analysis of Neanderthal DNA from the northern Caucasus Nature 404:490-493[ISI][Medline]

    Pacher M., 2000 Taphonomische untersuchugen der Höhlenbären-Fundstellen in der Schwabenreith-Höhle bei Lunz am See (Niederösterreich) Beit. Paläont 25:11-85

    Stuart A. J., 1991 Mammalian extinctions in the late Pleistocene of northern Eurasia and North America Biol. Rev. Camb. Philos. Soc 66:453-562[ISI][Medline]

    Taberlet P., J. Bouvet, 1994 Mitochondrial DNA polymorphism, phylogeography, and conservation genetics of the brown bear, Ursus arctos, in Europe Proc. R. Soc. Lond. B 255:195-200[ISI][Medline]

    Vila C., J. A. Leonard, A. Götherström, S. Marklund, K. Sandberg, K. Liden, R. K. Wayne, H. Ellegren, 2001 Widespread origins of domestic horse lineages Science 291:474-477[Abstract/Free Full Text]

    Weber D. S., B. S. Stewart, J. C. Garza, N. Lehman, 2000 An empirical genetic assessment of the severity of the northern elephant seal population bottleneck Curr. Biol 10:1287-1290[ISI][Medline]

Accepted for publication July 15, 2002.