*CNRS UMR 5534, Centre de Genetique Moleculaire et Cellulaire, Universite Claude Bernard Lyon 1, Villeurbanne, France;
Archeologie Andennaise, Sclayn, Belgium;
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
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Abstract |
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Introduction |
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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 1994
). 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 1976
; Stuart 1991
). 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 1.21.6 MYA (Hänni et al. 1994
; Loreille et al. 2001
).
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 1968
; Pacher 2000
). 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 1996
, 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.
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Material and Methods |
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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 min50 to 54°C, 45 s72°C, 45 s) on a Master Gradient apparatus (Ependorf®). Primers designed to amplify only short overlapping fragments in mtDNA CR (92206 bp, fig. 1 ) were described elsewhere (Loreille et al. 2001
). Three independent blanks were carried out for each set of PCR experiment as reported in Loreille et al. 2001
.
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Sequence Analyses
Our 21 sequences were compared with the published sequences. In earlier studies (Hänni et al. 1994
; Loreille et al. 2001
), 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 2001
) and 12 sequences of 26.5 to over 50-KY-old samples from nine European caves (Hofreiter et al. 2001a
). 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 1996
). All the phylogenies were computed with the Phylo_win program (Galtier, Gouy, and Gautier 1996
). 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 1994
). 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.
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Results |
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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. 2001a
). Such damage-induced errors may blur the genetic message preserved in fossils by adding artifactual polymorphism (Cooper and Poinar 2000
). 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 H3H1 and DF311DR500, DF311DR500, and H16143H16299, 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 GCAT 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. 2001a
). 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.
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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 1994
). 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 (40130 KYA). In France, contemporary caves located in close proximity also possess members of those two haplogroups (40 KYA in Prélétang and 2535 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 (90130 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 ).
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Discussion |
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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. 1994
; Hofreiter et al. 2001a
; Kühn et al. 2001
; Loreille et al. 2001
), 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 1214 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 1989
). 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 1989
). 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 1999
).
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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 1994
; Hewitt 2000
). 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 1991
). 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%, 90130 KYA) and 1A episodes (1.1%, 4045 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 (TC 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)
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)
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.
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Appendix A |
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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 1992
, 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 1999
), magnetic susceptibility and the thermoluminescence dating of a top-layer calcite to 100 KY BP (Huxtable and Aitken 1992
), 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 1998
). 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 1998
). Such a palaeoenvironmental interpretation is confirmed by the analysis of carbon and nitrogen isotopic compositions of bone collagen of large mammals (Bocherens et al. 1997
).
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Acknowledgements |
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Footnotes |
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Abbreviations: CR, mtDNA control region; BP, before present; KYA, thousand years ago; KY, thousand years.
Keywords: Ursus spelaeus
ancient DNA
population genetics
climatic change
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
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