Deciphering the complete mitochondrial genome and phylogeny of the extinct cave bear in the Paleolithic painted cave of Chauvet

Abstract

Retrieving a large amount of genetic information from extinct species was demonstrated feasible, but complete mitochondrial genome sequences have only been deciphered for the moa, a bird that became extinct a few hundred years ago, and for Pleistocene species, such as the woolly mammoth and the mastodon, both of which could be studied from animals embedded in permafrost. To enlarge the diversity of mitochondrial genomes available for Pleistocene species, we turned to the cave bear (Ursus spelaeus), whose only remains consist of skeletal elements. We collected bone samples from the Paleolithic painted cave of Chauvet-Pont d'Arc (France), which displays the earliest known human drawings, and contains thousands of bear remains. We selected a cave bear sternebra, radiocarbon dated to 32,000 years before present, from which we generated overlapping DNA fragments assembling into a 16,810-base pair mitochondrial genome. Together with the first mitochondrial genome for the brown bear western lineage, this study provides a statistically secured molecular phylogeny assessing the cave bear as a sister taxon to the brown bear and polar bear clade, with a divergence inferred to 1.6 million years ago. With the first mitochondrial genome for a Pleistocene carnivore to be delivered, our study establishes the Chauvet-Pont d'Arc Cave as a new reservoir for Paleogenetic studies. These molecular data enable establishing the chronology of bear speciation, and provide a helpful resource to rescue for genetic analysis archeological samples initially diagnosed as devoid of amplifiable DNA.

Fig. 1.

Bear bone sample and archaeological context. (A) Geographical localization of the Chauvet-Pont d'Arc Cave. (B) Cave topography. Red and black characters refer to the color of rock art pictures in the entry and deep sectors, respectively. (C) The sector of the Gallery of the Cross-Hatches from which Us18 (purple arrow) was retrieved.

Fig. 2.

Gel electrophoresis analysis of mitochondrial genome fragments generated by PCR from Us18. Variable amounts of the DNA extract (from 0.1 to 2.0 μl) were amplified using primer pair # 236, predicting a 117-bp DNA fragment. The total reaction volume was electrophoresed through an acrylamide gel stained with SYBR Green I. Negative controls included reactions carried out on a mock extract (Mock) or in the absence of any extract (H₂O). Molecular weight marker (M.W.) corresponds to λBstEII digest.

Fig. 3.

Mutational saturation analysis of the complete mitochondrial genome dataset. The y axis shows the observed number of differences between pair of species sequences. The x axis shows the inferred number of substitutions between the same two sequences in a Maximum Parsimony tree determined using Patristic software. The straight line represents the case for which there is no saturation, with no reversion occurring in the sequences.

Fig. 4.

Molecular phylogeny inferred from complete mitochondrial genome sequences. Tree construction was performed by MrBayes analysis, using the giant panda (Ailuropoda melanoleuca) as an outgroup. The posterior probability value (×100) of each node is indicated in black, and the scale for genetic distance is shown at the bottom of the figure. The same tree topology was obtained using three other methods, and bootstrap values are indicated with colored characters for PhyML (blue), maximum parsimony (red), and neighbor joining (green) analysis. The Ursus spealeus and Ursus arctos western lineage (west) sequences are from this study. GenBank accession numbers (from top to bottom) for the other sequences are as follows: EF196663, EF196662, AJ428577, AF303111, AF303110, AF303109, DQ402478, EF076773, EF196661, EF196664, and EF196665.

Fig. 5.

Phylogeny and divergence times determined using the mitochondrial genome sequence of the cave bear and of eight extant bears. Divergence times were calculated using BEAST software with the splits between the giant panda and Ursidae and between Ursinae and Tremarctinidae set to 12 and 6 million years (MY), respectively. Age for each node and 95% credibility intervals are as follows: 1, 6.3 MY (5.4–7.2); 2, 3.0 MY (2.2–3.8); 3, 2.8 MY (2.1–3.5); 4, 2.4 MY (1.7–3); 5, 2.1 MY (1.4–2.7); 6, 1.6 MY (1–2.1); 7, 0.6 MY (0.3–0.8); and 8, 0.4 MY (0.2–0.5).