Little is known about the population history of Neandertals over the hundreds of thousands of years of their existence. We retrieved nuclear genomic sequences from two Neandertals, one from Hohlenstein-Stadel Cave in Germany and the other from Scladina Cave in Belgium, who lived around 120,000 years ago. Despite the deeply divergent mitochondrial lineage present in the former individual, both Neandertals are genetically closer to later Neandertals from Europe than to a roughly contemporaneous individual from Siberia. That the Hohlenstein-Stadel and Scladina individuals lived around the time of their most recent common ancestor with later Neandertals suggests that all later Neandertals trace at least part of their ancestry back to these early European Neandertals.

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Elucidating when Neanderthal populations disappeared from Eurasia is a key question in paleoanthropology, and Belgium is one of the key regions for studying the Middle to Upper Paleolithic transition. Previous radiocarbon dating placed the Spy Neanderthals among the latest surviving Neanderthals in Northwest Europe with reported dates as young as 23,880 ± 240 B.P. (OxA-8912). Questions were raised, however, regarding the reliability of these dates. Soil contamination and carbon-based conservation products are known to cause problems during the radiocarbon dating of bulk collagen samples. Employing a compound-specific approach that is today the most efficient in removing contamination and ancient genomic analysis, we demonstrate here that previous dates produced on Neanderthal specimens from Spy were inaccurately young by up to 10,000 y due to the presence of unremoved contamination. Our compound-specific radiocarbon dates on the Neanderthals from Spy and those from Engis and Fonds-de-Forêt demonstrate that they disappeared from Northwest Europe at 44,200 to 40,600 cal B.P. (at 95.4% probability), much earlier than previously suggested. Our data contribute significantly to refining models for Neanderthal disappearance in Europe and, more broadly, show that chronometric models regarding the appearance or disappearance of animal or hominin groups should be based only on radiocarbon dates obtained using robust pretreatment methods.

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Ancient preserved molecules offer the opportunity of gaining a deeper knowledge on their biological past. However, the development of a proteomic workflow remains a challenge. The analysis of fossils must involve a low quantity of material to avoid damaging the samples. In this study an enhanced proteomic protocol was applied to 5‐milligram samples of about 130,000‐year‐old mammalian bones ranging from the end of the Middle Pleistocene up to the earlier Upper Pleistocene, excavated from Scladina Cave (Sclayn, Belgium). Using sequence homology with modern sequences, a biological classification was successfully achieved and the associated taxonomic ranks to each bone were identified consistently with the information gained from osteomorphological studies and palaeoenvironmental and palaeodietary data. Amino acid substitutions on collagens were identified, thus providing new information on extinct species sequences and helping in taxonomy‐based clustering. Considering samples with no osteomorphological information, such as two fragments of bone retouchers, proteomics successfully identified the families providing paleontologists new information on these objects. Combining osteomorphology studies and amino acid variations identified by proteomics, one of the retouchers was potentially identified as belonging to the Ursus spelaeus species.