Exceptionally high δ¹⁵N values in collagen single amino acids confirm Neandertals as high-trophic level carnivores

Identifying past hominin diets is a key to understanding adaptation and biological evolution. Bone collagen isotope studies have added much to the discussion of Neandertal subsistence strategies, providing direct measures of diet. Neandertals consistently show very elevated nitrogen isotope values. These values have been seen as the signature of a top-level carnivore diet, but this interpretation was recently challenged by a number of additional theories. We here apply compound-specific isotope analysis of carbon and nitrogen in bone collagen single amino acids of two Neandertals. These Neandertals had the highest nitrogen isotope ratios of bulk collagen measured so far, and our study confirms that these values can be most parsimoniously explained by a carnivorous diet.Isotope and archeological analyses of Paleolithic food webs have suggested that Neandertal subsistence relied mainly on the consumption of large herbivores. This conclusion was primarily based on elevated nitrogen isotope ratios in Neandertal bone collagen and has been significantly debated. This discussion relies on the observation that similar high nitrogen isotopes values could also be the result of the consumption of mammoths, young animals, putrid meat, cooked food, freshwater fish, carnivores, or mushrooms. Recently, compound-specific C and N isotope analyses of bone collagen amino acids have been demonstrated to add significantly more information about trophic levels and aquatic food consumption. We undertook single amino acid C and N isotope analysis on two Neandertals, which were characterized by exceptionally high N isotope ratios in their bulk bone or tooth collagen. We report here both C and N isotope ratios on single amino acids of collagen samples for these two Neandertals and associated fauna. The samples come from two sites dating to the Middle to Upper Paleolithic transition period (Les Cottés and Grotte du Renne, France). Our results reinforce the interpretation of Neandertal dietary adaptations as successful top-level carnivores, even after the arrival of modern humans in Europe. They also demonstrate that high δ15N values of bone collagen can solely be explained by mammal meat consumption, as supported by archeological and zooarcheological evidence, without necessarily invoking explanations including the processing of food (cooking, fermenting), the consumption of mammoths or young mammals, or additional (freshwater fish, mushrooms) dietary protein sources