Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition

Abstract

Previous dating of the Vi-207 and Vi-208 Neanderthal remains from Vindija Cave (Croatia) led to the suggestion that Neanderthals survived there as recently as 28,000-29,000 B.P. Subsequent dating yielded older dates, interpreted as ages of at least ∼32,500 B.P. We have redated these same specimens using an approach based on the extraction of the amino acid hydroxyproline, using preparative high-performance liquid chromatography (Prep-HPLC). This method is more efficient in eliminating modern contamination in the bone collagen. The revised dates are older than 40,000 B.P., suggesting the Vindija Neanderthals did not live more recently than others across Europe, and probably predate the arrival of anatomically modern humans in Eastern Europe. We applied zooarchaeology by mass spectrometry (ZooMS) to find additional hominin remains. We identified one bone that is Neanderthal, based on its mitochondrial DNA, and dated it directly to 46,200 ± 1,500 B.P. We also attempted to date six early Upper Paleolithic bone points from stratigraphic units G₁, Fd/d+G₁ and Fd/d, Fd. One bone artifact gave a date of 29,500 ± 400 B.P., while the remainder yielded no collagen. We additionally dated animal bone samples from units G₁ and G₁-G₃ These dates suggest a co-occurrence of early Upper Paleolithic osseous artifacts, particularly split-based points, alongside the remains of Neanderthals is a result of postdepositional mixing, rather than an association between the two groups, although more work is required to show this definitively.

Keywords: DNA analysis; Middle to Upper Paleolithic transition; Vindija Cave (Croatia); single-compound AMS dating; zooarchaeology by mass spectrometry.

Fig. 1.

(A) High-resolution photographs of the Vi-*28 Neanderthal bone found using ZooMS. The bone yields evidence for a probable cut and gauge marks (right upper part of the bone). The picture was taken after the bone had undergone sampling for ZooMS and before sampling for aDNA, radiocarbon, and stable isotope analysis. (B) MALDI-TOF mass spectrum of digested collagen from the Vi-*28 bone. All tagged peaks (A, B, C, D, and F) denote sequence-matched peptides observed in human collagen (27, 28).

Fig. 2.

Bayesian age model showing the calibrated HYP ages of the four Neanderthal samples from Vindija Cave. The model is a simple phase model in OxCal 4.3 (47), in which all F14C determinations are assumed to have no relative order. “Start” and “End” correspond to the boundaries calculated by the model. The calibration curve of Reimer et al. (48) was used to calibrate the results. Details can be found in the SI Appendix.