Radiocarbon determinations and analytical data from Vindija faunal remains and bone point Conclusions Single-amino acid AMS dating of the Vindija Neanderthals has yielded results that are substantially older than the previous ages that were initially obtained.
The results suggest this group was not a late-surviving refugial Neanderthal population, as previously thought, and means the group almost certainly did not overlap with early anatomically modern humans in this part of Europe. Despite our best attempts, we were not able to date the bone industry associated with the archaeology of level G1.
The one date we obtained from a later stratigraphic unit was younger than 30, B. The dating of other faunal materials from level G1 highlighted a significant range in age, which could indicate a perturbation of the general sequence.
The question, then, of whether some of the points could have been produced by Neanderthals remains open; however, it is parsimonious to conclude that the split-based point at least must have a maximum age of 32,—34, B. Our perception of the biological transition between Neanderthals and modern humans has changed radically during the last decade. On the basis of our hydroxyproline dates and the DNA results, the Vindija Neanderthals date before the period when the first modern humans arrived into Europe and interbred with Neanderthals.
We used collagen peptide mass fingerprinting or ZooMS to analyze preserved type 1 collagen COL1 from small unidentified bone fragments from the Vindija site. COL1 is characterized by three polypeptide alpha chains, and within these chains there are small differences in the amino acid sequences among different species.
These offer the possibility of identification of species-specific amino acid markers. We used two different methods to prepare the samples for AMS dating. First, samples from artifacts were pretreated following the routine ORAU procedure, comprising a decalcification, base wash, reacidification, gelatinization, and ultrafiltration coded AF in the ORAU , as described by Brock et al.
Sample P produced too little collagen for ultrafiltration, so the treatment was halted after gelatinization coded AG. The four human bone samples were dated using the single amino acid radiocarbon dating method developed at ORAU This method involves separation of the underivatized amino acids from hydrolyzed bone collagen samples using preparative HPLC. Details of the method are described in the SI Appendix. This pretreatment approach is the most efficient technique to remove contaminants, including conservation materials unless collagen-based glue has been applied.
DNA extraction and library preparation. DNA extraction was performed using a method for ultrashort DNA fragment retrieval 33 with modifications The bone powder of Vi and Vi was treated with 0. Libraries were amplified and labeled with a pair of unique index sequences 37 , Enrichment of the mitochondrial DNA and sequencing.
Amplified libraries were enriched for human mitochondrial DNA, using a bead-based hybridization capture 39 with modifications Enriched libraries were sequenced on an Illumina MiSeq platform with a double index configuration Analyses were restricted to sequences with perfect index combinations.
PCR duplicates were removed using bam-rmdup https: To reconstruct the mitochondrial genomes of the three previously unanalyzed hominin samples, DNA sequences were realigned to the Vindija Data were processed as described earlier.
Data analysis was performed as previously published ref. Acknowledgments We thank Fred H. The authors declare no conflict of interest. This article contains supporting information online at www.
Freely available online through the PNAS open access option.