It makes for a tragic story: one of the last surviving populations of Neanderthals huddled in a cave some 32,000 years ago as modern humans invaded their homeland of Europe and drove them to extinction. Previous studies of Neanderthal and human bones in a cave in Croatia seemed to tell just that tale, but a team from Oxford has analyzed the bones using a new radiocarbon dating technique and found the Neanderthal remains were too old to fit the narrative.

Vindija Cave in northern Croatia has been an archaeological gold mine for decades. Thousands of specimens of animals and hominids have been uncovered, spanning a period of tens of thousands of years, and some of our key knowledge about Neanderthals, including their genome, comes from discoveries made on this site.

The fossil record as a whole seems to suggest that Neanderthals disappeared about 40,000 years ago, but past analyses of the Vindija bones has suggested that the species was living there as recently as 32,000 years ago. That would make them one of the last surviving groups as modern humans swept throughout Europe – assuming, of course, that the estimate was accurate. But using a new dating method, the Oxford team believes that this population weren't outliers after all.

"The Vindija Neanderthals have, for decades, been considered to be a late-surviving, refugial population of humans, that overlapped with, and survived alongside, early modern human colonizers in Europe," says Tom Higham, lead researcher on the project. "Our new single amino acid results show that this was not correct and demonstrates, once again, the crucial importance of reliable chronology in archaeology."

The team's new technique is designed to reduce the chances of modern DNA contaminating the samples and returning false readings. The single compound method, as they call it, is somewhat self-explanatory: only one amino acid, called hydroxyproline (HYP), is extracted from the collagen preserved inside the bone and tested. Focusing on this one amino acid allows researchers to tune out modern contaminants of other compounds.

Using this technique, the Oxford team re-examined three Neanderthal bone samples previously discovered in Vindija Cave, and found that all of them were more than 40,000 years old. That extra 8,000 years over previous estimates places the specimens back in the widely-accepted window of Neanderthal occupation, and suggests that they might not have encountered modern humans after all.

"Our previous research has shown that Neanderthals in Europe did not survive after 40,000 years ago, so the Vindija Neanderthals were not a refugial group, rather they were present just before modern humans began to penetrate Europe for the first time," says Higham.

Other studies conducted on the bones backed up the new dates. DNA analyses showed that there was no modern human DNA in the Neanderthals, confirming they didn't cross-breed and probably didn't cross paths. Another new technique, called Zooarchaeology by Mass Spectrometry (ZooMS), picked out a fourth Neanderthal bone fragment among unidentified bones from Vindija, and also found it was over 40,000 years old.

"The research we have conducted shows the great benefits of developing improved chemical methods for dating prehistoric material that has been contaminated, either in the site after burial, or in the museum or laboratory for conservation purposes," says Thibaut Devièse, first author of the study. "We think that all human bones from the Palaeolithic period ought to be dated using this technology due to impact of even small amounts of contamination from modern times. In addition, our multi-analytical approach, using radiocarbon, ZooMS and DNA analysis shows that these methods can significantly help in understanding human occupation of Palaeolithic sites."

The research was published in the journal PNAS.

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