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Vindija Neanderthals press release Sept. 4 2017. Link to full story here.

Late surviving Neanderthals from Croatia were much older than previously thought, according to new research from the University of Oxford. Previous research suggested that the ‘Vindija Neanderthals’ living in Vindija Cave in northern Croatia lived as recently as 32,000 years ago. This would have made them amongst the last known surviving Neanderthals and implied that Neanderthals and modern humans lived in central Europe at the same time. They were therefore known as a ‘refugial’ group.

But a new radiocarbon dating method using single amino acids, developed by scientists at the Oxford Radiocarbon Accelerator Unit, has found that these human remains were actually more than 8,000 years older than this initial estimate. This dates them to just before the arrival of modern humans in Europe.

“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 colonisers in Europe’, said Professor Tom Higham, who leads the PalaeoChron research group at the University of Oxford. “Our new single amino acid results show that this was not correct and demonstrates, once again, the crucial importance of reliable chronology in archaeology. 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.”

The research has been published in the journal PNAS.

To make the discovery, the Oxford team had to improve the methods of purification for bone samples prior to radiocarbon dating. They developed the “single compound method” for radiocarbon dating of bones. This method relies on extracting just one of the amino acids from the collagen present in the bone. The amino acid hydroxyproline (HYP), which accounts for 13% of the carbon in mammalian collagen, was targeted by the researchers. Dating this amino acid allows for the drastic improvement in the removal of modern contaminants from the specimens. Using the single compound dating approach, the Oxford team re-dated three Neanderthal bone samples from Vindija. All of the dates were older than 40,000 years BP, much older than the dates previously obtained, suggesting that there was still a significant amount of unremoved

contamination in the original measurements. A fourth Neanderthal bone fragment from the Vindija collections in Zagreb was discovered using a novel technique called ‘Zooarchaeology by Mass Spectrometry’ or ZooMS, at the University of Manchester by study co-authors Cara Kubiak and Dr Michael Buckley. It also dated to the same period. ZooMS is being used by the team to identify hominin bones from amongst thousands of previously unidentified bones in Palaeolithic archaeological sites.

DNA analyses were also performed on the hominin bones by the team of Professor Svante Pääbo at the Max-Planck Institute for Evolutionary Anthropology in Leipzig, Germany. DNA analysis has shown that there is no modern human DNA in the Vindija Neanderthals, and supports the radiocarbon dating in suggesting that the Neanderthals at the site were not contemporaries of early modern humans in Europe.

Dr Thibaut Devièse, first author on the new paper, said: “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. 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.”

PRESS RELEASE 29 March 2016

Novel collagen fingerprinting identifies new Neanderthal bone among 2,000 fragments at Denisova Cave

Scientists from the universities of Oxford and Manchester have used a new molecular fingerprinting technique to identify one Neanderthal bone from around 2,000 bone fragments. All the tiny pieces of bone were recovered from a key archaeological site, Denisova Cave in Russia, with the remaining fragments found to be from animal species like mammoths, woolly rhino, wolf and reindeer. It is the first time that researchers have identified traces of an extinct human from an archaeological site using a technique called ‘Zooarchaeology by Mass Spectrometry’ or ZooMS. From just a microscopic sample of bone, their analysis revealed the collagen peptide sequences in the bone that mark out one species from another. Their paper, which appears in the journal Scientific Reports, suggests that ZooMS has huge potential to increase our understanding of human evolution, including the amount of interbreeding that went on between our closely related cousins and modern humans.

The international research team was led by Professor Thomas Higham and his student Sam Brown of the University of Oxford, with the developer of the ZooMS method, Dr Michael Buckley from the University of Manchester; the Max Planck Institute in Leipzig; Cranfield University, UK; and the Institute of Archaeology and Ethnography, Russia. The sequences of collagen peptides in bone differ in tiny ways between different animal species. The team profiled the sequences using microscopic samples from 2,300 unidentified bone fragments from the site. They then compared the sequences obtained against a reference library of peptides from known animal species.

On discovering that one 2.5 cm long piece of bone had a clear human fingerprint, Sam Brown said: ‘When the ZooMS results showed that there was a human fingerprint among the bones I was extremely excited. After a lot of hard work, finding this tiny bone which yields so much information about our human past was just fantastic. The bone itself is not exceptional in any way and would otherwise be missed by anyone looking for possible human bones amongst the dozens of fragments that we have from the site.’

Study co-author Professor Svante Pääbo and his group from the Max Planck Institute in Leipzig found that the bone belonged to a Neanderthal on the basis of its mitochrondrial genome. The results suggest that this Neanderthal was most closely related to other Neanderthals in the Altai region and more distantly related to those further to the west. The Neanderthal bone was also radiocarbon dated and shown to be more than 50,000 years old, as expected based on its deep position in the site. Acid etching on the surface of the bone also shows that it probably passed through the stomach of a hyaena for a short time before it was deposited in the cave sediments, says the paper.

Denisova Cave in the Russian Altai region is a key site for archaeologists wanting to understand the nature of evolution over the last 100,000 years. Its cold climate means that bones from the cave are often exceptionally well-preserved in their DNA and collagen, which is ideal for genetics and radiocarbon dating. In 2010, Pääbo and his team discovered a new species of human, the so-called ‘Denisovans’ at the site, using state-of-the-art genetic methods. His team have also found that Denisovans, Neanderthals and modern humans interbred periodically with one another in Eurasia and Europe.

During the Ice Age, there were periods when the cave was also occupied by carnivores such as hyaenas and wolves that crunched the bones into tiny pieces. This is why more than 95% of the bone fragments excavated were difficult to identify.

Professor Thomas Higham said: ‘This is a real breakthrough, showing that we can now use bioarchaeological methods like ZooMS to search the archaeological record and find even tiny fossil remains, where there are proteins that survive. In the Palaeolithic period, where we have Neanderthals, Denisovans and modern humans, this is potentially very important because if the fragments that we recover are big enough then we can date and analyse the DNA from the same bone. One of the big challenges is in understanding what happened when modern humans and Neanderthals met. We want to know over what period of time and where this happened. Fossils are the key, but for modern humans they are extremely rare in archaeological sites. We hope that more work like this will yield more human bone remains.’

Dr Michael Buckley said: ‘This really was a case of looking for a needle in a haystack, but the collagen fingerprinting method is very robust and tiny amounts of material are all that are needed. New developments underway in my lab are further refining the technique and enabling a higher throughput of samples to be achieved. We are planning on widening the search for human remains from many other sites in the future.’

Full press release here.

Link to the paper here.

[above] New Neanderthal bone fragment found using ZooMS at the Denisova Cave site. The bone has also yielded a complete mtDNA sequence.

[above] Samantha Brown, whose MSc dissertation research led to the current paper. Sam analysed over 2000 bone fragments, identifying each to species. One of the bones turned out to be a Neanderthal.

[left] The bone came from a corpus of excavated bone from the site of Denisova Cave, in the east gallery of the site.

[above] Mike Buckley with the Matrix-assisted Laser Desorption/Ionisation Mass Spectrometer (MALDI-MS) used for the collagen fingerprinting.

PRESS RELEASE 21 August 2014

Neanderthals and modern humans ‘overlapped’ in Europe for up to 5,400 years

Extinction date of European Neanderthals confirmed at 39,000-41,000 years ago

6-year study published in Nature

The high-precision dating of materials from 40 archaeological sites, from Russia to Spain, reveals that the disappearance of Neanderthals from Europe took place around 40,000 years ago. Rather than a rapid replacement of European Neanderthals by anatomically modern humans, the study led by Professor Tom Higham of the University of Oxford, published in Nature, supports a more complex picture characterized by a biological and cultural mosaic that lasted for several thousand years.

Determining the spatial and temporal relationships between Neanderthals and early modern humans is critical to the understanding of the processes underlying and reasons for the disappearance of Neanderthals. However, technical challenges have hindered the reliable dating of the period, as samples older than about 50,000 years preserve too little carbon-14 for conventional radiocarbon dating to yield accurate results.

Higham and colleagues used improved sample processing and accelerator-mass-spectrometry radiocarbon dating to analyse bone samples and items from the Mousterian and Châtelperronian stone-tool industries, which have been associated with Neanderthals, and from Uluzzian sites, which are currently thought to have been made by modern humans. The results suggest that the Neanderthal disappearance and the end of the Mousterian technocomplex occurred between 41,000 and 39,000 years ago, across sites ranging from the Black Sea to the Atlantic Coast. The findings also reveal a temporal overlap between Neanderthals and modern humans of 2,600 to 5,400 years, allowing ample time for possible cultural — and maybe also genetic — exchanges between the two groups.

Full press release here.

Link to the paper here.

See Chris Stringer of the Natural History Museum talking about the research here

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