Cave discovery holds clues to earliest Homo sapiens in Europe

Ancient DNA from 46,000-year-old bone fragments and a tooth reveals this group likely overlapped with Neanderthals for thousands of years.

People squatting in a cave with face masks on, digging through dirt and clay.
Excavations in Initial Upper Paleolithic Layer I at Bacho Kiro Cave in Bulgaria. Four Homo sapiens bones were recovered from this layer, along with a rich stone tool assemblage, animal bones, bone tools, and pendants. (Pre-pandemic image: Tsenka Tsanova, MPI EVA Leipzig, License: CC-BY-SA 2.0)

In a cave in Bulgaria, Penn Museum researcher Zeljko Rezek and colleagues discovered the oldest known unequivocally defined remains of Homo sapiens in Europe, dating back nearly 46,000 years. That’s about 5,000 years older than the previous recordholder and points to a time when Neanderthals still existed in the region, meaning the two likely overlapped for several thousand years. 

What’s more, based on preserved mitochondrial DNA, the researchers found that this group of H. sapiens seems not to have contributed genetically to later H. sapiens of the region. “Nuclear DNA analysis is in progress, but if this is the case, then contrary to previous beliefs, our species was not able to survive in all environments,” says Rezek, who earned his Ph.D. in anthropology from Penn in 2015. “That goes against previous narratives saying that one thing that sets us apart from other hominin species is behavioral flexibility to a variety of situations. Now we know that this may not be the case.” 

The research team, which included scientists from Germany’s Max Planck Institute for Evolutionary Anthropology, where Rezek is also a researcher, as well as the Bulgarian Academy of Sciences and others, published two papers on their findings, one in Nature, a second in Nature Ecology & Evolution

Bulgaria’s Bacho Kiro Cave, located in the Balkans and today a tourist attraction as well as an archaeological site, had been excavated twice before, in the 1930s and again in the 1970s. Although the latter work yielded stone and bone tools and personal ornaments made of animal teeth thought to represent Europe’s first wave of H. sapiens, no reliable dating had come from what was found at the site, nor was it clear which species left this record. 

A person using a tool during an archaeological dig in a cave. In front, on dirt, sit about 30 samples placed on top of small white sheets of paper. Behind are plastic bags to hold the findings.
An excavator records artifacts, each marked with a colored pin. The bags with barcodes will hold individual artifacts once all positions have been recorded. (Pre-pandemic image: Zeljko Rezek, MPI EVA Leipzig, License: CCBY-SA 2.0)

In 2015, the group that included Rezek returned to Bacho Kiro armed with advanced tools like the ability to analyze ancient DNA, something not around 40 years ago. Their aim: to re-excavate one sector and excavate another for the first time.  

For this type of work, bigger bone fragments are easier to find. Larger pieces also make it easier to tell whether remains are human or another animal. “That was not the case here,” Rezek says. In the layer where the scientists were working, the fragments were already broken into inch-size pieces, and because the cave is often damp, they were mostly in clayish sediment, which makes them hard to identify during excavation.

Beyond that, for the purpose of DNA and other analyses, it is best not to clean them before they reach the lab. Yet within these conditions, Rezek and colleagues discovered a human tooth and human bone fragments that provide new information about the earliest H. sapiens in Europe. 

To pinpoint the origins of what they’d found in the field, the researchers brought more than 1,200 fragments back to the lab, where they removed the outermost layer of the bones and investigated the composition and form of their collagen, the protein found in skin and bone. “Based on that, you can determine which animal genera, sometimes even which species, it is, including whether it’s human,” Rezek says. Five, including a fully intact second lower molar, turned out to be human remains. 

From there, ancient DNA analysis told the research team that the tooth and one of the bones came either from the same person or from two maternally related individuals. “Such high-resolution results of ancient DNA analysis do not happen often. You really have to have good preservation at the site and excavate in the right area,” he says. “The other three fragments were from different individuals.” 

Three rows of bones and stone tools on a white background. In the middle is a measurement showing 1 centimeter length.
Personal ornaments and bone tools from Bacho Kiro Cave in Bulgaria, artifacts that are attributed to Homo sapiens and which date to approximately 45,000 years ago. (Image: Rosen Spasov and Geoff Smith, MPI EVA Leipzig, License: CC-BY-SA 2.0)

The question then became, when did these early humans live? Radiocarbon dating, which calculates how much time has passed by determining how much of the carbon-14 isotope remains, put the bones at somewhere between 43,650 and 45,820 years old. That’s the Middle to Upper Paleolithic period, when Neanderthals still endured in Europe and western and central Asia. But these remains undoubtedly came from H. sapiens, meaning the two groups likely co-existed for about 5,000 years. 

“One of the main questions in paleoanthropology is how much did other hominins like Neanderthals and Denisovans genetically contribute to us? Was there any tradition, not just in terms of genetics but also stone and bone technology or culture, like using and adapting to the landscape?” Rezek says. 

An overlap of several thousand years in Europe likely means H. sapiens and Neanderthals did share a space, he adds, and other genetic studies have already shown that they had to meet in certain regions. But only now are details of their interactions coming to light. All of this is part of investigating the dynamics between genetic, cultural, and ecological inheritances that shaped the early groups of our species biologically and behaviorally, says Rezek.

Getting that granular requires more work and more physical evidence, plus using nuclear DNA to corroborate what’s already been found. The team will likely have at least one more field season at Bacho Kiro Cave and investigate some other area caves, though given the coronavirus pandemic, they don’t yet know when that will take place. 

Despite what’s left to do, the recent findings lay the groundwork for unpacking these other hypotheses, Rezek says. “A few bigger notions have emerged from these results that we didn’t think about before. If it’s true that this group did not contribute genetically to later Homo sapiens in this region, for example, that could indicate that as a species, Homo sapiens weren’t always adaptable to all environmental and social pressures, as we like to often think, and that our emergence and dispersal is actually a complex story.” 

Funding for the research came from the Max Planck Society, the European Research Council, ETH Zürich, and the Collège de France.

Zeljko Rezek is a consulting scholar at the Penn Museum and a research scientist at the Max Planck Institute for Evolutionary Anthropology. He earned his Ph.D. from the Department of Anthropology in the School of Arts & Sciences at the University of Pennsylvania