"The strength of ancient biomolecules is that they allow us to look beyond what is simply visible on the bones," said Biancamaria Bonucci, a junior research fellow in ancient DNA at the University of Tartu. Many diseases, for example, leave no traces on bones and can only be detected by studying ancient DNA and proteins. Written historical sources are also often incomplete and the presence of a particular disease in a certain region may only come to light through biomolecular research.

In her recently defended doctoral thesis, Bonucci studied ancient proteins and ancient DNA in a variety of materials. She paid particular attention to materials surrounding bones and teeth, including soil deposits accumulated on bones and dental calculus. "Soil actually contains a great deal of information about the skeleton, the individual and the burial environment. It can reveal stories that have remained hidden until now," Bonucci said.

Unwritten plague

Previous ancient DNA studies have focused primarily on the lives of individual people. The ancient DNA and proteins examined by Biancamaria Bonucci add a new layer to existing knowledge by providing context about the world surrounding ancient people. Ancient biomolecules can offer clues about what domestic animals people kept, what species they came into contact with, what diseases they suffered from and what the local climate was like during their lifetimes.

For example, one surprising finding in Bonucci's research was that a soil sample contained DNA from a lineage of black rats (Rattus rattus) that is now extinct in England. "What was important was that both the DNA and the proteins told the same story. In addition to the rat's genetic material, we found many proteins characteristic of that rat in the sediment. This allowed us to confirm that it was indeed this particular species, which is no longer found in England," Bonucci said.

In another study, she used dental remains to investigate infectious diseases that afflicted residents of Sint-Truiden, a medieval and early modern town in what is now Belgium. The composition of the samples indicated that the hepatitis B virus and the louse-borne, fever-causing bacterium Borrelia recurrentis, among others, once circulated in the town. "What was remarkable was that we also found traces of the plague bacterium Yersinia pestis dating to the 14th century, even though it was not mentioned in any chronicles," Bonucci added.

While plague victims were typically buried in mass graves, in Sint-Truiden, signs of plague were found in people laid to rest in ordinary individual graves. "These people may have been infected but died so quickly that we simply don't know the exact circumstances. In any case, the finding was surprising and added a new piece of information to the historical record," Bonucci said.

In another case study, she and her colleagues examined Bronze Age remains from Grotta della Spinoza in Italy. "These were not conventional burials, but rather scattered human bones from which we extracted DNA," Bonucci explained.

The remains of one of the cave's occupants showed traces of several viruses as well as both HBV and plague. "This individual is the earliest known carrier of Yersinia pestis in Southern and Western Europe," Bonucci said. According to her, this further confirms that ancient DNA can provide valuable information about periods for which no written sources exist.

The finding also opens up avenues for further research as it is not known exactly how the plague bacterium spread so long ago. Today, it is transmitted by rats and fleas, but ancient strains of the bacterium lacked the genes that would have allowed them to spread via fleas. "We found the plague bacterium alongside the zoonotic bacterium Erysipelothrix rhusiopathiae. We can therefore infer that they may have become infected through direct contact with animals, for example by eating meat from certain animals," Bonucci said.

Some finds should not be cleaned

According to Bonucci, her doctoral thesis had two aims: On the one hand, she demonstrated that ancient molecules are worth studying in materials originating outside the human body. On the other, she gained new insights into the history of specific archaeological sites. "Molecular studies give us much more information not only about the individual, but also about the environment that surrounded them and the diseases they likely suffered from," she said.

While the ability of teeth and bones to preserve DNA is well known, dental calculus and sediments accumulated on bones have received little previous study as repositories of information. Bonucci set out to fill that gap. "For example, we didn't find much human DNA in dental calculus. However, we did find a great deal of microbial DNA — specifically, DNA from the microbes that live in our mouths," she explained.

One study, however, showed that soil sediment covering a bone contained DNA from the same person as the bone itself. The exact origin of the sediment is unknown, but Bonucci believes it, too, forms after the body decomposes and preserves information in a similar way to dental calculus. "Unlike sediment, dental calculus forms during a person's lifetime and later fossilizes. It was very surprising that preservation in these two materials was so similar," she said.

In light of her doctoral research, she recommends that archaeologists avoid cleaning all material off finds during excavations. It is also worth preserving a certain amount of local soil and sediment. "I would recommend that archaeologists keep the soil and ask one of their geneticist friends to analyze it," Bonucci said.

According to Bonucci, the study's most striking findings — the extinct rat lineage in England and evidence of the plague bacterium's early spread in Europe — also indicate that the historical relationship between humans and animals deserves greater attention. "Based on ancient biomolecules, we can infer that many diseases in prehistoric times originated precisely from our closer contact with animals," she said.

She also urged researchers not to overlook ethical considerations when studying people and animals that died long ago. While research involving living people is subject to oversight by ethics committees, the situation is more complicated when it comes to ancient material. "Ancient material is a finite resource. We can't keep taking tooth and bone samples from these people indefinitely. We also need to understand that a person is more than just their body — we can find a person's DNA in the soil as well," Bonucci emphasized.

Biancamaria Bonucci defended her doctoral thesis in gene technology, "Reading the archaeological record through ancient biomolecules: preservation, disease landscapes and human-microbe interactions in the past," at the University of Tartu on August 14. Her supervisors were Professor Kristiina Tambets and Antonio de Dios Martinez of the University of Tartu and Christiana Lyn Scheib of the University of Cambridge. The opponent was Associate Professor Camilla Speller of the University of British Columbia.

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