Archaeology Magazine - September/October 2016 - 41

while you're still alive. But it wasn't clear until very recently- through Warinner's work and parallel projects in labs in Chile, Australia, and Denmark-that calculus traps or preserves DNA at all. "Dental calculus is really an unlikely hero," she says. Warinner, who had excavated the earliest known epidemic mass graves in Mesoamerica early in her career, began to see the potential in calculus in 2007, when she worked at the Smithsonian. A colleague there was Amanda Henry, now at the Max Planck Institute in Leipzig, who searches calculus for trapped plant fragments, such as phytoliths and starch grains. By 2010, Warinner was a postdoctoral researcher studying ancient disease and diet at the Institute of Evolutionary Medicine at the University of Zurich. When she tried to replicate Henry's methods there, she had trouble counting plant granules. "There were so many bacteria that were getting in the way," she says, "but I had a kind of aha moment." Other research groups were exploring the same idea, but at the time nothing had been published, other than a review article from the 1950s that stated that there was no DNA in calculus. She saw an opportunity to test her hypothesis that it could be a source of ancient microbial DNA, but it was a risky, expensive proposition for a postdoc. "People thought it was just a nuts, nuts idea," Warinner says. "I was sort of doing microbiome research without knowing the word." "In German we say überflieger; that means someone who flies higher than the others. I could immediately see her potential," says Frank Rühli, director of the Institute. But the calculus idea didn't seem promising. "I was a bit reluctant at the very first moment." Warinner's first few attempts in 2010 failed. She then got a more sensitive fluorometer, and it still didn't appear to be working. "I saw a message that I didn't even know existed," she says. "It said 'Error, DNA too high.'" So she diluted the sample 50 to 100 times. "I started realizing, as I started quantifying, that I had just discovered the richest source of ancient DNA ever described in an archaeological sample." There was more DNA in her sample than there is in fresh liver tissue. In the few years before these first experiments, several scientific currents were converging. In 2008, Lewis, who didn't yet know Warinner, published some of the first reports of the ancient microbiome from coprolites. The same year, the Human Microbiome Project (HMP), a multi-institution effort to catalogue the biota, was established with funding from the National Institutes of Health. This placed the term and its connection to human health in wide view. Microbes had long been seen only as biological villains to be exterminated, but with the launch of the HMP came the recognition that bacteria could be helpful and even essential to multicellular life and its processes. This was a seismic change. Perhaps most importantly, genetic research itself was undergoing a revolution. Gone were the days of laboriously generating one DNA sequence at a time. The new wave of technology, called next-generation sequencing (NGS), was becoming widely available, and is able to create hundreds of millions of sequences at once. It opened the door for "shotgun metagenomics," or amplifying and sequencing all the DNA archaeology.org from all the genes from all the organisms in a sample. NGS has made it possible, in a single pass, to describe entire microbial communities or reconstruct the full genome of a microbial species. It increased exponentially the amount of data researchers could gather. According to Warinner, "We were just racing as fast as we could to keep up with all of this." NGS, however, was expensive-the necessary reagents alone cost $13,000 for the experiment that Warinner planned to conduct. She was terrified, and thought at the time, "It's a moon shot. If it works, it's awesome. If it doesn't, I'm hosed." D alheim is a small town near Lichtenau, Germany, where in 1989 and 1990 the Westphalian Museum of Archaeology excavated the site of a medieval monastery, parish church, and convent. Remains from the 151 burials they uncovered were stored at the University of Mainz until the school needed the space and planned to incinerate and bury them. Rühli offered to bring the skeletal material to Zurich for research in 2010. "A big challenge when you're trying to develop a new method is just having material to practice on," says Warinner. While she was conducting her work on the Dalheim samples, several other groups were operating with the same idea. In 2012, researchers from the University of Chile were the first to publish the basic identification of DNA in archaeological dental calculus. A group led by Alan Cooper of the University of Adelaide was the first to apply NGS to it in 2013. Following these reports, the Dalheim burials offered Warinner an opportunity to produce the first shotgun metagenomic analysis and characterize complete ancient oral microbial communities. The Dalheim study stands now as the most forceful declaration of the possibilities of dental calculus, with orders of magnitude more data than had been reported before. The experiment resulted, Warinner says, in hundreds of millions of sequences. It took three years to analyze it all. Warinner and her coauthors-32 in total from a broad range of disciplines-catalogued, from the mouths of four medieval individuals, 40 opportunistic pathogens, including species associated with cardiovascular disease, meningitis, and pneumonia, as well as what might be the oral ancestor of modern gonorrhea. They sequenced the entire genome of Tannarella forsythia, a cause of periodontal disease. They saw dietary DNA from pigs, cruciferous vegetables, and bread wheat. They looked for proteins as well, and found ones associated with pathogen virulence, others produced by the human immune system, and beta-lactoglobulin, a durable dairy protein. Among the genes identified, oddly, are ones associated with microbial antibiotic resistance-hundreds of years before the advent of antibiotic drugs. The expert on the subject for the study, Lars Hansen of Aarhus University in Denmark, says that the cellular machinery that creates antibiotic resistance can serve other purposes in cells. The modern phenomenon, he explains, comes from increased availability and expression of these existing mechanisms. "It is the first time [antibiotic resistance sequences] have been found in an ancient humanassociated context," says Hansen. "It shows that these building blocks are basically everywhere." 41 http://www.archaeology.org

Table of Contents for the Digital Edition of Archaeology Magazine - September/October 2016