The Carthaginian general Hannibal is remembered for his march across the Alps with thirty-seven elephants, but scholars have long disputed exactly which route he took over the mountains. Illustration courtesy Universal History Archive / UIG / Getty

More than two thousand years ago, thirty-seven elephants from heat-shimmering latitudes ascended Europe’s highest mountain range, tramped though snow and across ice, and breathed the thin air of high altitudes. Those that survived the perilous journey met with a bitter winter and war, as the Carthaginian general Hannibal, who had urged them through the Alps, battled the emergent Roman Republic.

Hannibal, who had travelled from Spain with some fifty thousand men, fifteen thousand horses and mules, and, famously, the elephants, was attempting to deter an attack on Carthage by Rome. His approach from across a protective mountain barrier utterly surprised his enemies, whom he parried up and down the Italian peninsula for nearly fifteen years. After losing many men, his brother, and some of the territory he had won, he returned home in 203 B.C. to defend Carthage, and was defeated the following year. Carthage was ultimately destroyed by Rome half a century later.

The unlikely image of the elephants, those naked mammoths, picking their way through frigid, jagged peaks and along narrow defiles, clearly contributes to an ongoing fascination with Hannibal. Many narrative gaps keep his story alive as well, leaving room for lively, persistent dispute and imaginings. Historians and others have argued about whether the strategic genius studied Greek military maneuvers, what species of elephant he travelled with, and if the pachyderms crossed the Rhône River on rafts or simply waded across it.

Perhaps the most animated controversy centers on the last leg of the surreptitious invasion: the path across the Alps. Over the years, experts have proposed, and sparred over, six principal routes. “The number of publications on Hannibal over the Alps is mind-boggling,” Eve MacDonald, a historian at the University of Reading and the author of “Hannibal: A Hellinistic Life,” said. “Hannibal inspires a Boys’ Own adventure in the modern imagination.”

Last month, a team of scientists published a two-part study suggesting that Hannibal and his ever-dwindling company of soldiers and animals took a southerly course, travelling by way of the Col de la Traversette, a nearly ten-thousand-foot pass. The authors point to geological evidence—some already published—and to new microbiological evidence: genetic fragments from bacteria common to manure. The bacterial bits were found in a layer of possibly hoof-roiled sediment that might date to around 218 B.C., the year of Hannibal’s traverse.

The report appeared in the journal Archaeometry, which has published several Hannibal-in-the-Alps studies in the past decade, mostly by the lead author of the new work, William Mahaney, a physical geographer emeritus at York University, in Toronto. Mahaney has been seeking the Alpine route since 2002, using satellite images and field observations to find geological features or evidence supporting an account by the Greek historian Polybius. About six years ago, Mahaney settled on the Col de la Traversette as the course most consistent with Polybius’ account and expanded his ground-truthing efforts at the favored site.

In 2011 and 2013, members of his twenty-five-person team—which includes geologists, a chemist, and the microbiologist Chris Allen, from Queen’s University Belfast—travelled to the Col de la Traversette, scouting for a spot that could have been a watering hole during Hannibal’s era. They settled on one, took sediments from two cores and a trench, and dated and analyzed them. Fifteen or so inches down, they found a layer of “churned” soil they call the MAD, for “mass animal deposition”—a euphemism for dung.

In that layer, they discovered genetic material from several types of bacteria, including a high concentration of DNA fragments from Clostridia, which is frequently found in feces. Clostridia_ _are pervasive in soil as well, but the team also detected fatty compounds that come from the gut. That combination, Allen said, is what they would expect to see in a place where a great number—hundreds if not thousands—of mammals had defecated. “We have looked at Clostridia as an indicator of an archeological event,” he said, “and I don’t think anyone has tried that before, to my knowledge.”

Aspects of the findings are not convincing to some archaeologists. Andrew Wilson, of the Institute of Archaeology at the University of Oxford, noted in an e-mail that the authors’ date range seems too wide. “This suggests to me that the MAD layer accumulated over a considerable period of time—several centuries—perhaps from animals drinking at the mire, rather than in a single event during the passage of Hannibal’s army,” Wilson wrote.

Mahaney and Allen said that dating churned soils was a challenge, but they are confident that the range of dates they see overlaps with Hannibal’s travels. Allen and others are now determining if the bacteria are indeed from horses—or from humans. They recently found an equine tapeworm egg in the sediments, which they hope will indicate where the horse that dropped it came from. But there’s been no elephant tapeworm, so far. “I am very skeptical that we will find any evidence of elephants,” Allen said. “The thing about elephants is that horses and elephants don’t mix. . . . It could be that those few elephants were nowhere near this mire.” The team is also planning to analyze soil collected last summer for all the animal and plant DNA it can detect, not just for specific microbial genes.

For some observers, the appeal of the new work is not that it may be right about Hannibal but that it reveals the promise of using microbiology and genetics more widely in archeology. “It is exactly where we should be going, using updated scientific methods to answer these ancient questions,” Mark Pallen, a professor of microbial genomics at the University of Warwick, said. The ability to test soil directly for genetic material has extended archeology beyond the quest for the usual biological suspects, such as microscopic fossils. “The whole business of looking at sediments is bubbling up now—it is taking off because of advances in DNA sequencing,” Pallen said. “There is a realization that the environment is full of DNA . . . and you can detect it in sediments even in the absence of fossil remains.”

Pallen and his colleagues recently examined soils submerged off the Isle of Wight some eight thousand years ago, when sea levels rose during the early Holocene, giving rise to the English Channel. They found wheat DNA—present some two thousand years before the grain is known to have been cultivated on the British mainland. The finding, published in Science last year, suggested that an extensive trade and social network between Europe and Britain existed much earlier than historians thought.

The practice of searching soil or other substrates for plant and animal genes—a field known as environmental DNA—grew, in great part, out of experiments by Eske Willerslev, an evolutionary geneticist at the University of Copenhagen and the University of Cambridge. In 2003, he analyzed samples of Siberian permafrost on a hunch that he might find ancient DNA—an experiment he said bemused and amused his colleagues. “And bam! Out comes woolly mammoth and bison. It was just remarkable, really,” Willerslev said. He then tested sediments from a New Zealand cave to see if ancient DNA survived when not frozen—and out came moa. Conservation biologists adopted the method to monitor biodiversity. “Instead of people going out and identifying every single species of plant in an area, you take some environmental samples, and then you just screen it,” Willerslev said. “The approach has completely bloomed in modern settings,” he added. “In the ancient settings, it hasn’t exploded as much.”