First there was a massive mastodon tusk, etched with deep grooves. Then there was a knife fragment and a scattering of stone artifacts.

Now researchers excavating an underwater sinkhole in Florida report that they have evidence of one of the oldest archaeological sites in the Americas, a discovery that is helping to shatter long-held assumptions about our continent’s first people.

For decades, archaeologists believed humans first colonized the Americas 13,000 years ago. They called this group the Clovis culture for the distinctive stone tools first discovered in Clovis, New Mexico. Researchers who claimed to have discovered more ancient human artifacts were vigorously rejected.

The Florida sinkhole, known as the Page-Ladson site, was one casualty of this unshakeable “Clovis first” paradigm. Between 1983 and 1997, investigators diving at this nine-metre-deep spot in the Aucilla River discovered a mastodon tusk that appeared to have been butchered. They also found eight stone artifacts and the remains of what might have been a domestic dog. Radiocarbon dating indicated the objects were 14,400 years old, more than a millennia older than Clovis.

The findings were challenged, and the site was dismissed. It “was an impossible age for the scientific community to accept at the time,” said Jessi Halligan, a professor of anthropology at Florida State University.

Yet evidence that people inhabited the Americas before the Clovis culture kept mounting. Recent analysis of ancient and modern indigenous people’s genomes predicted that people came to the New World earlier than 13,000 years ago. A handful of archaeological sites stringing from Chile to Oregon support this, but they are very rare and the evidence from them heavily scrutinized.

“We can come up with genetic dates and models, but it’s important to actually find these sites on the ground,” said Ripan Malhi, a molecular anthropologist at the University of Illinois at Urbana-Champaign involved in the genetic research.

So in 2012, researchers returned to Page-Ladson, located where Florida turns west into its panhandle. “Our main research question was very simple: is there an older-than-Clovis cultural layer at this site?” said Halligan, co-principal investigator and lead diver for the project. “These excavations were successful beyond our dreams.”

Over three seasons, the archaeologists carefully excavated the site anew, using laser levels, vacuum suction tools and other techniques to leave little room for error at the difficult site. Most underwater archaeology is focused on shipwrecks. The excavation of a river sinkhole posed unusual challenges.

“It’s unique in the sense that it is essentially a terrestrial or dry land dig, but underwater,” said Brendan Fenerty, a Canadian researcher who was part of the dive team and is now a geosciences doctoral student at the University of Arizona. They set up a typical excavation grid at the bottom of the river, where almost no sunlight penetrated because of the organic material in the water. “You can imagine how long it can take, excavating an area centimeter by centimeter underwater.”

As the team sampled successive layers of sediments, they found new stone tools, including a knife, and mastodon dung. But the real work came in the lab. Because the stakes for a pre-Clovis archaeological site are so high, they needed a rock-solid “geological context:” radiocarbon dates for the sediment levels that sat in a neat chronology, not mixed by turbulent waters or any other natural event over thousands of years.

In a paper in the journal Science Advances published online Friday, the researchers reported their findings: human artifacts from the Page-Ladson site sit in a sediment layer that dates to 14,550 years old. That makes it the oldest unambiguous archaeological site in the southeastern U.S., and the oldest underwater site anywhere in the New World. The date of the site is also nearly the same as that of another pre-Clovis site in Chile known as Monte Verde, suggesting both the northern and southern hemispheres of the continent were populated by this very early date.

“The record of human habitation in the Americas between 14,000 and 15,000 years ago is sparse, but it is real,” said Michael Waters, director of the Center for the Study of the First Americans at Texas A&M University and co-principal investigator of the project.

Beyond the simple fact of its very ancient date, the Page-Ladson site holds interesting clues about the life of these first nations. The cut mastodon tusk suggests that people and megafauna co-existed for perhaps 2,000 years, adding kindling to a raging debate about whether human hunting drove to extinction the giant animals of this period, including mammoths, mastodons, and giant sloths, beavers and camels.

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The presence of what may be a domestic dog is also unusual, and more research will be conducted on the bones: the team hopes to extract ancient DNA from the remains and analyze it.

“It’s an extremely exciting period of time right now, with more information coming available every year. And this is just really solid work,” said Dennis Jenkins, a University of Oregon archaeologist involved in the research on the Paisley Caves, another pre-Clovis site.

The researchers believe more archaeological sites with evidence of pre-Clovis people may be lying underwater: sea levels were much lower in this period. The Page-Ladson sinkhole, for example, would have been a pond surrounded by dry grasslands at the time.

“I think this is hopefully going be the impetus to get further research done on these underwater archaeological sites,” said Fenerty. “These are unparalleled opportunities to really explore change through time, not just in people’s behaviours and adaptations, but we can actually tackle the fundamental issues: when did people get here?”