Wild feral horses have roamed freely across an island off the coast of Maryland and Virginia for hundreds of years, but exactly how they got there has remained a mystery. Now, in a new study, ancient DNA extracted from a 16th century tooth suggest that the old folk tales claiming that horses were marooned on Assateague following a Spanish shipwreck are likely true.

An abandoned Caribbean colony unearthed centuries after it had been forgotten and a case of mistaken identity with the tooth—long thought to be from a cow—have conspired to rewrite the history of that barrier island 1,000 miles away.

Those seemingly unrelated threads were woven together when Nicolas Delsol, a postdoctoral researcher at the Florida Museum of Natural History, set out to analyze ancient DNA recovered from cow bones found in archaeological sites. Delsol wanted to understand how cattle were domesticated in the Americas, and the genetic information preserved in centuries-old teeth held the answer. But they also held a surprise.

"It was a serendipitous finding," he said. "I was sequencing mitochondrial DNA from fossil cow teeth for my Ph.D. and realized something was very different with one of the specimens when I analyzed the sequences."

The specimen in question, a fragment of an adult molar, wasn't a cow tooth at all but instead once belonged to a horse. According to a study published last week in the journal PLOS ONE, the DNA obtained from the tooth is also the oldest ever sequenced for a domesticated horse from the Americas.

The tooth was excavated from one of Spain's first colonized settlements. Located on the island of Hispaniola, the town of Puerto Real was established in 1507 and served for decades as the last port of call for ships sailing from the Caribbean. But rampant piracy and the rise of illegal trade in the 16th century forced the Spanish to consolidate their power elsewhere on the island, and in 1578, residents were ordered to evacuate Puerto Real.

The remnants of the once-bustling port were inadvertently rediscovered by a medical missionary in 1975 and archaeological excavations of the site led by the Florida Museum were carried out until 1990.

Horse fossils are incredibly rare from the time period, primarily due to the way Spanish colonialists valued their livestock.

"Horses were reserved for individuals of high status, and owning one was a sign of prestige," he said. "There are full-page descriptions of horses in the documents that chronicle the arrival of Cortés in Mexico, demonstrating how important they were to the Spanish."

In contrast, cows were used as a source of meat and leather, and their bones were regularly discarded in communal waste piles called middens. But one community's trash is an archaeologist's treasure, as the refuse from middens often confers the clearest glimpse into what people ate and how they lived.

The specimen's biggest surprise wasn't revealed until Delsol compared its DNA with that of modern horses from around the world. Given that the Spanish brought their horses from the Iberian Peninsula in southern Europe, he expected horses still living in that region would be the closest living relatives of the 500-year-old Puerto Real specimen.

Instead, Delsol found its next of kin over 1,000 miles north of Hispaniola, on the island of Assateague off the coast of Maryland and Virginia. Feral horses have roamed freely across the long stretch of barrier island for hundreds of years, but exactly how they got there has remained a mystery.

According to the National Park Service, which manages the northern half of Assateague, the likeliest explanation is that the horses were brought over in the 1600s by English colonists from the mainland in an attempt to evade livestock taxes and fencing laws. Others believe the feral herds descended from horses that survived the shipwreck of a Spanish galleon and swam to shore—a theory popularized in the 1947 children's novel Misty of Chincoteague. The book was later adapted to film, helping spread the shipwreck legend to an even wider audience.

Until now, there has been little evidence to support either theory. Proponents of the shipwreck theory claim it would be unlikely that English colonists would lose track of valuable livestock, while those in favor of an English origin of the herds point to the lack of sunken vessels nearby and the omission of feral horses in historical records of the region.

The results of the DNA analysis, however, unequivocally point to Spanish explorers as being the source of the horses on Assateague.

The feral herds on Assateague weren't the only horses to revert back to their wild heritage after arriving in the Americas. Colonists from all over Europe brought with them horses of various breeds and pedigrees, some of which bucked their bonds and escaped into the surrounding countryside.

Today, the U.S. Bureau of Land Management estimates there are roughly 86,000 wild horses across the country, most of which are located in western states, such as Nevada and Utah. Delsol hopes that future ancient DNA studies will help decode the complex history of equine introductions and migrations that occurred over the last several centuries and offer a clearer understanding of today's diversity of wild and domesticated horses.

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