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It's the coldest case in American history.

The settlers who inhabited the 16th century North Carolina colony of Roanoke mysteriously disappeared centuries ago, leaving behind only two clues: the words "Croatoan" and "Cro" carved into a fort's gatepost and a nearby tree.

Many conspiracy theories have been concocted as to what happened in 1590, a mere three years after the colonists arrived in North America, but none have proven fruitful. Until now. Technological advances and the discovery of a cover-up on an ancient map have let researchers unearth new clues that may help bring an end to the mystery of America's lost colony.

Researchers began reinvestigating the mysterious disappearance after they noticed two strange patches on a long-forgotten map of the area called "La Virginea Pars" drawn by the colony's governor John White. Researchers at the First Colony Foundation in Durham, N.C., believed the two patches might be covering up something revealing.

The map was analyzed by scientists at the British Museum, who discovered a small red-and-blue symbol.

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"Our best idea is that parts of [Sir Walter] Raleigh's exploration in North America were a state secret, and the map 'cover-up' was an effort to keep information from the public and from foreign agents," historian and principal investigator Eric Klingelhofer of Mercer University in Macon, Ga., told National Geographic, which partially funded the effort.

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Historians believe that the symbol may have been the location of a fort the settlers fled to, running from violence or disease.

"It's obvious that that's the only way they could have survived. No single Indian tribe or village could have supported them ... They were over a hundred people," Klingelhofer said.

The current theory is that the colonists fled 50 miles south to Hatteras Island, then known as Croatoan Island. Klingelhofer suggests they may have gone in a different direction.

He believes the settlers traveled west via the Albermarle Sound to the Chowan River where there might have been a protected inlet occupied by a friendly tribe.

"It's a very strategic place, right at the end of Albemarle Sound," he said. "You can go north up the Chowan River to Virginia or west to the Blue Ridge Mountains. They were big trading partners with other Native American tribes."

Once the researchers uncovered the secrets of the "La Virginea Pars" map, they scheduled a trip to visit the area along with the help of magnetometers and ground-penetrating radar (GPR).

"What we do is we get the oldest maps we can find—so we can get a historic sense of what was there and what's there now—and orient them," said research associate Malcolm LeCompte at Elizabeth City State University in North Carolina, who was responsible for the GPR.

He looked for similarities between the old map and the current topography. The researchers than used GPR, which sends radio waves into the ground and measures the echo of the signals that bounce off of objects underground.

LeCompte and his team found a previously undiscovered pattern that indicated the possibility of multiple wooden structures approximately 3 feet underground.

"I don't know if it's one or a group [of structures]," he said, adding that they "could be joined or they could be close together."

The mere presence of the buried structure indicates that there was a colonial presence in the area. However, while the new information has begun to give archaeologists a clearer view as to what might have happened to the Roanoke colony, there are still pieces to the puzzle that remain unfound. What's the next step in solving this age-old mystery?

"We have to go in and dig some holes, I guess," Swindell said.