Ancient bones discovered under a parking lot have been confirmed as those of the medieval king Richard III, through a DNA test that also raises questions about the legitimacy of Henry VIII and other famous English royals.

The team of genetics detectives reported Tuesday that DNA from the skeleton shows that the bones were Richard III's, with a probability of 99.9994 percent. This is the first genetic identification of a particular individual so long after death—527 years.

Archaeologists had peeled back a parking lot in 2012 to excavate the skeleton, which was among buried relics of the Greyfriars Friary in Leicester, England, long the reputed burial site of Richard III. (See "The Real Richard III.")

Most people know the hunched-shouldered king through Shakespeare's play Richard III, in which the maligned ruler utters such memorable lines as "Now is the winter of our discontent/Made glorious summer by this son of York," and "A horse! A horse! My kingdom for a horse!"

Earlier this year, a forensic study of the remains revealed that the doomed king—the last English monarch to die in combat—suffered 11 wounds at the time of his death, in a 1485 battle with the Tudors that ended England's War of the Roses

But there had been lingering questions about whether the skeleton was really that of Richard III.

"The evidence directly indicates that these are the remains of Richard III," says geneticist Turi King of the University of Leicester in the U.K., who led the team reporting the results in the journal Nature Communications. (Related: "Richard III Mania: Understanding a Kingly Obsession.")

The scientists examined DNA inherited along maternal lines, known as mitochondrial DNA, from two distantly related modern-day relatives of Richard III's sister. That DNA is a near perfect match for the maternal genes of the hunchbacked skeleton buried at the friary. What's more, the DNA was "unusual," King adds, containing stretches that don't quite match anything in registries of European genes.

A statistical analysis led by David Balding and Mark Thomas of University College London took those genetic results and calculated the chances that a man of Richard III's age with battle wounds and a curved spine could turn up at Greyfriars and not be the slain king. They conservatively estimated that chance at 6.7 million to 1.

"It is surprising how many people initially argued that these skeletal remains weren't those of Richard III," says bioanthropologist Piers Mitchell of the U.K.'s University of Cambridge, who was not part of the study team. "Well, here it is."

In 2012 archaeologists peeled back a parking lot to excavate this skeleton, buried among relics of the Greyfriars Friary in Leicester, England. Photograph by University of Leicester

View Images In 2012 archaeologists peeled back a parking lot to excavate this skeleton, buried among relics of the Greyfriars Friary in Leicester, England. Photograph by University of Leicester

Risqué Royalty

However, Richard III's Y chromosome, which is inherited along the paternal lines, seems to have turned up some dirt on ancient aristocrats.

Because Richard III died without leaving any male heirs, the researchers had to trace his lineage back in time to find an ancestor of his who had inherited the same Y chromosome paternally and who had modern-day descendants. They found five men living today who are paternally descended from Richard III's great uncle, John of Gaunt, who died in 1399.

All five of those men should have inherited the same Y chromosome as Richard III through their more recent ancestor, the fifth Duke of Beaufort, who died in 1803. Thus they also should have the same Y chromosome as Richard III. Or so the researchers thought.

Yet none of the men had the same Y chromosome as Richard III, and only four of them had descended from the duke. This isn't too surprising, King says, given estimates of false-paternity rates, meaning "when someone's father is not who we think is their father."

The paternity problems don't shake the statistical probability that the Greyfriars skeleton belongs to Richard III, say the study authors.

But they say the Y chromosome finding "could be of key historical significance." False paternity in John of Gaunt's family could mean that Plantagenet kings such as Henry V had no genetic claim to their thrones. The study states, "This would also hold true, indirectly, for the entire Tudor line," including Elizabeth I and Henry VIII.

Still, the genes can't reveal exactly when the break in paternity occurred. And fortunately for today's royal-watchers, Queen Elizabeth II descended from a different family line.

Portrait of a King

The genes on Richard III's Y chromosome were unusual in English families and are seen more often in the Mediterranean, King notes, though Mark Thomas cautions about ascribing geographic provenances to chromosomes or genes.

Though the study doesn't say anything about the genetic health of Richard III, who was afflicted with scoliosis, it does say there's a 95 percent chance that he had blue eyes and a 77 percent chance that he had brown hair as a child. That closely matches his appearance in a Society of Antiquaries of London portrait from the early 1500s.

When this genetic evidence is added to all the other findings, including the shape of his back and the injuries he sustained in battle, Mitchell says, "now those performing Shakespeare's play about Richard III will have all the evidence they need to make it as authentic as possible."