In one sense, it was case closed, according to researchers, who on Tuesday published “overwhelming” evidence that they had indeed found the remains of King Richard III under a municipal parking lot. But in solving that mystery, they opened up another. When they compared Richard’s DNA with that of five living relatives on his paternal side, it did not match, indicating that one of his kin had been cuckolded somewhere along the line. Depending on when this false-paternity event occurred, certain English monarchs, including Richard III himself, may not have had a valid claim to the throne.

After only two years in power, King Richard III of England was killed in 1485 at the Battle of Bosworth Field, when a force loyal to his rival, Henry Tudor (the future Henry VII), crushed his reckless cavalry charge. Richard’s death at age 32 marked the end of the 330-year-old Plantagenet dynasty and the beginning of the reign of the Tudors, who vilified him as brutal and corrupt. (William Shakespeare cemented his tyrannical reputation with the play Richard III, known for such lines as “A horse! A horse! My kingdom for a horse!”) According to historical records, Richard was interred in the medieval church of the Grey Friars, located in Leicester, a city about 100 miles north of London. But in 1538, Tudor King Henry VIII ordered the friary dissolved, and its buildings were torn down. Thus, Richard became one of the few English monarchs with an unknown burial place.

Centuries passed without him popping up. Nonetheless, clues abounded about the friary’s location, such as the existence of a Leicester street named Friar Lane, and in 2012 researchers launched an effort to solve what they described as a “missing person’s case.” Despite having only enough funding to excavate a tiny percentage of the site, they were able to locate the friary’s church in a most unregal location: underneath a municipal parking lot. They then continued digging and within days had exhumed two skeletons, one of them male, age 30-34, with severe scoliosis and numerous battle injuries. Radiocarbon dating placed the time of death at between 1456 and 1530, and additional tests showed that he ate like royalty, with a diet of swan, heron and fish, not to mention copious amounts of alcohol. Based on these non-genetic results, plus preliminary DNA testing, the researchers announced in February 2013 that the remains belonged to Richard “beyond reasonable doubt.”

More DNA testing followed, culminating in a paper published Tuesday in the journal Nature Communications, which claimed that there is over a 99.9994 percent probability that the bones are Richard’s. “The chances it’s not him are less than 1 in 100,000,” Turi King, a genetics expert at the University of Leicester, who led the international research team, said via Skype. She and her colleague Kevin Schürer, also at the University of Leicester, recalled doing a celebratory dance upon first realizing the extent of their discovery. They even determined that Richard almost certainly had blue eyes and that he likely had blond hair as well, at least in childhood. This more or less matches up with one of the earliest known paintings of him, completed around 1510, which currently hangs in the Society of Antiquaries of London.

Richard III left no heirs. But the researchers were able to compare DNA extracted from the teeth and femur of his supposed remains with that of two living relatives on his maternal side, who descended from his sister, Anne of York. The DNA matched up almost perfectly, and the two distantly related descendants, a Canadian and an Australian, both of whom now live in London, have since become friends. “It’s very rare that you have a chance to put 14th cousins, twice removed, together,” Schürer laughed. The researchers also compared Richard’s DNA with that of five living genealogical relatives on his paternal side. This time, however, there was no match at all, signifying the likely presence of at least one cuckolded husband somewhere between King Edward III, who lived from 1312 to 1377, and the 5th Duke of Beaufort, who lived from 1744 to 1803—a span of 19 generations.

A false-paternity event, in which a man thinks a genetically unrelated child is his own, in 13 of those 19 generations would not affect royal descent in any way. But if it occurred during the other six, then there could be implications for the historical monarchy. For example, if John of Gaunt, a son of Edward III, was illegitimate, then his son Henry IV would have no right to the throne, nor would his direct descendants Henry V and Henry VI. The Tudors would also be affected, since their claim to power relied partly on their descent from John of Gaunt. Meanwhile, if Edmund of Langley, another of Edward III’s sons, was illegitimate, then Richard III would have no right to the throne.

The researchers expressed no surprise at these results, pointing out that false-paternity events occur relatively commonly among both royals and the general populace. In fact, Richard III became king only by claiming that his nephews were illegitimate. As of now, no one knows where along his family tree the false-paternity event, or events, occurred. Schürer asserted that it would be worthwhile investigating further, but that this would probably require the exhumation of more bodies. “Of course, you can’t just go around digging people up,” he said. “It’s fairly unlikely that we would be given permission.”