Scientists studying the DNA of Richard III, the Plantagenet king whose body was found buried beneath a Leicester car park, have revealed that there was marital infidelity among his aristocratic relatives.

Paternity tests on the DNA of a living male-line descendant of Richard III’s royal lineage have failed to find a match, which points to some royal person being born on the wrong side of the bed sheets, the researchers revealed.

The latest research into the DNA extracted from the 15th century skeleton shows unequivocally that the remains belong to Richard III, who died in the battle of Bosworth in 1485, said Turi King of Leicester University, the lead author of the study published in Nature Communications.

“Even with our highly conservative analysis, the evidence is overwhelming that these are indeed the remains of King Richard III, thereby closing an over 500-year-old missing person’s case,” Dr King said.

Mitochondrial DNA extracted from the skeleton, which is passed on exclusively through the maternal line, matches precisely with two female-line descendants alive today, who are both 14th cousins twice removed from Richard III, Dr King said.

In pictures: The remains of King Richard III Show all 8 1 /8 In pictures: The remains of King Richard III In pictures: The remains of King Richard III King Richard III The Plantagenet Alliance wants the remains to be buried at York Minster, claiming that was the wish 'of the last medieval king of England' PA In pictures: The remains of King Richard III King Richard III A picture shows a scale model showing the design for the tomb that will house the remains of medieval English king Richard III as it is unveiled at a press conference at Leicester Cathedral in Leicester, central England on June 16, 2014. British judges on on May 23 finally ended a bitter debate over the burial of king Richard III, ruling that his remains should be laid to rest at Leicester Caathedral the city where they were found under a car park. AFP PHOTO/PAUL ELLISPAUL ELLIS/AFP/Getty Images In pictures: The remains of King Richard III King Richard III Richard III Society member Philippa Langley and society Chairman Dr Phil Stone stand besides a facial reconstruction of King Richard III in London. After carrying out a series scientific investigations on remains found in a car park in Leicester, the University of Leicester announced that they were those of King Richard III In pictures: The remains of King Richard III King Richard III The first major production of the play since the discovery of the king’s remains will use the new archaeological evidence on the stage In pictures: The remains of King Richard III King Richard III A facial reconstruction of King Richard III is displayed on 5 February 2013 at a news conference in central London. The reconstruction is based on a CT scan of human remains found in a council car park in Leicester which are believed to belong to the last of the Plantagenet monarchs of Britain who was killed at the battle of Bosworth in 1485 Reuters In pictures: The remains of King Richard III King Richard III Students at the University of Leicester were involved in the excavation of Richard III’s remains. On 4 February 2013, scientific tests confirmed that the battle-scarred skeleton with spinal curvature dug up from underneath a council car park was that of the last English king to die in battle. He had been buried five centuries ago but all physical trace had long since been lost Getty Images In pictures: The remains of King Richard III King Richard III Remains found in trench one of the Grey Friars dig University of Leicester/Rex Features In pictures: The remains of King Richard III King Richard III The remains of King Richard III were found in a hastily dug, untidy grave, researchers have revealed University of Leicester

These findings, combined with other circumstantial evidence, give a probability of the skeleton belonging to Richard as 99.9994 per cent, the scientists said.

However, DNA analysis of the Y chromosome of Richard did not match a living descendant of Henry, fifth Duke of Beaufort, who was supposed to be related to Richard through the male line, Dr King said.

“Doing the Y-chromosome research I found there wasn’t a link between Richard III and the male-line relatives who are alive today. So clearly although the genealogy says they are male-line related, the DNA is telling us that there has been a non-paternity or break in that Y-chromosome chain between the 5th Duke of Beaufort, who is their common male-line ancestor, and Richard III, which is 19 generations between them,” she said.

This suggests that the male-line lineage descended from Richard’s great-great grandfather, Edward III, was broken at some point in history, calling into question various claims to the throne – including that of the Tudors, said Kevin Shurer, professor of local history at Leicester University.

“The fact that we have a break means just that: we have a break. It doesn’t mean the skeleton isn’t Richard because what we have is the very strong evidence triangulated with two living-day relatives with the mitochondrial DNA,” Professor Shurer said.

“So that in itself is very strong compelling evidence that the skeleton is Richard III. The break however does raise other questions, more of a historical nature than is this skeleton Richard,” he said.

It is not unusual for find that the male line is broken in DNA cases looking back over several generations. Scientists estimate that the rate of non-paternity is about two per cent. However the discovery could have wider implications.

“Depending on which link in the chain is broken, and of course there is the possibility that there could be more than one break, this asks questions about the Plantagenets and of course the claims to the throne of both the Houses of York and Lancaster,” Professor Shurer said.

“The critical [links in the chain] are between Edward III and his son John of Gaunt. If the break occurs here then this asks questions about the legitimacy of John’s son Henry IV and his subsequent heirs, Henry V and Henry VI,” he said.

“In addition, since the Tudor dynasty was also descended from John of Gaunt there is an indirect influence over the Tudors' claim to the throne,” he added.

The genetic discovery related to King Richard III raises a question mark over the current Queen's royal heritage (PA)

“Likewise if the break is in the part of the chain from Edward III to Richard III this would also ask questions about the legitimacy of the claims of both Richard and of course his brother Edward – but we have absolutely no proof of that,” he emphasised.