He is one of the nation’s most notorious monarchs and Richard III is still creating controversy more than 500 years after his death.

Genetic analysis of a battle-scarred skeleton - discovered under a council car park in Leicester three years ago - has confirmed that it did indeed belong to the last Plantagenet king.

But it may have also exposed skeletons in the closet of the British aristocracy - undermining the Tudor dynasty - and could even raise a question mark over the current Queen’s royal heritage.

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Researchers considered genetic material from two female-line descendants of Richard's sister Anne of York - a woman named Wendy Duldig and a man named Michael Ibsen (both pictured)

Down the line: An international team of researchers, led by Leicester University, pieced together sections Richard III’s family tree, right down to relatives who are alive today (pictured)

DNA analysis has revealed a break in the male line of Richard’s family tree. It means one of Richard III’s male relatives was cuckolded – leading to his wife to give birth to another man’s child.

Researchers also considered genetic material from two female-line descendants of Richard's sister Anne of York - a man named Michael Ibsen and a woman, Wendy Duldig.

They were able to confirm that Ms Duldig is a niece of Richard III, 18 times removed, while Michael Ibsen is Richard III's nephew, 16 times removed.

Depending on who was unfaithful in the family tree, it could have far-reaching consequences. The discovery was made by international researchers trying to prove the skeleton really was Richard III.

They pieced together sections Richard III’s family tree, right down to Ms Duldig and Mr Ibsen. The pair are 14th cousins twice removed, and their last common ancestor lived in the 16th century.

Genetic analysis of a battle-scarred skeleton (pictured) discovered under a council car park in Leicester three years ago has confirmed it did indeed belong to the last Plantagenet king

DNA analysis revealed that one of Richard III’s male relatives was cuckolded - leading to his wife giving birth to another man’s child. The king's skeleton, which was discovered under a car park, is shown

Connection: Researchers found a link between Richard III and Michael Ibsen (left) and Wendy Duldig (right)

The team - led by Leicester University - then compared the DNA of these living relations with genetic material extracted from the 15th century king’s teeth and bones.

Their analysis shows that DNA passed down on the maternal side matches that of living relatives, but genetic information passed down on the male side does not.

Given the wealth of other details linking the body to Richard III, the scientists conclude that infidelity is the most likely explanation. It isn’t clear where in the family tree it occurred.

But if it occurred close to the top of the tree, near Richard III, it could be of ‘key historical significance regarding royal succession’.

Of particular interest is the link between Edward III and his son John of Gaunt. If John of Gaunt was actually another man’s son, the Tudors’ right to the throne is threatened.

Analysis of the Y chromosome, which is passed down the generations from father to son, revealed a break in the male lineage (pictured). It isn’t clear where in the family tree it occurred

Analysis showed Richard III almost certainly had blue eyes and blond hair when he was young and it got darker as he grew older. This portrait shows the king with light brown hair and blue eyes

This is because Henry VII, the founder of the Tudor dynasty, claimed that his royal blood came from John of Gaunt. Henry’s mother, Margaret Beaufort, was John of Gaunt’s great-granddaughter.

WHAT DID RICHARD III LOOK LIKE? The genetic analysis showed that Richard III almost certainly had blue eyes and likely had blonde hair - although it may have darkened with age. Richard III was depicted by William Shakespeare as a tyrannical hunchback who murdered two princes in the Tower of London. But scans of the king's skeleton show he wasn't a hunchback, but only had a slight deformity that would have barely affected his appearance or prowess on the field of battle. The notorious description by Shakespeare of a ‘hunchback toad’, a Machiavellian villain suffering a twisted body, a limp and withered arm, was almost certainly an attempt to blacken his reputation, experts claim. Instead, a 3D reconstruction of the king’s spine shows 65 to 85 degrees of ‘scoliosis’, or sideways bending of his spine to the right. The condition, which would have developed in his early teens, means he was very far from being a hunchback. Despite having one shoulder slightly higher than the other and a short trunk in comparison with his arms and legs, there is no evidence he walked with a limp. Advertisement

If John of Gaunt was not Edward III’s son, and so did not have royal blood, the ‘claim of the Tudor dynasty would be brought into question’, the researchers said.

This is important because the current Queen can her ancestry back to Henry VII, via James I and Mary, Queen of Scots.

The same break in the chain would threaten the claims of the Lancastrian kings Henry IV, Henry V and Henry VI, while a different break in the chain would mean that Richard III’s own claim on the crown would be illegitimate, the journal Nature Communications reports.

Professor Schürer said that while ‘some may wish to’ use his results to speculate on the Queen’s right to rule, it wasn’t for him to do so.

He said that royal succession takes ‘many twists and turns’ and added that Henry Tudor took the throne by force – by defeating Richard III at the Battle of Bosworth in 1485.

The professor said that it is also much more likely that the break in the chain occurred lower down the line – and so would have no effect on royal succession.

He added: ‘This study has closed one door but its opened up another interesting one to which we don’t necessarily have the answers at this particular point.

‘There will be a lot of interest in trying to identify who the prime suspect is. All of the men here are suspects. In fact, it’s not the men it’s their wives who we are talking about. These things happen.

Skull: Samples of mitochondrial DNA - a type of genetic material passed through the female line - proved to be a match to Richard III’s eldest sister Anne of York

DNA analysis of the descendants of Henry Somerset, 5th Duke of Beaufort has suggested that at some point between Edward III, who died in 1377, and the 5th Duke of Beaufort, who died in 1803, there was a break in the male lineage as the wives of one of the men listed above were unfaithful to them. If that occurred before Edmund, the 2nd Duke of Somerset, who was born in 1406, then it could call in to question the Tudors claim to the throne, and so the current Royal family whose lineage is traced back to the Tudors through Henry VI. However, the break could also have occurred on the York side of the family tree and so Richard III's claim could also have been illegitimate

Professor Kevin Schürer from the University of Leicester said that while ‘some may wish to’ use his results to speculate on the Queen’s (pictured) right to rule, it wasn’t for him to do so

Dr Steven Gunn, an Oxford University historian and leading expert on the Tudors, said that there is a lot more to being accepted as monarch than simple bloodline and the research is unlikely to have any implications for the House of Windsor.

He added that it is possible that the cuckolded man knew his son wasn’t his own but kept it secret out of shame. Having a son to pass land down to is also important in aristocrats’ eyes.

The analysis also showed that Richard III almost certainly had blue eyes and likely had blond hair – although it may have darkened with age.

When the genetic analysis is combined with other information, including the skeleton’s battle scars, twisted spine and age, there is at least a 99.999 per cent probability that it belonged to Richard III.

RESEARCH SHOWS KING RICHARD MAY HAVE HAD MIDLANDS ACCENT King Richard III would have sounded more like a Brummie than a northerner, according to a language expert. Dr Philip Shaw, from the University of Leicester's School of English, used two letters penned by the last king of the Plantagenet line more than 500 years ago to try to piece together what the monarch would have sounded like. He studied the king's use of grammar and spelling in postscripts on the letters. Despite being the patriarch of the House of York, the king's accent 'could probably associate more or less with the West Midlands' than from Yorkshire or the North of England, said Dr Shaw. 'But that's an accent you might well see in London - an educated London accent,' he said. 'Possibly even a northern one but there are no northern symptoms, so there's nothing to suggest a Yorkshire accent in the way that he writes, I'm sorry to say for anyone who associates him with Yorkshire.' The first letter was written in 1469 before Richard became king - and well before his death at the Battle of Bosworth Field in 1485 - and is an urgent request for a £100 loan, while he rode to put down a rising in Yorkshire. The second letter from 1483 was written following his ascent to the throne, penned during a rebellion by the Duke of Buckingham. Advertisement

Of particular interest is the link between Edward III and his son John of Gaunt (illustrated). If John of Gaunt was actually another man’s son, the Tudors’ right to the throne is threatened

Richard III was depicted by William Shakespeare as a tyrannical hunchback who murdered two princes in the Tower of London.

He ruled for just two years before being killed at the Battle of Bosworth – ending the Wars of the Roses and the Plantagenet dynasty.

His naked body was paraded through Leicester and dumped in shallow grave where it remained for more than 500 years.

Simon Chaplin, director of culture and society at the Wellcome Trust, which co-funded the research, said: 'Adding this information to a wealth of existing material about Richard III further highlights the ways in which studying human remains can inform our understanding of the past, and we look forward to learning more about Richard for many years to come.'