Experts are almost 100 per cent sure that the skeleton with a twisted spine found in a Leicester car park in 2012 is that of the last Plantagenet king.
Now new research has found a chink in the Tudor ancestry of Queen Elizabeth II whose right to the throne can be traced all the way back to King Henry VII, via James I and Mary Queen of Scots.
Previous DNA analysis had determined two female-line relatives of King Richard III still living and five other male-line relatives that have little royal significance.
But new evidence released today shows a break in the male 'Y chromosome' line - a newly discovered illegitimacy - which brings into question the entire history of the British monarchy since the reign of Henry IV.
The research questions the historic legitimacy concerning the descent of Edward III to his son John of Gaunt and also his two grandsons, John Beaufort, Earl of Somerset and Henry IV, the first Lancastrian King.
It centres around John of Gaunt, who was Tudor King Henry VII's great great grandfather and ancestor of the Queen.
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Richard III was connected to these lineages through his great grandfather Edmund, Duke of York - John of Gaunt’s brother.
Prof Schurer, pro-vice chancellor of the University of Leicester, said: “We don’t know where the break is, but if there’s one particular link that has more significance than any other, it has to be the link between Edward III and his son John of Gaunt.
“John of Gaunt was the father of Henry IV, so if John of Gaunt was not actually the child of Edward III, arguably Henry IV had no legitimate right to the throne, and therefore neither did Henry V, Henry VI, and, indirectly, the Tudors.”
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Writing in the journal Nature Communications, the scientists said the claim to the crown of the “entire Tudor dynasty” partly rested on its members’ descent from John of Gaunt.
They added: “The claim of the Tudor dynasty would also be brought into question if the false paternity occurred between John of Gaunt and his son, John Beaufort, Earl of Somerset.”
Richard III was killed at the Battle of Bosworth in 1485, the last significant clash between the forces of the Houses of Lancaster and York in the War of the Roses.
Tudor and Plantagenet royal family tree
According to historical records he was buried in Grey Friars Church, Leicester, which once stood on the site of the car park where his bones were found.
Examination of the skeleton showed that it had a twisted spine rather than the hunchback for which Richard III was famous. Although he would have walked with one shoulder higher than the other, his deformity could easily have been concealed beneath clothing and armour.
The genetic analysis showed a 96 per cent probability that Richard had blue eyes and a 77 per cent likelihood that he was blond, at least in childhood. It was possible that his hair colour may have darkened with age, said the scientists.
His appearance was probably similar to that depicted in an early portrait held by the Society of Antiquaries in London.
In their paper, the researchers compared the investigation to a missing person case that becomes more difficult over time - in this case, 527 years.
Geneticist Dr Turi King, from the University of Leicester, said: “What we have concluded is that there is, at its most conservative, a 99.999 per cent probability that these are indeed the remains of Richard III. The evidence is overwhelming.