Shot by the Bolsheviks: A photograph of the Romanov family captured in 1913, five years before their death. Tsar Nicholas II and his wife Alexandra are in the middle, with their son Alexei. The four girls are (left to right) Maria, Tatiana, Olga and Anastasia.
BRISBANE: Forensic DNA testing has finally confirmed that bodies found in a field near Yerkaterinburg, Russia, are two of the missing children of Russia's last Tsar, killed following the 1917 revolution.
The bodies are almost certainly the crown prince, Alexei Romanov, and one of his sisters, according to a report today in the journal PLoS ONE.
The discovery puts to rest the persistent rumour that one of the royal children escaped the execution of Tsar Nicholas II and his family by a Bolshevik firing squad in 1918. Since that time, more then 200 people have claimed to be one of the Romanov children.
"None of the family survived the execution"
"Here we are able to give a full account of all of the Romanov family and can conclude that none of the family survived the execution," the authors said.
Human remains from a mass grave outside Yerkaterinburg were shown to be those of the Tsar, his wife Alexandra, and three of their five children in a 1994 Nature Genetics paper.
At the time, the authors, from Britain's Forensic Science Service and the Engelhardt Institute of Moscow, concluded that the missing remains were likely to be the prince and one of his sisters.
The new material – teeth and bone fragments from a male and a female, discovered 70 m from the original gravesite – was tested with forensic DNA techniques by two independent laboratories, the Armed Forces DNA Identification Laboratory (AFDIL) in Rockville, U.S., and the Institute of Legal Medicine in Innsbruck, Austria.
Blood-stained shirt
The DNA tests confirmed that the new bodies belong to people closely related to those found in the larger mass grave, and have also linked the family to living relatives of both the Tsar and his wife, said lead author and AFDIL geneticist Mike Coble.
The researchers were also able to now prove that one body from the mass grave is definitely Tsar Nicholas II, Coble said, by comparing DNA from the bones to DNA from a shirt that was stained with the Tsar's blood during an assassination attempt in 1891.
The researchers took a three-pronged approach to the DNA analysis by examining the autosomal DNA, mitochondrial DNA (mtDNA), and Y-chromosome DNA of the new remains. Autosomal DNA is found in the regular chromosomes in the cell's nucleus, and is inherited from both the mother and the father.
Autosomal and mtDNA from the mass grave's bodies had been tested for the 1994 paper, Coble said, but this was the first time the Y-chromosome DNA had ever been examined.
The researchers linked the two new bodies to the Tsar's family by examining 16 highly-variable sections of DNA called short tandem repeats (STR) in the autosomal DNA extracted from each body, Coble said.
Every person's STR profile is a mix of their parent's profiles, allowing the technique to be used for paternity testing. The STR profiles of the two bodies were a mix of the profiles from the Tsar and Tsarina's remains, proving that the new bodies are their children, Coble said.
Mitochondrial DNA is inherited only from the mother, Coble said. The mtDNA of the new bodies matched that from Alexandra's body, and also matched the mtDNA of a living relative, Prince Philip (the U.K.'s Duke of Edinburgh), whose maternal grandmother, Victoria of Hesse, was Alexandra's sister and shared her mtDNA sequence.
Descendent in the male line
The Y-chromosome is inherited only along a father's line. The researchers found that the male body's Y-STR profile was a perfect match for both the Tsar and another living relative, Prince Andrew Andreeivich Romanov, who is descended in the male line from Tsar's paternal great-grandfather, Tsar Nicholas I.
The DNA in the remains from both graves was unusually well-preserved, Coble said, probably because the ground at Yerkaterinburg is normally frozen from September to April every year.
However, the researchers are still unable to prove which of the daughters was in the second grave, because they have no reference DNA like the Tsar's bloodstained shirt for the children.
Angela van Daal, a forensics professor at Bond University on the Gold Coast, Australia, commented that the research made great use of forensic techniques that are used in modern-day cases. "These are [now] very standard techniques," she said. "It's nice work."
