“I will tell them that the play is over.”
—Helen Hayes as Maria Feodorovna in the 1956 movie Anastasia

It is a mystery of the twentieth century that captured the imaginations of writers, readers and movie goers. It is a mystery that generated over 200 claims of false identity. It is a mystery that inspired an Oscar-winning performance on the silver screen.

Did any of the children of Czar Nicolas II, the last imperial ruler of Russia, escape execution by the Bolshevik Secret Police?

Twenty-first century science has provided the final curtain call for this story, and the play is, indeed, over.

In a paper published in the Proceedings of the National Academy of Sciences, USA , scientific collaborators from the United States, Russia and Canada have “established beyond reasonable doubt” that remains found in two graves in the Ural region of Russia are those of the last Russian Emperor and his family.

In 1917 Nicholas II abdicated his throne, and he and his wife, Alexandra, and four children were placed under house arrest during the Russian Revolution. On July 17, 1918, the family was executed by a Bolshevik firing squad led by Yakov Yurovsky. To prevent identification of the remains, the bodies were burned and may have been doused with sulfuric acid.

The first grave site was discovered in 1991, and forensic studies indicated that the remains were those of Nicholas II, his wife and three of the daughters. In 2007, a second grave site was discovered that contained bone fragments of two burned skeletons: one of a boy and one of a young woman.

In the work published in the 2008 PNAS paper, Evgeny I. Rogaev and colleagues analyzed mitochondrial and nuclear DNA sequences amplified from the charred bones in the graves and from a blood-stained shirt of Nicholas II, which had been preserved at the Hermitage Museum in St. Petersburg. (The shirt was a family relic from an 1891 assassination attempt that Nicholas II had survived.) They also analyzed DNA from individuals descended through the maternal and paternal lineages of the European royal families.

Mitochondria are organelles within cells that are responsible for energy production. Mitochondria contain their own DNA molecules, and they are inherited from the mother. In this study the researchers were able to obtain complete mitochondrial (cmt) DNA sequences from the charred bone fragments and from living relatives from two branches of the maternal lineage of Queen Victoria. (Queen Victoria was the grandmother of Tsarina Alexandra.) Their analyses showed a perfect match between the bone fragments from the female remains in the second grave, the fragments in the first grave, which were believed to be those of Tasrina Alexandra, and the two living descendants.

The researchers also were able to reconstruct the cmt DNA from the remains believed to be those of Czar Nicholas II, and they were able to match that sequence to a reference sample from a maternal descendant of Empress Maria Feodorovna, the mother of Nicholas II. Futhermore, DNA from the blood-stained shirt of Nicholas II provided both mitochondrial and Y-chromosome matches to the putative remains of Nicholas II in the first grave. The chances that DNA from the bone sample belongs to Nicholas II rather than an unrelated individual are on the order of sextillions (>10²³) to one.

To identify the bone fragments from the male child in the second grave, researchers turned to analysis of Y chromosome sequences. There are several living descendants from paternal lineages of Nicholas I who would share the Y haplotype with Nicholas II and Prince Alexei. DNA from the bone fragments from the first grave believed to be those of Nicholas II and the second grave believed to be those of Alexei matched that of the living relatives of Nicholas I.

Additionally, analyses of multiple short tandem repeats (STRs), indicate that the bone fragments found in the second grave are not identical to, but show kinship with, the remains found in the first grave. When all of the data are considered together, the genotyping indicates that the remains in the two graves are from one biological family (father, mother, four daughters and a son), and that the father is Nicholas II and the mother is Alexandra.

Therefore, the scientists conclude that no one from the family of Nicholas II Romanov survived the massacre on July 17, 1918.

Rogaev, E., Grigorenko, A., Moliaka, Y., Faskhutdinova, G., Goltsov, A., Lahti, A., Hildebrandt, C., Kittler, E., & Morozova, I. (2009). Genomic identification in the historical case of the Nicholas II royal family Proceedings of the National Academy of Sciences, 106 (13), 5258-5263 DOI: 10.1073/pnas.0811190106