The Romanovs: The Final Chapter (16 page)

Because of the age and deteriorated condition of the Romanov bones, Dr. Gill and Dr. Ivanov faced a task radically more difficult than in any previous DNA typing examination. In a sterile environment, they began by grinding away one millimeter of the contaminated outer surfaces of the bones with sand wheels attached to a high-speed electric drill. The remaining bone was frozen in liquid nitrogen, then pulverized to a fine powder and dissolved in various solutions, then centrifuged to release a microscopic quantity of DNA. So paltry and degraded, in fact, were the sample yields that Gill and Ivanov applied an even more recently developed technique called PCR (polymerase chain reaction), in which selected relevant sections of base pair strands are chemically duplicated over and over in a test tube to provide sufficient quantities of DNA material for scientists to study.

Using nuclear DNA, the Aldermaston team first turned to determining the sex of each of the skeletons. A gene on the X chromosome (females have two X’s) is six base pairs longer than the similar gene on the Y (males have one X and one Y) chromosome. Using PCR, the scientists could obtain sufficient material to measure and determine this
six-base-pair difference. The result was a confirmation of the anthropological findings of Abramov and Maples: there were four males and five females. Next, still using nuclear DNA and studying base pair sequences, Gill and Ivanov tested all nine for a family relationship. Short tandem repeat (called STR.) sequences are natural base pair repetitions in certain hypervariable regions of a chromosome—say, T, A, T, T—occurring over and over again. Within a family, these sequences and the number of repetitions tend to be constant; a different sequence or different number of repetitions in each individual sample would indicate that no family group was present. Again, the results were what was to be expected if the bones had come from the Imperial party. In Gill’s words: “Skeletons 3 through 7 exhibited patterns which would be expected in a family group where 4 and 7 were the parents of children 3, 5, and 6.” The other four adults were excluded as possible parents. Further, Gill’s report continued, “If these remains are the Romanovs then … test data indicated that one of the daughters and the Tsarevich Alexis were missing from the grave.” Other tests established paternity. STR DNA patterns from Body No. 4 were found in No. 3, No. 5, and No. 6; thus, the adult male presumed to be Nicholas was confirmed to be the father of the three young women. This was as far as Gill and Ivanov could go using the small quantity of degraded nuclear DNA available. They had established a party of four males and five females. They had established a family: a father, a mother, their three daughters. But to identify these men and women—to give them names—they had to try another tack.

Fortunately, a second form of DNA is available in human cells. Called mitochondrial DNA, it appears plentifully in units outside the nucleus which function as power stations for the cell. Mitochondrial DNA is inherited independently of nuclear DNA, and whereas nuclear DNA is inherited half from the mother, half from the father, mitochondrial DNA is inherited exclusively from the mother. From mother to daughter, it is transmitted intact, “passing from generation to generation unchanging, like a time machine,” said Gill. “The same genetic code would be shared by mother, grandmother, great-grandmother, great-great-grandmother, and so on.” At all points in this chain, sons possess mitochondrial DNA received from their
mothers, but sons cannot pass this mitochondrial DNA along to their daughters or sons. Thus, as a tool for establishing identity, mitochondrial DNA can be used to identify a woman anywhere in a vertical chain of women descended from one another. And it can identify a son of one of these women. But it cannot continue through the male line; with sons the chain is broken.

Gill and Ivanov extracted mitochondrial DNA from the nine bone samples brought from Russia. The extracts were amplified to workable quantities by using PCR. To their delight, the quality of the DNA sequences obtained, said Gill, was “generally comparable to that produced from fresh blood samples.” Focusing on two different stretches of the DNA sequence normally hypervariable between different humans, and deriving between 634 and 782 base pair letters for each of the nine subjects, the scientists achieved DNA profiles for all nine of the bone samples they possessed.

Next, they needed contemporary DNA to make comparisons. The search for living relatives began. People at the FSS and the Home Office drew books from libraries and pored over genealogical trees. Someone drew up a list of names of people who would be scientifically suitable and might be approached. In the case of the Empress Alexandra, finding a genetically useful living relative was easy. Alexandra’s older sister, Princess Victoria of Battenberg, had a daughter, who became Princess Alice of Greece. Princess Alice, in turn, produced four daughters and a son. In 1993, only one of these daughters, Princess Sophie of Hanover, was living. The son was Prince Philip, who became Duke of Edinburgh and the consort of Queen Elizabeth II of England. Prince Philip, Empress Alexandra’s grandnephew, was perfectly suited for a mitochondrial DNA comparison with bone material of the murdered Russian empress. Accordingly, Dr. Thompson, director of the FSS, wrote to Buckingham Palace and asked whether the prince would be willing to help. Philip agreed, and a test tube filled with his blood soon made its way to Aldermaston. The testing was done in those parts of the mitochondrial DNA sequence where the greatest variety between family groups occurs. By November, Gill and Ivanov had results: the match was perfect; the sequence of DNA base pairs between the mother, the three
young women, and Prince Philip was identical. Gill and Ivanov knew that they had located the remains of Alexandra Feodorovna and three of her four daughters.

Confirming the presence of Tsar Nicholas II was far more difficult. The search for DNA material to compare with that extracted from the femur of Body No. 4 was widespread, prolonged, and, in several instances, controversial. The first attempts were made by Pavel Ivanov. It occurred to him that Nicholas II’s younger brother Grand Duke George, who died in 1899 of tuberculosis at the age of twenty-eight, was buried in the mausoleum of the Romanovs, the Cathedral of St. Peter and St. Paul in St. Petersburg. Comparison of DNA between brothers would nicely suffice. From England, Ivanov contacted Anatoly Sobchak, the mayor of St. Petersburg, and Vladimir Soloviev, who would become the investigator assigned to the Romanov case. “They protested that it would be too expensive,” Ivanov recalled. “ ‘The tombs in the fortress are made of Italian marble.… You must break it.… Who will pay for this?’ And so on.” For eight months, Ivanov persisted, and, at one point, Mstislav Rostropovich, the cellist and conductor, who is a friend of Sobchak, seemed about to pay for the exhumation of Grand Duke George.

Before this happened, however, Rostropovich told Ivanov that he was about to set out on a visit to Japan. Ivanov, still in England, remembered that in 1892 Nicholas II as tsarevich had visited Japan. In Otsu, the heir to the Russian throne suddenly had been attacked by a sword-wielding Japanese. The blow, aimed at his head, glanced off his forehead, bringing a gush of blood but failing to bite deep. The wound was bound with a handkerchief. For one hundred years, a museum in Otsu had kept the blood-soaked handkerchief in a small box. For DNA comparison purposes, nothing could provide more accurate positive identity than achieving a match between bone material of unknown origin and blood from a known person. Ivanov was eager to go to Japan, but, as always, “there was no money. The English said, Why should we pay for this? The Russians said, We have no money.” Eventually, Rostropovich arranged for Ivanov’s trip. “It was the money we were going to use to dig up George,” Ivanov said. “So, instead of George, we did Japan.”

The Japanese were not anxious to give up or even to disturb the handkerchief, but Rostropovich spoke to his friend the emperor of Japan, and the emperor spoke to the relevant authorities. When Ivanov arrived he was permitted to remove and take with him a strip of the handkerchief three inches long and one eighth of an inch wide. Unfortunately, back in Gill’s laboratory in England, Ivanov ran into difficulties. “The handkerchief had been handled by too many people,” he said. “Cells slough off from fingers. There was a lot of blood on the handkerchief, but who knows how much of it was Nicholas’s? And there was a lot of dust and dirt. It would be impossible to say that any result you got from that handkerchief was reliable. There were too many other possible contaminants.”

Having failed with both George and Japan, Ivanov came up with a third possible source of DNA for comparison to the piece of the presumed tsar’s femur at Aldermaston. In 1916, Nicholas II’s younger sister Grand Duchess Olga married Colonel Nicholas Kulikovsky, a commoner. With Kulikovsky, Olga had two sons, Tikhon, born in 1917, and Guri, born in 1919. In 1948, Olga and her family moved to Canada, where Kulikovsky bought a farm and raised cattle and pigs. Guri Kulikovsky died, but in 1992, when Gill and Ivanov began their work together, Tikhon, at seventy-five, was living in retirement in Toronto. He was, by that time, Tsar Nicholas II’s only living nephew and, as such, the best available source for comparative mitochondrial DNA. If the femur from Body No. 4 had belonged to Nicholas II, it should match perfectly with DNA from Tikhon Kulikovsky.

Mr. Kulikovsky, however, refused to cooperate. When Ivanov wrote to him, explaining the purposes of the investigation and asking for a blood sample, he received no reply. Ivanov tried again through Bishop Basil Rodzianko of the Orthodox Church in America, and, finally, through Metropolitan Vitaly, the head of the Russian Orthodox Church Abroad. Ultimately, Kulikovsky replied to Ivanov. “He told me he believed this whole bones business was a hoax,” Ivanov recalled. “He said, ‘How can you, a Russian man, be working in England, which was so cruel to the tsar and to the Russian monarchy?’ He said, ‘For political reasons, I will never give you a sample of my blood or hair or anything.’ ” Ivanov was disappointed, but he did not
give up. “At that time, it was critical,” Ivanov said. “He was the closest relative. I spent a lot of my own money talking with him and his wife by telephone, assuring them that I was not a KGB agent. And they replied, ‘Then probably the only reason for your investigation is to prove that Tikhon Nicholaevich is not of royal blood.’ ” Ivanov gave up. “Okay, so we forgot about this Tikhon,” he said. “And after we published our work, some people wrote that our analysis was not accurate because we didn’t use the blood of Tikhon Kulikovsky. The fact is that his blood is no longer necessary. We found two other relatives. They gave us their blood, and we had everything we needed for our research.”

To locate the other two relatives, the Aldermaston genealogists looked again at the family tree. Because the chain of similar mitochondrial DNA is repeated indefinitely down through generations of females, they focused on the women closest by blood to Tsar Nicholas II. Beginning with his mother, Dowager Empress Marie, they found an unbroken line of five generations of mothers and daughters leading to a contemporary descendant willing to help. The tsar’s sister Grand Duchess Xenia had one daughter, Princess Irina. This Irina married Prince Felix Yussoupov, famous for having murdered Rasputin. Irina and Felix produced one child, a daughter, also named Irina. This second Irina married Count Nicholas Sheremetyev, with whom she had one child, a daughter, Xenia. Upon her marriage, young Countess Xenia Sheremetyeva became Xenia Sfiris. Now in her early fifties, Mrs. Sfiris lives in Athens and Paris, and it was in Athens that she received the FSS’s appeal for help. An exuberant, warmhearted woman, she agreed immediately. Following instructions, she pricked her finger, let some blood run into a paper handkerchief, where it dried, put the handkerchief in an envelope, and took it to the British Embassy. From there, it went, via diplomatic pouch, to Aldermaston.

The other donor of DNA material given to identify Nicholas II was found on what must seem an infinitely remote branch of the massive European royal family tree. Nevertheless, although the line stretched back over six generations, the connection was as reliable and productive as it was in the case of Mrs. Sfiris. James George Alexander Bannerman Carnegie, third Duke of Fife, Earl Macduff, and Lord
Carnegie, is a sixty-six-year-old Scottish nobleman and farmer who descends from a common female ancestor of Tsar Nicholas II. She was Louise of Hesse-Cassel, a German princess who married King Christian IX of Denmark. One of her daughters became Empress Marie Feodorovna of Russia, the mother of Nicholas II. Another, older daughter, Alexandra, married the Prince of Wales, later King Edward VII. Queen Alexandra’s daughter Louise married the first Duke, of Fife. In 1929, Louise’s daughter Maud produced James, who, in 1959, succeeded to the title. The duke was willing to donate blood but, not wishing to incur publicity, made it a condition that he remain anonymous. Inevitably, in the course of time and with an investigation of this significance, knowledge leaked out.

As Gill and Ivanov expected, Xenia Sfiris’s mitochondrial DNA matched perfectly with that of the Duke of Fife. But when the matching 782 base pair letters lengths of the Greek woman and the Scottish peer were compared to the same section in the mitochondrial DNA extracted from the presumed tsar, there was a mismatch. A single letter was different. At a position numbered 16169, Xenia Sfiris and the Duke of Fife had a T; in this position Nicholas had a C. The other 781 pairs were in identical sequence. To check their data, Gill and Ivanov did a second mitochondrial DNA extraction from the bone believed to be the tsar’s. They cloned the DNA in this region after amplifying it with PCR and then transformed the product into
E. coli
bacteria. When fresh sequencing of these new clones was performed, seven of the clones were found to have a T at position 16169, thus matching Mrs. Sfiris and the Duke of Fife. But twenty-eight clones still presented the single spelling mistake, a mismatching C. The Aldermaston scientists concluded that Tsar Nicholas II had possessed two forms of mitochondrial DNA, one of which matched his relatives exactly; the other, at a single point, did not. This rare condition is known as heteroplasmy.