The Seven Daughters of Eve

by Bryan Sykes

  The discovery of the skeletons at least promised to introduce some objective evidence into the debate. The proof of the execution story depended entirely on showing that the remains taken from the pit really were those of the Romanovs. The location at least tallied with some contemporary accounts that told of the bodies being loaded on to a truck and driven into the woods on the outskirts of the town. According to these accounts, the executioners panicked when their truck became stuck in the mud, and they threw the bodies into a hastily dug pit before dousing them with sulphuric acid in a vain attempt to remove all features which could be used for identification.

  When all the recovered bones were assembled, it soon became clear that these were the remains of only nine bodies, two fewer than there should have been if all the victims of the massacre had been buried in the same grave. After the long and painstaking process of refitting more than eight hundred bones and rebuilding the shattered skulls that had been crushed by the rifle butts of the burial detachment, it was concluded from the skeletons that the nine bodies were those of the Tsar and Tsarina; three of their five children – Maria, Tatiana and Olga; their physician, Dr Eugeny Botkin; and three servants, Alexei Trupp the valet, Ivan Kharitonov the cook and Anna Demidova the Tsarina’s maid. There was no sign of the bodies of the youngest daughter Anastasia, nor of the Crown Prince, the Tsarevich Alexei. Other than these reconstructions, what further tests could be done on the remains to confirm their identity?

  We had already published a paper in 1989 showing that DNA could be extracted from much older bones than these, so it was only natural to try to get DNA from the Ekaterinburg remains in the hope of confirming that these were the Romanovs. The work was carried out by the Russian Academy of Sciences and the British Forensic Science Service. First they used conventional forensic genetic fingerprints to identify the sex of the skeletons and to confirm that they did indeed include a family group of two parents and three children. DNA from the remains presumed to be those of Dr Botkin and the servants showed that they were unrelated to the family group or to each other. So far, everything fitted in well with the conclusions of the bone experts.

  These scientists also succeeded in recovering mitochondrial DNA from the bones, and came up with two different sets of sequences from the family group. The female adult, the presumed Tsarina, and all three children had an identical mitochondrial DNA sequence. The male adult in the family group, the presumed Tsar, had a different sequence. This was exactly what you would expect from a family. All three children had inherited their mother’s mitochondrial DNA sequence while the father, who had got his from his own mother, had not passed it on to any of his children. However, on its own, extracting the mitochondrial DNA and sequencing it did not identify this family as the Romanovs – any family would show the same pattern of identity between mother and offspring, with the father showing a different sequence. The only way of proving which family this was was to locate living relatives of the Tsar and Tsarina who were connected to the dead Russians through a series of entirely maternal links. They didn’t have to be especially close relatives; the real power of mitochondrial DNA is that it is not diluted by distance. So long as the connections are exclusively maternal and not disrupted by a father–child link, then the mitochondrial DNA will be identical.

  Fortunately, it was possible to trace living direct maternal relatives of both the Tsar and the Tsarina. The Tsar had an unbroken maternal connection through his grandmother Louise of Hesse-Cassel, the Queen of Denmark, to a Count Nicolai Trubetskoy, seventy years old and living in peaceful retirement on the Côte d’Azur after a lifetime as a merchant banker. The Tsarina could trace a direct maternal link through her sister Princess Victoria of Hesse to His Royal Highness Prince Philip, the Duke of Edinburgh, the husband of Queen Elizabeth II. After several rounds of discreet negotiation both men agreed to provide a small blood sample from which their DNA could be extracted. What would they show?

  The notation everyone uses to compare mitochondrial DNA sequences involves quoting differences from a set reference sequence, in fact the very first mitochondrial DNA to be entirely sequenced, by a team from Cambridge in 1981. In this notation, a DNA sequence which differs from the reference sequence at the fifteenth and one hundredth positions in the 500 base control region segment is abbreviated to 15, 100. The sequence from the Duke of Edinburgh was 111, 357 using this notation. At all the other 498 positions along the 500 base stretch, the Duke’s sequence was exactly the same as the reference sequence.

  It is always much harder to get a complete sequence in one go from ancient DNA than from a modern sample. The strands are fragmented by the ageing process, so even the relatively short 500 base segment of the control region has to be built up in overlapping stages of a hundred bases or so. This is a laborious process, but eventually the sequences of the presumed Tsarina and her three children were typed. They all had exactly the same sequence of 111, 357. They were all an exact match with the Duke of Edinburgh.

  The same, however, was not true for the adult male, the presumed Tsar. He was not an exact match with Count Trubetskoy. Whereas the Trubetskoy sequence was 126, 169, 294, 296, the presumed Tsar’s DNA had mutations at only 126, 294 and 296 – very similar but not identical. This was a definite setback. There was so much circumstantial evidence connecting the bodies with the Romanovs, and there was the exact match of the females with the Duke of Edinburgh. But there is no point in doing a genetic test if you don’t take notice of the result. A close match is not an exact match. And if the maternal connection over six generations with Count Trubetskoy was unbroken, the match would be exact.

  Was there a chance that the Count was not really a relative of the Tsar, even though the family tree had recorded him as such? If so, there would have to have been a break somewhere along the line going back from the Tsar to Louise of Hesse-Cassel and then down to Count Trubetskoy. It would mean, in fact, that one of the people on this line had a different mother from the woman recorded on the pedigree. This is always a possibility – there could have been an adoption or a mix-up at the birth – but these are only remote possibilities. If it were a paternal line that was being followed it would be different. A child can easily have a different biological father from the man married to his mother; but such mis-identification is much more unlikely down the maternal line. After all, both mother and baby have to be present at the birth. The only formal conclusion that could be reached was that this was not the Tsar; and so, that since the conventional genetic fingerprints had already identified him as the father of the three children found in the grave, this was not the grave of the Romanovs after all.

  But even though the mitochondrial DNA sequences of Trubetskoy and the male skeleton were not exactly the same, they were very close; and so near a miss invited further thought. They both shared three mutations at positions 126, 294 and 296. Trubetskoy had another one at position 169. Was it possible that there had been an error in reading the sequence of the ‘Tsar’s’ mitochondrial DNA? The team went back to the original trace from the sequencing machine and looked very closely at the readings at position 169 for the ‘Tsar’s’ sample. The trace itself looks like four superimposed lines of different colours, representing the readout from four separate channels which detect the four DNA bases: red for T, black for G, blue for C and green for A. While Trubetskoy’s trace showed a clear red peak at position 169 corresponding to the mutation T, the ‘Tsar’s’ trace at the same position showed the blue peak for C, the same as the reference sequence. But underneath the blue peak was a small red blip. Could it be that the ‘Tsar’s’ DNA was a mixture of two mitochondrial DNA sequences, the main one with the sequence 126, 294, 296 and another, much smaller, with the same sequence plus the mutation at position 169? There was one way to find out, and that was to clone it.

  Cloning is the only way to separate the different DNA molecules in a mixture. Briefly, it involves tricking bacteria into accepting just a single molecule of DNA and then copying it as if it were their own.
Getting DNA into bacteria is a very inefficient process; only one in a million accepts it. Still, if just a couple of dozen bacteria can be persuaded to take in the DNA, they can be treated in such a way that the only bacteria to survive and grow as colonies on a culture dish are the ones with the extra DNA. They can then be picked off and the DNA sequenced. Within each colony, all the DNA will be copies of the original molecule that was accepted. If there is a mixture of two different DNA molecules to start with, some of the colonies will have one type and some will have the other. The scientists managed to create twenty-eight clones containing mitochondrial DNA from the ‘Tsar’. When each of these was individually sequenced, twenty-one contained the main sequence 126, 294, 296 read from the original trace, without the mutation at 169. But the DNA from seven clones did contain the additional 169 mutation, making it absolutely identical to Count Trubetskoy’s.

  What the researchers had stumbled across was the very rare state where a new mutation, in this case at position 169, is part way to becoming established. This state, formally called heteroplasmy, had scarcely ever been observed before and was very little understood. As we will see in a later chapter, we know a lot more about heteroplasmy now; in 1994, when the paper on the ‘Romanov’ remains was published, it was a novelty. But it did get the researchers off the hook. Here was the evidence they needed that there was indeed a continuous maternal link between the bones of the Ekaterinburg ‘Tsar’ and a living relative of Tsar Nicholas II.

  The mitochondrial DNA matches were certainly good evidence to support the case that the Ekaterinburg bones were the remains of the Romanovs. But was it proof? Proof can never be absolute. It is always relative. In the case of the Romanovs the degree of certainty could be given a mathematical form depending on how common these mitochondrial sequences are in Europe. In those early days of the research we didn’t know many European sequences, so it was hard to know how strong the evidence was. Now we have far more sequences to compare, and we know that the Duke of Edinburgh’s sequence (111, 357) is actually extremely rare: it has not been found again in over six thousand Europeans. Since it has not been seen elsewhere, we cannot accurately estimate its frequency, but it is very unlikely to be higher than one in a thousand. This means there is, at most, a one in a thousand chance that the mitochondrial DNA sequence from a European picked at random would match the Duke of Edinburgh. So there was still a very small chance that the female Ekaterinburg bones did not belong to the Tsarina and her children at all, but to another family who just happened to have the same mitochondrial DNA as the Duke of Edinburgh. The Trubetskoy sequence (126, 169, 294, 296) is again very rare and has not been seen in six thousand modern Europeans. However, the Tsar’s main sequence (126, 294, 296) is much more frequent, with just under one in a hundred Europeans matching it exactly. So, once again there was a small but finite chance that the bones of the adult male were not the Tsar’s but those of someone else who just happened to match.

  Though the DNA matches gave a pretty high level of proof already, there is a further level to consider. We have not yet taken into consideration the fact that the two sets of matching sequences were found in the same grave and came from the parents of the three children, according to the DNA fingerprints. How does that affect the result? The answer is that it makes the level of proof that these really were the Romanov bones very high indeed. The probability of getting matches to both sets of mitochondrial DNA sequences just by chance is the mathematical product of the individual probabilities. That is one in a thousand multiplied by one in a hundred, which comes to the vanishingly small figure of one in a hundred thousand. Add to that the circumstantial evidence which led to the discovery of the grave and the evidence of bullet wounds, and the proof climbs even higher towards 100 per cent.

  But one mystery remained. Only five Romanov bodies were ever found – two adults and three girls. Formally, one might take the view that this is evidence against the remains being those of the Romanovs at all. But it tallies with persistent rumours that some of the children had escaped execution. A Soviet announcement that only the Tsar himself had died and that the rest of the family had been sent to a safe place was swiftly followed by the appearance of all too obvious impostors. For a while, every town in Siberia, then in the hands of the White Russians and not the Bolsheviks, had its own ‘Grand Duchesses’ and ‘Crown Princes’. Most were obvious frauds, but some managed to do well out of the deception for a while. One enterprising businessman even ran a regular export service, persuading local millionaires to part with their cash to help him send the imperial refugees abroad to safety. His accomplice, playing the part of one or other rescued ‘Grand Duchess’, even allowed the entranced sponsors to kiss her hand as she bade a tearful last farewell to her beloved country.

  The Tsar’s mother, the Dowager Empress Marie Fedorovna, exiled in Copenhagen, did more than anyone to keep alive the myth that her family had survived, refusing to accept that they had died right up to her own death in 1928. Throughout the last ten years of her life she was challenged to accept the claims of numerous pretenders as her grandchildren. By far the most persistent of these claims was that of the woman who became known as Anna Anderson. It began when a young woman jumped from a bridge into the Landwehr canal in Berlin in February 1919, seven months after the Ekaterinburg massacre. She was rescued, but resolutely refused to reveal her identity and was confined to a mental hospital as ‘Fräulein Unbekannt’ – the unknown lady. One of her fellow inmates, Clara Peuthert, became convinced, from an account of the massacre in a Berlin newspaper, that this withdrawn and uncommunicative patient was none other than the Grand Duchess Tatiana, the second of the Tsar’s four daughters. After she was released from the asylum, Clara Peuthert championed Fräulein Unbekannt’s case among the White Russian émigrés in Berlin. Using these contacts, she arranged a visit by the Tsarina’s former lady-in-waiting, Baroness Buxhoeveden. This was the first of many often disastrous encounters with people anxious to establish the real identity of the ‘survivor’ that went on for most of the rest of her life. On this occasion, Fräulein Unbekannt hid under the bedclothes. The redoubtable Baroness pulled the sheets aside and dragged her out of bed. She could not possibly be Tatiana, exclaimed the Baroness. She was far too short. This rather obvious disqualification only made Fräulein Unbekannt declare that she had not actually said she was Tatiana, who was, in fact, the tallest of the Tsar’s daughters. At only 5ft 2in, Fräulein Unbekannt was much more the size of Anastasia. And so that is who she claimed to be for the rest of her life, taking the name Anna as an abbreviation of Anastasia and adding Anderson many years later to confuse local journalists during her stay at a hotel on Long Island, New York.

  Anna Anderson’s pathetic life, spent in hospitals and the homes of her supporters, came to an end in 1984 near Charlottesville in Virginia. If she were Anastasia she would have been eighty-three years old. Over the years she became embroiled in unending legal battles between her supporters and those who wanted her claim dismissed. Her opponents were accused of wanting to prove the death of the Tsar’s entire family so that they could benefit from money the Romanovs had deposited in overseas bank accounts; her supporters were accused of coveting those fortunes for themselves. Throughout all this conflict and controversy, Anna Anderson herself never vigorously prosecuted her claim. Whenever there was a chance to impress one of the Tsar’s relatives who had been persuaded to visit her, she would be untalkative and uncooperative, refusing to answer questions and often hiding in her room. While this behaviour annulled her claim in the eyes of her detractors, it was her very reluctance to press her case, coupled with an absolute self-belief that she was the Grand Duchess Anastasia, that convinced her supporters. The matter was never conclusively settled during her lifetime, and she passed away with her claim neither validated nor disproved. Fortunately for her, she died before the cold eye of genetics could be turned on the case. If she had lived another few years, like her contemporary Queen Elizabeth, the Queen Mother, who is still alive at
the age of 100, then her lifetime of deception would have been mercilessly exposed.

  In a thrilling piece of detective work, mitochondrial DNA was recovered from a stored biopsy from Anna Anderson, taken when she was in hospital for an operation to remove a bowel obstruction in 1979. It had a completely different sequence from the Tsarina’s DNA. Anna Anderson could not possibly have been Anastasia. A test that had taken a month at most to perform had demolished at a stroke one of the most enduring and romantic sagas that had gripped the world from one end of the twentieth century to the other. Such is the power of DNA to dispel myths – even those we might prefer to have believed.

  The sequence from Anna Anderson’s biopsy did, however, match a living maternal relative of one Franziska Schanzkowska, a patient in a Berlin mental home who disappeared in 1919 shortly before ‘Anastasia’ made her appearance in the same city. Opponents of Anna Anderson’s claim had always suspected her to be Franziska Schanzkowska, and not Anastasia as she claimed. DNA proved them right.

  So the mystery of Anastasia lives on. In our laboratory we have more than once been asked to examine the DNA of further claimants. Sadly, none of them has passed the scrutiny of the DNA test. In the 1956 film Anastasia, written as a romantic fiction rather than as a true record of events, the Dowager Empress Marie Fedorovna puts Anastasia, played by Ingrid Bergman, through a series of tests to prove whether she is her grand-daughter. She eventually accepts the young woman, and the film ends happily. It would not have, had DNA been around. But the film also brought its own reward for Anna Anderson, who received a share of the royalties.