However, they soon discovered that there was a way to obtain DNA from Marie-Antoinette and her family. The Empress Maria-Theresa, Marie-Antoinette’s mother, had kept a rosary, which was thought to contain a lock of hair of each of her children. This rosary, which the empress had used in her prayers for keeping count of her devotions, was now held in the Elisabethinen convent in Klagenfurt, Austria. It was here that her oldest daughter, the invalid, Maria-Anna, had spent the later years of her life. When she died in 1789, the monastic order had received a number of her belongings, including her mother’s bead string. Cassiman and Petrie obtained permission to test samples of this hair and, in March 1995, Cassiman set off for Klagenfurt.
The rosary was very ornate, shiny black beads interlaced with decorated gold medallions. It had not been opened for two hundred years. “We had to do this in a scientific way with as little chance as possible of contaminating the hairs with modern DNA,” explains Cassiman. “The whole thing
was very well planned; there were a number of witnesses. The archivist at Klagenfurt authenticated the rosary. A jeweller was brought in to open it because the people at Klagenfurt did not want to destroy the clasp.” There were sixteen medallions, one for each of Maria-Theresa’s children. Each medallion had an inscription of the name of the child. Marie-Antoinette’s hair was deemed too precious, but the convent did give permission for hairs of two of her sisters to be taken.
Before Cassiman’s eyes, the first medallion was opened. It was a tense moment; nobody really knew what was inside each medallion hiding its two-hundred-year-old secret. Cassiman could see a tiny metal grid that kept the hairs in place. When this filter was lifted the medallion was full of light brown hairs. They had been cut to the right length and carefully pasted in place, one by one. These hairs were from another of Marie-Antoinette’s older sisters: Johanna-Gabriela, who died in 1762 at the age of twelve. When Cassiman saw the fragile state of the hairs in the rosary he was doubtful about achieving success but was intrigued by the challenge.
The next medallion was the same: painstakingly filled with brown hairs of yet another sister, Maria-Josepha, the ill-fated beauty who had died suddenly of smallpox in 1767 just before her wedding at the age of sixteen. The empress had ordered Maria-Josepha to pray in the royal crypt beside a relative who had just died of smallpox, but the coffin lid had not been properly fastened and this intimate proximity was enough to ensure that the unseen deadly virus would accompany her dutiful prayers and exact a terrible price for her obedience. Cassiman selected two or three hairs from Johanna-Gabriela and Maria-Josepha, carefully placed them in sterile containers and conveyed them himself back to his laboratory in Leuven.
Meanwhile in Holland, Hans Petrie was making headway tracking down locks of Marie-Antoinette’s own hair. A collector near Nijmegen had seen their newspaper advertisements and phoned Petrie to tell him that there was a collection of curios from Marie-Antoinette in the museum of the University of Nijmegen. Petrie went immediately to find out what was there. To his great delight he found many evocative trinkets, including
samples of her hair, carefully set in a frame decorated with the lilies of the Bourbons. The hairs were held firmly in place by silk thread, fastened by a seal bearing the handwriting of Henri, the Comte de Chambord, declaring that if the seal and thread were intact, the hair was authentic. The documents with the frame showed that these hairs were originally owned by the dauphin’s sister, Marie-Thérèse, The memento had been passed down in the family until eventually sold to a Dutch collector at an auction in France and, in time, had been bequeathed to the university.
At around the same time, a second source of Marie-Antoinette’s hair came to light in another private collection in Cannes in southern France, owned by the Marquise Jane de Bernardières. These locks of hair were dried in a medallion and their origin could only be established from oral testimony passed down through the generations of the families who owned the piece. Nonetheless, when these light brown hairs arrived in a sealed envelope from southern France, Cassiman could see they looked very similar to the hairs from Holland.
By 1995, however, despite their success in gathering hairs from Marie-Antoinette and her maternal relatives scattered all over Europe, the scientific team had run into a problem. They had taken no less than sixty-two different extracts of Naundorff’s hair. Yet each time they were frustrated to find they obtained a different mtDNA sequence. The DNA in Naundorff’s hairs was too contaminated and too degraded to give a reproducible result. In Holland, Petrie was aware of the disappointment. Knowing that they now had hairs from Marie-Antoinette and her sisters, he was determined to find another source of biological material from Naundorff.
Petrie knew from the official description of Naundorff’s exhumation in 1950 that in addition to a lock of his hair, a bone was also taken from his coffin, the humerus, or upper arm bone. At the time, this had been used to determine whether Naundorff’s s death was due to poisoning, but Petrie could not trace what happened to this bone afterward. He tried many different museums and collections, but after a three-year search the trail went cold. In desperation, he went back to the main Dutch forensic laboratory in The
Hague. Petrie had already spoken to this laboratory once and had been assured that Naundorff’s bone was not there. He decided to try one last time—and this time he was lucky.
Naundorff’s arm bone, sawn in two, was indeed waiting to be discovered and rescued from such an unsuitable royal resting place, in a neatly labelled sealed jar in the Dutch forensic laboratory at Rijswijk. It was not on display, but kept in the archives of the pathology department, a mere curio among countless other curios that had accumulated over the years. Cassiman’s colleague, Ronny Decorte, hurried to Holland to collect the crucial specimen, and samples of Naundorff’s bone and the Habsburg hairs were also sent to a second genetics laboratory in Nantes, France, where tests were to be carried out independently for comparison.
In the dedicated extraction laboratory, the surface layers of Naundorff’s arm bone were removed with a sterile saw to ensure there was no contamination from handling the bone. Els Jehaes, who was working on her Ph.D., undertook much of the testing on Naundorff. Carefully, she removed four internal segments of bone, barely a centimeter each. These were placed in liquid nitrogen and ground to a powder in a freezer mill in preparation for DNA extraction. The fine grey dust of his bones now held the key to whether this was, indeed, the arm bone of the ill-fated and perhaps much-maligned prince.
“We repeated the extraction several times—thirteen in all,” says Cassiman. “Not all the sequences were complete; there were some that were contaminated, but we got an identical sequence nine times.” If a result is reproducible several different times, from different bone samples, he explains, this helps to confirm the authenticity of the sequence. What is more, the samples of bone analyzed in the laboratory in Nantes produced the exact same sequence: the unique trace of Naundorff’s mitochondrial DNA.
This sequence had to be compared to that obtained from the hairs of Marie-Antoinette and her maternal relatives. When they looked at these hairs under the electron microscope it was clear that some were damaged; the outer envelope of the hair was frayed or broken. There were, however, some hairs that were still intact. Starting with the young aunts of Louis
XVII—hairs from Johanna-Gabriela and Marie-Josepha—the scientific team successfully extracted four DNA samples. Of these, to their delight, three sequences were identical: the unique trace of the maternal mtDNA sequence of the Habsburg family. It was the first time his team had successfully extracted an mtDNA sequence just from a sample of hair.
However, when these mtDNA sequences from Marie-Antoinette’s sisters were compared to those from Naundorff’s bone—there was a surprise. In hypervariable region 1 in the mtDNA there was only one nucleotide difference at position 16260. “One nucleotide difference is insufficient to exclude Naundorff as a son of Marie-Antoinette,” Cassiman said. “We had a problem. One difference is not enough to conclude anything. We didn’t have enough proof.”
To obtain more evidence, they analyzed the mtDNA at another region, between hypervariable regions 1 and 2. This part is less variable between individuals and is usually not analyzed. However, they did find one other difference, at position 16519. With two nucleotide differences between Naundorff’s bone and Marie-Antoinette’s sisters, this strongly favored the conclusion that Naundorff was not Louis XVII.
“The chances that Naundorff was indeed a son of Marie-Antoinette were now very slim,” says Cassiman, “although I wouldn’t say he was completely excluded on the basis of this result.” They went in search of more proof. Cassiman’s first step was to check that the mtDNA sequences obtained for Marie-Antoinette’s two sisters were authentic. Attempts to obtain DNA for comparison from hair samples of other descendents of the Empress Maria-Theresa proved unsuccessful; however, he did have the hairs that were thought to have come from Marie-Antoinette herself. “These were important because there was fairly good evidence that this was indeed hair from Marie-Antoinette,” says Cassiman. “If the sequence from these hairs had been different from her sisters, that would have been worrying.” They tested both the Marie-Antoinette hairs from Cannes and from Nijmegen and successfully obtained an unambiguous sequence identical to her sisters’ hair. He now had a third confirmation of the Habsburg sequence.
There was one final check. They knew that Marie-Antoinette had living
maternal relatives who could provide fresh DNA for a comparison: Queen Anna of Romania and her brother, André de Bourbon-Parme. Cassiman wrote to Queen Anna explaining the tests and requesting a sample of her blood; the team in Nantes obtained samples of hair from her brother. Both these mtDNA sequences proved to be identical. Comparison of their mtDNA sequence with that of Naundorff revealed that there were four nucleotide differences between these living relatives and Naundorff’s mtDNA. This made it even more unlikely that Naundorff could be a relative.
“The result, however, was troublesome,” admits Cassiman. Although the living Habsburg relatives had the same two differences in their mtDNA as the hairs of Marie-Antoinette and her two sisters, in addition there were two further differences in their mtDNA. This was puzzling, since the results should have been identical. Studies on mtDNA had established that mutations usually occur only once in thirty-three generations. This suggested there had been two mutations in the nine generations that separated Marie-Antoinette and her two sisters from Queen Anna and her brother. Although this was possible, it was very rare. “This result bothered me and we hoped to find an explanation,” said Cassiman. “We believed in the conclusion that we had to rule Naundorff out, but we were left with this problem of two additional nucleotide differences. That frustrated me—not that we did not solve the Naundorff problem—but that we did not get clean results. As scientists we want one hundred percent.”
Cassiman and his team wrote up their results for publication in the
European Journal of Human Genetics
in 1998. Since Naundorff’s bone showed two nucleotide differences from the sequences of the two aunts and the mother of Louis XVII and four differences from the sequences of living maternal relatives, he concluded, as had the French courts in 1851, 1872 and 1954, that “it becomes very unlikely that Karl Wilhelm Naundorff is the son of Marie-Antoinette.” After a lifetime of acting the part of the prince, of looking like the prince, there, in the center of his being, the genetic essence of his bones denied it all. Naundorff was a great impostor, betrayed, apparently, by his arm bone.
Naundorff’s descendents, however, do not accept this scientific evidence.
They want Naundorff’s grave in Holland exhumed once more to obtain more bones for testing. “The only way now that the relatives can still claim that Naundorff was the son of Marie-Antoinette,” says Cassiman, “is if they can prove that the bone taken from his coffin in 1950 is
not
his bone!” Ever more convoluted explanations were needed for Naundorff to be the missing prince. Cassiman told them, “If you want to exhume, fine, go ahead. But the story is finished. There are no grounds to take it any further.” There was just one outstanding question. If Naundorff was not the missing prince, then who was?
With Naundorff’s claim out of the picture, Cassiman suddenly found he was a target for other hopeful counts and princes, descendents of the numerous pretenders. All the old romantic escapades and escape stories to shame Hollywood were resurrected. “There was a Belgian count who visited me who had contacts in Argentina,” recalls Cassiman. He believed the dauphin had fled from France and died in Argentina where his skeleton was buried in a mass grave. “They had been dowsing with a pendent over the grave to try to establish which of these skeletons might be the lost dauphin. They had even carried out tests on a skeleton with strange results which they wanted me to look at.” On another occasion, he received a call from a woman who claimed to be a descendent of Marie-Thérèse. According to her account, Marie-Thérèse escaped and a substitute girl was locked in the Tower who later went on to live in Austria and marry the Duc d’Angoulême. The real princess fled to Poland where she had children and her descendents now lived in Munich. Cassiman shook his head at these fanciful stories.
In the case of the Russian royal family, the British forensic team had been able to positively identify the skeletons found in Siberia. They found that one woman and three of her children in the grave were maternally related to Prince Philip, the duke of Edinburgh, a grand-nephew of Czarina Alexandra. This genetic evidence, taken with the historical record, provided convincing proof that this miscellaneous collection of bones was, in fact, the once all-powerful czarina and three of her beautiful daughters. However, genetic tests on bones thought to be from Czar Nicholas himself yielded a
rare anomaly and proved inconclusive. The case was only resolved when the Russian authorities finally agreed to exhume the czar’s brother, Georgij Romanov. Tests showed he had exactly the same rare anomaly, providing almost 100 percent confirmation that it was indeed the czar in the grave. In 1998, the Russian royal family had a state funeral in St. Petersburg. The genetics in the czar’s case had led to a satisfactory conclusion—but how could Cassiman resolve the case of the dauphin?