Old Bones

DNA and Skeletons

The Romanovs

On June 5, 1995, Pavel Ivanov, a 42-year-old molecular geneticist who was working at the Engelhardt Institute of Molecular Biology in Moscow, arrived at the United States Armed Forces Institute of Pathology in Rockville, Maryland, with an unusual package: a piece of the femur and a piece of the tibia of Grand Duke Georgij, the younger brother of Imperial Russia's last tsar, Nicholas II. Eleven months earlier, under the watchful eyes of bishops of the Russian Orthodox Church, forensic scientists had removed some of the Grand Duke's remains (he died of tuberculosis at the age of 28 in 1899) from his Italian marble coffin in the Cathedral of St. Peter and St. Paul in Petersburg. Ivanov also carried two other curious and precious items: a slender section of a handkerchief from a museum in Japan that is reported to be stained with the blood of Nicholas II, and a strand of hair from a locket that for decades reposed in a St. Petersburg palace and is said to have been cut from Nicholas II when he was a child. The Russian scientist hoped that DNA analysis of these artifacts would close one of the most persistent questions in modern Russian history: What really happened to Nicholas II and his family?

The Russian Orthodox Church had permitted the exhumation because it needed irrefutable evidence to corroborate the dramatic reports that the skeletal remains of the last Romanov tsar and his family had been identified. If DNA analysis of Grand Duke Georgij's mitochondrial DNA (see below), which would be extracted from bone samples for which the authenticity was beyond doubt, indicated a match with the mitochondrial DNA extracted from the bones alleged to be those of Nicholas II, it would establish to a certainty that the nine fragmented skeletons that Geli Ryabov, a filmmaker, and Alexander Avdonin, a geologist, had located in a shallow grave in Yekaterinburg in the Ural mountains included five members of the imperial family, all of whom had long been believed to have been assassinated in compliance with an order issued by Lenin in July, 1917.

According to the official report filed in Moscow in 1917 by Yakov Yurovsky, the man who carried out the execution, his squad had killed eleven people: the tsar, his wife, their five children, three servants, and the family physician. The discrepancy between that number and the nine skeletons found in 1995 could be explained by the fact that Yurovsky reported that he had incinerated two of the bodies before deciding to bury the rest. Of course, the discrepancy in the number of skeletons also indirectly supported the long popular rumor that Anastasia, one of the tsar's daughters, had miraculously escaped the death squad.

Confirmation of the identity of the skeletons would be an important historic and political event. Among other things, it would provide the historical evidence needed by the Russian Orthodox Church to reinter the last of the ruling Romanovs, this time with full religious rites. A mass funeral for the tsar's family would rivet the nation and further strengthen the position of the Orthodox Church in post-Communist Russia.

By the summer of 1995, efforts to confirm the identity of the Yekaterinburg massacre—by merely looking at the skulls and bones, one could see evidence of crushing blows, bayonet thrusts, and bullet wounds—had become mired in politics. The grave had been rediscovered in 1979, but it was only in 1991 after the collapse of the Soviet Union that the government permitted the exhumation, which was carried out the day after Yeltsin's inauguration. For a full year, Dr. Sergei Abramov, a forensic scientist and bone expert in Moscow, worked to reassemble 700 bones and several hundred fragments into nine skeletons. After painstaking analysis, he was convinced that the remains were of Tsar Nicholas II, his family, and servants, but he realized that the only way to close this extraordinary moment in 20th century history was to have other experts confirm his conclusions.

In the summer of 1992, Dr. William Maples, a forensic pathologist from the University of Florida, and fellow scientists who are experts in dental identification and hair analysis went to Yekaterinburg at the invitation of local authorities to study the skeletons. Just a few weeks earlier, acting on behalf of the Russian Ministry of Health in Moscow, Dr. Ivanov had reached an agreement with the forensic section of the British Home Office to have one of their scientists, Dr. Peter Gill, undertake two tasks: determine whether the skeletons were part of a family group, and, if they were, determine whether DNA analysis of the remains matched that of DNA from living descendants of the imperial family. Prince Philip, Duke of Edinburgh, consort to Queen Elizabeth II, is the grandnephew of Empress Alexandra, the tsar's wife. Because an unbroken female line connects Prince Philip to Alexandra and her children, his DNA could be used to confirm the identity of the skeletons. He agreed to provide a blood sample.

Efforts to extract DNA from old bones and use it to establish identity began in 1984 when Russell Higuchi, one of the scientists who developed PCR (polymerase chain reaction, a technology that enables one to amplify large amounts of DNA from exquisitely small initial samples), successfully retrieved mitochondrial DNA sequences from the remains of a 140-year-old quagga, a recently extinct species that looked like a cross between a horse and a zebra. A year later, an Italian scientist reported cloning DNA from an Egyptian mummy, and the race was on. One spectacular event was the successful cloning of human DNA from 91 well-preserved brains found among the 177 skeletons buried about 7,000 years ago in a heavily silted lake in Florida. That site is thought to have been used by a primitive culture for ritual sacrifice. The remains were well-preserved, in part because of the low oxygen conditions in the lake bed. By 1992, forensic scientists and molecular anthropologists had sufficient experience with ancient DNA that they were eager for almost any challenge, especially if large bone samples were available.

From September 1992 until early 1994, Dr. Gill and his team labored to extract DNA from fragments of the femurs of each of the skeletons and, by comparing the sequences of the DNA letters, use it to determine which, if any, of the skeletons belonged to the same family. Using the small amount of nuclear DNA he was able to extract, Gill proved that one of the skeletons of the adult males in the group had to be the remains of the father of the three young women whose remains were also in this grisly collection. This fit the profile of the Romanov family, but there were still several issues to be resolved, and there was not enough nuclear DNA to complete the molecular analysis.

The group turned to mitochondrial DNA. Mitochondria are tiny structures that reside in the cytoplasm that surrounds the cell's nucleus. They each contain a small circular genome (16,569 bases) that codes for key genes involved in making proteins that are critically important for operating the cell's power plant. Evolutionary biologists speculate that mitochondria are the ancient remnants of a subcellular organism or a simple bacterium that colonized higher organisms hundreds of millions of years ago, and that over the eons made itself indispensable. Only over the last decade or so have we come to realize that there is a whole class of genetic diseases that are caused by mutations in the mitochondrial genes and that are not inherited according to Mendelian laws.

Because it is not found in sperm, mitochondrial DNA is inherited strictly matrilineally. It has been transmitted from mother to offspring throughout the history of our species. Thus, mitochondrial DNA provides scientists with an unusually powerful tool to study the origin and spread of the human family. By comparing differences in the sequence of mito-chondrial DNA, scientists can estimate when different human groups diverged. The greater the differences, the more ancient the divergence. In effect, mitochondrial DNA constitutes a kind of biological record of the human diaspora.

One recent scientific report was popularly interpreted to show that all humans could theoretically trace their ancestry to a common maternal relative who lived in Africa about 200,000 years ago. The journalistic announcement that there really was a human Eve grew out of study of seven regions of the mitochondrial DNA taken from 147 persons who had been born in five distinct geographic regions of the world. The scientists used the variations in the patterns among the different persons to construct a phylogenetic tree, a diagram that defines degrees of relatedness according to the amount of divergence (essentially, the accumulation of differing mutations over time) between the mitochondrial DNA sequences. As one moves down the tree, its branches converge on a trunk—one ancient mitochondrial DNA pattern, the pattern from which all others diverged. The woman from whom this mitochondrial pattern was obtained is, arguably, the oldest true female ancestor to whom we are all related.

The Russian skeletons yielded abundant mitochondrial DNA. Analysis of the samples from the skeletons presumed to be Empress Alexandra and three of her four daughters perfectly matched the mitochondrial sequence of the sample donated by Prince Philip. The next task was to find living relatives of Nicholas II so the forensic scientists could attempt to establish his identity. This was not so easy, but they eventually located two living relatives, Xenia Cheremeteff-Sfiri, a direct female descendant of the tsar's sister, and James Carnegie, the Third Duke of Fife in Scotland, who descends from a line that is connected to Nicholas II through his grandmother. Both agreed to donate blood for analysis. The mitochondrial DNA samples taken from these two matched each other perfectly but, to Gill's surprise, they did not match with the sample from what was presumed to be Nicholas II. When he sequenced the DNA in the putative tsar's mitochondrial DNA, Dr. Gill found a mixture of the DNA letters C and T at position 16,169. In the mitochondrial DNA taken from Xenia Cheremeteff-Sfiri and James Carnegie, the DNA at position 16,169 is always a T. There can be more than one type of mitochondrial DNA in the cytoplasm of a single cell because hundreds of these subcellular structures are transmitted via the human egg through the generations, and mutations can arise in any one of them.

Probing further, Gill and his team found that Nicholas II had two forms of mitochondrial DNA (a condition called heteroplasmy), which is not uncommon, but which in this instance cast a tiny shadow of doubt on the argument that the remains were those of the tsar. Although the scientists continued to seek other ways to establish identity, they were ready to make their case. In July, 1995, they announced that their DNA studies established at a level of certainty greater than 98.5% that the bones were of the tsar and his family. Dr. William Maples and his colleagues were quick to challenge the assertion, arguing that the results of the DNA studies done on the remains could have arisen if two samples had been contaminated. This made officials in the Russian Orthodox Church unwilling to accept the evidence as definitive, and led them to acquiesce to the exhumation of Nicholas's brother, Georgij.

In the fall of 1995, another round of DNA tests confirmed that the remains presumed to be Nicholas II were in fact those of a brother to Georgij. Ivanov and colleagues at the DNA identification laboratory operated by the U.S. Armed Forces Institute of Pathology showed that the Grand Duke's mitochondrial DNA matched that of the putative tsar's exactly, even to the point of having a mixture of C and T at position 16,169. Thus, the heteroplasmy that thwarted the first effort to prove identity became the crucial proof in the second attempt. The reason that Countess

Xenia's mitochondrial DNA did not match at that position is easily explained by a "bottleneck" hypothesis. Simply put, only some of the many mitochondria in a human egg are transmitted to the next generation. If there are two different mitochondrial populations (as defined by slight differences in DNA sequence), then chance alone can account for one relative having one lineage while another did not.

One part of the story, the best known and most popular, still needed resolution. Until she died in a Virginia nursing home in 1984, a woman named Anna Anderson claimed she was in fact Anastasia, Nicholas II's youngest daughter, who had escaped the firing squad's bullets. In 1979, Anna Anderson underwent surgery, and a bit of her intestine was stored in a pathology department in a Virginia hospital. Using that sample, Dr. Gill's team showed absolutely that she was not a daughter of the tsar, but that she was related to the maternal grandnephew of a Franziska Schangkowska. The woman who had spent her life claiming she was the tsar's daughter was, as had long been thought, actually born into a Polish working-class family.

Perhaps she would have been happy to learn that we probably all number a royal ancestor or two in our family tree—if we go back far enough to look. Consider. We have two parents, four grandparents, eight great-grandparents and sixteen great-great grandparents. If we go back just 20 generations, about 400 years, and if we assume no inbreeding, we can theoretically claim relatedness with 2 to the 20th power ancestors, some 1,048,576 persons, one of whom might well have been part of a royal family ... or at least well off.

Proceed back just 10 generations more, another 200-250 years, and one could theoretically claim 1,000,000,000 ancestors. But this number far exceeds the total number of people who then lived on the planet. How can we reconcile the theoretical prediction and the reality? In fact, one need go back little more than about 10 generations before discovering that one is related distantly to all sorts of people including, perhaps, the very neighbors one most detests. Like it or not, we really are a human family. We share our ancestors. Go back far enough and you will find many great-great-grandparents to whom you are related through multiple other relatives. If the technologies for extracting DNA from old bones and studying it to determine relatedness improve much more, it will be possible to connect almost any recent human fossil (say within 5000 years) we find to a living descendant. Indeed, we are nearly there.

Cheddar Man

In 1903, cavers exploring Cheddar Gorge in southwest England discovered a remarkably intact skeleton of a man that, on the basis of archaeological study of artifacts in the cave, appeared to have lived more than 5000 years ago. Later on, carbon dating, a measure of radioactive decay in the bones, pinpointed his death at about 7150 B.C. In 1995, a British newspaper intrigued by the work of Bryan Sykes, an Oxford molecular biologist who is an expert on ancient DNA and who was among the first to show that it could be obtained from old bones, asked him to try to find a living descendant of Cheddar Man, as the fossil is fondly called. Sykes, who had just performed the DNA studies of the 4000-year-old Ice Man discovered in a glacier on the Italian-Austrian border in the summer of 1995, took the challenge.

Typically, the curators were stingy. They permitted Sykes to remove a single tooth from the fossil jaw. Using a drill, he and his team retrieved a core sample from the region of the tooth known as the dentine. They found a surprisingly large amount of DNA, which after weeks of laborious study they were able to characterize with confidence. Having constructed the mitochondrial DNA profile of the fossil, they were ready to look for relatives. The search was based on some simple logic. They called for volunteers who to the best of their knowledge came from families that had lived in or near Cheddar for generations. After interviews, the team invited a mere 20 persons to give blood. To his immense surprise, Eldrian Targett, a local history teacher who lives just a mile from the caves, learned that he and the 9000-year-old fossil were both descendants of a common female relative. Targett can, for the moment at least, claim an ancestor older even than can members of the world's most studied royal families. When she learned that her husband was related to a caveman, Targett's wife's quipped, "Maybe this explains why he likes his steaks rare."

The Cheddar caves are owned by the Lord of Bath, one of England's richest men. Intrigued by the mitochondrial studies, the Lord asked Sykes to analyze his DNA as well. Over dinner in a Victorian era hotel, Professor Sykes, a blonde, ruddy-cheeked, puckish fellow, confided to me that the Lord's butler was more closely related to the ancient skeleton than was his master, who, one might surmise, had been eager to establish that he had a more ancient lineage than any other member of the House of Lords.

Sykes's success in establishing a family connection between a living Englishman and a 9000-year-old skeleton set off a media frenzy. The

British tabloids gave new dimensions to the definition of bad taste. One ran the story below photos of two topless models holding fake stone age axes. Even the normally staid science journals delighted in speculating how far back in time one could retrieve stretches of DNA that were sufficiently intact so that questions could be asked about the organisms from which they were derived. The work with Cheddar man fueled the fantasies that had been evoked by Jurassic Park.

The idea for that novel arose when Michael Crichton read reports that molecular biologists had successfully extracted DNA from the fossilized digestive tracts of insects that had been preserved intact for tens of millions of years in the hardened amber resin in which they had become stuck and perished. It is logical to suppose that the ancient mosquitoes fed on dinosaurs, so it is conceivable that their bellies might include sequences of dinosaur DNA. It is, however, an amazingly long leap to think that one could reconstruct a whole genome of an extinct creature by assembling hundreds of thousands of tiny fragments. It is next to impossible that all the sequence is present, even in tiny fragments, and even if it were, the task of assembling it is beyond current technology.

The reports of extracting DNA from the remains of organisms that died many millions of years ago may not even be accurate. Sykes is among those who dismiss most of the claims out of hand, attributing the reports at best to sloppy science and at worst to fraud. He points out that the laws of physical chemistry that govern the effects of the impact of air and water molecules (oxidation and hydrolysis) on DNA suggest an outer limit of about 100,000 years for the molecule to remain intact, unless, of course, it has been perfectly protected as in amber. In 1997, a group of British scientists reported that despite meticulous efforts they had been unable to obtain any DNA from a large series of amber specimens. Their report contrasted sharply with 13 of 14 earlier, but much smaller, efforts that had claimed success. Reviewing the earlier papers, Sykes concluded that the DNA that each researcher claimed to have found was actually a modern contaminant. In one case, it was almost certainly a fragment of human mitochondrial DNA. In another study that claimed to detect DNA from a 20-million-year-old fossilized magnolia leaf, Sykes argues that the DNA is from a modern magnolia. It appears that we are unlikely ever to retrieve and study long stretches of DNA from specimens much older than 100,000 years.

Yet, this is still a large enough historical window to let us explore an endless list of fascinating questions about our own past. For example, mi-tochondrial DNA is already being used to investigate much more important historical questions than whether the Lord of Bath is a member of the Cheddar cave family. In 1995, archaeologists in Honduras uncovered a royal tomb that they think contains the remains of Kinich Ah Pop, a 5th century ruler in the Mayan Copan dynasty. There is strong circumstantial evidence that the remains are those of Kinich, but to confirm his royal lineage, scientists will compare his DNA to that taken from skeletons for which the evidence of membership in Mayan aristocracy is even greater. In Spain art historians and geneticists hope to celebrate the 400th anniversary of the birth of the great artist, Diego Velazquez de Silva, by locating his tomb and reinterring his bones. Since early 1999 archaeologists have been digging at the Plaza de Ramales, the former site of the Church of San Juan where Velazquez was buried. They hope to find the remains of his tomb and verify the bones by comparing the DNA from bone fragments with that of known living descendants. Similar work is surely to be done in archaeological projects around the world for many years.

In 1999, Dr. David Goldstein, a geneticist at Oxford University who is attempting to develop genetic signatures of scattered Jewish populations in order to refine our understanding of the diaspora, reported a remarkable discovery. In southern Africa the Lemba, a black Bantu-speaking tribe, have an oral history that they were led out of Judea by a man named Buba. They practice many Jewish customs such as circumcision, and they shun pork and the flesh of any pig-like animal. Goldstein and his colleagues demonstrated that 9% of Lemba men have a DNA sequence on their Y chromosomes that is distinctive of the cohanim (the Jewish priests who are believed to be the descendants of Aaron, the brother of Moses). Interestingly, among those Lemba men in the most senior of their twelve groups, 53% have the cohanim DNA markers. The genetic evidence combined with the cultural history of the Lemba strongly support their claim to Jewish ancestry.

DNA analysis of old bones can even be used to establish cause of death, a form of sleuthing that can provide insight into historical documents. In Luke 4:27 it is written that while in the Jordan Valley, Christ cured a leper named Naaman the Syrian. In 1994, archaeologists uncovered bones from the ancient monastery of St. John the Baptist in a valley near Jericho. Carbon dating indicated that the bones are 1400 years old. Working at University College London, microbiologist John Stanford and surgeon Mark Spigelman extracted DNA from the dried-up marrow of a toe bone. Using DNA probes encoding sequences from Mycobacterium leprae, the bacterium that causes leprosy, the scientists demonstrated that the bone came from a man who had been afflicted with the disease. This effectively proves the presence of this disease in the Jordan Valley at about 600 A.D.

Spigelman argues that this bacterial sleuthing can answer other very important questions. For example, did syphilis originate in the Old World or the New World? For years, historians have asserted that the sailors on Columbus's ships were the vectors who carried the disease to the Americas. If DNA from the spirochete (the bug that causes syphilis has a corkscrew or spiral shape) could be detected in a New World skeleton that was unquestionably older than the 15th century, it would refute that claim.

Of far greater importance is the current effort to use molecular analysis to decipher the genetic code of the RNA virus (RNA is a molecule very similar to DNA that takes its molecular orders to the cytoplasm where it directs the assembly of proteins) that caused the 1918 flu pandemic which killed more than 600,000 Americans and as many as 25,000,000 persons around the globe. Because RNA viruses are extremely fragile when outside of their host cells, there are few places on earth where one might find an intact copy of the agent that caused death in 1918. One is some graves near the coal mining village of Longyearbyen in Norway.

In the winter of 1918, seven healthy young miners working there caught the flu, succumbed in a matter of days, and were promptly buried in the permafrost. Legend has it that dynamite was used to prepare their graves. Thinking that they might be able to track down intact particles of the flu virus, an international scientific team was recently granted permission by the Norwegian government to exhume one or more of the bodies and take tissue samples in which they will search for dormant virus. The hope is that if they find a complete flu genome, the scientists could infer why the virus was so deadly and set the foundation for developing a vaccine to protect the world from an attack by a similar agent in the future. One of the issues that Norway's public health authorities faced was whether there would be a danger of awakening a dormant virus of great lethality. Almost all virologists agreed that this is extremely unlikely, but few were willing to say it is impossible. In the end, the remote risk was viewed as far smaller than the potential benefits that might flow from the research.

Identity testing offers unending adventures to the DNA detectives. James Starrs, the forensic scientist who testified at the Baltimore trial concerning the remains of John Wilkes Booth, also turned up at an interesting trial in Missouri. History recounts that Jesse James, one of America's most notorious outlaws, died on April 3, 1882, when Bob Ford, a 21-year-old who had just joined the gang, killed him for the $20,000 bounty. For decades a few amateur historians have argued that James was too clever to be killed by Ford, and that the murder was a setup to cover James's getaway and retirement from outlaw ways. In 1995, the Missouri judge granted permission to the descendants of Jesse James to open his grave. On their behalf, Starrs compared DNA from a bone and from a lock of hair taken from the grave with DNA from two of James's descendants. Unfortunately, for those who would prefer a more romantic tale, the DNA tests showed that the skeleton is that of an ancestor of the living relatives, and thus must be the remains of the celebrated outlaw.

Ironically, other forces may be foreclosing the opportunity for anthropologists, archaeologists, and molecular biologists to use DNA to solve important questions. Just as we have acquired a greatly enhanced ability to study ancient DNA, there has emerged a strong trend to return remains that have been housed in museums for a century or more to native peoples for reburial. The Museum of Victoria in Australia recently gave back to aboriginal peoples remains that may be 15,000 years old. In 1991 the Smithsonian Institute returned 756 sets of skeletal remains and funerary objects to a tribe that lives on Kodiak Island in Alaska. In Israel a new law requires the reburial of all skeletons less than 5000 years old, a rule that some scientists argue ends anthropology in that nation. The new laws make no allowance for retaining a small bit of bone for DNA studies.

The decision to return remains is motivated in part by guilt over the many atrocities committed against indigenous peoples during the colonial era, a time when simplistic racial thinking was at its zenith. For example, the Australian Aborigines, whom 19th century anthropologists thought represented our missing link to the great apes, suffered untold insults ranging from grave robbing to murder as a means to collect specimens. Another force is surely our great sensitivity to the need to act as stewards of the planet and respect biological diversity in all forms. However, when remains are so old that there is no possible cultural connection with living persons, it seems fair to decide the fate of old bones by asking what use of them best serves the human family. The answer to that may sometimes be that greater good comes from studying them before reburying them.

In the summer of 1999, hikers crossing a glacier in western Canada discovered the remarkably intact remains of a man who almost certainly died before modern Europeans reached that region. If the body turns out to be extremely old (say several thousand years) DNA studies might shed light on the migration patterns from Asia to North America. In planning their studies of the body, scientists are working with representatives of two indigenous groups, both of whom assume the body may be that of an ancestor. In sharp contrast to other, similar situations, because of the respect shown by the scientists to the beliefs of the native Americans, the two tribes have cooperated with the research. It is hoped that this will provide a model for research with other discoveries.

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