A new study, however, is reopening the book.
A team led by Alec Knight, a senior scientist in the Stanford lab of anthropological sciences Assistant Professor Joanna Mountain, argues that previous DNA analyses of the purported Romanov remains - nine skeletons unearthed near Ekaterinburg in central Russia - are invalid. Knight and his colleagues base their claim on molecular and forensic inconsistencies they see in the original genetic tests, as well as their independent DNA analysis of the preserved finger of the late Grand Duchess Elisabeth - sister of Tsarina Alexandra, one of the 1918 victims - which failed to match the tsarina's own DNA. The Stanford team's findings are reported in the January/February issue of the Annals of Human Biology.
The original DNA analysis was arranged by the Russian government's Commission on the Identification of the Remains. As reported in Nature Genetics in 1994, Peter Gill of Britain's Forensic Science Service and Pavel Ivanov, a Russian geneticist from the Engelhardt Institute in Moscow, conducted a battery of experiments supporting the hypothesis that the Ekaterinburg bones belonged to the Romanovs. The team performed DNA-based sex testing and analyzed short sequences of DNA from the nucleus of bone cells to establish that the remains of the alleged tsar, tsarina and three daughters belonged to the same family.
To solidify these conclusions, Gill and Pavel also examined DNA from mitochondria, the energy-producing organelles within cells. Compared to DNA found inside the nucleus, mitochondrial DNA preserves well in bones and acquires mutations very slowly - making it a prized specimen for multigenerational forensic analysis. But there is a catch: Mitochondrial DNA is passed only along the maternal line. For the 1994 study, researchers determined the sequence of mitochondrial DNA fragments from the presumed Romanov skeletons and found that they matched DNA sequences obtained from blood samples of Britain's Prince Philip (Tsarina Alexandra's grandnephew) and two living relatives of the tsar's maternal grandmother.
Knight argues these results are too good to be true. In particular, he doubts the researchers could have obtained such long stretches of DNA sequence (a string of 1,223 bases, DNA's chemical building blocks) from old bones. Citing standards for ancient DNA analysis that were established several years after the 1994 publication, Knight contends that DNA from skeletal remains that spent over 70 years in a shallow, earthen grave would have degraded so severely that sequences longer than 250 bases would have been nearly impossible to recover in lab experiments.
''Based on what we know now, those bones were contaminated,'' Knight said. He considers the successful amplification of a 1,223-base sequence from all nine skeletons in the original study as ''certain evidence'' that the bone samples were tarnished with fresh, less-degraded DNA - perhaps from an individual who handled the samples.
As reported recently in Science, Gill maintains that his team's DNA analysis ''set the standard.'' He says that Knight's paper mischaracterizes his work and ''comes across as vindictive and political.''
Peter de Knijff, head of the Forensic Laboratory for DNA Research at Leiden University Medical Center in the Netherlands, agrees with Knight's assessment that the Gill-Ivanov study was ''unrealistically solid.''
De Knijff's qualms about the original study also stem from Ivanov's unwillingness to disclose results from his analysis of a blood-soaked handkerchief that Tsar Nicholas II used to treat a head wound suffered after he was struck by a would-be assassin in Japan in 1891. The handkerchief is a ''potentially pristine source of DNA of the last tsar,'' according to de Knijff, noting that Ivanov refused to disclose experimental details and claimed that the handkerchief DNA was degraded and hence analysis was unfeasible - an assertion that other scientists dispute.
These sorts of irregularities provided the original impetus for Knight's entry into the forensic debate. About three years ago, Daryl Litwin, an author of the Knight et al. paper who was studying law in Sacramento, proposed to Knight the idea for a re-analysis of the Gill-Ivanov data after reading Robert Massie's book The Romanovs: The Final Chapter. ''I just kept finding contradiction and discrepancy from point to point,'' Litwin said. ''I was left kind of befuddled.'' Before approaching Knight, Litwin discussed his ideas with a Russian history expert at the Hoover Institution at Stanford, who agreed that the Romanov verdict was worth re-examining.
Months later, Knight went to New York to procure a small wooden case containing a finger of Grand Duchess Elisabeth. Since the 1982 opening of Elisabeth's coffin in Jerusalem, the finger had been preserved in a reliquary at the New York home of Bishop Anthony Grabbe, president of the now-disbanded Orthodox Palestine Society.
Though Knight's trip was funded by the Russian Expert Commission Abroad - a group of about 20 scholars in the West and Russia who challenge the assertion that the bones are royal - Knight maintains that his experiments were unbiased. ''[The Commission Abroad] didn't do the science,'' he said. ''They just bought me the plane ticket and got me the sample. They had no control over the work.''
Nevertheless, some scientists - several of whom participated in the original DNA analyses - are unconvinced by Knight's conclusions. Mark Stoneking, a molecular anthropologist at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, concludes that ''it is certainly plausible that DNA preservation was sufficient to permit amplification of large fragments.''
Tom Parsons of the Armed Forces DNA Identification Laboratory in Rockville, Md., and Victor Weedn, a forensic scientist who established the U.S. military's DNA identification program, agree that the discovery of the Ekaterinburg remains in an area of permafrost explains how larger DNA fragments were stable enough to be recovered in the original analyses.
Knight agrees that frozen DNA is more stable but points out that Ekaterinburg - which is at the same latitude as Moscow and just north of Kazakhstan - can reach 100 degrees Fahrenheit in July and August.
Meanwhile, as scientists squabble over finer details of the forensic analysis, historians seem content with a more holistic view of the Romanov drama.
''There may be some ambiguity about which physical remains belong to whom, but this uncertainty doesn't really change our fundamental understanding of the Russian Revolution and the nature of Bolshevism,'' said Robert Crews, an assistant professor of history at Stanford.
Donald Ostrowski, a Russian historian at Harvard University, said he had doubts about the Ivanov and Gill analysis of the bones, so he ''just decided to eliminate it from [his] consideration of the historical evidence.'' Though he has concluded from probabilistic analysis of existing evidence that the bones belong to the Romanovs, Ostrowski remains open to hearing new evidence or re-analysis of old data. ''The case is by no means closed,'' he said.
Esther Landhuis is a science-writing intern at the Stanford News Service.
By Esther Landhuis
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