But not everyone was persuaded. Some insisted that the bodies couldn’t belong to the Romanovs, because there were only five related skeletons, not seven. In Japan, meanwhile, a forensic scientist, Tatsuo Nagai, performed DNA analysis on a handkerchief stained with Nicholas II’s blood after a would-be assassin attacked the czar with a sword in Oda, Japan, in 1890. Nagai and a Russian colleague reported in 1997 that mitochondrial DNA from the bloody handkerchief did not match that from the bones the experts had determined to be Nicholas’. (The results were never published in a peer-reviewed journal and were not replicated; the findings have not gained acceptance.) Compounding the confusion, a forensic scientist at Stanford University obtained a finger bone of Alexandra’s older sister, Elizabeth, who had been shot by Bolsheviks in July 1918 and tossed down a well. The mitochondrial DNA from the finger, he reported, was not consistent with DNA from the skeleton identified as that of Alexandra.
Those findings caused controversy, but scientists working with the Russian government contend that both the bloody handkerchief and the finger had been contaminated with DNA from other sources, throwing off the results. Using this 80-year-old bone as a reference, says Coble, “ignored the entirety of the evidence.”
President Boris Yeltsin and the Russian government agreed with Gill, Ivanov and the other forensic scientists. On July 17, 1998—the 80th anniversary of the killings—the remains that had first been uncovered in 1979 were interred beside other members of the Romanov dynasty in a chapel in St. Petersburg’s state-owned Peter and Paul Cathedral.
Russian Orthodox Church authorities insisted that the remains were not those of the Romanovs. The Russian Orthodox patriarch, Alexei—with the support of several key Romanov descendants—refused to attend the ceremony.
Ever since the Romanov bones came to light, Gribenyuk had yearned to locate the still-unrecovered remains of Maria and Alexei. Gribenyuk suspected that the czar’s daughter and son were buried near the timber-covered grave that held the other Romanovs. In 2007, he put together a team of a half-dozen amateur forensic sleuths and headed for the Old Koptyaki Road. On their third search of the area, on July 29, 2007, they located some 40 bone fragments, buried in watery soil at a depth of about one and a half feet, 230 feet from the other members of the royal family.
Coble, the U.S. Army scientist, analyzed the bone fragments and extracted mitochondrial and nuclear DNA from both specimens. He compared the results with data from the remains attributed to Nicholas, Alexandra and their three daughters.
His analysis showed that mitochondrial DNA from the bone fragments of the unidentified boy and girl was distinctly similar to that from Czarina Alexandra. Further analysis using nuclear DNA—which, again, is inherited from both parents—indicated “it was four trillion times more likely” that the young female was a daughter of Nicholas and Alexandra than that she was unrelated, Coble says. Likewise, it was “80 trillion times more likely” that the boy was a Romanov rather than an unrelated male.
Coble and other scientists conducted an additional genetic test, involving analysis of markers on Y chromosomes—genetic material passed down through the paternal line. They compared the boy’s Y chromosome with those from the remains of Nicholas II as well as a living donor, Andrei Romanov, both of whom were descended from Czar Nicholas I. The testing, says Coble, “anchors Alexei to the czar and a living Romanov relative.”
Finally, Solovyev, the Moscow investigator, remembered that a bloody shirt worn by Nicholas on the day of the assassination attempt in Japan had been given, in the 1930s, to the Hermitage Museum in St. Petersburg. The shirt had not been seen for nearly 60 years. It was eventually traced to a storage-room drawer. Because of the age of the blood and the possibility of contamination, “I was absolutely skeptical [of getting a good DNA sample],” says Rogaev, of the University of Massachusetts. “But it worked even better than the bone samples.”
“This was the critical thing,” says Coble. “We now had a sample of the czar’s blood, and we had bone samples from after his death. We had living and post-mortem DNA. And they were a perfect match.”