The Shadow King

by Jo Marchant

  * James Harris was the only exception, concluding in 1980 that the mysterious KV55 monarch died in his thirties. Frustratingly, he never published the reasoning behind this conclusion.

  * Timmann and Meyer have an alternative theory for Tutankhamun’s death—sickle-cell disease (SCD). In SCD, a mutation in the gene for hemoglobin causes red blood cells to become rigid and sickle-shaped. Sufferers have severe anemia and often die young. The disorder can also cause bone necrosis like that seen in Tutankhamun’s mummy, when deformed blood cells get stuck in the tiny capillaries of the feet. Hawass’s team initially responded that the idea was “an interesting and plausible addition to the palette of potential disease diagnoses in Ancient Egyptian royalty that we are currently investigating.” Timmann and Meyer have developed a test for the SCD gene, so they offered to collaborate with the Egyptian team, but say they received a letter from Hawass that “somewhat roughly” declined any cooperation, and claimed to have ruled out SCD two years earlier.

  * Apparently, he’s using a bow rather than fishing rod because of the special royal status that this weapon had in ancient Egypt.

  CHAPTER FIFTEEN

  DNA DOWN THE RABBIT HOLE

  “IT’S SO FRUSTRATING,” says Eline Lorenzen, which is barely necessary as I can hear frustration if not anger dripping from every word as she speaks to me on the phone from her lab in Copenhagen. “Now everyone will be taught in school that King Tut died of malaria.”

  Lorenzen works at the Centre for GeoGenetics, part of Denmark’s Natural History Museum. Led by evolutionary biologist Eske Willerslev, it is a leading center for ancient DNA research, where researchers tease aging genetic material out of everything from killer whales to moas. Lorenzen herself studies the DNA of large prehistoric animals such as the mammoth and woolly rhino (extracted from remains found frozen in permafrost in places like Canada and Siberia), to investigate whether climate change or hunting by humans finished them off.*

  Together with Willerslev, she wrote the letter to JAMA that criticized Zink, Pusch, and Gad’s DNA analysis of the royal mummies.1 She tells me that she felt obliged to speak out about it after seeing the huge press coverage that their results gained. “This is not seen as a rigorous study,” she says. “When working with samples that are so well-known, it is important to convince readers that you have the right data. I am not convinced.”

  To find out if other experts agree with her, I start calling around. Although no one comes out and says the data on Tutankhamun and his family are definitely wrong, I have trouble finding anyone who believes them. “I’m very skeptical,” says Willerslev. The study “could do a much better job,” complains Svante Pääbo of the Max Planck Institute for Evolutionary Biology in Leipzig, one of the founders of the ancient DNA field. “I would be extremely cautious in using these data,” agrees Ian Barnes, an expert on the survival of ancient DNA, based at the Royal Holloway University of London. The DNA analysis of Tutankhamun and his family might have been a media sensation, but behind the headlines, many scientists seem to have written it off.

  Despite Zink’s track record in publishing DNA from Egyptian mummies going back five thousand years, the critics don’t believe that his team could possibly have detected the DNA that they claimed. And it’s not just Tutankhamun. Enter the world of ancient DNA and you are soon asked to choose between two alternate realities: one in which DNA analysis from Egyptian mummies is routine, and the other in which it is impossible. “The field is split absolutely in half,” says Tom Gilbert, a young professor who heads two research groups at Willerslev’s geogenetics center.

  One camp—the scientific mainstream, including the biggest labs such as Willerslev’s and Pääbo’s—argues that DNA from most Egyptian mummies simply doesn’t survive well enough to be studied. Meanwhile, the other camp has been happily publishing and building careers on this very DNA for years. Unable to resolve their differences, the two sides publish in different journals, attend different conferences, and refer to each other as “believers” and “skeptics”—when, that is, they’re not simply ignoring each other.

  To understand their feud, it helps to take a quick look at the history of the ancient DNA field. American biologist James Watson and English physicist Francis Crick famously worked out in the 1950s that DNA is made up of long strands twisted together in pairs to form a “double helix.” Each strand consists of a sequence of different molecular groups called bases, which have long unwieldy names but are given the letters A, C, G, and T for short. The order of these bases forms the instruction manual for how to build each living being, like a book written using a four-letter alphabet. Each base—in other words each letter in the sequence—is paired with a corresponding base in the opposite strand, forming what geneticists call a “base pair.”

  In the following decades, scientists worked out ways to manipulate DNA—cutting it into pieces, sticking it back together, reading its sequence, and so on. But it took until the 1980s before anyone managed to extract DNA from an organism that was long dead. This is because DNA degrades over time, with the long strands breaking up into smaller and smaller pieces, until eventually nothing readable remains. Studying DNA from ancient samples requires isolating and amplifying the tiny amounts of genetic material still left. The older the sample, the harder it is.

  In 1984, scientists from California extracted DNA from the quagga, a recently extinct relative of the horse.2 The DNA was from a museum specimen, just 140 years old. Most experts wrote off the idea of properly ancient DNA, but a young PhD student named Svante Pääbo, at the University of Uppsala in Sweden, was convinced it could be done. Behind his supervisor’s back, he persuaded an Egyptology professor at the university to help him take samples from some Egyptian mummies at the local museum in Uppsala, and at another in Berlin. He used a scalpel to remove little pieces of tissue from more than twenty mummies, then worked nights and weekends to isolate their DNA.

  This involved trying to purify DNA from the samples, then introducing it into bacteria. As the bacteria then divide and grow, so does the DNA, until there’s enough to analyze. This is called cloning, but it’s not to be confused with the procedure by which geneticists can clone entire organisms. All that is cloned in this case is a small piece of DNA.

  Pääbo’s mummies yielded no results—except for one. From a one-year-old boy who died more than two thousand years ago, he managed to clone several chunks of DNA. The student came clean to his supervisor, and was rewarded with a paper in Nature, one of the world’s most prestigious science journals. It was the second ancient DNA paper ever published, and the first to show the potential for analyzing DNA thousands of years old.3

  A few years later, the newly invented technique of PCR began to revolutionize the field. Because PCR can amplify any desired DNA sequence by copying it over and over again, ancient DNA researchers no longer had to worry about the tricky process of cloning. Even if their samples only contained a few molecules of DNA, they could use PCR to convert that into large amounts.

  This led to a burst of excitement, with papers triumphantly reporting the DNA of everything from prehistoric plants to insects preserved in amber. Most impressive of all, in 1994, was DNA from an 80 million-year-old dinosaur,4 published in the journal Science by Scott Woodward (the Mormon from Utah who shortly afterward missed out on DNA testing Tutankhamun’s mummy). It seemed that ancient DNA was about to provide an incredible, almost magical, window into past life on this planet.

  Then came the fall. Scientists started to realize that when applied to ancient remains, PCR comes with a huge downside. The method is susceptible to contamination with unwanted DNA at the best of times—it’s so sensitive that it can latch onto and amplify stray molecules of DNA from the surrounding environment, instead of from the sample. Usually, it’s fairly easy to control for this, but it turns out that the problem is magnified when you’re trying to amplify tiny amounts of old, broken-up DNA. The reaction works much better on modern DNA, so any trace of contamination—say, from
the skin cell of an archaeologist who handles a sample, or a speck of dust in the lab—can dwarf any ancient DNA present and wreck a result.

  When researchers started looking more closely at the DNA they had isolated from their ancient samples, they knew they had a huge problem. In most cases, the DNA they had reported so proudly wasn’t ancient at all. Woodward’s “dinosaur” DNA belonged to a modern human,5 as did Pääbo’s pioneering clone6 (in both cases the DNA probably came from the researchers themselves). In another study on monkey mummies, DNA assumed to be from the ancient monkeys turned out to belong to pigeons that nested in a storehouse where the mummies had once been kept.7

  The field had to start again. After much soul-searching, researchers introduced hugely strict criteria to minimize the risk of contamination. These include only using workspaces where DNA hasn’t been amplified before, reporting any contaminated results, sequencing amplified DNA to check its origin, and repeating all experiments in two independent labs.8

  Even with these precautions, several top researchers in the field, including Pääbo—who is now ancient DNA’s grand old man, helping to set the standards for everyone else—decided that it was still too risky to work on ancient human or microbial DNA. If a scientist manages to amplify and sequence mammoth or cave bear DNA, they can be fairly certain that it wasn’t just floating around in their lab. But if they retrieve DNA from a human or bacterium, they can’t be sure that it came from their ancient sample as opposed to from a modern person, or bacteria present in the environment. And if they can’t be sure, what is the point in doing the work? Labs such as Pääbo’s and Willerslev’s focused on other types of specimen, such as extinct prehistoric species for which there are no equivalents today.

  This left all work on DNA from human mummies extremely contentious. Egyptian mummies, however, were the most controversial of all. In addition to the problems with contamination, there were heated arguments over whether they even contain any DNA in the first place.

  As DNA degrades over time, it breaks into shorter and shorter pieces, and the rate at which this happens rises rapidly with temperature. To obtain a useful sequence using PCR, you need DNA fragments at least seventy base pairs long, and how long these survive in a sample depends largely on how hot it is. You can amplify DNA from a mammoth that has been frozen in permafrost for tens of thousands of years, whereas bones buried in the tropics for only a few centuries might yield nothing.

  In Egypt’s baking climate, the skeptics argue that the chance of amplifiable DNA surviving in three-thousand-year-old mummies like Tutankhamun and his family is vanishingly small. In 2005, Tom Gilbert and colleagues calculated that fragments a hundred base pairs long would survive only five hundred years or so at around 80° F, the estimated temperature of a sealed Egyptian tomb.9

  Another ancient DNA researcher, Franco Rollo of the University of Camerino in Italy, put this to the test.10 He checked pieces of papyrus of various ages, from modern samples to archaeological finds up to three thousand years old, preserved in similar conditions to mummies. He estimated that DNA fragments large enough to be amplified by PCR vanished after around six hundred years.

  Accordingly, once ancient DNA researchers started using rigorous controls against contamination, they had little luck getting DNA from Egyptian mummies. Rollo, who is best known for his work on Ötzi the Iceman, the five-thousand-year-old mummy found frozen in the Alps, drew a blank with two-thousand-year-old mummies from Saqqara in Egypt. He and his colleagues concluded: “In laboratories where rigorous criteria for the control of contamination are applied, the analysis of human and animal remains from Egyptian archaeological sites most frequently ends with the indication that no authentic DNA is left.”11

  “Preservation in most Egyptian mummies is clearly bad,” agrees Pääbo. In the past, he too has had little luck trying to get DNA from Egyptian mummies. In one study published in 1999, he tested 132 mummies but only managed to get DNA from two of the youngest ones.12 After such disappointing results, many researchers lost interest in Egyptian samples, and focused instead on remains that have been preserved in cold conditions.

  But here’s where the story gets weird. Throughout all of this, other scientists have been publishing papers on DNA extracted from Egyptian mummies up to five thousand years old, far older than Tutankhamun. These researchers are just as convinced that the DNA does survive, as the other experts are that it doesn’t. For example, Zink and his colleagues have reported ancient DNA from a range of disease-causing bacteria in hundreds of mummies, including the strains that cause tuberculosis (TB) and diphtheria, as well as the parasites responsible for malaria and leishmaniasis.13 Zink’s published work has led to insights into the past evolution of these pathogens that he hopes will help us to understand and predict the spread of these diseases, today and in the future.

  And in a high-profile study published in 2010, microbiologist Helen Donoghue of University College London reported tuberculosis DNA from Dr. Granville’s mummy (named after physician Augustus Bozzi Granville, who carried out the first mummy autopsy on it in front of fellows of the Royal Society in 1825).14 Donoghue says the idea that DNA can’t survive in Egyptian mummies is “rubbish,” and argues that theoretical and papyrus studies are of little relevance to what actually happens in mummies. She is studying early Christian mummies from Nubia, and says that around a third of them are testing positive for tuberculosis.

  Skeptics such as Ian Barnes and Tom Gilbert believe that the researchers claiming DNA from Egyptian mummies often confuse modern contamination for ancient DNA, just as the field’s pioneers did in the 1990s. Often these are groups who have moved into ancient DNA more recently from medical diagnostics, and critics feel they haven’t learned the lessons of those early mistakes. Barnes argues that some TB studies of ancient mummies report positive results “higher than if you contracted TB, turned up in a clinic, and got tested.” For malaria too, he points out that even for blood tests on living patients with an active outbreak of the disease, a PCR test only works about 75 percent of the time, so he finds it hard to believe that it could possibly work on bone samples from ancient mummies. Gilbert is slightly more blunt. “I’ve given up on the field a long time ago,” he says. “It’s full of crap.”

  Donoghue acknowledges that you have to be “super-careful” when studying ancient human DNA. But she insists that the studies on microbial DNA prove that genetic material does survive in these mummies, and points out that in some cases, the DNA results have been confirmed by detecting signs of lipids (fat molecules) from the pathogens concerned. She argues that the big labs have gone over the top in trying to compensate for the field’s difficult past, and counters that their criticisms have more to do with territorialism than valid scientific concerns. Zink agrees, adding that no matter how rigorous his studies, the skeptics refuse to believe his results. “It’s like a religious thing,” he says. “If our papers are reviewed by one of the other groups, you get revisions like, ‘I don’t believe it’s possible.’ It’s hard to argue with that.”

  By 2010, the two sides had pretty much stopped talking to each other. “There’s enough dead stuff around—you’re not obliged to get into anyone else’s area,” says Barnes. That year, ancient DNA researchers held two rival conferences: the skeptics’ International Symposium of Biomolecular Archaeology, in Copenhagen in September, and the believers’ International Conference on Ancient DNA, in Munich the next month. Hardly anyone went to both.

  This, then, is the tense atmosphere into which the royal mummy study dropped like a bomb. Zink and his team were studying by far the most controversial type of ancient DNA (human) on the most controversial possible subjects (Egyptian mummies). They also reported unprecedented success compared to even the most optimistic previous studies; not only did they retrieve DNA from every single mummy they tested, they were able to amplify relatively long fragments of DNA for their age, up to 250 base pairs, which even other believers describe as remarkable. And, of course, the study gained glo
wing media coverage, with its findings filtering down into living rooms and classrooms around the world. To say that it reignited tensions between the two camps would be something of an understatement.

  So who is right? Well, whether or not you believe the team’s results basically comes down to whether you think they have convincingly ruled out the possibility of contamination. Skeptics who have scrutinized the JAMA paper claim they haven’t.

  One concern is whether Gad took adequate precautions when he collected his bone samples from the royal mummies. “There is a complete lack of information about how this was done,” says Lorenzen. “It rings alarm bells.” With no details in the paper itself, DNA researchers worldwide were left in the surreal situation of trying to assess the quality of the work by watching the TV documentary. Lorenzen says that before writing her letter of complaint to JAMA, she emailed Zink and his colleagues several times asking for more details of their methods, but received no reply (a member of Zink’s team says this may have been because of her “un-collegiate” tone).

  Gad drilled deep inside the mummies’ bones, where contaminating DNA is less likely to reach. This isn’t a guarantee, however, and it’s debatable how effective it would be on Tutankhamun’s thin, fragmented skeleton, not to mention the tiny matchstick bones of the mummified fetuses. The team also tested the lab staff to rule out any contamination with their own DNA. But of course this doesn’t exclude DNA from all the people to have handled the mummies previously, from early archaeologists such as Gaston Maspero and Howard Carter to the workmen who moved Tutankhamun into his new case just a few months before the DNA samples were taken. Unlike Gad, none of these men wore gloves or masks. “You see people on TV handling samples with their bare hands, their sweat dripping onto the mummy,” notes Gilbert. “That’s a classic route of contamination.”