by Pbo, Svante
Johannes had come to our department as an undergraduate specializing in biochemistry. It soon turned out that he was not only very good at bench work but had good judgment and comprehension of all the complex experiments going on in the group. I always enjoyed talking with him, but as the months passed he seemed to bring me only bad news. None of the many extracts he prepared from various Neanderthal bones contained anything like the amount of Neanderthal DNA we had seen in Vi-80. Most of them contained no Neanderthal DNA at all, or so little that he could barely detect Neanderthal mtDNA by means of the PCR. We urgently needed more and better bones.
The obvious place to go was back to the Institute for Quaternary Paleontology and Geology in Zagreb, where the Vindija collections, including the remainder of the Vi-80 bone, were housed. In April 2006, I had written to the Zagreb institute. I said that we were interested in sampling the bone we called Vi-80{49} again and perhaps other bones excavated between 1974 and 1986 by Mirko Malez in Vindija Cave. Sadly, I learned that Maja Paunovic, with whom I had worked in 1999, had died. There was now no paleontologist in charge of the collection. The head of the institute was Milan Herak, an emeritus professor of geology at the University of Zagreb, who was eighty-nine and rarely if ever visited it. An elderly lady by the name of Dejana Brajković did the day-to-day work, together with Jadranka Lenardic, her younger assistant. I wrote a letter to both women, explaining that we would like to continue our successful collaboration on the Vindija collection—a collaboration that had already resulted in three high-profile publications. I suggested visiting them to discuss this and perhaps sample a few more of the bones. We agreed that I would visit Zagreb and give a seminar at the university on our work. But in May 2006, four days before Johannes and I were to leave for Zagreb, I received an e-mail saying that it would be impossible to sample any of the Vindija bones. The bones had to be “registered,” they said, and only after that event, at some undetermined time in the future, would it become possible to work with the bones. I sensed that someone else was behind this sudden turn of events. Their letter mentioned Jakov Radovčić, a famous paleontologist who curates the huge collection of much older Neanderthal bones found at Krapina housed in the Croatian Museum of Natural History in Zagreb. Although he had no formal authority over the Vindija collection, which belongs to the Croatian Academy of Sciences and Arts, I suspected that he wielded enough unofficial influence over the two women at the institute to have interfered with our arrangements. Still, I decided not to take no for an answer and go anyway. It seemed to me that the scientific promise of our project would be enough to persuade the people in Zagreb that our work should proceed.
Johannes and I arrived in Zagreb in early June and went directly to the institute, where I had spent considerable time with the late Maja Paunović several years before. It was still a rather dusty place, not exactly bristling with energy. Dejana Brajković and her assistant seemed nervous about our visit. They refused to let us see, let alone sample, the specimens and said that we would have to consult the Academy of Sciences and Arts before doing so. But after drinking coffee and chatting with them for a while, we were at least allowed to look at the bones. Some parts of the collection were in disarray, which may have contributed to their reluctance to allow us to work with it. I felt that establishing a proper catalog of the bones was a very good idea indeed. I was particularly attracted to a box of bones that Tim White, a well-known paleontologist at UC Berkeley, had set aside when he studied the collection a few years earlier. It contained fragments of bones that the excavator Mirko Malez had thought were from cave bears but that Tim thought could potentially stem from Neanderthals.
Looking at these bone fragments, I was reminded of something Tim had mentioned to me when we met at Berkeley a year earlier. The Vindija Neanderthal bones—all of them—were crushed into small fragments. This is typical of many, even most, sites where Neanderthal bones are found. Of course, it is not surprising that bones thousands of years old are not in good condition. But there are often cut marks on the bones where muscles and tendons had been attached as well as cut marks on the skulls. In short, the skeletons had clearly been deliberately de-fleshed, and bones containing marrow had been crushed, presumably to get to their nutritious contents. Tim had pointed out to me the similarity of this pattern of Neanderthal bone fragmentation to a gruesome Anasazi site from the American Southwest, where around AD 1100 some thirty men, women, and children had been butchered and cooked. He told me that the way in which many Neanderthal bones were crushed was similar to the way the bones of animals, such as deer, that were butchered by Neanderthals were crushed (see Figure 12.1). We will probably never know how common it was for Neanderthals to kill and eat other Neanderthals, or, indeed, whether these Neanderthal corpses might have been butchered and perhaps eaten as part of some mortuary ritual. But given that Neanderthal skeletons are found intact at some sites, and sometimes even positioned in ways that suggest deliberate burial, it seems likely that the Neanderthals in Vindija Cave had been unlucky enough to run into hungry neighbors.
Figure 12.1. The bone 33.16 from Vindija Cave that we used for sequencing the Neanderthal genome. It has been crushed, presumably to get to the nutritious marrow. Photo: Christine Verna, MPI-EVA.
Oddly, the fact that the Vindija Neanderthals were cannibalized, or at least de-fleshed, by other Neanderthals may be responsible for the fact that we had found rather a lot of Neanderthal DNA and relatively little bacterial DNA in at least some of the Vindija bone fragments. If the Neanderthal corpses had been buried, months would have passed before all its soft tissues were consumed by bacteria and other microorganisms. There would thus be ample time for bacteria to penetrate the bones, degrade the Neanderthal cells and their DNA, multiply, and eventually die themselves. Extracting DNA from such a bone would mostly yield DNA of microorganisms. If, on the other hand, the Neanderthal had been butchered, the bones crushed, gnawed, and sucked free of any meat and marrow before being tossed aside, some bone fragments would quickly dry out, limiting the chance for bacteria to multiply in them. Thus, we might have Neanderthal cannibalism to thank for the success of retrieving DNA in some specimens from Vindija.
All this went through my head as I looked in the box containing bones so crushed that it was impossible to tell whether they came from animals or Neanderthals. I turned to Dejana Brajković and asked if we could at least sample some of these fragments, whose source was so ambiguous. I argued that if DNA were preserved in them, we could determine what species they came from. But Brajković was adamant; we could not touch any of the bones. She said she had heard that in a few years one would be able to hold a sensor close to a bone and thereby determine its entire genome sequence; thus it was inadvisable to sacrifice even a tiny part of a bone fragment now. I agreed that techniques would certainly improve in the future but expressed polite doubt about whether we would live to see the advance she envisioned. Again, I suspected the influence of powers greater than hers. I said we would discuss our needs with the Croatian Academy and be in touch.
In the afternoon, we visited Jakov Radovčić at the Museum of Natural History. He appeared to be supportive of our project but expressed grave reservations about the sampling of any bones in either the Krapina or Vindija collections. I was sure we had not yet gotten to the bottom of things, and in a gloomy mood we returned to our small and scruffy hotel room. I lay on the bed, gazing at the paint peeling from the ceiling and feeling completely frustrated. As far as I knew, these were the bones containing the best Neanderthal DNA in the world. Many of them were of little or no morphological value, so fragmented that you couldn’t even tell whether they came from a Neanderthal, a cave bear, or some other animal. Yet some unknown person with influence over the people at the institute was apparently determined to make it impossible for us to work on them. Like a child denied his favorite candy, I felt like screaming and kicking, but my Swedish upbringing kept me from venting in such an obvious way. Instead, Johannes and I spent the evening in a bad restauran
t around the corner from our hotel, brooding about our mysterious enemy.
The next day I gave a talk before the medical faculty of the University of Zagreb about ancient DNA in general and our Neanderthal work in particular. It was well attended, and many of the students asked questions. It cheered me a bit that some young people in Zagreb were enthusiastic about science. In the evening, we had dinner with Pavao Rudan, a professor of anthropology at the university, who stems from an old family of landowners on the beautiful island of Hvar, off the Adriatic coast. He invited us to join him and his colleagues at a restaurant named Gallo, which turned out to be one of the best restaurants I have ever been to. Course after course of excellent seafood and creative Mediterranean dishes were served, along with good wines. The meal was rounded off by a wonderfully refreshing drink of fruit juice, champagne, and some other ingredient that I couldn’t make out. I felt slightly better. Then, Pavao started to talk about science. As it happened, my conversation with him was to improve my spirits in a much more lasting way than the exquisite dinner.
First, we talked about his work on small human populations on the Croatian islands. He was trying to find genes and lifestyle traits that contribute to common disorders, such as high blood pressure and heart disease. For many years, he had had grants from the United States, France and the UK for this project, which testified to his scientific credentials. I figured that he would recognize a good project when he heard about it, so I talked at length about our plans and our problems. Pavao listened to my plight with sympathy and was willing to help. Crucially, he knew how to navigate the Byzantine politics of Croatia. He told me that he had just been elected to the Croatian Academy of Sciences and Arts and would soon be inducted as a member. He suggested that we approach the project not just as a collaboration between our research group and the Zagreb institute where the Vindija collection was housed but as a collaboration between the Croatian Academy and another academy—that is, one I belonged to as a member.
I was in fact a member of several scholarly academies. Such memberships are honors that I had until then regarded as quite irrelevant to my everyday science. I never attended their meetings, having imagined that they consisted of earnest discussions attended by esteemed scientists well into their dotage. But now they suddenly seemed important. Which academy should we approach? I suggested the National Academy of Sciences in the United States, perhaps the most prestigious academy membership I held, but Pavao advised against it. He suggested we should rather approach some academy in Germany. We settled on the Berlin-Brandenburg Academy of Sciences and Humanities, of which I had been a member since 1999. He suggested that I approach the president of the Berlin Academy and ask him to write to the president of the Croatian Academy to propose our project as a collaboration. He also advised me to wait a few weeks, until he had been inducted into the Croatian Academy. Along with other sympathetic members, he could then put in a good word for the project with its president.
The next morning, Johannes and I flew back to Leipzig. I was feeling a bit more optimistic. We didn’t have the bones with us, as I had hoped, and persuading the Croatian Academy that it would be in the best interests of science to work with us remained a challenge. But with the help of Pavao, we might have a chance.
Back home, I immediately phoned Günter Stock, president of the Berlin-Brandenburg Academy. He listened intently and was ready to help; he liked the idea of strengthening ties with Croatia. With the help of his assistant for foreign relations, I drafted a letter from him to the president of the Croatian Academy, proposing the Neanderthal Genome Project as a collaboration between the two academies. We also suggested that we would be willing to support the establishment of a catalog of the Vindija collection, by donating a computer and resources for someone to do the work.
But I didn’t leave it at that; I wanted to do everything I could to overcome the mysterious resistance in Zagreb. One way to do this would be to involve all relevant parties there in the project. So I wrote to Jakov Radovčić and invited him to the upcoming July press conference with 454, suggesting that he present the paleontological aspects of Neanderthals to the press. He responded that he had other obligations that made it impossible for him to attend. I also contacted Frank Gannon, director of EMBO, the European Molecular Biology Organization, to which I also belonged, and asked him to contact Dragan Primorac, the Croatian minister of Science, Education and Sports, on our behalf. Dragan Primorac is an unlikely politician. He is a professor of forensic science at the University of Split in Croatia as well as an adjunct associate professor at Penn State University in the United States. Dragan, who has since become a friend, answered that he would put in a good word for our project with the academy. I had no idea whether all these initiatives would help our project, but I wanted to leave no stone unturned.
In the meantime, the letter from Professor Stock on behalf of the Berlin-Brandenburg Academy formally proposing the Neanderthal project, as well as a letter from me, had arrived at the academy in Zagreb. Pavao Rudan, asked by his colleagues for his opinion, suggested some conditions for the proposed collaboration: at least one Croatian co-author should appear on all papers we were to publish on the Vindija material, the Croatian Academy should be mentioned in the acknowledgments, and at least two scientists from Croatia should be invited to Leipzig each year as long as the project lasted. I agreed to these conditions and added that we, along with the Berlin Academy, would support the establishment of a catalog of the Vindija collection.
All this took time. Summer turned into fall, and fall turned into winter. I was meanwhile pursuing other promising Neanderthal sites, concentrating on places where our previous work had shown that DNA was preserved. The first and most obvious one was the Neander Valley site itself, where the type specimen had been found in 1856. At that time, the cave had not been scientifically excavated but was emptied out by quarry workers, who collected bones as they happened to notice them. Since then, the entire cave, as well as the small mountain where it was situated, had been quarried away for limestone. Frustratingly, many of the bones of the type specimen had never been collected. Some years earlier, Ralf Schmitz, with whom we had worked on the type specimen, had had the crazy but brilliant idea of trying to find the missing bones. After painstaking investigation of old maps, long walks in Neander Valley, and the exercise of a great deal of intuition, he had managed to find the place, then partly under a garage and car-repair shop, where much of the cave debris had been deposited 150 years ago. He began an excavation, and his efforts paid off handsomely in that he found not only fragments of the type-specimen individual but also bones from a second individual. In 2002, we retrieved mtDNA from this individual and published it with Ralf.{50} Now Johannes returned to the bits of samples we had left and did new DNA extractions, analyzing them with our new methods to look for nuclear DNA. The results were discouraging. The extracts contained between 0.2 percent and 0.5 percent Neanderthal DNA—not enough to sequence the genome.
Another site, Mezmaiskaya Cave in the northwestern Caucasus, had been excavated by an archaeologist couple, Lubov Golovanova and Vladimir Doronichev, who were based in St. Petersburg, Russia. They had found remains of a small Neanderthal child. This child had probably been deliberately buried, and not consumed, as all its bones were intact and found in their expected positions. An exciting aspect of this child was that while the Neanderthals we had analyzed up to that point were all about 40,000 years old, this baby was between 60,000 and 70,000 years old. Lubov and Vladimir had visited our institute, bringing with them a small piece of a rib from the child for us to analyze, and also a fragment of a Neanderthal skull found in a higher layer of the cave. When Johannes made extracts from these specimens, the rib turned out to contain 1.5 percent Neanderthal DNA. This was still not as much as we had hoped for; moreover, the rib was so tiny that we could never hope to get enough DNA for a genome sequence from it. But it might contribute some data to our efforts.
The third site we explored—El Sidrón—was in
Asturias, in northwestern Spain. I visited it in September 2007. When a child dreams of becoming a paleontologist, this is the type of site he or she imagines. El Sidrón is located in beautiful countryside. The cave entrance is small and hidden, and the cave has served as a refuge for people throughout the ages. In front of the entrance is a memorial to a fighter who hid there during the Spanish Civil War and was killed by the fascists. After crawling through the entrance, one walks about two hundred meters to a side gallery, twenty-eight meters long and twelve meters wide, on the right. There, Professor Marco de la Rasilla of the University of Oviedo and his collaborators and students excavate every summer. They have found bones from one Neanderthal infant, one juvenile, two adolescents, and four young adults. The long bones were crushed and full of cut marks. Only the bones of the hands were found together; they had been separated from the bodies and thrown to the side. Marco de la Rasilla believes that the body parts were disposed of in a small pond on the surface some 43,000 years ago and then washed into the cave.
New bones were being discovered at this site each summer, and we agreed that they should be collected for DNA analysis in ways that would maximize DNA preservation and minimize the chance of contamination by present-day human DNA. Working with Carles Lalueza-Fox, a molecular biologist at the University of Barcelona, and Antonio Rosas, a physical anthropologist at the National Museum of Natural Sciences in Madrid, the excavators equipped themselves with sterile gloves, clothing, face shields, and other tools typically used in our clean room. When they came across bones deemed suitable for DNA extraction, they donned the sterile outfits, removed the bones, and placed them directly in an icebox to freeze them. Back in Antonio’s lab in Madrid, computer tomography of the bones was performed to document their morphology. The bones were then sent, still frozen, to us in Leipzig. Almost no one had touched them since their discovery, and bacterial growth would also have been minimized. I had high hopes that when Johannes made extracts, they would contain a lot of Neanderthal DNA, but of all the DNA in the bones, just 0.1 to 0.4 percent came from Neanderthals. From none of these sites, nor several others that we tried with even less luck, did we find enough DNA for a Neanderthal genome sequence. Vindija Cave was the only site where we had so far found a bone that had anywhere near enough DNA. Yet in Zagreb, things moved at a glacial pace, if at all.
