Neanderthal Man (22 page)

In 2004, we had sequenced a part of the mtDNA from the very same Neanderthal bone, Vi-80, from which we had prepared test extracts for 454 and Eddy’s group. I suggested that we should look among the sequences we had gotten from 454. Surely some of those must overlap nucleotide positions that differed between this particular Neanderthal individual and present-day humans. This would tell us which fragments were unambiguously of Neanderthal origin and which were of modern human origin and would enable us to estimate directly the level of contamination in the actual final 454 data set. Frustratingly, Ed found that we did not have enough data in hand to do this. The sequences done by 454 contained only forty-one mtDNA fragments and none of them came from the part of the mtDNA genome that we had determined earlier from this or other Neanderthals. We checked the Berkeley data, but they were so scant that not even a single mtDNA fragment had been observed.

Happily, there was a solution: we had so much library left that we could simply sequence more DNA fragments. This should then yield fragments that could tell us whether we had contamination in the library or not. I contacted 454 and convinced the people there to quickly do more sequencing. They did six more runs in record speed, and as soon as the data were transferred to our server, Ed found six fragments that overlapped positions in the variable part of the mtDNA we had sequenced in 2004.

All six fragments matched the Neanderthal mtDNA and differed from present-­day human mtDNA! These were direct data suggesting that we had very little contamination in our sequences. Interestingly, these molecules, although clearly ancient, were not particularly short; four of them were 80 or more nucleotides long. This suggested that truly ancient DNA fragments were present also among the longer DNA fragments. Thus it was likely that the differences seen between short and long molecules were due to factors other than contamination. Ed was so elated that he ended the e-mail to the group describing these results with “I could kiss every one of you.”

We decided to go ahead with the
Nature
paper. Susan Ptak, a population geneticist in our group, sent a long technical e-mail to Eddy and Jim Noonan explaining why we felt that comparisons between long and short sequences were influenced by too many factors both known and unknown to represent strong evidence of contamination and explained why we trusted the direct mtDNA evidence more. She wrote: “Although there is indirect evidence which suggests some level of contamination, we now have a direct measure of the contamination rate in the final data set, which still suggests it is low.” We received no reply to this e-mail. Given the rather tense relationship that had developed between our groups, we did not find this too surprising.

This was a tremendously stressful incident. Ironically, as it turned out, both Eddy and we were right. The future would show that the data generated at 454 did contain contamination, but also that the indirect ways of detecting contamination via comparisons of long and short fragments were largely inadequate.

The two papers were published in
Nature
and
Science
on the 16th and 17th of November.
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There was the predictable excitement in the press, which I had by now gotten used to. In fact, I was much more preoccupied than excited. We had promised the world that we would sequence 3 billion base pairs of the Neanderthal genome within two years. Our paper ended with an estimate of what this would require—namely, about twenty grams of bone and six thousand runs on the 454 sequencing platform. We said that this was a daunting task, but added that technical improvements that would make the retrieval of DNA sequences on the order of ten times more efficient could “easily be envisioned.” The improvements we had in mind involved losing less material when making libraries for sequencing and taking advantage of secret future improvements to the 454 machines that Michael had revealed to us.

Things were looking up, but a major challenge still remained: finding good Neanderthal bones. The truth was that we did
not
have anywhere near twenty grams of Neanderthal bone of the quality of Vi-80, the bone we used in the test runs for the two papers. In fact, the piece we had left from Vi-80 weighed less than half a gram. I optimistically told myself that since one of the first Vindija bones we tried contained almost 4 percent Neanderthal DNA, surely we would find others that were equally good. Perhaps we would even find some that were better. I had to turn my full attention to this problem as soon as possible. First, however, I had to undertake a more unpleasant task: ending the collaboration with Eddy Rubin.

Terminating a scientific collaboration is often difficult, and it is even more so when a collaborator has become a personal friend. I had stayed with Eddy’s family in Berkeley; we had biked up the hills to his lab together; we had gone together to the theater in New York during Cold Spring Harbor meetings. I had always enjoyed his company. So I long pondered my e-mail to Eddy and wrote several drafts of it. I explained how I differed with him on bacterial cloning’s usefulness, and said that I felt that our communication, particularly on this point, had not been productive. I also noted that it now seemed that his group was trying to do the same things our group was trying to do, rather than working in a complementary way. For example, in our phone conferences, they had suggested that we send them our DNA extracts and the PTB reagent we had synthesized so that they could treat our extracts with our PTB. Neither I nor my group had been thrilled by this notion. I hoped I had expressed my reasons for not working together in a way that wasn’t hurtful or insulting, but it was still with some trepidation that I sent the e-mail. Eddy answered that he saw my points but that he continued to believe in the future potential for improvements and utility of bacterial libraries. I was relieved that he had taken my letter graciously, but we were now, clearly, competitors rather than collaborators.

The competition became apparent almost as soon as I turned my attention to the procurement of Neanderthal bones. Eddy was trying to obtain them too, I discovered, and from many of the same people we had worked with for years. In fact, I found out that already back in July,
Wired
magazine had published an article about Eddy’s Neanderthal efforts. The
Wired
piece ended with a quote from Eddy: “I need to get more bone. I’ll go to Russia with a pillowcase and an envelope full of euros and meet with guys who have big shoulder pads. Whatever it takes.”

_________________

Even before our
Nature
paper came out, Johannes Krause had begun preparing extracts from Neanderthal bones we had collected from Croatia and elsewhere in Europe over the years, hoping to find a bone that might contain as much or more Neanderthal DNA as Vi-80. Johannes was tall and blond, not so dissimilar to the German stereotype. He was also very intelligent. He was born and had grown up in Leinefelde, the very same town where in 1803 Johann Carl Fuhlrott had been born. Fuhlrott was the naturalist who in 1857, two years before Darwin published
The Origin of Species,
had suggested that the bones found in Neanderthal derived from a prehistoric form of humans. This was the first time anyone suggested that other forms of humans had existed before current humans and Fuhlrott was widely ridiculed for his idea, but he would be proven correct when additional Neanderthals were unearthed. Fuhlrott became a professor at the University of Tübingen, where today, appropriately, Johannes is a professor.

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
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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.