by Pbo, Svante
Figure 5.3. The right upper arm bone of the Neanderthal type specimen with the sample removed by Ralf Schmitz in 1996. Photo: R. W. Schmitz LVR-LandesMuseum Bonn.
Chapter 6
A Croatian Connection
________________________________
During the weeks and months after our publication of the Neanderthal mtDNA sequence, I reflected on what had led up to it. I had come a long way from my first attempts sixteen years earlier to extract DNA from a piece of dried calf’s liver from the supermarket. Now, for the first time, we had used ancient DNA to say something new and profound about human history. We had shown that the archetypical Neanderthal carried mitochondrial DNA very different from the mtDNA in people today, and that he or his relatives had not, before they became extinct, contributed their mtDNA to modern people. The achievement had required years of painstaking work to develop techniques to reliably determine DNA sequences from individuals long dead. Now that I had these techniques at my disposal, and a group of dedicated people able and willing to try new things, the biggest question was: Where should we go from here?
One task seemed of immediate importance: to determine mitochondrial DNA sequences from other Neanderthals. As long as we had studied only one individual, it remained possible that other Neanderthals carried mitochondrial genomes very different from the one from Neander Valley, perhaps even carrying mitochondrial genomes that were like those of present-day humans. Mitochondrial DNA sequences from additional Neanderthals would also reveal something about the genetic history of the Neanderthals themselves. Present-day humans, for example, have relatively little genetic mtDNA variation. If Neanderthals did, too, this would suggest that they had originated and expanded from a small population. If, on the other hand, they had as much mtDNA variation as any of the great apes have, this would suggest that over their history their numbers had never been very low. They would not have had such a dramatic history with ups and downs in population size as modern humans have. Matthias Krings, eager to follow up on his success with the iconic type specimen from Neander Valley, was keen to examine other Neanderthal specimens.
The major problem was getting access to fossils sufficiently well preserved for us to do work.
I thought a great deal about why we had been successful with the Neander Valley type specimen and came to realize that the fact that it had come from a limestone cave might be significant. Tomas Lindahl had taught me that acid conditions cause DNA strands to disintegrate, which was why the Bronze Age people found in acid bogs in northern Europe had never yielded any DNA. But when water passes over limestone, it becomes slightly alkaline. So I decided we should concentrate on Neanderthal remains unearthed in limestone caves.
Unfortunately, I had never paid much attention to the geological features of Europe in school. But I remembered the first anthropological conference I had ever attended, in Zagreb, in what was then Yugoslavia, in 1986. During the conference, we were taken on excursions to Krapina and Vindija, two sites where large amounts of Neanderthal bones had been found in caves. I made a quick search in the literature and confirmed that both Krapina and Vindija were limestone caves, which was promising. Promising as well was the presence of large numbers of animal bones, particularly of cave bears, in the caves. Cave bears, which were a large plant-eating species, became extinct shortly after 30,000 years ago, just like the Neanderthals. Their bones often abound in caves, often in circumstances suggesting that they died during hibernation. I was happy about the presence of cave-bear bones because they could possibly serve as a convenient tool to check whether DNA was preserved in the caves. If we could show that their bones contained DNA, this might be a good means to convince hesitant curators that they should allow us to try the much more valuable Neanderthal remains from the same cave. I decided to interest myself in cave-bear history, especially in the Balkans.
After a bloody war with Serbia, Zagreb had become the capital of the independent Republic of Croatia. The largest collection of Neanderthals there is from Krapina, in northern Croatia, where starting in 1899 the paleontologist Dragutin Gorjanović-Kramberger discovered more than eight hundred bones from some seventy-five Neanderthals—the richest cache of Neanderthals ever found. These bones are today housed in the Museum of Natural History in the medieval center of Zagreb. The other site, Vindija Cave (see Figure 6.1) in northwestern Croatia, was excavated by another Croatian paleontologist, Mirko Malez, in the late 1970s and early 1980s. He found bone fragments of several Neanderthals but no spectacular crania like those found in Krapina. Malez also found enormous amounts of cave-bear bones. His finds are housed in Zagreb, too, in the Institute for Quaternary Paleontology and Geology, which belongs to the Croatian Academy of Sciences and Arts. I arranged to visit both this institute and the Museum of Natural History. In August 1999, I arrived in Zagreb.
The Krapina Neanderthal collection was extremely impressive, but I was skeptical about its potential for DNA research. The bones were at least 120,000 years old and therefore older than anything we had found to yield DNA. The Vindija collection looked more promising. First of all, it was younger. Several layers in the excavation had yielded Neanderthal remains, but the uppermost and thus the youngest one to do so was between 30,000 or 40,000 years old—young, as far as Neanderthals go. I saw a second exciting feature of the Vindija collection: it was full to overflowing with ancient cave-bear bones. They were stored, according to bone type and layer, in innumerable paper sacks that were coming apart in the humidity of the Quaternary Institute’s basement. There were sacks full of ribs, others full of vertebrae, others of long bones, and yet others of foot bones. It was an ancient DNA gold mine.
Figure 6.1. Vindija Cave in Croatia. Photo: J. Krause, MPI-EVA.
In charge of the Vindija collection was Maja Paunovic, a woman of a certain age who spent her days in an institute without public exhibitions and with few facilities for doing research. She was friendly enough but understandably dour—no doubt aware that science had passed her by. I spent three days with Maja, going through the bones. She gave me cave-bear bones from several layers at the Vindija site as well as small samples of fifteen of the Neanderthal bones. This seemed exactly what we needed for the next step in our exploration of the genetic variation among Neanderthals. When I flew back to Munich I felt confident that we would make quick progress.
In the meantime, Matthias Krings had extended his sequencing of the Neanderthal type specimen to a second part of the mitochondrial genome. The results confirmed that this specimen’s mitochondrial DNA shared a common ancestor with present-day human mtDNAs about half a million years ago. But this was of course what we had expected, so the news felt slightly boring after the emotional high produced by the first Neanderthal sequences. Not surprisingly, he was eager to throw himself on the fifteen Neanderthal bone samples I had gotten from Maja in Zagreb.
We first analyzed their state of amino-acid preservation. Amino acids are the building blocks of proteins and can be analyzed from much smaller samples than are needed for DNA extractions. We had shown before that if we could not find an amino-acid profile suggesting that the samples contained collagen (the main protein in bones), and if the amino acids were not present largely in the chemical form in which they are built into proteins by living cells, then our chances of finding DNA were very small and there would be no point in destroying a larger piece of the bone in an attempt to extract it. Seven of the fifteen bones looked promising, with one that particularly stood out. We sent a piece of that bone for carbon dating and the result showed that it was 42,000 years old. Matthias made five DNA extracts and amplified the two mitochondrial segments he had studied in the type specimen. It worked nicely. He sequenced hundreds of clones, taking pains to ensure that every position was observed in at least two amplifications that, at my insistence, should come from different extracts, in order to make absolutely sure that they were totally independent of each other.
In March 2000, while Matthias was working on this, a paper that appeared in Natu
re took us by surprise. A group based in the UK had sequenced mtDNA from another Neanderthal, unearthed at Mezmaiskaya Cave in the northern Caucasus.{36} They had not applied all the technical approaches we advocated to make sure that the sequence was correct; for example, they had not cloned the PCR products. Nevertheless, the DNA sequence they found was almost identical to our type-specimen sequence from Neander Valley. Matthias, who had his sequences almost finished, was disappointed that he’d been beaten to the publication of the world’s second Neanderthal mtDNA sequences—especially since his progress had been slow due to all the precautions and checks on which I insisted. I sympathized with him, but I was also happy that our pioneer sequence from Neander Valley had been verified by a group working independently of us. Yet I did not quite agree with the commentary Nature published along with the paper, which said that this second Neanderthal sequence was “more important” than the first because it showed that the first was correct. I wrote that off as sour grapes on Nature’s part for not getting to publish the first Neanderthal sequence.
There was a consolation prize of sorts for Matthias. The second Neanderthal DNA not only served to confirm the results in our 1997 Cell paper, but now that we knew three sequences, including the one Matthias had determined from Vindija, it became possible to say something, albeit something tentative, about genetic variation among Neanderthals. Genetic theory holds that with just three sequences there is a 50 percent chance of sampling the deepest branch of a tree relating all the mitochondrial DNAs in a population. It turned out that 3.7 percent of the nucleotides in the segment that Matthias and the British group had sequenced differed among the three Neanderthals. For perspective, we wanted to compare this degree of variation to the variation in humans and the great apes. First, we used sequence data for the same segment determined by many other groups from 5,530 humans from all over the world. In order to make a fair comparison to the three Neanderthals, we sampled three randomly chosen humans many times, so that we could calculate an average of how many differences three humans carry in the same sequence. It was 3.4 percent, very similar to that for the three Neanderthals. There were 359 chimpanzee sequences available for the same mtDNA segment. When we sampled chimpanzees in the same way, they differed by an average of 14.8 percent, and for twenty-eight gorillas the corresponding value was 18.6 percent. So Neanderthals seemed to be different from the great apes in having little mtDNA variation, just like present-day humans. Obviously, it was risky to speculate from just three individuals, and from just mtDNA, so when we published these data later in 2000, in Nature Genetics, we stressed that it would be desirable to analyze more Neanderthals; nevertheless, we suggested that Neanderthals were probably similar to modern humans in having little genetic variation and that they had therefore expanded from a small population, just like us.{37}
Chapter 7
A New Home
__________________
Life is not an orderly thing. One morning, not long before our publication of the first Neanderthal mtDNA sequences in 1997, my secretary told me that an elderly professor had phoned asking for an appointment with me. He had told her that he wanted to discuss some plans for the future. I had no idea who he was but vaguely supposed he was a retired professor who wanted to share his crackpot ideas about human evolution with me. I was very wrong. What he had to say was very exciting.
He explained to me that he came on behalf of the Max Planck Society, or MPS for short, which supports basic research in Germany. Among its many efforts, the MPS had a program to build up world-class research in the former East Germany, which had been fused with West Germany seven years earlier. One guiding principle was to found new research institutes focusing on topics in which Germany was scientifically weak. An area of particular weakness was anthropology, and for a very good reason.
As do many contemporary German institutions, the MPS had a predecessor before the war. Its name was the Kaiser Wilhelm Society, and it was founded in 1911. The Kaiser Wilhelm Society had built up and supported institutes around eminent scientists such as Otto Hahn, Albert Einstein, Max Planck, and Werner Heisenberg, scientific giants active at a time when Germany was a scientifically dominant nation. That era came to an abrupt end when Hitler rose to power and the Nazis ousted many of the best scientists because they were Jewish. Although formally independent of the government, the Kaiser Wilhelm Society became part of the German war machine—doing, for example, weapons research. This was not surprising. Even worse was that through its Institute for Anthropology, Human Heredity, and Eugenics the Kaiser Wilhelm Society was actively involved in racial science and the crimes that grew out of that. In that institute, based in Berlin, people like Josef Mengele were scientific assistants while performing experiments on inmates at Auschwitz death camp, many of them children. Whereas Mengele was sentenced for his crimes after the war (although he had escaped to South America), his superiors at the Institute for Anthropology were never charged. On the contrary, some of them became professors at universities.
When the Max Planck Society was formed in 1946 as the successor of the Kaiser Wilhelm Society, anthropology was understandably a subject best avoided. In fact, as a result of what happened under Nazi rule, the entire field of anthropology had lost its status in Germany. It failed to attract funding, good students, and innovative researchers. Obviously, this was an area where Germany was scientifically weak, and my visitor said that the MPS had set up a committee to consider whether anthropology could be an area in which the MPS should found a new institute. He also indicated that there were different opinions as to whether this was a good idea given recent German history. Nevertheless, my visitor asked me if I would consider moving to such an institute should it be created. I was vaguely aware that the MPS commanded great resources, and that these resources had been augmented to build several new institutes in the east after reunification of the two German states. I was intrigued by the prospect of helping build a new institution, but I did not want to sound overenthusiastic and have them believe I would come under any circumstances. With this in mind I said that I would consider it if it meant that I would be able to influence how such an institute would be organized and function. The professor assured me that as a founding director I would have great freedom and influence. He suggested I come and present to the committee my views on how such an institute might be organized.
Some time later I received an invitation to hold a presentation in front of the committee. It would meet in Heidelberg and was made up of several foreign experts, headed by Sir Walter Bodmer, a human geneticist from Oxford and a specialist on the immune system. I presented those aspects of our work that I thought might be appropriate to an anthropology institute, focusing on the study of ancient DNA, especially Neanderthals, and the reconstruction of human history from genetic as well as linguistic relationships between human populations. In addition to my scientific presentation there were several informal discussions about whether anthropology was a topic that the MPS should engage in, given the dire history of the subject in Germany. Perhaps it was easier for me as a non-German born well after the war to have a relaxed attitude toward this. I felt that more than fifty years after the war, Germany could not allow itself to be inhibited in its scientific endeavors by its past crimes. We should neither forget history nor fail to learn from it, but we should also not be afraid to go forward. I think I even said that fifty years after his death, Hitler should not be allowed to dictate what we could or could not do. I stressed that in my opinion any new institute devoted to anthropology should not be a place where one philosophized about human history. It should do empirical science. Scientists who were to work there should collect real hard facts about human history and test their ideas against them.
I did not know how well my arguments went down with the committee. I returned to Munich and months went by, until I had almost forgotten about the whole thing. Then one day I received an invitation to meet with a new MPS committee that had been charged with actually founding an institute devoted
to anthropology. There followed a number of meetings with talks by different candidates. The fact that there were no traditions in the subject to build on either in the MPS or indeed in Germany turned out to be somewhat advantageous. It allowed us to discuss freely, unconstrained by academic traditions and preexisting structures, how one would organize a modern institute to study human history. The concept that emerged during our discussions was that of an institute not structured along the lines of academic disciplines but focused on a question: What makes humans unique? It would be an interdisciplinary institute where paleontologists, linguists, primatologists, psychologists, and geneticists would together work on this question. The framework within which one should ask this question was evolution. Ultimately, the goals should be to understand what had set humans on an evolutionary track so different from other primates. So it should be an institute in “evolutionary anthropology.”