Neanderthal Man (29 page)

I was skeptical of a Neanderthal contribution in part because I suspected that some biological problem might interfere with successful mating. Although Neanderthals and modern humans almost certainly had  sex with each other—after all, what humans groups don’t?—I sometimes wondered if perhaps some factor might have made the offspring less fertile. For example, humans have 23 pairs of chromosomes whereas chimpanzees and gorillas have 24 pairs. This is because one of our largest chromosomes, chromosome 2, is a fusion of two smaller chromosomes that still exist in the apes. Such rearrangements of chromosomes occasionally occur during evolution and generally are of no consequence for how the genome functions. But the hybrid offspring of individuals that have different numbers of chromosomes often have difficulty conceiving offspring themselves. If the fusion that created chromosome 2 occurred after modern humans diverged from Neanderthals, then perhaps we interbred with them but the children didn’t transmit any Neanderthal DNA because they couldn’t have children of their own. But these were just idle musings; now we hoped to actually find out for certain. And the best way to do this was to compare the Neanderthal genome to the genomes of present-day people to see if it was closer to people in Europe, where Neanderthals had lived, than to people in Africa, where they had never lived.

By October 2006, David and Nick were already deeply immersed in the project. They worked with Jim Mullikin, another member of our consortium. Jim was the head of DNA sequencing at the National Human Genome Research Institute (NHGRI) in Bethesda. He was soft-spoken and immensely helpful. In fact, he somehow reminded me of Winnie the Pooh, but a very, very competent version of the friendly bear. Jim had sequenced genomes from several modern Europeans and Africans. To compare these genomes with the Neanderthal genome, he identified positions where a nucleotide in one individual in such a pair differed from the other individual. Such positions, which, as noted, are called single nucleotide polymorphisms or SNPs, are the basis for almost all genetic analyses. I remembered how excited I was back in 1999 when Alex Greenwood found the first Ice Age SNP ever seen (see Chapter 9); he had retrieved nuclear DNA sequences from a mammoth and found a position where the two mammoth chromosomes differed from each other. Now we wanted to analyze the hundreds of thousands of such SNPs that had been identified in humans to see which versions the Neanderthals had carried about 40,000 years ago, long before the Ice Age when the mammoth had lived. Although we had worked toward this goal for many years, it still seemed like science fiction to me.

To use SNPs to look for traces of possible interbreeding between Neanderthals and modern humans, we went back to the logic underpinning an analysis we had done of the first Neanderthal mtDNA in 1996. At that time, we argued that since Neanderthals were known to have lived only in Europe and western Asia, we would expect any contribution of their mtDNA to have occurred there. Thus, if Neanderthals and modern humans had mixed, some Europeans would walk around with mtDNA that until some 30,000 years ago had been in a Neanderthal. So we would expect Neanderthal mtDNA to be on average more similar to that found in some Europeans than to that found in people in Africa. We had failed to see this and thus concluded that no mtDNA contribution had occurred. In the case of the nuclear genome, the same argument would hold; if there were no contribution from Neanderthals to present-day humans anywhere in the world, then on average, across many individuals and many SNPs in the genome, the Neanderthals should carry equally many nucleotide differences from all populations. If, on the other hand, they had contributed to some population, genomes in that population would on average be closer to the Neanderthal genome than to genomes in other populations. David, Nick, and Jim would therefore identify SNPs where one of the Africans Jim had sequenced happened to differ from one of the Europeans. They would then count how often the Neanderthal genome matched the African and European genomes, respectively. If Neanderthals were closer to the Europeans, that would indicate gene flow from Neanderthals into European ancestors.

In April 2007, in preparation for the Cold Spring Harbor Genome Meeting, Jim and David sent me their first analysis of the Neanderthal sequences we had generated with the 454 technique. To test the method, they had first analyzed a present-day European individual at SNPs where another European and an African were known to differ from each other. They found that the European matched the other European at 62 percent of the SNPs and the African at 38 percent of the SNPs. Thus, as we had expected, on average people from the same part of the world shared more SNP variants with each other than with people from other parts of the world. They were able to compare the Neanderthal sequence to 269 positions where the European and African individuals differed and they found that the Neanderthal matched the European at 134 positions and the African at 135 positions. This was as close to 50:50 as the data could possibly be and it perfectly fit my preconceived idea that there had been no admixture. I liked this result for another reason as well. It meant that what we had was  the DNA of a person who seemed to be equally related to Europeans and Africans. In short, there couldn’t be very much DNA contamination from present-day humans among our Neanderthal sequences, since any such contamination would likely have come from a European individual and therefore make the Neanderthal look closer to the European than to the African individual.

On May 8, 2007, the day before the meeting started, all the members of what was now officially called the Neanderthal Genome Analysis Consortium met for the first time at Cold Spring Harbor. I started the meeting by describing the tag that we had introduced to rule out any contamination that occurred after the libraries left our clean room. I also talked about the three archaeological sites (see Chapter 12) and the bones from which we now had generated data. We had 1.2 million nucleotides of Neanderthal DNA determined from Vindija with our new tagged library approach. We also had about 400,000 nucleotides from the type specimen from Neander Valley in Germany, the bone from which we had determined the mtDNA segment in 1997. Finally, we had 300,000 nucleotides from El Sidrón, the cave in Spain where Javier Fortea and his team had collected bones under sterile conditions for us.

My description of the Neanderthal sites was a welcome relief from the rather arcane technical discussion of how we had extracted and sequenced DNA from the bones and how these sequences might be analyzed. Everybody was impressed that the Neanderthal seemed to be equally distant from an African and a European individual, but David Reich correctly pointed out that with just 269 SNPs, we could only exclude a very large genetic contribution from Neanderthals into Europeans. In fact, the 90 percent confidence interval for the 49.8 percent estimate of the SNPs matching the European was 45.0 to 55.0 percent. This meant that, with 90 percent confidence to be correct, we could only say that Neanderthals hadn’t contributed more than 5 percent of the genome to Europeans. In other words, there was a 10 percent chance that Neanderthals had contributed more than 5 percent. This uncertainty drove home for me a powerful advantage of molecular genetic analyses over paleontological analyses. If we had been discussing the forms, shapes, holes, and ridges of Neanderthal bones, we couldn’t have made any realistic estimate of how sure we were of what we found. Neither could we have been confident of being able to collect more data to resolve the issue with greater confidence. With DNA, we could.

David had also used the SNPs Jim had detected in present-day humans in other analyses. He compared the DNA sequence for each SNP to  that of the chimpanzee to determine which of the two variants, or alleles, was ancestral, and which was derived. The further back in time the Neanderthal population became separated from modern human populations, the less often the Neanderthal would carry the newer, derived SNP alleles found in people today. When David analyzed 951 SNPs that had been discovered in Africans, he found that a present-day European carried derived alleles at 31.9 percent of the SNPs. When he analyzed our Neanderthal sequences, he found that they carried the derived alleles at 17.1 percent of the SNPs, about half as often as the present-day European. Given certain assumptions, such as constant population size over time, this suggested that the Neanderthals separated from Africans some 300,000 years ago. I was delighted by these results. The sequences we had determined clearly came from a creature with a history very different from that of people living today. However, David dampened my enthusiasm by again pointing out that we didn’t have very much data yet. In fact, the 90 percent confidence interval for the percentage of derived alleles in the Neanderthal ranged from 11 to 26 percent. Still, we were clearly on the right track.

After we shifted to using the Illumina sequencing machines and began generating DNA sequences at a much faster rate, our twice-per-month phone meetings with the consortium became longer and we started having them every week. In January 2009, as the AAAS meeting drew near, I pleaded with David and Nick to do a quick analysis of our 454 sequences, which represented about 20 percent of all our data. Although I still didn’t think there had been any interbreeding between Neanderthals and modern humans, I wanted David to come up with an estimate of how small any contribution by Neanderthals to Europeans could maximally be without our detecting it. In other words, how big a contribution could we exclude? That was the number I wanted to present at the press conference and the AAAS meeting.

On February 6, 2009, I received an e-mail from David. It said, “We now have strong evidence that the Neanderthal genome sequence is more closely related to non-Africans than to Africans.” I was totally taken aback. David found that our Neanderthal sequences matched Europeans at 51.3 percent of SNPs. This may not seem very different from 50 percent, but we now had so much data that the uncertainty was just 0.22 percent, which meant that even if we subtracted 0.22 from 51.3, we still had a number that was different from 50 percent. I realized I might have to revise my ideas  and concede that there had been genetic mixing between Neanderthals and the ancestors of Europeans. But there was another observation that made me wonder if there was something wrong with the analysis after all. When David compared the Chinese and African genomes, the Neanderthals matched the Chinese 51.54 percent of the time and the uncertainty was 0.28 percent despite the fact that there had never been Neanderthals in China. David himself was intrigued as well as worried by these results. We both agreed that this finding was potentially very exciting but also that the results had the potential to be spectacularly wrong. There was a frantic exchange of e-mails and David, Nick, and I agreed that we should keep our admixture result secret at the press conference and AAAS meeting. If we mentioned it, all the media would write about it. If it then later turned out to be due to some sort of error, we would look like idiots. Instead, I decided to talk about less hot topics in Chicago. Discussions about the potential admixture would have to be postponed to a meeting that the consortium would have in Croatia just after the AAAS meeting.

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Two days after returning from Chicago, I was again in an airplane, this time on my way to Zagreb to give a lecture on our project at the Croatian Academy of Sciences and Arts. The next day I flew south to Dubrovnik, where our consortium and our Croatian collaborators were to meet in a hotel on the coast outside the city. We were not there just to celebrate, but to hammer out how we would analyze and publish the Neanderthal genome.

But the flight to Dubrovnik didn’t go quite as planned. The Dubrovnik airport is squeezed between mountains and the sea and has a bad reputation for difficult side winds. It was at this airport that US Secretary of Commerce Ron Brown died in an airplane accident in 1996. The US Air Force investigation later attributed the crash to pilot error and a poorly designed landing approach. As we approached the airport it was windy and the plane was jumping around. The Croatian pilot, probably wisely, decided not to try to land. Instead he flew to Split, a city some 230 kilometers away. We arrived there late in the evening and were packed into an overcrowded bus that took us through the night to Dubrovnik. I was exhausted when our first session started at 9:00 a.m.

Despite how tired I was, I felt energized by the presence of almost all the twenty-five members of our analysis consortium in the conference room (see Figure 17.1). Together, we were now going to tease out the information in the 40,000-year-old DNA sequences we had determined. I gave the first talk, an overview of the data we now had in hand. This was followed by a technical presentation by Tomi about his library preparation. Ed described how we estimated the level of present-day human DNA contamination, the issue that had plagued our first paper back in 2006. Our “traditional” mtDNA analysis yielded an estimate of 0.3 percent. By the time of the meeting, we had also devised an additional analysis not based on mtDNA. It relied on using the large numbers of DNA fragments we had from certain portions of the genome—specifically, the sex chromosomes,  X and Y. Because females carry two X chromosomes while males carry one X chromosome and one Y chromosome, if a bone came from a female, we should find only X chromosome fragments and no Y chromosome fragments. Therefore, any Y chromosome fragments we detected in libraries derived from a female’s bone would be indicative of contamination by modern males.

This analysis, suggested during one of our Friday meetings in Leipzig, initially sounded simple. But as with so many of the things Ed did, it was not as straightforward as it seemed. The complication was that, although the X and Y chromosomes are morphologically distinct, some of their parts share a close evolutionary relationship. The DNA they share as a result of this relationship could confuse the analysis when we mapped our short DNA fragments. To avoid this problem, Ed identified 111,132 nucleotides on the Y chromosome that weren’t similar to anything else in the genome, even if these bits were fragmented into pieces as small as 30 nucleotides in length. When he looked among the Neanderthal DNA fragments, he found just four fragments carrying these Y chromosomal sequences; if the bones we used had all come from males, he would have expected to see 666 of them.