by Bryan Sykes
When a controversial paper is published it is not unusual for the scientific journal in which it appears to receive and publish a criticism from others in the field. This takes the form of a ‘Letter to the Editor’. The authors of the original paper are given the opportunity to respond, and if they do, both letters appear next to each other in the same issue of the journal. It was no surprise to learn that Cavalli-Sforza had composed such a criticism of our paper and that it had been accepted by the American Journal of Human Genetics. The editor sent us a copy of Luca’s letter with an invitation to reply to it.
The letter was a withering attack on mitochondria in general and on our interpretation of the control region sequence data in particular. It did, however, contain one very interesting statement that we had been waiting to hear. Although the overwhelming influence of the neolithic farmers on the make-up of the European gene pool was the main feature of Luca’s ‘demic diffusion/wave of advance’ model, no figures had ever been put on their overall genetic contribution. While we had estimated that roughly 20 per cent of modern Europeans traced their mitochondrial ancestors back to these early agriculturalists, there was no comparable figure from Luca’s work that we could use as a contrast. The assumption which most people had made was that the farmers had ‘overwhelmed’ the hunters. That was certainly how a generation of archaeologists had interpreted the ‘wave of advance’ model. But the scale of the immigration had never been quantified. There was probably no need. The model had gained its own momentum and everybody knew what it meant, or thought they did. But now, for the first time, Luca put a figure on the proportion of modern European genes contributed by farmers from the Near East. It was, according to the letter, roughly equal to the proportion of the genetic variation that contributed to the first principal component which tracked the cline of genes across Europe from the south-east to the north-west. And this was 26 per cent. No mathematical proof whatsoever accompanied the statement, but we weren’t going to complain about that. It was close enough to our estimate of about 20 per cent, derived from mitochondrial DNA analysis, that it looked as if there was little left to argue about.
Even though this was an important new announcement from Luca, we certainly needed to reply to his letter and the criticism of mitochondrial DNA that it contained. He had every right to be critical. It is perfectly reasonable to demand absolute clarification from anybody who is challenging a long-held view. Extraordinary claims, such as ours, demand extraordinary proof. Even so, we all felt under a lot of pressure. We were the new boys on the block up against the might of the Establishment. Nevertheless, I never doubted for a second that we were right. There was nothing for it but to answer the criticisms one by one.
We were confident that the first objection – that our chosen section of mitochondrial DNA, the control region, was so riddled with parallel mutations as to be completely unreliable – could be rebuffed. There are plenty of other base changes that can be used as molecular markers around the mitochondrial DNA circle. If we drew a new evolutionary tree using these other markers instead of the control region sequences, then one of two things would happen: either the clusters would match our own groupings or they would not. If they did match, then the control region must be reliable. If they didn’t match, then it wasn’t, and we might as well give up.
For this test we teamed up with Antonio Torroni, an Italian geneticist from Rome who had spent many years developing an intricate technical system for these other markers. He supplied us with samples he had already tested for us to sequence through the control region, and we in turn took our own sequenced samples to Rome to run through his system. The results couldn’t have been more encouraging. There was an almost exact fit between the clusters identified by Antonio’s markers and our own. The one or two minor incompatibilities were quickly resolved; those apart, the match was perfect – so much so, in fact, that we abandoned our own numerical classification for the clusters and adopted Antonio’s, based on letters of the alphabet. Now we had proof that the control region was not after all a fickle piece of DNA that could mislead and deceive but, once you got to know it, a faithful and reliable companion.
The mutation rate criticism was harder to address. It was certainly true that if we were using a gross under-estimate of the mutation rate then our cluster dates would be seriously adrift. If our estimates were out by a factor of ten, as some people suggested, then the ages of our clusters would fall from the Palaeolithic into the Neolithic and we could kiss our theory goodbye.
There are basically two ways of estimating a mutation rate. Either you can try to measure it by direct observations from one generation to the next, or you can see how many mutations have accumulated in two different groups – which could be tribes, or populations, or species – that have been separated for a known length of time. The very first estimate of the mutation rate, the speed of the molecular clock, was made by comparing the differences between humans and their closest relative, chimpanzees, and combining this with the time since they last shared a common ancestor, estimated at between four and six million years ago. Of course, precisely when that separation between the ancestors of humans and chimpanzees took place is not known, especially since there are no chimp fossils to help out. The other route that has been used is to estimate the mutation rate changes which have accumulated in native Americans, who first arrived on the continent about twelve thousand years ago. The remarkable thing is that both methods agree so well with each other and come out with a figure of around one mutation in twenty thousand years down a single maternal lineage. When tracking back to a common ancestor between two modern people, as I did when estimating the date of the common ancestor between myself and the Tsar, there are two lineages, each with a chance to mutate, going forward from our common ancestor to each of us. Only one mutation separates my control region sequence from the Tsar’s, but that mutation could have happened anywhere along the two maternal lineages leading from our common ancestor. At a rate of one mutation every twenty thousand years along a single lineage, that fixes the combined length of these two lineages to twenty thousand years. Since the Tsar and I are more or less contemporaries, the length of each lineage back to the common ancestor is therefore halved to ten thousand years. Our work in Polynesia had also shown an excellent agreement between the genetic and archaeological dates for settlement using this mutation rate. If the rate was wrong by a factor of ten in Europe, then it had to be wrong everywhere else. It would mean that chimps and humans diverged only 400,000–600,000 years ago, America was first settled only 1,200 years ago and Polynesia only 300 years ago – in fact after the Europeans got there. This was so obviously crazy that the rates we were using couldn’t be that far out.
Measuring mutation rates directly is a hard business. It means picking up a change between a mother and her child. We estimated that we would need to test a thousand pairs of parents and children to pick up a single new mutation. That was out of the question. Fortunately, the mutation process in mitochondria is a gradual one and, as it turned out, not too difficult to observe by a different route. Mutations happen in individual DNA molecules in individual mitochondria. However, in most people the DNA sequence of all the mitochondria in all the body cells is exactly the same. These two truths pose a paradox. A new mutation can only take place in one DNA molecule in one mitochondrion in one cell; so how does it manage to take over the whole body?
In order to be passed on to a new generation, a mutation has to occur in a female germline cell, one of the cells that divide to become eggs. Mutations also happen in other body cells – in skin, bone, blood, and so on – but, as these do not get passed on to the next generation, they play no part in the patterns of evolution. What seems to be happening is that each time a female germline cell divides it takes only a few mitochondria with it. If the mitochondrion with the new DNA mutation is one of the few to slip through this bottleneck then it can make up a much bigger proportion of the mitochondrial DNA in the new cells. When these cells divide there
is a chance that the new mutation will be further enriched, and so on.
There are only twenty-four cell divisions in the female germline between one generation and the next. These are twenty-four opportunities for enrichment of a new mutation; only rarely is this enough for a complete takeover in a single generation. The individual who grows from the fertilized egg will have a mixture of two mitochondrial sequences: the old one, which is the same as her mother’s, and the new one, which began as a new mutation somewhere in her mother’s germline cells.
We looked very hard at our sequencing results over the past few years, searching for the signs of mixed mitochondria within the same person. We found that about 1.5 per cent of people do indeed have a mixture of two different mitochondrial DNAs. We then tracked these mixtures through families and found that it took an average of six generations for a new mutation to establish itself and take over completely. Remember the unusual case of the Tsar, who had a mixture of two different mitochondria in his bone cells? It looks as if he was in the transitional state where a new mutation was struggling to get established; eventually it did, as we can see in the cells of his modern-day relatives like Count Trubetskoy. As far as we could tell from our experiments, there was no inevitability in this process; some new mutations appeared to be doing well for one or two generations, then slipped back into obscurity and disappeared. We were observing directly the appearance and spread of new mutations, and from these data we could make a separate estimate of the mutation rate, independent of the complications associated with the exact dating of past events like the evolutionary separation of humans and chimps. This independent estimate, though only approximate, matched the mutation rate we had been using. We had answered the second criticism. Mitochondrial DNA had survived with its reputation intact.
The points Luca had raised in his letter, and to which we had responded, were serious and valid questions to ask of a new technology, especially one that had rewritten the version of prehistory that had dominated thinking for so long. They needed to be addressed, and they were. What happened next threatened to discredit not only our studies in Europe but all the evolutionary work using mitochondrial DNA that had ever been done on humans. We had to deal with the spectre of recombination.
Briefly, what makes the chromosomes in the cell nucleus so difficult to use for tracing evolutionary histories is their habit of scrambling information at each generation. Until the germline cells are into their final division which produces the gametes (sperm or eggs), the chromosomes lead separate lives and don’t have a great deal to do with one another. However, in that final cell division, the pairs of chromosomes which have been inherited from each parent sidle up to one another, like mating earthworms, and start to exchange bits of DNA. After this canoodling they pull apart and go off to different gametes. But now they are no longer the same chromosomes but DNA mosaics. They have undergone what is called recombination. This is the ultimate genetic reason for sex itself, the potential for creating through recombination new and better gene arrangements that can advance evolution.
Recombination has its advantages for scientists. It has greatly helped the mapping of genes for serious inherited diseases on to specific chromosomes, and has been instrumental in unravelling the sequence of the entire human genome. But as far as tracing DNA through the generations is concerned, recombination is a very big nuisance. One of the features of mitochondrial DNA that have made it such a successful instrument for probing into the deep human past is that the information it brings us is not scrambled by recombination. The only differences between my mitochondrial sequence and that of my direct maternal ancestors are the changes that have been introduced over the millennia by mutation. With recombination, there would be the prospect of having not just one line of mitochondrial ancestors but dozens of them. Everything that had been assumed about mitochondrial genetics would be in doubt.
So, when two papers claiming evidence for mitochondrial recombination appeared in the March 1999 issue of the prestigious Proceedings of the Royal Society, they sent shock waves around the world. Editorials in the leading popular science journals, Science in Washington and Nature in London, immediately publicized this fundamental challenge to the authority of mitochondrial DNA. If recombination really was occurring, as these papers were suggesting, then it meant that all the work published over the previous decade on mitochondrial DNA in human evolution was completely undermined.
The wide publicity accorded to these articles was due not only to the claims they advanced but also to the great distinction of the author of one of them: John Maynard Smith, the undisputed doyen of British evolutionary biologists, the author of textbooks and other influential works, and still an active presence in his eighties. Condemnation by such an eminent figure, with no obvious axe to grind, spelled obliteration for us and everybody else in the field – if the claims for recombination could be substantiated. The substance of Maynard Smith’s largely theoretical argument was that there was too much variation in mitochondrial DNA to have arisen by mutation alone. It was not so much a proof of recombination as an elimination of other mechanisms that could account for what Maynard Smith saw as a higher than predicted number of mutations. The reasoning was reminiscent of Sherlock Holmes’ advice to Dr Watson in The Sign of Four: ‘When you have eliminated the impossible, whatever remains, however improbable, is the truth.’ But what made Maynard Smith’s argument so seductive was the announcement in an adjoining paper of actual evidence for recombination in mitochondria from the tiny and remote island of Nguna in the Pacific. And the leading author (one of six) of the second paper was Erika Hagelberg.
Erika, you will recall, had worked in my laboratory on the first recovery of DNA from human bone back in the late 1980s. She had since made a name for herself in the field of ancient DNA and become involved in some celebrated forensic cases, most famously when she and her colleagues had recovered DNA from the remains of Joseph Mengele, the infamous Nazi doctor who carried out unspeakable human experiments on prisoners in the Auschwitz extermination camp. With these and other cases under her belt she had built up a reputation as an imaginative scientist. However, despite occasional attempts on both our parts to heal the rift that had grown up during the difficult final days Erika spent in my laboratory, she and I had endured an uneasy relationship ever since. This tension added an extra dimension to the drama that was about to unfold.
The essence of Erika’s evidence for recombination was that a particular mitochondrial mutation, at position 76 in the control region, was cropping up in several different clusters on the small island of Nguna. Like the Maynard Smith paper that accompanied it, this wasn’t direct evidence for mitochondrial recombination. However, mutations at position 76 were exceedingly rare elsewhere in the world, so to find it frequently and in different clusters on the same island did deserve a special explanation. It would mean either that the mutation had happened spontaneously several different times in different clusters, which was extremely unlikely, or that a new mutation at 76 in one cluster had somehow spread to the others. And the only way for that to happen was by recombination.
For mitochondrial recombination to occur, two things have to happen. First, there needs to be a way for two circular mitochondrial DNA molecules to snuggle up to each other and exchange DNA. That didn’t seem too unlikely. There are about eight DNA molecules in each mitochondrion and they enjoy free access to each other. So it would not be hard for them to exchange DNA. More difficult to accept was that there had to be two very different mitochondrial genomes in the same cell. If all the mitochondria in the cell had exactly the same sequence, they could exchange DNA between themselves as much as they liked and it would not make any difference. All the mitochondria would still have the same DNA sequence. Only if there were two different mitochondria exchanging DNA would anything be noticed. So the Nguna observation demanded that there were, or had been in the past, people who had mixtures of mitochondria. One component of the mixture would have to be the DNA belonging to one cl
uster, let’s call it A, and with a mutation at position 76 in the control region. The other would be mitochondrial DNA from a completely different cluster, which we can call B, without the mutation at position 76. These two mitochondria would then exchange segments of DNA so that a piece from A, which included the mutation at position 76, ended up on B.
There was only one way to get two mitochondria from completely different clusters in a cell: one of them had to be coming not from the egg but from the sperm. So, if this claim of recombination turned out to be true, it would be a lethal double blow. Not only would it be impossible to trace mitochondrial lineages back in time because of the scrambling implicit in recombination, but it would also follow that mitochondrial inheritance was not, after all, exclusively maternal. No longer would it be safe to assume that our mitochondrial DNA had come from an ancestral line of mothers. It could have come from fathers as well. Something had to be done. We held an emergency meeting.
Vincent Macaulay, who trained as a physicist and was a formidable mathematician, and had joined the team two years previously, went off to check and recheck the sequence data used in the Maynard Smith paper. Incredibly, a lot of them were wrong. Either they had been incorrectly copied from the public databases, or the raw sequences themselves which had been deposited in these databases had mistakes in them (actually a common enough occurrence). The cumulative effect of both sorts of error made it look as though there were more mutations in the mitochondria than there really were. After correcting these mistakes in the data and redoing the Maynard Smith calculations, it was obvious that the force of the theoretical argument for recombination was seriously diluted. We wrote at once to Maynard Smith, who gracefully accepted the error.