by Bryan Sykes
The claim for recombination advanced by Erika Hagelberg was a more serious proposition. Even though it fell short of an actual proof of recombination, which would require a definition of the segments that had been exchanged between the two different mitochondria, it was still a piece of evidence that was hard to explain by any other mechanism. As far as I could see, it could only be wrong if there had been a massive systematic error in the sequencing of the Nguna samples. This seemed very unlikely, given that Erika was an experienced scientist who would be familiar with the rule that extraordinary claims needed extraordinary proofs. Conventionally, these sequences would have been repeated and checked several times before making such a radical claim that she must have realized would have such profound implications.
Nguna itself is a tiny island lying off Espirito Santo in Vanuatu, west of Fiji, and Vanuatu was one of the island groups which we had included in our earlier work on Polynesia. We had been given a few samples and, checking back, I found that four of them came from Nguna itself. In those days we did not report mutations lower than position 93, because the systems we used at the time sometimes gave unreliable readings below that. So it was no surprise that our computer records showed no mutations at the crucial site of position 76. However, we still kept the old X-ray films on which the sequence was displayed as a series of bands. By some miracle I managed to locate the Nguna plate dated 2 June 1992, and the quality was perfect. I could easily read the sequence down to 76 and beyond. There was no sign of a change at 76 in any of the samples. I went at once to my colleague in the Institute who had supplied me with the original blood samples and explained what I had found. He had some more from Nguna, and we tested those for the change at 76. Not one of them had it. It seemed incredible that we couldn’t find the 76 mutation in twenty samples from such a tiny island when Erika was reporting it in nearly half of hers from the same place.
The situation was serious enough to warrant contacting Erika, and I emailed her in Dunedin, New Zealand, where she had recently taken up a post at the University of Otago. Given our strained relationship, I was as diplomatic as possible and stuck to the point. I explained that we had found no sign of the crucial mutation at position 76 in samples from the same small island. Would she let me know the source of the relevant Nguna samples, and send me samples so that I could replicate her findings? She replied that she was sure of the sequences and would re-check the results as soon as she could, that the possibility of a sequencing mistake is always there but that she had been reassured by the sheer mass of data. Considering the gravity of the situation and the impact even the suspicion of mitochondrial recombination was having on the reputation of the field as a whole, I then made a second request for samples of the Nguna DNA. This is unusual but not unheard of. I mentioned earlier that whenever a scientific paper is published there is an implicit undertaking, where possible, to make the raw material available for verification. This principle is at the very foundation of scientific progress. Without independent verification, or at least the opportunity to do so, scientific results have no validity. In most cases an actual test is unnecessary because the findings are quickly overtaken by new results. But here we had a situation where an entire field had been threatened with extinction. The truth about the Nguna samples, whatever it was, had to come out. And quickly.
I am sad to report that my requests for samples to verify the Nguna sequences did not produce results. Nor did I know of other laboratories that had tried to contact Erika to replicate the results. In the meantime, the reputation of mitochondrial DNA as a reliable evolutionary tool was spiralling downwards. The undergraduates had heard all about it. In the 1999 biological anthropology exams at Oxford, the demise of mitochondria featured in many of the students’ answers. At a packed meeting in the zoology department at which some new work from Maynard Smith was being presented by one of his colleagues, I found myself in the distinctly uncomfortable position, during questions at the end of the lecture, of having to defend the reputation of mitochondria in front of an audience of very distinguished and influential evolutionary biologists who seemed only too eager to write it off.
I was pretty sure by now that Erika’s Nguna data were wrong. Still, it was no good my thinking that. It was not really much use publishing our own results from the same island, either. There would still be uncertainty, and the original paper would still stand. If it were wrong, then it had to be corrected in the scientific press by Erika herself. In the meantime, I had also contacted co-authors of the paper who cooperated as far as possible: but still no sign of the samples.
In September of 1999 there was to be a conference in Cambridge at which both Erika and I were down to speak. It was a conference about Europe, and I gave a paper early on about our European work. Erika had been invited to talk about the Pacific islands and, we all assumed, about mitochondrial recombination. Generally speaking, scientific conferences are intensely polite affairs. There is a brief introduction by the session chairman; the speaker comes to the front and presents the paper, usually illustrated by a few slides or overheads; there is polite applause, a few questions from the audience, perhaps a bit more applause; the chairman introduces the next speaker. On this occasion, by the time it came for Erika to speak, there was a tangible atmosphere of anticipation, the expectation of a showdown in the air. The audience was completely silent, not wanting to miss a single word.
Erika began by saying that she was not going to talk about recombination. A murmur of surprise spread round the audience. Why had she come halfway round the world to a meeting on the genetic history of Europe if not to talk about mitochondrial recombination? As she went through her text on other aspects of her work in the Pacific, I knew I had to ask her about her Nguna work during questions, even if it had not featured in the presentation itself. It was the only way to get the matter cleared up. Was she sticking to her story or not? As Erika finished speaking, I raised my hand and the chairman called me to put my question. I was very nervous indeed, and could feel my heart pounding. But the issue was so important that I pressed on, in as unemotional a tone as I could manage.
‘Erika,’ I began, ‘although you did not refer to this specifically in your talk, there has been, as you know, considerable interest in your claim of finding examples of mitochondrial recombination on the island of Nguna. As you also know, my laboratory did not find evidence for recombination in samples from the same small island. There has been a suggestion in the scientific press [which there had, and not by me] that there may be a systematic error in the DNA sequences which appeared in the article. How do you respond to this suggestion?’
She answered instantly that she had checked the sequences and stood by them.
I had to keep going. ‘In that case, Erika,’ I replied, ‘why have you refused my requests for samples of the original DNA so that the sequences could be independently verified?’
The entire conference hall froze into complete silence.
‘I did not refuse,’ she answered.
‘But you did not reply to my request, which amounts to the same thing,’ I argued.
This was turning into a Grade One row. Erika accused me of having not scientific but personal motives for pursuing the matter. Fortunately, before I could answer this charge, someone else asked a related question about the recombination data and got what seemed to me to be an equally unconvincing reply. And yet, though by now many in the audience must have had their doubts about her original paper, at the end of the meeting it was still standing. There was no retraction. Not yet.
After that conference, Erika came under pressure from some of her co-authors on the original paper to clarify the position. Eventually, she conceded that the sequences were indeed wrong and, in August 2000, nearly eighteen months after the first paper appeared, the correction was published. For some unexplained reason, the sequences from the first part of the control region had been shifted by ten bases. This is something that can happen if the sequencing machine is playing up. The base that the machi
ne had scored as a mutation at position 76 was actually the normal base for position 86. So there were no mutations at 76 after all. Getting to the truth had been an exhausting, unpleasant and distressing experience. Everyone makes mistakes. But to take so long to set the record straight on such an important issue with so many ramifications seems to me completely contrary to the spirit of scientific enquiry. But there it was. Mitochondria had survived the recombination scare.
12
CHEDDAR MAN SPEAKS
Although our scientific reasoning now appeared to be watertight, I was still nervous that there might be a flaw in our version of European prehistory that even our most persistent and vociferous critics had overlooked. They had done a good job in making us test and prove every conceivable aspect of our principal tool – mitochondrial DNA itself. We had checked and rechecked the mutation rate. We had spent weeks running different versions of our evolutionary network programs and they all gave the same results. We had ridden the storm of recombination. We still felt sure that main chapters of the genetic history of Europe were written in the time of the hunter–gatherers, long before the farmers arrived. To be sure, agriculture had added some important extra paragraphs; but it had definitely not erased the original text. We felt very confident that most living native Europeans traced their maternal ancestry back to the hunter–gatherers who lived before the dawn of the Neolithic and the coming of agriculture.
Nevertheless, even though we were very sure of our data and the way we had interpreted them, our conclusions were still only inferences about past events: inferences built on large amounts of data and robust statistical treatments, but inferences none the less. So I was still slightly anxious. Perhaps we had made a mistake about the dates. I didn’t think we had, but suppose we were out by a factor of two? Suppose that events we had dated to fifty thousand years ago actually took place only twenty-five thousand years ago? More importantly, suppose the dates for the major mitochondrial clusters which we had placed at or around the end of the last Ice Age, between fifteen and twenty thousand years ago, were out by the same factor and were really less than ten thousand years old? That would bring them too close for comfort to the Neolithic period, and mean that they might have been part of the wave of Near Eastern farmers after all.
What we needed was a direct test on DNA taken from a human fossil which was known to predate the arrival of farming. If we could only find DNA that fitted into one of these crucial clusters in the remains of a hunter who lived thousands of years before farming was ever thought of, then we would be home and dry. We would not need to rely exclusively on reconstructions from the modern sequences. We would have found the real thing in Palaeolithic Europe. These mitochondrial clusters then had to have arrived in Europe thousands of years before farming ever reached it, and our dates must be right. Conversely, if the DNA from a very old fossil was unlike anything we now found in Europe then we were on shaky ground. We could not then be sure that the ancestors of the major modern clusters were in Europe before farming.
Human remains from the Upper Palaeolithic are few and far between. For one thing, ten thousand years is a very long time, and only in the very best of circumstances do bones last that long. Any that do survive are jealously guarded specimens, and rightly so. We would have to make an exceptionally good case to persuade a curator to allow us to take a sample from such a rarity. In my favour I did at least have a track record in getting DNA out of old bones. With my colleagues, I was the first to do so, with the Abingdon bones in 1989, although in that case the material was only a few hundred years old. Our work a few years later on the Iceman had become widely known, and was well thought of. But that was a unique case – a completely frozen body. At five thousand years it was old, but not old enough to predate agriculture. Although the Iceman’s DNA belonged to one of the key clusters, it couldn’t be used to strengthen our case because he was living two thousand years after farming had reached the Alps. We were looking for remains that were at least twice as old as the Iceman. Even so, he was the oldest human by far to have had his DNA successfully extracted, and as a deep-frozen body he was an exceptional case. There was no assurance that an ordinary skeleton would retain its DNA for five thousand years, let alone ten thousand.
Although DNA is obviously a much tougher molecule than anyone ever thought when they were scared to take it out of the refrigerator for fear of its decomposing, it cannot survive very long on its own. It needs to be in a skeleton to survive for thousands of years. What distinguishes bones, and teeth, from all other tissues is the hard, calcium-based mineral, hydroxyapatite. This protects the proteins and the DNA from decay by shutting out the bacteria and fungi that feed on the soft tissue in the rest of the corpse. So long as the mineral is intact, there is a chance that the DNA will have escaped being gobbled up. Once the calcium goes, the DNA is exposed and soon disappears. Calcium is alkaline and survives much better in an alkaline soil than anywhere else. In neutral and particularly in acid soils, DNA is much shorter-lived. The spectacular peat-bog bodies of northern Europe, where even the hair and skin are intact, always have a collapsed and deflated look about them because the calcium in the bones has dissolved in the acid bog. A lot of the protein survives and is protected against decay by the acid, which kills bacteria and fungi. However, because of its molecular structure, DNA is cut to shreds by even dilute acid very quickly. So, unfortunately, bog bodies are not a good source of ancient DNA.
Heat is also bad news. Egyptian mummies were an early, high-profile target for those in search of ancient DNA and, sure enough, some was found. But these were the carefully embalmed bodies of the wealthy, sheltered from decay not only by the natural preservatives in the embalming fluid but also by a succession of wood and stone sarcophagi which sealed the body in an underground tomb away from the baking heat of the sun. There are thousands of much less elaborate burials for the less well off in shallow graves just beneath the sand; but, even though these mummies are only two or three thousand years old, they are almost totally devoid of protein or DNA. The inorganic calcium is unaffected by the heat, but the organic molecules are long gone, broken down and leached away by the scorching heat of the desert.
We knew, then, that we had to avoid burials in hot countries and acid soils, and so we turned our attention to the limestone caves of northern Europe. Within these caves the temperature remains cool and, importantly, constant throughout the year. The daily fluctuations of heat and cold in the Egyptian desert probably do more damage to the DNA than the heat alone. A cool, stable temperature was much more promising. But what really recommends limestone is the alkaline nature of the surroundings. Bone mineral and limestone are chemically very similar. They are both compounds of calcium. The water that drips its way through the caves, forming stalactites and stalagmites, and covering the walls in sheets of flowstone, is rich in dissolved calcium. There is calcium everywhere. A bone left in a limestone cave does not have its mineral leached away. And if the mineral stays, and the temperature isn’t too high, the DNA will stay as well.
The caves in Cheddar Gorge are the most famous in Britain. A small, winding road threads its way down from the top of the Mendip Hills about twenty miles west of Bath. At first it is like any other wooded valley in that part of the world. Ash and hawthorn trees flank the road and, in the spring, the woods are full of the white flowers and pungent smell of wild garlic. As you descend further, the sides of the valley get higher and higher and the trees retreat up the increasingly steep slopes until, only a couple of miles from the top, you are staring up at vast walls of limestone three hundred feet high. Except at the very bottom of the gorge there is no sign of the river which formed it. This disappeared underground long ago, where it dissolved caves and caverns out of the rock. As the roofs collapsed and collapsed again, so the gorge was formed. The newest caves are still there, not yet obliterated by the forces of water and gravity. In the bustling tourist town of Cheddar at the foot of the gorge, the caves are big business alongside the cheese for which
the town is famous. On the left hand side of the gorge, directly opposite the Cheddar Caves Fish and Chicken Bar, and with its entrance partially obscured by the Explorer’s Cafe-Bar and a shop, is the biggest and most spectacular cave of them all – Gough’s Cave. And in the museum near the entrance to the cave stands a cast of its most celebrated former inhabitant: Cheddar Man. He was excavated in 1903 and subsequently carbon-dated to about nine thousand years ago, at least three thousand years before farming reached Britain. The cast is a copy of the original skeleton, which is stored in the Natural History Museum in London, in the care of Chris Stringer, head of the Human Origins Group. I rang him and made an appointment.
I knew Chris by reputation and had met him once at a scientific conference in Sardinia. The Natural History Museum I had known since my childhood. It was always a treat for my brother and me to be taken there by my mother in the school holidays. As I made my way up towards the immense and towering Victorian Romanesque entrance I felt a real excitement to be going to the Museum again not as a schoolboy but as a professional scientist. To reach Chris Stringer’s office I had to walk past the skeleton of the huge dinosaur, Diplodocus, that dominates the magnificent entrance hall. Then I turned right into a wide corridor, its walls hung with the skeletons of Ichthyosaurs and other marine reptiles, still embedded in the blue clay of the Dorset cliffs where they were found. But when I went through the door into the palaeontology department, the atmosphere and the decor changed abruptly, from the dramatic to the professional. Row upon row of anonymous sliding cabinets concealed the treasures which lay catalogued within them. Chris Stringer’s modern office led off from this priceless yet strangely silent testament to the wonders of the natural world.
Over a mug of tea, it didn’t take long to explain my reason for wanting to sample human fossils from the Palaeolithic. He had read about the controversy which our work on European prehistory had sparked, and quickly saw the sense in testing the DNA from a pre-farming skeleton. He wanted to know what the chances were of our being able to recover any DNA if he were to give us permission to sample. I could not give a definite answer. After all, the Iceman was so unusual that I could not promise that because we had been successful with him we were guaranteed a good result with an unfrozen bone twice that age. Without that assurance Chris was understandably reluctant to give permission for us to take a destructive sample from something so precious as Cheddar Man. Remembering that we had also been successful with animal bones from the Mary Rose, I made a suggestion that I hoped would get us over this impasse. If there were any animal bones from Gough’s Cave of approximately the same age, could we try them? If that worked, we could be fairly confident that the conditions within the cave were good enough to preserve DNA for ten thousand years. Happily, there were scores of animal remains from Gough’s Cave and I went back to Oxford with a small piece of deer bone.
