Getting DNA out of an ancient bone was always going to be a huge challenge – DNA starts to fall apart after death – but Pääbo and his team had hoped that some tiny fragments might still be there. The extraction was done in a sterile room, to try to reduce the possibility of contamination with modern DNA. The bone sample was ground into powder and then the sample was treated to amplify up any DNA – by getting any fragments to make copies of themselves. Then the sequencing could start, and the results were quite stunning: when they compared the Neanderthal sequence with the equivalent mitochondrial DNA sequence from nearly 1000 modern humans, they found that it was distinctly different. The modern human mtDNA sequences differed from each other by an average of eight different base pairs out of almost four hundred. But the Neanderthal sequence had an average of twenty-six base pair differences compared with the modern human samples. This difference suggested that Neanderthal and modern human mtDNA had been evolving along separate pathways for about 600,000 years. Although this seems a very long time ago, compared with the dates of the earliest known Neanderthal (about 300,000 years ago) and the earliest known modern human (about 200,000 years ago) it still makes sense, as the lineages would have started to diverge within an ancestral population of Homo heidelbergensis.2
This result seems to support the theory of a recent African origin of modern humans, and a replacement of any earlier human populations. In contrast, the multiregional hypothesis suggests that archaic populations in Africa, Europe and Asia developed into modern human populations. A halfway house theory has modern humans originating in Africa, then spreading into Europe and Asia and interbreeding with existing archaic humans.
Pääbo’s findings suggested that the mitochondrial DNA lineages, at least, had separated (and stayed separate) hundreds of thousands of years before modern humans appeared in Europe. Even if you ignore the timings, then the multiregional model with hybridisation suggests that Neanderthals should be genetically closest to modern Europeans, but there was no evidence of this in the mitochondrial DNA: the Neanderthal sequence was equally different from all modern humans across the globe. Another study compared ancient DNA extracted from two 25,000-year-old European modern human fossils, and found that the Cro-Magnon mtDNA fell in the modern human range of variation, and was very different from the Neanderthal sequences.3
Looking at mtDNA variation as well as modelling the population expansion of modern humans in Europe, researchers in Switzerland came up with a maximum interbreeding rate between the two populations of less than 0.1 per cent. Statistically, this is so low as to be practically non-existent, and the Swiss scientists go as far as to say that this suggests the two species were biologically separate – and could not produce fertile offspring even if they had seized upon the chance to have sex with each other.4
So do these mtDNA results represent definitive evidence that the Neanderthals could not be counted as among the ancestors of modern Europeans? Well, they certainly seem to point in that direction, but, actually, it’s impossible to completely rule out any hybridisation between modern and archaic populations. Neanderthal genes could have entered the human gene pool, but those lineages might have died out, leaving no trace of them today. And what if only Neanderthal men, not women, had interbred with the incoming modern humans? That wouldn’t show up in the mitochondrial DNA – which is inherited only from the mother. So although these Neanderthal mtDNA studies are amazing, and suggest that hybridisation didn’t happen, they can’t rule it out. So would it be possible to probe further, to go after more Neanderthal DNA – perhaps nuclear DNA?
When Svante Pääbo was interviewed for Science magazine after the publication of the Neanderthal mtDNA paper in 1997, he was very pessimistic about the chances of anyone ever managing to recover and sequence nuclear DNA from Neanderthal bones.5 But just over a decade later, I was visiting his lab at the Max Planck Institute – and they were doing just that.
The genetics labs were just along the (very beautiful, sky-lit, gently curving) corridor from the bone lab. The Institute felt like a modern monastery, with an all-pervading calm and scholarly atmosphere. But instead of monks painstakingly copying out biblical passages, scientists were locked away in their high-tech scriptoria, sequencing the Neanderthal genome.
I met up with Ed Green, one of the geneticists hard at work on the Neanderthal Genome Project. Ed had brought along some casts of the original fossils from which DNA had been extracted.
‘How do you go about trying to extract DNA from these fossils?’ I asked Ed.
‘Well, the first thing is to find the fossil that has ancient DNA that can be extracted. Then the way it’s done is to simply to take a dentist’s drill, drill a bit, get some bone powder, and then use a standard extraction method where you bind DNA to silica beads.
‘Then the really fun part begins – trying to sequence this DNA, and see what is there. Is this DNA from the individual that owned this bone originally? Or DNA from bugs that have crawled into the bone since then?’
‘And presumably there’s quite a lot of modern human DNA knocking around as well – from the archaeologists who excavated them,’ I suggested. Ed agreed. He was very keen to encourage archaeologists to excavate fossils in a ‘sterile’ way today, but there were many bones that had been discovered decades ago, and handled by scores of archaeologists and curators.
The team had looked at more than seventy Neanderthal fossil bones, and tested them first to see if they were likely to contain any usable DNA by checking the condition of other organic molecules: amino acids. Six of the specimens had good levels of these protein building blocks, so there was a good(ish) chance that some DNA might be in them as well. They went on to extract DNA, but, always aware that this genetic material could come from modern people, they checked for contamination before going any further.
A sample from a fragment of Neanderthal bone from Vindija Cave in Croatia looked particularly promising. ‘Luckily for us, this shard of bone was not interesting enough morphologically to have been handled and looked at a lot – so this guy is nearly free from contamination by modern humans,’ said Ed.
So the geneticists chose to try out DNA sequencing procedure on the extract from the Vindija fossil. This technology is advancing at an astonishing rate. Inside insignificant looking white boxes in genetics labs there are small trays holding hundreds of wells of DNA fragments. And the genetic material they are dealing with is very fragmentary: over time, long stretches of DNA that start off with millions of base pairs become broken and broken again into short sections of just a few hundred or tens of base pairs each. So the process involved sequencing those fragments and then virtually sticking them back together. New technology meant that many different fragments could be sequenced at the same time. ‘The throughput for DNA sequencing is hundreds of times more than it was just three or four years ago,’ Ed told me.
He explained the sequencing method in a very visual way (considering you can’t actually open up the box and watch it in action). In each well, there were many copies of one strand of DNA, and the machine worked out the sequence by ‘asking’ each strand what nucleotide base (A, C, T or G) was next. It did this by flowing a solution over the wells containing each base in turn. If the ‘next’ base was T, the solutions of A, C and G would flow over uneventfully. When the solution containing T was introduced, enzymes would grab the base and at the same time emit a flash of light. This is called ‘pyrosequencing’. ‘Every flow, you’ve got different wells lighting up, like a firework display,’ said Ed. Every time a nucleotide solution passed through, some of the wells would answer ‘yes’ by emitting a flash. The machine cycled on and on, until all the strands in all the wells had been sequenced. This technique can read segments of 100–200 nucleotides in length: perfect when you’re looking at tiny fragments of an ancient genome.
Many of the sequences had turned out to be bacterial, but that’s exactly what the geneticists expected. But comparing the sequenced fragments with human, chimpanzee and mouse genome
s, a good percentage of them looked primate. Then came the work of assembling those sequenced fragments into longer pieces. Eventually, if they managed to extract enough fragments, the geneticists would be able to sequence the entire Neanderthal genome.6
Analysis of Neanderthal DNA should be able to cast light on many areas of enquiry, not only the question of hybridisation. By comparing the differences between Neanderthal and modern human DNA, the geneticists can estimate the time of the ‘split’ between the lineages. At the moment, in Leipzig, that’s looking as though it happened some time around 516,000 years ago. This is older than the split suggested by fossils, at about 400,000 years ago – but that’s unsurprising. The genetic split would have happened in a population that was still ‘together’.
This is ground-breaking science, so it’s not surprising that there are still problems that need to be ironed out. And probably the most tricky one is that problem of contamination with modern DNA, which could skew results. Pääbo’s Leipzig lab isn’t the only place where Neanderthal genome sequencing is going on. A team led by Edward Rubin, in California, are also at it – and they published their first chunk of Neanderthal sequence in the same week as Pääbo’s team. But they came up with different results and a different – even earlier – prediction for the divergence of Neanderthals and modern humans, of around 706,000 years ago.7 So it seems that, even with all that careful screening, some contamination may have crept in, explaining the earlier dates coming out of the Leipzig lab.8 With each lab acting as a check on the other, though, the scientists hope that they will be able to overcome these teething problems.9 The Californian dates may seem very early indeed, but it’s important to remember that this is the predicted date of divergence of the mtDNA lineages, not of the actual populations. Based on this genetic data, Rubin’s team estimated that the population split happened about 370,000 years ago, which is quite a good match with the fossil data.
Another potential application for ancient DNA is in identifying bone fragments that are too small to characterise on the basis of size and shape. In fact, this has already been applied to fossils from at least two sites. A child skeleton from Teshik Tash in Uzbekistan has often been held up as the most easterly example of a Neanderthal, but some have disputed its credentials. Even further east, bones and teeth from Okladnikov Cave in Siberia, found alongside Mousterian tools, were too broken up for it to be decided if they were modern human or Neanderthal. Genetics to the rescue, then. Scientists working in labs in Leipzig and in Lyons independently extracted and analysed the mtDNA from the bones from both sites. The results showed that the Teshik Tash child had Neanderthal mtDNA, and so did two of the bone fragments from Okladnikov.10 This study was very significant: it hugely extended the known range of Neanderthals to the east, right into Central Asia. Maybe they even got to Mongolia and China. Genetic analysis is clearly an exciting addition to the toolkit of the Palaeolithic archaeologist.
There is also exciting potential for finding out – at some point in the distant future, when we know a lot more about the functions of genes in us and other animals – more about Neanderthal biology.6 But even now we know that at least some Neanderthals possessed a version of a gene that probably gave them red hair. The gene in question is melanocortin 1 receptor (or ‘mc1r’). In modern humans today, mutations that impair the function of this receptor gene produce red hair and pale skin. A team of geneticists managed to extract DNA – including part of the mc1r gene, from two Neanderthal fossils, one from Spain and another from Italy. Both fossils contained a mutated version of the mc1r gene, different from any of the variants seen in modern humans. To see what effect this gene would have, scientists inserted it into cells in the lab and found that it had a partial loss of function – like the other variations in the mc1r gene that produce red hair in humans today.11 It is important to note that this is a different mutation from that in modern human redheads. It doesn’t imply any genetic mixing between Neanderthals and modern humans, and it certainly doesn’t suggest that the redheads among us are Neanderthals!
Another particular gene that has been identified in Neanderthals is FOXP2. This is a gene that has two specific differences in humans compared with other living primates. People missing out on those human-specific changes to FOXP2 have problems in both producing and understanding speech. Analysis of FOXP2 in living people suggested that it appeared and swept through the human population about 200,000 years ago, which seemed to fit quite well with the appearance of modern humans in Africa. It suggests that ‘modern’ language and symbolic behaviour are uniquely human attributes, with a biological basis. Eric Trinkaus took issue with this interpretation. He argued that there was evidence for symbolic behaviour in the Neanderthal archaeological record, with intentional burial, for instance. And he found it hard to imagine how complex subsistence strategies would have appeared – from around 800,000 years ago – without complex social communication. And yet the ‘human’ version of FOXP2 was initially estimated to have arisen well after the split between modern human and Neanderthal lineages.12 But a recent DNA study of two Spanish Neanderthal fossils showed that they both carried the ‘human’ form of FOXP2.13 For Trinkaus, this showed that the ‘much maligned Neanderthals’ had a degree of human behaviour that was reflected in the archaeological record but that he felt had often been played down. But how can we explain the same version of FOXP2 existing in both modern humans and Neanderthals? Either it is much older than the earlier studies suggested, and was present in the ancestors of modern humans and Neanderthals, or it has passed from one population to the other by gene flow. The latter seems very unlikely as no other genetic studies to date had produced any evidence of gene flow.13
But what about the ambitious Neanderthal Genome Project? Was there any evidence for hybridisation emerging from the nuclear DNA? The key to looking for evidence of hybridisation was to concentrate on genes or other bits of chromosomes that are specific to modern Europeans (and this is a tall order as most genetic differences are shared between populations across the globe rather than being specific to one area), keeping an eye out for these sequences in the Neanderthal genome. If any European-specific DNA sequences were found in Neanderthals, this would strongly imply that there had been some sharing of genes between Neanderthals and modern humans in Europe.
When I visited the Max Planck Institute in the early summer of 2008 Ed told me that they had managed to sequence about 5 per cent of the Neanderthal genome. I asked him a difficult question, considering that the Neanderthal Genome Project was still such a long way from completion: ‘If chimpanzees are about 1.3 per cent different from us, in terms of the sequence of DNA, do you have a feeling for how different the Neanderthal genome is going to be from ours?’
‘Yes, we do,’ he replied. ‘It’s looking about ten times closer than the chimpanzee. But Neanderthals are so closely related to us, it’s hard to speak in terms of percent differences. It really depends on which Neanderthal and which human you’re talking about.’
‘And have you seen any suggestion at all of hybridisation with modern humans?’ I asked.
‘No. There’s no evidence to date of any hybridisation between modern humans and Neanderthals,’ he replied. ‘But by the end of the summer we should have 65 per cent of the Neanderthal genome, so we’ll be able to give a much more definitive answer then.’
This question of what happened when modern humans walked into Neanderthal territory was fascinating. I asked Ed what he would have done if he’d met one of our cousins.
‘If I came face to face with a Neanderthal, the first thing I would do is ask for a DNA sample,’ said Ed, ever the scientist.
So far, then, Neanderthal genetics has shed light on how far this ancient species ranged across Europe and Asia, has shown that they possessed the same ‘language gene’ as modern humans (although it must be stressed that the development of language cannot be linked to just one gene), and that some of them had red hair. And, bearing in mind that there was still a lot of genome left to se
quence, there was no evidence – yet – for any mixing between Neanderthals and modern humans in Europe. (Nearly a year after I visited Leipzig, Svante Pääbo announced the completion of the first draft of the Neanderthal genome – 63 per cent of it, over three billion bases – at the annual meeting of the American Association for the Advancement of Science in Chicago. There was still no sign of interbreeding with modern humans.)14
But it’s also important to remember that the conclusions from genetic studies like this can never rule out any hybridisation. Perhaps it’s just that Neanderthal lineages have not survived to the present day, and maybe some Neanderthals had modern human genes – just not the ones whose genomes were being sequenced.
Does this make the whole endeavour futile? Far from it. If there is no evidence of mixing, then we can at least say that hybridisation didn’t happen at a level that we could consider to be significant, and so it cannot explain the apparent disappearance of Neanderthals from the fossil and archaeological record: they cannot have been absorbed and assimilated into ‘modern’ populations.
Thus far, all the genetic studies suggest that any hybridisation was, at the most, insignificant. And, actually, when you take a closer look at when and where Neanderthals and modern humans were living in Ice Age Europe, this makes some sense. There are only two areas where the dates for modern humans and Neanderthals actually coincide: in southern France and in south-west Iberia, in the period between 25,000 and 35,000 years ago.15 Even then, they could have missed each other by hundreds or thousands of years, so the opportunities for inter-species sex would have been extremely few and far between anyway. So it’s not really surprising that no ‘Neanderthal’ genes have been found in the modern gene pool – or vice versa.
The Incredible Human Journey Page 28