gene flow

I, for one, welcome my Neandertal ancestry.

It may not sound like a lot -- between 1 and 4 percent. But that's the equivalent of one great-great-great grandparent's DNA contribution. In the case of the Neandertal contribution, more than 1500 generations ago, it's an enduring legacy of an ancient group of people, spread across many lines of the genealogies of living people. Beyond their genealogical interest, Neandertal genes might have made a big difference to our evolutionary potential.

In case you wonder what the heck I'm talking about, here's the story: Two new papers in Science describe the full draft sequence of the Neandertal genome, and perform additional analyses to understand the pattern of adaptive evolution in the population ancestral to living people.

Richard Green and colleagues report on the genome, demonstrating very convincingly that present-day people have Neandertal ancestors. It is not entirely obvious when and where the gene flow between Neandertals and other ancient populations happened -- whether it was associated with the dispersal of most of our ancestry from Africa, or whether it may have been earlier. The gene flow was not limited to Europe, and evidence for Neandertal ancestry occurs in East Asian and Australasian populations.

The paper is full of other good stuff, including some evidence about which gene regions changed under selection in the ancestral human population.

Meanwhile, the second paper by Burbano and colleagues applies new microarray techniques to assess how much of the human legacy of amino acid changes has arisen in the latest, post-Neandertal period of our evolution.

So there's a lot about the pattern of evolution and gene flow leading to living people, and a lot about adaptive and functional evolution. That makes a lot for me to cover -- and while I have the papers a little early, time is short. Let's see how much I can help clarify what's in this new research.

If you had to sum up in a few words, what does this mean for paleoanthropology?

These scientists have given an immense gift to humanity.

I've been comparing it to the pictures of Earth that came back from Apollo 8. The Neandertal genome gives us a picture of ourselves, from the outside looking in. We can see, and now learn about, the essential genetic changes that make us human -- the things that made our emergence as a global species possible.

And in doing so, they've taken a forgotten group of people -- whom even most anthropologists had given up on -- and they've restored them to their rightful place in our heritage.

Beyond that, they've taken all of their data and deposited it in a public database, so that the rest of us can inspect them, replicate results, and learn new things from them. High school kids can download this stuff and do science fair projects on Neandertal genomics.

This is what anthropology ought to be.

What did they sequence?

The Max Planck group obtained most of their genomic sequence from three specimens from Vindija -- Vi33.16, Vi33.25, and Vi33.26. These are all postcranial fragments with minimal anatomical information. Green and colleagues were able to establish that the three bones represent different women, and that Vi33.16 and Vi33.26 may represent maternal relatives.

From these skeletons they got 5.3 billion bases of sequence. All this from an amount of bone powder about equal in mass to an aspirin pill.

Amazing. I mean, I know the folks at Max Planck are reading this. It's inspiring to see what they've been able to do. These are three pieces of barely diagnostic hominin bone, and they've obtained literally hundreds of times more information than we have ever gotten from the fossil record of Neandertals.

I'll describe the analyses of genetic similarity with humans in more detail below. As a brief summary, of those positions where the human genome differs from chimpanzees, Neandertals have the chimpanzee version around 12.7 percent of the time -- meaning that across the genome, a Neandertal and a human will share a genetic ancestor an average of around 800,000 years ago. This is a couple hundred thousand years higher than the same number if we compare two humans to each other. The higher age of genetic common ancestors reflects partial isolation between the Neandertal population and the African populations that gave rise to most of our current genetic variation.

The team were able to identify 111 candidate duplications, almost all of which have some evidence of copy number variation in humans or other primates. They tentatively show that Neandertals have a bit more copy number variation than present-day humans, and identify a few loci with substantially higher copy numbers in one group or the other.

A substantial part of the paper is dedicated to finding evidence of positive selection on the human lineage after the emergence of Neandertals. The idea is to look for fixed selective sweeps -- regions where humans are likely to have SNPs absent in Neandertals and a relatively shallow gene tree. They identify 212 regions like this -- as I discuss below, a surprisingly low number.

The second paper, by Hernán Burbano and colleagues, describes the application of a targeted microarray to probe Neandertal genetic samples for protein-coding variants that separate humans from chimpanzees. They identify 88 amino acid substitutions that seem fixed in the known sample of living humans, but not present in the Neandertal sequence. Those 88 are not necessarily all functionally important, although this list will include a number of "structural" genetic changes that make a difference to proteins expressed worldwide today. There is much to come in analyzing the categories and genes represented in both lists, which may tell us very interesting things about our Late Pleistocene evolution.

What is the evidence for interbreeding?

From their initial work sequencing the nuclear genome in Neandertals, the Max Planck group has followed a clever strategy: Don't look at the Neandertal sequence to see what humans share, look at human variation to see which version the Neandertal sequence has.

The strategy is smart because it helps to obviate some major problems with ancient DNA -- you don't have all the parts, and the parts you do have probably contain a lot of sequencing errors of various kinds. By looking first at sites that vary within humans (or, in some comparisons, between humans and chimpanzees), we can focus on a very simple question -- did the Neandertal have one version, or the other?

Applied to human variation today, there are several ways we might use a Neandertal genome test the hypothesis of no interbreeding. Green and colleagues focus on two complementary approaches.

1. If Neandertals contributed no genes to living populations, then they should be equally related to all living people, no matter where in the world those people live.

Green and colleagues show that the Neandertal genome is closer to some humans than others. People whose ancestry lies outside Africa are significantly more like Neandertals than are people who live in Africa today. In this study, the authors include whole genomes from people in France, China and Papua New Guinea outside Africa, and Yoruba and San inside Africa. The Africans are not as close to the Neandertal as any of the non-Africans.

That doesn't mean that non-Africans derive most of their genes from Neandertals -- in fact, as I describe below, the proportion is quite small. Living people are more like each other -- even non-Africans and Africans -- than any of them are like Neandertals.

The point is that despite this great similarity of living people, we have genetic variants that we share with the Neandertal genome, and that proportion is a lot higher outside Africa than inside it. The natural conclusion is the Neandertals contributed more genes to non-Africans than to Africans.

One thing is for sure: You can't explain this observation under the hypothesis that a small, African population expanded out of Africa without interbreeding with Neandertals along the way.

2. Look at the genes most likely to represent ancient population structure, the ones with deep roots outside Africa.

This is an idea that we came up with to look for genes in living humans that might have come in from Neandertals or other ancient populations (for example, we described it in our 2008 review). Look for the parts of the genome with the deepest genealogical roots outside of Africa. Those are candidates for Neandertal gene flow -- a high chance that one of the two sides of that deep root was present outside of Africa for hundreds of thousands of years.

Green and colleagues took this idea to the next level. They found parts of the genome where non-Africans have a deep root and Africans don't. Then they looked at the Neandertal sequence. Out of the 12 regions they identified with deep roots outside Africa, they found that the Neandertals had the deep, non-African specific version in 10 of those.

I mean, there's really not any other way you can explain this. We got those genes from Neandertals. Every one of those loci is a region where some people have a Neandertal-derived allele, and others don't. Those particular 10 loci are a small fraction of the overall Neandertal-derived element of our heritage -- because they used Perlegen SNPs to find them, they ended up with regions that are fairly long (100 kb or more in length). Those are probably all really interesting, but there will be more of them when we can reliably identify smaller segments with deep genealogies.

Could the results have been caused by contamination?

Green and colleagues are utterly convincing about the level of contamination in their sequence. They have employed several independent checks, all of which arrive at the same conclusion: The modern human contamination in almost all their comparisons is limited to significantly less than one percent -- and for autosomal sequence they can give a tight estimate of 0.7 percent contaminating sequence.

The methods that Green and colleagues used to test for a Neandertal contribution to non-African populations are not likely to be strongly influenced by contamination. The probe for deep roots in particular is extremely unlikely to be influenced by contamination in the Neandertal sequence.

The very low contamination rate, and methods that should be robust to some contamination, means that we can be very confident in their result.

How much Neandertal ancestry do we have?

The Neandertal contribution does not make up a major proportion of any population, even outside of Africa. Green and colleagues apply a population model that involves isolation between ancestral Neandertal and African populations, a dispersal from Africa into Eurasia, and subsequent mixture with the Neandertals. Under this model, the estimated fraction of Neandertal ancestry for non-African populations today is between 1 and 4 percent.

Now, let's put on our skeptics' hats. Is this the right model?

If Neandertal and African populations had not been isolated, then the amount of mixture after an out-of-Africa dispersal would be lower. On the other hand, the dispersing African population would already be part Neandertal, because of genetic mixture. The proportion of ancestry from ancestral Neandertals would be around the same amount, it would just be distributed across a longer time.

They did not examine the question of how much of the genome came in from Neandertals because of selection. The estimate they have, between 1 and 4 percent, is so high that this is not just a few genes introgressing in from Neandertals -- it is a big fraction of the neutral, non-coding part of the genome. So selection doesn't explain the similarity, nor can parallelism -- the similarity is genome-wide, not just coding or functional changes, and not as far as we know clustered into regions that might have hitchhiked with adaptive alleles.

But there's clearly a lot more to do, characterizing the functional implications of some regions, testing for selection, and finding Neandertal variants that might have reached very high frequencies in later populations. To the extent that selection has influenced the pattern, it will also throw off the simple population model. But it doesn't throw off the fraction of Neandertal ancestry -- if it's three percent, it doesn't matter whether it was selected or neutral, it's still three percent.

So the bottom line is, the fraction is going to be about right, regardless of the mechanism by which the genetic mixture happened.

Can we please take off our skeptics' hats? It's getting in the way of my Neandertal victory dance.

No. All the cool paleoanthropologists wear hats.

What about population structure within Africa? Could that explain the apparent Neandertal contribution?

We've known about the occasional deep-rooted genealogies outside Africa for a long time (and Jeff Wall's work, as an example among others, has explained that pattern as archaic human mixture into non-Africans). They've been talking about something like five percent of the human genome coming from admixture with ancient groups outside of Africa. So this shouldn't come as a shock.

Until now, though, it has been possible for some people to wave these results away. We didn't really know that any of those deep roots were in archaic humans, and after all, who's to say that they aren't variants that originated in Africa and have since been lost there, or that we haven't found them yet? African variation is great, and if you imagine that some variation might have once existed in northeastern Africa and was subsequently lost within African populations, that might look like admixture with archaic humans outside of Africa.

This line of argument is now special pleading. Why would we posit a cryptic mystery population in Africa, which happens to look genetically identical to Neandertals, but has subsequently disappeared? A big fraction of deep genealogies outside Africa really are in Neandertals. By far the simplest explanation is that today's non-Africans got them from ancient non-Africans. This is no surprise -- that's where the data have been pointing now for five years.

Yet Africans are a lot more diverse than other populations, and this diversity itself does reflect the dynamics of the ancient African population. The Neandertals aren't so different from that pattern that now still exists within Africa -- they're extending the notion that "modern" is something that's been evolving for a long time. I expect we'll be able to come to a better understanding of ancient population interactions within Africa, by understanding the parts of the genome that have come from Neandertals outside of Africa.

Could the gene flow be due to ancient interactions between West Asia and Africa?

Green and colleagues suggest that at most few genes from modern humans ended up in Neandertals.

That is, although they find lots of evidence of old-looking genes in us that are shared with the Neandertal genome, they find few cases of new-looking genes in us that are shared with that genome.

That might suggest several things about interactions between Africa and West Asia and Europe during the Middle to Late Pleistocene. For example, if there had been high gene flow from Africa into West Asia after the first appearance of a distinct Neandertal population, maybe 200,000 to 400,000 years ago, we might expect to find some new-looking genes in humans that Neandertals also got.

On the other hand, the data are from European Neandertals, who are at the end of a fairly long chain of populations from Northeast Africa. If gene flow had been ongoing into the Levant or further into West Asia during the last 200,000 years, it's not obvious how many of these genes would have made it into Europe. The rapid mitochondrial DNA coalescence of Neandertals does suggest substantial mobility in the population across Central Asia to Western Europe. But maybe that apparent dynamism had a boost from mtDNA selection.

So just on the data, I don't think we know yet whether this is gene flow in the Levant 200,000 or 100,000 years ago, or whether it's genes coming from West Asian Neandertals into dispersing Africans after 100,000 years ago. I expect all are likely. I have some ideas how to test some of these things, and we will get started immediately.

The lack of apparent mixture of "modern" genes into Neandertals -- what does it mean?

It means that a model of one-way gene flow from Neandertals into us can explain the pattern of genetic similarity.

The authors explain this as a function of population expansion. The expanding population (us) picks up some Neandertal genes that expand in numbers, while the contracting population (Neandertals) doesn't have a chance to pick up as many genes because it is declining in numbers. That model seems plausible, particularly in comparison with historical cases of population contact.

On the other hand, the three Neandertals from which most of the genome sequence was derived all date to before 40,000 years ago. There weren't any modern humans around for them to have interacted with around Vindija at that time. So should we be surprised that they don't have genes of modern humans?

A more interesting question was posed to me by a very sharp journalist: What would we expect the result to have been if they had sequenced a Near Eastern Neandertal, like Amud, for example?

The answer seems obvious -- the admixture fraction should have been higher. That population, which is the most likely to have been the source of mixture, must have been somewhat genetically different from the European Neandertals. Any extent of genetic differentiation between them would make the European Neandertals look less like non-Africans today than the Near Eastern ones.

I'll have more to say about these Near Eastern Neandertals in the next few days.

But wait a minute. I thought the mitochondrial DNA proved that Neandertals are extinct!

Selection. Selection. Selection.

I've been saying it for years. I've published it. Will you learn to listen to me, already?

The mtDNA of Neandertals is gone because it conferred some disadvantage. There are many reasons to suspect this -- the Neandertal variation is itself apparently recently derived; the human variation is clearly in disequilibrium, especially outside Africa; the mtDNA genes affect functions that differ greatly in Neandertal and recent populations, including energetics, longevity, and brain; there are clear signs of mtDNA selection in many recent human populations.

Mitochondrial DNA is useful for a lot of reasons, but nobody should ever have relied on it alone as evidence of Neandertal population dynamics.

Is it really true that there is no variation in Neandertal ancestry outside Africa?

The comparisons in the paper are highly convincing because of the sheer amount of sequence taken from the sampled individuals. A single gene locus from an individual may be unrepresentative of the person's population, but averaged across the whole genome, the difference between two people from distant populations is very, very close to the difference between the two populations.

But they sampled very few individuals. So we are left with a question -- do we really know we've sampled variation outside Africa enough to make regional estimates of Neandertal gene flow?

I think we could do better with more genomes. For example, when it comes to finding deep genealogies, we need to be able to find shorter regions than the ones used by Green and colleagues. That will expand the sample of candidate loci, and will catch some Neandertal-derived genes that we're missing now. Moreover, if gene flow was really around 1-4 percent, many SNPs that came in from Neandertals will be rare enough to be missing from the big SNP genotyping samples. We may find some variants with whole-genome sequencing on larger samples that will be worth examining.

But most important, we'll be able to develop strategies based on this success to find ancient population structure involving groups where we don't yet have the DNA -- like populations of South and East Asia. Some of those may give us the chance to test those methods soon, as for the Denisova individual.

Is this multiregional evolution, or just out-of-Africa with some leakage of earlier Eurasian genes?

Out-of-Africa movement was a major mechanism of recent human evolution. The genetic ancestry of living people is multiregional.

I see no contradiction between those statements. From now on, we are all multiregionalists trying to explain the out-of-Africa pattern.

There was clearly a dispersal of African genes into the rest of the world during the Late Pleistocene, sometime between 50,000 and 100,000 years ago. Living people everywhere on Earth derive more than 90 percent of their genes from African populations who lived 100,000 years ago. That much is plain.

(Why did I not write "more than 96 percent?" See below.)

These genetic observations require some kind of out-of-Africa event. This event was not limited to a few genes, and selection of a few genes even with substantial hitchhiking of surrounding genome cannot account for the pattern. There must have been some kind of demographic expansion including African-derived populations and preferentially excluding the genes of Eurasian populations like the Neandertals. Selection on a gene network might have mediated the expansion, as suggested by Eswaran (2002). Or the expansion might have been culturally or technologically mediated, as many other people have suggested.

Those are hypotheses about mechanisms. How did it come to be that living people trace the overwhelming majority of their ancestry to Africa within the last 100,000 years? These explanations may answer that question.

The present study shows that Neandertals were at a minimum partially isolated from their contemporaries in Africa, and that the genetic divergence between those populations was larger than the genetic differences between European, Asian, and African populations today.

Yet those Neandertals are among our ancestors. Late Pleistocene humans had multiregional origins, and the evolution of the Neandertals was itself a case of relatively recent population dispersal from Africa or West Asia. Human and Neandertal genes mostly derive from common genetic ancestors between 400,000 and a million years ago -- much, much later than the initial habitation of Eurasia 1.8 million years ago.

But 1-4 percent is so minor, can it be an important part of our evolution?

There are three things you have to ask about the fraction of Neandertal ancestry.

1. How much gene flow would it take to guarantee that anything adaptive in the Neandertal population survived into later people?

The answer to that question is simple -- it takes a few dozen matings to get most adaptive genes into our population. If there was a lot of interference with the genetic background, it might take more -- just to make sure that the advantageous alleles had a chance to be de-linked from the genetic background.

If Neandertals are one percent of the ancestry of non-Africans, we can be very sure that any gene in a Neandertal that had adaptive value in the later population is here now. That means they were important in an evolutionary sense.

2. What fraction of the human population 50,000 years ago were Neandertals?

This is very important -- when it comes to neutral genetic loci, the essential question is how much the Neandertals may be underrepresented today relative to their numbers in the past. Is three percent too low? It seems very unlikely that the fraction of Neandertals compared to the rest of humans was as high as 10 percent -- we know that Africa already had a large population 50,000 years ago, and everything we know about Neandertals suggests a very low population density, an effective size much smaller than 10,000 individuals. Were five percent of the people on Earth 50,000 years ago Neandertals?

We don't really know the answers, but now we have a chance to test hypotheses about ancient population size and expansion in Neandertals. My point at the moment is only this: If today Neandertal genes make up only one percent of the gene pool of the 5 billion people outside Africa, that's the genetic equivalent of 50 million Neandertals.

In relative terms, their contribution to our population may be a reduction from their fraction of the Late Pleistocene population. Not that great a reduction, not a massive crash to zero. A reduction in the wake of the out-of-Africa movement, possibly from five percent to three.

You might think the answer to this is obviously zero. But in genetic terms, we can ask, how many times has the average Neandertal-derived gene been replicated in our present gene pool? Those aren't Neandertal individuals -- that is, a forensic anthropologist wouldn't classify them as Neandertals. They're the genetic equivalent.

The answer to this is also simple: In absolute terms, the Neandertals are here around us, yawping from the rooftops.

There are more than five billion people living outside of Africa today. If they are one percent Neandertal, that's the genetic equivalent of fifty million Neandertals walking the Earth around us.

Does that sound minor? If I told you that your average gene would be replicated into fifty million copies in the future, would you be satisfied? Maybe your ambition is greater, but I think the Neandertals have done very well for themselves.

Does this mean that Neandertals belong in our species, Homo sapiens?

Yes.

Interbreeding with fertile offspring in nature. That's the biological species concept.

Now, some paleontologists might still disagree -- maintaining that species are units that can be distinguished morphologically, or by one or more derived features, or any number of other definitions. That's fine with me, as long as they're clear. But understand: It does define all non-Africans today as an interspecific hybrid population.

So maybe they want to rethink that one?

If Eurasians got less than 4 percent from Neandertals, doesn't that mean that they got more than 96 percent from Africa?

I look at the 1-4 percent estimate as a minimum, for several reasons. As I'll note below, this estimate mainly refers to the excess Neandertal ancestry outside Africa, which means there may be some additional amount that both recent African and non-African populations share.

But more important, Neandertals weren't the only people living in Eurasia 100,000 years ago. China didn't have Neandertals, nor did Southeast Asia and Java. India was full of hominins, which might or might not have shared substantial genetic similarity with Neandertals. They're close enough to the known Neandertal range to speculate that they may have been close, but the only available fossil, the Middle Pleistocene Narmada skull, is not very informative. Any of these populations might have been genetically different from Neandertals, and might have also contributed genes to present-day human populations -- genes that wouldn't show up by scanning the Neandertal genome.

The recent genetic sequencing of the Denisova pinky (a.k.a. the X-woman) from the Altai Mountains reminds us that these populations outside of Africa may have been quite a bit closer to us, genetically, than we might have expected from the 1.8-million-year record of humans outside Africa. These populations were dynamic in ways that many paleoanthropologists haven't yet appreciated.

Do living Africans have Neandertal ancestry, too?

I think that the present study doesn't have the power to answer this question, at least with the design that the authors used. The fact that living Africans are less genetically similar to the Neandertals is extremely important evidence of the Neandertals' genetic contribution to populations outside Africa. But it doesn't bear on how much back-migration into Africa may have happened.

We know that the answer is nonzero, because Africa has received immigrants from other parts of the world during historic times. The same genetic patterns that reflect population contacts up and down the East African coast, and across the Sahara into West Africa, show the possible conduits for the flow of Neandertal-derived genes into African populations.

But how much genetic dispersal into Africa happened in LSA or late MSA times? Mitochondrial and Y chromosome distributions in Northeast Africa suggest there was been some. Nevertheless, Africa would have been a very difficult place to return, for humans who had begun adapting to different ecological and disease environment.

I think that some Neandertal genes might have made it back into Africa, even in ancient times, but I wouldn't be surprised if that number was small.

The big shoe left to drop is the extent of population differentiation within Africa during MSA times. So far we've seen hints that these populations might have been nearly as differentiated from each other as they were from Neandertals, with substantial gene flow homogenizing them in the last 30,000 years. This paper includes an additional Bushman genome, after the four published earlier this year. Comparing that new genome to the Neandertals, its modal difference from the human reference (Hg18) genome is between the other humans and the Neandertal. Not quite halfway between, but nearly so. There's a lot of genomic variation within Africa, and exploring the population history that explains that variation may turn up some surprises.

What about recent selection?

One of the really exciting aspects of this work is that both Green and colleagues and Burbano and colleagues look for things that all humans today share but Neandertals lack.

You might call these "the genes that make us modern," although functionally we have little idea what any of them do.

Both papers show one thing that is extremely interesting: There aren't very many such genetic changes.

Burbano and colleagues put together a microarray including all the amino acid changes inferred to have happened on the human lineage. They used this to genotype the Neandertal DNA, and show that out of more than 10,000 amino acid changes that happened in human evolution, only 88 of them are shared by humans today but not present in the Neandertals.

That's amazingly few.

Green and colleagues did a similar exercise, except they went looking for "selective sweeps" in the ancestors of today's' humans. These are regions of the genome that have an unusually low amount of incomplete lineage sorting with Neandertals, and therefore represent shallow genealogies for all living people. They identify 212 regions that seem to be new selected genes present in humans and not in Neandertals. This number is probably fairly close to the real number of selected changes in the ancestry of modern humans, because it includes non-coding changes that might have been selected.

Again, that's really a small number. We have roughly 200,000-300,000 years for these to have occurred on the human lineage -- after the inferred population divergence with Neandertals, but early enough that one of these selected genes could reach fixation in the expanding and dispersing human population. That makes roughly one selected substitution per 1000 years.

Which is more or less the rate that we infer by comparing humans and chimpanzees. What this means is simple: The origin of modern humans was nothing special, in adaptive terms. To the extent that we can see adaptive genetic changes, they happened at the basic long-term rate that they happened during the rest of our evolution.

Now from my perspective, this means something even more interesting. In our earlier work, we inferred a recent acceleration of human evolution from living human populations. That is a measure of the number of new selected mutations that have arisen very recently, within the last 40,000 years. And most of those happened within the past 10,000 years.

In that short time period, more than a couple thousand selected changes arose in the different human populations we surveyed. We demonstrated that this was a genuine acceleration, because it is much higher than the rate that could have occurred across human evolution, from the human-chimpanzee ancestor.

What we now know is that this is a genuine acceleration compared to the evolution of modern humans, within the last couple hundred thousand years.

Our recent evolution, after the dispersal of human populations across the world, was much faster than the evolution of Late Pleistocene populations. In adaptive terms, it is really true -- we're more different from early "modern" humans today, than they were from Neandertals. Possibly many times more different.

More?

That's what I have time for now, if I want to get this posted. There is much, much more to say on the topic, and you can bet it will be all Neandertals all the time here for the foreseeable future.

References:

Green RE and many others. 2010. A draft sequence of the Neandertal genome. Science (in press) doi:10.1126/science.1188021

Burbano HA and many others. 2010. Targeted investigation of the Neandertal genome by array-based sequence capture. Science (in press) doi:10.1126/science.1188046