recent selection

I have had a New York Review of Books essay by Richard Lewontin, titled, "Why Darwin?" on my desktop for a week without getting to the last section of it.

Like many essays in the NY Review of Books, Lewontin's shoehorns small points from the books into an argument of his own. As you might guess from the title, Lewontin's theme is that Darwin has been overrated -- a result of biologists overemphasizing a "great man" story of the history of their science, and an unjustified belief in the ubiquity and power of natural selection. Lewontin mobilizes his argument against Jerry Coyne's Why Evolution Is True.

I don't really find the "pluralist versus adaptationist" debate very interesting. Despite the vocal complaints of some, I can't ever seem to locate the mythical "adaptationists" who deny that non-adaptive evolution ever happens. So the "debate" always comes down to whether particular adaptive hypotheses are true. Since no scientific hypothesis is true a priori, and since "those adaptationists are always saying stupid things" is not a scientific argument, I don't see the point.

Still, I meant to get to the last section of Lewontin's essay, and this morning I finally read it. To close his case for the weakness of natural selection, Lewontin turns to another new book by Greg Gibson, titled, It Takes a Genome: How a Clash Between Our Genes and Modern Life Is Making Us Sick. The book is an extended account of "diseases of civilization", a topic that I discussed here last week ("Arrested adaptation and the 'diseases of civilization'"). Here's a passage from the book's promotional material (on the Amazon page):

In It Takes a Genome, Greg Gibson posits a revolutionary new hypothesis: Our genome is out of equilibrium, both with itself and its environment. Simply put, our genes aren’t coping well with modern culture. Our bodies were never designed to subsist on fat and sugary foods; our immune systems weren’t designed for today’s clean, bland environments; our minds weren’t designed to process hard-edged, artificial electronic inputs from dawn ‘til midnight. And that’s why so many of us suffer from chronic diseases that barely touched our ancestors.

Set aside for a moment how "revolutionary" this hypothesis is -- I'll revisit the idea in another post. The question is whether this mismatch between our environments and our genetic variation means that human evolution "stopped" or that we are still "adapted to the Pleistocene". As I pointed out in my earlier post, both propositions are true: human populations are mismatched with their current environments, and human populations have been recently adapting very rapidly to new environments. Here's what I wrote last week:

[M]any of today's chronic diseases reflect the reaction of human biology to novel environments for which our genes are not well adapted. But we don't need to exaggerate the slowness of human evolution to arrive at that conclusion. Recent rapid evolution of humans does not mean that humans are perfectly adapted to the present. Far from it -- if human populations have undergone rapid genetic changes into the past thousand years, it is a strong sign that fitness has not yet maximized in the post-agricultural environment.

I can contrast my point of view with Richard Lewontin's, who perfectly reiterates the "human evolution stopped in the Pleistocene" version of events.

An important property of adaptive evolution is that it is usually a slow process. Certainly there are cases where a single genetic change can mean the difference between life and death in a hostile environment. The classic cases are the mutations that give pathogenic microorganisms the ability to resist antibiotics or mutations that allow crops to resist pathogens, for example insects or herbicides. But these are not representative models for how species adapt, by accumulation of mutations of small effect, to changes in food availability, temperature modifications, and the thousand shocks that flesh is heir to. The usual small differences in fitness among genotypes are therefore manifest as detectable evolutionary change only after thousands of generations.

This deliberate tempo has presented the human species with a problem of adaptation. With a human generation of about twenty-five years, there have been roughly only one hundred generations since the founding of the Roman Republic. Yet the changes in the human environment caused by changes in human activity have been enormous. Changes in diet, habitation, working conditions, the pollution of air and water, and especially the considerable increase of lifespan that result in major alterations and breakdowns in the bodily machinery have all been too rapid for genetic adaptation.

Notice the false premises: Adaptive evolution is "usually a slow process." Species adapt by "accumulation of mutations of small effect." It's as if he were transported back in time to 1908 where no one had heard of the breeder's equation.

There's nothing impossible about long series of small changes. But they are not the only mode of adaptation, or even the most likely one. Populations with additive genetic variation that correlates with fitness will change rapidly under selection. The structure of the additive variation may lead to strong selection on one gene of large effect, or selection in parallel across many genes of varying effects. Series of small changes may be required for some adaptations, but a rapid environmental change (as Lewontin observes for humans) may cause bursts of rapid changes in allele frequencies.

To maintain the slowness of human evolution, Lewontin must do three things:

1. Assume humans are genetically uniform.

2. Where humans obviously are not uniform, argue that variations are uncorrelated with fitness.

3. Ignore any historical or genetic evidence that might contradict 1 and 2.

Keeping in mind the short length of this section of the essay, Lewontin does manage all three of these conditions.

I think it's downright sneaky the way Lewontin reinforces the assumption of human genetic uniformity. He refers to "the human genotype" as if there were only one! By emphasizing that "parts of the human genome are out of correspondence with modern life", he precludes the possibility that some human genomes may be more in correspondence than others. Sure, if humans share a single genome, they can't possibly differ in any adaptive way.

But diversity is the reality. Examples of recent human evolution are fixtures in biology textbooks, from sickle-cell to lactase persistence. These are traits that have rapidly changed in frequency during the last 2500 years, due to changes in recent human environments -- disease for the former, diet for the latter. These rapid transformations in precisely those that Lewontin says are impossible -- environmental changes being "too rapid for genetic adaptation." A number of morphological changes are also evident when comparing archaeological and recent skeletal samples in many parts of the world. Somehow the relevance of these recent changes goes unmentioned in the essay.

One of the best-characterized examples of evolution in recent populations is the rapid Holocene evolution of pigmentation phenotypes. It's a textbook example of human variation, and several adaptive hypotheses may explain it. So pigmentation would seem an unlikely example of how human evolution has been too slow to cope with the environment. But Lewontin finds a way:

[H]igh doses of solar radiation that is experienced by surfers on the California beaches might induce an eventually fatal skin cancer, but the cancer death almost always occurs well after reproductive age, so there is no opportunity for selection to act.

I agree that current patterns of cancer mortality of light-skinned surfers may have little impact on their fitness. In other words, this chronic disease is a sign of an environmental "mismatch" that future genetic evolution is unlikely to erase.

But why turn to false arguments about the speed of evolution to make this point? Surely Lewontin knows that "reproductive age" in humans is not synchronous with reproductive effort? Skin cancer is one of the earliest-killing cancers, with a good fraction of victims dying at ages when they might otherwise be helping raise their kids or grandchidlren. Lewontin must also know that human populations vary greatly in their skin cancer susceptibility, and that some surfers (the dark pigmented ones) have lower skin cancer rates after the same sun exposure. Skin cancer may or may not be the best explanation for dark pigmentation in low-latitude human populations (there are others, none mutually exclusive), but this example works strongly against Lewontin's claims that natural selection is "slow" and that human environmental changes have been "too rapid for genetic adaptation." We aren't perfectly adapted today, and the rate of our evolution in the recent past was very fast.

References:

Lewontin RC. 2009. Why Darwin? New York Review of Books 56(9) May 28, 2009. Online

After the last post, you might wonder what the big deal is about these information theoretic measures of linkage. After all, we’ve got lots of other measures of linkage to choose in population genetics, with many years of theory behind them. The basic conclusion about genetic drift was that it adds mutual information to samples over short regions, but that recombination over longer areas washes it out. If the net effect is no linkage, why would we bother to come up with some non-standard linkage measure?

One answer: If the existing linkage measures were so great for testing neutrality, then we might expect some of the recent genome-wide selection scans to have used them. But they didn’t – instead we have several partially incompatible methods, all of which eschew the usual measures of linkage.

I’m not going to summarize all the reasons for this, at least not yet. But there is one thing that the current positive selection tests have in common: they all attempt to quantify the decay of linkage across long strings of loci. Each of them—LRH, iHS, LDD—distills into a single number the pattern of reduction of linkage across physical distance.

After the last post, that may seem odd. If we know the physical distance (or more to the point, the map distance), we can predict the amount of linkage under drift. That really ought to be enough to test neutrality. Yet the existing tests insist on a second dimension: the rate of decline of linkage. Partly, that’s because these tests try to separate positive selection from other things that violate neutrality, like inversions. But it also reflects a real limitation of the methods: they ignore some of the information available in the data, and thereby limit their power to test the hypothesis of genetic drift based on linkage. So they must turn to the rate of linkage decay as a test of drift.

The trouble with homozygosity

Massive genotyping projects are not sequencing projects. They provide long lists of genotypes, not sequences. If you’ve got a long list of genotypes, one of the measures of variation immediately available to you is homozygosity. Homozygosity among multiple sites, or joint homozygosity is not mathematically the same as the mutual information between sites, but it also is increased by linkage. To the extent that genetic drift predicts the distribution of long-range linkage, you can test neutrality by looking at the joint homozygosity of two or more sites. For example, the extended haplotype heterozygosity (EHH) is the probability that a pair of gametes that share a single core haplotype will also share identical haplotypes at longer distances. Homozygosity itself is not a test of linkage—for that we have to combine the probabilities of identity of all alleles in some way.

Homozygosity-based tests of neutrality set up this combination as a ratio: the ratio of homozygosity for one allele or core haplotype versus the homozygosity of other alleles or haplotypes. That procedure throws away a lot of information carried by the fraction of haplotypes that are nonidentical at distance. Since that fraction of non-homozygote mutual information, unlike homozygosity, actually increases with distance, it may in some circumstances provide a more powerful test of neutrality. The use of a ratio of homozygosity also reduces the chance to detect certain classes of non-neutral loci—most obviously, the ones that have two or more selected alleles.

To put some numbers to these ideas, let’s consider we have two loci, A and B. The alleles of A are a₁ and a₂, B has b₁ and b₂. Each of these alleles has an associated frequency, for example p(a₁), and the frequency of the haplotype a₁b₁ will be written p(a₁b₁). If A and B are independent (unlinked) then the expected value of p(a₁b₁) would simply be p(a₁)p(b₁).

The usual measure of linkage, D, is calculated as

(1)

It thus involves the frequencies of all four possible haplotypes. At linkage equilibrium, the two terms are expected to be the same, so that D = 0.

Several other measures of linkage are in common use (and sometimes lead to confusion). There are several reasons why you might want a different measure, and one of the biggest is that D depends on the frequencies of the alleles a₁ and b₁—low-frequency polymorphisms will have systematically lower linkage values for the same evolutionary scenario. A very good thing about D is that it does not require us to know the frequencies of the individual alleles a₁ or b₁, only the frequencies of their joint haplotypes. But the expectation that the products in the equation are equal depends on a 2 × 2 contingency table. Three-, four- or more-locus haplotypes demand some other measure.

Using the same terminology, the homozygosity of the haplotype a₁b₁ is given as p(a₁b₁)². By itself, this tells us nothing at all about linkage. If we combine it with the homozygosity of one or the other allele, (for example with the difference p(a₁)²- p(a₁b₁)², we will have a measure of the reduction in homozygosity with distance. That reduction in homozygosity still doesn’t tell us about linkage, without considering p(b₁). But if we scale the reduction by taking it as a ratio with the reduction in homozygosity of one or more other haplotypes, those other haplotypes give us indirect information about p(b₁). So we have an indirect measure of linkage, based on the relative rate of linkage decay.

Relying on the rate of decay of linkage isn’t such a bad idea if we don’t know the rate of recombination between markers. In that case, we can use the rate of decay of homozygosity of other haplotypes as a scaling factor. But there is a problem lurking: The decay of homozygosity around a marker or core haplotype depends on its frequency. Under drift, higher-frequency haplotypes decay over shorter distances. So our test will be biased in a way that depends on the frequency spectrum of haplotypes.

It seems much more direct to estimate the mutual information between A and B. We don’t need to use an indirect linkage measure when we have all the frequencies needed to calculate a direct measure. Mutual information is a useful measure because it extends easily to many loci. But it has a disadvantage: it requires us to obtain an independent estimate of the recombination fraction between our markers.

Maybe that’s not such a problem. High-resolution recombination maps are available for the HapMap markers. If we use those maps, then we can test neutrality with markers at fixed map distances instead of physical distances. That ought to let us easily quantify the genome-wide fraction of mutual information that originated in a given time interval in the past. But we’ll have to remain cautious, since the map distances are worked out under the assumption of neutrality.

Why multilocus haplotypes are useful

Why do we care about many loci, when we can test neutrality using only one? Suppose we’re interested in the mutual information across a 500 kb stretch of the genome. We could pick a single marker on each end of the 500 kb, and measure the mutual information between them. That’s basically what I did in the case of drift in the last post, “When genetic drift reduces entropy”.

That isn’t slicing the sample very finely. Suppose that our two markers both have major alleles at 80 percent. That makes the expected frequency of the most common joint haplotype 64 percent, and the least common 4 percent. In our sample of 120 gametes, we’ll conclude that the two are significantly associated if those values go to around 70 and 10 percent, respectively. That’s like salting the sample with seven or eight copies of the rare haplotype.

On the other hand, we could salt our sample with more than a dozen copies, evenly split between the two intermediate-frequency haplotypes, and never get a significant result. Even if the expected rare haplotype were completely absent, it wouldn’t be enough to reject the hypothesis of random association, much less the hypothesis of genetic drift. Natural selection can cover its tracks, if two or more linked haplotypes have both increased in frequency.

Is this likely to be a problem very often? Past some distance, positive selection loses its impact on linked haplotypes, because there’s too much recombination. Near a selected site, linkage is strong enough that there will usually be a single major haplotype hitchhiking upward. In between, there may be minor haplotypes hitchhiking more weakly but we will generally be able to look closer to find a major haplotype. But if there are multiple haplotypes under selection, perhaps because more than one adaptive mutation have occurred, the locus may well look completely neutral.

An example in human populations may be MC1R. Harding et al. (2000) sequenced the gene in 106 Africans and over 356 Europeans. They found a diverse array of amino acid mutations in Europeans that were absent in their African sample, concluding that purifying selection on skin pigmentation had prevented such mutations from becoming common in Africa. In contrast, purifying selection was relaxed in Europe, allowing several loss-of-function variants to increase in frequency. They found no statistical evidence for positive selection on these loss-of-function variants in Europe. Likewise, no other recent tests for positive selection have shown MC1R to stand out as selected.

But wait a minute. Where did all those European redheads come from?

Harding and colleagues found five different coding polymorphisms with frequencies more than 7 percent in Europe, but completely absent in their sample of Africans. That adds up to roughly half the copies of MC1R in Europe, all derived alleles. That kind of emergence of new alleles doesn’t happen by chance, at least not in a population history like Europe’s. But none of the usual tests will reject neutrality when five different alleles have increased under selection. And individually, each of these alleles is too rare to register a rejection of neutrality, at least, if we apply the usual tests.

What makes this gene so troublesome? There’s no problem estimating the frequency of any single derived allele in Europe, and it’s easy to figure out its extended homozygosity. But if we then compare that to other alleles at the same locus, well, some of them have long haplotypes, too. So none of them have an unusual pattern of LD decay compared to the others.

What to do? We could try to get a much larger genetic sample, or a large sample of individuals from a part of Europe where one of these alleles is especially common.

But since I’m lazy, I figure I’ll just consider the mutual information carried by haplotypes of multiple loci. Individually, no single allele may have a frequency high enough to reliably show linkage in a two-locus comparison. Individually, no single allele will reject neutrality by a homozygosity-based test, because the other selected haplotypes decay over very long areas, making the ratio for any single haplotype insignificant. Collectively, the selected haplotypes may carry linkage over long distances. Together they will reject neutrality, even if individually they don’t.

But first, we need to know the distribution of multilocus mutual information under neutrality.

Degrees of freedom

If you can calculate a χ² and its degrees of freedom, you can skip next four paragraphs.

In the second post I showed that the mutual information is distributed as a χ². That’s a statistical distribution with one parameter—the degrees of freedom. The concept of degrees of freedom is one of the trickiest definitions in statistics. Roughly, it means the number of independent factors that contribute to a single observation.

Let’s take the case of a contingency table, where we have a number of rows, r, and a number of columns, c, where the total number of cells is r ×c. If all the cells were independently distributed, then the number in each cell is expected to be the fraction in its row times the fraction in its column. That’s the expected value. If you take the number you observed in a cell, subtract the number you expected, square that difference, divide it by the expected value, and add up the result for every cell in the table, that’s the χ² statistic. This statistic is high when the observed numbers are far from the expected ones. It’s low when they are close to expected.

For a 2 × 2 table, we can ask, how many ways are there for the χ² statistic to be high? Any one of the cells may have an unusually large number of observations, so we might be tempted to say there are four different ways to get a high value. But it’s obvious that if one value is too high, the value in the same row and the other column must be too low. Likewise, the observed number in the other row and the same column must be too low. And if both those diagonal cells are too low, well then the observed value in the cell diagonal to our high cell must also be high. In other words, all four cells depend on each other. Push one, and you push them all. So there’s really only one axis along which the cells can vary—one degree of freedom.

With a 3 × 2 table, things are a little different. We can hold one row entirely constant, and just change the proportions in the other two. If one cell has a high observed value, that might be because both of the other rows were shortchanged. Or it might be just a single row that’s low, the other being high as well. That makes two distinct ways that we could get a high χ² statistic, two degrees of freedom. In general, there are (r - 1)(c - 1) degrees of freedom in a contingency table. It’s always one less than the number of rows or columns because at least one row and column must be shortchanged when a cell is higher than expected.

Degrees of freedom with comparisons of multiple haplotypes

From the previous post, you might have anticipated how we could do simulations of drift in samples of multiple markers. For the following, I’m going to examine the mutual information between two sets of three SNP markers. That is, I have a stretch of chromosome roughly 50 kb long from end to end (or more properly, 0.05 cM), with three markers tightly linked at either end. Using the same methods as the previous post, I’m applying the same three-stage population history with genetic drift only to these six markers.

Here are the results for 10,000 trials:

That doesn’t look very much like the chart that I got in the previous post, using the same population history. Here is that one:

What’s going on? That red line in the second chart is the χ² distribution with one degree of freedom. The first chart doesn’t match that at all.

Why not? The second chart shows the mutual information between two markers, with two alleles each. That’s one degree of freedom. The first chart shows the mutual information between two sets of three markers. Three markers give the possibility of eight distinct haplotypes (2³). So we have an 8 × 8 contingency table, with 49 degrees of freedom. Here is that distribution, in red, along with the result of our 10,000 simulated samples:

Except, oops, it doesn’t look like 49 degrees of freedom either! What the heck is going on?

If you’ll remember from the second post in the series, our empirical distribution isn’t going to fit a chi-square unless we have enough observations. Sure, in theory, three markers could give us 8 haplotypes. But in practice, the three markers on each end of our 50 kb region are so tightly linked that we get many fewer haplotypes. Sometimes we get only three or four haplotypes on each end. If each set of three markers were independent of the other, they might fit a chi-square with four degrees of freedom, or nine, or six or twelve. In fact, the mutual information observed in these 10,000 simulations accords with a mixture of distributions. Here are the number of degrees of freedom in the 10,000 simulations, as a histogram:

Most of the trials have six or nine degrees of freedom, with 12 being a substantial proportion as well. No primes, there, except for 2 and 3. Only a tiny fraction of trials have as many as 20 degrees of freedom between the 3-marker sets. None get anywhere close to the theoretical 49, because the three-marker sets are too tightly linked to express all possible haplotypes.

So if we want a theoretical distribution to match to our simulated one, we are going to need a mixture of different χ² in proportion to the number represented in the simulated set. Here’s that comparison:

The red line is a mixed distribution, in which different χ² distributions are blended in the proportions represented by the histogram above. That’s the distribution of mutual information between the two marker sets, conditioned on the haplotypes actually present in each set, under the hypothesis of independence. The simulations lie to the right of this distribution, meaning that small-sample bias and genetic drift have added mutual information to the simulations, compared to the expectation in the absence of linkage.

The only difference between these simulations and the ones in the previous post is the number of markers. Here, we’re looking at mutual information between two three-marker sets; there, we were looking at mutual information between two one-marker loci. Both examples used the same distance and the same population history. Genetic drift has had a similar effect—in both sets of trials, there is a slight excess of cases with high mutual information. With both, there are around twice as many at the tail as expected if the markers were independent.

This leads to a couple of conclusions:

1. Under this population history, genetic drift is not sufficient to cause very large amounts of mutual information to be shared by distant sites.
2. A slight alteration to the χ² test—for example, adding some proportion to the critical values—might allow us to test the hypothesis of neutrality for any given case without the necessity of all those simulations.

If we were to add more and more markers, we would eventually find that the bias in mutual information due to small sample size would get bigger. So there’s some maximum number of markers giving useful information in any given sample. But I just picked 3 markers out of a hat. Adding more markers, up to some point, should increase our ability to see rare haplotypes that might diverge from the expectations of genetic drift.

Next: Conditional mutual information: finding the haplotypes that explain linkage disequilibrium

References

Colin Renfrew is an archaeologist, in recent years well-known for his work on Neolithic Europeans and Indo-European origins. Last week, someone pointed me to his recent book, Prehistory: The Making of the Human Mind. I read a short review somewhere, but I've lost the link!

The book was first published in 2007, so its writing would have predated the publication of recent scans of the genome for selection. Renfrew of course has his own distinctive point of view, and he is not himself a geneticist. However, he has worked to integrate his work with genetic insights, interacts closely with many geneticists, and even coined the term, archaeogenetics, to describe a certain kind of gene-driven investigation of population history. So he's no neophyte when it comes to how geneticists describe the evolution of recent human populations.

A number of passages of the book are very interesting, from the perspective of the conventional wisdom about recent human evolution. I wanted to cite these paragraphs from page 92:

The genetic composition of living humans at birth (the human genotype) is closely similar from individual to individual today. That was an underlying assumption of the Human Genome Project and it is being further researched in studies of human genetic diversity. We are all truly born much the same. Moreover a child born today, in the twenty-first century of the Common Era, would be very little different in its DNA -- i.e., in the genotype, and hence in innate capacities -- from one born 60,000 years ago.

Then on page 93, after some additional discussion of Neandertal genetic results:

The implication here must be that the changes in human behaviour and life that have taken place since that time [between 60,000 and 100,000 years ago], and all the behavoural diversity that has emerged -- sedentism, cities, writing, warfare -- are not in any way determined by the very limited genetic changes which, as we understand the matter, distinguish us from our ancestors of 60,000 years ago. So the differences in human behaviour that we see now, when contrasted with the more limited range of behaviours then, are not to be explained by any inherent or emerging genetic differences. Modern molecular genetics suggests that, apart from the normal distribution range present in all populations in matters such as IQ, all humans are born equal.

This represents a widespread point of view, one with a long pedigree in archaeology and human genetics (refer also to my post on Ashley Montagu). Renfrew quite clearly claimed that human evolution stopped once humans became "modern". He emphasizes this point as the basis of a "paradox" -- the observation that no large anatomical changes correlate with the increase in archaeological complexity of the last 30,000 years.

I believe there is no paradox: rapid archaeological change certainly is no proof of evolutionary stasis!