selection

With draft sequences of genomes from several Neandertals and from Denisova, we can begin to investigate known human variations that affect phenotypes. In practice, this is a very simple approach -- take alleles that we know exist in recent human populations, and see if they are in the DNA sequences of these ancient people. My lab has been following this line of research, trying to get information about aspects of biology that are not evident from the skeleton. The immune system is one of the most fascinating, both because of its extensive variation in living people, and because we might be able to test hypotheses about the diseases and parasites that ancient humans faced.

Today Science has released an early manuscript edition of a paper by Laurent Abi-Rached and colleagues (bibliographic information not yet available), which identifies the HLA class-I alleles present in the three highest-coverage Neandertal genomes from Vindija (Vi 33.16, 33.25, and 33.26) and the Denisova pinky genome. The paper is very brief and fairly straightforward, providing provisional HLA class 1 allele types for these individuals, discussing possible haplotype associations among these alleles that may have been in the ancient genomes, and providing the frequency of those alleles in present-day human populations.

These archaic individuals carried HLA types that are presently rare in Africa and more common outside of Africa, supporting the hypothesis that these alleles in living people originated in those archaic populations. The linkage between alleles at different HLA class-I genes also supports that hypothesis. The present immune system biology of humans was strongly shaped by the interaction of different regional populations of archaic humans.

The title of the paper calls this "multiregional admixture", and the word "introgression" appears 8 times. Good for us!

(This is the point where I grumble about the lack of citations in this paper....OK, done grumbling.)

Selected genes may have a very different pattern from neutral genes

This paper is the first demonstration that gene variants of functional importance were not only inherited from Neandertals and Denisovans but were valuable and selected in later populations.

We already knew that humans today have gene variants from these archaic humans. Neandertal genes presently account for around 3 percent of the genomes of people outside Subsaharan Africa. My lab has been studying the pattern of frequency of these genes ("Europe and China have different Neandertal genes"). Most of the genes shared between the Neandertal genome and living people outside Africa are presently very rare -- most occur only in a single individual in our sample of Europeans and Chinese people, for example.

These HLA class-I alleles are different. Some of them are quite common today. If they came from the Neandertals and Denisovans -- that is, if they were not present in the African people who make up most of our ancestry genome-wide -- then these alleles must have increased quite a lot during the recent evolution of people outside Africa.

The best explanation for the large increase in frequency of these genes in modern human populations is selection. If readers want to get an introduction to the scientific literature on the topic of functional genes, I can suggest a detailed review paper I wrote with Greg Cochran on the dynamics of introgression and selection as applied to Neandertals [1], and a review paper we wrote in Trends in Genetics about identifying genes in living humans that that may have come from archaic populations [2]. In both papers, we discuss the dynamics of functional genes that may be affected by selection in modern human populations and how they differ from the predictions for neutral loci affected only by genetic drift. The new paper by Abi-Rached and colleagues follows on that line of inquiry.

I think the hypothesis of adaptive introgression is very likely, and that we shouldn't be at all surprised that the immune system might house many good examples of it.

A look at the most extreme examples, involving the Denisova genome, shows the extent that these functional genes might reflect introgression well beyond that indicated by most of the genome. The HLA class-I alleles present in the Denisova genome are most common today in South Asia (HLA-C*12:02, HLA-C*15 which is also common in Australia) and Southeast Asia (HLA-A*11). These regions of the Old World have no substantial evidence of Denisova inheritance across their genomes. Yet they may very well have substantial frequencies (up to 48 percent for HLA-A*11) of HLA class-I alleles from the archaic Denisovan population.

Reasons to be cautious

This is the point where I have to make a note of caution. Even though I personally think it is likely that these HLA alleles really did introgress into the modern population from Neandertals and Denisovans, their geographic pattern really isn't enough to demonstrate this without question.

Reports earlier this summer described some of the work this group was doing on HLA class-I loci, including a public lecture by PI Peter Parham. I noted at the time that the geographic distribution of the alleles mentioned in that lecture seemed a mismatch for the hypothesis of a Denisovan origin for the alleles ("The immune systems of archaic humans"). For example:

HLA-A*11 is very common in Papua New Guinea, but it is also very common in north India and in China. These two areas otherwise show no significant evidence of Denisova ancestry. We might conclude that the HLA-A gene just has an unusually high level of introgression into Asian populations, not typical of the genome as a whole. That's certainly possible. But without finding any substantial number of derived mutations in the HLA-A*11 variant in the Denisova genome and in living Asians, it is hard to rule out that the sharing of HLA-A*11 in all these populations is just coincidence.

Of course, if the allele were absent in Africa, that would weigh in favor of the idea it is shared by Late Pleistocene interbreeding outside Africa. But HLA-A*11 is in Africa, just very rare. And it's in Europe. This is the kind of locus that is difficult to interpret: if it has any tiny disadvantage against malaria, for instance, its rarity in Africa is easily explained as a function of recent evolution, while its presence almost everywhere outside Africa would be no surprise even if there were never any interbreeding.

The story of HLA-C*12:02 is similar. It's common in PNG, but also broadly across South Asia and into Iran, areas where no substantial evidence of Denisovan ancestry has been demonstrated.

Introgression under selection is a good hypothesis for why these alleles should be so much more broadly distributed than the evidence from the rest of the genome. But introgression isn't the only explanation, because the alleles might have been retained by balancing selection, with recombinant haplotypes suppressed by purifying selection. We might use haplotype age to test the hypothesis. If the alleles were retained by ILS, they would look much older than if they came in from an archaic population by introgression. But as I'll describe below, in this case we actually have the opposite problem: these haplotypes look too young to have come in by introgression, likely a consequence of selection long after the Neandertals and Denisovans had contributed their genes to us.

The curious case of HLA-B*73

If I agree that the results of this paper are pretty likely, why am I still cautious? Well, the most confusing thing in this paper is an allele described in great detail that they didn't find in the archaic genomes. And I know from experience that not finding things is a pretty common occurrence when we go looking for odd things that might have come from Neandertals.

There's a detective story here, that probably explains the initial interest of this group in the Neandertal genome, but that just didn't pan out in their search through the archaic genomes. The allele is HLA-B*73.

Parham and colleagues [3] first characterized this allele, which is remarkably different from other HLA-B alleles. Homologs of HLA-B*73 are present in living apes, suggesting that the different human alleles originated before we diverged from gorillas. The retention of such an ancient allele in humans isn't a surprise in the HLA system, because many very divergent alleles have been kept in the population across evolutionary time by balancing selection. What's a bit surprising about HLA-B*73 is its limited diversity in living people. It appears to have persisted in humans throughout our evolution, but people today who carry the allele have very similar sequences, and it is nearly always linked to one single allele at the nearby gene, HLA-C (HLA-C*15). Also, the allele is very rare inside Africa and reaches its highest frequency in West Asia., where it occurs in only 4.5 percent of people. Because of this strange pattern, Parham and colleagues suggested that the allele may have been inherited from Neandertals.

When I was in graduate school working on modern human origins, I took a special interest in genes that had this pattern of variation. HLA-B*73 was not the only one, there are others.

The variation of the HLA-B*73 allele and its association with HLA-C*15 correspond very well to the predictions we presented in our paper on identifying introgression from archaic humans [2]. It's a highly divergent allele in humans compared to others, and it appears not to have recombined much with nearby genes, suggesting it was sequestered in another population through much of the diversification of present-day HLA alleles. But the HLA system is actually a rotten place to look for this kind of evidence, because there are many, many instances where ancient alleles have been retained in human populations by balancing selection. As we pointed out in 2008, a deep root to the gene tree and a rarity of recombination can be good evidence of introgression, but balancing selection and inhibitions to recombination are alternatives to introgression for explaining this pattern of variation.

There's no necessary contradiction between the two processes, and ancient DNA in this case could establish that the allele was both under selection and came from archaic humans. The problem: they didn't find the allele in the archaic genomes.

So why did they spend so much time in this paper discussing this allele? My guess is that they were surprised not to find it. But they did find HLA-C*15 in the Denisova genome, which is often linked to HLA-B*73 in living people who carry it. That makes for an indirect argument:

C*12:02 and C*15 were formed before the Out-of-Africa migration (Fig. 2H and fig. S15) and exhibit much higher haplotype diversity in Asia than in Africa (fig. S16), contrasting with the usually higher African genetic diversity (20). These properties fit with C*12:02 and C*15 having been introduced to modern humans through admixture with Denisovans in west Asia, with later spreading to Africa (21, 22) (Fig. 1F and fig. S11 for C*15). Given our minimal sampling of the Denisovan population it is remarkable that C*15:05 and C*12:02 are the two modern HLA-C alleles in strongest LD with B*73 (Fig. 1E). Although B*73 was not carried by the Denisovan individual studied, the presence of these two associated HLA-C alleles provide strong circumstantial evidence that B*73 was passed from Denisovans to modern humans.

I would go one simpler: Given that HLA-B*73 is most common today in West Asia, I suspect it came from West Asian Neandertals. There's no reason why the HLA genes of European Neandertals should have been identical to West Asian Neandertals. Today's Europeans are different from today's West Asians in the frequencies of these alleles, so why not in the past as well? For that matter, we really only have two alleles from European Neandertals for HLA-B (since the paper finds that

Why do the Vindija Neandertals all have the same HLA types?

It's a pretty good question. The paper cannot distinguish the genotypes from these three individuals. That's not the same as saying they're exactly the same type, since the sequences are very low coverage, but probably they were. Here's what the paper says:

Genome-wide analysis showing three Vindija Neandertals exhibited limited genetic diversity (3) is reflected in our HLA analysis: each individual has the same HLA class I alleles (fig. S17). Because these HLA identities could not be the consequence of modern human DNA contamination of Neandertal samples, which is <1% (3), they indicate these individuals likely belonged to a small and isolated population (fig. S18).

Still, I think this indicates a pretty high degree of inbreeding among these individuals. I wonder what the organ registry for Neandertals would have looked like.

(Not so) final words

I have more to write on the topic of linkage disequilibrium among these genes. The rate of recombination between HLA-B and HLA-C is high enough that a haplotype between these genes should have mostly decayed in the time since our mixture with archaic humans. HLA-C and HLA-A are an order of magnitude further apart, so linkage between alleles of these genes should have been totally erased in the time since any archaic admixture.

That means that the extended haplotypes reported in this study must reflect selection in the period since the population mixture and introgression. The story isn't a simple case of inheritance from archaic humans, it is rather more complex. But more on that later.

I think this paper confirms that it will be really productive to look at archaic genomes for variants present in living humans. Identifying modern human alleles in a Neandertal isn't really very exciting science, though. I've been doing this on my blog for a year now. It's a tricky job to type these HLA alleles, compared to genotyping many other genes, as we discovered. Still, I never really expected that reporting on genotypes in the public domain would be sufficient to get printed in Science.

Still, this set of three genes is particularly interesting. And the paper does add evidence from one additional locus, KIR3DS1, which also has the pattern where an allele rare in Africa but common in Asia is present in the Denisova genome.

If it turns out that we have widespread adaptive introgression in Asia today from Denisovans, that will change the game of studying the origins of these populations. Based on the genome-wide comparison, it looks like the genetic interaction that led to the habitation of Asia did not involve Denisovans, who contributed only to populations at the most eastern extreme of habitation in island Southeast Asia. But the only Denisovans we know about lived near the geographic center of the Asian landmass, not at the extreme southeastern extreme.

The HLA pattern may suggest a more widespread pattern of mixture across Asia, which was later overwritten by population movements of people who didn't have Denisovan ancestry. That means that the habitation of Asia was a process of successive migrations and replacements, which imperfectly covered up the evidence of archaic intermixture. The genes that remain as signs of this intermixture are those that had selective advantages in later populations.

References

I've written about the study of selection on human mtDNA many times, and discussed the signs that Neandertal mtDNA may have disappeared because of selection.

I love how larger samples are starting to get zoologists to test the neutral hypothesis much more widely. This week, a new paper in Biology Letters by Andrew Foote and colleagues [1] shows that different populations of killer whales. They find possible evidence for positive selection on amino acid-coding variants in cytochrome b in two Antarctic populations.

Here's the last paragraph of the paper. This isn't totally clear without the context (describing the whale populations) but it gives the best short summary of the complexity that was found.

Based on morphological differences [21] and reciprocal monophyly of the mitogenome sequences [12], it has been suggested that type B and type C are distinct species. Positive selection on the cytochrome b could therefore be caused by adaptive divergence relating to a combination of variables that influence metabolic requirements, such as body size or diet; type C is a fish-eating dwarf form of killer whale, whereas type B is one of the largest forms of killer whale and primarily feeds upon seals [21,22] (J. W. Durban & R. L. Pitman 2010, unpublished data). However, the amino acid changes in both ecotypes could be the result of parallel evolution owing to environmental conditions such as oxygen concentration or sea temperature. Both type B and type C at least seasonally inhabit Antarctic pack ice, and both have been sighted over-wintering in the pack ice [21]. The third Antarctic ecotype, for which we found no evidence of positive selection, inhabits the offshore ice-free waters during the austral summer and over-winters at lower latitudes [21]. However, the mutations are in the opposite direction for each ecotype, suggesting that divergent evolution may be more likely. The two changes were private alleles within type B and type C, respectively, and neither substitution was found in the reconstructed ancestral sequence (electronic supplementary material), suggesting that each mutation has occurred and become fixed and almost fixed, respectively, since type B and type C diverged from their most recent common ancestor, approximately 0.15 Ma [12]. Therefore, the ancestral form may not have been subject to the same selective pressures.

Some thoughts:

1. We know how well "reciprocal monophyly" has turned out for human and Neandertal mtDNA genomes...

2. It's interesting how much play there seems to be in the mitochondrial genome. Lots of ways to change and have small phenotypic effects that may be adaptive in one or another ecology. The system as a whole is relatively robust to many mtDNA changes.

3. Many years ago, whale mtDNA was being explained in very similar ways to humans -- a matter of small effective size, in this case exacerbated by matrilineal pod structure. Might well be for many kinds of whales, but selection makes the story more complex.

References

I wrote early this week about Hopi Hoekstra's work on pigmentation evolution in mice ("The color of mice"). The linked article focusing on this empirical work didn't mention her interesting involvement in the debate over the nature and importance of gene regulation as a target of selection.

I wanted to point out some articles on the topic, by Hoekstra and others, because more and more, paleoanthropological hypotheses are being found to involve the evolution of gene regulation. I'll just note a couple of examples (diet and pigmentation), and hint that there are more coming in the next year.

If the topic of gene regulatory evolution, or cis-regulation in particular, are obscure, let me recommend a 2008 primer article by Wisconsin geneticist Sean B. Carroll: ("Evo-Devo and an Expanding Evolutionary Synthesis: A Genetic Theory of Morphological Evolution "). Carroll is probably the most well-known advocate of an "evo-devo" perspective on morphological evolution, and in particular the hypothesis that most morphological evolution may be explained by changes to cis-regulation -- "self-acting" sequence elements that affect gene transcription, such as promoter or enhancer elements.

In a 2007 commentary, Hoekstra and Jerry Coyne presented a critique of the idea that cis-regulation is a central mechanism of adaptation. Here's a quote from their conclusion (Hoekstra and Coyne 2007:1006):

While the study of cis-regulatory evolution is an important endeavor, justifiably championed by [evo-devotee Sean B.] Carroll and others, our survey of the theory and empirical data shows that the widespread enthusiasm for the importance of cis-regulatory change in evolution is at best premature. Analyzing the verbal theory, one finds no compelling reason to draw a distinction between the genetic basis of anatomical versus physiological evolution. Nor is there good reason to accept the a priori argument that—for either anatomy or physiology—changes in cis-regulatory genes are more likely to be fixed in evolution than are changes in the coding region of genes.

Everyone agrees that changes in cis-regulation, trans-regulation and good old-fashioned changes to protein sequences may all be selected, and there are examples of each being involved in the evolution of new adaptive phenotypes. So at that level, the theoretical disagreement is relatively sterile -- all of them are possible and cases are known for each.

The question is whether any of them account for a preponderance of adaptive evolution. Is there anything special about cis-regulation, or any other kind of change? Are they coequal, do they occur in proportion to the number of regulatory elements, amino acid-coding positions, gene duplications? Do any of them release constraints on adaptive changes, allowing more rapid evolution?

Why does anybody care? Well, there is a mercenary answer: They all have their own empirical research agendas to look out for, and some of them work mainly with experimental models and techniques effective for studying cis-regulation, others on trans-regulation and still others on classical polymorphisms. To me, these are totally boring topics, since I'm not hoping for any funding to do molecular work on gene regulation. Hopefully, the funding conflict will become less important as genomic methods get cheaper. Of course, when it's no longer difficult to find out the answers, we'll have a decent survey of empirical cases!

Search strategies. A second explanation for why we should care is a practical one. Now that we are able to get genomes from any species we like, the question arises: what is a sensible strategy for forming hypotheses about adaptive (and non-adaptive) change? What should we be looking for?

Coyne and Hoekstra wrote a 2007 perspective on an article about amylase adaptive evolution inhuman populations. They returned to the issue of whether we should expect a predominant target of adaptive change: cis-regulatory or so-called "structural" mutations to coding sequences.

The amylase results [showing adaptive change in gene regulation by duplication] follow a related study on the genetics of human dietary differences. In 2006, Tishkoff and colleagues [11] identified a mutation in the upstream regulatory region of the gene for lactase, an enzyme important for digesting milk, in pastoral African populations. Using an in vitro system, they showed that this mutation could increase gene expression. The relevant mutation, however, is not a duplication, but probably a change in cis-regulation. (An independent cis-regulatory mutation at this locus, also conferring lactose tolerance, was identified earlier in European populations [12].)

Even in the simplest cases of adaptation, then — increased enzyme production to handle new diets — evolution works in multiple ways. Obviously, no amount of a priori speculation will tell us which sorts of mutations will be important; the answer, unfortunately, requires meticulous, case-by-case analysis of putative adaptations.

Humans may be a poor model organism for considering this question. For one thing, we have a large store of loss-of-function mutations that have been selected for resistance to disease. The same thing probably occurs in other species, but the exceptional number of new diseases in humans may tilt the scales in favor of "structural" mutations.

Still, these diet-related examples show pretty clearly that multiple mechanisms of gene regulation may be targets of recent selection. That's also evident when we consider human pigmentation variation, a system that is relatively well-understood now from a genetic perspective, for the same reason that Hoekstra's deer mice pigment variations are tractable. In a genome-wide context, it now looks like cis-regulation has been a frequent target of recent adaptive evolution (Kudaravalli et al. 2009), but most of the well-studied examples of recent adaptive change are amino-acid coding

The breadth of pleiotropy. Pleiotropy ought to impede adaptation. If genes are solving multiple problems -- by interacting with distinct functional networks -- then changes that make one function better may often make others worse. If genes interact widely enough, then optimization becomes extremely difficult or impossible -- what Stuart Kauffman (1993) called a "complexity catastrophe." Make a system complicated enough, and the probability falls to nil that a random change might improve it.

If evolution were generally mutation-limited in this way, you might well expect to see a highly modularized system of gene regulation evolve, and that's precisely the argument for the cis-regulatory evo-devo model. I don't have an opinion on the general question of how often adaptations may make use of this modular system of regulation as opposed to trans-regulation, duplication, or straight-on coding substitutions. It seems like toolkit genes, which are both highly conserved and strongly pleiotropic, may have evolved by altering cis-regulation more often than other means. Those genes have highly modularized "cassettes" of cis-regulatory elements that control their expression in different contexts. One regulatory element can change without necessarily impeding the function of the gene in other contexts.

Empirical pigmentation research helps to illuminate the dispersal (and limits to dispersal) of recently selected mutations. That makes it a very relevant model system for understanding recent human evolution. Many human (and Neandertal) mutations to MC1R are trans-regulatory -- by altering the sequence of the hormone receptor, these mutations downregulate the pathway that converts pheomelanin to eumelanin. The nature of this regulatory change is structural -- it's an actual change in the gene product that affects pigmentation.

From the deeper perspective of paleoanthropology, the evolution of form is the central topic. How did the distinctive conformation of the bipedal pelvis evolve? Some paleoanthropologists have already laid out scenarios in which morphological evolution took a small number of very broad changes -- pelvis, spine, and femora as integrated units that may have had correlated effects on arms and other morphological structures. Such hypotheses make the background assumption of strong pleiotropy on a hard-to-explore adaptive landscape. If human evolution was a product of a few hard-to-get mutations, which might not have happened at all, then our emergence was contingent on a series of unlikely events.

Pleiotropic constraints may help to explain why we see a pulse of rapid adaptive evolution in humans along with population growth. Adaptive mutations rarely appeared during the earlier Pleistocene, making many possible changes were mutation-limited. If certain kinds of regulatory changes were very easily evolvable, then we might expect to see a different pattern of recent evolution.

Still, most folks don't think very much about pleiotropy as a constraint on human evolutionary change. The "one gene, one trait" model is really universal out there. Certainly, if you ask people, they'll give you the textbook answer -- genes have more than one function; phenotypes are influenced by many genes. But I can't tell you how many times I've heard people refer to "skin color genes" as if they did nothing else.

References:

Carroll SB. 2008. Evo-Devo and an Expanding Evolutionary Synthesis: A Genetic Theory of Morphological Evolution. Cell 134:25-36. doi:10.1016/j.cell.2008.06.030

Coyne JA, Hoekstra HE. 2007. Evolution of protein expression: New genes for a new diet. Curr Biol 17:R1014-R1016. doi:10.1016/j.cub.2007.10.009

Hoekstra HE, Coyne JA. 2007. The locus of evolution: Evo devo and the genetics of adaptation. Evolution 61:995-1016. doi:10.1111/j.1558-5646.2007.00105.x

Kauffman SA. 1993. The Origins of Order: Self-Organization and Selection in Evolution. Oxford University Press, New York.

Kudaravalli S, Veyrieras J-B, Stranger BE, Dermitzakis ET, Pritchard JK. 2009. Gene expression levels are a target of recent natural selection in the human genome. Mol Biol Evol 26:649-658. doi:10.1093/molbev/msn289