altitude

We don't really know the extent of territory that might have been occupied by the population represented by the Denisova genome. The signs of mixture into the Melanesian/New Guinea population suggests that the Denisova individual shared many genes with people who lived somewhere along the South or Southeast Asian coast. Denisova itself, however, is in the Altai Mountains.

Last week I wrote some thoughts about the possible introgression of HLA alleles from Denisovans into more recent populations. HLA genes pose many problems for testing this hypothesis -- including the difficulty of identifying the alleles in a low-coverage genome and the high chance of incomplete lineage sorting of ancient alleles in recent populations. Other parts of the genome in principle may be much easier to find evidence of introgression.

If an allele that originated in Denisovans had some advantage in later populations, it might today be found very widely spread across Asian populations, even if the amount of Denisovan ancestry in most of these populations is very small. This was the theme of my paper with Gregory Cochran several years ago [1] ("The inevitability of introgression"). The probability that a single copy of an advantageous allele will survive and increase in the population is roughly 2s, where s is the fitness advantage in a heterozygote carrying the allele. A relatively small number of copies of an allele might have entered a recent human population by introgression from some ancient population, but these few copies would have a high likelihood of surviving and increasing in frequency, possibly toward fixation. HLA alleles could easily be in this category, but the challenges identifying them and high chance of ILS make the hypothesis hard to test.

Another strategy is to identify genes that have been selected in recent populations and see if the linked haplotype shows up in the Denisova genome. Recently, several studies have attempted to identify genes related to high altitude adaptation in Tibetans. At least some Denisovans lived in the mountainous areas of central Asia, and so I'm curious whether they might have some alleles adapted to this environment. The Altai are not nearly as high as the Tibetan plateau (in fact Denisova itself is not much higher than western Kansas), and we don't know how long Denisovan people might have been resident in Central Asia, but if we're looking for selected alleles there are some strong candidates in this category of genes.

So let's look at some of them. All positions here are mapped to the hg18 human genome assembly.

Yi and colleagues [2] find a strong frequency difference between China and Tibet for a SNP in EPAS1, at chr2:46441523. The derived allele, G, has a frequency of 87% in their Tibetan sample but only 9% in their Chinese sample (and zero in Denmark). The Denisova genome is represented by two reads at this site, both C, the ancestral allele. We don't necessarily have to accept that this is a functional site, but as the marker most strongly differentiating the high altitude population it would likely be closely linked to any functional variant. So the Denisova allele suggests that this ancient individual lacked whatever functional variant might currently be common in Tibetans for this gene.

Simonson and colleagues [3] took a different approach, focusing on candidate genes that they argued a priori were likely to be involved in adaptation to hypoxia because of their physiological role. They evaluated these genes for evidence of positive selection in Tibetans, finding several candidate haplotypes for recent adaptive evolution to high altitude.

For each of five genes, they identified a three-locus "core selection haplotype" that shows signs of selection within Tibet. The purpose of these three-SNP haplotypes was to examine the correlation of haplotypes and phenotypes in a sample of people where physiological data were taken. So they are intended as tags, not as comprehensive and unique identifiers of the candidates at the genetic level. But the three-locus haplotypes are the only ones reported in the supplement to the paper, so that's what I have to compare.

EGLN1: The three-allele candidate selected haplotype consists of A at chr1:229793717, T at chr1:229667980 and T at chr1:229665156. Denisova apparently has the selected haplotype with A at chr1:229793717 (2/2 reads), T at chr1:229667980 (3/3 reads) and T at chr1:229665156 (1/1 reads). However, it is not obvious whether this is significant. All three alleles on the candidate selected haplotype are the ancestral (present in chimpanzees and gorillas) alleles, which are much more likely to show up in the archaic genomes than derived alleles. These ancestral alleles are also present in several of the whole genomes provided along with the Denisova sequence reads. So it's not clear to me how good a candidate for selection the haplotype really is.

CYP17A1: Here the three-allele candidate selected haplotype includes G at chr10:104568521, G at chr10:104594906, and C at chr10:104517420. Denisova has C (5/5 reads, ancestral), T (4/4 reads, ancestral), and C (3/3 reads, ancestral). Again, Denisova has the all-ancestral haplotype here, but in this case it is not the selection candidate.

PTEN: The selected candidate haplotype is G at chr10:89770364, C at chr10:89790851 and C at chr10:89778618. Denisova has G (5/5 reads, ancestral), T (2/2 reads, derived), and C (4/4 reads, ancestral). Not selected.

I always find it interesting when the Denisova genome has a derived allele at an interesting site -- it is the shared derived alleles between these archaic genomes and living people that constitute evidence of genetic persistence of the archaic people. No single site carries that information (any one allele may be shared by incomplete lineage sorting), but I still like to note them. The Papuan and half the Native American, Sardinian and Mongolian reads share the derived T at chr10:89790851 with Denisova.

HMOX2: The candidate selected haplotype has C at chr16:4456093, T at chr16:4465266, T at chr16:4442515. Denisova has this candidate selected haplotype: C (3/3 reads, ancestral), T (4/4 reads, ancestral), T (5/5 reads, ancestral). That haplotype may also be in the Cambodian whole genome accompanying the Denisova data, and can't be ruled out for the Mongolian. Again, the all-ancestral haplotype and wider distribution argue against the hypothesis that this haplotype was specifically selected in Tibet.

PPARA: The core candidate selected haplotype has A at chr22:44827140, C at chr22:44832376 and T at chr22:44842095. Denisova has A (8/8 reads, ancestral), A (5/5 reads, ancestral), and C (2/2 reads, ancestral). Notice again, Denisova has the all-ancestral haplotype. As an ancient sequence, we are finding this is the usual case, human-derived alleles are just rarer in this genome.

OK, where are we? Out of six genes that are candidates for selection on altitude adaptation in Tibetans, the Denisova genome has two -- at ELGN1 and HMOX2. In both cases, the core selected haplotype consists entirely of ancestral alleles, and so I think they are actually poor evidence of introgression on the surface. I would test them by looking at more SNPs linked to the presumed selected haplotype, hoping to find some derived SNPs shared by the Denisovan genome and the presumed selected haplotypes. Unfortunately, publications do not yet routinely report long haplotypes, so it will take some more digging to test these cases.

References

Did the altitude of the Tibetan plateau lead to the fastest instance of human adaptation yet known?

That's the claim in the new paper by Xin Yi and colleagues [1]:

Given our estimate that Han and Tibetans diverged 2750 years ago and experienced subsequent migration, it appears that our focal SNP at EPAS1 may have experienced a faster rate of frequency change than even the lactase persistence allele in northern Europe, which rose in frequency over the course of about 7500 years (26). EPAS1 may therefore represent the strongest instance of natural selection documented in a human population, and variation at this gene appears to have had important consequences for human survival and/or reproduction in the Tibetan region.

I have a significant criticism of that conclusion, but first I want to say I think this is really cool work. They sequenced 50 whole exomes of people of Tibetan ancestry. An exome is the coding fraction of the genome, leaving out the non-coding stuff. This let them do a genome-wide association including every SNP they found. As it turns out, the key gene (EPAS1) has no coding SNPs that differentiate strongly in these samples. It's an intronic SNP that shows a really large frequency difference (87% in Tibetans, 9% in Han Chinese). That's a really big difference.

And it takes a big difference to test neutrality in this sample. Fifty exomes is a whole lot of sequencing, but it's really a small sample for finding selection. It takes a really big frequency change to exceed chance. Besides that, most new adaptive mutations will be missed because they haven't gotten off the ground yet. Finding one major allele that correlates strongly with population, and then doing the work to show its association with red blood cell production, that's all pretty neat stuff. This paper should be added to the paper last month by Cynthia Beall and colleagues [2], who also found an association with Tibetans and made a functional link with high altitude adaptation. This gene is part of the system that adapts people to hypoxia in the Tibet/Nepal area, although it certainly does not act alone and we don't yet know how the system works. It's a solid first step.

OK, so what's my problem with the paper? Hypoxia is a strong selective agent, affecting performance, health, and -- maybe most important -- birth weight. As soon as people began living on the Tibetan Plateau, they were in a compromised environment. That makes this a really great example of recent selection associated with a novel environment. But the archaeological evidence suggests that people have been living in this environment for a lot longer than 3000 years. The population model in the paper is a mess.

People have been living on the Tibetan Plateau for more than 15,000 years. They may have occupied the area intermittently before the Last Glacial Maximum, and certainly were in nearby medium-altitude areas of northwestern China before that time. The Paleolithic-era occupation of northeastern highland Tibet was reviewed by Madsen and colleagues [3] and Brantingham and colleagues [4]. Aldenderfer [5] reviewed what is known about Neolithic-era occupation of highland Tibet. Sites with ceramics, evidence of sedentary village occupation and domesticated animals occur between 4000 and 6500 calendar years B.P. That doesn't mean that today's Tibetan population derives entirely from these early Neolithic settlers or the even earlier Paleolithic occupants. But the archaeological record does show that the opportunity for genetic adaptation would have been present long before 3000 years ago.

So there's a potential inconsistency. The inconsistency could be resolved by recognizing that selection is stochastic. Selection cannot start changing the frequency of an allele until after the mutation has occurred.

The following passage comes from Nicholas Wade's account of the research, in the NY Times. Wade also picked up on the problem with the demography in the paper, and probed the authors about it:

Geneticists have a more elastic view of dates than do archaeologists, and the estimate of a Han-Tibetan population split at 3,000 years ago could probably have been adjusted to 6,000 if the geneticists had taken any account of any other kind of evidence.

Rasmus Nielsen, a Danish researcher at the University of California, Berkeley, did the statistical calculations for the Beijing study. “We feel fairly confident that something on the order of 3,000 years is correct,” he said. But in a later e-mail message, Dr. Nielsen said, “I cannot with confidence rule out that the divergence time is 6,000 instead of 3,000.”

There is similar flexibility in the estimates of population sizes. The Beijing team calculates that at the time of divergence there were only 288 Han Chinese and 22,642 Tibetans. These estimates have bewildered archaeologists, given that rice cultivation in southern China started 10,000 years ago and that there was an extensive civilization by 3,000 years ago. Dr. Nielsen said that the figure of 288 people was meant simply to indicate a bottleneck in the Han population, meaning a time when it was very small, and that this bottleneck could just as easily have occurred 10,000 years ago.

I think that's totally remarkable. "Geneticists have a more elastic view of dates than do archaeologists"! I think that phrase should be framed and hung in every classroom teaching anthropological genetics.

Look at the expansion model. In what universe were there only 288 ancestors of Han Chinese people in the last 3000 years? We're talking about the late Bronze Age, here! This is just after the end of the Shang Dynasty, whose capital at Anyang had a walled area of 1000 hectares. That's 1000 soccer pitches full of city, within an empire that spanned the northern half of China.

It is completely lame to claim that the model could represent a bottleneck as long ago as 10,000 years. You see, the size of the population determines the rate of differentiation under genetic drift. If the population was big, it shouldn't have changed very fast, so the present populations shouldn't be very different. Putting it into numbers, if there hasn't been a bottleneck for 10,000 years, then the divergence must be a lot older than 3000 years. Probably older than 10,000 years.

These hypotheses can be tested directly with genetics, and the data are certainly rich enough now to do it. If they point to a genetic bottleneck in China during the last 10,000 years, we should be very, very surprised. Because then who was farming all the millet and rice, and domesticating pigs?

Does it matter? For EPAS1, the timing really doesn't affect the interpretation of selection -- there's no way that drift made the populations as different as they are for this one locus. But it seems clear that this is not a new mutation because it has no long, linked haplotype around it that also differs in frequency in the two populations. Selection on a standing variant is indeed newsworthy, as these are hard to find. Since we don't have a long haplotype to date, the only way that we can estimate the timing of selection is with the population model. Use the wrong model, and you get the wrong time. That is probably what has happened here.

Also, using this weird population model vastly increases the chance that genetic drift could cause large frequency changes in Tibet or China. This makes us much less likely to recognize genes that really have been subject to selection in either population. With respect to EPAS1 the test is conservative, but the genome-wide comparison will miss a lot of genes and give less significant p-values to others. It's a waste, because it means that we have to collect that much more data to get the same result.

UPDATE (2010-07-06): Rasmus Nielsen has written me to clarify his remarks to the Times and give more information about the demographic model in the paper. I have posted his full remarks along with some comments of my own. It is well worth reading.

References