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slc24a5

SLC24A5 alleles

Many years ago I was perplexed by particular patterns in some genes which have been subject to very strong selection. In particular, the locus SLC24A5 has been subject to a powerful sweep over the last 10,000 years across Western Eurasia, to near total fixation in Europe, but still at high frequencies as far south as India. Yet the derived variant is relatively uncommon in East Asia. Groups which carry the West Eurasian variant, such as the Uyghurs, almost certainly obtained it through admixture processes over the last 10,000 years (in the case of the Uyghurs and various northeast Eurasian ethnicities such as the Mongols, this admixture from West Eurasians is mostly in historical time over the past 2,000 years).

The common sense explanation is that vast regions of interior Eurasia were not highly populated for tens of thousands of years. Even after the Ice Age retreated the Eurasian interior would have been particular inhospitable. Though maps of human migration show where humans have lived at some frequency all across the world, they do not usually show any sign of the density. If densities were low enough in the inter-montane zones of Inner Asia, then for all practical purposes the idea of isolation-by-distance gene flow may not have held for the two antipodes of Eurasia for much of the Pleistocene and early Holocene. So have things changed? I believe so. And it comes down to agriculture, which enabled much higher population densities in areas which were previously simply not feasible areas for hunter-gatherers.

A new paper in Science outlines this for Tibet, Agriculture facilitated permanent human occupation of the Tibetan Plateau after 3600 B.P.. I’ll quote the relevant sections of the paper:

On the basis of the above evidence, the prehistoric human occupation of the NETP can be subdivided into three phases. During the first phase (pre–5200 cal yr B.P.), hunter-gatherers made occasional forays to altitudes reaching above 4300 masl, presumably tracking game. During the second phase (5200 to 3600 cal yr B.P.), a longstanding tradition of millet farming that had become widely established along the middle and lower reaches of the Yellow River extended upstream into the NETP. Millet farming had spread across the Loess Plateau after 5900 cal yr B.P. (17) and subsequently spread across these lower reaches of the NETP from 5200 cal yr B.P. Toward the end of the second phase (4000 and 3600 cal yr B.P.), two significant additions are observed in the crop repertoire (text S4 and fig. S6). The North Chinese crops of broomcorn and foxtail millet were joined or displaced on some sites by the principal cereals of the Fertile Crescent, barley and wheat. There has been much interest in the chronology and consequences of the meeting of east and west staple crops in prehistory (1820). Here, its notable consequence was to facilitate the sustained settlement of the Tibetan Plateau’s higher altitudes. The importation of wheat and barley enabled human communities to adapt to the harsher conditions of higher altitudes in the Tibetan Plateau, a possibility raised in previous studies (15, 21).

The key addition was barley. During phase three, from around 3600 cal yr B.P., sites can be divided into those that lie above or below 2500 masl. In the lower-altitude group, the longstanding crops, broomcorn and foxtail millet, are joined by barley as a third component in an otherwise traditional dietary repertoire. In the higher-altitude group, however, the frost-sensitive millet is absent, and the cold-tolerant barley has moved to a primary position (Fig. 2D). Alongside the presence of wheat (also relatively cold-tolerant) and sheep, the diet at these high altitudes has clearly been transformed, but in a manner that enabled sustained settlement at unprecedented altitudes.

There’s been a lot of interesting work on the genetics of Tibetans recently, from altitude adaptation from archaics, to the inference that a great deal of Tibetan ancestry is actually shared with the Han and other lowland groups in the past three to four thousand years. These results make more sense if you realize that the arrival of more advanced agricultural techniques reshaped the possibilities of habitation for humans at higher densities. In fact, it is almost certainly no coincidence that it is during the period of agriculture that the great fusions between the disparate “branches” of the human family tree came back together; higher population densities across huge areas mean that de facto gene flow no go zones disappeared.

 
• Category: Science • Tags: Genetics, Tibet 
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Credit: Albozagros


The genetics and history of Tibet are fascinating to many. To be honest the primary reason here is elevation. The Tibetan plateau has served as a fortress for populations who have adapted biologically and culturally to the extreme conditions. Naturally this means that there has been a fair amount of population genetics on Tibetans, as hypoxia is a side effect of high altitude living which dramatically impacts fitness. I have discussed papers on this topic before. And I will probably talk more about it in the future, considering rumblings at ASHG 2012.

But to understand the character of the effect of natural selection on a population it is often very important to keep in mind the phylogenetic context. By this, I mean that evolutionary processes occur over history, and those historical events shape the course of subsequent of phenomena. Concretely, to understand how the Tibetans came to be adapted to high altitudes one must understand who they are related to, and what their long term history is. There is a paper in Molecular Biology and Evolution which attempts to do just that, Genetic evidence of Paleolithic colonization and Neolithic expansion of modern humans on the Tibetan Plateau:

Tibetans live on the highest plateau in the world, their current population size is nearly 5 million, and most of them live at an altitude exceeding 3,500 meters. Therefore, the Tibetan Plateau is a remarkable area for cultural and biological studies of human population history. However, the chronological profile of the Tibetan Plateau’s colonization remains an unsolved question of human prehistory. To reconstruct the prehistoric colonization and demographic history of modern humans on the Tibetan Plateau, we systematically sampled 6,109 Tibetan individuals from 41 geographic populations across the entire region of the Tibetan Plateau and analyzed the phylogeographic patterns of both paternal (n = 2,354) and maternal (n = 6,109) lineages as well as genome-wide SNP markers (n = 50) in Tibetan populations. We found that there have been two distinct, major prehistoric migrations of modern humans into the Tibetan Plateau. The first migration was marked by ancient Tibetan genetic signatures dated to around 30,000 years ago, indicating that the initial peopling of the Tibetan Plateau by modern humans occurred during the Upper Paleolithic rather than Neolithic. We also found evidences for relatively young (only 7-10 thousand years old) shared Y chromosome and mitochondrial DNA haplotypes between Tibetans and Han Chinese, suggesting a second wave of migration during the early Neolithic. Collectively, the genetic data indicate that Tibetans have been adapted to a high altitude environment since initial colonization of the Tibetan Plateau in the early Upper Paleolithic, before the Last Glacial Maximum, followed by a rapid population expansion that coincided with the establishment of farming and yak pastoralism on the Plateau in the early Neolithic.

The two major salient points I think need emphasis are:

1) Massive sample sizes for mtDNA and lesser extent Y chromosomal linages

2) Tibetans are a compound of agriculturalists who arrived onto the plateau >10,000 years, and, hunter-gatherers who date back to the Paleolithic

Citation: Cai, Xiaoyun, et al. “Human migration through bottlenecks from Southeast Asia into East Asia during Last Glacial Maximum revealed by Y chromosomes.” PloS one 6.8 (2011): e24282.

There are many issues with this paper that bother me. The broadest interpretation of their thesis is one I find creditable, but in the details I’m left skeptical, confused, and more curious than when I began. Also, I need to add that I talked to the people who presented a poster on this paper at ASHG 2012, though I do not know if they were the authors. They seemed nice, but, also not necessarily totally focused on the questions they were exploring, as opposed to obtaining huge sample sizes and applying standard methods to them. Speak of which, the first thing that jumps out is that their sample is skewed toward what is today Tibet proper, the autonomous province. But Tibetan people have historically lived as far as Sichuan. Only 50% of ethnic Tibetans live in the autonomous region, but well over 90% of their samples are from this area. In terms of exploring adaptation to altitude this is fine, but if you are going to do phylogeography you need better geographical coverage I would think.

But that’s only a minor aside. The bulk of the paper consists of a laundry list of Y and mtDNA haplogroups, and coalescence times. Some of the results are very persuasive to me. There are some Y lineages which exhibit a “star shaped” phylogeny, which usually connotes a recent rapid population expansion. Using other methods the authors have inferred that there was indeed an expansion of population after the introduction of agriculture >10,000 years ago. There is no great reason on prior grounds to be skeptical of this finding. Nevertheless, drilling down produces great confusions, and I am not sure that the coalescence times and phylogenies actually mean what the authors assume they mean.

For example, here is a standard sort of analysis presented in this paper:

We identified a molecular signature of recent population expansion during the early Neolithic time in both paternal (Y-chromosomal D3a-P47 and O3a3c1-M117) and maternal (M9a1a and M9a1b1) lineages (10-7 kya) (table 1). The detailed analysis of haplotype sharing and time of divergence between Tibetans and Han Chinese suggests that the Neolithic population expansion on the Plateau was likely caused by the dispersal of the earliest Neolithic Han Chinese agriculturalists originating about 10 kya in what is now northwestern China….

O3a3c1-M117 is present at frequencies of nearly ~30%, and is connected with the Chinese as you can see above. This dovetails with other recent research which imply relatively recent common ancestors between Tibetans and Chinese. This result can be reconciled to the presence of Paleolithic roots via the fact that admixed populations will give you average results between the two extremes. The problem I have is that I am skeptical that Han Chinese existed 10,000 years ago, just as I am skeptical that Greeks existed 10,000 years ago.

Citation: Cai, Xiaoyun, et al. “Human migration through bottlenecks from Southeast Asia into East Asia during Last Glacial Maximum revealed by Y chromosomes.” PloS one 6.8 (2011): e24282.

A quick literature search yields the fact that M117 is modal in particular non-Han ethnic groups resident in southern China and northern Southeast Asia. I am not here proposing that the Hmong introduced M117 to the Tibetans. Rather, I am suggesting that we best be careful in assuming that we know the ethnic distribution of genetic haplogroups 6,500 years before there were any written records from a given region! To me the fact that there is a putative Sino-Tibetan group of languages is strongly indicative of diversification >10,000 years, not the existence of a Han ethnicity ~10,000 year ago. The historical records are clear that ~3,000 years ago the Yangzi river, now the informal dividing line between North China and South China, was the boundary of the zone where Han were demographically dominant. And even then there were clearly pockets of “barbarian” people on the North China plain itself! It simply does not stand up to the test of basic plausibility that the agricultural expansions ~10,000 years B.P. were Han as we would understand Han. The demographic and cultural dominance of the Han in Northeast Asia is a phenomenon of the last 3,000 years, perhaps 4,000 most generously (South China became Sinicized to some extent after the fall of the Latter Han Dynasty ~200 AD, and especially the Tang period ~600-950 AD).

Much of the argumentation is creaky because of these anachronistic assumptions and the casual inferences of contemporary haplogroup frequencies back toward ancient geographical demographic distributions. Ancient DNA has highlighted the danger of this in Europe, and that should update our priors as to the robustness of this sort of analysis. For example, the authors are curious as to the lack of structure of Y chromosomal lineages, combined with the fact of their deep coalescence times across Tibet. Why is this an issue? Because if these Y chromosomal lineages are Paleolithic, then the deep converges across the branches should also correspondent to geographic differences. But they don’t. To me the simplest explanation is that the last 10,000 years have seen a great deal of population movement, and sharply differentiated populations were brought together as agriculture opened up the Tibetan plateau. This presents a problem though with inferring ancient geographic connections from present distributions, since it opens up the possibility of migration, and radical genetic-demographic turnover.

Overall I would say that this paper is interesting and useful, but you should read it closely and not take the author’s inferences too much to heart. Those inferences are grounded in assumptions which may be built on false foundations.

Addendum: Also, a “gap” on a PCA plot does not necessarily mean long term isolation, as they say in the text. It might simply be a function of inadequate sampling. See above. There are many unsupported assertions such as that. But, I would like to add that the authors found a large number “exotic” haplogroups in Lhasa itself, which aligns with what we know about the cultural history of Tibet. Tibetan Buddhism actually is influenced more by extinct variants of South Asian (particularly, Bengali) Buddhism, rather than Chinese Buddhism. Though the demographic pump along the Himalayan border seems to go from the highlands to the lowlands, there were exceptions. And these exceptions tended to be found in Lhasa.

Citation: Cai, Xiaoyun, et al. “Human migration through bottlenecks from Southeast Asia into East Asia during Last Glacial Maximum revealed by Y chromosomes.” PloS one 6.8 (2011): e24282.

(Republished from Discover/GNXP by permission of author or representative)
 
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Indian_subcontinent

One of the great geological landmarks on earth are the Himalayas. Not only are the Himalayas of importance in the domain of physical geography, but they are important in human geography as well. Just as South Asians and non-South Asians agree that the valley of the Indus and its tributaries bound the west of the Indian cultural world, so the Himalayas bound it on the north. Unlike many pre-modern constructions, such as the eastern boundary of Europe, the northern limit of South Asia is relatively clear and distinct. It is stark on a relief map; the flat Gangetic plain gives way to mountains. And it is stark a cultural map, the languages of northern India give way to those of the world of Tibet. The religion of northern India gives way to the Buddhism of Tibet. In terms of human geography I believe that one can argue that the Himalayan fringe around South Asia exhibits the greatest change of ancestrally informative gene frequencies over the smallest distance when you exclude those regions separated by water barriers. Unlike the Sahara the transect from the northern India to Chinese Tibet at any given point along the border is permanently inhabited, albeit sparsely at the heights.

ResearchBlogging.org And yet despite the geographical barriers people and ideas did move across the Himalaya. The cultural influences upon Tibet from India are obvious. The script of Tibet is derived from India, while its form of Buddhism is the direct descendant of the last efflorescence of that religion in northern India. But while culture moved north, I do not see much evidence genetically that South Asians have been significant as an influence. This is somewhat shocking when you realize these two facts: the population of the Tibetan Autonomous Region is on the order of 5-6 million, while that of northern South Asia around ~1 billion (including Pakistan and Bangladesh). A 200-fold difference. And yet there is evidence of admixture between the two groups exactly where you’d expect: in Nepal. Below is a figure from a recent paper which shows how South and East Asian populations relate to each other. I’ve highlighted the Nepali groups, which span the two larger classes:


tibetsou1

tibetsouth2From the above figure it’s clear that there is considerable admixture among the Indo-European populations of Nepal with a Tibetan element. The Magar are a tribe which is representative of Tibet, with little South Asian genetic input presumably. The Newar are the Nepalese hybrids par excellence. To a great extent they can be viewed as the indigenous peoples of the Kathmandu region at the heart of modern Nepal. Their language is of Tibetan affinity, and yet it is heavily overlain with an Indo-Aryan aspect, and seems to have within it an ancient Austro-Asiatic substrate. Though predominantly Hindu today, the Newar have a substantial Buddhist minority whose roots may go back to the original Mahayana traditions which were once prominent in northern India. The Brahmin and Chetri groups are upper caste communities who claim provenance from the north Indian plain. Some of these upper caste groups in Nepal are of recent vintage, having fled the Islamic conquests of the Gangetic plain within the last 1,000 years. And yet even they have obvious Tibetan admixture. This should make one cautious about the excessive claims to genetic purity which South Asian caste groups make.

But admixture of a Tibetan or East Asian component in South Asia is not limited to Nepal. I have reedited a figure from a 2006 paper on Indian Americans which shows the inferred components of ancestry of various language-groups. It is clear that the northeastern groups, Bengalis, Assamese, and Oriya, have an affinity to East Asians. This is not just ancient east Eurasian ancestry, the “Ancestral South Indians” hypothesized in Reich et al.. The South Indian groups (which I have excised from the figure) do not exhibit the same level of elevation of the ancestral quantum dominant among the Han Chinese in the bar plot. In fact the Reich et al. paper also reported evidence of an eastern ancestral element in some of the Munda speaking groups of northeast India. This stands to reason as the Munda are a South Asian branch of the Austro-Asiatic family of Southeast Asia. But much of it may also be more recent, as groups such as the Ahom of Assam and the Chakma of Bangladesh seem to have arrived from Burma of late.

So we see that genes do flow around the margins of South Asia, and into it. And yet Tibet seems oddly insulated. Why? Because of adaptation. Like water, it seems in this case genes tend to flow downhill, not up, and the reason is likely the fitness differentials between lowland and highland populations along the slope of the Great Himalayas. A new paper in PNAS explores the issue by examining genetic variation among Indians, Tibetans, and worldwide populations, in relation to hypoxia implicated loci. EGLN1 involvement in high-altitude adaptation revealed through genetic analysis of extreme constitution types defined in Ayurveda:

It is being realized that identification of subgroups within normal controls corresponding to contrasting disease susceptibility is likely to lead to more effective predictive marker discovery. We have previously used the Ayurvedic concept of Prakriti, which relates to phenotypic differences in normal individuals, including response to external environment as well as susceptibility to diseases, to explore molecular differences between three contrasting Prakriti types: Vata, Pitta, and Kapha. EGLN1 was one among 251 differentially expressed genes between the Prakriti types. In the present study, we report a link between high-altitude adaptation and common variations rs479200 (C/T) and rs480902 (T/C) in the EGLN1 gene. Furthermore, the TT genotype of rs479200, which was more frequent in Kapha types and correlated with higher expression of EGLN1, was associated with patients suffering from high-altitude pulmonary edema, whereas it was present at a significantly lower frequency in Pitta and nearly absent in natives of high altitude. Analysis of Human Genome Diversity Panel-Centre d’Etude du Polymorphisme Humain (HGDP-CEPH) and Indian Genome Variation Consortium panels showed that disparate genetic lineages at high altitudes share the same ancestral allele (T) of rs480902 that is overrepresented in Pitta and positively correlated with altitude globally (P< 0.001), including in India. Thus, EGLN1 polymorphisms are associated with high-altitude adaptation, and a genotype rare in highlanders but overrepresented in a subgroup of normal lowlanders discernable by Ayurveda may confer increased risk for high-altitude pulmonary edema.

The paper itself is a follow up to a previous work attempting to see if there was a sense to the classification of constitutions found within Ayurvedic medicine. Like Chinese medicine this is a non-Western tradition which has different philosophical roots and axioms (Galenic medicine might be analogous). But in theory all medical traditions emerged to battle illness, so their target was unitary, the ailments which plague the human body. Therefore one might suppose that in fact there would be some sense in any long-standing medical tradition which has any empirical grounding, because human biology is relatively invariant. It is the relative clause which is of interest for the purposes of this paper, because the authors show how the classifications of Ayurvedic medicine seem to comport with the recent genetic evidence of high altitude adaptation! Specifically they found that particular Ayurvedic classes of individuals who seem to have negative reactions to high altitude exposure in the form of hypoxia tend to be carriers of particular EGLN1 genotypes.

I will at this point observe that since I don’t know much about Ayurveda I won’t address or cover that issue in detail. The paper is Open Access so you can read it yourself. So let’s move to the genetics. EGLN1 should be familiar to you by now. It’s cropped up repeatedly over the past year in studies of high altitude adaptation. It is a locus which seems to be a target of selection in both the peoples of the Andes and Tibet. Additionally, it has a peculiar aspect where the ancestral variant, the one found most frequently within Africa, seems to be the target of selection for altitude adaptation outside of Africa.

The slideshow below is an overview of the primary figures within this paper.


[zenphotopress album=197 sort=sort_order number=6]


What do we take away from this? Well, one aspect which I think is important to emphasize is that genetic background matters, and there’s much we don’t know. In the conclusion the authors note that the altitude adaptation papers which I alluded to above were not published when the manuscript was being written, so they were not privy to the rather repeated robust evidence that EGLN1 has been the target of natural selection, and that variation on the locus is correlated with variation in adaptation to higher altitudes. The widespread coverage of populations in this paper seems to almost obscure as much as highlight. What has African variation to do with this after all? Additionally one must always remember that one given marker on a gene which shows a correlation does not entail functional causation. We saw this with the markers which seemed to predict the odds of an individual of European ancestry having blue eyes; it turned out that the markers themselves were simply strongly associated with another SNP which was probably the real functional root behind the difference in phenotype.

Due to the replication of EGLN1 in both Andeans and Tibetans I am moderately confident that variation on this gene does have something to due with high altitude adaptation. What I am curious about is the fact that the ancestral alleles in many cases seem to be driven up on frequency. Is there an interaction between the genetic background of non-Africans and the SNPs in question which make it beneficial toward altitude adaptation? Was there an initial relaxation of function as human populations moved out of Africa, which was slammed back on at high altitudes? There does seem a correlation within South Asian populations between hypoxia and high altitudes and particular variants on EGLN1. Focusing just on this region we can draw some reasonable inferences, but taking a bigger picture view and encompassing the whole world we’re confronted with a rather more confused, and perhaps more interesting, picture.

Back to the specific issue of the lack of South Asian imprint on the genes of Tibetan peoples, I think one can chalk this up to the fact that humans are animals, and so we’re constrained by geography and biology. Tibetans can operate efficiently at lower altitudes, and so have mixed with South Asians in these regions. In contrast, South Asians can not operate at higher altitudes, and so the impact on Tibetans was purely cultural, and not genetic. More broadly this may also point to long term geopolitical implications: the Han Chinese demographic domination of Tibet is always going to be a matter of water flowing uphill. Unless of course we flesh out the genetic architecture of these traits well enough that the Chinese government knows exactly which individuals among the 1.2 billion Han population would be most biologically prepared to reside in the Tibetan Autonomous Region, and so can proactively recruit them to settle in Lhasa and other strategic locations.

Citation: Shilpi Aggarwal, Sapna Negi, Pankaj Jha, Prashant K. Singh, Tsering Stobdan, M. A. Qadar Pasha, Saurabh Ghosh, Anurag Agrawal, Indian Genome Variation Consortium, Bhavana Prasher, & Mitali Mukerji (2010). EGLN1 involvement in high-altitude adaptation revealed through genetic analysis of extreme constitution types defined in Ayurveda PNAS : 10.1073/pnas.1006108107

Image Credit: Wikimedia Commons

(Republished from Discover/GNXP by permission of author or representative)
 
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I said yesterday I would say a bit more about the new paper on rapid recent high altitude adaptation among the Tibetans when I’d read the paper. Well, I’ve read it now. Sequencing of 50 Human Exomes Reveals Adaptation to High Altitude:

Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP’s association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.

The exome is just the protein-coding part of the genome; so they’re focusing ostensibly on functionally relevant single nucleotide polymorphisms (SNPs). About a month and a half ago a similar paper on Tibetan high altitude adaptations was published in Science (I posted on that too), but their methodology was somewhat different. That group was looking at a set of genes, candidates, which they’d assume might have been under selection and so have functional significance in explaining Tibetan vs. non-Tibetan phenotypes at high altitudes. This second paper takes a more bottom up approach, scanning the genome of Tibetans and Han Chinese, and trying to spotlight regions which exhibit a great deal of between population variance, far greater than one might presume from the total genome genetic distances.

As to that last point…the timing of this has been causing a major problem with archaeologists. The supplements lays out the details a bit more than the press reports, so below is figure 2:


tibhan

It looks like to get a better sense of the model you’ll have to read the cited paper, and I’m not sure that that will satisfy the archaeologists. They did use a large number of neutral markers though, so I’m not too worried about biases in their data set. Some have been confused about the population numbers, but this value in a population genetic context can be counterintuitive, especially over the long term (low values are given much more weight than high values). The small Han value can be easily made less confusing when you consider a massive demographic expansion from a small founder group, as well as persist long term biases in reproductive value within the population (e.g., some males in a given generation are way more fecund than others through polygyny). A higher N for Tibetans may be explained by a more stable population where diverse subsets and across individuals the reproductive value may be more equitable. In other words, an effective population size is a statistic which is bundling together a lot of evolutionary history, and is not a simple measure of perceived census sizes (the Tibetans may also be something of a melange of a diverse set of ancient groups which took refuge in the highlands, while the Han are the descendants of early adopters of agriculture which expanded demographically; so they’re opposite ends of the demographic tunnel).

The time of divergence of a little under 3,000 years is important for the rest of the paper, so I suppose other workers had better replicate their findings in the future. Figure 1 is rather striking, so let’s jump to it:

tibhan2

This chart is simply showing frequencies of SNPs in Tibetans and Han. The two are obviously correlated, as evident by the diagonal. Shading indicates the density of the number of SNPs at a given position. Look to the bottom right, and you see the gene around which much of the paper hinges, EPAS1. It’s an enormous outlier, with SNPs where Tibetans and Han differ a great deal. This is important in regards to looking for genes which may drive adaptation to higher altitudes; if you don’t have different genes then you don’t have different traits. If the Tibetans and Han diverged ~3,000 years ago, then those adaptations may be recent and would have emerged through rapid allele frequency changes (though they observe that it may be drawn from standing variation). The researchers didn’t go looking for EPAS1 as such, rather, it came looking for them. What does it do? From the text:

EPAS1 is also known as hypoxia-inducible factor 2{alpha} (HIF-2{alpha}). The HIF family of transcription factors consist of two subunits, with three alternate {alpha} subunits (HIF-1{alpha}, HIF-2{alpha}/EPAS1, HIF-3{alpha}) that dimerize with a β subunit encoded by ARNT or ARNT2. HIF-1{alpha} and EPAS1 each act on a unique set of regulatory targets…and the narrower expression profile of EPAS1 includes adult and fetal lung, placenta, and vascular endothelial cells…A protein-stabilizing mutation in EPAS1 is associated with erythrocytosis…suggesting a link between EPAS1 and the regulation of red blood cell production.

Next, they dig into the functional significant of EPAS1 variants, in the literature, and in their current sample:

Associations between SNPs at EPAS1 and athletic performance have been demonstrated…Our data set contains a different set of SNPs, and we conducted association testing on the SNP with the most extreme frequency difference, located just upstream of the sixth exon. Alleles at this SNP tested for association with blood-related phenotypes showed no relationship with oxygen saturation. However, significant associations were discovered for erythrocyte count (F test P = 0.00141) and for hemoglobin concentration (F test P = 0.00131), with significant or marginally significant P values for both traits when each village was tested separately (table S5). Comparison of the EPAS1 SNP to genotype data from 48 unlinked SNPs confirmed that its P value is a strong outlier (5) (fig. S4).

The allele at high frequency in the Tibetan sample was associated with lower erythrocyte quantities and correspondingly lower hemoglobin levels…Because elevated erythrocyte production is a common response to hypoxic stress, it may be that carriers of the “Tibetan” allele of EPAS1 are able to maintain sufficient oxygenation of tissues at high altitude without the need for increased erythrocyte levels. Thus, the hematological differences observed here may not represent the phenotypic target of selection and could instead reflect a side effect of EPAS1-mediated adaptation to hypoxic conditions. Although the precise physiological mechanism remains to be discovered, our results suggest that the allele targeted by selection is likely to confer a functionally relevant adaptation to the hypoxic environment of high altitude.

There are random anomalies in nature, but it seems too perfect that this is the outlier in allele frequencies across two populations which differ in adaptations which relate to many of the traits above.

tibhan3OK, so they found an outlier SNP. The gene seems to have a reasonable probability of being involved in functional pathways relevant to altitude adaptation. But so far we’ve been focusing on the Tibetan-Han difference. If the two populations separated about 3,000 years ago one assumes that genes with SNPs with huge F sts, where most of the variation can be partitioned between the groups, not within them, are good candidates for having been driven by selection. But it would be nice to compare with an outgroup. So they compared the Tibetans and Hans with the Danes, who are an outgroup who separated from the East Asian cluster about one order of magnitude further back in time (~30,000 years). Next they generated a “population branch statistic,” (PBS), from the the F st data (see the supplements). Basically you’re getting a value which describes allele frequency differences normalized to the expected genetic distance as known from population history. I’ve extracted out Panel B from figure 2. T = Tibetans, H = Han, and D = Danes. The smaller tree represents genome average PBS values. It’s what you’d expect, the Danes are the outgroup. Over time genetic difference builds up because of separation between the groups. The Han and Tibetans are very close, as you’d expect from genetically similar populations. But look at the larger tree, the Tibetans are the outgroup by a mile! The Danes and Han differ far less from each other on EPAS1 than they do from the Tibetans. This seems like a clear deviation from the level of allele frequency difference one might be able to generate by neutral random walk processes.

EPAS1 isn’t the only gene which they found, but it was the most significant, and illustrates the nature of the methodological orientation of this group. Sift through the genome and look for something which is totally unexpected, and put a focus on the peculiar diamond in the rough and see what it can tell you. They conclude with the big picture:

Of the genes identified here, only EGLN1 was mentioned in a recent SNP variation study in Andean highlanders (24). This result is consistent with the physiological differences observed between Tibetan and Andean populations…suggesting that these populations have taken largely distinct evolutionary paths in altitude adaptation.

Several loci previously studied in Himalayan populations showed no signs of selection in our data set…whereas EPAS1 has not been a focus of previous altitude research. Although EPAS1 may play an important role in the oxygen regulation pathway, this gene was identified on the basis of a noncandidate population genomic survey for natural selection, illustrating the utility of evolutionary inference in revealing functionally important loci.

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…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.

Natural selection is somewhat stochastic; it can take different tacks to the same process because it doesn’t have infinite power in its search algorithm. Given enough time and gene flow no doubt adaptations would homogenize and converge upon a perfect optimum, but given enough time the universe will devolve into heat death. Evolution has to operate extemporaneously for eternity because the conditions are ever changing. Second, the big headline grabbing assertion about EPAS1 being the strongest instance of natural selection needs to be moduled by the fact that the conclusion was generated assuming the validity of the inferences of a particular model, and models can be wrong. It does seem like the evolutionary change is likely to be recent, I doubt they’d be off by an order of magnitude. But for lactase persistence we’ve extracted genetic material from ancient remains. The conclusion then is much more concrete in this case. Until we get remains from ancient Tibetans and can infer their allele frequencies, there will be some asymmetry in the confidence with which we can make a claim as to when the selection event began.

Citation: Yi, X., Liang, Y., Huerta-Sanchez, E., Jin, X., Cuo, Z., Pool, J., Xu, X., Jiang, H., Vinckenbosch, N., Korneliussen, T., Zheng, H., Liu, T., He, W., Li, K., Luo, R., Nie, X., Wu, H., Zhao, M., Cao, H., Zou, J., Shan, Y., Li, S., Yang, Q., Asan, ., Ni, P., Tian, G., Xu, J., Liu, X., Jiang, T., Wu, R., Zhou, G., Tang, M., Qin, J., Wang, T., Feng, S., Li, G., Huasang, ., Luosang, J., Wang, W., Chen, F., Wang, Y., Zheng, X., Li, Z., Bianba, Z., Yang, G., Wang, X., Tang, S., Gao, G., Chen, Y., Luo, Z., Gusang, L., Cao, Z., Zhang, Q., Ouyang, W., Ren, X., Liang, H., Zheng, H., Huang, Y., Li, J., Bolund, L., Kristiansen, K., Li, Y., Zhang, Y., Zhang, X., Li, R., Li, S., Yang, H., Nielsen, R., Wang, J., & Wang, J. (2010). Sequencing of 50 Human Exomes Reveals Adaptation to High Altitude Science, 329 (5987), 75-78 DOI: 10.1126/science.1190371

(Republished from Discover/GNXP by permission of author or representative)
 
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443px-PaldenLhamoNick Wade in The New York Times is reporting on a new paper which will come out in Science tomorrow which investigates the evolution of genes implicated in adaption to higher altitudes among Tibets. I’ve posted on the genetics of this topic before, it obviously is of great interest. The major new finding is that these adaptations seem to have spread among Tibetans very recently, on the order of 3,000 years or so. Here’s the relevant section on the genetic architecture:

The Beijing team analyzed the 3 percent of the human genome in which known genes lie in 50 Tibetans from two villages at an altitude of 14,000 feet and in 40 Han Chinese from Beijing, which is 160 feet above sea level. Many genes exist in a population in alternative versions. The scientists found some 30 genes in which a version rare among the Han had become common among the Tibetans. The most striking instance was a version of a gene possessed by 9 percent of Han but 87 percent of Tibetans.

Such an enormous difference indicates that the version typical among Tibetans is being strongly favored by natural selection. In other words, its owners are evidently leaving more children than those with different versions of the gene.

The gene in question is known as hypoxia-inducible factor 2-alpha, or HIF-2a, and the Tibetans with the favored version have fewer red blood cells and hence less hemoglobin in their blood.

But the more confused issue is the accuracy of the time timing:

The three new reports agree in finding the Tibetans’ version of the gene has been favored by natural selection. But the Beijing Genome Institute’s calculation that the Tibetan and Han populations split apart only 3,000 years ago is less likely to be accepted. Archaeologists believe the Tibetan plateau has been inhabited for at least 7,000 years and maybe for as long as 21,000 years.

“The separation of Tibetans and Hans at 3,000 years ago is simply not tenable by anything we know from the historical, archaeological or linguistic record,” said Mark Aldenderfer, a Tibetan expert at the University of California, Merced.

Dr. Aldenderfer said that there had probably been many migrations onto the Tibetan plateau, and that there was indirect evidence that pastoralists had entered the plateau from the north-northeast around 6,000 years ago. Earlier genetic studies have found that Tibetans are more similar to northern Han than to those from southern China, and have some admixtuWell, re of genes from Central Asia, he said.

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 or later if the geneticists had taken any account of any other kind of evidence.

Rasmus Nielsen, a Danish researcher currently at the University of California at 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 he explained that “I cannot with confidence rule out that the divergence time is 6,000 instead of 3,000.”

I haven’t seen the paper, but I assume that the 3,000 years figure was derived from the point at which selective sweeps started diverging the genetic architecture of adaptations related to altitude phenotypes. But there is probably a pretty large confidence interval here, and previous estimates have been revised upward (though Nielsen is one of the best in the game, and he sounds relatively confident). I don’t see why the length of human habitation is that relevant, populations move, or are replaced. Archaeologists simply have a very strong bias against population movements, and tend to see cultural continuity in their interpretation of physical remains. Very similar things were asserted by archaeologists when the first genetic evidence on the exogenous origin of the Etruscans came to light. I assume there are many scholarly careers which have been based on fleshing out the cultural continuities of societies and cultures on the Tibetan plateau, and the idea that contemporary Tibetans are newcomers would overturn that.

More to say tomorrow when the paper comes out. But ScienceDaily has some more details:

The widespread mutation in Tibetans is near a gene called EPAS1, a so-called “super athlete gene” identified several years ago and named because some variants of the gene are associated with improved athletic performance, Nielsen said. The gene codes for a protein involved in sensing oxygen levels and perhaps balancing aerobic and anaerobic metabolism.

“We can’t distinguish intermixing and replacement,” Nielsen said. “The Han Chinese and Tibetans are as different from one another as if the Han completely replaced the Tibetans about 3,000 years ago.”

The Tibetan and Han Chinese genomes are essentially identical in terms of the frequency of polymorphisms in the roughly 20,000 genes, though some 30 genes stood out because of dramatic differences between the Tibetans and the Han.
“We made a list of the genes that changed the most,” Nielsen said, “and what was fascinating was that, bing!, at the top of that list was a gene that had changed very strongly, and it was related to the response to oxygen.”

The SNP with the most dramatic change in frequency, from 9 percent in Han Chinese to 87 percent in Tibetans, was associated with lower red blood cell count and lower hemoglobin levels in Tibetans. That variation occurred near a gene called EPAS1, which earlier studies suggest is involved in regulating hemoglobin in the blood as a response to oxygen levels. The mutation may be in a transcription factor that regulates the activity of EPAS1.

Tibetans carrying only one allele with this mutation had about the same hemoglobin concentration as Han Chinese, but those with two mutated alleles had significantly lower hemoglobin concentration. However, they all have about the same oxygen concentration in the blood. For some reason, individuals with two copies of the mutation function well in high altitude with relatively low hemoglobin concentration in their blood. The mutation seems to provide an alternative inborn mechanism for dealing with the low oxygen levels, Nielsen said.

I’m intrigued that this seems to express recessively in terms of the trait-value they looked at. I wonder if there are other fitness benefits in the heterozygote state which allowed it to increase in frequency rapidly so that the benefits in the homozygote state could express.

Image Credit: Wikimedia

(Republished from Discover/GNXP by permission of author or representative)
 
• Category: History, Science • Tags: Genetics, Tibet 
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443px-PaldenLhamoYou probably are aware that different populations have different tolerances for high altitudes. Himalayan sherpas aren’t useful just because they have skills derived from their culture, they’re actually rather well adapted to high altitudes because of their biology. Additionally, different groups seem to have adapted to higher altitudes independently, exhibiting convergent evolution. But in terms of physiological function they aren’t all created equal, at least in relation to the solutions which they’ve come to to make functioning at high altitudes bearable. In particular, it seems that the adaptations of the peoples of Tibet are superior than those of the peoples of the Andes. Superior in that the Andean solution is more brute force than the Tibetan one, producing greater side effects, such as lower birth weight in infants (and so higher mortality and lower fitness).

The Andean region today is dominated by indigenous people, and Spanish is not the lingua franca of the highlands as it is everyone in in the former colonial domains of Spain in the New World. This is largely a function of biology; as in the lowlands of South America the Andean peoples were decimated by disease upon first contact (plague was spreading across the Inca Empire when Pizzaro arrived with his soldiers). But unlike the lowland societies the Andeans had nature on their side: people of mixed or European ancestry are less well adapted to high altitudes and women without tolerance of the environment still have higher miscarriage rates.

So despite the suboptimal nature of the Andean adaptations vis-a-vis the Tibetan ones, they are certainly better than nothing, and in a relative sense have been very conducive to higher reproductive fitness. And yet why might the Andeans have kludgier adaptations than Tibetans? One variable to consider is time. The probability is that the New World was populated by humans only for the past ~10,000-15,000 years or so, with an outside chance of ~20,000 years (if you trust a particular interpretation of the genetic data, which you probably shouldn’t). By contrast, modern humans have had a presence in the center of Eurasia for ~30,000 years. Generally when populations are exposed to new selective regime the initial adaptations are drastic and exhibit major functional downsides, but they’re much better than the status quo (remember, fitness is relative). Over time genetic modifications mask the deleterious byproducts of the genetic change which emerged initially to deal with the new environment. In other words, selection perfects design over time in a classic Fisherian sense as the genetic architecture converges upon the fitness optimum.*

Another parameter may be the variation available within the population, as the power of selection is proportional to the amount of genetic variation, all things equal. The peoples of the New World tend to be genetically somewhat homogeneous, probably due to the fact that they went through a bottleneck across Berengia, and that they’re already sampled from the terminus of the Old World. A physical anthropologist once told me that the tribes of the Amazon still resemble Siberians in their build. It may be that it takes a homogeneous population with little extant variation a long time indeed to shift trait value toward a local ecological optimum (tropical Amerindians are leaner and less stocky than closely related northern populations, just not particularly in relation to other tropical populations). In contrast, populations in the center of Eurasia have access to a great deal of genetic variation because they’re in proximity to many distinctive groups (the Uyghurs for example are a recent hybrid population with European, South Asian and East Asian ancestry).

So that’s the theoretical backdrop for the differences in adaptations. Shifting to the how the adaptations play out concretely, some aspects of the physiology of Tibetan tolerance of high altitudes are mysterious, but one curious trait is that they actually have lower levels of hemoglobin than one would expect. Andean groups have elevated hemoglobin levels, which is the expected “brute force” response. Interestingly it seems that evolution given less time or stabilizing at a physiologically less optimal equilibrium is more comprehensible to humans! Nature is often more creative than us. In contrast the Tibetan adaptations are more subtle, though interestingly their elevated nitric acid levels may facilitate better blood flow. Though the inheritance patterns of the trait had been observed, the genetic mechanism underpinning it has not been elucidated. Now a new paper in Science identifies some candidate genes for the various physiological quirks of Tibetans by comparing them with their neighbors, and looking at the phenotype in different genotypes with the Tibetan population. Genetic Evidence for High-Altitude Adaptation in Tibet:

Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. We report genome-wide scans that reveal positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia response pathways in humans.

Here’s what they did. First, Tibetans are adapted to higher altitudes, Chinese and Japanese are not. The three groups are relatively close genetically in terms of ancestry, so the key is to look for signatures of positive selection in regions of the genome which have been identified as possible candidates in terms of functional significance in relation to pathways which may modulate the traits of interest. After finding potential regions of the genome possibly under selection in Tibetans but not the lowland groups, they fixed upon variants which are at moderate frequencies in Tibetans and noted how the genes track changes in the trait.

This figure from the supplements shows how the populations are related genetically:

tib1

In a worldwide context the three groups are pretty close, but they also don’t overlap. The main issue I would have with this presentation is that the Chinese data is from the HapMap, and they’re from Beijing. This has then a northeast Chinese genetic skew (I know that people who live in Beijing may come from elsewhere, but recent work which examines Chinese phylogeography indicates that the Beijing sample is not geographically diversified), while ethnic Tibetans overlap a great deal with Han populations in the west of China proper. In other words, I wouldn’t be surprised if the separation between Han and Tibetan was far less if you took the Chinese samples from Sichuan or Gansu, where Han and Tibetans have lived near each other for thousands of years.

tib2But these issues of phylogenetic difference apart, we know for a fact that lowland groups do not have the adaptations which are distinctive to the Tibetans. To look for genetic differences they focused on 247 loci, some from the HIF pathway, which is important for oxygen homeostasis, as well genes from Gene Ontology categories which might be relevant to altitude adaptations. Table 1 has the breakdown by category.

Across these regions of the genome they performed two haplotype based tests which detect natural selection, EHH and iHS. Both of these tests basically find regions of the genome which have reduced variation because of a selective sweep, whereby selection at a specific region of the genome has the effect of dragging along large neutral segments adjacent to the original copy of the favored variant. EHH is geared toward detection of sweeps which have nearly reached fixation, in other words the derived variant has nearly replaced the ancestral after a bout of natural selection. iHS is better at picking up sweeps which have not resulted in the fixation of the derived variant. The paper A Map of Recent Positive Selection in the Human Genome outlines the differences between EHH and iHS in more detail. They looked at the three populations and wanted to find regions of the genome where Tibetans, but not the other two groups, were subject to natural selection as defined by positive signatures with EHH and iHS. They scanned over 200 kb windows of the genome, and found that 10 of their candidate genes were in regions where Tibetans came up positive for EHH and iHS, but the other groups did not. Since these tests do produce false positives they ran the same procedure on 240 random candidate genes (7 genes were in regions where Chinese and Japanese came up positive, so these were removed from the set of candidates), and came up with average EHH and iHS positive hits of ~2.7 and ~1.4 genes after one million resamplings (specifically, these are genes where Tibetans were positive, the other groups negative). Their candidate genes focused on altitude related physiological pathways yielded 6 for EHH and 5 for iHS (one gene came up positive for both tests, so 10 total). This indicates to them these are not false positives, something made more plausible by the fact that we know that Tibetans are biologically adapted to higher altitudes and we have an expectation that these genes are more likely than random expectation to have a relationship to altitude adaptations.

Finally, they decided to look at two genes with allelic variants which exist at moderate frequencies in Tibetans, EGLN1 and PPARA. The procedure is simple, you have three genotypes, and you see if there are differences across the 31 individuals by genotype in terms of phenotype. In this case you want to look at hemoglobin concentration, where those who are well adapted have lower concentrations. Figure 3 is rather striking:

tib3

Even with the small sample sizes the genotypic effect jumps out at you. This isn’t too surprising, previous work has shown that these traits are highly heritable, and that they vary within the Tibetan population. There’s apparently a sex difference in terms of hemoglobin levels, so they did a regression analysis, and it illustrates how strong the genetic effect from these alleles are:

tib4

My main question: why do Tibetans still have variation on these genes after all this time? Shouldn’t they be well adapted to high altitudes by now? A prosaic answer may be that the Tibetans have mixed with other populations recently, and so have added heterozygosity through admixture. But there are several loci here which are fixed in Tibetans, and not the HapMap Chinese and Japanese. For admixture to be a good explanation one presumes that the groups with which the Tibetans mixed would have been fixed for those genes as well, but not the ones at moderate frequencies. This may be true, but it seems more likely that admixture alone can not explain this pattern. As the Andean example suggests adaptation to high altitudes is not easy or simple. Until better options arrive on the scene, kludges will suffice. It may be that the Tibetans are still going through the sieve of selection, and will continue to do so for the near future. Or, there may be balancing dynamics on the genes which exhibit heterozygosity, so that fixation is prevented.

No matter what the truth turns out to be, this is surely just the beginning. A deeper investigation of the genetic architecture of Andeans and Ethiopians, both of which have their own independent adaptations, will no doubt tell us more. Finally, I wonder if these high altitude adaptations have fitness costs which we’re not cognizant of, but which Tibetans living in India may have some sense of.

Citation: Tatum S. Simonson, Yingzhong Yang, Chad D. Huff, Haixia Yun, Ga Qin, David J. Witherspoon, Zhenzhong Bai, Felipe R. Lorenzo, Jinchuan Xing, Lynn B. Jorde, Josef T. Prchal, & RiLi Ge (2010). Genetic Evidence for High-Altitude Adaptation in Tibet Science : 10.1126/science.1189406

* Additionally, it may be that archaic hominin groups were resident in the Himalaya for nearly one million years. Neandertal admixture evidence in Eurasians should change our priors when evaluating the possibility for adaptive introgression on locally beneficial alleles.

Image Credit: Wikimedia Commons

(Republished from Discover/GNXP by permission of author or representative)
 
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