[~ERSPECTIVES M o l e c u l a r evidence indicates that the lineages leading to humans and to African apes separated some five to seven million years agoLZ. Paleontological data suggest that the hominid lineage included several species of Australopitbecus and of Homo before it produced the modern H. sapien# -S. Our last ancestor is believed to have been H. erectus from whom H. sapiens began to evolve some| 500000 to 300000 years ago 6. Paleontology, however, does not reveal much about the population aspect of these seven million yearn of human evohation. How large was the population from which H. sapiens sprang? Indeed, how many individuals provided the foundations for the hominid lineage? Was there a 'bottleneck', a drastic reduction in population size, at the beginning of each new human species? Or, alternatively, was each species founded by a large gene pool provided by the preceding species in the evolutionary line?

The Eve myth The results of the recent studies on mitochondrial DNA 7 have been widely interpreted in editorials and by the popular press as providing evidence for the origin of the human race from a single female, 'Eve', who lived in Africa some 200 000 years ago 8.9. This interpretation is, however, based on a misunderstanding, on a confusion of gene trees with genealogical trees of individualsm. While it may be true that all mitochondrial DNA present in the human population today originated from a single female 200 000 years ago (although some investigators dispute this contentionlO, it by no means follows that this female was our common mother, as far as the thousands of other genes are concerned. These genes might be traced to different mothers and fathers who lived at different times and in different places in our past. The

The major histocompatibilitycomplex and humanevolution JAN KLEIN,JUTrAGUTKNECHTAND NORBERTFISCHER Many alleles at the human majorhistocompatibility complex (Hlai) loci diverged before the divergence of humans and great apes from a common ancestor. Thisfact puts a lower limit on the size of the bottleneck in human evolution: the genus Homo must have beenfounded by no less than ten and probab~ by more than l O000 individuals. mitochondrial DNA data, therefore, do not allow one to make any statements about the size and composition of the founding population.

MHCpolymorphism There is, however, one genetic system tha: does provide information about the population size in speciation, including human speciation - the major histocompatibility complex (MHC). The human MHC, referred to as the HLA (human leukocyte antigen) complex, consists of 30-35 loci that fall into two classes: some 16-20 class I, and 14 class II loci (Fig. 1) ]2. Each of the two classes can be divided into two subclasses, A and B, coding for 0t and ~ polypeptide chains, respectively. An HLA molecule is either a class I or class II cx]] heterodimer. All subcla~ A and all class II subclass B loci are closely linked on chromosome 6; the single class I subclass B locus (]32-'microglobulin, B2m) is on human chromosome 15. The subclasses can be divided into families so that loci

Other

Unrelated loci I

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. "i l

class I loci I Class

3 Families 3 Subclass and loci ..................... Chromosome 6

DR i

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1301 1302 1601 1002 2701 2702 2703 2704 2705 2706 2707

'

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3502 3883 3504 3701 380J 3001 6101 4102 4201 4202 4~01 44102 4001 480,1

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0102 0201 0202 0203 0204 0205 0301 0302 11ol 1102 1103 2301 2001 2402 2403 2ao4 2501 2601 2602

2001 2002 2~0= 2902 3001 3002 3003 3101 52ol 330X 3302 3401 3402 3501 8301 5001 6801 6001 7101

Alleles

FIGH The HLA complex - organization and polymorphism. The assignment of alleles at the DQAI and DQBI loci is based mainly on Tcell typing. Additional alleles (variants of the major alleles) have been identified biochemically but these have not yet been sorted out with respect to the locus and major alleles to which "h~y belonq. The assignment of alleles at the DPBI locus is also based on T-cell typing only. The assignment of alleles at all other loci is based on serology (the first two digits) as well as biochemistry and/or T-cell typing (the last two digits'). The last two-digit assignments should not be regarded as official: they are used here only to indicate the extent of the HL4 polymorphism. TIG JANUARY1990 VOL.6 NO~ 1 © 19¢X) Elsevier S c i e n c e . ublisllers I J d (L'K) 01(x8 - 9479 90 S02.00

~'~ERSPECTIVES

Phylogenetic tree of human HLA-DRB1and one chimpanzee ChLA-DRB1 allele constructed by the method of Saitou and Nei.~'. The tree is rooted by one mouse (H-2Ed6) allele. The first two digits in each allelic designation indicate the 'type' - the group to which a given allele belongs. The 'subtype' should normally be designated by another m'o digits (as in Fig. 1) but since no official agreement has yet been reached as far as the designation of individual subtypes is concerned, we use either cell line designations or t~i~et names (in parentheses). The numbers on the individual branches indicate genetic distances. Disproportion in branch length is indicated by 7/'. The origin of the sequences is given in Ref. 21.

FIGM IO(I~A)I) 01(1568) %"'% Io 01(LG2) ~,

0903

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within each family are more closely related to one another than they are to loci in other families. The HLA class II families are designated DN, DO, DP, DQ and DR; the HLA class I families remain to be identified. If there is more than one locus in a given family, the individual loci are numbered sequentially (e.g. DRBI, DRB2, DRB3; DQA1, DQA2, etc.). In each HLA class, some loci are highly polymorphic while others are either mono- or oligomorphic. The polymorphism of the HLA loci is unique in its extent and in the genetic distance between individual alleles. Between 6 and 57 alleles have been identified at the different polymorphic loci (Fig. 1), each allele occurring in at least some human populations at appreciable frequencies; but the numbers may eventually exceed 100, at least for some of the loci t.~. More remarkable, however, is the fact that some of the alleles (and they are true alleles rather than pseudoalleles)lq differ by as many as 90 or more nucleotide substitutions and that their products, the allomorphs, may differ by 20-30 amino acid substitutions.

t,

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convergent evolution, which is ruled out by comparisons of sites that evolve free of selection pressure), one must conclude that part of the HLA polymorphism predates the separation of lineages leading to humans and chimpanzees.

the trans.species theory Some ten years ago, we proposed that these unusual features of the MHC polymorphism could be explained by postulating that the alleles are older than the speciesl~.16. This proposal ts now supported by a rapidly growing body of data accumulated from studies of both primate and rodent MHCstV-2.~. Figure 2 provides one example of such evidence. Here, sequences of human and chimpanzee alleles at the class II DRBI locus have been compared and a phylogenetic tree has been constructed on the basis of the genetic distances between them z~. The remarkable thing about this tree is that the chim,:~/mzee allele is on the human branches rather than on a branch of its own. The implication of this unusual distribution is that some of the human alleles are mote closely related to particular chimpanzee alleles than they are to other human alleles. To explain such relationships (barring

Polymorphism and the neutral theory The notion that much of the MHC poiymorphism must have predated speciation also fits in with considerations based on the neutral theory of molecular evolution 22. One of the tenets of this th- ory is that the average number of generations required to fix one mutation is four times greater than the effective population size, N~.. If we assume that N~. is equal to 104 in hominid evolution 2-~, then we come to the conclusion that 4 x 104 or 40000 generations elapsed from the time a mutation emerged in a population to the time of its fixation. If we take one generation in hominid evolution to bc equal to 20 years 24, we must expect that a mutation ~lceded, on average, 20 x 40 000 or 0.8 million years for fixation. Since Homo sapiens is presumably less than 0.8 million years old, many of the polymorphisms now present in the human population must have arisen before its emergence. If thi~ consideration applies to any neutrally evolving gene, it must do so even more to the synonymous sites in HLA genes because of their greater polymorphism in comparison to other genes. Another tenet of the neutral theory of molecular evolution is that mutations at neutral sites are fixed with the regularity of a clock2L Although there is still much debate about the general validity of the mol~:cular clockZS, a certain constancy of evo~utionary rates in some lineages, at least over certain time periods, is undeniable 26. The molecular clock is normally applied to comparisons of genes in different species. There is, however, no reason why it could not also be applied to alleles at the same locus, such as those present at some of the HLA loci. Instead of fixation of mutations, considered in inter-species comparisons, one can use

T1GJANUARY 1990 VOL 6 NO. 1

m

'~ERSPECTIVES

T , ~ u 1. Calculated age o f s o m e o f the HLA-DRBI alleles HLA.IIRBI alleles compared t

1

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.0084 +_.0060 .O1Z7 +_.0073 .0256 +_.0105 .800 +_.0192 .0084 +_.0060 .0235 +_.0168 .OO72 +_.O3OO .0438 *_.022O :~'s9044+_.0307 .0801 +_.0192 .0588 +-.0243 .0852 +_.0327 .0585 _+.0153 .0712 _+.0172 .0757 +_.0188 .0898 +_.0206

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2

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aThe HIA-DRB1 symbols are omitted for brevity. The symbols in parentheses indicate the histogenetic (Dw) types and/or the designation of the cell line from which the particular gene was isolated.The origin of the sequences is given in Ref. 37. bCalculated by Kimura'sz7 two-parameter method as explained in the text.

the attainment of polymorphic frequencies of alleles in intra-species comparisons. Assuming that these frequencies are attained with the constancy of the molecular clock, one can use the genetic distance between alleles (the average number of nucleotide substitutions per site) to calculate the time of divergence of these alleles, just as in inter-species comparisons one uses genetic distance to calculate the time of divergence of genes. Age of HLA alleles We have applied these considerations to the HLA alleles to obtain a rough idea of their age and hence also of the number of alleles that pcedate hominid ~peciations. To this end, we used the formula developed by Kimura27 for inter-species gene comparisons, which takes into account .~nequal distribution of transitions and transversions among mutations:

Ks -- -(1/4)In(1-2P-2Q)(1-2P-2R)(1-2Q-2R) In this formula, ~ is the genetic distance between synonymous sites, P is the frequency of sites showing transition-type differences, Q is the frequency of homologous sites occupied by TA, AT, CG or GC pairs, and R is the frequency of homologous sites occupied by TG, GT, CA or AC pairs. The time of divergence is then given by thc fi ,rmula T-

Ks

2~ where l,.sis the mean evolutionary rate. Li and his colleagues z8 calculated ~ for chimpanzee-human comparisons to be equal to 1.2 x 10-9 substitutior~s per site per year. Sakoyama and his colleagues29 obtained a value of ~..56 x 10-9 per site per year for comparisons betv-eer~ Old Wodd monkeys and humans. Using these

two values of ~ we have calculated the age of the HLA alleles. The results of these calculations are given in Table 1 for some of the DRB1 alleles. The youngest allele in Table 1 is 2.7 million years old , indicating that all the alleles in the set are older than H. sapiens. While we are aware of the numerous sources of errors possibly flawing such calculations, not the least significant among which is the large standard deviation of each value, we believe, nevertheless, that the calculations demonstrate one point: the antiquity of the alleles. These calculations suggest that most of the allelic branches of the tree in Fig. 2 separated at least 5-15 million years agog0; the main branches may have separated even earlier, perhaps 15-35 million years ago, some of them at the time when Catarrhines (Old World monkeys, apes and humans) and Platyrrhines (New World monkeys, marmosets, tamarins and capuchinlike monkeys) split off from an ancestral stock and the radiation of modern primates began31. These deductions are supported by direct sequence comparisons among the MHCs of apes, Old World monkeys, and New World monkeys (Z. Zhu, F. Figueroa and J. Klein, unpublished). Similar situations also exist among rodents, in which available evidence indicates that some of the alleles of the house mouse and the brown rat have a common ancestry that goes back more than ten million years 19. Of course, not all the HLA alleles are this old. Once most of the alleles have been sequenced, they will probably form a spectrum of ages, with a few alleles generated after speciation, others predating the emergence of H. sapiens from H. erectus, others still predating the human-chimpanzee split, and a few allelic branches going right back to the time when

WIGJANUS," 1990 VOL.6 NO. 1

m

I'~ERSPECTIVES anthropoid primates began to emerge. The sequence data are still rather incomplete for firm estimates of the numbers of alleles in the different categories to be made. We estimate tentatively that no fewer than 30 HLA-DRB1, 10-20 HLA-DQB1, 20 HLA-A and 40 HLAB alleles predated the emergence of H. sapiens.

The size of the founding population The implications of these findings are obvious. To pass, say, 20 alleles from one species to the next in the evolutionary line, one needs a minimum of ten individuals, each heterozygous at the given MHC locus and each heterozygote different from the other nine. There is no way of passing all the 20 alleles through a single pair of individuals, the founding father and mother of the new species. The notion of there being a single Eve who was the first 'lucky' mother of us all 200 000 years ago can therefore be discarded - unless one wanted to postulate that the single mother mated with nine different males, each heterozygous for different HLA alleles, or some equally preposterous suggestion. It is, however, extremely unlikely that the founding population consisted of a mere ten individuals. The population size must have been much larger than that for the following reasons. First, had a band of only ten individuals separated from the main population of H. erectus to found a new species, it is unlikely that all of them would have been heterozygous for different allelic pairs; if some of the individuals had shared alleles among themselves, the founding population would have had to be larger than ten. Second, the estimate of ten individuals is based on the consideration of a single HLA locus; in reality,

2Or.,'

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,

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2 0 0

i 10000

i i i i i 20000 30000 t, oooo 50000 60000 Number of generotions

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pica Computer simulation of the loss of alleles in a population consisting of 10 000 individuals over 65 000 generations. The following conditions apply. Alleles are initially present at equal frequencies; individuals mate randomly; there is no loss or gain of alleles through migration or mutation; homozygotes have the same probability of mating as heterozygotes; size of the population does not change. These conditions were chosen to minimize allelic losses; most other conditions would have led to an even more rapid disappearance of alleles from the population. This sort of simulation is standard in population genetics and was done here to underscore the fact that even a population of this size cannot retain long-term polymorphisms. The computer program used in the simulation is available on request.

zo

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,

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6 4 2 .................................................. 0 I I 0 0 100t00 20000' 301000 40'000 50000 60'000 70000 Humberof generations

FIGlll Computer simulation of the loss of alleles in a population consisting of 1000 individuals. Here the conditions are the same as those in Fig. 3 except that in three of the four runs, heterozygotes have a higher probability (0.3, 0.2 or 0.1) of mating than do homozygotes. however, there are at least four or five loci that are highly polymorphic and for all of these there is evidence of polymorphisms older than the species. Only if the founding individuals bore different alleles and were heterozygous at all the polymorphic loci would they have endowed the new species with ancestral forms of all the extant alleles. This, however, is not likely to have happened. More realistically, if one individual bore a/b alleles at locus 1 and also a/b alleles at locus 2, another individual may have borne c/d alleles at locus 1 but a/b alleles at locus 2 . Third, a population of ten individuals would be subject to random drift which would normally lead to a loss of most of the polymorphisms. To avoid such a loss, the polymorphism would have to be under very strong selection pressure (only heterozygotes would survive or participate in reproduction). Although evidence for positive selection acting on the polymorphic MHC loci is growin#Z,33, the postulated selection pressure could not have prevented the loss of some alleles by random drift. All of these considerations lead to the conclusion that the size of the founding population of H. sapiens must have been much larger than ten. But how much larger was the population? Figure 3 shows what happens under computersimulated conditions in a population consisting of 10 000 individuals when no selection pressure is acting on the MHC genes, when the individuals mate randomly, and there is no migration or mutation. Under these conditions, most of the 20 alleles initially present in the population at equal frequencies are lost by drift over a period of 10 000 generations - the estimated separation time between H. sapiens and H. erectus. A similar loss occurs even when the initial population contains fewer alleles at higher frequencies. Hence, without positive selection acting on the MHC loci, most of the polymorphism is rapidly lost from a population, even when the population size never drops below 10 000 individuals. The situation changes in the presence of selection pressure, but even here the requirement for a large gene pool remains. Figure 4,

T1GJANUARY1990 VOL.6 NO. 1

m

~I~ERSPECTIVES foi example, simulates events in a population 1000 individuals strong, in which heterozygotes have a greater chance of reproducing than homozygotes. Even with a selective advantage of 0.3, most of the initial 20 alleles are lost from the population during the first 1000 generations. The simulated conditions are of course unrealistic, but the introduction of parameters that would bring the simulations closer to reality would result in an even more rapid elimination of polymorphism from the populations. For example, if our ancestors Eved, as they probably did, in small tribes rather than in large, randomly breeding populations, the effects of random drift on the loss of alleles would have been even more dramatic. We must assume therefore that during the entire evolutionary history of our species from a common ancestor with the chimpanzee, the population size has probably not dropped below 10000 individuals. (Nei and Graur3'i have reached a similar estimate for the long-term effective population size in humans using data on the extent of protein polymorphism.)

Population paleogenetics Similar considerations apply to other species in which extensive I**HC polymorphism has been demonstrated. In these species, too, the concept of founding pairs or founding small groups of individuals must be abandoned. The transition from one species to another has not occurred through small bottlenecks but through populations of considerable size. The study of HLA polymorphism can also be applied to more recent events in the history of the human species, such as the colonization of new lands. Our knowledge of the HLA polymorphism in Australian Aborigines and in American Indians is still too fragmentary to allow realistic estimates of the size of the bands that colonized Australia about 40000 years ago and crossed the Bering strait into Alaska some 15 000 years ago.*S. But here, too, even the limited knowledge we have suggests that these colonizations occurred either in several waves of smaller bands or by a band of considerable size. And once again, it should be possible to make similar inferences in other species as well as far as colonization events are concerned. The MHC polymorphism thus provides a new means of tracing the population history of a species. It provides information that may one day become the basis for the creation of a new discipline - population paleogenetics.

Acknowledgements We thank Ms Lynne Yakes for editorial assistance. Our experimental work cited in this communication was supported in part by grant No. RO1 A123667 from the National Institutes of Health.

5 Andrews, P. (1986) ColdSpring HarborSymp. Quant. Biol. 51,419--428 6 Stringer, C.B. and Andrews, P. (1988) Science 239, 1263-1268 7 Cann, R.L., Stoneking, M. and Wilson, A.C. (1987) Nature 325, 31-36 8 Wainscoat, J. (1987) Nature 325, 13 9 Cann, R.L. (1987) The Sciences, Sept./Oct., 30-37 10 Pamilo, P. and Nei, M. (1988) Mol. Biol. Evol. 5, 568-583 11 Excoffier, L. and Langaney, A. (1989) Am.J. Hum. Genet. 44, 73-435 12 Klein, J. (1986) Natural History of the Major Histocompatibtlity Complex, John Wiley & Sons 13 Dupont, B. (ed.) (1989) Immunobiology ofHLA, Vols 1 and 2, Springer-Veflag 14 Klein, J. (1989) lmmunolop~ 2 (suppl.), 36-39 15 Klein, J. (1980) in Immunology 80 (Fougereau, M. and Dausset, J., eds), pp. 239-253, Academic Press 16 Arden, B. and Klein, J. (1982) Proc. NatlAcad. Sci. USA 79, 2342-2346 17 Mayer, W.E. et al. (1988) EMBOJ. 7, 2765-2774 18 McConnell, T.J., Talbot, W.S., Mclndoe, R.A. and Wakeland, E.K. (1988) Nature 332, 651-654 19 Figueroa, F., Gtinther, E. and Klein, J. (1988) Nature 335, 265-267 20 Lawlor, D.A. et al. (1988) Nature 335, 268-271 21 Fan, W. et al. (1989) Hum. Immunol. 26, 107-121 22 Kimum, M. (1983) The Neutral Theory of Molecular Evolution, Cambridge University Press 23 Nei, M and l~oychoudhury, A.K. (1982) Evol. Biol. 14, 1-59 24 Nei, M. (1985) in Population Genetics and Molecular Evolution (Ohta, T. and Aoki, K., eds), Japan sci. Soc. Press (Springer-Verlag) 25 Gillespie, J.H. (1987) Oxford Sum. Evol. Biol. 4, 10-37 26 Nei, M. (1987) Molecular Evolutionary Genetics, Columbia University Press 27 Kimura, M. (1981) Proc. Natl Acad. Sci. USA 78, 454--458 28 Li, W-H., Tanimura, M. and Sharp, P.M. (1987) J. Mol. Evol. 25, 330-342 29 Sakoyama, Y. et al. (1987) Proc. Natl Acad. Sci. USA 84, 1080-1084 30 Klein, J. et al. Progr. A l l e ~ (in press) 31 Ciochon, R.L and Fleagle, J.G. (eds) (1987) Primate Evolution and Human Origins, Aldine de Gruyter 32 Hughes, A.L. and Nei, M. (1988) Nature 335, 167-170 33 Hughes, A.L. and Nei, M. (1989) Proc. NatlAcad. Sci. USA 86, 958-962 34 Nei, M. and Graur, D. (1984) Evol. Biol. 17, 73-118 35 Mascie-Taylor, C.G.N. and Lasker, G.W. (eds) (1988) Biological Aspects of Human Migration, Cambridge University Press 36 Saitou, N. and Nei, M. (1987) Mol. BioL Evol. 4, 406-425 37 Klein, J. el al. (1990) in Current Concepts in lmmunogenetics (Srivastava, R., ed.), Verlag Chemie

References

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1 Wilson, A.C. and Sarich, V.M. (1969) Proc. NatlAcad. Sci. USA 63, 1088-1093 2 Sibley, C.G. and Ahlquist, J.E. (1984) J. Mol. Evol. 20, 2-15 3 Lewin, R. (1989) Human Evolution. An Illustrated Introduction (2nd Edn), Blackwell Scientific Publications 4 Leakey, R.E. and Lewin, R. (1977) Origins, E.P. Dutton

17400 Tg~INGEN, FRG; N. FISCHERIS IN THEEBERHARDKARLS

~

BIOtOGI~ ABTEILf)NG IMMUNGEN~TIK, CORRENSSTg 42,

UNIVERSIT~T T~BINGEN, INSTilI3T F~}R BIOLOGIE ~, AUF DER MORGENST£LL~ 7400 Tf)BINGEN, FRG,'J. KLEIN IS ALSO 1N THE DEPARTMENT OF MICROBIOLOGYAND IMMUNOLOGY, UNIVERSITY OF MIAMI SCHOOL OF MEDICINE, P O BOX 016960, MIAMI,

FL 33101, USA.

TIGJANUARY1990 rOE. 6 r~o. 1

m