- Email: [email protected]
Neutral theory of molecular evolution
767
Neutral theory of molecular evolution Naoyuki Takahata DNA sequence Kimura’s
neutral
data are generally theory
with a great deal of controversy clocks,
DNA polymorphism,
genealogy.
Although remain
mutation,
unsettled.
about
and gene
recombination,
Of particular
need for more knowledge
to molecular
as a guiding
principle, and
importance
the function
is the
and structure
of
molecules.
Addresses The Graduate University for Advanced Studies, Hayama, Kanagawa 240-01,
Japan; e-mail: [email protected]
Current
Opinion
0 Current
Biology
in Genetics Ltd ISSN
& Development
1996,
6:767-772
0959-437X
Abbreviations most recent
common
ancestor
MRCA PBR PIV
peptide-binding region primate immunodeficiency
SI
self-incompatibillty
virus
Introduction The neutral theory posits that the great majority of evolutionary change at the molecular level is caused not by Darwinian selection but by the random fixation of selectively neutral mutations [l]. h,Iany people, including Darwin himself, have thought independently of the same idea but it was not until Kimura’s forcible advocacy [l] and his methodical presentation of one argument after another in successive papers (for review see [2]) that the idea made a major impact on evolutionary thinking. The proposal of the neutral theory and the subsequent pros and cons of this hypothesis were indispensable fuel in the propulsion of population genetics in rhe 1970s and promoted the establishment of molecular evoluGon and molecular taxonomy. Kimura favored the method of diffusion approximations-a mathematical means of describing gene frequency changes caused by stochastic evolutionary forces, such as random genetic drift-and applied it to significant problems concerning gene
frequency
and
amino
acid
sequence
data.
clock
Under the terms of the theory of neutrality, the substitution rate of molecules equals the neutral mutation race. Conversely, if the neutral mutation rate is constant
and often
evolution,
serves
Molecular
as favouring
dissent
with respect
adaptive
the theory
many issues concerning selection
interpreted
but not without
The
nature of both methodology and data in such analyses has recently changed dramatically; the neutral theory has been reformulated and many statistical tesrs have been devised by the method of gene genealogy, rhe family relationships among genes. In addition, a wealth of DNA sequence dara, sometimes together with detailed information about: the function and structure of molecules, has become available. These advances have permitted us to look at the neutral theory from much deeper viewpoints than ever before. In this review, I assess the current scatus of the neutral theory.
among different lineages, the substitution rate must also be constant. In fact, the rate of molecular evolution is remarkably constant compared with the rate of morphological evolution [3] and this constancy (molecular clock) was regarded as evidence for neutrality [Z]. It has become increasingly clear, however, that either the amino acid or nucleotide substitution rate varies among different taxonomic groups and not to a small extent [4]. It is conceivable that the mutation rate per se can differ among different taxonomic groups, presumably because of differences in molecular devices for DNA replication, DNA damage, and proof-reading. Haldane suggested high mutability in the male to be a general property of human and perhaps other vertebrate genes but he added that “it is difficult to see how this could be proved or disproved for many years to come” [5]. Indeed, it took 40 years for Haldane’s perceptive suggestion to become resurrected as a hypothesis of male-driven molecular evolution [6]. Now, several DNA sequences of X- and Y-linked homologues are available and their analysis has confirmed the validity of his hypothesis. The male/female ratio of nucleotide substitution rate is estimated as -6 and 2 in human and rodents, respectively [7’,8]. As each ratio is close to the ratio of the number of male/female germ-cell divisions per generation, nucleotide substitutions may be largely replication-dependent. It is argued, however, that body temperature [9] and metabolic rate [lo’] must have some bearing on the substitution rate. If high body temperature-as in birds and mammals-imposes functional constraints [4,9], it would lower the proportion of neutral mutation and result in a slow substitution rate. On the other hand, if a high metabolic rate tends to accumulate oxygen radicals, damage to DNA followed by error-prone repair may increase the rate of nucleotide substitution [lo’]. Actually, there is a counter example: the domestic chicken has low rates of nucleotide substitution relative to mammals, despite similarly high specific metabolic rates [ll’]. As a strong selective constraint in high body temperature and high mutation rate with high metabolic rate counteract each other, their effects on the rate of nucleotide substitution may have been cancelled out, if not exactly. Of greater controversy is the issue of whether molecules evolve at a constant rate over single years or generations. The observed rate constancy is apparent in units of years [3,4] rather than in units of generations. Nevertheless,
766
Genomes and evolution
the per-year
rate is faster for those
substitution
with a short generation generation time [12,13*]. [14] that the mutation
time Kimura
than those has therefore
rate per year should
organisms of a long suggested
be higher
for
polymorphism
in a selectively
rate of recombination positive or negative
neutral
region
with
is therefore an indicator selection at linked loci,
a low
of either although
short-lived than for long-lived organisms (the ‘generation time’ effect). There is indeed the tendency that the
different modes of selection result in distinguishable allele frequency spectra [Zl’]. Another line of study has been concerned with a correlation between intraspecific DNA
shorter the generation time, the larger the number of the male germ-cell divisions per year. In mice and rats, this
polymorphism and interspecific correlation must be positive
number is -100 and in humans accord with the observed faster
rate remains constant in time. Alternatively, one may test whether or not the ratio of DNA polymorphism to sequence divergence is the same in regions differing in the degree of selective constraint or the neutral mutation rate [ZZ]. This test was applied to several loci of D. melanogaster and its sibling species. Although the ratio varies from locus to locus and a departure from neutrality is suggested, it is not clear which specific loci are responsible for it. The number of loci available at present is rather limited in Drosophda species-24 loci for D. meianogaster and 12 loci for D. Gnu/am [23*] - therefore it is still difficult to find a neutral locus to use as a standard in this kind of test.
it is -10 [8], which is in rate of silent nucleotide
substitution in rodents than in humans. As for amino acid substitution, the generation time effect is less apparent and rate constancy is still controversial. To examine the accuracy of the molecular clock, Kimura also studied the mean and variance of the number of amino acid substitutions [14]. The variance/mean ratio is expected to be 1 under a simple (Poisson) substitution model; the estimated ratio is >1 for some genes. Kimura regarded this observation as insignificant, whereas Gillespie [15] pointed out that the ratio had been grossly underestimated and the actual molecular clock may be an episodic clock, with short bursts of rapid evolution followed by long periods of slow evolution. Subsequently, several other models were proposed to account for the estimated ratio [16’] but it was pointed out that the previously suggested means and distributions of the ratio estimators were highly inaccurate, particularly under a non-star phylogeny and may not be suitable to test the molecular clock hypothesis [17]. This result is in direct opposition to the previous analysis which led to the concept of the episodic clock.
DNA polymorphism Under neutrality, the extent of DNA polymorphism maintained in a population is proportional to the product of the effective population size and the neutral mutation rate per generation. The larger the size and the less the selective constraint, the higher the DNA polymorphism. As the effective population size reflects the demographic history of an organism, DNA polymorphism provides a means for inferring the history. Furthermore, as DNA polymorphism can be measured separately in various genomic
regions,
study the neutral the relative roles DNA sequences.
such
as coding
and noncoding,
one can
theory rigorously and directly examine of various evolutionary forces acting on
One line of study has focussed on a positive correlation between the levels of DNA polymorphism and recombination rate in Drosoph/a mdanogaster [18”]. This correlation is expected because DNA polymorphism in a region is affected by natural selection operating at linked loci and recombination diminishes this linkage effect. Advantageous mutations at linked loci sweep out the polymorphism in a neutral region on their way to fixation [19*] and deleterious mutations also reduce the polymorphism on their way to elimination [ZO’]. Low
sequence divergence; the if the neutral mutation
A species that has been examined more extensively for DNA polymorphism than Drosophila is the human. For the human, -65 loci are available for intraspecific comparison, including five pseudogenes, a few X- or Y-linked loci, and eight extremely polymorphic major histocompatibility complex loci Mzc; some of these can be compared with those of non-human primates. It has been long known from electrophoretic surveys of proteins that the human is the most polymorphic of the primates [24,25], and perhaps even among vertebrates [26]. In contrast, the human is less polymorphic than the chimpanzee at the DNA level [27]. The discordance between human protein and DNA polymorphisms is puzzling (281 but there is no strong evidence that human genetic data other than .lZhc are incompatible with neutrality. LJnder simple models of neutrality, it is possible to convert DNA polymorphism to the effective size of the extant population and separately to estimate, from sequence divergence, the species divergence time and the effective size of the common ancestral population [29-l. The estimated size of the human population is -10000 for the past one million years and -100000 before human and chimpanzee divergence in evolution 4.6 million years ago [30*]. Since the proposal of the replacement hypothesis on the basis of mitochondrial DNA ([31]; see also review by Stoneking and Soodyall, this issue, pp 731-736), the origin of modern humans has been debated extensively in light of new genetic data and the neutral theory [32”].
Adaptive evolution The neutral theory does not exclude the existence of positively selected mutations but assumes that these have played no major roles in molecular evolution. Within the past year, many authors have examined this assumption largely on the basis of comparisons between synonymous and nonsynonymous substitutions. Evidence for adaptive evolution has been obtained in HIV [33’,34”], PIV
Neutral theory of molecular evolution Takahata
[35*],
the
proB
gene
of
Neisseria gonovhoeae
and
N.
of pairwise
differences.
Although
this kind of study,
769
based
menirrgitidis [36’], the gamete recognition protein [37’], al-proceinase inhibitors in mammals [38*], the type I interferon gene family [39’], and so on (see [40’] for other instances). The most convincing evidence has come from self-incompatibility (SI) S loci in flowering plants and Alhc loci in vertebrates. Each of these loci usually exhibits extraordinarily high levels of allelic diversity, even
on a population genetics model, can be applied to iVhc sequence data (Y Satta, personal communication) and can be used to argue against the contribution of recombination to DRB allelic diversity [47’], more direct evidence is also welcome. Zangenberg eta/. [48”] have therefore analyzed
distributions of allele frequency, and long persistence of allelic lineages-all of which are incompatible with neu-
estimate of such events is about one out of 10000 sperm. It appears that recombination occurs more frequently at
trality and suggest the operation of some form of balancing selection. These two examples are convincing because not only is the departure from neutrality statistically significant but it is also directly related to the function of the gene product. Kinase or ribonuclease activity has been implicated in the S locus product in two major groups [41”]. The expression of S results in strong disassortative mating and therefore maintains a large number of alleles for a long time. Likewise, peptide presentation to T cells is a primary role of Mhc molecules and this function is thought to be a direct target of natural selection (see [42*] for review]).
DPB than at DRB and has contributed to allelic diversity. If this is the case, however, it is clear that additional evolutionary forces must have played important roles in the diversification of >lOO DRB alleles; high recombination rates do not appear to be always positively associated with high allelic diversity at Mhc loci.
Classical diallelic crosses have identified >lOO S alleles in Brassicaceae and a molecular approach has confirmed an enormously high diversity [43*]. Richman et nl. [44’] have reported the first direct determination through the polymerase chain reaction procedure of S locus genotypes of plants sampled from two natural populations of Solaaum carolinerue and, in a different study, Richman et al. [45**] have carried out a similar study for Phwalis crassifoiia. Although the estimated number of S alleles is not enormously large in these populations (13-15 for S. calaliwense and 4344 for J! uassifolia), long persistence of allelic lineages is apparent in both cases. By using a generalized least-squares method for estimating the rate and time of S allele divergences, Uyenoyama [46’] has demonstrated that the high levels of sequence diversicy in the sporophytic SI system of Brassica reflect ancient allelic divergence.
variant DPB sequences which appear to have arisen by gene conversion in the sperm of a heterozygous male. The
Gene genealogy
and the coalescent
With the accumulation of sequenced DNA and the reconstruction of ancestral gene relationships, the neutral theory has been reformulated in terms of gene genealogy. When a gene is multiplied more than once and the products are transmitted by luck to later generations, such a multiplication appears in a diagram of genealogy as a coalescence when looked ac back in time. To reconstruct gene genealogy, it is necessary to determine the timing of coalescence or the number of distinct ancestral genes at a given time in the past. Coalescence theory was developed originally to describe the family relationships of neutral genes sampled from a single panmictic (randomly mating) equilibrium population but the theory was soon extended to orthologous genes sampled from different species. Coalescence theory has not only reproduced the main results of neutral theory in a much simpler way than the method of diffusion approximations but has also shown wide applicability to DN.4 sequence data. A number of theoretical recently. A coalescence
advancements have been made theory has been developed for
neutral genes sampled with a deterministically
from a population that varying size [49] and
evolves from a
Although not all human ,Ifhr sequences deposited in GenBank necessarily differ at the amino acid level in the peptide-binding region (PBR), there are 67, 149, 39, 179, 29, and 69 alleles at the class I d, B, C,
population experiencing a recent colonization event [SO’]. Likewise, the coalescence process of two genes has been formulated in a non-equilibrium population consisting of demes under extinction and recolonization and has been
class II DRB, DQB, and DPB loci, respectively (42.1. Balancing selection is effective in enhancing genetic diversity but it alone may not easily explain such large numbers of alleles unless one assumes that the effective population size is unrealistically large or that the mutation rate is exceptionally high. An exploration was therefore made of the role of recombination (Y Satta, personal communication). Intra-exonic recombination in the PBR can increase the number of alleles substantially without changing the nucleotide substitution rate enhanced by balancing selection. Frequent intra-exonic recombination may decrease the mean number of nucleotide differences between alleles and change the shape of the distribution
used to explore various demographic situations that might have occurred in the lineage leading to modern humans [30-l. The first theory for a population with varying size has been used to study gene genealogy within an allelic class that arose by a unique mutation at some time in the past [Sl’]. Furthermore, the variance formulae of pairwise nucleotide differences of genes have been obtained for two partially isolated populations [S2*] and the greater variance expected under migration relative to recent isolation has been used to distinguish between the two situations [53*]. Coalescence approaches have been also used in other problems in population genetics, such as those concerning microsatellite allele frequencies
770
Genomes and evolution
in subdivided populations repetitive elements under
[.54] and short the ‘master copy’
interspersed model [SS].
As for human evolution, many authors have addressed the age of the most recent common ancestor (hIRC.4) on the basis of nucleotide differences in a sample of genes, including mtDNA sequences [56], ALU sequences inserted in the Y chromosome [57”], and available nuclear DNA sequences [29’]. Although these studies have consistently implicated recent origins for human polymorphisms, there is controversy over the estimate made from the absence of variation in 38 human ZFY genes [W]; the original estimate is too rough and a more rigorous estimate has been obtained on the basis of joint probability distribution of the number of segregating sites and the age of the hlRCA [59’]. The genealogical behaviour of neutral genes and the superposition of nucleotide substitutions on the genealogical tree are now well investigated in a variety of situations. Finally, one complication connected with the neutral theory is worth coverage. Kimura was once sympathetic to the ‘slightly deleterious’ hypothesis of Ohta when criticisms against the neutral theory were most intense and he himself proposed the ‘effectively neutral’ model in 1986 [Z]. Nevertheless, in his last few years, Kimura abandoned this ad/zoc model and returned to the original neutral theory. It was at this time that Ohta renamed her previously proposed hypothesis as ‘nearly neutral’. Although ‘slightly deleterious’ and ‘nearly neutral’ sound similar, there is an important difference: under a model of near-neutrality, one half of the mutations are implicitly assumed to be advantageous [16*,60*]. Needless to say, the concept of near-neutrality is no longer the same as that of Kimura’s neutrality.
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*
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within the annual period of review,
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Conclusions Because of its simplicity and value as a basis for prediction, Kimura’s neutral theory has been tested extensively. Whether the theory is accepted or rejected by particular tests, it has without question served as a fundamental guiding principle in the study of molecular evolution. Although there is agreement about the importance of random genetic drift in molecular evolution, there is obvious disagreement about the relative roles of other especially natural selection. It is evolutionary forces, important to keep in mind that convincing arguments for the theory of S and hlhc molecule evolution have been possible only by knowledge, albeit imperfect, of their function and structure which could be related directly to the causes
of natural
selecrion.
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Wolinsky SM, Korber BTM, Neumann AU, Daniels M, Kunstman KJ, Whetsell Al, Furtado MR, Cao Y, Ho DD, Safrit JT, Koup RA: Adaptive evolution of human immunodeficiencv virus tvoe1 dking the natural course of infection. Science 1996,” 272537-547. This paper examines the hypothesis that the loss of CD4 T cells in HIV-1 infected individuals is primarily caused by increasing antigenic diversity [35-I. No such evidence is found, but adaptive evolution of virus populations under selective constraints is suggested. 34.
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Kreitman M, Akashi H: Molecular evidence for natural selection. Annu Rev Ecol Syst 1995, 26:403-432. ;he authors argue that no current theories can account for all aspects of molecular evolution, and calls for a comprehensive theory. This conclusion is based largely on statistical tests of Drosophila genetic data. To demonstrate natural selection at the molecular level convincingly, however, we need to integrate into the framework of population genetics more information about the function and structure of molecules. 41.
Dodds PN, Clarke AE, Newbigin E: A molecular perspective on pollination in flowering plants. Cell 1996, 85:141-l 44. Ee molecular basis of self-incompatibility in Brassicaceae and Solanaceae are concisely reviewed here, concluding that there is a need to identify factors controlling pollen phenotype and the nature of the interactlons between stylar and pollen components. 42. Parham P, Ohta T: Population biology of antigen presentation . by MHC class I molecules. Science 1996, 272:67-74. A review of both experimental and theoretical population genetics studies of extraordinarily high diversity at the MHC. A significant, though controversial, role of interallelic recombination and conversion is emphasized in the process of generating class I MHC diversity. The contention that many South Amerindian 5 alleles derive from founding alleles by interallelic conversion may be regarded as tentative until a thorough survey is made of Asian populations. 43. .
Hinata K, Watanabe M, Yamakawa S, Satta Y, lsogai A: Evolutionary aspects of the S-related genes of the brassica self-incompatibility system: synonymous and nonsynonymous base substitutions. Genetics 1995, 140:1099-l 104.
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Analysis of all available DNA sequences for SRK (S-receptor kinase), SLG (S-locus glycoprotein), and two S-related genes (SRA and SRB) show the order of gene duplication and early SLG allelic divergences. Richman AD, Kao TH, Schaeffer SW, Uyenoyama MK: Sallele sequence diversity in natural populations of Solanum carolineme (Horsenettle). Heredity 1995, 75:405-415. Despite a rather small number of S alleles maintained in two natural populations, numerous sequence differences are found, presumably reflecting trans.species maintenance of polymorphism. 44. .
Richman AD, Uyenoyama MK, Kohn JR: Contrasting patterns of allelic diversity and gene genealogy at the self-incompatibility locus in two species of Solanaceae. Science 1996, 273:1212-1216. 12 and 26 alleles recovered from Solanum caroolinense and Physalis crassifolia populations are used to infer their demographic histories and modes of speciation. 45. ..
Uyenoyama MK: A generalized statistical estimate for the origin of sporophytic self-incompatibility. Genetics 1995, 139:975-992. Amino acid sequences of the proteins encoded by SLG and related genes were analyzed by a newly developed least-squares method to infer the age of the SI system in Brassica and the trans-specific evolution of S alleles.
52. .
Wakeley J: The variance of pairwise nucleotide differences in two populations with migration. Theor Pop/ Viol 1996, 49:39-57. The variance formulae of nucleotide differences between genes are obtained in this study when two equilibrium populations exchange genes. Migration results in large standard errors on estimates of population genetic parameters, on the basis of which the author makes an interesting suggestion to distinguish between alternative demographic situations. 53. .
Wakeley J: Distinguishing migration from isolation using the variance of pairwise differences. Theor Pop/ Biol 1996, 49:369-366. Ongoing gene flow between populations and a recent population split have similar -but not exactly the same - effects on gene genealogy. This paper suggests one way to distinguish between these two situations. The test statistic developed here is applied to mitochondrial stickleback DNA sequence data and suggests ongoing migration. 54.
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Tachida H: A population genetic study of the evolution of SINES. II. Sequence evolution under the master copy model. Genetics 1996, 143:1033-l 042.
56.
Horai S, Hayasaka K, Kondo R, Tsugane K, Takahata T: Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc Nat/ Acad SC; USA 1995, 92:532-536.
46. .
Klein J, O’hUigin C: Class II B Mhc motifs in an evolutionary perspective. lmmunol Rev 1995, 1143:69-l 11. ;wo possible explanations for the sharing of sequences motifs by different species - recombination and convergence - are discussed and forceful arguments for the latter are presented. The recurrent occurrence and/or long persistence of particular motifs in the peptide-binding region may result from the specific association between host and parasite.
47.
Zangenberg G, Huang MM, Arnheim N, Erlich H: New HLA-DPBI alleles generated by interallelic gene conversion detected by analysis of sperm. Nat Genet 1995, 10:407-414. This paper presents the first direct measurements of the rate of new variant LIP57 sequences generated by gene conversion between two parental alleles. The estimated conversion tract is 54-l 32 bp. 48. ..
49.
Griffiths RC, Tavare S: Sampling theory for neutral alleles in a varying environment. Phi/ Trans R Sot Land 5 1994, 344:403-410.
Austerlitz F, Jung-Muller B, Godelle B, Gouyon PH: Evolution of coalescence times, genetic diversity and structure during colonization. Theor Popul Biol 1996, in press. Under a non-equilibrium model of geographically arranged colonies, the authors analyze mean coalescence times and two other related quantities. A recent colonization process, which is common in temperate forest trees, is the main focus. 50. .
Slatkin M: Gene genealogy within mutant allelic classes. Genetics 1996, 143:579-587. 1 description of the genealogical process within an allelic class which arose by a unique mutational event is developed on the basis of the coalescence theory for a population of varying size [491. 51.
subdivision based Genetics 1996,
57. Hammer MF: A recent common ancestry for human Y .. chromosomes. Nature 1995, 378376-378. Interesting sequence analysis on a polymorphic A/u element which was inserted into the human Y chromosome after the divergence of humans and chimpanzees. This paper also summarizes the extent of DNA polymorphism and sequence divergences at various loci, providing no evidence for recent selective sweeps in the human population. Dorit RL, Akashi H, Gilbert W: Absence of polymorphism at the ZFY locus on the human Y chromosome. Science 1995, 268:1163-l 185. In a world-wide sample of 36 human males, no variation in a 729 bp intron of the ZFY gene is found. As for the estimate of the age of the MRCA, several comments were made recently [59’1. 56. .
Fu YX, Li WH: Estimating the age of the common ancestor of men from the ZFY intron. Science 1996, 272:1356-l 357. ;he posterior probability of the age of the MRCA is derived, given that there is no variation in the sample, a suggestion of an age compatible with other estimates. 59.
Gillespie JH: On Ohta’s hypothesis: most amino acid substitutions are deleterious. I MO/ Evol 1995, 40:64-69. ;\ model of near-neutrality is shown here to be incompatible with most
of the
major
by ex-
60.
features
clusively the slightly developing
of protein
deleterious deleterious the
evolution
mutations. theory
near-neutral
but
This who theory.
not
with
is an ironic
subsequently
a model paper
from
abandoned
of evolution the
proponent
it [16’],
recently
of
Neutral theory of molecular evolution Naoyuki Takahata DNA sequence Kimura’s
neutral
data are generally theory
with a great deal of controversy clocks,
DNA polymorphism,
genealogy.
Although remain
mutation,
unsettled.
about
and gene
recombination,
Of particular
need for more knowledge
to molecular
as a guiding
principle, and
importance
the function
is the
and structure
of
molecules.
Addresses The Graduate University for Advanced Studies, Hayama, Kanagawa 240-01,
Japan; e-mail: [email protected]
Current
Opinion
0 Current
Biology
in Genetics Ltd ISSN
& Development
1996,
6:767-772
0959-437X
Abbreviations most recent
common
ancestor
MRCA PBR PIV
peptide-binding region primate immunodeficiency
SI
self-incompatibillty
virus
Introduction The neutral theory posits that the great majority of evolutionary change at the molecular level is caused not by Darwinian selection but by the random fixation of selectively neutral mutations [l]. h,Iany people, including Darwin himself, have thought independently of the same idea but it was not until Kimura’s forcible advocacy [l] and his methodical presentation of one argument after another in successive papers (for review see [2]) that the idea made a major impact on evolutionary thinking. The proposal of the neutral theory and the subsequent pros and cons of this hypothesis were indispensable fuel in the propulsion of population genetics in rhe 1970s and promoted the establishment of molecular evoluGon and molecular taxonomy. Kimura favored the method of diffusion approximations-a mathematical means of describing gene frequency changes caused by stochastic evolutionary forces, such as random genetic drift-and applied it to significant problems concerning gene
frequency
and
amino
acid
sequence
data.
clock
Under the terms of the theory of neutrality, the substitution rate of molecules equals the neutral mutation race. Conversely, if the neutral mutation rate is constant
and often
evolution,
serves
Molecular
as favouring
dissent
with respect
adaptive
the theory
many issues concerning selection
interpreted
but not without
The
nature of both methodology and data in such analyses has recently changed dramatically; the neutral theory has been reformulated and many statistical tesrs have been devised by the method of gene genealogy, rhe family relationships among genes. In addition, a wealth of DNA sequence dara, sometimes together with detailed information about: the function and structure of molecules, has become available. These advances have permitted us to look at the neutral theory from much deeper viewpoints than ever before. In this review, I assess the current scatus of the neutral theory.
among different lineages, the substitution rate must also be constant. In fact, the rate of molecular evolution is remarkably constant compared with the rate of morphological evolution [3] and this constancy (molecular clock) was regarded as evidence for neutrality [Z]. It has become increasingly clear, however, that either the amino acid or nucleotide substitution rate varies among different taxonomic groups and not to a small extent [4]. It is conceivable that the mutation rate per se can differ among different taxonomic groups, presumably because of differences in molecular devices for DNA replication, DNA damage, and proof-reading. Haldane suggested high mutability in the male to be a general property of human and perhaps other vertebrate genes but he added that “it is difficult to see how this could be proved or disproved for many years to come” [5]. Indeed, it took 40 years for Haldane’s perceptive suggestion to become resurrected as a hypothesis of male-driven molecular evolution [6]. Now, several DNA sequences of X- and Y-linked homologues are available and their analysis has confirmed the validity of his hypothesis. The male/female ratio of nucleotide substitution rate is estimated as -6 and 2 in human and rodents, respectively [7’,8]. As each ratio is close to the ratio of the number of male/female germ-cell divisions per generation, nucleotide substitutions may be largely replication-dependent. It is argued, however, that body temperature [9] and metabolic rate [lo’] must have some bearing on the substitution rate. If high body temperature-as in birds and mammals-imposes functional constraints [4,9], it would lower the proportion of neutral mutation and result in a slow substitution rate. On the other hand, if a high metabolic rate tends to accumulate oxygen radicals, damage to DNA followed by error-prone repair may increase the rate of nucleotide substitution [lo’]. Actually, there is a counter example: the domestic chicken has low rates of nucleotide substitution relative to mammals, despite similarly high specific metabolic rates [ll’]. As a strong selective constraint in high body temperature and high mutation rate with high metabolic rate counteract each other, their effects on the rate of nucleotide substitution may have been cancelled out, if not exactly. Of greater controversy is the issue of whether molecules evolve at a constant rate over single years or generations. The observed rate constancy is apparent in units of years [3,4] rather than in units of generations. Nevertheless,
766
Genomes and evolution
the per-year
rate is faster for those
substitution
with a short generation generation time [12,13*]. [14] that the mutation
time Kimura
than those has therefore
rate per year should
organisms of a long suggested
be higher
for
polymorphism
in a selectively
rate of recombination positive or negative
neutral
region
with
is therefore an indicator selection at linked loci,
a low
of either although
short-lived than for long-lived organisms (the ‘generation time’ effect). There is indeed the tendency that the
different modes of selection result in distinguishable allele frequency spectra [Zl’]. Another line of study has been concerned with a correlation between intraspecific DNA
shorter the generation time, the larger the number of the male germ-cell divisions per year. In mice and rats, this
polymorphism and interspecific correlation must be positive
number is -100 and in humans accord with the observed faster
rate remains constant in time. Alternatively, one may test whether or not the ratio of DNA polymorphism to sequence divergence is the same in regions differing in the degree of selective constraint or the neutral mutation rate [ZZ]. This test was applied to several loci of D. melanogaster and its sibling species. Although the ratio varies from locus to locus and a departure from neutrality is suggested, it is not clear which specific loci are responsible for it. The number of loci available at present is rather limited in Drosophda species-24 loci for D. meianogaster and 12 loci for D. Gnu/am [23*] - therefore it is still difficult to find a neutral locus to use as a standard in this kind of test.
it is -10 [8], which is in rate of silent nucleotide
substitution in rodents than in humans. As for amino acid substitution, the generation time effect is less apparent and rate constancy is still controversial. To examine the accuracy of the molecular clock, Kimura also studied the mean and variance of the number of amino acid substitutions [14]. The variance/mean ratio is expected to be 1 under a simple (Poisson) substitution model; the estimated ratio is >1 for some genes. Kimura regarded this observation as insignificant, whereas Gillespie [15] pointed out that the ratio had been grossly underestimated and the actual molecular clock may be an episodic clock, with short bursts of rapid evolution followed by long periods of slow evolution. Subsequently, several other models were proposed to account for the estimated ratio [16’] but it was pointed out that the previously suggested means and distributions of the ratio estimators were highly inaccurate, particularly under a non-star phylogeny and may not be suitable to test the molecular clock hypothesis [17]. This result is in direct opposition to the previous analysis which led to the concept of the episodic clock.
DNA polymorphism Under neutrality, the extent of DNA polymorphism maintained in a population is proportional to the product of the effective population size and the neutral mutation rate per generation. The larger the size and the less the selective constraint, the higher the DNA polymorphism. As the effective population size reflects the demographic history of an organism, DNA polymorphism provides a means for inferring the history. Furthermore, as DNA polymorphism can be measured separately in various genomic
regions,
study the neutral the relative roles DNA sequences.
such
as coding
and noncoding,
one can
theory rigorously and directly examine of various evolutionary forces acting on
One line of study has focussed on a positive correlation between the levels of DNA polymorphism and recombination rate in Drosoph/a mdanogaster [18”]. This correlation is expected because DNA polymorphism in a region is affected by natural selection operating at linked loci and recombination diminishes this linkage effect. Advantageous mutations at linked loci sweep out the polymorphism in a neutral region on their way to fixation [19*] and deleterious mutations also reduce the polymorphism on their way to elimination [ZO’]. Low
sequence divergence; the if the neutral mutation
A species that has been examined more extensively for DNA polymorphism than Drosophila is the human. For the human, -65 loci are available for intraspecific comparison, including five pseudogenes, a few X- or Y-linked loci, and eight extremely polymorphic major histocompatibility complex loci Mzc; some of these can be compared with those of non-human primates. It has been long known from electrophoretic surveys of proteins that the human is the most polymorphic of the primates [24,25], and perhaps even among vertebrates [26]. In contrast, the human is less polymorphic than the chimpanzee at the DNA level [27]. The discordance between human protein and DNA polymorphisms is puzzling (281 but there is no strong evidence that human genetic data other than .lZhc are incompatible with neutrality. LJnder simple models of neutrality, it is possible to convert DNA polymorphism to the effective size of the extant population and separately to estimate, from sequence divergence, the species divergence time and the effective size of the common ancestral population [29-l. The estimated size of the human population is -10000 for the past one million years and -100000 before human and chimpanzee divergence in evolution 4.6 million years ago [30*]. Since the proposal of the replacement hypothesis on the basis of mitochondrial DNA ([31]; see also review by Stoneking and Soodyall, this issue, pp 731-736), the origin of modern humans has been debated extensively in light of new genetic data and the neutral theory [32”].
Adaptive evolution The neutral theory does not exclude the existence of positively selected mutations but assumes that these have played no major roles in molecular evolution. Within the past year, many authors have examined this assumption largely on the basis of comparisons between synonymous and nonsynonymous substitutions. Evidence for adaptive evolution has been obtained in HIV [33’,34”], PIV
Neutral theory of molecular evolution Takahata
[35*],
the
proB
gene
of
Neisseria gonovhoeae
and
N.
of pairwise
differences.
Although
this kind of study,
769
based
menirrgitidis [36’], the gamete recognition protein [37’], al-proceinase inhibitors in mammals [38*], the type I interferon gene family [39’], and so on (see [40’] for other instances). The most convincing evidence has come from self-incompatibility (SI) S loci in flowering plants and Alhc loci in vertebrates. Each of these loci usually exhibits extraordinarily high levels of allelic diversity, even
on a population genetics model, can be applied to iVhc sequence data (Y Satta, personal communication) and can be used to argue against the contribution of recombination to DRB allelic diversity [47’], more direct evidence is also welcome. Zangenberg eta/. [48”] have therefore analyzed
distributions of allele frequency, and long persistence of allelic lineages-all of which are incompatible with neu-
estimate of such events is about one out of 10000 sperm. It appears that recombination occurs more frequently at
trality and suggest the operation of some form of balancing selection. These two examples are convincing because not only is the departure from neutrality statistically significant but it is also directly related to the function of the gene product. Kinase or ribonuclease activity has been implicated in the S locus product in two major groups [41”]. The expression of S results in strong disassortative mating and therefore maintains a large number of alleles for a long time. Likewise, peptide presentation to T cells is a primary role of Mhc molecules and this function is thought to be a direct target of natural selection (see [42*] for review]).
DPB than at DRB and has contributed to allelic diversity. If this is the case, however, it is clear that additional evolutionary forces must have played important roles in the diversification of >lOO DRB alleles; high recombination rates do not appear to be always positively associated with high allelic diversity at Mhc loci.
Classical diallelic crosses have identified >lOO S alleles in Brassicaceae and a molecular approach has confirmed an enormously high diversity [43*]. Richman et nl. [44’] have reported the first direct determination through the polymerase chain reaction procedure of S locus genotypes of plants sampled from two natural populations of Solaaum carolinerue and, in a different study, Richman et al. [45**] have carried out a similar study for Phwalis crassifoiia. Although the estimated number of S alleles is not enormously large in these populations (13-15 for S. calaliwense and 4344 for J! uassifolia), long persistence of allelic lineages is apparent in both cases. By using a generalized least-squares method for estimating the rate and time of S allele divergences, Uyenoyama [46’] has demonstrated that the high levels of sequence diversicy in the sporophytic SI system of Brassica reflect ancient allelic divergence.
variant DPB sequences which appear to have arisen by gene conversion in the sperm of a heterozygous male. The
Gene genealogy
and the coalescent
With the accumulation of sequenced DNA and the reconstruction of ancestral gene relationships, the neutral theory has been reformulated in terms of gene genealogy. When a gene is multiplied more than once and the products are transmitted by luck to later generations, such a multiplication appears in a diagram of genealogy as a coalescence when looked ac back in time. To reconstruct gene genealogy, it is necessary to determine the timing of coalescence or the number of distinct ancestral genes at a given time in the past. Coalescence theory was developed originally to describe the family relationships of neutral genes sampled from a single panmictic (randomly mating) equilibrium population but the theory was soon extended to orthologous genes sampled from different species. Coalescence theory has not only reproduced the main results of neutral theory in a much simpler way than the method of diffusion approximations but has also shown wide applicability to DN.4 sequence data. A number of theoretical recently. A coalescence
advancements have been made theory has been developed for
neutral genes sampled with a deterministically
from a population that varying size [49] and
evolves from a
Although not all human ,Ifhr sequences deposited in GenBank necessarily differ at the amino acid level in the peptide-binding region (PBR), there are 67, 149, 39, 179, 29, and 69 alleles at the class I d, B, C,
population experiencing a recent colonization event [SO’]. Likewise, the coalescence process of two genes has been formulated in a non-equilibrium population consisting of demes under extinction and recolonization and has been
class II DRB, DQB, and DPB loci, respectively (42.1. Balancing selection is effective in enhancing genetic diversity but it alone may not easily explain such large numbers of alleles unless one assumes that the effective population size is unrealistically large or that the mutation rate is exceptionally high. An exploration was therefore made of the role of recombination (Y Satta, personal communication). Intra-exonic recombination in the PBR can increase the number of alleles substantially without changing the nucleotide substitution rate enhanced by balancing selection. Frequent intra-exonic recombination may decrease the mean number of nucleotide differences between alleles and change the shape of the distribution
used to explore various demographic situations that might have occurred in the lineage leading to modern humans [30-l. The first theory for a population with varying size has been used to study gene genealogy within an allelic class that arose by a unique mutation at some time in the past [Sl’]. Furthermore, the variance formulae of pairwise nucleotide differences of genes have been obtained for two partially isolated populations [S2*] and the greater variance expected under migration relative to recent isolation has been used to distinguish between the two situations [53*]. Coalescence approaches have been also used in other problems in population genetics, such as those concerning microsatellite allele frequencies
770
Genomes and evolution
in subdivided populations repetitive elements under
[.54] and short the ‘master copy’
interspersed model [SS].
As for human evolution, many authors have addressed the age of the most recent common ancestor (hIRC.4) on the basis of nucleotide differences in a sample of genes, including mtDNA sequences [56], ALU sequences inserted in the Y chromosome [57”], and available nuclear DNA sequences [29’]. Although these studies have consistently implicated recent origins for human polymorphisms, there is controversy over the estimate made from the absence of variation in 38 human ZFY genes [W]; the original estimate is too rough and a more rigorous estimate has been obtained on the basis of joint probability distribution of the number of segregating sites and the age of the hlRCA [59’]. The genealogical behaviour of neutral genes and the superposition of nucleotide substitutions on the genealogical tree are now well investigated in a variety of situations. Finally, one complication connected with the neutral theory is worth coverage. Kimura was once sympathetic to the ‘slightly deleterious’ hypothesis of Ohta when criticisms against the neutral theory were most intense and he himself proposed the ‘effectively neutral’ model in 1986 [Z]. Nevertheless, in his last few years, Kimura abandoned this ad/zoc model and returned to the original neutral theory. It was at this time that Ohta renamed her previously proposed hypothesis as ‘nearly neutral’. Although ‘slightly deleterious’ and ‘nearly neutral’ sound similar, there is an important difference: under a model of near-neutrality, one half of the mutations are implicitly assumed to be advantageous [16*,60*]. Needless to say, the concept of near-neutrality is no longer the same as that of Kimura’s neutrality.
References
and recommended
Papers of particular interest, published have been highlighted as: . l
*
reading
within the annual period of review,
of special interest of outstanding interest
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Conclusions Because of its simplicity and value as a basis for prediction, Kimura’s neutral theory has been tested extensively. Whether the theory is accepted or rejected by particular tests, it has without question served as a fundamental guiding principle in the study of molecular evolution. Although there is agreement about the importance of random genetic drift in molecular evolution, there is obvious disagreement about the relative roles of other especially natural selection. It is evolutionary forces, important to keep in mind that convincing arguments for the theory of S and hlhc molecule evolution have been possible only by knowledge, albeit imperfect, of their function and structure which could be related directly to the causes
of natural
selecrion.
Acknowledgements I
thank hlk: SuggcStionS.
~I~entr~ama
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and
by
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Takahata N, Satta Y, Klein J: Divergence time and population size in the lineage leading to modern humans. Theor Popul Biol 1995, 48:198-221. The authors use a maximum likelihood method to estimate simultaneously the species divergence time and the common ancestral population size from interspecific sequence divergence. This paper focuses on the phylogenetic relationship between human, chimpanzee and gorilla, concluding that the closest relative to the human is the chimpanzee rather than the gorilla. The authors also show, using autosomal DNA sequence data, that the effective size of the human population was -100 000 before it diverged from the chimpanzee 4.6 million years ago. 30. Takahata N: A genetic perspective on the origin and history of . humans. Annu Rev Ecol Syst 1995, 26:343-372. A review of approaches to molecular anthropology on the basis of models of population genetics. Topics include the demographic history of primates in the Miocene and Pliocene eras, the problem of the apparent trichotomy among human, chimpanzee and gorilla, and the debate over the uniregional versus multiregional origin of modern humans. 31.
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32. Cavalli-Sforza LL, Menozzi P, Piazza A: The History and Geography .. of Human Genes. New Jersey: Princeton University Press; 1995. A multi-disciplmary work on our rapidly vanishing genetic heritage which integrates population genetics, molecular genetics, geography, ecology, archaeology, and physical anthropology. The book contains the most extensive genetic data currently available and shows nicely how they are analyzed to elucidate human evolution. The expansion of prehistoric humans or natural selection for particular alleles is evident through the 518 genetic maps found in the index.
theory
of molecular
evolution
Takahata
771
33. .
Wolinsky SM, Korber BTM, Neumann AU, Daniels M, Kunstman KJ, Whetsell Al, Furtado MR, Cao Y, Ho DD, Safrit JT, Koup RA: Adaptive evolution of human immunodeficiencv virus tvoe1 dking the natural course of infection. Science 1996,” 272537-547. This paper examines the hypothesis that the loss of CD4 T cells in HIV-1 infected individuals is primarily caused by increasing antigenic diversity [35-I. No such evidence is found, but adaptive evolution of virus populations under selective constraints is suggested. 34.
Nowak M, McMichael Al: How HIV defeats the immune system. Sci Am 1995, 273:42-49. 2 evolutionary model to explain the combat between the generalist HIV army and specialist immune army offers a possible explanation for why some patients progress to AIDS more quickly than do others. The model suggests positive selection for viral diversity in epitopes recognized by immune forces. 35. .
Mindell DP: Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees. Proc Nat/ Acad Sci USA 1996, 93:3284-3288. A phylogenetic analysis of primate immunodeficiency virus genes, showing enhanced nonsynonymous substitution rates in its new host species, humans. This tendency is most conspicuous at the env gpl20 locus which encodes for surface antigens. It is unclear, however, why the virus protein is positively selected only in the new host. 36. .
Smith NH, Maynard Smith J, Spratt BG: Sequence evolution of the porB gene of Neisseria gonorrhoeare and Neisseria meningitidis: evidence of positive Darwinian selection. MO/ Biol Evol 1995, 12:363-370. Evidence of an accelerated nonsynonymous rate in the major outer membrane protein, as expected, under positive selection in response to pressure from the immune system. This is found in N. gonorrhoeae but not in N. meningitidis. The loop region of this gene product in these bacteria which serves as epitopes may be subjected to different immune responses. 37. .
Metz EC, Palumbi SR: Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin. MO/ Biol Evol 1996, 13:397-406. This paper shows an accelerated nonsynonymous rate in a gamete recognition protein and high levels of polymorphism in contrast to no polymorphism in abalone lysin; however, the cause for this discrepancy remains speculative. 38. .
Goodwin RL, Baumann H, Berger FG: Patterns of divergence during evolution of al-proteinase inhibitors in mammals. MO/ Biol Evol 1996, 13:346-358. Evidence for an accelerated nonsynonymous rate within the reactive centre of the serpin protein. The accelerated rate is, however, found in murids only. In primates and artiodactyls, the same gene is subjected to purifying selection. It is interesting to elucidate how such species-specificity arises, 39. Hughes AL: The evolution of the type I interferon gene family . in mammals. J MO/ Evol 1995, 41:539-548. Interferon-a and interferon-p are subject to negative selection, but an excess of radical amino acid changes in a domain of interferon-w relative to conservative changes suggests positive selection. 40.
Kreitman M, Akashi H: Molecular evidence for natural selection. Annu Rev Ecol Syst 1995, 26:403-432. ;he authors argue that no current theories can account for all aspects of molecular evolution, and calls for a comprehensive theory. This conclusion is based largely on statistical tests of Drosophila genetic data. To demonstrate natural selection at the molecular level convincingly, however, we need to integrate into the framework of population genetics more information about the function and structure of molecules. 41.
Dodds PN, Clarke AE, Newbigin E: A molecular perspective on pollination in flowering plants. Cell 1996, 85:141-l 44. Ee molecular basis of self-incompatibility in Brassicaceae and Solanaceae are concisely reviewed here, concluding that there is a need to identify factors controlling pollen phenotype and the nature of the interactlons between stylar and pollen components. 42. Parham P, Ohta T: Population biology of antigen presentation . by MHC class I molecules. Science 1996, 272:67-74. A review of both experimental and theoretical population genetics studies of extraordinarily high diversity at the MHC. A significant, though controversial, role of interallelic recombination and conversion is emphasized in the process of generating class I MHC diversity. The contention that many South Amerindian 5 alleles derive from founding alleles by interallelic conversion may be regarded as tentative until a thorough survey is made of Asian populations. 43. .
Hinata K, Watanabe M, Yamakawa S, Satta Y, lsogai A: Evolutionary aspects of the S-related genes of the brassica self-incompatibility system: synonymous and nonsynonymous base substitutions. Genetics 1995, 140:1099-l 104.
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Genomes and evolution
Analysis of all available DNA sequences for SRK (S-receptor kinase), SLG (S-locus glycoprotein), and two S-related genes (SRA and SRB) show the order of gene duplication and early SLG allelic divergences. Richman AD, Kao TH, Schaeffer SW, Uyenoyama MK: Sallele sequence diversity in natural populations of Solanum carolineme (Horsenettle). Heredity 1995, 75:405-415. Despite a rather small number of S alleles maintained in two natural populations, numerous sequence differences are found, presumably reflecting trans.species maintenance of polymorphism. 44. .
Richman AD, Uyenoyama MK, Kohn JR: Contrasting patterns of allelic diversity and gene genealogy at the self-incompatibility locus in two species of Solanaceae. Science 1996, 273:1212-1216. 12 and 26 alleles recovered from Solanum caroolinense and Physalis crassifolia populations are used to infer their demographic histories and modes of speciation. 45. ..
Uyenoyama MK: A generalized statistical estimate for the origin of sporophytic self-incompatibility. Genetics 1995, 139:975-992. Amino acid sequences of the proteins encoded by SLG and related genes were analyzed by a newly developed least-squares method to infer the age of the SI system in Brassica and the trans-specific evolution of S alleles.
52. .
Wakeley J: The variance of pairwise nucleotide differences in two populations with migration. Theor Pop/ Viol 1996, 49:39-57. The variance formulae of nucleotide differences between genes are obtained in this study when two equilibrium populations exchange genes. Migration results in large standard errors on estimates of population genetic parameters, on the basis of which the author makes an interesting suggestion to distinguish between alternative demographic situations. 53. .
Wakeley J: Distinguishing migration from isolation using the variance of pairwise differences. Theor Pop/ Biol 1996, 49:369-366. Ongoing gene flow between populations and a recent population split have similar -but not exactly the same - effects on gene genealogy. This paper suggests one way to distinguish between these two situations. The test statistic developed here is applied to mitochondrial stickleback DNA sequence data and suggests ongoing migration. 54.
Slatkin M: A measure of population on microsatellite allele frequencies. 139:3457-3462.
55.
Tachida H: A population genetic study of the evolution of SINES. II. Sequence evolution under the master copy model. Genetics 1996, 143:1033-l 042.
56.
Horai S, Hayasaka K, Kondo R, Tsugane K, Takahata T: Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc Nat/ Acad SC; USA 1995, 92:532-536.
46. .
Klein J, O’hUigin C: Class II B Mhc motifs in an evolutionary perspective. lmmunol Rev 1995, 1143:69-l 11. ;wo possible explanations for the sharing of sequences motifs by different species - recombination and convergence - are discussed and forceful arguments for the latter are presented. The recurrent occurrence and/or long persistence of particular motifs in the peptide-binding region may result from the specific association between host and parasite.
47.
Zangenberg G, Huang MM, Arnheim N, Erlich H: New HLA-DPBI alleles generated by interallelic gene conversion detected by analysis of sperm. Nat Genet 1995, 10:407-414. This paper presents the first direct measurements of the rate of new variant LIP57 sequences generated by gene conversion between two parental alleles. The estimated conversion tract is 54-l 32 bp. 48. ..
49.
Griffiths RC, Tavare S: Sampling theory for neutral alleles in a varying environment. Phi/ Trans R Sot Land 5 1994, 344:403-410.
Austerlitz F, Jung-Muller B, Godelle B, Gouyon PH: Evolution of coalescence times, genetic diversity and structure during colonization. Theor Popul Biol 1996, in press. Under a non-equilibrium model of geographically arranged colonies, the authors analyze mean coalescence times and two other related quantities. A recent colonization process, which is common in temperate forest trees, is the main focus. 50. .
Slatkin M: Gene genealogy within mutant allelic classes. Genetics 1996, 143:579-587. 1 description of the genealogical process within an allelic class which arose by a unique mutational event is developed on the basis of the coalescence theory for a population of varying size [491. 51.
subdivision based Genetics 1996,
57. Hammer MF: A recent common ancestry for human Y .. chromosomes. Nature 1995, 378376-378. Interesting sequence analysis on a polymorphic A/u element which was inserted into the human Y chromosome after the divergence of humans and chimpanzees. This paper also summarizes the extent of DNA polymorphism and sequence divergences at various loci, providing no evidence for recent selective sweeps in the human population. Dorit RL, Akashi H, Gilbert W: Absence of polymorphism at the ZFY locus on the human Y chromosome. Science 1995, 268:1163-l 185. In a world-wide sample of 36 human males, no variation in a 729 bp intron of the ZFY gene is found. As for the estimate of the age of the MRCA, several comments were made recently [59’1. 56. .
Fu YX, Li WH: Estimating the age of the common ancestor of men from the ZFY intron. Science 1996, 272:1356-l 357. ;he posterior probability of the age of the MRCA is derived, given that there is no variation in the sample, a suggestion of an age compatible with other estimates. 59.
Gillespie JH: On Ohta’s hypothesis: most amino acid substitutions are deleterious. I MO/ Evol 1995, 40:64-69. ;\ model of near-neutrality is shown here to be incompatible with most
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