Proc. Nati. Acad. Sci. USA Vol. 88, pp. 5%9-5973, July 1991 Evolution Recent development of the neutral theory viewed from the Wrightian tradition of theoretical population genetics* (molecular evolution/population genetics/macroevolution) MOToo KIMURA National Institute of Genetics, Mishima, 411 Japan Contributed by Motoo Kimura, April 1, 1991

ABSTRACT In contrast to the Darwinian theory of evo- continued mutation pressure (for details, see ref. 8). This lution by natural selection, the neutral theory emphasizes the view is in sharp contrast to the traditional neo-Darwinian great importance of random genetic drift (due to rinite popu- (i.e., the synthetic) theory ofevolution, which claims that the lation size) and mutation pressure as the main causes of spreading of mutants within the species in the course of molecular evolution. In this paper, after a brief review of the evolution can occur only with the help of positive natural neutral theory, recent data strongly supporting the neutral selection. theory are presented. Also discussed are such topics as com- The neutral theory also asserts that most intraspecific pensatory neutral evolution and an approach to a unified variability at the molecular level (including protein and DNA understanding of molecular and phenotypic evolution. It is polymorphisms) is selectively neutral, and it is maintained in concluded that random genetic drift acting on selectively the species by the balance between mutational input and neutral mutants must have played some very important role in random extinction. In other words, the neutral theory regards organic evolution, including the origin of life and macroevo- protein and DNA polymorphisms as a transient phase of lution. molecular evolution (9) and rejects the notion that the ma- jority of such polymorphisms are adaptive and actively The late Professor Sewall Wright was my idol when I was maintained in the species by some form of balancing selec- young. Soon after graduating from Kyoto University, I read tion. Wright's 1931 classic "Evolution in Mendelian populations" The neutral theory differs from traditional theories of (1) and his subsequent papers on random genetic drift and the evolution in that it is quantitative-namely, we can derive distribution of gene frequencies [such as refs. 2-5; see also simple formulae for such quantities as the rate of evolution Provine (6) for additional references]. These papers im- and the amount of intraspecific variability-and in that we pressed me deeply and, in fact, inspired me to become a can check the validity of the formulae by comparing theo- theoretical population geneticist. Without this foundation I retical predictions with actual data. would never have been able to propose the neutral theory or First, let us consider the cumulative process in which to incorporate new knowledge from molecular genetics into neutral mutants are substituted sequentially at a given locus the framework of population genetics. or site through random genetic drift under continued input of When the neutral theory was proposed (7), the only avail- new mutants. Then we have for the rate of evolution per able data consisted ofamino acid sequences ofa few proteins generation the formula in related organisms, such as hemoglobin molecules in some vertebrate species. Also, genetic variability at the molecular kg= Vo, [1] level could be inferred only from electrophoretic data on where kg represents the long-term average per generation of enzyme polymorphisms for a few species such as fruit flies the number of mutants that spread through the population and humans. Resolution ofthe ensuing controversy regarding and vo is the rate of production of neutral mutants per locus the pros and cons ofthe neutral theory ofmolecular evolution (or site) per generation. This formula is based on the well- was much limited by nonavailability of DNA data. The known property (7, 10) that, for neutral mutations, the situation has changed dramatically with the emergence of long-term rate of mutant substitution is equal to the mutation DNA sequence data, which resulted from the development of rate. rapid DNA sequencing techniques. I am glad to note that, If we denote by VT the total mutation rate, and iffo is the following this development, strong evidence for the neutral fraction of neutral mutations at the time of occurrence, so theory has accumulated steadily with the passage of time, that = VO/VT, then Eq. 1 may be rewritten as particularly during the past decade. fo In this paper I shall review recent developments of mo- kg = VTfO. [2] lecular evolutionary studies from the standpoint of the neu- tral theory. I shall also discuss some neutralist views on Advantageous mutations may occur, but the neutral theory evolution in general. assumes that they are so rare that they may be neglected in our quantitative consideration. Thus, (1 - fo) represents the Neutral Theory of Molecular Evolution fraction of definitely deleterious mutants that are eliminated from the population without contributing to either evolution According to the neutral theory, the great majority of evo- or polymorphism, even though the selective disadvantages lutionary mutant substitutions at the molecular level are involved may be very small in the ordinary sense. The above caused by random fixation, through sampling drift, of selec- formulation has a remarkable simplicity in that the evolu- tively neutral (i.e., selectively equivalent) mutants under tionary rate (on the long-term basis) is independent of pop- ulation size and environmental conditions of each organism. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" *This paper is an extended version of my talk presented at the Sewall in accordance with 18 U.S.C. §1734 solely to indicate this fact. Wright Centennial Symposium, Madison, WI, June 8-9, 1990.

5969 Downloaded by guest on October 2, 2021 5970 Evolution: Kimura Proc. Natl. Acad. Sci. USA 88 (1991) In molecular evolutionary studies, it is customary to mea- than in rodents (16, 17). According to my estimate (18), the sure the evolutionary rate in terms of years (i.e., taking one mouse line evolves faster than the human line with respect to year as the unit length of time) rather than in generations. amino acid replacements per year in hemoglobin a and X3 Therefore, it is convenient to modify Eq. 2 so that it gives the chains by a factor of 34.5/14.6, or approximately 2.4. Note evolutionary rate per year. that, from the standpoint ofthe neutral theory, the difference of the evolutionary rates in these lines is caused by the k, = (vT/g)fO. [31 difference in their neutral mutation rates per year. Thus, In this formula, g stands for the generation span (in years), assuming an average generation span of 20 years for the and VT/g is the total mutation rate per year. human lineage, the total mutation rate per generation is Next, let us consider intraspecific variability, assuming neutral mutations. For the average heterozygosity per nucle- VT = (3 X 109) x (5 X 10 9) X 20 2.4, otide site we have the following formula: or VT = 125.0, which means that the total number of new mutations per generation due to base substitutions amounts H = 4NeVonuc), [4] to 125 per gamete, and twice as many per zygote. where Ne is the effective population size and VO(nuc) is the In the above calculation, we assumed that the mouse line neutral mutation rate per nucleotide site. I first derived this evolved 2.4 times faster than the human line. It is possible, formula by using the "infinite site model" (11). and also likely, however, that this is an underestimate. In fact, Li and Tanimura (19) obtained a result that the rate of Some Recent Data Supporting the Neutral Theory of synonymous substitutions in rodents is about 7 times higher Molecular Evolution than that in higher primates. If we adopt this estimate, we obtain VT 43, which is about 1/3 of the above estimate. Still, The first definitive evidence supporting the neutral theory this is a very high value when compared with the traditional was the discovery that synonymous base substitutions, estimates of the genomic mutation rate. From the consider- which do not cause amino acid changes, almost always occur ation ofgenetic load (cf. ref. 20), the mutational load becomes at much higher rate than nonsynonymous-that is, amino- intolerably high unless the great majority (say, 99.5%) of acid-altering-substitutions. It was also found that evolution- them are selectively neutral (i.e., nondeleterious). ary base substitutions at other "silent" sites, such as introns, An equally interesting example suggesting neutral evolu- occur at comparably high rates. These observations suggest tion is the recent observation that the evolutionary rate of the that molecular changes that are less likely to be subjected to eye lens protein aA-crystallin has been much enhanced in the natural selection occur more frequently in evolution and blind mole rat, Spalax ehrenbergi (21). This animal is com- therefore show higher evolutionary rates. This is easy to pletely blind and is adapted to a burrowing subterranean way understand from the neutral theory, because such changes of life (and possibly has been for the last 25 million years, are more likely to turn out to be nondeleterious (i.e., selec- according to fossil evidence). Although this animal is com- tively neutral) and therefore fo in Eq. 2 is larger for them. pletely blind, the crystallins are still expressed in the atro- More than a decade and a half ago, in collaboration with phied lens cells. Generally speaking, aA-crystallin is a slowly Ohta, I enumerated (12) five principles that govern molecular evolving protein, with an average replacement rate of about evolution, one ofwhich states that functionally less important 0.3 x 10-9 per amino acid site per year in rodents and other molecules or parts of a molecule evolve (in terms of mutant vertebrates. In the mole rat lineage, however, this rate has substitutions) faster than more important ones. When this increased severalfold. In this case, although the rate is much principle was proposed, accompanied by its neutralist expla- increased, its maximum estimate is still only 'A5 of the nation, much opposition was voiced by the neo-Darwinian observed rate of the globin pseudogene. This is quite under- establishment, but I am glad to note that it has become a part standable because in this animal the aA-crystallin gene is still of common knowledge among molecular biologists, even if expressed (i.e., transcribed and translated), and even if the few of them seem to realize that its theoretical basis stems the from the neutral theory. It is now a routine practice to search eyes are no longer used for vision, vestigial amounts of for various signals by comparing a relevant region of homol- protein still exist in the body, so that some selective con- ogous DNA sequences of diverse organisms and to pick out straint should still remain (i.e., fo < 1). a constant or "consensus" pattern, but to disregard variable According to the neutral theory, mutation pressure plays a parts as unimportant. predominant role in molecular evolution. In recent years, I once predicted (13), on the basis of the neutral theory much evidence corroborating this has been added. One of the (i.e., using Eq. 2), that the maximal evolutionary rate is set most remarkable examples demonstrating this is the very by the mutation rate (kg . VT) and that the maximal rate is rapid evolutionary change observed in RNA viruses such as attained when all the mutations are selectively neutral (i.e., influenza viruses, which are known to have very high muta- whenfo = 1). A few years later, this prediction was dramat- tion rates: genes of RNA viruses show evolutionary rates per ically vindicated by the discovery of very high evolutionary year roughly a million times those ofDNA organisms (22,23). rates for pseudogenes (or "dead" genes), which have lost It is remarkable that, in this case, synonymous substitutions their function (14, 15). What is especially interesting, as also predominate over nonsynonymous substitutions, similar revealed by the studies of pseudogenes, is that the rates of to what has been found in genes of DNA-containing orga- substitution are equally high in all three codon positions. The nisms. A recent analysis of data on influenza A virus evolu- estimated rate in globin pseudogenes is about k = 5 x 10-9 tion by Gojobori et al. (24) also confirmed the existence of substitutions per nucleotide site per year. very clear, clocklike progression of base substitutions, in If the neutralist interpretation of the high evolutionary rate which the rate of synonymous substitutions (13.1 X 10-3 per of the mouse globin pseudogene is correct, it will enable us site per year) is about 3.5 times that of nonsynonymous to estimate the total mutation rate due to base substitutions substitutions (3.6 x 10-3 per site per year). These observa- per gamete per generation in humans. For this purpose, we tions can readily be explained by the neutral theory by noting assume that the human genome consists of 3 x 109 nucleotide that, in Eq. 2, the value of VT is about a million times higher sites, and we take into account the fact that the rate of in the RNA genome than in the DNA genome, while the molecular evolution per year is significantly lower in humans values offo remain roughly the same. Downloaded by guest on October 2, 2021 Evolution: Kimura Proc. Natl. Acad. Sci. USA 88 (1991) 5971

Compensatory Neutral Evolution mutants exists, the deleterious effect of mutations is sup- pressed (i.e., mutants become neutral), and this enhances the I propose that, in addition to neutral mutations, "compen- rate of evolution. If we use Wright's terminology, the above satory neutral mutations" (25) also play an important role in phenomenon of compensatory neutral evolution represents a molecular evolution. By compensatory neutral mutations, I situation in which a species can readily move, on the surface mean a pair of mutations at different loci or sites that are of selective values, from one peak to a nearby, equally deleterious individually but restore normal fitness in combi- adapted, peak, passing through a deep valley by mutation nation. This type of epistatic interaction is most likely to arise pressure and random drift. between two codons coding for two amino acids that are located close to each other in the three-dimensionally folded Discussion structure of a protein. In fact, several cases have been reported so far, suggesting strongly that such an interaction Compared with evolution at the phenotypic level, molecular does occur, including the report of Yanofsky et al. (26) on evolution is characterized by two outstanding features (28). mutational changes in tryptophan synthetase A protein. The first is the constancy of the rate-i.e., for each protein Since the interacting codons in these cases are contained in or gene region, the rate of amino acid or nucleotide substi- a single gene region, they must be very tightly linked. tution is approximately constant per site per year (hence the I have studied (25) the population dynamics ofevolutionary term "molecular evolutionary clock"). The second is the substitutions of compensatory mutants by making use of the "conservative nature" of the changes-i.e., functionally less diffusion equation method. Through this study a remarkable important molecules, or portions of molecules, evolve faster phenomenon was disclosed. The double mutants can easily than more important ones. become fixed in the population by random drift under con- As to the first feature (i.e., constancy of the rate), this may tinued mutation pressure if the two sites are very tightly be explained by the neutral theory by assuming that VT/g linked, even when the single mutants are definitely deleteri- remains the same (constant) among diverse lineages and over ous. time for a given protein or gene, for which fo is assumed to Let us consider a pair of alleles A and A' at the first site and be constant. In other words, the theory assumes that for a alleles B and B' at the second site. We assume that mutation given gene, the production of neutral mutations per year is occurs irreversibly from wild-type allele A to mutant allele A' nearly constant among diverse organisms whose generation at the rate v per generation, and similarly from B to B' at the spans are very different. Note that "mutation" here refers to same rate v. Consider a random-mating diploid population of changes that lead to DNA base replacements but not to lethal effective size Ne (or a haploid population of effective size or "visible" changes. These latter types of mutations, whose 2Ne). To simplify the mathematical treatment, we adopt a incidence has been known to be generation dependent, are haploid selection model and assign relative fitnesses 1, 1 - s', now suspected to be largely caused or controlled by various 1 - s', and 1, respectively, to the four haploid genotypes AB, movable genetic elements such as transposons and insertion A'B, AB', and A'B', where s' is the selection coefficient sequences in the genome (see ref. 28 for additional references against the single mutants (s' > 0). The problem here is to on this subject). On the other hand, it is likely that errors in determine how long it takes for the double mutant A'B' to DNA replication and repair are the main causes of DNA become fixed (i.e., to reach 100% frequency) in the popula- changes that are responsible for molecular evolution. Thus, tion, starting from the state in which the population consists the mutation rate for nucleotide substitutions may depend on exclusively of the wild-type AB. the number of cell divisions in the germ lines, particularly in Since the mathematical treatment in this case contains the male line (29), and this will make the molecular mutation approximations, I performed extensive Monte Carlo simula- rate roughly proportional to years. Experimental studies on tion experiments to supplement the analytical treatments. To this subject are much needed. show the remarkable property of compensatory changes As to the second feature (i.e., the conservative nature), it under very tight linkage, I would like to give a few examples. can easily be understood from the neutral theory, because the Assuming 2Nev = 1, namely, assuming the occurrence of one less drastic or more conservative the mutational change, the mutation per locus in the population (if N, = N) in each more likely it is to turn out to be nondeleterious, and generation, we have T7 8N, when 4Nes' = 36 under therefore selectively neutral. This means that for more con- complete linkage, whereas we have roughly == 800Ne for the servative changes the values of fo in Eq. 2 are larger. As same 4Nes' value under free recombination. More generally, mentioned already, it is now a common practice among the average time until fixation of double mutants (i.e., T) is molecular biologists to search for various signals by compar- not excessively long under complete linkage even when the ing homologous DNA sequences of diverse organisms and to 4Nes' value is several hundred. For example, assuming 2Nev pick out "consensus" patterns as important while disregard- = 1, we have T = 54N, for 4Nes' = 400, and T = 128N, for ing variable parts as unimportant. 4Nes' = 1000, under complete linkage. These values are not From the standpoint of the neutral theory, a universally unrealistically long compared with T -5AN for 4Ns' = 0. valid and exact molecular evolutionary clock would exist The amazing efficiency of compensatory neutral change only if, for a given molecule, the mutation rate for neutral when the linkage is very tight becomes even more apparent alleles per year (vo/g) were exactly equal among all orga- when we compare this with the fixation time of a deleterious nisms at all times (which is rather unlikely in nature). Thus, mutant at a single locus without the benefit of the compen- any deviation from the exact equality of neutral mutation rate satory effect. In the single-locus case, T 100Ne for 4Nes' per year makes the molecular clock less exact. In other = 9, T > 1000Ne for 4Nes' = 13, and T is practically infinite words, the variance of the evolutionary rates among different for 4Nes' > 40, again assuming 4Nev = 1. Recently, Ohta (27) lineages for a given molecule may tend to become larger than investigated the role of compensatory mutations in evolution expected from the simple Poisson distribution, as often noted by gene duplication. She has shown that the average time in actual observations. Gillespie (30, 31), who criticizes the until fixation of the double mutant (A'B') is much shorter neutral theory on the basis of such observations, claims that under gene duplication, particularly when 2Nev is much less a model of evolution, which he calls the "episodic model," than unity (even compared with the case of complete link- can fit the data better. His model is based on the idea that age). Note that in her model, relaxation of selective con- molecular evolution is episodic, with short bursts of rapid straint due to the existence of redundant gene copies is substitution being separated by long periods of no substitu- assumed, namely, when at least one gene copy that is free of tion. According to him, each environmental change presents Downloaded by guest on October 2, 2021 5972 Evolution: Kimura Proc. Natl. Acad. Sci. USA 88 (1991) a challenge to the species that may be met by amino acid emphasize the importance of a condition that I call "libera- substitutions caused by positive natural selection. I think that tion from selective constraint" as a main cause of macroev- Gillespie's theory is highly unrealistic in that it assumes that olution. If a species is confined for a long time to a constant the numbers of episodes in different lineages (which must ecological niche with all other potentially available niches experience different environments) follow the same proba- being occupied by other species, further adaptive shift be- bility distribution. It is also highly problematical to assume comes impossible. Only when new vacant niches are pre- that natural selection acts in such a way that the number of sented will the possibility for macroevolution to occur be mutant substitutions per episode follows the same probability open. This phenomenon is clearly shown by spectacular distribution for all episodes in all lineages. In his theory, evolution at the early Cambrian (often called "Cambrian natural selection is invoked arbitrarily to fit the data, while explosion"), where large-scale evolutionary experiments neglecting all the effects ofthe mutation rate, population size, were performed in the then-new way of life as multicellular and selective constraint. If it turns out that difference of organisms (40). Recently, Gould (41), in his book Wonderful evolutionary rates among lineages is mainly caused by dif- Life, tells the fascinating story about many bizarre animals ferences of VT/-gi.e., mutation rate in terms of base sub- that lived in the Cambrian sea, as revealed by the studies of stitutions per site per year-Gillespie's "episodic clock" the Burgess Shale fossils. Similarly, explosive diversification theory breaks down completely. of mammals in the early Cenozoic, immediately after the The predominant role played by mutation pressure in extinction of the then-dominant dinosaurs, is well known. molecular evolution, quite in line with the neutral theory, has What I want to emphasize is that relaxation of natural become increasingly evident from recent studies. One of the selection is the prerequisite for new evolutionary progress. In most remarkable examples demonstrating this is the very other words, "liberation from selective constraint" enables rapid evolutionary change observed in genes of RNA viruses, extensive neutral evolution to occur, creating new variants, which are known to have very high mutation rates. As some of which turn out to be useful in a new environment. pointed out already, this can readily be explained by noting Based on these considerations, I recently proposed (42) a that in Eq. 2 the value of VT is about a million times higher in hypothesis that I called the "four-stage scenario" theory of RNA genomes than in DNA genomes. macroevolution. According to this theory, macroevolution The concept of mutation-driven neutral evolution is also consists of the following four steps. (i) A population is useful for understanding the remarkable evolution of the liberated from the preexisting selective constraint. (it) There deviant coding system recently discovered by Osawa's group is a sudden increase or boom of neutral variations under (32-34) in the A+T-rich bacterial species Mycoplasma capri- relaxed selection. In this stage, gene duplication in addition colum (for details on neutralist interpretation on the mech- to point mutation must play a very important role in produc- anism of evolution involved, see refs. 35 and 36). ing genetic variations. Needless to say, their fate is largely If the neutral theory is valid so that the great majority of determined by random drift. (iii) The latent selection poten- evolutionary changes at the molecular level are controlled by tial of some of the neutral mutants is realized, an occurrence random genetic drift under continued input of mutations, it is that I have termed (see ref. 8) the Dykhuizen-Hartl effect. In likely that selectively neutral changes have played an impor- other words, some of the accumulated neutral mutants (at the tant role in the origin of life and also in phenotypic evolution. phenotypic level) turn out to be useful in a new environment, In fact, Dyson (37) proposed a theory on the origin of life which the species then exploits. (iv) Intergroup competition in which he assumes that neutral evolution was prevalent at and individual selection lead to extensive adaptive evolution, the time when life was originating. According to the Dyson creating a radically different taxonomic group adapted to a theory, an active protein evolved first in an Oparin-type newly opened ecological niche. A coherent mathematical primitive cell through a process similar to random frequency model of this hypothesis is yet to be developed, but if the drift in a finite population. The theory assumes also that the above theory turns out to be essentially valid, the importance RNA gene emerged later in the cell as a parasite. What of the neutral theory as an evolutionary paradigm will be pleased me is that in developing his theory, Dyson found both much enhanced. the main idea of the neutral theory and my diffusion equation In conclusion, I would like to emphasize the importance of method (given in ref. 38) very useful. Irrespective of whether random genetic drift as a major cause of evolution. We must Dyson's theory is valid or not, I believe that chance in the be liberated, so to speak, from the selective constraint posed form of random frequency drift acting on neutral changes by the neo-Darwinian (or the synthetic) theory of evolution. must have played some very important role in the origin of Wright, in his later years, used to claim that he had never life. attributed any significance to random drift except as an agent Finally, I would like to discuss briefly the problem How to bring about shift of adaptive peaks. As shown in Provine's can we understand evolution at two levels-that is, molecular recent book (6), however, Wright in his papers of the early and phenotypic-in a unified way? It is generally believed 1930s used to attach much more weight to random drift. that, in contrast to the neutralist view of molecular evolution, Personally, I was mainly influenced by Wright's earlier evolutionary changes at the phenotypic level are almost papers, so that he is truly the forerunner in whose footsteps exclusively adaptive and caused by Darwinian positive se- I have followed. I admire him very deeply. lection. However, I think that even at the phenotypic level, there must be many changes that are so nearly neutral that This paper is dedicated to the memory of Sewall Wright. I thank random drift plays a significant role, particularly with respect Drs. J. F. Crow and K. Aoki for having gone over the manuscript, to "quantitative characters." I have shown (39) that if a large or parts thereof, to suggest improved presentation. This work was number of segregating nucleotide sites each with a small supported in part by a grant-in-aid from the Ministry of Education effect are involved in a quantitative character subject to and Culture of Japan. This is contribution number 1848 from the stabilizing selection, the average selection coefficient per National Institute of Genetics, Mishima, Japan. mutant becomes exceedingly small. Under such a condition, extensive neutral evolution can occur through 1. Wright, S. (1931) Genetics 16, 97-159. random drift. 2. Wright, S. (1937) Proc. Natl. Acad. Sci. USA 23, 307-320. Note that stabilizing selection is the most prevalent type of 3. Wright, S. (1938) Proc. Natl. Acad. Sci. USA 24, 253-259. selection in nature. 4. Wright, S. (1942) Bull. Am. Math. Soc. 48, 223-246. Paleontological studies have revealed that big evolutionary 5. Wright, S. (1945) Proc. Nati. Acad. Sci. USA 31, 383-389. changes usually result from exploitation of a new ecological 6. Provine, W. B. (1986) Sewall Wright and Evolutionary Biology niche by a species. In this connection, I would like to (Univ. of Chicago Press, Chicago). Downloaded by guest on October 2, 2021 Evolution: Kimura Proc. Natl. Acad. Sci. USA 88 (1991) 5973

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