Motoo Kimura - random genetic drift prevails

TIBS - July 1976

N 152

DISCUSSION FORM Molecular evolution -

Roger Milkman - selection is the major determinant

Motoo Kimura -random drift prevails

genetic

Roger Milkman is Professor of Zoology at the University of Iowa, Iowa City. Injluencedby long-standing interests in development and physiology, his work in evolutionary genetics centers on three areas: the convergent effects of genes, the genetic structure of species and the basic rules of natural selection.

Motoo Kimura is head of the Department of Population Genetics at the National Institute of Genetics, Japan, and has worked for over 20 years on the mathematical theory of population genetics. In his spare time he is an enthusiastic breeder of Paphiopedilum (Lady’s Slipper) orchid.

The role of selection in the evolution of protein molecules is a subject of major importance and recent controversy. Selectionists view amino acid substitutions as unit responses to selection. Neutralists view the same amino acid substitutions as, largely, evolutionary noise. Let me begin by asserting that this controversy centers on a reasonable question; it is not the clash of restricted outlooks. Neutralists believe in natural selection; they recognize the advantage of the opposable thumb, and they understand in principle its genetic basis. What they do not know is the number of amino acid substitutions required to make a thumb opposable, and neither does anyone else. In the absence of a general quantitative relationship between amino acid substitutions and phenotypic changes at higher levels, it is not self-evident that all, or even a substantial proportion of amino acid substitutions are connected in any way with useful traits. Selectionists, for their part, understand stochastic processes. They recognize that mutations are random events and that, since populations are finite and are sampled by reproduction in every generation, allele frequencies are subject to random genetic drift. But, having seen again and again the adaptive significance of properties at every level of biological organization, from the Bohr effect to the red spot on a Herring Gull’s bill, they have become convinced that ‘if it’s there, it’s good’. Evolution does not entail natural selection; it is merely the history of life. We all agree that all living things are highly adapted and that these adaptations result from natural selection. We agree that most mutations are bad, and cast out. So here are the two positions: the selectionists believe that what is not bad and cast out is good and maintained; the neutralists believe, or believed, that a substantial proportion of mutations result in alleles that are not so bad as to be cast out and not so good as to be favored. So variety remains. This is not necessarily because differences do not exist; selection is limited, and the subject of its limitation is where the controversy begins. One view, the multiplicative fitness model, holds that selection operates successively and independently on each of several thousand genes. This model ignores the convergence of genetic effects

I accept that Darwin’s theory of natural selection is a great unifying principle in biology. What I am opposing is a naive panselectionism and its intrusion into the realm of molecular evolutionary studies. Before I present my rebuttal to Roger Milkman’s criticisms, let me review quickly the opposing claims made by neutralists and selectionists. Neutralists claim that the amino acid and nucleotide changes that accumulate within the species in the course of evolution are mainly due to random fixation of selectively neutral mutants. They also regard the enzyme polymorphism as a phase of molecular evolution. On the other hand, selectionists claim that practically all evolutionary amino acid and nucleotide substitutions are due to positive Darwinian selection. As to genetic polymorphisms, selectionists resort to other types of selection and claim that they are actively maintained by ‘some sort of balancing selection’. The detailed mechanism involved is not usually specified, and selectionist’s claims are elusive, but it is generally understood that overdominance (heterozygote advantage) is the most important type of selection. One of the characteristics of molecular evolution is its very high overall rate when the whole genome is considered. It amounts to at least six-‘nucleotide substitutions per genome per year in mammals. To explain such a high rate by positive natural selection that acts independently to each locus, an astronomical cost of selection is required (as Milkman mentions) and this presents a dilemma. This was one of the main reasons why I proposed the neutral theory in 1968 [l]. Milkman and other try to avoid the dilemma by resorting to a type of selection called ‘truncation selection’ (as explained in his article). This is a clever idea, but as far as I know, there is no actual evidence for it. Furthermore, experimental studies by Terumi Mukai and others, using the fruit fly Drosophilu, show that fitness of individuals as a quantitative character has extremely low heritability (H2=0.004) [2]. This means that environmental effects predominate in determining individual’s titness score, so that truncation selection does not work without entailing very severe culling (i.e. heavy cost). So, truncation

TIBS - July 1976 Roger Milkman on cells, tissues and organs, not to mention the entire organism.

I venture to say that historians of science will be at a loss, fifty years from now, to understand how this incredible error has persisted. Application of this model leads to the computation of an astronomical cost (in deaths, or reduced reproduction) of selection and necessitates the conclusion that few gene loci can be subject to selection at any one time. Most genie polymorphism, most protein polymorphism and most protein evolution could not be the result of selection. The correct view, I think, is that individuals are ranked competitively as to overall phenotype, and those favored by genes, environment and luck gain the available places, just as in animal or plant breeding, though not quite so rigidly. According to this model, substantial selection is easily possible at hundreds of loci at once. It takes very little selection to obliterate random genetic drift, and so the advantageous choice will be made almost every time. But what if there really is no advantage to one amino acid over another at a certain position? Obviously selection does not operate and the tield is left to random genetic drift. Since we cannot determine directly how often a substitution is truly neutral, we must look to the predictions of the neutral hypothesis. They are simple: when a steady state has been reached (after N, generations), the effective number of alleles n, at a locus will be equal to 4N,u+ 1. N, is the effective population size (ordinarily at least 0.1 of the number of individuals) and u is the mutation rate, per generation, between adaptively equivalent alleles. The effective number of alleles is the reciprocal of the probability of drawing the same allele twice successively (in diploids, the frequency of homozygotes). Since very few species have reached the steady state (a substantial temporary decrease in population size forces you to start over), the prediction is hard to test. Escherichia coli, however, has been at a population size well over lOi for at least 4x lOlo generations, or 4 x 10’ years (the horse’s evolution took 6 x lo7 years). It is found in most mammals all over the world. Since u could hardly be less than 1 . 10 -R, the equation predicts 400 for n,- 1. In contrast, I have found, by electrophoresis at five loci, values between 0 and 1. I have comirmed this finding for two loci by isoelectric focusing [l]. Moreover, the commonest mobility class had a frequency at each locus of more than 0.60, and almost all mobility classes were separated by empty classes. In all, 829 clones of E. coli from all over the world were tested. The commonest class in every place was the same, and many of the less frequent variants were widely distributed [2,3]. To me the clearest case of all was made by Richard Ambier, who sequenced cytochrome c from nine Pseudomonas aeruginosa cultures of diverse natural origins. Eight were identical, the ninth differed by one amino acid. Clearly P. aeruginosa is very choosy, that is selective [4]. The neutralist counter-arguments are mainly ad hoc. When n, is deficient compared to neutralist predictions, the deficiency is attributed to a recent bottleneck in population size. The notion that amino acid substitutions in proteins cannot cause fractional charge changes in the pH range common to electrophoresis (7-9, or so) is absurd to any biochemist and refuted by some of the earliest studies, but it has been espoused by some neutralists because it reduces markedly the number of mobility classes expected. (There is still a 30-100 fold disparity between the E. cofi data and the prediction, however!) Suggestions that man’s E. coli strains have suddenly taken over in all mammals are unreasonable. Additional enzyme variants have been found within mobility classes, most notably thermostability variants, some of which

Mot00 Kimura

selection, even if practised in nature, is of no help in avoiding the dilemma. As to enzyme polymorphisms in Escherichia coli, Milkman’s observation is most illuminating [3]. If-it is valid (and I believe it is), his observation immediately destroys the most cherished view of selectionists that overdominance is the prevailing nechanism for the maintenance of enzyme polymorphisms (note that E. coli is a haploid organism so heterozygote advantage does not work). Instead of mentioning this side of the story, Milkman uses his observation to attack the neutral theory saying that the neutralist’s prediction n,=4N,u+ 1 does not conform to the observation. The formula was derived under the assumption (‘model of infinite alleles’) that every mutation leads to a new, not preexisting neutral allele and that all the different alleles can be discriminated [4]. His main point is that the population size (NJ of E. coli on the earth is so immense that it is unlikely that 4N,u should ever stay between 0 and 1. In fact, the observed values of n,- 1 (averaged over many loci) in various organisms so far studied, including Drosophila and man, all come inside this interval, despite the enormous difference of their population sizes. This presents a difficulty to the neutral theory and particularly to the infinite allele model. In my opinion, neither selectionist can offer a satisfactory explanation, except to claim that a balancing selection acts in such a way that the level of genetic variability is kept roughly the same among diverse organisms - the most ad hoc argument imaginable! The most plausible (but admittedly tentative) explanation, I believe, comes from the theory put forward by Tomoko Ohta as an extension of the neutral theory [5]. According to her theory, the ‘neutral mutants’ are not stYictly neutral but are in fact very slightly deleterious, with coefficients of negative selection (denoted by s) comparable to mutation rates (say, one in 10,000 or less). These mutants behave in practically a neutral manner in relatively small populations such as most mammalian species. However, the number of ‘neutral allelic states’ diminishes as the population size increases, because effectiveness of negative selection increases with Na. In extremely large populations, such as E. coli and some neotropical DrosoDhila, the neutral allelic states are narrowed down to a very small number, with the value of n, becoming very large for them. I have to add that the neutral theory has many useful aspects in describing and predicting evolution and variation at the molecular level. For example, instead of splitting the observed value of n,- 1 or 4N,u into its components, if we treat it as one parameter we can predict fairly accurately the distribution of allelic frequencies within a species. Particularly in man and Drosophila, for which extensive data are available, the theoretical distributions agree quite well with observed distributions, except for an excess of rare alleles (contrary to that which might be expected if the majority of variants are actively kept by balancing selection). As to the evolutionary rates, there is now growing evidence that functionally less important molecules or parts of one molecule (in other words, those with less constraint in keeping molecular structure and function) evolve faster than more important ones in terms of mutant substitutions. Also, substitutions that disrupt less the existing structure and function of a molecule (conservative substitutions) occur more frequently in evolution than more disruptive ones. The evidence now is so overwhelming that no one can dismiss these observations. From the neutralist viewpoint, they can be explained easily by assuming that the probability of a random mutational change being neutral (not harmful) is higher in a functionally

N 154

TIBS - July I976

Roger Milkman have been mapped to the appropriate locus. Whether these are maintained by selection, rather than by drift or by recombination between alleles differing at two sites, is another question. It must be remembered that the neutral hypothesis arose to explain electrophoretic variation - while not correct at this level, it may well be at others, notably synonymous variation among codons. Abundant additional evidence, mostly circumstantial, opposes the neutral hypothesis in a great variety of organisms: disparities in patterns of geographic allele-frequency variation from locus to locus; relative independence of genetic variation on population size; and correlations between allele frequencies and certain environmental variables, notably temperature. Yet the explicit ‘disproofs’ of the neutral hypothesis are few. One conclusion remains: the neutralists, led by Kimura, have made a great contribution to evolutionary genetics by sculpting away some of the dogma from the theory of natural selection and forcing its proof at the level of proteins’ primary structure.

Motoo Kimura less important molecule or a part of one molecule and therefore the evolutionary rate in it is higher. The observation that synonymous or silent substitutions (at the third position of the codon) occur more frequently in evolution per nucleotide site than missense substitutions can be explained in a similar way. On the other hand, from the selectionist viewpoint, rapidly evolving molecules or parts of a molecule have some important unknown functions and that they are undergoing rapid adaptive modifications by accumulating definitely advantageous mutations. For me at least, such an interpretation is unnatural, and when used to explain the prevalence of synonymous substitutions in evolution, it is manifestly inadequate. I admit that the neutral theory is not perfect, and I do not want to claim that it can explain all observations. Nevertheless, the fact that the theory works in not a few cases to derive predictions that are fairly close to actual observations show that it is at least a useful scientific hypothesis. References

References

1 Kimura,

1 Milkman, R. and Koehler, R. (1976) Biochem. Genei. 14, 517-522 2 Milkman, R. (1975) in Isozymes, Vol. IV, Genetics and Evolufion (Marker?, C. L., ed.), pp. 273-285, Academic Press, New York 3 Milkman, R. (1973) Science 182, 1024-1026 4 Ambler, R. P. (1974) Biochem. J. 137, 3-14

2 3 4 5

M. (1968)Nature 2 17, 624-626 Mukai, T., Schaffer, H. E. and Cockerham, C. C. (1972) Genetics 72. 763-769 Milkman, R. (1973) Science 182, 1024-1026 Kimura, M. and Crow, J. F. (1964) Generics 49, 725-738 Ohta, T. (1974) Mature 252, 351-354

The debate is now open - readers’commentswhichmay bepublishedas letters to the editorare welcome.They should be sent to the StaffEditor, 58 MuswellHillRoad, London,NJ0 ??

LETTERS TOTHE EDITOR Orbitals, conformation and biological activity Drug conformations SIR: It is obviously true, as Horn and Kennard (TIBS, May p. N 106) point out, that no amount of knowledge of the structure and conformation of a drug molecule will tell us anything of the chemical nature of its receptor. A detailed knowledge of the conformation of the drug when bound to its receptor would, however, allow us to place constraints on the geometric disposition of those groups on the receptor which

interact with the drug. The extent to which one believes that such a knowledge of the conformation in the bound state can be obtained from studies of the free drug molecule depends on one’s conceptual model of the binding process. I would agree with Kier (TIBS, April p. N 80) and Horn and Kennard that an enzyme-substrate complex may not be a good model for a drug-receptor complex. However, this should not lead us to ignore all other protein-ligand complexes - in fact the vast majority of the detailed structural information we have on enzyme-ligand interactions necessarily relates to complexes in which no covalent changes are taking place. If one accepts that many drug receptors are proteins, then at the least an examination of enzyme-ligand complexes will give one a feeling for what can happen when a drug binds to its receptor.

Kier’s model for the drug-receptor interaction, the ‘weak interaction hypothesis’, presents a number of problems. As Richards (TIBS, June p. N 131) point out, it is difficult to account for the considerable specificity of the drug-receptor interaction on the basis of weak, long-range forces. In addition, Kier’s statement that ‘a “bound” or “complexed” state is not necessary to alter materially the receptor’ must be examined in the light of the energetics of the system. He points out that, as the drug approaches the receptor, a significant interaction energy develops before the conformation of either component is perturbed. This must necessarily be the case, since the energy required for the conformational perturbation arises from the interaction energy. Indeed, the conformation can only be perturbed if the interaction energy is greater in the ‘new’ conformation. On this basis one would clearly expect that the interaction will proceed to a ‘bound’ or ‘complexed’ state. A ‘hit-and-run’ model of the kind implied by Kier is at best energetically implausible. In a real situation it is of course possible (indeed likely) that more than one form of the complex exists, and the biological effect need not necessarily be associated with the thermodynamically most stable form of the complex. The essential fact remains that if the interaction energy in