Neutral Theory - University of Michigan

6/30/2016 Neutral Theory Evolutionary Biology Oxford Bibliographies

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Neutral TheoryJianzhi Zhang

LAST MODIFIED: 28 JUNE 2016DOI: 10.1093/OBO/97801999417280081

Introduction

The neutral theory, formally known as the neutral theory of molecular evolution, was independently proposed by M. Kimura in 1968 (seeKimura 1983, cited under General Overviews, the Neutral Hypothesis of Phenotypic Evolution, and Outstanding Questions; Kimura 1994,cited under General Overviews; and Kimura 1968, cited under Origin of the Theory) and J. L. King and T. H. Jukes in 1969 (see King andJukes 1969, cited under Origin of the Theory). The chief tenet of the theory is that most genetic differences between species andpolymorphisms within species are selectively neutral and result from mutation and genetic drift. This view sharply contrasts that of neoDarwinists, who maintain that most of these variations are adaptive. While the neutral theory has been controversial since its debut, theneutralistselectionist debate has been largely healthy in that it greatly stimulated the development of rigorous neutrality tests, and it isnow customary to invoke adaptive explanations only when a null hypothesis of neutrality is rejected. The neutral theory has been revisedand broadened in a number of ways since the 1990s, including the emphasis of nearly neutral mutations in the nearly neutral theory,expansion to evolutionary explanations of genomic architectures, and application to phenotypic evolution. While these studies are notwithout contention, the idea that genetic drift, rather than adaptation, could explain origins of nonrandom patterns in biology has hadprofound impacts on many biologists’ view of evolution and the living world. The neutral theory remains the sole paradigmchangingconceptual revolution in evolutionary biology since the maturation of the neoDarwinism in the 1950s.

General Overviews

There are a few books on the neutral theory, and Kimura 1983 remains the most comprehensive and authoritative book on the topic.Kimura 1994 is an edited volume of Kimura’s original papers on neutral theory and his underlying population genetic work. Crow andKimura 1970 is a classic textbook of population genetic theory and deals with many fundamental concepts; it is highly useful forunderstanding the population genetic basis of the neutral theory. Ohta 2009 is a Japanese book on nearly neutral theory, which assertsthat most intraspecific and interspecific genetic variations are not strictly neutral but are nearly neutral. Lynch 2007 argues that fixationsof slightly deleterious mutations by drift led to the origins of various complex genomic architectures. Nei 2013 argues for the role ofmutation and drift in evolution in general. Textbooks of molecular evolution that contain lucid discussions about the neutral theory andrelated topics include Nei 1987 and Li 1997.

Crow, J. F., and M. Kimura. 1970. An introduction to population genetics theory. Caldwell, NJ: Blackburn.

A classic textbook of theoretical population genetics, it provides many theoretical results pertaining to the neutral theory.

Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge, UK: Cambridge Univ. Press.

In this single most important book of evolutionary biology since the 1960s, the neutral theory is lucidly, comprehensively, and forcefullyargued for by its chief advocate.

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Kimura, M. 1994. Population genetics, molecular evolution, and the neutral theory: Selected papers. Chicago: Univ. of ChicagoPress.

An edited volume of Kimura’s original papers, many key to the development of the neutral theory.

Li, W.H. 1997. Molecular evolution. Sunderland, MA: Sinauer.

A lucid and comprehensive textbook on molecular evolution, it discusses many topics highly relevant to neutral theory, such asnucleotide and aminoacid substitution rates and the molecular clock.

Lynch, M. 2007. The origins of genome architecture. Sunderland, MA: Sinauer.

The role of nearly neutral mutations in the origins of genomic complexity is forcefully argued for in this monograph.

Nei, M. 1987. Molecular evolutionary genetics. New York: Columbia Univ. Press.

A pioneering textbook of molecular evolutionary genetics by a longtime supporter of the neutral theory.

Nei, M. 2013. Mutationdriven evolution. Oxford: Oxford Univ. Press.

The author proposes a central role of mutation (and drift) rather than selection in evolution in general.

Ohta, T. 2009. Nearly neutral theory of molecular evolution: Evolutionary models of selection and chance. Tokyo: Kodansha.

A text on the nearly neutral theory, written by the proposer of the theory. In Japanese.

Origin of the Theory

Lewontin 1974 suggests that neutral theory is a continuation of the classical view of population genetics in the classical versus balancecontroversy on the maintenance of genetic polymorphism. Dietrich 1994 argues that Lewontin’s view is at best partially correct, becausethe then knowledge of molecular biology provided more evidence for neutral theory. In any case, Kimura 1968 reports that the total rateof nucleotide substitution in a mammalian genome, extrapolated from three proteins, far exceeds the upper limit of adaptive evolutionsuggested by Haldane 1957 (cited under Initial Response to the Neutral Theory). This observation led Kimura to propose that mostgenetic changes have been fixed by random drift rather than positive Darwinian selection. Kimura 1968 further states that the largeamounts of fruit fly (Lewontin and Hubby 1966) and human (Harris 1966) genetic polymorphism discovered by using protein gelelectrophoresis is consistent with the hypothesis that natural polymorphisms are largely neutral. Summarizing evidence from molecularbiology, King and Jukes 1969 also argue that most evolutionary changes in DNA sequence are neutral. While the idea of neutralevolution of DNA sequences was previously published, for example, in Sueoka 1962 and Freese 1962, Kimura 1968 and King andJukes 1969 have certainly caught much wider attention and are commonly regarded as the origin of neutral theory, a response thatmay be in part owing to the provocative title of King and Jukes 1969.

Dietrich, M. R. 1994. The origins of the neutral theory of molecular evolution. Journal of the History of Biology 27:21–59.

A science historian’s reconstruction of the origin of the neutral theory; the reconstruction emphasizes the critical role of molecular biology.
















Freese, E. 1962. On the evolution of base composition at DNA. Journal of Theoretical Biology 3:82–101.

Shows the variation of base composition among different species and suggests that many nucleotide changes have no significantselective effect.

Harris, H. 1966. Enzyme polymorphisms in man. Proceedings of the Royal Society of London B: Biological Sciences 164:298–310.

One of the first papers revealing high levels of proteinpolymorphism in humans.

Kimura, M. 1968. Evolutionary rate at the molecular level. Nature 217:624–626.

The nucleotide replacement rate per genome per generation was found to be too high to have been driven by positive selection,prompting the author to hypothesize that most nucleotide replacements must have been neutral. This article is widely regarded as thedebut of the neutral theory.

King, J. L., and T. H. Jukes. 1969. NonDarwinian evolution. Science 164:788–798.

The authors argue for neutral evolution of DNA sequences on the basis of the then knowledge of molecular biology, largelycomplementary to the evidence used in Kimura 1968.

Lewontin, R. C. 1974. The genetic basis of evolutionary change. New York: Columbia Univ. Press.

In this classic book, Lewontin discusses evolutionary explanations of the high genetic polymorphisms revealed by protein gelelectrophoresis.

Lewontin, R. C., and J. L. Hubby. 1966. A molecular approach to the study of genic heterozygosity in natural populations: II. Amountof variation and degree of heterozygosity in natural populations of Drosophila pseudoobscura. Genetics 54:595–609.

One of the first papers to reveal high intraspecific protein polymorphisms by gel electrophoresis.

Sueoka, N. 1962. On the genetic basis of variation and heterogeneity of DNA base composition. Proceedings of the NationalAcademy of Sciences of the United States of America 48:582–592.

The author explains the large variation in base composition among bacteria by mutational pressure without the involvement of selection.

Initial Response to the Neutral Theory

The neutral theory received strong opposition from neoDarwinists immediately after its publication. Maynard Smith 1968 shows that,under truncation selection, the upper limit of the rate of adaptive evolution can be several orders of magnitude higher than thatsuggested in Haldane 1957 and is not necessarily inconsistent with the observed nucleotide substitution rate. A similar argument ismade in King 1967, Milkman 1967, and Sved, et al. 1967 that the observation in Lewontin and Hubby 1966 (cited under Origin of theTheory) of a high heterozygosity of fruitfly isozymes could be explained by overdominant selection without causing too high asegregational load. Here, segregational load refers to the genetic load (i.e., reduction of population mean fitness compared with thefittest genotype in the population) caused by genes segregating from advantageous heterozygotes to less fit homozygotes. Clarke 1970and Richmond 1970 assert that some of the phenomena King and Jukes use in supporting neutral evolution are better explained bynatural selection. For example, King and Jukes argue that synonymous substitutions, which do not alter protein sequence, are most likelyneutral. But, Clarke and Richmond argue that synonymous codon usage is potentially selected to optimize translational efficiency. There












were some misunderstandings about the neutral theory in its infancy. Without realizing that the neutral theory allows the predominantrole of purifying selection in molecular evolution, some authors, e.g., those of Clarke 1970 and Richmond 1970, have used the inferenceof purifying selection to argue against neutral theory. It is the limited role of positive selection in molecular evolution that distinguishesneural theory from neoDarwinism. Some authors, such as the author of Gatlin 1976, regarded nonrandom genome sequences asevidence against neutral theory, but nonrandom patterns could be due to mutational bias and/or differential purifying selection.

Clarke, B. 1970. Darwinian evolution of proteins. Science 168:1009–1011.

The author argues that the evolution of proteins is driven by natural selection rather than genetic drift.

Gatlin, L. L. 1976. Counterexamples to a neutralist hypothesis. Journal of Molecular Evolution 7:185–195.

The author mistakenly argues against the neutral theory because she is confusing mutational bias with selection and positive selectionwith negative selection.

Haldane, J. B. S. 1957. The cost of natural selection. Journal of Genetics 55:511–524.

Considering the limited reproductive rate and the cost of replacing an amino acid with a fitter one, the author proposes that the rate ofadaptive evolution cannot exceed one per 300 generations in mammals.

King, J. L. 1967. Continuously distributed factors affecting fitness. Genetics 55:483–492.

One of the first papers proposing the possibility of maintaining many balanced polymorphisms without a high segregational load.

Maynard Smith, J. 1968. “Haldane’s dilemma” and the rate of evolution. Nature 219:1114–1116.

Demonstrates that truncation selection substantially reduces the substitution load calculated by Haldane 1957 and thus allows adaptiveaminoacid replacements that are as rapid as what Kimura 1968 (cited under Origin of the Theory) observed.

Milkman, R. D. 1967. Heterosis as a major cause of heterozygosity in nature. Genetics 55:493–495.

Argues that multiple balanced genetic polymorphisms would not lead to unbearably high segregational load because selection acts onindividual organisms rather than individual genes.

Richmond, R. C. 1970. NonDarwinian evolution: A critique. Nature 225:1025–1028.

The author criticizes the neutral theory by asserting the role of natural selection in molecular evolution.

Sved, J. A., T. E. Reed, and W. F. Bodmer. 1967. The number of balanced polymorphisms that can be maintained in a naturalpopulation. Genetics 55:469–481.

Argues that a large number of polymorphisms could be maintained by balancing selection without imposing an unbearablesegregational load.

Evidence for the Neutral Theory








Over the years, the neutral theory has been supported by a number of lines of evidence. First, Zuckerkandl and Pauling 1965 proposesthe concept of “molecular clock” based on the authors’ observation that the aminoacid substitution rate per year for a protein is more orless constant across different evolutionary lineages. Because the rate of neutral substitution equals the rate of neutral mutation, neutraltheory can explain the molecularclock phenomenon if the neutral mutation rate is constant per year. By contrast, under neoDarwinism,the substitution rate is determined by the product of the effective population size, the selection coefficient, and the rate of adaptivemutation and is thus unlikely to be constant across different evolutionary lineages. Therefore, the molecularclock phenomenon isconsidered one of the strongest pieces of evidence for the neutral theory. Second, neutral theory predicts that the evolutionary rate of agene or a site increases as its functional constraint reduces, because a reduced functional constraint means an increased fraction ofneutral mutations. By contrast, neoDarwinism predicts that the evolutionary rate of a gene or a site increases as its functional importanceincreases, because high functional importance means high intensity of positive selection. Kimura and Ohta 1974 shows that thefunctionally important and constrained histone H4 evolves much more slowly than the functionally relatively unimportant andunconstrained fibrinopeptides, consistent with the prediction of the neutral theory. Because synonymous sites are functionally lessconstrained than nonsynonymous sites, the preponderance of substitutions per synonymous site in the evolution of proteincoding genesalso supports the neutral theory, as Kimura 1977 argues. Similarly, Li, et al. 1981 shows exceedingly rapid evolution of functionlessgenes known as pseudogenes, compared with functional genes, strongly supporting the neutral theory. Third, Nei and Graur 1984analyzes intraspecific protein polymorphisms measured by gel electrophoresis from seventyseven species. The authors report that thedata are generally consistent with the prediction of neutral theory under population bottlenecks but are incompatible with the model offrequent overdominant selection. More recent evidence is reviewed in Kimura 1991, Takahata 1996, and Nei, et al. 2010.

Kimura, M. 1977. Preponderance of synonymous changes as evidence for the neutral theory of molecular evolution. Nature267:275–276.

Argues that faster evolution at functionally less constrained synonymous sites than functionally more constrained nonsynonymous sitessupports the neutral theory.

Kimura, M. 1991. The neutral theory of molecular evolution: A review of recent evidence. Japanese Journal of Genetics 66:367–386.

An update on the evidence for the neutral theory since the publication of Kimura 1983 (cited under General Overviews, the NeutralHypothesis of Phenotypic Evolution, and Outstanding Questions).

Kimura, M., and T. Ohta. 1974. On some principles governing molecular evolution. Proceedings of the National Academy ofSciences of the United States of America 71:2848–2852.

This influential paper summarizes five fundamental rules of molecular evolution.

Li, W. H., T. Gojobori, and M. Nei. 1981. Pseudogenes as a paradigm of neutral evolution. Nature 292:237–239.

Shows that the extremely rapid evolution of functionless pseudogenes compared with functional genes lends the strongest support to theneutral theory.

Nei, M., and D. Graur. 1984. Extent of protein polymorphism and the neutral mutation theory. Evolutionary Biology 17:73–118.

Concludes that protein polymorphism levels are consistent with the neutral prediction with frequent population bottlenecks.

Nei, M., Y. Suzuki, and M. Nozawa. 2010. The neutral theory of molecular evolution in the genomic era. Annual Review of Genomicsand Human Genetics 11:265–289.

An update on the neutral theory in the context of genomic data.












Takahata, N. 1996. Neutral theory of molecular evolution. Current Opinion in Genetics & Development 6:767–772.

An update on the neutral theory.

Zuckerkandl, E., and L. Pauling. 1965. Evolutionary divergence and convergence in proteins. In Evolving Genes and Proteins.Edited by V. Bryson and H. J. Vogel. New York: Academic Press.

An insightful paper reporting an approximately constant rate of proteinsequence evolution known as the molecular clock.

NeutralistSelectionist Debate

The neutralistselectionist debate started immediately after the debut of the neutral theory in 1968 and has lasted to date. There are fourkey arguments against neutral theory. First, it was argued that the substitution rate of a protein is far from constant and that thisinconstancy was considered to reflect the action of positive selection. Gillespie 1991 summarizes this line of argument. A genomic studyin mammals in Kim and Yi 2008, however, finds no evidence for the overdispersed molecular clock for nonsynonymous substitutions.Second, mutation rate is commonly believed to be constant per generation, rather than per year. Thus, it has been argued that theobserved molecular clock per year cannot be explained by the neutral theory. Ohta developed the nearly neutral theory that can explainwhy the protein evolutionary rate is approximately constant per year, as reviewed in Akashi, et al. 2012. Third, synonymous substitutionswere initially believed to be the best example of neutral changes, but Ikemura 1981 finds that codons with high cognatetRNAconcentrations are used more often than other synonymous codons of the same amino acid, suggesting that synonymous substitutionsare subject to natural selection in relation to translation. Fourth, according to the neutral theory, the amount of genetic polymorphism in apopulation increases with the effective population size, but the lack of populations with very high protein polymorphisms, referred to as“invariance of heterozygosity” in Lewontin 1974 (cited under Origin of the Theory), poses a challenge to the neutral theory. Thisphenomenon is explained by frequent population bottlenecks in Nei and Graur 1984 and stronger selection against slightly deleteriousmutations in larger populations in Akashi, et al. 2012. A direct debate between a neutralist and a selectionist can be found in Ohta 1996and Kreitman 1996. Golding 1994 is an edited volume that presents empirical data supporting nonneutral evolution and statisticalmethods for testing neutrality.

Akashi, H., N. Osada, and T. Ohta. 2012. Weak selection and protein evolution. Genetics 192:15–31.

A thorough review of the nearly neutral theory in the context of proteinsequence evolution.

Gillespie, J. H. 1991. The causes of molecular evolution. New York: Oxford Univ. Press.

The author argues that episodic positive selection rather than fixation of neutral mutations by genetic drift explains molecular evolution.

Golding, B. 1994. Nonneutral evolution: Theories and molecular data. New York: Springer.

This edited volume contains empirical data that support the action of natural selection as well as statistical methods that can be used totest the null hypothesis of neutrality.

Ikemura, T. 1981. Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respectivecodons in its protein genes: A proposal for a synonymous codon choice that is optimal for the E. coli translational system. Journalof Molecular Biology 151:389–409.

Finds that codon usage is correlated with the cognate tRNA abundance, suggesting an adaptive nature of biased synonymous codonusage.













Kim, S. H., and S. V. Yi. 2008. Mammalian nonsynonymous sites are not overdispersed: Comparative genomic analysis of index ofdispersion of mammalian proteins. Molecular Biology and Evolution 25:634–642.

A reanalysis, using genomic data, on the overdispersion of molecular clock.

Kreitman, M. 1996. The neutral theory is dead. Long live the neutral theory. Bioessays 18:678–683.

An argument against the neutral theory in a debate with Ohta 1996. Specifically, Kreitman argues that the neutral theory serves as auseful null hypothesis that is nevertheless rejected by empirical data.

Nei, M., and D. Graur. 1984. Extent of protein polymorphism and the neutral mutation theory. Evolutionary Biology 17:73–118.

Explains the phenomenon of invariance of heterozygosity by frequent population bottlenecks.

Ohta, T. 1996. The current significance and standing of neutral and nearly neutral theories. Bioessays 18:673–677.

An argument for the neutral and nearly neutral theories in a debate with Kreitman 1996. Specifically, Ohta defends the nearly neutraltheory, arguing that many data that are seemingly incompatible with the neutral theory are explainable by the nearly neutral theory.

The Nearly Neutral Theory

Kimura defined neutral mutations by |2Ns|<<1, where N is the effective population size and s is the selection coefficient. The nearlyneutral theory was mainly developed by Ohta and she defines nearly neutral mutations by |Ns|~1. Unlike neutral mutations, whose fate isindependent of effective population size, the fate of nearly neutral mutations depends on the effective population size. As reviewed inAkashi, et al. 2012, the nearly neutral theory is able to answer almost each of selectionists’ challenges mentioned in NeutralistSelectionist Debate. Ohta 1973 is often considered the origin of the theory, which was initially named the slightly deleterious mutationtheory. The theory was originally proposed in Ohta 1972a to explain why the protein evolutionary rate is approximately constant per year,while the DNA evolutionary rate shows a generation time effect (i.e., higher rates for species with shorter generations). The nearly neutraltheory predicts a negative correlation between the protein evolutionary rate and population size, a correlation verified in Ohta 1972b andOhta 1995, and was later confirmed in genomescale analysis, for example, by Rhesus Macaque Genome Sequencing and AnalysisConsortium 2007. A comparison between conservative and radical nonsynonymous substitution rates in Zhang 2000 also supports thenearly neutral theory. Comparisons between freeliving bacteria and related endosymbiotic bacteria in molecular evolution rates inMoran 1996 strongly support the theory.

Akashi, H., N. Osada, and T. Ohta. 2012. Weak selection and protein evolution. Genetics 192:15–31.

A comprehensive summary of the nearly neutral theory.

Moran, N. A. 1996. Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. Proceedings of the National Academy ofSciences of the United States of America 93:2873–2878.

Contends that accelerated gene evolution of endosymbiotic bacteria, which have small population sizes, supports the nearly neutraltheory.

Ohta, T. 1972a. Evolutionary rate of cistrons and DNA divergence. Journal of Molecular Evolution 1:150–157.













Explains why the protein evolutionary rate is approximately constant per year, while the DNA evolutionary rate is approximately constantper generation.

Ohta, T. 1972b. Population size and rate of evolution. Journal of Molecular Evolution 1:305–314.

Suggests that protein evolution is faster in smaller populations.

Ohta, T. 1973. Slightly deleterious mutant substitutions in evolution. Nature 246:96–98.

Argues for the importance of slightly deleterious mutations in evolution.

Ohta, T. 1995. Synonymous and nonsynonymous substitutions in mammalian genes and the nearly neutral theory. Journal ofMolecular Evolution 40:56–63.

Analysis of fortynine mammalian genes shows a higher nonsynonymoustosynonymous substitution rate ratio in species with smallpopulations than those with large populations, supporting the nearly neutral theory.

Rhesus Macaque Genome Sequencing and Analysis Consortium 2007. Evolutionary and biomedical insights from the rhesusmacaque genome. Science 316:222–234.

Confirms that genomic data show a higher nonsynonymoustosynonymous substitution rate ratio in primates (with small populations)than rodents (with large populations), consistent with the nearly neutral theory.

Zhang, J. 2000. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. Journalof Molecular Evolution 50:56–68.

Shows that the rate ratio of radicaltoconservative nonsynonymous substitutions is higher in species with smaller effective populationsizes, consistent with the nearly neutral theory.

Neutrality Tests

While the general validity of the neutral theory remains controversial, almost nobody denies the importance of neutrality as a nullhypothesis in the study of evolutionary forces. Many tests of the neutral hypothesis have been developed. They are generally classifiedinto three groups, depending on whether the genetic variation considered is divergence, polymorphism, or both. It should be noted thatmany neutrality tests applied to individual genes have low statistical power such that failure to reject the neutral hypothesis may notequal a complete lack of selection.

Divergence Data of DNA Sequences

The standard method of testing the neutral hypothesis by using divergence data compares the number of synonymous substitutions persynonymous site (d ) with the corresponding number of nonsynonymous substitutions per nonsynonymous site (d ). Under the nullhypothesis of neutrality, d = d . A significantly greater d than d indicates positive selection, whereas a significantly smaller d than dindicates negative selection. Among the commonly used methods for estimating d and d are those developed in Nei and Gojobori1986, Li 1993, and Goldman and Yang 1994. Synonymous and nonsynonymous substitution rates can also be compared in one or morebranches in a phylogeny instead of between two extant sequences; e.g., see Zhang, et al. 1998 and Yang 1998. Furthermore,synonymous and nonsynonymous substitution rates can be compared for a subset of codons instead of the entire gene; e.g., see Suzukiand Gojobori 1999 and Yang, et al. 2000. Finally, synonymous and nonsynonymous substitution rates can be compared for a subset of

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codons in one or more branches in a tree, as in Zhang, et al. 2005.

Goldman, N., and Z. Yang. 1994. A codonbased model of nucleotide substitution for proteincoding DNA sequences. MolecularBiology and Evolution 11:725–736.

A likelihoodbased method for estimating synonymous and nonsynonymous substitution rates that has become the standard method ingenomescale analysis.

Li, W. H. 1993. Unbiased estimation of the rates of synonymous and nonsynonymous substitution. Journal of Molecular Evolution36:96–99.

Develops a method for estimating synonymous and nonsynonymous substitution rates.

Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotidesubstitutions. Molecular Biology and Evolution 3:418–426.

Develops one of the simplest and most widely used methods for estimating synonymous and nonsynonymous substitution rates.

Suzuki, Y., and T. Gojobori. 1999. A method for detecting positive selection at single amino acid sites. Molecular Biology andEvolution 16:1315–1328.

Proposes a counting method for testing the neutral hypothesis for a subset of codons along a phylogeny.

Yang, Z. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. MolecularBiology and Evolution 15:568–573.

Proposes a likelihoodbased method for testing the neutral hypothesis in a set of branches in a phylogeny.

Yang, Z., R. Nielsen, N. Goldman, and A. M. Pedersen. 2000. Codonsubstitution models for heterogeneous selection pressure atamino acid sites. Genetics 155:431–449.

Develops a method for detecting positive selection acting on a subset of codons in a gene.

Zhang, J., H. F. Rosenberg, and M. Nei. 1998. Positive Darwinian selection after gene duplication in primate ribonuclease genes.Proceedings of the National Academy of Sciences of the United States of America 95:3708–3713.

Proposes methods for estimating synonymous and nonsynonymous substitution rates and testing the neutral hypothesis in one or morebranches in a phylogeny.

Zhang, J., R. Nielsen, and Z. Yang. 2005. Evaluation of an improved branchsite likelihood method for detecting positive selection atthe molecular level. Molecular Biology and Evolution 22:2472–2479.

Presents a likelihood method for detecting positive selection acting on a subset of codons in a subset of branches of a phylogeny.

Polymorphism Data of DNA Sequences



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The neutral hypothesis can also be tested by using polymorphism data. The most widely used methods that depend on frequencies ofvariants at polymorphic nucleotide sites include those developed in Tajima 1989, Fu and Li 1993, and Fay and Wu 2000. Some othermethods require additional information on the linkage phase among variant sites and score a haplotype as an allele such as thosedeveloped in Ewens 1972, Watterson 1978, and Slatkin 1994. There are also methods that depend on patterns of linkage disequilibriumsuch as the extended haplotype homozygosity (EHH) test developed in Sabeti, et al. 2002 and its extension proposed in Voight, et al.2006. Different tests have different properties in terms of the power and accuracy in detecting positive or purifying selection.

Ewens, W. J. 1972. The sampling theory of selectively neutral alleles. Theoretical Population Biology 3:87–112.

This classic paper reports the probabilities associated with counts of how many different alleles are observed a given number of times ina sample under the infinite allele model with no selection.

Fay, J. C., and C. I. Wu. 2000. Hitchhiking under positive Darwinian selection. Genetics 155:1405–1413.

Adaptation is tested against the null hypothesis of neutrality by comparing two measures of DNA sequence polymorphism.

Fu, Y. X., and W. H. Li. 1993. Statistical tests of neutrality of mutations. Genetics 133:693–709.

The null hypothesis of neutrality in a constantsize population is tested by comparing measures of DNA sequence polymorphism that areexpected to be equal under the null hypothesis.

Sabeti, P. C., D. E. Reich, J. M. Higgins, et al. 2002. Detecting recent positive selection in the human genome from haplotypestructure. Nature 419:832–837.

Proposes the extended haplotype homozygosity (EHH) test of neutrality.

Slatkin, M. 1994. An exact test for neutrality based on the Ewens sampling distribution. Genet Res 64:71–74.

A neutrality test based on Ewens’ prediction of sampling distribution.

Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585–595.

The neutrality hypothesis is tested by comparing two measures of DNA sequence polymorphism that should be equal under neutrality ina constantsize population.

Voight, B. F., S. Kudaravalli, X. Wen, and J. K. Pritchard. 2006. A map of recent positive selection in the human genome. PLOSBiology 4:e72.

Proposes a modified version of the EHH test named the iHS (integrated haplotype score) test.

Watterson, G. A. 1978. The homozygosity test of neutrality. Genetics 88:405–417.

Presents a test of neutrality by comparing the observed and expected homozygosity under neutrality.

Polymorphism and Divergence Data











The neutral theory can also be tested by comparing polymorphism and divergence data. Hudson, et al. 1987 proposes such a test bycomparing the ratio of divergence and polymorphism between two loci. Neutrality is rejected (i.e., at least one locus is under purifying orpositive selection) when this ratio is unequal between two loci. McDonald and Kreitman 1991 revises the test in Hudson, et al. 1987 bycomparing synonymous sites and nonsynonymous sites of the same gene instead of comparing two loci. Under the assumption thatsynonymous sites are neutral, a rejection of the null hypothesis by the McDonaldKreitman test may indicate the action of positionselection for or negative selection against nonsynonymous substitutions. Specifically, when the ratio between the number of interspecificnonsynonymous differences (Dn) and the number of intraspecific nonsynonymous polymorphisms (Pn) significantly exceeds thecorresponding synonymous ratio (Ds/Ps), positive selection for interspecific nonsynonymous differences is inferred. By contrast, whenthe nonsynonymous ratio is significantly smaller than the synonymous ratio, negative selection against interspecific nonsynonymousdifferences is inferred. Because many lowfrequency polymorphisms are deleterious, Fay, et al. 2002 proposes to remove lowfrequency(e.g., less than 10 percent) derived alleles from the counts of polymorphisms in the McDonaldKreitman test to improve its power indetecting positive selection.

Fay, J. C., G. J. Wyckoff, and C. I. Wu. 2002. Testing the neutral theory of molecular evolution with genomic data from Drosophila.Nature 415:1024–1026.

Proposes a modification of the McDonaldKreitman test (McDonald and Kreitman 1991) to improve the power of detecting positiveselection in the presence of deleterious mutations.

Hudson, R. R., M. Kreitman, and M. Aguade. 1987. A test of neutral molecular evolution based on nucleotide data. Genetics116:153–159.

Neutrality is tested by comparing the divergence to polymorphism ratio between two loci.

McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351:652–654.

One of the most widely used tests of neutrality, it compares nonsynonymous and synonymous variations between and within species at agene.

Adaptive Molecular Evolution

Numerous tests of the hypothesis of neutral evolution have been conducted for DNA sequences. In a number of cases, neutrality isrejected in favor of adaptation driven by positive Darwinian selection. The best studied type of molecular adaptation, investigated inmany individual genes as well as genomes, involves beneficial nonsynonymous nucleotide changes. Genomewide fraction of aminoacid substitutions that are adaptive has been estimated for some species. In addition, other types of molecular adaptations, such asthose involving gene duplication or gene loss, have also been discovered. It should be noted that neutral theory allows the prominentrole of positive selection in the evolution of a minority of genes. Thus, rejecting the neutral hypothesis for a small fraction of genes doesnot refute the neutral theory.

Adaptive Protein Evolution Detected by Neutrality Tests from Studies of Individual Genes

A number authors reported the rejection of the neutral hypothesis by using the methods described in Neutrality Tests. Hughes and Nei1988 shows that d is significantly greater than d in the antigen recognition regions of the human majorhistocompatibilitycomplex(MHC) genes, suggesting the action of positive selection, which is most likely related to the immune function of MHC. Numerous geneshave since been shown to be subject to recurrent or episodic positive selection on nonsynonymous changes. Bestknown examplesinclude the hemagglutinin gene in human influenza viruses reported in Bush, et al. 1999; abalone sperm lysin reported in Lee, et al.1995; Drosophila “speciation genes” Odysseus reported in Ting, et al. 1998 and Nup96 reported in Presgraves, et al. 2003; colobinemonkey pancreatic ribonuclease gene reported in Zhang, et al. 2002b; and human speech gene FOXP2 studied in Enard, et al. 2002and Zhang, et al. 2002a.

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Bush, R. M., W. M. Fitch, C. A. Bender, and N. J. Cox. 1999. Positive selection on the H3 hemagglutinin gene of human influenzavirus A. Molecular Biology and Evolution 16:1457–1465.

Aminoacid sites in influenza hemagglutinin that are subject to recurrent positive selection are identified.

Enard, W., M. Przeworski, S. E. Fisher, et al. 2002. Molecular evolution of FOXP2, a gene involved in speech and language. Nature418:869–872.

A gene critical to human speech is found to have evolved under positive selection during human origins.

Hughes, A. L., and M. Nei. 1988. Pattern of nucleotide substitution at major histocompatibility complex class I loci revealsoverdominant selection. Nature 335:167–170.

Discovers one of the first examples of molecular adaptation.

Lee, Y. H., T. Ota, and V. D. Vacquier. 1995. Positive selection is a general phenomenon in the evolution of abalone sperm lysin.Molecular Biology and Evolution 12:231–238.

Reports very high d /d ratios between sperm lysins of different abalones.

Presgraves, D. C., L. Balagopalan, S. M. Abmayr, and H. A. Orr. 2003. Adaptive evolution drives divergence of a hybrid inviabilitygene between two species of Drosophila. Nature 423:715–719.

Reports that adaptive divergence of a nuclear pore protein underlies the hybrid inviability between two Drosophila species.

Ting, C. T., S. C. Tsaur, M. L. Wu, and C. I. Wu. 1998. A rapidly evolving homeobox at the site of a hybrid sterility gene. Science282:1501–1504.

Reports that rapid evolution of a homeobox gene is responsible in part for hybrid sterility in Drosophila.

Zhang, J., D. M. Webb, and O. Podlaha. 2002a. Accelerated protein evolution and origins of humanspecific features: Foxp2 as anexample. Genetics 162:1825–1835.

Reports substantial acceleration and action of positive selection in the evolution of a human speech gene.

Zhang, J., Y. P. Zhang, and H. F. Rosenberg. 2002b. Adaptive evolution of a duplicated pancreatic ribonuclease gene in a leafeatingmonkey. Nature Genetics 30:411–415.

Reports that adaptive evolution of a duplicate pancreatic ribonuclease gene followed the evolution of leaf eating in colobine monkeys.

Adaptive Protein Evolution Detected by Neutrality Tests from Genomic Studies

Identifying genes subject to positive selection using the methods described in Neutrality Tests has been a routine practice in comparativegenomic studies. One of the first genomewide scans of positive selection, Clark, et al. 2003 reports positively selected genes in thehuman lineage since its divergence from the chimpanzee lineage about six million years ago. Sabeti, et al. 2007 reports genes subject tocomparatively recent position selection within humans. Bakewell, et al. 2007 reports that more genes underwent positive selection inchimpanzees than in humans since their separation, probably because of a larger effective population size and hence more effective

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selection in the former than the latter. Qiu, et al. 2012 reports positively selected genes related to hypoxia and hence highaltitudeadaptation in the yak genome. The authors of Zhang, et al. 2014 have detected numerous bird genes under positive selection. Mackay,et al. 2012 identifies positively selected genes in the fruit fly Drosophila melanogaster. Huang, et al. 2012 identifies fiftyfive selectivesweeps during rice domestication. Raffaele, et al. 2010 reports positively selected genes in the fungal pathogen Phytophthora infestansthat causes potato blight, a serious potato disease. These experiments are just a few examples of a very large number of genome scansof positive selection.

Bakewell, M. A., P. Shi, and J. Zhang. 2007. More genes underwent positive selection in chimpanzee evolution than in humanevolution. Proceedings of the National Academy of Sciences of the United States of America 104:7489–7494.

Reports more positively selected genes in chimpanzee evolution than in human evolution.

Clark, A. G., S. Glanowski, R. Nielsen, et al. 2003. Inferring nonneutral evolution from humanchimpmouse orthologous gene trios.Science 302:1960–1963.

Infers positive selection for hundreds of genes in the human branch after the humanchimpanzee separation.

Huang, X., N. Kurata, X. Wei, et al. 2012. A map of rice genome variation reveals the origin of cultivated rice. Nature 490:497–501.

Identifies fiftyfive selective sweeps during rice domestication from the genome sequences of 446 geographically diverse accessions ofwild rice and 1,083 cultivated varieties.

Mackay, T. F., S. Richards, E. A. Stone, et al. 2012. The Drosophila melanogaster Genetic Reference Panel. Nature 482:173–178.

Identifies positively selected genes in the genome sequences of 168 lines from one outbred fruitfly population.

Qiu, Q., G. Zhang, T. Ma, et al. 2012. The yak genome and adaptation to life at high altitude. Nature Genetics 44:946–949.

A comparison between yak and cow genomes revealing that positively selected genes in the yak lineage are enriched in functionalcategories and pathways related to hypoxia and nutrition metabolism.

Raffaele, S., R. A. Farrer, L. M. Cano, et al. 2010. Genome evolution following host jumps in the Irish potato famine pathogen lineage.Science 330:1540–1543.

Positively selected genes are identified from the Irishpotatofamine pathogen.

Sabeti, P. C., P. Varilly, B. Fry, et al. 2007. Genomewide detection and characterization of positive selection in human populations.Nature 449:913–918.

More than 300 positively selected regions of the human genome are identified on the basis of polymorphism data.

Zhang, G., C. Li, Q. Li, et al. 2014. Comparative genomics reveals insights into avian genome evolution and adaptation. Science346:1311–1320.

A comparison of fortyeight bird genomes that detects many genes under positive selection.








Fraction of Adaptive AminoAcid Substitutions

When the author of Kimura 1968 (cited under Origin of the Theory) inferred the rate of aminoacid substitution per generation permammalian genome by using only three proteins, he was unaware that only 1.5 percent of the mammalian genome is protein coding andmistakenly assumed that this number is 100 percent. On the basis of the estimate of evolutionary rates from thousands of proteins, Zhang2010 calculates that Haldane’s upper limit of the rate of adaptive evolution (Haldane 1957, cited under Initial Response to the NeutralTheory) is not violated even when 33 percent of aminoacid substitutions in mammals are adaptive. Smith and EyreWalker 2002proposes that one can estimate the fraction of aminoacid substitutions that are adaptive by α=1−(DsPn)/(DnPs) (see Neutrality Tests:Polymorphism and Divergence Data for notation). EyreWalker 2006 reviews the estimates of α in a number of species, finding that itvaries from nearly zero in humans and Arabidopsis to over 50 percent in Drosophila and some microbes and viruses. By modifying theMcDonaldKreitman test (McDonald and Kreitman 1991, cited under Neutrality Tests: Polymorphism and Divergence Data), the authorsof Bustamante, et al. 2002 develop a hierarchical Bayesian method to estimate the mean scaled selection coefficient and its variance fornonsynonymous substitutions of each gene. The authors of Sawyer, et al. 2003 further develop this method to allow variation in selectioncoefficient among nonsynonymous substitutions within a gene. Using this method, Sawyer, et al. 2007 shows that approximately 95percent of aminoacid substitutions in Drosophila is adaptive. The finding in Drosophila and several microbes that most aminoacidsubstitutions in a genome have been driven by positive selection seriously challenges the neutral theory.

Bustamante, C. D., R. Nielsen, S. A. Sawyer, K. M. Olsen, M. D. Purugganan, and D. L. Hartl. 2002. The cost of inbreeding inArabidopsis. Nature 416:531–534.

Develops a method for estimating the distribution of selection coefficient from polymorphism and divergence data.

EyreWalker, A. 2006. The genomic rate of adaptive evolution. Trends in Ecology & Evolution 21:569–575.

A review of the fraction of adaptive aminoacid substitutions in multiple species.

Sawyer, S. A., R. J. Kulathinal, C. D. Bustamante, and D. L. Hartl. 2003. Bayesian analysis suggests that most amino acidreplacements in Drosophila are driven by positive selection. Journal of Molecular Evolution 57.Suppl. 1: S154–S164.

Develops a Bayesian randomeffect model to estimate the distribution of selection coefficient from polymorphism and divergence data.

Sawyer, S. A., J. Parsch, Z. Zhang, and D. L. Hartl. 2007. Prevalence of positive selection among nearly neutral amino acidreplacements in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 104:6504–6510.

Estimates that 95 percent of aminoacid substitutions in Drosophila are adaptive.

Smith, N. G., and A. EyreWalker. 2002. Adaptive protein evolution in Drosophila. Nature 415:1022–1024.

First estimation, using relatively large data, of the fraction of adaptive aminoacid substitutions.

Zhang, J. 2010. Evolutionary genetics: Progress and challenges. In Evolution since Darwin: The First 150 Years. Edited by M. A.Bell, D. J. Futuyma, W. F. Eanes, J. S. Levinton. Sunderland, MA: Sinauer.

A review of the progress of evolutionary genetics from the 1950s to the 2000s.

Other Types of Molecular Adaptation Detected by Neutrality Tests

In addition to the adaptive evolution of protein sequences, adaptive evolution of promoters that affect gene expression has also been












reported. The best known examples include the human prodynorphin gene promoter reported in Rockman, et al. 2005 and humanlactase gene promoter reported in Tishkoff, et al. 2007. The authors of Haygood, et al. 2007 have conducted a genomewide scan ofhuman promoters to detect signals of positive selection. Gene loss may also be subject to positive selection, as reported in Wang, et al.2006 for human CASPASE12 and reported in MacArthur, et al. 2007 for human ACTN3. In addition to point mutations,insertions/deletions may be subject to positive selection, as reported in Podlaha and Zhang 2003 for mammalian CATSPER1, via thecomparison between insertion/deletion substitution rates in coding and noncoding regions.

Haygood, R., O. Fedrigo, B. Hanson, K. D. Yokoyama, and G. A. Wray. 2007. Promoter regions of many neural and nutritionrelatedgenes have experienced positive selection during human evolution. Nature Genetics 39:1140–1144.

Reports genomic scan of positive selection in human promoters.

MacArthur, D. G., J. T. Seto, J. M. Raftery, et al. 2007. Loss of ACTN3 gene function alters mouse muscle metabolism and showsevidence of positive selection in humans. Nature Genetics 39:1261–1265.

Reports that lossoffunction mutants of ACTN3 are positively selected in humans.

Podlaha, O., and J. Zhang. 2003. Positive selection on proteinlength in the evolution of a primate sperm ion channel. Proceedingsof the National Academy of Sciences of the United States of America 100:12241–12246.

Reports greater insertion/deletion substitution rates in the coding sequence of a cation channel regulating sperm motility than inpresumably neutral regions.

Rockman, M. V., M. W. Hahn, N. Soranzo, F. Zimprich, D. B. Goldstein, and G. A. Wray. 2005. Ancient and recent positive selectiontransformed opioid cisregulation in humans. PLOS Biology 3:e387.

Presents evidence for the action of positive selection on cisregulation of human prodynorphin.

Tishkoff, S. A., F. A. Reed, A. Ranciaro, et al. 2007. Convergent adaptation of human lactase persistence in Africa and Europe.Nature Genetics 39:31–40.

Reports adaptive evolution of the lactase promoter in Africans.

Wang, X., W. E. Grus, and J. Zhang. 2006. Gene losses during human origins. PLOS Biology 4:e52.

Presents population genetic evidence for the action of positive selection on a null allele of human CASPASE12.

Molecular Adaptations Revealed by Other Methods

In addition to the methods mentioned in Neutrality Tests, several other methods have been designed to detect positive selection inmolecular evolution. First, parallel and convergent aminoacid substitutions have been reported in some proteins. The authors of Zhangand Kumar 1997 have developed a statistical method to test whether the observed numbers of parallel and convergent substitutionsexceed the neutral expectations. An example of parallel proteinsequence evolution can be found in Li, et al. 2010 on the hearing genePrestin of echolocating bats and whales. Christin, et al. 2010 reviews empirical evidence for parallel and convergent molecularevolution. Second, gene expression noise, defined by the variation in the mRNA or protein expression level of a gene among isogenicindividuals in the same environment, has been shown to be ubiquitous. Zhang, et al. 2009 proposes a theory on how a high level ofgene expression noise may be advantageous for some genes under certain conditions and provides evidence that yeast plasmamembrane transporters are subject to positive selection for elevated expression noise. In principle, nothing rejects neutrality more













convincingly than the demonstration that a substitution improves organismal fitness, but because it is usually difficult to know the exactgenetic background and environmental context in which the substitution occurred, fitness data might or might not be relevant.Nevertheless, when combined with other information such as gene expressional or functional changes and/or population geneticevidence, fitness data can be quite powerful. For example, Qian and Zhang 2014 shows by using fitness data and a series of geneexpression and protein function data that gene duplication frequently leads to adaptation. This finding complements an earlier casestudy in Hittinger and Carroll 2007, showing, on the basis of expressional, functional, and fitness data that the duplication of a key playerin the yeast galactose use pathway was adaptive. Beyond the single gene level, it has been argued that the expansions/contractions ofcertain gene families were subject to positive selection; e.g., see Demuth, et al. 2006 for a genomic study of mammalian gene familiesand Shi and Zhang 2007 for a case study of vertebrate vomeronasal receptor gene families.

Christin, P. A., D. M. Weinreich, and G. Besnard. 2010. Causes and evolutionary significance of genetic convergence. Trends inGenetics 26:400–405.

A review of various types of molecular convergence in evolution.

Demuth, J. P., T. De Bie, J. E. Stajich, N. Cristianini, and M. W. Hahn. 2006. The evolution of mammalian gene families. PLOS One1:e85.

A genomic analysis demonstrating the potential role of positive selection in some gene family expansions.

Hittinger, C. T., and S. B. Carroll. 2007. Gene duplication and the adaptive evolution of a classic genetic switch. Nature 449:677–681

A detailed analysis of expression, function, and fitness that demonstrates the adaptive evolution of a key regulator of the yeast galactoseuse pathway.

Li, Y., Z. Liu, P. Shi, and J. Zhang. 2010. The hearing gene Prestin unites echolocating bats and whales. Current Biology 20:R55–R56

A case of adaptive convergent evolution of a hearing protein in echolocating mammals.

Qian, W., and J. Zhang. 2014. Genomic evidence for adaptation by gene duplication. Genome Research 24:1356–1362.

Genomic analysis of the fitness of yeast strains with one or two genes deleted demonstrates that gene duplication facilitates adaptation.

Shi, P., and J. Zhang. 2007. Comparative genomic analysis identifies an evolutionary shift of vomeronasal receptor generepertoires in the vertebrate transition from water to land. Genome Research 17:166–174.

An analysis of vertebrate vomeronasal receptor genes suggests the involvement of positive selection in gene family size expansion.

Zhang, J., and S. Kumar. 1997. Detection of convergent and parallel evolution at the amino acid sequence level. Molecular Biologyand Evolution 14:527–536.

Develops a statistical test of adaptive convergent evolution of protein sequences.

Zhang, Z., W. Qian, and J. Zhang. 2009. Positive selection for elevated gene expression noise in yeast. Molecular Systems Biology5:299.

Evidence that positive selection drives the elevation of expression noise of a small group of yeast genes.







Neutral Evolution of Genomic Architecture

One of the most important extensions of the (nearly) neutral theory of molecular evolution is its application to the study of the origin ofgenomic architecture, including gene number, intron content, transposable element content, gene structure, etc. As the primary advocate,Lynch 2007 summarizes evidence for the role of nearly neutral mutations in the origin of genomic complexity. The chief tenet of thisthesis, more formally referred to as the mutationalhazard hypothesis, is that many complex elements of the genome, such as introns, areslightly deleterious. As the organism size increases in longterm evolution, the effective population size decreases, making it possible forslightly deleterious mutations to fix by chance. These elements may be subsequently tinkered by evolution to provide apparent utility,such as the existence of enhancers in introns. One good example is the evolution by gene duplication. It was commonly believed thatgene duplication leads to the origin of new gene functions. Force, et al. 1999 proposes that duplicate genes are initially retainedbecause of subfunctionalization (i.e., subdivision of ancestral functions to daughter genes) via degenerate mutations rather thanneofunctionalization (i.e., acquisition of new functions) by advantageous mutations. In other words, gene number in a genome canincrease via a pure neutral process. Based on an analysis of proteinprotein interactions and amongtissue gene expressions, He andZhang 2005 provides empirical evidence that subfunctionalization plays the major role in the initial fixation and retention of duplicates,whereas neofunctionalization likely occurs more slowly. Qian, et al. 2010 finds that a duplicate gene tends to have a lower expressionthan its progenitor gene, supporting subfunctionalization with regard to the expression level. Lynch and Conery 2003 provides evidencethat genome size increases as the effective population size decreases, and that intron number and size, halflife of duplicate genes, andnumber of transposable elements all increase with genome size. Lynch’s theory is not without controversy. For instance, Daubin andMoran 2004 argues that bacterial genome size and effective population size are actually positively correlated rather than negativelycorrelated, because genome size tends to decrease under relaxed selection as a result of mutational deletion bias in bacteria. Whitneyand Garland 2010 asserts that Lynch and Conery failed to correct for phylogenetic nonindependence in their statistical analysis; allsignificant correlations reported in Lynch and Conery 2003 are gone after the correction. Lynch 2011 rebuts the need for correctingphylogenetic nonindependence in this case and argues that the correction was not done properly.

Daubin, V., and N. A. Moran. 2004. Comment on “The origins of genome complexity.” Science 306:978.

The authors contend that, in bacteria, genomic complexity decreases rather than increases with the reduction of effective population size.

Force, A., M. Lynch, F. B. Pickett, A. Amores, Y. L. Yan, and J. Postlethwait. 1999. Preservation of duplicate genes bycomplementary, degenerative mutations. Genetics 151:1531–1545.

Subfunctionalization is proposed as an alternative to neofunctionalization in explaining the retention of duplicate genes.

He, X., and J. Zhang. 2005. Rapid subfunctionalization accompanied by prolonged and substantial neofunctionalization in duplicategene evolution. Genetics 169:1157–1164.

Analysis of yeast proteinprotein interaction data and human tissue geneexpression data supports the role of subfunctionalization induplicategene retention, but also identifies substantial neofunctionalization, which occurs more slowly than subfunctionalization.

Lynch, M. 2007. The origins of genome architecture. Sunderland, MA: Sinauer.

Extending the (nearly) neutral theory to the evolution of genomic architecture, this is one of the most influential books on evolutionarybiology since Kimura 1983 (cited under General Overviews, the Neutral Hypothesis of Phenotypic Evolution, and OutstandingQuestions).

Lynch, M. 2011. Statistical inference on the mechanisms of genome evolution. PLOS Genet 7:e1001389.

Lynch’s rebuttal of the view in Whitney and Garland 2010.














Lynch, M., and J. S. Conery. 2003. The origins of genome complexity. Science 302:1401–1404.

Proposes that a reduction in effective population size facilitated the fixation of slightly deleterious mutations and led to genomiccomplexity.

Qian, W., B. Y. Liao, A. Y. Chang, and J. Zhang. 2010. Maintenance of duplicate genes and their functional redundancy by reducedexpression. Trends in Genetics 26:425–430.

Reports that reduction of expression level after gene duplication, a special type of subfunctionalization, is prevalent in yeast andmammals and explains the long retention of duplicate genes.

Whitney, K. D., and T. Garland Jr. 2010. Did genetic drift drive increases in genome complexity? PLOS Genet 6:e1001080.

Questions Lynch and Conery’s statistical analysis (Lynch and Conery 2003) that was reported to support the relationship betweeneffective population size and genomic complexity.

The Neutral Hypothesis of Phenotypic Evolution

The author of Kimura 1983 clearly limits his neutral theory to molecular evolution. However, at least as a null hypothesis, neutralityapplies to phenotypic evolution such as the evolution of morphological and physiological traits. Several statistical methods have beendeveloped to test the neutral hypothesis for phenotypic traits, including, for example, Lande 1976, Chakraborty and Nei 1982, Lynch andHill 1986, Turelli, et al. 1988, and Spitze 1993. A major problem in testing the neutral hypothesis of phenotypic evolution is that thepublished phenotypic data are highly biased as a result of a general interest in adaptive traits. Nei 2007 proposes that the neutral theoryalso applies to phenotypic traits in general.

Chakraborty, R., and M. Nei. 1982. Genetic differentiation of quantitative characters between populations or species:1. Mutationand random genetic drift. Genetical Research 39:303–314.

It is proposed that the ratio of interpopulational genetic variance to intrapopulational genetic variance of a quantitative trait be used fortesting the neutral evolution hypothesis of the trait.


The most comprehensive and authoritative book on the neutral theory of molecular evolution.

Lande, R. 1976. Natural selection and random genetic drift in phenotypic evolution. Evolution 30:314–334.

The author derives the expected amount of phenotypic evolution in a given time when the trait is not under selection.

Lynch, M., and W. G. Hill. 1986. Phenotypic evolution by neutral mutation. Evolution 40:915–935.

Proposes a neutrality test for phenotypic traits by comparing within and betweenpopulation genetic variances of the traits.

Nei, M. 2007. The new mutation theory of phenotypic evolution. Proceedings of the National Academy of Sciences of the UnitedStates of America 104:12235–12242.











Proposes that mutation and drift play more important roles than positive selection in phenotypic evolution.

Spitze, K. 1993. Population structure in Daphnia obtuse: Quantitative genetic and allozymic variation. Genetics 135:367–374.

Proposes a neutrality test of phenotypic traits by comparing Q of phenotypes with F of neutral loci.

Turelli, M., J. H. Gillespie, and R. Lande. 1988. Rate tests for selection on quantitative characters during macroevolution andmicroevolution. Evolution 42:1085–1089.

Proposes a test of neutral evolution of quantitative traits, on the basis of the neutral expectation in the rate of phenotypic divergence.

Outstanding Questions

An important question regarding the neutral theory is the definition of neutral mutations. Kimura 1983 defines neutral mutations as thosewith selection coefficients << 1/(2N), where N is the effective population size. In many unicellular organisms, N exceeds 10 . Thus,mutations with selection coefficients not much smaller than 5×10 would be nonneutral by Kimura’s definition. Nei, et al. 2010 suggeststhat the definition be loosened to selection coefficients <1/√2N, which equals 2.2×10 when N=10 . The authors further suggest that, forpractical purpose, any selection coefficient < 0.001 be considered more or less neutral, because of expected environmental fluctuationover generations and the stochasticity in progeny number of a given genotype. The change of the definition, if accepted, will certainlyalter the prevalence of neutral evolution. As mentioned, the fraction of adaptive aminoacid substitutions has been estimated for anumber of species. But the estimators are dependent on certain simplifying assumptions such as the constancy in population size for theevolutionary history concerned for most estimators. How often these assumptions are violated and how much the violations affect theestimates will be an important subject of future study. Apart from protein coding regions, for which the neutral theory was initiallyproposed, it is important to ask what fraction of nucleotide substitutions in noncoding regions have been driven by positive selection.Andolfatto 2005 suggests that a large fraction of nucleotide substitutions in noncoding regions of the Drosophila genome are adaptive.Whether this result applies to other species, especially mammals, which have a much larger noncoding fraction of the genome, isunclear. Thanks to rapid technological development, many previously hidden variations at the molecular level have now been revealed.Most of these variations are currently explained as adaptations, but the possibility of neutrality has started to be considered. Oneexample is the finding in Xu and Zhang 2014 that, contrary to the initial belief by many, human coding RNA editing is generallynonadaptive. The neutralistselectionist debate started in the late 1960s, but strongly opposing views can still be found in the modernliterature. For example, Lynch 2007 criticizes the default use of adaptation to explain almost any aspect of biodiversity and argues thatadaptation is not only unnecessary but also insufficient for explaining many observations, whereas Hahn 2008 argues that the neutralhypothesis has been rejected by such overwhelming molecular evidence that its continued use as a null hypothesis is not onlydisingenuous but also harmful to the field of molecular evolution. The debate is unlikely to be resolved soon.

Andolfatto, P. 2005. Adaptive evolution of noncoding DNA in Drosophila. Nature 437:1149–1152.

Reports that Drosophila population genomic data suggest a role of positive selection in many noncoding changes.

Hahn, M. W. 2008. Toward a selection theory of molecular evolution. Evolution 62:255–265.

Calls for a selection theory of molecular evolution because the author sees overwhelming evidence against the neutral hypothesis.


A classic book on the neutral theory authored by the chief proponent of the theory.

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Lynch, M. 2007. The frailty of adaptive hypotheses for the origins of organismal complexity. Proceedings of the National Academyof Sciences of the United States of America 104.Suppl. 1: 8597–8604.

Criticizes the default use of adaptive explanation of almost any aspect of biodiversity and suggests that adaptation is neither necessarynor sufficient to explain many phenomena.

Nei, M., Y. Suzuki, and M. Nozawa. 2010. The neutral theory of molecular evolution in the genomic era. Annual Review of Genomicsand Human Genetics 11:265–289.

A review of the neutral theory in the context of genomic data, it also redefines neutrality.

Xu, G., and J. Zhang. 2014. Human coding RNA editing is generally nonadaptive. Proceedings of the National Academy of Sciencesof the United States of America 111:3769–3774.

An analysis of genomewide coding RNA editing data from humans suggests that most of the editing is nonadaptive and reflectsmistakes made by editing enzymes.

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