Molecular Evolution Activities

Testing Neutrality and Selection

The Neutral Theory of Molecular Evolution

Tests of Neutrality and Selection

In 1971, Motoo Kimura and Tomoko Ohta claimed that one of the principle virtues of the neutral theory was that it generated testable predictions and in so doing promised to "emancipate" biologists from "naïve panselectionism" (Kimura and Ohta 1971, 469). Using the diffusion equation method, Kimura and Ohta generated a number of quantitative predictions for neutral alleles. For instance, in 1969, Kimura and Tomoko Ohta calculated the average number of generations until a neutral mutant was either lost or fixed in a finite population (Kimura and Ohta 1969 a and b). The average time to fixation was 4Ne. These kinds of quantitative predictions coupled with the promise of electrophoretic data in the late 1960s spurred Crow and others to argue in favor of the neutral theory; not as a correct theory, but as a highly testable theory (Crow 1969). Selectionist critics such as G. Ledyard Stebbins, Richard Lewontin, and Francisco Ayala were less convinced that decisive tests could be conducted and deployed Popperian ideas of falsifiability to assail neutralist hypotheses (Stebbins and Lewontin 1972, Ayala et al.1974). Both attitudes were justified. The neutral theory did indeed make a large number of testable quantitative predictions, but most of the tests to detect neutrality and selection from 1970 until 1985 were not considered decisive.

Testing the neutral theory ran afoul of two main problems. On the one hand, statistical tests, such as the one proposed by Warren Ewens in 1972, were very promising, but turned out to have little statistical power. It was not possible to reject the neutral null hypothesis using the Ewens test, for instance. On the other hand, non-statistical tests were disputed and considered indecisive. For instance, Francisco Ayala's group used data about electrophoretic varaibility in natural populations of Drosophila to test a range of neutralist predictions. Using a measure of heterozygosity predicted using an infinite alleles model, Ayala's and his coworkers noted that the frequency distribution of heterozygous loci was signficantly different. Instead of the predicted distribution clustered around the average heterozygosity of 0.177, the distribution was fairly even except for an excess of loci with very little heterozygosity. This excess of rare alleles was seen as an explanatory failure of the neutral theory (Ayala et al. 1974, 378). In response, Jack King noted that many of the assumptions made in the infinite alleles could be the source for the rare alleles discrepancy. Others noted that the infinite alleles model was not appropriate for electrophoretic data, since electrophoretic classes probably encompassed many allelic differences (King 1976). Ayala and his coworkers were sensitive to these criticisms and discussed mutation models for electromorphs as well as the models' assumptions. In the course of the give and take over these models, Kimura and Ohta began to advocate a larger role for slightly deleterious mutants whose frequencies would still be subject to drift. Others, such as Masatoshi Nei, began to emphasize, shifting population dynamics such as those resulting from population bottlenecks. These kinds of results were influential, in that they drove revisions of both the neutralist and selectionist models, but they were not decisive in terms of resolving the dispute between neutralists and selectionists.

The availability of DNA sequence data after 1985 represents a significant turning point for the neutral theory and its tests. Using DNA sequence data, Martin Kreitman and others devised statistical tests that could statistically distinguish between neutrality and selection. These statistical tests subject a neutral null hypothesis to rejection (Kretiman 2000). The success of this method has been hailed by Jim Crow as one of the most important events in the history of molecular evolution. However, it is important to note that the frequent rejection of neutral null hypotheses does not necessarily imply the demise of the neutral theory. The relationship between statistical null hypotheses and the neutral theory is complicated by the introduction of new neutral models of sequence evolution, new data about DNA sequences, and new methods, such as coalescents, which make neutral assumptions.


Francisco Ayala, Martin Tracey, Lorraine Barr, John McDonald, and Santiago Perez-Salas, 1974, “Genetic Variation in Natural Populations of Five Drosophila Species and the Hypothesis of the Selective Neutrality of Protein Polymorphisms,” Genetics 77: 343-384.

James Crow, 1969 “Molecular Genetics and Population Genetics,” Proceedings of the Twelfth International Congress of Genetics Vol 3: 105-113.

Kimura, M., 1969, "The Rate of Molecular Evolution Considered from the Standpoint of Population Genetics," Proceedings of the National Academy of Sciences, 63, 1181-1188.

Kimura, M. and Ohta, T. 1971. Protein polymorphism as a phase in molecular evolution. Nature 229: 467-469.

King, J. 1976 “Progress in the Neutral Mutation Random Drift Controversy,” Federation Proceedings 35: 2087-91.

Kreitman, M. 2000. Methods to Detect Selection in Populations with Application to the Human. Annu. Rev. Genomics. Hum. Genet. 1: 539-559.

Lewontin R. C. and Hubby, J. L. 1966. Molecular Approach to the Study of Genic Heterozygosity in Natural Populations. II. Amount of Variation and Degree of Heterozygosity in Natural Populations of Drosophila pseudoobscura, Genetics 54: 595-609.

Lewontin, R. C. 1974. The Genetic Basis of Evolutionary Change. Columbia Univ. Press, New York.

Stebbin, G. L. and Lewontin, R. C. 1972. Comparative Evolution at the Level of Molecules, Organisms, and Populations. Pp. 23-42 in LeCam, L. et al eds. Proceedings of the Sixth Berkeley Symposium on Mathematical Statistics. Volume V: Darwinian, Neo-Darwinian, and Non-Darwinian Evolution. Univ, of California Press, Berkeley.


Author(s) Dates Description

W. Ewens

  • Interview
1972 Distribution of allele frequencies; actual and effective numbers of alleles
R. C. Lewontin &
J. Krakauer
1973 Heterogeneity among loci as a test of selection

T. Yamazaki &
T. Maruyama

1972-1974 Relationship between gene frequency and heterozygosity; actual and effective numbers of alleles
M. Nei, P. Fuerst, &
P. Chakaraborty
1976-1978 Gene frequency and heterozygosity
G. Watterson 1977-1978 Distribution of allele frequencies
R. Hudson, M. Kreitman, & M. Aguade 1987

Within v. Between Species (two loci)

Citation analysis

F. Tajima 1989 Tajima's D -- Within Species

J. McDonald &
M. Kreitman


Within v. Between Species (synonymous v. non-synonymous)

Citation analysis



  • Topics
    • Overview of Tests
    • Richard Lewontin's presentation on Statistical Testing in Molecular Evolution
  • Interviews
    • Martin Kreitman and Richard Lewontin
    • Warren Ewens and Anya Plutynski