McDonald–Kreitman test

The McDonald–Kreitman test is a statistical test often used by evolution and population biologists to detect and measure the amount of adaptive evolution within a species by determining whether adaptive evolution has occurred, and the proportion of substitutions that resulted from positive selection (also known as directional selection). To do this, the McDonald–Kreitman test compares the amount of variation within a species (polymorphism) to the divergence between species (substitutions) at two types of sites, neutral and nonneutral. A substitution refers to a nucleotide that is fixed within one species, but a different nucleotide is fixed within a second species at the same base pair of homologous DNA sequences. A site is nonneutral if it is either advantageous or deleterious. The two types of sites can be either synonymous or nonsynonymous within a protein-coding region. In a protein-coding sequence of DNA, a site is synonymous if a point mutation at that site would not change the amino acid, also known as a silent mutation. Because the mutation did not result in a change in the amino acid that was originally coded for by the protein-coding sequence, the phenotype, or the observable trait, of the organism is generally unchanged by the silent mutation. A site in a protein-coding sequence of DNA is nonsynonymous if a point mutation at that site results in a change in the amino acid, resulting in a change in the organism's phenotype. Typically, silent mutations in protein-coding regions are used as the 'control' in the McDonald–Kreitman test. The McDonald–Kreitman test is a statistical test often used by evolution and population biologists to detect and measure the amount of adaptive evolution within a species by determining whether adaptive evolution has occurred, and the proportion of substitutions that resulted from positive selection (also known as directional selection). To do this, the McDonald–Kreitman test compares the amount of variation within a species (polymorphism) to the divergence between species (substitutions) at two types of sites, neutral and nonneutral. A substitution refers to a nucleotide that is fixed within one species, but a different nucleotide is fixed within a second species at the same base pair of homologous DNA sequences. A site is nonneutral if it is either advantageous or deleterious. The two types of sites can be either synonymous or nonsynonymous within a protein-coding region. In a protein-coding sequence of DNA, a site is synonymous if a point mutation at that site would not change the amino acid, also known as a silent mutation. Because the mutation did not result in a change in the amino acid that was originally coded for by the protein-coding sequence, the phenotype, or the observable trait, of the organism is generally unchanged by the silent mutation. A site in a protein-coding sequence of DNA is nonsynonymous if a point mutation at that site results in a change in the amino acid, resulting in a change in the organism's phenotype. Typically, silent mutations in protein-coding regions are used as the 'control' in the McDonald–Kreitman test. In 1991, John H. McDonald and Martin Kreitman derived the McDonald–Kreitman test while performing an experiment with Drosophila (fruit flies) and their differences in amino acid sequence of the alcohol dehydrogenase gene. McDonald and Kreitman proposed this method to estimate the proportion of substitutions that are fixed by positive selection rather than by genetic drift. In order to set up the McDonald–Kreitman test, we must first set up a two-way contingency table of our data on the species being investigated as shown below: To quantify the values for Ds, Dn, Ps, and Pn, you count the number of differences in the protein-coding region for each type of variable in the contingency table. The null hypothesis of the McDonald–Kreitman test is that the ratio of nonsynonymous to synonymous variation within a species is going to equal the ratio of nonsynonymous to synonymous variation between species (i.e. Dn/Ds = Pn/Ps). When positive or negative selection (natural selection) influences nonsynonymous variation, the ratios will no longer equal. The ratio of nonsynonymous to synonymous variation between species is going to be lower than the ratio of nonsynonymous to synonymous variation within species (i.e. Dn/Ds < Pn/Ps) when negative selection is at work, and deleterious mutations strongly affect polymorphism. The ratio of nonsynonymous to synonymous variation within species is lower than the ratio of nonsynonymous to synonymous variation between species (i.e. Dn/Ds > Pn/Ps) when we observe positive selection. Since mutations under positive selection spread through a population rapidly, they don't contribute to polymorphism but do have an effect on divergence. Using an equation derived by Smith and Eyre-Walker, we can estimate the proportion of base substitutions fixed by natural selection, α, using the following formula: Alpha represents the proportion of substitutions driven by positive selection. Alpha can be equal to any number between -∞ and 1. Negative values of alpha are produced by sampling error or violations of the model, such as the segregation of slightly deleterious amino acid mutations. Similar to above, our null hypothesis here is that α=0, and we expect Dn/Ds to equal Pn/Ps. The neutrality index (NI) quantifies the direction and degree of departure from neutrality (where Pn/Ps and Dn/Ds ratios equal). When assuming that silent mutations are neutral, a neutrality index greater than 1 (i.e. NI > 1) indicates negative selection is at work, resulting in an excess of amino acid polymorphism. This occurs because natural selection is favoring the purifying selection, and the weeding out of deleterious alleles. Because silent mutations are neutral, a neutrality index lower than 1 (i.e. NI < 1) indicates an excess of nonsilent divergence, which occurs when positive selection is at work in the population. When positive selection is acting on the species, natural selection favors a specific phenotype over other phenotypes, and the favored phenotype begins to go to fixation in the species as the allele frequency for that phenotype increases. To find the neutrality index, we can use the following equation: One drawback of performing a McDonald–Kreitman test is that the test is vulnerable to error, as with every other statistical test. Many factors can contribute to errors in estimates of the level of adaptive evolution, including presence of slightly deleterious mutations, variation of mutation rates across the genome, variation in coalescent histories across the genome, and changes in the effective population size. All these factors result in α being underestimated. However, according to research done by Charlesworth (2008), Andolfatto(2008), and Eyre-Walker(2006), none of these factors are significant enough to make scientists believe the McDonald–Kreitman test is unreliable, except for the presence of slightly deleterious mutations in species.