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.
Since mutations under positive selection spread through a population rapidly, they don't contribute to polymorphism but do have an effect on divergence.
Negative values of alpha are produced by sampling error or violations of the model, such as the segregation of slightly deleterious amino acid mutations.
[11] However, according to research done by Charlesworth (2008),[3] Andolfatto(2008),[12] and Eyre-Walker(2006),[8] 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.
[13] The presence of slightly deleterious mutations is strongly linked to genes that have experienced the greatest reduction in effective population size.
[14] This means that soon after a recent reduction in effective population size in a species has occurred, such as a bottleneck, we observe a larger presence of slightly deleterious mutations in the protein-coding regions.
[15] We can make a direct connection with the increase in number of slightly deleterious mutations and the recent decrease in effective population size.
[14] For more information on why population size affects the tendency of slightly deleterious mutations to increase in frequency, refer to the article Nearly neutral theory of molecular evolution.
[16] However, the McDonald Kreitman test is very vulnerable to type I error, because of the many factors that can lead to the accidental rejection of the true null hypothesis.
Such factors include variation in recombination rate, nonequilibrium demography, small sample sizes, and in comparisons involving more recently diverged species.
This possible adjustment of the McDonald–Kreitman test includes removing polymorphisms below a specific value from the data set to improve and increase the number of substitutions that occurred due to adaptive evolution.