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A comparison of nonsynonymous and synonymous polymorphism and divergence. Under a simple neutral model their ratios match. An excess of nonsynonymous divergence can support adaptive fixation, but mildly deleterious polymorphism, changing demography and site-class assumptions affect interpretation.^[ref]

Sequence divergence alone does not identify its cause. A difference can arise through mutation and spread through genetic drift, natural selection, or both. The useful comparison is with an explicit neutral expectation that accounts for shared ancestry, mutation rate and demographic history, rather than treating every conspicuous genomic difference as an adaptation.

First establish orthology, align homologous sequence accurately, and sample several individuals from each species. An apparent fixed difference in one pair of genomes may actually be segregating variation, an assembly error or comparison of paralogues. An outgroup can help polarize changes, although ancestral polymorphism and introgression can make different genomic regions have different histories. Estimate population size changes, migration and relatedness from many suitably chosen loci before interpreting an exceptional region.

For coding sequence, compare nonsynonymous and synonymous substitution rates per eligible site through the nonsynonymous-to-synonymous substitution rate ratio. A ratio greater than one in an appropriate branch or site model supports adaptive amino-acid substitution; a low average ratio indicates pervasive constraint but can hide positive selection at a few sites. A rise toward one can reflect relaxed purifying selection rather than adaptation. Synonymous sites are useful controls, not guaranteed neutral standards: codon usage, splicing regulation and mutation-context effects can matter.

Within-species polymorphism adds an independent timescale. In the McDonald-Kreitman test, let $D_N,D_S$ be nonsynonymous and synonymous fixed differences and $P_N,P_S$ their polymorphic counterparts. A simple neutral model predicts matching ratios. An excess $D_N/D_S>P_N/P_S$ is consistent with adaptive fixation, and the idealized estimatorestimates the adaptive fraction of nonsynonymous divergence. John H. McDonald and Martin Kreitman introduced this logic with the Drosophila Adh locus. Slightly deleterious variants inflate $P_N$, demographic changes affect which variants persist, and low counts make the ratio unstable; the model and uncertainty must therefore be examined rather than interpreting every positive estimate literally.^[ref]

Other signatures are complementary. A recent selective sweep can reduce linked diversity and distort the site frequency spectrum and linkage disequilibrium. Excess differentiation measured by FST can suggest geographically divergent selection, but can also result from reduced within-population diversity or population history. Background selection can mimic some diversity reductions. Comparisons with matched recombination and mutation environments, demographic simulations and several independent populations help discriminate these explanations. Long-maintained variation can instead leave signatures of balancing selection.

Noncoding differences require their own functional tests. A conserved regulatory element with an accelerated lineage-specific substitution rate is a candidate for altered function, but altered mutation bias or GC-biased gene conversion can also produce rapid sequence change. Reporter assays, allele replacement and measurements of expression, phenotype and fitness connect a candidate to a mechanism. Demonstrating an effect on expression does not by itself establish an advantage in the historical environment.

The strongest inference combines an appropriate population-genetic null, agreement among independent signatures, and a functional explanation connected to reproductive success. Conversely, failure to reject neutrality means that the evidence does not discriminate the models at the available resolution; it does not prove that a difference has no effect.