Questions: The Neutral Theory of Molecular Evolution

Question 1

A comparison of two species reveals a Ka/Ks ratio of 0.04 for a particular gene. What is the most likely interpretation?

• A. The gene is evolving neutrally — drift drives both synonymous and non-synonymous changes equally
• B. Positive selection is actively driving amino acid changes to fixation
• C. Strong purifying selection is eliminating most non-synonymous mutations before they fix
• D. The mutation rate for this gene is unusually high relative to the genome average

Answer: (C)

Ka/Ks (or dN/dS) compares the rate of non-synonymous (amino-acid-changing) substitutions to synonymous (silent) substitutions. A ratio much less than 1 — like 0.04 — means non-synonymous changes accumulate far more slowly than synonymous ones, indicating that most amino acid changes are harmful and being eliminated by purifying selection. A ratio near 1 suggests neutrality; a ratio greater than 1 indicates positive selection driving amino acid changes faster than the neutral baseline.

Question 2

Two mammalian species diverged 50 million years ago. Neutral theory predicts that the number of synonymous substitutions accumulated since divergence depends primarily on which factor?

• A. The effective population sizes of both lineages
• B. The per-generation neutral mutation rate
• C. The generation times of both species
• D. The ecological niches and selective pressures each lineage faced

Answer: (B)

This is neutral theory's most elegant result. The rate at which neutral mutations fix equals the mutation rate μ — population size cancels out because larger populations produce more mutations (2Nμ per generation) but each individual mutation has a smaller fixation probability (1/2N). So the substitution rate per year depends on μ and generation time, not population size or ecology. This is the mathematical foundation of the molecular clock: two lineages accumulate neutral substitutions at roughly the mutation rate, regardless of their very different population histories.

Question 3

Synonymous (silent) substitutions accumulate faster than non-synonymous substitutions in most genes because they are largely free from natural selection.

• T. True
• F. False

Answer: True

This pattern — higher synonymous than non-synonymous divergence — is one of the strongest pieces of evidence for neutral theory. Synonymous changes do not alter the amino acid sequence, so most are invisible to selection and drift freely to fixation at the mutation rate. Non-synonymous changes alter the protein, and most such changes are deleterious, so they are eliminated by purifying selection before they can fix. The excess of synonymous over non-synonymous divergence is exactly what neutral theory predicts and is observed across essentially all protein-coding genes studied.

Question 4

The neutral theory of molecular evolution claims that natural selection plays no important role in shaping molecular sequences.

• T. True
• F. False

Answer: False

This is the most common misconception about neutral theory. Kimura did not claim selection is unimportant — he claimed that *most* molecular variation and substitutions are neutral and driven by drift. Selection plays a crucial role as a *filter*, removing the many harmful mutations that arise. What neutral theory challenges is the selectionist view that most substitutions are *adaptive* (driven by positive selection). The neutral theory says: most fixed differences are neutral; selection's main molecular role is negative (purifying), not positive.

Question 5

Why does population size cancel out in the neutral theory's prediction of substitution rate? Why is this surprising, and what does it imply?

Model answer: The fixation probability of a new neutral mutation is 1/(2N) (its initial frequency in a diploid population of size N). But the rate of new neutral mutations entering the population per generation is 2Nμ. Multiplying these: substitution rate = 2Nμ × 1/(2N) = μ. Population size cancels exactly. This is surprising because population size powerfully affects drift — large populations fix random variants less often per mutation — but that effect is exactly offset by producing more mutations. The implication is that neutral evolutionary rate is a clock set by mutation rate alone, independent of ecology, demography, or population dynamics.

The molecular clock hypothesis follows directly from this result. If neutral mutations accumulate at rate μ regardless of population size, then DNA sequence divergence between two lineages is approximately proportional to time since their common ancestor (assuming constant μ). This makes molecular data useful for dating evolutionary events. The caveat is that generation time and mutation rate can differ between lineages, requiring calibration — but the population-size independence is what makes the clock concept coherent in the first place.