Questions: Molecular Evolution and Molecular Clocks

Question 1

A gene is found to have a dN/dS ratio of 3.2, where dN is the rate of nonsynonymous substitutions and dS is the rate of synonymous substitutions. What does this indicate?

• A. The gene is under strong purifying selection
• B. The gene is evolving neutrally
• C. The gene is under positive (diversifying) selection
• D. The gene has a higher-than-average mutation rate

Answer: (C)

Under purifying selection, harmful nonsynonymous changes are removed, so dN < dS and dN/dS < 1. Under strict neutrality, dN ≈ dS and dN/dS ≈ 1. A dN/dS ratio > 1 means nonsynonymous substitutions are accumulating faster than synonymous ones — more amino acid changes are being fixed than expected by chance alone, indicating that natural selection is actively favoring protein sequence change. This is the signature of positive selection.

Question 2

Molecular clocks provide a reliable, constant rate of DNA substitution that can be used to date divergence events without any external calibration.

• T. True
• F. False

Answer: False

Molecular clocks are not perfectly constant — substitution rates vary across lineages (due to differences in generation time, metabolic rate, and population size) and across sites within a gene. Reliable molecular clock estimates require calibration using at least one independently dated event, typically from the fossil record or a geological event (e.g., a land bridge formation). Uncalibrated clocks can produce divergence estimates that are off by tens of millions of years.

Question 3

Explain why synonymous substitutions accumulate faster than nonsynonymous substitutions in most protein-coding genes.

Model answer: Synonymous (silent) substitutions change the DNA sequence but not the amino acid sequence of the encoded protein, due to redundancy in the genetic code. Because they do not alter protein function, they are largely invisible to natural selection and accumulate at a rate close to the mutation rate. Nonsynonymous substitutions change the amino acid sequence and therefore affect protein structure and function. Most such changes are harmful and are removed by purifying selection before they can spread through the population, reducing the observed substitution rate. Only rarely do nonsynonymous changes improve fitness and get fixed.

This question connects to the neutral theory: the substitution rate equals the mutation rate only for truly neutral changes. Synonymous substitutions approximate neutrality because the protein is unchanged. Most nonsynonymous substitutions are subject to selection — usually negative — which slows their fixation rate far below the underlying mutation rate. The dN/dS ratio formalizes this comparison and is one of the most widely used tests for natural selection at the molecular level.