Questions: Molecular Evolution Basics for Bioinformatics

Question 1

Why does raw percent identity between two sequences underestimate the true evolutionary distance?

• A. Because alignment algorithms introduce errors
• B. Because some mutations are silent and not counted
• C. Because multiple substitutions can occur at the same site, and only the most recent change is visible
• D. Because gaps are not counted as differences

Answer: (C)

Over evolutionary time, the same nucleotide position can mutate multiple times. A site that changed from A to G and then from G to C appears as an A-to-C change — two events counted as one difference. A site that changed from A to G and back to A appears unchanged — two events counted as zero differences. These multiple hits cause percent identity to plateau around 25% for DNA (random expectation for 4 bases), even as true divergence continues to increase. Substitution models like Jukes-Cantor correct for this by estimating the actual number of substitutions per site.

Question 2

Orthologs always have the same function in different species.

• T. True
• F. False

Answer: False

Orthologs (genes that diverged by speciation) tend to retain similar functions, and this is often a useful working assumption. But function can diverge after speciation due to changes in regulatory context, protein-protein interactions, or adaptive evolution in one lineage. Furthermore, paralogs (genes that diverged by duplication) can also retain similar function, or one copy may neofunctionalize or subfunctionalize. The ortholog conjecture — that orthologs are more functionally similar than paralogs — is a useful heuristic, not an absolute rule.

Question 3

Explain the biological significance of finding a dN/dS ratio significantly greater than 1 for a gene.

Model answer: A dN/dS ratio greater than 1 means nonsynonymous (amino acid-changing) substitutions are being fixed at a higher rate than synonymous (silent) substitutions. Since synonymous substitutions approximate the neutral mutation rate, a ratio above 1 indicates that natural selection is actively favoring amino acid changes — positive (diversifying) selection. This signature often occurs in genes involved in immune defense, reproduction, or host-pathogen interactions, where there is an evolutionary advantage to rapid protein sequence change.

Most genes have dN/dS well below 1, indicating purifying selection removes most amino acid changes. A ratio near 1 suggests neutral evolution (relaxed constraint). Values significantly above 1 are relatively rare genome-wide but point to genes of particular evolutionary interest. Branch-specific and site-specific models can localize positive selection to particular lineages or amino acid positions.