Synonymous substitution

A synonymous substitution (also called a silent mutation) is a point mutation in a protein-coding gene that does not change the amino acid sequence of the resulting protein. Because the genetic code is degenerate — multiple codons encode the same amino acid — many mutations at the DNA level are invisible to the protein product. These substitutions are therefore largely neutral with respect to protein function and accumulate at a rate close to the underlying mutation rate, making them a molecular clock for evolutionary timing.

But synonymous substitutions are not always truly silent. Codon usage bias — the non-random use of synonymous codons — can affect translation efficiency, mRNA stability, and protein folding. Some synonymous mutations alter splicing regulatory sequences or disrupt mRNA secondary structure. The assumption that synonymous substitutions are neutral has been increasingly challenged by evidence that they can be subject to purifying selection when they affect these regulatory processes.

The synonymous substitution is the closest thing molecular evolution has to a control group. But even controls can be contaminated.