The neutral-mutation hypothesis is the idea that evolutionary change at the molecular level takes place primarily through the fixation of neutral mutations by genetic drift. The rate at which one neutral mutation replaces another depends only on the mutation rate, which should be fairly constant for any particular gene and the protein it encodes. If the rate at which a protein evolves is roughly constant over time, the amount of molecular change that a protein has undergone can be used as a molecular clock to date evolutionary events.

For example, we could examine the enzyme cytochrome c in two organisms known from fossil evidence to have had a common ancestor 400 million years ago. By determining the number of differences between the two organisms in their cytochrome c amino acid sequences, we could calculate the number of substitutions that have occurred per amino acid site. The occurrence of 20 amino acid substitutions since the two organisms diverged indicates an average rate of 5 substitutions per 100 million years. Knowing how fast the molecular clock ticks allows us to use molecular changes in cytochrome c to date other evolutionary events: if we found that cytochrome c in two other organisms differed by 15 amino acid substitutions, our molecular clock would suggest that they diverged some 300 million years ago. If we assumed some error in our estimate of the rate of amino acid substitution, statistical analysis would show that the true divergence time might range from 160 million to 440 million years ago.

The molecular clock was proposed by Emile Zuckerkandl and Linus Pauling in 1965 as a possible means of dating evolutionary events on the basis of molecules in present-day organisms. A number of studies have examined the rate of evolutionary change in proteins (Figure 18.18) and in genes, and the molecular clock has been widely used to date evolutionary events when the fossil record is absent or ambiguous. For example, researchers used a molecular clock to estimate when Darwin’s finches diverged from a common ancestor that originally colonized the Galápagos Islands. This clock was based on DNA sequence differences in the cytochrome b gene. The researchers concluded that the ancestor of Darwin’s finches arrived in the Galápagos and begin diverging some 2 million–3 million years ago. The results of several studies have shown, however, that the molecular clock does not always tick at a constant rate, particularly over shorter time periods, and this method remains controversial.

492