Since Darwin's day, science has made astounding advances in the ways in which it can study organisms. One of the most useful advances has been the development of molecular biology. In this field, scientists look at the proteins and other molecules that control life processes. While these molecules can evolve just as an entire organism can, some important molecules are highly conserved among species. The slight changes that occur over time in these conserved molecules, which are often called molecular clocks, can help shed light on past evolutionary events.
The key to using biological molecules as molecular clocks is the hypothesis of neutral evolution. This hypothesis states that most of the variability in molecular structure does not affect the molecule's functionality. This is because most of the variability occurs outside of the functional regions of the molecule. Changes that do not affect functionality are called "neutral substitutions" and their accumulation is not affected by natural selection. As a result, neutral substitutions occur at a fairly regular rate, though that rate is different for different molecules.
Not every molecule makes a good molecular clock, however. To serve as a molecular clock, a molecule must meet two requirements: 1) it must be present in all of the organisms being studied; 2) it must be under strong functional constraint so that the functional regions are highly conserved. Examples of molecules that have been used to study evolution are cytochrome c, which is vital to the respiratory pathway, and ribosomal RNA, which performs protein synthesis.
Once a good molecular clock is identified, using it to compare species is fairly simple. The most complicated step is the comparison of molecular sequences. The sequences of the molecule in the different species must be compared so that the number of amino acid or nucleic acid bases that differ can be counted. This number is then plotted against the rate at which the molecule is known to undergo neutral base pair substitutions to determine the point at which two species last shared a common ancestor. Depending on the rate of substitution, molecules may be used to determine ancient relationships or relatively recent ones. Ribosomal RNA has a very slow rate of substitution, so it is most commonly used in conjunction with fossil information to determine relationships between extremely ancient species.