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.
