Molecular Clock

How do researchers figure out when the common ancestor of different organisms lived? How do they know the chronological order of evolutionary events?

Fig. 1. The graph above is a very schematic representation of a molecular clock. It represents the number of mutations that occurred to the gene at different points in time, expressed as millions of years in the past.

In this article, we will discuss the origin of the molecular clock hypothesis, the definition of the molecular clock, some examples of how a molecular clock can be used in constructing phylogeny, and the limitations of using the molecular clock.

Molecular Clock Concept

The origin of the molecular clock hypothesis

In 1965, the proponents of the molecular clock hypothesis Zuckerkandl and Pauling observed that the constant accumulation of amino acid substitutions in haemoglobin was similar to the regular ‘ticks’ of a clock.

From this observation, they thought that it was possible that a molecular evolutionary clock–which describes changes in amino acids over time since the divergence of species– also existed.

The molecular clock hypothesis argues that DNA and protein sequences mutate at a constant rate over time among different organisms and that the number of genetic differences between organisms can give us an estimation of when they last shared a common ancestor.

Molecular clock definition

Today, the molecular clock is a method used to estimate the amount of time needed for a certain amount of evolutionary change. This is done by analyzing biomolecular data, such as the number of changes or substitutions in nucleotide sequences of DNA and RNA, or the amino acid sequence of proteins. Substitution is a type of mutation where one nucleotide is replaced by another.

Assuming that the nucleotide or amino acid sequences mutate at a constant rate, the number of substitutions over time is equivalent to the evolutionary rate. For this reason, the molecular clock is also known as the gene clock or the evolutionary clock.

Example of a molecular clock diagram

Below is an example of a molecular clock diagram. It shows how quickly CCDC92, a protein-coding gene, changes by graphing the number of amino acid substitutions per millions of years. As points of comparison, it also shows the rate of change in Fibrinogen (a protein with a higher mutation rate) and Cytochrome C (a protein with a lower rate of change).

Fig 2. Molecular clock diagram showing the amino acid substitutions per millions of years to show the rate at which the gene CCDC92 changes.

How are DNA mutations used in molecular clocks?

Mutations may be harmful, beneficial, or neutral. Harmful mutations have a negative impact on an organism's evolutionary fitness, or its ability to survive and reproduce. On the contrary, beneficial mutations have a positive impact on an organism's evolutionary fitness. Most mutations are neutral: they have no effect on an organism’s evolutionary fitness.

Because neutral mutations have no effect on evolutionary fitness, their frequency in the succeeding generations of the population is determined by chance rather than natural selection. This means that all neutral mutations have an equal chance of undergoing substitution. As such, the substitution rate for neutral mutations is equal to the mutation rate.

Neutral mutations are used for molecular clocks because they tend to accumulate at a constant rate over time.

If a gene's specific amino acid sequence is necessary for survival, the majority of mutations will be harmful, with only a few neutral; such genes take a long time to change. On the other hand, if a gene's amino acid sequence is not as essential, fewer mutations will be harmful, and more will be neutral; such genes change at a faster rate. The molecular clock of a gene can be calibrated by corroborating the number of substitutions with dates from the fossil record that are known as diverging points.

The process of calculating a molecular clock can be summed up as follows:

1. Estimate the number of substitutions in the nucleotide or amino acid sequences.

2. Using the fossil record, determine the date when the organisms being studied last shared a common ancestor.

3. Estimate the number of substitutions in the nucleotide or amino acid sequences per unit of time. This will be our evolutionary rate.

4. Using the evolutionary rate, calculate the time of divergence for the new sequences.

Let’s say the evolutionary rate of a species is 2 mutations every million years. If there are 10 mutations in the nucleotide or amino acid sequence being studied, then the sequences must have diverged 5 million years ago.

Example of how molecular clocks are used

Molecular clocks can be used to determine when different species last shared a common ancestor and to put evolutionary events in chronological order, both of which are essential to the construction of phylogenetic trees.

In addition to dating evolutionary changes, molecular clocks are also useful for studying species that do not fossilize well. For example, using molecular clock analyses, researchers found that animals and fungi last shared a common ancestor more than a billion years ago. This kind of information is difficult to obtain from the fossil record because the oldest fossils of fungi–which do not fossilize well because they are soft–can be dated only as far back as about 460 million years ago.

Limitations of molecular clocks

As previously mentioned, molecular clocks work under the assumption that genetic changes (in DNA, RNA, or protein sequences) occur at a fixed rate. Limitations of molecular clocks include:

• DNA, RNA, or protein sequences may change at irregular bursts instead of at a constant rate.

• Some DNA, RNA, or protein sequences may appear to change at a smooth average rate but actually have some deviations from that average rate.

• As a result of natural selection, some genetic changes are favoured over others.

• The same DNA, RNA, or protein sequence substitutions may be occurring at different rates in different organisms.

• Some DNA, RNA, or protein sequences evolve faster than others.

In addition, estimates may be contested when molecular clocks are used to date evolutionary divergences that took place beyond what is documented by the fossil record. Molecular clocks have been used to estimate dates of evolutionary divergence that took place billions of years ago, but the fossil record extends back to only around 550 million years ago.

These limitations can be resolved in some calibrating molecular clocks using data on the evolutionary rate of genes in various taxa. In other circumstances, it is helpful to use a large number of genes rather than just one or two. Natural selection or other circumstances may cause fluctuations in evolutionary rate, but by studying multiple genes, these fluctuations may be averaged out. As such, despite its limitations, molecular clocks can still be useful in determining evolutionary relationships when used carefully.