Box 1 | The earliest uses of the molecular clock

From the following article:

Molecular clocks: four decades of evolution

Sudhir Kumar

Nature Reviews Genetics 6, 654-662 (August 2005)


In 1962, Zuckerkandl and Pauling3 estimated the time of divergence of four members of the haemoglobin gene family (alpha, beta, gamma and delta) by assuming an approximate molecular clock. This was calibrated using the number of observed sequence differences (D) between the horse and human alpha-haemoglobin proteins and the divergence time between the two species (T), which is based on the fossil record. They took a pair-wise approach to estimating divergence times, which is shown schematically for alpha- and beta-haemoglobin in panel a. The molecular-clock calibration was carried out by dividing twice the known divergence time by the amount of sequence divergence (2T/D); the factor of 2 is used here because D is equal to the sum of divergence from the common ancestor to the two descendents. This calibration was then used to convert other measurements of protein sequence differences to time. For example, the formula t = d (T/D) gives the time when the alpha- and beta-chains diverged, where d is the amount of sequence difference between alpha- and beta-chains in humans. The time estimate obtained will have the same units as the time used for clock calibrations (in this case, millions of years).

Molecular clocks: four decades of evolution 

Zuckerkandl and Pauling also estimated the timing of the human–gorilla divergence using alpha- and beta-chains separately (panel b). They calculated the molecular-clock calibration to be 11 to 18 million years (Myr) per amino-acid substitution, based on the observation of 18 differences between human and horse alpha-haemoglobin proteins and the assumption that these two species diverged 100–160 million years ago (Mya). Using an average calibration of 14.5 Myr per substitution, the human–gorilla divergence was dated to have occurred 14.5 and 7.25 Mya by alpha- and beta-chains, because human and gorilla show two and one differences in these chains, respectively. Therefore, Zuckerkandl and Pauling3 reported a mean date of 11 Mya for the human–gorilla divergence from an analysis of the two proteins. One year later, Margoliash5 used the same calibration point to estimate multiple species divergence times. These estimates were based on single, slowly evolving proteins and were therefore not very accurate. In 1965, Zuckerkandl and Pauling9 predicted that the accuracy of molecular clocks would be improved by using many proteins of different types. Over the past decade, a large number of proteins have been analysed to estimate divergence times among the principal groups of mammals and among animal phyla35, 39, 60.