Evolutionary Biology Molecular Clocks
Charles D. Bell
  • LAST REVIEWED: 24 November 2021
  • LAST MODIFIED: 13 January 2014
  • DOI: 10.1093/obo/9780199941728-0048


The concept of a “molecular clock” was originally suggested by Emile Zuckerkandl and Linus Pauling based on their observations of amino acid substitutions between hemoglobin sequences. The authors proposed that amino acid differences in a protein should accumulate at, more or less, a uniform rate across different species. That is, differences between sequences would accumulate in a linear fashion. In addition, they suggested that this uniform rate of a specific protein would be approximately constant, not just over evolutionary time, but also across different lineages or taxonomic groups. One major issue of using sequence data to infer absolute divergence times is how to disentangle time from evolutionary rates. Because of this, the absolute time since the last common ancestor for species must then be calculated by calibrations based on paleontological evidence. Although Zuckerkandl and Pauling provided evidence for a linear relationship between the accumulation of amino acid differences and evolutionary time, they did not provide an explanation for “why” they observed this pattern. Kimura’s neutral theory of molecular evolution provided an explanation of why macromolecules might be evolving in a clock-like fashion. This theory proposed that most of the substitutions that we observe in molecular data (and the variation we see within species at the molecular level) is due to the fixation of these changes that are neutral or nearly neutral with respect to selection. This theory also provided an important null model of molecular evolution, but was not without its critics. Over the years, the ability to estimate divergence times among species in this manor has been met with great skepticism. Despite these concerns, the use of molecular clock methods has seen a renewed interest in the past fifteen years. With this renewed interest, there has also been great effort made to modify the assumptions of a “strict” clock to account for rate variation among lineages. These are a general class of methods often referred to as “relaxed” clocks. These methods have become increasingly more statistical and have a strong foundation in molecular evolution and systematics. Regardless of methodology, molecular dating relies on two processes: (1) estimating substitution rates among sequences and (2) calibrating substitution rates with independent evidence to convert estimated genetic distances (usually in units of substitutions per site) to absolute ages. This is most commonly achieved by first estimating a phylogeny with branch lengths (in units of substitutions per site), then adjusting these branch lengths so that they are proportional to time (often called a chronogram or time tree). Next, independent paleontological evidence is used to calibrate the relative chronogram to generate a chronogram with absolute ages. This is done by assigning an age (either fixed, minimum, or maximum) to one or more nodes within the tree, then extrapolating ages for all the other nodes in the tree.

General Overviews

Several recent reviews of rate variation and molecular clocks have been provided in Sanderson 1998 and Sanderson, et al. 2004. Magallón 2004; Rutschmann 2006; and Lanfear, et al. 2010 have all provided reviews of the various molecular clock methods, especially “relaxed clock” methods. Swofford, et al. 1996; Li 1997; and Felsenstein 2004 all provide extremely detailed summaries on the estimation of rates of substitutions from nucleotide sequences. Gillespie 1991 has also provided an excellent overview concerning the theoretical aspects of molecular evolution. With respect to fossil calibrations, Forest 2009 has provided an excellent overview on this topic.

  • Felsenstein, J. 2004. Inferring phylogenies. Sunderland, MA: Sinauer.

    This is an encyclopedic work of all things phylogeny. It is probably the most comprehensive resource for phylogenetics to date.

  • Forest, F. 2009. Calibrating the tree of life: Fossils molecules, and evolutionary time scales. Annals of Botany 104:789–794.

    DOI: 10.1093/aob/mcp192

    The author provides a comprehensive discussion of use of the fossil record as a source of independent information in the calibration of molecular phylogenies.

  • Gillespie, J. H. 1991. The Causes of Molecular Evolution. New York: Oxford Univ. Press.

    This is an excellent source book for the theoretical basis of molecular evolution. It does a great job of describing the statistical foundations of the study of rates of molecular evolution. This book provides a great background to many of the controversies associated with the molecular clock and neutral theory.

  • Lanfear, R., J. J. Welch, and L. Bromham. 2010. Watching the clock: Studying variation in rates of molecular evolution between species. Trends in Ecology and Evolution 25:495–503.

    DOI: 10.1016/j.tree.2010.06.007

    Excellent summary of how to calculate rates of molecular evolution from sequence data. Provides much of the background on why we might see variation in rates across taxa.

  • Li, W. -H. 1997. Molecular Evolution. Sunderland, MA: Sinauer.

    This book is a great general resource about all sorts of topics in the field of molecular evolution.

  • Magallón, S. A. 2004. Dating lineages: Molecular and paleontological approaches to the temporal framework of clades. International Journal of Plant Sciences 165:S7–S21.

    This article provides a superb overview of various aspects of using molecular sequence data to date lineages and provides a very detailed overview of various methods that are used to date lineages, especially “relaxed clock” methods.

  • Rutschmann, F. 2006. Molecular dating of phylogenies: A brief summary of current methods that estimate divergence times. Diversity and Distributions 12:35–48.

    DOI: 10.1111/j.1366-9516.2006.00210.x

    This article provides another detailed review of many of the current methods used to infer divergence times from molecular sequence data.

  • Sanderson, M. J. 1998. Estimating rate and time in molecular phylogenies: Beyond the molecular clock? In Molecular systematics of plants II: DNA sequencing. Edited by D. E. Soltis, P. S. Soltis, and J. J. Doyle, 242–262. Boston: Kluwer Academic.

    DOI: 10.1007/978-1-4615-5419-6

    Sanderson provides an excellent and detailed summary of the use of molecular sequence data to date lineages, as well as the ways in which the clock has been refined over the years to deal with rate heterogeneity among lineages.

  • Sanderson, M. J., J. L. Thorne, N. Wikstrom, and K. Bremer. 2004. Molecular evidence on plant divergence times. American Journal of Botany 91:1656–1665.

    DOI: 10.3732/ajb.91.10.1656

    Although plant-specific with respect to empirical studies, the authors provide a detailed summary of many of the issues associated with dating lineages in the absence of a strict molecular clock.

  • Swofford, D. L., G. J. Olsen, P. J. Waddell, and D. M. Hillis. 1996. Phylogenetic inference. In Molecular systematics. 2d ed. Edited by D. M. Hillis, C. Moritz, and B. K. Mable, 407–514. Sunderland, MA: Sinauer.

    This is an excellent overview of molecular systematics and phylogenetic inference. Provides detailed information about the variety of optimality criteria used to infer phylogenies, as well as how trees are searched using algorithmic and heuristic methodologies.

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