M.Tevfik Dorak, M.D., Ph.D.
The controversial hypothesis of molecular clock (MC) is a consequence of the neutral theory of evolution. It holds that in any given DNA sequence, mutations accumulate at an approximately constant rate as long as the DNA sequence retains its original functions. The difference between the sequences of a DNA segment (or protein) in two species would then be proportional to the time since the species diverged from a common ancestor (coalescence time). This time may be measured in arbitrary units and then it can be calibrated in millions of years for any given gene if the fossil record of that species happens to be rich. MCs do not behave metronomically, i.e., neutral mutations do not yield literally constant rates of molecular change but are expected to yield constant average rates of change over a long period of time (known as ‘stochastically constant’ rates). The concept of MC fits well with Kimura’s neutrality theory because the rate of neutral evolution in genetic sequences is equal to the mutation rate of neutral alleles. Even where selection operates, an averaging of selection coefficients over long periods could also provide a MC for times of divergence. Undeniably, different DNA sequences (or proteins) or even different parts of the same gene (or protein) evolve at markedly different rates. Different functional constraints on structure and varying intensities of selectional forces as well as generation time (the rate of meiotic production of gametes) are the major sources of variation in evolutionary rates. In general, rRNA evolves slowly and mtDNA rapidly. Among other proteins, fibrinopeptide changes relatively rapidly and is useful for studying closely related species but cytochrome c is useful for studying the whole period of life on earth. Fast mutating sequences cannot be used to go back in evolutionary time because the mutations effectively randomize the sequences. It is also possible that reverse mutations will occur and the comparisons will be very difficult. Introns and pseudogenes evolve rapidly and nondegenerate sites in protein coding sequences (exons) slowly. Molecular differences between species are used to infer phylogenetic relationships. Molecular evolution from living fossils provides an example that constant rate of molecular evolution occurs independent of morphological evolution.
A MC must be calibrated first and this requires a reliable fossil record. Only after this calibration, a MC can be used for phylogenetic inference. A conventional calibration for evolutionary rate of animal mtDNA is about 2% sequence divergence per million years between pairs of lineages separated for less than 10 million years (or 20x10-9 substitutions per site per year). Beyond 15-20 million years, mtDNA sequence divergence begins to plateau, presumably as the genome becomes saturated with substitutions at variable sites. 16S rRNA gene, however, has an evolutionary rate of 1% sequence divergence per 50 million years. Although mean evolutionary rates in the nuclear genome may vary among taxa, they do so in a consistent fashion. For example, molecular evolution appears slower in primates than in rodents and especially slow in hominoids. Using the highly conserved protein cytochrome c sequences, it has been found that the two most divergent species by far are two species of ascomycetes (fungus) that separated twice as long ago as the ancestors of mammals separated from the ancestors of insects.
To test a MC, a relative rate test that does not depend on absolute divergence times is used. Each test requires at least two related species (A and B) and an outside reference species (C) known to have branched off prior to the separation of A and B. Theoretically, the distances (A to C) and (B to C) estimated by the MC should be similar. If there is a statistically significant difference, then this particular MC is unreliable for this taxa. Many relative rate tests have been conducted for molecular data from different species. Uniform average rates of DNA clocks in birds, rats, mice and hamsters have been established. The relative rate test does show up some discrepancies in molecular rates in many cases. This is not surprising as many allele frequencies are clearly modulated by natural selection. The main advantage of today’s favorite phylogenetic analysis method, cladistics, is that it does not rely on MCs.
Microsatellite loci have properties that make them suitable for dating evolutionary events. The mutation rate is so high (5.6 x 10-4 in dinucleotide microsatellites) that a reasonable number of mutational events can occur within the short time of modern human evolution. For CA dinucleotide microsatellites, the average estimate for mutation rate is about 1/5000 per generation. The application of the microsatellite data to human population studies gave an estimate of 156 (95 to 290) thousand years for separation of Africans and non-Africans.
Since most molecular systems evolve at heterogeneous rates across most taxa, if precise clocks exist, they are local rather than universal. MCs keep far from perfect time, but in certain cases (where they have been tested and approved), they provide invaluable information in phylogenetic studies.
M.Tevfik Dorak, MD, PhD
Last updated 9 January 2007