The molecular clock is a technique that uses the mutation rate of
biomolecules to deduce the time in prehistory when two or more life
forms diverged. The biomolecular data used for such calculations are
usually nucleotide sequences for
DNA or amino acid sequences for
proteins. The benchmarks for determining the mutation rate are often
fossil or archaeological dates. The molecular clock was first tested
in 1962 on the hemoglobin protein variants of various animals, and is
commonly used in molecular evolution to estimate times of speciation
or radiation. It is sometimes called a gene clock or an evolutionary
1 Early discovery and genetic equidistance
2 Relationship with neutral theory
3.1 Node calibration
3.2 Tip calibration
3.2.1 Total evidence dating
4 Non-constant rate of molecular clock
6 See also
8 Further reading
9 External links
Early discovery and genetic equidistance
The notion of the existence of a so-called "molecular clock" was first
attributed to Émile Zuckerkandl and
Linus Pauling who, in 1962,
noticed that the number of amino acid differences in hemoglobin
between different lineages changes roughly linearly with time, as
estimated from fossil evidence. They generalized this observation
to assert that the rate of evolutionary change of any specified
protein was approximately constant over time and over different
lineages (based on the molecular clock hypothesis (MCH)).
The genetic equidistance phenomenon was first noted in 1963 by Emanuel
Margoliash, who wrote: "It appears that the number of residue
differences between cytochrome c of any two species is mostly
conditioned by the time elapsed since the lines of evolution leading
to these two species originally diverged. If this is correct, the
cytochrome c of all mammals should be equally different from the
cytochrome c of all birds. Since fish diverges from the main stem of
vertebrate evolution earlier than either birds or mammals, the
cytochrome c of both mammals and birds should be equally different
from the cytochrome c of fish. Similarly, all vertebrate cytochrome c
should be equally different from the yeast protein." For example,
the difference between the cytochrome c of a carp and a frog, turtle,
chicken, rabbit, and horse is a very constant 13% to 14%. Similarly,
the difference between the cytochrome c of a bacterium and yeast,
wheat, moth, tuna, pigeon, and horse ranges from 64% to 69%. Together
with the work of
Emile Zuckerkandl and Linus Pauling, the genetic
equidistance result directly led to the formal postulation of the
molecular clock hypothesis in the early 1960s.
Relationship with neutral theory
The observation of a clock-like rate of molecular change was
originally purely phenomenological. Later, the work of Motoo Kimura
developed the neutral theory of molecular evolution, which predicted a
molecular clock. Let there be N individuals, and to keep this
calculation simple, let the individuals be haploid (i.e. have one copy
of each gene). Let the rate of neutral mutations (i.e. mutations with
no effect on fitness) in a new individual be
. The probability that this new mutation will become fixed in the
population is then 1/N, since each copy of the gene is as good as any
other. Every generation, each individual can have new mutations, so
N new neutral mutations in the population as a whole. That means that
new neutral mutations will become fixed. If most changes seen during
molecular evolution are neutral, then fixations in a population will
accumulate at a clock-rate that is equal to the rate of neutral
mutations in an individual.
The molecular clock alone can only say that one time period is twice
as long as another: it cannot assign concrete dates. For viral
phylogenetics and ancient
DNA studies—two areas of evolutionary
biology where it is possible to sample sequences over an evolutionary
timescale—the dates of the intermediate samples can be used to more
precisely calibrate the molecular clock. However, most phylogenies
require that the molecular clock be calibrated against independent
evidence about dates, such as the fossil record. There are two
general methods for calibrating the molecular clock using fossil data:
node calibration and tip calibration.
Sometimes referred to as node dating, node calibration is a method for
phylogeny calibration that is done by placing fossil constraints at
nodes. A node calibration fossil is the oldest discovered
representative of that clade, which is used to constrain its minimum
age. Due to the fragmentary nature of the fossil record, the true most
recent common ancestor of a clade will likely never be found. In
order to account for this in node calibration analyses, a maximum
clade age must be estimated. Determining the maximum clade age is
challenging because it relies on negative evidence—the absence of
older fossils in that clade. There are a number of methods for
deriving the maximum clade age using birth-death models, fossil
stratigraphic distribution analyses, or taphonomic controls.
Alternatively, instead of a maximum and a minimum, a prior probability
of the divergence time can be established and used to calibrate the
clock. There are several prior probability distributions including
normal, lognormal, exponential, gamma, uniform, etc.) that can be used
to express the probability of the true age of divergence relative to
the age of the fossil; however, there are very few methods for
estimating the shape and parameters of the probability distribution
empirically. The placement of calibration nodes on the tree informs
the placement of the unconstrained nodes, giving divergence date
estimates across the phylogeny. Historical methods of clock
calibration could only make use of a single fossil constraint
(non-parametric rate smoothing), while modern analyses (BEAST
and r8s) allow for the use of multiple fossils to calibrate the
molecular clock. Simulation studies have shown that increasing the
number of fossil constraints increases the accuracy of divergence time
Sometimes referred to as tip dating, tip calibration is a method of
molecular clock calibration in which fossils are treated as taxa and
placed on the tips of the tree. This is achieved by creating a matrix
that includes a molecular dataset for the extant taxa along with a
morphological dataset for both the extinct and the extant taxa.
Unlike node calibration, this method reconstructs the tree topology
and places the fossils simultaneously. Molecular and morphological
models work together simultaneously, allowing morphology to inform the
placement of fossils. Tip calibration makes use of all relevant
fossil taxa during clock calibration, rather than relying on only the
oldest fossil of each clade. This method does not rely on the
interpretation of negative evidence to infer maximum clade ages.
Total evidence dating
This approach to tip calibration goes a step further by simultaneously
estimating fossil placement, topology, and the evolutionary timescale.
In this method, the age of a fossil can inform its phylogenetic
position in addition to morphology. By allowing all aspects of tree
reconstruction to occur simultaneously, the risk of biased results is
decreased. This approach has been improved upon by pairing it with
different models. One current method of molecular clock calibration is
total evidence dating paired with the fossilized birth-death (FBD)
model and a model of morphological evolution. The FBD model is
novel in that it allows for “sampled ancestors,” which are fossil
taxa that are the direct ancestor of a living taxon or lineage. This
allows fossils to be placed on a branch above an extant organism,
rather than being confined to the tips.
Bayesian methods can provide more appropriate estimates of divergence
times, especially if large datasets—such as those yielded by
Non-constant rate of molecular clock
Sometimes only a single divergence date can be estimated from fossils,
with all other dates inferred from that. Other sets of species have
abundant fossils available, allowing the MCH of constant divergence
rates to be tested.
DNA sequences experiencing low levels of negative
selection showed divergence rates of 0.7–0.8% per
bacteria, mammals, invertebrates, and plants. In the same study,
genomic regions experiencing very high negative or purifying selection
(encoding rRNA) were considerably slower (1% per 50 Myr).
In addition to such variation in rate with genomic position, since the
early 1990s variation among taxa has proven fertile ground for
research too, even over comparatively short periods of
evolutionary time (for example mockingbirds). Tube-nosed seabirds
have molecular clocks that on average run at half speed of many other
birds, possibly due to long generation times, and many turtles
have a molecular clock running at one-eighth the speed it does in
small mammals, or even slower. Effects of small population size
are also likely to confound molecular clock analyses. Researchers such
as Francisco Ayala have more fundamentally challenged the molecular
clock hypothesis. According to Ayala's 1999 study, five
factors combine to limit the application of molecular clock models:
Changing generation times (If the rate of new mutations depends at
least partly on the number of generations rather than the number of
Population size (
Genetic drift is stronger in small populations, and
so more mutations are effectively neutral)
Species-specific differences (due to differing metabolism, ecology,
evolutionary history, ...)
Change in function of the protein studied (can be avoided in closely
related species by utilizing non-coding
DNA sequences or emphasizing
Changes in the intensity of natural selection.
Woody bamboos (tribes
Arundinarieae and Bambuseae) have long
generation times and lower mutation rates, as expressed by short
branches in the phylogenetic tree, than the fast-evolving herbaceous
Molecular clock users have developed workaround solutions using a
number of statistical approaches including maximum likelihood
techniques and later Bayesian modeling. In particular, models that
take into account rate variation across lineages have been proposed in
order to obtain better estimates of divergence times. These models are
called relaxed molecular clocks because they represent an
intermediate position between the 'strict' molecular clock hypothesis
and Joseph Felsenstein's many-rates model and are made possible
through MCMC techniques that explore a weighted range of tree
topologies and simultaneously estimate parameters of the chosen
substitution model. It must be remembered that divergence dates
inferred using a molecular clock are based on statistical inference
and not on direct evidence.
The molecular clock runs into particular challenges at very short and
very long timescales. At long timescales, the problem is saturation.
When enough time has passed, many sites have undergone more than one
change, but it is impossible to detect more than one. This means that
the observed number of changes is no longer linear with time, but
instead flattens out. Even at intermediate genetic distances, with
phylogenetic data still sufficient to estimate topology, signal for
the overall scale of the tree can be weak under complex likelihood
models, leading to highly uncertain molecular clock estimates.
At very short time scales, many differences between samples do not
represent fixation of different sequences in the different
populations. Instead, they represent alternative alleles that were
both present as part of a polymorphism in the common ancestor. The
inclusion of differences that have not yet become fixed leads to a
potentially dramatic inflation of the apparent rate of the molecular
clock at very short timescales.
The molecular clock technique is an important tool in molecular
systematics, the use of molecular genetics information to determine
the correct scientific classification of organisms or to study
variation in selective forces. Knowledge of approximately constant
rate of molecular evolution in particular sets of lineages also
facilitates establishing the dates of phylogenetic events, including
those not documented by fossils, such as the divergence of living taxa
and the formation of the phylogenetic tree. In these
cases—especially over long stretches of time—the limitations of
MCH (above) must be considered; such estimates may be off by 50% or
Human mitochondrial molecular clock
Mitochondrial Eve and Y-chromosomal Adam
Neutral theory of molecular evolution
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Allan Wilson and the molecular clock
Molecular clock explanation of the molecular equidistance phenomenon
Clade service for the molecular tree of life
Ab urbe condita
Anno Domini / Common Era
Hindu units of time
Hindu units of time (Yuga)
Canon of Kings
Lists of kings
Pre-Julian / Julian
Old Style and New Style dates
Adoption of the Gregorian calendar
Astronomical year numbering
Chinese sexagenary cycle
ISO week date
Winter count (Plains Indians)
Geological history of Earth
Geological time units
Global Standard Stratigraphic Age (GSSA)
Global Boundary Stratotype Section and Point (GSSP)
Law of superposition
Amino acid racemisation
Terminus post quem