Molecular Clocks

One of the easiest ways to obtain information about the evolutionary relationships of different alleles is to calculate the extent to which two sequences differ from one another (generally referred to as sequence divergence). This is most easily presented as the percentage of variable sites, although more complex models take into account mutational processes, for example by differentially weighting transitions versus transversions, or synonymous versus non-synonymous substitutions (Kimura, 1980). The similarity of two sequences provides us with some information about how long ago they diverged from one another because, generally speaking, similar sequences will have diverged recently whereas dissimilar sequences have been evolutionarily independent for a relatively long period of time. We may be able to acquire even more precise information about the time since sequences diverged from one another if we apply what is known as a molecular clock.

The idea of molecular clocks was introduced in the 1960s (Zuckerkandl and Pauling, 1965), based on the hypothesis that DNA sequences evolve at roughly constant rates and therefore the dissimilarity of two sequences can be used to calculate the amount of time that has passed since they diverged from one another. Molecular clocks have been used to date both ancient events, such as the emergence of ancestral mammals several millions of years before dinosaurs became extinct (Kumar and Hedges, 1998), and also more recent events, such as the splitting of the circumarctic-alpine plant Saxifraga oppositifolia into two subspecies approximately 3-5 million years ago (Abbott and Comes, 2004).

The calibration of molecular clocks is based on the approximate date when two genetic lineages diverged from one another. This date should ideally be obtained from information that is independent of molecular data, for example the fossil record or a known geological event such as the emergence of an island. The next step is to calculate the amount of sequence divergence that has occurred since that time. By dividing the estimated time since the lineages diverged by the amount of sequence divergence that has since taken place, we obtain an estimate of the rate at which molecular evolution is occurring, in ohter words the rate at which the molecular clock is ticking. Molecular clocks are usually represented as the percentage of base pairs that are expected to change every million years. If we sequence a gene from two species that were separated 500 000 years ago and we find that 490 out of 500 bp are still the same, the molecular clock would be calibrated as 10/500 = 2 per cent per 500 000 years, or 4 per cent per million years.

The most widely cited molecular clock is a 'universal' mtDNA clock of approximately 2 per cent sequence divergence every million years (Brown et al., 1982). This was originally calculated using data from primates and has since been extrapolated to a wide range of taxonomic groups. In recent years, however, it has become increasingly apparent that the idea of a 'universal' clock is something of a fallacy because evolutionary rates differ within DNA regions (e.g. synonymous versus non-synonymous substitutions), between DNA regions, and also between taxonomic groups. Different mutation rates have been calculated for numerous species that were separated by geological events of a known age, such as the emergence of the Isthmus of Panama that divided the Pacific Ocean from the Atlantic Ocean and the Caribbean Sea approximately 3 million years ago. Subsequent population divergence on either side of the Isthmus has led to a number of sister species known as geminate species. A comparison of sequences from geminate shark species that were separated by the Isthmus of Panama revealed nucleotide substitution rates in the mitochondrial cytochrome b and cytochrome oxidase I genes that are seven or eight times slower than in primates (Martin, Naylor and Palumbi, 1992). Although there are no set rules, mutation rates in mtDNA seem to vary according to a number of taxonomic variables, including thermal habit, generation time and metabolic rates (Martin and Palumbi, 1993; Rand, 1994). Researchers therefore now prefer to use a molecular clock that has been calibrated within the taxonomic group and gene region that

Table 5.1 Some examples of molecular clocks that have been calculated for various genomic regions in a variety of species. Each of these clocks was calibrated from the amount of time that has passed since species diverged from one another, which in turn was inferred from independent data such as the timing of a known geological event

Table 5.1 Some examples of molecular clocks that have been calculated for various genomic regions in a variety of species. Each of these clocks was calibrated from the amount of time that has passed since species diverged from one another, which in turn was inferred from independent data such as the timing of a known geological event

Species

DNA sequence

Sequence divergence rate (% per million years)

Method of calibration

Reference

Sorex shrews

Cytochrome b

1.36

Fossil record

Fumagalli

(Soricidae)

(mtDNA)

etat (1999)

Diatoms

Small subunit

0.04-0.06

Fossil record

Kooistra and

(bacillariophyta)

ribosomal RNA

Medlin (1996)

Taiwanese

Cytochrome b

1.1

Age of

Creer et dl.

bamboo viper

(mtDNA)

Taiwan

(2004)

(Trimeresurus

stejnegeri)

Geminate

ND2 (mtDNA)

1.3

Time since

Bermingham,

marine fishes

the Isthmus

McCafferty

of Panama

and Martin

emerged

(1997)

Hawaiian

Alcohol

1.2

Age of

Bishop and

Drosophila

dehydrogenase

Hawaiian

Hunt (1988)

gene (Adh)

islands

California newt

Cytochrome b

0.8

Fossil record

Tan and

(Taricha torosa)

(mtDNA)

Wake (1995)

Marine

Cytochrome

2.4

Time since

Hellberg and

gastropods

oxidase

the Isthmus

Vacquier

Tegula viridula

subunit

of Panama

(1999)

and T. verrucosa

I (mtDNA)

emerged

they are studying, instead of a so-called universal clock. Some examples of the molecular clocks that appear in the literature are shown in Table 5.1.

Some of the best examples of molecular clocks come from species that are endemic to oceanic islands. The Hawaiian islands are volcanic in origin and their ages have been estimated using potassium--argon (K--Ar) dating. This method, which is accurate on rocks older than 100 000 years, relies on the principle that the radioactive isotope of potassium (K-40) in rocks decays to argon gas (Ar-40) at a known rate. The proportion of K-40 to Ar-40 in a sample of volcanic rock therefore provides an estimate of when this rock was formed. Such K-Ar dating has revealed that the islands in the Hawaiian archipelago are arranged from the oldest at the northwest of the array to the youngest at the far southeast. Within the main Hawaiian Islands, Hawaii is approximately 0.43 million years old, Oahu is around 3.7 million years old and Kauai emerged approximately 5.1 million years ago (Carson and Clague, 1995).

Fleischer, McIntosh and Tarr (1998) superimposed these geological ages onto phylogenetic trees to calibrate the rates of sequence divergence in several endemic taxa. This provided them with molecular clocks of 1.9 per cent per million years for the yolk protein gene in Drosophila, 1.6 per cent per million years for the cytochrome b gene in Hawaiian honeycreeper birds (Drepananidae), and a variable rate of 2.4-10.2 per cent per million years for parts of the mitochondrial 12S and 16S rRNA and tRNA valine in Laupala crickets. The authors stressed that these estimates were based on a number of assumptions, including the establishment of populations very near to the time at which individual islands were formed, and there having been very little subsequent movement between populations. The surprisingly high rates for a ribosomal-RNA encoding gene that were calculated for Laupala crickets suggested that in this species at least one or more of the assumptions were not met.

There are two final points worth noting about molecular clocks. First, the rate at which a sequence evolves is not necessarily constant through time; in some cases, mutation rates are relatively rapid in newly diverged taxa but then slow down over time (Mindell and Honeycutt, 1990). Second, although many of the estimates presented in this section may appear very similar, a difference in mutation rates of only 0.5 per cent per million years can have a significant impact on the estimated timing of evolutionary events. If the sequences of two species diverged by 5 per cent then this would translate into a 5-million-year separation according to a clock of 1 per cent per million years, but a 10-million-year separation according to a clock of 0.5 per cent per million years. Molecular clocks remain widespread in the literature but are also highly contentious. In fact, some researchers have argued that we may never achieve molecular clocks that are sufficiently reliable to allow us to date past events (Graur and Martin, 2004). Molecular clocks should therefore be interpreted with caution and ideally should be based on accurately dated geological events or fossils, and be calibrated specifically for the gene region and taxonomic group that is being studied.

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