Neutral mutations and evolutionary clocks

The primary proponents of neutral mutations and evolutionary clock theories were Motoo Kimura (1924-1994) of the National Institute of Genetics of Japan and Masatoshi Nei (1931-2023) of the University of Texas. They asserted that most polymorphisms that occur at the molecular level are selectively neutral. Two major categories of polymorphisms are involved. The first is attributable to changes in the nucleotide sequences of deoxyribonucleic acid (DNA). The second is isozymic variation, detectable by protein electrophoresis. Isozymic variation is usually caused by changes in the amino acid composition of the protein. Since amino acids are determined by the genetic code, this type of variation also ultimately depends on changes in DNA. The proponents of the neutral theory admit that most of the evolution that occurs on the nonmolecular level, such as changes in morphological and behavioral traits, is attributable to natural selection. However, they believe most of the changes result from chance, or genetic drift, on the molecular level.

Selection Versus Chance

Extensive variability has been found for both DNA sequences and isozymes within most natural populations. Isozymic polymorphism, in which two or more variants of an enzyme occur within the same species, ranges from approximately 15 percent in mammals to approximately 40 percent in invertebrates. Isozymic variation, even within the same individual, ranges from about 4 percent in mammals to 14 percent in some insects. Variability in DNA sequences among individuals of the same species is even higher than those found for isozymes. Proponents of the neutral hypothesis hold that evolutionary changes in species occur at the molecular level and that these levels of variability are too high to be attributable to selection, but instead, most variability at the molecular level is attributable to genetic drift. The result, they say, has been a large amount of enzyme and DNA variability that is selectively neutral. The molecular mutations are neutral in the sense that their contributions to an organism’s fitness are so small that their occurrence is attributable more to chance than to natural selection. Selectionists do not believe that most molecular mutations are neutral; they assume that most are harmful and are eliminated by natural selection. Proponents of the neutrality theory believe that changes in DNA and amino acid sequences are generally neutral, consisting primarily of the gradual random replacement of functionally equivalent alleles. Some examples of neutral mutations include eye color, ear lobe appearance, and fur color.

Although the neutral theory can explain much about molecular evolution, there are some issues that remain subjects of intense debate. It is known that some protein and gene variation is not neutral but, instead, under certain conditions, conveys selective advantages or disadvantages. In some organisms (for example, the Japanese macaque), there also appear to be more rare alleles than would be predicted by the neutrality hypothesis.

The Hypothesis of a Molecular “Clock”

If the neutrality theory of molecular evolution is correct, then changes in base sequences of DNA could act as evolutionary “clocks.” This theory holds that because mutations change the DNA in all lineages of organisms at steady rates over long periods, a clock-like relationship can be established between mutation and elapsed time. The number of base substitutions in the DNA is directly proportional to the length of time since evolutionary divergence between two or more species. The idea of molecular evolutionary clocks was forwarded in the early 1960s by Linus Pauling (1901-1994) and Emile Zuckerkandl (1922-2013).

The molecular clock postulated by the neutrality hypothesis is not like an ordinary timepiece, which measures time in exact intervals. Rather, molecular changes occur as a stochastic clock, such as occurs during radioactive decay. Although there is some variability for this type of clock (it is slower or faster during some periods than others), it would be expected to keep relatively accurate time over millions of years. A potential problem arises, however, because the rate of “ticking” for the molecular clock is not the same at every position along the DNA molecule. The rate has been shown to be slow for DNA sequences that directly affect the function of a protein (for example, those at an enzyme’s active site), while the rate of change has been faster for positions on the DNA that are selectively neutral, that is, where they have little or no impact on the protein’s function.

In a molecular clock, the number of changes in a DNA or protein molecule are the “ticks” of the clock. The number of “ticks” estimates the extent of genetic differences between two species. With this knowledge, scientists can reconstruct phylogenies. The phylogenies are usually depicted as branching patterns based on differences in DNA base-pair or amino acid sequences. They depict not only the order of descent but also the degree of relatedness.

When choosing alternative phylogenetic hypotheses, biologists usually follow the principle of Occam’s razor—the simplest theory is chosen over more complex ones. Thus, the phylogenetic tree that requires the fewest mutations is preferred over those that require more mutations. By “calibrating” the molecular clock with other independent events, such as those obtained from the fossil record, the actual chronological times of divergence can be estimated.

For example, humans and horses differ by eighteen amino acids in the alpha chain of the blood protein hemoglobin. It has been estimated from the fossil record that humans and horses diverged from a common ancestor approximately ninety million years ago. Other evidence suggests that half the substitutions took place since the time of divergence. Since nine amino acids have changed over a ninety-million-year period, the rate of amino acid substitution would equal approximately one every ninety million years.

Since mutation rates are known to be different for different genes, the ticking rate for different genes or proteins would not necessarily be the same. For example, the rate of substitution for the genes coding for the protein histone H4 is lower than that for the genes coding for the protein gamma interferon. Yet, when nucleotide substitution rates are averaged for various proteins, there does appear to be a marked uniformity in the rate of molecular change over time. The ticking of a number of clocks can be averaged, leading to more accurate estimates for divergence times.

Advantages and Disadvantages of the Theory

Much of the early work was done on sequence changes in proteins; however, there is a drawback to protein clocks. Their usefulness is limited because the genetic material itself is not being examined but, rather, a product coded by genes. This means that some of the changes in the genetic material may not be detected. For example, because of the redundancy of the genetic code, several changes could occur in the nucleotide sequence of DNA that would not result in changes in the amino acid composition in a protein. Consequently, there has been great interest in directly examining the DNA itself.

Because of the advent of recombinant DNA techniques, molecular clocks can be based on changes in the genetic material. DNA-DNA hybridization also involves the comparison of DNA sequences, although on a broader scale. The DNA-DNA hybridization technique is attractive because it effectively compares very large numbers of nucleotide sites, each of which is effectively a single data point. One of the criticisms of molecular clocks is that most genes have not been found to “tick” with perfect regularity over long periods of time. During some periods, the rate of change (primarily because of mutation) may be fast, while at other periods, it may be significantly slower. By comparing very large numbers of nucleotides, which represent many genes, DNA-DNA hybridization measures the average rates of change, which will produce more uniform estimates.

The concept of a molecular clock has been criticized on many grounds. First, it assumes that evolution in macromolecules proceeds at an approximately regular pace, whereas morphological evolution is usually recognized as occurring irregularly. It is also clear that the clock can tick at different rates among different macromolecules, whether they be proteins or DNA. Another problem is that the rate of the molecular clock varies among taxonomic groups. For example, the insulin gene has evolved much more rapidly in the evolutionary line, leading to the guinea pig than in some other evolutionary lines. There are also notable differences among different parts of molecules. This variability was evident when sequences were compared among the first molecules examined in the light of the molecular clock hypothesis, notably hemoglobin molecules and cytochrome c. Another criticism is that a number of processes, known collectively as molecular drive, perturb the clock.

Some data suggest that nucleotide substitution rates in eucaryotes and procaryotes are as different as bacteria, flowering plants, and vertebrates, which are remarkably similar. For example, the average substitution rate at “silent sites” (where mutation in the DNA produces no change in the amino acid encoded) is 0.7 to 0.8 percent per million years in bacteria like Escherichia coli, 0.9 percent for mammals, and 1 percent for plants. This relatively equal substitution rate across broad taxonomic categories would support the concept of the constancy of molecular clocks. However, other data notes that the nucleotide substitution rate varies not only across species but also across areas of each animal’s genome. Larger species populations have lower substitution later than small populations. Also, free-living organisms have slower substitution rates than organisms like fungi.

Testing the Hypothesis

Several types of molecular clocks have been hypothesized. In the first group are techniques that directly estimate differences in the sequences of nucleic acids that make up DNA or in amino acid sequences (which are determined by DNA). Other methods are less direct, such as DNA-DNA hybridizations and immunological techniques. All the techniques ultimately assay genetic differences caused by base pair changes in DNA. Sequence comparisons and DNA-DNA hybridization techniques are now used more extensively.

In sequence comparisons, nucleic acid replacement in DNA or amino acid replacement in proteins are compared between species. Nucleic acid substitutions can be assayed by using restriction enzymes that only recognize specific base sequences or by direct sequencing. Amino acid substitutions can be assayed by traditional biochemical techniques, through automated sequencers, or by mass spectrometers. In both types of sequence analysis, the assumption is made that the greater the number of substitutions, the greater the evolutionary distance between the species. In DNA-DNA hybridization, DNA molecules from individuals from two species are separated into individual strands at high temperatures. The strands are then mixed at a lower temperature. This promotes the joining of the strands from the different organisms. The extent of rejoining (how tightly they bond together) will be dependent on the degree of nucleotide pairing that occurs. If the nucleotide sequences between the two species are very similar, the DNA strands will bond very tightly; if there is little similarity, the bonds will be weak. The extent of bonding is measured by the temperature at which the new DNA duplex dissociates, or “melts.” The higher the melting temperature, the greater the nucleotide similarity between the DNA strands from different species. The nucleotide similarity is presumed to be related to the evolutionary distance between the species.

Uses of the Theory

The use of molecules as clocks, despite their imperfect nature, has proven to be a valuable tool for inferring phylogenetic relationships among species and in estimating their times of divergence. Molecular data can be used independent of morphological and behavioral data for establishing evolutionary relationships. Similarly, divergence times estimated from the fossil record can be clarified using molecular clock data. Molecular clocks have had a significant impact on evolutionary studies of organisms ranging from bacteria to humans, and molecular data have been instrumental in changing some long-held phylogenetic views. For example, the data obtained from DNA-DNA hybridizations in birds have forced a major revision in bird taxonomy. Molecular clocks have been used to assign time scales to a large number of phylogenies. Some of the phylogenies are wide-ranging; approximate times of evolutionary divergence have been assigned to vertebrate species as diverse as sharks, newts, kangaroos, and humans. Others are more specific. Some of the best-known (and most controversial) work has been done on primates. In one set of experiments, the amino acid differences of serum albumin (a blood protein) were measured among different species of primates. By comparing the albumins of species whose divergence times were known from fossil evidence, researchers were able to “calibrate,” or calculate, the mean rate of change for serum albumin. Previously, most anthropologists believed that humans and apes had diverged approximately twenty-five million years ago; the DNA-DNA hybridization data suggest a much more recent date of approximately five million years. Subsequent DNA studies have confirmed the latter estimate. This led to a reevaluation of the primate fossil record and of the way in which primates have evolved, including humans.

Another group of researchers has used DNA-DNA hybridization data to calculate the divergence dates among different primate species. After calibrating the molecular clock with dates previously established from the fossil record, they estimated the following approximate divergence dates: Old World monkeys, 30 million years ago; gibbons, 20 million; orangutans, 15 million; gorillas, 7.7 to 11 million; and chimpanzees and humans, 5.5 to 7.7 million years ago. In contrast to earlier work, they concluded that humans and chimpanzees are genetically closer to each other than either are to gorillas. As with the serum albumin data, these new estimates of times and order of divergence led to a reexamination of primate evolution.

The neutrality theory of molecular evolution is criticized by some scientists as a null hypothesis of no effect, but with the appropriate application under specific conditions, the theory is useful. Evolution research is largely based on fossil evidence, and the neutrality theory allows scientists to establish the framework for estimating points in time based on the last known ancestor of the species in question. It also has applications in the evolution of genome architecture.

Principal Terms

Allele: One of two or more alternate gene forms of a single gene locus

Amino Acid: An organic molecule with an attached nitrogen group that is the building block of polypeptides

Electrophoresis: A technique for separating molecules when they are placed in an electrical field; the separation is usually based on their charge and weight

Genetic Code: The three-nucleotide base sequences (codons) that specify each of the twenty types of amino acids; there can be more than one codon for a particular amino acid

Nucleic Acid: An organic acid chain or sequence of nucleotides, such as DNA or RNA

Phylogeny: The evolutionary history of taxa, such as species or groups of species; order of descent and the relationships among the groups are depicted

Polymorphic: A genotype or phenotype that occurs in more than one form in a population

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