Molecular clock hypothesis

SIGNIFICANCE: The molecular clock hypothesis (MCH) proposes that the rates of amino acid changes in proteins and nucleotide changes in DNA are approximately constant over time. When first proposed, it was immediately embraced by many evolutionists as a way to determine the age of various evolutionary lineages. After more protein sequences were analyzed, however, many examples were found to be inconsistent with the MCH. The theory has generated a great deal of controversy among evolutionists, and although it is now generally accepted that many genes do not change at constant rates, methods are still being developed to determine the ages of lineages based on amino acid and nucleotide substitutions.

History

In 1962 Émile Zuckerkandl and Linus Pauling published evidence that the rate of amino acid substitution in proteins is constant over time. In 1965, after several protein sequences (cytochrome c, hemoglobin, and fibrinopeptides) seemed to show this pattern, they proposed the molecular clock hypothesis (MCH). According to their hypothesis, mutations leading to changes in the amino acid sequence of a protein should occur at a constant rate over time, rather than per generation, as previously assumed. In other words, if the sequence of cytochrome c were determined 1,000,000 years ago, 500,000 years ago, and in the present, the rate of amino acid substitution would be the same between the first two samples as it would be between the second and third. To state this more accurately, they considered the rate approximately constant, which means that one protein may display some variation, but if the average rates of change for several were considered as a group, they would be constant.

Importance of the Molecular Clock Hypothesis

The evolutionary importance of the MCH was almost immediately apparent. Paleontologists had long determined the ages of fossils using radiometric dating techniques, but determining the date of a fossil was not the same as determining how long ago flowering plants diverged (evolved from) the other vascular plants, for example. Using the MCH, researchers could compare the amino acid sequences of a protein in a flowering plant and another vascular plant, and if the substitution rate (that is, substitutions per unit of time) was known, they could determine how long ago these two plants diverged. The MCH held great promise for solving many of the questions about when various groups of organisms diverged from their common ancestors. To “calibrate” the clock—that is, to determine the rate of amino acid substitutions—all that was needed were the sequences of some taxa and a reliable age for fossils considered to represent the common ancestor to the taxa. Once this clock had been calibrated, other taxa that might not be as well represented in the fossil record could be studied, and their time of divergence could be determined as well.

As more data accumulated over the next twenty years, it was discovered that amino acid substitutions in many proteins were not as clocklike as hoped. Rates over time seemed to slow down and speed up, and there was no predictable pattern to the changes. In fact, the same proteins in different evolutionary lineages often “ticked” at different rates.

The Neutral Theory

During the time that more and more proteins were being sequenced, DNA sequencing gradually began to dominate. One of the theories about why the MCH did not seem to be working was that protein sequences were constrained by natural selection. The intensity of natural selection has always been assumed to vary over time, and if this is true, then amino acid substitution rates should also increase and decrease as some kind of function of the pressure exerted by natural selection. DNA sequences were quickly hailed as the solution to this problem. In 1968, biologist Motoo Kimura proposed the neutral theory of molecular evolution, in which he proposed that any nucleotide substitution in DNA that occurred in a noncoding region, or that did not change the amino acid sequence in the gene’s product, would be unaffected by natural selection. He suggested that because of this, neutral mutations (nucleotide substitutions) would be free to take place without being weeded out by selection.

The strength of the neutral theory was that, unlike mutations that affect the amino acid sequence, neutral mutations should occur at a constant rate over time. Therefore, Kimura predicted that the MCH would be valid for neutral mutations. Most eukaryotic genomes are riddled with sequences, like introns or highly repetitive DNA, that have no apparent function and can therefore be assumed to be prone to neutral mutations. Even within the coding regions (exons) of expressed genes, the third position of many codons can be changed without affecting the amino acid for which it codes. A number of evolutionists expressed skepticism concerning the neutral theory, arguing that there is probably no truly neutral mutation.

As DNA sequences were decoded, much the same story emerged as for protein sequences. Whether or not neutral mutations exist, nucleotide substitutions that were assumed to be neutral ticked no better. In the 1980s the controversy over the MCH reached its height, and most evolutionists were forced to conclude that very few genes, or neutral sequences, behaved like a clock. Even those that did behave like clocks did not tick at the same rate in all lineages, and even worse, some genes ticked more or less steadily in some lineages and very erratically in others. Comparisons among the many amino acid and nucleotide sequences revealed another surprise: Amino acid sequences tended, on average, to be more reliable than nucleotide sequences.

Beyond the Molecular Clock

Since the 1980s, the MCH has fallen into disfavor among most evolutionists, but attempts to use amino acid and nucleotide sequences to estimate evolutionary ages are still being made. In a few cases, often in closely related taxa, the MCH works, but other approaches are used more often. Many of these approaches attempt to take into account the highly variable substitution rates among different lineages and over time. Rather than using a single protein or DNA sequence, as was attempted when the MCH was first developed, they use several in the same analysis. Data analysis relies on complex, and sometimes esoteric, statistical algorithms that often require considerable computational power.

Today, the research community is in disagreement when it comes to post-MCH methods. There are several alternative approaches, and some that represent blended approaches, and agreement is far from being achieved. It is hoped that as more data are collected and analyzed, a coherent approach will be developed.

Key Terms

• codona three-letter nucleotide sequence in RNA or DNA that codes for a specific amino acid; a gene is composed of a long string of codons
• intronan intervening sequence in a eukaryotic gene (generally there are several to many per gene) that is removed when it is transcribed into messenger RNA (mRNA); introns are assumed to have no function and therefore mutations in them are often considered neutral
• neutral mutationa mutation in a gene which is considered to have a negligible effect on the fitness of the organism
• phylogenyoften called an evolutionary tree, the branching patterns that show evolutionary relationships, with the taxa on the ends of the branches
• taxon (pl.taxa) a general term used by evolutionists to refer to a type of organism at any taxonomic rank in a classification of organisms

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