Zoology 510, Class Notes for Ridley, Chapter 7
Molecular Evolution and the Neutral Theory

• "In a species with a million individuals, ...it takes roughly a million generations for genetic drift to change allele frequencies appreciably.  There is no conceivable way of verifying that genetic drift changes allele frequencies in most natural populations. Our understanding that it does in entirely theoretical."
• "[T]he common thread throughout the book is what Gillespie calls 'the main obsession of our field,' the recurring question, 'Why is there so much genetic variation in natural populations?'"
• "Population genetics remains the central intellectual connection between genetics and evolution."

Brief Outline

• Introductory comments
• Overview
• Checklist of important concepts and terms
• Historical references

• Section-by-section comments
  • 7.1. Two competing explanations.
  • 7.2. Rates of molecular evolution.
  • 7.3. Genetic load.
  • 7.4. The 'cost' of natural selection.
  • 7.5. Test 1 (part 1).
  • 7.6. Test 1 (part 2).
  • 7.7. Hard vs. soft selection; selection on many loci.
  • 7.8. Neither case can be refuted by Test 1.
  • 7.9. Consistency of rate for molecular evolution.
    • Box 7.1. Relative rate test.
  • 7.10. The molecular clock.
    • Box 7.2. Ohta's "Nearly Neutral Theory".
  • 7.11. Generation time effect.
    • Box 7.3. Fisher's model of adaptive evolution.
  • 7.12. Selection on protein structure.
  • 7.13. Correlation of evolutionary rate with heterozygosity.
  • 7.14. Inconclusive conclusion.

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Introductory comments.

• Protein and DNA sequences generally differ when compared in related species, with the number of differences approximately correlated with phylogenetic distance. To what extent is this molecular evolution driven by natural selection, and to what extent is it driven by mutation and genetic drift?  This question remains controversial in current evolutionary theory.  
• We shall be considering molecular evolution in terms of the processes that act to affect gene frequencies.  You might check here for a quick list of these several processes (use the "back" button to return here), or review Chapters 5 and 6.  You should also review molecular mechanisms of mutation (Chapter 2, sections 2.4 - 2.5).
• Let's be perfectly clear.  No one disputes that natural selection is responsible for adaptation.  And no one disputes that drift can cause molecular evolution.  The controversy concerns the relative importance of these two real mechanisms as a necessary explanation for most molecular evolution.
• Nor is it adequate simply to say that selection and drift are both important, since many other significant questions hinge on whether one or the other of these two predominates.
• Since this issue remains unsettled, we will be considering not only the two competing hypotheses, but also the observations and tests which attempt to distinguish between the two alternatives.
• Ridley's presentation roughly tracks the history of the controversy, with argument and counterargument as they were introduced over the past few decades.

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Overview.

• Read the entire chapter for its overall structure before concentrating on the several significant concepts that are explained along the way.
• This chapter presents a controversy via a point/counterpoint form, without final resolution.

• The central problem is introduced (7.1).
• Background concepts are introduced.
  • Rate of molecular evolution (7.2)
  • Genetic load (7.3).
  • Cost of selection (7.4).

• Test 1.
  • An argument against selection is presented.
    • The cost of selection appears too high for selection to be the cause for all gene replacement (7.5).
    • The segregational load appears too high for polymorphism to be maintained by heterozygote advantage (7.6).
  • The argument is countered (7.7).
    • Soft selection can reduce the cost of selection (7.7.1).
    • If fitness effects are not independent for various loci, segregational load is reduced (7.7.2).
    • Frequency dependent selection can maintain polymorphism without genetic load.(7.7.2).
  • Summary of Test 1.  Critical evidence is not available to discriminate between the two arguments (7.8).

• Test 2.
  • Another argument against selection is presented.
    • A molecular clock is consistent with neutral theory but is not expected under selection (7.9).
  • Counterargument.
    • The molecular clock appears to follow calendar time, not generation time as expected by neutral theory (7.10.1).
  • Counter to the counterargument.
    • The nearly-neutral mutation rate may be effectively higher in species with long generation time, moving the clock closer to calendar time (7.10.1, Box 7.2).
    • Selection does not predict either a calendar or a generation time clock.
  • Silent DNA substitutions fit neutral theory better than do amino acid substitutions in proteins.

• Test 3.
  • Different parts of proteins evolve at different rates, depending on functional constraints (7.11.1) .
  • Both theories can, with some effort, explain the observations (7.11.2) .
  • Different parts of genes, and different codon sites, evolve at different rates (7.11.3) .
  • Neither theory can completely explain patterns in the data (7.11.4) .

• Test 4.
  • Correlation of rate of change with heterozygosity does not give a clear result. (7.13) .

• "The controversy is not settled (7.14) .

CHECK LIST of important CONCEPTS and TERMS

• Neutral Theory controversy
• Mutation
  • Neutral mutation
  • Deleterious mutation (negative selection coefficient)
  • Favorable mutation (positive selection coefficient)
  • Frequency distribution for mutation rate vs. selection coefficients
• Protein sequence evolution
  • Amino acid substitution
• DNA sequence evolution
  • Base-pair substitution
  • Silent substitution
  • Replacement substitution (meaningful substitution)
• Rate of molecular evolution (rate of substitution)
  • Mutation rate
  • Substitution rate
• Genetic load
  • Optimal fitness
  • Mean fitness
• Cost of selection
  • Substitutional load
  • Segregational load
  • Hard and soft selection
  • Multiplicative (independent) fitnesses
• Molecular Clock
  • Relative rate test
  • Generation time effect
  • Functional constraint effect
  • Pseudogene
• Slightly deleterious mutations (the "Nearly Neutral Theory")
• Fisher's model of adaptive evolution
• Codon usage bias
• Correlation of heterozygosity with evolutionary rate

Chapter 7, Section-by-Section Comments

7.1. "Neutral drift and natural selection can both hypothetically explain molecular evolution."

• This section plainly lays out the basic controversy.
• Molecular variation is observed between homologous proteins.  Do these differences reflect:
  • Adaptive optimization of each variant by natural selection?
           OR
  • Random shuffling by genetic drift among adaptively equivalent variants?

• The presumed distribution of selection coefficients among mutations is one part of the controversy.  Ignoring substantially deleterious mutations (which everyone agrees can occur, and will be eliminated by selection), does the normal supply of mutation include:
  • A multitude of possibilities for adaptive improvement?
           OR
  • A variety of adaptively equivalent mutants?

• "Pan-neutralism" is not a serious contender, but is mentioned here to clarify the contrast between the two major competing theories.
• The hypothesis that neutral mutation and drift together can account for most molecular evolution is called the Neutral Theory.  The formulation of this influential concept in the 1960s and 70s is most closely associated with the name Motoo Kimura.  T. Ohta later introduced the significant modification known as the "nearly neutral theory".

7.2. "The rates of molecular evolution and amounts of genetic variation can be measured."

• The rate of molecular evolution can be measured for both protein sequences and DNA sequences, by counting sequence differences between two related species and dividing by twice the time since they diverged from a common ancestor.  (Why twice the time?  Because differences could accumulate independently in each species since divergence.)
• Genetic variation is measured as proportion of polymorphic loci within species.

7.3. "A population in which not all individuals have the optimal genotype is said to have a genetic load."

• Here Ridley introduces the standard symbol, w, for fitness.
• Genetic load is defined as optimal fitness minus mean fitness.  Since mean fitness is the proportion of survivors, genetic load is the fraction of the population removed by selection.  Optimal fitness is to be understood not as some imagined perfect adaptation, but as the fitness of the actual genotype that has the highest fitness of all available genotypes.
• The genetic load always exists as the result of deleterious mutation is called mutational load.  (The equation derived for mutational load, L = 2m, applies only for the given case of a single-locus, two-allele, recurrent dominant deleterious mutation.)
• A standing genetic load can also result from heterozygote advantage (as we shall see in section 7.6), where it is called segregational load.
• Genetic load is also incurred, temporarily, whenever a better allele (which allows for a higher optimal fitness) is introduced into a population.  The load decreases as the advantageous allele increases in frequency, until the new allele is fixed.

7.4. "Haldane described a 'cost' of natural selection."

• J.B.S. Haldane, you will recall, was one of the founders of the "Modern Synthesis".
• The cost of selection is related to the idea of genetic load.  (Note that Ridley's math for calculating cost, C, specifies a haploid case for simple computation, and is not meant to be a general model.)
• Whenever a "new, improved" allele is introduced into a population, there will be a genetic load associated with selection against the "old, inferior" allele.
• The cost of selection is the accumulated genetic load over all the generations that pass until the "new, improved" allele is fixed.
• It might not be quite obvious that a "unit" for the cost of selection (e.g., Table 7.3) is the size of the standing population.  For example, a cost of 4 means that over the many generations it takes to substitute one allele for another, the number of individuals removed by selection equals four times the standing population size.
• If one knows how many alleles have been substituted over time (as one can simply by counting sequence differences between species, see 7.2), one can calculate the cost that must have been incurred if selection were responsible for each substitution.
• We will see later in this chapter (7.7) that the very idea of the cost of selection assumes that most excess reproductive capacity is just waste rather than loss to selection, such that any selection actually reduces the population below its capacity at optimal fitness

7.5. "Test 1 (part 1):  the rates of molecular evolution were argued to be too fast to be explained by natural selection because the implied cost of selection would be too high."

• Here we apply the idea of genetic load and cost of selection.
• Since we can measure the rate of gene substitution (7.2), we can calculate the overall cost of selection if all of these substitutions resulted from selection.
• M. Kimura (the founder of neutral theory), calculating from Haldane's model, showed that the cost of selection, if all observed substitutions in molecular sequences resulted from selection, would far exceed the available number of organisms.  However, there is no genetic load when neutral alleles drift to fixation.
• Two conclusions might be reached from this result.
  
  • Selection is not the right explanation for most molecular substitutions.
               OR
  • Some assumptions in Haldane's model are not appropriate.
• Kimura chose the first conclusion, and gave us the neutral theory.  With a high enough rate for neutral mutation, genetic drift can explain molecular evolution.

7.6. "Test 1 (part 2):  the degree of genetic variation in populations was argued to be too high to be explained by natural selection because the implied segregational load would be too high.."

• If genetic variation were maintained by heterozygote advantage, genetic load would result from selection against the homozygotes.  This is called segregational load.
• Kimura calculated, if all of the many numerous molecular polymorphisms that can be observed were independently maintained by heterozygote advantage, the total mortality resulting from this segretional load would be impossibly high.  But random genetic drift of neutral mutations can maintain polymorphism with no genetic load.
• Again, two conclusions might be reached.
  • Selection must not be the right explanation for most molecular polymorphism.
               OR
  • Some assumptions about segregational load involving multiple loci may not be appropriate.
• Again, Kimura chose the first conclusion and gave us the neutral theory.
• However, the observed frequency of molecular polymorphism is not nearly as high as naively expected from neutral theory (Fig. 7.3, p. 164).

7.7. "Some possible answers to the genetic load problem."

• 7.7.1. "Natural selection can operate without running up an impossible genetic load if it is soft rather than hard."
  • Hard selection is defined as selection which reduces standing population size.
  • Soft selection is defined as selection which does NOT reduce standing population size, but occurs entirely within the excess reproductive capacity of the population.
  • Note that with hard selection, most "excess" reproductive capacity essentially goes to waste.  With soft selection, selection occurs within this excess capacity so population size is not reduced by selection.
  • You should understand Ridley's hypothetical examples of soft and hard selection, as well as the explanatory graphs of Fig. 7.4 on p. 166.
  • The argument regarding cost of selection assumed hard selection.
  • If selection is soft, then the cost of selection poses no problem for selection as an explanation for molecular evolution at observed rates.
  • Note that the possibility of soft selection in no way refutes the neutral theory, it merely preserves the possibility that selection might still be a viable explanation.

• 7.7.2. "Natural selection can act jointly on many genetic loci."
  • The argument regarding segregational load assumed that selection must operate independently on each locus.
  • If loci are not independent (technically, if loci do not have multiplicative effects on fitness), then the segregational load of many suboptimal alleles can be greatly reduced.
  • Again, note that the possibility of non-independent effects on fitness in no way refutes the neutral theory but merely preserves the possibility that selection might still be a viable explanation.

  • An additional point is introduced at the end of this section.  Selection can maintain polymorphism not only by heterozygote advantage but also by frequency-dependent selection.  And, unlike heterozygote advantage, there is no necessary cost associated with frequency-dependent selection.

• Make sure you understand how the previous sections (7.2 through 7.7) lead to this conclusion.
• "A neutralist could 'explain' almost any evolutionary rate or heterozygosity by positing suitable values for N or u" (effective population size and mutation rate).
• "The selectionist can [also] explain almost any observation by inventing suitable values for unknown numbers."

7.9. "Test 2:  the rates of molecular evolution are arguably too constant for a process controlled by natural selection."

• Rates of molecular evolution are surprisingly steady across related lineages, at least when homologous molecules are being compared.
• This result is called the molecular clock, although the term can be misleading.
• Although this result does not contradict a selectionist model, it was NOT expected from a selectionist model.
• This result IS expected from the Neutral Theory.
  • The comparison of alpha and beta hemoglobins among human, shark and carp is intended to highlight this surprising result.  However, the argument presented is incomplete.  To support the conclusion that differences between the alpha and beta forms have separately accumulated at the same rate in human, shark, and carp lineages, we need not only data showing how many substitutions have happened but also data to show when they occurred.  (From the data given, it could be possible that the observed substitutions all happened immediately after the globin gene duplicated but before the human, shark and carp lineages had diverged.)  If we can assume a consistent molecular clock for this gene, then all three lineages show similar rates of change (since similar numbers of substitutions have occurred).  Tabulation of alpha-alpha differences and beta-beta differences between the various pairs of species (not shown by Ridley) do support this conclusion.

Box 7.1. "The Relative Rate Test"

• Make sure you understand the basic logic for the relative rate test.  Later (Chapter 17) it will be useful for understanding the construction of phylogenetic trees from molecular sequence data.
• Note that Fig. B7.2 graphs a hypothetical possibility, not real data.

• 7.10.1. "The protein molecular clock runs relative to absolute time, not generation time."
  • Understand the distinction between absolute time and generation time.
  • A molecular clock is expected to count generation time because known mutational mechanisms seem to associate with DNA replication rather than with simple passage of time.  DNA is generally replicated less often in species with long generation times.
  • The molecular clock for protein evolution actually matches absolute time better than generation time.
  • This result was not only not expected from initial neutral theory, but could be seen as contradiction of neutral theory.  However, see Box 7.2.

• 7.10.2. "DNA sequences can now be used to test for a generation time effect in the molecular clock."
  • The molecular clock for silent substitutions in DNA evolution does show a generation time effect.
  • So, in this case where mutations are expected to be neutral in fact, the neutral theory does seem to fit.
  • "Perhaps the silent parts of the DNA do, in fact, evolve neutrally." (p. 178)

Box 7.2. "Slightly Deleterious Mutations in the Neutral Theory"

• This box introduces Ohta's "Nearly Neutral Theory".
• Basically, this theory offers a friendly modification of neutral theory to correct for the generation time effect.
• Creatures with longer generation times generally have relatively smaller population sizes.  With small population sizes, slightly deleterious mutations (which would be effectively selected against in large populations) will behave by drifting like effectively neutral mutations.  Thus as generation time increases, its effect on clock rate will be compensated by an increase in the rate of effectively neutral mutations
• Note that Figure B7.3 doesn't make much sense.  The caption is reasonable, but doesn't apply meaningfully to the graphs.

7.11. "Evolutionary rate and functional constraint."

• 7.11.1. "Test 3:  the more functionally constrained parts of proteins evolve at slower rates."
  • The basic idea in this section is that different parts of proteins, and different parts of genes, differ in how much change can be tolerated without deleterious effect.  If little change can be tolerated, that region is said to be "constrained".

• 7.11.2. "Both natural selection and neutral drift can explain the trend."
  • Under neutral theory, the rate of neutral mutations relative to the rate of deleterious mutations will be lower in the the more constrained regions, so neutral substitutions will accumulate more slowly.
  • Under selectionist theory, favorable mutations are less likely in constrained sites (because in such sites any change is likely to have a large effect), so selectively advantageous small changes are more likely in less constrained locations.
  • The argument and counterargument can become more subtle than this.

• 7.11.3. "Silent sites in the DNA evolve more rapidly than replacement sites."
  • If there were such a thing as a totally unconstrained sequence, where any mutation would be neutral, then the substitution rate at that site would indicate the actual mutation rate.  (Remember, from Chapter 6, section 6.4, the average rate of substitution by drift equals the mutation rate, for truly neutral mutations).
  • To a first approximation, silent codon substitutions and substitutions in pseudogenes might be expected to be totally unconstrained.
  • The observed more-rapid evolution in silent sites and pseudogenes IS expected by neutral theory.
  • Significant differences between between different pseudogenes or between the silent sites of different genes (Table 7.8 and Figure 7.10) are NOT expected by neutral theory.  These differences suggest that substitutions in such sites are constrained after all, and hence selection must be acting on such sites.

• 7.11.4. "Codon usages are biased."
  • If silent substitutions were unconstrained (i.e., if all synonymous codons were functionally equivalent), then only random variation should appear in the numbers of synonymous codons.  "In fact, consistent biases appear."
  • Something, maybe selection in relation to DNA secondary structure, maybe selection in relation to available tRNAs, maybe directional mutation pressure, must be preventing silent substitutions from evolving simply by drift.

Box 7.3. "Fisher's Model of Adaptive Evolution"

• In evolution, small steps are more likely to be successful than large steps, as a general rule.  This is because small steps must move either toward or away from improvement, while large steps can miss the mark in any direction.
• Therefore, most evolutionary changes will (must) be small.
• This is most readily seen in terms of the adaptive landscape, Fig. B7.4, p. 182.

7.12. "DNA sequences provide strong evidence for natural selection on protein structure."

• Note that the Adh (alcohol dehydrogenase) example does not offer clear evidence against neutral theory.  It merely affirms that amino acid sequence is highly constrained for this locus, while silent substitutions (as expected) are not so highly constrained.  Recall that, in neutral theory, functional constraint means neutral mutation rate is reduced so evolutionary rate is also reduced.
• Neutral theory does not deny that selection operates to resist deleterious changes; it only presumes that most molecular changes that do evolve result from neutral drift rather than advantageous mutations.
• Ridley's reference to Figure 7.1a is only accurate in part.  The Adh example does imply a paucity of neutral amino-acid-substitution mutations at this locus, as shown in the cartoon graph.  But the example does not indicate a significant number of mutations with positive selection coefficients.

7.13. "Test 4:  are the rates of evolutionary change of molecules correlated with their heterozygosities?"

• In the neutral theory, the rate of evolutionary change (nucleotide substitution) should be equal to the neutral mutation rate (see Chapter 6, section 6.4, p. 140), so the ratio of substitution rates for two sequences should be the same as the ratio of mutation rates.
• If the the neutral mutation rates vary because of functional constraints, so also should the evolutionary rates.
• In the neutral theory, the ratio of heterozygosities for two sequences should also be (approximately) proportional to the ratio of neutral mutation rates (derivable from Equation 6.7, p. 146).
• Therefore, rates of change due to neutral drift should correlate with heterozygosities due to neutral drift, and the ratios should be equal, regardless of functional constraint.
• Selection offers no reason to expect such a correlation, except by chance.
• Data from this test are ambiguous.

7.14. "The analysis of DNA sequences is only the most recent stage in a long controversy."

• This chapter has introduced the Neutral Theory controversy.
• But this chapter also introduces a number of other important concepts, as noted in the Checklist above.

Historical references:

• "The Neutral Theory of Molecular Evolution", by Thomas H. Jukes, 2000, Genetics 154 956-958, online.
• "Thomas H. Jukes (1906-1999)", by James F. Crow, 2000, Genetics 154 955-956, online.

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