20

Evolution of Genes and Traits

WORKING WITH THE FIGURES

1. Examining Figure 20-4, explain why the rate of evolution at nonsynonymous

sites is lower. Do you expect this to be true only of globin genes or of most
genes?

Answer: Both types of mutations are expected to arise at the same rate.
However, synonymous substitutions do not change the amino acid sequence
of a protein and generally have no phenotypic effect. They are therefore not
subject to natural selection and are expected to accumulate freely. On the
other hand, nonsynonymous substitutions do change the amino acid sequence
and potentially have a wide range of affects on phenotype. These effects are
subject to natural selection. Most nonsynonymous substitutions will be at
least slightly harmful and selected against, reducing their frequency, while a
few will be nearly neutral or advantageous; so nonsynonymous mutations will
tend to accumulate much more slowly and be fixed at a lower rate than
synonymous ones. This general tendency would not change from gene to
gene, so the effect would be expected to occur for most genes.

2. In Figure 20-7, the overall survival rates of three genotypes are plotted.
Explain the reasons for the differences between the three survival curves.

Answer: The decrease in survivorship common to all three genotypes could be

due to any of many factors affecting early childhood mortality. The
differences between the genotypes can be explained as differences in
resistance to infection by malaria parasites (P. falciparum), an important
source of early childhood mortality in areas where malaria is common. The
graph indicates that SS genotypes suffer the greatest mortality, likely due to
the consequences of red blood cell sickling in homozygotes. Untreated, this is
frequently a lethal condition. AA has the second highest mortality. AA
homozygotes have no sickling but are subject to high childhood mortality
from infection by malaria parasites. AS has the lowest mortality because
heterozygotes are partially resistant to infection by the malarial parasite and
do not exhibit harmful sickling of red blood cells. There are at least two
466 Chapter Twenty

possible explanations for a decrease in the difference between AS and AA as

the children age. First, malaria mortality is highest among young children,
who are more susceptible because their natural immunity has not yet
developed. Second, AA genotypes that survive may have acquired immunity
to infection and are less likely to be infected as adults. In areas where malaria
is common, mortality from the disease drops dramatically as children age and
is uncommon among adults.

3. From Table 20-4, would you expect the noncoding mutation g4205a to be
fixed before or after the coding mutation G238S in a population of bacteria
evolving resistance to the antibiotic cefotaxime? Give at least two reasons for
your answer.

Answer: The noncoding mutation g4205a would be fixed after the coding
mutation G238S. First, the noncoding mutation had a positive effect on only
8 alleles, whereas the coding mutation had a positive effect on all 16. Also,
the coding mutation had a positive effect independently of the state of other
genes. This gives it a strong advantage. Second, the noncoding mutation had a
negative effect on 2 other alleles, increasing its disadvantage relative to the
coding gene. The strength of the advantage of the coding gene is seen in the
mean proportional increase column—the increase in the coding mutation is
almost 3 orders of magnitude greater than the noncoding gene.

4. Examining Table 20-5, what do you think would be the order of mutations
fixed during selection in a third evolving virus line? Would the mutations
become fixed in the same order as the TX or ID virus?

Answer: The differences in the order of mutations fixed between the two
different lines indicates that the order is not predictable—even though several
of the fixed mutations were the same in the two lines, the mutations were not
fixed in the same order. It is likely that the mutations in a third virus line
would be fixed in a different order but involve at least some of the same
mutations, for example those that were the same between TX and ID. It also
seems likely that the mutations in a third line would involve the A and F
proteins, since the fixed mutations commonly occur in those two.

5. Using Figure 20-17, explain how the mutation in the GATA sequence of the
Duffy gene imparts resistance to P. vivax infection.

Answer: The GATA sequence activates Duffy expression that is specific to

red blood cells, which results in Duffy protein on the surface of red blood
cells. The malarial parasite, P.vivax, invades red blood cells by using Duffy
protein as a recognition site. The mutation GACA prevents binding of the
GATA1 protein to the enhancer and prevents expression of Duffy protein on
 Chapter Twenty 467

the red blood cells, but not on other cells. The lack of Duffy protein means
that P. vivax are no longer able to recognize and infect the red blood cells, and
resistance to malaria is conferred.

6. In Figure 20-18, what is the evidence that polyploid formation has been
important in plant evolution?

Answer: Polyploidy doubles the diploid chromosome number, which

automatically produces an even haploid number. In the absence of
polyploidy, the haploid number would be expected to be randomly distributed
between even and odd. The high frequency of even haploid chromosome
numbers compared to odd numbers indicates that polyploidy is common and
likely important in plant evolution.

BASIC PROBLEMS
7. Compare Darwin’s description of natural selection as quoted on page 730 with
Wallace’s description of the tendency of varieties to depart from the original
type quoted on page 731. What ideas do they have in common?

Answer: The ideas of Darwin and Wallace were remarkably similar. Both
authors recognized a struggle for existence and that survival and reproduction
were not assured. Both also recognized the existence of variations that could
confer a reproductive advantage. Finally, both realized that if beneficial
variations were passed to the offspring they would increase in frequency over
time.

8. What are the three principles of the theory of evolution by natural selection?

Answer: The three principles are: (1) organisms within a species vary from
one another, (2) the variation is heritable, and (3) different types leave
different numbers of offspring in future generations.

9. Why was the neutral theory of molecular evolution a revolutionary idea?

Answer: Before the neutral theory was developed, evolutionary biologists

considered all change to be due to natural selection. In the absence of
molecular data, mutations were thought of as beneficial or harmful, even if
only slightly so, and so subject to natural selection. It was difficult to conceive
that modification of a highly specific enzyme, for example, could be neutral.
With a more complete understanding of the genetic code and the molecular
basis for mutation and protein function came the surprising realization that
neutral or nearly neutral mutations could arise. Neutral theory established the
even more revolutionary idea that evolution of neutral genes by genetic drift
468 Chapter Twenty

was not only possible but common, and that most amino acid differences
between species had not arisen through natural selection of adaptive variation
but through genetic drift of neutral alleles.

10. What would you predict to be the relative rate of synonymous and
nonsynonymous substitutions in a globin pseudogene?

Answer: A pseudogene is a nonfunctional duplication of an active gene.

Because it is nonfunctional, changes in the pseudogene are not subject to
natural selection—all amino acid substitutions are neutral. As a result, there
should be no difference in the rate of synonymous and nonsynonymous
substitutions in a globin pseudogene or any other pseudogene.

11. Are AS heterozygotes completely resistant to malarial infection? Explain the

evidence for your answer.

Answer: AS heterozygotes are not completely resistant to malarial infection.

The primary evidence for this is that AS heterozygotes have lower rates of
infection than AA homozygotes, but still may be infected by the parasite. One
study showed a 27.9 percent rate of infection for AS and a 45.7 percent rate
for AA. Other indirect evidence indicates mortality among AS heterozygotes
associated with the infection. Malaria is a primary source of early child
mortality where it is present, so complete resistance would be expected to
greatly increase survivorship for AS heterozygotes. However, survivorship
curves between AA and AS indicate a relatively moderate difference.
Additionally, the selective advantage for the AS genotype calculated using the
Hardy-Weinberg equilibrium suggests a moderate advantage, inconsistent
with complete resistance to a disease that is often lethal.

CHALLENGING PROBLEMS
Unpacking the Problem

12. If the mutation rate to a new allele is 10–5, how large must isolated
populations be to prevent chance differentiation among them in the frequency
of this allele?

Answer: A population will not differentiate from other populations by local

inbreeding if

µ ≥ 1/N
so
N ≥ 1/µ
N ≥ 105
 Chapter Twenty 469

13. Glucose-6-phosphate dehydrogenase (G6PD) is a critical enzyme involved in

the metabolism of glucose, especially in red blood cells. Deficiencies in the
enzyme are the most common human enzyme defect and occur at a high
frequency in Tanzanian children.

a. Offer one hypothesis for the high incidence of G6PD mutations in

Tanzanian children.
b. How would you test your hypothesis further?
c. Scores of different G6PD mutations affecting enzyme function have
been found in human populations. Offer one explanation for the
abundance of different G6PD mutations.

Answer:
a. One hypothesis is that G6PD mutations affect the metabolism of glucose in
red blood cells in such a way that they reduce infection by malarial parasites,
which are common in Tanzania and a significant source of childhood
mortality. It is possible, for example, that malaria parasites cannot reproduce
in a red blood cell deficient for glucose metabolism. It is also possible that
G6PD deficiency modifies the cell in some way that prevents infection.

b. This hypothesis could be tested by comparing G6PD mutants with wild types
and measuring the rate of infection by parasites. If the mutations confer
resistance, infection rates should be lower.

c. The abundance of many G6PD mutations could be explained in at least two

different ways. One is that any of a wide variety of mutations is capable of
conferring resistance to parasitic infection and that different mutations have
arisen and spread in different populations (or lineages). In this case, the
variety of different mutations would be due to the randomness of the initial
mutation, but their spread would be due to a selective advantage. It would be
predicted from this hypothesis that G6PD variation would be greatest in
populations with higher rates of parasitic infection. Another explanation is
that existing G6PD mutations have a weak effect on fitness, or are neutral—
G6PD metabolic function may not be particularly sensitive to amino acid
changes. This seems unlikely given the importance of G6PD in glucose
metabolism, but some G6PD mutations may have minimal effects on enzyme
activity, or there may be other enzymes or biochemical pathways that can
compensate for reduction of G6PD activity. In this case, the various mutants
would have spread by drift, according to the neutral theory of evolution, and
G6PD variation would be widespread, rather than limited to certain
populations. Of course, both of these means could contribute to the existing
variety of G6PD mutations.

14. Large differences in HbS frequencies among Kenyan and Ugandan tribes had
been noted in surveys conducted by researchers other than Tony Allison.
These researchers offered alternative explanations different from the malarial
470 Chapter Twenty

linkage proposed by Allison. Offer one counterargument to, or experimental

test for, the following alternative hypotheses:

a. The mutation rate is higher in certain tribes.

b. There is a low degree of genetic mixing among tribes, so the allele
rose to high frequency through inbreeding in certain tribes.

Answer:
a. That mutation rates are higher in some tribes than others is very
unlikely, but this could be tested by comparing the synonymous
substitutions in the hemoglobin gene among the different tribes. This has
the advantage of targeting the hemoglobin molecule specifically, but the
number of substitutions in hemoglobin might be too low to be
informative. A better alternative would be to measure mutation rates in
noncoding DNA sequences such as intergenic spacers or introns. The
most useful sequences would be those near or within the hemoglobin
gene.

b. The S allele is common in geographical areas that have a high incidence

of malaria and much less common in areas without malaria, which
argues against the inbreeding hypothesis. However, the hypothesis could
be tested directly. The inbreeding hypothesis predicts that areas of high
frequencies of the S allele (and areas of high malaria) should be areas of
high inbreeding. A molecular analysis of neutral variation (at several
loci) for different tribes would allow an estimate of the extent of
inbreeding within the different populations and provide a test of the
inbreeding hypothesis.

15. How many potential evolutionary paths are there for an allele to evolve six
different mutations? Seven different mutations? Ten different mutations?

Answer: The number of potential evolutionary pathways is n!, where n is the

number of different mutations. For six mutations it would be 6! or 6  5  4 
3  2  1 = 720. For seven mutations, it would be 5,040, and for ten
mutations it would be 3,628,800.

16. The MC1R gene affects skin and hair color in humans. There are at least 13
polymorphisms of the gene in European and Asian populations, 10 of which
are nonsynonymous. In Africans, there are at least 5 polymorphisms of the
gene, none of which are nonsynonymous. What might be one explanation for
the differences in MC1R variation between Africans and non-Africans?

Answer: When amino acid changes have been driven by positive adaptive
selection, there should be an excess of nonsynonymous changes. The MC1R
gene (melanocortin 1 receptor) encodes a key protein controlling the amount
 Chapter Twenty 471

of melanin in skin and hair. Asian and European populations appear to have
experienced positive adaptive selection for more lightly pigmented skin
relative to their African counterparts. By contrast, since high levels of melanin
protect from damaging UV rays, purifying selection is likely to have
maintained the sequence of MC1R in Africans.

17. Opsin proteins detect light in photoreceptor cells of the eye and are required
for color vision. The nocturnal owl monkey, the nocturnal bush baby, and the
subterranean blind mole rat have different mutations in an opsin gene that
render it nonfunctional. Explain why all three species can tolerate mutations
in this gene that operates in most other mammals.

Answer: Both the owl monkey and bush baby are nocturnal while the
subterranean mole rat is blind. Genes necessary for color vision are therefore
not necessary for these three species. When species shift their habitats or life
styles, mutations that cause inactivation of genes whose functions are no
longer necessary is expected.

18. Full or partial limblessness has evolved many times in vertebrates (snakes,
lizards, manatees, whales). Do you expect the mutations that occurred in the
evolution of limblessness to be in the coding or noncoding sequences of
toolkit genes? Why?

Answer: Noncoding sequences. A major constraint on gene evolution is the

potential pleiotropic effects of mutations in coding regions. These can be
circumvented by mutations in regulatory sequences which play a major role in
the evolution of body form. Changes in noncoding sequences provide a
mechanism for altering one aspect of gene expression while preserving the
role of pleiotropic proteins in other essential developmental processes.

19. Several Drosophila species with unspotted wings are descended from a
spotted ancestor. Would you predict the loss of spot formation to entail coding
or noncoding changes in pigmentation genes? How would you test which is
the case?

Answer: Noncoding changes. The difference in pigment expression between

spotted and unspotted species of Drosophila is more likely due to the
differences in cis-acting regulatory sequences that regulate the pigment gene.
Mutations in coding regions might be expected to be highly pleiotropic. To
test this, the activity of the cis-acting regulatory sequences from the different
species could be tested by placing them upstream of a reporter gene and then
placing the constructs into D. melanogaster. The regulatory sequences from a
spotted species should drive high levels of reporter gene expression in just a
472 Chapter Twenty

spot while those from unspotted species should drive low-level expression of
the reporter gene across the whole wing blade.

20. It has been claimed here that “evolution repeats itself.” What is the evidence
for this claim from

a. the analysis of HbS alleles?

b. the analysis of antibiotic resistance in bacteria?
c. the analysis of experimentally selected bacteriophage X174?
d. the analysis of Oca2 mutations in cave fish?
e. the analysis of stickleback Pitx1 loci?

Answer:
a. The analysis of HbS alleles shows that this allele has arisen independently
five times in areas where malaria is common. In this case, evolution has
repeated itself.

b. The analysis of antibiotic resistance in bacteria indicates that only a small

proportion of the potential evolutionary pathways to resistance will
actually be used. This reduces the number of available pathways and
increases the chances of the same pathway occurring twice, but it also
shows that many different pathways still remain.

c. The experimentally selected bacteriophage had several of the same

substitutions, but they occurred in different orders. Also several
substitutions were different. In cases like this, the pathway differs even if
the outcome is the same, and evolution cannot be considered to have
repeated itself.

d. Lack of pigmentation in different populations of cave fish was found to be

due to mutations (deletions) in the Oca2 gene, but the deletions were in
different places. Again, the outcome was the same but the process differed
so evolution did not repeat.

e. Loss of spines due to deletions in the Pitx1 locus, a regulatory sequence,

has evolved independently in many populations of stickleback fish.
However, the size and location of the deletions differed in different
populations.

The overall pattern is that evolution can repeat itself in a general way, but
rarely follows exactly the same process. For example an evolutionary result is
unlikely to occur in the same way when multiple mutations are involved—
when the evolutionary pathway is complex. When the evolutionary pathway is
simpler, involving only one gene, it is more likely that mutations in that gene
will arise repeatedly and spread when they confer an advantage.
 Chapter Twenty 473

21. What is the molecular evidence that natural selection includes the “rejection
of injurious change”?

Answer: Purifying selection, or the “rejection of injurious variations,” as

Darwin termed it, explains why many protein sequences are unchanged, or
nearly so, over vast spans of evolutionary time. A lower-than-expected ratio
of nonsynonymous to synonymous changes is a signature of purifying
selection. For example, a set of about 500 genes that exist is all Domains-of-
life encode proteins whose sequences have been largely conserved over three
billion years of evolution. To preserve such sequences, variants that have
arisen at random in individuals in tens of millions of species have been
rejected by selection over and over again.

22. What are three alternative fates of a new gene duplicate?

Answer: First, a new gene duplication can become a pseudogene through

complete inactivation. Pseudogenes have no function and are invisible to
natural selection, so these genes will accumulate mutations relatively rapidly.
Second, the duplicate can remain active and a new or modified function for
the protein can evolve (neofunctionalization). An example of this is seen in
human hemoglobin coding genes. Finally, the duplicate can become
subfunctionalized, in which the original gene function is split between the
original gene and the duplicate. The evolution of regulatory sequences may
facilitate this path.

23. What is the evidence that gene duplication has been the source of the  and ß
gene families for human hemoglobin?

Answer: The  and ß gene families show remarkable amino acid sequence
similarities (see table in the companion text). Within each gene family,
sequence similarities are greater and, in some cases, member genes have
identical intron-exon structure.

24. DNA-sequencing studies for a gene in two closely related species produce the
following numbers of sites that vary:

Synonymous polymorphisms 50
Nonsynonymous species differences 2
Synonymous species differences 18
Nonsynonymous polymorphisms 20
474 Chapter Twenty

Does this result support neutral evolution of the gene? Does it support an
adaptive replacement of amino acids? What explanation would you offer for
the observations?

Answer: For polymorphic sites within a species, let nonsynonymous = a and

synonymous = b. For polymorphic sites between the species, let
nonsynonymous = c and synonymous = d. If divergence is due to neutral
evolution, then

a/b = c/d

If divergence is due to selection, then

a/b < c/d

However, in this example, a/b = 20/50 > c/d = 2/18, which fits neither
expectation.

Because the ratio of nonsynonymous to synonymous polymorphisms (a/b) is

relatively high, the gene being studied may encode a protein tolerant of
substitution (like fibrinopeptides, discussed in the companion text). The
relatively fewer species differences may suggest that speciation was a recent
event, so few polymorphisms have been fixed in one species that are not
variants in the other.

25. In humans, two genes encoding the opsin visual pigments that are sensitive to
green and red wavelengths of light are found adjacent to one another on the X
chromosome. They encode proteins that are 96 percent identical. Nonprimate
mammals possess just one gene encoding an opsin sensitive to the red/green
wavelength.

a. Offer one explanation for the presence of the two opsin genes on the
human X chromosome.
b. How would you test your explanation further and pinpoint when in
evolutionary history the second gene arose?

Answer:
a. One way the two genes could arise is a duplication of the opsin gene
sometime after the divergence of primates from other mammals. Since both
of these genes are functional, this would be an example of
neofunctionalization. Another way would be transposition, through the
movement of a transposable element. A third way would involve
retrotransposition.

b. A molecular analysis of the genes could differentiate between these three

alternatives. The three mechanisms for existence of the two genes would
have very different molecular characteristics. A direct duplication would
 Chapter Twenty 475

produce an identical copy of the gene. Transposable elements have

characteristic sequences that could be detected, either by DNA sequencing
or analysis by polymerase chain reaction. Retrotransposition produces a
duplicate that lacks introns. The timing of this event could be roughly
estimated by looking for the two duplicate genes in other primates. The
order of divergence of the primates is known, so the timing of the
duplication could be inferred from its first appearance in other primates. A
comparison of synonymous substitutions in the two genes would also
provide information about the timing of the duplication—most would be the
same, but the different ones would have occurred since the duplication. A
greater number would indicate a more distant divergence.

26. About 9 percent of Caucasian males are color-blind and cannot distinguish
red-colored from green-colored objects.

a. Offer one genetic model for color blindness.

b. Explain why and how color blindness has reached a frequency of 9
percent in this population.

Answer:
a. Color blindness could result from a mutation in any of several steps
involved in the perception of color. One would be a mutation in a gene
controlling light absorbing pigments in photoreceptors, so that both “red”
cones and “green” cones respond to the same wavelength of light. Another
could be a mutation in the way the information is processed by the nervous
system, so that “red” and “green” are processed the same way and become
indistinguishable.

b. Color blindness may be evolutionarily neutral. In that case its frequency of

9 percent would have been achieved by drift. However, it is also possible
that inability to distinguish certain colors leads to greater ability to
distinguish among others, or to greater visual acuity. Effectively
neutralizing some cones, for example, may place a greater emphasis on
rods, which are capable of greater acuity.