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Annu. Rev. Ecol. Syst. 2001. 32:183217


c 2001 by Annual Reviews. All rights reserved
Copyright

APPLIED EVOLUTION
J. J. Bull1 and H. A. Wichman2
1

Section of Integrative Biology, Institute of Cellular and Molecular Biology, University


of Texas, Austin, Texas 78712-1023; e-mail: bull@bull.biosci.utexas.edu
2
Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-3051;
e-mail: hwichman@uidaho.edu

Key Words artificial selection, directed evolution, phylogenetics, resistance,


evolutionary computation
Abstract Evolutionary biology is widely perceived as a discipline with relevance
that lies purely in academia. Until recently, that perception was largely true, except
for the often neglected role of evolutionary biology in the improvement of agricultural crops and animals. In the past two decades, however, evolutionary biology has
assumed a broad relevance extending far outside its original bounds. Phylogenetics,
the study of Darwins theory of descent with modification, is now the foundation of
disease tracking and of the identification of species in medical, pharmacological, or
conservation settings. It further underlies bioinformatics approaches to the analysis of
genomes. Darwins evolution by natural selection is being used in many contexts,
from the design of biotechnology protocols to create new drugs and industrial enzymes,
to the avoidance of resistant pests and microbes, to the development of new computer
technologies. These examples present opportunities for education of the public and for
nontraditional career paths in evolutionary biology. They also provide new research
material for people trained in classical approaches.

OVERVIEW
Evolutionary biology has undergone an expansion and transformation in the past
few decades. Despite occasional claims to the contrary, the big changes in evolutionary biology have come from improvements in understanding mechanisms that
are fully compatible with Darwinism; descent with modification and natural selection are still the conceptual foundations of the discipline. For example, a veritable
explosion of studies estimating the relationships among different species has refined our understanding of evolutionary history, but the modern version of the tree
of life has many similarities to old ones and certainly supports a Darwinian model.
The sequencing of genes and genomes has yielded insights into genetic mechanisms underlying evolution, and we are even progressing toward a genetic understanding of the major developmental and morphological transitionsadvances
that augment earlier ideas about these transitions. Theories based on natural selection have led to revolutions in understanding behavior, parasitism, and a wealth of
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genetic and physiological mechanisms that benefit neither the individual nor the
population.
Some of the revolution in evolution has occurred outside the traditional academic boundaries of evolutionary biology. Those who started as evolutionary biologists two or more decades ago are beholding a transformation of the field into one of
broad social relevance. Much of the biotechnology industry is concerned with creating biological molecules that have specific functions. This goal-oriented enterprise
has quickly embraced evolutionary principles to direct the evolution of molecules
in test tubes and, in so doing, has profoundly expanded the horizon and relevance
of evolutionary biology. Evolutionary principles are suddenly the material of multimillion dollar patents, leading industrial biochemists to new drugs and other
commercial molecules. On a different front, the medical establishment, after long
ignoring evolution, is faced with an onslaught of drug-resistant microbes, has seen
monkey viruses jump into humans and accelerate into epidemics, and must now
use evolutionary principles to understand the worldwide dynamics of pathogens.
The theme that unites the examples in this paper is that evolution and evolutionary biology are socially relevant. There are two main reasons for writing such
a paper. First, public perception of evolutionary biology is not up to date with the
discipline. Evolution is still a bad word to many people, not only because it is
perceived as conflicting with some religious views, but also because it is widely
viewed as an irrelevant science with no social value. Acceptance of evolutionary biology is far more likely when the public realizes that it holds the key to
many social improvements [a view that motivated G.C. Williams in his work on
applications of evolutionary biology to medicine (Nesse & Williams, 1994; G.C.
Williams, personal communication)]. As evolutionary biologists, we need to use
these examples of relevance when explaining evolution to our students and the
public. A second reason for writing this paper is that historical inertia in the training of evolutionary biologists has resulted in a lack of exposure to socially relevant
applications. Individuals trained as evolutionary biologists will have much to offer
in solving these problems, but they need to be aware of these applications. Career
opportunities for evolutionary biologists may already be more plentiful outside
academia than inside it.
This paper is an introduction to some examples of socially relevant evolutionary biology. We have chosen topics with which we are familiar and in which
evolutionary biology has already been used to produce an outcome or to affect a
policy: phylogenetics, artificial selection (in biotechnology), resistance management, and computation. Other applications of evolutionary principles to socially
relevant problems include Darwinian medicine (Lappe 1994, Nesse & Williams
1994, Trevathan et al. 1999), infectious diseases (Ewald 1994, Morse 1994), and
human impact on evolution (Palumbi 2001). An excellent overview of the social relevance of evolution has been assembled and endorsed by eight scientific
societies (Futuyma 1999).
The topics in this review fit logically into the conceptual framework of evolutionary biology. The first two sections are based on Darwins theory of descent

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with modification: The first is about estimating evolutionary history for biomedical
and other applications, and the second discusses the role of evolutionary models in
interpreting the molecular variation seen in the burgeoning genome databases. The
last three sections are based on the neo-Darwinian framework for natural selection.
The first describes examples in which humans have modified the elements of natural selection to produce various biological commodities. The next is somewhat
the reverse of that, in which an understanding of natural selection is recruited to
try to block evolution. The last section is a brief introduction to uses of models of
natural selection in designing computer programs.

PHYLOGENETICS: USING THE TREE OF LIFE


The premise that all life shares common ancestry has been a central tenet of
evolutionary biology since Darwin. If we go back far enough in time, the genealogy
of every organism alive today can be traced to a point that unites it with the
genealogy of any other organism alive today. By definition, closely related species
have recent common ancestors, whereas the common ancestors of distantly related
species go far back in time. Furthermore, the process is continual. Todays species
are themselves comprised of lineages that continue to diverge.
Phylogenetics is the study of these evolutionary genealogies. Despite the antiquity of the common-ancestry principle, the field of phylogenetics has matured immensely in the past 10 to 15 years, owing to advances in computer technology and
DNA sequencing as well as to the development of explicit theories and methodologies for phylogenetic reconstruction. Most phylogenetic methods use DNA
sequences, protein sequences, or RNA sequences, but some methods can use morphological data, which are vital to the analysis of fossils. Not only do methods differ
in the types of data that can be analyzed, methods using the same types of data may
also differ in the assumptions used to convert the data into evolutionary history.
The output of a phylogenetic analysis is a branching tree that represents the
evolutionary history of the lineages being studied. The tree provides not only a
nested hierarchy of common ancestors going back in time, but also quantitative
information on the amount of change between the different points in the tree. The
tree may incorporate taxa whose common ancestors reach back over a billion years,
or the tree may be limited to a group of viruses whose common ancestor existed
only weeks or months ago. The applications of phylogenetic methods to socially
relevant problems likewise occur at several timescales.

Disease Tracking: Molecular Epidemiology


Phylogenetics has become indispensable in identifying disease reservoirs and in
tracking the step-by-step transmission of some viruses. The conceptual basis of
this work is as follows. We want to know the source of a virus infecting person X.
Suppose that four different possible sources have been identified: A, B, C, and D.

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These four sources could be different infected individuals who had contact with X,
they could be different geographic locations that person X visited, or they could
be four species of mammals living in the village of person X that are sometimes
infected with the type of virus in X. The problem in figuring out the source is that
none of the viruses in A, B, C, and D will necessarily have the same sequence as
the virus in X. Phylogenetic analysis gets around that problem by establishing the
evolutionary relatedness of viruses from each of the possible sources (Figure 1).
The analysis not only indicates which of the viruses in AD are most similar to
that in X (source D in Figure 1), it also indicates how closely related the viruses
are and, hence, whether the source might be other than AD.
Phylogenetic analysis is now a standard part of any disease epidemiology. Below
we offer a few of the many applications.

Figure 1 The virus acquired by individual X is compared by phylogenetic methods


with viruses from four possible sources (AD). The context for this could be any of the
following. (a) AD are different individuals infected with HIV who had sex or shared
needles with individual X, and we are trying to find out who transmitted the virus
to X. (b) AD are different mammal species with rabies viruses circulating in their
populations, and X is a human who died of rabies. Which mammal species transmitted
the infection? (c) AD are mice with hantavirus from New Mexico, Texas, California,
and Nevada. X is a Texas resident who recently traveled across the United States and
became infected with hantavirus. Where did X contract the virus? In this hypothetical
example, the virus in X is most closely related to the virus from source D, which gives
information about the likely source (and place) of transmission.

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ERADICATION OF WILD-TYPE POLIOVIRUS FROM THE AMERICAS Poliomyelitis is a


paralyzing, occasionally fatal, disease caused by an RNA virus. On recovery from
infection, a person carries lifetime immunity, but there are three forms of the virus,
and immunity to one form does not confer immunity to the others. Less than 1%
of those infected by the virus actually develop the disease, but the incidence of
disease per infection is thought to increase with age. It has thus been speculated
that the widespread outbreaks of polio disease that occurred in the first part of the
twentieth century were a consequence of social hygiene, because improvements in
social hygiene delayed the average age of infection (Nathanson et al. 1995, Garrett
2000).
Over 38,000 cases of poliomyelitis were reported in the United States in 1954.
The first vaccine was approved a year later, and within 2 decades, the native polio
virus was thought to have been eradicated from the Americas. The only known
host for poliovirus was human beings, making eradication seem a feasible goal.
However, isolated cases of polio continue to occur in the Americas. With the
exception of an outbreak in a religious community that avoided vaccines and a
recent outbreak in the Dominican Republic (Greensfelder 2000), these isolated
cases failed to materialize into epidemics because the population maintained high
levels of vaccination (which continues today), but they raised the possibility that
native polio strains might still be present. The alternative explanation for these
sporadic cases was either nonnative poliovirus introduced from parts of the world
where it was still endemic or a vaccine strain. The vaccine in use after 1961 was a
live, attenuated virus that was capable not only of transmission, but also of evolving
into a more virulent form. Phylogenetic analysis showed that the viruses that gave
rise to isolated cases and epidemics were invariably either vaccine derived or
wild strains originating from outside the Americas; no native, American virus has
been found, and it is presumed to have been eradicated (Rico-Hesse et al. 1987).
An assault to eradicate poliovirus worldwide continues to this day, aided by the
knowledge that the virus does not lay hidden in nonhuman reservoirs.
ORIGIN OF HIV The most notorious human infectious disease to have arisen in
the 1900s is AIDS (acquired immune deficiency syndrome). Unknown until about
1981, 20 years later it was estimated that more than 30 million people were infected
worldwide and more than 16 million had already died. This disease is caused by the
retrovirus HIV (human immunodeficiency virus), which exists in two basic forms,
HIV-1 and HIV-2. Although the HIV epidemic is now driven by human-human
transmission, phylogenetic evidence indicates that HIV-1 originally came from
chimpanzees and HIV-2 (which is less abundant and less often fatal than HIV-1)
came from a type of monkey known as the sooty mangabey (Gao et al. 1999, Hahn
et al. 2000). Phylogenetic evidence supports multiple introductions of HIV-1 and
HIV-2 into humans this century (Korber et al. 2000).
HIV TRANSMISSION BETWEEN PEOPLE One of HIVs unusual properties is that it
evolves rapidly, even for a virus. That property is an unfortunate one from the

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perspective of curing an infection or creating a vaccine, because the virus evolves


quickly in response to drug treatment and immune attack (Coffin 1996, Colgrove &
Japour 1999, Mosier 2000). However, its rapid evolution enables fine-scale analysis
with molecular epidemiology that is not possible with most other viruses. Starting
from a single virus in a person, the infection will blossom into a miniature tree of
life, the viral lines ever expanding as the infection continues. Consequently, two
viruses from one person usually have different genome sequences, but sequences of
virus from one person will be more similar to each other than they are to sequences
of viruses from other people. This property potentially allows determination of the
individual who transmitted the virus.
One of the early analyses of HIV transmission revolved around a Florida dentist
with AIDS whose patients exhibited an unusually high incidence of HIV infection
(10 patients were eventually discovered to be HIV+). By itself, the clustering of
HIV+ patients associated with this dentist might have been regarded as evidence of
dentist-to-patient transmission, but this cluster was discovered in the earliest days
of understanding HIV, and the possibility of unknown routes of infection had to be
considered. If the dentist was not the source of the infection, it was important to
discover the source to halt further transmissions. On the other hand, if the dentist
was the source, the implications for health care practices were enormous. The
stakes were very high either way. Phylogenetic analysis suggested that the patient
viruses were close relatives of the dentists virus in all but two cases, and those
two patients had other known risk factors (Ou et al. 1992, Hillis & Huelsenbeck
1994, Hillis et al. 1994). Thus the dentist was likely the source of infection for the
eight patients with no known risk factors.
Perhaps the most sensational case of HIV molecular epidemiology was a criminal case in Lafayette, LA. A physician was accused of injecting his former
mistress with blood containing HIV. He had been giving her vitamin B injections, and it was supposedly the final injection in August 1994 that contained
the blood with HIV. When the woman was diagnosed with HIV and hepatitis
C in December 1994, she suspected the physicians injection as the source; at
the time of a blood donation in April 1994, she had been negative for both
viruses. This case was unusual in that the person infecting the woman (the physician) was not himself infected, so it was necessary both to locate the patient
whose HIV infected the woman and to demonstrate that the physician had access to this patients blood. Records were discovered in the physicians office
indicating that blood had been drawn from two patients during the week in question; one patient was previously known to be HIV+ and the other positive for
hepatitis-C. Phylogenetic analyses showed that the HIV sequences of the exmistress clustered within those of the patient with HIV, supporting her story (State
of Louisiana Criminal Dockett #96CR73313; D. Hillis, personal communication).
The physician was convicted of attempted second-degree murder and sentenced
to a 50-year term in prison in this first use of phylogenetics in a U.S. criminal
court.

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Predicting Evolution
A novel step in phylogenetic analysispredicting evolutionwas introduced in
work on influenza A, the respiratory virus that causes flu epidemics every year.
At any one time, several influenza types are circulating in the human population.
A viral type is defined by how the virus reacts with a set of antibodies (Webster
1993). Two viral proteins, hemagglutinin and neuraminidase, determine the viral
type.
For some decades, influenza isolates have been stored by the Centers for Disease
Control; these isolates include lineages that are now extinct in human populations.
When techniques were developed to obtain viral gene sequences, it was possible
not only to estimate the evolutionary history from currently circulating lineages,
but also to use the stored isolates to sequence extinct types to obtain detailed insight
into ancestral states. An unusual property of influenza discovered from this work is
that the level of sequence diversity has not progressively expanded over time (Fitch
et al. 1997, Bush et al. 1999). At any point over the past 15 years, variation existed
around a recent common ancestor that was never more than a few years old. Thus,
viral extinctions occurred as fast as new types evolved, with only one ultimate
winner. Each winner would continue to generate new variants that competed in
the population, and only one of them would win, and so on. The phylogeny of this
process was a tree with a long trunk and lots of short (dead-end) side branches.
The study that predicted influenza used the hemagglutinin gene for a single
viral type, HA3. The fact that only one lineage of HA3 survives means that it may
be possible to predict which of the variants circulating in a population will give
rise to the viruses of the future. Fitch et al. (1997) observed rapid evolution at a few
residues in the hemagglutinin gene. This finding was facilitated by the analysis of
historical samples, because the repeated evolution at common sites had otherwise
erased earlier evolution at those sites. Those rapidly evolving residues provided an
obvious yardstick by which to compare different viruses: Of the viruses present at
any one time, the ones with the most evolution at those sites were the candidates
as progenitors of future lineages. Bush et al. (1999) developed this predictive
statistic and applied it retrospectively to the existing data. The prediction worked
exceedingly well, although it awaits a truly a priori test (which will certainly be
carried out in the coming years).
Will this method lead to better predictions for flu vaccines? Not necessarily.
The difficulty in deciding each year which flu vaccine to produce lies in predicting
which of the circulating viral types will infect the most people. The phylogenetic
prediction is, instead, of which viral type will prevail in the long term and, thus,
covers a longer timescale and a smaller magnitude of viral variation than is necessary for vaccine design. Furthermore, the worldwide pandemics of influenza (that
infect and kill more people than average) are caused by a fundamentally different
type of sequence change than was studied in the HA3 predictions (Webster 1993).
However, the phylogenetic prediction method points toward a new level of utility
for this kind of evolutionary biology.

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Species Identification on the Tree of Life:


Ribotyping and Other Methods
The cells of all organisms from bacteria to humans (but not viruses) carry genes
that build ribosomes, the scaffolding to assemble proteins. Parts of the ribosome
are proteins, and other parts (rRNA) are RNA molecules whose ancestry may be
traced all the way back to the hypothetical RNA world, before DNA was the
genetic material. Portions of these rRNA genes evolve slowly, so their sequences
are very similar even between distantly related organisms; other portions are more
variable. The essential nature of rRNA genes combined with the wide range of
variation in rates of evolution across different stretches of the molecules makes
them ideally suited for estimating the tree of life. This approach to assessing the
diversity of and relationships among all life on the same scale was pioneered by
Woese (2000). This tree of life shows three main domains: Bacteria, Archaea, and
Eukarya. The impressive diversity of plants and animals is confined to a modest
branch in one of these three domains, the Eukarya (Figure 2).
Over the years, the rRNA tree of life has been filled in densely, covering thousands of taxa. Of course, the relationships of many of the taxa in this tree of life
are consistent with earlier theories, but many taxa have been added whose relationships were previously obscure. Equally important is the fact that this tree provides a universal standard for comparing any life form to any other (which was
not possible when using morphology, for example). The utility of the rRNA tree
of life has been facilitated by the development of different sets of polymerase
chain reaction (PCR) primers that can be used to amplify parts of rRNA genes
from any organism or amplify them from only specific taxa. It is now routine to
acquire DNA or RNA from an unknown taxon and to use its rRNA sequences to
identify it, or at least identify its closest known relatives. This technique (known
as ribotyping) can be extremely useful because it allows identification of the different species of organisms present in a community, even though it may be impossible to culture the organisms or recognize them by any other method (Pace
1997).
Ribotyping is not the only molecular method used for species identification. Perhaps the main advantage of ribotyping is that it can be used across the full spectrum
of life (excepting viruses) without knowledge of the organisms being typed. But
other methods may be preferred when working within narrow taxonomic groups
(e.g., mammals, or at a finer level, whales). Thus, noncoding regions of mitochondrial DNA are often more sensitive than rRNA sequences in distinguishing
closely related species and subspecies, because these mitochondrial sequences
evolve faster. These sensitive methods are not only useful in identifying a species,
they may also provide information about the geographic location of the taxon as
well (the DNA zip codes of Baker 1994).
USING THE TREE OF LIFE: PATHOGEN IDENTIFICATION AND DISEASE ETIOLOGY Several diseases have no known infectious causes. Ribotyping and other genotyping

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Figure 2 A tree of life, based on 18S rRNA sequences. The tree shows the three domains
(Bacteria, Archaea, and Eukarya) as well as many representatives in each domain. Note what
little divergence is represented by the animals and plants relative to the entire tree. The 18S
sequences from hundreds of life forms (but not viruses) have been obtained and can be resolved on this tree, with closest relatives sharing the most recent common ancestors (the
figure is simply too crowded to portray the many species that have been analyzed in this
fashion). The 18S sequences from samples of unknown identity can thus be mapped onto
this tree to discover what types of life forms were present in the sample. (Figure courtesy of
Mitch Sogin.)

methods offer a way of identifying whether diseased tissues are associated with
any microbe (Relman & Falkow 1992, Relman 1998). If the causative agent is
a previously unknown species, evolutionary biology plays a role in identifying
what type of organism it is, i.e., in identifying its closest known relatives. Once a
suspect microbe has been identified, steps can be taken to treat with appropriate
drugs. Had this approach been available at the time, it might have greatly facilitated
the discovery that ulcers were associated with a bacterium. Relman (1999) listed
five infectious diseases with causative agents recently discovered by genotyping
methods. An extension of this approach is to use ribotyping to assess the microbial
composition of a community of organisms, as in animal guts and other passages.
Changes in the composition of a community may foreshadow a predisposition to
disease or the onset of disease (Kroes et al. 1999).

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USING THE TREE OF LIFE: CONSERVATION AND FORENSICS Concern for endangered species has led to widespread adoption of laws limiting the harvesting,
marketing, and even possession of tissues and other products of those organisms.
Although some derivatives of a species are unmistakable (e.g., a sea turtle shell,
an elephant tusk), others are not so obvious. In many cases, an endangered species
has close relatives that are not endangered and are legitimately marketed. If the
harvesting occurs at a remote site, far from the eyes of concerned observers, by the
time the meat of an endangered species reaches market, it may be indistinguishable from the meat of a legal species. Using genotypic methods, Baker & Palumbi
(1996) and Baker et al. (2000) reported the sale of meat from endangered species of
whales in Asian markets. Ribotyping and similar molecular methods allow simple
and portable means of identifying endangered species in the marketplace.

GENOMICS AND BIOINFORMATICS


From the perspective of human health, a major goal of genomics work is to understand not only the function of human genes, but also the impact of mutations in
those genes, and how drugs can be designed to modify or repair those functions.
Yet we humans are neither sufficiently genetically variable nor amenable to experimentation that the function of most genes could be ascertained from just our
species. An immeasurable benefit to understanding human genetics comes from
work on other speciesmodel organisms. As we now know, work on the genetics
of other eukaryotes can often be extrapolated to humans. One recent example is
the identification of a human gene responsible for a sleep disorder based on the
Drosophila circadian clock gene per (Toh et al. 2001).
The extrapolation of information between species will accelerate now that
complete genome sequences are available for many species. Rapid advances in
biotechnology have taken us from the first complete sequence of a small DNA
viral genome (Sanger et al. 1977) to the complete sequence of the human genome
in less than 25 years (Venter et al. 2001). During the same period, the number
of transistors per computer processor has increased 6000-fold. The marriage of
increased computing power and large biological data sets has spawned the field
of bioinformatics, which is dominated by the analysis of nucleic acid and protein
data. Bioinformatics is firmly rooted in evolutionary biology. From the initial step
of carrying out a BLAST search (Altschul et al. 1990) of a database to identify
related sequences, to multiple sequence alignment (Higgins & Sharp 1988), to
the identification of orthologous genes in other species (DeBry & Seldin 1996,
Tatusov et al. 1997, Chambers et al. 2000), bioinformatics is a comparative exercise, and descent with modification is one of its inherent and most essential
assumptions.
Molecular evolution, specifically sequence divergence, is both a plus and a
minus in bioinformatics. A gene in humans and its counterpart in yeast will not
have the same DNA sequence even if the function of both has remained the same

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since their common ancestor. Divergence can be so great that it is difficult to


align or even recognize homologous genes. However, once alignments of homologous genes are achieved, evolutionary divergence can be very informative. The
divergence of genes with the same function is the evolutionary equivalent of an experiment in which gene positions are mutated to identify the functionally important
onesneutral or nearly neutral changes accumulate over evolutionary time, but
changes that disrupt gene function are weeded out by the filter of natural selection.
The concept that the most highly conserved amino acid residues are important for
gene function is firmly entrenched in molecular biology (Benner 1995, Golding &
Dean 1998). But not all residues that are important for function are highly
conservedadaptive evolution is achieved by genetic change. Statistical signatures
of past adaptive evolution can be recognized as high rates of nonsynonymous to
synonymous substitutions among homologous sequences (McDonald & Kreitman
1991, Boyd & Hartl 1998, Crill et al. 2000) or as correlated changes between
different residues (Gutell et al. 2000).

ARTIFICIAL SELECTION
Artificial Selection in the Past
Nearly all the common animals and plants we use today were domesticated thousands of years ago, some (sheep, goats, dogs, wheat, and rice) at least 9000 years
ago. Domestication probably started as a process of taming, then of captive breeding, and finally of selecting for specific traits. These early domestications may well
have been the first experiments in applied evolution. Their impact was so profound
as to make civilization possible by enabling societies to switch from hunting and
gathering to agriculture (Ucko & Dimbleby 1969, Clutton-Brock 1999). Although
few new species have been domesticated in the past millennium, we have continued to refine the old ones. The success of artificial selection is evident in its
ultimate creation of selected phenotypes well outside the extremes of the original
species. For example, Chihuahuas, Saint Bernards, pit bulls, and golden retrievers
differ in both appearance and behavior, and nothing like any of these breeds would
have been found in a population of wild dogs. Fruits of modern strains of domestic
plants are much larger than those of their ancestors; corn (maize) is profoundly
different from its ancestor, teosinte.
A model of evolution by natural or artificial selection has three components:
(a) variation, (b) inheritance, and (c) differential reproductive success (Lewontin
1970). Darwins theory was formulated largely on an understanding of variation
and differential reproductive success but a relatively poor understanding of inheritance. The inheritance void began to be filled early in the twentieth century with
the rediscovery of Mendels work, enhanced (in the West) by a close association
of evolutionary biology with agriculture to improve methods of artificial selection. This productive marriage continues today with the use quantitative trait locus
(QTL) mapping in both fields.

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Another cultural relationship between evolutionary theory, genetics and agriculture, was not so productive, however. In the 1930s, Trofim Lysenko rejected
Darwins theory of natural selection in favor Lamarcks theory of the inheritance
of acquired traits. Lysenkos subsequent rise in power as the head of the Soviet
Ministry of Agriculture directly destroyed genetic research and decimated agriculture in the Soviet Union (Soyfer 1994, Garrett 2000). The history of Lysenkoism
highlights the danger of letting political and other nonscientific ideology dictate
scientific practice.
Historically, artificial selection was a manipulation of differential reproductive success. Parents closest to the desired phenotype were chosen to produce the
next generation, and individuals that fell short of the ideal were omitted from the
breeding population. The components of variation and inheritance were present
but were often not manipulated from their natural states, except for the occasional
introduction of novel strains or wild relatives into the breeding stock. Mutation
rates were seldom, if ever, purposely manipulated, but it is possible that some of
the wide crosses used to introduce variation into the breeding population could
have increased mutation rate by inducing transposition or other mutagenic mechanisms. Although the mechanisms of inheritance in artificial selection were not
fundamentally different from inheritance during natural selection, the manipulation of crosses frequently increased the level of inbreeding and thus the ability to
select for the expression of recessive traits.
Artificial selection moved into a new realm with biotechnology (Kauffman
1993). In contrast to agriculture, biotechnology specializes in evolving small
things, i.e., molecules and microbes. Evolutionary methods in biotechnology have
much in common with classical artificial selection. The same three factors
variation, inheritance, and differential reproductive successare manipulated. But
it is no longer the artificial selection of old. The methods used in biotechnology,
including DNA sequencing of the entire evolved genome, determination of molecular structures, and monitoring gene expression levels, provide unparalleled levels
of analysis of evolution. The combination of experiments, replication, productoriented research, and analysis of results has allowed rapid attainment of a new
level of scientific accomplishment in evolutionary biology.

Directed Evolution
Various protocols based on evolution are currently used to fashion nucleic acids
(ribozymes and aptamers) or proteins with specific functions. The directed evolution of novel biological pathways and evolutionary engineering of whole genomes
may not be far off. These accomplishments were made possible by changing
the components of variation, inheritance, and differential reproductive success.
Nature comes close to some of these methodologies, and indeed, several are
borrowed from nature, using microbes and various types of parastic genetic elements. Biotechnology has, nonetheless, created unnatural means of evolving
molecules.

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Perhaps the most extreme form of directed evolution is the in vitro replication of nucleic acids outside of life-forms. The earliest system of this sort was
Spiegelmans replication of the Q genome with the Q RNA-dependent RNA
polymerase (Spiegelman et al. 1968). Q is a phage whose genome encodes four
genes, one being the polymerase enzyme that makes RNA copies of the phages
RNA genome. By isolating the phages replication enzyme and then placing the
RNA genome in a cocktail of nucleotides plus the enzyme, the genome was replicated on its own. In that environment none of its genes were being expressedthe
genome was just a molecule that was evolving to copy itself in an environment
where its genes were irrelevant. It rapidly evolved to a small size.
The Spiegelman in vitro system did not allow evolution to be directed toward any
goal other than fast self-replication. Perhaps the first purely in vitro system that did
allow directed evolution was the Systematic Evolution of Ligands by EXponential
enrichment (SELEX) system that was simultaneously developed by two different
labs (Ellington & Szostak 1990, Tuerk & Gold 1990). This elegant system allowed
one to start with a synthesized pool of nucleic acids (Figure 3). The ends of those
nucleic acids were constant regions, the sequences of which matched sequences
of primers for PCR amplification. The middle regions were randomized. This pool
of molecules was then washed across many copies of a particular target molecule
(e.g., a protein). The molecules that bound the target stayed behind, and the rest
were washed off. The bound molecules were then eluted into a separate tube,
amplified by PCR, and passed through the cycle again. In this way, a nucleic acidbinding species (aptamer) could be obtained for any particular target molecule
(Tuerk & Gold 1990, Gold et al. 1995, Osborne & Ellington 1997, Famulok &
Mayer 1999).
A variety of interesting variations on this theme has since been developed.
Beaudry & Joyce (1992) used an in vitro selection and amplification scheme to
modify the function of a ribozyme, an RNA molecule with enzymatic activity.
The starting ribozyme cleaved RNA molecules, but directed evolution produced
a ribozyme that cleaved DNA molecules. Breaker & Joyce (1994) then evolved
DNA molecules that could cleave RNA. These methods were similar to the SELEX
method in using PCR to amplify molecules that survived the selection, but the
methods differed from SELEX in the selection itself. Table 1 lists the impressive
variety of unnatural ribozymes (not known from nature) that have been evolved
by these methods. Although these few examples highlight nucleic acids, directed
evolution also manipulates proteins and entire microbes. In many ways, these
new technologies are so different from natural evolution as to warrant a special
term (e.g., techno-evolution or technovolution?). However, the term directed
evolution, which is now in wide use, does have the advantage of conveying the
message that these socially beneficial applications are based on the foundations of
standard evolutionary biology.
DIFFERENTIAL REPRODUCTIVE SUCCESS: REPRODUCTION Reproduction of the
things being selected is essential to evolutionary progress. In the past, one had

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Figure 3 SELEX, a purely in vitro, nonliving scheme of evolution by natural selection. (1) A heterogeneous pool of oligonucleotides (the squiggles at the top) is passed
through a column of anchored target molecules (dark ovals). All oligos have identical
sequences on their ends but vary in the sequences of their middle region. (2) Oligo
variants whose sequences facilitate binding remain behind in the column, whereas
variants that do not bind pass through and are washed away. (3) Binding oligos are
eluted, by changing the salt concentration or pH of the solution. (4) The oligos from
step 3 are amplified by PCR. (5) The cycle is repeated by passing the amplified pool of
oligos over the column. After a few cycles, the oligo sequences remaining are limited
to those capable of binding the target molecules.

to work with an organism capable of reproducing, but now we can literally create
and then evolve molecules that reproduce themselves in the right cocktail of enzymes and other nutrients. The most common method of reproducing nucleic acid
molecules is PCR. A similar but less popular method is self-sustained sequence
replication (3SR). In PCR, DNA molecules are the parents, and their progeny are
complementary DNA molecules. In 3SR, RNA molecules are the parents, cDNA
molecules are the progeny, and transcription of these DNA molecules creates
RNA copies as the grandchildren. PCR uses synchronized cycles of reproduction,
in which all molecules in the tube reproduce once and then stop until the next cycle

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TABLE 1 Unnatural
ribozymes evolved by directed
evolutiona
Ribosyl 20 -Omediated cleavage
Mg2+-dependent cleavage
Pb2+-dependent cleavage
Ribosyl 20 ,30 -cyclic P hydrolysis
Pb2+ dependent
RNA ligation
30 ,50 Ligation (class 1)
20 ,50 Ligation
50 ,50 Ligation
RNA phosphorylation
Class 1 (ATP-S)
Class 1 (ATP)
Self-aminoacylation
Acyl transfer reaction
Acyl transfer
Aminoacyl transfer
Self-nitrogen alkylation
Suflur alkylation
Biphenyl isomerization
Prophyrin metalation
a

RNA molecules with these activities are


unknown from nature. (From Jaeger 1997.)

is initiated. 3SR is a method in which everything happens continuously (Fahy et al.


1991).
For many purposes, the directed evolution of proteins and many other types of
molecules still requires an organism. A gene is expressed in an organism (typically
a bacterium or yeast), but the gene may be removed from that organism and
subjected to various manipulations before being returned to the same or a different
organism. Biotechnology thus allows a gene to hitchhike as part of an organism but
then be divorced from that organism at will. However, two new methods allow in
vitro translation to couple a protein to its mRNA, enabling proteins to be evolved
in a SELEX-like fashion (Wilson et al. 2001). Limited forms of peptide selfreplication have also been produced, although the chemistry of the peptide copying
mechanisms is fundamentally different from that of nucleic acids (Lee et al. 1997).
DIFFERENTIAL REPRODUCTIVE SUCCESS: DIFFERENTIAL SUCCESS A challenge in
carrying out directed evolution is to come up with a powerful method to manipulate the differential reproductive success of the molecules under selection.
The goal is to choose genes whose phenotypes offer the greatest improvement as

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parents for the next round of selection. This can be done directly through selection
or indirectly through screening. True selection occurs when molecules with the
desired phenotypes reproduce faster than molecules with less desired phenotypes;
in the extreme case, molecules with the desired phenotypes are the only ones to
survive. In screening, the good and bad survive; the phenotype of each molecule
is individually assessedfor example, through a colorimetric enzyme assayand
genes for the most desired molecules are chosen as parents for the next generation.
The distinction between selection and screening in directed evolution thus appears
to be functionally the same as between natural selection and artificial selection
(D. Futuyma, personal communication). True selection is generally much more
powerful than screening because the number of variant molecules that can be subjected to selection is generally much larger than for screening (Arnold & Volkov
1999). However, schemes for true selection can be difficult to design.
The form of selection used in much of biotechnology is what quantitative geneticists refer to as truncation selectionreproducing only those molecules that
meet a certain standard, in the same way that a livestock breeder mates only animals that achieve a certain weight or fat content. In biotechnology, the selection
might be determined by the ability of a bacterial cell to grow on a toxic or antibiotic
substrate. In other cases, as in aptamer selection, reproductive success is based on
adherence to a target molecule. In some protocols for DNA enzyme evolution,
reproductive success is determined in the reverse fashionescaping from a bound
state is the gateway to future reproduction.
One of the advantages of evolution in biotechnology is that truncation selection
can achieve astronomical levels. The limiting factors in any truncation selection
are (a) the range of variation, which is limited by the number of different genotypes
that exist, and (b) the fecundity of those organisms between bouts of selection. If
an asexual organism produces only two offspring between bouts of selection, for
example, then a 50% culling is the long-term upper limit, whereas if the fecundity
is 1000, then a 99.9% culling is possible. The latter allows much more rapid evolution, genetic variation permitting. Methods such as cloning genes into selectable
bacterial plasmids and PCR allow amplifications (reproduction) of as high as 109
between bouts of selection. Such levels could be achieved only in special cases of
natural selection (e.g., with microbes evolving in a new environment or invading
a new host species).
VARIATION AND INHERITANCE: ELEVATING LEVELS OF GENETIC VARIATION Perhaps
the greatest deviations from natural evolutionary processes used in biotechnology
have come from manipulating genetic variation. These advances include elevating
genetic variation and recombining molecules.

The rate of evolution in many experiments is limited by genetic variation, largely because the high levels of truncation selection that may be applied
can take advantage of levels of variation that lie orders of magnitude beyond anything natural. To raise mutation rates, a variety of approaches have been developed.

MUTATION

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The most extreme is simply to incorporate high levels of variation in synthesized


molecules. Pools of oligonucleotides can be synthesized with arbitrary base compositions at specific sites. These molecules can be used as the direct target of
selection (as in aptamer evolution studies) or as material cloned into vectors that
then becomes translated into protein. At the extreme, the sequence of the initial
selective population is completely randomized and all bases have equal frequencies. Current technology limits the pool size to about 1016, so an exhaustive search
of deep random is limited to a sequence of 27 bases (Landweber 1999). When
larger stretches of randomized sequences are used, this limitation means that independent experiments using the same protocol may arrive at different solutions
because only a small fraction of the possible sequences are present in the initial
pool of molecules. Evolution in these cases is dominated by the tyranny of small
motifs pattern (Ellington 1994), such that any solutions to the selective challenge
that are specified by a small number of residues will certainly be present in the
pool of randomized molecules, whereas solutions specified by a large number
of residues may not. So the evolved solutions tend to be the simpler ones, not
necessarily the best ones.
Oligo synthesis is not the only means of elevating mutation rates. If molecules
are not synthesized, then they are copied from preexisting templates. Natural enzymes and processes are invariably used for this replication, and their inherent error
rates are usually much lower than wanted. Other methods of generating variation
rely on inflating the error rate during replication or introducing errors into the template itself. The standard method of elevating whole-organism mutation rates has
been to expose the organism to a chemical or physical mutagen. That method does
not allow precise control over the genomic locations of the mutations, although it
allows some control over the types of genetic changes, because different agents
tend to cause different types of mutations. More recent methods involve inflating
the error rates during replication: (a) PCR with unnatural bases, with asymmetric
base compositions, and/or with manganese; (b) amplification that involves alternately copying between DNA and RNA because the transcription step (DNA into
RNA) is highly error prone (e.g., Fromant et al. 1995).
RECOMBINATION One of the most powerful techniques used in directed evolution is recombination among variants of the same or similar molecules. Several
methods for performing in vitro recombination are now routine (known in the
field as gene shuffling or molecular breeding). Pim Stemmer of Maxygen developed the first purely in vitro shuffling method (Stemmer 1994), which involves
pooling DNA templates, nicking them to cut strands, denaturing them, and letting them come back together before patching them up. Molecules require only
short regions of identity to realign, so recombination (strand exchange) can occur
between molecules that differ substantially. The variants to be shuffled can be
those already improved during earlier rounds of selection, or they can be naturally occurring homologous genes from a diversity of speciesfamily shuffling
(Crameri et al. 1998). Family shuffling has the same advantage as increasing genetic

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variation by introducing novel strains or wild relatives into the genetic stock: Natural selection has already weeded out the deleterious mutations from these variants.
In at least some cases family shuffling radically accelerates the process of directed evolution. For example, when cephalosporinase genes from four microbial
species were independently evolved through one round of DNA shuffling, they
showed up to an eightfold improvement in their resistance to the antibiotic moxalactam. A single cycle of family shuffling that combined all four genes yielded
a 540-fold improvement in resistance. The evolved gene showing the greatest increase in resistance was made of eight fragments from three of the four genes and
had an additional 33 amino acid substitutions (Crameri et al. 1998).
VARIATION AND INHERITANCE: VARIANTS WITH REDUCED CONSTRAINTS Reproductive success is an ultimate necessity in any form of evolution by natural or
artificial selection. Yet in a narrow sense, reproduction may have nothing to do
with the phenotype sought by artificial selection. The reproductive requirement
often limits the progress that can be achieved in artificial selection. For example,
most people prefer to eat seedless watermelons, but seeds are needed for the next
generation. (Seedless watermelons are created as triploid hybrids from crosses
between different strains.) One of the big successes in biotechnology has been the
reduction of such constraints, so that molecules with desired phenotypes can be
propagated without concern for their correlated negative effects on reproduction.
Several tricks facilitate the divorce between reproductive constraints and selection
on the desired phenotype.
REPRODUCTION BY PCR When a nucleic acid (RNA or DNA) molecule itself is
selected for the phenotype, as in aptamer or ribozyme evolution, amplification
by PCR is not only the easiest method of reproducing the molecule, it also entails minimal constraints. The ends of the molecule must match primer sequences,
but intervening sequences are largely irrelevant. Earlier methods of amplification
(cloning) incorporated the DNA molecule in a plasmid, and a plasmids reproduction is tied to that of its host. Not only was cloning a cumbersome method
of amplification, if the cloned sequences were incompatible with cell growth, the
amplification would not work. PCR is a nonselective amplification method, such
that the entire pool of nucleic acids (with suitable ends) experiences minimal evolution during the amplification (Bull & Pease 1995). Thus with PCR, differential
reproductive success can be virtually removed from the amplification/reproduction
stage.
LIMITING THE DELETERIOUS CONSEQUENCES OF PHENOTYPE EXPRESSION In many
cases, the desired phenotype requires protein expression. The standard way to
create proteins is to put the gene inside cells and let the cellular machinery build
the protein. This approach can be a problem if the protein is incompatible with
cellular function. A compatibility problem can be overcome by placing the gene in

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vectors that offer control over gene expression. Gene expression can be suppressed
until the host has reproduced, so that even if the cell is killed by expression, the
DNA vector from selected colonies can be recovered and put into new cells. The
new methods for in vitro translation and coupling of the peptide to its mRNA
avoid the problem entirely, however (Wilson et al. 2001). Another mechanism
for limiting deleterious consequences applies at the protein level instead of the
genome level. When evolving peptide sequences, it is often desirable to couple
the evolvable peptide with a reporter peptide (for screening or perhaps as part of
the selection). Activity of a reporter peptide will be incompatible with sequence
insertions at many positions, but there are often regions that can tolerate small
or even large peptide additions without destroying activity, such as protein ends
or loops. Thus a fusion protein is created that has both the reporter activity and a
variable region that can be selected for a different function. This rationale underlies
the highly successful methods of the yeast two-hybrid assay (Chien et al. 1991),
phage display (Smith 1985), and some other systems.
VARIATION AND INHERITANCE: UNNATURAL MOLECULES The synthesis of unnatural molecules, which is now routine chemistry, has affected artificial selection in
two ways: New types of molecules can be evolved, and old types of molecules
can be evolved toward new targets. Replication of new kinds of nucleic acids
is possible with modified bases, sometimes known as base analogs. The ribonucleotides in natural RNA contain the bases adenine, uracil, guanine, and cytosine.
Several types of modified bases have been incorporated into ribonucleotides, and
a few types have been incorporated into deoxyribonucleotides. These modified
bases have novel groups attached that do not interfere with hydrogen bonding
but that can alter the characteristics of the nucleic acid in other ways. Provided
the polymerase will accept the base analog, it is straightforward to create a mix
of bases that will result in a nucleic acid with one or more of the natural bases
replaced by the analog. These nucleic acids may then be subjected to the usual
selections, in the hope that the product will be superior to the natural nucleic acids
(Tarasow et al. 1997, Wiegand et al. 1997, Sakthivel & Barbas 1998, Battersby et al.
1999).
Novel molecules may also serve as selective agents. This phenomenon has been
visited many times in human history, as pesticides were applied to control pests or
synthetic drugs were used to treat microbes, and the offended organisms responded
with their own evolution (see section below). One of the more interesting uses of
novel molecules as a selective agent is based on the symmetry of mirror-image
molecules. Life uses the L-forms of amino acids and D-forms of nucleic acids,
but the mirror image forms of both can be synthesized chemically. The novel
method uses a synthesized D-peptide as a target against a pool of D-nucleic acids as
potential aptamers; the D-peptide is the unnatural form, whereas the D-nucleic acids
are of the natural form so that they can be evolved through directed evolution. When
a successful D-aptamer is evolved, its L-form is synthesized (which is unnatural).

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Because of the mirror-image symmetry, the synthetic L-aptamer recognizes the


L-peptide that is found in nature. This two-step technique was used to create an
L-form DNA aptamer that inhibits vasopressin and, unlike the D-aptamer, was
resistant to nuclease degradation (Klussmann et al. 1996, Williams et al. 1997).

Examples of Directed Evolution


A DNA ENZYME TO LIMIT ARTERIAL DAMAGE FROM ANGIOPLASTY Santoro & Joyce
(1997) evolved a short DNA molecule that cleaved a specific site in a target RNA
molecule. From a sequence comparison between this DNA enzyme and its target,
the DNA enzyme appeared to contain a catalytic core flanked on both sides by
regions that formed Watson-Crick base pairs with the target RNA. This discovery
suggested that a DNA enzyme could be synthesized against almost any RNA
merely by changing its flanking sequences to complement the RNA sequence at
the desired target. In one application, the DNA enzyme was designed to cleave the
mRNA of the Egr-1 gene, whose expression causes unwanted proliferation of the
arterial wall in response to damage from angioplasty (balloon inflation). Arterial
proliferation is counterproductive because the goal of angioplasty is to expand the
arterial canal, and the proliferation narrows it. Tests in a rat model showed that the
DNA enzyme had the desired effect of reducing arterial proliferation (Santiago
et al. 1999).
ENZYMES FOR HOUSEHOLD AND AGRICULTURAL USE The year 2000 marked the
5th Annual World Congress on Enzyme Technologies, organized by the IBC (International Business Communications). The sponsors were the biotech companies
Maxygen, Genencor, Diversa, and Thermogen. This meeting was dominated by
talks on using directed evolution to improve enzyme performance in specific settings. One industrial goal is improved cellulase activity. Agricultural waste in the
form of corn stalks and other plant material offers a potential windfall of ethanol
production, if cellulose can be digested with cheap enzymes. Another goal is
the improvement of enzymes (proteases, lipases) to use in laundry detergents to
remove stains and other dirt. Cellulases, proteases, and lipases have been isolated from numerous organisms, but their activity levels are too low under applied
conditions to justify their use. For example, naturally occurring proteases do not
function in warm, soapy water. Directed evolution is being used to improve that
performance [for an introduction to this large area of research, see Marrs et al.
(1999), Schmidt-Dannert & Arnold (1999), Voigt et al. (2000)].
EVOLVING PEPTIDES THAT LIMIT CRYSTAL SIZE Phage display is the insertion of
peptide sequences into coat proteins of a bacteriophage (bacterial virus) so that the
peptide insert is able to contact surfaces in the phages environment. The size and
location of the peptide insert is chosen so that it permits phage reproduction. With
randomized inserts, a phage display library may contain billions of different insert
sequences that can be selected for binding to many types of substrates. Using a

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commercial phage display library, Whaley et al. (2000) recovered peptide epitopes
that bound gallium arsenate crystals; some epitopes discriminated different forms
of the crystal. The peptide insert was a mere 12 amino acids long, yet it seemed
that crystal recognition could be achieved with even fewer than 12 amino acids.
This discovery may be an important step in miniaturizing the manufacture of
semiconductors (nanotechnology) because by binding the crystal lattice, these
peptides may offer a simple way of reproducibly controlling crystal growth.
BLOCKING HIV EXPRESSION Many drugs have been developed to suppress HIV,
but evolution of HIV resistance to these drugs fuels a continual demand for new
drugs. Some recent approaches to inhibit HIV use technologies involving RNA
molecules. These new methods include antisense RNA (RNAs that are complementary to the single-stranded viral mRNAs), ribozymes (RNA enzymes that cleave
the viral mRNAs), and aptamers that bind HIV proteins. The antisense RNAs and
ribozymes can be developed merely from an understanding of HIV genome sequences, whereas the aptamers need to be evolved in a SELEX-like manner. When
different agents created by these three methods were compared for their efficacy
against HIV in cell culture, successful inhibition was obtained only with aptamers
(Good et al. 1997). Assuming that an anti-HIV aptamer could become an effective drug in vivo, the encouraging aspect of this result is that new aptamers could
possibly be evolved each time the virus evolved resistance to the old aptamers.

Other Approaches
Evolution is just one of several technology-driven methods for modifying molecules
for specific uses. Methods for protein improvement are generally divided into approaches known as rational design and irrational design (Arnold 1997, Arnold &
Volkov 1999). Rational design consists of protein engineering by modification of
specific amino acid residues based on knowledge of protein structure and mechanistic details. Although this approach is potentially powerful, we are currently
far from having enough information about most proteins to apply rational design,
and in general we lack the deep understanding of protein structure and function
that would allow us to predict the outcome of specific amino acid substitutions
(Tobin et al. 2000). The irrational approaches of directed evolution do not require such detailed knowledge because they rely on selection to reach the desired
outcome.
These applications in biotechnology not only offer a new relevance for evolutionary biology, they also provide new opportunities for biologists with classical
training in evolution. The industrial approach of large-scale experiments with a
focus on products and detailed molecular analysis is often done in ignorance of
the underlying classical framework. A truly golden opportunity lies ahead for the
marriage of classical theory with industrial interests. What level of recombination
and mutagenesis is optimal? How rugged is the fitness landscape? It should be possible to build a new level of evolutionary theory and help industry reach its goals

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in this new area of directed evolution, allowing for appropriate accommodation of


the sometimes conflicting goals between industry and academia.

RESISTANCE MANAGEMENT
Many organisms (including humans) have been creative in developing chemistry to
deter or kill unwanted competitors. For humans, the pests may be cancer cells, insects, mites, worms, weeds, or microbes. As is known all too well, virtually all our
attempts to control pests with chemical agents have led to resistance to those agents
(Garrett 1994). The evolution of resistance is so routine that it seems inevitable,
notwithstanding a few noteworthy examples, such as the American chestnut, in
which an entire species with billions of individuals apparently failed to evolve resistance to an invading pathogen (Newhouse 1990). The question is how an understanding of evolution can help us retard or even prevent the evolution of resistance.
The evolution of resistance is not a new problem. It accompanied the introduction of antibiotics and pesticides 50 years ago. Attention to this problem from
evolutionary biologists has lagged, however. Part of the reason for this slow response may have been the seemingly limitless supply of new chemicals. When the
first antibiotics were isolated from nature, a virtual windfall of different drugs was
found. The evolution of resistance was inconsequential when new drugs became
available faster than old ones failed. Likewise, new pesticides may have seemed
easy to engineer in the days before government regulation. Now, however, many
of the old technologies have reached limits, and the cost of obtaining government
approval is enormous (e.g., on the order of half a billion dollars for a new drug).
There are thus ample incentives to use chemicals wisely and prolong the lives of
what works now.
One role of evolutionary biologists is to educate the public. A widespread
misconception concerning antibiotics is that an individual who abuses them will
develop a tolerance, so the drugs will no longer work for them personally. That
is, some people mistakenly assume that the persons body changes in response to
antibiotic misuse. The true problem is that misuse of antibiotics encourages evolution in the bacterium so that it is no longer affected by the drug. If the bacterium
spreads, then people contracting it are at risk of an untreatable infection, no matter how conscientious they have been in their past use of antibiotics. Resistance
quickly becomes a global problem caused by evolution.
The evolution of resistance is affected by the manner in which we apply the
toxins. These factors are under our control and allow us at least to impede the
evolution of resistance.

The Right Dose


No doubt the most widely acknowledged factor influencing the evolution of resistance is the dose. Many Americans, at least, are aware of the admonition to

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take the full course of prescribed antibiotics, lest the infection return in a more
resistant, less easily treated form (this is especially a problem with tuberculosis
infections, in which eradication of the bacterium from a person requires months of
treatment). Evolutionary biologists since Darwin have been aware that weak selection can ultimately yield phenotypes that lie well outside the range of phenotypes
currently in the population, whereas immediate selection for those extremes would
fail and thereby extinguish the population. A low dose of a druglow enough to
allow survival of some sensitive individualsis a form of weak selection. Its main
detriment is that it favors individuals with partial resistance; as partial resistance
evolves, full resistance is more easily attained. A second complication with a low
dose is that it leaves sensitive survivors that may mutate to resistance before further doses are applied. The evolution of bacterial resistance is now a factor when
recommending levels of some antibiotics (Blondeau et al. 2001), and variations in
dose, both temporally and within the patient, are used to help understand the evolution of resistance (Baquero & Negri 1997, Baquero et al. 1997, Blondeau et al.
2001). Dose is likewise a consideration when planting insect-toxic, transgenic
plants to avoid insect resistance (see below).

Selective Application
Larger populations are more likely than small ones to contain resistant genotypes,
so limiting the size of the population treated reduces the chance that resistance
will evolve. A simple way to limit the treated population yet still be effective
is to treat only those pests causing damage. With antimicrobial agents, selective
application would consist of treating only those patients manifesting an infection;
with pesticides, selective application would consist of spraying only those crops
experiencing economic injury levels of pests. Although this model is simple in
principle, for social and technical reasons it is often difficult to institute. With antibiotic treatment of bacterial infections, a strict adherence to selective application
would mean that patients are not given a drug until their infecting microbe has
been diagnosed as a strain sensitive to the drug. This practice is neither patientfriendly nor safe in all cases, as an infection can worsen during the time required for
diagnosis. Even when it is clear that antibiotic treatment is unwarranted, a patients
demands for a drug may override any physician concern for the eventual evolution
of resistance. The problem is a classic example of a phenomenon described by
Hardin (1968), Tragedy of the Commons, in that the cost/benefit ratio from the
individuals perspective favors antibiotic overuse, in opposition to the common
good (Palumbi 2001). Industrial concerns further contribute to antibiotic overuse
in our environment because antibiotic food supplements yield faster livestock
growth. The agricultural interest of greater meat production has so far been the
victor over proposals to restrict antibiotics in livestock food, despite clear evidence
that antibiotic food supplements encourage the evolution of antibiotic resistance
in human pathogens (Garrett 1994).

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Does limited use (selective application) reduce resistance levels? It is of course


clear that antibiotic use led directly to the widespread evolution of antibiotic resistance in many bacteria, as indicated by geographic and temporal correlations
between drug resistance and antibiotic use (Garrett 1994, 2000; Granizo et al.
2000). Limiting antibiotic use during the past four decades would at least have
slowed the rate at which resistance evolved. But selective application also causes
current resistance levels to drop (Cristino 1999).

Combination Therapy
Simultaneous application of multiple agents may extinguish a small population,
even when high doses of single agents would not work, for the same reason that
high doses of a single agent are better than low doses: The chance that resistant
individuals occur in the initial population decreases with the magnitude of the
imposed mortality. If resistance to a single agent can be conferred by a single
mutation, then multiple agents may offer the advantage that no single mutation
confers complete resistance. Perhaps the original use of combination therapy was
in the treatment of tuberculosis with the first antibiotics (Ryan 1993). Treatment
with a single antibiotic resulted in the evolution of drug resistance within the
patient. Simultaneous treatment with a combination of three drugs allowed the
infection to be cured and the within-patient evolution of resistance prevented.
More recently, the simultaneous use of multiple drugs to treat HIV infections
has resulted in prolonged avoidance of AIDS because the virus is so effectively
curtailed with the harsh treatment (Coffin 1995, Matsushita 2000). The long-term
success of many antiviral vaccines may likewise stem from the immunity generated
against multiple targets through both the humoral and cell-mediated components.
With other goals, however, simultaneous, multidrug treatment may not be the best
practice (Bonhoeffer et al. 1997).

Charting the Course of Resistance


When more drugs are available than can be given to a single patient, the choice of
which drugs to take should be based on the evolution of resistance. Screening for
preexisting resistance to the drugs can ensure that the combination of drugs administered is maximally effective. For example, anti-HIV drugs (of which there are now
many) are so harsh on the patient that doses and numbers of drugs are often based on
patient tolerance levels. There are three major classes of anti-HIV drugs (protease
inhibitors and two kinds of reverse-transcriptase inhibitors), but many drug options
exist within each class. A patient on highly active antiretroviral treatment will take
one drug from each class to maximize the number of viral targets, but within each
class, the drug that is taken is somewhat optional. Protocols are now being tested
in which a patients viral population is assessed for resistance mutations, with the
drug choice based on that genetic information (OMeara et al. 2001, MacArthur
2000). It is obvious that this kind of approach requires understanding the molecular
basis of resistance. It also requires a technical means of assessing low levels of

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resistance in the target population (e.g., on the order of 1% or even 0.1%), as such
levels are not easily detectable yet can change rapidly in response to selection.

Refugia
One of the more exciting new developments in agriculture is the engineering
of transgenic crops expressing a gene from the bacterium Bacillus thuringiensis
(Bt), the protein of which is highly toxic to butterflies and moths. This toxin (Bt)
has long been a favorite of organic farmers, used as a spray prior to transgenic
technology, because it selectively kills only a small subset of insects and typically
does not harm the parasitic and predatory control agents. Corn and cotton are two
major Bt transgenic crops in wide use now, and the potential for reducing pesticide
applications to the environment from them alone is impressive. Despite a public
backlash against genetically modified foods and the consequent withdrawal of one
Bt corn strain from the market, approximately one quarter of the U. S. corn crop is
expected to be Bt transgenic in 2001 (F. Gould, personal communication). There
is thus a huge market for Bt strains.
Alleles for Bt resistance are already found at moderate frequencies in several
pest populations, in some cases at frequencies greater than 10% (Gould et al.
1997, Tabashnik et al. 2000). These pests are thus poised for a rapid response
to Bt transgenic plants. Fortunately, levels of resistant insects are much lower
than levels of resistant alleles because the resistance alleles are recessive, hence
resistant insects occur at the square of the allele frequency. These surprisingly high
frequencies of resistance alleles are unexplained in at least some species, because
there is no known history of exposure to agricultural Bt toxin (F. Gould, personal
communication).
The commercial implications of Bt resistance are obvious and have inspired
the U. S. Department of Agriculture and seed companies to anticipate and slow its
evolution (Gould 1998). An antiresistance evolution strategy is being employed
that depends on recessive resistance. Farmers growing Bt crops are mandated to
plant a certain amount of nonBt crops as welluntil recently 4%; now it is higher.
These nonBt crops are planted in separate fields (refugia) close to the Bt crops,
to provide a safe haven for pests that are not resistant to the toxin.
Refugia impact the evolution of resistance to Bt as follows. Pests in the refugia
survive regardless of their resistance to the toxin. Pests in the Bt fields survive only
if they are resistant, i.e., homozygous for the resistance allele. As long as resistance
alleles are uncommon, most insects will be sensitive. Thus, refugia will produce
many more insects than the Bt fields. With random mating between pests born in
the refugia and pests born in the Bt fields, most offspring will be sensitive to the
toxin (because they are either homozygous sensitive or heterozygous). Refugia
will maintain resistance alleles at low frequencies for long periods of time because
they continually dilute the resistance genes and keep them from being selected.
Resistance will eventually ascend, but the ascent is greatly delayed with refugia
(Figure 4). This result is a special case of the population genetics principle that

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Figure 4 Effect of refugia on the delay of Bacillus thuringiensis (Bt) resistance evolution.
Three curves of gene frequency evolution are shown: (a) no refugia, (b) 1% refugia, and (c)
4% refugia. The evolutionary response to no refugia is immediate, because the only survivors
are recessive homozygotes. The ascent of the resistance allele is progressively delayed with
larger refugia because refugia are not selective for genotype and thus weaken selection for
the recessive, resistance allele. These simulations assumed complete mixing of individuals
between refugia and Bt crops when mating and when ovipositing. They also assumed that the
only source of selection was mortality of all heterozygotes and of sensitive homozygotes in
Bt fields, that generations were discrete, and that the initial frequency of the resistance allele
was 0.001. The effect of refugia would be modified to the extent that these assumptions do not
hold, especially the recessivity of the resistance phenotype and the mixing. Also, the curves
give only allele frequencies; the actual numbers of surviving pests would be an important
factor to consider in the economic injury to a crop, but that factor is not included here. These
curves were calculated by iteration of the equation p0 = (p2Waa + pqWAa)/(p2Waa + 2pqWAa
+ q2WAA), where p is the frequency of the recessive resistance allele in generation t, and p0
is its frequency in generation t + 1. The fitness values were determined as Waa = 1, WAa =
WAA = R, where R is the proportion of crops in the refugia.

weak selection on a recessive trait is ineffective when the recessive allele is rare
(Crow & Kimura 1970); the strength of selection decreases with refugium size.
One reason nonBt crops are not mixed in fields with Bt crops is to prevent insects
from moving between the two types of plants and thus experiencing intermediate
doses of the toxin (which might reduce the recessivity of the resistance alleles).

Evolvable Drugs
Biotechnology offers new drugs from new classes of molecules but it also offers
new drugs from some old moleculesproteins and nucleic acids. (Some types

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of RNA-based drugs were discussed above in a paragraph about blocking HIV.)


Proteins and nucleic acids in essence have their own genome, so theoretically it
should be possible to evolve those drugs. Evolving a drug might provide a way
of producing new forms of the drug that overcome pests resistant to the original
form of that drug. At the moment, this approach is futuristic. It is analogous to
the use of combinatorial chemistry to produce new generations of antibiotics by
modifying subgroups of old drugs. Drug evolution was attempted in a model system of antisense RNA used to inhibit a virus (Bull et al. 1998). Antisense nucleic
acids work on the simple principle of being complementary to a target sequence
(in this case a regulatory sequence of the virus) and thus blocking gene expression.
If the target sequence in the virus evolves to a resistant form, antisense molecules
complementary to the new target are then easily created. This arms race can
thus potentially be continued indefinitely because a new antisense RNA can be
created for every step the virus takes in evolving resistance. The empirical test of
this theory supported some of the assumptions but not all: New antisense RNAs
could be created that inhibited individual viruses resistant to the original antisense
RNA. However, the viral population exhibited a variety of escape mutants, and
no single new antisense RNA could control the resistant viral population. Resistance polymorphism in the viral population thus thwarted control by the second
generation of drugs.
Other variations of this approach have also been developed, again without
success. Djordjevic & Klaenhammer (1997) and Bull et al. (2001) each developed
a suicide plasmid cassette against a bacterial virus. Each plasmid contained a toxic
gene downstream of a promoter expressed by the virus. Infection expressed the
toxic gene, which, in turn, killed the infection. A priori, it seemed likely that an
arms race could be waged against the virus by inserting a new toxic gene into the
cassette each time the virus evolved resistance to the old toxic gene. However, in
both systems the virus evolved resistance by a change in its transcriptional activity,
selectively reducing expression from the plasmid regardless of which toxic gene
was carried by the plasmid. In a further attempt to keep pace with the resistant
virus, Djordjevic & Klaenhammer (1997) created a new plasmid that contained
multiple copies of the viral promoter; this plasmid partially restored inhibition
against the virus, although at some cost to the uninfected cell (perhaps through
low levels of constitutive expression).
It is not clear how widely the principle of drug evolution can be applied to
block resistance. Drugs that share a common molecular ancestor will likely use
the same molecular target. If evolution in the pest can protect the target and render
it inaccessible, then drug evolution will fail. The case of nonnucleoside reversetranscriptase inhibitors (NNRTI) in HIV provides an analogy to drug evolution.
Various NNRTI drugs have been developed, but they all function by similar mechanisms, binding to the same pocket in the viral reverse transcriptase (Emini 1996).
Resistance to one of these drugs often confers resistance to several of the others. If
one regards the different NNRTI drugs as the equivalents of evolved drugs, then an
evolved drug in this case affords much less protection against a resistant virus than

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does a drug using a different mechanism of inhibition. All the different evolved
forms of a drug may use the same basic mechanism of inhibition, just as different
NNRTI drugs use similar mechanisms, in which case viral resistance to one drug
will at least partly overlap with resistance to the others.

EVOLUTIONARY COMPUTATION
Introduction
Evolution is not restricted to biological systems, nor has the benefit of using evolution to solve problems been limited to biology. Although application of evolutionary principles to computer programming preceded the work of Fogel et al. (1966),
their book brought the idea to the forefront. The goal of this work was loftythat
through a replication of specific aspects of evolution, means will be found for
the generation of an artificially intelligent automata . . . capable of solving problems in previously undiscovered ways. The field gained sophistication almost as
quickly as its biological counterpart, and by the mid-1970s, Holland (1975) was
discussing coadapted gene sets and epistatic interactions in the context of computation. Many concepts and much of the language of evolutionary computation (EC)
are borrowed from the biological world. Bits are loci, potential solutions to a
problem, are termed individuals, chromosomes, or genomes, and they are changed
by mutation and recombination. A collection of variant solutions, termed a
population, is subjected to selection by imposing some measure of fitness. The
best solutions are selected as parents for the next generation, they reproduce
with mutation and recombination to produce a new population that is subjected to
selection, and so on.
EC, a subfield of artificial intelligence, exists in several forms that differ in the
representation of the problem that is evolved and the mechanisms of evolution employed (Foster 2001). For example, in genetic programming, executable programs
themselves are evolved, whereas in genetic algorithms, the parameters that describe
a potential solution to a problem are evolved (Koza 1992). Although recombination is commonly employed in genetic algorithms and genetic programming, it is
generally not employed in the other two EC varieties, evolutionary programming
and evolutionary strategies, which instead rely on large-scale mutations.
Like studies of the evolution of biological systems, research in the area of EC
makes use of model systems. In this case the models are problems to be solved.
These include the traveling salesman problem, an NP-complete problem (i.e.,
a class of computationally difficult problems) in which one tries to determine the
shortest route for a salesman traveling between a large number of cities. Another
interesting model problem is the iterated Prisoners Dilemma, in which one
decides when it is best to cooperate and when it is best to defect in a series of
confrontations. This problem has been explored from the perspective of the evolution of cooperation (Axelrod & Hamilton 1981) and is now a classroom standard
in EC (Vogel 1995). Another common model problem in genetic programming

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is symbolic regression, where one must evolve a program to reproduce the inputoutput behavior of an unknown function given only randomly selected inputs and
function values.
Evolved programs have interesting properties that in some cases are shared with
biological systems. For example, evolution of a genetic program via recombination
is accompanied by code bloat (Langdon et al. 1999). Like biological genomes,
such computer genomes almost always accumulate junk, and it is not easy to
separate the functional components of the genome from the nonfunctional ones. In
fact, a defense that removes junk from the genome will lead to the evolution of code
that evades the editing algorithm (Soule & Foster 1998). However, genome size
can be constrained directly by charging a fitness penalty for larger genomes. This is
similar to biological systems in which microbial genomes maintain more streamlined genomes than those of most multicellular organisms, presumably because of
selection for rapid replication.

What Kinds of Problems are Best Explored with EC?


Although in principle evolutionary algorithms can be applied to any computational
problem, they are best suited to very difficult problemsproblems to which there
are no other known efficient solutions. As with biological evolution, EC may not
find the optimal solution because it is a stochastic approximation algorithm. For
problems with a very large potential solution space, such as the traveling salesman
problem in which the number of possible routes through N cities is N!, EC will
not explore all possible solutions. But if the topology of the fitness landscape is
rough, evolution is an efficient way to find and explore fitness peaks.
Biological problems are naturals for EC. On one hand, this approach can be
used to build tools to solve difficult problems, such as predicting protein or RNA
folding, inferring phylogenies (Lewis 1998), or aligning DNA or protein sequences
(Notredame & Higgins 1996). On the other hand, it can be used to model complex
biological systems, such as the immune system, ecosystems, or cells (Adami 1998).
Genetic algorithms have found applications for some very practical problems,
such as scheduling and constructing complex timetables. In such cases, there may
be other methods that find equally good or even better solutions, but perhaps not
with the same flexibility and speed. Genetic algorithms have also been applied
to nonlinear filtering problems, such as processing signals from radar, sonar, and
GPS satellites (Whitely et al. 2000).
Evolved programs tend to be more robust than user-created (written) programs.
They can withstand more damage without total failure, a property that is analogous
to biological homeostasis. The basis for robustness is not always clear, but at least in
some cases, it is not merely redundancy in evolved programs (Masner et al. 2000).
Different user-created programs may tend to use the same approach to a particular
problem, but evolved solutions will tend to be unique. An important potential
application of the robustness and the unique nature of evolved programs is fault
tolerance. Where computer failure can have catastrophic consequences (i.e., on a

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space shuttle or the navigation system of an airplane), agreement between multiple


independent programs is used to reduce the probability of failure of the system.
But if all the programs have the same underlying fault because they were written
according to the same programming principles, agreement may not assure against
failure. Evolved programs may provide greater fault tolerance than do written
programs. And it is not out of the question to imagine programs that evolve to
repair themselves when they detect a fault (J.A. Foster, personal communication).

CONCLUSION
The examples presented here are but a small fraction of the applications of evolutionary biology to socially relevant issues. We have limited this review to examples in which evolutionary principles and methods have actually been used to
solve problems. An even richer variety of problems is being approached with evolutionary perspectives, and some of these efforts are likely to yield fruit in the near
future.
A research focus on applied evolution will perhaps prove to be an ephemeral
one. It does not offer a conceptual organization for a discipline as broad as evolutionary biology. New applications will, of course, continue to arise and prosper, and
applications may be useful in garnering funding and influencing students career
choices. But at the moment, the defining basis for applied evolution is ignorance.
Many people, both scientists and the public, are unaware that evolutionary biology
has become very relevant, yet attacks to suppress the teaching of evolution receive
widespread support at the local level. We hope that ignorance of applications will
be short-lived, as texts and the news media begin to disseminate the examples, and
evolutionary biology should emerge in the future with widespread public acceptance. This review has focused on positive applications of evolution, but it is also
important to understand these potential applications to guard against other uses
of this technology, such as production of biological weapons of mass destruction
or the evolution of damaging computer viruses. The time has arrived for wide
public understanding of the importance and relevance of evolutionary biology in
everyday lives.
ACKNOWLEDGMENTS
Many of the examples in this paper were identified through discussions with colleagues. We thank D. Hillis, F. Gould, A. Ellington, M. Robertson, B. Levin, L.
Ancel, L. Vawter, W. Maddison, I. Matsumura, S. Palumbi, D. Futuyma, R. Bush,
M. Courtney, I. Eckstranol, and J. Foster for discussions, references, or, in some
cases, personal accounts. Mitch Sogin provided Figure 2. Editorial comments of
D. Futuyma and D. Fautin helped improve the prose. The topics in this paper also
overlap with a symposium we organized on applied evolution at the 2000 meetings of the Society for the Study of Evolution (Bloomington). We also are very

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grateful for the support of the NIH (GM38737 and GM 57756) and the NSF (DEB
9726902) during the time we prepared this review.
Visit the Annual Reviews home page at www.AnnualReviews.org

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