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Experimental evolution is the use of experiments or controlled field manipulations to explore evolutionary dynamics.[1] Evolution may be observed in the laboratory as populations adapt to new environmental conditions and/or change by such stochastic processes as random genetic drift. With modern molecular tools, it is possible to pinpoint the mutations that selection acts upon, what brought about the adaptations, and to find out how exactly these mutations work. Because of the large number of generations required for adaptation to occur, evolution experiments are typically carried out with microorganisms such as bacteria, yeast or viruses, or other organisms with rapid generation times.[1][2][3] However, laboratory studies with foxes[4] and with rodents (see below) have shown that notable adaptations can occur within as few as 10-20 generations and experiments with wild guppies have observed adaptations within comparable numbers of generations.[5] More recently, using experimental evolution followed by whole genome pooled sequencing, an approach known as Evolve and Resequence (E&R) [6], allows researchers to keep a record of virtually every change taking place in the genomes of populations under experimental evolution across generations.[7] Experimental evolution is used for multiple purposes, both in basic research such as direct testing of evolutionary theory and in applied research for medical and biotechnological applications.

History

Domestication and breeding

Unwittingly, humans have carried out evolution experiments for as long as they have been domesticating plants and animals. Selective breeding of plants and animals has led to varieties that differ dramatically from their original wild-type ancestors. Examples are the cabbage varieties, maize, or the large number of different dog breeds. The power of human breeding to create varieties with extreme differences from a single species was already recognized by Charles Darwin. In fact, he started out his book The Origin of Species with a chapter on variation in domestic animals. In this chapter, Darwin discussed in particular the pigeon.

Early experimental evolution

One of the first to carry out a controlled evolution experiment was William Dallinger. In the late 19th century, he cultivated small unicellular organisms in a custom-built incubator over a time period of seven years (1880–1886). Dallinger slowly increased the temperature of the incubator from an initial 60 °F up to 158 °F. The early cultures had shown clear signs of distress at a temperature of 73 °F, and were certainly not capable of surviving at 158 °F. The organisms Dallinger had in his incubator at the end of the experiment, on the other hand, were perfectly fine at 158 °F. However, these organisms would no longer grow at the initial 60 °F. Dallinger concluded that he had found evidence for Darwinian adaptation in his incubator, and that the organisms had adapted to live in a high-temperature environment. Unfortunately, Dallinger's incubator was accidentally destroyed in 1886, and Dallinger could not continue this line of research. From the 1880s to 1980, experimental evolution was intermittently practiced by a variety of evolutionary biologists, including the highly influential Theodosius Dobzhansky. Like other experimental research in evolutionary biology during this period, much of this work lacked extensive replication and was carried out only for relatively short periods of evolutionary time.

Modern Experimental Evolution

Experiments where populations were subjected to controlled environments and phenotypic parameters were measured across generations were carried out throughout the 20th century. However, it was the advent of molecular biology and later genomics that led to an explosion of research in experimental evolution by allowing researchers to test the genetic background of observed changes and to track genetic changes with no apparent effect on phenotypes. Today, experimental evolution is carried out using a large variety of organism such as viruses, bacteria, eukaryotic microorganisms, vertebrate and invertebrate animals and even artificial life. The array of questions addressed is vast, ranging from fundamental questions regarding many different aspects of evolutionary theory to application in medicine, agriculture and other technological developments.

Long-term experimental evolution

Evolution experiments are generally designed with specific aims and are accordingly limited in time and scope. However, on February 15, 1988, Richard Lenski started a long-term evolution experiment with the bacterium E. coli. This experiment in contrast to other evolution experiments has the aim to track all changes that take place in adapting populations. Such an experimental approach allows researchers to address questions that had not been initially envisioned. One example happened when one of the test populations suddenly developed the ability to metabolize citrate from the growth medium aerobically and showed greatly increased growth. This provided a dramatic observation of evolution in action. The experiment continues to this day, and is now the longest-running controlled evolution experiment ever undertaken. Since the inception of the experiment, the bacteria have grown for more than 60,000 generations. Lenski and colleagues regularly publish updates on the status of the experiments.[24]

Field experiments

In order to maintain controlled conditions most experimental evolution is carried out under laboratorial conditions. However, some field experiments were carried out under semi-controlled conditions. These experiments are particularly helpful when trying to reproduce the conditions in which a certain trait evolved. An experiment on temperature adaptation in stickleback fishes showed that marine sticklebacks could adapt in only three generations to 2.5 degrees Celsius lower temperatures. This adaptation to cold-tolerance mimicked the natural adaptation that freshwater stickleback populations underwent when adapting to freshwater conditions.

Evolve and resequence

The advent of cheap Next-Generation Sequencing (NGS) techniques allowed a new approach to experimental evolution called Evolve and Resequence (E&R). This type of experiments is normally initiated with a limited number of genotypes that are fully or patially sequenced. After a set number of generations a round of resequencing is done with the goal of identifying mutations ocurrying in the know genomic sequences. E&R experiments involve subjecting experimental populations to some sort of selective pressure throughout the number of generations that the experiment lasts. NGS allows then to monitor genetic changes that occur in these populations in real-time, thus offering a link between phenotypic and molecular evolution .

Study systems

The choice of model organisms in evolution experiments is informed by the specific question that the researcher wishes to address and convenience. The greatest limiting factors in experimental evolution are time as evolutionary processes are slow, and the ability to keep the study population under a controlled environment.

Microorganisms

Microorganisms are extensively used in experimental evolution experiments given their convenience as model organisms with short-generation times, very large populations sizes and ease to maintain in laboratorial conditions. Thus, microorganisms are particularly suited to answer questions regarding general principles of evolutionary theory. The bacterium Escherichia coli is the model organism used in Lenski’s long-term evolution experiment and it has been used to test vast array evolutionary hypothesis such as: large vs. small-effect mutations as the basis for adaptation, adaptation to fluctuating environments, repeatability of evolution, and models of host-parasite dynamics where E. coli is used in combination with bacteriophages, among others. Pseudomonas sp. bacteria are also widely used in experiments trying to elucidate the genetic basis of adaptation and social evolution. Myxobacteria are a group of bacteria that is used for tests of kin selection and social evolution hypotheses. Eukaryotic microorganisms are also used in experimental evolution in particular yeast, which has been useful in tests of hypothesis regarding sexual selection and mating system evolution, social evolution and repeatability of evolution.

Invertebrate model organisms

Invertebrate model organisms offer relatively short-generation times and easy lab maintenance. In addition, having been extensively studied in the fields of molecular and developmental biology a wide array of genetic manipulation techniques is available along with a diversity of well-known genomes. Thus, organisms such as Drosophila fruit flies, Caenorhabditis roundworms and Daphnia crustaceans are suited to test general hypotheses of evolutionary theory in multicellular organisms and are particularly useful to address some specific questions. Fruit flies are the most widely used multicellular model organism in experimental evolution. Experiments with fruit flies address a wide range of questions including: mutation and adaptation, role of genetic drift, sexual selection, evolution of life-history traits, behavioral evolution, speciation, repeatability and reversibility of evolution and aging. Caenorhabditis elegans is used particularly for studies of mating system evolution, as its hermaphrodites reproduce both by outcrossing with males and by self-fertilization. Daphnia with its well-known ecology and large diversity of parasites has been of particular use for studies on the dynamics of host-parasite coevolution.

Vertebrates and plants

So-called higher organisms, in particular vascular plants and vertebrate animals generally have far longer generation times and smaller population sizes and are therefore considered to be less convenient and more burdensome in a classical experimental evolution setup. However, studies addressing specific questions have used such organisms. Laboratory mice are of particular usefulness to address questions regarding complex behavior and nervous system evolution. Stickleback fishes were used in field semi-controlled experiments to address questions regarding ecological adaptation. Vascular plants are seldom used in experimental evolution setups but nonetheless some studies used the diversity of plant reproductive systems to address questions regarding their evolution.

Artificial life

Ever since the advent of computers the possibility of digitally generated replicating units fascinated evolutionary biologists. Artificial life offers the possibility of testing the viability of evolutionary hypothesis and estimating parameters such selection coefficients, mutation rates and others, without constraints. In addition, evolutionary concepts are also used to expand the realm of possibilities with artificial life in applied fields such as robotics, software development and artificial inteligence.

Applications

Study of adaptation

One of the main topics addressed by experimental evolution is adaptation to new environments. Typically, populations are subjected to suboptimal environmental conditions (temperature, nutrition, and other multiple stress factors) and fitness is measured across generations. Then, variation in fitness is linked to detected mutations. This type of experiments allows the detection of specific mutations underlying adaptive evolution and permits the investigation of general rules of adaptation. For example, studies with E. coli indicate that adaptation to new environments is more likely to happen through a large number of mutations each with a small fitness effect (Perfeito et al., 2007). In another study using E. coli it was shown that different beneficial mutations can have negative epistasis effects, i.e. the combined fitness in the presence of several of these mutations is lower than the sum of the individual fitness benefits that each mutation could bring on its own (Khan et al. 2011). One other important result regarding the dynamics of adaption is the realization that adaptation to a specific environment might entail negative fitness effects, i.e. trade-offs, if the adapted genotype finds itself in an opposite environment. For example, E. coli strains adapted to high temperatures have lower fitness than non-evolved in strains when placed at lower temperatures (Bennett and Lenski 2007), moths adapted to resisting insecticides have lower fitness in insecticide-free environments (Boivin et al, 2003) or fruit flies adapted to low-density populations lose out in competition with non-adapted genotypes when they find themselves under high-density conditions (Mueller et al, 1991).

Test of evolutionary theory

Experimental evolution offers researchers a framework within which tests can be made of evolutionary processes that are proposed theoretically.

One such example is to test the effects of exposition to constant environments in contrast to fluctuating ones. Several predictions have been experimentally proven, such as: the advantage of generalist and specialist genotypes in fluctuating and constant environments, respectively, the emergence of bet-hedging strategies in unstable and unpredictable environments, and the induction of adaptive radiation due to spatial heterogeneity of the environment.

Kin selection and cooperation has been another field of interest in experimental evolution. Work on the social bacterium Mixococcus xanthus has shown the importance of relatedness and kin discrimination in the emergence of socially cooperative behavior and in the maintenance of such cooperative populations against the emergence of cheaters, i.e. from individuals that retain high fitness benefits from the cooperative population but fail to cooperate.

Sexual conflict theory predicts that the fitness interests of each sex are contradictory and experiments with fruit flies have shown that natural selection favors the emergence of male traits that are deleterious to their female mates.

Sex evolution theory shows that sex (outcrossing with a mate) is costly in terms of fitness when compared to other reproductive modes (parthenogenesis, self-fertilization). Evolution of sex predicts that sex is maintained because it allows the maintenance and emergence of high-diversity levels, rendering sexual populations more adaptable. In experiments with the hermaphrodite C. elegans outcrossing with males was shown to promote higher genetic diversity and adaptation to new environments when compared with the alternative reproductive strategy available, self-fertilization.

Evolution was shown to be repeatable, with fruit fly populations subjected independently to the same selective pressures having evolved the same phenotypic adaptations that were paralleled by identical genetic changes, in a proof of principle of the theory of parallel evolution. On the other hand, also using fruit flies, evolution was shown to be reversible. Evolved populations were selected to re-adapt to their ancestral environment and after a few generations the re-adapted populations had fitness levels comparable to their early ancestors. However, the genetic changes were different, meaning the reversed evolve flies are different from both their direct evolved ancestors and from their earlier non-evolved ancestors with whom they share the similar phenotypes.

Aging

One specific subject that was the object of studies in experimental evolution was the evolution of senescence or aging. Using fruit flies these studies showed that senescence – the decline in surviving and fecundity that accompanies advancing age – is associated to pleiotropic effects of adaptations that favor fecundity in an earlier age (Rose and Charlesworth, 1980). Furthermore, subsequent studies showed that putting fruit fly populations under selective pressure for late fecundity had the associated effect of increasing life-span (Rose, 1984). Thus, as predicted theoreticaly, a correlation was found between age of fecundity and senescence.

Applications to medicine and technology

Experimental evolution is used not only to address questions regarding fundamental questions in evolutionary biology but also to test hypotheses and applications in medicine and other technologies. Vaccine development is a case in point. Attenuated versions of pathogens that cause diseases such as polio, tuberculosis, measles, mumps and others, were serially passed in other hosts until their adaptation to the new host finally resulted in diminished pathogenic effects in humans. Other applications include the development of biological pest controls for agriculture, biofuel generation and even software development and robotics.