Evolutionary ecology of learning: insights from fruit flies

Ecologically and evolutionarily oriented research on learning has traditionally been carried out on vertebrates and bees. While less sophisticated than those animals, fruit flies (Drosophila) are capable of several forms of learning, and have the advantage of a short generation time, which makes them an ideal system for experimental evolution studies. This review summarizes the insights into evolutionary questions about learning gained in the last decade from evolutionary experiments on Drosophila. These experiments demonstrate that Drosophila has the genetic potential to evolve a substantially improved learning performance in ecologically relevant learning tasks. In at least one set of selected populations, the improved learning generalized to a task other than that used to impose selection, involving a different behavior, different stimuli, and a different sensory channel for the aversive reinforcement. This improvement in learning ability was associated with reductions in other fitness-related traits, such as larval competitive ability and lifespan, pointing to evolutionary trade-offs for improved learning. These trade-offs were confirmed by other evolutionary experiments where a reduction in learning performance was observed as a correlated response to selection for tolerance to larval nutritional stress or for delayed aging. Such trade-offs could be one reason why fruit flies have not fully used up their evolutionary potential for learning. Finally, another evolutionary experiment with Drosophila provided the first direct evidence for the long-standing idea that learning can under some circumstances accelerate and in others slow down genetically based evolutionary change. These results demonstrate the usefulness of fruit flies as a model system to address evolutionary questions about learning.     

No trade-off between learning ability and parasitoid resistance in Drosophila melanogaster

Learning ability and immunity to parasites are linked at the physiological level in several insect species. The aim of this work was to investigate the relationship between learning and immunity at an evolutionary level. We tested whether selection for improved learning ability in Drosophila melanogaster led to changes in parasitoid resistance as a correlated response. Similarly, we assayed whether selection for better parasitoid resistance led to a change in learning ability. There was no significant difference between selected and control lines in either case; the estimated confidence intervals for the differences indicate that a trade-off relationship is unlikely.     

LIFE-HISTORY CONSEQUENCES OF ADAPTATION TO LARVAL NUTRITIONAL STRESS IN DROSOPHILA

Many animal species face periods of chronic nutritional stress during which the individuals must continue to develop, grow, and/or reproduce despite low quantity or quality of food. Here, we use experimental evolution to study adaptation to such chronic nutritional stress in six replicate Drosophila melanogaster populations selected for the ability to survive and develop within a limited time on a very poor larval food. In unselected control populations, this poor food resulted in 20% lower egg‐to‐adult viability, 70% longer egg‐to‐adult development, and 50% lower adult body weight (compared to the standard food on which the flies were normally maintained). The evolutionary changes associated with adaptation to the poor food were assayed by comparing the selected and control lines in a common environment for different traits after 29–64 generations of selection. The selected populations evolved improved egg‐to‐adult viability and faster development on poor food. Even though the adult dry weight of selected flies when raised on the poor food was lower than that of controls, their average larval growth rate was higher. No differences in proportional pupal lipid content were observed. When raised on the standard food, the selected flies showed the same egg‐to‐adult viability and the same resistance to larval heat and cold shock as the controls and a slightly shorter developmental time. However, despite only 4% shorter development time, the adults of selected populations raised on the standard food were 13% smaller and showed 20% lower early‐life fecundity than the controls, with no differences in life span. The selected flies also turned out less tolerant to adult malnutrition. Thus, fruit flies have the genetic potential to adapt to poor larval food, with no detectable loss of larval performance on the standard food. However, adaptation to larval nutritional stress is associated with trade‐offs with adult fitness components, including adult tolerance to nutritional stress.     

Learning ability and longevity: a symmetrical evolutionary trade-off in Drosophila

Learning ability can be substantially improved by artificial selection in animals ranging from Drosophila to rats. Thus these species have not used their evolutionary potential with respect to learning ability, despite intuitively expected and experimentally demonstrated adaptive advantages of learning. This suggests that learning is costly, but this notion has rarely been tested. Here we report correlated responses of life-history traits to selection for improved learning in Drosophila melanogaster. Replicate populations selected for improved learning lived on average 15% shorter than the corresponding unselected control populations. They also showed a minor reduction in fecundity late in life and possibly a minor increase in dry adult mass. Selection for improved learning had no effect on egg-to-adult viability, development rate, or desiccation resistance. Because shortened longevity was the strongest correlated response to selection for improved learning, we also measured learning ability in another set of replicate populations that had been selected for extended longevity. In a classical olfactory conditioning assay, these long-lived flies showed an almost 40% reduction in learning ability early in life. This effect disappeared with age. Our results suggest a symmetrical evolutionary trade-off between learning ability and longevity in Drosophila.     

THE RESPONSES OF DROSOPHILA MELANOGASTER TO ARTIFICIAL SELECTION ON BODY WEIGHT AND ITS PHENOTYPIC PLASTICITY IN TWO LARVAL FOOD ENVIRONMENTS

To investigate the potential response to natural selection of reaction norms for age and size at maturity, fresh body weight at eclosion was mass selected under rich and poor larval food conditions in Drosophila melanogaster. The sensitivity of dry weight at eclosion to the difference between rich and poor larval food was selected using differences in sensitivities among families. For both experiments, the correlated response to selection of age at eclosion was examined. The flies were derived from wild populations and had been mass cultured in the lab for more than six months before the experiments started. These flies responded to selection on body weight upwards and downwards on both rich and poor larval food. Selection on increased or decreased sensitivity of body weight was also successful in at least one direction. Sensitivity was reduced by selection upwards in a poor environment and downwards in a rich environment.     

Genetically idiosyncratic responses of Drosophila melanogaster populations to selection for improved learning ability

To what extent is adaptive evolution over short timescales repeatable? To address this question, we studied the performance of crosses between replicate Drosophila melanogaster lines previously subject to selection for improved learning response in the context of oviposition substrate choice. Of the 10 pairwise F₁ crosses among the five selection lines, four performed in the original learning assay similarly to the parental lines, whereas the remaining six showed learning scores significantly below the average of the parental lines. In particular, four F₁ crosses (three involving the same line) showed no detectable learning, on a par with unselected control lines. This indicates that the response to selection in some lines involved allelic substitutions at different loci. Additional assays of crosses between two selection lines indicated that the loss of performance in hybrids generalized to another type of learning assay, and held for both short‐ and long‐term memory. Joint analysis of first‐ and second‐generation crosses between these two lines supported the hypothesis that the response to selection in these different lines was based on the spread of recessive alleles at different loci. These results show that the evolutionary trajectories of populations of the same origin subject to uniform selection may sometimes diverge over very short evolutionary timescales.     

Rapid responses to a strong experimental selection for heat hardening in the invasive whitefly Bemisia tabaci MEAM 1

Adaptation to new environments is an important issue for invasive species as colonization depends on evolvability in their new distribution range. Here, we considered the case of the whitefly Bemisia tabaci MEAM 1 (Gennadius) (Hemiptera: Aleyrodidae), a pest that has recently invaded Colombia and where thermal adaptation has been proposed to explain its colonizing ability. An experimental evolution study was conducted to assess the evolutionary potential of B. tabaci in relation to its upper thermal limits, to explain its rapid adaptation during post‐invasion periods. Selection for hardening capacity was conducted in four whitefly populations. We measured thermal responses in relation to fitness components (survival, fecundity, and viability) for 5–7 generations under a strong selection regime. Heat hardening responded rapidly in both sexes. This was expressed as an increase in survival, but not in fecundity or viability. These results suggest that thermal responses for heat hardening are not correlated and evolve independently. Increased survival after few generations of selection points to high adaptive potential in this insect, which leads to rapid post‐invasion adaptation. Our study can help to predict population responses to environmental change and explain the colonizing ability of this pest.     

Selection for associative learning of color stimuli reveals correlated evolution of this learning ability across multiple stimuli and rewards

We are only starting to understand how variation in cognitive ability can result from local adaptations to environmental conditions. A major question in this regard is to what extent selection on cognitive ability in a specific context affects that ability in general through correlated evolution. To address this question, we performed artificial selection on visual associative learning in female Nasonia vitripennis wasps. Using appetitive conditioning in which a visual stimulus was offered in association with a host reward, the ability to learn visual associations was enhanced within 10 generations of selection. To test for correlated evolution affecting this form of learning, the ability to readily form learned associations in females was also tested using an olfactory instead of a visual stimulus in the appetitive conditioning. Additionally, we assessed whether the improved associative learning ability was expressed across sexes by color‐conditioning males with a mating reward. Both females and males from the selected lines consistently demonstrated an increased associative learning ability compared to the control lines, independent of learning context or conditioned stimulus. No difference in relative volume of brain neuropils was detected between the selected and control lines.     

The effect of learning on experimental evolution of resource preference in Drosophila melanogaster

Abstract Learning is thought to be adaptive in variable environments, whereas constant, predictable environments are supposed to favor unconditional, genetically fixed responses. A dichotomous view of behavior as either learned or innate ignores a potential evolutionary interaction between the learned and innate components of a behavioral response. We addressed this interaction in the context of oviposition substrate choice in Drosophila melanogaster, asking two main questions. First, will learning also evolve in a constant environment in which it always pays to show the same choice? Second, how does an opportunity to learn affect the evolution of the innate (genetic) component of oviposition substrate choice? We exposed experimental populations to four selection regimes, involving selection on oviposition substrate preference (an orange versus a pineapple medium). In two selection regimes the flies were selected for preference either for the orange medium, or for the pineapple medium. In the remaining two selection regimes the flies were also selected for preference for either orange or pineapple, but additionally could use past experience (aversion learning) to decide which medium it paid to avoid. Lines exposed to the latter selection regimes evolved improved learning ability, indicating that learning may be advantageous even if the same behavioral response is favored every generation. Furthermore, of the two selection regimes that favored oviposition on the pineapple medium, the regime that allowed for learning led to the evolution of a stronger innate preference for pineapple, than the regime that did not allow for learning. In contrast, of the two regimes that selected for oviposition on the orange medium, the one that allowed for learning led to a smaller evolutionary change of the innate preference. Thus, an opportunity to learn facilitated the evolution of innate preference under selection for preference for pineapple, but hindered it under selection for preference for orange. We discuss possible mechanisms for this effect.     

Evolutionary adaptation to environmental stressors: a common response at the proteomic level

Mechanistic trade‐offs between traits under selection can shape and constrain evolutionary adaptation to environmental stressors. However, our knowledge of the quantitative and qualitative overlap in the molecular machinery among stress tolerance traits is highly restricted by the challenges of comparing and interpreting data between separate studies and laboratories, as well as to extrapolating between different levels of biological organization. We investigated the expression of the constitutive proteome (833 proteins) of 35 Drosophila melanogaster replicate populations artificially selected for increased resistance to six different environmental stressors. The evolved proteomes were significantly differentiated from replicated control lines. A targeted analysis of the constitutive proteomes revealed a regime‐specific selection response among heat‐shock proteins, which provides evidence that selection also adjusts the constitutive expression of these molecular chaperones. Although the selection response in some proteins was regime specific, the results were dominated by evidence for a “common stress response.” With the exception of high temperature survival, we found no evidence for negative correlations between environmental stress resistance traits, meaning that evolutionary adaptation is not constrained by mechanistic trade‐offs in regulation of functional important proteins. Instead, standing genetic variation and genetic trade‐offs outside regulatory domains likely constrain the evolutionary responses in natural populations.     

Adaptation of Drosophila to a novel laboratory environment reveals temporally heterogeneous trajectories of selected alleles

The genomic basis of adaptation to novel environments is a fundamental problem in evolutionary biology that has gained additional importance in the light of the recent global change discussion. Here, we combined laboratory natural selection (experimental evolution) in Drosophila melanogaster with genome‐wide next generation sequencing of DNA pools (Pool‐Seq) to identify alleles that are favourable in a novel laboratory environment and traced their trajectories during the adaptive process. Already after 15 generations, we identified a pronounced genomic response to selection, with almost 5000 single nucleotide polymorphisms (SNP; genome‐wide false discovery rates < 0.005%) deviating from neutral expectation. Importantly, the evolutionary trajectories of the selected alleles were heterogeneous, with the alleles falling into two distinct classes: (i) alleles that continuously rise in frequency; and (ii) alleles that at first increase rapidly but whose frequencies then reach a plateau. Our data thus suggest that the genomic response to selection can involve a large number of selected SNPs that show unexpectedly complex evolutionary trajectories, possibly due to nonadditive effects.     

Higher parasite resistance in Daphnia populations with recent epidemics

Natural populations often show genetic variation in parasite resistance, forming the basis for evolutionary response to selection imposed by parasitism. We investigated whether previous epidemics selected for higher resistance to novel parasite isolates in a Daphnia galeata–microparasite system by comparing susceptibility of host clones from populations with varying epidemic history. We manipulated resource availability to evaluate whether diet influences Daphnia susceptibility as epidemics are common in nutrient‐rich lakes. Exposing clones from 10 lakes under two food treatments to an allopatric protozoan parasite, we found that Daphnia originating from lakes (mainly nutrient rich) with previous epidemics better resist infection. Despite this result, there was a tendency of higher susceptibility in the low food treatment, suggesting that higher resistance of clones from populations with epidemic background is not directly caused by lake nutrient level. Rather, our results imply that host populations respond to parasite‐mediated selection by evolving higher parasite resistance.     

Within- and trans-generational plasticity affects the opportunity for selection in barbed goatgrass (Aegilops triuncialis)

• Premise of the study: Environments are composed of selective agents, and environments may also modify the efficacy of these agents. Environments affect the rate of maximum evolutionary change by influencing variation in relative fitness (i.e., the opportunity for selection, or I). Within- and transgenerational plastic environmental responses may affect I, speeding or slowing processes of local adaptation. • Methods: We determined whether environmental factors affected the opportunity for selection (I) in Aegilops triuncialis (barbed goatgrass) by measuring I as a within- and transgenerational plastic response to two maternal glasshouse environments (serpentine/dry and loam/moist). We also determined whether this species’ two most common genetic lineages (determined by DNA microsatellite length polymorphism) varied in response to glasshouse treatments. • Key Results: Opportunity for selection was less for plants grown in the dry serpentine environment than for plants grown in the moist loam environment. This response varied between genetic lineages. The east lineage exhibited a within-generation response to the dry serpentine environment. For both seed mass and average seed weight in this lineage, the opportunity for selection was lower in dry serpentine than in moist loam. The west lineage had a transgenerational response to the dry serpentine such that the opportunity for selection for seed number and seed mass was lower for plants produced by mothers grown in dry serpentine than for plants produced by mothers in moist loam. • Conclusions: Phenotypic variation in relative fitness is constrained by the dry serpentine environment, which leads to lower evolvability in this environment. Within- and transgenerational effects of the environment may slow local adaptation to serpentine soils.     

THE GENETIC BASIS OF DIFFERENCES IN GROWTH AND BEHAVIOR OF SPECIALIST AND GENERALIST HERBIVORE SPECIES: SELECTION ON HYBRIDS OF HELIOTHIS VIRESCENS AND HELIOTHIS SUBFLEXA (LEPIDOPTERA)

Two species of moths and their hybrids were studied to determine the genetic basis of host range. One species, Heliothis virescens (HV), has a broad host range and is an agricultural pest on cotton (Malvaceae), soybean (Fabaceae), and tobacco (Solanaceae). The other species, Heliothis subflexa (HS), has a narrow host range, feeding on plants in the genus Physalis (Solanaceae). Experiments were done to determine whether the abilities to feed on cotton, soybean, and tobacco were each under separate genetic control in HV (genetically uncorrelated) or whether feeding on all three hosts was genetically correlated. By repeatedly backcrossing hybrids to HS while selecting for high larval survival and weight on soybean, loci conferring the ability to feed on soybean were moved from HV into the genetic background of the specialist, HS. After six generations of selection on soybean we tested the selected line on soybean, cotton, Physalis, and tobacco to determine if ability to feed on soybean was correlated with feeding ability on these other hosts. We found no evidence to support the hypothesis that feeding on all three hosts was correlated and controlled by a single locus. Compared to HS, the survival and weight of the selected line were significantly higher on soybean, similar on tobacco and Physalis, and slightly, but not significantly, higher on cotton. We also conducted a behavioral choice test to determine if larval preference for soybean was correlated with preference for other hosts. The selected line larvae had higher preference for soybean and cotton than HS. Preference for tobacco was equal in the selected line and in HS. These results support the trends seen in survival and growth tests where performances on soybean and cotton appeared partially correlated and performances on soybean and tobacco were uncorrelated. In conclusion, the generalist, HV, did not have a single set of loci that governed feeding on soybean, cotton and tobacco. It is likely that some of the loci governing performance and preference for soybean also contributed to performance and preference for cotton and that the soybean loci examined did not interact negatively to lower performance and preference for Physalis, the specialist's host. This approach can be applied more widely to address evolutionary questions about host range and other ecological traits.     

Direct and correlated responses to chill‐coma recovery selection in Drosophila buzzatii

Chill‐coma recovery (CCR) is an important trait for thermal adaptation in insects. Multiple phenotypes could be affected by selection on CCR if the trait is genetically correlated with other adaptive traits. To test for heritable (co‐)variation in CCR, we examined direct and correlated responses to bi‐directional selection on CCR. Drosophila buzzatii Patterson & Wheeler (Diptera: Drosophilidae) was artificially selected for decreased and increased recovery time following exposure to 0 °C. After 18 selected generations, the selection response in CCR was significant but qualitatively asymmetric, with replicated lines for slow CCR showing the highest response. Knockdown resistance to high temperature was not affected by CCR selection. Starvation resistance in the adult fly showed no clear pattern of correlated responses to CCR selection. Selection on CCR had no impact on developmental time and body size. Chill‐coma recovery shows no apparent genetic trade‐offs with any of the multiple traits included in this study. These results are largely consistent with recent studies on clines in D. buzzatii, which showed that CCR is not across‐population correlated with other clinally varying traits of thermal adaptation. Cold adaptation may evolve toward increased cold resistance independent of upper thermal limits.     

Food availability and parasite infection influence the correlated responses of life history traits to selection for age at pupation in the mosquito Aedes aegypti

We selected six lines of mosquito Aedes aegypti for earlier or later pupation and measured the correlated responses of several life history traits: adult size, two fecundity measures and pre-adult survival. We further examined the influence of two environmental parameters – larval food availability and infection by the microsporidian parasite Edhazardia aedis– on the correlated responses. Pre-adult survival did not respond to selection for age at pupation in any environment. For all of the other traits, the environment influenced the correlated response, though the contribution of the different environmental aspects differed among traits. While the correlated response of adult size was influenced only by larval food availability, the likelihood that a female laid eggs was influenced by parasite infection, and the correlated response of the number of eggs was influenced by the interaction of the two environmental parameters. Generally, a deteriorating environment moved the correlated response from one favouring later pupation to one favouring earlier pupation. Larval food availability and parasite infection also influenced the association between the mean wing length and fecundity of the selected lines. At high food availability, there was a positive relationship between adult size and fecundity, while infected mosquitoes reared at low food availability showed the opposite trend. We discuss these results in light of the coevolutionary potential of the host–parasite interaction.     

RESPONSES AND CORRELATED RESPONSES TO ARTIFICIAL SELECTION ON THORAX LENGTH IN DROSOPHILA MELANOGASTER

Two sets of four replicate lines of Drosophila melanogaster were selected for large and small thorax with controls. F, progeny of crosses between the selected lines within each size category showed (a) a reduction in preadult viability in large lines relative to control and small lines when they were cultured at medium or high density in competition with a standard mutant marked competitor stock, and (b) an increase in larval development time in large lines relative to control and small lines. Natural selection for increased body size in adults may therefore be opposed by adverse effects on larval viability. The results are discussed in terms of the developmental mechanisms probably responsible for the change in body size. The preadult survival of the large and control lines was measured at three different temperatures, and there was no evidence for a significant interaction between size and temperature. The observed evolutionary increase in body size in response to reduced temperature in Drosophila must therefore involve either different genes from those subject to selection for size at a single temperature, or a fitness component other than preadult survival. There was no significant asymmetry in response to selection, and thorax length showed heterosis in crosses between the selected lines.     

NATURAL SELECTION FOR GRAZER RESISTANCE TO TOXIC CYANOBACTERIA: EVOLUTION OF PHENOTYPIC PLASTICITY?

Abstract We studied the selection response of the freshwater grazing zooplankter, Daphnia galeata, to increased abundance of cyanobacteria in its environment. Cyanobacteria are a poor‐quality and often toxic food. Distinct genotypes of D. galeata were hatched from diapausing eggs extracted from three time horizons in the sediments of Lake Constance, Europe, covering the period 1962 to 1997, a time of change in both the prevalence of planktonic cyanobacteria and levels of phosphorus pollution. We assessed whether the grazers evolved to become more resistant to dietary cyanobacteria by exposing genetically distinct clones to two diets, one composed only of the nutritious green alga, Scenedesmus obliquus (good food), and the other a mixture of S. obliquus and the toxic cyanobacterium Microcystis aeruginosa (poor food). Genotype performance was measured as the specific rate of weight gain from neonate to maturity (g_j). We evaluated evolutionary change in the Daphnia population using an analysis of reaction norms based on relative (log‐transformed) changes in g_j. Log(g_j) is a measure of the proportional effect of dietary cyanobacteria on other fitness components of the Daphnia phenotype. For comparison, we also analyze absolute (i.e., nontransformed) changes in g_j and discuss the interpretations of the two approaches. Statistical results using a general linear model demonstrate a significant effect of genotype (showing differences in g_j among genotypes), a significant genotype X food‐type interaction (showing differences in phenotypic plasticity among genotypes), and, in the case of log‐transformed data, a significant sediment‐genotype‐age X food‐type interaction. The latter shows that phenotypic plasticity evolved over the period studied. Two constraints act on response to selection in the D. galeata‐Lake Constance system. First, g_j on a diet containing poor food is highly correlated with g_j on a diet of good food, thus evolving resistance also meant evolving an increase in g_j on both diets. Second, because genotypes with a high g_j also grow to a large adult body size, which in turn increases Daphnia vulnerability to fish predation, we suggest that selection only acted to favor genotypes possessing a high potential g_j after cyanobacteria became prevalent. The presence of cyanobacteria depressed realized g_j and led to animals of small adult body size even if their genotypes had the potential for high g_j and large size. With realized g_j reduced, genotypes with an inherently high value could be selected even in the presence of predatory fish. The joint action of selection by dietary cyanobacteria and vulnerability to fish predation provides an explanation for the observed evolution of resistance to poor food through reduced phenotypic plasticity.     

Complex patterns of local adaptation in heat tolerance in Drosophila simulans from eastern Australia

Latitudinal clines are considered a powerful means of investigating evolutionary responses to climatic selection in nature. However, most clinal studies of climatic adaptation in Drosophila have involved species that contain cosmopolitan inversion polymorphisms that show clinal patterns themselves, making it difficult to determine whether the traits or inversions are under selection. Further, although climatic selection is unlikely to act on only one life stage in metamorphic organisms, a few studies have examined clinal patterns across life stages. Finally, clinal patterns of heat tolerance may also depend on the assay used. To unravel these potentially confounding effects on clinal patterns of thermal tolerance, we examined adult and larval heat tolerance traits in populations of Drosophila simulans from eastern Australia using static and dynamic (ramping 0.06 °C min^?1) assays. We also used microsatellites markers to clarify whether demographic factors or selection are responsible for population differentiation along clines. Significant cubic clinal patterns were observed for adult static basal, hardened and dynamic heat knockdown time and static basal heat survival in larvae. In contrast, static, hardened larval heat survival increased linearly with latitude whereas no clinal association was found for larval ramping survival. Significant associations between adult and larval traits and climatic variables, and low population differentiation at microsatellite loci, suggest a role for climatic selection, rather than demographic processes, in generating these clinal patterns. Our results suggest that adaptation to thermal stress may be species and life‐stage specific, complicating our efforts to understand the evolutionary responses to selection for increasing thermotolerance.     

Experimental social evolution with Myxococcus xanthus

Genetically-based social behaviors are subject to evolutionary change in response to natural selection. Numerous microbial systems provide not only the opportunity to understand the genetic mechanisms underlying specific social interactions, but also to observe evolutionary changes in sociality over short time periods. Here we summarize experiments in which behaviors of the social bacterium Myxococcus xanthus changed extensively during evolutionary adaptation to two relatively asocial laboratory environments. M. xanthus moves cooperatively, exhibits cooperative multicellular development upon starvation and also appears to prey cooperatively on other bacteria. Replicate populations of M. xanthus were evolved in both structured (agar plate) and unstructured (liquid) environments that contained abundant resources. The importance of social cooperation for evolutionary fitness in these habitats was limited by the absence of positive selection for starvation-induced spore production or predatory efficiency. Evolved populations showed major losses in all measured categories of social proficiency- motility, predation, fruiting ability, and sporulation. Moreover, several evolved genotypes were observed to exploit the social behavior of their ancestral parent when mixed together during the developmental process. These experiments that resulted in both socially defective and socially exploitative genotypes demonstrate the power of laboratory selection experiments for studying social evolution at the microbial level. Results from additional selection experiments that place positive selection pressure on social phenotypes can be integrated with direct study of natural populations to increase our understanding of principles that underlie the evolution of microbial social behavior. This revised version was published online in August 2006 with corrections to the Cover Date.