Specificity of genome evolution in experimental populations of Escherichia coli evolved at different temperatures.

Isolated populations derived from a common ancestor are expected to diverge genetically and phenotypically as they adapt to different local environments. To examine this process, 30 populations of Escherichia coli were evolved for 2,000 generations, with six in each of five different thermal regimes: constant 20 °C, 32 °C, 37 °C, 42 °C, and daily alternations between 32 °C and 42 °C. Here, we sequenced the genomes of one endpoint clone from each population to test whether the history of adaptation in different thermal regimes was evident at the genomic level. The evolved strains had accumulated ∼5.3 mutations, on average, and exhibited distinct signatures of adaptation to the different environments. On average, two strains that evolved under the same regime exhibited ∼17% overlap in which genes were mutated, whereas pairs that evolved under different conditions shared only ∼4%. For example, all six strains evolved at 32 °C had mutations in nadR, whereas none of the other 24 strains did. However, a population evolved at 37 °C for an additional 18,000 generations eventually accumulated mutations in the signature genes strongly associated with adaptation to the other temperature regimes. Two mutations that arose in one temperature treatment tended to be beneficial when tested in the others, although less so than in the regime in which they evolved. These findings demonstrate that genomic signatures of adaptation can be highly specific, even with respect to subtle environmental differences, but that this imprint may become obscured over longer timescales as populations continue to change and adapt to the shared features of their environments.

Different tradeoffs result from alternate genetic adaptations to a common environment.

Fitness tradeoffs are often assumed by evolutionary theory, yet little is known about the frequency of fitness tradeoffs during stress adaptation. Even less is known about the genetic factors that confer these tradeoffs and whether alternative adaptive mutations yield contrasting tradeoff dynamics. We addressed these issues using 114 clones of Escherichia coli that were evolved independently for 2,000 generations under thermal stress (42.2 °C). For each clone, we measured their fitness relative to the ancestral clone at 37 °C and 20 °C. Tradeoffs were common at 37 °C but more prevalent at 20 °C, where 56% of clones were outperformed by the ancestor. We also characterized the upper and lower thermal boundaries of each clone. All clones shifted their upper boundary to at least 45 °C; roughly half increased their lower niche boundary concomitantly, representing a shift of thermal niche. The remaining clones expanded their thermal niche by increasing their upper limit without a commensurate increase of lower limit. We associated these niche dynamics with genotypes and confirmed associations by engineering single mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase, and the rho gene, which encodes a termination factor. Single mutations in the rpoB gene exhibit antagonistic pleiotropy, with fitness tradeoffs at 18 °C and fitness benefits at 42.2 °C. In contrast, a mutation within the rho transcriptional terminator, which defines an alternative adaptive pathway from that of rpoB, had no demonstrable effect on fitness at 18 °C. This study suggests that two different genetic pathways toward high-temperature adaptation have contrasting effects with respect to thermal tradeoffs.

Physiology: postprandial cardiac hypertrophy in pythons.

Oxygen consumption by carnivorous reptiles increases enormously after they have eaten a large meal in order to meet metabolic demands, and this places an extra load on the cardiovascular system. Here we show that there is an extraordinarily rapid 40% increase in ventricular muscle mass in Burmese pythons (Python molurus) a mere 48 hours after feeding, which results from increased gene expression of muscle-contractile proteins. As this fully reversible hypertrophy occurs naturally, it could provide a useful model for investigating the mechanisms that lead to cardiac growth in other animals.

Microbial experimental evolution.

Microbes have been widely used in experimental evolutionary studies because they possess a variety of valuable traits that facilitate large-scale experimentation. Many replicated populations can be cultured in the laboratory simultaneously along with appropriate controls. Short generation times and large population sizes make microbes ideal experimental subjects, ensuring that many spontaneous mutations occur every generation and that adaptive variants can spread rapidly through a population. Another highly useful experimental feature is the ability to preserve and store ancestral and evolutionarily derived clones. These can be revived in parallel to allow the direct measurement of the competitive fitness of a descendant compared with its ancestor. The extent of adaptation can thereby be measured quantitatively and compared statistically by direct competition among derived groups and with the ancestor. Thus, fitness and adaptation need not be matters of qualitative speculation, but are quantitatively measurable variables in these systems. Replication allows the quantification of heterogeneity in responses to imposed selection and thereby statistical distinction between changes that are systematic responses to the selective regimen and those that are specific to individual populations.

Evolutionary adaptation to environmental pH in experimental lineages of Escherichia coli.

This study uses the enteric bacterium Escherichia coli as an experimental system to examine evolutionary responses of bacteria to an environmental acidic-alkaline range between pH 5.3 and 7.8 (15-5000 nM [H(+)]). Our goal was both to test general hypotheses about adaptation to abiotic variables and to provide insights into how coliform organisms might respond to changing conditions inside and outside of hosts. Six replicate lines of E. coli evolved for 2000 generations at one of four different constant pH conditions: pH 5.3, 6.3, 7.0, or 7.8. Direct adaptation to the evolutionary environment, as well as correlated changes in other environments, was measured as a change in fitness relative to the ancestor in direct competition experiments. The pH 5.3 group had the highest fitness gains, with a highly significant increase of 20%. The pH 7.8 group had far less significant gains and much higher variance among its lines. Analysis of individual lines within these two groups revealed complex patterns of adaptation: all of the pH 5.3 lines exhibited trade-offs (reduced fitness in another environment), but only 33% of the pH 7.8 lines showed such trade-offs and one of the pH 7.8 lines demonstrated exaptation by improving fitness in the pH 5.3 environment. Although there was also prevalent exaptation in other groups to the acidic environment, there were no such cases of exaptation to alkalinity. Comparison across the entire experimental pH range revealed that the most acidic lines, the pH 5.3 group, were all specialists, in contrast to the pH 6.3 lines, which were almost all generalists. That is, although none of the pH 5.3 lines showed any correlated fitness gains, all of the pH 6.3 lines did.

An experimental evolutionary study on adaptation to temporally fluctuating pH in Escherichia coli.

In this study, we use the bacterium Escherichia coli to examine evolutionary responses to environmental acidity fluctuating temporally among pH 5.3, 6.3, 7.0, and 7.8 (5,000-15 nM [H(+)]). Two experimental protocols of temporal variation were used. One group (six replicate lines) of populations evolved for 2,000 generations during exposure to a cycled regime fluctuating daily between pH 5.3 and 7.8. The other group (also in six replicate lines) evolved during exposure for 2,000 generations to a randomly shifting regime fluctuating stochastically each day among pH 5.3, 6.3, 7.0, and 7.8. Adaptation to these fluctuating acidity regimes was measured as a change in fitness relative to the common ancestor by direct competition experiments in both constant and fluctuating pH regimes. For comparisons with constant pH evolution, a group evolved at a constant pH of 5.3 and another group evolved at pH 7.8 were also tested. This study initiated the first long-term laboratory natural selection experiment on adaptation to variable acidity and addressed key questions concerning patterns of adaptation (trade-offs, specialists, generalists, plasticity, transitions, and acclimation) in temporally fluctuating environments.

Coadaptation: a unifying principle in evolutionary thermal biology.

Over the last 50 yr, thermal biology has shifted from a largely physiological science to a more integrated science of behavior, physiology, ecology, and evolution. Today, the mechanisms that underlie responses to environmental temperature are being scrutinized at levels ranging from genes to organisms. From these investigations, a theory of thermal adaptation has emerged that describes the evolution of thermoregulation, thermal sensitivity, and thermal acclimation. We review and integrate current models to form a conceptual model of coadaptation. We argue that major advances will require a quantitative theory of coadaptation that predicts which strategies should evolve in specific thermal environments. Simply combining current models, however, is insufficient to understand the responses of organisms to thermal heterogeneity; a theory of coadaptation must also consider the biotic interactions that influence the net benefits of behavioral and physiological strategies. Such a theory will be challenging to develop because each organism's perception of and response to thermal heterogeneity depends on its size, mobility, and life span. Despite the challenges facing thermal biologists, we have never been more pressed to explain the diversity of strategies that organisms use to cope with thermal heterogeneity and to predict the consequences of thermal change for the diversity of communities.

Trade-offs in thermal adaptation: the need for a molecular to ecological integration.

Through functional analyses, integrative physiology is able to link molecular biology with ecology as well as evolutionary biology and is thereby expected to provide access to the evolution of molecular, cellular, and organismic functions; the genetic basis of adaptability; and the shaping of ecological patterns. This paper compiles several exemplary studies of thermal physiology and ecology, carried out at various levels of biological organization from single genes (proteins) to ecosystems. In each of those examples, trade-offs and constraints in thermal adaptation are addressed; these trade-offs and constraints may limit species' distribution and define their level of fitness. For a more comprehensive understanding, the paper sets out to elaborate the functional and conceptual connections among these independent studies and the various organizational levels addressed. This effort illustrates the need for an overarching concept of thermal adaptation that encompasses molecular, organellar, cellular, and whole-organism information as well as the mechanistic links between fitness, ecological success, and organismal physiology. For this data, the hypothesis of oxygen- and capacity-limited thermal tolerance in animals provides such a conceptual framework and allows interpreting the mechanisms of thermal limitation of animals as relevant at the ecological level. While, ideally, evolutionary studies over multiple generations, illustrated by an example study in bacteria, are necessary to test the validity of such complex concepts and underlying hypotheses, animal physiology frequently is constrained to functional studies within one generation. Comparisons of populations in a latitudinal cline, closely related species from different climates, and ontogenetic stages from riverine clines illustrate how evolutionary information can still be gained. An understanding of temperature-dependent shifts in energy turnover, associated with adjustments in aerobic scope and performance, will result. This understanding builds on a mechanistic analysis of the width and location of thermal windows on the temperature scale and also on study of the functional properties of relevant proteins and associated gene expression mechanisms.

Changes in gene expression following high-temperature adaptation in experimentally evolved populations of E. coli.

Transcription profiling (quantitative analysis of RNA abundance) can provide a genome-wide picture of gene expression changes that accompany organismal adaptation to a new environment. Here, we used DNA microarrays to characterize genome-wide changes in transcript abundance in three replicate lines of the bacterium E. coli grown for 2,000 generations at a stressful high temperature (41.5 degrees C). Across these lines, 12% of genes significantly changed expression during high-temperature adaptation; the majority of these changes (55%) were less than twofold increments or decrements. Thirty-nine genes, four times the number expected by chance alone, exhibited moderately or highly replicated expression changes across lines. Expression changes within a priori defined functional categories showed an even greater level of replication than did individual genes. Expression changes in the phenotypically defined stress genes and adaptation functional categories were important in evolutionary adaptation to high temperature.

Evolutionary changes in heat-inducible gene expression in lines of Escherichia coli adapted to high temperature.

The involvement of heat-inducible genes, including the heat-shock genes, in the acute response to temperature stress is well established. However, their importance in genetic adaptation to long-term temperature stress is less clear. Here we use high-density arrays to examine changes in expression for 35 heat-inducible genes in three independent lines of Escherichia coli that evolved at high temperature (41.5 degrees C) for 2,000 generations. These lines exhibited significant changes in heat-inducible gene expression relative to their ancestor, including parallel changes in fkpA, gapA, and hslT. As a group, the heat-inducible genes were significantly more likely than noncandidate genes to have evolved changes in expression. Genes encoding molecular chaperones and ATP-dependent proteases, key components of the cytoplasmic stress response, exhibit relatively little expression change; whereas genes with periplasmic functions exhibit significant expression changes suggesting a key role for the extracytoplasmic stress response in the adaptation to high temperature. Following acclimation at 41.5 degrees C, two of the three lines exhibited significantly improved survival at 50 degrees C, indicating changes in inducible thermotolerance. Thus evolution at high temperature led to significant changes at the molecular level in heat-inducible gene expression and at the organismal level in inducible thermotolerance and fitness.

Rapid evolution of escape ability in Trinidadian guppies (Poecilia reticulata).

Predators are widely assumed to create selection that shapes the evolution of prey escape abilities. However, this assumption is difficult to test directly due to the challenge of recording both predation and its evolutionary consequences in the wild. We examined these events by studying natural and experimental populations of Trinidadian guppies, Poecilia reticulata, which occur in distinct high-predation and low-predation environments within streams. Importantly, in the last two decades several populations of guppies have been experimentally introduced from one type of predatory environment into the other, allowing measurements of the consequences of change. We used this system to test two hypotheses: First, that changes in predatory environments create phenotypic selection favoring changes in escape ability of guppies, and second, that this selection can result in rapid evolution. For the first test we compared escape ability of wild caught guppies from high- versus low-predation environments by measuring survival rates during staged encounters with a major predator, the pike cichlid Crenicichla alta. We used guppies from three streams, comparing two within-stream pairs of natural populations and three within-stream pairs of an introduced population versus its natural source population. In every comparison, guppies from the high-predation population showed higher survival. These multiple, parallel divergences in guppy survival phenotype suggest that predatory environment does create selection of escape ability. We tested our second hypothesis by rearing guppies in common garden conditions in the laboratory, then repeating the earlier experiments using the F2 generation. As before, each comparison resulted in higher survival of guppies descended from the high-predation populations, demonstrating that population differences in escape ability have a genetic basis. These results also show that escape ability can evolve very rapidly in nature, that is, within 26-36 generations in the introduced populations. Interestingly, we found rapid evolutionary loss of escape ability in populations introduced into low-predation environments, suggesting that steep fitness trade-offs may influence the evolution of escape traits.

Eat and run: prioritization of oxygen delivery during elevated metabolic states.

The principal function of the cardiopulmonary system is the matching of oxygen and carbon dioxide transport to the metabolic requirements of different tissues. Increased oxygen demands (VO2), for example during physical activity, result in a rapid compensatory increase in cardiac output and redistribution of blood flow to the appropriate skeletal muscles. These cardiovascular changes are matched by suitable ventilatory increments. This matching of cardiopulmonary performance and metabolism during activity has been demonstrated in a number of different taxa, and is universal among vertebrates. In some animals, large increments in aerobic metabolism may also be associated with physiological states other than activity. In particular, VO2 may increase following feeding due to the energy requiring processes associated with prey handling, digestion and ensuing protein synthesis. This large increase in VO2 is termed "specific dynamic action" (SDA). In reptiles, the increase in VO2 during SDA may be 3-40-fold above resting values, peaking 24-36 h following ingestion, and remaining elevated for up to 7 days. In addition to the increased metabolic demands, digestion is associated with secretion of H+ into the stomach, resulting in a large metabolic alkalosis (alkaline tide) and a near doubling in plasma [HCO3-]. During digestion then, the cardiopulmonary system must meet the simultaneous challenges of an elevated oxygen demand and a pronounced metabolic alkalosis. This paper will compare and contrast the patterns of cardiopulmonary response to similar metabolic increments in these different physiological states (exercise and/or digestion) in a variety of reptiles, including the Burmese python, Python morulus, savannah monitor lizard, Varanus exanthematicus, and American alligator Alligator mississipiensis.

Population density and energetics of lizards on a tropical island.

1. Population density, biomass, thermal relations and energetics of three common species of lizards (Anolis bonairensis, Cnemidophorus murinus, Gonatodes antillensis) were measured in a thornscrub community on the arid Caribbean island of Bonaire. 2. Population density and biomass estimates of these populations were 1318, 561, and 4200 individuals/ha and 4.2, 15.4, and 3.5 kg/ha, respectively. Although these densities are not exceptional for other Carribbean islands, they greatly exceed lizard densities reported for mainland communities. 3. Mean diurnal body temperatures are 33.4° C for Anolis, 40.4° C for Cnemidophorus, and 34.5° C for Gonatodes. Nocturnal temperatures average 27° C for all species. 4. Resting rates of oxygen consumption for all species were measured at naturally experienced diurnal and nocturnal temperatures. The values were used to calculate Minimal and more realistic Field Active estimates of the respiratory energy utilization of these lizard populations. 5. Minimal estimates of energy expenditure are 326, 950, and 268 kJ/(haxday) for Anolis, Cnemidophorus, and Gonatodes, and Field Active estimates are 693, 2510, and 379 kJ/(haxday), respectively. 6. These estimates greatly exceed values previously reported for other lizard populations. They also exceed reported values for the respiratory metabolism of populations of small mammals in temperate regions. 7. These values are probably not atypical of other tropical insular lizard populations, and the significance of these animals to energy flow in these communities has not generally been appreciated.

Experimental evolution and the Krogh principle: generating biological novelty for functional and genetic analyses.

August Krogh counseled the careful selection of the best subject organism on which to undertake mechanistic physiological research. But what if an organism with the desired properties does not exist? It is now within our power to engineer organisms genetically to achieve novel combinations of traits. I propose that it is a logical extension of the Krogh principle that we use biological methodologies to create novel organisms ideally suited for particular physiological studies. Transgenics may first come to mind as the method for such transformations, but here I suggest that an alternative and complementary technique for generating biological novelty is experimental evolution. The latter has several advantages, including modification of multiple characters in one experiment, the production of advantageous traits, the testing of evolutionary hypotheses, and the identification of previously unsuspected factors involved in adaptation. Three experiments are reviewed, each of which examined the evolution of different physiological characters in different environments and organisms: locomotor performance in mice, desiccation tolerance in fruit flies, and high temperature adaptation in bacteria. While diverse in experimental type and subject, all resulted in the successful production of new variants with enhanced function in their new environments. Each experiment successfully tested hypotheses concerning physiological evolution, and in each case, unanticipated results emerged, which suggests previously unsuspected adaptive pathways and mechanisms. In addition, replicate populations in each experiment adjusted to their common environments by several different means, which indicates that physiological evolution may follow diverse stochastic pathways during adaptation. Experimental evolution can be a valuable method to produce and investigate new physiological variants and traits. The choice of experimental subjects, according to the Krogh principle, is no longer limited to currently existing organisms but is open to our imaginations and our ingenuity.

Thermal acclimation effects differ between voluntary, maximum, and critical swimming velocities in two cyprinid fishes.

Temperature acclimation may be a critical component of the locomotor physiology and ecology of ectothermic animals, particularly those living in eurythermal environments. Several studies of fish report striking acclimation of biochemical and kinetic properties in isolated muscle. However, the relatively few studies of whole-animal performance report variable acclimation responses. We test the hypothesis that different types of whole-animal locomotion will respond differently to temperature acclimation, probably due to divergent physiological bases of locomotion. We studied two cyprinid fishes, tinfoil barbs (Puntius schwanenfeldii) and river barbels (Barbus barbus). Study fish were acclimated to either cold or warm temperatures for at least 6 wk and then assayed at four test temperatures for three types of swimming performance. We measured voluntary swimming velocity to estimate routine locomotor behavior, maximum fast start velocity to estimate anaerobic capacity, and critical swimming velocity to estimate primarily aerobic capacity. All three performance measures showed some acute thermal dependence, generally a positive correlation between swimming speed and test temperature. However, each performance measure responded quite differently to acclimation. Critical speeds acclimated strongly, maximum speeds not at all, and voluntary speeds uniquely in each species. Thus we conclude that long-term temperature exposure can have very different consequences for different types of locomotion, consistent with our hypothesis. The data also address previous hypotheses that predict that polyploid and eurythermal fish will have greater acclimation abilities than other fish, due to increased genetic flexibility and ecological selection, respectively. Our results conflict with these predictions. River barbels are eurythermal polyploids and tinfoil barbs stenothermal diploids, yet voluntary swimming acclimated strongly in tinfoil barbs and minimally in river barbels, and acclimation was otherwise comparable.

The molecular diversity of adaptive convergence.

To estimate the number and diversity of beneficial mutations, we experimentally evolved 115 populations of Escherichia coli to 42.2°C for 2000 generations and sequenced one genome from each population. We identified 1331 total mutations, affecting more than 600 different sites. Few mutations were shared among replicates, but a strong pattern of convergence emerged at the level of genes, operons, and functional complexes. Our experiment uncovered a set of primary functional targets of high temperature, but we estimate that many other beneficial mutations could contribute to similar adaptive outcomes. We inferred the pervasive presence of epistasis among beneficial mutations, which shaped adaptive trajectories into at least two distinct pathways involving mutations either in the RNA polymerase complex or the termination factor rho.

The energetic advantages of slug specialization in garter snakes (genus Thamnophis).

We tested the hypothesis that dietary specialization by foraging garter snakes is accompanied by increased assimilation efficiency on specialist prey items. Our comparison included two closely related garter snake species considered to be slug specialists (Thamnophis ordinoides and Thamnophis elegans terrestris), one fish specialist (Thamnophis couchii), and one diet generalist (Thamnophis elegans elegans). Our results suggest that slug specialists have an energetic advantage over non-slug-eating snakes when both eat slugs. Slug specialists T. ordinoides and T. e. terrestris both have higher assimilation and net assimilation efficiencies when eating slugs than do generalists T. e. elegans and T. couchii. The slug specialists did not experience decreased efficiency when eating fish. Therefore, there was no apparent digestive trade-off for the slug specialists when eating other prey.

Bart's familiar quotations: the enduring biological wisdom of George A. Bartholomew.

George A. Bartholomew was one of the most influential organismal biologists of the twentieth century. His insights and research were fundamental to the establishment and growth of physiological ecology and evolutionary physiology. In the process of fostering that area of science, he created a body of literature that is striking in the clarity of its thought and presentation. Here we present some of his most insightful and important quotations, group them thematically, and comment on their original context and their continuing relevance.

An experimental test of evolutionary trade-offs during temperature adaptation.

We used experimental evolution to test directly the important and commonplace evolutionary hypothesis that adaptation, increased fitness within the selective environment, is accompanied by trade-off, a loss of fitness in other nonselective environments. Specifically, we determined whether trade-offs at high temperature generally and necessarily accompany genetic adaptation to low temperature. We measured the relative fitness increment of 24 lineages of the bacterium Escherichia coli evolved for 2,000 generations at 20 degrees C and the relative fitness decrement of these lines at 40 degrees C. Trade-offs at the higher temperature were examined for their generality, universality, quantitative relationship, and historical contingency. Considering all 24 lines as a group, a significant decline in fitness was found at 40 degrees C (mean decline = 9.4%), indicating the generality of the trade-off effect. However, in a lineage-by-lineage analysis, only 15 of 24 showed a significant trade-off, and one lineage increased fitness at high temperature. Thus, although general, trade-offs were not universal. Furthermore, there was no quantitative association between the magnitude of adaptive fitness increment at 20 degrees C and fitness decline at 40 degrees C, and no effect of lineages' historical thermal environment on either their improvement at 20 degrees C or the extent of their trade-off at high temperature. We do not yet know the underlying mechanisms responsible for the trade-off, but they are sufficiently prevalent to drive a general effect. However, approximately one-third of the experimental lineages achieved low-temperature adaptation without detectable high-temperature trade-offs; therefore, it cannot be necessary that every change conferring benefit in cold environments has a negative effect on function in warmer environments.

Postprandial increase of oleoylethanolamide mobilization in small intestine of the Burmese python (Python molurus).

Oleoylethanolamide (OEA) is an endogenous lipid mediator that inhibits feeding in rats and mice by activating the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In rodents, intestinal OEA levels increase about threefold upon refeeding, a response that may contribute to the induction of between-meal satiety. Here, we examined whether feeding-induced OEA mobilization also occurs in Burmese pythons (Python molurus), a species of ambush-hunting snakes that consume huge meals after months of fasting and undergo massive feeding-dependent changes in gastrointestinal hormonal release and gut morphology. Using liquid chromatography/mass spectrometry (LC/MS), we measured OEA levels in the gastrointestinal tract of fasted (28 days) and fed (48 h after feeding) pythons. We observed a nearly 300-fold increase in OEA levels in the small intestine of fed compared with fasted animals (322 +/- 121 vs. 1 +/- 1 pmol/mg protein, n = 3-4). In situ OEA biosynthesis was suggested by the concomitant increase of N-acyl phosphatidylethanolamine species that serve as potential biosynthetic precursors for OEA. Furthermore, we observed a concomitant increase in saturated, mono- and diunsaturated, but not polyunsaturated fatty-acid ethanolamides (FAE) in the small intestine of fed pythons. The identification of OEA and other FAEs in the gastrointestinal tract of Python molurus suggests that this class of lipid messengers may be widespread among vertebrate groups and may represent an evolutionarily ancient means of regulating energy intake.