Proc. Natl. Acad. Sci. USAVol. 91, pp. 1917-1921, March 1994Evolution

Temperature acclimation and competitive fitness: An experimentaltest of the beneficial acclimation assumption

(physiologcal ecology/thermotolerance/heat shock response/bacteria/Eschenckia colt)

ARMAND M. LEROI*t, ALBERT F. BENNETT*, AND RICHARD E. LENSKI*§*Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92717; and tCenter for Microbial Ecology, Michigan State University,East Lansing, MI 48824-1325

Communicated by Walter M. Fitch, December 7, 1993

ABSTRACT Phenotypic acclimation is generally assumedto confer an advantage in the environment that stimulates theresponse. To test this beneficial aclimation assumption explic-itly, we investigated the consequences of temperature acclima-tion for the fitness ofEscherichia coliat two temperatures, 32?Cand 41.50C. Both temperatures permit growth and long-termpersistence of the genotypes in serial culture. We found thatprior acclimation to 3rC, relative to acclimation to 41.5'C,enhanced fitness at 32C, consistent with the assumption. Butprior acclimation to 41.5C actually reduced fitness at 41.50C,relative to acclimation to 32rC. Hence, the assumption thatacclimation always confers an advantage is demonstrated to befalse. Acclimation to 41.50C did, however, improve survival at500C, a lethal temperature. This protective response has beenshown to be associated with the induction of stress proteins.The reduced competitive fitness caused by acclimation at41.5°C may reflect a physiological burden associated withexpression of stress proteins when they are not needed toprevent lethal damage. Whatever the cause, acclimation to thehigher temperature decreased competitive fitness at that tem-perature.

Organisms respond to environmental change by differentmechanisms over different time scales. An individual orga-nism may acclimate phenotypically, without any change in itsgenotype. Although acclimation usually occurs within anindividual's lifetime, its effects may sometimes persist forseveral generations (e.g., ref. 1). Populations of organismsalso adapt genetically by natural selection, which requires achange in genotypic frequencies. The ability to acclimate issubject to genetic influences and may itself therefore evolve.We have been performing a series of experiments to examineboth short-term phenotypic acclimation to environmentaltemperature and long-term evolutionary modification ofthese acclimatory responses in the bacterium Escherichiacoli. Here we report experiments that measure directly thefitness consequences of phenotypic acclimation to changingtemperatures.Temperature acclimation may involve alteration of many

different physiological processes. Because acclimatory re-sponses are common and frequently homeostatic (2-4), bi-ologists have usually assumed that acclimatory responses areadaptive; i.e., that these responses benefit the organisms thatmanifest them (e.g., refs. 5-10). We call this the beneficialacclimation assumption. Stated another way, this assumptionasserts that acclimation to a particular environment gives anorganism a performance advantage in that environment overanother organism that has not had the opportunity to accli-mate to that particular environment. Indeed, some authors(e.g., ref. 10) restrict the definition of acclimation to phe-nomena having beneficial effects, although how these bene-

fits are to be judged is not stipulated. We regard the pre-sumption of benefit to be an empirical issue that needsexperimental verification.That acclimatory responses are beneficial is not axiomat-

ically true. Gould and Lewontin (11) argued that not allgenetic differences between populations or species are adap-tive and that "panglossian" explanations often ignore otherplausible explanations for such differences. Similarly, not allphenotypic changes that take place within individual orga-nisms in response to changing environments should be pre-sumed to be beneficial, and alternative explanations must beconsidered. Some phenotypic changes may be inconsequen-tial, while others may even be disadvantageous, either be-cause they have not previously been subject to selection orbecause they are functionally coupled to other more impor-tant phenotypic changes that are adaptive.The beneficial acclimation assumption has not previously

been subjected to direct test by a rigorous manipulativeexperiment, and we sought to do that in this study. It is ourview that any benefit associated with acclimation must ulti-mately be judged in terms of its impact on differentialreproduction, or Darwinian fitness. Other presumed "bene-fits" to the individual organism (e.g., increased growth rateor efficiency of energy utilization) are irrelevant evolution-arily unless they enhance fitness, because the individualorganism is only temporary. An assessment of the impact ofphenotypic acclimation on fitness, however, is often imprac-tical, because differential reproductive success is extremelydifficult to measure for most organisms. According to theKrogh principle (12, 13), a biological phenomenon should bestudied in that system in which it is most easily and rigorouslyexamined. Here, we experimentally test the beneficial accli-mation assumption, using a system in which measuring theeffect of acclimatory responses on fitness is both simple anddirect. With populations of bacteria, one can simply anddirectly measure the relative fitness of two competitors, onepossessing and one lacking a trait of interest (but otherwiseidentical), over several generations. The two competitorsshare a pool of limiting resources. The rate of change in theirrelative abundance is used to compute their relative fitnessand hence to infer the effect of the trait of interest on fitnessin the environment in which the experiment is conducted.Such experiments have been performed with E. coli for manytraits to test a variety of ecological and evolutionary hypoth-eses (see refs. 14-16 for reviews).The experiments reported here are unusual insofar as the

competitors are genetically identical (except for a neutralmarker that permits competitors to be distinguished) butdiffer in their acclimation state. The traits of interest, there-fore, are the sets of phenotypic attributes that are generated

tPresent address: Department of Molecular Genetics, Albert Ein-stein College of Medicine, 1300 Morris Park Avenue, Bronx, NY10461.§To whom reprint requests should be addressed.

1917

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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by acclimation to different environments. These differencesenable us to test directly the beneficial acclimation assump-tion. We do so by placing separate populations of a singlebacterial clone into two different thermal environments: 320Cand 41.50C. The bacteria grow in those environments forseveral generations, thereby allowing acclimation to thesedifferent temperatures. We then observe the growth of themarked lines in competition, so that fitness is assessed foreach acclimation state in both its "own" environment (i.e.,the temperature to which it has acclimated) and in the otherenvironment (i.e., the temperature to which it has not accli-mated). According to the beneficial acclimation assumption,the bacteria acclimated to each temperature should havegreater reproductive success in competition at the sametemperature than those acclimated to the different tempera-ture. In a companion study (30), we examine the evolution ofthese acclimatory responses during long-term propagation inan environment consisting of a daily alternation betweenthese two temperatures.

MATERIALS AND METHODS

Bacterial Strains. These experiments were conducted witha genotype of E. coli B isolated from a population that hadbeen maintained for 2000 generations at 370C (17). During this

time, the population was propagated by serial dilution in a

glucose-limited minimal medium, with %6.7 cell generationsper day. This genotype, designated REL1206, is unable toutilize arabinose for growth (Aral, but an Ara+ mutant(REL1207) was obtained by plating cells on minimal arabi-nose medium (18). These two variants, Ara+ and Ara-, arereadily distinguished by colony coloration on tetrazolium/arabinose (TA) indicator agar, allowing their abundances tobe enumerated simultaneously in competition experiments(19). The effective neutrality of the marker state in theglucose-limited minimal medium was verified at the temper-atures used in these experiments (ref. 18; see Table 1, rows1 and 2). These marked clones also served as the foundinggenotypes for a separate experiment on genetic adaptation todifferent thermal regimes (18, 20-22).Medium and Cultpre Conditions. Experiments were per-

formed in DM medium (23) supplemented with 0.002 pg ofthiamin hydrochloride and 25 pg of glucose per ml. Culturesconsisted of 10 ml of medium in 50-ml Erlenmeyer flasks,placed in shaking incubators (120 rpm) at the indicatedtemperatures. These conditions were identical, but for tem-perature, to the historical environment of the genotype usedin this study (see above).

Fitness Assays and Definition. Fitness assays consisted oftwo sequential steps, acclimation and competition. In theacclimation step, two bacterial populations were separatelygrown for 24 hr in DM medium at either 32.00C or 41.50C(±0.50(). During this step, the bacteria underwent severalcell generations and, in so doing, exhausted the limitingglucose. The bacteria also may have undergone phenotypicchanges caused by their exposure to one temperature or theother; these acclimatory responses may have occurred duringgrowth, stationary phase, or both. In the competition step,aliquots of the two separately acclimated and reciprocallymarked bacterial populations were simultaneously diluted(1:200 each) into a common flask of fresh DM medium,whereupon an initial sample was plated on TA indicator agar.This mixed population was then incubated for 24 hr at either320C or 41.50C, at which time a final sample was plated on TAagar. During the competition step, the mixed population alsoreached stationary phase after exhausting the available glu-cose.

In another experiment, the acclimation and competitionsteps were carried out in a series of flasks placed along athermal gradient from 41.30C to 42.20C (+0.20C), which was

stably maintained in a shaking incubator (21). Previousresults indicated that fitness measurements involving thegenotype used in this study are quite sensitive to even slightdifferences in temperature in this range (21).The relative fitness, W, ofthe differentially acclimated (and

reciprocally marked) populations can be calculated from thechange in their relative abundances between the initial andfinal samples in the competition step:

W1=l4200oe32/N'2)where N indicates population density, subscripts 32 and 42denote acclimation to lower and higher temperatures, respec-tively, and superscripts i and f indicate initial and finalsamples, respectively. Thus defined, W is equivalent to aratio of the realized Malthusian parameters of the two pop-ulations during competition with one another in the experi-mental environment (17). A value ofW = 1 indicates that thetwo competitors had equal fitness. Unequal fitness mayreflect a difference in survival and/or reproductive successduring any phase of the competition step (i.e., lag, growth,and stationary phases).Note that we express fitness of the population acclimated

to 41-420C relative to the population acclimated to 32(C,irrespective ofwhether the competition step is at 320C or thehigher temperature. Therefore, according to the beneficialacclimation assumption, we expect W < 1 when competitiontakes place at 320C and W > 1 when competition is at41-420C.

Thermotolerance. To examine differential thermotoleranceconferred by acclimation, death rates for bacteria acclimatedto 320C and 41.50C were measured at a high temperaturelethal to this clone (21). Following the same protocol used forthe acclimation step of the fitness assay, stationary-phasebacterial populations were transferred directly into a waterbath maintained at 50 ± 1.00C. Samples from these popula-tions were obtained after 0, 15, 30, 60, 90, 120, 180, and 240min and plated to determine the number of viable bacteria(colony-forming units). The natural logarithm of this numberwas regressed against time spent at 500C to calculate thedeath rate of the population.

RESULTSDuring competition at 320(C, bacteria that were acclimated to320C had a clear fitness advantage relative to those that wereacclimated to 41.50C, irrespective of the marker state of theclones acclimated to their respective temperatures (Table 1,rows 3 and 4). This result therefore accords well with the

Table 1. Effect of acclimation temperature on competitive fitnessat 320C and 41.50CAcclimationtemperature, Competition

OC temperature, FitnessAn+ Ara4 OC n Mean SE Pt

32 32 32 10 1.006* 0.013 0.62942 42 42 10 0.993* 0.012 0.58832 41.5 32 15 0.938§ 0.012 <0.00141.5 32 32 15 O.903§ 0.011 <0.00132 41.5 41.5 15 0.840§ 0.012 <0.00141.5 32 41.5 15 0.813§ 0.012 <0.001*Ara+ and Ara- refer to genotypic variants with alternative arabi-nose-utilization marker states.

tTwo-tailed probability of rejecting the null hypothesis that meanfitness equals 1, based on t test with n - 1 degrees of freedom.*Mean fitness of Ara+ relative to Ara- (data from ref. 18).Mean fitness of the 41.50C-acclimated variant relative to the 320C-acclimated variant.

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Table 2. Effect of acclimation temperature (320C or 41.50C) ondeath rates at lethal high temperature

Period at 500C Death rate at 500C, min-'during which death ratewas measured, min 320C 41.50C t P

0-240 0.027 0.016 11.60 <0.0010-60 0.006 0.005 1.42 0.93190-240 0.038 0.023 10.32 <0.001

2 8 0.

-_&"W.W_,W _._&I _-_ __-

Death rates were calculated by linear regression oflog(proportion.0 [- of viable cells) versus time. Regressions were performed separatelyI J

for each replicate using all eight time points, and then using only the41.2 41.4 41.6 41.8 42.0 42.2 first four or last four points. Six independent replicates were per-formed at each assay temperature. P values are one-tailed probabil-

Temperature, 0C ities of rejecting the null hypothesis of no effect of acclimationtemperature, based on t test with ni + n2 - 2 = 10 df.

FIG. 1. Effect on competitive fitness of acclimation to hightemperatures relative to acclimation at 320C. The experimentaltemperature was applied during the acclimation step (to one com-petitor only) as well as during the competition step of the fitnessassay. A relative fitness (W) < 1 indicates that prior acclimation tothe high temperature had a deleterious effect on fitness at thattemperature, relative to acclimation at 320C.

beneficial acclimation assumption.According to the beneficial acclimation assumption, it is

also expected that bacteria acclimated to 41.50C will be morefit than bacteria acclimated to 320C (i.e., W > 1), whencompetition takes place at 41.50C. In fact, the opposite resultwas obtained (Table 1, rows 5 and 6): the 320C-acclimatedcells were significantly more fit than their 41.50C-acclimatedcounterparts in competition at 41.50C. Again, the result isindependent of the marker state of the differentially accli-mated competitors. Therefore, acclimation to high tempera-ture actually reduced competitive fitness at high temperature,contrary to the beneficial acclimation assumption.To examine the effect of high-temperature acclimation on

competitive fitness in more detail, we measured the fitnessconsequences of acclimation and subsequent competitionalong a thermal gradient from 41.30C to 42.20C, relative to acompetitor conditioned at 320C (Fig. 1); 42.20C is at the upperlimit for population persistence of this genotype in the serialculture regime used in this study (21). Between 41.30C and41.70C, acclimation to these temperatures was moderatelydisadvantageous relative to acclimation at 320C (mean W =0.924- SE = 0.018. P < 0.001. based on two-tailed t test with

n - 1 = 23 df), consistent with the results in Table 1. Between41.9"C and 42.20C, the deleterious effects of acclimation tohigh temperature became even more severe, so that in somereplicates, the high-temperature-acclimated population hadnot even begun to grow by the time that the 320C-acclimatedcells consumed the limiting glucose (i.e., W 0). Evidently,the deleterious effect of high-temperature acclimation oncompetitive fitness has a strong thermal dependence. How-ever, it is not caused by the lethality of the high temperatureper se, since the genotype used in these experiments cansustain itself indefinitely by sufficient growth to offset serialdilution and death even at these higher temperatures (21).Although acclimation to 41.50C unexpectedly reduced

competitive fitness at that temperature, such acclimation didconfer thermotolerance to even more extreme temperatures.The 41.50C-acclimated bacteria had significantly lower deathrates when subsequently exposed to 500( than did bacteriaacclimated to 320( (Fig. 2 and Table 2). Indeed, a reductionin death rate at a lethally high temperature following expo-sure to a high but permissive temperature fulfills the opera-tional definition of the heat shock response (24, 25). It istherefore clear that some physiological adjustments to hightemperature did occur during the acclimatory phase at41.50C, even though these adjustments did not improvecompetitive fitness at that temperature.

DISCUSSIONThe beneficial acclimation assumption predicts that bacteria

0 , acclimated to a particular temperature should be significantly8* 8 more fit at that temperature than bacteria acclimated to some

other temperature. One ofour two reciprocal acclimation and0 competition experiments gave that result: 320C-acclimated

-2- bacteria were more fit than 41.50C-acclimated bacteria insubsequent competition at 32°(. However, the 32(C-acclimated bacteria also were more fit than the 41.5(C-acclimated bacteria at 41.50C, contrary to the beneficial

-4- acclimation assumption.o We must therefore reject the generality of the beneficial

acclimation assumption: a fitness benefit may or may notresult from acclimation. In fact, fitness in a particular envi-ronment may actually decrease because ofacclimation to thatenvironment. A single negative example is sufficient to falsifythe generality of a hypothesis, and we found such an excep-

-8- tion in the first explicit test of this one.0 60 120 180 240 How can we be sure that these effects-both beneficial and

adverse-are caused by phenotypic acclimation rather thanTime, mmn genotypic adaptation? We have previously studied the ge-

2. Effect ofacclimation temperature on death rate at a lethal netic adaptation of this same bacterial strain to these sameture, 500(2. Open circles represent cells acclimated at 320(; thermal regimes (18, 20). At 320C, no significant genetic

rircles represent cells acclimated at 41.5(2. Each point repre adaptation was observed for >400 generations (60 days),-he mean of six replicate assays; for statistical analysis, see whereas the beneficial acclimatory effect reported here is2. manifest in 6-7 generations (1 day). At 41.50C, we observed

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genetic adaptation within 200 generations (30 days), but notwithin 100 generations. Again, the acclimatory effect of41.50C is manifest in only 1 day, and it has the opposite effectofgenetic adaptation, leading to a decrement in performance.The fitness differences reported here are due to phenotypicacclimation rather than genetic adaptation.How are the fitness effects associated with acclimation

manifest during competition? The serial dilution regime usedin this study encompasses a series of more or less distinctphases of population growth. A competition experiment isbegun by placing two competitors, taken from stationary-phase cultures, into fresh medium. The bacteria begin aperiod of metabolic activation and synthesis prior to thecommencement of cell division (lag phase). They then entera period of rapid cell division (growth phase). Eventually thebacteria exhaust the limiting supply of glucose so that theycannot grow and may experience some death (stationaryphase). The transitions between these phases need not besharp: for example, populations may comprise both growingand nongrowing cells during the early or late phases ofgrowth. Higher relative fitness in a competition experimentmust be mediated through better performance in one or moreof these phases of population growth dynamics. That is, ashorter lag phase, a higher rate of growth, or reducedmortality during starvation will, alone or in combination,increase reproductive success and hence competitive fitness.From an evolutionary perspective, it is immaterial which of

these phases is the locus of differential performance. Suchdifferences may have straightforward physiological or bio-chemical explanations, but their simplicity does not detractfrom their profound fitness effect. We have not examinedwhich population phases are responsible for the fitnessdifferences observed in these experiments. Had we per-formed similar experiments using bacterial populations ingrowth phase only, the beneficial acclimation assumptionmight have been rejected simply because bacteria acclimatedto 320C continued to grow at the rate characteristic of thattemperature for some time after they were shifted to thehigher temperature. However, the unexpected disadvantageof acclimation to high temperature cannot be caused bycontinuation of more rapid growth by the population accli-mated to 320C, because neither population was growing at thestart of the competition step (each having reached stationaryphase during its separate acclimation treatment). We suspectthat a shorter lag phase for the 320C-acclimated bacteria wasat least partly responsible for their greater fitness at bothtemperatures, because an acclimatory response to a previousenvironment will eventually wear off in a new environment.However, we cannot exclude the possibility that the accli-mation treatments also influence subsequent growth rate oreven survival in stationary phase.How can the observed loss offitness caused by acclimation

to high temperature be reconciled with the many studies ofbacteria as well as eukaryotic species (5-10, 24-26) that havereported improved functional capacities resulting from high-temperature acclimation? In these other studies, the func-tional capacities usually considered are not growth rate orfitness at the permissive high temperatures that induce theacclimatory response, but rather death rate under moreextreme and typically lethal conditions (e.g., increased crit-ical thermal maxima). We also observed a reduction in deathrate, measured at a lethally high temperature, followingacclimation of the bacteria to a permissive high temperature(Fig. 2 and Table 2), and hence our results are in factconcordant with these other studies.

It is possible that both the loss of competitive fitness at41.5A C and the improved survival at 50(2C of the 41.5CAacclimated bacteria are consequences of a single physiolog-ical process. One candidate is the heat shock response, whichin E. coli entails the induction of an assemblage of stress

proteins at high, but sublethal, temperatures (24-29). Theexpression of these proteins is associated with reducedmortality at lethal temperatures, although the precise causalrelationship between these phenomena is unclear (25, 26, 28,29). Expression of at least some heat shock proteins is anincreasing function of temperature in E. coli, and 420C issufficient for their induction (24, 25). The competitive dis-advantage of cells acclimated to 41.50C and slightly highertemperatures might therefore reflect the physiological burdenassociated with the elevated expression of stress proteinswhen they are not actually needed for protection againstlethal temperature. But heat shock proteins are not the onlyplausible explanation for the decreased fitness of the 41.50C-acclimated group. Another possibility, for instance, is themore rapid degradation of some growth-essential proteinduring stationary phase at higher temperatures, which couldextend the subsequent lag phase upon transfer to freshmedium at both low and high temperatures. However, in-duction of the heat shock response could simultaneouslyaccount for both the fitness decrement at 41-420C and theimproved thermotolerance at 50(C.Whatever the precise physiological basis of these effects,

the bacteria clearly undergo significant phenotypic changesduring acclimation to high temperatures. However, thesephenotypic changes are beneficial only in other, even hotter,environments, and they are actually disadvantageous at thegrowth-permissive temperatures that stimulate them. In thiscase, acclimation seems to take the form ofhdging a bet thatthe environment will continue to become even hotter, ratherthan remaining at a high but tolerable temperature. Perhapsdefense against further temperature increments has beenmore important in the evolutionary history of these bacteriathan maintaining competitive ability in stressful but nonlethaltemperatures. In this regard, it is interesting that thermotol-erance (i.e., improved survival upon exposure to 50(C)declines significantly when these bacteria are serially prop-agated at 41.5°C for up to 14 days (A. Cullum, A.F.B., andR.E.L., unpublished data). Such a trend suggests that theheat shock response is turned down when a hih but growth-permissive temperature is sustaine4, rather than being fol-lowed by a lethally high temperature.Although 320C and 420C are symmetric around the tem-

perature (370() at which these bacteria had been propagatedfor the preceding 2000 generations, there are several objec-tive criteria by which 420C may be considered the morestressful temperature (22). (i) Relative to 37T, 420C reducesboth maal growth rate and yield, whereas 320C reducesonly the former. (ii) Forty-two degrees Celsius is within-0.5°( of the critical thermal limit for extinction of thebacterial population under our standard culture conditions,whereas 320( is >100C away from both the upper and lowerthermal limits. (iii) Whereas 420C induces expression ofstress proteins that mediate a protective response, 320C doesnot. Our a priori expectation was that acclimation to astressful environment would be more important than accli-mation to a nonstressful environment for competitive fitnessin the respective environments. However, the opposite resultwas obtained. Whether this unexpected result is itself gen-eralizable remains to be seen. But in light of our results andthe possible explanations for them, it now seems plausible tous that acclimation to a stressful but nonlethal environmentmay often be disadvantageous to the organism in that envi-ronment. That is, induction of a protective response to alethal environment may require that an organism be reason-ably vigorous, and so, such responses may need to beinduced before conditions become too damaging. However,there are physiological costs associated with the inductionand expression of the stress response, and these costs maytranslate into reduced reproductive success if the environ-ment does not progress to the damaging state. Under this

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interpretation, the acclimatory response of the bacteria tohigh temperature would in fact be beneficial in certainsequences of environments, even if not the one used in ourexperiments. This possibility raises interesting questionsabout the sequences of thermal environments that E. colitypically experiences in nature.

It is important to emphasize that although 41.50C is stress-ful to the bacterial genotype used in these experiments, it isnot lethal: in fact, one can maintain growing and viablepopulations indefinitely at temperatures up to about 42.20C(21, 22). That is, fitness does not steadily decrease over timeand populations do not eventually go extinct at this temper-ature. Therefore, the depression in competitive fitnesscaused by acclimation to high temperature is not simply aninconsequential character that is correlated with lethality,such as a lobster turning red when placed in boiling water.We do not know whether our results are specific to these

temperatures and this particular bacterial genotype orwhether similar results might be obtained for the effects ofthermal acclimation with other genotypes and species andeven for acclimation to other types of environmental change.Only a mixture of further experimental and comparativestudies can reveal the breadth of applicability of theseobservations. We have, however, disproved the generality ofthe beneficial acclimation assumption. Whatever the precisemechanistic basis, our results demonstrate that phenotypicchanges arising during acclimation to a particular environ-ment cannot automatically be assumed to increase fitness inthat same environment. Moreover, the phenotypic changesthat occur during acclimation may have dramatically differ-ent fitness consequences depending on which components offitness are assayed and in what environment the assay isperformed.

We thank Sue Simpson and John Mittler for help in the laboratoryand useful discussions. We thank Jim Bull, Walter Fitch, Ray Huey,and anonymous reviewers for their comments on the manuscript.This work was funded by National Science Foundation GrantsDEB-9249916 (to R.E.L.), IBN-9208662 (to A.F.B. and R.E.L.), andBIR-9120006 (Center for Microbial Ecology).

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