406 An Experimentl Evolutionry Study on Adpttion to Temporlly Flututing ph in Esherihi oli Brdley S. Hughes* Alistir J. Cullum Alert F. Bennett Deprtment of Eology nd Evolutionry Biology, University of Cliforni, Irvine, Cliforni 92697-2525 Aepted 3/12/2007; Eletronilly Pulished 5/2/2007 ABSTRACT In this study, we use the terium Esherihi oli to exmine evolutionry responses to environmentl idity flututing temporlly mong ph 5.3, 6.3, 7.0, nd 7.8 (5,000 15 nm [H ]). Two experimentl protools of temporl vrition were used. One group (six replite lines) of popultions evolved for 2,000 genertions during exposure to yled regime flututing dily etween ph 5.3 nd 7.8. The other group (lso in six replite lines) evolved during exposure for 2,000 genertions to rndomly shifting regime flututing stohstilly eh dy mong ph 5.3, 6.3, 7.0, nd 7.8. Adpttion to these flututing idity regimes ws mesured s hnge in fitness reltive to the ommon nestor y diret ompetition experiments in oth onstnt nd flututing ph regimes. For omprisons with onstnt ph evolution, group evolved t onstnt ph of 5.3 nd nother group evolved t ph 7.8 were lso tested. This study initited the first long-term lortory nturl seletion experiment on dpttion to vrile idity nd ddressed key questions onerning ptterns of dpttion (trde-offs, speilists, generlists, plstiity, trnsitions, nd limtion) in temporlly flututing environments. Introdution In our ever-hnging world, evolutionry physiologists re interested in understnding how orgnisms funtion in vrile environments nd how popultions of orgnisms dpt evolutionrily to environmentl flutution. Although there is strong empiril support for the rgument tht environmentl heterogeneity does indeed represent generl explntion for * Corresponding uthor; e-mil: hughes@ui.edu. Physiologil nd Biohemil Zoology 80(4):406 421. 2007. 2007 y The University of Chigo. All rights reserved. 1522-2152/2007/8004-6155$15.00 diversity in nturl systems, there re omprtively few dt on the response of nturl seletion to environmentl vriility (Kssen 2002). A ommon hypothesis is tht nturl seletion within vrile environments will rete generlists, while speilists will evolve in environments tht sty more onstnt in spe nd time (e.g., Levins 1968; Huey nd Sltkin 1976; Huey nd Hertz 1984; Lynh nd Griel 1987). The use of temporl vrition in seril trnsfer regimes used to propgte teril popultions in th ulture is nlogous to type of sesonl environment nd llows for the empiril study of the environmentl onditions promoting the evolution of speilists versus generlists (e.g., Riney et l. 2000). Experimentl evolution is n effetive lortory tehnique for exmining dpttion to ioti environments, sine it ffords rigorous regultion nd ontrol of environmentl onditions. Mny replite popultions n e exposed simultneously to novel stressful environments, nd evolutionry hnges n e oserved susequently over numer of genertions (Rose et l. 1996; Bennett nd Lenski 1999; Bennett 2003). Experimentl evolutionry methods hve previously een pplied to studying dpttion to temperture (e.g., Huey 1991; Huey et l. 1991; Bennett et l. 1992), while here we expnd the sope of experimentl evolution to inlude idity s n environmentl vrile. The ph ws seleted s n experimentl vrile euse of the importne of this environmentl stressor to enteri teri suh s Esherihi oli. While mny orgnisms my not experiene hnging environmentl idity nd lklinity t stressful levels, vrition in ph is iologilly signifint environmentl stress for enteri teri. The nturl history of these teri suggests tht they my spend hundreds or thousnds of genertions in the reltively neutrl environment of the olon, puntuted y rief exposures to extreme idity during pssge through the stomh, followed y modest lklinity in the smll intestine during oloniztion of new host. With modern sewge hndling (or mishndling), enteri teri my lso experiene periods of exposure to the oen, with ph ner 8.0, efore infeting new host. Thus, over mroevolutionry timesles, E. oli hve experiened ountless itertions of ph flutution etween idi nd si environments, nd tolerne to high- nd low-ph environments s well s to rpid trnsitions etween these onditions is fundmentl to the survivl of this speies. Known mehnisms of id nd lkline resistne in E. oli were previously desried y Hughes et l. (2007). Physiologilly, E. oli re often onsidered neutrophiles tht grow est
Evolution in Temporlly Flututing ph in Esherihi oli 407 t neutrl ph, lthough they n lso grow in moderte id or se. The growth of E. oli t ph 5.0 lowers ytoplsmi ph y 0.6 units (Hikey nd Hirshfield 1990), whih n lter tlyti properties of regultory enzymes nd memrne funtions (Somero 1986). Different physiologil ptterns in growth in the presene of glutmte or rginine revel tht E. oli n reverse the eletril memrne potentil to mke the inside of the ell positively hrged, whih is the sme strtegy tht is used y vrious idophiles to void ellulr dmge through protein denturing, depurintion of DNA, nd dmge to memrnes (Rihrd nd Foster 2004). During lkline stress, the peptidoglyn lyer of grm-negtive teri, wekened y high ph, my e less ple of preventing the ytoplsmi memrne from ursting in E. oli t ph 10 (Mendon et l. 1994). Although muh less is known out lkline stress resistne, it my involve protein repir of l-isosprtyl protein roxyl methyltrnsferse (Hiks et l. 2005). While E. oli my e ple of survivl through rief exposure to extreme idity or lklinity, it is inple of growing during suh onditions. The ph limits for growth of E. oli K-12 re etween 5.0 nd 9.0 (Zilerstein et l. 1984) nd etween 4.8 nd 8.4 for our nestrl strin of E. oli B (A. J. Cullum, unpulished dt). In the sttionry phse, however, E. oli n survive environments elow ph 2, tht is, more thn 1,000 times [H ] eyond its lower growth limit, while lethl se stress ours t ph 10, only single ph unit eyond the upper growth limit. In this evolution experiment, we onfined our seletive environments within the growth limits of these teri, etween ph 5.3 nd 7.8, providing signifint rnge of idi exposures (more thn 300 times [H ]) tht still permit dequte growth for regenertion in seril dilution ulture. This study initited the first long-term lortory nturl seletion experiment on dpttion to vrile ph nd ddressed severl questions onerning ptterns of dpttion in temporlly flututing environments. Temporl vrition of ph in this evolutionry system llowed us to ddress multiple hypotheses relting to evolution in vrile environments. The ssertion tht the redth of dpttion evolves to mth the degree of environmentl vrition (Vi nd Lnde 1985; Futuym nd Moreno 1988; Sheiner 1993) ws exmined through ssessment of the differentil ptterns of fitness trde-offs in dpttion to flututing versus onstnt id environments. Trde-offs re often ssumed to e hrteristi of nihe speilists, while lk of trde-offs is thought to e hllmrk of nihe generlists. It hs een shown empirilly, however, tht the ommon theoretil expettion of the generl neessity of trde-offs is not universl djunt of dpttion to temperture (e.g., Bennett et l. 1992; Bennett nd Lenski 1993; Mongold et l. 1996; Portner et l. 2006), so n investigtion of the presene of trde-offs during dpttion to onstnt nd vrile idity is of interest nd provides n dditionl test of this onept. Evolution of generlists in vrile environments is sometimes hypothesized to involve n dptive ost ( jk-of-ll-trdes, mster of none ; see Huey nd Hertz 1984; Joshi nd Thompson 1995; Vi et l. 1995; Fry 1996; Whitlok 1996; DeWitt et l. 1998). This potentil ost ws diretly ddressed in our system y ompring the verge fitness of ph speilists with tht of groups evolved in environments of vrile ph. The hypothesis tht evolution in temporlly vrying environment inreses phenotypi flexiility for mking trnsitions (e.g., Levins 1968, 1969; Feder 1978; Tsuji 1988) ws not supported in therml evolution experiments (e.g., Leroi et l. 1994). This hypothesis ws reexmined here in the sme teril system ut with ph s the environmentl vrile. Finlly, in regrd to limtion, numerous therml studies of enefiil limtion (Huey nd Kingsolver 1989; Rie nd Bzzz 1989; Kres nd Loeshke 1994; Leroi et l. 1994; Kingsolver 1995; Shmitt et l. 1995; Zmudio et l. 1995; Dudley nd Shmitt 1996; Huey nd Berrign 1996; Bennett nd Lenski 1997; Woods 1999; Giert et l. 2001; Gilhrist nd Huey 2001; Huey et l. 2002; Grlnd nd Kelly 2006; Terlnhe nd Chown 2006) hve demonstrted mix of results, inditing lk of generlity for the enefit of limtion. The seemingly prdoxil result of Leroi et l. (1994) tht limtion enefit delines during evolution in vrile environment my e less ounterintuitive if one onsiders how teri in flututing environment must often ompete without the enefit of n limtion period. Suh relentless seletion for the ility to trnsition to new environments without the id of limtion might elipse the dptive importne of mintining n ility to enefit from limtion. We tested the hypothesis tht propgtion in vrile environments tully results in the evolution of limtion insensitivity when ompred with evolution in onstnt environments. In ontrst, enefiil limtion under historil onditions plys onsistent role in therml environments in E. oli (Bennett nd Lenski 1997), nd we lso exmined this feture in ph evolution. Here we used mesurements of reltive fitness from four groups, eh onsisting of six independent lines, with eh evolved in one of the following onditions: onstnt id (A), onstnt se (B), rndomly flututing (R), or yling (C) ph. After either 1,000 genertions of evolution in onstnt ph or 2,000 genertions of evolution in flututing ph, these groups were ssessed for their evolved performne in four different environments to (1) determine the extent of dpttion of the vrious groups in eh ph regime ondition, (2) hrterize trde-off ptterns ssoited with the evolution of generlists or speilists in temporlly flututing nd onstnt ph regimes, (3) ompre evolutionry fitness responses in rndomly versus regulrly yled ph regimes, (4) ssess the extent to whih there ws dpttion to trnsitions etween the id nd lkline onditions rther thn only dpttion to stti ph onditions, nd (5) test the enefit of limtion preeding fitness ompetitions.
408 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Mteril nd Methods Bteri The teril study system in this experiment hs een previously desried (Bennett et l. 1992; Hughes et l. 2007). Briefly, the nestrl strin of Esherihi oli used to found the 24 lines of this study is sexul, prototrophi (ut originlly inple of growing on l-rinose [Ar ]), nd sent of plsmids or funtionl teriophges, nd it ws isolted s lone from one of 12 popultions tht were prt of n erlier experimentl evolutionry study (Lenski et l. 1991). During this erlier study, our nestrl linege ws llowed to evolve for 2,000 genertions (300 d) in stndrd lortory environment of ph 7.0 nd temperture of 37 C in regimen of dily seril dilution of 1 : 100 (0.1 ml in 9.9 ml) in stndrd Dvis miniml medium (DM; Crlton nd Brown 1981). Most of the fitness inrese during this lortory propgtion hppened within the first 1,000 genertions, so it ws inferred tht fter 2,000 genertions in this environment our nestor ws lredy reltively well dpted to the si ulture onditions of the evolutionry regimes, exept for the differenes in ph levels we would mnipulte in this experiment. This ssumption ws verified in susequent experiments, in whih fitness inresed minimlly during susequent 2,000 genertions of ontinued exposure to this environment (Bennett et l. 1992; Hughes et l. 2007). Two mrker sttes differing in ility ( ) to utilize the sugr rinose llow visul differentition etween two lineges ompeting diretly ginst eh other in the sme flsk, when they re oppositely mrked (Lenski et l. 1991). Hughes et l. (2007) verified the neutrlity of this mrker gene over the rnge of idities used in this study. The two distint (, ) nestrl genotypes s well s the lineges we evolved were stored t 80 C nd ould e revived t ny time for use in future experiments. Culture Conditions Culture onditions of our evolution experiments were identil to those previously desried (Bennett et l. 1992; Hughes et l. 2007), exept for the modifition of ph in the medi. Temperture ws mintined t onstnt 37 C. s were revived from the freezer y initil inoultion into Luri roth, ut otherwise propgtion ws in DM, modified y vrying proportions of the mono- nd disi potssium phosphte present in the uffer system. The DM mixtures used for our experimentl regimes hd H onentrtion levels of 5,000 nm [H ] (ph 5.3), 500 nm [H ] (ph 6.3), 100 nm [H ] (ph 7.0), nd 15 nm [H ] (7.8 ph), ll 0.1 ph units. Although in nonuffered medi t high ell densities teril metolism will shift ph signifintly, the low densities nd uffered medi of our experiments restrited suh ph shifts to no more thn 0.1 ph unit. Propgtion proeeded through dily seril trnsfer of 0.1 ml of eh ulture into 9.9 ml of fresh medium, uffered to the pproprite ph levels, nd these dily trnsfers effetively diluted popultion ounts y 100-fold, so tht the teril popultion grew y 100-fold ( 6.64 genertions) to regin its sttionry phse density. Competition ssys were performed through differentil enumertion of olonies grown on TA gr pltes, ontining triphenyltetrzolium hloride nd rinose, with inution t 37 C. Evolving s Six lines were reted within eh of the four evolutionry ph regime groups for totl of 24 experimentl lines. The C group ws lternted dily for 300 d ( 2,000 genertions) etween ph 5.3 nd 7.8, spending 24 h t 5,000 nm [H ] (ph 5.3) nd the next 24 h t 15 nm [H ] (ph 7.8) for n rupt shift of more thn 300 times [H ]. The R group ws stohstilly hnged dily for 300 d ( 2,000 genertions) mong four levels of ph (5.3, 6.3, 7.0, nd 7.8). Thus, the C group evolved for totl of 150 d in ph 5.3 nd 150 d in ph 7.8, nd the R group spent rndom mount of time, stohstilly pprohing totl of 150 d in idi onditions (ph 5.3 nd 6.3) nd 150 d in neutrl or si onditions (ph 7.0 nd 7.8). The A nd B groups used in these experiments were eh propgted in onstnt ph onditions for 150 d ( 1,000 genertions), with ph 5.3 for the A group nd ph 7.8 for the B group. We hose to use these 1,000 genertion lines for the A nd B groups euse we wnted to e le to ddress the question of whether the C nd R groups ould dpt s quikly (hnge in fitness per genertion) to eh ph s the A nd B groups ould to their respetive ph s, despite the potentil pleiotropi hllenges the C nd R groups fed in hving to dpt to dditionl ph environments t the sme time. Thus, lthough the C nd R groups hve spent more totl genertions in the generl experimentl environment thn the A nd B lines, the genertions spent in ny given environment (either id or se) re the sme for ll groups. (The A nd B groups were lso llowed to evolve for n dditionl 1,000 genertions in their respetive environments, nd dpttion of these groups fter 2,000 genertions hs een reported previously [Hughes et l. 2007].) In order to determine whether our hoie of the 1,000- versus 2,000-genertion smples might hve influened the outome of the omprisons in this study, we ompred the 1,000- nd 2,000-genertion reltive fitness vlues for eh of the two groups in oth ph 5.3 nd 7.8 onstnt onditions. In no se did the two genertions differ signifintly, nd so we elieve tht the nlyses reported here would hve hd similr outomes regrdless of the genertion of the A nd B groups used. Hlf of the lines in eh group originted from the Ar form of the nestor, whih is inple of rinose use, nd the other hlf from the rinose-utilizing Ar form isolted s spontneous muttion vi high-volume TA plting (Lenski et l. 1991); these two forms of the nestor re otherwise
Evolution in Temporlly Flututing ph in Esherihi oli 409 Tle 1: Evolved fitness of groups tested in different ph regimes Group nd Test Regime Men SE Reltive Fitness P (Two Tiled) C: Constnt ph 7.8 1.147.022.001 Constnt ph 5.3 1.188.009!.001 ph 5.3 r 7.8 1.143.028.004 ph 7.8 r 5.3 1.108.013!.001 ph 5.3 r 7.8 r 5.3 1.120.008!.001 R: Constnt ph 7.8 1.035.021.156 Constnt ph 5.3 1.142.014!.001 ph 5.3 r 7.8 1.073.030.060 ph 7.8 r 5.3 1.103.016.001 ph 5.3 r 7.8 r 5.3 1.088.022.011 B: Constnt ph 7.8 1.010.033.782 Constnt ph 5.3.809.062.027 ph 5.3 r 7.8 1.175.033.003 ph 7.8 r 5.3.824.065.042 ph 5.3 r 7.8 r 5.3.972.040.518 A: Constnt ph 7.8.708.144.098 Constnt ph 5.3 1.276.030!.001 ph 5.3 r 7.8.098.233.012 ph 7.8 r 5.3.939.090.527 ph 5.3 r 7.8 r 5.3.704.125.063 Note. Vlues shown in old represent the ondition(s) in whih the group evolved. Men SE vlues of fitness reltive to the nestor sed on six replite lines in eh experimentl group. Two-tiled proilities were lulted using the t distriution with n 1 p 5 df; the null hypothesis is tht the men fitness equls 1. identil nd show no fitness differenes in DM. Individul lines were identified y the revited group nme, or to identify the mrker stte, nd replite numer. As n exmple, the C group is omprised of the C 1, C 2, C 3, C 1, C 2, nd C 3 lines. Smples of eh evolving popultion were plted every 100 or 200 genertions, nd single olony ws rndomly seleted. These smpled lonl isoltes were then grown to high density in glyerol-ontining suspension medium nd frozen t 80 C for storge to llow lter revivl for experimentl nlysis. (Mixed popultion smples ontining the entire geneti diversity within line were lso frozen nd stored for future experimenttion with the full diversity of the popultion ut were not ssessed in this study.) Given the initil homogeneity of the lines nd the sexulity of the terium, the evolutionry responses to our ph regimes must hve een the result of muttions tht ourred de novo within the lines. Mesurements of Reltive Fitness Using the A, B, C, nd R groups isolted fter their evolutionry regimes were ompleted, mesurements of reltive fitness for eh of the 24 evolved lines were mde in vrious ph regimes following the methods of Bennett et l. (1992). Fitness ws determined through diret ompetition etween eh evolved line nd the reiprolly mrked (, ) nestrl genotype in single flsk. Different ompetitions were onduted in regimes of onstnt ph 5.3 nd onstnt ph 7.8 nd through ph 5.3 r 7.8 r 5.3 yle refleting the evolutionry onditions of the C group. In ll of the ompetitions, the two ompeting strins were revived from 80 C storge nd seprtely inoulted into stndrd nutrient-rih Luri roth ulture medium, with ph of 7.0 nd temperture of 37 C, for 24 h of growth. The lines were then eh trnsferred into the nestrl ph 7.0 Dvis miniml medi for the seond dy s growth. For the onstnt regimes of ph 5.3 nd 7.8, lines were seprtely exposed on the third dy to the ph environment in whih they would eventully ompete to provide opportunity for phenotypi limtion to the test regime efore ompetition. On the fourth dy of the onstnt regime ssys, ompetitions were initited y trnsfer mixing together 50 ml of the ompeting evolved line nd nestrl line into 9.9 ml of the sme test ph medi nd were then inuted under stndrd onditions for 24 h. A ensus of initil nd finl popultion density of eh ompetitor ws tken y plting diluted smples on TA gr, on whih the two ompetitors ould e distinguished nd enumerted through the differentil olony olor fforded y the reiprol neutrl mrker. The reltive fitness of the evolved line ws lulted s the rtio of the numer of its doulings ompred with the doulings of the nestor during the 100- fold omined popultion growth of the ompetition period (Lenski 1988, 1988; Bennett et l. 1990; Lenski et l. 1991). For the yled ompetition regimes, the ph of the third-dy medi ws set t 5.3. The fourth-dy trnsfer ws mde y omining oth nestor nd evolved line into ph 7.8 uffered medi to egin the ompetition, with initil ounts otined y immedite plting. On the fifth dy, seond ensus ws tken, nd the ompetitors were trnsferred k into ph 5.3 medi, nd 24 h lter, on the sixth dy, finl plting ws done to omplete the ompetition mesurements. The intermedite plting permitted the ssessment of limtion stte on fitness. Six simultneous replites were run for eh line in eh ompetition regime. Sttistil Anlysis We ompred the fitness mesurements produed in vrious onditions etween nd mong oth evolved individul lines nd groups using onservtive pprohes to test our priori hypotheses, nd while hving exmined only smll suset of the mny omprisons possile, we hve presented the results
410 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Figure 1. Men fitness responses (reltive to the ommon nestor) of the experimentlly evolved groups re shown with SE rs. A vlue of 1.0 (dshed line) represents no hnge reltive to the nestor. Groups with signifintly inresed reltive fitness re shown s lk rs, groups with signifintly deresed fitness re shown s white rs, nd groups with no signifint hnge re shown s gry rs. The C group evolved for 2,000 genertions in ph yled etween 5.3 nd 7.8; the R group evolved for 2,000 genertions in ph flututing rndomly mong 5.3, 6.3, 7.0, nd 7.8; the B group evolved for 1,000 genertions in onstnt si ph of 7.8; nd the A group evolved for 1,000 genertions in onstnt idi ph of 5.3. After evolution ws ompleted, eh of the four groups ws tested under five ph regimes, s designted y the lels ove the rs. of ll omprisons mde. Signifine of evolved reltive fitness mens were nlyzed y t distriutions ompred with null hypothesis vlue of 1, representing fitness equl to the nestor, nd were ssessed for signifine using two-tiled proilities nd n 1 p 5 df. The sttistil inferenes for testing our hy- potheses were sed on the numer of independent lines or replitions to estlish the degrees of freedom, whih were df p n 1 p 5, nd djustments to the degrees of freedom were mde for unequl vrine nd omprisons of lrger sets of groups. Comprisons of different groups were nlyzed to report differenes nd stndrd error of the differenes using the unpired two-tiled t-test, with null hypothesis tht the differene is 0, ssuming unequl vrine nd the speifi degrees of freedom listed in eh tle. Proility nlysis of trnsitionl dpttion, with stndrd errors for the mens, ws ompred within groups in different onditions, using t distriutions with the null hypothesis of 1 for fitness nd 0 for differene in fitness. Alimtion enefit ws nlyzed using pired omprisons of the sme group mesured in different onditions with the t-test for mthed pirs, with null hypothesis vlue of 0 nd df p n 1 p 5. The Shpiro-Wilk W- test ws employed to detet possile nonnormlity of mthed pir differenes, ut no prolemti distriutions were found. The sttistil inferenes for testing our hypotheses ll used the numer of independent lines or replitions to estlish the degrees of freedom, whih were df p n 1 p 5 in ll ses. Results Ptterns of Adpttion Ptterns of dpttion, s mesured y reltive fitness, re summrized sttistilly in Tle 1 nd grphilly in Figure 1, with referene to the speifi ph regimes of eh group s evolution nd the onditions under whih fitness ws ssyed. Blk rs (Fig. 1) indite signifint fitness gins reltive to the ommon nestor, while open rs indite signifint fitness losses. Note tht the fitness of group in prtiulr ph n hnge drstilly depending on the ph the group experiened the previous dy, tht is, whether the fitness ws mesured in onstnt or trnsitionl regime. The C group hd signifintly improved fitness in ll regimes, mesured in onstnt ph 5.3 nd 7.8, in trnsitioning ph
Evolution in Temporlly Flututing ph in Esherihi oli 411 Tle 2: Cost of plstiity in generlists versus speilists Comprison Test Regime Differene SE df P Cost C (A nd B) d ph 5.3 nd 7.8.025.047 12.50.608 A No R (A nd B) d ph 5.3 nd 7.8.054.046 11.96.300 A No C (A nd B) d ph 5.3 nd 5.3 r 7.8.060.032 16.00.077 A No R (A nd B) d ph 5.3 nd 5.3 r 7.8.118.030 15.59.001 A Yes e C R ph 5.3 nd 7.8.078.016 16.001 B Differene SE etween the smple mens of the generlist nd speilist fitness vlues. Two-tiled proilities for differenes were lulted using the t-test, ssuming equl (supersript B) or unequl (supersript A) vrines; n p 6 for C or R, nd n p 12 for the omined A nd B. Degrees of freedom s shown. Indites whether there is fitness ost to eing generlist. d Comprisons re etween the reltive fitness vlues of the six lines of the generlist group shown (C or R) nd the reltive fitness vlues of the 12 lines mking up the two speilist groups (A nd B). The single vlue used for eh generlist line is the men of its fitness sores in the two test regimes shown. The vlue used for eh speilist line is the single vlue mesured in onstnt ph 5.3 (A group), onstnt ph 7.8, or trnsitionl ph 5.3 r 7.8 (B group), s shown. e Comprison is etween the reltive fitness vlues of the six lines of the C group nd the six lines of the R group. The single vlue used for eh line is the men of its fitness sores in the two test regimes shown. 5.3 r 7.8 (fitness mesured in ph 7.8 fter trnsfer from ph 5.3) nd ph 7.8 r 5.3, nd in the overll ph 5.3 r 7.8 r 5.3 trnsitionl yle (fitness mesured over 2 d). Adpttion of the R group produed signifint fitness gins only in ph 5.3, ph 7.8 r 5.3, nd ph 5.3 r 7.8 r 5.3 ut not in onstnt ph 7.8 or trnsitionl ph 5.3 r 7.8 (Fig. 1, gry rs). The B group demonstrted surprising pttern, with no hnge in fitness in the onstnt ph 7.8 onditions ut signifint fitness gin in the trnsition from id to se, ph 5.3 r 7.8 ondition. (Note tht the onstnt se group hd not signifintly inresed fitness in its seletion environment of ph 7.8 even fter 2,000 genertions of seletion [Hughes et l. 2007].) Fitness lso did not hnge for the B group in the ph 5.3 r 7.8 r 5.3 yle. There ws signifint loss in fitness for the group when tested in oth onstnt id ph 5.3 nd trnsition from se to id, ph 7.8 r 5.3. The A evolved group developed highly signifint fitness gin of 28% in onstnt ph 5.3 ut showed no signifint gin in fitness when mesured in the idi onditions fter trnsitioning from se, ph 7.8 r 5.3, demonstrting the potentil differene etween onstnt nd trnsitionl regimes. The A group showed no hnge in reltive fitness in the yling ph 5.3 r 7.8 r 5.3 onditions. Also, lthough the A group s 30% loss of fitness in onstnt ph 7.8 ws not sttistilly signifint, euse of high vrine mong the A lines, the A group displyed signifint nd very sustntil 90% loss of fitness in the trnsitionl ph 5.3 r 7.8 test. Generlists, Speilists, nd Trde-Offs in Evolution to Flututing versus Constnt ph Further nlysis of the vrious evolved fitness mesurements (Tle 1; Fig. 1) reveled interesting ptterns involving trdeoffs, whih re defined here s signifint loss of fitness in one or more ph onditions ompnying signifint gin of fitness in one or more seletive ph onditions. These trdeoff ptterns indite the evolution of speilists, defined here s group exhiiting signifint inrese in fitness with ompnied trde-offs, or generlists, whih re defined here s Tle 3: Comprisons of fitness in C versus R groups Test Regime Mens C Group R Group Reltive Fitness Differene SE df P (Two Tiled) Constnt ph 7.8 1.147 1.035.112.031 9.983.005 Constnt ph 5.3 1.188 1.142.045.017 8.678.028 ph 5.3 r 7.8 1.143 1.073.070.041 9.969.120 ph 7.8 r 5.3 1.108 1.103.004.020 9.680.834 ph 5.3 r 7.8 r 5.3 1.120 1.087.033.024 6.434.209 Differene SE in reltive fitness etween the experimentl groups, sed on six replite lines in eh experimentl group ompred etween the two regimes. Two-tiled proilities were lulted using t-tests, ssuming unequl vrine, with the null hypothesis tht the men differene equls 0. Degrees of freedom s shown.
412 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Tle 4: Constnt ph fitness versus trnsitionl fitness of the A group lines Mens Constnt ph 7.8 r 7.8 () Constnt ph 5.3 r 5.3 () Trnsition ph 5.3 r 7.8 () Trnsition ph 7.8 r 5.3 (d) Trnsitionl Fitness Differentil ( d) ( ) A 1.781 1.313.250.808 1.536 A 2.918 1.271.316.849 1.656 A 3.047 1.297.240.704.880 A 1.632 1.383.275.869 1.420 A 2 1.042 1.206.805 1.307.136 A 3.828 1.182.862 1.094.054 Group mens.708 1.276.098.938.947 SE.144.030.233.090.290 P.097!.001.012.526.022 Note. Vlues shown in old represent the group mens. The A group evolved in regime of onstnt ph 5.3. Shown for eh of the four ph regimes tested is the men reltive fitness of eh line, sed on six replites, plus the group men lulted from the six line mens. SE of the group men (olumns d) nd SE of the men differene (right olumn). Two-tiled proilities sed on t distriutions, with n 1 p 5 df nd the null hypothesis tht men fitness equls 1 (olumns d) or differene equls 0 (right olumn). group exhiiting higher verge fitness thn the nestor in t lest one regime without signifint trde-offs in ny of the other regimes tested. By these riteri, the B group onsists of speilists, with signifint fitness gins from trnsitioning into lklinity, ph 5.3 r 7.8, ompnied y signifint fitness losses in oth onstnt ph 5.3 nd trnsition into id, ph 7.8 r 5.3. Likewise, the A group lso onsists of speilists, with fitness loss in the trnsition to lklinity, ph 5.3 r 7.8. In ontrst, the two groups evolved in vrile ph regimes, R nd C, oth exhiited generlist fitness ptterns, with neither group hving ny signifint loss in fitness in ny of the regimes ssyed. Thus, evolution in oth of the flututing ph regimes produed generlists, while oth onstnt ph regimes produed speilists. It hs een ommon ssumption tht the plstiity of generlist must ome with ost in performne when diretly ompred with speilist in the speilist s ntive environment. In ssessing whether the jk-of-ll-trdes ws tully Tle 5: Constnt ph fitness versus trnsitionl fitness of the B group lines Mens Constnt ph 7.8 r 7.8 () Constnt ph 5.3 r 5.3 () Trnsition ph 5.3 r 7.8 () Trnsition ph 7.8 r 5.3 (d) Trnsitionl Fitness Differentil ( d) ( ) B 1.971.951 1.184.966.228 B 2 1.144.778 1.284.628.010 B 3 1.007.959 1.190.959.183 B 1.974.819 1.129.921.257 B 2 1.056.543 1.044.627.072 B 3.906.805 1.217.844.350 Group mens 1.010.809 1.175.824.179 SE.033.062.033.065.053 P.782.027.003.042.020 Note. Vlues shown in old represent the group mens. The B group evolved in regime of onstnt ph 7.8. Shown for eh of the four ph regimes tested is the men reltive fitness of eh line, sed on six replites, plus the group men lulted from the six line mens. SE of the group men (olumns d) nd SE of the men differene (right olumn). Two-tiled proilities sed on t distriutions, with n 1 p 5 df nd the null hypothesis tht men fitness equls 1 (olumns d) or differene equls 0 (right olumn).
Evolution in Temporlly Flututing ph in Esherihi oli 413 Tle 6: Constnt ph fitness versus trnsitionl fitness of the R group lines Mens Constnt ph 7.8 r 7.8 () Constnt ph 5.3 r 5.3 () Trnsition ph 5.3 r 7.8 () Trnsition ph 7.8 r 5.3 (d) Trnsitionl Fitness Differentil ( d) ( ) R 1 1.033 1.148 1.048 1.068.065 R 2 1.017 1.146 1.068 1.124.029 R 3 1.028 1.162 1.187 1.164.161 R 1.966 1.184.960 1.067.124 R 2 1.042 1.080 1.087 1.080.046 R 3 1.126 1.134 1.085 1.118.058 Group mens 1.035 1.142 1.073 1.103.002 SE.021.014.030.016.041 P.156!.001.060!.001.966 Note. Vlues shown in old represent the group mens. The R group evolved in regime of rndomly flututing ph environments. Shown for eh of the four ph regimes tested is the men reltive fitness of eh line, sed on six replites, plus the group men lulted from the six line mens. SE of the group men (olumns d) nd SE of the men differene (right olumn). Two-tiled proilities sed on t distriutions, with n 1 p 5 df nd the null hypothesis tht men fitness equls 1 (olumns d) or differene equls 0 (right olumn). mster of none, we mesured the ost to eing generlist when ompeting in the speilists regimes. The fitness vlues of the speilist lines, eh mesured in the regime in whih it evolved (i.e., the fitness of the A group in onstnt ph 5.3 nd the B group in onstnt ph 7.8), were pooled nd then ompred with the fitness of the generlists, mesured in eh of the two regimes nd then verged for eh line, using t-test. The C nd R groups were eh ompred with the speilists seprtely, s shown in the first nd seond rows of Tle 2, nd in neither se ws there signifint ost to eing generlist. A slightly different set of vlues ould e used in these omprisons if one onsidered it importnt tht the B group did not tully evolve improved fitness in the onstnt 7.8 ph regime in whih it evolved ut insted showed fitness gin only in the trnsitionl ph 5.3 r 7.8 regime. We therefore lso ompred the speilists nd generlists for the omintion of onstnt ph 5.3 nd trnsitionl ph 5.3 r 7.8 environments, Tle 7: Constnt ph fitness versus trnsitionl fitness of the C group lines Mens Constnt ph 7.8 r 7.8 () Constnt ph 5.3 r 5.3 () Trnsition ph 5.3 r 7.8 () Trnsition ph 7.8 r 5.3 (d) Trnsitionl Fitness Differentil ( d) ( ) C 1 1.208 1.187 1.173 1.089.134 C 2 1.177 1.177 1.155 1.120.079 C 3 1.066 1.213 1.252 1.056.030 C 1 1.097 1.148 1.045 1.126.073 C 2 1.163 1.196 1.119 1.149.093 C 3 1.173 1.204 1.112 1.107.160 Group mens 1.147 1.188 1.143 1.108.084 SE.022.009.028.013.010 P.001!.001.004!.001.025 Note. Vlues shown in old represent the group mens. The C group evolved in regime yling etween ph 5.3 nd 7.8. Shown for eh of the four ph regimes tested is the men reltive fitness of eh line, sed on six replites, plus the group men lulted from the six line mens. SE of the group men (olumns d) nd SE of the men differene (right olumn). Two-tiled proilities sed on t distriutions, with n 1 p 5 df nd the null hypothesis tht men fitness equls 1 (olumns d) or differene equls 0 (right olumn).
414 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Tle 8: Adpttion to omplete yled regime in vrile versus onstnt groups Groups Compred Mens Vrile Groups Tested in ph 5.3 r 7.8 r 5.3 Constnt Groups Tested in ph 5.3 r 7.8 r 5.3 Trnsitionl Advntge of Vrile Evolution Perentge Chnge ( Differene SE) P (Two Tiled) C A 1.120.704 59 (.417.125).020 C B 1.120.972 15 (.148.041).013 R A 1.087.704 54 (.384.027).027 R B 1.087.972 12 (.115.046).036 Perentge differene lulted y dividing vrile y onstnt group reltive fitness (nd solute differene with SE of the differene) tested in the sme overll regime, sed on six replite lines in eh experimentl group. Two-tiled proilities were lulted using t-test, ssuming unequl vrine, with the null hypothesis tht the men differene equls 0, nd n1 n2 p 12 with 5.046 df (first row), 5.347 df (seond row), 5.315 df (third row), nd 7.795 df (fourth row), when djusted for unequl vrine. gin using the fitness of the A lines in the former nd the B lines in the ltter s the pooled speilist vlues, nd tested ginst the fitness of eh set of generlist lines verged for the two environments. As shown in rows three nd four of Tle 2, the C group still showed no signifint ost, ut the R group did revel signifint ost ompred with the speilist in this prtiulr ondition. Overll, however, these omprisons suggest tht the jk-of-ll-trdes my e mster of t lest some s well. Evolution in Cyled versus Rndom ph Flutution Regimes Beuse the seletive regimes of the C nd R groups did differ, despite oth eing lssified s generlists, we lso ompred the overll fitness of the two groups diretly in mnner similr to the generlist-speilist omprisons ove. Agin we lulted for eh line the men of its fitness vlues in the onstnt ph 5.3 nd 7.8 regimes nd then ompred the six vlues for eh group y t-test. The R group hd signifintly greter ost of plstiity, with reltive fitness 0.078 lower thn the C group. The generlist ptterns of the C nd R groups were lso not synonymous in other respets (Tle 1; Fig. 1). The R group showed no signifint gins under some regimes (i.e., onstnt ph 7.8 nd ph 5.3 r 7.8). However, the C group hd signifintly higher fitness thn the nestor in every regime tested nd therefore ould e lssified s superior generlist. The C group fitness ws lso higher thn the R group in every regime tested (Tle 3), ut only the onstnt ph 5.3 nd 7.8 regimes produed sttistilly signifint differenes. At onstnt ph 7.8, there ws highly signifint fitness dvntge of 0.011 for the C group over the R group, while t onstnt ph 5.3, the dvntge ws only 0.050. In ll of the flututing ph ssys, the yled nd rndom groups were sttistilly indistinguishle. Adpttion to Trnsitions versus Constnt Components in the ph Environment To ssess the extent to whih there ws dpttion y the C nd R lines to the rupt trnsitions etween id nd lkline onditions rther thn only dpttion to the two ph environments per se, omprisons of reltive fitness performne etween trnsitionl (yled) nd onstnt regimes were mde for eh line in ll four groups (Tles 4 7). These omprisons thus llowed us to exmine the role tht periods of flutution themselves ply in evolution to hnging environments. The trnsitionl fitness differentils reported in the lst olumns of Tles 4 7 were lulted for eh line y sutrting reltive fitness performne mesurements in the two onstnt regimes from those mesurements in the two trnsitionl regimes. The A group, evolved in onstnt id onditions, exhiited sig- Tle 9: Benefit of limtion to si ph for group A Mens Alimted ph 7.8 r 7.8 Nonlimted ph 5.3 r 7.8 Alimtion Benefit (Differene) A 1.781.250 1.031 A 2.918.316 1.234 A 3.047.240.287 A 1.632.275.907 A 2 1.041.804.236 A 3.828.862.034 Group mens.708.098.610 SE.209 95% CI.538 P.033 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the
Evolution in Temporlly Flututing ph in Esherihi oli 415 Tle 10: Benefit of limtion to id ph for group A Mens Alimted ph 5.3 r 5.3 Nonlimted ph 7.8 r 5.3 Alimtion Benefit (Differene) A 1 1.313.808.505 A 2 1.271.849.422 A 3 1.298.704.593 A 1 1.383.869.514 A 2 1.206 1.307.101 A 3 1.182 1.094.088 Group mens 1.276.939.337 SE.113 95% CI.292 P.031 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the nifint nd detrimentl trnsitionl fitness differentil of 0.95, demonstrting very sustntil loss of fitness in trnsitions ompred with its fitness in onstnt regimes (Tle 4). In ontrst, the B group, evolved in onstnt si onditions, showed signifint 0.18 trnsitionl fitness differentil, inditing its sustntil gin of fitness in trnsitions ompred with its fitness in onstnt regimes (Tle 5). This mrked improvement t trnsitioning ws due lmost exlusively to n improvement t trnsitioning from ph 5.3 to 7.8, while the A group s loss of fitness ws more evenly shred etween oth trnsitions. The R group, evolved in rndomly flututing ph, exhiited no signifint differene etween its performne in onstnt nd flututing onditions nd thus demonstrted no trnsitionl fitness differentil (Tle 6). However, the C group, evolved in yling of the extreme ph vlues, showed signifintly lower fitness in trnsitions ompred with its fitness in onstnt onditions (Tle 7), with fitness differentil of 0.08. It should e noted, however, tht this negtive trnsitionl fitness differentil in the C group is not due to ny loss of fitness in the trnsitionl onditions; in ft, the C group showed signifint fitness gins in oth trnsitions, ut the gins in the onstnt regimes were even greter nd hene resulted in the negtive differentil. In the R group, on the other hnd, there ws no signifint dpttion to the onstnt ph 7.8 regime, nd this redued the differentil. A further nlysis of omprtive dpttion to overll trnsitions, shown in Tle 8, determined the overll fitness gined in the omined trnsitions y the groups evolved in flututing regimes ompred with those evolved in onstnt ph. This overll trnsitionl dvntge ws lulted s the differene etween groups in their overll performne in the ph 5.3 r 7.8 r 5.3 yled regime. While this wider view of the dpttion pttern ould not disern the more speifi ftors using the evolved fitness, it provided hrteriztion of performne in long-term trnsitioning environments, whih we refer to s trnsitionl dvntge. Both flututing groups evolved sustntil nd similr performne dvntges in the yled regime when ompred with the onstnt ph evolved lines. The C group hd the lrgest trnsitionl dvntge of 59% over the A group nd signifint dvntge of 15% over the B group. The R group hd signifint dvntge of 54% over the A group nd signifint dvntge of 12% over the B group. Evidently, seletion in vrile environments did in ft produe signifint improvements in the overll ility to trnsition mong the omintion of environments. The Benefiil Alimtion Hypothesis To test the potentil enefit of limtion preeding ompetition, the reltive fitness of eh line within eh group ws ompred etween onditions hving dy of limtion to the test regime nd not hving limtion to the test ph (Tles 9 16). In the test with limtion, on the dy efore ompetition, the ph ws the sme s it ws on the ompetition dy, designted y id limtion of ph 5.3 r 5.3 or se limtion of ph 7.8 r 7.8. The nonlimted test pled the ompetitors in different ph ondition during the dy efore the ompetition, suh s ph 7.8 r 5.3 or ph 5.3 r 7.8. This study found no generl sttistil support for the universlity of enefit from limtion when exmined rodly y omining ll of the tested groups, lthough individul ses of enefit from limtion were lerly evidened. Finer-sle nlysis of the enefit of limtion, lulted s the differ- Tle 11: Benefit of limtion to si ph for group B Mens Alimted ph 7.8 r 7.8 Nonlimted ph 5.3 r 7.8 Alimtion Benefit (Differene) B 1.971 1.184.213 B 2 1.143 1.284.140 B 3 1.007 1.190.183 B 1.974 1.129.155 B 2 1.056 1.044.012 B 3.906 1.217.311 Group mens 1.010 1.175.165 SE.043 95% CI.111 P.012 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the
416 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Tle 12: Benefit of limtion to id ph for group B Mens Alimted ph 5.3 r 5.3 Nonlimted ph 7.8 r 5.3 Alimtion Benefit (Differene) B 1.951.966.015 B 2.778.628.150 B 3.959.959!.001 B 1.819.921.102 B 2.543.627.085 B 3.805.844.039 Group mens.809.824.015 SE.037 95% CI.094 P.700 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the ene of the limtion minus the nonlimtion fitness responses, reveled interesting ptterns mong ll four groups. The onstnt id evolved group A hd very sustntil nd signifint improvement in reltive fitness of 0.61 (Tle 9) from limtion to lklinity nd lso sustntil nd signifint fitness improvement of 0.34 (Tle 10) from limtion to id. In ontrst, the onstnt se evolved group B hd signifint detriment to fitness of 0.16 (Tle 11) from limtion to lklinity nd no signifint enefit from limtion to id (Tle 12). The rndom flututing ph evolved group R hd no signifint improvement due to limtion in either id or se (Tles 13, 14). In the yled ph evolved group C, there ws no signifint effet of limtion to si onditions (Tle 15) ut signifint enefit to fitness of 0.08 resulting from limtion to id (Tle 16). Evidently, the flututing seletive environments produed little or no limtion enefit ompred with the onstnt evolutionry onditions. The hypothesis for expeting enefit from limtion to the historil evolutionry regime hd mixed support y results for the A group nd ws unsupported y our B group. The A group, whih ws historilly dpted to ph 5.3, hd lrge loss to fitness of 0.61 fter limtion t the historil ph (ph 5.3 r 7.8) ompred with the nonhistoril limtion (ph 7.8 r 7.8; Tle 9). It lso experiened fitness gin of 0.34 fter historil limtion (ph 5.3 r 5.3) ompred with the nonhistoril limtion (ph 7.8 r 5.3; Tle 10). The B group, whih ws historilly dpted to ph 7.8, hd fitness gin of 0.16 following limtion t the historil ph (ph 7.8 r 7.8) ompred with the nonhistoril limtion (ph 5.3 r 7.8; Tle 11), while it hd no signifint differene etween the historil (ph 7.8 r 5.3) nd nonhistoril (ph 5.3 r 5.3) limtion ondition (Tle 12). While we hd no historil test for the rndom group, with ertin vets, the yled group ould potentilly e onsidered, if it were ssumed tht the histori environment for the yled group ould ppropritely e hrterized s the trnsitionl ph 7.8 r 5.3 test, sine this test ourred t the end omponent of ph 5.3 r 7.8 r 5.3 yle. If this yling tretment ws n pproprite proxy for yled history, while the nonhistoril omponent ws ph 5.3 r 5.3 (Tle 16), then the C group s signifint loss of fitness of 0.08 would lso e disordnt with the historil enefit hypothesis. Disussion Generlly, our study showed tht (1) distint ptterns of dpttion ourred in yling ph, rndomly flututing ph, onstnt id, nd onstnt se environments; (2) generlists evolved in vrile ph environments, nd speilists evolved in onstnt ph environments, lthough there were not neessrily osts to the generlist when tested in speilized environments; (3) with vrile environments, the yled group hd higher fitness ompred with the rndomly flututed group; (4) dpttion to trnsitions ourred in the onstnt se regime, yet it ws sustntilly redued in the onstnt id regime, nd, in omprison to oth onstnt regimes, the vrile regimes displyed signifint dvntge in dpttion to trnsitions; nd (5) the enefit of limtion ws not universlly supported, even when nlyzed y historil ontext. Interprettion of some results re lso onsidered in light of the nturl history of Esherihi oli, lthough ssertions regrding mroevolutionry implitions nnot e onlusive from this lortory-sed miroevolution study lone, sine Tle 13: Benefit of limtion to si ph for group R Mens Alimted ph 7.8 r 7.8 Nonlimted ph 5.3 r 7.8 Alimtion Benefit (Differene) R 1 1.033 1.048.015 R 2 1.017 1.068.051 R 3 1.028 1.187.159 R 1.966.960.006 R 2 1.042 1.087.045 R 3 1.126 1.085.041 Group mens 1.035 1.073.037 SE.028 95% CI.072 P.243 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the
Evolution in Temporlly Flututing ph in Esherihi oli 417 Tle 14: Benefit of limtion to id ph for group R Mens Alimted ph 5.3 r 5.3 Nonlimted ph 7.8 r 5.3 Alimtion Benefit (Differene) R 1 1.148 1.068.080 R 2 1.146 1.124.022 R 3 1.162 1.164.002 R 1 1.184 1.067.118 R 2 1.080 1.080.000 R 3 1.134 1.118.017 Group mens 1.142 1.103.039 SE.020 95% CI.051 P.107 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the rel-world onditions involve vstly greter environmentl omplexity nd evolutionry time lines. Ptterns of Adpttion The mere ourrene of fitness inrements due to evolutionry dpttion is fundmentl expettion of evolutionry iology nd is not prtiulrly interesting in nd of itself. Wht is interesting here is tht the omplex ptterns of dpttion in the vrious ph regimes were so different mong the groups nd reveled the first empiril hrteriztion of the intriies of evolution in response to vrile ph. One of the more surprising ptterns ws the ontrst etween id nd se dpttion. In id, the A, C, nd R groups eh hd signifint fitness gins, with tht of A eing gretest, while the generlly opposite pttern ourred in se, with only the C group produing ny signifint fitness gins. Interestingly, even fter 300 d (2,000 genertions) of onstnt exposure to lkline onditions, there ws no signifint improvement in fitness y the B lines (Hughes et l. 2007), while in this study, only 1,000 genertions of exposure to lkline onditions produed highly signifint 15% fitness gins in the C group. Apprently, the yling ph produed seletive pressure for improvement in onstnt lkline environments tht the onstnt environment itself did not provide, whih my indite further omplexity or onstrints on evolution of the underlying mehnisms of lkline resistne. Interestingly, the rndom flutution ws not dequte seletion for suh se fitness improvements ut only the extreme nd regulr yling of the C group. Generlists, Speilists, nd Trde-Offs in Evolution to Flututing versus Constnt ph Both the A nd B onstnt ph groups in this study hd orrelted fitness derements in nonseletion environments, lssifying them s speilists on their respetive nihes of id nd se nd suggesting trde-offs in fitness during evolution. Both C nd R vrile ph groups evolved to e generlists, s in previous vrile therml evolution experiment (Bennett nd Lenski 1993) tht showed tht the sme nestrl line s used here evolved into therml generlists in vrile therml environment. Perhps of more interest thn the mere pperne of generlists ws the reltive ost of eing jk-of-ll-trdes generlist versus speilist, nd prtiulrly whether there ws neessrily ost to the generlist genotype when tested in unvrying environments. Our experiments were le to ddress this question diretly. Assuming tht the C nd R group generlists displyed inresed phenotypi plstiity, s evidened y their signifint inreses of fitness in the vrile test environments, we were le to mesure the ost to eing generlist nd therey the ost of plstiity y ompring the verge fitness of generlists to tht of speilists in the ph regimes in whih the speilists evolved. Neither the C nor R group showed signifint osts (i.e., redued fitness) in these onstnt ph onditions when ompred with the speilists. Curiously, the B group did not improve fitness in its evolutionry regime of onstnt ph 7.8, yet it did improve in the novel regime of ph 5.3 r 7.8. Beuse of this ourrene of expttion (i.e., predpttion), in whih the B group evolved inresed fitness to ondition (ph 5.3 r 7.8) other thn the ondition for whih it ws seleted (onstnt ph 7.8), we lso Tle 15: Benefit of limtion to si ph for group C Mens Alimted ph 7.8 r 7.8 Nonlimted ph 5.3 r 7.8 Alimtion Benefit (Differene) C 1 1.208 1.173.035 C 2 1.177 1.156.021 C 3 1.066 1.252.187 C 1 1.097 1.046.051 C 2 1.163 1.119.045 C 3 1.173 1.112.061 Group mens 1.147 1.143.004 SE.039 95% CI.099 P.914 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the
418 B. S. Hughes, A. J. Cullum, nd A. F. Bennett Tle 16: Benefit of limtion to id ph for group C Mens Alimted ph 5.3 r 5.3 Nonlimted ph 7.8 r 5.3 Alimtion Benefit (Differene) C 1 1.188 1.089.099 C 2 1.178 1.120.057 C 3 1.213 1.056.156 C 1 1.148 1.126.021 C 2 1.196 1.149.048 C 3 1.204 1.107.097 Group mens 1.188 1.108.080 SE.020 95% CI.050 P.010 Note. Vlues shown in old represent the group mens. SE of the men differene. Confidene intervl (CI) for the group men differene. Two-tiled proilities sed on t distriutions, with n 1 p 5 null hypothesis tht the men differene equls 0. df nd the ompred the two generlist groups with the B group in this trnsitionl environment. Although we found signifint ost to eing generlist for the R group under these onditions, we still found no signifint ost for the C. The generl outome for these experiments, then, is tht jk-of-ll-trdes my still e mster of mny. Evolution in Cyled versus Rndom ph Flutution Regimes The higher fitness gins of the yled C group, superior generlist, ompred with the rndom R group, generlist with fewer signifint gins, my support the preditions tht plstiity is most fvored when seletion ts eqully strongly ross hitts, ll hitts re eqully regulr, nd interhitt vriility is high (Sheiner 1993; Grlnd nd Kelly 2006). The R group s regime, whih flututed stohstilly mong ph 5.3, 6.3, 7.0, nd 7.8, inluded environments with reltively weker seletion nd hd lower verge environmentl vriility when ompred with the yled regime, whih flututed regulrly etween the two extremes of ph 5.3 nd 7.8. However, suh onlusion would hve to ignore the distintion etween the types of environments tht produed signifintly different fitness etween the C nd R groups. Sine only the onstnt ph regimes (ph 5.3 nd 7.8) reveled sttistilly signifint differenes etween the yled nd rndom groups, while ll omprisons in the trnsitionl test regimes (ph 5.3 r 7.8, ph 7.8 r 5.3, ph 5.3 r 7.8 r 5.3) were not signifint, it ould e inferred tht plstiity ws not enhned y the inresed regulrity nd intensity of the yled regime versus the rndom one. However, the R group hd signifintly greter ost of plstiity thn the C group, offering some support for effiieny spets of the evolved plstiity hypothesis. It my lso suggest tht this re of theory ould e verified further y omprtive nlysis of dpttion to trnsitionl nd onstnt ph onditions. Adpttion to Trnsitions versus Constnt Components in the ph Environment It hs een repetedly rgued tht temporlly vrile environments should selet for inresed phenotypi flexiility (Levins 1968, 1969; Feder 1978; Tsuji 1988), lthough this question hs reeived very little empiril ttention. Typilly, environmentl heterogeneity or environmentl stress does indue phenotypi plstiity (Hrshmn et l. 1999; Wilson nd Frnklin 2002; Berrign nd Sheiner 2004; Griel 2005). The forml theoretil pprohes of quntittive geneti models, optimlity models, nd gmeti models ll suggest tht dptive plstiity will evolve with environmentl heterogeneity (Sheiner 1993). More speifilly, the evolution of plstiity is suspeted to depend on the speed of temporl environmentl hnges, the preditility of the temporl heterogeneity, nd the durtion of the heterogeneity (Grlnd nd Kelly 2006). The hypothesis tht evolution in temporlly vrying environment would inrese phenotypi flexiility for mking trnsitions ws not supported y vrile therml evolution (Leroi et l. 1994), yet when it ws reexmined here in the sme teril system, dpted insted to environmentl ph, highly signifint support ws found. Our investigtion used two pprohes to mesure whether temporlly vrying environments evolved higher phenotypi plstiity to trnsitions or whether the dpttion merely improved performne in the onstnt omponents. The first pproh involved within-group omprisons etween onstnt nd trnsitionl fitness to rrive t trnsitionl dpttion for eh group, whih ws mrkedly different for eh group. Surprisingly, trnsitionl dpttion showed the most hnges in the onstnt evolution groups, with the gretest gin of 0.18 with the B group nd the gretest loss of 0.95 in the A group, while the vrile R group hd no signifint hnge, nd the C group hd only modest 0.08 derement (Tles 4 7). By this mesure, the onstnt se evolution of the B group hd the only demonstrted trnsitionl dpttion tht ws not elipsed y dpttion to the onstnt omponents. The onstnt id evolution (A) produed huge redutions in trnsitionl fitness ompred with the dptive gins it produed in the onstnt omponent. From this omprison, it ould e onluded tht differenes etween trnsitionl nd onstnt evolution were of greter mgnitude in evolution in onstnt environments thn in vrile environments. However, this pproh lone does not inlude the reltive overll trnsitionl dpttion differenes etween these two types of evolution environments. So, our seond pproh mde roder mong-group omprison of fitness in the omintion of trnsitions etween the vrile nd onstnt groups performnes in the overll trnsitionl test yle ph