Appl. Environ. Microbiol. 2000 Miller 4222 9

10.1128/AEM.66.10.4222-4229.2000. 2000, 66(10):4222. DOI:Appl. Environ. Microbiol.

Scott R. Miller and Richard W. Castenholz

SynechococcusCyanobacteria of the Genus Evolution of Thermotolerance in Hot Spring

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Oct. 2000, p. 42224229 Vol. 66, No. 10

Copyright 2000, American Society for Microbiology. All Rights Reserved.

Evolution of Thermotolerance in Hot Spring Cyanobacteria ofthe Genus Synechococcus

SCOTT R. MILLER* AND RICHARD W. CASTENHOLZ

Department of Biology, University of Oregon, Eugene, Oregon 97403

Received 24 January 2000/Accepted 19 July 2000

The extension of ecological tolerance limits may be an important mechanism by which microorganisms adaptto novel environments, but it may come at the evolutionary cost of reduced performance under ancestralconditions. We combined a comparative physiological approach with phylogenetic analyses to study theevolution of thermotolerance in hot spring cyanobacteria of the genus Synechococcus. Among the 20 laboratoryclones of Synechococcus isolated from collections made along an Oregon hot spring thermal gradient, fourdifferent 16S rRNA gene sequences were identified. Phylogenies constructed by using the sequence dataindicated that the clones were polyphyletic but that three of the four sequence groups formed a clade.Differences in thermotolerance were observed for clones with different 16S rRNA gene sequences, and com-parison of these physiological differences within a phylogenetic framework provided evidence that morethermotolerant lineages of Synechococcus evolved from less thermotolerant ancestors. The extension of thethermal limit in these bacteria was correlated with a reduction in the breadth of the temperature range forgrowth, which provides evidence that enhanced thermotolerance has come at the evolutionary cost of increasedthermal specialization. This study illustrates the utility of using phylogenetic comparative methods to inves-tigate how evolutionary processes have shaped historical patterns of ecological diversification in microorgan-isms.

Has adaptation to novel environments by the extension oftolerance limits historically been an important mechanism bywhich the ecological requirements of microorganisms have di-verged? While the breadth of physiological diversity exhibitedby microorganisms suggests that this may be the case, therelevance of this mechanism is yet to be firmly established.Similarly, little is known about the consequences of this modeof adaptation for fitness in ancestral environments. For exam-ple, are there trade-offs or costs of adaptation such that thereis a correlated loss in performance under ancestral conditions?Providing answers to the above questions may help microbiol-ogists to better make sense of extant microbial diversity and itsdistribution on the planet.

The cyanobacteria provide an excellent system for testinghypotheses concerning the evolution of tolerance. During itsevolutionary history, this ancient lineage of oxygen-evolving,photoautotrophic bacteria has established itself in diverseaquatic and terrestrial habitats exhibiting wide ranges in tem-perature, salinity, water potential, pH, and irradiance (36). Anexample of ecological diversification in the cyanobacteria is theinvasion of alkaline hot spring habitats across western NorthAmerica, Asia, Africa, and possibly Europe by members of thegenus Synechococcus (8). Hot spring outflows typically exhibitmarked temperature gradients, and microbial communitiescontaining Synechococcus generally develop in these systems attemperatures between ;45 and 73C, the thermal maximumfor photosynthetic life (3, 4). Peary and Castenholz (26) dem-onstrated with laboratory isolates that at least four tempera-ture strains of Synechococcus inhabited a single channel atHunters Hot Springs in Oregon. It was later hypothesized thatmore thermotolerant Synechococcus strains evolved from lessthermotolerant ancestors growing at lower temperatures (5). It

was not possible to test this hypothesis, however, because thedata required to establish the branching order of the temper-ature strains in a phylogeny (i.e., their order of evolutionarydivergence) were not available.

Conversely, substantial cyanobacterial molecular diversityhas been identified among environmental 16S rRNA andrRNA gene fragment sequences recovered from microbial matcommunities in Octopus Spring in Yellowstone National Park(32). Among these, phylogenetically similar sequences whichclearly belong to Synechococcus have distribution patterns con-sistent with the hypothesis that genotypically distinct lines havediverged in temperature range (11, 13). However, while it ispossible to gain an understanding of genealogical patternsfrom these molecular data, the lack of phenotypic data for theorganisms limits the testing of evolutionary ecological hypoth-eses.

The ability to infer the patterns and processes of past eco-logical diversification therefore depends upon both the avail-ability of comparative phenotypic data for the study organismsand an understanding of their phylogenetic relatedness. Wehave combined comparative physiology of laboratory isolateswith phylogenetic approaches in order to investigate the evo-lution of thermotolerance in Synechococcus from Hunters HotSprings in Oregon. We first reconstructed molecular phylog-enies to infer the evolutionary relationships of Synechococcustemperature strains isolated in laboratory culture from Hunt-ers Hot Springs. We next collected comparative growth ratedata for these isolates at a series of temperatures in order todistinguish four groups of strains based on both identity of 16SrRNA gene sequence data and thermotolerance characteris-tics. From the phylogeny we then determined the order ofevolutionary divergence of groups of strains with different ther-motolerances to test the hypothesis that more thermotolerantstrains of Synechococcus evolved from less thermotolerant an-cestors. Finally, we incorporated phylogenetic information intoa statistical analysis of the comparative physiological data in

* Corresponding author. Present address: Mailstop 239-4, NASAAmes Research Center, Moffett Field, CA 94035. Phone: (650) 604-6052. Fax: (650) 604-1088. E-mail: [email protected].

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order to reveal possible phenotypic trade-offs during the evo-lution of increased thermotolerance.

MATERIALS AND METHODS

Sample collection, enrichment, and clone isolation. Hunters Hot Springscomprises a cluster of several alkaline thermal sources located ;3 km north ofLakeview, Oreg. Between 1994 and 1998, several collections of microbial matwere taken at four temperatures (45, 55, 65, and 70C) along one of the outflowchannels (Jacks Spring). For each collection, the upper, Synechococcus-contain-ing component of the mat was harvested with a sterile 10-ml syringe, transferredto a sterile 20-ml scintillation vial, and stored at ambient temperature in the darkuntil it was taken to the University of Oregon.

The general procedure used for enrichment and isolation of Synechococcusclones in laboratory culture was as follows. Each sample was homogenized in thelaboratory with a sterile syringe, and the density of Synechococcus cells in theresulting cell suspension was estimated with a hemocytometer. The suspensionwas diluted serially by successive transfers of 1 ml of suspension into 9 ml ofliquid BG11 medium (7) to create a series of suspensions ranging in density from101 to 107 cells ml21. Aliquots (1 ml) from each tube in the series were used toinoculate individual liquid enrichment flasks (75 ml) containing D medium andBG11 medium (7). Most of the enrichment flasks were incubated at the collec-tion temperature; the only exceptions were the flasks prepared from 70C col-lections, which were incubated at 67C. This temperature had previously beenfound to be the upper limit of the optimal temperature range for laboratorycultures of the high-temperature form of Synechococcus cf. lividus (22). Tominimize evaporation during enrichment, isolation, and maintenance of Syn-echococcus cultures at 65 and 67C, metal-capped Bellco flasks were used. En-richments grown at 45 and 55C were incubated in incubators with an irradianceof 120 microeinsteins m22 s21 (provided by cool-white fluorescent lamps), while65 and 67C enrichments were incubated in water baths with an irradiance of 30microeinsteins m22 s21 (provided by very-high-output, cool-white fluorescentlamps).

Isolation attempts were made with both the most- and least-diluted positiveenrichments in a series. Synechococcus clones were isolated from 45 and 55Cenrichments after at least three rounds of picking and restreaking of isolatedcolonies on 1.5% agar plates containing the appropriate medium. Clones wereobtained from 65 and 67C enrichments by using the filter isolation method ofMeeks and Castenholz (22). Laboratory clones were maintained under the sameconditions as the enrichments.

The isolation procedure was different for one 55C clone (OH2). In this case,1 drop of a homogenized cell suspension made from a collection taken in March1996 was spread on an agar plate. Individual Synechococcus cells were visualizedunder a dissecting microscope, transferred to test tubes containing 7 ml of eitherD or BG11 medium, and incubated as described above. Ten isolations wereattempted for each medium. One of the BG11 tubes was successful, and cloneOH2 was obtained from this tube after repeated plating as described above.

DNA isolation, amplification, and sequencing. Genomic DNA of clones wereisolated as described by Pitcher et al. (27). Fragments (ca. 950 bp) of the 16SrRNA gene were amplified from these DNA preparations. This gene was chosenbecause the database for it is the most extensive database among those forcyanobacterial loci, which facilitated adequate taxon sampling and placement ofthe Synechococcus strains within the context of the cyanobacterial phylogeny.The 50-ml amplification reaction mixtures contained 10 mM Tris-HCl (pH 8.3),50 mM KCl, each deoxynucleoside triphosphate at a concentration of 200 mM,1.12 mM MgCl2, 1.25 U of Taq polymerase (Perkin-Elmer), ;10 ng of genomicDNA, 0.2 mM primer CYA359F (25), and 0.2 mM primer PLG2.3 (59CTTCA[C/T]G[C/T]AGGCGAGTTGCAGC39), a modification of PLG2.1 (31). Thereaction conditions used were 40 cycles of 94C for 1 min, 58C for 1 min, and72C for 2 min. Amplification products were purified with a QIAquick-spin PCRpurification kit (Qiagen), and cleaned amplification products were directly se-quenced with an ABI Prism 377 sequencer at the DNA Sequencing Facility,University of Oregon.

Sequence alignment. 16S rRNA gene sequences were preliminarily alignedwith Clustal W1.6 (29). Since the stem-loop structure of the mature 16S rRNAmolecule contains information with respect to nucleotide positional homology,secondary structure was then syntactically imposed on each sequence for thefragment spanning Escherichia coli nucleotide positions 360 to 1326 as describedby Titus and Frost (30). Previously published secondary-structure models aidedidentification of stem-encoding nucleotides of cyanobacterial and outgroup se-quences (14, 37). A final pairwise alignment was made by using Malign 2.7 (35)with a gap cost to substitution cost of 3. Missing data were treated as unknownnucleotides.

Phylogeny reconstruction. Phylogenetic trees were constructed by using threeoptimality criteria. A neighbor-joining tree was built with MEGA 1.01 (20). Forcalculation of a distance matrix, Kimuras two-parameter model was assumed(19), and nucleotide positions containing gaps or missing data were deleted in apairwise fashion. The tree was inferred with pairwise deletion of gaps and with1,000 bootstrap pseudoreplicates. A maximum-parsimony phylogeny was con-structed by using PAUP 3.1.1 (28). A heuristic search was performed by using thetree bisection-reconnection branch-swapping algorithm. Starting trees were ob-tained by stepwise addition of sequences and 10 replications of random sequence

addition. The analysis was bootstrap pseudoreplicated 100 times. A maximum-likelihood tree was created with the dnaml program in PHYLIP 3.5c (10) byusing a transition-to-transversion ratio of 2, sequential addition of sequences,and 100 bootstrap pseudoreplicates. All trees were rooted with Aquifex pyrophi-lus.

Determination of clone growth temperature ranges. Exponential growth rateswere estimated at intervals along a clones thermal range for growth by using thefollowing method. Beginning at the clones maintenance temperature, the growthrate was determined as described below. The experimental temperature was thenshifted up, usually by 5C, and cells were transferred to fresh medium as de-scribed below. Near the predicted upper thermal limit for the clone, based ondata for temperature strains of Synechococcus from a previous study (26), themagnitude of the shift was decreased in order to resolve differences amongclones with respect to thermal maxima. Growth rates were again estimated, andthis procedure was repeated until the clones lethal temperature was reached, asindicated by a lack of growth and cell bleaching. To evaluate clone performanceat temperatures below that used for routine maintenance, an analogous proce-dure was employed with thermal down-shifts accompanying clone transfer.

For clones of 16S rRNA gene sequence group I (Table 1), the experiment wasinitiated by inoculating duplicate flasks with cells from an early-stationary-phasebatch culture grown under standard maintenance conditions. Cells were concen-trated by centrifugation and resuspended in fresh medium to an A750 of ;0.75.Aliquots (1 ml) of this suspension were delivered to duplicate 125-ml Erlenmeyerflasks containing 75 ml of the appropriate medium to obtain an initial experi-mental A750 of ;0.01. The flasks were supplemented with 1 ml of 0.5 MNaHCO3, previously found to stimulate growth in several Synechococcus clones(unpublished data), and were incubated at the experimental temperature under120 microeinsteins of cool-white fluorescent light m22 s21. The positions of theflasks in the incubator were randomized every 24 h. The population densities inthe flasks were monitored spectrophotometrically by determining the increase inA750. Between four and five generations of exponential population growth weresupported under the above conditions. The generation time during exponentialgrowth was estimated by determining log 2/b, where b is the slope of logarith-mically transformed A750 data regressed on time (in hours). This value wastransformed and reported as number of population doublings per day. Theexperimental temperature was then shifted up or down as described above. After4 h, one of the experimental replicates was randomly chosen and transferred toduplicate flasks as described above. The flasks were then used to determine thegrowth rate at the new temperature.

The procedure used to determine the growth rates of clones belonging to allother 16S rRNA sequence groups (Table 1) was the same as the proceduredescribed above except that no bicarbonate was added to the experimental flasks.This was because bicarbonate had an unexpected deleterious effect on the growthrates of clones isolated from the 70C collections (sequence group IV) (Table 1).Due to the difference in procedure, the growth rate data obtained for the groupI clones were not included in statistical analyses of data obtained for clonesbelonging to the other 16S rRNA gene sequence groups.

Statistical analyses. Growth rate data were analyzed with analysis of variance(ANOVA) models, and pairwise comparisons of means were made by usingBonferroni tests with a significance level of a 5 0.05. All analyses were done withSPSS Base 8.0 (SPSS Inc.).

Correlations between thermotolerance traits were estimated after the datawere transformed by using the method of independent contrasts (9), a phyloge-netic comparative method for comparing two or more continuously distributedvariables. Phylogenetic comparative methods (16, 21) incorporate informationfrom phylogenies into the analysis of comparative phenotypic data both to ensurethat the data meet the assumptions of the statistical methods used to analyzethem and to facilitate the investigation of past evolution by using data collectedfrom extant organisms. The method of independent contrasts accounts for thestatistical problem of nonindependence of comparative data which arises due toshared evolutionary histories of taxa. It assumes a Brownian motion model ofphenotypic evolution, i.e., that the phylogeny branch lengths in units of sequencedivergence are proportional to the expected amount of phenotypic divergence.Brownian motion models the pattern of phenotypic change expected for char-acters evolving under random genetic drift or directional selection (15). Toestimate the expected amount of phenotypic change, we used the branch lengthsinferred from our phylogeny reconstructions. Correlations between the contrastswere then estimated, and t tests with 2 degrees of freedom were used to testwhether estimated correlation coefficients were significantly different from zeroat the a 5 0.05 level.

Nucleotide sequence accession numbers. The 16S rRNA gene sequence dataobtained for the Synechococcus strains used in this study have been deposited inthe GenBank database under accession numbers AF285244 to AF285253 (groupI), AF285240 to AF285243 (group II), AF285259 (group III), AF285254 toAF285258 (Group IV), and AF285260 (Synechococcus sp. strain SH-94-5).

The GenBank nucleotide sequence accession numbers for the taxa used inphylogenetic analyses are as follows: Aquifex pyrophilus, M83548; Escherichiacoli, J01859; Bacillus subtilis, X60646; Synechococcus sp. strain PCC 6307,AF001477; Synechococcus sp. strain PCC 6301, X03538; Leptolyngbya sp. strainPCC 73110, X84810; Microcoleus sp. strain PCC 7420, X70770; Mastigocladussp. strain PCC 7518, X68780; Nostoc sp. strain PCC 7120, X59559; Nostoc sp.strain PCC 73102, AF027655; Prochloron sp., X63141; Pleurocapsa sp. strain PCC

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7516, X78681; Synechococcus sp. strain PCC 7002, D88289; Spirulina sp. strainPCC 6313, X75044; Microcystis sp. strain PCC 7941, U40340; Synechococcus sp.strain PCC 6803, D64000; and Synechococcus sp. strain C9, L35481 to L35483).Sequences for Chamaesiphon sp. strain PCC 7430, Chroococcidiopsis sp. strainPCC 7203, and Gloeobacter PCC 7421 were obtained from the Ribosomal Da-tabase Project II (http://www.cme.msu.edu/RDP).

RESULTS

Synechococcus clones isolated from Hunters Hot Springs.Twenty Synechococcus clones were isolated in two kinds ofmineral salts media from samples collected at four tempera-tures at Hunters Hot Springs (Table 1). Isolates were obtainedfrom both diluted and undiluted enrichments (Table 1). Six-teen of these clones were obtained from along the temperaturegradient of a single outflow channel (Jacks Stream). ClonesOH12, OH16, and OH19 were derived from an adjacent site atHunters Hot Springs sampled in October 1994. At this timecentral Oregon was experiencing drought conditions, andJacks Stream was dry at temperatures above ;45C. By thefollowing collection date in May 1995, the source of JacksStream was flowing again, and a microbial mat had becomereestablished at higher temperatures.

16S rRNA gene sequence groups of Hunters Hot SpringsSynechococcus clones. All of the clones isolated in this study fallinto four groups based on 100% sequence identity of an;950-bp fragment of the 16S rRNA gene. All Synechococcusclones isolated from 45C collections, as well as some clonesobtained from 55C collections, have identical sequences(group I) (Table 1). Clones representing two additional se-quence groups, groups II and III, were isolated from 55Ccollections, while all isolates from samples collected at 65C orabove have identical sequences (group IV) (Table 1).

While the 16S rRNA gene sequence data for group I Syn-echococcus isolates exhibit no more than 89.2% identity to thedata for all other sequence groups obtained in this study, thelatter groups exhibit lower levels of sequence divergence.Groups II and IV differ at 44 (4.7%) of the 944 nucleotide

positions used for phylogenetic analysis. The level of dissimi-larity between groups II and III is 3.2% (30 of 944 positions),and the level of dissimilarity between groups III and IV is 1.8%(17 of 944 positions).

In addition, none of the sequences of the Hunters HotSprings isolates is identical to any environmentally derivedcyanobacterial 16S rRNA gene sequence from Octopus Springin Yellowstone National Park (11, 13, 34), although the groupII, III, and IV sequences exhibit a high degree of similarity tothese sequences. If comparisons are restricted to a 174-bpfragment (E. coli nucleotide positions 1146 to 1319) for whichdata are available for all of the Octopus Spring sequences,group II Synechococcus differs from sequence OSB by fournucleotides. Group III Synechococcus differs from OSA by asingle nucleotide, whereas group IV Synechococcus differsfrom OSA9 at two positions.

Phylogenetic reconstructions. Cyanobacterial 16S rRNAgene sequence phylogenies were inferred by using maximum-parsimony, neighbor-joining, and maximum-likelihood meth-ods (Fig. 1). While there are topological differences among thethree phylogenies, they are similar in several important re-spects. First, Synechococcus clones belonging to sequencegroups II, III, and IV, which were all derived from collectionsobtained at 55C or above, form a clade in all three trees. Thetopology of this clade is consistent for all methods and isstrongly supported by bootstrap analysis data in all cases (Fig.1). The maximum-likelihood analysis, which provides errorestimates for the estimated evolutionary distances betweentaxa, further supports the inferred structure of this clade. Theestimated evolutionary distance between group II and IV Syn-echococcus clones is 0.051 substitution per site, with a 95%confidence interval of (0.024,0.079). The estimated evolution-ary distance between group II and III Synechococcus clones is0.038 substitution per site, with a 95% confidence interval of(0.017,0.061), while the distance between group III and IVSynechococcus clones was estimated to be 0.018 substitutionper site, with a 95% confidence interval of (0.007,0.031). The

TABLE 1. 16S rRNA gene sequence groups of Synechococcus strains isolated from Hunters Hot Springs in Oregon

Sequence groupa Clone

Collectionb Isolation

Temp (C) Date Inoculum(no. of cells) Medium Temp (C)

I OH5 45 1 Oct. 1994 10 BG11 45OH9 45 1 Oct. 1994 10 D 45OH10 45 30 May 1995 105 BG11 45OH13 45 1 Oct. 1994 10 BG11 45OH14 45 30 May 1995 10 BG11 45OH18 45 1 Oct. 1994 10 D 45OH12 55 1 Oct. 1994 106 BG11 55OH16 55 1 Oct. 1994 106 BG11 55OH19 55 1 Oct. 1994 106 BG11 55OH26 55 9 Sept. 1998 104 BG11 55

II OH4 55 30 May 1995 10 BG11 55OH20 55 30 May 1995 107 BG11 55OH31 55 9 Sept. 1998 104 D 55OH32 55 9 Sept. 1998 102 BG11 55

III OH2 55 9 Mar. 1996 1c BG11 55IV OH28 70 9 Sept. 1998 107 D 67

OH29 70 9 Sept. 1998 107 D 67OH30 70 20 Mar. 1998 107 BG11 67OH33 65 9 Sept. 1998 104 D 65OH34 65 9 Sept. 1998 104 D 65

a Clones belonging to the same sequence group have identical sequences.b Most samples were obtained from Jacks Spring; the only exception was the 55C sample collected on 1 October 1994, which was collected from an adjacent outflow.c See Materials and Methods.

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fact that none of the confidence intervals overlaps zero pro-vides strong evidence that a statistically significant evolutionarydistance separates each pair of taxa and, therefore, that the16S rRNA gene fragment used in this study contains sufficientgenetic variation to resolve the phylogeny of the Hunters HotSprings sequence groups.

In addition, sequence groups II, III, and IV are part of alarger clade which includes Synechococcus sp. strains SH-94-5and C9 (Fig. 1). The former strain was isolated from a sample

collected at 50C from South Harney Hot Springs in Oregon,;150 km northeast of Hunters Hot Springs (unpublisheddata), while the latter strain was isolated from Octopus Springin Yellowstone National Park (12). Group I Synechococcusclones from Hunters Hot Springs fall outside this clade in allthree phylogenies (Fig. 1). This indicates that Synechococcusclones collected along the Hunters Hot Springs environmentaltemperature gradient are not monophyletic (i.e., they do notshare a common ancestor to the exclusion of all other taxa) andthat Synechococcus clones from samples collected at tempera-tures greater than 55C were not derived from an ancestorresembling the group I Synechococcus clones predominant atlower temperatures. The fact that the genus Synechococcus, amorphologically defined taxonomic unit encompassing a di-verse assemblage of taxa ranging from the hot spring repre-sentatives to open-ocean picoplankton, is not a natural grouphas long been recognized by cyanobacterial taxonomists, as hasthe need for revision of the classification scheme of theseunicellular organisms to reflect their phylogeny (33).

Temperature range of group I Synechococcus clones. Growthrate data were collected at temperatures between 30 and 60Cfor five group I clones isolated from 45C collections (Table 2).No differences were found among the clones in terms ofgrowth temperature range; the highest temperature for growthwas estimated to be 57C, and the lower limit was less than30C (Table 2). However, there was variation in the meangrowth rate among temperatures (Table 3). One-way ANOVAswere performed for each temperature treatment to partitionthis variation into among-clone and within-clone components.No clone effect was found at temperatures between 40 and55C (Table 3). The estimated mean growth rates at these

FIG. 1. Cyanobacterial phylogenies inferred from ;950 bp of 16S rRNAgene sequence data by using neighbor-joining (A), maximum-parsimony (B), andmaximum-likelihood (C) methods. Synechococcus clones from Hunters HotSprings are indicated by capital letters. A value at a node indicates the percent-age of the time that the taxa to the right of the node formed a clade for 100 (Aand C) or 1,000 (B) bootstrap pseudoreplicates. Only bootstrap values greaterthan 50% are indicated.


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temperatures (Table 3) could therefore be directly comparedby using Bonferroni tests. The growth rate estimates increasedin the order 55C , 40C (550C) , 45C, which indicates thatthe optimal growth temperature of these group I clones was45C. The relative amount of variation in the growth rate (i.e.,the coefficient of variation) increased at the temperature rangeextremes, and differences among clones explained much of thisvariation, as indicated by the high R2 values (Table 3). Theexistence of genetic variation in the growth rate among clonesat the extremes of the temperature range may indicate that theorganisms do not typically experience these temperatures insitu; i.e., this genetic variation is not expressed phenotypicallyand can therefore accumulate without being subject to naturalselection. Otherwise, it would be expected that this geneticdiversity would be reduced by natural selection, as appears tobe the case at intermediate temperatures, including the opti-mal temperature (Table 3). An alternative explanation for thispattern is that the variation was generated by mutation duringlaboratory cultivation. Distinguishing between these two hy-potheses will require further experimentation.

Some growth rate data were also determined for two groupI clones isolated from 55C collections (OH16 and OH26)(Table 2). These clones did not show any significant differencesin performance compared with the 45C clones, as determinedby Bonferroni tests comparing growth estimates obtained at 55and 57C for these clones with growth estimates obtained at 55and 57C for 45C clones. The demonstration that these clonesalso could not survive at temperatures above 57C indicatesthat although they were collected from samples having a tem-

perature higher than the temperatures of the samples thatyielded the other group I clones examined, they do not have anextended thermal limit.

Temperature range diversification in the group II-groupIII-group IV clade. The two group II Synechococcus cloneswhich were examined (OH4 and OH20) grew at temperaturesbetween 40 and 61C (Table 4). ANOVA with clone and tem-perature as factors indicated that there was no difference be-tween the clones in the exponential growth rate (F[1,16] 5 0.02;P 5 0.89) and no clone-by-temperature interaction (F[7,16] 50.77; P 5 0.62); i.e., the two clones had the same temperatureresponse profile. Growth data for these clones were thereforepooled, and estimated growth rates at different temperatureswere compared with Bonferroni tests. The mean growth rateincreased in the order 40C , 61C (5 45C 5 50C 5 55C) ,57C, which indicates that the optimum temperature forgrowth of these clones is 57C.

The single group III Synechococcus clone obtained in culture(OH2) grew at temperatures between 45 and 63C (Table 4).This clones upper and lower thermal limits are thus higherthan those of the group II clones. However, no detectabledifference in optimal temperature or in maximum growth ratewas observed between group II and III Synechococcus clones(Table 4).

Group IV Synechococcus clones exhibited the greatest ther-motolerance. Growth of the three clones tested occurred attemperatures between 55 and 70C, and optimal growth oc-curred at 65C, although the value was not significantly differ-ent from the value obtained at 60 or 70C (Table 4).

Growth rates were also determined for Synechococcus sp.strains SH-94-5 and C9 (12) (C9 was a gift from David Ward,Montana State University), taxa belonging to the inferred sis-ter group of Synechococcus groups II, III, and IV. Thesestrains, which are ;1% divergent in the 16S rRNA gene frag-ment used for phylogenetic analysis, did not exhibit statisticallysignificant differences in their responses to temperature, asindicated by the lack of a clone effect (F[1,22] 5 0.15; P 5 0.71)or a clone-temperature interaction (F[5,22] 5 1.03; P 5 0.41) inan ANOVA. Growth was observed at temperatures between 30and 55C (Table 4). Both clones died at 57C and therefore hada lower maximum temperature than the group I clones.

Evolution of enhanced thermotolerance in Synechococcus.The phylogenetic branching order of lineages which have dif-ferent phenotypes for a particular trait provides informationon the direction of phenotypic change during cladogenesis(21). For the clade comprising group II, III, and IV Synecho-coccus clones and their inferred sister taxa (Fig. 1), the leastthermotolerant lineages are basal, while isolates with greaterthermotolerance branch later along the phylogeny (Fig. 2).

TABLE 2. Exponential growth rates of group I Synechococcus clones at different temperatures

Temp (C)Exponential growth rate (no. of doublings day21) ofa:

Clone OH5 Clone OH9 Clone OH10 Clone OH14 Clone OH18 Clone OH16 Clone OH26

30 0.28 6 0.036 0.27 6 0.020 0.67 6 0.012 0.75 6 0.012 0.76 6 0.020 NDb ND35 1.34 6 0.124 1.05 6 0.032 1.55 6 0.080 1.43 6 0.080 1.81 6 0.080 ND ND40 1.84 6 0.108 1.92 6 0.064 2.10 6 0.088 1.94 6 0.056 2.19 6 0.112 ND ND45 2.24 6 0.124 2.51 6 0.151 2.40 6 0.148 2.42 6 0.044 2.85 6 0.044 ND ND50 2.20 6 0.036 2.20 6 0.032 2.26 6 0.120 2.05 6 0.036 1.93 6 0.223 ND ND55 1.43 6 0.036 2.05 6 0.183 1.46 6 0.187 1.65 6 0.020 1.49 6 0.144 2.18 6 0.052 1.93 6 0.02457 0.66 6 0.000 1.24 6 0.205 1.17 6 0.144 1.40 6 0.104 2.00 6 0.355 1.25 6 0.084 1.36 6 0.06460 0 0 0 0 0 0 0

a Mean 6 standard error.b ND, not determined.

TABLE 3. Summary statistics for growth rate data and ANOVAmodels for group I Synechococcus clones grown at

different temperatures

Temp(C)

No. of doublingsday21a

Coefficient ofvariationa

ANOVA modelb

Fc R2

30 0.55 6 0.075 44.5 6 9.71 149.4*** 0.99235 1.44 6 0.086 19.4 6 4.24 17.1** 0.93240 2.00 6 0.052 8.5 6 1.85 2.7 045 2.48 6 0.078 10.1 6 2.21 4.1 050 2.13 6 0.056 8.5 6 1.86 1.4 055 1.62 6 0.091 18.2 6 3.97 3.0 057 1.30 6 0.157 39.4 6 8.59 6.0* 0.829

a Mean 6 standard error.b One-way ANOVA was used to model the effect of the clone on the observed

variation in the growth rate at each temperature. Nonsignificant F values and R2

values of zero indicate no effect.c Significance levels are based on an F[4,5] distribution: p, P , 0.05; pp, P ,

0.01; ppp, P , 0.001.


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This pattern of diversification supports the hypothesis thatSynechococcus strains capable of growing at increasingly highertemperatures evolved from less thermotolerant ancestors. Thisis the pattern of diversification expected if there has been anadaptive radiation of Synechococcus temperature strains up thethermal gradient in response to past directional selection alonga hot spring outflow channel. The common ancestor of thesethree groups, however, was not group I-like in terms of itsphylogenetic status, and a comparable radiation has not oc-curred in the group I Synechococcus lineage (Fig. 1; Table 2).

Evidence for trade-offs. The observation that more thermo-philic lineages of Synechococcus have lost the ability to grow atlower temperatures (Fig. 2) is evidence that there have beenevolutionary costs of adapting to high temperatures. Wewished to determine whether there may have been additionaltrade-offs during the evolution of thermotolerance in thisclade. This can be assessed by testing whether different aspectsof thermal performance are negatively correlated across taxa(18). However, because the study organisms are related to eachother to different degrees due to common ancestry, phenotypicdata collected from them may be nonindependent and conse-quently must first be transformed to meet the assumption ofcorrelational analysis that data points are independent. Weused Felsensteins (9) method of independent contrasts (seeMaterials and Methods) to transform the data prior to esti-mating correlations.

We estimated correlation coefficients for the following ther-mal performance traits: optimal temperature, maximum tem-perature, maximum growth rate, and temperature range. The

TA

BL

E4.

Grow

thrates

ofgroup

II,III,andIV

Synechococcusclones

andinferred

sistertaxa

atdifferenttem

peratures

Tem

p(C

)

Grow

thrates

(no.ofdoublings

day2

1) a

Group

IIclones

Group

IIIclone

Group

IVclones

Sistertaxa

OH

4O

H20

PoolO

H2

OH

28O

H29

OH

30Pool

SH-94-5

C9

30N

Db

ND

ND

ND

ND

ND

ND

ND

0.376

0.0200.20

60.020

350

00

ND

ND

ND

ND

ND

0.576

0.0080.60

60.020

400.42

60.064

0.676

0.0400.55

60.078

0N

DN

DN

DN

D1.20

60.016

0.946

0.06445

0.836

0.1590.94

60.108

0.886

0.0840.74

60.012

ND

ND

ND

ND

1.086

0.3591.43

60.303

50N

DN

DN

DN

D0

00

0N

DN

D55

1.116

0.1241.09

60.028

1.106

0.0521.36

60.175

0.266

0.0400.45

60.016

0.366

0.0960.36

60.045

1.056

0.1080.91

60.088

571.73

60.207

1.576

0.0521.65

60.099

1.606

0.028N

DN

DN

DN

D0

061

0.996

0.1950.81

60.231

0.906

0.1341.36

60.012

0.636

0.0160.55

60.060

0.416

0.0800.53

60.045

ND

ND

630

00

0.936

0.056N

DN

DN

DN

DN

DN

D65

ND

ND

ND

00.64

60.028

0.746

0.0720.77

60.020

0.726

0.032N

DN

D70

ND

ND

ND

ND

0.696

0.0600.54

60.179

0.696

0.0400.64

60.059

ND

ND

73N

DN

DN

DN

D0

00

0N

DN

D

aM

ean6

standarderror.

bN

D,not

determined.

FIG. 2. Phylogenetic pattern of thermotolerance diversification in the groupII-group III-group IV Synechococcus clade.


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values for each sequence group (Table 5) were derived fromthe growth rate data (Table 4). The optimal temperature is thetemperature at which the maximum growth rate was estimated,the maximum temperature is the highest observed temperaturefor growth, the maximum growth rate is the growth rate esti-mated at the optimal temperature, and the temperature rangeis the estimated difference between the observed maximumtemperature and minimum temperature. Independent con-trasts for the above traits were estimated separately by usingbranch length data from the three cyanobacterial phylogenies(Fig. 1), and correlation coefficients were then estimated forpairs of contrasts (Table 5). In all cases, significant negativecorrelations were found between an organisms maximum tem-perature and its temperature range. This indicates that en-hanced thermotolerance may have come at the cost of in-creased thermal specialization. The maximum growth rate waspositively correlated with the temperature range in all threeanalyses and was negatively correlated with the maximum tem-perature in the neighbor-joining analysis (Table 5). These re-sults suggest that a reduced maximum growth rate may alsohave been a consequence of evolved thermotolerance.

DISCUSSION

A hot spring outflow produces a thermal gradient whichdirectly selects for enhanced performance at higher tempera-tures. Phylogenetic analyses of ;950 nucleotides of the 16SrRNA gene were able to resolve a clade of increasingly morethermotolerant lineages of the cyanobacterium Synechococcuswhich have radiated in response to this directional selection toexploit previously inhospitable thermal niches (Fig. 2). Whileextension of the maximum temperature was integral to theevolution of thermotolerance in Synechococcus, there was notnecessarily a corresponding increase in the optimal tempera-ture, as evidenced by the lack of a detectable difference in thistrait between group II clones and group III clone OH2 (Tables4 and 5). The minimum temperature, however, has clearlyshifted to higher values along with the maximum temperature(Fig. 2). Although we cannot infer the underlying mecha-nism(s) for this apparent trade-off, this is the expected patternin the case of antagonistic pleiotropy in which there has beenevolution of a gene or genes influencing performance at bothends of the thermal tolerance range (17). These genes couldinclude many enzyme-encoding loci whose products are subjectto thermodynamic constraints on performance (1). Becauseperformance at lower temperatures decreased faster than per-formance at higher temperatures increased, the net evolution-ary result has been a reduction in the breadth of the temper-ature range (Table 5), which further indicates that adaptation

of Synechococcus to higher temperatures has come at the costof increased thermal specialization.

The increase in thermal specialization may be related to thebiogeographical structure suggested by the lack of identitybetween the 16S rRNA gene sequences of group II, III, and IVSynechococcus clones and clones from Octopus Spring in Yel-lowstone National Park. Hot spring Synechococcus isolateshave low tolerance for freezing and desiccation, factors likelyto be important in dispersal (6, 8). The more rapid loss oftolerance to lower temperatures during adaptation to highertemperatures may have limited the ability of cells to survivedispersal between habitats. Increased ecological specializationmay therefore be an agent of geographical isolation.

It is worthwhile to compare the evolutionary trends revealedby this study with results obtained with E. coli lines selected forenhanced thermotolerance. A clone was more likely to extendits thermal limit if it was exposed to a lethal temperature(44C) than if it was exposed to a high but nonlethal temper-ature (42C) (2, 24). However, clones previously propagated at42C tended to be more predisposed to evolving an extendedthermal limit upon lethal selection than were clones formerlymaintained at the ancestral temperature, 37C (24). An anal-ogous predisposition for more thermotolerant lines to be de-rived from lines with a previous history of exposure to elevatedtemperatures has been recorded in the topology of the Syn-echococcus clade (Fig. 2). On the other hand, in contrast to thepattern suggested by our comparative data (Table 5), Mongoldet al. (24) found that extension of the thermal limit in E. coliwas not necessarily accompanied by a trade-off with perfor-mance at the lower thermal limit.

Directional selection still acts on Synechococcus at the ther-mal limit in nature, yet there is no evidence of positive popu-lation growth at temperatures above 73C (3, 4). Since manynonphotosynthetic prokaryotes can grow at temperatureshigher than 73C, it has been suggested that the lack of aresponse to selection may be due to intrinsic limits in thestability of the photosynthetic apparatus (3). However, studieson a Synechococcus isolate from Hunters Hot Springs with agroup IV-like phenotype indicated that carbon reduction wasmore thermolabile than photosynthetic oxygen evolution (23).Clearly, more information is needed to determine the factor(s)underlying the upper temperature limit for photosynthesis.Having established a phylogenetic framework in which to studythermotolerance in Synechococcus, we are now in a position totrace the mechanistic history of biochemical adaptation in thisclade and to investigate the possible evolutionary constraintswhich have prevented a further response to natural selection.

TABLE 5. Analysis of correlated character evolution during the diversification of temperature range by Synechococcusa

Characterb Group II Group III Group IV CloneSH-94-5 Clone C9

Correlation coefficientsc

Maximum parsimony Neighbor joining Maximum likelihood

Tmax Ratemax Range Tmax Ratemax Range Tmax Ratemax Range

Topt 57 57 65 40 45 0.80 20.52 20.73 0.81 20.56 20.75 0.79 20.53 20.73Tmax 61 63 70 55 55 20.87 20.96* 20.89* 20.97* 20.88 20.97*Ratemax 1.65 1.60 0.72 1.20 1.43 0.96* 0.96* 0.96*Range 21 18 15 25 25

a Phenotypic data for the five sequence groups were transformed into four independent contrasts as described by Felsenstein (9). Branch length data from the threephylogenies in Fig. 1 were used in separate transformations, and correlations were determined for contrast pairs.

b Optimal temperature (Topt), maximum temperature (Tmax), and temperature range (Range) are expressed in degrees Centigrade. The units for maximum growthrate (Ratemax) are number of doublings per day. Character values were estimated from Table 4 as described in the text.

c An asterisk indicates that the correlation coefficient is significantly different from zero at the a 5 0.05 level as determined by a t test with df 5 2.


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ACKNOWLEDGMENTS

We thank Emlia Martins, Stephen Giovannoni, Doug Gordon,Kevin Vergin, and Tracie-Lynn Nadeau for training, advice, and valu-able discussions. We also thank two anonymous reviewers for theircomments and suggestions.

This work was supported by National Science Foundation grantIBN-9630674 to R.W.C. and by a National Science Foundation train-ing grant to study the genetic mechanisms of evolution to S.R.M.

REFERENCES

1. Alexandrov, Y. Y. 1977. Cells, molecules and temperature. Springer-Verlag,New York, N.Y.

2. Bennett, A. F., and R. E. Lenski. 1993. Evolutionary adaptation to temper-ature. II. Thermal niches of experimental lines of Escherichia coli. Evolution47:112.

3. Brock, T. D. 1967. Micro-organisms adapted to high temperatures. Nature214:882885.

4. Castenholz, R. W. 1969. Thermophilic blue-green algae and the thermalenvironment. Bacteriol. Rev. 33:476504.

5. Castenholz, R. W. 1973. Ecology of blue-green algae in hot springs, p.379414. In N. G. Carr and B. A. Whitton (ed.), The biology of blue-greenalgae. Blackwell Scientific Publications, Oxford, United Kingdom.

6. Castenholz, R. W. 1978. The biogeography of hot spring algae throughenrichment cultures. Mitt. Int. Ver. Limnol. 21:296315.

7. Castenholz, R. W. 1988. Culturing methods for cyanobacteria. MethodsEnzymol. 167:6893.

8. Castenholz, R. W. 1996. Endemism and biodiversity of thermophilic cya-nobacteria. Nova Hedwigia 112:3347.

9. Felsenstein, J. 1985. Phylogenies and the comparative method. Am. Nat.125:115.

10. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c.Department of Genetics, University of Washington, Seattle.

11. Ferris, M. J., G. Muyzer, and D. M. Ward. 1996. Denaturing gradient gelelectrophoresis profiles of 16S rRNA-defined populations inhabiting a hotspring microbial mat community. Appl. Environ. Microbiol. 62:340346.

12. Ferris, M. J., A. L. Ruff-Roberts, E. D. Kopczynski, M. M. Bateson, andD. M. Ward. 1996. Enrichment culture and microscopy conceal diversethermophilic Synechococcus populations in a single hot spring microbial mathabitat. Appl. Environ. Microbiol. 62:10451050.

13. Ferris, M. J., and D. M. Ward. 1997. Seasonal distributions of dominant 16SrRNA-defined populations in a hot spring microbial mat examined by de-naturing gradient gel electrophoresis. Appl. Environ. Microbiol. 63:13751381.

14. Gutell, R. R., B. Weiser, C. R. Woese, and H. F. Noller. 1985. Comparativeanatomy of 16S-like ribosomal RNA. Prog. Nucleic Acids Res. Mol. Biol.32:155216.

15. Hansen, T. F., and E. P. Martins. 1996. Translating between microevolu-tionary process and macroevolutionary patterns: the correlation structure ofinterspecific data. Evolution 50:14041417.

16. Harvey, P. H., and M. D. Pagel. 1991. The comparative method in evolu-tionary biology. Oxford University Press, Oxford, United Kingdom.

17. Huey, R. B., and J. G. Kingsolver. 1989. Evolution of thermal sensitivity ofectotherm performance. Trends Ecol. Evol. 4:131135.

18. Huey, R. B., and J. G. Kingsolver. 1993. Evolution of resistance to hightemperature in ectotherms. Am. Nat. 142:S21S46.

19. Kimura, M. 1980. A simple method for estimating evolutionary rate of basesubstitution through comparative studies of nucleotide sequences. J. Mol.Evol. 16:111120.

20. Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: Molecular EvolutionaryGenetics Analysis, version 1.0. The Pennsylvania University, University Park.

21. Martins, E. P., and T. F. Hansen. 1996. Phylogenetic comparative methods:the statistical analysis of interspecific data, p. 2275. In E. P. Martins (ed.),Phylogenies and the comparative method in animal behavior. Oxford Uni-versity Press, Oxford, United Kingdom.

22. Meeks, J. C., and R. W. Castenholz. 1971. Growth and photosynthesis in anextreme thermophile, Synechococcus lividus (Cyanophyta). Arch. Mikrobiol.78:2541.

23. Meeks, J. C., and R. W. Castenholz. 1978. Photosynthetic properties of theextreme thermophile Synechococcus lividus. II. Stoichiometry between oxy-gen evolution and CO2 assimilation. J. Therm. Biol. 3:1924.

24. Mongold, J. A., A. F. Bennett, and R. E. Lenski. 1999. Evolutionary adap-tation to temperature. VII. Extension of the upper thermal limit of Esche-richia coli. Evolution 53:386394.

25. Nubel, U., F. Garcia-Pichel, and G. Muyzer. 1997. PCR primers to amplify16S rRNA genes from cyanobacteria. Appl. Environ. Microbiol. 63:33273332.

26. Peary, J. A., and R. W. Castenholz. 1964. Temperature strains of a thermo-philic blue-green alga. Nature 202:720721.

27. Pitcher, D. G., N. A. Saunders, and R. J. Owen. 1989. Rapid extraction ofbacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol.8:151156.

28. Swofford, D. L. 1996. PAUP 3.1.1. Sinauer Associates, Sunderland, Mass.29. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:

improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 22:46734680.

30. Titus, T. A., and D. R. Frost. 1996. Molecular homology assessment andphylogeny in the lizard family Opluridae (Squamata: Iguania). Mol. Phylo-genet. Evol. 6:4962.

31. Urbach, E., D. Robertson, and S. W. Chisholm. 1992. Multiple evolutionaryorigins of prochlorophytes within the cyanobacterial radiation. Nature 355:267269.

32. Ward, D. M., M. J. Ferris, S. C. Nold, and M. M. Bateson. 1998. A naturalview of microbial diversity within hot spring cyanobacterial mat communi-ties. Microbiol. Mol. Biol. Rev. 62:13531370.

33. Waterbury, J. B., and R. Rippka. 1989. Subsection I. Order Chroococcales, p.17281746. In J. G. Holt, J. T. Staley, M. P. Bryant, and N. Pfennig (ed.),Bergeys manual of systematic bacteriology, vol. 3. The Williams and WilkinsCo., Baltimore, Md.

34. Weller, R., M. M. Bateson, B. K. Heimbuch, E. D. Kopczynski, and D. M.Ward. 1992. Uncultivated cyanobacteria, Chloroflexus-like inhabitants, andspirochete-like inhabitants of a hot spring microbial mat. Appl. Environ.Microbiol. 58:39643969.

35. Wheeler, W., and D. Gladstein. 1994. MALIGN: a multiple sequence align-ment program. J. Hered. 85:417418.

36. Whitton, B. A., and M. Potts (ed.). 1999. Ecology of cyanobacteria: theirdiversity in time and space. Kluwer Academic Publishers, Dordrecht, TheNetherlands.

37. Wilmotte, A., G. Van der Auwera, and R. De Wachter. 1993. Structure of the16S ribosomal RNA of the thermophilic cyanobacterium ChlorogloeopsisHTF (Mastigocladus laminosus HTF) strain PCC 7518, and phylogeneticanalysis. FEBS Lett. 317:96100.


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