evolutionary relationships among and greenchloroplasts · evolutionary relationships...

Vol. 170, No. 8JOURNAL OF BACTERIOLOGY, Aug. 1988, p. 3584-35920021-9193/88/083584-09$02.00/0Copyright C) 1988, American Society for Microbiology

Evolutionary Relationships among Cyanobacteriaand Green Chloroplasts

STEPHEN J. GIOVANNONI, SEAN TURNER, GARY J. OLSEN,t SUSAN BARNS,§DAVID J. LANE,§ AND NORMAN R. PACE*

Department of Biology and Institute for Molecular and Cellular Biology,Indiana University, Bloomington, Indiana 47405

Received 25 January 1988/Accepted 20 May 1988

The 16S rRNAs from 29 cyanobacteria and the cyanelle of the phytoflagellate Cyanophora paradoxa were

partially sequenced by a dideoxynucleotide-terminated, primer extension method. A least-squares distancematrix analysis was used to infer phylogenetic trees that include green chloroplasts (those of euglenoids, green

algae, and higher plants). The results indicate that many diverse forms of cyanobacteria diverged within a shortspan of evolutionary distance. Evolutionary depth within the surveyed cyanobacteria is substantially less thanthat separating the major eubacterial taxa, as though cyanobacterial diversification occurred significantly afterthe appearance of the major eubacterial groups. Three of the five taxonomic sections defined by Rippka et al.(R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier, J. Gen. Microbiol. 111:1-61,1979) (sections II [pleurocapsalean], IV [heterocystous, filamentous, nonbranching], and V [heterocystous,filamentous, branching]) are phylogenetically coherent. However, the other two sections (I [unicellular] and III[nonheterocystous, filamentous]) are intermixed and hence are not natural groupings. Our results not onlysupport the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherentphylogenetic group but also suggest that the chloroplast lineage, which includes the cyanelle of C. paradoxa, isnot just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.

The cyanobacteria are one of the most morphologicallydiverse and conspicuously successful procaryotic groups. Itis generally believed that the cyanobacteria were the firstmajor group of phototrophs to arise with a two-stage photo-synthetic pathway capable of oxidizing water to producemolecular oxygen. Geochemical and fossil evidence indi-cates that in the Precambrian Era they caused the transitionin the Earth's atmosphere from its primordial, anaerobicstate to its current, aerobic condition (19, 36, 43). Moreover,molecular phylogenetic analysis of c-type cytochrome andrRNA sequences have established a relationship betweencyanobacteria and the green (euglenoids, green algae, andhigher plants) and red (rhodophyte) chloroplasts, thus sup-porting the procaryotic origins of chloroplasts.Because of their ubiquity, rRNA sequences are particu-

larly useful for establishing evolutionary relationshipsamong diverse organisms. Woese and colleagues, usingpartial (RNase Tl-generated oligonucleotide catalogs) andcomplete 16S rRNA sequences, have defined about 10 majordivisions (phyla) of eubacteria (45). The cyanobacteria areone of these phyla. However, too few strains (eight) ofcyanobacteria had been investigated to develop a compre-hensive overview of the diversity of the group.We have used a method for directly sequencing 16S rRNA

to explore the evolutionary relationships among 30 represen-tatives of the diverse cyanobacterial groups, including thephotosynthetic organelle of the phytoflagellate Cyanophoraparadoxa. The results shed new light on the relative ages of

* Corresponding author.t Present address: Department of Microbiology, Oregon State

University, Corvallis, OR 97331.t Present address: Department of Microbiology, University of

Illinois, Urbana, IL 61801.§ Present address: Gene-Trak Systems, Framingham, MA 01701.

the cyanobacteria and other eubacterial lineages and on theorigins of chloroplasts.

MATERIALS AND METHODS

Organisms and cultivation. Cultures or cell paste of allPasteur Culture Collection strains were provided by John B.Waterbury (Woods Hole Oceanographic Institution). AllCastenholz Culture Collection strains were supplied as cul-tures by Richard H. Castenholz (University of Oregon).Cultures of Oscillatoria limnetica (Solar Lake) and Micro-coleus sp. strain 10 mfx were from Yehuda Cohen (H.Steinitz Marine Biology Laboratory, Eilat, Israel). Cultureswere maintained in BG-11 medium (34). C. paradoxa cya-nelle RNA, provided by Stuart Maxwell (North CarolinaState University) and Jessup M. Shively (Clemson Univer-sity), was prepared as described by Starnes et al. (40).

Preparation of RNA for sequencing. High-molecular-weight cellular RNAs were prepared for sequencing aspreviously described (25). Briefly, cell pellets (0.3 to 3.0 g[wet weight]) were removed from storage at -70°C, thawed,and lysed by one or two passages through a French pressurecell. RNA was purified by extraction with phenol, precip-itated with ethanol, and suspended. Overnight precipitationfrom 1.0 M NaCl at 0°C was used to prepare high-molecular-weight RNA.RNA sequencing. Dideoxynucleotide-terminated sequenc-

ing, using reverse transcriptase and synthetic oligodeoxynu-cleotide primers complementary to conserved 16S rRNAsequences, was carried out as described by Lane et al. (25;D. J. Lane, K. G. Field, G. J. Olsen, and N. R. Pace,Methods Enzymol., in press). Three "universal' smallsubunit rRNA sequencing primers (complementary to Esch-erichia coli 16S rRNA sequence positions 519 to 536, 907 to926, and 1392 to 1406) were used to determine ca. 1,000nucleotides of each rRNA sequence. The sequences have

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PHYLOGENY OF CYANOBACTERIA AND CHLOROPLASTS

been deposited with GenBank and are available from theauthors upon request.

Sequence analysis. Sequences were manually aligned onthe basis of conserved sequence and secondary structuralelements. Regions of ambiguous sequence alignment wereomitted from subsequent analyses. For each pair of se-quences, the number of nucleotide differences was used toestimate the average number of fixed point mutations persequence position that has accumulated since their diver-gence (22). This is called the "evolutionary distance" sepa-rating the contemporary sequences (Fig. 1 and 2, lower left).The statistical uncertainty of the pairwise evolutionary dis-tance estimates was determined by the method of Kimuraand Ohta (23) (Fig. 1 and 2, upper right). A least-squaresmethod was used to infer the phylogenetic tree most con-sistent with the pairwise distance estimates and their statis-tical uncertainties (30).

RESULTS AND DISCUSSIONRelationships among free-living cyanobacteria. The unroot-

ed phylogenetic tree in Fig. 3 broadly depicts evolutionaryrelationships of oxygenic, phototrophic bacteria and organ-elles to other representatives of the three primary lines ofdescent: the archaebacteria, the eucaryotes, and the eubac-teria. The diversity among cyanobacteria and chloroplasts isindicated by the depth of the hatching. The cyanobacteriaand chloroplasts together make up 1 of approximately 10major eubacterial taxa (not all of which are represented inthis tree). The rRNA sequence diversity within the cyano-bacterial lineage is substantially less than the separationsbetween the major eubacterial taxa. In contrast, the se-quence diversity observed within most other eubacterialrRNA phyla is nearly equal to interphylum evolutionarydistances (45). Interphylum evolutionary distances for rep-resentative eubacteria vary from 0.19 to 0.30 fixed mutationsper sequence position (Fig. 1). Intraphylum evolutionarydistances among the cyanobacteria are typically muchsmaller, about 0.15 mutations per sequence position. Thus,relatively close phylogenetic relationships underlie the ex-traordinary morphological diversity of cyanobacteria.The phylogenetic tree in Fig. 4 depicts inferred relation-

ships among the nonheterocystous cyanobacteria inspected.A diverse representation of cyanobacteria was selected forsequencing on the basis of available taxonomic, morpholog-ical, and physiological information. Many of the lineagesbranch at similar depths in the cyanobacterial tree, lendingthe tree a fan-like aspect. This result indicates that manymodern cyanobacterial lineages arose in an expansive evo-lutionary radiation. The nearly equivalent depths of many ofthese branchings render their detailed relationships(branching orders) unresolved. This is because the uncer-tainty in the evolutionary distances separating pairs oforganisms increases with the distance, a consequence ofuncertainty in the number of multiple mutations of individualresidues. The statistical errors for the calculated evolution-ary distances separating pairs of organisms are included inFig. 2. The relative positions of the nodes in the tree aremore accurate than suggested by Fig. 2, because the uncer-tainties are not independent and some of the uncertaintyapplies only to the lengths of peripheral branches, not to thelengths of the branchings deeper in the tree. Although theprecise branching order of the short segments near the rootof the cyanobacterial tree is uncertain, significant relation-ships emerge.The morphological complexity of cyanobacteria served as

a primary basis of the classification system of Rippka et al.

(34), which divides the cyanobacteria into five sections. Theunicellular cyanobacteria constitute section I. Members ofsection II, the pleurocapsalean cyanobacteria, share a com-mon developmental feature, the capacity of large cells tosubdivide internally, producing numerous smaller cells(baeocytes). Section III incorporates the filamentous, non-heterocystous organisms. Sections IV and V are, respec-tively, the nonbranching and branching filamentous formsthat are capable of forming heterocysts (specialized, nitro-gen-fixing cells).The deepest branches so far in the cyanobacterial tree are

represented by Gloeobacter violaceus (section I) and twoclosely related strains of the genus Pseudanabaena (sectionIII) (Fig. 4). G. violaceus is unique among cyanobacteriabecause of its lack of thylakoids and the unusual structure ofits phycobilisomes. The photosynthetic reaction centers arecontained in the cytoplasmic membrane with the phycobili-somes arranged in an underlying cortical layer (17). Theearly divergence of this organism from the main cyanobac-terial lineage is consistent with this remarkably differentlight-harvesting apparatus. The result suggests that the com-mon ancestor of the modern cyanobacteria may have lackedthylakoids. Unlike G. violaceus, the Pseudanabaena strainsinspected have no obvious characteristics to suggest an earlyevolutionary divergence from the main cyanobacterial line.Biochemical and ultrastructural studies of these strains mayreveal additional features that distinguish them from theother cyanobacteria and may clarify the phylogenetic rela-tions among them (15, 16).The unicellular cyanobacteria of section I and the nonhe-

terocystous, filamentous organisms of section III are dis-persed throughout the tree (Fig. 4), indicating that thesemorphotypes have multiple evolutionary origins. Thus, tax-onomic classifications based principally on morphology donot necessarily reflect phylogenetic relationships.The orientation of cell division planes has been used as a

further basis for subdividing the unicellular cyanobacteriainto several genera. An earlier study of RNase T1-generated16S rRNA oligonucleotide catalogs appeared to support thisdefinition of natural relationships (5). However, the addi-tional sequence data gathered during the present study led usto conclude that the various modes of cell division are notwell correlated with phylogenetic groups. An example inFig. 4 is the local cluster that contains Gloeothece sp. strainPCC 6501 (cell division in one plane), Synechocystis sp.strain PCC 6308 (cell division in two planes), and Synechocys-tis (Eucapsis) sp. strain PCC 6906 and Gloeocapsa sp. strainPCC 73106 (cell division in three planes). This cluster alsocontains the section III filamentous cyanobacterium Spiru-lina sp. strain PCC 6313 (cell division in one plane). Othercellular properties are consistent with the conclusion thatdivision planes do not define phylogenetic groups. Forinstance, the DNA base compositions of unicellular cyano-bacteria that divide in one plane span a broad range (35 to 71mol% G+C) (20). Phylogenetic studies that include addi-tional genera are likely to uncover still greater diversityamong unicellular cyanobacteria.Even a conspicuous feature such as the oscillatorian

morphotype may not indicate a relatedness group, to theexclusion of other organisms. Although some clustering ofOscillatoria spp. is indicated (e.g., 0. limnetica and PCC7515), other Oscillatoria spp. are scattered throughout thetree (e.g., 0. amphigranulata). Some physiological traits areconsistent with the rRNA sequence diversity seen in thisgroup. For instance, both 0. limnetica and 0. amphigranu-lata are able to use sulfide, as well as water, as a source of

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ARCHAEBACTERIA

Selflbus soffatcus

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EUKARYOTES EUBACTERIAFIG. 3. Unrooted phylogenetic tree illustrating evolutionary relationships among diverse 16S-like rRNAs (adapted from data in reference

30). The hatched area depicts evolutionary diversity among oxygenic, phototrophic bacteria and organelles. Segment lengths are proportionalto evolutionary distances. The scale bar corresponds to 0.1 fixed point mutations per sequence position. This topology is based on an analysisof 920 nucleotides from complete sequences.

electrons for noncyclic photosynthesis. However, the detailsof sulfide adaptation in these two strains differ (8, 9), andthey are not close relatives (Fig. 4). The LPP subgroup offilamentous cyanobacteria (section III) was provisionallycreated to encompass organisms which did not fit strictlywithin the morphological definitions of the genera Oscillato-ria, Pseudanabaena, and Spirulina (34). Here the group isrepresented by Lyngbya sp. strain PCC 7419, Plectonemasp. strain PCC 73110, and Phormidium sp. strain PCC 7375,which do not cluster phylogenetically (Fig. 4). A Lyngbyasp. (LPP group A; sheathed, nonmotile except for hormogo-nia) branches deeply but appears to be related to a phyloge-netic subgroup containing other filamentous genera, includ-ing two Oscillatoria spp. and a Microcoleous sp.The pleurocapsalean organisms (section II) studied so far

constitute a coherent, phylogenetic subgroup, to the exclu-sion of other cyanobacteria (Fig. 4). Although only three ofsix genera of this section have been analyzed, all the strainsin the section have very similar DNA base compositions(16). This and their common mode of reproduction distin-guish them from all other cyanobacteria, suggesting thatreproduction by multiple fission (baeocyte formation) is ofmonophyletic origin.

Similarly, the members of sections IV and V, the hete-rocystous, filamentous forms, constitute a distinct phyloge-netic group. The organisms of section V are a subgrouparising from within the lines of section IV (Fig. 5). Thisconfirms similar conclusions drawn from DNA-DNA hybrid-ization data (24). The organisms of sections IV and V areamong the most morphologically diverse of the cyanobacte-ria. Aside from heterocysts, many strains also produceakinetes, "resting" cells that develop as a result of insuffi-cient light or other factors (35). The strains shown in Fig. 5that produce hormogonia as part of their developmentalcycle (Scytonema sp. strain PCC 7110, Chlorogloeopsis sp.strain PCC 6718, Fischerella sp. strain PCC 7414, andCalothrix sp. strain PCC 7102) branch more deeply in the

heterocystous forms than do strains of those genera that donot produce hormogonia (Anabaena sp. strain PCC 7122 andNodularia sp. strain PCC 73104).

Origin of photosynthetic organelles. The present sequencecomparisons confirm the conclusion of Bonen et al. (3-5),based on RNase T1-generated oligonucleotide catalogs, thatthe cyanobacteria and green chloroplasts form a naturalgroup (Fig. 6). However, in contrast to the earlier clusteranalysis of oligonucleotide catalogs, which indicated that theprogenitor of the green chloroplasts diverged before theradiation that gave rise to the diversity seen in modemfree-living cyanobacteria (3-5), the present analysis of con-tinuous sequences included the green chloroplasts wellwithin the cyanobacterial radiation. That is, the green chlo-roplast line of descent should be viewed as one of thecyanobacterial sublines. While the present study included agreater diversity of cyanobacteria than previous analyses,this alone does not explain the different placement of thegreen chloroplasts. The interpretation of the origin of chlo-roplasts on the basis of their 16S rRNA sequences iscomplicated by their large amount of evolutionary diver-gence relative to the other cyanobacterial sublines. Becauseof undercompensation for multiple mutations, rapidly evolv-ing lineages can branch spuriously deeply in inferred phylo-genetic tree topologies (30). Green chloroplast 16S rRNAsequences from the following organisms were analyzed: Zeamays (maize), Nicotiana tabacum (tobacco), Marchantiapolymorpha (liverwort), Chlamydomonas reinhardii (greenalga), and Euglena gracilis (euglenoid). The analysis indi-cated that these sequences are specifically related. Thesequence from M. polymorpha showed the lowest averagerate of sequence change for the group and hence was chosenfor comparison in Fig. 6. Cluster analysis, as previously usedto establish the close relationship of chloroplasts and cya-nobacteria, is particularly susceptible to artifacts in branch-ing order that arise from different rates of change amongcompared sequences (10). The least-squares distance matrix

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-. Pseudanabaena PCC 6903Pseudanaboena "gleata CCC OL-75-PS

oo->~ Gloeobacter 'violaceus' PCC 7421

o Synechococcus "lividus' CCC Y7C-S^ Synechococcus PCC 6301 CAnacystis nidulansl)Oscillator/a amphigranulata" CCC NZ-concert-Oa

Plectonema "boryanum" PCC 73110

Oscllatoria PCC 6304 (Microcoleus vaginatus")Chamaesiphon PCC 7430

Phormidium "ectocarpi* PCC 7375Synechocystis PCC 6906 ("Eucapsis" sp.)

Synechocystis PCC 6308

Gloeothece PCC 6501

Spiruilna PCC 6313Gloeocapse PCC 73106

Democarpa PCC 7437- ~ . Pleurocapsa PCC 7321

Myxosarcina PCC 7312

I 00.04 0.06

Oscillatoria Vlimnetica' (Solar Lake)0 mfx

Oscillatoria PCC 7515

Anabeene "cylindrica" PCC 7122

0.08

FIG. 4. Rooted-tree topology illustrating evolutionary relationships among 16S rRNAs from cyanobacteria. Evolutionary distances are

proportional to the horizontal component of segment length in this representation. A. tumefaciens, B. subtilis, and P. testosteroni 16S rRNAsequences were used to locate the root. Only one heterocystous cyanobacterium, Anabaena sp. strain PCC 7122, is included in this tree. Thescale is in units of fixed point mutations per sequence position.

method used here is less sensitive to this problem, and itincludes the chloroplast ancestry within the cyanobacterialradiation. Thus, it seems necessary to include the chloro-plasts, if the cyanobacteria are to be considered a holophy-letic group, i.e., a group consisting of a common ancestorand all lineages derived from it (1).

In many respects the photosynthetic organelle (cyanelle)of the flagellate C. paradoxa resembles a cyanobacteriummore than a green chloroplast: it contains phycobilin an-tenna pigments, has a rudimentary peptidoglycan wall, lackschlorophyll b, and lacks the double membrane and thylakoidarrangement of chloroplasts. Thus, it is frequently supposedthat cyanelles arose independently of the chloroplasts as arelatively recent endosymbiosis between a eucaryote and acyanobacterium (18). Our results are consistent with such aview but nevertheless indicate a specific relationship be-tween the C. paradoxa cyanelle and the green chloroplasts(Fig. 6) (28). We offer two alternative explanations for thisrelationship. (i) The common ancestor of the cyanelle andgreen chloroplast lineages may have been a free-livingcyanobacterium that independently initiated the two en-dosymbioses. In this case, the chlorophyll b light-harvestingmechanism would have arisen in the chloroplast lineage, ineither the free-living or symbiotic state. (ii) Both lineagesmay derive from a single endosymbiotic event, in which casechlorophyll b would have originated in the chloroplast

progenitor during its endosymbiotic state. The former theorymight suggest that certain procaryotic lines are predisposedtoward symbioses, a possibility that finds a parallel in thelineage that contains mitochondria, plant parasites such as

Agrobacterium tumefaciens (46), and a rickettsia (44). Thelatter possibility implies multiple, independent origins for thechlorophyll b light-harvesting mechanism, which is alsoknown to exist in symbiotic and free-living procaryotes(prochlorophytes) (7, 27).Timing of events in cyanobacterial evolution. Although the

cyanobacteria often are cited as a particularly ancient group,the sequence similarities of their rRNAs to one another andto those of other eubacteria show that the other majoreubacterial taxa diverged significantly before the diversifica-tion of the modern cyanobacteria. Among these other eu-

bacterial taxa are the family Chloroflexaceae (Fig. 3, Ther-momicrobium), which diverge from the main eubacteriallineage substantially more deeply than do the cyanobacteria(31). Obligately anaerobic, phototrophic Chloroflexus spp.are known to form laminated microbial mats and are mor-phologically similar to microfossils in the earliest knownstromatolites (2, 12). These considerations caution againstthe interpretation of the earliest microbial fossils as cyano-bacterial in origin (37). Interpretations of microfossil evi-dence are frequently based upon the assumption that mor-

phology is phylogenetically conserved. As seen in the extant

0- 0.02

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Pseudanabaena PCC 6903Gloeobacter "violaceus" PCC 7421

Synechococcus PCC 6301 (Anacystis nidulans")Synechocystis PCC 6308

- Gloeothece PCC 6501e Gloeocapsa PCC 73106Myxosarcin8 PCC 7312

Oscillatoria PCC 7515

Scytonema PCC 7110

- Fischerella PCC 7414 (Mastigocladus laminosus")Chlorogloeopsis 'fritschil' PCC 6718

Calothrix "desertica" PCC 7102Nostoc PCC 73102

~ Nodularia PCC 73104Anabaena "cylindrica" PCC 7122L~~~~~~~~~~~~~~~~~~.__.,. __-

0 0.02 0.04 0.06 0.08FIG. 5. Rooted-tree topology illustrating evolutionary relationships among 16S rRNAs from heterocystous cyanobacteria. The presenta-

tion is as described in the legend to Fig. 4.

procaryotes, morphology correlates imperfectly with phylo-geny, suggesting that convergent evolution of morphologicalcharacteristics among procaryotes is a common theme (33,39).Because the 16S rRNAs of different organisms accumulate

mutations at different rates, evolutionary distances cannotbe accurately calibrated in terms of time. Hence, we do notattempt to infer the length of time that passed between thedivergence of the major eubacterial phyla and the floweringof cyanobacterial diversity. It is clear, however, that cyano-bacterial diversification occurred within a relatively shortspan of molecular evolutionary distance. Although theevents responsible for this apparent burst of evolution in thecyanobacterial line of descent are uncertain, as the firstorganism to exploit water as an electron donor for photo-synthesis, the common ancestor of the cyanobacteria andchloroplasts had available a novel and profoundly fertilephysiological niche. We suggest that rapid diversification

ensued, leaving its record in the molecular relationshipsobserved here.The molecular phylogenetic data suggest that the hete-

rocystous cyanobacteria arose significantly after the appear-ance of other cyanobacterial lines. The oxygenic nature ofcyanobacterial photosynthesis would have provided selec-tive pressure for the evolution of a mechanism for seques-tering the oxygen-sensitive process of N2 reduction, i.e., theheterocyst. Aerobic nitrogen fixation is not limited to theheterocystous cyanobacteria; it also occurs in vegetativecells of some unicellular cyanobacteria (Synechococcus spp.[26] and Gloeothece spp. [34]) and in other eubacterial phyla.These lineages separate earlier in the inferred phylogenythan do the heterocystous forms, so it is likely that the latterhad no role in the origin of biological nitrogen fixation.

This study provides the most detailed phylogenetic dataavailable on the evolution of oxygenic photosynthesis. Fun-damental questions remain to be answered. Do green chlo-

Pseudanabaena "galeata" CCC OL-75-PS/ Gloeobacter Nviolaceus" PCC 7421

Synechococcus PCC 6301 (Anacystis nidulans")Plectonema 'boryanum' PCC 73110

Synechocystis PCC 6308

Cyanophora paradoxa cyanelleliverwort chloroplast

Oscillatoria "limnetlca (Solar Lake)/-Mcrocoleus 10 mfx

"

~~~ Oscillatoria PCC 7515PCC 7419

L0 0.02 0.04 0.06

Anabaena 'cyl/ndrica' PCC 7122I I-1

0.08 0.1 0 I

FIG. 6. Rooted-tree topology illustrating the relationships of green chloroplasts and cyanelle 16S rRNAs to cyanobacterial 16S rRNAs.The presentation is as described in the legend to Fig. 4. See text for discussion.

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roplasts share a common ancestor with modern prochlo-rophytes? Do rhodophyte, chlorophyte, chrysophyte, andcryptomonad chloroplasts have monophyletic or polyphyle-tic origins? As further phylogenetic and biochemical dataaccumulate, an integrated view of this complex evolutionaryhistory will emerge.

ACKNOWLEDGMENTS

We thank John Waterbury, Richard Castenholz, and YehudaCohen for the strains used in this study and Jessup Shively andStewart Maxwell for C. paradoxa cyanelle RNA.

This work was supported by Public Health Service grantGM34527 to N.R.P. from the National Institutes of Health, NationalScience Foundation grant BSR 8600170 to S.J.G., and Office ofNaval Research grants N14-86-K-268 to G.J.O. and N14-87-K-0813to N.R.P.

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