JOURNAL OF BACTERIOLOGY, Aug. 1988, p. 3584-3592 Vol. 170, No. 8 0021-9193/88/083584-09$02.00/0 Copyright C) 1988, American Society for Microbiology

Evolutionary Relationships among and Green 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 distance matrix 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 short span of evolutionary distance. Evolutionary depth within the surveyed cyanobacteria is substantially less than that separating the major eubacterial taxa, as though cyanobacterial diversification occurred significantly after the 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 only support the conclusion of previous workers that the cyanobacteria and green chloroplasts form a coherent phylogenetic group but also suggest that the lineage, which includes the cyanelle of C. paradoxa, is not just a sister group to the free-living forms but rather is contained within the cyanobacterial radiation.

The cyanobacteria are one of the most morphologically the cyanobacteria and other eubacterial lineages and on the diverse and conspicuously successful procaryotic groups. It origins of chloroplasts. is generally believed that the cyanobacteria were the first major group of phototrophs to arise with a two-stage photo- synthetic pathway capable of oxidizing water to produce MATERIALS AND METHODS molecular oxygen. Geochemical and fossil evidence indi- Organisms and cultivation. Cultures or cell paste of all cates that in the Precambrian Era they caused the transition Pasteur Culture Collection strains were provided by John B. in the Earth's atmosphere from its primordial, anaerobic Waterbury (Woods Hole Oceanographic Institution). All state to its current, aerobic condition (19, 36, 43). Moreover, Castenholz Culture Collection strains were supplied as cul- molecular phylogenetic analysis of c-type cytochrome and tures by Richard H. Castenholz (University of Oregon). rRNA sequences have established a relationship between Cultures of Oscillatoria limnetica (Solar Lake) and Micro- cyanobacteria and the green (euglenoids, green algae, and coleus sp. strain 10 mfx were from Yehuda Cohen (H. higher plants) and red (rhodophyte) chloroplasts, thus sup- Steinitz Marine Biology Laboratory, Eilat, Israel). Cultures porting the procaryotic origins of chloroplasts. were maintained in BG-11 medium (34). C. paradoxa cya- Because of their ubiquity, rRNA sequences are particu- nelle RNA, provided by Stuart Maxwell (North Carolina larly useful for establishing evolutionary relationships State University) and Jessup M. Shively (Clemson Univer- among diverse organisms. Woese and colleagues, using sity), was prepared as described by Starnes et al. (40). partial (RNase Tl-generated oligonucleotide catalogs) and Preparation of RNA for sequencing. High-molecular- complete 16S rRNA sequences, have defined about 10 major weight cellular RNAs were prepared for sequencing as divisions (phyla) of eubacteria (45). The cyanobacteria are previously described (25). Briefly, cell pellets (0.3 to 3.0 g one of these phyla. However, too few strains (eight) of [wet weight]) were removed from storage at -70°C, thawed, cyanobacteria had been investigated to develop a compre- and lysed by one or two passages through a French pressure hensive overview of the diversity of the group. cell. RNA was purified by extraction with phenol, precip- We have used a method for directly sequencing 16S rRNA itated with ethanol, and suspended. Overnight precipitation to explore the evolutionary relationships among 30 represen- from 1.0 M NaCl at 0°C was used to prepare high-molecular- tatives of the diverse cyanobacterial groups, including the weight RNA. photosynthetic organelle of the phytoflagellate Cyanophora RNA sequencing. Dideoxynucleotide-terminated sequenc- paradoxa. The results shed new light on the relative ages of ing, using reverse transcriptase and synthetic oligodeoxynu- cleotide primers complementary to conserved 16S rRNA sequences, was carried out as described by Lane et al. (25; D. J. K. G. G. J. and N. R. * Corresponding author. Lane, Field, Olsen, Pace, t Present address: Department of Microbiology, Oregon State Methods Enzymol., in press). Three "universal' small University, Corvallis, OR 97331. subunit rRNA sequencing primers (complementary to Esch- t Present address: Department of Microbiology, University of erichia coli 16S rRNA sequence positions 519 to 536, 907 to Illinois, Urbana, IL 61801. 926, and 1392 to 1406) were used to determine ca. 1,000 § Present address: Gene-Trak Systems, Framingham, MA 01701. nucleotides of each rRNA sequence. The sequences have 3584 VOL. 170, 1988 PHYLOGENY OF CYANOBACTERIA AND CHLOROPLASTS 3585 been deposited with GenBank and are available from the (34), which divides the cyanobacteria into five sections. The authors upon request. unicellular cyanobacteria constitute section I. Members of Sequence analysis. Sequences were manually aligned on section II, the pleurocapsalean cyanobacteria, share a com- the basis of conserved sequence and secondary structural mon developmental feature, the capacity of large cells to elements. Regions of ambiguous sequence alignment were subdivide internally, producing numerous smaller cells omitted from subsequent analyses. For each pair of se- (baeocytes). Section III incorporates the filamentous, non- quences, the number of nucleotide differences was used to heterocystous organisms. Sections IV and V are, respec- estimate the average number of fixed point mutations per tively, the nonbranching and branching filamentous forms sequence position that has accumulated since their diver- that are capable of forming heterocysts (specialized, nitro- gence (22). This is called the "evolutionary distance" sepa- gen-fixing cells). rating the contemporary sequences (Fig. 1 and 2, lower left). The deepest branches so far in the cyanobacterial tree are The statistical uncertainty of the pairwise evolutionary dis- represented by violaceus (section I) and two tance estimates was determined by the method of Kimura closely related strains of the genus Pseudanabaena (section and Ohta (23) (Fig. 1 and 2, upper right). A least-squares III) (Fig. 4). G. violaceus is unique among cyanobacteria method was used to infer the phylogenetic tree most con- because of its lack of and the unusual structure of sistent with the pairwise distance estimates and their statis- its . The photosynthetic reaction centers are tical uncertainties (30). contained in the cytoplasmic membrane with the phycobili- somes arranged in an underlying cortical layer (17). The RESULTS AND DISCUSSION early divergence of this organism from the main cyanobac- Relationships among free-living cyanobacteria. The unroot- terial lineage is consistent with this remarkably different ed phylogenetic tree in Fig. 3 broadly depicts evolutionary light-harvesting apparatus. The result suggests that the com- relationships of oxygenic, phototrophic and organ- mon ancestor of the modern cyanobacteria may have lacked elles to other representatives of the three primary lines of thylakoids. Unlike G. violaceus, the Pseudanabaena strains descent: the archaebacteria, the eucaryotes, and the eubac- inspected have no obvious characteristics to suggest an early teria. The diversity among cyanobacteria and chloroplasts is evolutionary divergence from the main cyanobacterial line. indicated by the depth of the hatching. The cyanobacteria Biochemical and ultrastructural studies of these strains may and chloroplasts together make up 1 of approximately 10 reveal additional features that distinguish them from the major eubacterial taxa (not all of which are represented in other cyanobacteria and may clarify the phylogenetic rela- this tree). The rRNA sequence diversity within the cyano- tions among them (15, 16). bacterial lineage is substantially less than the separations The unicellular cyanobacteria of section I and the nonhe- between the major eubacterial taxa. In contrast, the se- terocystous, filamentous organisms of section III are dis- quence diversity observed within most other eubacterial persed throughout the tree (Fig. 4), indicating that these rRNA phyla is nearly equal to interphylum evolutionary morphotypes have multiple evolutionary origins. Thus, tax- distances (45). Interphylum evolutionary distances for rep- onomic classifications based principally on morphology do resentative eubacteria vary from 0.19 to 0.30 fixed mutations not necessarily reflect phylogenetic relationships. per sequence position (Fig. 1). Intraphylum evolutionary The orientation of cell division planes has been used as a distances among the cyanobacteria are typically much further basis for subdividing the unicellular cyanobacteria smaller, about 0.15 mutations per sequence position. Thus, into several genera. An earlier study of RNase T1-generated relatively close phylogenetic relationships underlie the ex- 16S rRNA oligonucleotide catalogs appeared to support this traordinary morphological diversity of cyanobacteria. definition of natural relationships (5). However, the addi- The phylogenetic tree in Fig. 4 depicts inferred relation- tional sequence data gathered during the present study led us ships among the nonheterocystous cyanobacteria inspected. to conclude that the various modes of cell division are not A diverse representation of cyanobacteria was selected for well correlated with phylogenetic groups. An example in sequencing on the basis of available taxonomic, morpholog- Fig. 4 is the local cluster that contains Gloeothece sp. strain ical, and physiological information. Many of the lineages PCC 6501 (cell division in one plane), Synechocystis sp. branch at similar depths in the cyanobacterial tree, lending strain PCC 6308 (cell division in two planes), and Synechocys- the tree a fan-like aspect. This result indicates that many tis (Eucapsis) sp. strain PCC 6906 and Gloeocapsa sp. strain modern cyanobacterial lineages arose in an expansive evo- PCC 73106 (cell division in three planes). This cluster also lutionary radiation. The nearly equivalent depths of many of contains the section III filamentous cyanobacterium Spiru- these branchings render their detailed relationships lina sp. strain PCC 6313 (cell division in one plane). Other (branching orders) unresolved. This is because the uncer- cellular properties are consistent with the conclusion that tainty in the evolutionary distances separating pairs of division planes do not define phylogenetic groups. For organisms increases with the distance, a consequence of instance, the DNA base compositions of unicellular cyano- uncertainty in the number of multiple mutations of individual bacteria that divide in one plane span a broad range (35 to 71 residues. The statistical errors for the calculated evolution- mol% G+C) (20). Phylogenetic studies that include addi- ary distances separating pairs of organisms are included in tional genera are likely to uncover still greater diversity Fig. 2. The relative positions of the nodes in the tree are among unicellular cyanobacteria. more accurate than suggested by Fig. 2, because the uncer- Even a conspicuous feature such as the oscillatorian tainties are not independent and some of the uncertainty morphotype may not indicate a relatedness group, to the applies only to the lengths of peripheral branches, not to the exclusion of other organisms. Although some clustering of lengths of the branchings deeper in the tree. Although the Oscillatoria spp. is indicated (e.g., 0. limnetica and PCC precise branching order of the short segments near the root 7515), other Oscillatoria spp. are scattered throughout the of the cyanobacterial tree is uncertain, significant relation- tree (e.g., 0. amphigranulata). Some physiological traits are ships emerge. consistent with the rRNA sequence diversity seen in this The morphological complexity of cyanobacteria served as group. For instance, both 0. limnetica and 0. amphigranu- a primary basis of the classification system of Rippka et al. lata are able to use sulfide, as well as water, as a source of 3586 GIOVANNONI ET AL. J. BACTERIOL.

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0-'p1 EUKARYOTES EUBACTERIA FIG. 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 proportional to evolutionary distances. The scale bar corresponds to 0.1 fixed point mutations per sequence position. This topology is based on an analysis of 920 nucleotides from complete sequences. electrons for noncyclic . However, the details heterocystous forms than do strains of those genera that do of sulfide adaptation in these two strains differ (8, 9), and not produce hormogonia (Anabaena sp. strain PCC 7122 and they are not close relatives (Fig. 4). The LPP subgroup of Nodularia sp. strain PCC 73104). filamentous cyanobacteria (section III) was provisionally Origin of photosynthetic organelles. The present sequence created to encompass organisms which did not fit strictly comparisons confirm the conclusion of Bonen et al. (3-5), within the morphological definitions of the genera Oscillato- based on RNase T1-generated oligonucleotide catalogs, that ria, Pseudanabaena, and Spirulina (34). Here the group is the cyanobacteria and green chloroplasts form a natural represented by Lyngbya sp. strain PCC 7419, Plectonema group (Fig. 6). However, in contrast to the earlier cluster sp. strain PCC 73110, and Phormidium sp. strain PCC 7375, analysis of oligonucleotide catalogs, which indicated that the which do not cluster phylogenetically (Fig. 4). A Lyngbya progenitor of the green chloroplasts diverged before the sp. (LPP group A; sheathed, nonmotile except for hormogo- radiation that gave rise to the diversity seen in modem nia) branches deeply but appears to be related to a phyloge- free-living cyanobacteria (3-5), the present analysis of con- netic subgroup containing other filamentous genera, includ- tinuous sequences included the green chloroplasts well ing two Oscillatoria spp. and a Microcoleous sp. within the cyanobacterial radiation. That is, the green chlo- The pleurocapsalean organisms (section II) studied so far roplast line of descent should be viewed as one of the constitute a coherent, phylogenetic subgroup, to the exclu- cyanobacterial sublines. While the present study included a sion of other cyanobacteria (Fig. 4). Although only three of greater diversity of cyanobacteria than previous analyses, six genera of this section have been analyzed, all the strains this alone does not explain the different placement of the in the section have very similar DNA base compositions green chloroplasts. The interpretation of the origin of chlo- (16). This and their common mode of reproduction distin- roplasts on the basis of their 16S rRNA sequences is guish them from all other cyanobacteria, suggesting that complicated by their large amount of evolutionary diver- reproduction by multiple fission (baeocyte formation) is of gence relative to the other cyanobacterial sublines. Because monophyletic origin. of undercompensation for multiple mutations, rapidly evolv- Similarly, the members of sections IV and V, the hete- ing lineages can branch spuriously deeply in inferred phylo- rocystous, filamentous forms, constitute a distinct phyloge- genetic tree topologies (30). Green chloroplast 16S rRNA netic group. The organisms of section V are a subgroup sequences from the following organisms were analyzed: Zea arising from within the lines of section IV (Fig. 5). This mays (maize), Nicotiana tabacum (tobacco), Marchantia confirms similar conclusions drawn from DNA-DNA hybrid- polymorpha (liverwort), Chlamydomonas reinhardii (green ization data (24). The organisms of sections IV and V are alga), and Euglena gracilis (euglenoid). The analysis indi- among the most morphologically diverse of the cyanobacte- cated that these sequences are specifically related. The ria. Aside from heterocysts, many strains also produce sequence from M. polymorpha showed the lowest average akinetes, "resting" cells that develop as a result of insuffi- rate of sequence change for the group and hence was chosen cient light or other factors (35). The strains shown in Fig. 5 for comparison in Fig. 6. Cluster analysis, as previously used that produce hormogonia as part of their developmental to establish the close relationship of chloroplasts and cya- cycle (Scytonema sp. strain PCC 7110, Chlorogloeopsis sp. nobacteria, is particularly susceptible to artifacts in branch- strain PCC 6718, Fischerella sp. strain PCC 7414, and ing order that arise from different rates of change among Calothrix sp. strain PCC 7102) branch more deeply in the compared sequences (10). The least-squares distance matrix VOL. 170, 1988 PHYLOGENY OF CYANOBACTERIA AND CHLOROPLASTS 3589

-. Pseudanabaena PCC 6903 Pseudanaboena "gleata CCC OL-75-PS oo->~ Gloeobacter 'violaceus' PCC 7421

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Oscllatoria PCC 6304 (Microcoleus vaginatus") Chamaesiphon PCC 7430 Phormidium "ectocarpi* PCC 7375 Synechocystis PCC 6906 ("Eucapsis" sp.) Synechocystis PCC 6308 Gloeothece PCC 6501 Spiruilna PCC 6313 Gloeocapse PCC 73106 Democarpa PCC 7437 - ~ . Pleurocapsa PCC 7321 Myxosarcina PCC 7312

Oscillatoria Vlimnetica' (Solar Lake) 0 mfx

Oscillatoria PCC 7515

Anabeene "cylindrica" PCC 7122

I 0 0- 0.02 0.04 0.06 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 rRNA sequences were used to locate the root. Only one heterocystous cyanobacterium, Anabaena sp. strain PCC 7122, is included in this tree. The scale is in units of fixed point mutations per sequence position.

method used here is less sensitive to this problem, and it progenitor during its endosymbiotic state. The former theory includes the chloroplast ancestry within the cyanobacterial might suggest that certain procaryotic lines are predisposed radiation. Thus, it seems necessary to include the chloro- toward symbioses, a possibility that finds a parallel in the plasts, if the cyanobacteria are to be considered a holophy- lineage that contains mitochondria, plant parasites such as letic group, i.e., a group consisting of a common ancestor Agrobacterium tumefaciens (46), and a rickettsia (44). The and all lineages derived from it (1). latter possibility implies multiple, independent origins for the In many respects the photosynthetic organelle (cyanelle) chlorophyll b light-harvesting mechanism, which is also of the flagellate C. paradoxa resembles a cyanobacterium known to exist in symbiotic and free-living procaryotes more than a green chloroplast: it contains phycobilin an- (prochlorophytes) (7, 27). tenna pigments, has a rudimentary peptidoglycan wall, lacks Timing of events in cyanobacterial evolution. Although the chlorophyll b, and lacks the double membrane and cyanobacteria often are cited as a particularly ancient group, arrangement of chloroplasts. Thus, it is frequently supposed the sequence similarities of their rRNAs to one another and that cyanelles arose independently of the chloroplasts as a to those of other eubacteria show that the other major relatively recent endosymbiosis between a eucaryote and a eubacterial taxa diverged significantly before the diversifica- cyanobacterium (18). Our results are consistent with such a tion of the modern cyanobacteria. Among these other eu- view but nevertheless indicate a specific relationship be- bacterial taxa are the family Chloroflexaceae (Fig. 3, Ther- tween the C. paradoxa cyanelle and the green chloroplasts momicrobium), which diverge from the main eubacterial (Fig. 6) (28). We offer two alternative explanations for this lineage substantially more deeply than do the cyanobacteria relationship. (i) The common ancestor of the cyanelle and (31). Obligately anaerobic, phototrophic Chloroflexus spp. green chloroplast lineages may have been a free-living are known to form laminated microbial mats and are mor- cyanobacterium that independently initiated the two en- phologically similar to microfossils in the earliest known dosymbioses. In this case, the chlorophyll b light-harvesting stromatolites (2, 12). These considerations caution against mechanism would have arisen in the chloroplast lineage, in the interpretation of the earliest microbial fossils as cyano- either the free-living or symbiotic state. (ii) Both lineages bacterial in origin (37). Interpretations of microfossil evi- may derive from a single endosymbiotic event, in which case dence are frequently based upon the assumption that mor- chlorophyll b would have originated in the chloroplast phology is phylogenetically conserved. As seen in the extant 3590 GIOVANNONI ET AL. J. BACTERIOL.

Pseudanabaena PCC 6903 Gloeobacter "violaceus" PCC 7421 Synechococcus PCC 6301 (Anacystis nidulans") Synechocystis PCC 6308

- Gloeothece PCC 6501 e Gloeocapsa PCC 73106 Myxosarcin8 PCC 7312

Oscillatoria PCC 7515 Scytonema PCC 7110 - Fischerella PCC 7414 (Mastigocladus laminosus") Chlorogloeopsis 'fritschil' PCC 6718 Calothrix "desertica" PCC 7102 Nostoc PCC 73102 ~ Nodularia PCC 73104 Anabaena PCC 7122 L~~~~~~~~~~~~~~~~~~.__.,."cylindrica"__- 0 0.02 0.04 0.06 0.08 FIG. 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- ensued, leaving its record in the molecular relationships geny, suggesting that convergent evolution of morphological observed here. characteristics among procaryotes is a common theme (33, The molecular phylogenetic data suggest that the hete- 39). rocystous cyanobacteria arose significantly after the appear- Because the 16S rRNAs of different organisms accumulate ance of other cyanobacterial lines. The oxygenic nature of mutations at different rates, evolutionary distances cannot cyanobacterial photosynthesis would have provided selec- be accurately calibrated in terms of time. Hence, we do not tive pressure for the evolution of a mechanism for seques- attempt to infer the length of time that passed between the tering the oxygen-sensitive process of N2 reduction, i.e., the divergence of the major eubacterial phyla and the flowering heterocyst. Aerobic nitrogen fixation is not limited to the of cyanobacterial diversity. It is clear, however, that cyano- heterocystous cyanobacteria; it also occurs in vegetative bacterial diversification occurred within a relatively short cells of some unicellular cyanobacteria (Synechococcus spp. span of molecular evolutionary distance. Although the [26] and Gloeothece spp. [34]) and in other eubacterial phyla. events responsible for this apparent burst of evolution in the These lineages separate earlier in the inferred phylogeny cyanobacterial line of descent are uncertain, as the first than do the heterocystous forms, so it is likely that the latter organism to exploit water as an electron donor for photo- had no role in the origin of biological nitrogen fixation. synthesis, the common ancestor of the cyanobacteria and This study provides the most detailed phylogenetic data chloroplasts had available a novel and profoundly fertile available on the evolution of oxygenic photosynthesis. Fun- physiological niche. We suggest that rapid diversification 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 cyanelle liverwort chloroplast Oscillatoria "limnetlca (Solar Lake) /-Mcrocoleus 10 mfx " ~~~ Oscillatoria PCC 7515 PCC 7419

Anabaena 'cyl/ndrica' PCC 7122 L I I-1 0 0.02 0.04 0.06 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. VOL. 170, 1988 PHYLOGENY OF CYANOBACTERIA AND CHLOROPLASTS 3591 roplasts share a common ancestor with modern prochlo- Arch. Microbiol. 129:181-189. rophytes? Do rhodophyte, chlorophyte, chrysophyte, and 18. Hall, W. T., and G. Claus. 1963. Ultrastructural studies on the cryptomonad chloroplasts have monophyletic or polyphyle- blue-green algal symbiont in Cyanophora paradoxa Korschi- tic origins? As further phylogenetic and biochemical data koff. J. Cell Biol. 19:551-563. accumulate, an integrated view 19. Hayes, J. M. 1983. Geochemical evidence bearing on the origin of this complex evolutionary of aerobiosis, a speculative hypothesis, p. 291-300. In J. W. history will emerge. 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