1 NIOO-Centre for Estuarine and Marine Ecology, NL-4400 AC Yerseke, The Netherlands
2 Lehrstuhl für Physiologie und Biochemie der Pflanzen, Universität Konstanz, D-78457 Konstanz, Germany
Correspondence
Anneliese Ernst
a.ernst{at}nioo.knaw.nl
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: CCA, complementary chromatic adaptation; ITS, internal transcribed spacer; PC, phycocyanin; PE, phycoerythrin
The GenBank accession numbers for sequences reported in this paper are AF317071AF317079, AF330246AF330254 and AF466990AF467000.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic traits often reflect characteristics of a corresponding selective force. For example, light quality (i.e. light of limited range of light wavelengths) varies with depth of the water column, with distance from the coast and also with biotic factors. Light quality is also known to affect the synthesis of phycobiliproteins, which allows cyanobacteria capable of CCA to acclimate to the ambient light climate (for review see Grossman et al., 1993). However, most of the Synechococcus-type cyanobacteria of the autotrophic picoplankton produce either PE or PC as major light-harvesting pigments. Thus, they cannot acclimate to light by synthesis of complementary pigments. In this case, light quality is thought to influence the abundance and distribution of the different pigment types in the ecosystem. For example, PC-rich strains, which absorb mainly red light, occur frequently in shallow and eutrophic ecosystems, where mixing depth is limited either physically or by very stable stratification (Vörös et al., 1991
). On the other hand, PE-rich strains, which can take advantage of blue light penetrating deeply in clear water columns, dominate in the autotrophic picoplankton of deep oligotrophic and mesotrophic lakes and in the open ocean (Pick, 1991
; Wood et al., 1998
).
The PE- and PC-rich picocyanobacteria described in this study have been isolated from several deep subalpine lakes (central Europe), Lake Biwa (Japan), Lake Balaton (Hungary) and the Baltic Sea, a large brackish water ecosystem. In all these ecosystems, both pigment types coexist and dominate the autotrophic picoplankton. Physiological studies showed that apart from the obvious difference in the major accessory pigments, PC and PE, the strains also differed in carotenoid content, light stress tolerance, response to nutrient limitation and surface structures (for review see Postius & Ernst, 1999). In addition, most freshwater isolates have previously been characterized as distinct genotypes by a genomic fingerprinting technique, RFLP of psbA genes (
Ernst et al., 1995; Postius et al., 1996
). However, the phylogenetic relationships of these strains have not been examined. Phylogenetic relationships are often inferred from comparison of coding and non-coding sequences in the ribosomal operon. In cyanobacteria, this operon includes genes encoding 16S rRNA, 23S rRNA and 5S rRNA and none, one or two tRNAs (Iteman et al., 2000
). 16S rRNA has been frequently used to establish a gene-based phylogeny of cyanobacteria (
Giovannoni et al., 1988; Wilmotte et al., 1992
, 1993
, 1994
; Nelissen et al., 1996
; Turner, 1997
; Honda et al., 1999
; Lyra et al., 2001
; Robertson et al., 2001
). However, 16S rRNA sequences often do not allow discrimination of bacterial strains at the subgeneric level (Fox et al., 1992
). Therefore, the less conserved internal transcribed spacer (ITS) sequences, which are removed from the primary transcript during rRNA processing, have also been used for bacterial identification, typing and evolutionary studies (Gürtler & Stanisich, 1996
). In several studies the amplified 16S23S rDNA spacer (ITS-1) of cyanobacteria has been used to genetically characterize strains by PCR-RFLP (
Lu et al., 1997; West & Adams, 1997; Scheldeman et al., 1999
) or by sequence analyses (Wilmotte et al., 1994
; Otsuka et al., 1999
; Boyer et al., 2001
; Rocap et al., 2002
). These studies indicated the presence of multiple ribosomal operons with differences in ITS-1 in some strains. Genome analysis confirmed that cyanobacteria (including Prochlorococcus spp.) comprise one, two or four ribosomal operons [Kaneko et al., 1996
; see also ongoing sequencing projects at the DOE Joint Genome Institute (http://www.jgi.doe.gov./JGI_microbial/html/index.html) and at the Kazusa DNA research institute (http://www.kazusa.or.jp/cyano/)]. As the number of ribosomal operons can vary among phylogenetically closely related cyanobacteria (i.e. among different Prochlorococcus spp.), this information may be an important criterion in strain characterization.
In this study, we analysed the 16S rRNA and ITS-1 of 19 picocyanobacteria that were isolated from different sources and that differed in genotype (RFLP of psbA genes) and/or phenotype. For 10 additional isolates from the same ecosystems only ITS-1 sequences were determined. The number of ribosomal operons per genome was examined by hybridization of genomic DNA with probes targeting 16S rDNA and ITS-1 in Southern blots. The analysis of the sequences and the deduced phylogenetic assignment of strains did not support our original distinction of isolates according to phenotype, i.e. pigmentation. Rather, the genetic and phenotypic diversification indicated that selection processes may have shaped local populations leading to similar phenotypes in different phylogenetic lineages. Therefore, we discuss the possibility that diversification among Synechococcus spp. is caused by ecosystem-specific adaptive radiations.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Picocyanobacteria from the Baltic Sea were obtained from 10 m water depth in August 1996 at sampling sites in the Bornholm Sea (surface salinity 9 g l-1; strains BS 4, BS 5, BS 7, BS 8) and in June 1999 in the Gotland Sea (salinity 7 g l-1; strain BS 20). Original samples contained untreated seawater (sampling 1996) or a fraction that had passed a 3 µm filter and remained on a 0·2 µm filter (sampling 1999). The original material was cultivated in a growth medium prepared from 1 part ASN III and 3 parts BG11. Successively, seven strains were isolated that were able to form colonies on ASN/BG11 solidified with 0·7 % agarose (Sigma).
Synechococcus rubescens SAG B 3.81, originating from Lake Zurich (Chang, 1980), and Synechococcus sp. SAG 1403-1 (synonyms Anacystis nidulans and Synechococcus PCC 6301) were obtained from Sammlung von Algenkulturen Göttingen (SAG, Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Untere Karspüle 2, 37073 Göttingen, Germany). Synechococcus sp. SAG 1403-1 will be addressed under its former name, Anacystis nidulans, to emphasize its distinct phylogenetic origin. Both strains were maintained in BG11.
DNA preparation and Southern analysis.
Two to three weeks after inoculation of 40 ml batches containing BG11, cultures were harvested by centrifugation (7 min, 7000 g), frozen in liquid nitrogen and stored at -20 °C. Genomic DNA was extracted according to Postius et al. (1996). For RFLP analysis, 510 µg genomic DNA was completely digested with different restriction endonucleases (Boehringer), separated by agarose gel electrophoresis and blotted on Hybond (Amersham) membranes (Ernst et al., 1995
). Restriction fragments were probed with a 16S rDNA fragment labelled with UTP-digoxigenin in a PCR reaction with the primer pair P16S3p/P16S4m or the primer pair PITS2/PITS4 (see Table 1
) using DNA of S. rubescens as template. Labelled fragments and DIG-labelled molecular size markers (Boehringer) on Southern blots were detected using chemiluminescent substrates of alkaline phosphatase.
|
DNA sequencing.
The PCR fragments were sequenced by a non-radioactive version of the dideoxynucleotide chain-termination method (Sanger et al., 1977), employing the GATC 1500 System for direct blotting electrophoresis as described by Pohl & Maier (1995)
. Non-radioactive labels were introduced in cyclic sequencing reactions either via DIG-labelled primers PITS1PITS8, using the DIG- Sequencing Kit, including Taq polymerase (Boehringer) as described by Brass et al. (1996)
, or via Biotin-labelled dideoxynucleotides using ThermoSequenase (Amersham-Buchler; reaction kit provided by GATC) as described by Neuschaefer-Rube et al. (2000)
. Ribosomal sequences of strains isolated from the Baltic Sea and from the littoral zone of Lake Constance were obtained with an ABI sequencer (GATC).
Sequence alignment and analysis.
The sequences were assembled, analysed and aligned using programs of the software package PCGENE, version 6.7, from IntelliGenetics (Mountain View, CA, USA). For sequence comparison, sequences were searched in GenBank using the alignment tools provided by Altschul et al. (1990). Preliminary sequence data of several unicellular cyanobacteria were obtained from the DOE Joint Genome Institute (JGI) at http://www.jgi.doe.gov./JGI_microbial/html/index.html The sequences were aligned using CLUSTALX, originally developed by Higgins & Sharp (1988)
. 16S rRNA and ITS-1 sequences were analysed separately. For 16S rRNA analysis, the 20 nt encoded by the primer P16SAnf were excluded, resulting in analysis of 1456 nt (98 %) of 16S rRNA. Aligned sequences were corrected manually using the program GENEDOC (http://www.psc.edu/biomed/genedoc/). For analysis of ITS-1, sequences encoding 16S and 23S rRNA were excluded. Genetic distances were calculated and corrected for multiple base exchanges by the method of Jukes & Cantor (1969)
. The phylogenetic trees were reconstructed by the neighbour-joining method of Saitou & Nei (1987)
, including bootstrap analysis (Felsenstein, 1985
) as provided by TREECON (Van de Peer & De Wachter, 1994
). Secondary structures of segments of ITS-1 were analysed using the program RNADRAW V1.1 of Ole Mazura (ole{at}mango.mef.ki.se).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Because of extended length and sequence divergence between the ITS-1 sequences of different 16S rRNA-defined lineages, sequences were initially aligned per lineage. Within these clusters the largest variability was observed among strains assigned to the C. gracile cluster (see above). The length of ITS-1 varied between 976 and 1033 nt and the maximum divergence in pairwise comparison was 22·9 %. In contrast, ITS-1 of stains of the subalpine cluster I, which exhibited a similar number of variable bases in 16S rRNA, contained 911914 nt with only 5·0 % difference in pairwise comparison. The ITS-1 sequences of the two clustered strains from Lake Biwa, LB B3 and LB G2 (length 812/813 nt), and two strains of the subalpine cluster II, BO 0014/BO 8805 (length 847/851 nt), differed at 5·8 and 11·2 % of aligned positions, respectively. The least divergence was observed among ITS-1 sequences of six isolates from the Bornholm Sea. The PC- and PE-rich strains of this cluster exhibited only 12 variable positions in 887 nt of ITS-1 with a maximum of 1·0 % sequence divergence between two strains.
A low degree of sequence conservation and extended insertions/deletions in the ITS-1 complicated the alignment of ITS-1 sequences of the five lineages and the construction of a phylogenetic tree (Fig. 4). Unambiguous alignment of all strains, including a unique isolate with the shortest ITS-1 sequence in this study, strain LB P1 (703 nt), was possible only for the two tRNAs, for about 30 nt adjacent to the 16S rDNA and the target sites of primers PITS5/6 and PITS7/8 (Fig. 1
). We tried to improve the alignment using structures in the non-coding sections of the sequence that were identified by Iteman et al. (2000)
. Domains D2 and D4, which form the base of highly complex stemloop structures carrying the 16S rRNA and the 23S rRNA, were located in the target sequence of primers PITS5/6 and of primers PITS7/8, respectively. The latter also contained the antiterminator site, box B of the spacer. No other common structures were detected. The lack of conserved elements in this long spacer sequence led to deep branching in the phylogenetic inference with low statistical support of the arrangement of the lineages by bootstrap analysis (Fig. 4
).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Initially, most of the isolated strains were typed and identified using the monocistronic psbA genes, a gene family encoding a protein of photosystem II, as targets in Southern analyses (for review see Postius & Ernst, 1999). By selecting restriction enzymes that do not cut within the probed area we showed that each genome of the picocyanobacteria analysed contained two ribosomal operons (Fig. 5
). PCR amplifications, restriction analysis of the ITS-1 and results of direct sequence analysis indicated that in most cases both ribosomal operons comprised identical sequences. This is consistent with results of genome analysis of so far three other unicellular cyanobacteria, Synechocystis sp. PCC 6803 (Kaneko et al., 1996
), Synechococcus WH 8102 and Prochlorococcus marinus MIT 9313. However, it should be noted that the closely related P. marinus MED4 possesses only one ribosomal operon (see http://www.jgi.doe.gov./JGI_microbial/html/index.html). Even with two ribosomal operons present, genomic typing with targets in this polycistronic operon provided much less resolution (Fig. 5
) than that achieved with psbA genes (cf. Postius & Ernst, 1999
).
We also examined restriction typing of amplified fragments of the less conserved non-coding sequences of ITS-1. The length of ITS-1 of the Synechococcus isolates described in this study varied between 704 nt in strain LB P1 (Lake Biwa) and 1034 nt in strain BGS 171 (Lake Balaton). This range was larger than the length polymorphism observed among marine Synechococcus spp. (747810 nt) and Prochlorococcus spp. (537829 nt; Rocap et al., 2002). In total the length polymorphism of ITS-1 in this strictly monophyletic group of organism ranges from 537 to 1034 nt and hence, is larger than that of all other major cyanobacterial lineages, for which ITS-1 sequences between 283 and 648 nt have been reported (Itemann et al., 2000
; Boyer et al., 2001
). Restriction of this long intergenic region produced a characteristic pattern of restriction fragments for each of the lineages studied (Fig. 6b
). Nevertheless, this method was in most cases not suitable to distinguish genotypes and ecotypes within these lineages.
Strain cluster descriptions and taxonomic considerations
Based on a sequence identity of >99 % in 16S rRNA, we assigned six PC-rich isolates, five from Lake Constance, one from Lake Balaton, and one PE-rich isolate from the brackish Baltic Sea, to a cluster with the type strain C. gracile PCC 6307T. Strains of this cluster are non-motile, obligately photoautotrophic, freshwater strains with a high DNA G+C content and peripheral thylakoids. Similar strains have been isolated from temperate zone lakes in Europe, America (Rippka et al., 1979) and Japan (Robertson et al., 2001
). Phylogenetic inference from 16S rRNA and ITS-1 also assigned two strains from brackish ecosystems, Synechococcus sp. PCC 7009 (Rippka et al., 1979
) and BS 20 (this study), and although the majority of isolated strains use the blue pigment PC as the major accessory pigment, there are also two red, PE-rich strains in this lineage (strains BS 20, this study; and PS-715, Robertson et al., 2001
).
All PE-rich strains isolated from the pelagic zones of the subalpine Lake Constance, Lake Zurich and Lake Maggiore form a tight cluster in 16S rRNA as well as in ITS-1 inferred phylogenetic analyses: subalpine cluster I. S. rubescens SAG B3.81, which was the first PE-rich Synechococcus species isolated (Chang, 1980), is suggested as type strain of this cluster. All strains, except strain BO 8807, exhibit a coccoid morphology and lack particular surface structures. Strain BO 8807, which also confers a distinguished position in 16S rRNA and ITS-1 sequence analysis, exhibits elongated cells that are completely covered by an S-layer formed by a glycosylated protein (Ernst et al., 1992
, 1996
). This demonstrates that phylogenetically closely related strains can exhibit major morphological and biochemical variations apart from variations in accessory pigments.
Two clusters were formed by a single PE-rich and a single PC-rich isolate co-existing in the same ecosystem. The PE-rich strain Synechococcus sp BO 0014 was isolated from artificial substratum (tile) deposited for 6 weeks in the littoral zone of Lake Constance, while the phylogenetically closely related PC-rich strain BO 8805 was isolated from the pelagic zone of this lake. Accordingly, a PE-rich isolate LB B3 clustered with the PC-rich strain LB G2 from Lake Biwa, Japan. Both clusters are paraphyletic to the above-mentioned clusters, but their relative positions remained ambiguous in all analyses (Figs 3 and 4).
The Bornholm Sea cluster contained three PC-rich and three PE-rich isolates that differ in cell morphology and growth characteristics (B. E. M. Schaub, S. Vegos, U. I. A. Wollenzien, L. A. Stal & A. Ernst, unpublished results), but exhibit very few differences in ITS-1 sequences. The morphological differences observed in this cluster indicate that phenotypic diversification can occur with significantly higher frequency than fixation of mutations in the non-coding sections of the ribosomal operon. In the 16S rRNA-inferred phylogenetic analysis the Bornholm Sea cluster showed a weak affinity to two halotolerant PC-rich strains from coastal origins, Synechococcus spp. PCC 7001 and PCC 9005, originally assigned to the marine Synechococcus cluster B (Waterbury & Rippka, 1989). This taxonomic assignment was revised recently to a newly established form-genus Cyanobium Rippka & Cohen-Bazier 1983, characterized by a higher G+C content (6671 mol%), but a more limited range of cell diameters (0·81·4 µm) than the cyanobacteria that remained provisionally assigned to the form-genus Synechococcus (Rippka et al., 2001
).
The newly established form-genus Cyanobium comprises two physiologically distinct groups of strains: those not capable of sustained growth in a marine medium (cluster 1, type strains C. gracile PCC 6307T) and strains with a higher salt tolerance (cluster 2; Rippka et al., 2001). As far as morphological and ultrastructural data are available (Chang, 1980
; Ernst et al., 1992
, 2000
) all picocyanobacteria described in this study conform to the criteria of Cyanobium. However, whether they exhibit a high DNA G+C content remains difficult to prove, because, with the notable exception of subspecies of C. gracile, many freshwater strains do not form isolated colonies on agarose amended with BG11 and, therefore, are difficult to obtain as axenic cultures. Phylogenetic analysis of the 16S rRNA concurred with the taxonomic definition, but received a low bootstrap support in our analysis (Fig. 3
). Thus, the reassignment of all Synechococcus-type cyanobacteria from the autotrophic picoplankton of freshwater and brackish ecosystems to one genus, Cyanobium, remains debatable. As a putative genus, Cyanobium is paraphyletic to three other putative genera formed by the Antarctic Synechococcus strains (Vincent et al., 2000
), the marine Synechococcus spp. and the marine Prochlorococcus spp. Together these four genera seem to dominate the autotrophic picoplankton of marine, brackish and freshwater ecosystems. Within these genera, the small divergence in 16S rRNA often limited further resolution (Fig. 3
). Supplementary phylogenetic information obtained from ITS-1 provided convincing evidence for the existence of different lineages in this clade representing species and subspecies in a taxonomic context (Fig. 4
)
Ecosystem-dependent adaptive radiations
The data presented in this study show that many closely related genotypes, classified as subspecies of Cyanobium spp. can coexist in a particular ecosystem. We emphasized that in four of the five paraphyletic strain clusters (i.e. species) the subspecies comprise two pigment variants, the PE-rich and the PC-rich strains (see Fig. 4). From this observation we conclude that adaptation in lineages unable to acclimate to different light quality by synthesis of complementary pigments (CCA) can occur by loss or gain of genes required to synthesize PE, the outermost pigment of the phycobilisome. This may be caused by point mutations, by extended deletions or by recombination with intact genetic information provided from a closely related organism by horizontal gene transfer. Evidence for the exchange of genetic material between closely related strains was demonstrated in the ribosomal operon (see Fig. 2
) but evidence also comes from other cyanobacteria (Barker et al., 2001
).
Pigmentation as well as cell-surface structures are characteristics that determine the ecophysiology of strains and changes are expected to create new niches for the organism. Mutation and selection can therefore lead to an adaptive radiation of strains within a particular ecosystem. Recent adaptive radiations are characterized by marginal differences in 16S rRNA sequences, limited divergence in ITS-1 and very high bootstrap support in distance-matrix-based phylogenies (Figs 3 and 4) as well as in maximum-likelihood trees (data not shown). Putative endemic radiations were demonstrated in the subalpine clusters I and II and in the Bornholm Sea cluster (Figs 3 and 4
). A different genetic structure in ITS-1 was observed among strains assigned to the C. gracile cluster. Strains of this cluster were isolated all over the world and the sequence divergence in ITS-1 between isolates from Lake Constance was larger than between strains isolated from other ecosystems (Fig. 3
). The lack of a geographic coherence in the genetic diversification of this lineage is not consistent with a hypothesis of endemic radiations. Interestingly, cluster-specific DGGE revealed that isolates of the subalpine cluster I are abundant in the autotrophic picoplankton of Lake Constance (Becker et al., 2002
), while those assigned to the universal species C. gracile remained below the detection limit (S. Becker, A. K. Singh, C. Postius & A. Ernst, unpublished results). Thus, an adaptive radiation may have allowed the subalpine cluster I to gain dominance in the pelagic autotrophic picoplankton, but not C. gracile.
Recent adaptive radiations may also be seen in the genetic structure of marine Synechococcus spp. and Prochlorococcus spp. (Rocap et al., 2002). However, while some of these marine clusters can be associated with a particular phenotype, such as motility, chromatic adaptation or lack of phycourobilin, or differ in chlorophyll composition, phenotypic diversification among the most recently diversified genotypes, which is prerequisite for the hypothesis of an adaptive radiation, has not been reported. Nevertheless, the similarities in the genetic structure of different genera of the autotrophic picoplankton of marine, brackish and freshwater ecosystems are intriguing and inevitably lead to the question what has caused the sudden boosts in diversification in particular lineages, while others seemingly remained unaffected?
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barker, G. L. A., Handley, B. A., Vacharapiyasophon, P., Stevens, J. R. & Hayes, P. K. (2001). Allele-specific PCR shows that genetic exchange occurs among genetically diverse Nodularia (cyanobacteria) filaments in the Baltic Sea. Microbiology 146, 28652875.
Becker, S., Fahrbach, M., Böger, P. & Ernst, A. (2002). Quantitative tracing, by Taq nuclease assays, of a Synechococcus ecotype in a highly diversified natural population. Appl Environ Microbiol 68, 44864494.
Boone, R., Castenholz, R. W. & Garrity, G. M. (editors) (2001). Bergey's Manual of Systematic Bacteriology, 2nd edn, Vol. 1. New York: Springer.
Boyer, S. L., Flechtner, V. R. & Johansen, J. R. (2001). Is the 16S23S rRNA internal transcribed spacer region a good tool for use in molecular systematics and population genetics? A case study in cyanobacteria. Mol Biol Evol 18, 10571069.
Brass, S., Ernst, A. & Böger, P. (1996). An insertion element prevents phycobilisome synthesis in N2-fixing Synechocystis sp. strain BO 8402. Appl Environ Microbiol 62, 19641968.[Abstract]
Callieri, C., Amicucci, E., Bertoni, R. & Vörös, L. (1996). Fluorometric characterization of two picocyanobacteria strains from lakes of different underwater light quality. Int Rev Gesamten Hydrobiol 81, 1323.
Chang, T.-P. (1980). Zwei neue Synechococcus-Arten aus dem Zürichsee. Schweiz Z Hydrol 42, 247254.
Ernst, A., Sandmann, G., Postius, C., Brass, S., Kenter, U. & Böger, P. (1992). Cyanobacterial picoplankton from Lake Constance: II. classification of isolates by cell morphology and pigment composition. Bot Acta 105, 161167.
Ernst, A., Marschall, P. &, Postius. C. (1995). Genetic diversity among Synechococcus spp. (cyanobacteria) isolated from the pelagial of Lake Constance. FEMS Microbiol Ecol 17, 197204.[CrossRef]
Ernst, A., Postius, C. & Böger, P. (1996). Glycosylated surface proteins reflect genetic diversity among Synechococcus spp. of Lake Constance. Arch Hydrobiol 48, 16.
Ernst, A., Becker K. Hennes, S. & Postius, C. (2000). Is there a succession in the autotrophic picoplankton of temperate zone lakes? In Microbial Biosystems: New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology, pp. 623629. Edited by C. R. Bell, M. Brylinski, P. Johnson-Green. Halifax, Canada: Atlantic Canada Society for Microbial Ecology.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783791.
Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P., Jr (1992). How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166170.[Abstract]
Garcia-Pichel, F., Nübel, U. & Muyzer, G. (1998). The phylogeny of unicellular, extremely halotolerant cyanobacteria. Arch Microbiol 169, 469482.[CrossRef][Medline]
Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J. & Pace, N. R. (1988). Evolutionary relationships among cyanobacteria and green chloroplasts. J Bacteriol 170, 35843592.[Medline]
Grossman, A. R., Schaefer, M. R., Chiang, G. G. & Collier, J. L. (1993). The phycobilisome, a light-harvesting complex responsive to environmental conditions. Microbiol Rev 57, 725749.[Abstract]
Gürtler, V. & Stanisich, V. A. (1996). New approaches to typing and identification of bacteria using the 16S23S rDNA spacer region. Microbiology 142, 316.[Medline]
Herdman, M., Janvier, J. B., Waterbury, J. B., Rippka, R., Stanier, R. Y. & Mandel, M. (1979). Deoxyribonucleic acid base composition of cyanobacteria. J Gen Microbiol 111, 6371.
Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237244.[CrossRef][Medline]
Honda, D., Yokota, A. & Sugiyama, J. (1999). Detection of seven major evolutionary lineages in cyanobacteria based on 16S rRNA gene sequence analysis with new sequences of five marine Synechococcus strains. J Mol Evol 48, 723739.[Medline]
Iteman, I., Rippka, R., Tandeau de Marsac, N. & Herdman, M. (2000). Comparison of conserved structural and regulatory domains within divergent 16S rRNA23S rRNA spacer sequences of cyanobacteria. Microbiology 146, 12751286.
Jain, R., Rivera, M. C. & Lake, J. A. (1999). Horizontal gene transfer among genomes: the complexity hypothesis. Proc Natl Acad Sci U S A 96, 38013806.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, Vol. III, pp. 21132. Edited by H. N. Muntu. New York: Academic Press.
Kaneko, T., Sato, S., Kotani, H. & 21 other authors (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3, 109136.[Medline]
Lu, W., Evans, E. H., McColl, S. K. & Saunders, V. A. (1997). Identification of cyanobacteria by polymorphisms of PCR-amplified ribosomal DNA spacer region. FEMS Microbiol Lett 153, 141149.[CrossRef]
Lyra, C., Suomalainen, S., Gugger, M., Vezie, C., Sundman, P., Paulin, L. & Sivonen, K. (2001). Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. Int J Syst Evol Microbiol 51, 513526.[Abstract]
Maeda, H., Kawai, A. & Tilzer, M. M. (1992). The water bloom of cyanobacterial picoplankton in Lake Biwa, Japan. Hydrobiologia 24, 93103.
Miller, S. R. & Castenholz, R. W. (2000). Evolution of thermotolerance in hot spring cyanobacteria of the genus Synechococcus. Appl Environ Microbiol 66, 42224229.
Mullis, K. B. & Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol 155, 335350.[Medline]
Nelissen, B., De Baere, R., Wilmotte, A. & De Wachter, R. (1996). Phylogenetic relationships of nonaxenic filamentous cyanobacterial strains based on 16S rRNA sequence analysis. J Mol Evol 42, 194200.[Medline]
Neuschaefer-Rube, O., Westermann, M., Bluggel, M., Meyer, H. E. & Ernst, A. (2000). The blue-colored linker polypeptide L55 is a fusion protein of phycobiliproteins in the cyanobacterium Synechocystis sp. strain BO 8402. Eur J Biochem 267, 36233632.
Otsuka, S., Suda, S., Li, R., Watanabe, M., Oyaizu, H., Matsumoto, S. & Watanabe, M. M. (1999). Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence. FEMS Microbiol Lett 172, 1521.[CrossRef][Medline]
Palenik, B. & Swift, H. (1996). Cyanobacterial evolution and prochlorophyte diversity as seen in DNA-dependent RNA polymerase gene sequences. J Phycol 32, 638646.
Pick, F. R. (1991). The abundance and composition of freshwater picocyanobacteria in relation to light penetration. Limnol Oceanogr 36, 14571462.
Pohl, T. M. & Maier, E. (1995). Sequencing 500 kb of yeast DNA using a GATC1500 direct blotting electrophoresis system. BioTechniques 19, 482486.[Medline]
Postius, C. & Ernst, A. (1999). Mechanisms of dominance: coexistence of picocyanobacterial genotypes in a freshwater ecosystem. Arch Microbiol 172, 6975.[CrossRef][Medline]
Postius, C., Ernst, A., Kenter, U. & Böger, P. (1996). Persistence and genetic diversity among strains of phycoerythrin-rich cyanobacteria from the picoplankton of Lake Constance. J Plankton Res 18, 11591166.[Abstract]
Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111, 161.
Rippka, R., Castenholz, R. W. & Herdman, M. (2001). Form-genus IV. Cyanobium Rippka and Cohen-Bazire 1983. In Bergey's Manual of Systematic Bacteriology, 2nd edn, Vol. 1, pp. 498499. Edited by D. R. Boone, R. W. Castenholz & G. M. Garrity. New York: Springer.
Robertson, B. R., Tezuka, N. & Watanabe, M. M. (2001). Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content. Int J Syst Evol Microbiol 51, 861871.[Abstract]
Rocap, G., Distel, L., Waterbury, J. B. & Chrisholm, S. W. (2002). Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S23S ribosomal DNA internal transcribed spacer sequences. Appl Environ Microbiol 68, 11801191.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406425.[Abstract]
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 54635467.[Abstract]
Scheldeman, P. B., Baurain, D., Bouhy, R., Scott, M., Muhling, M., Whitton, B. A., Belay, A. & Wilmotte, A. (1999). Arthrospira (Spirulina) strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer. FEMS Microbiol Lett 172, 213222.[CrossRef][Medline]
Stockner, J. G., Callieri, C. & Cronberg, C. (2000). Picoplankton and other non-bloom-forming cyanobacteria in lakes. In The Ecology of Cyanobacteria, pp. 195231. Edited by B. A. Whillon & M. Potts. Dordrecht: Kluwer.
Toledo, G., Palenik, B. & Brahamsha, B. (1999). Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group. Appl Environ Microbiol 65, 62475251.
Turner, S. (1997). Molecular systematics of oxygenic photosynthetic bacteria. Plant Syst Evol 11 suppl., 1352.
Urbach, E., Scanlan, D. J., Distel, D. L., Waterbury, J. B. & Chisholm, S. W. (1998). Rapid diversification of marine picoplankton with dissimilar light harvesting structures inferred from sequences of Prochlorococcus and Synechococcus (cyanobacteria). J Mol Evol 46, 188201.[Medline]
Van de Peer, Y. & De, Wachter, R. (1994). TREECON: a software package for the construction and drawing of evolutionary trees. Comput Appl Biosci 9, 177182.
Vincent, W. F., Bowman, J. B., Powell, L. M. & McMeekin, T. A. (2000). Phylogenetic diversity of picocyanobacteria in Arctic and Antarctic ecosystems. In Microbial Biosystems: New Frontiers. Proceedings of the 8th International Symposium on Microbial Ecology, pp. 317322. Edited by C. R. Bell, M. Brylinski, P. Johnson-Green. Halifax, Canada: Atlantic Canada Society for Microbial Ecology.
Vörös, L., Gulyas, P. & Nemeth, J. (1991). Occurrence, dynamics and production of picoplankton in Hungarian shallow lakes. Int Rev Gesamten Hydrobiol 76, 617629.
Ward, D. M., Ferris, M. J., Nold, S. C. & Bateson, M. M. (1998). A natural view of microbial biodiversity within hot spring cyanobacterial mat communities. Microbiol Mol Biol Rev 62, 13531370.
Waterbury, J. B. & Rippka, R. (1989). Subsection I. Order Chroococcales Wettstein 1924, emend. Rippka et al., 1979. In Bergey's Manual of Systematic Bacteriology, vol. 3, pp. 17281746. Edited by J. T. Staley, M. P. Bryant, N. Pfennig & J. G. Holt. Baltimore: Williams & Wilkins.
West, N. J. & Adams, D. G. (1997). Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site. Appl Environ Micriobiol 63, 44794484.[Abstract]
Wilmotte, A., Turner, S., Van de Peer, Y. & Pace, N. R. (1992). Taxonomic study of marine Oscillatorian strains (cyanobacteria) with narrow trichomes. II. Nucleotide sequence analysis of the 16S ribosomal RNA. J Phycol 28, 828838.
Wilmotte, A., Van der Auwera, G. & De Wachter, R. (1993). Structure of the 16S ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF (Mastigocladus laminosus HTF) strain PCC 7518 and phylogenetic analysis. FEBS Lett 317, 96100.[CrossRef][Medline]
Wilmotte, A., Neefs, J. M. & De Wachter, R. (1994). Evolutionary affiliation of the marine nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067, derived by 16S ribosomal RNA sequence analysis. Microbiology 140, 21592164.[Abstract]
Wood, A. M., Phinney, D. A. & Jentsch, C. S. (1998). Water column transparency and the distribution of spectrally distinct forms of phycoerythrin-containing organisms. Mar Ecol Prog Ser 162, 2531.
Received 17 January 2002;
revised 9 September 2002;
accepted 16 October 2002.