Evolution of branching filamentous cyanobacteria: Molecular-phylogenetic analyses of stigonematalean species

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1 Evolution of branching filamentous cyanobacteria: Molecular-phylogenetic analyses of stigonematalean species Akiko Tomitani Research Program for Paleoenvironment, Institute for Research on Earth Evolution (IFREE) 1. Introduction Modern cyanobacteria form a morphologically diversified group. In traditional taxonomy, morphological characters are used to divide them into five subsections [Rippka et al., 1979; Castenholz, 2001]. Cyanobacteria of subsection I (formerly order Chroococcales) and II (Pleurocapsales) are unicellular coccoids. Those of subsections III to V have filamentous forms with varying morphological complexity. Filaments of subsection III (Oscillatoriales) have only vegetative cells, whereas in subsections IV (Nostocales) and V (Stigonematales) vegetative cells can differentiate into heterocysts or akinetes depending on growth conditions. Heterocystous species are divided into two subgroups, subsections IV and V. Subsection IV consists of heterocystous cyanobacteria whose cells always divide along with the longitudinal axis of a trichome and therefore filaments are uniseriate and one-dimensional. In cyanobacteria of subsection V, some cells in a trichome have additional division plane(s) and their filaments exhibit true-branchings in all members and are multiceriate in some cases. Cyanobacteria of subsection V present the highest degree of developmental complexity in prokaryotes, characterized by cell differentiation and true-branching filaments. Elucidation of how cyanobacterial complex morphology evolved is a key to an understanding not only of their evolutionary history but also of prokaryotic developmental systems. Many Precambrian microfossils have been linked to modern cyanobacteria by comparative study between fossil and extant taxa [Knoll and Golubic, 1992]. However, geological records are always accompanied by incomplete preservation and the evolutionary history of cyanobacteria has remained unclear. In the past few decades, spectacular progress has occurred in molecular biology, and molecular phylogeny has become a powerful tool in elucidating biological evolution, owing to tremendous accumulation of sequence data and splendit progress in computational analyses. Molecular data have been used to reconstruct cyanobacterial phylogeny so far. Most are based on 16S rrna sequences [e.g., Giovannoni et al., 1988; Turner et al., 1999; Wilmotte et al., 2001; Gugger et al., 2004]. Those studies suggested monophyly of heterocystous clade (subsection IV and V) and that of cyanobacteria bearing true branching (V). Some phylogenetic trees are constructed based on other gene sequences, such as nifh [Zehr et al., 1997] or nifd [Henson et al., 2004]. However, the nifh and nifd analyses do not support monophyletic origin of stigonematalean taxa. Moreover, most of the phylogenies presented to date contain only two genera (Chlorogloeopsis, Fischerella) of subsection V, due to lack of axenic cultures, and could not resolve the evolutionary history of branching filamentous cyanobacteria. Here the author carried out a molecular phylogenetic study of stigonematalean cyanobacteria to examine how morphologically complex cyanobacteria evolved. Seven strains distributed five genera of subsection V were chosen to represent morphological variety of the group. Multiple genes (16S rrna, hetr) were sequenced and analyzed to clarify phylogeny. 2. Materials and methods 2.1 Gene isolation and sequencing Seven cyanobacterial strains of subsection V were obtained from Culture Collection of Algae at the University of Göttingen (SAG collection, Germany) (Tab.1). Total genomic DNAs were extracted by a standard method [Asubel et al., 1987]. 16S rrna was amplified using a pair of primers designed specially for cyanobacteria [Urbach et al., 1992]. A pair of primers for hetr were designed based on conserved regions between the hetr sequences of Nostoc (formerly described as Anabaena) sp. PCC7120 (GenBank accession number L02522) and of Nostoc punctiforme (AF318069). Polymerase chain reactions (PCRs) were performed in a GeneAmp PCR System 9700 (Applied Biosystems, USA) using the GeneAmp Gold PCR Reagent kit (Applied biosystems, USA). Amplified PCR products were cloned using TOPO TA cloning kit (Invitrogen, USA). Both strands of a minimum of 3 clones from each PCR product were sequenced with the Big Dye Terminators Cycle Sequencing kit version 3.1 (Applied Biosystems, USA) using a DNA sequencer (3100 Genetic Analyser, Applied Biosystems, USA). 2.2 Phylogenetic analyses. 16S rrna sequences of cyanobacteria (accession no. AB003165, AB039009, AB39006, AF001480, AF027655, AF053398, AF091108, AF091150, AF132777, AF132783, AF132785, AF132789, AF132790, AF132792, AF132933, AJ544077, AJ544086, AJ544087, D64000, D83715, U40340, X59559, X63141, X70770, X78680) and that of Agrobacterium (D14500) were obtained from GenBank. Sequences of two hetr genes of two nostocalean cyanobacteria (M37779, AF318069) and of two hetr-like genes of two oscillatorialeans (AF410433, AF410432) were also obtained from GenBank. The nucleotide sequences were aligned using CLUSTAL X [Jeanmougin et al., 1998] together with manual refinement. Phylogenetic trees were generated using PAUP* (version 4.0b10) [Swofford, 2002]. The Hasegawa Kishino Yabe 1985 model was used as a nucleotide substitution model for the neighbor joining (NJ) and maximum likelihood (ML) analyses. Each calculation based on the maximum parsimony (MP) method was performed by heuristic search of 100 replicates with TBR (tree bisection and reconnection) option. ML analyses of thirty-one 16S rrna 1

2 sequences were carried out by heuristic search with NNI (nearestneighbor interchange) option using the NJ and MP results as starting trees. To reconstruct phylogeny based on 11 sequences of either 16S rrna or hetr, the ML calculations were executed by heuristic search with TBR option, using obtained results from NJ and MP analyses as starting trees. Base frequencies and transition transversion ratio were estimated based on each data set. 3. Results 16S rrna sequences obtained in this study consist of 1147 to 1149 bp nucleotides. To examine monophyly of subsection V, the stigonematalean 16S rrna sequences obtained were aligned together with known 16S rrna sequences of 26 other cyanobacteria and that of Agrobacterium as an outgroup (data not shown). All the sites where gaps existed or where sequences were ambiguous were excluded and 1120 positions were used for calculation. Phylogenetic analyses show that heterocyst- and akinete-bearing cyanobacteria (subsection IV, V) form a monophyletic clade (Fig. 1). Monophyly is supported by the ML analysis, as well as by the NJ and MP methods, with high bootstrap values [Felsenstein, 1985] of 100% and 97%, respectively. The 16S rrna trees constructed by all the three methods also supports the view that the members of subsection V form a monophyletic cluster within the heterocystous clade, with high bootstrap values of 99 (NJ) and 93% (MP). Further more, internal tree topology within subsection V is common to the phylogenetic trees constructed by all the three methods. In contrast to monophyly of heterocustous taxa, filamentous cyanobacteria without cell differentiation (subsection III) mix with unicellular members (I, II). Three pleurocapsalean cyanobacteria form a monophyletic clade with a conserved internal tree topology and have the chlorophyll b-containing Prochloron as the closest relative to the cluster. To further examine relationships between cyanobacteria of subsection V, partial hetr sequences were amplified and sequenced. All of the obtained hetr sequences consist of 742 bp nucleotides. They were aligned with the hetr sequences of two nostocalean Nostoc PCC7120 and N. punctiforme ATCC29133, whose genomes are completely sequenced, and hetr-like genes of the oscillatorialeans Leptolyngbya (formerly described as Plectonema) PCC73110 and Trichodesmium IMS101 (data not shown). No gene known so far has significant similarity to hetr and so an outgroup was not included in the calculation. All the sites where gaps existed or where sequences were ambiguous were excluded and 735 positions were used for calculation. Phylogenetic trees contructed by the three methods supported the same tree topology (Fig. 2a), except that Westiellopsis SAG16.93 forms a cluster with Nostochopsis in the MP and ML trees. To compare the phylogenies inferred from hetr and 16S rrna, 16S rrna sequences obtained here were analyzed together with known 16S rrna genes of the same cyanobacterial strains used in the hetr analysis (Fig. 2b). Overall tree topology is mostly common to the trees constructed by three analytical methods, except that Nostoc PCC7120 and N. punctiforme form a monophyletic cluster in the NJ tree. 4. Discussion The 16S rrna phylogeny of thirty cyanobacteria allocated to all the five subsections (Fig. 1) supports the view that heterocystand akinete- bearing cyanobacteria (subsections IV and V) are monophyletic in origin, consistent with the former 16S rrna phylogenies. It also suggests that heterocystous cyanobacteria with true-branching filaments (subsection V) are monophyletic. hetr plays a key role in the early stage of heterocyst differentiation [Wolk et al., 1994] and is unique to filamentous cyanobacteria. Accordingly, hetr should provide a good genetic marker for investigations into the relationship within the heterocystous clade. Interestingly, genes homologous to hetr have been detected from some non-heterocystous nitrogen-fixing filamentous cyanobacteria (e.g. Leptolyngbya PCC73110, Trichodesmium IMS101) though their function is not yet certain [Janson et al., 1998]. The phylogeny inferred from eleven hetr sequences (Fig. 2a) supports almost the same tree topology as 16S rrna (Fig. 2b), except that Westiellopsis SAG16.93 forms a cluster with Nostochopsis in the hetr tree. Both 16S rrna and hetr analyses show that Chlorogloeopsis, which is characterized by absence of distinct branches, has a basal position within the stigonematalean clade. Fischerella, Nostochopsis and Wesiellopsis, whose filaments exhibit T -type branching [Golubic et al., 1996], form a cluster within subsection V. Type of branching forms can be a criterion for grouping stigonematalean genera into broader subgroups. Fischerella SAG46.79, 2027 and Westiellopsis SAG23.96 appeared to have the identical or nearly identical sequences of both 16S rrna and hetr genes (99.7 to 100% similarity and sequences are different only where nucleotides are degenerate in the determined sequences in this study), suggesting that they are conspecific strains. Inconsistency of genetic information and morphological classification was also indicated within some strains of the genera Fischerella and Nostochopsis [Gugger et al., 2004]. Considering polyphyletic presence of the two Wesiellopsis species, which are assigned to the same species by the SAG culture collection, in both 16S rrna and hetr trees (Fig. 2a, b), reexamination of cyanobacteria of the genera Fischerella, Nostochopsis and Wesiellopsis would be necessary. 5. Conclusion Molecular phylogenetic analyses were carried out on 16S rrna and hetr sequences of seven strains allocated to 5 genera of filamentous cyanobacteria bearing true branching. Monophyletic origin of the stigonematalean taxa was supported by phylogenetic analysis of 16S rrna isolated from the cyanobacteria of subsection V together with those of subsection I to IV. It also supported monophyly of the heterocystous taxa, consistent with previous studies on the cyanobacterial 16S rrna sequences. Detailed genetic analyses of the stigonematalean strains using 16S rrna and hetr revealed that Chlorogloeopsis has the basal position and that the species bearing T -type branching form a monophyletic cluster in subsection V. Although there exist number of stigonematalean taxa that were not examined, the phylogenetic analyses here suggested that branching type could be used to group stigonematalean genera into broader subgroups within subsection V. Phylogenetic relationships inferred from molecular data here are not compatible with traditional classification in the case of the genera Fischerella and Wesiellopsis. It is necessary to revise the existing cyanobacterial taxonomy by integrating morphological, physiological and genetic information on, at least, those two genera. Further research on the evolution of branching-filamentous cyanobacteria will provide background knowledge for understand- 2

3 ing how prokaryotic multicellularity evolved. Acknowledgements. The author thanks Masaru Kawato (XBR, JAMSTEC) for his technical support in DNA sequencing. This work was partly supported by Fujiwara Natural History Foundation to A. T. Bryant, pp , Kluwer Acad. Pub., Netherlands, Zehr, J. P., M. T. Mellon, W. D. Hiorns, Phylogeny of cyanobacteial nifh genes: evolutionary implication and potential applications to natural assemblages, Microbiol., 143, , References Asubel, F. M., R. Brent, R. E. Kingston, D. Moore,J. G. Seidmann, J. A. Smith, and K. Sturuhl (Eds.), Current Protcols in Molecular Biology, 2.4, Wiley, New York, Castenholz, R. W., General characteristics of the cyanobacteria, In Bergey's Manual of Systematic Bacteriology, Vol.1, 2nd ed., Ed., D. R. Boone and R. W. Castenholz, pp , Springer-Verlag, New York, Felsenstein, J., Confidence limits on phylogenies : An approach using the bootstrap, Evolution, 39, , Giovannoni, S. J., S. Turner, G. J. Olsen, S. Barns, D. J. Lane and N. R. Pace, Evolutionary relationships among cyanobacteria and green chloroplasts, J. Bacteriol. 170, , Golubic, S., M. Hernandez-Marine, L. Hoffmann, Developmental aspects of branching in filamentous Cyanophyta/Cyanobacteria, Algological Studies, 83, , Gugger, M. F. and L. Hoffmann, Polyphyly of true branching cyanobacteria (Stigonematales), Int. J. Sys. Evol. Microbiol., 54, , Henson, B. J., S. M. Hesselbrock, L. E. Watson, S. R. Barnum, Molecular phylogeny of the heterocystous cyanobacteria (subsections IV and V) based on nifd, Int. J. Syst. Evol. Microbiol. 54, , 2004.ß Janson, S., A. Matveyev and B. Bergman, The presence and expression of hetr in the non-heterocystous cyanobacterium Symploca PCC 8002, FEMS Microbiol. Lett., 168, , Jeanmougin, F., J.D. Thompson, M. Gouy, D. G. Higgins and T. J. Gibson, Multiple sequence alignment with Clustal X. Trends Biochem. Sci., 23, , Knoll, A.H. and S. Golubic, Living and Proterozoic cyanobacteria, In Early Organic Evolution: Implications for Mineral and Energy Resources, Ed., Schidlowski, M. et al.., pp , Springer- Verlag, Berlin, Rippka, R. J., J. B. Deruelles, J. B. Waterbury, M. Herdman & R. Y. Stanier, Generic assignments, strain histories and properties of pure cultures of cyanobacteria, J. Gen. Microbiol., 111, 1-61, Swofford, D. L., PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), ver. 4., Sinauer Associates, Sunderland, MA, Turner, S., K. M. Prayer, V. P. Miao and J. D. Palmer, Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rrna sequence analysis, J. Eukaryot. Microbiol., 46, , Urbach, E., D. L. Robertson and S. W. Chisholm, Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation, Nature, 355, , Wilmotte, A. and M. Herdman, Phylogenetic relationships among the cyanobacteria based on 16S rrna sequences, In Bergey's Manual of Systematic Bacteriology, Vol.1, 2nd ed.,, Ed., D. R. Boone and R. W. Castenholz, pp , Springer-Verlag, New York, Wolk, C. P., A. Ernst and J. Elhai, Heterocyst metabolism and development, In The Molecular Biology of Cyanobacteria, Ed., D. 3

4 Figure 1. The phylogenetic relationships of cyanobacteria inferred from 16S rrna sequences. The tree was constructed by ML method. Roman numerals denote cyanobacterial subsections I to V. The sequences obtained in this study are indicated in bold type. Numbers at each branch point are the bootstrap values for percentages of 1000 replicate trees calculated by NJ (upper) and MP (lower) methods. Only values above 50% are shown. The arrowheads represent the deepest roots determined using Agrobacterium as an outgroup for 16S rrna. 4

5 Figure 2. The phylogenetic relationships of stigonematalean cyanobacteria inferred from (a) hetr and (b) 16S rrna sequences. The trees were constructed by ML method. Numbers at each branch point are the bootstrap values for percentages of 1000 replicate trees calculated by NJ (upper) and MP (lower) methods. Only values above 50% are shown. The trees are unrooted. Table 1. Cyanobacterial strains used in this study. Taxon Strain Origin Chlorogloeopsis fritschii SAG a Garden soil, Allahabad, India. Fischerella spec. SAG Surtsey near Iceland. Fischerella muscicola SAG 2027 Rice field near Allahabad, India. Mastigocladus laminosus SAG 4.84 Thermal spring of Reyhjanes/Isafjord, Iceland. Nostochopsis lobatus SAG 2.97 Cryptoendolithic in sandstone, Northern Province, South Africa. Westiellopsis prolifica SAG Aldabra Atoll, Seychelles. Westiellopsis prolifica SAG Saline soil of ph 9.5, Karnal, India. 5

The evolutionary diversification of cyanobacteria: Molecular phylogenetic and paleontological perspectives

The evolutionary diversification of cyanobacteria: Molecular phylogenetic and paleontological perspectives Akiko Tomitani, Andrew H. Knoll, Colleen M. Cavanaugh, and Terufumi Ohno The Kyoto University