Genetic Diversity of Brazilian Cyanobacteria Revealed by Phylogenetic Analysis

Cyanobacteria are prokaryotic microorganisms with a long evolutionary history. They are usually aquatic, performe oxigenic photosynthesis and have been responsible for initial rise of atmospheric O2. Cyanobacteria are predominant in the phytoplankton of continental waters, reaching an ample diversity of shapes due to their morphological, biochemical and physiological adaptabilities acquired all along their evolutionary history. Some cyanobacteria, like the Microcystis, Cylindrospermopsis, Anabaena, Aphanizomenon and Planktothrix, could give rise to blooms with the liberation of a wide range of toxins.

cpcA) and the intervening intergenic spacer (IGS) show variations in their sequences which are capable of differentiating genotypes below the generic level. Besides, they are relatively large-sized in comparison with other genes encoding for photosynthetic pigments (~700-800bp), belonging to all cyanobacteria and they are almost totally restricted to this group of organism when in freshwater ecosystems (Barker et al., 2000;Bittencourt-Oliveira et al., 2001, 2009bBolch et al., 1996;Dyble et al., 2002;Haverkamp et al., , 2009Neilan et al., 1995;Tan et al., 2010;Wu et al., 2010).
Geitlerinema amphibium and G. unigranulatum are morphologically similar to each other. According to Romo et al. (1993) and Komárek & Anagnostidis (2005), they could be differentiated only by their dimensions and the number of cyanophycin granules close to the cross-walls. However, Bittencourt-Oliveira et al. (2009b) used transmission electronic and optical microscopy to study strains of these two morphospecies showing that there is a large overlap between them, in both cell dimensions and the number of granules per cell. Therefore, the authors concluded that it was not possible to distinguish G. amphibium from G unigranulatum by means of morphological data.
Cyanobacterial blooms of the genus Microcystis (Chroococcales, Cyanobacteria) are of serious ecological and public health concern due to their ability to dominate the planktonic environment and produce toxins. These toxins can affect aquatic and terrestrial organisms and humans. M. aeruginosa (Kützing) Kützing, M. ichthyoblabe Kützing, M. novacekii (Komárek) Compère, M. flos-aquae (Wittrock) Kirchner ex Forti and M. viridis (A. Braun in Rabenhorst) Lemmermann are commonly reported species causing hepatotoxicity and odor problems in lakes and water supply systems (Carmichael, 1996;Codd et al., 1999). However, in tropical regions such as in Brazil, M. panniformis is also a potential microcystin-producing morphospecies. It is morphologically characterized by flattened irregular colonies and it is closely related to M. aeruginosa morphospecies (Bittencourt-Oliveira et al., 2005).
In the same way that happens to other species of the genus, M. panniformis colonies can have morphologically different stages during their life cycle, which make difficult to define and to establish taxonomic limits for its identification (Bittencourt-Oliveira, 2000;Otsuka et al., 2000Otsuka et al., , 2001. Furthermore, it has been observed that in Microcystis populations a genotype could represent more than one morphotype , or that distinct morphotypes could represent a single genotype (Bittencourt-Oliveira et al., 2001;Hannde et al., 2007). HIP1 sequences is a powerful tool to study genetic diversity of cyanobacteria strains or closely related taxa, and it was used by Bittencourt-Oliveira et al. (2007a, 2007b to investigate M. panniformis, G. amphibium and G. unigranulatum. However, it is recommended that studies on molecular phylogeny do confirm, by the use of DNA sequences, previous findings which have used fingerprinting techniques like HIP1.
The goal of this study was the investigation of Geitlerinema amphibium, G. unigranulatum and Microcystis panniformis taxonomic position, using the phycocyanin gene partial sequencing for the build up of phylogenetic trees.

Field sampling, isolation and growth conditions
Sequences from 14 clonal and non-axenic strains of Geitlerinema and 17 of Microcystis from the Brazilian Cyanobacteria Collection of the University of São Paulo (BCCUSP; previously named FCLA), were used in this study ( Figure 1, Table 1). These strains were isolated from aquatic habitats situated in localities of Brazil. One strain of Geitlerinema amphibium (BCCUSP31) was donated by Dr. Romo from University of Valencia, Spain. For isolation purpose one individual colony or thricome was removed by micromanipulation techniques with Pasteur pipettes at magnifications of 100 -400X. Each isolate was washed, by transferring it through several consecutive drops of water until all other microorganisms be removed, and subsequently transferred to glass tubes containing 10 ml of BG-11 medium (Rippka et al., 1979). All strains were maintained in incubators at 21°C ± 1ºC and 30 ± 5 μmol photons . m -2 . s -1 (photometer Li-Cor mod. 250), under a 14:10 hour light:dark photoperiod. The cultures are maintained at the Brazilian Cyanobacteria Collection of the University of São Paulo, Brazil (BCCUSP). The Spanish strain (BCCUSP 31) was acclimatized for three months in the BCCUSP at the same conditions as for those before the beginning of the experiment.   Tillett et al. (2001).

www.intechopen.com
Another ten sequences selected form the GenBank were also included in the analysis.

DNA extraction
DNA was extracted from fresh cells harvested at the exponential phase. Total genomic DNA was extracted using the commercial kit Gnome DNA (BIO 101, Vista, CA, USA) according to the manufacturer's instructions or according to the procedures described in Bittencourt-Oliveira et al. (2010).

PCR amplification
Amplifications were carried out in the thermocycler GeneAmp 2400 or GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The reaction were performed using 2.5 to 10 ng of DNA, 20 µM of each oligonucleotide primer in a total volume of 25 µL and 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ) with buffer containing 1.5 mM MgCl 2 and 200 M of each dNTP (Boehringer-Mannheim, Mannheim, Germany).
Amplifications for the intergenic spacer and flanking regions from cpcBA-phycocyanin operon were accomplished with the primers PC -F and PC -R described by Neilan et al. (1995) using the same cycling parameters conditions according to Bolch et al. (1996). Control reactions were carried out by using the same reaction conditions and primer without DNA, and no PCR products were detected on agarose electrophoresis. All PCR reactions were repeated at least five times.
Amplification products were visualized by electrophoresis on 0,7% agarose gels stained with ethidium bromide. PCR products were purified using the Purelink Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. When necessary, bands were extracted and purified from the gel using the QIAquick kit (Qiagen, Hilden, Germany) as recommended by the manufacturer.

Sequencing and phylogenetic analyses
The amplified fragments were directly sequenced using the forward and reverse primers with ABI Prism Big Dye Terminator Cycle Sequencing Ready Kit (Applied Biosystems, Foster City, CA, USA) and 3100 ABI sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. To avoid errors from PCR at least four separated amplification reactions were pooled for sequencing. The sequencing was repeated on independent PCR products. The PCRs products were sequenced on both strands at least three times.
Automated base calls for both strands were checked by manual inspection and ambiguous calls and conflicts resolved by alignment and comparison using BioEdit program (Hall, 1999) to establish a consensus sequence for each strain. Consensus sequences were aligned using ClustalW in BioEdit program (Hall, 1999) and were manually inspected. Spirulina subsalsa PD2002/gca (accession number AY575949) and Cyanothece sp. ATCC51142 (accession number CP000806) were used as outgroups for Geitlerinema and Microcystis analysis respectively.

www.intechopen.com
Evolution distances between the sequences were calculated by P distance in the program MEGA 4.0.2 (Tamura et al., 2007).
Phylogenetic analyses were performed with MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003). An appropriate evolution model was selected using MrModeltest 2.2 (Nylander, 2004) under the Akaike Information Criterion. For the Bayesian analysis two runs of four Markov chains over 5,000,000 generations sampling every 100 generations was employed. The initial 2,500 generations were discarded as burn-in. For all analyses, posterior probability values were considered low up to 70%, moderate from 71% to 90%, and high above 90%.

Geitlerinema amphibium
The phylogenetic tree including Geitlerinema species showed that Geitlerinema amphibium and G. unigranulatum are not genetically separated from each other ( Figure 2).
The phylogenetic tree showed two Clades (I and II) ( Figure 2). The Clade I encompasses three strains, one of G. amphibium separated from the other two of G. unigranulatum . These strains showed the region corresponding to the IGS with 83bp, shorter than the remaining sequences which contained up to 298bp. The evolutionary distance calculated by P distance between the strains from Clade I and II was up to 0.098, while among strains included on Clade II it did not exceed 0.056. This is a demonstration that the strains included in Clade I were genetically diverse from the others. Bittencourt-Oliveira et al. (2009b) had shown that strains BCCUSP352 and BCCUSP94, belonging to Clade I, did not exhibit differences regarding cellular morphology and ultrastructure which would allow distinguish them from the other strains of Geitlerinema.
The inclusion of G. amphibium BCCUSP80 in the present study reinforced the argument that these taxons are distinct species, maybe even of distinct genera because of the IGS size difference taxa.
The Clade II encompassed all the remaining strains and could be subdivided into two smaller groups (A and B). The group A included only strains of the morphospecies G. amphibuim , while the group B was equally constituted by strains from G. amphibuim or from G. unigranulatum, besides two strains of "Oscillatoria sp. G. amphibium and G. unigranulatum show overlapping morphological characteristics which make difficult the taxonomic discrimination (in the identifying sense). According to Komárek & Anagnostidis (2005), the variation interval of the G. amphibium cellular width ranges from (1) 1.8 to 3 (3.5-4) μm, whereas for G. unigranulatum, it ranges from 0.8 to 2.4 μm.

www.intechopen.com
According to the data taken by Bittencourt-Oliveira et al. (2009b), the measurements of cell lengths in strains attributable to G. amphibium and G. unigranulatum show complete overlap of maximum, minimum, and mean values. Accordingly, only when the length by width ratio (L:W) is taken into account the distinction is more accentuated.
One or two cyanophycin granules per cell (less frequently three) were positioned near the cross walls in strains attributed to both species (Figure 3). Therefore, based on cellular morphology, only through the length to width ratio was it possible to differentiate G. amphibium from G. unigranulatum. Given their quite uniform morphology and the occurrence of these taxa in the same habitat, it would be nearly impossible to distinguish them in nature. The localization and number of granules, as well as ultrastructural data, did not aid in species discrimination either. Therefore, those characteristics could not be used as diacritical features. The obtained results by Bittencourt-Oliveira et al. (2009b) did not show ultrastructural differences between G. amphibium and G. unigranulatum strains, except for the BCCUSP96 which in some trichomes exhibited slightly thickening of the apical cell, slightly folded cellular wall, thylakoids with invaginations and unidentified granules. BCCUSP96 strain was unique in having four granules per cell in some trichomes and the highest cell length-to-width ratio (more details, see Bittencourt-Oliveira et al. 2009b). We have observed in the present study that G. amphibium and G. unigranulatum are not genetically separated from each other. The morphospecies are mixed in the phylogenetic tree and they could not be distinguished as monophyletic entities. Our findings reinforce that they should be considered synonyms as previously stated in Bittencourt-Oliveira et al.  Fig. 4. Bayesian phylogenetic tree with Microcystis aeruginosa and M. panniformis strains. The tree was generated using intergenic spacer and flanking regions from cpcBA-phycocyanin operon. For the Bayesian analysis two runs of four Markov chains over 5,000,000 generations sampling every 100 generations was employed. The initial 2,500 generations were discarded as burn-in. Posterior probability (x100) is shown on each branch when higher them 70. Cyanothece sp. ATCC 51142 (CP000806) used as outgroup. The bar represents 0.08 substitutions. Strains in boldface were sequenced in this work. www.intechopen.com

Microcystis panniformis
Similarly as in the strains of Geitlerinema, Microcystis panniformis strains did not form a Clade isolated from M. aeruginosa in the phylogenetic tree generated by means of the cphycocyanin genes cpcB, cpcA sequences and the intervening intergenic spacer (cpcBA-IGS) (Figure 4).
Two major groups in the Microcystis phylogenetic tree were formed (Clade I and II), constituted by strains of M. panniformis and M aeruginosa. We observed from the topology tree that some strains of M. panniformis are genetically more close to those identified as M. aeruginosa than others from their same morphospecies.
The posterior probability was moderate for both Clades and only the M. aeruginosa BCCUSP232 strain, situated as sister group of Clade II, did not shown relevant Bayesian support value for its position.
Traditional taxonomy of Microcystis based on morphologic criteria has been questioned by several authors because of the gap lack in the variations observed in the nature and in laboratory conditions. Earlier infraspecific studies of Microcystis concluded that morphology does not correlate with molecular data (Bittencourt-Oliveira et al., 2001;Kurmayer et al., 2003;Otsuka et al., 1999aOtsuka et al., , 1999bOtsuka et al., , 2000Otsuka et al., , 2001Wu et al., 2007).
Our results indicated that M. panniformis could also be considered synonym of M. aeruginosa. The similarity found between sequences of M. panniformis and M. aeruginosa coming from diverse regions all over the world, led us to stress the cosmopolitan character of the species, with strains showing ample geographical distribution in both South and North hemispheres. These results corroborate similar previous findings (Bittencourt-Oliveira et al., 2001, 2007bOtsuka et al., 1999aOtsuka et al., , 1999bOtsuka et al., , 2000Otsuka et al., , 2001.

Conclusion
We conclude that G. unigranulatum and M. panniformis should be considered as synonyms of G. amphibium and M. aeruginosa, respectively, since they do not represent genetically isolated Clades inside those genera. Anagnostidis, K. & Komárek, J. (1985) Modern approach to the classification of the Cyanophytes 1-Introduction. Algological Studies, Vol. 38/39, (January, 1985) Komárek, J. (1990). Modern approach to the classification system of Cyanophytes, 5 -Stigonematales. Algological Studies, vol. 59, (January, 1990), pp.1-73, ISSN 1864Konopka, A.;Handley, B.A. & Hayes, P.K. (2000). Genetic variation in Aphanizomenon (cyanobacteria) colonies from the Baltic Sea and North America. Journal of Phycology, Vol.36, No. 5, (October, 2000),  (1996). Genetic characterization of strains of cyanobacteria using PCR-RFLP of the cpcBA intergenic spacer and flanking regions. Journal of Phycology, Vol.32, No.3, (June, 1996) Genetic Diversity in Microorganisms presents chapters revealing the magnitude of genetic diversity of microorganisms living in different environmental conditions. The complexity and diversity of microbial populations is by far the highest among all living organisms. The diversity of microbial communities and their ecologic roles are being explored in soil, water, on plants and in animals, and in extreme environments such as the arctic deep-sea vents or high saline lakes. The increasing availability of PCR-based molecular markers allows the detailed analyses and evaluation of genetic diversity in microorganisms. The purpose of the book is to provide a glimpse into the dynamic process of genetic diversity of microorganisms by presenting the thoughts of scientists who are engaged in the generation of new ideas and techniques employed for the assessment of genetic diversity, often from very different perspectives. The book should prove useful to students, researchers, and experts in the area of microbial phylogeny, genetic diversity, and molecular biology.