Polyphasic Characterization of the Genus Anabaena from the Gulf of Gdańsk| 65 Group of Organisms

Oceanological and Hydrobiological Studies International Journal of Oceanography and Hydrobiology

Volume 41, Issue 3

ISSN 1730-413X (64–78) eISSN 1897-3191 2012

DOI: 10.2478/s13545-012-0029-8 Received: November 10, 2011 Original research paper Accepted: February 08, 2012

INTRODUCTION

Polyphasic characterization of the genus Cyanobacteria are a very important component of the phytoplankton in the brackish waters of the Anabaena from the Gulf of Gdańsk Gulf of Gdańsk. The environmental conditions of (Southern Baltic Sea). Application of the Baltic Sea are defined by the inflow of fresh DGGE in taxonomic studies on waters from rivers and by the limited inflow of saline environmental samples waters from the North Sea. Without the constant, but small influx of saline water through the Danish straits, the Baltic Sea would have been transformed

into a huge freshwater lake long ago. A clear salinity Grzegorz Siedlewicz, Karolina Forycka, * gradient exists from the almost oceanic conditions in Waldemar Surosz the northern Kattegat to the almost freshwater conditions in the northern Bothnian Bay, where frequent cyanobacteria blooms are observed (Pliński, Department of Marine Biology and Ecology Jóźwiak 1999). The first taxonomic studies of the Institute of Oceanography, University of Gdańsk cyanobacteria from the Gulf of Gdansk date from Al. Piłsudskiego 46, 81-378 Gdynia, Poland the early twentieth century. Lakowitz (1907) described 15 species of cyanobacteria. Populations of Anabaena commonly occur in the plankton of fresh and salt waters, and they often Key words: Cyanobacteria, Anabaena, Gulf of accompany other cyanobacterial species during water Gdańsk, Baltic Sea, polyphasic approach blooms. The genus Anabaena is characterized by straight or spiral single trichomes that often gather in jelly-like clusters. They are capable of producing Abstract heterocysts and akinetes. The presence of these specific cells, and their layout in the trichome is a The aim of this paper was a taxonomic verification of cyanobacteria of the genus Anabaena that occur in the Gulf of significant taxonomic feature (Pliński, Komárek Gdańsk. Classical taxonomic methods were combined with 2007). They are potentially toxic and can produce modern molecular taxonomic methods (polyphasic approach). cyanotoxins from the neurotoxin (anatoxin and Analysis of the species diversity of cyanobacteria from the genus saxitoxins) and hepatotoxin (microcystins) groups Anabaena was based on the microscopic analysis and on the analysis of 16S rRNA and ITS region. Comparison of the (Vaitomaa et al. 2003). The analysis of the obtained results with sequences in GenBank showed 97.8-100% morphological structure of Anabaena specimens similarity for 16S rRNA and 98.8-100% similarity in the case of indicates that the vegetative cells of certain species the ITS fragment. Similarity of the 16S rRNA and ITS sequence from the Gulf of Gdańsk are characterized by similar of 98.5% seems to be sufficient to determine the species. shapes and sizes (Kobos 2008). Morphological and molecular studies on Anabaena strains from various geographical regions conducted by Rippka et al. (2001) confirmed the taxonomic diversity of this * Corresponding author: [email protected]

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 65 group of organisms. denatured, the PCR products continue running Taxonomic verification of the Anabaena genus through the gel as a single-stranded DNA, but the that occurs in the Gulf of Gdańsk was the primary fragments have to remain precisely where they objective of this paper. The morphological studies denatured. To achieve this, the so-called GC clamp is took into consideration the structure (size and shape attached to prevent the complete denaturation. This of cells and their relative position in relation to each GC clamp is a string of 40–60 nucleotides composed other and in the filament or in colonies) according to only of guanine and cytosine, and it is attached to the criteria of classical taxonomy (Starmach 1966; one of the PCR primers. PCR with a GC clamp Anagnostidis, Komárek 1985, 1999; Komárek, results in a product with one end having a very high Anagnostidis 1989; Li, Watanabe 2000; Pliński, denaturation temperature. A PCR product running Komárek 2007). At the same time, molecular studies through DGGE gel will, therefore, partially denature. were performed on environmental samples without The GC clamp remains double stranded. The prior isolation of cyanobacterial strains. The fragment will form a Y-shaped piece of DNA that combination of classic and molecular methods will stick firmly at its position in the gel (Muyzer et al. provides a more realistic picture of evolutionary 1993). It is anticipated that this approach will relations. DNA-based typing methods are widely contribute to the understanding of the genetic known to provide information for classification of diversity of cyanobacteria (Anabaena) populations. cyanobacteria into different genera and species, and The aim of this study was a taxonomic can potentially resolve differences among strains of a verification of cyanobacteria from the genus given species. Therefore, newly-isolated strains must Anabaena from the Gulf of Gdańsk (Southern Baltic be classified on the basis of the polyphasic approach. Sea). Additionally, if required, the previously classified organisms can be reclassified based on these MATERIALS AND METHODS techniques in order to obtain information about their actual position in the bacterial world. Thus, current Samples of Anabaena populations were collected techniques enable microbiologists to decipher the at selected stations in the Gulf of Gdańsk in summer natural phylogenetic relationships between microbial months 2006 (Fig. 1) with horizontal tows made at organisms (Prakash et al. 2007). The Polymerase depths of 0-10 m with a Copenhagen-type plankton Chain Reaction (PCR) was used in the molecular net. Samples for morphological studies were studies, which permitted selective amplification of preserved with Lugol’s iodine, and samples for the studied DNA region of cyanobacteria. DGGE molecular studies were preserved with F6 lysis buffer was also employed and permitted the comparison of prepared with a base of guanidine (Boom et al. 1990). closely related organisms and identification of very subtle genetic differences. DGGE is a robust method for point mutation detection that has been widely used for many years. It is a PCR-based method, in which the altered denaturation temperature of a PCR product with a mutation is compared to a wild-type product. PCR performed on the DNA of a specimen with a point mutation in one of two genes will lead to a mixture of different products. PCR products from both the wild-type gene and the mutated gene are formed, and are known as homoduplex products. There is, however, a subtle difference in the melting temperature between these two products. Another type of product, heteroduplexes, consisting of a wild- type strand combined with a mutant strand of DNA, is also formed during the last cycle of the reaction. During electrophoresis, the PCR products run Fig. 1. The Gulf of Gdańsk with sample collection through the gel as a double-stranded DNA until they stations. SW − Świbno (54°22N, 18°57E); M6 − Mechelinki (54°40N, 18°40E); reach the point where they start to denature. Once K5 − Gdańsk (54°25N, 18°38E); M1 – Mechelinki (54°38N, 18°32E).

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66 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz

The material thus collected was subjected to Table 1 morphological and genetic analysis. The amounts of reagents used for DGGE. Microscopy The denaturing gradient for the 16S rRNA and the ITS fragment The reagents 25% (200 ml) 45% (200 ml) 65% (200 ml) 15% (200 ml) Morphological characteristics of specimens TAE 50% (ml) 4 4 4 1 from the genus Anabaena were observed with a Urea (g) 21.0 39.6 54.5 - Formamide (ml) 20.0 37.2 52.0 - Nikon Eclipse E600 light microscope fitted with a Akrylamide 40% (ml) 41.3 41.3 60.0 25.0 Digital Sight DS-V1 camera and an integrated Mili-Q (ml) fill up to 200.0 to 200.0 to 200.0 to 50.0 program (NIS-Elements), which was used to measure the length and width of vegetative cells, heterocysts gel from the cartridge, it was stained with a solution and akinetes. A scanning electron microscope (SEM) of 50 ml of TE (pH 7.4) and of 5 µl of SYBR Gold was used for analyzing the morphological analysis of Nucleic Acid Gel Stain. The gel was left under a the A. flos-aquae species more accurately. Samples for cover for 15 minutes (this was repeated twice). After SEM analysis were prepared following the procedure staining, the gel was rinsed with a considerable described by Luft (1961) and Surosz, Palinska (2004). amount of Mili-Q water and transferred to a UV The DSM 940 scanning electron microscope used for transilluminator. The removal of the gel with visible the analysis was manufactured by Zeiss. DNA separation was analyzed using QUANT 5.0 SOFTWARE. DNA isolation, PCR, DGGE Polyacrylamide gel fragments were excised from the DNA using a sterile scalpel under a UV The ribosomal DNA of cyanobacterial cells was transilluminator, placed in Eppendorf-type centrifuge isolated using the protocol in the Genomic Midi AX tubes containing 200 μl of Mili-Q water, and then kit manufactured by Heliconius A&A Biotechnology incubated for 24 hours. The suspension was (http://www.aabiot.com). centrifuged for 15 minutes at 13,000 rpm. The Characteristic fragments of 16S rRNA and 16S- supernatant was poured into new Eppendorf-type 23S rRNA (ITS) were studied in this paper. The pairs tubes. The samples were stored at a temperature of - of primers used to study the 16S rRNA fragment 20°C. Afterward, the products obtained were re- were CYA106F/CYA781R and amplified, using the same primers. After re- CYA359F/CYA781R (Nübel et al. 1997), while the amplification, the PCR products were analyzed analysis of 16S-23S rRNA (ITS) was based on primer electrophoretically on 1% agarose gel in the presence pairs 16CITS/23CITS (Neilan 2002, Laamanen et al. of ethidium bromide. Electrophoretic separation 2002) and another pair specific to the genus Anabaena lasted for an hour at 120 V. To determine the – Ana2 (F)/Ana4 (R) (Fergusson, Saint 2000). PCR product size accurately, a Lambda DNA/Eco47I conditions were as described in Palińska and Surosz (AvaII) molecular weight marker was separated (2008). DNA separation with DGGE was performed simultaneously. The next stage was the purification using an INGENYphorU-2 apparatus. of DNA from agarose using a kit for isolation of Polyacrylamide gel electrophoresis was conducted on DNA from agarose gels (similar to Gel-Out and a denaturing gradient that was prepared for the Clean-up manufactured by Heliconius A&A procedure (Table 1), and to which the following Biotechnology (http://www.aabiot.com)). reagents were added: 46 µl of catalyst N,N,N’,N’, - tetramethylethylenediamine (TEMED) and 129 µl of Sequencing and phylogenetic analysis ammonium persulfate (APS) (1 g APS per 10 ml water). Values of these reagents for 15% Samples for sequencing were prepared polyacrylamide without denaturing substances were according to the protocol of the Nucleic Acids 29 µl and 80 µl. 25 ml of denaturing polyacrylamide Analysis Laboratory of the Pomeranian Science and and 7 ml of 15% solid polyacrylamide were used with Technology Park (http://www.ppnt.gdynia.pl) using single gel (Laamanen et al. 2002, Hongmei et al. 4 μl of each primer pair in a concentration of 1 2005). The reaction was conducted at 58°C for 17 pmol/1 µl, which corresponded to 11 μl of purified hours at a voltage of 140 V. Samples and molecular DNA of a known concentration. Samples thus weight standards (PCR products) of 7-8 µl were prepared were transferred to an Applied Biosystem placed in each of the chambers. After removing the ABI PRISM 3130 type 4-capillary DNA sequencer

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 67 for sequencing. The DNA sequence obtained was Considerable differences in cell size were found compared to the full 16S rRNA and ITS sequence in the studied material. The sizes of the vegetative available from the GenBank database cells were within the ranges of 2.5 - 13.4 µm for A. (http://www.ebi.ac.uk/clustalw/index.html). flos-aquae (dominant at station GG-SW), 4.1 - 8.1 for Sequences at the nucleotide level were analyzed with A. spiroides (dominant at station GG-M1 ), 6.2 - 11.3 the Clustal W and Genedoc programs. Dendrograms for A. lemmermannii (dominant at station K5), and 6.3 of the sequences compared were compiled. The - 12.3 for A. circinalis (dominant at station GG-M6). Anabaena genome sequence database was searched Curved trichomes with irregular twists in free- with NCBI BLASTN, version 2.2.10 floating colonies were observed in all the species (http://www.ncbi.nlm.nih.gov/BLAST/). except Anabaena spiroides (Fig. 2, 3). The Anabaena Phylogenetic analysis of the Anabaena genus was species whose structure was characterized by a spiral conducted on the sequences obtained using the filament and spherical akinetes located within a programs from the PHYLIP package, version 3.5 certain distance from the heterocysts was classified as (Felsenstein 1993) and Mega4, version 4.0 (Tamura et Anabaena spiroides. The analyses of some al. 2007; http://www.megasoftware.net). phytoplankton samples indicated that this species also has spherical akinetes situated in the immediate RESULTS vicinity of the heterocysts. Loosely twisted trichomes with cylindrical and Morphological studies barrel-shaped cells were observed in A. lemmermannii (Fig. 2d, 2e, 2f, 3a), while spiral trichomes with The results of morphometric measurements are spherical and slightly barrel-shaped cells were found presented in Table 2 and in Figures 2 (LM) and 3 in A. circinalis (Fig. 2h, 3f). (SEM). A. spiroides was characterized by long trichomes Single trichomes of various Anabaena species with regular coils with a diameter range of 18.8- were observed during the summer in the waters of 30.7μm (Fig. 2a, 2g, 3d). The vegetative cells of this the Gulf of Gdańsk, and they constituted 10% of the species were spherical. On the other hand, A. flos- phytoplankton species. In August 2006, mass aquae was characterized by irregularly twisted occurrences of the following Anabaena species were trichomes forming small aggregations (Fig. 2b, 2c, observed: A. flos-aquae, A. lemmermannii, A. spiroides, 3b, 3e). The vegetative cells of this species were A. circinalis. These comprised approximately 50% of spherical and oval. the phytoplankton species composition. Specimens The smallest heterocysts, with a length of of the genus Anabaena were observed at the majority 5.2 µm, were observed in A. flos-aquae and the largest, of the monitoring stations; however, these species with a length of 27.3 µm, in A. circinalis. The largest did not occur individually, and were usually akinetes were found in A. lemmermannii and were accompanied by Nodularia spumigena and 36.9 µm, while the smallest ones were found in A. Aphanizomenon flos-aquae. flos-aquae and were 17.1 µm in length. Fully-

Table 2

Phenotypic characteristics of the genus Anabaena. Spirals (μm) Vegetative cells (μm) Heterocytes (μm) Akinetes (μm) Strain Distance between spirals Diameter Length Width Length Width Length Width 2.5 - 13.4 2.5 - 8.3 5.2 - 19.4 4.7 - 8.8 17.1 - 27.2 16.5 - 25.3 Anabaena flos-aquae 20.5 - 37.2 30.8 - 59.5 Ellipsoidal or cylindrical, Spherical to barrel-shaped Spherical to shortly ellipsoidal slightly curved, distant from heterocytes 7.1 - 12.3 6.3 - 8.5 19.9 - 27.3 6.6 - 17.8 23.4 - 33.7 18.1 - 28.0 Anabaena circinalis 40.6 - 81.4 96.0 - 127.0 Spherical Spherical Ellipsoidal with conical ends 7.5 - 11.3 6.2 - 10.1 7.6 - 13.9 5.1 - 10.9 19.2 - 36.9 18.0 - 34.9 Anabaena lemmermannii - - Cylindrical with rounded ends, Cylindrical with rounded ends, barrel shaped to spherical Spherical or ellipsoidal slightly curved 6.2 - 8.1 4.1 - 8.0 10.7 - 24.3 6.4 - 20.1 17.3 - 29.8 16.2 - 23.5 Anabaena spiroides 8.9 - 18.9 18.8 - 30.7 Oval to rounded Oval Elipsoidal

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68 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz

Fig. 2. Morphotypic properties of studied Anabaena: (A) A. spiroides, (B-C) A. flos-aquae, (D-F) A. lemmermannii, (G) A. spiroides, (H) A. circinalis; H- heterocyst, A – akinete, V – vegetative cells.

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 69

Fig. 3. Scanning electron microscopy (SEM) microphotographs of Anabaena: (A) A. lemmermannii, (B) A. flos-aquae, (C) A. circinalis, (D) A. spiroides, (E) A. flos-aquae, (F) A. circinalis; A – akinete, H – heterocyst. Magnification: (A-E) – 2000×, (F) – 1000×.

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70 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz developed trichomes sometimes contained single A) spherical, oval-shaped intercalary heterocysts (A. circinalis), two or three spherical heterocysts (A. lemmermannii, A. flos-aquae), or several ellipsoid heterocysts (A. spiroides). Clusters of slightly curved cylindrical akinetes were observed in A. lemmermannii (Fig. 2d), and single, elongated oval intercalary akinetes were found in A. circinalis, (Fig. 2h, 3c, 3f), while single or double akinetes with elongated oval, kidney-like shapes were found in A. flos-aquae, and B) single elongated oval ones − in A. circinalis (Fig. 2b, 2c, 2h, 3b, 3f). The SEM images illustrate Anabaena sp., the typical form of A. flos-aquae with irregularly bent or twisted trichomes. Akinetes in this form were almost always separated from the heterocyst by two vegetative cells. The forms of Anabaena spiroides were Fig. 4. A – Reamplification products of 16S rRNA, B – characterized by more or less regularly spiral-shaped Reamplification products of 16S-23S rRNA (ITS). trichomes. The next trichomes of A. lemmermannii were irregular. In many trichomes, several akinetes were assembled in one location, and they formed the center of colonies comprising heterocysts surrounded by akinetes. Forms with intermediate morphology, compared to the species described above, were also observed in the samples examined. Some trichomes exhibited features of both Anabaena flos-aquae and Anabaena lemmermannii. The akinetes of these forms were either distant from or close to the heterocysts. Fig. 5. 16S rRNA reamplification products of Anabaena colonies. 1 – Anabaena lemmermannii, 2 – Molecular studies A. flos-aquae, 6 – A. spiroides, 7-8 – A. circinalis.

Figures 4 and 5 illustrate agarose gels, while Figure 6 illustrates polyacrylamide gels with environmental cyanobacteria samples. Separated DNA bands of cyanobacteria from environmental samples corresponded to the following species: 1 – A. flos-aquae; 2 – A. circinalis; 3 – A. lemmermannii; 4 – A. spiroides (Fig. 6). The bands marked with an arrow in Figure 5 illustrate isolated and re-amplified cyanobacterial DNA from environmental samples: the 16 S rRNA fragment is approximately 750 bp long (Fig. 4a), ITS1 is approximately 370 bp long, while ITS2 is approximately 600 bp long (Fig. 4b). Figure 6 presents the melting curves of PCR products from Anabaena species obtained with primers CYA (approximately 750 bp), and 16 CITS and 23 CITS (approximately 300-400 bp). Fig. 6. DGGE amplification products: A) 16S rRNA ; B) 16S-23S rRNA (ITS). 1 – Anabaena flos-aquae, 2 – A. The obtained 16S rRNA and ITS sequences circinalis, 3 – Anabaena lemmermannii, 4 – A. were compared to the 16S rRNA sequences of spiroides. particular Anabaena species from the GenBank

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 71 database, and then a phylogenetic analysis of the circinalis AJ293176; A. flos-aquae GG-SW and strains species of the genus Anabaena was performed. A. flos-aquae NRC44-1, A. flos-aquae 1tu35s12; and A. The phylogenetic analysis of Anabaena sequence lemmermannii GG-K5 and strains A. lemmermannii indicated that there are four genotypes: A. spiroides; NIVA-CYA 281/1, A. lemmermannii BC Ana 0010, A. circinalis; A. flos-aquae; A. lemmermannii. The first were identical with a similarity of 100% (Tables 3 and genotype includes strains of A. flos-aquae, the second 4). The nucleotide sequence of A. lemmermannii GG- one – A. spiroides, the third one – A. lemmermannii and K5 and A. lemmermannii BC Ana 0017, and the the fourth one – A. circinalis. The relatedness of the sequence of A. spiroides GG-M1 and A. spiroides sequences obtained was 97.8-100%. Nucleotide NIES-76 2, differed to the greatest extent, and their sequences of A. spiroides GG-M1 and strains A. relatedness was 98.8% and 97.8%, respectively spiroides NIES-76, A. spiroides AJ294540; A. circinalis (Figures 7a and 7b). GG-M6 and strains A. circinalis 0tu25s6, Anabaena cf.

Table 3

Sequence similarity (%) of 16S rRNA gene fragments of studied Anabaena GG to other cyanobacterial sequences from GenBank. Strain assignment Strain no. Accession no. Origin Identity GG-SW NRC44-1 AF247596 Lake Australia 100% Beltran et al. 2000 Anabaena flos-aquae 37 AJ293102 Lake Aydat (France) 99.8% Gugger et al. 2002 1tu35s12 AJ630423 Lake Tuusulanjarvi (Finland) 99.8% Rajaniemi et al. 2005 GG-M6 0tu25s6 AJ630412 100% Rajaniemi et al. 2005 Anabaena cf. circinalis Lake Tuusulanjarvi (Finland) 1tu28s13 p AJ630411 99.8% Rajaniemi et al. 2005 GG-K5 NIVA-CYA 281/1 AJ630412 Lake (Norway) 100% Rudi et al. 1997 Anabaena lemmermannii 1tu32s11 AJ630424 Lake Tuusulanjarvi (Finland) 99.8% Rajaniemi et al. 2005 GG-M1 NIES-76 AB047104 100% Oikawa et al. 2001 Anabaena spiroides Lake (Japan) NIES-76 2 AY701561 97.8% Li et al. 2000

Table 4

ITS sequence similarity (%) of studied Anabaena GG to other cyanobacterial sequences from GenBank. Strain assignment Strain no. Accession no. Origin Identity GG-SW 1tu35s12 Z82797 100% 1tu30s4 AJ630422 Lake Tuusulanjarvi (Finland) 99.9% Anabaena flos-aquae (Rajaniemi et al. 2005) 1tu31s11 AJ630419 99.8% NIVA-CYA83/1 AJ133158 Gulf of Finland (Baltic Sea) 99.8% GG-M1 Anabaena spiroides - AJ294540 Lake Aydat (France) 100% (Gugger et al. 2002) Uncultured Nostocaceae DGGE gel band 39 - EF150975 Lake (Greece) 98.8% (Sainis Valeri 2006) GG-M6 Anabaena cf. circinalis - AJ293176 100% Lake Aydat (France) (Gugger et al. 2002) Anabaena sp. 299 - - 95.6% GG-K5 Anabaena lemmermannii BC Ana 0010 AY887007 100% Cotswold Water Park (UK) (El Semary 2005) Anabaena lemmermannii BC Ana 0017 AY887008 98.8% Anabaena cf. lemmermannii - AJ293101 Lake Aydat (France) 91.6% (Gugger et al. 2002)

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72 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz

A) The forms and dimensions of vegetative cells and akinetes, as well as the relative location of akinetes toward heterocytes are important for the identification of Anabaena species. On the other hand, the form of trichomes, such as straight or coiled, and the dimensions of coils, do not reflect the phylogeny (Tuji, Niiyama 2010). In the case of Anabaena, it is very difficult to establish a simple criterion that would make it possible to classify the studied species accurately (Hindák et al. 2003). The results of morphometric measurements of the fresh samples that were collected for the current study

B) were similar to those presented by Li and Watanabe (2000). Komárek and Zapomělová (2007) noted morphological similarities among the four Anabaena species. According to these authors, the following are especially difficult to distinguish: A. circinalis, A. spiroides, and A. flos-aquae. The shapes and sizes of the vegetative cells are similar and differences are primarily in the shape and position of the trichome. The trichomes of the A. circinalis material analyzed in the current study were spun into regular coils, while those of A. spiroides were spun into regular, free- floating coils. On the other hand, the trichomes of Fig. 7. A) – 16S rRNA neighbor-joining tree, B) – 16S- A. flos-aquae were irregularly coiled, tangled, and 23S rRNA (ITS) neighbor-joining tree. sometimes loosely curved. Substantial differences in the cell size were

DISCUSSION noted in the study material depending on the sampling site. The size of vegetative cells exceeded The study presented in this paper is the first the ranges presented in the identification key for attempt to perform a multifaceted characterization of cyanobacteria from the Gulf of Gdańsk (Pliński, the genus Anabaena from the Gulf of Gdańsk. Komárek 2007). Vegetative cells and heterocysts, and In order to identify the extent of biodiversity akinetes of particular Anabaena species did not show within cyanobacteria, new conceptions of 'species' any specificity; their shapes and sizes were similar. must be adopted, which will provide the theoretical Thus, it was difficult to identify them solely on the rules and pragmatic criteria for describing the basis of morphometric analysis when using some increasing numbers of taxa as polyphasic data on environmental tests. cyanobacteria become available. The current authors Trichome length was very diverse. No regularity reject the species concepts based on phenetic was noted in the distance between heterocysts; similarity since they are incorrect according to therefore, this was not regarded as a taxonomic modern systematic standards. The authors also reject feature characteristic of the species studied. the Biological Species Concept since it does not For example, observations by Ward (2006) apply to asexual organisms. Four phylogenetic challenge the traditional paradigms about prokaryotic species concepts seem to be good options for species and call for questioning the evolutionary cyanobacteria; they are 1) the Evolutionary Species ecological theory, because the genome-based Concept, 2) the Ecotypic Species Concept, 3) the methods are developed for the high-resolution Phylogenetic Species Concept, and 4) the analysis of species as basic units comprising Monophyletic Species Concept. The Ecotypic microbial communities, and for investigating how Species Concept is appropriate when only species such units coordinate physiological activities within morphology and ecology are known and molecular the guilds of communities. data are not available (Johansen, Casamatta 2005). According to observations by Sáez and Lozano

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 73

(2005), more must be learned about the biology of responses to temperature and light by various the taxa involved, not only for its own sake, but also representatives of the same group of Anabaena for the confirmation of the authenticity of their morphospecies. cryptic status. Could, for example, some of the Specimens of Anabaena circinalis and Anabaena sexual/asexual alternating species be genetically flos-aquae occurring in lakes were characterized by a clonal instead of cryptic? This has been proposed for larger number of heterocysts and akinetes in the many parasitic protozoa, despite their sexuality. trichomes. The shape of the vegetative cells and Additionally, instead of concentrating on particular heterocysts were spherical, and similar in both cases, systematic and quantitative comparisons across species. The akinetes of A. circinalis were slightly different taxa and habitats are also necessary in the more elongated than those of A. flos-aquae (Nayak et search for conditions in which cryptic species thrive al. 2007, Kobos 2008). and decrypt their nature while pursuing their causes. It was observed that trichomes of Anabaena The planktonic species of the cyanobacterial lemmermannii from the Gulf of Gdańsk were genus Anabaena represent a special generic entity, significantly shorter than those of the same species which is genetically and morphologically different occurring in freshwater environments (Kobos 2008). from the typical (benthic) segment of the genus Saline stress might be one of the factors that results Anabaena that is based on the species Anabaena in shorter Anabaena filaments in salt waters, as oscillarioides Bornet ex Bornet et Flahault. This group observed during laboratory studies on N. spumigena of planktonic species should be classified in a special (Mazur-Marzec et al. 2006). The greater water mass subgenus of Dolichospermum Thwaites ex Wittrock et dynamics in the sea might also cause the breakdown Nordstedt 1889 with the species Anabaena flos-aquae of cyanobacterial aggregates in this environment. It is [Lyngbye] Brébisson ex Bornet et Flahault 1888, believed that the occurrence of A. lemmermannii in the which should be moved to the level of genus form of free-floating filaments instead of larger cell (Wacklin et al. 2009). The subgenus Dolichospermum clusters hinders the formation of blooms of this comprises species of two morphological types with species in the Baltic Sea (Hajdu et al. 2007). coiled and straight trichomes (Komárek, Zapomělová Phenotypic variability might be caused by several 2007). Komárek and Zapomělová (2007) included a environmental factors, such as salinity, pH, and review of species with coiled trichomes in the first nutrient and chemical substances (Lang et al. 1987; part of this list for practical reasons. In their article, Kearns, Hunter 2001), temperature, UV radiation, morphospecies are included that occur in nature water circulation, or the presence of heavy metals mainly with straight trichomes. In contrast, (Mischke 2003). Intra- and inter-specific chemical Kozhevnikov and Kozhevnikova (2009) and Wacklin signals allow bacteria to respond to environmental et al. (2009) described the size and shape of conditions by regulation of gene transcriptions. In vegetative cells, the size of akinetes and heterocysts, cyanobacteria, gene products and the presence of and the position of akinetes from the cultured fixed nitrogen regulate the heterocyst frequency. material that nearly correspond to the isolates in the A taxonomic system based on morphological current study that were obtained from the material features based on the comparative analysis of the collected from natural populations. nucleotide sequence encoding a smaller subunit of The study by Zapomělová et al. (2010) suggests ribosome 16S rRNA is currently being completed that Anabaena strains of even the same and verified (Rudi et al. 2000; Castenholz 2001; Lyra morphospecies can thrive in a wide range of et al. 2001; Iteman et al. 2002; Komárek, temperature and light conditions. Temperature and Zapomělová 2007; Rajaniemi et al. 2005; Willame et light preferences were specific to each strain, and did al. 2006; Palinska, Surosz 2008). Rudi et al. (1997) not reflect the taxonomic affiliations of the studied claim that on the basis of a genetic platform common Anabaena strains. The current results are supported to a species, extremely varied phenotypes by similar conclusions reported by Rapala and candevelop, depending on the environmental Sivonen (1998). Their study focuses on strain-specific conditions in which a given organism develops and differences in the growth rates of various Anabaena lives. The characteristics of the cyanobacterial genera strains as a function of temperature and light. Thus, examined in this study are described by Rippka et al. inter-strain variability can be expected when there are (2001) temperature and light preferences. On the contrary, The identification of these organisms is largely Stulp and Stam (1985) report similar growth based on morphological features, which are useful

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74 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz for broad classification to the genus level, but which assessment of Anabaena sp. community structure was can be inconclusive due to either their occasional investigated. The results indicated that the increased absence or phenotypic changes under different electrophoresis run time resulted in dissimilar environmental conditions. However, there is an profiles, which were likely due to the instability of the obvious need to integrate diverse datasets for the denaturing gradient. For optimal DGGE separation, species-level identification of Anabaena sp. The incorporating a 40-bp GC-rich clamp proved present study focused on evaluating the utility of necessary to obtain the optimal resolution of the such an approach for the genus Anabaena. These fragments in the denaturing gradient. The banding morphological features were preserved in different pattern provided a profile of the Anabaena sp. light and temperature conditions (Stulp, Stam 1985) populations so the relative intensity of each band and and even in brackish water. In planktonic Anabaena its position most likely represented the relative strains, trichomes varied from coiled to straight. abundance of particular species in this population. The strain of Anabaena circinalis had significantly However, bands at identical positions in the wider trichomes, heterocysts and akinetes than DGGE gel are not necessarily derived from the same strains of Anabaena flos-aquae. The phylogenetic species (Muyzer et al. 1993). As DGGE analysis is relationship of the studied strains did not follow the highly sensitive for detecting sequence differences, current taxonomic classification by Komárek and the possibility that chimeric genes are created in the Anagnostidis (1989) or Bergey’s Manual of course of PCR can be excluded. Additionally, Systematic Bacteriology (Rippka et al. 2001); thus, a Sivonen et al. (2007) used the DGGE community revision of the taxonomy of these anabaenoid strains fingerprinting method to separate partial 16S rRNA is needed. genes amplified from environmental DNA by Molecular studies carried out for this paper universal primers, but their study focused on the involved determination of the 16S rRNA and 16S- bacterioplankton community composition in 23S rRNA sequence (ITS). In all the environmental brackish surface waters in the Baltic Proper and the tests, two clear ITS bands were obtained for each Gulf of Finland. To identify the organisms, DGGE species, with one clear 16S rRNA band. In order to bands were sequenced subsequently, and the determine the similarity of species, phylogenetic sequences retrieved were compared to those in the analysis was conducted based on the nucleotide database. sequence of PCR products. Phylogenetic analysis based on comparing the Thanks to the application of DGGE, it was full sequence of genes encoding 16S rRNA with a possible to compare closely related organisms of the length of approximately 800 bp indicated that there same genus and to detect very subtle genetic was a similarity of Anabaena species with strains from differences (Settanni et al. 2006). Similarly, Boutte et GenBank at 97.8%-100%. Analysis of 370 bp of the al. (2006), using DGGE and primers CYA359F and ITS fragment of the studied organisms revealed a CYA781(a) and CYA781(b) in their studies on similarity range of 98.8%-100%. The sequenced filamentous cyanobacteria of the Aphanizomenon fragments of 16S-23S rRNA differed by about 25-40 genus and unicellular cyanobacteria of the nucleotides from the freshwater and saltwater strains Synechococcus genus, detected genetic differences in the available from GenBank, which might have been the 16S rRNA fragment. result of single base mutations. There are two main considerations for using Four species of cyanobacteria from the genus primers specific to cyanobacteria in DGGE analyses. Anabaena occurring in the Gulf of Gdańsk were First, specific primers prevent amplification of the identified based on molecular analysis. As abundant DNA of noncyanobacterial microbes in demonstrated previously with other organisms field samples. The resulting DGGE profiles are less (Iteman et al. 2002), intra-genomic 16S rRNA complex than those generated with general bacterial sequence heterogeneity has implications for the primers; hence, detection of cyanobacteria, which are inference of phylogenetic relationships of less abundant or have lower amplification heterocystous, planktonic cyanobacteria. efficiencies, is more feasible. Second, characterization A similar method for assessing the homology of of cultures by DGGE, but also by restriction enzyme bacteria, cyanobacteria, and other organisms was digestion or sequencing, is not possible when DNA employed by Ludwig and Klenk (2001), Garrity and from contaminants is coamplified (Janse et al. 2003). Holt (2001), Skotarczak and Cichocka (2001), The impact of DGGE run time on the Skotarczak et al. (2003), Taton et al. (2003), and

Polyphasic characterization of the genus Anabaena from the Gulf of Gdańsk| 75

Gkelis et al. (2005). Theses authors recommend using Ebers 2006). caution when revising the similarity between groups With the improvement of molecular techniques of studied cyanobacteria and their phylogenesis. They and the increased amounts of data obtained through noted that certain features can evolve through their application, it is evident that the similarity random mutations and natural selection. Their work threshold required for classification of a given species on pure cultures or clones isolated previously from must be raised. Due to the high level of the environment demonstrated polymorphism of the conservativeness of gene 16S rRNA, it is 16S-23S rRNA intergenic spacer region (ITS). Their recommended to employ additional molecular studies provided molecular evidence for the variety markers to supplement analysis based on 16S rRNA. of cyanobacteria in the studied region, which was A significant aspect of the current study was the much greater than previously believed. While application of the molecular key for cyanobacteria studying an Anabaena circinalis population, Fergusson identification directly to environmental samples and Saint (2000) observed differences in their without prior isolation or cultivation. Analysis of the morphology. In order to obtain full taxonomic 16S rRNA and the ITS fragment of the obtained verification of the species studied, they conducted sequences made it possible to confirm the existence molecular analysis based on the sequence of 16S of four genotypes corresponding to four Anabaena rRNA and the rpoC1 gene, which made it possible to species in particular environmental samples. confirm the species of a given strain; however, in the Barker et al. (1999), Lehtimäki et al. (2000), and current study only two genetic markers, 16S rRNA Lyra et al. (2001, 2005) identified cyanobacteria in the and ITS, were used for verification of the taxonomic Baltic Sea in a similar way. Not only did they study species of Anabaena and the phylogenetic the genus Anabaena, but they also addressed the relationships of the Anabaena strains. Similar morphological and genetic analysis of strains of the conclusions are reported by Iteman et al. (2002). genera Nodularia, Aphanizomenon, and Microcystis. They According to Stackebrandt et al. (2002), primers developed a molecular method based on PCR-RFLP that are universal for the amplification of the studied analysis of the fragment 16s rRNA, rpoB which they gene fragment should be used for bacteria. These used to create a molecular identification key for authors also suggest that in order to establish the particular genera. Moreover, they suggested that similarity between groups of organisms, the structure more than one marker should be used since more of a given population should be taken into account, reliable results can be achieved using a minimum of as well as hybridization, divergence, mutation, two markers. reproduction, and ecological conditions. According Additionally, the current study indicated that to their study, based on an almost complete sequence more genetic markers should be used; however, using of genes encoding 16S rRNA, the degree of similarity more than two markers is too expensive and too between various species is labile, and in order to time-consuming. establish the final threshold, the studied gene must Relatedness studies of the two genera Anabaena be thoroughly analyzed. In their papers published in and Aphanizomenon indicated 97% relatedness despite 1994 and 1995, the authors suggest that 97% the fact they are separate genera (Gugger et al. 2002, similarity can be regarded as a sufficient criterion for Willame et al. 2006). This indicates that the threshold classification of a given species. However, in later of identity is arbitrary and cannot be used universally publications (2003 and 2004), this position is revised, for all species. and the authors conclude that the probability ought The topology of the phylogenetic tree to be over 97%. This conclusion is based on the constructed based on the full 16S rRNA and ITS thorough phylogenetic analysis of selected sequence made it possible to establish the relatedness Procaryota groups. Not only did these authors study of these organisms and their origin from one ribosomal RNA, but they also aligned the segments descendant. As in the current study, Wilmotte and of the genome that were similar and then conducted Golubić (1991) published a distance tree constructed analyses of conservative genes, e.g. the rpoC1 gene, from all available, complete cyanobacterial sequences; which encodes the γ subunit of RNA polymerase however, partial sequences have been identified for dependent on DNA. On the other hand, recent many more cyanobacterial strains. In future studies reports on the relatedness of organisms raise the of 16S rRNA of other closely related organisms, it threshold necessary for an organism to be classified might be helpful to stabilize the position of the strain as a particular species to 98.7-99% (Stackebrandt, studied in the distance tree. Thus, it might be

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76 | Grzegorz Siedlewicz, Karolina Forycka, Waldemar Surosz interesting to assess the genotypic relationships of the EMBL/GenBank/DDBJ databases, NUCLEOTIDE this enigmatic strain to other cyanobacteria using 16S SEQUENCE. Unpublished. NCBI Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package) version rRNA sequence analysis. 3.5c, Distributed by the author, Department of Genetics, It is probably risky to determine the taxonomic University of Washington, Seattle, classification of a species based only on phenotypic (http://evolution.genetics.washington.edu/phylip.html) or genotypic features. Genotype differences between Fergusson, K.M. & Saint, C. P. (2000). Molecular phylogeny of Anabaena circinalis and its identification in environmental species tend to be small; and it is sometimes difficult samples by PCR. Appl. Environ. Microbiol. 66, 4145-4148 to detect them during such studies. Phenotype Garrity, G.M. & Holt, J.G. (2001). The road map to the Manual. In differences arise primarily from the reaction of an Bergey’s Manual of Systematic Bacteriology (pp. 119-141). Springer, organism to changing environments (Palińska, Surosz New York 2008). Therefore, for the purposes of full taxonomic Gkelis, S., Rajaniemi, P., Vardaka, E., Moustaka-Gouni, M., Lanaras, T. & Sivonen, K. (2005). Limnothrix redekei (Van verification of the genus Anabaena studied, it is Goor) (Cyanobacteria) strains from Lake Kastoria, Greece advisable to apply the polyphasic approach, with form a separate phylogenetic group. Microb. Ecol. 49, 176–182 which it is possible to draw the most precise Gugger, M., Lyra, C., Henriksen, P., Coute´, A., Humbert, J.-F. & conclusions concerning the phylogenesis of the Sivonen, S. (2002). Phylogenetic comparison of the cyanobacterial genera Anabaena and Aphanizomenon, Int. J. studied cyanobacteria genus. Syst. Evol. Microbiol. 52, 1867 – 1880 In conclusion, a similarity of 98.5% for the 16S Hajdu, S., Höglander, H. & Larsson, U. (2007). rRNA and ITS sequence appears to be sufficient for Phytoplanktonvertical distributions and composition in the classification of cyanobacterial species from the Baltic Sea cyanobacterial blooms. Harmful Algae. 6, 187–205 Anabaena genus from the Gulf of Gdansk. Hindák, F., Šmarda J. & Komárek J. (2003). Nodularia moravica spec. nova, a new benthic freshwater nostocalean species (Cyanophyta/Cyanobacteria/Cyanoprokaryota). Algol. Stud. ACKNOWLEDGMENTS 109, 241 - 253 Hongmei, J., Aitchison, J. C., Lacap, D. C., Peerapornpisal, Y., We thank Dr. Sylwia Jafra from the Sompong, U. & Pointing, S. B. (2005). Community phylogenetic analysis of moderately thermophilic Intercollegiate Faculty of Biotechnology UG-MUG, cyanobacterial mats from China, the Philippines and Poland, for her help at different stages of the present Thailand. Extremophiles. 9, 325 - 32 study and Dr. Katarzyna A. Palińska from Oldenburg Iteman, I., Rippka, R., Tandeau de Marsac N. & Herman, M. 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