INTRODUCTION

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The genus Nodularia (Nostocales) consists of filamentous heterocystous nitrogen-fixing cyanobacteria, which are found in brackish water and freshwater, as well as in terrestrial environments, worldwide (2, 7, 17, 35). Planktic Nodularia form mass occurrences, especially in warm surface waters with a low N:P ratio (18, 21, 34). In the Baltic Sea, the blooms are common in late summer and may cover areas in excess of 60,000 km² (16).

Nodularin toxin is commonly detected in cyanobacterial blooms containing Nodularia spp. (2, 42), and Nodularia isolates from the blooms usually produce nodularin (6, 20, 42). Nodularin is a cyclic pentapeptide hepatotoxin with a 50% lethal dose of 50 to 70 µg kg¹ when tested intraperitoneally in mice (39, 42). Furthermore, nodularin is an inhibitor of protein phosphatases 1 and 2A and a potent tumor promoter (36). Toxic blooms of Nodularia have been associated with poisonings of domestic animals in different parts of the world (see, for example, references 13, 23, and 29). A few nontoxic strains of Nodularia have been isolated from the plankton of the Baltic Sea (20). In physiologic experiments, the nontoxic strains never produced nodularin, whereas the nodularin-producing strains were toxic in all test conditions (19).

Traditionally, Nodularia have been classified on the basis of the morphology of the different types of cells (vegetative cells, heterocytes, and akinetes), on their ability to produce gas vesicles (structures essential for providing buoyancy), on nodularin production, on ultrastructural features of the cells, and on ecological characteristics (17). The genus Nodularia was recently divided into seven species (17). Four species (Nodularia spumigena, N. baltica, N. litorea, and N. crassa) are planktic, with the capability to produce gas vesicles. Three of the species (N. harveyana, N. sphaerocarpa, and N. willei) lack gas vesicles and grow in benthic, periphytic, or soil habitats. Phylogenetically, on the basis of the 16S rRNA gene, the genus Nodularia is most closely related to the genera Anabaena, Nostoc, and Cylindrospermum (22, 44, 45). Methods involving the whole genome (20) and 16S rRNA sequences (20, 26) have indicated the close overall relatedness of Nodularia strains and also distinguished the nodularin-producing strains from the nontoxic ones. In addition, putative peptide synthetase and polyketide synthetase sequences distinguished the toxic from the nontoxic strains (25). Nodularia isolates from the Baltic Sea plankton were assigned to three groups based on the 16S-23S rRNA internally transcribed spacer (ITS) sequences, to three groups on the basis of the sequences from the phycocyanin encoding operon intergenic spacer and flanking regions (PC-IGS), and two groups based on the intergenic region between genes encoding gas vesicle proteins (gvpA-IGS) (3). Data from RAPD [random(ly) amplified polymorphic DNA]-PCR and PC-IGS sequences have suggested that hierarchical patterns of genetic variation within Nodularia isolates exist on both regional and global scales (6).

From both an ecological and a human point of view it would be valuable to understand which conditions in nature favor the occurrence of nodularin-producing and nontoxic strains. Analysis methods, such as high-pressure liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), or the mouse bioassay enable detection of hepatotoxins from water samples. However, single strains or filaments of Nodularia spp. cannot be assessed. For the detection of individual strains of Nodularia in the field, toxicologic and molecular information derived from laboratory isolates can be extended to the natural populations by detecting predetermined alleles by PCR amplification and subsequent molecular analyses of Nodularia filaments.

In the present study, 18 cultured Nodularia strains from planktic, benthic, and soil habitats were characterized by phenotypic and molecular approaches. In terms of classical taxonomy (17), the examined strains were defined as Nodularia sp., N. spumigena, N. baltica, N. harveyana, and N. sphaerocarpa. The characterization was performed in order to find morphologic or molecular markers for discriminating nodularin-producing from nontoxic strains of Nodularia in water samples. In addition, a field survey was undertaken in the Gulf of Finland to assess the composition of the Nodularia populations.

	MATERIALS AND METHODS

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Nodularia strains. Eighteen monospecific strains of Nodularia were used in the study (Table 1). They were grown in liquid Z8 medium without nitrogen and with added salt (43). The strains N. harveyana Hübel 1983/300 and N. baltica Hübel 1988/306a and Hübel 1988/306b had been used in the intrageneric evaluation of Nodularia taxonomy (17). The species description of N. baltica (17) is partly based on strain Hübel 1988/306, which was later on divided into strains Hübel 1988/306a and Hübel 1988/306b.

Phenotypic characteristics of the strains. The sizes of vegetative cells and heterocytes were measured from 3-week-old cultures, and the akinetes were measured from 3.5-month-old cultures. Examination was carried out with a Leica Aristoplan phase-contrast microscope. Thirty cells were measured; however, when the akinetes were rare, the measurements were made with fifteen cells. The presence or absence of gas vesicles and the shapes of terminal cells were recorded. The averages and standard deviations of the sizes of the different kinds of cells were calculated for each Nodularia strain. Photographs of 3-week-old strains were taken with a Polaroid digital microscope camera fitted to a Leitz DM IRB phase-contrast microscope.

Preparation of template and PCR. PCR amplifications were performed alternatively with DNA or 2 µl of culture as a template. The DNA was extracted as described by Golden et al. (12) without further purification. The 2-µl aliquot from a culture was boiled for 10 min prior to PCR, and polymerase was added after the denaturation step (hot start).

Sequencing and construction of phylogenetic trees. The template for DNA sequencing of the PC-IGS region was purified from the PCR products with Wizard PCR Preps DNA purification kit (Promega, Madison, Wis.). The ITS1-S PCR products were first separated by gel electrophoresis, and the smallest band (ITS1-S) was excised from the gel and subsequently purified with the Wizard PCR Preps DNA purification kit. The ITS1-S and PC-IGS sequences were resolved on both strands by using the same primers as for PCR. For sequencing, 5 to 20 ng of DNA was applied in a PCR with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit according to the manufacturer's instructions (PE Applied Biosystems, Foster City, Calif.). The sequencing was carried out on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). The ITS1-S and PC-IGS sequences of each strain were checked by aligning the forward and reverse sequences with PILEUP of the GCG package version 10.1 (Genetics Computer Group, Madison, Wis.) and manual editing using the GeneDoc multiple sequence alignment editor.

Sampling in the Gulf of Finland and determination of the PC-IGS allele. Sampling for the determination of the PC-IGS allele of single Nodularia filaments was performed from the research vessel Aranda (Finnish Institute of Marine Research) between 7 and 11 August 2000. Plankton samples were obtained with a 100-µm-mesh-size plankton net from surface water (0 to 4 m) at six sampling stations in the Gulf of Finland (the Baltic Sea). Nodularia filaments were picked and treated as described by Barker et al. (4) with slight modifications. The washed filaments were transferred to PCR tubes containing 8 µl of 10× DyNAzyme II DNA polymerase buffer and 16 µl of sterile water. After immersion in boiling water for 5 min to lyse the cells, the 345 sample tubes were stored at 20°C until use as templates in the PCR amplifications. Positive control samples (nodularin-producing strain AV3 and nontoxic strain UP16f) were treated similarly to the field samples, and the negative controls contained no templates.

	RESULTS

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Phenotypic features of the strains. Eight Nodularia strains (Hübel 1988/306a, Hübel 1988/306b, Hübel 1987/310, Hübel 1987/311, NSPI-05, NSOR-12, GR8b, and AV63) had discoid cells in unbroken, straight, or slightly curved trichomes (Fig. 1a to g and k). The trichomes of F81, AV3, and HEM were straight and fragmented, and the cells were spherical rather than discoid (Fig. 1i, j, and l). Strains BY1, UP16a, UP16f, HKVV, PCC 73104/1, Hübel 1983/300, and PCC7804 had unbroken and straight or flexuous trichomes with roundish to barrel-like cells (Fig. 1h and m to r).

View larger version (169K): [in this window] [in a new window]   FIG. 1.   Photomicrographs of Nodularia strains used in the study. The heterocytes are indicated with black arrows and the akinetes with white ones. Panels: a, Hübel 1988/306a; b, Hübel 1988/306b; c, Hübel 1987/310; d, Hübel 1987/311; e, NSPI-05; f, NSOR-12; g, GR8b; h, BY1; i, F81; j, AV3; k, AV63; l, HEM; m, UP16a; n, UP16f; o, HKVV; p, PCC73104/1; q, Hübel 1983/300; and r, PCC7804. The scale in all figures corresponds to 10 µm.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   First two components of the PCA on the average dimensions of the vegetative cells, heterocytes, and akinetes of Nodularia strains and those of the species descriptions of N. spumigena, N. baltica, N. litorea, N. harveyana, and N. sphaerocarpa as given in Komárek et al. (17). Together, PC1 and PC2 accounted for 79% of the variability in the data.

The ITS1 region of Nodularia and its phylogeny. Amplification with primers 16CITS and 23CITS yielded two ITS1 amplification products from each Nodularia strain. In most strains, the length of the larger product (ITS1-L) was ca. 900 bp and that of the smaller product (ITS1-S) ca. 550 bp (data not shown). Strain Hübel 1983/300 had shorter ITS1-L and ITS1-S fragments of 870 and 470 bp, respectively. Without the flanking 16S and 23S rRNA regions, the ITS1-S sequence lengths varied from 277 bp (strain Hübel 1983/300) to 356 bp (NSPI-05). The alignment contained 360 positions.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Neighbor-joining tree on the ITS1-S sequences of the Nodularia isolates with Baltic Sea reference sequences N. spumigena BC Nod-9427 and N. litorea BC Nod-9408 (3) and Nostoc sp. strain PCC7120 as an outgroup. Species designations are based on morphologic criteria described by Komárek et al. (17). Nontoxic strains are denoted by an asterisk, and the main clades are indicated with letters from A to D. The scale bar corresponds to 10% difference of 360 nucleotides and only bootstrap values of >80% are shown.

PC-IGS region in Nodularia strains and its phylogeny. The primers PCF and PCR gave a single amplification product of ca. 670 bp from each Nodularia strain, and 506 to 508 nucleotides were unambiguously resolved from each strain. The alignment of the Nodularia PC-IGS sequences with the reference sequences and the outgroup contained 512 positions.

View larger version (33K): [in this window] [in a new window]   FIG. 4.   Neighbor-joining tree on the phycocyanin operon (PC-IGS) sequences of the Nodularia isolates with Baltic Sea reference sequences BC Nod-9401, BC Nod-9427, and BC Nod-9402 (14) and Nostoc sp. PCC7120 as outgroup. Species designations are based on morphologic criteria described by Komárek et al. (17). Nontoxic strains are denoted by an asterisk, and the main clades are indicated with letters from A to D. The scale bar corresponds to 10% difference of 499 nucleotides, and only bootstrap values of >80% are shown.

Discrimination of PC-IGS alleles and their detection in the Gulf of Finland of the Baltic Sea. Based on both the ITS1-S and the PC-IGS sequences, the nontoxic N. sphaerocarpa and N. harveyana strains were different from the nodularin-producing strains (Fig. 3 and 4). Consequently, the sequence information was used for discrimination of potentially toxic Nodularia strains from the nontoxic ones. The PC-IGS sequences of the planktic nodularin-producing strains contained a recognition site (5'-GGCC-3') for HaeIII at positions 118 to 121 and 528 to 531. The enzyme thus produced three fragments (100, 190, and 380 bp) from the PC-IGS amplicons of the planktic nodularin-producing strains. HaeIII digestion of the PC-IGS PCR products from the nontoxic strains HKVV, UP16a, UP16f, PCC73104/1, and Hübel 1983/300 and the toxic strain PCC7804 produced no restriction fragments (data not shown).

1

View larger version (78K): [in this window] [in a new window]   FIG. 5.   Steps of the analysis of the PC-IGS allele of Nodularia filaments collected at station Kasuuni in the Gulf of Finland. (A) Agarose gel electrophoresis of PC-IGS PCR products of Nodularia filaments 1 to 15 collected at station Kasuuni (+, positive control, nontoxic strain UP16a; ++, positive control, nodularin-producing strain AV3; , negative control with no template; M, 100-bp marker). (B) Agarose gel electrophoresis of the restriction fragments produced by HaeIII digestion of PC-IGS PCR products shown in panel A (+, positive control, nontoxic strain UP16a; ++, positive control, toxic strain AV3; M, 100-bp marker).

	DISCUSSION

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The nodularin-producing and nontoxic Nodularia strains in culture had different ITS1-S and PC-IGS alleles. Consequently, PC-IGS PCR and HaeIII digestion was used to assign Nodularia filaments collected from plankton in the Gulf of Finland to one of the two types of PC-IGS alleles: the one found in the nodularin-producing cultures or the other present in nontoxic cultures of Nodularia. All 186 Nodularia filaments from the Gulf of Finland contained the PC-IGS allele found in nodularin-producing cultures. The result suggests that all of the examined planktic Nodularia were nodularin producing. At the time of our study, 0.2 to 6.0 mg of nodularin g (dry weight) of net plankton¹ was measured by HPLC at 16 stations in the Gulf of Finland and the northern Baltic Proper (H. Kankaanpää et al., unpublished data), supporting our observations.

Morphologic characteristics were ambiguous in terms of distinguishing the nontoxic Nodularia strains from the nodularin-producing strains. The two most distinct characteristics of the nontoxic strains were the lack of gas vesicles and the large spherical akinetes in series in N. sphaerocarpa strains. However, the presence of gas vesicles is a facultative feature in planktic Nodularia (17), as was demonstrated by two isolates in the present study, and therefore cannot be considered as a feasible marker. The akinetes, although distinct in N. sphaerocarpa strains, are rarely observed in natural populations of Nodularia in Baltic Sea plankton (see, for example, reference 1). An experienced microscopist is likely to distinguish the planktic Nodularia strains from the benthic, periphytic, or soil Nodularia strains. However, PC-IGS PCR from single filaments and subsequent digestion with HaeIII provided an alternative and straightforward method for distinguishing between the planktic and the benthic strains. Furthermore, it allowed the detection in nature of the PC-IGS alleles, which are found in nodularin-producing isolates.

Nontoxic Nodularia strains originally isolated from plankton of the Baltic Sea were identified as benthic species N. sphaerocarpa according to the classical taxonomy (20; this study). The nontoxic N. sphaerocarpa- or N. harveyana-type Nodularia strains were not found in plankton of the Gulf of Finland by the PC-IGS PCR and HaeIII digestion method. These observations imply that the nontoxic N. sphaerocarpa strains, which in three cases were isolated in cultures from the Baltic Sea plankton, occur there only sporadically. Water masses transport loose periphytic and benthic microorganisms from coastal areas to the open sea, and occasionally these organisms can be found in plankton (see, for example, reference 9). Two of the nontoxic N. sphaerocarpa-resembling strains (UP16a and UP16f) were isolated from an open sea population on the same day, while the exact origin of HKVV is uncertain ("Stockholm archipelago water" [40]). Strain PCC73104/1, on the other hand, originates in soil, which is mentioned as a potential habitat for N. sphaerocarpa (17). It is possible that the filaments collected in the Baltic Sea plankton and isolated in culture (HKVV, UP16a, and UP16f) originally grew in the drainage area of the Baltic Sea, either in freshwater or in flooded soil, and that they were transported to the open sea by water. However, the true habitat of N. sphaerocarpa in the Baltic Sea area remains to be determined.

In general, nodularin production seems to be inherent for planktic Nodularia strains, whereas nontoxicity may be perceived as an exception (2, 6, 20, 25, 26, 42; this study). To date, only two nontoxic planktic N. spumigena strains (NSBL-03 and NSBL-05), which originated in an Australian inland lake, have been reported (6). Accordingly, strains with benthic or soil origin are in principle nontoxic (6, 20, 25, 26; this study). N. sphaerocarpa and N. harveyana resembling strain PCC7804, which was isolated in a thermal spring in France in 1966, produces mainly a nodularin variant (5) and is an exception among the benthic strains. To date, however, the number of examined benthic strains is limited and new isolates from benthic, periphytic, and soil habitats should be examined in order to draw further conclusions about their toxicologic and ecological properties.

Hayes and Barker (14) suspected the poor condition of Nodularia filaments to be the reason for ambiguous PCR results in an allele-specific PCR. In our study, the Nodularia population appeared to be senescent at station Jussarö, and only 13% of the samples were successfully amplified. At station LL13, the PCR yield was also poor,but the Nodularia filaments appeared to be in good condition. Therefore, poor quality or possibly a low quantity of the template, as implied by the condition of the filaments, does not seem to explain the variability in PCR success. Drastic sequence differences in the template at the different stations also seem unlikely. A more plausible explanation for the low PCR success could be the effect of storage time since the samples with low PCR yield were the last ones to be processed after almost 2 months of storage at 20°C.

When isolated in culture, cyanobacteria often change morphologically and lose features, which are typical for organisms occurring in natural habitats (see, for example, references 8 and 38). Morphologic features can vary remarkably even among genotypically (16S rRNA) identical cyanobacterial strains (38). The planktic Nodularia usually lose the coiling of the filaments and sometimes also gas vesicles, as do strains BY1 and HEM. The distinct mucilaginous sheaths covering the filaments may be greatly diminished or lost (35). Moreover, the cell dimensions seem to change with time and prevailing growth conditions. The same strains growing under different conditions have differed remarkably in cell dimensions (6, 20; this study). The average lengths and widths of the vegetative cells and heterocytes, respectively, of the strain BY1 varied between the three studies: 3.9 by 5.7 µm and 4.8 by 7.0 µm (this study), 4.4 by 5.1 µm and 5.1 by 6.1 µm (20), and 4.1 by 8.2 µm and 4.5 by 9.1 µm (6). The strains in the present study and that by Lehtimäki et al. (20) were grown in different light conditions. In general, the differences in cell dimensions are likely to be due to differences in growth media, ambient temperatures, and light environments. This plasticity suggests that the growth environment has a pronounced effect on the cell dimensions. Contrasting results have also been reported, since only little variation in cell and heterocyte characteristics under different light, temperature, pH, and salinity conditions was noted by Nordin and Stein (35). However, the changes in the cell dimensions, together with the loss of diacritical features such as gas vesicles, renders species identification of the strains difficult and unreliable. It also impedes the use of morphologic features for the detection of strains with defined physiological characteristics, such as toxin production, in nature.

In the present study, the shift in cell widths was notable, especially in the case of strains Hübel 1988/306a and Hübel 1988/306b, which were used as material for the description of the species N. baltica (17). According to our results, these strains were morphologically closest to the species N. spumigena, indicating rather significant changes in cell dimensions. A shift in the trichome widths of cultured N. baltica and N. litorea toward the intermediate size of N. spumigena was also observed by Komárek et al. (17).

The ecological background of Nodularia strains seemed to define their phylogenetic clustering in the present study, a finding which is in accordance with previous results (20, 26). Based on both the ITS1 and the PC-IGS regions, strain Hübel 1983/300 N. harveyana was clearly different from all of the other strains, thus supporting its genetic divergence from the other Nodularia isolates. N. harveyana inhabits benthic and periphytic environments of saline and brackish waters (17), and consequently its genetic differentiation from ecologically different Nodularia is reasonable. The nontoxic N. sphaerocarpa strains, on the other hand, were distinctly differentiated from the nodularin-producing planktic strains on the basis of the ITS1-S. Based on the PC-IGS region, the N. sphaerocarpa strains clustered separately but were closely related to the planktic strains. The divergence of nontoxic N. sphaerocarpa from the nodularin-producing strains has been shown by 16S rRNA sequences (20, 26) and whole-genome techniques (20). In addition, physiological experiments have indicated differences between nodularin-producing planktic Nodularia and nontoxic N. sphaerocarpa strains (19, 27).

The results presented here indicate no consistent genetic differentiation between the three planktic species of N. baltica, N. spumigena, and N. litorea, which have been described on the basis of morphologic and ultrastructural (i.e., the size and density of the gas vesicles) criteria (17). The strains BY1 N. baltica and GR8b N. spumigena had 100% identical ITS1-S and the PC-IGS sequences (this study), as well as 16S rRNA sequences (20). Clustering of the strains Hübel 1987/306a and Hübel 1987/306b, which were used as material for the description of the species N. baltica (17), with N. spumigena strains was consistent. In addition, reference N. litorea ITS1-S and PC-IGS sequences (3) were identical to sequences from N. spumigena and N. baltica strains. It is highly unlikely that 16S rRNA, which is the most relevant region of the genome in terms of taxonomy and more conserved than the ITS1-S and PC-IGS, would reveal differentiation between the three species if the more variable ITS1-S and PC-IGS regions are identical. Molecular and phenotypic characterizations of Nodularia strains from the Baltic Sea plankton have been unable to reveal a consistent grouping of Nodularia in three different types, which could be considered as three different species (3, 4, 20; this study). Moreover, the most important phenotypic characteristic underlying the species descriptions of N. baltica, N. spumigena, and N. litorea, i.e., the trichome width, seems to be unstable in cultures (3, 20; this study), and intermediate forms have been found in nature as well (7). The view that there is only one planktic Nodularia species in the Baltic Sea has been presented (3, 4). The data shown here, which covers also the morphologically defined species N. baltica, is in agreement with the above view that in the Baltic only one planktic species, N. spumigena, instead of three species, is genetically justified (3, 4). However, it is worth noting that one genotype (16S rRNA) may produce different morphotypes (38), which probably is the case with the planktic Nodularia of the Baltic Sea.

The importance of comparing several regions of the genome in the study of organismal relationships has been stressed (37). The results obtained from the ITS1-S region in the present study were in good agreement with those for the 16S rRNA except for strain NSOR-12 (20, 26). With both regions of the genome, the nontoxic strains were separated from the nodularin-producing planktic strains (20, 26; this study). Grouping of the strains on the basis of the PC-IGS region was slightly different from the grouping based on the ITS1-S (this study) and the 16S rRNA (20). Especially, the planktic strains were differently positioned since they were separated into two clusters in the PC-IGS tree. Of the three regions of the genome, the ITS1-S was most polymorphic, e.g., similarities between the strains PCC73104/1 and GR8b were 91.8% (ITS1-S), 96.7% (PC-IGS), and 99.4% (16S rRNA [20]). However, among the strains from the plankton of the Baltic Sea, the PC-IGS region contained more variation than the ITS1-S.

Within the planktic Nodularia populations of the Baltic Sea, genetic exchange has been shown to most likely occur by allele-specific PCR on ITS1, PC-IGS, and gas vacuole protein A intergenic spacer regions (gvpA-IGS) (4). Already, Barker et al. (3) noted different grouping of the studied Nodularia strains on the basis of the different regions of the genome (ITS1-S, PC-IGS, and gvpA-IGS), and these authors suspected genetic exchange. In the present study, grouping of the planktic strains from the Baltic Sea was also different on the basis of the ITS1-S and the PC-IGS regions. The most striking discrepancy in the clustering on the basis of the ITS1-S and PC-IGS was noted in strains NSOR-12 and PCC7804. Most of the variation accounting for the divergence of NSOR-12 and PCC7804 from one another and the other strains was found in the 3' end of the ITS1-S, in a region spanning ca. 100 nucleotides. In general, the 3' end of the ITS1 region is highly variable in cyanobacteria, and it has been shown that much of the ITS1 length differences between cyanobacteria are due to variability in that region (15).

Within the genus Nodularia, toxin production has been restricted mainly to one phylogenetic cluster containing planktic strains (20, 26). In the genus Anabaena, the neurotoxic and the hepatotoxic strains were differentiated on the basis of the 16S rRNA (22). In addition, other physiological traits, such as extreme halotolerance in Halothece spp. (11), differential light adaptations in Prochlorococcus spp. (28), and thermotolerance in Synechococcus spp. (24), have been confined to phylogenetically defined groups. However, in other cyanobacterial genera, e.g., Microcystis (22, 32) and Planktothrix (22), no correlation between phylogenetic clustering and toxin production capability has been found. Nodularin is produced nonribosomally by a complex multienzyme system (33). In the future, detection of the genes responsible for nodularin synthesis (see, for example, reference 25), and studies on their expression are likely to shed more light on the features of nodularin production of Nodularia strains in nature.

In this study, we investigated phenotypic features and two variable regions of the genome, the ITS1-S and the PC-IGS, in 13 nodularin-producing and 5 nontoxic Nodularia strains. We found that the nodularin-producing and nontoxic Nodularia strains carried distinct ITS1-S and PC-IGS alleles. Consequently, we detected the two types of PC-IGS alleles in Nodularia filaments collected from plankton in the Gulf of Finland. Our results suggested that all of the analyzed Nodularia filaments from the Baltic Sea represented the nodularin-producing type. Furthermore, our results from the ITS1-S and PC-IGS regions supported a previous suggestion by Barker et al. (3, 4) that only one planktic Nodularia species is genetically justifiable in the Baltic Sea.