Hydrobiologia (2010) 639:99-113 DOI 10.1007/sl0750-009-0028-y PHYTOPLANKTON Polyphasic characterization of eight planktonic Anabaena strains (Cyanobacteria) with reference to the variability of 61 Anabaena populations observed in the field Eliška Zapomělová • Klára Řeháková • Jitka Jezberová • Jaroslava Komárkova Published online: 18 December 2009 © Springer Science+Business Media B.V. 2009 Abstract The plasticity of morphological features used for single morphospecies identification was studied under varied experimental conditions (temperature, light, nitrogen, phosphorus) in eight planktonic Anabaena strains. The strains represented all of the morphospecies with coiled trichomes commonly occurring in Central Europe (two strains of A. mendo-tae & A. sigmoidea complex, two A. lemmermannii strains, two A. flos-aquae strains, and two strains of A. circinalis &A. crassa complex). Significant effects of the growth conditions on vegetative cell dimensions were observed in seven strains, and P concentration Guest editors: T. Zohary, J. Padisäk & L. Naselli-Flores / Phytoplankton in the Physical Environment: Papers from the 15th Workshop of the International Association for Phytoplankton Taxonomy and Ecology (IAP), held at the Ramot Holiday Resort on the Golan Heights, Israel, 23-30 November 2008 E. Zapomělová (El) • K. Řeháková • J. Jezberová • J. Komárkova Institute of Hydrobiology, Biology Centre of AS CR, Na Sádkách 7, 37005 Ceske Budějovice, Czech Republic e-mail: eliska.zapomelova@seznam.cz E. Zapomělová • J. Komárkova Faculty of Science, University of South Bohemia, Branišovská 31, 37005 Ceske Budějovice, Czech Republic K. Řeháková • J. Komárkova Institute of Botany, AS CR, Dukelská 135, 37982 Trebon, Czech Republic was the main influencing factor in most cases (six strains). Significant effect of an environmental factor (P) on akinete morphology was found in only one strain. Experimentally assessed temperature and light growth optima were specific for each strain and were not consistent with the taxonomic affiliation of the strains. Morphologies of the Anabaena strains studied were compared with the field morphologies of 61 Anabaena populations of eight morphospecies observed in the Czech Republic. The range of morphological variability of single strains under the experimental conditions spanned the total variability of the populations of relevant morphospecies observed in the field. Delimitations and proper descriptions of the morphospecies are discussed in the light of partial 16S rRNA gene sequences of the studied strains. Keywords Anabaena ■ Taxonomy • Identification • Morphological variability • 16S rRNA gene Introduction The genus Anabaena belongs to the most frequent bloom-forming cyanobacterial genera, affecting water quality worldwide and represents a potential risk due to the production of a range of bioactive toxic compounds (Rapala & Sivonen, 1998; Lyra et al., 2001). Detailed knowledge of the biology, morphological variability, toxicity, and phylogenetic Springer 100 Hydrobiologia (2010) 639:99-113 status of various Anabaena morphospecies is therefore required. Planktonic Anabaena species represent a widely diversified group of cyanobacteria, encompassing around 80 freshwater morphospecies (Komárek, 1996). Nevertheless, the proper description and classification of these morphospecies has not yet been satisfactorily resolved and many questions remain unanswered. So far published studies on morphological features of Anabaena have been limited either on morphology in cultures (Stulp, 1982; Stulp & Stam, 1982, 1984a, b, 1985; Li et al., 2000; Zapomělová et al., 2008a) or onto one-shot field observations (e.g., Komárek, 1958; Hill, 1976a, b, c; Hickel, 1982; Cronberg & Komárkova, 1988; Komárkova, 1988; Komárková-Legnerová & Cronberg, 1992; Komárková-Legne-rová & Eloranta, 1992; Padisák & Kovács, 1997; Hindák, 2000; Zapomělová et al., 2007). These approaches, when they are used separately, have serious disadvantages. Observation in the field does not provide complete information on morphological plasticity related to the variety of environmental conditions or growth phases. On the other hand, it has been known for a long time that long-term cultivation can cause substantial morphological changes that do not correspond to the natural conditions (Anand, 1988). Thus, confusions and misidentifications can arise when cyanobacteria are identified according to the morphology later during the cultivation (Komárek & Anagnostidis, 1989). In spite of this, nobody has tried to combine both approaches in the research of Anabaena, i.e., field observation, subsequent isolation, and short-term cultivation under varied experimental conditions. Even the most recent studies applying a polyphasic approach in Anabaena classification (Gugger et al., 2002; Rajaniemi et al., 2005a, b; Willame et al., 2006) evaluated morphological features mainly or entirely on strains in cultures. Nevertheless, the enormous value of these studies resides in the combination of morphological and molecular approach that they applied for the first time on a higher number of Anabaena isolates. They demonstrated very high 16S rRNA gene, rpoB and rbcLX sequence similarity between various Anabaena morphotypes. However, the correlation and discussion of morphological plasticity of single Anabaena strains under varied environmental conditions and their phylogenetic characteristics are still missing. Consequently, we have described the morphological plasticity of eight planktonic Anabaena strains of four morphospecies under a variety of experimental conditions. The results were compared with the field morphologies of 61 Anabaena populations observed in the Czech Republic, including the original populations from which the selected strains were isolated. Delimitations of single morphospecies were then discussed in the light of the ecological parameters (temperature and light growth optima) and phylogenetic affiliations based on their 16S rRNA gene sequences. Materials and methods Strains and cultivation The eight Anabaena strains studied (Fig. 1) were unialgal, clonal, but not axenic. They were isolated in 2004 from different localities in the Czech Republic and cultivated in WC medium (Guillard & Lorenzen, 1972) at 21°C and the light intensity of 70 umol m"2 s"1 (16:8 L:D cycle). Cross gradients of light and temperature Cross gradients (Kviderova & Lukavsky, 2001) were used to test the effect of light and temperature on the cyanobacterial morphology and to determine the growth optima of the strains studied. For the morphological experiments, the strains were exposed in sterile culture plates (9 x 12 cm , 12 wells, 6.5 ml each) to nine different combinations of light and temperature of cross gradients (Fig. 2a) for 10 days. The temperature ranged from 10 to 28°C and the Fig. 1 Microphotographs of the studied Anabaena strains, a-c ► A. mendotae/A. sigmoidea strain 04-11 from the fishpond Cernis. d-f A. mendotae/A. sigmoidea strain 04-45 from the fishpond Svet. g-i A. lemmermannii strain 04-33 from the Orlik reservoir, j-1 A. lemmermannii strain 04-42 from the fishpond Svet. m-o A. flos-aquae strain 04-40 from the Skalka reservoir, p-r A. flos-aquae strain 04-53 from the fishpond Svarcenberk. s-u A. circinalis/A. crassa strain 04-26 from the Jesenice reservoir, v-x A. circinalis/A. crassa strain 04-28 from the fishpond Hodejovicky. Images a, d, g, j, m, p, s, and v illustrate the morphology of the strains in the original populations in the field. Magnification of all microphotographs is identical; the scale bar in the lower right corner represents 10 um Springer 102 Hydrobiologia (2010) 639:99-113 Fig. 2 Design of the cross-gradient experiments, a Evaluation of morphological variability of the strains in relation to light and temperature, b Determination of light and temperature growth optima of the strains. Gradients of light intensity and temperature are indicated with arrows range of light intensity, provided by sodium vapor lamps, was 20-750 umol m-2 s_1. To estimate the temperature and light growth optima of the strains, a modified design of the cross-gradient experiments was used (Fig. 2b). Identical volumes of stirred dense batch culture were inoculated into sterile culture plates containing fresh WC medium (9 x 12 cm , 6 wells, 16 ml each). The plates were then exposed to 25 positions of the cross-table. The temperature range was 6-34°C, and the light intensity was 20-750 umol m-2 s_1. The experiments were terminated in the exponential phase of growth of the Table 1 Modifications of WC medium used in the experiments Medium type P (ug 1 ') N (jj.g 1 ') WC0xP 0 8 x 103 WCq.001 xP 8 x io-1 8 x 103 WCo.ixP 8 x 101 8 x 103 WClxP = WClxN = WC 8 x 102 8 x 103 wc10xP 8 x 103 8 x 103 wc0xN 8 x 102 0 WCq.001 xN 8 x 102 8 x 10° WCo.lxN 8 x 102 8 x 102 WCiOxN 8 x 102 8 x 104 "Starvation medium" 0 0 fastest growing cultures. Chlorophyll a concentrations were determined spectrophotometrically after acetone extraction (Lorenzen, 1967) and compared among the positions of cross gradients. Concentration series of nitrogen and phosphorus Modified types of WC medium containing different concentrations of nitrogen (N) and phosphorus (P) (Table 1) were used to test the effect of nutrients on the cyanobacterial morphology. The concentration series were designed with respect to N and P concentrations commonly occurring in fishponds and reservoirs of the Czech Republic (Znachor et al., 2006). To force the strains to deplete their intracellular nutrient reserves, cyanobacterial bio-mass was incubated in a modified WC medium without N and P ("starvation medium," Table 1) for 7 days prior to the experiment. Equimolar concentrations of KC1 were added to the "starvation medium," WC0xP, WC0001xP, and WC01xP, to retain the original K+ concentration. Morphometry Microphotographs of at least 30 fresh trichomes per each natural population or per each experimental Springer Hydrobiologia (2010) 639:99-113 103 treatment were taken with a digital camera (Olympus DP 70, magnification 400x). Dimensions of all cell types were measured (five vegetative cells per trichome measured in 30 trichomes and as many heterocytes and akinetes as was possible to find in each sample). Length:width ratios of vegetative cells, heterocytes, and akinetes were computed to roughly characterize the cell shapes. All size measurements were performed using image analysis (Olympus DP Soft). Statistical analyses The effects of light, temperature, P, and N on the morphometric characteristics were tested by redundancy analysis (RDA) with Forward selection. Monte-Carlo permutation test was used for calculation of P-values. The data were centered and standardized. Morphologies of the studied strains under varied experimental conditions and field morphologies of the 61 Anabaena populations (including those from which the studied strains were isolated) were compared by principal component analysis (PCA). These statistical analyses were performed using the program CANOCO (Ter Braak & Smilauer, 1998), and ordination diagrams were created using CanoDraw software (Smilauer, 1992). Basic statistical characteristics such as average values, 25 and 75% percentiles, and extreme values were computed for each morphological feature. Box-whisker plots were created by the GraphPad Prism program (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The light and temperature growth optima of the strains were derived from the surface plots, which were created using the program Statistica (Anonymous, 1996). Phylogenetic study The biomass was harvested in the exponential phase of growth by repeated centrifugation, during which the trichomes were washed several times with the physiological solution (NaCl solution, concentration 1 g 1_1) to remove mucilaginous substances. The biomass samples were stored at —20°C until DNA extractions. DNA was extracted using UltraClean™ Microbial DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA). The 16S rRNA gene and ITS region were amplified with primers 16S27F and 23S30R (Taton et al., 2003). Amplification was carried out as follows: one cycle of 5 min at 94°C; 10 cycles of 45 s at 94°C, 45 s at 57°C, and 2 min at 72°C; 25 cycles of 45 s at 94°C, 45 s at 54°C, and 2 min at 72°C; and a final elongation step of 7 min at 72°C. PCR product was used as a template for sequencing with primers 16S27F, 23S30R (Taton et al., 2003), primer cAlaR (Wilmotte et al., 1994), primers WAW1486R and Primer 14 (Wilmotte et al., 1993), and primer CYA781F(a) (Niibel et al., 1997). Sequences were aligned in the program ARB (http://www.arb-home.de). The alignment was edited manually and ambiguous bases were removed. For the phylogenetic analysis, trees were built with the Neighbor-joining method (NJ) (Saitou & Nei, 1987) and the Maximum Parsimony (MP) algorithm in the PHYLIP program (Felsenstein, 2004). Five-hundred bootstrap replicates were performed both for NJ and MP analyses. Nucleotide sequences were deposited at GenBank under the accession numbers FM242083-FM242088. In addition to the strains whose accession numbers are given in the phylogenetic tree, following sequences were used for the construction of the phylogenetic trees: AJ293112, AJ293115, AJ293116, AJ630413, AJ630415, AJ630417, AJ630430, AJ630432, AF247572, AF247575, AF247576, AF247584, AJ 293126, AJ293127, AJ630441, AJ630418, AJ293111, AJ293107, AJ630409, AJ630445, AJ133154, AJ293124, AJ293131, AJ293104, AJ133159, EF568912, EF547196, AJ293106, AJ293113, AJ133156, AJ630419, AJ630422, AJ630423, AY196087, AY196088, AJ630457, AJ630458, AJ630428, AJ133161, AF160256, AJ133181, AY038033, and AY038036. Results Morphological plasticity of the strains with reference to the variability of 61 Anabaena populations observed in the field Morphometrical characteristics of the eight studied Anabaena strains were measured under various temperature, light intensity, and concentration of N and P (Table 2). These data were compared with field morphologies of 61 Anabaena populations, including those from which the studied strains were isolated. PCA has demonstrated that the studied strains were able to span the total variability of the populations of Springer 104 Hydrobiologia (2010) 639:99-113 o 3 Oil > 3 J3 BO 60 T3 60 Oil ' 1 On o O 00 00 t-; cn 00 t-; vo —i on in rn oo O ^h^h^hooovoooo^h oo" in in \o o of oC —3 o ^ CI (N ^ ^ 00 00 *= 5- oS 4. ? 3- T TT T OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus b ?10 i 9 £ 8 T3 "55 6 -o CD 5 > "5 ~ V > 2 OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus i T OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus Fig. 4 Vegetative cell dimensions of the A. mendotae/A. sigmoidea strain 04-45 under various P concentrations, a Vegetative cell length, b Vegetative cell width, c Length:width ratio of vegetative cells. Whiskers represent minimal and maximal values, boxes symbolize 25 and 75% percentiles, and lines inside boxes show mean values Analogously, batch cultures in the strain A. mendotae/A. sigmoidea 04-45 were observed several times, where only akinetes adjacent to heterocytes formed, although the prevailing akinete position of this strain and of its original field population was remote from heterocytes. Akinetes of the strain A. mendotae/A. sigmoidea 04-11 and of its original field population were steadily remote from heterocytes, both during the experiments and the 4-year cultivation. Trichome coiling Alternating occurrence of regularly (helixes) and irregularly (clumps) coiled trichomes was observed in the majority of the strains (Fig. 1), except for the strains A. circinalis/A. crassa 04-26 and 04-28, where the predominant type of the trichome coiling was regular. Proportional abundance of regularly and irregularly coiled trichomes differed both among the strains and among various experimental treatments of the same strain. Nevertheless, the effect of experimental conditions on the regularity of trichome coiling was not demonstrated in any of the strains studied. Temperature and light preferences of the strains The temperature and light growth optima of the Anabaena strains studied were rather diverse, ranging from 13.5 to 28°C and from 80 to 360 umol m"2 s"1, respectively. All of the strains were able to grow in the whole range of experimental temperature (10-28°C), but some of them did not survive extreme light Springer Hydrobiologia (2010) 639:99-113 107 Fig. 5 Vegetative cell dimensions of the A. flos-aquae strain 04-53 in various positions of cross gradients of light and temperature. Light intensity is symbolized by shading (750 umol m~2 s-1, plain boxes; 200 umol m~2 s-1, simple shading; 20 umol m~2 s_1, double shading). Whiskers represent minimal and maximal values, boxes symbolize 25 and 75% percentiles, and lines inside boxes show mean values, a Vegetative cell length, b Vegetative cell width, c Length:width ratio of vegetative cells. Microphotographs show differences in vegetative cell dimensions of the strain 04-53 grown at 750 umol m~2 s-1 (d-e) and 20 umol m~2 s-1 (f-g). Scale bars represent 10 um 30 ?25 £ 20 CT 2 15 0) a> I 10 re 5 •—•11 - IL T Iio- OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus 12 ~11 Í10 S 9 * 8 0 1 7 re 6 - I 3.5 O 3.0 H 2 » .= £ 2.5H ts °> I OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus OxP 0.001xP 0.1xP 1xP 10xP concentration series of phosphorus Fig. 6 Akinete dimensions of the A. flos-aquae strain 04-53 under various P concentrations, a Akinete length, b Akinete width, c Length:width ratio of akinetes. Whiskers represent minimal and maximal values, boxes symbolize 25 and 75% percentiles, and lines inside boxes show mean values intensities (20 and 750 umol m-2 s_1). The temperature and light preferences were specific for each strain and were not consistent with the taxonomie affiliation of the strains (Table 4). Phylogenetic relationships of the strains The eight studied Anabaena strains were placed into three different clusters in the distance tree of partial 16S rRNA gene sequences (Fig. 7). Cluster A contained both A. circinalis/A. crassa strains (04-26 and 04-28), showing 100% identity. Besides, A. flos-aquae 04-53 was also affiliated to cluster A. The strains A. medotae/A. sigmoidea 04-11 and 04-45 and A. lemmermannii 04-33 appeared together in cluster B with high bootstrap support. The strains A. lemmermannii 04-42 and A. flos-aquae 04-40 were situated in cluster C. <£) Sprin: ger 108 Hydrobiologia (2010) 639:99-113 Table 4 Temperature and light optima of the studied Anabaena strains Morphospecies affiliation Strain Temperature (°C) Light (urnol m 2 s ') Anabaena mendotae/A. sigmoidea 04-11 18.5-21.0-23.5 80-150-220 A. mendotae/A. sigmoidea 04-45 16.0-18.5-21.0 140-200-260 A. lemmermannii 04-33 18.5-22.0-25.5 120-210-300 A. lemmermannii 04-42 13.5-16.0-18.5 190-230-270 A. flos-aquae 04-40 19.5-22.5-25.5 220-250-280 A. flos-aquae 04-53 25.0-27.0-29.0 110-140-170 A. circinalis/A. crassa 04-26 17.5-20.0-22.5 220-290-360 A. circinalis/A. crassa 04-28 22.0-25.0-28.0 100-160-210 The range of the values represents the experimentally determined growth optima; their median values are in bold Fig. 7 Neighbor-joining tree based on 16S rRNA gene sequences (1,340 bp) showing the clustering of studied Anabaena strains. Numbers near the nodes indicate the bootstrap values over 50% for NJ and MP analyses. An. Anabaena, Aph. Aphanizomenon, inch including, OTU operational taxonomic unit, spec. div. different species, filled diamond studied Anabaena strains . An. circinalis, An. crassa, An. planclonica, 10 OTU's An. spec div; incl An. circinalis04-26 I and 04-28 (AM940218, AM940219) ♦♦ - An. spiioides PMC9702 (AJ293118) - An.smithii 1tu39s8 (AJ63C436) - An. sigmoidea 0tu38s4 (AJ630435) - An. flos-aquae 04-53 (FM242088) + - An.planclonica 1tj36s8 (AJ630433) 4 OTU's An. circinalis -Australian cluster 3 OTU's Aphanizomenon spec. div. Anabaena sp. BIR300 (EF568908) __ An. mendotae. Aph. gracile. An. circinalis var. macrospora; I 9 OTU's| incl. An. mendotae 04-11 and 04^5{FM242083, FM242084),I and An. lemmermannii 04-33 (FM242085) ♦ gj 87/731-Aph. cf.gracile 271 (AJ293125) I An. cl.cyiindhca PMC9705 (AJ293119) -Anabaena sp. BIR25 (EF568914) 1 An. lemmermannii; I _^OTUJsJ incl. An. lemmermannii04-42(FM242086) + I - An. lemmermannii 1tu32s11 (AJ630424) I - An. flos-aquae 04-40 (FM242087) + Q | 97/70 6 OTU's Anabaena spec, div Aph. issalschenkoi An augstumalis. An oscillahoides. An pergii, Trichormus variabilis I Nostoc sp., Nodularia sp., Anabaenopsis sp., Cyanospira hppkae - Synechococciis sp. 0tu28s07 (AM259221) Discussion The range of morphological variability of single Anabaena strains A comparison was drawn between the morphologies of Anabaena isolates exposed to varied experimental conditions and the populations from the field conditions. This is the first direct evidence for the potential of a single cyanobacterial strain to span the total variability of all relevant morphospecies or morphospecies complexes. These findings were consistent in all of the eight strains studied, representing distinctly different Anabaena morphospecies, which possibly indicates that they may be generalized throughout the entire group of the planktonic Anabaena. Our present conclusions are in a good agreement with previous suggestions that only larger morphological complexes can be delimited within the group of planktonic Anabaena since the traditional morphospecies are not distinctly defined by their morphological characteristics (Zapomelova et al., 2007). The only reported morphospecies that appears to be clearly defined by both the morphological and phylogenetic characteristics was A. compacta (Nyg-aard) Hickel (Rajaniemi et al., 2005a, b). It was Springer Hydrobiologia (2010) 639:99-113 109 therefore excluded from this study, since no transitions to other morphospecies were expected. Despite this, there may be a reason for keeping the narrow traditional morphospecies and their names as ecomorphs. More studies are to be conducted to clarify the relationships between single morphological features and environmental factors. The ecomorphs could be then used for estimation of environmental conditions in the field. Taxonomie implications Significant relationships between environmental factors and taxonomically important morphological features were demonstrated and described for the first time within the genus Anabaena. Only a few studies focusing on these problems within the genus Anabaena have been published so far (Stulp, 1982; Stulp & Stam, 1984b, 1985; Zapomělová et al., 2008a); however, none of them detected significant responses in Anabaena morphology related to variable growth conditions. On the contrary, it was demonstrated on other genera that some of the features which have been traditionally used in cyanobacterial classification varied under different culture conditions (e.g., dimensions and length:width ratios of vegetative cells and appearance of colonies of chroococcacean cyanobacteria; Chang, 1988; Doers & Parker, 1988; Wilmotte, 1988; Palinska et al., 1996; Saker & Neilan, 2001). This study has revealed some limitations in the classification of planktonic Anabaena and has indicated future directions for research of particular morphological groups within this genus. Anabaena mendotae Trelease 1889 vs. A. sigmoidea Nygaard 1949 Modifications of vegetative cell morphology under various P concentrations are important mainly in connection with the morphospecies complex A. sigmoidea & A. mendotae. Vegetative cell length, the length: width ratio, and their general shape are traditionally regarded as distinguishing between these two taxa, together with the dimensions of the akinetes (Komárek, 1996; Komárek & Zapomělová, 2007). Morphometry parameters observed in the strains 04-11 and 04-45 during the experiments spanned the ranges of both A. mendotae and A. sigmoidea (Table 2), which was supported also by the genetic data. Both these strains displayed very high 16S rRNA gene sequence similarity and appeared together in one cluster, although the field morphology of the strain 04-11 corresponded more with the description of A. mendotae, and the strain 04-45 was identified as A. sigmoidea (Fig. 1, a vs. d). Investigation of higher number of A. mendotae and A. sigmoidea strains would be required to clarify the status of these morphospecies. Anabaena circinalis Rabenhorst ex Bornet et Flahault 1888 vs. A. crassa (Lemmermann) Komárková-Legnerová et Cronberg 1992 Similarly, our experiments demonstrated that the studied A. circinalis strains (04-26, 04-28) were able to span the variability of both A. circinalis and A. crassa, as they were originally described. Trichome widths and coil diameters are considered the main distinguishing criteria for these species (Komárek, 1958; Komárková-Legnerová & Cronberg, 1992; Komárková-Legnerová & Eloranta, 1992). The two strains studied displayed a wide range of features, corresponding with the description of both A. circinalis and A. crassa (Table 2; in detail described in Zapomělová et al., 2008b). Continuous variability of trichome widths and coil diameters was reported also from 13 field populations of A. circinalis and A. crassa studied in the Czech Republic (Zapomělová et al., 2007). Broader morphological and molecular revision of this morphospecies complex would be necessary to elucidate the taxonomie validity of the taxa A. circinalis and A. crassa. Anabaena flos-aquae complex The two studied A. flos-aquae strains (04-40, 04-53) exhibited marked differences in 16S rRNA gene sequences. They were located in distinctly separate subclusters in the phylogenetic tree based on the 16S rRNA gene. From a morphological point of view, these strains differed mainly in the shape and size of the akinetes and in the trichome coiling morphology (Table 2; Fig. 1, m-o vs. p-r). Ambiguities in the definition of the morphospecies A. flos-aquae were previously pointed out by Zapomělová et al. (2007). The concepts of A. flos-aquae (Lyngbye) Brébisson ex Bornet et Flahault 1888, A. spiroides Klebahn Springer 110 Hydrobiologia (2010) 639:99-113 1895, A. perturbata Hill 1976, and similar morpho-species differ among various authors and the shifts in these concepts can also be observed during the history of the cyanobacterial taxonomy. The definition of A. flos-aquae is rather vague, encompassing wide range of morphotypes with rounded vegetative cells, approximately 4-8 um in diameter, and with kidney shaped or cylindrical akinetes (Komárek & Zapomělová, 2007), i.e., also A. perturbata and A. spiroides. Morphological heterogeneity within A. flos-aquae complex is evident also from the herein presented PCA (Fig. 3). Furthermore, obvious differences among various A. flos-aquae strains were previously shown by Rajaniemi et al. (2005a) at the genetic level (16S rRNA gene, rpoB and rbcLX sequences). Both the morphological and molecular analyses of more strains from this species complex are highly required to revise the status of the A. flos-aquae taxon. Anabaena lemmermannii Richter in Lemmermann 1903 vs. A. mendotae & A. sigmoidea Very important finding of this study is the relation between the akinete position stability and the phylo-genetic affiliation of the A. lemmermannii-like strains 04-33 and 04-42. Based on the 16S rRNA gene sequences, the strain 04-33 with variable akinete position clustered together with the strains from A. mendotae & A. sigmoidea group. From those strains, occasional dominance of akinetes adjacent to the heterocytes was observed in the strain 04-45. On the other hand, the strain 04-42, with the akinetes steadily adjacent to the heterocytes, grouped together with some other A. lemmermannii strains from Genbank into a separate cluster. The validity of A. lemmermannii was challenged by Zapomělová et al. (2007), who pointed out the enormous morphological heterogeneity within this taxon. The only identification feature of this morphospecies considered is the position of akinetes adjacent to heterocytes, while the shape and dimensions of the vegetative cells are highly variable (Komárkova, 1988; Zapomělová et al., 2007). Nevertheless, the genetic cluster comprising the strain 04-42 appears to be clearly separate and corresponds exactly to the main A. lemmermannii cluster of Gugger et al. (2002) and Rajaniemi et al. (2005a, b). A. mendotae and A. sigmoidea strains constitute a distinct, highly consistent cluster together with the strain 04-33. This cluster agrees exactly with the A. cf. lemmermannii strain PH262 of Gugger et al. (2002), which exhibited outlying position in the phylogenetic tree and probably represented the A. lemmermannii-like strain with unstable akinete position. For the present, A. lemmermannii appears to be a valid taxon, which should be, however, verified by thorough analyses of more A. lemmermannii strains. Moreover, some other phenotypic features will then have to be found allowing its identification, since the determination of the stability of akinete position would be extremely problematic in natural populations. Experimental conditions and the ecological context Phosphorus Phosphorus concentration was the main factor influencing the morphology of vegetative cells, which can be easily explained by reduced division rate and consequential cell elongation under the P limitation. Significant differences in the trichome width were previously reported also from P-limited and non-limited populations of Cylindrospermopsis raciborskii (Komárkova et al., 1999; Shafik et al., 2003). Extreme modification of morphology under the P-limiting conditions was observed in the strain 04-26, where the elongation of vegetative cells resulted in the trichome straightening (Zapomělová et al., 2008b). Light intensity Anabena flos-aquae 04-53 was the only strain that showed significant response of the vegetative cell dimensions toward varied light intensity. The majority of the strains studied did not exhibit any response to the light, which corresponded well with the conclusions of Stulp & Stam (1985). Variations in cell sizes and shapes induced by changes in light intensity were reported from different cyanobacterial strains (Wyman & Fay, 1997), but their trends were not consistent, indicating that the response may be strain specific. Influence of light on trichome coiling was reported from the oscillatoriacean cyanobacte-rium Arthrospira fusiformis (Bai & Seshadri, 1980) but has never been demonstrated in Nostocales, Springer Hydrobiologia (2010) 639:99-113 111 although cases of trichome straightening are known from Anabaena cultures (Booker & Walsby, 1979; Hickel, 1982; Zapomelova, 2004; Zapomelova et al., 2008a). Temperature A significant effect of temperature was found in the strain 04-26, whose vegetative cells and heterocytes were bigger (both lengths and widths) at higher temperature values. A potential explanation of these cells' enlargement may be the accumulation of metabolic products resulting from enhanced metabolic rates (Robarts & Zohary, 1987). Nitrogen Insignificant effect of low N concentrations can be easily explained by the N2 fixing ability of Anabaena (heterocytes). However, our study also demonstrated that the high N concentrations achievable in the field had no effect on Anabaena morphology. This is in disagreement with the results of Saker & Neilan (2001), who found significant morphological variability (dimensions of vegetative cells and heterocytes) in heterocytous cyanobacterium Cylin-drospermopsis raciborskii exposed to different sources and concentrations of N. The growth demands These experiments suggest that Anabaena strains of even the same morphospecies are able to prosper in a wide range of temperature and light conditions. The temperature and light preferences were specific for each strain and did not reflect the taxonomic affiliations of the Anabaena strains studied. Our results are supported by similar conclusions of Rapala & Sivonen (1998), who focused on strain-specific differences in growth rates of various Anabaena strains as a function of temperature and light. Thus, interstrain variability may be expected in temperature and light preferences. 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