JPR Advance Access originally published online on February 18, 2009
Journal of Plankton Research 2009 31(5):465-480; doi:10.1093/plankt/fbp011
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Genetic and morphologic characterization of four putative cylindrospermopsin producing species of the cyanobacterial genera Anabaena and Aphanizomenon
1 Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department Ecology of Stratified Lakes, Alte Fischerhütte 2, 16775 Stechlin, Germany 2 Department of Biology, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, 0316 Oslo, Norway 3 Cooperative Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia, Australia 4 School of Pharmacy and Medical Sciences, University of South Australia, GPO Box 2471, Adelaide, South Australia 5001, Australia 5 Departamento de Biología, C/Darwin, 2, Universidad Autónoma de Madrid, 28049 Madrid, Spain 6 Israel Oceanographic and Limnological Research, Yigal Allon Kinneret Limnological Laboratory, PO Box 447, 14950 Migdal, Israel 7 Department of Botany, Government College, Ajmer-305 001, Rajasthan, India
* CORRESPONDING AUTHOR: anke.stuken{at}web.de
Received on September 24, 2008; accepted on January 24, 2009
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Cylindrospermopsin (CYN) is a potent hepatotoxic alkaloid that has been detected in freshwater samples worldwide and is produced by a number of cyanobacterial species, mainly of the genera Cylindrospermopsis, Aphanizomenon and Anabaena. Cylindrospermopsis raciborskii is a morphologically distinctive species which forms a genetically well-defined cluster. In contrast, some species within Aphanizomenon and Anabaena are morphologically not clearly assignable to either genera and both genera are polyphyletic. In the Cylindrospermopsis cluster CYN producing and non-producing strains co-occur, but it is not known if CYN producing and non-producing strains are closely related in Anabaena and Aphanizomenon. Here we attempt to disentangle the phylogenetic relationships of four taxa of the genera Anabaena and Aphanizomenon, some of which are known as CYN producers. We have sequenced and phylogenetically analysed partial sequences of the 16S rRNA gene, cpcBA-IGS and rpoC1 from 31 cyanobacteria isolates of the genera Aphanizomenon and Anabaena and have documented morphotypic characteristics of new and recently isolated strains. Our results do not corroborate the separation of Aph. gracile and Aph. flos-aquae into separate species. In contrast, they support the distinction of Ana. bergii and Aph. ovalisporum into distinct taxa. Further, Ana. bergii is most likely not a CYN producing species.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Cylindrospermopsin (CYN) is a potent hepatotoxic alkaloid that is produced by a number of cyanobacterial species. The toxin or cyanobacterial strains producing it have been reported from Australia and New Zealand (e.g. McGregor and Fabbro, 2000
; Stirling and Quilliam, 2001), South and North America (Carmichael et al., 2001; Burns et al., 2002), Asia (e.g. Harada et al., 1994; Chonudomkul et al., 2004) and Europe (e.g. Fastner et al., 2003; Manti et al., 2005; Quesada et al., 2006). The recommended drinking water guideline of 1 µg L–1 (Humpage and Falconer, 2003; Sukenik et al., 2006) is frequently exceeded in these water bodies (e.g. McGregor and Fabbro, 2000; Burns et al., 2002; Rücker et al., 2007).
All CYN producing cyanobacterial strains that have been isolated so far belong either to the order Nostocales or to the order Stigonematales. Both orders include filamentous heterocystous cyanobacteria that are capable of forming akinetes. Cells of the former order divide in one plane only, whereas those of the later divide in more than one plane. This distinction is not supported by phylogenetic analyses; instead, it has been suggested that all heterocyst-forming cyanobacteria form a monophyletic group and that the branching patterns within this group are polyphyletic (Gugger and Hoffmann, 2004).
The majority of CYN producing strains belong to the Nostocales genera Cylindrospermopsis (Ohtani et al., 1992; Saker and Eaglesham, 1999; Saker et al., 1999a, b; Li et al., 2001; Chonudomkul et al., 2004), Aphanizomenon (Banker et al., 1997; Preußel et al., 2006; Wormer et al., 2008; Yilmaz et al., 2008) or Anabaena (Schembri et al., 2001; Spoof et al., 2006). Cylindrospermopsis strains have been shown to form a well-supported monophyletic group within the heterocystous cyanobacteria, whereas strains of Anabaena and Aphanizomenon have been shown to be intermixed and polyphyletic (Lyra et al., 2001; Gugger et al., 2002; Iteman et al., 2002; Rajaniemi et al., 2005). Studies of the Cylindrospermopsis cluster alone have revealed that strains group according to geographic origin (Dyble et al., 2002; Neilan et al., 2003; Gugger et al., 2005; Haande et al., 2008). In addition, toxin synthesis appears to be geographically depended, too; only Australian (e.g. Saker and Eaglesham, 1999; Saker et al., 1999a, b) and Asian (Li et al., 2001; Chonudomkul et al., 2004) isolates have been shown to produce CYN, whereas none of the European (e.g. Fastner et al., 2003; Saker et al., 2003; Briand et al., 2004), African (Berger et al., 2006; Haande et al., 2008) or American (Yilmaz et al., 2008) C. raciborskii isolates produces the toxin.
The genera Anabaena and Aphanizomenon include species that are neither phylogenetically nor morphologically clearly separable. The criteria used for generic classification and species definition are not always reliable and several species have been described, which show characteristics intermediate between both genera (Komárek and Kovacik, 1989). This ambiguous distinction has also been reported for the two putative CYN producing species Aphanizomenon ovalisporum Forti and Anabaena bergii Ostenfeld. It can be difficult to distinguish between these two species based on morphologic characteristics (Bazzichelli and Abdelahad, 1994; Yilmaz et al., 2008). It has been suggested that with so many shared characteristics, these species may instead be morphotypes of a single species (Komarék and Ettl, 1958). Although this suggestion has been supported by phylogenetic analyses of several different DNA sequences from the Australian Ana. bergii strain ANA283A (Fergusson and Saint, 2000; Wilson et al., 2000; Kellmann et al., 2006), ANA283A remains the only Ana. bergii strain for which sequences are publicly available. A paucity of sequence data is also apparent for Aph. ovalisporum. There are currently only four 16S rRNA gene sequences of which two are of the same strain (ILC146), one is a short (477 bp) sequence from an Australian isolate and the fourth a full-length sequence of the American isolate FAS-AP1. This isolate has morphologically been described as Aph. ovalisporum but the 16S rRNA gene sequence is more similar to the Ana. bergii ANA283A than to the Aph. ovalisporum ILC146 sequence (Yilmaz et al., 2008).
Cyanobacteria strain designation is based on morphological descriptions, but unfortunately no morphologic documentation has been provided for the Ana. bergii strain ANA283A or for any other Ana. bergii strain analysed for CYN production (Wilson et al., 2000; Schembri et al., 2001, Fergusson and Saint, 2003; Rasmussen et al., 2007, 2008). In contrast, detailed morphologic descriptions or pictures have been provided for CYN-producing strains designated as Aph. ovalisporum (Banker et al., 1997; Shaw et al., 1999; Wormer et al., 2008; Yilmaz et al., 2008).
Thus, it is unclear if the Ana. bergii and Aph. ovalisporum strains, for which a close genetic relatedness has been established, also resemble each other morphologically.
The occurrence of CYN in water bodies cannot always be clearly linked with the presence of putative CYN producing species (e.g. Carmichael et al. 2001; Fastner et al., 2003; Rücker et al., 2007; Yilmaz et al., 2008). Aph. flos-aquae is the only clearly identified CYN producer in German waters (Preußel et al., 2006; Rücker et al., 2007), but its presence could not always explain the occurrence of the toxin (Rücker et al., 2007). This might either be due to the co-occurrence of CYN-producing and non-producing strains of the same taxon, or to the presence of additional, so far unidentified CYN producing species, most likely of the genera Aphanizomenon or Anabaena (Fastner et al., 2007; Rücker et al., 2007). Two potential candidates are Ana. bergii and A. aphanizomenoides (Forti) Horecká and Komárek, which have recently been reported for the first time from German water bodies (Stüken et al., 2006). Aphanizomenon gracile Lemmerman has been put forward as a third candidate, because the highest correlation coefficients were observed between the biovolume of this taxon and the concentration of CYN in a range of water bodies (Rücker et al., 2007; Wiedner et al., 2008).
In this study, we attempt to disentangle the phylogenetic relationships of 31 strains belonging to the genera Anabaena and Aphanizomenon, some of which are known as CYN producers. Morphological comparisons were additionally undertaken between species within the genera, isolated from four different continents, to investigate any reconciliation between molecular and morphological characters.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Strain isolation, cultivation and documentation
All strains are listed in Table I. The German strains were isolated according to Preußel et al. (2006), the Spanish strains to Wormer et al. (2008) and the Australian strains to Andersen and Kawachi (2005). The Israeli Aph. ovalisporum culture KA1.1 is a re-isolate of the strain ILC-146, originally isolated by Banker et al. (1997). The Spanish strains were grown in BG110 under continuous illumination at 30 µmol photons m–2 s–1 and 28°C. All German strains, strain KA1.1. and PMC215.03 were grown under continuous shake and a 12/12 h dark/light cycle, at 20°C and an illumination of 65 µmol photons m–2 s–1. The Australian isolates were maintained under similar conditions with the exception that they were not shaken and maintained at 18°C in Artificial Seawater Medium-1 (ASM-1). The strains were uni-cyanobacterial but not axenic.
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Photographs and measurements were taken with an Olympus BX51 system microscope at 400 and 1000x magnification with the Cell D software from Olympus Soft Imaging Solutions GmbH, Münster, Germany.
DNA isolation, PCR amplification and sequencing
From the Australian isolates, DNA was isolated using the QIAamp® DNA minikit (Qiagen®, Hilden, Germany) according to the manufacturer's tissue protocols. The UltraCleanTM kit (MO BIO laboratories, Inc.) was used to extract DNA from UAM290 and UAM291. The remaining DNA extractions were carried out using the Dynabeads® DNAdirectTM Universal kit (DYNAL Biotech ASA, Oslo, Norway). PCR reactions were performed using a PTC-200 Peltier thermal cycler and carried out in final volumes of 50 µL. Reaction mixture contained 10–50 ng of genomic DNA, 1 x PCR-buffer (Qiagen®), 2.5 mM MgCl2, 20 pmol of each primer, 200 µM of each dNTP and 1.5 U Qiataq DNA Polymerase (Qiagen®). All PCRs had an initial heat activation of 96°C/300 s and PCR and subsequent cycles were begun with a denaturation step of 94°C/30 s. Primers, annealing times and temperatures are given in Table II. Primers rpoC1-1 and rpoC1-T were used in combination with the following touchdown PCR: 95°C/60 s, 11x (94°C/15 s, 64–53°C/ 30 s [1°C reduced each cycle], 72°/60 s), 30x (94°C/15 s, 53°C/30 s, 72°C/60 s), 72°C/600 s. RpoC1-amplification of the German and the Senegalese Ana. bergii strains resulted in multiple banding patterns. Bands of the approximate right size (635 bp) were excised from the gel, purified with the QIAquick Gel Extraction Kit (Qiagen) and cloned using the TOPO TA cloning kit (pCR® 2.1-TOPO® vector, Invitrogen) according to the manufacturer's protocol. All PCR-products were purified using the QIAquick PCR purification kit (Qiagen®), and sequenced with Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems, Darmstadt) on an ABI Prism 3100-Avant Genetic Analyzer according to the manufacturers' protocols. Both DNA strands were sequenced, and several internal primers were used to sequence the 16S rRNA gene (Table II). Accession numbers are listed as Supplementary information.
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Sequence alignment and phylogenetic analyses
Sequences were aligned with the software MEGA, version 4 (Tamura et al., 2007). Alignments were checked manually. All codon positions were included but positions containing gaps and missing data were eliminated. The final alignment lengths common to all sequences were: 16S rRNA gene 1059 bp, cpcBA 405 bp (excluding the IGS) and rpoC1 541 bp.
Three different tree construction methods were used to compute evolutionary distances: Neighbour-Joining (NJ; Saitou and Nei, 1987) using the Jukes-Cantor method (Jukes and Cantor, 1969) was computed with MEGA, Maximum Parsimony (MP) was computed using PAUP 4 beta 10 WIN (Swafford, 1998) and Maximum Likelihood (ML) using the software TREEFINDER version of June 2008 and the parameters suggested by the software for the best fit model (Jobb, 2008). One thousand bootstrap values were generated for each analysis.
CYN analyses of the German and the Senegalese Ana. bergii strains were carried out as described in Preußel et al. (2006), those of strains UAM290 and UAM291 according to Quesada et al. (2006).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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General phylogenetic patterns
The phylogenetic relatedness of 31 cyanobacterial strains identified as Ana. bergii, Aph. ovalisporum, Aph. aphanizomenoides, Aph. flos-aquae and Aph. sp. was studied by analysing the DNA sequence similarity of three different gene fragments; of the 16S ribosomal RNA gene (Fig. 1), of the phycocyanin operon (cpcBA-IGS; Fig. 2) and of the gamma subunit of the cyanobacteria DNA-dependent RNA polymerase (rpoC1, Fig. 3).
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CYN not detected.
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The overall branching pattern was not always consistent and well supported between the different tree constructing methods used. These inconsistencies in branching commonly occur in closely related taxa. However, stable groups of sequences, which consistently occur at the tips of branches regardless of the tree constructing method used, indicate relatedness and true phylogenetic significance (Wilmotte and Herdman, 2001
). The strains analysed in the present study always formed four groups (Figs 1
–3). Group I contained all CYN producing and non-producing Aph. flos-aquae and Aph. sp. strains and clustered with or close to Anabaena and Aphanizomenon sequences obtained from Genbank. Group II was comprised of the two Australian Ana. bergii strains ANA283B and ANA283D, all Australian and German Aph. aphanizomenoides strains, and two A. kisseleviana and one A. flos-aquae strains isolated from Japan. None of the Ana. bergii and Aph. aphanizomenoides strains of this group produces CYN (Table I). Group III contained all German and the African Ana. bergii strains, as well as the four non-CYN producing Australian Ana. bergii strains ANA366A, ANA 360B, ANA360H and ANA360D. This was true for the 16S rRNA gene and cpcBA-IGS phylogenies. The amplification and sequencing of the rpoC1 product failed for most strains of this group, despite trying several different protocols (R. Campbell and A. Stüken, unpublished results). Thus, group III in the rpoC1 phylogeny only consisted of the Ana. bergii strains ZIE26AB and PMC215.03 (Fig. 3). Group IV comprised all known Aph. ovalisporum strains and the two Australian CYN producing Ana. bergii strains ANA366B and ANA283A. All strains in group IV produce CYN.
The shortest branch lengths were observed in the 16S rRNA gene phylogeny, whereas those of the cpcBA-IGS and rpoC1 phylogenies were longer and of similar length (Figs 1–3). The intergenetic spacer (IGS) between the cpcB and cpcA subunits of the phycocyanin operon was not unambiguously alignable and was analysed separately. It was well conserved within each group (96–100% sequence similarity) and the combination of its length and GC content was distinct for each group (Fig. 2). The IGS analyses thus substantiated the results from 16S rRNA gene, cpcBA-IGS and rpoC1 phylogenies. Similar results have been obtained from IGS analyses of picocyanobacteria (Crosbie et al., 2003), Oscillatoriales (Manen and Falquet, 2002) and Nostocales (Dyble et al., 2002).
CYN producing and non-producing Aph. sp. strains
The CYN producing and non-producing German Aph. sp. strains shared a high 16S rRNA sequence similarity (99.7–100%, 1074 bp) and clustered with the Aph. flos-aquae type strain PCC7905, the A. gracile strain 1tu26s16s and the Aphanizomenon sp. strain TR 183 (Fig. 1). None of the 16S rDNA, the cpcBA-IGS or the rpoC1 analyses separated strains designated as Aph. flos-aquae, Aph. gracile and Aph. sp. (Figs 2 and 3). Similar results were obtained from phylogenetic studies based on longer 16S rRNA gene sequences (Lyra et al., 2001; Gugger et al., 2002; Iteman et al., 2002; Rajaniemi et al., 2005), the internal transcribed spacer region between the 16S and 23S rRNA gene (ITS1; Gugger et al., 2002; Iteman et al., 2002), the RubisCo region (rbcLX; Gugger et al., 2002; Rajaniemi et al., 2005) and the β subunit of the RNA polymerase (rpoB; Rajaniemi et al., 2005). Aph. flos-aquae and Aph. gracile were not clearly separated in any of these studies.
Using morphological criteria, Komárek and Kovacik (Komárek and Kovacik, 1989) have previously compared four Aphanizomenon species, including Aph. flos-aquae and Aph. gracile. According to these authors, A. flos-aquae trichomes are between 4.3 and 7.8 µm wide and have more or less the same width along the whole trichome. The terminal cells are cylindrical, distinctly elongated and intensely vacuolated. In contrast, trichomes of Aph. gracile are narrower, 2.3–3.7 µm and obligatory narrow towards the ends. The terminal cells exhibit a characteristic "capitate" morphology and are not significantly vacuolated or elongated. Aph. flos-aquae trichomes typically form fascicle-like colonies, whereas Aph. gracile trichomes occur solitarily (Komárek and Kovacik, 1989).
Natural cyanobacteria populations contain filaments with morphological variations intermediate between these two morphotypes, and the differentiation between Aph. gracile and Aph. flos-aquae in field populations remains debatable (e.g. Baker, 1981; Kohl et al., 1985). Nutrients also have an influence on cell width; availability of NH4+ results in widening of cells, whereas P-limitation results in narrowing (Kohl et al., 1985). Further, strains of Aph. flos-aquae have frequently been observed to loose their colony forming habit under culture conditions (e.g. Kohl et al., 1985), including the Aph. flos-aquae reference strain PCC7905 (Rippka et al., 2001). The CYN producing Aph. sp. strains 22D11, 10E6 and 10E9 have originally been identified as Aph. flos-aquae (Preußel et al., 2006). However, none of these strains forms colonies under culture conditions (picture in Preußel et al., 2006) and the terminal cells, even though cylindrical, are only occasionally distinctly elongated and vacuolated. None of the strains has trichomes that obligatorily narrow towards the ends or terminal cells that exhibited a characteristic "capitate" morphology. The vegetative cells of strain 22D11 are 5.0 ± 1.35 µm wide and 10.1 ± 2.4 µm long (n = 192; mean ± SD), whereas they are 3.35 ± 0.64 µm wide and 7.0 ± 1.53 µm long in strain 10E6 (n = 245) and 3.3 ± 0.8 µm wide and 6.0 ± 1.3 µm long in strain 10E9 (n = 343). Based on these cell dimensions and according to Komárek and Kovacik (Komárek and Kovacik, 1989), strain 22D11 should be designated as Aph. flos-aquae and strain 10E6 and 10E9 as Aph. gracile. However, as none of the three cultures shows the typical morphologies associated with either Aph. flos-aquae (colony formation, elongated and vacuolated terminal cells) or with Aph. gracile (trichomes narrowing towards the end, capitate terminal cells) and as all of the strains are genetically very similar based on the fragments investigated during this study, it is not possible to clearly assign any of these three strains to either Aph. flos-aquae or Aph. gracile. Thus, we have decided to refer to strains 10E6, 10E9 and 22D11 as Aph. sp. instead of Aph. flos-aquae. None of the other four Aphanizomenon strains (30D11, 06D11, 14E6 or 16D11) formed colonies under culture conditions or showed other distinct morphological characteristics associated with either Aph. flos-aquae or Aph. gracile. Thus, these strains were designated as Aph. sp. as well. Pictures of the typical strain morphology of the German Aphanizomenon strains of group I are depicted in Fig. 4.
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Non-CYN producing Aph. aphanizomenoides and Ana. bergii strains
Group II clustered separately from the remaining groups described in this study and was well supported (Figs 1–3). The strains contained within this group shared a high 16S rRNA gene sequence similarity (99.3–100%, 1074 bp).
The strain NIES73 is named Aph. flos-aquae in the Genbank records from which the DNA sequences of this strain have been taken (AY701573
[GenBank]
, AY702243
[GenBank]
), but the same strain has been described as A. oumiana previously (Watanabe, 1996; Table III). This strain and the two Ana. kisseleviana strains included in the cpcBA-IGS and 16S rDNA phylogenies have previously been shown to have very similar base compositions (Li and Watanabe, 2002). Aph. aphanizomenoides, Ana. kisseleviana and Ana. oumiana share one distinct morphological characteristic: they all have spherical akinetes adjacent to their heterocysts, either on one or on both sides (Horecká and Komárek, 1979; Watanabe, 1996; Li et al., 2000; Table III). Akinete parameters have been shown to be useful for the classification of selected Anabaena and Aphanizomenon strains (Rajaniemi et al., 2005). The main morphological difference between the three species is that Ana. oumiana has coiled (Watanabe, 1996) and Aph. aphanizomenoides and Ana. kisseleviana have straight trichomes (Horecká and Komárek, 1979; Li et al., 2000). Similar differences in filament form are also known from other species of the order Nostocales. Strains of C. raciborskii can have almost identical 16S rRNA gene sequences, while their filament morphologies differ significantly; filaments may either be straight or coiled (Saker et al., 1999a). Thus, strains with very different morphology but with high genetic similarity have been assigned to one species, in this case to C. raciborskii. The different filament forms are referred to as different morphotypes.
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The present analyses show that Aph. aphanizomenoides, Ana. kisseleviana and Ana. oumiana are phylogenetically closely related and suggest that they are morphotypes of the same species. However, due to the absence of a current clear species definition in cyanobacteria, a re-classification is not attempted. Rather, future revisions of the cyanobacterial taxonomy should take the knowledge of the genetic relatedness of these types into consideration.
The two German Aph. aphanizomenoides strains were isolated from the small (0.35 km2), relatively shallow (max. depth 13 m) eutrophic lake Heiliger See near Potsdam, Germany in 2004 (Table I). Both strains are morphologically very similar (Table III). Pictures of strain 22F6.1 and of a German Aph. aphanizomenoides filament from a natural population are depicted in Fig. 4.
The Australian Aph. aphanizomenoides and the Ana. bergii of this cluster were isolated from five different water bodies in South Australia and New South Wales in 1992 (Table I). No pictures of the initial cultures are available. Morphologic observations of the current cultures have revealed that they have lost virtually all of their diagnostic characteristics. Based on the genetic markers used in this study, all of the Australian Aph. aphanizomenoides and the two Ana. bergii ANA283B and ANA283D are more closely related to the two German Aph. aphanizomenoides and the Japanese Ana. oumiana and Ana. kissleviana strains than to the remaining Ana. bergii strains.
Non-CYN producing Ana. bergii and CYN producing Aph. ovalisporum and Ana. bergii strains
Groups III and IV consistently clustered closely together (Figs 1–3). Group III was comprised of non-CYN producing Ana. bergii strains, whereas group IV consisted of CYN producing Aph. ovalisporum and Ana. bergii strains. The sequence similarities within each group were high. In group III, the 16S rRNA gene fragments were 99.6–100% similar; they were identical for all sequences of group IV.
Ana. bergii and Aph. ovalisporum are morphologically very similar (Komárek and Ettl, 1958). They have straight to slightly curved free-floating unbranched trichomes, sometimes hyaline apical cells, intercalary spherical to broadly oval heterocysts and elliptical to spherical akinetes, which are usually intercalary and distant from the heterocysts. Dimensions of vegetative cells, heterocysts and akinetes reported for different populations of Ana. bergii and Aph. ovalisporum vary and overlap (Table IV). However, vegetative cells were generally wider than long or maximally as long as wide and distinctly constricted at the cross walls in all populations designated as Ana. bergii. Further, two different kinds of apical cells have been observed in Ana. bergii trichomes; bluntly rounded and conically shaped, elongated cells (Table IV). Apart from the Florida isolate FAS-AP1, all strains and population identified as Aph. ovalisporum had vegetative cells that were at least as long as wide, but generally longer than wide. The cell lengths were often not well defined in and the apical cells were bluntly rounded (Table IV). Thus, Aph. ovalisporum and Ana. bergii may be morphologically separated based on the length–width ratio of the cells and the shape of the apical cells. Representative pictures of Ana. bergii and Aph. ovalisporum isolates and field samples are depicted in Fig. 4.
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The Australian Ana. bergii isolates ANA283A and ANA366B are the only Ana. bergii isolates that produced detectable amounts of CYN (Table I). Unfortunately, no pictures of the initial cultures are available to compare the original morphologies of these isolates to the Ana. bergii and Aph. ovalisporum strains and populations listed in Table IV. Genetically, ANA283A and ANA366B cluster distinct from the non-CYN producing Ana. bergii isolates and together with the CYN-producing isolates identified as Aph. ovalisporum. In fact, the cpcBA-IGS and the 16S rRNA gene sequences were identical, and the rpoC1 sequences highly similar. Based on this genetic information, we suggest that ANA283A and ANA366B have been misclassified. The same suggestion has previously been put forward for ANA283A, but no details of the analyses have been given (Rasmussen et al., 2007
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Our results reveal conflicts between the taxonomy and phylogeny of the isolates investigated, showing that the genetic relatedness and presumptive evolutionary relationship are not reflected in the systematic naming of these isolates. In particular, our results do not support the separation of Aph. gracile and Aph. flos-aquae into different species. Isolates assigned to either taxon were genetically highly similar at all loci investigated and, under culture conditions, not morphologically separable. Similarly, Aph. aphanizomenoides, Ana. kisselevina and Ana. oumina might be considered morphotypes of the same species. In contrast, our results suggest that Ana. bergii and Aph. ovalisporum are closely related but genetically and morphologically distinct taxa. Based on genetic analyses, the two isolates ANA283A and ANA366B initially identified as Ana. bergii, should be re-named as Aph. ovalisporum.
None of the Aph. aphanizomenoides isolates from Germany and Australia in group II, nor any of the Ana. bergii isolates from Germany, Australia and Senegal in group III produced CYN. Judging from this sample, it is unlikely that either of these taxa is a major CYN producer. In contrast, all of the Aph. ovalisporum strains isolated until now have been shown to produce the toxin, and thus, caution is warranted when this taxon appears in a water body.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data can be found online at http://plankt.oxfordjournals.org
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FUNDING |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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The study was funded by the Kompetenzzentrum Wasser Berlin with financial support of Veolia Water, and by a Marie Curie stipend to A.S.
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ACKNOWLEDGEMENTS |
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We thank Monika Degebrodt, for her expert assistance with culturing and sequencing, Karina Preußel, Aaron Kaplan and Cécile Bernard for generously providing the German Aphanizomenon strains, strain KA1.1 and strain PMC215.03, respectively. Further, we are very thankful to Samuel Cirés and David Carrasco for providing the UAM290 and 291 strains and making the DNA extractions and measurements of these strains, and to Martin Beck for isolating the German Ana. bergii strains. We thank Jutta Fastner for kindly undertaking the CYN analyses of the German and Senegalese strains, Bagmi Pattanaik for taking cell measurements of some of the German isolates, Peter Baker of the AWQC for use of his culture collection, photographs and expert assistance in morphological discussions and Helgard Täuscher for providing the Aph. aphanizomenoides picture of the German environmental sample. We are indebted to Paul Rasmussen for constructive discussions throughout the study and helpful comments on the manuscript.
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Notes |
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Corresponding editor: William Li
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAFUNDINGREFERENCES |
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