Abstract In this study, ten cyanobacterial strainsassigned to the oscillatorian species Phormidium autum-nale have been characterized using a polyphasic approachby comparing phenotypic and molecular characteristics.The phenotypic analysis dealt with cell and Wlament mor-phology, ultrastructure, and pigment content. The molecu-lar phylogenetic analyses were based on sequences of the16S rRNA gene and the adjacent intergenic transcribedspacer (ITS). The strains were quite homogenous in theirmorphologic features. Their thylakoids showed a stacked orfascicular pattern. Some, but not all strains contained phy-coerythrin. Only one strain (P. autumnale UTCC 476) devi-ated signiWcantly in its phenotype by lacking a calyptra. Inneighbour-joining and maximum Parsimony trees most 16SrRNA sequences were located on a single well-deWnedbranch, which, however, also harboured sequencesassigned to other cyanobacterial genera. Two strains (P.autumnale UTCC 476 and P. autumnale UTEX 1580) werefound on distant branches. The presence of phycoerythrinwas not correlated with the strains’ position in the phyloge-netic trees. Our results reconWrm that the morphospecies P.autumnale and the Phormidium group in general are notphylogenetically coherent and require revision. However,as indicated by sequence similarities most of the strainsassigned to P. autumnale except P. autumnale UTCC 476and P. autumnale UTEX 1580 are phylogenetically relatedand might belong to a single genus.
Cyanobacteria are among all known microorganisms themost widespread, diverse and abundant (Whitton 1992). Themetabolic property of oxygenic photosynthesis, which char-acterizes cyanobacteria and their sharing of ecologicalniches with eukaryotic algae prompted their treatment in thephycological circles, where they were often called blue–green algae, although their prokaryotic nature, akin to bacte-ria, has been recognized for over a century. High phenotypicdiversity of cyanobacteria is expressed by their morphologi-cal, biochemical and physiological properties, which enablethem to settle and persist in a wide range of habitats. Theirstructural complexity, unusual for prokaryotes, promptedtheir taxonomic distinction based on phenotypic, mostlymorphological properties, including colony formation, mor-phology of cells and extracellular envelopes, pigmentation,development and reproduction and, to a lesser extent, physi-ological and biochemical properties. Similarly as in all otherorganismal groups, various cyanobacterial properties arephenotypic expressions of particular genotypes.
The order Oscillatoriales, as recognized by Anagnostidisand Komárek (1988), comprises Wlamentous cyanobacterialacking true branching, heterocysts and akinetes. However,the heterogeneity of the majority of genera of the traditionalfamily Oscillatoriaceae was discussed repeatedly (Geitler1932; Rippka et al. 1979; Anagnostidis and Komárek 1988;Komárek and Anagnostidis 2005).
Traditionally, the presence of sheaths was used as a maindiacritical feature in the systematic classiWcation of oscilla-torialean genera (Frémy 1934; Geitler 1932; Desikachary
Communicated by Mary Allen.
K. A. Palinska (&) · J. MarquardtDepartment of Geomicrobiology, ICBM, Carl von Ossietzky University of Oldenburg, Carl-von-Ossietzky-Str., 9-11, 26111 Oldenburg, Germanye-mail: [email protected]
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1959; Bourrelly 1970; Komárek and Anagnostidis 2005).However, the ability of sheath formation is common inmany genera and proved to be depended on environmentaland cultural conditions (e.g. Palinska et al. 1996; Rippkaet al. 1979; Stal and Krumbein 1985). Thus sheath forma-tion should be only an additional character in connectionwith other features.
Phormidium autumnale (Agardhi) Trevisan ex Gomont1892 was chosen for this study because this taxon wasreported to be the most widespread cyanobacterial speciesall over the world (Geitler 1932). P. autumnale was evenfound to be the most common cyanobacterium in Antarc-tica where it lives in similar ecological niches as in otherregions (Komárek 1999). P. autumnale belongs to a groupof cyanobacteria growing commonly in brooks and rivers,sometimes also in other biotopes as lake shores, humidsoils, various springs, wet rocks, etc. Their morphologicalvariability depends on the conditions at diVerent localities.P. autumnale is characterized by more or less isodiametriccells, trichomes shortly or gradually narrowed towards theends, and the presence of a calyptra, a thickened cap on theouter cell wall of the apical cells. The taxon comprisesnumerous morphotypes and ecotypes, from which severalwere also described as individual species (Komárek 1972,1999; Kann and Komárek 1970). Komárek (1972)described several strains of P. autumnale, which diVer fromeach other in their morphological variation ranges; theycannot be classiWed as a single taxon.
Thus, this species is truly a frequent, ecologically amor-phous taxon, which in addition has an unusually broad mor-phological and physiological species description. Due tothe potentially high degree of environmentally inducedmorphological variability, high-resolution molecular tech-niques are invaluable for resolving questions pertaining tothe systematics and cosmopolitism of this organism andgenerally of all Phormidium-like strains (see Turner 1997;Wilmotte and Herdman 2001; Marquardt and Palinska2007). Therefore, the conWrmation of the taxonomic statusof Phormidium-like strains by molecular methods isrequired. The aim of this study was to prove if the “mor-phospecies” P. autumnale represents a genetically coherentgroup or is artiWcial with respect to phylogenetic relation-ships and may be referred to as form-species that ultimatelywill have to be redeWned on the basis of the morphologicaland genetic properties of type species.
Materials and methods
Cyanobacterial material and culture conditions
We investigated the following P. autumnale (Agardhi) Tre-visan ex Gomont 1892 strains: CCALA 143, CCALA 144,
CCALA 145, CCALA 697, and CCALA 757 from the Cul-ture Collection of Autotrophic Microorganisms (Trebon,Czech Republic), SAG 35.90 and SAG 78.79 from theSammlung von Algenkulturen Göttingen (Göttingen, Ger-many), CCAP 1462/6 from the Culture Collection of Algaeand Protozoa (DunstaVnage Marine Laboratory, Oban,UK), UTCC 579 from the University of Toronto CultureCollection (Toronto, Canada), and UTEX 1580 from theCulture Collection of Algae at the University of Texas(Austin, TX, USA). CCALA 145 and SAG 35.90 aredescended from the same isolate. The original habitats ofthe strains are listed in Table 1. All the strains are availablefrom the appropriate culture collection upon request. Thestrains were cultured in 50 ml Erlenmeyer Xasks at ambientlight and temperature in BG11 medium (Rippka et al.1979). The studied cultures were not axenic.
Pigment extraction and spectroscopy
Cyanobacterial cultures were centrifuged for 5 min at12,000g in a microcentrifuge and resuspended in 50 mMTris/HCl (pH 8.0) with 250 mM NaCl and 10 mM EDTA.Cells were disrupted by sonication for 5–10 min on ice witha Branson SoniWer Cell Disruptor B15. Triton X-100 wasadded to a Wnal concentration of 0.5% and the samples wereincubated at 28°C for 30 min. After centrifugation at12,000g for 5 min, absorbance spectra of the supernatantwere recorded with a Hitachi U-3000 spectrophotometerfrom 400 to 750 nm.
Light microscopy, scanning electron microscopy and ultrastructural studies
Light and scanning electron microscopy (SEM) were usedto study cell sizes, forms, the degree of constriction at celljunctions, and the presence of a calyptra. Light microscopystudies were performed with a Zeiss Axioskop 50 micro-scope equipped with transmitted light, phase contrast, andNomarski interference contrast illumination. Photomicro-graphs were taken using a Nikon Digital Camera DXM1200. For scanning electron microscopy (SEM), sampleswere Wxed in 4% glutaraldehyde in 0.1 M sodium phos-phate buVer (pH 7.2), and dehydrated through a series ofethanol–water solutions. The samples were then critical-point dried in a Balzers Union (CPD 010) apparatus beforegold sputtering (SCD 030, Balzers Union). The sampleswere examined with a ZEISS DSM 940 or a Hitachi S-450scanning electron microscope operated at 10 or 20 kV andwith working distances of 7–9 mm.
For ultrastructural studies by transmission electronmicroscopy (TEM), samples were prepared and embeddedaccording to the procedures described by Surosz and Palinska(2004). The approximately 1-week-old cultures were
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washed twice in 0.1 M sodium phosphate buVer (PB),pH 7.5. The Wxation was performed with 3% glutaralde-hyde in PB, the cells were postWxed with 2% osmiumtetroxide in PB. After dehydration through a series ofethanol–water solutions starting with 10% of ethanol andended with two washes in pure ethanol, cells were embed-ded in Epon (Spurr 1969). Sections, cut with diamondknives, were post-stained with uranyl salts and examinedwith a Zeiss EM 109 or EM 902A transmission electronmicroscope.
Molecular techniques and phylogenetic analyses
Genomic DNA was isolated as previously described (Mar-quardt and Palinska 2007). The intergenic transcribedspacer (ITS) between the 16S and 23S rRNA genes wasampliWed with primers 322 and 340 (Iteman et al. 2000).Partial 16S rRNA genes were ampliWed with the primersPLG1.1 and PLG2.1 described by Nadeau et al. (2001),except for UTCC 579 where the primer PLG2.1 did notbind. Here, a larger DNA fragment was ampliWed withprimers PLG1.1 and 340. PCR conditions were asdescribed in Marquardt and Palinska (2007). The sampleswere directly sequenced in both directions by a commercialsequencing laboratory. The primers were the same as forthe ampliWcation combined with F primer Phor-580 and Rprimer Phor-710 (Marquardt and Palinska 2007) for 16SrRNA and primers 323 (Iteman et al. 2000), ITS 300 andtRNA-F (Marquardt and Palinska 2007) for ITS fragments.The GenBank accession numbers of sequences determinedin this study are given in Table 1. Sequence similaritieswere calculated online with ClustalW (Higgins et al. 1994)at http://www.ebi.ac.uk/clustalw/index.html. Phylogeneticanalysis of the 16S rRNA gene was performed with thehelp of the software ARB (Ludwig et al. 2004, availableonline at http://www.arb-home.de) using the “ssu_jan04_corr_opt.arb” database. A fragment of approximately925 nt (corresponding to E. coli K12 16S rRNA residues269–1,215 and to residues 1,003,153–1,004,077 of thegenome of Anabaena variabilis ATCC 29413, GenBankacc. no. NC007413) was aligned to the 16S rRNA align-ment of the ARB database with the integrated aligner of thesoftware using other cyanobacteria as reference organisms.The alignment was checked by eye and corrected manually.The aligned sequences were included into the cyanobacte-rial sub-tree (containing 1,315 sequences) of the Parsimonytree “tree_1000_jan05” of the ARB database using thecyanobacterial Wlter with the “Parsimony (Quick AddMarked)” function of the software. Additionally, oursequences and selected GenBank entries were alignedonline with clustalW at default conWguration and the 925 ntfragment given above was used for the calculation of aNeighbor-Joining tree (Jukes and Cantor distance estimation,T
all sequence position considered, but insertions and dele-tions not taken into account, 2,000 bootstrap replicates)with the software TREECON for Windows 1.3b (Van dePeer and De Wachter 1994). E. coli was used as outgroupand positioned with the “Single sequence (forced)” method.
Results
Pigment composition
We studied ten cyanobacterial strains assigned to the spe-cies P. autumnale. Data for an 11th P. autumnale strain,CCAP 1462/10, were published earlier in Marquardt andPalinska (2007). All strains were bluish-green. Absor-bance spectra showed Chl a and phycocyanin as majorpigments. Additional phycoerythrin was found in Wvestrains and it was also present in strain CCAP 1462/10(Table 1). Phycoerythrin was not detectable in strainsCCALA 143, CCALA 697, CCALA 757, UTCC 579, andUTEX 1580.
Morphology and ultrastructure
The morphology of the strains was analyzed by lightmicroscopy and SEM (Figs. 1, 2). Results of these investi-gations are compiled in Table 1. The average Wlamentdiameters ranged from 3.4 to 6.3 nm. Single cells of theWlaments were short (length/diameter <1) to disc-shaped.The cell length within a single strain was quite variable,e.g. between 1.7 and 4.4 nm in UTCC 579. Short, disc-shaped cells could be found especially close to the Wlamenttip whereas longer cells usually were observed in the mid-dle region of the Wlament. Constrictions between the cellswere missing in most cases. Only UTEX 1580 and CCALA145 showed weak constrictions that were not visible in allWlaments (Fig. 2c, j).
In our TEM investigation we focused on the orientationof thylakoid membranes, which are the most conspicuousstructures within the cells. The results are compiled inTable 1. Peripheral thylakoids running parallel to the cyto-plasmic membrane as well as radially arranged thylakoidswere never found. In most cases, the thylakoids were
Fig. 1 Photomicrographs of ten P. autumnale strains examined: CCALA 143 (a), CCALA 144 (b), CCALA 145 (c), CCALA 697 (d), CCALA 757 (e), SAG 35.90 (f), SAG 78.79 (g), CCAP 1462/6 (h), UTCC 579 (i), and UTEX 1580 (j). The arrows indicate the calyptra. All strains are shown in the same magniW-cation. Bar 20 �m
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organized in stacks lying irregularly within the cells (Fig. 3a).Some strains (CCALA 143, CCALA 757 and UTEX 1580)rather showed a fascicular thylakoid pattern (sensu Casam-atta et al. 2005) where the thylakoids are arranged in fasci-cles running parallel to the longitudinal axis of the cell(Fig. 3b). However, also in these strains occasionally thyla-koid stacks running in a diVerent direction were found,
while some cells of strains with irregular oriented thylakoidstacks showed a rather fascicular pattern (Fig. 3c).
All strains but UTCC 579 had a calyptra (Fig. 3d, arrowsin Figs. 1, 2), which tends to be stripped oV during prepara-tion for SEM (cf. Fig. 2e, f) and leaves a ring-shaped inci-sion around the apical cell (Fig. 2c, e, f, g). In UTCC 579,Wlament tips were rather pointed and bent (Fig. 2i).
Fig. 2 Scanning electron micrographs of ten P. autumnale strains examined: CCALA 143 (a), CCALA 144 (b), CCALA 145 (c), CCALA 697 (d), CCALA 757 (e), SAG 35.90 (f), SAG 78.79 (g), CCAP 1462/6 (h), UTCC 579 (i), and UTEX 1580 (j). The arrows indicate the calyptra which is partially ripped oV in (e) and (f). Bars 5 �m
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Molecular phylogeny
We analysed a part of the of the 16S rRNA gene (corre-sponding to E. coli K12 16S rRNA residues 269–1,215and to residues 1,003,153–1,004,077 of the genome ofA. variabilis ATCC 29413, GenBank acc. no. NC007413) and the entire ITS region of the P. autumnalestrains. Data for CCAP 1462/10 were published earlier inMarquardt and Palinska (2007). All 16S rRNA sequenceswere 925 nt long, except that of UTEX 1580 (924 nt) andthose of CCALA 143 and CCALA 757 (937 nt). The ITSvaried in length between 452 and 582 nt (Table 1). Mostof them were in the range of 556–582 nt, only those ofUTCC 579 and UTEX 1580 were signiWcantly smaller,509 and 452 nt, respectively. All of them harboredtRNAIle and tRNAAla genes, except the one in UTEX1580 which contained only the tRNAIle gene. The strainsCCALA 144, CCALA 145, SAG 35.90, SAG 78.79 andCCAP 1462/6 showed identical 16S rRNA sequences(Table 2). In the case of CCALA 144, CCALA 145 andSAG 35.90 the ITS sequences were also identical oralmost identical.
All 16S rRNA sequences shared more than 95% pair-wise similarity except those of UTCC 579 and UTEX 1580whose degrees of similarity with other sequences were inthe range of 91.0–93.0%. Sequence homologies from 95.0to 97.5% were found between CCALA 757 and CCALA143 (99.4% pairwise similarity) and the remainingsequences. All other sequences shared more than 97.5%pairwise similarity. In the case of ITS, all sequencesshowed more than 80% homology except UTCC 579 andUTEX 1580 where the value ranged from 47.8 to 58.6%.
Using the ARB software, we added our 16S rRNAsequences to a cyanobacterial Parsimony tree (containing1,345 entries) constructed from the cyanobacterial subtreeof “tree_1000_jan05” of the ARB database, which was sup-plemented with 30 additional cyanobacterial sequences(Marquardt and Palinska 2007). Due to its large size, theentire phylogenetic tree cannot be shown here. A partialtree with selected strains is given in Fig. 4. The 16S rRNAsequence data for these strains were also used to calculate aNeighbor-Joining tree with the TREECON software(Fig. 5). The absolute topology of the inferred trees diVersslightly, especially in the partitions deWned by the deeper
Fig. 3 Transmission electron micrographs of some of the examined P. autumnale strains in longitudinal sections. Strain CCALA 145 (a) harbours stacks of thylakoids arranged irregu-larly within the cells, strain UTEX 1580 (b) shows a fascicu-lar pattern with all thylakoids running parallel to the longitudi-nal axis of the Wlament. In the Wl-ament of strain SAG 78.79 in c, thylakoids are arranged in a fas-cicular manner in the two cells to the left, but irregularly stacked in the cell to the right. The cell in between shows a transitional pattern. d Longitudinal section through the apical cell of strain SAG 78.79 with a prominent calyptra
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branches where the bootstrap values in the Neighbor-Join-ing tree are small. However, the two trees are very similarregarding the position of the P. autumnale strains. Allstrains but UTCC 579 and UTEX 1580 cluster on a singlebranch supported by 99% bootstrap value. P. autumnaleUTEX 1580 clusters together with P. tergestinum CCALA155. These two organisms showed a sequence homology of99.8%. UTCC 579 stands somewhat isolated. In the Parsi-mony tree, one of the closest neighbours of UTCC 579 is P.animale CCALA 140. These strains share a pairwisesequence homology of 92.2%. However, the other investi-gated strains also do not form an exclusive P. autumnalecluster, but are intermixed with other cyanobacteria asMicrocoleus vaginatus or Tychonema bourellyi.
Discussion
In a previous study (Marquardt and Palinska 2007), weinvestigated 30 cyanobacterial strains assigned to the Pho-rmidium group. We found a strong diversity of morpholog-ical and ultrastructural traits as cell size, cell shape, andthylakoid arrangement combined with large phylogeneticdistances. The organisms examined in the present study, alltraditionally assigned to the species P. autumnale, showeda relative homogenous morphology. The average Wlamentwidth varies between 3.4 and 6.3 nm, but these variationsare small compared to 0.7–6.3 nm, as found in the previousstudy for the whole spectrum of Phormidium-like cyano-bacteria. Furthermore, cell size may vary not only betweenstrains, but also within the same strain. For strains CCALA145 and SAG 35.90 deriving from the same isolate andwith identical genotype our measurements varied by0.6 nm. The diVerences might be due to varying availabilityof nutrients or light (Cermeno et al. 2006; Komárek 1972;Prézelin et al. 1987). Cell length and consequently cellshape showed some variations as well, but in no case cylin-drical cells (cell length/diameter >1) typical for some otherstrains assigned to the genus Phormidium were found.
In some strains, occasionally weak constrictions at celljunctions were found which were missing in other strains.These diVerences, however, might be explained by diVerentcell turgor. Also the thylakoid arrangement showed a ratherhomogenous pattern. In no case were peripheral thylakoidsfound which are typical for the majority of cyanobacteria.Thylakoids were either arranged in stacks lying irregularlyin the cells or in a fascicular manner running parallel to thelong axis of the Wlament. But these two types cannot alwaysbe clearly distinguished because there are transitionsbetween them and both can be found in diVerent cells of thesame Wlament. All strains but UTCC 579 contained a calyp-tra, and only by this characteristic could this strain be mor-phologically distinguished from the others.T
able
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C
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A
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8722
CC
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A
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8.79
A
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LA
697
A
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CC
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A
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158
0 A
M77
8713
CC
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43 A
M77
8706
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.184
.4
84.5
84.5
87.4
87.2
81.8
82.7
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57.7
CC
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57 A
M77
8705
99.4
–
82.1
82.1
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86.4
86.1
80.2
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.792
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.556
.0
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.284
.258
.356
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.499
.499
.499
.499
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.9
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92.9
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93.0
92.0
–
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332 Arch Microbiol (2008) 189:325–335
The morphological homogeneity of studied P. autum-nale strains is reXected in the phylogenetic trees. Most ofthe strains examined cluster on a single branch together
with Microcoleus species and Tychonema bourellyi. Aclose relationship to these species was also observed byComte et al. (2007) and Willame et al. (2006) for other
Fig. 4 Maximum Parsimony tree based on a partial 16S rRNA genesequences (approximately 925 bp) as inferred with the ARB software(Ludwig et al. 2004). GenBank accession numbers are in square
brackets. The strains examined in this study are marked with asterisks.Their accession numbers can be found in Table 1. Bar: substitutionsper site
Fig. 5 Neighbor-joining tree based on partial 16S rRNA gene se-quence (925 bp) as inferred with the TREECON software (Van de Peerand De Wachter 1994). GenBank accession numbers are in squarebrackets. The strains examined in this study are marked with asterisks.
Their accession numbers can be found in Table 1. The numbers at thenodes show bootstrap support as percentages based on 2,000 resam-pled data matrices. Only bootstrap values greater than 60% are given.Bar: substitutions per site
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Arch Microbiol (2008) 189:325–335 333
cyanobacterial strains assigned to P. autumnale. Also mor-phogically, these species are very similar. According toKomárek and Anagnostidis (2005), cells of T. bourellyi andP. autumnale are isodiametric and 4–6.3 �m wide. The api-cal cells are rounded and often possess a calyptra (Lund1955). Both species are reported in the plankton of lakes inEurope. There are also very strong morphological similari-ties between P.autumnale and Microcoleus vaginatus. Thetrichome morphology in both species is almost identical(Komarek and Anagnostidis 2005; Boyer et al. 2002;Garcia-Pichel et al. 2001). As in P. autumnale the trichomesare 3–7 �m wide, not constricted at the cross walls, andusually attenuated at the ends. Apical cells are capitatedand rounded with a calyptra. M. vaginatus was reported inmarine, brackish, freshwater and terrestrial environments.However, the typical M. vaginatus is probably only terres-trial and occurs in desert soils as reported for M. vaginatusPCC9802. However, the ultrastructure of the species shouldbe diVerent. Komárek and Anagnostidis (2005) reportedwidened thylakoids for T. bourellyi in contrast to a radialarrangement of thylakoids in P. autumnale. To our knowl-edge there is no report about the thylakoid arrangement inMicrocoleus vaginatus. We did not notice any vacuoliza-tion and keritomization of the chromoplasm in any of ourstrains, but we neither found a radial thylakoid arrange-ment. According to this feature, none of our strains actuallybelongs to the genus Phormidium as deWned by Anagnosti-dis and Komárek (1988). In our recent study with cyano-bacteria assigned to the genus Phormidium only one strain,P. animale CCALA 140, showed a radial thylakoidarrangement (Marquardt and Palinska 2007). However, aradial pattern is just a special case of a fascicular arrange-ment, and as stated above, the thylakoid pattern may varyeven within a single Wlament. Thus phenotypic criteriaseem to be too uncertain and overlapping for a clear distinc-tion between P. autumnale, T. bourellyi and Microcoleusspecies.
The strains UTEX 1580 and UTCC 579 show a greatphylogenetic distance from all other strains examined. In thecase of UTCC 579, this is accompanied with the morpholog-ical diVerence mentioned above and thus this strain is proba-bly misclassiWed. Similarly, Pseudanabaena tremula UTCC471 has until recently been classiWed as P. autumnale (and isstill found under this name in the UTCC catalog). The incor-rect classiWcation of the strain was noticed by Casamattaet al. (2005) who renamed it based on morphological char-acteristics. In our trees Pseudanabaena tremula UTCC 471is found on a branch distant from the P. autumnale strainsexamined here. The distant position of UTEX 1580 is inconcordance with the results of Comte et al. (2007). Also intheir Neighbour-Joining tree the strains Arct-Ph5 and Ant-Ph86 cluster with Microcoleus antarcticus UTCC 474 andT. bourellyi, while UTEX 1580 is found on a diVerent
branch. Arct-Ph5 and CCALA 697 are probably identicalsince the statements of their origin in the CCALA catalogand in Comte et al. (2007) are matching and they share iden-tical 16S rRNA sequences. Ant-Ph86 shows highest similar-ity with CCAP 1462/10. UTEX 1580 is phylogeneticallydistant from the majority of the P. autumnale strains. SinceUTEX 1580 was the Wrst cyanobacterium assigned to thisspecies whose 16S rRNA sequence was deposited in theGenBank database, it is found in many phylogenetic trees asa representative of P. autumnale (e.g. in Casamatta et al.2005; Comte et al. 2007; Warren-Rhodes et al. 2006; Fioreet al. 2005). Furthermore, UTEX 1580 is a strain often usedin physiological studies (e.g. Nobles et al. 2001; Gleasonand Paulson 1984). We strongly discourage to use strainsUTCC 579 and UTEX 1580 in laboratory studies as typicalrepresentatives of P. autumnale.
Although most of the examined strains are phylogeneti-cally closely related they might belong to diVerent species.16S rRNA sequence homologies of more than 97.5%, avalue often used as threshold for bacterial species deWnition(Palinska et al. 1996; Rajaniemi et al. 2005), was found forstrains CCALA 757 and CCALA 143 as well as strainsCCALA 144, CCALA 145, SAG 35.90, SAG 78.79, CCAP1462/6, CCALA 697 and CCAP 1462/10. The degree ofsimilarity between these two groups is above 95%, whichhas been suggested as a minimum 16S rRNA sequencehomology for congeneric bacterial species (Stackebrandtand Goebel 1994). Thus, according to these criteria, theymight represent two species of one genus. However, a clas-siWcation based solely on phylogenetic trees inferred frommolecular sequence data can be misleading as well. Foxet al. (1992) found that Bacillus strains with more than 99%16S rRNA similarity had less than 70% DNA–DNAhybridization, the most commonly accepted benchmarkused to distinguish between bacterial species (Stackebrandtand Goebel 1994). Therefore, the strains may even belongto more than two diVerent species. Most probably the three(two of the four strains are of identical origin) strains withidentical 16S rRNA sequences should be placed into a sin-gle species since they also have very similar ITS sequences.CCALA 143 and CCALA 757 might be more closelyrelated to M. vaginatus with which they share more than99% 16S rRNA sequence homology. The homologybetween UTCC 579 and UTEX 1580 and the other exam-ined strains is signiWcantly below 95%, and consequentlythey should be placed into diVerent genera.
The clustering of the strains showed no correlation withtheir geographic origin or the presence of phycoerythrin, asit was already observed for other members of the Phormi-dium group (Marquardt and Palinska 2007). In the presentstudy, strains possessing phycoerythrin were not closelyrelated and appeared in diVerent clusters, which also con-tained strains without phycoerythin. Hence, the presence of
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phycoerythrin appears unsuitable as criterion for a system-atic revision. However, it should be noted that strains whichseem to be very closely related as CCALA 757 andCCALA 143 on the one hand, and CCALA 144, CCALA145/SAG 35.90, SAG 78.79, and CCAP 1462/6 on theother hand show the same phenotype regarding the pres-ence of phycoerythrin. Our Wnding of a close phylogeneticrelationship of strains from distant regions (e.g. CCALA143 from Slovakia and CCALA 757 from China) is inagreement with the cosmopolitan distribution of manycyanobacterial species (e.g. Mullins et al. 1995; Wilmotteet al. 1997; Garcia-Pichel et al. 1996). However, it is strik-ing that among the strains with identical 16S rRNA thosewith identical ITS sequences (CCALA 145/SAG 35.90 andCCALA 144) came from Switzerland, while CCAPA 1462/6 with a slightly diVerent ITS sequence was isolated inFrance. This might be coincidental, but it might also reXectthe expansion of this 16S rRNA genotype.
Our study points again to the necessity of a revision notonly of the species P. autumnale but also of the genus Pho-rmidium as a whole. After such a revision, the strains exam-ined here should be placed into diVerent, perhaps novelgenera. Since T. bourellyi CCAP 1459/11b was proposed asa type strain under the rules of the Bacteriological Code bySuda et al. (2002), all P. autumnale strains found on thesame branch of the phylogenetic trees might be transferredto the genus Tychonema. However, since the taxon Tycho-nema was introduced quite recently by Anagnostidis andKomárek (1988), older genus names might have priorityaccording to the Botanical Code. Which of the strains cur-rently assigned to P. autumnale should keep the speciesepithet “autumnale” cannot be decided, since there is nomolecular study on the botanical type specimen of P.autumnale currently available. Molecular data gained fromthe P. autumnale exsiccatum originally described by Agar-dhi and deposited in a herbarium would help to Wnd theclosest relatives among the recent strains. This approachwas recently done for some other cyanobacteria (Palinskaet al. 2006).
Acknowledgments This work was supported by DFG, grant PA842/1–3. Authors wish to thank Sophie Martyna and Heike Oeltjen forthe help by the scanning and transmission electron microscopy.
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