“Oscillatoria Simplicissima” Isolated from the Vaal River, South Africa, As Planktothrix Pseudagardhii ☆ ⁎ K.R

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South African Journal of Botany 74 (2008) 101–110 www.elsevier.com/locate/sajb

Re-identification of “Oscillatoria simplicissima” isolated from the Vaal River, South Africa, as Planktothrix pseudagardhii ☆ ⁎ K.R. Conradie, S. Du Plessis , A. Venter

School of Environmental Sciences and Development, Department of Botany, North-West University (Potchefstroom Campus), Potchefstroom 2520, South Africa Received 25 October 2006; received in revised form 24 August 2007; accepted 7 September 2007

Abstract

Nearly complete 16S ribosomal RNA sequences from a strain isolated in the Vaal River, South Africa, commonly identified as “Oscillatoria simplicissima sensu PIETERSE & STEYNBERG 1993”, as well as Planktothrix pseudagardhii NIVA CYA 153 were determined. Phylogenetic analysis of these species, together with P. pseudagardhii NIES 845T, Planktothrix agardhii NIES 204T and other closely related strains, showed that this South African strain of “O. simplicissima” closely clusters with P. pseudagardhii NIVA CYA 153, P. pseudagardhii NIES 845T and a group of P. pseudagardhii available on the database. A polyphasic approach was used to re-identify this isolate of “O. simplicissima”.A comparative morphological study between it and P. pseudagardhii NIVA CYA 153 revealed no clearly defined structural differences. Morphological studies indicated that cells of the “O. simplicissima” isolate have clear gas vesicles, which further suggests that it must be renamed to “Planktothrix pseudagardhii SUDA et M.M. WATANABE in SUDA et al. [Suda, S., Watanabe, M.M., Otsuka, S., Mahakahant, A., Yongmanitchai, W., Nopartnaraporn, N., Liu, Y., Day, J.G., 2002. Taxonomic revision of water-bloom-forming species of oscillatoroid cyanobacteria. International Journal of Systematic and Evolutionary Microbiology 52, 1577–1595.]”. © 2007 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Cyanobacteria; Morphology; Oscillatoria sp.; Phylogeny; Planktothrix sp.; 16S rRNA

1. Introduction In South Africa there is a real need to specifically identify bloom formers and to develop an early warning system for In 1993, Pieterse and Steynberg reported the occurrence of bloom formation. Cyanobacteria represent only a small “Oscillatoria simplicissima” Gomont for the first time in South proportion of all algal groups present in the Vaal River, but Africa. Since then, it has been encountered more frequently and they are probably one of the most problematic because of their has been described as a major bloom former by Janse Van toxin-producing, filter-clogging, scum-forming, unaesthetic Vuuren (2001) and Kruskopf (2002) in the Midvaal region. properties (Venter et al., 2003). The taxonomy of the Venter et al. (2003) completed a detailed taxonomic study on Cyanophyceae has often been revised since the last century as this organism. As a bloom former in the Vaal River, it is of great a consequence of their relative morphological simplicity and, importance to all involved in water purification and water thus, restricted set of useful characters for classification quality, as it is known to have the potential to be able to secrete (Wilmotte et al., 1992). Furthermore, the stability of phenotypic toxins and produce an unpleasant odour and taste in raw and characters and their use in taxonomy have been questioned by a treated water, resulting in a general decline of the water quality number of authors (reviewed by Anagnostidis and Komárek, (Tanskanen, 1990; Leppänen et al., 1995). 1988; Suda et al., 2002). The morphology of cyanobacteria in laboratory cultures is often considerably altered from the original morphology of environmental isolates (Neilan, 1995) and morphological and ☆ The EMBL accession number for the partial 16S rRNA gene sequence of Planktothrix pseudagardhii is AM236076. cytological characters do not necessarily reflect evolution (Rudi ⁎ Corresponding author. Tel.: +27 18 2992508. et al., 1997). Researchers' inability to grow certain organisms in E-mail address: [email protected] (S. Du Plessis). the laboratory (Ferris et al., 1996), and misidentifications of

0254-6299/$ - see front matter © 2007 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2007.09.003 102 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110 strains in culture collections (Garcia-Pichel et al., 1996) make it trated by filtration of 30 ml culture onto glass fiber filters, which difficult in many cases to apply taxonomic assignments based were then washed three times with 10 ml of a solution on cultures to field populations (Nübel et al., 1997). Molecular containing 50 mM Tris–HCL, pH 8.0, 5 mM EDTA and 50 mM structures and sequences are generally more revealing of NaCl to reduce extracellular polysaccharides. A modified evolutionary relationships than are classical phenotypes (par- method of Fiore et al. (2000) was followed for the purification ticularly so among micro-organisms) (Woese et al., 1990). of DNA. Consequently, the basis for the definition of taxa has progres- The DNA was resuspended in 500 μl of TE buffer (10 mM sively shifted from the organismal to the cellular to the molec- Tris–HCL, pH 8.0, 1 mM EDTA). A final concentration of ular level. According to Komarek (2005), the Bacteriological 750 mM ammonium acetate was added to the DNA solution and Botanical nomenclatoric codes are not quite satisfactory for followed by a chloroform extraction (1:1). After centrifugation cyanobacteria and the result is that only few cyanobacterial taxa the DNA in the supernatant was precipitated overnight and can be considered as “validly described” (Oren, 2004; Oren and washed with ethanol. The DNA was dried and diluted in TE Tindall, 2005). buffer (10 mM Tris–HCL, pH 8.0, 1 mM EDTA). The purity of For classifying cyanobacteria, a phylogenetic system based the DNA was assessed by A260/A280 ratio measurement using on the 16S rRNA gene (rDNA) sequence information retrieved a spectrophotometer (Spectronic, Genesys 2) and the DNA from organisms in pure cultures has been developed (Giovan- concentration was determined. noni et al., 1988). The small subunit rRNA (16S rRNA) represents the best-studied sequence (Giovannoni et al., 1988; 2.3. PCR amplification Nelissen et al., 1994, 1995, 1996; Schmidt et al., 1991). Sequences of 16S rRNA genes are independent of cultivation or The primers were synthesized commercially (Roche) growth conditions and can be retrieved by PCR from small (Table 1). PCR amplification of a cyanobacterial 16S rRNA amounts of DNA extracted from laboratory cultures or natural gene plus the ITS was performed in a 50 μl (total volume) environments (Giovannoni et al., 1990). reaction mixture containing 0.5 μl of DNA, 1×Super Taq Plus The aim of this study was to identify the harmful bloom- PCR buffer (Southern Cross Biotechnology), each deoxynu- forming cyanobacterium isolated from the Vaal River, wrongly cleotide triphosphate at a concentration of 0.2 mM, 0.5 μM identified as “O. simplicissima”, on a molecular and morpho- primer 16S27F, 0.5 μM primer 23S30R and 1 mg of bovine logical basis. serum albumin. Amplification was carried out with a PCR Thermal cycler 2. Materials and methods (PCR Express, Hybaid) as follows: To minimise non-specific annealing of the primers to non-target DNA, 1 U of SuperTaq 2.1. Strain and strain cultivation DNA polymerase (Southern Cross Biotechnology) was added to the reaction mixture after the initial denaturation step (5 min at Four strains of cyanobacteria, isolated in South Africa, were 94 °C), at 80 °C. Ten cycles of 45 s at 94 °C, 45 s at 57 °C used to compile a phylogeny based on nearly complete 16S followed, and 2 min at 68 °C; 25 cycles of 45 s at 92 °C, 45 s at rDNA sequences: the presumed Oscillatoria simplicissima (“O. 54 °C, and 2 min at 68 °C; and a final elongation step of 7 min at simplicissima”), two different cultures of Spirulina sp. Turpin 68 °C. (1829) and Gomont (1892) and Arthrospira sp. Stizenberger PCR products were run on a 1.5% agarose gel, the PCR (1852) and Gomont (1892). These organisms were chosen fragments cut out of the gel, and purified with an agarose gel because of their regional abundance in the Vaal River and their DNA extraction kit (Boehringer Mannheim) and subsequently potential to produce toxins. Two additional strains were used as used for sequencing. references, one of Planktothrix pseudagardhii (Suda et al., 2002), NIVA CYA 153 from the Norwegian culture collection 2.4. Sequencing and the other of Planktothrix sp. 126/8, provided by K. Sivonen (University of Helsinki, Helsinki, Finland). Sequencing of “O. simplicissima”, P. pseudagardhii NIVA Cultures of “O. simplicissima” and of P. pseudagardhii were CYA 153 and Planktothrix sp. 126/8 was done automatically on established in glass flasks containing 100 ml of EM medium a DNA sequencer, using cyclic sequencing by University of (Venter, 2000), and strains of Arthrospira and Spirulina were Cape Town (UCT). The primers used for sequencing of the 16S cultivated in glass flasks containing 100 ml of Zarrouk medium rRNA gene are shown in Table 2. (Fox, 1996) at a constant-temperature (24 °C) and light (20 μmol m− 2 s− 1). Cultures were routinely checked for purity by microscopic examination. Table 1 The primers used for amplification of the cyanobacterial 16S rRNA plus ITS 2.2. DNA purification Primer Sequence (5′ to 3′) Target site Reference 16S27F AGA GTT TGA TCC 7–27 Wilmotte et al. (1993) The sample preparation did not discriminate between the TGG CTC AG different types of fresh material from the different species 23S30R CTT CGC CTC TGT 30–52 Taton et al. (2003) and tested. Two weeks after inoculation, algal cells were concen- GTG CCT AGG T references therein K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110 103

2.5. Alignment and phylogenetic analysis For light microscopic studies, eight-day old living cultures of “O. simplicissima” and P. pseudagardhii were filtered to The 16S rDNA sequences were aligned and analysed using increase cell density and studied under the light microscope. the multiple sequence alignment program PhredPhrap (linux) For the confocal microscope studies, samples of eight-day (http://www.phrap.org/phredphrapconsed.html). Publicly avail- old cultures were fixed in 2% formaldehyde for 24 h. The able sequences deposited on the NCBI database (http://www. samples were stained with “Nile Red” (for phospholipids) and ncbi.nlm.nih.gov/blast/Blast.cgi) with similarities greater than studied and digital images captured using a PCM 2000 confocal 96% (closely related strains) to “O. simplicissima” were also microscope connected to a Nikon (TE300) microscope, with a aligned, giving a total of fifty strains. The strains used in the 60×/1.40 Apo Planar oil objective. The system used the present study are listed in Table 3. The computer-generated helium–neon ion laser to excite the “Nile Red” colourant at alignments were refined manually. The sequences were then 505 nm. Emission waves were gained at 565 nm. exported to BioEdit (Windows) (http://www.mbio.ncsu.edu) and gaps removed as suggested by Suda et al. (2002) to obtain a 3. Results and discussion set containing 1269 positions that was used for phylogenetic analysis of the 50 cyanobacterial strains. The cyanobacterium The filamentous cyanobacteria that first appeared in the Vaal Gloeobacter violaceus (PCC 7421) (AF132790) was used as River during the early nineties was originally identified by outgroup to root the tree. Synechococcus (single cell) and Pieterse and Steynberg (1993) and Venter et al. (2003) as O. Lyngbya (filamentous) were used to test the monophyly of the simplicissima under the order Nostocales, family Oscillator- ingroup and to give better resolution between the groups in the iaceae, according to the criteria of Desikachary (1959). tree. A cladogram (Fig. 1) based on the 16S rDNA sequences All phylogenetic analyses were done using NONA via revealed two distinct groups, the filamentous and the single WINCLADA version 1.00.08. (Nixon, 2002). Cladograms were celled cyanobacteria. The filamentous group is divided into four generated with equally weighted heuristic searches using tree distinct clades, namely, a cluster of the family Pseudanabaena- bisection–reconnection (TBR) branch-swapping algorithm and ceae, a cluster with Oscillatoria princeps, a cluster with Ar- a random addition sequence with 1000 replicates (steepest throspira/Lyngbya and a cluster with Planktothrix species. The descent option in effect). Uninformative characters were last cluster is clearly divided into two subclusters, P. deactivated, and the consensus was strict. Reliability of internal pseudagardhii and P. agardhii/rubescens. nodes of trees was ascertained by 1000 bootstrap replications. In the first cluster, the strains labeled as Oscillatoria The influence of gap characters on phylogenetic inference (AJ133106) and O. neglecta (AB003168) according to the was investigated by conducting an additional parsimony BLAST database do no longer belong to the genus “Oscilla- analysis with gap information included as coded characters toria”. O. neglecta is classified according to the modern system (http://www.home.duq.edu/~youngnd/GapCoder). The boot- to the genus Jaaginema, which is close to Leptolyngbya strap values for this tree were lower and we maintained the (Anagnostidis and Komárek, 1988). All strains from this cluster data matrix without gaps. belong to the family Pseudanabaenaceae, not Oscillatoriaceae. “Spirulina platensis” belongs to the genus Arthrospira, not 2.6. Microscopic study Spirulina (Ballot et al., 2002). The strains named “Spirulina BFN” (Fig. 1), which are included into the cluster together with Strains of both “O. simplicissima” and P. pseudagardhii, Arthrospira are clearly Arthrospira, both of these genera are NIVA CYA 153 cultures were grown at three different quite different according to molecular and cytological criteria temperatures (18 °C, 25 °C and 30 °C) each at three different (Nelissen et al., 1994). light intensities (5, 10 and 20 μmol m− 2 s− 1). Six replicates of In the last cluster, molecular sequencing indicates that all the each treatment were grown for each strain. strains belong to the genus Planktothrix, the species of which are only slightly distinguishable according to phenotype but are Table 2 clearly divided into two clusters namely the pseudagardhii and The primers used for sequencing the 16S rRNA genomic fragment the agardhii/rubescens clusters. Similar results were found by Primer Sequence (5′ to 3′) Target site Reference Humbert and Le Berre (2001). These authors found no polymorphism between P. agardhii/rubescens using partial 16S27F AGA GTT TGA TCC 7–27 Wilmotte et al. (1993) TGG CTC AG 16S rDNA sequences. Rudi et al. (1998) suggested that CYA781R(a) GAC TAC TGG GGT 781–805 Nübel et al. (1997) sequence homogeneity can be due to frequent transfers of ATC TAA TCC genetic material between strains. Humbert and Le Berre (2001) CYA781R(b) CAT T 781–805 Nübel et al. (1997) subsequently also suggested that recombination occurred – 16S727R GAC TAC AGG GGT 727 741 Wilmotte et al. (1993) between these species (P. agardhii/rubescens), and that the ATC TAA TCC CYA1514R CTT T 1494–1514 Taton et al. (2003) and existence of recombinations between the strains suggest that RGG ATT AGA references therein they are conspecific (Humbert and Le Berre, 2001). TAC CCC O. princeps (AB045961T)andPlanktothrix agardhii GTA CGG CTA CCT (AB045954T) NIES 204 (Anagnostidis and Komárek, 1988) TGT TAC GAC were selected as reference strains of the genera Oscillatoria and 104 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110

Table 3 Strains used in the cladistic analysis and listed in the cladogram Taxon Accession number Location of isolation Reference Base pairs Arthrospira – Carolina Carolina™ Science & Math. ER-15-1721. www.carolina.com 1350 Arthrospira fusiformis AF260510 Taxon: 54297 Li, R., Debella, H.J. & Carmichael, W.W. Taxonomic re-evaluation 1294 Strain Ethi-B2 of species of Arthrospira (Cyanobacteria). Unpublished. Arthrospira maxima AF260509 Taxon: 129910 Li, R., Debella, H.J. & Carmichael, W.W. Taxonomic re-evaluation 1293 Strain Ethi-A2 of species of Arthrospira (Cyanobacteria). Unpublished. Arthrospira sp. X70769 Taxon: 35824 Nelissen et al. (1994) 1959 Strain PCC 8005 Arthrospira sp. AF329392 Taxon: 153965 Mao, Y., Yang, G., Zhang, B. & Zhang, X. Application of the sequences 1963 Strain FACHB438 analysis of the 16S rRNA gene and ITS of 16S-23S rRNA to the systematic study of the genus Arthrospira and Spirulina. Unpublished. Gloeobacter violaceus AF132790 Strain PCC 7421 Turner et al. (1999) 1407 Leptolyngbya sp. AB039012 Taxon: 118166 Ishida et al. (2001) 1440 Strain: PCC 7104 Lyngbya aestuarii AJ000714 Taxon: 65095 Nübel et al. (1997), Garcia-Pichel et al. (1998) 1451 Strain PCC 7419 Lyngbya aestuarii AB039013 Taxon: 118322 Ishida et al. (2001) 1432 Strain PCC 7419 Lyngbya hieronymusii AB045906 China: Lake Dalai, Suda et al. (2002) 1368 Inner Mongolia Strain CN4-3 Microcystis (Loch Vaal) – Loch Vaal: South Africa 1485 Microcystis sp. (UV 027) – Strain UV 027 1290 Oscillatoria neglecta AB003168 Taxon: 71189 Ishida et al. (1997) 1438 Strain=“M-82” Oscillatoria princepsT AB045961 Thailand: Hao Phya, Suda et al. (2002) 1367 Bangkok Strain NIVA CYA 150 Oscillatoria simplicissima AM236076 Vaal River Venter (2000) 1350 Oscillatoria sp. AJ133106 Netherlands: Lake Zwart G., Van Agterveld M., Gons H., van der Werff I. & Hagen F. 1410 Loosdrecht Yanobacterial diversity in a shallow eutrophic lake. Unpublished. Planktothrix sp. AB045914 Finland: Lake Langsjon, Suda et al. (2002) 1494 Aland NIVA CYA 126 Planktothrix agardhii X84811 Taxon: 1160 Nelissen et al. (1996) 1463 Strain CYA 18 Planktothrix agardhii AB045958 Netherlands: Veluwemeer Suda et al. (2002) 1373 Strain NIES 596 Planktothrix agardhii AB045915 Finland: Lake Vesijari, Lahti Suda et al. (2002) 1371 Strain NIVA CYA 127 Planktothrix agardhii AB045918 Norway: Lake Suda et al. (2002) 1369 Ogderen, Akershus Strain NIVA CYA 133 Planktothrix agardhii AB045938 Norway: Lake Suda et al. (2002) 1369 Steinsfjorden, Buskerud Strain NIVA CYA 56/3 Planktothrix agardhii AB045935 Norway: Lake Suda et al. (2002) 1369 Kolbotnvatnet, Akershus Strain NIVA CYA 34 Planktothrix agardhii AB045947 Sweden: Lake Oren, Suda et al. (2002) 1371 Atvidanberg, Ostergotland Strain NIVA CYA 88/3 Planktothrix agardhii AB045924 United Kingdom: Suda et al. (2002) 1370 Windermere, Cumbria Strain NIVA CYA 168 Planktothrix mougeotii AB045971 Thailand: Nakhon Pathon Suda et al. (2002) 1360 Strain TR1-5 Planktothrix mougeotii AB045969 Thailand: Bangkok Suda et al. (2002) 1370 Strain TK4-5 Planktothrix pseudagardhii ABO45922 Chao Phya, Bangkok, Suda et al. (2002) 1544 Thailand by R. Strain NIVA CYA 153 Planktothrix pseudagardhii ABO45922 Thailand: Chao Phya, Suda et al. (2002) 1352 (NIVA 153) Bangkok Strain NIVA CYA 153 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110 105

Table 3 (continued) Taxon Accession number Location of isolation Reference Base pairs Planktothrix pseudagardhii AB045907 China: Lake Dalai, Inner Suda et al. (2002) 1355 Mongolia Strain CW4-5 Planktothrix pseudagardhii AB045966 Thailand: King Palace, Suda et al. (2002) 1355 Bangkok Strain T19-6'-8 Planktothrix AB045968 Thailand: Bangkok Suda et al. (2002) 1355 pseudagardhiiT Strain “T1-8-4” Planktothrix rubescens AB045921 Denmark: Lake almind so Suda et al. (2002) 1370 Strain NIVA CYA 151 Planktothrix rubescens AB045959 Norway: Lake Gjersjoen Suda et al. (2002) 1369 Strain NIES 610 Planktothrix rubescens AB045946 Sweden: Lake Levrasjon, Suda et al. (2002) 1369 Skane Strain NIVA CYA 87 Planktothrix rubescens AB045950 Norway: Lake Suda et al. (2002) 1370 Steinsfjorden, Buskerud Strain NIVA CYA 97/5 Planktothrix rubescens AB045937 Norway: Lake Suda et al. (2002) 1369 Steubsfjorden, Buskerud Strain NIVA CYA 55 Planktothrix rubescens AB045934 Norway: Lake Suda et al. (2002) 1370 Kolbotnvatnet, Akershus Strain NIVA CYA 320 Planktothrix rubescens AJ132250 Switzerland Beard et al. (1999) 1443 Strain BC-Pla 9401 Planktothrix sp. AJ133168 Taxon: 213625 Lyra et al. (2001) 1446 Strain NIVA-CYA 127 Planktothrix sp. AJ133166 Taxon 213624 Lyra et al. (2001) 1448 Strain NIVA CYA 126 Planktothrix sp. AJ133169 Taxon: 213626 Lyra et al. (2001) 1446 Strain NIVA CYA 128/R Spirulina platensis AB074508 Taxon: 1156 Seo, P. & Yokota, A. The phylogenetic relationships of 1441 cyanobacteria inferred from gyrB sequences. Unpublished. Strain IAM M-135 Spirulina sp. (BFN) – Kamferpan, Kimberley Roos, J. (unpublished) 1393 Spirulina sp. (G) – Kamferpan, Kimberley Jordaan, G. (unpublished) 1356 Synechococcus sp. AF330251 Germany: Lake Constance Ernst et al. (2003) 2309 Strain BO0014 Synechococcus sp. AY172832 Taxon: 69042 Fuller et al. (2003) 1440 Strain WH5701 Synechococcus sp. AY151249 Taxon: 210768 Crosbie et al. (2003) 1552 Isolate MW101C3 Synechococcus sp. AY151251 Taxon: 210717 Crosbie et al. (2003) 1554 Isolate MW97C4

Planktothrix respectively and P. pseudagardhii (AB045968T) The “O. simplicissima” isolate from the Vaal River, had (Suda, personal communication) were selected as reference nearly identical sequence of 16S rDNA with that of the strain of the species P. pseudagardhii according to findings by reference strain P. pseudagardhii and forms part of a distinct P. Suda et al. (2002). Our findings corroborate those of Suda et al. pseudagardhii clade (Fig. 1). Computation was performed at (2002), that species of Oscillatoria are polyphyletic and none NCBI using the BLAST network service (http://www.ncbi.nlm. are related to the reference strains. The bacteriological requested nih.gov/blast/Blast.cgi) and the sequences of the 16S rDNA of limit of similarity below 95% is clearly expressed between these all representatives within this clade show a 99% similarity. The entities. Suda et al. (2002) suggested that O. princeps should high bootstrap values for that clade strongly support the re- not be listed under the genus name Oscillatoria and that their identification of the organism found in the Vaal River as P. generic name should be changed, as proposed previously by pseudagardhii. Anagnostidis and Komárek (1988) on the basis of purely Use of 16S rDNA sequence analyses is a powerful tool for phenotypic characteristics. This is also clearly supported by our taxonomy; however, characterisation based on the sequence findings (Fig. 1). alone is not enough for the classification of species of some 106 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110

Fig. 1. Cladogram based on partial 16S rRNA gene sequences. Gloeobacter violaceus (PCC 7421) sequence was used as an outgroup. Bootstrap values are indicated at the nodes. Abbreviations: Table 3.

organisms, especially those that are closely related species measuring the degree of relatedness between highly related (Suda et al., 2002). For example, P. agardhii and Planktothrix organisms. However it is interesting that at sequence homology rubescens could not be separated by 16S rDNA sequence values below about 97.5%, it is unlikely that two organisms analysis (Humbert and Le Berre, 2001; Suda et al., 2002) have more than 60–70% DNA similarity and hence that they are morphology, fatty acid composition, or G+C content, but were related at the species level (Stackebrandt and Goebel, 1994). On distinguishable by phycobilin pigment composition, growth at the other hand, Suda et al. (2002) reported that the closely different temperatures and salinities and DNA–DNA hybridisa- related species P. agardhii and P. pseudagardhii were well tion (Suda et al., 2002). The resolution power of DNA defined by 16S rDNA sequence analysis, although they shared hybridisation is significantly higher than that of sequence the same morphology as well as fatty acid composition, analysis and DNA hybridisation remains the optimal method for phycobilin pigment and G+C content. The present results K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110 107

(Fig. 1) confirm this as P. agardhii clearly clustered separately (aerotopes) are present in the trichomes of this family and the from the group P. pseudagardhii. These results therefore, once sheath, the production of which is dependent on environmental again emphasize the use of a polyphasic approach in identifying conditions, remains open at the ends and contains one or more cyanobacterial strains. trichomes (Anagnostidis and Komárek, 1988). In the genus To verify the molecular results, a comparative morphological Phormidium, the trichomes are slightly to intensely waved or study was also done between “O. simplicissima” and P. coiled (Anagnostidis and Komárek, 1988). These phenotypical pseudagardhii NIVA CYA 153 under different environmental characteristics furthermore clearly exclude the current material conditions. Venter et al. (2003) found that “O. simplicissima” of “O. simplicissima” also from the family Phormidiaceae and has typical characteristics of the Oscillatoriaceae as described the genus Phormidium. by Geitler (1932) and Desikachary (1959). This purely clarifies Anagnostidis and Komárek (1988) established the new that “O. simplicissima” is a filamentous bacterium with genus Planktothrix for gas-vacuolated Oscillatoria species. uniseriate cells that are not constricted at the cross walls. According to Suda et al. (2002), gas vesicles in P. pseuda- Terminal cells are hemispherical with a slightly thickened gardhii are relatively large and scattered at the periphery of the membrane on the outer cell envelope. The trichomes are not cells. Venter et al. (2003) found that gas vesicles in “O. attenuated or capitated at the apices. Venter et al. (2003) found simplicissima” formed conspicuous beehive-like structures in gas vesicles (aerotopes) present in the trichome and found that the older cells of filaments but that filaments grown in culture the straight, unbranched trichome is covered with a thin hyaline for a long time lose these gas vesicles. Our finding showed that sheath, which is in contrast with the description by Anagnos- gas vesicles are present in both “O. simplicissima” (Fig. 3a) as tidis and Komárek (1988) of the genus Oscillatoria. well as P. pseudagardhii even under different light and The family Oscillatoriaceae, as reviewed by Anagnostidis temperature conditions (Fig. 3b). and Komárek (1988), is characterised by straight or slightly The average width of the filament of P. pseudagardhii waved trichomes. One or more trichomes can be present in (3.05 μm) was found to be narrower than that of “O. a sheath. The trichomes have a thicker outer cell wall some- simplicissima” (3.65 μm) (Figs. 2 and 3). Cells of P. times with a calyptra and the trichomes are rarely solitary and pseudagardhii are 3.0–6.4 μm wide and 1.2–4.2 μm long mainly compact. In the genus Oscillatoria, the trichomes are (Suda et al., 2002), but Desikachary (1959), Drouet (1968) and usually without sheaths, but a sheath can occur in suboptimal Anagnostidis and Komárek (1988) respectively reported widths conditions, and no gas vesicles are present. Trichomes of of 8–9 μm, 1.5–8 μm and 1–12 μm in this species. P. the current material, similar to what was found by Venter pseudagardhii NIVA 153 has a width of 2–4 μm. Venter et al. et al. (2003) were always straight, with only one trichome in (2003) found that the width of “O. simplicissima” cells varies each sheath, and the trichomes were solitary. Gas vesicles between 8.0 and 12.0 μm, while current observations range were present. Thus this species cannot be classified as a from 3 to 5 μm. It is thus clear that width does not characterise member of the genus Oscillatoria under the family Oscil- this species. latoriaceae according to the criteria of Anagnostidis and The following findings furthermore illustrate why morphol- Komárek (1988). ogy alone cannot be used to distinguish between closely related Anagnostidis and Komárek (1988) however reassigned O. cyanobacterial species: The trichomes of Planktothrix are simplicissima to Phormidium simplicissimum, in the family sometimes attenuated towards the end and apical cells show Phormidiaceae, which is characterised by slightly or intensely variable shapes, such as rounded, tapered, bluntly conical, waved or coiled trichomes that form mats. No gas vesicles occasionally capitate, with and without calyptra (Suda et al.,

Fig. 2. Light micrograph of a) O. simplicissima and b) P. pseudagardhii, grown at 25 °C with a light intensity of 20 μmol m− 2 s− 1. Scalebar=10 μm. 108 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110

Fig. 3. Confocal micrograph of a) O. simplicissma and b) P. pseudagardhii grown at 25 °C with a light intensity of 20 μmol m−2 s− 1. Scalebar=5 μm.

2002). Trichomes of Planktothrix are slightly tapering or not according to Rule 16, Note 2 (De Vos and Trüper, 2000). P. tapering to the end, end cells (when fully developed) with agardhii (Gomont) Anagnostidis and Komárek (1988) is the calyptra or with thickened outer cell (Anagnostidis and only Planktothrix validly published under the International Komárek, 1988). According to Venter et al. (2003) the trichome Code of Botanical Nomenclature. of “O. simplicissima” is not attenuated or capitated at the apex In spite of this, we suggested that “O. simplicissima” must and the terminal cells are hemispherical with a slightly then be reassigned to the name of P. pseudagardhii according to thickened outer cell wall on the outer cell envelope. Our results the results of the 16S rDNA sequencing. This follows after the indicate that the trichomes are hemispherical to the end without still ongoing dispute that exists in the international community a calyptra. No transient forms have been observed. on whether the cyanobacteria should be described and The cross walls of P. agardhii may be slightly constricted or not published as blue-green algae under the International Code of constricted at the end (Suda et al., 2002; Anagnostidis and Botanical Nomenclature or be recognized as having been Komárek, 1988). Venter et al. (2003) found that the cells are not published under the Bacteriological Code. constricted at the cross walls. Our results indicate that filaments In June 1985 the Subcommittee on the taxonomic of may be constricted or not constricted under different environmental Phototrophic Bacteria proposed that names of cyanobacteria conditions. Fig. 3a shows a filament of “O. simplicissima” of which described and validly published as blue-green algae under the the cells are constricted at the cross walls. The filaments were International Code of Botanical Nomenclature are recognized as grown at 18 °C and a light intensity of 20 μmol m−2 s−1. having been published under the Bacteriological Code (1990 No clear differences between “O. simplicissima” and P. Revision) (Trüper, 1986). pseudagardhii NIVA CYA 153 were found using different In September 1986, the Judicial Commission agreed unan- microscopy techniques, which further supports results obtained imously to recommend the following to the ICSB (now ICSP): from the 16S rRNA study. According to Komárek (2005) the those names of taxa of Cyanobacteria and Cyanophyta that are molecular (phylogenetic) data should be accepted as a basic valid under the Botanical Code be considered valid under the criterion for taxonomical studies. Bacteriological Code for the purpose of preparing an acceptance In conclusion, the presented morphological results such as list comparable to the Approved Lists of Bacterial Names the presence of gas vacuoles and especially the molecular (Jones, 1987). However, “Declarations of intent” by the Judicial results which indicate that the isolate formerly known as “O. Commission were not taken up by the ICSB (now ICSP). In simplicissima” grouped with the genus, Planktothrix and more 2003, the “Subcommittee on the taxonomy of phototrophic precisely the species of P. pseudagardhii, suggested that “O. bacteria” (Imhoff and Madigan, 2004) proposed the preparation simplicissima” (sensu Pieterse and Steynberg, 1993; Venter et of a list of approved names of cyanobacteria under the al., 2003) should be reassigned to P. pseudagardhii under the Bacteriological Code and a small committee was appointed to order Oscillatoriales, family Phormidiaceae and subfamily prepare such a list. However, such a list is not yet available. Phormidioideae (Suda, personal communication). P. pseuda- Consequently, names of cyanobacteria described and validly gardhii Suda and Watanabe (2002) is however illegitimate published as blue-green algae under the International Code of because the genus name Planktothrix has never been validly Botanical Nomenclature have no standing in bacterial nomen- published under the Rules of the Bacteriological Code (1990 clature, unless they are again described under the Rules of the Revision) and the species was therefore not validly published Bacteriological Code (1990 revision). K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110 109

Acknowledgements Humbert, J.F., Le Berre, B., 2001. Genetic diversity in two species of freshwater cyanobacteria, Planktothrix (Oscillatoria) rubescens and P. agardhii. Archiv fur Hydrobiologie 150, 197–206. We are grateful to the WRC for the funding of this project. Imhoff, J.F., Madigan, M.T., 2004. International Committee on Systematics of We also thank Prof. Kaarina Sivonen and Dr. Ilona Oksanen of Prokaryotes Subcommittee on the taxonomy of phototrophic bacteria. University of Helsinki for their help with the methods used and Minutes of the Meetings, 27 August 2003, Tokyo, Japan. International for providing the strain of Planktothrix sp. NIVA 126/8 and Journal of Systematic and Evolutionary Microbiology 54, 1001–1003. Prof. Suda for advice and suggestions. Ishida, T., Yokota, A., Sugiyama, J., 1997. Phylogenetic relationships of filamentous cyanobacterial taxa inferred from 16S rRNA sequence divergence. Journal of General and Applied Microbiology 43, 237–241. References Ishida, T., Watanabe, M.M., Sugiyama, J., Yokota, A., 2001. Evidence for polyphyletic origin of the members of the orders of Oscillatoriales and Anagnostidis, K., Komárek, J., 1988. Modern approach to the classification Pleurocapsales as determined by 16S rDNA analysis. FEMS Microbiology system of cyanophytes 3. Oscillatoriales. Archiv fur Hydrobiologie 80, Letters 201, 79–82. 327–472. Janse van Vuuren, S., 2001. Environmental Variables and the development of Ballot, A., Krienitz, L., Kotut, K., Wiegand, C., Metcalf, J.S., Codd, G.A., phytoplankton assemblages in the Vaal River between the Rand Water Pflugmacher, S., 2002. Cyanobacteria and cyanobacterial toxins in three Barrage and Balkfontein. PhD Thesis, PU for CHE, Potchefstroom, alkaline Rift V alley lakes of Kenya — Lakes Booria, Nakuru and pp. 370. Elmenteita. Journal of Plankton Research 26, 925–935. Jones, D., 1987. Judicial Commission of the International Committee on Beard, S.J., Handley, B.A., Hayes, P.K., Walsby, A.E., 1999. The diversity of Systematic Bacteriology. Minutes of the meeting, 5 September 1986, gas vesicle genes in Planktothrix rubescens from Lake Zurich. Microbiology Manchester, United Kingdom. International Journal of Systematic Bacter- 145, 2757–2768. iology 37, 85–87. Crosbie, N.D., Pockl, M., Weisse, T., 2003. Dispersal and phylogenetic diversity Komárek, J., 2005. The modern classification of cyanoprokaryotes (Cyano- of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA- bacteria). Oceanological and Hydrobiological Studies 34, 5–17. intergenic spacer sequence analyses. Applied and Environmental Microbi- Kruskopf, M.M., 2002. Phosphatase activities of riverine phytoplankton ology 69, 5716–5721. in the Vaal river (South Africa), Physiological responses of nuisance spe- Desikachary, T.V., 1959. Cyanophyta. ICAR Monographs on Algae. Indian cies to different nutrient regimes. Ph.D. Thesis, PU for CHE, Potchefstroom, Council of Agricultural Research, New Delhi, p. 686. pp. 255. De Vos, P., Trüper, H.G., 2000. Judicial Commission of the International Leppänen, J.M., Rantajärvi, E., Hällfors, S., Kruskopf, M., Laine, V., 1995. Committee on Systematic Bacteriology. IXth International (IUMS) Unattended monitoring of potentially toxic phytoplankton species in the Congress of Bacteriology and Applied Microbiology. Minutes of the Baltic Sea in 1993. Journal of Plant Research 17, 891–902. Meetings, 14, 15 and 18 August 1999, Sydney, Australia. International Lyra,C.,Suomalainen,S.,Gugger,M.,Vezie,C.,Sundman,P.,Paulin,L., Journal of Systematic and Evolutionary Microbiology 50, 2239–2244. Sivonen, K., 2001. Molecular characterization of planktic cyanobacteria Drouet, F., 1968. Classification of the Oscillatoriaceae. The Academy of of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. Natural Sciences of Philadelphia, Pennsylvania, p. 370. International Journal of Systematic and Evolutionary Microbiology 51, Ernst, A., Becker, S., Wollenzien, U.I.A., Postius, C., 2003. Ecosystem- 513–526. dependent adaptive radiations of picocyanobacteria inferred from 16S rRNA Neilan, B.A., 1995. Identification and phylogenetic analysis of toxigenic and ITS-1 sequence analysis. Microbiology 149, 217–228. cyanobacteria by multiplex randomly amplified polymorphic DNA PCR. Ferris, M.J., Ruff-Roberts, A.L., Kopczynski, E.D., Bateson, M.M., Ward, D.M., Applied and Environmental Microbiology 61, 2286–2291. 1996. Enrichment culture and microscopy conceal diverse thermophilic Sy- Nelissen, B., Wilmotte, A., Neefs, J.M., De Wachter, R., 1994. Phylogenetic nechococcus populations in single hot spring microbial mat habitat. Applied relationships among filamentous helical cyanobacteria investigated on the and Environmental Microbiology 62, 1045–1050. basis of 16S ribosomal RNA gene sequence analysis. Systematic and Fiore, M.F., Moon, D.H., Tsai, S.M., Lee, H., Trevors, J.T., 2000. Miniprep Applied Microbiology 17, 206–210. DNA isolation from unicellular and filamentous cyanobacteria. Journal of Nelissen, B., Van de Peer, Y., Wilmotte, A., De Wachter, R., 1995. An early Microbiological Methods 39, 159–169. origin of plastids within the cyanobacterial divergence is suggested by Fox, R.D., 1996. Spirulina: Production and potential. Edisud, Aix-en-Provence, evolutionary trees based on complete 16S rRNA sequences. Molecular France, p. 232. Biology and Evolution 12, 1166–1173. Fuller, N.J., Marie, D., Partensky, F., Vaulot, D., Post, A.F., Scanlan, D.J., 2003. Nelissen, B.J.M., De Baere, R., Wilmotte, A., De Wachter, R., 1996. Phylogenetic Clade-specific 16S ribosomal DNA oligonucleotides reveal the predomi- relationship of nonaxenic filamentous cyanobacterial strains based on 16S nance of a single marine Synechococcus clade throughout a stratified water rRNA sequence analysis. Journal of Molecular Evolution 42, 194–200. column in the Red Sea. Applied and Environmental Microbiology 69, Nixon, K.C., 2002. WinClada ver. 1.00.08 Published by the author, Ithaca, NY. 2430–2443. (http://www.cladistics.com). Garcia-Pichel, F., Prufert-Bebout, L., Muyzer, G., 1996. Phenotypic and Nübel, U., Garcia-Pichel, F., Muyzer, G., 1997. PCR primers to amplify 16S phylogenetic analyses show Microcoleus chthonoplastes to be a cosmopolitan rRNA genes from cyanobacteria. Applied and Environmental Microbiology cyanobacterium. Applied and Environmental Microbiology 62, 3284–3291. 63, 3327–3332. Garcia-Pichel, F., Nuebel, U., Muyzer, G., 1998. The phylogeny of unicellular, Oren, A., 2004. A proposal for further integration of the cyanobacteria under the extremely halotolerant cyanobacteria. Archives of Microbiology 169, 469–482. Bacteriological code. International Journal of Systematic and Evolutionary Geitler, L., 1932. Kryptogamen – Flora von Deutschland, Österreich und der Microbiology 54, 1895–1902. Schweiz. Bnd. 14. Cyanophyceae von Europa. Akademische Verlagsgesell- Oren, A., Tindall, B.J., 2005. Nomenclature of the cyanophyta/cyanobacteria/ shaft, Leipzig. cyanoprokaryotes under the International Code of Nomenclature of Giovannoni, S.J., Turner, S., Olsen, F.J., Barns, S., Lane, D.J., Pace, N.R., 1988. Procaryotes. Algological Studies 117, 39–52. Evolutionary relationships among cyanobacteria and green chloroplasts. Pieterse, A.J.H., Steynberg, M.C., 1993. Water quality and algal growth in the Journal of Bacteriology 170, 3584–3592. Loch Vaal. Report on a consultation project for Rand Water, Department of Giovannoni, S.J., DeLong, E.F., Schmidt, T.M., Pace, N.R., 1990. Tangential Botany and Genetics, UOFS, Rand Water, Vereeniging, p. 19. flow filtration and preliminary phylogenetic analysis of marine picoplank- Rudi, K., Skulberg, O.M., Larsen, F., Jakobsen, K.S., 1997. Strain character- ton. Applied and Environmental Microbiology 56, 2572–2575. ization and classification of oxyphotobacteria in clone cultures on the basis Gomont, M.M., 1892. Monograpie des Oscillariées (Nostocacées homocystées). of 16S rRNA sequences from the variable regions V6, V7 and V8. Applied Annales des Sciences Naturelles. Botanique Seria 7 15, 263–368; 16, 91–264. and Environmental Microbiology 63, 2593–2599. 110 K.R. Conradie et al. / South African Journal of Botany 74 (2008) 101–110

Rudi, K., Skulberg, O.M., Jakobsen, K.S., 1998. Evolution of cyanobacteria by discussion workshop: taxonomy of cyanobacteria, 17 to 19 June 1985, Paris, exchange of genetic material among phyletically related strains. Journal of France. International Journal of Systematic Bacteriology 36, 114–115. Bacteriology 180, 3453–3461. Turner, S., Pryer, K.M., Miao, V.P., Palmer, J.D., 1999. Investigating deep Schmidt, T.M., DeLong, E.F., Pace, N.R., 1991. Analysis of a marine picoplankton phylogenetic relationships among cyanobacteria and plastids by small community by 16S rRNA gene cloning and sequencing. Journal of Bacteriology subunit rRNA sequence analysis. Journal of Eukaryotic Microbiology 46, 173, 4371–4378. 327–338. Stackebrandt, E., Goebel, B.M., 1994. Taxonomic note: a place for DNA–DNA Turpin, P.J.F., 1892. Spirulina oscillariodide. Dictionnarire des Sciences reassociation and 16S rRNA sequence analysis in the present species Naturelles. F.G. Lévrault, Paris. definition in bacteriology. International Journal of Systematic Bacteriology Venter, A., 2000. The physiological characteristics of Oscillatoria simplicis- 44, 846–849. sima. PhD dissertation. Potchefstroom University, South Africa, pp. 140. Stizenberger, E., 1852. Spirulina und Arthrospira (nov. gen.). Hedwigia 1, Venter, A., Jordaan, A., Pieterse, A.J.H., 2003. Oscillatoria simplicissima:a 32–34. taxonomical study. Water S.A. 29, 101–104. Suda, S., Watanabe, M.M., Otsuka, S., Mahakahant, A., Yongmanitchai, W., Wilmotte, A., Turner, S., Van de Peer, Y., Pace, N.R., 1992. Taxonomic study of Nopartnaraporn, N., Liu, Y., Day, J.G., 2002. Taxonomic revision of water- marine Oscillatoriacean strains (cyanobacteria) with narrow trichomes. II. bloom-forming species of oscillatoroid cyanobacteria. International Journal Nucleotide sequence analysis of the 16S ribosomal RNA. Journal of of Systematic and Evolutionary Microbiology 52, 1577–1595. Phycology 28, 828–838. Tanskanen, S., 1990. Itämeressä esiintyvät myrkylliset sini-ja panssarisiimale- Wilmotte, A., Van der Auwera, G., De Wachter, R., 1993. Structure of the 16S vät. Meri 18, 1–45 (in Finnish). ribosomal RNA of the thermophilic cyanobacterium Chlorogloeopsis HTF Taton, A., Grubisic, S., Brambilla, E., De Wit, R., Wilmotte, A., 2003. (‘Mastigocladus laminosus HTF’) strain PCC7518, and phylogenetic Cyanobacterial diversity in natural and artificial microbial mats of lake analysis. FEBS Letters 317, 96–100. Fryxell (McMurdo dry valleys, Antarctica): a morphological and molecular Woese, C.R., Kandler, O., Wheelis, M.L., 1990. Towards a natural system of approach. Applied and Environmental Microbiology 69, 5157–5169. organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Trüper, H.G., 1986. International Committee on Systematic Bacteriology Proceedings of the National Academy of Sciences of the United States of subcommittee on the taxonomy of phototrophic bacteria. Minutes of the America 87, 4576–4579.

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