Genus Microcystis Kuetzing ex Lemmermann (Chroococcales, Cyanoprokaryota) from India

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1 (Chroococcales, Cyanoprokaryota) from India Richa Tandon,* Sarika Kesarwani, Arti Mishra, 1, Anupam Dikshit and G.L. Tiwari Department of Botany, University of Allahabad, Allahabad , India 1 Department of Botany, Govt. P.G. College, Satna, M.P., India *Corresponding author: drrichatandon@gmail.com Tel: Abstract Microcystis Kuetzing ex Lemmermann is well known planktonic genus of coccoid (Chroococcales) cyanoprokaryota. It produces dense water blooms and certain of them are highly toxic. Based on collections from various localities of Uttar Pradesh during last over one decade ( ), we have described seven species, of which five are new records of India. The study include key of all the species, brief description, figures and photomicrographs. Incidence of toxicity of bloom causing death of animals or birds was never observed by the local inhabitants, although they were continuously consuming the blooms. M. aeruginosa has been isolated in BG 11 medium and it did not show structure of typical colonies found in natural habitat, indicating that shape of the colony is influenced by environmental features and interaction of associated microbes; which cannot be conferred on the basis of molecular studies only. Therefore, morpho-taxonomic studies are important to have exact influence about the phenotypic expression of genomic or proteomic variations in normal as well as under stress conditions. Keywords: Chroococcales, Microcystis, water blooms. Introduction Morpho-taxonomy facilitates many workers to contribute specifically to the knowledge of micro-organisms growing even in remote localities. However, if one happened to get some different organism from the view point of structural, ecological, biochemical or biotechnological usefulness, it need to be investigated by polyphasic approach including all available techniques and parameters to understand the evolutionary diversity of the organism to utilize its capabilities. Morpho-taxonomy is the well defined approach to identify the closely linked species and predict their position in systematics The study of phenotypic variation is a must to correlate and exactly postulate the genotypic changes. Even for proteomics one has to take the phenotypic variations under considerations. In our endeavour of preparing detailed information on morphology and life history of various cyanobacteria growing in Uttar Pradesh, we have published several accounts on genera like Aphanothece (Kant et al., 2005a, 2006), Asterocapsa (Kant et al., 2005b), Gloeocapsa (Kant et al., 2005c), Chroococcus (Kant et al., 2008), Bacularia (Kesarwani et al., 2008), Gomphosphaeria (Dwivedi et al., 2010), Chamaecalyx (Tandon et al., 2012), Radaisia (Tandon et al., 2014a) and Pleurocapsa (Tandon et al., 2014b, 2016). The present communication deals with the genus Microcystis of the family Microcystaceae. The family Microcystaceae of the order Chroococcales (Cyanoprokaryota) as described by Komarek & Anagnostidis (1998) is characterized by the colonies known to have cell division in three planes and where daughter cells always grow into original size before the next division takes place. The family includes four genera viz, Chondrocystis, Eucapsis, Gloeocapsa and Microcystis. The first two genera are rarely reported. Gloeocapsa is occasionally reported but not clearly defined as it is often associated with Chroococcus (Starmach, 1966; Boone & Castenholz, 2001). Microcystis is a cosmopolitan genus, found mostly in fresh water bodies and known to have fifty one morpho-species at the global level (Komarek & Anagnostidis, 1998; McGregor, 2013; Guiry & Guiry, 2014). All species are planktonic and certain species produce dense blooms in eutrophic water bodies and some of them are reported to be highly toxic. Our survey of literature indicated the occurrence of seventeen species in India, including five species reported here for the first time from India. Material and Methods The collection of Microcystis was made from various localities of Uttar Pradesh, India. The locality included: Allahabad, Varanasi, Jaunpur, Pratapgarh, Mirzapur, Chitrakoot, Raebareli, Fatehpur and Kaushambi. One species viz. M. aeruginosa was isolated and maintained in BG 11 medium (Stanier et al., 1971) for the last over one decade. Photographs were taken under Leica DMLB trinocular microscope with DC 300 camera and its quin imaging system. Observations 4

2 Colonies free-floating, minute or large, spherical, oval, lobate or irregular, with or without holes (air spaces), clathrate or composed of sub-colonies. Mucilage fine, hyaline, homogenous, diffluent or distinct and distinctly delimited or refractive in some species. Cells loosely or densely arranged, spherical, gas vacuoles present, cell divisions occur in three planes at right angle to each other, cells grow into the original size and shape before next division. Morphologically different micro or macroscopic stages may occur during the growth cycle of the organism. The cells always produce numerous gas vesicles. The gas vesicles are reversible and control buoyancy of colonies in water bodies. Many unidentifiable colonies with atypical stages or transient forms commonly occur in planktonic samples and such unclear stages usually appear in the beginning and at the end of growing period. Key characters of morpho-species of Microcystis Kuetzing ex Lemmermann Many morpho-species have been described in traditional literature. Morphological parameters which have been used to distinguish different species of Microcystis include the following: (i) Colonies: various shapes, spherical, irregular, lobate, flat or three dimensional clathrate colonies, micro or macroscopic at older stages. (ii) Colonial boundaries: diffluent, indistinct, or distinctly demarcated as thin or thick hyaline or coloured envelopes, may be with gelatinous tubular protuberances or refractive margins. (iii) Presence or absence of air spaces (holes) in colonies. (iv) Cell size, density of aggregations inside the colonies, presence and abundance of gasvesicles or aerotopes in the cells. List of Taxa of Microcystis Kuetzing ex Lemmermann from India 1. Microcystis aeruginosa Kuetz.: Martens, 1870* 2. M. bengalensis Banerji, 1936* 3. M. densa West, G.S.: Thomas & Gonz., 1965a* 4. M. firma (Breb. & Lenorm.) Schmidle : Thomas & Gonz., 1965b* 5. M. flos-aquae (Wittr.) Kirchn. : Bruhl & Biswas, 1926* 6. M. lamelliformis Holsinger : Barhate & Tarar, 1984* 7. M. protocystis Crow : Banerji, 1936* 8. M. pseudofilamentosa Crow : Banerji, 1936* 9. M. ramosa Bharadwaja, 1935* 10. M. robusta (Clark) Nyg. : Biswas, 1927* 11. M. scripta (Richt.) Lemm. : Banerji, 1936* 12. M. viridis (A. Br.) Lemm. : Banerji, 1936* 13. M. wesenbergii (Komarek) in Kondrateva 1968 # 14. M. panniformis Komarek et al 2002# 15. M. botrys Teiling 1942# 16. M. novacekii (Komarek) Compere 1974# 17. M. smithii Komarek et Anagnostidis 1995# *Marked reference is for the first report from India #Marked species are being reported for the first time from India in the present study Key to the species of Microcystis Kuetzing ex Lemmermann included in the study A. Colonies irregular in outline. B A. Colonies more or less spherical. D B. Colonies enveloped by distinctly delimited and refractive mucilage; colonies lobate or perforate M. wesenbergii B. Colonies enveloped by diffluent or indistinct mucilage. C C. Colonies irregular, elongate, lobate, later clathrate M. aeruginosa C. Colonies in the shape of a flattened discs forming thin often folded film; not clathrate.. M. panniformis D. Mucilaginous margin wide, distinct with radial, hyaline tubular gelatinous protuberances; cells 6-7µm in diameter M. botrys D. Mucilage with wide margin, without such protuberances.. E 5

E. Colonies with cuboid clusters of cells. M. novacekii E. Colonies without cuboid clusters F F. Colonies with densely packed cells M. flos-aquae F. Colonies with loosely arranged cells.. M. smithii Plate 1 Explanation of Figures 1-7 Microcystis Kuetzing ex Lemmermann.

3 E. Colonies with cuboid clusters of cells. M. novacekii E. Colonies without cuboid clusters F F. Colonies with densely packed cells M. flos-aquae F. Colonies with loosely arranged cells.. M. smithii Plate 1 Explanation of Figures 1-7 Microcystis Kuetzing ex Lemmermann. 1. M. wesenbergii. 2. M. Aeruginosa. 3. M. panniformis. 4. M. botrys. 5. M. novacekii. 6. M. flos-aquae. 7. M. smithii Scale 10µm for figure 4; Scale 20µm for figs. 5 & 7; Scale 25µm for figs. 1 & 3; Scale 50µm for figs. 2 & 6. 6

4 Plate 2 Explanation of Figures 8-17 Microcystis Kuetzing ex Lemmermann. 8 & 9. M. wesenbergii M. Aeruginosa. 14 & 15. M. panniformis. 16 & 17. M. botrys 7

Plate 3 Explanation of Figures 18-24 Microcystis Kuetzing ex Lemmermann 18 & 19. M. aeruginosa (in culture).

5 Plate 3 Explanation of Figures Microcystis Kuetzing ex Lemmermann 18 & 19. M. aeruginosa (in culture). 20. M. novacekii. 21 & 22. M. flos-aquae. 23 & 24. M. smithii 8

Plate 4 Explanation of Figures 25-30 Microcystis Kuetzing ex Lemmermann 25 & 26. Growth of M. aeruginosa in culture. 27. Birds and a buffalo in bloom of M. aeruginosa. 28. Dense bloom of M.

6 Plate 4 Explanation of Figures Microcystis Kuetzing ex Lemmermann 25 & 26. Growth of M. aeruginosa in culture. 27. Birds and a buffalo in bloom of M. aeruginosa. 28. Dense bloom of M. aeruginosa in nature. 29 & 30. Amoebae engulfing the cells of M. aeruginosa (Phagocytosis) Description of seven species of Microcystis Kuetzing ex Lemmermann 1. M. wesen-bergii (Komarek) Komarek in Kondrateva (Plate 1, Fig.1; Plate 2, Figs. 8 & 9) Komarek & Anagnostidis, Chroococcales, 1998, P. 232, Fig. 305 McGregor, Chroococcales, 2013, P. 63, Pl. 20 E, Fig. 41G Colonies micro to macro-scopic, spherical to elongated and lobed, often with air spaces, occasionally composed of sub-colonies; mucilage hyaline, distinctly delimited, firm conspicuous and distinctly refractive in outline; cells spherical, packed with aerotopes, µm in diameter. Habitat: muddy water puddles Occurrence: frequently in colder months (November-January), Allahabad and Pratapgarh 2. M. aeruginosa Kuetzing (Plate 1, Fig. 2; Plate 2, Figs ; Plate 3, Figs. 18 & 19) 9

7 Desikachary, Cyanophyta, 1959, P. 93, Pl.17, Figs. 1, 2, 6; Pl.18, Fig. 10 Komarek & Anagnostidis, Chroococcales, 1998, P. 232, Fig. 304 McGregor, Chroococcales, 2013, P. 62, Pl. 20 A, Figs. 39I, J, K Colonies mucilaginous micro to macro-scopic, spherical to elongate or clathrate in various shapes; mucilage colourless, diffluent not well defined; cells numerous densely packed, spherical or hemispherical after division, µm in diameter with conspicuous aerotopes. Habitat: eutrophic water bodies Occurrence: common all round the year, Allahabad, Jaunpur and Varanasi 3. M. panniformis Komarek et al. (Plate 1, Fig. 3; Plate 2, Figs. 14 & 15) McGregor, Chroococcales, 2013, P. 63, Pl. 20 D, Figs. 41B, C, D Colonies in the shape of a flattened discs and may form thin often folded film; mucilage wide, colourless; cells spherical with distinct aerotopes, 3-5 µm in diameter, evenly distributed throughout the colony or form small aggregations. Habitat: eutrophic water bodies, mixed with M. flos-aquae Occurrence: rare during summer month (March-June), Allahabad and Mirzapur 4. M. botrys Teiling (Plate 1, Fig. 4; Plate 2, Figs. 16 & 17) Komarek & Anagnostidis, Chroococcales, 1998, P. 228, Fig. 298 McGregor, Chroococcales, 2013, P. 62, Pl. 20 B, Figs. 40A-D Colonies mucilaginous, micro to macro-scopic, essentially spherical, with irregularly and densely aggregated cells; mucilage thick, wide, colourless, sometimes indistinct but structured and forming a broad margin (10-20 µm) around the colony, semiglobose or tubular gelatinous projections, radially arranged; cells spherical, with aerotopes, 6-7 µm in diameter. Habitat: small ditches Occurrence: rare (September-October), Kaushambi and Fatehpur 5. M. novacekii (Komarek) Compere (Plate 1, Fig. 5; Plate 3, Fig. 20) Komarek & Anagnostidis, Chroococcales, 1998, P. 230, Fig. 302 Colonies micro to macro-scopic, spherical or lobed, composed of packet-like colonies with densely arranged cells, occasionally scattered cells are present around the clustered cells; mucilage hyaline and distinctly delimited around the colonial lobes; cells spherical filled with aerotopes, 3.5-5µm in diameter. Habitat: cemented tanks Occurrence: occasionally (September-October), Allahabad and Chitrakoot 6. M. flos-aquae Kuetzing (Wittrock) Kirchner (Plate 1, Fig. 6; Plate 3, Figs. 21 & 22) Desikachary, Cyanophyta, 1959, P. 94, Pl.17, Fig. 11; Pl.18, Fig. 11 Komarek & Anagnostidis, Chroococcales, 1998, P. 228, Figs. 300, 301 McGregor, Chroococcales, 2013, P. 62, Pl. 20 C, Figs. 40E-H Colonies microscopic, spherical or irregular in outline; cells more or less densely aggregated, occasionally with small air spaces (holes); mucilage diffluent and hyaline, envelopes do not exceed the margin of cell clusters; cells spherical with numerous aerotopes µm in diameter. Habitat: eutrophic water bodies Occurrence: all round the year; Allahabad, Varanasi, Chitrakoot, Kaushambi and Raibareli 7. M. smithii Komarek et Anagnostidis (Plate 1, Fig. 7; Plate 3, Figs. 23 & 24) Komarek & Anagnostidis, Chroococcales, 1998, P. 228, Fig. 299 Colonies free-floating, spherical or slightly irregular, later gelatinous, never clathrate, with scattered, rarely densely arranged cells; mucilage fine, colourless, more or less distinctly delimited or diffuse, homogenous, without refractive margin; cells loosely arranged throughout the colony, single or in pairs after division, spherical with several brownish aerotopes in each cell, µm in diameter. Habitat: cemented tanks mixed with Chlorococcalean green algae Occurrence: occasionally (August-October), Varanasi Growth in culture: Microcystis is generally considered as difficult to isolate in culture, but repeated efforts of streaking the cells of M. aeruginosa on agar plates resulted into uni-species growth of the organism. It appeared that young cells were able to propagate easily. It grows well in BG 11 liquid or solid medium and cells are uniformly distributed in a formless mucilaginous matrix (Plate 3, Figs. 18 & 19). They never form typical colonies as found in nature. Although shape of the colony, demarcation of colonial boundary, pattern of cell aggregation, presence of air 10

8 spaces have been considered as important characters for identification of species collected from natural habitat. It appears quite possible that specific factors of habitat including environmental conditions and also interaction with other associated organisms including bacteria inhabiting the mucilaginous matrix may be responsible for special morphology of colonies and this need to be worked out. If liquid cultures are not disturbed for a fortnight or so, a translucent green precipitate-like irregular clusters of cells may occur in the medium (Plate 4, Figs. 25 & 26). The cultures need to be transferred in fresh medium at an interval of days otherwise it turned into whitish milky in appearance and ultimately get degenerated. The isolate of M. aeruginosa (AU 1502) is being maintained in culture condition for the last one decade (temperature C; light lux; 12h: 12h LD). It is difficult to find the pattern of cell division in growth of natural habitat but was clearly seen in cells of culture medium (Plate 3, Fig. 19). Although in literature (Komarek & Anagnostidis, 1998), pattern of three plane cell division is mentioned for the family Microcystaceae but we could see only two plane division at right angle to each other in the present Microcystis. If it is proved, it may need to be placed in its own independent family. It appears that the cell division in Microcystis is a synchronized process and occur quickly at some specific slot of time and all this need to be varified. Toxic species: M. aeruginosa and M. flos-aquae are the most common species and frequently found in eutrophic water bodies often connected with sewage drains. In the present study birds and domestic animals including cows and buffaloes were found drinking and waddling in water bodies containing dense water bloom of M. aeruginosa (Plate 4, Figs. 27 & 28), but they were never harmed in our knowledge. This may be due to the fact that toxin producing strains are geographically restricted. During microscopic observations, we also found that numerous Amoebae were engulfing the cells of Microcystis (Phagocytosis) and thus further indicating that species is probably not toxic (Plate 4, Figs. 29 & 30). Discussion and conclusion Morphological features of colonies and cells have always been used as criteria for identification of different species of the genus Microcystis (Komarek & Anagnostidis, 1998). Genetically Microcystis is consistently monophyletic and species are closely related (Komarek et al., 2014). However, growth under culture condition never results into formation of typical colonies as found in natural habitat. Morphology of colony in nature is the result of various growth factors present in natural habitat and they are absent in culture condition. Therefore, it is suggested that traditional species with distinct phenotypic and ecological features cannot be ignored for identification (Komarek & Komarkova, 2002). At the global level, 51 species (Guiry & Guiry, 2014) and India level 17 species are known. In the present work seven species have been described with their typical features collected from the state Uttar Pradesh. Two species viz. M. aeruginosa and M. flos-aquae are the most frequently recorded in water blooms and five other species viz. M. wesenbergii, M. panniformis, M. botrys, M. novacekii, M. smithii are new report from India. Ten other species viz. M. bengalensis, M. densa M. firma M. lamelliformis, M. protocystis, M. pseudofilamentosa, M. ramosa, M. robusta, M. scripta and M. viridis although known from India, were not encountered in the present work. However, M. lamelliformis, M. ramosa, M. densa and M. firma have been considered doubtful and need revision (Komarek & Anagnostidis, 1998). M. aeruginosa growing in this area (U.P.) was not found to be toxic and may be related to geographical distribution. In several species of Microcystis found in North America toxicity has not been recorded (Komarek, 2003). Acknowledgements Authors are thankful to Head, Department of Botany, University of Allahabad for providing facilities and to the Department of Science and Technology, Government of India, New Delhi, for providing financial assistance. References Banerji, J.C Studies on the Myxophyceae of Lower Bengal-I. J. Indian Bot. Soc. 15: Barhate, V.P. and J.L. Tarar, The algal flora of Tapti water works, Bhusawal, India. J. Plant Nature 1(2): Bharadwaja, Y The Myxophyceae of United Provinces, India-I. Proc. Indian Acad. Sci. B 2(1): Biswas, K Aquatic vegetation of Bengal in relation to supply of oxygen to the water. J. 11

9 Dept. Sci., Calcutta University, 8: Boone, D.R. and R.W. Castenholz, Bergey s Manual of Systematic Bacteriology. Second Edition Vol I, Springer-Verlag, New York, Bruhl, P. and K. Biswas, Algae of the Loktak lake. Mem. Asiatic Soc., Bengal, 8(5): Compere, P Algues De La Region Du Lac Tchad II Cyanophycees- Cah ORSTOM, Ser. Hydrobiologia 8(3-4): Desikachary, T.V Cyanophyta. ICAR, New Delhi, pp Dwivedi, V.K., R. Tandon and G.L. Tiwari, Polymorphic stages of the fresh water blue- Green alga, Gomphoshaeria aponina. Algae 25(3): Guiry, M.D. and G.M. Guiry, Algaebase. World-Wide Electronic Publication. National University of Ireland, Galway. Algaebase org; searched on 24 July Kant, R., O.N. Tiwari, R. Tandon and G.L. Tiwari, 2005a. Morphology, growth and perennation in Aphanothece, Cyanoprokaryote. Geophytology 35(1&2): Kant, R., O.N. Tiwari, R. Tandon and G.L. Tiwari, 2005b. Adaptive mechanism in the developmental stages of an aerophytic Cyanoprocaryote, Asterocapsa Chu: a survival factor. Natl. Acad. Sci. Letter 28(11&12): Kant, R., O.N. Tiwari, R. Tandon and G.L. Tiwari, 2005c. Growth pattern, structure, reproduction and perennation in Gloeocapsa decorticans (A. Br.) Richter. Bionature 25(1&2): Kant, R., O.N. Tiwari, R. Tandon and G.L. Tiwari, On the validity of the genus Aphanothece Nägeli, Chroococcales, Cyanoprokaryota. J. Indian Bot. Soc., 85: Kant, R., R. Tandon, V.K. Dwivedi and G.L. Tiwari, Growth pattern and new developmental stages in Chroococcus (Chroococcales, Cyanobacteria) under culture conditions. Geophytology 37: Kesarwani, S., V.K. Dwivedi, R. Tandon and R. Kant, A new record of coccoid Cyanobacateria genus Bacularia Borzi, Cyanoprokaryota from India. Natl. Acad. Sci. Lett., 31(5&6): Komarek, J Coccoid and colonial cyanobacteria In; Fresh-water algae of North-America, edited by Wehr, J.D. and R.G. Sheath. Elsvier Science, USA, pp Komarek, J. and J. Komarkova, Review of the European Microcystis morphospecies (cyanoprokaryotes) from nature. Czech Phycology Olomouc, 2: Komarek, J., J. Kastovsky, J. Mares and J.R. Johansen, Taxonomic classification of Cyanoprokaryotes (Cyanobacterial genera) 2014, using a polyphasic approach. Preslia 86: Komarek, J., J. Komarkova-Legnerova, C.L. Sant anna, M.T.P. Azevedo and P.A.C. Senna, Two common Microcystis species (Chroococcales, Cyanobacteria) from tropical America, including M. panniformis sp nov. Cryptogamie Algol. 23(2): Komarek, J. and K. Anagnostidis, Nomenclatural novelties in Chroococcalean Cyanoprokaryotes- Preslia Praha 67: Komarek, J. and K. Anagnostidis, Cyanoprokaryota. Chroococcales-Sü Wasserflora Von Mitteluropa, Spektrum Akademischer Verlag, Heidelberg, pp Kondrateva, N.V Sin o-zeleni Vodorosti Cyanophyta (Blue-green algae- Cyanophyta)- In: Vizn Prisnov Vodorost Ukr Rsr 1, 2: 524 pp., Naukova Dumka, Kiev. Martens, G.V A third list of Bengal Algae, determined by G.V. Martens, communicated by Mr. S. Kurz. Proc. Asiatic Soc., Bengal 39: McGregor Glenn, B Freshwater Cyanobacteria of North-Eastern Australia: 2. Chroococcales. Phytotaxa 133(1): Stanier, R.Y.R., M.M. Kunisawa and G. Cohen-Bazire, Purification and properties of unicellular blue-green algae (Order Chroococcales). Bact. Rev. 35: Starmach, K Cyanophyta-Sinice.- Flora Slodkow Polski 2: 753 pp, Pan Panstw Wyd. Nauk, Warszawa. Tandon, R., R. Kant, V.K. Dwivedi and G.L. Tiwari, 2014a. Morphological distinction of Cyanodermatium (Hydrococcaceae) and Radaisia (Hyellaceae) of Chroococcales 12

10 (Cyanoprokaryota). Proceedings Natl. Acad. Sci., 84b (III): Tandon, R., R. Kant, O.N. Tiwari and G.L. Tiwari, 2014b: On growth, reproduction and perennation of Pleurocapsa aurantiaca Geitler, Hyellaceae, Cyanoprokaryota. Phykos, 44 (2): Tandon, R., S. Kesarwani and G.L. Tiwari, 2016: Baeocytes and pseudo-filamentous stages of Pleurocapsa concharum Hansgirg (Cyanobacteria, Pleurocapsales) in culture. Nova Hedwigia, 102(3-4): Tandon, R., V.K. Dwivedi, R. Kant and G.L. Tiwari, On the morphology of a rare coccoid Cyanoprokaryote, Chamaecalyx swirenkoi, Chroococcales from India. J. Indian Bot. Soc., 91(4): Teiling, E Schwedische planktonalgen 3 Neue Oder Wenig Bekannte Formen-Bot. Notiser, Thomas, J., E.A. Gonzalves, 1965a. Thermal algae of Western India-I (Algae of the hot spring at Akloli and Ganesh Puri). Hydrobiologia 25: Thomas, J., E.A. Gonzalves, 1965b. Thermal algae of Western India-II (Algae of the hot spring at Palli). Hydrobiologia 25:

On growth, reproduction and perennation of Pleurocapsa aurantiaca Geitler, Hyellaceae, Cyanoprokaryota

On growth, reproduction and perennation of Pleurocapsa aurantiaca Geitler, Hyellaceae, Cyanoprokaryota Richa Tandon*, Rama Kant 1, O.N.Tiwari 2 and G.L. Tiwari Department of Botany, University of Allahabad,