SEM study of planktonic chlorophytes from the aquatic habitat of the Indian Sundarbans and their conservation status

Scanning electron microscopy (SEM) is the most modern technique for plankton research. The present paper deals with the taxonomy and morphology of some rare and endangered planktonic chlorophytes in relation to scanning electron microscopy. Water samples from the distinct water body of the Sundarbans have been concentrated and examined by scanning electron microscopy. A total of 45 species, of which 17 species of Scenedesmaceae, 10 species each of Hydrodictyaceae and Desmidiaceae, five species of Chlorococcaceae, two species of Selenastraceae and only one species of Chlorellaceae were recorded from the study site. Some species were recorded as new and rare from the study area. About 18 species including nine extremely rare, seven occasional, six frequent, four sporadic and one abundant was recorded in the present study. A detailed taxonomic description with line drawings is also included in the present communication.


Communication SEM study of planktonic chlorophytes from the aquatic habitat of the Indian Sundarbans and their conservation status
Gour Gopal Satpati & Ruma Pal 26 October 2019 | Vol. 11 | No. 13 | Pages: 14722-14744 DOI: 10.11609/jott.4422.11.13.14722-14744 INTRODUCTION Planktonic chlorophytes play an important role in the aquatic ecosystem. They act as the primary producer of the aquatic food chain. Several reports have emphasized the importance of small planktonic chlorophytes in the aquatic habitat such as pond (Anand 1998), river, and sea (Vaulot et al. 2008). They also function as the progenitor of higher plants because of their photosynthetic pigments, starch as storage reserve food and chloroplast morphology (Lewis & McCourt 2004). They possess simple structural organization and reproduction. They also belong to unicellular and multicellular form.
The planktonic flora of the Indian Sundarbans was greatly diversified due to salinity gradient. Phytoplanktons play an important role in protecting the mangrove vegetation of the planet. In our earlier reports a few micro-and macro-chlorophytes were found from different habitats of the Indian Sundarbans (Satpati & Pal 2015. The habitats such as mud surface, tree bark, pneumatophore surface, stone surface, forest floor, water surface, and stilt root surface were surveyed for filamentous algae collection. A few reports were also available from other parts of the Sundarbans (Prain 1903;Naskar & Santra 1986;Sanyal & Bal 1986;Maity et al. 1987;Pal et al. 1988; Santra & Pal 1988;Santra et al. 1991;Mandal & Naskar 1994;Mukhopadhyay & Pal 2002). Changes in atmospheric temperature and global warming remarkably affect mangrove vegetation including micro-and macro-flora. The algal species are diminishing with changes in temperature and increased salinity. Micro-and pico-planktons are becoming rare and endangered and not functioning as primary producer of the ecosystem.
The present study was undertaken in order to identify some rare and endangered planktonic chlorophytes in different water bodies of the Indian Sundarbans. All taxa were documented on SEM and illustrations were also made for the same.

Study area
The Sundarbans is the largest tiger inhabiting mangrove biosphere reserve in India and also a world heritage site designated by UNESCO. It is the largest chunk of mangrove ecosystem of the World encompassing many islands and rivers interconnected with creeks and canals. The deltaic appearance of the mangrove is formed by the confluence of three rivers, Ganges, Brahmaputra and Meghna in the Bay of Bengal. The major part of the Sundarbans (60%) lies in the Bangladesh and the remaining portion (40%) in India. The Hooghly River flows over India's state of West Bengal comprising mudflats, multiple tidal streams, open and closed mangrove systems. The Indian part of the Sundarbans is distinctive in terms of its vegetation, marine ecosystem and salinity. Continuous inundation of saline water into the fresh water ecosystem, greatly affect the floral diversity. The study area lies between 21. 516-22.883 0 N and 88.616-89.150 0 E of the southeastern part of Bay of Bengal (Image 1).

Sampling site
The sampling site varies from fresh water to brackish water. A total number of 23 sites were studied in detail. All the sampling stations belong to the 24 Parganas (South and North) of the state of West Bengal, India. The name of the sampling sites and their physico-chemical parameters are given in Table 1.

Collection of samples
The phytoplanktons were collected from aquatic habitats during tidal action and also from the brackish water areas with the help of a truncated plankton net of 25μ mesh size. The samples thus collected were thoroughly washed with running tap water or saline water and then with double distilled water to remove soil particles and other debris. The sample material was then washed with phosphate buffer saline (PBS) 2-3 times and centrifuged at 8000rpm ).

Measurement of Physicochemical Parameters
Physicochemical parameters like air and surface water temperature, pH, and salinity were recorded using digital thermometer (Eurolab ST9269B), pH meter (Eco testr) and Refractometer (Erma, Japan).

Preparation of voucher specimens
Samples were preserved in 4% (v/v) formalin and stored as voucher specimen in Calcutta University Herbarium (CUH) for further study.

Scanning Electron Microscopy (SEM)
One drop of washed material was put on a glass cover slip (Blue Star) and dried at 20°C. The samples were repeatedly washed with ethanol grade and dried at room temperature. After complete dehydration the cover slips were placed on carbon tape and put in Quorum (Q 150 TES) gold coater to coat the samples with gold. The scanning electron microscopic (SEM) images have been taken with Carl Zeiss EVO 18 (EDS 8100) microscope and Zeiss Inca Penta FETX 3 (Oxford instruments) attachment. Photographs were taken in different magnifications.

Camera Lucida drawing
The hand drawing was made under a compound microscope with the help of a prism and 0.1-and 0.2-Rotring isograph pen (Germany). The drawing was done on transparent tracing paper (A4 size). The cellular details of vegetative and reproductive parts of different species were outlined and scale measurement was given under 10X, 40X and 100X objectives with proper magnification. The cell length and breadth was measured with ocular lens as ocular division (O.D.) and standardized using stage micrometer.

Stauridium tetras (Ehrenberg) Hegewald var. apiculatum (Fritsch) Keshri et Mallick comb. nov.
(Image 2E and Figure 1C) Synonym: Pediastrum tetras (Ehrenberg) Ralfs var. apiculatum Fritsch. [Keshri & Mallick 2013]. Coenobia 4-celled, less rectangular with cells without intercellular spaces; marginal cells divided into two lobes with deep linear to cuneate incision on the outer side reaching the middle of the cell and are trapezoidal in shape; each lobe further divided into two lobes terminating in an apical nodular thickening; cells 5-15 μ in diameter and colony of four cells up to 14-28 μ in diameter. Cell wall ultrastructure varies being irregular net-like or warty.
Occurrence: aquatic; voucher number: CUH/Al/MW-199. Figure 1D) [Prescott 1982;Kim & Kim 2012]. Coenobia nearly entire, with minute interstices formed by the retuse margins; Coenobia oblong to nearly star shaped. Coenobia 8-32 celled with deep narrow sinus forming two major lobes, lobes incised to form bluntly rounded lobules. The sinus outwardly closed due to the contact of two central lobules. The ultrastructure of cell wall shows dotted appearance having minute pores.
Coenobia 16-celled, arranged more or less compactly, semicircular in outline. The outer margin is smooth, concave and extended into two blunt tapering processes. Cells 10-15 μ in diameter and coenobia are 40-80 μ in diameter. The ultrastructure of cell wall is smooth with tiny pores.
Coenobia entire with minute interstices. Central cells packed and peripheral cells with two minute lobes; margin concave between two lobes. Cell wall smooth and with reticulate ridges. The ultrastructure of cell wall shows tiny pores. Cells are 8-12 μ in diameter.

Pediastrum integrum Nägeli (Image 2I and
Coenobia 4, 8, 16 and 32 celled, without or little perforations. Cell wall reticulates with tiny granules. Shapes of the inner cells are similar to the peripheral cells. Outer margins of the peripheral cells with two truncate short processes. The tip of the processes is unequal. Coenobia 14-18 μ in diameter and cells are 4-8 μ in diameter.

Parapediastrum biradiatum (Meyen) E. Hegewald
(Image 2J and Figure 1H  Coenobia oval or circular, 8-celled, marginal cells are deeply incised and form two lobes, each lobes truncate, generally further divided into two lobes and are trapezoidal in shape, inner cells 4-6 sided with a single linear or cuneate incision; cells 5-8 μ in diameter, eight celled colonies 12-18 μ in diameter. The ultrastructure of cells and coenobia shows presence of granules throughout. The cell surface is well wrinkled and folded.

Desmodesmus bicaudatus (Dedusenko) P.M. Tsarenko (Image 3C and
Occurrence: aquatic; voucher number: CUH/Al/MW-186. Coenobia composed of four ovate, oblong cells arranged in a single series; the outer and inner cells with longitudinal teeth; apices of all cells bearing 3-4 small teeth. Cells are 6-10 μ in length and 2-4 μ in width. The electron microscopic study revealed presence of beads like structure throughout the cell wall.
Coenobia composed of four cells and arranged in single series. Cells are elongated, ellipsoid; each cell with abundant uneven spines; each pole of the individual cell contains 3-6 uneven spines; each cell contains a longitudinal ridge. Cells are 4-8 μ in length and 3-5 μ in width. The sculptured cell wall with folded margin and granules are shown under scanning electron microscope.

Journal of Threatened
Coenobia 2-4 celled, cylindrical, sometimes ellipsoid arranged in a single series. Cells are 6-12 μ long and 2-4 μ width; cells contain long spines at two opposite poles. Each cell contains a longitudinal ridge covered by small teeth. Under electron microscope the cell wall showed several granules and spines.
Coenobia 2-celled, arranged in a single row, cells cylindrical, bean shaped. Cells are 6-10 μ long and 2-4 μ width. Cell contains numerous small spines throughout the body. Under electron microscope the cell wall of each cell showed convoluted margins with numerous granules.
Coenobia composed of 4 cells; cells alternately arranged in a single series; cells are ellipsoid to cylindrical, 4-6 μ long and 1-3 μ width; each cell with 1-4 small spines and teeth. Under electron microscope the cells showed wavy margins on the cell wall and a few apertures.
Coenobia composed of 2-4 celled arranged in a single series; cells 2-6 μ long and 1.5-3 μ width; cells with long spines at the apices. Cell wall is granulated and slightly folded as shown under electron microscope.
Occurrence: aquatic; voucher number: CUH/Al/MW-168. Coenobia 2-celled arranged in a single row; cells 6-8 μ in length and 2-3 μ width; apices of the cells contain 2-4 small teeth or spines. The cell surface showed numerous small teeth and granules under scanning electron microscope.

Desmodesmus subspicatus (Chodat) E. Hegewald &
Occurrence: aquatic; voucher number: CUH/Al/MW-169. Coenobia composed of four cells arranged in a single row; cells 6-10 μ in length and 1-2 μ in width; apices of each cell with 1-4 small teeth and with a longitudinal median ridge extending between the apices of each cell. Median ridge of each cells are surrounded by folded margins. The outer two cells are covered with crown like structure shown under electron microscope.

Scenedesmus acutiformis Schröder (Image 4D and
Coenobia of 2-celled arranged in a single row; cells 10-16 μ long and 6-8 μ broad; each cell having 2-3 facial longitudinal ridges covered by folded margins. The crown-like folded cell wall with smooth surface was shown under scanning electron microscope.

Acutodesmus acuminatus (Lagerheim) Tsarenko
(Image 4E and Figure 2I Coenobia composed of two cells arranged in a curved series; cells strongly lunate with sharply pointed apices; cells 12-20 μ long and 2-4 μ width; the concave faces of the cells directed outward. The smooth cell wall is observed under electron microscope. Occurrence: aquatic; voucher number: CUH/Al/MW-195. Figure  3A)

Scenedesmus magnus Meyen (Image 4F and
Synonym: Desmodesmus magnus (Meyen) Tsarenko; Scenedesmus longus Meyen [Prescott 1982;John & Tsarenko 2002]. Coenobia composed of four cells arranged in a single row; cells cylindrical 2-6 μ long and 1-3 μ width; apices of both inner and outer end of each cell with 1-2 sharp spines. Cells are compactly arranged and contain a median ridge. The convoluted cell wall with tiny pores was observed under electron microscope.

Scenedesmus raciborskii
Coenobia 2-celled arranged in a single row; cells elliptical or spindle shaped 6-8 μ long and 2-4 μ width; the cells are swollen in the middle and tapered at the two ends. Fine ridges and folds were found on the cell wall under electron microscope.
Free living, unicellular, green, cells are solitary or sometimes in colonial form; striking variation in size shows between various cells when the alga grows in an expanded stratum, young cells are thin walled and spherical or somewhat compressed, old cells have thick walls that are often irregular in outline, chloroplasts of young cells are parietal massive cups, completely filling the cell except for a small hyaline region at one side, they contain one pyrenoid, as a cell increases in size, the chloroplast usually becomes diffuse and contains several pyrenoids, young cells are 50-125 μ in diameter and mature cells are 120-210 μ in diameter. Under electron microscope several groves and ridges were found on the cell surface. Many tiny pores were also observed on the cell walls.

Tetraëdron minimum (A. Braun) Hansgirg (Image 5C-D and
Cell flat, tetragonal, the angles rounded and without spines and processes, sometimes very minute process were found on each angles; cell margin concave; cells 8-16 μ in diameter. Various apertures and undulating margins were observed under scanning electron microscope.

Tetraëdron trigonum (Nägeli) Hansgirg (Image 5E
and Figure 3H-I) Basionym: Polyedrium trigonum Nägeli [Prescott 1982;Day et al. 1995;Hu & Wei 2006]. Cell triangular, the angles narrower and tapering at each corner, each angle was terminated to a small spine or processes, each arm of the triangle is straight, margins convex; cells 12-20 μ in diameter. Smooth and wavy cell surface was observed under electron microscope. Occurrence: aquatic; voucher number: CUH/Al/MW-178. Figure 3J) Synonym: T. trigonum var. gracile (Reinsch) DeToni Basionym: Polyedrium gracile Reinsch [Prescott 1982;Day et al. 1995;Hu & Wei 2006]. Cell triangular, the angles narrower and more curved like starfish, the angles tapering and terminated to spines; cells 10-22 μ in diameter; the arms of triangle are not straight and form V-shaped structure. The electron micrograph showed wrinkled margins and a triangular ridge on the cell surface.
Cells are found in colonies; cells are sickle shaped and in irregular arrangement; cells 2-8 μ in diameter; apices of the cells are sharply pointed. Electron micrograph showed folded and wrinkled cell surface.
Cell solitary, green, longer than broad, small spine like projections are found on the surface of the cells; sinus narrow and linear; cells 18-26 μ long and 13-17 μ broad. The convoluted cell surface with wrinkled margins was observed under scanning electron microscope.
Cell solitary, green, semi-cells trapezi-form, basal angles broadly rounded, four very small spines like projections or processes were shown at each corner of the cell; the margins were denticulate; cells 18-22 μ long and 12-16 μ broad. Ornamented cells with wavy margin were observed under scanning electron microscope.

DISCUSSION
The present investigation reveals that the Indian Sundarbans exhibit rare green planktonic diversity. A few studies on planktonic chlorophytes and diatoms were found in the Gangetic belt and Bhagirathi-Hooghly estuary of the southern coast (Mukhopadhyay & Pal 2002;Chowdhury & Pal 2008). Mukhopadhyay & Pal (2002) have reported nine species of chlorophytes and five species of rhodophytes from the estuarine and coastal region of Bay of Bengal. A detailed systematic account of 19 genera and 32 species of diatoms has been done so far from the coastal belt (Chowdhury & Pal 2008). A new planktonic diatom, Cocconeis gracilariensis was investigated from the brackish water ecosystem of the Indian Sundarbans as epiphytic on Gracilaria sp. . Continuous inundation of marine water in the freshwater ecosystem is the major problem for diminishing these planktonic chlorophytes. A total number of 46 taxa belonging to six groups have been reported from the Sundarban estuarine ecosystem (Manna et al. 2010). They have reported two green algal taxa Chlorella and Dunaliella in association with cyanobacteria and diatom assemblages. The conservation of these planktonic chlorophytes is suggested to protect the primary food chain of aquatic ecosystem. A few strains were maintained in the laboratory in isolated condition with accession number. Planktonic Eugenophytes were also reported and conserved from different brackish water habitats of the Indian Sundarbans . A total of 41 species of euglenoids were reported in our previous study . Some work was available on planktonic diatoms from the Sundarbans ecoregion (Choudhury & Bhadury 2014; ). Most of the work was conducted in the Bangladesh region of the Sundarbans mangrove (Aziz & Rahman 2011). Based on previous literature, Sarkar (2011) has reported 166 species of phytoplanktons from estuarine ecosystem and associated brackish water wetlands of the Indian Sundarbans. Most of the work has been done on cyanobacteria, diatoms and filamentous chlorophytes. In this study the present group highlighted on rare green planktonic chlorophytes which were not reported in earlier studies.
Riverine fresh water run-off and tidal influx of marine water are two antagonistic hydrological processes resulting in dynamic changes in phytoplanktons and their community structure. The continuous anthropogenic perturbations, nutrient overload, increasing human population density, globalization and economic development causes vulnerability of phytoplanktons in estuarine ecosystems (Roshith et al. 2018). Temporal succession of phytoplankton assemblages of Sundarbans' mangrove was reported in a tidal creek system of the Sundarbans mangrove (Bhattacharjee et al. 2013). The phytoplankton assemblage depends on the physicochemical parameters of water and nutrient availability. The tropical and sub-tropical coastal ecosystems of the World serve as a great carbon sink due to the presence of mangroves and phytoplanktons. The biogeochemistry of carbon regulated by the key functions of genes present in the phytoplanktons reveals to illustrate their diversity (Bhattacharjee et al. 2013). The availability of phytoplanktons and mangroves helps to maintain the aquatic food chains of the coastal environments. Roshith et al. (2018) have reported the most updated information on the green phytoplanktons of Hooghly-Matla estuary. They have reported about 44 species of green chlorophytes of which 32 belong to Chlorophyceae, 11 belong to Trebouxiophyceae and 1 to Prasinophyceae. The Indian part is still less explored and more work is needed to investigate the different areas of the Sundarbans. The detailed morphological study of