A Prospective Study on Morphological Identification and Characterization of Freshwater Green Algae Based on the Microscopic Technique in District Rawalpindi

| The current study was carried out to isolate, identify and characterized fresh water green algae species from ecologically diverse habitats of Tehsil Gujar Khan, District Rawalpindi. A microscopic image data was used to identify algal species. A total of 30 species were recorded that belonged to 4 orders, 11 families, and 14 genera. Clamydomonas reinardtii, Acutodesmus obliquus and Cosmarium isthmocondrum are new record from Pakistan. Among identified taxa, Scenedesmus was the dominant genus with 8 species and their contribution was 26.6%. The 2 nd most dominant genus was Cosmarium with 6 species and their contribution was 20%. The other genera Chlorococcum , Ankistrodesmus, Coelastrum, and Closterium were (6.6%). Some genera represented only one (3.3%) species that were Chlamydomonas, Eudorina, Tetraspora, Chlorella, Westella, Pediastrum, Acutodesmus, and Stigeoclonium . Hence, the current study reveals that Gujar Khan, District Rawalpindi is a rich source


Introduction
G reen algae are one of the most diverse groups of algae, with at least 7000 species (Nelson and Garcia-Pichel, 2021).Green algae are characterized by several distinct features including filaments, colonies, branched, motile, non-motile, and bladelike thallus (Leliaert et al., 2012).Chloroplast contained chlorophyll a, b and accessory pigments including carotenes and xanthophylls which are the same pigments.They synthesize the same type of carbohydrates during photosynthesis as terrestrial plants (Nzoiwu et al., 2017).
Algal taxonomy is a key discipline in phycology and is critical for algal genetics, physiology, ecology, applied phycology, and particularly bioassessment.Taxonomic identification is the most common analysis and hypothesis-testing endeavor in science.Errors of identification are often related to the inherent problem of small organisms with morphologies that are difficult to distinguish without research-grade microscopes and taxonomic expertise in phycology (Manoylov, 2014).
Microalgae are a diverse group of aquatic photosynthetic organisms having variations in cell morphologies, life cycle, and growth patterns, and favoring a variety of habitats (Alam et al., 2019).Green algae may be found in land areas in large numbers, although certain species have specific ecological requirements.For example, flagellated chlorophytes are common in nutrientrich standing waters.Filamentous conjugating green algae are present in the stagnant water of roadside ditches and ponds as well as the littoral zones of lakes where they can form free-floating mats or mix with other algae in attached or floating masses.Desmids are more prevalent in low-conductance ponds and streams with moderate nutrient levels and they frequently mix with macrophytes (Haworth, 2016).Some algal species have the ability to face stressful environments and some species are unable to adopt themselves a stress environment.Some algal species can face stressful environments and some species are unable to adapt themselves to a stressful environment (Mukhtar et al., 2021).Many green algae may grow heterotrophically in the dark using an external source of organic carbon and this growth is faster than pure autotrophic conditions (Bell, 2013;Fan et al., 2012).In mountains, biodiversity and distribution of green algae are fragmented and overall poor.Some environments, such as the snowpack at high elevations ( Jacquemin et al., 2019), have gained more attention (Hoham and Remias, 2020).

Physiological features
Rawalpindi city is a capital of Pakistan.It is situated in the northernmost part of Punjab and comprised an area of 5,286 km² with latitude 33.626057° N and its longitude 73.071442° E (Shabbir and Ahmad, 2015).Its elevation is 508 m.The distance between Rawalpindi to Gujar Khan is 44km and 861.9 meters.The average rainfall is 1,346.8millimeters (53.02 inc), most of which falls in the monsoon season (Rehman et al., 2021).

Study area
Green algae samples were collected from different stations of Potohar Gujar Khan Tehsil of district Rawalpindi.All samples were collected during the mid of February and the start of October 2019.Its latitude is 33.2616°N and longitude is 73.3058°E with 461 m elevation.Samples were collected from polluted (ditches, stagnant water, and dam) and nonpolluted (Streams, running water, ponds and tube wells) sites.Five different sites of Gujar Khan selected for the collection of algal samples were Takia baba Rahim shah, village Sukho, Dehra Muslim, village Cheena, and Village Arrha.

Collection of samples
All algal samples were in liquid form.Forceps, gloves, falcon tubes, notebook, EC meter, thermometer, barometer, polythene bags, permanent marker, and glass jars were used for collection purpose.The field data of collected samples are mentioned in Table 1.During the collection of samples, different parameters like the color of the substrate, temperature, humidity, vegetation, and habitat were recorded on the spot.The samples were brought to the laboratory and electrical conductivity (EC) and pH were measured.The collected samples were transferred to conical flasks containing 4% formalin and stored in a growth chamber at 24-25 o C.

Microscopic observations
The collected samples were taxonomically investigated under a light microscope using different magnifications i.e., 10X, 40X, and 100X.To visualize and characterize green algae each specimen was mounted on a glass slide with the help of a dropper or micropipette.The glass slide was covered with a cover slip.A little drop of distilled water was added to the slide to remove dehydration Immersion oil was also used to observe the slide at 100X, and that oil drop was placed upon the cover slip.The dimensions of each species i.e., length and width were noted by using the calibrated eyepiece.All the morphological characters were recorded such as color, shape, pyrenoid, filamentous, branched, unbranched, unicellular, arrangement of chloroplast, and presence or absence of mucilage sheath.Each species was photographed using a camera and species were recognized by comparing them to legitimate literature.Identification was done by using authentic literature (Beherepatil and Deore, 2013;Rai, 2013b;Hegewald, 1997;Jena and Adhikary, 2007;Leghari, 2001b;Aquino et al., 2016;Vijayan et al., 2015;Ramos et al., 2015;Goldstein, 2015;Bhakta et al., 2011) and databases like algae base, UTEX, protist.com, and SAG.
Culturing procedure for algal growth Different species of green algae require different nutrient media and different pH for optimum growth under laboratory conditions.Each purified sample were inoculated in BG11 (Blue-green algae media) (George et al., 2014), BBM (Bold Basal Media), and MBBM (Modified Bold Basal Media) (Bischoff, 1963).Algal samples were kept in the growth chamber for incubation at a temperature of about 25-28°C with continuous illumination at 2500-3000 lux.When algal cultures attained exponential growth, then they were transferred to 500 mL Erlenmeyer flasks with fresh media (Gerloff et al., 1950).

Growth response on different culturing media
Green algae species showed different growth on different media.Three media were used for the isolation and purification of algal species.The growth of green algae was maximum on BBM and MBBM media.While on BG-11 media, it showed minimum or no growth.The growth response of green algae on different culturing media is given in Table 3.  Taxonomic characters: 4 cells present in mucilage, ellipsoidal in shape, visible pyrenoid, pyrenoid is present at the posterior end of the cell, width is 7.8 µm, green in colour, flagella at the anterior end, single chloroplast is present in cell, light green in colour Figure 1a.

Local distribution: New to Pakistan
Novelty: Clamydomonas reinardtii has not been isolated and describe previously from Pakistan and it is the new addition to algal flora of Pakistan.
Taxonomic characters: Colony of 14 cells present in mucilage, colony is elliptical in shape, cells are spherical in shape, cup-shaped chloroplast, pyrenoid is present, and colony is 52 µm in diameter Figure 1b.
Taxonomic characters: Cell wall smooth, chloroplast scattered throughout cell, pyrenoid is present, single pyrenoid, and cells are spherical in shape, 12µm in diameter, without mucilage, multinucleate Figure 1d.
Taxonomic characters: Colonial form diameter is 23.4 µm, cup shaped chloroplast is present, single, or parietal pyrenoid is visible under microscope, circular in shape, elliptical or oval in shape, unicellular, green in colour, thin cell wall, young cells are spherical in shape Figure 1f.
Colony of cells, single chloroplast is present, pyrenoid is not visible under microscope, cells are spherical in shape, cell wall is present, width is 31 µm, light green in colour, cells are arranged in groups Figure 1g.
Taxonomic characters: Each cell attached with protrusion of cell wall, plate-like structure, cells are spherical in shape, chloroplast is present, single pyrenoid, 28.6 µm width, without mucilage, colonies of cells range from 8 to 32 cells Figure 1i.
Local distribution: New to Pakistan.
Novelty: Coelastrum pseudomicroporum has not been isolated and describe previously from Pakistan and it is the new addition to algal flora of Pakistan.
Taxonomic characters: Cells attached with protrusion of cell wall, without mucilage, colonies of cell range from 8-32 cells, single pyrenoid, chloroplast is present, width 22 µm, without mucilage, spherical cells, free-living, colony have triangular holes Figure 1j.

Scenedesmus denticulatus var. linearis Hansgirg 1888: 268
Taxonomic characters: Colony of 4 cells, cells are cylindrical and ovid, cells with rounded ends and arranged in linear shape, cells having straight spines, 4.5-5 µm broad, 8 µm long, cells arranged slightly in zigzag pattern, dented projection at both ends of the cell Figure 2b.
Taxonomic characters: Colony of 4 cells, curved colony, cells with sharp pointed ends, cell wall without spines, 13 µm long, 7.8 µm broad, cells are slightly curved in shape, cells are arranged in a linear or irregular form, single chloroplast, paranoid is visible Figure 2e.

Scenedesmus dimorphus (Turpin) Kützing 1834: 608
Taxonomic characters: Colony of 8 cells, cells arranged in irregular series, internal cells less curved.cells apices sharp, without spines, external cells more curved in shape, visible paranoids, chloroplast present in the whole cell, cells are 15 µm long, 3.5µm broad Figure 2f.

Scenedesmus quadricauda var. bicaudatus
Hansgirg 1890: 9 ( Jena and Adhikary, 2007) Taxonomic characters: Colony of 2, 4, 8 cells, cells arranged in a linear shape, cells are rigid at the middle, long spines, cells are 2.5µm broad, 13 µm long, and spines are 17 µm long, Spines are larger than cells, Cells are ovoid or cylindrical in shape, Internal cells have no spines, smooth cell wall Figure 2h.

Ankistrodesmus braunii (Nägeli) Lemmermann 1908: 168
Taxonomic characters: Cells are solitary, no visible pyrenoids, pointed ends, chloroplast scattered at the center of the cell, cells are straight or in a curved shape, 2-5 µm long and 4.5 µm broad, cells are relatively broad, lateral view convex but irregularly Figure 2j.
Taxonomic Characters: Colony of 2-32 cells, pyrenoids are visible, pyrenoid is present at the center of the chloroplast, cells are in leaf-shaped, 26 µm long, 7.1 µm broad, coenobium is flower-shaped, pointed ends Figure 2k.
Local distribution: New to Pakistan.
Novelty: Acutodesmus obliquus has not been isolated and describe previously from Pakistan and it is the new addition to algal flora of Pakistan.
Taxonomic characters: Filamentous algae, light green in colour, main filaments form branches, end of filaments have pointed ends, width of the main filament is 7.8 µm, width of branches is 5.2 µm, branches are alternate, sharp ends, cells are present in the main axis Figure 2l.
Taxonomic characters: Body is slightly curved, rounded ends, pyrenoid is present, 273µm length, 13 µm width, chloroplast is present, empty spaces are present in chloroplast, attenuated toward apices, rounded apices, smooth cell wall, axial chloroplast is present Figure 2n.
Taxonomic characters: Pyrenoid is visible, one pyrenoid per semi-cell, 23µm long, and 18 µm broad, no mucilage is present, and 2 semi cells are connected from the base, deeply constricted in the middle, linear sinus, closed, lateral view elliptical in shape, cells are 1.5 times longer than broad Figure 2o.
Local distribution: New to Pakistan.
Novelty: Cosmarium isthmocondrum has not been isolated and describe previously from Pakistan and it is the new addition to algal flora of Pakistan.2q.

Cosmarium impressulum var. suborthogonum (Raciborski) Taft 1945: 195
Taxonomic characters: Cells are 1.5 times longer than broad, deeply constricted in the middle, linear sinus, semi-cells rectangular in shape, cell wall thicker, apical view is elliptical in shape, lateral view is circular, one pyrenoid is present in the chloroplast, length is 29µm, width is 18 µm, Isthmus is 6 µm Figure 2r.
A total of thirty different species of green algae were found to be unicellular, colonial, filamentous, branched filamentous and irregular forms.All of these were collected from different habitats.This is the first taxonomic investigation of green algae from different habitats including streams, ponds, ditches, and tube well.et al., 2010).Moreover, (Leghari, 2001a) described green filamentous algae from Sindh's lakes and ponds revealing 31 kinds of Chlorophyta from new water and Riverin lakes.
The genus Scenedesmus was dominant in all ponds and ditches.The presence of Scenedesmus sp.indicates that ponds and ditches have a high level of organic compounds and nutrient content because species of this genus prefer these conditions for their growth.These species are thought to be indicators of highly organic contaminated water.In some studies, similar results were explained about the Scenedesmus sp.diversity in highly contaminated organic water (Verma et al., 2012) and (Singh et al., 2013).
Results of our study showed that all 30 species were found in polluted and non-polluted water.From these species Chlamydomonas reinardtii, Acutodesmus obliquus and Cosmarium isthmocondrum found in our collection, are being reported here for the first time from Pakistan.These sites have some common species and have unique adaptive features which make them able to survive in both types of conditions.Similar studies were conducted by Barinova et al. (2006a) and explored 145 algal species from Alexander River (Central Israel), and Barinova et al. (2006b) reported 126 algal species from Hadera River, Israel depicting that the algae are the indicators of environmental conditions.
It is primarily of ecological, biotechnological, and commercial importance to isolate, cultivate and identify green algae from different habitats.The present work also investigated the response of green algae on three different media namely, BG-11 BBM and MBBM.Results of media response showed that BBM and MBBM media support better growth as compared to BG11 media.In this study, green algae were isolated using BBM, a standard chemically defined medium.In this medium, we were able to isolate thirty different species of green algae.We recognize that some genera are difficult to cultivate in this medium, thus we claim that our study is representative of Gujar Khan's overall green algal biodiversity (Lloyd et al., 2021).The study revealed that sampling areas with varied ecological variables play important role in influencing the distribution and diversity of subaerial algal communities.Hence, most of the readily responding types are potential candidates for biomass production and research for high value compounds deemed industrially important.

Conclusions and Recommendations
The present study depicts that Tehsil Gujar Khan, District Rawalpindi is a rich source of freshwater green algae.The most dominant genus in this region is Scenedesmus and the 2 nd most dominant genus is Cosmarium.Species of other genera like Chlorococcum, Ankistrodesmus, Coelastrum, Closterium, Chlamydomonas, Eudorina, Tetraspora, Chlorella, Westella, Pediastrum, Acutodesmus, and Stigeoclonium also prevail in this area.Three species Chlamydomonas reinardtii, Acutodesmus obliquus and Cosmarium isthmocondrum are new record to the algal flora of Pakistan.From this research it is also inferred that green algae prefer culturing on BBM and MBBM.In the district of Rawalpindi many other aquatic sites are that are yet to be explored.Exploration of remaining sites would provide a chance to record new species that might be new to alga flora of Pakistan or new to algal flora of the world.As a result, it is important to maintain algal diversity in local habitats and to conduct more systematic research on them which can only be done after the understanding of ecology and habitats of distinct algal flora.These discoveries will be useful in future research to explore freshwater green algae in Gujar khan, District Rawalpindi.
Colony of 2, 4, 8 cells, rounded apices, cells are ovoid and oblong, without spines, cells are 4.5 µm broad, 12 µm long, all cells in equal length, cells are arranged in a linear row, Internal cells are larger than external ones, internal cells narrower with rounded ends Figure 2c.

Table 1 :
Field data of collected green algae samples.

Table 2 :
Classification of collected algal species.

Table 3 :
Growth response of different green algal species.