Bangladesh J. Plant Taxon. 30(2): 283-286, 2023 (December) DOI: https://doi.org/10.3329/bjpt.v30i2.70504 © 2023 Bangladesh Association of Plant Taxonomists - Short communication STUDY OF NITELLA HYALINA (CHARALES) BASED ON OOSPORE WALL ORNAMENTATION SABRINA NAZ*, MD. MONIRUZZAMAN 1, NASRIN JAHAN DIBA 2, ARFATUN NAHAR CHOWDHURY 1 AND SHAH MD. GOLAM GOUSUL AZAM 3 *Institute of Environmental Science, University of Rajshahi, Rajshahi-6205, Bangladesh. Keywords: Nitella hyalina; Oospores; SEM; Characeae; Bangladesh. Charophytes are ancestors of land plants (Laurin-Lemay et al., 2012) and multi-cellular, branched, macroscopic filaments from a few centimeters (cm) to several meters in length with colorless rhizoid (Urbanik and Kwiatkowski, 2019). The main filaments are organized into short nodes forming whorls of branches, and much longer internodal cells (Schubert, 2014). General morphology varies with environmental conditions such as temperature, depth of the water, light levels, and amount of wave action (Naz et al., 2011). In reproductive structure, oospores are multilayered, pigmented and thick-walled female sex organ (Ahmadi et al., 2012), Differential deposition in this layer of the wall is frequently sculptured and forms specific oospores wall ornamentation (Ray et al., 2001). On the different oospores characters, the ornamentation pattern is considered to be an important taxonomic marker because of its conservative nature ( Urbaniak and Blaženčić, 2012; Casanova and Karol, 2008). Nitella hyalina (DC.) Ag. is a cosmopolitan species and occurs between 70° N and 40° S on all continents (Naz et al., 2011), but seems always to be restricted to few locations ( Krause, 1997). The aim of this study is to determine the type, size and ornamentation of the o pores of Nitella hyalina (DC.) Ag. using SEM. Nitella hyalina (DC.) Ag. were collected by hand from a depth of 15-20 cm in July, 2020 from the river Padma near the Rajshahi city corporation area (24°22´0″ N, 88º 36´0″ E), in Bangladesh. Specimens were conserved under Ex situ conditions for the SEM study and identified by relevant cited literature (Naz et al. 2011, Wood and Imahori 1965). The oospores were collected from the living plants, choosing only matured oospores (dark colored or black). Oogonia were placed in a plastic pot containing 10-20 ml of distilled water (DW) about 15 days. As a result, the tube cells were removed from the oogonia. Oospores were washed several times in DW. The selected mature oospores were placed in a 1:9 solution of liquid detergent in water and kept for 12 hrs in an oven maintained at 50 °C. The oospores were again washed several times in DW. Then, oospores were centrifuged at 3000 rpm for 5 min with DW. Oospores were treated with glacial acetic acid (100%) for 10 min and centrifuged at 3000 rpm for 5 min and the acid was decanted off. The oospores were prepared by following (John and Moore, 1987) and discarded the unwanted material from the upper portion of the eppendorf tube. Glacial acetic acid (100%) was added for the second times and again centrifuged at 3000 rpm for 5 min and discard the unwanted material from the upper portion of the Eppendorf tube. Following washing and centrifuging the oospores were then passed through serial grades of alcohol (40% to absolute alcohol) for dehydration and finally stored in absolute alcohol (96%). Acetolysed and thoroughly cleaned mature oospores were mounted on specimen stubs having double-sided sticky tapes. The *Corresponding author. E-mail: drsabrina_naz@ru.ac.bd 1Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Rajshahi-6206, Bangladesh. 2Baneswar Government College, Baneswar-6260, Rajshahi, Bangladesh. 3Sarraan English Editing Services, 116 W University Pkwy, Baltimore MD 21210 USA. mailto:drsabrina_naz@ru.ac.bd 284 NAZ et al. oospores were coated with gold-palladium by a Sputter-coater and observed with a JEOL JSM- 6490 LA at an accelerated voltage of 15 kv. The SEM photographs were taken whenever desired at different magnifications. Morphological features of oospores of Nitella hyalina (DC.) Ag. were studied based on 20 oospores preserved in 70% ethanol in pyrex glass bottle for further study. Largest polar axis (LPA, length) and the largest equatorial diameter (LED, width) were measured and then isopolarity index (ISI= LPA/LED×100) was calculated. The number of ridge, width of fossa, distance from apical pole to LED (AND) and anisopolarity index (ANI=AND/LPA×100) were also examined as earlier described (Horn af Rantzien, 1956). The SEM photographs were taken at Centre for Advanced Research in Sciences (CARS) at University of Dhaka, Dhaka in Bangladesh. All statistical computations (Basic descriptive statistics and Regression Analysis) were performed with the use of IBM SPSS Statistics 20 version. No work had been done earlier on the dimension of oospores at the population level in Bangladesh using SEM. The present investigation revealed that the oospores wall ornamentation was found to have a bright brown color with a prominent ridges and membrane minutely granule (Fig. 1a-c) similar to the findings of De Winton et al. (2007). However, the patterns are characterized by a shape, slightly roughened ornamentation due to the presence of numerous irregular pits and pores, the weakly developed striae and small ribbon-like structure found on the striae (Fig. 1a-c), but were often completely or was partially detached during cleaning. The projections on the striae were absent as previously reported for Nitella hyalina (Urbaniak and Blaženčić, 2012). Under LM, the fossa wall was finely granular, but under (SEM), it was fibrous, Fig. 1(a-c). A general view of the oospores showing well developed striae and minutely fibrous fossa wall. as previously reported ( Casanova and Karol, 2008; De Winton et al., 2007; Sakayama et al., 2005). The oospores were ellipsoidal (ISI index 103.818-187.208) and had an ovoid to ellipsoidal shape (ANI index 64.676-53.487). LPA ranged from 64.65 - 97.78, with an average of 80.949±7.477 m, LED range appeared to 41.12 to 76.28 with an average of 66.614±8.214 m. There were 8 ridges on the oospores surface; the mean width of fossa was 8.909±1.607 m, as they can be from 6.83 to 13.16 m; the coefficient of variation ranged from 9.237% for oospores length to 18.041% for width of fossa. The mean of apical pole (AND) to LED was 40.9033.273 m, range of the same was found to be 34.325 to 48.89 and coefficient of variation was 8.002% (Table 1). Ahmadi et al., 2012, described that the mean value of LPA, LED, ISI-index, width of fossa and number of ridges are respectively 269.13±1, 208.06±3, 1.29±1, 31.53±1, 8±0.7, which assumes that our samples were small in size than them. The mean value of the Macedonian populations of Nitella hyalina (DC.) Ag. LPA, LED, number of ridges, width of fossa, ISI index was 330±17.8, 294±8.8, 6±0.5, 45±6.2, 1.16±0.05, respectively. The findings detected from Balkan Charophytes were dissimilar to our populations (Urbaniak and Blaženčić, 2012) which is also similar to our findings. The Pearson’s correlation between LED and LPA was given STUDY OF NITELLA HYALINA (CHARALES) BASED ON OOSPORE WALL 285 Table 1. Descriptive statistics of the oospores of Nitella hyalina (DC.) Ag. Features Mean SD Median Min. Max. V (%) LPA 80.950 7.477 81.120 64.650 97.780 9.237 LED 66.614 8.214 86.275 41.120 76.280 12.331 No. of ridges 8 00 8 8 8 0.00 Width of fossa 8.910 1.607 8.595 6.830 13.160 18.041 ISI index 122.926 16.530 119.368 103.818 187.208 13.447 AND 40.903 3.273 41.278 34.325 48.890 8.002 ANI index 50.605 1.500 50.039 64.676 53.490 2.964 AND-distance from apical pole to LED; ANI Index-Anisopolarity Index AND/LPAx100; ISI=Isopolarity Index LPA/LED*100; LED-Largest Equatorial Diameter; LPA-Largest Polar Axis; Max.-maximum; Min.- minimum; SD-Standard Deviation; V-Variation coefficient. Table 2. Pearson’s correlation between LED and LPA. r t-test p.value 95% CI 0.633 3.472 0.003 (0.265, 0.840) Predictors: (Constant), LPA, Dependent Variable: LED Table 3. Simple linear regression analysis of LED on LPA. Model Estimate Standard error t-test p.value R-square Intercept 10.30 16.288 0.632 0.535 0.401 LPA 0.696 0.200 3.471 0.003 F-value 12.05 0.003 (a) (b) Fig. 2 (a-b). a) Normal Probability Plot (N= 20) showing the deviation from expected and Observed data; b) Histogram between LPA and LED showing the mean and SD value of the population. (r=0.633), R square = 0.401, which implied that only 40.1% of the LED was explained by the LPA (Table 2). The ANOVA analysis showed that the F-value was 12.051 and p-value (significance value) is 0.003 (p<0.05), which indicated that regression equation was statistically significant in 286 NAZ et al. linear relation (Table 3). Table 3 is also, provided the qualification of the relationship between LPA and LED. With every increase of one unit in LPA, the LED (on the average) increased by 0.696 (95% CI 0.265 to 0.840) units, p<0.05. As indicated in Fig. 2(a), most splashes were close to diagonal, which indicated that standardized residuals were obeyed the normal distribution and Fig. 2(b) histograms indicated that the distribution of the residual satisfies the normality assumption. Acknowledgement We acknowledge the guidance and support of Dr. Moniruzzaman Khondker, Professor, Department of Botany, University of Dhaka during carried out the research work. References Ahmadi, A., Riahi, H., Sheidai, M. and Van Raam. J.C. 2012. A study of the oospore characteristics in some Charophytes (Characeae) of Iran. Nova Hedwigia 94: 487–504. Casanova, M.T. and K.G. Karol 2008. Monoecious Nitella species (Characeae, Charophyta) from south- eastern mainland Australia, including Nitella paludigenasp. nov. – Aust. Sys. Bot. 21: 201–216. De Winton, M.D., Dugdale, T.M. and Clayyon, J.S. 2007. An identification key for oospores of the extant charophytes of New Zealand. New Zealand Journal of Botany, 45(3), pp.463-476. 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