Phytoplankton adaptation strategies under the influence of climatic changes and anthropogenic pressure on the Black Sea coastal ecosystems on the example Sevastopol Bay

Some ways of the Black Sea coastal waters phytoplankton community adaptation to changes in water temperature, nutrients concentration and anthropogenic pollution have been identified on the example of the Sevastopol Bay. The increase in water temperature and decrease in nutrient content in the studied waters during 2000 – 2014 caused a gradual decrease in the chlorophyll a concentrations, total phytoplankton biomass and its re-composition, predominantly in the summer and autumn periods. The phytoplankton restructuring was predominantly reflected by a decrease in relative diatoms contribution in the total phytoplankton biomass and an increase in dinoflagellates contribution. Among the dominant diatoms species, the share of resistant species to high temperatures, pollution, low nitrate content in the water and microzooplankton grazing was increasing. An increase in nitrate concentration in the studied waters in 2020 led to increase in total phytoplankton biomass and a predominance of diatoms species, which under the stated conditions did not lead to bloom emergence as were regularly observed earlier in the Sevastopol region.


Introduction
One of the main ecological problems is an assessment of a current state and possible evolution path of marine ecosystems within the reigning conditions of global climate changes and constantly increasing anthropogenic pressures. A primary component of any aquatic ecosystem is phytoplankton, which variabilities determine the development and dynamics of all subsequent trophic levels.
Over the last century, due to climate changes, sea water temperature globally increased by approximately 1ºC (Häder & Gao 2015). From the middle of the 1990's, within the surface layer of the deep water areas of the Black Sea, temperature changes has been characterized by an upward trend (Oguz & Glibert 2007). As a result of temperature stratification increasing in the water column, nutrient supply from the depths to the photosynthesis zone gradually decreases (Mikaelyan et al., 2018). The phytoplankton biomass as well as the matter and energy flow from the phytoplankton to upper trophic levels is therefore significantly lower than previously observed.

Black Sea
In the coastal waters in the Crimean peninsula area, the multiannual unidirectional positive trend in the water temperature of the surface layer was also observed (Repetin 2012). Therefore, the studies directed at identifying changes occurring in the primary trophic links of these water areas ecosystems due to the warming effect, are extremely relevant. Moreover, aquatic organisms are exposed to the greatest anthropogenic impact in the coastal areas of the Black Sea.
The objective of this study was to identify some phytoplankton adaptation strategies under the influences of climatic change and anthropogenic pressures on the Black Sea coastal ecosystems on the example of Sevastopol Bay.

Material and Methods
The study is based on the results of the authors' own research carried out in the Sevastopol Bay ( Fig. 1) during the period 2000 to 2020. Water samples (5 -8 l) were collected from the 0 -0.5 m layer on a monthly basis. An exception was sampling encountered during research conducted in 2014, when it was carried out on a weekly basis. For determination of phytoplankton abundance, biomass and species composition, 2 -3 l samples of sea water were concentrated using track membranes (1 μm pore size) in an inverse filtering funnel. Condensed to 50 ml samples were fixed with neutralized 1% formaldehyde (final concentration in the sample). The abundance and linear dimensions of algae cells were determined in a 0.1 ml drop, placed into a Naujotte counting chamber, with three replications under a light microscope, ZEISS Primo Star. Linear measurements were converted to cell volume using appropriate geometric formulas.
Phytoplankton organic carbon concentrations were calculated from the average cell volumes for each species of diatoms and dinoflagellates using the equations presented in the work of Menden-Deuer and Lessard (2000). In cases of other algaethe equations of Strathmann (1967) were used. Only during 2014 the phytoplankton biomass was calculated based on the concentration of chlorophyll a and the ratio between organic carbon and chlorophyll a (C/Chl a). The С/Chl a data were obtained by us during 2000-2010 for the Black Sea coastal waters in the Sevastopol area (Stelmakh & Gorbunova 2018). Phytoplankton species identification was carried out using the manual of (Tomas 1997).
The phytoplankton growth rate and microzooplankton grazing were determined by the dilution method (Landry & Hasset 1982) with daily increases of chlorophyll a concentrations (Chl a) in experimental bottles exposed in a flowtype incubator with natural luminance. Five dilutions of the original sample were used (0.1; 0.25; 0.50; 0.75; 1.0). The initial concentration of Chl a was determined only for the undiluted samples, while, for the diluted samples it was calculated using the dilution factor (DF). The observed daily phytoplankton growth rate for each of the 5 dilutions (µDF) was calculated as: µDF = ln(Chl afinal/ Chl ainitial) (1).
The linear regression equations were calculated to estimate the interrelations between the observed phytoplankton growth rate (µDF) and the dilution factor (DF) as: where µ is true phytoplankton growth rate (day -1 ) and gthe zooplankton grazing rate (day -1 ). The nutrients concentrations in the water were determined by the methods described in previous studies (Stelmakh & Gorbunova 2019).
Chlorophyll a concentration was measured in the acetone extracts by applying the fluorimetric method and calculated according to the equation presented in the work (JGOFS Protocols 1994).
Statistical treatment of the data was carried out using the software Excel 2007 for Windows. The graphs were built using the Grafer 7 program. Map construction was carried out using the program Surfer 8.

Results
Winter period. Analysis of the results showed that in December -February, during the period from 2000 to 2006, the average water temperature gradually increased from 8 to 10 °C and remaining practically constant further on (Fig. 2 a). The nitrates concentration until 2006 amounted to an average from 2 to 13 µM, and then decreased to 0-2 µM (Fig. 2 b). The average phosphate concentrations during 2000-2006 were 0-0.20 µM, and from 2010-2014 it increased to 0.25-0.60 µM. Unidirectional changes in the silicon and ammonium concentrations were not observed. The average values of the first biogenic element were 2.20-6.50 µM, while the concentrations of the second one did not exceed 1 µM. Against this background, a gradual increase in the concentration of chlorophyll a from 0.5 to 2.5 mg· m -3 by 2011 was noticed followed by a decrease to 0.7 mg· m -3 by 2014. (Fig. 2 a). A similar pattern of change was obtained for phytoplankton biomass and the maximum average value observed was 120 mg C· m -3 and the minimum value was 25 mg C· m -3 . Chlorophyll and phytoplankton biomass maxima were seen at sufficiently high nitrates, silicon and phosphate concentrations (1.56, 6.50, and 0.22 µM, respectively). The phytoplankton biomass in the studied aquatic areas was presented by dinoflagellates and diatoms. Throughout the entire multiannual observations, the small species Skeletonema costatum (Grev.) Cleve and Chaetoceros socialis Laud were dominated among diatoms by biomass. Dinoflagellates were introduced mainly by representatives of two genera: Gymnodinium and Prorocentrum. The third significant group of algae were coccolithophorides, among which Emiliania huxleyi (Lohmann) Hay & Mohler dominated. The specific diatom biomass decreased unidirectionally from 70-88% during 2000-2003 to 37% in 2014. (Fig. 2 c), which is probably due to a decrease in the nitrate concentrations present in the environment. The contribution of dinoflagellates increased from 10-30% to 50%, and coccolithophoridesfrom 1 to 10%.
Spring period. Between March and May, the water temperature increased from 11-12 °C during 2000-2006 to 15 °C in 2014. (Fig. 2 d). Unidirectional changes in the nutrients concentrations were not observed. The maximum concentrations of silicon (7.50 µM) and phosphates (0.30 µM) were detected in 2003 ( Fig. 2 e). At the same time, a sufficiently high concentration of nitrate (4 µM) was observed, which probably served as a hydrochemical basis for the increase in the chlorophyll a concentrations to 1.6 mg·m -3 and phytoplankton biomass to 260 mg C· m -3 (Fig. 2 d). The low values of these parameters during 2000-2002 were probably due to low nitrate and phosphate concentrations in the environment. The decrease in the chlorophyll concentration and phytoplankton biomass after 2003 was probably caused by a decrease in the silicon content. During these years, the chlorophyll concentration was usually no higher than 1 mg· m -3 , and the phytoplankton biomass did not exceed 100 mg C· m -3 . The diatom contribution to the total phytoplankton biomass varied on average from 75-90% during 2000-2003 to 50% in 2014. (Fig. 2 f). At the same time, the share of dinoflagellates increased from 5-20% to 30%, and share of coccolithophoridesfrom 1 to 20%.  (2) and water temperature (3), b, econcentrations of nitrates (1), silicon (2), phosphates (3) and ammonium (4), c, f-diatoms contribution (1), dinoflagellates (2) and coccolithophorides (3) in the total phytoplankton biomass in Sevastopol Bay in winter and spring Chaetoceros curvisetus Cleve, C. socialis, and S. costatum dominated by biomass among diatoms during 2000-2006, whereas during 2010-2014, Pseudo-nitzschia delicatissima (Cleve) Heiden, S. costatum, and Cyclotella caspia Grunow dominated. Among the dinoflagellates, the species belonging to the genus Prorocentrum dominated. The restructuring of the phytoplankton taxonomic structure and its species composition during 2010-2014 caused a decrease in the phytoplankton specific growth rate, as well as the rate of its consumption by microzooplankton (Table 1). Table 1. Seasonal and interannual variability of phytoplankton biomass (B), the specific phytoplankton growth rate (µ), the rate of its microzooplankton grazing (g), and the relative share of net primary production consumed by microzooplankton (g/µ) in the surface layer of Sevastopol Bay in 2006-2020. Summer period. From 2000 to 2014, during the June -August period, a gradual increase in the seasonal average water temperature of the surface layer was observed from 21 to 26-27 °C (Fig. 3 a). The nitrate concentrations reached their maximum values (3.20 µM) in 2003, after which a gradual decrease was observed to 0.30-0.70 µM. At the same time, the amount of ammonium was 2 times higher (Fig. 3 b) The maximum chlorophyll a concentration, 1.91 mg· m -3 , was observed in 2003, after which it decreased to 1 mg· m -3 . Phytoplankton biomass was observed gradually decreasing from 350 to 130 mg C·m -3 during the entire study period (Fig. 3a). The dynamics of the relative diatoms biomass had a negative trend (Fig. 3 c) and as a result, this algae group proportion was only 18% by 2014. The dinoflagellates specific biomass dynamics was characterized by a positive trend and by the end of the study period, this value was equal to 67%. The coccolithophorides contribution also gradually increased and reached 15% in 2014. A decrease in the relative diatom contribution to the total phytoplankton biomass was accompanied by a change in the species composition. Thus, in 2002 and 2006, during the months June -August, representatives of the Chaetoceros genus prevailed among diatoms, primarily, C. socialis, as well as Coscinodiscus sp., Proboscia alata (Brightwell) Sundström, P. delicatissima, Licmophora ehrenbergii (Kützing) Grunow, Thalassionema nitzshioides Grun. and Cerataulina pelagica (Cleve) Hendey. Whereas in 2010 and 2014, representatives of the Chaetoceros genus, which for many previous decades developed massively, reaching the level of bloom in the studied waters, were not observed among the dominant species. Species such as Pseudosolenia calcaravis (Schultze) B. G. Sundström, Nitzschia tenuirostris Mer., Striatella interrupta (Ehrenberg) Heiberg and Cyclotella caspia Grunow predominated among diatoms. Dinoflagellates were introduced mainly by representatives of the Gynmodinium genus as well as Prorocentrum cordatum (Ostf.) Dodge, Prorocentrum micans Ehrenberg, Prorocentrum lima (Ehrenberg) Stein, Prorocentrum compressum (Bailey) Dodge. E. huxleyi dominated among coccolithophorides. During the 2010-2014 period, a significant decrease in the specific rate of phytoplankton grazing by microzooplankton in comparison to 2006 was determined. As a result, the primary production proportion consumed by microzooplankton (g/µ) decreased from 81 % in 2006 to 34 % in 2014 (Table 1).  Autumn period. Within the months of September -November, the water temperature gradually increased from 17 °C during 2001 to 18 °C during 2014. (Fig. 3 d). Against the background of slightly  (Fig. 3d). Phytoplankton biomass also changed in a similar way. In the first observational period, it was noted as 200-400 mg C· m -3 , while in the second period it decreased to 60-140 mg C·m -3 . Under these conditions, the diatom contribution in the total phytoplankton biomass decreased from 80-95% to 30-50% over the entire observation period. The contribution of dinoflagellates increased from 10-15% to 40-70%. The coccolithophorides contribution in the total phytoplankton biomass increased from 2 to 10% between 2000 and 2014 (Fig. 3 f). In the autumn phytoplankton during the entire period of research, among diatoms species such as P. delicatissima, Dactyliosolen fragilissimus (Bergon) Hasle and C. pelagica dominated; among dinoflagellates representatives of the Prorocentrum and Ceratium genus dominated.
The winterspring and summer periods in 2020. The studies carried out in the winter and summer of 2020 in Sevastopol Bay indicated that during these periods there was a decrease in water temperature in comparison to 2010 and 2014. (Fig. 2 a, 3 a). Only during the spring period the average water temperature was similar as in 2010, amounting to 12.8 °C (Fig. 2 d). An increase in the nitrates and silicon concentrations within the environment was determined in comparison to 2014. This was accompanied by an increase in diatom contribution in the total phytoplankton biomass to 65-78%, and a decrease in the relative dinoflagellates biomass to 21-33%, and coccolithophorides to 1-5 % (2 c, f, 3 c). However, among the dominant species of algae, as in 2010-2014, there were no representatives of the Chaetoceros genus, which (until 2007) caused blooms over prolonged periods of time moreover, none of the three main taxonomic groups of algae reached these levels of bloom, including coccolithophoride Emiliania huxleyi. In July and August 2020, Pseudosolenia calcar-avis, one of the largest marine diatoms, dominated in phytoplankton. In the summer 2020 the maximum average seasonal phytoplankton biomass (274 mg C· m -3 ) was recorded for the entire multiannual observation period. Its value was 1.7 times higher than the corresponding value obtained in 2014. This parameter was also significantly higher in the spring period than the one for an equal period in 2014 (table 1). However, in the spring period of 2020 the primary production share consumed by microzooplankton, was 79 %, while in the summer it decreased to 32 %.

Discussion
The marine phytoplankton dynamics is determined by the combined influence of abiotic and biotic environmental factors. Climate and increased anthropogenic impacts affect these factors, which leads to changes in the phytoplankton structure and its abundance (Winder & Sommer 2012). Multiannual observations are required to identify the main trends in the phytoplankton community in the Black Sea coastal areas. In the Russian sector of the Black Sea, such activities are carried out in the North-Eastern regions, and they indicate a decrease in the phytoplankton abundance and biomass during recent years, changes in its species composition in the bays of the resort cities of Anapa and Gelendzhik (Yasakova 2014) are noted. Analysis of the multiannual phytoplankton dynamics in Sevastopol Bay, performed for the period 1940-2001, indicates that there are no unidirectional changes in the average annual values of phytoplankton biomass at this time. The consistency of the taxonomic composition of phytoplankton's dominant species during a number of decades was observed, which, according to the authors, indicates a significant degree of resistance of this community to anthropogenic, climatic and biotic influences within this region (Polikarpov et al. 2003) however, over a period of the past 20 years, significant changes in hydrochemical, physicochemical and some biological characteristics have been observed in the Sevastopol Bay ecosystem (Orekhova & Varenik 2018). These changes led to the development of hypoxia and anaerobic conditions within the upper layer of bottom sediments and natural regimes as well as natural cycles of transformation. These changes probably affected plankton communities as well.
Our analysis indicated that from 2000 to 2014, in the Sevastopol Bay, unidirectional changes in phytoplankton were observed in summer and autumn, when the temperature stratification of the water column is most pronounced. During the study period, the average seasonal chlorophyll a concentrations and total phytoplankton biomass decreased by 2-5 times. This was due to the low nitrates and silicon concentrations in water after 2006, as well as a decrease in the relative diatom contribution in the total phytoplankton biomass in favor of dinoflagellates and coccolithophorides. One of the features of dinoflagellates is that they have significantly lower (2-3 times) in comparison to diatoms specific chlorophyll a concentrations in cells and low values of specific growth rate (Mansurova 2013) as well as relatively low efficiency of photosystem 2 (Akimov & Solomonova 2019). Therefore, with other conditions equal, they provide a lower increase in the chlorophyll concentration and phytoplankton biomass. A decrease in the diatom contribution to the total phytoplankton biomass was observed in the Bay practically during all seasons. One of the main reasons for such changes in the phytoplankton taxonomic composition is due to a decrease in the nitrates and silicon compounds content in the environment. These nutrients are necessary for intensive diatom development, while dinoflagellates and coccolithophorides grow well on ammonium nitrogen with the absence of silicon and nitrates (Glibert et al. 2016). It is quite natural that in the winterspring period and during the summer of 2020, diatoms prevailed by the biomass again. The increase in their contribution is due to a significant increase in the nitrates and silicon concentrations in the waters of the Sevastopol Bay as a result of increased effluent discharge from household drains as well as the runoff of the river Chernaya.
The low relative diatom biomass during the summer of 2010-2014 compared to the previous period (2000)(2001)(2002)(2003)(2004)(2005)(2006) is due not only to a decrease in the nutrients content, but also to the effects of high temperature. The direct negative impact of the surface water layer high summer temperature on diatoms, which has increased by 6 °C over 15 years, is probably due to the fact that the maximum growth temperature, after which the degradation of their representatives, in particular S. costatum and C. curvisetus, is 23-25 °C. While for dinoflagellates, this parameter is 4-6 °C higher. Although some diatoms species isolated from Black Sea plankton, such as Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle and Cylindrotheca closterium (Ehrenberg) Reimann & J. C. Lewin, which in their temperature resistance do not differ from dinoflagellates (Akimov & Solomonova 2019). Perhaps, due to this reason, during the summer periods 2010-2014, within the studied Bay, previously dominating diatoms, such as representatives of the Chaetoceros genus, were represented in low quantity. At the same time, other species such as P. calcar-avis and N. tenuirostris became predominant.
The indirect negative impact of high temperatures on diatoms in summer is probably due to the fact that the Bay, with the contribution of household effluent, receive a large amount of organic forms of nitrogen, which in the process of transformation provides ammonium nitrogen. Its content in the Sevastopol Bay in the summer of 2010-2014 exceeded the content of nitrates by about 2 times. It is known that this nitrogen form, even at low concentrations consisted of tenths of µM, and it inhibits the nitrate absorption by microalgae (Lomas & Glibert, 1999). As a result, limitation on the growth of diatoms by nitrogen may increase, since it is nitrates that are necessary for their intensive growth. Another possible reason for a decrease in the diatoms contribution in phytoplankton at high temperatures may be an increase in the rate of microzooplankton grazing on phytoplankton as the ambient temperature increases as these heterotrophic organisms are sensitive to temperature (Allen et al. 2005). Microzooplankton is the main consumer of smalland medium-sized diatom cells in the sea (Sherr & Sherr 2007;Stelmakh & Georgieva 2014) and therefore, their insignificant contribution in the total phytoplankton biomass in the summer of 2014 and 2020 led to a decrease in the relative share of primary production consumed by microzooplankton to 32-34 %.