Phytoplankton of Šasko Lake and the first record of invasive toxin-producing cyanobacterium Cylindrospermopsis raciborskii ( Woloszynska ) Seenayya et Subba Raju , 1972 in

Shallow lakes are specific aquatic systems sensitive to eutrophication, due to the constant exchange of nutrients between sediments and water column and less capacity to buffer external nutrient loading. The aim of this work was to analyze phytoplankton community in Šasko Lake after the gap of almost 40 years. A total of 88 phytoplankton taxa belonging to seven divisions were identified in Šasko Lake during the warm period of year (spring and summer) in 2016. Phytoplankton composition and abundance didn’t show spatial heterogeneity, due to the small surface of the lake and wind action, but seasonal differences were observed. Spring period characterized numerical dominance of B and E functional groups of phytoplankton (codons), while summer period characterized higher species diversity and abundance in comparison to spring and dominance of B, P, Sn and H1 codons. Results showed significant increase in phytoplankton abundance (for about two orders of magnitude) as well as the increased percentage share of cyanobacteria in comparison to former investigations. Besides, for the first time, invasive, potentially toxic cyanobacteria Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba-Raju was recorded in Montenegro.


Introduction
Phytoplankton are the main primary producers in the pelagic zone (Kawecka and Eloranta 1994).Most of the phytoplankton taxa are sensitive bioindicators since their metabolism and abundance changes quickly in response to environmental changes (Padisak et al. 2006).According to Reynolds et al. (2002) and Padisak et al. (2009), functional classification approach proved to be more useful for the ecological assessment in comparison to the frequently applied taxonomic grouping of phytoplankton.Functional classification assumes grouping together phytoplankton species with similar sensitivities and tolerances to different combinations of physical, chemical and biological properties of lakes.
The annual variation of predominant phytoplankton species in lakes is driven mainly by a seasonal change of temperature, light availability, retention time, nutrient load and grazing pressure (Reynolds, 1986).Phytoplankton development in temperate lakes often follows a predictable seasonal succession as described in the PEG model (Sommer et al. 1986).However, in shallow lakes, species dominance patterns vary greatly and frequently show discrepancies from the model.One of the main reasons is polymictic nature of the shallow lakes, as winds frequently mix the whole water column preventing the formation of stable stratification (Wiedner et al. 2002).
Although shallow lakes are common in the Mediterranean region, there is still a lack of data that limits our understanding of their ecology and functioning (Beklioglu et al, 2007;Özen et al, 2010).Most of them are small and isolated (Casado & Montes, 1995).Besides, these lakes, unlike similar ones in northern parts of Europe, are often characterized by substantial fluctuations in water levels that have significant influence on ecological processes in lakes.Šasko Lake is a shallow lowland lake located in southeast Montenegro.The last phytoplankton investigation of this lake dates from 40 years ago (Petković & Petković 1971;1978).One of the distinct signs of eutrophication in lakes is a shift in phytoplankton species composition.Unfortunately, due to the lack of continuous investigations of Montenegrin lakes, literature concerning the eventual changes in phytoplankton structure and trophic conditions is missing.Therefore, the main aim of this study was to determine current qualitative and quantitative structure of the phytoplankton community of Šasko Lake.In addition, some comparisons to former phytoplankton data are made.Understanding phytoplankton community structure and dynamic of shallow lakes provides insight into the overall ecology of these lakes, what is the base for their future conservation and management..

Study area
Šasko Lake is lowland lake (1 m altitude) with Mediterranean climate, located in southeast Montenegro, near the town Ulcinj.The mean annual depth of the lake is 3.5 m, the maximal depth (8 m) is registered in sub lacustrine spring Begovo oko (southern shore), while the maximal depth in central part of the lake is 6.5 meters.The lake has a surface area that fluctuates seasonally from approximately 3.6 km 2 (dry summer season) to 5.3 km 2 (winter and spring).Beside the spring Begovo oko, lake receives water from the small river Medjurijec and has a specific connection with Bojana River.During the wet season and high water level of the Bojana River, lake receives water from the river, while during the dry season, the communication is interrupted or water in channel changes direction flowing from the lake to the Bojana River.

Sampling and analyses
Phytoplankton samples were collected from six sampling points (Figure 1) in May and August of 2016, in order to cover spring and summer season, since the effects of eutrophication are the most pronounced in warm period of the year.Samples for qualitative analyses were taken with a plankton net (25 μm pore diameter) drawn from the bottom to the top of the water column and fixed with formalin.For quantitative analyses, samples were collected using Ruttner hydrobiological bottle and transported to the laboratory for the counting of phytoplankton.Phytoplankton abundance was determined using the sedimentation method (Utermohl, 1958) and expressed as cell/L.Taxa were divided into divisions: Cyanobacteria, Bacillariophyta, Chlorophyta, Chrysophyta, Dinophyta, Euglenophyta and Xanthophyta (Reynolds, 2006).The phytoplankton species were classified into functional groups (codons) according to Reynolds et al. (2002) and Padisak et al. (2009).

Results and discussion
The phytoplankton composition of Šasko Lake revealed the presence of 88 taxa, with Chlorophyta being best represented (33 taxa), followed by Bacillariophyta (20 taxa).Cyanobacteria (12 taxa) and Euglenophyta (11 taxa) were moderately represented.Less well represented were: Dinophyta (8 taxa), Chrysophyta (3 taxa) and Xanthophyta (1 taxon), as shown in Figure 2. List of taxa recorded in Šasko Lake in two investigated periods (May and August), together with corresponding functional group (codon) is given in Table 1.The phytoplankton community of Šasko Lake consisted of two ecological formations: typical pelagic populations (euplanktonic forms) and tychoplanktonic populations (benthic/epiphytic forms), represented in the general structure of the phytoplankton community with a ratio of 9:1.In case of small lakes the surface/volume ratio is considerably higher in comparison to large lakes (Wetzel, 2001).Therefore, the ratio of benthic habitats is high and phytoplankton community usually contains detached taxa of benthic and epiphytic origin that remain in suspension (meroplankton).Such taxa were mostly represented with MP and T functional groups (codons) in Šasko Lake, with percentage share of about 9%.These taxa are adapted to turbid water and tolerant of both low and high light conditions.
Former investigation of Šasko Lake (Petković & Petković, 1978) reported higher species richness (total of 214 taxa) due to the distinctly higher number of pennate diatoms.That investigation covered a threeyear cycle and beside pelagic samples, it included also littoral samples.For that reason, a lot of registered taxa originated from the lake bottom and aquatic macrophyts and significantly contributed to higher species richness in comparison to present results.Therefore, lower species richness recorded in present investigation of the lake, was a result of different sampling methodology and shorter research period rather than consequence of some ecological changes in the lake.In contrast to previous study, in present research we didn't register typical marine/brackish taxa such as: Campylodiscus noricus Ehrenberg ex Kutzing, Bacillaria paradoxa Gmelin, Entomoneis alata Ehrenberg and Entomoneis paludosa (W.Smith) Reimer, but some of the recorded species, such as Amphipleura pelucida Kutzing, Prorocentrum mikans Ehrenberg, Acanthoceras zachariasii (Brun) Simonsen and Gyrosygma acuminatum (Kutzing) Rabenhorst could be considered as brackishness indicators.However, these species were not abundant and occurred only sporadically.* New taxa for Šasko Lake, ** New taxa for Montenegro There were no significant spatial differences in phytoplankton composition and abundance among stations or sampled depths during periods of investigation.The absence of horizontal differences may be attributed to a small distance between sampling points (i.e.small size of the lake) and thus the similar physico-chemical conditions that resulted in uniform phytoplankton community structure in different parts of the lake.On the other side, the absence of vertical differences (i.e.phytoplankton distributed uniformly throughout the water column) was mainly due to lake shallowness and frequent water mixing from surface to the bottom.
During this study, 17 functional groups (codons) were identified (Table 1): 5 groups represented the Cyanobacteria (H1, Sn, M, MP and Lo), 10 groups represented the Chlorophyta (G, F, J, N, T, X1, X2, X3, MP, W1), 6 groups represented the Bacillariophyta (A, B, C, D, P and MP), while single group represented Dynophyta (Lo), Euglenophyta (W1) and Chrysophyta (E).There were differences in phytoplankton composition between two investigated seasons (Table 1).Spring (May) was characterized with lower species richness (31 taxa) and most of recorded taxa were members of B, E, F and Lo codons (centric diatoms, chrysophytes, some chlorococcaleans and dinoflagelates): mainly oligo-to mesotrophic species that prefer lower water temperature.Codons B, E and F refer to species adapted to well mixed water columns because these species have high sinking rates and therefore need turbulences to keep them in the photic layer.Codon Lo contains motile species adapted to various habitats: deep and shallow, oligo to eutrophic, medium to large lakes (Padisak, 2006).
On the other side, summer (August) characterized significantly higher species richness (87 taxa), with greens (mainly Chlorococcales) being best represented, followed with bluegreens, pennate diatoms and euglenoids (Table 1).The highest stability of the phytoplankton community as well as the highest species richness are usual for summer period, reflecting rather well the physical and chemical conditions in lakes (Padisak et al. 2006).Most of the taxa recorded in summer belonged to J, H1, MP and W1 codons: species adapted to shallow eutrophic lakes (in case of codon W1 also lakes rich in organic matter).
The total phytoplankton abundance in Šasko Lake ranged from 8.6 x 10 4 ind/L (May) to 5.2 x 10 5 ind/L (August).In shallow lakes, the lowest water level and water exchange rate and consequently autochthonous enrichment in nutrients is characteristic for summer period and that usually leads to increase in phytoplankton abundance.In other words, phytoplankton abundance is positively correlated to water residence time (Søballe & Kimmel, 1987).
In May, diatoms numerically dominated the phytoplankton community with mean abundance of 7.4 x 10 4 ind/L, making 86% of the total phytoplankton abundance.Although centric diatoms were present with small number of species, they had much higher abundance in comparison to pennate diatoms in lake.The dominant diatoms were representatives of codon B: Pantocsekiella ocellata (Pantocsek) Kiss&Acs and P. costei (Druart et F.Straub) Kiss&Ács.Small centric diatoms are one of the best-adapted algal groups to turbulent and turbid systems (Reynolds, 2006).Because of the high sinking rate, these taxa are sensitive to the onset of stratification and usually dominant under well mixed conditions (Reynolds et al. 2002).As rstrategists, these species have high reproductive rate and efficiently exploit available light and nutrients, so they are predominantly characteristic for oligo-to mesotrophic conditions (Padisak, 2009).
Chrysophytes were the second algal group in terms of abundance in spring, with 1.2 x 10 4 ind/L, making 13.9% of the total phytoplankton abundance.The major representatives, Dinobryon divergens Imhof and D. sociale Ehrenberg, are assorted into functional group E (Reynolds et al. 2002;Padisak et al. 2009).This group is characteristic for small, shallow, oligotrophic to mesotrophic lakes.As mixotrophs, can supplement nutrient uptake by the phagotrophic ingestion of bacteria (Kamjunke et al. 2007).Apart of diatoms and chrysophytes, other algal groups had negligibly small abundance in spring (400-600 ind/L).
In summer (August), diatoms abundance (2.3 x 10 5 ind/L) was higher in comparison to spring, but they were present with lower percentage share (44.2%).The most abundant diatom species were Fragilaria crotonensis Kitton (codon P) and Pantocsekiella costei (codon B) which are typical inhabitants of eutrophic and mesotrophic waters respectively.Their high abundance in summer reflects polymictic nature of the Šasko Lake, since they are typical for well-mixed environments.
Cyanobacteria was the second group in terms of abundance in August, with density of 1.4 x 10 5 ind/L (26.9%).Cyanobacteria are tolerant of broad ranges of environmental gradient and pollution and strongly driven by physical factors such as local weather conditions.The dominant cyanobacteria and at the same time the most abundant species in summer samples was Cylindrospermopsis raciborskii known to produce different kinds of neurotoxins, hepatotoxins and skin-irritating substances.C. raciborskii wasn't recorded in Šasko Lake in former investigations and this is de facto the first record of this species in Montenegro.Trichomes of C. raciborskii were solitary, straight or mildly bent (Figure 3), ranging from 69 to 115 µm (average length 105 µm), with generally cylindrical vegetative cells (3-7.8 µm length and 2-3.5 µm width) and terminal heterocysts (4.5-9.2 µm length and 2.4-3.9 µm width).The absence of akinetes suggests favorable conditions for development of this species.C. raciborskii, as a representative of codon Sn, is adapted to warm and mixed environments.It may produce blooms when the following conditions are met: high water temperature and irradiance, low water level and poor water exchange (Berger et al. 2006, Burford & Davis, 2011;Buford et al. 2014).According to data from the Institute of Hydrometeorology and Seismology of Montenegro (http://www.meteo.co.me), extremely hot weather (62 days with air temperature above 30°C) accompanied to low precipitation in August 2016.(41.2 mm), resulted in relatively long period of high water temperatures, low water level and increase of water residence time in the Šasko Lake.These conditions probably favored summer development of C. raciborskii in the lake.This species is not recorded at all in the neighboring Skadar Lake for instance, although it is also shallow lowland lake under same climate conditions.Having in mind that C. raciborskii is sensitive to flushing, the absence of this species in Skadar Lake might be explained with the short water residence time (about 100 days, Scarbøvic et al, 2014), while the Šasko Lake represents almost the closed system during the summer, being completely separated from Bojana River.Cylindrospermopsis raciborskii was reported to be tropical in origin (Padisak, 1997), but currently it is distributed throughout most of Europe (Borics et al. 2000;Saker et al. 2003;Fastner et al. 2003;Briand et al. 2004;Manti et al. 2005;Stuken et al. 2006;Moustaka-Gouni et al. 2009) and reported in all continents except Antarctic, so it can be considered as cosmopolitan.The main mechanisms that can explain the intercontinental dispersal of this species are migratory birds and unintentional human transport (Atkinson, 1972(Atkinson, , 1980)).The occurrence of Cylindrospermopsis raciborskii in Šasko Lake further extends its known geographical distribution.
Dinophyta had the abundance of 4.7 x 10 4 ind/L (9%), with domination of Peridinium cinctum (Muller) Ehrenberg and Peridiniopsis cuningtonii Lemmerman.These species are motile mixotrophs what gives them more opportunities under light or nutrient limitation.As members of codon Lo, they occur in wide range of habitats comprising deep and shallow, oligo-to eutrophic, medium-to-large lakes (Padiśak et al. 2009).
Although Chlorophyta was the group with the highest number of detected species, they represented only 7% of total phytoplankton abundance in August (3.7 x 10 4 ind/L).Within this group, Chlorococcales (Coelastrum, Scenedesmus, Pediastrum and Oocystis) from codon J as well as Pandorina morum (codon G) were the most abundant greens in the lake.Padiśak et al. (2009) indicated that codons J and G are usually abundant in shallow, nutrient-rich systems.
Euglenophyta were present with 2.1 x 10 4 ind/L (4%), and the most abundant species were Euglena proxima Dangeard and E. granulata (Klebs) Schmitz.Euglenoid species (codon W1) have mixotrophic nutrition i.e. can use organic matter as the source of energy.Therefore, they are typical for water rich in organic matter and are indicators of higher saprobity level.The least abundant algal group in August was Chrysophyta, with 5 x 10 3 ind/L (0.9%).
The influence of various factors on phytoplankton seasonal dynamic highly depends on the type of the lake, with physical i.e. meteorological and hydrological factors (temperature, irradiance, water level, residence time, water column stability etc) being the most important and chemical (nutrient level) and biotic factors (grazing) being of lesser importance in small and shallow lakes (Bouvy, 2006;Reynolds, 2006).The absence of vertical heterogeneity, numerical predominance of taxa adapted to well-mixed conditions in both investigated seasons, as well as significant proportion of meroplanktonic taxa (MP codon) indicate generally well-mixed conditions in the Šasko Lake.On the other side, although vertical stratification is primarily a depth-dependent feature, it can sometimes periodically occur also in shallow and small water bodies (Borics et al. 2011) and support development of motile algae that can migrate vertically to avoid photoinhibition (example of codon Lo in Šasko Lake) and bloom-forming cyanobacteria with buoyancy-regulation (codons H1 and M in Šasko Lake) that permit these organisms access to optimal light and nutrient conditions (Krasznai et al. 2010).Thus, development of the species favored by water column stability, might indicate occasional occurrence of stagnation periods (steady states) in Šasko Lake during summer, in spite of the small size and depth of the lake.However, significantly lower abundance of these species in comparison to those adapted to water mixing suggests that well-mixed conditions prevail in Šasko lake during summer period.
In comparison to former phytoplankton investigations of Šasko Lake (Petković & Petković, 1971;1978;Petković, 1979), several differences were observed.In qualitative composition of phytoplankton in former research, diatoms were dominant group with 47% of all registered species, followed with Chlorophyta (29%).In present research the situation was the opposite: Chlorophyta with 37% was the most diverse group, followed with Bacillariophyta (23%).Beside increase in proportion of Chlorophyta, there is also observed increase in Cyanobacteria proportion: from 7.2% (former investigation) to 14% (current investigation).Furthermore, in former phytoplankton investigations, the density of phytoplankton was in range from 3 x 10 3 ind/L (spring) to 9 x 10 3 ind/L (summer), what was for about two orders of magnitude lower value in comparison to current data.In addition, the most abundant species in summer samples of current research was cyanobacterium C. raciborskii, while the former investigations showed numerical domination of diatoms (mainly species from genera Cyclotella, Synedra and Asterionella) and low abundance of cyanobacteria (mainly Merismopedia and Anabaena) in phytoplankton community.The increased contribution of greens and especially blue-greens in the phytoplankton community is usually considered as symptom of eutrophication of the lake (Carlson 1977;Carlson & Simpson, 1996;Reynolds, 2006;Jekatierynczuk-Rudczyk et al. 2014).Furthermore, the increase in phytoplankton abundance is also frequently cited in the literature as the first sign of eutrophication, leading to formation of phytoplankton blooms in case of significant progression of eutrophication process (Padisak et al. 2006;Zhang et al. 2009;Pasztaleniec & Poniewozik, 2010).Despite the fact that abundance of cyanobacterium C. raciborskii in Šasko Lake didn't reach the bloom values, development of this invasive species capable for production of very harmful cyanotoxins, may cause a displacement of native phytoplankton species as well as deterioration of water quality.Therefore, all the observed differences in phytoplankton community of Šasko Lake suggest the increase in trophic level of the lake over the period of last 40 years, but this assumption should be taken with caution and checked trough the further more detailed monitoring of Šasko Lake, including physicochemical parameters as well as other elements of the pelagic biocenosis.

Table 1 .
List of taxa recorded in Šasko Lake in two investigated periods in 2016.(May and August), together with corresponding functional group (codon).