Diversity of Cyanobacteria in the Zasavica River , Serbia

Cyanobacteria are ancient organisms that are capable of colonizing different habitats in various climatic zones due to their plasticity and rapid accommodation. They are a widely studied group of microorganisms due to the presence of many potentially toxic and invasive species. The aim of this research was a diversity exploration of the freshwater Cyanobacteria in the Zasavica River, which is part of the Special Nature Reserve “Zasavica” in Serbia. Organisms were sampled once a month at two study sites during one year. Phytoplankton and metaphyton analysis showed the presence of 50 freshwater cyanobacterial taxa, of which 12 are new taxa for Serbia. Three invasive and potentially toxic species (Cylindrospermopsis raciborskii, Sphaerospermopsis aphanizomenoides and Raphidiopsis mediterranea) were recorded only in metaphyton in April at one site. It can be expected that, if conditions change, this species can migrate and form phytoplankton blooms.


INTRODUCTION
Cyanobacteria, also known as blue-green algae, are ancient, cosmopolitan inhabitants of fresh, brackish and marine environments, of soil and moist surfaces.Their plasticity and rapid accommodation make them very successful organisms that are able to colonize different habitats in various climatic zones.Cyanobacteria are a natural component of phytoplankton in most of the world's surface waters.They fulfill key roles in the biogeochemical cycling of matter and in the structure, maintenance and biodiversity of microbial and higher organism communities (Codd et al., 2005).However, high cyanobacterial biomass has long been recognized mainly as a cause of taste and odor problems in freshwater bodies, but it also contributes to aesthetic problems, reduces recreational use, etc.The increase in human population and the consequent intensification of agriculture and industry along with deficient water management lead to the enhancement of eutrophication in many freshwater bodies (De Figueiredo et al., 2004).The occurrence of phytoplanktonic blooms, caused by the appearance of cyanobacteria in high biomass, is also becoming more frequent worldwide.Many cyanobacterial species produce toxins that cause mortality or illness in freshwater organisms, livestock and even humans (Chorus at al., 2000;Codd, 2000;Duy et al., 2000;Hitzfeld et al., 2000;Carmichael, 2001;De Figueiredo et al., 2004;Svirčev et al., 2009;Karadžić, 2011).The presence of algal toxins in the aquatic environment (Svirčev at al., 2007(Svirčev at al., , 2009(Svirčev at al., , 2013) ) has been recognized as a serious problem and is of important socioeconomic concern in many places worldwide.Knowledge about the function of these species, their toxins and the distribution of particular compounds that cyanobacteria of different genotypes, morphotypes and ecology produce, is important for hydrobiology and water quality monitoring, as well as theoretical studies about diversity and chemotaxonomy (Hrouzek, 2010).Noteworthy is that not all cyanobacterial blooms are toxic, neither are all strains within one species.Toxic and non-toxic strains show no predictable difference in appearance (Spoof et al., 2003).Environmental conditions such as higher temperature and pH values, low turbulence and high nutrient inputs (particularly phosphorus, as well as nitrogen) favor the development of planktonic cyanobacteria.Some advantageous characteristics, such as lower nutrient requirements and buoyancy regulation in the water column for achieving better light and nutrient level conditions, make some species dominant at the surface water layer (De Figueiredo et al., 2004).
There are numerous papers from the previous floristic, taxonomic and ecological studies in Serbia that contain information about taxa from Cyanobacteria division.The presence of Cyanobacteria in Serbia is first mentioned in a hydrobiology study by Schaarschmidt (1883).
The aim of this research was to explore the diversity of freshwater cyanobacteria in the River Zasavica with special attention to the presence of potentially toxic species.

MATERIALS AND METHODS
The Zasavica River is the center of the Special Nature Reserve "Zasavica" in Serbia (Predojević et al., 2014;Predojević et al., 2015) which has very high biodiversity.This river can be characterized as both flowing and standing water according to its properties.
The main physical parameters were measured using the following standard analytical methods and instruments: a multi digital thermometer for measuring the temperature, ECTestr 11+ multi range for electrical conductivity and WTW multi 3430 multiparameter meter for measuring pH value and dissolved oxygen.The transparency of the river was measured by Secchi disc.
The samples for qualitative phytoplankton analysis were collected at these two study sites by towing a plankton net (pore diameter 22 µm) through the open water.In the field, phytoplankton samples were placed in a plastic bottle (0.1 L) and preserved with Lugol's solution.One more sample that was taken for qualitative analysis was not preserved immediately, but after exploration on the same day of sampling.The metaphyton samples were collected by towing a plastic bottle (0.1 L) through the water near the bank, among the submerged vegetation.These samples were fixed with Lugol's solution immediately.
Phytoplankton samples for quantitative analysis were collected using a Ruttner bottle (1 L volume), after which they were put in 1 L plastic bottles and fixed with Lugol's solution.Phytoplankton quantitative analysis was performed with a Leica inverted microscope using the Utermöhl method (Utermöhl, 1958).The results were expressed as the number of cells mL -1 and number of individuals mL -1 .
All samples were analyzed and stored in the Institute of Botany and Botanical Garden "Jevremovac" at the Faculty of Biology, University of Belgrade.
Table 2.The list of all identified Cyanobacteria taxa from December 2012 to November 2013 in the phytoplankton and metaphyton of the Zasavica River.The new taxa for Serbia are marked with an asterisk.

Physicochemical analysis
Results of physicochemical analysis during the study period are given in Table 1.From these results it can be concluded that the Mostić site is characterized by fewer temperature fluctuations.At both sites, the higher the temperature, the greater the abundance of Cyanobacteria.Water level was low, but variable.According to values of pH of the water, it can be concluded that the water of the Zasavica River is slightly alkaline.The values of water conductivity of the Zasavica River can be characterized as medium-high.

Biological analysis
Exploration of all samples of phytoplankton and metaphyton from both study sites showed the presence of about 450 taxa from seven divisions: Cyanobacteria, Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta.From the total number of taxa, there are 50 cyanobacterial taxa (Table 2) in the Zasavica River.From the total number of recorded taxa, 12 are new for Serbia and they are marked with an asterisk in Table 2. Some of the photographs of cyanobacterial taxa are given in Fig. 1.
At the Molo site, cyanobacterial abundance ranged from a minimal value of 0 individuals mL -1 in January and April, to maximal value of 302 individuals mL -1 in August (Fig. 2a).Cyanobacterial abundance expressed as number of cells mL -1 on the same site shows the same pattern, minimal value of 0 cell mL -1 in January and April and maximal value of 6 838 cell mL -1 in August (Fig. 2b).At the Mostić site, the number of individuals mL -1 ranged from 0.8 individuals mL -1 in December, to 528 individuals mL -1 in July (Fig. 2a).Minimal abundance at Mostić expressed as the number of cells mL -1 was in December (11.2 cell mL -1 ) while maximal abundance was in August (13 888 cell mL - 1 ) like at the Molo site (Fig. 2b).The percentage of Cyanobacteria in the phytoplankton of the Zasavica River compared to the other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) from December 2012 to November 2013, when the abundance is expressed as a number of individuals mL -1 , showed the highest values during summer months (Fig. 3).The percentage of Cyanobacteria (when the abundance is expressed as the number of cells mL -1 ) in the phytoplankton of the Zasavica River compared to the other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) during the study period showed the same pattern, but higher values (Fig. 4).A dense development of submerged (Ceratophyllum sp.) and floating (Salvinia natans) plants was noticed from April to July at both study sites.In addition, when examining the phytoplankton and metaphyton samples a lot of grazing zooplankters were noticed per sample.
A total of 50 freshwater cyanobacterial taxa from 27 genera belonging to the orders Chroococcales, Oscillatoriales and Nostocales were recorded during this research.Since this is almost one-sixth of all taxa recorded in Serbia found in Zasavica River, it could be concluded that this is a locality with high diversity of freshwater cyanobacteria.However, it is documented that there are localities with even higher cyanobacterial diversity in Serbia (for example the River Ponjavica with 76 cyanobacterial taxa detected (Karadžić et al., 2013)).The River Zasavica, which is characterized both as flowing and standing water, provides a suitable habitat for the development of phytoplankton.In addition, during the study period transparency was very high, often to the bottom, which also favored phytoplankton development.
These three invasive and potentially toxic species did not cause any bloom although conditions were favorable, which is probably due to the stable state among phytoplankton and submerged vegetation.Submerged vegetation was well developed from April to July as were grazing zooplankters.Grazing zooplankters that are especially numerous within the submerged vegetation probably go out at night to graze on any developing phytoplankton.This keeps the water clear and provides good light conditions for the submerged vegetation.It seems that the submerged vegetation provides refuge for the animals against fish predation and allows large and effective grazer communities to coexist with the fish.In the places where such refuges are absent, for example, in the turbid basin, fish remove most of the grazers.This allows the development of phytoplankton algae, which reduce the light that is needed for developing plants (Moss, 1998).Finally, plant-dominated communities that have important roles in this river can be lost if the artificial eutrophication becomes higher and more rapid, because then the phytoplankton will be dominant.The thick layer of sludge at the bottom of the river and the depth of the river of about 1.1 m at medium water level, which is lower than the depth recorded about 10 years ago (2.5 m) (Stanković, 2006), provide good proof for the natural process of eutrophication.In an alternative stable state with phytoplankton dominance, it can be expected that potentially toxic and invasive cyanobacteria taxa from metaphyton will form blooms.Because of this, further monitoring is recommended.
They had the highest abundance and highest percentage compared to taxa from other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) during the summer months.When the abundance was expressed as the number of cells mL -1 , the percentage of Cyanobacteria as compared to other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) was much higher than when the percentage of abundance was expressed as the number of individuals mL -1 .This is due to the morphological properties of cyanobacterial taxa.Most of the recorded cyanobacterial taxa are trichal or colonial, which is the reason why the number of cells per individual is sometimes very high.Their high abundance is in positive correlation with low values of dissolved oxygen and high water temperature.
The diversity of cyanobacteria in the River Zasavica has changed over the last twenty years.Some of the taxa have disappeared while some new, potentially toxic and invasive, have appeared.The ecosystem is under self-control for now.If the anthropogenic impact in the future remains insignificant, changes in cyanobacterial diversity and abundance in the phytoplankton can be considered a consequence of the natural process of succession and aging of this ecosystem.

Fig. 2 .
Fig. 2. Cyanobacterial abundances expressed as (a) number of individuals mL -1 and (b) number of cells mL -1 , for both sites during the study period.

Fig. 3 .
Fig. 3.The percentage of the Cyanobacteria division in the phytoplankton of the Zasavica River compared to the other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) from December 2012 to November 2013, when the abundance was expressed as a number of individuals mL -1 .

Fig. 4 .
Fig. 4. The percentage of the Cyanobacteria division in the phytoplankton of the Zasavica River compared to the other divisions (Bacillariophyta, Chlorophyta, Cryptophyta, Chrysophyta, Euglenophyta and Pyrrophyta) during the study period, when the abundance was expressed as the number of cells mL -1 .

Table 1 .
Results of physicochemical analysis during the study period (from December 2012 to November 2013) at both study sites.