DIVERSITY OF CHRYSOPHYCEAE ( HETEROCONTOPHYTA ) IN THE ZASAVICA RIVER ( SERBIA )

Detailed studies of the Chrysophyceae class of algae have not been undertaken in Serbia thus far. The golden algae usually occur during the winter and spring months. Chrysophyceae of the Zasavica River in Serbia were studied at two localities from December 2012 to Jun 2013. In our research, 26 taxa were recorded and the genus with the highest diversity was Mallomonas (15 species). The most abundant species during the whole study period were Synura uvella, Dinobryon divergens and Dinobryon sociale. At the beginning of summer, Chrysophyceae disappeared from the phytoplankton community.


INTRODUCTION
The Zasavica River is part of a Special Nature Reserve "Zasavica", which covers the area of north Mačva, east of the Drina River, and south of the Sava River.It is located in the municipality of Sremska Mitrovica and Bogatić.Zasavica occupies an area of 109 km 2 .The river basin is 5-6 km wide and consists of the Zasavica River and its tributaries, Batara and Žurava.The river is a 33.1 km long, with a maximum width of up to 80 m.Its depth at medium water level is 2.5 m (Stanković, 2006).The river is mostly surrounded by marshes, which indicates that the Zasavica River is powered by groundwater from the Drina and Sava rivers and therefore the Zasavica can be classified as both flowing and standing waters.
The Special Nature Reserve "Zasavica" was created in 1997 and placed under state protection as a category I natural treasure of exquisite value.The Reserve is characterized by high biodiversity.
Chrysophyceae is a heterogenic group of algae.Most are free-swimming and unicellular, but there are also colonial and filamentous forms.They possess one or two chloroplasts that contain chlorophyll a and chlorophyll c, and pigment fucoxanthin, which gives Chrysophyceae its golden-brown or brown-green color.Some species contain contractile vacuoles or stigma.Mobile forms have two flagella, commonly one active and one passive.Reproduction in Chrysophyceae can be sexual, which is rare, and asexual, by forming spores and by cell division.They are found in both marine and fresh waters.
There are numerous floristic, taxonomic and ecological studies in Serbia with only sporadic informa-tion about Chrysophyceae.However, none of them have dealt with this class specifically.The first paper to note the presence of Chrysophyceae in Serbia is the hydrobiology study of Daićko Lake by Košanin (1908a).

MATERIALS AND METHODS
Samples from the Zasavica River for physicochemical and phytoplankton analyses were taken once a month from December 2012 to June 2013 on two study sites, "Molo" (44º57'26.14" N and 19º31'37.58" E) and "Mostić" (44º56'59.26" N and 19º29'38.84"E).
In the field, water temperature, pH values, water conductivity, depth of the river and transparency were measured at the time when quality and quantity samples were collected.The main physical parameters were measured applying standard analytical methods and instruments: temperature with a multi digital thermometer, electrical conductivity with ECTestr 11+ multi range and pH value with a WTW multi 3430 pH-meter.The transparency of the river was measured by Secchi disc.
The samples for quality analysis were collected at the two study sites by using a plankton net (pore diameter of 22 µm).In the field, phytoplankton samples were placed in 100-ml plastic bottles.Algae were investigated directly from the non-preserved samples using a Zeiss AxioImager M1 microscope equipped with a camera system and software AxioVision 4.8, so some of the taxa were photographed.Taxonomic identifications of Chrysophyceae were made according to widely used taxonomic keys (Starmach, 1980;Starmach, 1985;John, 2002).After investigation, the samples were preserved with Lugol's solution.
Phytoplankton samples for quantitative analysis were collected using a Ruttner bottle (1 l volume).Samples were preserved at once in Lugol's solution in 1-l plastic bottles.Quantitative analysis of phytoplankton was made by using the Utermöhl method (Utermöhl, 1958) with a Leica inverted microscope.Results are expressed as number of cells per ml and number of individuals per ml.
All samples were analyzed and stored in the Institute of Botany and Jevremovac Botanical Garden, Faculty of Biology, University of Belgrade, Belgrade.

Physicochemical analysis
During the study period, the water level was variable, mostly low.From December 2012 to June 2013, the water level increased.The highest transparency and depth were recorded in May (170 cm) at both study sites.The lowest transparency (80 cm) was detected in December at the "Mostić" site where the smallest depth (90 cm) was also recorded in the same month.The lowest transparency (90 cm) was recorded in February at the "Molo" site, and the smallest depth (100 cm) was detected in December at the same study site.
Water temperature varied from 0.6ºC in December 2012 to 22.6ºC in June 2013 at the "Molo" site, while the "Mostić" site had less temperature fluctuations during the study period (from 2.1ºC to 21.2ºC).Ice cover was detected at both study sites in December 2012.The thickness of the ice was 7 cm at the "Molo" site, while at the "Mostić" site it was 10 cm.A thicker layer of ice provides better insulation, so the number of Chrysophyceae taxa was higher at the "Mostić" site than at the "Molo" site in this month due to a higher water temperature.
The water had pH values varying from 7.6 to 8.82, so the water of the Zasavica River is slightly alkaline which represents the appropriate habitat for Chrysophyceae (Starmach, 1985).The values of water conductivity of the Zasavica River can be characterized as medium-high (from 250 µS/cm to 820 µS/cm), and they rose from December 2012 to June 2013.

Phytoplankton analysis
In the examination of the algological samples from the Zasavica River, 26 taxa were identified from the class Chrysophyceae.The list of all identified Chrys-ophyceae taxa in the Zasavica River is given in Table 1 and some of photos are in Fig. 1.The genus Mallomonas had the highest number of taxa had (15 species), followed by the genus Dinobryon (3 species).
From the total number of taxa there were 24 present at the "Molo" site and 20 taxa at the "Mostić" site.The highest diversity of this class per single sample was recorded at the "Molo" site in March 2013 with 19 taxa, from which the highest percent (47%) belonged to the genus Mallomonas.From the total number of taxa found in the Zasavica River, 16 are new to the algal flora of Serbia (they are marked with an asterisk in Table 1).The most frequently found species were Synura uvella (during the whole study period) and Dinobryon sociale (during the whole study period except June).It was noted that Synura uvella was not present at the "Molo" site in December when the water temperature was 0.6º C.
The class Chrysophyceae is quantitatively very important in the Zasavica phytoplankton community.This class was the most abundant in December 2012 at the "Mostić" site, contributing to 58.3% (Fig. 2) from the total number of cells per liter of phytoplankton.The highest abundance of taxa belonging to this class was in winter and spring months due to the physicochemical conditions, which coincides with their ecology (Starmach, 1985).
The maximal abundance (1 861 200 cells/l) in the Zasavica River was recorded in March 2013 at the "Molo" site.The abundance of Chrysophyceae (Fig. 3) ranged from a minimal 0 cell/l in June 2013 (at both sites) to a maximal 1 861 200 cells/l in March 2013 (at the "Molo" site) and 1 164 000 cells/l in February 2013 (at the "Mostić" site).
The presence of a significant number of zygotes of the genera Dinobryon and Kephyrion in winter months indicates that it is the period of their intensive reproduction.With higher water temperatures, the diversity of this class declines, so only one taxon (Synura uvella) was recorded in June in a very low abundance.The most abundant species was Synura uvella (598 000 cells/l) in March 2013 at the "Molo" site and it was quantitatively dominant in all the investigated samples from this site.It was noted that Synura uvella was also dominant at the "Mostić" site during the whole study period, except February 2013, when Uroglena skujae, with an abundance of 532 000 cells/l, was dominant.In addition, Dinobryon sociale and Dinobryon divergens were quantitatively significant during this study.

Fig. 2 .
Fig. 2. Percentage of class Chrysophyceae present in the phytoplankton of the Zasavica River from December 2012 to June 2013.

Fig. 3 .
Fig. 3. Quantitative presence of class Chrysophyceae in the phytoplankton of the Zasavica River from December 2012 to June 2013.

Table 1 .
The list of all identified Chrysophyceae taxa from present (at two study sites from December 2012 to Jun 2013) and previous studies in the Zasavica River.All new Chrysophyceae taxa for the algal flora of Serbia are marked with asterisk.