DIATOM SPECIES COMPOSITION AND THEIR SEASONAL DYNAMICS IN THE TIMOK RIVER BASIN

The aim of this paper was to present the composition and seasonal dynamics of epilthic diatoms in the Timok River basin. The diatom samples were collected along the Timok River basin in March, May, August and November 2017. Permanent diatom slides were prepared after oxidizing the organic material by the hydrogen peroxide (H2O2). The cleaned diatom materials were mounted on permanent slides using ZRAX glue. A total of 85 taxa were identified in the Timok River Basin. The benthic diatom taxa belong to 31 genera with the highest diversity observed within Navicula Bory (15), Nitzschia Hassall (12) and Gomphonema Ehrenberg (8). Quantitative analysis showed that in all seasons, Achnanthidium minutissimum and Amphora pediculus were dominant taxa. In May, they were joined by the Achnanthidium pyrenaicum, Gomphonema olivaceum and Ulnaria ulna; in August by the Cocconeis placentula, Denticula kuetzingii, Melosira varians, Navicula metareichardtiana, Nitzschia capitellata, N. fonticola and N. palea; in November by the D. kuetzingii, G. olivaceum, G. pumilum, N. metareichardtiana, N. veneta, Nitzschia capitellata and N. palea and in March by A. pyrenaicum, Diatoma moniliformis, G. olivaceum, N. palea and U. ulna. Our research is the floristical and ecological study of benthic diatoms in this basin and can form the groundwork for further research work.


INTRODUCTION
Diatoms  are a species rich group of eukaryotic unicellular microalgae and one of the most common organic matters in aquatic ecosystem (Wojtal, 2009). They play an important role the primary producers in freshwater and marine environments (Sarthou et al., 2005;Ishii et al., 2011). Diatoms are found in all aquatic habitats and in moist terrestrial habitats (Mann & Vanormelingen, 2013). They are cosmopolitan, with a wide geographical distribution and well-known autecology of most species (Wu et al, 2011;Van Dam et al., 1994). The taxonomic structure of diatom communities in rivers and streams is conditioned by water temperature, nutrient concentration, water flow, amount of light, grazing effect, substrate type and size (Wu et al., 2016). The impact of topography (altitude, longitude and latitude), geographically climatic characteristics (Tison et al., 2005), as well as glaciation (Stevenson et al., 1996), geomorphological characteristics and land use (Leland & Porter, 2000) must certainly not be neglected. The distribution, quality and quantity of diatom taxa on a temporal scale also depend on the characteristics of the species (growth rate, size) and the ecological niche of the species, since species of a broad ecological spectrum are more able to tolerate fluctuation of ecological factors in habitats than species with narrow ecological valences. The main study objectives were to present the floristic richness and seasonal dynamics of diatoms in the Timok River basin. * Corresponding author: nikola.djukic@pr.ac.rs

MATERIAL AND METHODS
The Timok is a across-border river. Most of the basin is located in eastern Serbia (98 %), while the smaller part is in the territory of Bulgaria (Paunović et al., 2008). It belongs to the Black Sea basin. The Timok River is formed by the joining of the Beli Timok and Crni Timok rivers downstream of the town Zaječar. The Beli Timok is formed by the joining of the Svrljiški Timok and Trgoviški Timok rivers on the northern outskirts of the town Knjaţevac. Timok is the largest river of eastern Serbia, the right and last tributary of the Danube in Serbia, which flows into the Danube at 845.65 river km and not far from the mouth of the Timok river is the border of Serbia and Bulgaria. In the area of the Timok River basin, the so-called Timok river system is being developed for water supply for hydroelectric power plants, supply for settlements and water industry, irrigation, and tourist valorization of watercourses (Paunović et al., 2008).

BIOLOGY
The sampling was conducted during four seasons (March, May, August and November 2017) from 8 localities ( Figure 1, Table 1) covering evenly the whole length of of the watercourse. Benthic diatoms were collected from a depth of 20-30 cm by brushing the stones with a toothbrush following Taylor et al. (2005). The samples were immediately fixed in 4% formaldehyde (HCHO).  At each site pH, conductivity, temperature and salinity were measured using a multiparameter device (PCSTester 35K), which was calibrated beforehand.
Diatom frustules were cleaned using 10% cold hydrochloric acid (HCL) and 30% hydrogen peroxide (H 2 O 2 ) to remove organic matter from the sample according to the standard method described by Taylor et al. (2005). All samples were washed several times with distilled water, then the material was mounted in ZRAX glue and permanent slides were made. The relative abundance (%) of the identified taxa was determined by counting 400 valves on each slide on the same microscope that used for qualitative analysis of diatoms, and at 1000 x magnification (EN 14407, 2014).
In order to analyse the diversity of epilithic diatom assemblages at different sampling sites and in different seasons, Shannon's Diversity Index was computed along with Pielou's evenness index following the calculations described in Heip et al. (1998).
A total of 85 taxa were identified in the Timok River basin. The benthic diatom taxa of the Timok River basin belong to 31 genera with the highest diversity observed in Navicula (15), Nitzschia (12) and Gomphonema (8). The largest number of taxa was identified at the CT8 site (44)  Smith were the most frequent taxa in the Timok basin, represented in over 75% of the samples. The same taxa are also dominant.   The Timok River basin is dominated by taxa that often occur in samples, as well as a large number of taxa that occur sporadically, which is typical of many rivers (Szabóet et al., 2005;Chatháin & Harrington, 2008;Makovinska & Hlubikova, 2015). A. minutissimum and A. pediculus were dominant taxa in all four seasons. These taxa are cosmopolitan and have widespread distribution in the epilithic communities of diatoms of many rivers (Potapova & Charles, 2003;Bere & Tundisi, 2011). A. minutissimum is a widespread taxon in acidic, base, oligo-to hypereutrophic waters (Van Dam et al., 1994;Wojtal & Sobczyk, 2006). A. pediculus is a widespread freshwater species that occasionally inhabits eutrophic and moderately polluted waters (Levkov, 2009). It is an alkalophilic species, which often occurs in waters with moderate conductivity (Van Dam et al., 1994), and is absent from acidic habitats with low electrolyte concentration (Lange-Bertalot et al., 2017). The most abundant genera were:Navicula, Gomphonemaa nd Nitzschia. This is to be expected, since researches in Serbia (Andrejić et al., 2012;Vidaković et al., 2015;Jakovljević et al., 2016aJakovljević et al., , 2016bVasiljević et al., 2017) and other countries (Hlúbiková et al., 2009;Sevindik & Kucuk, 2016;Noga et al., 2016) have shown that these three genera are dominant in the epilithic community.
In November, the values of the diversity index at almost all localities were above 3, and it was observed that the diversity of codominant taxa in the community increased and their distribution was the most uniform. The Pielou's evenness index (Pielou, 1966) follow the upward trend, ie. decreases in the value of the diversity index per season (Figure 3). Research of diatoms of the Velika Morava in Serbia has also shown the highest number of diatom taxa in the autumn (Vasiljević, 2017). The diversity index, as well as the structure of dominant taxa, 4 O N L I N E F I R S T BIOLOGY suggests a very similar community along the Timok River Basin with noticeable seasonal changes.

CONCLUSION
In this study, it was aimed to explore the composition and seasonal abundance of diatoms in the Timok River basin.
In terms of benthic diatom communities, previous data from the Timok River Basin is scattered. The presented data increase our knowledge of the river system, which is important for further prediction of diatoms as bioindicators; however, further fundamental investigations are necessary since the diatom microflora in this part of the Balkan Peninsula have been insufficiently studied regardless of a relatively long period of research.