SEASONAL DYNAMICS OF EPILITHIC DIATOM COMMUNITY FROM THE VRLA RIVER (SOUTHEASTERN SERBIA)

Seasonal analysis of epilithic diatom community from the Vrla River was performed in four seasons a year (six times) at six sampling sites. Between the second (VR2) and the third sampling site (VR3) was located a trout fish farm. The highest number of diatom taxa was recorded in spring (149) and the lowest in autumn (93). The number of taxa that were found to be dominant during all four seasons was twenty-four. The relationship between 45 the best fitted diatom taxa, which showed conspicuous seasonal dynamics, and seasons in which these taxa were recorded was illustrated using canonical correspondence analysis (CCA). The large number of taxa was identified in all four seasons or in winter period only. Few taxa were present only in spring and summer.


INTRODUCTION
Diatoms play a key role in aquatic ecosystems for millions of years, as one of the most important primary producers on Earth. This group of algae is a valuable resource of very important chemical compounds, therefore has found application in the food, chemical and pharmaceutical industry, as well as the synthesis of biofuels (BOZARTH et al., 2009). Their application in paleoecology and, in recent years, in forensics is also very important (KALE and KARTHICK, 2015;WAGNER et al., 2017). However, diatoms have been widely used in systemic monitoring of surface waters, which is necessary, according to the Water Framework Directive (WFD, 2000) (SIMIĆ et al., 2018). Also, ecological status assessment of surface waters in Serbia is performed by studying diatoms, calculating the value of two diatom indices (OFFICIAL GAZETTE OF THE REPUBLIC OF SERBIA 74/2011). Temporal and spatial dynamics research of diatom communities is obligatory to understand the structure and dynamics of aquatic ecosystems, but also a required and effective tool for ecological status assessment (HWANG et al., 2011). The study of diatoms in rivers and streams mainly referred to their role in monitoring, while the temporal and spatial dynamics of communities were less studied (LAVOIE et al., 2008). Also, their seasonal and annual dynamics were often studied separately (SOININEN and ELORANTA, 2004).
Numerous studies from Europe and North America on the seasonal dynamics of algae indicate regular seasonal patterns in rivers and streams in the temperate climate zone (LEIRA and SABATER, 2005;KÖSTER and PIENITZ, 2006). Diatoms are dominant during the winter and continue to be the main component of the algal flora during the spring and early summer, although the composition of the species changes. Other groups of algae become dominant during the summer. Diatoms' diversity and biomass of rivers in the temperate climate zone, are the greatest during the spring, while the second maximum usually occurs in the autumn. In these rivers, seasonal changes are primarily due to differences in light availability and nitrogen concentration (ALLAN and CASTILLO, 2007).
A large number of environmental factors and their interactions affect the diatom community structure and composition in rivers and streams (KOEDOODER et al., 2019). These are complex and dynamic aquatic ecosystems in which physical and chemical parameters vary, affecting the living world in them in different ways (LENGYEL, 2016). Physical and chemical parameters that most affect the composition of diatom communities are chemical properties of water (especially pH, ion, and nutrient concentration), substrate composition, flow rate, light, water temperature and grazing. Most of these factors depend on climate, geology, topography, land use and other relief characteristics, which explains the similarity of the composition of diatom communities in regions with similar ecological conditions (BERE and TUNDISI, 2009).
The temporal dynamics of diatom communities also depends on the species diversity in the community (MYKRÄ et al., 2011). Thus, in communities with high diversity, abundance of some species can vary greatly, without a huge impact on the community composition and biomass. In low-diversity communities, small changes of diversity have a major impact on community composition. Productivity and ecosystem size can also affect the temporal dynamics of diatom communities (KOEDOODER et al., 2019). There is a difference in the composition of diatom communities in rivers and streams of different productivity. If the composition of diatom communities is observed at regional level, local ecological parameters directly affect the composition of diatom communities. However, many factors and processes (e.g., land use and ecosystem interactions) operate within larger geographical areas and affect conditions at the local level (LAMBERTI et al., 2010). So, if the influence of factors within different spatial units is not considered, important ecological information can be missed, thus alleviating the significance of some biotic and abiotic factors on the temporal dynamics of diatom communities.
Diatom study of the Vrla River has been referred to water quality assessment (JAKOVLJEVIĆ et al., , 2016b and diversity (JAKOVLJEVIĆ, 2015; JAKOVLJEVIĆ and KRIZMANIĆ, 2015; KRIZMANIĆ et al., 2016) without temporal and spatial analysis of community structure and composition.
Seasonal study of epilithic diatom community from the Vrla River aimed to: (1) compare diversity of diatom taxa throughout the seasons at investigated sites, (2) determine dominant diatom taxa in each season and (3) examine the seasonal persistence and stability of diatom community.

Study area and location data
The Vrla River is located in Southeastern Serbia (

Diatom sampling
Epilithic diatom samples were collected by removing from stones with a toothbrush and were placed in a plastic bottles (100 ml volume) according to following standard (EN 13946, 2015). The algological materials were preserved in 4% formaldehyde. A total of 36 epilithic diatom samples were collected from May 2011 to March 2012 during four seasons (six months) at six sampling sites.

Laboratory method
Laboratory processing of diatoms, which aims to remove organic content, was carried out applying the standard method (KRAMMER and LANGE- BERTALOT, 1986). This method involves the use of hot H2SO4 and KMnPO4 to obtain cleaned diatom frustules. In this way, during microscopy, different details of the diatoms cell wall become visible, which is necessary for their identification. Before microscopic analysis, permanent diatom slides were made. Prepared material was air-drying on cover glasses and mounted in Naphrax ® synthetic medium.

Microscopic, data and statistical analysis
Microscopic analysis of permanent diatom slides was performed using a 1000 x and 1600 x magnifications of Carl Zeiss AxioImager.M1 microscope, with AxioCam MRc5 camera and AxioVision 4.8 software.

RESULTS
The number of recorded taxa throughout all seasons and at all sampling sites is shown in Figure 2. By analyzing diversity for each season, considering all sampling sites, the highest diversity of diatoms was observed in spring and the lowest in autumn. The number of taxa that were found to be dominant during all four seasons was twenty-four. Their relative abundance was greater than 5% at least at one site and one season.
A total of 149 diatom taxa were recorded in the Vrla River during the spring season The number of dominant taxa, in the Vrla River during the spring season, was 14. Their relative abundance was greater than 5% at least at one site (  (Table 1)  The presence of 16 taxa with a relative abundance greater than 5% at least at one site was determined in summer ( Table 2). Of these, 12 taxa occurred at all sites. Achnanthidium subatomus (Hust.  Table 2).
Diversity of diatom taxa in autumn (November 2011) period was much lower than in the previous two seasons and included a total of 93 taxa (Figure 2). VR6 sampling site characterized by the highest number of taxa (71 taxa) and VR5 the lowest (50 taxa). The number of taxa with a relative abundance greater than 5% at least at one site was 14 (Table 3). Of these, 12 taxa occurred at all 6 sites. Nitzschia soratensis was the dominant taxon at 4 sites (VR3, VR4, VR5 and VR6) with a relative abundance of 11.91% to 40.2%. Achnanthidium subatomus was dominant taxon at VR1 (14.64%) and Cocconeis lineata at VR2 (14.89%) ( Table 3).
By analyzing diatom taxa richness in winter period (March 2012), the number of recorded taxa was 121 ( Figure 2). VR6 sampling site stood out as the site with the largest number of taxa (81 taxa) and VR1 with the lowest (46 taxa). The other three sites were characterized by a similar number of taxa (VR2 -62 taxa, VR3 -64 taxa and VR4 -58 taxa). A total of 16 dominant diatom taxa were identified (   (*) -Taxa with a relative abundance greater than 5% at least at one site are shown in Table 1; (+) -Relative abundance of taxon less than 5%; (/) -Taxon not recorded.  (*) -Taxa with a relative abundance greater than 5% at least at one site are shown in Table 1; (+) -Relative abundance of taxon less than 5%; (/) -Taxon not recorded. (*) -Taxa with a relative abundance greater than 5% at least at one site are shown in Table 1; (+) -Relative abundance of taxon less than 5%; (/) -Taxon not recorded.

DISCUSSION
The highest number of diatom taxa from the epilithic diatom community of the Vrla River (Southeastern Serbia) was recorded in spring and the lowest in autumn (Figure 1). SOININEN and ELORANTA (2004) have indicated that seasonal dynamics of diatoms depends greatly on the type of community. They pointed that epilithic diatom communities showed greater seasonal dynamics than epipelic. They recorded the highest number of diatom taxa in the epilithic communities of three boreal rivers in South Finland in September and the lowest in August. Our results are in accordance with generally accepted view that the number of diatom taxa decreases significantly in autumn and winter, while maximum diversity is reached in spring and late summer (ALLAN and CASTILLO, 2007).
Achnanthidium minutissimum, Gomphonema elegantissimum and Nitzschia soratensis were distinguished as dominant taxa in epilithic diatom community of the Vrla River during the spring period (Table 1). A. minutissimum is a taxon of wide ecological valence, which successfully inhabits both oligotrophic and eutrophic waters (LOBO et al., 2004). Nevertheless, this taxon is usually dominant in oligotrophic waters, while its relative abundance significantly reduced in eutrophic waters (KELLY et al., 2007). The dominance of this taxon in our study is expected and confirms the good ecological status of the Vrla River (JAKOVLJEVIĆ  et al., 2016). G. elegantissimum is a taxon characteristic of oligo-to weakly eutrophic running water with a carbonate base (LANGE-BERTALOT et al., 2017). Considering the composition of the substrate, as well as the ecological status of the Vrla River (JAKOVLJEVIĆ et al., 2016), dominance of this species was also expected in our study. N. soratensis, which occured as a dominant taxon in spring, has so far recorded only in fresh water with low temperature and pH > 8 (TROBAJO et al., 2012). However, our study showed this is not always the case, since this species was dominant at sites VR4 and VR5 in May 2012 (Table 1) where moderate temperature and pH < 8 were measured (JAKOVLJEVIĆ et al., 2016).
In addition to these three taxa, Achnanthidium subatomus, Cocconeis lineata and C. pseudolineata were dominant taxa in epilithic diatom community during the summer period ( Table 2). According to VAN DAM et al. (1994), these two species of the genus Cocconeis are indicators of mesotrophic and eutrophic waters. Epilithic diatom communities research of streams in Sardinia testifies about the dominance of A. subatomus in winter (LAI et al., 2016). The authors pointed out there is no information on the preferences of this taxon when it comes to organic matter, still it is known to occur successfully in oligo-to mesotrophic waters. In our study, A. subatomus distinguished as the dominant taxon during three periods (summer, autumn and winter) (Tables 2, 3, 4). Nevertheless, this taxon reached the highest relative abundance during the summer ( Table 2).
Taxa that were dominant during spring and summer also dominated in autumn and winter periods. Hannaea arcus and Nitzschia pura were exceptions, which dominated only in the winter (Table 4). In general, even though winter was not characterized with the highest number of taxa recorded, it was the most distinguished season according to the species composition ( Figure 3). H. arcus is a taxon generally considered to be oligosaprobic and tolerates a certain level of organic pollution (LANGE-BERTALOT et al., 2017). Thus, common to all dominant taxa of the Vrla River is that they are oligosaprobes, which are widely distributed in oligo-to slightly eutrophic running waters. In general, the structure and dynamics of the epilithic diatom community from the Vrla River faithfully reflects the good ecological status of this river, which is confirmed by calculating four diatom indices (IPS, IBD, CEE and DESCY) (JAKOVLJEVIĆ et al., 2016). As mentioned before, a lot of taxa were present in all four seasons or in winter period only (Figure 3). This probably indicates that in addition to many physicochemical parameters, the seasonal dynamics of diatoms is also greatly influenced by microclimatic factors. VIRTANEN (2015) emphasized that the impacts of climate changes on the temporal dynamics of diatom communities will increase in the future, changing patterns of their composition.

CONCLUSION
Our results confirm that diatoms diversity is the highest during the spring in rivers in the temperate climate zone. As a result of seasonal sampling in the Vrla River a total of 149 diatom taxa were recorded during the spring, followed by summer (140), winter (121) and autumn (93) season. The large number of taxa (24) was identified in all four seasons. Few taxa were present only in spring and summer period (3) while a lot of taxa were present in winter period only (18). Results of our research indicate that study of the temporal dynamics of diatom communities in freshwaters is necessary for proper monitoring and protection of water bodies. Seasonal variation of diatom community composition was conspicuous and probably linked to seasonal variation of many physical and chemical parameters, so further, more detailed research of diatom communities should be conducted having in mind that microclimatic characteristics also play important role. It is clear that more attention should be paid in the future to the study of temporal dynamics of diatom communities in freshwaters.