Periphytic Ciliate Communities in Lake Ecosystem of Temperate Riverine Floodplain: Variability in Taxonomic and Functional Composition and Diversity with Seasons and Hydrological Changes

Abstract

Periphytic ciliate communities of riverine floodplains have hardly been studied, although they play an important role in aquatic food webs and contribute to the overall ecosystem functioning. In this study we analyzed the taxonomic and functional composition and diversity of these communities across all seasons and hydrological phases. The study was conducted in a floodplain lake, a part of the large natural Danube floodplain, from February 2015 to September 2016. We found that higher temperature and hydrologically stable conditions during the lake isolation phase, when a high amount of suspended food is available, result in the highest ciliate abundances and dominance of relatively large suspension-feeding ciliates, mainly peritrichs, which could serve as good bioindicators for detecting disturbances in river-floodplain ecosystems. During the flow pulse phase, associated with lower temperatures, and during the phase of extreme floods, when the availability of suspended food was largely reduced, small surface-feeding ciliates prevailed in the periphyton. Further, while the total ciliate abundance was the lowest, the highest taxonomic and functional diversity was found, especially during an intermediate level of hydrological connectivity (flow pulse). Our results confirm the importance of different levels of hydrological connectivity for maintaining biodiversity in riverine floodplains and add to a growing awareness of the need to preserve the natural hydrological regimes of large rivers.

1. Introduction

Periphytic or biofilm communities are complex microecosystems composed of both primary producers and various consumers living in a heterogenous matrix of extracellular polymeric substances, detrital material, and inorganic particles [1,2,3]. Ciliates (Ciliophora), the group of phagotrophic protists, often dominate periphytic communities and are usually represented by high abundance and diversity [4,5,6]. Because they utilize food particles of various sizes, ranging from bacteria to small metazoans, and thus provide food for organisms at higher trophic levels, ciliates serve as important mediators of carbon and energy flux within aquatic food webs [4,7,8]. Being the main consumers of bacteria and algae, they play a key role in controlling their populations in freshwater habitats [7,9,10].

An integrative approach that captures both taxonomic and functional diversity is increasingly used in the study of aquatic communities, as it provides a deeper understanding of the relationships between community structure and ecosystem functions, and leads to a comprehensive view of ecosystem biodiversity [11,12,13]. Functional diversity studies are usually based on the analysis of species functional traits [14,15]. One of the most acceptable definitions of functional traits is one that describes them as morphological (e.g., body size, length), physiological (e.g., basal metabolic rate), or behavioral (e.g., feeding strategy, predator evasion strategies) characteristics of organisms that affect not only their fitness but also their response to the environment and/or their impact on ecosystems [16]. Since microbial communities are known to have a high degree of functional redundancy, arranging ciliates into functional groups based on their functional traits allows for a better assessment of their response to environmental changes [17]. Traditionally, ciliate functional feeding groups based on food sources [18,19,20] and feeding strategies have been used to understand changes in trophic structure and assess water quality in freshwater ecosystems [17,21], as well as their preferred habitat and locomotion [22]. For instance, sessile filter feeders (suspension-feeding ciliates such as peritrichs) can consume considerable amounts of planktonic pico- and nano-sized food particles (bacteria and small algae) [5,6,23], and several studies pointed out that these ciliates, which often prevail in mature periphytic communities, play a major role in importing organic matter from planktonic to the periphytic food web, thus contributing to the coupling of pelagic and benthic habitats [6,24,25]. Motile ciliates that feed on surface-associated food items can affect the periphyton morphology through grazing on the periphyton matrix, thereby altering the periphyton spatial heterogeneity and affecting the population dynamics of other periphytic microorganisms, and they contribute to the carbon flux in the periphyton [20,26,27].

Taxonomic and functional composition and diversity of periphytic ciliate communities are driven by environmental factors such as temperature, hydrology, water chemistry, grazing pressure, and structure of the planktonic communities [4,24,28,29,30]. Responses of ciliate communities to environmental changes are best studied after the community has reached equilibrium and become stable [25,31], which usually occurs in period of two weeks or even earlier in nutrient-rich ecosystems [32,33,34]. As opposed to early phases of development, mature periphytic communities are largely controlled by resource availability and biotic interactions [35,36]. In freshwater habitats of temperate regions, seasonal changes in environmental conditions, especially light and temperature, can significantly alter the structure of periphytic communities and the taxonomic and functional composition of ciliates in periphyton [4,24,37]. Riverine floodplains represent complex and highly dynamic ecosystems characterized by high productivity and biodiversity. In these systems, hydrological connectivity, enabled by the expansion and contraction of surface waters (flow and flood pulses), is considered a key process allowing the exchange of energy and matter (including biota) between the main river channel and floodplain water bodies and maintenance of biodiversity [38,39,40]. Floods in these systems represent a variably strong disturbance that can modify or even reset environmental conditions in a floodplain water body, disrupting the community development and leading to an arrangement comparable to early successional stages [40,41]. Tockner et al. [38] emphasize that in river-floodplain systems of temperate regions, hydrological regimes and temperature represent the two principle driving variables of biota.

In the Kopački Rit Nature Park, a floodplain area of the Danube River, hydrological changes significantly modify the structure of planktonic [42,43] and periphytic communities [44,45]. Flood pulses strongly disturb planktonic communities in this floodplain area, probably due to the dilution and washout effect of the floodwaters [42,43,46]. The periphytic ciliate communities in Kopački Rit are also strongly affected by hydrological variability [47]. We found that flooding events cause a shift from a community dominated by sessile, suspension-feeding ciliates to one dominated by small, motile, surface-feeding taxa. Our findings are consistent with the predictions of the intermediate disturbance hypothesis (IDH), which also states that the species diversity is highest at intermediate disturbance frequency or intensity due to the coexistence of a variety of taxa [48]. However, ecosystems exposed to high or very low levels of disturbance are characterized by low species diversity, due to dominance of rapid colonizers tolerating the disturbance at high and strong competitors controlling the resources at low disturbance levels. This hypothesis can also be applied to floodplain ecosystems, where the highest species diversity can be expected during an intermediate level of hydrological connectivity [11,40].

The seasonal dynamics of periphytic ciliate communities and influence of different levels of hydrological connectivity on community dynamics have not been sufficiently studied, both in the Kopački Rit floodplain and in other river-floodplain ecosystems, while studies of their functional diversity are completely lacking. Therefore, we conducted an 18-month study in a temperate floodplain lake, covering all seasons and hydrological phases (isolation, flow pulse, and flood pulse), as well as both taxonomic and functional aspects of community analysis. The primary objectives of the study were to (1) determine the taxonomic composition and diversity of the mature (stable) periphytic ciliate communities across all seasons and hydrological phases, (2) analyze their functional traits and assess functional diversity, and to (3) determine the environmental factors that significantly influence periphytic ciliate communities, their taxonomic and functional diversity, across different seasons. We hypothesized that communities will be driven primarily by temperature variations (i.e., seasonality), whereby higher temperatures would stimulate ciliate growth rates leading to higher values of total abundance. However, communities will also be significantly controlled by hydrological conditions and resource availability (suspended vs. surface-associated food items) which are highly interrelated in Lake Sakadaš [47]. Accordingly, we tested the hypothesis that during the phase of lake isolation (without disturbance) characterized by the high amounts of suspended food particles, periphytic communities will be characterized by high ciliate abundances, particularly by relatively large, suspension-feeding ciliates (filter feeders), representing mature periphytic ciliate communities. Further, we anticipated that flood pulses, especially extreme ones, as a strong disturbance will reduce the availability of suspended food particles, thus reducing ciliate abundances and favoring small, fast-growing, surface-feeding ciliates, which are considered highly resistant to disturbances. Isolation phase (without disturbance) and flood pulse phase (high levels of disturbance) will support low taxonomic diversity, and consequently low functional diversity of ciliates. The highest taxonomic and functional diversity are expected during an intermediate level of disturbance, i.e., flow pulse phase.

2. Materials and Methods

2.1. Study Site

The present study was performed in one of the largest natural floodplain areas placed in the central section of the Danube River, between 1383 and 1410 river km, in the north-east of Croatia (Figure 1). The Kopački Rit floodplain is a protected area, having the status of a nature park, while its international significance was confirmed by the inclusion on the Ramsar List of Wetlands of International Importance. A sampling station was in Lake Sakadaš, the deepest water depression in this area, in a small bay in the eastern part of the lake, near the shore (Figure 1a). A mean depth of Lake Sakadaš is about 5 m, while a surface area covers about 0.15 km². In recent decades, it has been classified as a eutrophic-hypertrophic system [49,50].

Lake Sakadaš, positioned in the western part of the Kopački Rit, is hydrologically connected to the River Danube via approximately 10 km long channel system (Figure 1a), and Danube water level oscillations primarily determine the hydrological conditions in the lake [51]. When Danube water level at the Apatin gauge station (1401.4 river km) is below 1.67 m, the lake is isolated from the river channel. Hydrological connectivity between the lake and the river is established at a water level of 1.67 m when flow pulse begins, leading to the increase in lake depth [52]. The phase of flood pulse, during which the water level rises above bank level and inundates the surrounding terrestrial area, occurs at Danube water levels above 3 m [51]. While the phase of lake isolation is characterized by the most stable conditions stimulating the development of lake communities, flood pulses represent a strong disturbance that considerably modify environmental conditions in the lake, consequently disrupting the structure of lake communities [42,46,47]. Floods may occur at any time of the year, yet they are typical for the spring and early summer period due to more intense snowmelt in the Alps [53]. Minor floods (as categorized by Mihaljević and Stević [54]) inundate only the lowermost terrain in the floodplain (Figure 1b), while extremely high floods inundate almost the entire floodplain area (Figure 1c) [55].

Figure 1. Maps of the study area (Kopački Rit floodplain) at the Danube water level: (a) below 1.5 m (phase of the Lake Sakadaš isolation); (b) between 2.5 and 3.5 m (flow pulse to minor flooding); (c) around 5 m (major flooding during which most of the area is inundated (modified from Mihaljević and Stević [54] and Schwarz [55]). Position of the Kopački Rit in Croatia is marked with a black dot, while the border of the floodplain is marked with a dotted line. The sampling station in the Lake Sakadaš is marked with a gray point. A channel system connecting the Lake Sakadaš and the Danube is signified.

Figure 1. Maps of the study area (Kopački Rit floodplain) at the Danube water level: (a) below 1.5 m (phase of the Lake Sakadaš isolation); (b) between 2.5 and 3.5 m (flow pulse to minor flooding); (c) around 5 m (major flooding during which most of the area is inundated (modified from Mihaljević and Stević [54] and Schwarz [55]). Position of the Kopački Rit in Croatia is marked with a black dot, while the border of the floodplain is marked with a dotted line. The sampling station in the Lake Sakadaš is marked with a gray point. A channel system connecting the Lake Sakadaš and the Danube is signified.

2.2. Experimental Design and Sampling Strategy

In order to collect data on periphytic ciliates within all seasons and during different hydrological conditions, samples were collected monthly over an 18-month period, from February 2015 to September 2016. The sampling was not performed in January and February 2016 because the lake was covered with ice at that time.

Glass microscope slides (7.6 × 2.6 cm) were used as artificial substrates for periphyton development. Prior to immersion in the lake, glass slides were cleaned by washing in detergent, 1 M of hydrochloric acid, and finally in distilled water. A plastic microscope slide box was modified to carry vertically placed slides, which were exposed in the lake at a depth of 20 cm beneath the water surface. The slide box was attached to a wooden frame which was placed above the water surface using buoys, while stone blocks on the lake bottom served to anchor the frame. This allowed the frame to stay on the surface during the water level oscillations, so that the glass slides could always be positioned at the same 20 cm depth. Periphyton samples were randomly collected after approximately four weeks of substrate exposition, which was period sufficient for the development of mature (stable) ciliate community [34]. On each sampling date three replicates were taken. Glass slides with periphyton were placed separately in plastic vessels with water from the sampling site and transported to the laboratory in a portable cooler, which allowed live material for microscopic examination.

Environmental parameters including water temperature (WT), dissolved oxygen concentration, pH, and conductivity (COND) were measured in situ using a portable multimeter WTW Multi 340i (Wissenschaftlich-Technische Werkstätten, Weilheim, Germany). Due to electrode failure, dissolved oxygen values were not measured in May and June 2015. Water transparency (SD) was measured using a Secchi disc, while lake depth (D) was determined with a weighted rope. The water level of the Danube (DWL) was recorded at the Apatin gauge station at 1401.4 river km. Assuming that recent water level changes, preceding the sampling, have the greatest impact on lake communities, mean water level values for a period of seven days before sampling were taken into account [43,46]. In that way the effects of hydrological changes on periphytic ciliate communities in this highly dynamic system could have been determined more accurately. Concurrently with the periphyton sampling, water samples (1 L) were collected at a depth of 20 cm for the laboratory analyses of nutrients, chlorophyll a, and suspended solids.

2.3. Laboratory Analyses

The concentration of nutrients (ammonium (NH₄⁺), nitrates (NO₃⁻), nitrites (NO₂⁻), total nitrogen (TN), and total phosphorus (TP)) in water was determined according to APHA [56]. Chlorophyll a concentration in water samples (Chl a W) was determined spectrophotometrically according to Unesco [57] and Strickland and Parsons [58] as an indicator of phytoplankton biomass, and thus a surrogate for ciliate food availability. The amount of total suspended solids (TSS), particulate inorganic matter (PIM), and particulate organic matter (POM) in water was determined according to Luef et al. [59].

Periphyton from each glass slide (total surface area of 39.52 cm²) was scraped off using a razor blade and separated for different analyses. One part, consisting of periphyton taken from the surface area of 9.88 cm², was suspended in 100 mL of tap water, followed by filtration over a glass microfiber filter for the analysis of chlorophyll a (Chl a P), chlorophyll b (Chl b P) and chlorophyll c (Chl c P) in periphyton, using the same protocol as for the Chl a W analysis. Chl a is present in all algae and in Cyanobacteria, thus indicating their biomass in the periphyton. Chl b indicates the presence of green algae, while Chl c, as one of the major pigment of diatoms, indicates their abundance in the periphyton [60]. The second part of the periphyton sample, also taken from the surface area of 9.88 cm² but from the opposite side of the glass slide, was used for the analysis of periphytic biomass (dry weight (DW)—inorganic plus organic matter; ash weight (AW)—inorganic matter; ash-free dry weight (AFDW)—organic matter) [56]. Each sample was weighed after it had been dried for 24 h at 105 °C to a constant weight (DW), and reweighed after combustion in a muffle furnace (Nabertherm, LE040K1RN) at 500 °C for 1 h (AW). The loss in weight after ignition is referred to as AFDW.

The remaining sample, consisting of periphyton taken from both sides of the glass slide (9.88 cm² from one side and 9.88 cm² from the other side), was used for the microscopic analysis of the ciliate community. For this analysis, periphyton was scraped off into 30 mL lake water and gently mixed. Ciliates were identified and counted in two or more 50 μL subsamples taken from each homogenized periphyton suspension (the number of subsamples depended on the appearance of new species in the sample—the analysis was completed when no new species were observed). Ciliates were examined live under the Olympus BX51 microscope at magnifications 100×–1000×. The nuclear staining method was applied for more precise determination of species [61]. Some specimens were photodocumented with the Olympus CAMEDIA C-4040z for later confirmation of species identification. Taxonomic composition of ciliate communities was determined with the use of general identification keys [61,62,63,64,65], while the classification follows that of Adl et al. [66]. Five functional traits were selected to describe the functional diversity of the ciliate communities (mainly those traits that reflect trophic interactions and may influence ecosystem processes) which were later used as a grouping mechanism for various functional groups: (1) preferred food; (2) feeding mode; (3) motility/movement type; (4) origin of food items; and (5) body size [13,67]. A detailed overview of the functional trait categorization can be found in Table S1. Literature sources [8,20,26,61,68,69,70,71,72] and personal observations during the microscopic examination were taken into consideration in the analysis of species functional traits. The abundance of ciliates was calculated from three replicate slides at each sampling occasion and expressed as ind. cm⁻², whereby in the colony-forming peritrichs every zooid was counted [28]. Ciliates that had a relative abundance >5% on a sampling date were defined as dominant and were used for describing seasonal variability in the taxonomic composition of ciliate community.

2.4. Statistical Analysis

The relationships between environmental variables, as well as between environmental variables and periphytic biomass, periphytic chlorophyll concentrations, and ciliate community (total ciliate abundance, taxonomic groups, dominant taxa, and functional groups) were investigated using a nonparametric correlation coefficient (Spearman correlation, Rs) calculated in software Statistica v. 13.5 (Statsoft, Inc.). The relationship between environmental parameters (except dissolved oxygen) and hydrological phases was analyzed using principal component analysis (PCA), which works most effectively when the data are as near as possible to normality [73]. Therefore, NH₄⁺, NO₃⁻, NO₂⁻, TP, Chl a W, and PIM (variables which did not follow normal distribution (p < 0.05) according to the Shapiro Wilk W test for normality performed in Statistica v. 13.5), were log-transformed prior to the analysis, and all variables were normalized prior to analysis. To test the significant differences between hydrological phases considering environmental parameters, a one-way analysis of similarity (ANOSIM) was applied, based on Euclidean distance matrix after log transformation of data which did not follow normal distribution. One-way ANOSIM was also used to analyze the significant differences in periphytic biomass and chlorophyll concentration values between seasons and hydrological phases, by applying the Euclidean distance matrix with log transformation of data. Taxonomic diversity of ciliate communities was measured using two classical diversity indices, species richness (S), and Shannon–Wiener diversity index (H′) [74]. Functional diversity was quantified using two indices: functional dispersion (FDis), an abundance-weighted measure of species’ distribution in a multidimensional trait space [75,76] and RaoQ quadratic diversity (RaoQ) coefficient, a measure of trait convergence/divergence patterns compared to random expectation [77]. One-way ANOSIM was used to test the significant differences between ciliate communities of different seasons and hydrological phases (by applying the Bray–Curtis similarity matrix with log transformation of species and functional groups abundance data), as well as significant differences in taxonomic and functional diversity indices between seasons and hydrological phases (by applying the Euclidean distance matrix with log transformation of data). PCA, ANOSIM, and calculation of taxonomic diversity indices were performed in the PRIMER 6 software [73], while FDis and RaoQ values were computed using R v.4.1.1. [78].

The potential influence of environmental factors on taxonomic and functional composition of periphytic ciliate communities across different seasons was examined using a multivariate ordination method, redundancy analysis (RDA), performed in CANOCO for Windows version 4.5 software package [79]. RDA was chosen according to the initially performed detrended correspondence analysis (DCA). In DCA, the value of the longest gradient was lower than 4.0, suggesting the linear method as appropriate ordination technique [80]. To obtain a normal distribution, ciliate abundance and environmental data were log-transformed prior to RDA. The manual forward selection and Monte Carlo test with 499 unrestricted permutations were used to identify the significant (p < 0.05) environmental factors. In order to minimize the influence of rare taxa, only dominant ciliate taxa with relative abundance greater than 5% on a sampling date were included in the analysis.

3. Results

3.1. Environmental Parameters

3.1.1. Hydrological Conditions

The water level of the Danube fluctuated considerably during the study period, resulting in the alternating phases of lake isolation, flow pulse, and flood pulse. A detailed overview of the Danube water level oscillations during the study period is given in Figure 2.

3.1.2. Abiotic and Biotic Water Parameters

Abiotic and biotic water parameters during the study period showed seasonal changes, but were also linked to hydrological conditions (Figure 3). PCA with environmental data allowed clear separation of samples by seasons and hydrological conditions (Figure 4). The flood pulse phases (spring and early summer of 2015 and late spring and summer of 2016) were marked by the highest values of lake depth and water transparency, and the lowest concentrations of Chl a and suspended solids (TSS, PIM, and POM) in water. Further, considerably low concentrations of nutrients were recorded at the time of flood pulses (Figure 3). Substantially high water transparency was also recorded in early winter of 2015 when flow pulse occurred prior to the sampling (Figure 2) and Chl a concentration in water considerably decreased (Figure 3). Samples from late spring of 2015 and early summer of 2016, marked by the long-lasting flooding phase with extremely high flood pulses, were more widely separated in the PCA ordination. Water temperature showed a typical seasonal pattern of temperate waters, with maximum values during summer months and minimum values in winter months. The highest concentrations of Chl a, suspended solids, NH₄⁺, and NO₂⁻ in water were recorded in late summer and early and late autumn of 2015, as well as in early autumn of 2016, when lake was mostly isolated from the river. The highest concentrations of total nitrogen, total phosphorus, and NO₃⁻ were found at the time of flow pulse (the exception was in August 2015 when lake was isolated). The highest conductivity values were recorded during the phases of flow pulse (late winter and early spring of 2015 and spring of 2016), while the lowest values were found during the extremely high flood pulses (late spring of 2015 and early summer of 2016). The pH values were above 7 during the entire study period, indicating alkaline conditions in the lake (Figure 3).

The phases of lake isolation and flood pulse significantly differed considering the abiotic and biotic water parameters (one-way ANOSIM R = 0.443, p = 0.009). Danube water level positively correlated with the lake depth (Rs = 0.79, p < 0.001) and water transparency (Rs = 0.53, p < 0.05), while it showed negative correlation with Chl a in water (Rs = −0.53, p < 0.05), TSS (Rs = −0.50, p < 0.05), PIM (Rs = −0.55, p < 0.05), NH₄⁺ (Rs = −0.57, p < 0.05), total phosphorus (Rs = −0.50, p < 0.05), and conductivity (Rs = −0.48, p < 0.05). Conductivity was also in significant negative correlation with water temperature (Rs = −0.52, p < 0.05). Significant positive correlation was found between the concentrations of Chl a and POM in water (Rs = 0.71, p < 0.01).

3.2. Periphytic Biomass and Chlorophyll Concentrations

Seasonal differences in the periphytic biomass values (DW, AW, and AFDW) were confirmed by the ANOSIM, which indicated that there was a significant difference between winter and summer (one-way ANOSIM R = 0.237, p = 0.015), as well as between winter and autumn (ANOSIM R = 0.237, p = 0.041). However, biomass values did not significantly differ between hydrological phases (p > 0.05). The highest values of periphytic biomass were recorded in spring and summer of 2015 (Figure 5a), with substantially high values also found in early autumn in both years, as well as in early summer of 2016. Late autumn and winter months of 2015 and early spring of 2016 were characterized by the lowest values of biomass. Considerably low biomass values were also detected in mid-summer of 2016 (Figure 5a). DW, AW, and AFDW showed significant positive correlation with water temperature (Rs = 0.67, p < 0.01, Rs = 0.61, p < 0.01, and Rs = 0.68, p < 0.01, respectively), confirming seasonal patterns. AFDW also positively correlated with POM in water (Rs = 0.51, p < 0.05), while it showed negative correlation with water transparency (Rs = −0.56, p < 0.05).

ANOSIM confirmed seasonal differences in the periphytic chlorophyll concentrations (Chl a P, Chl b P, and Chl c P), with spring and autumn being significantly different (one-way ANOSIM R = 0.484, p = 0.001), as well as spring and summer (ANOSIM R = 0.398, p = 0.001) and winter and autumn (ANOSIM R = 0.186, p = 0.048). However, no statistically significant differences in chlorophyll concentrations were found between hydrological phases (p > 0.05). Chlorophyll concentrations in periphyton were highest in summer and autumn months, while the lowest concentrations were found in spring. Throughout the study, higher values of Chl c P were recorded compared to Chl b P (except in mid-summer of 2016), indicating higher biomass of diatoms than green algae in periphyton (Figure 5b). Chl a P, Chl b P, and Chl c P positively correlated with NH₄⁺ concentration in water (Rs = 0.68, p < 0.01, Rs = 0.54, p < 0.05 and Rs = 0.65, p < 0.01, respectively) and concentration of Chl a in water (Rs = 0.61, p < 0.01, Rs = 0.49, p < 0.05, and Rs = 0.53, p < 0.05, respectively).

3.3. Periphytic Ciliate Communities

3.3.1. Taxonomic Composition, Diversity and Abundance

A total of 110 ciliate taxa were identified in this study and assigned to 16 taxonomic groups (Figure 6 and Figure 7, Table S1). Among them, 27 taxa had relative abundance above 5% and are regarded as dominant ciliate representatives (Figure 7c). Peritrichs represented the most diverse taxonomic group with a total of 23 identified taxa throughout the study. They were followed by haptorians (18 taxa) and hypotrichs (16 taxa) (Figure 6b). The largest contribution of peritrichs to the total number of taxa was detected in summer and early autumn, while haptorians and hypotrichs had a more significant contribution in winter, spring, and autumn months (Figure 6b).

Taxonomic diversity indices (species richness and Shannon–Wiener diversity) showed similar trend during the study period (Figure 6a). ANOSIM revealed significant differences in Shannon–Wiener diversity index between hydrological phases of flow pulse and isolation (one-way ANOSIM R = 0.163, p = 0.015). Values of diversity indices were generally higher during periods of lower water temperature and hydrological phases of flow pulse and extremely high flood pulse, while lower values of indices were characteristic for periods of higher water temperature and hydrological phase of lake isolation (Figure 6a).

According to the ANOSIM, total ciliate abundance significantly differed between spring and summer season (one-way ANOSIM R = 0.128, p = 0.021), as well as between hydrological phases of flow pulse and isolation (ANOSIM R = 0.149, p = 0.014). The highest ciliate abundances in periphyton were recorded in summer of 2015 during the lake isolation, with high values also observed in late spring of 2016 following short-term flood pulse, and in early autumn of 2016 after short-term flow pulse. The lowest values were observed in phases of extreme flooding (late spring of 2015 and early summer of 2016) and flow pulse (mid-autumn of 2015 and spring of 2016) (Figure 7a). Total ciliate abundance was significantly positively correlated with POM (Rs = 0.56, p < 0.05) and AFDW (Rs = 0.56, p < 0.05).

Peritrichs were the most abundant ciliates in periphytic communities in almost all seasons (from mid-spring to early winter of 2015 and from mid-spring to late summer of 2016, with the exception of mid-summer 2016), accounting for >50% of the total abundance (Figure 7b). The highest proportion of peritrichs in total abundance (near or above 80%) was found in spring, summer, and late autumn of 2015, as well as in late spring of 2016. The lowest proportion of peritrichs in total abundance (<20%) was observed in late winter and early spring of 2015, while in early spring, mid-summer, and early autumn of 2016, their proportion was slightly below 50% (Figure 7b). In spring and summer of 2015, peritrichs were dominated by solitary species of genus Vorticella (V. aquadulcis-complex, V. campanula, V. convallaria-complex, and V. picta, with the last two species represented as Vorticella spp.) (Figure 7c). In summer, the abundance of colony-forming peritrichs (Epistylis chrysemydis and E. entzii) increased, and they became the main contributors to peritrich abundance in late summer and early autumn. In mid-autumn, both Vorticella and Epistylis contributed to the peritrich dominance, while in late autumn and early winter V. campanula was the most abundant in the community. In 2016, peritrichs were mainly dominated by Vorticella species, with Epistylis species also contributing to the peritrich abundance in mid-summer and early autumn (Figure 7c).

When the proportion of peritrichs in total abundance was lower or close to 50%, a more diverse ciliate community was observed, and taxonomic groups such as cyrtophorids, euplotids, heterotrichs, hypotrichs, prostomatids, scuticociliates, and suctorians substantially contributed to the total abundance of ciliates in periphyton (Figure 6a and Figure 7b). In late winter and early spring of 2015 scuticociliates Cinetochilum margaritaceum, Ctedoctema acanthocryptum, Cyclidium glaucoma, and Pseudocohnilembus pusillus, prostomatid Coleps spp., hypotrichs Holosticha pullaster and Tachysoma pellionellum, euplotids Aspidisca spp. and Euplotes spp., as well as cyrtophorids Trithigmostoma sp. and Dysteria fluviatilis, mainly characterized the periphytic ciliate community (Figure 7c). The proportion of heterotrich ciliate Stentor roeselii in the total abundance was considerable in summer and early autumn months, while suctorians (e.g., Tokophrya quadripartita) were the most abundant during summer and autumn (Figure 7b,c). In early winter (December 2015), although peritrichs accounted for nearly 60% of total abundance, a notable proportion of scuticociliates Calyptotricha pleuronemoides and C. margaritaceum, prostomatid Coleps spp. and hypotrich H. pullaster was observed in the total abundance. In the spring of 2016 euplotids Aspidisca spp. and Euplotes spp., scuticociliates C. pleuronemoides, C. margaritaceum, and C. acanthocryptum, and hypotrich T. pellionellum were found to substantially contribute to ciliate abundance. In early summer of 2016, despite the peritrich dominance, a notable proportion of hypotrich Chaetospira muelleri, haptorian Litonotus lamella, and scuticociliates C. pleuronemoides, C. margaritaceum, and C. acanthocryptum in the total ciliate abundance was recorded. Cyrtophorid Pseudochilodonopsis spp. was found to largely contribute to the total abundance in early autumn of 2016 (Figure 7b,c). In general, groups such as cyrtophorids, euplotids, haptorians, hypotrichs, prostomatids, and scuticociliates had higher share in the periphyton when low values of total ciliate abundance were recorded, i.e., during colder months, especially those accompanied by the conditions of flow pulse, as well as during warmer months accompanied by the conditions of extremely high floods (Figure 7a,b).

3.3.2. Functional Trait Composition and Functional Diversity

In nearly all seasons periphytic communities were dominated by medium-sized attached (sessile) ciliates feeding mainly on suspended food items (fine filter feeders). The main food of periphytic ciliates were bacteria and bacteria/algae (excluding diatoms). The highest abundances of these functional groups were recorded during warmer months and more stable hydrological conditions (Figure 8a–d). Large omnivorous fine to coarse filter-feeding ciliates had the highest proportion in total abundance during the summer and early autumn months, with omnivores and fine to coarse filter feeders also largely contributing to total abundance during winter and spring season (Figure 8a,b,d). Predacious diffusion-feeding ciliates mainly characterized summer and autumn communities (Figure 8a,b). Raptorial feeders hunting algae (including diatoms) as well as crawling ciliates substantially contributed to total abundance in early autumn of 2016, while omnivorous raptorial feeders and those capturing bacteria/algae and bacteria had high proportion in total abundance in winter and early spring of 2015 (Figure 8b,c). These seasons were also characterized by a high share of small crawling (surface-feeding) and free-swimming (both surface- and suspension-feeding) ciliates in periphyton (Figure 8c,d). In general, more diverse functional groups were present during colder seasons and hydrological phases of flow pulse and extremely high flood pulse, when lower values of total ciliate abundance were found (Figure 8a–d).

Functional diversity indices (FDis and RaoQ) substantially oscillated throughout the study period, displaying similar trend (Figure 8e,f). RaoQ significantly differed between winter and summer season (one-way ANOSIM R = 0.276, p = 0.007). Significant differences in FDis and RaoQ values were found between hydrological phases of flow pulse and isolation (ANOSIM R = 0.556, p = 0.001 and R = 0.517, p = 0.001, respectively) and flow pulse and flood pulse (ANOSIM R = 0.261, p = 0.001 and R = 0.195, p = 0.001, respectively). Generally, higher FDis and RaoQ values were found at lower water temperature and during the phases of flow pulse and extremely high floods, while lower values of indices were found at higher water temperature and during the phase of lake isolation (Figure 8e,f).

3.3.3. Seasonal Patterns of Taxonomic Composition Related to Environmental Conditions

In terms of taxonomic composition and abundance, ciliate communities significantly differed between the seasons (one-way ANOSIM Global R = 0.475, p = 0.001), as well as between hydrological phases (Global R = 0.207, p = 0.001). The significant difference was found between each pair of seasons (p < 0.01) and each pair of hydrological phases (p < 0.01). A significant relationship between dominant ciliate taxa and environmental variables was indicated by the high species-environment correlations for axes 1 (0.928) and 2 (0.966) in the RDA. The two main axes explained 36.4% of the variance in the species data (Figure 9a,b). RDA showed that the summer and autumn communities, established mainly during the phases of lake isolation and flow pulse and characterized by dominant peritrich, heterotrich, and suctorian taxa, were associated with the high concentration of Chl b in periphyton and high water temperature, as well as with the low water transparency. Spring and summer communities formed during the phase of flood pulse and dominated by peritrich (Vorticella) species, as well as hypotrich, haptorian, and scuticociliate species, were related to high Danube water level, transparency, and water temperature, and low conductivity. Ciliate communities developed in winter and early spring months mainly during the phase of flow pulse were driven by the high conductivity and water transparency, and low water temperature. These communities were mainly characterized by scuticociliate, prostomatid, hypotrich, euplotid, as well as cyrtophorid taxa (Figure 9a,b). The significant relationships between ciliate taxonomic groups/dominant taxa and environmental variables are also confirmed by Spearman correlation coefficients (Table S2).

3.3.4. Seasonal Patterns of Functional Trait Composition Related to Environmental Conditions

ANOSIM, based on the functional trait composition and the abundance of functional groups, showed that periphytic ciliate communities significantly differed between the seasons (Global R = 0.241, p = 0.001) and hydrological phases (Global R = 0.127, p = 0.001). The significant difference was found between each pair of seasons (p < 0.05) except winter and spring, as well as between each pair of hydrological phases (p < 0.05). In RDA, a significant relationship between ciliate functional groups and environmental variables was confirmed by high correlations between functional groups and environmental variables for axes 1 (0.847) and 2 (0.889). The two main axes explained 32.8% of the variance in the functional trait data (Figure 9c,d). According to the RDA, summer and autumn communities developed primarily during the phases of lake isolation and flow pulse, were related to the high concentration of Chl b in periphyton and low water transparency. These communities were dominated by medium- and large-sized attached (sessile) ciliates, which feed mainly by filtering bacteria and bacteria/algae suspended in water (fine filter feeders and fine to coarse filter feeders) or by intercepting swimming prey (diffusion feeders). Predacious and omnivorous ciliates were characteristic for these communities. The winter and spring communities, especially those developed during the phase of flow pulse, were characterized by small motile (crawling and free-swimming) ciliates. Surface-feeding and surface-/suspension-feeding taxa dominated in this period, and raptorial feeding was the common feeding mode. These communities were associated with high conductivity and water transparency (Figure 9c,d). The significant relationships between ciliate functional groups and environmental variables are also confirmed by Spearman correlation coefficients (Table S2).

4. Discussion

The present study confirmed high ciliate diversity and abundance in periphytic communities of temperate riverine floodplains [36,47]. In the investigated eutrophic/hypertrophic Lake Sakadaš, diversity of periphytic ciliate community was much higher than for example in eutrophic lake in Poland [19]. This study also showed that periphytic ciliate communities differ significantly between seasons as well as hydrological phases when considering both the taxonomic and functional community composition and diversity. Hence seasonal variability, i.e., temperature oscillations, coupled with changes in the hydrological regime, are considered as major factors influencing the structure of periphytic ciliate communities in temperate floodplain lake ecosystem. Such importance of the interaction between temperature and hydrological conditions in the structuring of biotic communities in temperate riverine floodplains has been emphasized earlier by Tockner et al. [38]. Another important factor for shaping ciliate communities was the resource availability, which is highly controlled by hydrological conditions, as well as temperature variability. Due to fluctuations in these environmental factors, ciliate abundances, taxonomic and functional composition and diversity changed considerably throughout the study, affecting the ecological role that these organisms play in a floodplain lake ecosystem.

Changes in the hydrological regime are recognized as one of the most important factors affecting aquatic communities in floodplain ecosystems [40,41,81], and our results confirm this. The phases of lake isolation, flow pulse, and flood pulse alternated in the lake throughout the study (Figure 2) resulting in considerable changes in lake conditions (Figure 3) and in the origin and quantity of food sources. During the phase of flow pulse, which could be considered as the phase of intermediate level of hydrological connectivity [11,40], constantly oscillating water level resulted in the highest conductivity values, as well as high concentrations of nutrients (total nitrogen, total phosphorus, and nitrates) (Figure 3 and Figure 4), which may have been imported by the river water inflow [50,81]. Galir Balkić and Ternjej [82] found similar conditions in the Lake Sakadaš under flow pulse. The dilution effects of floodwaters during flooding conditions were noticeable, also noted in the study conducted by Mihaljević et al. [42]. This effect is detectable, among other parameters, by nutrient, Chl a and POM dynamics. Accordingly, the phases of lake isolation and flood pulse differed the most when considering the abiotic and biotic water parameters. While the isolation phase was marked by the highest concentrations of Chl a, suspended particles (TSS, PIM, and POM), ammonium and nitrites in the water, as well as the lowest values of lake depth and water transparency, the flood pulse phase (especially phase of extremely high flooding) was characterized by completely opposite trend (Figure 3 and Figure 4). In Danubian floodplain in Austria, Hein et al. [83] recognized hydrological connectivity between a river and its floodplain as a key factor influencing the sources of POM: during high levels of hydrological connectivity the POM imported from the river to the floodplain is largely composed of detrital (non-living) carbon and is of limited biological availability but, as connectivity decreases, autochthonous production by plankton becomes the dominant source of POM in the floodplain. Similar results were reported by Tockner et al. [81]. Since we observed that the increase in POM concentration coincides with the increase in Chl a concentration (Figure 3) proved by statistically significant positive correlation, we assume that planktonic algae also constitute a large part of POM in Lake Sakadaš. The isolation phase of Lake Sakadaš was previously reported as the most stable hydrological phase, supporting the development of different lake communities (e.g., phytoplankton and bacterioplankton) [42,43] and the concentrations of POM are therefore expectedly high during this phase. In such conditions, a large amount of suspended food is available for periphytic ciliates, especially for attached filter feeders [6,24], leading to high total ciliate abundances, as was detected in the present study.

The highest ciliate abundances in periphyton recorded in summer of 2015 coincide with the increase in Chl a and POM concentration in water, i.e., the isolation phase (Figure 3 and Figure 7a). The values of Chl a concentration in water recorded in the present study confirm the pattern in the phytoplankton growth already recognized in the Lake Sakadaš, with the highest phytoplankton abundances observed during more stable phase of lake isolation and the lowest during the flooding phase [42,54]. We suggest that the high abundances of planktonic algae combined with the lower water levels and highest values of water temperature led to the high abundances of ciliates in periphyton. Such a response of periphytic ciliates towards phytoplankton abundance has been reported earlier in River Rhine [6,24], whereby Kathol et al. [24] demonstrated that the availability of phytoplankton has a major influence on seasonal patterns in periphytic ciliate communities. Furthermore, the observed functional composition of the community (Figure 8a) implies that planktonic bacteria are an important source of food for periphytic ciliates, along with algae (not including diatoms). Studies on bacterioplankton community conducted in the same lake showed that the highest abundances of planktonic bacteria are found during hydrologically more stable phases [43,46]. These findings, accompanied by the results of the present study, suggest that suspended bacteria and algae represent the major food sources for periphytic ciliates, stimulating ciliate growth and increasing their abundance in the studied system. Since flood pulses regularly disrupt communities of planktonic microorganisms in Lake Sakadaš, most likely due to the dilution and washout effects of the floodwaters [42,43,46,54], a decrease in the periphytic ciliate abundance is expected during the flood pulse phases as well. Indeed, in our study the lowest ciliate abundances were recorded when extremely high flood pulses preceded the periphyton sampling (Figure 7a) leading to low concentrations of Chl a and POM in water and consequently a decrease in ciliate abundances. The intensity of the flood pulse highly defines the structure of periphytic ciliate communities, as observed during the extremely high flooding events in the course of our study. Although we expected to find high ciliate abundances in these seasons due to high values of water temperature, which generally stimulates ciliate growth, the lowest abundances were recorded, most likely due to the severe disturbance caused by the extreme floods [25,84]. Hence, the general effect of temperature on periphytic ciliates, and accordingly seasonal patterns in periphytic ciliate communities, can be significantly altered by interactions with the hydrological regime. Furthermore, we found that lower intensity floods did not have such an effect on the community. The low ciliate abundances recorded in mid-autumn of 2015 and spring 2016 during the phase of flow pulse (Figure 7a) could be explained by the lower temperature values during these seasons which resulted in the decrease of ciliate feeding and growth rates, slowing down their response to the already reduced resource availability [6,10].

Peritrich populations were well developed throughout the study, with solitary Vorticella species and colony-forming Epistylis as numerically dominant in the periphyton (Figure 7b,c). Peritrichs contributed most to the total ciliate abundance as well as high abundance of sessile, medium-sized, fine filter feeders in mature periphytic communities in almost all seasons. Evidently, the conditions in this temperate floodplain lake promote the growth of these suspension-feeding ciliates which is congruent to our previous findings [34,36,47]. Peritrichs are commonly represented with high abundances in periphytic assemblages of various aquatic ecosystems [8,28,32,85]. Additionally, since these ciliates are successful competitors, it is expected that they develop abundant populations in mature periphytic communities [6,24,25]. According to our results, the optimal conditions for peritrichs were high values of water temperature, chlorophyll concentration, and organic matter content in periphyton, along with the high concentration of Chl a and organic particles in water and low values of water transparency (Figure 9a,b, Table S2). Therefore, the highest abundances were found during the warm seasons and during the phase of lake isolation, i.e., in hydrologically most stable phase, when high amounts of food (such as suspended bacteria and algae) are available. As typical representatives of pico-nanophagous ciliates [23] and fine filter feeders [71,72], peritrichs show a preference for small-sized food particles, mainly bacteria (<2 μm) but also nano-sized algae (2–20 μm) [5,86] such as small green algae [6]. RDA analysis related dominant peritrich species with the Chl b concentration in periphyton (Figure 9a,b), indicating that suspension-feeding ciliates can also utilize small-sized periphytic green algae [7]. Surprisingly, a substantially high ciliate abundance was also recorded in late spring of 2016 during the moderate flood pulse (Figure 7a), with peritrichs (especially Vorticella campanula), contributing mainly to this peak (Figure 7b,c). We assume that the substantial increase in water temperature at that time (Figure 3) stimulated peritrich growth, as higher ambient temperatures have been shown to compensate for negative disturbance effects on ciliate abundance in mature communities [25]. In addition, this flood pulse was of moderate intensity and therefore did not interfere with the effect of temperature on feeding capacity and growth rates as was the case during extreme floods. Furthermore, nanophytoplankton abundance and biomass of planktonic bacteria, which are less sensitive to hydrological disturbances [43], could represent an important additional food source for peritrichs stimulating their development during the phase of moderate flood pulse. Seasonal distribution of the two dominant peritrich genera Vorticella and Epistylis was also detected in the periphyton from the River Rhine [24], with higher abundance of Vorticella species recorded in winter periphyton samples when planktonic bacteria prevailed, and Epistylis species in summer samples when planktonic algae were the most abundant. Although some literature findings [42,43,54] imply possibility of contrary patterns with higher bacterial abundance in late spring and summer and higher phytoplankton abundance during summer and autumn, it is yet not clear how exactly the presence and shifts in the abundance of planktonic microorganisms affect peritrich species. In addition to peritrichs, summer and autumn periphytic communities were characterized by large, sessile, suspension-feeding heterotrichs (Stentor roeselii) and medium-sized, sessile, suspension-feeding suctorians (Figure 7b,c and Figure 8c,d), which were stimulated by similar environmental conditions as peritrichs (Figure 9). These ciliates contributed to the higher proportion of omnivorous fine to coarse filter feeders and predacious diffusion feeders in periphyton (Figure 8a,b). Most likely, higher resource availability during warm seasons supported the growth of these ciliates [47].

During colder seasons, especially those accompanied by the conditions of flow pulse, as well as during warmer seasons accompanied by the conditions of extreme floods, when values of total ciliate abundance were generally lower as well as peritrich contribution to the total abundance, species belonging to groups such as cyrtophorids, euplotids, haptorians, hypotrichs, prostomatids and scuticociliates were more represented in the periphyton (Figure 7), which resulted in the altered functional trait composition (Figure 8a–d). RDA ordination associated those communities with parameters that indicate reduced planktonic food sources. Such conditions of reduced availability of suspended food particles, especially in combination with lower values of water temperature, favored surface-feeding and surface-/suspension-feeding ciliates as well as raptorial feeders (Figure 9c), which rely mostly on surface-associated food items. This suggests that during the increased hydrological connectivity, when lake planktonic communities are disturbed, periphyton becomes a more significant food source for periphytic ciliates. As periphytic biomass was not affected by hydrology in our study (Figure 5), it could serve as important food source for ciliate communities during higher water levels. Further, small motile (crawling and free-swimming) specimens characterized periphytic ciliate communities during these seasons and hydrological conditions (Figure 8c,d). Species belonging to these functional groups represent good colonizers with high growth rates and are characteristic for the early phases of community development [34,36], but also for disturbed systems in which disturbance events reduce the number of organisms and lead to community rearrangement [84]. Since floods in riverine floodplain systems can act as a disturbance of varying severity which can disrupt the community development and its arrangement [11,40,41], such share of functional groups was expected. Strüder–Kypke [32] found similar seasonal distribution of ciliates in periphytic communities of dystrophic lakes.

Taxonomic diversity values matched those of functional diversity throughout the study. While the highest diversity (taxonomic and functional) values were recorded during the seasons marked by the lower values of water temperature and hydrological phases of flow pulse and extremely high flood pulse, the opposite was observed for the phase of lake isolation and higher water temperature (Figure 6a and Figure 8e,f). Therefore, high diversity coincided with the low numbers of peritrichs, which as highly competitive ciliates control the resources and dominate in mature periphytic communities [6,24,25]. Disturbance represents an important factor affecting diversity patterns [87], and in river-floodplain ecosystems disturbances in the form of natural floods are crucial for the maintenance of biodiversity [11,88]. Hydrological variability shapes diverse habitat structures in the floodplain and leads to the high spatio-temporal heterogeneity, thus creating the basis for high biodiversity [40]. Tockner et al. [89] emphasized the importance of different levels of hydrological connectivity for the maintenance of high biodiversity in the Danube floodplain in Austria, whereby the highest diversity of various groups of aquatic organisms was found at an intermediate level of connectivity. Similar findings on the diversity patterns were reported by Galir Balkić and Ternjej [82] for planktonic crustaceans in the Lake Sakadaš. The authors found that the diversity of planktonic Cladocera and Copepoda decreases with the decreasing water levels. Our results, which are analogous to those of zooplankton research, support the hypothesis which states that the species diversity is highest at intermediate levels of disturbance frequency or intensity [48], which is expected in floodplain ecosystems at intermediate levels of hydrological connectivity [11,40]. However, we found that floods of extremely high intensity cause the major turnovers in taxonomic and functional composition in ciliate communities, also leading to the high diversity of periphytic ciliate communities. In such conditions highly competitive peritrichs, otherwise dominant ciliates in periphyton, can be overpowered by other species with different functional traits. According to Dunck et al. [90], flood pulses favor the appearance of new species with different functional traits through increasing the variety of niches and facilitating the organisms’ dispersion. However, low values of functional diversity indices during warmer seasons and hydrologically more stable phase of lake isolation indicate a low degree of niche differentiation and high competition for resources among dominant, functionally similar species [91], i.e., indicate high functional redundancy [92]. The coexistence of species with similar functional traits, as a result of environmental limitations, leads to reduced ecosystem function as well as higher susceptibility to disturbance [13]. Therefore, our results suggest that peritrich ciliates that dominate in periphytic communities during stable phase of lake isolation could serve as good bioindicators for detecting the disturbance presence in river-floodplain ecosystems.

5. Conclusions

The results of the present study suggest that seasonality, i.e., temperature variations, coupled with the changes in hydrological regime, represent principle driving variables of periphytic ciliate communities in a lake ecosystem of temperate riverine floodplain. Hydrological changes influence periphytic ciliates mainly through reshaping the lake communities at lower trophic levels and consequently altering the food source for ciliates. High water temperature combined with hydrologically most stable environment and high availability of suspended food (lake isolation phase), supports the highest ciliate abundances and domination of highly competitive peritrich ciliates, i.e., fine filter feeders that have strong preference for suspended bacteria and nano-sized algae. Therefore, periphytic ciliates might play an important role in importing planktonic production into the periphytic food web, as well as transferring bacterial and algal biomass to higher trophic levels. However, to determine more precisely the strength of the trophic link between periphytic ciliates and lake planktonic communities, it is necessary to include quantitative data on planktonic microorganisms in the future research. In addition, peritrich ciliates might serve as good bioindicators for detecting the disturbance presence in river-floodplain ecosystems.

In the presence of extreme floods, when the availability of suspended food is highly reduced, ciliate abundance decreases and small surface-feeding species, which as good colonizers with high growth rates are more resilient to disturbance, become more represented in periphyton. These ciliates are also characteristic for seasons marked by the lower temperature values and hydrological phase of flow pulse. As expected, we found the highest taxonomic and functional diversity during this hydrological phase, i.e., during an intermediate level of hydrological connectivity. However, high diversity also characterized the phase of extremely high flood pulse. These results confirm the importance of different levels of hydrological connectivity for the maintenance of high biodiversity in river-floodplain ecosystems, most probably through increasing the variety of niches and facilitating the organisms’ dispersion, and emphasize the need for the protection of river hydrological regime in its natural state.