Diversity of the Rhopalodiaceae Diatoms (Bacillariophyta) on Macrophytes of Different Architecture in Small and Shallow Oxbow Lakes (SE Poland)

The research was conducted in July 2018 on six oxbow lakes of the San River. A total of 21 samples from various macrophyte species were collected. Over 400 species of diatoms were identified in the analyzed materials, 9 of which belonged to the Rhopalodiaceae family. Most of these Epithemia and Rhopalodia species developed as dominants in most of the studied samples. One of reported species – Epithemia selengaensis Vishnyakov, Kulikovskiy & Genkal was first time reported from Poland. The aim of the study was to present the taxonomic diversity of the representatives of the Rhopalodiaceae family growing different macrophyte species in oxbow lakes in the summer aspect.


INTRODUCTION
Communities of primary producers (macrophytes, phytoplankton and benthic algae) play a crucial role in supporting various food webs within the aquatic ecosystem (Špoljar et al. 2017). Nevertheless, the benthic algal communities (including epiphytic) represent an important component of food webs as primary producers, particularly in oligo-mesotrophic shallow lakes or littoral zones, where high water transparency results in higher light availability for microorganisms living below the water surface (Sierszen et al. 2006). Apart from its ecological role, algal epiphytic communities are also used in the assessment of the ecological status of aquatic ecosystems. It applies primarily to diatoms, which fulfill many the requirements of being useful indicator organisms in running waters, although several studies showed that the diatom analyses may also be applied to determine the ecological status of lentic ecosystems, especially shallow lakes (Kitner, Poulícková 2003, Cejudo-Figueiras et al. 2011, Blanco et al. 2014, MacDougall et al. 2017. It seems to be broadly accepted that the bioassessment methods based on epiphytic diatoms may be also effective for tracking the past changes under the environmental conditions of lakes or even small ponds (Wiklund et al. 2010, Emson et al. 2018. The studies concerning epiphytic diatoms in small and shallow water bodies like ponds or oxbow lakes are relatively scarce, despite the fact that small waterbodies play an important role in the structure of biodiversity on both landscape and species level (Scheffer et al. 2006, Cereghino et al. 2008). The importance of oxbow lakes in Europe as biodiversity hot-spots was acknowledged by EC Habitats Directive 92/43/EEC; thus, they became protected under the name "Habitat 3150" (Nowak et al. 2015). Their presence in the river valleys enriches biodiversity as habitats form protected, rare, valuable and disappearing species of vascular plants (Michalska-Hejduk et al. 2009) but also other organisms. In oxbow lakes, which usually are small and shallow water bodies, macrophytes play a crucial role in the ecosystem functioning; thus, the structure of aquatic plants is the main indicator which is used in the bioassessement of their conservation status (Wilk-Woźniak et al. 2019). As other shallow lake ecosystems with differentiated macrophyte architecture they offer an excellent habitat for epiphytic diatom assemblages (Letáková et al. 2018).
Rhopalodiaceae (Karsten) Topachevs'kyj and Oksiyuk (Round et al. 1990) are one of only two families within the Rhopalodiales order. Among several small genera only two: Rhopalodia and Epithemia contain several dozen of species. The most characteristic feature of species within this two genera is the presence of 2-5 cyanobacterial endosymbionths (Geitler 1977, Janson 2002. The cell walls of both genera are similar in symmetry, being strongly dorsiventral. The raphe of Rhopalodia species is positioned on the dorsal side, in a shallow keel, whereas the raphe of the species within the Epithemia genus is positioned along the ventral margin, with simple or slightly expanded raphe endings. In contrast to Epithemia, Rhopalodia cells have no girdle bands (Round et al. 1990, Taylor, Cocquyt 2016. Most of the Epithemia species are cosmopolitan and have worldwide distribution, although generally prefer temperate climates. They are widespread in almost all freshwaters, growing on submerged vegetation or more seldom in epilithic assemblages (Sims 1983, Krammer, Lange-Bertalot 1988. The Epithemia species reaches the maximum abundance especially in those microhabitats where phosphorus is relatively more available (DeYoe 1992, Kociolek et al. 2015).
The representatives of the Rhopalodiaceae family, due to the large cell size and wide geographical distribution, are well known species, often described more than 100 years ago. However, the taxa belonging to the two discussed genera, are usually identified only to the species level, based on a broad species concept. As a result the real diversity and within the family is often underestimated.
The paper discusses the variability and occurrence of the diatom taxa from the Rhopalodiaceae family from six oxbow lakes in the San river catchment area. All noted species developed on the submerged macrophytes. Additional information about morphology, ecology of Rhopalodia and Epithemia taxa based on observed populations was provided.

STUDY AREA
The San River, the largest of Carpathian rivers, is 458 km long. It is also one of the main tributaries of Vistula River. Oxbow lakes are very numerous in the San catchment area, especially along the middle and lower sections of the river.
The study was conducted in six oxbow lakes formed by the San River (south-eastern Poland) in its middle (sites P1, P2, M1, H1) and lower (Z1, Z2) course ( Fig. 1). First two (P1 and P2) are located in the outskirts of the Przemyśl city (49°47'N, 22°46'E). These water bodies with an area of 0.22 and 0.11 ha, are part of a larger group, located close to the San River, in the vicinity of allotment gardens. The area of both water bodies is overgrown with aquatic and waterside vegetation. Another two study sites (H1 and M1) are situated east of Przemyśl, close to the state border with Ukraine. The largest of studied oxbow lakes in the village Hurko (H1), with an area of ca. 10 ha, is used for fishery. The site M1 is a part of vast park surrounding the old historical palace in Medyka village, thus playing a role of a landscape pond. Both lakes (H1, M1), being under the influence of the human activity, have rather scarce aquatic vegetation, which was removed during its management. During the field studies in these lakes, a cyanobacterial bloom was observed. The sites Z1 and Z2, situated in the lower course of the San River have the most natural character. They are located within the NATURA 2000 Special Area of Conservation "Dolina Dolnego Sanu" (PLH180020), situated between Stalowa Wola (50°34'N, 22°03'E) and Sandomierz (50°40'N, 21°44'E), close to the village Zaleszany. Both sites are part of socalled "Old San", the largest of the river-beds in the lower course of the San River, with several arms stretched between two present river beds (San and Łęg rivers) (

METHODS
The diatom material was collected in July 2018 from six different oxbow lakes in four localities. The samples were taken from dominant macrophytes (floating leaves (FLM), emergent (EM) and submerged plants (SM). A total of 21 samples were collected, separately for each of the macrophyte species (Table 1). The plant species identification was based on Kłosowski, Kłosowski (2010).
Water temperature, oxygen concentration, pH and conductivity were measured in situ during sampling, with the use of a MARTINI PH65 meter, a MARTINI EC59 meter and a HANNA HI 9146 oxygen meter, respectively. An additional water sample for the chemical analyses (listed in Table 2) was taken from the central part of each oxbow lake. Those analyses were performed using a DIONEX ICS-5000 + DC Thermo scientific ion chromatograph in a laboratory.
The diatom samples were cleaned by adding 30% H 2 O 2 and heating until the organic matter is digested (1-2 h). The reaction was completed by the addition of HCl. Following digestion and centrifugation (5 times, 5 minutes at 2500 rpm) with distilled water, the resulting cleaned material was mounted in Pleurax (refractive index 1.75). The diatoms were identified and counted under a Carl Zeiss Axio Imager A2 light microscope (LM) equipped with Differential Interference Contrast (Nomarski) optics, under 1000× magnification. The diatom images were captured using a Zeiss

RESULTS
The values of physical and chemical parameters measured in the study are shown in Table 2. Temperature (23.8-26.3 ºC) and pH values (7.4-8.5) fell within the ranges expected for the oxbow lakes in Central Europe in summer. Some differentiation among the studied lakes were observed in electrolytic conductivity. The lowest values of conductivity were measured at sites Z1 and Z2 in Zaleszany (347-367 µS cm -1 ). The rest of lakes had conductivity in the range 500-565 µS cm -1 except P2 site in Przemyśl, where twice as high values of this parameter were found, as compared to Z1 and Z2 sites ( Table 2). The water in all studied lakes was rich in chlorides (17.72-67.54 mg l -1 ) and calcium (26.5-83.3 mg l -1 ) but had very low values (below the limit of determination) of nitrates and phosphates (with the exception of site M1).
More than 400 diatom taxa were identified in 21 samples. The number of taxa noted in each sample ranged from 40 (sample 2018/152 collected from Potamogeton lucens at site P1) to 137  (Table 3). Among all recorded taxa, nine belong to the Rhopalodiaceae family, from which seven Epithemia and two Rhopalodia taxa were noted in all studied samples, but in variable abundance (Table 3). At the sites devoid of their "semi-natural" character (H1, M1), the Rhopalodiaceae taxa were noted only occasionally, whereas in macrophyte dominated oxbow lakes of more "natural" character (P1, P2, Z1, Z2), they developed more numerously. The diatoms from that family dominated the assemblages in some cases: in six from twenty one studied samples, Rhopalodiaceae reached together more than 50% off all counted valves. Among them, in the sample collected from Myriophyllum spicatum (2018/135), the diatom community was entirely dominated by Epithemia and Rhopalodia taxa. However, no preferences of individual taxa in relation to macrophytes were observed.
Below, the population characteristics of Rhopalodiaceae taxa found in the study are presented.

DISCUSSION
The oxbow lakes can develop as a result of isolation/disconnection from meander necks or separation from rivers by riverside embankments during freshets (Żmudziński et al. 2002, Dembowska, Napiórkowski 2012). The oxbow lake has many important functions in river valley ecosystems, where together with rivers and their catchments it contributes to the natural diversity of those areas. Apart their hydrological and microclimatic role of retention reservoirs, oxbow lakes form a suitable and supportive environment for the growth of water and hygrophilous flora, as well as valuable habitat for wildlife (Glińska-Lewczuk 2009, Koc et al. 2009).
In general, in all studied oxbow lakes more or less similar values of water temperature, pH and conductivity were noted, which is in accordance with the results presented by Michalska-Hejduk et al. (2009) in the same locality. Only the oxygen content was higher (2.52-4.21 mg/l) than in our samples, which is probably connected with differences in the weather conditions and water temperature. Oxbow lakes are considered as natural eutrophic water bodies. However, as a result of macrophyte and phytoplankton activities, the levels of soluble mineral forms of nutrients in such lakes can be very low, as it was noted in the studied lakes (Reddy et al. 1989, Olguín et al. 2007, Ozimek et al. 1990, Upadhyaya et al. 2007). The low values of the analyzed ions might also be a result of the oxbow separation from the river, as it was revealed by Glińska-Lewczuk (2009). In her study, in the comparison of two types of oxbow lakes: lentic and lotic, it was demonstrated that various ions are withdrawn from the system. The oxbow lakes separated from the river showed significantly lower values of nitrates and nitrites. In lentic oxbow lakes, isolated from the river, the matter cycling (mainly organic) influences the change and development of the specific macrophytes species composition. The plant species in this type of habitat are well adapted to the anoxic conditions and decreased productivity (Glińska-Lewczuk 2009). From seven identified in this study Epithemia species, E. selengaensis was reported for the first time from Poland. The species was described in 2014 from Selenga River delta in Russia, but was also observed in the samples from lakes in Mongolia and Eastern Baikal Region (Vishnyakov et al. 2014). The species was also recorded on macrophytes in various types of water in Turkey (C. Solak -pers. comm.). E. selengaensis belongs to E. adnata group but can be easily distinguished on the basis of roundly capitate apices. Other Rhopalodiaceae noted during our study belong to commonly reported taxa. Unfortunately, all of them are identified in wide species concept, very often only to the species level. They are widespread in fresh and brackish waters and prefer waters rich in calcium, meso-to eutrophic. They occur mainly in epiphytic, more seldom in epil- The studied taxa were found on each of the analyzed macrophytes. The most numerous population was formed by Epithemia adnata s.l., following by E. turgida and Rhopalodia gibba s.l. The semi-natural oxbow lakes (i.e. in Przemyśl city) overgrown by various macrophytes favor the development of epiphytic diatom species. The stagnant, freshwater ecosystems is Europe are very often overgrown by macrophytes. Among the epiphytic diatom assemblages growing on various macrophytic plants, many authors report the dominance of species from the Rhopalodiaceae, especially E.adnata s.l., E. sorex, R.gibba (Kuczyńska-Kippen et al. 2005, Laugaste, Reunanen 2005, Toporowska et al. 2008). In the samples taken from the lakes where the cyanobacterial bloom persisted (site H1, M1), the discussed species developed rarely and assemblages were manly dominated by typically epiphytic taxa. On stems of Phragmites australis and on Ceratophylum demersum the most common taxa were: Cocconeis placentula Ehrenberg, C. lineata Ehrenberg and Cymbella subleptoceros Krammer. In plant community "Lemno-Spirodeletum" diatom assemblages was dominated by C. lineata Ehrenberg, Halamphora veneta (Kützing) Levkov and Lemnicola hungarica (Grunow) Round & Basson. Moreover, Lemnicola hungarica is reported as a species associated with various Lemnaceae species (Buczkó 2007, Emson et al. 2018).
In addition to the dominance of macrophytes, also the nitrogen limitation tends to give preferential conditions for the Rhopalodiaceae diatoms (Fairchild et al. 1985, Hill, Knight 1988, Peterson, Grim 1992). This species have endosymbionts capable of fixing the atmospheric nitrogen. Marks, Lowe (1993) reported that E. adnata is a nitrogen specialist, often dominates epiphytic microhabitats where the ratio of available nitrogen to phosphorous might be relatively low.
The Epithemia and Rhopalodia genera contain mainly the species considered as polytypic, containing many varieties. However, in most commonly used keys (Hofmann et al. 2011, Lange-Bertalot et al. 2017, only the broad species concept is given for most of them, resulting in poor understanding of ecological preferences and the distribution of individual taxa of the Epithemia and Rhopalodia genera. Therefore, the actual diversity of the Rhopalodiaceae family is also overlooked.