Diatoms (Bacillariophyta) from the Genus Eunotia and Pinnularia Developing on Soils in the Open Landscape of the Low Beskids

In the areas of medium mountains, including the landscape of the Low Beskids, turf communities guarantee the biodiversity increasing the value of open landscape and protecting soil from erosion. The aim of this study was to show the diversity of the diatom communities from the Eunotia and Pinnularia genera developing on soils under the conditions of varied landscape use (meadows, pastures, fallow lands). The research was carried out within the catchment area of the Ryjak stream in the Magura National Park, together with the buffer zone at three sites. The water content varied in the studied soils with fluctuating moisture content depending on the season and the research year. On the other hand, the pH values indicated high acidification of the studied soils. The diatomaceous analysis showed numerous occurrences of the Eunotia (12 taxa) and Pinnularia (18 taxa) genera. The acid pH of the soil undoubtedly favored the development of diatoms of these genera. Most species of the Eunotia and Pinnularia genera usually developed individually, except for Pinnularia borealis Ehrenberg and P. obscura Krasske. They are typically soil species; therefore, they often dominated in the material and occurred in most of the research seasons. Apart from cosmopolitan diatoms, rare or endangered taxa from the Red List of the algae in Poland (Siemińska et al. 2006) were also found on soils. Four species belonged to the endangered category (E): Eunotia botuliformis F. Wild, Nörpel & Lange-Bert., Pinnularia nodosa (Ehrenberg) Smith, P. schoenfelderi and P. viridiformis Krammer. Canonical analysis (CCA) demonstrated a rather low statistical significance of the impact of environmental parameters (pH and humidity) on the differentiation of diatom communities. It only confirmed the fact that diatoms develop better in the environment with permanent or at least periodic moisture, compared to the dry habitats.


INTRODUCTION
The open landscape, which is generally the case of the Low Beskids, was created by the natural forces and as such is also subjected to slow, natural transformations. The human activity resulting from e.g. construction of rural settlements, farming, etc. also has slight impact on its transformations (Patoczka 2016). The most of agricultural lands in the Low Beskids and in the entire Polish Carpathians are located along the river valleys. Turf assemblages in river valleys guarantee biodiversity, improve the landscape values and have a protective function against surface runoff (Żyszkowska 2003, Jankowska-Huflejt 2007). The characteristic soil types forming the landscape of the middle mountains (including the Low Beskids) are the initial soils, the occurrence of which is, however, mostly limited to dorsal rocky outcrops. The soil cover structure is dominated by brown soils, among them eutrophic brown soils (Skiba 2006).
Soil algae are very important group of soil microorganisms that can be used as an excellent tool for testing the quality of the environment. Due to the large amplitude of adaptation to changing environmental conditions, they are good indicators of environmental quality and their activity is very important for soil functioning (Metting 1981, Gajda & Przewłoka 2012).
The algae in terrestrial habitats have been under research worldwide for many years. Attempts are being made to use algae as indicators of the changes in the soil environment resulting from the use of fertilizers, pesticides, tillage systems and varieties of arable crops (Gollerbach & Sztina 1969, Sieminiak 1998, Zancan et al. 2006, Adesalu et al. 2015). Many scientific works concern the structure and species composition of cyanobacteria and green algae (Durrel 1959, Cameron 1960 The aim of this work was to show the diversity of the diatom communities from the Eunotia and Pinnularia genera growing on soils under diverse landscape use (meadows, pastures, fallow lands) in the Low Beskids.

STUDY AREA
The research was carried out in the Magura National Park with a buffer zone, located in the mountainous landscape of the Low Beskids with rich flora and fauna, usually associated with meadow, pasture and forest habitats. The area is covered by special operating principles defined by a special nature protection zone. The cultural landscape is formed by the remains of former Lemko villages. Numerous churches and remains of residential buildings are a reminiscence of the past culture.
The catchment area of the Ryjak stream is 43.6 km 2 and the natural and forest landscape predominates in this area. In the stream valley, there are permanent grasslands, i.e. meadows and pastures, on ranker soils with browning characteristics SQbr (site R1) and on eutrophic brown soil BEgg (sites R2, R3) (Skiba 2006, Szewczyk 2017. A total of 3 study sites were designated on soils with different uses, within the catchment area of the Ryjak stream: 1) the first site R1 on a pasture (N21,465655; E49,425374), 2) the second one R2 on a meadow (N21,433175; E49,458211) and the third site R3 on fallow land (N21,428535; E49,483552).
The R1 site was exposed and sunny, the soil was covered with low turf (grazed by cattle), stamped with animal hooves. The soil was often dry during the vegetation season. The R2 site was permanently shaded, the soil was overgrown with meadow vegetation (typical for communities in the order Arrhenatheretalia elatioris), mown once a year or less frequently. After heavy rainfall, the groundwater pools overgrowthd with mosses in some places. The R3 site can be described as intermediate between the pasture (R1) and the meadow (R2), with unevenly turf soil (ruderal plants grew in some places, and soil was exposed in other).

METHODS
The material for diatom research and soil physicochemical analysis was collected from three sites (R1-R3) twice a year (from spring 2013 to spring 2019). The sites were designated on the soils in the catchment area of the upper, middle and lower section of the Ryjak stream. The date and method of soil sampling for the physicochemical analyses was established on the basis of the recommendations of the National Chemical and Agricultural Station in Warsaw (https://schr.gov.pl/).
The soil material was taken from pasture, fallow land and meadow, from a 5-15 cm layer, using a metal spatula, at three randomly selected sites, and an in situ combined sample was created in 1-liter plastic bags. At the same time, the soil samples were taken into 100 cm 3 metal cylinders from the surface soil layer (5-15 cm) to examine the level of soil moisture.
The diatomaceous material was collected randomly at three selected points in each site (exposed, moss-covered, greened), by cutting off a 10 cm diameter disc to the Petri dish with a selected fragment of the surface soil layer (0-2 cm).
Then, a combined sample was prepared in the laboratory, ready for preparation, by taking the samples with a scalpel (about 1 cm 3 ). The materials were collected according to the methodology provided by Stanek-Tarkowska & Noga (2012b) and Stanek-Tarkowska et al. (2013).
In the laboratory, the pH was measured using the potentiometric method in a soil solution with distilled water (pH in H 2 O) (Karczewska & Kabała 2008) and the percentage moisture content after drying at 105°C for 72 hours.
The diatomaceous material was macerated for several days in a mixture of K 2 Cr 2 O 7 potassium dichromate and concentrated sulfuric acid VI H 2 SO 4 (so-called 'chromianka') in a 3:1 ratio. The material was then centrifuged in a Thermo Scientific Megafuge 8 laboratory centrifuge at 2,500 r × min − 1 for 5 minutes. Microscopic slides were prepared from the obtained solution, which were closed in Pleurax synthetic resin (refractive index 1.75). The method of diatom purification and the preparation of permanent slides were adopted according to Kawecka (2012).
The microscopic slides were determined under a Carl Zeiss Axio Imager A2 light microscope using Nomarski interference differential contrast (DIC) and a 1.4 planapochromatic lens at 1000 × magnification. The diatom micrographs were taken with using a light microscope (Carl Zeiss AxioCam ICc 5 camera) and a HITACHI SU8010 scanning electron microscope. The diatoms were identified using the following keys: Krammer & Lange-Bertalot (1986, 1991, Krammer (2000), The number of individual diatom taxa was obtained by counting the valves in each preparation, in individual fields of view of the microscope, until a total of about 300 valves was obtained.
On the basis of the Red List of the algae in Poland (Siemińska et al. 2006) species with different degrees of threat were distinguished. The statistical analysis of the impact of environmental variables on the differentiation of communities was performed in the Canoco 5.0.3 program (Ter Braak, Šmilauer 2012).

RESULTS
The water content in the studied soils of different usage types was variable, and fluctuated from 5.6% to 61.9% depending on the season and the research year. In the first site, the moisture content ranged from 10.4% in spring 2019 to 54.8% in autumn 2017, in the second sitebetween 15.5% in spring 2019 and 61.9% in autumn 2016, while in the third site -between 5.6% in spring 2015 and 52.1% in autumn 2017 ( Table 1).
The pH values indicated high acidification of the studied soils, because most soils were characterized by very acid and acid pH(pH: 4.4-5.6). Only at the third site, in the years 2013-2014, the soil had a neutral pH (pH: 6.2-6.8), Table 1.
In order to investigate the impact of pH and habitat moisture on the differentiation of diatom communities, canonical analysis (CCA) was performed. These variables are statistically significant (p <0.05). The analysis did not reveal high statistical significance (for the pH it explains 6% variability, for humidity: 4.7%).
The relationships between the environmental parameters and the diatom communities at individual sites are shown in Figure 4. The analysis shows that the communities were more affected by humidity in 2016-2018 in most seasons. It was also associated with greater development of taxa such as Pinnularia borealis and Pinnularia obscura in these years (Table 2), especially in autumn. This is also confirmed by the level of soil moisture, which in these seasons oscillated between 32.1 and 49.2% in spring and between 49.0 and 61.9% in autumn (Table 1). Similar values were also recorded at individual sites in 2014 and 2015, but no increase in the number of taxa was observed, including the most numerous ones i.e. Pinnularia borealis and P. obscura.

DISCUSSION
The soils at the studied sites in the Magura National Park and the buffer zone were usually characterized by a high level of moisture, especially in autumn. The lowest moisture content was found in soils in the spring of 2015 and the autumn of 2013. In the first case, the material was collected in late spring, when there was no rainfall for a long time, while 2013 was designated  Numerous development of Pinnularia borealis and P. obscura on soils in the Low Beskids in many research seasons, definitely confirms that they are typically soil species. Other diatoms of the Pinnularia genus usually occurred individually and the number of some species increased slightly on rare occasions (up to a dozen or several dozen cells on the site). The performed statistical analysis showed a certain correlation between the numerous development of these species at the studied sites and the increase in the level of moisture. Foets et al. (2020) indicated that diatoms have a number of features, including morphological ones that allow them to adapt to dynamic (daily, seasonal) changes in the environment, e.g. to the changes in moisture content. Therefore, in order to clarify this statistical relationship, further in-depth research on the ecology of these species is needed. Species such as: Pinnularia frequentis, P. isselana or P. marchica were described for the first time in 2000, and their ecology (among others due to the rare occurrence) is still poorly understood (Krammer 2000). This research shows that they can also successfully grow on soils, especially those covered with mosses, but they do not form mass populations. Pinnularia isselana is known for oligotrophic waters with low electrolyte content (Krammer 2000, Hofmann et al. 2011). On the investigated soils, it occurred most frequently on the R3 site (28 valves) in 2018 at low pH and high moisture.
Many diatoms, both of the Pinnularia and Eunotia genera, reached smaller cell sizes in the material compared to the literature data. Especially Eunotia curtagrunowii often appeared in the form of very small specimens and many valves were shorter and narrower compared to the dimensions contained in the key (Lange-Bertalot et al. 2011). Eunotia curtagrunowii in the designated sites was one of the most frequently identified diatoms of its genus. Moreover, Holarctic is one of the most common species of the Eunotia genus. It prefers oligotrophic springs, marginal parts of peat bogs, dystrophic lakes and wet bryophytes. In all habitats, it occurs at low pH and low electrolyte content (Lange-Bertalot et al. 2011). On the studied soils, it was most abundant on the R2 site, which was covered with mosses in places. E. dorofeyukae, which in the studied material was designated as Eunotia cf. dorofeyukae, is characterized by a similar ecology due to high morphological variability and the presence of much narrower valves in most of the cells found (5.8-7.8 μm and not 7-8.5 μm as reported by Lange-Bertalot et al. 2011). The species requires further investigation in the future, but the observations carried out so far indicate that in a soil environment with unstable conditions (e.g. frequent lack of water or even prolonged drought), many cells reach much smaller sizes and become clearly dwarfish.
In addition to cosmopolitan diatoms, widespread in various habitats, there are also rare or endangered taxa, included in the Polish Red List of the algae in Poland (Siemińska et al. 2006 (Wojtal 2013). It occurs individually in the Podkarpackie region in aquatic habitats (Noga et al. 2014b); so far, it has not been identified on soils.
Two Pinnularia species -P. schoenfelderi and P. viridiformis -are probably cosmopolitan diatoms, dispersed in oligo-to dystrophic waters, with low to medium electrolyte content ( as endangered (category E). However, the research conducted in recent years in south-eastern Poland shows that the species are commonly found in different types of habitats, usually individually, while P. schoenfelderi is more often developing outside the aquatic environment, especially on soils. For this reason, it appears that these diatoms should not be assigned to the category of endangered species, but recognized as rare species. However, further research is needed, especially in various terrestrial habitats, which would allow for changing the threat category and extending the ecological preferences of these species.
Pinnularia nodosa -a species widespread in Central Europe in flowing waters, peat bogs and ecosystems with low electrolyte content and pH below 5.5 -was also found in the endangered category (E). It rarely develops in large numbers, especially in periodically wet or humid environ- An interesting species is Pinnularia cuneola, which occurred individually only on site R3. The species is known from a few locations in the world, including puddles in Germany, Alpine lakes and the USA (Krammer & Lange-Bertalot 1986, Krammer 2000. Pinnularia cuneola is mentioned in the taxa lists from Romania (Caraus 2017) and the Netherlands (Veen et al. 2015). It was also found in a small puddle in Stalowa Wola (Rybak et al. 2019). Until now, the species has not been reported in soils, and studies have shown that it can also develop in this habitat, especially on sthe oils covered with mosses.
The conducted studies showed a large variety of diatom communities developing in the terrestrial environment, under the conditions of varied landscape use. The natural landscape of the Low Beskids is conducive to the occurrence of many common species, but is also a refuge for rare species, not yet recorded or even considered as endangered. The diatom studies and laboratory analyses of environmental parameters have shown a great need to expand the studies on the diatom communities of terrestrial habitats. Other authors also believe that the current studies conducted on the terrestrial diatom communities are still incomplete ).