Valuable habitats of protected areas in southern Poland – a source of rare and poorly known diatom species

The peatbogs and natural upper sections of streams in national parks in southeastern Poland represent unique study areas for research on freshwater diatom diversity. During studies conducted on diatoms in three Polish national parks, many little-known, very rare, and endangered species were noted. For most of the taxa presented in this article, especially from the Adlafia, Eunotia, and Placogeia genera, only single individuals have been observed, and only from a few localities worldwide. Moreover, this is the first presentation of SEM photodocumentation and descriptions for Eunotia minutula Grunow and Fallacia sublucidula (Hustedt) D. G. Mann. Based on both light and scanning electron microscopy, detailed descriptions of morphological characteristics, ecological notes, and new localities are presented for the following species: Adlafia langebertalotii Monnier & Ector, Caloneis undulata (Gregory) Krammer, Eunotia fennica (Hustedt) Lange-Bertalot, E. glacialifalsa Lange-Bertalot, E. groenlandica (Grunow) Nörpel-Schempp & Lange-Bertalot, E. minutula Grunow, E. neocompacta Mayama var. neocompacta, E. superpaludosa Lange-Bertalot, Fallacia sublucidula (Hustedt) D. G. Mann, Pinnularia rhombarea Krammer, P. similiformis Krammer, Placogeia gereckei (Cantonati & Lange-Bertalot) Bukhtiyarova, and Sellaphora vitabunda (Hustedt) D. G. Mann.


Introduction
Traditionally, most diatoms have been described as cosmopolitan species [1], although the biogeographic distribution of several species has been defined [2,3]. Recently, however, many new rare, invasive, exotic species with very limited distribution were described not only in Europe, but also globally [4][5][6][7]. Based on the niche theory, several groups have assumed that rare species have narrow environmental requirements and could, therefore, be used as indicators of environmental conditions [8,9].
Southeastern Poland comprises different geographical regions: lowlands in the north, low hills in the central region, and mountainous areas in the south. This part of Poland is characterized by a large variety of habitats, i.e., mountain and submountain valleys with streams (often forested), medium and large rivers (meandering, oxbow lakes), lakes, ponds, peatbogs, and swamps. The high proportion of forest areas, with their natural character and low level of anthropogenic impact on a national scale, makes this area unique. Many protected areas are often home to rare and endangered species spring flooding may increase the concentration of nitrate ions. Generally, the waters of the studied areas can be considered clean, and the adverse water quality changes observed in some seasons are caused by small water flows during rainless periods and a large supply of organic matter in fall (fallen leaves). Additionally, in periods of little atmospheric precipitation, water quality in peatlands may deteriorate because of the mineralization of organic matter [22,26,28,29].
The samples were collected in 2012, 2013, and 2016 within the boundaries of the Roztocze National Park, in 2013 and 2014 in the Magura National Park, and from 2013 to 2015 in peatbogs and small streams and rivers in the Bieszczady National Park. Sampling for diatom and water analysis was always carried out in spring (March-May) and fall (September-October). In the streams and rivers, the samples were collected from stones and submerged mosses (if present at the sampling site). In the peatbogs, the samples were collected from small depressions with water or from mosses. Diatom samples were preserved in a 4% formalin solution. To obtain pure diatom valves, part of the collected material was digested in a chromic acid cleaning mixture (a mixture of sulfuric acid and potassium dichromate at a ratio of 3:1) and then washed in a centrifuge at 2,500 rpm. Diatoms were mounted in Pleurax resin (refractive index 1.75). Laboratory processing of diatoms was carried out using Kawecka [30] methods. All slides and material are stored at the Podkarpackie Innovative Research Center of the Environment (PIRCE) at the University of Rzeszów, Poland.
Chemical analyses were performed using a Thermo Scientific DIONEX ICS-5000+DC device in the Faculty Laboratory for Analysis of Environmental Health and Materials of Agricultural Origin at the University of Rzeszów. Diatoms were identified using both Nikon ECLIPSE 80i and Carl Zeiss Axio Imager A2 light microscopes (LM) with a Plan Apochromatic ×100 objective with differential interference contrast (DIC) for oil immersion. The diatoms were identified from the following available literature: Krammer and Lange-Bertalot [31][32][33][34], Krammer [35], Hofmann et al. [36], Lange-Bertalot et al. [37], Bąk et al. [38], and Monnier et al. [39].
For SEM observations, the samples were coated with gold using a Turbo-Pumped Sputter Coater Quorum Q 150OT ES and observed under a Hitachi SU 8010 at the PIRCE, University of Rzeszów.
The diatom species composition of the samples was determined by counting specimens in randomly selected fields of view under a light microscope. The number of valves counted was 400. Species comprising 5% or more of each assemblage were defined as abundant.

Results
The studies were conducted in two different oligotrophic habitats in southern Poland -the upper sections of watercourses and shallow depressions in peatbogs. Studied watercourses were characterized by usually alkaline to circumneutral pH (6.4-8.8) and low to slightly high conductivity (78-530 µS cm −1 ) (Tab. 1 and Tab. 2). Acidic pH (<4) was noted only in the peatbogs (Site 1 and 7). The ion content was usually low or very low, often below the limit of quantification, indicating pure, oligotrophic waters. Elevated biogenic values (mostly nitrates) were found mainly in the spring (4.76-10.0 mg L −1 ) (Tab. 1, Tab. 2).
During this survey, 13 rare diatom taxa were observed, of which eight were new to Poland. Detailed ecological and morphological notes for these taxa (which are not provided in the literature) are presented below and in Tab. Notes. Knowledge about the ecological preferences of this species is limited. Our studies have shown that the species tolerates a wide range of pH (5.5-8.2) but is also abundant at pH < 6 and medium electrolyte content (254-302 μS cm −1 ). More valves (2-3% of the assemblage) were observed during autumn in the Krupiec stream, on a sandy substrate with high water nitrate content (9.4 mg L −1 ). The bottom of the stream was covered in a large amount of organic matter, especially fallen leaves. At the other sites where A. langebertalotii was found (Tab. 3), the nutrient content was low and the calcium content often exceeded 40 mg L −1 .

Distribution in Poland.
Placogeia gereckei is a new species for Poland.

Notes.
Placogeia gereckei was only observed in the upper, shaded parts of small streams with rapid currents, among mosses, in oligotrophic conditions, most often when the pH was close to neutral and with medium conductivity. Fallen leaves in the streams resulted in an increase in organic matter at these sampling sites. Type of substrate. Always developed on stones.

Distribution in Poland.
Species known only from Kortowskie Lake in northern Poland [40] and Eemian freshwater sediments near Wroclaw [41,42].
Notes. Sellaphora vitabunda was identified individually from both the Wisłoka and Kłopotnica sites. It only developed abundantly (1.7%) at one site on the Wisłoka River,  below a dam reservoir, in August 2013. It was always found in water with an alkaline pH and high electrolyte content in oligo-to mesotrophic conditions. All sites where S. vitabunda was found were located in flowing waters.
Type of substrate. The species developed mainly among mosses growing on stones, very rarely it occurred on stones.

Distribution in Poland.
Species occurred in small numbers in periphyton on the Kraków-Częstochowska Upland [43,44] and in the Gulf of Gdańsk [45].
Notes. Single valves of F. sublucidula were observed only in the upper shaded sections of small streams, most commonly at alkaline pH (7.5-8.4) and medium conductivity under oligo-to mesotrophic conditions. Most of the cells were found among mosses growing on stones, partly or completely submerged in water. Type of substrate. This species occurred in organic sediments in small and shallow depressions and among the mosses in peatbogs. It was noted in streams among mosses growing on stones partly or completely submerged in water.

Distribution in Poland.
Eunotia groenlandica has been so far listed only from the Roztoka stream, from the Tatra Mountains as Eunotia fallax var. groenlandica [54].
Notes. The species was found rarely both in peatbogs and in the upper sections of small streams. In the streams, single valves were most often observed on moss covered stones partially or completely submerged in water. In the peatbogs the species was observed in oligotrophic waters with low pH (3.0-4.5), while in streams, it was found in circumneutral or slightly alkaline pH (6.3-8.6). The conductivity value was low or average.

Type of substrate.
Organic sediments in small and shallow peat pits.

Distribution in Poland.
Eunotia neocompacta var. neocompacta is a new species for the Polish diatom flora.
Notes. The species was always found in acidic pH (below 3.5) and low to medium conductivity, only in the Międzyrzeki peatbog.

Type of substrate.
Organic sediments in small and shallow peat pits.

Distribution in Poland.
Eunotia superpaludosa has not have been reported from Poland.
Notes. Few frustules of E. superpaludosa were found only in two sites in the Międzyrzeki peatbog, so it is difficult on this basis to draw conclusions about the ecology and occurrence of this species. This taxon always occurred in low pH (under 3.5) and low to moderate conductivity.

Distribution in Poland. Pinnularia rhombarea was not reported for Polish territory.
Notes. The species was observed only in peatbogs, more often in the Międzyrzeki (up to 5% of all counted valves), under oligotrophic conditions, always at a pH below 4. It has not been found in flowing waters, even those flowing from peatlands.

Type of substrate.
Organic sediments in small and shallow peat pits.

Distribution in Poland. Pinnularia similiformis is new to Poland.
Notes. Only a few specimens were stated on the Międzyrzeki peatbog in oligotrophic and acidic waters.

Discussion
The waters of the studied watercourses and peatbogs were usually oligotrophic, with a low ion content. Nutrient values were high only in spring (April 2013), which was related to the rapid melting of the snow cover and surface run-off from surrounding fields, meadows, and pastures that had been fertilized with manure the previous fall (especially in the Magura National Park). Within the area of the Roztocze National Park, high nutrient values are observed mainly in fall, when organic matter (mostly fallen leaves and branches) enriches the stream beds. Many rivers and streams in the Magura and the Bieszczady national parks are characterized by average and high concentrations of calcium ions, often >40 mg L −1 , due to the presence of Carpathian flysch in the bedrock [23,26]. Watercourse habitats were considerably more diverse (from more than 100 to more than 250 taxa per site in the studied streams) than peatbogs (50-100 taxa at the site) (T. Noga, unpublished data in preparation). Four species were distinguished from the watercourses. Two of them, Adlafia langebertalotii and Placogeia gereckei, have only been reported from Europe. Adlafia langebertalotii was described from rivers in Luxembourg by Monnier et al. [39] as an aerophilous diatom, occurring mainly in small rivers with low mineralization and organic pollution levels. Recently, these taxa have also been noted in France [55] and Italy [56]. Placogeia gereckei was described from the Dolomiti Bellunesi National Park in the southeastern Alps, Italy. Placogeia gereckei was characteristically found only on wet stones (epilithic taxon) covered by leaf litter. The species was found in small mountain springs located at low elevations (<1,000 m a.s.l.), with an extremely low discharge and always in very shaded sites. The species occurred under oligotrophic conditions, where the pH was alkaline (8.0) and conductivity was low [57]. The species was later reported several times from sources in the Alps [58][59][60], and recently in Serbia in two small mountain rivers, in the epilithon and epibryon [61]. The species developed in oligotrophic conditions in the upper, shaded parts of small streams with a rapid current, and among mosses. Placogeia gereckei was reported by Cantonati and Lange-Bertalot [57] and Vidakovic et al. [61] under similar conditions. As the species usually develops in the upper reaches of streams or sources, it may be a good indicator species for unpolluted, oligotrophic waters.
In contrast, two other species, Fallacia sublucidula and Sellaphora vitabunda, are widespread, but often reported only in different checklists, meaning knowledge about their ecology is limited. Little is known about the ecology and occurrence of Fallacia sublucidula, which is present throughout both the lowlands and highlands [31,38]. Fallacia sublucidula is usually only mentioned in lists of species from Great Britain [62], Germany [10,63,64], Macedonia [65], the Netherlands [66], Romania [67,68], and North America [69]. Fallacia sublucidula probably has a wide development spectrum. Lange-Bertalot [10] described it as an eutraphentic species, which does not quite match the conditions in which it was observed in this study. It is also likely to be an aerophytic species, mainly because it was found on mosses growing on stones, often extending beyond the water surface, depending on water levels in the watercourses.
Krammer and Lange-Bertalot [31] reported that Sellaphora vitabunda has been reported from Europe and Northern America. This species is widespread and often grows in oligo-to mesotrophic lakes with varying amounts of electrolytes. According to Lange-Bertalot et al. [70] and Bąk et al. [38], it is a poorly known species and often mistaken with for Sellaphora verecundiae. It develops in alkaline lakes and lowlands and is relatively rare in the Alps. There is no evidence for the occurrence of this species in flowing waters. Hällfors [71] reported S. vitabunda from the Gulf of Finland (Baltic Sea) and characterized it as a freshwater species that does not tolerate full salinity. According to Lange-Bertalot [10], it is a very endangered (Category 2) species in Germany. Sellaphora vitabunda has a wide global distribution but is usually only mentioned in species lists. Only Kaczmarska [72] gives a detailed morphological description of the species, together with SEM micrographs with descriptions. Sellaphora vitabunda is reported from Europe including the Baltic Sea [71], Great Britain [62,73,74], Germany [10,63,70], Ireland [75,77], the Netherlands [66], Macedonia [65], Romania [67,68,77], Slovakia [78], Spain [79,80], Turkey [81], and Iceland [82], and from other continents: North America [69,[83][84][85], South America [86], Africa [87], Asia [88,89], and Australia (Tasmania) [90]. To date, the species has only been described from lakes [31,38,70], but recent studies have shown that S. vitabunda can also develop in flowing waters if the run of the water is slowed down by natural or artificial barriers (e.g., dams and beaver ponds).
Nine species were distinguished from the peatbog. Most rare or new species were found in the Międzyrzeki peatbog in the Roztocze National Park, showing that protected areas have proved to be a refuge for rare and poorly known diatom species.
Some species, especially from the genus Eunotia, Pinnularia, and Caloneis undulata, are associated with oligotrophic and acidic habitats, with a low electrolyte content [31,35,37]. These are often peatland areas that in many countries, including Poland, represent only a small percentage of the different types of habitat and are still poorly studied. Species of the genus Eunotia -typical of oligotrophic, low-mineral aquatic habitats -are especially rare and threatened to different degrees in Europe. Many of them prefer acidic water and are referred to as acidiobiontic or acidophilous [10,[91][92][93].
In this study, single specimens were found in the oligotrophic, acidic waters of the Międzyrzeki peatbog, under conditions like those described in the literature [36,37,52].
Eunotia minutula is a rare species in Europe, occurring in oligotrophic or dystrophic habitats [37]. Among other places, it was reported from the Netherlands [66], Portugal [95], and Asia [96].
The third species, E. superpaludosa, was described recently by Lange-Bertalot et al. [37], but its occurrence in Europe and North America is not precisely defined due to its similarity to many species of the genus Eunotia, including E. exigua, E. fallax, E. paludosa, and E. nymanniana. Eunotia superpaludosa occurred in ombrotrophic peatbogs with Sphagnum spp. and with other acidobiontic diatoms, including E. paludosa and E. fennica. The species was reported from the Netherlands [66] and several other places.
The other two taxa from the genus Eunotia (E. glacialifalsa and E. groenlandica) were previously reported from Poland, but usually from only a few sites.
Eunotia glacialifalsa is a very scattered species in Central Europe, never forming a large population. It develops in acidic waters, poor in electrolytes, as well as alkaline, oligotrophic waters buffered by carbonates. It is often found in standing waters. However, this species occurs not only in oligotrophic environments, but also tolerates mesotrophic conditions [36][37][38]. It is very rare in the Holarctic region, and has been reported from the Netherlands [66], Belgium [102], and peatbogs in northern Mongolia [101], the Czech Republic, and Denmark [103].
The distribution of Eunotia groenlandica is not precisely defined due to it being confused with E. fallax or E. pseudogroenlandica. Lange-Bertalot et al. [37] found E. groenlandica in various regions of Eurasia and North America. It prefers low pH, low conductivity, and semiaquatic habitats on intermittently wet silicate rocks and bryophytes. This species is known from Europe as Eunotia fallax var. groenlandica, mostly from Great Britain [62], the Netherlands [66], Germany [63], and Slovakia [78]. It was also recorded from North America [83,104], the Czech Republic [94], the Netherlands [66], and Asia [89]. Eunotia groenlandica seems to prefer semiaquatic habitats, as was reported by Lange-Bertalot et al. [37], especially habitats among mosses. It was found in both peatbogs and streams. It is classified as vulnerable (VU) in the "Red list of Bulgarian algae" [13].
Two species from the genus Pinnularia, P. rhombarea and P. similiformis, have also never been reported from Poland. Both taxa prefer cold, oligotrophic waters with a low electrolyte content [35]. Pinnularia rhombarea is more developed in the northern and subarctic regions than in Central Europe [35]. On the "Red list of Bulgarian algae", it is considered an endangered species (EN) [13]. The species is mentioned in the list of species of many European countries, including the Netherlands [66], Romania [67,68], and Bulgaria [13], as well as North America [69], South America [105,106], and Asia [88,89,96,101,107,108]. Oligotrophic waters with a low electrolyte content promote the growth of this species, as also reported by Krammer [35]. Research has shown that P. rhombarea is a peatbog species which prefers small ponds with stagnant water.
Pinnularia similiformis is a cosmopolitan and aerophilic species that develops in oligotrophic, electrolyte-poor, oxygen-rich moorland waters. It is common in Northern Europe, the Alps, and in northern Germany [10,35,109]. Pinnularia similiformis is also known from Chile [110], the Netherlands [66], and Russia [89], among other countries.
Caloneis undulata, the last of the discussed species, has been recorded mainly from the Northern Hemisphere and is considered nordic-alpine. It occurs in oligo-dystrophic waters with low electrolyte content. In Central Europe, C. undulata has been found in mountain areas, usually as single individuals [31,38]. According to Foged [52], it is a cosmopolitan species, oligohalobe and circumneutral. This species was identified as oligotrophic and classified as vulnerable (V) in the German red list of algae [10]. Caloneis undulata has been reported from many places worldwide, but usually it is only listed. It was identified in the Arctic [96] and Iceland [82] as Pinnularia undulata. It is often found in Europe, including in Great Britain [62,73,74], Germany [10], Ireland [75,76], the Netherlands [66], Romania [67,68,77], and Spain [79,111]. Caloneis undulata also occurs in other continents, including North America [52,69,83,112,113], Asia [88,89,96,107,114,115], and even in Australia [90]. This study and data from the literature confirm that the species develops in oligotrophic waters and often in peatbogs, but usually as single individuals. This paper presents rare diatom species, which are often very small, making them difficult to identify. The discussed taxa occur very rarely in specific, uncommon, vulnerable habitats (i.e., unpolluted headwaters and peatbogs) which in many cases are endangered. Consequently, most of these diatom taxa can also be considered as endangered. Most of them, i.e., Adlafia langebertalotii, Eunotia fennica, E. minutula, E. neocompacta var. neocompacta, E. superpaludosa, Pinnularia rhombarea, P. similiformis, and Placogeia gereckei, have not previously been reported from Poland. Two of the mentioned species, Fallacia sublucidula or Sellaphora vitabunda, are often described from Europe and other continents; however, they are usually only mentioned on diatom lists, likely because of their small size and scattered, rare occurrences, as well as the difficulty associated with full SEM documentation. Here, a detailed description of the cell structure with SEM pictures is presented for Fallacia sublucidula. To date, only Wojtal and Sobczyk [43] have presented a specimen of F. sublucidula photographed under SEM in their work, but without visible details of the cell structure. Species like Adlafia langebertalotii, Eunotia minutula, E. superpaludosa, or Placogeia gereckei have only been reported from a few sites and their occurrence and ecology are still poorly known. They were recently described as new to science [37,39,57], and are therefore also not included in national red lists of algae. Every new report on the occurrence and ecological conditions where they develop allows the extension and valuable supplementation of autecological characteristics.