**2. Materials and Methods**

The Nesamovyte Lake is one of the highest mountain waterbodies of its type in Ukraine, located in the central, highest mountainous part of the Eastern (Ukrainian) Carpathians– Polonyno-Chornogora geomorphological area [68], belonging to Carpathian-Danube algofloristic sub-provinces according to algofloristic zoning of Ukraine [69]. The lake is located in the Carpathian National Nature Park (48◦07 36.6 N, 24◦32 26.4 E) and is situated in the glacial mountain trench on the eastern slope of Mount Turkul (Chornohora Ridge, Eastern Carpathians, and is approximately 5 km from the highest mountain in Ukraine—Hoverla) at an altitude of 1 748 m above sea level (a.s.l.) belongs to the water basin of the Prut River (Figure 1). According to the origin of the lake, it can be called a polymictic tarn that refers the following explanation 'a proglacial mountain lake, pond or pool, formed in a cirque excavated by a glacier' [70].

**Figure 1.** Map of the Carpathian region and the location of the Nesamovyte Lake. (**A**)—the Carpathian Mountains with its Eastern part the Ukrainian Carpathians. (**B**)—map-scheme of Chornohora Massif with the location of the lake. (**C**)—littoral zone of the lake.

The lake covers an area of about 0.4 hectares (about 88 × 45 m) and has a depth of about 2 m and, according to the EC Water Framework Directive [29], belongs to the category of small and very small lakes. In general, the lake water is generated by rain and melting snow with a sandy muddy bottom (rocky from geological rocks of sandy flysch near the southern and western coast and sandy-muddy—in the northern part), it has a long period of freezing (October-May up to a depth of 1.5 m). It belongs to the cold climatic zone of the Carpathians. The summer water temperature normally is 18.3–(12.4) ±3.2 ◦C. The lake has a slow underground runoff in the north-western direction, through a narrow channel on the alloy (swamp intrusion). This part of the lake is intensively overgrown by *Carex rostrata* Stokes and *Sphagnum* spp. Other moss-like organisms and sedge-sphagnum alloy cover the surface of the lake up to 35 cm from the shore and occupies about 40% of the lake bed area and reaches 1 m in thickness. The water in the lake corresponds now to the hydrocarbonate-sulphate-sodium class (Table 1) and belongs to zones of saprobity from oligo- to β-mesosaprobic [71–75]. The hydrochemical formula of water of the lake [76] is as follows: M0.018 SO4 HCO3 42/Na 79 Ca 21. According to EUNIS classification, [77], the Nesamovyte lake belongs to biotopes C1.1 or B1.1.1 (permanent oligotrophic lakes, ponds, puddles).

The material for this study was built on the analysis of the first record (1910–1920) for the Nesamovyte Lake published by Wołoszy ´nska, work with samples from 1967–1978 (collectors Prof. Z.I. Asaul and Prof. G.M. Palamar-Mordvintseva) and our previous investigations (2013–2018).

The species list provided by Wołoszy ´nska (collected in 1910) [67] was carefully analysed and describes the period between 1910–1920.

From 1967 to 1978, samples from Algoteca funds of M.G. Kholodny Institute of Botany of NAS of Ukraine (AKW)—NN 16855–16898 (samples from the year 1967–1978, collectors Prof. Z.I. Asaul and Prof. G.M. Palamar-Mordvintseva) were studied (44 samples). In addition, the data based on these samples published from the period of 1967–1978 [2–5] were included in the current analysis.

To characterize the modern period (2013–2021), 18 samples of net plankton (50–100 L of water filtered out), periphyton from vascular plants (dead and living parts of herbaceous plants (*Carex rostrata*) and pine branches (*Pinus mugo* Turra) and squeezes from the moss (*Sphagnum* spp.) were collected along the perimeter of the lake in August 2013–2018. To study the species composition of *Bacillariophyta*, the method of forming a combined sample from different studied substrates was used. This study is based on the living algal material from plankton (algal cultures, with the addition of BBM-medium) [78] and samples that were fixed with 4% formaldehyde solution.

The obtained results are comparable because almost identical methods for collecting and fixation of the algal material were used in all the studied periods (1910–1920, 1967–1978, 2013–2021).

The algae were studied and identified using light (LM) and scanning electron (SEM) microscopy, the permanent slides of diatoms were made according to the standard procedure using 35% H2O2 [79]. For LM investigations, the diatoms were fixed in the synthetic mounting medium Naphrax (refractive index 1.74) and investigated under a BX-53 microscope (Olympus, Tokyo, Japan). The slides are stored in the Algotheca of M.G. Kholodny Institute of Botany, NAS of Ukraine (AKW). For SEM analysis, the samples were put to specimen stubs, dried, covered with gold (10–20 nm) JFC-1600 sputter coater, and examined in a scanning electron microscope JSM-6060LA (JEOL, Tokyo, Japan) at the Institute of Botany in the Center of Electron Microscopy. The resulting micrographs were processed using the software packages Axiovision 4.3.7. (Carl Zeiss MicroImaging GmbH, Jena, Germany) and GIMP 2.8.10.m (Free Software Foundation, Inc., Boston, MA, USA).


1.

Identification of the species diversity was carried out according the Süßwasserflora von Mitteleuropa [80–85] with some newer updates from Diatoms of Europe [86–89], Diatomeen im Süßwasser-Benthos von Mitteleuropa [90] and Flora of algae of Ukraine [91–97] as well as with updates from [98–102], and electronic resources [103]. The identified taxa, as well as all algal species lists from previous years of studies of the territory were validated using the AlgaeBase system [104] and "Algae of Ukraine . . . " [105] monographic series.

Ecological bioindicator species analysis was based both on the historical data (1910– 1920 and 1967–1978 collections) and the results of our studies (2013–2021) [2–4,61,63,65,67, 106,107]. The following ecological characteristics were used: Habitat preference, streaming and oxygenation, pH [108], salinity, trophic state and сlass of organic pollution [109–112]. Identification of organic pollution by the values of saprobity indices and indicator groups, equated to water quality classes [113,114]. Considering the groups of indicators that we identified, the intervals of the quality classes were distributed as follows: I—0–0.5 (x, x-o); II—0.6–1.5 (o-x, x-b, o, o-b), III—(b-o, o-a, b, b-a) and IV—(a) [112,115–117]. Ecological features of the species were presented according to [112,117,118].

The Cluster analysis of algal composition was carried out using the Paleontological Statistics Software (PAST, Palaeontological Association, Hammer & Harper, Oslo, Denmark, Galway, Norway, Denmark, Ireland) to measure the degree to which species composition was similar among the studied periods (1910–1920, 1967–1978, 2013–2021) [119]. For this purpose, the presence/absence of data were used in the meaning of the Sørencen coefficient, calculated in the program as Bray-Curtis Similarity index [120].
