*4.2. Comparison between Studied Periods of Species Composition of Algae during the 100-Year Investigated Period*

The total dataset regarding the composition of algae covering a 100-year period of investigations presents unique information for a comparative study between the different decades of studies and reveals some defining features in each. The low similarity between years of studies could be a result of the changes that happened with the Nesamovyte lake. Ecological analysis allows us to determine possible reasons for such changes. Some similarity between 2013–2021 and 1910–1920, is in contrast to the 1967–1978 years of investigation (Figure 3) and is attributable to the nature of the study of species diversity during the middle period. The coverage of only two taxonomic groups of algae—Euglenozoa [2] and Desmidiales [3] were conducted during 1967–1978, while the studies of the species diversity at the beginning of ХХth century and ХХIst centuries covered the diversity of all taxonomic groups of algae.

The species composition of the lake during 1910–1920 revealed the presence of specific 27 alpine Holarctic species of desmids and coccoid green algae. It also confirmed uniqueness as well as some affinity to the "Montane-Alpine" component of the species composition of the studied "flora" of the Eastern Carpathians with similar lakes of the Tatra, Sudetes and Alps. At the same time, the author provides the common species lists of algae with the lakes of Tatra, Sudetes and Alps (*Cylindrocystis brebissonii* (Ralfs) De Bary, *Penium cylindrus* Brébisson ex Ralfs (=*Penium cylindrus* var. *subtruncatum* Schmidle), *Tetmemorus laevis* Ralfs ex Ralfs (=*T. laevis* var. *ornatus* Schmidle), *Tetmemorus brebissonii* Ralfs var. *minor* De Bary, *Actinotaenium cuccurbita* (Brébisson ex Ralfs) Teiling ex R ˚užiˇcka (=*Cosmarium cucurbita* Brébisson var. *cucurbita* et var. *attenuatum* G.S. West), *Euastrum montanum* West et G.S. West (=*Cosmarium subreinschii* Schmidle var. *boldtianum* Schmidle), *Euastrum insigne* Hassall ex Ralfs, *Staurastrum scabrum* Brébisson, *S. muricatiforme* Schmidle and other) and alpine holarctic species of charophytes (desmids) and green (coccoid) algae—*Cosmarium nasutum* Nordstedt f. *tatrica* Gutwinski, *C. staurastriforme* Gutwinski, *C. polonicum* Raciborski (=C. vogesiacum Lemaire), *Euastrum aboense* Elfving, *E. binale* (Turpin) Ralfs var. *papilliferum* Gutwinski, *Pediastrum tricornutum* Borge var. *alpinum* Schmidle, *Teilingia granulata* (J.Roy & Bisset) Bourrelly (=*Sphaerozosma granulatum* Roy et Bisset var. *trigranulatum* West), *Staurastrum subavicula* (West) West et G.S. West f. *tyrolense* (Schmidle) G.W.Prescott, C.E.M.Bicudo & W.C.Vinyard (=*Staurastrum vastum* Schmidle var. *tyrolense* Schmidle), *Tortitaenia alpina* (Schmidle) Brook [67] (p. 147).

The studies during 1967–1978 [2–4] showed certain changes in the taxonomic structure of the "flora" and the importance of the following groups: euglenoid algae in different ecotopes of the lake as well as the distinctive high diversity of desmids [3,61].

The modern species composition of algae in the Nesamovyte Lake (2013–2021) has extremely high rates of species diversity noted for Bacillariophyta. This fact makes the modern period significantly different from the two previous periods of studying the floristic composition of algae during 1910–1920 [67] and 1967–1978 [2–4], i.e., 100 and 50 years ago respectively [11]. However, this fact can probably be explained by the current purposeful study of the species composition of diatoms and apparently the influence of a set of ecological and geographical reasons for such transformation, in particular the possible settlement of various ecotopes of the lake by the widespread forms as well as mesotrophic and eutrophic species of the plains area. The reason for the detected diversity of diatoms can possibly be explained by the change in the hydrochemical parameters of the lake. Comparisons by our study with the previous studies as well as inter-comparison of the previous studies revealed more than an 8-fold increase in TDS level over the past 50 years (12–98.6 mg L−1) and a 4-fold increase in the number of sulfates (up to 15.0 mg L<sup>−</sup>1). Species of this taxonomic group were found to be resistant to heavy metal ions Cd— 0.26 mg L<sup>−</sup>1, Cr—2.37 mg L−1, Pb—0.39 mg L−<sup>1</sup> [70,72,74]. Furthermore, high values of the diversity of Bacillariophyta as the leading taxonomic group is distinctive of the highlands of Europe [14,26,52,54,55,60] and correspond to our obtained results. Besides, oligotrophic lakes of the alpine zone are refugium for the conservation of rare and conditionally endemic species [15,39,126], and therefore the presence of European and regionally rare species of Bacillariophyta in the ecotopes of the Nesamovyte Lake is an additional argument for the protection and preservation of the ecosystem of this lake and its diversity as a habitat for their existence.

Materials used in our study for the diversity of the "green" phyla algal flora (Charophyta and Chlorophyta) of this lake during the last decade of the XXI confirm the richness of their species composition and floristic importance in the ecosystem of the lake (Table 2) compared to the period of 50 (1967–1978) and 100 years [3,4,67]. The species composition of Chlorophyta in alpine lakes is characterized by a low level of diversity in general, but often reveals features of uniqueness and ecological specificity [34,49,58,127], which was reported at the beginning of the twentieth century at the first stage of investigations of algae in oligotrophic lake Nesamovyte [61,67]. The findings of noted algae from "green" phyla indicate the probable degradation of the alpine oligotrophic ecosystem of the lake with the earliest possible data (early twentieth century), its transformation to oligo-mesotrophic status nowadays and the settlement of its ecotopes by common species with a wide ecological amplitude [11,61,66,121].

In addition, the comparison of the species diversity of algae in the beginning of ХХwith a modern period revealed changes in the "Montane" complex. This resulted in the disappearance of the majority (over 80%) of the taxa of the "Montane-Alpine" complex that is common for high-altitude lakes of the Alpine-Carpathian region and suggested the mentioned above degradation of the ecosystem. Besides, this fact once again proved the loss of the alpine oligotrophic indicator species [15,126]. However, the presence of a unique algal composition typical for alpine lakes in Europe—a complex of conditionally arctic algae species, including diatoms was noted [26,39,67,121].

The change in ecological conditions became first evident in the 1960s during focused investigations of different ecotopes of the lake for Euglenozoa and Cyanobacteria [2,61,121]. These groups were considered as the typical representatives of the flora in alpine lakes of Europe and indicated a change of the ecosystem of lakes with high levels of organic pollution. This fact confirms a probable increase of trophic state in the Nesamovyte Lake [124]. Such evidence may indicate a degradation of the ecosystem of the highaltitude oligotrophic lake, its transformation to the mesotrophic type and its settlement by representatives with a wide ecological amplitude that started somewhere in between the two studied periods from 1910–1920 to 1967–1978 [61]. And nowadays, the confirmation of such changes in the ecosystem, its violation and increase in trophic state is in our opinion supported by the fact of "blooming" of the lake with green colonial coccoid algae *Botryococcus terribilis*, which was previously noted only in mesotrophic lakes in Australia [128].

An analysis of geographically close and hydrologically related lakes of the Chornogora mountain group (Figure 5) testified to its high level of floristic originality, the pronounced difference between algal communities and uniqueness. The group of lakes (Nesamovyte, Bolotne Oko, Tsyclop) is characterized by the distinct species composition (in particular the leading taxonomic groups—green, charophyte algae and diatoms). In turn, they are close because of the presence of a significant number of regionally rare species. That could also serve as an additional argument for the protection and preservation of the ecosystem of this waterbody [61,63,65].

#### *4.3. Ecological Characteristics of the Nesamovyte Lake*

Our ecological investigations published in previous works [61,65] were based on the analysis of selected groups of algae, and the complex species composition is characterized for the first time.

Indication of habitat preferences (with reference to the type of substrate) and changes in the amount of P-B group gives us an assumption that some hydrological changes in the ecosystem of the Nesamovyte Lake has occurred between 1910–1920 and 1967–1978. In turn, the same changes also appeared in the modern period. Such conclusion can be connected also with the focus of investigation into some groups of algae and thus it should be checked by way of further analysis.

Streaming and oxygenation analysis, in the meaning of oxygen concentration and preference of species to mixing of water in lake, also could confirm the changes in oxygen concentration are due to hydrological or hydromorphological changes [112]. However, the small amount of indicator taxa suggests caution before coming to any conclusions. Even though the amount of pseudoaerial forms seems quite strange for the result of the aquatic

investigation and may be connected with the sampling method. The water was sampled in a littoral zone, which is characterized by wave activity or in the ebb and flow zone where high humidity creates certain conditions for the development of a high number of pseudoaerial forms. Analysis of this group of indicators in 1967–1978 showed absence of the organisms that favour streaming water with a high level of oxygenation. This fact can be connected with a focused study of conjugates, however further analysis of indicator taxa in the modern period (2013–2021) testified the stable presence of organisms that favour standing water with low oxygenation. Thus, it also provides a thesis that some changes with the ecosystem of the lake happened.

The characteristics of pH indicator taxa between different years of investigations revealed some changes in water pH. The prevailing of acidophiles that could characterize the acidic conditions in 1910–1920 indicates a marsh type in the studied waterbody (one of the banks had marsh waters) or input of such type of waters into it [112], because this group of algae is characterized by the ability to survive under pH of 5–6. During the 1967–1978 period, the water pH was on the same level, however, the absence of alkaliphiles reveals some changes that have happened at that time. And in the modern period, both hydrological and ecological conditions of the lake have significantly changed. The evidence of it is the high amount of alkaliphiles, testifying to the change of рНto the low-alkaline side under рН7–8. However, the low number of acidophil taxa is still explained by the presence of marsh type on one bank in the Nesamovyte Lake confirmed by a swampy area with overgrowth of sedge-sphagnum flotation (personal observation). The conducted analysis indicates that the area with marsh conditions has decreased.

Indicator characteristics of salinity displays chloride concentration in the Nesamovyte Lake which allows us to characterize their concentration in the waterbody [112]. As noticeable for all studied periods, the salinity level in the Nesamovyte lake was stable and did not change significantly.

Trophic state analysis [111] indicated changes from an almost minimal level of trophism to some increase in 1967–1978 and a far worst position in 2013–2021. The emergence of hypereutraphetic (he) and an increase in the number of eutraphetic (e) species characterizes the general trend towards the deterioration of the trophic state of the investigated lake. The possible reason for this could be connected to the increasing tourism near the lake.

Characterization of water saprobity indicators as well as saprobity indices made it possible to characterize organic pollution and correlate it with the classification adopted in Ukraine, highlighting water quality classes for the Nesamovyte Lake [113,114]. It revealed the changes in water quality from class I in 1910–1920 to a degradation of the ecological condition of the lake confirmed by the changes in the proportion between classes and appearance of indicators of class IV in 1967–1978 with the same tendency for the deterioration in the modern period.

Thus, the conducted investigation testified a gradual increase in organic pollution from 1910–1920 to the modern period which was also underlined earlier during ecological analyses of the selected taxonomic groups [61,65].
