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

Comparative floristic analyses for three periods of investigations showing the similarity of algae composition is presented in Figure 3. For building the graph, the species lists for the periods 1910–1920, 1967–1978 and 2013–2021 were compared. The graph demonstrates a low similarity between the studied periods, however, some higher similarity for 2013–2021 and 1910–1920 can be noted in contrast to the middle period of the investigations.

**Figure 3.** Bray-Curtis tree of species composition comparison in three periods of the Nesamovyte Lake study (1910–1920; 1967–1978; 2013–2021).

The first studies of the species composition of algae in this lake were conducted in the early twentieth century by Professor J. Wołoszy ´nska (based on the 1910 samples of Prof. M. Raciborski) [67]. With the help of this study, the presence of only 67 species (70 inft), in particular Bacillariophyta (15–16 inft), Charophyta (43–45 inft), Chlorophyta (4), Miozoa (Dinophyta) (2), Ochrophyta (Chrysophyceae) (1) and Cyanobacteria (2) were noted.

Leading taxonomic groups by species richness were charophytes (desmids—64.1%) and diatoms (23.4%), combining more than 87% of the whole composition of algae of this waterbody. At the same time, the author noted that diatoms do not make a "*significant contribution to the diversity of species composition of the lake*" ([67], p. 144) (probably meaning a much lower percentage of diatoms). Some distinguishing features for the uniqueness of the diatom composition at the genera level—*Eunotia*, *Pinnularia* and *Neidium* Pfitzer were underlined.

Half a century later (1967–1978 studied period) the targeted studies of species diversity of euglenoid and desmids algae [2–4] from different ecotopes of the lake revealed 102 species of algae. The core divisions of Charophyta (66) and Euglenozoa (9), were added by different divisions presented by Cyanobacteria (3), Cryptophyta (1), Ochrophyta (2), Miozoa (2), Chlorophyta (4) and Bacillariophyta (15) (Table 2).

Our investigations of the modern composition of algae in the Nesamovyte Lake (2013–2021) confirm its species diversity (165 species—168 inft, belonging to 80 genera, 47 families, 28 orders, 11 classes and seven divisions—Table 2) and provide insights into the taxonomic composition and leading complexes of the species. According to the results of this study, the species composition was presented by Bacillariophyta (115 species—117 inft, namely ~70% of total species composition), Charophyta (24–25, ~15%) and Chlorophyta (12, ~7.3%), sparse—Euglenozoa (5–8, ~5.0%) and the lower—Cyanobacteria (3, ~1.8%) and Cryptophyta (2 > 1%).

The basis of the species composition of Bacillariophyta is formed by the following orders: *Naviculales* (38.8%), *Cymbellales* (21.5%), *Achnanthales* (10.3%), *Eunotiales* (8.6%), *Fragilariales* (5.2%), and among families—*Naviculaceae* (12.1%), *Cymbellaceae* (12.1%), *Pinnulariaceae* (11.3%), *Gomphonemataceae* (9.5%), *Eunotiaceae* (8.6%), *Achnanthaceae* (7.7%) [61,65,121]. These families comprise more than 61% of the species composition of diatoms in the Nesamovyte Lake. The genera characterized by high species diversity are the following: *Pinnularia* (12 species (14 inft)—12.1%), *Navicula* (11 species—9.5%), *Eunotia* (10 species— 8.6%), *Gomphonema* (10 species—8.6%), *Encyonema* (5 species—4.3%). The complex of leading species of diatoms from different ecotopes (mainly plankton) of the lake with high quantitative indicators of development was formed by *Tabellaria flocculosa* (Roth) Kützing, *Eunotia minor* (Kützing) Grunow and *Frustulia crassinervia* (Brébisson) Lange-Bertalot et Krammer. Their abundance varied from 4 to 5 according to Starmach scale [122]. In addition, the regionally rare species of the flora of Ukraine, which are known to be from this lake were revealed—*Cymbella lange-bertalotii* Krammer, *Encyonema neogracile* Krammer, *Eunotia tetraodon* Ehrenberg, *Pinnularia macilenta* Ehrenberg, *P. subanglica* Krammer (Figure 4), *Skabitchewskia peragalloi* (Brun & Héribaud) Kuliskovskiy & Lange-Bertalot, and *Pinnularia falaiseana* Krammer. Some of them have a pronounced disjunctive distribution in the world and are considered as rare species [65].

A high variety of modern species composition found for Bacillariophyta [65] contrasts sharply with the data at the beginning of the XXth century [67], when only 16 species were reported (17 inft). Moreover, for the modern period of studies, the presence of arctic diatom species that also are regionally rare for Ukraine [105], and in particular, *Cavinula pseudoscutiformis* (Hustedt) D.G. Mann & Stickle, *Pinnularia rhombarea* Krammer, *P. rupestris* Hantzsch, *P. subanglica* Krammer were found (Figure 4).

According to the results of the "green" phyla algae flora (Charophyta and Chlorophyta), the comparison modern and studied periods of 50 (1967–1978) and 100 years [3,4,67] were conducted. A decrease in the species diversity of Charophyta (from 43 species (45 inft) in 1910–1920 to 27 (31)—in 1967–1978 and up to 24 (25) nowadays) and increase of Chlorophyta (four species—13 species, correspondingly for 1910–2021) were noticed [61,121]. Representatives of the classes Zygnemaphyceae (14.6%) and Chlorophyceae (7.3%) comprise about one-fifth of all the algal species in the Nesamovyte Lake nowadays. A significant role in forming this diversity belongs to the representatives of the order Desmidiales (11.6%), and species diversity of *Zygnematales* and *Sphaeropleales*—low and is between 3.7% and 3.0%, correspondingly. Among genera by species diversity differ *Euastrum* Ehrenberg ex Ralfs and *Staurodesmus* Teiling ex Compere (five species—3.0%, each of them). Eighteen (18) genera are represented only by one species each. In turn, compared to the beginning of the ХХth century, the presence of species of genera has not been confirmed for *Actinotaenium* (Nägeli) Teiling, *Cylindrocystis* Meneghini ex De Bary, *Micrasterias* Meneghini ex De Bary, *Netrium* (Nägeli) Itzigsohn & Rothe, *Teilingia* Bourrelly and *Coelastrum* Nägeli, and for half a century (1967–1978)—species of genera *Penium* Brébisson ex Ralfs, *Sphaerozosma* Corda ex Ralfs, *Spirotaenia* Brébisson and *Tortitaenia* Brook.

At the same time, rare species of genera have now been identified: *Hyalotheca* Ehrenberg ex Ralfs, *Euastrum* Ehrenberg ex Ralfs and *Tetmemorus* Ralfs ex Ralfs. These species are inherent to the flora of water bodies in mountainous regions in general [34]. During the current research period (2013–2021), the presence of rare coccoid green algae (in particular, *Pediastrum braunii* Wartmann = *P. tricornutum* Borge var. *alpinum* Schmidle) had also been unconfirmed. In addition to rare forms of these three genera, the following algae species were found now for Nesamovyte lake the first time: representatives of filamentous charophyte algae and mucilage-forming green coccoid, flagellar algae and cyanobacteria (*Mougeotia* C. Agardh, *Spirogyra* Link, *Zygnema* C. Agardh, *Oedogonium* Link ex Hirn, *Botryococcus* Kützing, *Chlamydomonas* Ehrenberg, *Mucidosphaerium* C. Bock, Pröschold & Krienitz, *Chlorella* Beijerinck, *Mychonastes* P.D. Simpson & S.D. Van Valkenburg, *Westella* De Wildeman, *Anabaena* Bory ex Bornet & Flahault).

**Figure 4.** Regionally rare diatom species from the Nesamovyte Lake: 1–2, 12–14—*Pinnularia rhombarea,* 3–4, 15–17— *P. rupestris*, 5–7, 11—*Cavinula pseudoscutiformis,* 8–10—*P. subanglica*.

According to the results of comparative analysis of species composition of Charophyta division in the Nesamovyte Lake for 100 years [67], a change in the complex of leading groups of Desmidiales/Zygnematales according to quantitative characteristics (frequency of occurrence) was observed. The communities *Cylindrocystis brebissonii* (Ralfs) De Bary, *Actinotaenium cucurbita* (Brébisson ex Ralfs) Teiling ex R ˚užiˇcka, *Cosmarium staurastriforme* Gutwinski, *C. venustum* (Brébisson) W. Archer var. *excavatum* (Eichler et Gutwinski) West et G.S. West, *Euastrum insigne* Hassall ex Ralfs, *E. humerosum* Ralfs var. *humerosum* and var. *subintermedium* Schröder, *E. didelta* (Turpin) Ralfs, *Staurastrum muricatiforme* Schmidle in 1910–1920, have changed to the similar *Staurastrum senarium* Ralfs f. *senarium* and f. *tatricum* Raciborski, *Euastrum pinnatum* Ralfs, *E. humerosum* var. *humerosum* and var. *affine* (Ralfs) Raciborski, *E. didelta* Ralfs, *E. amoenum* F. Gay in 60–70-es of ХХ[3]. The modern grouping of these algae has happened in the beginning of the ХХIst century and the community was formed by *Hyalotheca dissiliens* Brébisson ex Ralfs, *Netrium digitus* (Ehrenberg ex Ralfs) Itzigsohn et Rothe emend. Ohtani, *Euastrum humerosum* var. affine, *E. ansatum* Ehrenberg ex Ralfs and *Staurastrum polytrichum* (Perty) Rabenhorst.

The comparison between the modern period and total list of algae showed that the species diversity of algae nowadays makes up over 70% of the total number of the found species and is characterized by an increase in the number of widespread forms. At present, the basis of the taxonomic and species diversity of the lake is being formed by Bacillariophyta and Charophyta, which in total exceeds about 57% of the genera amount and over 84% of the species and infraspecific composition of algae. Less diversely represented are Chlorophyta and Euglenozoa (>6.0%), and Cyanobacteria and Ochrophyta—are quite low (>2.1%). The dynamics of change in the composition of the algae over the period of 100-years shows an increase of Bacillariophyta (from ~23% to above 70%) and the change in the leading taxonomic group—Charophyta.

The appearance and floristic significance of Euglenozoa (~5.0%) over the last halfcentury—as one of the indicators to the increased degree of the trophic state of the waterbody—was also recorded. In turn, the preservation of secondary importance in terms of species composition groups as Cyanobacteria, Ochrophyta and Cryptophyta were noted. Representatives of Miozoa (Dinophyta) according to comparison with the latest investigations were not identified. In addition, the presence of widespread species of filamentous charophytes and mucilage forming green coccoid and filamentous algae has been noted (*Mougeotia*, *Spirogyra*, *Zygnema*, *Oedogonium*, *Botryococcus*, *Chlamydomonas*, *Mucidosphaerium*, *Chlorella*, *Mychonastes*). Also, some members of Euglenozoa group were found [61].

The "blooming" in the water of the Nesamovyte lake was also tested. It is assumed, that the blooming is caused by the mass development of green colonial coccoid algae *Botryococcus terribilis* Komarek et Marvan (Trebouxiales, Trebouxiophyceae), and which was noted for the first time during the summer of 2015 [106,123].

Our previous investigations [61,63,65] for the comparison of the floral community similar to the Chornogora mountain group revealed a low level of similarity and spatial separation among the lakes of the group (Figure 5). However, the Jaccard coefficient with 43% distinguished a group of lakes—Nesamovyte, Bolotne Oko and Tsyclop with similar algal composition.

**Figure 5.** Dendrogram of floristic similarity of species composition of algae in the Nesamovyte Lake with regionally close lakes of the Chornogora mountain group (Jaccard coefficient).

#### *3.3. Ecological Characteristics of the Nesamovyte Lake Due to Algal Preferences*

Environmental analysis is based on the types of indicators, which are grouped according to the following characteristics: habitat preferences, streaming and oxygenation, pH, salinity, trophic state, organic pollution (water quality class) (Figure 6). With the help of ecological preferences of species grouped by the previously mentioned time intervals (I— 1910–1920; II—1967–1978; III—2013–2021), the ecological characteristics of these periods are also presented.

Characteristics of habitat preferences are determined on a base of indicator species (according to [112]). From 1910 to 1920, species preferring the attached to the substrate way of existence, were formed by benthic species (B) that amounted to 19 taxa or 43.2% (from the total number of indicators of habitat preference) and plankto-benthic (P–B)—15 taxa or 34.1%. For 1967–1978, 17 benthic (B) taxa dominated, composing 53.1% of the total amount of indicators of habitat preferences. However, the amount of plankto-benthic (P–B) taxa were less—eight taxa or 25%. During the modern period (2013–2021), the benthic algae (B) prevailed composing 61 taxa or 50% from total indicator number of habitat preferences, also plankto-benthic (P–B)—47 taxa or 38.5%. Over the studied periods, the restructuring of dominant groups of habitat preference indicators were reported.

Streaming and oxygenation analysis for 1910–1920 revealed the indicators of mediummobile waters, medium enriched with oxygen or standing-streaming (st-str) composing 10 taxa or 45.5% from the total number of indicators of this ecological preference. Somewhat less amount (seven taxa or 31.8%) was formed by aerophytic forms (ae) that are also called pseudoaerial species. For 1967–1978, indicators of standing-streaming (st-str) prevailed and their amount was 53.8% or seven taxa. Analysing the modern period (2013–2021), indicators of standing-streaming (st-str) waters, also prevailed and their amount equalled 58 taxa-indicators (conjugates, diatoms and green algae), composing 58% from the total amount of taxa of indicators of this group.

**Figure 6.** Bioindication plots for ecological analysis of *Habitat preference* (Habitat): P—planktonic, P-B—plankto-benthic, B—benthic; *Streaming and oxygenation* (Oxygen): st—organisms that favour standing water with low oxygenation, st-str—organisms that favour low-flow, moderately oxygenated water; str—organisms that favour streaming water with a high level of oxygenation, aer—aerophyles; *pH*: acf—acidophiles, ind—pH-indifferents; alf—alkaliphiles; alb—alkalibiontes; *Salinity*: hb—halophobes, i—indifferents, hl—halophiles, mh—mesohalobes, *Trophic state* (Trophy): ot—oligotraphentes, o-m—oligo-mesotraphentes, m—mesotraphentes; me—meso-eutraphentes; e– eutraphentes; o-e—from oligo- to eutraphentes; he—hypereutraphentes and *Class of water quality* (according to organic pollution) in the Nesamovyte Lake (for the periods: I—1910–1920, II—1967–1978, III—2013–2021.

Indication of рH (according to [108,112]) for 1910–1920 revealed the prevalence of acidophils, which included 30 taxa, composing 68.2% of the total amount of indicators of this group. For 1967–1978, acidophils also prevailed containing 22 taxa, that was equal to 73.3% from the total amount of the taxa. For modern period (after 2013), the indifferents (ind) prevailed and their amount equalled 50 taxa or 43.1% with the relatively large number of alkaliphiles (alf), composing 31 taxa or 26.7%. Noteworthy is the number of acidophilic (acf) species that is formed by 29 taxa or 25% of the total number of indicators of this group for this period of investigations.

The salinity indicators (according to [109,112]) covered from 8.9% to 60.5% of the total amount of species for each studied period. During all the years of investigations, the significant prevailing of indifferents (i) was recorded.

The trophic state of the Nesamovyte Lake in 1910–1920 indicated prevailing of oligomesotraphentic (o-m) indicators that covered 21 taxa or 48.8% from the total amount of taxa for this year. In 1967–1978, the mesotraphentic (m) indicator taxa, which numbered 12 taxa or 48% along with oligo-mesotraphentic (o-m) composing 11 taxa or 44% prevailed. During the modern period (2013–2021), the following groups formed majority of indicators: oligo-mesotraphentic (o-m): 26 taxa or 24.1%, meso-eutraphentic (me): 25 taxa or 23.1% and oligotraphentic (ot): 24 taxa or 22.2%. It is also worth noting the presence of hypereutraphetic (he) species as well as an increase in the number of eutraphetic (e) species for this period that was not recorded from the earliest periods.

Characterization of organic pollution (saprobity) for 1910–1920, indicated class I water quality formed by 17 indicator taxa composing 44.7% from all groups of indicators for this period. In 1967–1978, indicators of class I predominated (11 taxa or 39.3% of indicators of characterizing parameter of this year). However, the presence of indicators of class IV water quality was also noticed. During the modern period, indicators of class II of water quality (46 taxa or 39% of indicators characterizing this parameter for this year) prevailed with the presence of indicators of the class IV quality.
