Environmental Factors Structuring Diatom Diversity of the Protected High Mountain Lakes in the Kaçkar Mountains National Park (Rize, Turkey)

: The altitude of the habitat is one of the important regulators of species survival. Kaçkar Mountains National Park is located in the Eastern Black Sea region of Turkey. This is the first study on the benthic diatom flora of the high mountain lakes in Kaçkar Mountains National Park, which is situated between 2782 and 3075 m a


Introduction
Siliceous crystal mountain ranges are among the highest-altitude regions in the world.This feature allows them to harbor pristine biodiversity, provide recreation areas, and maintain healthy water resources.Therefore, they hold a prominent position among natural areas that need protection [1].
High mountain lakes are habitats where a limited number of species live, as they generally have low nutrient and ion concentrations [2].They are also sensitive to climate change, dissolved organic carbon, and nitrogen inputs [3,4].Increases in air temperature and changes in snow and ice cover on mountains in some regions have changed the functioning, diversity, and productivity of these lakes [5][6][7].Despite these effects, high mountain lakes are still considered as undisturbed ecosystems [8].
Biodiversity, which bears the evidence of evolutionary processes, plays an important role in the maintenance of ecological functions and the stability of the ecosystem [9].At the same time, biodiversity is also used to evaluate the environmental status of aquatic ecosystems [10].Diatoms, which have very important ecological functions, contribute 20-25% to the world's global primary production, carbon fixation, and oxygen release to the atmosphere, thanks to their photosynthetic activities [11,12].In addition, the annual amount of carbon fixation by diatoms represents 40% of total primary production in seawater, an amount equivalent to the total amount fixed by all terrestrial tropical forests [13].Therefore, examining the diatom communities in the sediments of high mountain lakes and using the obtained information in the creation of diatom-based biomonitoring programs will be one of the most effective ways to understand the response of these lakes to climatic change [14].
The need to study the species richness and ecology of benthic diatoms becomes more obvious as data accumulate in the biomonitoring system of high mountain lake ecosystems of the Eastern Black Sea region.Studies have been carried out on the benthic diatom flora of high mountain lakes and the physico-chemical properties of their waters in the region since 1990 [15].However, until now, no information has been obtained about the diatom flora of the high mountain lakes in the Kaçkar Mountains National Park.
The aim of this work was to describe the diversity of benthic diatoms collected from 14 lakes and a pond in the Kaçkar Mountains National Park, located in the Eastern Black Sea region of Turkey, and their relationship with environmental factors.Additionally, it determines the rarity and frequencies of diatom species based on the number of studied lakes where species were found.The area is classified under Turkey's climate classification system [19] as being under the influence of the Eastern Black Sea climate.This climate is characterized by cool summers, temperate winters, and rainfall throughout the year.In the lower, northern part of the Kaçkar Mountains National Park, such as around the Ayder Plateau, temperatures range from 0 °C to 4 °C during the winter months and rise above 18 °C in the summer.At an altitude of over 3000 m a.s.l. in the southern mountains of the park, temperatures drop to −6 °C in winter and range from 6 to 9 °C in summer.High alpine areas are typically covered in snow from late September to mid-May.The park receives an annual average precipitation of over 2000 mm.The park is composed of four major soil groups: high mountain meadow soils, limeless brown forest soil, red yellow soils, and gray brown soils [16].The park area has significant biodiversity in terms of flora and fauna.Davis [20] identifies the research area as part of the Colchis (Colchis) region of the Euro-Siberian floristic region.The forest belt contains broad-leaved conifers, Fagus orientalis, Castanea sativa, and Carpinus sp.The alpine zone has a rich vegetation with many endemic and relict species.This location in Turkey is unique, as it is the only place where Rhododendrons grow at an altitude of 3000 m above sea level.The study area is one of three significant routes for bird migration, with the northeast-south migration route being the most important for daytime raptors in the Western Palearctic [21].

Methods of Sampling and Laboratory Studies
A total of 39 samples of epipelic, epilithic, and epiphytic algae were collected from 14 lakes and a pond on 19 July, 28 August, and 10 September 2020 (Figure 2, Table 1).Due to the inaccessibility of the lakes, 2 or 3 fouling samples were taken from each.Epipelic algae were collected from the sediment surface of all studied waters using a glass pipe 1 m long and 0.8 cm in diameter.Epilithic samples from 2 or 3 stones were collected only

Methods of Sampling and Laboratory Studies
A total of 39 samples of epipelic, epilithic, and epiphytic algae were collected from 14 lakes and a pond on 19 July, 28 August, and 10 September 2020 (Figure 2, Table 1).Due to the inaccessibility of the lakes, 2 or 3 fouling samples were taken from each.Epipelic algae were collected from the sediment surface of all studied waters using a glass pipe 1 m long and 0.8 cm in diameter.Epilithic samples from 2 or 3 stones were collected only from TSL-1, TSL-2, BDL, and KVL lakes.Randomly selected stones were scraped off with a toothbrush and the suspension was placed in plastic bottles.
Epiphytic species were collected by squeezing out from the macrophytes (Potamogeton sp. and Juncus sp.), where several parts of submerged plants were manually squeezed out, and the suspension was placed in a test tube found in KVL and AP [22,23].All samples were preserved in 100 mL plastic bottles with 4% (v/v) formaldehyde.In the field, we measured in three repetitions the water temperature, dissolved oxygen, conductivity, and pH using Thermo Orion-4-Star pH and YSI-55 portable meters for all sampling points, excluding AP pond, where variables were not tested because the water sample was lost.The DSI General Directorate Laboratories DSI 22nd Regional Directorate Quality Control and Laboratory Branch Office conducted analyses of variables other than temperature, dissolved oxygen, conductivity, and pH: potassium, total hardness, calcium, magnesium, ammonium, chlorine, nitrate, nitrite, and phosphate hydrochemical parameters.Diatom samples were acid-purified using H 2 SO 4 and HNO 3 in the lab, followed by washing with distilled water [22].The cleaned diatom shells were then placed in Naphrax ® .The diatoms were examined (up to 400 shells per slide) and photographed using a Leica DM 2500 light microscope and a Leica DFC 290 camera (Leica, Wetzlar, Germany).
Bioindication methods were used to assess the ecological state of lake ecosystems [38].For this purpose, the distribution of species with certain ecological preferences was identified across intervals of environmental factors [39].Then, data on the abundance of species in a particular lake were summarized for each indicator group.The distribution of the number of species with the same indicator properties was plotted by ecological groups for each environmental variable.The class of water quality indicators of organic pollution was grouped by the range of the species-specific index of saprobity S: class 1, S = 0.0-0.5;class 2, S = 0.5-1.5;class 3, S = 1.5-2.5;class 4, S = 2.5-3.5 [39].In total, groups of indicators for nine environmental and ecosystem variables (substrate preferences, temperature, oxygen, salinity, pH, organic pollution by Watanabe system [40], organic pollution by Sládeček system [41], autotrophy-heterotrophy nutrition type, trophic state) were used for analysis in this study.The arrangement of groups of indicators for each environmental variable on the histogram was in increasing order of the indicated variable.
Ecologies 2024, 5, FOR PEER REVIEW 4 from TSL-1, TSL-2, BDL, and KVL lakes.Randomly selected stones were scraped off with a toothbrush and the suspension was placed in plastic bottles.Epiphytic species were collected by squeezing out from the macrophytes (Potamogeton sp. and Juncus sp.), where several parts of submerged plants were manually squeezed out, and the suspension was placed in a test tube found in KVL and AP [22,23].All samples were preserved in 100 mL plastic bottles with 4% (v/v) formaldehyde.In the field, we measured in three repetitions the water temperature, dissolved oxygen, conductivity, and pH using Thermo Orion-4-Star pH and YSI-55 portable meters for all

Lake with Abbreviation
Geographic Coordinates Altitude (m) Area, km 2   Kapılı Lake-1 (KPL-1) 40 Bray-Curtis analysis was performed using BioDiversity Pro 9.0 and a similarity tree was constructed [42].Pearson correlation coefficients were calculated using [43].Correlation analysis of species data in each lake was performed as a network graph in JASP (Jeffrey's Amazing Statistics Program 0.16.4) statistics botnet package with R [44].Threedimensional (3D) surface plots of the number of species versus individual parameters were constructed in Statistica 12.0 using the distance-weighted least squares method.For comparison, for each 3D graph, one main parameter (species number) and two others are selected, within which the program calculates probable changes in the main parameter.Thus, the resulting graph shows the trends in each of the related parameters.From here, extreme values may appear that are not real, but only reflect trends for a given distribution.The graph can be interpreted as a trend of changes (increases or decreases) in the values of the main parameter (z-axis) when the other two parameters (x-and y-axis) change.Redundancy discriminant analysis (RDA) to calculate the relationship between biological dominant variables and environmental variables was performed using the CANOCO program [45].

Physical and Chemical Properties of Waters
The water temperatures of the studied lakes fluctuated between 7.1 and 22.5 • C. While the pH values of the waters were determined to be between 6.10 and 8.21, the dissolved oxygen values were measured to be between 8.02 and 9.17 mg L −1 .Total dissolved solids were 9.92-44.27mg L −1 and electrical conductivity values were found as 14.1-71.4µSm cm −1 .Total hardness was measured in the range of 17.59-38.84mg L −1 in the KPL-1,2,3,4,5, BDL, and ML.The amount of nitrate detected in the studied lakes was in the range of 0.207-0.575mg L −1 , excluding lakes KPL-1, VKL, and KVL, where the nitrates were very low.Nitrite was detected as 0.020 mg L −1 in ML and TSL-1, while phosphate was detected as 0.113 mg L −1 only in KPL-5.Potassium, calcium, magnesium, ammonium, and chlorine values of the waters were also determined and are represented in Table 2, with some indeterminate values as a result of fresh soft water.
Note: (-): Could not be detected as below the determination level.Variables in AP pond were not tested because water sample was lost.

Floristic Composition and Diversity of Diatoms
A total of 84 species and intraspecific taxa of Bacillariophyta, belonging to three classes, 15 orders, 25 families, and 42 genera were identified in 14 lakes and the pond (Appendix A Figures A1-A4).While the two classes (Mediophyceae, Coscinodiscophyceae, four species in each class) represent a very small part of the benthic diatom flora (9.52%), class Bacillarophyceae formed the basis of the species richness of the flora (76 species, 90.47%).The top two orders of the flora composition of benthic diatoms included Naviculales (35 species) and Cymbellales (10 species).Among the dominant families were Pinnulariaceae (thirteen species), Naviculaceae (eight species), Surirellaceae (seven species), Cymbellaceae (five species), and Eunotiaceae (five species).The main part of the benthic diatom flora was formed by genera Pinnularia (thirteen species), Eunotia (five species), Navicula (five species), and Frustulia (four species) (Appendix A Table A1).One diatom species (Eunotia cristagalli) was identified for the first time in the freshwater diatom flora of Turkey.It is marked with an asterisk (*) in Appendix A Tables A1 and A2.About 8.33% of the diatom species were found in more than 80% of investigated lakes (VF), whereas 72.61% were found in less than 40% of the investigated lakes (VR, R).The representation ratios of the species in the frequent (F) and common (C) groups are the same (eight species in each group, 9.52%).Thirty diatom species (35.71% of the flora) were identified in only one lake each.
On the other hand, Iconella capronii is the only species identified in all studied lakes and the pond.Likewise, Didymosphenia geminata and Encyonema minutum were observed in 15 habitats.The Caloneis silicula, Navicula cryptocephala, Pinnularia interrupta, and P. major were found in 13 lakes (Appendix A Table A1; Appendix A Figures A1-A4).
When the benthic diatom flora of the studied lakes were compared, it was observed that the species diversity and relative abundances were different.In particular, the epilithic diatom species richness (81 species) was higher than that of epipelic and epiphytic diatoms.With regard to the geographical distribution of species, a significant part of the flora consisting of cosmopolitan species is of alpine and subalpine origin.The flora also includes northern alpine and alpine diatom species [24-35].

Bioindicators
The distribution of indicator properties of diatom species in studied lakes is represented in Appendix A Table A2 and summarized in Appendix A Table A3.It can be seen that the total number of diatom species in each lake strongly correlated with the abundance scores, R = 0.995, p < 0.0001 (Figure 3).The dashed trend lines show a decrease in both variables with increasing altitude of the lake.The organic pollution evidence is crucial for the protected lakes, and in the Kaçkar Mountains National Park, we can observe that the calculated Index of Saprobity (S) reflects clear waters in the studied lakes, falling within Class 2 or 3 of water quality (Figure 3), with a tendency for a slight decrease with increasing altitude.
The distribution of each group of indicators is represented in Figures 4 and 5. Benthic species dominated the examined samples.The plankto-benthic species were also observed (Figure 4a).It can be seen that the percentage of benthic inhabitants slightly increased with altitude from 60% in KRDL to 70% in AL1, but it appears to be rather stable in the lake community (Figure 4a).Among the indicators of water temperature belonging to four groups, species characteristic of temperate conditions strongly predominate in the surveyed lakes (Figure 4b).Three ecological groups of diatom species were found in relation to oxygenation, water velocity, and oxygen saturation.In the studied lakes, the species characteristics for standing water prevail (Figure 4c).The results of the pH bioindication show that indifferent species (45.45%) are common in the Kaçkar Mountains Natural Park.It was followed by alkaliphile (35.06%) and acidophile species (15.58%), respectively (Figure 4d).Additionally, alkalibionte species were also seen in VKL and KPL-3 lakes (Appendix A Table A3).As a whole, indicator groups consisting of indifferent alkaliphiles and acidophiles comprised 96.09% of the indicator species in each lake community (Figure 4d).abundance scores, R= 0.995, p < 0.0001 (Figure 3).The dashed trend lines show a decrease in both variables with increasing altitude of the lake.The organic pollution evidence is crucial for the protected lakes, and in the Kaçkar Mountains National Park, we can observe that the calculated Index of Saprobity (S) reflects clear waters in the studied lakes, falling within Class 2 or 3 of water quality (Figure 3), with a tendency for a slight decrease with increasing altitude.The distribution of each group of indicators is represented in Figures 4 and 5. Benthic species dominated the examined samples.The plankto-benthic species were also observed (Figure 4a).It can be seen that the percentage of benthic inhabitants slightly increased with altitude from 60% in KRDL to 70% in AL1, but it appears to be rather stable in the lake community (Figure 4a).Among the indicators of water temperature belonging to four groups, species characteristic of temperate conditions strongly predominate in the surveyed lakes (Figure 4b).Three ecological groups of diatom species were found in relation to oxygenation, water velocity, and oxygen saturation.In the studied lakes, the species characteristics for standing water prevail (Figure 4c).The results of the pH bioindication show that indifferent species (45.45%) are common in the Kaçkar Mountains Natural Park.It was followed by alkaliphile (35.06%) and acidophile species (15.58%), respectively (Figure 4d).Additionally, alkalibionte species were also seen in VKL and KPL-3 lakes (Appendix A Table A3).As a whole, indicator groups consisting of indifferent alkaliphiles and acidophiles comprised 96.09% of the indicator species in each lake community (Figure 4d).Salinity is a crucial component of the total ion content in water, influencing the algal community.Bioindication based on water salinity reveals that the "indifferent" group of species dominates in all studied lakes.The other groups were oligohalobes/halophobes, halophiles, and mesohalobes (Figure 5a).Light and temperature are the basic climatic variables affecting photosynthesis, so these global climatic factors also define life and evolution [4].Photosynthetic diatoms prevail in communities of all studied lakes (Figure 5b) and strongly prevail with increasing altitude.Salinity is a crucial component of the total ion content in water, influencing the algal community.Bioindication based on water salinity reveals that the "indifferent" group of species dominates in all studied lakes.The other groups were oligohalobes/halophobes, halophiles, and mesohalobes (Figure 5a).Light and temperature are the basic climatic variables affecting photosynthesis, so these global climatic factors also define life and evolution [4].Photosynthetic diatoms prevail in communities of all studied lakes (Figure 5b) and strongly prevail with increasing altitude.

Comparative Analysis
A JASP Network plot of bioindicator correlation of the studied lakes in the Kaçkar Mountains National Park was constructed based on Appendix A Table A3.Four clusters can be seen in Figure 6a.Cluster 1, marked with a black dotted line, unites lakes AL-1 and KPL-3,4, the structure of diversity of which is similar, and the species composition is not high, as seen in Appendix A Table A3.These three lakes are located above all the others and are part of the southern group of lakes, and their floras are dominated by indicators of class 2 clean waters.Cluster 2, outlined by a red dotted line, consists of the floras of four lakes, the indicators of which occupy an intermediate position between the high mountain lakes of cluster 1 and the rest.Cluster 3 includes five lakes, among which are low-lying lakes with higher temperature and conductivity.The three lakes of cluster 4 have high species richness and are located in the group of southern and middle lakes.In general, the analysis did not reveal a strict relationship to groups of lakes and to altitude, but revealed a noticeable role of species richness as a grouping factor.We supplemented the current analysis with a comparison with the floras of the lakes of the Artabel Park [47] located in The bioindication results of organic pollution obtained from the Sladecek's [41] systems are shown in Figure 5c.Organic pollution indicator groups of Watanabe's [40] method are not presented on the histogram but can be seen in Appendix A Table A3 and demonstrated the same results as Sládeček's method.While the indicators of Class 2 in Sládeček's [41] system contained a significant portion of the diatom community in all the studied waters, Class 3 was second.While Class 1 was represented in the KVL and KPL-1 lakes, Class 4 was also represented in the AP, KRDL, VKL, BDL, KPL-5, TSL-2, ML, KPL-2, and KPL-1.KPL-1 was the only lake where all class indicators were found.The indicators for water pollution in Class 5 were not identified.Figure 5c illustrates a decrease in Class 4 indicators with an increase in altitude, accompanied by a rising percentage of indicators corresponding to Class 3 water quality.
The trophic state of the lake usually correlates to organic matter content [46].Oligotrophic diatom species constitute 15.47% of all diatom species and dominate the diatom communities of the studied lakes.They are followed by oligo-mesotrophic (13.09%) and eutrophic (11.90%) diatom species, respectively.In total, they comprise 40.46% of all diatom species.Additionally, mesotrophic, meso-eutrophic, and oligo-eutrophic diatoms were recorded, representing a smaller share in the diatom community (5.95%) (Figure 5d).

Comparative Analysis
A JASP Network plot of bioindicator correlation of the studied lakes in the Kaçkar Mountains National Park was constructed based on Appendix A Table A3.Four clusters can be seen in Figure 6a.Cluster 1, marked with a black dotted line, unites lakes AL-1 and KPL-3,4, the structure of diversity of which is similar, and the species composition is not high, as seen in Appendix A Table A3.These three lakes are located above all the others and are part of the southern group of lakes, and their floras are dominated by indicators of class 2 clean waters.Cluster 2, outlined by a red dotted line, consists of the floras of four lakes, the indicators of which occupy an intermediate position between the high mountain lakes of cluster 1 and the rest.Cluster 3 includes five lakes, among which are low-lying lakes with higher temperature and conductivity.The three lakes of cluster 4 have high species richness and are located in the group of southern and middle lakes.In general, the analysis did not reveal a strict relationship to groups of lakes and to altitude, but revealed a noticeable role of species richness as a grouping factor.We supplemented the current analysis with a comparison with the floras of the lakes of the Artabel Park [47] located in the mountainous region of Turkey, but somewhat to the west, and also included in the analysis two previously studied lakes of the Kaçkar part [15].Figure 6b shows that the lake communities are grouped according to territorial characteristics, corresponding to two groups of protected areas, while the floras of two previously studied lakes located on the low spurs of the mountains in the Kaçkar Park are included in the western group of lakes of the Artabel Park, emphasizing the high individuality of those studied in this study of 14 lakes and ponds.
Ecologies 2024, 5, FOR PEER REVIEW 11 the mountainous region of Turkey, but somewhat to the west, and also included in the analysis two previously studied lakes of the Kaçkar part [15].Figure 6b shows that the lake communities are grouped according to territorial characteristics, corresponding to two groups of protected areas, while the floras of two previously studied lakes located on the low spurs of the mountains in the Kaçkar Park are included in the western group of lakes of the Artabel Park, emphasizing the high individuality of those studied in this study of 14 lakes and ponds.The comparison of the species richness from Appendix A Table A1 by the Bray-Curtis calculation of similarity for diatom species composition shows two groups of lakes (Figure 7), outlined by a dashed line and represented by different colors.It can be observed that the smaller group of lakes includes only KPL lakes 3, 4, 5, and AL 1 and 2. All other diatom communities are outlined by the largest cluster.Both type analyses (JASP and Bray-Curtis) show similar results, because the smallest group also included the lakes with low species richness of diatoms placed at high altitude.The comparison of the species richness from Appendix A Table A1 by the Bray-Curtis calculation of similarity for diatom species composition shows two groups of lakes (Figure 7), outlined by a dashed line and represented by different colors.It can be observed that the smaller group of lakes includes only KPL lakes 3, 4, 5, and AL 1 and 2. All other diatom communities are outlined by the largest cluster.Both type analyses (JASP and Bray-Curtis) show similar results, because the smallest group also included the lakes with low species richness of diatoms placed at high altitude.
calculation of similarity for diatom species composition shows two groups of lakes (Figure 7), outlined by a dashed line and represented by different colors.It can be observed that the smaller group of lakes includes only KPL lakes 3, 4, 5, and AL 1 and 2. All other diatom communities are outlined by the largest cluster.Both type analyses (JASP and Bray-Curtis) show similar results, because the smallest group also included the lakes with low species richness of diatoms placed at high altitude.

Species-Environment Relationships
RDA plots of the relationship analysis of dominated ecological groups and environmental variables are represented in Figure 8a,b and in the correlation matrix in Appendix A Table A4.Data from fourteen lakes were used for this analysis, with seven environmental data points as independent variables and nine biological data points as dependent variables.Despite the fact that the test indicators are far from ideal (945 permutation; Eigenvalue = 0.488; p-value = 0.5), since this is a complex natural system of lakes, one can focus on the general trends in the dependence of the activity of indicator groups in the available environmental indicators.Figure 8a shows that the majority of indicators are combined into one set related to the increase in dissolved ions and oxygen concentration.The other set of environmental variables represents a negative influence on most indicator groups and includes ammonia, pH, and temperature variables.It is remarkable that one of the most species-rich indicator groups of oligotrophic waters stays in opposition to the Index Saprobity S value.An increase in the Index Saprobity S value is typically associated with eutrophication.Figure 8b reflects the lakes related to the groups of variables described above.So, ammonia is an important factor for KVL lakes, favoring oligotrophic species domination.The increasing water pH influenced diversity mostly in AL-1, while a decrease in pH is important for diatom species abundance in TSL-2 lake when total salt concentration was increased.
The relationships between species richness in the diatom community of the protected lakes in the Kaçkar Mountains National Park and the major climatic and water property variables were studied with the 3D plots in the Statistica program.Figures 9-12 show the surface plots in which the dependent variable was the number of diatom species in the community of 14 lakes and the independent variable for each plot was the lake altitude, but the third variable for each plot was different.Figure 9a shows an increase in the number of diatom species in the community when water conductivity and the lake altitude were low.The different tendency can be seen in relation to dissolved oxygen (Figure 9b) when species richness decreased in high-altitude lakes where DO increased.
in opposition to the Index Saprobity S value.An increase in the Index Saprobity S value is typically associated with eutrophication.Figure 8b reflects the lakes related to the groups of variables described above.So, ammonia is an important factor for KVL lakes, favoring oligotrophic species domination.The increasing water pH influenced diversity mostly in AL-1, while a decrease in pH is important for diatom species abundance in TSL-2 lake when total salt concentration was increased.The relationships between species richness in the diatom community of the protected lakes in the Kaçkar Mountains National Park and the major climatic and water property variables were studied with the 3D plots in the Statistica program.Figures 9-12 show the surface plots in which the dependent variable was the number of diatom species in the community of 14 lakes and the independent variable for each plot was the lake altitude, but the third variable for each plot was different.Figure 9a shows an increase in the number of diatom species in the community when water conductivity and the lake altitude were low.The different tendency can be seen in relation to dissolved oxygen (Figure 9b) when species richness decreased in high-altitude lakes where DO increased.Figure 10a demonstrates the relationships of water pH that fluctuated between 6.0 and 8.4, in which, at the low-altitude lakes, the species number increased.In contrast, the temperature surface has a two-wave shape that reflects the complex relation of species richness with this environmental factor.In any case, species richness increased with the highest temperature in the low-altitude lakes (Figure10b).Figure 10a demonstrates the relationships of water pH that fluctuated between 6.0 and 8.4, in which, at the low-altitude lakes, the species number increased.In contrast, the temperature surface has a two-wave shape that reflects the complex relation of species richness with this environmental factor.In any case, species richness increased with the highest temperature in the low-altitude lakes (Figure 10b).Index saprobity S is evidence of organic pollution, which is an important factor for the protected lakes' diversity.Figure 11a demonstrates that the relationships between species richness in the diatom community and organic pollution are complex, showing a two-wave pattern.But in general, there is an increase in species richness in the group of "low"-altitude lakes with an increase in organic pollution.The ammonium impact is reflected in the surface of Figure 11b when increasing, which stimulates the species richness in both high-altitude and low-altitude lakes.
Figure 10a demonstrates the relationships of water pH that fluctuated between 6.0 and 8.4, in which, at the low-altitude lakes, the species number increased.In contrast, the temperature surface has a two-wave shape that reflects the complex relation of species richness with this environmental factor.In any case, species richness increased with the highest temperature in the low-altitude lakes (Figure10b).Index saprobity S is evidence of organic pollution, which is an important factor for the protected lakes' diversity.Figure 11a demonstrates that the relationships between species richness in the diatom community and organic pollution are complex, showing a two-wave pattern.But in general, there is an increase in species richness in the group of "low"-altitude lakes with an increase in organic pollution.The ammonium impact is reflected in the surface of Figure 11b when increasing, which stimulates the species richness in both high-altitude and low-altitude lakes.There was a special part of the 3D analysis in which we tried to reveal the relationships of species richness with the calculated index of species number per lake area, and the lake area as an environmental factor for the Kaçkar Mountains National Park lakes.So, Figure 12a shows that the number of diatom species is decreasing in the lakes at low altitude and that have a large surface area.This may be the result of insufficient research, but in any case, it pointed to potential future research in the diatom studies in the protected areas.At the same time, Figure 12b shows an increase in the number of species per lake surface area in 'lowland' lakes if the Sp/Area index has low values.However, the left side of Figure 12b shows an increasing trend for both the number of species per lake area and species richness in high-altitude lakes if efforts are made to deplete diatom diversity in the Kaçkar Mountains National Park in future studies.There was a special part of the 3D analysis in which we tried to reveal the relationships of species richness with the calculated index of species number per lake area, and the lake area as an environmental factor for the Kaçkar Mountains National Park lakes.So, Figure 12a shows that the number of diatom species is decreasing in the lakes at low altitude and that have a large surface area.This may be the result of insufficient research, but in any case, it pointed to potential future research in the diatom studies in the protected areas.At the same time, Figure 12b shows an increase in the number of species per lake surface area in 'lowland' lakes if the Sp/Area index has low values.However, the left side of Figure 12b shows an increasing trend for both the number of species per lake area and species richness in high-altitude lakes if efforts are made to deplete diatom diversity in the Kaçkar Mountains National Park in future studies.
but in any case, it pointed to potential future research in the diatom studies in the protected areas.At the same time, Figure 12b shows an increase in the number of species per lake surface area in 'lowland' lakes if the Sp/Area index has low values.However, the left side of Figure 12b shows an increasing trend for both the number of species per lake area and species richness in high-altitude lakes if efforts are made to deplete diatom diversity in the Kaçkar Mountains National Park in future studies.

Discussion
We investigated the diatom communities of 14 high mountain lakes and a pond in Kaçkar Mountains National Park and their relationship with environmental factors.The chemistry of the studied lakes characterizes their waters as fresh and soft, slightly

Discussion
We investigated the diatom communities of 14 high mountain lakes and a pond in Kaçkar Mountains National Park and their relationship with environmental factors.The chemistry of the studied lakes characterizes their waters as fresh and soft, slightly saturated with salts, which unites them with the group of high mountain lake Artabel [47], but significantly distinguishes them from the sulfate alkaline lake Great Lota [48].
We identified 84 taxa of epipelic, epilithic, and epiphytic diatoms.The families (Pinnulariaceae, Naviculaceae, Surirellaceae, Cymbellaceae and Eunotiaceae) and genera (Pinnularia, Eunotia, Navicula, and Frustulia) that stand out in the flora are also characteristic members of other high mountain lakes that have been identified in the region.The flora, which generally consists of common species, is also similar to the diatom communities of the alpine and subalpine lakes in the region [15].We suppose that the macro-climatic conditions, the similarity of the land structure, and the lake characteristics are the factors in the formation of this situation.
The common species were Caloneis silicula, Encyonema minutum, Iconella capronii, Navicula cryptocephala, and Pinnularia interrupta (Appendix A Table A1).These species occur in most of the lakes studied and thus may be indicators of the ecological health of the park as a whole (Appendix A Table A2).The most abundant and common species were benthic autotrophes, indicative for temperate well-oxygenated waters with circumneutral and low-alkaline pH and with low organic content of Class 2 of water quality.
The noticeable presence of species of the genus Pinnularia in the studied flora of the lakes of the park attracted attention, since these species are inherent in the floras of high-mountain and high-latitude reservoirs with fresh, slightly acidic waters and low electrical conductivity [46,49,50], which is confirmed by our chemical analyses (Table 2), that is, they allow us to classify the studied lakes as high mountain habitats not subject to anthropogenic influence.Thus, the ecological conditions of the waters form a suitable environment for the development of representatives of Pinnularia species.In addition, species of the genus Pinnularia are also known to inhabit extreme habitats, including highlands and arctic [49], indicators of diversity hotspots in the Ecotones, which are a border area of different landscapes [51].Pinnularia interrupta and P. maior, which were found in 14 of the investigated lakes and pond, were recorded very frequently (VF) (86.66%) and were among the important species in the diatom flora of the park (Appendix A Table A1).Pinnularia interrupta occurs in low-mineral-content, circumneutral, oligosaprobic, and oligo-mesotrophic waters, while P. maior prefers low-mineral-content, circumneutral, βmesosaprobic, and meso-eutrophic waters [34,38,46].In addition, Pinnularia borealis and P. viridis species were also recorded as common (C).
Caloneis silicula is usually found in alkaliphilic, oligosaprobic, and meso-eutrophic waters, and in littoral areas of freshwater habitats with moderate electrolyte content [31,38,46], whereas Patrick and Reimer [34] state that the species has a wide ecological tolerance.Didymosphenia geminata prefers cool, low-conductivity, and circumneutral-pH waters [35,38,46].Encyonema minutum, which has a wide geographical distribution, prefers oligo-mesotrophic freshwater habitats with medium electrolyte content, and circumneutral-pH waters [31,34,46].According to Krammer and Lange-Bertalot [28], Iconella capronii is a cosmopolitan benthic form common throughout Europe and generally prefers meso-eutrophic waters with moderate to high electrolyte levels.In addition, Van Dam et al. [46] states that the species has alkaliphilic and oligosaprobic ecological properties.Navicula cryptocephala, which has a wide ecological tolerance, is found in oligo-eutrophic and eutrophic-polytrophic freshwater habitats with poor electrolyte content, and circumneutral-pH and alkaline waters.At the same time, this species also tolerates saprobic levels exceeding beta-alphamesosaprobic [31,34,38,46].The detected physico-chemical properties in the studied lakes support the above-mentioned references.
A comparison of the influence of individual environmental parameters on the diatom communities of 14 lakes showed that the flora consists of diatom taxa that are influenced by the ionic composition of water and habitat altitude, which was demonstrated by RDA and 3D plot anslysis.Both statistical methods (JASP and Bray-Curtis) helped to reveal the high role of the lake altitude in the formation of diatom communities.At the same time, comparison with the other lake systems in the protected areas in the north of Turkey show high individuality of diatom community content of each natural reserve.
Eutrophication and acidification are among the important problems of high mountain lakes [52,53].The bioindication and chemical data obtained from the research showed us that these problems do not exist in the lakes studied.In addition, the low species richness of the identified diatoms and the absence of a pronounced domination in the communities are among the notable features of the detected flora, and these features are inherent for the intact ecosystems of high mountain lakes [54][55][56].
In Appendix A Table A4, a strong correlation was observed between DO, conductivity, and Cl ions.This may be why we found a correlation between DO and species richness, but it may also be why the correlation does not actually occur.Table 2 shows that DO values do not change much and fluctuate in the range of 7.98-9.17,especially in lakes such as TSL-1, AL-2, and BDL, located at altitudes above 2900 m.However, the results of bioindication confirm an increase in the number of indicators requiring increased oxygen content in water.At the same time, we do not forget that chemical analyses of oxygen content were carried out in the laboratory; therefore, they are subject to changes during transportation from the inaccessible research area, where sampling was carried out simultaneously.But the presence of certain indicator species is not instantaneous but is the result of the development of a biological system in given living conditions, and therefore integrates the chemical parameters inherent in lake waters over a long period.Thus, bioindicators confirm the connection between the saturation of lake water with oxygen and the altitude of the lake and the species composition of its communities, even though these changes are not statistically significant.Thus, bioindicator analysis allows us to conclude that the lakes of the park were clean, mesotrophic, and of class 2 water quality.The aquatic inhabitants of the lakes developed in well-oxygenated waters of moderate temperature, and the number of benthic species increased slightly with altitude.
With increasing lake altitude, an increase in the proportion of autotrophic species in communities was observed.At the same time, the number of indicators of slightly alkaline waters decreased slightly with altitude.The low degree of the studied lakes' diatom endemism and rarity [15, 23,49] was revealed for this first study, which proved to be an insufficient study in the park, on the one hand, and stimulates future research on the other.

Conclusions
The species composition, dominant species, and prominent families and genera determined in this study are characteristic of the benthic diatom composition of high mountain lakes.Very frequent (VF) species comprised only 8.33% of the species composition, while very rare (VR) species comprised 57.14%.The composition of very frequent (VF) species is not rich (seven species).
The data we obtained from the research show that the physico-chemical properties of the waters, environmental conditions, and the lake altitude are effective in shaping the benthic diatom flora and in the distribution of diatoms.Future research in the Kaçkar Mountains National Park should include a greater number of high mountain lakes, so that deeper information about the distribution of diatoms within the park should be obtained.Continued research is all the more important, as our analysis reveals a trend for diatom diversity to increase in high mountain lakes if research is expanded.This study is the first on the benthic diatom flora of the high mountain lakes in Kaçkar Mountains National Park and therefore constitutes a starting point for the creation of diatom-based biomonitoring programs for further investigation on diatom-environment relationships and implementation of sustainable management plans.

Figure 3 .
Figure 3. Distribution of diatom species richness, abundance sum of scores, and Index of Saprobity S value over studied lakes in the Kaçkar Mountains National Park, 2022.The order of studied lakes is according to increasing lake altitude.Trend lines are dashed lines.

Figure 3 .
Figure 3. Distribution of diatom species richness, abundance sum of scores, and Index of Saprobity S value over studied lakes in the Kaçkar Mountains National Park, 2022.The order of studied lakes is according to increasing lake altitude.Trend lines are dashed lines.

Figure 4 .
Figure 4. Distribution of indicator species by water temperature, oxygen, pH, and habitat preferences for benthic communities in the Kaçkar Mountains National Park.Habitat (a): P-B-planktobenthic, B-benthic; temperature preferences (b): cool-cool water, temp-temperate, eterm-eurythermic, warm-warm water; oxygenation and water moving (c): str-streaming water, st-strlow streaming water; pH preference groups (d): alb-alkalibiontes; alf-alkaliphiles, ind-indifferent; acf-acidophiles.The lakes order is by increasing altitude.The indicator group order is according to increase in the indicated variable value.

Figure 4 .
Figure 4. Distribution of indicator species by water temperature, oxygen, pH, and habitat preferences for benthic communities in the Kaçkar Mountains National Park.Habitat (a): P-B-plankto-benthic, B-benthic; temperature preferences (b): cool-cool water, temp-temperate, eterm-eurythermic, warm-warm water; oxygenation and water moving (c): str-streaming water, st-str-low streaming water; pH preference groups (d): alb-alkalibiontes; alf-alkaliphiles, ind-indifferent; acf-acidophiles.The lakes order is by increasing altitude.The indicator group order is according to increase in the indicated variable value.

Figure 5 .
Figure 5. Distribution of indicators of salinity, nutrition type, class of water quality, and trophic state for benthic communities in the Kaçkar Mountains National Park.Abbreviations of ecological groups are given in Appendix A Table A2.Salinity ecological groups (a): hb-oligohalobes/halophobes, i-oligohalobes/indifferent, hl-halophiles; mh-mesohalobes, oh-oligohalobes of wide spectrum with optimum as indifferent.Nitrogen uptake metabolism (autotrophy-heterotrophy) (b): ats-nitrogen-autotrophic taxa, tolerating very small concentrations of organically bound nitrogen; ate-nitrogen-autotrophic taxa, tolerating elevated concentrations of organically bound nitrogen.The water quality class is determined as the sum of indicators whose species-specific index saprobity S from Appendix A Table A2 is within the range of each class.Classes of water quality colored in EU color code (c).Trophic state indicators (d): ot-oligotraphentic; om-oligomesotraphentic; m-mesotraphentic; me-mesoeutraphentic; e-eutraphentic; o-e-hypereutraphentic.The lakes are ordered by increasing altitude, and the indicator groups are arranged in increasing order of the indicated variable value.

Figure 5 .
Figure 5. Distribution of indicators of salinity, nutrition type, class of water quality, and trophic state for benthic communities in the Kaçkar Mountains National Park.Abbreviations of ecological groups are given in Appendix A Table A2.Salinity ecological groups (a): hb-oligohalobes/halophobes, i-oligohalobes/indifferent, hl-halophiles; mh-mesohalobes, oh-oligohalobes of wide spectrum with optimum as indifferent.Nitrogen uptake metabolism (autotrophy-heterotrophy) (b): ats-nitrogen -autotrophic taxa, tolerating very small concentrations of organically bound nitrogen; ate-nitrogenautotrophic taxa, tolerating elevated concentrations of organically bound nitrogen.The water quality class is determined as the sum of indicators whose species-specific index saprobity S from Appendix A Table A2 is within the range of each class.Classes of water quality colored in EU color code (c).Trophic state indicators (d): ot-oligotraphentic; om-oligomesotraphentic; m-mesotraphentic; me-mesoeutraphentic; e-eutraphentic; o-e-hypereutraphentic.The lakes are ordered by increasing altitude, and the indicator groups are arranged in increasing order of the indicated variable value.

Figure 6 .
Figure 6.JASP Network plot of diatom bioindicator correlation (a) in the communities of the lakes of the Kaçkar Mountains National Park, p < 0.5, calculated based on Appendix A Table A3, and plot of comparison of the studied lake communities (Kachkar-15), previous studied diatom communities in Kaçkar Mountains National Park (Kachkar-2) [15] and in Artabel [47] (b).Major groups of the lakes abbreviated and colored in the legend.Blue lines are positive correlations, while red lines are negative correlations.The line thickness reflects the value of correlation.Dashed line outlined different clusters with numbers of 1-4 on Figure (a) and 1-2 on Figure (b).

Figure 6 .
Figure 6.JASP Network plot of diatom bioindicator correlation (a) in the communities of the lakes of the Kaçkar Mountains National Park, p < 0.5, calculated based on Appendix A Table A3, and plot of comparison of the studied lake communities (Kachkar-15), previous studied diatom communities in Kaçkar Mountains National Park (Kachkar-2) [15] and in Artabel [47] (b).Major groups of the lakes abbreviated and colored in the legend.Blue lines are positive correlations, while red lines are negative correlations.The line thickness reflects the value of correlation.Dashed line outlined different clusters with numbers of 1-4 on Figure (a) and 1-2 on Figure (b).

Figure 7 .
Figure 7. Tree of Bray-Curtis similarity analysis of diatom species composition in communities of the Kaçkar Mountains National Park, 2020.Clusters are outlined by dashed lines and represented by different colors.

Figure 8 .
Figure 8. RDA plots for dominated groups of species indicators and environmental variables in 11 studied lakes based on data from Tables1 and 2, and Appendix A Tables A1 and A4 in the Kaçkar Mountains National Park.RDA plot for species richness, sum of scores and environmental variables (a).RDA plot for environmental variables in the studied lakes (b).

Figure 8 .
Figure 8. RDA plots for dominated groups of species indicators and environmental variables in 11 studied lakes based on data from Tables1 and 2, and Appendix A Tables A1 and A4 in the Kaçkar Mountains National Park.RDA plot for species richness, sum of scores and environmental variables (a).RDA plot for environmental variables in the studied lakes (b).

Ecologies 2024, 5 ,Figure 9 .
Figure 9. Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water conductivity (a), and lake altitude and dissolved oxygen (DO) (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Figure 9 .
Figure 9. Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water conductivity (a), and lake altitude and dissolved oxygen (DO) (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Figure 10 .
Figure 10.Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water pH (a), species richness in diatom community in relation to lake altitude and temperature (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Figure 10 . 14 Figure 11 .
Figure 10.Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water pH (a), species richness in diatom community in relation to lake altitude and temperature (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.Ecologies 2024, 5, FOR PEER REVIEW 14

Figure 11 .
Figure 11.Three-dimesional surface plots of species richness in diatom community in relation to lake altitude and Index Saprobity S (a), and lake altitude and ammonium (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Figure 12 .
Figure 12.Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water area (a), and lake altitude and Index of Species per Area (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Figure 12 .
Figure 12.Three-dimensional surface plots of species richness in diatom community in relation to lake altitude and water area (a), and lake altitude and Index of Species per Area (b) in the protected lakes in the Kaçkar Mountains National Park, 2020.

Table 1 .
Geographic coordinates, altitude, and surface area information of the studied lakes in Kaçkar Mountains National Park, 2020.

Table A2 .
Indicator properties of diatom species in the lakes of the Kaçkar Mountains National Park, in summer and autumun of 2020.

Table A4 .
RDA correlation matrix for biological and environmental variable relationships of diatom communities in 14 lakes of the Kaçkar Mountains National Park.