Bangladesh J. Plant Taxon. 28(2): 413‒428, 2021 (December) DOI: https://doi.org/10.3329/bjpt.v28i2.57137 © 2021 Bangladesh Association of Plant Taxonomists CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS OF THERMAL SPRINGS IN THE KÜTAHYA PROVINCE OF TURKEY SEVİLAY ÖZTÜRK* Department of Biology, Faculty of Sciences and Letters, Manisa Celal Bayar University, Manisa, Turkey Keywords: Biodiversity; Cyanobacteria; Thermal springs; RDA analyses; Turkey. Abstract Thermal springs are very difficult environments for organisms due to the high temperature, and physicochemical parameters. Cyanobacteria, which are photosynthetic prokaryotes, are best adapted to these environments. Kütahya is an important thermal area in Turkey. The aim of the study was to determine the cyanobacterial flora with a morphologic and ecologic approach in the 11 thermal. The physicochemical properties of the thermal springs in Kütahya province were measured. The thermal springs are alkaline (pH6) with an average temperature of 52°C. As a result, 54 cyanobacteria taxa were identified. Oscillatoriales were the predominant order in terms of taxa diversity (24 taxa) and biomass size. Statistical analyses were conducted to reveal the physicochemical properties of the thermal springs and the distribution of cyanobacteria in detail. According to these analyses, the thermal springs were classified into two main groups with a Piper. As a result of the RDA analysis under CANOCO 5.0, the total variation was 55.45455, and the first two axes explained a total of 57.43% of the variance. There was a significant difference (P0.001) in the comparison of the physicochemical parameters including pH, EC, TDS, and temperature values of the thermal springs in the Kruskal Wallis tests. Introduction Cyanobacteria are ecologically important because of their role in oxygen production and in assimilation of carbon and nitrogen. Although life is difficult in thermal springs, the cyanobacteria are the most adapted organisms for this environment. Cyanobacteria are the most commonly reported microbial groups constituting thermophilic mats and considered the major primary producers in these type of habitats (Castenholz, 1973). Due to their abilities, the determination of diversity of cyanobacteria in thermal springs is gaining importance. Studies of thermal springs allow us to know which cyanobacterial taxa can adapt to the thermal environment. The diversity of cyanobacteria in thermal springs depends on two basic factors: i) the temperature of the thermal spring, ii) the dissolved chemicals in the thermal spring. There are more than 600 thermal springs in Turkey (Özsahin and Kaymaz, 2013). Despite this, studies of the biology of thermal springs are very limited (Adıgüzel et al., 2009; Yedier et al., 2016). Biodiversity studies of thermal algae in Turkey began in Pamukkale with collected algae (Regel and Skuja, 1937) and continued with Güner (1966, 1967, 1970); Aysel et al. (1992); Pentecost et al., (1997); Ünal (1996); Ulcay Öztürk et al., (2006; 2007); Yüksel et al., (2009); Demirel and Sukatar, (2011); Ulcay and Kurt, (2014a,b,c), Altunoz et al., (2016); Öztürk Ulcay and Kurt (2017); Öztürk Ulcay et al., (2017); Kalkan et al., (2020) and Öztürk, (2020). Corresponding author, E-mail: seviozturk@yahoo.com https://doi.org/10.3329/bjpt.v28i2.5713 mailto:seviozturk@yahoo.com 414 ÖZTÜRK Numerous cyanobacteria taxa have been reported in thermal springs throughout the world. Regarding their morphotypes characteristics, Sompong et al. (2005) identified 19 genera and 36 cyanobacteria taxa from nine thermal springs (3080°C) in northern Thailand. Debnath et al. (2009) reported 18 taxa distributed in 12 genera at three geothermal springs in Bakreswar in India. In total, 43 taxa belonging to 20 genera of the planktonic cyanobacteria were identified at four hot springs in Iran by Heidari et al. (2013). Roy et al. (2015) identified 16 taxa spread over 14 genera in the Bakreswar geothermal springs in India. Based on morphology, the distribution of 31 cyanobacteria taxa (3038.2°C) from Thermopylae thermal spring in Greece were identified by Kanellopoulos et al. (2016). Singh et al. (2018b) reported 22 taxa under 11 genera based on the morphology at nine thermal springs in the northwestern Himalayas. The aim of the study was to determine the cyanobacterial flora of thermal springs in the Kütahya province, an important thermal area in Turkey including morphological and ecological aspects. In this context, the results obtained by morphological methods have been studied in an attempt to determine species diversity. Additionally, the thermal springs in Kütahya province measured to determine their physicochemical properties. The piper diagram provided ease in classification and comparison of the thermal springs in Kütahya province where the anion and cation of the water taken from 11 thermal springs was compared. In addition to all this, the relationship between the physicochemical parameters, the sampling sites, and the taxa were explored with the redundancy analysis (RDA) (CANOCO 5.0.). Kruskal-Wallis tests were applied under Statistical Package for the Social Sciences (SPSS) to reveal the statistical significance of differences or similarities of the physicochemical parameters and the taxa numbers of the sampling sites. Materials and Methods Sampling sites Kütahya is situated on major fault lines in the western Anatolian region of Turkey. In this study, a large number of sampling sites with different physicochemical characteristics were selected from 11 thermal springs (Fig. 1). These sites were scattered over an area of approximately 2500 km2 ranging from an altitude of 588 m to 1462 m, and most of them had thermal spring facilities like spas or thermal hotels. Physicochemical characteristics of the thermal springs Water samples were collected in sterile glass bottles from the sampling sites while collecting cyanobacteria samples. The temperature (T-°C), pH, conductivity (EC- mS/cm), and total dissolved solids (TDS-mg/l) were measured using a Hanna HI 9812-5 Portable pH/EC/TDS/Temperature Meter (Europa-Romania) in-situ. The water samples were labelled and transported to the laboratory for chemical analysis. Fluoride (F), chloride (Cl), bromine (Br), nitrite (NO2 ), nitrate (NO3 ), phosphate (PO4 3), sulphate (SO4 2) analyses were performed by DIONEX ICS-5000 Ion Chromatography/ppm. Other chemical analyses were performed by Perkin Elemer Optima 8000/mg/L for other chemical factors including calcium (Ca), ferrous (Fe2), potassium (K), magnesium (Mg), sodium (Na), silicon (Si), and manganese (Mn). Ammonium (NH4 ) analyses were performed by Nesslerizasyon/ppm. All chemical analysis were performed at the Manisa Celal Bayar University-Applied Science Research Center (Manisa, Turkey). A Piper diagram provides convenience in the classification and comparison of natural springs. The similarities and differences of these thermal springs were investigated with the Piper diagram, and the eleven springs were classified according to their chemical composition with the Piper CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 415 diagram using GW Chart Software (USGS) and Microsoft Excel 2016 (Piper, 1944; Winston, 2020). Fig. 1. Thermal Spring Sample Site Names and Locations in Kütahya Province. T1- Gediz-Ilica (38°56'22"N 29°15'31"E), T2- Gediz-Murat Mountain (38°57'19"N 29°37'14"E), T3- Tavsanli-Göbel (39°29'51"N 29°26'17"E), T4- Esire (39°12'08"N 29°16'53"E), T5- Sarpasan (39°12'10"N 29°16'40"E), T6- Hisarcik-Hamam (39°12'07"N 29°16'35"E), T7- Hisarcik-Sefaköy (39°10'33"N 29°15'37"E), T8- Günlüce-Dereli (39°27'46"N 29°15'55"E), T9- Emet (39°20'32"N 29°15'12"E), T10- Simav-Eynal (39°07'38"N 28°59'33"E), T11- Naşa (39°08'37"N 28°57'39"E). Sampling and identification of cyanobacteria Cyanobacteria samples were collected between February 2014 and January 2015. Collected samples were placed in 50 ml falcon tubes for morphological identification. All samples were labeled and transported to the laboratory. Collected samples were divided into two parts in the laboratory, one used in direct observations, and the other part fixed with 4% formalin solution to prevent degradation of the characteristics of the taxa. Microscopic studies were conducted in the laboratory using an Olympus BX 50 (phase- contrast) microscope, and taxonomical characteristics were determined and photographed using the Sony DSC-TX7 camera for morphological identification. The identification of the taxa was made according to previous studies including Komárek and Anagnostidis (2000, 2005), John et al., (2002) and Komárek, (2013). The nomenclature was checked on the AlgaeBase database (Guiry and Guiry, 2021). 416 ÖZTÜRK Statistical analysis The relationship between the physicochemical variables of the thermal springs and the distribution of cyanobacteria taxa was assessed by redundancy analysis (RDA) and detrended correspondence analysis (DCA). The analysis was carried out using CANOCO 5.0 software for Windows (Ter Braak and Smilauer, 2012). Initially, a DCA was performed to determine the gradient length and which model (linear or unimodal) the studied gradient is suitable for. According to the DCA results, it was seen that the available data was suitable for RDA analysis. To obtain gradients not associated with the coverable, a forward selection of the environmental variables was performed. Physicochemical properties were determined through a Monte Carlo test (499 permutations), taking into account all canonical axes. Kruskal-Wallis tests were performed using SPSS 20.00 software to determine whether the differences (in terms of physicochemical parameters) between the thermal springs were statistically significant. Kruskal-Wallis is a non-parametric test and is used for multiple data comparisons. The Kruskal-Wallis test was used to determine the importance of pH, EC, TDS, and temperature values of the eleven thermal springs. Results and Discussion Physicochemical characteristics of the thermal springs Some chemical parameters of the eleven thermal springs in Kütahya as well as the annual average pH, temperature, and EC and TDS measurements are shown in Table 1. The results indicate that the thermal springs were alkaline (pH6), the average temperature was 52°C, and they were transparent. In addition, it was found that nutrient elements were high, and sulphate and ammonia were below the measurable values in the thermal springs. These physicochemical parameters explain the abundance of cyanobacteria diversity in the thermal springs. In the Piper diagram, which is the most acceptable method in classifying and comparing natural springs and ground waters, anions and cations are shown in two separate triangles while all ions are shown from a quadrilateral, and this diagram makes classification and comparison of waters easier (Piper, 1944). Based on the Piper diagram, the thermal springs of the Kütahya province were classified into two main groups, one (T2, T4, T5, T6, T7, T9) Ca-Mg-SO4, the other one (T1, T3, T8, T10, T11) Na-HCO3-SO4 (Fig. 2). Cyanobacteria taxa Collected samples were identified based on morphological characteristics. As a result, the 54 cyanobacteria taxa identified were distributed in five orders (Table 2). Among the identified taxa, Oscillatoriales were dominant with 25 taxa. In this study, Pseudanabaena minima had the highest diversity in the thermal springs of Kütahya. Among the sampling sites, Gediz Ilıca (T1) with 10 taxa and Naşa (T11) with one taxon represented the highest and the lowest species diversity, respectively. The most abundant genera was Leptolyngbya, which almost dominated in the nine thermal springs. The most common taxon was Pseudanabaena minima, which was identified from the three thermal springs. Statistical analysis The relationships between the physicochemical parameters, the sampling sites, and the taxa were explored with an RDA using CANOCO 5.0 software for Windows. Firstly, DCA was performed to find a suitable analysis and gradient lengths were assessed (Axis 1: 0.00; Axis 2: 0.00). Among the physicochemical parameters analyzed, five were included in the forward selection (temperature, pH, TDS, EC, and PO4). In the RDA, the physicochemical parameters (T, pH, TDS, EC, and PO4), the sampling sites, and the taxa were used as explanatory variables. The CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 417 418 ÖZTÜRK significance of their effect was supported by a Monte Carlo permutation test (499 permutations, F- ratio = 1.3, P-value = 0.026). As a result of the RDA analysis, the total variation was 55.45455, and the first two axes explained a total of 57.43% of the variance (Fig. 3). Fig. 2. Piper diagram showing the anions-cations and comparison of the thermal springs. In the comparison of physicochemical parameters that cause species diversity and the taxa differences of the thermal springs, the question of whether the difference between them was significant with the Kruskal Wallis tests was examined. There was a significant difference (P0.001) in the comparison of physicochemical parameters including pH, EC, TDS, and temperature values of the thermal springs in this study (Fig. 4). Kruskal Wallis tests showed variations in the physicochemical parameters of the thermal springs and in the cyanobacteria diversity (Fig. 5). The frequency of distribution of taxa according to pH, TDS, T, and EC values can be seen in Fig. 6 (P0.001). Besides, the manganese (Mn) values of the sampling sites were compared with the Kruskal-Wallis test in SPSS (P0.001) as a remarkable value (Fig. 7). As a result, 54 cyanobacteria taxa were identified based on morphological characteristics. The physicochemical properties of the thermal springs were measured. In addition, statistical analyses were made to reveal in detail the physicochemical properties of the thermal springs and their comparisons with the cyanobacterial flora. The piper diagram was provided for convenience in the classification and comparison of thermal springs in the Kütahya province; thus, the anions and cations of the water taken from the CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 419 thermal springs (Fig. 2) were compared. According to the Piper diagram, sampling sites T1, T3, T8, T10, and T11 were classified as a Na-HCO3-SO4 type, and sampling sites T2, T4, T5, T6, T7, and T9 were classified as a Ca-Mg-SO4 type. In addition, these thermal springs in Kütahya were classified with the Piper diagram by different researchers (Gemici et al., 2004; Güneş, 2006; Bello et al., 2014). In the literature, it is noteworthy that the Piper diagram has been used less frequently in the determination of cyanobacteria in thermal springs (Singh et al., 2018b). Table 2. Cyanobacteria taxa and sampling sites. Taxa code Cyanobacteria taxa Thermal spring Chroococcales 1 Gloeocapsa sp. T6 2 Gloeocapsopsis cyanea (Krieger) Komárek & Anagnostidis T3, T10 3 Chroococcus membraninus (Meneghini) Nägeli T9 4 Cyanosarcina thermalis (Hindák) Kovácik T6 Synechococcales 5 Anathece clathrata (West & G.S.West) Komárek, Kastovsky & Jezberová T1 6 Arthronema sp. T5 7 Romeria chlorina Böcher T1 8 Planktolyngbya contorta (Lemmermann) Anagnostidis & Komárek T1 9 Leptolyngbya boryana (Gomont) Anagnostidis & Komárek T3 10 L. tenerrima (Hansgirg) Komárek T8 11 L. gelatinosa (Woronichin) Anagnostidis & Komárek T7,T10 12 L. granulifera (J.J.Copeland) Anagnostidis T1 13 L. thermarum (Woronichin) Anagnostidis & Komárek T10 14 Leptolyngbya sp. 1 T6 15 Leptolyngbya sp. 2 T5 16 Leptolyngbya sp. 3 T4 17 Leptolyngbya sp. 4 T4 18 Pseudanabaena minima (G.S.An) Anagnostidis T1,T7,T10 19 P. lonchoides Anagnostidis T8 20 P. thermalis Anagnostidis T10 21 P. limnetica (Lemmermann) Komárek T10 22 Pseudanabaena sp. T5 23 Limnothrix mirabilis (Böcher) Anagnostidis T1 24 Trichocoleus sociatus (West & G.S.West) Anagnostidis T2 Spirulinales 25 Spirulina subsalsa Oerstedt ex Gomont T1 26 S. subtilissima Kützing ex Gomont T9 27 S. labyrinthiformis Gomont T5 Oscillatoriales 28 Geitlerinema nematodes (Skuja) Anagnostidis T1 29 Anagnostidinema amphibium (C.Agardh ex Gomont) Strunecký, Bohunická, J.R.Johansen & J.Komárek T5 30 Planktothrix clathrata (Skuja) Anagnostidis & Komárek T8 420 ÖZTÜRK Table 2 Contd. Taxa code Cyanobacteria taxa Thermal spring 31 Microcoleus autumnalis (Gomont) Strunecky, Komárek & J.R.Johansen T3 32 M. lacustris Farlow ex Gomont T8 33 M. paludosus Gomont T2 34 Kamptonema jasorvense (Vouk) Strunecký, Komárek & J.Smarda T1 35 K. okenii (C.Agardh ex Gomont) Strunecký, Komárek & J.Smarda T9,T10 36 K. cortianum (Meneghini ex Gomont) Strunecký, Komárek & J.Smarda T9,T10 37 Phormidium incrustatum Gomont ex Gomont T8 38 P. terebriforme (C.Agardh ex Gomont) Anagnostidis & Komárek T5 39 P. thermobium Anagnostidis T7,T8 40 P. chalybeum (Mertens ex Gomont) Anagnostidis & Komárek T9 41 Phormidium sp. T4 42 Oscillatoria subcapitata Ponomarev ex Elenkin T8 43 O. proboscidea Gomont T1,T5 44 O. princeps Vaucher ex Gomont T10,T11 45 O. subbrevis Schmidle T5 46 O. curviceps C.Agardh ex Gomont T2 47 O. sancta Kützing ex Gomont T8 48 Lyngbya martensiana Meneghini ex Gomont T3 49 L. thermalis Kützing ex Gomont T2 50 Limnoraphis hieronymusii (Lemmermann) J.Komárek, E.Zapomelová, J.Smarda, J.Kopecký, E.Rejmánková, J.Woodhouse, B.A.Neilan & J.Komárková T4 51 Blennothrix sp. T4 Nostocales 52 Nostoc sp. T7 53 Calothrix sp. T3 54 Hapalosiphon sp. T10 Although the T1 and T2 springs appear to be close to each other, the T2 source is located at a much higher altitude than the others. When the physicochemical parameters of the T1 and T2 are examined (Table 1), it is seen that the pH and Mg values are similar. However, the other parameters, particularly the temperature, are quite different. Based on their own studies, Singh et al. (2018b) stated that the close proximity of the hot springs does not mean that they may have similar physical and chemical characteristics. However, the physicochemical parameters of the T4, T5, T6, and T7 springs are quite similar, so they are seen to be in the same class in the Piper diagram and close to each other in the study area (Fig. 2). It has been reported that there are different species compositions in different thermal springs depending on the substratum and the physicochemical parameters of springs (Ward and Castenholz, 2000; Papke et al., 2003). In the literature, there are many studies of the cyanobacterial flora in thermal springs (Sompong et al., 2005; Debnath et al., 2009; Heidari et al., 2013; Roy et al., 2015; Kanellopoulos et al., 2016; Singh et al., 2018a, Singh et al., 2018b). When compared with the literature, it may be seen that more cyanobacteria taxa were determined in this study. The main reason for this may be that there are high numbers of thermal springs and sampling sites in this study. Another possible reason may be that the thermal springs in the sampling area have different physicochemical properties. CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 421 Fig. 3. RDA Diagram showing the relationship between the cyanobacteria taxa (with full triangle), the thermal springs (with full circle), and the physicochemical variables of thermal water (with arrow) [the cyanobacteria taxa code and the thermal springs where they were sampled are given in Fig. 1 and Table 2] T: temperature, EC: conductivity, TDS: total dissolved solid, and PO4 3:phosphate. Fig. 4. Comparison of the pH, TDS, T, and EC values of the sampling sites with the Kruskal-Wallis test in SPSS (P0.001). 422 ÖZTÜRK Fig. 5. Comparison of the number of taxa and the sampling sites with the Kruskal-Wallis test in SPSS. Fig. 6. Frequency of pH, TDS, T, and EC between the taxa (P0.001). CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 423 Fig. 7. Comparison of the manganese (Mn) values of the sampling sites with Kruskal-Wallis test in SPSS (P0.001). Oscillatoriales were a predominant order with taxa diversity (24 taxa) in this study. Similarly, a major component of the thermal spring’s cyanobacterial flora worldwide belongs to order Oscillatoriales (Pentecost et al., 1997; Sompong et al., 2005; Mcgregor and Rasmussen, 2008; Ionescu et al., 2010; Arman et al., 2014). Nevertheless, Leptolyngbya (order Synechococcales) were determined frequently in the thermal springs of the Kütahya province. Also, this taxon is one of the most frequently reported taxa observed in thermal springs (Ulcay Öztürk et al., 2006; Mcgregor and Rasmussen, 2008). Commonly identified Pseudanabaena and Spirulina taxa in this study were also reported in other thermal springs (Heidari et al., 2013; Arman et al., 2014; Roy et al., 2015). According to the RDA analysis, the affinity of Spirulina subsalsa and Spirulina subtilissima with temperature was completely different in this study. While S. subsalsa was related to temperature, S. subtilissima was not. Krienitz et al. (2003) reported that S. subsalsa and S. subtilissima are closely related and have a wide ecological distribution. In addition, both taxa occur in thermal springs and in mesophilic brackish and marine habitats (Geitler, 1932). According to the results of the RDA analysis, the presence of cyanobacteria taxa in thermal springs was related to the physicochemical parameters (Fig. 3). The RDA analysis showed that the most highly determining factor affecting the distribution of the taxa is the temperature (T) in this study (Fig. 3). Similarly, Roy et al. (2015) reported that temperature has been one of the most important factors as far as the distribution and diversity of cyanobacteria are concerned in geothermal springs. Also, there are a lot of studies concerning this subject in the literature (Sompong et al., 2005; Debnath et al., 2009; Singh et al., 2018b). Pseudanabaena thermalis was collected at 50°C and below from sampling site T10. Similarly, this taxon was collected by McGregor and Rasmussen (2008) at 48.6°C from Innot Hot Springs in Australia. Planktolyngbya contorta was clearly associated with temperature and PO4 values in this study. Similarly, P. contorta was sampled in the thermal springs in Himachal Pradesh, India by Singh et al. (2018a). 424 ÖZTÜRK Lyngbya thermalis was sampled in the form of dark green and thin mats at 36°C (close to spring mouth) and at 29°C (where the water was discharged) from the sampling site T2. Similarly, Lukavsky et al. (2011) reported that a deep blue-green growth washed directly with water of 43°C was colonized with L. thermalis; in addition, L. thermalis also dominated near the outlets of hot water of 22°C. Arman et al. (2014) sampled L. thermalis at two different thermal springs from a temperatures range of 3742°C. Castenholz (1969; 1973) stated that changes in species composition with concomitant changes of temperature occurred along the gradient from the mouth of the thermal springs. Based on the RDA analysis, Gloeocapsa sp., Cyanosarcina thermalis, Leptolyngbya sp. 1, and Nostoc sp. had an affinity with low pH, T, TDS, EC, and PO4 in this study (sampling sites T6 and T7). Despite the results of the RDA analysis, C. thermalis was sampled at 4238°C from the T6 in this study. Actually, C. thermalis is known as a common taxon at thermal springs (Rueda and Monroy, 2009; Komárek and Anagnostidis, 2000; Arman et al., 2014; Šaraba and Krunić, 2017). It has been reported that Spirulina labyrinthiformis has a high tolerance for sulfides (Pentecost and Coletta, 2007; Ward et al., 2012). S. labyrinthiformis was not collected in sampling sites with higher sulfate values, but it was sampled from sampling site T5 with 410.52 ppm SO4 -2 in this study (Tables 1 and 2). Similarly, Pentecost and Coletta (2007) reported that the dominance of this taxon might be related to its tolerance of dissolved sulfide although Spirulina is scarce in sampling sites with the highest sulfide. In some hot springs in Yellowstone Park (52°C or below), a sulfide- utilizing S. labyrinthiformis morphotype predominates near the sulfide-rich source (Ward et al., 2012). Temperature, in combination with the availability of combined nitrogen, phosphorus and other nutrients, and/or a concentration of free sulfide also determines the cyanobacteria composition (Ward and Castenholz, 2000; Singh et al., 2018b). Sulfide rich thermal springs usually contain sulfide tolerant and sulfide utilizing Oscillatoria (Castenholz and Utkilen, 1984; Ward and Castenholz, 2000; Singh et al., 2018b). Oscillatoria princeps were sampled from sampling sites T10 and T11 with sulfate values of 587.78 and 436.13 ppm SO4 -2. In the literature, O. princeps has similarly been sampled from high sulphate values in thermal springs (Heidari et al., 2013; Arman et al., 2014). However, this taxon has been recorded in thermal springs with relatively low sulphate values (Debnath et al., 2009; Roy et al., 2015). In addition, O. princeps was the only taxon detected in sampling site T1, and it had formed large mats. Also, this taxon was sampled from T10 (Table 2). According to the literature, O. princeps is perhaps cosmopolite (not marine) (Komárek and Anagnostidis, 2005). When compared to the physicochemical parameters of the sampling sites, the high manganese (Mn) value of T11 drew attention. A comparison of the Mn values of the sampling sites with the Kruskal-Wallis test showed significant differences (P0.001) (Fig. 7). The reason it was the only taxon in T11 may be that O. princeps can tolerate a high manganese value. Mn is an essential micronutrient that may become toxic if present at a high concentration (Moura et al. 2019). Ward and Castenholz (2000) and Sompong et al. (2005) reported that pH is as important as temperature for cyanobacteria in thermal springs. In the RDA analysis, a negative correlation of Pseudanabaena limnetica with EC and a positive correlation with pH was determined in this study. In contrast, Altunöz et al. (2016) stated that P. limnetica has the highest affinity with EC and a negative correlation with other environmental variables including pH. Cyanobacteria can be considered alkaline since they grow optimally between pH 7.5 and above (Brock, 1973). Gloeocapsopsis cyanea was sampled from the two sampling sites with the lowest and the highest pH values in this study (T3, pH 6.7; T10, pH 8.7). In conclusion, it can be concluded that the CYANOBACTERIAL DIVERSITY AND PHYSICOCHEMICAL CHARACTERISTICS 425 ecological valence of the taxon for the pH demand is wide. In the literature, this taxon was reported from different environments (Lamprinou et al., 2012; Arman et al., 2014; Ozturk Ulcay et al., 2017; Davydov, 2018). Also, Grimmett and Lebkuecher (2017) reported that G. cyanea was among the taxa determined as potential indicators of nutrient-rich areas based on their own data. Phormidium incrustatum is known as the common taxa of the limestone/travertine/calcareous substrata (Pentecost, 2005; Couradeau et al., 2013; Kanellopoulos et al., 2016). P. incrustatum was sampled from T8, one of the stations with high carbonate and bicarbonate values in this study. Kanellopoulos et al. (2016) noted that trichomes of P. incrustatum are surrounded by a firm sheath of extracellular polymeric substances (EPS), constituting the locus of intensive calcification. Because many taxa cannot tolerate high temperatures, thermal springs are extreme habitats for living organisms (Ozturk Ulcay and Kurt, 2017). Thermal springs are very difficult environments for organisms because of high temperatures and physicochemical parameters. Yet, thermal springs create special living environments. The group that has best adapted to these environments is cyanobacteria, which are photosynthetic prokaryotes. In this case, it is important to determine the diversity of cyanobacteria. However, in the literature, no sufficient study of the biodiversity of thermal springs in Turkey was found. The Kütahya province is very rich in thermal springs, and the primary objective of this study was to determine the cyanobacterial diversity in these springs. In this study, numerous different statistical analyses were performed to reveal the physicochemical properties of the thermal springs and compare them with the cyanobacterial flora. Comparison between the physicochemical parameters and the cyanobacteria taxa was made with RDA analysis, the Piper diagram, and Kruskal Wallis tests. Since physicochemical parameters are important in understanding the ecology of aquatic habitats, many parameters were measured in this study. It is important to perform these statistical analyses to understand the ecology of the thermal springs and the cyanobacteria that prefer these environments. Acknowledgements I am appreciate the contributions of Dr. Oğuz KURT (PhD; Manisa Celal Bayar University, Turkey). This work was supported by the MCBU BAP, Manisa, Turkey for research, under the project FEF 2013107. References Adiguzel, A., Ozkan. H., Baris, O., Inan, K., Gulluce, M. and Sahin, F. 2009. Identification and characterization of thermophilic bacteria isolated from hot springs in Turkey. Journal of Microbiological Methods 79(3): 321–328. https://doi.org/10.1016/j.mimet.2009.09.026 Altunöz, M., Obali, O., Atici, T. and Arru, L. 2016. 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