Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing

microorganisms Article Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing Can Akpolat 1, Ana Beatriz Fernández 2, Pinar Caglayan 1, Baris Calli 3, Meral Birbir 1,* and Antonio Ventosa 4,*   Citation: Akpolat, C.; Fernández, A.B.; Caglayan, P.; Calli, B.; Birbir, M.; Ventosa, A. Prokaryotic Communities in the Thalassohaline Tuz Lake, Deep Zone, and Kayacik, Kaldirim and Yavsan Salterns (Turkey) Assessed by 16S rRNA Amplicon Sequencing. Microorganisms 2021,9, 1525. https:// doi.org/10.3390/microorganisms9071525 Academic Editor: Ricardo Amils Received: 20 June 2021 Accepted: 13 July 2021 Published: 17 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Biology, Faculty of Arts and Sciences, Marmara University, 34722 Istanbul, Turkey; [email protected] (C.A.); pinar[email protected] (P.C.) 2 Institute for Multidisciplinary Research in Applied Biology-IMAB, Universidad Publica de Navarra, Multilva, 31006 Navarra, Spain; [email protected] 3Department of Environmental Engineering, Faculty of Engineering, Marmara University, 34722 Istanbul, Turkey; [email protected] 4 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, 41004 Sevilla, Spain *Correspondence: [email protected] (M.B.); [email protected] (A.V.); Tel.: +90-216-777-3200 (M.B.); +34-954-556-765 (A.V.) Abstract: Prokaryotic communities and physico-chemical characteristics of 30 brine samples from the thalassohaline Tuz Lake (Salt Lake), Deep Zone, Kayacik, Kaldirim, and Yavsan salterns (Turkey) were analyzed using 16S rRNA amplicon sequencing and standard methods, respectively. Archaea (98.41% of reads) was found to dominate in these habitats in contrast to the domain Bacteria (1.38% of reads). Representatives of the phylum Euryarchaeota were detected as the most predominant, while 59.48% and 1.32% of reads, respectively, were assigned to 18 archaeal genera, 19 bacterial genera, 10 archaeal genera, and one bacterial genus that were determined to be present, with more than 1% sequences in the samples. They were the archaeal genera Haloquadratum,Haloarcula,Halorhabdus,Natronomonas, Halosimplex,Halomicrobium,Halorubrum,Halonotius,Halolamina,Halobacterium, and Salinibacter within the domain Bacteria. The genera Haloquadratum and Halorhabdus were found in all sampling sites. While Haloquadratum,Haloarcula, and Halorhabdus were the most abundant genera, two uncultured Tuz Lake Halobacteria (TLHs) 1 and 2 were detected in high abundance, and an additional uncultured haloarchaeal TLH-3 was found as a minor abundant uncultured taxon. Their future isolation in pure culture would permit us to expand our knowledge on hypersaline thalassohaline habitats, as well as their ecological role and biomedical and biotechnological potential applications. Keywords: Tuz Lake (Salt Lake); thalassohaline lakes; salterns; metagenomics; extremophiles; 16S rRNA amplicon sequencing; physico-chemical analyses 1. Introduction Hypersaline habitats such as salt lakes, saline soils, solar salterns, hypersaline soda lakes, salt mines, and deep-sea and oil reservoir brines are typical extreme environments in which not only NaCl, but other environmental factors, such as pH, temperature, nutrients, radiation, pressure or presence of heavy metals and other toxic compounds, limit their microbiota [1–8]. Hypersaline aquatic environments are classified as thalassohaline habitats; those derived from marine origin having relative proportions of salts according to those of seawater, and athalassohaline aquatic systems, with salts proportions very different from seawater, reflecting their non-marine and very different geological origins [ 1 , 2 , 4 ]. Most studies on hypersaline lakes have been focused on athalassohaline lakes, such as the Dead Sea, hypersaline and alkaline (soda) lakes in Antarctica, East African lakes such as Lake Magadi and the lakes of Wadi Natrun, in Australia or China, especially in Inner Microorganisms 2021,9, 1525. https://doi.org/10.3390/microorganisms9071525 https://www.mdpi.com/journal/microorganisms Microorganisms 2021,9, 1525 2 of 18 Mongolia region, and deep-sea lakes [ 1 , 2 , 8 – 14 ]. These environments, which host a wide variety of microorganisms belonging to Archaea and Bacteria, have attracted considerable attention in recent years in terms of the microbial diversity and their adaptations to high salt concentrations, as well as on the biotechnological and industrial applications of these extremophilic microorganisms [ 5 , 15 – 17 ]. However, relatively little is known about the prokaryotic diversity in thalassohaline lakes, including the hypersaline Tuz Lake and its associated salterns, in Turkey. The hypersaline Tuz Lake (in Turkish Tuz Gölü), located in the Konya Closed Basin on the Central Anatolian Peninsula, is the largest hypersaline lake in Turkey, with a maximum surface area of 1665 km 2 [ 18 ]. It has an average altitude of 905 m and is surrounded by the Kızılırmak massif in the east, Obruk Plateau in the south, Cihanbeyli Plateau in the west, and the Haymana Plateau in the north [ 19 ]. Tuz Lake constitutes the Main Zone (a water body with a depth of about 70 cm in the spring) and a separate area of the lake, designated as the Deep Zone (a water body with a depth exceeding 100 cm in the spring), is found in the mid-east part of the main area of the lake [ 20 , 21 ]. In addition, on the shore of Tuz Lake, there are several salterns that are the major source for salt production in Turkey, estimated to meet 70% of the salt needs of this country [ 22 , 23 ]. Kayacik saltern, Kaldirim saltern, and Yavsan saltern are located in the mid-east, north-east, and western parts of the lake, respectively (Figure 1). The lake is a closed water basin where the water partly filters into the soil and partly evaporates. The lake is mostly fed by underground water and partially by the small rivers that come from Konya Closed Basin and Obruk Plateau. In summer, most of the incoming streams dry up and do not reach the lake. Due to the excessive evaporation, the lake dries out almost completely and about 30 cm thick salt layers are formed. The average salt content and density of the lake water are 32.4% and 2–2.5 g/cm 3 , respectively [ 24 ]. The annual average temperature is 12 ◦ C (1.6 ◦ C in winter and 22.2 ◦ C in summer, respectively), and the mean annual precipitation is 324 mm/m 2 . Tuz Lake and the surrounding area have been determined as the most arid regions in Turkey. Microorganisms 2021, 9, 1525 2 of 19 Sea, hypersaline and alkaline (soda) lakes in Antarctica, East African lakes such as Lake Magadi and the lakes of Wadi Natrun, in Australia or China, especially in Inner Mongolia region, and deep-sea lakes [1,2,8–14]. These environments, which host a wide variety of microorganisms belonging to Archaea and Bacteria, have attracted considerable attention in recent years in terms of the microbial diversity and their adaptations to high salt concentrations, as well as on the biotechnological and industrial applications of these extremophilic microorganisms [5,15–17]. However, relatively little is known about the prokaryotic diversity in thalassohaline lakes, including the hypersaline Tuz Lake and its associated salterns, in Turkey. The hypersaline Tuz Lake (in Turkish Tuz Gölü), located in the Konya Closed Basin on the Central Anatolian Peninsula, is the largest hypersaline lake in Turkey, with a maximum surface area of 1665 km 2 [18]. It has an average altitude of 905 m and is surrounded by the Kızılırmak massif in the east, Obruk Plateau in the south, Cihanbeyli Plateau in the west, and the Haymana Plateau in the north [19]. Tuz Lake constitutes the Main Zone (a water body with a depth of about 70 cm in the spring) and a separate area of the lake, designated as the Deep Zone (a water body with a depth exceeding 100 cm in the spring), is found in the mid-east part of the main area of the lake [20,21]. In addition, on the shore of Tuz Lake, there are several salterns that are the major source for salt production in Turkey, estimated to meet 70% of the salt needs of this country [22,23]. Kayacik saltern, Kaldirim saltern, and Yavsan saltern are located in the mid-east, north-east, and western parts of the lake, respectively (Figure 1). The lake is a closed water basin where the water partly filters into the soil and partly evaporates. The lake is mostly fed by underground water and partially by the small rivers that come from Konya Closed Basin and Obruk Plateau. In summer, most of the incoming streams dry up and do not reach the lake. Due to the excessive evaporation, the lake dries out almost completely and about 30 cm thick salt layers are formed. The average salt content and density of the lake water are 32.4% and 2–2.5 g/cm 3 , respectively [24]. The annual average temperature is 12 °C (1.6 °C in winter and 22.2 °C in summer, respectively), and the mean annual precipitation is 324 mm/m 2 . Tuz Lake and the surrounding area have been determined as the most arid regions in Turkey. Figure 1. Locations of the sampling sites and 30 brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns in Turkey. Figure 1. Locations of the sampling sites and 30 brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns in Turkey. Some pollution load reaches the Tuz Lake from various sources located around the lake, especially sulfate-rich wastewater from a sodium sulfate manufacturing plant in the southwest, and partially treated domestic wastewater discharged into the small creeks Microorganisms 2021,9, 1525 3 of 18 feeding the lake, and to a lesser extent the metal residue particles of the mining activities and fertilizer and pesticide residues originating from the agricultural activities in the region [19]. The prokaryotic diversity of Tuz Lake has been mostly investigated using culturedependent methods, permitting the isolation of moderately halophilic bacterial and extremely halophilic archaeal strains that produced lipases and proteases, and to a lower extent cellulase and β -galactosidase enzymes [ 22 , 25 – 28 ]. More recently, the archaeal and bacterial diversity of the lake, using culture-independent methods, has been investigated [ 18 , 29 – 32 ]. A higher percentage of the archaeal community than that of the bacterial community was detected by FISH. Based on the 16S rRNA gene library data, Haloquadratum and Salinibacter ruber related phylotypes were found as the predominant microorganisms [ 29 ]. In another culture-independent study carried out in Tuz Lake, in the five brine samples studied, 29 archaeal and 23 bacterial OTUs were detected using 16S rDNA metabarcoding. The OTUs of Haloquadratum walsbyi and the genus Salinibacter were found as the most predominant in these samples [ 30 ]. Moreover, the bacterial composition of Tuz Lake was examined by the V3–V4 variable regions of bacterial 16S rRNA gene. Firmicutes, Fusobacteria, and Proteobacteria were determined as the most abundant bacterial phyla in two brine samples of the lake [31]. In a recent study, the bacterial and archaeal diversity found in 11 sediment samples collected from Cihanbeyli and ¸Sereflikoçhisar regions of Tuz Lake were examined by the comparison of the V4 region of 16S rRNA gene, using Illumina MiSeq sequencing platform and cloning. While Gammaproteobacteria,Bipolaricaulia,Desulfovibrionia, candidate division MSBL-1, Bacteroidia, and Desulfobacteria were detected as the most abundant classes, Gemmatimonadota_c, Rhodothermia,Alphaproteobacteria,Methanonatronarchaeia,Thermoplasmata, Halanaerobiia,Desulfobulbia, and Bacilli were found as low abundance classes [18]. Overall, previous microbiological studies using culture-independent analyses were limited to a small number of samples and sampling areas compared to the great extent of the lake or were restricted to the study of bacteria or sediments of Tuz Lake [18,29–31] . In this study, we have determined the composition and abundance of prokaryotic communities present in the brines of different sampling sites of Tuz Lake, including the Deep Zone, as well as the three salterns, Kayacik, Kaldirim, and Yavsan that are associated with the lake, through 16S rRNA gene amplicon sequencing. 2. Materials and Methods 2.1. Brine Sample Collection Thirty brine samples were collected in July 2017 from 15 different sampling sites (2 brine samples per site), as shown in Figure 1. The Deep Zone, with a depth of about 1 m, is located at the east of Tuz Lake and was previously part of the lake. However, drought has caused a permanent separation of this area from Tuz Lake. Kayacik, Kaldirim, and Yavsan salterns are located on the shores of the Tuz Lake. The samples were coded according to the sampling sites: Tuz Lake (TL1A–TL1B, TL2A–TL2B, TL3A–TL3B, TL4A–TL4B, TL5A–TL5B, TL6A–TL6B, TL7A–TL7B), Deep Zone (TL8A–TL8B, TL9A–TL9B), Kayacik saltern (KYS1A–KYS1B, KYS2A–KYS2B), Kaldirim saltern (KS1A–KS1B, KS2A–KS2B), and Yavsan saltern (YS1A–YS1B, YS2A–YS2B). The brine samples were collected in sterile bottles, carried to the laboratory on ice in the dark, and processed immediately (Figure 1). 2.2. Physico-Chemical Analyses In-situ measurements of temperature and pH of the brine samples were performed using a portable pH meter (PT 10, Sartorius Professional Meter PP–50 AG, Göttingen, Germany). The salinity of the brine samples was measured using a hand refractometer (Reef Octopus, China). Chloride concentrations of the brines were determined by argentometric method [33,34]. Sodium, potassium, magnesium, and calcium concentrations in the brine samples were determined using a flame atomic absorption spectroscopy (FAAS) (Perkin Microorganisms 2021,9, 1525 4 of 18 Elmer, AAS 400, Waltham, MA, USA) via air-acetylene flame [ 35 ]. Linear ranges used in the analyses were 10–100 mg L −1 for Na + , 1–20 mg L −1 for K + , 0.1–2 mg L −1 for Mg 2+ , and 20–100 mg L−1for Ca2+. 2.3. DNA Extraction from the Brine Samples A total of 1.5 L of the brine sample was sequentially filtered through 5 µ m and 0.2 µ m pore size filters, using a peristaltic pump [ 36 – 38 ]. Cells obtained from the filters were resuspended in 2.5 M NaCl, then separately placed into sterile tubes and centrifuged at 13,000 rpm for 30 min. The environmental DNA was extracted from each sample using the PowerSoil DNA isolation kit (Qiagen, USA), according to the manufacturer’s protocol [ 38 ]. The quantity and quality of the extracted DNA were analyzed by a Nanodrop spectrophotometer (NanoDrop 2000) at 260/280 nm and 260/230 nm, and by agarose gel electrophoresis. The extracted DNA samples were stored at − 20 ◦ C until further processing [39]. 2.4. 16S rRNA Amplicon Processing from the Extracted DNA Samples The V6–V8 variable regions of archaeal and bacterial 16S rRNA genes of the extracted DNA belonging to the brine samples were amplified using forward primer 926F (5 0 - AAACTYAAAKGAATTGRCGG-3 0 ) and reverse primer 1392R (5 0 -ACGGGCGGTGTGTRC- 3 0 ) [ 40 ]. PCR amplification was performed using the HotStarTaq Plus Master Mix (Qiagen, USA). The following PCR conditions were used: initial denaturation at 94 ◦ C for 3 min, followed by 28 cycles at 94 ◦ C for 30 s, at 53 ◦ C for 40 s, and 72 ◦ C for 60 s, followed by final extension step at 72 ◦ C for 5 min. After the PCR amplification step, PCR products were run on 2% agarose gel. The concentration of each PCR product was measured and pooled equimolarly. The pooled amplicons were purified using AMPure XP (Beckman Coulter, Brea, CA, USA) magnetic beads. The pooled and purified amplicons were subjected to NGS library preparation protocol for Illumina. Sequencing was performed on the Illumina MiSeq platform with 300 bp paired-end chemistry (Macrogen Inc., Seoul, Korea). 2.5. Bioinformatic Analysis The quality of the raw data was evaluated using the FastQC program [ 41 ]. 16S rRNA gene amplicon data were merged and analyzed with the QIIME program [ 42 ]. Demultiplexing was performed using barcode sequences specific to each sample. Then, the Illumina MiSeq reads were quality filtered by removing low-quality or ambiguous reads. All barcode and primer sequences were removed and the reads were clustered at 97% identity in operational taxonomic units (OTUs) using UCLUST [ 43 ]. A representative sequence for each OTU was selected and all representative OTU sequences were aligned with PyNAST v1.2.2 [ 44 ] using the Greengenes (GreenGenes v13.8) database. Chimeric fragments were detected with the ChimeraSlayer algorithm [ 45 ] and all chimeric reads were excluded. In addition, OTUs with only one read were discarded. Then, the OTU table was formed, including information of read counts of each OTU belonging to the sample. Taxonomic classification of the OTU table was performed using the UCLUST program and Greengenes database. Alpha diversity analyses were also performed with the QIIME program using the OTU table and diversity indexes (PD Whole Tree, Chao1, Observed OTUs, Shannon, Simpson, and Dominance) were calculated. Beta diversity analyses were performed using weighted and unweighted unifrac metrics and visualized as principal component analysis (PCA) graphs. Rarefaction curves were generated by QIIME pipeline v1.9.1 [42]. 2.6. Statistical Analyses In the present study, statistical correlations among the abundance of archaeal and bacterial genera were performed using the R software. The correlation among different variables was analyzed by the “cor” function using the R software. Principal component analysis was made by the “prcomp” function [46]. Microorganisms 2021,9, 1525 5 of 18 2.7. Sequencing Data All sequence data obtained in this study are freely available at the European Nucleotide Archive (ENA)/NCBI under the accession number PRJNA705280. 3. Results 3.1. Physico-Chemical Analyses The 30 brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns were analyzed to determine the physico-chemical characteristics (Table S1). High salinity and near-neutral pH values were observed in all brine samples. Salinity of the samples from Tuz Lake and Deep Zone ranged from 30 to 38%, with intermediate values of 32, 34, and 36% from several sampling sites. The salinity of the samples from Kayacik, Kaldirim, and Yavsan salterns was also very high, with percentages of 30–32, 32–36, and 32%, respectively. The pH of the samples ranged between 6.9 (only two sampling sites) and 7.4, and the temperature of the brine samples was very constant, ranging from 25 to 27 ◦ C, except for some of Tuz Lake samples and all Deep Zone samples for which the temperature was 22 and 24 ◦ C. Na + and Cl − contents were fairly high in all brine samples. Although K + and Mg 2+ contents were found as moderate, low Ca 2+ concentrations were determined for all samples studied (Table S1). Overall, these data, especially the neutral pH and high content of Na + and Cl − concentrations, in contrast to divalent cations, corroborate the thalassohaline character of Tuz Lake and the salterns investigated in this study. 3.2. Alpha Diversity and Richness Metrics In the present study, a total of 1,282,558 16S rRNA gene sequences obtained from the 30 brine samples were clustered into 17,488 OTUs at 97% sequence similarity. YS2A sample, obtained from Yavsan saltern, had the maximal values according to both, the number of sequences (97,361) and OTUs (5725). In contrast, the Tuz Lake and Deep Zone samples TL4A and TL8B had minimal values in terms of the sequence count (26,179) and OTU number (707), respectively. Although samples from Tuz Lake and Deep Zone showed slopes on the rarefaction curves close to zero or gentle slope, samples from Kayacik, Kaldirim, and Yavsan salterns had raised slopes, suggesting that the deep sequencing was insufficient to cover the diversity in these few samples (Figure S1). To examine the diversity and richness in Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns, observed OTUs, PD whole tree, Chao1, Shannon, Simpson, and Dominance indexes were calculated. Chao1 and Shannon indexes indicated that the highest diversities were detected in TL2 and KS1 sampling regions and the lowest diversities were detected in TL8–TL9 sampling sites corresponding to the Deep Zone (Table 1). Brine samples of the Deep Zone had lower OTU numbers than those of brine samples of Tuz Lake and its salterns, and consequently, lower prokaryotic diversity was detected at this region because of a large dominance of a few OTUs (Table 1). Table 1. Prokaryotic richness and diversity estimates, based on 97% OTU clusters of Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. The diversity indexes: PD Whole Tree, Chao1, Shanon, Simpson, and Dominance were calculated. Sample PD Whole Tree Chao 1 Observed OTUs Shannon Simpson Dominance Tuz Lake TL1A 39.02 2879.40 1517.00 6.22 0.94 0.06 TL1B 41.00 3388.46 1753.00 6.09 0.94 0.06 TL2A 77.94 8354.98 3799.00 7.50 0.95 0.05 TL2B 88.63 9402.42 4539.00 7.82 0.96 0.04 TL3A 56.38 5378.22 2756.00 6.31 0.90 0.10 TL3B 60.63 5983.23 2881.00 7.23 0.95 0.05 TL4A 53.08 3761.75 1936.00 6.75 0.95 0.05 Microorganisms 2021,9, 1525 6 of 18 Table 1. Cont. Sample PD Whole Tree Chao 1 Observed OTUs Shannon Simpson Dominance TL4B 59.47 3958.28 2357.00 7.23 0.96 0.04 TL5A 64.97 6757.91 3134.00 8.11 0.98 0.02 TL5B 76.81 7325.52 3930.00 8.46 0.98 0.02 TL6A 57.09 5566.74 2674.00 5.38 0.77 0.23 TL6B 69.76 7010.20 3450.00 6.15 0.84 0.16 TL7A 51.98 4779.82 2134.00 5.73 0.89 0.11 TL7B 53.02 4555.30 2241.00 5.24 0.80 0.20 Deep Zone TL8A 24.94 1805.01 914.00 4.43 0.82 0.18 TL8B 20.61 1480.57 707.00 4.29 0.79 0.21 TL9A 23.76 1684.02 883.00 4.65 0.85 0.15 TL9B 26.09 2007.02 1008.00 4.55 0.83 0.17 Kayacik saltern KYS1A 71.63 8049.00 3488.00 6.98 0.91 0.09 KYS1B 58.10 6263.30 2768.00 6.94 0.91 0.09 KYS2A 40.67 3763.57 1812.00 5.52 0.90 0.10 KYS2B 34.26 2669.73 1453.00 5.31 0.90 0.10 Kaldirim saltern KS1A 78.75 8057.13 4026.00 8.27 0.98 0.02 KS1B 81.74 8825.45 4242.00 8.19 0.97 0.03 KS2A 78.01 8276.09 3692.00 7.24 0.94 0.06 KS2B 67.12 6241.22 2900.00 6.66 0.91 0.09 Yavsan saltern YS1A 32.29 2572.08 1321.00 5.16 0.82 0.18 YS1B 70.16 6340.94 3174.00 5.98 0.85 0.15 YS2A 104.00 10,259.51 5725.00 7.17 0.92 0.08 YS2B 85.79 8352.18 4468.00 7.28 0.92 0.08 3.3. Prokaryotic Communities in Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan Salterns Our analyses indicated that most 16S rRNA gene sequences were classified within the domain Archaea (98.41%) and clustered into 16,939 OTUs. In contrast, only a few sequences could be assigned to the domain Bacteria (1.38%) and they clustered into 218 OTUs. Fifty-one OTUs belonging to the archaeal class Halobacteria and one OTU belonging to the bacterial class Rhodothermia, family Salinibacteraceae were determined in all the brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. The archaeal phylum Euryarchaeota (93.76–99.94%, with a mean of 98.41 ± 0.27%), and the bacterial phyla Rhodothermaeota (0.02–5.52%, 1.28 ± 0.25%), Proteobacteria ( 0.00–0.83% , 0.06 ± 0.03%), Firmicutes (0.00–0.33%, 0.02 ± 0.01%), and Lentisphaerae (0.00–0.11%, 0.009 ±0.005% ) were found in the samples. In addition, the archaeal phylum Parvarchaeota (0.00–0.003%) and the bacterial phyla Actinobacteria (0.00–0.008%, 0.001 ± 0.0003%), Spirochaetes ( 0.00–0.004% ) and Cyanobacteria (0.00–0.01%, 0.001 ± 0.0005%) were detected at extremely low abundance in a few brine samples. According to these data, Euryarchaeota, distantly followed by Rhodothermaeota, were the most abundant phyla determined in all brine samples studied (Figure 2). The phyla composition detected in all brine samples of Tuz Lake, and Kayacik, Kaldirim and Yavsan salterns were fairly similar, except for the Deep Zone (Table S2). It is noteworthy that the two brine replicates collected per site shared a quite similar phyla composition (Figure 2). The highest percentage of Euryarchaeota was detected at the brine samples of the Deep Zone. Rhodothermaeota was found slightly more abundant in some locations of Tuz Lake (TL2, TL3, TL4, TL6, and TL7 sampling sites), Kayacik saltern (KYS1 samples), Kaldirim saltern (KS1 and KS2 samples), and Yavsan saltern (YS1 and YS2 samples) (Figure 2). Microorganisms 2021,9, 1525 7 of 18 Microorganisms 2021, 9, 1525 7 of 19 0.005%) were found in the samples. In addition, the archaeal phylum Parvarchaeota (0.00– 0.003%) and the bacterial phyla Actinobacteria (0.00–0.008%, 0.001 ± 0.0003%), Spirochaetes (0.00–0.004%) and Cyanobacteria (0.00–0.01%, 0.001 ± 0.0005%) were detected at extremely low abundance in a few brine samples. According to these data, Euryarchaeota, distantly followed by Rhodothermaeota, were the most abundant phyla determined in all brine samples studied (Figure 2). Figure 2. Phyla distributions of prokaryotic communities determined in brine samples of Tuz Lake (TL1A to TL7B), Deep Zone (TL8A to TL9B), and Kayacik (KYS1A to KYS2B), Kaldirim (KS1A to KS2B), and Yavsan (YS1A to YS2B) salterns. The phyla composition detected in all brine samples of Tuz Lake, and Kayacik, Kaldirim and Yavsan salterns were fairly similar, except for the Deep Zone (Table S2). It is noteworthy that the two brine replicates collected per site shared a quite similar phyla composition (Figure 2). The highest percentage of Euryarchaeota was detected at the brine samples of the Deep Zone. Rhodothermaeota was found slightly more abundant in some locations of Tuz Lake (TL2, TL3, TL4, TL6, and TL7 sampling sites), Kayacik saltern (KYS1 samples), Kaldirim saltern (KS1 and KS2 samples), and Yavsan saltern (YS1 and YS2 samples) (Figure 2). At genus level, most of the sequences were classified in the phylum Euryarchaeota, being the OTU classified in the genus Haloquadratum remarkably abundant in most of the samples (Figure 3). However, a large number of sequences clustered into OTUs that could not be assigned to any known genera (39% of sequences). Thus, only 61% of sequences obtained in this study were assigned or closely related to previously described genera. Two uncultured OTUs were well represented in these samples and both belonged or were related to the class Halobacteria. Besides, the genera Haloarcula and Halorhabdus also recruited many sequences in these environments. In contrast, only 1.38% of sequences were assigned to Bacteria and most of them were related to the genus Salinibacter (Table 2 and Figure 3). Figure 2. Phyla distributions of prokaryotic communities determined in brine samples of Tuz Lake (TL1A to TL7B), Deep Zone (TL8A to TL9B), and Kayacik (KYS1A to KYS2B), Kaldirim (KS1A to KS2B), and Yavsan (YS1A to YS2B) salterns. At genus level, most of the sequences were classified in the phylum Euryarchaeota, being the OTU classified in the genus Haloquadratum remarkably abundant in most of the samples (Figure 3). However, a large number of sequences clustered into OTUs that could not be assigned to any known genera (39% of sequences). Thus, only 61% of sequences obtained in this study were assigned or closely related to previously described genera. Two uncultured OTUs were well represented in these samples and both belonged or were related to the class Halobacteria. Besides, the genera Haloarcula and Halorhabdus also recruited many sequences in these environments. In contrast, only 1.38% of sequences were assigned to Bacteria and most of them were related to the genus Salinibacter (Table 2 and Figure 3). Table 2. Taxonomic distribution at the genus level, with more than 0.3% sequences detected in the brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. Genus/Taxon Phylum Tuz Lake Deep Zone Kayacik Saltern Kaldirim Saltern Yavsan Saltern Uncultured Halobacteria TLH-1 Euryarchaeota 24.61 ±3.20 5.43 ±0.28 10.65 ±2.66 31.77 ±4.86 17.93 ±2.12 Haloquadratum Euryarchaeota 23.99 ±4.25 47.32 ±1.74 31.33 ±2.78 16.57 ±3.18 41.30 ±3.60 Haloarcula Euryarchaeota 14.36 ±4.32 0.03 ±0.00 1.80 ±0.79 14.52 ±6.39 5.46 ±1.42 Uncultured Halobacteria TLH-2 Euryarchaeota 13.98 ±1.04 22.99 ±1.78 36.18 ±5.85 14.14 ±1.51 16.40 ±2.16 Halorhabdus Euryarchaeota 6.90 ±0.93 23.76 ±1.95 14.22 ±2.93 6.40 ±0.69 8.82 ±1.78 Halorubrum Euryarchaeota 3.87 ±1.34 0.15 ±0.02 1.80 ±0.42 2.07 ±0.46 2.26 ±0.60 Halonotius Euryarchaeota 3.25 ±0.66 0.02 ±0.00 0.96 ±0.41 4.69 ±1.65 1.50 ±0.51 Natronomonas Euryarchaeota 3.00 ±1.55 0.02 ±0.00 0.30 ±0.15 2.99 ±1.39 0.81 ±0.24 Halolamina Euryarchaeota 1.73 ±0.91 <0.01 0.27 ±0.23 2.15 ±0.17 0.39 ±0.24 Salinibacter Rhodothermaeota 1.54 ±0.37 0.09 ±0.03 0.88 ±0.42 1.09 ±0.16 2.10 ±1.13 Halobacterium Euryarchaeota 1.19 ±0.53 0.01 ±0.00 0.04 ±0.02 0.44 ±0.19 0.51 ±0.27 Halosimplex Euryarchaeota 0.58 ±0.11 0.08 ±0.01 0.37 ±0.14 1.34 ±0.43 0.67 ±0.23 Halomicrobium Euryarchaeota 0.23 ±0.05 0.01 ±0.00 0.17 ±0.05 0.84 ±0.36 0.30 ±0.14 Haloplanus Euryarchaeota 0.16 ±0.04 0.01 ±0.00 0.46 ±0.11 0.42 ±0.09 0.60 ±0.11 Uncultured Halobacteria TLH-3 Euryarchaeota 0.07 ±0.02 0.00 ±0.00 0.06 ±0.03 0.22 ±0.05 0.34 ±0.14 Microorganisms 2021,9, 1525 8 of 18 Microorganisms 2021, 9, 1525 8 of 19 Figure 3. Taxonomic profiles at the genus level of prokaryotic communities detected in brine samples of Tuz Lake (TL1A to TL7B), Deep Zone (TL8A to TL9B), and Kayacik (KYS1A to KYS2B), Kaldirim (KS1A to KS2B), and Yavsan (YS1A to YS2B) salterns. Each OTU contains at least 1% 16S rRNA sequence. Table 2. Taxonomic distribution at the genus level, with more than 0.3% sequences detected in the brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. Genus/Taxon Phylum Tuz Lake Deep Zone Kayacik Saltern Kaldirim Saltern Yavsan Saltern Uncultured Halobacteria TLH-1 Euryarchaeota 24.61 ± 3.20 5.43 ± 0.28 10.65 ± 2.66 31.77 ± 4.86 17.93 ± 2.12 Haloquadratum Euryarchaeota 23.99 ± 4.25 47.32 ± 1.74 31.33 ± 2.78 16.57 ± 3.18 41.30 ± 3.60 Haloarcula Euryarchaeota 14.36 ± 4.32 0.03 ± 0.00 1.80 ± 0.79 14.52 ± 6.39 5.46 ± 1.42 Uncultured Halobacteria TLH-2 Euryarchaeota 13.98 ± 1.04 22.99 ± 1.78 36.18 ± 5.85 14.14 ± 1.51 16.40 ± 2.16 Halorhabdus Euryarchaeota 6.90 ± 0.93 23.76 ± 1.95 14.22 ± 2.93 6.40 ± 0.69 8.82 ± 1.78 Halorubrum Euryarchaeota 3.87 ± 1.34 0.15 ± 0.02 1.80 ± 0.42 2.07 ± 0.46 2.26 ± 0.60 Halonotius Euryarchaeota 3.25 ± 0.66 0.02 ± 0.00 0.96 ± 0.41 4.69 ± 1.65 1.50 ± 0.51 Natronomonas Euryarchaeota 3.00 ± 1.55 0.02 ± 0.00 0.30 ± 0.15 2.99 ± 1.39 0.81 ± 0.24 Halolamina Euryarchaeota 1.73 ± 0.91 <0.01 0.27 ± 0.23 2.15 ± 0.17 0.39 ± 0.24 Salinibacter Rhodothermaeota 1.54 ± 0.37 0.09 ± 0.03 0.88 ± 0.42 1.09 ± 0.16 2.10 ± 1.13 Halobacterium Euryarchaeota 1.19 ± 0.53 0.01 ± 0.00 0.04 ± 0.02 0.44 ± 0.19 0.51 ± 0.27 Halosimplex Euryarchaeota 0.58 ± 0.11 0.08 ± 0.01 0.37 ± 0.14 1.34 ± 0.43 0.67 ± 0.23 Halomicrobium Euryarchaeota 0.23 ± 0.05 0.01 ± 0.00 0.17 ± 0.05 0.84 ± 0.36 0.30 ± 0.14 Haloplanus Euryarchaeota 0.16 ± 0.04 0.01 ± 0.00 0.46 ± 0.11 0.42 ± 0.09 0.60 ± 0.11 Figure 3. Taxonomic profiles at the genus level of prokaryotic communities detected in brine samples of Tuz Lake (TL1A to TL7B), Deep Zone (TL8A to TL9B), and Kayacik (KYS1A to KYS2B), Kaldirim (KS1A to KS2B), and Yavsan (YS1A to YS2B) salterns. Each OTU contains at least 1% 16S rRNA sequence. Haloquadratum (23.99%) and Haloarcula (14.36%) were determined as the most predominant genera in the brine samples of Tuz Lake, with the uncultured Halobacteria TLH-1 (24.61%) as the most abundant taxon, and lower percentages for the representatives of the uncultured Halobacteria TLH-2 (13.98%) and the archaeal genera Halorhabdus (6.90%), Halorubrum (3.87%), Halonotius (3.25%), Natronomonas (3.00%), Halolamina (1.73%), and Halobacterium (1.19%), and the bacterial genus Salinibacter (1.54%) among those genera represented by at least 1% of the sequences (Table 2). The Deep Zone samples were highlighted by a large presence of Haloquadratum (47.32%), followed by Halorhabdus (23.76%) and the uncultured Halobacteria TLH-2 (22.99%). This last scenario is also repeated in Kayacik saltern samples, but in this case, the uncultured Halobacteria TLH-2 was more remarkable in these samples (36.18%) than in the others. A similar composition was found in Yavsan saltern samples, but also uncultured Halobacteria TLH-1 showed a great abundance (17.93%). In Kaldirim saltern, the most abundant taxon was the uncultured Halobacteria TLH-1, followed by Haloquadratum and the uncultured Halobacteria TLH-2. Finally, in contrast to Deep Zone and the other salterns, the genus Haloarcula was well represented in KS1 samples from Kaldirim saltern (Table 2 and Figure 3). Microorganisms 2021,9, 1525 9 of 18 In summary, concerning the domain Archaea, the genus Haloquadratum was always present and represented the most abundant taxon on most sampling sites (with percentages ranging from 47.32 to 16.57%), while on the domain Bacteria, the genus Salinibacter was the only representative present at relatively low proportions (Table 2). Finally, two unknown and not yet isolated taxa, designated as uncultured Halobacteria TLH-1 and TLH-2, were predominant on Tuz Lake and Kaldirim saltern, and on Kayacik saltern, respectively (Table 2). 3.4. Principal Components Analysis (PCA) Unweighted and weighted UniFrac, which are measurements of β -diversity, compare prokaryotic communities based on the phylogenetic relationships of the Archaea and Bacteria contained within communities. The PCA plot provides an overview visualization of the relationships of the communities among the samples [ 47 – 49 ]. UniFrac distances were generated using weighted and unweighted methods to compare the detected archaeal and bacterial communities of the brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns in terms of diversity among the samples and plotted them on PCA plots (Figure 4). Microorganisms 2021, 9, 1525 10 of 19 Figure 4. Principal components analysis (PCA) of the brine samples of Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. (A) Unweighted UniFrac, (B) Weighted UniFrac. Figure 4 shows that the samples from each site were grouped relatively close to each other, except the sample YS1A with a more limited taxonomic diversity than the other samples from Yavsan saltern and the samples from Tuz Lake that were taken from different distant places due to the great extension of this lake. Especially, TL1 brine samples showed a greater difference in their variance compared to the rest of the brine samples collected from Tuz Lake. A higher variance (84.1%) was obtained from the weighted UniFrac PCA than the unweighted UniFrac PCA using the first two principal components. In this plot, five brine samples of Tuz Lake (TL1A, TL1B, TL4A, TL5A, and TL5B), and one brine sample of Yavsan (YS1A) saltern were not grouped closely with the other brine samples (Figure 4B). 3.5. Correlations among Genera The genera (>1% sequences) detected in the brine samples collected from Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns and their statistical correlations were generated using R software version 3.6.2 (Figure 5). Diversity and abundance of the microbial communities changed according to positive and negative correlations among archaeal genera found in each sampling site (p < 0.05). Positive strong correlations were found between Halonotius and the uncultured Halobacteria TLH-1 (r = 0.917); genera Halorubrum and Natronomonas (r = 0.857); and genera Halolamina and Halobacterium (r = 0.676). Moreover, the genus Haloquadratum was found positively correlated with the genus Halorhabdus (r = 0.582) and the uncultured Halobacteria TLH-2 (r = 0.159). However, the increased presence of Haloquadratum, Halorhabdus, and the uncultured Halobacteria TLH-2 were negatively correlated with the abundance of the genera Haloarcula, Halobacterium, Halolamina, Halomicrobium, Halonotius, Halorubrum, Halosimplex, Natronomonas and the uncultured Halobacteria TLH-1. Although these genera had negative correlations with Figure 4. Principal components analysis (PCA) of the brine samples of Tuz Lake, Deep Zone, and Kayacik, Kaldirim, and Yavsan salterns. (A) Unweighted UniFrac, (B) Weighted UniFrac. Figure 4shows that the samples from each site were grouped relatively close to each other, except the sample YS1A with a more limited taxonomic diversity than the other samples from Yavsan saltern and the samples from Tuz Lake that were taken from different distant places due to the great extension of this lake. Especially, TL1 brine samples showed a greater difference in their variance compared to the rest of the brine samples collected from Tuz Lake. A higher variance (84.1%) was obtained from the weighted UniFrac PCA than the unweighted UniFrac PCA using the first two principal components. In this plot, five brine samples of Tuz Lake (TL1A, TL1B, TL4A, TL5A, and TL5B), and one brine sample of Yavsan (YS1A) saltern were not grouped closely with the other brine samples (Figure 4B). Microorganisms 2021,9, 1525 16 of 18 18. Çınar, S.; Mutlu, M.B. Prokaryotic community compositions of the hypersaline sediments of Tuz Lake demonstrated by cloning and high-throughput sequencing. 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