Introduction

Pristine, cold oligotrophic lakes like those in Chilean Patagonia are characterized by low temperature (values below 10 °C) and concentrations of nutrients and dissolved organic carbon, determining a low primary productivity and species diversity [3, 14, 31, 50]. Chilean Patagonian lakes are of glacial origin with different geomorphological characteristics and marked seasonal temperature variations (minimum of 4 °C in winter, and maximum of 20 °C in summer) [12, 47]. In winter, lacustrine surfaces are in part covered by a thin iced layer, which may affect all ecological conditions. As remote sites, the cold Chilean lakes are environments with little anthropogenic impact and therefore still harbor pristine biotopes [3, 31].

Cold environments are colonized by different microorganisms well adapted to the fluctuations in temperatures and nutrients availability, playing a relevant role in biogeochemical cycling and in functioning of aquatic environments [32, 35, 37, 40, 41, 48, 49]. Because only a minor fraction of microbial communities in natural environments is culturable [35, 50], different techniques including molecular, structural, genetic, and metabolic approaches have been applied to analyze the prokaryotic community structure and composition and to understand the role of microorganisms in different ecosystems. Among classical molecular analyses, based on 16S rRNA gene sequence analysis, the denaturing gradient gel electrophoresis (DGGE) has been commonly applied to investigate the prokaryotic community structure and composition in different aquatic environments [33, 43]. In addition, fluorescent in situ hybridization (FISH) using specific probes is commonly used to identify active members of the microbial community, obtaining a direct measure of their relative abundance [4, 51].

Prokaryotic communities in cold environments, frequently subjected to freezing periods, have to cope with low temperature, water, and nutrient availability [41]. Schiaffino et al. [44] reported that temperature is an important factor in shaping the prokaryotic abundances of Argentinean Patagonia lakes. The distribution patterns of major bacterial and archaeal groups in these lakes showed that Bacteria possessed similar abundances in late winter or in spring, whereas Archaea varied markedly in summer, spring, and winter, suggesting that the two prokaryotic domains could have different temporal ecological strategies in these lakes [44]. Similar bacterial communities were observed in three cold and nutrient-poor lakes in the Chilean Patagonia (Alto Reino, Las Dos Torres, and Venus), as a consequence of similar capability in responding to different environmental stressors [31].

The remote location of Patagonian lakes could be considered an important geographical factor in determining the characteristic diversity of microbial communities in each aquatic ecosystem [17, 31, 44]. In addition, these aquatic environments could be exposed to global climate changes and to human activities that, as main stressors, could modify the microbial community structure and composition. Therefore, Patagonian lakes represent suitable environments for evaluating changes in the prokaryotic community composition and diversity associated with pronounced environmental and anthropogenic perturbations.

To obtain more information on the microbiota of remote, lacustrine ecosystems in Chilean Patagonia, in this study water and sediment samples were collected in winter and summer periods, from three cold, oligotrophic Patagonian lakes (Témpanos, Las Torres and Mercedes) located in the northern region of Aysén (Chile), along a longitudinal gradient (150 km) (Fig. 1).

Fig. 1
figure 1

Map of the Chilean Aysén Region and site location of Témpanos, Las Torres, and Mercedes lakes

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The aim of this work is to evaluate the prokaryotic abundance, structure, and composition in these Patagonian lakes, using molecular approaches based on 16S rRNA FISH and PCR-DGGE techniques, and to test the importance of local environmental factors on the natural communities.

Materials and Methods

Study Sites and Sampling

Témpanos lake (44°27′39.5″S; 72°31′41.74″W) is located in the Queulat National Park and is close to a glacier, with an area of 1.34 km2 and an altitude of 115 m above the sea level (a.s.l.). Las Torres lake (45°43′56.96″S; 72°12′50.68″W) has an area of 3.3 km2 and an altitude of 267 m (a.s.l.). Mercedes lake (45°43′59.02″S, 72°12′50.35″W) has an area of 0.19 km2 and an altitude of 267 m (a.s.l.).

Sampling was performed in winter (August 2010) and summer (January 2011) seasons. Surface water samples were collected manually approximately 50 m from the shore. Surface sediment samples (500 g) were collected by a Van Veen grab sampler at approximately 20-m depth in Témpanos and Las Torres, and at 10-m depth in Mercedes. Samples were immediately placed in sterile polyethylene bags (Whirlpak). After collection, water and sediment subsamples for microbial abundance estimation were fixed with formaldehyde 2% (v/v; final concentration), whereas subsamples for DNA extraction were directly stored at −20 °C until further processing.

Chemical Characterization of Water and Sediment Samples

Values of pH and temperature in water were recorded in situ by a portable multiparametric probe (Hanna HI 9812), and dissolved oxygen (DO) was measured using an Oxygen Meter (Cole Palmer, 5946-75). Total Organic Carbon (TOC) content of water samples was analyzed by a Shimadzu TOC analyzer Model 5050. Concentrations of total phosphorous (TP) and total nitrogen (TN) were determined according to standard methods [1].

Total organic matter in sediment samples was estimated by the loss-on-ignition technique, following the method described previously [11] and then converted to TOC according to Schumacher et al. [45]. In addition, the samples were independently analyzed for TP and TN using Kjeldahl digestion followed by colorimetric detection by molecular absorption spectrophotometry, according to standard methods [1].

In order to evaluate the input of contaminants from human activities, organochlorine and polychlorinated biphenyls pesticides were analyzed in samples collected from the three lakes, according to the methods reported by Barra et al. [6]. Briefly, sediment samples (2 g) were mixed with anhydrous sodium sulfate and then extracted by accelerated solvent extraction with acetone:cyclohexane (1:1) at 150 °C, 14 MPa for 5 min (three times). Extracts were concentrated to 0.2 ml and then cleaned up in a Florisil column (10-mm i.d., 3-g Florisil). The conditions of the performed GC/MS analysis were column from Machery Nagel (Optima d6, 60 m, 0.25 mm, 0.25 lm film thickness). The temperature program was 80 °C, then 3 min isothermal, 15 °C/min to 160 °C, and then 3 °C/min to 280 °C hold for 12 min. Quantification was performed with the external standard method with six calibration levels. Water samples (500 ml) were analyzed with liquid–liquid extraction followed by GC–MS for PCBs. The extracts were concentrated to 1 ml and analyzed by GC/MS as described above.

Prokaryotic Cell Abundance

The LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes, Eugene, OR), based on cell membrane integrity, was used to distinguish live from dead cells, and their sum was indicated as total counts (TC). Living cells (LC) with intact membranes stain green with SYTO®9, while dead cells with damaged membranes, which are considered to be dead, stain red with propidium iodide [23].

Aliquots of each water sample were concentrated onto black polycarbonate membrane filters (0.2-µm pore size, 25-mm diameter, Nuclepore Corporation, Pleasanton, USA). To detach cells from sediment samples, 1 g of each sample was diluted (1:10 w/v) in sterilized phosphate buffer solution (PBS) (130-mM NaCl; 7-mM Na2HPO4; 3-mM NaH2PO4, pH 7.4) containing Tween 20 (0.5%, v/v; Research Organics Inc.) and sodium pyrophosphate (0.1%, v/v; Sigma-Aldrich), and then sonicated in an ice-water bath three times for 10 s with 20 s interval between cycles using a Brandelin SonoPlus HD 200 (Electronic, Berlin) [26]. Cells from sediments were finally concentrated onto black polycarbonate membrane filters (0.2-µm pore size, 25-mm diameter). After staining, the filtered cells were counted using epifluorescence microscopy (Olympus BX-60M, at 1000× magnification) under ultraviolet light (HG 100 W). The microscope was equipped with a halogen (HG 100 W) light. A G 330–385 exciter filter, an FT 400 chromatic beam splitter, and an LP 420 barrier filter were used for direct counts.

Fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes was used to estimate the abundance of microorganisms ascribed to Bacteria and Archaea domains, according to the protocol previously reported [39]. Cells were concentrated from samples on white polycarbonate filters (0.2-μm pore size, 25-mm diameter) and subsequently fixed for 30 min at room temperature by overlaying filters with a freshly prepared paraformaldehyde (final concentration, 4%) phosphate-buffered saline (PBS; 130-mM NaCl, 10-mM Na2HPO4, and 10-mM NaH2PO4, pH 7.4) solution (3 ml). The fixative was removed by applying vacuum, and filters were washed twice with 3 ml of PBS and, finally, with distilled water. Filters were air-dried and stored in a sterile Petri dish at −20 °C until processing. Filter sections were hybridized with Cyanine-labeled (Cy3) oligonucleotide probe EUB338 and ARCH915 (ThermoHybaid, Interactiva Division, Ulm, Germany) to enumerate bacterial and archaeal cells. Hybridization was performed at 46 °C for 2 h in a hybridization oven with continuous rotation. The filter sections were washed in 10 ml of hybridization buffer and incubated at 48 °C for 15 min. Subsequently, filter sections were mounted on glass slides with Citifluor AF1 (Citifluor Ltd., Canterbury, UK). For each sample, between 50 and 200 cells were then counted under epifluorescence, using Olympus BX60 microscope, equipped with an appropriate filter set for Cy3.

Genomic DNA Extraction and Denaturing Gradient Gel Electrophoresis Analysis

Each water sample (500 ml) was pre-filtered through 2.0-µm pore size membrane filters and concentrated onto 0.22-µm cellulose ester membranes (GSWP04700 Millipore), and genomic DNA was extracted using the Power Soil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer’s instructions.

DNA from each sediment sample (5 g) was extracted using the UltraClean Soils DNA kit (MoBio Laboratories, Carlsbad, CA, USA), according to the manufacturer’s instructions. DNA concentrations and purity were checked using a NanoDrop ND-1000, UV–vis spectrophotometer (NanoDrop Technologies, USA).

Bacterial 16S rRNA genes were amplified with eubacterial primer set, forward primer (Escherichia coli positions 9–27: 5′-GAGTTTGATCCTGGCTCAG-3′), and reverse primer (E. coli positions 1542-1525: 5′-AGAAAGGAGGTGATCCAGCC-3′). In order to avoid chimeric formation during the PCR amplification process, we chose to amplify the V3 region of bacterial 16S rRNA by a nested PCR using primers 341f-GCclamp (5′-CCTACGGGAGGCAGCAG-3′) and 534r (5′-ATTACCGCGGCTGCTGG-3′), as previously described [33].

Archaeal 16S rRNA genes were amplified using the Archaea-specific primers 21f (5′-TTCCGGTTGATCCYGCCGGA-3′) and 958r (5′-YCCGGCGTTGAMTCCAATT-3′), in PCR conditions described previously [18]. To amplify the V3 region of the Archaea 16S rRNA gene, a second nested PCR was performed using the Archaea-specific primers set, ARC344-GC (5′-ACGGGGCGCAGCAGGCGCGA-3′) and ARC517r (5′-ATTACCGCGGCTGCTGG-3′) [42].

Bacterial and archaeal PCR products were resolved in polyacrylamide gels according to Muyzer et al. [33] and Casamayor et al. [13], respectively, using a DGGE-2001 system (CBS Scientific Company, CA, USA). All visible bands in DGGE gels were excised, and the small blocks of gel were placed into 50 µl of HypureTM molecular biology grade water (nuclease-free) (Thermo scientific) at +4 °C overnight to allow diffusion of DNA out the gel blocks. The eluate (1 µl) was used as template DNA in a reamplification PCR, using primers and reaction conditions above described.

Sequencing of DGGE bands was performed with primers 341 f for Bacteria and ARC344f for Archaea at Macrogen laboratories (Macrogen Inc., Seoul, Korea). A nucleotide BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) was performed to obtain sequences with the greatest significant alignment.

The partial 16S rRNA gene sequences were deposited in the GenBank database under accession numbers KU912443–KU942475.

Statistical Analysis

DGGE band position and intensity were determined using GELCOMPAR II software package (Applied Maths) and were manually modified. To compare bacterial and archaeal community across all samples, Sørensen similarity coefficient (S s) was applied to the banding patterns and Nonmetric Multi-Dimensional Scaling diagrams were obtained using the PRIMER 6.1.12 (Primer-E, Ltd). The total number of bands in each line was expressed as phylotype richness (R). The relative intensity was used to calculate the Shannon diversity (H’) and the evenness (E) indices, in order to describe possible changes in the DGGE profiles among samples from the same lake (alpha diversity), and among lakes (beta diversity) in the two seasons.

Pearson’s correlation analysis was applied to determine the links between prokaryotic abundances (Bacteria and Archaea), diversity indices (R, H’ and E), and the relative abundance of the dominant bacterial and archaeal groups, as derived by DGGE analysis, with the environmental parameters retrieved from water (pH, TOC, TP, and TN) and sediment (TOC, TP, and TN) samples. Principal Component Analyses (PCAs) were also performed on data from environmental factors and microbial community properties, using PRIMER 6.1.12. A (natural) logarithmic was employed to equalize variances and normalize the data prior to these analyses.

Results

Physical and Chemical Characteristics of the Lakes

Physical and chemical parameters recorded in water and sediment samples collected from the three Chilean Patagonia lakes are reported in Table 1. Values of pH (4.0–5.0) were constantly acidic in all samples, with a slight increase in winter. Temperature recorded in winter was 4 °C in all samples, while in summer ranged from 8.3 (in Témpanos) to 16.3 (in Mercedes). Dissolved oxygen ranged from 8.0 to 9.0 mg l− 1. TOC content in water was higher in winter, when abundant rains provided terrestrial inputs of organic matter, than in summer. Minimum and maximum TOC values were both recorded from Témpanos. TP concentrations were high in Témpanos in both seasons, and TN levels were greater in summer than in winter. The highest TN concentration was recorded in samples from Mercedes.

Table 1 Physical and chemical parameters in water and sediment samples, in winter and summer periods, collected from the three lakes of Chilean Patagonia
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Sediments from the three lakes were characterized by muddy textures (mud and sand). TOC values were higher in sediments from Las Torres, in both seasons, than those recorded at Mercedes and Témpanos lakes (Table 1). The highest TP was observed in samples from Témpanos in summer. High TN abundances were recorded from Las Torres collected in both seasons, and the lowest was from Témpanos in summer.

Polychlorinated biphenyl and organochlorine pesticides were not detected in any water and sediment samples.

Prokaryotic Cell Abundance

Total counts (TC) from samples, obtained after live/dead staining, were one order of magnitude higher in sediment than in water samples and also in summer when compared to winter (Table 2). The percentage of living cells (LC) with respect to TC was always higher in sediment than in water samples. The lowest contribution of LC to TC was observed in waters collected from Las Torres and Témpanos in winter.

Table 2 Abundances (cells ml− 1 or g− 1) of prokaryotic cells (TC), living cells, and of hybridized cells with probes for Bacteria (EUB338) and Archaea (ARCH915) in water and sediment samples, in winter and summer periods, collected from the three Chilean Patagonia lakes
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As resulted by FISH, Bacteria always numerically dominated over Archaea (Table 2). Archaeal contribution (ranging from 0.01 to 1.1%) was higher in water than in sediment, and also higher in winter than in summer.

Bacterial DGGE Analyses

Bacterial DGGE profiles and the analysis of the bacterial community structure are reported in Fig. 2. The DGGE banding patterns of Bacteria are presented for water and sediment samples, in winter (w) and summer (s) periods (Fig. 2a). The total number of bands identified in each profile, ranging from 12 to 22 per sample, was lower in water (101) than in sediment (109) samples. When comparing per season, the number of bands was slightly higher in summer (106) than in winter (104). Based on the presence and absence of bands in each lane, S s were calculated from the DGGE profiles. Overall, bacterial populations occurring in water samples were more similar to each other (30.3–72.7% S s) than those in sediments (13.3–57.1% S s). The water samples from Témpanos (Tw and Ts), as well as the sediment samples from Mercedes (Ms) and Las Torres (LTw), were clearly different from the remaining samples, which clustered together to varying degrees (Fig. 2b).

Fig. 2
figure 2

Bacterial DGGE profiles obtained from water and sediment samples (a), and nonmetric multidimensional scaling diagrams (b) representing the bacterial community structure in Las Torres (LT), Mercedes (M), and Témpanos (T) lakes, in winter (w) and summer (s) periods. Sequenced bands are indicated by numbers

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Bacterial Phylogeny

A total of 21 excised bands from bacterial DGGE profiles were sequenced to allow their phylogenetic affiliation. The levels of 16S rRNA sequence identity to the most closely related Bacteria are reported in Table 3. None of the bacterial bands was unique to one of the three studied lakes, indicating that common phylotypes were present in all of them, even if different arrangements in bacterial aggregation were observed. After BLAST search, only sequences from five bands (2, 5, 6, 7 and 12) showed high similarity (≥97%) with sequences deposited in the database. Almost all sequences were related to Proteobacteria, mainly consisting of the Beta- (bands 1–5, 7–9, 11–13, 17, 20, and 21), Alpha- (bands 6, 16, 18, and 19), and Gamma- (bands 14 and 15) proteobacterial subclasses. The only sequence related to Firmicutes (band 10) was dominant in all sediments, but occurred also in waters from Témpanos and Las Torres. Sequences belonging to Betaproteobacteria dominated in all fingerprints, but differed in their relative occurrence in sediment and in water samples. Janthinobacterium sp. (band 2) was exclusively retrieved in sediments. Some sequences (bands 1, 3, 9, 17, and 21) were more frequently found in sediments, while others (bands 4, 5, and 13) in waters, and still others (bands 7, 8, 9, 11, 12, and 20) were common to water and sediment samples. The sequence related to Sphingopyxis alaskensis (band 6) (Alphaproteobacteria) was retrieved more frequently in water than in sediment samples, while sequences referred to the two different species of Methylobacterium (bands 16 and 19) were more frequent in sediments. Sequences from bands 14 and 15 were assigned to two different species of Pseudomonas (Gammaproteobacteria), which were found more frequently in sediments than in waters.

Table 3 Blast results of bacterial sequences derived from excised DGGE bands from water and sediment samples, in winter and summer periods, of Las Torres (LT), Mercedes (M), and Témpanos (T) lakes
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Archaeal DGGE Analyses

Archaeal DGGE profiles in water and sediment samples collected in winter and summer and the analysis of the archaeal community structure are reported in Fig. 3. A total number of 90 archaeal phylotypes was observed, ranging from 4 to 14 per sample (Fig. 3a). Archaeal richness was slightly higher in sediments (49) than in waters (41), and in summer (53) rather than in winter (37). Based on the presence and absence of bands in each lane, S s were calculated for the DGGE profiles. Archaeal populations from sediments were more similar to each other (12.5–80% S s) than those from waters (14.3–61.5% S s). Sediments collected from Mercedes and Las Torres in winter were more similar to each other (Fig. 3b).

Fig. 3
figure 3

Archaeal DGGE profiles obtained from water and sediment samples (a), and nonmetric multidimensional scaling diagrams (b) representing the archaeal community structure in Las Torres (LT), Mercedes (M), and Témpanos (T) lakes, in winter (w) and summer (s) periods. Sequenced bands are indicated by numbers

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Archaeal Phylogeny

A total of 16 DGGE bands were sequenced. Most sequences (12/16) were assigned to uncultured deposited clones and the remaining four could not be affiliated with any archaeal phyla (Table 4). From the twelve sequences, nine of them were related to Euryarchaeota and the remaining three to Crenarchaeota. Euryarchaeotal sequences were mainly referred to methanogens within the order of Methanosarcinales, including members of the families Methanosaetaceae (bands 2 and 6) and Methanosarcinaceae (bands 5 and 13). Sequences obtained from bands 2 and 6 were affiliated with two different Methanosaeta clones. Band 2 was mainly retrieved in sediments from Las Torres and Mercedes, in both seasonal periods. Band 6 was present only in waters from Las Torres and Mercedes in winter, while in Témpanos was present in both water and sediment samples, only in summer. The sequence from band 5, affiliated with a Methanosarcina clone, was retrieved in water samples from Las Torres and Mercedes in both seasons, while that from band 13, related to a Methanosarcina clone, was only found in sediments from Las Torres in winter. Band 14, referred to Thermoplasmata, was retrieved only from sediments collected from Las Torres in summer. Among Crenarchaeota, band 1 was common to all samples, while bands 10 and 11 were retrieved in sediments from all lakes, but in different periods.

Table 4 Blast results of archaeal sequences derived from excised DGGE bands from water and sediment samples, in winter and summer periods, of Las Torres (LT), Mercedes (M), and Témpanos (T) lakes
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Comparison of Prokaryotic Communities

Diversity indices (R, H’, and, E), as derived by DGGE profiles, from the same lake (alpha diversity) and among lakes (beta diversity) in water and sediments, are reported in Fig. 4. Bacterial H’ slightly varied among samples (from 2.3 to 2.9) and was higher in samples collected in summer than in winter. Bacteria from Las Torres showed the smallest R and H’ values, while the highest were observed in samples collected from Témpanos. Bacterial R and H’ were positively correlated (P < 0.01) in waters, while no correlation was observed in sediments. Overall, bacterial E slightly differed among samples (ranging from 0.80 to 0.87), indicating that bacterial populations were homogenously distributed in each sample.

Fig. 4
figure 4

Comparison of Bacteria and Archaea diversity indices (R, H’, and E) in water and sediment samples collected from Las Torres (LT), Mercedes (M), and Témpanos (T) lakes in winter (w) and summer (s) periods

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Conversely to Bacteria, the smallest archaeal R index was present in water samples collected in winter at Témpanos, and the highest at Las Torres, and Mercedes in summer. H’ greatly varied among samples (from 1.35 to 2.52), being higher in sediments collected in summer. Differently to Bacteria, archaeal R and H’ were positively correlated (P < 0.05) in sediments, but not in waters. Archaeal evenness (E) showed greater variations in water (from 0.77 to 0.97) than in sediment (from 0.82 to 0.91) samples, and it was positively correlated to richness (R) (P < 0.05) in sediments but not in waters.

When the relative abundances of dominant bacterial phylotypes were analyzed (Fig. 5), water samples from Las Torres and Témpanos lakes collected in winter showed the highest diversities. Alphaproteobacteria were more abundant in Mercedes. Phylotypes of Euryarchaeota were more abundant in water of Las Torres in winter, whereas those of Crenarchaeota were prevalent in sediment samples from Témpanos lake.

Fig. 5
figure 5

Relative abundance of dominant bacterial and archaeal phylotypes retrieved by DGGE in samples collected from Las Torres, Mercedes, and Témpanos lakes

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PCA analyses, based on the physical and chemical properties of water and sediment samples, the cell abundance of Bacteria and Archaea, their diversity indices, and main phylotypes, were performed to identify groups of samples with similar community composition and to find their relationships with environmental variables (Fig. 6). The two main components explained 66.4% of the total variance in water samples (Fig. 6a), and 69.1% in sediment samples (Fig. 6b).

Fig. 6
figure 6

Principal component analyses, based on physico-chemical variables (pH, TOC, TP, and TN), the relative cell abundance of Bacteria (Bac) and Archaea (Arch), their diversity indices (R, H’ and E), and most significant phylotypes in water (a) and sediment (b) samples collected from the three Chilean Patagonia lakes, recorded in winter (w, in blue) and summer (s, in red). Las Torres (LT), Mercedes (M), Témpanos (T), Bacterial phyla/classes: Alphaproteobacteria (Alpha), Betaproteobacteria (Beta), Gammaproteobacteria (Gamma), Firmicutes (Firmi). Archaeal phyla/genera: Euryarchaeota (Eury), Crenarchaeota (Cren), Methanosacina (Methsarcina), and Methanosaeta (Methsaeta)

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In water samples, Axis 1 (explaining 34.7% of the variance) was strongly associated in the positive side with pH, Euryarchaeota and Methanosaeta, whereas it was related to all bacterial diversity indices (H’, R, and E) in the negative side. Axis 2 (31.7% of the variance) was mainly associated with archaeal richness (RA) and diversity (H’A) in the positive side, while with TOC, Alphaproteobacteria and Firmicutes in the negative side.

In sediments (Fig. 6b), Axis 1 (36.6% of the variance) was strongly associated with archaeal abundance (Arch) and richness (RA) in the positive side, whereas with bacterial abundance (Bac) in the negative side. Instead, Axis 2 (33.1%) was strongly related to a combination of all environmental variables (TOC, TN, and TP) in the positive quadrant and to Firmicutes and Betaproteobacteria in the negative side. Sediments collected in winter from the three lakes appeared completely separated from those collected in summer by Axis 2, suggesting a strong influence of seasonality on sediment rather than on water samples.

Témpanos and Mercedes lakes, with the highest negative scores for Axis 1, appeared more closely clustered in summer than in winter, mainly due to the bacterial community features in waters (Fig. 6a), and to those of archaeal community in sediments (Fig. 6b). Although distantly, in winter Las Torres and Mercedes lakes appeared clearly separated from Témpanos lake by Axis 2 (Fig. 6a) in waters, and Axis 1 (Fig. 6b) in sediments.

PCA clusters appeared quite different to those occurred in the NMDS plots (Figs. 2, 3), indicating that a combination of environmental factors and limnological characteristics shapes the prokaryotic abundance and diversity in the three lakes.

Discussion

The cold, oligotrophic Chilean Patagonia lakes are located in land-protected areas and some of them were previously described as pristine environments [3, 8, 31]. The absence of polychlorinated biphenyls and organochlorine pesticides in water and sediment samples collected from the studied lakes suggests low human activity [3, 6, 31, 50]. Although the low amounts of nutrients were indicative of oligotrophic environments, all lakes showed an increase in TOC in winter, as consequence of abundant rainfalls providing terrestrial inputs of organic matter [47]. However, anthropogenic activities in this area could expose them to the risk of altering their natural conditions.

Due to the different lake locations, and surrounding vegetation, the limnological properties appeared different in the three lakes. Lake Témpanos, located most northerly and closer to a glacier, showed less variation in temperatures recorded in winter and in summer than those from the other two lakes. Similarly to Témpanos, Mercedes lake, the smallest of the three studied, is partially covered by ice in winter. As a consequence of volcanic activity and the geologic composition of the area, pH values registered in the three lakes were always acidic [3]. Chaiten and Hudson are active volcanos separated by approximately 340 km, and the three lakes considered in this study are located between both volcanos. Besoain et al. [7] indicated that the eruptive activity of Hudson volcano in 1991 deposited 2500 kg/ha of sulfured volcanic ashes and the pH of affected waters decreased. Gammons et al. [20] reported that the hyper-acidic (pH 2.68) conditions of Caviahue lake, located in the northern Argentinean Patagonia (37.5°S, 71°W), are due to the presence of ions and rare earth elements incorporated by the eruptive activity of the Copahue volcano.

Prokaryotic abundances, dominated numerically by Bacteria, were quite similar in the three studied lakes, being higher in sediment than in water samples, and in summer than in winter. These results were similar to those of other cold oligotrophic lakes, also including those from Patagonia [2, 3, 25, 31, 44]. Although Archaea represent a minor component, their percentage contribution to the total prokaryotic community was highest in water samples collected in winter, when lacustrine surface were partially covered by a thin layer of ice. Recent investigations in Argentinean Patagonia lakes also reported that relative abundances of Bacteria and Archaea showed a marked seasonality, with peaks of Bacteria in summer and those of Archaea in winter [44].

To compare the prokaryotic communities from the three lakes, we also analyzed the intrinsic genetic diversity by the fingerprinting method based on PCR-DGGE of 16S rRNA, which reveals prominent populations associated with different samples and sampling dates. Moreover, sequencing analysis of the most prominent bands was used to assess their potential affiliation to other cultured and uncultured prokaryotes from other environments. Although there were not many differences in the DGGE number of bands between water and sediment samples from the three lakes, a higher richness (numbers of bands) was found in sediments than in waters, and in summer than in winter samples, for both Bacteria and Archaea. Bacterial communities consisted of more numerous and more diverse populations with respect to those of Archaea. As indicated by the index Shannon H’, bacterial diversity showed a large variation among the examined samples from the three lakes (Fig. 4), and it was higher in summer than in winter. Bacterial evenness slightly differed among samples, indicating that bacterial populations were homogenously distributed in each sample. Interestingly, evenness of Archaea was greater than that of Bacteria, with higher values in water rather than in sediment samples.

As resulted by phylogenetic analysis, the closest relatives associated with Proteobacteria, mainly consisting of Beta-, Alpha-, and Gammaproteobacteria, and Firmicutes, were chemoorganotrophs or aerobic heterotrophs. These putative metabolisms were highly dependent on organic matter, which explains the relationship between TOC with bacterial community composition (Fig. 6).

Even if Proteobacteria was the largest phylum, the major subclasses and their proportions varied among the different lakes. Βetaproteobacteria constituted the most prominent bacterial group in the prokaryotic communities of the three Patagonian lakes. Similar results have been reported in different cold oligotrophic polar environments [22, 38], as well as in small oligotrophic mountain lakes [24, 53]. Among Betaproteobacteria, the genus Herminiimonas was often found in cold aquatic ecosystems, including the species H. glaciei able to live in ice, as miniaturized cells for extended periods of time [30]. Since they may compete effectively in their habitat, the capacity of certain psychrophiles to grow at higher temperature might be ecologically relevant. This may be the case of sequences related to Janthinobacterium sp., often retrieved in different environments, even if several species have been isolated from cold Antarctic habitats [49]. Similarly, the eurypsychrophilic alphaproteobacterium Sphingopyxis alaskensis, isolated from cold waters (4–10 °C) at the North Pacific Ocean near Alaska and Japan, is able to grow faster at higher temperatures [15]. Also, Sphingopyxis chilensis, isolated from the Bio Bio river in the central Chile, is able to grow at 30 °C [21].

Members of the genus Methylobacterium (Alphaproteobacteria) are able to grow on single-carbon compounds, such as CH4 or CH3OH, as a sole carbon and energy source [34], and their presence in the studied lakes suggests the production of organic matter from simple carbon compounds. Although Actinobacteria are recognized as ubiquitous and dominant in oligotrophic lakes [36], sequences related to this phylum were not detected in the samples from any of the three Patagonian lakes here studied. In fact, Aguayo et al. [3] suggested that an increase of Actinobacteria in Patagonian lakes could be indicative of alteration in the lacustrine homeostasis.

Phylogenetic information on archaeal communities in cold freshwater lakes is still very limited. Most of the archaeal sequences were affiliated with Euryarchaeota and the remaining with Crenarchaeota, as also reported in samples obtained from high-altitude lakes [38]. A large number of sequences could not be attributed to the phylum level, suggesting the presence of new phylotypes in these lakes. Overall, diversity indices for the water archaeal community appeared influenced by pH and TN. The highest archaeal richness and diversity were found at Mercedes and Las Torres lakes, where high TN concentrations were recorded in both seasons. Seasonal correlation between nitrogen compounds and Archaea were previously reported in the surface waters of Spanish alpine lakes [5], suggesting therefore a pivotal role of Archaea also in the N biogeochemistry of Chilean oligotrophic lakes.

Euryarchaeotal sequences were mainly related to methanogens belonging to the order Methanosarcinales, including the genera Methanosarcina and Methanosaeta, which catalyze the terminal phase of the degradation of organic matter in anoxic environments [27], but also in oxic lake waters [9]. These methanogens, retrieved as predominant in sediment samples from Las Torres and Mercedes, have been frequently observed in superficial zone of freshwater sediments [10, 16, 28, 46, 52], and also from different cold habitats, such as basal ice layers of glaciers and Antarctic lakes [14, 19]. Thermoplasmata, usually found as a minor group in freshwater lake sediments [29], were retrieved in this study only from sediments of Las Torres, suggesting their potential role in the methanogenic activities.

The prokaryotic community from water and sediment samples from the three Chilean Patagonian lakes appeared different in winter and summer, demonstrating that a close relationship exists among microbial community composition and the limnology of the lakes. Bacteria and Archaea could play different biogeochemical roles in waters and sediments in response to seasonal changes.

The prokaryotic community composition from Témpanos lake, located most northerly and closer to a glacier, greatly differed with respect to the other two lakes. The highest bacterial diversity was detected in this lake, mainly related to betaproteobacterial phylotypes, whereas Alphaproteobacteria were distinctive of Mercedes lake. Archaeal diversity was mainly related to euryarchaeotal methanogens, affiliated with members of the order Methanosarcinales, in samples from Mercedes and Las Torres, and to Crenarchaeota from Témpanos. These findings indicate that the different site location, the proximity to the glacier, and the seasonal availability of nutrients shape the composition of the prokaryotic communities in the three cold, oligotrophic Chilean Patagonian lakes. The absence of pesticides was considered as indicative of low human influences. As micro-scale indicators, the prokaryotic community characteristics registered at the present conditions can be used to follow the microbial responses to potential global changes and anthropogenic impacts.