Experimental design

Three lagoons located in the Yungay region of the hyperarid core of the Atacama Desert were sampled on November 11, 2017 (coordinates detailed in Table 2). Water samples were taken with sterile gloves and sterile 50 ml falcon tubes, and kept at room temperature for further processing. For all experiments, at least triplicates were analyzed, and in most cases up to 10 samples per lagoon were analyzed.

Ion Chromatography

Water samples were loaded into a Metrohm 861 Advanced compact ion chromatographer IC (Metrohm AG, Herisau, Switzerland) by an automatic loader, undiluted or at different dilution values, depending on the expected ion concentration. Samples were diluted in IC-grade water (Sigma Aldrich). For all anions, the column Metrosep A supp 7–250 was used with 3.6 mM sodium carbonate (NaCO 3 ) as eluent. Each sample was measured three times, and each measurement at a different dilution, to take the values that best fitted the calibration curve. The measurement error of the equipment for replicate samples was less than 1%. The instrument was calibrated with a multi-anionic solution with 6-point concentrations curve for each anion and detection limits at few ppb level for all of them.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Quantitative analysis of Mn, Cu, Co, Cd, Ba, Na, Mg, K, Ca, Fe, Ni, Zn, and As were performed using a PerkinElmer NexION 2000 ICP-MS (PerkinElmer Inc.) using the conditions shown in Table S2. A semiquantitative analysis using 47 elements as external standards, detailed in Table S3, was previously performed to determine concentration levels prior to quantification. The quadrupole cell allows the adjustment of ion transmission of any isotope without affecting other masses. This capability was applied to Na and K, since both elements have low ionization potentials, high isotopic abundances and are present at high levels in the water samples analyzed. The electronic dilutions conditions are detailed in Table S4. The instrument was tuned to maximum sensitivity and the lowest background, oxides and double charge ion prior to the analysis using a solution containing 1 μg/L Be, Ce, Fe, In, Li, Mg, Pb and U. Samples were taken up by an ASX-500 CETAC Autosampler and on-line addition of internal standard. Helium (99.9999%) was used as collision gas (KED mode) to remove possible polyatomic interferences. Gas conditions are detailed in Table S5. External calibration involved eight solutions from 1 to 5000 ppb, prepared diluting 1000 ppm monoelemental standard solution (SCP Science) in volumetric flask in 1% nitric solution prepared with high purity MilliQ system water and Suprapur grade nitric acid (Merck). All external calibration equations achieved a minimum correlation coefficient of 0.999. Solutions were prepared by diluting 0.02 ml of each lagoon sample to 10 ml with maximum accuracy. A Quality control standard containing 100 ppb of all elements was measured at the end, to control the standard recovery and equipment deviation.

X-Ray Diffraction

In order to characterize the mineralogical composition of the lagoons, water samples from all lagoons were inspected by X-Ray Diffraction, using a Bruker D8 Eco Advance with Cu Kα radiation (λ = 1.542 Å) and Lynxeye XE-T linear detector. The X-ray generator was set to an acceleration voltage of 40 kV and a filament emission of 25 mA. Samples were scanned between 5° (2θ) and 70° (2θ) using a step size of 0.1° (2θ) and a count time of 1 s, using the Bragg–Brentano geometry. The analysis of the XRD diffraction spectra (diffractograms) of powdered crystals obtained from the evaporation of 5 ml of the large and medium sized lagoons was performed with the DIFFRAC.EVA software (Bruker AXS).

GC-MS Analysis

The three lipidic fractions (non-polar, acid and polar fraction) present in water samples of the lagoons were analyzed by gas chromatography mass spectrometry using a 6850 GC system coupled to a 5975 VL MSD with a triple axis detector (Agilent Technologies), operating with electron ionization at 70 eV and scanning from m/z 50 to 650. The analytes were injected (1 μl) and separated on a HP-5MS column (30 m × 0.25 mm i.d. ×0.25 um film thickness) using He as a carrier gas at 1.1 ml min−1. For the non-polar fraction, the oven temperature was programmed to increase from 50 C to 130 °C at a rate of 20 °C/min, then to 300 °C at 6 °C/min (held 20 min). For the acid fraction the oven temperature was programmed from 70 °C to 130 °C, at 20 °C/min; and to 300 °C at 10 °C/min (held 10 min). For the polar fraction, the oven temperature program was the same as for the acid fraction, but the oven was held for 15 min at 300 °C. Injector temperature was 290 °C, transfer line 300 °C and MS source at 240 °C. Compound identification was based on the comparison of mass spectra and/or reference compounds, and compounds were quantified using external calibration curves. External standards of n-alkanes (C 10 to C 40 ), FAMEs (C 8 to C 24 ), alcohols (C 10 , C 14 , C 18 , C 20 ) and branched isoprenoids (2,6,10-trimethyl-docosane, crocetane, pristane, phytane, squalane and squalene) were injected to obtain calibration curves. Recoveries of the internal standards averaged 75 ± 15%.

Illumina NGS-Based 16S rRNA Sequencing

DNA extraction: Samples from the lagoons were centrifuged for 5 min at 13000 rpm, and the supernatants carefully discarded in order to collect the resultant pellets. DNA was extracted from these pellets using the DNeasy PowerSoil Kit according the manufacturer instructions, except that at the cell lysis step, one pulse of 2 minutes was used in a FastPrep-24 5 G homogenizer (MP Biomedicals), to better preserve DNA integrity.

Purified DNAs were then amplified in a first PCR of 30 cycles with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs) in the presence of 100 nM primers for 16S amplification (5′-ACACTGACGACATGGTTCTACACCTACGGGNGGCWGCAG-3′ and 5′-TACGGTAGCAGAGACTTGGTCTGACTACHVGGGTATCTAATCC-3′, these primers amplify the V3-V4 region of 16S). After the first PCR, a second PCR of 15 cycles was performed with Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs) in the presence of 400 nM of primers 5′-AATGATACGGCGACCACCGAGATCTACACTGACGACATGGTTCTACA-3′ and 5′-CAAGCAGAAGACGGCATACGAGAT-[10 nucleotides barcode]-TACGGTAGCAGAGACTTGGTCT-3′) of the Access Array Barcode Library for Illumina Sequencers (Fluidigm).

The obtained amplicons were validated and quantified by a Bioanalyzer, and an equimolecular pool was purified using AMPure beads and titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit for Light Cycler 480” and a reference standard for quantification. The pool of amplicons was denatured prior to be seeded on a flowcell at a density of 10pM, where clusters were formed and sequenced using a “MiSeq Reagent Nano Kit v2”, in a 2 × 250 pair-end sequencing run on a MiSeq sequencer”.

The obtained raw sequences were processed in MOTHUR software v.1.40.0 (ref.43), using a custom script based upon MiSeq SOP (ref.44). Sequence reads were clustered into OTUs (Operational Taxonomic Units) at the 97% similarity level. Datasets were rarefied independently by random selection to even sequencing depth, corresponding to the lesser number of sequences found in the samples (60673 reads). Taxonomic affinities for the reads were assigned by comparison of OTUs representative sequences against RDP (RDP reference files v.16; release 11 (ref.45)) and against nr/nt (NBCI) databases. OTU’s affinities reported as ‘cyanobacteria/chloroplast’ were further assigned a taxonomic identity by comparing them against EMBL, Greengenes and SILVA databases for identification. Sequences assigned to mitochondria or chloroplasts were removed from further analyses as contaminants, as fluorescence microscopy did not detect chlorophyll autofluorescence in any of the sampled lagoons.

Cultivation and identification of isolates

One ml of each lagoon was aerobically incubated at room temperature (~25 °C) in Petri dishes containing agar and three different growing media: Luria Broth (Sigma), Nutrient agar (Pronadisa) and Marine Media (Conda). Growth was followed during 2 weeks.

DNA extraction from isolates

DNA was extracted as detailed for Illumina NGS-Based 16S rRNA Sequencing.

ERIC-PCR fingerprinting

This technique was used in order to detect the number of unique isolates from all the colonies that grew in marine media. ERIC-PCR uses specific primers that amplify ERIC (Entero-Bacterial Repetitive Intergenic Consensus) sequences, giving as a result a number of bands of different sizes that is unique for each bacterial species. ERIC PCR was first used to characterize enteric species46, but was subsequently found to be useful for other types of bacteria too21. DNA was amplified using the GoTaq Green Master Mix (Promega), using the primers ERIC2 5′AAGTAAGTGACTGGGGTGAGCG3′ and ERIC1R 5′-ATGTAAGCTCCTGGGGATTCAC-3′. PCR conditions used were: 95 °C for 2 min, 92 °C for 30 s, and 35 cycles of (92 °C for 30 s, 48 °C for 80 s, and 65 °C for 108 s), followed by 68 °C for 8 min. The resultant reaction was visualized in a 2% agarose gel at 50 V. Based on the number and molecular weight of bands of the isolates observed in 2% TAE agarose gels, only a single isolate was detected.

16S rRNA amplification and sequencing of distinct isolates

16S rRNA of isolates was amplified using the GoTaq Green Master Mix (Promega) and the primers Bac8f AGAGTTTGATCATGGCTCAG and UN1541 AAGGAGGTGATCCAACC. PCR conditions used were: 95 °C for 5 min, and 25 cycles of (95 °C for 40 s, 55 °C for 2 min, 72 °C for 1 min) followed by 72 °C for 7 min. The resultant reaction was visualized in a 2% agarose TAE gel at 50 V.

The automated sequencing of the resulting PCR products was conducted by Macrogen DNA Sequencing Inc. (Seoul, Korea).

Isolates Phylogeny

Closest species of the isolate obtained was determined by analyzing the 16S rRNA gene sequences obtained using the Megablast option for highly similar sequences of the BLASTN algorithm against the National Centre for Biotechnology Information nonredundant database (www.ncbi.nlm.nih.gov).

Phylogenetic analysis of 16S rRNA gene sequences were aligned by multiple sequence comparison by log-expectation (MUSCLE)47, analyzed with jModelTest48 and then by Phylip NJ49, all tools of the BOSQUE phylogenetic analysis software50, as similarly performed in previous works5,21.

Transmission Electron Microscopy

As the objective was to examine the aspect of the species detected by 16S rRNA, we analyzed samples from the small and large lagoons, because we knew that all four species found were present in the large lagoon. Therefore, for convenience, we examined the large and small lagoon samples only, knowing that the medium sized lagoon samples were an intermediate situation.

For negative staining, a solution of sodium phosphotungstate (Sigma, ref, P-6395) was employed. 200 mesh copper grids covered with formvar and reinforced with carbon were used. Cultures were centrifuged and washed twice with ammonium acetate, pH 7, 0,1 M. Pellets were resuspended in ammonium acetate solution, until the concentration of cells was adequate to produce no excess material at the time of observation. Sodium phosphotungstate salt was prepared at 1% (dry wt/vol), and its pH adjusted to 7 with NaOH. A volume of cells were mixed with the sodium phosphotungstate solution. Small drops of this mixture were placed on Parafilm and the grids were floated on it for 5 minutes. Then, the remaining material in the grids was dried, and the grids were placed for five additional minutes on a drop of distilled water. Finally, grids were removed, allowed to dry and observed by Transmission Electron Microscopy (TEM) (JEOL, JEM-2100 instrument with a LaB6 filament, operating at 200 kV acceleration potential).

The characterization of different bacterial morphotypes was achieved by the determination of distinct and different micromorphologies, as detailed in ref.51. Briefly, the four detected morphotypes were found by examining all TEM micrographs obtained and inspecting for unique morphologies (i.e., size, shape, presence or absence of flagella, number of flagella, position of insertion of the flagella, presence/absence of electron-dense bodies and its location). In order to confirm these findings, we then compared these morphotypes with the morphologies reported for the species identified by 16 rRNA, finding a coincidence for all four cases.

It is unlikely that cyanobacteria could have not been observed due to 16S primer region mismatches and in addition preferentially lost during the microscopy sample preparation procedure, because no cyanobacteria were observed after a detailed inspection by bright field microscopy of water samples taken directly from the analyzed lagoons. They were not observed by TEM and they did not appear in the molecular data, so we are confident on this negative result.

Lipid Extraction, Fractionation and Analysis

Water samples were filtered through a GFF pre-cleaned filter, then extracted with a mixture of dichloromethane/methanol (DCM/MeOH, 3:1, v/v) with an ultrasound apparatus (3 × 30 min cycles at room temperature). Internal standards (tetracosane-D 50 , myristic acid D 27 , 2-hexadecanol) were added prior to extraction. Total lipids extracts were concentrated using rotary evaporation to 2 ml. After this step, activated Cu was added and left overnight for elemental sulfur removal. The extracted sample was separated in three fractions using a Bond-elute column chromatography (Bond phase NH 2 , 500 mg, 40 μm particle size). The neutral lipid fraction was obtained by eluting with 15 ml DCM/2-propanol (2:1, v/v), the acid fraction with 15 ml of acetic acid (2%) in diethyl ether, and the phospholipid fraction with 15 ml of methanol. Further separation of the neutral lipid fraction was completed using 0.5 g of alumina in a Pasteur pipe. The non-polar fraction was obtained by eluting 4.5 ml of hexane/DCM (9:1, v/v), and the polar fraction with 3 ml of DCM/methanol (1:1, v/v). The acid fraction was derivatized with BF 3 in methanol, and the polar fraction with N,O-bis (trimethylsilyl) trifuoroacetamide (BSTFA).

Fluorescence Sandwich Microarray Immunoassay with a Life Detector Chip (LDChip)

A LDChip containing 200 antibodies to crude lysates of bacterial and archaeal strains, as well as to key proteins and peptides from different universal metabolisms as nitrogen and carbon fixation, iron metabolism (oxidation, reduction, storage), sulfur oxidation, or methanogenesis, was used to detect and profile microbial markers in the three studied lagoons. The water samples were processed and analyzed by multiplex sandwich microarray immunoassay with LDChip as described previously27. The anti-cyanobacterial antibodies are specific52. A confirmation of this result is that the amplification of 16S rRNA with cyanobacteria-specific primer and the microscopy analysis of the samples in search of cyanobacteria turned both negative.