Sediments from the Laguna Lejía terrace revealed a saline, sulfur-rich environment with large-scale water level fluctuations between ca. 10.3 and 11 kyr BP
The geochemistry, mineralogy, and radiocarbon dating of the Laguna Lejía terrace allowed the reconstruction of the hydrogeochemical history of the lake over the last millennia. Conventional 14C ages (i.e., raw ages) of the sedimentary organic matter were similar along the 1 m high terrace profile, with the middle layer (C; ~22,000 year BP) being older than the upper (F; ~21,500 year BP) and the lower (A; ~21,000 year). These relatively high radiocarbon ages and their inversion with depth suggest a reservoir effect that could overestimate the actual age of the sediments. Conventional radiocarbon ages of another 4 m high terrace from Laguna Lejía were lower (11,700 − 15,490 year BP) than those of our samples, but showed a similar age inversion with depth (Grosjean, 1994; Grosjean et al., 1995; Geyh et al., 1999) consistent with changes in the degree of a reservoir effect as lake water level fluctuated (Geyh et al., 1999). Based on the location of our 1 m high profile relative to the 4 m high of the total terrace (Grosjean, 1994; Geyh et al., 1999), our sediment samples correspond to the lithologic units II and III and should have 14C-corrected ages between 10,300 and 11,000 year BP (Figure S1). Given this chronostratigraphic relationship, we applied a reservoir effect correction of ~ 9,900 − 12,300 years, resulting in 14C-corrected ages of ~ 11,000 year BP for sample A (bottom), ~ 10,650 year BP for sample C (middle), and ~ 10,300 year BP for sample F (top). If the hypothesis of the reservoir effect is the factor explaining the relatively high ages and their inversion with depth, then the section of the terrace studied here correspond to a time interval of ca. 700 years.
The geochemistry of the Laguna Lejía terrace provided evidence for a sulfur-rich saline environment influenced by the volcanic activity and climatic conditions of the region. The Laguna Lejía is located in a volcanic area near the Lascar volcano (Cabrol et al., 2009), which is currently active (Gaete et al., 2020) and emits high concentrations of SO2, HCl, H2S and HF (Tassi et al., 2009). The low precipitation (< 200 mm year− 1) and high evaporation rates (1,500 mm year− 1) of the region (Grosjean et al., 1995; Risacher et al., 1999) favour the evaporation of the water and, consequently, the concentration of salts. For instance, the most abundant elements and the pH in the Laguna Lejía terrace are consistent with the lake water chemistry reported in Grosjean et al. (1995) (pH of 8.7 and high concentrations of SO42− (28 g L− 1), Cl− (9.9 g L− 1), Mg2+ (5.9 g L− 1), and Na+ (1.6 g L− 1)). In addition, metals and metalloid elements detected in different concentrations in the terrace (i.e., Li, B, F, and As) can also be explained as a result of the volcanic activity or weathering of extensive volcanic rocks (Kurth et al., 2017; Saona-Acuña et al., 2020; Álvarez-Amado et al., 2022).
Differences in the concentration of elements, mineralogy, lipid biomarkers, TOC and TN along the Lejía terrace profile suggested large-scale water level fluctuations over the ~ 700 years. Samples A, C, E, and F had a relatively higher proportion of magnesium calcite and halite than B and D (Fig. 2a). This mineralogical composition in A, C, E, and F coincided with a higher concentration of elements (e.g., Cl, Na, SO42−-S and Mg), lipid biomarkers, TOC, and TN than in B and D (Fig. 2b), suggesting periods of desiccation (A, C, and E-F) and water recharge events (B and D). As water evaporates, chemical species (Cl−, Na+, Mg+ 2, CO32−, etc.) and organic matter (i.e., TOC and lipid biomarkers) tend to concentrate in the water column of the lake and accumulate in the sediments. In contrast, lake water recharge dilutes inorganic and organic material in the water column and accumulates relatively less in the sediments. Interestingly, Geyh et al. (1999) and Grosjean et al. (1995) described three large-scale water level fluctuations (25 m to 7 m) in the Laguna Lejía between 10,300 and 11,000 year BP. According to our geochemical and mineralogical results, five periods of alternating desiccation (A, C, and E-F) and water recharge events (B and F) might have occurred in the Laguna Lejía during these ~ 700 years.
Laguna Lejía terrace preserves aquatic/terrestrial plant remnants and harbors metabolically active microorganisms
The higher proportion of polar and acidic lipid families over the non-polar ones in all sediment layers suggested a well-preserved biomass in the terrace. The n-alkanes are the most resistant of the three lipid families to chemical alteration over time (Brocks and Summons, 2003), whereas n-fatty acids and n-alkanols contain functional groups (i.e., carboxylic and hydroxyl, respectively) that are more prone to decay. The higher relative abundance of functionalized (n-fatty acids and n-alkanols) over non-functionalized (n-alkanes) hydrocarbons in all sediment layers (Fig. 3c) suggested good preservation of lipids in the Laguna Lejía terrace.
The molecular distribution of the major lipid families (i.e., n-alkanes, n-fatty acids, n-alkanols and sterols) revealed preservation of ancient biomass from aquatic and terrestrial sources. Despite the taxonomic specificity of lipid biomarkers is limited (i.e., they can only be assigned to a general group of organisms), their stability in the geological record for billions of years (Brocks and Pearson, 2005; Vinnichenko et al., 2020) makes them relevant biomolecules for paleobiological reconstructions (Sánchez-García et al., 2021; Lezcano et al., 2022). The identification of lipid biomarkers attributed to aquatic macrophytes (C21 n-alkane; Figure S2) (Ficken et al., 2000) and terrestrial plants (C27 n-alkane, C24 n-fatty acid, and/or C22 n-alkanols; Figure S3 and S4) (Eglinton and Hamilton, 1967) suggested the presence of ancient plant biomass inhabiting the lake or its surroundings when the water level was higher. The presence of phytosterols (campesterol, stigmasterol, and β-sitosterol) from mostly vascular plants (Goad and Akihisa, 1997) in all samples also supported this notion and is consistent with the vegetation associated to the Laguna Lejía (Muñoz-Pedreros et al., 2018).
A prominent peak at C16 within the n-fatty acids distribution also hinted towards a contribution from bacteria in the terrace (Lezcano et al., 2022; Megevand et al., 2022). This bacterial community in the Laguna Lejía terrace was presumably active at the time of collection according to in silico growth predictions of MAGs (average iRep values between 1.3–1.4 in samples A-D and F, and 2.4 in sample E) (Figs. 7 and 8). The replication index is calculated based on the DNA sequence coverage at the origin over the terminus of replication (Brown et al., 2017). Therefore, iRep values greater than 1 indicate replication of genomes, and values greater than 2 may point out multiple replication forks. Certain bacterial MAGs have values close to 1 (e.g., 1.09 in sample C), suggesting slow replication rates. By contrast, the high iRep values in some of the MAGs, especially in sample E (i.e., up to 4.7 in E) (Fig. 8), may be explained by the relatively high concentration of key nutrients in this sediment layer, such as SO42−-S or NO3−-N, that may promote bacterial growth.
The possibility that a fraction of the microbial biomass (DNA and lipids) detected in the Lejía terrace was ancient may not be ruled out, as ancient samples tens to billion years old usually contain traces of DNA, proteins and lipids of microbial origin (Lezcano et al., 2019), especially if they are well insulated from UV radiation and high temperatures (Willerslev and Cooper, 2005), as it the case of the Laguna Lejía terrace.
Desiccation of the Laguna Lejía provided new ecological niche opportunities for microorganisms
The prokaryotic community composition of the Laguna Lejía terrace differed from that reported in the water and wet sediments of the lake in previous studies, suggesting that lake desiccation resulted into a new ecological niche for microorganisms adapted to drier conditions. The bacterial community of the wet sediments showed Bacteroidota, Firmicutes, Spirochaeota and Proteobacteria as the most abundant phyla (Demergasso et al., 2010; Mandakovic et al., 2018b), while the dried terrace was dominated by Actinobacteriota, Proteobacteria, Chloroflexota, and the Candidate Phylum Patescibacteria (or CPR) (Fig. 4a). Although Actinobacteriota is a common phylum in aquatic and terrestrial habitats worldwide, its dominance in the terrace (23–40%) compared to the wet sediments (~ 2.5%) (Mandakovic et al., 2018b), and its common presence in soils and rocks of the Atacama Desert (Idris et al., 2017; Meslier et al., 2018; Sánchez-García et al., 2023), supports a microbial community shift likely driven by extended periods of drought during water level fluctuations.
In contrast to Actinobacteriota, the high proportion of CPR bacteria found in the Lejía terrace (up to 15%) has not been reported in Atacama sediments or rocks, but detected in smaller relative abundances in Lomas Bayas, María Elena and Yungay (Schulze-Makuch et al., 2018), and in the Salar de Llamara (Finstad et al., 2017; Rasuk et al., 2020). The relatively high proportion of CPR, and the presence of Nanoarchaeota (DPANN superphylum) in the Laguna Lejía terrace suggested extensive microbial interactions, potentially even between bacterial and archaeal communities (Kuroda et al., 2022). The CPR bacteria are a group of ultra-small, genome-reduced bacteria with host-associated lifestyles (Chaudhari et al., 2021), either parasitic (Moreira et al., 2021) or symbiotic (Nelson and Stegen, 2015), to meet their metabolic requirements (Luef et al., 2015). Similarly, Nanoarchaeota are also small-size, genome-reduced archaea that are parasitic or symbiotic to other archaea (Castelle and Banfield, 2018; Adam et al., 2022), for instance, of the Thermoproteota phylum (Munson-McGee et al., 2015), which is also present in the Laguna Lejía sediments.
The low relationship between the prokaryotic community structure and the geochemistry and mineralogy of sediment layers suggested that biological interactions (e.g., symbiosis and competition) or abiotic variables not measured here (e.g., moisture, temperature, redox potential or physical structure of the sediment) could have played a decisive role on the microbial community structure of the Laguna Lejía terrace. Previous works on Atacama rocks showed that the physical structure better determined the microbial community composition and abundance than the chemistry of lithic substrates (Meslier et al., 2018; Casero et al., 2021). There are, however, a few exceptions to the low relationship found here. Actinobacteriota, mainly represented in the Lejía terrace by the orders Gaiellales, Solirubrobacterales, and UBA5794, were positively correlated with the anorthoclase and anorthite feldspars (Table S5). To our knowledge, no previous studies have correlated Actinobacteriota with feldspars more than with other minerals. In fact, Gaiellales and Solirubrobacterales are widely distributed in extreme aquatic and terrestrial ecosystems with different substrates (Crits-Christoph et al., 2013; Casero et al., 2020; Chen et al., 2021).
The geochemistry of the Laguna Lejía terrace exerted selective pressure on microbial communities able to metabolize CO, S, N, As and halogenated compounds
The prokaryotic community of the Lejía terrace is adapted to the particular geochemistry of the lake, and shows a wide metabolic potential involved in S and N biogeochemical cycles. The high relative abundance of predicted enzymes for assimilatory and dissimilatory sulfate reduction, as well as sulfur and thiosulfate oxidation in all sediment layers (Fig. 4b) is consistent with the high SO42−-S concentrations along the terrace profile (5–20 mg · g− 1 dw). For instance, the detection of sulfate adenylyltransferases (e.g., CysN) and sulfite reductases (e.g., CysJ) (Table S6) suggested that microorganisms had the potential to transform sulfate to sulfide as a previous step for the synthesis of Cys amino acids (assimilatory) (Wu et al., 2021). In addition, the sulfate adenylyltransferase (Sat), adenylyl-sulfate reductases (AprA and AprB), and sulfite reductases (DrsA and DsrB) suggested the potential of the prokaryotic community to first reduce sulfate to sulfite, and then reduce sulfite to hydrogen sulfide (dissimilatory) (Anantharaman et al., 2018). In addition to sulfur reduction, the prokaryotic community also had six sox genes (soxXYZABC) that encode for a complete thiosulfate-oxidizing enzyme system (Dahl and Friedrich, 2008). This sulfur oxidation may be coupled, but not necessarily, to nitrate reduction in the Lejía prokaryotic community, based on the prediction of nitrate reductases (e.g., NarG or NapA) (Smith et al., 2007). In addition, the annotation of enzymes for nitrite reduction to ammonia (e.g., NrfA), denitrification (e.g., NirS, NirK, NorB, NosZ), and nitrification (AmoA, AmoB and AmoC) (Smith et al., 2007; Levy-Booth et al., 2014) suggested the potential of the prokaryotic community for a complete N cycle.
The high relative abundance and wide distribution of predicted enzymes related to arsenic and halogenated compounds in the Lejía prokaryotic community (Fig. 4b) suggested that arsenic and halogenated compounds are important selective pressures for microorganisms. The annotation of enzymes involved in the arsenate (As(V)) reduction as a detoxification mechanism (e.g., ArsA, ArsB, ArsC, ArsH, Acr3) in all sediment layers (Table S6) is consistent with the detection of As in the Lejía terrace profile (2–7 µg · g− 1 dw). ArsC is involved in the reduction of arsenate to arsenite (As(III)), which is then pumped out of the cell with the efflux pumps ArsB and Acr3 (Amend et al., 2014; Ordoñez et al., 2015). Also, the prediction of arsenite oxidases (AoxA and AoxB) suggested the potential of Lejía prokaryotes to oxidate arsenite to arsenate. The potential to transform arsenate or arsenite either as a detoxification mechanism or energy gain has also been reported in other Andean environments (Ordoñez et al., 2015; Kurth et al., 2017; Saona et al., 2019). Unlike previous studies, the As cycle was surprisingly the most abundant metabolism in the Lejía prokaryotic community, reaching up to 27% of the total predicted proteins in sample D. In addition, the annotation of several haloalkane and haloacetate dehalogenases (DehH and DhaA) in the samples suggested the ability of the Lejía prokaryotic community to transform halogenated compounds (Janssen et al., 1994).
The coexistence of aerobic and anaerobic carbon fixation pathways by the Lejía prokaryotic community indicated a variety of microconditions in the terrace and suggested autotrophic metabolism diversification to optimize available resources. The most abundant autotrophic metabolism was the photosynthetic assimilation of CO2 through the CBB cycle, based on the relative abundance of predicted Rubisco enzymes (RbcL and RbcS) (Table S6) (Hügler and Sievert, 2011). The bulk carbon isotopic composition of the six Lejía samples (δ13C from − 17‰ to -22‰) also supports the relevance of CBB in the terrace (Hayes, 2001). These δ13C values could also imply a contribution from the reductive tricarboxylic acid (rTCA) (Preuß et al., 1989), but the absence of detection of any of the key genes associated with rTCA rules it out as a relevant CO2 fixation pathway in this environment. Besides Cyanobacteria, other bacteria present in the Lejía sediments, such as α-, β- and γ-Proteobacteria (Hügler and Sievert, 2011), may explain the dominance of the CBB cycle. The autotrophic community of the Laguna Lejía terrace were also involved in other aerobic and anaerobic CO2 fixation pathways, based on the prediction of enzymes for 3-HP/4-HB (e.g., 3-hydroxypropionyl-CoA dehydratase), and WL (e.g., anaerobic CO dehydrogenases) pathways (Hügler and Sievert, 2011).
Besides CO2 fixation, the high relative abundance of predicted CO dehydrogenases (CoxL, CoxM and CoxS) in the Lejía prokaryotic community suggested CO oxidation as a determining trait for their survival in the organic carbon-depleted terrace. CO has been previously hypothesized to serve as a supplemental carbon and energy source in oligotrophic habitats (Ji et al., 2017). The low TOC concentration in the Lejía terrace (0.1–1.2%) and the high relative abundance of predicted CO dehydrogenases supported CO oxidation as a carbon and energy source in this organic carbon-depleted environment.
Novelty of genomes from the Laguna Lejía terrace and expansion of metabolic capabilities to unexpected microbial taxa
The 591 MAGs reconstructed from the prokaryotic community of the Laguna Lejía terrace (Fig. 5) provided information on the metabolic potentials of yet uncultivated microorganisms from Andean ecosystems. The identification of bacterial MAGs not assigned to any known class, order, or family, as well as the large proportion of bacterial and archaeal MAGs unclassified to the genus (64% and 16%, respectively) or species (99.7% and 74%, respectively) level (Fig. 6), revealed the novelty of the recovered MAGs and evidenced the large amount of microbial dark matter in the Laguna Lejía ecosystem. For instance, the percentage of species novelty of the Lejía prokaryotic MAGs was 98.8% (considering bacteria and archaea together), and was higher than the 83% found in the Arctic ocean, calculated with the same methodology as here (Royo-Llonch et al., 2021). The absence of genomes in the GTDB similar to those recovered from the Lejía terrace showed up the need for microbial isolations from extreme environments and highlights the potential of omics techniques to gain insights into the functional diversity of microbial dark matter, expanding our view of the limits of life on Earth and providing biotechnological opportunities (Idris et al., 2017; Jiao et al., 2021).
The most frequent and widespread metabolic potentials among the Lejía MAGs were those related to the S and N cycles, CO oxidation, and transformation of As, Cu and halogenated compounds. The presence of many of these metabolic capabilities among phylogenetically distant bacteria (Fig. 7) suggested gene acquisition by horizontal gene transfer. For instance, the ubiquitous presence of arsenic genes in the Lejía MAGs (i.e., all phyla except Cyanobacteria) and the reported location of ars operon in transposons (Andres and Bertin, 2016) suggested horizontal gene transfer as a mechanism to facilitate bacterial adaptation to the particular Laguna Lejía chemical environment that requires in-depth study. Similarly, copper resistance proteins (Cop and Cus) may also be associated with mobile elements (Richard et al., 2017), and genes for haloalkane dehalogensases (DehH or DhaA) (Janssen et al., 1994) and nitrate reductases (Nap and Nar) (Moreno-Vivián et al., 1999; Stolz and Basu, 2002) can be localized on plasmids and thus transfer laterally.
The broad redox capacity for N and S compounds as well as varied CO2 fixation pathways in phylogenetically distant bacteria and archaea from Laguna Lejía extends metabolic capabilities to previously unknown phyla. As Nar proteins can act as nitrite oxidorreductases, their prediction in phylogenetically distinct bacteria beyond Nitrospirota, Chloroflexota and Proteobacteria (Sorokin et al., 2012) may extend the list of nitrite oxidizers known so far. Moreover, the prediction of ammonia monooxygenase (AmoC) in an unknown Actinobacteriota (class UBA4738) may expand the catalogue of ammonium oxidizers, dominated in the Lejía samples by the archaeal family Nitrosopumilaceae (already described as ammonium oxidizers (Stahl and de la Torre, 2012)) (Table S7). While assimilatory sulfate reduction is frequent in all microorganisms, the dissimilatory pathway has only been described in Firmicutes, Proteobacteria (δ-Proteobacteria), Nitrospirae, and Euryarchaeota (Thauer et al., 2007). Therefore, the prediction of adenylylsulfate reductases in Bacteroidota and α- and γ-Proteobacteria in Lejía MAGs may expand the list of dissimilatory sulfate reducers known so far. In addition, sulfur and thiosulfate oxidation was also widespread across MAGs, some belonging to already described phyla, such as Cyanobacteria or Proteobacteria (e.g., Rhodospirillales or Pseudomonadales; Table S7) (Friedrich, 1997; Friedrich et al., 2001), but also to potentially new taxa, such as Methylomirabilota (Rokubacteriales). The annotation of phosphoribulokinases (prk) in one Planctomycetota (Phycisphaerales) and Gemmatimonadota (order SG8-23), and a ribulose-bisphosphate carboxylase (Rubisco) in a Nanoarchaeota (Pacearchaeales), may also extend the list of microorganisms fixing CO2 through the CBB cycle known so far (Garritano et al., 2022). Alternatively, the prediction of these enzymes could be associated with a metabolism different from CBB, as occurs in the reductive hexulose-phosphase pathway mediated by Rubisco and prk in methanogenic archaea (Kono et al., 2017).
The expansion of genetic diversity and potential metabolisms involved in the biogeochemical cycles of carbon, nitrogen and sulfur to unexpected microorganisms in the ancient sediments of the Laguna Lejía terrace contribute to our knowledge on the limits of life on Earth and on the habitability of similar present-day planetary settings. Our results demonstrate that bacterial and archaeal communities can thrive and adapt to changing environmental conditions over thousands − and perhaps millions − of years within a well-structured, slightly moist sediments. The microbial genetic and metabolic diversity in the terrace of the Laguna Lejía supports Martian craters that served as paleolake basins (Carr, 2012) as strategic landing sites for missions aimed at searching for signs of life, such the Mars 2020 mission, which landed in the Jezero crater delta to collect rock and regolith samples for possible return to Earth (Scheller et al., 2022; Tait et al., 2022).