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Hyperdiverse archaea near life limits at the polyextreme geothermal Dallol area

Abstract

Microbial life has adapted to various individual extreme conditions; yet, organisms simultaneously adapted to very low pH, high salt and high temperature are unknown. We combined environmental 16S/18S ribosomal RNA gene metabarcoding, cultural approaches, fluorescence-activated cell sorting, scanning electron microscopy and chemical analyses to study samples along such unique polyextreme gradients in the Dallol–Danakil area in Ethiopia. We identified two physicochemical barriers to life in the presence of surface liquid water defined by (1) high chaotropicity–low water activity in Mg2+/Ca2+-dominated brines and (2) hyperacidity–salt combinations (pH ~0/NaCl-dominated salt saturation). When detected, life was dominated by highly diverse ultrasmall archaea that were widely distributed across phyla with and without previously known halophilic members. We hypothesize that a high cytoplasmic K+-level was an original archaeal adaptation to hyperthermophily, subsequently exapted during several transitions to extreme halophily. We detect active silica encrustment/fossilization of cells but also abiotic biomorphs of varied chemistry. Our work helps circumscribing habitability and calls for cautionary interpretations of morphological biosignatures on Earth and beyond.

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Fig. 1: Overview of sampling sites at the polyextreme geothermal field of Dallol and its surroundings in the Danakil Depression, Ethiopia.
Fig. 2: Physicochemical features of liquid samples from the Dallol area.
Fig. 3: Distribution and diversity of prokaryotes in samples from the Dallol dome and surrounding areas based on 16S rRNA gene metabarcoding data.
Fig. 4: SEM pictures and chemical maps of cells and abiotic biomorphs identified in samples from the Dallol region.

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Data availability

Sanger sequences have been deposited in GenBank (National Center for Biotechnology Information) with accession numbers MK894601–MK894820 and Illumina sequences in GenBank Short Read Archive with BioProject number PRJNA541281.

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Acknowledgements

We are grateful to O. Grunewald for co-organizing the Dallol expeditions, documenting field research and providing drone images, and also to Jean-Marie Hullot (in memoriam), Françoise Brenckmann and the Fondation Iris for funding the first field trip. We thank L. Cantamessa for the in situ logistics and discussions about local history. We acknowledge M. Tafari (Mekelle University), A. A. Aliyu and the Afar authorities for local assistance, as well as the Ethiopian army and the Afar police for providing security. We thank J. Barthélémy, E. Kotopoulou and J. Garcia-Ruiz for help and discussions during field trips. We thank H. Timpano and the UNICELL platform for cell sorting; A. Gutiérrez-Preciado for bioinformatic assistance; A. Kish and C. Faveau for allowing us to measure water activity of selected samples at the Muséum National d’Histoire Naturelle; E. Viollier for discussion on chemical analyses; C. Gille for help with cultures; G. Billo for script help to treat SEM pictures; and J. T. Díaz and P. T. Sanz for advice on statistical analyses. This research was funded by the French CNRS (National Center for Scientific Research) basic annual funding, the CNRS programme TELLUS INTERRVIE and the European Research Council (ERC) under the European Union’s Seventh Framework Programme (ERC grant no. 322669 to P.L.-G.). We thank the European COST Action TD1308 Origins for funding a short stay of A.I.L.-A. in Orsay. J.B. was financed by the French Ministry of National Education, Research and Technology.

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Contributions

P.L.-G. and D.M. designed and supervised the research. P.L.-G. organized the scientific expeditions. J.B., P.L.-G., D.M., L.J. and J.M.L.-G. collected samples and took measurements in situ. J.B., P.L.-G. and P.B. carried out molecular biology analyses. J.B., A.I.L.-A. and D.M. performed culture, chemistry analyses and water–salt related measurements. A.I.L.-A. and J.B. performed statistical analyses. J.B., G.R. and D.M. analysed metabarcoding data. K.B. performed SEM and EDX analyses. J.M.L.-G. mapped geothermal activity and georeferenced all samples. L.J. and J.B. performed FACS-derived analyses. P.L.-G. and J.B. wrote the manuscript. All authors read and commented on the manuscript.

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Correspondence to Purificación López-García.

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Extended data

Extended Data Fig. 1 Aerial view of the main sampling sites in the Dallol area.

a, Dallol dome summit showing the acidic green-yellow-brown coloured hydrothermal ponds and active degassing areas during our 2017 sampling trip; the orange-shaded area shows the active hydrothermal zone in January 2016. b, Dallol West salt canyons and Black Mountain area. c, Black Lake. d, Yellow Lake and surroundings. Names of samples and sampling sites are indicated. The size of circles is proportional to the water volume collected or filtered for subsequent analyses. Aerial photographs were taken from a drone by O. Grunewald, except b, which is a Google Earth aerial image (09/03/2016) obtained by the Sentinel satellite (ESA Copernicus program) provided by Image © 2019 CNES/Airbus.

Extended Data Fig. 2 Views of different sampling sites in the Dallol dome and surroundings in the Danakil Depression.

a, DAL4 sampling site ponds; b, DAL5 pond and active degassing area; c, active hydrothermal springs in DAL9 ponds; d, in situ cell‐trap filtration at the 7DA7 sampling area; e, 7DA9 sampling site; f,7DA10 ponds showing increasingly darker and brownish colours along the oxidation gradient; g, water samples from the different 7DA10 ponds; h, DAL8 mineral precipitates; i,’proto-soil’-like salt crust (7YL-S1) near the Yellow Lake; j, Yellow Lake showing active degassing; k, YL3, salt-mud volcano in the Yellow Lake area; l, ‘Little Dallol’ hydrothermal very active area in 2016 on the way to the Black Mountain (in the distance; inlet, chimney emitting hydrocarbon‐rich fluids at 110 °C); m, Black Lake; n, PSBL2 (Black Lake area ponds); o, wet salt plain, influenced by hydrothermal activity, corresponding to PS3 sample area; p, the cave in the salt canyons where Gt, 7Gt and 8Gt samples were collected; q, salt canyons; r, Assale (Karum) lake. Sample names starting by 7 indicate collection in 2017. Pictures from all other samples/sampling sites were taken during the 2016 expedition.

Extended Data Fig. 3 List and description of samples from the Dallol area analysed in this study and type of analyses performed.

DO, dissolved oxygen; ORP, oxido-reduction potential; SEM–/EDXS, scanning electron microscopy/energy-dispersive x-ray spectrometry; FACS, fluorescence-activated cell sorting analysis; n.a., not applicable; n.d. not determined. Refractometry-derived salinity refers to the percentage (w/v) of local salt composition (see Supplementary Tables 1 and 3 for elementary and ionic analyses) measured in situ. Salinity was also directly measured by weighting the total solids (dry weight experimentally measured in triplicates; SD, standard deviation).

Extended Data Fig. 4 Principal Component Analyses (PCA) of Dallol area sampling sites as a function of physicochemical parameters.

PCA of 29 samples according to their chemical composition; only relatively abundant elements (see Supplementary Table 1) are included in the analysis. A summary of this analysis is shown in Fig. 2f. b, PCA including the same variables as Fig. 2f but additionally including dissolved oxygen (DO). Measured parameters on site can be found in Extended Data Fig. 3. Coloured zones in PCA analyses correspond to the three major chemical zones identified in this study.

Extended Data Fig. 5 Chaotropicity, ionic strength and water activity for a selection of samples of the Dallol area.

Chaotropicity was measured experimentally (see Methods) and also calculated, together with ionic strength values were from dominant Na, K, Mg, Ca, Fe chemistry data; water activity values were measured using a probe (see Methods). Known limits for life for each parameter are listed at the top of the table. Samples beyond that threshold for one or more of those parameters are shaded in grey.

Extended Data Fig. 6 Sequence data and diversity measurements.

*Contaminant sequences included sequences identified in negative controls and/or high similarity to human-associated bacteria; s.e., standard error. Eventual mitochondrial and chloroplast 16S rRNA gene sequences were also removed at this step.

Extended Data Fig. 7 Phylogenetic tree of bacterial 16S rRNA gene sequences showing the phylogenetic placement of OTUs identified in the different Dallol area samples.

Sequences derived from metabarcoding studies are represented by blue lines (Illumina sequences); those derived from cloning and Sanger sequencing of environmental samples, cultures and FACS-sorted cells are labelled with a red dot. Reference sequences are in black. Concentric circles around the tree indicate the presence/absence of the corresponding OTUs in different groups of samples (groups shown in Fig. 3a). Only sequences not deemed contaminant (see Supplementary Table 5) were included in the tree. The full tree is provided as Supplementary Data 1.

Extended Data Fig. 8 Eukaryotic presence, diversity and relative abundance in Dallol area samples.

Histogram showing the phylogenetic affiliation and abundance of 18S rRNA gene amplicon reads of eukaryotes (upper panel) obtained with universal eukaryotic primers and the associated OTU diversity (lower panel). Only a few samples yielded amplicons; negative PCR controls were always negative. Sequences corresponding to macroscopic plants and fungi (probably derived from pollen or spores) were considered contaminant (light grey). The phylogenetic affiliation of dominant eukaryotic groups is colour-coded.

Extended Data Fig. 9 Multiparametric fluorescence analyses and fluorescence-activated cell sorting (FACS) analyses of representative Dallol area samples.

a, effect of DNA fluorescent dyes on background fluorescence emission; natural (sterile medium-only) and DNA dye-induced fluorescence in the sterile hypersaline SALT-YE medium used to dilute/sort Dallol samples. Fluorescence is plotted against the size of the analysed particles (forward scatter); events concentration is colour-coded, red being high concentration and blue, low concentration. DRAQ5 and SYTO13 introduced less background and were chosen for FACS of natural samples. The approximate background threshold (ca. 102) is indicated by a broken grey line. b, multiparametric fluorescence analyses of different Dallol samples before (left panels) and after (right panels) adding fluorescent DNA dyes. Events (particles) above background (red squares) were FACS-sorted and filtered on 0.1 µm pore-size filters prior to SEM observations. c, SEM photographs showing examples of sorted particles. Cells are observed in samples PS, Gt and 7Gt; halite crystals in 7DA7 and amorphous mineral particles in 7DA9 and 7YL. Arrows indicate ultrasmall cells. The scale bar is 1 µm.

Extended Data Fig. 10 Mineral phases observed by SEM-EDX in precipitates of typical abiotic morphology and ‘biomorphs’.

Biomorphs correspond to rounded-shaped crystalline morphs resembling cell structures (cocci, rods) and compatible with cellular sizes. Observed dominant phases are highlighted in bold.

Supplementary Information

Supplementary Information

Supplementary Figs. 1 and 2 and Tables 1–4.

Reporting Summary

41559_2019_1005_MOESM3_ESM.xlsx

Supplementary Table 5 Identification, phylogenetic affinity and relative abundance of prokaryotic OTUs. a, OTUs identified in samples that yielded amplicons in direct (PS3, PS, Gt, 7 Gt, 7 Gt-pp, Ass, Ass-PJ) PCR amplifications. b, OTUs identified in samples that yielded amplicons in seminested PCR amplification reactions. c, List of contaminant OTUs identified in ‘negative’ controls of nested PCR reactions. d, List of OTUs removed as potential contaminants owing to their similarity to typical dust/soil bacterial contaminants or human-associated biota. id, identifier; bh, best hit; db, database; cmr, cleaned merged reads; cult, cultivated organism; env, environmental organism; seq, sequence; pciden, percentage of identity; pcqcov; percentage of coverage with query sequence; mism, mismatch; aln, alignment; len, length. Supplementary Table 5 sections a to d are provided in independent sheets for readability.

41559_2019_1005_MOESM4_ESM.xlsx

Supplementary Table 6 Identification, phylogenetic affinity and relative abundance of eukaryotic OTU from Dallol area samples. a, Eukaryotic OTU corresponding to protists thriving in Danakil samples; only a small subset of samples yielded 18S rRNA gene amplicons. b, Potential contaminant sequences (likely dispersal of pollen/spores) (see sheet 6b). id, identifier; bh, best hit; db, database; cmr, cleaned merged reads; cult, cultivated organism; env, environmental organism; seq, sequence; pciden, percentage of identity; pcqcov; percentage of coverage with query sequence; mism, mismatch; aln, alignment; len, length.

Supplementary Dataset 1

Full tree of archaeal 16S rRNA gene fragments in Newick format.

Supplementary Dataset 2

Full tree of bacterial 16S rRNA gene fragments in Newick format.

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Belilla, J., Moreira, D., Jardillier, L. et al. Hyperdiverse archaea near life limits at the polyextreme geothermal Dallol area. Nat Ecol Evol 3, 1552–1561 (2019). https://doi.org/10.1038/s41559-019-1005-0

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