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Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments

Nature Microbiology volume 9, pages 964–975 (2024)Cite this article

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Abstract

Extremely halophilic archaea (Haloarchaea, Nanohaloarchaeota, Methanonatronarchaeia and Halarchaeoplasmatales) thrive in saturating salt concentrations where they must maintain osmotic equilibrium with their environment. The evolutionary history of adaptations enabling salt tolerance remains poorly understood, in particular because the phylogeny of several lineages is conflicting. Here we present a resolved phylogeny of extremely halophilic archaea obtained using improved taxon sampling and state-of-the-art phylogenetic approaches designed to cope with the strong compositional biases of their proteomes. We describe two uncultured lineages, Afararchaeaceae and Asbonarchaeaceae, which break the long branches at the base of Haloarchaea and Nanohaloarchaeota, respectively. We obtained 13 metagenome-assembled genomes (MAGs) of these archaea from metagenomes of hypersaline aquatic systems of the Danakil Depression (Ethiopia). Our phylogenomic analyses including these taxa show that at least four independent adaptations to extreme halophily occurred during archaeal evolution. Gene-tree/species-tree reconciliation suggests that gene duplication and horizontal gene transfer played an important role in this process, for example, by spreading key genes (such as those encoding potassium transporters) across extremely halophilic lineages.

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Fig. 1: Phylogenetic position and metabolic potential of the families Afararchaeaceae and Asbonarchaeaceae.
Fig. 2: Protein amino acid compositional biases in extremely halophilic archaeal lineages.
Fig. 3: Maximum likelihood phylogeny of archaea, including the Afararchaeaceae and Asbonarchaeaceae.
Fig. 4: Schematic representation of the tree reconciliation analysis based on the NM species tree.

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Data availability

The MAGs reported in this study have been deposited in GenBank under BioProject number PRJNA901412. All raw data underlying phylogenomic analyses (raw and processed alignments and corresponding phylogenetic trees) and all predicted proteomes have been deposited in Figshare (https://figshare.com/s/353259800b42a4e190eb). Additional data were obtained from public databases, including GTDB (https://gtdb.ecogenomic.org/), Pfam (http://pfam.xfam.org/), COG (https://www.ncbi.nlm.nih.gov/research/cog), RefSeq (https://www.ncbi.nlm.nih.gov/refseq/), eggNOG (http://eggnog5.embl.de/#/app/home), the Genomic Catalog of Earth’s Microbiomes (https://genome.jgi.doe.gov/portal/GEMs/GEMs.home.html), the Global Microbial Gene Catalog (https://gmgc.embl.de/), the Unified Human Gastrointestinal Genome collection (http://ftp.ebi.ac.uk/pub/databases/metagenomics/mgnify_genomes/), the Ocean Microbiomics Database (https://microbiomics.io/ocean/) and PDB (https://www.rcsb.org/).

Code availability

Custom code used for data analysis is available on GitHub at https://github.com/bbaker567/phylogenetics.

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Acknowledgements

D.M. and L.E were supported by grants from the European Research Council (ERC Advanced grant 787904 and ERC Starting grant 803151, respectively). This work was also supported by the Moore-Simons Project Call on the Origin of the Eukaryotic Cell, Simons Foundation 812811 (A.J.R, E.S. and L.E.), Moore Foundation GBMF9739 (P.L.-G.), and ANR DArchFolds ANR-22-CE02-0012-02 (D.M., P.L.-G. and L.E.). A.R.d.R. was supported by ‘la Caixa’ Foundation (ID 100010434, fellowship code LCF/BQ/DI18/11660009, the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No. 713673) and by an EMBO Scientific Exchange Grant. We thank P. Deschamps for help in managing our bioinformatic cluster; A. Oren for advice on taxonomic descriptions; and the Iris Foundation for the continuous support of our work on the microbial diversity of the Danakil Depression.

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Authors and Affiliations

  1. Ecologie Systématique Evolution, CNRS, Université Paris-Saclay, AgroParisTech, Gif-sur-Yvette, France

    Brittany A. Baker, Ana Gutiérrez-Preciado, Purificación López-García, Laura Eme & David Moreira

  2. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), Madrid, Spain

    Álvaro Rodríguez del Río & Jaime Huerta-Cepas

  3. Institute for Comparative Genomics, Dalhousie University, Halifax, Nova Scotia, Canada

    Charley G. P. McCarthy, Edward Susko & Andrew J. Roger

  4. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada

    Charley G. P. McCarthy & Andrew J. Roger

  5. Department of Mathematics and Statistics, Dalhousie University, Halifax, Nova Scotia, Canada

    Edward Susko

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Contributions

D.M., P.L.-G. and L.E. designed the study. A.G.-P. and B.A.B. annotated the archaeal MAGs. A.R.d.R., B.A.B. and J.H.-C. studied the protein families. C.G.P.M., A.J.R. and E.S. conceived the binomial methods to identify significant shifts in amino acid composition, and E.S. implemented the changes of the GFmix model in the GFmix software. B.A.B., L.E., D.M., C.G.P.M., A.J.R. and E.S. carried out phylogenetic analyses. B.A.B., L.E., P.L.-G. and D.M. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Laura Eme or David Moreira.

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Nature Microbiology thanks Aharon Oren, Tom Williams and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Schematic tree showing the phylogenetic position of extremely halophilic archaeal groups (colored branches) proposed in previous articles.

Branches that have been found at different places in the tree of archaea are indicated with dashed lines (Narasingarao et al. 20123, Rinke et al. 20136, Sorokin et al. 20177, Aouad et al. 201844, Aouad et al. 20198, Martijn et al. 202010).

Extended Data Fig. 2 Number and isoelectric point of novel gene families identified in the Asbonarchaeaceae and Afararchaeaceae MAGs.

(a) The average number of novel genes in the nine asbonarchaeal and four afarachaeal MAGs described in this study. Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (where IQR is the interquartile range) and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge, while data beyond the end of the whiskers are outlying points that are plotted individually. (b) The isoelectric point of these novel proteins (solid lines) compared to the average isoelectric point of the whole proteomes (dashed lines).

Extended Data Fig. 3 Maximum likelihood phylogeny of 192 archaea based on the NM dataset.

The ML tree was inferred with the LG + C60 + F + Γ4 model of sequence evolution with 1,000 ultrafast bootstraps as implemented via IQ-TREE. The scale bar indicates the expected average number of substitutions per site. Extremely halophilic archaea are indicated in color.

Extended Data Fig. 4 Maximum likelihood phylogeny of 192 archaea based on the RP dataset.

The ML tree was inferred with the LG + C60 + F + Γ4 model of sequence evolution with 1,000 ultrafast bootstraps as implemented via IQ-TREE. The scale bar indicates the expected average number of substitutions per site. Extremely halophilic archaea are indicated in color.

Extended Data Fig. 5 Halophilic-specific amino acid compositional biases along the phylogeny of 192 archaeal taxa.

(a) The ratio of [D + E/I + K] amino acids of 192 archaeal taxa was calculated along the untreated NM and RP alignments (39,385 and 6,792 amino acid positions, respectively). (b) 10% of the most biased sites (that is, those with the highest ratio) were removed from the NM and RP alignments. Distinct halophilic clades are indicated in color, including the Nanosalinaceae (sand), Asbonarchaeaceae (wine), Halarchaeoplasmatales (cyan), Methanonatronarchaeia (rose), Afarachaeaceae (green), and Haloarchaea (indigo). The scale bar indicates the expected average number of substitutions per site.

Extended Data Fig. 6 Impact of compositional bias on the phylogeny of archaeal ATP synthase.

Maximum likelihood phylogenetic trees based on the concatenation of ATP synthase subunits A and B (a) before and (b) after removal of 15% of sites with the highest D + E/I + K ratio. Notice the shift in the position of the Nanosalinaceae+Asbonarchaeaceae group. The trees were reconstructed using the LG + C60 + F + Γ4 model of sequence evolution. Numbers at branches indicate 1,000 ultrafast bootstrap support values. Only values > 70% are indicated. The scale bar indicates the expected average number of substitutions per site. (c) D + E/I + K ratio for all sites in the ATP synthase subunits A and B dataset ordered from highest to lowest values.

Extended Data Fig. 7 Heat map of the number of gene duplications, transfers, originations, and losses in various archaeal halophilic lineages according to their COG classification.

The counts were obtained using the amalgamated likelihood estimation (ALE) tree reconciliation method on the set of 17,288 orthologous genes present in the 192-taxa genomic dataset for several nodes within the (a) Euryarchaeota and the (b) DPANN archaea (see Methods). Node numbers correspond to the nodes in the complete tree shown in Supplementary Fig. 18.

Extended Data Fig. 8 Maximum likelihood trees showing cases of horizontal gene transfer involving archaeal halophilic lineages.

(a) NhaP-type Na + /H+ and K + /H+ antiporters. (b) choline dehydrogenase BetA. The trees were constructed with the LG + C60 + F + Γ4 model of sequence evolution. Dashed branches have been shortened to half of their actual length. The scale bar indicates the expected average number of substitutions per site.

Supplementary information

Supplementary Information

Supplementary Figs. 1–41.

Reporting Summary

Supplementary Video 1

Comparison of acidic (D + E) versus hydrophobic (I + K) amino acids on the large ribosomal subunits of Haloarcula marismortui and Methanothermococcus thermautotrophicus.

Supplementary Table 1

Genome statistics for the Afararchaeaceae, Asbonarchaeaceae and Chewarchaeaceae MAGs. The location of the lakes where the MAGs came from, the completeness and redundancy, the %GC, estimated genome size, number of contigs and the N50 are included. Supplementary Table 2 Average amino acid identity (AAI), average nucleotide identity (ANI), and RED values for the Afararchaeaceae and Asbonarchaeaceae MAGs. Supplementary Table 3 Major biosynthetic pathways identified in the most complete afararchaeal MAG (DAL-WCL_na_97C3R). Supplementary Table 4 Major biosynthetic pathways identified in the most complete asbonarchaeal MAG (DAL-WCL_45_84C1R). Supplementary Table 5 Novel gene families identified in the Afararchaeaceae MAGs. A link to a graphical interface is provided to explore the synteny and neighbourhood of the novel gene families. Supplementary Table 6 Novel gene families identified in the Asbonarchaeaceae MAGs. A link to a graphical interface is provided to explore the synteny and neighbourhood of the novel gene families. Supplementary Table 7 Gene annotations for the 136 NM dataset markers based on predictions from eggNOG-mapper. Supplementary Table 8 Proteomes used for phylogenomic datasets and gene family clustering. Taxonomy, assembly characteristics and source databases are listed for all taxa included in the species trees. Metadata information was collected from GTDB r207. Supplementary Table 9 Gene annotations of orthologous groups (OGs) specifically mentioned in the ALE analysis section. For each OG, the raw relative frequencies of the duplication, transfer, origination and loss events are reported, as well as the presence probability in the halophilic nodes of interest.

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Baker, B.A., Gutiérrez-Preciado, A., Rodríguez del Río, Á. et al. Expanded phylogeny of extremely halophilic archaea shows multiple independent adaptations to hypersaline environments. Nat Microbiol 9, 964–975 (2024). https://doi.org/10.1038/s41564-024-01647-4

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