# Evolutionary placement of Methanonatronarchaeia

Monique Aouad, Guillaume Borrel, Céline Brochier-Armanet, Simonetta Gribaldo

► To cite this version:

Monique Aouad, Guillaume Borrel, Céline Brochier-Armanet, Simonetta Gribaldo. Evolutionary placement of Methanonatronarchaeia. Nature Microbiology, 2019, 10.1038/s41564-019-0359-z. pasteur-02059654

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Submitted on 6 Mar 2019

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1 **Subject ontology**

2 [URI /631/326/26/2526]

3 [URI /631/326/171]

4

5

6 **Evolutionary placement of Methanonatronarchaeia**

8

9 Monique Aouad¹#, Guillaume Borrel²#, Céline Brochier-Armanet¹, and Simonetta Gribaldo²

10 ¹Univ Lyon, Université Lyon 1, CNRS, UMR5558, Laboratoire de Biométrie et Biologie

11 Évolutive, 43 bd du 11 novembre 1918, F-69622 Villeurbanne, France.

12 ²Institut Pasteur, Unit Evolutionary Biology of the Microbial Cell, Department of Microbiology,

13 15 rue du Dr Roux, 75015 Paris, France.

14 # these authors contributed equally to this work

15 Correspondence:

16 celine.brochier-armanet@univ-lyon1.fr

17 simonetta.gribaldo@pasteur.fr

18

19 **Methanonatronarchaeia**, a newly discovered archaeal lineage of extremely halophilic

20 methanogens, were proposed to represent an evolutionary intermediate between

21 archaeal methanogens and the extremely halophilic *Halobacteria*. Here, we show that

22 the sistership between *Methanonatronarchaeia* and *Halobacteria* results from a tree

23 reconstruction artefact and that the divergence of *Methanonatronarchaeia* is in fact

24 much deeper. This sheds a new light on the adaptation to extreme halophilic lifestyle

25 in archaea and on the evolution of methanogenesis.

26

27 Sorokin and colleagues recently reported the identification of *Methanonatronarchaeia*, a

28 fascinating archaeal lineage of extremely halophilic, moderately thermophilic, methyl-

29 reducing methanogens¹,². Similar to most recently discovered methanogens,

30 *Methanonatronarchaeia* perform methanogenesis based on H₂ and methyl compounds, a

31 metabolism not previously reported from hypersaline environments. Together with

32 *Halobacteria* and *Nanohaloarchaea*³, *Methanonatronarchaeia* represent the third discovered

33 lineage of extreme halophilic archaea and the most halophilic methanogens ever found. They

34 have likely adapted to this lifestyle by employing a salt-in osmoprotection strategy¹, unlike

35 previously known halophilic methanogens and similarly to the two other extreme halophilic

36 archaeal lineages⁴. Moreover, *Methanonatronarchaeia* rely on cytochromes for

37 methanogenesis¹, a characteristic previously thought to be restricted to the

38 *Methanosarcinales*⁵. A maximum Likelihood (ML) phylogenetic analysis of a supermatrix

39 gathering ribosomal proteins indicated *Methanonatronarchaeia* as the closest relatives to

40 *Halobacteria* (Fig. 1A, red branches)¹. They were therefore proposed to be evolutionary

41 intermediates on the path from methanogens to extreme halophiles¹. However, multiple

42 substitutions occurring at the same site in sequences can mask the original phylogenetic

43 signal and provoke tree reconstruction artefacts⁶, a phenomenon particularly evident in

44 lineages that adapted to extreme salinity⁷.

45
To test the phylogenetic position of *Methanonatronarchaeia*, we reanalyzed the original supermatrix of ribosomal proteins used by Sorokin et al.¹, through the progressive removal of the fastest evolving sites, a method that is frequently used to reduce artefacts linked to multiple substitutions⁷. This analysis, both by ML and Bayesian approaches including non-homogeneous evolutionary models, shows that the clustering of *Halobacteria* and *Methanonatronarchaeia* (Fig. 1B-C, red line) was recovered only when the fastest evolving sites are included in the analysis, while the progressive removal of these sites shifted the position of *Methanonatronarchaeia* away from *Halobacteria* and to a deeper branching position at the base of the superclass 'Methanotecta'⁸ (Fig. 1B-C, green line). This placement is also consistently and robustly recovered when *Methanonatronarchaeia* were included in two recently published supermatrices comprising a larger number of markers⁶ (over 250 conserved protein families) or a larger taxonomic sampling of the Methanotecta⁹ (including ANME1, Syntrophoarchaeales, Methanoliparia, and a third *Methanonatronarchaeia* member). In contrast with the dataset of Sorokin et al.¹, the new placement of *Methanonatronarchaeia* was robust to the removal of the fastest-evolving sites for both these supermatrices (Fig. 1D-G).

Our analyses indicate that the placement of *Methanonatronarchaeia* as the methanogenic closest relatives of *Halobacteria* proposed in Sorokin et al.¹ is likely the consequence of a tree reconstruction artefact induced by a multiple substitution-bias which is particulary strong in their ribosomal protein dataset, but not in the other two datasets. The alternative position of the *Methanonatronarchaeia* disclosed here provides a new perspective on the evolution of this fascinating lineage. For example, it indicates that their adaptation to extreme halophily would have occurred independently from the *Halobacteria*. Moreover, following the recent proposal for the placement of *Nanohaloarchaea* as sister to the *Methanocellales*⁶, the salt-in strategy used for thriving in hypersaline environments would have emerged three times independently in the *Archaea*, a remarkable example of convergent evolution for adaptation to similar environments. Finally, the new placement of *Methanonatronarchaeia* is highly relevant for the evolution and diversity of methanogenesis, as their characteristics may reflect those of the methanogenic ancestor of the whole 'Methanotecta' superclass. For example, the fact that *Methanonatronarchaeia* rely on cytochromes for methanogenesis³ raises the question of whether this feature may be ancestral to all Class II methanogens and was retained only in *Methanosarcinales* while *Methanomicrobiales*, *Methanocellales* and *Methanoflorentaceae* shifted secondarily to methanogenesis without cytochromes, or if instead it emerged twice independently.

The current pace in the acquisition of genomic data and the discovery of new lineages⁸,¹⁰ will certainly allow to tackle these fundamental questions in the evolution and ecology of methanogens and of *Archaea* in general.

## References

1) Sorokin et al. (2017) *Nat Microbiol* 2:17081.

2) Sorokin et al. (2018) *Int J Syst Evol Microbiol* 68(7):2199-2208.

3) Narasingarao et al. (2012) *ISME J* 6, 81-93

4) Oren (2008) *Saline Syst* 4, 2.

5) Thauer et al. (2008) *Nat Rev Microbiol* 6(8):579-91.

6) Delsuc (2005) *Nat Rev Genet* 6(5):361-75.

7) Aouad et al. (2018) *Mol Phylogenet Evol* 127:46-54

8) Adam et al. (2017) *ISME J* (11):2407-2425.

9) Borrel et al. *Nat Microbiol* (accepted).

10) Spang et al. (2017) *Science* 357(6351).

Correspondence and request for materials should be addressed to Simonetta Gribaldo (simonetta.gribaldo@pasteur.fr) or Céline Brochier-Armanet (celine.brochier-armanet@univ-lyon1.fr).
101 Acknowledgements

102 S.G., G.B., and C.B.A acknowledge funding from the French National Agency for Research,
103 Grant ArchEvol (ANR-16-CE02-0005-01). M.A. is funded by a doctoral fellowship from the
104 Région Rhône-Alpes-ARC 1 Santé. We thank the PRABI (Pôle Rhône-Alpes de
105 Bioinformatique) and the computational and storage services (TARS cluster) provided by the
106 IT department at Institut Pasteur, Paris.

107

108 Author contributions

109 S.G. and C.B.A. supervised the study. M.A. and G.B. assembled the datasets and performed
110 all analyses. All authors analysed the data and wrote the manuscript.

111

112 Competing interests

113 The authors declare no competing interest.

114

115 Legend of Fig. 1

116 (A) Schematic phylogeny of the *Archaea*, with a focus on the 'Methanotecta' superclass⁸.
117 Dotted lines indicate two alternative branchings of *Methanonatronarchaeia*: as the sister-
118 lineage of *Halobacteria* (red) or at the base of 'Methanotecta' (green).

119 (B-G): Impact on the placement of *Methanonatronarchaeia* of the progressive removal of the
120 fastest-evolving sites from the three analysed supermatrices (see Supplementary Information
121 (SI) for details). (B-C): the supermatrix of ribosomal proteins (8,072 amino acid positions)
122 derived from Sorokin et al.¹, (D-E): the supermatrix, derived from Adam et al.⁸ (40 conserved
123 protein families, 9,228 amino acid positions), and (F-G): the supermatrix derived from Aouad
124 et al.⁶ (258 conserved protein families, 62,398 amino acid positions).

125 The x-axis indicates the percentage of amino acid positions of the supermatrices that were
126 kept for phylogenetic analyses during the progressive removal of the fastest evolving sites.
127 The y-axis corresponds to bootstrap values associated to the ML trees inferred using the
128 LG+G4 evolutionary model (B, D, and F) or the PMSF+LG+G4 evolutionary model (G), or to
129 posterior probabilities associated to the Bayesian trees inferred with the CAT+GTR+G4
130 evolutionary model (C, and E). The green and red lines shown on these graphs correspond
131 to the bootstrap values and posterior probabilities supporting the two alternative placements
132 of *Methanonatronarchaeia* as illustrated in Figure 1A. In all trees, the clustering of
133 *Methanonatronarchaeia* with 'Methanotecta' was strongly supported, excluding the branching
134 of *Methanonatronarchaeia* elsewhere in the archaeal phylogeny. For two supermatrices on
135 panel C (86, 82, indicated by an asterisk), *Methanonatronarchaeia* branched in-between
136 *Archaeoglobales* and 'Ca. Methanophagales' (ANME-1). All trees and corresponding
137 supermatrices are provided in Supplementary Information (SI).
A

Other Archaea

Diaforarchaea

Methanonatronarchaeia

Archaeoglobales

'Ca. Methanophagales' (ANME-1)

Class II methanogens

Methanosarcinales

Methanomicrobiales

Methanocellales

Methanonatronarchaeia

Halobacteriales

Methanotecta

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