Members of the class Candidatus Ordosarchaeia imply an alternative evolutionary scenario from methanogens to haloarchaea

Abstract The origin of methanogenesis can be traced to the common ancestor of non-DPANN archaea, whereas haloarchaea (or Halobacteria) are believed to have evolved from a methanogenic ancestor through multiple evolutionary events. However, due to the accelerated evolution and compositional bias of proteins adapting to hypersaline habitats, Halobacteria exhibit substantial evolutionary divergence from methanogens, and the identification of the closest methanogen (either Methanonatronarchaeia or other taxa) to Halobacteria remains a subject of debate. Here, we obtained five metagenome-assembled genomes with high completeness from soda-saline lakes on the Ordos Plateau in Inner Mongolia, China, and we proposed the name Candidatus Ordosarchaeia for this novel class. Phylogenetic analyses revealed that Ca. Ordosarchaeia is firmly positioned near the median position between the Methanonatronarchaeia and Halobacteria–Hikarchaeia lineages. Functional predictions supported the transitional status of Ca. Ordosarchaeia with the metabolic potential of nonmethanogenic and aerobic chemoheterotrophy, as did remnants of the gene sequences of methylamine/dimethylamine/trimethylamine metabolism and coenzyme M biosynthesis. Based on the similarity of the methyl-coenzyme M reductase genes mcrBGADC in Methanonatronarchaeia with the phylogenetically distant methanogens, an alternative evolutionary scenario is proposed, in which Methanonatronarchaeia, Ca. Ordosarchaeia, Ca. Hikarchaeia, and Halobacteria share a common ancestor that initially lost mcr genes. However, certain members of Methanonatronarchaeia subsequently acquired mcr genes through horizontal gene transfer from distantly related methanogens. This hypothesis is supported by amalgamated likelihood estimation, phylogenetic analysis, and gene arrangement patterns. Altogether, Ca. Ordosarchaeia genomes clarify the sisterhood of Methanonatronarchaeia with Halobacteria and provide new insights into the evolution from methanogens to haloarchaea.


Supplementary results on other features of the gene content of Ca. Ordosarchaeia
In the five representative MAGs of Ca.Ordosarchaeia obtained in the research, most of the genes of the archaeal DNA replication complex were annotated (Table S6).In addition, the encoding gene of the bacterial DNA primase DnaG was found.Four genomes contain the genes of Cdc6-related protein, which participates in the replication initiation of chromosomes and megaplasmids.
The members of Ca.Ordosarchaeia harbor the encoding genes of diverse DNA repair systems (Table S6).Homologous recombination (single-stranded DNA-specific exonuclease RecJ, recombinase RecA/RadA, and archaeal type of Holliday junction resolvase) and base excision repair (DNA glycosylases MutY and AlkA) might play a role considering the presence of the relevant marker genes.The encoding gene of deoxyribodipyrimidine photolyase repairing ultraviolet damage in DNA was predicted.
However, the encoding genes of MutSLH, UvrABC, and DNA end-binding protein Ku functioning in mismatch repair, nucleotide excision repair, and non-homologous endjoining, respectively, were not found.In fact, the uvrABC genes of nucleotide excision repair are also absent in Methanonatronarchaeia, whereas they are widely distributed in Halobacteria, Ca.Hikarchaeia, Methanomicrobia, and some other classes.
The genomes of Ods04 and Ods05 contain the sequences of ssDNA and dsDNA phages (Table S5).Correspondingly, the two genomes lack a restriction-modification system and CRISPR-Cas system.Ods04 harbors the encoding gene of SoFic protein, which is involved in posttranslational AMPylation (i.e., AMPylase).In fact, SoFic is involved in diverse molecular activities and biological functions.The physiological function of SoFic in Ods04 remains unclear.
The genome of Ods01 harbors the type II restriction-modification system, whereas Ods02 has diverse defense systems including the type I and II restriction-modification systems and the subtype I-B CRISPR-Cas system.The defense systems might protect the hosts from infection by phages.
The sparse existence of the encoding genes of transposases in the genomes of Ca.
Ordosarchaeia species indicates the possibility of the presence of inserted sequences (Table S6).S1).S2.The detailed parameters are described in the Methods.

Fig. S1
The number at the node represents the percentage of the posterior probability of each partition.Some branches are collapsed, and the number in the bracket represents the number of genomes.The colored line displays the presence of functional genes in the genomes.The order of the genes from left to right and their functions are listed in Table S6a.The accession numbers of the genomes in the phylum Halobacteriota are listed in Table S1.S7.The detailed approaches are described in the Methods.
Fig. S1 Relative abundance and hypersaline adaptation strategy of Ca.Ordosarchaeia in soda-saline lake or enrichment samples.

Fig. S2
Fig. S2 Phylogenetic analyses of the classes in the phylum Halobacteriota with different trimming methods.

Fig. S3
Fig. S3Bayes tree reconstruction of the classes in the phylum Halobacteriota.

Fig. S4
Fig. S4 Phylogenetic analyses of the classes in the phylum Halobacteriota with the removals of the aspartate and glutamate residues.

Fig. S6
Fig.S6  Species tree of ALE analysis with node numbers shown.
Fig. S1 Relative abundance and hypersaline adaptation strategy of Ca.Ordosarchaeia in soda-saline lakes or enrichment samples.a. Relative abundance of obtained Ca.Ordosarchaeia is expressed as rpkm in sodasaline lakes (brine, surface sediment, and deep sediment samples) and the enrichment cultures of deep sediments with different conditions (no substrate, formate, and acetate added).Dot size presents the logarithm of the rpkm.The metagenomic sequencing data of samples could be found via the BioProject identifiers provided in the Data

Fig. S2
Fig. S2 Phylogenetic analyses of the classes in the phylum Halobacteriota with different trimming methods.The multiple sequence alignment (MSA) of the 53 archaeal marker proteins prior to maximum-likelihood tree reconstruction (also the LG+F+G4 model) was trimmed using BMGE (a) and ClipKIT (b) for the removal of phylogenetically uninformative fast-evolving sites.The number at the node represents the percentage of >70% ultrafast bootstrap support after 1,000 iterations.Some branches are collapsed, and the number in the bracket represents the number of sequences.Both trees reveal the same topology at the class level as that based on the untrimmed MSA in Fig. 2a.

Fig. S3
Fig. S3 Bayes tree reconstruction of the classes in the phylum Halobacteriota.The phylogenomic trees are based on the 53 archaeal marker proteins, and the reference members are listed in TableS2.The detailed parameters are described in the Methods.

Fig. S6
Fig.S6  Species tree of ALE analysis with node numbers shown.The species tree was extracted from the ALE analyses to show node numbers.A maximum-likelihood tree was read as input for the ALE analysis.During the ALE analysis, only the topology was used and the branch lengths were not used.

Fig. S7
Fig. S7 Phylogenetic analyses of McrBG subunits.Two trees show different subunits: a. McrB and homolog (LG+F+I+G4 model); b.McrG and homolog (LG+I+G4 model).The circle at the node signifies an ultrafast bootstrap support of >70% based on 1 000 iterations.Some branches are collapsed, and the number in the bracket represents the number of sequences.The red label indicates that the sequences were affiliated with lineages of the phylum Halobacteriota.All the sequences are listed in TableS7.The detailed approaches are described in the Methods.

Table S1
Statistic and genomic summary of Ca.Ordosarchaeia and related lineages in the phylum Halobacteriota Table S2 Reference genomes for phylogenetic and comparative genomic analyses

Table S4
Statistic summary of Ca.Ordosarchaeia-related samples

Table S5
Functional assignment of the encoding genes of five Ca.Ordosarchaeia genomes using arCOG and eggNOG

Table S6
Functional genes of Ca.Ordosarchaeia and reference lineages as well as ancestral and evolutionary inference

Table S7
Summary of methanogenesis-related marker proteins in archaea

Table S8
Genes of interest as well as up-and down-stream genes in the genomes of representative members

Table S9 Halobacteria
taxa that maintain the cells intact under nonsaline conditions