Analysis of nearly 3000 archaeal genomes from terrestrial geothermal springs sheds light on interconnected biogeochemical processes.

Terrestrial geothermal springs are physicochemically diverse and host abundant populations of Archaea. However, the diversity, functionality, and geological influences of these Archaea are not well understood. Here we explore the genomic diversity of Archaea in 152 metagenomes from 48 geothermal springs in Tengchong, China, collected from 2016 to 2021. Our dataset is comprised of 2949 archaeal metagenome-assembled genomes spanning 12 phyla and 392 newly identified species, which increases the known species diversity of Archaea by ~48.6%. The structures and potential functions of the archaeal communities are strongly influenced by temperature and pH, with high-temperature acidic and alkaline springs favoring archaeal abundance over Bacteria. Genome-resolved metagenomics and metatranscriptomics provide insights into the potential ecological niches of these Archaea and their potential roles in carbon, sulfur, nitrogen, and hydrogen metabolism. Furthermore, our findings illustrate the interplay of competition and cooperation among Archaea in biogeochemical cycles, possibly arising from overlapping functional niches and metabolic handoffs. Taken together, our study expands the genomic diversity of Archaea inhabiting geothermal springs and provides a foundation for more incisive study of biogeochemical processes mediated by Archaea in geothermal ecosystems.

Temperature, pH, and oxygen availability contributed to the functional differentiation of ancient Nitrososphaeria.

Ammonia-oxidizing Nitrososphaeria are among the most abundant archaea on Earth and have profound impacts on the biogeochemical cycles of carbon and nitrogen. In contrast to these well-studied ammonia-oxidizing archaea (AOA), deep-branching non-AOA within this class remain poorly characterized because of a low number of genome representatives. Here, we reconstructed 128 Nitrososphaeria metagenome-assembled genomes from acid mine drainage and hot spring sediment metagenomes. Comparative genomics revealed that extant non-AOA are functionally diverse, with capacity for carbon fixation, carbon monoxide oxidation, methanogenesis, and respiratory pathways including oxygen, nitrate, sulfur, or sulfate, as potential terminal electron acceptors. Despite their diverse anaerobic pathways, evolutionary history inference suggested that the common ancestor of Nitrososphaeria was likely an aerobic thermophile. We further surmise that the functional differentiation of Nitrososphaeria was primarily shaped by oxygen, pH, and temperature, with the acquisition of pathways for carbon, nitrogen, and sulfur metabolism. Our study provides a more holistic and less biased understanding of the diversity, ecology, and deep evolution of the globally abundant Nitrososphaeria.

Metagenomic Discovery of "Candidatus Parvarchaeales"-Related Lineages Sheds Light on Adaptation and Diversification from Neutral-Thermal to Acidic-Mesothermal Environments.

"Candidatus Parvarchaeales" microbes, representing a DPANN archaeal group with limited metabolic potential and reliance on hosts for their growth, were initially found in acid mine drainage (AMD). Due to the lack of representatives, however, their ecological roles and adaptation to extreme habitats such as AMD as well as how they diverge across the lineage remain largely unexplored. By applying genome-resolved metagenomics, 28 Parvarchaeales-associated metagenome-assembled genomes (MAGs) representing two orders and five genera were recovered. Among them, we identified three new genera and proposed the names "Candidatus Jingweiarchaeum," "Candidatus Haiyanarchaeum," and "Candidatus Rehaiarchaeum," with the former two belonging to a new order, "Candidatus Jingweiarchaeales." Further analyses of the metabolic potentials revealed substantial niche differentiation between Jingweiarchaeales and Parvarchaeales. Jingweiarchaeales may rely on fermentation, salvage pathways, partial glycolysis, and the pentose phosphate pathway (PPP) for energy conservation reservation, while the metabolic potentials of Parvarchaeales might be more versatile. Comparative genomic analyses suggested that Jingweiarchaeales favor habitats with higher temperatures and that Parvarchaeales are better adapted to acidic environments. We further revealed that the thermal adaptation of these lineages, especially Haiyanarchaeum, might rely on genomic features such as the usage of specific amino acids, genome streamlining, and hyperthermophile featured genes such as rgy. Notably, the adaptation of Parvarchaeales to acidic environments was possibly driven by horizontal gene transfer (HGT). The reconstruction of ancestral states demonstrated that both may have originated from thermal and neutral environments and later spread to mesothermal and acidic environments. These evolutionary processes may also be accompanied by adaptation to oxygen-rich environments via HGT. IMPORTANCE "Candidatus Parvarchaeales" microbes may represent a lineage uniquely distributed in extreme environments such as AMD and hot springs. However, little is known about the strategies and processes of how they adapted to these extreme environments. By the discovery of potential new order-level lineages, "Ca. Jingweiarchaeales," and in-depth comparative genomic analysis, we unveiled the functional differentiation of these lineages. Furthermore, we show that the adaptation of these lineages to high-temperature and acidic environments was driven by different strategies, with the former relying more on genomic characteristics such as genome streamlining and amino acid compositions and the latter relying more on the acquisition of genes associated with acid tolerance. Finally, by the reconstruction of the ancestral states of the optimal growth temperature (OGT) and isoelectric point (pI), we showed the potential evolutionary process of Parvarchaeales-related lineages with regard to the shift from the high-temperature environment of their common ancestors to low-temperature (potentially acidic) environments.

Panguiarchaeum symbiosum, a potential hyperthermophilic symbiont in the TACK superphylum.

The biology of Korarchaeia remains elusive due to the lack of genome representatives. Here, we reconstruct 10 closely related metagenome-assembled genomes from hot spring habitats and place them into a single species, proposed herein as Panguiarchaeum symbiosum. Functional investigation suggests that Panguiarchaeum symbiosum is strictly anaerobic and grows exclusively in thermal habitats by fermenting peptides coupled with sulfide and hydrogen production to dispose of electrons. Due to its inability to biosynthesize archaeal membranes, amino acids, and purines, this species likely exists in a symbiotic lifestyle similar to DPANN archaea. Population metagenomics and metatranscriptomic analyses demonstrated that genes associated with amino acid/peptide uptake and cell attachment exhibited positive selection and were highly expressed, supporting the proposed proteolytic catabolism and symbiotic lifestyle. Our study sheds light on the metabolism, evolution, and potential symbiotic lifestyle of Panguiarchaeum symbiosum, which may be a unique host-dependent archaeon within the TACK superphylum.

Functional differentiation determines the molecular basis of the symbiotic lifestyle of Ca. Nanohaloarchaeota.

BACKGROUND: Candidatus Nanohaloarchaeota, an archaeal phylum within the DPANN superphylum, is characterized by limited metabolic capabilities and limited phylogenetic diversity and until recently has been considered to exclusively inhabit hypersaline environments due to an obligate association with Halobacteria. Aside from hypersaline environments, Ca. Nanohaloarchaeota can also have been discovered from deep-subsurface marine sediments. RESULTS: Three metagenome-assembled genomes (MAGs) representing a new order within the Ca. Nanohaloarchaeota were reconstructed from a stratified salt crust and proposed to represent a novel order, Nucleotidisoterales. Genomic features reveal them to be anaerobes capable of catabolizing nucleotides by coupling nucleotide salvage pathways with lower glycolysis to yield free energy. Comparative genomics demonstrated that these and other Ca. Nanohaloarchaeota inhabiting saline habitats use a "salt-in" strategy to maintain osmotic pressure based on the high proportion of acidic amino acids. In contrast, previously described Ca. Nanohaloarchaeota MAGs from geothermal environments were enriched with basic amino acids to counter heat stress. Evolutionary history reconstruction revealed that functional differentiation of energy conservation strategies drove diversification within Ca. Nanohaloarchaeota, further leading to shifts in the catabolic strategy from nucleotide degradation within deeper lineages to polysaccharide degradation within shallow lineages. CONCLUSIONS: This study provides deeper insight into the ecological functions and evolution of the expanded phylum Ca. Nanohaloarchaeota and further advances our understanding on the functional and genetic associations between potential symbionts and hosts. Video Abstract.

Deciphering Symbiotic Interactions of "Candidatus Aenigmarchaeota" with Inferred Horizontal Gene Transfers and Co-occurrence Networks.

"Candidatus Aenigmarchaeota" ("Ca. Aenigmarchaeota") represents one of the earliest proposed evolutionary branches within the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, their ecological roles and potential host-symbiont interactions are still poorly understood. Here, eight metagenome-assembled genomes (MAGs) were reconstructed from hot spring ecosystems, and further in-depth comparative and evolutionary genomic analyses were conducted on these MAGs and other genomes downloaded from public databases. Although with limited metabolic capacities, we reported that "Ca. Aenigmarchaeota" in thermal environments harbor more genes related to carbohydrate metabolism than "Ca. Aenigmarchaeota" in nonthermal environments. Evolutionary analyses suggested that members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota contribute substantially to the niche expansion of "Ca. Aenigmarchaeota" via horizontal gene transfer (HGT), especially genes related to virus defense and stress responses. Based on co-occurrence network results and recent genetic exchanges among community members, we conjectured that "Ca. Aenigmarchaeota" may be symbionts associated with one MAG affiliated with the genus Pyrobaculum, though host specificity might be wide and variable across different "Ca. Aenigmarchaeota" organisms. This study provides significant insight into possible DPANN-host interactions and ecological roles of "Ca. Aenigmarchaeota." IMPORTANCE Recent advances in sequencing technology promoted the blowout discovery of super tiny microbes in the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, and Nanohaloarchaeota (DPANN) superphylum. However, the unculturable properties of the majority of microbes impeded our investigation of their behavior and symbiotic lifestyle in the corresponding community. By integrating horizontal gene transfer (HGT) detection and co-occurrence network analysis on "Candidatus Aenigmarchaeota" ("Ca. Aenigmarchaeota"), we made one of the first attempts to infer their putative interaction partners and further decipher the potential functional and genetic interactions between the symbionts. We revealed that HGTs contributed by members from the Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota (TACK) superphylum and Euryarchaeota conferred "Ca. Aenigmarchaeota" with the ability to survive under different environmental stresses, such as virus infection, high temperature, and oxidative stress. This study demonstrates that the interaction partners might be inferable by applying informatics analyses on metagenomic sequencing data.

Comparative Genomics Reveals Thermal Adaptation and a High Metabolic Diversity in "Candidatus Bathyarchaeia".

"Candidatus Bathyarchaeia" is a phylogenetically diverse and widely distributed lineage often in high abundance in anoxic submarine sediments; however, their evolution and ecological roles in terrestrial geothermal habitats are poorly understood. In the present study, 35 Ca. Bathyarchaeia metagenome-assembled genomes (MAGs) were recovered from hot spring sediments in Tibet and Yunnan, China. Phylogenetic analysis revealed all MAGs of Ca. Bathyarchaeia can be classified into 7 orders and 15 families. Among them, 4 families have been first discovered in the present study, significantly expanding the known diversity of Ca. Bathyarchaeia. Comparative genomics demonstrated Ca. Bathyarchaeia MAGs from thermal habitats to encode a large variety of genes related to carbohydrate degradation, which are likely a metabolic adaptation of these organisms to a lifestyle at high temperatures. At least two families are potential methanogens/alkanotrophs, indicating a potential for the catalysis of short-chain hydrocarbons. Three MAGs from Family-7.3 are identified as alkanotrophs due to the detection of an Mcr complex. Family-2 contains the largest number of genes relevant to alkyl-CoM transformation, indicating the potential for methylotrophic methanogenesis, although their evolutionary history suggests the ancestor of Ca. Bathyarchaeia was unable to metabolize alkanes. Subsequent lineages have acquired the ability via horizontal gene transfer. Overall, our study significantly expands our knowledge and understanding of the metabolic capabilities, habitat adaptations, and evolution of Ca. Bathyarchaeia in thermal environments. IMPORTANCE Ca. Bathyarchaeia MAGs from terrestrial hot spring habitats are poorly revealed, though they have been studied extensively in marine ecosystems. In this study, we uncovered the metabolic capabilities and ecological role of Ca. Bathyarchaeia in hot springs and give a comprehensive comparative analysis between thermal and nonthermal habitats to reveal the thermal adaptability of Ca. Bathyarchaeia. Also, we attempt to determine the evolutionary history of methane/alkane metabolism in Ca. Bathyarchaeia, since it appears to be the first archaea beyond Euryarchaeota which contains the mcrABG genes. The reclassification of Ca. Bathyarchaeia and significant genomic differences among different lineages largely expand our knowledge on these cosmopolitan archaea, which will be beneficial in guiding the future studies.

Insights into the ecological roles and evolution of methyl-coenzyme M reductase-containing hot spring Archaea.

Several recent studies have shown the presence of genes for the key enzyme associated with archaeal methane/alkane metabolism, methyl-coenzyme M reductase (Mcr), in metagenome-assembled genomes (MAGs) divergent to existing archaeal lineages. Here, we study the mcr-containing archaeal MAGs from several hot springs, which reveal further expansion in the diversity of archaeal organisms performing methane/alkane metabolism. Significantly, an MAG basal to organisms from the phylum Thaumarchaeota that contains mcr genes, but not those for ammonia oxidation or aerobic metabolism, is identified. Together, our phylogenetic analyses and ancestral state reconstructions suggest a mostly vertical evolution of mcrABG genes among methanogens and methanotrophs, along with frequent horizontal gene transfer of mcr genes between alkanotrophs. Analysis of all mcr-containing archaeal MAGs/genomes suggests a hydrothermal origin for these microorganisms based on optimal growth temperature predictions. These results also suggest methane/alkane oxidation or methanogenesis at high temperature likely existed in a common archaeal ancestor.

Genomic inference of the metabolism and evolution of the archaeal phylum Aigarchaeota.

Microbes of the phylum Aigarchaeota are widely distributed in geothermal environments, but their physiological and ecological roles are poorly understood. Here we analyze six Aigarchaeota metagenomic bins from two circumneutral hot springs in Tengchong, China, to reveal that they are either strict or facultative anaerobes, and most are chemolithotrophs that can perform sulfide oxidation. Applying comparative genomics to the Thaumarchaeota and Aigarchaeota, we find that they both originated from thermal habitats, sharing 1154 genes with their common ancestor. Horizontal gene transfer played a crucial role in shaping genetic diversity of Aigarchaeota and led to functional partitioning and ecological divergence among sympatric microbes, as several key functional innovations were endowed by Bacteria, including dissimilatory sulfite reduction and possibly carbon monoxide oxidation. Our study expands our knowledge of the possible ecological roles of the Aigarchaeota and clarifies their evolutionary relationship to their sister lineage Thaumarchaeota.