Ecological drivers of Promethearchaeati’s specialization for deep subsurface environments

Abstract

Promethearchaeati, a recently discovered kingdom of archaea, shares striking similarities with eukaryotes, including genes for cellular complexity previously thought unique to eukaryotes. Despite these features, Promethearchaeati thrive almost exclusively in stable subsurface environments, unlike their eukaryotic relatives that thrive on Earth’s surface. Here we explore why these microbes remain confined to the subsurface, examining their slow growth, fragile cellular structures, and dependence on microbial partners. We discuss how their eukaryotic-like traits might function in subsurface ecosystems and why these traits may be selective in these types of ecosystems.

Promethearchaeati are archaea with eukaryote-like genes, yet they live mainly in stable subsurface habitats. Their slow growth, fragile cells, and reliance on partners may explain why these traits are favored underground but not at Earth’s surface.

Introduction

Promethearchaeati is a kingdom of archaea (formerly the Asgard archaea superphylum) with a high degree of phylogenetic and functional similarity to eukaryotes^1,2. In fact, many phylogenetic analyses suggest that the eukaryotes branch out of one of the subgroups (Heimdallarchaeota or Heimdallarchaeia) of the Promethearchaeati archaea^2–5. Promethearchaeati are so phylogenetically divergent from all previously discovered organisms that the current taxonomic system has not easily included them. However, they differ from eukaryotes in that they are primarily found in subsurface environments, where they are often one of the most abundant clades present⁵. Here, we examine what is known about their physiology and ecology and suggest possibilities for why these features may explain their apparent preference for subsurface environments, which tend to be very stable over time.

We use the definition of subsurface provided in Ruff et al. 2024, as soils, rocks, or sediments deeper than a meter below Earth’s land surface or deeper than 100 cm meters below the seafloor (cmbsf)⁶. The marine subsurface, in particular, contains a vast microbial ecosystem, with estimates of ~3 × 1029 living cells⁷. Since Promethearchaeati comprise such a large part of this ecosystem, they may drive much of its ecological functioning and biogeochemical cycling. In turn, this environment seems to be particularly important for these archaea, since they are rarely found in terrestrial, freshwater, or surficial environments. However, the fact that Promethearchaeati are primarily associated with marine subsurface environments might be an important clue for determining their ecological and physiological roles in Earth’s ecosystems.

The production of many diverse metagenome-assembled genomes (MAGs) from Promehearchaeati, as well as the isolation of the first strains from some of its subgroups: Prometharchaeia (formerly Lokiarchaeia), Odinarchaeia, and Hodarchaeales, has been steadily increasing the information that is available about the environmental biogeochemical and ecological functions of this kingdom^1,8,9. Here, we review how the inferred physiology of these organisms may relate to the fact that they are commonly only abundant in marine sedimentary systems. This may also begin to explain why they have not shared the eukaryotes’ level of success on Earth’s surface.

Background on Promethearchaeati

Promethearchaeati were first identified through 16S rRNA gene amplicons from deep-sea marine sediments. Early literature identifying these groups via 16S rRNA gene sequences named them Marine Benthic Group B (MBG-B), Ancient Archaeal Group (AAG), Deep Sea Archaeal Group (DSAG), and Marine Hydrothermal Vent Group (MHVG)^10–12. They had to be given these new names because they did not fit cleanly into any known taxa of microorganisms. These initial studies were exclusively conducted in deep-sea environments, and they established that this group of 16S rRNA gene sequences were basal to those of the rest of the archaea, making their exact phylogenetic placement difficult. The first images of Promethearchaeati were obtained by making small DNA probes with fluorescent tags for fluorescent in situ hybridization (FISH). This work showed that MBG-B/DSAG cells were very small (200–400 nm in diameter) and tended to cluster together with other cells¹³. The first nearly-complete genomes for Promethearchaeati were obtained from MAGs binned from shotgun metagenomic assemblies from environmental samples, giving them the name Lokiarchaeota, since they were obtained from deep-sea sediments near the Loki’s Castle hydrothermal vent field². The steadily growing library of MAGs, largely obtained from environmental samples, have identified classes/phyla within the Promethearchaeati, which until recently have been called the Asgard archaea. The placement of the highly diverse subgroups within it is in flux, with these subgroups alternately being referred to as phyla, subphyla, or classes^3,5,14,15. As of this perspective, the Genome Taxonomy Database (GTDB) lists the following classes: Baldrarchaeia, Heimdallarchaeia, Hermodarchaeia, Jordarchaeia, Lokiarchaeia (Promethearchaeia), Njordarchaeia, Odinarchaeia, Sifarchaeia, Thorarchaeia, and Wukongarchaeia. However, there are some discrepancies between the classification provided by GTDB and that of the National Center for Biotechnology Information (NCBI). NCBI includes the aforementioned classes under the phylum level, while also recognizing additional groups such as Borrarchaeia, Gerdarchaeia, Helarchaeia, Hodarchaeia, and Kariarchaeia. In contrast, GTDB classifies Hodarchaeia as an order under Heimdallarchaeia, whereas Kariarchaeia is classified as a family. As more MAGs and genomes become available, it is likely that these taxonomic classifications will be further updated and refined.

To further support the hypotheses derived from the analysis of MAGs, techniques such as environmental proteomics, metatranscriptomics, metabolomics, or the incorporation of chemically labeled substrates and geochemical measurements during incubations of environmental samples have been conducted. To date, Promethearchaeati has only been analyzed in conjunction with environmental metatranscriptomic data^16,17. Even with this limited data set, these analyses generally support the idea that the capabilities inferred from Promethearchaeati genomes may reflect their functional roles in nature. One nearly-pure culture, Promethearchaeum syntrophicum (MK-D1), has recently been obtained by incubating methane seep fluids for five years in a downflow hanging sponge bioreactor followed by an additional seven years of in vitro enrichment⁸. This isolation marked the first opportunity to directly observe and analyze the cellular morphology of members from this superphylum. This was followed by a second isolated culture, ʻCandidatus Lokiarchaeum ossiferumʼ, that was obtained by enriching sediments from an estuary with a mix of complex organics over the course of several months⁹. Recently, two additional strains belonging to the order ʻHodarchaealesʼ, ʻCandidatus Margulisarchaeum peptidophilaʼ (HC1) and ʻCandidatus Flexarchaeum multiprotrusionisʼ (SC1), have been successfully isolated by enriching biofilms from a gas production well¹. This has enabled the first direct physiological assessments of this kingdom, although their extremely slow growth rate, with doubling times ranging from ~ 7 to 25 days, limits the rate of experimentation on these organisms.

Environmental distribution

A key difference between Promethearchaeati and other archaea is that most cultured phyla of archaea, such as Euryarchaeota, Thermoplasmatota, Thermoprotatota, and Crenarchaeota, are broadly distributed in all Earth environments; however, Promethearchaeati appear to be abundant mainly in subsurface environments, buried under marine sediments or deep terrestrial and marine aquifers (Fig. 1)^18–20. A study of the relative abundance of all archaea and bacteria in marine sediments of the Southwest Atlantic Slope found that Promethearchaeati dominated the community at 200 cm sediment depth, but were absent in the upper 15 cm²¹.

Fig. 1. Habitat diversity of Promethearchaeati MAGs recovered from different environments (Modified from Appler, 2026).

The distribution pattern of Promethearchaeati across multiple habitats as well as the total number of associated MAGs (pink circle). Guaymas Basin is represented separately to highlight its genomic diversity^5,22.

Promethearchaeia, originally referred to as MBG-B and DSAG, was the first class within Promethearchaeati to be identified through environmental 16S rRNA gene amplicon surveys and was found in microbiomes from deep anoxic sediments from both coastal and deep-sea sites^2,9,12,23. A survey of archaeal groups across different types of sediments found that it is abundant in globally diverse deep marine sediments, but absent in surficial anoxic environments^6,24. Heimdallarchaeia, previously known as AAG and MHVG, is also primarily reported from deep-sea marine sediments but in some coastal sediments as well^25–27. Thorarchaeia displays a broader environmental distribution, being found in marine, lake, and estuarine sediments^25–29. The more recently-discovered Promethearchaeati such as Sifarchaeia, Hermodarchaeia, Odinarchaeia, Helarchaeia, and Baldarchaeia, are also found in subsurface coastal and marine habitats^28,30–33. Most of the Promethearchaeiti MAGs and 16S rRNA gene sequences have come from saline sediments^2,5,20,27,34. Although most samples containing Promethearchaeiti originate from lower-salinity environments (<38 psu), Promethearchaeiti comprise a larger share of the archaeal community, predominated by Lokiarchaeota and Thorarchaeota, under extreme salinity conditions (>38 psu)²⁰. It is exceedingly rare to find Promethearchaeati in pelagic marine environments or environments that are fully freshwater. However, a Heimdallarchaeia MAG was reconstructed from metagenomic data obtained in the Red Sea water column³⁵. One study found Thorarchaeia in freshwater wetland soil³⁶; however, the sediments were in a mudflat and salts may have been present in the soils. Currently, no MAGs of Promethearchaeati have been obtained from surficial soils or host-associated microbiomes despite a broad body of literature that has been conducted for these environments using many of the same primer sets and search methods that have successfully located them in marine sediments.

Functional and morphological similarities of Promethearchaeati to Eukaryotes

Some defining features of eukaryotes include membrane-bound compartmentalization of organelles, a well-defined nuclear envelope, and the ability to define and change their cellular shape, many of which are made possibly by eukaryotic signature proteins (ESPs), which are found only in Eukarya^37–39. The Promethearchaeati are the first-discovered group of non-eukaryotes that appear to share some characteristics of ESPs, with evidence from genomes, heterologous gene expression of Promethearchaeati-derived genes, microscopy, and culture-based growth studies^3,9,32,40,41. ESPs include proteins important for key eukaryotic cell functions related to membranes, cytoskeleton, the nucleus, and protein synthesis⁴². Among the genes encoding ESPs in Promethearchaeati genomes are those involved in intracellular trafficking, secretion, vesicular transport, post-translational modification, protein turnover, and chaperoning⁵. Additionally, MAGs from Heimdallarchaeia and Odinarchaeia encode homologs of the eukaryotic ubiquitin modifier systems as well as the Endosomal Sorting Complex Required for Transport (ESCRT)-mediated protein degradation pathway³². The ubiquitin and the ESCRT pathways work together to target and deliver damaged intracellular proteins for degradation to either the vacuole or lysosome of eukaryotic cells⁴³. Notably, some ESCRT-III proteins of both Promethearchaeati and human lineages exhibit conserved chromatin/DNA binding properties and interspecies interactions highlighting their close evolutionary relationship⁴⁴. In the genomes of Odinarchaeia and Heimdallarchaeia, the ubiquitin modifier system and ESCRT pathway are situated adjacent to each other within the same genomic region, suggesting that these systems operate in tandem, although colocalization does not always imply coordination³².

Eukaryotic and prokaryotic ribosomes usually supplement a common core rRNA structure with expansion segments of longer stretches of rRNA, however only eukaryotes contain Supersized Expanded Sites, which are larger than 100 nucleotides. Recently, Supersized Expanded Sites have been identified on the large ribosomal subunits of both Promethearchaeia and Heimdallarchaeia ribosomes⁴⁵. Eme and colleagues suggested that eukaryotes are not only phylogenetically positioned within the Promethearchaeati clade, but more specifically form a sister lineage to the newly discovered ʻHodarchaealesʼ, a proposed order within the Heimdallarchaeia class³.

Further evidence of Promethearchaeota’s eukaryotic-like features comes from their actin and profilin proteins. These proteins play a pivotal role in actin polymerization and depolymerization, processes essential for eukaryotic cell movement and shape. Promethearchaeati profilins have exhibited sensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that regulates actin nucleation in eukaryotes. While they share ancestry with their eukaryotic counterparts, these profilins form a distinct clade based on structure-based sequence alignments⁴⁰. This divergence suggests that Promethearchaeati profilins have unique structural adaptations relative to those of eukaryotes, but the clear homology with eukaryotic profilins suggests they may have similar roles in cellular processes. Promethearchaeati also appear to possess calcium-dependent regulatory mechanisms for actin dynamics, like those found in eukaryotes⁴⁶. Several potential eukaryotic-like calcium channels have been identified within Promethearchaeati genomes, suggesting the possibility of calcium-mediated regulation of actin dynamics. Calcium-controlled actin filament disassembly has been observed in the gelsolin proteins of Promethearchaeia, Heimdallarchaeia, and Thorarchaeia, signaling that calcium-dependent gelsolin may have been present in the last common ancestor of Promethearchaeati. Furthermore, in the absence of calcium ions, Heimdallarchaeia and MK-D1 gelsolin were unable to sequester actin monomers, further supporting the calcium dependency of actin sequestration by these proteins⁴⁶.

In addition, Promethearchaeati have characteristics that set them apart from the other archaea. Their small cell size and tendency to aggregate with other cells was first noted in 2005 through FISH of cells in their natural sediment matrix¹³. Currently, all isolates form actin-based protrusions of various lengths stemming from their membrane. This feature is hypothesized to be a universal characteristic of the Promethearchaeati, as all members have Lokiactin, a eukaryotic-like actin homolog, conserved within their genome⁴⁷. The function of the protrusions remains unknown; however, it has been proposed to have had a pivotal role in cell-to-cell interactions that led to eukaryogenesis^8,9. This is in contrast to the barbed appendages found in the archaeon ʻCandidatus Altiarchaeum hamiconexumʼ, which is in a different kingdom than the Promethearchaeati. ʻCa. Altiarchaeum hamiconexumʼ possess highly complex cell surface appendages called ‘hami’ that carry barbed hooks at their distant end. Unlike the Promethearchaeati protrusions, hami do not appear to contain cytoplasmic intrusions, suggesting that physical attachment may be the key function of hami, nor do they contain cytoskeletal elements.⁴⁸. In summary, Promethearchaeati may share some functional similarities with essential eukaryotic processes, distinct from those found in other archaea. Beyond simply supporting Promethearchaeati’s role in eukaryogenesis, these features may be important to Promethearchaeati’s continued success in Earth’s environments.

The high degree of similarity between Promethearchaeati and eukaryotes makes it particularly surprising that the eukaryotes are only found in low abundance in deep marine sediments^49–51, whereas this environment appears to be the primary environment for Promethearchaeati.

Inferred metabolism

The breadth of metabolic potential for Promethearchaeati and their ecological role in the environment are still poorly-understood; however, ongoing discoveries suggest they encompass a range of metabolic capabilities⁵². Metagenomic analyses have suggested that many Promethearchaeati are anaerobic and live heterotrophic or chemolithotrophic lifestyles. Some have fermentative pathways and/or the ability to perform anaerobic hydrocarbon, nitrogen, or sulfur cycling.^4,8,27. Methanogenesis is commonly associated with subsurface archaea; however, to date, no known Promethearchaeati species possess a complete pathway necessary for methane production, nor is there any geochemical or incubation evidence that they produce methane. Promethearchaeati may have syntrophic relationships with methanogens, with the first isolated member, P. syntropicum’, being grown in co-culture with Methanogenium sp.^3,5,8. Recently, newly proposed Promethearchaeati, Atabeyarchaeia and Freyarchaeia, have the metabolic capacity to utilize methylated compounds as a carbon source¹⁷.

Thorarchaeia have the genomic potential for protein degradation and assimilation. Their genomes also suggest that they can fix inorganic carbon, reduce sulfur/sulfate, and produce acetate^27,28. Like Thorarchaeia, Promethearchaeia are characterized by their capacity to degrade and assimilate proteins as well. Both encode a complete archaeal Wood–Ljungdahl pathway in many of their genomes and could possibly grow lithoautotrophically using H₂ as an electron donor^23,27,28,53. MAGs for Heimdallarchaeia seem to suggest that this class encompasses a variety of metabolisms using a range of organic substrates, with previous studies describing this phylum as polyphyletic². Heimdallarchaeia also has the capacity to use proteins and fatty acids as a source of carbon like the others, but it also encodes enzymes from the beta-oxidation pathway, suggesting that it can use short-chain fatty acids as both an energy and carbon source^53,54. Helarchaeia has variants of the methyl-COM reductase-like complex that grants it the potential to syntrophically oxidize short-chain fatty acids anaerobically with sulfate-reducing bacteria^4,55. MAGs of Sifarchaeia suggest that these archaea are heterotrophic and have an anaerobic lifestyle. Additionally, their metabolic capacity includes the production of various fermentation products, the ability to respire sulfate to sulfite and nitrite to ammonia, and the utilization of C₁ to C₃ compounds to synthesize acetyl coenzyme A (acetyl-CoA)⁵⁴.

Similar metabolisms are present in other bacterial and archaeal phyla that are abundant in both surface and subsurface environments^{11,21,26,30,56–66}, suggesting that Promethearchaeati’s metabolic capabilities per se do not confine this group to the subsurface.

Why are modern Promethearchaeti lineages primarily found in the subsurface?

It is tempting to suggest that Promethearchaeati are largely relegated to the subsurface because they are strict anaerobes that must be protected from the oxygen in Earth’s atmosphere. However, Earth’s surface contains many anoxic environments, such as animal guts, soils, wastewater treatment plants, landfills, and rice paddies, yet Promethearchaeati are rarely, if ever, found in these environments. Other strictly anaerobic Archaea, most notably the methanogens, have been highly successful in colonizing these environments⁶⁷. Additionally, some branches of Hodarchaeales contain genes indicative of oxygen respiration^1,22, Atabeyaracheia and Freyarchaeia encode genes for clade I catalases, iron/manganese superoxide dismutase, and a catalase-peroxidase that is specific to Freyarchaeia¹⁷. So, not all Promethearchaeati may be strict anaerobes. Also, the subsurface may in fact, contain small amounts of oxygen generated from dark oxygen production metabolisms⁶⁸. Therefore, anoxia is not likely to be the sole factor keeping Promethearchaeati in the subsurface.

In addition to being largely anoxic, much of the subsurface is also both physically and chemically stable, where microbial cells forgo the energetic gains of high substrate turnover while gaining the advantage of living in a stable environment with little threat of being washed out of the system or subjected to physical disturbance for thousands to billions of years^69,70. Earth’s surface, on the other hand, is continually in motion, mixing with the atmosphere and fast-moving multicellular organisms. Perhaps modern lineages of Promethearchaeati have successfully used the stability of the subsurface to their advantage. Below, we review features that could allow Promethearchaeati to use the type of highly stable conditions found in deep sediments or rock fractures to their advantage.

First, extremely slow growth, which also seems to be universal among the Promethearchaeati, would favor deep subsurface environments where cells do not need to continuously multiply to compensate for cells that washed from the system. All four enrichments have average doubling times of several weeks, and Promethearchaeati are normally in subsurface ecosystems where the energy delivery rate is too low to support significant cell division^6,69. Imachi et al. 2025, noted that increasing substrate concentration failed to accelerate cell division in Hodarchaeales isolates. Instead, cells channeled the extra energy into increasing cell size/volume and more protrusions, suggesting they invest more effort into existing cells than in cell division. Although some slow-growing organisms can be found in oligotrophic surface environments, slow growth would also be an advantage in a stable sedimentary environment that does not require fast growth to compensate for turn-over rate⁷¹.

Second, Promethearchaeati’s eukaryotic-like cytoskeletal elements that form arm-like cytoplasmic protrusions have proved to be fragile. All four enrichments have these structures and all MAGs of Promethearchaeati have genes for them. Attempts to “plunge-freeze” “Ca. Lokiarchaeum ossiferum” to prepare them for cryo-electron tomography (cryo-ET) failed due to their physical fragility⁹. Although ʻCa. Margulisarchaeum peptidophilaʼ and ʻCa. Felxarchaeum multiprotrusionisʼ did not fall apart during plunge-freezing, their protrusions fell off when treated with formaldehyde¹. If the fragility while undergoing laboratory procedures indicates a fragility in nature, then intolerance to rough conditions could be a reason they are not commonly found in surface environments. Subsurface environments (many of which contain stable accumulations of sediments) could be a beneficial place for a fragile cell to reside. Sediment pore size decreases with depth in marine sediments due to compaction over time. The subsurface can provide small and stable pore spaces between sediment grains or small fractures, which may provide an ideal location to house and support these cellular protrusions, providing a sort of geological analog to an exoskeleton^72,73.

The ecological success of Promethearchaeati in the subsurface may be due to their small cell size (~0.5 μm), which would allow them to inhabit smaller pore spaces at greater depths. Promethearchaeati generally occupy deeper subsurface environments where overlying sediment compaction leads to the restriction of pore connectivity⁶. However, the problem with these small pore spaces is that they are bounded by even smaller pore throat sizes, restricting the flow of materials and cells through the sediments⁷³. This means that even if small cells can fit in the pore spaces, they become physically isolated from any potential syntrophic partners, nutrients, or waste removal. We propose that it is therefore possible that Promethearchaeati use their cytoplasmic protrusions to overcome these physical constraints by sending the protrusions through pore throats to reach adjacent pore spaces (Fig. 2). If Promethearchaeati use these protrusions to pass nutrients to their syntrophic partners, which has yet to be directly shown experimentally, then this adaptation may enable nutrient acquisition from adjacent pores or facilitate proposed syntrophic interactions, as in the relationship with MC1, SC1, and their methanogenic partners. These protrusions might be damaged or destroyed in physically dynamic locations common on Earth’s surface, such as animal guts undergoing peristalsis, rapidly churning waters, or sediments in waste treatment plants. If Promethearchaeati cannot live without the protrusions, then it may be hard for them to compete with more physically robust organisms in these environments. An exception might be Heimdallarchaeia, which has been reported from an aquatic environment³⁵.

Fig. 2. Potential subsurface adaptation through cellular extensions.

Proposed mechanism of syntrophic interactions between Promethearchaeati (red) and methanogens (gray) in the subsurface. The illustration depicts how Promethearchaeati may utilize cytoplasmic extensions to traverse pore throat connections and enable nutrient exchange with neighboring cells in adjacent pore spaces.

Third, Promethearchaeati seem unable to grow without a partner. Both MK-D1 and L. ossiferum were successfully enriched by growing syntrophically with both a sulfate-reducing bacterium of the Halodesulvibrio genus and a hydrogenotrophic methanogen of the Methanogenium genus. Both MC1 and SC1 are dependent on a methanogen as well¹. There are other archaea whose growth are completely dependent on their host, such as some members of the DPANN group and Nanoarchaeum equitans with its host Ignicoccus hospitalis^74–76. This possible dependency on a symbiotic partner, and on fragile protrusions necessary for that syntrophy, may make it difficult to survive in turbulent environments where cells essential for syntrophy might be pushed away from each other.

It seems that the only relatives of the Promethearchaeati that survive in great quantities in surface environments are those that gained mitochondria and became the eukaryotes⁵³. Mitochondria create an excess of energy via aerobic respiration that powers the complex machinery of the eukaryotic cell. By generating high energy outputs, mitochondria may have facilitated the use of ESPs for the more complex functions that are necessary for maintaining complicated structures in physically demanding environments. This may also explain why Promethearchaeati are primarily located in the subsurface; they didn’t reap the benefit of mitochondria that could have facilitated their expansion beyond the subsurface. Of course, mitochondria are not required to live in surface environments, plenty of bacteria and archaea attest to this; however, they may be required for organisms on the Promethearchaeati and eukaryote branch of life to adapt to living in the more turbulent surface world.

Conclusions

These observations present a picture of Promethearchaeati as primarily subsurface organisms that occupy a unique position in the evolutionary timeline between prokaryotes and eukaryotes. They are unique among prokaryotes for having cytoskeletal structures and ESPs resembling those of eukaryotes. These structures may have provided fitness advantages allowing the persistence of Promethearchaeati in subsurface environments billions of years after the divergence of the last eukaryotic common ancestor. If so, then their dependence on the physical structures and stability of subsurface environments may explain why Promethearchaeati are rarely found in other environment types. This kingdom represents an exciting new area of study in microbial evolution, with recent discoveries steadily challenging what we thought we knew about archaeal diversity.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

L.S. conceived the perspective and wrote the original draft of the manuscript. L.S. and K.L. reviewed and revised the manuscript. Both authors approved the final version of the manuscript.

Peer review

Peer review information

Communications Biology thanks Masaru Nobu and the other, anonymous, reviewer for their contribution to the peer review of this work. Primary Handling Editor: Tobias Goris. A peer review file is available.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-026-09786-6.