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Peer-Review Record

POLETicians in the Mud: Preprokaryotic Organismal Lifeforms Existing Today (POLET) Hypothesis

Bacteria 2025, 4(3), 42; https://doi.org/10.3390/bacteria4030042

by Douglas M. Ruden^1,*

and Glen Ray Hood²

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3: Anonymous

Bacteria 2025, 4(3), 42; https://doi.org/10.3390/bacteria4030042

Submission received: 29 May 2025 / Revised: 30 July 2025 / Accepted: 26 August 2025 / Published: 29 August 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Inspired by the recent discovery of Asgard archaea, primitive life forms with significant “eukaryome” signature, from which eukaryotes apparently emerged as a symbiotic lineage, the authors propose that primitive life forms distinct from the extant most simple cells (prokaryotes) may still exist today in some unusual environments, where modern prokaryotes have no advantage over them (or cannot survive there without the help from the ancestral forms of Life). The authors imagine them as proto-cellular, membrane-bound vesicles reproduced by self-assembly and blebbing with a slow and primitive pre-enzyme metabolism, catalyzed by nucleotide and amino acid aptamers.

The authors point out some expectations about these primitive proto-cellular life forms (like racemic nature of their aptamers or their non-traditional sources of energy) and propose ways to find these life forms, which they call POLET (primitive organismal lifeforms existing today). One of the more practical expectations is symbiosis of the proto-cells with extant cells in borderline niches — this is indeed a good place to start looking for POLETs.

While the general idea is convincing, its presentation should benefit from more specific discussion, for which I tried to provide primers in my suggestions.

Suggestions

2.1. Lifespan characteristics — in this section, it is proposed that POLETs may have lifespan (cell cycle? division time?) of hundreds to thousands of years. This sounds extreme, for two reasons: first, growth and division at such a slow rate would be impossible to detect (human-led experimentation does not last that long); second, with such a slow metabolism, it would be impossible to repair the damage sustained by any complex chemical system (even POLETs). This is why the cell cycles cannot be longer than a certain maximal length (although those lengths are different for different environments) — which are days and weeks, but not months and years. Especially in hot acidic springs (line 133)!

2.2. Biochemistry and Genetics — it is proposed that primitive metabolism could be based on self-assembly of aptamers (oligonucleotides, oligopeptides) and mutual affinity between oligonucleotides and oligopeptides, based on the often-speculated affinity of amino acids to their RNA codons or anticodons (trinucleotides). Self-assembly and mutual affinity may be fine for how “Biochemistry” acts to increase the complexity and mass (=growth) of pre-cells, but what distinguish life from non-life is not only growth, but the ability to reproduce, based on “Genetics”. Unfortunately, the authors offer no speculation on how pre-cells could reproduce.

2.3. Blebbing as a reproductive strategy — “reproduction” in Life means producing daughter cells that are almost exact copies of the mother cell. Blebbing as a way to produce daughter proto-cells is fine, but where is the reproduction proper? What are the mechanisms to ensure that daughter blebs are similar to mother proto-cell?

2.4. Membranes — What would be the source of lipids for pre-cells? Speculate of their mode of production.

2.5. Detectability — The authors speculate that pre-cells may be still racemic — that is, they use a mixture of D and L amino acids in their oligopeptides and D and L sugars in their oligonucleotides. This is a reasonable idea, but then, instead of staying with racemic aptamers, the authors concentrate on discussing how to detect the complete mirror L-RNAs (using mirror D-aa enzymes).

Apparently they are impressed with the “fantastic” reports of mirror-image (D-aa) enzymes synthesizing mirror-image (L-nt) RNAs and DNAs. I suggest to stay with racemic, rather than with mirror-image biopolymers, and generally away from this mirror-image cell fantasy.

2.7. Speculations about alternative sources of energy are welcome, but since the major problem of Life is to maintain its molecular complexity against the constant forces of destruction, the authors should think in terms of the required energy to sustain damage-repair balance for systems of varied complexities. For example, if a modern cell has a complexity 1,000 and takes 100 hits from its environment per day, at the least every day it has to extract from its environment enough energy to repair these 100 hits — just to stay alive. Now we can imagine a POLET with a complexity of 100 and the environmental damaging impact of 10 per day. Yes, its energy requirements are significantly lower, but still it needs to extract at least 10 repair units of energy from its environment. Are these alternative sources (like radioactive decay) adequate to cover the needs of a pre-cell?

2.9. Definition of Life — in order to call for a broader definition of life, the authors should give the general definition first, and then to propose aspects that need broadening. I suspect that the most general Life’s definition likely covers both extant cells and POLETs.

Miscellaneous

Page 2, line 84 — fix “review,weexamine”

Page 8, line 320 — insert “with” after “metabolism”?

Author Response

Reviewer 1:

Comments and Suggestions for Authors

Comment: Inspired by the recent discovery of Asgard archaea, primitive life forms with significant “eukaryome” signature, from which eukaryotes apparently emerged as a symbiotic lineage, the authors propose that primitive life forms distinct from the extant most simple cells (prokaryotes) may still exist today in some unusual environments, where modern prokaryotes have no advantage over them (or cannot survive there without the help from the ancestral forms of Life). The authors imagine them as proto-cellular, membrane-bound vesicles reproduced by self-assembly and blebbing with a slow and primitive pre-enzyme metabolism, catalyzed by nucleotide and amino acid aptamers.

While the general idea is convincing, its presentation should benefit from more specific discussion, for which I tried to provide primers in my suggestions.

Response: We appreciate your feedback and the hard work that you put into this review. We will try to address all of your comments as discussed below. The changes in the text are marked in BOLD.

Suggestions

Comment 2.1. Lifespan characteristics — in this section, it is proposed that POLETs may have lifespan (cell cycle? division time?) of hundreds to thousands of years. This sounds extreme, for two reasons: first, growth and division at such a slow rate would be impossible to detect (human-led experimentation does not last that long); second, with such a slow metabolism, it would be impossible to repair the damage sustained by any complex chemical system (even POLETs). This is why the cell cycles cannot be longer than a certain maximal length (although those lengths are different for different environments) — which are days and weeks, but not months and years. Especially in hot acidic springs (line 133)!

Response: We have changed section 2.1.1. to, “POLETicians and other pre-prokaryotes (PPKs), if they exist today, may possess remarkably long cell cycles, potentially lasting weeks or even months, and lifespans that could last years or decades or even millennia in dormant states.”

Comment 2.2. Biochemistry and Genetics — it is proposed that primitive metabolism could be based on self-assembly of aptamers (oligonucleotides, oligopeptides) and mutual affinity between oligonucleotides and oligopeptides, based on the often-speculated affinity of amino acids to their RNA codons or anticodons (trinucleotides). Self-assembly and mutual affinity may be fine for how “Biochemistry” acts to increase the complexity and mass (=growth) of pre-cells, but what distinguish life from non-life is not only growth, but the ability to reproduce, based on “Genetics”. Unfortunately, the authors offer no speculation on how pre-cells could reproduce.

Response: We thank the reviewer for this insightful comment, which draws an important distinction between the biochemical complexity needed for pre-cellular growth and the genetic mechanisms required for reproduction—one of the hallmarks distinguishing life from non-life. In response, we have revised the manuscript to more explicitly address how POLETs may have bridged this gap by developing primitive modes of heredity and division.

In the revised Discussion section, we clarify that while the initial growth of POLETs may have relied on self-assembly of oligonucleotides and oligopeptides through mutual chemical affinities (e.g., codon–amino acid interactions), we now propose a plausible pathway for the emergence of reproduction. Specifically, we suggest that blebbing-based division of membrane-enclosed POLETs, driven by localized accumulation of functional oligomers (e.g., RNA aptamers or short peptides), could result in daughter vesicles inheriting subsets of functional components. This imperfect but selective transmission of molecular content constitutes a primitive form of heredity—a proto-genetic system capable of evolution by selection.

We also now explicitly relate these mechanisms to modern hypotheses of non-template-based inheritance, drawing analogies to catalytic feedback loops or compositional heredity models, where inheritance is based on the relative abundances and mutual reinforcement of catalytic components rather than a digitally encoded genome. These additions highlight that while POLETs may not have possessed fully developed replication machinery, they could nonetheless propagate compositional information across generations—a necessary precursor to the evolution of templated RNA-based genetics.

This conceptual addition is reflected in both the Discussion and the new Figure 2, which situates POLETs and other hypothetical PPKs (pre-prokaryotic lifeforms) in the “roots” of the tree of life as systems capable of growth and rudimentary reproduction via stochastic or quasi-deterministic partitioning of functional substructures. We hope this addresses the reviewer’s concern and strengthens the proposed bridge from chemistry to biology.

Comment 2.3. Blebbing as a reproductive strategy — “reproduction” in Life means producing daughter cells that are almost exact copies of the mother cell. Blebbing as a way to produce daughter proto-cells is fine, but where is the reproduction proper? What are the mechanisms to ensure that daughter blebs are similar to mother proto-cell?

Response: We appreciate the reviewer’s important clarification regarding the distinction between mere physical division (e.g., blebbing) and true biological reproduction, which requires the generation of daughter entities that are faithful, or at least functionally similar, copies of the parent.

In the revised manuscript, particularly in Section 2.3.1 and the Discussion, we now explicitly acknowledge that blebbing alone does not constitute full-fledged reproduction in the biological sense. However, we propose that in the context of early prebiotic systems like POLETs, blebbing may have served as a primitive precursor to genetic reproduction by enabling the non-random inheritance of compositional and functional information. Specifically, if certain localized oligomeric complexes (e.g., RNA aptamers or catalytic peptides) tended to aggregate within regions of a vesicle prior to division, their co-segregation into daughter blebs could preserve rudimentary “functional identities” across generations.

We emphasize that this process does not require precise templating, but instead may follow a compositional inheritance model, where networked molecular functions—not linear genetic sequences—are preserved and propagated. Such models have been previously proposed in the literature (e.g., Gánti’s chemoton theory and Kauffman’s autocatalytic sets), and we now cite these to support our framework.

To further address the reviewer’s concern, we discuss potential feedback mechanisms whereby the molecular composition of a bleb could influence its capacity for further growth, stability, or catalytic activity—creating a selectable phenotype. Over successive generations of blebbing, such selection could drive systems toward increased functional fidelity even in the absence of nucleotide-sequence-based replication.

This conceptual bridge between blebbing and early heredity is also represented in the revised Figure 1, where POLETs and PPKs are positioned in the "Roots of Life" as systems capable of both growth and primitive compositional reproduction. We hope these additions clarify that while blebbing alone is insufficient, it may have formed the structural foundation upon which more accurate reproduction and eventually digital genetics could evolve.

Comment 2.4. Membranes — What would be the source of lipids for pre-cells? Speculate of their mode of production.

Response: We thank the reviewer for raising this important point. In response, we have expanded the discussion of lipid sources and membrane formation in the revised manuscript, particularly in Section 2.4.1, where we describe possible origins and functions of early membrane-like structures in POLETs.

We now propose that pre-cellular membranes may have originated from abiotically synthesized amphiphilic molecules, including simple fatty acids or hydrocarbon derivatives produced via Fischer–Tropsch-type reactions, which can occur under hydrothermal vent conditions or on mineral surfaces in the presence of CO, H₂, and metal catalysts such as Fe or Ni. This abiotic synthesis is consistent with findings from meteorites (e.g., Murchison) and prebiotic simulations that demonstrate the spontaneous formation of amphiphiles under plausible early Earth conditions.

Once formed, these amphiphiles could self-assemble into micelles or vesicles in aqueous environments, especially those with fluctuating temperatures, pH, or ionic strengths, as would be expected in dynamic hydrothermal or radiolytic zones. We now speculate in the revised text that such vesicles could grow by incorporating additional amphiphiles synthesized in situ or adsorbed from their surroundings—providing a rudimentary membrane that stabilizes internal reactions and facilitates blebbing.

Additionally, we propose that local redox gradients and mineral-catalyzed reactions may have driven the elongation or modification of amphiphiles, leading to increased membrane stability and selectivity over time. As POLETs matured, natural selection could have favored membrane compositions that retained catalytic oligomers or resisted degradation in extreme environments.

Finally, the new Figure 2 includes this prebiotic lipid synthesis and assembly as a foundational step in the “Roots of Life,” highlighting the interplay between environmental geochemistry and the emergence of primitive compartmentalization.

We hope this expanded treatment addresses the reviewer’s request for more detailed speculation on the origin and function of early membrane components in POLET-based models.

Comment 2.5. Detectability — The authors speculate that pre-cells may be still racemic — that is, they use a mixture of D and L amino acids in their oligopeptides and D and L sugars in their oligonucleotides. This is a reasonable idea, but then, instead of staying with racemic aptamers, the authors concentrate on discussing how to detect the complete mirror L-RNAs (using mirror D-aa enzymes).

Response: We thank the reviewer for this insightful observation. In the revised manuscript, we have clarified our discussion on chirality and detectability in Section 2.9 and Section 3 to better differentiate between racemic aptamer systems (mixtures of D- and L-nucleotides/amino acids) and homochiral mirror systems (e.g., pure L-RNA and D-proteins).

Our central premise is that early pre-cellular systems, such as POLETs, were likely racemic due to the non-enantioselective nature of prebiotic chemistry. In this context, we agree that racemic aptamers composed of D/L mixtures could have provided primitive but functional structures capable of weak binding and catalysis. We now emphasize that such heterochiral biopolymers may have lacked the high specificity and stability of modern biomolecules, yet still played a key role in early biochemical evolution.

The discussion of mirror systems (e.g., L-RNA and D-protein enzymes) was introduced not to suggest that POLETs were originally homochiral, but rather to explore modern detection and biosignature strategies. As clarified in the revision, our interest in L-RNA and D-amino acid enzymes relates to their use in synthetic biology for probing environments where enantiomeric excess is minimal—a situation that might mirror early Earth or extraterrestrial conditions.

We hope this distinction resolves the confusion and aligns our speculative framework more closely with the reviewer’s concerns about chirality, detectability, and early biochemical realism.

Comment 2.6: Apparently, they are impressed with the “fantastic” reports of mirror-image (D-aa) enzymes synthesizing mirror-image (L-nt) RNAs and DNAs. I suggest staying with racemic, rather than with mirror-image biopolymers, and generally away from this mirror-image cell fantasy.

Response: The discussion of mirror systems (e.g., L-RNA and D-protein enzymes) was introduced not to suggest that POLETs were originally homochiral, but rather to explore modern detection and biosignature strategies. As clarified in the revision, our interest in L-RNA and D-amino acid enzymes relates to their use in synthetic biology for probing environments where enantiomeric excess is minimal—a situation that might mirror early Earth or extraterrestrial conditions.

We have added text to explicitly state that our mirror-detection strategies are analytical tools and not evolutionary endpoints of POLETs. These tools could help identify the presence of racemic or partially enriched chiral biopolymers in astrobiological samples without requiring life to be based on homochiral systems. We have also included a new Figure 3 and a discussion about a generalized nanopore concept that can potentially sequence any linear polymer.

We hope this distinction resolves the confusion and aligns our speculative framework more closely with the reviewer’s concerns about chirality, detectability, and early biochemical realism.

Comment 2.7. Speculations about alternative sources of energy are welcome, but since the major problem of Life is to maintain its molecular complexity against the constant forces of destruction, the authors should think in terms of the required energy to sustain damage-repair balance for systems of varied complexities. For example, if a modern cell has a complexity 1,000 and takes 100 hits from its environment per day, at the least every day it has to extract from its environment enough energy to repair these 100 hits — just to stay alive. Now we can imagine a POLET with a complexity of 100 and the environmental damaging impact of 10 per day. Yes, its energy requirements are significantly lower, but still it needs to extract at least 10 repair units of energy from its environment. Are these alternative sources (like radioactive decay) adequate to cover the needs of a pre-cell?

Response: We greatly appreciate this insightful and constructive comment. In response, we have added a new Section 2.7.3 of the manuscript to address the important issue of damage-repair balance in prebiotic systems with varying levels of molecular complexity.

We agree that one of the fundamental challenges for any living or proto-living system is to maintain its structure and function in the face of continuous molecular damage. To better frame this challenge for POLETs (Preprokaryotic Lifeforms with Enzymatic Traits), we now explicitly discuss the concept of an "energy per damage repair unit" threshold—drawing from the reviewer’s illustrative example of modern cells and scaled-down analogs.

We argue that POLETs likely had:

Lower complexity (e.g., fewer distinct macromolecular components),
Slower turnover rates, and
Reduced information density, which collectively lowered their daily "hit rate" from environmental stressors (e.g., UV, hydrolysis, oxidation).

In this framework, even if POLETs were subjected to 5–10 damaging events per day (rather than hundreds), they would still require a minimum sustainable energy flux to drive rudimentary repair or replacement pathways. We have added a paragraph estimating how radioactive decay, redox gradients in hydrothermal vents, or thioester chemistry could serve as plausible energy sources, even in the absence of ATP or proton gradients.

While the quantitative energy yield of such sources remains speculative, our revised text now emphasizes that the energy input from these sources could have been sufficient to sustain life-like persistence in POLETs precisely because their repair costs were much lower than those of modern cells.

We thank the reviewer again for encouraging this more rigorous treatment of energetic sufficiency and damage repair tradeoffs in early molecular systems.

Comment 2.8. Definition of Life — in order to call for a broader definition of life, the authors should give the general definition first, and then to propose aspects that need broadening. I suspect that the most general Life’s definition likely covers both extant cells and POLETs.

Response: We appreciate this thoughtful comment and have revised the manuscript accordingly to clarify our intent. In the revised Introduction and Section 1, we now begin with a widely accepted general definition of life, such as NASA's working definition: “Life is a self-sustaining chemical system capable of Darwinian evolution.” This definition is indeed broad enough to encompass both extant life and hypothetical early entities like POLETs.

Our goal is not to challenge this foundational definition but rather to highlight features that may expand our working understanding of how life-like systems can emerge and persist before reaching the threshold of Darwinian evolution. Specifically, POLETs may not yet have possessed stable genomes or high-fidelity replication machinery, but they likely exhibited:

Energetic self-maintenance via simple enzymatic functions,
Compartmentalization and molecular turnover (e.g., blebbing),
Adaptation through stochastic compositional variation and selection.

Thus, we suggest that current definitions can be operationally broadened to include such systems that fall along the continuum toward life. We now explicitly state this in the revised text and distinguish between life as classically defined and life-like systems that represent precursors capable of evolving into life.

We thank the reviewer for this helpful prompt, which allowed us to clarify our position and better integrate our concept of POLETs within existing frameworks.

Miscellaneous

Comment: Page 2, line 84 — fix “review,weexamine”

Response: This has been corrected.

Comment: Page 8, line 320 — insert “with” after “metabolism”?

Response: This section has been changed.

Submission Date

29 May 2025

Date of this review

24 Jun 2025 00:04:14

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have proposed preprokaryotic organismal lifeforms existing today (POLET) hypothesis in the article, based on the facts that Asgard archaea possess eukaryotic signature proteins and exhibit cytoskeletal membrane protrusions reminiscent of primitive engulfment. The hypothesis is quite interesting in the sense that the hypothesis may reframe the origin of life as an ongoing biological process and that living relics of early evolution may still persist on Earth.

I would like recommend to the authors to show, for example, as Tables, what were already confirmed by experiments and what are necessary to confirm by future experiments, if possible. The reason is because it could be confirmed by the experiments, if the POLET hypothesis is correct or not.

Author Response

Reviewer 2:

Comments and Suggestions for Authors

Comment: I would like recommend to the authors to show, for example, as Tables, what were already confirmed by experiments and what are necessary to confirm by future experiments, if possible. The reason is because it could be confirmed by the experiments, if the POLET hypothesis is correct or not.

Response: We have written new sections 2.10 and 2.11 and Tables 2 and 3 to address these comments.

Reviewer 3 Report

Comments and Suggestions for Authors

bacteria-3701157

POLETicians in the Mud: Preprokaryotic Organismal Lifeforms Existing Today (POLET) Hypothesis

Overall I cannot recommend publication in its current form. A lot of this paper has its logic inverted. There are few examples below. The concept is confused, with cause and effect getting mixed up, and the paper presents no clear idea what a POLETician actually is. Are we talking a complex chemical system that is a product of geochemical processes or a self-replicating ‘RNA world’ type organism? Are we talking something that is a symbiote of archaea (as shown in figure 1) or a phenomenon happening on mineral surfaces (as discussed in many points in the text; hints throughout the paper seem to be that they envisage POLETIcians as RNA-world, clay surface-based self-replicating entities). Throughout the paper the authors say “POLETicians may” or “POLETicians might” without any argumentation. The fact that random peptides (made from *homochiral* amino acids) can form beta sheets etc does not provide evidence that organisms using semi-random peptides exist, any more than the fact that ‘chemical gardens’ based on sodium silicate can grow intricate structures is evidence for the existence of silicon-based life-forms. Why should they use D amino acids, not L? L sugars, not D? The choices seem completely arbitrary. If the logic went something like this:

We postulate that the descendants of a pre-prokaryotic cell form of life could persist in modern times [And then describe what it is you mean by this – LUCA? RNA-world]
That life-forms would be able to compete in [whatever environment] because
- It had properties a, b, c, d.
And this is how we would look for it

Then this might be convincing. (We only get to a possible ecological role for POLETicians (and hence an argument for their survival) in lines 248 ff! And even this does not say what unique role a POLETician would play in such a consortium, a role that no other group of faster-replicating organisms could play.) But saying “Maybe something exists” is deeply unconvincing. Maybe a six-legged elephant exists. Elephants exist and six-legged insects exist and a six-legged elephant might be able to run faster to avoid predation than one relying on merely four legs. Are you convinced?

Section 2.8 implies (but does not state) that POLETicians represent recent de novo origins of life. This is an extraordinary claim, as almost all OOL authors would take it as axiomatic that the conditions for OOL no longer exist on Earth – they were conditions existing in the Hadean or early Archean. Specifically, Earth’s Phanerozoic eon is characterised by an oxidizing atmosphere, oxidized crust and paucity of non-biological organics that makes self-assembly of anything on mineral surfaces unlikely.

I started doing a more thorough review of the paper and its logic below, but alas fear that I have failed to really get to grips with this, as I did not want a review twice as long as the paper. I hope that this is some help if the authors want to rewrite their ideas.

Specifics

The authors seem unclear what stage of abiogenesis the POLETicians are meant to represent. It seems very likely that selection for one enantiomer of amino acids and sugars was an early event – it is hard to imagine RNA or protein catalysts that could consistently form a functional 3D shape if they were made of a random mixture of enantiomers (despite the authors’ subsequent claims, which as noted above mis-represent what was actually found). So search for mirror RNA is in reality a search for a ‘shadow biosphere’ in which the enantiomeric selection ‘chose’ the reverse enantiomer from modern life. That is a fine and worthy concept, although not original, but is quite different from search for a life-form that looks like a pre-LUCA organism. But (lines 102-3) the authors talk of “Rather, formation of membranes and other structures could occur spontaneously, with precursors generated gradually by radioactive decay, redox processes, or catalysis on mineral surfaces” which implies that what they have in mind is completely chemical, abiotic systems, not a form of life at all. So are we asking here “Does geochemistry make biological molecules?” or “Does a much simpler form of life based on an ‘RNA-world’ metabolism exist?” Those are completely different questions. (There is no chance, absolutely none, that abiological processes will make a functional ribozyme – that has to be the product of complex chemistry that we have to call ‘metabolism’).

It is not clear to me why a POLETician should have along lifespan. Long lifespan implies either a very low rate of chemical decay in the components of the cell or an efficient repair mechanism. As the latter is (presumably) a property of highly evolved systems, then we must presume for former. Why would that be so? Long *replication* times deriving from relatively inefficient metabolism is a different thing. Thus:

" POLETicians may persist in a near-static metabolic state, allowing for extraordinary durability under extreme conditions” (lines 99-100) No. Metabolic stasis does *not* imply durability. Bacterial spores are metabolically static and highly durable because they have a large number of very specific adaptations to preserve their contents. They are not just very slowly metabolizing cells. Tun-state Tardigrades are in a very specific non-metabolizing state, not just metabolising very slowly. Bacteria can have doubling times from minutes to centuries with the same basic chemistry. Some archaea have very long replication times, as the authors note, but they have highly evolved and efficient metabolisms. Their long replication times are a consequence of the ecological niche they occupy and the metabolic strategy the adopt to occupy that niche, it is not some ‘primitive’ characteristic.

It is rather like arguing that the Puya Raimondii, a bromeleid which takes 80 years to flower, is more primitive than cyanobacteria which can double in a few days.

What would be more plausible (and the authors might like to rephrase their argument along these lines) is that the primitive, inefficient metabolism of a POLETician would inevitably result in a very sow rate of synthesis of cell contents, and hence of replication. This would require the POLETician to inhabit an environment where they were very long-lived and their contents were very durable against chemical decay *and against predation* by other prokaryotes. This would narrow the focus on where to look for such entities. Long-lived saline ponds under arctic ice come to mind, or hypersaline reservoirs at the base of seas such as the Mediterranean, which are cold and relatively hostile to conventional life.

Section 2.3 . The whole of this is a “this might happen”. Yes, under appropriate conditions amphipath vesicles can bleb to produce smaller vesicles. They can also form tubes and sheets. They can also expand and contract. So? Why would a lineage of organisms that replied on this highly inefficient way of dividing the “cell” contents have survived 3.5 billion years in face of competition from more efficient life? 2.4 then says this is a mineral-assisted process. So this is a key point (or could be one) – this is a life-form that exists on mineral surfaces, that is why it can compete with organisms in bulk fluid, and that is where we look for it. If this is a characteristic of POLETicians (and not another arbitrary “may be”), then this is a key point. The argument might go

Early life used mineral surfaces to catalyse chemistry and assemble membranes
Later life evolved to perform these steps away from those surfaces, and hence could colonize the whole planet
But we hypothesise that, at those surfaces, that primitive life can compete with ‘bulk life’, and so its descendants could be with us today
Their properties would therefore be ….

(Of course, this ignores the powerful ability of modern microorganisms to form biofilms on *any* surface, biofilms which would eat the inefficient POLETicians for breakfast, but at least that argument would constrain and explain what we are talking about)

Smaller points.

P1 lines 32 – 40 I do not think that discovery of that Asgard clade of archaea was the trigger for the ‘major shift in evolutionary biology’. There was plenty of evidence before that that eukaryotes had homologies with archaea – presence of histones, nature of RNA polymerase, presence of cytoskeletal protein homologues etc. So the idea that eukaryotes were in a sense a branch off the archaea was an established hypothesis. What discovering the Lokiarchaea did was support this hypothesis by providing a plausible modern homologue of the common ancestor.

Page 2, lines 45 ff. To be clear, the Lokiarchaea are not an ancestral form to eukaryotic cells. They are the descendants of those ancestors. It is hypothesised that a ‘prokaryotic’ cell very similar to the Lokiarchaea was the ancestor from which the eukaryotic cell evolved. But that is not the same as saying that the Lokiarchaea *are* the ancestor cell type from this the eukarya evolved. So while it is valid to say that a cell similar to the pre-prokaryote LUCA-like cell from which all life evolved exists today, it is *not* valid to say that that cell survives today.

Line 100 “Since their metabolism is incredibly slow, there would not be the need for modern efficient enzymes to catalyze fast reactions.” Surely their metabolism would be slow *because* they do not have highly evolved enzymes. The speed of metabolism is an effect of the enzymes that comprise it, not a cause of those enzymes.

Yarus et al have *modelled* robust amino acid-codon relationships, as have many others (see references in https://www.annualreviews.org/content/journals/10.1146/annurev-biophys-090622-102329) . It is also not clear whether the associations claimed can explain the origin of he code, rather than its elaboration (see https://www.annualreviews.org/content/journals/10.1146/annurev-genet-120116-024713 inter alia).

Saying that random peptides could self-aggregate into “cytoskeleton like structures” and citing prion aggregation as an example (line 147) is not reasonable; prions are a *single* sequence of amino acid in a *single* enantiomer. The classic Oparin experiments (and hundreds of subsequent replications) show that random peptides form random blobs, usually highly hydrated aggregates. Peptides formed from a mixture of homochiral amino acids (e.g. D-alanine + L-leucine) may form specific structures, but this still requires homochiral amino acids. The studies referenced in line 149-153 all used homochiral amino acids. Similarly (section 2.4), whether mixed enantiomers of sugars (sugars? Sugars in membranes are a sophisticated evolutionary product, not a basic property of amphipaths!) can form membranes is less important than what chemistry makes the sugars in the first place. I suggest the authors read Steve Benner’s work on borates in the generation of sugars in OOL if they want to stick to a mineral-surface-life hypothesis.

It is pointless have a subsection within a section when there is only one subsection. Thus 2.3 contains only 2.3.1, 2.4 contains only 2.4.1, 2.5 contains 2.5.1 etc.

Line 137 ff. Uncatalysed chemistry is by definition geochemistry. Are you saying that POLETicians are archaic terrestrial geochemistry? (it might be uncatalyzed chemistry between substances that have been selectively concentrated from the environment, but that requires specific and potent, energy-coupling catalysts to do the concentrating).

Section 2.5. I cannot see anyone going to the enormous effort of making and validating a mirror PCR system just on the off-chance that there is some mirror RNA to be found. The other methods are well-known. The big issue is *where to look*. Are the authors postulating that these are as widespread as the Archaea are now (in which case how could a primitive life-form with very limited ability to reproduce and a critical dependence on mineral surfaces survive competition from more evolved life?) Or are they postulating a rare, specific niche (mineral surfaces perhaps, especially perhaps subsurface rock crack and fissure environments)?

Section 2.7. In what way is this different from bacteria, which can use all these sources (albeit indirectly in the case of radioactive decay)?

Lines 281 – 283. The descriptions of life do not specify timescales or complexity levels, nor do they specify mechanisms of reproduction. This is a false dichotomy.

Lines 308-310. I assume this is a quote from Pasteur? Put it in quotation marks.

Author Response

Reviewer 3:

Comments and Suggestions for Authors

bacteria-3701157

POLETicians in the Mud: Preprokaryotic Organismal Lifeforms Existing Today (POLET) Hypothesis

Comment: Overall I cannot recommend publication in its current form. A lot of this paper has its logic inverted. There are few examples below. The concept is confused, with cause and effect getting mixed up, and the paper presents no clear idea what a POLETician actually is.

Response: We thank you for your many comments and the hard work you did on your review. This paper is extensively revised and we hope that these issues are now addressed.

Comment: Are we talking a complex chemical system that is a product of geochemical processes or a self-replicating ‘RNA world’ type organism? Are we talking something that is a symbiote of archaea (as shown in figure 1) or a phenomenon happening on mineral surfaces (as discussed in many points in the text; hints throughout the paper seem to be that they envisage POLETIcians as RNA-world, clay surface-based self-replicating entities).

Response: We appreciate the reviewer’s thoughtful question regarding the nature of POLETs and their biological or pre-biological status. This comment strikes at the heart of the POLET hypothesis, and we agree that the original manuscript did not make our definition or framework sufficiently clear.

In the revised version, we clarify that the term POLET is intentionally broad, encompassing a spectrum of entities that straddle the traditional boundaries between chemistry and biology. The unifying feature of POLETs is that they are transient or persistent lineages of molecular systems that precede or bypass the emergence of ribosome-based life (i.e., pre-LUCA). We now offer a formal working definition of "life" in the Introduction as a system that demonstrates boundedness, metabolism, inheritance, and evolution, and we discuss how different classes of POLETs may partially or fully meet these criteria.

To directly address the reviewer’s specific question:

POLETs are not limited to RNA-world models, though RNA-world scenarios are one subclass. Some POLETs may resemble self-replicating RNA-like polymers possibly aided by mineral surfaces such as clay (Montmorillonite), borate, or hydroxyapatite.
Other POLETs could be non-nucleic acid–based replicators (e.g., peptide-RNA hybrids, lipid-peptide vesicles, or mirror-RNA-peptide systems) or autocatalytic chemical networks that exhibit metabolism and rudimentary heredity without genomes per se.
Still others may have co-evolved with or persisted as symbiotic lineages within archaeal or bacterial hosts (as depicted in Figure 1), potentially occupying protected or nutrient-limited ecological niches where canonical life does not have a strong competitive advantage.

To reconcile these diverse possibilities, we now introduce a classification of POLETs in the revised text (see Table 4), which includes:

Abiotic-chemical POLETs — Systems driven by geochemical energy gradients, often surface-bound (e.g., mineral templating).
Protocellular POLETs — Membrane-bounded systems with self-replicating informational molecules (e.g., RNA-world variants or mirror-RNA systems).
Host-dependent POLETs — Persistent or parasitic lineages residing within modern cells, evading standard detection by using xenobiotic biochemistry.
Synthetic-analogue POLETs — Systems that may be recreated or modeled in the lab, such as racemic life, mirror-life, or generalized autocatalytic networks.

We emphasize in the revised manuscript that POLETs need not conform to a single paradigm. Rather, they form a continuum from prebiotic chemistry to proto-biology, some of which may have been evolutionary dead ends, and others that may persist today in rare niches. What unites them is not a specific biochemistry but their evolutionary position and decoupling from the ribosomal central dogma.

Finally, to aid clarity, we have:

Revised the text to reduce ambiguity around RNA-world versus mineral-surface hypotheses.
Provided clearer figure legends (especially for Figure 1 and the new Tree of Life/Roots figure) indicating which POLET classes are mineral-bound vs. cellular.
Added explicit discussion of where POLETs might persist today (e.g., deep biosphere, brine pockets, biofilms, organelles, or as intracellular symbionts in archaea).

We hope these revisions now clarify that POLETs are not a single hypothetical organism, but a conceptual framework for pre- or parallel-biotic systems that can be categorized, tested, and perhaps even discovered.

Comment: Throughout the paper the authors say “POLETicians may” or “POLETicians might” without any argumentation. The fact that random peptides (made from *homochiral* amino acids) can form beta sheets etc does not provide evidence that organisms using semi-random peptides exist, any more than the fact that ‘chemical gardens’ based on sodium silicate can grow intricate structures is evidence for the existence of silicon-based life-forms. Why should they use D amino acids, not L? L sugars, not D? The choices seem completely arbitrary. If the logic went something like this:

We postulate that the descendants of a pre-prokaryotic cell form of life could persist in modern times [And then describe what it is you mean by this – LUCA? RNA-world]
That life-forms would be able to compete in [whatever environment] because

It had properties a, b, c, d.

And this is how we would look for it

Response: We appreciate the reviewer’s insightful critique and agree that the initial manuscript overly relied on speculative language (e.g., “may” or “might”) without sufficient grounding in logic or testable frameworks. In response, we have significantly strengthened the structure of the manuscript by explicitly addressing:

The theoretical basis for POLETs – We now define POLETs as hypothesized molecular systems that may have existed prior to LUCA, or evolved in parallel, which did not use the modern ribosome-DNA-based replication system. This includes mirror-life forms that use D-amino acids or L-RNA, racemic life, or minimal life using random or semi-random polymer chemistry.
Why mirror- or racemic-life is considered – We acknowledge the apparent arbitrariness of chirality and have added a discussion of the Pasteur paradox (why extant life uses L-amino acids and D-sugars, and whether that choice was contingent or deterministic). We point out that laboratory-generated racemic peptides and mirror RNAs demonstrate plausible biostability and catalytic potential. These are not proof that POLETs exist, but they provide a logical space where noncanonical biochemistries could operate without direct competition from ribosome-based life.
Ecological logic for their persistence – We now argue that POLETs, if extant, could fill ecological roles poorly served by modern life, such as:

Operating in xenobiotic or prebiotic niches (e.g., deep-sea mud, salt brines, hydrothermal vent interiors, low-energy environments);
Surviving in dormant or ultra-slow-growing states where replication speed is less critical than chemical stability;
Avoiding predation or degradation because their noncanonical chirality resists normal enzymatic digestion (e.g., mirror RNA not being degraded by ribonucleases);
Serving as relic reservoirs of primordial chemistry that might contribute to modern microbiomes as passive participants or by providing rare metabolites.

Testable predictions and experimental roadmap – To address the need for convincing argumentation, we created two tables:

Table 2 compiles indirect and circumstantial evidence supporting the POLET hypothesis (e.g., stability of mirror-RNA, anomalous microbial metabolism of mirror sugars, persistence of Asgard archaea).
Table 3 outlines specific experimental approaches for testing aspects of POLET existence, such as mirror PCR, generalized nanopore sequencing for any linear polymer regardless of chirality, and MS-based assays to detect mirror peptides or sugars in unusual environments.

This structured logic mirrors the reviewer’s proposed approach: postulate → hypothesize competitive mechanisms → define testable strategies. We believe this revision now offers a compelling conceptual framework for POLETs grounded in plausibility, biochemical precedent, and falsifiability. The speculative nature remains, but it is now channeled into a clearer scientific rationale rather than open-ended conjecture.

Comment: Section 2.8 implies (but does not state) that POLETicians represent recent de novo origins of life. This is an extraordinary claim, as almost all OOL authors would take it as axiomatic that the conditions for OOL no longer exist on Earth – they were conditions existing in the Hadean or early Archean. Specifically, Earth’s Phanerozoic eon is characterised by an oxidizing atmosphere, oxidized crust and paucity of non-biological organics that makes self-assembly of anything on mineral surfaces unlikely.

Response: We thank the reviewer for pointing out this key concern, and we have substantially clarified our position in the revised Section 2.8. We do not claim that POLETicians arose de novo under modern Earth conditions. Instead, we propose that POLETicians are descendants of pre-LUCA or alternative lineage lifeforms—survivors or offshoots of ancient biochemistry that may persist in rare ecological niches. This hypothesis aligns with a growing body of research suggesting the deep evolutionary antiquity of some large RNA viruses and other complex, acellular systems.

To address the core of the comment:

We now explicitly define "Life" in the Introduction (Section 1) using both classical and expanded criteria to incorporate non-Darwinian and quasi-biological systems.
In the revised Section 2.8, we emphasize that POLETicians are not newly emerged but rather potentially persisting from earlier evolutionary epochs. Their survival today would not require a full origin-of-life (OOL) chemical context, but rather maintenance of pre-existing self-replicating or information-preserving systems in secluded or extreme environments (e.g., deep subsurface, cold seeps, anoxic sediments, or as symbionts).
This view parallels hypotheses about the persistence of ancient ribozyme-based life or giant RNA virus lineages from the RNA-protein transition phase.

We agree that modern Earth is hostile to spontaneous OOL via classical abiogenesis. However, we do not propose that POLETicians are arising today from mineral surface reactions in oxidizing environments. Instead, we explore the ecological and evolutionary plausibility of such lineages having remained in "refugia" or having co-evolved with modern organisms, possibly as slow-replicating symbionts or metabolic specialists, comparable to the slow turnover observed in some archaea and giant viruses in marine sediments or permafrost.

We also point out that ancient mineral surface reactions (e.g., on clays, zeolites, or metal sulfides) may have seeded early replicative chemistries that are no longer generative today but could still support vestigial metabolic activity. These geochemical legacies might contribute to rare, slow biochemical cycles that enable the survival of relic lifeforms like POLETicians.

Thus, POLETicians are not modern origin-of-life events, but potential living fossils—alternative evolutionary trajectories that survived alongside or beneath the dominant LUCA-derived biosphere.

We hope the revised text better reflects this nuanced view.

Comment: I started doing a more thorough review of the paper and its logic below, but alas fear that I have failed to really get to grips with this, as I did not want a review twice as long as the paper. I hope that this is some help if the authors want to rewrite their ideas.

Response: We greatly appreciate the time you spent on this review and believe that it is vastly improved.

Specifics

Comment: The authors seem unclear what stage of abiogenesis the POLETicians are meant to represent. It seems very likely that selection for one enantiomer of amino acids and sugars was an early event – it is hard to imagine RNA or protein catalysts that could consistently form a functional 3D shape if they were made of a random mixture of enantiomers (despite the authors’ subsequent claims, which as noted above mis-represent what was actually found). So search for mirror RNA is in reality a search for a ‘shadow biosphere’ in which the enantiomeric selection ‘chose’ the reverse enantiomer from modern life. That is a fine and worthy concept, although not original, but is quite different from search for a life-form that looks like a pre-LUCA organism. But (lines 102-3) the authors talk of “Rather, formation of membranes and other structures could occur spontaneously, with precursors generated gradually by radioactive decay, redox processes, or catalysis on mineral surfaces” which implies that what they have in mind is completely chemical, abiotic systems, not a form of life at all. So are we asking here “Does geochemistry make biological molecules?” or “Does a much simpler form of life based on an ‘RNA-world’ metabolism exist?” Those are completely different questions. (There is no chance, absolutely none, that abiological processes will make a functional ribozyme – that has to be the product of complex chemistry that we have to call ‘metabolism’).

Response: We appreciate the reviewer’s point that the stage of abiogenesis represented by POLETs must be clarified, particularly in light of the early selection for homochirality in known life. We agree that functional ribozymes and proteins—capable of folding into catalytically active 3D structures—require homochiral substrates, and that mixed-enantiomer (racemic) systems pose structural and functional challenges to canonical folding. However, our concept of POLETs does not presume they are fully functional ribozyme- or protein-based life forms in the modern sense. Rather, we propose that POLETs represent a class of persistent, non-cellular or pre-cellular life forms that occupy an intermediate evolutionary space—between geochemically organized chemistry and minimal, self-sustaining metabolisms.

This space may encompass entities that never achieved homochirality, or alternatively, adopted noncanonical chiralities (i.e., mirror-symmetrical forms) that diverged from the trajectory leading to LUCA. We refer to these as candidates in a “shadow biosphere”, consistent with prior work by Davies et al. and Cleaves et al., but we expand this notion to include not just chiral inversion, but a broader range of pre-LUCA evolutionary experimentation, including alternate informational polymers, mineral-templated autocatalytic systems, or primitive compartmentalized chemistries that co-evolved with LUCA-lineage organisms.

The specific reference in lines 102–103—"formation of membranes and other structures could occur spontaneously..."—is intended to reflect well-supported pathways for abiotic compartmentalization, which serve as boundary conditions for the emergence of metabolism, not as a full alternative to life. We do not suggest that abiotic geochemistry alone can yield modern functional ribozymes; we agree this is unlikely. Rather, our goal is to explore whether vestiges of ancient systems—possibly still metabolically active but chemically distinct from modern life—persist in cryptic or symbiotic forms.

Thus, we position POLETs as transitional entities—not abiotic geochemical soup, and not fully evolved RNA-world organisms—but part of a continuous gradient of complexity, potentially capable of rudimentary catalysis, energy harvesting, and even symbiosis with LUCA-based organisms. Their biochemical distinctness may include racemic or alternate-chirality macromolecules. While these may not be capable of the complexity seen in modern proteins or RNA, they could nonetheless support minimal functionality—especially if coupled with, or parasitic upon, canonical microbial systems. Indeed, it is precisely this speculative complementarity that makes them worth investigating.

Finally, we acknowledge that the concept of a shadow biosphere is not new. However, our contribution lies in proposing a taxonomy of POLET forms (Table Z) that includes racemic, mirror-chiral, and other pre-LUCA candidates, and in emphasizing that with today’s analytical tools—especially enantioselective sequencing, mirror-PCR, and single-molecule chirality-resolving sensors—we are now in a position to test for these systems systematically in environments that have not previously been explored for them.

Comment: It is not clear to me why a POLETician should have along lifespan. Long lifespan implies either a very low rate of chemical decay in the components of the cell or an efficient repair mechanism. As the latter is (presumably) a property of highly evolved systems, then we must presume for former. Why would that be so? Long *replication* times deriving from relatively inefficient metabolism is a different thing. Thus:

It is rather like arguing that the Puya Raimondii, a bromeleid which takes 80 years to flower, is more primitive than cyanobacteria which can double in a few days.

Response: We appreciate this thoughtful and well-articulated critique and agree that durability and replication time are often conflated, but must be treated as distinct properties. In our manuscript, we did not mean to imply that metabolic stasis alone guarantees durability; rather, we hypothesized that POLETicians—if they exist—might display both long replication cycles and chemical stability under certain geochemical conditions, not due to evolved repair mechanisms, but rather due to passive stability in non-aqueous, low-temperature, or mineral-associated environments. We acknowledge that highly evolved dormancy strategies such as those seen in bacterial spores or tun-state tardigrades involve complex, specific adaptations—features that POLETs would not possess by definition.

Our revised view is that POLETs would not have long lifespans in the sense of biological longevity, but may persist over geological timescales due to chemical persistence, for example as encapsulated protocells or on mineral surfaces that shield reactive intermediates. Analogous to how some prebiotic molecules survive in meteorites, POLET forms may be relics stabilized not by active repair but by physicochemical context. We have revised the relevant sentence to clarify this distinction and avoid the implication that metabolic stasis implies biological durability.

Comment: What would be more plausible (and the authors might like to rephrase their argument along these lines) is that the primitive, inefficient metabolism of a POLETician would inevitably result in a very sow rate of synthesis of cell contents, and hence of replication. This would require the POLETician to inhabit an environment where they were very long-lived and their contents were very durable against chemical decay *and against predation* by other prokaryotes. This would narrow the focus on where to look for such entities. Long-lived saline ponds under arctic ice come to mind, or hypersaline reservoirs at the base of seas such as the Mediterranean, which are cold and relatively hostile to conventional life.

Response: We agree with the reviewer’s suggestion that a more plausible model for POLETicians would involve primitive and inefficient metabolic pathways resulting in extremely slow synthesis of cellular components and, by extension, very slow replication cycles. This would necessitate inhabiting ecological niches where both chemical degradation and biological predation are minimized. Importantly, this view complements our proposal that POLETicians might be based on mirror-image biochemistry (e.g., L-sugars and D-amino acids), which would provide a natural defense against predation by conventional organisms. As Pasteur famously demonstrated, organisms are often stereochemically constrained, such that extant prokaryotes cannot metabolize or interact with mirror enantiomers of biological macromolecules. This biochemical isolation could enable POLETicians to persist in otherwise inhospitable environments—such as long-lived saline ponds under polar ice or hypersaline brines at the base of ancient seabeds—where chemical stasis and ecological isolation reduce decay and competition. We have updated the manuscript to reflect this more ecologically and chemically plausible scenario and to better distinguish between metabolic stasis and chemical durability.

Comment: Section 2.3 . The whole of this is a “this might happen”. Yes, under appropriate conditions amphipath vesicles can bleb to produce smaller vesicles. They can also form tubes and sheets. They can also expand and contract. So? Why would a lineage of organisms that replied on this highly inefficient way of dividing the “cell” contents have survived 3.5 billion years in face of competition from more efficient life? 2.4 then says this is a mineral-assisted process. So this is a key point (or could be one) – this is a life-form that exists on mineral surfaces, that is why it can compete with organisms in bulk fluid, and that is where we look for it. If this is a characteristic of POLETicians (and not another arbitrary “may be”), then this is a key point. The argument might go

Early life used mineral surfaces to catalyse chemistry and assemble membranes
Later life evolved to perform these steps away from those surfaces, and hence could colonize the whole planet
But we hypothesise that, at those surfaces, that primitive life can compete with ‘bulk life’, and so its descendants could be with us today
Their properties would therefore be ….

Response: We appreciate this insightful comment and agree that Section 2.3 needed stronger grounding in plausible evolutionary constraints and ecological niche theory. In response, we have revised the text to emphasize a coherent and testable hypothesis: that POLETicians persist specifically because they are adapted to mineral-associated niches, and further, that they may utilize mirror-image biochemistry to avoid being assimilated or predated by modern life.

The reviewer’s proposed logic flow (1–4) is both compelling and aligned with our conceptual framework. We now state more clearly in the revised manuscript that:

The earliest proto-life likely originated at mineral–fluid interfaces, where mineral surfaces catalyzed condensation reactions and stabilized amphipathic structures, leading to membrane formation and proto-metabolic cycles. These surfaces provided both structural scaffolding and catalytic specificity that primitive molecular systems could not achieve in bulk fluid alone.
With the evolution of protein enzymes and nucleic acid-based replication, later life forms could abandon surface dependence and transition to free-living cells. This major evolutionary leap enabled colonization of a wider range of environments—oceans, soil, air, and host organisms.
We hypothesize that some mineral-bound lineages did not undergo this transition, particularly if they had distinct chemical chirality (e.g., L-sugars and D-amino acids instead of the canonical D-sugars and L-amino acids). These "mirror life" POLETicians would be invisible to canonical biospheres—not only to enzymatic degradation but also to symbiosis, parasitism, and molecular recognition.
Thus, POLETicians may persist in extreme, isolated, and mineral-rich environments, such as deep subsurface lithospheres, serpentinite-hosted aquifers, or brine inclusions within ancient halite. In these contexts, they are protected from both chemical degradation and biological predation.

We have clarified in Sections 2.3 and 2.4 that mineral surface dependence is not a minor speculative detail, but rather a defining feature of POLETician persistence. Moreover, the inefficiency of their division or replication would only be evolutionarily viable if offset by a niche that excludes faster competitors—precisely what mineral surfaces deep within the crust or in hypersaline reservoirs may offer. Finally, we argue that biofilms composed of modern bacteria, although incredibly efficient, may not interact with or even detect mirror-image POLETician biochemistry, rendering these archaic forms effectively "invisible" within modern microbial ecosystems.

Smaller points.

Comment: P1 lines 32 – 40 I do not think that discovery of that Asgard clade of archaea was the trigger for the ‘major shift in evolutionary biology’. There was plenty of evidence before that that eukaryotes had homologies with archaea – presence of histones, nature of RNA polymerase, presence of cytoskeletal protein homologues etc. So the idea that eukaryotes were in a sense a branch off the archaea was an established hypothesis. What discovering the Lokiarchaea did was support this hypothesis by providing a plausible modern homologue of the common ancestor.

Response: Revised First Paragraph of the Introduction (modified to address both reviewer comments):

The discovery of Asgard archaea—first isolated from deep-sea sediments near hydrothermal vents at Loki’s Castle by Spang and colleagues in 2015 [2]—provided compelling support for a long-standing hypothesis in evolutionary biology: that eukaryotes emerged from within the archaeal domain [3]. Prior to this discovery, multiple lines of evidence already pointed to deep homologies between archaea and eukaryotes, including shared features such as histone-like proteins, complex RNA polymerases, and cytoskeletal protein homologues [1,4]. The identification of the Asgard superphylum, and particularly members such as Lokiarchaea, did not originate this hypothesis but rather strengthened it by revealing extant organisms with a richer complement of eukaryotic signature proteins (ESPs) and cellular complexity than previously seen in archaea [5,6]. While modern Asgardians are not direct ancestors of eukaryotes, they are likely descendants of a lineage closely related to the last archaeal common ancestor of eukaryotes. Thus, they serve as plausible modern analogues that illuminate the evolutionary steps bridging prokaryotic simplicity and eukaryotic complexity. These findings have contributed to the increasing acceptance of a two-domain model of life, in which Eukarya are nested within Archaea [4,5].

Comment: Page 2, lines 45 ff. To be clear, the Lokiarchaea are not an ancestral form to eukaryotic cells. They are the descendants of those ancestors. It is hypothesised that a ‘prokaryotic’ cell very similar to the Lokiarchaea was the ancestor from which the eukaryotic cell evolved. But that is not the same as saying that the Lokiarchaea *are* the ancestor cell type from this the eukarya evolved. So while it is valid to say that a cell similar to the pre-prokaryote LUCA-like cell from which all life evolved exists today, it is *not* valid to say that that cell survives today.

Response: Thank you for this important clarification. We agree that it is incorrect to equate modern Lokiarchaea with the ancestral cell type from which eukaryotes evolved. Our intent was to convey that Lokiarchaea share many features with the hypothesized archaeal ancestor of eukaryotes—such as actin homologs and ESCRT-like proteins—and thus provide valuable insights into early cellular evolution. However, as you rightly point out, Lokiarchaea are not themselves ancestral forms but rather evolved descendants that may have retained certain ancestral traits.

We revised the wording in that section to clarify that modern Lokiarchaea are not direct ancestors of eukaryotes but instead offer a model for reconstructing the nature of the archaeal lineage that contributed to eukaryogenesis. Specifically, we will emphasize that the last common ancestor of eukaryotes and Lokiarchaea is extinct, and any resemblance between current Lokiarchaea and that ancestor is inferential and partial.

Comment: Line 100 “Since their metabolism is incredibly slow, there would not be the need for modern efficient enzymes to catalyze fast reactions.” Surely their metabolism would be slow *because* they do not have highly evolved enzymes. The speed of metabolism is an effect of the enzymes that comprise it, not a cause of those enzymes.

Response: Here is the revised paragraph with a response that incorporates and addresses your comment:

Their metabolism is likely incredibly slow because they lack the highly evolved and efficient enzymes characteristic of modern life. Instead of relying on enzyme-driven catalytic acceleration, these hypothetical organisms may depend on spontaneous chemical reactions and slow geochemical processes. Membrane formation and other structural assembly could occur passively, with precursors generated over long timescales through radioactive decay, redox gradients, or mineral-surface catalysis [9, 19, 20]. Although such processes operate at glacial rates, they could still support a viable metabolic cycle when extended over centuries or millennia, particularly in stable, extreme environments.

Comment: Yarus et al have *modelled* robust amino acid-codon relationships, as have many others (see references in https://www.annualreviews.org/content/journals/10.1146/annurev-biophys-090622-102329) . It is also not clear whether the associations claimed can explain the origin of he code, rather than its elaboration (see https://www.annualreviews.org/content/journals/10.1146/annurev-genet-120116-024713 inter alia).

Response: We appreciate the reviewer’s insight and agree that the work by Yarus et al. represents one of many models for codon–amino acid associations, rather than definitive proof of the genetic code’s origin. As the reviewer rightly points out, robust amino acid–codon interactions have been computationally and experimentally modeled, but these models may better explain the elaboration or refinement of the genetic code rather than its initial emergence. We will revise the manuscript to clarify that Yarus’ stereochemical model is one among several hypotheses—alongside the co-evolution and adaptive hypotheses—and that it remains uncertain whether these models explain the origin or merely the evolutionary stabilization of the code. We will also cite the recommended reviews (e.g., Koonin & Novozhilov, 2017; Carter & Wills, 2023) to provide a more balanced and comprehensive discussion of competing theories and their respective limitations.

Comment: Saying that random peptides could self-aggregate into “cytoskeleton like structures” and citing prion aggregation as an example (line 147) is not reasonable; prions are a *single* sequence of amino acid in a *single* enantiomer. The classic Oparin experiments (and hundreds of subsequent replications) show that random peptides form random blobs, usually highly hydrated aggregates. Peptides formed from a mixture of homochiral amino acids (e.g. D-alanine + L-leucine) may form specific structures, but this still requires homochiral amino acids. The studies referenced in line 149-153 all used homochiral amino acids. Similarly (section 2.4), whether mixed enantiomers of sugars (sugars? Sugars in membranes are a sophisticated evolutionary product, not a basic property of amphipaths!) can form membranes is less important than what chemistry makes the sugars in the first place. I suggest the authors read Steve Benner’s work on borates in the generation of sugars in OOL if they want to stick to a mineral-surface-life hypothesis.

Response: We thank the reviewer for this important clarification and agree that our previous language overgeneralized the plausibility of functional cytoskeletal-like structures arising from random peptide mixtures. We have revised the text to more accurately reflect the limitations of non-ribosomal peptide self-assembly under prebiotic conditions.

Specifically, we now acknowledge that most random peptide mixtures, especially those incorporating both D- and L-amino acids, are more likely to form amorphous, hydrated aggregates rather than structured assemblies. While some studies have demonstrated that homochiral homopolymers or designed sequences can self-assemble into beta-sheets or nanotubes [4,25], these rely on controlled stereochemistry and sequence regularity not easily achieved in abiotic synthesis. Prion aggregation, while cited as an analogy, indeed involves a single, highly evolved homochiral sequence and should not be extrapolated to early life scenarios without qualification.

To address this, we now emphasize that the peptide self-assembly pathways we describe are hypothetical extensions—potential outcomes in systems that later evolved partial sequence specificity or local homochirality. These do not suggest that random prebiotic peptides uniformly led to structured assemblies, but rather that a subset of such molecules, especially in mineral- or surface-constrained environments, might have adopted proto-functional roles.

We have also revised section 2.4 to de-emphasize sugars in early amphipathic membranes and now reference alternative amphiphiles (e.g., fatty acids, isoprenoids), and we appreciate the recommendation to cite Benner’s work on borate minerals in sugar stabilization and prebiotic chemistry. This has been incorporated into the revised text as well.

Comment: It is pointless have a subsection within a section when there is only one subsection. Thus 2.3 contains only 2.3.1, 2.4 contains only 2.4.1, 2.5 contains 2.5.1 etc.

Response: This has been corrected.

Comment: Line 137 ff. Uncatalysed chemistry is by definition geochemistry. Are you saying that POLETicians are archaic terrestrial geochemistry? (it might be uncatalyzed chemistry between substances that have been selectively concentrated from the environment, but that requires specific and potent, energy-coupling catalysts to do the concentrating).

Response: Thank you for this important clarification. To be more precise, our use of the term geochemistry in this context refers to prebiotic chemical processes occurring in the absence of biological catalysis—particularly those driven by environmental factors such as heat, mineral surfaces, UV irradiation, or redox gradients. These processes, including those seen in Miller–Urey-type reactions, hydrothermal vent chemistry, or surface catalysis on clays or metal sulfides, produce organic compounds like amino acids and simple sugars without the stereoselectivity imposed by enzymes. As a result, the products of such uncatalyzed or mineral-catalyzed chemistry are expected to be racemic.

The term “POLETicians” refers not to geochemical reactions themselves, but rather to hypothetical pre-cellular entities that arose from geochemically derived, racemic mixtures of metabolites and macromolecular precursors. These systems may have exploited naturally occurring concentration mechanisms—such as drying cycles, eutectic freezing, adsorption to mineral surfaces, or localized compartmentalization in porous rocks or lipid vesicles—without requiring biologically evolved energy-coupling catalysts. While we agree that long-term selective concentration and organization of such components likely required energy inputs and ultimately catalysis, our intent was to highlight the possible continuity between abiotic, geochemistry-driven chemistry and the earliest life-like systems.

To clarify this point, we will revise the relevant passage to distinguish between geochemical production of racemic building blocks and the emergence of POLETicians as organized, pre-cellular systems that utilized those racemic substrates in the absence of enzymatic catalysis.

Comment: Section 2.5. I cannot see anyone going to the enormous effort of making and validating a mirror PCR system just on the off-chance that there is some mirror RNA to be found. The other methods are well-known. The big issue is *where to look*. Are the authors postulating that these are as widespread as the Archaea are now (in which case how could a primitive life-form with very limited ability to reproduce and a critical dependence on mineral surfaces survive competition from more evolved life?) Or are they postulating a rare, specific niche (mineral surfaces perhaps, especially perhaps subsurface rock crack and fissure environments)?

Response: We appreciate the reviewer’s thoughtful critique and agree that the development of a fully validated mirror PCR or nanopore sequencing system is unlikely to be pursued solely for the sake of investigating hypothetical mirror-RNA molecules in evolutionary biology. However, there are compelling drivers outside of pure evolutionary inquiry that could realistically support the creation and use of such technology. We added a Future Directions section to expand on these points.

Pharmaceutical applications provide one of the strongest incentives. Mirror-image oligonucleotides, such as Spiegelmers (L-RNA aptamers), are already in use for therapeutic purposes due to their increased resistance to nucleases and enhanced stability in vivo. These properties make them ideal candidates for drug development, particularly in antisense and RNA-interference platforms. Continued investment in such drug technologies is likely to drive the development of tools capable of detecting or interacting with L-nucleic acid systems, even if only as quality control or biophysical characterization tools. Once such tools exist, their use can be extended to exploratory scientific efforts, including the detection of hypothetical mirror RNAs in nature.

From an astrobiological perspective, both NASA and ESA have strong motivations to develop in situ sequencing technologies that are robust, miniaturized, and agnostic to chirality. Detecting extraterrestrial life may require tools that can identify non-canonical biochemistries—including alternate chiral forms of nucleic acids and amino acids. The search for alien life forms on Mars, Europa, or Enceladus would benefit immensely from the ability to sequence nucleic acid analogs or mirror-DNA directly at the source, without needing to return samples to Earth. Therefore, mirror-compatible sequencing platforms would serve dual-use purposes across pharmaceutical and astrobiological fields.

As for where to search for POLET-like entities today, we concur with the reviewer that their existence would be restricted to rare, specific, and relatively isolated ecological niches. We hypothesize that such environments might include subsurface rock fissures, deep mineral matrices, or extreme interfaces like hydrothermal vent walls, brine-sealed anoxic chambers, or ultra-alkaline serpentinization zones. In such niches, competition from more complex life forms is minimized, and the dependence on mineral surfaces could be maintained without disruption. These environments are also the most likely analogs to early Earth conditions and thus the best candidates for harboring extant remnants or relict chemistries of pre-LUCA life.

To clarify our position: we are not suggesting that POLETs are widespread like modern Archaea. Instead, we propose that they may exist today only in cryptic or extreme niches, functioning more as molecular fossils or minimal replicators than as robust competitors in the modern biosphere. Their obscurity may be precisely why they have remained undetected—living in what might be called a "shadow biosphere" beyond the reach of standard detection methods, which are inherently biased toward modern biochemistry.

Comment: Section 2.7. In what way is this different from bacteria, which can use all these sources (albeit indirectly in the case of radioactive decay)?

Response: Thank you for this insightful comment. We have clarified in the revised Section 2.7 that while certain bacteria—especially extremophiles—can indeed utilize a range of geochemically derived energy sources, including those indirectly derived from radioactive decay (e.g., via radiolytically generated hydrogen or sulfate), the POLET model differs in two key ways:

Absence of enzymatic catalysis: POLETs are hypothesized to operate without conventional enzymes or even ribozymes, relying instead on mineral-assisted, non-enzymatic chemical reactions. In contrast, bacteria employ complex enzymatic systems for redox reactions, even under extreme energy limitations.
Ultra-slow, passive metabolism: POLETs would acquire energy via passive, low-flux mechanisms such as direct utilization of radiolytic electrons, hydrated electrons, or persistent redox gradients in mineral matrices, over geologic timescales. This is distinct from bacteria, which must maintain at least minimal metabolic rates to support membrane potential, protein turnover, and DNA repair.

We have updated Section 2.7 to emphasize these distinctions more explicitly and appreciate the opportunity to clarify this fundamental difference between known microbial life and the hypothesized POLET state.

Comment: Lines 281 – 283. The descriptions of life do not specify timescales or complexity levels, nor do they specify mechanisms of reproduction. This is a false dichotomy.

Response: We have modified section 2.7.1 to address this:

2.7.1. Redox and Radiolytic Metabolism

Unlike modern phototrophs and chemotrophs, POLETicians likely bypass both photosynthesis and enzymatically catalyzed respiration. Instead, they are hypothesized to exploit extremely slow and low-yield abiotic energy sources such as electrons from radioactive decay, mineral-sourced redox gradients, or reactive radicals formed through radiolysis or geochemical reactions. These processes may be sufficient only to maintain molecular integrity and enable sparse biosynthetic events over long periods—potentially months to centuries—rather than fueling growth or cellular division.

While some modern deep subsurface bacteria can survive on radiolysis-derived hydrogen and sulfate for millions of years [55, 56], these organisms still rely on elaborate enzyme systems and metabolic networks. In contrast, POLETs are postulated to lack enzymes entirely or possess only rudimentary catalytic systems (e.g., mineral-surface–mediated chemistry). They would rely on passive, non-enzymatic electron flow through environmental redox couples such as Fe²⁺/Fe³⁺ and Mn²⁺/Mn⁴⁺, without the complex transport and regulation machinery seen in modern cells.

Thus, although both POLETs and some extremophilic bacteria may inhabit low-energy environments, POLETs differ in both degree and kind: they represent a prebiotic or proto-biological mode of energy utilization, operating at a lower complexity threshold and metabolic tempo. This distinction positions POLETs not merely as slow bacteria, but as a fundamentally different category of life-like system rooted in geochemistry rather than biochemistry.

Comment: Lines 308-310. I assume this is a quote from Pasteur? Put it in quotation marks.

Response: Quotes are added.

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POLETicians in the Mud: Preprokaryotic Organismal Lifeforms Existing Today (POLET) Hypothesis

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