We appreciate the three reviews and are thankful for their helpful comments! Our answers to the reviewers’ comments are included here in italics:

Reviewer 1:

The manuscript by Franz et al. (The Volyn biota (Ukraine) – 1.5 Ga old (micro)fossils in  preservation, a spotlight on the ‘boring billion’) presents a very compelling dataset on a unique fossil assemblage of most probably fungal origin.

Overall, the manuscript is well written and the data are convincing, particularly the SEM images. However, I feel that in some cases the interpretations are not supported by the data. My main concern is the interpretation that this fossil assemblage represents a deep biosphere community.

I was not able to follow the author’s argument about the 150 m depth. Perhaps a cross section of the geologic structure could clarify this issue. Currently I do not see any compelling evidence for the claim that these organisms thrived at a depth of 150 m, in fact, I even see evidence for the opposite. In line 502f the authors interpret the intact ends of the filaments as indications for growth in a soft sediment or floating organisms. It is pretty hard to imagine floating organisms in depths >100 m unless there are fractures through which water can flow freely, like for example in Karst areas. Also, how should one imagine soft sediment at great depth, assuming compaction due to overburden?   If the sediment is so soft that soft filaments can push themselves into it, then there are no fractures with floating water. And these filaments are much too large to assume that they can float freely through the pore space.

We will include in the Introduction a paragraph, describing the geological situation, possibly with a sketch similar to the summary figure from the first publication. Kerite has not been found outside the cavities, which are in the core of the pegmatite. It exists as fine fiber masses between fragments of the wall of the cavities and as larger masses hanging freely on the walls, attached to feldspar and often around topaz. Concerning the term ‘floating’ the sketch should also make it clear that we have to distinguish between sessile and non-sessile organisms. We suggest to avoid ‘floating’ and use ‘non-sessile’ instead.

Although I am not a microbiologist myself, reading the term “methanogenic bacteria” makes me cringe. There are no methanogenic bacteria, only archaea. Please change throughout the text. Also, what do the authors mean with “anoxygenic”? I assume that “anoxic” is meant, at least that is the only term that would make sense to me. There are several other instances where I got the impression that the authors’ expertise lies outside the field of microbiology and thus, I would strongly recommend to include a microbiologist in the revision.

We agree and will rephrase the text! The reviewer is also correct in assuming that none of the author team is a microbiologist, so we are very happy with any comments concerning the nomenclature and wording in our text! We will use the more general terms suggested by reviewer 2 of methanogens or methanogenic archaea.

Given the geologic evidence it seems plausible that the fossil assemblage formed after the GOE and that atmospheric oxygen levels were in the single percent range around 1.5 Gy. However, I am at a complete loss as to how this fact can be used to infer that there were oxygen-depending ecosystems at great depth at that time. There isn’t even good evidence for such ecosystems today, with oxygen concentrations between 5 and 10 times higher. And coming back to my argument in the previous paragraph, how should such an ecosystem have looked like? Again, fractures that allow for rapid flow of water into great depths are highly unlikely in soft sediment and in soft, fine-grained sediment diffusion is the dominant mode of transport. It is not even remotely imaginable that oxygen diffused to 150 m depth, given its low concentration at the surface (a good example is given in Roy et al. Aerobic Microbial Respiration in 86-Million-Year-Old Deep-Sea Red Clay. Science. 2012;336(6083):922-5.)

This should also be clear, when we include a sketch about the situation, what we assume as a geyser system, where there is a clear connection between surface waters (saturated with oxygen of the atmospheric oxygen level at the time) and hydrothermal waters in depth. There is no need for diffusive transport. We will also make it clear how the depth of the ecosystem is estimated: The current situation of approximately 150 m depth of the mining and sampling, and the intrusion depth of approximately 2 km (with reference to the literature, based on fluid inclusion data).

Another point that argues against a deep biosphere setting is the mentioning of palynomorphs in line 668. How do should palynomorphs reach a depth of 150 m? Of course, they could have been deposited at the surface and then eventually got buried, but then they must be (much?) older than the estimated 1.5 Gy. This argument does seem to be out of place here.

We wrote that there is “little similarity to morphologies of the Volyn biota”, so this paragraph is intended to say that neither palynomorphs nor the Changcheng biota nor vase-shaped metazoan microfossils are good analogues for the Volyn biota. We will rephrase the introductory sentence to this paragraph.

Another statement that I am somewhat inclined to believe, but would like to see substantiated, is in line 484. Why does the age ague for microorganisms? The following sentences meander around, but the fundamental question here would be the first occurrence of eukaryotes.

We will introduce the term ‘eukaryotes’ in this paragraph and delete the first sentence. See also the comment of reviewer 2, to include this term into the title.

The argument in line 710 ff does not really fit the scope of this study. There was subseafloor life much earlier in the Archean, but the cited study (Cavalazzi et al., 2021) describes a hydrothermal system that was most probably quite near to or even at the sediment surface and not buried at 150 m depth. While there are reports of abundant fungi in modern hydrothermal systems, none go as deep as 150 m. So I doubt that the Cavalazzi reference is useful here.

The main point of this sentence is the difference between ‘continental’ and ‘seafloor’, and we only want to emphasize that from the continental environment a deep biosphere of this age has not yet been described. We intend to leave the reference in the text.

The mentioning of radioactivity as a potential energy source is intriguing, but like before, I would strongly recommend involving a microbiologist in the revision. Yes, there are highly radiation-resistant prokaryotes and even fungi, and given that the ecosystem was hosted in a U-Th-K-rich granitic-pegmatitic system, radiation must have played a role in shaping the community. However, I do not really see how this entire paragraph connects to the main study other than stating that life was possible despite high levels of radiation.

The last paragraph about the radiation is intended to provoke a discussion from microbiologists about this possibility. So, we suggest to leave it more or less like it is.

In closing I think that the SEM study is remarkable and warrants publication, but the interpretation that the fossil assemblage represents a terrestrial deep biosphere ecosystem is not well supported to say the least. I suggest major revisions and either removal of the deep biosphere part or much stronger arguments to support these claims.

Reviewer 2:

The manuscript entitled “The Volyn biota (Ukraine) – 1.5 Ga old (micro)fossils in 3D-preservation, a spotlight on the ‘boring billion’“ reports about micrometer sized silicified filamentous fossils found in pegmatite cavities in the Precambrian Korosten pluton.

The authors describe these fossilized filaments using plenty of SEM images and accomplished by D13C isotope fraction patterns. Based on there description, they interpreted these fossils being of probable fungal origin.

Overall, the manuscript is well written and these almost perfectly preserved Precambrian fossils are worth publishing in every case.

However, I recommend some additions to the manuscript. I agree with the first anonymous reviewer (Rev1), that there is no clear description in this manuscript, why these fossils belong to a deep biosphere community. Reading the cited literature, i.e. the previously published paper about the Volyn biota, of really deep environment at about 2-2.5 km depth becomes clearer. Maybe a few more sentences describing this environment will help the read to follow in the current manuscript.

See our comment to reviewer 1 on this aspect!

The microbial descriptions should be adapted to general terms: The Methanobacteria are a class of Euryarchaeota. As other classes of Euryarchaeota are methanogenic too, I would stick to the more general term of methanogens or methanogenic archaea, as the major process is relevant here and not the specific class.

We agree and will rephrase accordingly!

The authors mainly compare the filamentous structures to fungi like organisms. Indeed, numerous studies about recent (Sohlberg et al 2015 Revealing the unexplored fungal communities in deep groundwater) and ancient (Drake et al 2017 NCOMMS anaerobic consortia of fungi and SRB in deep granite fractures) continental deep biosphere highlight the abundance and diversity of fungi and or fungi-bacteria communities. I do not doubt this interpretation; however, I miss a more diverse discussion, either about abiogenic filamentous structures that in part resemble biotic ones (Mc Mahon 2019 deepest purported fossils may be iron-mineralized chemical gardens) but also the consideration of Myxomycetes. These protozoans are likely to have evolved already back then and some of the fossil structures shown in this manuscript are very similar to the myxomycetes morphologies and the formation of chitosan and similar amino sugars (Schnittler et al. 2012.Myxocetes -Myxomycete-like Organisms, e.g. Fig 4-2).

We are thankful for the hints to the literature and will extend the discussion, especially to the abiogenic filaments, which was the first interpretation in the literature. We will also consider the references given by reviewer 3 to this point!

I also encourage the authors to speculate more about the lifestyle and metabolism of these organisms. Even if the fungal-like organisms may eat the biofilm, as the authors say – for such high amounts of kerite – there must be a reasonable nutrient and or organic matter supply to such depth which so far remains rather unclear. As groundwater aquifers are oxygen depleted/ or lack oxygen completely – how do these early eukaryotes survive?

One last point – we seem to have here perfectly preserved 1.5 Ga old complex eukaryotes – this is worth mentioning in the title.

In the title, we will exchange the word ‘(micro)fossils’ for ‘eukaryotes’.

I really would like to see this manuscript being published - therefore I suggest “minor revisions”  by taking the above mentioned comments into account.

Reviewer 3:

The authors report possible microbial structures, presumably remains of putative fungal hyphae from 1.5 Ga old granitic pegmatites of the Korosten Pluton (NW Ukrainian Shield). The preserved structures are silicified and show predominantly filament-like structures of various diameters up to 200µm. They interpret these structures as remnants of a Proterozoic deep biosphere.

The manuscript is well structured and the analytical methods used are state of the art for such studies. The SEM images are impressive and allow a critical view of the observed structures. Some of the structures shown are putative reminiscent of mineralized fungal hyphae filaments, but no segmentations are seen that would be expected in hyphal filaments of higher fungi, though in Zygomycete-like organisms (fungal-like protists) non-segmented filaments also occur. They analyzed in one filament possible chitin-remains which may support a Zygomycete affinity. However, many of the figured SEM pictures are enigmatic for me.

The presented isotope data are a bit problematic. Stable N isotopes are convincing but the stable C-isotopes are more problematic – there are no “methangenic bacteria”, only methanogenic archaea are known. Methanogenic archaea are not “anoxygenic” (-anoxygenic bacteria are e.g. photosynthetic S-purple bacteria) they harbor anoxic/anaerobic environments. It is not clear for me what they have measured organic matter from the filaments? or only from kerite? Some AOM archaea are also filamentous. AOM/ANME communities are present in the deep biosphere and I would expect these microbes in such an environment (Drake et al 2017), also in Proterozoic time.

C-isotopes and nomenclature about methanogenic bacteria – see our comments to reviewers 1 and 2. We measured whole samples (see sample list), i.e. the bulk of an assemblage collected from one point in the pegmatites (said in lines 401-403). In continuation of our study we intend to determine C- and N-isotopes from individual fossils and also check for variation of isotopes within a fossil with the ion probe.

Unfortunately the authors do not exactly describe how they obtained the filamentous structures from the pegmatites. The preserved putative fossil filaments are well preserved and I have problems to understand how they prepared it to get out of the rock.

There is no need for separation of the fossils from the rock (lines 91-92), see the comment above to reviewer 1, the fossils are freely exposed in the cavities. We will extend this description and also refer to the first published study about the fossilization process.

The authors interpret the perfect preservation of the filaments with intact ends as a hint for growth in a soft clay-rich sediment or as floating organisms (line 499-503). From the figures I cannot see any remains of a soft-sediment and floating is also not a good explanation. Biofilm-like fungal structures normally need firm substrates, may be the inner layer of small rock fractures or other types of open pore space, seen in granite fractures from the Black Forest (Triberg Granite, see in Gadd 2006, Fungi in Biochemical Cycles, Drake et al 2017) and former gas bubbles from pillow basalts (Peckmann et al 2008, Ivarsson et al 2015). In these particular cases the pore space was filled with fluids, enriched with various types of nutrients. Many of the figured structures are very strange and remain to figures I have seen in the publication of Nims et al 2021, Rouillard et al 2018 interpreted as pseudo fossils –biomorphs. Especially the paper of Rouillard et al 2018 explains best how these structures are formed and we should be very careful in a biological interpretation of such structures.

See our comment to reviewer 2 concerning abiotic origin; we will extend the discussion. However, we are convinced that our observations about the inner structures with systematic N-S-O distribution, the inclusion of nanocrystals of Bi-S-Te, the large variation in morphology (not only filamentous), and the preservation of sheath-like structures is a good indication for a biogenic origin.

In lines 668-675 they interpret some of the structures as palynomorphs. 1.5 Ga old palynomorphs are rare and look like simple acritarchs and I cannot recognize it in the figured pics.

See our comment to reviewer 1 about the palynomorphs, our wording seemed to provoke the misunderstanding.

Another aspect is, that the putative microbial/fungal fossils are may be much younger as 1.5 Ga. The filaments are spatially related between crystal boundaries (Fig.2). The preservation and overall shape of the structures are too “perfect”.

Our argument for the age of ca. 1.5 Ga is presented in lines 65-69, and we don’t see another possibility to explain the fossils as younger. We will extend the description in more detail.

I suggest major revision! My recommendation is to invest more time in comparing the found structures with further examples of so-called deep biosphere biota, putative pseudo fossils (biomorpha), and also the aspect, that these structures were later formed than 1.5 Ga. From a methodological viewpoint I also recommend to use Raman techniques and, if possible, electron microprobe + µ-xrf mappings on polished surfaces/thin section for a more advanced microfacies analyses.

We intend to continue with the study and will try to find biomarkers (in cooperation with C. Heim, Köln) and using the ion-probe for C-N-insitu determination (in cooperation with A. Hertwig, Heidelberg). Ongoing investigations about the Bi-S-Te nanominerals with TEM are in progress with R. Wirth (Potsdam). We will also explore the possibility to use Ramanspectroscopy. µ-XRF mappings have less resolution than the microprobe mapping, so we don’t see the advantage of this mapping.

The preservation of the putative microbial fossils is very good, and if these structures are pristine this contribution would be a milestone in the understanding of the “boring billion”.