Reviewer 1,

We are very grateful for the detailed, thoughtful and helpful comments. Your comments have been extremely helpful in identifying areas for improvement and clarification. Below, please find our point-by-point response and how we intend to address both your general and your specific comments.

General points:

● First, the determination of BHP concentrations is complicated by the certainly very different responses of individual BHP during ionisation and decay during mass spectrometric analysis. The authors correctly describe this. However, they withdraw from this and write that they cannot make any (semi-)quantitative statements. In practice, however, they do it indirectly themselves by adding up the peak responses (in relation to TOC) in Figure 3, for example. If no quantitative comparisons are permitted, a presentation that totals up to 100 % is also out of the question. I think it should be possible to use this data and also make a semi-quantitative statement on the relative concentration of at least the majority of BHPs versus time/depth (with mentioning of the restriction). I would find such a curve helpful as insert to Figure 3 (as a or b). Of course, it would also be interesting to see whether this is consistent with other studies that have analysed (fewer) BHPs, but where there is a large overlap in the BHP biomarkers used and most common?

Thank you for this comment. In our manuscript we do make “semi-quantitative” interpretations, as that is one of the few ways that we can discuss this dataset. This type of data is very similar to DNA datasets, which are also often discussed in the context of relative abundances even though this does not directly reflect the actual abundances of species present in a dataset. We think it is important to highlight the limitations of the dataset, even though we do rely on “semi-quantitative” interpretations. We will rephrase the sentences discussing these limitations in the methods section to make this clearer. Further, we will include a second subplot in Figure 3 to show “total BHP concentrations.” However, we would like to refrain from making comparisons with previous studies about total BHP concentrations as these studies used either GC-MS or HPLC-APCI-MS quantifications whereas our study uses an UHPLC-ESI-HRMS system. The ionisation efficiency in all three systems differs, and unfortunately, we do not have any standards that would allow us to make this comparison.

● Second, there seem to be only a few errors or ambiguities here, but the authors should check this again very carefully. For example, the term "anhydrous-" BHT is sometimes used and sometimes "anhydro-" BHT.

Thank you for identifying this. We will change “anydrous-BHT” to “anhydro-BHT” throughout the text

● Furthermore, it remains unclear to me what "BHT" means in Figure 3, for example? In S4, the course of BHT-22S is shown. Is the former a sum of the different BHT isomers? Please do not take this as a request to show all isomers. This would complicate the manuscript even more. However, how certain is the structural elucidation of "BHT-22S" really? Are there, for example, co-elutions with BHP extracts from reference organisms? Me-adenosylhopaneHG-diMe exists twice in Figure 4, for example (in addition to 2-Me and 3- Me). Are these isomers? Which ones are meant in the text when the compound is mentioned? Please double-check everything again. Further, a correct "...may be..." to a structure proposal in a cited study ("BHT-22S") does not seem to be discussed here and the structure or interpretation of the structure is simply adopted (text and Fig. S4). Of course, it is not possible to simply repeat the very extensive structuralelucidations that colleagues from Strasbourg in particular carried out on individual BHPs in the 1980s and 1990s. Nevertheless, the limitations of structure elucidation with MS-(MS) should not be forgotten and uncertainties should be described to remind readers to this limitation.

Thank you for pointing this out. BHT in Figure 3 is meant to represent regular BHT (BHT-34S) and BHT-x is shown separately in the figure. We will change the label in the figure caption from BHT to BHT-34S.

We agree with the reviewer that using a MS-(MS) based system we are limited in our structural identifications, and as we state in the methods sections these are “tentative” identifications. Our structural identifications are based on retention times, exact masses, and fragmentation spectra, as well as comparisons with previous studies. In the case of the BHT isomers, the identification of these compounds and distinguishing the various isomers on a UHPLC system was extensively discussed in Schwartz-Narbonne et al. (2020) and Hopmans et al. (2021). Identification of the different isomers was determined based on retention times as described in Cutmore et al. (2025), which was found to be reliable when using a UHPLC system (Schwartz-Narbonne et al., 2020; Hopmans et al., 2021). We will add a short sentence to the methods section that clarifies these limitations and that we relied on previous studies for our structural elucidations.

Thank you for catching this mistake with the labelling of Me-adenosylhopaneHG-diMe. The two Me- adenosylhopaneHG-diMes in Fig. 4 refer to two different isomers with unknown Me positions, therefore, we will change this to “early” and “late” isomers in Figures 3, 4, and S8 as well as in Table S1.

● Third, as a reader, I miss take-home messages in some places in the sub-chapters. In some cases, relatively long explanations with pros/cons/exceptions are described (please utilise the potential for shortening here). However, a quintessence is sometimes missing. One example is the long block of text on "BHPs associated with the N-cycle". As a reader, I would expect a clear categorisation of whether the data fully correspond to the interpretation in Cutmore et al. (2025), whether they are helpful and supportive for the other paper or whether they contradict aspects of it. What is new and different in this manuscript compared to Cutmore et al 2025?

Thank you for this comment. We will shorten this chapter and reference the CoP paper rather than outline the findings in detail. We will also clearly show what is new research/conclusions and outline how the new findings support the interpretations of Cutmore et al., 2025. Finally, we will ensure there is a concluding sentence.

● Fourth, illustrations are also a matter of taste. Nevertheless, I find it unfortunate and urge the authors to reconsider whether a form of representation can be found that shows the temporal change in a uniform way. In other words: In Figures 1 and 3, time is represented from top to bottom (similar to the core). A form of representation that I welcome and find understandable. In Figure 4 and 5 (and in the supplement), on the other hand, time is shown from left to right. As I said, it is also a matter of taste, but a change in the basic presentation of data versus depth/time within a study makes it unnecessarily difficult for readers.

We appreciate the reviewer’s comments regarding the consistency of time-axis orientation across figures. We fully agree that clarity and ease of interpretation are important, and we understand the concern that changes in the basic presentation of data can make comparisons more challenging. In Figures 4 and 5 (and in the Supplement), we chose to present the paleo records with time on the horizontal axis, as this is thestandard convention within palaeoclimate literature. To explore the reviewer’s suggestion, we tested an alternative version of Figure 3 with a horizontal time axis for visual consistency. However, due to the complexity and density of information in that figure, this layout significantly reduced readability and made the data more difficult to interpret.

● Finally, in general, the authors demonstrate a great deal of expertise and generally cite existing studies that have worked on similar issues (including with BHP, albeit with fewer structures) in a good way. Nevertheless, at one point or another I would expect categorisations as to where there is clear support from the new data and where there is not.

Thank you for this comment. We will make sure we have concluding sentences and make it clear throughout the manuscript where our study is supporting existing information, and where we are putting forward new theories.

Specific points:

● Line 44: I think early studies by Helen Talbot should be mentioned in these citations of important papers (from 2003 or similar). Her work was methodologically groundbreaking, but also contributed to the understanding of source organisms and biogeochemical processes.

We’re grateful for your recommendation. We will mention these key studies by Talbot et al. (Talbot et al., 2003, Organic Geochemistry; Talbot & Farimond, 2007, Organic Geochemistry)

● Line 88: Please replace "compounds" with "BHPs".

Thank you for this. We will replace this with BHPs.

● Line 97: mbsl must be explained.

Thank you for picking this up. We will make sure this acronym is explained as metres below sea level

● Line 130: See general note. This is understandable, but if you take a very strict view, then BHPs can also not be shown in a comparative graph (e.g. Fig. 3). However, this greatly reduces the informative value, as only the progressions of individual connections can then be considered. I suggest looking for a way to show at least the total BHPs over time in a semi-quantitative way.

As discussed above, we will amend the text to highlight that although our data is only “semi-quantitative”, it is still valuable to show major changes in BHP abundances as it demonstrates changes in BHP distributions during the key periods of the Black Sea. We will also add a subplot to Figure 3 to show changes in total response units of all BHPs per gram TOC. Comparisons of individual BHP abundances are shown in the supplementary which we will make sure are clearly referred to in the text when necessary.

● Line 173: See above. Is it really BHT-22S? The illustration only says BHT. It seems to me that the latter is better documented.

Thank you for noticing this. It is regular BHT (BHT-34S) that we have identified based on the mass spectral information. Consequently, we will change ‘BHT’ to ‘BHT-34S’ in Fig. 3 to improve clarity.

● Line 197: See above. This statement refers to "absolute abundances of BHP", but this is not shown at all. Please find a way and discuss the result comparatively in this chapter.

We will rephrase this and refer to the supplementary graphs where this is shown.

● Line 211: Please change to "...suggesting rapid early diagenetic modifications of BHPs."

We will rephrase the sentence accordingly.

● Line 229ff: This is a good example of a summarising sentence at the end of a discussion. However, what is missing here is that diagenetic changes in the BHP play a subordinate role (as this appears the opinion of the authors!?). It would be interesting to look at the ratio of individual unsaturated to saturated BHPs assuming that some are diagenetic products of the other. At least any diagenetic-looking change would hint at respective relationships. Does this support the statement?

Unfortunately, we cannot clearly state that diagenetic changes in BHPs play a subordinate role as we are only looking at a proportion of potential diagenetic BHPs as discussed in our response to reviewer 2. Although a comparison of unsaturated aminotriol to aminotriol was done in Blumenberg et al. (2009) and was used as a first order interpretation for preservation, we feel that this is complicated by the fact that not all saturated BHPs are diagenetic products of unsaturated BHPs.

● Line 237: "later"

Thank you for picking this up, we will make this suggested change.

● Line 279ff: "As future studies discover more..." sounds odd. Please check and consider rewriting.

We will rephrase the sentence as follows: “As ongoing research continues to explore the sources of Nu- BHPs”

● Lines 334ff and 380ff: I think it is very unlikely that a relevant amount of BHP comes from sulphate-reducing bacteria. For the lacustrine phase, this can easily be explained by the general absence of relevant sulphate in the water. It would be good if the authors could find a way a shorter way to have this component-specific discussion of why the one is somewhat unlikely, though not impossible, and so on. Similar to the authors, I think that neither the transport of biomass from the sulphidic zone nor relevant biomass in the sediment plays a major role. The text and a (shorter) length should express that.

Thank you for this feedback, we will shorten this section.

● Line 356: Aminotriol seems so unspecific to me that it could be produced by all kinds of bacteria.

We agree that aminotriol is found in too many bacteria to make this conclusion, so will remove this from the manuscript.

● Lines 359-366: It is not entirely clear what is meant here. Is there now a peak for methanotrophic bacteria in the transition phase? Does this show aminotetrol and pentol? The study cannot contribute anything to the anaerobic oxidation of methane and the topic should therefore only be mentioned briefly.

We will amend our phrasing to more clearly state that we observe MOB-related BHPs throughout the transition phase (i.e., aminotetrol and ethenolamine-BHpentol). Although there is only a small peak in these BHPs near the beginning of the transition phase, they are present throughout this time period. We will remove the sentence on anaerobic methane oxidation.

● Line 366: The data does not show a "distinct increase" for me. Please describe a little better. Figure 5 shows a peak before the transition phase and, in comparison, slightly increased values in marine phase II.

Thank you for pointing this out. We agree this is confusing, as ethenolamine-BHpentol, ethenolamine-BHhexol and propenolamine-BHT all show an increase, while the aminotetrol and aminopentol show a more complex change. We will rephrase this sentence to make it less confusing and more accurate.

● Line 441: This statement is difficult to understand based on the data. Aminotetrol and -pentol appear to fluctuate strongly (Figure 5).

We will rephrase this section to make it clear that we are talking about absolute abundance being higher during the early marine phase compared to the later marine phase.

● Figure 1: Please write the approximate ages (in ka) next to the models for the three phases.

Thank you for this suggestion, we will include the ages in this figure.

● Figure 3: Many BHP structures are colour-coded in the figure. It is sometimes difficult to make an assignment and it is even impossible without colour (black/white). Perhaps the authors could write the abbreviations of the BHP on the main horizontal bars to at least make these clearly comprehensible?

We appreciate the suggestion. To maintain the visual clarity of Figure 3 and highlight broad temporal patterns, we prefer not to add text to the bars, as it may make the figure overly cluttered. We will make sure the colours used in this figure are appropriate for a black/white figure. The detailed information on individual BHPs is provided in the supplementary material and will be referenced more clearly in the main text. We have also added a supplementary figure showing diagenetic products to help visualize changes in specific BHPs more clearly. Based on the reviewer’s previous comments we will add a subplot to this figure to show “total BHP abundance” and a supplemental spreadsheet containing all the data so readers can confirm trends shown in the figures.

● BHP figures (Figure 5, but also in the supplement). Please insert a 0-line (y) for each BHP so that it is easier to see when data points differ from 0We thank the reviewer for the suggestion to add a zero-line to each BHP plot in Figure 5 and the Supplement.

We understand that such a reference line can help readers quickly assess deviations from zero, however, after testing this addition, we found that including a zero-line for each compound made the figures appear overly cluttered, particularly given the number of compounds and the existing visual elements (e.g., shaded backgrounds denoting the marine, transition, and lacustrine phases). We feel that the current design—with the shaded intervals so the reader is able to quickly assess the patterns of each compound during the key periods—already provides sufficient context for interpreting the trends in BHP concentrations over time without compromising visual clarity. Each individual BHP is also shown in the supplement for ease of readability, and the full BHP dataset (peak area per g of TOC) will also be made publicly available in a supplemental spreadsheet with this manuscript.

Reviewer 2,

Thank you for the comprehensive and helpful comments. We greatly appreciate the time and effort you invested in reviewing our manuscript. Please find our point-by-point response and how we intend to address your general, specific, and technical comments, below:

●The role of degradation products of originally produced BHPs is also discussed in one chapter (4.2) in this paper, however, it only includes very few hopanoids, like anhydro BHT, anhydro BHpentol, as well as some BHtriols. These compounds are only very early degradation products, and only allow a small portion of the degradation story of hopanoids. Since the authors would like to report about these signatures, I suggest to modify the presentation of these compounds in Figure 3. For me it was not easy to see the diagenetic products in this figure. I suggest to make a new group, or to show the diagenetic BHPs with hatches, points or whatever, additionally to the colour code already available. So everyone can see at first sight which BHPs are diagenetic BHPs. More suggestions can be found in the general comments.

We are grateful for your recommendation. We will add a new supplementary figure to show changes in the diagenetic products to help visualize changes in these BHPs more clearly and refer to this figure in chapter 4.2.

Specific comments and Technical Comments

● Line 31: Please provide some percentages of diagenetic products. It is not really surprising that diagenetic products like anhydro-BHT can be found already in the water column?

Thank you for this suggestion, but we feel that adding this would overly emphasise the relative abundance of diagenetic products. BHP quantification in this study is “semi-quantitative” as we lack the standards to account for differences in ionisation efficiencies between different types of BHPs. Further, the ionisations of the diagenetic products will likely differ greatly from the potential precursor BHPs. This may lead to false trust in the absolute amount of diagenetic products. Instead we would prefer to highlight general changes and trends in the early diagenetic BHPs, and we rely on previous work by Blumenberg et al (2009) for quantitative estimates of diagenetic products in the Black Sea record.

● Line 54: …oxidizing…

Thank you for noticing this, we will make the change.

● Line 65: This is true, but is limited by the stability of the BHPs, which is already demonstrated by the presence of early degradation products. The authors, however, do not mention the possibility, that at least a certain percentage of the BHPs is not in the easy extractable fraction, but may be preserved as macromolecules, especially under euxinic conditions as organic sulfur compounds. This is not the topic of this paper, but should at least be mentioned, that only very early degradation products are monitored, so it is rather an incomplete observation and discussion of degradation products of BHPs. This must be clarified in chapter 4.2. Secondly, as already mentioned by the authors, the record of many of the measured BHPs is very limited over time, and can only be found in sediments a little older than 1 million years.

We will add a sentence to section 4.2 to highlight that this study only focuses on early “polyol” degradation products of BHPs and that potentially S-bound BHPs were not extracted. We will also mention that this paper does not discuss the additional degradation processes of BHPs towards more stable hopanoid products (as these were not analysed using our method). As to the reviewer's second point, we agree that BHPs have so far only been identified in continuous paleo-records that span the last 1 million years (e.g., Talbot et al. 2014) and are likely more degraded on longer timescales. However, as our study only looks at BHPs over the last 20ka we do not think this is a major limitation in our study. We will add the word “recent” before "geological record” in the text.

● Line 79: replace ‘enhanced’ by ‘increased’. This should be done all through the manuscript.

We will make the change throughout the manuscript.

● General comment Chapter 4.2: As mentioned in the general comments above the diagenetic products here are only covering a very small portion of the diagenetic pathway of BHPs. Blumenberg et al. (2009) tried to unravel this problem already in their manuscript and their findings should be mentioned more clearly, especially when discussing the degradation products here.

We will add a clearer discussion on the diagenetic products as described by Blumenberg et al. (2009).

● Line 212: As much as I can remember, Kusch et al., 2022 suggested that selective degradation is rather unlikely from their results. Moreover, aminotriol has been found to be transformed into a tentatively identified anhydro-BHT-like degradation product by Eickhoff et al., (2014). These authors proposed that in temperature/pressure experiments of R. Palustris TIE-1 aminotriol is transferred into these N-containing anhydro-BHTs. Have you tried to search for these compounds? I am not sure if they ever were identified in the environment, and most likely their precursors are less abundant than the anhydro-BHT precursors. I am just asking out of curiosity.

Our interpretation of the discussion of selective degradation in Kusch et al. (2022) is that (in section 5.4 of Kusch et al. (2022)), the authors suggest that one possible explanation for the changes they see between the “fluff layers” of the sediments and the first few centimetres, is likely associated with either selective degradation or preservation of the most abundant BHPs in the sediment (i.e., BHT-CE, BHT glucosamine, BHT pentose, BHT, and aminotriol). Although this is likely not the sole reason for the observed changes in BHP composition, the authors observe an associated increase in anhydro-BHT at the sediment surface (Kusch et al., 2022).

Thank you for asking about N-containing anhydro-BHTs. In our screening, we did not detect any N-containing anhydro-BHTs in this record.

● Line 217: I am not sure if everyone is aware of the BHtriols, so the papers first reporting about them must be mentioned, as well as the environments they were found (e.g. Watson and Farrimond, 2000 or papers from Lago di Cadagno). As much as I can remember, they are especially found in lacustrine samples, which seems to be true also for the Black Sea samples.

Thank you for the suggestion. We will add these key references and mention the previous environments in which BHtriols have been found.

● The potential sources of the degradation products anhydro BHT have been at least partially identified in culture and P/T experiments, as cited by the authors, but to my knowledge BHtriol and other related compounds were never produced in any experiments, so not a lot is known about them. Maybe I overlooked this in the literature, but if there is anything known it should be discussed as well. Most likely, both anhydroBHT and BHtriol seem to be derived from BHPs with four functional groups, whether this was a compound like adenosylhopane or just BHT or methoxy BHT, is unclear. BHPs with 5 or 6 functional groups are rather unlikely, since they had two additional hydroxy groups at carbons 31 and 30. In Watson and Farrimond (2000) they also showed diols and triols with hydroxy groups at positions 30 and 31. If there are BHPs with functional groups at C-30 and C-31, I would expect to find such degraded BHPs as well. Is this the case? Were any of these ‘degraded’ hopanols introduced by Watson and Farrimond (2000) found?

To our knowledge, Rodier et al. (1999) and Watson & Farrimond (2000) are the only previous studies on these compounds. As suggested by the reviewer, we searched for the additional diols and triols identified in Watson & Farrimond (2000); we were only able to tentatively identify one of the triols with 32 carbons (C32H56O3) in the lake phase. It is possible that other diols or triols were present during the lake phase, however, the fragmentation patterns were not clear enough for us to assign these compounds. We will mention this in the text.

● Figure 3: The figure is very nice and it’s easy to follow the various colour codes. However, when reading the text I found it rather difficult to identify the diagenetic BHPs. Maybe it is possible to make either a separate group of columns only for these BHPs, or alternatively the degraded BHPs could be marked by hatching, so everyone can see immediately what are diagenetic BHPs. See my general comments.

We thank the reviewer for their suggestion, we will add an additional figure that shows the diagenetic BHPs to the supplementary material.

● Line 239: I am confused by this reference. The element and GDGT distributions are both from Hopmans et al. (2004)? Which data are from Yang and personal communication? Element data are also provided in Cutmore et al. (2025). Please clarify. Further, the authors should refer here to their figure 5, where crenarchaeol is displayed. I suggest to include the BIT curve next to the crenarchaeol as well, so anyone can follow better what the authors are talking about.

Thank you for identifying this. We will alter this sentence to improve clarity, to ensure the reader is aware that the elemental and crenarchaeol records are from Cutmore et al., 2025 and the BIT index is from Yang (personal communication). The BIT curve is a key part of an upcoming manuscript by Yang et al., so is unable to be shown in this study.

● Line 252 ff.: Is there anything known about potential producers of 2Me-adenosylhopaneHG-diMe? What is the basis for the interpretation, that this compound is from bacteria thriving in the water column? Its interpretation as indicator of warming is rather speculative. I am not fully convinced and suggest to tone this down.

There are currently no known producers for 2Me-adenosylhopaneHG-diMe, although this is part of ongoing work at NIOZ. The hypothesised production of 2Me-adenosylhopaneHG-diMe in the water column was based on previous observations in a lake, where 2Me-adenosylhopaneHG-diMe increased with depth (Richter et al., 2023). Further, in the Black Sea record, 2Me-adenosylhopaneHG-diMe abundances vary independently of other nucleoside BHP distributions before the transition phase (Fig. S8 & S9), and also differs from other proxies that are indicative of soil inputs during this time period (Fig. 5). Thus, we speculate that 2Me-adenosylhopaneHG-diMe is derived from an alternative source during this time period. We have added a short sentence to clarify this point in the manuscript and we will remove the section that suggests it is associated with warming temperatures.

● Lines 261, 270: It is okay to take data from another paper (Ti/Ca, K values), but they need to be cited, or shown in figures as well. Such information is important. I know it has been done somewhere else, but need to be provided whenever needed throughout the text.

Thank you for pointing this out. We will add the citation (Cutmore et al., 2025) every time these records are mentioned in the text.

● Line 287: This sentence is confusing. It reads like the Anammox are expanding from the sediment to the water column. Re-write the sentence.

To make sure this is clearer, we will rephrase this sentence as follows: “which enabled anammox bacteria to inhabit both the anoxic sediments and overlying water column”

● Line 295 ff.: There is a lot of speculation about the source of BHT-CE. Nitrososphaerota are suggested as producers. Is it known that these bacteria can produce BHT-CE? Refer to a paper, or explain that this is a speculation based on whatever. BHT-CE and potential sources seem to be puzzling, also in other studies. It is intriguing, that both crenarchaeol and BHT-CE really seem to correlate well over the entire core, which is surprising, because Thaumarchaeota and the potential BHT-CE producers are possibly not thriving in exactly the same environment. BHT-CE are either known from soil, peats etc. produced by unknown anaerobic bacteria, or methylotrophic bacteria.

Thank you for these comments. We will make it clearer in the manuscript that Nitrososphaerota are archaea and therefore not suggested as producers of BHT-CE. The suggestion is that the dominant bacterial producer of BHT-CE was coupled either to the archaea Nitrososphaerota or to the first step in the nitrification process. We will make sure this explanation is clearer. We are also extremely surprised and interested by this coupling and it certainly would be a great area to explore in future studies.

● Line 312 ff.: The old reports of 2-methyl hopanoids produced by marine cyanobacteria are outdated, and have been disproved, rather other producers are more likely. Finally, the authors decide to assign them potentially to heterocyst cyanobacteria? Do HGs produce 2-methyl hopanoids at all? This suggestion must be confirmed by any data reported in the past. Numerous studies analysed a great variety of cyanbacteria starting from Talbot et al., 2008, and it was the major topic of the paper by Naafs et al., 2022 as well, to verify sources of 2-methyl hopanes in various environments. Findings presented in these papers need to be discussed and included also here.

Thank you for these suggestions. We will make it clearer in our manuscript that the reports of 2-methyl hopanoids produced by marine cyanobacteria were initial propositions by early studies, and that, since then, there has been development and new theories of who is producing these BHPs. Furthermore, we will add information about the heterocystous cyanobacteria that have been shown to produce 2-MeBHT (Nostoc muscorum, Calothrix sp. and Chlorogloeopsis fritschii) from these key studies.

● Chapter 4.4: It is great to discuss the sources in detail, but especially for the BHP inventory tentatively associated with the N-cycle, there is a lot of overlap with the paper published by the same authors last year in Climate of the Past. I am aware that this paper is supposed to cover all potential sources of BHPs and need to include also the BHPs associated with the nitrogen cycle, but I suggest to shorten this chapter and refer to the Climate of the past paper.

We’re grateful for your recommendation. We will shorten this chapter to ensure there is no overlap, and we will ensure the CoP is referenced.

● In line 338 a beta,alpha-diploptene is used as evidence for MOB? This is not really a convincing evidence. Diploptene is known from many bacteria, but especially ANNAMOX can also produce diploptene and cannot be used as sole argument for MOB, especially because Schwartz-Narbonne et al., 2023 showed as well strong 13C-depletions for ANAMMOX lipids, and values of MOB may vary as well, as shown in a compilation of MOB signatures in methane seeps (Cordova-Gonzalez et al., 2020). The same is true for the source assignment of such hopanoids, also BHPs.

We agree that this is not the strongest argument; however, this is not our sole argument and is rather used as independent evidence from previous research by Blumenberg et al. (2009) to further support our data that MOBs were present in the Black Sea during the lacustrine phase.

● Line 344/345: Be more precise with the MOB. MOB can thrive in various environments but in the setting described here they are especially abundant at the chemocline, better oxyclines. If MOB are prominent in the sediments under fully oxic conditions, this needs to be further discussed. There are reports of MOB in methane seeps, especially in seep carbonates formed in anoxic sedimentary conditions, but microaerophilic niches where MOBs can thrive (see Cordova-Gonzalez et al., 2020 and references therein). It must be further discussed, if there is additional evidence from other lipids, that methane was oxidized in the lacustrine phase. If you discuss sedimentary sources of MOB BHPs, these occurrences must be (at least shortly) further discussed.

We agree with the reviewer that MOBs in lakes are especially abundant near the oxycline, however, MOBs are known to thrive in fully mixed lakes (i.e., when the water column is oxic) at the sediment-water interface (see Hanson & Hanson, 1996 and references therein), but have also recently been detected in anoxic lake sediments (e.g., Martinez-Cruz et al., 2017). Thus, a sedimentary source of BHPs during the lake phase cannot be excluded, although we agree with the reviewer that this is likely a minor source of BHPs relative to the water column. Further work on BHPs in lakes is needed to distinguish these sources, and as far as we know, there are currently no additional studies on this topic. We will add a few references from modern lake studies to highlight sedimentary BHPs from MOB as a potential source.

● Line 363: The citation of Zhu et al., 2024 is tricky. The report of 13C-depleted carbon isotopes is true only for archaeols, but not for the GDGTs Caldarchaeol and Crenarchaeol, respectively acyclic biphytane and tricyclic biphytane. These two GDGTs show isotope values characteristic for marine planktic Thaumarchaeota. Isotopes of monocyclic and bicyclic biphytane, which are very abundant in ANMEs were not shown by Zhu et al., 2024. In deeper sediment, usually ANME-1 consortia are prevailing, and they produce especially GDGT- 1 and GDGT-2, leaving behind isotopically depleted monocyclic and acyclic biphytanes after ether- cleavage. If ANMEs would have been important, they would have had some influence on the water column derived signature for the caldarchaeol, whereas the crenarchaeol should remain unchanged. This also questions the interpretation of the BHP signatures without isotopes, whether these are either signatures of bacteria from the chemocline or diagenetic sedimentary sources. One way to test the potential abundance of ANMEs in your sediments would be to calculate the methane index from the GDGTs (Zhang et al., 2011). Since Crenarchaeol has been measured, all other GDGTs were measured as well, I guess.

We will remove this sentence from the manuscript, as we agree with the reviewer that preservation may be an issue. The methane index in the Black Sea is complicated as any lipids produced in the bottom waters during the transition or marine phase do not necessarily get transported to the sediment. Thus, we will refrain from discussing this in the manuscript.

● Line 391: No, BHT-CE is definitely not only produced by SRB, the same is true for aminotetrol and aminotriol. Sure, Desulfovibrio can make these BHPs, but compared to contents in MOBs, these two are very minor BHPs, although the results from cultures are very limited. This should be mentioned here. The Talbot and Farrimond (2007) is a very good review, but more recent findings of BHT-CE and BHT-22S must be included and also discussed, such as Eickhoff et al. (2013), who report about BHT-CE in Geobacter, which also could be potential producers, or bacteria like M. oxyfera and related (e.g. Kool et al., 2012), but the latter were already mentioned above.

We’re grateful for your recommendation. We will rephrase this section and add these references.

● Line 403: unclear wording. It reads like the rates of MOB in the anoxic water column are higher as in the oxycline, but here MOB vs. AOM is meant. Re-write this sentence.

Thank you for this comment, we will rephrase the sentence as follows: “MOB are present at the oxic-anoxic transition zone of the modern-day Black Sea water column; however, their associated rates of aerobic methane oxidation are several orders of magnitude lower than the anaerobic oxidation of methane (AOM) occurring in the anoxic water column”

● Line 411, 412: Yes, this is true. They could not find any evidence for AOM, but honestly, they did not show any isotope data of the characteristic ANME biomarkers such as GDGT-2 (monocycic biphytane after ether cleavage) and archaeols, especially hydroxyarchaeols. See my comment for line 363. But in general I agree.

Thank you for the comment, we have removed the discussion at line 363 as detailed in our previous response but will leave this in as a concluding discussion.

● Figure 3: It’s in places confusing, a) when it comes to the discussion of the diagenetic BHPs (see my comments above), but especially for some of the less abundant BHPs, which are not so easy to be identified.

Thank you for the suggestion. We have added a supplementary figure to show changes in the diagenetic products to help visualize changes in these BHPs more clearly. Additionally, we will make it clearer throughout the text that the information on individual BHPs is provided in the supplementary material. Due to the large number of graphs, it is not possible to add these to the main manuscript.

● Figure 4: The numeration must be modified. The figure in the upper left corner needs a letter, too, even though it is just showing the same information as shown in the three graphs on the right hand side. Otherwise, the caption is incomplete.

Thank you, we will edit the graph and the caption accordingly.

● Figure 5 (new figure 6): This is a good way to show the potential candidates for specific groups of bacteria. I suggest to show the structures of the displayed BHPs and also the creanarchaeol, so everyone can see the major differences of the various BHPs and their potential producers. The readership of biogeosciences may not be aware of the various BHPs and other molecules and would be a valuable addition.

We are grateful for your recommendation. We will move Supplementary Figure 1 (illustrating the BHP structures) to the figures of the main manuscript. To this diagram we will also add the structures of Crenarchaeol and isorenieratene.