Please refer to the attached PDF for my review of your manuscript.

In the manuscript “Archaeal lipostratigraphy of the Scotian Slope shallow sediments, Atlantic Canada” Ahangarian et al. present the archaeal lipidomes in a wide range of sediments taken across the Scotian Slope. I was impressed by the large dataset which covers such a broad geographical range. I am generally accepting of the methods utilized and the lipid data itself looks interesting and valid. However, my concern about the manuscript is in the interpretation and discussion of the data. I feel that authors have not sufficiently reviewed the literature on archaea and archaeal lipids, which in turn has had a considerable effect on how they interpret their data.

One major issue is that the authors have made the assumption that glycosidic GDGTs are indicative of living archaea. This has been shown extensively to not be true, both experimentally and in the environment. Glycosidic GDGTs are known to be preserved for thousands and even millions of years. GDGTs with phospho-bound head groups are more accurate markers of living or recently living cells. However, the authors do not detect phospho-bound GDGTs, only some phospho-bound archaeols. The authors have failed to correctly interpret some of the literature they cite, such as the review of Schouten et al. (2013) which clearly states that “these IPLs may be of fossil origin, as degradation of ether lipids with a sugar head group, or even IPL GDGTs in general can proceed much more slowly than that of regular bacterial ester-bound IPLs.”. I draw the authors’ attention to more recent work that also shows that glycosidic IPL GDGTs cannot be used as markers for living archaea (Wu et al., 2019) and ask that the reviewers read this and the literature cited in its introduction. As a result of this error, amongst others listed below, the authors should go back and fully rewrite their manuscript to take this into account, carefully reexamining their assertions from their data about “living, fossil and degraded lipids”. Indeed, it would seem possible (or even probable) that the vast majority of the GDGTs detected originate from the overlying water column, not from the sediment itself. This is going to have considerable effect on the manuscript and hence I suggest major revisions to accomplish this.  

Overall, I find it quite risky that the authors using the profiles of the photosynthetic pigment chl-a and its alteration product OH-chl-a in order to interpret the lipid data. The lipid extraction method used is not designed for pigment extraction, nor is the analysis method. As this pigment is notoriously easy to destroy with light, heat or acid, there is a risk that the depth profile of these compounds is an effect of variable extraction efficiency. The authors need to examine experimentally how rigorous their extraction methodology is for chlorophyll. As I stated above, OH-chl-a is an alteration product of chl-a (often formed during extraction), so I would not describe it as a photosynthetic pigment (Steele et al., 2018).

Another issue in terms of the authors’ interpretation of the literature, is the assignment of GDDs as GDGT degradation products. This is indeed the conclusion of Hingley et al. (2024), however the other article cited, (Mitrović et al., 2023), makes a quite different statement “it seems unlikely that GDDs are a direct diagenetic product of GDGTs. Overall, the observations from our study support the theory of a joint cellular origin for GDGTs and GDDs.”. It is ok if the authors of this manuscript to not agree with Mitrović et al. (2023), but they should not incorrectly present the conclusions of that study.

The authors also need to address the language used throughout. During revision this manuscript should be carefully read through and checked. As I have asked for major revisions, I will not do a line-by-line list of edits, as so much is likely to change. However here are some edits I would draw to the authors' attention.

Writing general – when rewriting this should be tightened up with fewer words and sharper sentences.

Line 21 and in more places throughout the manuscript (e.g. line 64). It’s not clear here what the authors mean here by deep. Is that deep sea or deep sediment.

Line 53. Thaumarchaeota were reclassed a few years ago as Nitrososphaerota (Rinke et al., 2021). I’m not suggesting that you use this newer term throughout the manuscript, but it should be mentioned here.

Line 74. I think you should redefine all abbreviations in the introduction and not rely on them being defined in the abstract.– e.g. IPL and CL

Table 1 – this needs to be improved visually and a lot of the information (such as sediment extracted and TLE weight) moved to a supplementary table.

Figure 1 (and others) – seems very fuzzy.

Line 236. This isomer of crenarchaeol is not a regioisomer and is hence no longer called this (cf. Sinninghe Damsté et al., 2018). Please read the up-to-date literature on this subject.

Entire section 4.1. Many of lines read poorly and should be rewritten for clarity. Additionally, the authors emit some significant recent work on the GDGTs of Thaumarchaeota, e.g. Elling et al. (2017) and Bale et al. (2019) and on hydroxy GDGTs e.g. (Varma et al., 2024).

Articles mentioned in the review

Bale, N.J., Palatinszky, M., Rijpstra, W.I.C., Herbold, C.W., Wagner, M., Sinninghe Damsté, J.S., 2019. Membrane Lipid Composition of the Moderately Thermophilic Ammonia-Oxidizing Archaeon “Candidatus Nitrosotenuis uzonensis” at Different Growth Temperatures. Applied and Environmental Microbiology 85. doi:10.1128/AEM.01332-19

Elling, F.J., Könneke, M., Nicol, G.W., Stieglmeier, M., Bayer, B., Spieck, E., de la Torre, J.R., Becker, K.W., Thomm, M., Prosser, J.I., Herndl, G.J., Schleper, C., Hinrichs, K.-U., 2017. Chemotaxonomic characterisation of the thaumarchaeal lipidome. Environmental Microbiology 19, 2681–2700.

Hingley, J.S., Martins, C.C., Walker-Trivett, C., Adams, J.K., Naeher, S., Häggi, C., Feakins, S.J., Naafs, B.D.A., 2024. The global distribution of Isoprenoidal Glycerol Dialkyl Diethers (isoGDDs) is consistent with a predominant degradation origin. Organic Geochemistry 192, 104782.

Mitrović, D., Hopmans, E.C., Bale, N.J., Richter, N., Amaral-Zettler, L.A., Baxter, A.J., Peterse, F., Miguel Raposeiro, P., Gonçalves, V., Cristina Costa, A., Schouten, S., 2023. Isoprenoidal GDGTs and GDDs associated with anoxic lacustrine environments. Organic Geochemistry 178, 104582.

Rinke, C., Chuvochina, M., Mussig, A.J., Chaumeil, P.-A., Davín, A.A., Waite, D.W., Whitman, W.B., Parks, D.H., Hugenholtz, P., 2021. A standardized archaeal taxonomy for the Genome Taxonomy Database. Nature Microbiology 6, 946–959.

Schouten, S., Hopmans, E.C., Sinninghe Damsté, J.S., 2013. The organic geochemistry of glycerol dialkyl glycerol tetraether lipids: A review. Organic Geochemistry 54, 19–61.

Sinninghe Damsté, J.S., Rijpstra, W.I.C., Hopmans, E.C., den Uijl, M.J., Weijers, J.W.H., Schouten, S., 2018. The enigmatic structure of the crenarchaeol isomer. Organic Geochemistry 124, 22–28.

Steele, D.J., Kimmance, S.A., Franklin, D.J., Airs, R.L., 2018. Occurrence of chlorophyll allomers during virus-induced mortality and population decline in the ubiquitous picoeukaryote Ostreococcus tauri. Environmental Microbiology 20, 588–601.

Varma, D., Villanueva, L., Bale, N.J., Offre, P., Reichart, G.-J., Schouten, S., 2024. Controls on the composition of hydroxylated isoprenoidal glycerol dialkyl glycerol tetraethers (isoGDGTs) in cultivated ammonia-oxidizing Thaumarchaeota. Biogeosciences 21, 4875–4888.

Wu, W., Xu, Y., Hou, S., Dong, L., Liu, H., Wang, H., Liu, W., Zhang, C., 2019. Origin and preservation of archaeal intact polar tetraether lipids in deeply buried sediments from the South China Sea. Deep Sea Research Part I: Oceanographic Research Papers 152, 103107.