Dear Editor,

We thank the reviewer for their positive evaluation of our manuscript. Below we address all their comments point by point. In summary, we aim to follow essentially all their suggestions in our revision of the manuscript.

Sincerely, also on behalf of my co-authors,

Yannick Bats, MSc.

This is a clear very well written paper looking at an interesting and important aspect of  paleo science.

I offer a couple of minor comments,:

The discussion from line 400 about the differences between sporopollenin from seed plant and non-seed plants seems to be resented with too much certainty given  the very limit amount of taxa currently evaluated.  I would recommend this statement be revised o reflect uncertainty.

Authors’ response:

We agree that we cannot generalize based on our limited data. Therefore, we will add the following statement: "However, considering the limited amount of taxa studied here, more research is needed to confirm this.”

I think the section 4.5 should be expanded and a more detailed  comparison made to data from the experiments carried out in this study and to experiments that have sort to tease out changes in chemistry by simulating thermal maturation and maybe  this could be extended to look at fossils. This would give a more rounded approach to the discussion and make the paper more useful to the broader bio-geoscience community.  

Authors’ response:

To accommodate this point, we will expand the text in section 4.5. to include a better comparison with artificial maturation experiments, and to discuss the use and effects of acetolysis more in depth.

With regard to figures I would encourage the authors to reconsider their colour palette choices and avoid red green color combinations given the prevalence of red/ green color vision deficiency. Authors’ response: We agree and will optimize colors used.

I wonder if figure two might be better plotted as a difference from untreated for each taxa.

Authors’ response: We have experimented with replotting Figure 2 with values as a difference from untreated, but all of our efforts resulted in less clear figures. We therefore respectfully decided to retain the current version, but with optimized colors.

Dear Editor,

We thank the reviewer for their positive evaluation of our manuscript. Below we address all their comments point by point. In summary, we aim to follow essentially all their suggestions in our revision of the manuscript.

Sincerely, also on behalf of my co-authors,

Yannick Bats, MSc.

This is an interesting and well-written paper, focusing on sporopollenin δ13C measurements across a series of taxa, in relation to different laboratory processing procedures and analytical approaches (EA-IRMS vs LA-IRMS).

I think the paper is broadly publishable as is it. The only part I am less convinced by is section 4.5 'Implications for fossil sporomorphs'. The authors treat acetolysis as if it efficiently isolates the sporopollenin wall with no additional effects, but previous studies have shown that acetolysis also alters the sporopollenin: Lutzke et al. (2020) and Wang et al. (2023) showed that acetolysis reduces the phenolic content of sporopollenin, and Amundsen et al. (1997) and Loader and Hemming (2000) showed a considerable decrease in δ13C values with acetolysis, including (in Loader and Hemming) in relation to a different sporopollenin isolation approach (sulphuric acid - so it’s not just that the δ13C value goes down because the sporopollenin is being isolated, the acetolysis is actually doing something else as well). Dominguez et al. (1998) and Jardine et al. (2015, 2017, 2021, 2023) have also used FTIR to show that new peaks appear with acetolysis that are not produced by other processing/isolation approaches. So acetolysis is useful for removing labile compounds and leaving the sporomorph exine behind for morphological analyses, but from a chemical point of view it is does change things, and I would be wary of saying that it produces something similar to diagenetically altered sporomorphs from the geological record (the authors also need to be careful here - do they mean relatively recently buried sporomorphs, where we might expect the labile compounds to have degraded but the sporopollenin to be more or less unchanged, or geological samples where the sporopollenin has repolymerised?).

Authors’ response: We thank the reviewer for these thoughtful remarks and suggestions on relevant literature and incorporated these in the manuscript. As suggested, we will change section 4.5. to include a more thorough discussion on the use and effects of acetolysis, and provide some examples of other methods of isolating sporopollenin.

In terms of recommendations for comparing modern and fossil sporomorphs, the authors also need to keep in mind that other approaches for isolating sporopollenin have been suggested. Li et al. (2019) and Lutzke et al. (2020) used a combination of enzymes and solvents, for example, and Loader and Hemming (2000) used sulphuric acid. Jardine et al. (2023) carried out a comparison of these and other methods from the perspective of FTIR and chemotaxonomy. I don’t suggest that the authors add these approaches to this study, but some comments in the discussion to point out that other methods are available and could be compared in future research would be a useful addition.

There are three papers that I have mentioned here but are not cited in the paper, which I think the authors would do well to read and incorporate into the text:

Domínguez, E., J. A. Mercado, M. A. Quesada, and A. Heredia. 1998. Isolation of intact pollen exine using anhydrous hydrogen fluoride. Grana 37(2):93-96.

Jardine, P. E., M. S. Kent, W. T. Fraser, K. H. Knorr, and B. H. Lomax. 2023. Uncovering a phylogenetic signal in plant biopolymer chemistry: a comparison of sporopollenin isolation approaches for use in palynological research. Palaeontology 66(6): e12683.

Wang, T., Bell, B.A., Fletcher, W.J., Ryan, P.A., and Wogelius, R.A. 2023. Influence of common palynological extraction treatments on ultraviolet absorbing compounds (UACs) in sub-fossil pollen and spores observed in FTIR spectra. Frontiers in Ecology and Evolution 11:1096099.

Authors’ response: We will modify section 4.5 to improve comparison to studies that assessed the molecular and isotopic effects of thermal maturation, to draw more solid conclusions on the implications of our study on fossil sporomorphs, following the reviewer’s suggestion.

And finally, one minor comment:

Lines 42 - 43: exine isn’t a common term for sporopollenin - the exine is the outer wall of pollen and spores, and is composed of sporopollenin. I suggest rephrasing this sentence.

Authors’ response:

We wil adapt to: “Organic microfossils are comprised of organic macromolecular structures such as sporopollenin (the main component of exine – the outer wall of pollen and spores), residual labile compounds (e.g., polysaccharides, proteins) and diagenetically produced ‘geopolymers’ …”