| 26 Mar 2026
Tracking sulfate, magnesium, phosphorus and amorphous phases in Rosalina-like benthic foraminifera
Abstract. In this study, we combined high resolution Transmission Electron Microscopy (TEM) bright field and High-Angle Annular Dark-Field Scanning TEM imaging with nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) analyses of Rosalina like foraminifera cells cultured in 33S-labeled seawater to understand the origin of sulfur in the test and the cytoplasm, its interaction with the ultrastructure of the test and the co-distribution of sulfur with phosphorus, calcium and magnesium. Test chambers that grew in 33S-labeled seawater revealed that at least 1/3 of the sulfur incorporated is directly taken from seawater sulfate. Our observations reveal a global co-occurrence of P, S and Mg within the previous two last chambers of the test, that all appear more concentrated in two areas, mostly near the Primary Organic sheet (POS) and in the Outer Calcitic Layer (OCL) that corresponds to the calcite added during the growth of the last chamber. Less crystalline grains can be found in the P, Mg and S-richest part of the POS and within OCLs. We interpret these grains as ACC (Amorphous Calcium Carbonate), in association with organic matter and possibly ACP (Amorphous Calcium Phosphate) likely formed or assembled at the sites of calcification, revealing a complex interplay between S, Mg and P. This interplay could indicate an inorganic cause for S and P enrichments, co-occuring with Mg enrichments and amorphous phases. Finally, ~0.1 to ~3 µm Ca-rich grains are detected in the cytoplasm. The biggest ones can be interpreted as ACC vesicles, the smaller ones are Ca and P rich and can be interpreted as autophagosomes or acidocalcisomes, the latter also containing labeled sulfur. Labeled sulfur is also found within smaller vesicles in the cytoplasm and represents overall 10 to 25 % of the sulfur we observe. Though we cannot confirm the occurrence of an assimilatory pathway for sulfur in foraminifera, the clear presence of seawater sulfate in the cell demonstrates that sulfate is involved in many aspects of foraminifera biological activity, in close association with Ca-transportation, Mg and P distribution, and likely with ACC formation and its evolution to calcite.
The manuscript, "Tracking sulfate, magnesium, phosphorus and amorphous phases in Rosalina-like benthic foraminifera", by Paris et al., presents results of correlative TEM and NanoSIMS imaging to track the distribution of sulfur, phosphorus, calcium and magnesium in cultured foraminiferal cells. The foraminifera were cultured within 33S-labeled seawater to track the incorporation of inorganic sulfate into the test walls.
The paper is generally well-written and the results are novel and exciting. Nevertheless, there are some serious issues with the interpretation of the results. While the interpretation of the sulfur distribution is generally fine, I have problems to agree with the interpretations about the phosphorus distribution. Many studies showed that inorganic phosphorus is completely discriminated in foraminiferal calcite, which is different to corals (see Science paper by Boyle, 2006). Most of the P in foraminiferal calcite is associated with the organic matrix and efficiently removed by bleaching of the tests and in my opinion the results of the study by Paris et al. show exactly this. In addition, there are certain other major points of revision that are in described in detail below. In my opinion, the paper is definitely suited for Biogeosciences, but it needs substantial major revisions before publication.
Below you can find my major points of revision:
1.: As mentioned above, the interpretation of the P distributions in the test walls are problematic and not really supported by the results of this study. Previous studies showed that P associated with foraminiferal calcite either is located in inorganic coatings (Boyle, 2006) or sits in the organic matrix of the test walls (Geerken et al., 2019; Glock et al., 2019). Nevertheless, the authors interpret their results as evidence for P that is bound in the calcite lattice. This is questionable, especially by looking at the plots in figure 4. Figure 4b shows the plot of P vs. CN. CN tracks the distribution of nitrogen in the test and is basically a proxy for organic matter in the test. The plot shows that there are two or three slightly different phosphorus bearing phases in the test but in all these phases P is strongly correlated with organic matter. While this is similar to the correlation between S and CN (Fig. 4a), the intercept with the CN axis for all three different phases is zero or close to zero for the correlation between P and CN (Fig. 4b). This is not the case for the correlation between S and CN where the intercept indicates always significant S concentrations even when nitrogen is absent. In addition, the plot in Fig. 4g indicates, that S and P certainly are sitting in different phases, when normalized to CN. To really show that there is inorganic P within the calcite, I would have recommended to bleach the shells before the analyses to remove the organic matter in the walls. As it is now, I really think the authors have to do some major revisions to their interpretation here. Also, I have a bit of doubt about the interpretation of amorphous calcium phosphate (ACP) phases in the test walls.
Then, the authors found some P-hotspots within the cells, which are partly associated with Ca and Mg. These have been interpreted as acidocalcisomes that have recently been found in foraminifera. In this context, those results could potentially be exciting. Nevertheless, there are some issues with the methodology, since classical drying for the preparation of thin sections for TEM, that the authors used, usually removes soluble content and dissolved ions from the cells (Ayache et al., 2010). This is also stated by the authors in line 174, although there is a reference missing there. That is also the reason why traditional fixation is not recommended, if acidocalcisomes are supposed to be visualized (Goodenough et al., 2019). Goodenough et al. (2019) recommend cryo-fixation to preserve acidocalcisomes and dissolved content. The only study that found evidence for acidocalcisomes in foraminifera so far also used cryo-fixation (Glock et al., 2025). In addition, the authors used a phosphate buffer solution solution for the cell fixation (line 171), which potentially could create artificial P hotspots in the foraminifera. In line 372 it is written: “More phosphorus and sulfur are observed in the organic matter located close to the test compared to the center of the cell.” Could this maybe be an indication that the P originates from the buffer but did not intrude deeper into the cell? Nevertheless, there could be a slight possibility that the P hotspots in the cell are no artifacts, since they have a similar size and shape as the recently found P-bearing structures in foraminifera. This would have been incredibly lucky since acidocalcisomes in foraminifera have not been found yet by using classical TEM-fixation methods. Therefore, I would recommend to tone down the interpretation of the P hotspots in the cell and recommend to add a discussion of the possible artifacts, related to the preparation method of the thin sections.
The next point is not that severe but should maybe be considered by the authors: I was a bit puzzled by the use of the term “Rosalina-like foraminifera”. Either the species that the authors cultured is a Rosalina or not. I can imagine, where this is coming from: Most likely a foraminiferal species that has been optically identified as a Rosalina species has been cultured and later genetic analyses revealed that it is actually not a Rosalina. In this case, I would recommend to give this species (and genus) a name, rather then to use the term “Rosalina-like foraminifera”, which is objectively not correct and misleading.
Moderate points of revision:
The introduction appears to be a bit long and detailed. It almost reads like a review. I have the feeling that the introduction can be shortened quite a bit and/or some parts could be moved to appropriate sections in the discussion. This is not completely obligatory, since Biogeosciences does not have a length limit but keep in mind that the length could deter some readers to read the text in detail.
Minor points of revision:
Line 62: “CO2“ instead of “CO2”.
Line 70: Add “a” before sulfate reduction pathway.
Line 113: See Boyle (2006) regarding phosphorus discrimination in foraminiferal calcite in contrast to incorporation of P in aragonite of corals.
Line 171: If the authors used phosphate buffer for cell fixation: Couldn´t P distribution in the cell be partly artificial? P in acidocalcisomes is usually lost during cell fixation, based on dehydration that removes free ions in the cell and cryo-fixation is the preferred method (see longer comment above).
Line 220: This transparency regarding the problem of god standard materials is really appreciated. This is often a problem. I think it is indeed very unlikely that the strong fractionation of the S incorporated into the test is caused by artifacts from machine drifts.
Line 244-245: “The laterally heterogeneous lightness or darkness of the grey in the outer calcitic layers is intriguing.” In my opinion this sentence is a bit redundant and intriguing is a bit to pictorial for a scientific paper.
Line 262: “chemically lighter” sounds a bit strange. Do the authors mean that the material is “composed out of lighter elements” or that it is “isotopically lighter”?
Line 264: “show” instead of “shows” (plural)
Line 271: Again “chemically lighter”. See above. Just “lighter” would be also enough, I guess.
Line 302-303: I would write “to the strongest P, S and Mg enrichments in relation to CN” instead of “to the strongest P, S and Mg enrichments compared to CN”.
Line 331: I don´t see that those distributions are very similar in fig. 5. Actually at the edges the distributions are very different. P gets really high at the edges and Mg not. S is in between. Also the minima and maxima in the inner parts are sometimes more or sometimes less pronounced in the different elements. I would suggest to show a correlation plot and test if the correlation is significant. This could very well be the case but it is very hard to see in fig. 5.
Line 351 and 352: Replace “foraminifera” with “foraminifer” in both cases, since it is used for singular here and not for plural.
Line 396: The cited study doesn´t really show “Ca-phosphate” but most likely polyphosphate granules and gels that are associated with Ca in addition to dissolved pyrophosphate.
Line 443: Maybe use “primarily” instead of “first”?
Line 444: Replace “foraminifera” with “foraminifer”, since it is used for singular here and not for plural.
Line 460: “but also to…”. To what? I have the feeling a word is missing in this sentence.
Section 4.1.1: There is a difference between precipitation of calcite and ion exchange between the lattice of already formed calcite in the surrounding medium. I have the feeling that both processes are a bit mixed up in this section.
Sections 4.1.1-4.1.3: I don´t really understand, why these three previously proposed biomineralization mechanisms have to be discussed and reviewed in such a detail in this section. They are partly not really related to the authors results.
Line 510: I think this sentence could be formulated a bit better.
Line 512 and following: Could the mixing of the labeled with the unlabeled P be related to mixture of unlabeled organic S with the labeled inorganic S that is incorporated into the test walls? Would the percentage fit to the plot in fig. 4a? For example in phase 1 it looks like up to 30-50% of the sulfur would sit in the organics. This would roughly fit to your 1/3 of non-labeled S.
Line 514: There is a word missing before “than”. More than? Less than?
Line 528-530: What would that mean? This section is a bit confusing.
Line 567: The use of “on the other hand” needs also “on the one hand”.
Line 581: “Locally dark local”? Please reformulate.
Line 581-591: See my long comment above about the association of P to organic matter in foraminiferal tests and the discrimination of inorganic P in the calcite lattice. I really do not understand why the authors interpret their results as an evidence for inorganic phosphorus in the test. In my opinion all their results show the opposite and former literature is completely ignored. Especially the older works of Boyle (2006) and the studies that are cited within…
Line 606: Why co-distribution? P and S are not even clearly correlated when they are normalized to CN (the organics). They are only co-correlated, if they are not normalized to CN, because both elements are at least partly associated to organic matter (P more and S to a lesser content).
Line 608: Phospho-aminoacids are not really polyphosphates. Polyphosphates are chained phosphate molecules, which could be completely inorganic or have an organic rest such in ATP.
Line 616: I cannot agree with this hypothesis and don´t see that the data are justifying this hypothesis.
Line 619: See above: The patterns are not similar, if normalized to CN. Thus, P and S are only co-correlated in the organic content.
Line 689: “plays” instead of “play” at the end of the line (singular).
Line 698: How can you design an observation?
References from that review that are not cited in the paper:
Ayache, J., Beaunier, L., Boumendil, J., Ehret, G. & Laub, D. in Sample Preparation Handbook for Transmission Electron Microscopy: Methodology (eds Ayache, J. et al.) 125–170 (Springer, 2010).
Edward A. Boyle: A Direct Proxy for Oceanic Phosphorus? Science 312, 1758-1759 (2006). DOI:10.1126/science.1129723