Overview: 

de Vries et al. present research on the distinct life cycle of Coccolithus braarudii, a species of coccolithophores, focusing on the trade-offs between its haploid and diploid phases. They examined how these phases respond to environmental factors such as light, temperature and nutrients. With the laboratory experiments they showed similarities in cell size, nitrogen requirements, uptake rates, and optimal temperature and light conditions between the phases. However, differences were noted in coccosphere size, maximum growth rates, and nitrogen quotas. Authors offered the explanation  that trade-off is observed between maximum growth rate and nitrogen quota, with the haploid phase favouring higher growth rates and lower nitrogen storage, while the diploid phase shows the opposite pattern. They also run model simulations that indicated that trade-off allows C. braarudii to more effectively utilise varying nitrogen conditions. The ability of the diploid phase to store more nitrogen proves beneficial when nitrogen is intermittently available, while the higher growth rate of the haploid phase is advantageous when nitrogen is constantly available. This study suggests that the trade-off between nitrogen storage and maximum growth rate is a critical factor determining the distribution and functionality of the C. braarudii life cycle.

The results of this study contribute to understanding of how haplo-diplontic life cycle works and I believe it is an important addition to the scientific literature. The paper introduces the concept of trade-off between maximum growth rate and nitrogen quota in the haploid and diploid phases of C. braarudii, which has not been identified previously and the models used to test the tradeoffs under different environmental conditions also provide new tools for the field.

However, I do have some major and minor comments and concerns that are provided below. In terms of the writing, it is recommended to revise both the results section and subsequent discussion. The results section currently contains significant conclusions and comparisons, going beyond the objective presentation of the findings. Therefore, it would be beneficial to adjust the content to focus on a more objective reporting of the results. Similarly, the discussion section should be modified to reflect a balanced and unbiased analysis rather than incorporating conclusions that extend beyond the presented data.

Main remarks:

Abstract

Considering the strong photoinhibition observed in figure 6b under depleted conditions, the authors' claim that HOLs prefer high light and low nutrients becomes questionable. The pronounced differences between high/low nutrient conditions and between HOL/HET phases are noteworthy enough to merit inclusion in the abstract.

Introduction

Ls 276, 277, 278: The use of the broad term "nutrient" instead of the specific term "nitrogen" throughout the paper may lead to potential confusion. This generalisation might hold true for nitrogen but not necessarily for other nutrients such as phosphorus. I recommend considering the use of "nitrogen-depleted" specifically in the results section, where other nutrients remain present, and then extrapolating to the term "nutrients" in the discussion to encompass a broader context.

L 135-136: The assumption made regarding DNA content is a bit concerning. It is reasonable only if the G2 phase is significantly longer than the G1 phase during the cell cycle and if the stationary phase primarily consists of G1 cells. It is essential to have supporting information to justify this assumption. Without such information, any calculation could potentially be true. For example, if the stationary phase primarily consists of cells in G2, the average DNA content could be even higher than that of a dividing population. Furthermore, if the G1 phase is longer than G2, the situation becomes even more complex. It is important to thoroughly discuss this issue and, if uncertainty exists, take it into account in both the assumption and subsequent models. 

L 280: Please exclude the latter part of the sentence: "Furthermore, the HOL phases show highly reduced ETR during photosynthesis, especially when exposed to high light." This statement holds true only under depleted conditions.

Figure 1

In Figure 1e, the chromosomes of the HET phase represent metaphase chromosomes, consisting of two chromatids, indicating a genome still at the haploid state (n). The 2n condition, on the other hand, would typically represent pairs of chromosomes, which can be illustrated side by side but should not be linked by their centromeres.

Considering the abundance of figures in the paper, it might be appropriate to consider moving the entire Figure 1 to supplemental material, as Figure 9 provides more comprehensive information. Additionally, certain differences observed in Figure 1, such as cell size, contradict the observations, which could potentially lead to confusion or misinterpretation.

Material and methods

L 9: Regarding cell size, was the difference between the presence and absence of a coccolith within the cell considered? Were the cell sizes estimated throughout experiments? During the exponential phase, significant coccolith calcification occurs, which could potentially impact the conclusions made. It would be valuable to investigate and account for any potential influence of coccolith presence on cell size when drawing conclusions. Additionally, please provide information on how many cells were counted per image/strain/condition?

L 118: Although I am not an expert in quotas, I find it surprising that the estimation of Qmax and Qmin is based on completely distinct methods. Please provide the specific equations used for calculating Qmax and Qmin in the given context, especially as these are a major part of modelling efforts.

L 125-129: The values of Fv/Fm and ETR are "computed" rather than directly measured, particularly ETR, which relies on parameters that are subject to certain hypotheses, such as the efficiency of light capture (cell concentration, antenna size, etc.). While it is appropriate to present the differences in ETR in the results section, the underlying cause and significance of these differences should be thoroughly discussed.

L 126: In section 2.2 on nutrient limitation, it is mentioned that the "deplete" concentration is 20 μM. Therefore, the condition indicated as 220.5 μM should be referred to as "replete." The same clarification should be made in the legend of Figure 7.

Results.

Several portions of the text extensively discuss the findings and would be better placed in the discussion section. While not explicitly mentioning all of them, it is crucial for the authors to consider that the results section should solely describe the outcomes of the data analysis. Conclusions, comparisons, and in-depth discussions should be reserved for the dedicated discussion section.

L 251-254: This paragraph belongs in the discussion section.

L 253: Cell surface (no “ ’s ”)

L257: Duplicate: cycle cycle 

L 259: The phrase "unlike temperature" is awkwardly worded. Both for temperature and light, the differences are not life cycle phase-specific. 

L 263-269 This section belongs in the discussion section.

L 270-271 This sentence belongs in the discussion section.

Figure 3: Could you please provide the information on the number of cells counted for each condition in this section here? Additionally, remove the conclusions "Both the HOL and HET …” from the figure legend and especially if mentioning significantly provide the appropriate data supporting this wording. It would be more appropriate to include a statistical test and describe that.

L 276:  Please insert “maximal”, as in: “similar maximal photosynthetic efficiency”

L 286: What is “Fig. A2” ?

L 292-294: This sentence belongs in the discussion section. In addition: considering the DNA content and its potential "cost," I find it very speculative, as evolutionary pressure in such cases would likely lead to the development of more compact genomes. However, it appears that this is not the case here, and therefore, the connection between DNA content and its cost might be somewhat far-fetched. - Discuss.

Figure 5

The summarizing Figure 5 is interesting. It would be helpful to include the conditions associated with each maxima.

Figure 6:

a) Consider renaming Fv/Fm as "Maximal yield" or a similar term, as it represents an estimation of the maximal yield, but theoretical, since it is measured in the “dark”(F0).

b) Remove "light inhibition" from the title.

Clarify whether the average values presented are based on all HET/HOL strains or just one of each. This information is important.

Provide details on the duration of cell exposure to each light level. Are different samples used at each level?

In Figure 6b, ensure consistency with the positioning of deplete and replete conditions. Currently, they are presented on the left and right sides in (a) but are opposite in (b), which could be misleading.

L 301-302, what is “Figure A1” ?

Additionally, the results of Table 1 and Figure 8 are briefly described and may benefit from a more comprehensive explanation.

Discussion

Please revise the write-up, with the intention of systematically integrating the parts that were previously discussed in the results section.

General Comments:

This manuscript considers the trait trade-offs of the haploid and diploid life phases of Coccolithophores under different environmental conditions (e.g.changes in light, nitrogen and temperature) and provides important contributions to the literature.

HOL type strains appear more sensitive to nitrogen stress (in terms of Fv/Fm and ETR), although under replete conditions HOL strains have higher max growth rates/Fv/Fm/ETR. While HOL strains have higher max growth rates, their nitrogen content per cell is lower under N replete conditions. Nitrogen deplete conditions reduce N content in both life cycle types to similar levels, but the allocation of N quota to DNA content is increased in HET strains.

Experiments using a chemostat model are used to determine if trait-tradeoffs confer competitive advantages to HOL vs HET under varying nutrient supply. The authors conclude that higher growth rates and smaller nitrogen storage seen in the haploid phase are advantageous under consistent nitrogen supply, while larger Qmax with lower growth rates is advantageous under conditions of sporadic nitrogen input. This work has applicability to informing ecosystem models with life cycle distinctions in coccolithophore populations.

Overall, this manuscript presents data comparing coccolithophore stains and life cycle types under difference environmental conditions, and shows that there are clear differences in the physiological response and growth advantages of HOL vs HET lifestyles. Furthermore, modeling these trait differences provides a unique tool for broader applicability across time and space.

However, the photosynthetic response data needs more explanation and discussion, as low Fv/Fm can imply nutrient stress but is sensitive to nutrient history and length of acclimation to new nutrient regimes. Further detail of culture methods during these experiments would be helpful. The chemostat modeling experiments also require clarification regarding the sensitivity experimental methods (not the model construction), and better explanation of the results when presenting Fig. 8.

Figures 1 and 9 are beautiful, but may contain more than the scope of the data presented in this paper. And in a similar sense, the discussion section should remain more closely linked to data presented in the results section. Some comments on other related topics are appropriate at the end of the section, but should be minor additions to the core discussion of the data presented.

Specific Comments:

Line 10/11: What is the difference between nitrogen requirement and nitrogen quota?

Line 14: what model? Hasn't been introduced yet…

Line 41/43/47: C. braarudii, already stated full name in line 37

Line 53: Match body text and figure text more closely? ie genome N content,  transport proteins (nitrogen uptake?)…

Line 60: …for changes in coccolithophore traits in response to light, nutrients….

Line 67: Section 2.1 and 2.2 are both experimental?: perhaps 2.1 is specified as strain comparison experiments and 2.2 is nutrient limitation experiments? Or combine all the culture conditions under one section here? This feels like a helpful clarification for when you present the results that switch back and forth between comparing differences in strains, life-cycle phases and nutrient status…

Line 74-77: Does the use of HOL vs HET strains isolated from mostly coastal locations influence the ability to fully understand “low nutrient/high light/stratification adapted” vs “turbulence adapted” life cycles? i.e. how do these strains compare to more open ocean strains? and is there comparison of light cycles collected from a single location?

Line 78: It may be helpful to use “nitrogen” in place to “nutrient” to clearly specify that these experiments are nitrogen limitation experiments. Perhaps the model equations below could remain generalized to any “nutrient”, but once your data is applied, I think using “nitrogen” may be more appropriate/specific.

Line 80: 20uM NO3 is usually a quite adequate amount of N in the surface ocean, where max concentrations may only reach 35uM below the nitracline, and could be considered replete in other studies. I’m guessing early exponential nitrogen growth physiology would be similar in both your deplete and replete conditions and differences would be seen in a batch culture only once nitrogen begins to be much lower.

Line 82: Triplicate bottles? Culture volumes? Vessel type? temperature control method? More detail on culture methods would be helpful.

Line 95: BMG?

Line 102/192: It would be nice to see that raw cell density data over time in the supplement/appendix… how many days does it take to get to 10-20k cells?

Line 120: Q^max is also divided by cell count? Q^min calculation uses initial media nitrogen concentration? unclear

Line 125: Citation for fluorescence. Parkhill 2001? Falkowski?

Line 136: Is there further explanation of this assumption or a citation? Assuming half the DNA content is assuming ALL replete cells are actively in the DNA replication stage…?.

Line 163: units for P and N and Q

Line 167:  is Q^N the same as Q? Specify if N in equations is for nutrients or nitrogen.

Line 169: Flynn 2002?

Line 200: What is m^inf ?

Line 213: Is E.hux spelled out earlier in manuscript?

Line 226: mortality term (m, + units)

Line 228: redefine terms that have i subscripts (?), or define i once at the start of the chemostat model section.

Line 237: “Using the chemostat model, we….”

Line 237: does this mean main difference between experimental nutrient treatments?

Line 238-242: Some of the justification and theory of this model experiment may be better in the discussion section? Then, this model experiment needs more explaination. I am having trouble interpreting Fig. 8 when I don't know how long the model was run, what an “input scenario” is or how you determine and implement “metabolic cost” differences.

Line 248: The cell size and coccosphere size were measured in replete and depleted nitrogen conditions (methods), so is this showing average across all nitrogen treatments? Or is this under culture maintenance conditions (15C + 50 mE m­^-2s^-1) or respective temp and light optima? clarify

Line 253: Would a larger coccosphere volume have a higher metabolic cost for production? It may not influence uptake rates, but would it possibly influence growth rates in addition to grazing susceptibility?

Line 259: Are average maximum growth rates observed between HET and HOL strains statistically different? Sort of looks like HOL growth rates have higher maxima within your treatment ranges for both temp and light….  (oops, addressed in next section!)

Line 262: Was there a reason for not testing higher irradiances in the lab? Eg. artificial lighting can only reach 150?

Fig.4: Not sure box plots are appropriate for n=3??

Line 275: Are the Fv/Fm tests conducted in the first transfer generation into N depleted media? I think there are difference in observable change in Fv/Fm based on transient changes in N supply vs balanced growth when cells are fully acclimated to low N supply…. (Parkhill?)

Line 282: fewer resources? Is Fv/Fm measured during exponential growth phase? I don't think changes in Fv/Fm due to nutrient stress occur until very low level of N….? Are lower growth rates in HOL relevant to the higher Fv/Fm and ETR measured in N depleted cultures?

Fig. 6 put Depleted and Replete panels on consistent side in both a and b

Line 300: What do you mean by “no apparent difference”? KQ and mmax bars look different in A1….show stats of some kind? I also still want to see the raw cell abundance data that these growth rates were estimated from…

Fig 7. replete quota is Q^max

Lines 306-310: I think these comparisons to the literature should be in the discussion section.

Table 1: Order strains in consistent order with other figures. Is this caption correct? I thought Table A1 had the model parameters used. This table is the parameter value estimates from your lab data?

Line 311: “Trait trade-offs” section feels like discussion.

Line 320: I think you are saying “competitive advantage” to mean the relative abundances of HOL vs HET cells? But it is unclear. This section needs better explanation of the results (possibly clarification in the methods section too). How much nitrogen is in a pulse? Does it go to zero between pulses? What is relative Qmax? How much nitrogen is supplied under continuous supply? Low nitrogen concentrations should also influence abundances of low vs high growth rate strains, I don't see how this is ruled out by your study.

Line 345: Turbulence is not really part of this dataset…

Line 352: What’s an “extended maximum uptake rate”?

Line 354: When what is similar?

Line 356: Explain what Fv/Fm and ETR tell you about photosynthetic ability either here or in the Fv./Fm/ETR methods section.

Figure 9. Are motility and calcification data part of this paper?

 “-a” twice in second caption sentence.

Technical Corrections:

Line 79: “…modifying the K/2 media from an initial NO₃ concentration of 220.5 mM down to 20 mM.”

Line 86: no new paragraph

Line 89: “Cell size was measured for each stain in both nutrient replete and nutrient depleted cultures”

Line 95: “…growth rates were estimated using change in cell abundance over time as estimated using…”

or change “growth rate” to “cell abundance” and leave end of paragraph as is.

Line 112: temperate-sensitivity

Line 125: Walz WATER-PAM (?) Pulse-amplitude modulated

Line 125: nutrient replete and nutrient depleted

Line 126: PAM is not a measurement “…nitrogen replete experimental cultures grown with 220 mM NO₃, Fv/Fm and ETR were measured….”

Line 127: “…For the nitrogen depleted experimental cultures,”……..”and Fv/Fm and ETR were measured once cells…”

Line 135: replete

Line 181/185/189/205/210/etc.: subscript K_N in equation and text

Line 241: -it

Line 225: phytoplankton abundance of both HET and HOL strains

Line 275: which temp and light conditions are the nutrient experiments conducted at, assuming respective temp and light optima, but unclear? Fig. 6b is showing the average of both HET and HOL strains…

Line 276: depleted, replete

Fig 6 – why are RCC1200 and RCC3779 not shown in panel a?