Does size matter? Pico-phytoplankton cell size affects biomass distribution and nutrient limitation in the oligotrophic Eastern Mediterranean Sea

Albagly, Elad; Rahav, Eyal

doi:10.5194/egusphere-2026-934

Preprints

https://doi.org/10.5194/egusphere-2026-934

Preprints

12 Mar 2026

| 12 Mar 2026

Does size matter? Pico-phytoplankton cell size affects biomass distribution and nutrient limitation in the oligotrophic Eastern Mediterranean Sea

Elad Albagly and Eyal Rahav

Abstract. The Eastern Mediterranean Sea (EMS) is one of the most oligotrophic marine environments in the world, characterized by extreme nutrient scarcity and strong water-column stratification. In such systems, pico-phytoplankton typically dominate primary production, yet how cell size structures biomass distribution and nutrient limitation remains poorly constrained. Here, we examined the spatial and vertical variability of pico-phytoplankton cell size, abundance, and nutrient status in the easternmost Mediterranean Sea during oligotrophic summer stratification. Using flow cytometry and microscopy, we quantified the cell volumes of Prochlorococcus, Synechococcus, and pico-eukaryotes and combined these with ambient nutrient concentrations to estimate cellular nutrient quotas and Resource Supply Indices (RSI). All three groups exhibited increasing cell size with depth and from offshore to coastal waters, coinciding with higher nutrient availability and chlorophyll concentrations. Synechococcus and pico-eukaryotes were consistently larger in coastal and deeper waters, whereas Prochlorococcus maintained small and relatively constant cell size across environments. RSI analysis revealed widespread nitrogen limitation for Synechococcus and pico-eukaryotes (RSIₙ<1), while phosphorus was generally sufficient (RSIₚ>1). In contrast, Prochlorococcus remained largely unconstrained by either nitrogen or phosphorus, reflecting its low cellular nutrient demand and streamlined physiology. These results demonstrate that cell size is a powerful integrator of environmental forcing and ecological strategy in oligotrophic seas. The dominance of small Prochlorococcus cells under extreme nutrient scarcity imply how stable ocean stratification and nutrient decline may reshape microbial communities and biogeochemical cycling in the future oligotrophic oceans.

Received: 17 Feb 2026 – Discussion started: 12 Mar 2026

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Elad Albagly and Eyal Rahav

Status: closed

RC1:
'Comment on egusphere-2026-934', Anonymous Referee #1, 02 May 2026
This manuscript investigates the spatial and vertical variability of pico-phytoplankton cell size, abundance, and nutrient status in the oligotrophic Eastern Mediterranean Sea (EMS). Using flow cytometry combined with microscopy, the authors quantify cell volumes of Prochlorococcus, Synechococcus, and pico-eukaryotes, and apply a Resource Supply Index (RSI) framework to assess theoretical nutrient sufficiency for each group. The dataset is solid, the sampling design covers a meaningful coastal–offshore gradient, and the integration of cell-size measurements with stoichiometric demand calculations is a valuable contribution to phytoplankton ecology in this understudied zone.
That said, several aspects require substantial revision. My main concerns are: (i) the causal framing in the title and abstract overstates what the data can demonstrate; (ii) the methodological description of cell-size determination has gaps that affect confidence in the results; (iii) the link between cell size and nutrient limitation is conceptually muddled, since the observed differences appear to reflect taxonomic/genetic traits more than size per se; and (iv) several figures, calculations, and references contain inconsistencies or potential errors. In addition, the overall writing quality is uneven, with grammatical issues, awkward phrasing, and inconsistent terminology throughout, and the manuscript would benefit from careful language editing. I therefore recommend major revision, with the specific points detailed below.
Major Comments
Causal claim in the title is not supported by the evidence. The title claims that cell size "affects biomass distribution." If "biomass distribution" refers to the chlorophyll a distribution shown in Figure 2D, the causal logic remains problematic: the manuscript demonstrates that chlorophyll, cell size, and nutrients all co-vary along the same coastal–offshore and vertical gradients, but such co-variation does not establish that cell size affects biomass. Could the authors clarify the basis for the causal claim in the title, or revise the wording to a more correlative framing?

Methodological gaps in cell-size determination. The authors state that "a calibration curve relating light scatter to particle size" was constructed (lines 145–146, Figure S1A), but Figure S1A actually shows only an FSC–SSC bead distribution plot, with no calibration curve linking either signal to particle size; the actual size-conversion procedure is therefore not shown. In addition, several critical details are missing for the cross-validation in Figure S1B: were the flow cytometry size readings group-specific (i.e., separated for Synechococcus, Prochlorococcus, and pico-eukaryotes)? Were the corresponding microscopy measurements also group-resolved? Was microscopy performed under brightfield or epifluorescence? Resolving pico-sized cells (<2–3 µm) typically requires 1000× magnification rather than the 400× reported here, which raises concerns about measurement reliability. I recommend that the authors include a representative microscopy image of an in situ sample in the Supplementary Information to allow readers to evaluate the quality of the size measurements.

The link between cell size and nutrient limitation is not logically established. The title claims that cell size affects nutrient limitation, but the results actually demonstrate differences in N-limitation status among the three pico-phytoplankton groups: Prochlorococcus is not N-limited, whereas Synechococcus and pico-eukaryotes are. This pattern reflects taxonomic differences in nutrient limitation, not a cell-size effect per se. Indeed, the authors themselves attribute Prochlorococcus's lack of N-limitation to its inability to assimilate nitrate, a genetic/physiological trait rather than a consequence of small cell size. How, then, is the causal chain "cell size → nutrient limitation" justified? Could the authors clarify the logical basis for this claim, or revise the framing accordingly?

Minor Comments
Abstract, L15: "structures" overstates a causal role that is not fully supported by the data. Consider softening the wording.

Keywords: "Cell-size" should be "Cell size".

Graphical abstract: (1) Pico-eukaryotes should not be depicted as a single uniform morphotype, as this group encompasses multiple taxonomically diverse phyla rather than a single genus. (2) The intended meaning of "Ambient nutrients" as the x-axis is not entirely clear to readers; please clarify in the legend or revise the axis.

Lines 43–49: The first sentence introduces pico-phytoplankton as the subject, but the following sentence uses "they" in a way that appears to refer to pico-phytoplankton, whereas the cited 50% global primary production estimate actually refers to phytoplankton as a whole, not pico-phytoplankton specifically. Please clarify the subject and revise to avoid this misattribution.

Lines 50–53: The introduction of pico-eukaryotes and prokaryotes in this passage feels somewhat abrupt and may confuse readers. Please rephrase to improve the logical flow.

Lines 89–96: A short methodological summary at the end of the Introduction would improve logical flow and help orient the reader before the Results.

Line 95: The phrase "suggesting other controls of this cyanobacterial genus in the EMS" is speculative and unsupported at this point in the Introduction. Consider removing it or rephrasing more neutrally, and reserve mechanistic interpretation for the Discussion.

Lines 104–105: Photic-layer depth varies among stations; please clarify how "180 m" was defined and whether it applies to all stations.

Line 108: "analyzed" would be more accurately replaced with "collected".

Line 125: "ml" should be written as "mL" to follow standard SI unit conventions; please apply this correction consistently throughout the manuscript.

Line 131: "Bacterial" appears inappropriate in this context, since the samples are used to enumerate pico-phytoplankton.

Line 132: The final glutaraldehyde concentration is reported as "0.2%", but adding 6 µL of 50% glutaraldehyde to 1.8 mL of sample yields ~0.167%. Please correct to "0.167%" or add "~" before "0.2%".

Lines 138–140: Two issues with this sentence: (1) the logic is somewhat confused, as thawing in the 37 °C water bath occurs prior to analysis, but determining pico-phytoplankton abundance is the analysis itself, not a step prior to it; (2) the sentence states only that abundance was determined, but FSC and SSC signals were also recorded.

Line 166: "Cell diameters" would be more accurately described as "equivalent spherical diameter (ESD)".

Calibration range in Figure S1B: The regression between flow-cytometry- and microscopy-derived cell volumes appears to span only ~0.8–3.6 µm³, which corresponds to the Synechococcus and pico-eukaryote size range, and excludes Prochlorococcus (reported here at ~0.51–0.74 µm³). The calibration therefore does not validate FCM-derived size estimates across the full size range of the dataset. The authors should explicitly acknowledge this limitation.

Equation numbering: Please move equation numbers from the left to the right side, following standard convention.

Line 204: The abbreviation "Low-Nutrient Low-Chlorophyll (LNLC)" is introduced for the first time in the Results section heading, but it is not mentioned in the Introduction.

Lines 209–211: The authors report that NO₂+NO₃ and PO₄ concentrations were frequently below detection, but the manuscript does not specify how these values were handled in subsequent analyses (e.g., RSI calculations). Please clarify.

Line 218: An N:P ratio >16:1 conventionally indicates P limitation, not N limitation as stated. Please reconsider this inference and the connector "In agreement" that follows.

Lines 249–251: This sentence is grammatically awkward ("larger than of Synechococcus and Prochlorococcus"), and the use of "respectively" with three values mapped to two named comparators is confusing.

Line 250: The authors report a mean Prochlorococcus cell volume of ~0.6 µm³ and state that this is comparable to values from the NPSG, citing Winter et al. (2025). However, Winter et al. (2025) actually report a Prochlorococcus equivalent spherical diameter (ESD) of 0.6 µm at Station ALOHA, i.e., a linear dimension, not a volume. A 0.6 µm ESD corresponds to a cell volume of only ~0.11 µm³. Please verify and correct accordingly.

Line 255: Two points: (1) the manuscript does not measure non-pico-sized phytoplankton, so dominance cannot be established from the present data and should perhaps not be presented as a finding of this study; (2) the term "water masses" may be inappropriate here, since no formal water-mass analysis (e.g., T–S diagrams) was performed.

Line 318: Attributing larger coastal cell sizes directly to "human-induced perturbations" skips a key step in the causal chain. Please clarify the intermediate mechanism.

Lines 339–347: The EMS is described as one of the most P-depleted marine systems globally, yet none of the three groups shows P limitation in the RSI analysis. The current explanation is somewhat brief.

Line 365: The mention of grazer visibility appears abruptly in the Discussion without prior framing.

Lines 458–462: The authors recommend applying size-adjusted, depth-resolved carbon conversion factors, but do not apply these values in the present study. Could the authors clarify why these factors were not used here to derive the biomass estimates?

An interesting point that warrants further discussion: Although the RSI analysis indicates that Prochlorococcus is not N-limited whereas Synechococcus and pico-eukaryotes are, Figure 3 shows that in the upper 100 m Prochlorococcus abundance is actually lower than that of Synechococcus. This is a counterintuitive and ecologically interesting pattern, in which the supposedly nutrient-limited group outnumbers the unconstrained one. The authors may wish to expand the discussion to address this apparent paradox.

Figure 1: (1) The y-axis label appears partially obscured. (2) Please verify that "bottom depth" is the appropriate terminology for the colour bar. (3) The caption states 15 sampling stations, but only 14 points appear visible on the map; please reconcile this discrepancy.

Figure 2: Labeling Panel B as "dissolved NO₂+NO₃" while Panel C is simply "PO₄" is inconsistent, given that both nutrients underwent identical 0.45 µm filtration. Please harmonize the labels.

Figure 3: "Picoeukaryotes" in the figure is inconsistent with "pico-eukaryotes" used in the main text; please standardise.

Figure 3C: In typical oligotrophic systems, Prochlorococcus surface abundances are generally close to the profile maximum (e.g., Vaulot & Marie, 1999, J. Geophys. Res. Oceans, 104(C2), 3297–3310). However, Figure 3C shows surface Prochlorococcus abundances substantially lower than at the DCM. One plausible explanation is that surface Prochlorococcus cells exhibit weak red fluorescence due to photoacclimation, making them difficult to distinguish from instrument noise in flow cytometry. Alternatively, this may genuinely reflect the local hydrography. The authors should clarify which interpretation applies and, ideally, include representative FCM cytograms (particularly from the surface layer) in the Supplementary Information to allow readers to evaluate the gating and noise discrimination.

Figure 4: (1) The Methods do not specify how "coastal" and "offshore" stations were defined; please add this criterion. (2) The x-axis label "Phytoplankton group" could be changed to "Pico-phytoplankton groups" or removed.

Figure 5: A key conclusion of the manuscript is that, in contrast to Synechococcus and pico-eukaryotes, Prochlorococcus is not N-limited (lines 288–294). However, as currently presented, Figure 5C appears essentially identical to Figure 5A, and would instead suggest that Prochlorococcus is also N-limited. Could the authors check whether this is a figure placement error, or clarify the intended interpretation?

Figure 5 (labeling): As in Figure 3, please consider labeling the three pico-phytoplankton groups (Synechococcus, Prochlorococcus, pico-eukaryotes) on the right side of the panels to improve readability.

ODV figures (Figures 2, 3, 5): The decimal formatting of axis tick labels is inconsistent (e.g., "33.5", "34", "34.5"). Please standardise to a uniform number of decimal places (e.g., "33.0", "33.5", "34.0", "34.5") across all ODV-generated panels.

Inconsistent terminology: The manuscript uses inconsistent forms (e.g., "chlorophyll-a", "Chlorophyll.a"). Please standardise to a single format throughout.

Typographic consistency: Throughout the manuscript (e.g., lines 42, 116, 206–208), hyphens (-) are used where en-dashes or minus signs would be more appropriate. Please correct accordingly.

Manuscript formatting: Paragraphs in the main text and reference list lack first-line indentation or clear inter-paragraph spacing, which somewhat impairs readability. Please consider adjusting the formatting.

Reference list formatting: Several inconsistencies are present, including but not limited to: line 502, "Deep. Res. Part I" should be "Deep-Sea Res. Part I"; lines 489 and 494, "CO2" should be "CO₂" with subscript; lines 497–499, journal name appears to be missing. Please carefully proofread the entire reference list and correct such errors.
Citation: https://doi.org/10.5194/egusphere-2026-934-RC1
- AC1: 'Reply on RC1', Eyal Rahav, 23 Jun 2026
  
  Dear Reviewer,
  Many thanks for the time and effort you invested in reviewing our manuscript. Your suggestions and comments have significantly improved the manuscript, both scientifically and in terms of clarity and readability.
  Please find our detailed response in the attached file.
  
  Citation: https://doi.org/10.5194/egusphere-2026-934-AC1
RC2:
'Comment on egusphere-2026-934', Anonymous Referee #2, 12 Jun 2026

General comments
This study focuses on variability along an environmental gradient from coastal to offshore areas and with depth, examining the main photoautotrophic picoplankton groups (Synechococcus, Prochlorococcus and picoeukaryotes) in an oligotrophic sea.
Although cell size variability in picoplankton is a relevant topic in microbial ecology, there appears to be a mismatch between the Introduction, Discussion and Conclusion, making the key message of the study unclear. It is not evident whether the main objective is to assess (i) variability in cell sizes across environmental gradients in oligotrophic conditions or (ii) the use of different conversions factors depending on depth for biomass estimation.
In addition, the Introduction should be more clearly emphasize the role of small cell size and its implications for overall community performance, rather than focusing mainly on comparisons between small and large cells. The Material and Methods section should also be reorganized to clearly describe the steps used to determine biovolume, as this represents a central component of the study. Finally, the Discussion should be revised to reduce speculative statements and better align with the presented results and main objectives of the work.

Specific comments.
# Title
Lines 1 and 2: The term pico-phytoplankton may be misleading if the study includes, as not all cyanobacteria are typically considered phytoplankton in the strict taxonomic sense. I recommend replacing “pico-phytoplankton” with “photoautotrophic picoplankton” to more accurately encompass both eukaryotic and cyanobacteria.

# Abstract
Line 31: In this sentence, it is unclear whether the authors use the word “stable” to refer to both ocean stratification and nutrient decline, or only to ocean stratification. If it refers to both, the sentence is difficult to understand because “stable” and “decline” covey contrasting meanings. In that case, I suggest replacing “stable” with “persistence”
Graphical abstract: The meaning of the X and ✓ symbols is not clear…DO they indicate the presence/absence of cells, an increase/decrease in cell size, or another response variable? Please clarify in the figure

# Introduction
Line 39: What do the author mean by “community structure”? Community structure is generally understood as a combination of multiple phytoplankton aspects (cell size, taxonomy, distribution of taxa abundance, functional traits…and other characteristics).
Please rewrite the sentence to clarify which components(s) of community structure are being considered in this study.
Line 41: This sentence is vague and should be revised to provide a clearer and stronger statement. Please rephrase to improve precision and clarity.
Line 42: The terms “low-productivity” and “oligotrophic” are largely redundant, as oligotrophic systems are, by definition, characterized by low productivity. Please consider using only on of these terms to avoid redundancy
Line 42: The same as lines 1 and 2
Lines 42:43: Please delete the phrase ….”dominate the autotrophic community” and the sentence “Pico-phytoplankton are small-sized phytoplankton”…., as this information is redundant and already mentioned in the previous sentence.
Line 47: Why do the authors place “biological pump” in quotation marks? Please clarify whether this is intended and revise if necessary for consistency and clarity. The same in line 60.
Line 48: Please add a more recent reference to support this statement.
Line 54: Replace “Pico-phytoplankton” with “photoautotrophic picoplankton”
Line 59: What do the author mean by “trophic pathways”. Please clarify the term.
Line 63: What do the author mean by “phytoplankton cellular composition”. Please clarify the term.
Line 76: This is the first time Eastern Mediterranean Sea (EMS) is mentioned in the main text. Please define the abbreviations at first use.
Lines 94-95: This is the first time nitrogen (N) and phosphorous (P) are mentioned in the main text. Please define the abbreviations at first use.

# Materials and Methods
Line 104: Replace …”seawater was sampled”… with …”seawater was collected”…
Line 107 -109: Please remove this sentence., as the information is repeated in the following sentences.
Figure 1: Replace “Bottom depth” with just “Depth” for simplicity

Line 125: This sentence is hard to follow. I suggest revising the beginning as follows:
“For determination of chlorophyll-a (chl-a), seawater samples (300 mL) were filtered through …”
Line 126: Please delete for chlorophyll-a (chl-a) analysis.
End of chlorophyll section: The authors do not clearly describe how Chla concentrations were determined. Please include the reference followed (e.g., Ritchie or another standard protocol).
Line 130: Replace the title by “ Photoautotrophic picoplankton analysis”
Line 131: Please replace “Bacterial abudance samples” with a clearer methodological description, e.g.: “Water samples (1.8 mL) were fixed in …” followed by the fixation details. Please also specify the type of containers used e.g., cryotubes/cryovials) to improve clarity and reproducibility.
Line 133:Replace “put” with “stored”.
Line 133: Specify the type of analysis: “Flow cytometry analysis….”
Line 135: Remove “…syringe-based fluidic system and…”. It is not necessary to mention.
Lines 136-138: Caution: In routine flow cytometry analysis, taxonomic groups are not specifically identified, only broad groups, populations or assemblages are resolved.
Please replace “Taxonomic discrimination was…” and the following sentences with: “Photoautotrophic picoplankton groups were identified based on their autofluorescence. Cells were excited at (add lasers of the flow cytometer) and three populations were distinguished according to light forward scatter (FSC, proxy of cell size), light side scatter (SSC, a poxy of cell granularity or complexity), and red and orange fluorescence bands (proxy for chlorophyll and phycoerythrin respectively) (ref).
After this part, the authors should clearly specify which three groups were distinguishable according to the differences in their chlorophyll and phycoerythrin fluorescence signals, FSC and SSC (ref. ref)”.
Lines 138-140: Authors should specify with more details about the conditions of the flow cytometer while collect the event (i.e. flow rate, how many events were recorded, if dilution was needed, and finally how the authors determined the abundance of the assemblages.
Lines 141-154: This is the core of the M&M section and the main goal of the paper. The authors need to be precise in this part. For example…it is confused what is “the cell size distribution” for the authors…It should be simply as “Cell size of the three photoautotrophic picoplankton groups”. Also…since they already mention FSC and SSC signals…not need to mention flow cytometry nether side scatter.
Lines 142-145: It is confused how the instrument was calibrated with the mixture of beads and then use the same beads to build the calibration curve. The mixture of beads was running each day of the samples analysis? How many times was collected this mixture?
Line 145: When the authors mention “flow cytometer settings”, these should be specified earlier in the Methods section (see lines 138-140). Please ensure that all relevant instrument settings are clearly described at first mention to improve clarity and reproducibility.
In this section, where the authors describe how cell sizes of the autotrophic picoplankton groups were determined, the method for calculating biovolume (BV, um3) is missing. Please include a clear description of how BV was estimated for each assemblage. It is the core of the paper.
Caution: SSC is not a proxy of volume…It is proxy for cell granularity and complexity. You need extra calculations to determine the biovolume (BV, um3) of the cells.
Line 147: Include “water” between random and samples.
Lines 147-154: This part of the microscopy analysis is difficult to follow. Please rewrite this section to improve clarity, structure and readability, ensuring that the methodological steps are clearly and logically presented.
Line 155: Please remove the abbreviations from the title and keep the terms in full, to improve clarity.
Line 158: What do the authors mean by “these matrixes? Please clarify which specific matrices are being referred, as the term is unclear in this context.
Lines 159-160: These sentences need to be rephrased and starting with: “The minimum cellular content of a nutrient (Q_min) is defined as the minimum amount required to sustain basic cellular functions and balanced growth under ambient nutrient conditions. In contrast, the maxima cellular content of a nutrient (Q_max) is defined as….
Line 161: Delete “support”
Line 166: This information should be moved to Section 2.4, after indicating that cell size (um) was determined from the calibration curve. Please also clarify that these measurements were subsequently used to estimate biovolume (BV) in the section Cellular nutrient quotas (Qmin and Qmax) and resource supply index (RSI).
Line 166: Delete this sentence from this part.
Line 167: The authors should start the sentence with Biovolume (BV, um3) was calculated assuming spherical cell as shown in Eq.1:
where d is the cell diameter (um) determined from FSC values.
CAUTION: In seawater samples, side scatter (SSC) values are commonly used to determine biovolume estimations, whereas forward scatter (FSC) is less reliable due to variability in light scattering associated with differences in refractive index in natural seawater. Did the authors try to calculate the biovolume with SSC and compare with FSC?
Lines 179-180: What the authors mean with…” if all cells reached Qmax”. What if the cells do not reach Qmax?

Line 182: Remove “Where, “
Lines 187-188: The authors should rephrase this sentence to improve clarity and readability.
Line 189: What do the authors mean by “ standardized environmental matrix”? Please clarify whether this refers to scaled/normalization data (e.g., z-score standardization, range scaling from 0 to 1, or another information). This needs to be explicitly defined to avoid ambiguity.

# Results
Line 204: Please remove the abbreviation form the title
Line 206: How the authors determined sigma-theta (density)? Please clarify.
Line 218: If N:P ratio was often higher than the 16:1 ‘Redfield ratio’, this suggests phosphorous limitation rather than nitrogen limitation. Please rephase this sentence.
Line 221: Remove “Where” at the beginning please.
Line 222: The authors should use the term Deep Chlorophyll Maxima (DCM) with caution and provide a clear justification for its use, given that the reported chlorophyll-a concentration is very low ( ~0.3 μg L-1). Please clarify whether these values truly correspond to a DCM or rather to a weak subsurface chlorophyll maximum.
Line 225: Why do the authors use the term “Low-Nutrient Low-Chlorophyll” in the title and here, if it is not used elsewhere in the text? Please clarify.
Lines 230-231: Please remove this sentence, as it repeats information already said.
Lines 232-233: I suggest expressing the numbers of picoplankton abundance in scientific notation (e.g, 2x 10⁴ ,3 x 10⁶) to improve readability.

Figure 4: What do the authors mean by “single-cell volumes”? Please clarify how do these volumes were estimated: were they calculated individually for each cell of each group within a sample and then averaged per sample, or were they derived from mean FSC values per group per sample? This methodological aspect should be clearly described in the Materials and Methods section to ensure reproducibility. If volume were determined form the FCS values average, single-cell should be removed from the figure caption.
Line 276: This is a title for the discussion part.
Lines 277-283: This section, as currently written, is not appropriate for the Results section, as it primarily contains interpretation rather than the presentation of results. The content would be more suitable for the Discussion section. Please consider revised and rewrite it accordingly.
The Figure 7 is not mentioned in the Results section. Please consider describing the main findings associated with this figure in the text. In addition, I recommend using different colors and/or symbols to represent sampling sites according to their distance from the coast and depth, different color arrows and vectors for environmental variables and for the sizes of the three photoautotrophic picoplankton groups respectively. All together would improve the readability and interpretation of the figure.
# Discussion
Lies 314-315: This sentence is part of the Results section.
Lines 317-321. This sentence appears to be based on speculation rather than on observations or evidence from the study site. Please revise the statement to more closely reflect the results obtained in this study or provide supporting evidence for the interpretation.
Line 328: What do the authors mean with “size structuring”? Please clarify.
Line 333: what do the authors mean with “adaptive trait”? Please clarify
Line 333: Please replace “chronic” with “persistence”.
Line 346: What do the authors mean with “to begin with”? Please consider rephrase the sentence.
Lines 334 and 348. The authors discuss differences reported in the literature between small and large Synechococcus. However, it is unclear how this distinction applies to the present dataset. Were both small and large Synechococcus populations identified in this study? If so, please specify in which samples or regions they were observed and how they were distinguished? Otherwise, the relevance of this discussion to the current results should be clarified.
Lines 377-380: These sentences are part of the results section.
Line 385: Please replace “machinery” with “capacity”.
Line 391: The use of the term “evolutionary optimization” appears inappropriate and speculative in the context of this study. Please clarify why the authors infer that this process is occurring in their dataset and provide supporting evidence. If no direct evidence is available, the statement should be rephrased to avoid overinterpretation of the results.
Line 394: What do the authors mean by “intermediate response”? Please clarify this term, as its meaning is not clear in the current context.
Line 398: What do the authors mean by “pico-eukaryotes respond dynamically”? Please clarify this term, as its meaning is not clear in the current context.
Lines 399-400.: What do the authors want to say in this sentence? Please consider rephrase the sentence.
Lines 410-446 The final part of the Discussions speculative and does not clearly convey the main message of the paper. IN addition, it is not well connected to the biomass calculations mentioned in the conclusions. Please revise and restructure this section to better integrate the interpretation with the key results, particularly the biomass estimates, and ensure that the main takeaway of the study is clearly articulated.
# Conclusions:
The authors introduce a new aspect related to biomass estimation and the of different conversion factors. However, it is unclear why the authors did not test biomass calculations sung both a single conversion factor and depth-specific conversions factors, to evaluate potential differences. Please clarify this methodological choice and its implications for the results.

General typos:
Please replace “ml” with “mL” throughout the manuscript.
Please remove “Where,..“ from the beginning of these sentences. This structure is not used in proper English syntax.
Please remove the unnecessary quotation marks throughout the manuscript. For example, in line 384 ( “allows”) and line 390 (“tendency”9, the quotation marks do not appear to serve a specific purpose. Please review the manuscript and remove similar unnecessary quotation marks for consistency and readability.

Citation: https://doi.org/10.5194/egusphere-2026-934-RC2
- AC2: 'Reply on RC2', Eyal Rahav, 23 Jun 2026
  
  Dear Reviewer,
  Many thanks for the time and effort you invested in reviewing our manuscript. Your suggestions and comments have significantly improved the revised maunscipt, both scientifically and in terms of clarity and readability.
  Please find our detailed response in the attached file.
  
  Citation: https://doi.org/10.5194/egusphere-2026-934-AC2

Status: closed

RC1:
'Comment on egusphere-2026-934', Anonymous Referee #1, 02 May 2026
This manuscript investigates the spatial and vertical variability of pico-phytoplankton cell size, abundance, and nutrient status in the oligotrophic Eastern Mediterranean Sea (EMS). Using flow cytometry combined with microscopy, the authors quantify cell volumes of Prochlorococcus, Synechococcus, and pico-eukaryotes, and apply a Resource Supply Index (RSI) framework to assess theoretical nutrient sufficiency for each group. The dataset is solid, the sampling design covers a meaningful coastal–offshore gradient, and the integration of cell-size measurements with stoichiometric demand calculations is a valuable contribution to phytoplankton ecology in this understudied zone.
That said, several aspects require substantial revision. My main concerns are: (i) the causal framing in the title and abstract overstates what the data can demonstrate; (ii) the methodological description of cell-size determination has gaps that affect confidence in the results; (iii) the link between cell size and nutrient limitation is conceptually muddled, since the observed differences appear to reflect taxonomic/genetic traits more than size per se; and (iv) several figures, calculations, and references contain inconsistencies or potential errors. In addition, the overall writing quality is uneven, with grammatical issues, awkward phrasing, and inconsistent terminology throughout, and the manuscript would benefit from careful language editing. I therefore recommend major revision, with the specific points detailed below.
Major Comments
Causal claim in the title is not supported by the evidence. The title claims that cell size "affects biomass distribution." If "biomass distribution" refers to the chlorophyll a distribution shown in Figure 2D, the causal logic remains problematic: the manuscript demonstrates that chlorophyll, cell size, and nutrients all co-vary along the same coastal–offshore and vertical gradients, but such co-variation does not establish that cell size affects biomass. Could the authors clarify the basis for the causal claim in the title, or revise the wording to a more correlative framing?

Methodological gaps in cell-size determination. The authors state that "a calibration curve relating light scatter to particle size" was constructed (lines 145–146, Figure S1A), but Figure S1A actually shows only an FSC–SSC bead distribution plot, with no calibration curve linking either signal to particle size; the actual size-conversion procedure is therefore not shown. In addition, several critical details are missing for the cross-validation in Figure S1B: were the flow cytometry size readings group-specific (i.e., separated for Synechococcus, Prochlorococcus, and pico-eukaryotes)? Were the corresponding microscopy measurements also group-resolved? Was microscopy performed under brightfield or epifluorescence? Resolving pico-sized cells (<2–3 µm) typically requires 1000× magnification rather than the 400× reported here, which raises concerns about measurement reliability. I recommend that the authors include a representative microscopy image of an in situ sample in the Supplementary Information to allow readers to evaluate the quality of the size measurements.

The link between cell size and nutrient limitation is not logically established. The title claims that cell size affects nutrient limitation, but the results actually demonstrate differences in N-limitation status among the three pico-phytoplankton groups: Prochlorococcus is not N-limited, whereas Synechococcus and pico-eukaryotes are. This pattern reflects taxonomic differences in nutrient limitation, not a cell-size effect per se. Indeed, the authors themselves attribute Prochlorococcus's lack of N-limitation to its inability to assimilate nitrate, a genetic/physiological trait rather than a consequence of small cell size. How, then, is the causal chain "cell size → nutrient limitation" justified? Could the authors clarify the logical basis for this claim, or revise the framing accordingly?

Minor Comments
Abstract, L15: "structures" overstates a causal role that is not fully supported by the data. Consider softening the wording.

Keywords: "Cell-size" should be "Cell size".

Graphical abstract: (1) Pico-eukaryotes should not be depicted as a single uniform morphotype, as this group encompasses multiple taxonomically diverse phyla rather than a single genus. (2) The intended meaning of "Ambient nutrients" as the x-axis is not entirely clear to readers; please clarify in the legend or revise the axis.

Lines 43–49: The first sentence introduces pico-phytoplankton as the subject, but the following sentence uses "they" in a way that appears to refer to pico-phytoplankton, whereas the cited 50% global primary production estimate actually refers to phytoplankton as a whole, not pico-phytoplankton specifically. Please clarify the subject and revise to avoid this misattribution.

Lines 50–53: The introduction of pico-eukaryotes and prokaryotes in this passage feels somewhat abrupt and may confuse readers. Please rephrase to improve the logical flow.

Lines 89–96: A short methodological summary at the end of the Introduction would improve logical flow and help orient the reader before the Results.

Line 95: The phrase "suggesting other controls of this cyanobacterial genus in the EMS" is speculative and unsupported at this point in the Introduction. Consider removing it or rephrasing more neutrally, and reserve mechanistic interpretation for the Discussion.

Lines 104–105: Photic-layer depth varies among stations; please clarify how "180 m" was defined and whether it applies to all stations.

Line 108: "analyzed" would be more accurately replaced with "collected".

Line 125: "ml" should be written as "mL" to follow standard SI unit conventions; please apply this correction consistently throughout the manuscript.

Line 131: "Bacterial" appears inappropriate in this context, since the samples are used to enumerate pico-phytoplankton.

Line 132: The final glutaraldehyde concentration is reported as "0.2%", but adding 6 µL of 50% glutaraldehyde to 1.8 mL of sample yields ~0.167%. Please correct to "0.167%" or add "~" before "0.2%".

Lines 138–140: Two issues with this sentence: (1) the logic is somewhat confused, as thawing in the 37 °C water bath occurs prior to analysis, but determining pico-phytoplankton abundance is the analysis itself, not a step prior to it; (2) the sentence states only that abundance was determined, but FSC and SSC signals were also recorded.

Line 166: "Cell diameters" would be more accurately described as "equivalent spherical diameter (ESD)".

Calibration range in Figure S1B: The regression between flow-cytometry- and microscopy-derived cell volumes appears to span only ~0.8–3.6 µm³, which corresponds to the Synechococcus and pico-eukaryote size range, and excludes Prochlorococcus (reported here at ~0.51–0.74 µm³). The calibration therefore does not validate FCM-derived size estimates across the full size range of the dataset. The authors should explicitly acknowledge this limitation.

Equation numbering: Please move equation numbers from the left to the right side, following standard convention.

Line 204: The abbreviation "Low-Nutrient Low-Chlorophyll (LNLC)" is introduced for the first time in the Results section heading, but it is not mentioned in the Introduction.

Lines 209–211: The authors report that NO₂+NO₃ and PO₄ concentrations were frequently below detection, but the manuscript does not specify how these values were handled in subsequent analyses (e.g., RSI calculations). Please clarify.

Line 218: An N:P ratio >16:1 conventionally indicates P limitation, not N limitation as stated. Please reconsider this inference and the connector "In agreement" that follows.

Lines 249–251: This sentence is grammatically awkward ("larger than of Synechococcus and Prochlorococcus"), and the use of "respectively" with three values mapped to two named comparators is confusing.

Line 250: The authors report a mean Prochlorococcus cell volume of ~0.6 µm³ and state that this is comparable to values from the NPSG, citing Winter et al. (2025). However, Winter et al. (2025) actually report a Prochlorococcus equivalent spherical diameter (ESD) of 0.6 µm at Station ALOHA, i.e., a linear dimension, not a volume. A 0.6 µm ESD corresponds to a cell volume of only ~0.11 µm³. Please verify and correct accordingly.

Line 255: Two points: (1) the manuscript does not measure non-pico-sized phytoplankton, so dominance cannot be established from the present data and should perhaps not be presented as a finding of this study; (2) the term "water masses" may be inappropriate here, since no formal water-mass analysis (e.g., T–S diagrams) was performed.

Line 318: Attributing larger coastal cell sizes directly to "human-induced perturbations" skips a key step in the causal chain. Please clarify the intermediate mechanism.

Lines 339–347: The EMS is described as one of the most P-depleted marine systems globally, yet none of the three groups shows P limitation in the RSI analysis. The current explanation is somewhat brief.

Line 365: The mention of grazer visibility appears abruptly in the Discussion without prior framing.

Lines 458–462: The authors recommend applying size-adjusted, depth-resolved carbon conversion factors, but do not apply these values in the present study. Could the authors clarify why these factors were not used here to derive the biomass estimates?

An interesting point that warrants further discussion: Although the RSI analysis indicates that Prochlorococcus is not N-limited whereas Synechococcus and pico-eukaryotes are, Figure 3 shows that in the upper 100 m Prochlorococcus abundance is actually lower than that of Synechococcus. This is a counterintuitive and ecologically interesting pattern, in which the supposedly nutrient-limited group outnumbers the unconstrained one. The authors may wish to expand the discussion to address this apparent paradox.

Figure 1: (1) The y-axis label appears partially obscured. (2) Please verify that "bottom depth" is the appropriate terminology for the colour bar. (3) The caption states 15 sampling stations, but only 14 points appear visible on the map; please reconcile this discrepancy.

Figure 2: Labeling Panel B as "dissolved NO₂+NO₃" while Panel C is simply "PO₄" is inconsistent, given that both nutrients underwent identical 0.45 µm filtration. Please harmonize the labels.

Figure 3: "Picoeukaryotes" in the figure is inconsistent with "pico-eukaryotes" used in the main text; please standardise.

Figure 3C: In typical oligotrophic systems, Prochlorococcus surface abundances are generally close to the profile maximum (e.g., Vaulot & Marie, 1999, J. Geophys. Res. Oceans, 104(C2), 3297–3310). However, Figure 3C shows surface Prochlorococcus abundances substantially lower than at the DCM. One plausible explanation is that surface Prochlorococcus cells exhibit weak red fluorescence due to photoacclimation, making them difficult to distinguish from instrument noise in flow cytometry. Alternatively, this may genuinely reflect the local hydrography. The authors should clarify which interpretation applies and, ideally, include representative FCM cytograms (particularly from the surface layer) in the Supplementary Information to allow readers to evaluate the gating and noise discrimination.

Figure 4: (1) The Methods do not specify how "coastal" and "offshore" stations were defined; please add this criterion. (2) The x-axis label "Phytoplankton group" could be changed to "Pico-phytoplankton groups" or removed.

Figure 5: A key conclusion of the manuscript is that, in contrast to Synechococcus and pico-eukaryotes, Prochlorococcus is not N-limited (lines 288–294). However, as currently presented, Figure 5C appears essentially identical to Figure 5A, and would instead suggest that Prochlorococcus is also N-limited. Could the authors check whether this is a figure placement error, or clarify the intended interpretation?

Figure 5 (labeling): As in Figure 3, please consider labeling the three pico-phytoplankton groups (Synechococcus, Prochlorococcus, pico-eukaryotes) on the right side of the panels to improve readability.

ODV figures (Figures 2, 3, 5): The decimal formatting of axis tick labels is inconsistent (e.g., "33.5", "34", "34.5"). Please standardise to a uniform number of decimal places (e.g., "33.0", "33.5", "34.0", "34.5") across all ODV-generated panels.

Inconsistent terminology: The manuscript uses inconsistent forms (e.g., "chlorophyll-a", "Chlorophyll.a"). Please standardise to a single format throughout.

Typographic consistency: Throughout the manuscript (e.g., lines 42, 116, 206–208), hyphens (-) are used where en-dashes or minus signs would be more appropriate. Please correct accordingly.

Manuscript formatting: Paragraphs in the main text and reference list lack first-line indentation or clear inter-paragraph spacing, which somewhat impairs readability. Please consider adjusting the formatting.

Reference list formatting: Several inconsistencies are present, including but not limited to: line 502, "Deep. Res. Part I" should be "Deep-Sea Res. Part I"; lines 489 and 494, "CO2" should be "CO₂" with subscript; lines 497–499, journal name appears to be missing. Please carefully proofread the entire reference list and correct such errors.
Citation: https://doi.org/10.5194/egusphere-2026-934-RC1
- AC1: 'Reply on RC1', Eyal Rahav, 23 Jun 2026
  
  Dear Reviewer,
  Many thanks for the time and effort you invested in reviewing our manuscript. Your suggestions and comments have significantly improved the manuscript, both scientifically and in terms of clarity and readability.
  Please find our detailed response in the attached file.
  
  Citation: https://doi.org/10.5194/egusphere-2026-934-AC1
RC2:
'Comment on egusphere-2026-934', Anonymous Referee #2, 12 Jun 2026

General comments
This study focuses on variability along an environmental gradient from coastal to offshore areas and with depth, examining the main photoautotrophic picoplankton groups (Synechococcus, Prochlorococcus and picoeukaryotes) in an oligotrophic sea.
Although cell size variability in picoplankton is a relevant topic in microbial ecology, there appears to be a mismatch between the Introduction, Discussion and Conclusion, making the key message of the study unclear. It is not evident whether the main objective is to assess (i) variability in cell sizes across environmental gradients in oligotrophic conditions or (ii) the use of different conversions factors depending on depth for biomass estimation.
In addition, the Introduction should be more clearly emphasize the role of small cell size and its implications for overall community performance, rather than focusing mainly on comparisons between small and large cells. The Material and Methods section should also be reorganized to clearly describe the steps used to determine biovolume, as this represents a central component of the study. Finally, the Discussion should be revised to reduce speculative statements and better align with the presented results and main objectives of the work.

Specific comments.
# Title
Lines 1 and 2: The term pico-phytoplankton may be misleading if the study includes, as not all cyanobacteria are typically considered phytoplankton in the strict taxonomic sense. I recommend replacing “pico-phytoplankton” with “photoautotrophic picoplankton” to more accurately encompass both eukaryotic and cyanobacteria.

# Abstract
Line 31: In this sentence, it is unclear whether the authors use the word “stable” to refer to both ocean stratification and nutrient decline, or only to ocean stratification. If it refers to both, the sentence is difficult to understand because “stable” and “decline” covey contrasting meanings. In that case, I suggest replacing “stable” with “persistence”
Graphical abstract: The meaning of the X and ✓ symbols is not clear…DO they indicate the presence/absence of cells, an increase/decrease in cell size, or another response variable? Please clarify in the figure

# Introduction
Line 39: What do the author mean by “community structure”? Community structure is generally understood as a combination of multiple phytoplankton aspects (cell size, taxonomy, distribution of taxa abundance, functional traits…and other characteristics).
Please rewrite the sentence to clarify which components(s) of community structure are being considered in this study.
Line 41: This sentence is vague and should be revised to provide a clearer and stronger statement. Please rephrase to improve precision and clarity.
Line 42: The terms “low-productivity” and “oligotrophic” are largely redundant, as oligotrophic systems are, by definition, characterized by low productivity. Please consider using only on of these terms to avoid redundancy
Line 42: The same as lines 1 and 2
Lines 42:43: Please delete the phrase ….”dominate the autotrophic community” and the sentence “Pico-phytoplankton are small-sized phytoplankton”…., as this information is redundant and already mentioned in the previous sentence.
Line 47: Why do the authors place “biological pump” in quotation marks? Please clarify whether this is intended and revise if necessary for consistency and clarity. The same in line 60.
Line 48: Please add a more recent reference to support this statement.
Line 54: Replace “Pico-phytoplankton” with “photoautotrophic picoplankton”
Line 59: What do the author mean by “trophic pathways”. Please clarify the term.
Line 63: What do the author mean by “phytoplankton cellular composition”. Please clarify the term.
Line 76: This is the first time Eastern Mediterranean Sea (EMS) is mentioned in the main text. Please define the abbreviations at first use.
Lines 94-95: This is the first time nitrogen (N) and phosphorous (P) are mentioned in the main text. Please define the abbreviations at first use.

# Materials and Methods
Line 104: Replace …”seawater was sampled”… with …”seawater was collected”…
Line 107 -109: Please remove this sentence., as the information is repeated in the following sentences.
Figure 1: Replace “Bottom depth” with just “Depth” for simplicity

Line 125: This sentence is hard to follow. I suggest revising the beginning as follows:
“For determination of chlorophyll-a (chl-a), seawater samples (300 mL) were filtered through …”
Line 126: Please delete for chlorophyll-a (chl-a) analysis.
End of chlorophyll section: The authors do not clearly describe how Chla concentrations were determined. Please include the reference followed (e.g., Ritchie or another standard protocol).
Line 130: Replace the title by “ Photoautotrophic picoplankton analysis”
Line 131: Please replace “Bacterial abudance samples” with a clearer methodological description, e.g.: “Water samples (1.8 mL) were fixed in …” followed by the fixation details. Please also specify the type of containers used e.g., cryotubes/cryovials) to improve clarity and reproducibility.
Line 133:Replace “put” with “stored”.
Line 133: Specify the type of analysis: “Flow cytometry analysis….”
Line 135: Remove “…syringe-based fluidic system and…”. It is not necessary to mention.
Lines 136-138: Caution: In routine flow cytometry analysis, taxonomic groups are not specifically identified, only broad groups, populations or assemblages are resolved.
Please replace “Taxonomic discrimination was…” and the following sentences with: “Photoautotrophic picoplankton groups were identified based on their autofluorescence. Cells were excited at (add lasers of the flow cytometer) and three populations were distinguished according to light forward scatter (FSC, proxy of cell size), light side scatter (SSC, a poxy of cell granularity or complexity), and red and orange fluorescence bands (proxy for chlorophyll and phycoerythrin respectively) (ref).
After this part, the authors should clearly specify which three groups were distinguishable according to the differences in their chlorophyll and phycoerythrin fluorescence signals, FSC and SSC (ref. ref)”.
Lines 138-140: Authors should specify with more details about the conditions of the flow cytometer while collect the event (i.e. flow rate, how many events were recorded, if dilution was needed, and finally how the authors determined the abundance of the assemblages.
Lines 141-154: This is the core of the M&M section and the main goal of the paper. The authors need to be precise in this part. For example…it is confused what is “the cell size distribution” for the authors…It should be simply as “Cell size of the three photoautotrophic picoplankton groups”. Also…since they already mention FSC and SSC signals…not need to mention flow cytometry nether side scatter.
Lines 142-145: It is confused how the instrument was calibrated with the mixture of beads and then use the same beads to build the calibration curve. The mixture of beads was running each day of the samples analysis? How many times was collected this mixture?
Line 145: When the authors mention “flow cytometer settings”, these should be specified earlier in the Methods section (see lines 138-140). Please ensure that all relevant instrument settings are clearly described at first mention to improve clarity and reproducibility.
In this section, where the authors describe how cell sizes of the autotrophic picoplankton groups were determined, the method for calculating biovolume (BV, um3) is missing. Please include a clear description of how BV was estimated for each assemblage. It is the core of the paper.
Caution: SSC is not a proxy of volume…It is proxy for cell granularity and complexity. You need extra calculations to determine the biovolume (BV, um3) of the cells.
Line 147: Include “water” between random and samples.
Lines 147-154: This part of the microscopy analysis is difficult to follow. Please rewrite this section to improve clarity, structure and readability, ensuring that the methodological steps are clearly and logically presented.
Line 155: Please remove the abbreviations from the title and keep the terms in full, to improve clarity.
Line 158: What do the authors mean by “these matrixes? Please clarify which specific matrices are being referred, as the term is unclear in this context.
Lines 159-160: These sentences need to be rephrased and starting with: “The minimum cellular content of a nutrient (Q_min) is defined as the minimum amount required to sustain basic cellular functions and balanced growth under ambient nutrient conditions. In contrast, the maxima cellular content of a nutrient (Q_max) is defined as….
Line 161: Delete “support”
Line 166: This information should be moved to Section 2.4, after indicating that cell size (um) was determined from the calibration curve. Please also clarify that these measurements were subsequently used to estimate biovolume (BV) in the section Cellular nutrient quotas (Qmin and Qmax) and resource supply index (RSI).
Line 166: Delete this sentence from this part.
Line 167: The authors should start the sentence with Biovolume (BV, um3) was calculated assuming spherical cell as shown in Eq.1:
where d is the cell diameter (um) determined from FSC values.
CAUTION: In seawater samples, side scatter (SSC) values are commonly used to determine biovolume estimations, whereas forward scatter (FSC) is less reliable due to variability in light scattering associated with differences in refractive index in natural seawater. Did the authors try to calculate the biovolume with SSC and compare with FSC?
Lines 179-180: What the authors mean with…” if all cells reached Qmax”. What if the cells do not reach Qmax?

Line 182: Remove “Where, “
Lines 187-188: The authors should rephrase this sentence to improve clarity and readability.
Line 189: What do the authors mean by “ standardized environmental matrix”? Please clarify whether this refers to scaled/normalization data (e.g., z-score standardization, range scaling from 0 to 1, or another information). This needs to be explicitly defined to avoid ambiguity.

# Results
Line 204: Please remove the abbreviation form the title
Line 206: How the authors determined sigma-theta (density)? Please clarify.
Line 218: If N:P ratio was often higher than the 16:1 ‘Redfield ratio’, this suggests phosphorous limitation rather than nitrogen limitation. Please rephase this sentence.
Line 221: Remove “Where” at the beginning please.
Line 222: The authors should use the term Deep Chlorophyll Maxima (DCM) with caution and provide a clear justification for its use, given that the reported chlorophyll-a concentration is very low ( ~0.3 μg L-1). Please clarify whether these values truly correspond to a DCM or rather to a weak subsurface chlorophyll maximum.
Line 225: Why do the authors use the term “Low-Nutrient Low-Chlorophyll” in the title and here, if it is not used elsewhere in the text? Please clarify.
Lines 230-231: Please remove this sentence, as it repeats information already said.
Lines 232-233: I suggest expressing the numbers of picoplankton abundance in scientific notation (e.g, 2x 10⁴ ,3 x 10⁶) to improve readability.

Figure 4: What do the authors mean by “single-cell volumes”? Please clarify how do these volumes were estimated: were they calculated individually for each cell of each group within a sample and then averaged per sample, or were they derived from mean FSC values per group per sample? This methodological aspect should be clearly described in the Materials and Methods section to ensure reproducibility. If volume were determined form the FCS values average, single-cell should be removed from the figure caption.
Line 276: This is a title for the discussion part.
Lines 277-283: This section, as currently written, is not appropriate for the Results section, as it primarily contains interpretation rather than the presentation of results. The content would be more suitable for the Discussion section. Please consider revised and rewrite it accordingly.
The Figure 7 is not mentioned in the Results section. Please consider describing the main findings associated with this figure in the text. In addition, I recommend using different colors and/or symbols to represent sampling sites according to their distance from the coast and depth, different color arrows and vectors for environmental variables and for the sizes of the three photoautotrophic picoplankton groups respectively. All together would improve the readability and interpretation of the figure.
# Discussion
Lies 314-315: This sentence is part of the Results section.
Lines 317-321. This sentence appears to be based on speculation rather than on observations or evidence from the study site. Please revise the statement to more closely reflect the results obtained in this study or provide supporting evidence for the interpretation.
Line 328: What do the authors mean with “size structuring”? Please clarify.
Line 333: what do the authors mean with “adaptive trait”? Please clarify
Line 333: Please replace “chronic” with “persistence”.
Line 346: What do the authors mean with “to begin with”? Please consider rephrase the sentence.
Lines 334 and 348. The authors discuss differences reported in the literature between small and large Synechococcus. However, it is unclear how this distinction applies to the present dataset. Were both small and large Synechococcus populations identified in this study? If so, please specify in which samples or regions they were observed and how they were distinguished? Otherwise, the relevance of this discussion to the current results should be clarified.
Lines 377-380: These sentences are part of the results section.
Line 385: Please replace “machinery” with “capacity”.
Line 391: The use of the term “evolutionary optimization” appears inappropriate and speculative in the context of this study. Please clarify why the authors infer that this process is occurring in their dataset and provide supporting evidence. If no direct evidence is available, the statement should be rephrased to avoid overinterpretation of the results.
Line 394: What do the authors mean by “intermediate response”? Please clarify this term, as its meaning is not clear in the current context.
Line 398: What do the authors mean by “pico-eukaryotes respond dynamically”? Please clarify this term, as its meaning is not clear in the current context.
Lines 399-400.: What do the authors want to say in this sentence? Please consider rephrase the sentence.
Lines 410-446 The final part of the Discussions speculative and does not clearly convey the main message of the paper. IN addition, it is not well connected to the biomass calculations mentioned in the conclusions. Please revise and restructure this section to better integrate the interpretation with the key results, particularly the biomass estimates, and ensure that the main takeaway of the study is clearly articulated.
# Conclusions:
The authors introduce a new aspect related to biomass estimation and the of different conversion factors. However, it is unclear why the authors did not test biomass calculations sung both a single conversion factor and depth-specific conversions factors, to evaluate potential differences. Please clarify this methodological choice and its implications for the results.

General typos:
Please replace “ml” with “mL” throughout the manuscript.
Please remove “Where,..“ from the beginning of these sentences. This structure is not used in proper English syntax.
Please remove the unnecessary quotation marks throughout the manuscript. For example, in line 384 ( “allows”) and line 390 (“tendency”9, the quotation marks do not appear to serve a specific purpose. Please review the manuscript and remove similar unnecessary quotation marks for consistency and readability.

Citation: https://doi.org/10.5194/egusphere-2026-934-RC2
- AC2: 'Reply on RC2', Eyal Rahav, 23 Jun 2026
  
  Dear Reviewer,
  Many thanks for the time and effort you invested in reviewing our manuscript. Your suggestions and comments have significantly improved the revised maunscipt, both scientifically and in terms of clarity and readability.
  Please find our detailed response in the attached file.
  
  Citation: https://doi.org/10.5194/egusphere-2026-934-AC2

Elad Albagly and Eyal Rahav

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Elad Albagly and Eyal Rahav

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Short summary

Photosynthetic microbes form the base of ocean food webs, yet their size is often assumed to be constant. We show that in the nutrient-poor water Eastern Mediterranean Sea, these microbes become larger with depth and vary from coast to open sea. These subtle size shifts change how much carbon they store, meaning common methods can underestimate ocean carbon stocks. Accounting for size improves estimates and strengthens predictions of how stratified oceans function under climate change.


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