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

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

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12 Mar 2026

| 12 Mar 2026

Status: this preprint is open for discussion and under review for Biogeosciences (BG).

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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RC1:
'Comment on egusphere-2026-934', Anonymous Referee #1, 02 May 2026 reply
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.

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Citation: https://doi.org/10.5194/egusphere-2026-934-RC1

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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