Phytochromes facilitate social behaviour in marine diatoms

Domain: biology
Published: 2026-03-10
Editorial outcome: major_revision

Abstract
The phytochrome superfamily comprises photosensory proteins that enable organisms to perceive changes in light intensity and quality and is widespread across plants, fungi, algae, and microbes. In terrestrial plants, phytochromes sense red and far-red light to regulate key developmental and physiological processes. In marine environments, however, where red and far-red wavelengths penetrate only the upper few meters of water, the function of phytochromes has remained unclear. Recent work shows that diatom phytochromes exhibit photoreversible responses across a broad spectral range, extending beyond red and far-red, suggesting a role in underwater light sensing. Here, we examine the role of phytochromes in light perception and collective behavior in the marine diatom Phaeodactylum tricornutum. Comparing wild-type and phytochrome knockout strains under different light wavelengths reveals that activation of phytochromes by blue or far-red light synchronizes cell movements into a coordinated “wobbling dance.” This behavior is absent in phytochrome-deficient mutants, demonstrating the essential role of phytochromes. Our results further suggest that this collective motion involves intercellular communication, potentially mediated by variable red and far-red autofluorescence. Together, these findings uncover a previously unrecognized light-driven social behavior in marine diatoms and highlight the ecological significance of phytochrome-mediated communication in microbial communities.

Introduction
Photosynthesis is an essential biological process that sustains life on Earth by enabling autotrophic organisms to transform solar energy into chemical energy, which in turn serves as a vital source of sustenance for other living organisms. The effectiveness of photosynthesis relies, among other factors, on the ability of autotrophs to adapt to fluctuating environmental sunlight conditions. To this end, they have evolved highly specialized sensory molecules that allow the perception of incoming environmental signals and transduction into biochemical outputs that shape organism responses1,2.

Phytochromes are a group of photoreceptor chromoproteins that enable plants and some photosynthetic and non-photosynthetic microbes to detect and adapt to changes in light intensity and quality3. In terrestrial plants, phytochromes (PHYs) are capable of sensing light in the red (R=660nm) and far-red (FR=780nm) regions of the spectrum4. PHYs provide essential plasticity to plant survival by activating specific signaling cascades and enabling the active adjustment of vital processes such as germination, growth, development, phototropism, and associated metabolic pathways5,6. Plant PHYs are also capable of perceiving changes in the R/FR ratio of ambient light caused by neighboring vegetation, initiating shade avoidance and acclimation reactions7. In some human pathogenic bacteria, PHYs-mediated light signals also integrate quorum-sensing responses to control collective behaviors and diverse lifestyles8.

PHYs have also been recently found in a broad range of aquatic microorganisms. Their discovery first came as a surprise because R and FR wavebands derived from sunlight radiation are scarce in aquatic environments, due to their rapid attenuation by water absorption9,10, opening questions about the mechanisms of action and function of these photoreceptors11. The more recent characterization of phytochromes from diverse eukaryotic algae provided some answers to this conundrum. Extensive spectral tuning and blue-shifted absorption spectra have been described in PHYs of some prasinophyte and glaucophyte species, leading to the hypothesis of an adaptation of the photoreceptor absorption properties to the blue-rich aquatic environment12. On the contrary, conserved R and FR light absorption spectra have been described for the phytochrome of diatoms 13, one of the most prominent and diversified phytoplankton groups in the ocean, which are distributed worldwide and play a key role in global biogeochemical cycles14,15. Unforeseen roles for R and FR PHYs-mediated detection have been hypothesized, such as the perception of fluorescent light emitted by nearby photosynthetic congeners, potentially providing valuable information on cell densities, for example, during algal blooms13,16,17, and stimulating various aspects of their cellular metabolism, such as iron uptake17. Recent studies, however, indicate that the Diatom Phytochromes (DPH), despite the conserved R and FR light absorption13, can also exhibit photoreversible responses across the whole available underwater light spectra, suggesting an additional potential role of DPH as an optical depth sensor18 but not ruling out other functional roles.

Diatom lifestyle diversity and survival success in contemporary oceans are attributed to their exceptional ability to adapt to highly dynamic aquatic environments using efficient mechanisms to respond to environmental changes19–22. For example, several diatom species can cope with highly variable light conditions, suggesting that diatoms are capable of perceiving, responding, and likely, anticipating light variations and that they possess suited underlying molecular systems mediating light responses23,24. Recent studies have also shown that several pennate marine diatoms are indeed capable of perceiving and using external light signals to synchronize intracellular processes driving their unsteady sinking behavior 25,26. However, the molecular mechanisms underpinning these photoresponses and the role of PHYs in light-mediated behavior at the population level are not yet established.

Here, we use several independent P. tricornutum dph-knockout lines, together with laser diffractometry, to evaluate the role of photosensing in the light-mediated development of collective cell behavior27–30. We experimentally demonstrate that photoreception by DPH is required to establish a coordinated social pattern in populations of P. tricornutum suspended in the water column. The possibility that microbial organisms use endogenous light signals to perceive and communicate with neighboring homologous cells for the development and propagation of coordinated behavior is also discussed.

Results
Pelagic diatoms with elongated shapes characteristically display coherent vertical cell orientation and synchronized wobbling when sinking under continuous illumination 25,26,31. In the case of nearly elongated cells, this behavior can be characterized by laser diffractometry (LD) using the relative variation of two size bands associated with the major and minor cell axis lengths30,31. Due to the 2D projection of the LD measurements, the Ratio between these signals is a scalar proxy for the mean orientation of the cell population (see M&M). As shown in Figure 1 (see also Fig. S1; Supplementary Data 1, 5), in the diatom P. tricornutum, the Ratio time series displays a pronounced wavelength dependence of synchronized wobbling, with strong responses in the B and FR regions, but minimal responses elsewhere in the light spectrum. This spectral dependence is consistent with that described by Duchêne et al.18 for DPH-mediated gene expression (Fig. 1B). No coherent wobbling is observed in dph-knockout strains (5D1 and 4F1), while their respective control lines (Tc5A1 and Tc4E1, transformed but non-mutated in DPH13) exhibit a synchronized collective response under B light, which is indistinguishable from that observed in the wild-type (WT Pt1; Fig. S2; Supplementary Data 6). These results strongly support that DPH photoperception is strictly required for light-induced synchronized cell wobbling.

A key feature of PHY-mediated control is its ability to scale the magnitude of a response according to the wavelength ratio. Since, in P. tricornutum, DPH was shown to be activated by B or FR and inactivated by R light18, we tested whether the described collective behavior is hindered by changing the relative intensity of B and R light. Starting with the intensity of B light used in Fig. 1, we simulated the effect of decreasing depth in the water column by increasing R light (shallower waters), and observed inhibition of the synchronized wobbling amplitude (Fig. 2, see also Fig. S3; Supplementary Data 2, 7) while wobbling frequency was unaffected (Fig. 2A). A similar response was observed in experiments performed with varying FR to R ratios (Fig. S4; Supplementary Data 8). To confirm that this was a response to R light and not just the consequence of modified light quantity, we also performed control experiments with increased B (or FR) light intensity and observed no change in the response (Fig. 2B and Fig. S4). Our results suggest that, similarly to the proposed in situ modulation of DPH activation by optical depth throughout the water column18, synchronized wobbling is also modulated by blue-to-red ratio. As a consequence, it would be favored at depth, an environment dominated by B light.

While the triggering mechanisms of the described cell behavior rely on ambient B light availability and detection by DPH, the mechanism through which independent cells freely suspended in the water column coordinate their movements remains challenging. Font-Muñoz et al.25 framed the question within the context of the theory of weakly coupled oscillators for phase locking32, which requires a harmonic coupling signal compatible with the cell wobbling frequency (ω; Fig. 3; Supplementary Data 3). It can be speculated that either hydrodynamic physical disturbances in the flow field surrounding individual cells or the modulation of unknown dissolved infochemicals govern these cell-to-cell interactions33–35. However, these are unlikely to generate coherent forcing at the involved spatial and temporal scales. Alternatively, B light-excited cell autofluorescence emitted in the R and FR spectral regions provides a plausible explanation for intercellular communication, inducing the observed synchronized movements. Autofluorescence emission by photosystem II is a by-product of photosynthesis that is cost-efficient and almost instantaneous, and depending on cell morphology, can provide a cell density-dependent but directional signal of the correct frequency16,17. Although in the sea the intensity of fluorescence emission is always very small compared to the ambient light, it could still convey precious information and be perceivable locally by neighbor cells as a weak coupling signal above the R light background. Indeed, the measured emitted chlorophyll fluorescence signal in each arbitrary direction is modulated by the cell's relative orientation, thus exhibiting a time modulation at the exact frequency of the cell's wobbling oscillation, as displayed in Figure 3. This periodic light signal comprises both R and FR spectral components, whose temporal signals are highly similar (Fig. 3B). It is worth noting that while dph-knockout strains autofluoresce with the same spectral qualities as WT -at room temperature-, they do not exhibit a population-level coherent fluorescence signal modulation. It should also be stressed that illuminating an algal suspension with an exogenous monochromatic light does not guarantee a homologous light environment since both Raman scattering and, more intensely, chlorophyll autofluorescence cause the emission of photons shifted in frequency relative to the incident light, thereby modifying the spectral composition of the surrounding light field16,36. Exogenous light and chlorophyll-emitted light signals cannot easily be decoupled. This opens the question of whether DPH mediates cellular responses only to exogenous ambient light or whether they are also involved in the perception of time-modulated autofluorescence.

The idea that PHYs enable algae to sense neighboring cells through the perception of cell autofluorescence is not novel13,16 but, to the best of our knowledge, has yet to be confirmed. We set out to test this hypothesis by interrogating cells only with pulsed R and FR light that mimic the measured fluorescence signal naturally emitted by P. tricornutum cells (see Fig. 3A). In Figure 4, we show a striking contrast in the response of the WT and the dph-knockout strains to the same pulsed stimulus, both under R and FR illumination. While WT and Tc cells are readily entrained by pulsed light, the wobbling of dph-knockout strains remains largely incoherent (Fig. 4A and B; Supplementary Data 4). These results indicate that DPHs are not only essential for triggering collective behavior under constant light illumination, they are also key to the synchronization process, potentially implying their involvement in the ability of cells to perceive the light emission of their neighboring congeners and thereby contribute to the observed collective behavior by propagating the signals in the population. Full characterization of this function and its ecological relevance remains to be elucidated.

Finally, it is important to note that, while a collective photoresponse is not elicited by illumination under constant R light in our experiments (see Fig. 1C), a periodic modulation of the signal with frequencies around natural wobbling frequencies (~140s) suffices to induce strongly coherent cell responses (Fig. 4C). In fact, these are as coherent as those observed under constant B (or FR) illumination (Fig. 4D). Notably, DPH-knockout strains do not exhibit collective responses in either case. This differential photoresponse, not observed at any other wavelength, hints at a possible unknown DPH signal transduction pathway and PHYs-facilitated mechanism for light perception under quickly fluctuating light environments.

We have found that phytochromes are involved in light-driven coordinated diatom wobbling, a collective behavior modulated by the effective wavebands activating DPH 18, and therefore enhanced in B light-enriched environments. Although the function of this collective dynamics in the dilute marine environment is still unclear, we can speculate it could influence key ecological processes such as sedimentation rates, light harvesting, or increased cell-cell contacts, thereby favoring sexual reproduction in pelagic environments 25,26, especially in the deep, non-turbulent layers of the water column 31,37,38. The analysis presented here immediately suggests a number of specific experimental investigations. Chief among them are detailed studies of DPH involvement in the photoresponse to pulsed R light. This can be tested with experiments at the single-cell level in which photoresponses to exogenous light can be readily distinguished from signaling from neighboring cells. Open issues include the origins of this regulation, the comprehensive characterization of the perceived stimuli, along with the possible interplay of these processes with other biotic and abiotic signals, as those observed in the quorum sensing of bacteria due to high cell densities39. Additionally, our results under pulsed R and FR light suggest that oscillations of autofluorescence by cell wobbling could be involved in the propagation of the DPH-mediated response. Nonetheless, earlier observations of collective oscillations in pennate diatom species that are not known to express DPHs25, suggest that DPH may function in coordination with other, yet undiscovered photoreceptors. Overall, our findings provide strong evidence of the relevance of light signalling in shaping microorganism interactions and social behavior, and, at a higher hierarchical level, as a structuring element of life in the ocean.

Methods
Cell cultures
The wild-type Phaeodactylum tricornutum cells and transgenic lines were grown in f/2 medium40 at 18 °C under a 12-h-light/12-h-dark cycle and 80 mol photons m-2 s-1, in algal incubators. The cultures were transferred once a week (dilution factor x8) to keep the cells healthy and in their exponential growth phase at maximum concentrations. All the experiments were done at a concentration of 3x108 cells/L, with cells in the exponential phase of growth, each experiment was carried out in triplicate. DPH knockout lines were obtained by TALEN-mediated genome editing as described in Fortunato et al.13. Their respective DPH non-mutated lines (derived from the same cell transformed with TALEN vectors but not having undergone a mutagenic event on the DPH gene) were used as a control for DPH-specific phenotype 13.

Experimental set-up
Experiments were carried out in an in-house built set-up for the concomitant measurement of particle size distribution, orientation, and chlorophyll autofluorescence as in Font-Muñoz et al. (2021)25. The small volume ~100 ml flow-through chamber of a LISST-100x laser diffractometer (Sequoia Scientific) was adapted to include an additional light-line source. This light was modulated both spectrally, using a monochromator, and temporally, and aligned orthogonally to the laser diffraction axis. Two Thorlabs PM16-130 power meters were integrated into the setup to monitor this secondary illumination. The modified system enables the investigation of a broad spectrum of experimental conditions, providing simultaneous sub-second resolution measurements of both cell orientation and light emission 25.

Before each experiment, the LISST-100X chamber was pre-cleaned and filled with the desired v/v concentration of cell culture. Agitation was induced by a 2 cm long magnetic bar powered by the built-in speed controller of the chamber at minimum speed. Experiments consisted of an initial mixing phase where agitation was turned on (100 s) followed by a sedimentation phase where the system could evolve without any external disturbance.

Diatom response to light: to study the effect of light conditions on cell orientations the cells and collective behavior were illuminated with continuous light of different wavelengths (430, 530, 617, 660 and 780 nm, mod. M430L5, M530L4, M617L5, M660L4 and M780L3 from Thorlabs) with an intensity of 8 𝜇mol photons m-2 s-1 for 2h.

Diatom response to composed blue and red light: To study the responses of cells to composed continuous red and blue light, cells were illuminated with 430 nm at 8 μmol photons m-2 s-1 and 660 nm at different intensities (2, 4, and 6 μmol photons m-2 s-1). We performed experiments using monochromatic blue light at the same total intensities (10, 12 and 14 μmol photons m-2 s-1 ) as a control. We used a long-pass dichroic mirror 550 nm cut-on (DMLP550 from Thorlabs) to combine the two light sources.

Diatom response to pulsed red and far-red light: to study the response of P. tricornutum to pulsed red light, cells were illuminated by a 660 nm and 780 nm LED (M660L4-C and M780L3, Thorlabs) through a neutral density filter (ND10A Thorlabs) used to attenuate light intensity down to a maximum value of ~8 μmol photons m-2 s-1. This intensity was then modulated sinusoidally with periods of cell wobbling natural frequency (~140 s) by coupling the controller triggering the excitation LED source to a pulse generator.

Fluorescence signal variations measurements: variations in the cells' chlorophyll autofluorescence were measured by illuminating the culture with an excitation blue light (430 nm at 30 µmol photons m-2 s-1) for 45 min while the cells settled without disturbance. The emitted R and FR light coming from the cells was measured every second by the orthogonal power-meters (PM16-130 from Thorlabs) via high-pass R and FR filters (FGL645M, Thorlabs; 𝜆 > 645nm and FGL715 M, Thorlabs; 𝜆 >715nm).

Laser diffractometry
Laser diffractometry (LD) measurements were used to obtain the particle volume concentration by size ranges (i.e., volume of particles in the seawater per unit volume of seawater) using a technique based on laser diffraction theory. The LISST-100X employed in this study uses a 670 nm collimated laser beam to illuminate the suspended particles and a 32-ring detector measures the intensity of the scattered light, corresponding to 32 different size classes logarithmically spaced. The angular pattern of optical scattering depends on the physical characteristics of the particles (e.g., size, shape and orientation), and is used to calculate the particle volume concentration in these size classes 41. LD can be used to adequately characterize the different dimensions of non-spherical particles in specific orientations28,29,31. Using this property, a method to infer the preferential orientation of particles in a suspension has been recently described 30. Hereby, we use this method to characterize the preferential orientation of diatoms under distinct lighting conditions: LD measurements of nearly spheroidal cells are interpreted in terms of the relative variation of two size bands representing the known major and minor cell axes (r1 and r2) of P. tricornutum (major axis VD2=7.33-14 μm and minor axis VD1=2.5- 3.5 μm) 2. As cells orient in different directions, the signal in the size bands representing these axes presents an opposite behavior. Hence, we can confidently compute the ratio between the signal from each of these two size bands, Ratio(t)=(𝛴VD1)/(𝛴VD2), as a scalar proxy for cell orientation25,30,31.

Determination of spectral peak
We have used a standard and robust criterion to discriminate and characterize the main component of each power spectrum calculated from the Fast Fourier Transform (FFT) of the time-series of the Ratio. The criterion is based on conventional signal processing tools designed to locate peaks and arrange them by their relative prominence (Pro) in the signal (Pro= 6.2±2.12; 2.4±0.95; 2.38±0.88; 0.73±0.26; 5.12±2.23, for the different wavelengths of the experiment, respectively). For signals with a variance of the relative peak prominence above one, std (Pro)>1 (i.e., when a main peak can be isolated), we stick to a Gaussian fit to the main peak. For std (Pro)<1 (i.e., when no peak stands out in a statistically significant way above the others), a single Gaussian is fitted across the full spectrum. Although a refined analysis of the secondary peaks might be of eventual interest, we do not consider that it has an impact on the results presented in this manuscript, so we defer this to future work.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Manuscript#: NCOMMS-24-74582A-Z\nThe authors tested the cell response of Pheodactylum tricornutum as a representative for pelagic diatoms with an elongated\nshape, to varies wavelength in regard their synchronized wobbling activity. Their results show that the strongest response\nwas observed at blue (430 nm) and far red(780nm) when the diatom phytochrome encoding gene isn’t knocked out. They\nfurther show that the cell response to blue and far-red light can be inhibited by red light (660nm), while the impact depends\non the red to blue light ratio.\nThe present study is the logical consequence of the authors previous work, where they have established the laser\ndiffractometry allowing them to determine the cell orientation within the water column.\nmajor comments:\nL130: Have you tested the ratio impact of FR and R as well? If yes, what were the results? If not tested, why not?\nL 170: Why not repeat the experiment on a low cell density culture or on a single cell level?\nL: 154 ff: this is a weak point of the hypothesis and should be tested. What is the minimal intensity which a cell can detect\nand how much do they approximately emit and how close they would need to be to each other in order to be able to receive\nthe FR from one cell to another. How likely is it that it serves as an intracellular signal?\nL 189ff. Maybe I simply didn’t understand the last experiment, but I have trouble to follow the conclusions. While not pulsed\nred light seems to inhibit cell wobbling, pulsed red lights seems to trigger it?\nMinor comments:\n• Fig 1.\no legend title: ‘continuous light experiment’ is not accurate as it can refer to either wavelength of exposure time. Please\nrephrase it, e.g. continuous exposure to various wavelengths.\no 1.A and S1 and all following figures. Nice idea to represent the wavelength in the corresponding colors. However,\nchoosing black for FR isn’t very intuitive. May you want to change it to a dark red, eg. As in panel 1 C?\no 1A. what rare the dotted and what the solid line stand for? WT and KO? Or is the solid line a fit of the scatterplot? In the\nlatter case:\nthe fit for430 and 530 doesn’t seem to respect the real shape of the data points. While 430 shows another peak (even though\nlower) around 350 s 530nm peaks at 150s and not at 200 as the fitted line suggests. While it doesn’t contradict the finding\nthat 430 and 780nm result in the highest wobbling response, 430nm also shows a clear interaction. The sentence in L 199\n“minimal response elsewhere” thus is not correct and needs to be rephrased accordingly.\no 1B Legend text and figure could be better connected to each other. While the graphs show delta Abs the legend text talks\nabout DPH de-/activation and uses a color-code, which are not defined Green= activated? Blue deactivated? Furthermore,\nthe abbreviations ‘Pfr and Pr’ are nowhere defined in the MS. Are those data of WT or KO?\no 1C what does Pk/FWHM stand for? Unit? (please define)\n• Fig2.\no Legend title: ’continuous’ blue and red light. The word doesn’t make sense like that. Do you mean continuously changing\nratio?\no Panel A&B, highest y axis value cut off\n• Fig 4.\no Panel D. please define the abbreviation within the graph in the figure legend. E.g. What does 5D1 for?\n• References:\no Number 26. Correct title from pennates to pennate\n• L446: symbol for micro is not shown\no The Materials and methods partly “copy paste from: 1st author previous article: Pelagic diatoms communicate through\nsynchronized beacon natural fluorescence signaling (DOI: 10.1126/sciadv.abj5230\no The used methodology is new to me and seems to be quite sophisticated.\no The used unit Einstein to express the light intensity is not accepted as an SI unit and outdated, please substitute with µmol\nquanta (or photons) m-2 s-1\no The article would benefit from a conceptual diagram to help understand the set-up.\no I miss some further details:\nFor how long the cells were the cells exposed to the different wavelength during the different experiments?\nL110: How have the authors dealt with the other morphotypes of Pheodactylum as the triradiate form?\nL113: please define the “two size bands”\nL454: ‘This intensity was then modulated sinusoidally with periods of cell wobbling natural frequency (~140 s)’. It is not\nexplained how the authors did that.", "reviewer_2": "Reviewer #2a (early career co-reviewer):\nGeneral comments:\nThis is a highly interesting and well-structured study. The article is clear and concise, making complex concepts accessible\nwhile maintaining scientific rigor. One particularly intriguing question raised is whether DPH mediates cellular responses\nonly to exogenous ambient light or if it is also involved in the perception of autofluorescence. This is a fascinating\nhypothesis, and the approach used to test it, as well as the results obtained, is particularly interesting. The experimental\nsetup is straightforward and leads to clear results regarding the involvement of phytochromes in the wobbling dance in\nresponse to specific wavelengths. However, as explicitly mentioned in the article, further investigation and additional\nevidence are required to confirm and fully understand the role of intracellular communication. The Materials and Methods\nsection is well described, ensuring good reproducibility. However, more transparency regarding data processing would be\nbeneficial (see individual comments for details).\nThree key findings stand out and are clearly highlighted:\n- The implication of phytochromes in the synchronization of cell movements.\n- The response to red, far-red, and blue light, indicating a qualitative aspect of behavioral signaling.\n- The response of phytochromes to autofluorescence signals from diatoms, potentially suggesting intercellular\ncommunication.\nOverall, this is an innovative and valuable study that contributes to a better understanding of how diatoms use different\nwavelengths and how they might communicate through light signaling. It opens exciting avenues for future research on\nphotoreception and cellular interactions in microalgae.\nSpecific comments:\nNeed for standardization of irradiance units (PPFD):\nIn the supplementary materials and methods, there is a typo in the \"cell culture\" paragraph: \"and 80 µm-2 s-1\". Additionally,\nfor irradiance values, it would be preferable to use the unit \"µmol photons·m⁻²·s⁻¹\", which is more commonly used in\nphotophysiology, rather than \"µE·m⁻²·s⁻¹ ».\nThe use of the unit \"1mW\" (appearing multiple times, e.g., in Fig. 3, Fig. 2B, and the supplementary \"Fluorescence signal\nvariations measurements\") is uncommon, as it refers to power rather than irradiance?\nIn Figure S1B, why are the ratios not displayed for all wavelengths, as they are in Figure S1A?\nI am not entirely sure how Figures 1 and S1 correspond. How are the data transformed to go from Ratios to Power?\nThe period of cell wobbling natural frequency is given as ~140 s. How was this value determined? Was it measured\nexperimentally on your cells, or is it taken from the literature?\nIn the main text, Figure S3 is not well integrated and could benefit from additional context. For example:\n\"Starting with the intensity of B light used in Figure 1, we simulated sinking in the water column by increasing R light (Fig.\nS3) and observed inhibition of the synchronized wobbling amplitude (Fig. 2), while wobbling frequency remained unaffected\n(Fig. 2A). »\nProvide more transparency on data usage and processing, particularly regarding the number of individuals\nobserved/included for Figures 2A and 4C.\nTypo in \"autofluoresce\" in the Results and Discussion section.\n\nReviewer #2b (senior reviewer):\nThe study by Front-Munoz et al. investigates the role of phytochromes in marine diatoms. It is particularly well-conducted,\nwith high-quality and inspiring results. The study clearly demonstrates the role of diatom phytochromes (DPH) in detecting\nlight signals and the behavioral adaptations resulting from this detection in diatoms. The DPH gene appears to be central to\nthe synchronized response of diatoms to light signals. This is, therefore, an excellent study on the perception of light quantity\nand quality in Phaeodactylum tricornutum and the triggering effect of DPH at the community level. That being said, I have\nsome comments on certain aspects of the study that seem unclear to me, as well as others where I believe the results may\nhave been over-interpreted. These points are detailed in the specific comments below.\nIn general, I find that:\nThe results are not always well explained in the methodology, making it difficult to read and interpret certain graphs. In\naddition, the study claims to demonstrate that phytochromes are involved in an intercellular social behavioral response,\nwhich would indeed be a novel finding. However, on this second point, I remain skeptical after reading the manuscript.\nEven though the results are exciting and may seem convincing—particularly Figure 4, which shows diatom responses to pulsed\nlight, I believe there is a lack of direct evidence that diatom autofluorescence indeed serves as a communication signal\nbetween cells, driving the observed behaviors. The authors themselves appear to be cautious in their manuscript when\nestablishing this direct link (e.g. L38: potentially mediated, L104: the possibility of endogenous light signal, L150: a plausible\nexplanation, L183: potentially implying, ...). Thus, while the content of the article is not in question, the title could\nperhaps better reflect its findings.\nSpecific comments:\nL57: It would be nice to define in the introduction the wavelength of the R and FR region.\nL110-119 (+figure1): The principle of laser diffractometry is well explained (L110-116), but its connection to Figure 1 is\nunclear to me. I may have missed something, but unfortunately, the Methods & Materials section in the Supplementary\nMaterial does not provide additional clarification. I suggest improving the explanation in the Methods & Materials on how the\nPower Spectrum (Figure 1A) and the Time Series Ratio (Figure S1A) are related. Additionally, please clarify what Pk/FWHM\nrepresents in Figure 1C. It would also be helpful to specify in the Figure 1 caption the differences between the solid and\ndashed lines—are they meant to represent WT vs. KO?\nLines 136-139: The inclusion of the vertical distribution of the Red:Blue light ratio in the sea (figS3) is a strong point, making\nthe extrapolation of the results to natural environments very convincing.\nLines 147-154: In this section, the authors analyze the synchronized response of diatoms to light signals. It would be helpful\nto clarify at this stage whether diatom autofluorescence is produced in the R or FR region (or both) when excited in the B,\nas this information is only provided later (L178, Figure 3). Including it earlier could better support the authors' hypothesis.\nThat being said, in this section, the authors dismiss the possibility of coordinated behavior driven by info-chemicals to introduce\nthe autofluorescence hypothesis. While the autofluorescence hypothesis is intriguing, at this stage of the manuscript, it is\nsufficient to establish that light acts as a trigger—via DPH—to induce a behavioral response at the community level. Since\nthe hypothesis of autofluorescence as an intercellular signal is introduced later with the pulsed light experiments (L189,\nFigure 4), it may be premature to bring it up as early as L150.\nL154-156: intensity of fluorescence in the sea => This is very interesting and brings the question of the distance at which the\nautofluorescence of the Chlorophylle a could be detected by other cells in nature. Is Phaeodactylum tricornutum widely\ndistributed in several different ecosystems, or rather restricted to clear, oligotrophic or non turbid waters ?\nL158-162 & Fig3 : The manuscript states that the measured chlorophyll fluorescence includes both R and FR components.\nHowever, in Figure 3, it is mentioned that the Red fluorescence corresponds to a long-pass filter that collects light >645 nm.\nThis implies that the R signal actually represents R + FR, while the FR signal refers only to FR wavelengths (> 715nm). If\nthis is correct, it makes sense that the emitted R+FR and FR signals produce similar results, as the R+FR signal would be\nlargely dominated by FR fluorescence.\nL180-181: The wobbling motility of diatoms is described as \"incoherent\" in dph-KO cells, but this point needs further\nclarification. When comparing the power spectrum curves in Figure 4A&B (pulsed light) with Figure 1A (continuous light), I\ndon’t see a significant difference. What are the main distinctions? What exactly is considered \"incoherent\" here? The\nauthors then emphasize that DPH is essential for triggering collective behavior(which the study clearly demonstrates).\nHowever, they also reintroduce the hypothesis of autofluorescence as an intercellular signal. This needs a more detailed\nexplanation because it is not immediately clear how Figure 4A&B supports the autofluorescence hypothesis.\nL189-197: From my perspective, this paragraph is the only one that supports the autofluorescence hypothesis. It is indeed\nintriguing that R light does not induce movement when provided continuously (Fig1C) but does when introduced at a period\nmatching the wobbling frequency (Fig4C). This serves as an argument in favor of the autofluorescence hypothesis, even\nthough the direct relationship between autofluorescence and movement is not explicitly demonstrated in this case. Thus, I\nsuggest presenting this result earlier in the manuscript.\nL221: \"strong evidence of the relevance of light signaling (remove communication)\"\nM&M: Continuous light experiments were conducted by exposing the cells to different wavelengths for 2 hours. Meanwhile,\nlaser diffractometry measurements—central to establishing time series ratios and power spectra—were performed using a\nLISST-100X, which operates at 670 nm (R light). Looking at Figures S1 and 2, it appears that the measurement duration\nexceeds 3000 seconds (i.e., more than 50 minutes). This raises the question of how the LISST-100X, which exposes the\ncells to red light for a relatively long period, aligns with the experimental treatments that are supposed to provide only blue\nand FR light. Could you clarify how this potential interference is accounted for?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nThe authors tested the cell response of Pheodactylum tricornutum as a representative for pelagic diatoms\nwith an elongated shape, to varies wavelength in regard their synchronized wobbling activity. Their results\nshow that the strongest response was observed at blue (430 nm) and far red(780nm) when the diatom\nphytochrome encoding gene isn’t knocked out. They further show that the cell response to blue and far-red\nlight can be inhibited by red light (660nm), while the impact depends on the red to blue light ratio.\nThe present study is the logical consequence of the authors previous work, where they have established\nthe laser diffractometry allowing them to determine the cell orientation within the water column.\nmajor comments:\nL130: Have you tested the ratio impact of FR and R as well? If yes, what were the results? If not tested,\nwhy not?\nIn the first version of the manuscript, we analyzed the impact of B/R ratios as the most relevant ones in the\nmarine environment (associated for instance with varying depth). The Reviewer 1 is right that, if light-\ninduced synchronized wobbling is mediated by DPH, the induction by FR should also be counteracted by\nred light. As suggested by Reviewer 1, we have included new data also showing the impact of varying FR\nto R ratios. The results indicate a similar behavior to that shown in Fig2b; i.e. a decaying oscillation\namplitude with lower FR to R ratio. The analysis confirms the involvement of the diatom phytochrome\nphoto perception in the analyzed processes, and is coherent with the mode of action of DPH recently\ndescribed in Duchene et al. (2025)). The new data are now included in figure S4 in the Sup. Mat. In the\ntext, we have added the following sentence to line 133: “a similar response was observed in experiments\nperformed with varying FR to R ratios”.\nL: 154 ff: this is a weak point of the hypothesis and should be tested.\n‘The capacity of diatoms to detect and respond to weak but characteristic fluorescence emission of\nneighboring cells is already demonstrated in the work of Liu et al. (2021), where this signal is shown to\ntrigger cellular responses such as algal iron uptake’. We expand on this in the following comments.\nWhat is the minimal intensity which a cell can detect?\nWe did not test the minimal intensity required to trigger wobbling. Photoreceptors are in fact capable of\ndetecting single-photons (see Hegemann 2008), a common feature of many photochemical processes.\nHowever, it is indeed an interesting question whether there is a minimum intensity threshold (or cell-to-\ncell distance) to initiate, after detection, a signal transduction pathway leading to a measurable cellular\nphotoresponse. Duchene et al measure that phytochrome-dependent gene expression changes are induced\nby intensities of 1µmol photon/m2/s, or even lower (0.1 µmol/m2/s) under blue light. From Liu et al.\n(2021) (see their figures 5 and 6) we also know that’s the case for endogenously emitted chlorophyll\nautofluorescence with emission intensities probed on populations densities similar to those explored in our\nwork. Although we cannot yet pinpoint the actual value of that threshold for the triggering of cell\noscillations, we can at the very least provide an upper bound based on those previous observations.\nhow much do they approximately emit? Measured fluorescence emission at the used cell concentration is\nshown in Fig. 3A and correspond to an intensity about 10^(-4) that used for excitation. This is also\nconsistent with previous measurements By Liu et al. (2021).\nhow close they would need to be to each other in order to be able to receive the FR from one cell to\nanother?\nAs previously mentioned, light perception occurs at the single-photon level and does not depend on the\ndistance to the source. What may depend on the distance between cells is whether the perceived signal is\nsufficient to elicit a photoresponse. We estimate a maximum effective separation of approximately 100\nµm, typical of dense suspensions. Given a typical fluorescence yield of 1%, the fluorescence emitted by a\nsingle neighboring cell would contribute approximately 0.0002–0.00007 µmol m⁻² s⁻¹. However, this is a\ncollective process, and a cell receives fluorescent light from a surrounding constellation of neighboring\ncells, as illustrated in the schematic below (adapted from Liu et al.). When this collective effect is taken\ninto account, the light intensity at a distance of 100 µm can reach values on the order of 0.1 µmol m⁻² s⁻¹.\nFig. Schematic illustration of the mathematical model for estimating the chlorophyll fluorescence photon\nflux density (CFPFD) received per cell. (a) Two adjacent cells. (b) Two cells with cellular distance at 10\nμm. (c) Two cells with cellular distance at 100 μm. Schematic illustration of the mathematical model for\nestimating the chlorophyll fluorescence photon flux (CFPF) during diatom blooms in oceans. (a) Evenly\ndistributed cells in real water. The cell in the center can receive chlorophyll fluorescence from the whole\nspace. (b) Percentage of CFPF versus upper limit of the integral.\nHow likely is it that it serves as an (inter)cellular signal?\nIn the manuscript, we do not taxatively state that fluorescence serves as “the” intercellular signal, but\nrather propose it is a plausible signal. We consider that the cumulative evidence provided by the\nexperimental results shown here, together with previously published results, supports this statement:\ni) Photoresponse spectra partially overlap with that of natural autofluorescence.\nii) For RL, there is a strong photoresponse for time-periodic excitation with periods close to those of the\nnatural cell wobbling.\niii) Experiments like those performed by Liu et al. (2021) demonstrate the cells' capability to respond to\nfluorescence emissions by neighbouring cells.\nUnfortunately, direct evidence is not currently available as it would require experiments where cell\nfluorescence emission is blocked while perception is maintained, which remains technically challenging in\na cell population.\nL 170: Why not repeat the experiment on a low cell density culture or on a single cell level?\nThe sentence could be interpreted to refer to a denso-dependent signal, which is not the case. Performing\nexperiments at different cell densities wouldn’t solve the technical challenge previously referred to, as\nemission and perception aren’t easily decoupled. Moreover, all the potential coupling factors\n(hydrodynamic coupling, chemical signaling, and auto-fluorescence signaling) would depend on the\naverage distance between cells (i.e., density); such an experiment would therefore not help discriminate\nbetween the different coupling factors. We have modified the sentence to avoid misinterpretations. It now\nreads: “Exogenous light and chlorophyll-emitted light signals cannot easily be decoupled.”\nL 189ff. Maybe I simply didn’t understand the last experiment, but I have trouble to follow the\nconclusions. While not pulsed red light seems to inhibit, pulsed red lights seems to trigger it? This is\ncorrectly understood. The experiments indeed show that pulsed red light (RL) at frequencies comparable\nto natural wobbling (periods ~100 s) elicits a strong photoresponse. In contrast, cells do not exhibit a\nphotoresponse under continuous RL exposure. This observation supports the hypothesis that cell\nautofluorescence may act as an intercellular signal. It is also consistent with previous findings on cell\nwobbling under non-stationary illumination (see Font-Muñoz et al., Sci. Adv., 2021). The mechanisms\nunderlying the responses to continuous and pulsed light may involve distinct pathways, as we now\nemphasize in the main text.\nMinor comments:\n• Fig 1. In to legend title: ‘continuous light experiment’ is not accurate as it can refer to either wavelength\nof exposure time. Please rephrase it, e.g. continuous exposure to various wavelengths. Done.\no 1.A and S1 and all following figures. Nice idea to represent the wavelength in the corresponding colors.\nHowever, choosing black for FR isn’t very intuitive. May you want to change it to a dark red, eg. As in\npanel 1 C? Done.\no 1A. what are the dotted and what the solid line stand for? WT and KO? Or is the solid line a fit of the\nscatterplot? The latter.\nIn the latter case:\nthe fit for 430 and 530 doesn’t seem to respect the real shape of the data points. While 430 shows another peak (even though\nlower) around 350 s 530nm peaks at 150s and not at 200 as the fitted line suggests. While it doesn’t contradict the finding\nthat 430 and 780nm result in the highest wobbling response, 530nm also shows a clear interaction. The sentence in L 119 “minimal response elsewhere” thus is not correct and needs to be rephrased accordingly.\nWhile we agree with the reviewer that there are indeed multiple peaks in all cases, we have used a\nstandard and robust criteria to discriminate and characterize the main component of each power spectrum.\nThe criteria is based on conventional signal processing tools designed to locate peaks and arrange them by\ntheir relative prominence (Pro) in the signal. For signals with a variance of the relative peak prominence\nabove one, std(Pro)>1 (i.e. when a main peak can clearly be isolated), we stick to a Gaussian fit to the\nmain peak. For std(Pro)<1 (i.e. when no peak stands out in a statistically significant way above the others)\na single Gaussian is fitted across the full spectrum. Although a refined analysis of the secondary peaks\nmight be of eventual interest, we do not consider that it has an impact on the results presented in this\nmanuscript, so we defer this to future work. This is now better explained in the Materials and Methods\nsection of the Supplementary Material where we also include the values of “peak prominence” for each\ncase.\no 1B Legend text and figure could be better connected to each other. While the graphs show delta Abs the\nlegend text talks about DPH de-/activation and uses a color-code, which are not defined Green= activated?\nBlue deactivated? Furthermore, the abbreviations ‘Pfr and Pr’ are nowhere defined in the MS. We have\nnow modified the figure caption to correct for all these discrepancies.\nAre those data of WT or KO? WT\no 1C what does Pk/FWHM stand for? Unit? (please define) Pk/FWHM stand for peak amplitude (Pk) of\nthe power spectrum (shown in fig 1A) normalized by its full width at half maximum (FWHM). These are\ngiven in arbitrary units. To help the readers we now showcase an example of both variables for one of the\npower spectra in panel A.\n• Fig2.\no Legend title: ’continuous’ blue and red light. The word doesn’t make sense like that. Do you mean\ncontinuously changing ratio? The referee is right as this, once again, refers to “continuous exposure”. We\nhave modified the text to reflect that.\no Panel A&B, highest y axis value cut off. That is true and it has been corrected in the resubmitted version\nof the figure.\n• Fig 4.\no Panel D. please define the abbreviation within the graph in the figure legend. E.g. What does 5D1 for?\nThese abbreviations correspond to the different strains used in our experiments. We have now clarified\nthis in the caption.\n• References:\no Number 26. Correct title from pennates to pennate. Done.\n• L446: symbol for micro is not shown. Done.\no The Materials and methods partly “copy paste from: 1st author previous article: Pelagic diatoms\ncommunicate through synchronized beacon natural fluorescence signaling (DOI: 10.1126/sciadv.abj5230)\nWe have now shorten the section and refer the readers to Font-Muñoz et al. Sci. Adv. (2021).\no The used methodology is new to me and seems to be quite sophisticated.\no The used unit Einstein to express the light intensity is not accepted as an SI unit and outdated, please\nsubstitute with µmol quanta (or photons) m-2 s-1. Done.\no The article would benefit from a conceptual diagram to help understand the set-up.\nADD A modified version in supp here again to make the reviewer HAPPY and simplify the reading!!\nThe experimental set-up used in this work was developed by our group a few years ago and was already\nused in previous work. We refer the interested readers to Fig. S1 in Font-Muñoz et al. Sci. Adv. (2021).\nDifferences rely on the use of different excitation spectral bands and phytoplankton strains. This is now\ncommented in the Materials and Methods section of the Supplementary Materials.\no I miss some further details:\nFor how long were the cells exposed to the different wavelength during the different experiments? Cells\nwere exposed to light for 2 hours. This information is now available in the Materials and Methods section\nof the Supplementary Materials.\nL110: How have the authors dealt with the other morphotypes of Pheodactylum as the triradiate form?\nOur laboratory cultures are maintained under conditions that exclusively promote the fusiform\nmorphotype. Fusiform cells are typically obtained by culturing under white light and maintaining the\ncultures in the exponential growth phase.\nL113: please define the “two size bands” Done.\nL454: ‘This intensity was then modulated sinusoidally with periods of cell wobbling natural frequency\n(~140 s)’. It is not explained how the authors did that. We coupled the controller triggering the excitation\nLED source to a pulse generator. This is now described in the Materials and Methods section of the\nSupplementary Materials.\nReviewer #2 (Remarks to the Author):\n(review split between senior co-reviewer comments and Early-career co-reviewer comments)\n• Reviewer #2a (early career co-reviewer):\nGeneral comments:\nThis is a highly interesting and well-structured study. The article is clear and concise, making complex\nconcepts accessible while maintaining scientific rigor. One particularly intriguing question raised is\nwhether DPH mediates cellular responses only to exogenous ambient light or if it is also involved in the\nperception of autofluorescence. This is a fascinating hypothesis, and the approach used to test it, as well as\nthe results obtained, is particularly interesting. The experimental setup is straightforward and leads to clear\nresults regarding the involvement of phytochromes in the wobbling dance in response to specific\nwavelengths. However, as explicitly mentioned in the article, further investigation and additional evidence\nare required to confirm and fully understand the role of intracellular communication. The Materials and\nMethods section is well described, ensuring good reproducibility. However, more transparency regarding\ndata processing would be beneficial (see individual comments for details).\nThree key findings stand out and are clearly highlighted:\n- The implication of phytochromes in the synchronization of cell movements.\n- The response to red, far-red, and blue light, indicating a qualitative aspect of behavioral signaling.\n- The response of phytochromes to autofluorescence signals from diatoms, potentially suggesting\nintercellular communication.\nOverall, this is an innovative and valuable study that contributes to a better understanding of how diatoms\nuse different wavelengths and how they might communicate through light signaling. It opens exciting\navenues for future research on photoreception and cellular interactions in microalgae.\nWe thank the reviewer for the general remarks on the manuscript and address the specific comments\nbelow.\nSpecific comments:\nNeed for standardization of irradiance units (PPFD):\nIn the supplementary materials and methods, there is a typo in the \"cell culture\" paragraph: \"and 80 µm-2\ns-1\". Additionally, for irradiance values, it would be preferable to use the unit \"µmol photons·m⁻²·s⁻¹\",\nwhich is more commonly used in photophysiology, rather than \"µE·m⁻²·s⁻¹ ». Done.\nThe use of the unit \"1mW\" (appearing multiple times, e.g., in Fig. 3, Fig. 2B, and the supplementary\n\"Fluorescence signal variations measurements\") is uncommon, as it refers to power rather than irradiance?\nWe agree with the referee and have now transformed all units to irradiance.\nIn Figure S1B, why are the ratios not displayed for all wavelengths, as they are in Figure S1A?\nExperiments with dph knockouts were only performed for the three wavelengths for which a\nphotoresponse in the WT was observed.\nI am not entirely sure how Figures 1 and S1 correspond. How are the data transformed to go from Ratios\nto Power? The power spectra shown in fig. 1A are estimated using fast Fourier transform (FFT) of the\ntime series of the Ratios shown in fig. S1\nThe period of cell wobbling natural frequency is given as ~140 s. How was this value determined? Was it\nmeasured experimentally on your cells, or is it taken from the literature? The wobbling natural frequency\nwas estimated from our experiments by locating the position of the main peak in the power spectra\npresented in Fig. 3B (also now indicated for the power spectra shown in fig 1A)\nIn the main text, Figure S3 is not well integrated and could benefit from additional context. For example:\n\"Starting with the intensity of B light used in Figure 1, we simulated sinking in the water column by\nincreasing R light (Fig. S3) and observed inhibition of the synchronized wobbling amplitude (Fig. 2),\nwhile wobbling frequency remained unaffected (Fig. 2A). » Done.\nProvide more transparency on data usage and processing, particularly regarding the number of individuals\nobserved/included for Figures 2A and 4C. The bars in figure 2A show the mean plus/minus standard\ndeviation of all individual experiments (n=4) performed with varying irradiance (shown in panel B).\nThose in figure 4D also show the mean plus/minus standard deviation of all performed experiments with\neither responsive (WT and Tc) or knockout strains, respectively.\nTypo in \"autofluoresce\" in the Results and Discussion section. Corrected.\n• Reviewer #2b (senior reviewer):\nThe study by Front-Munoz et al. investigates the role of phytochromes in marine diatoms. It is particularly\nwell-conducted, with high-quality and inspiring results. The study clearly demonstrates the role of diatom\nphytochromes (DPH) in detecting light signals and the behavioral adaptations resulting from this detection\nin diatoms. The DPH gene appears to be central to the synchronized response of diatoms to light signals.\nThis is, therefore, an excellent study on the perception of light quantity and quality in Phaeodactylum\ntricornutum and the triggering effect of DPH at the community level. That being said, I have some\ncomments on certain aspects of the study that seem unclear to me, as well as others where I believe the\nresults may have been over-interpreted. These points are detailed in the specific comments below.\nWe thank the reviewer for the general remarks on the manuscript and address the specific comments\nbelow.\nIn general, I find that:\nThe results are not always well explained in the methodology, making it difficult to read and interpret\ncertain graphs. In addition, the study claims to demonstrate that phytochromes are involved in an\nintercellular social behavioral response, which would indeed be a novel finding. However, on this second\npoint, I remain skeptical after reading the manuscript.\nEven though the results are exciting and may seem convincing—particularly Figure 4, which shows diatom responses to pulsed\nlight, I believe there is a lack of direct evidence that diatom autofluorescence indeed serves as a communication signal\nbetween cells, driving the observed behaviors.\nIn the manuscript, we do not taxatively state that fluorescence serves as “the” intercellular signal, but\nrather propose it is a plausible signal. We consider that the cumulative evidence provided by the\nexperimental results shown here, together with previously published results, supports this statement:\ni) Photoresponse spectra partially overlap with that of natural autofluorescence.\nii) For RL, there is a strong photoresponse for time-periodic excitation with periods close to those of the\nnatural cell wobbling.\niii) Experiments like those performed by Liu et al. (2021) demonstrate the cells' capability to respond to\nfluorescence emissions by neighbouring cells.\nUnfortunately, direct evidence is not currently available as it would require experiments where cell\nfluorescence emission is blocked while perception is maintained, which remains technically challenging in\na cell population.\nThe authors themselves appear to be cautious in their manuscript when establishing this direct link (e.g.\nL38: potentially mediated, L104: the possibility of endogenous light signal, L150: a plausible explanation,\nL183: potentially implying, ...). Thus, while the content of the article is not in question, the title could\nperhaps better reflect its findings.\nWe respectfully disagree with the referee on this point. The current title does not suggest the direct link\nbetween cell autofluorescence and collective behaviour. Instead, it focuses on the role of DPH in\ncollective responses, which we believe it is well established by our findings.\nSpecific comments:\nL57: It would be nice to define in the introduction the wavelength of the R and FR region. Done. We have\nnow included it in the second paragraph of the introduction.\nL110-119 (+figure1): The principle of laser diffractometry is well explained (L110-116), but its\nconnection to Figure 1 is unclear to me. I may have missed something, but unfortunately, the Methods &\nMaterials section in the Supplementary Material does not provide additional clarification. We have modified\nthe Methods section now to include further details on our estimates of the power spectra of the Ratio signals.\nAdditionally, please clarify what Pk/FWHM represents in Figure 1C. Done. We have modified fig 1A and\nthe figure caption to indicate what Pk (peak height) and FWHM (full width at half maximum) stand for.\nIt would also be helpful to specify in the Figure 1 caption the differences between the solid and\ndashed lines—are they meant to represent WT vs. KO? There are no dashed lines in fig1. We guess the referee is\nthinking about the dotted lines in panel A, which correspond to the actual data points; the solid lines\ninstead are gaussian fits to those points, as indicated now in the modified caption.\nLines 136-139: The inclusion of the vertical distribution of the Red:Blue light ratio in the sea (figS3) is a\nstrong point, making the extrapolation of the results to natural environments very convincing. We thank\nthe reviewer for this appreciation of the relevance of our findings to natural environments.\nLines 147-154: In this section, the authors analyze the synchronized response of diatoms to light signals. It\nwould be helpful to clarify at this stage whether diatom autofluorescence is produced in the R or FR\nregion (or both) when excited in the B, as this information is only provided later (L178, Figure 3).\nIncluding it earlier could better support the authors' hypothesis. As suggested by the reviewer, we now\nmention in this paragraph the wavelength bands of the emitted autofluorescence.\nThat being said, in this section, the authors dismiss the possibility of coordinated behavior driven by info-\nchemicals to introduce the autofluorescence hypothesis. While the autofluorescence hypothesis is\nintriguing, at this stage of the manuscript, it is sufficient to establish that light acts as a trigger—via\nDPH—to induce a behavioral response at the community level. Since the hypothesis of autofluorescence\nas an intercellular signal is introduced later with the pulsed light experiments (L189, Figure 4), it may be\npremature to bring it up as early as L150.\nThe focus of our manuscript is on the relevance of DPH in triggering collective behavior. Our experiments\nfirmly demonstrate this role. We further speculate that fluorescence light emission and unknown\ncomponent downstream of DPH signalling could be involved in the measured photoresponse. That said,\nwe want to stress we do not intend to discard either physical disturbances or dissolved infochemicals as\npotential cell-to-cell coupling signals, but rather hypothesize fluorescence could be an additional\ninteraction mechanism compatible with all current observations. As already mentioned above, we cannot\ndirectly test this hypothesis, but we can provide additional evidence (including some new experimental\nresults; e.g. figure 4) that supports it. Based on this argument, we believe the reader would better\ndiscriminate between speculative discussion and proven facts with the current logical structure.\nL154-156: intensity of fluorescence in the sea => This is very interesting and brings the question of the\ndistance at which the autofluorescence of the Chlorophylle a could be detected by other cells in nature.\nLight perception occurs at the single-photon level and does not depend on the distance to the source. What\nmay depend on the distance between cells is whether the perceived signal is sufficient to elicit a\nphotoresponse. We estimate a maximum effective separation of approximately 100 µm, typical of dense\nsuspensions. Given a typical fluorescence yield of 1%, the fluorescence emitted by a single neighboring\ncell would contribute approximately 0.0002–0.00007 µmol m⁻² s⁻¹. However, this is a collective process,\nand a cell receives fluorescent light from a surrounding constellation of neighboring\ncells, as illustrated in the schematic below (adapted from Liu et al.). When this collective effect is taken\ninto account, the light intensity at a distance of 100 µm can reach values on the order of 0.1 µmol m⁻² s⁻¹.\nFig. Schematic illustration of the mathematical model for estimating the chlorophyll fluorescence photon\nflux density (CFPFD) received per cell. (a) Two adjacent cells. (b) Two cells with cellular distance at 10\nμm. (c) Two cells with cellular distance at 100 μm. Schematic illustration of the mathematical model for\nestimating the chlorophyll fluorescence photon flux (CFPF) during diatom blooms in oceans. (a) Evenly\ndistributed cells in real water. The cell in the center can receive chlorophyll fluorescence from the whole\nspace. (b) Percentage of CFPF versus upper limit of the integral.\nIs Phaeodactylum tricornutum widely distributed in several different ecosystems, or rather restricted to\nclear, oligotrophic or non turbid waters? P. tricornutum, is a marine pennate diatom commonly found in\ncoastal waters, including highly productive areas like estuaries, rock pools, and shallow waters exposing\nthe species to important fluctuations in light intensity and salinity.\nL158-162 & Fig3 : The manuscript states that the measured chlorophyll fluorescence includes both R and\nFR components. However, in Figure 3, it is mentioned that the Red fluorescence corresponds to a long-\npass filter that collects light >645 nm. This implies that the R signal actually represents R + FR, while the\nFR signal refers only to FR wavelengths (> 715nm). If this is correct, it makes sense that the emitted\nR+FR and FR signals produce similar results, as the R+FR signal would be largely dominated by FR\nfluorescence.\nWe agree with the reviewer that what we show in figure 3A is either the FR or R+FR components of the\nemitted signal. However, while the frequency of the measured oscillations is similar, note that the\namplitudes are not (see left and right vertical axis range). In particular, the R+FR signal (i.e. >645nm)\ndoubles in amplitude the FR signal (>715nm), clearly demonstrating the coherent contribution of the R\nsignal to the overall emission.\nL180-181: The wobbling motility of diatoms is described as \"incoherent\" in dph-KO cells, but this point\nneeds further clarification. When comparing the power spectrum curves in Figure 4A&B (pulsed light)\nwith Figure 1A (continuous light), I don’t see a significant difference. What are the main distinctions?\nWhile in figure 1A each power spectrum corresponds to a distinct excitation wavelength, all curves shown\nin fig 4A (or correspondingly in 4B) represent the power spectrum for a single excitation wavelength (FR\nor R, respectively) but different strains (WT, KOs and Tc). Although the power spectra for WT and Tc\nstrains are indeed similar to that of Figure 1 for FR light, it is not the case for R light: the WT line shows a\nclear peak at ~140s in pulse R but not in continuous red light (Fig 1). The power spectrum of the WT in\ncontinuous R (Fig 1, dashed red line) rather resembles that of DPH KO mutants under pulse R light\n(dashed lines in Fig. 4B).\nWhat exactly is considered \"incoherent\" here? A lack of a clear peak in the power spectrum of the\noscillatory signal. That’s the case for certain excitation wavelength for the WT strain under continuous\nexposure (fig 1A) but also for the knock-outs under pulsed light excitation in the R and FR (fig 4A & B).\nThe authors then emphasize that DPH is essential for triggering collective behavior (which the study\nclearly demonstrates). However, they also reintroduce the hypothesis of autofluorescence as an\nintercellular signal. This needs a more detailed explanation because it is not immediately clear how Figure\n4A&B supports the autofluorescence hypothesis.\nThe experiments performed under pulsed excitation light, the results of which are presented in fig 4, are\nintended to mimic the response of a population to an oscillatory fluorescence signal at the natural\ncharacteristic frequency of cell wobbling. As the measured R and FR autofluorescence emissions oscillate\nwith a well-defined frequency (see fig 3), we interrogated cells for their photoresponse to exogenous light\nwith the same characteristics. The results presented in Figure 4 show that a clear photoresponse exists in\nthis case, supporting the hypothesis that fluorescence emission can trigger collective behavior mediated\neither directly by DPH or indirectly through an unknown component downstream of DPH signalling. We\nhave now expanded the paragraph to explain more thoroughly the connection between these experiments\nand this hypothesis.\nL189-197: From my perspective, this paragraph is the only one that supports the autofluorescence\nhypothesis. It is indeed intriguing that R light does not induce movement when provided continuously\n(Fig1C) but does when introduced at a period matching the wobbling frequency (Fig4C). This serves as an\nargument in favor of the autofluorescence hypothesis, even\nthough the direct relationship between autofluorescence and movement is not explicitly demonstrated in this case. Thus, I\nsuggest presenting this result earlier in the manuscript.\nWe agree with the reviewer but decided to remain cautious about the mechanisms involved in the\ncommunication process. While we strongly believe there are several arguments pointing to the relevance\nof cell autofluorescence in this process, direct proof is still lacking. This by no means belittles the direct\nproof we do provide on the relevance of DPH for collective behaviour. Hence, we would prefer to keep\nthis as the main focus of our manuscript while keeping the light-mediated interaction as a working\nhypothesis.\nL221: \"strong evidence of the relevance of light signaling (remove communication)\" Done.\nM&M: Continuous light experiments were conducted by exposing the cells to different wavelengths for 2\nhours. Meanwhile, laser diffractometry measurements—central to establishing time series ratios and\npower spectra—were performed using a LISST-100X, which operates at 670 nm (R light). Looking at\nFigures S1 and 2, it appears that the measurement duration exceeds 3000 seconds (i.e., more than 50\nminutes). This raises the question of how the LISST-100X, which exposes the cells to red light for a\nrelatively long period, aligns with the experimental treatments that are supposed to provide only blue\nand FR light. Could you clarify how this potential interference is accounted for?\nThe laser from the LISST-100X diffractometer operates by pulsing short measuring pulses synchronized\nwith the sensors’ electronics. These pulses, although emitting in the R as correctly pointed out by the\nreviewer, are of extremely short duration compared to natural wobbling periods (ms vs. ~100s). We have\nmeasured no photoresponse from cells exposed only to this measuring light. Moreover, even in the\nunlikely event that there might be a weak response below our sensitivity, this induced photoresponse\nwould be present as a background signal for all our experiments and would not be relevant for the\nmeasured relative responses and the extracted conclusions."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Reviewer #2a (early career co-reviewer):\nMy comments have been considered, and the revised version of the manuscript generally addresses the critiques raised\nduring the first round, which improves the clarity, methodological rigor, and overall structure of the manuscript. However,\nsome imprecisions remain.\nGeneral comments:\nThere are numerous typographical errors throughout the document, particularly missing spaces between numerical values\nand their units. While spaces are sometimes used correctly, they are frequently omitted, and the manuscript\nrequires consistent homogenization.\nThere is a lack of transparency regarding the number of biological replicates used to compute means and standard\ndeviations throughout the manuscript. This information is not provided in the supplementary Materials and Methods section\nnor in the figure legends. The authors should clearly the number of replicates (n = ?) used for the following figures: Figure\n1C, 2A, 4C, 4D, S2A, S2B, S5A, and S5B.\nSpecific comments:\nL113: The detailed technical description of the two size bands (7.33–14 μm and 2.5–3.5 μm) used in the laser diffractometry\n(LD) measurement seems superfluous for the Results and Discussion section, and the mention of “two size bands” is\nsomewhat redundant with the following sentence L116. Wouldn’t it be more appropriate to move this explanation to the\nsupplementary Materials and Methods section on laser diffractometry? Doing so would help create a more concise and\nfocused paragraph?\nFigure 1A: I still have some uncertainties regarding the application of a Gaussian fit to the full spectrum when no single peak\nis clearly dominant (std(Pro) < 1). Is this approach truly justified? What is its relevance and robustness?\nMoreover, is there a statistical or physiological justification for using the std(Pro) > 1 threshold as a criterion for this data?\nFinally, it is good that arrows for both FWHM and Pk were added to the graph to improve readability. However, the arrow for\nPk is not visually optimal, as it appears somewhat floating within the graph. Although it is mentioned in the legend, it would\nbe better to find a clearer visual solution to link the \"Pk\" label to its arrow, similar to the \"FWHM\" arrow.\nTypo:\nL173: « autofluoresce » instead autofluorescence\nL224 and 229: « signalling » instead signaling\nL429: « cells/L » unit instead cells mL⁻¹\nL511: « impa » instead impact\nFigure 4A: add space between period and (s) on x axis\n\nReviewer #2b (senior co-reviewer):\nAfter thoroughly reviewing the revised manuscript, I can confirm that all of my previous comments and suggestions have\nbeen satisfactorily addressed by the authors. I also fully agree with the observations and recommendations made by my co-\nreviewer.\nRegarding Reviewer #1’s comments on the experimental design—specifically the impact of the FR and R ratios, which were\npreviously identified as a weak point in the study—I find that the authors have now addressed this concern convincingly.\nThey provided new data demonstrating the effects of varying FR-to-R ratios, and the results show a behavior similar to that\nobserved with the B/R ratio. This addition strengthens the overall validity of the experimental design.\nAt this stage, I have no further comments or suggestions concerning the manuscript.\nHowever, I remain curious about 2 points :\n1- In the continuous light experiment (Fig. 1A), the power spectrum of the ratio still shows a weaker, yet seemingly\nsignificant, signal in the green, orange, and red wavelength ranges, with a longer period (in seconds). To me, this suggests\nthat the “wobbling” becomes weaker and occurs at a higher frequency as one moves from the blue toward the red region of\nthe light spectrum (with the exception of FR of course). I am wondering whether the signals recorded between approximately\n200 and 400 seconds (depending on the wavelength) correspond to different wobbling frequencies, or whether they might\ninstead result from the Brownian motion of the cells. In the latter case, would one expect such an effect to vary with\nwavelength?\n2- In line with this question, regarding Figure 4 (“Pulsed light experiments”): does the fact that the authors selected a pulsing\nfrequency of approximately 150 s—which corresponds to the main signal observed under continuous B and FR light—\nintroduce a potential element of circular reasoning? I am inclined to think not, since Figure 4A indeed shows a power\nspectrum peak around 150 s under pulsed R light. However, I would appreciate confirmation of this interpretation, or at least\nclarification on how to interpret the weaker signals observed at higher frequencies.\nOverall, this study is excellent and highly stimulating, although at times somewhat challenging to follow—even though the\nauthors have made substantial improvements compared with the previous version. The methodology is very specific and\ncomplex, and the constraints of the manuscript’s length make it difficult to provide a fully comprehensive\ndescription without referring the reader to earlier publications where the methods are explained in greater detail. While this\napproach is understandable, it does make the reader’s (and reviewer’s) task more demanding, as one must frequently cross-reference\nmultiple papers to fully grasp the experimental procedures—sometimes leaving the impression that one must take certain\nmethodological aspects on trust rather than direct verification.\nThat said, at this stage, I do not consider it necessary for the authors to include additional methodological details in the\ncurrent version of the manuscript. Perhaps, I could invite them to pay extra attention with the notation throughout the\nmanuscript. For instance, Peak amplitude of the power spectrum is noted \"Pk\" in figure 1A and P(omega) in fig4C,D.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "RESPONSE TO REVIEWERS’ COMMENTS\nReviewer #2 (Remarks to the Author): (review split between senior co-reviewer\ncomments and Early-career co-reviewer comments)\nReviewer #2a (early career co-reviewer):\nMy comments have been considered, and the revised version of the manuscript\ngenerally addresses the critiques raised during the first round, which improves\nthe clarity, methodological rigor, and overall structure of the manuscript.\nHowever, some imprecisions remain.\nGeneral comments:\nThere are numerous typographical errors throughout the document, particularly\nmissing spaces between numerical values and their units. While spaces are\nsometimes used correctly, they are frequently omitted, and the manuscript\nrequires consistent homogenization.\nWe thank the reviewer for the positive feedback and constructive remarks. We\nhave carefully revised the manuscript to correct all typographical\ninconsistencies, including missing spaces between numerical values and units,\nwhich have now been homogenized throughout.\nThere is a lack of transparency regarding the number of biological replicates\nused to compute means and standard deviations throughout the manuscript.\nThis information is not provided in the supplementary Materials and Methods\nsection nor in the figure legends. The authors should clearly the number of\nreplicates (n = ?) used for the following figures: Figure 1C, 2A, 4C, 4D, S2A,\nS2B, S5A, and S5B.\nWe thank the reviewer for noting this omission. The number of replicates is\nnow indicated in all relevant figure legends and in the Supplementary Materials\nand Methods section.\nSpecific comments:\nL113: The detailed technical description of the two size bands (7.33–14 μm and\n2.5–3.5 μm) used in the laser diffractometry (LD) measurement seems\nsuperfluous for the Results and Discussion section, and the mention of “two\nsize bands” is somewhat redundant with the following sentence L116. Wouldn’t\nit be more appropriate to move this explanation to the supplementary Materials\nand Methods section on laser diffractometry? Doing so would help create a\nmore concise and focused paragraph?\nWe thank the reviewer for this suggestion. The detailed description of the two\nsize bands has been removed from the Results and Discussion and moved to the\nMaterials and Methods section to improve clarity and avoid redundancy.\nFigure 1A: I still have some uncertainties regarding the application of a\nGaussian fit to the full spectrum when no single peak is clearly dominant\n(std(Pro) < 1). Is this approach truly justified? What is its relevance and\nrobustness?\nWe thank the reviewer for this constructive and insightful comment. The\ncriterion based on the standard deviation of peak prominence (std(Pro) > 1) was\nselected as a simple, quantitative, and statistically robust approach to determine\nwhether a single oscillation mode dominates the power spectrum.\nPeak prominence (Pro) quantifies the relative contrast of each spectral peak\nwith respect to its neighboring minima, enabling reliable identification of\ndominant, isolated modes. In our dataset, cases where std(Pro) > 1 consistently\ncorrespond to spectra with a clearly dominant peak, whereas std(Pro) < 1\nindicates that multiple peaks contribute comparably to the overall signal. In the\nlatter case, a Gaussian fit over the full spectrum is therefore appropriate to\ncapture its broadband character.\nThis approach is further supported by an independent, complementary\ncriterion—identifying the dominant peak as an outlier relative to the\ndistribution of all peak prominences (P_max > mean(Pro) + 2*std(Pro)). Both\ncriteria yielded identical classifications across all spectra, robustly\ndistinguishing single-peak from multi-peak or flatter spectral profiles.\nAccordingly, applying a Gaussian fit when std(Pro) < 1 is statistically justified\nand accurately reflects the underlying signal structure, as further confirmed by\nagreement with the outlier-based metric. Importantly, as shown in Figures R1\nand R2, the overall results remain consistent regardless of whether this criterion\nis applied. Even when spectra are fitted uniformly with either a single Gaussian\nor multiple Gaussians, a clear separation persists between responses under blue\nand far-red illumination (which show strong signals) and those at other\nwavelengths (where the responses are markedly weaker or absent).\nFigure R1. A) Power spectra and their corresponding Gaussian fits obtained by fitting each spectrum with\na single Gaussian. B) Ratio between the height of the Gaussian and its FWHM (full width at half maximum).\nFigure R2 A) Power spectra and their corresponding Gaussian fits obtained by fitting each spectrum with\nmultiple Gaussians; the highest Gaussian is highlighted in the figure. B) Ratio between the height of the\nhighest Gaussian in each fit and its FWHM (full width at half maximum).\nMoreover, is there a statistical or physiological justification for using the\nstd(Pro) > 1 threshold as a criterion for this data?\nWe thank the reviewer for this point. The threshold std(Pro) > 1 is data-driven\nand was validated against an independent outlier-based criterion (P_max >\nmean(Pro) + 2*std(Pro)), which produced identical classifications across all\nspectra.\nFinally, it is good that arrows for both FWHM and Pk were added to the graph\nto improve readability. However, the arrow for Pk is not visually optimal, as it\nappears somewhat floating within the graph. Although it is mentioned in the\nlegend, it would be better to find a clearer visual solution to link the \"Pk\" label\nto its arrow, similar to the \"FWHM\" arrow.\nWe thank the reviewer for this helpful remark. We have adjusted the position\nof the Pk arrow and its label to improve visual clarity and consistency with the\nFWHM indication.\nTypo:\nL173: « autofluoresce » instead autofluorescence\nL224 and 229: « signalling » instead signaling\nL429: « cells/L » unit instead cells mL⁻¹\nL511: « impa » instead impact\nFigure 4A: add space between period and (s) on x axis\nWe thank the reviewer for pointing out these typographical and formatting\nissues. All have been corrected in the revised manuscript\nReviewer #2b (senior co-reviewer):\nAfter thoroughly reviewing the revised manuscript, I can confirm that all of my\nprevious comments and suggestions have been satisfactorily addressed by the\nauthors. I also fully agree with the observations and recommendations made by\nmy co-reviewer.\nRegarding Reviewer #1’s comments on the experimental design—specifically\nthe impact of the FR and R ratios, which were previously identified as a weak\npoint in the study—I find that the authors have now addressed this concern\nconvincingly. They provided new data demonstrating the effects of varying FR-\nto-R ratios, and the results show a behavior similar to that observed with the\nB/R ratio. This addition strengthens the overall validity of the experimental\ndesign.\nWe thank the reviewer for their careful evaluation and positive feedback. We\nare pleased that the additional data on FR-to-R ratios satisfactorily address\nprevious concerns and further support the study.\nAt this stage, I have no further comments or suggestions concerning the\nmanuscript.\nHowever, I remain curious about 2 points:\n1- In the continuous light experiment (Fig. 1A), the power spectrum of the ratio\nstill shows a weaker, yet seemingly significant, signal in the green, orange, and\nred wavelength ranges, with a longer period (in seconds). To me, this suggests\nthat the “wobbling” becomes weaker and occurs at a higher frequency as one\nmoves from the blue toward the red region of the light spectrum (with the\nexception of FR of course). I am wondering whether the signals recorded\nbetween approximately 200 and 400 seconds (depending on the wavelength)\ncorrespond to different wobbling frequencies, or whether they might instead\nresult from the Brownian motion of the cells. In the latter case, would one\nexpect such an effect to vary with wavelength?\nWe thank the reviewer for this insightful observation. In the green, orange, and\nred experiments, the power spectra exhibit multiple peaks of comparable\namplitude, indicating that no single oscillation frequency clearly dominates\nunder these conditions. By contrast, the spectra at 430 nm and 780 nm show a\nsingle, well-defined peak with a pronounced amplitude, suggesting that most\ncells oscillate coherently at a similar frequency under these wavelengths.\nWhile one could hypothesize that the multiple peaks arise from distinct subsets\nof cells oscillating at different frequencies, we cannot confirm this directly. It\nalso seems unlikely that these features result from Brownian motion, as random\ndisplacements would not produce reproducible, well-defined oscillatory\nsignals. Overall, the most parsimonious interpretation is that the oscillatory\nresponse is weaker and less coherent in the green, orange, and red ranges\ncompared with the blue and far-red conditions.\n2- In line with this question, regarding Figure 4 (“Pulsed light experiments”):\ndoes the fact that the authors selected a pulsing frequency of approximately 150\ns—which corresponds to the main signal observed under continuous B and FR\nlight—introduce a potential element of circular reasoning? I am inclined to\nthink not, since Figure 4A indeed shows a power spectrum peak around 150 s\nunder pulsed R light. However, I would appreciate confirmation of this\ninterpretation, or at least clarification on how to interpret the weaker signals\nobserved at higher frequencies.\nThe period of the pulsed light (in both the red and far-red experiments) was\ndetermined based on the main oscillation period observed in the collective\nfluorescence emission of a cell suspension, as shown in Figure 3B. Our aim was\nto reproduce the oscillatory signal that a cell population naturally generates\nunder continuous illumination.\nTherefore, rather than representing a case of circular reasoning, this\nexperimental design allowed us to test whether the cells could synchronize with\nan external periodic light signal and whether this behavior might be mediated\nby phytochromes. The fact that cells respond to and synchronize with this\nsignal—even beyond the natural oscillation period of the population (see, for\nexample, Figure 3 in Font-Muñoz et al., 2021)—suggests that light can act as\nan effective coupling mechanism capable of synchronizing the individual\nwobbling motions of the cells.\nOverall, this study is excellent and highly stimulating, although at times\nsomewhat challenging to follow—even though the authors have made\nsubstantial improvements compared with the previous version. The\nmethodology is very specific and complex, and the constraints of the\nmanuscript’s length make it difficult to provide a fully comprehensive\ndescription without referring the reader to earlier publications where the\nmethods are explained in greater detail. While this approach is understandable,\nit does make the reader’s (and reviewer’s) task more demanding, as one must\nfrequently cross-reference multiple papers to fully grasp the experimental\nprocedures—sometimes leaving the impression that one must take certain\nmethodological aspects on trust rather than direct verification.\nWe thank the reviewer for their encouraging assessment of our study and for\nrecognizing the improvements made in the revised manuscript. We also\nappreciate their understanding of the complexity of our methodology.\nWe acknowledge that, given the constraints of manuscript length, full\nmethodological details must be referred to in earlier publications. We hope that\nthe explanations provided here allow readers to follow the key aspects of the\nexperimental and analytical procedures. We are grateful for the reviewer’s\nthoughtful feedback and their appreciation of the stimulating nature of our\nwork.\nThat said, at this stage, I do not consider it necessary for the authors to include\nadditional methodological details in the current version of the manuscript.\nPerhaps, I could invite them to pay extra attention with the notation throughout\nthe manuscript. For instance, Peak amplitude of the power spectrum is noted\n\"Pk\" in figure 1A and P(omega) in fig4C,D.\nWe thank the reviewer for this helpful suggestion. We have carefully reviewed\nthe manuscript to ensure consistency in the notation of peak amplitude\nthroughout. Specifically, we have standardized the labeling of the power\nspectrum peaks, making it uniform across all figures. In addition, we have\nreorganized Figure S2 in the Supplementary Material to enhance clarity and\ncoherence.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate\ntraining in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, {"round": 3, "reviewer_comments": {"reviewer_2": "After thoroughly reviewing the revised manuscript, I (and the early-career researcher) can confirm that all of our previous comments and suggestions have been satisfactorily addressed by the authors."}, "author_response": ""}]

Decision letter