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  1. Evaluation Summary:

    This paper will be of interest to marine biologists, and has particular relevance to those studying symbiotic corals. The use of compelling experimental optical measurements performed in situ allows testing of previous predictions on protein-based pigments that are found in many coral species. Specifically, the study analyzes the role of two classes of pigments, the Red Fluorescent Proteins (RFPs) and the Chromo proteins. It provides direct measurement data that suggest that RFPs can indeed provide additional light to the symbionts by converting the prevalent blue-green light at depth in orange-red light that penetrates more in the tissues of the polyps, thus increasing the number of photons available for photosynthesis. The authors also provide evidence based on light measurement for a possible photoprotective role of Chromoproteins, although the study does not yet provide any direct evidence for an ecological benefit of such light conversion/light protective functions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Scleratinian corals, fundamental species in the ocean for their structuring of habitats that host a diversity of up to 30% of marine known species, prevalently rely on phosynthases of their phototrophic symbionts for their energy budget at shallow depths. Possible adaptive strategies to deal with the low light regimes of deeper layer have been recurrently studied also to assess the balance between the resources provided by the phototrophic symbionts and those coming from direct feeding on suspended preys.

    Most corals synthetize photoconvertible Red Fluorescent Proteins, whose role has not yet fully assessed but it has been prevalently ascribed to photoprotection. Recent, more refined measurements on bio-optical properties and responses on a few species have casted doubts upon previous conclusions that they are not involved in photocapture of PAR.

    The authors utilize advanced bio-optical observations to provide support to the hypothesis that photoconvertible red fluorescent proteins (pcRFPs) synthetized by scleratinian corals may optimize the photon flux towards the hosted phototrophic symbionts by converting the prevalent blue-green light of deeper layers to red-orange light that penetrates more in the polyps' tissues.

    They show that the more penetrating in the tissue yellow-orange band produced by the pcRFP after absorption of blue-green light, can account to up to 100% of the available light in that band within the tissue, even though with a photon flux in the order of, or smaller than, 0.1 µE m-2 s-1, for an external photon flux ≲ 20 µE m-2 s-1. This photon flux could convey additional photons to symbionts located deeper in the tissue which may be shaded by the symbionts closer to the periphery of the polyp thus optimizing, together with internal scattering, light distribution within the polyp.

    In parallel, they show that Chromoproteins (CP) photoprotect corals in shallow, high irradiance waters and favors the recolonization by the symbionts.

    Indeed, their measurements confirm that green-orange light deriving from pcRFPs are present in the deeper parts of polyps tissues and that can account for up to 100% of the available light in that wave band but there is no estimate on the relevance of this additional source of energy for the overall energy budget of the corals. Their results also characterize the opposite effect on the penetration of blue vs. orange red band in absence or presence of chromophoric proteins.

    The hypothesis on possible role of pcGFPs as photoconverters to supply PAR to symbionts, despite several previous studies had rejected this possibility, has already been proposed by Smith et al, 2017 (cited by the authors), who supported their inference by the mapping the fluorescence of symbionts' chlorophyll in the tissue. This could also motivate the seemingly better, adaptation of orange fluorescent corals at low light regimes.

    Also the photoprotective role of CPs has been studied and characterized, among the others, by Smith et al, 2013 (also cited by the authors).

    The methods used by the authors, and their results are robust but there are some areas for improvement in the present version, because: 1. it does not add significant insight on the effective role of pcRFP in respect to what discussed by Smith et al, 2017; 2. it does not assess how relevant is this additional source of energy for the organismal nutritional budget; 3. it does not provide any physiological/molecular information which could support the link between pcRFPs internal stock and light quantity and quality availability.

  3. Reviewer #2 (Public Review):

    In this well-written paper, Bollati et al investigate the role of two types of protein-based pigments found in coral species in modulating the spectral distribution of light throughout the coral depth: green to red photoconvertible fluorescent proteins (PCFP), and chromoproteins (CP). The functional role of such proteins in reducing ("sun cream effect") or enhancing the amount of light penetrating through corals, notably affecting the efficiency of photosynthesis by coral symbionts, has long been hypothesized. The present study now uses artificial light sources, optical filters, optical micro-sensors and a spectrometer to probe spectral light distributions and intensities in situ at variable depth throughout coral thickness and under a variety of conditions. In doing so the authors are able to quantify the modifications of light intensity distribution imparted by the protein-based pigments. Overall the results are in line with simple intuition. In the case of PCFP containing corals, it is found that in shallow waters, where the sunlight spectrum is essentially preserved, PCFPs exert no significant influence on light distribution throughout coral thickness. On the contrary, in deeper waters where only blue-green light penetrates, the PCFPs enhance the availability of orange-red light by fluorescence emission. By combining lab measurements with an available dataset of downwelling spectral irradiance in the Red Sea, the authors arrive at the spectacular conclusion that at depths beyond ~90 m, practically all of the orange red light available emanates from PCFPs fluorescence, despite the fact that the absolute amount of available light in that spectral range becomes minute.

    In the case of CP expressing corals in shallow waters, the study is less extensive, but a clear shielding effect of orange light by the upregulated CP is observed, likely to be significant in mitigating the consequences of coral bleaching.

    Overall, the work is well conducted and the paper is very instructive, at least to nonspecialists like this reviewer. This reviewer has no expertise in marine biology nor in how to handle corals, but it appears that the manuscript would deserve a more thorough description of the technological challenge of measuring light irradiance in situ with the described microsensors at well-defined depths within the investigated corals. In fact, the experimental data presented in this work appear to be relatively limited in scope, as for example no functional studies are conducted to backup the non-surprising but still speculative conclusions of the paper in terms of coral-symbionts physiology (such as e.g PCFPs found in corals thriving at mesophotic depths facilitate photosynthesis by coral symbionts found in deep coral tissue layers by shifting available blue-green light to orange-red light, able to penetrate deeper, but also less absorbed by chlorophyll-a).

    The statistical significance of the presented data may not be sufficiently discussed. For example in Figure 1a, is the lower scalar irradiance by the converted coral, as compared to the unconverted coral, significant or not? If yes, why? In figure 2a (which has no error bars) is the apparent slight reduction in the 514/ 582 nm ratio at deep layers for the most photoconverted coral (dark red line) significant or not? If yes, why? In Figure 3d and e (again no error bars), is the slight increase of green and red fluorescence emission in the deeper coral layers by unconverted PCFPs significant or not, and if yes why? In figure 4, to judge the significance of the quantitative evaluation, it would be important to show a comparison between the irradiance spectra of the Red Sea (figure 4A) with the lab-based spectra of figure S1.

    One aspect which is left unanswered is the exact balance of green versus red PCFPs in physiological conditions, which depends on the PCFPs expression turnover combined with the kinetic of green to red photoconversion by near UV light. Such data might be hard to obtain, but at least the issue could be discussed more in depth. Alongside, the notion of incomplete photoconversion, well known by biophysicists using PCFPs in advanced microscopy, would be worth discussing. Has nature designed PCFPs to only partially photoconvert, so as to maintain a right balance of green and red monomers? Further, it would be interesting to evaluate the results presented in e.g. Figure 4b, in view of the known extinction coefficients and fluorescence quantum yields of PCFPs.

    This being said, the paper does not try to oversell the recorded data and their consequences for an advanced understanding of coral physiology. Rather, based on these data, it provides a well-balanced and highly interesting discussion.

  4. Reviewer #3 (Public Review):

    The manuscript is well written and nicely presented but the Materials and Methods section is very weak. A detailed explanation on the experimental design and setup is currently missing. The authors need to be very clear on the number of specimens they measured in each analysis. Moreover, while the results of this study are relevant, they should not be generalized, as analyses were conducted, for all the investigated species, on fragments derived from the same mother colony kept in aquaria for 10 years, thus excluding the natural variability in a natural population.

    References in the Introduction are a bit outdated, the reference list should be double checked (e.g., some references are incomplete) and page numbers should be included.