Photoautotrophic Euendoliths and Their Complex Ecological Effects in Marine Bioengineered Ecosystems

Round 1

Reviewer 1 Report

This ms is a complete and exhaustive milestone review on euendolithic and their roles for marine ecosystems. It will be a milestone reference on the topic. As a non-specialist, I read this review with interest and have with difficulty managed to find a couple of suggestions of minimal value. The authors are free to consider these minor corrections or not. The review can be published as is.

L219. condition 1 and 2 are the same. So, finally how many conditions need to be fulfil?

Fig. 2. In the scheme it has been suggested that some Active transport of HCO3 are necessary to the functioning of the Calcium Pump. I would like to read if they presence have been revealed  (and for what organism/species) or it is just an hypothesis.

L537. I think the Authors misunderstood or simplified the mechanism of tissue retraction on corals. On most of them, tissue do not retract at all. It happen only in the presence of a major stress (I observed that on Med corals during thermal stress) and for some solitary corals such as the Desmophyllum studied in  ref 185. So, this sentence should be changed to specify that euendoliths can colonize exposed skeleton in some species while for some others they can access to the skeleton from the base of the recruit/colony when the tissue cover new substrate where the endoliths are, so they are covered firt by the tissue, then by the skeleton. Ref 166 exlain this mechanism.

571. While the terms azooxathellate and zooxanthellate corals have not been revised, it is more corret to refere to the zooxanthellae as Symbiodiniaceae (as revised by La Jeunesse (2018?).

Finally, Paragraph 6. It could be opportune to cite the fact that the increase in the euendolithic boring on corals will fragilize the whole reefs making them less resistant to protect coastal area, and lagoons, especially during cyclones and extreme events, with serious consequences for the ecosystems and humans!

That's all. I congratulate the authors for this nice job.

Riccardo Rodolfo-Metalpa

Author Response

Dear Dr Rodolfo-Metalpa,

Thank you so much for your review ! Here is my point-by-point response to your comments.

Point 1: L219. condition 1 and 2 are the same. So, finally how many conditions need to be fulfil?

Response 1: Indeed, I made a mistake during the formatting of the document and copy/paste Condition 2 twice. Here is the right Condition 1:

The dissolution process is thermodynamically unfavourable, as it mainly occurs in waters saturated with calcium carbonate (i.e., calcite and aragonite). Excavation then becomes an ATP-driven active process with an energetic cost;

Point 2: Fig. 2. In the scheme it has been suggested that some active transport of HCO₃^- is necessary to the functioning of the Calcium Pump. I would like to read if they presence have been revealed (and for what organism/species) or it is just a hypothesis.

Response 2: When boring into calcium carbonates, the Calcium Pump involves the excavation of Ca²⁺ from the substrate coupled with a necessary counter-transport of electrons H⁺ (red transporters). The electron released interact with the remaining carbonate CO₃^2- to form HCO₃^-. Photosynthetic organisms generally use CO₂, or under low CO₂ concentrations, HCO₃^-, which is intracellularly transformed into CO₂ (Merz-Preiβ, 2000). The uptake of HCO₃^- from the surrounding seawater by the cyanobacterial cell is part of the carbon-concentrating mechanism (CCM - dotted lines, see Reinfelder (2011)).

I haven’t done extensive research on the CCM, as I thought it was outside the scope of the review. Espie et al. (1991) found evidence of active HCO₃^- transport in other cyanobacteria and Guida (2016) mentioned the presence of bicarbonate transporter in Mastigocoleus testarum, but Tribollet et al. (2009) suggests that euendolithic Chlorophyta, such as Ostreobium quekettii, do not possess such a mechanism.

Espie, G.S.; Miller, A.G.; Kandasamy, R.A.; Canvin, D.T. Active HCO 3− Transport in Cyanobacteria. Can. J. Bot. 1991, 69, 936–944, doi:10.1139/b91-120.

Guida, B.S. Unique Cellular, Physiological, and Metabolic Adaptations to the Euendolithic Lifestyle in a Boring Cyanobacterium. Doctor of Philosophy, Arizona State University, 2016.

Merz-Preiβ, M. Calcification in Cyanobacteria. In Microbial Sediments; Riding, R.E., Awramik, S.M., Eds.; Springer: Berlin, Heidelberg, 2000; pp. 50–56 ISBN 978-3-642-08275-7.

Reinfelder, J.R. Carbon Concentrating Mechanisms in Eukaryotic Marine Phytoplankton. Annu. Rev. Mar. Sci. 2011, 3, 291–315, doi:10.1146/annurev-marine-120709-142720.

Tribollet, A.; Godinot, C.; Atkinson, M.; Langdon, C. Effects of Elevated pCO2 on Dissolution of Coral Carbonates by Microbial Euendoliths. Global Biogeochem. Cycles 2009, 23, n/a-n/a, doi:10.1029/2008GB003286.

I modified the legend of the figure as follow, to make it clearer and add some of the above references:

Figure 2. (right panel). Proposed “Calcium Pump” transport mechanism within the trichome of euendolithic cyanobacteria (both filamentous and pseudofilamentous), with the inferred distributions of transporter components (legend in the upper left corner) and the potential fates of the calcite dissolution products (Ca2+ and CO32-). The calcium transport unit consists of repeating individual bipolar cells, where one pole is specialized for calcium uptake and the opposite for calcium extrusion. Conversion of carbonate ions CO32- released from calcite into HCO3- is promoted by the counter-transported protons H⁺. HCO3– is then actively transported within the cell and converted into CO2 through the carbon-concentrating mechanisms and can then be used in photosynthesis [89]. (left panel) Holistic calcium ion localization during boring, with relative calcium concentrations within each compartment indicated. Calcicytes allow a higher proportion of cells within the trichome to remain photosynthetically active by controlling intrafilamentous calcium flow (modified from [90]).

Point 3: L537. I think the Authors misunderstood or simplified the mechanism of tissue retraction on corals. On most of them, tissue do not retract at all. It happen only in the presence of a major stress (I observed that on Med corals during thermal stress) and for some solitary corals such as the Desmophyllum studied in ref 185. So, this sentence should be changed to specify that euendoliths can colonize exposed skeleton in some species while for some others they can access to the skeleton from the base of the recruit/colony when the tissue cover new substrate where the endoliths are, so they are covered first by the tissue, then by the skeleton. Ref 166 explain this mechanism.

Response 3: I modified the paragraph as below.

In live corals, the colonization of the skeleton by euendoliths from the water column is prevented by the polyp tissue [120,180]. In young coral recruits, the entire corallite (i.e., part of the skeleton elaborated by a single polyp) is tightly covered with polyp tissue [127,166,184], which efficiently protects the underlying calcareous skeleton from infestation by other life stages of endolithic organisms from the water column (e.g., propagules, epilithic biofilms), thanks to its superficial mucus and cnidocysts. In some instances, the polyp tissue retracts towards the proximal portions of the coral colony, either temporarily when corals are under major thermal stress [185] or permanently in the case of solitary corals [186], leaving the skeleton unprotected. Photoautotrophic euendoliths therefore have the ability to colonize the coral skeleton from its base, as soon as the larvae settle on an already infested substrate [166], or to enter exposed skeleton through lateral fissures or when the polyp tissue retracts [185,186].

Point 4: L571. While the terms azooxathellate and zooxanthellate corals have not been revised, it is more corret to refere to the zooxanthellae as Symbiodiniaceae (as revised by La Jeunesse (2018?).

Response 4: I added the name in brackets in the text with the reference you advised after the first occurrence of ‘zooxanthellate corals’. I prefer to keep the remaining text referring to zooxanthellate and azooxanthellate corals for readability.

Point 5: Finally, Paragraph 6. It could be opportune to cite the fact that the increase in the euendolithic boring on corals will fragilize the whole reefs making them less resistant to protect coastal area, and lagoons, especially during cyclones and extreme events, with serious consequences for the ecosystems and humans!

Response 5: This concept was already present in the paragraph but I agree that it wasn’t the best wording.

Before: If rates of bioerosion exceed accretion rates, the increased porosity of individual coral skeletons and the inevitable acceleration of degradation of the reef framework [118,148] increase coral susceptibility to damages by cyclones and storms [212], by El Niño events [217,218] and by predators, such as the crown-of-thorns starfish [12].

Now: Severe euendolithic infestation of individual coral skeletons can fragilize whole coral reefs, increasing their susceptibility to damage by cyclones and storms [212], by El Niño events [217,218] and by predators, such as the crown-of-thorns starfish [12], thus diminishing the coastal protection they offer to other ecosystems and mankind.

Reviewer 2 Report

This is a useful and nicely written review focused on phototrophic marine endolithic microorganisms. I am sure that it will be a valuable contribution to the scientific literature on this topic. The authors refer to a large number of individual studies dealing with this peculiar marine microhabitat and I did not find significant omissions in their list of references.

Abstract is written in a concise style and sums up properly the contents of the review. My only point is that the phrase "we address the nature and diversity of euendoliths" should include the word "marine" sa the current phrasing might confuse a reader who might think that the review deals with endolithic microorganisms in a wider range of habitats.

I consider the division of the article into individual chapters to be coherent and logically interrelated. In particular, I appreciated the chapter dealing with endolithic assemblages in the context of current changes related to the anthropogenic activities.

My subsequent comments and critique point to several aspects of the text that might be modified or supplemented in the revision:

1) The authors never mentioned that among "chlorophytes" the marine boring microalgae almost exclusively belong to the class Ulvophyceae. At least, it should be mentioned when referring to Ostreobium as one of the prime endolithic members of this lineage.

2) l. 96-99: "Red algae from the genera Porphyra C. Agardh (1824) and Bangia Lyngbye (1819) are endolithic during the early stages of their life cycle, called the conchocelis phases, but become epilithic during the adult stage [43,44]."

It should be mentioned, however, that "Conchocelis" stages do not exclusively occur as endoliths, they may readily be found as epilitihc or epizoic (on bivalve shells) biofilms, too. In addition, Porphyra and Bangia are not exclusively limited to epilihic assemblages. They may definitely occur in a wider range of coastal microhabitats.

3) "More recently, [159] revealed"

I suggest to include a full name of the first author here and the number of this reference at the end of the sentence.

4) l. 542: "In live crustose coralline algae (CCA)"

At this point, it should be mentioned that taxa belonging to "CCA" indeed almost entirely belong to the subclass Corallinophycidae of red algae.

Author Response

Good morning,

Thank you very much for your comments! Please find below our point-by-point response.

Point 1: Abstract is written in a concise style and sums up properly the contents of the review. My only point is that the phrase "we address the nature and diversity of euendoliths" should include the word "marine" so the current phrasing might confuse a reader who might think that the review deals with endolithic microorganisms in a wider range of habitats.

Response 1: Done.

Point 2: 1) The authors never mentioned that among "chlorophytes" the marine boring microalgae almost exclusively belong to the class Ulvophyceae. At least, it should be mentioned when referring to Ostreobium as one of the prime endolithic members of this lineage.

Response 2: I added this detail here, as follow:

Moreover, the use of eDNA metabarcoding, in combination with other techniques, such as microscopy, spectrophotometry and cultivation, has revealed previously undisclosed diversity of prokaryotic and eukaryotic endolithic organisms [52,64,67] and can help resolve their phylogenetic history [64]. For example, euendolithic green algae almost exclusively belong to the class Ulvophyceae, in which the ability to bore evolved independently over 20 times [64], while the cyanobacterium Acaryochloris marina Miyashita & Chihara (2003) has been recorded for the first time in the skeleton of live corals using eDNA [64] and produces chlorophyll-d, allowing it to use far-red light for photosynthesis and thus to occupy niches depleted of visible light [72].

Point 3: 2) l. 96-99: "Red algae from the genera Porphyra C. Agardh (1824) and Bangia Lyngbye (1819) are endolithic during the early stages of their life cycle, called the conchocelis phases, but become epilithic during the adult stage [43,44]."

It should be mentioned, however, that "Conchocelis" stages do not exclusively occur as endoliths, they may readily be found as epilitihc or epizoic (on bivalve shells) biofilms, too. In addition, Porphyra and Bangia are not exclusively limited to epilihic assemblages. They may definitely occur in a wider range of coastal microhabitats.

Response 3: I included this detail as follow:

Some red algae from the genera Porphyra C. Agardh (1824) and Bangia Lyngbye (1819) can exhibit an endolithic lifestyle during the early stages of their life cycle, called the conchocelis phase, while occurring in a wide variety of habitats as adults [43,44].

Point 4: 3) "More recently, [159] revealed"

I suggest to include a full name of the first author here and the number of this reference at the end of the sentence.

Response 4: Agree, full name of first author was also included when other similar occurrences appeared in the manuscript.

Point 5: 4) l. 542: "In live crustose coralline algae (CCA)"

At this point, it should be mentioned that taxa belonging to "CCA" indeed almost entirely belong to the subclass Corallinophycidae of red algae.

Response 5: I added this detail in brackets for readability.