|The 13C/12C ratios (= δ13C) are indicative rather than definitive proxy of carbon use physiology (Giordano, Beardall & Raven 2005).|
δ13C values can vary depending on several variables such that inconclusive δ13C signatures have been reported in several seaweed species (See Roleda and Hurd 2012, page 412). In this study, were there any widely distributed species collected? Did the δ13C varied with collection sites (tidal level and latitude), season, and collection years?
For some key species, the δ13C data could have been backed up in combination with other techniques such as pH drift experiment, and/or HCO3 utilization pathway inhibitors (Fernandez et al. 2014, 2015), which are relatively easy to do, to categorically establish their carbon use physiology. For example, δ13C in combination with pH drift experiments, HCO3-using macroalgae can shift seawater pH to 9.0 or higher and will have corresponding δ13C values less negative than -30‰. Conversely, CO2-using species have δ13C values more negative than -30‰ and will be unable to raise pH above a critical value of pH 9 (Maberly et al. 1992). Unfortunately, this may not be possible anymore. Otherwise, additional data will make this paper better- be critical on the use, significance, and limitations of solely using δ13C as a proxy for carbon use physiology.
When additional data is not possible, the authors are encouraged to give emphasis on the limitations of the study. Despite the huge dataset and corresponding statistical analyses, the significant correlations are meaningless when they do not have physiological and ecological relevance. For example, why would morphology determine carbon use physiology? Is there a specific morphology that would tend to be strictly CO2-user rather than mix HCO3/CO2-user or strictly HCO3-user?
Environmental conditions may change δ13C values but should not in that instance change carbon use physiology, which is most likely inherently species-specific. A change in δ13C signature within the range specific to a carbon use strategy (e.g. mix HCO3/CO2-user) may indicate the presence of the CCM capacity of algae but may not indicate preferential uptake of certain Ci species (HCO3 or CO2), which could be measured using membrane-inlet mass spectroscopy (MIMS). In extreme cases, some species have been reported to have totally different δ13C values representing different carbon use strategy. Can these cases be attributed to incorrect species ID from different studies? In this regard, correct species ID is of utmost importance specially those cryptic and morphologically simple but phenotypically plastic taxa. These species may require molecular ID. How well was Ulva discriminated based on morphology? See Roleda and Heesch 2021 Food Chemistry and references therein on problems related on Ulva ID. The Ulva LPP (linza-procera-prolifera) complex is another issue to contend with (Shimada et al. 2008, Herrero et al. 2020).
Throughout the manuscript, the authors should be clear whether the increase/decrease, change, or variability in δ13C refers to change in carbon use? Or shift within a specific carbon use strategy. Then what is the significance of this shift or variability? How does it explains lines 683-685: “Filamentous uniseriate and pluriseriate with erect thallus and C-Tubular) and genus (e.g., Colpomenia, Padina, Polysiphonia and Gracilaria) revealed that certain life forms are better monitors explaining the variability of δ13C-macroalgal (and D13C values) than others.” It is not convincing that there is biological and ecological basis or support that δ13C variability and therefore carbon use physiology is controlled by morphology. For example, within the genus Halimeda, different species (relatively the same morphology?) measured different carbon use physiology based on pH drift experiment (Narvarte et al. 2020).
The title can be made more interesting. So, what does this variability suggest?
Specimen were collected during spring (March-April) and dry season (nominally from November to May) from 2009 to 2014. When were the environmental parameters measured? E.g. collected only during the sampling? For example, was the pH measurements snapshot? Or daily average? Does the temperature category represent daily average or seasonal average? Is there interannual variations?
What does “composite samples” means? For each species (big or small), was the whole plant (rhizoid/holdfast, stipe, and blade) analysed as one unit? Please specify. Otherwise, what are the implications of analysing only parts or the whole plant.
How different is the 4th strategy of DIC uptake from the other three? Have not encountered this classification before (please cite a reference). Is this only for calcifying species? What type of calcification? For example, were both Amphiroa and Padina classified under the same category?
Line 711: “CO2 carbon mechanism”?
The English language can still be significantly improved.