Multi-Biomarker Analysis Uncovers High Spatio-Temporal Stability of a Subarctic Rhodolith (Lithothamnion glaciale) Bed Food Web

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review for the paper “Multi-biomarker analysis uncovers high spatio-temporal stability of a subarctic rhodolith (Lithothamnion glaciale) bed food web” by Sean Hacker Teper and co-authors submitted to “Diversity”.

 

The authors conducted a study on the food web associated with a bed of the rhodolith Lithothamnion glaciale in southeastern Newfoundland, Canada. They used lipid and fatty acid analyses, as well as stable isotope analysis, to uncover the food web dynamics within the study site over a 9-month period. The authors discovered seasonal variations between the spring and autumn communities. They also revealed that the food web consists of three trophic levels. The authors concluded that this system exhibits spatio-temporal stability. These findings could have significant implications for studying similar sub-arctic food webs and monitoring food web dynamics. The authors utilized standard methods to collect and process samples, as well as appropriate statistical methods for data analysis. The authors' data supports all conclusions made in the study. The paper is well-written and well-illustrated, requiring only minor revisions before final acceptance.

 

Recommendations.

 

In Section 2.7, the authors should provide detailed information about the chromatograph used to study fatty acids, as well as a thorough description of the temperature regimes.

 

In Section 2.10.3, the authors should specify which post hoc test was used for multiple comparisons (from Line 474).

 

Table 1 should also be included. The table should include the range (min-max) for each parameter.

 

 Lines 363-366. It is unclear whether the authors accounted for Type I error when conducting multiple correlation analyses.

 

Consider replacing the header 'Significance' with 'Factor' in Tables 2 and 3.

 

Section 4.1. When discussing the differences in benthic biomass between the two studied sites, it is important to consider the role of salinity. The authors noted that the North site is affected by a freshwater inflow, which may result in lower salinity levels compared to the Southern site.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This is a review of the manuscript  diversity-2977210 "Multi-biomarker analysis uncovers high spatio-temporal stability of a subarctic rhodolith (Lithothamnion glaciale) bed food web".

I found the topic very interesting and I was very excited about this manuscript when reading the introduction. However, after reading the methods and results, I was disappointed with the overall analysis and results presentation. I think the authors spent so many resources on sampling and processing samples/laboratory analysis, but the results and overall interpretations are shallow compared to the potential of their data. Please, see my comments below; I hope this helps you improve the manuscript and make it more attractive to the public.

 

OVERALL: Great idea, many results, but not so good analysis and results presentation.

INTRODUCTION: very clear and explicit on the topic to be evaluated.

 

METHODS: Needs more clarification on the analysis, and a better explanation of which analysis was used for which hypothesis. Also, many better statistical analyses are available to analyze your data. You have great potential with this amazing dataset, but it was under-explored. The numbers below express the line number on the manuscript.

 

86 - please, provide a map instead and with, preferentially, a picture of the rhodolite beds

88 - that is why is good to have your own map - you are trying to detect spatial variability, so it is good to show de reader the environmental/morphological characteristics of each site.

105 - call in the text and explain this figure - to show the reader the environmental characteristics of your sites. did not notice the call for Fig. 1b

149 – please, be more specific.

177 – ‘lipid classe’- please, describe them.

Statistical analysis in general: TESTING FOR ALL LIPIDS, FATTY ACIDS, AND STABLE ISOTOPES - why so many PERMANOVAS instead of using all variables together to see patterns? Also, why not include an nMDS maybe GLMs would be good to evaluate the relationship of isotopic values to spatial, temporal, and species components.

 

RESULTS: not the best presentation. All important results are presented in table format in the supplementary material (which I need to discover by myself because the text is described as Table A, B, C… not as supplementary material). Instead of tables, you should consider summarizing results as figures, joining the biomarkers that trace terrestrial vs pelagic vs benthic (or any other categories that are important for your interpretation), and plotting the percentages for each species as boxplots, bar graphs, and any graph that helps the interpretation. Also, VERY IMPORTANT, that this should be in the main manuscript, not as supplementary material. The results section is very hard to read because it is a report of lots of percentual and acronyms that make the reader tired and confused. Consider presenting the results in an easier and better way. Also, many environmental data were collected and not correlated to the biomarkers and species. Why is that?

 

288-289 - insert test value

304-306 (and all Other cases below) - please insert the value of the test, which test was used?

335 – add test in methods

340 - maybe some info about each class, which ones are more terrestrial, which are marine/ pelagic x benthic  - would be good to interpret the results

372 - make a table indicating each biomarker would be very helpful (and what they trace – terrestrial, pelagic, etc)

Figure 3 b – this is a table, not a figure, and it should be its own table in the manuscript (comment above)

405 – table d ??

419 – standardize decimal cases

Table 2 - did not understand the significance column. also, which analysis was being tested here? Please, report the analysis properly. (similar Table 3)

439 – ‘was less carbon-13 depleted ‘ – confusing, replace by  ‘more d13C enriched’

441 – that is not how it works.  ‘increased the d15N’ - not trophic position. Kelp is a primary producer and is a baseline for the system.

445 – ‘least depleted’ – replaced by ‘enriched’

446 - Table F ???

449 - did you calculate the trophic position to say that?

Figure 4  - can you make the grouping more informative by establishing when the samples were collected?  - i.e., including season and spatial differentiation of each group

Table 4 – very confusing, why subgroups? Also, present the test results instead of letters.

Tabe 5 -  what was the methodology for this calculation? Please, describe in the methods section

 

DISCUSSION: it is ok, but certainly can be improved, it will need to be reviewed when using the appropriate analysis in the results section.

467 – where are the environmental variables in your results section? not in the analyses you presented…

491 - why did not you calculate the actual trophic positions of each species? By using d15N you could easily calculate that (see Fry 2006, Post 2002). Even better, you could use a Bayesian approach and calculate the TP using tRophicPosition package on R (Quezada-Romanelli et al. 2018). You have all baselines, d15N, and d13C to make your estimates more robust...

510 – example of info good in methods

545 - reference for this Citation

558 - why this result was not presented? where are chla and other environmental variables in your results?

570 - a figure showing the seasonal patterns on increase vs decrease of fatty acids derived from terrestrial vs pelagic vs benthic would be fundamental to help understand your results better.

574 – about blooming season - not in results, why is that?

580 – ‘doubled’ but from 1% to 2% - still very low to consider an important increase.

587-588 - a strong conclusion with not-so-strong results.

591-591 – cite the conceptual figure here (fig 5)

614-615 - not explored in the results, need to show this

629, 635, and below – please, do not use the term ‘signature’ for isotopes; stable isotopes change seasonally, and spatially, and 'signature' is something that do not change. Use isotopic values, isotopic ratios, and isotopic composition instead.

640-641 - do not agree with this statement. If the species feed on the same trophic level, their d15N in the same system would overlap - you need to analyze d13C together to say they are feeding on the same resource. d15N indicated mostly they feed in the same level/position. Also, you found no difference in the abundance of organisms, so there is no indication of competition - contrary, there is an indication of the availability of resources, the abundance of that, they do not need to share, they can consume similar sources due to the abundance o the systems - partitioning.

643-644 - maybe is just a variability of local sources and the groups are from different locations and seasons. stable isotope values vary spatiotemporally, in special POM and SOM due to environmental conditions and nutrient input. so the groups could be showing spatial differences, considering polychaetes are residents and would reflect local isotopic values. consider revising the identity of species in each group - eating site and season they were collected and grouped.

651 - how about using mixing models to better analyze your data and get robust conclusions?

Suggested reading:

·         Fry B (2006) Stable isotope ecology. Springer, New York, NY

·         Lanari M, Possamai B, Garcia AM, Copertino M (2021) Seasonal and El Nino Southern Oscillation-driven variations

·         in isotopic and elemental patterns among estuarine primary producers: implications for ecological studies. Hydrobiologia 848: 593−611

·         Post DM (2002) Using stable isotopes to estimate trophic position: models, methods, and assumptions. Ecology 83: 703−718

 

·         Quezada-Romegialli C, Jackson AL, Hayden B, Kahilainen KK, Lopes C, Harrod C (2018) tRophicPosition, an R package for the Bayesian estimation of trophic position from consumer stable isotope ratios. Methods Ecol Evol 9: 1592−1599

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

In the reviewed paper the results of the comprehensive study of subarctic rhodolith Lithothamnion glaciale bed food web by multi-biomarker analysis are summarized. The MS is important to better understanding the unique ecosystems of subarctic rhodolith and marine biodiversity in total. The objectives of the study, methodology, results and discussion described in great detail. The results of the research presented in the tables and clear figures. It is necessary to note, that results of the study summarized in the schematic partial food web. The conclusions of the MS are consistent.  

I can recommend MS to publication after some corrections.

Major comments:

1. You provide very detail study and have got very interesting results. However, in some parts of the MS the data  are hard to perceive, for example in sections 3.3 (lines 368-390, 424-427), 3.4 (lines 468-476). Please, divide this part of the paper into paragraphs. This will make it easier to understand.

2. Add recent publication (2019-2024 years) in reference list (at least 40). Now from 101 reference only 12 published in 2019-2024.

Minor comments:

Title and further: Is it necessary to always use term “rhodolith (Lithothamnion glaciale)”? Please use synonyms for this.

Lines 2, 91- 96 and further: Add the authors of the taxa at the first mention.

Lines 14-15, 105-110: I think, that it is better to replace the square brackets with round ones. References to the literature are indicated in square brackets.

Line 58: Delete the dash at the end of the sentence.

Lines 60, 79-80: Try to use another way to write “rhodolith (Lithothamnion glaciale) bed”.

Lines 197, 204, 212, 214, 216: Add equations numbers according to journal template.

Line 706: Correct yellow insertion.

 

Reference: Correct reference according to journal template (change color of references from blue to black, correct the font size). 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments.

Author Response

We thank Reviewer 1 for their continued efforts in reviewing our manuscript and appreciate the time and care they have taken to provide feedback. We are pleased that they are fully satisfied with the revised manuscript and recommend it for publication in its current form.

Reviewer 2 Report

Comments and Suggestions for Authors

Unfortunately, no changes have been detected since my last revision, so I need to stand with my last comments. Almost none of my comments were accepted in this review and I did not change my opinion, so there is not much I can add here. I think all important results are in the appendix and should be moved to the main manuscript; also the analysis used here could be improved. 

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors  corrected the MS according to reviewer suggestions. I can recommend it for publication.

Author Response

We thank Reviewer 3 for their continued efforts in reviewing our manuscript and appreciate the time and care they have taken to provide feedback. We are pleased that they are fully satisfied with the revised manuscript and recommend it for publication in its current form.