Stable Isotope Analysis of Food Web Structure and the Contribution of Carbon Sources in the Sea Adjacent to the Miaodao Archipelago (China)

Round 1

Reviewer 1 Report

Review comments

Basic reporting

This manuscript explores the benthic food web in the sea adjacent to the Miaodao Archipelago by analyzing stable isotopes in producers and consumers. It has a good feature, including the functional feeding groups approach and the estimated scaled trophic level. It is somehow well written and organized; however, it suffers from somewhat weak methodological features that do not allow proper statistical treatment of the data to assess whether the patterns observed hold any generality. 

Below please find specific comments requiring major revision before the manuscript could be accepted for publication.

Comment 1: Abstract (Line 15-20) disappoints with the focus. The authors need to think about what they are trying to say in the initial lines and set out the study's aims. For example, the authors briefly mentioned the sea adjacent's importance as a carbon sink area (in the abstract). However, the manuscript does not provide more information regarding the importance of amounts of carbon and implications in the benthic food web, the trophic structure of trophic diversity of sources, or carbon flow in the food web.

Comment 2: Please re-write the abstract in about 200 words. I advise authors to omit smaller details, limiting to the major findings.

Comment 3: Line 19 Basic carbon sources determine the food web structure. What does it mean 'basic carbon sources'? Please clarify. If not, then you should remove this sentence from the abstract.

Comment 4: Lines 20-21 In this study, we divided the primary producers (phytoplankton, macroalgae, suspended particulate organic matter, and substrate organic matter) and consumers (invertebrates, omnivores, benthivores, and piscivores) …Why did you divide producers and consumers? Alternatively, do you mean 'determinate'? I think the sentence's meaning is that you determinate the producers and consumers.

Comment 5: Line 24: Here again, 'basic carbon sources.' It is better to use basal carbon sources or primary carbon sources in the whole manuscript than basic carbon sources.

Comment 6: Line 32 keywords; please add: benthic food web, isotopic niche, trophic level, and delete food web structure, SIAR.

Comment 7: Line 46-47; please modify as follow …' Studies have shown that many aquatic plants are the trophic foundation of the food web of ecosystems such as freshwater, salt marshes, estuaries, and shallow water coastal habitats [8,9]'.

Comment 8: Line 55; 'the importance of macroalgae in the benthic food web has been rarely studied. Do you mean in the Miaodao Archipelago or in general? If it is in general, please add some references, especially studies that have been done, for example, in subpolar regions.

Comment 9: Line 61. attempted? 

Comment 10: Line 71. Delete methods

Comment 11: Line 74-75: Miaodao Archipelago .…' has extremely rich biological resources [13]. Like which resources? Then -…. 'The sea area is greatly affected by human activities and has obvious sea-land interaction [14]. Could you provide more information for the readers here? Especially if they are not familiar with the study area.

Comment 12: Line 77-78: Random sampling method? Please add this information. 

Comment 13: Figure 1 Line 82-83. The map shows 30 sampling stations and not 60 stations, as mentioned in line 78. How many samples were collected in each station? Is this information necessary for the study? I guess so since the abstract is mentioned 'certain spatial characteristics.' However, the authors do not address spatial variability in the food web in the study.

Comment 14: Line 84. Please provide details about the dimensions of the grab sediment sampler.

Comment 15: Line 107. Please add the measurements units to body length and weight.

Comment 16: Line 110. The macroalgae were washed.

Comment 17: Line 109. 2.2 Stable isotope analysis. It could be nice that authors provide a Table or Suppl. Material about a list of samples type, number of samples, treatment, feeding group, isotopic data (mean, sd).

Comment 18: Line 140. Please provide the correct reference and add it to the references list (Hussey et al., 2014).

Comment 19: Line 162. Could you provide a reference?

Comment 20: Line 163-167. Is this the first stable isotope study in the area? Is there no more information about prey importance for consumers, which can help choose the baseline? What are the isotopic values of zooplankton?

Comment 21: Line 168. I found that the worst flaw in the manuscript is the data analysis. In section 2.3. statistical analysis needs to be improved. There is some information regarding methodological analysis in the results section, which should be written. My major concern with the manuscript deals with mixing model development for the stable isotopes data. For the relative contributions of organic matter from different sources to each consumer species, the authors used the SIAR analysis in R. However, the SIAR package has been unsupported for years now and defunct. I believe the MixSIAR mixing model (Stock et al. 2018) should improve the relative contribution of organic matter to the food web or simmer for a lighter package for basic mixing models akin to SIAR, so re-do analysis must be done using MixSiar or SIMMR (simmr). By doing a new analysis, the authors can also explore the possibility of two sources as food web bases.

Comment 22: Regarding Table 1. Why were only fish consumers classified in the dietary division and not the rest of the consumers? This is confusing to me. In my opinion, the correct term to use here is not dietary division but a functional feeding group or guild.

Comment 23: Line 194-199. δ13C and δ15N values can vary significantly in primary producers over Spatio-temporal scales. The authors only found significant differences in δ13C and δ15N values of the invertebrates and fishes, but not in macroalgae? I am unsure whether the strategy used by authors to investigate statistical differences with the Kruskal-Wallis test is correct. As shown in Table 2, the number of samples is highly variable, so I recommend to check the permutation test (PERMANOVA) rather than K-W to compare. 

Comment 24: Line 197. Table 2 in capital.

Comment 25: Line 202-208. The figure shows the benthic trophic structure in the sea adjacent to the Miaodao Archipelago and not the isotope niches. For that, the authors should estimate them by the SIBER approach. Please re-write the results in the correct way of what the figure shows. Also, the difference between isotopic niche and trophic niche should not be mentioned, and the reference of Jackson et al. (2011). In my opinion, this sentence should be included in the data analysis section.

Comment 26: Line 202-203. Figure 2. Please re-write the legend. For example, Stable isotope biplot (δ13C and δ15N ‰, mean ± SD) for the benthic food web (basal carbon sources and consumers) in the sea adjacent to the Miaodao Archipelago. Symbols represent the different functional groups (xxx, etc.). The dashed ellipse represents the main carbon sources (primary producers); Green and the inverted triangle represent macroalgae, in which S. horueri, G. turuturu, and U. pertuca are the main macroalgae. The error bar is the 95% confidence interval. The figure indicates two expected isotope patterns (expected marine-terrestrial patterns and expected C3-C4 plant patterns). 

Comment 27: Line 213. Please provide the isotopic values of all primary producers and consumers in a Table or Suppl. Material.

Comment 28: In section 3.2 Trophic level. Please include the standard deviation value of the estimates of trophic level. How were tested differences in trophic levels? Please provide details in the statistical analysis section. 

Comment 29: In section 3.3, Basic carbon source contribution. See comment 21 regarding SIAR analysis and results. The change of isotopic data analysis to a new mixing model may change considerable the results section and the relative contribution of sources.

Comment 30: In section 3.4, Isotopic niche width. Line 251-253. This sentence should be included in the data analysis of the material and methods section. Also, the authors should provide in the results the values of the niche areas by groups, the percentage of overlap. In addition, I recommend to use SEAc for small sample size, and it should be described in the data analysis the complete SIBER details.

Comment 31: In section 4.1. The authors divide between consumers and sources. Why do they do that? To divide is not the correct form, and also, the explanation of what authors do should be included in the methodology section. Please re-write the discussion section according to the new analysis request. 

Comment 32: In section 4.3. The authors should emphasize what it means, the importance of macroalgae as the main carbon source, and the ecological meaning of overlap. The discussion section here should be re-written and contrasted with the literature.

Comment 33: In section 4.4. The authors should re-write the discussion. What does it mean that fishes have higher trophic positions or occupy different isotopic niches? Why do piscivores have narrow niches? Many questions left the study open. The authors can discuss them in this section, contrast with the literature, and probably give more insight regarding fauna conservation. 

Comment 34: In section 5. Authors appeal to the uncertainty of the mixing model and seasonal differences. The isotopic data should be improved with the new model to make correct statements and conclusions.

Comment 35: Please revise the complete references list and the Journal format since many errors are being made. For example, 1) journal name should be in Italic, 2) species name in line 453: Mytilus edulis should be in Italic, and so on. Also, some journals have the complete name, and others with the abbreviation name. 

I hope all this information will provide helpful for your work!

Kind Regards

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

Review on “Stable isotope analysis of food web structure and the contribution of carbon sources in the sea adjacent to the Miaodao Archipelago (China)”

This study applied stable carbon and nitrogen isotope analyses to coastal food webs in the Bohai Sea, China. The authors found that different carbon sources contribute differently to different types of consumers, which can be largely explained by their ecology and behavior. The manuscript has been already well written and I have only one minor comment and some editorial suggestions as follows:

The authors tried to extend their finding to the context of global carbon cycle, as macroalgae would efficiently draw down atmospheric CO2, which is transferred into consumers at higher trophic positions. One of the concluding statements found in lines 386-387 ‘Through fisheries, humans remove the carbon accumulated in consumers from the ocean’ seems to suggest that the coastal fishery should be promoted under the name of ‘carbon sink fisheries’ (line 387). Although the next sentence identifies this is the case for the mixed culture of shellfish and macroalgae, some readers could misunderstand this as the more fisheries catch, the more CO2 sequestered from the atmosphere. This may result in misleading of a wrong conclusion that the overexploitation of fisheries resources is recommended by scientists to reduce atmospheric CO2. As suggested by numerous studies including this study, the ecosystem is not that simple, and greater care must be taken when the discussion goes to applications and politics. The statement in lines 386-387 also conflicts with the previous paragraph where the importance of ecosystem conservations was discussed. Please consider revision.

L80: ‘substrate organic matter’ sounds unclear to me and would be better to say ‘sedimentary organic matter’

L149: More explanation needed for what these beta0 and beta1 are

L222: ‘P<0.05’ what kind of statistical test was made here?

L251-253: These sentences should be moved to Methods

L255: standard ellipse area (SEA)

L275: terrestrial organic matter (TOM)

L285: Delete (values)

L303: What is ‘influencing substances’?

L306: ‘affect’ should be rephrased with ‘decrease’

L313: Are these numbers permil?

Table 1: Isn’t any of these fish planktivores?

Table 2: Are zooplankton included in invertebrates? If so, it would be better to separate zooplankton and benthos as their isotope values should be very different

Figs 1, 7 and 9: miles should be changed to kilometers

Fig. 2: I understand zooplankton TL was set at 2. But how large was their isotopic variation? If it was significantly large, then the following TL calculation would be severely affected

L322: Delete (values)

L324: ‘significantly higher’ what kind of statistical test was made here?

L329-331: Unclear sentence and thus needs revision

L355: ‘micro food ring’ should be rephrased with ‘microbial loop’

Author Response

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear author and co-authors,

Your manuscript is much more improved, and I appreciate your effort with it. The discussion section is much more interesting with all the new information you added on it. However, some issues I will report below to this new version, and they should be addressed before publication.

Line 193; R (SIAR) package in R 4.0.5 can be used to estimate. Please use the past sentence: was used to estimate...

I agree with your answer about the degree of uncertainty that can have Bayesian mixing models for stable isotopes analyses and may be the 'real representativeness' of a food source such as macroalgae, can bias your data. However, it is no longer acceptable to use SIAR, and I appeal that the best option for your data analysis is the SIMMR (simmr) mixing model. This new package is the updated version of SIAR, and it will also improve your figures to a more nicer view of the carbon food sources' contribution to the consumer's species.

Lines 194 – 197: In order to further explore the reasons for the differences in the use of basic carbon sources by different functional feeding groups, their niche relationships were compared. The study used SIBER (in SIAR package) to calculate the niche repeatability of consumers.

Please re-considered why do you use the SIBER analysis. It is not correct to mentioned that the reason was to check differences in the use of basic carbon sources. The SIBER analysis is used to predict the niche size of the community, that is the main reason why you calculated or estimated the TA, SEA, SEAB, etc. parameters. Please, re-write this section according to the publication of Jackson, A.L., Inger, R., Parnell, A.C., Bearhop, S., 2011. Comparing isotopic niche widths among and within communities: SIBER - Stable Isotope Bayesian Ellipses in R: Bayesian isotopic niche metrics. Journal of Animal Ecology. 2011, 80, 595–602. 590 https://doi.org/DOI:10.1111/j.1365-2656.2011.01806.x

Line 303. SEA(Standard EllipSP Area), Please correct to Standard Ellipse Area

Line 315. Table 3. Please re-write the correct legend of table. For example, summary statistics of SIBER analysis for each group, etc…

Line 500. I agree that future studies can considered to explore mixing models such as MixSIAR (but see my comments above to better use simmr).

Thank you!

Kind Regards

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.docx