Diet and Trophic Structure of the Fish Community in a Small Sub-Tropical Lake in Central Mexico

Round 1

Reviewer 1 Report

Keywords could be improved; 'native species' is vague; add 'fish' and main families of fishes e.g. Goodeidae or Cyprinodontiformes. 

Introduction has a good selection of references, leading to 3 hypotheses examined later.

Methods: Lake Zacapu seems like a headwater system in the Lerma; suggest enlarge description for non-Mexican readers. The map looks like an endorheic basin; add the Lerma outflow.  State that abiotic variables were determined elsewhere, if this is correct, and give reference (e.g. 36?). Diets are well analysed.

Results are generally well described. It would help readers if fish genera and families were provided at the first mention (e.g. line 120, or Table 2), and perhaps also whether they are endemic, native or non-native. To this reader, invertebrates were grouped rather strangely (e.g. why were only chironomids separated from all other insects?). Why combine gastropods and bivalves, when their feeding methods are so different?  PSIRI results are clear, but trophic levels tend to merge or overlap in reality. 

Table 2: abbreviations are rather difficult to read, but this may look better if the Table is reset. You used about 2% (about 110 fish) from each season for isotope analysis; was this adequate?

Line 349, Fig. 6: the complex coloured layers of the Bayesian model need further explanation. 

Discussion and conclusions are generally clear. Stomach contents complement stable isotope results; both give different information, including behavioural. Three of the goodeids are recently discovered endemics, closely related, and their diets closely overlap; perhaps amplify this finding.

Line 409: the evidence that resources are so abundant is not clear from the article. Line 416 suggests that hypothesis 3 only found trophic overlaps in 2 species, C. idella and S. lermae but this is not obvious in Table 4; please clarify this.

Line 437: is ref. 67 the most appropriate here for lake size?

Line 446: Interesting that isotope records in Lake Zacapu didn't correlate with previous feeding information in Mexico, suggesting opportunism and food switching.

Line 470: the conclusion that food webs didn't differ between sampling sites, 'likely reflecting high abundance of aquatic macroinvertebrates', needs supporting.

References show good geographic and date range (a few are classics; most are since 2000), though large arctic lakes may have less relevance to this study.

English is good. Spelling not rigorously checked, but I noted  'Hyalella' (line 133 and 390), 'trophic' line 365), 'ample' (line 392), and 'Potamogeton' (line 419).

Author Response

Comment 1.- Keywords could be improved; 'native species' is vague; add 'fish' and main families of fishes e.g. Goodeidae or Cyprinodontiformes. 

Reply 1.- we changed the keywords as recommended. “Fish and Goodeidae”

Comment 2.- Introduction has a good selection of references, leading to 3 hypotheses examined later.

Reply 2.- we appreciate your comment.

Comment 3.- Methods: Lake Zacapu seems like a headwater system in the Lerma; suggest enlarge description for non-Mexican readers. The map looks like an endorheic basin; add the Lerma outflow.  State that abiotic variables were determined elsewhere, if this is correct, and give reference (e.g. 36?). Diets are well analysed.

Reply 3.- The description of Lake Zacapu for non-Mexican readers are in lines 91 to 106. We modified the map. The abiotic variables were measured at the same points as fish samples and in the same season of the year, lines 107 to 117.

Comment 4.- Results are generally well described. It would help readers if fish genera and families were provided at the first mention (e.g. line 120, or Table 2), and perhaps also whether they are endemic, native or non-native. To this reader, invertebrates were grouped rather strangely (e.g. why were only chironomids separated from all other insects?). Why combine gastropods and bivalves, when their feeding methods are so different?  PSIRI results are clear, but trophic levels tend to merge or overlap in reality. 

Reply 4.- we added the genera, the families of the species, and the origin of the species (native, endemic, and nonnative) in table 2 as recommended Chironomids are separated from the other insects because it was an item that gave higher values of the PSIRI index for some species and the fish did not consume other insects. We have now separated gastropods from bivalves, they are now in two different columns as requested. 

Comment 5.- Table 2: abbreviations are rather difficult to read, but this may look better if the Table is reset. You used about 2% (about 110 fish) from each season for isotope analysis; was this adequate?

Reply 5.- we modified table 2 for better reading. Yes, the isotope analyzed were conducted based on 112 fish from the wet season and 118 fish samples from the dry season, as now explained in lines 161 to 164. Following the criteria of other studies based in stable isotopes three to five individuals of each species are enough to represent their diet (e.g. Mercado-Silva et al., 2015; Córdova-Tapia et al, 2015)

Comment 6.- Line 349, Fig. 6: the complex coloured layers of the Bayesian model need further explanation. 

Reply 6.- we now explain the color layers of the Bayesian model, lines 359 to 361.

Comment 7.- Discussion and conclusions are generally clear. Stomach contents complement stable isotope results; both give different information, including behavioural. Three of the goodeids are recently discovered endemics, closely related, and their diets closely overlap; perhaps amplify this finding.

Reply 7.- only two endemic species are from Goodeidae family, the third species is from the Cyprinidae family, we extended the text in lines 414 to 417. 

Comment 8.- Line 409: the evidence that resources are so abundant is not clear from the article.

Reply 8.- unfortunately macroinvertebrates have been poorly studies, the only study that exists is now cited. Line 426. Our judgement is based on 1) short hydraulic retention time leading to continuous nutrient addition and high productivity (river like), 2) little terrestrial content in the stomachs and 3) visual judgement when sampling macroinvertebrates and the paper cited. We admit that this is not quantitative evidence and have softened the sentence on resource abundance in the text.

Comment 9.-Line 416 suggests that hypothesis 3 only found trophic overlaps in 2 species, C. idella and S. lermae but this is not obvious in Table 4; please clarify this.

Reply 9.- we clarified the third hypothesis; we found a higher overlap between native species than native versus nonnative species. Line 418 to 426.

Comment 10.- Line 437: is ref. 67 the most appropriate here for lake size?

Reply 10.- Point taken. We added another reference (68) dealing with a subtropical shallow lake.

Comment 11.- Line 446: Interesting that isotope records in Lake Zacapu didn't correlate with previous feeding information in Mexico, suggesting opportunism and food switching.

Reply 11.- We appreciate your comment, Lake Zacapu differ from other aquatic system in Mexico, as most of these are in critical condition with a high percent of human activities involved, while Zacapu lake is one of the most well conserved in the country (lines 78 to 82). 

Comment 12.- Line 470: the conclusion that food webs didn't differ between sampling sites, 'likely reflecting high abundance of aquatic macroinvertebrates', needs supporting

Reply 12.- We added the reference that supports this high abundance of macroinvertebrates and softened the sentence further-see also reply to comment 8.

Comment 13.- References show good geographic and date range (a few are classics; most are since 2000), though large arctic lakes may have less relevance to this study.

Reply 13.- we appreciate your comment.

Comment 14.- English is good. Spelling not rigorously checked, but I noted  'Hyalella' (line 133 and 390), 'trophic' line 365), 'ample' (line 392), and 'Potamogeton' (line 419).

Reply 14.- we corrected the words and checked all the document for the correct grammar.

References

1. Mercado-Silva, N.; Lyons, J.; Moncayo-Estrada, R.; Gesundheit, P.; Krabbenhoft, T.J.; Powell, D. L.; Piller, K.R. Stable isotope evidence for trophic overlap of sympatric Mexican Lake Chapala silversides (Teleostei: Atherinopsidae: Chirostoma spp.). Ichthyol. 2015, 13(2), 389–400. https://doi.org/10.1590/1982-0224-20140079
2. Córdova-Tapia, F., Contreras, M., Zambrano, L. Trophic niche overlap between native and non-native fishes. Hydrobiologia 2015, 746:291-301. 10.1007/s10750-014-1944-z

Author Response File: Author Response.docx

Reviewer 2 Report

It is necessary to present data and discuss the following two contents.

1. What are the environmental differences between the two seasons during the study period? It is necessary to present data to explain this in Materials and Methods. And the authors need some clarification on what it means to compare two seasons.

2. The discussion needs an explanation of the environmental factors that control Lake's environment.

Author Response

1. What are the environmental differences between the two seasons during the study period? It is necessary to present data to explain this in Materials and Methods. And the authors need some clarification on what it means to compare two seasons.

Reply 1.- we appreciate your review in our document. During the present study no significant differences between the two seasons for temperature (x2 = 3.6, p = 0.056), dissolved oxygen (x2 = 0.2, p = 0.599), TDS (x2 = 0.8, p = 0.369), conductivity (x2 = 1.8, p = 0.170), NO₃ (x2 = 2.6, p = 0.102), and NH₄ (x2 = 2.2, p = 0.134), however, we found significant differences in pH (x2 = 6.4, p = 0.01129) and NH₃ (x2 = 6.1, p = 0.012). We did not find significant differences among the sites for any of the variables. This information is given in the manuscript, lines 228 to 231. In materials and methods, we explained the data analysis of the environmental variables of the lake (lines 166 to 170). We compared two seasons. Other studies from the lake, we now refer to have reported that there are no significant differences between the environmental variables in different points of the lake, or in different months of the year (Valencia-Vargas and Escalera-Vazquez, 2019; Ramírez-García et al., 2021; Ramírez-Garcia et al., 2022). Lake Zacapu showed homogeneous environmental characteristics throughout the system, and most environmental variables have remained in the same range for decades (1995 to 2019). Now referred to in the text. Therefore, we concentrated the study to the two contrasting seasons and found that there were no significant differences in the diet of the species between the two seasons. We now clarified the approach selected in the methods (lines 112 to 117). 

2. The discussion needs an explanation of the environmental factors that control Lake's environment.

Reply 2.- we added an explanation of the factors that’s control lakes environmental variables, lines 393 to 397.

As in other studies of Lake Zacapu [35, 36, 41], we found homogeneous environmental conditions, both spatially and temporally, reflecting continuous circulation with little zonation due to the shallowness of the system, high capacity for hydraulic renewal, and small spatial dimensions [33]. Only ammonia and pH differed slightly between the two seasons (Table 1).

References

1. Ramírez-García, A.; Moncayo-Estrada, R.; González-Cárdenas, J.J.; Domínguez-Domínguez, O. Reproductive cycle of native viviparous fish species (Actinopterygii: Cyprinodontiformes: Goodeidae) in a subtropical Mexican lake. Ichthyol. 2021, 19(4), e210105. https://doi.org/10.1590/1982-0224-2021-0105
2. Ramírez-García, A.; Domínguez-Domínguez, O.; De La Cruz-Agüero, J.; López-López, E.; Moncayo-Estrada, R. Temporary changes in the structure of the fish community separated by decades in a small subtropical lake. Freshw. Fish 2022, 32(1), 53–63. http://dx.doi.org/10.1111/eff.12669
3. Valencia-Vargas, R.; Escalera-Vázquez, L.H. Abundancia de la salamandra Ambystoma andersoni con relación a la dinámica estacional y heterogeneidad espacial en el lago de Zacapu, Michoacán, México. Mex. Biodivers. 2021, 92, e923283 3. https://doi.org/10.22201/ib.20078706e.2021.92.3283

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have worked on the trophic structure and niche overlap of the aquatic fish species in the Zacapu lake in Mexico. the authors have also used the stable isotops for the same.

The work done by the authors are interesting and presented in a nice way. I just wanted to know why the authors have not used other food web models for trophic structure and prey-predator overlap for the comparison (e.g. ecopath model).

The minor comments are mentioned in the MS.

Comments for author File: Comments.pdf

Author Response

Comment 1.- The work done by the authors are interesting and presented in a nice way. I just wanted to know why the authors have not used other food web models for trophic structure and prey-predator overlap for the comparison (e.g. ecopath model).

Reply 1: we appreciate your review of our manuscript. There is a wide variety of tools (models) applicable for trophic levels studies, however, we focus on the comparison of stable isotopes with stomach content as in other studies (e.g. Peta et al., 2020; Choi et al., 2021; Harbison et al., 2022; Klarian et al., 2022). The Stable Isotope Analysis (SIA) of carbon (δ¹³C) provides information of food resources and nitrogen (δ¹⁵N) on trophic position. This method is a powerful tool for measuring the trophic structure by describing the relative trophic positions of consumers within a community and the contributions of distinct basal resources (Svanbäck et al., 2015). The gut content analysis usually complements the stable isotope methodology (e.g., Laske et al., 2018), as also discussed in the paper. Stable isotopes and gut content analyses complement each other to gain more insight into the feeding relationships of fish species and the degree of dietary resource sharing (Mwijage et al., 2018).

All tools have strength and weaknesses. Ecopath, Ecosim and Ecospace are valuable tools that could have been used, but they are sensitive to the right identity of diet composition for species or functional group, and to give the correct flow of energy across trophic levels especially if fish shift niche during ontogeny (Pauly et al., 2000; Steenbeek et l., 2018). We think our approach is sufficient for this study, as we use two robust and complementary methods- stomach content analysis and stable isotopes.

The minor comments are mentioned in the MS.

Comment 1.- Line 41 Fish trophic structure or ecosystem trophic strcture

Reply 1.- we modified the sentences as recommended.

Comment 2.- line 117 to 120 Ethical approval for fish sample testing is required.

Reply 2.- we added the ethical approval for fish samples. Line 128-129: The fish were euthanized with an overdose of benzocaine.

Comment 3.- table 1, it should be DO

Reply 3.- changed.

References

Choi, B., Kim, W. S., Ji, C. W., Kim, M. S., & Kwak, I. S. (2021). Application of combined analyses of stable isotopes and stomach contents for understanding ontogenetic niche shifts in Silver Croaker (Pennahia argentata). International Journal of Environmental Research and Public Health, 18(8), 4073.

Harbison, T., Rogers, M., & Henkel, S. (2022). Assessing commercial fishery bait in Dungeness crab Cancer magister feeding ecology: δ13C and δ15N stable isotope and gut content analysis. Marine Ecology Progress Series, 700, 125-143.

Klarian, S. A., Schultz, E. T., Hernández, M. F., Valdes, J. A., Fernandoy, F., Barros, M. E., ... & Arancibia, H. (2022). Stomach contents and stable isotope analysis reveal ontogenetic shifts and spatial variability in Brama australis diet. Environmental Biology of Fishes, 1-10.

Laske, S.M., Rosenberger, A.E., Wipfli, M.S., Zimmerman, C.E., 2018, Generalist feeding strategies in Arctic freshwater fish: A mechanism for dealing with extreme environments, Ecology of Freshwater Fish, 27(3): 767–784. DOI: 10.1111/eff.12391

Mwijage, A.P., Shilla, D.A., Machiwa, J.F., 2018, Differences in trophic resources and niches of two juvenile predatory species in three Pangani estuarine zones, Tanzania: Stomach contents and stable isotope approaches, Journal of Biological Research, (Greece), 25(1):1–16. DOI: 10.1186/s40709-018-0084-4

Pauly, D., Christensen, V., and Walters, C. 2000. Ecopath, Ecosim, and Ecospace as tools for evaluating ecosystems impacts of fisheries. Journal of marine Science, 57: 696-706.

Petta, J. C., Shipley, O. N., Wintner, S. P., Cliff, G., Dicken, M. L., & Hussey, N. E. (2020). Are you really what you eat? Stomach content analysis and stable isotope ratios do not uniformly estimate dietary niche characteristics in three marine predators. Oecologia, 192, 1111-1126.

Steenbeek, J., Corrales, X., Platts, M., & Coll, M. (2018). Ecosampler: a new approach to assessing parameter uncertainty in Ecopath with Ecosim. SoftwareX, 7, 198-204.

Svanbäck, R., Quevedo, M., Olsson, J., Eklöv, P., 2015, Individuals in food webs: the relationships between trophic position, omnivory and among-individual diet variation, Oecologia, 178 (1): 103–104.

Weidner, T.A., Hirons, A.C., Leavitt, A., Kerstetter, D.W., 2017, Combined gut-content and stable isotope trophic analysis of the pelagic stingray Pteroplaytrygon violacea (Bonaparte, 1832) diet from the western North Atlantic Ocean, Journal of Applied Ichthyology, 33(3):386–394

Author Response File: Author Response.docx