Interactive Effects of Rising Temperature, Elevated CO2 and Herbivory on the Growth and Stoichiometry of a Submerged Macrophyte Vallisneria natans
Round 1
Reviewer 1 Report
Thank you for the opportunity to review this ms! It is an interesting and important study, which have explored how increased temperature, elevated CO2, snail herbivory and their interactions impacted on the growth and stoichiometric traits of a typical submerged macrophyte Vallisneria natans. The authors further analyzed how temperature and CO2 impacts the growth and stoichiometric traits of the herbivorous snail Radix. In general, the experiment is well performed and the study is well interpreted. However, I think there are still some parts of the ms can be improved before accept.
Title
The authors used warming, however, only two different temperatures were used in this study. I would suggest to correct it to rising temperature, as warming seems to be rising temperature above the fluctuated ambient temperature, not a constant temperature.
The authors indicated interactions in the title, however, I could not find how they quantified interactions in the ms. I would suggest to focus on the macrophyte, as the design of the experiment is a full factorial design with three factors crossed.
The impacts on snails are interesting but not many significant effects were found, and should not be the emphasis for this paper.
The title can be corrected to: Interactive effects of rising temperature, elevated CO2 and herbivory on the growth and stoichiometry of a submerged macrophyte Vallisneria natans
Abstract
The description of the results should be reorganized according to the new title.
Line 11 Herbivory interaction
Line 12 nutrient cycle
Introduction
Line 28 Submerged plants are a group of ecological groups ?
Line 33 herbivory should not be a key reason for submerged macrophyte decline, as it is just a normal phenomenon in nature, but other factors, such as eutrophication.
Line 34 affecting to?
Line 42 Ocean temperature? This reference is not the best as your study is in freshwater ecosystem.
Line 49, 55, 57, 74, 78…… the format of citations are not correct
Materials and Methods
Line 94 need a citation
Line 97 the temperatures were set to 27 and 23 ̊C
Line 101, what are the shape and size of the pots?
The abbreviation of the eight treatments can be clearer: C for control, low temperature, low CO2 and without herbivory; Tem for rising temperature; CO2 for elevated CO2; Her for herbivory.
Statistical analysis
Not sure why use a two-way Anova after the three-way anova, I would suggest to use posthoc tests after the three-way anova to distinguish the differences among the eight groups.
Figure 2 Should the low temperature put in front of the high temperature treatments? Same for CO2 and herbivory. And differences among the groups can be indicated by different letters. Same for Figure 3, 4 and 5.
Figure 3, 4, 5 The scales of the y-axis can be narrowed to make the bars more comparable.
The ms should be thoroughly checked and corrected by a more sophisticated researcher.
Author Response
Response to Reviewer 1 Comments
Point 1: The authors used warming, however, only two different temperatures were used in this study. I would suggest to correct it to rising temperature, as warming seems to be rising temperature above the fluctuated ambient temperature, not a constant temperature.
Response 1: Thanks for your suggestions. We correct "warming" to "rising temperature".
Point 2: The authors indicated interactions in the title, however, I could not find how they quantified interactions in the ms. I would suggest to focus on the macrophyte, as the design of the experiment is a full factorial design with three factors crossed.
Response 2: Thanks for your suggestions. We have made revision.
Point 3: The impacts on snails are interesting but not many significant effects were found, and should not be the emphasis for this paper.
Response 3: Thanks for your suggestions. We have made revision.
Point 4: The title can be corrected to: Interactive effects of rising temperature, elevated CO2 and herbivory on the growth and stoichiometry of a submerged macrophyte Vallisneria natans.
Response 4: Thanks for your suggestions. We have corrected the title to: "Interactive effects of rising temperature, elevated CO2 and herbivory on the growth and stoichiometry of a submerged macrophyte Vallisneria natans."
Point 5: The description of the results in abstract should be reorganized according to the new title.
Response 5: Thanks for your suggestions. We have revised "Abstract " according to the new title.
Point 6: Line 11 Herbivory interaction
Response 6: Thanks for your suggestions. We have made revision.
Point 7: Line 12 nutrient cycle
Response 7: Thanks for your suggestions. We have made revision.
Point 8: Line 28 Submerged plants are a group of ecological groups?
Response 8: Thanks for your suggestions. We have revised this sentence: "Submerged plants play an important role in the stability of structure and function of aquatic ecosystem."
Point 9: Line 33 herbivory should not be a key reason for submerged macrophyte decline, as it is just a normal phenomenon in nature, but other factors, such as eutrophication.
Response 9: Thanks for your suggestions. We have revised this sentence: "submerged plants in shallow lakes have experienced a decline in recent decades for many reasons, including climate change and eutrophication." And we also replaced the reference No. 8.
- Xing, W.; Wu, H.; Hao, B.; Liu, G. Stoichiometric Characteristics and Responses of Submerged Macrophytes to Eutrophication in Lakes along the Middle and Lower Reaches of the Yangtze River. Ecol. Eng. 2013, 54, 16–21, doi:10.1016/j.ecoleng.2013.01.026.
Point 10: Line 34 affecting to?
Response 10: Thanks for your suggestions. We have revised this sentence: " Climate changes are affecting submerged plants and mainly shown in the increase of temperature and atmospheric CO2 concentration."
Point 11: Line 42 Ocean temperature? This reference is not the best as your study is in freshwater ecosystem.
Response 11: Thanks for your suggestions. We have revised this sentence: " In addition, studies have predicted that the temperatures would increase more than 2°C due to greenhouse gas effects in 2100." And the reference (IPCC. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (Eds.)]. IPCC, Geneva, Switzerland; 151 pp.) is suitable.
Point 12: Line 49, 55, 57, 74, 78…… the format of citations are not correct.
Response 12: Thanks for your suggestions. We have made revision in the ms.
Point 13: Line 94 need a citation
Response 13: Thanks for your suggestions. We added two new references.
- He, L.; Zhu, T.; Wu, Y.; Li, W.; Zhang, H.; Zhang, X.; Cao, T.; Ni, L.; Hilt, S. Littoral Slope, Water Depth and Alternative Response Strategies to Light Attenuation Shape the Distribution of Submerged Macrophytes in a Mesotrophic Lake. Front. Plant Sci. 2019, 10, doi:10.3389/fpls.2019.00169.
- Stift, M.; Michel, E.; Sitnikova, T.Y.; Mamonova, E.Y.; Sherbakov, D.Y. Palaearctic Gastropod Gains a Foothold in the Dominion of Endemics: Range Expansion and Morphological Change of Lymnaea (Radix) auricularia in Lake Baikal. Hydrobiologia 2004, 513, 101–108, doi:10.1023/B:hydr.0000018175.37771.d6.
Point 14: Line 97 the temperatures were set to 27 and 23 ̊C
Response 14: Thanks for your suggestions. We have made revision.
Point 15: Line 101, what are the shape and size of the pots?
Response 15: Thanks for your suggestions. We have revised this sentence: " 64 plastic pots (d = 23 cm, h = 9 cm) were divided into 8 CO2 and temperature treatments "
Point 16: The abbreviation of the eight treatments can be clearer: C for control, low temperature, low CO2 and without herbivory; Tem for rising temperature; CO2 for elevated CO2; Her for herbivory.
Response 16: Thanks for your suggestions. We still retain the original abbreviations. Although these abbreviations are not clearer than the abbreviations you proposed, they may be easier to organize and explain in the chart.
Point 17: Not sure why use a two-way Anova after the three-way anova, I would suggest to use posthoc tests after the three-way anova to distinguish the differences among the eight groups.
Response 17: In general, when we perform three-way ANOVA, we will adopt the following process: (1) Determine whether there is a three-factor interaction. (2) if there is a three-factor interaction, determine whether there is a simple two-factor interaction; if there is no three-factor interaction, then determine whether there is a two-factor interaction. (3) if there is a simple two-factor interaction, determine whether there is a simple single effect; if there is no simple two-factor interaction, end the analysis; if there are two factors interaction, determine whether there is a separate effect; if there is no two-factor interaction, end the analysis; (4) If there is a simple separate effect, then judge whether the simple pairwise comparison is meaningful.
We used posthoc tests after the three-way anova, and performed a paired comparative analysis and corrected it with the Bonferroni method.
Point 18: Should the low temperature put in front of the high temperature treatments? Same for CO2 and herbivory. And differences among the groups can be indicated by different letters. Same for Figure 3, 4 and 5.
Response 18: Thanks for your suggestions. We have revised Figure 2, 3, 4 and 5.
Point 19: Figure 3, 4, 5 The scales of the y-axis can be narrowed to make the bars more comparable.
Response 19: Thanks for your suggestions. We have adjusted the scale of the y-axis of Figure 2, 3, 4 and 5.
Point 20: The ms should be thoroughly checked and corrected by a more sophisticated researcher.
Response 20: Thanks for your suggestions. We have checked and corrected the ms again.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments for authors:
The authors explored the effects of the increase in temperature and CO2 concentration on a submerged plant, herbivorous snail, and their herbivory relationship, and found that the increase in temperature and elevated CO2 could affect the relationships. This paper deals with relevant and interesting topic and is potentially a valuable contribution for understanding effects of global climate changes on plant-animal interactions. However, there are numerous problems, some of them major, that need to be addressed. The following is my concerns.
1. The introduction seems to be out of focus possibly because there are many small paragraphs on temperature and CO2 effects. I recommend that the authors combine some introduction paragraphs.
2. The authors need to provide testable hypothesis and prediction we can follow in the introduction. I recommend that the authors predict how the increases in temperature and CO2 concentrations could affect the herbivory relationships in the introduction.
3. The authors used parametric statistical analyses (e.g., ANOVA) in this paper; however, I am wondering whether the data have normal distributions or not. Thus, I recommend that authors explain if the statistical analyses used for this paper are appropriate for the experimental data.
4. In the discussion, the authors need to better clarify whether there were significant effects of temperature and CO2 concentrations on the submerged plant, snail, and the herbivory relationships based on the results of their experiments.
5. The conservation/management suggestions are limited in this study. I recommend that the authors write the more specific conservation/management strategies based on your results.
Author Response
Response to Reviewer 2 Comments
Point 1: The authors explored the effects of the increase in temperature and CO2 concentration on a submerged plant, herbivorous snail, and their herbivory relationship, and found that the increase in temperature and elevated CO2 could affect the relationships. This paper deals with relevant and interesting topic and is potentially a valuable contribution for understanding effects of global climate changes on plant-animal interactions. However, there are numerous problems, some of them major, that need to be addressed. The following is my concerns.
Response 1: Thanks for your suggestions. We have checked and corrected the ms again.
Point 2: The introduction seems to be out of focus possibly because there are many small paragraphs on temperature and CO2 effects. I recommend that the authors combine some introduction paragraphs.
Response 2: Thanks for your suggestions. We have combined some introduction paragraphs.
Point 3: The authors need to provide testable hypothesis and prediction we can follow in the introduction. I recommend that the authors predict how the increases in temperature and CO2 concentrations could affect the herbivory relationships in the introduction.
Response 3: Thanks for your suggestions. We have revised the last paragraph of the introduction: " Whether the responses of submerged macrophytes to climate changes, which include rising temperature and elevated CO2 concentrations, and herbivory, which include herbivorous fish and snails, etc., remains unclear. And the effects of climate change factors and herbivory on submerged macrophytes may be additive, multiplicative, synergistic, or antagonistic [40–43]. Addressing this will get a better understanding of the response of submerged macrophytes to climate changes and herbivory, as well as aid prediction the impact of climate change on freshwater ecosystems. Here, we studied the response of a submerged macrophytes (Vallisneria natans L.) to rising temperature, elevated CO2 concentrations and herbivory by an herbivorous snail (Radix auricularia L.). Specifically, we (1) assessed the effects of rising temperature, elevated CO2 concentrations and herbivory on the growth and stoichiometry of V. natans; (2) explored the effects of rising temperature, elevated CO2 concentrations on the growth and stoichiometry of R. auricularia; and (3) generated new information that could be used to aid prediction the impact of climate change on freshwater ecosystems."
Point 4: The authors used parametric statistical analyses (e.g., ANOVA) in this paper; however, I am wondering whether the data have normal distributions or not. Thus, I recommend that authors explain if the statistical analyses used for this paper are appropriate for the experimental data.
Response 4: Sorry for the unclear expression. We have made revision: " If the data did not satisfy homogeneity of variances or normal distribution of residuals, they were log transformed before analysis."
Point 5: In the discussion, the authors need to better clarify whether there were significant effects of temperature and CO2 concentrations on the submerged plant, snail, and the herbivory relationships based on the results of their experiments.
Response 5: Thanks for your suggestions. In major revision, we have corrected the title to: "Interactive effects of rising temperature, elevated CO2 and herbivory on the growth and stoichiometry of a submerged macrophyte Vallisneria natans.", and focus on the submerged macrophyte.
Point 6: The conservation/management suggestions are limited in this study. I recommend that the authors write the more specific conservation/management strategies based on your results.
Response 6: Thanks for your suggestions and sorry for the unclear expression. This study focused on the effects of climate change (rising temperature and elevated CO2) and herbivory on submerged plants, in order to aid prediction of the impact of climate change on freshwater ecosystems.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript has been improved substantially! Thanks for answering all the questions! However, there are still a few points needs to be further clarified.
Line 22-25 “rising temperature, elevated CO2 concen-22 trations and herbivory”, these are repeated in the same sentence.
Line 86-90 As suggested by the other reviewer, these are not hypotheses, please consider revision.
Statistical part
The authors first use three-way ANOVA, then two-way ANOVA, if there was a three-way interaction. Are you doing model selection or just want to indicate interactions of factors? More clarifications are needed and strong literature support should be provided for your statistics.
Results and discussion
The authors have found the growth of the plant (leaf number and biomasses) was inhibited with rising temperature, this is not consistent with others’ study, more discussion should be added.
Author Response
Response to Reviewer 1 Comments
Point 1: Line 22-25 “rising temperature, elevated CO2 concentrations and herbivory”, these are repeated in the same sentence.
Response 1: Thanks for your suggestions. We correct this sentence.
Point 2: Line 86-90 As suggested by the other reviewer, these are not hypotheses, please consider revision.
Response 2: Thanks for your suggestions. The Reviewer 2 comment is that “The authors need to provide testable hypothesis and prediction we can follow in the introduction. I recommend that the authors predict how the increases in temperature and CO2 concentrations could affect the herbivory relationships in the introduction. “. So, we revised our introduction. However, the effects of climate change factors and herbivory on submerged macrophytes may be additive, multiplicative, synergistic, or antagonistic., we didn’t 't give a clear hypothesis.
Point 3: The authors first use three-way ANOVA, then two-way ANOVA, if there was a three-way interaction. Are you doing model selection or just want to indicate interactions of factors? More clarifications are needed and strong literature support should be provided for your statistics.
Response 3: Thanks for your suggestions. We used three-way ANOVA (Full factorial model) to analysis our data and added a new strong literature to support our statistical analysis method.
- McCarthy, T.M.; Fisher, W.A. Multiple Predator‐avoidance Behaviours of the Freshwater Snail Physella heterostropha pomila : Responses Vary with Risk. Freshw. Biol. 2000, 44, 387–397, doi:10.1046/j.1365-2427.2000.00576.x.
Point 4: The authors have found the growth of the plant (leaf number and biomasses) was inhibited with rising temperature, this is not consistent with others’ study, more discussion should be added.
Response 4: Thanks for your suggestions. Some studies have shown that some submerged plants are suitable for growth at 15 °C -25 °C, and the growth rate becomes significantly slower after exceeding 25 °C. However, due to the different species and experimental conditions, the results are different, so we could only infer that this result may be caused by species-specificity of aquatic plants in response to raised temperature.
Author Response File: Author Response.pdf
Reviewer 2 Report
I think that the authors respond to my comments well. I recommend that the authors check the manuscript again to make sure if there are minor mistakes in the manuscript. Thank you for giving me the opportunity for reviewing this manuscript.
Author Response
Response to Reviewer 2 Comments
Point 1: I think that the authors respond to my comments well. I recommend that the authors check the manuscript again to make sure if there are minor mistakes in the manuscript. Thank you for giving me the opportunity for reviewing this manuscript.
Response 1: Thanks for your suggestions. We have checked and corrected the ms again.
Author Response File: 
Author Response.docx
