Leaf Physiological Responses and Early Senescence Are Linked to Reflectance Spectra in Salt-Sensitive Coastal Tree Species

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Anderson et al sought to answer two primary questions: (1) Which common taxa found in freshwater forested wetlands of the southeastern U.S. are the most and least sensitive to elevated saltwater exposure? And, (2) what physiological responses are reliable indicators of plant performance when exposed to moderate pulses of seawater? The results showed that Acer rubrum was the most sensitive, and Juniperus virginia the most tolerant species to moderate levels of soil salinity after two exposures of seawater. Remotely sensed techniques might be used to provide early warning signals of salinity stress by detecting A. rubrum leaf spectral reflectance. The description of the experimental design, plant treatments, sample preparation, measurement methods, and data analysis are detailed. The interpretation of the data and the conclusion are appropriate. I therefore would only ask for a few minor revisions.

 

L429-430, “Interestingly, maximum soil salinity was greater (p < 0.01) in conifers (max = 0.66-0.79 ug Na⁺ g^-1) compared to hardwoods (max = 0.42-0.59 ug Na⁺ g^-1) (Table 1).” ug--->μg; In Table 1, mg/L should be converted to μg/g.

 

Table A1, please list the data for each of the 7 treatments (0.5-6 ppt) separately, rather than combining them on a single line.

 

L461, T. distichum? ---> P. taeda

 

L461, L464, Figure A5--->Figure A9. Or, merge Figure A9 into Figure A5

 

L478-L480, “The two evergreen tree species, J. virginiana and P. taeda, had different specific leaf area (SLA) compared to all other species. A. rubrum and Q. nigra had similar SLA, as did N. sylvatica and T. distichum (Figure A7).” In the control condition?

 

L478-490, should be rephrased more clearly. The ≥3ppt in the text seems to correspond to 4-6 ppt (high) but not 3 ppt (mid) in Figure A7. The resolution of this figure is poor. Cannot distinguish between asterisks and black points. And I don't see any brackets, any letters to indicate the significance of the difference. In addition, is the SLA unit cm²/g? Modify the title of the vertical coordinate in Figure B

 

L501, italic T. distichum

 

L502, “and a mean decrease in leaf %C”. leaf %C ---> C:N?

 

L503, “Additional biomass and growth rate results can be found in Appendix A.” which part of Appendix A?

 

Figure 4, The cell color does not match the annotations

 

Figure 5A and B are identical to Figure A10B and D. Replace Figure 5 with Figure A10. Alternatively, modify Figure A10

 

L561, 3.3--->3.4

Author Response

Comment 1: L429-430, “Interestingly, maximum soil salinity was greater (p < 0.01) in conifers (max = 0.66-0.79 ug Na⁺ g^-1) compared to hardwoods (max = 0.42-0.59 ug Na⁺ g^-1) (Table 1).” ug--->μg; In Table 1, mg/L should be converted to μg/g.

Response 1: We have scanned the entire document to ensure all units as ug are replaced with μg.

Comment 2: Table A1, please list the data for each of the 7 treatments (0.5-6 ppt) separately, rather than combining them on a single line.

Response 2: We have added basin water chemistry summary data to Table A1 for the treatments not previously presented. In addition, we changed the caption text and row titles to add clarity to what the values are showing, for example, the salinity stock use for dilutions in each of the seven salinity treatments. We agree that this addition is helpful and augments Figure 1A, which shows additional data over the course of the experiment in visual form.

Comment 3: L461, T. distichum? ---> P. taeda

Response 3: We have replaced T. distichum with the correct species name, P. taeda as shown in Figure A5.

Comment 4: L461, L464, Figure A5 ---> Figure A9. Or, merge Figure A9 into Figure A5

Response 4: We have corrected the referenced figure to Figure A6 to reflect the figure order as it appears in the manuscript. Subsequent figures A6, A7, and A8 titles were also changed to A7, A8, and A9 respectively.

Comment 5: L478-L480, “The two evergreen tree species, J. virginiana and P. taeda, had different specific leaf area (SLA) compared to all other species. A. rubrum and Q. nigra had similar SLA, as did N. sylvatica and T. distichum (Figure A7).” In the control condition?

Response 5: We have added clarity to this sentence to ensure readers understand we are referring to control trees not exposed to pulses of seawater.

Comment 6: L478-490, should be rephrased more clearly. The ≥3ppt in the text seems to correspond to 4-6 ppt (high) but not 3 ppt (mid) in Figure A7. The resolution of this figure is poor. Cannot distinguish between asterisks and black points. And I don't see any brackets, any letters to indicate the significance of the difference. In addition, is the SLA unit cm²/g? Modify the title of the vertical coordinate in Figure B

Response 6: We have rewritten this paragraph to improve clarity for the reader. Results for SLA and LDMC were broken into two paragraphs. We have also replaced the figure to incorporate the asterisks that were missing to ensure they are distinguishable from the outliers to match the figures caption. We have also corrected the y-axis label for the right LDMC panel.

Comment 7: L501, italic T. distichum

Response 7: Corrected

Comment 8: L502, “and a mean decrease in leaf %C”. leaf %C ---> C:N?

Response 8: We have replaced %C with C:N to reflect the results in Figure 4..

Comment 9: L503, “Additional biomass and growth rate results can be found in Appendix A.” which part of Appendix A?

Response 9: We have deleted this sentence since we had decided to not include any additional figures in the appendix to reduce confusion of the existing information within the results.

Comment 10: Figure 4, The cell color does not match the annotations

Response10: We have revisited Figure 4 to ensure the colors mentioned in the caption, annotations match what is shown in the figure.

Comment 11: Figure 5A and B are identical to Figure A10B and D. Replace Figure 5 with Figure A10. Alternatively, modify Figure A10

Response 11: We have removed the replicated panels in Figure A10 that were already presented in Figure 5 to ensure that only the hardwood species are shown in the Appendix. We wanted to include two panels in Figure 5 to highlight the physiological impacts of osmotic potential on Pinus taeda.

Comment 12: L561, 3.3--->3.4

Response 12: We corrected the subheading number to 3.4.

Reviewer 2 Report

Comments and Suggestions for Authors

The huge effort put into experimental set-up, data collection, analysis, and graphic presentation for this manuscript is unfortunately not reflected in its organization. I recommend moderate-to-major revision.

General comments

There are frequent references in the text to various graphs in the plethora of data in the appendix, suggesting that some of the appended graphs belong in the article itself.

There are still more male scientists than female, and at least 10% of men are red (orange)-green colorblind. Please make sure that contrasting data cited in the text is not invisible to a section of the readers. Use different symbols or lines to distinguish data, or choose other contrasting colors.

Please adhere to the Rule of the Decimal. If there are 3 or more digits to the left of the decimal, then no digits should be to the right of the decimal. Two digits to the left of the decimal allows only one digit to the right of the decimal. One or no digits to the left allows two digits to the right. If there is a need for more than two digits to the right of the decimal to make matters clear, then in most cases  the number should be multiplied by at least 100 for clarity of presentation.

Most researchers who read this journal think that 'ppt'  means 'precipitate' or is a typo for 'ppm'. That marine researchers think that it means 'parts per thousand' should be pointed out in the abstract, as well as in the introduction.

Fig 1 Can you cite some marsh species? 

line 123 divide the trees into conifers/hardwoods at this point

159 consider dropping Q. nigra altogether-- did you have any experiment with n=3 where all trees were the same age and condition?

169-70 Why not use actual seawater? why repeat yourself in line 290-91?  I understand that Na is dangerous to plants, but your Cl levels are way higher, and are likely more responsible for the noted effects. Please consider stressing that point.

Fig. 2 Not at all clear. How did you separate the water treatments in the basin, and why did you need one basin for three water treatments when three separate basins would be more sensible (although you would then have many many more containers to deal with)? How many trees were in each basin? You illustrate 6, but that's per water quality, not per statistical unit. 

214 to end of mss. See Rule of Decimal Place

249 you say you only looked at leaf loss, but then detail a lot more data than just that.

369 Tukey adjustment? You mean Tukey test for mean separation?

384 Hedge's g is all well and good, but it would be better to report the mean values from the treatments themselves in a table, with Hedge's g as a mean separation. I like the heatmap with Hedge's g, but I want to know the actual, not just relative, differences.

Fig. 3 Way way way too busy. In addition to the earlier comment about colorblindness, graphs b & c are way to small in size and in font used,. In general, graphing data where n=1 or if there are no indications of variability  is not a sustainable idea. Just show 0 and 3 ppt, and put other data in a table, if you must. 

Table 1 -- statistics?

Fig 4 still need averages, not just max and min

Fig 6 Better to show all species, as in A15. Graph is not needed, mean values and range are needed.

ref 34 -- fix the citation, unless JSTOR is paying you for advertising.

Fig A6 if you intend to publish it, pls add scale bar. The plant in Pic 5 looks very different in size and leaf shape/size to the other 4 plants in the figure.

Author Response

Comment 1: Fig 1 Can you cite some marsh species? 

Response 1: Our primary goal for presenting Figure 1 was to highlight where our study tend to occur in freshwater forest wetlands in relation to shrub-scrub and brackish marshes, however, to provide plant species that reflect the entire gradient, we have included several examples of shrub and marsh species to further highlight other commonly occurring taxa in the Coastal Plain of the Southeastern United States.

Comment 2: line 123 divide the trees into conifers/hardwoods at this point

Response 2: Beginning to decipher hardwoods and conifer species in the experiment will add clarify throughout. We have grouped the six species into hardwoods and conifers in the last paragraph of the introduction and made the appropriate changes throughout the document.

Comment 3: 159 consider dropping Q. nigra altogether-- did you have any experiment with n=3 where all trees were the same age and condition?

Response 3: This is an excellent point, and we also considered dropping Q. nigra; however, since there is a lack of quantitative assessment on the salt tolerance Q. nigra, we thought it would be helpful to the literature on the species even with less replication. Additionally, since salt pulses were added in the basins where all species were grown, and seawater ions were taken up by each species differently, we also thought it was important to report.

Comment 4: 169-70 Why not use actual seawater? why repeat yourself in line 290-91?  I understand that Na is dangerous to plants, but your Cl levels are way higher, and are likely more responsible for the noted effects. Please consider stressing that point.

Response 4: Using actual seawater and diluting it to the treatment concentrations would have been an excellent way to dose the trees. Since our primary goal was to isolate the salinization concentrations, we decided to use Instant Ocean (a commonly used product for experiments similar to this study) to standardize the treatments and minimize the possible introduction of various concentrations of pollutants, nutrients that may introduce outside impacts to plant performance and physiological performance. To ensure this is clear, we have included additional language so the readers know this was our primary consideration for using Instead Ocean. We have removed repeated information: “dissolved Instant Ocean^â, Spectrum Brands, Inc., Blacksburg VA, USA.” We stressed the point that sodium is the second most dominant ion in seawater behind chloride. This description was added to line 147-148 when introducing the dominance of the study species in North Carolina wetlands related to sodium concentration, which was us instead of chloride since soil concentrations of chloride were measured at all study sites (Andreson et al. 2022), however, they tested the correlation of sodium and chloride. Readers can also see that chloride is the leading contributor to salinity since we have reported concentrations in Table A1.

Comment 5: Fig. 2 Not at all clear. How did you separate the water treatments in the basin, and why did you need one basin for three water treatments when three separate basins would be more sensible (although you would then have many many more containers to deal with)? How many trees were in each basin? You illustrate 6, but that's per water quality, not per statistical unit. 

Response 5: We had a total of 22 basin, each with six trees. Each basin was assigned only one treatment. Soil salinity was measured in each of the six tree pots, and groundwater quality was measured in the water for each basin. We have added clarity in the caption and in the primary text for interpretation wherever possible.

Comment 6: 214 to end of mss. See Rule of Decimal Place

Response 6: We have corrected all numbers in the text and tables to follow the Rule of the Decimal Place as suggested.

Comment 7: 249 you say you only looked at leaf loss, but then detail a lot more data than just that.

Response 7: We removed this line since we decided to report more functional trait and biomass results in the manuscript.

Comment 8: 369 Tukey adjustment? You mean Tukey test for mean separation?

Response 8: Yes, correct. We have made this change in the text.

Comment 9: 384 Hedge's g is all well and good, but it would be better to report the mean values from the treatments themselves in a table, with Hedge's g as a mean separation. I like the heatmap with Hedge's g, but I want to know the actual, not just relative, differences.

Response 9: Figure 4 was created to present readers with a concise figure that summarize many results in a relatively easy to digest visualization. We agree that presenting the mean values for the two primary treatments (controls and 3 ppt) would augment Figure 4 to give readers access to the actual values and differences between treatments. Therefore, we added Table A3 in the appendix with the mean and standard deviation values for all six species.

Comment 10: Fig. 3 Way way way too busy. In addition to the earlier comment about colorblindness, graphs b & c are way to small in size and in font used,. In general, graphing data where n=1 or if there are no indications of variability is not a sustainable idea. Just show 0 and 3 ppt, and put other data in a table, if you must. 

Response 10: We agree that the size of figures is not sufficient especially to read the text and interpret the noted differences in the line and point graphs. We have stacked the three figures to each panel is more easily visualized as larger images, increased the size of the axis titles, and replace the green for controls with blue. We agree there is a lot of data shown here, but the figure size improves the legibility, and the non-replicated treatment trends are for readers to visualize the concentration over time in specific conductance for each treat in the basins and the soil at two depths.

Comment 11: Table 1 -- statistics?

Response 11: are unsure which statistics you think would be important to show. However, additional soil concentration statistics are reported in Table A2 which is mentioned on line 435. We have added a note in the caption that Table A2 has additional soils statistics.

Comment 12: Fig 4 still need averages, not just max and min

Response 12: We ran separate statistics for each of the twelve parameters shown in Figure 4 as mentioned in section 2.5. Statistical analysis. Our goal for using effect sizes was to largely summarize performance responses for each species in a concise figure. The values displayed in the heat map figure are single output values from Hedge’s g analysis based on control and 3ppt mean values.

Comment 13: Fig 6 Better to show all species, as in A15. Graph is not needed, mean values and range are needed.

Response 13: To improve interpretation, we decided to show Acer rubrum (as the most sensitive species related to spectral reflectance), 

Comment 14: ref 34 -- fix the citation, unless JSTOR is paying you for advertising.

Response 14: Edited citation to remove reference of JSTOR.

Comment 15: Fig A6 if you intend to publish it, pls add scale bar. The plant in Pic 5 looks very different in size and leaf shape/size to the other 4 plants in the figure.

Response 15: Although this figure highlights several aspects how T. distichum leaves changed and were dropped when exposed to 6ppt, we did not take the photos with a scale bar at the end of the experiment, and so we have decide to remove the figure. We have also adjusted the figure numbers in the appendix to reflect this change.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

the current study aimed to study salinity effects on coastal plants and could be considered of great importance. Overall, the study is great but some points should be considered to improve the study.

All comments were presented through the attached.

key points:

Abstract should give a total view of the study therefore add some key points of M&M as well as most results. then give a final conclusion based on ur achievements (recommendation for coastal plants)

Intro must have some key points regarding the studied plants. add them and some previous studies regarding salinity effects on them

M&M mostly were enough

results was presented in good manner as well

discussion also could be explained as good but could be improved by adding some litreture and the possible reasons for the results in some points

No conclusion was presented; strongly recommend to have the section or at least a paragraph giving final points

some minor points regarding English and scientific names should be considered

Comments for author File: Comments.pdf

Comments on the Quality of English Language

some minor points should be considered. overall, English was well and MS presented in good manner.

Author Response

Comment 1: Abstract should give a total view of the study therefore add some key points of M&M as well as most results. then give a final conclusion based on ur achievements (recommendation for coastal plants)

Response 1: We feel that the abstract covers all the mentioned elements of the manuscript. We have added a few additional details to highlight other reviewer’s concerns.

Comment 2: Intro must have some key points regarding the studied plants. add them and some previous studies regarding salinity effects on them

Response 2: The introduction covers several studies that have investigated several of the tree species (e.g.,Taxodium distichum, and Pinus taeda) we included in our experiment, however, several species we studied (e.g., Juniperus virginiana, and Acer rubrum) have very little known about their salt tolerance at lower levels of salinity relevant to field conditions. We include multiple key studies while maintaining a relatively concise introduction focused on our research questions.

Comment 3: M&M mostly were enough

Response 3: We feel that our Materials and Methods cover what was conducted in the study with the appropriate details.

Comment 4: results was presented in good manner as well

Response 4: We agree.

Comment 5: discussion also could be explained as good but could be improved by adding some litreture and the possible reasons for the results in some points

Response 5: We have revisited the discussion to consider highlighting additional outcomes from the results and/or literature that might strengthen the narrative.

Comment 6: No conclusion was presented; strongly recommend to have the section or at least a paragraph giving final points

Response 6: We had considered adding a conclusion, but our aim for the opening two paragraphs of the discussion were to highlight the major takeaways in the opening paragraphs of the discussion. However, we added a short paragraph reiterating the primary finds.

Comment 7: some minor points regarding English and scientific names should be considered

Response 7: We have reviewed all scientific names and grammar throughout the document for consistency and accuracy.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have done a large amount of work over a relatively short period of time (6 months). I have larger concerns (presentation of graphs in tiny unreadable fonts, reliance on single replication for presentation of data, use of Q. nigra in non-statistical amounts) and smaller ones (the first author misspelled his own name in a caption, there are incomplete sentences). Addressing the first concerns will result in a slightly different manuscript, addressing the secondary ones will make the manuscript more reliable. The first author seemingly insists on using n=1 as sufficient sampling, and varying amounts of Q nigra of varying ages as representative of the species. Statistics mandates that the first is just not so, while physiology mandates that the second is to be avoided. The organization of the 22 boxes used for modeling exposure to drought is still not clear-- if there are only 6 seedlings per box, how are they divided among 3 levels of irrigation? There are still too many graphs in both the manuscript and the supplementary material. Care should be taken to put supplementary material into the manuscript itself if such material is cited more than once.  The data themselves are interesting, but the material still has to be honed so that the intended audience can benefit from the authors' efforts.

Comments on the Quality of English Language

English is OK, although even one typo or fragmented sentence is one too many in an era of spellcheck and computer-assisted grammar checks.

Author Response

Comment 1: presentation of graphs in tiny unreadable fonts,

Response 1: The reviews concern regarding the readability of fonts in data visualization encouraged us to review each figure and replace any figures with poor image quality with higher resolution images. This included the primary figures in the manuscript and figures in the appendix. Several figures required increasing text size to improved legibility appropriate for the manuscript.  

Comment 2: reliance on single replication for presentation of data,

Response 2: We certainly agree that a single individual (n=1) is insufficient and unacceptable for attempting any statistical analyses. We believe there may be a misunderstanding of the experimental design. We have reviewed the methods and schematic in Figure 2 to edit for increased clarity. Due to the relatively complex design of the experiment, we intentionally included Lines 180-183 where we indicate the goal of the non-replicated trees exposed to intermediate salinity treatments compared to the control (coastal wetland surface water only) to the 3 ppt salinity treatment. In all of the figures that show a non-replicated intermediate treatment, we have added some clarification to the text (where figures are referenced, and in the caption) to ensure the reader knows intermediate treatments were binned, and no statistical analyses were conducted on non-replicated treatments alone. For example, in the caption of Figure 3 (line 419), we have noted all the grey lines are non-replicated treatments, and the figure is only displaying the trends of the raw monitoring data in the groundwater and soil at two depths, and no statistical models were conducted. We also offer intentional language on lines 396-397 mentioning the result of only the control and 3ppt treatments. In version 3, we have added additional context to figures A3, A4, A5, A6, A7, A10, A11, and A12 to make sure the reader is aware that we use the intermediate treatments by binning the three treatments (0.5, 1, and 2 ppt; and 4, 5, and 6 ppt)  for assessing broader trends in the differences in soil nutrients across all treatments. We have removed Figure A8 to ensure we do not misrepresent the results since it does show the 5 ppt treatment in addition to the replicated controls and 3 ppt salinity treatment. This figure was intended for data exploration only, and not for primary outcomes of the study, and needed to be removed. However, we kept the results of control and 3ppt in the results section (line 499) to report how the percentage of leaf carbon and nitrogen differ between the two replicated treatments of the experiment. We have also changed lines 432-434 where we mentioned a significant differences in soil moisture in non-replicated results of 0.5 ppt and 6 ppt treatment bins. We have removed mention of significant differences when referring to non-replicated salinity treatment, and removed Figures A1 and A2 per the reviewer suggestions since they simply display raw data and are not crucial for the study outcomes highlighted in this manuscript. These data are already summarized in Table A1 and Table A2.

Comment 3: Concerned with use of Q. nigra in non-statistical amounts; The first author seemingly insists on using n=1 as sufficient sampling, and varying amounts of Q nigra of varying ages as representative of the species. Statistics mandates that the first is just not so, while physiology mandates that the second is to be avoided.

Response 3: We have double checked the replication of Q. nigra as shown in Figure 2. We have also added clarification of when Q. nigra seedlings (grown from seed) we used in the study. As described above, we only conducted statistical analyses with the intermediate treatments if all non-replicated treatments were binned into low (0.5, 1, and 2ppt) or high (4, 5, 6 ppt) salinity. Certainly, we did not have enough replication for binning N. sylvatica. We added clarification in the methods section (lines 162-167) to add more transparency to what individual trees were used. Additionally, we removed portions of the results (e.g., line 530-535) that directly mention trends in non-replicated treatments, since without more replication, we can not say for certain what the species response is to the treatment without more individuals being tested.

Comment 4: There are incomplete sentences; English is OK, although even one typo or fragmented sentence is one too many in an era of spellcheck and computer-assisted grammar checks.

Response 4: After each edit of the manuscript in this round of revisions, we ran a spelling and grammar check on the entire document and corrected any suggested edits. We anticipate any misspellings are corrected and have reviewed the paper extensively for any sentences that were fragments are now corrected and highlighted to show the changes.

Comment 5: The organization of the 22 boxes used for modeling exposure to drought is still not clear-- if there are only 6 seedlings per box, how are they divided among 3 levels of irrigation?

Response 5: We have come to realize that the conceptual diagram and caption in Figure 2 is giving more detail than the reader needs for the results and conclusion gleamed from this paper. We believe this will add clarity. Water levels in the basins were kept between 8-16cm from the surface for the entirety of the experiment. As it was written in the last draft, we were offering the reader too much information regarding our questions of how root biomass changed by depth and treatment, however, this was not part of the current manuscripts aims. We have removed any language discussing soil saturation depths outside of where the water levels were kept throughout the experiment. It seems these changes will fix the misconception that drought was a part of this experiment. Tree species response to drought was not a question we sought to investigate in this study as we sought to isolate the impacts of low concentrations of salinity.

Comment 6: There are still too many graphs in both the manuscript and the supplementary material. Care should be taken to put supplementary material into the manuscript itself if such material is cited more than once. 

Response 6: We have made sure that all figures in the appendix are helpful to presenting the outcomes of the study, but not distracting from the primary message(s). Now, each figure in the appendix  is only referenced once, and we did not move any new figures to the manuscript since we feel as though they are supplementary, yet helpful for readers to have access to seeing the visual representation of the results we mention in the paper. We feel that each graphic in the primary text is critical to the manuscript. Our aim was to only have five figures for readability and reasonable length, but decided to include the conceptual diagram in Figure 1 to acclimate the reader to the problem and the questions we sought to answer.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did their best to concern the comments.

Author Response

Comment 1: The introduction could be improved to provide sufficient background and include all relevant references.

Responses 1: We have reviewed the introduction again to consider any necessary deletions or additions to improve clarity of the manuscripts background, questions, and methods for answering the questions outlined therein.