Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazer

Michelle Helmer; Desiree Helmer; Elizabeth Yohannes; Jason Newton; Daniel R. Dietrich; Dominik Martin-Creuzburg

doi:10.1371/journal.pone.0306173

Peer Review History

Original SubmissionMarch 11, 2024
27 May 2024 Decision Letter - Lee W Cooper, Editor PONE-D-24-09860Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazerPLOS ONE Dear Dr. Martin-Creuzburg, Thank you for submitting your manuscript to PLOS ONE. I want to start by apologizing for the length of time it took to have your manuscript reviewed. I contacted a number of potential reviewers, but it took a significant amount of time to identify two reviewers who were willing and able to review the manuscript. Having said that, these two reviewers judged that your manuscript will be suitable for publication after some minor changes are made. Both reviewers mentioned the number of significant units you are reporting stable isotope data for, meaning that the resolution reported is better than the analytical precision. Another joint comment was about the nitrogen isotope composition of nitrate often being below zero. Reviewer #2 also had a specific concern regarding the turnover time it takes to replace half of Daphnia’s tissue N and that it should not depend on the magnitude of difference in stable isotope signatures between consumer and food source. (Point #1). Both reviewers also made critical comments regarding Figure 3. I want to take this opportunity, therefore to invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 11 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: No ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is an interesting paper that details variations in the nitrogen isotope composition of cyanobacteria and Daphnia in controlled experiments that varied concentrations of nitrate, which provides insights on nitrate utilization relative to organic nitrogen generated through nitrogen fixation. I don’t think the paper explicitly proves that the nitrate starved experiments resulted in increases in nitrogen fixation, but these seem like reasonable assumptions, and perhaps a cleverly designed enriched isotope experiment would be of value as a follow-up. The changes observed in the feeding experiments are relatively small, a few per mil, but have consequences for understanding variation in nitrogen isotope ratios within foodwebs. The non-linear change observed in nitrogen isotope fractionation relative to nitrate concentrations is a particularly interesting finding. There is also an interesting investigation of organic material turnover time in Daphnia that could be emphasized more in the abstract. The discussion is particularly well-written and provides good context for the data presented by citing a number of prior studies. Line 61. I am not sure I would say that 15N is an “excellent” tracer---nitrogen cycling is complex from the standpoint of isotopic fractionation. On the next line it is stated that diatomic nitrogen gas is isotopically depleted in 15N relative to nitrate, but this isn’t universally true. Atmospheric nitrate is commonly depleted in 15N relative to N2, so if nitrate in aqueous sources has been in part derived from atmospheric deposition or as a result of nitrification in soils, it is possible that the nitrate will have negative delta values relative to N2 gas. One recent paper that shows this is Matiatos, I., Wassenaar, L.I., Monteiro, L.R. et al. Global patterns of nitrate isotope composition in rivers and adjacent aquifers reveal reactive nitrogen cascading. Commun Earth Environ 2, 52 (2021). https://doi.org/10.1038/s43247-021-00121-x Line 67-68. Again, the full range of nitrogen isotope composition of nitrate is not acknowledged here. The paper cited, Soto et al. 2019, is a study of a single lake basin in Canada, and doesn’t provide a global overview of the full range of nitrate isotopic compositions possible. I don’t think it makes the follow-on statements untrue about the potential use of 15N as a tracer, but the introduction doesn’t convey the full complexity of isotopic transformations that are possible in the nitrogen cycle. Line 149. Isotope masses on the delta values need to be superscripted. Was the isotopic composition of the sodium nitrate used in the study measured, and was it homogeneous? Line 209. Since the relationship between the nitrate concentration and the nitrogen isotope composition is not linear (Figure 1a), is it appropriate to report a Pearson correlation coefficient? Doesn’t that reflect a linear correlation that clearly isn’t present here? Line 211. Since the reported precision for the nitrogen isotope measurements is 0.15 per mil, reporting data to two significant digits, e.g. -0.72 is not consistent with that reported precision. The data reported throughout the manuscript are reported to two decimal places. Figure 3b. Since there isn’t an obvious correlation between DIN and the carbon isotope composition, the sinusoidal function that is plotted on the figure doesn’t really have any meaning. Line 258 Treatment should be treatments Table 3. Again, as with the nitrogen isotope data, since the precision is 0.09 per mil, (~0.1 per mil), it is dubious to use two decimal places to report the data. Some editing of the bibliography would be of benefit. For example, superscripts and subscripts where needed, e.g. Baker et al. 2018, Fry and Sherr, 1989, Loick-Wilde et al. 2012, McClelland et al. 2003, Oelbermann and Scheu, 2002; italicization of genus and species names, e.g. Bradburn et al. 2012; capitalization of proper nouns, e.g. Ambrose and DeNiro reference; inconsistent capitalization of all words in an article title, e.g. Karlson et al. 2014, Meeks et al. 1983; more bibliographic information needed for an edited book, e.g. Montoya, 2008. Reviewer #2: This manuscript presents experimental results showing how the freshwater diazotroph Trichormus variabilis switches from N-fixation to use of nitrate when cultured with increasing nitrate concentration. Changes in the stable N isotope ratios of Trichormus in different nitrate treatments were used to demonstrate the switch. Stable isotope analyses were also used to show how changes in stable N isotope values in Trichormus are passed on to a consumer (Daphnia). Stable carbon isotope ratios were also measured in this study, though the focus is primarily on nitrogen. The experiments present in this paper were well designed and well executed, and the resultant data are instructive. The results largely support expectations based on previous work, but controlled laboratory studies of N switching by diazotrophs and stable isotopic relationships between diazotrophs and their consumers are nonetheless useful as a basis for refining our ability to interpret field observations. Some suggestions to improve the manuscript are provided below. 1) While most of the findings make sense, I’m a bit perplexed by the findings related to stable N isotope equilibration rates between Daphnia and Trichormus in the different treatments. The time it takes to replace half of Daphnia’s tissue N with the new food source signature should not depend on the magnitude of difference in stable isotope signatures between the consumer and its food source. Are you suggesting that somehow N from Trichormus grown with increasing amounts of nitrate becomes more easily assimilated by Daphnia? Or is this result related to the precision of the isotope measurements? The smaller the difference in isotopic value between food source and consumer, the more uncertain the estimate of tissue turnover time becomes (the error surrounding the measurements becomes an increasing proportion of the difference between them). In the case of Trichormus grown with 28 mg/L DIN (Table 2, bottom row), the initial and equilibrated isotope values are essentially the same. How can an equilibration time even be calculated when this is the case? This part of the manuscript needs some clarification. 2) Throughout paper – Reporting results to two places after the decimal point indicates a level of precision that is not supported. Rounding to one place after the decimal would be more appropriate. 3) Lines 61-62. The blanket statement that N2 is depleted in 15N compared to nitrate is not correct. Stable N isotope values in rainwater nitrate are often below zero. 4) I suggest deleting Figure 3. Since any potential linkage between Daphnia and DIN concentration is via their consumption of Trichormus, Figure 2 should is sufficient. Plotting Daphnia values versus DIN concentrations suggests a direct linkage that does not exist. ****** 6. 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Revision 1
7 Jun 2024 Author Response Response to Reviewers – Helmer et al. Reviewer #1: This is an interesting paper that details variations in the nitrogen isotope composition of cyanobacteria and Daphnia in controlled experiments that varied concentrations of nitrate, which provides insights on nitrate utilization relative to organic nitrogen generated through nitrogen fixation. I don’t think the paper explicitly proves that the nitrate starved experiments resulted in increases in nitrogen fixation, but these seem like reasonable assumptions, and perhaps a cleverly designed enriched isotope experiment would be of value as a follow-up. The changes observed in the feeding experiments are relatively small, a few per mil, but have consequences for understanding variation in nitrogen isotope ratios within food webs. The non-linear change observed in nitrogen isotope fractionation relative to nitrate concentrations is a particularly interesting finding. There is also an interesting investigation of organic material turnover time in Daphnia that could be emphasized more in the abstract. The discussion is particularly well-written and provides good context for the data presented by citing a number of prior studies. Response: Thank you for this very positive feedback. As suggested, we now emphasize our results on nitrogen turnover rates already in the abstract by adding: ‘Nitrogen isotope turnover rates of Daphnia were highest in the absence of DIN as a consequence of N2 fixation and resulting depletion in 15N at the primary producer level.’ Line 61. I am not sure I would say that 15N is an “excellent” tracer---nitrogen cycling is complex from the standpoint of isotopic fractionation. On the next line it is stated that diatomic nitrogen gas is isotopically depleted in 15N relative to nitrate, but this isn’t universally true. Atmospheric nitrate is commonly depleted in 15N relative to N2, so if nitrate in aqueous sources has been in part derived from atmospheric deposition or as a result of nitrification in soils, it is possible that the nitrate will have negative delta values relative to N2 gas. One recent paper that shows this is Matiatos, I., Wassenaar, L.I., Monteiro, L.R. et al. Global patterns of nitrate isotope composition in rivers and adjacent aquifers reveal reactive nitrogen cascading. Commun Earth Environ 2, 52 (2021). https://doi.org/10.1038/s43247-021-00121-x Response: Thank you for pointing out the interesting paper, which we are now also citing and thus highlighting the complexity of possible isotopic fractionations that are happening in the nitrogen cycle. Line 67-68. Again, the full range of nitrogen isotope composition of nitrate is not acknowledged here. The paper cited, Soto et al. 2019, is a study of a single lake basin in Canada, and doesn’t provide a global overview of the full range of nitrate isotopic compositions possible. I don’t think it makes the follow-on statements untrue about the potential use of 15N as a tracer, but the introduction doesn’t convey the full complexity of isotopic transformations that are possible in the nitrogen cycle. Response: Please, see above Line 149. Isotope masses on the delta values need to be superscripted. Was the isotopic composition of the sodium nitrate used in the study measured, and was it homogeneous? Response: Has been changed. Yes, the isotopic composition of sodium nitrate was homogeneous. Line 209. Since the relationship between the nitrate concentration and the nitrogen isotope composition is not linear (Figure 1a), is it appropriate to report a Pearson correlation coefficient? Doesn’t that reflect a linear correlation that clearly isn’t present here? Line 211. Since the reported precision for the nitrogen isotope measurements is 0.15 per mil, reporting data to two significant digits, e.g. -0.72 is not consistent with that reported precision. The data reported throughout the manuscript are reported to two decimal places. Response: That’s right. We used the asymptotic regression model to describe the non-linear relationship between DIN and delta15N, as described in the method section. The Pearson statistics was erroneously reported here and has now been removed. Thanks for pointing this out. We agree with the comment on the reported precision and removed the second decimal place of the delta values throughout the manuscript. Figure 3b. Since there isn’t an obvious correlation between DIN and the carbon isotope composition, the sinusoidal function that is plotted on the figure doesn’t really have any meaning. Response: Yes, probably not. We removed the function from the Figure 3b. Line 258 Treatment should be treatments Response: Has been changed Table 3. Again, as with the nitrogen isotope data, since the precision is 0.09 per mil, (~0.1 per mil), it is dubious to use two decimal places to report the data. Response: Has been changed Some editing of the bibliography would be of benefit. For example, superscripts and subscripts where needed, e.g. Baker et al. 2018, Fry and Sherr, 1989, Loick-Wilde et al. 2012, McClelland et al. 2003, Oelbermann and Scheu, 2002; italicization of genus and species names, e.g. Bradburn et al. 2012; capitalization of proper nouns, e.g. Ambrose and DeNiro reference; inconsistent capitalization of all words in an article title, e.g. Karlson et al. 2014, Meeks et al. 1983; more bibliographic information needed for an edited book, e.g. Montoya, 2008. Response: The reference list has been revised Reviewer #2: This manuscript presents experimental results showing how the freshwater diazotroph Trichormus variabilis switches from N-fixation to use of nitrate when cultured with increasing nitrate concentration. Changes in the stable N isotope ratios of Trichormus in different nitrate treatments were used to demonstrate the switch. Stable isotope analyses were also used to show how changes in stable N isotope values in Trichormus are passed on to a consumer (Daphnia). Stable carbon isotope ratios were also measured in this study, though the focus is primarily on nitrogen. The experiments present in this paper were well designed and well executed, and the resultant data are instructive. The results largely support expectations based on previous work, but controlled laboratory studies of N switching by diazotrophs and stable isotopic relationships between diazotrophs and their consumers are nonetheless useful as a basis for refining our ability to interpret field observations. Some suggestions to improve the manuscript are provided below. Response: Thank you for this positive feedback. 1) While most of the findings make sense, I’m a bit perplexed by the findings related to stable N isotope equilibration rates between Daphnia and Trichormus in the different treatments. The time it takes to replace half of Daphnia’s tissue N with the new food source signature should not depend on the magnitude of difference in stable isotope signatures between the consumer and its food source. Are you suggesting that somehow N from Trichormus grown with increasing amounts of nitrate becomes more easily assimilated by Daphnia? Or is this result related to the precision of the isotope measurements? The smaller the difference in isotopic value between food source and consumer, the more uncertain the estimate of tissue turnover time becomes (the error surrounding the measurements becomes an increasing proportion of the difference between them). In the case of Trichormus grown with 28 mg/L DIN (Table 2, bottom row), the initial and equilibrated isotope values are essentially the same. How can an equilibration time even be calculated when this is the case? This part of the manuscript needs some clarification. Response: Well, the switch from N2 fixation to DIN uptake resulted in a positive relationship between delta15N values of Trichormus and the concentration of DIN, because N2 was depleted in 15N relative to DIN. Thus, the differences in delta15N values between Trichormus and Daphnia become smaller with increasing DIN concentration in the Trichormus growth medium and, consequently, it takes longer after die diet switch until isotopic equilibrium in Daphnia is achieved at low DIN than at high DIN concentrations. We used the exponential model of Hobson and Clark (1992) to calculate these turnover rates (https://doi.org/10.2307/1368807). The data reported in Table 2 in the bottom row were from a linear decay model (as was explained in the Table caption) that was used for the highest DIN concentration, because the exponential decay model used for the lower DIN concentrations could not be applied here. However, we agree that due to the lack of differences between the initial and equilibrated isotope values, it makes no sense to calculate an equilibration time at the highest DIN concentration and have therefore deleted the corresponding values from the table. 2) Throughout paper – Reporting results to two places after the decimal point indicates a level of precision that is not supported. Rounding to one place after the decimal would be more appropriate. Response: We agree. Has been changed. 3) Lines 61-62. The blanket statement that N2 is depleted in 15N compared to nitrate is not correct. Stable N isotope values in rainwater nitrate are often below zero. Response: This issue has been also raised by Reviewer 1 with reference to Matiatos et al. 2021, showing that negative delta15N values of nitrate may result from atmospheric nitrate deposition or nitrification in soil (https://doi.org/10.1038/s43247-021-00121-x). We are now citing this paper and included a respective statement on the complexity of isotopic transformations within the nitrogen cycle. 4) I suggest deleting Figure 3. Since any potential linkage between Daphnia and DIN concentration is via their consumption of Trichormus, Figure 2 should is sufficient. Plotting Daphnia values versus DIN concentrations suggests a direct linkage that does not exist. Response: That's right, the link between Daphnia and DIN is indirect via the consumption of Trichormus. Nevertheless, we find this relationship interesting, as it illustrates that the effect of DIN availability and thus the switch from nitrogen fixation to DIN uptake is also reflected at the consumer level, which might be of interest also for future field studies. Reviewer 1 suggested to remove the rather meaningless function from Figure 3b (delta13C), and we followed this suggestion. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306173.r002
10 Jun 2024 Decision Letter - Lee W Cooper, Editor PONE-D-24-09860R1Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazerPLOS ONE Dear Dr. Martin-Creuzburg, Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 25 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Sincerely, Lee Cooper Additional Editor Comments: I appreciate the careful attention to the reviewer recommendations and I am ready to accept the paper for publication, and offer my congratulations. I did see a couple of reference issues that might be best taken care of now though. References 22 and 39 have more than 6 authors, and the "Vancouver" style that PLOS One uses calls for "et al" to be placed after the sixth author. For these two references, the sixth author is followed by "u.a." which my spouse, who has German heritage confirms means "et al" in German. Could you please change those two references, and simply re-submit the paper without a rebuttal letter, and I will make sure the next version goes on for editorial acceptance to the journal office? I apologize, but I can't make those simple changes using the editor's platform, at least I don't think so. I appreciate again your efforts to address the reviewer concerns, and look forward to receiving a revised version of the manuscript. [Note: HTML markup is below. Please do not edit.] [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] - - - - - For journal use only: PONEDEC3 https://doi.org/10.1371/journal.pone.0306173.r003
Revision 2
11 Jun 2024 Author Response Dear Dr. Cooper, I uploaded a new file "Manuscript_ref revised" with the requested changes to References 22 and 39. I apologize for not spotting this while revsing the manuscript. Best wishes Dominik Martin-Creuzburg Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0306173.r004
12 Jun 2024 Decision Letter - Lee W Cooper, Editor Dissolved nitrogen uptake versus nitrogen fixation: Mode of nitrogen acquisition affects stable isotope signatures of a diazotrophic cyanobacterium and its grazer PONE-D-24-09860R2 Dear Dr. Martin-Creuzburg, Thank you for addressing my final concerns with the two references. I'm pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing any required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Lee W Cooper, Ph.D. Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306173.r005
Formally Accepted
24 Jun 2024 Acceptance Letter - Lee W Cooper, Editor PONE-D-24-09860R2 PLOS ONE Dear Dr. Martin-Creuzburg, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Lee W Cooper Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306173.r006

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