Green Infrastructure Microbial Community Response to Simulated Pulse Precipitation Events in the Semi-Arid Western United States

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Some corrections in text

Check reference again

It can be publish in the journal

Comments for author File: Comments.pdf

Author Response

General comments:

Some corrections in text

Please see responses to specific comments below.

Check reference again

We found some errors in the references list that we have now rectified. Thank you for bringing this to our attention.

It can be publish in the journal

We hope the manuscript is accepted based on the changes made in response to reviewer comments.

Specific Comments:

Line 53-55 – add reference

The statement in that line is a widely recognized phenomenon. Nonetheless, we added the following citations:

Collins, S.L.; Sinsabaugh, R.L.; Crenshaw, C.; Green, L.; Porras-Alfaro, A.; Stursova, M.; Zeglin, L.H. Pulse Dynamics and Microbial Processes in Aridland Ecosystems. Journal of Ecology 2008, 96, 413–420, doi: 10.1111/j.1365-2745.2008.01362x.

Sinsabaugh, R.L.; Shah, J.J.F. Ecoenzymatic Stoichiometry and Ecological Theory. Annu Rev Ecol Evol Syst 2012, 43, 313–343.

Lines 87-89 – add reference

We did not add a citation to this statement as it is our premise.

Line 98 - ????

According to the Merriam-Webster dictionary of English language, ‘bunchgrass’ is a categorical term referencing grasses whose growth form is a tuft. The term is commonly used to reference grasses in the western U.S. and is found as a term without definition in many scientific papers of the western U.S. As such, we did not elect to define it or the term ‘shrub’ that precedes it.  

Lines 262-285, 309-322, 389-396 – please rewrite

The request to rewrite these sections of text is appreciated but too vague to know how to improve upon the existing text. In addition, Reviewer 3 complimented us on the text of the Methods. We feared making changes to the text that may displease Reviewer 3 (and potentially other reviewers) without clear direction.

Line 203 – Add reference for plant names

We added a sentence after the listing of plant common and scientific names as follows:

“Common and scientific names for all plants are based on listings found within the U.S. Department of Agriculture PLANTS database [36].”

Citation 36 is:

United States Department of Agriculture Natural Resources Conservation Service. USDA Plants Database. Available online: https://plants.sc.egov.usda.gov/home (accessed on 14 May 2020).

Figure 1 caption – Add reference

We are unclear about the comment to add a reference in the caption. No photo credits are needed, as all photos were taken by the project team. Nonetheless, this caption was edited to provide more detail as suggested by another reviewer.

Figure 3 caption – recheck again

We are not clear what ‘recheck again’ is implying. We added site names and experimental pulse event month as headers above columns, similar to other figures. We also edited this caption for clarity.

References – check

We found slight errors in the references list and fixed them. Thank you for bringing this to our attention.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses the issue of soil and green infrastructure processes under different plant compositions under semi-arid conditions during two simulated rainfall events. The authors compare soil moisture, soil pH, organic matter content, organic and inorganic N, 4 enzymes and microbial N and C using statistical methods. The methods of investigation are adequately detailed and the results are presented in an appropriate form without the need for further elaboration. The conclusion of the work is supported by the results obtained. The literature used is relevant to the problem addressed, mostly published after 2000.
Specific comment:
Figure 1: I assume that plot 1 and plot 2 are reversed. If not, it would be useful to add why they are interchanged with the other plots (4, 5, 7 and 8).

Author Response

General comments:

The manuscript addresses the issue of soil and green infrastructure processes under different plant compositions under semi-arid conditions during two simulated rainfall events. The authors compare soil moisture, soil pH, organic matter content, organic and inorganic N, 4 enzymes and microbial N and C using statistical methods. The methods of investigation are adequately detailed and the results are presented in an appropriate form without the need for further elaboration. The conclusion of the work is supported by the results obtained. The literature used is relevant to the problem addressed, mostly published after 2000.

Thank you for this synthesis of our work.

Specific comment:

Figure 1: I assume that plot 1 and plot 2 are reversed. If not, it would be useful to add why they are interchanged with the other plots (4, 5, 7 and 8).

We included more text in the figure caption to explain the distribution of plant treatments within study plots.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is aimed at solving the important and urgent problem of replacing natural ecosystem functions with artificial green infrastructure in the urban environment. The authors used modern research methods, including experiment, laboratory and statistical analysis. The data obtained can undoubtedly be useful for creating SGI in arid regions. However, some of the authors' conclusions regarding the influence of plant diversity on the microbial community require clarification. Firstly, the identified differences between GIRF plots without plants and with plants cannot be interpreted as the influence of plant diversity on the soil community. These results show the influence of the presence of plants as such, regardless of their diversity. Secondly, it would be useful to emphasize that the authors did not find significant effects of species number on microbial community in the experimental plots, while enzyme activity in a natural meadow differs significantly from experimental plots, which may, among other things, also be a result of the influence of higher species diversity.

There are also a number of smaller shortcomings in the article, listed below.

Line 24. I recommend emphasizing here that you did not find the effect of plant diversity on the functioning of the microbial community in the experiment. The natural grassland community you studied, which probably contains more species than the experimental communities, is functionally different from them, for example, in ecoenzyme activity.

Lines 27-28. Your results show that the natural meadow differs significantly from experimental communities in response of ecoenzyme activity to the addition of water.

Line 91. Reference [5]. After 2001, the BEF theory and the results of its testing in both experimental and natural systems have advanced significantly. A number of works have been published summarizing these results. This issue was included in the IPBES reports. References to more recent publications would be appropriate here.

Lines 118-126. This paragraph seems more appropriate for the Methods section.

Lines 177-178. It's a little unclear. Were there 34 plants in each diverse plot and 24-25 plants in each grass plot?

Line 202. Approximate values of species richness at studies natural meadow would be useful as you are discussing influence of species richness on microbial community. Of course, it is impossible to compare a natural meadow and experimental plots, but this would give an idea of how much they differ in species richness.

Line207-208. Figure 1.

-        Please, indicate that the white rectangle without a corner is the state of Utah. Not all readers have detailed knowledge of US geography. And, it seems, it is more useful for readers to show the location of the study area not in the state of Utah, but in the United States.

-        Photos of the plots should be larger. So, I can't see anything.

Line 224. I think the diagram S1 should be moved to the main text, since it is basically important for understanding the study.

Line 269. The subheading “2.4.3. Lab Processing” looks illogical given the presence of section “2.5. Lab Methodology”.

Lines 484-487. Figure 2. I advise to clarify the figure caption. The graphs in the bottom row don’t show changes, but all values at once.

Line 496. Check the figure number 3.2A, please.

Lines 591-592. Fig. 4F shows that the increase in AP activity in the meadow was significant.

Line 808. Fig. 7 shows no sign of an increase in nitrogen content in the meadow.

Lines 853-855. In the case of soil moisture (Fig. 2), differences were found between GIRF plots without plants and with plants, and there is no difference between grass and diverse plots. Thus, this result does not indicate the influence of plant diversity on the soil community. It shows the influence of the presence of plants as such, regardless of their diversity.

Lines 856-867. It would be useful to emphasize here that you did not find significant effects of species number on microbial community in the experimental plots. At the same time, enzyme activity in the natural meadow differs significantly from experimental plots, which may, among other things, also be a result of the influence of higher species diversity.

Lines 976-979. Your results show that artificial communities differ from natural ones in some important ways, such as the response of enzyme activity to the addition of water.

Author Response

General comments:

The article is aimed at solving the important and urgent problem of replacing natural ecosystem functions with artificial green infrastructure in the urban environment. The authors used modern research methods, including experiment, laboratory and statistical analysis. The data obtained can undoubtedly be useful for creating SGI in arid regions. However, some of the authors' conclusions regarding the influence of plant diversity on the microbial community require clarification. Firstly, the identified differences between GIRF plots without plants and with plants cannot be interpreted as the influence of plant diversity on the soil community. These results show the influence of the presence of plants as such, regardless of their diversity.

We appreciate this nuanced perspective and have added detail to the text of the manuscript to address this point. Please see responses to Reviewer 3 – Specific Comments below for more details.

Secondly, it would be useful to emphasize that the authors did not find significant effects of species number on microbial community in the experimental plots, while enzyme activity in a natural meadow differs significantly from experimental plots, which may, among other things, also be a result of the influence of higher species diversity.

Species richness and species identity at the reference meadow was similar to the species richness within ‘grass’ plots at GIRF, although plant cover differed between the two sites. However, these details were unclear in the initial submission of manuscript. We have clarified species richness and plant cover in the Methods and revisited the difference in plant cover in the Discussion. Please see responses to Reviewer 3 – Specific Comments below for more details.

We also included text to the manuscript Methods section better defining why data from the reference meadow was included in the present study as follows:

“Todd’s Meadow (TM), a montane grassland area in the Red Butte Canyon Research Natural Area, served as a comparative non-SGI reference site used to ascertain if responses observed within SGI are apparent in an established natural ecosystem with similar plant species and species richness as one of the GIRF plant treatments.”

Specific comments:

There are also a number of smaller shortcomings in the article, listed below.

Line 24. I recommend emphasizing here that you did not find the effect of plant diversity on the functioning of the microbial community in the experiment. The natural grassland community you studied, which probably contains more species than the experimental communities, is functionally different from them, for example, in ecoenzyme activity.

We have included mention in the abstract that plant diversity at GIRF did not have an effect on ecoenzyme activities. It has resulted in an Abstract length in excess of 2 words that we hope the Editor will allow.

Lines 27-28. Your results show that the natural meadow differs significantly from experimental communities in response of ecoenzyme activity to the addition of water.

The reviewer’s point is valid. Yet, the majority of studied variable responses were similar in trend, if not magnitude, between the SGI facility plots and the reference meadow plots. Indeed, differences in the relative rates of hydrolytic vs. oxidative enzymes existed, but these differences did not translate into differences in microbial growth (i.e., biomas C content) or N uptake (i.e., biomass N content) or a significant shift in soil N pools. As such, we left these details to the text of the manuscript given the limited length of the Abstract set by the journal. We added the word ‘largely’ before ‘comparable’ to the last sentence of the Abstract as a qualifier, resulting in an Abstract length in excess of 3 word that we hope the Editor will allow.

Line 91. Reference [5]. After 2001, the BEF theory and the results of its testing in both experimental and natural systems have advanced significantly. A number of works have been published summarizing these results. This issue was included in the IPBES reports. References to more recent publications would be appropriate here.

This is a very valid point, especially given BEF is a major framework for the study. We have added four additional, more recent citations. These include two reviews of current knowledge that have contributed to IPBES reports over the years.

Hooper, D.U.; Chapin, F.S.; Ewel, J.J.; Hector, A.; Inchausti, P.; Lavorel, S.; Lawton, J.H.; Lodge, D.M.; Loreau, M.; Naeem, S.; et al. Effects of Biodiversity on Ecosystem Functioning: A Consensus of Current Knowledge. Ecol Monogr 2005, 75, 3–35, doi:10.1890/04-0922.

de Bello, F.; Lavorel, S.; Díaz, S.; Harrington, R.; Cornelissen, J.H.C.; Bardgett, R.D.; Berg, M.P.; Cipriotti, P.; Feld, C.K.; Hering, D.; et al. Towards an Assessment of Multiple Ecosystem Processes and Services via Functional Traits. Biodivers Conserv 2010, 19, 2873–2893, doi:10.1007/s10531-010-9850-9.

Cardinale, B.J.; Duffy, J.E.; Gonzalez, A.; Hooper, D.U.; Perrings, C.; Venail, P.; Narwani, A.; MacE, G.M.; Tilman, D.; Wardle, D.A.; et al. Biodiversity Loss and Its Impact on Humanity. Nature 2012, 486, 59–67, doi: 10.1038.nature11148.

Oliver, T.H.; Heard, M.S.; Isaac, N.J.B.; Roy, D.B.; Procter, D.; Eigenbrod, F.; Freckleton, R.; Hector, A.; Orme, C.D.L.; Petchey, O.L.; et al. Biodiversity and Resilience of Ecosystem Functions. Trends Ecol Evol 2015, 30, 673–684, doi: 10.1016/j.tree.2015.08.009.

Lines 118-126. This paragraph seems more appropriate for the Methods section.

Agreed. We moved the vast bulk of this text to the Methods as suggested.

Lines 177-178. It's a little unclear. Were there 34 plants in each diverse plot and 24-25 plants in each grass plot?

We edited the text in question for increased clarity. Specifically, we included the species richness of each plant treatment in addition to the enumeration of plant number per plot.  We also added the desired plant cover of each plot to the following sentence to better clarify why there were differences in the number of plants established in plots of different plant diversity treatments. The text now reads:

“Diverse treatments contained 11 species with a total of 34 plants per plot and grass treatments contained 3 species with a total of 24-25 plants per plot (Table S1). The number of plants in these plots varied due to differences in aboveground biomass between grasses and shrubs. Plantings aimed to achieve a similar percentage of aboveground cover (~50% of plot area).”

Line 202. Approximate values of species richness at studies natural meadow would be useful as you are discussing influence of species richness on microbial community. Of course, it is impossible to compare a natural meadow and experimental plots, but this would give an idea of how much they differ in species richness.

We both clarified and added more detail about species richness and plant cover in this section of the Methods as follows:

“We established three study plots with dimensions 3 m x 4 m (12 m²) within a 48m² (12m long, 4m wide) area of Todd’s Meadow dominated by blue wildrye (Elymus glaucus), Kentucky bluegrass (Poa pratensis), and cheatgrass (Bromus tectorum; Figure 1) [39]. This species composition was similar to grass plots at GIRF. Forb species within these plots were rare (i.e., 1 or 2 per plot). Unlike GIRF, plant cover was close to 100% in all plots.”

Line207-208. Figure 1.

Please, indicate that the white rectangle without a corner is the state of Utah. Not all readers have detailed knowledge of US geography. And, it seems, it is more useful for readers to show the location of the study area not in the state of Utah, but in the United States.

We added a label showing it is the state of UT in the United States, but refrained from showing a map of the US because the study site locations within UT would then be more difficult to discern. Readers can easily find where the state of UT is located within the US via the internet.

Photos of the plots should be larger. So, I can't see anything.

We removed one photo from beneath the Todd’s Meadow plots, as it only shows the climate station but may confuse readers regarding the plant composition of plots. This also allowed us to increase the size of the photos below the study site plot diagrams for both GIRF and Todd’s Meadow. We added note in caption indicating that photos below plots show landscape views of each site.

Line 224. I think the diagram S1 should be moved to the main text, since it is basically important for understanding the study.

Earlier, unsubmitted versions of our manuscript included Figure S1 in the main text. We decided to move it to the supplementary information prior to submission to Water because it seemed redundant to Figures 8-10 in the manuscript. We also recognized that the main text already has a lot of figures. We agree with the reviewer that this figure helps conceptualize our study, but still feel it is appropriate in the supplement as it will still be available to readers and limits manuscript length.

Line 269. The subheading “2.4.3. Lab Processing” looks illogical given the presence of section “2.5. Lab Methodology”.

We reorganized the Methods section and renumbered sections appropriately to make the suggested change. We agree the previous organization was illogical.

Lines 484-487. Figure 2. I advise to clarify the figure caption. The graphs in the bottom row don’t show changes, but all values at once.

We edited the caption for Figure 2 (and all figures) for greater clarity.

Line 496. Check the figure number 3.2A, please.

We fixed figure reference typo to now accurately reference a figure in the supplementary information.

Lines 591-592. Fig. 4F shows that the increase in AP activity in the meadow was significant.

The trend in Figure 4F appears significant, yet we found weak evidence of significance (p = 0.08). This is why no lines with asterisks appear in Figure 4F.

Line 808. Fig. 7 shows no sign of an increase in nitrogen content in the meadow.

We found an increase in soil inorganic (Fig. 7F) but not organic N (Fig. 7C) pools at the reference meadow. However, we acknowledge the previous text in this section was unclear. This paragraph now reads:

“Soil moisture is key driver of microbial processes in semi-arid to arid ecosystems [11] and proves to be an important factor governing rates of N cycling and removal from SGI in both field [17,63] and mesocosm studies [15,64,65]. Yet, SGI systems also display a wide range of variability with respect to rates of N cycling because factors other than moisture availability also regulate N pools and fluxes in SGI soils [66]. Such variability was apparent in the present study where we observed modest increases in pools of soil proteins at both GIRF in September 2020 and at Todd’s Meadow, as well as elevated organic N at GIRF in September 2020, and elevated inorganic N at Todd’s Meadow in response to pulse events. In contrast, pools and fluxes of soil N were largely unresponsive to wetting at GIRF in June 2021. Furthermore, proteolytic rate was invariant amongst all pulse events.”

Lines 853-855. In the case of soil moisture (Fig. 2), differences were found between GIRF plots without plants and with plants, and there is no difference between grass and diverse plots. Thus, this result does not indicate the influence of plant diversity on the soil community. It shows the influence of the presence of plants as such, regardless of their diversity.

This is a valid point. We omitted mention of plant diversity effects on gravimetric soil moisture in the first sentence of this paragraph. The first three sentences of this paragraph are now more accurate:

“In our study, differences in plant diversity at GIRF resulted in significant differences with respect to pre-pulse values of microbial C:N ratios and the content of soil organic matter, proteins, and N pools. However, differences amongst plant treatments were inconsistent between pulse events. In the case of soil moisture, differences were related to the presence of plants, rather than plant diversity.”

Lines 856-867. It would be useful to emphasize here that you did not find significant effects of species number on microbial community in the experimental plots. At the same time, enzyme activity in the natural meadow differs significantly from experimental plots, which may, among other things, also be a result of the influence of higher species diversity.

We made several changes throughout the text to clarify the conception that species richness differed between Todd’s Meadow and the grass plots, while still acknowledging differences in plant cover that could affect organic matter accumulation, with cascading effects on ecoenzyme activity rates. As to the line in question, we clarified that this sentence was in reference to observations at GIRF alone.

Lines 976-979. Your results show that artificial communities differ from natural ones in some important ways, such as the response of enzyme activity to the addition of water.

We edited the final sentence of the manuscript to provide more detail and nuance to our conclusion as follows:

“We conclude that microbial community N acquisition and soil N dynamics in SGI of semi-arid to arid ecosystems that infiltrate water infrequently and dry rapidly behave similarly to regional vegetated communities when soil media composition is comparable to neighboring natural areas (i.e., low in organic matter and nutrient content, basic pH), despite differences in ecoenzyme activity rates mediating N availability and utilization.”

Reviewer 4 Report

Comments and Suggestions for Authors

The idea of the study is very interesting. The manuscript is generally well organized and all stages are presented in a very methodological way.

 

Main comments and suggestions for Authors:

 

1. The title is clear. Key words are well selected and in line with the topic and the study itself.

The Abstract is rather general but the main results are presented.

2. The section of Introduction is developed and includes wide presentation of the background related to the topic. The Authors focused on main and important aspects for their study. The literature used is much developed and in line with presented aspects, also based on current studies.

The main aim of the study is rather hidden in the part preceding the formulation of research questions, while it must be clearly formulated – this element must be improved. The 3 research questions are clear Authors included also rather wide explanation related to their expectations, what is a bit surprising, and indicates the range of expected results before their presentation.

 

3. Section 2. Materials and Methods is well divided into subsections. The study area is well presented and described. The used methods are also clearly presented with references to literature, what is easy to repeat by other scientists.

 

4. Section 3. Results is a strong part of the manuscript and contains key information; main results were divided into aspects and logically presented.  

 

5. Section 4. Discussion is clearly related to the results obtained. The Authors discuss them in a very methodological manner following the answers for the main 3 research questions. A certain inaccuracy is that these 3 questions included in the subsection titles are formulated a bit differently than in the Introduction part - they should be identical in both parts of the manuscript. The relations to other current studies are reach. Some limitations of the conducted study could be also added in my opinion.

 

6. The Conclusions are supported by the results. Some limitations of the study could be also formulated in my opinion.

 

Summing up, the presented study is valuable and its presentation is very well organized and argued. Just small improvement is related to the formulation more clearly the main aim of the study, and the formulation of research questions in the Discussion section should be identical to those in the Introduction section.

I can recommend the publication of the manuscript after very small improvement related to the above-mentioned elements.

Author Response

General comments:

The idea of the study is very interesting. The manuscript is generally well organized and all stages are presented in a very methodological way.

Thank you.

Specific comments:

Main comments and suggestions for Authors:

 

1. The title is clear. Key words are well selected and in line with the topic and the study itself.

The Abstract is rather general but the main results are presented.

Thank you. We would like to include more detail to the Abstract but are limited by the 200 word count limit.

2. The section of Introduction is developed and includes wide presentation of the background related to the topic. The Authors focused on main and important aspects for their study. The literature used is much developed and in line with presented aspects, also based on current studies.

Thank you.

The main aim of the study is rather hidden in the part preceding the formulation of research questions, while it must be clearly formulated – this element must be improved.

We now include an aims statement immediately preceding the research questions as follows:

“This study aimed to elucidate the relative role of plant diversity vs. ecological stoichiometry for controlling microbial community and soil N responses to pulsed water events in SGI of a semi-arid region experiencing a period of extended drought. Specifically, we ask: (followed by research questions)”

The 3 research questions are clear 

Thank you.

Authors included also rather wide explanation related to their expectations, what is a bit surprising, and indicates the range of expected results before their presentation.

The reviewer’s comment about the range of explanations reflects our recognition of the ecological complexity of both designed and natural systems. We also wanted readers to realize we had a competing set of alternative hypotheses reflecting this complexity.  

3. Section 2. Materials and Methods is well divided into subsections. The study area is well presented and described. The used methods are also clearly presented with references to literature, what is easy to repeat by other scientists.

Thank you.

4. Section 3. Results is a strong part of the manuscript and contains key information; main results were divided into aspects and logically presented.  

Thank you.

5. Section 4. Discussion is clearly related to the results obtained. The Authors discuss them in a very methodological manner following the answers for the main 3 research questions. A certain inaccuracy is that these 3 questions included in the subsection titles are formulated a bit differently than in the Introduction part - they should be identical in both parts of the manuscript. The relations to other current studies are reach.

The research questions and headers in the Discussion now match.

Some limitations of the conducted study could be also added in my opinion.

The section of the Discussion formerly labeled ‘Caveats’ is now labeled ‘Study Limitations’, given that describing study limitations was the intent of the paragraph. Labelling this section ‘Caveats’ apparently made that unclear.

6. The Conclusions are supported by the results. 

Thank you.

Some limitations of the study could be also formulated in my opinion.

In addition to the section in the Discussion on ‘Study Limitations’, we added two sentences in the conclusions about BEF and ES not being mutually exclusive, with a supporting citation. This section now reads:

“We utilized biodiversity-ecosystem function (BEF) and ecological stoichiometry (ES) theories as frameworks to assess the dominant mechanisms controlling microbial community responses to precipitation pulses, and subsequent effects on nutrient retention and cycling in bioswales planted with three vegetation treatments and a montane grassland meadow within a semi-arid environment. We found greater support for the ecological stoichiometry of microbial biomass and resource supply (as inferred by ecoenzymatic activity) vs. plant diversity for controlling N dynamics in SGI post experimental wetting. However, biodiversity effects may have been masked by similarities in plant cover at GIRF, and ostensibly biomass production, resulting in inconsistencies between plant treatments with regards to organic matter availability that was more directly linked to N response variables. Indeed, the two theoretical frameworks are not mutually exclusive. There is growing recognition that ecological stoichiometry represents a mechanistic link between biodiversity and ecosystem function across levels of biological organization [82]. SGI systems represent laboratories within human landscapes in which this linkage can be further tested.”

The citation we added for this section is:

Hillebrand, H.; Cowles, J.M.; Lewandowska, A.; Van de Waal, D.B.; Plum, C. Think Ratio! A Stoichiometric View on Biodiversity-Ecosystem Functioning Research. Basic Appl Ecol 2014, 15, 465–474, doi:10.1016/j.base.2014.06.003.

We also noted earlier in the discussion (present in original submission) that more in situ studies are needed to test our ideas. Finally, we believe the caveats in our final sentence related to the context of our study (i.e., systems infiltrating water infrequenty, drying rapidly, and having similar soil matrices (low organic matter and nutrient content, basic pH)) sets limits on the generalizations that can be made from our study.

Summing up, the presented study is valuable and its presentation is very well organized and argued. Just small improvement is related to the formulation more clearly the main aim of the study, and the formulation of research questions in the Discussion section should be identical to those in the Introduction section.

Thank you. We believe we have addressed all concerns.

I can recommend the publication of the manuscript after very small improvement related to the above-mentioned elements.

We hope the manuscript is accepted based on the changes made in response to reviewer comments