Responses of Fine Root Functional Traits to Soil Nutrient Limitations in a Karst Ecosystem of Southwest China

Round  1

Reviewer 1 Report

Great job authors. The manuscript is well written and well structured. Please find my specific questions on methodology, and line 234 of Figure 1 and some minor editorials comments throughout the attached file. 

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

Thank you very much for reviewing our manuscript (forests-380262). 

The comments from you were very helpful. We took your comments into consideration in preparing the revised manuscript. The revisions had been marked in the file named “manuscript (marked)”. The responses to the comment and suggestion were listed as below.

Thank you again for the helpful comments. If you have any further suggestions, please do not hesitate to contact us.

Yours sincerely Kelin Wang

 10 NOV. 2018

Responses to reviewer (Q refers to the question and R for the response)

Responses to reviewer #1

Q1: Whould be great to have a photo of the studied shrub and tree species in Karst ecosystem, if available.

R1: Thanks for your advice. I am very sorry that we could not take any photos of the studied shrub and tree species since 2014. But, you can get their informations from websites as follows.

Alchornea trewioides (Shrub): http://www.plantphoto.cn/tu/720504

Ligustrum sinense (Shrub): http://www.plantphoto.cn/tu/4650856

Celtis biondii (Tree): http://www.plantphoto.cn/tu/4260885

Pteroceltis tatarinowii (Tree): http://www.plantphoto.cn/tu/88157

Q2: Any picture from the Karst landscape?

R2: Thanks for your helpful advice. I had provided the figure in the revised manuscript (Figure1).

Q3: What kind of soil sampler you used?

R3: Thanks for your helpful advice. Fine roots and soils were collected from the topsoil at 0–15 cm depth with the excavation method. If we selected one target plant, we would dig out the fine roots and soils from the topsoil at 0–15 cm depth. The fine roots were then lightly and carefully hand shaken to remove bilk soil after loosening. The remaining soil on the roots, i.e., the rhizosphere soil, was also lightly loosened (to minimize the losing of fine roots in the sampled soil) and carefully brushed away in the field. (Lines 145-151 in the revised manuscript).

Q4: Why 87 samples? how you decide this sample size is enough for your study in Karst lands?

R4: Thanks for your helpful advice. In the present study, we wanted to select four dominant species, and to collect four replicate soil samples of each species in lower, middle, and upper slope in two seasons. The total amounts of sample were collected based on this regulation, as four plant species × three slope positions × two seasons × four replicates (three replicates if we could not find a fourth individual at a particular site). That was our first try to study the response of fine root functional traits to environment changes. This study, we had collected 87 rhizosphere soil samples, 87 bulk soil samples, and 87 fine root samples. I am very sorry that it is very difficult to answer you whether or not the sample size is enough.

Other errors had also been corrected in the revised manuscript

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors, although I appreciate all the effort done I must recognize that the work cannot be published in the present form. There are several issues that needs to be fixed and that I have addressed in the attached file. In particular, severa references are missing in my opinion concerning important advances in fine root dynamics and their importance for forest restoration strategies. The introduction is not including the state of the art for soil N and P in the rhizosphere and non-rhizosphere. The Mat&Met section is insufficient and need to be more deeply explained as well as need to be reformatted. Also it is not clear how measurements were done, if soil analysis were related to both sampling point. Also there are several mistakes concerning SRL. It is not clear what does it means non growing seasons and so on. Finally I would like to underline that relationship tbetween fine roots and soil nutrient availability is not clear. It seems that the control is missing or at least a sort of pattern of nutrients and fine root to be related each other. Soil analysis seems to be done only at one sampling point. You need to better explaint eh relationship with soil nutrient and roots. Comments in the attached file will help to work on these issues.  

Comments for author File: Comments.docx

Author Response

Dear reviewer,

Thank you very much for reviewing our manuscript (forests-380262). 

The comments from you were very helpful. We took your comments into consideration in preparing the revised manuscript. The revisions had been marked in the file named “manuscript (marked)”. The responses to the comment and suggestion were listed as below.

Thank you again for the helpful comments. If you have any further suggestions, please do not hesitate to contact us.

Yours sincerely Kelin Wang

 10 NOV. 2018

Responses to reviewer (Q refers to the question and R for the response)

Responses to reviewer #2

Q1: Please refer each reference to the specific issue (i.e. acquisition strategies (1), enhanced genome size (2) etc.

R1: Thanks for your helpful advice. We had been modified them. (Lines 53-57 in the revised manuscript).

Q2: This issue of fine root plasticity has been widely investigate by Montagnoli et al. specifically in 2012 (Ecol Res), 2014 (Eur J For Res) and 2019 (ForEco). Please pay attention to these paper and include them in the citation. Water limitation is also leading to plasticity in roots and often water limitation is the cause of nutrient limitation.

R2: Thanks for your helpful advice. I had read and cited your papers. And, I had overwritten the introduction section. (Lines 58-100 in the revised manuscript).

Q3: Same here. You might have read the papers from Montagnoli et al. in relation to plant strategies and fine roots.

R3: Thanks for your helpful advice. I had read and cited your papers. And, I had overwritten the introduction section. (Lines 58-100 in the revised manuscript).

Q4: What do you mean with vegetation restoration? Please explain better, is that referred to Forest restoration strategies? Afforestation or reforestation? Assisted migration? On this purpose you might have a look to Montagnoli et al. 2018 (Plant Biosystems) and Chiatante et al. 2015 (J of Plant Research) concerning the importance of studying morphological traits of seedling of different species for forest restoration activities.

R4: Thanks for your helpful advice. I had overwritten the introduction section. (Lines 58-100 in the revised manuscript).

Q5: This part of the paper work must be explained in the Introduction. Can you please explain and refer to other work the issue of using oxalic acid, and what do you mean with rhizosphere? Moreover, what do you mean with non-growing seasons. These species have evergreen or deciduous habit? Why did you measure nitrogen content in the roots tissues. See Terzaghi et al. 2013-2016

R5: Thanks for your helpful advice. I had overwritten the introduction section. (Lines 58-100 in revised manuscript). And, I had re-analyzed our database. Please see the changes of Table 1-3, figure 3-5 in the revised manuscript.

Q6: Sampling:In general this sub-section is not clear. Please be more specific and detail how you made these things. Also how did you sort roots from the soil core? On a sieve? 2 mm sieve? By hand, or just washed them automatically? This is important because you could have miss some of the fine roots in the sampled soil.

Please specify how you did the sampling. Which method? The soil coring technique?

R6: Thanks for your helpful advice. In this study, the fine roots and soils of each individual plant of each species were collected from the topsoil at 0–15 cm depth with the excavation method and the intact segments were extracted with minimal breakage. The fine roots were then lightly and carefully hand shaken to remove bilk soil after loosening. The remaining soil on the roots was also lightly loosened (to minimize the losing of fine roots in the sampled soil) and carefully brushed away in the field. Please see lines 136-161 in the revised manuscript.

Q7: I don’t understand if the non rhizosphere soil has been collected only once or two times. Table 1 indicate only one sampling point.

R7: Thanks for your helpful advice. Bulk soils (non rhizosphere soils) had also been collected two times. We calculated the average values in the original manuscript. We had re-analyzed our database. Please see the Table 1 in the revised manuscript.

Q8: Resuts: The first results sub-section must be ”Soil chemical characteristics” rhizosphere and non-rhizosphere. If this is the source of the plasticity in the plant root. This is missing!

R8: Thanks for your helpful advice. We had re-analyzed our database and had overwritten this part. Please see the Table 1 and lines 216-223 in the revised manuscript.

Q9: Resuts: This is obvious: if non-growing season is considered as the vegetative rest period (you must explain it earlier in the manuscript).

R9: Thanks for your helpful advice. We had explained the non-growing season and growing season in lines 137-141 (Sampling section).

Q10: Discussion: This is true, but is also strictly related to the water content in the soil. Low water low nutrients higher SRL higher root tip number. Again see Montagnoli’s works

This is depending on plant strategy adoption: intensive or extensive. Facing drought or avoiding drought.

In Amendola et al. 2017 was demonstrated that when soil water content is higher root growth is mainly radial thus SRL is lower. Please specify and refer to that paper.

(Season) This need a better fusion with the discussion of drought and my comments. However, although you have measured soil chemicals you did not measure soil water content. Have you measured it?

R10: Thanks for your helpful advice. Based on your suggestions, we collected database of soil temperature and moisture from one automatic monitoring system in each site. Please see lines 197-199 and figure 2. We had overwritten the discussion part. Please see lines 292-316 in the revised manuscript.

Q11: (Soil nutrients)There is a big flaw in the paper. You related the root dynamics to soil nutrient content and availability but you don’t have the control. To assert that the dynamics is influenced by nutrient starvation or abundance you need to have measured fine root dynamics of the same species with control nutrient conditions. Or you should monitor the nutrient change in time and relate that to the fine root dynamics. Did you measure the soil nutrients changing in time? I would like to see this in table 1 and then see a clearer relationship for each of the root traits with this per species

R11: Thanks for your helpful advice. Based on your suggestions, we had overwritten the discussion part. Please see lines 317-345 in the revised manuscript.

Other errors had also been corrected in the revised manuscript.

Round  2

Reviewer 2 Report

Line 80 increasing soil temperature and decreasing soil water content. This sentence has to be changes as follow

Thus, the SRL is inversely related with the increasing interactions of soil nutrients availabilities, water, and temperature.

Although the introduction section has been improved (Montagnoli et al. 2018 For Eco is still missing see line 79 page 2), the part concerning oxalic acid and Nitrogen concentration in root tissues is still missing. You need to better explain why it is important to measure such traits (Terzaghi et al. 2013 and 2016 can help with this)

Sampling operation was conducted on lower, middle, and upper slope positions (what does it means?-explain in the text and add a sampling scheme)

I think that even if English language is of good level there are several little issues and redundancy that need to be carefully corrected throughout the manuscript. I suggest, for a high quality manuscript, a professional text revising.

“with the excavation method” I still don’t understand what is the excavation method. Please explain what you did. We excavated the root system ….HOW? with a brush with a shovel with the air spade…..??

“And carefully brushed away in the field”.

I think this is misleading. You brushed the rhizosphere soil into the bags!

“In the laboratory, fine root samples were washed and then frozen for analysis at a later date” (which temperature ?? -20°C ?)

“We had explained the non-growing season and growing season in lines 137-141 (Sampling section)”. I am not sure if non-growing season is a vegetative rest. Indeed, according to your data SOIL temperature is around 12 °C and this might be still growth for evergreen species. Thus, I suggest changing this terminology. You can say during the summer and winter season, or maximum and minimum temperature of the season and specify that your species are broadleaved and thus they enter in the vegetative rest period.

I think you have to specify that there is a winter or that the weather in winter is characterized by very low temperature and no-growth may occur. Also, if your species are broad-leaved and /or evergreen might differ. Please detail this in the manuscript.

Soil water content and temperature, is to be included in the results section (Figure 1). Please provide a subsection in the results where these data are described.

“We hypothesized that (1) the fine roots of shrubs would have lower SRL, RT/RB, and Nroot than do trees;”

I wonder on which background you base this hypothesis. You might explain it in the introduction.

Excuse me but this is not clear to me

3) these parameters would have positive relationships with increasing soil nutrients.

It has been found that plants with water and nutrient shortage increase the SRL since need to produce more fine root length with finer roots for the same biomass. How you hypotheses is this the opposite? Provide exact background in the introduction to support this.

In Table 1 the statistical difference between values measure in growing season and non-growing seasons should be indicated (x,y,z??)

Same in Table 2

In Figure 3. What does the asterisks mean? Can we see the P values for each interpolation line? If the room is not graphically enough you can leave out the R = Same Figure 4.

The discussion section has been much improved. Since your results are in contrast with most of the previous work, they are interesting and need to be stressed as you species belongs to different plant strategy.

Author Response

Response to Reviewer #2 comments

Please see the file attached

Author Response File: Author Response.docx