Increasing Wood δ¹⁵N in Response to Pig Manure Application

Round 1

Reviewer 1 Report

Agreed, anthropogenic alteration of global cycling of nitrogen is an important ecological topic, and ecosystem (forest) responses to increased availability of fixed nitrogen may vary by site or even by region.  Therefore, research such as described here that explains site details of nitrogen cycling should be encouraged.  Some comments on this ms:

Table 1: Lat-long waypoints of the study subjects is ok, but is it too much to ask for a map figure showing the study subjects in relationship to the N source?

Figure 1: NH4 didn’t differ significantly between treatment vs control for Aleppo?  Those value ranges appear non-overlapping.

Figure 3: Odd that control almond trees suddenly grew large rings for 2020.  An explanation for this finding (lines 350-352) is proposed, but how to know for sure why this occurred?  If it were related to N availability, it would essentially be a reversed response to N fertilization, not exactly intuitive.

Line 358: Speaking of expanding this research, what about measuring more than one time period of tree rings for N isotopes?  Having a times series of N values to compare against production data from pig farming could be pretty interesting.

Author Response

Agreed, anthropogenic alteration of global cycling of nitrogen is an important ecological topic, and ecosystem (forest) responses to increased availability of fixed nitrogen may vary by site or even by region.  Therefore, research such as described here that explains site details of nitrogen cycling should be encouraged.  

• We thank you for finding merit on our study.

Table 1: Lat-long waypoints of the study subjects is ok, but is it too much to ask for a map figure showing the study subjects in relationship to the N source?

• We added a map in the Supplementary Material.

Figure 1: NH4 didn’t differ significantly between treatment vs control for Aleppo?  Those value ranges appear non-overlapping.

• The variability in NH4 values of the two groups explains the non-significance of the comparison which gives a  p value of 0.21.

Figure 3: Odd that control almond trees suddenly grew large rings for 2020.  An explanation for this finding (lines 350-352) is proposed, but how to know for sure why this occurred?  If it were related to N availability, it would essentially be a reversed response to N fertilization, not exactly intuitive.

• We agree on your comment. We also propose on the ms that the supply of N may have lead to imbalances in other nutrients such as P or some kind of soil pollution. Nevertheless, this shows that an elevated N supply is not necessarily related to improved radial growth.

Line 358: Speaking of expanding this research, what about measuring more than one time period of tree rings for N isotopes?  Having a times series of N values to compare against production data from pig farming could be pretty interesting.

• We fully agree on your view. However, the application of N-enriched manure is relatively recent in the study stands. So, extending the reconstruction of N isotopes values for more than 1 decade is not necessary in the study site, but it would be interesting in regions with long and intense pig manure application on agroforestry systems.   

 

 

Reviewer 2 Report

Through experiments, the authors found higher δ15N values in tree-ring wood of manure-applied trees of two tree species (Aleppo pine, almond tree) indicating increased N supply relative to demand, despite this did not translate into growth enhancement, which had certain research value.But there are the following problems

1.In the section of 3.1,Please list the calculation formulas for these indicators,such as Sum of squares, R2 etc..

2. In the section of 4，The manuscript needs to pay more attention to the difference between the evaluation indicators and others, mainly because it needs better discussion - this is the weakest part of the manuscript.

Author Response

1.In the section of 3.1,Please list the calculation formulas for these indicators,such as Sum of squares, R2 etc..

• Done, we added the formulas in section 2.5 when describing the PERMANOVA.

2. In the section of 4，The manuscript needs to pay more attention to the difference between the evaluation indicators and others, mainly because it needs better discussion - this is the weakest part of the manuscript.

• Done, we revised the section and improved it.

Reviewer 3 Report

Pig manure is a major source of nitrogen (N) pollution of surface and ground waters in some European regions. N is a key element for the functioning of terrestrial ecosystems which is obtained through biological N fixation. Here, the authors explore how pig manure application affected: soil physico-chemical features, soil microbiota mass, tree radial growth, N concentration in leaves and wood, and tree-ring δ¹⁵N in wood. They found similar growth between fertilized and non-fertilized pine trees, but lower growth in fertilized almond trees. Leaf N concentrations decreased but ¹⁵N wood increased in trees subjected to pig manure application. Pig manure application alters the N cycling in the soil and within trees.

In general, this article needs to be modified:

1. Add a figure about the geographical location of the study area.

2. There are too many sentences using “we” in the article, which need to be modified.

3. The vertical coordinate font in Figure 2b is too small, so it should be appropriately enlarged.

4. “Pinus halepensis” and “Prunus dulcis” in the text, figures and tables should all be spelled in full or abbreviated as “P. halepensis” and “P. dulcis”.

5. Are the tree-ring width data the raw ring width or the detrended ring width?

6. “P” and “K” in the abstract need to be explained.

7. Figure 3b, why is the basal area increment in 2020 so different between the control (C) and manure-applied (M) trees?

Author Response

Regarding the first commented 4 issues, we added a map of the study area in the supporting information; rephrased many sentences starting by "we"; enlarged fonts in Fig 2b and spelled the species' names (which are abbreviated most of the times excepting the first time they are cited).

1. Add a figure about the geographical location of the study area.
2. There are too many sentences using “we” in the article, which need to be modified.
3. The vertical coordinate font in Figure 2b is too small, so it should be appropriately enlarged.
4. “Pinus halepensis” and “Prunus dulcis” in the text, figures and tables should all be spelled in full or abbreviated as “P. halepensis” and “P. dulcis”.

Regarding the last 3 issues, 

1. we used either tree-ring width or basal area increment data, as explained in the ms. We never used detrended ring-width indices which are more appropiate to assess climate-growth relationships.
2. P and K were explained in the abstract.
3. Fig. 3b: because C trees grew more whereas M trees showed low growth rates.