Influence of Leaf Litter and Humus Composition on the Development of Black Spruce Seedlings: A Greenhouse Experimentation
Round 1
Reviewer 1 Report
This paper presents the results of an extensive but in fact very simple greenhouse experiment in which spruce seedlings were grown in various substrates (various kinds of humus originating from different habitats), covered by various kinds of leaf litter.
The preparation of the experiment as well as its course and results have been presented in great detail in the article, and some results have been included in Supplementary Materials.
The detailed descriptions and the multitude of data presented in the tables and graphs make it difficult to find and follow the main scientific issues in this article.
Some key information is also lost in the excess of information, e.g. there is no clear description in the section “2. Materials and methods” how the leaf litter was added (put on the surface or mixed with humus?). Admittedly, from the description given in L. 232, one can guess that it was crushed on the surface, but this needs to be clarified earlier.
The study indicated the possibility of natural enrichment of forest humus in Black spruce stands by the maintenance of broadleaf species, which I find an important conclusion that is worth publishing. In my opinion, however, the discussion should more strongly emphasize the crucial results and arguments that led to such a conclusion.
I would like to add that the data collected in this paper may constitute an interesting reference database on elementary composition of various kinds of humus and spruce seedlings. This is another reason why I consider the paper worth publishing.
Though, I have found several shortcomings in the manuscript that definitely require revision or explanation. Below, there are some more important comments:
Major results have been presented in the text and in the tables with unjustified precision (!). The number of digits presented should be related to the assessed uncertainty of measurements. Usually, presenting more than three significant digits is unjustified and should be considered as a serious mistake. Please, check this matter in the entire text and in the tables.
L. 115. Do plant species belong to abiotic conditions?
L. 133-136. This fragment should be moved to introduction
L. 125-126. The issue of susceptibility to paludification process should be explained and commented more thoroughly. Note that hydrology and soil air-water properties of particular habitats are crucial for development of humus properties. Therefore, each sampling site should be described more in detail in this matter. Consider the comparison of humus current moisture at the time of sampling.
L. 127. “experience” should be replaced by “experiment”
L. 143-4. I wonder if the removal of the substrate from seedling roots and cleaning the rooting system did not adversely affect rhizospheric properties of seedlings, such as mycorrhiza, etc.…
L. 163. The methods used for the measurement of soil pH and percent moisture at 2 cm and 8 cm depth from the soil surface should be described
L. 173. A reference should be provided for the digestion with H2SO4, as some elements can form insoluble compounds with this acid
L. 200. Moisture rather than humidity
L. 230-31. Unclear sentence. Please reword. Was the effect of amount of litter added very weak, whereas the effect of humus amount was much stronger?
L. 461. More acids soil – should be corrected
There are some typos in the tables.
Table 3. This table should be definitely moved to the Supplementary Materials.
Table 5. It would be useful to indicate the significance of the pH differences in the variants with litter in relation to the control
Fig. 2 and 3. The boxplots are quite difficult to follow. It is not clear what the individual elements of diagrams mean: blocks, error bars, dots. Additionally, I would recommend to clearly write (in the explanations to the graphs) how the significance of differences was estimated.
Author Response
Dear Editor,
We appreciate the reviewers for their generous contributions and corrections on our research entitled “Influence of leaf litter and humus composition on the development of black spruce seedlings: a greenhouse experimentation”.
Please find below our comments and responses reviewed by all of us authors, considering all your comments and suggestions.
Your comments and suggestions are in black, and the authors' comments and responses are in blue. The attached documents contain all the changes in the "track change" tool.
Reviewer comments and suggestions for the authors:
- This paper presents the results of an extensive but in fact very simple greenhouse experiment in which spruce seedlings were grown in various substrates (various kinds of humus originating from different habitats), covered by various kinds of leaf litter. The preparation of the experiment as well as its course and results have been presented in great detail in the article, and some results have been included in Supplementary Materials. The detailed descriptions and the multitude of data presented in the tables and graphs make it difficult to find and follow the main scientific issues in this article.
In particular, the main scientific issues of this study focused on comparing not only the growth, but also the development, of black spruce seedlings in 4 different types of humus on the effect of short-term fertilization of 5 different types of litter present in the spruce-moss forest domain. We focused on analyzing the effect of the presence of broadleaf species in predominant stands of black spruce. For this we target on the following variables:
- Leaf litter and humus nutrients, before and after treatment;
- General black spruce seedling development (i.e., aerial parts of B. spruce seedlings: Stem growth, stem weight and needles weight, and; Root development: root weight and root length).
- Physicochemical characteristics of humus (pH and moisture content)
- Nutrient concentrations in the foliage of black spruce seedlings after the experiment (to check the nutritional absorption of the seedlings).
- Some key information is also lost in the excess of information, e.g. there is no clear description in the section “2. Materials and methods” how the leaf litter was added (put on the surface or mixed with humus?). Admittedly, from the description given in L. 232, one can guess that it was crushed on the surface, but this needs to be clarified earlier.
You made a good comment, thank you. We clarified this procedure. Leaf litter from each of the 5 tree species was dried, ground, separated in proportionate amounts, and added to the surface of the humus treatments containers (i.e., L. 186).
- The study indicated the possibility of natural enrichment of forest humus in Black spruce stands by the maintenance of broadleaf species, which I find an important conclusion that is worth publishing. In my opinion, however, the discussion should more strongly emphasize the crucial results and arguments that led to such a conclusion.
Thank you for your comment. However, as seen in the discussion, our results supported our hypothesis and demonstrated a better performance for black spruce seedlings grown in humus from mixed stands and from pure shudder stands compared to humus from pure black spruces and from substrate. nursery (e.g., L. 1260). We highlighted the relationships of primary macronutrients with different types of humus when subjected to the effects of crushed leaf litter treatments (e.g., L.1269-1286). We also highlighted the relationship between the increase and/or maintenance of pH for humus from mixed and pure trembling stands (e.g., L.1296-1300). We also approached that the effect of humus from mixed stands and pure aspen were more accentuated in relation to the effect of the addition of crushed broadleaf leaf litter, when analyzing the significance at the level of seedling growth (e.g., L.1302-1307). In addition, we address the possibility of the quantitative influence of leaf litter on soil moisture can influence the development of seedlings (e.g., L. 1312-1315).
- I would like to add that the data collected in this paper may constitute an interesting reference database on elementary composition of various kinds of humus and spruce seedlings. This is another reason why I consider the paper worth publishing.
Thank you very much. We worked very hard on this research, this comment is very gratifying.
- Though, I have found several shortcomings in the manuscript that definitely require revision or explanation. Below, there are some more important comments: Major results have been presented in the text and in the tables with unjustified precision (!). The number of digits presented should be related to the assessed uncertainty of measurements. Usually, presenting more than three significant digits is unjustified and should be considered as a serious mistake. Please, check this matter in the entire text and in the tables.
We sincerely apologize for this serious mistake and thank you for your observation. We made the necessary corrections.
- 115. Do plant species belong to abiotic conditions?
We added the following sentence to address this question. The origin of the seeds that formed the black spruce seedlings is located in regions of close latitudes to where the humus was collected, although distant longitudes (-68.341; 48.619), in 2nd generation orchard of Québec MFFP (e.g., L. 161-163).
- 133-136. This fragment should be moved to introduction.
Done. Thank you.
- 125-126. The issue of susceptibility to paludification process should be explained and commented more thoroughly. Note that hydrology and soil air-water properties of particular habitats are crucial for development of humus properties. Therefore, each sampling site should be described more in detail in this matter. Consider the comparison of humus current moisture at the time of sampling.
We have added details about the paludification process as suggested (e.g., L. 61-75), as well as we added more specifically information about the humus collection in stands (e.g., L. 149-151).
- 127. “experience” should be replaced by “experiment”
Correction performed. Thank you.
- 143-4. I wonder if the removal of the substrate from seedling roots and cleaning the rooting system did not adversely affect rhizosphere properties of seedlings, such as mycorrhiza, etc.…
"The substrate from all seedling roots was removed, the rooting system was gently washed and carefully cleaned to remove all residues avoiding affecting the rhizo-sphere" (e.g., L163-165). We remove all substrate in a standardized way for all seedlings, to avoid influence of nursery soils on inserted treatments, avoiding any damage to the plant.
- 163. The methods used for the measurement of soil pH and percent moisture at 2 cm and 8 cm depth from the soil surface should be described
We added information on the pH meter + moisture meter used: "Roots and central stems of the 720 plants were measured before being transplanted in-to the substrate treatments, along with soil pH and percent moisture at 2 cm and 8 cm depth from the soil surface. Every 25 days plants had their central stem height measured along with soil pH and percent moisture, also at 2 cm and 8 cm depth from the soil surface, via the Esimen Digital Plant Test, 12 hours after watering" (e.g., L. 191-194).
- 173. A reference should be provided for the digestion with H2SO4, as some elements can form insoluble compounds with this acid
Correction performed (e.g., L. 201-205). Thank you.
Hossner, L.R. Dissolution for Total Elemental Analysis. Methods Soil Anal. Part 3 Chem. Methods 1996, 5, 49–64 (e.g., 1563).
Jones Jr, J.B.; Case, V.W. Sampling, Handling, and Analyzing Plant Tissue Samples. Soil Test. Plant Anal. 1990, 3, 389–427 (e.g., L.1564).
- 200. Moisture rather than humidity
Correction performed (e.g., L. 230). Thank you.
- 230-31. Unclear sentence. Please reword. Was the effect of amount of litter added very weak, whereas the effect of humus amount was much stronger?
Yes. L. 262-264: “Although the triple interaction was significant between the treatments, the effect of amount of litter added had a very weak impact on seedling growth compared to humus amount and litter type” (Table S2).
In addition, L. 1302-1311 "Although significant, the F values associated with the amount of litter added to humus treatments and type of litter treatments were among the lowest; in addition, the interactions between the amount of litter and other factors were not significant. There-fore, the amount of litter added effect was very weak compared to the other factors. For all these reasons, litter quantitative effects were not noticed for seedling growth variables. However, the amount of litter had a positive effect on substrate moisture. Thus, the finding in this study that different leaf litter species also have an effect, albeit relatively small, on aerial and underground development of B. spruce seedlings, supports our hypothesis that different leaf litter species have a certain effect on the development of B. spruce seedlings when added to different humus".
- 461. More acids soil – should be corrected
Correction performed. Thank you.
- There are some typos in the tables.
Correction performed. Thank you.
- Table 3. This table should be definitely moved to the Supplementary Materials.
The table was moved to the Supplementary Materials. Thank you.
- Table 5. It would be useful to indicate the significance of the pH differences in the variants with litter in relation to the control
Correction performed. Thank you.
- 2 and 3. The boxplots are quite difficult to follow. It is not clear what the individual elements of diagrams mean: blocks, error bars, dots. Additionally, I would recommend to clearly write (in the explanations to the graphs) how the significance of differences was estimated.
Correction performed. Thank you.
The authors have read and approved the final version revised of the manuscript submitted.
Thank you for your consideration.
Sincerely,
Maísa De Noronha
Université du Québec en Abitibi-Témiscamingue
Rock Ouimet
Ministère des Forêts, de la Faune et des Parcs du Québec
Martin Barrette
Ministère des Forêts, de la Faune et des Parcs du Québec
Alain Leduc
Université du Québec à Montréal
Yves Bergeron
Université du Québec en Abitibi-Témiscamingue
Université du Québec à Montréal
26 October 2022
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript “Influence of leaf litter and humus composition on the development of black spruce seedlings: a greenhouse experimentation” concerns a diligently performed experiment focused on the effects of different growth substrates, specifically different humus types, on the growth performances of Picea mariana (black spruce) seedlings. Particularly, a greenhouse experiment has been developed in order to compare the growth of seedlings belonging to a black spruce provenance, transplanted in cells filled with humus of pure black spruce, pure trembling aspen, mixed black spruce with trembling aspen and the normal peat used for nursery purposes. Furthermore, in order to test what they call the short-term effect linked to different types of leaf litter, the authors were able to enrich the experimental design by adding leaf litter from 5 tree species to the different growth substrates (humus): Populus tremuloides, Alnus spp., Betula papyrifera, Salix spp. and Picea mariana.
Overall, this work brings novelty and support towards new models of silvicultural management. Especially, managing mixed conifers and broadleaves stands could favour the recruitment of black spruce through the amelioration of both soil litter and humus. The manuscript is well written and basically mature for publication.
Some minor comments are appended below.
R47-51. Please, elucidate better the important concepts on the specific effects on soil properties and fertility. Especially, it would be beneficial to explain better the meaning of wet humus as well as that of the paludification.
R104. Do you mean silt and clay or you mean mud just to indicate general sediments with texture other than clay?
R142 Declaring here 4 type of humus is a bit confusing. As far as I can understand you consider the nursery peat as the 4th humus type. Then, the question is: is peat a humus kind? I would answer "it is not". Another confusion to be fixed is to let the reader clearly understanding if the pots/cells are entirely filled with a humus type to compose the growth substrate.
R142-143. Here it is good you declare the Black spruce provenance. Otherwise, it would be even better to provide some information more on the geographical features of the provenance. Especially, I suggest to add the provenance location on the maps in fig. 1. Thus everyone could realize if a geographical congruence exists among the humus sampling sites and the seedlings’ provenance.
R128 vs R150 Sorry, the difference between the long-term influence and the short-term one is rather unclear. Aside the different types of collected litter, what logical issue is explored here? I believe beneficial deepening a little these aspect in Introduction.
Author Response
Dear Editor,
We appreciate the reviewers for their generous contributions and corrections on our research entitled “Influence of leaf litter and humus composition on the development of black spruce seedlings: a greenhouse experimentation”.
Please find below our comments and responses reviewed by all of us authors, considering all your comments and suggestions.
Your comments and suggestions are in black, and the authors' comments and responses are in blue. The attached documents contain all the changes in the "track change" tool.
Reviewer comments and suggestions for the authors:
- R47-51. Please, elucidate better the important concepts on the specific effects on soil properties and fertility. Especially, it would be beneficial to explain better the meaning of wet humus as well as that of the paludification.
You made a good comment, thank you. We clarified this procedure. We have added details about the paludification process as suggested (e.g., Lines 61-75), as well as we added more specifically information about the humus collection in stands (e.g., Lines 149-151).
- Do you mean silt and clay or you mean mud just to indicate general sediments with texture other than clay?
Thank you for your observation. We made the necessary corrections: the correct is silt (e.g., R. 120)
- R142 Declaring here 4 type of humus is a bit confusing. As far as I can understand you consider the nursery peat as the 4th humus type. Then, the question is: is peat a humus kind? I would answer "it is not". Another confusion to be fixed is to let the reader clearly understanding if the pots/cells are entirely filled with a humus type to compose the growth substrate.
Correction performed. Thank you. We added the following sentence to address this question: “The collected humus was transported to a greenhouse where they were mixed and sieved at 10 mm by each stand category (i.e., pure B. spruce; pure T. aspen and Mixed B. spruce – T. aspen). Sequentially, each of the three categories of humus and the peat substrate (i.e., crushed peat moss that was sieved and artificially fertilized, commonly used by tree nurseries for the production of containerized tree seedlings) (i.e., control treatment) was entirely added in containers with a capacity of approximately 462 cm³ each (e.g., every 180 cells in the containers were filled with one of the 4 types of humus, totaling 720 cells). Two-year-old B. spruce seedlings from the Grandes-Piles MFFP nursery (provenance: BS-V2-PLU-1-0) were transported to the greenhouse. (e.g., R. 153-160)”.
- R142-143. Here it is good you declare the Black spruce provenance. Otherwise, it would be even better to provide some information more on the geographical features of the provenance. Especially, I suggest to add the provenance location on the maps in fig. 1. Thus everyone could realize if a geographical congruence exists among the humus sampling sites and the seedlings’ provenance.
We agree that. We added the following sentence to address this question: “The origin of the seeds that formed these seedlings are located in regions of close latitudes, although distant longitudes (-68.341; 48.619), in 2nd generation orchard of Québec MFFP (e.g., R 161-163).
- R128 vs R150 Sorry, the difference between the long-term influence and the short-term one is rather unclear. Aside the different types of collected litter, what logical issue is explored here? I believe beneficial deepening a little these aspect in Introduction.
Thank you for your comment. We have added details about the long-term (i.e., humus) and short-term (i.e., leaf litter crushed added to cells surfaces – 150 days): “To evaluate the long-term influence of leaf litter on growth and development of B. spruce seedlings in different stands, the humic part of the forest floor (i.e., made up of litter incorporated into the soil decomposed over several years, commonly known as humus) was collected in the 3 selected stands (e.g., with trees around each humus col-lected point composed by B. spruce; pure T. aspen and Mixed B. spruce with T. aspen) in each of the 3 areas” (e.g., 144-149).; “It is crucial to know more about how the beneficial effects of leafy litter (i.e., short term effects) on different humus (i.e., long term effects) could justify the maintenance of deciduous trees in a silvicultural scenario in order to maintain or improve the pro-duction and growth of desired seedlings in these stands”. (e.g., R. 97-100).
The authors have read and approved the final version revised of the manuscript submitted.
Thank you for your consideration.
Sincerely,
Maísa De Noronha
Université du Québec en Abitibi-Témiscamingue
Rock Ouimet
Ministère des Forêts, de la Faune et des Parcs du Québec
Martin Barrette
Ministère des Forêts, de la Faune et des Parcs du Québec
Alain Leduc
Université du Québec à Montréal
Yves Bergeron
Université du Québec en Abitibi-Témiscamingue
Université du Québec à Montréal
27 October 2022
Author Response File: Author Response.docx
