Response of Oak and Maple Seed Germination and Seedling Growth to Different Manganese Fertilizers in a Cultured Substratum
Round 1
Reviewer 1 Report
In my opinion the manuscript has been corrected properly, although it still has minor errors:
Page 2 line 79 Fe.18 g/g/hm2 should be changed by 7.18 g g-1 hm-2
line 86 Linn. should be replaced by L.
I do not understand why in all text °C were replaced by oC? Sometimes Authors write for example 60 °C and sometimes 60°C
Page 3 Line 143 gram should be replaced by g
Author Response
Page 2 line 79 Fe.18 g/g/hm2 should be changed by 7.18 g g-1 hm-2
This has been corrected.
line 86 Linn. should be replaced by L.
This has been corrected.
I do not understand why in all text °C were replaced by oC? Sometimes Authors write for example 60 °C and sometimes 60°C
These were typos. These have been corrected.
Page 3 Line 143 gram should be replaced by g
This has been corrected.
Reviewer 2 Report
The study is well designed and the manuscript written very well. But the major drawbacks is the statistical analysis, which doesn't allow comparison among species. The authors compared species response in some places, and such comparison is allowed if two-way anova was conducted to determine significant differences between species, treatments and their interaction. With the current one-way anova, the authors can't claim differences between species are significant or not. Therefore, I suggest either to avoid species comparisons in the text or do two-anova and re-write the results.
There are some misplaced sentences. For instance L376 and L382-386 are not your results and should be moved to the discussion section.
The discussion is a bite broad and sometime unrelated to the results presented in this study. For example, L388-391 is not related to the work presented here. L444-446 is not supported by the results as there is no evidence of mycorrhizal benefits in this study as you didn't use soils beneath oak forests.
Figure 1: indicate the significant differences with letters
Author Response
The study is well designed and the manuscript written very well. But the major drawbacks is the statistical analysis, which doesn't allow comparison among species. The authors compared species response in some places, and such comparison is allowed if two-way anova was conducted to determine significant differences between species, treatments and their interaction. With the current one-way anova, the authors can't claim differences between species are significant or not. Therefore, I suggest either to avoid species comparisons in the text or do two-anova and re-write the results.
We would respectfully disagree with this idea. First, we do not indicate any significant or non-significant differences between species in the text. Due to the inherent differences in the morphological and physiological attributes of each species our intent was not to compare directly between species but rather examine the potential differences in the effects of different treatments within each species. Accordingly, figure 1 encapsulates the differences of treatments between species as it displays the percent change compared to the control, and places each species on a more equal comparison.
There are some misplaced sentences. For instance L376 and L382-386 are not your results and should be moved to the discussion section.
This has been accomplished and reworded to fit the repositioning.
The discussion is a bite broad and sometime unrelated to the results presented in this study. For example, L388-391 is not related to the work presented here. L444-446 is not supported by the results as there is no evidence of mycorrhizal benefits in this study as you didn't use soils beneath oak forests.
We would respectfully disagree that lines 388 – 391 are unrelated to the work presented. It is a discussion point related to the effects of treatments on acorn germination. However, we reworded to reflect the possibility of this effect rather than emphatically stating this effect on germination, which we did not study. Lines 444 – 446 were removed, reworded and moved to the introduction to provide information regarding the influence of Mn on ectomycorrhizal communities associated with oak.
Figure 1: indicate the significant differences with letters
The purpose of Figure 1 is to demonstrate the relative influence of treatments on seedling size. Using actual morphological characteristics (height, diameter) would not reveal the relative magnitude of treatment influence due to the inherent morphological differences between species (similar to comparing apples with oranges). Therefore, the percent change/increase caused by treatments compared to the control was used, and it was the mean result of each treatment used. This was not a pairwise comparison of individuals between species. Therefore, there are no statistical comparisons that can be made based on the percent change based on the means.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The subjects of the presented study were three tree species which are the competitors in North America oak forest ecosystems. Authors investigated germination and seedling development under Mn treatment. This element was applied in three various compounds. Manuscript could be accepted for publication but after major revision.
My comments are given below:
1. Title: In my opinion term: medium suggests artificial medium applied in tissue culture, I suggest to use term: substratum
2. Abstract
line 26: It should be written for what purpose the seedlings were divided
3. Materials and Methods
Authors should mark that they used in the experiment dormant seeds and explain why they stored seeds at low temperature for two months.
4. Results
Table 1 presents only percentage of germinated seeds but not a germination rate. The rate germination taking into account number of germinated seeds and length of the hypocotyls can be calculated using various methods.
The differences between means were marked with different letters using Duncan’s multiple range test. Unfortunately these signs are inconsistent. The alphabetical order of the letters should go according to the rising or with the decreasing value of the means. In many tables some data were not analyzed using Duncan’s test (there are no letters for numbers).
In Table 2 Authors used name “Red oak” while in text they write about northern red oak. Please use the same name consistently throughout the manuscript.
Line 210. The response (for what?) of the red maple seedling…..
Lines 210-213: The description of the results is not consistent with the data presented in Table 2.
5. Discussion
Figure 1 should be moved and described in the chapter Results. The data under columns should be deleted; the scale on the axis OY is enough. The statistical analysis of presented data is required.
Author Response
Reviewer 1
The subjects of the presented study were three tree species which are the competitors in North America oak forest ecosystems. Authors investigated germination and seedling development under Mn treatment. This element was applied in three various compounds. Manuscript could be accepted for publication but after major revision.
My comments are given below:
1. Title: In my opinion term: medium suggests artificial medium applied in tissue culture, I suggest to use term: substratum Completed
2. Abstract
line 26: It should be written for what purpose the seedlings were divided Completed
3. Materials and Methods
Authors should mark that they used in the experiment dormant seeds and explain why they stored seeds at low temperature for two months. Completed
4. Results
Table 1 presents only percentage of germinated seeds but not a germination rate. The rate germination taking into account number of germinated seeds and length of the hypocotyls can be calculated using various methods. We agree that it is inaccurate to refer to it as germination rate. Since we do not have the data to express as germination rate, the language was changed to reflect germination success based on the percentage of seeds germinated.
The differences between means were marked with different letters using Duncan’s multiple range test. Unfortunately these signs are inconsistent. The alphabetical order of the letters should go according to the rising or with the decreasing value of the means. In many tables some data were not analyzed using Duncan’s test (there are no letters for numbers). Duncan’s multiple range test was performed on all the data. Letters were placed only in columns where significant differences were found. However, to make it more clear, letters were placed in all columns with the same letter to indicate Duncan’s MRT was performed but no significant differences were found. Also, letters were arranged in alphabetical order according to increasing or decreasing values
In Table 2 Authors used name “Red oak” while in text they write about northern red oak. Please use the same name consistently throughout the manuscript. Completed
Line 210. The response (for what?) of the red maple seedling….. This has been clarified
Lines 210-213: The description of the results is not consistent with the data presented in Table 2. This problem has been rectified
5. Discussion
Figure 1 should be moved and described in the chapter Results. The data under columns should be deleted; the scale on the axis OY is enough. The statistical analysis of presented data is required. Figure has been moved and modified as suggested. However, since the figure shows percent changes compared to the control, and seedlings were not paired between treatments, no statistical analysis was performed.
Reviewer 2 Report
Dear Authors
Reading your paper was interesting. The practical contribution of article is great since the effects of Mn fertilizers on germination and seedling growth in two oak species and in their major competitor red maple were investigated.
In Material and Methods section, you have referred that for each treatment in each species there were three replications of ten seeds each (see lines 106 – 108). I consider that the sample of seeds subjected to germination is very small. According to ISTA, a sample of 400 seeds (4 replications of 100 seeds each) is used in a germination test. If 400 seeds are not available the number of seeds per replication is reduced and not the number of replications. Furthermore, information about the date of sowing, and the sowing depth have to be given. In lines 124-125, you have referred that the number of germinated seeds was recorded. I think that the number of emerged seedlings was counted. So, you have to refer the criterion for the seedlings counting (i.e., the appearance of epicotyl for the oaks and hypocotyl with the cotyledons for red maple above the soil surface). In case that the number of emerged seedlings was counted, it is more accurate for you to refer to seedling emergence (percentage) in the whole manuscript. Furthermore, the mean time to emergence (MTE) can be calculated for each treatment in each species. The MTE will give information about the effects of treatments on the rate of seedling emergence.
In Result section, there are some inconsistencies between tables and the text. You have to be more accurate in the presentations of results. Comments were made on the manuscript (attached file).
In the Discussion section, results are repeated. The discussion has to be limited to interpreting the data and explaining its significance. Furthermore, I suggest the effect of treatments on overall seedling size in each species using the Fig. 1 (lines 293-332) to be presented in the Result section (place Fig 1 in The Result section).
All the comments and questions which were made on the manuscript (attached file) have to be taken account and answered by authors.
Comments for author File: 
Comments.pdf
Author Response
In Material and Methods section, you have referred that for each treatment in each species there were three replications of ten seeds each (see lines 106 – 108). I consider that the sample of seeds subjected to germination is very small. According to ISTA, a sample of 400 seeds (4 replications of 100 seeds each) is used in a germination test. If 400 seeds are not available the number of seeds per replication is reduced and not the number of replications. Furthermore, information about the date of sowing, and the sowing depth have to be given[RW1] . In lines 124-125, you have referred that the number of germinated seeds was recorded. I think that the number of emerged seedlings was counted. So, you have to refer the criterion for the seedlings counting (i.e., the appearance of epicotyl for the oaks and hypocotyl with the cotyledons for red maple above the soil surface).[RW2] In case that the number of emerged seedlings was counted, it is more accurate for you to refer to seedling emergence (percentage) [RW3] in the whole manuscript. Furthermore, the mean time to emergence (MTE) can be calculated for each treatment in each species. The MTE will give information about the effects of treatments on the rate of seedling emergence.
The information regarding the sowing date and the fact that germination was determined by seedling emergence through the substrate surface has been included. Accordingly, the wording has been changed to reflect the fact that it is not a rate of germination; rather it is the percent of seeds that germinated. The primary author believes that the germination mean time would be synthetically affected by species' own physiological characteristics, treatments, culture medium, temperature and so on, not only the treatment element which we focused in our research. Accordingly, the MTE was not included.
In Result section, there are some inconsistencies between tables and the text. You have to be more accurate in the presentations of results. Comments were made on the manuscript (attached [RW4] file).
These inconsistencies were errors rightly pointed out within the manuscript, and have been corrected
In the Discussion section, results are repeated. The discussion has to be limited to interpreting the data and explaining its significance[RW5] . Furthermore, I suggest the effect of treatments on overall seedling size in each species using the Fig. 1 (lines 293-332) to be presented in the Result section (place Fig 1 in The Result section).[RW6]
The figure has been moved into the appropriate location within the Results section. The Discussion section has been rewritten to reflect more of the data interpretation and its significance. Also, some results that were previously presented in the Discussion section have been moved to the appropriate locations in the Results section.
All the comments and questions which were made on the manuscript (attached file) have to be taken account and answered by authors.
All comments/questions made directly in the manuscript have been accepted and appropriate corrections made.
Round 2
Reviewer 1 Report
Manuscript seems be well improved and can be accepted to publish. But please explain what is this unit 8.68g/hm2
Reviewer 2 Report
Dear Authors,
I appreciate your modifications in the manuscript but it has not been significantly improved, compared with the first version. There are the following problems in the revised manuscript:
· You have referred that “The remaining seeds were stratified by storing them in moist polyethylene bags at 3-5˚C for two months to assure the breaking of dormancy.” (lines 104-106). The acorns of Quercus species have not dormancy. The acorns were usually stored under appropriate (cold and moist) conditions until they used, in order to avoid desiccation which can reduce their viability. In contrast, for the seeds of Acer species which they exhibit dormancy (usually physiological dormancy) the cold moist stratification is necessary for dormancy breaking. The authors have to add a reference in order to support that a 2-month period of cold stratification results in completely dormancy breaking of Acer rubrum seeds.
· I insist that the size of seed sample that was used for each species was very small (3 replications of 10 seeds each). This comment was also made in the first version of the manuscript. However, it has not been replied.
· My comment on the analysis of data (if the percentages data were arc-sine square root transformed and if the data were checked for normality and homogeneity of variances) was not replied.
· In Result section of revised manuscript, you have not taken into account my comments. In many points of this section (these points have been commented in the first review), you have referred to values of various treatments as “the greatest” or “the highest” without these values to be statistically the greatest or the highest. These values are arithmetically only the greatest. So, a reader can be easily confused. In a scientific paper an appropriate language has to be used.
· You have referred that “However, fertilizer treatments did not produce significant differences (p < 0.05) on seedling basal diameter and leaf number among the three species.” Since, no significant differences were observed among fertilizer treatments the p has to be > 0.05. However, according to Table 2 in chestnut oak and red maple there are significant differences among fertilizer treatments in diameter and leaf number.
· You have referred that “Treatments used in this study had a significant effect on northern red oak biomass, except for the stem biomass (Table 3).” However according to the result of statistical analysis presented in Table 3, there is a significant effect of treatments on stem biomass of northern red oak.
· You have changed the data in Table 3 without to give any explanations. Furthermore, there are changes in the results of statistical analysis in all Tables. Have you changed the data or (and) the way of data analysis?
