Leaf Carbon, Nitrogen and Phosphorus Stoichiometry in a Pinus yunnanensis Forest in Southwest China

Round 1

Reviewer 1 Report

After carefully reading the manuscript "Leaf carbon, nitrogen and phosphorus stoichiometry in a Pinus Yunnanensis forest in Southwest China"  Below I send my considerations to the authors: - Abstract is very poorly constructed. It needs to clearly state the research hypotheses, objectives and experimental design. What were the variables evaluated? How can these results be applied to pine cultivation in China? - The Introduction is extremely superficial. It needs to be expanded. The authors need to mention under the cultivation of Pinus in China, which are the main species, which problems they have in relation to N and P deficiency that justify this research. It was not clear to me how important this study was. What are the hypotheses made? - Statistical analysis is very basic. Readers would benefit from seeing the whole picture through the use of multivariate statistics. I suggest that the authors use techniques such as principal component analysis and clustering to demonstrate the similarity between the evaluated species. - Improvement of manuscript statistics is necessary, including to present the results, which are extremely short.

Author Response

Response to Reviewer 1 Comments

Point 1: After carefully reading the manuscript "Leaf carbon, nitrogen and phosphorus stoichiometry in a Pinus Yunnanensis forest in Southwest China"  Below I send my considerations to the authors: - Abstract is very poorly constructed. It needs to clearly state the research hypotheses, objectives and experimental design. What were the variables evaluated? How can these results be applied to pine cultivation in China?

Response 1: Thanks for your valuable comment. According to your suggestions, We have corrected the Abstract as follows:

“Abstract: Pinus yunnanensis forest is a unique forest type in southwest China and one of the main forest types in Yunnan Province, which also has great ecological, economic and social significance. Understanding the changes in the stoichiometric characteristics is a key to study nutrient cycling, limiting factors and stability mechanisms of the forest ecosystem. However, the stoichiometric characteristics, stability of ecosystem of P. yunnanensis natural forests and whether they are limited by nutrients are still poor understood. Based on K-S test, ANOVA analysis and OLS regression analysis, we analyzed the concentrations of leaf C, N and P in 48 woody species of P. yunnanensis natural forests from 122 plots to explore the pattern of leaf C:N:P stoichiometry . Our result showed that the mean values of leaf C, N and P plus C: N, C:P and N: P for the 48 woody species were 451.12, 11.05, 1.11 mg/g and 45.03, 496.98, 11.27, respectively. The Coefficient of Variation of leaf C, N and P plus C: N, C: P and N: P were 5.29%, 36.75%, 51.53%, 29.63%, 43.46% and 41.68%, respectively. The geometric mean values of leaf N, P and N: P were 10.49 and 1.00 mg/g and 10.51, respectively. Leaf C and N, P relationships showed a significant negative correlation, but significant positive correlation between leaf N and P. There were significant differences in leaf N and C: N across functional groups. There were significant differences in leaf C and P between evergreen and deciduous, conifer and broadleaf trees. Significant differences in leaf C: P were only observed between evergreen and deciduous trees and leaf N: P between conifer and broadleaf trees. The relative low N: P in all trees sampled indicated that N was a limiting factor in the distribution of P. yunnanensis natural forests. However, the higher leaf C:N, C:P ratio indicated that the P. yunnanensis natural forest ecosystem was in a relatively stable state.”

Point 2: The Introduction is extremely superficial. It needs to be expanded. The authors need to mention under the cultivation of Pinus in China, which are the main species, which problems they have in relation to N and P deficiency that justify this research. It was not clear to me how important this study was. What are the hypotheses made?

Response 2: Thanks for your valuable comment. According to your suggestions, We have corrected the Introduction as follows:

“Nitrogen (N) and phosphorus (P) are fundamental elements required by plants, and are the basic chemical elements in a variety of proteins and genetic material. Carbon (C) is made through photosynthesis assimilation and is the energy source of various physi-ological and biochemical processes in plants [1]. The combined effects of C, N and P on plant growth are stronger when compared with other nutrient elements [2, 3]. Stoichio-metric ratios can reflect plant ecological strategies, for example, C: N and C: P ratios can reflect plant growth rates and the growth rates were related to N and P efficiency of plants. Plant N: P stoichiometry can reflect the N or P limitation in plant growth [4–9]. If lacking either N or P, the leaf traits relationships and photosynthesis would be changed, and plant growth rates would reduce [10, 11]. Leaf C, N, P content and C: N: P ratios are important plant functional traits that reflect nutrient use efficiency and nutrient limita-tions, which are crucial to ecosystem structure and functions [12]. Therefore, ecological stoichiometry provides us with a framework to discuss the circulation of matter, balance and coupling of nutrient elements in an ecosystem through the relationship of proportion of elements [12]. Han et al. (2005) comprehensively analyzed the patterns of leaf N and P stoichiometry in Chinese terrestrial 753 plant species [13]. Since then, ecological stoichiometry has rapidly developed in recent years in China. Research has focused on forest [14–16], grassland [17–19] and wetland ecosystems [20–22], and effect of fertilizers on N: P ratios [23, 24]. In general, the research has been broad, showing a trend of taking forest, the largest ecosystem on land, as the dominant. For example, Wu et al. (2017) used 348 plant species from 8 forest types of China to analyze the stoichiometry of N and P and found that the distribution of leaf N and P contents was significantly different among the 8 different forest types and leaf N and P generally increased with increasing latitude, except for a slight decrease in cold temperate zone [25]. Zhang et al. (2018) used 803 plant species to explore C:N:P stoichiometry in 9 natural forest ecosystems ranging from cold-temperate to tropical forests and found that the C:P and N:P ratio decreased with increasing latitude and the trend P limitation increased in lower latitude of China’s forests [26]. However, there is lack of research on ecological stoichiometry of a particular area of a specific type of forest ecosystem, especially ecological stoichiometry in natural Pinus yunnanensis forests, which have important ecological and economic value.

1. yunnanensis is a fast-growing native tree species in southwest China. The plateau in mid-Yunnan is the distribution center of P. yunnanensis. P. yunnanensis forests live mainly in river valleys between 700 and 3000m above sea level and are distributed from 23° to 30° N and 96° to 108° E [27]. The P. yunnanensis forests can provide lots of good quality wood, large quantities of industrial raw materials and a variety of habitat conditions and habitats for animals and plants, which play an important role in biodiversity protection, Between 1960-1980s, large areas of P. yunnanensis forests were destroyed by humans, and the great majority of P. yunnanensis forests were replaced by artificial forests, and thus P. yunnanensis natural forests have decline significantly [29].

To our knowledge, the stoichiometric pattern of P. yunnanensis natural forest is still poor understood. The main objectives of this study were to (1) investigate the leaf C, N, P, C: N ratios, C: P ratios and N: P ratios in P. yunnanensis forests at the community-level; (2) analyze the intrinsic relationships among leaf C, N and P; (3) determine whether the growth of P. yunnanensis natural forest is limited by N or P. Our results will be conducive to providing scientific basis for the evaluation of ecosystem service function and sustainable development of P. yunnanensis forests. This study can enrich the database of forest stoichiometry and help enhance the parameters of future ecological models.”

Point 3: Statistical analysis is very basic. Readers would benefit from seeing the whole picture through the use of multivariate statistics. I suggest that the authors use techniques such as principal component analysis and clustering to demonstrate the similarity between the evaluated species. - Improvement of manuscript statistics is necessary, including to present the results, which are extremely short.

Response 3: Thanks for your valuable comment. Because we only analyzed the stoichiometric characteristics of Pinus yunnanensis natural forests, but did not explore the effect of biotic and abiotic factors on leaf C, N ,P and C:N, C:P, N:P. So we believe that the current statistcal methods (K-S test, ANOVA analysis, OLS regression) are adequate. We will adopt the statistical methods you mentioned (principal component analysis and clustering) in future research.

Reviewer 2 Report

Dear authors,

Thanks for your good job! However, your must improve the paper. The following comments can help you:

Introduction:

• To some extent,………...... between these elements: Which studies? Please cite.
• Ecological stoichiometry has rapidly developed in recent years in China: Since when with the reference?
• In introduction you must focus on the stoichiometry in forest, however you write about the stoichiometry in all parts such as marine, lakes, grasslands and etc.
• In objectives, what do you mean by existing rules?
• The necessity of the study is not clear and did not explain well. Please write comprehensively for this part.
• The last line in introduction, our research……: this is a kind of implication? If yes, it is not enough and must be write in better way.

Material and Methods:

• The study sites were chosen in the natural forests of yunnanensis, where as in introduction you write about the plantation, afforestation and artificial forests. Please revise the introduction and write about natural forests more.
• Please write more about the study sites, including elevations, climate, soil, dominant species at each site, and etc. The reader must know about the similarities and differences between the study areas.
• Please clarify the aim of choosing these sites. Only natural forests of yunnanensis is not the good criteria to choose a study site.
• Table 1. Please arrange the table in alphabetical order (based on the families or species). Also, it would better if you separate evergreen and deciduous species.

 Results:

• Figure 2: the quality of the figure is not well. Specially numbers and text.
• Figure 3: Same as figure 2

Conclusion:

• The conclusion is written in summary. What is the implication of your study?
• What are the research limitations?

Author Response

Response to Reviewer 2 Comments

Introduction:

Point 1: To some extent,………...... between these elements: Which studies? Please cite.

Response 1: Thanks for your valuable comments. By looking at the context, we found that this sentence was not appropriate, so we replaced it with the following sentence:

“Leaf C, N, P content and C: N: P ratios are important plant functional traits that reflect nutrient use efficiency and nutrient limitations, which are crucial to ecosystem structure and functions [12]. Therefore, ecological stoichiometry provides us with a framework to discuss the circulation of matter, balance and coupling of nutrient elements in an ecosystem through the relationship of proportion of elements [12]”.

Point 2: Ecological stoichiometry has rapidly developed in recent years in China: Since when with the reference?

Response 2: Thanks for your valuable comments. By searching the literatures, we modified this sentence as follows:

“Han et al. (2005) comprehensively analyzed the patterns of leaf N and P stoichiometry in Chinese terrestrial 753 plant species [13]. Since then, ecological stoichiometry has rapidly developed in recent years in China.”

Point 3: In introduction you must focus on the stoichiometry in forest, however you write about the stoichiometry in all parts such as marine, lakes, grasslands and etc.

Response 3: Thanks for your valuable comment, we fully agree with the reviewer’s comment. So we added the content about stoichiometry in forest as follows:

“In general, the research has been broad, showing a trend of taking forest, the largest ecosystem on land, as the dominant. For example, Wu et al. (2017) used 348 plant species from 8 forest types of China to analyze the stoichiometry of N and P and found that the distribution of leaf N and P contents was significantly different among the 8 different forest types and leaf N and P generally increased with increasing latitude, except for a slight decrease in cold temperate zone [25]. Zhang et al. (2018) used 803 plant species to explore C:N:P stoichiometry in 9 natural forest ecosystems ranging from cold-temperate to tropical forests and found that the C:P and N:P ratio decreased with increasing latitude and the trend P limitation increased in lower latitude of China’s forests [26].”

Point 4: In objectives, what do you mean by existing rules?

Response 4: Thanks for your valuable comment. That’s not an accurate statement, so we corrected it as “intrinsic relationships”.

Point 5: The necessity of the study is not clear and did not explain well. Please write comprehensively for this part.

Response 5: Thanks for your comment. We fully agree with the reviewer’s suggestion. So we rewrote this part as follows:

“In general, the research has been broad, showing a trend of taking forest, the largest ecosystem on land, as the dominant. For example, Wu et al. (2017) used 348 plant species from 8 forest types of China to analyze the stoichiometry of N and P and found that the distribution of leaf N and P contents was significantly different among the 8 different forest types and leaf N and P generally increased with increasing latitude, except for a slight decrease in cold temperate zone [25]. Zhang et al. (2018) used 803 plant species to explore C:N:P stoichiometry in 9 natural forest ecosystems ranging from cold-temperate to tropical forests and found that the C:P and N:P ratio decreased with increasing latitude and the trend P limitation increased in lower latitude of China’s forests [26]. However, there is lack of research on ecological stoichiometry of a particular area of a specific type of forest ecosystem, especially ecological stoichiometry in natural Pinus yunnanensis forests, which have important ecological and economic value.

1. yunnanensis is a fast-growing native tree species in southwest China. The plateau in mid-Yunnan is the distribution center of P. yunnanensis. P. yunnanensis forests live mainly in river valleys between 700 and 3000m above sea level and are distributed from 23° to 30° N and 96° to 108° E [27]. The P. yunnanensis forests can provide lots of good quality wood, large quantities of industrial raw materials and a variety of habitat conditions and habitats for animals and plants, which play an important role in biodiversity protection, Between 1960-1980s, large areas of P. yunnanensis forests were destroyed by humans, and the great majority of P. yunnanensis forests were replaced by artificial forests, and thus P. yunnanensis natural forests have decline significantly [29].

To our knowledge, the stoichiometric pattern of P. yunnanensis natural forest is still poor understood. The main objectives of this study were to (1) investigate the leaf C, N, P, C: N ratios, C: P ratios and N: P ratios in P. yunnanensis forests at the community-level; (2) analyze the intrinsic relationships among leaf C, N and P; (3) determine whether the growth of P. yunnanensis natural forest is limited by N or P. Our results will be conducive to providing scientific basis for the evaluation of ecosystem service function and sustainable development of P. yunnanensis forests. This study can enrich the database of forest stoichiometry and help enhance the parameters of future ecological models.”

Point 6: The last line in introduction, our research……: this is a kind of implication? If yes, it is not enough and must be write in better way.

Response 6: Thanks for your valuable comment. We rewrote this sentence as follows:

“Our results will be conducive to providing scientific basis for the evaluation of ecosystem service function and sustainable development of P. yunnanensis forests. This study can enrich the database of forest stoichiometry and help enhance the parameters of future ecological models.”

Material and Methods:

Point 7: The study sites were chosen in the natural forests of yunnanensis, where as in introduction you write about the plantation, afforestation and artificial forests. Please revise the introduction and write about natural forests more.

Response 7: Thanks for your valuable comment. We have deleted the content about plantation, afforestation and artificial forests, and revised the introduction as follows:

“Nitrogen (N) and phosphorus (P) are fundamental elements required by plants, and are the basic chemical elements in a variety of proteins and genetic material. Carbon (C) is made through photosynthesis assimilation and is the energy source of various physi-ological and biochemical processes in plants [1]. The combined effects of C, N and P on plant growth are stronger when compared with other nutrient elements [2, 3]. Stoichio-metric ratios can reflect plant ecological strategies, for example, C: N and C: P ratios can reflect plant growth rates and the growth rates were related to N and P efficiency of plants. Plant N: P stoichiometry can reflect the N or P limitation in plant growth [4–9]. If lacking either N or P, the leaf traits relationships and photosynthesis would be changed, and plant growth rates would reduce [10, 11]. Leaf C, N, P content and C: N: P ratios are important plant functional traits that reflect nutrient use efficiency and nutrient limita-tions, which are crucial to ecosystem structure and functions [12]. Therefore, ecological stoichiometry provides us with a framework to discuss the circulation of matter, balance and coupling of nutrient elements in an ecosystem through the relationship of proportion of elements [12]. Han et al. (2005) comprehensively analyzed the patterns of leaf N and P stoichiometry in Chinese terrestrial 753 plant species [13]. Since then, ecological stoichiometry has rapidly developed in recent years in China. Research has focused on forest [14–16], grassland [17–19] and wetland ecosystems [20–22], and effect of fertilizers on N: P ratios [23, 24]. In general, the research has been broad, showing a trend of taking forest, the largest ecosystem on land, as the dominant. For example, Wu et al. (2017) used 348 plant species from 8 forest types of China to analyze the stoichiometry of N and P and found that the distribution of leaf N and P contents was significantly different among the 8 different forest types and leaf N and P generally increased with increasing latitude, except for a slight decrease in cold temperate zone [25]. Zhang et al. (2018) used 803 plant species to explore C:N:P stoichiometry in 9 natural forest ecosystems ranging from cold-temperate to tropical forests and found that the C:P and N:P ratio decreased with increasing latitude and the trend P limitation increased in lower latitude of China’s forests [26]. However, there is lack of research on ecological stoichiometry of a particular area of a specific type of forest ecosystem, especially ecological stoichiometry in natural Pinus yunnanensis forests, which have important ecological and economic value.

1. yunnanensis is a fast-growing native tree species in southwest China. The plateau in mid-Yunnan is the distribution center of P. yunnanensis. P. yunnanensis forests live mainly in river valleys between 700 and 3000m above sea level and are distributed from 23° to 30° N and 96° to 108° E [27]. The P. yunnanensis forests can provide lots of good quality wood, large quantities of industrial raw materials and a variety of habitat conditions and habitats for animals and plants, which play an important role in biodiversity protection, Between 1960-1980s, large areas of P. yunnanensis forests were destroyed by humans, and the great majority of P. yunnanensis forests were replaced by artificial forests, and thus P. yunnanensis natural forests have decline significantly [29].

To our knowledge, the stoichiometric pattern of P. yunnanensis natural forest is still poor understood. The main objectives of this study were to (1) investigate the leaf C, N, P, C: N ratios, C: P ratios and N: P ratios in P. yunnanensis forests at the community-level; (2) analyze the intrinsic relationships among leaf C, N and P; (3) determine whether the growth of P. yunnanensis natural

forest is limited by N or P. Our results will be conducive to providing scientific basis for the evaluation of ecosystem service function and sustainable development of P. yunnanensis forests. This study can enrich the database of forest stoichiometry and help enhance the parameters of future ecological models.”

Point 8: Please write more about the study sites, including elevations, climate, soil, dominant species at each site, and etc. The reader must know about the similarities and differences between the study areas.

Response 8: Thanks for your comment. We fully agree with the reviewer’s suggestion. We have added the content as follows:

“The site of Qiubei has a mean annual temperature ranged from 13.2 to 19.7℃, and a mean annual precipitation ranged from 1000 to 1270mm, with the altitude ranged from 1000 to 1500m. The main associated tree species are Lyonia ovalifolia, Quercus variabilis, Schima noronhae, Craibiodendron stellatum and so on. The site of Shidian has a mean annual temperature of 17.6℃, and a mean annual precipitation of 883.2 mm, with the altitude ranged from 1600 to 1900m. The main associated tree species are Schima noronhae, Pyrus pashia, Dodonaea viscosa and so on. The site of Shuangbai has a mean annual temperature of 15℃, and a mean annual precipitation of 927 mm, with the altitude ranged from 1900 to 2100m. The main associated tree species are Schima noronhae, Craibiodendron stellatum, Lyonia ovalifolia, Phyllanthus emblica and so on. The site of Yunlong has a mean annual temperature of 13.2℃, and a mean annual precipitation of 815.5 mm, with the altitude ranged from 2500 to 3000m. The main associated tree species are Lyonia ovalifolia, Pinus armandii, Rhododendron delavayi, Rhododendron decorum and so on. All the soil types are classified as hilly red soil.”

Point 9: Please clarify the aim of choosing these sites. Only natural forests of yunnanensis is not the good criteria to choose a study site.

Response 9: Thanks for your valuable comment. We added the reasons for choosing these sites as follows:

“These specific areas chosen are concentrated distributions of P. yunnanensis natural forest with low human disturbance” and “Such a wide geographical distribution provides us with a rare opportunity to examine the nutritional status and stoichiometric characteristics of P. yunnanensis natural forest on a regional scale”.

Point 10: Table 1. Please arrange the table in alphabetical order (based on the families or species). Also, it would better if you separate evergreen and deciduous species.

Response 10: Thanks for your valuable comment. We rearranged the table in alphabetical order based on the species.

Results:

Point 11: Figure 2: the quality of the figure is not well. Specially numbers and text. Figure 3: Same as figure 2.

Response 11: Thanks for your valuable comment. All the figures were corrected.

Conclusion:

Point 12: The conclusion is written in summary. What is the implication of your study?

Response 12: The implication of our study is as follows:

“According to our study, it was proved that P. yunnanensis natural forest was a stable ecosystem for the time being. In the P. yunnanensis natural forest, most plants of different functional types have different strategies for obtaining nutrients. Our results provide strong evidence that P. yunnanensis natural forests are limited by N.”

Point 13: What are the research limitations?

Response 13: Thanks for your valuable comment. We added the limitations as follows:

“However, this study did not analyze the influencing factors of ecological stoichiometric characteristics, such as climate and soil properties. Future study will focus on the response of ecological stoichiometric characteristics of P. yunnanensis natural forests to more environmental factors.”

Round 2

Reviewer 1 Report

The authors have made important changes to the manuscript and it can be accepted for publication in its current form.

Reviewer 2 Report

Dear Authors,

Congratulation for your effort. The paper improved very well.

Best regards,