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. 2022 Dec 2;17(12):e0278656. doi: 10.1371/journal.pone.0278656

Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants

Dong-Wei Yu 1, Su-Juan Duan 1, Xiao- Chao Zhang 2, Da-Qiu Yin 3, Shi-Jun Wang 3, Jin-Song Chen 1,*, Ning-Fei Lei 2,*
Editor: Xiao Guo4
PMCID: PMC9718409  PMID: 36459510

Abstract

Different nutrient supply brings about changes in leaf stoichiometry, which may affect growth rate and primary production of plants. Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide. A pot experiment was conducted by using three stoloniferous alien plants Wedelia trilobata, Alternanther philoxeroides and Hydrocotyle vulgaris to investigate effects of nutrient supply on their leaf stoichiometry and relative growth rate. Different nitrogen or phosphorus supply was applied in the experiment (N1:1 mmol L-1, N2:4 mmol L-1, and N3:8 mmol L-1, P1:0.15 mmol L-1, P2:0.6 mmol L-1 and P3:1.2 mmol L-1). Nitrogen and phosphorus concentrations in leaves of the three alien plants significantly increased with increase of nitrogen supply. With increase of phosphorus supply, nitrogen or phosphorus concentration of leaf was complex among the three alien plants. N:P ratio in leaf of the three alien plants subjected to different levels of nutrient supply was various. A positive correlation between relative growth rate and N:P ratio of the leaf is observed in W. trilobata and A. philoxeroides suffering from N-limitation. A similar pattern was not observed in Hydrocotyle vulgaris. We tentatively concluded that correlations between relative growth rate and N: P ratio of the leaf could be affected by species as well as nutrient supply. It is suggested that human activities, invasive history, local abundance of species et al maybe play an important role in the invasion of alien plants as well as relative growth rate.

Introduction

N:P ratio is a critical indicator of nutrient limitation (N vs P) in the terrestrial ecosystem [1]. Leaf stoichiometry can reflect nutrient allocation strategy, growth strategy and expanding ability of invasive plants [2]. Plant stoichiometry refers to balance and ratio of C, N, P in its issue or organ [3]. Primary production and litter decomposition of plants were significantly affected by C: N: P ratio of the leaf as well as community dynamics and nutrient cycling [4–9]. In addition, C: N: P ratio of the leaf was related to plant adaptation to specific environments [10,11]. N:P ratio of the leaf may be useful to assess nitrogen versus phosphorus limitation to primary production in terrestrial ecosystems [12–15].

Different nitrogen and phosphorus supply bring about changes of leaf stoichiometry, and these changes are various among different plants [16]. For example, with increase of nitrogen availability, phosphorus concentration in the leaf of desert grass Seriphidium korovinii and two alpine grasses significantly increased [17,18]. Meanwhile, an opposite pattern was observed in the phosphorus concentration of leaf [19–22]. With increase of nitrogen availability, nitrogen concentration in the leaf significantly increased [23,24]. In central Brazil, the similar pattern was not observed in four plants (Caryocar Brasiliense, Qualea parviflora, Schefflera macrocarpa and Ouratea hexasperma) grown on a dystrophic soil [25]. Nitrogen concentration in the leaf of Arabidopsis thaliana and desert grasses was not significantly affected by phosphorus supply [20,21]. With increase of phosphorus supply, nitrogen concentration in the leaf of the oak Quercus acutissima and shrub Rhus typhina significantly decreased [21,24]. With increase of phosphorus supply, phosphorus concentration in leaves of the alpine grassland significantly increased [18]. With increase of phosphorus supply, phosphorus concentration in the leaf of S. korovinii was various [20]. So, the effects of nitrogen and phosphorus supply on leaf stoichiometry of plants are still controversial.

With a lower N:P ratio, faster-growing organisms (such as microbes, zooplankton, arthropod and insect) often present greater tissue phosphorus concentration than slower-growing ones [26]. Relative growth rate of Betula pendula and Arabidopsis thaliana grown in P-limited conditions was negatively correlated with the N:P ratio of leaf. At the same time, a positive correlation between the N:P ratio of leaf and relative growth rate was detected in the two species suffering from N-limited conditions [16,27–29]. Relative growth rate of mangroves was not significantly affected by the N:P ratio of leaf [30]. More studies are needed to investigate the correlation between N:P ratios of leaves and relative growth rate, especially in vascular plants [27,30,31].

The invasion of alien plants severely threatens biodiversity and ecosystem worldwide [32–34]. Alien plants often accumulate more nitrogen and/or phosphorus than the co-occurring native ones[35,36]. The possible driving factor for the successful invasion of alien plants is their greater nutrient capture capacity than native ones[37,38]. However, the clear relationship between nutrient absorption capacity of alien plants and their expanding ability was not established in other studies[39,40]. Further studies are thus needed to clarify the correlations between leaf stoichiometry of alien plants and their growth performance.

Stoloniferous alien plants Wedelia trilobata, Alternanther philoxeroides and Hydrocotyle vulgaris originate from Europe and/or America respectively. The three alien plants are widely distributed in the south of the Yangtze River, China [41,42]. A pot experiment was conducted (1) to evaluate the effects of nutrient supply on leaf nitrogen and phosphorus concentrations of the three alien plants; (2) to explore the correlation between leaf N: P ratio of the three alien plants and their relative growth rate. Finally, our study aims to understand the potential influence of leaf stoichiometry on invasion of the three alien plants.

Materials and methods

Species

Wedelia trilobata, Alternanther philoxeroides and Hydrocotyle vulgaris are perennial stoloniferous herbs. Axillary buds on the vertical stem of W. trilobata may grow out to form stolon. Ramets with opposite leaves take root at the node of stolon, which forms a network above-ground [43]. W. trilobata is distributed predominantly in tropical and subtropical regions such as Asia and South America [42].

The stolon of Alternanther philoxeroides usually takes root when in contact with moist sub-stratum, forming a network of stolon [44]. A. philoxeroides is distributed in many areas of the world such as Australia, the United States, and China [45,46].

Each node along the stolon of Hydrocotyle vulgaris has the potential to produce an independent ramet that consists of a simple leaf and adventitious roots [47]. H. vulgaris is widely distributed in tropical and temperate regions such as Southeast Asia, Europe and North America.

In 2018, 10 original plants of W. trilobata were collected from Zhejiang Province, China. 20 original plants of A. philoxeroides and H. vulgaris were collected in Sichuan Province, China. All original plants were planted in a greenhouse located at Sichuan Normal University. The average temperature is about 25 ± 5°C in the greenhouse.

Experimental design

In September 2019, stolon internodes of the three alien plants without leaves were chosen. The stolon internodes were about 3-5cm length in size. Balance was used to measure their fresh weight. According to the ratio of dry to fresh weight, we could obtain initial biomass of each stolon internode [19]. The stolon internodes were respectively grown in a plastic pot (12 cm in upper edge diameter, 9 cm in bottom diameter and 10.5 cm in height) filled with perlite. Same volume of nutrient solution was added to each plastic pot every two days.

Three levels of nitrogen supply (N1: 1mmol L-1, N2:4 mmol L-1, and N3: 8 mmol L-1 nitrogen, added as NH4NO3) and three levels of phosphorus supply (P1:0.15 mmol L-1, P2:0.6 mmol L-1, and P3:1.2 mmol L-1 phosphorus, added KH2PO4 and NaH2PO4 as 1:1 ratio) were applied in the experiment (Table 1). In addition, macro- and microelements (such as 0.75 mM K2SO4, 0.65 mM MgSO4, 1 μM MnSO4, 0.1 μM CuSO4, 1 μM ZnSO4, 0.035 μM Na2MoO4, 0.1 mM Fe-EDTA, 0.01 mM H3BO3, and 2 mM CaCl2) were added to nutrient solution [16]. The pH of nutrient solution was adjusted to 5.8. The experiment was carried out in a greenhouse with 16-h light/8-h dark photoperiod and 25 ± 5°C. Each treatment was replicated six times. The experiment lasted for 60 days. To avoid potential effects of the micro-environment, the pots were randomly rearranged every two days.

Table 1. Nitrogen and phosphorus supply used in the experiment.

Level of nitrogen supply Level of phosphorus supply Nitrogen supply (mmol L-1) Phosphorus supply (mmol L-1) N:P (mol:mol)
Low nitrogen (N1) Low phosphorus (P1) 1 0.15 6.7
Intermediate phosphorus (P2) 1 0.6 1.7
High phosphorus (P3) 1 1.2 0.8
Intermediate nitrogen (N2) Low phosphorus (P1) 4 0.15 26.7
Intermediate phosphorus (P2) 4 0.6 6.7
High phosphorus (P3) 4 1.2 3.3
High nitrogen (N3) Low phosphorus (P1) 8 0.15 53.3
Intermediate phosphorus (P2) 8 0.6 13.3
High phosphorus (P3) 8 1.2 6.7
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At the end of the experiment, fully expanded and mature leaves were selected from regenerated clonal fragments and dried to constant weight. The leaf samples were powdered and sifted through 100-mesh. Mortar Autoanalyzer 3 system (Seal analytical, Germany) was used to measure the nitrogen and phosphorus concentrations of the leaf [48].

Finally, whole regenerated clonal fragments were harvested and dried to constant weight. Balance was used to determine their biomass.

The relative growth rate was calculated according to following formula:

Relativegrowthrate=Ln(Mt)‐Ln(M0)t (1)

In the experiment, M0 is the initial biomass of the stolon internode, Mt is the final biomass of regenerated clonal fragment, and t is duration of the experiment.

We estimate the balance of plant homeostasis by stoichiometric regulation coefficient H.

The stoichiometric regulation coefficient H was calculated according to the following formula:

H=Log10(x)Log10(y)‐Log10(c) (2)

In the experiment, x is N:P or C: N or C:P ratio of nutrition, y is N:P or C: N or C:P ratio of leaf, c is a constant[49].1/H is a slope value and it should be between 0 and 1. The classifications of H values are as follows: H>4(homeostasis), 4>H>2(weak homeostasis), 2>H>1.33 (weakly sensitive state), 1.33>H(sensitive state). But it is often understood as absolute homeostasis when the equation fitting the data is insignificant.

Statistical analyses

Two-way analysis of variance was performed to assess the effects of nitrogen, phosphorus supply and their interaction on leaf stoichiometry of the three alien plants. Linear regression was performed to explore the correlation between relative growth rate of the three alien plants and their leaf stoichiometry respectively. All analyses were conducted with SPSS 20.0 software (SPSS Inc).

Results

Nitrogen and phosphorus concentrations in the leaf of W. trilobata and H. vulgaris were significantly affected by nitrogen supply as well as interaction between nitrogen and phosphorus supply (Tables 2 and 3). Phosphorus concentration in the leaf of W. trilobata was significantly affected by phosphorus supply (Table 2). Nitrogen and phosphorus concentrations in the leaf of A. philoxeroides was significantly affected by nitrogen supply, phosphorus supply as well as interaction between nitrogen and phosphorus supply (Table 4).

Table 2. Effects (F value) of nitrogen and phosphorus supply and their interaction on the relative growth rate of W. trilobata, nitrogen and phosphorus concentrations, N: P ratio in its leaf.

Factors Relative growth rate
(g day -1)		 Nitrogen concentration
(mg g -1)		 Phosphorus concentration
(mg g -1)		 N:P ratio
Nitrogen supply 26.16*** 13.83*** 57.10*** 31.49***
phosphorus supply 0.76 2.36 99.44*** 30.25***
Interaction between nitrogen
and phosphorus supply 		2.36* 5.01** 4.60** 25.71***
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**P < 0.01

*P < 0.05, nsP ≥ 0.05.

Table 3. Effects (F value) of nitrogen and phosphorus supply and their interaction on relative growth rate of H. vulgaris, nitrogen and phosphorus concentrations, N: P ratio in its leaf.

Factors Relative growth rate
(g day -1)		 Nitrogen concentration
(mg g -1)		 Phosphorus concentration
(mg g -1)		 N:P ratio
Nitrogen supply 7.93** 281.40*** 30.43*** 348.79***
Phosphorus supply 12.08*** 2.22 2.48 25.08***
Interaction between nitrogen
and phosphorus supply 		0.61 9.16*** 9.07*** 0.72
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**P < 0.01

*P < 0.05, nsP ≥ 0.05.

Table 4. Effects (F value) of nitrogen and phosphorus supply and their interaction on the relative growth rate of A. philoxeroides, nitrogen and phosphorus concentrations, N: P ratio in its leaf.

Factors Relative growth rate
(g day -1)		 Nitrogen concentration
(mg g -1)		 Phosphorus concentration
(mg g -1)		 N:P ratio
Nitrogen supply 2.00 135.46*** 90.03*** 62.04***
Phosphorus supply 1.40 4.17* 7.12** 13.76***
Interaction between nitrogen and phosphorus supply 1.37 16.09*** 20.30*** 7.62***
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**P < 0.01

*P < 0.05, nsP ≥ 0.05.

With increase of nitrogen supply, nitrogen and phosphorus concentrations in the leaf of W. trilobata significantly increased (Figs 1A and 2A). Nitrogen concentration in the leaf of H. vulgaris significantly increased (Fig 1B). Phosphorus concentration in the leaf of H. vulgaris significantly increased (P2 or P3). Phosphorus concentration in the leaf of H. vulgaris was significantly higher in the treatment (N2P1) than in the treatment (N1P1). Then, phosphorus concentration in the leaf of H. vulgaris was significantly lower in the treatment (N3P1) than in the treatment (N2P1) (Fig 2B). Nitrogen concentration in the leaf of A. philoxeroides was significantly higher in treatment (N2P2) than in treatment (N1P2). Then, nitrogen concentration in the leaf of A. philoxeroides was significantly lower in the treatment (N3P2) than in the treatment (N2P2) (Fig 1C). Phosphorus concentration in the leaf of A. philoxeroides significantly increased (Fig 2C).

Fig 1. Nitrogen concentration in leaf of the three alien plants was subjected to different nutrient supplies.

Fig 1

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N1: Low nitrogen supply (1 mmol L-1); N2: Intermediate nitrogen supply (4 mmol L-1); N3: High nitrogen supply (8 mmol L-1); P1: Low phosphorus supply (0.15 mmol L-1); P2: Intermediate phosphorus supply (0.6 mmol L-1); P3: High phosphorus supply (1.2 mmol L-1). Values are means ± SE. Bars with different lowercase letters denote significant differences (P < 0.05).

Fig 2. Phosphorus concentration in leaf of the three alien plants was subjected to different nutrient supplies.

Fig 2

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N1: Low nitrogen supply (1 mmol L-1); N2: Intermediate nitrogen supply (4 mmol L-1); N3: High nitrogen supply (8 mmol L-1); P1: Low phosphorus supply (0.15 mmol L-1); P2: Intermediate phosphorus supply (0.6 mmol L-1); P3: High phosphorus supply (1.2 mmol L-1). Values are means ± SE. Bars with different lowercase letters denote significant differences< 0.05).

With increase of phosphorus supply, phosphorus concentration in the leaf of W. trilobata significantly increased (Fig 2A). With increase of phosphorus supply, nitrogen concentration in the leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N1 or N2). Nitrogen concentration in the leaf of A. philoxeroide was significantly lower in the treatment (N3P2) than in the treatment (N3P1). Then, nitrogen concentration in the leaf of A. philoxeroide was significantly higher in the treatment (N3P3) than in the treatment (N3P2) (Fig 1C). Phosphorus concentration in the leaf of A. philoxeroide was significantly lower in the treatment (N3P1) than in the treatment (N3P2). Then, phosphorus concentration in the leaf of A. philoxeroide was significantly higher in the treatment (N3P2) than in the treatment (N3P3). With increase of phosphorus supply, phosphorus concentration in the leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N2) (Fig 2C).

N:P ratio in leaf of W. trilobata or A. philoxeroides was significantly affected by nitrogen, phosphorus supply and interaction between nitrogen and phosphorus supply (Tables 2 and 4). With increase of nitrogen supply, N:P ratio in leaf significantly increased when W. trilobata was subjected to phosphorus supply (P1 or P2). N:P ratio of leaf was significantly lower in the treatment (N1P3) than in the treatment (N2P3). Then, N:P ratio of leaf was significantly higher in the treatment (N2P3) than in the treatment (N3P3). With increase of phosphorus supply, N:P ratio in leaf significantly decreased when W. trilobata was subjected to nitrogen supply (N1 or N3). N:P ratio of leaf was significantly lower in the treatment (N2P1) than in the treatment (N2P2). Then, N:P ratio of leaf was significantly higher in the treatment (N2P2) than in the treatment (N2P3) (Fig 3A).

Fig 3. P ratio in leaf of the three alien plants subjected to different nutrient supplies.

Fig 3

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N: N1: Low nitrogen supply (1 mmol L-1); N2: Intermediate nitrogen supply (4 mmol L-1); N3: High nitrogen supply (8 mmol L-1); P1: Low phosphorus supply (0.15 mmol L-1); P2: Intermediate phosphorus supply (0.6 mmol L-1); P3: High phosphorus supply (1.2 mmol L-1). Values are means ± SE. Bars with different lowercase letters denote significant differences (P < 0.05).

With increase of nitrogen supply, N:P ratio in leaf significantly increased when H. vulgaris was subjected to the same level of phosphorus supply (P1, P2, P3). With increase of phosphorus supply, N:P ratio in leaf significantly decreased when H. vulgaris was subjected to nitrogen supply (N2 or N3). N:P ratio of leaf was significantly higher in the treatment (N1P2) than in the treatment (N1P1). Then, N:P ratio of leaf was significantly lower in the treatment (N1P3) than in the treatment (N1P2) (Fig 3B).

With increase of nitrogen supply, N:P ratio in leaf significantly decreased when A. philoxeroides was subjected to phosphorus supply (P3). N:P ratio of leaf was significantly lower in the treatment (N1P1) than in the treatment (N2P1). Then, N:P ratio of leaf was significantly higher in the treatment (N2P1) than in the treatment (N3P1). N:P ratio of leaf was significantly lower in the treatment (N1P2) than in the treatment (N2P2). Then, N:P ratio of leaf was significantly higher in the treatment (N2P2) than in the treatment (N3P2). With increase of phosphorus supply, N:P ratio in leaf significantly decreased when A. philoxeroides was subjected to nitrogen supply (N2). N:P ratio of leaf was significantly lower in the treatment (N1P2) than in the treatment (N1P1). Then, N:P ratio of leaf was significantly higher in the treatment (N1P3) than in the treatment (N1P2). N:P ratio of leaf was significantly lower in the treatment (N3P2) than in the treatment (N3P1). Then, N:P ratio of leaf was significantly higher in the treatment (N3P3) than in the treatment (N3P2) (Fig 3C).

H(N:P) of W. trilobata and H. vulgaris was 2.78 and 6.25 respectively. The relationship between N:P ratio of leaf and N:P ratio in nutrition solution was not established in A. philoxeroide (Table 5). N:P ratio in leaf of W. trilobata and A. philoxeroides was positively correlated with their relative growth rate (Fig 4A and 4C). However, a similar correlation was not observed in H. vulgaris (Fig 4B).

Table 5. The homeostatic regulation coefficient (H) and parameters calculated from the linear regression for leaf ecological stoichiometry (N:P) of three alien plants with stolon and their congenerics.

Species Rate 1/H R2 H Stoichiometric homeostasis Relative growth rate
W. trilobata N:P 0.36*** 0.83 2.78*** Weak homeostasis 0.065±0.008 ***
H. vulgaris N:P 0.16** 0.67 6.25** homeostasis 0.077±0.007 ***
A.philoxeroides N:P -0.04 0.04 -25.00 absolute homeostasis 0.064±0.009 **
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***P<0.001

**P< 0.01

*P<0.05.

Fig 4. Relationship between the relative growth rate of the three alien plants and N:P ratios of their leaf.

Fig 4

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Discussion

Nitrogen or phosphorus concentration in leaf of the three alien plants significantly increased with increase of nitrogen supply, which is consistent with previous studies [50,51]. On the one hand, nitrogen addition increased the nitrogen concentration of leaf [52–55]. On the other hand, nitrogen addition exerted a positive effect on phosphatase activity, which enhances phosphorus acquisition of plants grown in high phosphorus availability of soil [56–58].

Nitrogen concentration in the leaf of W. trilobata and H. vulgaris was not significantly affected by phosphorus supply, which is consistent with the previous study [59]. With increase of phosphorus supply, a tendency of decreasing was observed in the nitrogen concentration of leaf when A. philoxeroides was subjected to nitrogen supply (N1 or N2). The possible explanation is that phosphorus addition incurred the use of internal nitrogen in issue or organ of plant [18]. As the same time, the tendency of decreasing first and then increasing was observed in the nitrogen concentration of leaf when A. philoxeroides was subjected to nitrogen supply (N3). The possible explanation is that efficiency and proficiency of N resorption significantly decreased in plants subjected to high nitrogen supply [18,59]. Phosphorus addition increased phosphorus concentration in leaves of alpine grassland [18]. Responses of phosphorus concentration in the three alien plants to phosphorus addition were complex, which is similar to the previous study [20].

Compared with those in previous studies, N: P ratio (<14) in leaf of the three alien plants was lower [27,60]. The possible explanation is that the three alien plants are suffering from N-limitation in the experiment [16,29,61]. N:P ratio in leaf was various when the three alien plants were subjected to different levels of nutrient supply. So, more studies are needed to explore the relationship between N:P ratio and nutrient supply.

In the experiment, relative growth rates of W. trilobata, A. philoxeroides and H. vulgaris were 0.065±0.008, 0.064±0.009 and 0.077±0.007 respectively. Based on their H(N:P), W. trilobata, A. philoxeroides and H. vulgaris were classified as weak homeostasis, absolute homeostasis and homeostasis respectively [62]. Stoichiometric homeostasis presented a positive influence on relative growth rate of plants growing in different nutrient availability [49]. Species dominance in Inner Mongolia grassland was positively correlated with their stoichiometric homeostasis [63,64]. So, further studies are needed to explore the relationship between stoichiometric homeostasis and growth performance of the plant.

Relationship between relative growth rate of plants and the leaf N: P ratio of its leaf was affected by nutrient limitation [16,28]. With nitrogen addition, a positive correlation between relative growth rate and N: P ratio of the leaf was observed in Arabidopsis thaliana suffering from N-limitation [16]. Negative correlation between relative growth rate and N: P ratio of the leaf was observed in N. tangutorum [49]. Correlations between relative growth rate and N: P ratio of leaf were different among W. trilobata, A. philoxeroides and H. vulgaris. We tentatively concluded that correlations between relative growth rate and N: P ratio of the leaf could be affected by species as well as nutrient supply.

W. trilobata is distributed in Guangxi Zhuang Autonomous Region, Guangdong Province, Fujian Province, Hainan Province, China. A. philoxeroides is distributed in Guangxi Zhuang Autonomous Region, Guangdong Province, Fujian Province, Sichuan Province, Hebei Province, Yunnan Province, Chongqing, Hunan Province, Jiangxi Province, Zhejiang Province, Hubei Province, Henan Province, Anhui Province, Jiangsu Province, Shandong Province, China. H. vulgaris is distributed in Yunnan province, China (https://doi.org/10.15468/39omei). In the non-native range, the relative growth rate of invasive plants (Agropyron cristatum and Bromus inermis) was higher than native plants (Elymus canadensis and Pascopyrum smithii) [65]. In addition, a high relative growth rate could partly explain the successful invasion of alien plants [66]. A similar pattern was not observed in the experiment. It is suggested that human activities, invasive history, local abundance of species et al maybe play an important role in invasion of alien plants as well as relative growth rate.

Supporting information

S1 Data

(XLSX)

Click here for additional data file. (14.9KB, xlsx)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the Specialized Fund for the Post-Disaster Reconstruction and Heritage Protection in Sichuan Province(No.5132202019000128) and Study and Application on Technological System of Ecological Repair for Surface Destroyed by Hydropower Station Engineering Located in Middle Reaches of Yaluzangbu River (SKLGP2021Z018). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Xiao Guo

11 Oct 2022

PONE-D-22-22959Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plantsPLOS ONE

Dear Dr. Chen,

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Additional Editor Comments:

The authors compared the leaf stoichiometry and relative growth rate of three stoloniferous alien plants in response to nutrient supply. The topic has potential and would be of interest to ecologists and Invasion biologists.

I agree with both reviewers and recommend minor revision.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the paper, the authors addressed the effects of nutrient supply on their leaf stoichiometry and relative growth rate. As I know, the researches on the correlationship bewteen leaf stoichiometry of alien clonal plants are scarce. In this sense, this manuscript is a very good paper. Additionally, the experiment is also well designed and results are also very interesting. However, there are a couple of points that will need to be added or improved.

Line 2, “change” should be “changes”.

Line 7-8， “leaf” should be “leaves”

Line 10，“level” should be “levels”。

Line 10 and Line 12，“correlationship” should be “correlation”。

Line 14， “may be play an important role….” should be “may play an important role….”or “maybe play an important role….”。

Line 20， “plant” should be “plants”。

Line 29， “similar pattern” should be “the similar pattern”.

Line 37， “such as microbe” should be “such as microbes”。

Line 49， “bewteen” should be “between”。

Line 81， “was applied in the experiment” should be “were applied in the experiment”。

Line 84， “pH of nutrient” should be “The pH of nutrient”。

Line 104-106， “The classification of H values is” should be “classifications of H values are”

Line 106-107, “it often is” should be “it is often”.

Line 106-107, “fit” should be “fitting”.

Line 146， Fig 1 “supply ” should be “supplies”。

Line 207-209，“decreased in plant subjected high” should be “decreased in plants subjected to high”.

Line 212, “Comparing with” should be “Compared with”.

Line 214, “N:P ratio in leaf of was” should be “N:P ratio in leaf was”.

Line 215-216, “more studies are need” should be “more studies are needed”.

Line 215-216, “between N:P ration” should be “between N:P ratio”.

Line 220-221, “presented positive influence” should be “presented a positive influence”.

Line 224, “growth rate of plant and its leaf N:P ratio” should be “growth rate of plants and the N:P ratio”.

Line 230, “nutrientsupply” should be “nutrients supply”.

Line 234, “Jinagxi Province” should be “Jiangxi”.

Line 240， “ may be play” should be “ may play” or “maybe play”.

Reviewer #2: The manuscript described leaf stoichiometry of three alien plant species subjected to different N/P availability. The results provided evidence on relationship between leaf stoichiometry and relative growth rate. However，correlations between relative growth rate and N: P ratio of the leaf could be affected by species as well as nutrient supply. Overall, the article is well organized. The study may be helpful to understand effects of leaf stoichiometry on invasion of the three alien plants. In addition, some modifications are still needed to improve quality of the manuscript.If possible, the authors might polish the language in the manuscript with the help of a native English speaker.

Abstract:

Line6: ”N1:1 mmol L-1” . The expression is not clear on what is added.

Line 7-9: Throughout the manuscript, author refer to nutrient ‘contents’. I think it would be more accurate to refer to nutrient ‘concentrations’ in the plant tissue.

Line 14: Local abundance of species was not studied and the grammar of this sentence has error.

Introduction:

Line 19-20: There are many other mechanisms by which plants adapt to nitrogen and phosphorus deficient soils. It would be useful to briefly discuss these before focusing on leaf stoichiometry.

Line 24-25：In introduction, leaf stoichiometry would be changed by different nitrogen and phosphorus supply. The pattern is inconsistent among different plants. It is suggested that author may reorganize the sentence to express clearly.

Line 48-49: The sentence is difficult to understand. It is suggested that author rewrite the sentence to express clearly.

Materials and methods:

Further detail on the three species is not enough. It is suggested that more details on the three species are needed, for example, their invasive potential in China.

Tables and Figures:

The units for relative growth rate (i.e. g g–1 d–1) should be g day–1 (growth per day)?

Language editing:

Line 2, change “change” into “changes”.

Line 7-8, change “leaf” into “leaves”

Line 10, change “level” into “levels”

Line 40, change “……, positive correlation between……” into “……, a positive correlation between……”

Line 43, change “……between N:P ratio of leaf and…..” into “…..between N:P ratios of leaves and…..”

Line 45, change “Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide.” into “The invasion of alien plants severely threatens biodiversity and ecosystem worldwide.”

Line 49，change “bewteen” into “between”

Line 65, change “stolen” into “stolon”.

Line 67, change “Each node along stolon of…..” into “Each node along the stolon of…..”.

Line 81，change “was applied in the experiment” into “were applied in the experiment”

Line 158, change “diffdifferences” into “differences”.

Line 158, change “Table 2 and 4” into “Tables 2 and 4”

Line 234, change “Jinagxi Province” into “Jiangxi”.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Attachment

Submitted filename: comments.docx

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PLoS One. 2022 Dec 2;17(12):e0278656. doi: 10.1371/journal.pone.0278656.r002

Author response to Decision Letter 0

30 Oct 2022

Dear Editors:

Thank for comments from two anonymous reviewers. Those comments are very valuable for revising and improving our manuscript. We have revised the manuscript according to comments. All changes have been marked in red. The responses to the reviewer’s comments are as following:

Reviewer#1:

1. Line 2, “change” should be “changes”. We change “change” into “changes”

2. Line 7-8， “leaf” should be “leaves”. We change “leaf” into “leaves”

3. Line 10，“level” should be “levels”. We change “level” into “levels”

4. Line 10 and Line 12，“correlationship” should be “correlation”. We change “correlationship” into “correlation”

5. Line 14， “may be play an important role….” should be “may play an important role….”or “maybe play an important role….”.We change “may be play an important role….” Into “maybe play an important role….”

6. Line 20， “plant” should be “plants”. We change “plant” into “plants”

7. Line 29， “similar pattern” should be “the similar pattern”. We change “similar pattern” into “the similar pattern”

8. Line 37， “such as microbe” should be “such as microbes”. We change “such as microbe” into “such as microbes”

9. Line 49， “bewteen” should be “between”. We change “bewteen” into “between”

10. Line 81， “was applied in the experiment” should be “were applied in the experiment”. We change “was applied in the experiment” into “were applied in the experiment”

11. Line 84， “pH of nutrient” should be “The pH of nutrient”. We change “pH of nutrient” into “The pH of nutrient”

12. Line 104-106， “The classification of H values is” should be “classifications of H values are”. We change “The classification of H values is” into “classifications of H values are”

13. Line 106-107, “it often is” should be “it is often”. We change “it often is” into “it is often”

14. Line 106-107, “fit” should be “fitting”. We change “fit” into “fitting”

15. Line 146， Fig 1 “supply ” should be “supplies”. We change “supply” into “supplies”

16. Line 207-209，“decreased in plant subjected high” should be “decreased in plants subjected to high”. We change “decreased in plant subjected high” into “decreased in plants subjected to high”

17. Line 212, “Comparing with” should be “Compared with”. We change “Comparing with” into “Compared with”

18. Line 214, “N:P ratio in leaf of was” should be “N:P ratio in leaf was”. We change “N:P ratio in leaf of was” into “N:P ratio in leaf was”.

19. Line 215-216, “more studies are need” should be “more studies are needed”. We change “more studies are need” into “more studies are needed”

20. Line 215-216, “between N:P ration” should be “between N:P ratio”. We change “between N:P ration” into “between N:P ratio”

21. Line 220-221, “presented positive influence” should be “presented a positive influence”. We change “presented positive influence” into “presented a positive influence”

22. Line 224, “growth rate of plant and its leaf N:P ratio” should be “growth rate of plants and the N:P ratio”. We change “growth rate of plant and its leaf N:P ratio” into “growth rate of plants and the N:P ratio”

23. Line 230, “nutrient supply” should be “nutrients supply”. We change “nutrient supply” into “nutrients supply”

24. Line 234, “Jinagxi Province” should be “Jiangxi”. We change “Jinagxi Province” into “Jiangxi”

25. Line 240， “ may be play” should be “ may play” or “maybe play”. We change “may be play” into “maybe play”

Reviewer#2:

1. Line6: “N1:1 mmol L-1”. The expression is not clear on what is added. Details on the nitrogen or phosphorus supply were mentioned in the sentence (line83-85).

2. Line 7-9: Throughout the manuscript, author refer to nutrient ‘contents’. I think it would be more accurate to refer to nutrient ‘concentrations’ in the plant tissue. We change ‘contents’ into ‘concentrations’

3. Line 14: Local abundance of species was not studied and the grammar of this sentence has error. Significant correlation between invasion ability and relative growth rate was not observed in the three alien plants. So, it is suggested that local abundance of species, human activities, invasive history et al may play an important role in invasion of alien plants as well as relative growth rate.

4. Line 19-20: There are many other mechanisms by which plants adapt to nitrogen and phosphorus deficient soils. It would be useful to briefly discuss these before focusing on leaf stoichiometry. N:P ratio is a critical indicator of nutrient limitation (N vs P) in the terrestrial ecosystem. Leaf stoichiometry can reflect nutrient allocation strategy, growth strategy and expanding ability of invasive plants.

5. Line 24-25：In introduction, leaf stoichiometry would be changed by different nitrogen and phosphorus supply. The pattern is inconsistent among different plants. It is suggested that author may reorganize the sentence to express clearly. We rewritten the sentence into “Different nitrogen and phosphorus supply bring about changes of leaf stoichiometry, and these changes are various among different plants”.

6. Line 48-49: The sentence is difficult to understand. It is suggested that author rewrite the sentence to express clearly. We rewritten the sentence into “However, the clear relationship between nutrient absorption capacity of alien plants and their expanding ability was not established in other studies”.

7. In the section of materials and methods, further detail on the three species is not enough. It is suggested that more details on the three species. More information on the three alien plants was supplemented in line54-56 and line237-242.

8. In the section of tables and figures, the units for relative growth rate (i.e. g g–1 d–1) should be g day–1 (growth per day)? We have made correction according to the comments.

9. Line 2, change “change” into “changes”. We change “change” into “changes”

10. Line 7-8, change “leaf” into “leaves”. We change “leaf” into “leaves”

11. Line 10, change “level” into “levels”. We change “level” into “levels”.

12. Line 40, change “……, positive correlation between……” into “……, a positive correlation between……”. We change “……, positive correlation between……” into “……, a positive correlation between……”.

13. Line 43, change “……between N:P ratio of leaf and…..” into “…..between N:P ratios of leaves and…..”. We change “……between N:P ratio of leaf and…..” into “…..between N:P ratios of leaves and…..”.

14. Line 45, change “Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide.” into “The invasion of alien plants severely threatens biodiversity and ecosystem worldwide.” We change “Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide.” into “The invasion of alien plants severely threatens biodiversity and ecosystem worldwide.”

15. Line 49，change “bewteen” into “between”. We change “bewteen” into “between”.

16. Line 65, change “stolen” into “stolon”. We change “stolen” into “stolon”.

17. Line 67, change “Each node along stolon of…..” into “Each node along the stolon of…..”. We change “Each node along stolon of…..” into “Each node along the stolon of…..”.

18. Line 81，change “was applied in the experiment” into “were applied in the experiment” We change “was applied in the experiment” into “were applied in the experiment”

19. Line 158, change “diffdifferences” into “differences”. We change “diffdifferences” into “differences”

20. Line 158, change “Table 2 and 4” into “Tables 2 and 4”. We change “Table 2 and 4” into “Tables 2 and 4”.

21. Line 234, change “Jinagxi Province” into “Jiangxi”. We change “Jinagxi Province” into “Jiangxi”.

We greatly appreciate help from you and two anonymous referees again. We hope that the revised manuscript may be acceptable for publication. We look forward to hearing from you.

Sincerely

Dongwei Yu

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (23.9KB, docx)

Decision Letter 1

Xiao Guo

15 Nov 2022

PONE-D-22-22959R1Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plantsPLOS ONE

Dear Dr. Chen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Please include the following items when submitting your revised manuscript:

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Xiao Guo, Ph.D.

Academic Editor

PLOS ONE

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #1: Manuscript PONE-D-22-22959R1 has been substantially improved after revision.

The authors have satisfactorily responded to most of the comments in my previous report.

Reviewer #2: All the comments have been addressed.The manuscript is relatively well-organized. I evaluated the manuscript and suggest for its publication

**********

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Reviewer #2: No

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PLoS One. 2022 Dec 2;17(12):e0278656. doi: 10.1371/journal.pone.0278656.r004

Author response to Decision Letter 1

18 Nov 2022

Dear Editors:

Thank for comments from two anonymous reviewers. Those comments are very valuable for revising and improving our manuscript. We have revised the manuscript according to comments. All changes have been marked in red. The responses to the reviewer’s comments are as following:

Reviewer#1:

1. Line 2, “change” should be “changes”. We change “change” into “changes”

2. Line 7-8， “leaf” should be “leaves”. We change “leaf” into “leaves”

3. Line 10，“level” should be “levels”. We change “level” into “levels”

4. Line 10 and Line 12，“correlationship” should be “correlation”. We change “correlationship” into “correlation”

5. Line 14， “may be play an important role….” should be “may play an important role….”or “maybe play an important role….”.We change “may be play an important role….” Into “maybe play an important role….”

6. Line 20， “plant” should be “plants”. We change “plant” into “plants”

7. Line 29， “similar pattern” should be “the similar pattern”. We change “similar pattern” into “the similar pattern”

8. Line 37， “such as microbe” should be “such as microbes”. We change “such as microbe” into “such as microbes”

9. Line 49， “bewteen” should be “between”. We change “bewteen” into “between”

10. Line 81， “was applied in the experiment” should be “were applied in the experiment”. We change “was applied in the experiment” into “were applied in the experiment”

11. Line 84， “pH of nutrient” should be “The pH of nutrient”. We change “pH of nutrient” into “The pH of nutrient”

12. Line 104-106， “The classification of H values is” should be “classifications of H values are”. We change “The classification of H values is” into “classifications of H values are”

13. Line 106-107, “it often is” should be “it is often”. We change “it often is” into “it is often”

14. Line 106-107, “fit” should be “fitting”. We change “fit” into “fitting”

15. Line 146， Fig 1 “supply ” should be “supplies”. We change “supply” into “supplies”

16. Line 207-209，“decreased in plant subjected high” should be “decreased in plants subjected to high”. We change “decreased in plant subjected high” into “decreased in plants subjected to high”

17. Line 212, “Comparing with” should be “Compared with”. We change “Comparing with” into “Compared with”

18. Line 214, “N:P ratio in leaf of was” should be “N:P ratio in leaf was”. We change “N:P ratio in leaf of was” into “N:P ratio in leaf was”.

19. Line 215-216, “more studies are need” should be “more studies are needed”. We change “more studies are need” into “more studies are needed”

20. Line 215-216, “between N:P ration” should be “between N:P ratio”. We change “between N:P ration” into “between N:P ratio”

21. Line 220-221, “presented positive influence” should be “presented a positive influence”. We change “presented positive influence” into “presented a positive influence”

22. Line 224, “growth rate of plant and its leaf N:P ratio” should be “growth rate of plants and the N:P ratio”. We change “growth rate of plant and its leaf N:P ratio” into “growth rate of plants and the N:P ratio”

23. Line 230, “nutrient supply” should be “nutrients supply”. We change “nutrient supply” into “nutrients supply”

24. Line 234, “Jinagxi Province” should be “Jiangxi”. We change “Jinagxi Province” into “Jiangxi”

25. Line 240， “ may be play” should be “ may play” or “maybe play”. We change “may be play” into “maybe play”

Reviewer#2:

1. Line6: “N1:1 mmol L-1”. The expression is not clear on what is added. Details on the nitrogen or phosphorus supply were mentioned in the sentence (line83-85).

2. Line 7-9: Throughout the manuscript, author refer to nutrient ‘contents’. I think it would be more accurate to refer to nutrient ‘concentrations’ in the plant tissue. We change ‘contents’ into ‘concentrations’

3. Line 14: Local abundance of species was not studied and the grammar of this sentence has error. Significant correlation between invasion ability and relative growth rate was not observed in the three alien plants. So, it is suggested that local abundance of species, human activities, invasive history et al may play an important role in invasion of alien plants as well as relative growth rate.

4. Line 19-20: There are many other mechanisms by which plants adapt to nitrogen and phosphorus deficient soils. It would be useful to briefly discuss these before focusing on leaf stoichiometry. N:P ratio is a critical indicator of nutrient limitation (N vs P) in the terrestrial ecosystem. Leaf stoichiometry can reflect nutrient allocation strategy, growth strategy and expanding ability of invasive plants.

5. Line 24-25：In introduction, leaf stoichiometry would be changed by different nitrogen and phosphorus supply. The pattern is inconsistent among different plants. It is suggested that author may reorganize the sentence to express clearly. We rewritten the sentence into “Different nitrogen and phosphorus supply bring about changes of leaf stoichiometry, and these changes are various among different plants”.

6. Line 48-49: The sentence is difficult to understand. It is suggested that author rewrite the sentence to express clearly. We rewritten the sentence into “However, the clear relationship between nutrient absorption capacity of alien plants and their expanding ability was not established in other studies”.

7. In the section of materials and methods, further detail on the three species is not enough. It is suggested that more details on the three species. More information on the three alien plants was supplemented in line54-56 and line237-242.

8. In the section of tables and figures, the units for relative growth rate (i.e. g g–1 d–1) should be g day–1 (growth per day)? We have made correction according to the comments.

9. Line 2, change “change” into “changes”. We change “change” into “changes”

10. Line 7-8, change “leaf” into “leaves”. We change “leaf” into “leaves”

11. Line 10, change “level” into “levels”. We change “level” into “levels”.

12. Line 40, change “……, positive correlation between……” into “……, a positive correlation between……”. We change “……, positive correlation between……” into “……, a positive correlation between……”.

13. Line 43, change “……between N:P ratio of leaf and…..” into “…..between N:P ratios of leaves and…..”. We change “……between N:P ratio of leaf and…..” into “…..between N:P ratios of leaves and…..”.

14. Line 45, change “Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide.” into “The invasion of alien plants severely threatens biodiversity and ecosystem worldwide.” We change “Invasion of alien plants is a severe threat to biodiversity and ecosystem worldwide.” into “The invasion of alien plants severely threatens biodiversity and ecosystem worldwide.”

15. Line 49，change “bewteen” into “between”. We change “bewteen” into “between”.

16. Line 65, change “stolen” into “stolon”. We change “stolen” into “stolon”.

17. Line 67, change “Each node along stolon of…..” into “Each node along the stolon of…..”. We change “Each node along stolon of…..” into “Each node along the stolon of…..”.

18. Line 81，change “was applied in the experiment” into “were applied in the experiment” We change “was applied in the experiment” into “were applied in the experiment”

19. Line 158, change “diffdifferences” into “differences”. We change “diffdifferences” into “differences”

20. Line 158, change “Table 2 and 4” into “Tables 2 and 4”. We change “Table 2 and 4” into “Tables 2 and 4”.

21. Line 234, change “Jinagxi Province” into “Jiangxi”. We change “Jinagxi Province” into “Jiangxi”.

We greatly appreciate help from you and two anonymous referees again. We hope that the revised manuscript may be acceptable for publication. We look forward to hearing from you.

Sincerely

Dongwei Yu

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (23.9KB, docx)

Decision Letter 2

Xiao Guo

22 Nov 2022

Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants

PONE-D-22-22959R2

Dear Dr. Chen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Xiao Guo, Ph.D.

Academic Editor

PLOS ONE

Acceptance letter

Xiao Guo

25 Nov 2022

PONE-D-22-22959R2

Effects of nutrient supply on leaf stoichiometry and relative growth rate of three stoloniferous alien plants

Dear Dr. Chen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Xiao Guo

Academic Editor

PLOS ONE

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