Elevated CO2 and Increased N Intensify Competition between Two Invasive Annual Plants in China

As multiple invaders often co-occur, understanding the interactions between different invasive species is important. Previous studies have reported on invasional meltdown and neutral and interference relationships between invasive species. However, interspecific interactions may vary with environmental change owing to the different responses of interacting invaders. To better understand the interaction of notorious invasive alien plants under CO2 enrichment and N deposition, the growth characteristics of common ragweed (Ambrosia artemisiifolia) and redroot pigweed (Amaranthus retroflexus) were studied when they were planted in monoculture (4Rag and 4Pig) or mixture (1Rag:3Pig, 2Rag:2Pig, 3Rag:1Pig) under four environmental treatments: elevated CO2, increased N, elevated CO2 + increased N and a control. Increased N positively affected almost all the traits (basal stem diameter, height, shoot biomass, root biomass and total biomass) of common ragweed, except for branch number and root-shoot ratio. But increased N only promoted redroot pigweed’s height and basal stem diameter. interspecific competition promoted basal stem diameter and number of branches but decreased root biomass of common ragweed, and the basal stem diameter was significantly higher in 1Rag:3Pig and 2Rag:2Pig compared to the other two treatments. interspecific competition inhibited almost all the characteristics of redroot pigweed. The interaction between elevated CO2 and increased N also increased the biomass characteristics (shoot biomass, root biomass and total biomass) of common ragweed. However, elevated CO2 inhibited the root biomass of redroot pigweed. The results indicated that common ragweed was a superior competitor under conditions of elevated CO2 and increased N. Moreover, environmental change might strengthen the super-invasive plant common ragweed’s competitive ability.

Understanding the interactive effect of elevated CO 2 and N deposition on invasive alien plants is important. Recent research demonstrated that elevated CO 2 and N deposition synergistically increased the performance (such as biomass and size, survival and reproduction, and photosynthetic rate) of invasive [4,17,18] or naturalized alien plants [6] Life 2022, 12, 1669 2 of 14 than native. Previous studies tested the effect of elevated CO 2 and N deposition on alien plants compared with native plants, but few studies have tested the effect among various invasive plants.
As biological invasions increase in frequency, most habitats are invaded by multiple invasive plants, and interactions between invasive species have attracted attention [19][20][21]. Thus far, three types of interactions have been described: (1) Establishment and impact of one alien invasive species can be facilitated by another invasive species, which is described as invasional meltdown [22]; (2) neutral interactions have also been described [23]; (3) invasion by one species can be negatively impacted by the presence of another invader, termed invasional interference [24][25][26]. Many authors have noted that a decline in one nonnative species results in a rapid increase in another, which indicates that competition among invasive plants may be common [21,27]. In some cases, these negative relationships may result in an invasive species replacing another invasive species, termed "over-invasion" [20]. For example, the replacement of Spartina anglica by S. alterniflora was found in China [28]. Interactions between invasive plants under elevated CO 2 and N deposition remain unclear.
Common ragweed and redroot pigweed are two notorious invasive species both native to North America. They are both included on the list published by the Ministry of Ecology and Environment of the People's Republic of China [29]. Common ragweed, an annual weed in crop fields, usually forms dense mono-specific stands and produces a considerable amount of pollen [30], and this weed is one of the most problematic aero-allergens [31]. It was introduced into China in the 1930s and, since then, it has spread across twenty provinces [32]. Redroot pigweed occurs in various habitats, including agricultural and ruderal habitats [25,33]. It has a negative impact on ecosystems and native species [34,35] and is regarded as the third most notorious weed in the world [25,36]. Redroot pigweed, introduced into China around 1905, has expanded its distribution in large areas [37]. These two invasive species often co-occur in crops or other habitats [38,39].
In the present study, we tested competitive interactions between these two invasive alien plants in monoculture and mixture under four environmental treatments: elevated CO 2 , increased N, elevated CO 2 and N, and a control. We aimed to answer the following questions: (1) do the growth characteristics of these two invasive alien plants respond to elevated CO 2, increased N, and replacement in the same way? (2) which invasive species is more competitive under elevated CO 2 , increased N deposition, and replacement? (3) how do elevated CO 2, N deposition, and replacement affect the reproduction of common ragweed?

Plant Materials
We collected seeds of common ragweed and redroot pigweed from Mentougou District in Beijing (100 km from the experiment site) in October 2013. The seeds were treated at low temperatures (−20 • C) for two months to break dormancy and then stored at room temperature in paper bags. The seeds of the two species were sown on 7 May 2014, at a depth of 2 cm in two field plots and then watered to field capacity once to stimulate germination. After three weeks, seedlings were transplanted into pots in the OTCs according to the experimental design.

Experimental Design
The experiment was conducted at the Chinese Research Academy of Environmental Sciences field laboratory, Shunyi District, Beijing, China (116.5875 • E, 40.19 • N), between 5 June and 8 October 2014.
We tested two CO 2 concentrations in our experiment: ambient CO 2 (375 ppm) concentrations and elevated CO 2 (700 ppm) based on IPCC (Intergovernmental Panel on Climate Change) [40]. Four pairs of open-top chambers (OTCs: 2.2 m in height with an octagonal ground surface area of 6.25 m 2 ) were used. During the experiment, pure CO 2 was continuously ventilated into the OTCs of elevated CO 2 treatment. Elevated CO 2 concentrations were measured at 5-min intervals by monitoring sensors (Qs100). In the elevated CO 2 Life 2022, 12, 1669 3 of 14 treatment, the achieved level was 695.00 ± 15.67 (mean ± SD) ppm. Ambient CO 2 and elevated CO 2 were randomly assigned to each pair. According to the increase in N deposition rates over the coming decades in China [41,42], two N levels were conducted: ambient N (0 addition) and increased (N 0. 8 g/pot).
Four seedlings were planted in each pot under the replacement design: 4Rag:0Pig, 1Rag:3Pig, 2Rag:2Pig, 3Rag:1Pig, and 0Rag:4Pig (where Rag and Pig denote common ragweed and redroot pigweed, respectively). Two N levels, and five replacement levels within each N level, produced 10 pots in each chamber. Thus, 80 pots (32 cm in diameter and 38 cm in depth) were randomly arranged within 8 OTC in the following experimental design: 2 CO 2 levels × 2 N levels × 5 replacement levels × 4 replicates ( Figure 1). During the growing season, N was equally divided eight times and uniformly applied in the form of NH 4 NO 3 solution, while the control pots were sprayed with the same volume of water.
We tested two CO2 concentrations in our experiment: ambient CO2 (375 ppm) concentrations and elevated CO2 (700 ppm) based on IPCC (Intergovernmental Panel on Climate Change) [40]. Four pairs of open-top chambers (OTCs: 2.2 m in height with an octagonal ground surface area of 6.25 m 2 ) were used. During the experiment, pure CO2 was continuously ventilated into the OTCs of elevated CO2 treatment. Elevated CO2 concentrations were measured at 5-min intervals by monitoring sensors (Qs100). In the elevated CO2 treatment, the achieved level was 695.00 ± 15.67 (mean ± SD) ppm. Ambient CO2 and elevated CO2 were randomly assigned to each pair. According to the increase in N deposition rates over the coming decades in China [41,42], two N levels were conducted: ambient N (0 addition) and increased (N 0. 8 g/pot).
Four seedlings were planted in each pot under the replacement design: 4Rag:0Pig, 1Rag:3Pig, 2Rag:2Pig, 3Rag:1Pig, and 0Rag:4Pig (where Rag and Pig denote common ragweed and redroot pigweed, respectively). Two N levels, and five replacement levels within each N level, produced 10 pots in each chamber. Thus, 80 pots (32 cm in diameter and 38 cm in depth) were randomly arranged within 8 OTC in the following experimental design: 2 CO2 levels × 2 N levels × 5 replacement levels × 4 replicates (Figure 1). During the growing season, N was equally divided eight times and uniformly applied in the form of NH4NO3 solution, while the control pots were sprayed with the same volume of water. To ensure homogeneity of the growing conditions, the pots were filled with a mixture of local soil (80%; collected at a depth of 3~15 cm from a weedy field neighboring the experiment base after excluding topsoil) and vermiculite (20%). Before transplanting, the following soil nutrient contents were measured: organic carbon, 8.4 g·kg −1 ; total N, 0.73 g·kg −1 ; ammonium N, 10.77 mg·kg −1 ; and nitrate N, 6.53 mg·kg −1 . One week after transplanting, the elevated CO2 and N enrichment treatments were initiated. Plants were watered weekly as needed throughout the experiment. In each OTC, pots were rearranged randomly within the chamber every month to minimize location-specific effects.

Measurements and Calculations
Plant height, basal stem diameter, and branch number per plant of common ragweed and redroot pigweed were measured in October. Shoots of each species in every pot were harvested from above the soil surface and stored in archival paper bags. We turned over the pot, removed the soil of roots with running water, carefully separated the roots of two species in every pot, and then stored them in paper bags. The shoots and roots were oven- To ensure homogeneity of the growing conditions, the pots were filled with a mixture of local soil (80%; collected at a depth of 3~15 cm from a weedy field neighboring the experiment base after excluding topsoil) and vermiculite (20%). Before transplanting, the following soil nutrient contents were measured: organic carbon, 8.4 g·kg −1 ; total N, 0.73 g·kg −1 ; ammonium N, 10.77 mg·kg −1 ; and nitrate N, 6.53 mg·kg −1 . One week after transplanting, the elevated CO 2 and N enrichment treatments were initiated. Plants were watered weekly as needed throughout the experiment. In each OTC, pots were rearranged randomly within the chamber every month to minimize location-specific effects.

Measurements and Calculations
Plant height, basal stem diameter, and branch number per plant of common ragweed and redroot pigweed were measured in October. Shoots of each species in every pot were harvested from above the soil surface and stored in archival paper bags. We turned over the pot, removed the soil of roots with running water, carefully separated the roots of two species in every pot, and then stored them in paper bags. The shoots and roots were oven-dried at 80 • C for 72 h. Shoot biomass and root biomass of each species in each pot were measured, and the total biomass of each species was calculated per pot.
The competitive ability of these two invasive plants was measured by relative yield. RY values > 1 indicate that one species does better when competing against the other species than when competing against itself [43]. RY values were calculated using the equation below [43,44].
where Y ij is the yield of species i in the presence of species j, p i is the proportion at which species i is sown, and Y i is the yield of species i in monoculture under the same CO 2 and N treatment as that for Y ij .
To analyze the impact of elevated CO 2 , increased N, and replacement on the reproduction of common ragweed, in each pot with common ragweed, seeds were collected by hand in October when the seeds were mature but had not yet begun to drop. The total seed number and the total seed weight were measured as seed yield.

Statistical Analysis
Split-plot ANOVA was employed to test the effects of CO 2 , N, and replacement on the plant performance and relative yield of common ragweed and redroot pigweed, with block as a random factor. When replacement level had a significant effect, significant differences between replacement levels were tested using the Tukey honesty significant difference post-hoc analyses (HSD) (p < 0.05). In all ANOVAs, data were log-transformed to conform to the assumptions of normality and homoscedasticity. All analyses were performed using IBM SPSS Statistics 19 (IBM, 2010, New York, NY, USA).

Growth Characteristics of Two Invasive Alien Plants
Results revealed that height and basal stem diameter of common ragweed were significantly enhanced by increased N in both monoculture and mixtures under both CO 2 concentrations (p < 0.001) (Figure 2a-d; Table 1). At the same time, increased N enhanced (p < 0.05) the height and basal stem diameter (p < 0.001) of redroot pigweed.
Species replacement (p < 0.001) also promoted the basal stem diameter of common ragweed, which was significantly larger in the mixture than in monoculture. The mean stem diameter of ragweed was the largest in the 1Rag:3Pig (p < 0.05, HSD test) (Figure 2a,b), and also significantly larger in the 2Rag:2Pig than in monoculture (p = 0.014, HSD test). Species replacement (p < 0.001) decreased the height and basal stem diameter of redroot pigweed (Figure 2a-d, Table 1). These two characteristics of redroot pigweed in monoculture were all significantly larger than all mixture treatments (p < 0.001, Figure 2a-d; HSD test). Species replacement increased the branch number of common ragweed (p < 0.001), but no branches were observed for redroot pigweed during the experiment.
The impacts of elevated CO 2 on these two invasive plants were not found. A significant interaction of impacts on redroot pigweed was detected in increased N and competition (p = 0.041), elevated CO 2 and species replacement (p = 0.019).

Biomass Characteristics of Two Invasive Alien Plants
Shoot biomass, root biomass, and total biomass of common ragweed were all significantly increased by N addition in both monoculture and mixtures under both CO2 Df represents degrees of freedom, * represents p < 0.05, ** represents p < 0.01

Biomass Characteristics of Two Invasive Alien Plants
Shoot biomass, root biomass, and total biomass of common ragweed were all significantly increased by N addition in both monoculture and mixtures under both CO 2 concentrations (Figure 3a-d; Table 2). No impacts of N addition were found in biomass characters of redroot pigweed.    Df represents degrees of freedom, * represents p < 0.05, ** represents p < 0.01

Relative Yields of Two Invasive Alien Species
Root biomass and the root-shoot ratio of common ragweed were affected by species replacement. Interspecific and intraspecific competition all impacted root biomass and were higher in monoculture (p = 0.001, HSD test) and decreased in 3Rag:1Pig (p = 0.020, HSD test) than in 1Rag:3Pig mixture. The root-shoot ratio of common ragweed was significantly lower in the 3Rag:1Pig mixture than in monoculture (p = 0.011, HSD test). Shoot biomass, root biomass, and total biomass of redroot pigweed were all significantly suppressed in the mixture compared with monoculture (p < 0.001) (Figure 3a-d; Table 2), and these characteristics were all decreased in 3Rag:1Pig than in the other two mixture controls (p < 0.05, HSD test). The root-shoot ratio of redroot pigweed was significantly higher in mixtures than in monoculture (p < 0.05, HSD test) (Figure 3e, f). Elevated CO 2 inhibited the root biomass (p = 0.011) of redroot pigweed in mixtures (Figure 3c, d). An interaction between elevated CO 2 and increased N promoted shoot biomass (p < 0.001), total biomass (p < 0.001), and root biomass (p = 0.03) of common ragweed.

Relative Yields of Two Invasive Alien Species
The relative yields of common ragweed were significantly higher than 1 (p < 0.001, t-test) irrespective of any treatments; while these values were significantly lower than 1 (p < 0.001, t-test) for redroot pigweed (Figure 2e,f). The highest relative yield (RY) values of common ragweed reached 4.72, and the mean values of relative yield were larger in 1Rag:3Pig compared to other replacement treatments (p < 0.001, HSD test), while these values in 2Rag:2Pig were higher than in 3Rag:1Pig (p = 0.001, HSD test). In contrast, the largest relative yield of redroot pigweed was only 0.87, and the mean values of relative yield were higher in 2Rag:2Pig compared to other replacement treatments; however, no significant difference was observed.

Reproductive Characteristics of Common Ragweed
Increased N enhanced the seed yield (p < 0.001; Table 3; Figure 4a,b) and decreased the seed mean weight (p = 0.001) of common ragweed (Figure 4c, d), while elevated CO 2 , competition, and interaction effects showed no significant difference on any of the abovementioned indices ( Figure 4; Table 3). Table 3. Results of three-way ANOVA for the seed characters of the alien invasive plants common ragweed according to CO 2 , N, and replacement levels: seed yield, seed mean weight.  The number of seeds (c,d) mean weight of seeds. On the horizontal axis, 1:3 refers to one common ragweed plant and three redroot pigweed plants, 2:2 refers to two common ragweed plants and two redroot pigweed plants, 3:1 refers to three common ragweed plants and one redroot pigweed plant, and 4:0 refers to four common ragweed plants.

Discussion
In the present study, we found common ragweed and redroot pigweed responded differently to elevated CO2, increased N, and species replacement. We revealed the interspecific competition between common ragweed and redroot pigweed under elevated CO2 and increased N.
Common ragweed showed an apparent competitive advantage over redroot pigweed under mixture treatments, where even one common ragweed plant could strongly inhibit redroot pigweed plants. Common ragweed and redroot pigweed may compete for limited resources. The tall statue of the common ragweed provided a decisive advantage in acquiring light [45]. Common ragweed was higher and more robust than redroot pigweed in mixture treatments (Figures 1 and 2). The height disadvantage of redroot pigweed under competition results in a decrease in its light-capturing capacity [46]. The decline in quality and quantity of light influenced the phenology of pigweed [35,47], so the growth of redroot pigweed was inhibited under competition. We also found the height of redroot pigweed are all positively related to the other characteristics. By comparing the competition between redroot pigweed and common lamb's quarters (Chenopodium album), Figure 4. Effects of elevated CO 2 , N addition and replacement levels on the reproductive characteristics of common ragweed. Bars show mean ± SD (4). (a,b) The number of seeds (c,d) mean weight of seeds. On the horizontal axis, 1:3 refers to one common ragweed plant and three redroot pigweed plants, 2:2 refers to two common ragweed plants and two redroot pigweed plants, 3:1 refers to three common ragweed plants and one redroot pigweed plant, and 4:0 refers to four common ragweed plants.

Discussion
In the present study, we found common ragweed and redroot pigweed responded differently to elevated CO 2 , increased N, and species replacement. We revealed the interspecific competition between common ragweed and redroot pigweed under elevated CO 2 and increased N.
Common ragweed showed an apparent competitive advantage over redroot pigweed under mixture treatments, where even one common ragweed plant could strongly inhibit redroot pigweed plants. Common ragweed and redroot pigweed may compete for limited resources. The tall statue of the common ragweed provided a decisive advantage in acquiring light [45]. Common ragweed was higher and more robust than redroot pigweed in mixture treatments (Figures 1 and 2). The height disadvantage of redroot pigweed under competition results in a decrease in its light-capturing capacity [46]. The decline in quality and quantity of light influenced the phenology of pigweed [35,47], so the growth of redroot pigweed was inhibited under competition. We also found the height of redroot pigweed are all positively related to the other characteristics. By comparing the competition between redroot pigweed and common lamb's quarters (Chenopodium album), it was also found that the species which shade the ground with a dense canopy will favorably compete [48]. In our study, we also found that branch numbers of common ragweed increased with competition, but no branches were observed in redroot pigweed. When two alien invasive plants live in the same habitat or require the same scarce resource [49,50], one species limiting the material or space of the other will result in competition.
Growth characteristics (except for branch number and root-shoot ratio) of common ragweed were enhanced by increased N in all treatments. Only the height and basal stem diameter of redroot pigweed increased in the mixtures under increased N (Figures 1 and 2). Our results are consistent with the premise that responsiveness considerably differs between species under N addition [51]. We also found that increased N and elevated CO 2 intensified the competitive advantage of common ragweed. For example, biomass characteristics of common ragweed were positively affected by the interaction of increased N and elevated CO 2 . Lack of an elevated CO 2 direct effect on these two invasive alien plants might be explained by indirect effects of CO 2 on N limitation, similar to Blumenthal et al. [52]. First, these two invasive alien plants all prefer high levels of nitrogen fertilizer. A previous study showed that the height and dry matter of common ragweed were increased by N addition in both greenhouse and field experiments [53]. Moreover, N addition stimulated the height of redroot pigweed [35] and shoot biomass and root biomass [51]. In the present study, the biomass of redroot pigweed did not increase under N addition in mixtures. Mainly because common ragweed can competitively pre-empt soil nitrogen from redroot pigweed. Secondly, the CO 2 response of species might depend on local resource availability in mixed-species competition [4]. Most plants exhibit a positive growth response to elevated CO 2 due to increased photosynthesis and/or efficient nutrient use when other factors (e.g., water and nutrients) are not limited [6,54]. Because most invasive plants are sensitive to N availability, the impacts of CO 2 on invasive plants varied with N levels [52]. Redroot pigweed increased biomass allocation to the roots to diminish aboveground competitive disadvantage, where plants allocate more biomass to roots to acquire the most limiting resource [55][56][57]. But elevated CO 2 inhibits this increase (Figure 2). A previous study on redroot pigweed revealed lower rates of photosynthesis and stomatal conductivity under elevated CO 2 compared to ambient in water stress treatments [58,59]. We speculate that there is an apparent limit of available N for redroot pigweed growth under competition. Third, C3 plants are thought to take more advantage of CO 2 enrichment than plants with a C4 [60,61]. In our study, common ragweed was a C3 plant and redroot was a C4 plant. Fourth, allelopathic effects also impacted interspecific competition. Bae et al. found that elevated CO 2 may enhance the allelopathic potential of common ragweed [62].
The competition outcome varies with the performance [45] and resource availability of neighboring species [63]. For example, Italian ryegrass has a competitive advantage over common ragweed [64], while redroot pigweed shows higher competitiveness than Phaseolus vulgaris [65]. Competitiveness decreased with an increasing density of common ragweed. The highest competitiveness was under 1Rag:3Pig, where the relative yield of common ragweed increased to 350%. Results indicated interspecific and intraspecific generality, as detected in other experiments [4]. It is also possible that release from intraspecific competition allows common ragweed to grow higher and more robust. In contrast, their biomass decreased when redroot pigweed was released from the intraspecific competition (Figures 1 and 2). Intra-or con-specific competition is always stronger than interspecific one [66,67]. Previous studies have shown that elevated CO 2 and N deposition might favor performances of invasive plants relative to that of native species [3]. Our results revealed elevated CO 2 and increased N prefer the super competitor when two notorious alien invasive plants grew together.

Conclusions
The purpose of this study was to understand the differences in growth characteristics of two alien invasive plants under elevated CO 2 , increased N, and species replacement. The results showed that the biomass of common ragweed was positively enhanced by increased N and species replacement, but redroot pigweed was negatively inhibited. The relative yield of these two notorious invasive plants revealed a competition interaction. In addition, our results revealed that common ragweed gained a more competitive advantage than redroot pigweed under increased N and elevated CO 2 . Our results indicated that common ragweed may be replaced redroot pigweed in heavily invaded regions. The competition or coexistence of redroot pigweed and common ragweed should be discussed under different invasion stages in the future, especially under environmental change. Moreover, understanding the interaction of invasive alien species will help us to manage multispecies invasions in the future.