Climate Change Hastens Invasional Interference Between Two Notorious Invasive Plants, Common Ragweed (Ambrosia Artemisiifolia) and Redroot Pigweed (Amaranthus Retroexus), in China

As multiple invaders co-occur in similar habitats, understanding the interactions between different invasive species is very important. Invasional meltdown and neutral and interference relationships have been reported. However, interspecic interactions may vary with environmental change due to the different responses of various invaders. To better understand the interaction of notorious invasive alien plants under global climate change, the growth characters of common ragweed (Ambrosia artemisiifolia) and redroot pigweed (Amaranthus retroexus) were compared when they were planted in monoculture or mixed culture under four environmental treatments: elevated CO 2 , enriched N, elevated CO 2 + enriched N and a control. The results showed that 1) the plant height, basal stem diameter, and shoot, root, and total biomass of common ragweed all consistently increased in response to the treatments, while the growth traits of redroot pigweed were all inhibited. A signicant CO 2 ×N interaction was found only for the shoot and total biomass of common ragweed. 2) Invasional interference between these two notorious alien invasive plants was discovered. Common ragweed consistently displayed an obvious competitive advantage over redroot pigweed regardless of treatment. 3) Elevated CO 2 and enriched N obviously changed the seed mass frequency distribution of common ragweed: elevated CO 2 increased the proportion of small seeds, while enriched N increased the proportion of large seeds. We conclude that common ragweed can outcompete redroot pigweed; moreover, elevated CO 2 and N addition hasten this competitive advantage.

Understanding how invasive plants respond to the interactive effect of elevated CO 2 and N deposition is important but still underappreciated. Plant growth and biomass accumulation can be enhanced by elevated CO 2 , and the rates of these processes may be constrained by soil N availability (Luo et al. 2004;Feng et al. 2015). The invasion of Eupatorium adenophorum may be exacerbated by CO 2 enrichment and N deposition (Lei et al. 2012).
Successful invasion by one plant not only is related to its adaptability to the environment but also depends on its ability to compete with other plants, including other invaders. As biological invasions increase in frequency, most habitats are invaded by multiple invasive plants, so interactions between different invasive species become more important (Shea and Chesson 2002). Invasive species can interact with each other (Russell et al. 2014; Kuebbing and Nunez 2014). One alien invasive species can be facilitated by another species, which is described as invasional meltdown (Simberloff and Von Holle 1999).
Most known cases of invasional meltdown involve plants and organisms at other trophic levels, and there are few examples involving plant-plant interactions (Simberloff 2006). Invasion by one species can be negatively impacted by the presence of another invader, known as invasional interference (Yang et al. 2011; Rauschert and Shea 2012), especially when they require similar niches (Rauschert and Shea 2016). In some cases, these negative relationships may result in an invasive species replacing another invasive species, known as "overinvasion", which is used to describe this phenomenon in an animal context (Russell et al. 2014). 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 (Kuebbing and Nunez 2014). Invasional interference and overinvasion are understudied in plants. For example, the replacement of Spartina anglica by Spartina alterni ora was deduced (Zhi et al. 2007) in China. It is also possible that the interactions are neutral, as supported by a meta-analysis on the interactions of invasive animals (Jackson 2015).
The types of interactions among plants depend on environmental, species and individual features (Callaway and Walker, 1997 (Sorte et al., 2013). However, different invasive plants are seldom compared in altered environments. Competitive ability varies with changes in environmental conditions; for example, an increased competitive advantage of invasive species has been attributed to higher N availability (Quinn et al., 2007). Variations in the environment can in uence the intensity of competitive interactions (James and Richards 2007) and competitive relationships of plants because competing species respond differently to environmental change (Miller et al. 2007;Mamolos 2006). Moreover, competition relationships and elevated CO 2 and N deposition often occur at the same time, and how these factors impact invasive species still needs to be determined.
To study the relationship of two notorious invasive plants, Ambrosia artemisiifolia (common ragweed) and Amaranthus retro exus (redroot pigweed), and the impacts of elevated CO 2 and N deposition on their relationship, we planted these two plants in monoculture and mixed culture under different environmental treatments, namely, elevated CO 2 , N deposition, elevated CO 2 + N deposition and a control. The growth characters, biomass and relative yield of the two invasive species were all measured, and we also examined the seed production of common ragweed under competition and elevated CO 2 and N deposition.
We aimed to answer the following questions: (1) Do these two invasive alien plants respond to elevated CO 2 , N deposition and competition in the same way?
(2) Does climate change impact the relationship between two invasive alien plants? (3) How do elevated CO 2 , N deposition and competition affect the reproduction of common ragweed?

Materials And Methods
Two notorious invasive species were selected: Ambrosia artemisiifolia (common ragweed) and Amaranthus retro exus (redroot pigweed), both of which are native to North America. Ambrosia artemisiifolia, which is an annual weed in crop elds, usually forms dense monospeci c stands and produces a considerable amount of pollen (Gentili et al. 2018), and this weed is one of the most problematic aero-allergens (Ziska and Caul eld 2000). It was introduced into China in the 1930s and has since spread in twenty provinces . Amaranthus retro exus occurs in various habitats, including agricultural and ruderal habitats (Valerio et  June 5 and October 8, 2014. Four chambers each were randomly selected to be maintained at one of two CO 2 concentration levels: ambient CO 2 (375 µl/L) and elevated CO 2 (700 µl/L). Ten plastic pots (32 cm in diameter and 38 cm in depth) were randomly arranged within each OTC in the following experimental design: 2 CO 2 levels × 2 N levels × 5 competition levels × 4 replicates. Thus, the experiment consisted of 80 pots in total. During the growing season, the total N addition levels were 0 and 0. 80384 g·pot − 1 (10 g·m − 2 ×0.080384 m 2 ·pot − 1 ). N was divided equally 8 times and uniformly applied in the form of NH 4 NO 3 solution, and the control pots were sprayed with the same volume of water. Five competition levels were assigned as follows: 4A:0Am (where A and Am denote common ragweed and redroot pigweed, respectively), 1A:3Am, 2A:2Am, 3A:1Am and 0A:4Am. Four plants were planted in each plot under the different competition levels.
The seeds were treated at low temperature (-20°C) for two months and then stored at room temperature in paper bags. The seeds of the two species were sown on May 7, 2014, at a depth of 2 cm in two eld plots and then watered to eld capacity once to stimulate germination. After three weeks, uniformly strong and tall seedlings were transplanted into pots in the OTCs according to the experimental design. To ensure homogeneity of the growing conditions, the pots were lled with a homogeneous mixture of local soil (80%; collected at depth of 3 ~ 15 cm from a weedy eld neighboring the experiment base after the exclusion of topsoil) and vermiculite (20%). Before transplantation, 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 transplantation, the elevated CO 2 and N enrichment treatments were initiated. Pots were watered weekly as needed throughout the experiment. In each OTC, pots were moved randomly every month. In the elevated CO 2 treatment, the achieved level was 695.00 ± 15.67 (mean ± SD) ppm CO 2 (measured at 5-min intervals).

Measurements and calculations
The plant height, basal stem diameter and branch number per plant of common ragweed and redroot pigweed were measured. The shoots of each species in every pot were harvested from above the soil surface and stored in archival paper bags. Roots and soil were removed from the pots, and the roots of each species in every pot were collected and stored in archival paper bags. The shoots and roots were oven-dried at 80°C for 72 h. The shoot biomass and the root biomass of each species in each pot were measured, and the total biomass of each species was calculated per pot.
Relative yield (RY) is a measure of the relative competitive abilities of two species. RY values > 1 indicate that one species does better when competing against the other species than when competing against itself. High RY values indicate a high degree of competitiveness of one species relative to that of the other where Y ij is the yield of species i in the presence of species j, p i is the proportion at which species i was 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 and N addition 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. To examine the impacts of CO 2 and N on the size of seeds of Ambrosia artemisiifolia, a total of 100 seeds were selected randomly from each pot of monocultured Ambrosia artemisiifolia; each seed was weighed and placed in one of four mass classes (< 2 mg, 2-5 mg, 5-8 mg and > 8 mg) on the basis of individual seed mass. The percent ratio of each class per pot was calculated, from which the seed mass frequency distributions were determined.

Statistical analysis
Three-way ANOVA was used to test the effects of CO 2 , N and competition on the growth performance and competitive ability of Ambrosia artemisiifolia and Amaranthus retro exus. When competition level had a signi cant effect, signi cant differences between competition levels were tested using the Tukey honesty signi cant difference post-hoc analyses (HSD) (P < 0.05). Two-way ANOVA was used to test the effects of CO 2 and N on the seed size of Ambrosia artemisiifolia. In all ANOVAs, data were log transformed where necessary to conform to the assumptions of normality and heteroscedasticity. All analyses were performed using IBM SPSS Statistics 19 (IBM, 2010).
Effects of CO 2 , N and competition on the growth characters of two invasive alien plants The height, basal stem diameter and branch number of common ragweed were signi cantly increased by N addition when the species was grown in monoculture or with redroot pigweed, whereas only the height of redroot pigweed was increased by N addition (Fig. 1). Common ragweed bene tted more from the presence of redroot pigweed than vice versa. There were no distinct differences in height or basal stem diameter between the two alien plants when grown in monoculture; however, these characters were markedly decreased in redroot pigweed (P < 0.001) and increased in common ragweed when these two species were grown together. The basal stem diameter of common ragweed in the 1A:3Am competition treatment was larger than that in 3A:1Am (P < 0.001) and slightly larger than that in 2A:2Am (P = 0.02). Nonsigni cant differences in the basal stem diameter of redroot pigweed were observed under different competition scenarios when the two alien plants were grown together. The branch number of common ragweed increased in response to competition (P < 0.001) and was not impacted by elevated CO 2 and N addition (Table 1). No change in branch number of redroot pigweed in response to competition was observed, even in the monoculture treatment. The interaction effects of different factors did not affect the growth characters of the two invasive plants. Effects of CO 2 , N and competition on the biomass of the two invasive alien plants The three-way ANOVA results showed that N addition consistently resulted in signi cant increases in the shoot, root, and total biomass of common ragweed at all competition levels (P < 0.001) ( Table 2; Fig. 2A, B, C). Elevated CO 2 increased the root biomass (P = 0.009) and the root-shoot ratio (P = 0.029) of common ragweed and inhibited the root biomass (P = 0.015) and the root-shoot ratio (P = 0.004) of redroot pigweed (Table 2). An interaction effect of elevated CO 2 and N addition was observed for the shoot biomass (P = 0.035) and total biomass (P = 0.042) of common ragweed. Competition notably decreased the shoot biomass and total biomass of redroot pigweed (P < 0.001) and differentially impacted the root biomass and the root-shoot ratio of these two invasive alien plants. The shoot biomass and total biomass of redroot pigweed under 3A:1Am were lower than those under 1A:3Am (P < 0.001), 2A:2Am (P < 0.05) and monoculture (P < 0.001). The root biomass of redroot pigweed under competition was lower than that under monoculture, with the root biomass under 3A:1Am lower than that under 1A:3Am (P = 0.006). The root-shoot ratio of redroot pigweed was signi cantly higher under monoculture than under 1A:3Am (P = 0.03) and 3A:1Am (P = 0.016). The root biomass of common ragweed under 1A:3Am was lower than that under 3A:1Am (P = 0.034) and monoculture (P = 0.001).
The root-shoot ratio of common ragweed under 1A:3Am was signi cantly lower than that under monoculture (P = 0.011).

Effects of CO 2 , N and competition on the RY of two invasive alien plants
The RY of common ragweed was higher than that of redroot pigweed, and the largest RY value of common ragweed reached 4.72; in contrast, the largest RY of redroot pigweed was only 0.87. The RY of common ragweed was enhanced by elevated CO 2 , N addition and competition (  Fig. 3). These results showed that common ragweed had an obvious competitive advantage over redroot pigweed under control conditions; even one common ragweed plant could strongly inhibit three redroot pigweed plants. Effects of CO 2 , N and competition on the reproductive characters of common ragweed N addition increased the seed yield (P < 0.001; Table 4; Fig. 4A) and decreased the seed mean weight (P = 0.003) of common ragweed. The seed number, seed total weight and seed mean weight were not signi cantly altered by elevated CO 2 , competition or their interaction. To examine the impacts of elevated CO 2 and N addition, seeds collected from monoculture were grouped into four mass levels. The results showed that climate change obviously changed the seed mass frequency distributions. Elevated N and CO 2 enhanced the degree of seed size variation, and higher proportions of larger seeds (mass > 8 mg) and smaller seeds (mass < 2 mg) were produced. Elevated N signi cantly increased the proportion of larger seeds from 3.29 ± 1.12 in the ambient N pots to 6.04 ± 1.33 in the enriched N pots (P < 0.001; Fig. 5) and decreased the proportion of smaller seeds from 3.34 ± 1.24 to 1.58 ± 0.80 (P = 0.014; Table 3). Elevated CO 2 increased the proportion of smaller seeds to 12.53 ± 5.64 (P = 0.001; Fig. 5). There was no signi cant N×CO 2 interaction effect on any of the abovementioned indices. Table 4 Results of three-way ANOVA for the seed number, seed total weight, and seed mean weight of common ragweed according to CO 2 , N and competition levels.

Negative interaction between common ragweed and redroot pigweed
In this experiment, common ragweed was clearly the superior competitor under the different treatment levels, which implied a negative interaction between these two notorious alien invasive species. Negative relationships between alien invasive species are common at broader taxonomic levels, such as in insects (Braks et al, 2004), mammals (Bailey 1993;Russell et al. 2014) and plants (Williams and Buxton, 1995;Kuebbing and Nunez 2014). Competition between two alien invasive species can occur with one species limiting the material, substance, or space of the other when they live in the same habitat or require the same scarce resource (Belote and Weltzin 2006; Griffen et al. 2008). In most cases, the alien invasive species belong to the same genus; however, in some cases, they are phylogenetically dissimilar (Kuebbing and Nunez, 2014). For example, the removal of water hyacinth, which increased the invasion of alligator weed, indicated that competition can occur between phylogenetically dissimilar species (Wundrow et al., 2012). Thus, phylogenetic relatedness is not always necessary for predicting interactions between invaders (Cahill et al., 2008).
We focused on the relationship between two dissimilar invasive species that occupy similar niches.

Climate change hastens the negative interaction
Alien species interactions will change in response to environmental alterations, such as climate change and N deposition (Tylianakis et al., 2008). Common ragweed bene ted more than redroot pigweed from elevated CO 2 and N addition, which resulted in the enhanced competitive ability of common ragweed.
N addition exerted a similar effect on the height of both common ragweed and redroot pigweed but contrasting effects on their biomass. We found that N addition increased the shoot, root and total biomass of common ragweed but had no impact on these characters in redroot pigweed (Fig. 2). The level of responsiveness differs considerably between species under N addition (Blackshaw et al. 2003). These results are consistent with the ndings of Leskovšek et al. (2012b) that the height and dry matter of common ragweed were increased by N addition in both greenhouse and eld experiments. N addition stimulated the height of redroot pigweed, which was also observed in other experiments (Wang et al. 2017), and no discernible effect on biomass was observed. Biomass allocation in response to nutrients was also different between the two invasive alien species. Compared with monoculture, 1A:3Am and 2A:2Am decreased biomass allocation to the roots in common ragweed, and 3A:1Am increased it (Fig. 2). Results is dissimilar to previous study showed that biomass allocation of ragweed to the roots at N addition levels exhibited a low plasticity (Leskovšek et al, 2012a), and mainly because in our study inter-and intra-species competition are all considered. This result can be explained by resource-limitation theory, where plants allocate more biomass to roots in order to acquire more of the most limiting resource (Poorter and Nagel 2000;). However, redroot pigweed allocated more biomass to the roots than to the shoots, as a clear decrease in shoot biomass and a slight decrease in root biomass were observed under competition (Fig. 2). The height disadvantage of redroot pigweed under competition results in a decrease in its light-capturing capacity (Wang et al., 2021), and root-foraging responses help it capture more nutrients (Keser et al. 2014(Keser et al. , 2015. Decreased growth and physiological performance of redroot pigweed was also observed under competition (Wang et al. 2016). N addition increased relative competition intensity measured according to most traits of common ragweed but decreased that for most traits of redroot pigweed when the two species were grown together. The negative interaction of these two species was ampli ed by N addition.
Elevated CO 2 increased the root biomass of common ragweed but decreased that of redroot pigweed. Most plants exhibit a positive growth response to elevated CO 2 due to increased photosynthesis and/or nutrient use e ciency when other factors (e.g., water and nutrients) are not limited (Kimball et al. 2002).
Some research has also indicated that elevated CO 2 signi cantly increases shoot, root, and total plant biomass; plant height; and seed mass (Wayne et al. 2002;Rogers et al. 2006;Runion et al. 2014). Our results indicated that common ragweed gains a competitive advantage under N addition, so a positive relationship was observed between root biomass and elevated CO 2 . Otherwise, we speculate that there is an obvious limit of available N for redroot pigweed growth in the experimental area, and the CO 2 effects on this weed are constrained by N limitation. We also observed that common ragweed grown in monoculture responded strongly to elevated CO 2 , and when grown under competition with redroot pigweed, it responded to elevated CO 2 with a slight increase, . Thus, we speculate that common ragweed invasion may be more serious than redroot pigweed invasion, as available N limitation is reduced by N deposition in our experimental area, whereas the positive effect of elevated CO 2 will be greater for common ragweed. Seed size/mass is highly in uential in determining seed germinability, seedling establishment and seedling growth, particularly in competitive environments (Stanton 1984 resources (Venable 1992) and generally disperse farther than large seeds (Cappuccino et al. 2002), which is an important life-history trait of invasive alien plants (Rejmanek and Richardson 1996). We speculate that more large seeds in common ragweed may improve its interspeci c competitive ability through rapid seedling growth during the early phase, while more small seeds would be bene cial for farther dispersal. Therefore, the intensi ed seed size variation resulting from elevated CO 2 and enriched N will promote the invasion of common ragweed. Of course, verifying this speculation will require more experiments to determine the exact relationships of seed size with seed germination, the seedling growth rate, interspeci c competitive ability, and seed dispersal.

Conclusion
Common ragweed showed a higher competitive ability than redroot pigweed under interspeci c competition, although no difference was observed under monoculture. The results revealed a negative interaction between these two notorious invasive species, which suggested invasional interference between them. In addition, elevated CO 2 and N addition increased the competitive ability of common ragweed but suppressed that of redroot pigweed, and simulated N deposition had a stronger effect than CO 2 on these two alien species. The results suggested that the competitive abilities of plants respond differently to climate change, N is a key factor in the invasion of these two species, and the role of elevated CO 2 may be impacted by insu cient N. Finally, N addition increased the proportion of smaller seeds of common ragweed, but elevated CO 2 increased the proportion of smaller seeds; thus, N should be bene cial for reproduction, and CO 2 favors the spread of common ragweed.