Effects of Parasitism on the Competitive Ability of Invasive and Native Species

Parasitic plants can often seriously harm host plants and, thus, alter competitive dominance between hosts and neighbouring species. However, whether and how parasitic plants differently affect the competitive abilities of invasive and the native plants have not been tested. In this study, we used Cuscuta grovonii as the parasitic plants and three invasive plants and three native plants as host plants. Host plants grown alone or in competition with Coix lacryma-jobi were either parasitized with Cuscuta grovonii or not parasitized. Parasitism caused similar damage to invasive and native plants when grown with Cuscuta grovonii alone but caused less damage to invasive species than native species when grown in competition. Parasitism increased the competitive ability of invasive plants but did not affect the competitive ability of native plants. In the absence of parasitism, the competitive ability of host plants was significantly negatively correlated with the competitive ability of Coix lacryma-jobi, but under parasitism, there was no significant relationship of the competitive ability between host and competitor plants. Our results indicated that parasitic plants can increase the competitive tolerance of invasive plants, but have no effect on native plants. Thus, parasitism may play an important role in the process of plant invasion.


Introduction
Plant invasion has caused serious economic losses and threatened the biodiversity of ecosystems [1,2] and studies on the mechanisms underlying the success of plant invasions have been an active research focus in ecology and environmental sciences. After arriving in a new habitat, alien plants re-establish new interactions with multitrophic organisms, which may have the potential to facilitate or hinder their invasion [3]. For example, herbivory can promote, inhibit, or have no effect on plant invasion [4][5][6][7]. Therefore, exploring the interactions between invasive species and their new interactors as well as their consequent ecological effects are of great importance to find effective ways to manage invasive plants.
Invasive plants often differ from native plants in their competitive ability [8][9][10], but their effects on native plants can be inconsistent. Some invasive plants are considered to be more competitive than native plants because of resource reallocation from defence to growth or competition (as characterized by the evolution of increased competitive ability (EICA) hypothesis) [11], strong allelopathy [12], or enhanced mutualism [13,14]. However, other studies showed that not all invasive plants are more competitive than native plants and the competitive ability of invasive plants can vary with competitors [15], herbivore stress, or local environments [16]. For example, Sakata and Craig [16] showed that herbivores differently affected the competitive abilities of invasive and native plants and reinforced the negative effects of exotic plants on native plants in herbivore-dense environments. Therefore, the biological control of invasive plants can be advanced by finding biotic factors that can shift the competitive hierarchy between native and invasive plants. Parasitic plants are heterotrophs that obtain part or all of their water, carbon, and nutrients from host plants via haustoria [17][18][19]. Parasitic plants can suppress the growth of invasive plants and have been considered effective biocontrol agents [18]. Parasitic plants often cause serious damage to the growth of host plants and, thus, alter the competitive dominance among host plants and neighbouring species [20,21]. For example, Li et al. [18] found that parasitism on the host species Mikania micrantha by the parasitic plant Cuscuta campestris decreased the growth of M. micrantha and facilitated the growth of neighbouring Coix lacryma-jobi plants. However, whether and how parasitic plants differently affect the competitive abilities of invasive plants and native plants have not been tested.
Invasive plants may respond to parasitism differently from native plants. On one hand, parasitic plants may have a larger impact on invasive species than native species, because invasive species generally have higher resource use efficiency and higher nutrient content than native species [22][23][24]; thus, parasites can acquire more resources from invasive plants than those from native plants [24]. On the other hand, invasive plants may have evolved lower resistance to parasitism than native plants in accordance with the EICA hypothesis [25,26]. In this study, we used Cuscuta grovonii as the parasitic plant to evaluate the competitive abilities of three invasive plants and three native plants in the absence or presence of parasitic plants. Cuscuta grovonii was chosen because it is holoparasite, which is common in the southeast of China [27]. We observed that C. grovonii can cause serious damage on some invasive and native plants in the field, including Solidago canadensis, Bidens pilosa, and Lepidium virginicum. Our objective was to test how the parasitic plant differently regulates the competition between invasive plants and native plants. Our hypotheses were as follows: (1) parasitic plants would cause more damage to invasive species than native species; (2) parasitic plants would decrease the competitive ability of invasive plants more than that of native plants. Our results not only enrich the understanding of the ecology of invasive plants but also provide a useful guide for the management of invasive plants.

Plant Species and Cultivation Conditions
We selected three invasive plant and three native plant species pairs belonging to three different families (Compositae, Amaranthaceae, Cruciferae, respectively) as host plants. The three invasive plants were Solidago canadensis, Alternanthera philoxeroides, and Lepidium virginicum. The three native plants were Ixeris polycephala, Achyranthes bidentata, and Cardamine hirsuta. The selection of pairs of invasive and native plant species in the same family was intended to control for phylogenetic effects. We also selected one native plant species Coix lacryma-jobi (hereinafter Coix) belonging to Poaceae as the native competitor in our study. The stems of C. grovonii were collected from a field population near Taizhou University, Taizhou, China.
The three paired invasive and native plants and Coix are common host plant species in field which can be parasitized by C. grovonii. Solidago canadensis is native to North America and was first introduced to China in the 1910s. It then rapidly spread along the southeast coast of China [28]. Alternanthera philoxeroides is native to South America and was widely introduced to Southern China [29]. Lepidium virginicum is native to North America and now mainly distributed in the east of China [30]. Ixeris polycephala, A. bidentata, C. hirsute, and Coix are common native plant species that are widely distributed in China (http://www.iplant.cn/frps, accessed on 3 November 2022). It is highly likely that these six host plants can compete with Coix in field.
Three-centimetre-long stem cuttings of Alternanthera philoxeroides were collected from a field near Taizhou University. The stem cuttings of A. philoxeroides were vertically inserted into a plastic box (60 cm × 30 cm × 20 cm) filled with peat and cultivated in a greenhouse. Seeds of the other six species were bought from a commercial supplier. On 20 June 2018, seeds of the plant species, except A. philoxeroides, were surface sterilized for 10 min in 5% sodium hypochlorite. After that, the sterilized seeds germinated in a sterilized mixture of vermiculite and peat moss (1:1, v:v).

Experiment Set-Up
The substrate used in this study was a mixture of peat, sand, and vermiculite (6:3:1, v:v:v). The peat had a pH of 6.2 and contained 635 g kg −1 organic matter, 8.43 g kg −1 total nitrogen, and 0.604 g kg −1 total phosphorus. On 31 July 2018, all germinated seedlings were transplanted into plastic pots (diameter × height, 17.5 cm × 14.6 cm) filled with substrate. Additionally, one-node stem cuttings of A. philoxeroides measuring 3.0 cm in length and with similar stem diameters were also planted vertically into the substrate in plastic pots.
This experiment had three factors with six replicates for each treatment: (1) six host plant species (three invasive and three native species); (2) with or without parasitism (P+ and P−); (3) two competition statuses (each of the six host species alone, with just one host seedling per pot, or in competition with one Coix seedling). Additionally, to test the competitive ability of Coix, one extra treatment that consisted of one Coix seedling cultured alone in one pot was also performed.
Four weeks after transplantation, one 10 cm long stem piece of C. grovonii was wound counter clockwise around the stems of host plants to allow the parasite to infect the host plants. Non-parasitized host plants were treated as controls [31]. The experiment was conducted in a greenhouse with 16 h of light and 8 h of dark at 22 • C (day) and 18 • C (night), respectively. Water was sprayed evenly onto the soil of each container every day.

Harvest
On 30 September 2018, the C. grovonii plants were harvested by detaching them from the host plants. After that, shoots of plants were cut 2 cm above the soil surface [32]. Aboveground biomass was dried at 60 • C to a constant mass (at least 72 h) before weighing.

Statistical Analysis
The relative competition intensity (RCI) index was assessed as the difference between biomass when host plants were grown alone and mean biomass when they were grown in competition divided by biomass when grown alone: (Biomass alone − Biomass competition )/Biomass alone [9]. To test the effect of invasive status of host plants (i.e., invasive vs. native) and parasitism (P+ vs. P−) on RCI of host plants or on RCI of Coix, we used linear mixed-effects models with host invasive status and parasitism as fixed factors and species as a random effect nested in host invasive status after the homogeneity test.
The deleterious effect (DE) index is defined as the difference between biomass without parasitism and mean biomass under parasitism divided by biomass without parasitism: (Biomass parasitized − Biomassun parasitized )/Biomassun parasitized . To test the effect of the invasive status of host plants (invasive vs. native) and competition status on the DE of C. grovonii on host plants, we also used linear mixed-effect models with host invasive status and competition status as fixed factors and species as a random effect nested in host invasive status after the homogeneity test.
Data were transformed, as necessary, using the appropriate transformations. Specifically, these were sine transformation for the RCI index, cube root transformation for the DE index, and natural logarithm (ln) transformation for C. grovonii biomass and for the ratio of C. grovonii biomass to host biomass.
Spearman's correlation analysis was used to assess the relationship between the RCI of host plants and the RCI of Coix in the absence or presence of parasitism. Additionally, a simple linear regression model (y = a + bx) was used to fit a straight line to the relationship between the DE index on invasive or native plants and the RCI index of invasive or native plants. To determine whether the slopes of the regression lines significantly differed between invasive and native plants, the homogeneity of the slopes (i.e., parallelism) was Life 2022, 12, 1800 4 of 10 tested by one-way ANCOVA [33]. SPSS (version 16.0; SPSS Inc., Chicago, IL, USA) was used for all statistical analyses and significance was assessed at a p < 0.05 threshold.

Competitive Ability of Host Plants and Coix
Parasitism significantly decreased the RCI of host plants (Table 1; Figure 1a). There was a significant interactive effect between parasitism and host invasive status. Specifically, parasitism significantly decreased the RCI of invasive plants, but did not significantly affect the RCI of native plants (Table 1; Figure 1b). In the absence of parasitism, the RCI of invasive plants was significantly higher than the RCI of native plants. However, in the presence of parasitism, there was no significant difference in RCI between invasive and native plants (Table 1; Figure 1b).

Deleterious Effect of Cuscuta grovonii on Host Plants
Competition significantly increased the DE of C. grovonii on host plants (Tab Figure 3a). There was a significant interactive effect between competition and inva status on the DE of C. grovonii on host plants. Competition significantly increased the of C. grovonii on invasive plants but did not significantly affect the DE of C. grovoni native plants (Figure 3b). When grown without competition, the damage caused b grovonii to invasive and native plants was not significantly different, but when grow competition, C. grovonii caused significantly more damage to invasive plants than na plants (Figure 3b).

Deleterious Effect of Cuscuta grovonii on Host Plants
Competition significantly increased the DE of C. grovonii on host plants (Table 2; Figure 3a). There was a significant interactive effect between competition and invasive status on the DE of C. grovonii on host plants. Competition significantly increased the DE of C. grovonii on invasive plants but did not significantly affect the DE of C. grovonii on native plants (Figure 3b). When grown without competition, the damage caused by C. grovonii to invasive and native plants was not significantly different, but when grown in competition, C. grovonii caused significantly more damage to invasive plants than native plants (Figure 3b). The DE of C. grovonii on invasive or native plants was significantly negatively correlated with the RCI of invasive or native plants. The slopes of the regression lines were significantly different between invasive and native plants (Figure 4).

The Biomass Ratio of Cuscuta grovonii to Host Plants
Neither competition nor host invasive status significantly affected the biomass o grovonii or the ratio of C. grovonii biomass to the biomass of host plants ( Table 3). The

The Biomass Ratio of Cuscuta grovonii to Host Plants
Neither competition nor host invasive status significantly affected the bioma grovonii or the ratio of C. grovonii biomass to the biomass of host plants (Table 3).

The Biomass Ratio of Cuscuta grovonii to Host Plants
Neither competition nor host invasive status significantly affected the biomass of C. grovonii or the ratio of C. grovonii biomass to the biomass of host plants ( Table 3). The ratio of C. grovonii biomass to host biomass varied among host plant species. The ratio of C. grovonii biomass was highest when parasitizing Achyranthes bidentata and Ixeris polycephala and was lowest when parasitizing Lepidium virginicum (Table 3; Figure S2).  Table 3. The effects of host invasive status (PS), species (S), and competition (C) on the biomass of Cuscuta grovonii and on the ratio of C. grovonii biomass to host biomass. p-values of statistical significance below 0.05 are indicated in bold.

Discussion
We found that C. grovonii parasitism differently affected the competitive abilities of invasive and native plants. Specifically, parasitism increased the effects of competition of invasive plants with Coix but did not affect the effects of competition of native plants with Coix. Our results were consistent with a previous study, showing that higher-trophic-level organisms (e.g., herbivores or parasites) can regulate the competition between invasive and native plant species [5]. The modulation of the competition of invasive and native plants caused by parasitism might influence the consequent process of plant invasion.
Non-native plants can use two strategies to gain a competitive advantage over native plants, neighbour tolerance and neighbour suppression [34,35]. Neighbour tolerance refers to the ability to tolerate unfavourable conditions associated with the presence of neighbours [34,36], while neighbour suppression refers to the ability to suppress neighbouring plants via superior resource acquisition and/or allelopathic activity [34,35,37]. In this study, we found that in the absence of parasitism, the competitive ability of host plants was significantly negatively correlated with the competitive ability of Coix, indicating that neighbour suppression may be the main competitive strategy between Coix and host plants in the absence of parasitism. However, under parasitism, the relationship of the competitive abilities between Coix and host plants was non-significant; additionally, parasitism increased the competitive ability of host plants, but had no effect on the competitive ability of Coix ( Figure 1a; Table 1). We hypothesize that parasitism may increase the competitive tolerance of host plants to Coix, because the competitive suppression of Coix by host plants was not influenced by parasitism. However, how parasitism affected the competitive tolerance of host plants merits further study.
In the present study, the competitive abilities of three invasive and native plant species pairs belonging to three families were compared. We found that the competitive ability of two invasive species was lower than their paired native plants in the same family (Solidago canadensis vs. I. polycephala; A. philoxeroides vs. A. bidentata), while that of invasive L. virginicum was similar to native Cardamine hirsuta ( Figure S1). When grown without parasitism, the competitive ability of invasive plants in our study was lower than that of native plants. This result was inconsistent with previous studies, that invasive plants have higher competitive abilities than native plants. For example, in the absence of a parasite, invasive plants S. canadensis and A. philoxeroides are strong competitors and can gradually form monocultures in their invaded ranges [38,39]. The invasive species L. virginicum grew larger and produced a higher biomass relative to competitors than the native species did [40]. However, currently, there are increasing studies finding that not all invasive plants were more competitive than native plants [5,41]. For example, Bossdorf et al. [42] found that performance was similar for Alliaria petiolata plants from native and invasive populations in the absence of competition, whereas native plants outperformed their invasive conspecifics when competing against each other. We speculated that the higher competitive abilities of native plants in our study may be owing to the identity of competitors [15,41,43]. For Life 2022, 12, 1800 8 of 10 example, by quantifying 48 pairs of native and alien plants, Zhang et al. [41] found that common alien plants are more competitive than rare natives but not common natives.
Meanwhile, we found that parasitism increased the competitive ability of invasive plants but had no significant effect on the competitive ability of native plants. This may be owing to C. grovonii causing less damage to invasive plants than native plants when under competition. A previous study showed that parasitism may cause more damage to invasive plants than native plants, as the invasive plants have higher resource use efficiency that could support more vigorous growth of parasitic plants [24]. However, several studies showed that invasive plants generally had higher resource use efficiency than native plants [23,44], which can depend on the identity of host plants and the environment [41,45]. More invasive and native plants should be examined in further research when testing the effect of parasitism on competitive ability. Another possible explanation for the present findings may be the invasive status of C. grovonii in China. Cuscuta gronovii, which is native to North America, was introduced to Beijing, China, in 2010 [27] and is now widely distributed throughout China. The potential co-evolution of C. gronovii and its invasive hosts might cause more damage via parasitism to invasive hosts than native hosts.
In this study, we finished the experiment after one-month parasitism, as we observed that C. grovonii had caused serious damage on host plants. The competition between host plants and competitors may be changed with time and with the developments of C. grovonii. Therefore, we think that studies with different parasitism levels and competition duration are needed in future studies.

Conclusions
Contrary to our hypotheses, we found that parasitic plants caused less damage to invasive plants than native plants and increased the competitive ability of invasive plants, but not native plants. Our results indicate that parasitic plants can dissimilarly affect the growth of invasive and native plants and may, thereby, alter the competitive dominance among invasive plants and native species. Our results provide an example of how parasitic plants change the structure and function of plant communities, especially invaded communities. Further studies focusing on more invasive and native plant pairs are merited.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12111800/s1, Figure S1: The relative competitive index (RCI) of the three family-matched pairs of invasive and native plants species in the presence or absence of Cuscuta grovonii parasitism. P−, without parasitism; P+, with parasitism. * indicates the RCI index of native and invasive plants belonging to the same family were significantly different (p < 0.05); Figure S2: The ratio of Cuscuta grovonii biomass to the biomass of host plants when parasitized by C. grovonii. LV, Lepidium virginicum; AP, Alternanthera philoxeroides; SC, Solidago canadensis; CH, Cardamine hirsuta; AB, Achyranthes bidentata; IP, Ixeris polycephala. Different lowercase letters indicate significant differences between different host plants  Data Availability Statement: The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.