A new strategy for controlling invasive weeds: selecting valuable native plants to defeat them

To explore replacement control of the invasive weed Ipomoea cairica, we studied the competitive effects of two valuable natives, Pueraria lobata and Paederia scandens, on growth and photosynthetic characteristics of I. cairica, in pot and field experiments. When I. cairica was planted in pots with P. lobata or P. scandens, its total biomass decreased by 68.7% and 45.8%, and its stem length by 33.3% and 34.1%, respectively. The two natives depressed growth of the weed by their strong effects on its photosynthetic characteristics, including suppression of leaf biomass and the abundance of the CO2-fixing enzyme RUBISCO. The field experiment demonstrated that sowing seeds of P. lobata or P. scandens in plots where the weed had been largely cleared produced 11.8-fold or 2.5-fold as much leaf biomass of the two natives, respectively, as the weed. Replacement control by valuable native species is potentially a feasible and sustainable means of suppressing I. cairica.


Results
Changes in plant growth and relative interaction index in a pot experiment. When the three plants were grown separately, the total biomass of each of the two native species was significantly greater than that of the invasive species, with P. lobata having by far the highest biomass among them ( Table 1). The total biomass of I. cairica grown in competition with P. lobata was significantly lower than that under intraspecific competition or when it was growing alone (Table 1). Conversely, the total biomass of P. lobata in competition with I. cairica was significantly greater than that under intraspecific competition, though lower than that when P. lobata was growing alone (Table 1). There was no significant difference between the total biomass of P. scandens when it was grown with I. cairica and when it was under intraspecific competition ( Table 1). The caulis length showed similar trends as total biomass, except that I. cairica had significantly greater caulis length when it grew alone compared to other treatments (Table 1). Furthermore, as compared with intraspecific competition, the root mass ratio (RMR) of I. cairica increased significantly when it was planted with one of the two native species, whereas the root mass ratio (RMR) of P. lobata decreased significantly when it was planted with I. cairica or P. scandens (Table 1).
Interactions between plants, in general, consisting of competition and facilitation, can be described by the relative interaction index (RII, defined in Methods) 21 . RII has values ranging from − 1 to + 1 and it is symmetrical around zero. A negative value indicates competition and a positive value indicates facilitation. Fig. 1a shows that the RII of I. cairica grown in the presence of P. lobata or P. scandens was negative (RII Ic(Pl) = − 0.280, RII Ic(Ps) = − 0.013, respectively), indicating that the two native species competed well with the alien species, and that the magnitude of the negative effect of P. lobata on I. cairica was larger than that of P. scandens on I. cairica (0.280 > 0.013). That is, P. lobata was a stronger competitor than P. scandens against I. cairica. Further, the effect of P. scandens on P. lobata was positive (RII Pl(Ps) = 0.112) (Fig. 1a), indicating that P. scandens could promote the growth of P. lobata, relative to growth in conditions of intraspecific interaction.
Regarding the impact by an alien species, I. cairica had a positive effect on P. lobata and P. scandens [RII Pl(Ic) = 0.203, RII Ps(Ic) = 0.036] (Fig. 1b), indicating that I. cairica could promote the growth of the two native species (relative to intraspecific interaction conditions). The effect of I. cairica on P. lobata was more positive than that of P. scandens on P. lobata (Fig. 1b) and P. lobata had a relatively small negative Scientific RepoRts | 5:11004 | DOi: 10.1038/srep11004 effect on P. scandens (Fig. 1a), indicating that the facilitation by the alien species was larger than the impact that the two natives had on each other. Changes in gas exchange parameters and chlorophyll fluorescence parameters in a pot experiment. When I. cairica grew alone or under intraspecific competition, the gas exchange parameters were often greater than those of I. cairica growing with P. lobata or P. scandens (Fig. 2), showing the superior photosynthetic ability of the invasive species in the absence of interspecific competition. The net photosynthetic rate P n , stomatal conductance G s , the intercellular CO 2 concentration C i and the transpiration rate T r decreased significantly when I. cairica was grown under interspecific competition with P. lobata or P. scandens (Fig. 2).
When I. cairica and P. lobata were grown alone, there were no significant differences between them in the maximal photochemical efficiency (F v /F m ) of dark-relaxed Photosystem II (the photosystem that splits water to evolve oxygen), the electron transport rate estimated by chlorophyll fluorescence (ETR) and the photochemical yield of Photosystem II under illumination (Φ PSII ). However, when I. cairica was grown with one of the two native species under interspecific competition, F v /F m , ETR and Φ PSII decreased significantly, being lower than when I. cairica was grown alone or under intraspecific competition (Table 2). Similarly, I. cairica grown with P. scandens or P. lobata exhibited a significantly lowered content of Rubisco (the enzyme complex that fixes CO 2 ) in the plant's leaves when compared to I. cairica under intraspecific competition or when grown alone ( Table 2). On the other hand, there was no significant difference in stomatal limitation between interspecific and intraspecific competition treatments. It appears that the lower net photosynthetic rate of I. cairica under interspecific competition could be attributed to a decrease in Rubisco content but not to a stomatal limitation factor.
Effects of replacement control in the field. Five months after sowing seeds of the two native species, the whole experiment plots were pictured in Fig. 3a. There was little recruitment of the invasive weed I. cairica in the plots replaced by the two native species (Fig. 3c,d). By contrast, there was much recruitment of I. cairica in the control plots (Fig. 3b), from which the aboveground biomass of I. cairica and most roots had been cleared five months before.
The biomass of roots, stems, leaves and flowers of the invasive species I. cairica decreased significantly in the plots replaced by the two native species, compared to the control plots (Table 3). P. lobata had the greatest total biomass: its aboveground biomass was considerably greater than that of I. cairica in any treatment, and its foliar biomass, in particular, was 2.2 times that of I. cairica in the control plots, though its root biomass was much lower ( Table 3). The caulis length of I. cairica also decreased significantly in Figure 2. Changes in gas exchange parameters (means ± SD, n = 6). Ic, Pl and Ps means I. cairica, P. lobata and P. scandens growing alone respectively; Ic(Ps) and Ic(Pl) means I. cairica growing with P. scandens and P. lobata, respectively, under interspecific competition; Ic(Ic) means I. cairica under intraspecific competition. P n -the net photosynthetic rate, G s -stomatal conductance, C i -the intercellular CO 2 concentration, and T r -the transpiration rate. Different letters above columns indicate significant differences between competition treatments according to the Least-Significant Difference test (LSD-test, P < 0.05).
the plots replaced by native species compared with the control plots. Replacement control caused a great reduction in total biomass and caulis length of the invasive species (Table 3).
Replacement control also led to a decline in the net photosynthetic rate (P n ) of I. cairica in the field (Fig. 4a). Compared with that in the control plots, the intercellular CO 2 concentration (Ci) of I. cairica under interspecific competition showed the reverse trend (Fig. 4c). Stomatal conductance (G s ) and the transpiration rate (T r ) of I. cairica were not significantly affected by competition from the two native species (Fig. 4b,d).
Changes in soil chemical characteristics showed that soil fertility improved in P. lobata and P. scandens plots. Total nitrogen (TN), NH 4 -N and soil organic matter (SOM) increased significantly in P. lobata and  Table 2. Changes in chlorophyll fluorescence parameters (means ± SD, n 1 = 5, n 2 = 6). * Ic, Pl and Ps means I. cairica, P. lobata and P. scandens, respectively, growing alone; Ic(Ps) and Ic(Pl) means I. cairica growing with P. scandens and P. lobata, respectively, under interspecific competition; Ic(Ic) means I. cairica under intraspecific competition. F v /F m means the maximal photochemical efficiency of PSII, ETR means total electron transport rate, and Φ PSII means the effective photochemical efficiency of PSII. ** Different letters within the same column indicate significant differences between competition treatments according to Least-Significant Difference test (LSD-test, P < 0.05). P. scandens plots compared with those in the control plots, highest in the nitrogen fixer, P. lobata, plots (Table 4).

Discussion
Interspecific competition has been reported to play an important role in determining the likelihood of success in the replacement control of invasive weeds 22,23 . However, when testing the hypothesis that a native species is a better competitor than an invasive species, simultaneous consideration of both the  Table 3. Changes in plant biomass and caulis length in the plots (means ± SD, n = 3, Size of each plot = 2. 0 m 2 ). * Ic(CK) means I. cairica recruiting in the control plots not replaced by any native species; Ic(Pl) means I. cairica recruiting in the plots replaced by the native species P. lobata; Ic(Ps) means I. cairica recruiting in the plots replaced by the native species P. scandens; Pl(Ic) means P. lobata in the same plots as Ic(Pl); Ps(Ic) means P. scandens in the same plots as Ic(Ps). ** Different letters within the same column indicate significant differences between replacement control treatments according to the Least-Significant Difference test (LSD-test, P < 0.05). Ic(CK) means I. cairica recruiting in the control plots not replaced by any native species; Ic(Pl) means I. cairica recruiting in the plots replaced by the native species P. lobata; Ic(Ps) means I. cairica recruiting in the plots replaced by the native species P. scandens; Pl(Ic) means P. lobata in the same plots as Ic(Pl); Ps(Ic) means P. scandens in the same plots as Ic(Ps). P n -the net photosynthetic rate, G s -stomatal conductance, C i -the intercellular CO 2 concentration, and T r -the transpiration rate. Different letters above columns indicate significant differences between competition treatments according to the Least-Significant Difference test (LSD-test, P < 0.05).
Scientific RepoRts | 5:11004 | DOi: 10.1038/srep11004 relative competitiveness of a native species against the invader, and the invader's relative impact on the native species has rarely been attempted 24 . If a native species is to be competitive, we expect it to reduce the growth of the invasive species, I. cairica, more than it could reduce the growth of another coexisting native. Indeed, this was observed in the pot experiments: the native species P. lobata significantly reduced I. cairica growth (RII Ic(Pl) = − 0.280), while the competition between the two natives gave positive or less negative RII values (Fig. 1a). With regard to the invader's relative impact, we expected that the negative effect of the invader on the natives would be less than that of the natives on the invader. Indeed, this was the outcome: I. cairica facilitated the growth of the two natives [RII Pl(Ic) = 0.203 and RII Ps(Ic) = 0.036] relative to growth under intraspecific competition (Fig. 1b). Therefore, at the level of the individual, the two native species have the potential to replace the invasive species I. cairica, with P. lobata having the greater control potential than P. scandens.
What underpins the competitiveness of the two native species, particularly P. lobata? Changes in the root mass ratio of P. lobata indicated that less biomass was allocated to roots and more biomass was allocated to shoots when P. lobata was in competition with I. cairica or P. scandens (Table 1). By contrast, the root mass ratio of I. cairica increased when the invasive weed was in competition with the two native species, as compared with growth in intraspecific competition conditions in the pot experiment (Table 1). Similarly, comparing Ic(Pl) and Pl(Ic) in the field experiment in Table 3, while the root biomass was similar, P. lobata had 11-fold more leaf biomass per plant, and five-fold more stems, compared with I. cairica. Similarly, P. scandens had 2.5-fold more leaf biomass and 5.5-fold more stems compared with I. cairica in interspecific competition conditions. Together, these effects imply that the two native species invested more biomass in light interception, thereby increasing total photosynthetic productivity.
Another factor that lowers the competitiveness of the invasive species, I. cairica, is that interspecific competition reduced its rate of photosynthesis per unit leaf area (Fig. 2), accompanied or caused by a decrease in Rubisco content (Table 2). Gas exchange parameters in the field experiment also showed that I. cairica had a lower P n in the presence of competition from the native species (Fig. 4a). Perhaps the competition for light resulted in partial shading of the I. cairica leaves by P. lobata or P. scandens leaves. A slightly lower growth irradiance to which I. cairica leaves were exposed would represent a lower-light environment which would give rise to a lower content of cytochrome bf (often a rate-limiting bottle-neck in electron flow from PS II to PS I) and a lower Rubisco content 25 . Since I. cairica has relatively high light requirements 15 , reduced light levels due to crowding could be the main reason for its reduction in photosynthetic rates in the presence of interspecific competition.
Another possible reason for the much greater amount of aboveground biomass of P. lobata growing under interspecific competition with I. cairica is its ability to fix atmospheric nitrogen 18 . Indeed, the total soil nitrogen was almost 10-fold higher in the P. lobata plot compared with the I. cairica plot (Table 4). P. scandens enriched soil nitrogen to an immediate extent, lower than that in the P. lobata plot (Table 4), probably because it is not a nitrogen-fixing plant. Its total biomass in a pot experiment was also intermediate [comparing Ic(Ic), Pl(Ic) and Ps(Ic) under competition conditions in Table 1], though this was not the case in the field experiment ( Table 3). All else being equal, using a native legume is a better option for the replacement control of an invasive weed.
Our results are consistent with another study of the competition effects between the native grass, Imperata cylindrica (Poaceae), and the invasive herb, Ageratina adenophora (Asteraceae). I. cylindrica had a higher competitive ability than A. adenophora, being able to heavily suppress the growth of A. adenophora by shoot competition 26 . Another example is the seedling competition between native cottonwood and exotic saltcedar; when native plants have rapid seedling establishment, they can compete with invasive weeds in re-vegetation projects 1 . Both Imperata cylindrica and Ageratina adenophora are herbaceous plants, while cottonwood and saltcedar are trees, each pair having the same life form, just as vine versus vine in our study.
Niche-based community assembly theory predicts that communities should be resistant to invasion by non-native species if they contain native species that have traits similar to the common non-natives [27][28][29][30] . In restoration, this concept may guide the selection of native plants, supporting the use of natives with traits similar to those of invaders 10,31 , since a resident species whose niche overlaps with that of an  Table 4. Changes of soil chemical characteristics in the field plots (means ± SD, n = 3). * Ic(CK) means I. cairica recruiting in the control plots not replaced by any native species; Pl means the plots replaced by the native species P. lobata; Ps means the plots replaced by the native species P. scandens; ** Different letters within the same column indicate significant differences between replacement control treatments according to the Least Significant Difference test (LSD-test, P < 0.05).
invading species will compete most effectively with the invader 32,33 . Therefore, we suggest that selection of a similar life form, sympatric congeners or the same habitat with the invasive plants should be regarded as the preferred option when choosing plant species to replace invasive species. Economic value and ecological security should also be considered, such that economically valuable native species should be given priority. Moreover, if the chosen native species have high seed yields, as is the case of P. lobata and P. scandens here 18,19 , easy and simple sowing methods will help in replacing the invasive species in the field. Replacement control does not result in environmental pollution or re-sprouting of the weeds as do chemical herbicides or mechanical removal, and it offers a safe, economical, and environmentally sustainable solution for weed management.
In conclusion, we have demonstrated that replacement control through planting valuable native species can be a potential means of preventing the invasive weed I. cairica from re-growing. Our results showed that the impact of a one-off replacement control was significant in the short term (about half a year). Further studies need to be conducted on the succession results of replacement control in the long term so as to provide a complete understanding of the ecological restoration of the invaded habitats.

Materials and Methods
Culture of plant materials in a pot experiment. Seeds of P. lobata and P. scandens were collected from the campus of South China Normal University at the end of 2008 (lat. 28°08′ N, long. 113°09′ E, elevation 65 m above sea level). In March 2009, seeds of the two native vines were sown in flat trays and put in an artificial climate incubator (day: 30 °C, 12 h, 65% humidity; night: 23 °C, 12 h, 50% humidity) to germinate before transplanting. Because of the extremely low production amount and viability of I. cairica seeds, most of its spread in China is due to vegetative growth. Therefore, I. cairica rhizomes collected in the Biological Garden at South China Normal University were selected as the experimental materials. To ensure that all material was of similar sprouting potential, rhizomes with similar diameter and of the same age were cut into 10 cm-long fragments, on which there were at least two nodes. Cuttings were grown in plastic cups (diameter 7 cm, height 8 cm) filled with sand, one cutting per cup, and watered every two days and fertilized with 100% Hoagland's nutrient solution once a week before transplanting.
Competition treatment in a pot experiment. In April 2009, three weeks after sowing and sprouting, seedlings of P. lobata and P. scandens, and the regenerated plantlets of I. cairica were transplanted outdoors into pots (diameter 18 cm, height 16 cm) filled with soil (pond mud:sand:humus = 1:1:1) at a naturally-lit experimental site in the Biological Garden from where the founding rhizome had originated. Nine competition treatments which included all possible pair-wise combinations of intraspecific and interspecific competition and no competition were replicated 12 times, as follows: (1) one seedling of I. cairica per pot, indicated by Ic; (2) one seedling of P. lobata per pot, indicated by Pl; (3) one seedling of P. scandens per pot, indicated by Ps; (4) two seedlings of I. cairica per pot, indicated by Ic(Ic); (5) two seedlings of P. lobata per pot, indicated by Pl(Pl); (6) two seedlings of P. scandens per pot, indicated by Ps(Ps); (7) one seedling of I. cairica and one of P. lobata per pot, indicated by Ic(Pl) or Pl(Ic), Ic(Pl) means I. cairica growing with P. lobata under interspecific competition and Pl(Ic) means P. lobata growing with I. cairica under interspecific competition; (8) one seedling of I. cairica and one of P. scandens per pot, indicated by Ic(Ps) or Ps(Ic), Ic(Ps) means I. cairica growing with P. scandens under interspecific competition and Ps(Ic) means P. scandens growing with I. cairica under interspecific competition; (9) one seedling of P. lobata and one of P. scandens per pot, indicated by Pl(Ps) or Ps(Pl), Pl(Ps) means P. lobata growing with P. scandens under interspecific competition and Ps(Pl) means P. scandens growing with P. lobata under interspecific competition.
Pots were watered when plants showed signs of drought stress, and were randomly moved every week to ensure that all the plants were growing under the same environmental conditions. A pergola was constructed for the plants to climb as they grew up. The average monthly temperatures during the experimental period, March to July 2009, were 20.2-28.8 °C. Measurements of gas exchange parameters in a pot experiment. At the same time as chlorophyll fluorescence measurements were made, gas exchange parameters were determined using the LI 6400 portable gas exchange system (LI-COR Inc., Lincoln, NB, USA). Measurements commenced at 8:00 a.m. and were completed within 2 h in full sunshine. PPFD of the natural light ranged from 800 to 1000 μ mol m −2 s −1 , ambient temperature ranged from 28 to 30 °C. CO 2 concentration inside the leaf chamber was maintained at 380 cm 3 m −3 through the CO 2 -controlling system of the LI-6400 attached to a portable CO 2 cylinder. The PPFD of 800 μ mol m −2 s −1 on the cuvette surface was provided by an LED source. Before taking readings, leaves were equilibrated under the artificial light conditions in the leaf chamber for at least 10 min. During measurements, the relative air humidity was about 75% and leaf temperature was maintained at 25 °C. Net photosynthetic rate (P n ), intercellular CO 2 concentration (C i ), stomatal conductance (G s ) and transpiration rate (T r ) were recorded. The stomatal limitation (L s ) was estimated as L s = 1 − C i /C a , where C a is the atmospheric CO 2 concentration 38-40 . Plant growth measurements in a pot experiment. In July 2009, when flowers started to appear, plants were harvested. After removing the cutting fragments of I. cairica rhizomes, the leaves, stems, and roots were separated from each plant and dried to a constant weight for at least 48 h at 60 °C and then weighed. The total biomass was the sum of leaves, stems and roots. Root mass ratio (RMR) was calculated as the biomass of root in proportion to the total biomass. Caulis length was measured with a roll ruler.

Measurements of chlorophyll fluorescence parameters in
To test if the two native species had a competitive ability superior to the alien, we considered both the native competition and the alien impact. First, with regard to the native competition, we tested whether the effects of the two natives on the alien were larger than (a) the effect of the alien on the natives and (b) the effects between the natives. Second, focusing on the alien impact we tested if the effect of the alien on the two natives was lower than the effects between the natives. A relative interaction index (RII) has been proposed by Armas et al. 21 to estimate the intensity of the effect of competition. RII is expressed as: where B w is the observed mass of the target plant when growing with another plant and B o is the mean mass achieved by the target plant in the absence of intra-or inter-specific interaction 21 . This index has revealed several advantages compared to other competition intensity indices such as the relative competition intensity 41,42 . The RII of a target plant ranges from − 1 for a plant completely out-competed by another plant to + 1 for a plant facilitated by another plant so much that its biomass under only intraspecific interaction is negligibly small by comparison. When interspecific interaction and intraspecific interactions have equal effects on the biomass of a target plant, according to our definition in Methods, RII = 0. A negative value indicates competition (i.e., growth of the target species is reduced) and a positive value indicates facilitation (i.e., growth of the target species is promoted). Considering the fact that plants always grow as a population and not as an individual, here we modify the definition of RII slightly, such that B o is the mean mass achieved by the target plant under intraspecific competition. In this definition of B o , RII = 0 when the interspecific interaction is identical with intraspecific interaction; it equals − 1 for a plant completely out-competed by another plant, and equals + 1 for a plant facilitated by another plant so much that its biomass in the presence of only intraspecific interaction is negligible by comparison. Replacement-control treatments in the field. In April 2011, one year after I. cairica had been growing, the plants aboveground and most roots in 9 plots were cleared to mimic the real situation when the plants were weeded out artificially. Of the 9 plots, 3 plots were used to sow seeds of P. lobata (63 seeds/2 m 2 ), 3 plots to sow seeds of P. scandens (63 seeds/2 m 2 ), and the remaining 3 plots as controls (no seeds were sown). Prior to sowing, the seeds of P. lobata and P. scandens were soaked in water for 3 hours in order to increase the sprouting rate. The field was watered once a day after sowing until the seedlings rose up. The seedlings were thinned to 21 plants/2 m 2 plot when they grew up to 20 cm high. After that, no water was added and the plants grew naturally. Approximately six months after replacement-control treatments, gas exchange parameters, plant biomass and soil chemical characteristics were measured.

Establishment of a natural population of
Gas exchange, plant growth and soil characteristics measurements in the field. Gas exchange measurements were measured on August 14, 2011. Procedures followed those of the pot experiment.
In September 2011, the plants were harvested. The leaves, stems, roots and flowers were separated from each plant and dried to a constant weight for at least 48 h at 60 °C and then weighed. The total biomass was the sum of leaves, stems, roots and flowers. Caulis length was measured with a roll ruler.
In addition, the surface soil (0-10 cm) in each plot was collected and soil chemical characteristics were measured. The soil organic matter (SOM) was determined using a K 2 Cr 2 O 7 -H 2 SO 4 oxidation method, total nitrogen (TN) was measured using the Kjeldahl method, and the available NH 4 -N and NO 3 -N were determined in fresh soil samples through steam distillation 43 . Statistical analysis. All statistical tests were performed using SPSS 11.5 software (SPSS Inc., USA).
Plant biomass variables, gas exchange parameters, the fluorescence variables and soil chemical characteristics were compared using one-way ANOVA, followed by least significant difference (LSD) tests at P < 0.05. All observations are independent of one another and scores in groups are normally distributed. A univariate F-test for each variable was used to interpret the respective effects. The equality of error variances was tested by using Levene's test and the error variance of the dependent variable was considered to be equal across groups when P > 0.05.