New Evidence for the Hypothesis of Reducing Natural Enemy Pressure of Eupatorium adenophorum: Solenopsis invicta Competing with Doxrylus orientalis to Feed on E. adenophorum ()
1. Introduction
Eupatorium adenophorum Sprengel (Asteraceae; hereafter Eupatorium) is a composite broad-leaved weed native to Central America, which has invaded more than 30 countries and regions with subtropical climates [1] . Eupatorium inhibits local plant growth with a great competitive advantage in the invaded area, causing significant damage to the local ecological and economic environment [2] [3] [4] . Increasing evidence shows that Eupatorium population faces an increasing pressure of natural enemies in the invaded habitats [2] [5] [6] [7] [8] , and a decreasing trend of invasiveness [7] .
The successful parasitism of the specific natural enemies Procecidochares utilis [9] [10] [11] reduced the reproductive capacity of Eupatorium and weakened its invasiveness to some extent. So far, the confirmed natural enemies of Eupatorium include Dorylus orientalis Westwood (Hymenoptera: Formicidae; hereafter Dorylus) [7] , and Orthezia quadrua (Homoptera: Ortheziidae) [8] . Niu Yanfen and her colleagues found that Acrydium japonicum Bolivar (Orthoptera: Tettigidae), larvae of Calimorpha albipuncta Wileman (Lepidoptera: Arctidae), Agriolimax sp (Stylommatophore: Agriolimacidae)] and one larvae each of Lepidoptera and Lepidop-tera, Lepidoptera, also ate Eupatorium [7] . This indicated that Eupatorium in Yunnan habitats had already faced great pressure from natural enemies. This study supports the hypothesis of reduced pressure on natural enemies of Eupatorium by reporting the phenomenon that Solenopsis invicta Buren (Membranoptera Formicidae; hereafter Solenopsis) competed fiercely with Dorylus for Eupatorium and the biomass of invasive Eupatorium populations was lower than those of the original populations in a homogeneous garden.
Solenopsis, also known as the red imported fire ant, is the most destructive and aggressive worldwide invasive ant [12] [13] . It is a social insect commonly creating colonies in the soil [14] . It is native to central South America and has invaded many countries, including the United States, Mexico, Australia, New Zealand, China, Malaysia, Singapore, and the West Indies [15] . Under current climatic conditions, in China, Hainan, Guangdong, Guangxi, Fujian, Zhejiang, Jiangsu, Anhui, Hubei, Hunan, Jiangxi, Guizhou, Yunnan, Chongqing, Sichuan, Henan, and Taiwan are potentially suitable areas [12] . In the invaded areas, it had a very negative effect on biological diversity, public safety, agriculture, and economics [14] [15] [16] . However, it competed with the deadly natural enemy of Eupatorium, Dorylus, to feed on Eupatorium, which shows that this species has a certain weakening effect on the invasion of Eupatorium.
2. Materials and Methods
2.1. Plant Materials
In cultivation experiment, seeds of Eupatorium were originally collected from their original (Mexico) and invasive (China) areas (Table 1) and planted for a generation at Qujing Normal University, Qujing City, Yunnan Province, Southwest China, and stored in 4˚C until used. On 10 August 2012, seeds from each population were sown in seedbeds. On 14 October 2012, when the seedlings were approximately 10 cm tall, vigorous seedlings with similar size were transplanted singly in soil in a common garden in Kunming University, Kunming City, Yunnan Province, Southwest China, where the mean annual temperature is 16.5˚C and the mean annual precipitation is 1450 mm, the frost free period is 278 d.
2.2. Experimental Design
In cultivation experiment, 10 populations of Eupatorium were cultivated. All plants were cultivated separately. 10 plants were used for each population, resulting in a total of 100 plants. Plants were watered via a trickle irrigation system every week. Populations were arranged in a completely randomized designed. Every two plants are spaced 40 cm apart in a community, and every two populations are spaced 60 cm apart. The roots of each plant are separated by plastic tubes (35 cm in width and 25 cm in depth) to prevent tangle. Hand weeding was
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Table 1. Background information on each site where the seeds of the crofton weed were collected in the native and invasive ranges.
MAT, mean annual temperature; MAP, mean annual precipitation.
performed when necessary.
On 20 March 2013, 156 days after being transplanted, plants were harvested and the total dry biomass was recorded for 7 plants of each population. Supporting organs (including stems, branches, and petioles) were severed at the soil surface, belowground tissue was harvested by washing soil off roots/rhizomes with water. Each plant parts were dried for 48 h at 60˚C until constant mass was achieved, and biomass determined by weighing to nearest 0.01 g. Total biomass was determined.
2.3. Observation of Natural Insect Enemy and Result
On March 3, 2015, it was observed that Dorylus attacking the wild population of Eupatorium on the outskirts of experimental base in Kunming University. Considering that was a familiar phenomenon, it attracted no extra attention. Two months later, researchers observed that there were many broken or complete bodies or dying Dorylus around Eupatorium which were once eaten by Dorylus (Figure 1), and a large number of Solenopsis were eating the epidermis and cortical stem tissue of Eupatorium (Figure 1). Solenopsis moved to another feeding position after eating up about 1 - 2 cm2 of non-lignified tissue of stems of Eupatorium. The width of each destroyed point did not exceed half of the perimeter of the stem (Figure 1). Solenopsis generally ate from the lower part of the stem of Eupatorium, moving upwards, thus leaving many mottled damage symptoms on the stem of Eupatorium. Each patch measured 3 - 5 cm in length and about 1 cm in width (Figure 1).
During the observation period, the diseased bodies or remains of Dorylus were moved out by Solenopsis from the caves under Eupatorium from time to
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Figure 1. Differences in total biomass of Eupatorium adenophorum Sprengel from native (Mexico, filled bars) and introduced (China, open bars) populations. Open bars and fillled bars depict means and SE for introduced population of introduced and native population, respectively. C1, C2, C3, C4, and C5 represent the five populations in the introduced area; M1, M2, M3, M4, and M5 represent the five populations in the introduced area; C and M bar depict means and SE across populations within each region.
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Figure 2. Solenopsis invicta (Buren) (Hymenoptera: Formicidae) eating living roots, stems, leaves of Eupatorium adenophorum Sprengel and driving away Dorylus orientalis Westwood. Aa, S. invicta attacking epidermis and cortex tissue of stems of E. adenophorum; (B) S. invicta (a) and D. orientalis (c) engaged in a fight; Cd, corpses of D. orientalis were moved out by S. invicta and put on the ground around E. adenophorum; Ab, imprints on the stems of A. adenophora eaten by S. invicta.
time and stacked on the soil surface within 40 cm around Eupatorium. To confirm whether the two kinds of ants attack each other, the researchers put two comparable-sized ants into a Petri dish and observed that the ants attacked each other violently, the fight ending with the failure of Dorylus, without exception. If the number of Solenopsis was increased, even if Solenopsis were smaller, Dorylus rarely won (Figure 2B). Solenopsis attacked all parts of Dorylus, and even bit the ventral handle of Dorylus until it died. In field investigations it was found that the place formerly occupied by Dorylus had been displaced by Solenopsis. The most typical example could be found around the roots of Conyza canadensis (L.) Cronq, Galinosoga parviflora Cav., Bidens pilosa L, and Eupatorium, which had been occupied by Dorylus. These phenomena not only showed that Solenopsis was an important natural enemy of Eupatorium, but also the fierce survival competition for eating Eupatorium between Dorylus and Solenopsi.
3. Statistical Analysis
Two-way nested ANOVA was used to test the biomass difference in Eupatorium from native and introduced ranges. Population origin (native vs introduced) was used as fixed factors; population is nested within origin as a random factor. These data were tested for normality and homogeneity of variance before analysis. To meet test assumptions, total biomass was lg10 transformed. Biomass date analyses were performed using the statistical software package SPSS22.0 (SPSS Inc, Chicago, IL).
A significant difference in variables between origins in common garden environments suggests genetically based changes in Eupatorium plants; compared with the original populations, the decrease in biomass in invasive populations indicates a decline in invasiveness of Eupatorium.
4. Results and Analysis
In cultivation experiment, there was no statistically significant differences in total biomass in Eupatorium from native (Mexico) and introduced (China) populations (Figure 1) in common garden. There was little evidence that invasive (China) populations had higher growth ability than native (Mexico) populations. On the contrary, the biomass of the invasive population of Eupatorium tends to be lower, although there was no statistically significant difference. In observation experiment, the competitive feeding behavior of Solenopsis and Dorylus on Eupatorium not only indicates that the red fire ant is a new natural enemy of Eupatorium, but also reveals complex interactions within the local herbivore community.
5. Discussion and Conclusion
The feeding of native enemies in Yunnan on Eupatorium may result from the adaptive evolution of this alien species. The unique geological history, geographical location, topography, and unique climate conditions make Yunnan rich in biodiversity [17] . High insect diversity may be the main reason for the increasing pressure of natural enemies of Eupatorium in Yunnan.
Local herbivorous insects can adapt to foreign plants and undergo adaptive evolution in behavior, physiology, and biochemistry, thereby increasing the feeding pressure on exotic plants [18] [19] . For example, Uroleucan ambrosiae only feeds on Ambrosia trifida L. in eastern North America, in order to adapt to the dispersion and unpredictability of the distribution of A. trifida in extreme arid regions, where it evolved into an oligophagous insect in western North America, feeding on several composite weeds [20] . In northern Argentina, the stem borer Apagomerella versicolor (Coleoptera: Cerambycidae, subribe Aerini) only feeds on Pluchea sagittalis Cabr, but in the central and southern regions, it feeds on seven species of composite plants, including Cerambycidae [21] .
Wang et al. (2009) [22] conducted research in Liangshan Prefecture, Sichuan Province, China, and found that the number of species and individuals of Arthropod (especially herbivorous insects) in Eupatorium community on abandoned land (equivalent to heavily invaded habitats) was more than those in forest area (equivalent to uninvaded habitats) and agroforestry ecotone (equivalent to moderately invaded habitats). Jiang et al (2017) [23] investigated the Qilin Mountain and its surrounding areas (24˚42'N, 102˚52'E, 1957 - 2015 m above sea level) of Chengjiang County, Yunnan Province, and found that the diversity of natural enemies in the heavily invaded communities by Eupatorium was higher than those in the moderately invaded and uninvaded communities. These phenomena indicated that the feeding habits of local natural enemies to Eupatorium are related to the degree of invasion. As the invasion time prolongs, the behavioral limitations of insects are broken and their physiological and biochemical adaptation to Eupatorium strengthen [18] . In this study, the strong competition of Dorylus and Solenopsis for eating Eupatorium showed that local omnivorous insects have established a stable feeding relationship to Eupatorium. With the strengthening of the feeding relationship of local insects to Eupatorium, the invasiveness of Eupatorium will gradually weaken, eventually leading to its naturalization as one of the common populations.
Acknowledgments
We thank Professor Xu Zhenghui of Southwest Forestry University for identifying Solenopsis invicta Buren and Dorylus orientalis Westwood.
Fund Projects
Southwest Wildlife Resources Protection Key Laboratory Open Fund Project of the Ministry of Education (XNYB17-7); Kunming Spring City Plan Youth Top Talent Project (C201914001).