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The Evolution of Increased Competitive Ability (EICA) hypothesis has been proposed as a primary mechanism of plant invasiveness (Blossey & Notzold, 1995), when introduced plants escape from their specialist herbivores and pathogens (Keane & Crawley, 2002). Based on a frequently assumed tradeoff between herbivore defense and plant growth (Coley et al., 1985; Herms & Mattson, 1992; Koricheva, 2002), the EICA hypothesis predicts that release from herbivory in introduced ranges selects for plant genotypes with reduced resource allocation to herbivore defense and improved competitive ability (CA) via increased vigor (Blossey & Notzold, 1995).
The EICA hypothesis has been traditionally tested by comparing the biomass production of native and introduced populations of plants in common garden experiments (Blossey & Notzold, 1995). Although several studies have demonstrated the evolution of increased plant growth of invasives (Blair & Wolfe, 2004; Hahn et al., 2012), the support for the hypothesis remains mixed (Bossdorf et al., 2005). An important criticism of this approach is that geographical comparisons alone do not test directly the causal link between herbivore release and the evolution of plant traits, as they can be confounded by additional environmental differences between populations (Colautti et al., 2009) and/or by founder effects (Bossdorf et al., 2005; Dlugosch & Parker, 2008; Keller & Taylor, 2008). For example, plants may rapidly adapt to abiotic environmental variations along the latitudinal gradient (Maron et al., 2007; Etterson et al., 2008). Thus, if native and introduced populations are sampled from different latitudinal ranges, observed differences in CA may arise from adaptation to a variety of environmental factors other than release from herbivory (Colautti et al., 2009). Founder effects may also obscure the effects of selection by herbivory if introduced populations are founded by a limited number of genotypes that differ in mean CA from the native populations (Dlugosch & Parker, 2008). In addition, previous EICA studies have often compared growth differences only in the absence of competition, but the growth under unchallenged conditions does not necessarily reflect plant performance in a naturally competitive environment faced by invasive populations (Bossdorf et al., 2005). Moreover, because the underlying mechanisms of competition may differ between intra- and inter-specific competition (Lankau, 2008), we expect independent evolution of CA for conspecific and heterospecific competitors. However, only a few studies have tested simultaneously both intra- and inter-specific CA (Barney et al., 2009).
To address these issues and to test directly the EICA hypothesis, we asked whether the exclusion of herbivory alone would result in the evolution of intra- and inter-specific CA in Solidago altissima, a perennial plant native to eastern North America. The plant species is particularly relevant because it is an aggressive invasive species in Japan, where specialist herbivores have been absent for > 100 yr (Ando et al., 2010). Moreover, release from herbivory in introduced ranges is likely to have had a strong impact on the evolutionary trajectory of invasive S. altissima populations, because the plants in native ranges encounter a diversity of highly damaging herbivores (Root & Cappuccino, 1992). To circumvent confounding factors associated with previous EICA studies, we used S. altissima plants from an artificial selection experiment in which the above-ground herbivore assemblage has been manipulated by insecticide treatment for 12 yr. We sampled multiple individual plants from insecticide-treated plots (hereafter called ‘H− plots’; n = 30 individuals sampled) and untreated control plots (‘H+ plots’; n = 29 individuals), and propagated clones from these individuals for a subsequent common garden experiment. By comparing CA of H− clones and H+ clones, we were able to examine evolutionary shifts in competitive phenotype as a result of selection from above-ground herbivores. Because S. altissima primarily reproduces vegetatively (see the 'Materials and Methods' section), evolution in this system is likely to occur predominantly via differential propagation and the mortality of different clones. A previous study examining a subset of clones used here found reduced constitutive resistance to a major herbivore, Trirhabda virgata, in H− clones relative to H+ clones (Bode & Kessler, 2012), suggesting that enemy release resulted in the evolution of plant defense traits in S. altissima.
Here, we tested experimentally the second prediction of the EICA hypothesis that release from herbivory would result in increased CA (Blossey & Notzold, 1995). We assessed the CA in a strict sense (Barney et al., 2009) by growing S. altissima either by itself or with conspecific or heterospecific competitors in a common garden, and assessing the interactions between clone origin (i.e. H− or H+) and competition treatments. Poa pratensis was chosen as a heterospecific competitor, as it is a common competitor of S. altissima in old fields (Carson & Root, 2000). We predicted that H− clones would be less affected than H+ clones by competition. We also expected that the degree to which inter- and intra-specific CA evolved would differ as a result of differential mechanisms through which S. altissima competes with conspecifics and heterospecifics. Solidago altissima produces allelopathic polyacetylene compounds, which inhibit the germination and growth of several heterospecific plants (Kobayashi et al., 1980; Sawabe et al., 1999, 2000; Johnson et al., 2010), but have no previously reported effects on conspecifics. If allelopathic compounds are primarily used for inter-specific competition, intense inter-specific competition would select for increased polyacetylene production, which may provide no advantage in intra-specific competition. Thus, we also explored the differential effect of a major polyacetylene, cis-dehydromatricaria ester (DME), on the germination and seedling growth of S. altissima and P. pratensis by growing them under various concentrations of DME.
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- Materials and Methods
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We used an experimental approach to test one of the main predictions of the EICA hypothesis, namely that release from herbivore pressure will lead to an evolution of increased CA (Blossey & Notzold, 1995). Solidago altissima plants used in our common garden experiment originated from long-term artificial selection plots, in which we manipulated the above-ground herbivore community, whilst controlling for other environmental factors, and using the same pool of starting plant genotypes as present in the old field. Thus, our approach addresses the limitations of traditional EICA studies, whose results are often confounded by additional environmental differences between sample populations and/or founder effects. Moreover, insecticide treatments are known to dramatically reduce herbivore damage on S. altissima and increase its stem density at an ecological time-scale (Carson & Root, 2000), mimicking the enemy-free environment in the introduced ranges. So far, such manipulative experiments to test the evolutionary consequences of herbivore exclusion on plant competitiveness are rare (Agrawal et al., 2012).
Our results partially support the EICA hypothesis: although clone origin did not affect leaf and ramet production in the absence of competitors, a significant interaction between clone origin and competition with P. pratensis suggested that H− clones were able to maintain a higher growth rate than H+ clones under inter-specific competition. The differences between H− and H+ clones indicate the evolutionary shift in inter-specific CA. By contrast, no such interaction was found when competing with conspecifics, indicating that intra-specific CA did not evolve. Although we observed an evolutionary shift in inter-specific CA, as measured by leaf and ramet production during the early growing season, the pattern did not translate into measures of end-of-season asexual (rhizome mass) and sexual (inflorescence mass) reproduction. This is surprising because previous experiments have shown that the inflorescence mass (Carson & Root, 2000) and rhizome production (Cain, 1990) are greater in plants from H− plots relative to those from H+ plots when they are measured in situ. The results may indicate that the removal of herbivores does not select for increased production of reproductive tissues, but rather selects for faster early growth, which could have significant fitness effects in a highly competitive environment. Because light availability is an important factor determining the success of understory plants (Carson & Root, 2000), rapid production of leaves may allow S. altissima plants to efficiently shade out other plants and obtain competitive advantages. In addition, Cain (1990) found that the survival of S. altissima stems in the field increases with plant size in the early growing season. Rapid ramet production also allows plants to grow laterally, thereby occupying more space for growth. Therefore, an increased rate of leaf and ramet production should allow clones to persist and to eventually dominate in highly competitive environments.
That we found the growth advantage of H− clones only under a competitive environment suggests that growth measures in the absence of competition do not necessarily reflect growth in competitively challenged environments, and emphasizes the need for the specific testing of plant CA. Previous studies have shown varying results: For instance, Barney et al. (2009) showed that invasive mugwort (Artemisia vulgaris) populations in North America produced more ramets and total biomass than native European populations, and the difference between continents was exacerbated in inter-specific competition with Solidago canadensis. Similarly, Bossdorf et al. (2004) found growth differences between native and invasive populations of Alliaria petiolata only in a competitive environment, but invasive populations evolved reduced CA relative to native populations, the opposite of the EICA prediction. Leger & Rice (2003), by contrast, found growth differences in invasive and native populations of California poppies (Eschscholzia californica) only in the absence of competition, indicating that CA, in a strict sense, did not evolve in invasive populations. Similarly, Blumenthal & Hufbauer (2007) found differential growth only in the absence of competition across 14 invasive plant species, and suggested that these species may be adapted to disturbed and noncompetitive environments in their introduced ranges. The different outcomes among these studies therefore seem to depend on the competitive environment in which newly established populations evolved. In our study, CA rather than growth in the absence of competitor was selected for, potentially because the initial herbivore exclusion experiment was conducted in an already established old field, where surviving S. altissima plants faced strong competition.
Interestingly, we observed an evolutionary shift in inter-specific CA, but not in intra-specific CA, suggesting that selection for inter-specific competition was stronger than that for intra-specific competition. In support of this hypothesis, Carson & Root (2000) found that herbivory suppresses the growth of S. altissima, which allows understory heterospecific competitors to increase in abundance. When herbivores were removed by insecticide, S. altissima initially competes with these understory plants more intensively than with conspecifics. Thus, the clones from H− plots may represent the winners of such inter-specific competition. Whether inter-specific CA is more likely than intra-specific CA to evolve in invasive populations is an interesting question, but few EICA studies so far have tested both inter- and intra-specific CA simultaneously (Barney et al., 2009).
Differences in the degree to which inter- and intra-specific CA evolve in S. altissima may be explained by mechanisms used by the plant to compete with conspecifics and heterospecifics. We found that H− clones, on average, produced twice as much DME as did H+ clones, a compound previously shown to be allelopathic to Oryza sativa (Kobayashi et al., 1980, 2004; Ito et al., 1998), Miscanthus sinensis, Ambrosia artemisiaefolia (Kobayashi et al., 1980), Lactuca sativa (Sawabe et al., 1999) and Asclepias syriaca (Johnson et al., 2010). Similar patterns were found for the other three polyacetylene compounds, which are also known to exhibit allelopathic properties (Sawabe et al., 2000). We cannot exclude experimentally the possibility that the root chemistry evolution resulted from a direct physiological effect of insecticide, as the effect of fenvalerate on root secondary metabolite production is unknown. However, our preliminary results from a geographic comparison showed congruent evolution of increased polyacetylene production in invasive Japanese genotypes relative to native North American genotypes (A. Uesugi, unpublished), suggesting that the insecticide itself is not the major driver of root chemistry evolution. Thus, herbivore exclusion is the most likely factor causing the evolution of increased polyacetylene production via a change in competitive environment in our experimental populations.
These evolutionary shifts in root chemistry coincided with an evolutionary increase in inter-specific CA, suggesting that allelopathy may play a direct role in inter-specific competition. Consistent with this hypothesis, we found that DME suppresses germination and seedling growth of P. pratensis at 24 and 48 ppm, similar to the active concentrations found in previous studies (Kobayashi et al., 1980, 2004; Ito et al., 1998; Sawabe et al., 1999; Johnson et al., 2010). Although these concentrations are slightly higher than the reported natural levels of DME in soil (c. 6 ppm; Kobayashi et al., 1980), a high concentration of DME within roots (up to 200 ppm) suggests that DME could effectively suppress the growth of competitors that come into contact. Moreover, these high concentrations of DME did not suppress the germination of S. altissima seeds, but enhanced seedling growth by inhibiting seed-borne pathogenic fungal growth, suggesting contrasting direct effects of DME on conspecific and heterospecific competitors. DME may also influence indirectly the growth of heterospecific competitors by changing the microbial community with which each is associated (Mummey & Rillig, 2006; Vogelsang & Bever, 2009; Zhang et al., 2010). In a closely related species, Solidago canadensis, the root exudate, including DME, inhibited colonization of mutualistic mycorrhizal fungi on competing species in invasive ranges (Lu et al., 1993; Zhang et al., 2010). Such allelopathic effects on microbial communities are not likely to influence the growth of conspecifics, which could explain the observed differences between inter- and intra-specific CA of H− clones in our experiment. Although our results are consistent with the hypothesis that selection for increased polyacetylene production mediates the evolution of increased CA against heterospecific competitors, a further study should examine the allelopathic effects of polyacetylenes in bioactive soils under natural conditions.
Using clones originating from an artificial herbivore exclusion experiment, this study provides a direct link between enemy release and the evolution of plant competitiveness implied in the EICA hypothesis (Blossey & Notzold, 1995). A previous study examining a subset of H− and H+ clones used here found that H− clones have evolved a reduced level of constitutive resistance to a major herbivore (Bode & Kessler, 2012). Our study further demonstrates that release from herbivory alone can result in the evolution of CA through increased growth under inter-specific competition mediated by the production of allelopathic compounds. The observed evolutionary shifts occurred within 12 yr of herbivore exclusion, suggesting a rapid evolution of plant competitive traits. A similar, rapid evolution of plant competitive and defense traits in response to experimental herbivore removal has been shown in Oenothera biennis (Agrawal et al., 2012), suggesting a vital role of insect herbivores in driving plant adaptation.
Would we expect a parallel evolutionary shift in comparisons between native and invasive populations of S. altissima? Our results suggest that release from herbivory in introduced ranges could lead to a rapid evolution of CA, which could potentially override the effects of additional environmental differences among populations and/or reduced genetic diversity caused by founder effects (Xu et al., 2010). Moreover, an evolutionary change in allelopathy may provide even greater competitive advantages for introduced populations if polyacetylenes function as novel weapons to which many competitors are not adapted (the Novel Weapon hypothesis; Callaway & Aschehoug, 2000; Qin et al., 2013). Although we observed only the evolution of inter-specific CA in this experiment, we expect intra-specific CA to also evolve if intra-specific competition eventually intensifies as S. altissima becomes dominant (e.g. density in invasive Japanese populations can be more than twice that of native North American populations; Carson & Root, 2000; Nishihiro et al., 2007). These temporal dynamics of selection in invasive populations were observed in Alliaria petiolata, where plants from newly colonized populations were more allelopathic to heterospecific competitors than were older populations (Lankau et al., 2009; Lankau, 2011). A future study will compare native and invasive populations of S. altissima with emphasis on both inter- and intra-specific CAs in a common garden experiment.