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Herbivores alter the fitness benefits of a plant–rhizobium mutualism
Acta Oecologica 37 (2011) 87e92
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Original article
Herbivores alter the fitness benefits of a planterhizobium mutualism Katy D. Heath*, Jennifer A. Lau 1 Department of Plant Biology, University of Minnesota, 250 Biological Science Center, 1445 Gortner Ave., St. Paul, MN 55108, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Accepted 14 December 2010 Available online 8 January 2011
Mutualisms are best understood from a community perspective, since third-party species have the potential to shift the costs and benefits in interspecific interactions. We manipulated plant genotypes, the presence of rhizobium mutualists, and the presence of a generalist herbivore and assessed the performance of all players in order to test whether antagonists might alter the fitness benefits of planterhizobium mutualism, and vice versa how mutualists might alter the fitness consequences of planteherbivore antagonism. We found that plants in our experiment formed more associations with rhizobia (root nodules) in the presence of herbivores, thereby increasing the fitness benefits of mutualism for rhizobia. In contrast, the effects of rhizobia on herbivores were weak. Our data support a community-dependent view of these ecological interactions, and suggest that consideration of the aboveground herbivore community can inform ecological and evolutionary studies of legumeerhizobium interactions. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Diffuse interaction Ecological cost Indirect effect Medicago truncatula Sinorhizobium meliloti Tri-trophic
1. Introduction The study of mutualisms typically focuses on pairwise interactions between two species (Stanton, 2003). However, species interactions are often diffuse, with the presence or abundance of other community members altering the intensity or fitness outcomes of the interaction (Bronstein and Barbosa, 2002; Strauss and Irwin, 2004; Johnson and Stinchcombe, 2007; Morris et al., 2007). As a result, the community context in which an interaction occurs can alter the ecological outcome of the interaction (e.g. whether they are mutualistic or parasitic, Thompson and Cunningham, 2002), the direction or intensity of natural selection acting on partners (Stinchcombe and Rausher, 2002; Rudgers and Strauss, 2004; Lau, 2008), or even the outcome of co-evolution (Galen and Cuba, 2001; Lau, 2006; reviewed in Bronstein and Barbosa, 2002; Strauss and Irwin, 2004). If such higher-order effects are common, then results from studies investigating pairwise interactions between species may not be robust in more complex communities (Wootton, 1994; McPeek and Miller, 1996; Miller and Travis, 1996; Bennett et al., 2006). Legumeerhizobium mutualisms, in which rhizobia fix nitrogen in exchange for the products of plant photosynthesis, are important
* Corresponding author. Present address: Department of Plant Biology, University of Illinois at Urbana-Champaign, 192 Edward R. Madigan Lab, 1201 W. Gregory Dr. Urbana, IL 61801, USA. Tel.: þ1 217 265 5473; fax: þ1 217 244 7246. E-mail addresses: [email protected] (K.D. Heath), [email protected] (J.A. Lau). 1 Present address: W. K. Kellogg Biological Station and Department of Plant Biology, Michigan State University, USA. 1146-609X/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2010.12.002
to the nitrogen economy of both natural and managed ecosystems (Vance, 2002). Recent work has shown that the fitness benefits to both partners depend on the abiotic nitrogen environment, as well as the particular genotypes of the interacting partners (Heath and Tiffin, 2007; Heath, 2010), and that the traits mediating the exchange of fitness benefits between partners are structured on a geographic scale (Parker, 1999; Heath, 2010). However, little is known about the potential for additional community members to affect planterhizobium interactions and, therefore, the potential for variation in community composition to contribute to the observed local or geographic genetic variation. Because natural selection will favor association with mutualist partners only as long as the fitness benefits of an interaction exceed costs, third-party fitness effects on either mutualist partner could have ramifications for the stability of the interactions (Gomulkiewicz et al., 2003), as well as the population densities/persistence of both partners (Bennett et al., 2006). Because of their respective impacts on plant resources, both herbivores and rhizobia might be expected to influence other ecological interactions involving their host plants, including those with each other. The direct effects of herbivores in decreasing plant growth and fitness are well-known (reviewed in Marquis, 1992). However, herbivores might also indirectly impact plant fitness by altering the costs or benefits of associating with rhizobia. For example, because herbivores consume leaf tissue, one might expect that increased herbivory will reduce the amount of excess plant carbon, thereby increasing the carbon costs of association with rhizobia. Aside from their beneficial effects on plant performance, rhizobia might alter the costs of herbivory for plants, perhaps by
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increasing tolerance by improving plant condition (Johnson et al., 1987). In addition to potential direct and indirect effects on the plant host, herbivores and rhizobia might also affect each other via their plant hosts. Herbivores might be predicted to decrease rhizobium fitness via their detrimental effect on photosynthesis and plant vigor (Borowicz, 1993). Conversely, rhizobia might be expected to improve the performance of insect herbivores via positive effects on plant quantity or quality (reduced C/N ratios), although rhizobium-induced changes in foliar chemistry sometimes involve changes in the form of available N and defense chemicals and so may not always improve plant quality for herbivores (e.g. Wilson and Stinner, 1984). Here we present the results of a greenhouse study in which we manipulated the abundance of rhizobial mutualists (Sinorhizobium meliloti) and the presence of a generalist herbivore (Spodoptera exigua) on plant genotypes from natural Medicago truncatula populations, in order to estimate how direct and indirect effects impact the performance of all three players. We asked whether (1) herbivores affect rhizobial fitness benefits; (2) rhizobia affect herbivore performance; and (3) herbivores and rhizobia independently or interactively impact plant fitness. In sum, our treatment combinations investigate how an aboveground antagonist impacts the benefits of belowground mutualism, and vice versa. 2. Materials and methods 2.1. Study system M. truncatula Gaertn. (Fabaceae), or barrel medic, is an annual legume native to the Mediterranean regions of Europe, Africa, and Asia, where it occurs in small populations along disturbed roadsides and agricultural hedgerows (Lesins and Lesins, 1979). M. truncatula and S. meliloti, its primary rhizobial symbiont, are a model system for understanding legume-rhizobium symbiosis (Cook et al., 1997). S. exigua (Lepidoptera: Noctuidae), the beet armyworm, is a generalist herbivore common in both North America and Europe, where it feeds on many plant families, including the Fabaceae. Plant genotypes were collected from natural populations within their native range in southern France (detailed in Heath, 2010). Offspring from four haphazardly selected maternal genotypes (maternal families) from each of two populations (Chat and Sals, w120 km apart) were used in this experiment. M. truncatula populations have been found to harbor few maternal haplotypes (Siol et al., 2008); therefore, even four plant genotypes likely represent a significant fraction of the extant genetic variation in a given population. The estimated rate of self-fertilization in nearby M. truncatula populations is w97.5% (Bonnin et al., 1996); therefore, offspring from the same maternal plant are expected to be nearly identical in genetic composition. Maternal families were allowed to self for one generation in the greenhouse to standardize potential maternal environmental effects. S. exigua eggs were obtained from laboratory colonies (Benzon Research, Carlisle, PA). The S. meliloti strain used in this experiment (Chat a) was isolated from soil collected from population Chat at the time of seed collection (strain isolation and characterization detailed in Heath, 2010). Prior work (Heath, 2010) has shown that this strain is mutualistic for plant genotypes collected from both Chat and Sals populations, but found no evidence of local co-adaptation. 2.2. Experimental design To investigate interactions between rhizobia, insect herbivores, and their plant hosts, we manipulated the presence of rhizobia and herbivores in a full factorial design. This resulted in four
treatments: two rhizobium treatments ( inoculation with S. meliloti) crossed with two herbivore treatments ( S. exigua). We used eight plant families (four from each of two natural populations). Each plant family treatment combination was replicated 10 times, for a total of 320 experimental plants. Although we used only one strain in our experiment, we note that intra-specific genetic variation is well-known to exist in both rhizobia (Heath, 2010) as well as in herbivores (Via, 1990) for their respective interactions with plants. Seeds of M. truncatula were manually scarified, sterilized (dipped in 70% EtOH, followed by 7 min in commercial bleach), and germinated in sterile petri plates in the dark at 4 C for three weeks. Seedlings were randomized into 656 mL Deepots (Stuewe & Sons Inc.) containing a steam-sterilized mixture of 1:1 Sunshine Mix #5 (Sun Gro, Bellevue, WA, USA) and Turface MVP (Profile Products LLC, Buffalo Grove, IL) and placed in the greenhouse under 16 h days. To manipulate the density of rhizobia, we grew S. meliloti for 48 h at 30 C in liquid modified arabinoseegluconate (MAG) media (van Berkum, 1990). We then adjusted the inoculum cell density to w106 cells/mL (based on OD670) by diluting liquid cultures with sterile water, and immediately inoculated each plant in the þrhizobium treatment with 1 mL inoculum. Plants were inoculated four days after transplant to the greenhouse. While many plants in the rhizobium treatment produced nodules, plants in the þrhizobium treatment produced many more nodules {nodule number increased from 17.5 3.3 to 48.3 3.3 with inoculation (P < 0.0001)}, although plants formed smaller nodules in the þrhizobium treatment {mean nodule length decreased from 2.68 0.08 to 2.41 0.08 mm (P ¼ 0.0011)}. This is consistent with a commonly-observed negative correlation between nodule number and nodule size (e.g. Mytton and Jones, 1971; Heath, 2010). These nodule numbers are consistent with observed field nodulation patterns in native M. truncatula populations (Mean 11.7 nodules per plant, range 0e67 nodules per plant) (M. Friesen, pers. comm.) To manipulate the presence of herbivores, S. exigua eggs were allowed to hatch at 37 C in the absence of artificial diet or leaf tissue. Three first-instar larvae were placed on each plant in the þherbivory treatment one month after planting. Insect mortality in the greenhouse was high; therefore, 11 days later, we re-distributed a second set of hatchlings across plants (5e6 larvae per plant) to ensure that all plants in the þherbivory treatment received adequate foliar damage. All surviving herbivores (with the exception of a single larva per plant which was removed for the herbivore growth bioassay, see below) remained on the plant for the remainder of the experiment, so a majority of plants in the þherbivory treatment received relatively consistent S. exigua damage for two to four weeks. All plants were bagged to prevent herbivore movement between plants in the þ and herbivory treatments. 2.3. Plant, rhizobium, and herbivore measurements We estimated the performance of all three partners (plant, rhizobium, and insect). We estimated early plant growth, before herbivory by S. exigua, by counting leaf number four weeks after transplanting. We harvested all plants 8 weeks after transplanting when plants were beginning to senesce, flowering on most individuals had slowed, and leaves had begun to brown. We counted total fruit number and weighed aboveground biomass (after drying for 3 days at 60 C) of each individual. In a previous experiment conducted in the same greenhouse environment, M. truncatula total seed number was positively correlated with fruit number (R ¼ 0.86; N ¼ 241 plants, P < 0.0001; Heath and Tiffin, 2007),
K.D. Heath, J.A. Lau / Acta Oecologica 37 (2011) 87e92
indicating that fruit number is a good estimate of fecundity for this species. We assessed the performance of S. exigua by removing, freezing, and weighing one S. exigua individual from each plant in the þherbivory treatment after three days of feeding. All larvae used in this herbivore performance bioassay came from the first cohort of larvae introduced onto the plants. Because larvae were not fed artificial diet between hatching and placement on plants, final biomass after the feeding assay can be used to estimate the impact of plant quality on herbivore performance. Such short-term (<five day) growth bioassays on early instar larvae are commonly used to estimate host plant quality and effects of host plant quality on early herbivore growth and performance (e.g. Agrawal, 2002; Kempel et al., 2010). Early in the experiment, plants experienced an unexpected outbreak of thrips. To explore the effects of thrips herbivory on our measured variables (see Analyses section below), we estimated the level of thrips herbivory on a per plant basis by counting the proportion of leaves on each plant with thrips damage (at 4 weeks after plants were transplanted). Finally, at harvest, we estimated rhizobium fitness benefits in multiple ways: as the total number of nodules produced on each plant, as well as two estimates of nodule size: mean length (mm), and weight of ten nodules per plant. Nodules were haphazardly selected from the plant root system, without respect to size, although the number of experimental units precluded a formal randomization scheme. Nevertheless, while bias in nodule selection (e.g. choosing larger nodules) could feasibly alter the mean nodule weight or size, it cannot account for differences between treatments and the significance of any model effects. Nodules were collected in 1.5 mL tubes and frozen until the time of measurement. Nodule size in Medicago is positively correlated with rhizobium reproduction inside the nodule (Heath and Tiffin, 2007; W.C. Ratcliff, pers. comm.). Nevertheless, the number of nodules has a larger impact on rhizobium fitness in the M. truncatulaeS. meliloti symbiosis (Heath and Tiffin, 2009), a finding that likely results from the exponential reproduction of rhizobial cells inside each nodule (Ratcliff et al., 2008). Therefore, nodule number in particular should be strongly correlated with the fitness benefits that rhizobia receive from plants.
2.4. Statistical analyses We used MANOVA (PROC GLM) and mixed-model ANOVA (PROC MIXED) to investigate how herbivory, rhizobium treatment, and plant family impact estimates of rhizobium fitness (nodule number, length, and weight) and plant fitness (biomass and fruit
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number) (SAS Institute, 2001). Rhizobium treatment, herbivore treatment, plant population, and all interactions were included as fixed factors, while block, plant family nested within population, and all interactions between plant family and the fixed factors were included as random factors. The significance of random effects was determined by comparing the log likelihood ratio of models differing by the inclusion of each random effect to a c2-distribution with one degree of freedom. All response variables were natural log-transformed for use in multiplicative models in tests for interactive effects on fitness (Hambäck and Beckerman, 2003; Wootton, 1994). Because we were concerned that the thrips outbreak may have compromised our herbivory treatments (i.e., most plants, including those in the eS. exigua treatments, experienced thrips herbivory), we also repeated all analyses including the proportion of leaves damaged by thrips as a covariate. Results were qualitatively similar, regardless of whether thrips damage was included as a covariate, suggesting that the thrips outbreak had minimal impacts on the observed treatment effects. Similar models were used to test for the effects of rhizobia and plant family on thrips damage and S. exigua growth. Final biomass of the bioassay larva (natural log-transformed) or the proportion of leaves with evidence of thrips damage (arcsin square root transformed) were the response variables; rhizobium treatment and plant population were included as fixed factors, and plant family (nested within plant population) and the plant family rhizobium interaction were included as random factors. Phenotypic correlations between all dependent variables in the experiment are presented in Appendix A in the electronic Supplementary material.
3. Results 3.1. Effect of herbivores on rhizobium fitness benefits Herbivory by S. exigua positively affected rhizobium fitness benefits in this experiment. The presence of S. exigua increased the number of nodules formed by plants (Table 1, Fig. 1), and the direction of this effect was consistent across plant populations and rhizobium inoculation treatments (not shown). By contrast, there were no significant effects of herbivory on nodule length or nodule weight (all P > 0.3). We also detected plant genetic variation (significant family effects, Table 1) for nodule number, nodule length, and nodule weight e indicating that plant families differ in the benefits they confer to their rhizobial mutualists. However, we found little evidence that variation among families mediated the effects of herbivory on rhizobium fitness (no significant family herbivory interactions, Table 1).
Table 1 MANOVA and mixed-model ANOVAs for the effects of rhizobium inoculation, herbivore presence and plant population on the fitness benefits to rhizobia (nodule number, nodule length, and nodule weight). The effects of plant family (nested within population), block, and all interactions with family were tested as random effects (values shown are c2). Significance indicated as follows: D P<0.10, * P<0.05, ** P<0.01, *** P<0.001, **** P< 0.0001. MANOVA
Rhizobia (R) Herbivory (H) Pop (P) RH RP HP RHP Family (Pop) Family R Family H Family R H Block
Nodule Number
Nodule length
Pillai
df
F
df
F
df
F
df
F
0.41 0.02 0.52 0.00 0.06 0.03 0.07 0.28 0.10 0.07 0.07 0.50
223 223 4 223 223 223 675 675 675 675 675 675
51.8**** 1.60 1.46 0.82 4.46** 2.29D 0.89 3.81**** 1.29 0.97 0.89 1.46**
280 270 6 276 289 290 278
161.20**** 7.78** 2.73 0.25 2.39 1.95 0.81
6 6 6 270 6 6 272
36.73** 1.22 0.40 0.94 8.30* 0.47 0.30
6 268 6 266 6 269 269
30.92** 0.29 0.11 0.07 1.75 0.21 0.68
12.6*** 0 0 0 14.8****
Nodule weight
21.3**** 0.7 0.5 0 0.1
2.9* 3.6* 0 0 2.0D
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30
- rhizobia + rhizobia
2.5
b
a
2
a
a
a
20
Biomass (g)
Number of nodules
25
b
15 10
1.5
1
0.5
5
0
0 - herbivores
+ herbivores
While herbivores benefited rhizobia, the interaction was asymmetrical: presence of rhizobia did not significantly affect either herbivore (S. exigua performance or thrips damage, all P > 0.5; Appendix B). We also did not detect any evidence for variation among plant populations or families in their effects on S. exigua performance or thrips damage (all P > 0.19; Appendix B).
3.3. Effects of herbivores and rhizobia on plants As expected, insect herbivores decreased fruit number in M. truncatula (Fig. 2b, Table 2), while rhizobia increased both plant biomass and fruit production (Fig. 2a,b, Table 2). We detected weak evidence for the interactive effect of rhizobia and herbivory on plants, although the interaction varied across populations (significant rhizobia herbivory population term in MANOVA, Table 2). Specifically, the presence of herbivores altered the benefits of rhizobia for plants from Chat, but not Sals, and the direction of the effect varied across performance measures (biomass vs. fruit number; results not shown). We detected much genetic variation among plant families (but not plant populations) in both biomass and fruit production, consistent with previous work on these genotypes (Heath, 2010). However, we found little evidence that plant genotype mediated the effects of herbivores or the interactive effects of herbivores and rhizobia on plant performance (Table 2).
a,b a,b
Number of fruits
3.2. Effects of rhizobia and plant genotype on herbivores
6
b 4
2
0
- herbivores
+ herbivores
Fig. 2. Effects of herbivory by S. exigua and rhizobium mutualists on the biomass (A), and fruit number (B) of M. truncatula plants. Shown are least-squares means SE.
4.1. Mechanisms mediating rhizobiumeherbivore interactions We hypothesized that, because herbivores remove leaf tissue and therefore reduce carbon acquisition (Borowicz, 1993; Stark and Kytoviita, 2005), their presence would negatively affect mutualism
Table 2 MANOVA and mixed-model ANOVAs for the effects of rhizobium inoculation, herbivore presence and plant population on plant growth (final biomass) and reproduction (fruit production). The effects of plant family (nested within population), block, and all interactions with family were tested as random effects (values shown are c2). Significance indicated as follows: D P<0.10, * P<0.05, ** P<0.01, *** P<0.001, **** P< 0.0001. MANOVA
4. Discussion Although the potential for the biotic environment to alter the dynamics of species interactions is well-appreciated (for recent reviews see Strauss and Irwin, 2004; Morris et al., 2007), few empirical data exist on the potential for third-parties to alter the costs and benefits of mutualistic interactions to both partners in ecological time. We studied how a herbivore antagonist influenced the interactions between M. truncatula and S. meliloti and found that herbivores increased the reproductive benefits to rhizobia. By contrast, we found little evidence that rhizobia influenced the outcome of a planteherbivore antagonism. Potential mechanisms for, and implications of, the observed results are discussed.
a
8
Fig. 1. Effects of herbivory by S. exigua on plant allocation to rhizobium mutualists (nodule number) for rhizobium-inoculated M. truncatula plants. Shown are leastsquares means SE.
Rhizobia (R) Herbivory (H) Pop (P) RH RP HP RHP Family (Pop) Family R Family H Family R H Block
Biomass
Fruit Number df
Pillai
df
F
df
0.11 0.05 0.31 0.02 0.02 0.02 0.03 0.27 0.04 0.04 0.04 0.43
241 241 5 241 241 241 241 241 484 484 484 484
14.6**** 6.42*** 1.14 2.01 2.20 2.62D 3.51* 6.22**** 0.77 0.92 0.84 2.13****
274 6 6 271 281 7 272
F 34.07**** 5.20D 0.46 2.74D 3.80D 1.47 1.79 27.2**** 0 0.3 0 35.0****
F
281 5.71* 6 7.24* 6 1.70 276 0.02 287 1.66 6 00.63 279 2.15 29.0**** 0 1.8D 0 14.2****
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benefits to rhizobia. Indeed, multiple studies of forage legumes have found that nodule number or size decreases after severe defoliation or grazing (Butler et al., 1959; Whiteman, 1970; Vance et al., 1979), suggesting that the removal of most or all aboveground biomass does limit resource availability to such a degree that plants cannot maintain or form nodules. By contrast, however, insect herbivores in our experiment increased the number of nodules by ca. 28%, and therefore the total number of rhizobium offspring resulting from symbiosis. Although the experiments are not precisely comparable, these results are in agreement with Dean et al. (2009), who found that higher aphid densities were associated with a rhizobium treatment in which plants also formed nodules sooner and exhibited a (non-significant) trend for increased nodulation. Several mechanisms might account for the positive effect of herbivory on rhizobia in our experiment. Herbivory might alter carbon allocation to roots vs. shoots (Babst et al., 2005; Henkes et al., 2008), or increase root exudates (Holland et al., 1996), which are necessary for attracting rhizobia and initiating symbiosis (reviewed in Cohn et al., 1998). In addition, given the physiological overlap between plant hormones active in defense and symbiotic signaling, it is also possible that induced plant defenses might disrupt the regulation of nodulation. Specifically, jasmonic acid (JA) is an important elicitor of plant defense, often increasing in response to herbivore damage (Dong, 1998; Howe and Jander, 2008), and more recently has been found to play a role in nodulation. The role of JA in nodulation is complex, and this is currently an active area of research (recently reviewed by Hause and Schaarschmidt, 2009). Nevertheless, it appears that, although exogenous root JA inhibits nodulation in synergy with ethylene, elevated shoot JA might actually promote nodulation, possibly in antagonism with ethylene and/or by altering of the frequency of the calcium spikes that precede nodulation (Sun et al., 2006; Hause and Schaarschmidt, 2009). Among other lines of evidence, the soybean nts mutant has high shoot JA and is also a supernodulator (Seo et al., 2007; Kinkema and Gresshoff, 2008). Independent of the particular physiology involved, it seems clear that such subtle, plant hormone-mediated effects should only be detected when plants are not severely carbon-limited. Thus, whether herbivores impact rhizobia negatively or positively might depend on the level of herbivory, as posited by Wearn and Gange (2007), albeit in the context of mycorrhizal interactions. While insect herbivores impacted the planterhizobium mutualism, we detected no overall effect of rhizobium treatment on the performance of the generalist S. exigua, or on plant damage received by thrips in an unexpected outbreak. These results were in spite of the fact that all plants were grown in very low nutrient soil and were not fertilized; therefore, rhizobia should have increased plant quality by increasing leaf nitrogen concentrations (Wilson and Stinner, 1984). By contrast, Dean et al. (2009) found that soybeans nodulated by a commercial inoculum, vs. naturallyoccurring soil strains, supported higher populations of specialist soybean aphids. Because coevolved specialist herbivores or different feeding guilds might show different responses to plant mutualists (Hartley and Gange, 2009), it might be important to note that, unlike Dean et al. (2009), we used a broad generalist folivore. Variation in response among different herbivore classes might be responsible for the wide range of effects observed in similar resource mutualisms (i.e., fungaleplanteherbivore interactions, recently reviewed by both Gehring and Bennett, 2009 and Hartley and Gange, 2009). For example, Gange et al. (2002) found that associations with arbuscular mycorrhizal fungi positively affected specialist, but not generalist, herbivores e likely because these fungi increase plant secondary metabolites that benefit specialists but are detrimental to generalists (Gange and West, 1994). It is also
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conceivable that effects on herbivores will depend on the particular rhizobium and plant genotypes involved in the interaction, since prior work has demonstrated that the fitness outcomes of M. truncatulaeS. meliloti interactions are dependent on the particular plant and rhizobium genotypes involved in the interaction (Heath and Tiffin, 2007; Heath, 2010). Only a single rhizobium strain was included in this study; however, it is possible that other strains could have greater impacts on plant tissue nutrient concentrations and, therefore, stronger plant-mediated indirect effects on herbivore growth and fitness. Unfortunately, without more studies on rhizobiumeplanteinsect interactions, we cannot yet assess how outcomes vary across inter- and intra-specific variation in the interacting taxa or whether the handful of results to date are consistent with the patterns emerging from studies of fungaleplanteinsect interactions. 4.2. Context-dependence e ecological effects In addition to their direct negative impact on plant growth and reproduction, herbivores might indirectly impact plants by altering the costs and benefits of mutualism with rhizobia (by increasing the number of nodules). Whether such an effect is positive or negative should, in turn, depend on many factors: e.g. the carbon/ nitrogen balance of the plant and the relative effectiveness of the available rhizobium strains. In the context of our study, increased nodulation was, on average, associated with higher plant performance (see Appendix A in electronic Supplementary material); however, genetic correlations suggest that supporting many nodules is costly for plants (Heath, 2010). Thus, increasing nodulation could increase both the costs and benefits of mutualism and could, therefore, potentially explain the lack of strong and consistent rhizobium herbivory interactions on plant fitness observed in this study, even though changes in nodule numbers were observed. While several previous studies have demonstrated direct effects of herbivores on plant population dynamics (Lau et al., 2008 and citations within), little research has addressed how herbivores might indirectly alter population growth rates by disrupting mutualisms, or by changing the costs and benefits of mutualism. Herbivore-induced changes in the fitness benefits of mutualism may result in changes in the population growth rates or abundance of one or both partners, and in ways not easily predicted by singlegeneration studies. For example, increased nodulation should increase standing densities of rhizobia in the soil. If plants are rhizobium-limited (e.g. Parker et al., 2006), this could benefit future plant generations. The long-term population consequences of diffuse interactions may be difficult to predict based on short-term changes in fitness or growth benefits observed in single-generation studies, which cannot account for changes in population densities of interacting species (see also Schmitz, 1998; Wootton, 2002). 5. Conclusions There has been increasing awareness in recent years of the importance of third-party interactions to population and community dynamics (e.g. Strauss and Irwin, 2004; Johnson and Stinchcombe, 2007; Morris et al., 2007). Most studies have been phyto-centric, though the effects of third-parties on all players are important in determining both ecological and evolutionary outcomes. Our data show that aboveground herbivores have the potential to increase rhizobium fitness benefits. In complex natural communities, where a given plant is likely to interact with a suite of mutualists, herbivores, and competitors, higher-order effects on partner fitness may be common. As a result, the outcomes of pairwise mutualistic interactions are likely to vary across communities that differ in composition.
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Acknowledgments We thank Molly Peterson for assistance with the greenhouse experiment, and two anonymous reviewers for helpful comments on an earlier version of the manuscript. This work was funded by an NSF Dissertation Improvement Grant DEB-0508305 to KDH. JAL was supported by NSF IOB 0620318 to P. Tiffin, R. Shaw, and P. Reich. This is KBS contribution no. 1459.
Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.actao.2010.12.002.
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Contents lists available at ScienceDirect
Acta Oecologica journal homepage: www.elsevier.com/locate/actoec
Original article
Herbivores alter the fitness benefits of a planterhizobium mutualism Katy D. Heath*, Jennifer A. Lau 1 Department of Plant Biology, University of Minnesota, 250 Biological Science Center, 1445 Gortner Ave., St. Paul, MN 55108, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 July 2010 Accepted 14 December 2010 Available online 8 January 2011
Mutualisms are best understood from a community perspective, since third-party species have the potential to shift the costs and benefits in interspecific interactions. We manipulated plant genotypes, the presence of rhizobium mutualists, and the presence of a generalist herbivore and assessed the performance of all players in order to test whether antagonists might alter the fitness benefits of planterhizobium mutualism, and vice versa how mutualists might alter the fitness consequences of planteherbivore antagonism. We found that plants in our experiment formed more associations with rhizobia (root nodules) in the presence of herbivores, thereby increasing the fitness benefits of mutualism for rhizobia. In contrast, the effects of rhizobia on herbivores were weak. Our data support a community-dependent view of these ecological interactions, and suggest that consideration of the aboveground herbivore community can inform ecological and evolutionary studies of legumeerhizobium interactions. Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: Diffuse interaction Ecological cost Indirect effect Medicago truncatula Sinorhizobium meliloti Tri-trophic
1. Introduction The study of mutualisms typically focuses on pairwise interactions between two species (Stanton, 2003). However, species interactions are often diffuse, with the presence or abundance of other community members altering the intensity or fitness outcomes of the interaction (Bronstein and Barbosa, 2002; Strauss and Irwin, 2004; Johnson and Stinchcombe, 2007; Morris et al., 2007). As a result, the community context in which an interaction occurs can alter the ecological outcome of the interaction (e.g. whether they are mutualistic or parasitic, Thompson and Cunningham, 2002), the direction or intensity of natural selection acting on partners (Stinchcombe and Rausher, 2002; Rudgers and Strauss, 2004; Lau, 2008), or even the outcome of co-evolution (Galen and Cuba, 2001; Lau, 2006; reviewed in Bronstein and Barbosa, 2002; Strauss and Irwin, 2004). If such higher-order effects are common, then results from studies investigating pairwise interactions between species may not be robust in more complex communities (Wootton, 1994; McPeek and Miller, 1996; Miller and Travis, 1996; Bennett et al., 2006). Legumeerhizobium mutualisms, in which rhizobia fix nitrogen in exchange for the products of plant photosynthesis, are important
* Corresponding author. Present address: Department of Plant Biology, University of Illinois at Urbana-Champaign, 192 Edward R. Madigan Lab, 1201 W. Gregory Dr. Urbana, IL 61801, USA. Tel.: þ1 217 265 5473; fax: þ1 217 244 7246. E-mail addresses: [email protected] (K.D. Heath), [email protected] (J.A. Lau). 1 Present address: W. K. Kellogg Biological Station and Department of Plant Biology, Michigan State University, USA. 1146-609X/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.actao.2010.12.002
to the nitrogen economy of both natural and managed ecosystems (Vance, 2002). Recent work has shown that the fitness benefits to both partners depend on the abiotic nitrogen environment, as well as the particular genotypes of the interacting partners (Heath and Tiffin, 2007; Heath, 2010), and that the traits mediating the exchange of fitness benefits between partners are structured on a geographic scale (Parker, 1999; Heath, 2010). However, little is known about the potential for additional community members to affect planterhizobium interactions and, therefore, the potential for variation in community composition to contribute to the observed local or geographic genetic variation. Because natural selection will favor association with mutualist partners only as long as the fitness benefits of an interaction exceed costs, third-party fitness effects on either mutualist partner could have ramifications for the stability of the interactions (Gomulkiewicz et al., 2003), as well as the population densities/persistence of both partners (Bennett et al., 2006). Because of their respective impacts on plant resources, both herbivores and rhizobia might be expected to influence other ecological interactions involving their host plants, including those with each other. The direct effects of herbivores in decreasing plant growth and fitness are well-known (reviewed in Marquis, 1992). However, herbivores might also indirectly impact plant fitness by altering the costs or benefits of associating with rhizobia. For example, because herbivores consume leaf tissue, one might expect that increased herbivory will reduce the amount of excess plant carbon, thereby increasing the carbon costs of association with rhizobia. Aside from their beneficial effects on plant performance, rhizobia might alter the costs of herbivory for plants, perhaps by
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increasing tolerance by improving plant condition (Johnson et al., 1987). In addition to potential direct and indirect effects on the plant host, herbivores and rhizobia might also affect each other via their plant hosts. Herbivores might be predicted to decrease rhizobium fitness via their detrimental effect on photosynthesis and plant vigor (Borowicz, 1993). Conversely, rhizobia might be expected to improve the performance of insect herbivores via positive effects on plant quantity or quality (reduced C/N ratios), although rhizobium-induced changes in foliar chemistry sometimes involve changes in the form of available N and defense chemicals and so may not always improve plant quality for herbivores (e.g. Wilson and Stinner, 1984). Here we present the results of a greenhouse study in which we manipulated the abundance of rhizobial mutualists (Sinorhizobium meliloti) and the presence of a generalist herbivore (Spodoptera exigua) on plant genotypes from natural Medicago truncatula populations, in order to estimate how direct and indirect effects impact the performance of all three players. We asked whether (1) herbivores affect rhizobial fitness benefits; (2) rhizobia affect herbivore performance; and (3) herbivores and rhizobia independently or interactively impact plant fitness. In sum, our treatment combinations investigate how an aboveground antagonist impacts the benefits of belowground mutualism, and vice versa. 2. Materials and methods 2.1. Study system M. truncatula Gaertn. (Fabaceae), or barrel medic, is an annual legume native to the Mediterranean regions of Europe, Africa, and Asia, where it occurs in small populations along disturbed roadsides and agricultural hedgerows (Lesins and Lesins, 1979). M. truncatula and S. meliloti, its primary rhizobial symbiont, are a model system for understanding legume-rhizobium symbiosis (Cook et al., 1997). S. exigua (Lepidoptera: Noctuidae), the beet armyworm, is a generalist herbivore common in both North America and Europe, where it feeds on many plant families, including the Fabaceae. Plant genotypes were collected from natural populations within their native range in southern France (detailed in Heath, 2010). Offspring from four haphazardly selected maternal genotypes (maternal families) from each of two populations (Chat and Sals, w120 km apart) were used in this experiment. M. truncatula populations have been found to harbor few maternal haplotypes (Siol et al., 2008); therefore, even four plant genotypes likely represent a significant fraction of the extant genetic variation in a given population. The estimated rate of self-fertilization in nearby M. truncatula populations is w97.5% (Bonnin et al., 1996); therefore, offspring from the same maternal plant are expected to be nearly identical in genetic composition. Maternal families were allowed to self for one generation in the greenhouse to standardize potential maternal environmental effects. S. exigua eggs were obtained from laboratory colonies (Benzon Research, Carlisle, PA). The S. meliloti strain used in this experiment (Chat a) was isolated from soil collected from population Chat at the time of seed collection (strain isolation and characterization detailed in Heath, 2010). Prior work (Heath, 2010) has shown that this strain is mutualistic for plant genotypes collected from both Chat and Sals populations, but found no evidence of local co-adaptation. 2.2. Experimental design To investigate interactions between rhizobia, insect herbivores, and their plant hosts, we manipulated the presence of rhizobia and herbivores in a full factorial design. This resulted in four
treatments: two rhizobium treatments ( inoculation with S. meliloti) crossed with two herbivore treatments ( S. exigua). We used eight plant families (four from each of two natural populations). Each plant family treatment combination was replicated 10 times, for a total of 320 experimental plants. Although we used only one strain in our experiment, we note that intra-specific genetic variation is well-known to exist in both rhizobia (Heath, 2010) as well as in herbivores (Via, 1990) for their respective interactions with plants. Seeds of M. truncatula were manually scarified, sterilized (dipped in 70% EtOH, followed by 7 min in commercial bleach), and germinated in sterile petri plates in the dark at 4 C for three weeks. Seedlings were randomized into 656 mL Deepots (Stuewe & Sons Inc.) containing a steam-sterilized mixture of 1:1 Sunshine Mix #5 (Sun Gro, Bellevue, WA, USA) and Turface MVP (Profile Products LLC, Buffalo Grove, IL) and placed in the greenhouse under 16 h days. To manipulate the density of rhizobia, we grew S. meliloti for 48 h at 30 C in liquid modified arabinoseegluconate (MAG) media (van Berkum, 1990). We then adjusted the inoculum cell density to w106 cells/mL (based on OD670) by diluting liquid cultures with sterile water, and immediately inoculated each plant in the þrhizobium treatment with 1 mL inoculum. Plants were inoculated four days after transplant to the greenhouse. While many plants in the rhizobium treatment produced nodules, plants in the þrhizobium treatment produced many more nodules {nodule number increased from 17.5 3.3 to 48.3 3.3 with inoculation (P < 0.0001)}, although plants formed smaller nodules in the þrhizobium treatment {mean nodule length decreased from 2.68 0.08 to 2.41 0.08 mm (P ¼ 0.0011)}. This is consistent with a commonly-observed negative correlation between nodule number and nodule size (e.g. Mytton and Jones, 1971; Heath, 2010). These nodule numbers are consistent with observed field nodulation patterns in native M. truncatula populations (Mean 11.7 nodules per plant, range 0e67 nodules per plant) (M. Friesen, pers. comm.) To manipulate the presence of herbivores, S. exigua eggs were allowed to hatch at 37 C in the absence of artificial diet or leaf tissue. Three first-instar larvae were placed on each plant in the þherbivory treatment one month after planting. Insect mortality in the greenhouse was high; therefore, 11 days later, we re-distributed a second set of hatchlings across plants (5e6 larvae per plant) to ensure that all plants in the þherbivory treatment received adequate foliar damage. All surviving herbivores (with the exception of a single larva per plant which was removed for the herbivore growth bioassay, see below) remained on the plant for the remainder of the experiment, so a majority of plants in the þherbivory treatment received relatively consistent S. exigua damage for two to four weeks. All plants were bagged to prevent herbivore movement between plants in the þ and herbivory treatments. 2.3. Plant, rhizobium, and herbivore measurements We estimated the performance of all three partners (plant, rhizobium, and insect). We estimated early plant growth, before herbivory by S. exigua, by counting leaf number four weeks after transplanting. We harvested all plants 8 weeks after transplanting when plants were beginning to senesce, flowering on most individuals had slowed, and leaves had begun to brown. We counted total fruit number and weighed aboveground biomass (after drying for 3 days at 60 C) of each individual. In a previous experiment conducted in the same greenhouse environment, M. truncatula total seed number was positively correlated with fruit number (R ¼ 0.86; N ¼ 241 plants, P < 0.0001; Heath and Tiffin, 2007),
K.D. Heath, J.A. Lau / Acta Oecologica 37 (2011) 87e92
indicating that fruit number is a good estimate of fecundity for this species. We assessed the performance of S. exigua by removing, freezing, and weighing one S. exigua individual from each plant in the þherbivory treatment after three days of feeding. All larvae used in this herbivore performance bioassay came from the first cohort of larvae introduced onto the plants. Because larvae were not fed artificial diet between hatching and placement on plants, final biomass after the feeding assay can be used to estimate the impact of plant quality on herbivore performance. Such short-term (<five day) growth bioassays on early instar larvae are commonly used to estimate host plant quality and effects of host plant quality on early herbivore growth and performance (e.g. Agrawal, 2002; Kempel et al., 2010). Early in the experiment, plants experienced an unexpected outbreak of thrips. To explore the effects of thrips herbivory on our measured variables (see Analyses section below), we estimated the level of thrips herbivory on a per plant basis by counting the proportion of leaves on each plant with thrips damage (at 4 weeks after plants were transplanted). Finally, at harvest, we estimated rhizobium fitness benefits in multiple ways: as the total number of nodules produced on each plant, as well as two estimates of nodule size: mean length (mm), and weight of ten nodules per plant. Nodules were haphazardly selected from the plant root system, without respect to size, although the number of experimental units precluded a formal randomization scheme. Nevertheless, while bias in nodule selection (e.g. choosing larger nodules) could feasibly alter the mean nodule weight or size, it cannot account for differences between treatments and the significance of any model effects. Nodules were collected in 1.5 mL tubes and frozen until the time of measurement. Nodule size in Medicago is positively correlated with rhizobium reproduction inside the nodule (Heath and Tiffin, 2007; W.C. Ratcliff, pers. comm.). Nevertheless, the number of nodules has a larger impact on rhizobium fitness in the M. truncatulaeS. meliloti symbiosis (Heath and Tiffin, 2009), a finding that likely results from the exponential reproduction of rhizobial cells inside each nodule (Ratcliff et al., 2008). Therefore, nodule number in particular should be strongly correlated with the fitness benefits that rhizobia receive from plants.
2.4. Statistical analyses We used MANOVA (PROC GLM) and mixed-model ANOVA (PROC MIXED) to investigate how herbivory, rhizobium treatment, and plant family impact estimates of rhizobium fitness (nodule number, length, and weight) and plant fitness (biomass and fruit
89
number) (SAS Institute, 2001). Rhizobium treatment, herbivore treatment, plant population, and all interactions were included as fixed factors, while block, plant family nested within population, and all interactions between plant family and the fixed factors were included as random factors. The significance of random effects was determined by comparing the log likelihood ratio of models differing by the inclusion of each random effect to a c2-distribution with one degree of freedom. All response variables were natural log-transformed for use in multiplicative models in tests for interactive effects on fitness (Hambäck and Beckerman, 2003; Wootton, 1994). Because we were concerned that the thrips outbreak may have compromised our herbivory treatments (i.e., most plants, including those in the eS. exigua treatments, experienced thrips herbivory), we also repeated all analyses including the proportion of leaves damaged by thrips as a covariate. Results were qualitatively similar, regardless of whether thrips damage was included as a covariate, suggesting that the thrips outbreak had minimal impacts on the observed treatment effects. Similar models were used to test for the effects of rhizobia and plant family on thrips damage and S. exigua growth. Final biomass of the bioassay larva (natural log-transformed) or the proportion of leaves with evidence of thrips damage (arcsin square root transformed) were the response variables; rhizobium treatment and plant population were included as fixed factors, and plant family (nested within plant population) and the plant family rhizobium interaction were included as random factors. Phenotypic correlations between all dependent variables in the experiment are presented in Appendix A in the electronic Supplementary material.
3. Results 3.1. Effect of herbivores on rhizobium fitness benefits Herbivory by S. exigua positively affected rhizobium fitness benefits in this experiment. The presence of S. exigua increased the number of nodules formed by plants (Table 1, Fig. 1), and the direction of this effect was consistent across plant populations and rhizobium inoculation treatments (not shown). By contrast, there were no significant effects of herbivory on nodule length or nodule weight (all P > 0.3). We also detected plant genetic variation (significant family effects, Table 1) for nodule number, nodule length, and nodule weight e indicating that plant families differ in the benefits they confer to their rhizobial mutualists. However, we found little evidence that variation among families mediated the effects of herbivory on rhizobium fitness (no significant family herbivory interactions, Table 1).
Table 1 MANOVA and mixed-model ANOVAs for the effects of rhizobium inoculation, herbivore presence and plant population on the fitness benefits to rhizobia (nodule number, nodule length, and nodule weight). The effects of plant family (nested within population), block, and all interactions with family were tested as random effects (values shown are c2). Significance indicated as follows: D P<0.10, * P<0.05, ** P<0.01, *** P<0.001, **** P< 0.0001. MANOVA
Rhizobia (R) Herbivory (H) Pop (P) RH RP HP RHP Family (Pop) Family R Family H Family R H Block
Nodule Number
Nodule length
Pillai
df
F
df
F
df
F
df
F
0.41 0.02 0.52 0.00 0.06 0.03 0.07 0.28 0.10 0.07 0.07 0.50
223 223 4 223 223 223 675 675 675 675 675 675
51.8**** 1.60 1.46 0.82 4.46** 2.29D 0.89 3.81**** 1.29 0.97 0.89 1.46**
280 270 6 276 289 290 278
161.20**** 7.78** 2.73 0.25 2.39 1.95 0.81
6 6 6 270 6 6 272
36.73** 1.22 0.40 0.94 8.30* 0.47 0.30
6 268 6 266 6 269 269
30.92** 0.29 0.11 0.07 1.75 0.21 0.68
12.6*** 0 0 0 14.8****
Nodule weight
21.3**** 0.7 0.5 0 0.1
2.9* 3.6* 0 0 2.0D
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30
- rhizobia + rhizobia
2.5
b
a
2
a
a
a
20
Biomass (g)
Number of nodules
25
b
15 10
1.5
1
0.5
5
0
0 - herbivores
+ herbivores
While herbivores benefited rhizobia, the interaction was asymmetrical: presence of rhizobia did not significantly affect either herbivore (S. exigua performance or thrips damage, all P > 0.5; Appendix B). We also did not detect any evidence for variation among plant populations or families in their effects on S. exigua performance or thrips damage (all P > 0.19; Appendix B).
3.3. Effects of herbivores and rhizobia on plants As expected, insect herbivores decreased fruit number in M. truncatula (Fig. 2b, Table 2), while rhizobia increased both plant biomass and fruit production (Fig. 2a,b, Table 2). We detected weak evidence for the interactive effect of rhizobia and herbivory on plants, although the interaction varied across populations (significant rhizobia herbivory population term in MANOVA, Table 2). Specifically, the presence of herbivores altered the benefits of rhizobia for plants from Chat, but not Sals, and the direction of the effect varied across performance measures (biomass vs. fruit number; results not shown). We detected much genetic variation among plant families (but not plant populations) in both biomass and fruit production, consistent with previous work on these genotypes (Heath, 2010). However, we found little evidence that plant genotype mediated the effects of herbivores or the interactive effects of herbivores and rhizobia on plant performance (Table 2).
a,b a,b
Number of fruits
3.2. Effects of rhizobia and plant genotype on herbivores
6
b 4
2
0
- herbivores
+ herbivores
Fig. 2. Effects of herbivory by S. exigua and rhizobium mutualists on the biomass (A), and fruit number (B) of M. truncatula plants. Shown are least-squares means SE.
4.1. Mechanisms mediating rhizobiumeherbivore interactions We hypothesized that, because herbivores remove leaf tissue and therefore reduce carbon acquisition (Borowicz, 1993; Stark and Kytoviita, 2005), their presence would negatively affect mutualism
Table 2 MANOVA and mixed-model ANOVAs for the effects of rhizobium inoculation, herbivore presence and plant population on plant growth (final biomass) and reproduction (fruit production). The effects of plant family (nested within population), block, and all interactions with family were tested as random effects (values shown are c2). Significance indicated as follows: D P<0.10, * P<0.05, ** P<0.01, *** P<0.001, **** P< 0.0001. MANOVA
4. Discussion Although the potential for the biotic environment to alter the dynamics of species interactions is well-appreciated (for recent reviews see Strauss and Irwin, 2004; Morris et al., 2007), few empirical data exist on the potential for third-parties to alter the costs and benefits of mutualistic interactions to both partners in ecological time. We studied how a herbivore antagonist influenced the interactions between M. truncatula and S. meliloti and found that herbivores increased the reproductive benefits to rhizobia. By contrast, we found little evidence that rhizobia influenced the outcome of a planteherbivore antagonism. Potential mechanisms for, and implications of, the observed results are discussed.
a
8
Fig. 1. Effects of herbivory by S. exigua on plant allocation to rhizobium mutualists (nodule number) for rhizobium-inoculated M. truncatula plants. Shown are leastsquares means SE.
Rhizobia (R) Herbivory (H) Pop (P) RH RP HP RHP Family (Pop) Family R Family H Family R H Block
Biomass
Fruit Number df
Pillai
df
F
df
0.11 0.05 0.31 0.02 0.02 0.02 0.03 0.27 0.04 0.04 0.04 0.43
241 241 5 241 241 241 241 241 484 484 484 484
14.6**** 6.42*** 1.14 2.01 2.20 2.62D 3.51* 6.22**** 0.77 0.92 0.84 2.13****
274 6 6 271 281 7 272
F 34.07**** 5.20D 0.46 2.74D 3.80D 1.47 1.79 27.2**** 0 0.3 0 35.0****
F
281 5.71* 6 7.24* 6 1.70 276 0.02 287 1.66 6 00.63 279 2.15 29.0**** 0 1.8D 0 14.2****
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benefits to rhizobia. Indeed, multiple studies of forage legumes have found that nodule number or size decreases after severe defoliation or grazing (Butler et al., 1959; Whiteman, 1970; Vance et al., 1979), suggesting that the removal of most or all aboveground biomass does limit resource availability to such a degree that plants cannot maintain or form nodules. By contrast, however, insect herbivores in our experiment increased the number of nodules by ca. 28%, and therefore the total number of rhizobium offspring resulting from symbiosis. Although the experiments are not precisely comparable, these results are in agreement with Dean et al. (2009), who found that higher aphid densities were associated with a rhizobium treatment in which plants also formed nodules sooner and exhibited a (non-significant) trend for increased nodulation. Several mechanisms might account for the positive effect of herbivory on rhizobia in our experiment. Herbivory might alter carbon allocation to roots vs. shoots (Babst et al., 2005; Henkes et al., 2008), or increase root exudates (Holland et al., 1996), which are necessary for attracting rhizobia and initiating symbiosis (reviewed in Cohn et al., 1998). In addition, given the physiological overlap between plant hormones active in defense and symbiotic signaling, it is also possible that induced plant defenses might disrupt the regulation of nodulation. Specifically, jasmonic acid (JA) is an important elicitor of plant defense, often increasing in response to herbivore damage (Dong, 1998; Howe and Jander, 2008), and more recently has been found to play a role in nodulation. The role of JA in nodulation is complex, and this is currently an active area of research (recently reviewed by Hause and Schaarschmidt, 2009). Nevertheless, it appears that, although exogenous root JA inhibits nodulation in synergy with ethylene, elevated shoot JA might actually promote nodulation, possibly in antagonism with ethylene and/or by altering of the frequency of the calcium spikes that precede nodulation (Sun et al., 2006; Hause and Schaarschmidt, 2009). Among other lines of evidence, the soybean nts mutant has high shoot JA and is also a supernodulator (Seo et al., 2007; Kinkema and Gresshoff, 2008). Independent of the particular physiology involved, it seems clear that such subtle, plant hormone-mediated effects should only be detected when plants are not severely carbon-limited. Thus, whether herbivores impact rhizobia negatively or positively might depend on the level of herbivory, as posited by Wearn and Gange (2007), albeit in the context of mycorrhizal interactions. While insect herbivores impacted the planterhizobium mutualism, we detected no overall effect of rhizobium treatment on the performance of the generalist S. exigua, or on plant damage received by thrips in an unexpected outbreak. These results were in spite of the fact that all plants were grown in very low nutrient soil and were not fertilized; therefore, rhizobia should have increased plant quality by increasing leaf nitrogen concentrations (Wilson and Stinner, 1984). By contrast, Dean et al. (2009) found that soybeans nodulated by a commercial inoculum, vs. naturallyoccurring soil strains, supported higher populations of specialist soybean aphids. Because coevolved specialist herbivores or different feeding guilds might show different responses to plant mutualists (Hartley and Gange, 2009), it might be important to note that, unlike Dean et al. (2009), we used a broad generalist folivore. Variation in response among different herbivore classes might be responsible for the wide range of effects observed in similar resource mutualisms (i.e., fungaleplanteherbivore interactions, recently reviewed by both Gehring and Bennett, 2009 and Hartley and Gange, 2009). For example, Gange et al. (2002) found that associations with arbuscular mycorrhizal fungi positively affected specialist, but not generalist, herbivores e likely because these fungi increase plant secondary metabolites that benefit specialists but are detrimental to generalists (Gange and West, 1994). It is also
91
conceivable that effects on herbivores will depend on the particular rhizobium and plant genotypes involved in the interaction, since prior work has demonstrated that the fitness outcomes of M. truncatulaeS. meliloti interactions are dependent on the particular plant and rhizobium genotypes involved in the interaction (Heath and Tiffin, 2007; Heath, 2010). Only a single rhizobium strain was included in this study; however, it is possible that other strains could have greater impacts on plant tissue nutrient concentrations and, therefore, stronger plant-mediated indirect effects on herbivore growth and fitness. Unfortunately, without more studies on rhizobiumeplanteinsect interactions, we cannot yet assess how outcomes vary across inter- and intra-specific variation in the interacting taxa or whether the handful of results to date are consistent with the patterns emerging from studies of fungaleplanteinsect interactions. 4.2. Context-dependence e ecological effects In addition to their direct negative impact on plant growth and reproduction, herbivores might indirectly impact plants by altering the costs and benefits of mutualism with rhizobia (by increasing the number of nodules). Whether such an effect is positive or negative should, in turn, depend on many factors: e.g. the carbon/ nitrogen balance of the plant and the relative effectiveness of the available rhizobium strains. In the context of our study, increased nodulation was, on average, associated with higher plant performance (see Appendix A in electronic Supplementary material); however, genetic correlations suggest that supporting many nodules is costly for plants (Heath, 2010). Thus, increasing nodulation could increase both the costs and benefits of mutualism and could, therefore, potentially explain the lack of strong and consistent rhizobium herbivory interactions on plant fitness observed in this study, even though changes in nodule numbers were observed. While several previous studies have demonstrated direct effects of herbivores on plant population dynamics (Lau et al., 2008 and citations within), little research has addressed how herbivores might indirectly alter population growth rates by disrupting mutualisms, or by changing the costs and benefits of mutualism. Herbivore-induced changes in the fitness benefits of mutualism may result in changes in the population growth rates or abundance of one or both partners, and in ways not easily predicted by singlegeneration studies. For example, increased nodulation should increase standing densities of rhizobia in the soil. If plants are rhizobium-limited (e.g. Parker et al., 2006), this could benefit future plant generations. The long-term population consequences of diffuse interactions may be difficult to predict based on short-term changes in fitness or growth benefits observed in single-generation studies, which cannot account for changes in population densities of interacting species (see also Schmitz, 1998; Wootton, 2002). 5. Conclusions There has been increasing awareness in recent years of the importance of third-party interactions to population and community dynamics (e.g. Strauss and Irwin, 2004; Johnson and Stinchcombe, 2007; Morris et al., 2007). Most studies have been phyto-centric, though the effects of third-parties on all players are important in determining both ecological and evolutionary outcomes. Our data show that aboveground herbivores have the potential to increase rhizobium fitness benefits. In complex natural communities, where a given plant is likely to interact with a suite of mutualists, herbivores, and competitors, higher-order effects on partner fitness may be common. As a result, the outcomes of pairwise mutualistic interactions are likely to vary across communities that differ in composition.
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Acknowledgments We thank Molly Peterson for assistance with the greenhouse experiment, and two anonymous reviewers for helpful comments on an earlier version of the manuscript. This work was funded by an NSF Dissertation Improvement Grant DEB-0508305 to KDH. JAL was supported by NSF IOB 0620318 to P. Tiffin, R. Shaw, and P. Reich. This is KBS contribution no. 1459.
Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.actao.2010.12.002.
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