And Belowground Damage on an Invasive Tree

A native plant competitor mediates the impact of above- and belowground damage on an invasive tree

Juli Carrillo1,3 and Evan Siemann2 1Department of Entomology, Purdue University, West Lafayette, Indiana 47907 USA 2Department of Biosciences, Rice University, Houston, Texas 77005 USA

Abstract. Plant competition may mediate the impacts of herbivory on invasive plant species through effects on plant growth and defense. This may predictably depend on whether herbivory occurs above or below ground and on relative plant competitive ability. We simulated the potential impact of above- or belowground damage by biocontrol agents on the growth of a woody invader (Chinese tallow tree, Triadica sebifera) through artificial herbivory, with or without competition with a native grass, little bluestem (Schizachyrium scoparium). We measured two defense responses of Triadica through quantifying constitutive and induced extrafloral nectar production and tolerance of above- and belowground damage (root and shoot biomass regrowth). We examined genetic variation in plant growth and defense across native (China) and invasive (United States) Triadica populations. With- out competition, aboveground damage had a greater impact than belowground damage on Triadica performance, whereas with competition and above- and belowground damage impacted Triadica similarly. Whole plant tolerance to damage below ground was negatively associated with tolerance to grass competitors indicating tradeoffs in the ability to tolerate herbivory vs. compete. Competition reduced investment in defensive extrafloral nectar (EFN) production. Aboveground damage inhibited rather than induced EFN production while belowground plant damage did not impact aboveground nectar production. We found some support for the evolution of increased competitive ability hypothesis for invasive plants as United States plants were larger than native China plants and were more plastic in their response to biotic stressors than China plants (they altered their root to shoot ratios dependent on herbivory and competition treatments). Our results indicate that habitat type and the presence of competitors may be a larger determinant of herbivory impact than feeding mode and suggest that integrated pest management strategies including competitive dynamics of recipient communities should be incorporated into biological control agent evaluation at earlier stages. Key words: aboveground–belowground interactions; biological control; Chinese tallow tree, Triadica sebifera; competition; extrafloral nectar; little bluestem, Schizachyrium scoparium; resistance; tolerance.

Introduction compensatory continuum hypothesis [Maschinski and Whitham 1989] and growth rate model [Hilbert et al. Herbivory and competition interact to influence plant 1981]). There has been support both for and against these performance (e.g., Agrawal 2004) and can promote scenarios (e.g., Hawkes and Sullivan 2001, Lieurance and species coexistence or exclusion (e.g., Carson and Root Cipollini 2013), which may be due to differences in the 2000, Hanley and Sykes 2009, Kim et al. 2013). This can limiting resource for plants and where herbivory occurs, occur, for example, through herbivory reducing the and for many plants, the presence and type of ability of plants to compete or by plant competitors competitors. inhibiting the ability of plants to defend against Specifically, simple models of plant resource compe- herbivores (e.g., Hambäck and Beckerman 2003). tition with two limiting resources (in this case above- and Fundamentally, this tension between plant competitive belowground resources, e.g., light and nitrogen), predict ability and defense against herbivory can be seen as a that where herbivores feed on target plants can shift the phenotypic response to a reduction in plant resources. balance of plant competitive dynamics (resource compe- However, for some plant defenses, including tolerating tition model; Siemann and Weisser 2004) and the ability some level of herbivore damage without reductions in of plants to tolerate herbivory (limiting resource model, plant fitness, it has been hypothesized that both high and LRM; Wise and Abrahamsom 2005, 2007). For example, low resource levels will improve plant defense (e.g., in a simple two-species model of resource competition, coexistence is assumed because the species differ in their Manuscript received 22 March 2016; accepted 5 April 2016. Corresponding Editor: R. A. Hufbauer. competitive ability for different resources (Siemann and 3 E-mail: [email protected] Weisser 2004). In this case, the resource competition

1 2 JULI CARRILLO AND EVAN SIEMANN Ecological Applications Vol. 0, No. 0 model predicts that aboveground damage on the puta- being tested for Triadica, including two multivoltine tively better aboveground competitor decreases the insects: an aboveground-feeding caterpillar (Gadirtha potential for coexistence, as damage above ground fusca; previously G. inexacta) and a flea beetle Bikasha( reduces or removes that species’ aboveground compet- collaris) that has both an aboveground feeding adult itive advantage, while damage below ground should have stage and a belowground feeding larval stage (e.g., Wang less of an impact on plant success. This focus on plant et al. 2012). Triadica is extremely tolerant of herbivory, competitors, and their most limiting resource, contrasts although native (Chinese) vs. invasive (United States) the LRM, which predicts that damage that inhibits the populations vary in the specificity of their tolerance ability of plants to uptake their most limiting resource, response to different aboveground-feeding herbivores independent of plant competitors (or perhaps, encom- (Carrillo et al. 2014). Increasing evidence of evolved passing all plant competitors), will reduce that plant’s increased tolerance of herbivory in invasive plant popula- ability to compensate (e.g., Wise and Abrahamson 2007). tions compared to their native counterparts (e.g., Carrillo However, only a handful of studies have examined plant et al. 2014 [T. sebifera]; Joshi and Tielborger 2012 tolerance responses across different competition regimes [Lythrum salicaria]; Wang et al. 2011 [T. sebifera]) neces- or with different herbivory types (e.g., apical vs. foliar sitates a greater understanding of this defense response damage; Dahlgren and Lehtilä 2015), and integration of to above- and belowground forms of damage, and its these models is lacking. interaction with the presence of plant competitors. Biological control of weedy or invasive plants could Invasive populations of Triadiaca have reduced direct, inform our understanding of herbivory–resource compe- chemical defenses (Wang et al. 2011, 2012), and reduced, tition interactions. Whereas classical biological control of yet retained, indirect defenses against herbivores (extra- weeds considers host plant specificity and traditionally floral nectar, EFN; Carrillo et al. 2012b). EFN can focuses on biocontrol impact in artificial monocultures attract predaceous ants which can provide protection to where the target weed is experimentally isolated from its plants (e.g., Heil 2015) and could potentially reduce recipient plant community (i.e., without competitors from population levels of biocontrol agents. It is unclear how the invasive range), a greater awareness of recipient com- EFN production will be impacted by plant competition. munities has been shown to improve control. For example, Importantly, evaluating potential biocontrol agent biological control of purple loosestrife (Lythrum salicaria) impacts solely with native populations would likely over- by Galerucella beetles was more likely to be successful in estimate the effects of biocontrol, therefore including sites with lower vs. higher fertility (Hovick and Carson invasive populations is necessary for accurate man- 2015), potentially indicating reduced tolerance of plants agement as they may differ in defense syndromes due to at lower fertility. Moreover, it is known that the presence divergent evolutionary history (e.g., Agrawal and of plant competitors can reduce the negative effect of Fishbein 2006). Although we predict that broad niche invaders through providing biotic resistance to invasion differences in resource utilization will determine target and reducing invader establishment and/or negative effect plant responses, genetic variation in competitive ability (Levine et al. 2004). Plant competitors may also inhibit and resistance and tolerance to above- and belowground the ability of plants to defend against introduced control herbivory may be important for target plant performance agents and magnify those agents’ impacts. For example, (Agrawal 2004). Therefore, we include both native and the combined effects of native herbivory and competition invasive populations of Triadica to examine evolved dif- by native thistle (Cirsium altissimum) resulted in strong ferences in competitive ability and herbivore defense that biotic resistance to exotic thistle (C. vulgare), and reduced may impact biocontrol success. invasive impact to the community (Suwa and Louda We evaluated the potential relative success of above- or 2012). Indeed, the mechanisms underlying plant com- belowground damage on the invasive success of a woody petitive and invasive success may make some biocontrol invader, Triadica, by simulating above- or belowground agents especially effective for particular competitive sce- herbivory when in competition with a native grass, little narios. One such case could be tree/grass competition bluestem (Schizachyrium scoparium) or when grown (e.g., woody encroachment into grasslands), where we singly. We also examined how damage and competition would predict that damage above ground would be more jointly influenced plant tolerance responses and the pro- effective than damage below ground in reducing tree per- duction of extrafloral nectar inTriadica as this may impact formance. This is because fine-rooted grasses can often future biocontrol success. We hypothesize that, in exploit belowground soil resources at a greater rate than accordance with the resource competition model (Siemann woody trees, that in contrast, eventually dominate above- and Weisser 2004), plants that are superior light compet- ground resource capture of light (e.g., Picon-Cochard itors but weaker soil-resource competitors will be more et al. 2006). impacted by aboveground feeding agents than below- Chinese tallow tree (Triadica sebifera, Euphorbiaceae; ground feeding agents when competing with a superior Triadica hereafter) is a devastating invader of the south- belowground competitor. We specifically predict that eastern United States and can quickly dominate natural Triadica, a superior light competitor, will be more areas including roadsides, forests, and endangered tall- impacted by aboveground feeding biocontrol agents when grass prairies. Several biocontrol agents are currently in competition with little bluestem grass, a putatively May 2016 PLANT COMPETITION MEDIATES DAMAGE IMPACT 3 better soil-resource competitor. Due to reduced resource belowground tissue (both), or did not cut them (control), availability, we predict that competition with grass will to simulate outbreak herbivory conditions. We sacrificed reduce the defensive (EFN) response of plants, but that in some realism and potential relevance by utilizing artificial general damage will induce defensive (EFN) responses. herbivory to control and impose simultaneous and equivalent amounts of relative damage above and below ground. This type of simultaneous control over damage amounts Materials and Methods above and below ground would be effectively impossible with real herbivores feeding naturally on plants that likely Study system vary in resistance across populations and above and below Triadica sebifera (L.) Small (Euphorbiaceae) is a dom- ground, and where belowground damage is hard to observe. inant, non-clonal tree species across the southeastern The aboveground damage treatment represented complete United States where it commonly occurs along roadsides, defoliation but left some stem tissue that could potentially along waterways, bottomland hardwood forests, and function in photosynthesis. We cleaned scissors between endangered coastal prairies. Triadica is virtually free of each plant with a 10% ethanol rinse and a kimwipe (Kimtech herbivore pests in its introduced range, but is attacked by Science, Kimberly Clark). After we recorded a seedling’s a diversity of specialist and generalist herbivores across height and root length and had simulated herbivory, we multiple feeding guilds both above and below ground in immediately transplanted the seedling into either a compe- its native range (e.g., Huang et al. 2014). Little bluestem tition pot or a no-competition control pot. For each of the grass, Schizachyrium scoparium, is dominant in coastal six populations from either the native or introduced ranges, prairie remnants and is widely distributed throughout the we planted four replicates per treatment combination when United States across a range of conditions. It is the most possible. However, some populations did not have enough likely competitor for Triadica invading coastal prairies seeds germinate to include in all possible combinations, so (Bruce, Cameron, and Harcombe 1995) and is frequently our total number of plants equaled 380 (vs. 384 for a fully used for revegetation or restoration seed mixes (USDA balanced design). We dried and weighed the biomass of the PLANTS database). Therefore, results for its competitive removed above- and belowground tissue to quantify ability against invasive Triadica may be generalizable to amount of tissue removed. other grass–tree interactions across the United States. We used this high level of herbivory for several reasons. We grew native and invasive Triadica populations (six First, the amount of aboveground damage approximates a populations each) with or without competition with little single bout of intense foliar defoliation, which is possible in bluestem and with or without simulated above- or the native range from a variety of herbivores (J. Ding, per- belowground herbivory to determine the effects of compe- sonal communication), including Gadirtha fusca, a potential tition, aboveground herbivory, belowground herbivory, biocontrol agent of Triadica (Pogue 2014). Second, attempts and their interactions on plant growth and defense to mechanically control Triadica include cutting or burning (2 × 2 × 2 = 8 treatment combinations per each of 12 plant the aboveground portion of the tree and some attempt at populations). Triadica propagates by seed and we collected root severing; despite these efforts, Triadica notoriously Triadica seeds in late November/early December 2010 recovers from massive tissue damage and tissue removal. across the native (China) and introduced (United States) Third, although it is unclear what typical herbivore damage ranges. We sampled six populations each (five to six trees rates are belowground for Triadica, reductions in root within each population) from across the native and intro- chemical defenses suggest invasive populations may be duced range and kept seeds in refrigerated storage until especially poorly defended against potential biocontrol spring 2011. We planted seeds on 5 April 2011 in forestry agents (Huang et al. 2012). In this regard, our experimental “cone-tainers” (Stuewe & Sons, Tangent, OR, USA) after damage treatment represents a best case scenario for feeding removing the non-provisioning waxy seed coat by scrubbing by potential biocontrol agents. with a mild detergent, and grew the seedlings in a climate Two weeks after the artificial herbivory treatments and controlled greenhouse until we began the experiment in after transplanting the seedlings, we recorded the height June 2011. For competition treatments, we scattered 1.6 g and leaf number of the Triadica seedling and the number of S. scoparium seeds across the soil surface of 1-gallon pots of extrafloral nectaries producing EFN. We recorded this (1 gallon = ~3.8 L) equipped with drip-emitter irrigation same data again in early September before harvesting the and containing a peat-based soil mixture (Pro-mix, Premier plants 9 weeks post-herbivory treatments. We separated Tech Horticulture, Quakertown, PA, USA). Schizachyrium leaf, stem, and root mass of Triadica, and above- and scoparium was allowed to grow for 1 week before we trans- belowground grass leaves and roots, then dried them and planted Triadica seedlings directly into the center of the recorded their masses. pots. Before transplanting, we recorded Triadica seedling height, number of leaves, and after washing all the soil from Statistical analyses the roots, the length of the roots. With scissors, we cut 90% of the aboveground tissue by height (leaf clip treatment, We examined differences in plant biomass above and Fig. 1A), cut 90% of the belowground tissue by length below ground (combined, separately, and the ratio of the (root clip treatment), cut 90% of both aboveground and two), final plant height and height growth (calculated as 4 JULI CARRILLO AND EVAN SIEMANN Ecological Applications Vol. 0, No. 0 Y Y Y the relative percentage change in height from the start of LRRG =[ln ( LC,G)+ln ( RC,G )+ln ( B,G) our experimental treatments until the end of the exper- +ln (Y )]−[ln (Y )+ln (Y ) iment), and EFN (the number of nectaries actively pro- CO,G LC,NG RC,NG Y Y ducing EFN/leaf) across our competition and artificial +ln ( B,NG)+ln ( CO,NG)] herbivory treatments and among native and invasive where Y represents the sample mean, the subscripts LC, populations with a mixed model ANOVA. The model RC, B, and CO refer to leaf clip, root clip, both, and included competition (grass or no grass), aboveground control treatments, respectively, and the subscripts g and herbivory (leaf clip or control), belowground herbivory ng refer to grass and no grass, respectively. (root clip or control), and plant population origin (China A positive LRR indicates overcompensation for a or United States) and their interactions as fixed factors. population, e.g. biomass greater than undamaged plants. We included plant population nested within plant popu- Negative LRR or LRR values indicate undercom- lation origin as a random factor and conservatively esti- LC RC pensation for populations, e.g. biomass less than mated differences between origins using the variation undamaged plants, with more negative values showing among populations for the main effect of origin and inter- less tolerant populations compared to populations with active effects including the origin term. Triadica plant LRR or LRR values closer to zero. Similarly, for mass (above-, belowground, and total) was log trans- LC RC LRR , more positive values indicate greater competitive formed to meet assumptions of normality and homoge- G tolerance while more negative values indicate less tol- neity of variance. Plant seedlings were randomly assigned erance of competition within a population. A zero LRR to experimental treatments, however a statistical associ- would indicate full compensation. ation in initial plant height and experimental treatment We performed an ANOVA on the LRRs with plant existed such that plants in our grass treatments and below- population origin (China or United States) as a main ground damage treatments began the experiment shorter effect to detect if native vs. invasive populations differed than our other plants, independent of plant population. from each other in their aboveground, belowground, or To control for this potential bias, we initially included whole plant tolerance response. We did this for either starting plant height as a covariate in our model. However, above- or belowground damage, or grass competitors the inclusion of this covariate did not change the signifi- (but not their interactive effects). We also examined cor- cance of any of the terms of our model except for one relations among the different tolerance biomass responses interactive term for total plant mass, so we excluded the (tolerance to aboveground damage, tolerance to below- covariate from our final analyses. When significant inter- ground damage, and tolerance to grass competitors) using active effects occurred, we examined differences among these LRRs both within and across native and invasive treatments combinations using adjusted means partial dif- populations and within biomass types (for above- vs. ference tests (P < 0.05). belowground biomass, and whole plant biomass across We examined whether native vs. invasive populations treatments). This analysis was used to detect tradeoffs in differed in their tolerance of competition, leaf clipping plant tolerance to multiple stressors, e.g. does tolerance and root clipping with a mixed model ANOVA. We first above ground trade off with tolerance below ground? Or, determined plant tolerances to each type of stress by cal- does tolerance to competitors reduce tolerance to her- culating the log response ratio of plant biomass (above-, bivory and does this depend on plant population origin? belowground, and whole plant biomass) to that treatment Next, we performed an ANOVA with plant origin and type for each of our 12 populations, i.e., tolerance to tolerance type as main effects and plant population as a herbivory relative to undamaged controls and or tol- random effect on the LRRs for whole plant tolerance erance to competition relative to non-competing control. with or without a grass competitor. In this analysis, tol- We determined plant tolerance by modifying the erance type had four possible designations: leaf clip in approach of Hawkes and Sullivan (2001), who calculated grass, root clip in grass, leaf clip without grass, root clip the compensatory responses of plants as log response without grass, calculated as follows: ratios (LRRs) equivalent of treatment effect sizes. To do this, we calculated LRRs for aboveground, belowground, Y Y and total plant biomass for each population using popu- LRRLC_G =[ln ( LC,G)+ln ( B,G)] lations means within each treatment (LRRs = 108 total, Y Y −[ln ( RC,G )+ln ( CO,G)] 9 per population [3 main effects × 3 biomass types])

LRR =[ln (Y )+ln (Y )] Y Y RC_G RC,G B,G LRRLC =[ln ( LC,G)+ln ( LC,NG) −[ln (Y )+ln (Y )] Y Y Y LC,G CO,G +ln ( B,G)+ln ( B,NG)]−[ln ( RC,G )

Y Y Y =[ Y + Y ] +ln ( RC,NG )+ln ( CO,G)+ln ( CO,NG)] LRRLC_NG ln ( LC,NG) ln ( B,NG) Y Y −[ln (Y )+ln (Y )] LRRRC =[ln ( RC,G )+ln ( RC,NG ) RC,NG CO,NG Y Y Y Y Y +ln ( B,G)+ln ( B,NG)]−[ln ( LC,G) LRRRC_NG =[ln ( RC,NG )+ln ( B,NG)] Y Y Y Y Y +ln ( LC,NG)+ln ( CO,G)+ln ( CO,NG)] −[ln ( LC,NG)+ln ( CO,NG)] May 2016 PLANT COMPETITION MEDIATES DAMAGE IMPACT 5

A 6 controlroot clip leaf clip both controlroot clip leaf clip both )

2 AG biomass (g

0 ) 2 China US

4 Root:Shoot values ± SEs reported below

BG biomass (g 0.90 0.98 0.69 0.77 1.09 0.95 1.39 0.94 0.89 0.98 0.74 0.74 1.41 1.69 0.75 0.90 (0.08) (0.08) (0.07) (0.08) (0.09)(0.08) (0.09) (0.08) (0.08) (0.08) (0.07) (0.07) (0.07) (0.08) (0.13) (0.09) 6 no grass grass

40 no grass grass

final height 30 starting height after leaf clip relative change in height

20 Plant height (cm)

0 l rol clip th ro clip th t bo af clip bo cont root leaf clip cont roo le

Fig. 1. (A) Total, above- and belowground biomass (AG and BG, respectively) of native (China) and invasive (United States) populations of Triadica across our treatments. Back-transformed means and standard error (SE) shown. Back-transformed mean root to shoot ratios are presented below bars with SE in parentheses. (B) Starting height of Triadica plants immediately following leaf clipping (or no leaf clip for controls) and final height at the end of the experiment. The relative change in height ([final height − starting height]/[starting height]) is indicated. All error bars show SE. Grass treatments are indicated with hatching.

Results 458% ± 17%, respectively; all means shown with standard error). In general, competition increased the relative Effects of plant competition amount of biomass allocated belowground compared to Competition with grass reduced total biomass of biomass allocated aboveground, i.e., root : shoot ratio Triadica plants by 77%, aboveground biomass by 78%, (grass vs. no grass, 1.09 ± 0.03 vs. 0.88 ± 0.03, respec- and belowground biomass by 77% (Table 1, Fig. 1A). tively; Table 1, Fig. 1A). Competition reduced the The main effect of competition reduced final plant height number of active extrafloral nectaries per leaf by 74% and decreased height growth (Fig. 1B; relative percentage and the total number of active extrafloral nectaries by change in height, grass vs. no grass 242% ± 16% vs. 85% (EFN/leaf, Table 1; total EFN, Fig. 2B). 6 JULI CARRILLO AND EVAN SIEMANN Ecological Applications Vol. 0, No. 0

Table 1. ANOVA of Triadica response to simulated herbivory and competition with grass.

Aboveground Belowground Term df Mass* (g) mass* (g) mass* (g) Root : shoot* EFN/leaf

Origin (O) 1, 10 8.34 (0.0162) 8.91 (0.0137) 8.42 (0.0158) 0.11 (0.7463) 0.00 (0.9776) Grass comp. (G) 1, 298 440.16 (<0.0001) 460.64 (<0.0001) 339.57 (<0.0001) 14.60 (0.0002) 26.65 (<0.0001) Leaf clip (LC) 1, 298 565.66 (<0.0001) 632.84 (<0.0001) 414.40 (<0.0001) 34.84 (<0.0001) 50.61 (<0.0001) Root clip (RC) 1, 298 96.19 (<0.0001) 63.71 (<0.0001) 114.50 (<0.0001) 30.23 (<0.0001) 2.44 (0.1195) O × G 1, 10 2.78 (0.1262) 2.30 (0.1604) 1.61 (0.2333) 0.77 (0.4018) 0.19 (0.6683) O × LC 1, 10 0.23 (0.6420) 0.13 (0.7300) 1.18 (0.3028) 2.84 (0.1227) 0.23 (0.6390) O × RC 1, 10 0.17 (0.6847) 0.01 (0.9086) 0.21 (0.6592) 0.40 (0.5414) 0.15 (0.7098) G × LC 1, 298 22.80 (<0.0001) 13.09 (0.0003) 32.66 (<0.0001) 19.00 (<0.0001) 12.87 (0.0004) G × RC 1, 298 5.20 (0.0233) 2.52 (0.1131) 6.13 (0.0138) 1.07 (0.3016) 1.62 (0.2044) RC × LC 1, 298 1.29 (0.2576) 1.60 (0.2069) 0.99 (0.3210) 0.30 (0.5840) 1.47 (0.2257) O × G × LC 1, 10 0.13 (0.7293) 0.37 (0.5560) 0.05 (0.8217) 0.01 (0.9051) 0.60 (0.4547) O × G × RC 1, 10 2.35 (0.1566) 5.50 (0.0410) 0.36 (0.5644) 8.00 (0.0179) 0.63 (0.4466) O × LC × RC 1, 10 0.08 (0.7767) 0.00 (0.9545) 0.02 (0.8894) 0.00 (0.9779) 0.01 (0.9447) G × LC × RC 1, 298 0.00 (0.9704) 0.10 (0.7553) 0.07 (0.7850) 1.12 (0.2913) 1.99 (0.1590) O × G × LC × RC 1, 10 2.77 (0.1268) 3.89 (0.0768) 0.39 (0.5459) 7.53 (0.0207) 0.04 (0.8370)

Notes: Total mass and above- and belowground biomass were log transformed. EFN refers to extrafloral nectaries. F-values reported with p-values in parentheses. Bolded values indicate significance atP < 0.05.

Effects of aboveground herbivory A 8 Simulated aboveground herbivory (leaf clipping) control 7 reduced total biomass of Triadica plants by 85%, above- A leaf clip ground biomass by 86%, and belowground biomass by ) 6 84% (Table 1, Figs. 1A and 2A). The main effect of leaf clipping reduced final plant height but increased relative 5 mass (g height growth (Fig. 1B; relative percentage change in 4 height, leaf clip vs. control was 614% ± 18% vs. 3

85% ± 16%, F1, 309 = 105.42, P < 0.0001). Leaf clipping Triadica increased root : shoot ratio (Table 1; leaf clip vs. control, 2 B 1.14 ± 0.03: 0.83 ± 0.03). The main effect of leaf clipping B 1 reduced EFN/leaf by 89% and total EFN produced by C plants by 91% (Table 1, Fig. 2B). 0 no grassgrass B Effects of belowground herbivory 50 s Simulated belowground herbivory (root clipping) ns reduced total biomass of Triadica plants by 42%, above- 40 ** ** ground biomass by 36%, and belowground biomass by 49% (Table 1, Figs. 1A and 2B). The main effect of root 30 clipping reduced final plant height and decreased height growth (Fig. 1B; relative percentage change in height for 20 root clip vs. control, 298% ± 17% vs. 402% ± 16%, respectively, F1, 309 = 24.66, P < 0.0001). Root clipping decreased root : shoot (Table 1; root clip vs. control, 0.86 ± 0.03 vs. 10 1.12 ± 0.03, respectively). Root clipping did not affect total number of active nectarie EFN/leaf production or total EFN (Table 1, Fig. 2B; 0 s s EFN/leaf root clip vs. control, 0.93 ± 0.12 vs. 0.65 ± 0.13, clip gras gras respectively). no leaf root clip no leaf clip no root clip

Interactions among treatments Fig. 2. (A) Total Triadica biomass (back transformed) across leaf clip and grass competition treatments. Bars with the Simulated above- and belowground herbivory inter- same letters were not significantly different in post hoc tests (P < 0.05). All means are shown with SE. (B) Reductions in acted to negatively impact belowground biomass of extrafloral nectar production due to main effects of grass Triadica, such that Triadica plants were largest in competitors, leaf clip, or root clip. ** P < 0.0001. May 2016 PLANT COMPETITION MEDIATES DAMAGE IMPACT 7 control treatments, followed by root clip only, leaf clip Effects of plant origin only, and root clip and leaf clip plants (Table 1, Figs. Plants from the native range (China) were smaller (less 1A, B and 2). However, leaf clip only plants had the biomass) than plants from the invasive range (United highest relative growth rate for height, followed by root States), both above and below ground (Table 1, Fig. 1A). clip/leaf clip plants, and then by control and root clip Plants from the native vs. the introduced ranges did not only plants, which did not differ from each other (Fig. differ overall in root : shoot nor in the EFN/leaf they pro- 1B, leaf clip × root clip: F = 16.78, P < 0.0001). 1, 309 duced (Table 1), nor plant height. United States and Simulated aboveground herbivory interacted with com- China plants did not differ in their tolerance responses petition such that their negative effects on total, above-, (biomass to simulated above- or belowground herbivory, and belowground biomass were less than additive the combination of the two, nor any combination of (Table 1, Fig. 1A). Likewise, belowground herbivory simulated herbivory with grass (Appendix S1) with one interacted with competition to impact total and below- exception: the aboveground tolerance response of United ground biomass, but not aboveground biomass (Table States populations was greater than China populations 1). Simulated aboveground herbivory and competition to the combined stress of root clipping and grass interacted to affect root : shoot such that it was signifi- competitors (F = 7.17, P = 0.03; Appendix S1). cantly higher in the leaf clip and grass treatment com- 1,9 pared to the all other treatments (Table 1; leaf clip and Tolerance and tradeoffs across responses above and below grass, no root clip vs. control and grass, no root clip: ground 1.55 ± 0.06 vs. 0.93 ± 0.05, respectively). Correspondingly, a significant interaction for leaf clip and competition Triadica tolerated belowground damage better than existed for plant height growth (Fig. 1B; leaf clip × com- aboveground damage in terms of biomass (Figs. 1A, B petition: F1, 309 = 23.21, P < 0.0001), as plants attained and 4). Tolerance to aboveground damage was greater a greater percentage of their initial experimental height for Triadica plants growing with grass (Fig. 4). This is when clipped above ground and grown without grass because within grass treatments, damaged plants were (773% ± 25%), followed by leaf clip/grass (456% ± 22%), closer in terms of biomass to non-damaged plants than then control/no grass (143% ± 20%), and finally by damaged plants were to non-damaged plants in the control/grass (28% ± 20%). A significant interaction no-grass treatments (Fig. 4). Triadica had greater existed among plant population origin, competition, tolerance to belowground herbivory without grass and simulated belowground herbivory for root : shoot competitors (Fig. 4). (Table 1, Fig. 3). The four-way interaction including Across all populations (native China and invasive simulated aboveground herbivory was also significant United States together), most tolerance responses to for root : shoot (Table 1). Simulated aboveground her- leaf clipping, root clipping, and grass competitors were bivory and competition with grass interacted to impact not correlated, e.g., aboveground biomass response to the number of active extrafloral nectaries produced per leaf clipping was unrelated to aboveground biomass leaf (EFN/leaf, Table 1) such that the combined response to root clipping (r = −0.009, P = 0.98; other reductive effects on EFN were less than additive but non-correlated tolerance responses not reported). Some bounded by zero. tolerance responses were positively correlated, typically

origin x grass x root clip ** no grass grass 1.6 US China control 1.4 A root clip 0 B 1.2 BC t BCD CD A 1.0 DE DE -2 E B 0.8 root:shoo

0.6 C Triadica 0.4 -4

D Plant population origin: F =0.06, P=0.82 0.2 1,10 lerance Score (Log Response Ratio) Stress type: F3,27=61.55, P<0.0001 To Origin x stress: F =0.44, P=0.73 0.0 3,10 -6 no grassgrass no grassgrass root clip leaf clip root clip leaf clip

Fig. 3. Root : shoot ratio of native (China) and invasive Fig. 4. Tolerance scores for the effect of leaf clipping vs. (United States) populations of Triadica across root clip and root clipping with or without a grass competitor. Means with SE grass competition treatments. Back-transformed means + SE show log response ratios for individual population whole plant are shown. Hatching indicates grass treatments. Bars marked biomass. Hatching indicates grass treatments. Bars marked with with the same upperacase letters were not significantly different the same uppercase letters were not significantly different in in post hoc tests (P < 0.05). post hoc tests (P < 0.05). 8 JULI CARRILLO AND EVAN SIEMANN Ecological Applications Vol. 0, No. 0 when experiencing the same stress: i.e., aboveground biomass reductions (Fig. 1A), so we recommend prior- biomass in response to leaf clipping was positively cor- itizing aboveground biocontrol agents for woody inva- related with belowground biomass response to leaf sives over those that feed only below ground. Competition clipping (r = 0.90, P < 0.0001), aboveground biomass with the relatively better belowground competitor (little response to grass competitors was positively correlated bluestem grass) magnified these effects, with plant with belowground biomass responses in to grass com- biomass higher but herbivory tolerance lower in the petitors (r = 0.69, P = 0.01), and aboveground biomass no-competition environments (Figs. 1A and 5). The response to root clipping was positively correlated with greater impact of above- vs. belowground damage for belowground biomass response to root clipping Triadica contrasts a recent meta-analysis of plant (r = 0.74, P = 0.01). A negative trend existed between responses to belowground herbivory, which found the aboveground biomass responses of plants to grass similar impacts and biomass responses to above- and competitors and the aboveground biomass responses of belowground damage for woody plants (Zvereva and plants to root clipping (r = −0.59, P = 0.05). Likewise, Kozlov 2012). However, within woody plants, that study aboveground and belowground biomass responses to predominantly considered evergreen trees (primarily root clipping were both negatively correlated with leaf-bearing citrus; Zvereva and Kozlov 2012), which belowground biomass response to grass competitors may explain the difference we observed. For a deciduous (r = −0.79, P = 0.002 and r = −0.86, P = 0.0002, tree such as Triadica, the loss of leaves may be compara- respectively). tively more harmful in terms of resource capture, as the Within either United States or Chinese populations, seasonal window for photosynthesis is shorter. In our these tolerance patterns were mostly consistent but with study, although belowground damage did not have as some exceptions. Within United States, but not China great an impact on plant success as aboveground damage, populations, plant biomass response above ground was competition with a grass competitor magnified the positively correlated to plant biomass response below impacts of belowground damage on plant biomass (Fig. ground when competing with grass (US, r = 0.69, 1A). Triadica could almost completely compensate in P = 0.01; China, r = 0.50, P = 0.34). Also, within United terms of biomass for the loss of 90% of its root tissue States but not China populations, when competing with compared to controls when not competing with grass, grass, plant biomass response aboveground was nega- however, Triadica had reduced compensatory ability tively correlated to plant biomass response belowground belowground when grass was present (Fig. 4). Indeed, we with root clipping (US, r = −0.86, P = 0.02; China, detected a tradeoff between the ability of Triadica to tol- r = −0.04, P = 0.96). Likewise, with root clipping, plant erate root clipping and to tolerate grass competitors in biomass response aboveground was negatively correlated terms of biomass produced (Fig. 5). This suggests evolu- with plant biomass response belowground with leaf tionary tradeoffs in the ability to endure belowground clipping for United States, but not China populations herbivory when coexisting with good belowground com- (US, r = −0.85, P = 0.03; China, r = −0.39, P = 0.56). petitors, and may indicate that plants can specialize on In three cases, the correlation across populations was either competitive or defensive ability. For Triadica or driven by significant associations for United States but not China populations: above- vs. belowground biomass 0.8 in response to grass competitors (US, r = 0.97, P = 0.0003; 2 Overall: r =0.72, F =22.60, P=0.001 0.6 1,9 China, r = 0.50, P = 0.34), above- vs. belowground 2 US:r =0.80, F =16.29, P=0.02 0.4 1,4 biomass in response to root clipping (US, r = 0.85, 2 China:r =0.64, F =5.27, P=0.11 P = 0.03; China, r = 0.72, P = 0.20), and belowground 0.2 1,3 biomass in response to grass vs. belowground biomass 0.0 in response to root clipping (US, r = −0.95, P = 0.001; -0.2 China, r = −0.76, P = 0.16). -0.4 For whole plant biomass responses, tolerance of grass -0.6 competitors was negatively correlated with tolerance to -0.8 root clipping across all populations. This was driven by -1.0 US populations a significant association within United States but not -1.2 China populations China populations (Fig. 5; US, r = −0.90, P = 0.02; -1.4 China, r = −0.80, P = 0.12). Tolerance to root clipping (log response ratio) -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 Tolerance to grass competitors (log response ratio)

Discussion Fig. 5. Tolerance of plants to root clipping is negatively correlated with tolerance of plants to grass competitors across We found that plant competition will likely mediate all populations, and within United States, but not China the impact of above- vs. belowground damage on plant populations. For each population, the log response ratio (LRR) success. As predicted by the resource competition model, is equivalent to the effect size for the treatment main effect on whole plant biomass and is used to determine tolerance to we found that aboveground damage had a greater impact herbivory relative to undamaged controls and or tolerance to than belowground damage on Triadica in terms of competition relative to non-competing controls. May 2016 PLANT COMPETITION MEDIATES DAMAGE IMPACT 9 other woody invaders expanding into open vegetation aboveground herbivory then in the alternative resource gaps or disturbed open ground, we predict that the com- scenario of relatively high aboveground resources with bined effects of integrated management through the relatively limited belowground resources (Wise and addition of both competitors and herbivores will be Abrahamsom 2005, 2007). greater than either alone. Competition with grass will likely reduce the ability of The compensatory continuum hypothesis predicts that Triadica to defend against native herbivores and any plants in low-resource environments will be less able to biocontrol agents that get introduced, as grass compet- compensate for herbivory than plants in high-resource itors reduced the number of active nectaries producing environments (e.g., Grime 1977 and reviewed in Wise extrafloral nectar (EFN; Table 1, Fig. 2B). Although and Abrahamsom 2007). We did not find this to be the little is known about the relative investment of plants in case due to differences in plant size between the high and EFN or other defense traits across different habitats, a low competition environments. Specifically, we found recent study with Mallotus japonicus found that some that, in general, plants were larger in the putatively defenses (trichomes, pellucid dots, and EFNs) had higher higher-resource, no-competition environment compared expression but lower ant attendance in open gaps (which to the putatively low-resource, grass-competition envi- included grasslands) vs. treefall gaps (Yamawo et al. ronment. However, tolerance to aboveground damage 2014). Native populations of Triadica did not produce was greater for Triadica plants growing in the low- more EFN (absolute or relative) than invasive popula- resource, grass-competition environment, as plants tions, contrary to expectations of the evolution of damaged and competing with grass were closer in size to increased competitive ability (EICA) hypothesis. But, non-damaged control plants than damaged plants were evidence for EICA in Triadica for EFN production is to non-damaged controls in the no-grass treatment (Figs. equivocal. One study showed greater constitutive and 1A and 4). Put another way, despite absolute biomass induced EFN production by native compared to invasive being larger in the high-resource, no-competition envi- populations (Carrillo et al. 2012a), while others show ronment, the proportional change in biomass was smaller decreased constitutive but increased induced EFN for for plants growing with grass than plants without grass, native populations (Wang et al. 2013), or equivalent EFN leading to higher tolerance of aboveground damage com- production across populations (Carrillo et al. 2012b). pared to controls within that treatment (Figs. 1 and 4). EFN is inducible in both native and invasive populations In contrast, another study examining compensatory of Triadica in response to chewing herbivory and clipping growth in two woody species (a tree, Brosimum ali- (e.g., Carrillo et al. 2012a, b) and several ant species tend castrum, and a liana, Vitis tiliifolia) found similar rates EFN in the invasive range of Triadica, including the of compensatory growth in terms of biomass across light highly aggressive red imported fire ant,Solenopsis invicta resource conditions (Ballina-Gómez et al. 2010), as (J. Carrillo, personal observation). In this study, we saw biomass responses to light were similar in damaged vs. inhibition of EFN in response to both grass competitors undamaged plants within each species. More comparable and the high levels of damage inflicted by aboveground to our study, Aranda et al. (2015) found that competition clipping, likely in response to the reduced biomass with pasture grasses reduced growth of seedlings of the available to produce new leaves and nectaries. invasive tree, Gleditsia triacanthos (honey locust), greater Interestingly, belowground damage did not induce EFN than defoliation alone, and that the negative impact of production (nor inhibit it), which intuitively makes sense defoliation was greater in the absence of competition. from a defense allocation perspective if EFN defenses These results, and our findings, better support the lim- target aboveground herbivores only. However, we have iting resource model (LRM) of plant tolerance of her- previously shown that belowground herbivory by larvae bivory (Wise and Abrahamsom 2005, 2007), as plants of the specialist flea beetle Bikasha( collaris, a potential were more likely limited by above- than belowground biocontrol agent for Triadica), does induce EFN, indi- resources in the no-competition environment, leading to cating there is some specificity to this defense response reduced tolerance to aboveground damage compared to (Huang et al. 2015). Intriguingly, B. collaris larvae the grass-competition environment where plants were co-occur with aboveground feeding adult beetles, so likely limited belowground. Competition with grass can belowground herbivory in that instance may be a reliable reduce both above- and belowground resources available indicator of aboveground herbivory and vice versa to trees but it is likely that belowground competition (Huang et al. 2012). reduced belowground resources more in our experiment Invasive (United States) populations were larger than that aboveground competition reduced aboveground native (China) populations (Fig. 1), providing additional resources, as has been demonstrated in other tree–grass support for the EICA hypothesis in invasive plants systems (e.g., Picon-Cochard et al. 2006). In the LRM, (Blossey and Nötzold 1995). Across all populations, tol- high levels of belowground resources lead to relative erance responses to grass competitors for plant biomass aboveground resource limitation for plants (Wise and was negatively correlated with tolerance responses to Abrahamsom 2005). Aboveground herbivory under this root clipping for plant biomass, but this was driven by a scenario would be more detrimental for plants than significant association within United States but not belowground herbivory and lead to reduced tolerance of China populations (Fig. 5). Additionally, invasive United 10 JULI CARRILLO AND EVAN SIEMANN Ecological Applications Vol. 0, No. 0

States populations tolerated the combined stress of identifying vulnerable plant parts, e.g., roots or shoots, belowground damage and competition with grass better to target and in the selection insect biocontrol agents than native China populations, indicating evolved differ- (Raghu and Dhileepan 2005). Although all tests to date ences in their responses to biotic interactions. Invasive of biocontrol agents for Triadica have focused on the populations appear to be more plastic in their response seedling and sapling stage (e.g., Huang et al. 2010, Wang to biotic stressors, as they invested more in roots in et al. 2012), the agents under consideration feed on all undamaged controls when in competition with grass than life stages of Triadica in its native range (E. Siemann, China plants, but less than China plants when roots were personal observation) and reproduction of trees may be clipped (Fig. 3). Others have shown that plant responses especially impacted by herbivory at later life stages (e.g., to the individual and simultaneous stress of competition Massad 2013). and herbivory can be phenotypically plastic and expe- Encouragingly, we found that competition with grass rience differential selection due to these stress combi- might be able to increase the impact of biocontrol agents nation (Boege 2010); our results show that genetic on invasive, woody Triadica, and directly suppressed variation exists in the degree of plasticity that may be biomass of Triadica. We assert that invasive species man- selected by biotic environment (e.g., native range vs. agement should integrate competitive dynamics of the introduced range). target species to better predict the efficacy of agents Tolerance in invasive plants may reduce the effec- within specific communities, and reduce potential non- tiveness of biocontrol agents (Wang et al. 2011). This is target effects and the release of potentially ineffective because for highly tolerant plant populations, biocontrol agents. For Triadica and other woody invasive plants, agents may be able to reach high population densities we recommend a combination of management but not reduce the biomass or reproductive fitness of approaches, including the restoration or addition of target plants. In our study, we found that tolerance was native competitors, particularly native grasses or forbs higher with competition, but competition directly that are relatively good belowground competitors and reduced plant biomass, both above and below ground, the introduction of a suitable biocontrol agent that and increased the relative amount of biomass Triadica targets the invasive plant’s area of competitive superi- allocated belowground (root : shoot; Table 1, Fig. 1A). It ority, e.g. aboveground resource capture. Alternately, for may be that competition with native grass or other a weedy or invasive grass or herbaceous species, including co-occurring plants may ameliorate the potential positive woody native species that are superior competitors for effects of increased tolerance for invasive plant popula- light and/or prioritizing belowground biocontrol agents tions. In another weedy system, de la Pena and Bonte in restoration efforts could prove successful. These guide- (2014) found that dandelion plants (Taraxacum offic- lines provide a contracted field of potential agents and inale) overcompensated with growth both above and plant competitors from which to choose from in initiating below ground after aboveground herbivory by a locust, or maintaining a management program which recognizes while belowground herbivory by root-knot nematodes the context dependency of competitive/herbivore interac- had no effect on plant growth. When the two herbivores tions (Schädler et al. 2007, Jing et al. 2015) and answers fed together, the production of plant trichomes was calls within the field to reduce “lottery” approaches to inhibited leading to greater leaf damage by locusts and agent introductions (Stephens et al. 2015). Woody plants combined herbivore feeding may have also inhibited the severely threaten grasslands and invade disproportion- compensatory growth response of plants. Antagonistic ately into disturbed and open habitats (e.g., Zalba and interactions among biocontrol agents have been exten- Villamil 2002); utilizing multiple top-down and bottom- sively reported elsewhere (Stephens et al. 2015); directed up approaches will be necessary to reduce their and other choice of agents that target valuable plant parts could invasive species’ ecological impacts. decrease indiscriminate release of multiple agents and potentially reduce antagonistic interaction among insect Acknowledgments herbivores used to control plants. Our study is limited by the fact that we simulated her- We thank Ian Kaplan for helpful comments and discussion. bivory on young tree seedlings instead of using natural J. Carrillo was supported by an NSF Postdoctoral Research Fellowship in Biology. This work was supported by US-NSF herbivores across a range of plant stages. Plants, including (DEB 0820560). 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Supporting Information Additional supporting information may be found in the online version of this article at http://onlinelibrary.wiley.com/ doi/10.1002/eap.1359/suppinfo

Data Availability Data associated with this paper have been deposited in Dryad: http://dx.doi.org/10.5061/dryad.j72c0