Biocontrol Science & Technology
For Peer Review Only
The lesser of two weevils: physiological responses of spotted knapweed ( Centaurea stoebe ) to above- and belowground herbivory by Larinus minutus and Cyphocleonus achates
Journal: Biocontrol Science & Technology
Manuscript ID: Draft
Manuscript Type: Research Article
Date Submitted by the n/a Author:
Complete List of Authors: Wooley, Stuart; California State University, Stanislaus, Biological Sciences Smith, Bonnie; California State University, Stanislaus, Biological Sciences King, Chad; California State University, Stanislaus, Biological Sciences Seastedt, Timothy; University of Colorado at Boulder, Ecology and Evolutionary Biology Knochel, David; University of Colorado at Boulder and Institute of Arctic and Alpine Research, Terrestrial Ecology
Plant physiology, Biological control, Centaurea stoebe L. ssp. Keywords: micranthos, Invasive plants, Larinus minutus, Cyphocleonus achates
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1 RESEARCH ARTICLE
2
3 The lesser of two weevils: physiological responses of spotted knapweed (Centaurea stoebe )
4 to above- and belowground herbivory by Larinus minutus and Cyphocleonus achates 5 For Peer Review Only 6 Authors: Stuart C. Wooley a, Bonnie Smith a, Chad King a, Timothy R. Seastedt b, David G.
7 Knochel b*
8
a 9 Department of Biological Sciences, California State University, Stanislaus, 1 University Circle,
10 Turlock, CA 95382
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b 12 Department of Ecology and Evolutionary Biology and Institute of Arctic and Alpine Research,
13 University of Colorado, Campus Box 450, Boulder, CO, 80309-0450, USA
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16 *Corresponding author: E-mail: [email protected]
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23 Abstract
24
25 The physiological responses of plants to variable levels of root and shoot herbivory in the field
26 may yield valuable insights regarding potential compensation or tolerance responses for 27 herbivory. In anFor infestation Peer of Centaurea Reviewstoebe (spotted knapweed) Only located in the Colorado 28 foothills, we measured physiology, biomass, and flower production of individual plants
29 undergoing isolated or simultaneous herbivory by the above- and belowground biological control
30 insects, Larinus minutus and Cyphocleonus achates . Over the growing season, net C assimilation
31 rate, transpiration, stomatal conductance, and Ci all decreased, while water use efficiency
32 increased. The reductions in plant physiology were found to be correlated to late-season plant
33 performance measures of aboveground biomass and flower production. The decrease in these
34 physiological traits was due to an increase in the intensity of Larinus damage over time; effects
35 of C. achates root damage to plant physiology, including transpiration were only marginally
36 significant. The effects of these two species on plant physiology were not interactive, and
37 Larinus minutus was found to exert larger negative effects on this invasive plant in terms of plant
38 physiology and potential reproductive output than C. achates . While previous studies have
39 shown C. achates to have significant negative effects on population densities of spotted
40 knapweed, the addition of Larinus minutus to the suite of insects used in biological control of
41 spotted knapweed should facilitate continued or enhanced reduction in densities of this noxious
42 weed.
43
44 Keywords : Plant physiology; Biological control; Centaurea stoebe L. ssp. micranthos ; Invasive
45 plants; Larinus minutus ; Cyphocleonus achates
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46 1. Introduction
47 Among the many dynamic interactions between plants, herbivores, and their external abiotic
48 conditions, plants employ multiple mechanisms to deal with herbivore damage, including altered
49 photosynthetic activity (Welter, 1989; Delaney, 2008), compensatory growth (Paige and 50 Whitham, 1987;For Trumble etPeer al., 1993), phenological Review escape (Coley Only and Barone, 1996; Saltz and 51 Ward, 2000), or resistance through chemical defense (Baldwin and Ohnmeiss, 1994; Stamp,
52 2003). These mechanisms of response to herbivory deserve further attention (Tiffin, 2000). The
53 direction and magnitude of responses to herbivory, and how those responses may influence plant
54 fitness, are also highly complex and site specific (Maschinski and Whitham, 1989; Trumble et
55 al., 1993; Stamp, 2003). The generalized plant responses to herbivory elucidated from short-term
56 experiments sometimes involve artificially imposed damage, or low to moderate levels of
57 herbivore damage inflicted in a single bout; however, in natural systems plants may undergo
58 chronic damage over a broader range of intensities to both root and foliar tissues by multiple
59 herbivores. In particular, introduced biological control insects released in an attempt to control
60 invasive plants may have large impacts since these species may lack predators and reach high
61 densities (e.g. Hunt-Joshi et al., 2004). Such is the case with spotted knapweed ( Centaurea
62 stoebe L. subsp. micranthos [Gugler] Hayek [Asteraceae]), also identified as C. maculosa and C.
63 biebersteinii (see Ochsmann 2001, Hufbauer and Sforza 2008), a plant that infests over two
64 million ha of range and pasture lands in North America, and causes a wide-range of economic
65 and ecological problems (Lacey, 1989; DiTomaso, 2000; Smith, 2008). The species is a short-
66 lived perennial that produces multiple flowering stems from June through the early fall, and that
67 is capable of producing tens of thousands of seeds per square meter at high densities and in the
68 absence of biological control insects (Sheley et al. 1998).
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69
70 Extensive research has been attempted to understand the biotic and abiotic factors related to C.
71 stoebe 's ability to invade, compete with, and attain dominance over other herbaceous vegetation
72 (e.g. Pokorny, 2005; Hill et al., 2006; Maron and Marler, 2008). A substantial number of reports 73 exist that suggestFor that biological Peer control insectsReview may directly or indireOnlyctly contribute to this 74 dominance (e.g., Pearson and Callaway, 2008), or contrarily, to its control and demise as a
75 dominant (Story et al., 2008; Knochel and Seastedt, 2010). Biological control studies provide
76 evidence that through tissue damage and reduction of reproductive potential, the root feeder,
77 Cyphocleonus achates Fahr. (Coleoptera: Curculionidae), flower head weevil Larinus minutus
78 Gyllenhal (Coleoptera: Curculionidae), and the gall fly species Urophora affinis Fairfield
79 (Diptera:Tephrididae), together have the ability to reduce populations of C. stoebe (Story et al.,
80 2006; 2008). Some evidence indicates that drought may also interact with root and foliage
81 herbivory to impact C. stoebe (Corn et al., 2006; Pearson and Fletcher, 2008), and that damage
82 by the root-feeding weevil, C. achates may contribute to population control by reducing adult
83 plant longevity and in some cases causing mortality (Cortilet and Northrop, 2006; Jacobs et al.,
84 2006; Michels et al., 2009). Cyphocleonus achates , a univoltine root weevil introduced to North
85 America in the late 1980’s, feeds on C. stoebe aboveground tissues from June-Sept as an adult
86 and females oviposit during the late summer and fall near the taproot crown. There, larvae
87 typically overwinter at first instar and continue development and the consumption of taproot
88 tissue the following spring, with emergence of adults in June and July (Corn et al., 2009).
89
90 The univoltine flower head weevil, Larinus minutus , overwinters as an adult in the soil and
91 emerges in late May-June to feed on stems, rosettes and cauline foliage. Females oviposit into
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92 open flowers, and larvae develop within the maturing flower head, where they consume
93 reproductive tissues and thereby greatly reduce seed production. Larinus minutus in the larval
94 stage are the primary impact to flowers—they feed within the maturing flower heads on achenes.
95 Larinus minutus effects on seed production were not considered in this experiment but are 96 reported elsewhereFor (Knochel Peer and Seastedt Review 2010), because our focus Only here was to determine the 97 relatively undocumented effects L. minutus in the adult life stage on other plant tissues. Adults
98 feed primarily on stem and foliar tissues, and although they do consume a small portion of inner
99 flower petals when preparing a location for oviposition within the top of the flower head, this
100 damage does not eliminate flower heads. In addition to these effects of larval seed predation
101 (Story et al., 2008; Knochel et al., 2010a), this species has recently been shown to reduce
102 flowering and aboveground biomass of C. stoebe through tissue damage, at least in Colorado
103 (Seastedt et al., 2007; Knochel et al., 2010b; Knochel and Seastedt, 2010). However, little is
104 known about how damage by L. minutus and C. achates to non-reproductive root and
105 aboveground tissues influences plant physiology and performance in the field.
106
107 This is important for several reasons. First, as noted above, studies have shown both positive and
108 negative effects of biological control agents on indices of plant fitness. Examining how a range
109 of damage intensity by L. minutus and C. achates affects C. stoebe physiology could improve our
110 understanding of plant compensation responses to introduced herbivores. Second, knowledge of
111 the effects and interactions of both insect species on individual plant physiology and
112 performance could improve our understanding of their potential role in controlling the plant’s
113 population-level dynamics. Third, measuring the physiological traits of C. stoebe plants in the
114 field experiencing a range of damage from multiple herbivores provides a first assessment of the
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115 effects of stem and foliar herbivory by L. minutus , and the potential interaction with C. achates .
116 Finally, this study may provide a mechanistic link between herbivory and plant performance.
117
118 We directly measured how isolated and simultaneous above- and belowground herbivory by 119 Larinus minutusFor and Cyphocleonus Peer achates Review, respectively, influences Only spotted knapweed 120 physiology and performance in a field infestation on the Colorado Front Range. We asked three
121 questions: 1) What is the relationship between spotted knapweed physiology and plant
122 performance outcomes in terms of biomass and potential reproductive output? 2) How does a
123 gradient in the intensity of herbivory by L. minutus and C. achates affect plant physiology and
124 performance? 3) Are the effects of C. achates root herbivory measurably superior to
125 aboveground foliar herbivory by Larinus minutus in terms of its effect on growth and potential
126 reproduction and is there and interaction between the two species?
127
128 2. Methods
129 2.1. Field site description
130 Field research occurred during the summer of 2009 approximately 15 km northwest of the city of
131 Boulder, CO (40°07’13.86’’N 105°19’26.34’’W), between 1865 and 2070 m elevation, in a
132 meadow surrounded by rugged 15 to 60 percent slopes and dominated by ponderosa pine ( Pinus
133 ponderosa Douglas Ex. Lawson). Soils at the site are part of the Fern Cliff-Allens Park-Rock
134 outcrop complex, and are composed of mixed loamy alluvium parent material, with a stony
135 sandy clay loam profile at 0 to 100 cm depth (NRCS, 2008). Mean annual air temperature is 6 to
136 8 degrees °C, with a frost-free period of 80 to 120 days. Precipitation during the course of this
137 study measured 104% of the 30 year average for June and July (NOAA, 2009). The study area
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138 has sustained moderate cattle grazing since the mid 1900’s, however cattle were not present on
139 the property during this study. Centaurea stoebe was accidentally introduced to this area in the
140 late 1980's, and since then has spread to over 40 ha of meadows, riparian areas, and forests. Also
141 present are other non-native species including dalmatian toadflax, Linaria dalmatica (L.) Mill., 142 sulfur cinquefoil,For Potentilla Peer recta L., and Reviewthe invasive cheatgrass, Only Bromus tectorum . Native 143 mixed grass prairie species also common in the area include sand dropseed, Sporobolus
144 cryptandrus (Torr.) A. Gray, blue grama, Bouteloua gracilis (HBK) Lag. ex Steud., and wild
145 bergamot, Monarda fistulosa L. Since 2001 we have observed and monitored specialist
146 biological control insects at the site (Knochel and Seastedt 2009). These include two gall flies of
147 the Urophora genus, and the seed head weevil Larinus minutus, which likely colonized the site
148 from nearby populations of Centaurea diffusa . These insects were supplemented during 2001-
149 2005 with releases of three thousand Larinus minutus weevils, and two thousand C. achates root
150 weevils.
151
152 2.2. Experimental design
153 The experimental meadow was at the core of a spotted knapweed infestation, located near the
154 stream channel of a steep gulch. Spotted knapweed stem densities in this area reach 50 stems/
155 m2. Four 50 m long transects were placed across this meadow and oriented east-west and parallel
156 to an ephemeral stream that ceased running in mid-July, and 48 spotted knapweed plants were
157 selected and tagged along the transects at 1m intervals. Target individuals were chosen that were
158 identifiable as a single plant with one taproot, and having dead stems from the previous year
159 (generally > 2 years old), that had initiated upright stem growth (bolted) from the rosette stage in
160 the spring of 2009. Thus, most plants were likely between 2-4 years of age.
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161
162 2.3. Insect census and damage estimates
163 All insect censuses and physiological measurements were taken for each target plant on June 26,
164 July 7 and July 20, 2009. On each date, the number of Larinus minutus adults present on each 165 plant was recorded.For Weevil Peer damage to stem Review and foliage tissues wasOnly visually estimated 166 independently by three people, with each person assigning a number describing the intensity of
167 defoliation between 0 (no damage) and 5 (removal of entire leaves and with the majority of each
168 stem length having tissues scraped-away). The average of the three independent estimates was
169 used as the damage value testing the relationship between damage and physiological traits. The
170 presence of C. achates and damage to the taproot was estimated when whole plants were
171 harvested upon termination of the experiment on July 20, 2009. At this time, the taproots were
172 carefully sectioned to count the number of larvae, pupae, or adult C. achates root weevils
173 present. Damage by C. achates was scored by the presence and number of individuals per root.
174 The total mass of C. achates was also measured, and was entered into the preliminary stepwise
175 regressions but not found to be a powerful explanatory variable. In previously published
176 research at this field site, we have found that seasonal insect attack by these two species is highly
177 variable, with plants sustaining variable seed head attack by L. minutus , as well as about 20% of
178 plants on average not undergoing attack by C. achates root weevils. Thus, although we did not
179 cage or manipulate insect attack or insect densities from naturally occurring levels at this field
180 site, our experimental design inherently allowed comparison of a wide range of attack by both
181 species: from very low L. minutus damage to heavier levels of damage, and of plants whose roots
182 had no C. achates weevil attack to roots with high densities.
183
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184 2.4. Photosynthetic gas exchange
185 Gas exchange measurements on spotted knapweed were taken using a portable closed-flow gas
186 exchange system (LI-6400 XTi. LI-COR, Inc., Lincoln, NE) with CO 2 controller set at 380 ppm
187 fitted with a standard 6-cm 2 leaf chamber and red-blue light source set at 1500 µmol·m -2·sec -1. 188 All measurementsFor were taken Peer between 1000 Review and 1400 hours under Only clear skies. Leaves were 189 chosen for measurement that best represented the majority of leaves on the plant or when few
190 leaves were present, the most suitable leaf for measurement was used. As such, on highly
191 damaged plants leaves with herbivore damage were measured because no leaves were present
192 without damage. However, every effort was made to select intact leaves for measurements.
193 Because knapweed leaf margins dissected and will not occupy completely the area of the
194 chamber, after taking each measurement, we calculated total leaf area by covering a leaf with a
195 clear acetate sheet with a 0.25cm 2 grid. The number of complete squares contained within the
196 area of the leaf that had been enclosed in the leaf chamber was recorded, with partial squares
197 considered 0.125cm 2. All the physiological variables dependent on leaf area were recalculated
198 using the leaf area determined with the grid. Carbon assimilation rate (A), transpiration rate (E),
199 stomatal conductance ( gs),and intercellular leaf [CO 2] ( Ci) were determined. Water use efficiency
200 (WUE) was calculated as A/E (µmol CO 2/mmol H 2O). By the end of the experiment, four plants
201 had either senesced, lost too much leaf tissue to herbivores for adequate physiological
202 measurement, or died and so were excluded from the statistical analyses.
203
204 2.5. Plant trait measurements
205 Centaurea stoebe plant trait measurements were taken on July 20, 2009 at the end of the
206 experiment. The number of flowers produced by each plant was counted, and plant stems were
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207 clipped at ground level and collected. Both coarse and fine roots were excavated, harvested and
208 transported to the lab. Some loss of fine roots occurred due to the difficulty of separating these
209 tissues from the soil, however, root harvest was equivalent across all experimental plants. Plant
210 tissues were dried at 60°C to a constant weight and weighed. Specific leaf area (SLA) was 211 determined by Formeasuring thePeer fresh leaf area Review (cm 2) divided by the Only dry leaf mass (mg). In most 212 cases, leaves used to calculate SLA were the same leaves used previously for photosynthesis
213 measurements. To determine C:N content, the leaf samples were ground in a Wiley mill (40
214 mesh screen), and 3-5 mg per plant were analyzed by combustion on a Carlo Erba Flash EA1110
215 CN analyzer (CE Elantech, Lakewood, New Jersey, USA) calibrated with an Atropine standard.
216
217 2.6. Statistical analysis
218 We used two different procedures in the data analysis. First, we broadly analyzed the data by
219 using an automated stepwise regression procedure that included all response and predictor
220 variables and combined both forward addition and backward elimination. We then used a manual
221 method with a more narrow set of variables based on the significance of initial results and the
222 factors that we considered most important for our biological system. Using this process, the
223 stepwise regression gave us a broad indication of the relationships between the various plant
224 physiological measurements or growth traits (e.g. biomass, flowers) and measured herbivore
225 traits (e.g., damage estimates and abundance of L. minutus per plant, number or mass of C.
226 achates per plant root). The effects of the two species of insects were considered together, and
227 then separately. When L. minutus and C. achates were considered together, the effects of L.
228 minutus often outweighed those of the root weevil, C. achates . For L. minutus , variables other
229 than the estimates of their damage intensity, or the density of C. achates , were not included .
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230 When the effects of C. achates were separately analyzed, the dry mass of root tissue was
231 included in the model to test effects of this insect while holding root mass constant (the insect
232 induces some enlargement of the root tissue when forming galls in the taproot, and, the insect
233 tends to be in higher abundance in larger plants), and to see whether the presence of C. achates 234 significantly reducesFor additional Peer unexplained Review error. The best model Only was the one with the smallest 235 AIC (Aikaike’s information criterion) which indicates a better fit of the model. Next, using a
236 subset of response variables that would minimize multicollinearity (Zar, 1999) in the regression
237 model and most effectively explain overall relationships between plant physiology and insect
238 attack, we created several models manually by entering predictor variables in the order we
239 thought they affected the response variables and also in the order that would control for the
240 variance used by the previously included variable. Once the models were determined, we report
241 the results of individual regressions and provide R 2, P and F-values. When graphing the results,
242 curvilinear lines were presented when these models provided a significantly better fit to the data
243 points. All statistical analyses were performed using SAS (SAS Inc, Cary NC). For comparisons
244 of physiology across time, we used repeated measures ANOVA, with date, as the main effect and
245 plant (the replicate) included as a random term, thereby providing the appropriate degrees of
246 freedom for the main effect of date (Gotelli and Ellison, 2004). All plant trait (biomass, flower
247 production) response data were collected only at the termination of the experiment, thus we did
248 not analyze L. minutus or C. achates effects for previous dates. For plant physiology, taken on
249 three separate dates, we first analyzed Larinus minutus damage across the three dates. As we
250 report, the effects of L. minutus on physiology intensified—the R2 value increased as the season
251 progressed, reflecting the increase in cumulative damage sustained by a majority of the
252 individual target plants. Our primary results then report the relationship between L. minutus
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253 damage and plant physiology measurements from the last date. This statistical method was used
254 because it allowed us to account for both L. minutus and C. achates on the last date, and we did
255 not have multiple measurements of C. achates over the season, but rather just one measurement
256 when upon excavation of the roots. 257 For Peer Review Only 258 3. Results
259 3.1. Physiological responses to herbivory
260 On the last sampling date in July, estimates of cumulative damage to stems and foliage by
261 Larinus minutus ranged from 0.5 (10 plants had very low damage with scores of 1.5 or less) to 5
262 (scores of 4 or 5 indicated large amounts of tissue removed), with an average damage estimate of
263 2.8 ± 0.2. Cyphocleonus achates densities ranged from zero to nine individuals per taproot, with
264 an average of 2.6 ± 0.4 (SE), and with 15 plants that were undamaged and contained no root
265 weevils. The majority of plants were damaged by at least one of the two species; however 7 of
266 the plants undamaged by C. achates also had very low cumulative damage by L. minutus.
267 Photosynthesis, transpiration, stomatal conductance, and C i all decreased 45-80% over time,
268 while WUE increased nearly 40% over the same time period (Table 1; Fig. 1). The decrease in
269 the same physiological traits was associated with leaf and stem herbivory by L. minutus (Fig. 2).
270 Carbon assimilation, stomatal conductance and transpiration all increased at intermediate levels
271 of L. minutus herbivory, then decreased as herbivore damage increased (Fig. 2). Transpiration
272 decreased in response to aboveground damage, but surprisingly, C. achates damage to roots
273 (estimated from C. achates census data) did not influence transpiration (Table 1), even as
274 precipitation decreased and temperatures increased.
275
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276 3.2. Plant performance measures and herbivore damage
277 At the time of plant harvest in July, before the emergence of C. achates weevils from the roots,
278 C. stoebe was in full flower but flower heads had not set seed. Stem and leaf damage increased
279 by 40% over time (Fig. 1), even though the total number of Larinus minutus abundance on 280 individual plantsFor did not increasePeer during theReview experiment (P = 0.13). Only Aboveground biomass 281 decreased as Larinus damage increased (Fig. 3). For each unit increase in estimated Larinus
282 canopy damage, flower production decreased by 8.3 flowers. Finally, Larinus damage increased
283 as the foliar C:N ratio increased.
284 Flower production and aboveground biomass were negatively influenced by C. achates weevil
285 density, controlling for root biomass as an additional parameter in the model (Table 1; Fig. 3).
286 Root biomass was positively correlated with C. achates root weevil numbers (Table 1), and was
287 negatively correlated with aboveground damage by Larinus (Table 1), suggesting that
288 aboveground foliar feeding decreased allocation of resources to roots (Fig. 3). However,
289 root:shoot ratios did not change in response to Larinus damage (F 1,42 = 2.14; P = 0.15), but did
290 increase as more C. achates adults were present (F 1, 41 = 9.92; P =0.003) in the roots. The SLA
2 291 decreased as the total mass of C. achates per root increased (F 1, 42 = 4.55 P = 0.039, R =0.10), but
292 was not correlated with C. achates density. The SLA decreased as Larinus damage increased (p
293 = 0.058), though not as a result of the interaction between both weevils (p = 0.55). Indeed, we
294 found no interaction between Larinus weevil damage and C. achates root weevil density on any
295 physiological or plant trait measurement (Table 1).
296
297 4. Discussion
298 The results from a number of studies suggest that Larinus and/or the Urophora gall flies can
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299 facilitate control of C. stoebe populations through direct reductions in seed production (Seastedt
300 et al., 2007; Story et al., 2008). Here, we report that in addition to its known effects as a seed
301 predator, Larinus may also substantially influence knapweed populations through the effects of
302 foliar herbivory that reduce aboveground biomass and flower production (Fig. 3). Damage from 303 Larinus was negativelyFor correlated Peer with the Review size of aboveground plantOnly tissues (i.e. smaller plants 304 had more damage); however, the numbers of Larinus weevils observed per plant was not
305 reflective of plant size. Thus, it seems that for a similar density of weevils, smaller plants either
306 sustained more tissue damage than larger ones, or the damage itself reduced the plant size, or
307 both. Heavily damaged plants had a considerable amount of stem and foliar tissues removed,
308 thus decreasing their aboveground biomass, and these plants also appeared to senesce earlier,
309 likely leading to reductions in overall reproductive output.
310 Larinus minutus fed chronically on individual plants over the experimental period, and thus our
311 observations of herbivory escalating over time reflect cumulative damage rather than an increase
312 in feeding (Fig. 1). For the physiological parameters we measured, increased damage by Larinus
313 was associated with decreases in plant photosynthetic rate, stomatal conductance, and
314 transpiration (Fig. 2), but not root:shoot ratio. The trends in the plant response to aboveground
315 herbivory suggest that at low to moderate aboveground herbivory, spotted knapweed plants
316 actually compensate (Welter, 1989; Meyer, 1998) for damage by increasing rates of
317 physiological activity. Higher than average precipitation in our study and better water relations
318 might also have driven the early apparent compensation for herbivory. In Amaranthus hybridus ,
319 photosynthesis increased in response to herbivory only when water was highly available
320 (Gassman, 2004). In our system, photosynthesis increased in response to herbivory at the same
321 time water was more available. As water availability declined over the season and herbivory
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322 increased, spotted knapweed’s ability to compensate declined and physiological responses were
323 depressed, including photosynthetic rates. Hill and Germino (2005) also showed that C. stoebe
324 photosynthetic rates decreased as water stress increased, and found that stem epidermal tissues
325 were critical for persistence of photosynthesis into the late summer, after the senescence of 326 rosette and caulineFor foliage. Peer We found similar Review results, with all knapweed Only physiological response 327 declining over the course of the season, suggesting that as water availability was reduced, plant
328 physiological activity also declined. Water-stress induced reductions in photosynthetic rates and
329 other physiological traits could explain why L. minutus damage to stem tissues resulted in the
330 substantial reductions of growth and flower production reported here. The declines in all plant
331 physiological activity over time (Fig 1) all indicate some kind of stress leading to a reduction in
332 growth and potentially in the ability of plants to compensate for herbivory. These results support
333 the idea of a compensatory continuum or optimization model of herbivory (McNaughton, 1979;
334 Maschinski and Whitham, 1989) that may interact with climatic factors. In other words, the
335 plant’s ability to tolerate herbivory declined in response to both increased damage from multiple
336 herbivore species as well as climatic conditions that may have led to greater water stress (Hill et
337 al., 2006).
338 Cumulative damage over the experimental period by Larinus was most severe on plants with a
339 higher foliar C:N ratio (Fig. 3). Thus, to meet its N needs Larinus appears to have increased
340 tissue consumption when tissue N was low. This increased herbivory may have occurred instead
341 of (or perhaps in addition to) Larinus also preferentially feeding on spotted knapweed plants
342 possessing tissues of higher nutritional quality (White, 1988). These results are similar to the
343 feeding patterns of another biological control root weevil reported by Van Hezewijk et al.
344 (2008), and suggest that in areas where soil resources are high, Larinus damage may be
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345 somewhat reduced. Not only would L. minutus damage be reduced in areas where soil resources
346 are higher, but also knapweed tolerance to herbivory would likely be higher (Cronin et al., 2010).
347 For smaller knapweed plants, L. minutus damage appeared to shorten their growing season (they
348 senesced earlier) thereby reducing their reproductive output. In contrast, for larger plants that 349 may better tolerateFor adult L.Peer minutus foliar Review damage, the overall impact Only of L. minutus to plant 350 fitness might be greater in terms of larval consumption of seeds.
351
352 Damage by the root weevil C. achates has also contributed to some of the reported population
353 declines of C. stoebe in North America via effects that reduce aboveground biomass and flower
354 production (Knochel and Seastedt, 2010). In contrast to the substantial impacts we found for
355 Larinus on plant physiology and performance, the impacts of C. achates on physiology were
356 largely insignificant. However, the root weevil did have some negative effects on flower
357 numbers and plant aboveground biomass (Fig. 3). Cyphocleonus achates presence decreased
358 flower production and aboveground biomass when controlling for the positive correlation of
359 weevils with root biomass, an effect primarily due to the accumulation of additional tissue that
360 forms around the damaged areas of the taproots (Fig. 3). In addition, plants typically endure
361 damage to the taproot over multiple years, thus the root weevil may also reduce the longevity of
362 plants and thereby influence lifetime reproductive output, an outcome we did not measure here.
363 Cyphocleonus achates has been shown to reduce C. stoebe dominance and has been reported to
364 significantly contribute to reductions in densities and abundance (e.g. Jacobs et al., 2006; Corn et
365 al., 2006). Thus, we hypothesized that heavy damage by this species to the plants' taproot would
366 have negative impacts on physiology, in particular transpiration and water use efficiency (WUE)
367 and provide a mechanistic explanation for some of its observed impacts the plants dominance or
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368 population densities. However, we detected no effects of belowground C. achates damage on C.
369 stoebe physiology, including its water use efficiency, a physiological parameter that Hill et al.
370 (2006) showed was advantageous to this invasive weed compared with native vegetation,
371 especially under dryer conditions. 372 For Peer Review Only 373 Thus, we speculate that C. achates damage to the root reduces spotted knapweed population
374 densities during dry years, but the plant exhibits some capabilities of increased persistence in wet
375 years. In other words, knapweed is tolerant of C. achates root damage when water is more
376 available. Our comparisons of spotted knapweed seed production in dry upland sites versus
377 wetter sites indicate that the plant appears to be able to avoid some of the negative effects of
378 biological control insects in areas where soil moisture is higher (unpublished results).
379 Interestingly, the majority of roots dissected for the presence of C. achates also contained a
380 fungus that had colonized the damaged taproot. The direct effects of this root fungus on plant
381 performance or its interactions with the root weevil, if any, remain unknown.
382 In some systems, simultaneous herbivory by multiple species on the root and shoot tissues of the
383 same plants results in interactions (Masters and Brown, 1992; James et al., 1992; Maron, 1998;
384 Masters et al., 2001; Bezemer et al., 2003; Soler et al., 2007), but not in others (Hunt-Joshi et al.,
385 2004; for a review see Denno et al.,1995; Blossey and Hunt-Joshi, 2003). Here, we found that
386 the abundance and damage inflicted by the aboveground feeder L. minutus were independent of
387 the infestation levels and damage to roots of the same plants by C. achates , and vice-versa .
388 Furthermore, the effects of C. achates and L. minutus on plant physiological response and plant
389 performance were not interactive. Although root feeding is known to influence aboveground
390 tissue quality (Kaplan et al., 2008), and potentially the feeding rates or amount of tissue
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391 consumed by foliar insects, we found no relationship between C. achates densities and foliar
392 C:N. In some plants, leaf N increases in response to herbivory (Gassman, 2004), but in our study
393 C:N ratios actually increased with Larinus herbivory, again suggesting that knapweed plants
394 were not able to tolerate or compensate for herbivory, especially as the season became 395 progressively drier.For Our resultsPeer here indicate Review a potentially larger Only and more important role of L. 396 minutus in the control of spotted knapweed than previously reported (Story et al. 2008; Knochel
397 and Seastedt, 2010b). This is perhaps due to inter-annual variability in Larinus densities, or even
398 due to the decline in absolute knapweed abundance at the study site. Regardless, this species
399 needs to be more thoroughly tested throughout spotted knapweed's range in North America.
400
401 5. Conclusions
402 Overall, damage to roots by C. achates had measurably smaller effects on spotted knapweed than
403 did the quite extensive effects on physiology and performance related to canopy damage by L.
404 minutus. Our results partially explain why L. minutus was dubbed a ‘silver bullet’ biological
405 control agent for diffuse knapweed (Myers, 2004; Myers et al., 2009). We provide a
406 physiological mechanism to explain the effects of Larinus on spotted knapweed, and we contend
407 that this insect species, when combined with plant competition and the negative effects of C.
408 achates reported here and elsewhere, may together provide a collective ‘silver bullet’ for
409 reductions of spotted knapweed populations in North America.
410
411 Acknowledgements
412 We thank Linda and Sergio Sanabria for use of their land to conduct research, as well as
413 laboratory assistance from Christopher Washenberger. Research was funded by the USDA
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414 Faculty and Student Team (FAST) program (grant number TEXE-2008-02095) under the
415 National Research Initiative of the Cooperative State Research, Education, and Extension
416 Service, and USDA NRI CSREES program (grant number 2009-55320-05072).
417 418 For Peer Review Only 419
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420 References
421
422 Baldwin, I.T., Ohnmeiss, T.E., 1994. Coordination of photosynthetic and alkaloidal responses to
423 damage in uninducible and inducible Nicotiana sylvestris . Ecology 75(4):1003-1014. 424 For Peer Review Only 425 Blossey, B. and Hunt-Joshi, T.R. 2003. Belowground herbivory by insects: influence on plants
426 and aboveground herbivores. Annual Review of Entomology 48: 521-47.
427
428 Coley, P.D. and Barone, J.A. 1996. Herbivory and plant defenses in tropical forests. Annual
429 Review of Ecology and Systematics 27:305-335.
430
431 Corn, J.G., Story, J.M., White, L.J., 2006. Impacts of the biological control agent C achates on
432 spotted knapweed, Centaurea maculosa , in experimental plots. Biological Control 37, 75–81.
433
434 Corn, J.G., Story, J.M., White, L.J., 2009. Comparison of larval development and overwintering
435 stages of the spotted knapweed biological control agents Agapeta zoegana
436 (Lepidoptera: Tortricidae) and Cyphocleonus achates (Coleoptera: Curculionidae) in Montana
437 versus Eastern Europe. Environmental Entomology 38:971 –976.
438
439 Cortilet, A.B., Northrop, N. 2006. Biological control of European buckthorn and spotted
440 knapweed. Minn Dept Agri Final Program Report, St Paul, MN
441 http://www.mda.state.mn.us/news/publications/pestsplants/weedcontrol/knapweedlcmrfinalrepor
442 t.pdf. Accessed 27 May 2008.
URL: http://mc.manuscriptcentral.com/cbst Page 21 of 32 Biocontrol Science & Technology
443
444 Cronin, J.P., Tonsor, S.J., Carson, W.P. 2010. A simultaneous test of trophic interaction models:
445 which vegetation characteristic explains herbivore control over plant community mass? Ecology
446 Letters 13:202-212. 447 For Peer Review Only 448 Delaney, K.J. 2008. Injured and uninjured leaf photosynthetic responses after mechanical injury
449 on Nerium oleander leaves, and Danaus plexippus herbivory on Asclepias curassavica leaves.
450 Plant Ecology 199:187-200.
451
452 DiTomaso, J.M. 2000. Invasive weeds in rangelands: species, impacts, and management. Weed
453 Science 48(2): 255-265.
454
455 Gassmann, A.J. 2004. Effect of photosynthetic efficiency and water availability on tolerance of
456 leaf removal in Amaranthus hybridus . Journal of Ecology 92:882-892.
457
458 Gotelli, N.J. and Ellison, A.M. 2004. A Primer of Ecological Statistics. Sinauer Associates, Inc.
459 Sunderland, MA.
460
461 Hill, J.P., Germino, M.J. 2005. Coordinated variation in ecophysiological properties among life
462 stages and tissue types in an invasive perennial forb of semiarid shrub steppe. Canadian Journal
463 of Botany 83(11): 1488-1495.
464
465 Hill J.P., Germino, M.J., Wraith, J.M. Olsen, B.E., Swan, M.B. 2006. Advantages in water
URL: http://mc.manuscriptcentral.com/cbst Biocontrol Science & Technology Page 22 of 32
466 relations contribute to greater photosynthesis in Centaurea maculosa compared with established
467 grasses. International Journal of Plant Science 167(2): 269-277
468
469 Hunt-Joshi, TR, Blossey, B, Root RB 2004. Root and leaf herbivory on Lythrum salicaria : 470 implications forFor plant performance Peer and communities. Review Ecological OnlyApplications 14(5): 1574-1589. 471
472 Jacobs, J.S., Sing, S.E., Martin, J.M. 2006. Influence of herbivory and competition on invasive
473 weed fitness: observed effects of Cyphocleonus achates (Coleoptera : Curculionidae) and grass-
474 seeding treatments on spotted knapweed performance. Environmental Entomology 35: 1590-
475 1596
476
477 Kaplan, I., Halitschke, R., Kessler A, Sardanelli S, Denno RF. 2008. Constitutive and induced
478 defenses to herbivory in above- and belowground plant tissues. Ecology 89(2):392-406.
479
480 Knochel, D.G. and Seastedt, T.R. 2009. Sustainable control of spotted knapweed, ( Centaurea
481 stoebe ). In: Inderjit (ed.), Management of Invasive Weeds, Springer Science + Business Media
482 B.V. pp. 211-225
483
484 Knochel, D.G. and Seastedt, T.R. 2010. Reconciling contradictory findings of herbivore impacts
485 on the growth and reproduction of spotted knapweed (C entaurea stoebe ). Ecological
486 Applications 20:1903-1927
487
488 Knochel DG, Flagg C, Seastedt TR 2010a Effects of plant competition, seed predation, and
URL: http://mc.manuscriptcentral.com/cbst Page 23 of 32 Biocontrol Science & Technology
489 nutrient limitation on seedling survivorship of spotted knapweed ( Centaurea stoebe ). Biological
490 Invasions, in press
491
492 Knochel, D.G., Monson, N.D., Seastedt T.R. 2010b. Additive effects of above- and belowground 493 herbivores on theFor dominance Peer of spotted knapweedReview ( Centaurea stoebe Only). Oecologia, in press 494
495 Lacey, J.R. 1989. Influence of spotted knapweed ( Centaurea maculosa ) on surface runoff and
496 sediment yield. Weed Technology 3:627-631.
497
498 Maron, J.L. 1998. Insect herbivory above- and belowground: individual and joint effects on plant
499 fitness. Ecology 79(4): 1281-1293.
500
501 Maron, J.L., Marler, M. 2008. Field-based competitive impacts between invaders and natives at
502 varying resource supply. Journal of Ecology 96:1187-1197.
503
504 Maschinski, J., Whitham, T.G. 1989. The continuum of plant responses to herbivory: the
505 influence of plant association, nutrient availability, and timing. The American Naturalist 134(1):
506 1-19.
507
508 Masters, G.J., Brown, V.K. 1992. Plant-mediated interactions between 2 spatially separated
509 insects. Functional Ecology 6(2): 175-179.
510
511 McNaughton, S.J. 1979. Grazing as an optimization process: grass ungulate relationships in the
URL: http://mc.manuscriptcentral.com/cbst Biocontrol Science & Technology Page 24 of 32
512 Serengeti. American Naturalist 113(5): 691-703
513
514 Meyer, G.A. 1998. Pattern of defoliation and its effect on photosynthesis and growth of
515 goldenrod. Functional Ecology 12:270-279. 516 For Peer Review Only 517 Michels, G.J. Jr, Carney, V.A., Jurovich, D., Kassymzhanova-Mirik, S., Jones, E., Barfot, K.,
518 Bible, J., Mirik, M., Best, S., Bustos, E., Karl, B., Jimenez, D. 2009. Biological control of
519 noxious weeds on federal installations in Colorado and Wyoming. Texas Agric. Experiment
520 Station 2009 Annual Report. URL:
521 http://amarillo.tamu.edu/programs/agrilife_programs/entomology_research/
522 entomology_research_colorado_weed_biocontrol.php Accessed 11 October 2009
523
524 Myers, J.H. 2004. A silver bullet in the biological control of diffuse knapweed. ESA 2004
525 Annual meeting abstract. URL: http://abstractscoallenpresscom/pweb/esa2004/
526 document/36421 Accessed 27 May 27, 2008
527
528 Myers, J.H., Jackson, C., Quinn, H., White, S., Cory, J.S. 2009. Successful biological control of
529 diffuse knapweed, Centaurea diffusa , in British Columbia, Canada. Biological Control 50:66-72.
530
531 NOAA 2009. National Oceanic and Atmospheric Administration Climate Diagnostics Center.
532 URL: http://www.cdc.noaa.gov/Boulder/Boulder.mm.precip.html (accessed 05/01/09).
533
534 NRCS 2008. National Resources Conservation Service, United States Department of
URL: http://mc.manuscriptcentral.com/cbst Page 25 of 32 Biocontrol Science & Technology
535 Agriculture, Web Soil Survey. URL: http://websoilsurvey.nrcs.usda.gov/ Accessed 09/30/2008.
536
537 Paige, K.N., Whitham, T.G. 1987. Overcompensation in response to mammalian herbivory: the
538 advantage of being eaten. American Naturalist 129:407-416. 539 For Peer Review Only 540 Pearson, D.E., Callaway, R.M. 2008. Weed biocontrol insects reduce native-plant recruitment
541 through second-order apparent competition. Ecological Applications 18(6): 1489-1500.
542
543 Pearson, D.E., Fletcher, R.J. 2008. Mitigating exotic impacts: restoring deer mouse populations
544 elevated by an exotic food subsidy. Ecological Applications 18(2): 321-334
545
546 Pokorny, M.L., Sheley, R.L., Zabinski, C.A., Engel, R.E., Svejcar, T.J., Borkowski, J.J. 2005.
547 Plant functional group diversity as a mechanism for invasion resistance. Restoration Ecology
548 13(3): 448-459
549
550 Saltz, D. and Ward, D. 2000. Responding to a three-pronged attack: desert lilies subject to
551 herbivory by dorcas gazelles. Plant Ecology 148(2):127-138.
552
553 SAS 2009. Statistical analysis system, proprietary software release v. 9.2. SAS Institute Inc.,
554 539 Cary, N.C.
555
556 Seastedt, T.R., Knochel, D.G., Garmoe, M., Shosky, S.A. 2007. Interactions and effects of
557 multiple biological control insects on diffuse and spotted knapweed in the Front Range of
URL: http://mc.manuscriptcentral.com/cbst Biocontrol Science & Technology Page 26 of 32
558 Colorado. Biological Control 42: 345-354
559
560 Sheley, R.L., Jacobs, J.S., Carpinelli, M.F. 1998. Distribution, biology, and management of
561 diffuse knapweed ( Centaurea diffusa ) and spotted knapweed ( Centaurea maculosa ). Weed 562 Technology 12:For 353-362 Peer Review Only 563 Soler, R., Bezemer, T.M., Cortesero, A.M., Van der Putten, W.H., Vet, L.E.M., Harvey, J.A.
564 2007. Impact of foliar herbivory on the development of a root-feeding insect and its parasitoid.
565 Oecologia 152(2): 257-264.
566
567 Stamp, N. 2003. Out of the quagmire of plant compensation responses. Quarterly Review
568 Biology 78(1)23-55.
569
570 Story, J.M., Callan, W., Corn, J.G., White, L.J., 2006. Decline of spotted knapweed density at
571 two sites in western Montana with large populations of the introduced root weevil, Cyphocleonus
572 achates (Fahraeus). Biological Control 38: 227–232
573
574 Story, J.M., Smith, L., Corn, J.G., White, L.J. 2008. Influence of seed head–attacking biological
575 control agents on spotted knapweed reproductive potential in western Montana over a 30-year
576 period. Environ. Entomol. 37(2): 510-519
577
578 Tiffin, P. 2000. Mechanisms of tolerance to herbivore damage: what do we know? Evolutionary
579 Ecology 14:1573-8477.
580
URL: http://mc.manuscriptcentral.com/cbst Page 27 of 32 Biocontrol Science & Technology
581 Trumble, J.T., Kolodny-Hirsch, D.M., Ting, I.P. 1993. Plant compensation for arthropod
582 herbivory. Annual Reviews of Entomology 38:93–119.
583
584 Welter, S. C. 1989. In: Insect-Plant Interactions, Bernays, E.A. (ed) (CRC, Boca Raton), pp. 585 135–151. For Peer Review Only 586
587 Van Hezewijk, B.H., De Clerck-Floate, R.A., Moyer, J.R. 2008. Effect of nitrogen on the
588 preference and performance of a biological control agent for an invasive plant. Biological
589 Control 46(3): 332-340
590
591 Zar, J.H. 1999. Biostatistical Analysis. 4 th ed. Prentice Hall, NJ.
URL: http://mc.manuscriptcentral.com/cbst Biocontrol Science & Technology Page 28 of 32
592 Figure legends
593
594 Figure 1 (a) Photosynthetic rate, (b) transpiration, (c) stomatal conductance, (d) Ci, (e) water use
595 efficiency and (f) L. minutus canopy damage measured from individual spotted knapweed plants 596 on three dates Forin Boulder County,Peer CO. Points Review depict the mean ± 1Only SE, different letters denote 597 significant differences between dates at P<0.05.
598
599
600 Figure 2 Negative polynomial regressions of (a) photosynthetic rate (A), (b) stomatal
601 conductance (log), and (c) transpiration (E) as a function of cumulative Larinus minutus damage
602 (data shown is from last measurement date only) to stem and foliar tissues of individual spotted
603 knapweed plants over a two month period.
604
605
606 Figure 3 Linear and polynomial regressions of insect and C. stoebe plant traits showing (a) foliar
607 C:N (b) flower production and (c) aboveground biomass (log) as a function of cumulative
608 Larinus minutus damage to stem and foliar tissues; and (d) root biomass as a function of C.
609 achates density. Panels (e) flower production and (f) aboveground biomass are partial regression
610 plots that depict the effects of C. achates root weevil density (log), holding root biomass
611 constant.
612
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Table 1 Repeated measures ANOVA across three sampling dates for (a) C. stoebe physiology (date), and regression results for insect effects on (a) physiology and (b) plant traits. Effects of foliar damage by Larinus minutus , and root damage by Cyphocleonus achates , or their interaction, are presented for measurements taken on the last sampling date. Significant effects are in bold type. Date Larinus minutus Cyphocleonus achates † Larinus x Cyphocleonus Variable Fordf FPeer P df ReviewF P df OnlyF P df F P (a) Physiology
Photosynthesis 2, 44 15.4 <0.001 1, 43 3.1 0.057 1, 39 0.73 0.398 1, 44 0.54 0.468
Transpiration 2, 46 49.1 <0.001 1, 43 8.1 0.001 1, 44 2.41 0.128 1, 44 0.01 0.931
Conductance 2, 46 43.3 <0.001 1, 43 7.56 0.002 1, 39 2.83 0.101 1, 44 0.08 0.775
Ci 2, 42 36.5 <0.001 1, 38 6.14 0.018 1, 39 0.30 0.588 1, 41 1.15 0.290
WUE 2, 46 18.1 <0.001 1, 47 0.03 0.967 1, 39 0.69 0.411 1, 42 0.77 0.385
Larinus damage 2, 47 35.0 <0.001 - - - 1, 48 0.10 0.75 - - -
(b) Plant traits
Foliar C:N - - - 1, 43 8.5 0.006 1, 39 2.39 0.130 1, 43 0.05 0.816
Flowers / plant † - - - 1, 46 43.8 <0.001 2, 47 11.36 <0.001 1, 47 0.05 0.823
Aboveground biomass † - - - 1, 38 14.1 <0.001 2, 48 26.9 <0.001 1, 48 0.05 0.825
Root biomass - - - 1, 38 11.5 0.002 1, 48 10.3 <0.002 1, 48 0.35 0.556
† For C. achates effects on flowers and aboveground biomass, log-transformed root biomass was included in the model, thus df =2.
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a) Photosynthesis d) Ci 22 A A net 350 Ci ) -1 -1 20 F 2,44F2, =138 15.39 = 15.39 F 2,135 = 10.10 s A F 2,42 = 10.10 P < 0.001
-2 A P < 0.001 300 PP <= >0.001 0.001
m 18
2 B
(molair) 250 16 2 B 14 200 B
A (µmol CO 12 150 —µmol CO i
10 100 (june 23) For(july 7) Peer(July 20) Review Only Transpiration (E) (june 23) (july 7) (July 20) 18 b) e) WUE Transpiration 2.5 WUE -1 A s F = 35.78 C F 2,138 = 35.78 -2 15 2,46 F = 18.05 B P =< >0.001 0.001 F2,46 2,136 = 18.05 PP <= 0.001> 0.001 B
O m O 12
2 2 O transpired transpired C O 9 2 A C 6 1.5
3 intake : H 2
0 1 1 (june 23) (july 7) (July 20) c) ConductanceConductance (gs) 3 f) Larinus damage BB F 2,138 = 44.44 F 2,46 = 44.44 P = > 0.001 ) (log) mmol ) (log) (E) H P < 0.001 F 2,47 = 9.79 -1 0.75 A 2.5 P < 0.001 s -2
0.5 A O m O 2 2 B CO damage canopy A C 0.25 1.5 (mol H s g
0 L. minutus 1 June(june 23) 23 July (july 7)7 July (July 2020) June(23 June 23 July (7 July 7 July (20 July 20
Figure 1
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35 a) Photosynthesis
) R2 = 0.17
1 2 - 30 R = 0.1694
s P = 0.014 2
- 25 m
2 20
CO 15
10
mol μ
( 5
net 0 -5 0 1For2 Peer3 4 Review5 Only
0.5 b) Conductance R2 = 0.30
0.4 R2P == 0.29860.010
)(log) A )(log)
1
- s
2 0.3
- O m O
2 0.2
0.1
(molH s g 0 0 1 2 3 4 5
20 c) Transpiration 1 - 2
s R = 0.310.3145 2
- 15 P < 0.001
O m O 2
H 10
mmol 5 (E) (E)
0 0 1 2 3 4 5 Larinus minutus damage
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40 a) 5 d) 2 2 R = 0.28 R = 0.1689 35 P < 0.001 4 30
25 3 20 15 2 Foliar% C:N 10 R2 = 0.17 1 P = 0.006
5 g (log) biomass Root 0 0 0 1 2 3 4 5 -1 0 1 2 3 4 5 6 7 8 9 For Peer ReviewCyphocleonus achates Only root -1 6 b) Larinus minutus damage e) R2 = 0.49 R2 = 0.34 5 P R<2 0.001= 0.49 2.00 P = <0.001 P < 0.001 4 0.75 3 -0.50 2 -1.75
1 Flowers per plantFlowers per (log) 0 -3.00 0 1 2 3 4 5 -1 0 1
5 c) 2 f) R2 R= 0.3195= 0.32 25 R2 = 0.52 4 P = 0.002 P = 0.026 14 3 03 2
-1 1 2
0 -12 Aboveground biomass g(log) biomassAboveground
0 1 2 3 4 5 plant per (log) Flowers g (log) biomass Aboveground -1 0 1 Larinus minutus damage 0 Cyphocleonus achates root -1 (log) -1 0 1 2 3 4 5 6 7 8 9
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