Ecological Costs of Plant Resistance to Herbivores in the Currency of Pollination Author(s): Sharon Y. Strauss, David H. Siemens, Meika B.

Decher, Thomas Mitchell-Olds Reviewed work(s): Source: Evolution, Vol. 53, No. 4 (Aug., 1999), pp. 1105-1113 Published by: Society for the Study of Evolution Stable URL: http://www.jstor.org/stable/2640815 . Accessed: 10/12/2011 11:21
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Evolution, 53(4), 1999, pp. 1105-1113

ECOLOGICAL COSTS OF PLANT RESISTANCE TO HERBIVORES IN THE CURRENCY OF POLLINATION

SHARON

Y.

@ edu 2E-mail: systrauss ucdavis. 3Max-Planck-Institute ChemicalEcology, Tatzendpromnenade 07745 Jena, Germany for 1A,

DAVID H. SIEMENS,3 MEIKA B. DECHER,1 AND THOMAS MITCHELL-OLDS3 STRAUSS,1,2 PopulationBiology, University California,Davis, California95616 lCenter for of

we be Abstract.-Inthispaper, examine howecologicalcostsofresistance might manifested through plantrelationships withpollinators. defensive If are into or compounds incorporated floralstructures if theyare sufficiently costlythat are to fewer rewards offered pollinators, pollinators maydiscriminate againstmoredefended plants.Here we consider whether selection increased for resistance herbivores to couldbe constrained opposingselection directional by through discrimination moredefended We of pollinator against plants. used artificial selection create to twopopulations Brassica concentrations consequently, to rapa plantsthathad highand low myrosinase and, highand low resistance fleabeetle of herbivores. measuredchangesin floralcharacters plantsin both damagedand undamagedstatesfromthese We resistances fleabeetleattack. also measured to We visitation plants, to populations withdifferent pollinator including of numbers pollinators measuresof visitquality(numbers flowers of and visitedand timespentper flower). Damage in fromherbivores resulted reducedpetal size, as did selectionforhigh resistance herbivores to laterin the plant of lifetime. addition, In floraldisplay(number open flowers) was also alteredby an interaction betweenthesetwo effects. traits translated overallgreater of low-resistance, into use Changesin floral undamaged plantsbased on total on of attracted plantsdid not differ to amountof timepollinators spentforaging plants.Total numbers pollinators on moretimeper flower plantsfromthe low-resistance amongtreatments; however, pollinators spentsignificantly and tended visitmoreflowers theseplantsas well. Previousworkby other on on population to investigators thesame taxa has shownthatlongervisittimesare associatedwithgreater male and femaleplantfitness. Because pollinator of initialnumbers pollinators notdiffer did betweenselectionregimes, palatability and/or amount rewards of offered are for by high-and low-resistance populations likelyto be responsible thesepatterns. Duringperiodsof pollinator less limitation, defended plantsmay have a selectiveadvantageand pollinator preferences may mediatedirectional if selectionimposedby herbivores. addition, pollinator In limitseed set in highly defended preferences plants,then to in lower seed set previously attributed allocationcosts of defensemay also reflect limitation greater pollinator to theseplantsrelative less defended plants. Keywords.-Allocationcosts,Brassica rapa,costsofresistance, ecologicalcosts,herbivores, myrosinase, pollinators. Received July 23, 1998. AcceptedMarch 12, 1999. Secondary compounds in plant tissues have long been known to govern patternsof attack from insect herbivores (De Candolle 1804; Brues 1924; Painter 1941). The diversificationof these compounds, in turn,may have been driven by selection from herbivorous species (Ehrlich and Raven 1964). Several studies have shown thatherbivorescan act as selective agents on resistance characters of theirhost plants (e.g., Berenbaum et al. 1986; Mauricio and Rausher 1997). In field studies, herbivores exerted directional selection for increased glucosinolate contentin naturalized populations of Arabidopsis thaliana (Mauricio and Rausher 1997). Therefore,traitsthatconferresistance to damaging herbivorescan be selectively advantageous. Despite the fact that many herbivores exert selection for increased resistance to attack, additive genetic variation in resistance to herbivorespersists in almost every naturalplant population studied (e.g., Fritz and Price 1988; Marquis 1990; Simms 1990; Strauss 1990; Karban 1992). Several hypotheses have been put forward to explain why directional segreatlection has not caused the fixationof alleles conferring est resistance. These hypotheses generallyrely on genotypeby-environment (G X E) interactionsarising from shifting herbivorepressures and/orcosts of producingplant defenses. Plants thathave invested resources into defensivecompounds are thoughtto be more fitin the presence of herbivores,but less fitthan undefended plants in enemy-freeenvironments, where defenses are costly and withoutbenefit(Simms 1992; Zangerl and Bazzaz 1992). The rate at which alleles will go to fixationwill be slowed by fluctuating abundances of herbivores, and in some cases, genetic variation in quantitative traitscan even be maintainedthroughsuch G X E interactions (Gillespie and Turelli 1989; Han and Lincoln 1994). Alternative hypotheses to explain the presence of allelic diversityin resistance traitssuggest thatthe costs and benefits are ecological and lie in the differing attack patternsof herbivorous species with respect to plant traits (Simms 1992; Giamoustaris and Mithen 1995). Some herbivores,oftenspecialists, are attracted to high concentrations of secondary compounds that serve as oviposition and feeding stimulants and thatoftenhave low inhibitory effectson the development of adapted herbivores. In contrast,more generalized herbivores frequently suffer on greatlyreduced performance plants with high concentrationsof secondary compounds (e.g., Carroll and Hoffman 1980; Giamoustaris and Mithen 1995). Thus, defense against one herbivore can lead to attack from others; again, the shiftingrelative abundances of these herbivores in environmentscan slow loss of variation in nonequilibrial plant populations over long periods of time. Recently,it has been proposed thatinteractions defended of plants may differfrom those of less defended plants with otherimportant such as mutualists(Fineblum and interactors, Rausher 1997; Strauss 1997). Fineblum and Rausher (1997) suggested thatif precursorsof defensive compounds are also precursorsin the petal pigmentationpathway, then response to selection fromherbivoresmay be constrainedby response to selection from pollinators that favor pigmented flowers

1105
?) 1999 The Society for the Study of Evolution. All rightsreserved.

1106

SHARON

Y. STRAUSS

ET AL.

and vice versa. In addition, if secondary compounds are also incorporatedinto floral structures, then defended plants may differ in their attractiveness or palatability to pollinators (Strauss 1997). Glucosinolates are presentin nonsepal floral parts of wild radish (Strauss and Kniskern,unpubl. data) and buds of Brassica spp. (4979 [mol/100 g dry weight; Rosa 1997), nicotine is found in corollas of wild tobacco (Euler and Baldwin 1996), and alkaloids in the nectar and corollas of jimson weed (Grant and Grant 1983). Because pollinators consume pollen and nectar,defensive chemicals may directly influence use of floral resources by pollinators. Investment in defenses such as secondary compounds may also affect pollinators indirectly,if costs of producing secondary compounds are reflectedin decreased allocation to plant attractiveness or rewards for pollinators (Agrawal et al. 1999). To date, examples of costs of defense against herbivores arising frompollinator preferenceshave rarely been sought (but see Fineblum and Rausher 1997) and have not yet been documented. In this study, we assess the potential for ecological costs of resistance in the currencyof the pollination biology of Brassica rapa, and the possibility that pollinator preferencesmay constrain the evolution of plant resistance to herbivores.Pollinators are especially important B. rapa for because it is an obligately outcrossingspecies. We determine whetherselection for resistance to herbivoreshas led to correlated changes in floral characters and subsequent differences in usage of plants by pollinators. Both herbivoredamage and pollinator limitationcan reduce plant fitness,especially in obligate outcrossers. The shiftingrelative impacts of pollinatorlimitationand herbivoredamage on plant fitness may provide a source of stabilizing selection thatconstrains directional selection for more defended or attractiveplants. Standard arguments about allocation trade-offscould also apply, if costs of defense come at the expense of attractiveness to or rewards for pollinators. Ultimately,we are interested in whether selection from pollinators could constrain the degree to which plants increase levels of defensive compounds in response to selection fromherbivores. Background Siemens and Mitchell-Olds (1996, 1998) have shown that resistance to flea beetle herbivores in B. rapa is influenced by total glucosinolate and myrosinase concentrations.Myrosinase is an enzyme that degrades glucosinolates into isothiocyanates and other bioactive forms. Two populations of plants were created using a disruptive selection regime to generate families that were either very high or very low in myrosinase content (see Methods). In the field, plants from high-myrosinasepopulations experienced significantlyless damage fromflea beetles than did those in the low-myrosinase population (Siemens and Mitchell-Olds 1996); we therefore referto plants fromthe high-myrosinasepopulation as having greaterresistance to flea beetles. In addition to potential constitutivedifferencesbetween susceptible and resistant populations, allocation to floraltraits may differafter damage, because both populations exhibit an inducible increase in total glucosinolate contentwithdamage (Siemens and Mitchell-Olds 1998). In a previous study, when plants fromthese populations were exposed to fungal

pathogens as well as damage frommoth larvae, inductionof glucosinolates in plants from the high-myrosinasepopulations was greaterthanthatof plants fromthe low-myrosinase population (Siemens and Mitchell-Olds 1998). Thus, damage effectsmay also reflecteffectsof selection for greaterresistance. We measured floral traits and pollinator responses to plants in high- and low-resistance populations, both in the presence and absence of herbivore damage.
MATERIALS AND METHODS

Seeds from45 maternalB. rapa plants were collected from a naturalized population growing in the BitterrootValley of Montana. Artificialselection for high and low foliar myrosinase concentrationwas performedfromthis bulk seed collection (for details, see Siemens and Mitchell-Olds 1998). In the firstgeneration,50 high- and 50 low-myrosinase plants were selected from a base population of 500 plants. In subsequent generations,50-high myrosinaseplants were selected from 250 plants in the high-resistance population, and 50 low-myrosinase plants were selected from 250 in the lowresistance population. To equalize environmentaleffects,the 250 high- and 250 low-myrosinase plants were grown randomly intermixedin the greenhouse each generation. Variance effectivepopulation size was 72, a size thatminimized effectsof random genetic drift. After three generations of selection, there was a twofold differencein myrosinase concentrationsbetween the populations (P = 0.009); parent-offspring regression showed a narrow-senseheritability myrosinase concentrationof h2 for = 0.35 (Siemens and Mitchell-Olds 1998). Plants in the selected populations still expressed concentrationsof myrosinase thatwere representedin the originalpopulation; therefore, our experiments examine potential selective pressures experienced by individuals toward the tails of the distribution within natural populations. A main assumption is that the original base population was in linkage equilibrium, so any correlated changes with divergence in myrosinase were not caused by chance association of alleles at different loci. In an obligately outcrossed species like B. rapa, this assumption is reasonable (Crow and Kimura 1970). For this study, 42 maternal families, 20 from the lowglucosinolate population and 22 fromthe high-glucosinolate population were grown in the greenhouse. Each family had between two and six full/half sibling replicates. Seeds were washed with dilute bleach solution and then bathed in gibberellic acid on filterpaper such that they all germinatedat once; thus, plants were equal-aged at the startof the experiment. At the four-leafrosette stage, half of the seedlings fromeach familywere randomlychosen to receive 50% leaf area removal of the firsttwo true leaves by Pieris rapae caterpillars. These caterpillars are natural herbivores of B. introducedhabitats. rapa in both native European and current, Caterpillars were placed in small clip cages and allowed to feed alongside the midrib of each damaged leaf. Clip cages alone did not affectplant reproductionand did not significantly increase levels of indole glucosinolates or total glucosinolates in studies of R. raphanistrun (Agrawal et al. 1999; Lehtila and Strauss 1999). This level of leaf area removal was small with respect to the lifetimeleaf production

COSTS OF RESISTANCE IN POLLINATION

1107

of the plant, comprising about 15% of the total leaf area of experience plants. Brassica rapa plants in the fieldfrequently this extent of damage (Pilson 1996; Siemens, pers. obs.). A damage effect reflects differences from undamaged plants arising from both leaf area loss and induction of increased myrosinase and glucosinolates afterdamage. Both of these effectsaltered allocation patternsto plant reproductiveparts (Agrawal et al. 1999). in a realted mustard,R. raphanistrum We recorded the days elapsed from germination to first flower,measured the petal lengths and widths of the third flowerfromeach and collected pollen fromthe fourth flower, plant. We collected half the anthers, one from a short and two from long stamens (mustards have a total of four long and two shortstamens per flower)and placed themin an open Eppendorf tube in a closed cabinet to dry. Aftertwo weeks, vials were sealed and samples were laterprocessed and counted using an Elzone particle counter (for procedures, see Young and Stanton 1990). We quantified total number of pollen grains produced per floweras well as measured modal pollen grain volume per flower. To measure pollinator response to these high and low myrosinase populations, we placed arrays of eight to 16 plants in the field over the course of 15 days. Within each array, therewere equal numbersof high- and low-myrosinasefamilies and one damaged and one undamaged sibling per family. Plants were linearly arranged in the field such thatall plants were equidistant froma naturallyoccurringpatch of blooming mustards; positions within the array were randomly asthathigh-and low-resistancefamsigned underthe constraint ily members alternated in the array. The number of plants observed per day was determinedby weather and numbers fromthe different families and treatments. of plants flowering For each plant in an array,we also recorded mean petal size of two open flowers,thenumberof open flowers,plant height, and the total numberof flowersproduced by plants untilthat date. Because all plants were of equal age, the latter measurementwas an indicator of differencesin floweringrate. have all been shown to affectpollinators of These attributes mustards(Stanton et al. 1986; Conner and Rush 1996; Strauss et al. 1996). Pollinators were classified into the following taxa: honeybees, native bees, syrphidflies,and others.These taxa represented42%, 42%, 7% and 9% of visitors,respectively.The mostlyP. rapae, bee"other" category included butterflies, tles, and other less abundant visitors. All members of the threemain groups of these floralvisitors as well as P. rapae fromour "other" category have been shown to be effective anothermustardwith similar pollinators of R. raphanistrum, floral morphology (Conner et al. 1995). Of these groups, native bees are primarilypollen foragers,with less than 5% of visits spentdrinking nectar(Strauss, unpubl. data); syrphid flies use pollen exclusively; and honeybees use both pollen and nectar produced by plants (Mohr and Jay 1988). Observations of damaged and undamaged siblings from the same families were conducted simultaneously and all plants were observed once in these arrays. Observations of high- and low-resistance families were alternatedand which was randomlyselected foreach population was observed first array. Every plant was observed for 12 min, and all pollinators visiting the plant during this interval were recorded.

In addition, the behavior of individual pollinators on the plants was followed. For the firstpollinator that arrived on the plant duringthe observation period, we recordedthenumber of flowers visited on the plant and the total visit time spent on the plant. We did this for each successive pollinator left afterthe first untilthe observation period was over.While observing these pollinators,we also kept trackof the number and identities of other pollinators visiting the plant during the observation period. We estimatedtotal numberof flowers visited per observation period by multiplyingthe numberof pollinators visiting plants of a given taxon by the average number of flowers visited per visit by that taxon and then summing across pollinator taxa. Similarly, the total time spent foraging by pollinators on plants was estimated by multiplyingthe average time spent by each pollinator by the number of that taxon visiting the plant and then summing over all pollinator taxa. These types of observations allowed us to determinetotal visitationduringthe observation period, as well as to assess visit quality (time and numberof flowers visited) fromthe observations of individual pollinators. Statistical Analyses We used MANOVA to examine responses of suites of plant traits to selection regime and damage. MANOVA is useful when sets of dependent variables are intercorrelated(as in floraltraits;Scheiner 1993). Response variables fortwo separate MANOVAs were: plant traits measured on the third/ fourthflower(petal size, total pollen production,pollen volume, and date firstflower was produced) and plant traits measured during trials (petal size, number of open flowers, and total number of flowersproduced to date). All response when necessary to variables were inspected and transformed meet assumptions of normality.The MANOVA model included damage and resistance (two levels each) as fixed effects, a plant family effectthat was a nested random factor withinresistance level, and all interactions.For all analyses of data taken duringobservation trials,we also included date as a block effect. Sample sizes were somewhat smaller for flowermeasurementsbeobservation trials than third/fourth cause we were constrained to use floweringpairs of sibling of damaged and undamaged plants with equal representation high- and low-resistance families. If a MANOVA was significant,we then conducted protected univariate tests to examine how each response variable was affectedby treatments (Scheiner 1993). To examine usage of plants by pollinators,we used a summary measurement:total time spent foragingon a plant. This in gauge is tied to both male and female fitness closely related mustards (Conner et al. 1995; see Discussion). In addition, we broke down this measure into its component parts: the total number of pollinators visiting plants, total number of flowers visited, and time spent per flower.These were analyzed using separate ANOVAs, with the same model as above. When considering aspects of visit quality in analyses, sample sizes included only plants that received visits from pollinators. For all analyses of data collected during trials, were never below 44 plants per sample sizes for treatments treatment.

1108

SHARON Y. STRAUSS ET AL.

traits.(a) Measurements fromthe fourth flower. of on reproductive (b) TABLE 1. Effects leaf damage and selectionfor resistance after MANOVAs (see planttraits univariate ANOVAs on individual during pollinator trials.Resultsfrom Measurements from flowers Results).Significant P-values (<0.10) are shownin bold.
(a) Third/fourth flower Petal size Source df F P No. pollen grains F P F Pollen size P Date of first flower F P

Damage Resistance* Family(Res) Damage X Res" Damage X Fain (Res) Error

1 1 38 1 38 152

16.30 0.09 2.84 0.05 1.21

0.001 0.80 0.001 0.87 0.27

2.95 0.43 1.71 2.36 1.02

0.08 0.62 0.01 0.12 0.44

0.07 0.35 0.87 0.05 0.97

0.97 0.60 0.68 0.83 0.53

1.67 0.32 0.94 0.78 0.91

0.20 0.55 0.57 0.55 0.32

(b) Plant traitsduringpollinatortrials Petal size Source df F P F No. open flowers P No. flowersproduced to date F P

Date Damage Resistance*

Family (Res)

Damage X Res** Damage X Fain (Res) Error

1 38 119

38

1 1 1

0.50 13.88 4.43 0.24 0.98

2.87

0.48 0.001 0.04 0.63 0.51

0.001

1.48 2.55 1.30 8.25 0.86

1.74

0.23 0.11 0.26

0.01

6.54 1.65 1.45 2.85 0.41

3.20

0.01 0.20 0.24

0.007 0.71

0.10 0.99

0.001

* Resistance MS tested over family (resistance) MS.

* Damage X resistance interaction MS tested over damage X family (resistance) MS.

RESULTS

Responses of Floral Characters to Selection for Resistance and Leaf Damage Measurementsfrom the Third and Fourth Flowers MANOVA showed that there were no effectsof selection for resistance on floral traitsof early flowers (P = 0.54 and interaction, 0.75 for resistance and treatment-by-resistance respectively)but thatearly leaf damage did affectfloraltraits (Wilk's X = 0.887, F = 4.97; df = 4, 149; P < 0.0009,). Subsequent univariate ANOVAs showed that damage significantlydecreased petal size and tended to decrease the but numberof pollen grainsproduced per flower, had no effect on pollen grain size or on the date the firstflower was produced (Table la, Fig. 1). These effects of damage on petal size and pollen number are consistentwith those found in a (Strauss et al. 1996; closely related species, R. raphanistrum Lehtila and Strauss 1999). Measurements of Plant Traits during Pollination Trials

Plant traits,except pollen production,were measured again during pollination trials, when plants had produced 50-70 flowers (approximately one-quarter of the total flower production per plant). These traitswere: petal size, total number of flowers produced to date (a phenological measurement), and numberof open flowerson a plant. Overall, we still found effectsof damage (P < 0.0009; Wilk's X = 0.87; significant F = 5.85; df = 3, 117); in addition, there was also a significantdamage-by-resistanceinteraction(P = 0.05; Wilk's X = 0.81; F = 2.80; df = 3, 35). Main effectsof resistance VisitationPatterns by Pollinators withRespect to Selection were nonsignificant(P = 0.16). From univariate analyses, and Damage Treatments with familyand we found thatpetal size varied significantly To determineeffectsof damage and selection forresistance was still reduced by early leaf damage (P < 0.001). In addition, petals on plants from high-resistancefamilies were to herbivores on use of plants by pollinators, we initially

significantlysmaller (P = 0.04) than those on plants from low-resistance families (Fig. 2, Table lb). Floral display, or the number of open flowers on a plant, was affectedby familyand by an interaction between damage and selection for resistance to herbivores (Fig. 2, Table lb). In the interaction, damage reduced the numberof open flowers in the low-resistance population, but did not affect the number of open flowers in the high-resistancepopulation. Flowering rate (phenology), as measured by the total number of flowers produced by plants up to the observation time, althoughdamwas not signficantly affectedby any treatment, aged, low-resistance plants tended to have a slower flower production rate (P = 0.10; Fig. 2, Table lb). In summary,although selection for resistance to flea beetles did not affectfloral traitsin the firstflowers produced, differencesbetween selection regimes arose later in the plant lifetime.Petal sizes were smaller forplants selected forhigh resistance than for those with low resistance to herbivores. In contrast to petal size, the number of open flowers was reduced in low-resistancefamilies afterdamage occurred,but not in high-resistance families. Thus, plants in damaged, high-resistance families tended to have smaller petals but more open flowers than damaged plants in low-resistance families. Both of these traits affect attractivenessto these groups of pollinators in mustards (Conner and Rush 1996; Strauss et al. 1996). Early damage caused overall decreases in petal size and pollen productionand exerted strongeffects on petal size thatpersisted until the end of the measurement period (at least 25% of all flowersproduced).

COSTS OF RESISTANCE IN POLLINATION

40
-_ _ _ _ _ _ _ _ _ _ _ _ _ _ _

1109

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and lighter bars are plantsin thelow-resistance population. population

Low High RESISTANCE LEVEL

FIG. 1. Measurements floraltraits of fromthe thirdand fourth flower flowers. bars represent means ( SE) forplantsin Patterned solid bars represent means (t? SE) for undamagedsiblingplants.Darkerbars are plants in the high-resistance damaged treatment;

40 -~35 .30 25 20

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2. Measurements of floral traits plantswhenpollinator of observations weremade approximately days after 10 original...measurements. Patterned represent bars mans (? SE) forplantsin damagedtreatment; barsrepresent eans (? SE) forundamaged solid siblin plans. Drkebar areplats i thehig-resstane ppulaion nd ightr bas ae plnts2n0te lo-resstace ppulaion

High Low~~~~~~......... RESISTANCE LEVEL~...........

1110
TABLE

SHARON Y. STRAUSS ET AL.

TABLE

2. Effects of damage and selection for resistance on the total time pollinators spent foraging on plants (log-transformed). SignificantP-values (<0.10) are shown in bold.
Source df MS F P

resistanceand leaf 3. Effectsof selectionforherbivore of on damageon components timespent pollinators plants:total by of number pollinators observation of per period,number flowers PSignificant visited per plant,time spentforaging per flower. values (<0.10) are shownin bold.
Total no. pollinators Source df F P No. flowers visited per plant F P Time foraging per flower F P

Date Damage Resistance* Family (Res) Damage X Res** Damage X Fam (Res) Error

1 1 1 38 1 38 114

0.13 1.89 1.51 1.95 3.57 0.82 1.36

0.10 1.39 0.78 1.43 4.37 0.60

0.75 0.24 0.38 0.08 0.04 0.96

sistance)MS.

* Resistance MS. MS testedoverfamily (resistance) ** Damage X resistance (reinteraction tested MS overdamage X family

examined one composite measure of plant usage-total time spent foragingby pollinators on plants-and then broke this measure down into its component parts. The total amount of time pollinators spent foraging on plants reflectedboth damage and selection for resistance (P = 0.04 for resistance-by-treatment interaction;Table 2, Fig. 3a). Pollinators spent more time on low-resistance, undam(a aged plants than on plants in other treatments priori one-

Date Damage Resistance* Family(Res) Damage X Res** Damage X Fam (Res) Error
sistance) MS.

1 1 1 38 1

1.23 2.89 0.09 1.05 0.00

0.27 0.09 0.76 0.41 0.97

0.40 3.36 0.59 1.64 4.09

0.52 0.07 0.45 0.02 0.05

0.03 0.08 4.14 0.96 0.43

0.87 0.78 0.05 0.55 0.45

38 0.39 0.99 114

0.61 0.96

0.95 0.56

* Resistance MS tested over family (resistance) MS. "* Damage X resistance interactionMS tested over damage X family (re-

Total time spent per plant is a functionof the number of pollinators that visit a plant, the time spent per flower,and the number of flowers visited. In this case, there were no tailed contrasts; df = 1, 114, P = 0.03 [low/damaged], P = significanteffectsof any treatmentson the total number of 0.03 [high/damaged] and P = 0.055 [high/undamaged]). pollinators visiting plants (Table 3, Fig. 3b), although damDamage reduced the amount of time pollinators spent for- aged plants tended to attractfewer visitors than undamaged aging on low-resistanceplants (P = 0.055), but not on high- plants (P = 0.09). However, therewas a significanteffectof resistance plants (P = 0.35). selection for resistance to herbivores on the amount of time
400
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FIG.

3 Polliator esponss masuredover te (t SE)for pants damaed tratmen; soli barsrepreent mens (tSE) n fr undaaged high-resistance population and lighter bars are plantsin the low-resistance..population.

RESISTANCE LEVEL (men t S.B.) t plant as

High....Low. RESISTANCELEVEL..... 12-m obsevatio perio.

......L...I

Paterned.ars.rpresen.mean iblin plant. Darer bas.are.lantsin.th

COSTS OF RESISTANCE IN POLLINATION

30 -_30 Honeybees 25 25 20
05 -

1111

bees Native

Q~~~~~~~~~~~ 20 U)~~~~~~~~~~~~~~~~~~~~~C CD~~~~~~~~~~~~~~~~~~~~~~a 15 0~~~~~~~~~~~~~~~~~~~~~

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~~

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High
FIG. 4.

Low

High

Low

RESISTANCE LEVEL

RESISTANCE LEVEL

Mean (+ SE) amount of time spent foraging per flower by each pollinator taxon. Patterned bars represent means (+ SE) for plants in damaged treatment;solid bars representmeans (+ SE) forundamaged sibling plants. Darker bars are plants in the high-resistance population and lighter bars are plants in the low resistance-population.

spent per flowerby pollinators (P = 0.05). Pollinators spent significantlymore time per flower on plants from the low resistance population than on plants fromthe high-resistance population. Together, these results suggest that plants are equally attractiveto pollinators before they begin to forage, but that low-resistance plants are more palatable or provide greaterrewards,once pollinators begin to forage on flowers. Both treatments affectedthe numberof flowsignificantly ers visited per plant. Overall, damaged plants tended to receive fewervisits (P = 0.07), and therewas also a significant interactionbetween selection for resistance and damage on numbers of flowers visited (P = 0.05). Plants in the lowresistance population had the greatestnumberof flowersvisited in the absence of damage and the least, once damaged. In contrast,damage did not affect the numbers of flowers visited in the high-resistancepopulation. In summary,both damage and selection regime affected how pollinators used plants. Although there did not appear to be differencesbetween selection regimes in their initial attractivenessto pollinators, pollinators spent less time foraging per flower on resistantplants. They spent most time on undamaged, low-resistance flowersand also visited more flowerson these plants. This result was especially strongfor native bees, who spent almost twice the amount of time on flowers of low-resistance, undamaged plants than in any of the treatments(Fig. 4). These native bees are almost exclu-

sively pollen foragerson B. rapa, and comprised 42% of the visitors to B. rapa. The differencein amount of pollen produced between high- and low-resistance populations is unlikely to be of the magnitudeof the difference visit lengths in to flowers;note thattherewas no statisticallysignificant difference in pollen productionbetween treatments days ear10 lier (Table 1). Even if low-resistance plants were producing more pollen duringour observation period, it is significantly highly unlikely that they were producing vastly greater amounts of pollen. These visitation patternsin bees suggest thatfloralrewards of undamaged, low-resistancepopulations may be more palatable to bees. It should be noted, however, that time spent per flower was highly idiosyncratic by pollinator taxon, although both bee taxa (and those accounting for more than 84% of the visits) spent most time on undamaged, low-resistance plants (Fig. 4). DISCUSSION Ecological costs may constrain the response to selection for increased plant resistance imposed by herbivores. Traditionally, these costs have been couched in terms of interactions between plants and varying pathogenic or herbivorous taxa. Most comparisons focus on the possibility of negative cross-resistance within a suite of similar interactors (e.g., pathogens [Mitchell-Olds et al. 1995], herbivores [Gia-

1112

SHARON

Y. STRAUSS

ET AL.

moustaris and Mithen 1995], or both [Karban et al. 1987; Siemens and Mitchell-Olds 1998]). We examined whether ecological costs of resistance to herbivores could also be manifested in mutualistic relationships between plants and pollinators. We foundthatpollinators spentless timeforagingon plants selected forgreaterresistance to flea beetles. In addition,both leaf damage and selection forgreaterresistance to herbivores altered petal size and floraldisplay, which serve as cues for pollinators in mustards (Conner and Rush 1996; Strauss et al. 1996). Recall that effectsof leaf damage include effects of defensive compounds as well, because damage induces greater concentrations of glucosinolates in both low- and high-resistance lines (Siemens and Mitchell-Olds 1996, 1998). How foraging behaviors of these pollinators affectplant fitness has been addressed extensivelyby Conner et al. (1995) in a related,obligately outcrossingmustard,R. raphanistrum. Raphanus raphanistrum and B. rapa have similar generalized floralmorphologyand share most of the same pollinator species in the field,especially the two main bee taxa. Pollen size is also similar in the two species, so thatresults fromradish are likely to be comparable to B. rapa. Conner et al. (1995) examined pollen removal and deposition in relation to visit durationfor syrphids,native bees, honeybees, and P. rapae butterflies. All taxa were effectivepollinators of R. raphanFor istrumn. native bees, pollen removal fromflowerswas an increasing functionof time spent per flower(across the visit durations we observed on B. rapa). Therefore, native bees that spend more time on undamaged, low-resistance flowers in our study are likely to remove more pollen from these flowers than from flowers in the other treatments.In wild radish, native bees were slightly more effectivepollinators than syrphidflies,as bee pollination resultedin slightlymore fruitsinitiatedand significantly more seeds per fruitthan fly pollination. Because theyare also numericallyveryabundant, bees are likely to be a very importantpollinator of B. rapa. For honeybees and butterflies, pollen deposition onto stigmas was also positively associated with visit time over visit durations we found for B. rapa. Differencesin visitationbetween defendedand undefended plants may be especially importantduring periods of pollinatorlimitation-in cool weatheror years withlow pollinator abundances. Little information available on how oftenpolis linator limitation occurs for B. rapa in the field; however, addition of honeybees boosted seed yield in turnipcrops (B. rapa) in India (Singh et al. 1987), and pollinators were considered limitingfor B. rapa in Finland (Varis 1997). The reduced seed set that is often found in well-defended plants is usually attributedto allocation costs of defense (Simms 1992; Zangerl and Bazzaz 1992; Bergelson and Purrington1996). Depending on the mechanism underlyingpollinator discrimination,however, differencesin seed set may not necessarily imply energetic limitation. If supplemental hand-pollination boosts seed set, then subsequent examination of quality and quantityof rewards is required. Costs of defense could still be allocation costs if rewards produced These by defendedplants were less abundantor less nutritive. true allocation costs would be indirectlymediated by pollinators that discriminate against plants with fewer or lower-

quality rewards. In contrast, if the direct incorporation of secondary compounds into pollen and/or nectar makes rewards less palatable, then these costs are not strictlyallocation costs, but rather pleiotropic effects associated with increased production of secondary compounds. Both causes of pollinator-mediatedreduced seed set could constrain the response to selection fromherbivoresin resistancecharacters, but the mechanisms are very differentand would require different types of counteradaptationsby the plant to bypass these constraints. Our results here are the firstto show that correlated responses of floraltraitswith selection forincreased resistance to herbivoresreduced quality of visitationby pollinators.The evolution of plant resistance traitsmay therefore not only be under selection from herbivores, but may also proceed in concert with selection from other interactorsthat constrain plant responses to herbivores.
ACKNOWLEDGMENTS

We thank A. Emerson for her help with pollinator observations. We also thank L. Adler, A. Agrawal, J. Conner, R. Grosberg,R. Karban, N. Williams and especially M. Rausher and an anonymous reviewer for their helpful comments on the manuscript.This work was supportedby National Science Foundation grant DEB-94-07362 to SYS, USDA grant 9337302-9572 to DHS; TMO was supported by the MaxPlanck-Gesellschaft.
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