FLORAL SCENT IN A WHOLE-PLANT CONTEXT ... fileFLORAL SCENT IN A WHOLE-PLANT CONTEXT Testingthepotentialforconﬂictingselectiononﬂoral chemicaltraitsbypollinatorsandherbivores: predictionsandcasestudy

FLORAL SCENT IN A WHOLE-PLANT CONTEXT

Testing the potential for conflicting selection on floral

chemical traits by pollinators and herbivores:

predictions and case study

Andre Kessler* and Rayko Halitschke

Department of Ecology and Evolutionary Biology, Cornell University, 445 Corson Hall, Ithaca, New York 14853, USA

Summary

1. There are myriad ways in which pollinators and herbivores can interact via the evolutionary

and behavioural responses of their host plants.

2. Given that both herbivores and pollinators consume and are dependent upon plant-derived

nutrients and secondary metabolites, and utilize plant signals, plant chemistry should be one of

the major factors mediating these interactions.

3. Here we build upon a conceptual framework for understanding plant-mediated interactions

of pollinators and herbivores. We focus on plant chemistry, in particular plant volatiles and aim

to unify hypotheses for plant defence and pollination. We make predictions for the evolutionary

outcomes of these interactions by hypothesizing that conflicting selection pressures from herbi-

vores and pollinators arise from the constraints imposed by plant chemistry.

4. We further hypothesize that plants could avoid conflicts between pollinator attraction and

herbivore defence through tissue-specific regulation of pollinator reward chemistry, as well as

herbivore-induced changes in flower chemistry and morphology.

5. Finally, we test aspects of our predictions in a case study using a wild tomato species, Solanum

peruvianum, to illustrate the diversity of tissue-specific and herbivore-induced differences in plant

chemistry that could influence herbivore and pollinator behaviour, and plant fitness.

Key-words: plant–insect interactions, coevolution, plant defences, induced responses to

herbivory, pollinator limitation

Introduction

Plants have evolved many ways of coping with their insect

attackers, including physical or chemical defences that

directly reduce herbivore performance and survival, indirect

defences that actively attract or facilitate the action of pre-

dators and parasitoids, as well as tolerance of herbivory

(Duffey & Stout 1996; Dicke & Van Loon 2000; Kessler &

Baldwin 2002).

Ehrlich & Raven (1964) first deduced that insect herbivory

had selected for chemically defended plants. In turn, high

plant secondary metabolite production would favour insect

herbivores that are resistant to plant defences. All hypothe-

ses that were derived from this original plant defence theory

assume both costs of tissue damage in the presence of herbi-

vores and costs of producing chemical defences in the

absence of herbivores. The latter can arise from the alloca-

tion of limited resources to defences at the expense of other

fitness-related traits (i.e. physiological costs, allocation costs,

opportunity costs) (Karban & Baldwin 1997; Heil & Baldwin

2002). Alternatively, the production of defensive compounds

could be ecologically costly if they compromise interactions

with mutualists such as pollinators. Evidence for ‘ecological

costs’ is beginning to accumulate, suggesting they are poten-

tially an important force in the evolution of chemical plant

defences (Strauss et al. 2002; Kessler & Halitschke 2007; Po-

elman, Van Loon & Dicke 2008).

Compromised interactions with pollinators are often cited

as significant and probably underestimated costs of plant sec-

ondary metabolite production and thus important factors

influencing selection on plant defensive traits (Lohmann,

Zangerl & Berenbaum 1996; Strauss & Armbruster 1997;

Strauss et al. 1999; Strauss &Whittall 2006; Adler 2007). The

reproductive success of a plant is determined both by invest-

ment in reproductive tissue as well as maintenance of a large

enough vegetative biomass to provide the necessary resources

for reproduction. As a result, pollinator attraction might be

compromised by chemical defences in two principal ways.*Correspondence author. E-mail: [email protected]

� 2009 The Authors. Journal compilation � 2009 British Ecological Society

Functional Ecology 2009, 23, 901–912 doi: 10.1111/j.1365-2435.2009.01639.x

First, allocation of resources to defence may come at the

expense of investment in reproductive tissues, and may

thereby reduce the attractiveness of flowers by reducing

flower size, display and reward (nectar or pollen) quality or

quantity (Mothershead &Marquis 2000). Second, a generally

higher production of defensive metabolites in the plant may

also be expressed in reproductive tissues and floral rewards,

and thereby reduce pollinator attraction or the number of

suitable pollinator species (Adler, Karban & Strauss 2001;

Adler et al. 2006; Kessler & Baldwin 2007; Kessler, Gase &

Baldwin 2008). In this scenario we can view toxic or deterrent

metabolites in pollinator rewards as costs of plant defences

that result from a pleiotropic effect of defence compound pro-

duction in the leaves and floral tissues that are under selection

by folivores and florivores respectively (Strauss et al. 1999;

Adler 2000). This pleiotropy hypothesis is a non-adaptive

explanation for why we find defensive secondary metabolites

in pollinator rewards, and contrasts with a number of other

hypotheses that assume that pollinators (Kessler & Baldwin

2007; Kessler, Gase & Baldwin 2008) and nectar robbers or

degrading microbes (Baker 1977; Rhoades & Bergdahl 1981)

are the primary selective agents on pollinator reward toxins

(for a review, see Adler 2000). The mechanisms described by

these hypotheses are not mutually exclusive but may repre-

sent the extremes of interactive continua that result from reci-

procal and potentially conflicting natural selection by

organisms interacting with plant chemistry and associated

floral traits.

Accepting that both herbivores and pollinators are agents

of selection on chemical floral traits allows for a number of

predictions concerning the circumstances that would favour

the evolution of certain combinations of reproductive and

defensive strategies and the composition of the pollinator

community in plant populations.

Strauss & Whittall (2006) predicted that floral trait

values depend on the relative strength of the selective

agents and on whether pollinator and non-pollinator

agents of selection inflict antagonistic or coincidental

selective pressure. Accordingly, pollinators and herbivores

would exert coincident selective effects favouring the

same trait optimum when they have opposing preferences

for floral traits. Alternatively, floral traits may reflect a

compromise between values maximizing plant fitness

through interactions with antagonists and those maximiz-

ing fitness through interactions with pollinators when

pollinators and herbivores share the same floral prefer-

ences. More specifically for defensive chemical traits, the

relative dependence of a plant’s reproductive success on

herbivory vs. pollinator availability should determine the

evolution of vegetative and floral secondary metabolite

production and may result in a correlation between

defensive and reproductive strategies among different

plant species. We are aware that few data exist with

which to test these predictions, but they provide a valu-

able opportunity to perform comparative studies under

various environmental conditions. As a first step, studies

are needed that measure selection on floral chemical

traits in the presence and absence of pollinators and her-

bivores respectively (e.g. island studies). Recent studies

revealed that herbivory can alter the strength of pollina-

tor selection on morphological traits, but such studies

have not been extended to include measuring selection

on chemical traits (Gomez 2003). Moreover, more studies

are needed that test (i) whether defensive secondary

metabolite content in the vegetative tissue is correlated

with that of the reward tissues (nectar and pollen) and

(ii) whether or not pollinator attraction is altered by her-

bivore-induced changes in plant chemistry. Along these

lines, such studies will need to address the possibility

that tissue-specific developmental regulation could side-

step the indirect selective pressures between pollinators

and herbivores (as there would be no cross-tissue corre-

lation). Ideally, plants could induce production of either

foliar and ⁄or floral chemicals (production of chemical

defences only when needed) in order to escape compro-

mising pollinator attraction with herbivore defences when

herbivores are absent. The available data on the direc-

tion and relative strength of natural selection on floral

chemical traits by pollinators and herbivores are very

limited (but see Mant, Peakall & Schiestl 2005; Zangerl,

Stanley & Berenbaum 2008). However, an increasing

number of studies report tissue-specific and induced pro-

duction of secondary metabolites. In the following two

sections we first review tissue-specific production of

defensive and signalling metabolites and herbivory-

induced plant responses as potential factors influencing

pollinator behaviour. We then introduce a case study

with a wild tomato, Solanum peruvianum, to test whether

allogamous plants will show (i) tissue-specific defensive

secondary metabolite production (allogamy ⁄ tissue specific-

ity hypothesis), or (ii) will induce changes in both leaf

and floral secondary metabolism (allogamy ⁄ inductionhypothesis) with consequences for pollinator behaviour.

Optimal defence and pollination

McKey (1974) was the first to suggest that if the resources

available for the production of chemical defences against

herbivores are limited, then the distribution of chemical

protection within the plant should parallel the ‘needs’ of

the plant as defined by the ‘value’ and ‘vulnerability’ of

the different tissues. This hypothesis was later termed

‘optimal defence theory’ according to which the ‘value’ of

a tissue is determined by its relative contribution to the

plant’s Darwinian fitness (Rhoades 1979). Resources for

the production of secondary metabolites as well as the

allocation of metabolites should be directed to the most

valuable tissues. Flower parts have a strong effect on

fitness and should thus be protected by relatively high

amounts of defensive plant metabolites compared to

leaves, and this prediction is supported by numerous stud-

ies (Catalfamo, Martin & Birecka 1982; Hartmann et al.

1989; Detzel & Wink 1993; Bravo & Copaja 2002; Frolich,

Hartmann & Ober 2006; Alves, Sartoratto & Trigo 2007;

� 2009 The Authors. Journal compilation � 2009 British Ecological Society, Functional Ecology, 23, 901–912

902 A. Kessler & R. Halitschke

Beninger et al. 2007; Frolich, Ober & Hartmann 2007; Ci-

rak et al. 2008; Zhang et al. 2008).

C O R R E L A T ED S E C O N D A R Y M E T AB O L I T E P R O D U C T I O N

I N F L O W E R S A N D L E AV E S

In a survey of 14 publications that reported quantitative mea-

sures of secondary metabolites in leaf and flower tissues, 13

studies reported significantly higher secondary metabolite

concentrations in flower tissues than in leaf tissues (Table S1,

Supporting information). Additionally, studies in which a

broader spectrum of compounds was analysed seem to find

compounds exclusively produced in either the flowers or the

leaves, suggesting a pattern of tissue-specific biosynthesis or

allocation (Bennett et al. 2006). Moreover, finer scale analy-

ses of different floral organs on the same flowers find the high-

est concentrations of defensive compounds in the stamen or

pistil tissues (Bravo & Copaja 2002). It is noteworthy that

none of these studies included a community wide or phyloge-

netic survey, reiterating the need for such studies to evaluate

the evolutionary significance of tissue-specific differences in

secondary metabolite production.

Does the presence of higher concentrations of defensive

secondary metabolites in rewarding floral tissues (nectaries

and anthers ⁄pollen) compromise interactions with pollina-

tors? Such pleiotropy-mediated correlations have been found

in a number of cases. For example, the concentration of the

deterrent and toxic alkaloid gelsemine in the nectar of Gelse-

mium sempervirens flowers is correlated with the concentra-

tion of gelsemine in the leaves. Gelsemine concentration in

floral nectar is also negatively correlated with pollinator

attraction, suggesting fitness costs of gelsemine production

through pollinator deterrence (Irwin & Adler 2006). Simi-

larly, the concentration of herbivore-defensive 1,2-saturated

pyrrolizidine alkaloids (T-and iso-phalaenopsine) that are

produced in every tissue of Phalaenopsis orchid hybrids is

highest in the pollinia (Frolich, Hartmann & Ober 2006). If

defences are optimized to maximize reproductive output of

the plant, one should also expect plants to be under selection

to limit the presence of toxic or deterrent secondary metabo-

lites in pollinator rewards. One principal way for the plant to

overcome this is the spatial regulation of the production

and ⁄or allocation of secondary metabolites and their secre-

tion into nectar and pollen.

D E F E N S I V E S E C O N D A R Y M E T AB O L I T E S I N N E C T A R

A N D P O L LE N

While the precise mechanisms of secondary metabolite allo-

cation ⁄production to nectar remain poorly understood

(Adler 2000), it is likely that secondary metabolites are trans-

ported into the nectar through mechanisms similar to those

known to mediate sugar and amino acid transport into nec-

tar, including both passive diffusion and active transport

(Luettge & Schnepf 1976; Fahn 1988). Evidence has been

found for both secretory mechanisms and, additionally, the

active resorption of nectar constituents (Nepi & Stpiczynska

2007, 2008) as well as the passive differential diffusion of flo-

ral volatiles into the nectar from surrounding tissues (Ra-

guso 2004). Transport of components within the nectaries,

the mechanism of active and passive secretion, and resorp-

tion all can influence the composition of nectar (Luettge &

Schnepf 1976). Passive vs. active transport for different com-

pounds (or the same compound in different species) may

reflect conflicting natural selection by both pollinators and

herbivores.

Given this diversity of mechanisms, it seems possible that

natural selection could affect different nectar constituents dif-

ferentially. Compounds that are passively transported into

the nectar are more likely subject to compromising selection

by both pollinators and herbivores while compounds that are

actively secreted into the nectar, under regulatory control, are

more likely subject to selection by pollinators and nectar rob-

bers only. As a consequence, plants which are under strong

selection by herbivores, and thus have high secondary metab-

olite production, also should be under strong selection to

tightly control secondary metabolites in floral rewards when

they depend on an allogamous reproductive strategy (see

example of S. peruvianum below). Alternatively, selection

might favour an evolutionary shift to an autogamous repro-

ductive strategy if such plants’ defences compromise their

interactions with pollinators, or themitigation of compromis-

ing selection by herbivores and pollinators through induced

defence metabolite production only when needed after attack

(see next section).

Making predictions about the allocation of plant

defences into pollen (compared to nectar) is even more dif-

ficult because we are aware of even fewer studies reporting

pollen secondary metabolites (Pernal & Currie 2002; Lon-

don-Shafir, Shafir & Eisikowitch 2003; Boppre, Colegate &

Edgar 2005). Nectar is exclusively a reward, whereas pol-

len is only a reward for pollinators if its provision as food

results in increased plant fitness, which likely results in

more diffuse selection on pollen chemical traits compared

with nectar chemical traits. For example, plants exclusively

pollinated by nectar-foraging hummingbirds are unlikely

to experience pollinator-mediated selection on pollen

chemistry. Accordingly, in plant taxa which have pollen as

the sole reward for pollinators (e.g. many Solanum and

most Begoniaceae), selective forces on pollen chemical

traits should be similar to those on nectar chemical traits.

In contrast, pollen defensive secondary metabolite content

should be relatively high in plants where pollen is not a

reward and plants produce both nectar and pollen (Fro-

lich, Hartmann & Ober 2006). As with nectar rewards,

future studies on pollen composition should focus on

multi-species and multi-tissue comparisons to more fully

explore the potential for conflict in plant–pollinator–herbi-

vore interactions.

Similar to leaf tissues, secondary metabolites in nectar

and pollen may have multiple functions, such as defence

(toxic or repellent) and interspecific signalling. Thus their

production may be correlated with other attributes of the

reward that are meaningful to pollinators. Flowers produce


Plant reproductive and defensive strategies 903

a large diversity of volatile compounds, many of which can

also be found in nectar. As with other secondary metabo-

lites, there is evidence for both a passive diffusion and

active secretions of these compounds from the secreting tis-

sues into the nectar (Raguso 2004). Although floral volatiles

may not have a direct defensive function, their value as a

signal is depending on the association with reward quality

by the pollinator (Wright & Schiestl, 2009) and thus should

be under very similar natural selection as nutrients and

defensive metabolites in pollinator rewards. This may be

why very similar hypotheses have been proposed for why

plants have volatile organic compounds (VOCs) as well as

non-volatile toxic or repellent secondary metabolites in pol-

linator rewards (Adler 2000; Raguso 2004).

Herbivore-induced plant responses andpollinator behaviour

The above examples illustrate the many ways by which her-

bivory can influence the interaction between plants and poll-

inators on an evolutionary scale. In addition, plants have a

vast array of phenotypically plastic responses to herbivory

that can influence their interactions with other organisms,

including pollinators, in ecological time. While phenotypic

plasticity itself is subject to natural selection, the extent to

which the plasticity of a trait influences selection on the same

trait is still debated (Agrawal 2001). An herbivore-induced

change that reduces pollinator attraction could be viewed as

an additional cost of inducible plant defences (Heil & Bald-

win 2002). This would make pollinator attraction an impor-

tant selective agent in a plant’s adaptive response to

herbivory. However, induced responses to herbivory could

also be viewed as adaptive strategies of plants to maximize

fitness under the altered environmental conditions of herbiv-

ory. The herbivory-induced accumulation of a defensive

metabolite can save the plant the costs of defences that

would accompany the constitutive production of this trait

when herbivores are absent, including the ecological cost of

being less attractive to pollinators. Plants induce defences in

response to an herbivore attack and can therewith limit the

potentially fitness-reducing removal of leaf tissue (Karban &

Baldwin 1997). Thus, induced plant responses that influence

pollinator behaviour could represent a mechanism to cope

with the compromising selection by herbivores and pollina-

tors. In the absence of herbivory, plants would escape the

antagonistically pleiotropic link between defensive and

reward secondary metabolite accumulation to allow maxi-

mal pollinator attraction. We suggest that the extent to

which induced plant responses to herbivory influence polli-

nator behaviour will depend on whether the benefits of

induction for herbivore resistance outweigh the costs of

deterring pollinators for plant fitness.

H E R B I V OR E D A M A GE AN D P O LL I N A T O R BE H A V I O U R

Evidence for the effects of herbivore damage itself on poll-

inators is found in an increasing number of recent studies

documenting the dramatic, immediate effects on a plant’s

interactions with pollinators (Strauss 1997; Adler & Bron-

stein 2004; Irwin, Adler & Brody 2004; Bronstein, Huxman

& Davidowitz 2007). To summarize, there are two principal

ways in which herbivory can influence pollinators: (i) imme-

diately, when pollinators avoid herbivore-damaged flowers

because of the directly altered floral display (Murawski

1987; Karban & Strauss 1993; Cunningham 1995; Krupnick

& Weis 1999; Krupnick, Weis & Campbell 1999; Adler,

Karban & Strauss 2001) or avoid herbivores on the flowers

(Lohmann, Zangerl & Berenbaum 1996), or (ii) subsequent

to the attack, when herbivores induce growth and ⁄or meta-

bolic changes in the plant that mediate the effects of herbiv-

ory on pollinators.

Herbivore-induced effects include reduced flower num-

bers (Karban & Strauss 1993; Lehtila & Strauss 1997;

Krupnick, Weis & Campbell 1999; Mothershead & Marquis

2000; Rathcke 2001), reduced flower size (Strauss, Conner

& Rush 1996; Lehtila & Strauss 1997; Mothershead & Mar-

quis 2000), the alteration of flowering phenology (Freeman,

Brody & Neefus 2003; Sharaf & Price 2004) and sexual

expression (Hendrix 1984; Mutikainen & Delph 1996;

Strauss 1997; Krupnick & Weis 1998; Strauss, Conner &

Lehtila 2001).

A meta-analysis of 16 studies that measured or manipu-

lated herbivory and analysed pollinator responses revealed a

dramatic negative effect of herbivory on pollinator behaviour

(Fig. 1, Table S2). Interestingly, the effects of herbivory on

pollinator behaviour differ, depending on where the plants

were damaged, e.g. flowers, shoots or roots (Fig. 1). The

strongest reduction in pollinator visitation was caused by

–1·5

–1·0

–0·5

0·0

0·5

1·0

1·5

Mea

n ef

fect

siz

e (d

+)

Flowern = 4

Leafn = 10

Rootn = 2

Fig. 1. Impact of herbivory on pollinator visitation. Hedge’s d-values

were calculated in a meta-analysis and the effect of flower, leaf and

root herbivory was calculated as mean effect sizes (d+). Sample sizes

n for each herbivory category are indicated and 95% bootstrap confi-

dence intervals are shown as error bars. Comparison of the impact of

the different herbivory categories by between-group heterogeneity

statistics revealed significant differences between the effects of flower,

leaf and root herbivory on pollinator visitation (Q = 8Æ4225,P = 0Æ015). Studies included are listed in Table S2.



damage inflicted directly to the floral tissue, whereas root her-

bivory actually resulted in increased pollinator attraction in

two published studies (Poveda et al. 2003, 2005). Most of

these studies only measure morphological changes in flowers

or inflorescences of herbivore-attacked plants, but do not

consider herbivore-induced changes in the chemistry of floral

rewards or display as alternative factors influencing pollina-

tor behaviour. However, relatively minor herbivore-damage

to leaf tissue may result in a more rapid and significant recon-

figuration of the plant’s secondary metabolism and reward

quality, while flower size and number remain constant.

H E R B I V OR E - I N D U C E D C H A N G E S I N P O L L I N A T O R

R E W A R D Q U A L I T Y

Recent studies find significant herbivory-induced changes

in the pollen and nectar reward quality and quantity of

attacked plants that can influence pollinator attraction and

constancy (see Quesada, Bollman & Stephenson 1995; Aizen

& Raffaele 1996; Strauss, Conner & Rush 1996; Krupnick,

Weis & Campbell 1999). Even small herbivory-induced

reductions in nectar production and concentration can signi-

ficantly affect pollinator visitation and may shorten or elimi-

nate visitations to flowers (Zimmerman 1983; Cresswell &

Galen 1991; Mitchell & Waser 1992; Ackerman, Rodrigues-

Robles &Melendez 1994; Hodges 1995). However, it remains

unknown how such herbivore-induced changes in pollinator

attraction affect male and female fitness of the studied plant

systems.

Similarly little is known about the mechanisms that lead to

induced reduction in pollinator reward quantity and quality.

Interestingly, and contrary to floral nectar, extrafloral nectar

production is usually increased in herbivore-attacked plants

that bear such nectaries (Heil et al. 2001), which suggests that

very similar damage and the elicited wound signalling path-

ways affect nectar production differentially in floral and ex-

trafloral nectaries. Wound-induced jasmonate signalling has

been shown to mediate the increased extrafloral nectar pro-

duction, but its effect on floral nectar production remains

unknown. Jasmonate signalling is known to be crucial for

induced plant defences to herbivory, such as increased sec-

ondary metabolite accumulation, and is also involved in

flower development (Creelman & Mullet 1997). Therefore,

many of the induced phenotypic changes in floral display and

pollinator reward quality and quantity could be subject to

jasmonate-mediated regulation. This can certainly be true for

induced changes in pollen and nectar quality. Pollen from sta-

minate flowers on partially defoliatedCucurbita texana plants

is less likely to sire seeds than pollen from undamaged plants,

demonstrating that leaf damage can not only decrease pollen

production but also pollen performance (Quesada, Bollman

& Stephenson 1995). If the reduced performance of pollen is a

result of the altered chemical composition of pollen, this may

affect the behaviour of pollen-collecting pollinators. A similar

reduction in pollen quality and size has been observed in her-

bivore-attacked Alstroemeria aurea (Aizen & Raffaele 1996,

1998) and Lobelia siphilitica plants (Mutikainen & Delph

1996). While we know very little about the secondary

metabolite content of pollen and even less about its herbiv-

ory-induced changes, recent studies find induced changes in

nectar constituents with ecological consequences. Adler et al.

(2006) found that leaf and nectar alkaloid production is phe-

notypically correlated in Nicotiana tabacum plants and that

herbivory induces an increased alkaloid production in nectar

that could decrease pollinator preferences (but see Baldwin

2007).

P O L L I N A T O R B E H A V I O U R A N D H E R B I VO R E - I N D U C E D

C H A N G E S I N V O C E M I S S I O N

Herbivore-induced changes in secondary metabolism also

alter the volatile signalling of plants in ways that can have

profound influence on pollinator attraction. There are only

few examples of herbivore-induced changes in floral volatile

emission but there is a large body of literature describing the

specifically induced production of leaf VOCs on herbivore-

attacked plants (reviewed in Janssen, Sabelis & Bruin 2002).

While these herbivore-induced leaf volatiles can function as

attractive signals for predators and parasitoids, and thus as

indirect defences, they may also have signalling function for

pollinators, as they affect the composition of the odour plume

emanating from the plant, as well as the context in which

flowers are perceived (Beker et al. 1989). Because induced

volatile signalling is correlated with changes in plant quality,

pollinators could use these same volatile signals to gain infor-

mation about the reward quality of the plant as well as about

the risks (predation) associated with approaching a plant if

previous herbivory attracted more predators that could pose

a risk to the pollinators (Dukas & Morse 2003; Munos &

Arroyo 2004). In the following case study we found such a

correlation between herbivore-induced floral and leaf volatile

emission and the behaviour of the pollinators as well as a

flower-specific secondary metabolite production, and discuss

these findings in the context of the plant’s reproductive

strategy.

Herbivory-induced pollinator limitation inSolanum peruvianum

The wild tomato plant S. peruvianum is native to the pacific

slopes of theAndeanmountains of SouthAmerica and occurs

in seasonal dry forest habitats. Solanum peruvianum, like all

tomato plants, does not produce floral nectar and the sole

reward for visiting pollinators is pollen stored inside the

anther cone surrounding the stigma. This morphological fea-

ture found inmost Solanum species limits the potential pollin-

ators to certain bees that are able to ‘buzz’ flowers to release

the pollen from the anther cone for collection (Luo, Zhang &

Renner 2008). In the native habitat of S. peruvianum in Peru,

we observed bees of the Apidae, Colletidae and Halictidae

families as the most abundant pollinators. While working on

a number of different native tomato species in Peru we

observed that leaf tissue damage by a diverse herbivore

community was generally correlated with reduced pollinator



visitation. This reduced pollination could be the result of an

altered pollinator behaviour in response to a changing reward

quality and ⁄or floral signalling in herbivore-attacked plants

compared with unattacked plants. Here we test the above

hypotheses by analysing pollinator behaviour and the volatile

and non-volatile secondary metabolite production in herbi-

vore-attacked and undamaged plants.

Materials and methods

Our initial field observations in Peru motivated the characterization

of the underlying mechanisms in more controlled manipulative

experiments. The study was performed in Ithaca, NY, rather than in

Peru in order to control plant history and development, and to

manipulate herbivore damage. Furthermore, the utilization of a single

pollinator species, Bombus impatiens (Apidae), reduced the complex-

ity of the study system and allowed a focused behavioural analysis.

Bombus impatiens is behaviourally and functionally very similar to the

bee species that we observed in the native habitats of S. peruvianum

and would allow subsequent insect physiological experiments that

we can not perform with the native bee species in the field.

To investigate the functional link between herbivory and pollina-

tion we planted 16 S. peruvianum plants (accession LA1616) in field

plots at Cornell University (Ithaca, NY, USA). The seeds were pro-

vided by the TGRC collection (UC Davis, CA, USA) and sown in

mid-April. Seedlings were grown in the greenhouse for 6 weeks before

they were planted into the field plots. All experiments were performed

on flowering plants at the end of September. For chemical analysis we

used tissue from flowers that opened at least 3 days after the initial

damage by an herbivore (10–15%of leaf tissue removed) or from sim-

ilarly developed flowers of undamaged plants.

As a first step to characterize the chemical profiles and to evaluate

the defensive and semiochemical function of volatile and non-volatile

compounds of foliar and floral tissue, we harvested leaves, anther

cones and pollen from individual plants. Samples were weighed, flash

frozen in liquid N2 and extracted with 80% MeOH. Phenolic com-

pounds were analysed by HPLC using a standard method (Keinanen,

Oldham&Baldwin 2001).

To analyse herbivore-induced resistance, six plants each were

exposed to herbivory by 6 third-instarManduca sexta larvae (10–15%

leaf tissue removed). Another six plants remained undamaged as

controls.After10 days larvaewere removedandnewM. sextaneonate

larvae were placed on plants of both treatment groups. The neonates

were allowed to feed for 7 days and were then removed and weighed.

To measure pollinator preferences, we generated arenas of individ-

ual plants with Manduca-attacked and undamaged branches. Previ-

ous experiments (A. Kessler, unpublished data) had shown that

branches on S. peruvianum plants respond independently to damage

and do not result in systemic plant responses that can be measured in

distant branches. Flower preference and remaining time on the flow-

ers newly opened after the damage were recorded for individual

B. impatiens bumblebees within the arena, so that each bee observa-

tion resulted in a proportion of visits to flowers in the two treatment

categories as well as an average residence time for each bee on flowers

of damaged and undamaged branches. A total of 12 bees with a mini-

mum of 12 flower visits within the experimental arena were recorded

and used in the analysis.

To determine whether a change in plant volatile emissions in

response to herbivory could be used by the pollinators as a cue to

avoid flowers on damaged plants, we collected VOCs from the

headspace of leaves and flowers on untreated and herbivore-damaged

S. peruvianum plants. VOCs were collected by enclosing single leaves

in 500 mL polyethylene cups and pulling air through ORBO-32

charcoal adsorbent tubes (Supelco, Bellefonte, PA, USA), using a

12 V vacuum pump (GAST�), Gast Manufacturing Inc., Benton

Harbor, MI, USA). The ORBO-32 tubes were desorbed with

dichloromethane and samples were analysed by GC-MS (Kessler &

Baldwin 2001). Selected ion chromatograms were integrated and

peak areas of individual compounds were normalized by the area of

the internal standard (tetraline).

Results and discussion

F L O R A L AN D V E GE T A T I VE SE C O N D A R Y M E T A B O L I T E

P R O D U C T I ON I N S . P E R U V I A N U M

Solanum peruvianum is an allogamous species and sexual

reproduction is fully dependent on bee-assisted cross-pollina-

tion (Rick 1963). Contrary to the ‘allogamy ⁄ tissue-specificityhypothesis’ our HPLC analysis found correlations between

the content of several non-volatile phenolic compounds in

leaves and pollen, including compounds with reported defen-

sive functions such as rutin (Spearman Rank Correlation,

Z ¼ 1Æ846, P ¼ 0Æ06) and chlorogenic acid (Spearman Rank

Correlation, Z ¼ 1Æ056, P ¼ 0Æ039) (Fig. 2); this result mir-

rors a similar study on the nectar of tobacco (Adler et al.

2006). However, a number of unidentified coumaroyl deriva-

tives were only present in pollen and anther cones, but not in

leaf tissue, while a number of caffeic acid derivatives (e.g.

neo-chlorogenic acid) were found only in leaf tissue but not in

pollen. This result suggests that the plants, at least in part, are

able to control secondary metabolite production or accumu-

lation in a tissue-specific manner.

H E R B I V OR E - I N D U C E D V O C E M I S S I O N O F

S . P ER U VI ANU M

A very similar conclusion can be drawn from the GC-MS

analysis of the volatile emissions of flowers and leaves

(Fig. 3). Several leaf- and flower-specific VOCs could be iden-

tified, while a large proportion of the headspace bouquet was

shared between leaves and flowers. Interestingly, the VOC

emission patterns of flowers and leaves ofManduca-attacked

plants differed significantly from those of undamaged plants

(Fig. 3, Table S3). Some of the compounds were induced in

both flowers and leaves, which, again suggests that leaf and

flower secondary metabolism are closely linked. However,

other compounds were leaf-specifically and flower-specifically

induced, which suggests that a common wound signal is

transmitted throughout the plant but triggers tissue-specific

responses.

H E R B I V OR Y - I N D U C E D C H A N G E S I N P O L L I N A T O R

B EH AV I O U R

The herbivory-induced changes in the secondary metabolism

of S. peruvianum were correlated with reductions in subse-

quent herbivory (Fig. 4a; Student’s t-test, t = 7Æ66,



P = 0Æ0003), pollinator visitation (Fig. 4c; Student’s t-test,

t = 10Æ32, P < 0Æ0001) and residence time (Fig. 4d; Stu-

dent’s t-test, t = 4Æ43, P = 0Æ0006). Hence, foliar herbivory

increased the resistance of the plant to the attacking herbivore

species and reduced its attractiveness to bumblebee pollina-

tors. These results complement findings of earlier studies with

similar negative effects of herbivory on pollinator attraction

(Fig. 1). However, our results differ from most previous

reports on changes in pollinator visitation, which have been

linked to changes in floral display as a result of reduced flower

number or size (Steets & Ashman 2004; Poveda et al. 2005;

Steets, Hamrick&Ashman 2006).We analysed inflorescences

on damaged and undamaged control branches and found no

changes in flower numbers (Student’s t-test, t = 1Æ7,P = 0Æ11, N = 10) and size (Student’s t-test, t = 0Æ9,P = 0Æ37, N = 10), suggesting that nutritional and ⁄or info-chemical changes in the floral tissue mediate the reduced pol-

linator attraction. Although floral chemical traits have been

linked to pollinator behaviour, only one other study so far

showed induced changes in nectar secondary metabolite con-

stituents with an effect on pollinators (Adler et al. 2006).

The avoidance behaviour of B. impatiens to flowers on

damaged plants in our study is strongly correlated with the

altered floral chemistry and is the major correlate we could

identify. Although this is strong evidence that the herbivore-

induced chemical changes in the plant caused this altered pol-

linator behaviour, we do not yet know why the pollinators

avoid the chemically altered flowers. We note that bumble-

bees barely land on flowers of damaged plants (Fig. 4c), sug-

gesting remote evaluation of their quality. The average

residence time on those flowers is less than 2 s, which is cer-

tainly not enough time for the typical ‘buzzing’ behaviour,

which takes longer than 3 s for B. impatiens (Fig. 4d). These

observations suggest that the pollinators use volatile and ⁄orcolour visual cues to differentiate between flowers on dam-

aged and undamaged plants. The latter seems less likely

because corollas and anther cones of damaged and undam-

aged plants showed no obvious colour differences in the visi-

ble light and UV spectrum in preliminary experiments (data

not shown). Whether or not bumblebees use visual or olfac-

tory cues to avoid flowers of damaged plants, we propose

three basic and non-mutually exclusive hypotheses for why

they avoid these flowers.

1. The cue is associated with pollen quality, which

decreases in flowers of damaged plants. An increased

(a)

0·0

0·2

0·4

0·6

0·8

1·0

Con

cent

ratio

n (µ

g/10

0 00

0 po

llen

grai

ns)

Leaf concentration µg/mg fresh mass

Chlorogenic acid

0 1 2 3 4 5 0·0 0·5 1·0 1·5 2·0 2·50·00

0·05

0·10

0·15

0·20

0·25Rutin

(b)

700600500400300200100

0

2500

2000

1500

Abs

orba

nce

A21

0nm

1000

500

1500

1000

800

600

400

200

0

0 5 10 15Retention time (min)

20

4

5

5

5

6 7

3

2

1

684

2

7

9

9

Pollen

Anther

Leaf

72

25 30

0

Fig. 2. Phenolic compound production in

leaf and floral tissue of Solanum peruvianum.

(a) Representative HPLC chromatograms of

leaf, anther cone and pollen samples

(1, neo-chlorogenic acid; 2, chlorogenic acid;

3, quercetin glycoside; 4, 7, 8, unidentified

flavonoids; 5, rutin; 6, quercetin glycoside; 9,

coumaroyl derivatives). (b) Correlation

between chlorogenic acid (y = 0Æ12x ) 0Æ06;r2 = 0Æ453) and rutin (y = 0Æ04x + 0Æ04;r2 = 0Æ212) concentrations in leaves and

pollen.



secondary metabolite production in the pollen could

decrease the pollen quality, but bees may not be able

to assess the quality of the pollen during foraging

(Roulston, Cane & Buchmann 2000). Unless the

ability to avoid pollen of lower quality (Robertson

et al. 1999) from herbivore-damaged plants is inher-

ited or there is a feedback loop from the offspring,

signal–quality association is unlikely to directly

explain the bumblebee preferences.

2. The cue is associated with increased predation pressure.

Herbivore-induced VOC emission can attract predators

and parasitoids to the attacked plant and may increase

predation pressure on mutualist insects such as pollina-

tors. In our experiments we observed no predators on the

plants or flowers, but ‘damaged plant odours’ may be gen-

erally associated with increased predation pressure in their

native habitats if the arthropod populations are strongly

‘top-down’ controlled.

3. The pollinator avoids an ‘unfamiliar’ floral cue. Bee spe-

cies typically have high flower constancy, the mechanisms

of which remain highly contested (Wright & Schiestl

2009). However, if bees are using VOC cues as part of their

5432105 1

2

43 7

6

43

Ion

inte

nsity

(kc

ount

s)Io

n in

tens

ity (

kcou

nts)

21054

3

2

1

0543210

10·0 12·5 15·0Retention time (min)

17·5 20·0

1211

10

98IS

IS

IS

Leaf; Manduca

Flower; Manduca

Flower; Control

Leaf; ControlIS

5

Fig. 3. Volatile organic compound emission

from leaves and flowers ofManduca-attacked

and undamaged (Control) plants of Solanum

peruvianum. Numbers designate identified

compounds that illustrate the difference

between leaf and flower as well as herbivore-

induced and un-attacked tissues. 1, a-pinene;2, unknown monoterpene; 3, b-myrcene; 4,

b-pinene; 5, unknown monoterpene; 6, limo-

nene; 7, cis-b-ocimene; IS, internal standard,

tetraline; 8, methyl salicylate; 9, 3,5-dimeth-

oxy toluene; 10, unknown sesquiterpenes;

11, 3,5-dimethoxy benzaldehyde ⁄ geranylacetone; 12, unknown sesquiterpene.

0·0

0·1

0·2

0·3

0·4

0·5

0·6

0·7

0·8

Pol

linat

or v

isita

tion

rate

Control Manduca damage0

5

10

15

20

25

30

Cat

erpi

llar

mas

s af

ter

7 da

ys (

mg)

Control Manduca damage

Manduca damage

0

1

2

3

4

5

6

Pol

linat

or v

isita

tion

time

(s)

Control

(a) (b)

(d)(c) Fig. 4. Induced resistance in Solanum

peruvianum. (a) Average Manduca sexta

caterpillar mass (+SEM) after 7 days of

feeding on undamaged control plants and

previously Manduca caterpillar-attacked

plants. (b) Bombus impatiens bumblebee

‘buzz’-pollinating a tomato flower. (c) Bom-

bus impatiens preference [average proportion

(+SEM) of undamaged control and Mandu-

ca-damaged flowers visited per observation]

for and (d) average visitation time (+SEM)

on flowers of plants that are attacked

by M. sexta caterpillars or undamaged

(Control).



search image to find flowers that were previously rewar-

ding, flowers on damaged plants could be avoided because

they emit a fundamentally different VOC bouquet. Floral

and vegetative VOC emissions are likely integrated by the

insect given their mutual proximity, which would mean

that an induced change in vegetative VOC emission alone

(floral emission unchanged) might be sufficient to trigger

the avoidance response in the bees. This hypothesis is

supported by preliminary data from another wild tomato

species, S. habrochaites, where only vegetative VOC emis-

sion changes in response to herbivory (floral VOC emis-

sion unchanged) but bees do also avoid flowers on

damaged plants.

H E R B I V OR Y - I N D U C E D C H A N G E S I N S . P E R U V I A N U M

H E R K OG A M Y

In our study the herbivore-induced changes in plant

metabolism were associated with reduced pollinator

attraction, which results in an herbivore-induced pollina-

tor limitation and thus in a potential ecological fitness

cost of plant defences. However, the plant may have

ways to alleviate these fitness costs by also changing

herkogamy (stigma–anther separation; SAS) in response

to herbivore damage. While floral display and the size of

the pollen-containing anther cones remained constant, a

significant reduction in pistil length was induced by

M. sexta herbivory (Fig. 5). This disproportionate change

in size of different flower parts results in a change in

SAS in flowers of damaged tomato plants. Stigma

exertion or SAS is associated with the evolution of

autogamy in the Lycopersicon section of the genus Sola-

num (Chen & Tanksley 2004) and has been identified as

a crucial factor determining selfing rates in several plant

genera including Mimulus (Carr, Fenster & Dudash

1997), Ipomoea (Parra-Tabla & Bullock 2005) and

Datura (Stone & Motten 2002). If the observed reduction

in SAS in wild tomato plants changes the selfing rate in

herbivore-damaged plants it could represent a mechanism

of reproductive assurance allowing the plant to alleviate

fitness consequences of the herbivory-induced pollinator

limitation. The adaptive function of this proposed mech-

anism should be highly dependent on the type of self-

fertilization barrier.

These hypotheses can be tested in detailed behavioural

and comparative field studies and allow analysing the

causal links between herbivory and reduced pollinator

attraction. Furthermore, such studies provide an oppor-

tunity to integrate functional floral morphology and

chemistry in a more balanced and dimensional concept

of floral ‘phenotype’. Additional chemical analyses of

pollinator reward quality and herbivore-induced changes,

in a comparative context, should allow us to identify

common patterns in the production of plant secondary

metabolites and their impact on the mediation of herbi-

vore–plant–pollinator interactions.

Conclusion

We have discussed predictions arising from theory on

how herbivores and pollinators interact to shape the evo-

lution of chemical floral traits. The literature analysis

and our data from S. peruvianum partially support the

allogamy ⁄ tissue specificity hypothesis. While a large pro-

portion of secondary metabolite production in vegetative

tissues is correlated with the content in the pollinator

reward, other proportions can also differ tissue-specifi-

cally. The second hypothesis (allogamy ⁄ induction hypoth-

esis) is supported by our data from S. peruvianum that

show that secondary metabolite production in floral tis-

sues can be induced by herbivory. The literature review

and our own findings identify major gaps in our under-

standing of how plant defence and pollination are coor-

dinated through secondary metabolism.

First, we need a more systematic comparative analysis

of pollinator reward chemistry and its relationship to

whole plant secondary metabolism. The analysis should

be performed across and within different taxonomic

groups in the light of the defensive and reproductive

strategies of each included species. In order to accom-

plish this goal, systematic studies of defence chemistry in

the vegetative and reproductive tissues of taxonomically

related groups should be integrated with detailed case

studies of the pollination and defence ecology of some

of the constituent species. At present, these two fields of

research are just beginning to connect (Adler & Irwin

2005; Irwin & Adler 2006; Andrews, Theis & Adler

2007; Bronstein, Huxman & Davidowitz 2007). While

making this connection, particular attention should be

paid to the identity and tissue-specific production of the

0

2

4

6

8

10

Pistil

Org

an le

ngth

(m

m)

Control Manduca damageManduca damage Control

Anther cone

*

Fig. 5. Herbivore-induced changes in herkogamy. Anther cone and

pistil length were measured in flowers on Manduca sexta-damaged

Solanum peruvianum plants (Manduca damage) and plants that

remained undamaged (Control). Only pistil length changed signifi-

cantly in response to Manduca attack (*Student’s t-test, t = )2Æ69,P = 0Æ02), while the size of the anther cone remained constant

(t = )0Æ313,P = 0Æ76).



compounds and compound classes that similarly or dif-

ferentially affect pollinator and herbivore behaviour (e.g.

is the production of certain compound classes more

likely than others to be selected on by both, herbivores

and pollinators). This will provide crucial information

about the phylogenetic constraints that may limit plants

in coping with compromising natural selection by herbi-

vores and pollinators. At this point we can only specu-

late on this question because studies of the natural

selection on floral chemical traits by pollinators and her-

bivores are almost entirely missing. Moreover, there is a

need to better understand the physiological mechanisms

of how defensive secondary metabolites are secreted into

the nectar and pollen rewards.

Second, we found only a few examples of herbivore-

induced changes in floral chemistry and their effects on

pollinators (Adler et al. 2006), although there are exam-

ples of changing nutrient content and quality in nectar

and pollen (Quesada, Bollman & Stephenson 1995; Aizen

& Raffaele 1996; Strauss, Conner & Rush 1996; Krup-

nick & Weis 1999). Here we propose induced changes in

whole plant and floral chemistry as a potential plant

strategy to alleviate fitness costs that might derive from

an antagonistic pleiotropic interaction between whole

plant and floral chemistry. Changes in plant VOC emis-

sion in response to herbivory may provide the informa-

tion for both herbivores and pollinators to assess the

quality of the plant as a food source. With respect to

plant defensive chemistry, pollinators are herbivores too

(Praz, Mueller & Dorn 2008), but we do not know the

extent to which pollinators use the same cues as herbi-

vores to assess plant quality, nor do we know much

about the reliability of plant VOC signals. Thus, it seems

important to consider both the herbivore-induced

changes in floral- as well as leaf-VOC emission as poten-

tially informative cues for pollinators. Studies of pheno-

typic plasticity of floral chemical traits in general, and

VOC signalling in particular (e.g. Majetic, Raguso &

Ashman 2009), should be included in the systematic

analysis of floral and defensive leaf chemistry and should

be viewed as interconnected entities.

Through their direct and immediate effects on fitness, poll-

inators and herbivores are the agents of selection that most

likely influence pollen and nectar chemistry in most cases.

Future studies that combine phylogenetic information,

whole-plant and pollinator reward chemistry, and functional

molecular biology in ecologically relevant settings (Kessler,

Gase & Baldwin 2008) will provide us with the tools to better

predict evolutionary outcomes between plants, pollinators

and herbivores.

Acknowledgements

We thank Robert A. Raguso, Lynn Adler, Anurag Agrawal, Stuart Campbell,

Katja Poveda, IanKaplan, Amy L. Parachnowitsch, Robert F. Bode and three

anonymous reviewers for helpful suggestions on the manuscript and inspiring

discussions when developing the ideas presented in this paper. The study was

funded by theNational Science Foundation (DEB-0717139).

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Received 8 June 2009; accepted 23 July 2009

Handling Editor: Robert Raguso

Supporting information

Additional Supporting Information may be found in the online ver-

sion of this article.

Table S1.Comparison of foliar and floral concentration of secondary

metabolites.

Table S2. Meta-analysis of the effects of herbivory on pollinator

attraction.

Table S3. Average emission (±SEM) of selected volatile organic

compounds from leafs and flowers of undamaged control andMand-

uca sexta-attacked Solanum peruvianum plants. The data were com-

pared with a two-way ANOVA and the effects of organ (leaves vs.

flowers), treatment (undamaged vs. Manduca-damaged) and

organ · treatment interactions are presented. Compounds are cate-

gorized as being emitted from leaves (L), flowers (F) or in response to

M. sexta hornworm damage (M).

Please note: Wiley-Blackwell is not responsible for the content or

functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.



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