Corolla Herbivory, Pollination Success and Fruit Predation in Complex Flowers:An Experimental Study with Linaria lilacina (Scrophulariaceae)

ALFONSO M. SANCHEZ-LAFUENTE*

Departamento de Biologıa Vegetal y Ecologıa, Facultad de Biologıa, Universidad de Sevilla, Avd. Reina Mercedes 9,41012 Sevilla, Spain

Received: 22 September 2006 Returned for revision: 16 October 2006 Accepted: 19 October 2006 Published electronically: 4 January 2007

† Background and Aims Herbivory on floral structures has been postulated to influence the evolution of floral traitsin some plant species, and may also be an important factor influencing the occurrence and outcome of subsequentbiotic interactions related to floral display. In particular, corolla herbivory may affect structures differentiallyinvolved in flower selection by pollinators and fruit predators (specifically, those ovopositing in ovaries prior tofruit development); hence floral herbivores may influence the relationships between these mutualistic and antagon-istic agents.† Methods The effects of corolla herbivory in Linaria lilacina (Scrophulariaceae), a plant species with complexflowers, were considered in relation to plant interactions with pollinators and fruit predators. Tests were made asto whether experimentally created differences in flower structure (resembling those occurring naturally) may trans-late into differences in reproductive output in terms of fruit or seed production.† Key Results Flowers with modified corollas, particularly those with lower lips removed, were less likely to beselected by pollinators than control flowers, and were less likely to be successfully visited and pollinated. As aconsequence, fruit production was also less likely in these modified flowers. However, none of the experimentaltreatments affected the likelihood of visitation by fruit predators.† Conclusions Since floral herbivory may affect pollinator visitation rates and reduce seed production, differencesamong plants in the proportion of flowers affected by herbivory and in the intensity of the damage inflicted onaffected flowers may result in different opportunities for reproduction for plants in different seasons.

Key words: Complex flowers, corolla herbivory, Linaria lilacina, pollination success, fruit predation.

INTRODUCTION

Floral morphology of complex flowers may have evolvedto accomplish two main, complementary, functions: first,to attract and permit access to efficient pollinators whilerestricting access to illegitimate or ineffective ones (whichcould negatively affect plant fitness via a reduction inmale and/or female reproductive success); and secondly,to protect organs related to sexual functions, and the costlyrewards produced for pollinator attraction, againstunwanted visitors (including robbers, herbivores and pre-dators). Under these assumptions, evolutionary biologistshave been trying to assess the adaptive value of floraltraits since the early 20th century using a wide variety ofapproaches, and numerous authors have presented resultssuggesting that specialized and complex floral mor-phologies may have evolved as a response to selectivepressures imposed by particular biotic agents, withspecial reference to pollinators (e.g. Campbell et al.,1991; Mitchell et al., 1998; Goldblatt and Manning, 2000;Wilson et al., 2004, among others; but see Herrera, 1996;Waser et al., 1996).

However, in the last decade, a number of studies havesuggested that form and integration of different traits incomplex flowers are not always the result of the prefer-ences of particular pollinators for certain floral phenotypes

(e.g. Herrera, 1996, 2001; Aigner, 2001), and that floraladaptations may not be exclusively induced by the mosteffective pollinators (sensu Stebbins, 1970; see alsoMayfield et al., 2001). In other words, selection may beachieved by a variable number of agents that differ in theirability as pollinators, which runs counter to the idea offlower specialization to a narrow range of pollinatorspecies (see Waser et al., 1996; Fenster et al., 2004).

Furthermore, even assuming selection by particular pol-linators on certain floral traits, it has been shown that post-pollination events (such us fruit or seed predation, or theaction of pathogens) may strongly influence the results ofthe pollination services (e.g. Mutikainen and Delph, 1996;Krupnick et al., 1999; Lethila and Strauss, 1999; Herrera,2000a; Mothershead and Marquis, 2000; Gomez, 2003;Cariveau et al., 2004). Thus, an increasing number ofpapers are currently aimed at studying the likelihood offloral evolution not exclusively based upon the interactionbetween plants and pollinators, but using a multilevelinteraction approach including several biotic agents (e.g.Strauss et al., 1996; Herrera et al., 2002; Strauss andIrwin, 2004; Newman and Thomson, 2005b).

Particularly interesting is the presumed influence ofherbivores (see Strauss, 1997). Since the effect ofherbivory on plant reproduction (either as a pre- or as apost-dispersive event) may be highly variable, dependingon the species inflicting or suffering the damage, and theenvironmental and biological conditions of the interacting* For correspondence. E-mail slafuente@us.es

# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

For Permissions, please email: journals.permissions@oxfordjournals.org

Annals of Botany 99: 355–364, 2007

doi:10.1093/aob/mcl267, available online at www.aob.oxfordjournals.org

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

agents, the results of the interaction may eventually bedetrimental (e.g. Herrera, 2000a), neutral (e.g. Sanchez-Lafuente, 2002) or even beneficial (e.g. Newman andThomson 2005a) for plant fitness.

Herbivory on floral structures has been postulated toinfluence the evolution of floral traits in some species (e.g.Galen and Cuba, 2001; Herrera et al., 2002). While fruitpredators may have a direct negative effect on plant fitnessby reducing the number of seeds available for dispersal,corolla herbivory may also be an important factor influen-cing the occurrence and outcome of subsequent bioticinteractions related to floral display (e.g. Strauss et al.,1996; Krupnik et al., 1999; Gomez, 2003). Thus, if corollaherbivory affects structures involved in flower selection bypollinators and fruit predators (specifically, those ovoposit-ing in ovaries prior to fruit development), it may deterboth these mutualists and antagonist agents, thus changingour predictions about the outcome of both interactors.

In this study, the functionality of the complex flower ofLinaria lilacina Lange (Scrophulariaceae) is considered inrelation to its interaction with pollinators, corolla herbi-vores and fruit predators, and an examination is made ofwhether experimental removal of flower parts to simulateherbivory may affect pollinator and herbivore visitationand plant female reproductive success. During thepre-dispersive stage, flowers of this plant species undergoherbivore damage affecting corolla structure, and fruitpredation affecting seed production. Floral herbivores maydamage the zygomorphic corolla (see Fig. 1A) in severalways, generally affecting the lower lips, but also the upperlips or both lips at the same time. While both upper andlower lips may have a role in pollinator attraction (standardin upper lips, nectar guides in lower lips), lower lips mayalso have a mechanical function to facilitate access to thecorolla throat by both pollinators and fruit predatorsovopositing in developing ovaries. Consequently, damageto the upper and/or lower lips may have different conse-quences for flower selection and likelihood of visitation.

Specifically, the following questions are addressed. (a)Are modified flowers less attractive to flower visitors thanintact ones? (b) Do modified flowers impose restrictions topollinators or fruit predators when visiting such flowers?(c) Do such eventual restrictions translate into limitation tofruit and seed production at flower and plant levels? (d ) Isthere a conflicting selection between mutualists and/orantagonists originating from corolla herbivory?

MATERIALS AND METHODS

Study species and site

Linaria lilacina Lange is a perennial herb endemic to theSubbetic mountains in the South of the Iberian Peninsula(Valdes, 1970; Saez and Crespo, 2005). Plants emergeevery season from mid to late winter and produce fromone to many simple or branched stems (mean length +s.d.: 22.07 + 7.65, n ¼ 68), with verticillate leaves, someof which may produce a variable number of flowers attheir top. Morphologically, flowers are zygomorphic(Fig. 1A), consisting of a closed, tubular corolla with

upper and lower lips (variable in colour, including white,blue or violet spots and yellow nectar guides) that obstructthe opening to the throat of the corolla. There is also aspur at the base of the lower lip which collects nectar pro-duced by a nectary located under the ovary inside thecorolla. Functionally, flowers are hermaphroditic and self-incompatible (A. M. Sanchez-Lafuente, unpubl. res.). Inmost cases, flowers are not dichogamous, but someslightly protogynous or protandrous flowers may be even-tually found (pers. obs.). The calyx is small and thepedicel is short. The fruit is a two-locule capsule, usuallydehiscing by valves.

This study was carried out in the Jaen mountains(Southern Spain), where plants usually grow on rockycliffs, generally oriented northwards. Two populationswere selected: Sillon del Rey (SR) and Otinar (OT), withapprox. 80 and 48 plants, respectively, separated byapprox. 3 km. In these populations, flowers are approx.23.01 + 1.62 mm long (mean + s.d.). Nectar length is7.01 + 1.01 mm (n ¼ 80). In the study area, floweringgenerally begins in January–February and, depending onthe population and weather conditions, it may extend for

FI G. 1. (A) Front and side view of a Linaria lilacina flower.(B) Schematic representation of the simulations of corolla herbivory. 1,upper lips removed (UP); 2, lower lips removed (LO); 3, both lips

removed (BL).

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria356

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

2–4 months. After successful pollination, the capsuledevelops containing numerous small brown, flattened,winged seeds. Fruit maturation takes between 18 and 24 d.

Experimental manipulations and natural incidenceof corolla herbivory

In January 2005, 31 experimental plants were randomlyselected in SR and 27 in OT. Manipulations were carriedout during the flowering peak when pollinators and seedpredators were most abundant (early-April to mid-May),and were designed to imitate the damage caused to corol-las by floral herbivores (currently, one unidentifiedGeometridae larva and a grasshopper are known toconsume floral tissues). These herbivores may damage thecorolla in several ways, affecting the upper and lower lipsalone or both at the same time. Nectar robbery has beenreported in several studies on L. vulgaris as a commontype of damage to flowers (e.g. Stout et al., 2000;Newman and Thomson, 2005a, b). Nevertheless, no evi-dence of nectar robbery was found at the study site, andthis does not seem to be an isolated example, since it isalso absent in other distant (.150 km) L. lilacina popu-lations (pers. obs.). For this reason, nectar robbery was notsimulated as it does not occur naturally. Consequently, thetype of floral herbivory simulated in this study is the onlytype of damage to reproductive tissues that was actuallyobserved at the site.

Damage to corollas was simulated by assigning flowersto one of the following treatments (see Fig. 1B): UP, theupper lips were gently removed with a pair of scissors(Fig. 1B-1); LO, the lower lips were removed in the sameway (Fig. 1B-2); BL, both upper and lower lips wereremoved (Fig. 1B-3); CTRL, flowers not suffering fromeither natural or experimental damage (i.e. resembling theabsence of corolla herbivores, taken as controls and pro-tected with bags when not under observation). All treat-ments were randomly replicated in at least four randomlyselected flowers within each plant (range 4–8 flowers pertreatment and plant, depending on flower availability andplant size). Treatments were applied approx. 1 d before theflowers opened (i.e. upper and lower lips expanded), andwere fully available to pollinators. Overall, 928 flowerswere tagged. Furthermore, 18 more plants in SR and 17 inOT were used to assess natural corolla herbivory duringthe study season. These plants were not manipulated, andwere used to record the type and abundance of the differ-ent corolla herbivores, total flower production, totalnumber of fully functional flowers damaged and type ofdamage inflicted (categorized as damage to lower lips, toupper lips or to both lips). Counts were made at weeklyintervals throughout the experimental study period.

The unexpected and particularly adverse winter con-ditions experienced during the study season in theMediterranean area in Southern Spain (unusual cold anddry weather during most of the late winter and spring)affected the phenology and physical conditions of manyplants in the study site. Thus, some of them did not flowerat all, while others were lost a few days after floweringbegan, or produced very few flowers. For these reasons,

the initial number of plants was reduced to 27 in SR and22 in OT; thus the number of tagged flowers was reducedby n ¼ 144. Further, in the plants left and due to suchadverse weather, n ¼ 307 flowers were lost before fruitmaturation. Thus, the final number of flowers consideredto assess fruit set and seed production was 477 (n ¼ 262in SR, range 52–76 flowers per treatment; and n ¼ 215 inOT, range 42–62 flowers per treatment).

Pollinator censuses and variables estimated

Use of the manipulated flowers by visitors was assessedby 5 min censuses conducted on all tagged plants in thetwo selected populations. Each day a plant was randomlychosen for the first count, then the rest were sequentiallymonitored several times a day. During each census, thespecies or genus of all visitors was noted, together withthe floral treatment assigned to the visited flowers. Floraldisplay (estimated as the number of functional flowers ineach plant) was recorded every day before censusesstarted. The total number of censuses was 681 (298 at SRand 383 at OT), with 136 censuses yielding at least onevisit (76 in OT and 60 in SR).

To assess whether experimental manipulations had anaffect on insect visitation, a check was first made as towhether potential pollinators attempted to visit experimen-tal flowers (i.e. whether corolla manipulation may beresponsible for differences in flower selection by visitors).A visitation attempt was considered as being when aninsect touched the corolla or landed on it. Secondly, anestimate was made of whether a visitation attempt resultedin a successful visitation (i.e. whether treatments imposedany limitations on insects actually to enter selectedflowers). To this end, the flower handling time (HT) wasrecorded as the time elapsed between when an insecttouched or landed on the flower and when it entered thecorolla. If the visitor was finally unable to enter thecorolla, this parameter estimates the time invested beforethe visitor rejected the flower (i.e. the visitation attemptfailed). Finally, the time invested inside the flower in suc-cessful visitations was also recorded. Time was recordedusing a digital stopwatch to the nearest 0.1 s. Overall, 528visitation attempts were analysed.

Fruit set, fruit predation and seed production

Pollination success was assessed by estimating fruit andseed production. All flowers were surveyed every third dayafter the experimental treatment was applied to check forovary enlargement (indicating a successful pollination).Thus, fruit production was considered as a binomial vari-able (fruit developed vs. not developed). Flowers withdeveloping fruits were collected before fruit dehiscence,and the number of seeds produced was counted. Plant size,in terms of total flower production, was recorded at theend of the season.

Developing fruits of L. lilacina are used by Gymnaetronsp. (Curculionidae, Coleoptera) weevils for ovopositionbefore corolla abscission. For this purpose, weevils enterinside the flower through the throat in order to access the

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria 357

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

ovary. In order to test the effect of these herbivores onseed production, half of the experimental plants weresprayed with Syngenta Karate King (a lambda-cyhalothrin-based insecticide) when flowers were no longer functional.Spray was applied every third day. Previous observationshave demonstrated that this product may be well toleratedby bees when diluted and applied according to the manu-facturer’s instructions, while it is effective against weevilattacks (although this effect may be highly variable). Fruitpredation was detected by the presence of a weevil larvainside the mature fruit.

Data analyses

For the purposes of this study, experimental treatment,pollinator group (see Results), population and their inter-actions were considered as main effects in generalizedlinear mixed models. Since treatments were replicatedwithin each plant, plants were used as a random groupingfactor (i.e. as blocks, e.g. Herrera, 2000b), to account fornon-independent data (see Pinheiro and Bates, 2000).Floral display (estimated as the daily number of functionalflowers per plant) was used as a covariate in analysesinvolving pollinator visitation parameters, while plant size(estimated as total flower production) was used in analysesinvolving fruit and seed production. To account for mul-tiple comparisons, P-values were calculated by modelbootstrapping with 1000 repetitions. Statistical analyseswere done using R 2.3.1. (R Development Core Team,2005).

Plant visitation was analysed by testing the likelihoodthat a plant was visited or not during a given census (bino-mial response: visited vs. not visited). Use and handling offlowers within plants were analysed by linear models withlogit link function, in order to test if experimental treat-ments imposed any limitation to insect visitors. Since theexperimental groups were eventually unbalanced due tothe flower losses mentioned above, the number of flowersleft in each experimental group and in each plant was usedas a factor weight. Fruit set and fruit predation events werealso analysed by testing the proportion of experimentalflowers developing fruit, the number of seeds produced perfruit and the likelihood that developing fruits wereattacked by predators.

RESULTS

Incidence of natural corolla herbivory in the studypopulations

In SR, damaged flowers were detected in ten of the 18plants used to asses natural incidence of corolla herbivory(55.55 %). In OT, 11 of the 17 plants used had damagedflowers (64.70 %). While the incidence of corolla herbiv-ory did nor vary between populations (F1,292 ¼ 0.59,P ¼ 0.44), the occurrence of the three types of damagediffered (F2,292 ¼ 9.37, P , 0.001). Such differences wereconsistent between populations (type � population inter-action: F2,292 ¼ 0.46, P ¼ 0.63). Thus, removal of thelower lips, and the lower lips plus part of the upper

lips, was the most frequent damage found (meanproportion + s.d.: 0.43 + 0.25 and 0.37 + 0.32, respect-ively; both populations pooled). Their occurrence wassignificantly higher than removal of the upper lips alone(0.07 + 0.12; both populations pooled; differences testedby post hoc Tukey tests).

Plant size (as measured by total flower production) hada significant effect on the absolute number of flowersaffected by corolla herbivory (F1,292 ¼ 28.66, P , 0.001;larger plants had comparatively more flowers affected;coefficient: 0.045 + 0.008), but not on the proportion offlowers damaged (F1,292 ¼ 3.25, P ¼ 0.08).

Flower visitors and overall plant visitation likelihood

Experimental plants were exclusively visited by Apismellifera (n ¼ 324 records), Anthophora dispar (n ¼ 112),Anthophora acervorum (n ¼ 29) and Bombus terrestris(n ¼ 63). No other pollinators have been recorded visitingL. lilacina plants during three consecutive study seasons(A. M. Sanchez-Lafuente, unpubl. res). Both Anthophoraspecies were pooled into a single group, given theirsimilar size, and similar behaviour when visitingL. lilacina flowers.

The relative abundance of the three pollinator groups dif-fered overall (F2,581 ¼ 4.80, P , 0.02) and between popu-lations (F1,581 ¼ 3.03, P , 0.03), most probably due todifferences in the relative abundance of A. mellifera.Nevertheless, there were no differences between populationsin the probability that individual plants were visited (i.e. polli-nators visiting any flower on a given plant; F1,581 ¼ 0.07,P ¼ 0.79), although a larger floral display contributed toexplain a higher visitation likelihood (F1,581 ¼ 36.27,P , 0.001; coefficient ¼ 0.015 + 0.004,+ s.e.).

However, the number of flowers in a row visited in aplant (i.e. ‘geitonogamous’ visitations) was the highest forA. mellifera (8.29 + 5.24) followed by B. terrestris(4.20 + 3.41) and Anthophora (2.33 + 1.20). Differencesbetween pollinator groups were significant (F2,581 ¼ 4.38,P , 0.04).

Visitation attempts and visitation success relatedto treatment and pollinator group

Flowers in the CTRL and UP groups received more visi-tation attempts than those in the BL and LO groups ( posthoc Tukey tests; Table 1 and Fig. 2A). The effect of thetreatment seemed to be consistent between populationsand for all pollinator groups. An overall effect of the polli-nator group was found, again most probably due to differ-ences in the relative abundance of A. mellifera and itsinfluence on visitation likelihood. Similarly, the interactionbetween pollinator group and population was presumablycaused by differences in the relative abundance ofA. mellifera between populations. Between-plant, variance(18.60) was considerably larger than that found withinplants (8.54), and there were significant differences in themagnitude of the treatment effect among plants (Table 1).Larger floral display was again related to more visitationattempts (coefficient ¼ 0.018 + 0.006, + s.e.).

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria358

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

A visitation attempt could be eventually successful (theflower is visited) or unsuccessful (the flower is rejected).Visitation attempts to control flowers were more likely tobe successful compared with flowers with modified corol-las ( post hoc Tukey tests; Table 1 and Fig. 2B). The likeli-hood that a visitation attempt resulted in a successfulvisitation did not vary among pollinator groups(A. mellifera ¼ 0.78 + 0.41; Anthophora ¼ 0.81 + 0.42;B. terrestris ¼ 0.73 + 0.44). Successful visitations weremore likely in the SR population (0.86 + 0.35) than in OT(0.67 + 0.47), while Anthophora achieved a higher visita-tion success in OT, and A. mellifera and B. terrestris inSR. Between-plant variance (1.09) was again larger thanthat found within plants (0.77). Differences in floraldisplay did not contribute to explain the likelihood of asuccessful visitation.

Considering the likelihood of flower selection for a visi-tation attempt, and the likelihood that a visitation attemptwas successful, flowers in the CTRL and UP groups werealmost twice as likely to be successfully visited (54 and40 %, respectively) than those in the LO and BL groups(25 and 26 %).

Differences in flower handling time between successfuland unsuccessful visitations

The difference in the time invested by visitors toachieve a successful visitation was strongly dependent onthe pollinator group and the treatment (Table 2A). Theoverall trend suggests that Anthophora invested less timein achieving a successful visitation than in rejecting aflower, while A. mellifera did the opposite, and no differ-ence was observed for B. terrestris (Fig. 3). The dispropor-tionate HT of A. mellifera (Fig. 3) seemed responsible forthe significant effects of pollinator group in the differentinteractions. However, the mean time that made the differ-ence between a flower being visited or rejected was veryshort (about 1 s; mean + s.d.: successful ¼ 7.03 + 4.04;unsuccessful ¼ 6.06 + 4.69).

Significant interactions may reflect that pollinator groupsare affected by the type of floral structure damaged in differ-ent ways, but only when flowers were rejected. However,considering successful visitations, the three manipulationsimposed similar limitations to pollinators, with A. melliferainvesting the most time, followed by B. terrestris andAnthophora (Fig. 3). Finally, the effect of floral display wassignificant, with flowers in plants with a larger floral displaybeing handled for longer (coefficient ¼ 0.014 + 0.004,+s.e.). Between-plant variance (28.04) was much largerthan within-plant variance (6.33).

The time spent inside a flower also varied among polli-nator groups (mean + s.d.: A. mellifera ¼ 6.34 + 4.35,Anthophora ¼ 2.03 + 1.22, B. terrestris ¼ 3.650 + 1.39;Table 2B) and between populations (mean + s.d.:

FI G. 2. (A) Mean number of flowers selected for a visitation attempt depending on the treatment (see Fig. 1 for abbreviations). All pollinators andpopulations pooled. Bars with different letters indicate significant differences. (B) Differences among pollinator groups and treatments in the likelihood

that a visitation attempt resulted in a successful visitation. Both populations pooled. Data are means +s.e.

TABLE 1. Results of the models testing for differences invisitation attempts (flower selection) and visitation success(flower visitation) related to experimental treatment,

pollinator group and study population

Fixed effects

Visitation attempts Visitation success

F3,438 P F3,408 P

Treatment (Tre) 3.25 0.03 14.69 0.001Pollinator class (Pol) 193.35 0.02 2.12 0.11Population (Pop) 1.72 0.26 5.29 0.02Tre � Pol 0.91 0.44 0.95 0.53Tre � Pop 2.28 0.11 2.36 0.09Pol � Pop 4.61 0.04 5.64 0.03Tre � Pol � Pop 2.56 0.10 1.32 0.32Floral display 6.09 0.01 0.29 0.59

Block effect L Ratio P L Ratio P

Plant 64.47 0.001 32.62 0.001

Individual plants were considered as blocks. P-values were calculatedby bootstrapping (1000 repetitions). Significant effects are presented inbold.

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria 359

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

SR ¼ 4.21 + 3.45, OT ¼ 3.05 + 3.17). The effect offloral display was again highly significant, with the dur-ation of a visit to flowers in plants with a larger floraldisplay being longer than those to plants with a smallerfloral display (coefficient + s.e. ¼ 0.022 + 0.006). Incontrast to previous findings, within-plant variance (37.48)was much higher than between-plant variance (6.89).

Effects of treatment on flower and plant visitation rates

From the data obtained on the number of within-plantand between-plant visits to flowers with different treat-ments‘, and the mean time invested to manipulate andvisit flowers with different treatments, an estimate wasmade of the likely ‘visitation rate’ of each pollinator groupon each flower depending on the treatment. Such visitationrates were defined according to two variables: number offlowers visited per minute (FVM) and number of plantsvisited per minute (PVM).

FVM varied depending on the treatment (F3,493 ¼141.98, P , 0.001), the pollinator group (F2,493 ¼ 301.03,P , 0.001) and the population (F1,493 ¼ 43.01,P , 0.001), although absolute differences between popu-lations were subtle. Thus, pollinators visiting flowers inthe CTRL group could achieve the highest flowervisitation rate, while those visiting BL groups wouldachieve the lowest (Fig. 4). As for pollinator groups,A. mellifera could visit significantly fewer flowers permin (8.68 + 4.76) than Anthophora (17.58 + 5.83) andB. terrestris (16.66 + 8.88). The interaction between treat-ment and pollinator was significant (F3,493 ¼ 3.43,P , 0.001), suggesting that the treatments affected visita-tion rates in different ways (Fig. 4). No other interactionwas significant, but the effect of floral display was signifi-cant (F1,493 ¼ 8.89, P , 0.004). Overall, flowers on plantswith larger floral display may receive fewer visits permin than those on plants with a smaller floral display(coefficient + s.e. ¼ –3.51 � 1024 + 2.47 � 1024).

Similar results were obtained from PVM. Pollinatorsvisiting flowers in the CTRL group would achieve thehighest plant visitation rates compared with those visitingother flowers (F3,493 ¼ 44.19, P , 0.001; Fig. 4). Again,pollinators differed in their ability in visiting plants(F2,493 ¼ 415.09, P , 0.001). While A. mellifera couldvisit on average 1.80 + 1.40 plants min21, Anthophoracould visit 7.90 + 4.78 and B. terrestris 4.52 + 3.29.The interaction between treatment and pollinator wasagain significant (F3,493 ¼ 3.75, P , 0.001); however, nodifferences were found between populations in plantvisitation rates (F1,493 ¼ 0.29, P ¼ 0.58). Floral displaywas significant in the same way as found above (F1,493 ¼6.45, P , 0.02, i.e. plant visitation rate was inverselyrelated to floral display; coefficient + s.e. ¼ –8.18 �1024 + 6.58 � 1024).

Effects of treatments on fruit set, fruit predationand seed production

Flowers in the CTRL and UP groups were compara-tively more likely to set fruit than those in the other

TABLE 2. (A) Linear model testing the differences in HT as afunction of the fate of the visitation attempts (i.e. whether avisitation attempt resulted in a flower visited or rejected),treatment, pollinator group and population; see also Fig. 3.

Effects

Handling time (HT)

F3,408 P

Successful/unsuccessful (Suc) 16.78 0.001Treatment (Tre) 172.87 0.001Pollinator class (Pol) 129.89 0.001Population (Pop) 25.73 0.001Suc � Tre 0.23 0.87Suc � Pol 54.14 0.001Tre � Pol 3.47 0.003Suc � Pop 0.69 0.40Suc � Tre � Pol 3.63 0.002Floral display 54.28 0.001

Block effect L Ratio P

Plant 122.04 0.001

TABLE 2. (B) Analyses of the time spent inside the flowerexperimental treatment, pollinator group and study

population.

Fixed effects

Time inside

F3,381 P

Treatment (Tre) 1.18 0.31Pollinator class (Pol) 22.78 0.001Population (Pop) 4.81 0.03Tre � Pol 0.61 0.72Tre � Pop 0.96 0.41Pol � Pop 0.60 0.55Tre � Pol � Pop 1.17 0.33Floral display 19.97 0.001

Block effect L Ratio P

Plant 12.42 0.001

FI G. 3. Differences in handling time between flowers visited (V, success-ful visitation attempt) and not visited (NV, failed visitation attempt) foreach pollinator group and treatment (see Fig. 1 for other abbreviations).

Both populations pooled. Data are means + s.e.

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria360

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

treatment groups (Table 3; CTRL ¼ 0.65 + 0.47; UP ¼0.61 + 0.49; LO ¼ 0.48 + 0.50; BL ¼ 0.38 + 0.49). Thetreatment effect was consistent between populations (nosignificant treatment � population interaction). Further,there were no differences between populations (Table 3),with flowers in OT having similar probabilities to set fruit(0.60 + 0.50) to those in SR (0.55 + 0.48). The effectof plant size was not significant, and the varianceexplained was larger between (0.92) than within plants(0.58). Fruit set was not affected by spraying, indicatingno detrimental effects of the insecticide used on pollina-tion services.

The likelihood of fruit predation did not depend on thetreatment, but was related to fruit set (i.e. developingfruits were subject to much more predation than those notdeveloping). In fact, it is possible that manyof the fruitswhich had been sujected to predation that were eventually

considered as not developing were actually successfullypollinated flowers damaged or aborted at very earlystages of fruit development. Fruits on sprayed plants weresignificantly less likely to suffer predation by weevils(Table 3; 0.26 + 0.14) than those on plants not sprayed(0.40 + 0.29). Again, no differences were found betweenpopulations regarding the significant effects. Between-plant variance was larger (0.87) than within-plant variance(0.33).

Finally, no effect of the treatment was detected on seedproduction per flower (24.78 + 10.39 seeds per fruit;Table 3), although significant differences were foundbetween populations. Thus, mean seed production perflower was higher in SR (28.20 + 8.64) compared withOT (20.53 + 10.85). Fruits that had been subject to preda-tion produced significantly fewer seeds (mean ¼ 11.61+ 9.78, + s.d.) than those that had not (27.26 + 8.77).

FI G. 4. Flower and plant visitation rates by each pollinator group depending on the treatment. See text for further considerations on these variables.Both populations pooled. Data are means + s.e. Horizontal lines indicate mean values across pollinator groups. See Fig. 1 for abbreviations.

TABLE 3. Differences in fruit set, fruit predation likelihood and seed production related to experimental treatment and studypopulation

Fixed effects

Fruit set Fruit predation Seed production

F3,408 P F3,408 P F3,392 P

Treatment (Tre) 5.58 0.003 1.83 0.24 0.11 0.98Population (Pop) 0.06 0.84 0.63 0.84 35.18 0.003Fruit set (FrS) – – 9.75 0.002 – –Sprayed 0.33 0.56 38.19 0.001 0.16 0.70Fruit predation (FrP) – – – – 15.54 0.001Tre � Pop 2.34 0.24 1.52 0.38 0.58 0.67Tre � FrS – – 0.38 0.82 – –Tre � FrP – – – – 1.09 0.36Plant size 0.20 0.65 2.89 0.08 0.41 0.52

Block effect L Ratio P L Ratio P L Ratio P

Plant 14.45 0.001 6.79 0.01 1.23 0.27

Individual plants were considered as blocks. P-values were calculated by bootstrapping (1000 repetitions). Significant effects are presented in bold.

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria 361

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

Further, seeds from fruits which hade undergone predationhave a very limited germination ability (pers. obs.). Theeffect of plant size was not significant, while the varianceexplained was much larger between (86.37) than withinplants (4.11).

DISCUSSION

One of the major aims of this study was to examinewhether artificial modifications of a complex flower, thatmimicked natural damage, may alter visitation ability bypollinators and fruit predators, and whether such eventuallimitations may affect plant fitness in terms of seed pro-duction. Among the most usual types of damage found inL. lilacina populations are those that were replicatedexperimentally in this study, affecting upper and/or lowercorolla lips in different ways. This study shows thatremoval of the lower lips (either alone or in combinationwith the upper lips) may have detrimental effects onflower selection and visitation by pollinators, and conse-quently on fruit set (see below), compared with intactflowers or with flowers with only the upper lips removed.However, none of the experimental manipulations affectedthe likelihood of fruit predation, thus suggesting thataccess to flowers of specialized weevils ovopositing indeveloping fruits is not limited by the corolla modifi-cations accounted for in this study. Thus, this studybelongs with those addressing the additive and negativeeffects of floral herbivory and fruit predation on plantfitness via a reduction in seed production (e.g. Mahoro,2003).

Effects of corolla herbivory on flower selection

Flowers with lower lips removed were overall less likelyto be selected for a visitation attempt than those showingno damage or damage to the upper lips, although the mag-nitude of the effect varied among plants. Further, the like-lihood that a visitation attempt was eventually successfulwas lower for flowers with lower lips removed, and thiseffect was consistent among plants. Consequently,L. lilacina flowers may be selected by pollinators accord-ing to cues related to the perception and use of certaincorolla traits. However, although phenotypic selection offloral traits has been commonly reported in the literature(e.g. Andersson and Widen, 1993; Herrera, 1993), recentstudies have suggested that visitors may not discriminateamong flowers based exclusively upon morphological cues(e.g. Herrera, 1996, 2001). There are two known limit-ations of the present study. First, it was not specificallydesigned to detect pollinator preferences according to pheno-typic differences among flowers. Hence, it is difficult toassess whether floral selection may be based exclusivelyupon pollinator perception of corolla traits or upon mech-anical limitations imposed by removal of the lower lip. Ifthe function of the lower corolla lips differs from that ofthe upper lips (see also Herrera, 2001), then pollinators’response may originate simply by the presence/absence ofsuitable structures on which to land. Alternatively, prefer-ences may be caused by the presence/absence of nectar

guides, located in the lower lips, and thus removed byexperimental manipulations. Coloured nectar guides(bluish-to-yellow; variation is common among popu-lations) are usually the first structures consumed by corollaherbivores. These results suggest a future direction in thestudy of the different functions of floral parts in thisspecies. Secondly, the possibility that flower damage maybe a direct cause of fruit losses was not explored.However, while green persistent sepals may contribute tofruit and seed development (Herrera, 2005), this role as aphotosynthetic tissue is unlikely in this particular case,since natural corolla abscission occurs 2–4 d after suc-cessful pollination, while fruit maturation takes 14–24 d.

Effects of corolla herbivory on flower handling

The effects of corolla herbivory may also be estimatedby analysing flower handling, which may be considered asan indirect estimator of the cost/benefit ratio to obtain afloral reward. Although floral herbivory may affect florallongevity (thus reducing the time the reward is availableto pollinators), no influence of the treatments was assumedon the amount or quality of the reward (samples takenfrom naturally damaged and undamaged flowers did notdiffer; A. M. Sanchez-Lafuente, unpubl. res.). HT toaccess flowers was strongly dependent on pollinator groupand treatment. This was particularly the case forA. mellifera, which disproportionately increased HT onmodified flowers (Fig. 4). This behaviour may beexplained by the absence of alternative flowering plants inthe study site during most of the L. lilacina floweringseason (except for Prunus dulcis, Rosaceae, heavily usedby honey bees while flowering), which forced bees to visitthe only flowering species available. Although HT wasshorter overall in rejected than in visited flowers for allpollinators (except for A. mellifera), there is a cost associ-ated with rejection, because visitors also invested sometime on rejected flowers. However, despite the increasedcost associated with handling of experimental flowers, allpollinator groups visited a similar proportion of flowers ineach treatment (no significant treatment � pollinator inter-action; Table 1). In other words, despite the significantdifferences among visitors in the cost associated with vis-iting flowers with modified corollas, all pollinators had thesame ability to visit experimental flowers.

Effects of corolla herbivory on flower and plantvisitation rates

Anthophora and B. terrestris were capable of muchhigher flower and plant visitation rates (for visitation ratesof different Bombus species to L. vulgaris see Stout et al.,2000; Newman and Thomson, 2005a) than A. mellifera,particularly on damaged flowers. However, the likelihoodof a successful visitation did not vary among pollinatorgroups, although available data suggest that there may bedifferences among them in their ability to set seeds(A. M. Sanchez-Lafuente, unpubl. res). Thus, these threepollinator groups vary in the number of flowers visited ina row within a given plant. Considering that L. lilacina is

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria362

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

self-incompatible, the higher number of ‘geitonogamous’visitations may render ineffective a number of visitations.As the likelihood of making ‘geitonogamous’ visitationsincreases, so does the likelihood of acting as an illegiti-mate pollinator, consuming rewards without successfullypollinating. Further, if plants in the study populations havea patchy distribution of genetic diversity (as found for aclosely related species, e.g. Torres et al., 2003; see alsoCresswell et al., 2002), pollinators visiting plants locatedwithin a short range may be more ineffective than thosevisiting plants separated by longer distances, as the risk ofconsanguinity may be inversely related to the distancebetween plants.

Effects of corolla herbivory on fruit predationand seed production

This study has shown that differences in experimentalmanipulations of corolla design, imitating natural floralherbivory, may cause differences in flower selection bypotential pollinators, and differences in fruit set amongplants. Since pollinator efficiency on a per fruit basis (asmeasured by the number of seeds per fruit) was similaramong flowers, regardless of the treatment, the overallconsequence is that individual differences in floral herbiv-ory may promote a variable reduction in plants’ overallseed production. Thus, differences in the proportion offlowers affected by herbivory, and in the intensity of thedamaged inflicted on affected flowers, may result in differ-ent opportunities for reproduction for plants in differentseasons.

Current data for the study species suggest that, whilefruit set is positively correlated with plant size (in terms oftotal flower production), fruit predation rates only increasemarginally as fruit set does (A. M. Sanchez-Lafuente,unpubl. res.). Some studies have suggested that seedpredation may have stronger direct effects on seed set thanpollination, and that plant size and the effects of seedpredators are generally positively correlated (see Cariveauet al., 2004). Fruit predation thus appears as a majorreason for seed losses in this species, as suggested byother related plant species in which pre-dispersal seed pre-dation is frequent (Cariveau et al., 2004), although largerplants may have an advantage over smaller ones, sincemore fruits may escape herbivory as plant size increases.

At least two factors may attenuate the antagonisticeffect of fruit predators on seed production: first, the flow-ering phenology. While this study was carried out duringthe peak of abundance of pollinators and herbivores forpractical reasons, observations suggest that early flowersmay escape from fruit predation at the cost of producingfewer seeds per fruit, since both pollinators and fruit pre-dators are scarce and limited by temperature (pers. obs.).In contrast, later flowers may benefit from a more abun-dant pollinator assemblage and produce more seeds perfruit, but at the cost of a higher predation likelihood.Secondly, within-plant variation in floral traits during theflowering season may influence temporal variation in seedproduction (e.g. Breadmore and Kirk, 1998) associatedwith differences in preferences and abundance of

pollinators and predators. The combined effect of flower-ing phenology and temporal variation in floral traits, onpollinator selection, fruit predation and maternal success isyet to be analysed.

The interaction between mutualisms and antagonisms

The interaction between pollinators and floral herbivoreshas been suggested to affect plant populations in bothdemographic and evolutionary terms (e.g. Galen andCuba, 2001; Kelly and Dyer, 2002), with examples of con-flicting selection between both interacting agents. Further,some hypotheses presume that correlated evolution oftraits favouring mutualistic relationships while avoidingantagonistic ones may play an important role in evolution-ary change (see Herrera et al., 2002). In the present study,a conflicting selection would be likely if traits contributingto herbivore avoidance also contributed to pollinator rejec-tion. Alternatively, if herbivores and pollinators areattracted by different traits, then individual plants exhibit-ing the ‘right’ trait combination to avoid herbivores whileattracting pollinators would be favoured. Although non-additive effects regarding different estimators of femalefecundity have been found between mutualistic and antag-onistic agents in plant populations (namely pollinators andherbivores; e.g. Herrera, 2000a; Gomez, 2005), studiesaddressing non-additive effects between antagonisticagents (i.e. herbivores) are rare (but see Ingvarsson andEricson, 2000; Frey, 2004). However, it was found thatboth herbivory events analysed may have additive effects,and there seems to be no conflict between the pre-dispersive herbivory events accounted for in this study.However, although the most frequent corolla damagefound in natural L. lilacina populations does not seem tohave any direct effect on the likelihood of fruit predation,corolla herbivory has detrimental effects on fruit set via areduction in visitation likelihood by pollinators; hencethere may be an indirect effect on fruit predation causedby a reduced pollination success in flowers with modifiedcorollas.

ACKNOWLEDGEMENTS

I am most grateful to J. Herrera, J. Alcantara, P. Rey andtwo anonymous reviewers for valuable comments on anearlier version of this manuscript, and to A. Simarro forher help during field work. This project is funded by theSpanish Ministerio de Educacion y Ciencia (grantBOS2003-00292).

LITERATURE CITED

Aigner PA. 2001. Optimality modelling and fitness trade-offs: whenshould plants become pollinator specialists? Oikos 95: 177–184.

Andersson S, Widen B. 1993. Pollinator-mediated selection on floraltraits in a synthetic population of Senecio integrifolius (Asteraceae).Oikos 66: 72–79.

Breadmore KN, Kirk WDJ. 1998. Factors affecting floral herbivory in alimestone grassland. Acta Oecologica 19: 501–506.

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria 363

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022

Campbell DR, Waser NM, Price MV, Lynch EA, Mitchell RJ. 1991.Components of phenotypic selection: pollen export and flowercorolla width in Ipomopsis aggregata. Evolution 45: 1458–1467.

Cariveau D, Irwin RE, Brody AK, Sevillano Garcıa-Mayeya L,Ohe A. 2004. Direct and indirect effects of pollinators and seedpredators to selection on plant and floral traits. Oikos 104: 15–26.

Cresswell JE, Osborne JL, Bell SA. 2002. A model of pollinator-mediated gene flow between plant populations with numerical sol-utions for bumblebees pollinating oilseed rape. Oikos 98: 375–384.

Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD.2004. Pollination syndromes and floral specialization. Annual Reviewof Ecology, Evolution and Systematics 35: 375–403.

Frey FM. 2004. Opposing natural selection from herbivores and patho-gens may maintain floral-color variation in Claytonia virginica(Portulacaceae). Evolution 58: 2426–2437.

Galen C, Cuba J. 2001. Down the tube: pollinators, predators, and theevolution of flower shape in the alpine skypilot, Polemonium visco-sum. Evolution 55: 1963–1971.

Goldblatt P, Manning JC. 2000. The long-proboscid fly pollinationsystem in southern Africa. Annals of the Missouri Botanical Garden87: 146–170.

Gomez JM. 2003. Herbivory reduces the strength of pollinator-mediatedselection in the Mediterranean herb Erysimum mediohispanicum:consequences for plant specialization. American Naturalist 162:242–256.

Gomez JM. 2005. Non-additive effects of herbivores and pollinators onErysimum mediohispanicum (Cruciferae) fitness. Oecologia 143:412–418.

Herrera CM. 1993. Selection on floral morphology and environmentaldeterminants of fecundity in a hawk moth-pollinated violet.Ecological Monographs 63: 251–275.

Herrera CM. 1996. Floral traits and plant adaptations to insect pollina-tors: a devil’s advocate approach. In: Lloyd DG, Barret SCH, eds.Floral biology. Studies on floral evolution in animal-pollinatedplants. New York: Chapman & Hall, 65–87.

Herrera CM. 2000a. Measuring the effects of pollinators and herbivores:evidence for non-additivity in a perennial herb. Ecology 81:2170–2176.

Herrera CM. 2000b. Flower-to-seedling consequences of different polli-nation regimes in an insect-pollinated shrub. Ecology 81: 15–29.

Herrera CM. 2001. Deconstruction of a floral phenotype: do pollinatorsselect for corolla integration in Lavandula latifolia? Journal ofEvolutionary Biology 14: 574–584.

Herrera CM. 2005. Post-floral perianth functionality: contribution ofpersistent sepals to seed development in Helleborus foetidus(Ranunculaceae). American Journal of Botany 92: 1486–1491.

Herrera CM, Medrano M, Rey PJ, Sanchez-Lafuente AM, GarcıaMB, Guitian J, et al. 2002. Interaction of pollinators and herbivoreson plant fitness suggests a pathway for correlated evolution ofmutualism- and antagonism-related traits. Proceedings of tneNational Academy of Sciences of the USA 99: 16823–16828.

Ingvarsson PK, Ericson L. 2000. Exploitative competition between twoseed parasites on the Common sedge, Carex nigra. Oikos 91: 362–370.

Kelly CA, Dyer RJ. 2002. Demographic consequences of inflorescence-feeding insects for Liatris cylindracea, an iteroparous perennial.Oecologia 132: 350–360.

Krupnick GA, Weis AE, Campbell DR. 1999. The consequences offloral herbivory for pollinator service to Isomeris arborea. Ecology80: 125–134.

Lethila K, Strauss SY. 1999. Effects of foliar herbivory on male andfemale reproductive traits of wild radish, Raphanus raphanistrum.Ecology 80: 116–124.

Mahoro S. 2003. Effects of flower and seed predators and pollinators onfruit production in two sequentially flowering congeners. PlantEcology 166: 37–48.

Mayfield MM, Waser NM, Price MV. 2001. Exploring the ‘most effec-tive pollinator principle’ with complex flowers: bumblebees andIpomopsis aggregata. Annals of Botany 88: 591–596.

Mitchell RJ, Shaw RG, Waser NM. 1998. Pollinator selection, quanti-tative genetics, and predicted evolutionary responses of floral traitsin Penstemon centranthifolius (Scrophulariaceae). InternationalJournal of Plant Sciences 159: 331–337.

Mothershead K, Marquis RJ. 2000. Fitness impacts of herbivorythrough indirect effects on plant–pollinator interactions inOenothera macrocarpa. Ecology 81: 30–40.

Mutikainen P, Delph LF. 1996. Effects of herbivory on male reproduc-tive success in plants. Oikos 75: 353–358.

Newman DA, Thomson JD. 2005a. Effects of nectar robbing on nectardynamics and bumblebee foraging strategies in Linaria vulgaris(Scrophulariaceae). Oikos 110: 309–320.

Newman DA, Thomson JD. 2005b. Interactions among nectar robbing,floral herbivory and ant protection in Linaria vulgaris. Oikos 110:497–506.

Pinheiro JC, Bates DM. 2000. Mixed-effects models in S and S-plus.New York: Springer Verlag.

R Development Core Team. 2005. R: a language and environmentfor statistical computing. R Foundation for Statistical Computing,Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org.

Saez L, Crespo MB. 2005. A taxonomic revision of the Linaria verticil-lata group (Antirrhineae, Scrophulariaceae). Botanical Journal ofthe Linnean Society 148: 229–244.

Sanchez-Lafuente AM. 2002. Floral variation in the generalist perennialherb Paeonia broteroi (Paeoniaceae): differences between regionswith different pollinators and herbivores. American Journal ofBotany 89: 1260–1269.

Stebbinns GL. 1970. Adaptive radiation of reproductive characteristics inangiosperms, I: pollination mechanisms. Annual Review of Ecologyand Systematics 1: 307–326.

Stout JC, Allen JA, Goulson D. 2000. Nectar robbing, forager efficiencyand seed set: bumblebee foraging on self incompatible plant Linariavulgaris (Scrophulariaceae). Acta Oecologica 21: 277–283.

Strauss SY. 1997. Floral character link herbivores, pollinators, and plantfitness. Ecology 78: 1640–1645.

Strauss SY, Irwin RE. 2004. Ecological and evolutionary consequencesof multispecies plant–animal interactions. Annual Review ofEcology, Evolution and Systematics 35: 435–466.

Strauss SY, Conner JK, Rush SL. 1996. Floral herbivory affects floralcharacters and plant attractiveness to pollinators: implications formale and female plant fitness. American Naturalist 147: 1098–1107.

Torres E, Iriondo JM, Escudero A, Perez C. 2003. Analysis of within-population spatial genetic structure in Antirrhinum microphyllum(Scrophulariaceae). American Journal of Botany 90: 1688–1695.

Valdes B. 1970. Revision de las especies europeas de Linaria con semi-llas aladas. Anales de la Universidad Hispalense no. 7, Universidadde Sevilla.

Waser NM, Chittka L, Price MV, Williams NM, Ollerton J. 1996.Generalization in pollination systems, and why it matters. Ecology77: 1043–1070.

Wilson P, Castellanos MC, Hogue JN, Thomson JD, Armbruster WS.2004. A multivariate search for pollination syndromes among penste-mons. Oikos 104: 345–361.

Sanchez-Lafuente — Corolla Herbivory and Pollination in Linaria364

Dow

nloaded from https://academ

ic.oup.com/aob/article/99/2/355/2391004 by guest on 29 M

arch 2022