Plant-animal interactions and seed output in twoinsect-pollinated herbs

Anna R. Waites

2005

Department of Ecology and Environmental ScienceUmeå UniversitySE-901 87 Umeå

Sweden

AKADEMISK AVHANDLING

Som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet förerhållande av filosofie doktorsexamen I ekologi kommer att offentligen

försvaras lördagen den 1 oktober, kl. 13.00 I lilla hörsalen, KBC.

Examinator: Professor Lars Ericson, Umeå Universitet

Opponent Dr. Ørjan Totland, Department of Ecology and Natural ResourceManagement, Norwegian University of Life Sciences, Ås, Norway.

ISBN: 91-7305-911-0© Anna R. Waites 2005Printed by: Media-Tryck

ORGANISATION

Umeå UniversityDept. of Ecology and Environmental ScienceSE-901 87 Umeå

DOCUMENT NAME

Doctoral DissertationDATE OF ISSUE

September 2005

AUTHOR: Anna R. Waites

TITLE: Plant-animal interactions and seed output in two insect-pollinated herbs

ABSTRACT I combined comparative and experimental studies in the field and in the greenhouse to examinefactors influencing reproductive success in two insect-pollinated herbs, the tristylous, self-incompatible perennial Lythrum salicaria and the self-compatible, biennial Pedicularis palustris.More specifically, I explored (i) the effects of plant population characteristics on the intensity andoutcome of interactions with pollinators and seed predators, (ii) whether flower morphology affectsduration of stigma receptivity, and (iii) whether damage-induced reduction in floral display reducespollinator visitation and increases pollen limitation. As predicted, the rate of pollinator visitation tended to increase and the number of flowers probedper plant tended to decrease with increasing population size in L. salicaria, but these relationshipsonly approached statistical significance. By taking advantage of the pollen size polymorphism thatis typical of many heterostylous plants, I could show that the number of compatible pollen grainsreceived increased with population size, and that this was associated with a reduction in pollenlimitation and increased seed output per flower. The deposition of high numbers of incompatibleconspecific and heterospecific pollen grains did not appear to reduce seed set. In P. palustris, fruit set and seed predation varied markedly among populations and years, butthis variation could only partly be explained by variation in population size. Fruit set was positivelyrelated to population size, and seed predation was negatively related to population size, in one ofthree years. Similarly, the level of pollen limitation, which was quantified in two years, variedamong populations, but was not related to population size, density or isolation. In L. salicaria, both the duration of stigma receptivity and the effect of prior self-pollination onseed output varied among style morphs. These differences may contribute to morph-specificdifferences in pollen limitation and seed production documented in the field. The results of a field experiment demonstrated that damage to the shoot apex may markedlyreduce fruit production in L. salicaria, and suggested that this is mainly because damage reducesflower number. I found no evidence that a reduced floral display increased the severity of pollenlimitation. Taken together, the results show that interactions with both pollinators and herbivores maymarkedly affect reproductive output in the two plant species, and that the intensity of bothmutualistic and antagonistic interactions vary considerably in space and time. Moreover, they areconsistent with the hypothesis that pollination success should depend less on population size in self-compatible than in self-incompatible plants.

KEYWORDS: plant reproduction, pollen limitation, pollinator behaviour, heterostyly, seedpredation, Lythrum salicaria, Pedicularis palustris, stigma receptivity, self-pollen

LANGUAGE: English ISBN: 91-7305-911-0 NUMBER OF PAGES

SIGNATURE DATE: July 19, 2005

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SAMMANFATTNING

Fragmentering, grad av isolering, växtpopulationers storlek och täthet är alla variablersom kan påverka populationsdynamiken hos växter. Effekten av dessapopulationsegenskaper är speciellt viktig att förstå för växter vars populationer ärfröbegränsade. Djurpollinerade växter är beroende av att pollinatören transporterarpollen från ståndare till märke. Pollinatörernas beteende, och därmed mängd ochkvaliteten av pollen som avsätts på märken, kan påverkas av populationsegenskaper.Detta kan i sin tur påverka växternas förmåga att producera bra avkomma.

Jag har kombinerat experimentella och jämförande studier, utförda i fält och i växthus,för att undersöka faktorer som påverkar frösattning hos två insektspollinerade växter:kärrspira (Pedicularis palustris) och fackelblomster (Lythrum salicaria). Mer specifiktså har jag undersökt (i) hur populationskaraktärer påverkar graden avpollenbegränsning, frösättning och fröpredation, (ii) om blommorfologi påverkar hurlänge märket är mottagligt för pollen och (iii) om minskning i antal blommor, som enkonsekvens av skada, minskar antalet pollinatörer och ökar pollenbegränsningen.

Fackelblomster är en flerårig växt. Den är heterostyl och producerar tre olika typer(morfer) av blommor. Placeringen av ståndarna och märket i blomman skiljer mellande tre morferna (se fig 2, sidan 11 i Summary). Märket kan vara placerat på kort,mellan eller lång nivå. I varje blomma finns två ståndarnivåer som är placerade påmotsvarande nivå som märkena hos de andra två morferna (tex korta morfen harmärke på kort nivå och ståndare på mellan och lång nivå). Full frösattning kan endastuppnås om pollen från motsvarande ståndarnivå når märket (dvs hos kort morf måstepollen från korta ståndare avsättas på det korta märket, detta pollen finns endast hosmellan och långa morfen).

Kärrspira är en tvåårig växt (vegetativ första året och blommande andra året). Den ärsjälvfertil och bildar därmed frön då självpollen avsätts på märket. Den är däremot intesjälvpollinerande utan en pollinatör krävs för att förflytta pollen från ståndaren tillmärket för att frön ska kunna bildas. Kärrspira får ofta frön uppätna avkärrspireblomvecklarens (Gynnidomorpha minimana) larv. Denna vecklare lägger äggi blommorna och larven utvecklas sedan i frökapslarna och äter av de mognande fröna.

Fackelblomster och kärrspira växer båda naturligt i olikstora populationer i Skeppsviksskärgård utanför Umeå. I detta område pollineras de båda växterna framförallt avhumlor.

I en tidigare studie av fackelblomster i Skeppsviks skärgård har man visat attpopulationens storlek är positivt relaterat till frösättningen. För att klargöramekanismerna bakom detta resultat använde jag mig av skillnader i pollenkornens

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storlek för att studera kvalitet och mängd pollen som avsätts på märket hos den långamorfen. Jag fann att den totala mängden av fackelblomsterpollen som avsätts påmärken inte varierar med populationsstorlek, men att andelen kompatibelt pollen(pollenkorn som kommer från motsvarande ståndarlängd och därmed ger upphov tillfrön) ökar med populationsstorlek. Det fanns en tendens till att antaletpollinatörsbesök ökade, medan antalet blommor besökta per planta minskade medökande populationsstorlek. På samma sätt ökade antalet frön per blomma ochpollenbegränsningen minskade med ökande populationsstorlek.

Hos kärrspira varierar fruktsättning och fröpredation mycket mellan populationer ochår, men denna variation kan bara delvis förklaras av populationsstorlek. I ett av tre årär fruktsättningen positivt relaterad till populationsstorlek medan fröpredation ärnegativt relaterat till populationsstorlek. Det verkar därför som att både pollinatöreroch fröpredatorer ofta kan hitta och därmed besöka växter även i de minsta och demest isolerade av mina studiepopulationer.

För växter som inte bildar frön från självpollen, kan självpollen förhindra korspollenatt befrukta fröämnen. Det kan ske genom att självpollen tar upp plats på märket ochhindrar korspollen att nå märket, men det kan också kemiskt blockera korspollen attgro på märket. En stor mängd inkompatibelt pollen avsätts på märket hosfackelblomster. Genom att jag utförde kontrollerade själv- och kors-pollineringar, fannjag att både den tid då märket är mottagligt för pollen och effekten av självpollenavsatt före korspollen, varierade mellan fackelblomsters tre morfer. Dessa skillnaderkan bidra till de skillnader som man tidigare funnit i frösättning och grad avpollenbegränsning mellan de tre morferna.

Växtätare kan indirekt påverka fröproduktionen hos växter genom att påverkainteraktionerna mellan pollinatörer och växter. Fackelblomster är ofta skadat av enskalbagge, Gallerucella calmariensis, som äter blad, blomknoppar och toppen avskott. I ett experiment där vi manuellt skadade toppen av skott för att simulera skadorav skalbaggen studerade jag hur minskad blomproduktion, som konsekvens av skada,påverkade pollinatörsbeteende och fröproduktion. Jag fann att skada av skott minskarfröproduktionen och att denna minskning i fröproduktion framförallt är en konsekvensav ett minskat antal blommor. Ett minskat antal blommor per planta påverkadedäremot inte graden av pollenbegränsning.

Sammantaget visar resultaten för dessa båda växter att interaktioner med pollinatöreroch växtätare kan ha stor påverkan på frösättningen. Styrkan av både samverkande ochmotverkande interaktioner varierar betydligt i tid och rum och visar på betydelsen avfleråriga studier. Dessa resultat stöder dessutom hypotesen om attpollineringsframgång borde bero mindre på populationsstorlek hos självfertila än hossjälvsterila växter.

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LIST OF PAPERS

This thesis is a summary and discussion of the following papers, which will bereferred to in the text by their Roman numerals.

I. Waites, A.R. & Ågren,. J. (2004) Pollinator visitation, stigmatic pollen loads, andamong-population variation in seed set in Lythrum salicaria. Journal of ecology,92, 512-526.

II. Waites, A.R. & Ågren, J. Stigma receptivity and effects of prior self-pollinationon seed set in tristylous Lythrum salicaria. Accepted subject to revisions inAmerican journal of botany.

III. Waites, A.R., Alcantara, J.M. & Ågren, J. Shoot apex damage, pollinatorvisitation and pollen limitation in the self-incompatible herb Lythrum salicaria: afield experiment. Manuscript.

IV. Waites, A.R. & Ågren, J. Spatio-temporal variation in fruit set, seed predationand seed output in the biennial herb Pedicularis palustris. Manuscript.

Paper I is published with the kind permission of the publishers.

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TABLE OF CONTENTS

INTRODUCTION .................................................................................................. 7Pollinators and population characteristics ....................................................... 7Pollinators and individual plant characteristics ............................................... 8

AIMS OF THE THESIS .......................................................................................... 9

METHODS ........................................................................................................... 10Study area ...................................................................................................... 10Study species ................................................................................................. 10Description of the studies ............................................................................... 12

Population characteristics and plant reproduction (Paper I, IV) ................ 12Stigma receptivity and effect of self-pollen deposition (Paper II) ............... 13Shoot apex damage and pollination success ............................................... 13

MAJOR RESULTS AND DISCUSSION ..................................................................... 14Population characteristics and plant reproduction (Paper I and IV) .............. 14Stigma receptivity and effect of self-pollen deposition (Paper II) ................. 15Shoot apex damage and pollination success (Paper III) ................................ 16

CONCLUSIONS .................................................................................................... 17

REFERENCES ...................................................................................................... 17

ACKNOWLEDGEMENTS ...................................................................................... 17

THANKS…. ........................................................................................................ 23

APPENDICES: PAPER I - IV

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INTRODUCTION

Changes in land use have fragmented many plant populations that previously spannedover large continuous areas (Vitousek 1994). Other plant populations have alwaysconsisted of small patches of plants scattered in a diverse landscape. The effects offragmentation, isolation, decreasing plant density and plant population size on plantreproductive success is important to understand, especially for understanding thedynamics of plant populations where recruitment is seed limited. Zoophilous plantsdepend on their animal vectors to transport pollen from anthers to stigma. Extendedareas of monocultures may affect the abundance of pollinating insects and this may inturn disrupt plant-pollinator interactions and reduce reproduction in fragmented plantpopulations (Rathcke and Jules 1993; Aizen and Fensinger 1994; Olesen and Jain1994; Cox and Elmqvist 2000). Although recent studies are questioning the view thathabitat fragmentation will result in a wide spread collapse of plant–pollinatorinteractions, they also point out that effects of fragmentation on the pollinating faunais context and species dependent (Westphal et al. 2003, Ghazoul 2005, see also reviewby Hobbs and Yates 2003).

Pollinators and population characteristics

Pollinator behavior and the quality and quantity of pollen deposited may be influencedby population characteristics, which in turn may affect plant reproductive output.Positive relationships between population size and/or density and plant reproductiveoutput have been documented in several studies, many of which attribute variation inplant reproductive output to variation in pollen quality or quantity (e.g. Jennersten1988; Lamont et al. 1993; Ågren 1996; Molano-Flores and Hendrix 1999; Morgan1999; Kéry et al. 2000; Brys et al. 2004 but see also Costin et al. 2001; Mustajärvi etal. 2001; Tomimatsu and Ohara 2002).

The quality and/or quantity of pollen received by an individual plant may vary withpopulation characteristics because of an associated change in pollinator visitation.Pollinator visitation rate may decrease with decreasing population size and density(Sih and Balteus 1987; Jennersten 1988; Waites and Ågren 2004) which in turn maydecrease the quantity of outcross pollen deposited on stigmas (Engel & Irwin 2003;Waites & Ågren 2004). At the same time the number of flowers visited per plant mayincrease with decreasing population size and density, resulting in an increase ingeitonogamous self-pollination (de Jong et al. 1993; Barrett et al. 1994; Hodges 1995).A small population size or density may also reduce pollinator constancy (Zimmerman1981, Chittika et al. 1997; Chittika et al. 1999) and therefore increase heterospecificpollen transfer.

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The effect of increased self-pollen deposition on plant reproductive output differsbetween self-compatible and self-incompatible plant species. In self-compatible plants,seeds can be formed after self-pollination, although in many cases outcross pollen willgive rise to seeds with higher germination and survival probabilities. In self-incompatible plants, seed production depends on the deposition of outcross pollen. Inself-incompatible plants both self-pollen and heterospecific pollen grains depositedmay have negative effects on the number of seeds produced because clogging of thestigma and the stylar tube, and incompatibility reactions can prevent outcross pollenfrom reaching the ovules (Thompson et al. 1981; Galen & Gregory 1989; Scribailo &Barrett 1994; Murphy 2000). In addition self-pollen grains deposited are unavailablefor pollen export to other plants and may therefore reduce male siring success (Harderand Wilson 1998).

Small populations may also be affected by genetic inbreeding, leading to reduced seedset (Ouburg et al. 1991; van Treuren et al. 1991; Lande 1994). It is therefore importantto combine surveys of seed production with pollinator observation, supplementalhand-pollination and other experiments to evaluate possible mechanisms behindvariation in seed production. There can also be considerable temporal variation in theeffect of population characteristics on plant reproduction (review by Hobbs and Yates2003) and studies conducted over several years are therefore warranted.

Pollinators and individual plant characteristics

Individual plant characteristics such as plant height, number of flowers per plant, andflower morphology may affect the proportion of self-pollen deposition. Heterostylousplants provide useful experimental systems for investigating how discrete variation inflower morphology and pollen-pistil interactions affect pollen transfer and seedproduction (e.g. Ganders 1979; Barrett and Glover 1985; Kohn and Barrett 1992;Harder and Barrett 1993; Pailler et al. 2002; Barrett et al. 2004; Ornelas et al. 2004).Heterostylous plants differ in the lengths of their stamens and styles, and may alsodiffer in the strength of the self-incompatibility reaction (Darwin 1877; Dulberger1992). Morph-specific differences in the positioning of the stigma should affect pollenreceipt because stigma position may influence both the duration of stigma receptivityand the efficiency of pollen transfer during pollinator visits. A stigma concealed insidethe corolla tube should be less exposed to sun, wind, and rain, and may thereforeremain receptive for longer than a stigma protruding out of the corolla tube. InSwedish populations of the tristylous, self-incompatible herb Lythrum salicaria, thelong-styled morph tends to produce fewer seeds per flower than the mid- and short-styled morphs, and this can at least partly be explained by differences in the degree ofpollen limitation (Ågren 1996; Ågren and Ericson 1996; I).

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Plant-pollinator interactions may be affected by other plant-animal interactions such asthose between plants and herbivores (Juenger and Bergelson 1997; Mothershead andMarquis 2000; Strauss and Irwin 2004). For example floral herbivores may reduce theattractiveness of damaged plants and thereby reduce reproductive fitness of thoseplants. Plants damaged by herbivores may also delay flowering and disrupt the timingof plant and pollinators (Junger and Bergelson 1997; Sharaf and Price 2004). Whenthe apical meristem on plants is grazed or attacked by inflorescence feeding insects,lateral branching often increases. A reduced number of flowers per plant as well asincreased lateral branching may affect pollinator behaviour and ultimatelyreproductive success. Reduced attractiveness in damaged plants may result in reducedpollinator visitation rates and thereby increased pollen limitation (e. g. Geber 1985;Ohashi and Yahara 2002; Mitchell et al. 2004). In counteraction, pollinators tend tovisit fewer flowers per plant in less attractive plants. This may reduce the amount ofgeitonogamus pollen deposited on the stigma. Reduced amounts of geitonogamuspollen grains could potentially reduce the negative effects of herbivory in self-incompatible plants by reducing pollen limitation caused by stigma clogging, or polleninterference (Auguspurger 1980; Gerber 1985; Klinkhammer et al 1989).

AIMS OF THE THESIS

The goal of this thesis was to examine mechanisms that determine reproductive fitnessin two insect-pollinated herbs. The two plant species differ in life history, but bothdepend on pollinators for pollen transfer and seed production. The main questionsaddressed in this thesis were:

1. Why does seed output increase with increasing population size in Lythrumsalicaria? (Paper I)

2. Does decreased population size, density and increased isolation affect seedpredation and reproductive output in Pedicularis palustris negatively? (Paper IV)

3. Do the duration of stigma receptivity or effects of self-pollination vary among style-morphs in Lythrum salicaria? (Paper II)

4. How does shoot apex damage affect floral display, pollinator visitation andreproductive output in L. salicaria? (Paper III)

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METHODS

Study area

The Skeppsvik archipelago, which is located in the Gulf of Bothnia, outside Umeå,northern Sweden (63°47’N, 20°37’E) consists of numerous islands of various size andage (Figure 1). Plant populations on the islands are naturally fragmented and vary insize. This creates an opportunity to examine effects of population size, isolation anddensity on pollinator behaviour, seed predation and plant reproductive fitness.

Figure 1. Pedicularis palustris and Lythrum salicaria populations studied in this thesis. Thepopulations are situated on the islands of the Skeppsviks archipelago outside Umeå, northern Sweden.Cut in picture of Sweden shows the location of the archipelago.

Study species

Purple loosestrife, Lythrum salicaria L. (Lythraceae) is a self-incompatible, tristylous,perennial herb, which is native to Eurasia and an invasive species in North America(Hultén & Fries 1986; Thompson et al. 1987). It produces three morphologicallydistinct mating types: plants are either long-styled, mid-styled, or short-styled (Figure2). Each flower of a given morph produces two anther whorls at levels correspondingto the levels of stigmas in flowers of the other two morphs (reciprocal herkogamy).Full seed set requires that pollen grains from anthers of corresponding height are

Pedicularis palustris Lythrum salicaria

0 1 2 Km

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deposited on the stigma (Darwin 1877; O’Neil 1994; Mal et al. 1999). Each plant canproduce one to several floral shoots. Flowers develop from leaf nodes in the upper partof the floral shoot. In Fennoscandia, L. salicaria flowers for 6-8 wk in July-August.The flowers are visited mainly by bumblebees, but also by lepidoptera, honey bees,solitary bees and syrphid flies (I). The fruit is a capsule, which matures 6-8 wk afterflowering.

Long - styled Mid - styled Short - styled

Figure 2. Tristyly in Lythrum salicaria. Flower morphs (long-, short, and mid- styled morph) differ intheir relative positioning of anthers and stigma. Arrows indicating stigma position.

Pedicularis palustris spp. palustris (Scrophulariaceae) is a self-compatible, hemi-parasitic herb that is found in fens and moist-wet meadows. In the study area, P.palustris is a strict biennial with no persistent seed bank (A.R. Waites unpublisheddata), and variation in seed production may therefore strongly influence populationdynamics. In this area, P. palustris flowers in June/July and the seeds mature a monthlater. The fruit forms a dry capsule. In the Skeppsvik archipelago, P. palustris is foundon shores where moderate ice movement reduces competition from shrubs and othersuccessional species. P. palustris is primarily pollinated by bumblebees (Kwak 1977;I V ). The tortricid moth Gynnidomorpha minimana (Caradja, 1916) (Insecta:Lepidoptera: Tortricidae) is the most important seed predator in the area (IV). G.minimana flies at dusk and lays eggs in the flower of the host plant, the larvae developand eat the seeds in the fruit that form (one to several larvae can be found in the samecapsule). They will then exit, either by penetrating the capsule wall or through the topof the capsule as it opens, and search for more seeds in other capsules (A. R. Waitespers. observation).

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Description of the studies

Population characteristics and plant reproduction (Papers I and IV)

Because changes in land use have led to increasing destruction and fragmentation ofnatural and semi-natural habitats for many plant species (Jennersten et al. 1992;Vitousek 1994; Fojt and Harding 1995), it is important to evaluate how populationsize, density and isolation affect plant reproductive fitness. The effects offragmentation on plant reproductive success may vary among plants with different lifecycle, pollination biology and breeding system. Fruit and seed set have been found tobe positively related to population size or negatively related to isolation in several self-incompatible plant species (Hobbs and Yates 2003), and in some cases experimentaldata indicate that these relationships are due to insufficient pollen transfer in small orisolated populations (Ågren 1996; Cunningham 2000; Waites and Ågren 2004; Wardand Johnson 2005). In self-compatible plants, pollination success should be lesssensitive to variation in population size, density and isolation than in self-incompatiblespecies, because automatic or pollinator-assisted self-pollination may keep fruit andseed set high when overall levels of visitation and pollen transfer among plants are low(Bond 1994; Kéry et al. 2000).

In the tristylous, self-incompatible herb Lythrum salicaria, a previous study reported apositive relationship between population size and seed set, and a negative relationshipbetween population size and degree of pollen limitation in the Skeppsvik archipelago,northern Sweden (Ågren 1996). To clarify the mechanisms behind these relationships,we took advantage of the pollen size polymorphism typical of heterostylous plants(Dulberger 1992), and documented among- and within-population variation in thequantity and composition of pollen loads on stigmas in L. salicaria. We examinedwhether pollinator visitation and pollen deposition vary with population size and canexplain variation in seed output and pollen limitation.

Plant population size, density and isolation may affect the intensity of both mutualisticand antagonistic biotic interactions influencing plant reproductive success. Therelationship between plant reproductive success and population size, density andisolation should depend on whether interactions with pollinators, antagonists, or bothvary with these population characteristics (Colling and Matthies 2004). If pollinatorvisitation increases with population size and density, plant fecundity may displaypositive density-dependence, and pollen limitation may increase the risk of localextinction of small, low-density populations (Kunin 1993; Groom 1998; Hackney andMcGraw 2001). On the other hand, if seed predation and herbivory increase withpopulation size and density, this may contribute to negative density-dependence ofplant fecundity, and small isolated populations may potentially escape attack

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altogether. In self-compatible Pedicularis palustris, we tested the hypotheses that fruitset and seed predation would increase, and pollen limitation decrease, with increasingpopulation size and density, and with decreasing isolation of populations.

Stigma receptivity and effects of self pollen deposition (Paper II)

In self-incompatible plants, self-pollen may interfere with the ability of compatiblepollen grains to reach the ovules physically through stigma clogging or throughincompatibility reactions. Many studies have shown that self-pollen may reduce seedset in self-incompatible plants (e.g. Campsis radicans Bertin and Sullivan 1988;Polemonium viscosum Galen et al. 1989; Ipomopsis aggregata Waser and Price 1991;Asclepias exaltata Broyles and Wyatt 1993; Burchardia umbellata Ramsey andVaughton 2000). Differences among style morphs in the extent to which self-pollendeposition interferes with the performance of compatible pollen could potentiallycontribute to morph-specific differences in seed output (Shore and Barrett 1984;Scribalio and Barrett 1994; Cesaro et al. 2004). In Swedish populations of thetristylous, self-incompatible herb Lythrum salicaria, the long-styled morph tends toproduce fewer seeds per flower than the mid- and short-styled morphs, and this can atleast partly be explained by differences in the degree of pollen limitation (Ågren 1996;Ågren and Ericson 1996). Paper I revealed that significant fraction of the pollendeposited on stigmas of the long-styled morph was incompatible. In paper II, weconduct controlled crosses to determine whether morph-specific differences in theduration of stigma receptivity and interference from self-pollen may contribute to theobserved variation in seed output and pollen limitation among style-morphs.

Shoot apex damage and pollination success

Herbivore damage can indirectly affect plant fecundity by influencing interactionsbetween plants and pollinators (Juenger and Bergelson 1997; Mothershead andMarquis 2000; Strauss and Irwin 2004). In the study area, the main herbivore ofLythrum salicaria is the chrysomelid beetle Galerucella calmariensis, which feeds onleaves, flower buds and shoot apices, and can markedly reduce seed output (Hambäcket al. 2000). In paper III, we set up an experimental population to examine how shootapex removal influences floral display, pollinator visitation, pollen limitation and seedoutput in L. salicaria. Floral display and pollinator visitation were monitored acrossthe flowering period, the level of pollen limitation was assessed through supplementalhand-pollination, and total fruit production was scored at fruit maturation.

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MAJOR RESULTS AND DISCUSSION

Population characteristics and plant reproduction (Papers I and IV)

Bumble bees were the dominating pollinators of both Lythrum salicaria (I) andPedicularis palustris (IV). The bumble bees were able to locate and visit plants evenin the smallest populations of both plant species.

In paper I, the rate of pollinator visitation and the number of flowers visited per plantvaried significantly among Lythrum salicaria populations. As predicted, the number ofvisits received per plant per unit time tended to increase, while the number of flowersvisited per plant tended to decrease with increasing population size. However,although the regressions of visitation rates on population size had slopes that wereconsistent with prediction, they only approached statistical significance. The absoluteand relative numbers of compatible pollen grains received increased with populationsize, indicating that the efficiency of outcross pollen transfer was higher in large thanin small populations. In contrast, the total number of L. salicaria pollen grainsreceived was not related to population size. The number of seeds per flower increasedand the degree of pollen limitation decreased with increasing population size, which isconsistent with patterns documented in a previous study (Ågren 1996). Seedproduction increased with the receipt of compatible pollen up to about 200 compatiblepollen grains received per flower, but was not related to the number of incompatibleconspecific or heterospecific pollen grains received. The relationships documentedbetween pollen deposition and seed production indicate that the number of compatiblepollen grains received is the most important aspect of the pollination environment foramong- and within-population variation in seed production in long-styled L. salicaria.The absence of a significant negative relationship between the number of incompatibleor heterospecific pollen received and seed set in the present study may suggest thatinterference from this portion of the pollen load was not sufficiently strong toinfluence seed set of long-styled plants in the study populations.

In the study of Pedicularis palustris (IV) we documented considerable spatial andtemporal variation in fruit set, seed predation, and seed output in this self-compatible,biennial herb. Although pollinator visitation and seed predation can be expected todepend on population characteristics such as size, density, and isolation, we foundvery limited support for the hypotheses that fruit set and seed predation wouldincrease, and pollen limitation decrease, with increasing population size and density,and with decreasing isolation of populations. In most years, even small, isolatedpopulations of P. palustris are apparently actively visited by pollinators and detectedby seed predators in the study area. Fruit set increased with population size in one ofthe three years. In that year, the positive relationship between population size and fruit

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set may have been caused by low pollinator activity in small populations. However, inthe following two years, fruit set was not significantly related to population size,density or isolation. Moreover, in the latter two years, seed output per flower waslimited by insufficient pollination in several populations, but the magnitude of pollenlimitation was not related to measures of population size, density or isolation. Theresults suggest that in these years, small, isolated populations were not necessarily lessvisited than large populations. A substantial fraction of fruits were attacked by seedpredators in all years, but levels of seed predation did not increase with population sizeor density. On the contrary, the proportion of fruits attacked by the tortricid mothGynnidomorpha minimana tended to decrease with population size in one of threeyears. This suggests that even small isolated populations are detected by the seedpredator, and that plants in small, isolated populations do not have a higher chance toescape seed predation.

Stigma receptivity and effects of self-pollen deposition (Paper II)

Both the duration of stigma receptivity and the effects of self-pollen deposition onseed set varied among style morphs in tristylous Lythrum salicaria (II). The resultssuggest that the relative seed output of the three style morphs is be influenced by therate at which compatible pollen is received, and by the relative timing of self- andlegitimate cross-pollination.

The negative effect of prior self-pollen deposition on seed set was stronger in theshort-styled morph than in the other style morphs, although the magnitude of thisdifference was contingent on the relative timing of the deposition of self and legitimatecross-pollen. Flowers of the short-styled morph remained receptive for longer if notpollinated than flowers of the other two style morphs. This is consistent with the ideathat the concealed stigma of the short-styled morph should dry out more slowly than astigma that is exposed outside the corolla. A long period of stigma receptivity shouldbe advantageous when pollinator visitation is low. To our knowledge, this is the firststudy comparing quantitatively the duration of stigma receptivity in the different stylemorphs of a heterostylous plant.

Morph-specific differences in seed output have been recorded both in the native and inthe introduced ranges of L. salicaria. Long-styled plants tend to produce fewer seedsand to be more strongly pollen-limited than mid-, and short-styled plants in severalSwedish populations (Ågren 1996; Ågren and Ericson 1996; I), while short-styledplants have been found to produce fewer seeds and to be more strongly pollen limitedin two North American populations (O’Neil 1992). The results of the present study(II) suggest that morph-specific differences in seed output may be observed even if the

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timing of self- and compatible- pollen deposition is the same in the three style morphs.When the rate of pollen deposition is low, the short-styled morph may have anadvantage because of its longer stigma receptivity. If self-pollen is regularly depositedbefore legitimate cross-pollen, the mid-styled morph should have an advantage. InSwedish L. salicaria populations, legitimate cross-pollen is often deposited atrelatively low rates as indicated by frequent pollen limitation (Ågren 1996; Ågren andEricson 1996; I). The likelihood that self-pollen is deposited prior to legitimate cross-pollen may be related to the size of the individual plant, but also to population size anddensity. Geitonogamous self-pollination is likely to increase with the number of openflowers (de Jong et al. 1993; Barrett et al. 1994; Karron et al. 2004), and may beparticularly high in small populations of low density (I).

Shoot apex damage and pollination success (Paper III)

Shoot apex removal reduced floral display, the rate of pollinator visitation and fruitproduction in L. salicaria. However, no significant effects of shoot apex removal onfruit set, seed output per fruit or pollen limitation were detected. This suggests thatdamage to the shoot apex reduces fruit output mainly because of effects on flowerformation. The visitation rate and the number of flowers probed per visit increasedwith the number of open flowers. This is consistent with observations in several otherplant species (e.g., Ohashi and Yahara 1998, 2002; Mitchell et al. 2004; see alsoreview by Ohashi and Yahara 2001).

Because pollinators probed fewer flowers on damaged than on undamaged plants, therisk of geitonogamous self-pollination should have been lower for damaged plants (cf.de Jong et al. 1993; Barrett et al. 1994; Karron et al. 2004). However, this was notreflected in a higher fruit set or seed output per fruit. There are several possiblereasons why differences in flower visitation did not give rise to differences in fruit setor seed output per fruit. First, controlled crosses indicate that the negative effects ofself-pollen deposition prior to cross-pollen deposition may be relatively limited unlessmore than an hour passes between the arrival of self- and cross-pollen (II). Second, itis possible that the difference in number of flowers visited per plant, was too small toappreciably increase geitonogamous self-pollination and associated negative effects onseed production.

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CONCLUSIONS

In this thesis I have shown that the effects of population characteristics on plantreproductive output vary among species, as well as the importance of long-termstudies. In Lythrum salicaria, the quality of the pollen deposited increased withincreasing population size and this was associated with decreased pollen limitation andincreased reproductive fitness.

In Pedicularis palustris, fruit set, seed predation and seed output and their relationshipwith population characteristics (size, density and isolation) varied considerably amongyears. Long-term studies are needed to appreciate fully the magnitude of this variationand its consequences for plant population dynamics.

I have experimentally shown that stigma receptivity and the effects of prior self-pollendeposition vary among style morphs and that this is contingent on the timing ofcompatible pollen deposition

Finally I have shown that shoot apex damage reduces flowering and fruit production,as well as influence pollinator visitation rates and the number of flowers probed pervisit in L. salicaria.

ACKNOWLEDGEMENTS

I would like to thank Jon Ågren and Julio Alcántara for providing helpful commentson earlir versions of this thesis. Financial support for the studies in this thesis werekindly provided by the Gunnar och Ruth Björkmans Foundation, J. C. Kempes MinnesStipendiefond and Hierta – Retzius stipendiefond to ARW and by grants from theSwedish Natural Sciences Research Council and the Swedish Council for Forestry andAgricultural Sciences to J.Å.

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THANKS….

A huge thank you to all the people who have helped me along the crooked path thatled to this book. To start at the beginning thank you Ronny for introducing me to plantpollinator interactions and then Umeå and Jon Ågren. Without you this would neverhad started. Jon for keeping a positive attitude, letting me explore my own ideas (andforcing me to choose when I wanted to do everything) and guiding me in the world ofresearch. Thanks to all of you that have been helping out in the field and to everyoneat the department for the good conversations that made it worthwhile taking a break.Lars Ericson who has always been there to help me out with practical things(especially when leaving Umeå and the country) and for teaching me how to find myway around the archipelago. Anita, Ulf, Ragna and Elsa for brightening up the days inUmeå with good food and company.

Thanks everyone in Melbourne for your help, making it possible for me to continueworking on my thesis. John Morgan who welcomed me at La Trobe University andkept my enthusiasm up by including me in ecological discussions, field trips and otherevents at the University. Lynise, Jody, and all other graduate students that took me outfor coffee and made me feel welcome and enjoy my stay. Bende, for helping me withthe kids, giving me yummy dinners and cups of coffee and for always being therewhen I needed you. Belinda, Sharon, Chris that took me for long horse back rides,talked horses and introduced me to Adult Riders.

Thanks Lena and who found me a place to sit and work in Lund at the department ofPhysical Geography and Ecosystems Analysis and for all you friendly people at thedepartment who made me feel welcome. Without your help I would probably still mesitting at home trying to write.

Last but absolutely not least I want to thank my family. My wonderful parents whohave helped me through the whole process of writing by being there, helping out withthe kids and helping us renovating all our houses. For always being there when I’veneeded you and supporting me and believing in me. Jack and Glenys who have helpedme with the kids and the property during our years in Melbourne. Thanks for the allcups of tea, yummy dinners and warm support. Johan, Anna, Sofia, Fredrik, Henrik,Karin, Antonio, Cathaysa and Jonay for great talks and fun over the years. Tony,Martin, Daniel, Tomas and ? for being the loves of my life, and giving my life truemeaning.