Competition of a Nectar-Robbing Bumble Bee with a Legitimate Forager and Its Consequences for Female Reproductive Success of Fuchsia Magellanica

University of Calgary PRISM: University of Calgary's Digital Repository

Graduate Studies The Vault: Electronic Theses and Dissertations

2018-09-14 Competition of a nectar-robbing bumble bee with a legitimate forager and its consequences for female reproductive success of Fuchsia magellanica

Rosenberger, Nick Martin

Rosenberger, N. M. (2018). Competition of a nectar-robbing bumble bee with a legitimate forager and its consequences for female reproductive success of Fuchsia magellanica (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/33042 http://hdl.handle.net/1880/108689 master thesis

University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY

Competition of a nectar-robbing bumble bee with a legitimate forager and its consequences for

female reproductive success of Fuchsia magellanica

Nick Martin Rosenberger

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN BIOLOGICAL SCIENCES

CALGARY, ALBERTA

SEPTEMBER, 2018

Abstract

In pollination systems, competition can cause floral visitors to adopt behaviors at high densities that may antagonize floral reproduction. I evaluated the density-dependence of nectar robbing by a short-tongued bumble bee, Bombus terrestris, and its consequences for both competition with an effective pollinator, Bombus dahlbomii, and female reproduction by the shrub Fuchsia magellanica. Daily sampling documented an abrupt, density-dependent transition from no robbing to almost exclusive robbing by B. terrestris. Robbing facilitated flower visitation by

B. terrestris while aggravating its competition with B. dahlbomii. Nectar depletion and flower damage caused by robbing reduced pollen receipt by F. magellanica flowers, depressed pollen- tube success and reduced fruit quantity and quality. This research demonstrates that by modifying floral conditions to suit their foraging needs nectar robbers can gain a competitive advantage over effective pollinators, possibly promoting their long-term decline, while also compromising reproduction by the affected plant species.

Keywords: competition, mutualism, pollination, nectar robbing, antagonism, density- dependence, invasive species.

Preface

This thesis is original, unpublished, independent work by the author, N. Rosenberger.

iii

Acknowledgements

This thesis was made possible by so many people from different aspects of both my personal and professional life, and to all involved I am eternally grateful.

I would like to thank my co-advisers Lawrence Harder and Marcelo Aizen. To Lawrence

I am grateful for his endless patience and thoughtful guidance throughout this long scientific process. He has made me both a more critical scientist but opened a broader world by helping me become a process-oriented ecologist. To Marcelo I am eternally grateful for his willingness and enthusiasm to help me pursue a dream project and for his kindness, support and excellent mentorship over our years of working together. I would also like to thank my committee members Ralph Cartar and John Post for their conversations and helpful feedback in the analytical and conceptual direction of the project. I thank my funding sources, National

Geographic and the National Science and Engineering Research Council of Canada for supporting my field work and lab work respectively.

I would like to thank my fellow members of the Harder Lab, Lauren Sawich, Ilona

Clocher and Colby Regal for their companionship and friendship through the graduate school process. I also thank my Argentine colleagues Agustín Sáez, Carolina Morales, Marina Strelin and Marina Arbetman for their kind support and friendship during my time in Argentina. In particular, Agustin and Carolina helped tremendously during the early phases of the project helping with study design and deal with logistics in the field and laboratory. I also thank Mora

Ibánez Molina for her invaluable aid in the laboratory and sorting out methods for sample processing.

I thank Rachel Dickson for her wonderful help and friendship in the field, and for enthusiastically embracing the hard work of such a rigorous long study. I also thank Sara Saez

for her help in the field at the beginning of the project. I thank the ranger in Puerto Blest, Sol

Hourmilougue for her logistical support and generosity to ensure the success of this project.

Furthermore, I thank Carlos Mayud for his friendship and for the many dinners he cooked in the

Estación Biológica, and for making sure I had not died in the field when I was out late into the evening during a thunder storm.

I thank my fellow students, Jessica Hopson, Kyle Wilson, Will Murphy, Emily Cribb,

Analisa Lazaro-Cote, Dan Wuitchik, Sara Smith and Danielle Clake for their friendship and continuous support. I especially would like to thank Louise Hahn for looking out for me during my time in graduate school and for her friendship. I would also like to thank my undergraduate mentors Daniel Pletscher and Frank Rosenzweig for their continued support of my research and scientific career beyond my undergraduate career.

I thank my parents Dana and Dave Joslyn, and Ed Rosenberger and Kate Catillaz for their love, always standing by my decision to pursue research and wholeheartedly supporting me in pursing my dreams. Finally, I thank my grandparents Lon and Zoe Richardson for their love, support and shining role in my life.

Table of Contents

Abstract ...... ii Preface ...... iii Acknowledgements ...... iv Table of Contents ...... vi List of Tables ...... viii List of Figures and Illustrations ...... ix

Chapter 1. Interactions in Pollination Systems ...... 1 1.1 Multiple outcomes of direct and indirect species interactions ...... 1 1.2 Pollinator-pollinator interactions ...... 2 1.3 Consequences for plant-pollinator mutualisms ...... 3 1.4 Objectives ...... 5

Chapter 2 – Density-dependent nectar robbing and consequences for competition between two bumble bee species ...... 6 2.1 Introduction ...... 6 2.2 Methods ...... 9 2.2.1 Study species and site...... 9 2.2.2 Plant surveys...... 10 2.2.3 Data analysis...... 12 2.3 Results ...... 15 2.3.1 Dynamics of flowering, bee visitation and the incidence of robbed flowers...... 15 2.3.2 Bee abundance...... 17 2.3.3 Incidence of robbed flowers...... 18 2.3.4 Flower visits...... 19 2.3.5 Flower visit behavior...... 20 2.4 Discussion ...... 21 2.4.1 Density-dependent interactions...... 23 2.4.2 Nectar robbing...... 24 2.4.3 Implications for nectar robbing invasion...... 25

Chapter 3 – Quantitative and qualitative effects of nectar robbing on female reproductive success of Fuchsia magellanica ...... 44 3.1 Introduction ...... 44 3.2 Methods ...... 48 3.2.1 Study species...... 48 3.2.2 Flower and fruit sampling...... 48 3.2.3 Pollen-tube quantification...... 50 3.2.4 Statistical analysis...... 51 3.3 Results ...... 53 3.3.1 Floral longevity...... 53 3.3.2 Pollination and pollen-tube success...... 54 3.3.3 Fruit production...... 55 3.4 Discussion ...... 55

3.4.1 Plant responses...... 56 3.4.2 Invasive robbing and female reproduction...... 59

Chapter 4 – Concluding Discussion...... 68 4.1 Temporal and geographic incidence of nectar robbing ...... 68 4.2 Hummingbirds and Fuchsia magellanica ...... 69 4.3 Long-term ecological and evolutionary implications ...... 70 4.4 Conservation implications ...... 71 4.5 Suggestions for future studies ...... 72

References ...... 74

vii

List of Tables

Table 3.1 – Estimated partial regression coefficients (b ± SE) from non-linear mixed models and generalized linear mixed models...... 60

Table 3.2 – Summary of contrasts for non-linear regressions of variation in the width of the pollen-tube in Fuchsia magellanica styles...... 62

viii

List of Figures

Figure 2.1. Examples of the study species and their interactions with Fuchsia magellanica ...... 32

Figure 2.2. Temporal patterns of (A) overall flowering phenology and least-squares mean ( SE) (B) dahl abundance, (C) terr abundance and (D) proportion of robbed flowers...... 34

Figure 2.3. Temporal patterns of the proportion of visits to Fuchsia magellanica flowers that involved the number of robbing visits and incidence of nectar robbing behavior for dahl and terr during the study period (A and B)...... 35

Figure 2.4. Overall least-squares mean (± SE) abundance of dahl and terr on Fucshia magellanica plants in four habitats during 10-min samples...... 36

Figure 2.5. Partial effects of continuous independent variables on the least-squares mean ( SE) numbers of Bombus dahlbomii and B. terrestris visiting Fucshia magellanica plants during 10-min samples ...... 37

Figure 2.6. Partial effects of interactions between independent variables on the incidence of nectar-robbed flowers...... 39

Figure 2.7. Partial effects of continuous independent variables on least-squares mean ( SE) visitation of Fuchsia magellanica plants by Bombus dahlbomii and B. terrestris during 10-min samples ...... 41

Figure 2.8. Partial effects of continuous effect for individual bee behavior and visits in different habitats (A), proportion of robbed flowers (B,C), and total daily terr abundance during 10-min plant samples...... 43

Figure 3.1 – Photomicrographs illustrating the measurement of pollen deposition on stigmas and variation in the width of the mass of pollen tubes...... 63

Figure 3.2 – Relations of least-squares mean (± SE) floral to (A) average daily temperature while individual flowers were open, and (B) the average proportion of robbed flowers on a plant while individual flowers were open...... 64

Figure 3.3 – Relations of the least-squares mean (± SE) proportion of stigma covered by pollen open-pollinated flowers ...... 65

Figure 3.4 – Relations of the width of the pollen-tube mass at the base of the style to the proportion of the stigma covered with pollen ...... 66

Figure 3.5 – Relations of the least-squares mean (± SE) of proportional fruit set ...... 67

Figure 3.6 – Relations of least-squares mean (± SE) fruit mass to (A) the width of the pollen- tube mass at the base of the style and (B) habitat...... 67

Chapter 1. Interactions in Pollination Systems

1.1 Multiple outcomes of direct and indirect species interactions

The multitude of ecological interactions tend to occur not simply as pairwise interactions

(Bronstein 1994, Wootton 1994). Instead, individuals engage in many interspecific interactions during their lives that range from antagonism to mutualism (Stanton 2003). This range of interactions can co-occur for individuals of each species involved, simultaneously influencing their fitness (Rodriguez-Cabal et al. 2013). Furthermore, the outcome of a direct interaction between species may indirectly influence the outcome of other species interactions (Wootton

1994, Rodriguez-Cabal et al. 2013). This linkage between species interactions is a pervasive feature of ecology and underscores the importance of evaluating species interactions beyond pairwise interactions. Particularly relevant in this context are mutualisms, as partners often also engage in antagonisms associated directly or indirectly with the mutualism (Stanton 2003).

Biotic pollination systems involve the direct and indirect interactions of plants and animal pollinators. Flower-visiting animals and plants can interact mutualistically (e.g. pollen transfer and floral reward) or antagonistically (e.g. removing floral resources without pollen transfer or not providing a reward) (Bronstein 2001a). Furthermore, pollinators can engage with other pollinators and plants with other plants either positively (i.e. facilitation) or antagonistically (i.e. competition) (Pyke 1984, Brown et al. 2002, Moeller 2004, Ghazoul 2006). To the extent that pollinators compete for floral resources, changes in their interactions and behaviors can affect pollination, potentially causing antagonism between pollinators to modify their mutualistic interactions with plants.

1.2 Pollinator-pollinator interactions

Pollinators are mobile animals motivated selfishly to visit flowers, often by the need to collect food, even if the interaction outcome is mutualistic (Pyke 1978). Like other animals, pollinators generally forage in manners that maximize their benefit-cost ratio (Schmid-Hempel et al. 1985, Rasheed and Harder 1997). In doing so, pollinators must contend, directly or indirectly, with other conspecific or heterospecific floral visitors for floral resources (Pyke 1982), perhaps modifying their foraging behavior (Thomson et al. 1987, Brosi and Briggs 2013, Thomson

2016). If competitors increase the cost or reduce the benefit of visiting a plant, then a visitor may shift to other floral species (Pyke 1982) or alter how they access floral resources (Newman and

Thomson 2005). If previous visitors have depleted resources from a plant’s flowers, a pollinator may avoid visiting it to search elsewhere for resources (Stout et al. 1998).

Whether nectar-feeding animals visit a plant species with tubular corollas (including nectar spurs) often depends on their proboscis length relative to the distance to nectar within flowers (Harder 1985). Most species can access shallow nectar, whereas only long-tongued species can access deeply seated nectar. Thus, a short-tongued visitor may reach nectar in tubular flowers only when it is abundant, whereas such flowers become unrewarding during competition with longer-tongued species that lower nectar levels (Pyke 1982, Irwin et al. 2010).

The intensity of resource competition depends on the density of conspecific and heterospecific individuals. Importantly, the density of heterospecific competitors can limit the number of conspecifics able to forage effectively (Richards et al. 2000, Svanbäck and Bolnick

2007). At high competitor densities, individuals may change their behavior to access resources more efficiently. In the context of nectar-feeders feeding on tubular flowers, individuals with short tongues may bypass the front nectar entrance and bite a hole in the side of the corolla

(primary nectar robbing) or use an existing hole (secondary nectar robbing) to access nectar

(Inouye 1978, 1980). Depending on the depth of nectar in unvisited flowers, a short-tongued flower visitor may rob consistently (nectar always inaccessible by front visits), or facultatively in response to competition with other visitors (Irwin et al. 2010, Richman et al. 2017a). Facultative robbing may alter competition with long-tongued competitors, both by allowing short-tongued foragers to extract a resource that is otherwise difficult to access, and by reducing the resources available to long-tongued foragers.

Like other foraging behaviors, robbing is likely to be adopted only when the benefits exceed the costs (Dedej and Delaplane 2005, Richman et al. 2017a). When short-tongued pollinators can access nectar from unvisited tubular flowers, this balance, and hence the incidence of robbing, may depend on the density of competitors for nectar, which can change during the flowering season (Ye et al. 2018). Correspondingly, the nature and consequences of interactions of flower visitors with each other and of plants and flower visitors, including robbers, is likely highly dynamic (Irwin and Maloof 2002). Such dynamism has received limited attention, but probably occurs commonly in pollination systems.

1.3 Consequences for plant-pollinator mutualisms

Being sessile, angiosperms depend on vectors to disperse their pollen and outcross. About

80-90% of flowering plants rely on animals to some degree for this purpose (Ollerton et al.

2011). Successful cross-pollen dispersal requires that pollen is removed from flowers, transported to conspecific plants and deposited on recipient stigmas. Pollen dispersal typically varies extensively among plants and their flowers (Herrera 2004, Richards et al. 2009, Schreiber et al. 2015). Often, pollen dispersal does not result in enough ovule fertilization for plants to mature as many seeds as possible, given the resources available for seed development (Ashman

et al. 2004). Such pollen limitation of seed production depends on both the quantity and quality of pollen dispersal (Aizen and Harder 2007).

Pollination success is highly context dependent and hence dynamic during flowering seasons (Aizen 2001). Particularly relevant features of the pollination environment include the number of potential mates, which determine the pool of available pollen, and pollinator abundance and behavior. These features generally have positive effects on pollination; however, some adverse effects become apparent when these features are disproportionate. For example, low pollinator availability may leave many flowers unvisited or inadequately pollinated, even though plants have potential mates (Hayter and Cresswell 2006). Changes in the availability of pollen, abundance of pollinators and their behavior can lead to diverse pollination outcomes

(Irwin and Brody 2000, Irwin et al. 2010, Morris et al. 2010). Hence, these relevant features of pollination are tightly linked, and their dynamics can strongly influence reproduction of flowering plants.

Nectar robbing can alter pollination and reproductive success in plants (Irwin et al. 2010).

The effects of nectar robbing vary extensively from positive, through neutral to negative, depending on context (Irwin et al. 2010). Generally, the direct pollination effects of nectar robbers are negative to neutral, if nectar robbers do not contact anthers and stigmas (Burkle et al.

2007, Irwin et al. 2010). More complex effects manifest through indirect changes in behavior by other visitors (Irwin and Maloof 2002). After visiting robbed flowers with depleted nectar, long- tongued visitors may fly farther among successively-visited plants in search of unrobbed flowers and visit fewer flowers per plant (Irwin 2000), reducing the incidence of self-pollination relative to cross-pollination. Depending on self-compatibility and the severity of inbreeding depression

(Lande and Schemske 1985), this response may increase the average quality of pollen delivered to some flowers (positive), while reducing overall pollen deposition (negative).

1.4 Objectives

In this thesis, I address the different roles of antagonism and mutualism in pollination systems and how interactions between antagonism and mutualism dynamically influence the pollinator behavior and its pollination consequences.

In Chapter 2 I examine the interactions between two bumble-bee species (Bombus dahlbomii and B. terrestris) with functionally dissimilar tongue lengths while visiting the deep- corolla flowers of Fuchsia magellanica. Specifically, I investigate how changes of the densities of the bee species both limit each other and alter their foraging under different resource conditions. Relevant foraging changes include nectar-robbing, a behavior that B. terrestris adopts commonly (Castro et al. 2008, Irwin et al. 2010).

In Chapter 3 I assess the consequences of density-dependent nectar robbing for reproductive success by Fuchsia magellanica. I specifically consider direct and indirect effects of robbing during successive reproductive stages, including floral lifespan, pollen receipt, pollen- tube development, and fruit production.

In Chapter 4 I synthesize the implications of the results in Chapter 2 and 3 and address broader consequences for ecological dynamics, including the ecological, evolutionary and conservation implications of community invasion by a robbing pollinator.

Chapter 2 – Density-dependent nectar robbing and consequences for competition between

two bumble bee species

2.1 Introduction

Individuals commonly compete with others of the same or different species for limited resources (Svanbäck and Bolnick 2007). Such competition has diverse effects, many of which arise as consequences of altered foraging associated with changes in the costs and benefits of searching for and acquiring resources (Richards et al. 2000). If a specific resource becomes unprofitable owing to competition, the individual may abandon it, or adopt a new behavior that allows them to extract resources more efficiently (Richards et al. 2000, Palmer et al. 2003).

Behavior shifts may exacerbate competition for other foragers, or under certain ecological conditions lead to stable coexistence of species (Richards et al. 2000, Palmer et al. 2003).

Importantly, the magnitude of changes in resource use depend on the extent of generalization or specialization of a species, as generalists have greater capacity to adopt other resources and behaviors than specialists (Svanbäck and Bolnick 2007).

Within generalist populations, individuals capable of using diverse resources may temporarily specialize on resources and partition use of alternative resources with conspecifics

(Araújo et al. 2011). The extent of individual specialization depends on competitor density

(Araújo et al. 2011). Under low density individuals are free to use preferred resources, whereas under high density they may need to diversify resource use to contend with competitors

(Fontaine et al. 2008, Araújo et al. 2011). Alternatively, the presence of heterospecific competitors can exacerbate resource competition and limit the generalization of conspecific individuals as they respond to both conspecific and heterospecific density (Svanbäck and

Bolnick 2007, Fontaine et al. 2008).

Intraspecific and interspecific competition and behavioral changes may be common features of plant-pollinator interactions (Bowers 1985, Balfour et al. 2015). Many animals visit flowers to obtain energy (nectar or oil), and/or protein (pollen: Roulston et al. 2000). To the extent that these resources serve as pollinator rewards for plants, their location is relatively obvious (advertised by floral signals) and they are relatively accessible once located (Willmer

2011). Consequently, a variety of animals may visit flowers of individual plant species (Waser et al. 1996, Vázquez and Aizen 2004), possibly competing for the resources that plants provide to facilitate pollination (e.g. nectar) (Pyke 1984, Thomson 2016).

Nectar competition can be quite dynamic, depending on the abundance and diversity of animals visiting a plant population, the abundance of flowers in the population and their rate of nectar replenishment, and the variety of other local nectar sources and their characteristics. The abundance and diversity of visitors determines nectar availability at any given moment in relation to the number of available flowers, which determines the maximum nectar resource available to visitors (i.e. all flowers full of nectar) (Pleasants 1981). Nectar replenishment can be stimulated by removal by visitors, which can also reduce nectar availability (standing crop) in the plant population (Pleasants 1981, Castellanos et al. 2002, Dreisig 2012). Importantly, different plant species flowering simultaneously in a community represent alternative nectar sources, although they may differ in quality, abundance and replenishment rates (Chalcoff et al.

2008).

The behavior used by flower visitors to access nectar can also influence competition for nectar, although this feature has received limited attention (Irwin et al. 2010). From a plant’s perspective, animals visit flowers “legitimately” when they probe the opening of the corolla

(front visit), contacting the anthers and stigma and effecting pollination (Irwin et al. 2010).

However, the tubular morphology of some flowers can restrict visitor access and reduce their nectar-foraging efficiency (Harder 1983). If a short-tongued visitor cannot reach nectar, so that the cost of visiting a flower outweighs the benefits (Morris et al. 2010), they may instead collect nectar through holes in the corolla tubes of flowers that they create themselves (primary nectar robbing), or that were made by previous visitors (secondary nectar robbing: Inouye 1980, Irwin et al. 2010). The incidence of robbing could vary with overall visitor density if short-tongued visitors can access nectar efficiently only when deep flowers are relatively full (Newman and

Thomson 2005). In such cases, robbing would allow short-tongued visitors to extract nectar, despite being competitively inferior to long-tongued visitor in obtaining nectar legitimately

(Harder 1986). Accordingly, nectar robbing by bumble bees generally coincides with their peak abundance and diminishing floral resources (Irwin and Maloof 2002, Newman and Thomson

2005). However, once robbed flowers exist in a plant population, they may provide long-tongued visitors an alternative foraging mode, secondary theft, again altering overall competition for nectar, depending on their ability to use robbing holes efficiently (Richman et al. 2017a).

Newman and Thomson (2005) observed that long-tongued bumble-bee species visiting Linaria vulgaris rarely used secondary robbing, even when more than 90% of flowers had robbing holes, because they extracted nectar more efficiently during front visits. In contrast, long-tongued species may become secondary robbers when it becomes more beneficial because of specific characteristics of competing species and floral morphology.

In this study I assess competition in a simple natural experiment involving two bumble- bee (Bombus) species, one short-tongued and the other long-tongued, visiting a plant species with long-tubed flowers. I specifically elaborate and tested predictions for two hypotheses:

1) Nectar robbing occurs as a density-dependent response due to nectar competition and tongue- length/nectar tube “mismatch”. Consequently, the incidence of nectar-robbed flowers and their use by flower visitors should vary positively with the abundance of the short-tongued, primary- robbing bee species and resource (flower) availability. This effect should intensify when long- tongued bees are abundant because they reduce nectar availability in flowers disproportionately, limiting nectar access by front-visiting short-tongued bees.

2) Nectar robbing relieves competition for short-tongued visitors but exacerbates competition for long-tongued visitors. Given competition, the local abundance, number of flowers visited and visiting behavior of one bee species on individual plants should vary negatively with the global and/or local abundance of the other species. If nectar-robbing influences these relations, the short-tongued species (primary robber) should affect the long-tongued species more than vice versa.

2.2 Methods

2.2.1 Study species and site

I studied interactions between Bombus dahlbomii Guérin-Méneville, 1835 and Bombus terrestris Linnaeus, 1758, visiting flowers of Fuchsia magellanica Lam. (Onagraceae) in northern Patagonia, Argentina. Bombus dahlbomii is the only native bumble bee in Patagonia and workers have proboscides with a mean (± SE) length of 11.10 ± 0.17 mm (Madjidian et al.

2008). In contrast, Bombus terrestris is a short-tongued species (mean proboscis length for workers = 6.3 ± 0.5 mm SE: Goulson et al. 2008). Bombus terrestris is a Eurasian species that escaped agricultural pollination in Chile and has spread throughout southern South America

(Morales and Aizen 2006). In its native and exotic ranges B. terrestris commonly acts as a primary and secondary nectar robber (De Palma et al. 2016), whereas B. dahlbomii queens have

been recorded nectar robbing only infrequently while visiting Campsidium valdivianum (Urcelay et al. 2006).

Fuchsia magellanica is a perennial shrub distributed in temperate regions of Chile and

Argentina along the Andes Mountains from 32.5° S to the Straits of Magellan (53.6° S) (Berry

1989). This species flowers continuously for up to 6 months during austral summer. Individual

F. magellanica flowers are pendent with an inferior ovary, a long basal nectar tube (range = 7-

15 mm, Berry 1989) and flaring distal corolla, and exerted stamens and style (Fig. 2.1A). After anthesis, flowers remain open for approximately 8 days before wilting (Rosenberger, N.M. unpublished data). During this period, they produce copious nectar (up to 40 μl, unpublished data). In addition to bumble bees, F. magellanica is pollinated by the hummingbird Sephanoides sephaniodes Lesson (Traveset et al. 1998). I do not consider this hummingbird further, as only

76 sightings were recorded during 129.5 observation h, compared to 1512 for B. dahlbomii, 1174 for B. terrestris. Hereafter, I refer to B. dahlbomii as dahl and B. terrestris as terr.

I collected data at Puerto Blest, Parque Nacional Nahuel Huapi, Rio Negro, Argentina

(-41.033333, -71.816667). Puerto Blest is located at the end of the western arm of Lago Nahuel

Huapi near the Andean continental divide. This site is occupied by Valdivian temperate rainforest, which at Puerto Blest is dominated by Nothofagus dombeyi (Mirb.) Oerst., along with a variety of hummingbird-pollinated shrubs and herbaceous species (Aizen and Rovere 2010).

2.2.2 Plant surveys

I haphazardly selected 22 F. magellanica plants throughout the study area to represent spatial variation, including four along the beach of Lago Nahuel Huapi, four in Nothofagus dombeyi forest, 10 along forest streams, and four in an open, disturbed site around the Puerto

Blest Hotel. The study plants varied in size, with the maximum number of simultaneously open

flowers ranging from 34 – 510. Maximum display size did not differ significantly among the habitats (F3,18 = 1.46, P > 0.25; generalized linear model with negative binomial distribution).

The 22 study plants were sampled every other day from December 1, 2015 to April 9, 2016 (11 plants one day and the remainder the next day). Two observers conducted the sampling, with each observer conducting approximately half of each day’s surveys. To prevent plants from being sampled at similar times of day, the order in which plants were surveyed alternated daily.

Surveys of individual plants occurred between 0700 h to 2000 h. I report here results from only

January 20 to April 8, 2016, when both bumble-bee species visited flowers. For the analyses of flower visitation and visit behavior, I also excluded surveys from January 21, March 17 and

April 7, when no bees were observed owing to inclement weather.

While surveying a plant, an observer counted all open flowers and, if some flowers were open, observed a subset of flowers for 10 min for flower visitors (i.e., total 110 observation min per day). The flowers observed during different surveys of individual plants were selected at varying locations and included flowers on one or more branches. As each visitor arrived at the observed flowers, the observer recorded its species and caste (e.g. worker, queen, male), how many flowers it visited, and the number of flowers from which it accessed nectar by probing the distal end of the floral tube (front visit; Fig. 2.1B) or through a hole in the side of the floral tube

(tube visit, or robbing; Fig. 2.1C,D). For tube visits I also recorded whether the visitor created the hole (primary robbing) or used an existing hole (secondary robbing). Observation of a visitor ceased when it left the observation flowers. Observers recorded each new visitor to the observation flowers until the 10-min survey ended.

In addition to observation flowers, other flowers were monitored to quantify the incidence of nectar robbing. Every 4 days an observer haphazardly selected five large flower

buds per plant and attached a uniquely numbered jeweler’s tag to each flower’s pedicle.

Everyday thereafter between anthesis and wilting whether a tagged flower had a hole in the nectar tube indicating that it had been subject to primary robbing was recorded.

2.2.3 Data analysis

Statistical analyses involved generalized linear and nonlinear mixed models as implemented in SAS/STAT 14.2 (GLIMMIX and NLMIXED procedures: SAS/STAT® 14.2

User’s Guide 2016). Analyses of daily bee abundance and the numbers of flowers that each bee visited from the front or robbing through the tube considered negative binomial distributions, whereas the analysis of the number of flowers visited per plant by individual bees involved a truncated negative binomial distribution. These three analyses involved ln link functions. The analysis of the proportion of robbed flowers involved a binomial distribution and logit link function. In addition to categorical and continuous independent variables (see below), all analyses included plant identity nested within habitat as a random effect. To assess observer bias, all analyses initially included observer identity and its interactions with other independent variables; however, they did not have statistically significant effects ( = 0.05) and so were excluded from the analyses reported below.

All analyses of abundance, overall flower visits and individual flower visits included bee species as a factor, with interactions between covariates to account for simultaneous variation between the species. Individual observations of the dependent variables in these analyses represented different counts: abundance – the number of bees of a given species observed per plant per day; visitation – the total number of observation flowers visited by individual bees on a specific plant per daily observation period; and visit behavior – the number of observation flowers visited by an individual bee on a specific plant using a particular behavior (front,

robbing) per daily observation period. These analyses initially included various independent variables, including bee species, an observation plant’s habitat, species x habitat, the daily numbers of observation and total flowers on the observation plant, the total daily abundances of each bee species observed during surveys on all study plants, and the interactions of the continuous covariates with species. The visitation and visit-behavior analyses also initially included the daily abundances of each bee species on the observation plant during flower surveys

(local abundance) and their interactions with species. As these analyses modeled variation in the ln mean of the dependent variable, all continuous covariates were ln-transformed and so the analyses assessed linear ln-ln relations (power functions) between dependent and continuous independent variables. Therefore, a partial regression coefficient of  = 1 indicates proportional variation of the dependent and independent variables,  < 1 indicates a decelerating relation, and

 > 1 indicates an accelerating relation. Some analyses also initially included the untransformed proportion of robbed flowers and its interaction with species. Note that analyses did not explicitly involve day within the study period: it was included implicitly in daily observations of total flower number and bee abundance.

Initial analysis of bee visitation revealed that the relation of the ln(number of flowers) visited by dahl to ln(total dahl abundance) decelerated more strongly than could be represented adequately with a ln-ln linear model. I therefore used a nonlinear model based on the approach described above but with the linear term  ·ln(total dahl abundance) for dahl visits replaced with

 (1-e·ln[total dahl abundance]), where  is the asymptotic ln(number of dahl visits) and  determines the rate of approach to the asymptote with increasing total dahl abundance.

As the visitation and visit-behavior analyses included only observations when individual bees visited at least one flower, the zero class of the assumed negative binomial distributions was

missing. I addressed this for the visit-behavior analyses by subtracting 1 from all observations.

For the visitation analysis the use of non-linear methods allowed fitting of a zero-truncated negative binomial distribution.

For the analysis for visit behavior, each continuous covariate was included in three-factor interactions with species (dahl vs. terr) and type of visit (front visit vs. robbing visit). This allowed estimation of specific partial regression coefficients for each species and their visit type simultaneously. Of the 1313 bees observed during surveys, only 33 switched between visit types during the same foraging bout. These individuals were omitted from the final analysis of individual bee flower visits due to the rarity of their transitions and to keep individual behavior consistent.

The analysis of the proportion of robbed flowers per plant considered the abundance and visitation by floral visitors, to assess the predictions that nectar robbing increases with the density of visitors, and the presence of long-tongued visitors lowers the density necessary to precipitate nectar robbing. Because of over-dispersion of the dependent variable compared to a binomial distribution the analysis used quasi-likelihood estimation (McCullagh and Nelder

1989). This analysis initially included the daily and past total abundances of dahl and terr (ln transformed after the addition of 1) and ln(daily total open flowers). Past abundance was the mean of the total abundances during the preceding two surveys (usually 2 and 4 days prior to the current sample). Interactions included in the analysis involved past and present conspecific ln(daily bee abundances +1), heterospecific past and present ln(daily bee abundance + 1) and ln(daily bee abundances + 1) and ln(daily total flowers). Each continuous covariate was also initially included in an interaction with habitat type.

I used backward elimination to identify the best-fitting models for each analysis. During the elimination process, nonsignificant ( = 0.05) individual independent variables could be excluded from an analysis only if they were not involved in a significant interaction.

To illustrate relations of dependent variables to independent variables I present partial effects that account for variation in other independent variables in a final statistical model. For categorical independent variables, I present least-squares means. For the effects of continuous independent variables, I illustrate adjusted observations that represent the predicted value based on the means of all but the focal independent variable(s) plus an observation’s residual. All results are presented back-transformed from the link scale, which results in asymmetrical standard errors.

2.3 Results

2.3.1 Dynamics of flowering, bee visitation and the incidence of robbed flowers

At least some of the 22 study Fuchsia plants produced flowers from mid-December until mid-April. Overall, flowering exhibited a bimodal pattern, peaking during late December and early January and again during early March (Fig. 2.2A). During this study of bumble-bee interactions (January 20 to April 8), flowering increased gradually until the peak in March and then decreased moderately until the study terminated (Fig. 2.2A).

Based on their occurrences on the studied Fuchsia plants, the two bumble-bee species exhibited somewhat different phenologies during the study period (Fig. 2.2B, C). Terr abundance peaked earliest, at the beginning of February, and then generally declined until late March, after which terr was not observed visiting Fuchsia (Fig. 2.2C). In contrast, relatively few dahl visited

Fuchsia until about March 20. Dahl then became the most common species visiting

F. magellanica, reaching peak abundance at the end of March and remaining relatively abundant until the end of the study (Fig. 2.2B).

Of the 4730 flower visits observed during flower sampling, 46.3% were front visits and

53.7% involved robbing. Among the 2542 robbing visits, only 32 (1.3%) involved primary robbing, so secondary robbed predominated. Observed primary robbing involved 28 visits by 20 terr and four visits by two dahl. The observed primary robbing by dahl occurred late during the sampling period (March 28 and 29), when terr had ceased visiting Fuchsia flowers.

The proportion of robbed flowers largely reflected the presence of terr and the 8-day average lifespan of individual flowers (Fig. 2.2D). None of the observation flowers were robbed until late January, when terr became increasingly abundant. The proportion of robbed flowers then increased rapidly, peaking on the same day as terr abundance (Fig. 2.2D). The proportion of robbed flowers remained relatively high while an average of at least one terr was observed per plant during sampling periods. When observed terr abundance fell below this level, the proportion of robbed flowers declined gradually, despite the peak in dahl abundance. Some robbed flowers persisted about 8 days after the last terr was observed visiting observation flowers. During the last observation day, no flowers had been robbed, despite the relatively high abundance of dahl.

The incidence of robbing visits relative to front visits by dahl and terr during observation periods varied during the study (Fig. 2.3). Both species made only front visits until February 1.

Thereafter, terr robbed Fuchsia flowers almost exclusively (Fig. 2.3B). In contrast, dahl rarely made robbing visits until early March, when they were temporarily its main type of visit (Fig.

2.3). After mid-March, the incidence of robbing visits by dahl declined consistently (Fig. 2.3A) in parallel with diminished terr abundance and fewer robbed flowers (Fig. 2.2B, D).

2.3.2 Bee abundance

Overall, both bee species were equally abundant during individual plant surveys (Table

2.1, species effect); however, their relative abundance differed somewhat among habitats (Table

2.1, species x habitat interaction; Fig. 2.4), after accounting for other influences. In general, more bees visited plants in open sites (beach and disturbed) than in shaded sites (forest and forest streams: Table 2.1, habitat effect). In open sites, both species were equally common on beach plants (t18 = 1.70, P > 0.6, Dunn-Šidák multiple comparison), whereas dahl was more than twice as abundant as terr on plants at the disturbed site (t18 = 27.0, P < 0.001). In shaded sites, both species were equally common on forest plants (t18 = 6.64, P > 0.05), whereas dahl was somewhat more abundant than terr on plants along forest streams (t18 = 20.9, P < 0.001). Bee abundance also varied significantly among plants within habitats (Table 2.1, among-plant variance); however, the among-plant variance components differed among habitats (Table 2.1, heterogeneous among-plant variance), being negligible for open habitats, but significant for shaded habitats.

The number of bees of each species observed during individual plant surveys also varied with a plant’s floral display, overall bee abundance and the proportion of robbed flowers (Table

2.1, 2.2). In general, the number of bees per survey varied positively, but weakly, with the number of observed flowers, with no difference between the species. For both species, more bees visited plants with larger displays (Fig. 2.5A, B); however, this effect was stronger for dahl than for terr (Table 2.1, Species x ln[Total flowers]). Unsurprisingly, the numbers of both species per plant also increased proportionally with their overall abundance on all study plants

(Fig. 2.5C, F: test of  = 1; dahl, t1519 = 1.31, P > 0.15; terr, t1519 = 1.51, P > 0.1). In contrast, the number of bees of one species tended to vary negatively with the total abundance of the other

species (Fig. 2.5D, E). These effects were weakly statistically significant if the analysis did not include the proportion of robbed flowers on focal plants (dahl, t1519 = 2.15, P < 0.05; terr, t1519 =

2.03, P < 0.05), but not when this covariate was included (Table 2.2). The proportion of robbed flowers had contrasting effects on the numbers of bees observed per plant (Table 2.1, Species x

Proportion of robbed flowers), being negative for dahl but positive for terr (Table 2.2, Fig. 2.5G,

H).

Given the daily measurements of the independent variables considered in the preceding analysis, the fitted regression relation can be used to characterize variation in the daily mean abundance of both bee species. As indicated by the correspondence between the prediction curves and observed means in Fig. 2.2B and 2.2C, the fitted regression relation accurately depicts daily and seasonal variation in the abundance of both species.

2.3.3 Incidence of robbed flowers

As the proportion of robbed flowers might influence the behavior of visiting bees, I consider the influences on the incidence of robbed flowers first to provide context for the analyses of visit number and type. Given that terr was the primary nectar robber, that its abundance had diverse effects on the incidence of robbed flowers (Table 2.1, Fig. 2.6A, B, D) is not surprising. Except at the disturbed site, the incidence of robbed flowers increased with daily total terr abundance (Fig. 2.6D). Daily total terr abundance also had interacting effects on the proportion of robbed flowers with daily total dahl abundance and past total terr abundance

(Table 2.1). Terr abundance affected the incidence of robbed flowers most strongly when dahl visited Fuchsia infrequently (Fig. 2.6A: i.e., during February and early March, Fig. 2.2, B, C), whereas its effect became largely neutral when terr abundance declined during late March (Fig.

2.2C) and dahl abundance increased (Fig. 2.2B). In addition, terr abundance had greatest

positive effect on the incidence of robbed flowers when terr had also been abundant recently, rather than when past terr abundance had been low (Fig. 2.6B). The incidence of nectar robbing also varied significantly among plants and with two additional interactions involving habitat

(Table 2.1). The proportion of robbed flowers varied positively with the total number of open flowers at open sites (beach and disturbed), but not at shaded sites (stream and forest) (Table 2.1,

2.2, Fig. 2.6E). Robbing incidence varied independently of daily dahl abundance at distributed and stream sites, but negatively for beach plants and positively for forest plants (Table 2.1, 2.2,

Fig. 2.6C).

As the incidence of nectar robbing and its associated independent covariates were measured daily, the fitted regression for the incidence of robbing can be used to depict daily variation in nectar robbing through time. The predicted daily means of nectar robbing closely match the observed means, so this model accurately characterizes seasonal variation in nectar robbing (Fig. 2.2D).

2.3.4 Flower visits

The number of flowers visited by bees during observation periods was subject to diverse influences (Table 2.1, Fig. 2.7). Unsurprisingly, individual bees visited more observation flowers when more flowers were observed (Table 2.2, Fig. 2.7J). Visitation also varied consistently among the studied plants within habitats (Table 2.1), but not among habitats. On average, individual dahl visited significantly more observation flowers than individual terr

(Table 2.1, species effect), with no difference among habitats. Dahl tended to visit fewer flowers on plants with many open flowers (Fig. 2.7G), whereas terr visitation did not vary significantly with floral display size (Fig. 2.7H: Table 2.1, Species x ln[Total flowers]). Visits by dahl varied weakly negatively with the proportion of robbed flowers, but terr visits did not (Table 2.1:

Proportion robbed flowers, t20 = 1.30, P > 0.2; Species x Proportion robbed flowers, t20 = 1.90, P

> 0.05).

Total and local bumble-bee density had contrasting effects on the number of observation flowers visited by individual bees for the two species. Combined visitation by both species varied positively with overall terr abundance (Fig. 2.7I), but not with overall dahl abundance. In general, flower visits by each species varied positively with its own abundance; however, the effect of conspecific density on visitation differed between the species (Table 2.2). For dahl, the presence of more than five conspecifics on the same plant had a decelerating effect on visitation

(Fig. 2.7A), as expected with intraspecific competition. In contrast, terr visitation increased in an accelerating manner with local conspecific density (Fig. 2.7D: one-tailed test of  = 1, t20 = 3.06,

P < 0.005), as expected with intraspecific facilitation. Local heterospecific density also had contrasting effects on visitation by the two species (Table 2.2, Fig. 2.7B, C). As expected for heterospecific competition, terr visitation declined with increasing numbers of simultaneously visiting dahl (Fig. 2.7B). In contrast, dahl visitation did not vary significantly with local terr abundance (Fig. 2.7C).

2.3.5 Flower visit behavior

The frequencies of front and robbing flower visits by individual bees differed among plants and habitats and varied with the proportion of robbed flowers and overall terr abundance

(Table 2.1, Fig. 2.8). Front and robbing visits occurred equally on beach plants, whereas front visits were relatively more frequent on forest plants, but less frequent on plants along streams and in the disturbed site (Fig. 2.8A). Individual bees generally made fewer front visits on plants with a high proportion of robbed flowers (Table 2.3, Fig. 2.8B), whereas robbing by individual bees did not vary significantly with the proportion of robbed flowers (Fig. 2.8C: Table 2.1, Type

of visit x Proportion of robbed flowers; Table 2.3). Similarly, the proportion of front visits, but not of robbing visits, varied significantly with overall terr abundance; however, the former effect differed between the bee species (Table 2.1, ln[Total terr. abundance + 1] x Type of visit x

Species; Table 2.3; Fig. 2.8D,E). In particular, dahl engaged in more front visits when terr was common (Fig 2.8D), whereas front visits by terr declined with overall terr abundance (Fig 2.8E).

2.4 Discussion

This study revealed diverse negative effects of each bumble bee species on the abundance and flower visitation of the other species, mediated in part by the alteration of nectar-access opportunities created by primary nectar robbing by terr. The two species exhibited contrasting seasonal phenologies as visitors of Fuchsia magellanica flowers, with terr abundance peaking earlier, prior to maximal Fuchsia flowering, and dahl abundance peaking somewhat after both terr and Fuchsia flowering (Fig. 2.2). As a consequence, total daily abundance of the two species varied negatively (Fig. 2.5D, E). In addition, the two species used the site types occupied by

Fuchsia somewhat differently (Fig. 2.4). These negative associations could represent intrinsic features for the two species, including different colony cycles and habitat preferences.

However, other evidence from this study demonstrates competitive effects between terr and dahl, which likely contributed to their negative associations. In particular, in the presence of many dahl on individual plants, terr visited fewer flowers (Fig. 2.7B). In contrast, local terr abundance did not directly affect the number of flowers visited by individual dahl (Fig. 2.7C), although dahl visited more flowers from the front when many terr were also present on the same plant (Fig. 2.8D). Terr also affected dahl visitation indirectly, as the latter varied negatively with the proportion of robbed flowers (Fig. 2.7E), which terr had likely created. Together, these

results suggest that competition underlies all aspects of the negative associations between terr and dahl.

This conclusion is consistent with those of other studies of bumble-bee competition

(Inoyue 1978, Pyke 1982, Bowers 1985). For example, Bowers (1985) observed that Bombus flavifrons in the Uinta Mountains of Northern Utah became numerically dominant to B. rufocinctus as a consequence of initiating colony and worker production 3 weeks before B. rufocinctus. When B. flavifrons was experimentally removed, B. rufocinctus shifted to flowers of plant species preferred by B. flavifrons. During my study, B. terrestris workers similarly emerged 2-3 weeks before B. dahlbomii and rapidly became numerically dominant (compare Fig.

2.2B and C). This contrast suggests that B. terrestris gained a competitive advantage over B. dahlbomii from its earlier colony development. Similarity in tongue length also promotes competition among bumble-bee species, leading Pyke (1982) to propose that two nectar-robbing species with similar tongue lengths could not co-exist. He suggested that nectar robbing occurred as an effect of displacement by superior competitors, as B. occidentalis was the only member of the bumble-bee assemblage that robbed flowers. However, his study did not consider overlap of primary and secondary nectar robbing, or the circumstances that cause short-tongued and long- tongued visitors to use the same resources. In the Rocky Mountains of Colorado, Inouye (1978) observed that bumble bees visited more flowers after ecologically similar species were removed, demonstrating competition. I observed a similar negative association of dahl abundance as terr abundance increased and then decreased during the study period. I now consider these results in detail by discussing implications for density-dependent interactions, nectar robbing and the implications of terr being an invasive species.

2.4.1 Density-dependent interactions

Resource density, as represented by total flower number, and bee density had pervasive effects on the dynamics of bee abundance and behavior. The abundances of both bee species varied positively with overall flower abundance, but in a decelerating manner. As flower abundance changed faster than bumble-bee colonies produce new individuals (e.g., Cnaani et al.

2002), this association suggests that bees that were previously visiting flowers of other plant species recruited to visiting Fuchsia flowers as they became abundant. This association would increase opportunities for bees of the same and different species to interact directly and indirectly.

The relations of flower visits per bee to conspecific density provides some evidence of intraspecific density effects. Terr abundance and behavior was subject to positive-density dependence. The number of terr visiting individual plants during sampling periods increased in an accelerating manner with total terr abundance (Fig. 2.5F), which also positively affected the number of flowers visited by individual terr (Fig. 2.7I). A key feature of these results is the generally positive association of the proportion of robbed flowers to terr abundance (Fig. 2.6A,

B, D), but a strongly negative association for the number of front visits per terr (Fig. 2.8E). Once terr became moderately frequent visitors of Fuchsia flowers, they collected nectar almost exclusively by (mostly secondary) robbing (Fig. 2.3B). As terr created the holes in corollas needed to access nectar by tube visits, the presence of more (primary-robbing) terr individuals likely facilitated flower visitation and numerical increase by terr. In contrast, dahl experienced negative effects of conspecific density. Specifically, the number of flowers visited by individual dahl increased in a decelerating manner with observed dahl abundance per plant (Fig. 2.7A), indicating intraspecific competition.

As outlined above, both dahl and terr apparently compete with the other species; however, the nature of their negative effects differed between the species. Competition by terr seems largely associated with its overall presence, as its effects on dahl abundance and behavior relate to total terr abundance (Fig. 2.5E, 2.8D), rather than its abundance on individual plants

(Fig. 2.7C), and to the number of robbed flowers (Fig. 2.5G, 2.7E). A particularly interesting result is that the number of front tube visits by dahl individuals increased with daily total terr abundance (Fig. 2.8D). This suggests that when terr was numerically dominant, dahl increasingly searched for unrobbed plants and visited more flowers to compensate for reduced nectar availability caused terr’s robbing and presence. In contrast to the global effects of terr on dahl, the number of flowers visited by terr declined with increasing dahl abundance during observations on individual plants (Fig. 2.7B), indicating behavioral aversion. This effect may partially counteract the interspecific competition experienced by dahl. Nevertheless, dahl seems competitively inferior to terr, especially as dahl also experienced intraspecific competition, unlike terr.

2.4.2 Nectar robbing

Based on the phenology of robbed flowers, terr switched to robbing precipitously during early February and then rarely used front visits (Fig. 2.2D, 2.3B). Although I observed few primary robbing visits, terr was likely responsible for creating robbed flowers, as dahl did not begin robbing until a week after terr, and terr was involved in most robbing visits until the second week of March (Fig 2.3A).

The results demonstrate the density-dependent nature of nectar robbing, as the incidence of robbing increased with both the current and past total abundance of terr (Fig. 2.6A, B, D). The effect of past terr abundance reflects the inertia of the presence of robbed flowers caused by the

protracted lifespan of flowers, which also resulted in robbed flowers remaining for a week after terr was not present. This inertia allowed dahl to continue secondary robbing in early April, despite terr’s absence (Fig. 2.3).

The presence of robbed flowers had several consequences for bee interactions. As explained above, robbing likely alleviates the effects of intraspecific competition by terr by making nectar more accessible. Robbing holes may also be beneficial during early phases of robbing for dahl by creating ecological opportunity. However, as terr abundance increased it became competitively superior. Indeed, the presence of robbed flowers had an overall negative effect on dahl abundance and visits, including stimulating fewer front flower visits (Fig. 2.5G,

2.7E, 2.8B).

2.4.3 Implications for nectar robbing invasion

The demonstrated superior competitive ability of terr over dahl is additionally significant because terr is a recent invader in Argentina. As terr has expanded throughout the Patagonian region of Chile and Argentina, it has rapidly displaced dahl (Aizen et al. in press). This dramatic ecological transition has been largely attributed to high parasite and disease loads in terr populations (Arbetman et al. 2013, Schmid-Hempel et al. 2014). However, the results of this study indicate that resource competition likely also contributes to the rapid extirpation and displacement of dahl by terr.

Table 2.1. Overall results of generalized linear and non-linear mixed models evaluating effects on the abundance and total visitation of each bumble-bee species, the visit behavior of individual bees, and the proportion of robbed flowers for individual plants. The analysis for visitation involved nonlinear methods to account for an asymptotic effect of dahl abundance on dahl visitation. Effect Abundance Visitation Visit behavior Robbing

Species F1,18 = 0.16 t20 = 6.74*** F1,19 = 0 .32

Habitat F3,18 = 10.59*** F3,17 = 3 .88* F3,18=2.55

Species x Habitat F3,18 = 9.87***

26 ln(Observed flowers) F1,1519 = 5.57* t20 = 11.75*** F1,1216 = 129.72***

ln(Observed flowers) x Species F1,1216 = 6.49*

ln(Total flowers) F1,1519 = 111.72*** t20 = 1.31 F1,1216 = 4.45*

Species x ln(Total flowers) F1,1519 = 5.16* t20 = 3.27** F1,1216 = 9.07**

ln(Total terr abundance +1) F1,1519 = 121.51*** t20 = 3.36** F1,1216 = 0.32 F1=3.51

Species x ln(Total terr abundance +1) F1,1519 = 159.32*** F1,1216 = 7.50**

ln(Total dahl abundance +1) F1,1519 = 37.82*** F1=9.38**

Species x ln(Total dahl abundance + 1) F1,1519 = 76.63***

Proportion robbed flowers F1,1519 = 0.73 t20 = 1.30 F1,1216 = 8.28**

Species x Prop. robbed flowers F1,1519 = 30.65*** t20 = 1.90 F1,1216 = 10.85**

ln(Local dahl abundance + 1) F1,1216 = 18.62***

ln(Local dahl abundance + 1) asymptote for B. t20 = 18.13***

dahlbomii

ln(Local dahl abundance + 1) nonlinear rate for t20 = 4.11***

B. dahlbomii

ln(Local dahl abundance + 1) for B. terrestris t20 = 3.61**

ln(Local terr abundance + 1) t20 = 10.33*** F1,1216 = 1.29

27 Species x ln(Local terr abundance + 1) t20 = 13.13*** F1,1216 = 1.76

Type of visit F1,17 = 9.37**

Species x Type of visit F1,7 = 0.14

Habitat x Type of visit F3,17 = 4.83*

ln(Observed flowers) x Type of visit F1,1216 = 6.15*

ln(Total terr. abundance + 1) x Type of visit F1,1216 = 5.98*

ln(Total terr. abundance + 1) x Type of visit x F1,1216 = 5.66*

Species

ln(Local terr abundance + 1) x Type of visit F1,1216 = 2.19

ln(Local terr abundance + 1) x Type of visit x F1,1216 = 3.88*

Species

ln(Total terr abundance +1) x ln(Total dahl F1 = 28.17***

abundance +1)

ln(Past total terr abundance + 1) F1 = 1.90

ln(Past total terr abundance + 1) x ln(Total terr F1 = 16.94***

abundance +1)

28 ln(Total dahl abundance +1) x Habitat F1 = 7.33***

ln(Past total terr abundance + 1) x Habitat F1 = 5.18**

ln(Daily total open flowers) x Habitat F1 = 3.44*

2 2 2 Among-plant variance χ4 = 102.8*** t20 =1.33 휒2 =10.00*** 휒1 =150.00***

2 Heterogeneous among-plant variance χ3 = 15.7**

* P < 0.05, ** P <0.01, *** P <0.001

Table 2.2. Partial regression coefficients (± SE) associated with continuous effects on the abundance and total visitation of each bumble-bee species, and the proportion of robbed flowers for individual plants (see Table 2.1), including effects that are common to both bee species and unique to each species. Dependent

variable Effect Common B. dahlbomii B. terrestris

Abundance Intercept -5.968 ± 0 .423*** -5.748 ± 0.496***

(ln link)

Observed flowers 0.184 ± 0.078 *

Total flowers 0.665 ± 0.068*** 0.447 ± 0.074***

Daily dahl abundance 0.893±0.082*** -0.143±0.088

Daily B. terrestris abundance -0.007±0.052 1.120±0.080***

Proportion of robbed flowers -0.563±0.177** 0.791±0.185***

Visitation Intercept -2.364±0.514*** -2.138±0.317***

(ln link)

ln(Observed flowers) 0.630±0.054***

ln(Total flowers) -0.171±0.054** 0.065±0.054

ln(Local dahl. abundance + 1) asymptote 5.166±0.285***

ln(Local dahl. abundance + 1) nonlinear rate -0.729±0.177***

ln(Local dahl. abundance + 1) -0.267±0.074***

ln(Local terr. abundance + 1) -0.053±0.070 1.252±0.083***

ln(Total terr. abundance + 1) 0.117±0.035***

Proportion of robbed flowers -0.253±0.105* 0.037±0.121

Robbed flowers Intercept -8.776±1.364***

(logit link)

30 ln(Daily total flowers) 0.922±0.173***

ln(Total dahl abundance + 1) 0.570±0.186**

ln(Total terr abundance + 1) 0.473±0.252

ln(Past total terr abundance + 1) 0.194±0.141

ln(Total dahl abundance + 1) x ln(Total terr -0.410±0.077***

abundance + 1)

ln(Total terr abundance + 1) x ln(Past total 0.283±0.069***

terr abundance + 1)

* P < 0.05, ** P <0.01, *** P <0.001

Table 2.3. Partial regression coefficients (± SE) for significant covariate effects (all ln-transformed) detected by a generalized linear mixed model evaluating effects on visitation behavior of individual bees (ln link function). Significant effects included a simple effect of B. dahlbomii abundance on individual plants, two-variable interactions of species with ln(observed flowers) and ln(total flowers) and between visit type and the proportion of robbed flowers, and the three-variable interaction of species and local and total terr abundance. Covariate

Local Local Total

Categorical B. dahlbomii Observed Proportion of B. terrestris B. terrestris

effect abundance flowers Total flowers robbed flowers abundance abundance

None -0.266±0.062

dahl 0.996±0.104*** -0.256±0.071***

terr 0.566±0.114*** 0.019±0.074

Front visits -0.799±0.198***

Robbing visits 0.067±0.166

dahl front visits -0.068±0.117 0.177±0.058**

dahl rob 0.031±0.155 0.195±0.100

terr front visits 0.595±0.342 -0.696±0.310*

terr rob -0.097±0.089 0.133±0.086

* P < 0.05, ** P <0.01, *** P <0.001

Figure 2.1. Examples of the study species and their interactions with Fuchsia magellanica, including (A) two pendent flowers of F. magellanica (credit: L.D. Harder), (B) a dahl making a front visit (credit: L.D. Harder), (C) a terr making a robbing visit (credit: M.A. Aizen), and (D) a dahl making a robbing visit (credit: N.M. Rosenberger). In (A) the left- hand flower is younger and in female phase, whereas the older right-hand flower has dehisced anthers.

A B

C D

Figure 2.2. Temporal patterns of (A) overall flowering by 22 Fuchsia magellanica plants and least-squares mean ( SE) (B) dahl abundance, (C) terr abundance and (D) proportion of robbed flowers. For panels B-D the solid line depicts the predicted mean based on the respective statistical analyses (which did not explicitly include date).

8 2000 A C

6 1500

1000 abundance 4

500 2 B. terrestris Number of open flowers of open Number 0 0 10 20 1 10 20 1 10 20 1 10 20 1 20 1 10 20 1 10 20 1 December January February March April January February March April

8 B 1.0 D 0.8 6 0.6

abundance 4 0.4 2 0.2

B. dahlbomii 0 0.0 20 1 10 20 1 10 20 1 flowers of robbed Proportion 20 1 10 20 1 10 20 1 January February March April January February March April Date Date

Figure 2.3. Temporal patterns of the (A) number and (B) proportion of observed visits to Fuchsia magellanica flowers that involved nectar robbing visits for dahl and terr during the study period.

40 A dahl 30 terr

10 Number of robbing visits robbing of Number

0 20 1 10 20 1 10 20 1 January February March April

1.0 B

0.8

0.6

0.4

0.2 Proportion of robbing visits robbing of Proportion 0.0 20 1 10 20 1 10 20 1 January February March April Date

Figure 2.4. Overall least-squares mean (± SE) abundance of dahl and terr on Fucshia magellanica plants in four habitats during 10-min samples. Based on the analysis that also accounted for the effects of continuous independent variables illustrated in Fig. 2.5 (also see Table 2.1). Means are back-transformed from ln values, hence the asymmetric standard errors.

Figure 2.5. Partial effects of continuous independent variables on the least-squares mean ( SE) numbers of Bombus dahlbomii (A, C, E, G) and B. terrestris (B, D, F, H) visiting Fucshia magellanica plants during 10-min samples, including: A, B the number of open flowers per plant; C, D the total number of dahl observed daily on all study plants; E, F the total number of terr observed daily on all study plants; and G, H the daily proportion of robbed flowers per plant. Panels depicting statistically significant effects include the fitted regression lines (see Table 2.2). Based on an analysis that also accounted for the habitat effects presented in Fig. 2.4 (also see Table 2.1). Means are back-transformed from ln values, hence the asymmetric standard errors.

Figure 2.6. Partial effects of interactions between independent variables on the daily incidence of nectar-robbed Fuchsia magellanica flowers, including (A) the total daily abundances of dahl and terr, (B) the daily past and current total abundances of terr, and of habitat with (C) past total terr abundance, (D) the number of open flowers and (E) total daily dahl abundance. In C-E black=beach, red=disturbed, blue=stream and green=forest.

A 1.0

0.8

0.6

0.4

0.2 Proportion of robbed flowers of robbed Proportion 0.0 40 50 Total 30 40 terr 20 30 abundance10 20 10 abundance 0 dahl 0 Total

B 1.0

0.8

0.6

0.4

0.2 Proportion of robbed flowers of robbed Proportion 0.0 40 10 Total 20 30 terr 30 20 abundance abundance 40 10 terr 50 0 Total past

Figure 2.7. Partial effects of continuous independent variables on least-squares mean ( SE) visitation of Fuchsia magellanica plants by Bombus dahlbomii (A, C, E, G) and B. terrestris (B, D, F, H) during 10-min samples, including: A, B the local number of dahl on a study plant; C, D the local number of terr on a study plant; E, F the daily proportion of robbed flowers per plant; G, H the number of open flowers on a study plant; and I, J the number of open flowers on a study plant. Panels depicting statistically significant effects present fitted regression lines (see Table 2.2). Means are back-transformed from ln values, hence the asymmetric standard errors.

Figure 2.8. Partial effects on individual bee behavior and visits including (A) habitat, (B) the proportion of robbed flowers, and (C) total daily terr abundance during 10-min plant samples. Means are back-transformed from ln values, hence the asymmetric standard errors.

Chapter 3 – Quantitative and qualitative effects of nectar robbing on female reproductive

success of Fuchsia magellanica

3.1 Introduction

Despite being mutualistic, plant-pollinator interactions involve self-interested partners

(Bronstein 2001a). Most animals visit flowers for food, especially nectar (energy) and pollen

(protein) (Willmer 2011). For a foraging animal to visit flowers of a particular plant species repeatedly, as needed for cross-pollination, it must procure enough food to outweigh the time and energy costs of searching and visiting (Morris et al. 2010). If one partner does not benefit from this interaction, an apparent mutualism shifts to antagonism (Morris et al. 2010, Aizen et al.

2014). Antagonism may be exacerbated as increased success by the antagonistic partner undercuts that of their partner (Bronstein 2001b). For example, plants may reduce reward production to improve resource allocation (Bronstein 2001a, 2001b), or visitors may damage flowers to collect otherwise unattainable floral resources (Hargreaves et al. 2009, Irwin et al.

2010, Morris et al. 2010). As each partner acts to maximize its own net benefits, the return is reduced for the partner, potentially crossing the interaction threshold from beneficial to detrimental (Bronstein 2001b).

Antagonism can arise in pollination systems that involve “poorly” suited partners

(Bronstein 2001a). Some poorly suited visitors may visit flowers for food without contacting floral organs, because they are too small or adopt positions and behaviors inconsistent with pollination, and so function as pollen or nectar thieves (Hargreaves et al. 2009, Irwin et al. 2010).

The most extreme form of theft occurs when visitors damage flowers to access pollen or nectar, and so act as robbers (Inouye 1980, Irwin et al. 2010). Although theft and robbing generally

benefit the animals involved, their effects for the affected plants can be quite heterogeneous, depending on circumstances.

The role of antagonism, specifically nectar robbing, has yet to be explored in the context the entire reproductive process (Irwin et al. 2010). Female reproduction begins with pollen receipt, which influences the number of pollen tubes that can fertilize ovules and subsequent seed and fruit set and quality (Aizen and Harder 2007). Pollen receipt depends on both the number of pollen grains delivered per pollinator and the number of pollinator visits during a flower’s life (Bell and Cresswell 1998). Pollen deposition per visit varies with the amount of pollen carried by a pollinator and the extent and duration of its contact with a flower’s stigma(s)

(Harder and Barrett 1996). Because nectar thieves and robbers often adopt positions that do not involve anther and stigma contact, they are often carry and deposit little pollen, and so are poor pollinators (Irwin et al. 2010). The number of pollinating visits received by flowers depends on overall pollinator abundance and a plant’s and flower’s attractiveness (Fontaine et al. 2008).

Nectar theft can affect attractiveness by damaging flowers and reducing nectar availability (Irwin

2000). In addition, if robbing shortens floral longevity (e.g., Zhang et al. 2011), it can limit opportunities for pollinator visits.

After arriving on a stigma, the success of pollen grains at germinating pollen tubes that grow down the style to fertilize the ovary depends on their quality, as it affects their intrinsic capacity and interaction with stylar tissue (Aizen and Harder 2007). Pollen tubes must contend with their own ecological environment, competing with other pollen tubes for limited space in the style and potentially inferior conditions in the stylar environment (Harder et al. 2016a).

Pollen-tube success may be further limited if physiological conditions within the stigma are sub- optimal, potentially owing to damage to flowers or localized herbivory (Harder et al. 2016b). If

pollen tubes successfully penetrate the base of the style, they can compete to fertilize ovules.

However, this does not guarantee that the embryo will grow into a seed, as sufficient resources are needed for seed and fruit development (Rosenheim et al. 2016). Fruits may or may not develop, depending on the overall success of the flower in pollination and the quality of pollination. The tubes of pollen that are of poor genetic quality may fail during germination and growth in pistil, or inbred embryos may fail owing to deleterious alleles. If the fruit does set, the range of pollen quality and subsequent zygote success will determine fruit quality and further resource investment (Harder et al. 2016a). Therefore, antagonism can influence success during all phases of female reproduction by plants.

The effects of nectar robbing have received more consideration than those of other antagonistic interactions in pollination systems (Irwin et al. 2010). Robbing can affect plant performance both directly and indirectly. Direct effects of nectar robbing can be related to the floral damage from robbing holes themselves (see Fig. 2.1D) and the subsequent costs of maintaining damaged flowers, or direct damage to the ovary (Traveset et al. 1998). Furthermore, this damage may alter physiological conditions in flowers to support pollen tube germination and success, as maintenance becomes costly (Harder et al. 2016b). Subsequent effects of nectar robbing damage on pollen tube success would expectantly lead to lowered fruit set and fruit quality. Such damage could also influence a flower’s ability to produce nectar if the nectary is damaged (Irwin et al. 2010). Other consequences, such as depressed fruit quality due to limited pollen tube success or resource investment, may occur later during fruit production, but these have not been examined. Indirect negative effects include altered pollinator behavior in response to reduced plant attractiveness and reduced pollen receipt due to less contact with floral reproductive organs (Irwin 2000). Conversely nectar robbing can improve outcrossing if

legitimate visitors visit fewer flowers per plant and move between plants more readily (Irwin

2000).

Studies of the impacts of nectar robbing generally examine pollination quantity, with less consideration of the qualitative effects of nectar robbing for pollination, pollen-tube success and fruit and seed production (Irwin et al. 2010). Qualitative effects could arise for self-compatible species if robbing changes the characteristics of pollen received by flowers, specifically the proportion of self-pollen (e.g., Irwin 2003). Tubes produced by self-pollen often survive poorer than cross-pollen tubes (e.g., Cruzan 1989, Aizen and Harder 2007, Harder et al. 2016a) and so have lower fertilization success (Harder et al. 2012). In addition, self-fertilized zygotes often have lower chance of developing into a viable seed than cross-fertilized seeds because of inbreeding depression (Charlesworth and Charlesworth 1987, Harder et al. 2012). Robbing could reduce the incidence of self-pollination among a plant’s flowers (geitonogamy) and associated self-fertilization if the associated nectar depletion causes effective pollinators to visit fewer flowers before leaving a plant (e.g., Fig. 2.8B). Thus, robbing could enhance average pollen and seed quality (Irwin and Brody 2000). However, if damage to flowers lowers the capacity for flowers to support pollen tubes, beneficial effects of higher quality pollen may be negated by changes in the stylar environment (Harder et al. 2016b).

In this chapter I examine the effects of nectar robbing for female performance of Fuchsia magellanica, including floral longevity, pollen receipt, pollen-tube success, fruit set and fruit mass (quality). Specifically, I assess the extent to which nectar robbing reduces reproduction by causing flower damage and by reducing pollen quantity and quality. Flower damage could affect reproduction by shortening floral longevity (e.g., Zhang et al. 2011) and diminishing a flower’s capacity to support pollen-tube growth. As robbers generally fail to contact floral sex organs,

they tend to be inefficient or ineffective pollinators. Furthermore, by affecting plant attractiveness and nectar availability, robbers could alter both the quantity and quality of visits by effective pollinators. This study examines all these possible consequences.

3.2 Methods

The research described in this chapter was conducted simultaneously with that reported in

Chapter 2 using the same 22 Fuchsia magellanica plants. Descriptions of the study species and site can be found in §2.2.1 and those of the daily plant and pollinator surveys can be found in

§2.2.2. Here I describe unique features of the methods relevant to my assessment of female reproductive success by Fuchsia magellanica.

3.2.1 Study species

Fuchsia magellanica produces long, pendent flowers with red calyxes and purple corollas

(Fig. 2.1A). Each flower has 8 exserted stamens (4 longer and 4 shorter). The pistil of each flower comprises an inferior ovary with 300-500 ovules and a long style that presents a four- lobed stigma beyond the anthers. Flowers remain open for 4-19 days and are protogynous with an approximately 4-day female phase before the anthers dehisce (Fig. 2.1A). I did not determine whether the latter phase is bisexual or the stigma is no longer receptive to pollen receipt. When each flower senesces, its style abscises naturally, so that styles can be collected for pollen-tube analysis without affecting subsequent fruit and seed development.

3.2.2 Flower and fruit sampling

This study considered two sets of flowers and their fruits: open-pollinated flowers, and flowers that were enclosed in fine-mesh pollinator-exclusion bags and subjected to specific pollination treatments. The open-pollinated flowers provided information on floral longevity and natural pollination and fruiting success. Every 4 days I tagged up to 5 flower buds on each plant,

depending on availability, using small jeweler’s tags attached to their pedicels. Plants were visited and the state of each tagged flower was recorded as “bud”, “open” or “senesced”. For

Open-pollinated flowers, I also recorded whether they had nectar-robbing holes, so that each flower was classified as robbed or unrobbed. At senescence, I collected each flower’s style and placed it in a 1.5-ml Eppendorf tube in 70% ethanol. I continued monitoring the ovaries of these flowers daily to determine their fruiting success (aborted or developed). If fruits failed, the ovary generally abscised within 10 days of flower senescence. I collected developed fruits just before maturity, when they were soft but had not abscised (some fruits fell before collection). Collected fruits were dried in an oven at 35 ºC for 24 h and then weighed.

The pollination treatments applied to bagged flowers assessed four aspects of reproductive capacity.

1) Cross pollination – Flowers were emasculated with fine forceps at anthesis and then hand- pollinated with pollen from non-study plants on their first and second days after anthesis.

Anthers were collected daily from 3-5 non-study plants, placed in a 1.5-ml Eppendorf tube and kept in a cool, dark location until use. Before pollination, I shook the Eppendorf tube to mix the pollen and then removed an anther with fine forceps and used it to apply pollen liberally to the stigma.

2) Self-pollination – On the first and second days after a flower opened I removed a dehisced anther from an untagged flower on the same plant with fine forceps and used it to pollinate the bagged flower.

3) Autonomous autogamy – Intact flowers were bagged, but not hand-pollinated, allowing only autonomous self-pollination.

4) Apomixis – Bagged flowers were emasculated with fine forceps to preclude autonomous self- pollination, so they could develop fruit only asexually.

Pollination bags were left on all experimental flowers until their corollas abscised. As for the open-pollinated flowers, I monitored the ovaries of the experimental flowers to assess fruit set.

Flowers subject to the autonomous-autogamy and emasculation treatments did not set fruit, whereas all experimental Cross- and Self-pollinated flowers set fruit.

3.2.3 Pollen-tube quantification

I used fluorescence microscopy to quantify pollen receipt and pollen-tube success in the sampled styles. Given that F. magellanica flowers produce up to 500 ovules, stigmas can receive many pollen grains and styles can support large numbers of pollen tubes, neither of which can be counted accurately. Instead, I used the proportion of the stigmatic surface covered by pollen grains as a measure of pollen receipt, and the width of the total mass of pollen tubes just distal to the base of the style as a measure of pollen-tube success. Collected styles were removed from the ethanol in which they were preserved and rinsed three times with distilled water. After the styles had rested for 3 h I rinsed them again and then placed them in 8 mol L-1

NaOH for 20 h to soften the stylar tissue. Styles were again rinsed three times in distilled water before being stained with aniline blue following Martin’s (1959) protocol. Stained styles rested in the aniline blue solution in a refrigerator for approximately 24 h before being placed on a slide and squashed with a cover slip. I squashed each style with equal pressure.

I examined the squashed styles with an Olympus BX 51 Epifluorescence microscope at

4x objective magnification and photographed them with a DigiRetina 16 (Tucsen Photonics,

Fuzhou, China) camera attached to the camera port of the microscope. Photographs were recorded in TIFF format (4608 x 3456 pixels) using image capture software (TCapture v3.9,

Tucsen Photonics 2017). Because of their large size, I took three photographs of each stigma. I used ImageJ v.1.52a (Schneider et al. 2012) to quantify pollen receipt and the width of the pollen-tube mass (e.g. Fig. 3.1). The proportion of pollen covering the stigmas was measured by converting the TIFF photos to RGB stack (e.g. Fig. 3.1A). Then I adjusted the image threshold so that area was represented by fluoresced pollen (e.g. Fig. 3.1B). Using the ImageJ measure function, I then calculated pollen cover and averaged the proportion of the stigma covered by pollen for the three photos. The width of the pollen-tube mass (in pixels) was measured from the style photographs (e.g. Fig. 3.1C and 3.1D) with the ImageJ length measure tool. If the pollen- tube mass had gaps, I excluded the space(s) from measurement.

3.2.4 Statistical analysis

Statistical analyses involved generalized linear and nonlinear mixed models, as implemented in SAS/STAT 14.2 (GLIMMIX and NLMIXED procedures: SAS/STAT® 14.2

User’s Guide 2016). As described below, the analyses of floral longevity and pollen-tube success were implemented using the NLMIXED procedure, but for different reasons. Generalized linear mixed models were used for the analyses of pollen receipt by open-pollinated plants (proportion of stigma covered by pollen: beta distribution, logit link function), proportional fruit set

(binomial distribution, logit link function) and fruit mass (normal distribution, identity link function). Initial covariates for the analysis of each flower’s pollen receipt included floral longevity and several plant variables measured during the flower’s life, including the average proportion of robbed flowers, total observed visits by each bee species and the average number of open flowers. For the analysis of proportional fruit set, initial covariates included the width of the pollen-tube mass, the average proportion of robbed flowers, the ln(sum of observed visits by each bee species) and the average number of open flowers. For the analysis of fruit mass (a

measure of fruit quality), initial independent variables included the width of the pollen-tube mass, whether the flower was robbed, habitat and the interactions of both factors with the width of the pollen-tube mass. This analysis included only open flowers. Another analysis was conducted to test the effects of Self- versus Cross-pollination on fruit mass, but the treatments did not differ (F1,17=1.68, P<0.3) and this result is not reported below. Each analysis included an initial test of habitat, but it significantly affected only fruit mass. All analyses included plant nested within habitat as a random effect. However, no significant among-plant variation was detected for pollen-tube success, so it was excluded from the final analyses.

Floral longevity was measured as a discrete variable (integer days); however, it was generally less variable than expected for a Poisson distribution. To accommodate this under- dispersion, I analyzed the influences on floral longevity with a double-Poisson distribution

(Efron 1986), using a ln-linear function of the covariates to characterize variation in the mean.

The NLMIXED procedure was used in this case because it allows explicit representation of the known likelihood function for any distribution. For the double-Poisson distribution, parameter  depicts under-dispersion ( > 1) or over-dispersion ( < 1) compared to a Poisson distribution.

For the analysis reported here,  = 2.687 (95% confidence interval = 2.245 - 3.128), indicating significant under-dispersion. Continuous independent variables for this analysis included the flower’s pollen receipt and several variables measured during a flower’s life, specifically average daily temperature and the plant variables total observed visits by each pollinator species, average proportion of robbed flowers and average number of open flowers. The initial model also included interactions between whether an individual flower was robbed or not and each covariate.

Pollen-tube success usually varies non-linearly with pollen receipt (Harder et al. 2016a, b), so I analyzed the effect of the proportion of the stigma covered by pollen (P) on the width of the tube mass at the base of the style (T) according to T = (1 – e-rP) where  is the asymptotic tube-mass width and r influences the rate of increase to the asymptote. This general model was used for two sets of analyses: one comparing Cross-, Self- and unrobbed Open-pollinated flowers; and the other comparing robbed and unrobbed Open-pollinated flowers. Whether separate parameters for the different classes of flowers fit better than common parameters was assessed with likelihood-ratio tests comparing full and reduced models. For the comparison of pollination treatments, the full model included separate estimates of . Parameter r was not estimated separately because hand-pollination for the Cross and Self treatments generally deposited abundant pollen, providing limited information for estimating this parameter.

Consequently, the estimate of r for this analysis depicts the approach to the asymptote based on unrobbed Open-pollinated flowers. For the comparison of unrobbed and robbed Open-pollinated flowers the full model allowed for separate estimates of both  and r.

3.3 Results

3.3.1 Floral longevity

Average floral longevity ranged from about 6 to 9 days and varied significantly with a plant’s average proportion of robbed flowers during the lives of sampled flowers, average daily temperature and whether a flower was robbed (Table 3.1, Fig. 3.2). Mean floral longevity declined approximately 3 days as the average proportion of robbed flowers on a plant increased from 0 to 1 (Fig. 3.2A). Overall, the average floral longevity of unrobbed flowers declined about

2 days over the 8 ºC range of variation in average daily temperature (Fig. 3.2B). In contrast,

floral longevity of robbed flowers did not vary with temperature, so that unrobbed flowers lasted longer than robbed flowers at lower temperatures (Fig. 3.2B).

3.3.2 Pollination and pollen-tube success

Pollen deposition (proportion of stigma covered by pollen) varied significantly with the average proportion of robbed flowers, flower visits by terr, the number of open flowers and floral longevity. The proportion of the stigma covered by pollen increased from 0.2 to 0.45 over the range of open flowers (Table 3.1, Fig. 3.3A) and from 0.2 to 0.4 over the 10-day range of floral longevity (Table 3.1 and Fig. 3.3B). In contrast, proportional stigma coverage decreased from 0.4 to 0.25 over the range of flower visits by terr, especially for visits to 0 to 30 flowers

(Table 3.1 and Fig. 3.3C). If the number of observed flower visits by terr was excluded from the model, adjusted mean proportional stigma coverage decreased from 0.4 to 0.3 as the average proportion of robbed flowers increased from 0 to 1 (Fig. 3.3D). This dependence suggests co- linearity between these covariates.

Pollen-tube success varied significantly with pollen deposition and differed among pollination treatments (Table 3.1 and Fig 3.4A). The asymptotes for the Cross- and Open- pollinated (unrobbed) flowers did not differ significantly, but both were significantly larger than that for Self-pollinated flowers (20.7%, and 12.1% respectively) (Table 3.2, Fig. 3.4A).

Pollen-tube success in Open-pollinated flowers also varied with pollen receipt, but nectar robbing altered this relation (Table 3.1 and Fig 3.4B). In unrobbed flowers, pollen-tube success increased more slowly with increased pollen receipt than in robbed flowers following limited pollination (Table 3.2), but the asymptote of pollen-tube success was 17% higher in unrobbed flowers (Table 3.2, Fig. 3.4B).

3.3.3 Fruit production

Proportional fruit set varied with the width of the pollen-tube mass at the base of the style and whether a flower was robbed or not (Table 3.1 and Fig. 3.5). Overall, a higher proportion of unrobbed flowers set fruit than for robbed flowers (Fig. 3.5A). This result reflected the high pollen-tube success of unrobbed flowers, as it was not evident if pollen-tube success was also included in the analysis (Fig. 3.5B). Proportional fruit set increased strongly from < 0.1 to 1 as the measured width of the pollen-tube mass increased from 0 to 3000 pixels (t183 = 2.29, P<0.05;

Table 3.1 and Fig. 3.5B).

Fruit mass (quality) differed among habitats and varied with the width of the pollen-tube mass at the base of the style, but this relation depended on whether flowers were robbed. Fruit mass increased approximately 50% faster with pollen-tube success in unrobbed flowers than in robbed flowers (Table 3.1 and Fig. 3.6A). Fruits were significantly heavier for beach plants than those in forest and along streams (Tukey’s Tests t18=3.31, P<0.01 and t18=2.49, P<0.05 respectively) but did not differ from those in the disturbed habitat (P < 0.05 in all cases, Fig.

3.6B). Fruit mass did not vary significantly among any other habitats.

3.4 Discussion

Nectar robbing had pervasive effects on the performance of Fuchsia magellanica flowers.

Compared to unrobbed flowers, robbed flowers wilted sooner than unrobbed flowers during cool periods (Fig. 3.2B) and wilted sooner when more flowers on a plant were robbed. Pollen receipt generally declined with increasing proportion of robbed flowers (Fig. 3.3D). Pollen tubes in the styles of robbed flowers also had higher success following limited pollen receipt than those in unrobbed flowers, whereas the pattern reversed following abundant receipt (Fig. 3.4B), indirectly affecting fruit set (Fig. 3.5B). In addition, robbed flowers with many pollen tubes produce lighter

fruits than unrobbed flowers with equivalent pollen-tube success (Fig. 3.6A). I now consider the likely causes and consequences of these robbing effects, as well as other influences on female reproduction by F. magellanica, before assessing the detrimental effects of the invasive bumble bee, terr, that was responsible for the damaging these flowers.

3.4.1 Plant responses

Flower lifespan decreased in response to increased temperature and increased nectar robbing. Temperature is known to influence floral lifespan strongly, generally shortening the time to senescence (Ashman 2004). However, the average proportion of robbed flowers open on a plant during a flower’s lifespan also had strong negative effects on floral lifespan. This effect may have occurred for several reasons. First, as nectar robbing damages flowers, plants may reduce resource allocation to robbed flowers. Second, damage causes flowers to release ethylene which accelerates senescence (Thompson et al. 1982, van Doorn 1997).

Pollen deposition varied with floral longevity, the average number of open flowers, the proportion of robbed flowers on a plant and the presence of nectar robbers. Pollen deposition increased with the average number of flowers open on a plant during its lifespan, which affects pollinator attraction (Ohashi and Yahara 2001; Fig. 3.3A). Pollen deposition also increased with floral lifespan (Fig. 3.3B), likely because flowers that remain open longer have more opportunity to be visited and receive pollen. However, pollen receipt varied negatively with flower visits by terr and with the average proportion of robbed flowers (Fig. 3.3C, D). These effects are related, as nectar robbing varied positively with changes in terr density (Fig. 2.1C, 2.6A, B). This association arises because most visits by terr involve nectar robbing (Fig. 2.3B), during which they do not contact stamens and stigmas. In addition, legitimate visitors may have sought resources at other plant species in response to changed resource conditions (Irwin 2000).

The association of the width of the pollen-tube mass at the base of the style to pollen receipt (Fig. 3.4) reveals key features of reproduction by F. magellanica, including the impact of nectar robbing. Self-pollen generally performed poorer in F. magellanica styles than cross- pollen. Pollen-tube success in styles of unrobbed Open-pollinated flowers was similar to that for

Cross-pollinated flowers (Table 3.2 and Fig. 3.4A). This similarity suggests that Open-pollinated flowers received mostly cross-pollen, rather than self-pollen. Comparison of robbed and unrobbed open-pollinated flowers revealed more complex results (Table 3.2 and Fig 3.4B). On one hand, pollen tubes in robbed flowers that received limited pollen were relatively more successful than equivalent unrobbed flowers. This result suggests that robbed flowers received higher quality pollen (Aizen and Harder 2007) and is consistent with the reduced number of flowers visited per bee (and hence lower geitonogamy) on heavily robbed plants (Fig. 2.8B). In contrast, asymptotic pollen-tube success was significantly lower in robbed flowers than in unrobbed flowers (Table 3.2 and Fig. 3.4B). This difference could arise if the flower damage inflicted by nectar robbing reduced the capacity of the stylar environment to support pollen-tube growth. Thus, nectar robbing may increase the proportion of cross-pollen received by flowers, while compromising the conditions for its maximal success.

The contrasting effects of robbing on pollen-tube success suggest an explanation for the heterogeneous effects of nectar robbing reported by previous studies (Irwin and Brody 2000,

Burkle et al. 2007, Irwin et al. 2010, Richman et al. 2017b). Robbing reduced pollen receipt and apparently enhanced pollen quality, but limited stylar capacity to support pollen tubes.

Depending on pollen receipt by robbed flowers of other species and their tolerance of damage, robbing effects could be generally positive, neutral (equally opposing beneficial and detrimental effects), or negative. This possibility has not been appreciated previously, because studies of

nectar robbing rarely assess effects on pollination quantity and have not considered pollination quality and its consequences for pollen-tube success.

Nectar robbing variously influenced fruiting success. Fruit set increased substantially with the width of the pollen-tube mass (Table 3.1 and Fig. 3.5B), suggesting that the number of pollen tubes penetrating the base of the style primarily determines ovule fertilization and fruit set

(see Cruzan 1989). In addition, fruit set decreased 20% when a flower was nectar robbed (Fig.

3.5A). This effect may result partially from resources costs incurred from the robbing itself. It may also reflect poor zygote fertilization due to reduced pollen-tube performance, causing plants to be less likely to invest in a fruit. Importantly, nectar robbing can also indirectly reduce pollen tube success, which would reduce fruit set.

Fruit mass was subject to diverse direct and indirect effects of nectar robbing. In unrobbed flowers, fruit mass increased substantially with pollen-tube success, but was lower in robbed flowers with the same width of pollen-tube mass (Fig. 3.6A). This response may be due to damage effects on ovule fertilization and/or embryo abortion. Fruits produced by plants along the more sunlit beach were slightly larger than for plants along streams and in forest. Another possibility is that dahl was more frequent along the beach compared to the stream and forest habitats (Fig. 2.4) and hence flowers received higher pollination.

The results of this study support aspects of indirect and direct antagonisms mediated by density-dependent behavior of pollinators; however, they are more nuanced and not directly aligned with my predictions. Direct effects are evident through reduced floral lifespan induced by nectar robbing and indirect effects through altered pollen deposition. The effects of robbing on pollen-tube success varied with pollen receipt, with indirect positive effects on pollen quality when pollination was limited, but negative following abundant pollination, likely caused by

direct flower damage. Post-fertilization effects of robbing include indirect reduction on fruit set and mass mediated by effects on pollen-tube success and direct effects of nectar robbing on fruit mass (Fig. 3.5B, 3.6A). Thus, the combination of results provides evidence in support of density- dependent antagonism acting directly and indirectly on this mutualism.

Density-dependent antagonism manifests both directly and indirectly in the F. magellanica pollination system. Robbing directly damages flowers, with subsequent effects on floral lifespan, pollen-tube success, fruit set and fruit quality. Meanwhile, indirect effects of robbing manifest through reduced pollen deposition but improved pollen quality. These effects depend on competition for nectar between visiting bumble-bee species. Arguably, because of the density-dependent shift to robbing, F. magellanica experiences density-dependent antagonism when terr visits its flowers frequently.

3.4.2 Invasion by a nectar robber and female plant reproduction

The negative effects of nectar robbing for female reproduction by Fuchsia magellanica are novel for this species, as terr has invaded Patagonia only recently (Schmid-Hempel et al.

2014). Nectar robbing has direct and indirect effects that manifest throughout the pollination process to limit female reproductive success of F. magellanica. Given the negative effects of nectar robbing, F. magellanica should have higher reproductive success in the absence of terr.

Being long-lived perennials, the plants in this study experienced very different pollination conditions prior to the terr invasion, and so likely lack adaptations that could confer resistance or tolerance to nectar robbing (Irwin et al. 2004, 2010). Other plant species with long-tubed flowers in Patagonia (and other invaded regions) may experience similar indirect and direct negative reproductive consequences in response to novel nectar robbers.

Table 3.1 – Estimated partial regression coefficients (b ± SE) from non-linear mixed models and generalized linear mixed models assessing influences of female reproductive success of Fuchsia magellanica. Dependent variable Effect Test statistics b ± SE

Floral longevity Intercept - Common t21= 19.77 2.486±0.126***

Intercept – Robbed t21= 9.18 2.154±0.237***

Intercept – Unrobbed t21= 33.09 2.817±0.085***

Average temperature X t21= 0.15 -0.0025±0.016

Robbed

Average temperature X t21= 7.48 -0.047±0.006***

Unrobbed

Average proportion of t21= 5.03 -0.334±0.066***

robbed flowers

Proportion of Intercept t21 = 5.06 -3.381±0.664*** stigma covered ln(Floral longevity) t172 = 2.60 0.750±0.288** by pollen ln(Average open flowers) t172 = 3.99 0.359±0.090***

ln(Sum terr visits) t172 = 3.09 -0.160±0.052***

Average proportion of t172 = 1.99 -0.400±0.201*

robbed flowers*

Width of pollen r t350 = 8.37 8.250 ± 0.986*** tube-tube mass Asymptote - Cross t350 = 37.71 2033.90 ± 64.529***

Asymptote – Self t350 = 26.12 1685.76 ± 77.346***

– comparison of Asymptote – Open t350 = 26.85 1890.27 ± 71.765*** treatments (unrobbed)

Width of r – unrobbed t318 = 7.01 7.241±1.033*** pollen-tube r – robbed t318 = 5.32 12.166±2.288*** mass – Asymptote – unrobbed t318 = 22.39 1974.77±88.212*** comparison of Asymptote – robbed t318 = 20.30 1687.90±83.145*** robbing effects

Fruit set Intercept t21= -4.75 -2.702±0.514***

Pollen tube width t186 = 6.45 0.002427±0.000355***

(pixels) t182 = -3.88 -2.322±0.599***

† Intercept t182 = -1.02 -0.620±0.610

Robbed†

Unrobbed†

Fruit mass Robbed Intercept t21 = 3.00 0.034±0.008**

(normal) Unrobbed Intercept t21 = 5.26 0.027±0.009***

-5 -6 Pollen tube mass (pixels) t105 = 6.05 1.400x10 ±4.498x10 **

X Robbed

-5 -6 Pollen tube mass (pixels) t105 = 3.05 3.000x10 ±5.023x10 ***

X Unrobbed

* P<0.05, ** P<0.01, *** P<0.001; † = Alternative model

Table 3.2 – Summary of contrasts for non-linear regressions of variation in the width of the pollen-tube mass in Fuchsia magellanica styles. The analysis of All pollination treatments considered all data and estimated separate asymptotes for each pollination treatment. The analysis for open-pollinated flowers compared both the rate of increase (r) to the asymptote and the asymptote between robbed and unrobbed flowers. Model Contrast Statistical test P

All pollination Asymptote – Cross vs t350 = 0.91 P=0.365 treatments Open (unrobbed)

Asymptote Cross vs t350 = 3.73 P=0.0002***

Self

Asymptote – Open t350 = 2.92 P=0.004**

unrobbed vs Self

Only open flowers Asymptote – Open t318 = 2.37 0.019*

(robbed) vs Open

(unrobbed)

r – Open (robbed) vs t318 = 1.97 0.050

Open (unrobbed)

* P<0.05, ** P<0.01, *** P<0.001

Figure 3.1 – Photomicrographs illustrating the measurement of pollen deposition on stigmas (A, raw; B, threshold transformed) and variation in the width of the mass of pollen tubes (C, wide tube mass; D, narrow tube mass). All photomicrographs are on the same pixel scale – the grey bars represent 1000 pixels at 4x objective magnification. A B

C D

Figure 3.2 – Relations of least-squares mean (± SE) floral longevity to (A) the average daily temperature and (B) the average proportion of robbed flowers on a plant while individual flowers were open. Means are back-transformed from ln values, hence the asymmetric standard errors.

11 A 11 B unrobbed 10 10 robbed 9 9

8 8

7 7

6 6

Floral longevity (days) longevity Floral 5 5

4 4 0.0 0.2 0.4 0.6 0.8 1.0 10 12 14 16 18

o Proportion of robbed flowers on plant Average daily temperature ( C)

Figure 3.3 – Relations of the least-squares mean (± SE) proportion of stigma covered by pollen for open-pollinated flowers to (A) the average number of flowers open on a plant during a flower’s life, (B) floral longevity, (C) the total terr visits to the plant observed during surveys while the flower was open and (D) the average proportion of robbed flowers on the plant during a flower’s life. Means are back-transformed from logit values, hence the asymmetric standard errors.

Figure 3.4 – Relations of the width of the pollen-tube mass at the base of the style to the proportion of the stigma covered with pollen for (A) Self-, Cross- and unrobbed Open- pollinated flowers, and (B) unrobbed and robbed Open-pollinated flowers.

Figure 3.5 – Relations of the least-squares mean (± SE) of proportional fruit set by open- pollinated flowers to (A) whether a flower was robbed or not and (B) the width of the pollen-tube mass at the base of the style. Means are back-transformed from logit values, hence the asymmetric standard errors.

Figure 3.6 – Relations of least-squares mean (± SE) fruit mass to (A) the width of the pollen-tube mass at the base of the style and (B) habitat. A B 0.09 unrobbed 0.15 robbed 0.08

0.10 0.07

0.05

0.06 Adjusted fruit mass (g) fruit mass Adjusted

0.00 0.05 0 500 1000 1500 2000 2500 3000 3500 Beach Disturbed Forest Stream

Width of pollen-tube mass (pixels) Habitat

Chapter 4 – Concluding Discussion

This study demonstrated density-dependent nectar robbing of Fuchsia magellanica flowers by a short-tongued bumble bee that caused a cascade of effects. As demonstrated in

Chapter 2, primary nectar robbing by Bombus terrestris changed competitive conditions for

Bombus dahlbomii, a long-tongued bee that does not act as primary robber, putting dahl at a competitive disadvantage. In response, some dahl may have shifted their visitation to plant species other than F. magellanica until terr abundance decreased, and many of those that continued visiting F. magellanica opportunistically became secondary nectar robbers, reducing their effectiveness as pollinators. In turn, Chapter 3 demonstrated that robbing had largely negative consequences for all phases of reproduction by F. magellanica, from floral condition and longevity to final fruit quality. In this chapter, I briefly address general implications of these results, including the importance of spatial and temporal variation of nectar robbing, the role of hummingbirds in the pollination of F. magellanica, potential long-term ecological and evolutionary consequences of nectar robbing for F. magellanica and conservation implications. I finish this chapter with some suggestions for future studies.

4.1 Temporal and geographic incidence of nectar robbing

Two previous studies have examined nectar robbing of Fuchsia magellanica (Combs

2011, Valdivia et al. 2016). Both studies considered variation in robbing intensity between sites based on single samples per site. These studies reported dichotomous spatial variation in robbing: either robbing was rare (0%-14% of flowers), or most flowers had been robbed (84%-

100% of flowers: Valdivia et al. 2016). These results may accurately characterize spatial variation in robbing of F. magellanica flowers owing to heterogeneity of the abundance of

Bombus terrestris, which has expanded its range extensively since its introduction to South

America in 1997 (Arbetman et al. 2013, Schmid-Hempel et al. 2014)

Alternatively, these results may reflect the confounding of single sampling dates at individual sites with temporal, density-dependent changes in nectar robbing within sites associated with the cycle of established Bombus terrestris colonies. During my study, the incidence of robbing switched in both directions between the two extremes observed in previous studies during a few days as terr abundance increased during early February and collapsed during late March (Fig. 2.1D). These results indicate that repeated sampling and consideration of temporal variation are needed to characterize accurately the incidence of nectar robbing and interaction dynamics between pollinators and plants.

4.2 Hummingbirds and Fuchsia magellanica

Based on its floral traits (pendent flowers with long, tubular corollas, exserted anthers and stigmas and copious nectar), Fuchsia magellanica seems adapted from hummingbird pollination. Consistent with this expectation, the hummingbird Sephanoides sephaniodes is a known visitor of Fuchsia magellanica (Traveset et al. 1998, Morales and Aizen 2006, Aizen and

Rovere 2010). However, during my study they visited infrequently, and perhaps only opportunistically in the presence of bumble-bee visitors. The presence of nectar-robbed flowers and associated reduced nectar availability can lower hummingbird visitation (Irwin 2000,

González and Valdivia 2005). As both dahl and terr visited F. magellanica intensively, reduced nectar availability may have caused the rarity of hummingbird visits. Furthermore, during the period that F. magellanica flowers many other “hummingbird-pollinated” species flower at and near the study site (Aizen and Rovere 2010). Many of these species may have been more suitable nectar sources if they were not visited by dahl and terr. Thus, the results presented in Chapter 2

may reveal only part of the competitive responses and community consequences caused by the presence of terr and its primary robbing of F. magellanica flowers.

4.3 Long-term ecological and evolutionary implications

In addition to the profound short-term implications on the reproductive success of flowering plants and legitimate native visitors reported in this thesis, invasive nectar robbers may provoke longer-term ecological and evolutionary effects. The sequential introduction of Bombus ruderatus and B. terrestris in Chile and their subsequent expansion rapidly extirpated

B. dahlbomii from most of its endemic range (Schmid-Hempel et al. 2014, Aizen et al. In press).

This rapid displacement of dahl complicates interpretation of the longer-term effects of nectar robbing. This study captured a unique snapshot during the transition of pollinator displacement.

Given that both the invader and resident species were present, this study was not representative of conditions and plant-pollinator interactions prior to invasion or of post-dahl pollination.

Unfortunately, during the summers since this study nectar robbing by terr at Puerto Blest has intensified and dahl has been observed only rarely (Aizen, M.A. pers. comm.).

Terr invasion could cause a variety of longer-term ecological consequences, depending on how it interacts with Fuchsia magellanica in the absence of dahl. In the absence of nectar robbing, dahl is an effective competitor, and its presence may limit exploitation of F. magellanica by terr. However, by removing nectar more efficiently dahl also lowers the nectar robbing threshold in F. magellanica. Thus, terr may be less likely to rob (or rob during a briefer period) in the absence of dahl and interspecific competition. However, the presence of robbing holes in F. magellanica flowers facilitates secondary robbing by terr. Furthermore, the absence of dahl may have two effects on reproduction by F. magellanica. Robbing by terr aggravates pollen limitation of fruit and seed production by F. magellanica. If nectar robbing occurred more

intensely or for a longer period, it might reduce reproductive benefits conferred by extensive flowering. This, in combination with the absence of legitimate visitation by dahl, could further reduce the benefits of extensive flowering.

Long-term evolutionary consequences of nectar robbing by terr for F. magellanica are also unclear. Given that nectar robbing reduced female reproductive success by F. magellanica, it may select for reduced flowering during peak abundance of terr and increased flowering earlier to increase its opportunities for pollination by hummingbirds. This response is feasible for

F. magellanica, given its protracted flowering. However, F. magellanica may be constrained by competition for pollination by opportunistic hummingbirds with other plant species (Aizen and

Rovere 2010). Whatever evolutionary responses robbing by invasive terr provokes, they will likely occur slowly, as F. magellanica is a long-lived perennial shrub and can live for decades

(Berry 1989).

4.4 Conservation implications

Bee species are increasingly used for agricultural pollination outside of their native range

(Aizen et al. In press). In particular, terr has also been introduced to Tasmania, Japan, New

Zealand and Israel for agricultural pollination (Goulson 2003, Aizen et al. In press), where it has also had generally negative impacts on native pollinators (Ishii 2013). These outcomes call for more stringent regulations limiting the use of commercialized nectar robbers outside of their range and development of more benign means of enhancing agricultural pollination. In most cases where terr has become invasive, the introduction has been accidental, but in Chile it has been intentional, and colonies continue to be introduced (Aizen et al. In press.). Given the evidence of its detrimental impacts, continuation of this practice is difficult to justify. An alternative approach that should have lesser negative consequences for native plants and

pollinators could involve domestication of native bee species for agricultural pollination (Aizen et al. In press).

The negative impacts of terr observed in this study demonstrate mechanisms by which invasive flower visitors can gain advantage over native pollinators, contributing to their displacement, and reducing reproductive success of the plants that they visit. If nectar robbing by invasive flower visitors also acts facilitatively in other pollination systems by liberating resources otherwise inaccessible to them, then other long-tongued visitors may also decline or be displaced. This could reduce pollinator diversity and cause declines in plant species with long- tubed flowers, depending on their life-histories. Furthermore, invasive visitors could facilitate disproportionate reproductive success between long-tubed and short-tubed flowering plant species (see Medel et al. 2018).

4.5 Suggestions for future studies

This case study adopted two methodological approaches that are seldom used in studies of plant-pollinator interactions: detailed temporal sampling and assessment of robbing consequences for most plant reproductive stages, rather than just pollinator visitation and seed or fruit production. Regular sampling during a species’ flowers period reveals insight into plant- pollinator interactions, including nectar robbing (also see Barrett 1980, Aizen 2001, Irwin and

Maloof 2002, Kudo and Kasagi 2005) that otherwise elude detection. Of particular relevance in this study was the ability to characterize the impacts of sudden shifts in ecological interactions. I also addressed how nectar robbing affects sequential reproductive processes, instead of just the final outcome. Doing so revealed previously undetected effects of nectar-robbing on pollen-tube performance, which is a key determinant of ovule fertilization and seed production. The new insights revealed by the combined application of these two approaches argue that they should be

incorporated more frequently in population and community studies of pollinator competition, plant-pollinator interaction, and plant reproduction.

References

Aizen, M. A. 2001. Flower sex ratio, pollinator abundance, and the seasonal pollination

dynamics of a protandrous plant. Ecology 82:127-144.

Aizen, M. A., and L. D. Harder. 2007. Expanding the limits of the pollen-limitation concept:

Effects of pollen quantity and quality. Ecology 88:271–281.

Aizen, M. A., and A. E. Rovere. 2010. Reproductive interactions mediated by flowering overlap

in a temperate hummingbird-plant assemblage. Oikos 119:696-706.

Aizen, M. A., C. Smith-Ramírez, C. L. Morales, L. Vieli, A. Sáez, R. M. Barahona-Segovia, M.

P. Arbetman, J. Montalva, L. A. Garibaldi, D. W. Inouye, and L. D. Harder. In press.

Coordinated species importation policies are needed to reduce serious invasions globally:

The case of alien bumblebees in South America. Journal of Applied Ecology.

Araújo, M. S., D. I. Bolnick, and C. A. Layman. 2011. The ecological causes of individual

specialisation. Ecology Letters 14:948–958.

Arbetman, M. P., I. Meeus, C. L. Morales, M. A. Aizen, and G. Smagghe. 2013. Alien parasite

hitchhikes to Patagonia on invasive bumblebee. Biological Invasions 15:489-494.

Ashman, T.L. 2004. Flower longevity. Plant Cell Death Processes, p349–362. L.D. Noodén,

Academic Press, Cambridge, Massechussets

Ashman, T.-L., T. M. Knight, J. A. Steets, P. Amarasekare, M. Burd, D. R. Campbell, M. R.

Dudash, M. O. Johnston, S. J. Mazer, R. J. Mitchell, M. T. Morgan, and W. G. Wilson.

2004. Pollen limitation of plant reproduction: Ecological and evolutionary causes and

consequences. Ecology 85:2408-2421.

Balfour, N. J., S. Gandy, and F. L. W. Ratnieks. 2015. Exploitative competition alters bee

foraging and flower choice. Behavioral Ecology and Sociobiology 69:1731–1738.

Barrett, S. C. H. 1980. Sexual reproduction in Eichhornia crassipes (Water Hyacinth). II. Seed

production. Journal of Applied Ecology 17:113-124.

Bell, S. A., and J. E. Cresswell. 1998. The phenology of gender in homogamous flowers:

temporal change in the residual sex function of flowers of oil-seed rape (Brassica napus).

Functional Ecology 12:298-306.

Berry, P. E. 1989. A systematic revision of Fuchsia Sect. Quelusia, Onagraceae. Annals of the

Missouri Botanical Garden 76:532-584.

Bowers, M. A. 1985. Experimental analyses of competition between two species of bumble bees

(Hymenoptera: Apidae). Oecologia 67:224–230.

Bronstein, J. L. 1994. Conditional outcomes in mutualistic interactions. Trends in Ecology and

Evolution 9:214–217.

Bronstein, J. L. 2001a. The exploitation of mutualisms. Ecology Letters 4:277–287.

Bronstein, J. L. 2001b. The costs of mutualism. American Zoologist 41:825–839.

Brosi, B. J., and H. M. Briggs. 2013. Single pollinator species losses reduce floral fidelity and

plant reproductive function. Proceedings of the National Academy of Sciences USA

110:13044–13048.

Brown, B. J., R. J. Mitchell, and S. A. Graham. 2002. Competition for pollination between an

invasive species (purple loosestrife) and a native congener. Ecology 83:2328–2336.

Burkle, L. A., R. E. Irwin, and D. A. Newman. 2007. Predicting the effects of nectar robbing on

plant reproduction: Implications of pollen limitation and plant mating system. American

Journal of Botany 94:1935–1943.

Castellanos, M. C., P. Wilson, and J. D. Thomson. 2002. Dynamic nectar replenishment in

flowers of Penstemon (Scrophulariaceae). American Journal of Botany 89:111–118.

Chalcoff, V.R., M.A. Aizen and L. Galetto. 2006. Nectar concentration and composition of 26

species from the temperate forest of South America. Annals of Botany 97:413-421.

Charlesworth, D., and B. Charlesworth. 1987. Inbreeding depression and its evolutionary

consequences. Annual Review of Ecology and Systematics 18:237-268.

Cnaani, J., R. Schmid-Hempel and J.O. Schmidt. 2002. Colony development, larval development

and worker reproduction in Bombus impatiens Cresson. Insectes Sociaux 49:164-170.

Cruzan, M. B. 1989. Pollen tube attrition in Erythronium grandiflorum. American Journal of

Botany 76:562-570.

De Palma, A., S. Abrahamczyk, M. A. Aizen, M. Albrecht, Y. Basset, A. Bates, R. J. Blake, C.

Boutin, R. Bugter, S. Connop, L. Cruz-López, S. A. Cunningham, B. Darvill, T. Diekötter,

S. Dorn, N. Downing, M. H. Entling, N. Farwig, A. Felicioli, S. J. Fonte, R. Fowler, M.

Franzén, D. Goulson, I. Grass, M. E. Hanley, S. D. Hendrix, F. Herrmann, F. Herzog, A.

Holzschuh, B. Jauker, M. Kessler, M. E. Knight, A. Kruess, P. Lavelle, V. Le Féon, P.

Lentini, L. A. Malone, J. Marshall, E. M. Pachón, Q. S. McFrederick, C. L. Morales, S.

Mudri-Stojnic, G. Nates-Parra, S. G. Nilsson, E. Öckinger, L. Osgathorpe, A. Parra-H, C.

A. Peres, A. S. Persson, T. Petanidou, K. Poveda, E. F. Power, M. Quaranta, C. Quintero, R.

Rader, M. H. Richards, T. Roulston, L. Rousseau, J. P. Sadler, U. Samnegård, N. A.

Schellhorn, C. Schüepp, O. Schweiger, A. H. Smith-Pardo, I. Steffan-Dewenter, J. C. Stout,

R. K. Tonietto, T. Tscharntke, J. M. Tylianakis, H. A. F. Verboven, C. H. Vergara, J.

Verhulst, C. Westphal, H. J. Yoon, and A. Purvis. 2016. Predicting bee community

responses to land-use changes: Effects of geographic and taxonomic biases. Scientific

Reports 6:31153.

Dedej, S., and K. S. Delaplane. 2005. Net energetic advantage drives honey bees (Apis mellifera

L) to nectar larceny in Vaccinium ashei Reade. Behavioral Ecology and Sociobiology

57:398–403.

Dreisig, H. 2012. How long to stay on a plant: The response of bumblebees to encountered

nectar levels. Arthropod-Plant Interactions 6:315–325.

Fontaine, C., C. L. Collin, and I. Dajoz. 2008. Generalist foraging of pollinators: Diet expansion

at high density. Journal of Ecology 96:1002–1010.

Ghazoul, J. 2006. Floral diversity and the facilitation of pollination. Journal of Ecology 94:295–

304.

González, P. L., and C. E. Valdivia. 2005. Direct and indirect effects of nectar robbing on the

polinating behaviour of Patagona gigas (Trochilidae). Biotropica 37:693–696.

Goulson, D. 2003. Effects of introduced bees on native ecosystems. Annual Review of Ecology,

Evolution, and Systematics 34:1–26.

Goulson, D., G. C. Lye, and B. Darvill. 2008. Diet breadth, coexistence and rarity in

bumblebees. Biodiversity and Conservation 17:3269–3288.

Harder, L. D. 1983. Flower handling efficiency of bumble bees: morphological aspects of

probing time. Oecologia 57:274–280.

Harder, L. D. 1985. Morphology as a predictor of flower choice by bumble bees. Ecology

66:198-210.

Harder, L. D. 1986. Effects of nectar concentration and flower depth on flower handling

efficiency of bumble bees. Oecologia 69:309–315.

Harder, L. D., M. A. Aizen, S. A. Richards, M. A. Joseph, and J. W. Busch. 2016a. Diverse

ecological relations of male gametophyte populations in stylar environments. American

Journal of Botany 103:484–497.

Harder, L.D., M.A. Aizen and S.A. Richards. 2016b. The population ecology of male

gametophytes: the link between pollination and seed production. Ecology Letters 19:497-

509.

Harder, L. D., and S. C. H. Barrett. 1996. Pollen dispersal and mating patterns in animal-

pollinated plants. Floral Biology, pp. 140-190. M. Percival, Chapman & Hall, New York.

Harder, L. D., N. Hobbhahn, and S. A. Richards. 2012. How depressed? Estimates of inbreeding

effects during seed development depend on reproductive conditions. Evolution 66:1375–

1386.

Hargreaves, A. L., L. D. Harder, and S. D. Johnson. 2009. Consumptive emasculation: The

ecological and evolutionary consequences of pollen theft. Biological Reviews 84:259–276.

Hayter, K. E., and J. E. Cresswell. 2006. The influence of pollinator abundance on the dynamics

and efficiency of pollination in agricultural Brassica napus: Implications for landscape-

scale gene dispersal. Journal of Applied Ecology 43:1196–1202.

Herrera, C. M. 2004. Distribution of pollen tubes : ecology labile in southern spatial mosaics

Spanish Lamiaceae. New Phytologist 161:473–484.

Inouye, D. W. 1978. Resource partitioning in bumblebees: Experimental studies of foraging

behavior. Ecology 59:672–678.

Inouye, D. W. 1980. The terminology of floral larceny. Ecology 61:1251–1253.

Irwin, R. E. 2000. Hummingbird avoidance of nectar-robbed plants: spatial location or visual

cues. Oikos 91:499-506.

Irwin, R. E. 2003. Impact of nectar robbing on estimates of pollen flow: Conceptual predictions

and empirical outcomes. Ecology 84:485–495.

Irwin, R. E., L. S. Adler, and A. K. Brody. 2004. The dual role of floral traits: Pollinator

attraction and plant defense. Ecology 65:1503-1511.

Irwin, R. E., and A. K. Brody. 2000. Consequences of nectar robbing for realized male function

in a hummingbird-pollinated plant. Ecology 81:2637-2643.

Irwin, R. E., J. L. Bronstein, J. S. Manson, and L. Richardson. 2010. Nectar robbing: Ecological

and evolutionary perspectives. Annual Review of Ecology, Evolution, and Systematics

41:271–292.

Irwin, R. E., and J. E. Maloof. 2002. Variation in nectar robbing over time, space, and species.

Oecologia 133:525–533.

Ishii, H. S. 2013. Community-dependent foraging habits of flower visitors: Cascading indirect

interactions among five bumble bee species. Ecological Research 28:603–613.

Kudo, G., and T. Kasagi. 2005. Microscale variations in the mating system and heterospecific

incompatibility mediated by pollination competition in alpine snowbed plants. Plant Species

Biology 20:93–103.

Lande, R. and D.W. Schemske. 1985. The evolution of self-fertilization and inbreeding

depression in plants. I. genetic models. Evolution 39:24-40.

Madjidian, J. A., C. L. Morales, and H. G. Smith. 2008. Displacement of a native by an alien

bumblebee: Lower pollinator efficiency overcome by overwhelmingly higher visitation

frequency. Oecologia 156:835–845.

Martin, F. W. 1959. Staining and observing pollen tubes in the style by means of fluorescence.

Biotechnic and Histochemistry 34:125–128.

McCullagh, P., and J.A. Nelder. 1989. Generalized linear models, 2nd ed. Chapman & Hall,

London, U.K.

Medel, R., C. González-Browne, D. Salazar, P. Ferrer, and M. Ehrenfeld. (2018). The most

effective pollinator principle applies to new invasive pollinators. Biology Letters 14:

20180132.

Moeller, D. A. 2004. Facilitative interactions among plants via shared pollinators. Ecology

85:3289–3301.

Morales, C. L., and M. A. Aizen. 2006. Invasive mutualisms and the structure of plant-pollinator

interactions in the temperate forests of north-west Patagonia, Argentina. Journal of Ecology

94:171-180.

Morris, W. F., D. P. Vazquez, and N. P. Chacoff. 2010. Benefit and cost curves for typical

pollination mutualisms. Ecology 91:1276–1285.

Newman, D. A., and J. D. Thomson. 2005. Effects of nectar robbing on nectar dynamics and

bumblebee foraging strategies in Linaria vulgaris (Scrophulariaceae). Oikos 110:309–320.

Ohashi, K., and T. Yahara. 2001. Behavioral responses of pollinators to variation in floral

display size and their influences on the evolution of floral traits. Cognitive Ecology of

Pollination, p274–296. J.D. Thomson and L. Chittka, Cambride University Press,

Cambridge, United Kingdom.

Ollerton, J., R. Winfree, and S. Tarrant. 2011. How many flowering plants are pollinated by

animals? Oikos 120:321–326.

Palmer, T. M., M. L. Stanton, and T. P. Young. 2003. Competition and coexistence: Exploring

mechanisms that restrict and maintain diversity within mutualist guilds. The American

Naturalist 162:63–79.

Pleasants, J. M. 1981. Bumblebee response to variation in nectar availability. Ecology 6:1648-

1661.

Pyke, G. H. 1978. Optimal foraging: Movement patterns of bumblebees between inflorescences.

Theoretical Population Biology 13:72–98.

Pyke, G. H. 1982. Local geographic distributions of bumblebees near Crested Butte, Colorado:

Competition and community structure. Ecology 63:555-573.

Pyke, G. 1984. Optimal foraging theory: A critical review. Annual Review of Ecology and

Systematics 15:523–575.

Rasheed, S. A., and L. D. Harder. 1997. Economic motivation for plant species preferences of

pollen-collecting bumble bees. Ecological Entomology 22:209–219.

Richards, S. A., R. M. Nisbet, W. G. Wilson, and H. P. Possingham. 2000. Grazers and diggers:

Exploitation competition and coexistence among foragers with different feeding strategies

on a single resource. The American Naturalist 155:266–279.

Richards, S. A., N. M. Williams, and L. D. Harder. 2009. Variation in pollination: Causes and

consequences for plant reproduction. The American Naturalist 174:382–398.

Richman, S. K., R. E. Irwin, and J. L. Bronstein. 2017. Foraging strategy predicts foraging

economy in a facultative secondary nectar robber. Oikos 126:1250–1257.

Richman, S. K., R. E. Irwin, C. J. Nelson, and J. L. Bronstein. 2017. Facilitated exploitation of

pollination mutualisms: fitness consequences for plants. Journal of Ecology 105:188-196.

Rodriguez-Cabal, M. A., M. N. Barrios-Garcia, G. C. Amico, M. A. Aizen, and N. J. Sanders.

2013. Node-by-node disassembly of a mutualistic interaction web driven by species

introductions. Proceedings of the National Academy of Sciences USA 110:16503–16507.

Rosenheim, J. A., S. J. Schreiber, and N. M. Williams. 2016. Does an “oversupply” of ovules

cause pollen limitation? New Phytologist 210:324–332.

Roulston, A. H., J. H. Cane, and S. L. Buchmann. 2000. What governs protein content of pollen:

pollinator preferences, pollen-pistil interaction, or phylogeny? Ecological Monographs

70:617-643.

SAS Institute Inc. 2016. SAS/STAT® 14.2 User’s Guide. Cary, North Carolina.

Schmid-Hempel, P., A. Kacelnik, and A. I. Houston. 1985. Honeybees maximize efficiency by

not filling their crop. Behavioral Ecology and Sociobiology 17:61–66.

Schmid-Hempel, R., M. Eckhardt, D. Goulson, D. Heinzmann, C. Lange, S. Plischuk, L. R.

Escudero, R. Salathé, J. J. Scriven, and P. Schmid-Hempel. 2014. The invasion of southern

South America by imported bumblebees and associated parasites. Journal of Animal

Ecology 83:823-837.

Schneider, C. A., W. S. Rasband, and K. W. Eliceiri. 2012. NIH Image to ImageJ: 25 years of

image analysis. Nature Methods 9:671-675.

Schreiber, S. J., J. A. Rosenheim, N. W. Williams, and L. D. Harder. 2015. Evolutionary and

ccological consequences of multiscale variation in pollen receipt for seed production. The

American Naturalist 185:14–29.

Stanton, M. L. 2003. Interacting guilds: Moving beyond the pairwise perspective on mutualisms.

The American Naturalist 162:10–23.

Stout, J. C., J. A. Allen, and D. Goulson. 1998. The influence of relative plant density and floral

morphological complexity on the behaviour of bumblebees. Oecologia 117:543–550.

Svanbäck, R., and D. I. Bolnick. 2007. Intraspecific competition drives increased resource use

diversity within a natural population. Proceedings of the Royal Society B: Biological

Sciences 274:839–844.

Thompson, J. E., S. Mayak, M. Shinitzky, and A. H. Halevy. 1982. Acceleration of membrane

senescence in cut carnation flowers by treatment with ethylene. Plant Physiology 69:859-

863.

Thomson, D. M. 2016. Local bumble bee decline linked to recovery of honey bees, drought

effects on floral resources. Ecology Letters 19:1247–1255.

Thomson, J. D., S. C. Peterson, and L. D. Harder. 1987. Response of traplining bumble bees to

competition experiments: shifts in feeding location and efficiency. Oecologia 71:295–300.

Traveset, A., M. F. Willson, and C. Sabag. 1998. Effect of nectar-robbing birds on fruit set of

Fuchsia magellanica in Tierra Del Fuego: A disrupted mutualism. Functional Ecology

12:459–464.

Tucsen Photonics. 2017. TCapture v3.9. Fuzhou, China.

Urcelay, C., C. L. Morales, and V. R. Chalcoff. 2006. Relationship between corolla length and

floral larceny in the South American hummingbird-pollinated Campsidium valdivianum

(Bignoniaceae). Annales Botanici Fennici 43:205–211.

Valdivia, C. E., J. P. Carroza, and J. I. Orellana. 2016. Geographic distribution and trait-

mediated causes of nectar robbing by the European bumblebee Bombus terrestris on the

Patagonian shrub Fuchsia magellanica. Flora: Morphology, Distribution, Functional

Ecology of Plants 225:30–36. van Doorn, W. G. 1997. Effects of pollination on floral attraction and longevity. Journal of

Experimental Botany 48:1615-1622.

Vázquez, D. P., and M. A. Aizen. 2004. Asymmetric specialization: A pervasive feature of plant-

pollinator interactions. Ecology 85:1251–1257.

Waser, N. M., L. Chittka, M. V Price, N. M. Williams, and J. Ollertons. 1996. Generalization in

pollination systems, and why it matters. Ecology 77:1043-1060.

Willmer, P. 2011. Pollination and Floral Ecology. Princeton University Press, Princeton, New

Jersey.

Wootton, J. T. 1994. The nature and consequences of indirect effects in ecological communities.

Annual Review of Ecology and Systematics 25:443–466.

Ye, Z. M., X. F. Jin, D. W. Inouye, Q. F. Wang, and C. F. Yang. 2018. Variation in composition

of two bumble bee species across communities affects nectar robbing but maintains

pollinator visitation rate to an alpine plant, Salvia przewalskii. Ecological Entomology

43:363–370.

Zhang, Y. W., J. M. Zhao, C. F. Yang, and W. R. Gituru. 2011. Behavioural differences between

male and female carpenter bees in nectar robbing and its effect on reproductive success in

Glechoma longituba (Lamiaceae). Plant Biology 13:25–32.