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Title The Causes and Consequences of Phenotypic Plasticity in Clarkia concinna (Onagraceae)

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Author Miller, Timothy Jordan

Publication Date 2016

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THE CAUSES AND CONSEQUENCES OF PHENOTYPIC PLASTICITY IN CLARKIA CONCINNA (ONAGRACEAE)

A dissertation submitted in partial satisfaction Of the requirements for the degree of

DOCTOR OF PHILOSOPHY

ECOLOGY AND EVOUTIONARY BIOLOGY

Timothy J. Miller

June 2016

This Dissertation of Timothy J. Miller is approved:

______Professor Kathleen M. Kay, Chair

______Professor James D Thomson

______Professor Ingrid M. Parker

______Professor John N. Thompson ______Tyrus Miller Vice Provost and Dean of Graduate Studies

Timothy J. Miller

2016 Table of Contents Page

List of Tables and Figures iv

Abstract v

Acknowledgements vii

General Introduction 1

Chapter 1: Maladaptive plasticity in response to soil moisture in Clarkia concinna 13

Bibliography 33

Tables and Figures 42

Chapter 2: Pollination of early flowers changes later floral and seed traits of Clarkia concinna 46

Bibliography 70

Tables and Figures 80

Chapter 3: Large floral displays increase self-pollination but do not affect visitation rates in Clarkia concinna 84

Bibliography 108

Tables and Figures 115

Synthesis 120

iii

List of Figures and Tables Page

Table 1.1 Description of study populations 42

Table 1.2 Response of ten traits to a drought treatment 43

Figure 1.1 Standardized trait scores and selection gradients 44

Figure 1.2 Effect of precipitation variation on total plasticity 45

Table 2.1 Response floral traits to pollination and abiotic manipulations 80

Figure 2.1 Selected floral trait responses 82

Figure 2.2 Effect of pollination on seed weight and number 83

Figure 3.1 Effect of floral display on consecutive pollinator visits 115

Figure 3.2 Effect of floral display on pollen pigment deposition 116

Figure 3.3 Effect of floral display on seed mass 117

Figure 3.4 Pollinator visitation rates to patches of differing size 118

Figure 3.5 Effect of patch size and floral display on pollination 119

Abstract:

The Causes and Consequences of Phenotypic Plasticity in Clarkia concinna

(Onagraceae)

Timothy J. Miller

Organisms can alter traits plastically in response to the biotic and abiotic environment in ways that potentially increase fitness, however not all plasticity is adaptive. Despite much study, the effects of plasticity on mating systems, species interactions, and responses to novel environments need further exploration. In this dissertation I study plasticity in an annual plant, Clarkia concinna (Onagraceae) by using a combination of greenhouse experiments and field observations and manipulations. In chapter one I investigate the plastic responses of multiple traits in a greenhouse droughting experiment, comparing the adaptive value of plasticity among plants from eight populations that varied widely in precipitation. Surprisingly, none of the measured traits show adaptive plasticity, and less plastic genotypes show a smaller reduction in fitness in the stressful drought treatment. Plants from locations with greater and more variable annual precipitation tend to show less plasticity in response to drought. In chapter two I focus on the causes of plasticity in floral traits and seed production. Hand-pollinating early flowers on a plant reduces total flower number as well as floral longevity in later flowers. This effect is independent of the abiotic resource conditions (water or nutrient availability). While pollen quality (self

v or outcross) does not affect floral traits, later fruits on plants with early outcrossed flowers have fewer seeds than fruits on plants with a background of selfing. In chapter three I examine the effects of floral display on pollination. Most pollinators were more likely to move pollen between flowers on the same plant when floral display size was large, increasing the deposition of self pollen. However, the predicted pollinator attraction benefit of large displays is absent in this species, even when there are few surrounding plants. Exploring plasticity in one species though the use of multiple techniques promotes a comprehensive understanding of the subject and leads me to propose potentially fruitful avenues of new research.

Acknowledgements:

This dissertation was only possible through the support of advisor, Dr.

Kathleen Kay, my dissertation committee, the EEB department, the greenhouse staff, undergraduate volunteers, reserve managers, native plant society member, my labmates, my friends, and my family. Thank you, all!

vii

General Introduction:

Phenotypic plasticity, or the ability of an organism to alter its phenotype in response to the environment, plays an important role in determining the fitness of a plant genotype (Woltereck 1909; Bradshaw 1965; Sultan 1987; Pigliucci 2001). The plasticity of a trait is subject to selection independently of the trait mean (Via and

Lande 1985; Scheiner 2002; Wund 2012), and may be a particularly important force in plant evolution (Schlichting 1986; Thompson 1991). Plants are modular organisms with repeated units (leaves, branches, flowers) that are often produced serially over protracted periods (Herrera 2009). Their indeterminate growth means plants can modify traits on later units in adaptive ways based on earlier environmental cues

(Sachs 2002). Additionally, sessile organisms such as plants can use plasticity as a means of coping with a potentially broader range of environmental conditions than those experienced by mobile life forms that can move to track favorable conditions

(Huey et al. 2002; Borges 2005). The high amounts of plasticity found in plants can have far-reaching consequences, potentially promoting colonization of novel habitats

(Baldwin 1896; Robinson and Dukas 1999; Yeh and Price 2004). Plasticity may also have a large influence on reproductive isolation and speciation (Fitzpatrick 2012), species interactions (Agrawal 2001), mating systems (Levin 2010; Peterson and Kay

2015), and life history (Sultan 2000). However, despite much theoretical and empirical study, connecting environmental variability to the adaptive value of plasticity in multiple traits remains difficult.

In this dissertation, I use an annual plant, Clarkia concinna (Onagraceae), to explore some of the causes and consequences of plasticity. First, I compare the magnitude and adaptive value of trait plasticity in response to a drought manipulation among eight populations that differ in their historical environmental variability. I predict plants from populations with more environmental variability will show greater adaptive plasticity for some traits and reduced maladaptive plasticity for others. Next,

I test how later floral traits and seed production would respond to early pollination. I expect plants would invest fewer resources in later flowers and fruits when they received early, high quality pollen, particularly when abiotic resources were limiting.

Finally, I study the effects of variation in floral display size on pollinator attraction and self-pollination. I predict large floral displays will increase insect visitation and self-pollination, especially when few other plants were nearby.

In chapter one, I test if more plastic plants tended to originate from more variable environments and have greater fitness in a stressful drought treatment. There are clear circumstances in which phenotypic plasticity is advantageous. If an individual can detect some aspect of their environment and alter physiological or morphological traits in response to that cue, it can potentially increase its fitness

(Pigliucci 1996; Gomez-Mestre and Jovani 2013}. Adaptive trait plasticity may provide the greatest fitness benefits when the environment is heterogeneous but can be predicted by recognizing an environmental cue (Ackerly et al. 2000; Scheiner

2013). Additionally, the plasticity of a trait can be most beneficial when not constrained by correlations with other traits (Lande and Arnold 1983; Arnold 1992).

Trait correlations may increase with increasing stress, limiting adaptive plasticity

(Tonsor and Scheiner 2007). However, not all plasticity is adaptive. Plasticity may be a maladaptive response to stressful conditions, particularly if these conditions are novel—not experienced in the recent evolutionary history of an organism (Pigliucci

2001; van Kleunen and Fischer 2005; Ghalambor et al. 2007).

I test three hypotheses in chapter one by subjecting plants from eight populations to a greenhouse watering treatment. First I test whether plasticity in response to drought in nine morphological traits was adaptive. All traits show large between-treatment differences in value, but surprisingly I find selection pressures were consistent between the environments. Genotypes that show less overall plasticity have a smaller decrease in fitness in the stressful drought treatment.

Therefore, much of the plasticity measured in this experiment is likely a maladaptive response to stressful conditions. Second, I expect correlations among traits would be stronger in the stressful drought treatment, indicating a potential constraint on adaptive plasticity. I did find stronger correlations in the drought treatment; however, since no traits were adaptive, there is no evidence for constraints on adaptive plasticity. Finally, I compare total plasticity among the eight populations, which varied widely in their annual precipitation regimes. Because the measured traits show maladaptive plasticity, I expect genotypes from locations with more variable precipitation to show less plasticity. I find a marginal support for this final prediction; however, a correlation between the mean annual precipitation amounts and the among year variability in precipitation makes generalization difficult. These findings indicate

3 that selection against maladaptive plasticity in response to stressful or novel conditions may be as important as selection for adaptive plasticity. Two features of this experiment may have promoted maladaptive over adaptive plasticity. Conditions in the greenhouse may not have mimicked natural drought conditions; adaptively plastic responses are less likely in response to such a novel treatment (Ghalambor et al 2007; Chevin et al. 2010), Additionally species such as Clarkia concinna with rapid, drought-avoiding life histories, may show less adaptive plasticity to water availability than drought tolerators (Ludlow 1989; Franks 2011).

In chapter two, I test for plastic responses in floral traits in response to early pollination. Mixed mating plants, such as Clarkia concinna, set seed through pollinator-mediated outcrossing and self-pollination (Goodwillie et al. 2005; Winn et al. 2011). While selfing can provide reproductive assurance and reduce the energetic costs of mating (Baker 1955; Lloyd and Schoen 1992), offspring of this species suffer substantial inbreeding depression under field conditions (Groom and Preuninger

2000), which may promote selection for selfing avoidance. Some plants can detect pollen deposition on the stigmas of early-opening flowers, initiating senescence in pollinated flowers (e. g. van Doorn 1997). Plants may be able to use the cue of early pollination to adjust traits in later flowers, maximizing resource allocation toward outcrossed offspring (Herrera 1991; Guitian and Navarro 1996).

I hand-pollinate early flowers with selfed or outcrossed pollen and measure the responses in the floral traits of later flowers. In addition, I manipulate the resource conditions to test if there was greater plasticity under water or nutrient limitation. As

4 expected, I find plastic responses in later flowers to these manipulations—floral longevity and total flower number are reduced on early-pollinated plants compared to unpollinated controls. However, there is no effect of pollen quality on floral traits— deposition of self or outcross pollen resulted in the same changes on later flowers.

Thus, the response is due to pollen receipt rather than pollen quality. While abiotic conditions also affect floral traits, they do so independently of the pollination treatment. Therefore, resource limitation may not be the main reason for the plastic changes in floral traits. Additionally, I test for the effects of pollen quality (self or outcross) on resource allocation towards seeds. Selfed seeds are lighter than outcrossed seeds regardless of the quality of pollen received by other flowers on the plant, likely due to inbreeding depression. However, more seeds are produced from later flowers on plants with a background of selfing than with a background of outcrossing, potentially indicating changes in resource allocation. In general, I find reductions in investment in later flowers and fruits in response to early pollen deposition in Clarkia concinna.

Whereas chapter two indicates the potential for plants to regulate their floral display (the number of simultaneously open flowers) by changing floral longevity, chapter three focuses on some of the consequences of the floral display plasticity.

Optimal floral display is often viewed as a tradeoff between pollinator attraction and avoidance of within-plant pollen movement. Larger displays may bring in more visitors but also increase the amount of pollinator-mediated geitonogamy—movement of pollen among flowers on the same plant (de Jong et al. 2003; Klinkhamer and de

Jong 1993; Finer and Morgan 2003). Several factors can affect the attraction/geitonogamy tradeoff, altering optimal floral display. If plants are not pollen limited, attraction is less important, potentially increasing selection for geitonogamy avoidance (Burd 1994; Ashman et al 2004; Knight et al 2005).

Additionally, pollinators differ in how they forage, and if pollinators move haphazardly among plants, geitonogamy is less of a problem (Whelan et al. 2009;

Gomez et al. 2010). Finally, when plants are more isolated, visitation may decrease and geitonogamy may increase (e. g. Kunin 1997; Duncan et al. 2004).

In chapter three, I test for evidence of the tradeoff between visitation rate and geitonogamy, investigating how the optimal floral display size might be affected by pollinator identity and the number of surrounding plants. I measure geitonogamy and visitation rate by observing pollinator movements, using fluorescent pigment to track pollen, and measuring seed production on plants with varying display sizes. All three methods show a pattern of increased geitonogamy in plants with larger displays, but fail to find any attraction benefits of large displays. Additionally, I test how the number of surrounding plants would influence patterns of visitation. While fewer pollinators visit plants in small patches than larger patches, display size does not affect visitation rate. Thus, Clarkia concinna makes larger floral displays than would be expected based on a tradeoff between visitation rate and geitonogamy alone. There are likely other factors that increase optimal display size. For instance, a quickly- drying environment may shorten the growing season. A reduced window in which to bloom would force plants to open a greater number of flowers at once in order to

6 maximize seed production despite the cost of increased geitonogamy and subsequently lower-quality offspring.

Taken together, this dissertation both adds support for widely-held hypotheses and finds some surprising patterns. As expected, I find populations and genotypes vary widely in the amount of trait plasticity they exhibit. I confirm the idea that plants can respond to the pollination environment in potentially adaptive ways. And I uphold the notion that larger displays increase geitonogamy. However, unexpectedly, none of the traits I measure in chapter 1 show adaptive plasticity to variation in drought. In chapter 2, plasticity in response to pollination surprisingly does not depend on abiotic resource levels, which would be expected under a resource allocation hypothesis. In chapter 3, contrary to theory, larger displays do not more attractive to pollinators, even in small patches of other plants. In these chapters, I interpret these findings with the goal of guiding research in productive new directions.

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Chapter 1

Maladaptive plasticity in response to soil moisture in Clarkia concinna

Abstract:

Natural selection might favor phenotypic plasticity in highly heterogeneous environments. However, organisms may exhibit maladaptive plasticity as a passive response to stressful or novel conditions. Stronger among-trait correlations in stressful environments may also limit the adaptive value of plastic responses.

Populations experiencing high amounts of environmental variability should experience selection for both increases in adaptive plasticity and decreases in maladaptive plasticity. I exposed eight populations of the annual plant, Clarkia concinna (Onagraceae) to a stressful greenhouse drought treatment and quantified the relationship between the plasticity of nine traits and fitness. Although population responses to dry conditions differed greatly, plasticity was generally maladaptive, and genotypes expressing less plasticity suffered smaller fitness reductions. Among-trait correlations were stronger in the dry treatment as predicted. However, correlations were not between maladaptive and adaptive responses, and thus did not constrain adaptive plasticity. Total plasticity was marginally lower in populations experiencing more heterogeneous annual precipitation regimes. These results indicate an important role of selection against maladaptive plastic responses in response to stressful conditions. Passive stress responses, novel environments, and rapid life histories may all reduce the adaptive value of plasticity.

Introduction:

Phenotypic plasticity is the tendency of a genotype to produce alternative phenotypes under different environmental conditions, and it has the potential to increase the fitness of an organism in the face of environmental variability (Bradshaw

1965; Sultan 1987; Schlichting and Pigliucci 1995; Whitman and Agrawal 2009). The relationship of a particular phenotypic trait with fitness can change depending on environmental context (Woltereck 1909; Lande and Arnold 1983). Trait plasticity is adaptive if the between-environment change in the direction of selection and the change in trait value match (Bradshaw 1965; Thompson 1991; Pigliucci 2001).

Plasticity for a trait can be heritable independently of the trait mean (Via & Lande

1985; Yeh and Price 2004; Kingsolver et al. 2001; de Jong 2005; Lind et al. 2007), and thus can respond to selection (reviewed in Scheiner 2002; Wund 2012). However, although plasticity is pervasive, reviews of plasticity studies find responses to environmental variation are frequently maladaptive (Pigliucci 2001; de Jong 2005; van Kleunen and Fischer 2005; Ghalambor et al. 2007). The ubiquity of maladaptive plasticity could result from passive responses to stressful environments, strong trait correlations, or lack of strong selection.

As plasticity refers to any environmentally induced change, a genotype may exhibit both active and passive plasticity (Kurashige and Callahan 2007; Whitman and Agarwal 2009). Active plasticity is a generally adaptive change in morphology caused in response to the detection of an environmental cue (van Kleunen et al.

2000). Active plasticity should evolve in traits with different selective pressures

14 between environments if the benefits of the phenotypic change outweigh the physiological costs of detection (Pigliucci 1996; Heschel et al. 2004; Gomez-Mestre and Jovani 2013). Well-studied examples of active plasticity with a demonstrated adaptive value include leaf shape in response to submersion in water (Cook &

Johnson 1968), shell shape in response to the chemical detection of predators (Lively

1986), and stem elongation in response to light quality (e.g. Schmitt et al. 2003). In contrast to these active responses to a cue, passive plasticity is a change in phenotype directly resulting from resource limitation under environmentally stressful conditions

(Sultan and Bazzaz 1993; Dorn et al. 2000; van Kleunen and Fischer 2005). Passive plasticity can also be adaptive in some conditions, however it is likely maladaptive if the trait is under similar selection across environments (David et al. 2004; Dudley

2004; Morris and Rogers 2013). For instance, plant size shows a positive correlation with fitness in most environments because larger plants generally produce more seeds

(e.g. Wolfe and Mazer 2005). If resources are limiting, plants may not grow as large, thus exhibiting passive maladaptive plasticity. Genotypes able to reduce maladaptive plasticity, for instance by maintaining larger sizes despite limiting resources, may have higher fitness in these stressful environments.

Because traits are rarely independent, correlations among traits can affect how selection acts on trait plasticity (Arnold 1992; Lande and Arnold 1983; Arnold 1992).

Studies comparing trait correlations among environments, populations, or related species frequently show changes in their strength and direction (e.g. Marshall et al.

1986, Schlichting and Levin 1990; Carroll et al. 2001; Caruso et al. 2006; Nicotra

2007; reviewed in Sgro and Hoffmann 2004). Stressful environments can increase tradeoffs in resource allocation, strengthening among-trait correlations (Tonsor and

Scheiner 2007). For example, empirical studies in plants have shown stronger correlations among floral and reproductive traits in low than high light (Callahan and

Waller 2000), in low than high nutrients (Waitt and Levin 1993), and in drought than benign conditions (Dudley 1996; Carroll et al. 2001). Increased correlation strengths may limit the range of potential phenotypes to only suboptimal combinations if these correlations occur between adaptive and maladaptive plastic responses. For instance,

Brock and Weining (2007) found the adaptive response of petiole elongation to low red:far red light ratios was correlated with a reduction in flower size. In primarily outcrossing species, smaller flowers often reduce seed set because they attract fewer pollinators. Thus the correlation would reduce the adaptive value of petiole elongation. Alternatively, correlated plastic responses between adaptive plastic responses may increase fitness in a stressful environment (Moran 1992; Parsons and

Robinson 2006). If the plant species in the previous example is self-compatible, smaller flowers may actually be beneficial in shaded environments with few pollinators, as they may promote self-pollination and ultimately increase seed set.

Selection on plasticity is stronger in some populations than others.

Populations experiencing greater temporal or spatial heterogeneity likely experience stronger selection on plasticity than populations in more constant environments

(Scheiner 2013). If plastic responses are adaptive, then populations experiencing high environmental variability may evolve higher plasticity (Sultan 1987; Ackerly et al.

2000). Conversely, less plastic genotypes would be favored in variable environments if plasticity is maladaptive (Grether 2005). Increasingly, studies compare plasticity between populations from heterogeneous and homogeneous environments (Sultan and

Bazzaz 1993; Donohue et al. 2001; Balaguer et al. 2001; Gianoli 2004; Nicotra et al.

2007; Lind et al. 2007; Baythavong 2011; Molina-Montenegro et al. 2010; Lazaro-

Nogal et al. 2015). These studies frequently find greater plasticity in genotypes from heterogeneous environments for at least some traits. However, the focal traits often explicitly show active plasticity with different fitness relationships among the treatments. It is less clear whether environmental heterogeneity can select against passive and potentially maladaptive plasticity.

In this study, I investigate plastic responses to a controlled watering treatment in eight populations of Clarkia concinna subsp. concinna (Onagraceae), comparing variation in plasticity among genotypes and populations. I test if plasticity tends to be adaptive or maladaptive by comparing between-treatment trait changes to the between-treatment differences in selection. I predict traits under different selection in the two environments will show the greatest plastic responses to the more stressful drought environment. Conversely, traits under similar selection in both environments should show less of a treatment effect, as this plasticity would be maladaptive. I compare among-trait correlation strengths between the environments, in order to test if among-trait correlations are stronger in the drought treatment. I regress the total plasticity of plants on the change in fitness between the treatments. If trait plasticities are generally adaptive, I expect more plastic genotypes will show a smaller reduction

17 in fitness in the stressful drought treatment. Alternatively, if plasticity is largely a maladaptive passive response to stressful conditions, low plasticity will predict smaller fitness reductions in the drought treatment. Finally, I compare total plasticity among populations that span the range of the species. If plasticity is generally adaptive, I expect higher plasticity in populations experiencing greater year-to-year environmental variability in precipitation. If plasticity is maladaptive, I expect the opposite pattern.

Methods:

Study System. Clarkia concinna is a self-compatible annual plant native to a variety of habitats throughout California’s Northern Coast Ranges with disjunct occurrences in the Northern Sierra Foothills. Individuals produce 1 to more than 100 four-merous, fuchsia, lobed and clawed flowers (average of 6 in natural populations) on leafy compound spikes. Flowers are protandrous and self-compatible, and fruits are elongate capsules containing an average of about 50 ovules (range 1-124, Groom

1998). The species occurs in a variety of habitats and natural populations show significant among population variation in all measured vegetative and floral traits

(unpublished data). Across the species range, mean precipitation during its growing season (October-June) varies from approximately 500 mm in the south east to 1800 mm in the northwest. Inter-annual variation in precipitation is also much higher in the north (standard deviation varying from approximately 150 to 450 mm). Within the

18 genus Clarkia, multiple lineages have adapted to faster-drying environments (Lewis

1962; Bartholomew et al. 1973; Allen et al. 1990; Eckhart et al. 2004). Populations and species in this genus that occur in drier regions tend to have shorter time to senescence, smaller, thicker leaves, and floral traits such as smaller size and reduction in herkogamy and dichogamy that increase self-fertilization (Lewis and Lewis 1955;

Vasek 1964; Vasek 1971; Dudley et al. 2007). In general, these traits can confer fitness advantages in dry environments (Eckhart et al. 2004; Moeller and Geber 2005;

Mazer et al. 2010).

Plasticity in traits associated with water use and phenology may be an important mechanism for Clarkia concinna to cope with annual heterogeneity in precipitation. Reliable environmental cues are a necessary condition for the evolution of adaptive plasticity (Schlichting and Pigliucci 1995; Pigliucci 2001; Scheiner 2013).

This species germinates at the beginning of the winter rains and blooms for a few weeks in May or June. As most rainfall occurs early in the growing season, relatively dry early soil moisture conditions likely indicate more severity of water stress as the plants flower. Because they have a less permanent seed bank than many annuals

(Lewis and Lewis 1955; Groom 1998), Clarkia may need to tolerate a larger amount of inter-annual environmental variation in order to ensure population persistence.

Adaptive plasticity may represent a potential mechanism that enables this persistence.

Experimental design. In the summer of 2011, I collected seeds from eight large populations (>500 plants) of C. concinna chosen to span the geographic range of the species (Table 1.1). For each population location, I used data from PRISM

Climate Group (Oregon State University, http://prism.oregonstate.edu), to calculate the mean and standard deviation of precipitation during the growing season of

Clarkia concinna (October-June) for the past 110 years. In each population, I collected 2-3 capsules from each of 30 haphazardly chosen plants spaced at least 3 meters apart. Clarkia concinna is fully self-compatible and can experience high levels of within-plant pollen movement (chapter 3). Seeds are gravity dispersed and nearby individuals are likely to be siblings. Therefore, seeds from each plant were at least maternal half-siblings and were likely more closely related. Although plasticity studies are traditionally conducted by placing clonal replicates in two or more environments, a less restrictive approach of comparing related individuals among treatments has been widely used when the focus of the study is broadly comparative

(Gianoli and Valladares 2012).

On August 13 and 14 of 2012, I planted seeds in 3.8 X 21 cm conetainers

(SC10 Super, Stuewe & Sons) containing four parts Pro-Mix HP Mycorrhizae potting soil to one part perlite. Fifteen replicate conetainers with five seeds each were planted per family. Germination was conducted in growth chambers with 10 hour 21° C days,

14 hour 13° C nights, and frequent watering. After germination, the containers were thinned to one randomly-chosen plant. The 15 families from each population with the highest germination rates were selected for the study, and moved to a greenhouse to begin the treatments. Each selected family included a maximum of twelve and a minimum of five replicates that were randomly assigned to the treatments. All families had at least one replicate that survived to flowering in both treatments (mean

= 4.8, st. dev ± 1.2). In total 1153 plants were included in the study. All plants were fertilized once (November 2, 2012) with a spray of 30 mL of Peters Professional 20-

20-20 General Purpose Fertilizer in 3 L of water.

I manipulated the soil moisture environment by inserting the conetainers into florist foam. The foam sat in a plastic tub containing approximately 2.5 cm of water and soaked up some of the water through capillary action (similar approach in Snow and Tingey 1985; Lambrecht et al. 2007). By altering the height that the pots sit above the surface of the water, I could keep soil moisture at two relatively constant levels. For the wet treatment, the bottom of the conetainer was inserted into the florist foam 0.5 centimeters below the level of the water, and for the dry treatment, the conetainer sat 5 centimeters above the maximum water level. Water was added to the trays every few days in the wet treatment and every week in the dry treatment. In late

November, I measured the percent soil moisture in all conetainers using a Procheck version 6 moisture probe (Decagon Devices). The treatments showed a large difference in percent soil moisture (drought 12.4% ± 3.8 SD, wet 21.0% ± 2.2 SD, t =

36.45, p < 0.0001), and plants in the dry treatment showed signs of drought stress such as leaf wilt and discoloration.

I surveyed all plants daily, and on each plant I measured ten traits including final number of flowers (flower number), my estimate of fitness. Seed number or total fruit weight are not an appropriate fitness measures for a greenhouse study, as they would only capture autogamous seed set. I chose nine additional traits that were likely to respond plastically to a watering treatment, but might differ in whether that

21 plasticity was adaptive. For instance, I expected stem diameter to show the same

(positive) relationship with my fitness estimate in both treatments, but I expected the relationship between specific leaf area and fitness to differ between treatments. I recorded the number of days from planting until the day that the first flower opened

(time to flowering), and the day that the lowest pair of leaves senesced (time to leaf drop). On the first flower day, I measured the stem diameter just above the soil surface with calipers (stem diameter), the height of the first flower above the soil surface (flower height) and collected the leaf subtending the open flower. I measured the specific leaf area (SLA) by tracing the outline of the collected leaf in ImageJ

(NIH, http://rsb.info.nih.gov/ij), calculating the area of the polygon, and dividing the value by the dry leaf mass. I took floral measurements on the second-from-lowest flower on the main stem. On the day the flower opened, I measured the length of the longest filament. On the day that the flower became female, I measured the corolla at its widest point (flower size) and the style length. I then subtracted the filament length from the style length (herkogamy) and subtracted the date the flower was female from the date the flower was male (dichogamy). After the last flower bloomed, I collected and dried the plants, removed all remaining fruits and leaves (most leaves had senesced) and separately weighed the above- and below-ground biomass. I then divided the belowground biomass by the above-ground biomass (root to shoot ratio).

Statistical Methods: Analyses were conducted in JMP Pro (version 11.2.0), unless otherwise noted. First, lognormally-distributed traits (flower height, SLA, root to shoot ratio, flower number) were log transformed to improve normality. In order

22 allow direct comparisons among variables, I then converted all trait values to z-scores by subtracting the grand mean from each value and then dividing by the standard deviation. I used a mixed-model ANOVA to evaluate whether each of the ten traits showed plasticity to the drought treatment with watering treatment (wet or dry), population, the random variable of family nested within population, and the two-way interaction of treatment and population were the predictive variables. Because I conducted multiple tests within each environment, I corrected the significance using a sequential Bonferroni criterion (k = 9). A significant interaction indicates among population variation in plasticity. I also performed an ANOVA on each trait in each population separately. Watering treatment and the random variable of family were the predictive variables.

I tested for environmental differences in selection gradients by performing a multiple regression analysis, testing for an effect of all nine traits on flower number

(my measure of fitness) in both environments in R version 2.7 (R development team).

The partial regression coefficients from this analysis are the selection gradients for each trait (Lande and Arnold 1983). Significant selection gradients indicate directional selection for the trait in that environment. Selection gradients account for correlations among traits and are directly comparable among traits and environments.

Selection gradients with different signs in the two environments would indicate the potential for adaptive plasticity. Changes in trait values (reaction norms) in the same direction as the change in selection gradient indicate adaptive plasticity. I also tested for stabilizing or disruptive selection of each trait in each environment by conducting

23 independent quadratic regressions. However, no quadratic terms were significant.

Because selection gradients differed substantially among populations, I also compared selection gradients between treatments within each population separately.

I compared the strength of correlations between the environments to test if trait correlations were stronger in the stressful dry treatment than the more benign wet treatment. I calculated the strength of each linear correlation (absolute value) for each pair of traits in each population and treatment and compared the correlation strength between the environments with a paired t-test.

I tested for a relationship between total plasticity and change in fitness. I calculated the total plasticity of a family by summing the absolute change in the mean family z-score across all 9 traits. Finally, I investigated the relationship between overall plasticity and source site environmental variability. I calculated the mean plasticity value across families for each population. I regressed the among-year standard deviation of precipitation against the total population plasticity against the during the Clarkia concinna growing season (October through June). A significant positive relationship would indicate that populations experiencing greater environmental variability have higher overall plasticity. I also regressed total population plasticity against mean annual precipitation and the coefficient of variation in order to compare the strength of potential predictors.

Results:

All ten traits including flower number (the fitness estimate) differed among

24 populations. Six of the traits including flower number differed between watering treatments. The effect of the watering treatment on specific leaf area, root to shoot ratio, and flower number depended on population (Table 1.2, Figure 1.1). Although not all traits showed an overall effect of watering, all traits were plastic in at least one population when populations were analyzed separately.

Selection gradients were similar in magnitude and direction in both wet and dry environments (Figure 1.1). In both environments, time to flowering, SLA, and root-to-shoot ratio showed negative selection gradients while stem diameter, herkogamy, and dichogamy showed positive selection gradients. Time to leaf drop, flower height, and flower size showed a non-significant selection gradient in one or both environments. Selection gradients were also similar in strength and direction between environments when populations were analyzed separately, as evidenced by the high correlation coefficient (0.58) between wet and dry selection gradients across the nine traits and eight populations (N = 72, p < 0.0001).

The strength of a particular correlation between two traits varied widely depending on both the population and the treatment. Pairwise correlations between traits were stronger in the dry treatment than the wet treatment (paired t-test with 36 pairwise comparisons across eight populations, t = 4.28, N = 288, p < 0.0001 mean correlation strength 0.22, in dry, 0.18 in wet). This pattern held for all eight populations when analyzed individually.

Families with greater overall plasticity had larger reductions in fitness in the

25 dry treatment (reduction in fitness = 0.241 + 0.107(sum absolute trait change), r2 =

0.07, N = 120, p = 0.003). Total population plasticity showed a marginally negative relationship with variability in precipitation (sum absolute trait change = 4.56 –

0.0023*(standard deviation precipitation), r2 = 0.43, N = 8, p = 0.076, Figure 1.2).

One population (MG) had the highest plasticity but occurred in an intermediately variable environment. Its exclusion greatly increased the relationship strength between variability in precipitation and total population plasticity (r2 = 0.84, p =

0.004). Variability in precipitation was a slightly better predictor of plasticity than mean precipitation (r2 = 0.40, p = 0.091), which was also much higher in northern populations. The high positive correlation between the standard deviation and mean of precipitation meant the coefficient of variation (standard deviation/mean) did not predict population plasticity (r2 = 0.15, p = 0.34).

Discussion:

All eight populations of Clarkia showed a large plastic response to a stressful drought treatment. Plasticity was maladaptive for all nine phenotypic traits, because the selection gradient did not differ between treatments. For some traits, particularly stem diameter and flower height, a lack of difference in selection between environments was expected. Plants that are taller and have larger stems tend to produce more flowers regardless of the environment. For other traits, the lack of a change in selection gradient was surprising. For instance, I expected early flowering

26 to be favored in the dry treatment but not the wet treatment, as previous studies in

Clarkia have demonstrated that early flowering is a drought adaptation (Eckhert et al.

2004; Mazer et al. 2010). However, genotypes that flowered earlier had more flowers regardless of treatment.

Positive selection on dichogamy in both environments was also unexpected given the known cost of producing large, long-lived flowers (Ashman and Shoen

1994; 1997). Families that produced more flowers tended to make larger, longer-lived flowers. These families may have globally higher fitness not only due to genetic differences such as being less inbred, but also due to maternal effects (Wolf and

Wade 2009) or epigenetic processes such as DNA methylation (Alonso et al. 2014).

Alternatively, if dichogamy is negatively correlated with autogamy (self-pollination within a flower), plants with lower dichogamy may allocate resources towards ripening selfed seeds rather than producing additional flowers, confounding my fitness estimates. This species does reduce flower number in response to early hand- pollination (chapter 2). Kay and Picklum (2013) found a non-significant negative correlation between dichogamy and autogamous seed set in this species. However, I did not find a correlation between dichogamy and autogamous fruit set, and the percentage of fruit set did not increase in the drought treatment despite lower dichogamy (about 50% of flowers autogomously produced fruits in both treatments).

Therefore, resource allocation likely cannot completely explain the relationship between dichogamy and flower number.

Although the plastic responses were maladaptive in a greenhouse setting,

27 plasticity in these traits may be more adaptive under field conditions. The greenhouse likely represents a novel environment, with conditions that fall outside of those experienced in recent evolutionary history. Organisms may be less likely to show adaptively plastic responses to novel environments (Rutherford and Lindquist 1998;

Ghalambor et al. 2007; Chevin et al. 2010). The greenhouse dry treatment involved subjecting plants to soil moisture levels that were chronically lower than the wet treatment throughout most of the lifespan of the plant. This difference in treatments would favor a drought tolerator strategy in the drought treatment, as tolerators are likely better able to deal with chronic stressors than avoiders (Levitt 1980; Ludlow

1989). However, a fast-cycling annual such as Clarkia concinna may follow a drought avoider strategy. Thus, I may have found adaptive plasticity by imposing a dry-down type of treatment (Franks 2011), rather than the continuous dry treatment that I imposed. Alternatively, avoiders and other taxa with fast lifecycles may be less adaptively plastic than tolerators in general.

The greenhouse also does not capture important environmental influences on fitness such as the pollinator environment and intra- and interspecific competition

(Moeller and Geber 2005). Selection gradients may be more likely to differ between more natural environments, allowing greater opportunity for adaptive plasticity. For instance, Clarkia growing dry environments tend to show a general reduction in flower size and herkogamy, leading to increased autogamous selfing rates (Holtsford and Ellestrand 1992; Kay and Picklum 2013). These environments have fewer pollinators (Dudley et al. 2007; Levin 2012), and thus passive plasticity in floral

28 morphology may increase fitness though reproductive assurance in a pollinator-poor environment (Fishman and Willis 2007).

Stronger correlations between adaptive and maladaptive traits under stressful conditions may to constrain the range of potential combinations of trait values, reducing the magnitude of the adaptively plastic response (Dudley 1996; Carroll et al.

2001; Gianoli and Palacio-Lopez 2009). Traits correlations were generally stronger in the drought treatment, as expected. However, as all measured plastic responses were maladaptive, I did not find evidence of correlations acting to constrain an adaptively plastic response.

The population of origin was the largest determinant of trait value, strength of trait correlations, and the relationship between a trait and fitness. Generally, plants from the same population but different treatments were more similar than plants from different populations in the same treatment. This large effect of population holds for many taxa (e.g. Schlichting and Levin 1990; Nicotra et al. 2007). Population size, genetic diversity, and location in the center or periphery of the geographic range may affect the overall plasticity of a population (Schlichting 1986; Berg et al. 2005; Magi et al. 2011). As these factors may co-vary with environmental heterogeneity, they need to be considered when making among-population comparisons of plasticity.

Because measured plasticity was maladaptive, it is logical that families exhibiting a smaller plastic response showed a smaller reduction in dry-treatment fitness. Unmeasured adaptive plasticity in physiological traits such as water use

29 efficiency may underlie the maintenance of a beneficial phenotype under stressful conditions (Dudley 1996; Caruso et al. 2006; Dudley et al. 2012). Families that expressed high adaptive plasticity in these physiological traits may have prevented the expression of maladaptive plasticity in morphological and timing traits (Morris and Rogers 2013).

Although the amount of plasticity varied greatly within a population, families that maintained a more similar phenotype under stressful conditions tended to be from more northern populations with higher inter-annual variation in precipitation. These populations also experienced higher amounts of rainfall on average, especially in wet years. It is noteworthy that the populations from drier locations showed the greatest maladaptive plastic response in the dry treatment. The most plastic population (MG) was much more plastic than predicted by precipitation variability or mean. This was the only study population originating from harsh serpentine soils. The physiological adaptations necessary to survive in this stressful local environment may limit the ability to avoid maladaptive plasticity to other stressful environmental conditions

(reviewed in Valladares et al. 2007).

Several studies that relate plasticity and environmental variability have found higher plasticity in heterogeneous environments (Donohue et al. 2001; Balaguer et al.

2001; Gianoli 2004; Lind et al. 2007; Baythavong 2011; Lazaro-Nogal et al. 2015).

In these studies, the fitness benefit of plasticity in the traits under consideration is often explicit. For instance, in the annual plant Impatiens capensis, plasticity in stem elongation in response to density is more adaptive in open sites than woodland sites,

30 and genotypes from open sites show greater plasticity (Donohue et al. 2000; 2001). In the frog Rana temporia, plasticity in development time is both more adaptive on islands with greater spatial heterogeneity in pool-drying regimes, and is higher there

(Lind et al. 2007). However, other studies find mixed relationships between population environmental variability and the adaptive value of plasticity (Nicotra et al. 2007) or no consistent among-population differences in adaptive plasticity across the measured traits (Sultan and Bazzaz 1993).

Although the idiosyncrasies of individual systems can make generalizations challenging, more among-trait comparisons of the adaptive value of plasticity are needed. A recent review of reciprocal transplant studies found that over half of measured traits were not plastic, and of the traits that were, almost a third showed maladaptive plasticity (Palacio-Lopez et al. 2015). This study may be less biased in estimating the frequency of adaptive plasticity than a review of the plasticity literature because the measured traits were not necessarily chosen with adaptive plasticity in mind. More comparative studies are needed to relate both the types of traits and the novelty of the environment to likelihood of adaptive plasticity. For instance, active developmental or regulatory responses to environmental conditions are predicted to be more adaptive than passive responses to stress (Pigliucci 1996; Pigliucci 2001;

Gomez-Mestre and Jovani 2013). An example of this pattern occurs in Arabidopsis, where active responses to a change in the ratio of red to far red light as perceived by the plant via phytochrome tended to be adaptive, while passive stress responses due to the reduction in photosynthetically active radiation were largely maladaptive (Dorn et

31 al. 2000). A more comprehensive understanding of plasticity may find that heterogeneous environments should select for both increases in adaptive plasticity and decreases in maladaptive plasticity.

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Wund, M. A. 2012. Assessing the impacts of phenotypic plasticity on evolution. Integrative and Comparative Biology 52:5-15.

Yeh, P. J., and T. D. Price. 2004. Adaptive phenotypic plasticity and the successful colonization of a novel environment. The American Naturalist 164:531-542.

Table 1.1. A description of the study populations, which were all located in California, USA, including location, elevation in meters, growing season (October through June) precipitation annual mean and standard deviation measured in millimeters, and a brief habitat description.

Table 1.2. The response of ten traits to a drought treatment including flower number, a proxy for fitness, as indicated by trait means (standard deviations) in both environments and the F-statistics from two-way ANOVAs.

dry mean wet mean trait treatment F population F interaction F (stdev) (stdev) time to flowering 111.0 (14.3) 111.9 (16.4) 1.33 50.67 0.84 time to leaf drop 112.7 (16.2) 113.8 (15.5) 2.16 80.09 1.43 stem diameter 1.79 (0.31) 1.86 (0.32) 7.81 16.52 1.38 flower height 211.7 (93.8) 244.4 (102.7) 24.59 168.1 2.05 SLA 16.7 (6.4) 14.8 (4.1) 38.53 13.06 3.98 flower size 58.2 (7.7) 60.1 (6.7) 14.05 126.19 2.17 herkogamy 3.07 (1.92) 3.27 (1.88) 2.58 104.42 0.76 dichogamy 4.50 (2.05) 5.14 (2.23) 38.78 197.11 2.48 root to shoot ratio 0.35 (0.17) 0.36 (0.16) 3.45 57.96 4.15 flower number 13.3 (7.1) 15.4 (7.2) 36.41 28.7 2.86

Note. Bold values are significant (p < 0.05) after Bonferroni correction. Population df = 7, treatment df = 1; interaction df = 7. N = 529 for dry and 622 for wet. Time to flowering, and time to leaf drop, and dichogamy measured in days. Stem diameter, flower height, flower size, and herkogamy measured in mm. SLA measured in mm2/g.

Figure 1.1. Standardized trait values (z-score) and selection gradients (partial correlation coefficients with each trait and flower number) in wet and dry treatments for the nine measured traits. Athough traits showed large differences between treatments, selection gradients remained relatively constant, resulting in maladaptive plastic responses.

Figure 1.2. Regression of the total standardized change in nine traits in response to a drought treatment against the variation in precipation that occurs during the growing season of Clarkia concinna. Points are population means of total plasticity (absolute value of change in z-scores summed across 9 traits; N = 15 families per population). Populations from more variable environments average lower plasticity with the exception of MG, a serpentine population with medium amounts of precipitation variation and high plasticity.

Chapter 2

Pollination of early flowers changes later floral and seed traits of Clarkia

concinna

Abstract:

Premise of Research: Plastic responses to local environmental variation are advantageous if selection is temporally variable. In flowering plants with mixed mating systems, the relative benefits of outcrossing and selfing depend on the availability of pollinators and abiotic resources, potentially favoring plasticity in mating system traits. Plants should produce traits that increase pollen receipt when abiotic resources are abundant, but pollinators scarce. Additionally, plants should invest more resources in later-developing outcrossed fruits when early fruits are selfed than when early fruits are also outcrossed.

Methodology We used a mixed-mating annual, Clarkia concinna, to test for plasticity in floral traits and seed production in response to pollination, drought, and fertilization. I predicted early receipt of outcross pollen would reduce resource allocation to later flowers and fruits, especially when abiotic resources were limiting.

Pivotal Results: Under field and greenhouse conditions, unpollinated later flowers on plants with hand-pollinated early flowers had shorter longevities than unpollinated later flowers on unmanipulated plants. Total flower number was greater on unpollinated plants in the greenhouse. Water and nutrient availability affected

46 floral traits, but did not alter the impacts of the pollination treatment. The number of seeds matured per focal fruit was greater when all other flowers on the plant were self-pollinated than when they were outcrossed, reflecting shifts in resource allocation. However, seeds were heavier when sired from outcross pollen regardless of the background pollination treatment.

Conclusions: Taken together, these experiments show Clarkia concinna responds to both early pollen deposition and abiotic cues by altering floral traits and seed production in putatively adaptive ways.

Introduction:

Populations frequently experience environmental variation over small spatial and temporal scales. The detection of local environmental cues may enable an organism to plastically alter traits, potentially increasing fitness (Bradshaw 1965;

Schlichting 1986; van Kleunen and Fischer 2005). In plants, traits that affect mating systems particularly may be subject to selection for plasticity. Biotically-pollinated hermaphroditic plants frequently have mixed mating systems, producing both outcrossed and selfed offspring (Goodwillie et al. 2005; Winn et al. 2011). When the most beneficial mating system depends on predictable variation in local environment, selection should favor mating system plasticity (Schoen and Lloyd 1984; Kalisz et al.

2004).

Both models and empirical data indicate that optimal outcrossing rates will depend on the environmental conditions experienced by a plant. Selfing can provide plants with reproductive assurance and an automatic transmission advantage as well as reducing the energetic costs of mating (Baker 1955, Lloyd and Schoen 1992).

Transitions from primarily outcrossing to primarily selfing have occurred repeatedly across the plant phylogeny (Stebbins 1974; Jain 1976; Barrett 2002). However, selfed offspring of mixed-mating taxa often suffer inbreeding depression (Charlesworth and

Charlesworth 1987; Goodwillie and Knight 2006). Models predict that, under stressful environments, traits that promote autogamous self-pollination can be under positive selection, even when inbreeding depression is high (Lloyd 1979; Schoen et al. 1996). Comparative studies among populations and species find support for these

48 models, and correlations between stressful conditions and selfing rates or traits that affect mating system are common (e.g. Vasek and Harding 1976; Holtsford and

Ellstrand 1992; Fausto et al. 2001; Armbruster et al. 2002; Mazer et al. 2004).

However, when the environment varies at small spatial or temporal scales, evolution of mean trait values may be less beneficial than plastic responses (Sultan 1987;

Thompson 1991; Whitman and Agrawal 2009; Baythavong 2011).

A few key floral traits have a large effect on mating system in self-compatible plants. Self-pollination can occur within or between flowers (autogamy and geitonogamy, respectively). Autogamy is largely determined by the amount of separation in the male and female functions of a flower in space (herkogamy) and time (dichogamy) (Lloyd and Webb 1986; Webb and Lloyd 1986). Geitonogamy is strongly influenced by number of flowers open at any one time (floral display) (De

Jong et al. 1993; Barrett 2003). Slowing the rate at which flowers open or decreasing floral longevity can reduce floral display and subsequently geitonogamy (Harder and

Johnson 2005). Flower size also impacts mating system—smaller flowers tend to have higher selfing rates (e.g. Ritland and Ritland 1989; Eckert and Geber 1999; Elle and Carney 2003). Flowers are modular structures, and can show large amounts of within-plant variability in these traits (Murren 2002; Herrera 2009). Because few plants open all flowers simultaneously, individuals have the opportunity to alter the traits of later-opening flowers in response to the fate of early flowers (Herrera 1991;

Guitian and Navarro 1996; Zhao et al. 2010). However, some traits, such as pollen to ovule ratio, may be set earlier in development and will be less able to respond to

49 environmental conditions than others (Lloyd 1980; Lloyd et al. 1980; Thomson

1989).

The floral traits that maximize fitness will depend on pollinator abundance.

Some plants can sense stigmatic pollen deposition, rapidly causing corolla senescence in pollinated flowers (Mayak and Halevy 1980; Arathi et al. 2002; Clark and Husband

2007; Jorgensen and Arathi 2013). Individuals can potentially use the cue of pollen deposition in early-opening flowers to alter traits in later flowers. If early visitation rates are high, then fewer resources need to be invested in pollinator attraction.

Therefore, we may expect reductions in size, longevity, and floral display when the majority of early opening flowers receive pollen. Conversely, attraction is at a premium in pollinator-poor environments (Primack 1985; Schoen and Ashman 1995), and while increased floral longevity and large floral displays promote geitonogamy, they also can increase overall visitation rates (Klinkhamer and de Jong 1993; Finer and Morgan 2003). This plastic response may run counter to selection for autogamy in chronically low-pollinator environments.

Mating system traits also respond plastically to the abiotic environment.

Stressful abiotic conditions may increase the costs of large or long-lived structures designed for pollinator attraction, indirectly selecting for decreased outcrossing rates.

For instance, plants subjected to drought treatments often show smaller or shorter- lived flowers as a way to reduce water loss, subsequently reducing herkogamy and dichogamy and increasing selfing rates (Holtsford and Ellstrand 1992; Schoen and

Ashman 1995; Runions and Geber 2000; Mazer et al. 2010; Halpern et al. 2010;

Jorgensen and Arathi 2013; Spigler and Kalisz 2013; Kay and Picklum 2013).

Because traits associated with pollinator attraction require more nutrient expenditure, selfing may also be indirectly favored when nutrients are limiting (Munoz et al. 2005;

Burkle and Irwin 2009). Alternatively, inbreeding depression may be stronger in stressful environments, increasing the fitness benefits of outcrossing and promoting the maintenance of outcrossing traits despite limiting resources (reviewed in

Armbruster and Reed 2005; Fox and Reed 2011). These plastic responses to the abiotic environment may interact with responses to biotic cues. Specifically, later flowers on early outcrossed plants may exhibit selfing traits most strongly when the abiotic environment is stressful. Plants experiencing water or nutrient limitation will be less able to take advantage of the continued receipt of high-quality pollen (Totland

2001; Caruso et al. 2005).

In addition to floral trait plasticity, plants may plastically alter the allocation of resources to ovules after fertilization. Many species overproduce ovules beyond what they can mature even when pollen is not limiting (Wiens 1984; Burd 1994), allowing the selective abortion of embryos sired by lower-quality mates (Lee 1984;

Montalvo 1992). Often plants mature fewer self-fertilized ovules than would be expected based on the amount of self pollen received (Jones 1994; Husband and

Schemske 1996). For instance, in Erythronium, when presented simultaneously with pollen from two pollen donors, seeds were more likely sired by pollen from distant

(and presumably less-related) plants than pollen from nearby plants or self pollen

(Rigney et al. 1993). It can be difficult to disentangle whether differences in seed

51 number or weight are the result of partial self-incompatibility, early-acting inbreeding depression, or preferential maternal allocation toward outcrossed seeds (Marshall and

Ellstrand 1986; Seavey and Bawa 1986; Korbecka et al. 2002; Goodwillie et al.

2005). Whole-plant pollination manipulations are one way to test whether selective allocation of resources toward outcrossed offspring is occurring (Zimmerman and

Pyke 1988; Lawrence 1993; Ashman et al. 2004). If seed weight and number are greater in later flowers when early-opening flowers on the plant are selfed than when they are outcrossed, this indicates an effect of maternal resource allocation. If seed weight and number are greater for outcrossed than selfed flowers regardless of the type of pollen received by other flowers, this indicates self-incompatibility or inbreeding depression.

We investigated the plastic responses of floral and seed traits to pollen deposition and resource availability in Clarkia concinna (Onagraceae). This species is a mixed-mating annual that shows both plasticity and among-population variation in floral traits that affect mating system, including size, herkogamy, dichogamy, and floral display (Kay and Picklum 2013; Miller unpublished data). I tested how Clarkia concinna responds to receipt of pollen on early flowers with three experiments. The first investigated the combined effects of water availability and pollination environment on floral traits in a greenhouse environment. The second tested the effects of nutrient availability and pollination on floral traits in a natural setting. The final experiment examined the effects of self and outcross pollen on seed traits in the greenhouse. Specifically, We asked: 1) Do plants respond plastically to the early

52 pollination environment by altering floral traits in later flowers? We expected plants receiving outcross pollen on early-opening flowers would reduce investment in attraction traits in later flowers compared to those that did not receive pollen. 2) Do the responses to the pollination environment depend on available resources? We predicted a larger effect of the pollination treatment in plants with less available water or nutrients. 3) Does pollen quality affect resource allocation toward offspring? We hypothesized that flowers receiving outcross pollen would produce more and heavier seeds, and this effect would be larger when other flowers on the plant received self pollen.

Material and Methods:

Study System: The genus Clarkia comprises 41 annual species, mainly native to western North America. Plants produce protandrous flowers on indeterminate compound spikes, with lower flowers on the inflorescence opening days to weeks before higher flowers. Most species are pollinated by solitary bees, including Clarkia concinna, which is also effectively pollinated by long-tongued flies, bumble bees, and honey bees (Miller et al. 2014). Although all species are self-compatible, there can be substantial differences in floral traits and outcrossing rates among closely related taxa and even among populations within taxa (Lewis and Lewis 1955). In Clarkia, as in other flowering plant clades, primarily selfing taxa have evolved repeatedly at the range margins of more outcrossing taxa (Lewis 1962; Vasek 1964; Bartholomew et al. 1973; Allen et al. 1990; Eckhart et al. 2004). Traits associated with high selfing rates in this genus include reduced time to flowering, smaller flowers with reduced

53 herkogamy, protandry, and longevity, and reduced flower number (Lewis & Lewis

1955; Vasek 1971; Holtsford and Ellstrand 1992; Runions and Geber 2000; Dudley et al. 2007;). These traits are often under strong directional selection in dry environments (Eckhart et al. 2004; Mazer et al. 2004; Mazer et al. 2010).

Additionally, individuals show plasticity in these floral traits in response to the abiotic environment (Vasek 1977). For instance, under low water availability, Clarkia concinna shows reduced dichogamy, flower size, and flower number as well as increased autogamous seed set (Kay and Picklum 2013; Miller unpublished data).

Moreover, the pollination environment influences selection on and plasticity of floral traits in the genus. Selection for reductions in dichogamy and herkogamy occur in small populations of Clarkia xantiana where few pollinators are present (Moeller and

Geber 2005). Floral longevity is shortened and seed production reduced in response to early pollen deposition on Clarkia tembloriensis flowers (Ashman and Schoen

1997). Clarkia gracilis produces more outcrossed than selfed offspring from flowers that received equal amounts of self and outcross pollen, potentially indicating cryptic self-incompatability (Jones 1994). Clarkia is therefore an ideal system to test for plastic responses of floral and seed traits in later flowers to the early pollination environment.

Floral trait response to pollination and drought: We tested the response of

Clarkia concinna flowers to the combined effects of water and pollinator availability by growing plants under controlled greenhouse conditions. We collected seeds in the summer of 2011 from four large populations (> 500 plants): 1) Knoxville Road in

Napa County, California, USA (38.497, -122.242; KN), 2) Lucas Valley Road in

Marin County (38.044, -122.612; LV), 3) Curry Cave Canyon Road in Contra Costa

County (37.855, -121.890; CC), and 4) Highway 116 near Old Cazedero Road in

Sonoma County (38.499, -123.006; OC). We collected 2-3 open-pollinated fruits from 30 haphazardly chosen plants spaced more than 3 meters apart. We planted 5 seeds from each maternal plant in 15 replicate containers (3.8 X 21 cm SC10 Super,

Stuewe & Sons) on August 13th and 14th of 2012. The soil was four parts Pro-Mix HP

Mycorrhizae potting soil to one part perlite. We allowed the seeds to germinate in growth chambers with 10 hour 21° C days, 14 hour 13° C nights, and frequent misting. We chose the 15 families with the highest germination rates (9-12 replicates per family), thinning conetainers to one randomly chosen plant. In total, 616 plants were included in the experiment.

After germination We moved the plants to a greenhouse and factorially manipulated the soil moisture and pollination environments. We controlled moisture by placing conetainers containing individual plants in blocks of florist’s foam. We created constant dry and benign treatments by altering the depth of the conetainers in the foam blocks and the frequency of watering. In both treatments the foam sat in a plastic tub containing approximately 2.5 cm of water and soaked up some of the water through capillary action (Snow and Tingey 1985; Lambrecht et al. 2007). For the benign treatment, the bottom of the conetainer was inserted into the florist foam

0.5 centimeters below the level of the water, and for the dry treatment, the conetainer sat 5 centimeters above the maximum water level. We added water every few days in

55 the wet treatment and every week in the dry treatment. All plants were fertilized once

(November 2, 2012) with a spray of 30 mL of Peters Professional 20-20-20 General

Purpose Fertilizer in 3 L of water. In late November, we measured the percent soil moisture in all conetainers using a Procheck version 6 moisture probe (Decagon

Devices) (soil moisture in dry: 12.6%  3.8 SD, benign 21.0%  2.2 SD, t = 33.85, N

= 616 p < 0.0001). Plants in the dry treatment differed from the benign treatment in a number of vegetative characteristics including overall size and specific leaf area

(unpublished data).

In both benign and dry treatments, we simulated environments with pollinators present (outcrossed) and with pollinators absent (unpollinated). For plants in the outcrossed treatment, the two earliest-blooming newly female flowers on each plant (the lowest two flowers on the main stem) received outcross pollen. We applied the pollen sequentially from non-focal flowers of two other plants in the same population by directly rubbing the anthers onto the receptive stigma. Plants in the unpollinated treatment were untouched.

We surveyed plants once daily, taking floral measurements on the 5th flower from the bottom of the main stem. The 5th flower was unmanipulated (not pollinated) in all treatments and generally entered female phase around a week after the second

(manipulated) flower (mean = 6.9 days, st dev = 3.4). Dichogamy was calculated as the first day of stigma receptivity minus the day of anther dehiscence. Floral longevity was calculated as the number of days elapsed between the corolla opening and senescing. On the first day of stigma receptivity, we measured flower size (width

56 of the corolla) and herkogamy (style length minus anther length). We subtracted the

5th flower size, herkogamy, and dichogamy from the same measures on the 2nd lowest flower (manipulated in the pollination treatment) to measure the change in these two values. These measures were not correlated. Using change in floral traits controlled for individual variation. However, we could not use change in longevity because the pollination treatment also affected the floral longevity of the second flower

(pollinated flowers wilted more quickly). After all flowers on the plant had opened, we counted the total number of flowers. Analyses were conducted in JMP Pro

(version 11.2.0). We compared the floral traits using mixed model ANOVAs with moisture conditions (benign or dry), pollination type (outcrossed or unpollinated), their interaction, and variables of family and population as random effects. We adjusted significance levels for multiple tests using the sequential Bonferroni correction.

Floral trait response to pollination and nutrient fertilization: We tested whether Clarkia concinna flower traits respond plastically to the combined effects of nutrient and pollinator availability by manipulating these factors under field conditions. A field experiment allowed me to test if any effects of early pollination

We observed in the greenhouse would hold in a more variable natural setting. We set up the experiment on May 2nd and 3rd, 2014 in an extremely large population (more than 100,000 plants) along Davis Creek in the Donald and Sylvia McLaughlin

Natural Reserve in Yolo County, California (38.86, -122.37). We used a blocked design (40 total cages), with each block consisting of three nearby plants with at least

57 five buds, matched for similar initial size. Cages were constructed using PVC pipes and connectors as a frame and white bridal veil, which was secured to the ground with metal stakes. Cage size and shape depended on plant spacing and microtopography, but cages were approximately 0.5 cubic meters and were large enough to ensure the three plants could grow to full size without touching the cage edges or each other. The vegetation, including additional C. concinna, was thinned inside the cage. Cages successfully kept out all potential pollinators, as evidenced by a lack of pollen on unpollinated flower stigmas.

The effect of nutrient limitation was tested by randomly assigning each cage as fertilized or unfertilized. Fertilized cages received 2 liters of water with 7.5 mL of

Miracle Grow all-purpose plant food [24% N, 8% P, 16% K] applied on May 3rd and again on May 11. Unfertilized controls received 2 liters of water on the same days.

The 3 plants in each cage were randomly assigned to one of three pollination treatments—outcrossed, selfed, and unpollinated. Pollinations were conducted daily on all open female phase flowers until the day the 5th flower was female. Flowers on plants in the outcrossed treatment received pollen on the first day of stigma receptivity sequentially from 2 plants at least 10 meters away. Donors were not reused. We applied pollen by directly rubbing the anthers of a male phase flower on the stigma. The 5th flower and all concurrently and subsequently female phase flowers were left untouched. Plants in the selfed treatment received pollen in the same manner, but then anthers came from the focal plant. The anthers of the 2nd or 5th

58 flower were not used for these pollinations. All flowers on plants in the unpollinated treatment were not touched.

As in the drought and pollination manipulation, we counted the total number of flowers, measured the 5th flower for floral longevity, and calculated the change in dichogamy, herkogamy, and flower size between the 2nd and 5th flower. Additionally, we recorded the number of open flowers daily for each plant (floral display). From this, we determined the maximum floral display as an absolute number of flowers and as a proportion of the total flowers produced. The first flower opened on May 12th and the experiment concluded June 11th. All plants in one fertilized cage experienced heavy herbivory from a Hyles lineata caterpillar and were excluded from analyses.

We analyzed each floral trait using a mixed model ANOVA with fertilization

(fertilized or unfertilized), pollination type (selfed, outcrossed, or unpollinated), and their interaction as fixed effects, and cage (block) as a random effect, adjusting significance levels with a sequential Bonferroni correction.

Seed trait response to pollination: We tested whether Clarkia concinna allocates resources differently to selfed and outcrossed ovules by performing an additional greenhouse manipulation of pollen source. In the summer of 2012, We collected seeds from 30 families along Knoxville Road in the Donald and Sylvia

McLaughlin Natural Reserve in Northern Napa County, California (38.857, -

122.407), using the methods described in experiment 1 (this population is ~3 km from the field experiment). On December 16, 2014, We planted several seeds from each family in six to ten replicate conetainers. We chose the 20 families with the highest

59 germination rates, thinned conetainers to 1 seedling, and moved them from the growth chamber to the greenhouse on January 26, 2015. All plants were watered as needed and fertilized once (February 24, 2015) with a spray of 30 mL of Peters

Professional 20-20-20 General Purpose Fertilizer.

To distinguish between the effects of resource allocation and early inbreeding depression, we independently manipulated the pollen source (self or outcross pollen) on a focal flower (focal treatment) as well as on all other flowers on the plant

(background treatment). We randomly assigned four plants from each family to receive one of four treatment combinations. Unselected plants from the same 20 families served as pollen donors. The focal flower was always the fifth lowest on the main stem and was never used as a pollen donor. The four treatment combinations were 1) All flowers on a plant (background and focal) were pollinated with self pollen on the first day they were female. 2) All flowers were pollinated with outcross pollen from 2 donor plants from different families. 3) Background flowers received self pollen while the focal flower received outcross pollen. 4) Background flowers received outcross pollen, while the focal flower received self pollen. We allowed all fruits to mature, ensuring seeds remained in the ripening capsule with a small piece of transparent adhesive tape around the top of the fruit. We then collected the capsules from the focal flower and manually counted viable and inviable seeds. Inviable seeds were lighter in color and smaller. We collectively weighed the viable seeds to the nearest tenth of a milligram. The majority of fruits from one family failed to mature across all treatments, and it was excluded from analyses, leaving 19 replicate

60 families. In order to test whether the background or focal pollen treatment had an effect on seed traits, we conducted mixed model ANOVAs with the fixed effects of focal treatment, background treatment, and their interaction, the random effect of family, and the response of seed number or weight per seed. We also tested the response of the percent ovules matured (number of viable seeds divided by the total ovules), but the results were similar to seed number, and are not presented.

Results:

Floral trait response to pollination and drought: Both the pollination treatment of early flowers and water availability in the greenhouse affected later floral traits. However, the effects of pollination did not depend on the water resource level

(table 2.1). Plants in the unpollinated treatment produced more flowers than the outcrossed treatment, and the 5th flower in these treatments (unpollinated across all treatments) had greater floral longevity (Figure 2.1a, b). There was a significantly greater decrease in herkogamy between the 2nd and 5th flower in the unpollinated treatment than in the outcrossed treatment, although the difference was small

(unpollinated = -0.19 mm  0.08 SE, outcrossed = -0.50 mm  0.08 SE). Pollination did not affect change in flower size or dichogamy. Plants in the benign moisture treatment produced more flowers and had greater floral longevity than plants in the dry treatment. Water availability did not affect the change in herkogamy, change in flower size, or dichogamy.

Floral trait response to pollination and nutrient fertilization: The experiment conducted in a natural population showed fewer floral trait responses to the manipulations than in the greenhouse (table 2.1). As in the greenhouse experiment, the effects of the abiotic and pollination treatment were independent. The floral longevity of the 5th flower (unpollinated across all treatments) tended to be greater when earlier flowers were also not pollinated than when they were pollinated with self or outcross pollen, but this effect was not significant after correcting for multiple tests (figure 2.1c). There were no additional effects of the pollination treatment in this study. Total flower number was marginally greater in plants given fertilizer (figure 2.1d). While maximum floral display was unaffected by nutrient availability, fertilized plants were able to maintain similar floral displays as unfertilized plants by simultaneously opening a smaller proportion of their total flowers by prolonging anthesis (figure 2.1e). Floral longevity and change in dichogamy, herkogamy, and flower size was unaffected by nutrient availability.

Seed trait response to pollination: The type of pollen received by both the focal and background flowers affected seed number and weight in somewhat different ways. The number of viable seeds produced by the focal flower (fifth flower from the bottom on the main stem) was greater when the other flowers on the plant received self pollen than with a background of outcross pollen (F1,54 = 7.39, P = 0.009, figure

2.2a). Seed number was not affected by whether the focal flower was selfed or outcrossed (focal F1,54 = 1.28, df = 1,54, P = 0.26; interaction F1,54 = 0.91, P = 0.35).

Conversely, the weight per seed was affected by the focal treatment—seeds were

62 heavier when the focal flower received outcross pollen (F1,54 = 8.30, P = 0.006, figure

2.2b), but was not affected by the background treatment (background F1,54 = 1.24, p =

0.27; interaction F1,54 = 0.02, P = 0.90).

Discussion

The floral and seed traits of later-opening, unpollinated Clarkia concinna flowers showed plastic responses to the pollination of earlier-opening flowers. The direction of the changes indicates reduced investment in later flowers and fruits when early flowers receive adequate amounts of outcross pollen. Specifically, early- outcrossed plants produced fewer total flowers and reduced floral longevity and seed number in later flowers than plants with less successful early flowers. Instead of investing additional resources on later reproductive effort, these plants may allocate these resources toward maturating high-quality fruits. However, unexpectedly, while both water and nutrient availability also affected floral traits, the shifts in resource allocation toward early-pollinated flowers were not greater when resources were more limiting.

The reduction in floral longevity in later flowers in response to early pollination is likely an adaptively plastic response, because it could reduce geitogamous pollination when pollinators are abundant, increasing offspring quality.

Physiological changes in pollinated flowers could provide a cue to reduce the longevity of later unpollinated flowers. Clarkia, as in other taxa, shows a within-

63 flower response to pollination, senescing flowers quickly after full pollen load receipt

(reviewed in van Doorn 1997; Ashman and Schoen 1997). Pollination-induced senescence is likely an oxidative response to interactions between the stigma and deposited pollen involving the hormones auxin and ethylene (O’Neill 1993; Attri et al. 2007).

In addition to reducing selfing rates, a plastic reduction in floral longevity would potentially allow plants to spend fewer resources on maintaining energetically expensive flowers once their function is complete (Ashman and Schoen 1997). Plants can allocate resources not spent on flowers toward improving offspring quantity or quality. For instance, early removal of corollas increased seed production of hand- pollinated flowers in both Cistus and Nigella (Andersson 2005; Teixido and

Valladares 2013). However, if resource allocation was important in driving floral trait plasticity, the response to pollination should be larger in the dry treatment, where resources were more limiting. Surprisingly, in this study, while the dry treatment did reduce floral longevity, the effect of pollination on longevity did not depend on water or nutrient availability. Collinsia heterophyla also shows reduced floral longevity under early pollination regardless of the moisture environment (Jorgenson and Arathi

2013).

The response of floral longevity to pollination was greater than that of dichogamy, although the two traits are correlated (Pearson’s correlation between dichogamy and floral longevity was 0.40 in field and 0.59 in greenhouse). The female phase was the main determinant of total floral longevity. Reducing time between

64 male and female function (dichogamy) would be maladaptive in a high pollination environment if it resulted in higher autogamous selfing rates (Brys et al. 2013).

Dichogamy in the absence of pollinators has only a marginal effect on autogamous selfing rates in Clarkia concinna, likely because if pollen is not removed, it still can be knocked onto the stigma as the flower senesces (Kay and Picklum 2013).

However, reductions in dichogamy could increase pollinator-mediated autogamy. It is possible that flowers would adjust dichogamy in response to pollen removal rather than pollen deposition. Pollen removal can affect dichogamy and floral longevity in some taxa (e.g. Devlin and Stephenson 1984; Evanhoe and Galloway 2002).

However, other species do not show this effect (e.g. Ishii and Sakai 2000). Although we did not rigorously test for an effect of pollen removal on floral traits, we did not observe changes in floral timing in the pollen donor plants in the two greenhouse experiments.

The lack of an effect of pollen quality (self or outcross) on floral traits matches results from other studies that measured the within-flower effects of pollination rather than measuring responses in later-opening flowers. Marquez and

Draper (2012) did not find a difference in floral longevity between self and outcross pollen receipt in the self-compatible Narcissus serotinus. Clark and Husband (2007) found greater floral longevity in selfed than outcrossed flowers in Chamerion angustifolium, but only for a narrow window of pollination timing. Species with stronger inbreeding depression or those that experience more variability in pollen quality than Clarkia concinna may show greater responses in floral traits to pollen

65 quality. For instance, flowers of taxa with pollinia, such as orchids and milkweeds, may frequently receive 100% self or outcross pollen, and selection in response to this binary variable may be stronger than in species with high pollen carryover and a relatively constant ratio of selfed to outcrossed pollen ratios in pollen loads.

Alternatively, species with self-incompatibility mechanisms may not plastically respond to the deposition of self pollen at all.

The reduction of flower production in early-pollinated greenhouse plants is consistent with the prediction of resource allocation toward high-quality fruits at the expense of later flowers. Ashman and Schoen (1997) altered the timing of pollination on early Clarkia temblorensis flowers and found early-pollinated flowers produced higher quality fruits and resulted in smaller later-opening flowers. They did not find an overall reduction in flower number in response to the timing of pollination and did not measure floral longevity in later flowers. Greater nutrient and water availability unsurprisingly increased the total number of Clarkia concinna flowers. However, despite almost doubling total flower number in the field, nutrient fertilization did not increase the maximum floral display. Thus Clarkia concinna can increase lifetime floral production without increasing geitonogamy by opening a smaller proportion of its flowers simultaneously. While larger floral displays often attract more pollinators

(reviewed in Ohashi and Yahara 1999), this does not appear to be the case in Clarkia concinna (Miller unpublished data). Flowers, at least in large patches of plants, are likely not pollen limited and display size doesn’t increase visitation rates even in small patches. As floral longevity was not affected by nutrient fertilization, Clarkia

66 concinna may maintain the advantage of a small display size by opening flowers at a slower rate.

In general, floral morphology changed less in response to the treatments than timing traits. Flowers were larger and had greater herkogamy in the wet and fertilized treatments (unpublished data), as in previous work in the genus (Vasek 1964; Vasek

1977; Eckhart et al. 2004). However, the change in these values between early and later flowers was not affected by abiotic conditions. Morphology may be determined earlier in development than phenological traits, and thus show greater constraints on plasticity in response to pollination (Lloyd 1980; Herrera 1991; Guitian and Navarro

1996). The buds of the 5th flower are relatively large when the first and second flower are blooming, and thus may grow to a predetermined size. Plants that form new buds after the first flowers open may be better able to alter floral morphology in response to the early pollination environment.

Unlike floral traits, seed traits were affected by pollen source. Seed number showed a plastic response to the background pollination treatment. Plants with self- pollinated early-opening flowers matured more seeds in later flowers than initially outcrossed plants, indicating more resources are available for maternal investment in later offspring when early seeds were lower quality. Mazer and Dawson (2001) found later opening Clarkia unguiculata flowers had reduced seed counts and ovule:pollen ratios as compared to earlier-opening outcrossed flowers. This indicates lower investment in female function in later flowers when pollen is not limiting. Runquist and Moeller (2013) compared whole plant and single flower manipulations of pollen

67 source in Clarkia xantiana ssp. parviflora, a primarily selfing subspecies. They did not find an effect of pollen source on seed number or evidence for resource reallocation. However, neither of these studies factorially manipulated the pollen treatment of early and later opening flowers. Moreover, among-flower resource allocation in response to pollen source should be greater in primarily outcrossing than selfing taxa because the latter species will likely show smaller differences in offspring quality (Husband and Schemske 1996)

Seed weight responded somewhat differently to pollination than seed number.

Selfed seeds weighed less than outcrossed seeds irrespective of the background pollination treatment, likely due to early-acting inbreeding depression. While the effect size is relatively small, even minor differences in seed weight can have large effects on offspring size and fitness (McGinley et al. 1987). The smaller size of selfed seeds may contribute to the substantial late-acting inbreeding depression Clarkia concinna experiences under field conditions (Groom 2000).

Overall, this study indicates Clarkia concinna can recognize the pollination environment by detecting the pollination status of early flowers and respond by altering floral and seed traits in later flowers. Floral longevity showed the greatest plastic response to early pollination. Longevity is a logical target of selection for increased plasticity, as it can affect both attraction and outcrossing rate and may be less developmentally constrained than morphological traits. While other studies have documented within-flower reductions in longevity to pollen receipt (e.g. Ashman and

Schoen 1997; Marquez and Draper 2012; Clark and Husband 2007), this study is

68 unique in finding similar effects in later, unmanipulated flowers. Another important finding is the contrast in plastic responses between pollen receipt (pollinated or unpollinated) and pollen quality (self or outcross). While floral traits responded to pollen receipt but not quality, seed number in later flowers did respond to early pollen quality. Later flowers had more seeds when early flowers were selfed than when they were outcrossed. The plastic responses in floral and seed traits of Clarkia concinna could be selectively favored given the temporal and spatial variability of pollination environments. Many biotically-pollinated plants experience variable pollinator environments, and may demonstrate similar patterns to those found here.

Comparisons among taxa with contrasting mating systems, life histories, or pollination syndromes would provide additional insight into the function and ubiquity of floral plasticity in response to the pollination environment.

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Table 1. Mixed model ANOVAs of floral trait responses to early pollination manipulation, abiotic manipulation, and their interaction

Pollination and Pollination and Source Drought Nutrient Fertilization F df N F df N Total Flowers Pollination 7.28* 1 648 6.32+ 2 117 Abiotic 6.57* 1 648 1.70 1 117 Pollination X Abiotic 0.08 1 648 0.25 2 117 Max Floral Display (Number) Pollination - - - 1.17 2 117 Abiotic - - - 1.40 1 117 Pollination X Abiotic - - - 1.02 2 117 Max Floral Display (Proportion) Pollination - - - 0.43 2 117 Abiotic - - - 27.74** 1 117 Pollination X Abiotic - - - 0.34 2 117 Floral Longevity Pollination 52.02** 1 616 3.91+ 2 110 Abiotic 55.65** 1 616 1.15 1 110 Pollination X Abiotic 0.03 1 616 0.23 2 110 Change in Dichogamy Pollination 1.54 1 616 0.98 2 110 Abiotic 0.50 1 616 0.22 1 110 Pollination X Abiotic 0.08 1 616 0.46 2 110

Change in Herkogamy Pollination 7.67* 1 622 1.99 2 108 Abiotic 0.04 1 622 0.15 1 108 Pollination X Abiotic 0.46 1 622 0.68 2 108 Change in Flower Size Pollination 0.00 1 622 2.62 2 111 Abiotic 2.25 1 622 0.01 1 111 Pollination X Abiotic 0.75 1 622 0.22 2 111

Note. Abiotic treatment was benign or dry in first model and fertilized or unfertilized in second model. Pollination treatment was outcrossed or unpollinated in first model and outcrossed, selfed, or unpollinated in second model. Random effects of population and family included in first model and family in second model. * indicates p < 0.05 after sequential Bonferroni correction. ** indicates p < 0.01. + indicates marginal significance.

Figure 2.1. Clarkia concinna alters floral traits in response to drought, nutrient, and pollination treatments. Left panels (a, b): trait responses of later flowers to greenhouse manipulation of water and pollination environment. The first 2 flowers on each plant were pollinated in the outcross treatment. Right panels (d, e, f): trait responses to a field manipulation of nutrients and pollination environment. All flowers that opened before the 5th flower on the main stem were pollinated in the outcross and self treatments. Dry conditions and pollination of early flowers reduced floral longevity (a, c) in the 5th flower (The 5th flower was unpollinated across all treatments). Dry conditions and early pollination reduced total flower number in the greenhouse (b), whereas fertilization marginally increased flower number in the field (d). Fertilized plants in the field opened proportionally fewer of their flowers simultaneously (e). Bars with the same letter are not significantly different. Vertical lines are  1 S.E.

Figure 2.2. Seed weight and number of the 5th open flower of Clarkia plants depend on the pollination treatment of all other flowers on the plant (background) and the pollination treatment it receives (focal). Flowers on plants with a background of outcrossing mature fewer seeds than flowers with a background of selfing, potentially reflecting resource allocation (a). Seeds produced from self pollen are lighter than those produced from outcross pollen, potentially indicating early-acting inbreeding depression. Bars with the same letter are not significantly different. Vertical lines are  1 S.E.

Chapter 3

Large floral displays increase self-pollination but do not affect visitation rates in

Clarkia concinna.

Abstract: Optimal floral display (the number of simultaneously open flowers) is frequently viewed as a balance between pollinator attraction and avoidance of pollinator-mediated interfloral selfing (geitonogamy). However, the most beneficial display size may be affected by pollinator abundance, pollinator identity, and other environmental variables. I determined the effects of individual floral display and patch size on pollination outcomes in natural populations of a mixed-mating annual plant, Clarkia concinna. I predicted that individual plants with larger floral displays would have both greater pollinator visitation and more geitonogamy. Additionally, I tested whether the effects of floral display would be greater in smaller patches of plants. Larger individual floral displays increased total geitonogamy, but the effect of display on geitonogamy differed among pollinator taxa. Visitation rates were lower in small patches for some pollinators. However, individual display size did not affect per flower visitation rate in small or large patches. Therefore, if we only consider the expected tradeoff between pollinator attraction and geitonogamy, the number of simultaneously open flowers appears excessive. In this species, large floral display size requires explanations beyond increased pollinator attraction, such as temporal

84 constraints on flowering imposed by a limited growing season and the benefits of additional low-quality offspring.

Introduction:

Biotically-pollinated plants face a dilemma. Individual plants that produce floral displays with many simultaneously open flowers can attract more pollinators, increasing per-flower visitation rates (reviewed by Ohashi and Yahara 1999).

However, larger displays promote pollinator movement between flowers on the same plant, increasing geitonogamy (within-plant transfer of self pollen; de Jong et al.

1993, Finer and Morgan 2003). Geitonogamy can negatively affect male fitness through the reduction of pollen export (Klinkhamer and de Jong 1993). In self- compatible species, these transitions also can result in higher selfing rates (Hessing

1988; Harder and Barrett 1995; Karron et al. 2004; Williams 2007). Geitonogamy decreases fitness through inbreeding depression in self-compatible plants and reduces seed set by clogging stigmas in self-incompatible species. Geitonogamy can be quite high in biotically-pollinated plants, with up to 30-90% of flowers receiving some geitonogamous pollen deposition (reviewed by Goodwillie et al. 2005; Kropf and

Renner 2008). Unlike other forms of self-pollination, geitonogamy will rarely or never be beneficial, even if inbreeding depression is weak, since it does not provide reproductive assurance in the absence of pollinators (Lloyd 1992). The tradeoff between increased attraction and increased geitonogamy may represent a driving force in the evolution of inflorescence structure and mating systems (Klinkhamer and de Jong 1993; Snow et al. 1996; Finer and Morgan 2003; Devaux et al. 2014).

However, not all plants experience this tradeoff. If larger floral displays do not increase per-flower visitation rates, then opening flowers over a longer period of time

86 in order to decrease geitonogamy may be favored. Some studies find no relationship or a negative relationship between the total number of flowers simultaneously open on an inflorescence (floral display size) and per-flower visitation rates, although per- plant visitation may be higher (e.g. Andersson 1988; Harder and Barrett 1995; Karron et al. 2004; Grindeland et al. 2005). Additionally, when many pollinators are present, pollen is often not limiting and the potential attraction benefit of a larger display is not necessary (Burd 1994; Ashman et al. 2004; Knight et al. 2005). Plants in large or dense patches can benefit from a large shared display by having higher total visitation rates and subsequently setting more seed (e.g. Dauber et al. 2010; Kunin 1997).

When pollinators visit plants with more or nearer neighbors, they make more among- plant transitions, decreasing geitonogamy (Klinkhamer et al. 1989; Duncan et al.

2004) and subsequently rates of self-fertilization regardless of the display size of individuals (Routley et al. 1999; Franceschinelli and Bawa 2000). Therefore, the effects of floral display size on pollen receipt and export may be more pronounced in small than large patches.

Pollinator taxa can differ in how they move through a patch, changing the relationships between floral display, visitation, and outcrossing rates (Thompson

2001; Burnett and Sweet 2006). Some species may move systematically through a patch, frequently visiting multiple flowers per plant. Others may be very choosy or haphazard in their visits, moving longer distances among flowers and making fewer transitions between flowers on the same plant (Whelan et al. 2009). Visits from systematically-foraging pollinators such as bumble bees could drive selection for

87 smaller displays, while choosy but far-moving pollinators such as hummingbirds could select for increased attraction. Pollinator traits, such as average pollen load and amount of pollen carryover, also differ among species, impacting geitonogamy and, subsequently, the optimal display strategy (Robertson 1992).

Pollinator communities and patch sizes can differ greatly, particularly at small temporal and spatial scales (e. g. Herrera 1988; Moeller 2005; Gomez et al. 2010).

While small-scale environmental variability may preclude local adaptation, plants can respond by adjusting floral traits through phenotypic plasticity (Bradshaw 1965;

Schlichting 1986; Sultan 1987). For instance, plants can alter their floral display by increasing floral longevity in response to pollinator-poor environments (van Doorn

1997; Harder and Johnson 2005) If the relative rates of geitonogamy and attraction depend on patch size and pollinator community, plastic changes in floral display may be adaptive.

Additional environmental factors beyond patch size and pollinator community may strongly influence floral display size, making the optimal size larger or smaller than would be predicted by the attraction/geitonogamy tradeoff alone (Elle and Hare

2002). It is possible that taxa with hard constraints on total flowering time, such as a rapidly drying edaphic environment or a short pollinator flight period, forcing plants to simultaneously open a higher proportion of flowers. Particularly if inbreeding depression is weak, the benefits of increasing offspring quantity though additional flower production may outweigh the costs that geitonogamy exacts via offspring quality. Selection to increase paternal reproductive success may result in what

88 appears to be excess floral production when viewed from the lens of maternal fitness

(Stanton et al. 1987; Paige et al. 2001; Barrett 2003). Alternatively, floral displays may be relatively smaller than predicted due to lack of available resources, herbivore avoidance, or physiological and evolutionary constraints.

I analyzed the effects of floral display on pollination using natural populations of Clarkia concinna (Onagraceae), a mixed-mating annual that shows wide variation in individual and population-level floral display. Flowers in this species are protandrous, opening as male and entering a female phase a few days later. While flowers mature sequentially from lower to higher positions, they are borne on branched inflorescences, such that a systematically foraging visitor would encounter a mix of male and female phase flowers. Floral displays at the study sites range from 1 to approximately 75 flowers with a mean around 5 and a median around 3. Bees, flies, and butterflies are all common visitors, most of which are more or less effective pollinators (Miller et al. 2014). All visitors fly between flowers, and their behavior does not qualitatively differ between flower phases (male or female) or types of transition (within or between plants). This species can plastically alter its floral display. I previously demonstrated that this species can plasticity reduce floral longevity in later flowers in response to pollen receipt, which will affect floral display size (chapter 2). Plants grow in dense, variously-sized patches, and experimental manipulation found individuals in small, isolated patches (less than 50 plants more than 10 meters away from other patches) receive less pollen and set fewer seeds than plants in large patches, and are more likely to go extinct (Groom 1998).

I tested for a tradeoff between pollinator attraction and geitonogamy avoidance by comparing total visitation rates and geitonogamy across floral display sizes. I predicted that larger floral displays would have both higher per-flower visitation rates and increased geitonogamy. I then investigated how patch size may affect this tradeoff. I predicted that flowers in small patches would have lower visitation rates from fewer pollinator taxa. Additionally, I tested whether patch size changes the effects of floral display. I factorially manipulated patch size and floral display and tested for differences in pollination success. I predicted that large floral displays would increase visitation more in small than large patches.

Methods

In the springs of 2014 and 2015, I conducted five studies on the floral display, patch size, and pollinator assemblage of Clarkia concinna in northern Napa, southeastern Lake, and northwestern Yolo Counties, California. Here, Clarkia concinna grows in serpentine chaparral and riparian woodland communities. Studies

1, 4, and 5 were conducted at several populations in both plant communities. Studies

2 and 3 occurred in a very large population (> 100,000 plants) growing in riparian woodland along Davis Creek, in the Donald and Sylvia McLaughlin Natural Reserve

(38.86, -122.37). This was the same location as a concurrently running pollination and fertilization experiment (chapter 2). All studies in both years were restricted to patches in peak flowering, to minimize temporal changes in pollinator abundance

(Thompson 1981; 1982). Studies 1, 2, and 3 looked for an overall effect of floral display on pollinator attraction and geitonogamy by comparing of pollinator movement, pollen analogue (pigment) deposition, and seed traits. Studies 4 and 5 tested whether the effect of floral display depended on patch size by comparing visitation and pollen deposition between small and large patches.

Study 1) How does floral display size affect pollinator movement? In order to compare pollinator movement among plants with different floral display sizes, I observed floral visitors to Clarkia concinna during peak flowering in 2014 (May 21st through June 8th). Observations took place throughout the day 7:40-19:20. Each observation began with a visitor arriving at a new plant and ended when it left the patch or stopped foraging for approximately 15 seconds. A visit included any contact with the interior surface of the corolla or reproductive parts. Most observed visitor taxa were found to be effective pollinators in a previous study (Miller et al. 2014). I counted the number of flowers visited per plant after the initial flower was visited, giving the total visits for which geitonogamy could occur (consecutive visits). Plants with one open flower could not have consecutive visits, and were not included. I used numbered flags to temporarily mark visited plants and counted the number open flowers per plant (floral display) after the completion of the foraging bout. Rare instances when visitors returned to the same flower or plant after visiting elsewhere were counted as separate visits. I identified all visitors to genus. Visitor preference for either male or female phase flowers could reduce the effectiveness of consecutive visits as a measure of geitonogamy. In initial observations, I generally did not observe

91 such bias in flower choice with the exception of two small solitary bee genera,

Cerotina and Lasioglossum. These bees were uncommon visitors that exclusively pollen-collected on male-phase flowers. Emasculation did not reduce visitation in a study of the related Clarkia xantiana, supporting the idea that visitors do not cue in on flower phase (Moeller et al. 2012). Floral display and consecutive visit data were approximately lognormally distributed, and were log transformed. I tested for a positive linear relationship between the log floral display and the log of consecutive visits. To test for among-species differences in this relationship, I selected the eight pollinator genera with more than 50 observations. I used a generalized linear model

(GLM) including the log of display size and visitor genera as the predictive variables

I modelled consecutive visits with a Poisson distribution and a log link function.

Study 2) How does floral display size affect pollen deposition? Although high numbers of within-plant transitions strongly indicate the potential for geitonogamy, high pollen carryover or erratic pollinator behavior may reduce the relative amount of within-plant pollen transfer (Geber 1985). I compared the amounts of self and outcross pollen movement across floral display sizes by manipulating display and comparing the deposition of fluorescent pollen pigment. From May 18th through June 7th, 2014, I selected 140 focal plants in large patches (greater than 250 open flowers) that had at least 6 male phase and 1 female phase flowers. I marked the most recently female phase flower on the main stem (highest) with thread, and removed any other female phase flowers on the plant by pinching off the perianth just above the ovary. I chose plants with no prior pollen deposition on the focal flower

92 stigma. Pollen in this species is large and colorful enough in this species that I could observe deposition with a hand lens. I varied floral display by leaving 1, 2, 4, or 6 of the male phase flowers on the focal plant and removing additional flowers. Although large plants can have greater than six simultaneously open male-phase flowers, the range of treatment display sizes was comparable with the majority of natural display sizes. I used a pipe cleaner to brush all anthers of the remaining male-phase flowers with blue, orange, green, yellow, or pink fine-grained florescent powdered pigment

(self pigment; Series JST-300, Radiant Color, Richmond, CA, USA). I coated the anthers of the 50 nearest male phase flowers with a different color of pigment

(outcross pigment). The surrounding treated flowers were within a one-meter radius of the focal plant. Replicates were at least 10 meters apart and pigment colors were changed between adjacent replicates. The presence of pigment did not appear to affect visitation—I observed all common visitors foraging freely on treated flowers.

Treatments were initiated between 7:00 and 12:00.

After 6-10 hours, I collected the focal stigma and quantified the amount of self and outcross pigment deposition the same evening using a dissecting microscope.

There was no effect of time on pollen deposition (r2 <0.01, N = 136, P = 0.81) Three stigmas were lost or eaten, resulting in sample sizes per treatment of 34 or 35. I separately ranked focal and surrounding pigment deposition as: no pigment (0), 1 fluorescent grain to approximately 5% stigma surface covered (1), 5% to 20% coverage (2), 20% to 40% coverage (3), and >40% coverage (4). Pigment as a pollen surrogate may overestimate pollen movement—its smaller size may result in higher

93 amounts of wind transfer (Thompson et al. 1986). However most pigment deposition was associated with pollen deposition, indicating the pigment is an adequate pollen surrogate. I tested whether the proportion of stigmas receiving any pigment depended on display size with 2 contingency test. For stigmas that received pigment (N = 105),

I compared self and outcross pigment scores among display sizes using Kruskal-

Wallis and post-hoc Steel-Dwass All Pairs tests (the nonparametric equivalent of a

Tukey test).

Study 3) How does floral display size affect seed size and number? If large floral displays increase the deposition of self pollen, then floral display may be negatively correlated with offspring quantity (fewer seeds matured) or quality

(smaller seeds) due to early-acting inbreeding depression. Alternatively, plants may be able to discriminate against self pollen, and display size will not affect seed set

(Jones 1994). I tested whether floral display affects seed weight or number, by selecting 130 total plants on May 19th and May 30th, 2014. Focal plants were separated by at least 5 meters and had one unpollinated freshly-female phase flower

(confirmed with a hand lens) and 6 or more male phase flowers. On each plant, I removed all other female phase flowers and left 0, 1, 2, 4, or 6 male phase flowers. I also removed buds that were likely to bloom while the focal flower was still open.

Because plant size and flower position affect seed traits in this species, I measured the basal stem diameter with calipers (a proxy for size), and the position of each focal flower along the main stem (with position 1 being the lowest and first to bloom).

After corollas senesced, I placed a small piece of transparent adhesive tape around the

94 top of the capsule to prevent seed loss. Mature fruits were collected on June 25th,

2014, and ripe seeds were counted and weighed collectively after air-drying. Twenty- four fruits were missing or eaten prior to collection, resulting in per-treatment sample sizes between 19 and 23 plants. I compared weight per seed and seed number among floral display classes with an ANCOVA, using stem diameter and stem position as covariates.

Study 4) How does patch size affect the pollinator environment? The previous three studies were conducted in relatively large patches of plants (hundreds to thousands of individuals). However, the relationship between display size, visitation, and geitonogamy may depend on patch size. Therefore, I tested for an effect of patch size on visitor frequency and identity with an observational study.

During peak flowering in 2015 (May 2nd to May 19th), I observed groups of 20 flowers centrally located in varying patch sizes for 121 20-minute observation periods between 10:00 and 16:30. Patches ranged from 20 to approximately 10,000 open flowers, and I did not reuse the same patch on the same day. Patches were groups of

Clarkia concinna more or less separated from others by at least ten meters. I identified all visitors to genus and counted the total flower visits and visits per taxa. I used a GLM to test for a relationship between the log of patch size and total visits. I modelled visits with a Poisson distribution and a log link function. I also included observation date as a predictive variable. In addition to total visits, I ran separate

GLMs for Apis and Bombylius, the two taxa with more than 100 visits in the data set.

Finally, for observation periods with at least one visitor (N = 87) I conducted a

95 regression analysis of the Shannon-Weiner species diversity index against the log of patch size, including date as a random variable.

Study 5) Do the effects of floral display size depend on patch size? If pollinators are less abundant in small patches, plants with small displays may be pollen limited in small, but not large patches. I manipulated both patch size and floral display to test for a significant interaction between these variables. From May 2nd to

May 17th, 2015, I selected single unpollinated (confirmed with a hand lens) freshly- female flowers on a total 204 focal plants with at least 6 male-phase flowers. For half the plants, I created very small patches (< 50 additional open flowers within 2 meters of the focal plant, mean 14.8, s.d. = 8.9). In order to avoid excessive destruction of plants, I used natural patchiness in plant distribution, supplemented by thinning excess plants within a 10-meter radius of a focal plant. Other focal plants were chosen in large patches (> 250 flowers). Focal plants in small and large patches received one of 3 display treatments: 1) all additional open flowers and large buds removed, 2) six unmanipulated male-phase flowers left on the plant, or 3) six male-phase flowers left on the plant, but emasculated by removing the anthers with forceps. For the treatments with six unmanipulated and six emasculated flowers, I maintained the display size until the focal flower senesced by removing flowers as they aged and controlling the number of newly opening flowers daily, emasculating them in bud in the emasculated treatment. Unlike studies 2 and 3, I emasculated the focal flower, ensuring any pollen deposition was pollinator mediated. Differences between the emasculated large display treatment (3) and the small display treatment (1) are

96 attributable to differences in display attraction. Differences between the emasculated large display treatment (3) and the unmanipulated large display treatment (2) are due to within plant pollinator movement.

An equal number of replicates per treatment (2 or 3) were initiated on each of

13 days between May 2nd to May 17th. Replicates were set up between 7:00 and

10:00, before the bulk of pollinator activity, and ran until the corolla senesced (1-5 days). Concurrently running replicates were spaced more than 10 meters apart. Four focal flowers were eaten, but they were replaced the next day, resulting in even sample sizes of 34 per treatment. I checked focal flowers for pollen deposition with a hand lens and corolla senescence 3 times per day, between 7:00 and 9:00, between

12:00 and 14:00, and between 17:00 and 19:00. I calculated the time to initial pollen deposition and time to corolla senescence to the nearest third of a day. Once the corolla wilted, I collected the stigma. The same evening, I applied basic fuchsin jelly

(Kearns and Inouye 1993) to a collected stigma, heated it, squashed it between two microsope slides, and counted the stained pollen grains under a dissecting microscope. I used Cox Proportional Hazards to test for an effect of patch size, display size, and their interaction on time to pollen deposition and time to senescence.

I also included date as an independent variable, as among-day differences in weather and location could affect the response variables. I conducted an ANOVA to test for an effect of patch size, display size, their interaction, and the random effect of date on the number pollen grains deposited on focal stigmas.

Results:

Study 1) How does floral display size affect pollinator movement? I observed 1422 visits to Clarkia concinna plants with 2 or more open flowers. Visits were made by sixteen genera, including bees, flies, butterflies, a diurnal hawkmoth

(Hemaris), and a hummingbird (Calypte). The log of consecutive visits showed a positive linear response to the log floral display size (r2 = 0.20, P <0.0001). Eight genera visited more than 50 plants with 2 or more open flowers (N = 1292 visits) and were included in the GLM. Of these, five were bees (Anthophora, Apis, Bombus,

Osmia, and Xylocopa) and three were flies (Bombylius, Eulonchus, and

Thevenetimyia). All except Apis are native insects, and all were observed depositing pollen on Clarkia concinna stigmas. In addition to legitimate visits, Apis and

Xylocopa occasionally made illegitimate visits, Apis by approaching the hypanthium opening from the side (nectar thieving), and Xylocopa by biting the base of the hypanthium (nectar robbing; Inouye 1980). All common bees and Thevenetimyia showed a similar positive relationship between within-plant transitions and floral display size (Figure 3.1; log(display size) 2 = 601.30, P < 0.0001). Bombylius and

Eulonchus showed different patterns of visitation from each other and from other pollinators (Bombylius Consecutive Visits = -0.499 + 0.473*log(Display Size), N =

251 2 = 66.65 P = 0.0025, P <0.0001; Eulonchus, Consecutive Visits = 0.093 +

0.074*log(Display Size), N = 106 2 = 23.67, P <0.0001) Bombylius made more consecutive visits to plants of a given display size than any other insect and showed the strongest positive relationship between consecutive visits and display size.

Eulonchus rarely made any consecutive visits to the same plant regardless of its floral display.

Study 2) How does floral display size affect pollen deposition? Of 136 focal stigmas, 47 (35%) received fluorescent pollen pigment from only surrounding plants (outcrossed), 14 (10%) only from other flowers on the focal plant (selfed), and

44 (32%) from both sources. Display size of the focal plant did not affect the likelihood of pigment receipt (2 = 0.45, P = 0.92), but did affect the pigment source

(Figure 3.2). Of the plants that received pigment, plants with 1 male phase flower received less self pigment that those with 4 or 6 male phase flowers (Kruskal-Wallis test 2 = 14.97, d.f. = 3 P = 0.002; post-hoc comparison 1 and 4, z = 3.30, P = 0.005; comparison of 1 and 6, z = 2.99, P = 0.02). Additionally, plants with 1 male phase flower received more outcross pigment than any other size (Kruskal-Wallis 2 =

17.05, d.f. = 3 P < 0.001; post-hoc comparison 1 and 2, z = 2.74, P = 0.03; comparison of 1 and 4, z = 3.29, P = 0.001; comparison of 1 and 6, z = 3.38, P =

0.004).

Study 3) How does floral display size affect seed size and number? Seed weight was affected by floral display size (F= 6.99, P < 0.0001, Figure 3.3). Focal flowers on plants with 0, 1, or 2 male-phase flowers had heavier seeds than plants with 4 and 6 male-phase flowers (Tukey’s HSD post hoc test). Stem diameter and stem position were significant covariates of seed weight, with heaver seeds on larger plants and lower stem positions (stem diameter F = 62.04, P < 0.0001; stem position

F = 28.48, P < 0.0001). The number of ripe seeds did not differ among floral display classes (F = 1.26, P = 0.29). Flowers on larger plants and on lower stem positions had more ripe seeds (stem diameter F = 40.96, P < 0.0001; stem position F = 39.72, P <

0.0001).

Study 4) How does patch size affect pollinator environment? In 40 hours of observing focal patches I recorded 660 visits made by twelve genera, all of which also visited Clarkia concinna in 2014 (study 1). The only common visitor in 2014 not observed in 2015 was the fly Thevenetimyia (which I never observed at other populations or in other years despite many observation hours, Miller 2014, unpublished data). The skipper butterfly Ochlodes was a more common visitor in

2015 than 2014. Total visitation showed a positive relationship with the log of patch size (GLM 2 = 43.96, N = 121, P < 0.0001, Figure 3.4). Date also had a significant effect on total visitation (2 =182.66, df = 15, P < 0.0001). This pattern was driven by Apis, which greatly increased in abundance with increasing patch size (GLM patch size 2 = 79.46, N = 121, P < 0.0001; date 2 =117.54.66, df = 15, P < 0.0001).

Overall diversity also increased with patch size (linear regression r2 = 0.38, N = 87, p

= 0.0003) The fly Eulonchus, the bees Andrena, Lasioglossum, Megachile, and the hummingbird Calypte all only visited flowers in medium and large patches, although they were not abundant enough to be analyzed separately. However, the most abundant visitor, the fly Bombylius, did not show an effect of patch size on visitation

(GLM patch size 2 = 0.84, N = 121, P < 0.35; date 2 =183.96, df = 15, P < 0.0001).

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Study 5) Do the effects of floral display size depend on patch size? All but one out of 204 stigmas on the emasculated focal flowers received conspecific pollen

(although only a few grains in some cases). Time to pollen deposition, time to corolla senescence and number of pollen grains deposited on the stigma all depended on patch size, but not on floral display size or their interaction (Figure 3.5). Time to pollen deposition in small patches was 40% longer than flowers in large patches (Cox

Proportional Hazards, N = 204 with 1 censored; patch 2 = 9.92, df = 1 P = 0.002; display 2 = 2.52, df = 2 P = 0.28; interaction 2 = 0.01, df = 2 P = 0.99; date 2 =

18.91 df = 12, P = 0.09 ). Flowers in small patches also wilted (Cox Proportional

Hazards, N = 204 with 0 censored; patch 2 = 4.24, df = 1 P = 0.039; display 2 =

1.31, df = 2 P = 0.52; interaction 2 = 0.02, df = 2 P = 0.99; date 2 = 97.19 df = 12,

P < 0.0001 ). Occurring in a small patch ultimately resulted in less pollen deposition

(ANOVA N = 204 patch F = 4.92, df = 1 P = 0.028, display F = 0.68, df = 2 P = 0.68 interaction F = 2.83, df = 2 P = 0.06); however, this effect size was small.

Discussion

Across three separate studies, I found evidence for a strong effect of floral display on geitonogamy in Clarkia concinna, matching patterns found in other systems (reviewed in Goodwillie et al. 2005; Kropf and Renner 2008). All pollinators except the long-tongued fly, Eulonchus, frequently made within-plant transitions, especially on plants with many open flowers (Study 1, Figure 3.1). The movement of

101 pollen analogue pigment indicates roughly half of all pollen deposition on plants with relatively large displays (four or more male-phase flowers) is due to geitonogamy

(Study 2, Figure 3.2). The positive relationship between geitonogamy and display size likely results in plants with larger displays producing smaller seeds due to early- acting inbreeding depression (Study 3, Figure 3.3). Large Clarkia concinna plants can have a dozen or more male-phase flowers open at once, potentially resulting in even higher geitonogamy than quantified in studies 2 and 3. Additional selfing can occur in this species through delayed autogamy (Kay and Picklum 2013).

Despite the likely history of high rates of selfing, inbreeding depression in

Clarkia concinna is substantial when measured under field conditions—averaging

0.76 for cumulative fitness (Groom 2000). Selfed offspring may be at a stronger disadvantage in large or dense patches of plants, where intraspecfic competition is high (Cheptou et al. 2001; Uyenoyama et al. 2003). Self pollen can have other effects besides the direct reduction of fitness due to inbreeding depression. A model by

Aizen and Harder (2007) postulates reductions in seed set due to pollen limitation may be caused more frequently by pollen quality than quantity. Asclepias inflorescences that received outcross pollinia set fewer fruits when they were exposed to additional self pollinia than when they were bagged, suggesting self pollen can interfere with outcrossing (Finer and Morgan 2003). Therefore, the high amounts of geitonogamy in Clarkia concinna with large displays may substantially reduce the fitness benefits of additional flowers.

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In addition to display size, patch size affected geitonogamy, with flowers in smaller patches receiving fewer visits from a less diverse assemblage of pollinators than flowers in larger patches (Studies 4 and 5, Figures 3.4 and 3.5). Groom (1998) also found lower visitation rates in experimentally created small, isolated patches of this species. In fact, her effect of patch size on pollen deposition was greater, with isolated plants showing pollen limitation. The high visitation rates (Figure 3.4) and high rates of pollen deposition (Figure 3.5) in this study suggest Clarkia concinna was likely not pollen limited. Flowers in small patches received 100 pollen grains on average, which is greater than the maximum of around 80 ovules produced per fruit.

Pollinator abundances can vary greatly among years, and perhaps pollinators were more abundant in 2014 and 2015 than during Groom’s experiments. However, even if pollen is not limiting, delayed pollen deposition and greater floral longevity can reduce fitness due to the increased resource costs of floral maintenance and the reduced reproductive capabilities of aging structures (Arathi et al. 2002; Ashman and

Schoen 1997; Ehrlen et al. 2015).

The differences in pollinator communities among patch sizes may have important fitness consequences. For instance, as Bombylius visits were not affected by patch size, they made up a proportionally higher percentage of visits to small patches. Bombylius moved the most systematically among flowers of all pollinators, visiting more flowers per plant and likely depositing the highest proportion of self pollen (Study 1, Figure 3.1). Thus, the difference in pollinator community may contribute to higher rates of selfing in small patches. Visitor identity can have large

103 impacts on mating system, even when multiple visitors are effective pollinators. For instance, the shrub Grevillea macleayana is visited by honeyeaters and introduced honey bees. Honeyeaters are more likely to travel far distances between visits, and sites with more honeyeaters have higher outcrossing rates (Whelan et al. 2009). In

Clarkia concinna, small patches have fewer available flowers, and so the proportion of within-plant transitions would also likely increase—a pattern found in other systems (e.g. Delmas et al. 2015). Delayed pollen receipt and increased self pollen deposition could combine to reduce seed production in small patches, contributing to the high small-patch extinction rates documented by Groom (1998; 2001).

In contrast to the strong effect of display on geitonogamy, I did not find a positive relationship between visitation and display size in any study. While pollen analogue pigment origin differed among display sizes, there was no difference in total pigment receipt, indicating all plants were roughly receiving the same amount of pollen (Study 2, Figure 3.2). Despite differences in seed weight, seed number was the same across display sizes (Study 3, Figure 3.3). If smaller displays are pollen limited due to reduced attraction, they should set fewer seeds per flower. When I explicitly tested the combined effect of patch size and floral display on visitation, floral display did not affect any of the three visitation measures, even in small patches (Study 5,

Figure 3.5). The lack of effect was runs contrary to the theory that larger floral display should be more attractive to pollinators (Ohashi and Yahara 1999). Some studies have failed to find the hypothesized relationship between visitation rates and display size in bumble bees, where patterns of visitation are most studied (e.g.

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Andersson 1988; Harder and Barrett 1995; Karron et al. 2004; Grindeland et al.

2005). In plants with a generalist pollination syndrome such as Clarkia concinna, some visitor taxa may show the relationship while others do not (Thompson 2001).

The lack of increased visitation to large displays indicates that a tradeoff between attraction and geitonogamy does not occur in this species. This begs the question of why Clarkia concinna simultaneously opens multiple flowers. One possibility is that my metrics overestimated geitonogamy by not accounting for high amounts of pollen carryover (e.g. Geber 1985). Additionally, many plants, including the congener Clarkia gracilis (Jones 1994) can discriminate against self pollen, leading to higher outcrossing rates than expected based on selfed pollen receipt (also see Rigney et al. 1993). However, neither of these possibilities fully explain why large displays are favored.

A more likely explanation for floral display size in Clarkia concinna is that there are additional ecological constraints placed on structural and temporal floral development. A protracted flowering period with fewer flowers open at once would reduce geitonogamy, the fast-drying habitat in which Clarkia concinna occurs may eliminate this option. The simultaneous production of additional flowers will increase the total seed production of a plant if total flowering time is limited due to environmental constraints. Thus, while increasing floral display may reduce the marginal fitness increase per seed produced, it will still increase total fitness. For a given bloom period, the relationship between floral display size and total female fitness would be an asymptotic function determined by the strength of inbreeding

105 depression, the relationship between geitonogamy and floral display size, the maximum number of flowers a plant is capable of producing given its available resources, and the flexibility it has in allocating these resources. The asymptote of this function may frequently occur at a display size beyond the maximum rate of floral production for a plant, given that this rate has some developmental constraints.

In other words, the strategy with the highest fitness may be to simultaneously produce as many flowers as possible, even at high rates of geitonogamy. A mathematical model would reveal the nature of this parameter space.

Additional considerations of optimal floral display include the role of male fitness and resource allocation. Male fitness can also be reduced by geitonogamous pollen movement, however larger floral displays may increase the total pollen export of a plant, especially if pollen carryover is high (Klinkhamer and de Jong 1993).

Therefore, selection on paternal fitness may result in larger optimal displays than maternal fitness (Stanton et al. 1987; Barrett 2003). A disproportionately high percent of total female fitness may typically come from the first few flowers to open, as this is when floral displays are small and geitonogamy is low. Later flowers may primarily increase fitness through male function. Early-opening flowers do tend to have more and heavier seeds (study 3). Similar patterns of temporal partitioning of male and female fitness occur in other taxa, such as lilies (Thomson 1989). Later

Clarkia concinna flowers may show increased investment in female function if early flowers are herbivorized or selfed (Lloyd 1980; Lloyd et al. 1980; Stephenson 1981,

Eriksson 1995). Greenhouse experiments demonstrated the total number of Clarkia

106 concinna flowers produced is greater when early flowers are left unpollinated, and the seeds per flower increase in later flowers when early flowers are selfed (chapter 2).

Conclusions: These five studies showed that geitonogamy is greatest in

Clarkia concinna plants with large floral displays and after visits from a more systematically-moving pollinator (Bombylius flies). They add to the growing evidence that geitonogamy is an important determinant of mating system and can have large impacts on pollen quality and seed set (Lloyd 1992; Goodwillie et al. 2005; Kropf and Renner 2008). However, they did not show evidence for a tradeoff between geitonogamy avoidance and visitation rates, because increasing display size did not increase visitation. The lack of a relationship between visitation rate and display size even occurred in small patches of plants where pollinators were less abundant.

Therefore, additional factors such as environmental constraints on flowering time and the role of male fitness are needed to explain large floral displays in Clarkia concinna.

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Figure 3.1. Effect of floral display on the number of consecutive visits to flowers on the same plant that could result in geitogonamy for the eight most common visitors to Clarkia concinna. Bombylius visited more flowers per plant than other taxa. Eulonchus made very few consecutive visits, regardless of display size. Other taxa did not differ from each other.

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Figure 3.2. Effect of floral display on florescent deposition on focal stigmas of Clarkia concinna. Pigment originated from male-phase flowers on the focal plant and from the 50 nearest male-phase flowers. Unique letters indicate differences in amount of pigment received within each type (self or outcross). Stigma coverage was ranked as: no pigment (0), 1 grain to 5% (1), 5% to 20% (2), 20% to 40% (3), and >40% (4). Display did not affect total pigment receipt. Stigmas with no pigment deposition are excluded. Vertical bars  1 SE.

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Figure 3.3. Mass per seed of Clarkia concinna flowers in female phase while plants had 0, 1, 2, 4, or 6 flowers in male-phase flowers. Unique letters indicate significantly different seed mass. Seeds were also heavier on larger plants and earlier-opening flowers. Seed number did not depend on floral display size. Vertical bars  1 SE.

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1.2

0.9 Calypte Eulonchus 0.6 other bees Apis

Visits/Flower/Hour 0.3 Ochlodes Bombylius

0 Small Medium Large Patch Size

Figure 3.4. Visits per flower per hour by various genera to Clarkia concinna. Total visitation rates and visitor diversity were lower in small patches. Size classifications here are small (20-100 flowers; 28 observation periods of 20 minutes) patches than those in medium (between 100 and 1000 flowers; 52 observation periods) and large (> 1000 flowers; 41 observation periods) patches. When analyzed separately, Bombylius did not show a relationship between visitations and patch size, while Apis showed a positive relationship. Date also affected visitation.

118 a)

Figure 3.5. Effect of patch size and floral display on three measures of pollination success. Focal flowers on plants in larger patches received pollen sooner (a), wilted slightly sooner (b), and received more pollen grains (c), regardless of display size. The date of treatment initiation also affected these responses.

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Synthesis: In this dissertation, I used Clarkia concinna as a model for understanding some of the causes and consequences of phenotypic plasticity. Genotypes that experience variable environments may benefit from the ability to plastically alter traits in order to match adaptive optima (Bradshaw 1965; Sultan 1987; Schlichting &

Pigliucci 1995; Whitman and Agrawal 2009). I compared plastic responses among populations in response to soil moisture conditions, and among individuals in response to soil moisture, nutrients, and pollination. I focused my efforts on understanding plasticity in floral traits because of their important role in species interactions and reproduction. Specifically, I compared later floral traits between plants with early pollinated flowers and unpollinated flowers. Additionally, I explored the consequences of variation in floral display for pollinator attraction and mating system.

An important theme of this dissertation was to understand the adaptive value of plasticity. In chapter one, I predicted some traits, such as stem diameter, will have the same relationship with fitness in both wet and dry conditions, and any plasticity would be a passive and potentially maladaptive stress response. I expected the relationship between a trait and fitness will differ between environments for other traits, such as specific leaf area, where a plant might respond to drought conditions by actively thickening its leaves. The distinction between active and passive plasticity has been made repeatedly, and while both types of plasticity are common and can be beneficial, the former is more likely to be an adaptive response (Smith-Gill 1983;

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Dorn et al. 2000; Pigliucci 2001; van Kleunen and Fischer 2005). Much classic work on plasticity has been conducted on active plastic responses, where researchers choose traits in which plasticity has clear adaptive value (e.g. Cook and Johnson

1968; Lively 1986; Schmitt et al. 2003). I did not find any evidence for adaptive plasticity in response to a stressful drought treatment. The lack of any adaptive plasticity in chapter one highlights the potential benefits to plasticity avoidance

(Dudley 2004; Morris and Rogers 2013).

The maladaptive plasticity I found in chapter one contrasts with the response of later floral traits to early pollination seen in chapter two, in which I found later flowers and fruits respond to earlier pollination. While I did not measure total fitness in these experiments, the direction and type of changes that later flowers and seeds exhibit makes adaptive sense. While plasticity in response to pollen receipt within a flower is well documented, my novel results showed the potential for longer term effects of early pollination on later flower and inflorescence structure and seed number. Investing resources in already fertilized seeds is a more conservative strategy than spending them on further pollinator attraction or ovule production. However, in chapter three, I found large floral displays do not necessarily attract more pollinators—a pattern that is not unique to this system (e.g. Andersson 1988; Harder and Barrett 1995; Karron et al. 2004; Grindeland et al. 2005). Only considering this result, selection should act to reduce floral display in order to avoid pollinator- mediated selfing (geitonogamy) regardless of the pollination environment, and plasticity in display size would be maladaptive (Klinkhamer and de Jong 1993;

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Goodwillie et al 2005; Kropf and Renner 2008). However, the determination of optimal display size for a given set of environmental conditions likely involves multiple factors that I did not explicitly measure, such as a limited bloom period, male fitness, and the benefits of additional, albeit low-quality offspring.

Habitat alteration and climate change increasingly subject plants to novel environments, where the adaptive value of plasticity is especially important to consider. Adaptive plasticity may aid in the establishment or persistence of populations preventing extinction until selection can act on mean trait values (Yeh and Price 2004; Nicotra et al. 2010; Chevin et al. 2010). However, without a recent history of selection in a given environment, plastic responses are less likely to be in an adaptive direction (Ghalambor et al. 2007; Chevin et al. 2010). The stressful drought condition that I impose on plants in the greenhouse represents a novel environment, and may be at least part of the reason plasticity was maladaptive. Novel biotic changes, such as the local extinction of a pollinator, could also lead to maladaptive plastic responses. The assumption that a particular plastic response has an adaptive explanation needs to be explicitly tested for each set of novel conditions.

One way maladaptive plasticity can be reduced is through through trait canalization, or the ability of a genotype to produce a consistent phenotype across a range of environmental conditions (Waddington 1942; Debat and David 2001; Flatt

2005). Genetic canalization of morphological traits may be the result of plasticity in physiological tolerance mechanisms (Stearns et al. 1995; Rutherford 2000; Morris and Rogers 2013). A recent review of plant reciprocal transplant studies found

122 surprisingly high amounts of both trait canalization and maladaptive plasticity

(Palacio-López et al. 2015). It may be that Clarkia concinna from populations with more variable annual precipitation experience selection for greater morphological trait canalization. The ability of fertilized Clarkia concinna plants to maintain a similar maximum floral display despite producing more flowers could also be seen as a form of trait canalization. Contrasting the relative benefits of plasticity versus canalization among types of traits or among taxa with disparate life history strategies may be a particularly fruitful research direction.

The bulk of this dissertation focused on floral and seed traits, as these are important targets of selection. Interestingly, there is some evidence that floral traits are less plastic than vegetative traits because plasticity in the former is constrained by strong phenotypic integration for both developmental and selective reasons (Berg

1960; Conner and Via 1993; Herrera 2001; Brock and Weinig 2007). However, as with leaves or branches, flowers and fruits are modular units, and thus plants should have the ability to sequentially modify the number, overall size, and timing of later flowers based on environmental cues (Herrera 2009). Additionally, because flowers are directly involved in reproduction, relatively small changes in floral phenology or morphology may have large effects on fitness.

In pollination studies, whole plant pollination manipulations are likely superior to single flower manipulations because of the ability of plants to reallocate resources (Knight et al. 2006). For instance, single flower manipulations can overestimate pollen limitation. In chapter 2, by applying pollination treatments to

123 early flowers and measuring the effect on later flowers, I observed that early pollination can affect resource allocation. In Clarkia concinna, pollen receipt on early flowers reduced investment in later flowers relative to unpollinated plants.

Additionally, outcross pollen receipt on early flowers reduced mature seed number in later flowers relative to self pollen receipt. The distinction between the effects of pollen quantity (number of grains deposited on the stigma) and pollen quality (self or outcross pollen) has been made previously (e.g. Zimmerman and Pyke 1998; Aizen and Harder 2007), but this work provides further support for the need to carefully separate them. Interestingly, a recent study in a selfing congener failed to find an effect of resource reallocation (Runquist and Moeller 2013). Because selfing species, suffer less inbreeding depression, may be less likely to reallocate resources based on pollen quality. Comparisons of resource reallocation in response to early pollination among taxa with different mating systems could be particularly beneficial.

The genus Clarkia has a long history of investigation into speciation, drought and pollinator adaptation, and mating system evolution, and this dissertation builds on this previous work (e.g. Lewis and Lewis 1955; Vasek and Harding 1976; Groom

1998; Mazer et al. 2004; Moeller and Geber 2005). Plastic responses to my drought treatment mirror the changes in mean trait values shown by populations or species of

Clarkia that occur in drier areas. For instance, Clarkia tembloriensis is a species with selfing traits, derived at the dry range margin of Clarkia unguiculata (Vasek and

Harding 1976). However, the direction of the plastic response to pollination runs counter to selection on mean trait values in pollinator-limited environments. For

124 instance, Clarkia xiantiana shows reductions in herkogamy and protandry in populations where pollinators, especially specialist bees, are rare (Moeller 2006;

Moeller et al. 2012). There may be an important difference between variability in pollinator service, and a chronically scarce pollinator environment. In the former case, plasticity to increase attractiveness when pollinators are rare may the best solution, particularly when, as is the case in Clarkia, delayed selfing can serve as a backup even in primarily outcrossing taxa (Lloyd 1979). However, if pollinators are always absent, we would expect selection for increased prior selfing, as selfing traits tend to reduce resource expenditure.

The results of this dissertation are especially transferrable to other mixed- mating annual plants, where total lifetime fitness is entirely related to mating success in one season. Additional life-history variables, such pollinator or habitat specialization may be important in influencing plasticity (Lortie and Aarssen 1996;

Gomez et al 2014). Carefully quantifying the causes and consequences of plasticity across a range of species and environmental conditions will allow us to better understand and predict how plants survive in a variable world.

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