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Electronic Theses, Treatises and Dissertations The Graduate School

2004 Inbreeding Depression and Evolution in the Perennial Herb Septemloba; and the Evolutionary Maintanence of Cleistogamy Christopher G. Oakley

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

INBREEDING DEPRESSION AND MATING SYSTEM EVOLUTION

IN THE PERENNIAL HERB VIOLA SEPTEMLOBA; AND THE

EVOLUTIONARY MAINTANENCE OF CLEISTOGAMY

By

CHRISTOPHER G. OAKLEY

A Thesis submitted to the Department of Biological Science In partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Fall Semester, 2004

The member of the Committee approve the thesis of Christopher G. Oakley defended on July 12, 2004.

Alice A. Winn Professor Directing Thesis

David Houle Committee Member

Joseph Travis Committee Member

Approved:

Timothy S. Moerland, Chair, Department of Biological Science

The Office of Graduate Studies has verified and approved the above named committee members.

ii

ACKNOWLEGEMENTS

I would like to especially thank Alice Winn, David Houle and Joseph Travis for their help and advice throughout the course of my studies. I would also like to thank Ken Moriuchi, Jean Burns Moriuchi, Karen Jacobsen, and David Low for help with the field and greenhouse experiments, and /or reviewing parts of this work, and a special thanks to the Florida State University presidential research fellowship for funding during a portion of my studies.

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TABLE OF CONTENTS

List of Tables ..…………………………………………………………………………v List of Figures ..………………………………………………………………………..vi Abstract ..………………………………………………………………………………vii

INTRODUCTION ………………………...…………………………………………….1

1. INBREEDING DEPRESSION AND MATING SYSTEM EVOLUTION IN THE CLEISTOGAMOUS PERENNIAL VIOLA SEPTEMLOBA ………………..…4

2. THE MAINTINANCE OF CLEISTOGAMY AS A MIXED-MATING SYSTEM ………………………………………………………………………………35

CONCLUSION ………………………………………………………………………..51

REFERENCES ……………………………………………………………………….53

BIOGRAPHICAL SKETCH ………………………………………………………….63

iv

LIST OF TABLES

Table 1.1………………………………………………………………………………..26

Table 1.2………………………………………………………………………………..27

Table 1.3………………………………………………………………………………..28

Table 1.4………………………………………………………………………………..28

Table 1.5………………………………………………………………………………..29

Table 1.6………………………………………………………………………………..29

Table 1.7………………………………………………………………………………..30

Table 2.1………………………………………………………………………………..49

Table 2.2………………………………………………………………………………..50

Table 2.3………………………………………………………………………………..50

v

LIST OF FIGURES

Figure 1.1……………………………………………………………………………….31

Figure 1.2……………………………………………………………………………….31

Figure 1.3……………………………………………………………………………….32

Figure 1.4……………………………………………………………………………….33

Figure 1.5……………………………………………………………………………….34

vi

ABSTRACT

The persistence of mixed mating systems in plants, in spite of theory suggesting that either complete outcrossing or selfing should evolve depending on the level of inbreeding depression, has become a classic puzzle in evolutionary biology. Despite the central role of inbreeding depression in the theory for the evolution of mating systems, the majority of published estimates of inbreeding depression are for annual species, are incomplete, or are measured in artificial environments, calling into question their general utility. I measured inbreeding depression in the field and greenhouse, for all life history stages including male and female reproductive fitness, of a perennial cleistogamous species. I found greater inbreeding depression in the greenhouse (33%) than in the field (11%), but in both cases, inbreeding depression was lower than the threshold required by models to promote outcrossing; however there was considerable variation in the magnitude of inbreeding depression expressed among maternal plants. Inbreeding depression alone is unlikely to be sufficient to explain the persistence of mixed mating in this species. Cleistogamy, a system of dimorphic flowering in which individuals produce both open flowers, and closed obligately selfing flowers, is a mixed mating system which appears to be stable. I reviewed mathematical and verbal models of the maintenance of cleistogamy, as well as relevant published empirical data. Mathematical models require an inherent advantage of CH outcrossing, but do not address the mechanism for such an advantage. I found that CL flowers are generally cheaper to produce, have a higher probability of fertilization, and produce progeny that experience relatively low levels of inbreeding depression. Verbal bet-hedging models provide the most general explanation for the maintenance of CH flower production, given the many advantages of CL reproduction. Future theoretical and empirical work should address how temporal

vii variation, in selection for genetically variable progeny, or in male outcrossing success may act to stabilize mixed CH/CL.

viii

INTRODUCTION

The mating system of a species or population, particularly the degree to which individuals mate with related vs. non-related individuals plays a key role in evolution, shaping the underlying population structure and patterns of genetic variation (reviewed in Jain 1976). Flowering plants in particular exhibit a tremendous diversity of mating strategies. Many are hermaphroditic and are therefore capable of self-fertilization, the most extreme form of mating with relatives. Hermaphroditic flowering plants have evolved many elaborate mechanisms to promote cross-fertilization, the benefits of which are believed to include increased genetic variability and consequently, the ability to adapt to new environments. Another genetic benefit of outcrossing is the avoidance of inbreeding depression, the reduction in fitness of selfed relative to outcrossed progeny (Darwin 1876, Charlesworth and Charlesworth 1987). There are however, many potential advantages to self-fertilization (reviewed in Jain 1976). Selfing may provide reproductive assurance, guaranteeing the ability to reproduce even when conditions for outcrossing are unfavorable (Darwin 1876). A selfing individual, that can still donate pollen for outcrossing, also has the advantage of transmitting more copies of its genes to the next generation than an obligate outcrosser (Fisher 1941, Nagylaki 1976). Habitually selfing species may also preserve beneficial gene combinations (Mather 1973), and may be able purge their genetic load of deleterious recessive alleles (Lloyd 1979, Lande and Schemske 1985, Charlesworth and Charlesworth 1987). Given these numerous advantages to selfing, it is not surprising that we observe a variety of mechanisms by which plants promote self-fertilization.

1 Over the course of evolutionary history, selfing species are thought to have arisen from outcrossing progenitors (reviewed in Jain 1976), and the continued evolution of the selfing rate in populations, the juxtaposition of the benefits of selfing against the negative effects of inbreeding depression, has become a classic puzzle in evolutionary biology. Basic theory suggests that in most cases, mixed selfing and outcrossing should not be evolutionarily stable, and that either complete selfing or complete outcrossing should evolve depending on the level of inbreeding depression (Maynard Smith 1978, Lloyd 1979, Feldman and Christiansen 1984, Lande and Schemske 1985, Charlesworth et al. 1990). The abundance of species with intermediate selfing rates remains largely a mystery (Schemske and Lande 1985, Barrett and Eckhardt 1990). Additional theory suggests that under certain conditions, mixed mating could be stable, but the data needed to evaluate the realism of such conditions is scarce (reviewed in Uyenoyama 1993). I present estimates of inbreeding depression for all life history stages, including male and female reproductive fitness, of a perennial plant species with a mixed mating system. Measures of inbreeding depression in the literature are numerous, but many are incomplete and/or were made in an artificial environment, and studies of perennials are not well represented. Previous empirical and theoretical work suggests that estimates of inbreeding depression may be greatly influenced by life history, and the environment in which it is measured (Husband and Schemske 1996, Morgan et al. 1997). If we are to begin to understand mating system evolution in nature, complete and accurate measures of inbreeding depression are a critical first step. I also present a review of studies on the evolutionary maintenance of cleistogamy. Cleistogamy, a system of dimorphic flowering in which individuals produce both open, potentially outcrossed, as well as closed, obligately selfing flowers, is probably the most clear-cut case of facilitated mixed-mating (Lord 1981). It is an interesting syndrome because the closed flowers are generally much cheaper to produce, giving yet another advantage to selfing, and making the persistence of outcrossing all the more puzzling. I review mathematical and

2 verbal models of the maintenance of mixed flower production, many of which weigh the costs of reproduction of each flower type against the relative fitness of progeny (as influenced by, for example, inbreeding depression of the selfed progeny) produced from each flower type. I summarize existing empirical data on relative costs and fitness benefits, and point out potential areas where our knowledge is deficient. I also suggest additional data that are needed, and what new approaches could advance our understanding of how this balance of dimorphic flower production is maintained over time.

3

CHAPTER 1

INBREEDING DEPRESSION AND MATING-SYSTEM EVOLUTION IN THE CLEISTOGAMOUS PERENNIAL VIOLA SEPTEMLOBA

Introduction

Since the time of Darwin, mating systems have been recognized as an important aspect of biology. The mating system, and more specifically the proportion of inbreeding vs. outbreeding, has profound effects on genetic variability, and therefore the potential for response to selection. These effects are pronounced in hermaphroditic organisms, such as many plants, which are capable of self-fertilization. Plant mating systems are of particular interest because of the wide variety of strategies that plants employ, including elaborate mechanisms to promote outcrossing, selfing, or in some cases, both. Though many forces are involved in the evolution of the selfing rate, there are two main opposing forces: the automatic transmission advantage of selfing, and inbreeding depression (Jain 1976, Maynard Smith 1978, Lloyd 1979, Lande and Schemske 1985, Charlesworth and Charlesworth 1987, Charlesworth et al. 1990). The most commonly invoked advantage of selfing is the automatic transmission advantage (Fisher 1941, Jain 1976), a selfing genotype has a 50% fitness advantage over an obligate outcrossing genotype, contributing pollen and ovules to selfing, as well as contributing pollen to outcrossing. This advantage however, is reduced with pollen discounting; when selfing reduces the amount of pollen a selfer can contribute to outcrossing (Nagylaki 1976, Feldman and Christiansen 1984, Holsinger et al. 1984).

4 Inbreeding depression, the reduction in fitness of selfed, relative to outcrossed progeny, is a key force opposing the evolution of selfing (Lloyd 1979, Lande and Schemske 1985, Charlesworth and Charlesworth 1987, Charlesworth et al. 1990). In simple models of the evolution of selfing, inbreeding depression (caused by deleterious, partially recessive alleles) must be greater than 50% in order to counter the automatic transmission advantage, and prevent selfing from evolving to fixation (Maynard Smith 1978, Lande and Schemske 1985). Other models suggest that this threshold may shift depending on the level of dominance of deleterious mutations, but that the end result will still be either complete selfing or complete outcrossing (Charlesworth et al. 1990). The abundance of plant species that have intermediate levels of selfing therefore, remains an evolutionary puzzle (Schemske and Lande1985, Barrett and Eckert 1990). Inbreeding depression may be due to partially recessive deleterious alleles, as discussed above, or it may be due to overdominance (Charlesworth and Charlesworth 1987). Predictions about the trajectory of mating system evolution are drastically different if overdominance is invoked. A number of genetic models predict stable mixed-mating, assuming overdominance for fitness and genetic associations between fitness loci and a mating system locus (Campbell 1986). Genotypes that are heterozygous at selfing loci (mixed-mating) are more likely to be heterozygous at fitness loci, and therefore more fit. Stable mixed-mating is most easily achieved for the case of symmetrical overdominance, or equal selection against the alternative homozygous genotypes (Charlesworth and Charlesworth 1987, 1990, Holsinger 1988 Uyenoyama and Waller 1991). This mechanism will result in an increase in inbreeding depression with increased generations of successive selfing (Maynard Smith 1977, Charlesworth and Charlesworth 1987, Latta and Ritland 1994), and may be detected empirically by greater inbreeding depression in lineages with higher genetically determined selfing rates (Chang and Rausher 1999). This is exactly the opposite of the expectation for inbreeding depression caused by partially recessive deleterious mutations, in which highly selfing lines are

5 expected to purge their genetic load resulting in lower levels of inbreeding depression (Lande and Schemske 1985, Charlesworth and Charlesworth 1987). A number of ecological models also predict that under certain conditions, mixed-mating may be stable. Some models incorporate ecological factors, such as the timing of self-fertilization relative to the opportunity for cross-fertilization. Whenever self-fertilization increases seed set over that achieved through outcrossing alone (reproductive assurance), it can be favored even with strong inbreeding depression. Therefore, a system of delayed selfing, where ovules not fertilized by outcrossing are subsequently selfed, will always be advantageous (Lloyd 1979). Other such ecological factors include a positive covariation of seed or pollen discounting with selfing rate. Seed discounting is analogous to pollen discounting (defined above), and is the decrease in outcrossed seeds produced as the result of selfing. It is proposed that this scenario will promote mixed- mating by favoring selfing up to a certain threshold rate, where fitness losses of discounting exceed fitness gains of selfing (Lloyd 1979, Holsinger 1991, Lloyd and Schoen 1992). Another, more recent, model predicts that with low overall inbreeding depression, greater inbreeding depression on female, relative to male function may promote stable mixed mating (Rausher and Chang 1999). Strong inbreeding depression on female function promotes selfing, but low inbreeding depression for male function means that selfed plants have higher fitness through outcrossing. This model is most plausible for habitual selfers because they maintain lower overall levels of inbreeding depression than do outcrossers (Husband and Schemske 1996). At present, there is insufficient data to evaluate all the conditions of models predicting stable mixed-mating. Inbreeding depression is one critical parameter common to many of these models (Charlesworth et al. 1990). Accurately estimating inbreeding depression under natural conditions is therefore a crucial first step toward evaluating the assumptions and predictions of models that could explain the persistence of mixed mating. There are numerous empirical estimates of inbreeding depression within the context of mating

6 systems (reviewed in Husband and Schemske 1996), but the completeness and accuracy of these estimates is questionable for a number of reasons. The vast majority of estimates of inbreeding depression come from greenhouse studies (reviewed in Husband and Schemske 1996, but see Schemske 1983, Schoen 1983, Dudash 1990, Johnston 1992, Ågren and Schemske 1993, del Castillo 1998, Chang and Rausher 1999, Stone and Motten 2002). Measuring fitness in the greenhouse, may underestimate (or overestimate) the magnitude of inbreeding depression (Husband and Schemske 1996). Environment has been shown to significantly affect the magnitude of inbreeding depression, which is often expected to be greater in harsher conditions, due to factors such as drought or increased competition (Dudash 1990, Wolfe 1993, Lu 2000, but see Ågren and Schemske 1993). The majority of estimates of inbreeding depression are for annual species (Husband and Schemske 1996), and studies of perennials often consider only early portions of the life cycle (but see Schemske 1983, Johnston 1992), and perennial plants are generally expected to express inbreeding depression in later life history stages (Husband and Schemske 1996). Furthermore, theory suggests that given the same level of expression of yearly inbreeding depression in later life history stages such as survival, a perennial life history will experience greater inbreeding depression than would an annual life history. This greater inbreeding depression would make the evolution of selfing more difficult for a perennial life history (Morgan et al. 1997). Although it is easier to obtain lifetime fitness measures of annual plants, making generalizations about the genetic basis of inbreeding depression, purging, or evolution of the selfing rate based primarily on estimates of annuals is tenuous given the predicted interaction between mating system evolution and life history. Effects on male fitness components (e.g. pollen production and viability) have rarely been included in estimates of inbreeding depression. Of the few studies that measured inbreeding depression for male fitness, most have found it to be significant (Willis 1993, Robertson et al. 1994, Carr and Dudash 1995, 1997, del Castillo 1998, Good-Avila et al. 2003), but see Chang and Rausher

7 (1999). Thus, overlooking the male component of inbreeding depression may lead to an underestimate of overall inbreeding depression, as well as obscuring our understanding of the relationship between inbreeding depression and stable mixed-mating. Though a history of selfing typically corresponds to decreased inbreeding depression at the species level (reviewed in Husband and Schemske 1996), suggesting a partially recessive basis to inbreeding depression, there is considerable variation in this relationship at the population level (Holtsford and Ellstrand 1990, Eckert and Barrett 1994, Latta and Ritland 1994, Johnston and Schoen 1996, Mayer et. al 1996), and we know little about the co-variation of inbreeding depression with selfing rates among lineages within a population. Several studies have found variation in inbreeding depression among lineages (Schemske 1983, Dudash 1990, Ågren and Schemske 1993, Norman et. al 1995, Pray and Goodnight 1995, Dudash et al. 1997, Carr et al. 1997, Mutikainen and Delph 1998, Rankin et. al 2002, Pico et al. 2003, but see Johnston 1992, Chang and Rausher 1999) and a few have compared inbreeding depression between classes of selfing rates (high and low) based on characters such as herkogamy (Carr et al. 1997, Chang and Rausher 1999, Rankin et al. 2002, Stone and Motten 2002), or gynodioecy (Mutikainen and Delph 1998). Because selfing rate is likely to be continuously distributed in many populations, to further elucidate the relationship between selfing rate and inbreeding depression we would really like to know the selfing rates of individuals within a population. Given the numerous advantages of selfing, and the existence of many species that exhibit mixed-mating, the maintenance of outcrossing remains one of the great unanswered questions in evolutionary biology. In this paper, my primary objective is obtaining a comprehensive and accurate measure of inbreeding depression for a perennial species, under natural conditions. Such estimates are conspicuously absent from the literature, and are of critical importance to begin to evaluate the conditions and assumptions of models that predict the persistence of mixed-mating. Comprehensive measures of inbreeding depression in perennial species are grossly underrepresented in the literature,

8 and recent theory (Morgan et al. 1997) predicts that these species are more likely to maintain outcrossing than are annuals, making them interesting candidates for the study of mating system evolution. Here I report measures of relative fitness of selfed and outcrossed progeny in the field for all life history stages, from seed set to flower production of a perennial species: A parallel greenhouse experiment permits a comparison of measures of inbreeding depression between natural and controlled conditions. My estimates include measures of inbreeding depression for both male and female fitness components, and I measured within-lineage inbreeding depression and examined its co-variance with an estimation of individual selfing rate.

Materials and Methods

Study system

Viola septemloba (Violaceae) is a perennial cleistogamous herb found throughout the under-story of southeastern coastal forests (Fernald 1970). It is a short-lived perennial, typically living up to 7 years or more. It produces two flower types: chasmogamous (CH) flowers in the early spring, and cleistogamous (CL) flowers in the late summer. The CH flowers are open and showy, with many specializations for outcrossing, including bilateral symmetry and nectar spurs, whereas the CL flowers are reduced in size, and obligately self in the bud. There are no differences between the two flower types in ovule number per flower or seed number per fruit, and there are no differences in the size or field germination rates of seeds produced by the two flower types (Winn, pers. com.). In the field, the plants rarely produce more than one chasmogamous flower at a time, and flower production is typically limited. For 225 reproductive field plants over two years, average annual CH and CL flower production was 0.60, and 0.51 respectively, and of the plants that produced at least one CH flower, the average annual CH and CL flower production was 1.02 and 1.06 respectively (Winn and Moriuchi, unpublished data).

9 Cleistogamous species differ from mixed-mating species with monomorphic flowers in several ways that influence mating system evolution. The most striking difference is that the CL flowers cannot donate pollen to outcrossing. Consequently, they experience complete pollen discounting, and the automatic transmission advantage is eliminated for CL selfing. The effects of inbreeding depression, however, may be ameliorated by the lower cost of production of the CL flowers relative to the CH flowers (Schemske 1978, Waller 1979). Another important point is that cleistogamous species are likely to have a more consistent history of selfing than mixed-mating species with monomorphic flowers. An advantage of CL species for estimating selfing rates is that, at least in species with low CH selfing rates, the ratio of CL seed to total seed production provides a measure of individual selfing rate.

Experimental design

In November 2001, I haphazardly collected 30 adult plants from Saint Marks National Wildlife Refuge in Wakulla County, Florida. All plants were collected at least 5 meters apart to help minimize relatedness. I transplanted these plants into 4-inch pots with a 3:1 mixture of potting soil and sand, and grew them in the greenhouse at Florida State University. Plants were watered every other day, and fertilized with a half strength solution of general purpose 20-20-20 water-soluble fertilizer every 2 weeks. These plants are the maternal parents for the greenhouse crosses (cross-location = greenhouse). In January 2002, I performed controlled outcross and self-pollinations on each maternal plant that produced CH flowers (26 of the 30 plants). For the outcross pollinations, I emasculated the recipient flowers 2 days prior to anthesis, and placed a mesh bag over the flower to prevent insect pollination. Two days later, I applied a controlled amount of pollen from a randomly chosen donor (from the maternal plants) to the stigma using a dissecting probe, and re-bagged the flower. For the self-pollinations, I placed a mesh bag over the flower 2 days prior to anthesis. Two days later, I emasculated the flower and applied pollen from its anthers to its stigma and replaced the mesh bag. I used the same flower as dam

10 and sire for the self-pollinations because the plants rarely produced more than one flower at a time. I conducted pollinations through March 2002 (74 total pollinations on 26 of the maternal plants), when chasmogamous flowering ceased. In February 2002 at St. Marks NWR, I haphazardly selected 92 naturally occurring adult plants to serve as maternal parents for the field crosses (cross- location = field). For approximately 1 month, I performed controlled pollinations as described above, on all plants that produced flowers (78 of the 92 plants). To determine if the process of emasculation resulted in accidental self- pollination, I emasculated and, without pollinating, re-bagged 62 flowers in the field and greenhouse in 2002. Because the emasculation regime differed between the self and outcross pollinations, I conducted a separate assay to measure the effect of differences in timing of emasculation between the cross and self-pollinations on fruit set. I preformed 74 controlled crosses, in which approximately half of the flowers were emasculated and bagged 2 days prior to pollination, and the other half were bagged 2 days prior to pollination, then emasculated immediately before pollination. I conducted these crosses in the field and greenhouse over the course of two flowering seasons. To estimate the rate of autogamous selfing, I measured fruit production for flowers (bagged to exclude pollinators) in the field and greenhouse for a total of 93 flowers over the course of 3 flowering seasons. I estimated individual selfing rates for the field maternal plants starting in the fall of 2002. I counted the total number of seeds produced via CL and CH flowering for 2 years, and estimated selfing rate as the ratio of CL seeds produced to overall seed production. Because the plants rarely produce more than one flower in the field at a time, and because the rate of autogamy is extremely low (see results), this is a fairly good estimate of within-lineage selfing rates.

11 Fruit and seed set on maternal plants

For all pollinations, I analyzed the effect of cross-location (greenhouse or field) cross type (self or outcross), and their interaction on probability of fruit set using a categorical model (Proc CATMOD, SAS Institute). For all successful pollinations, I collected seeds when the fruits dehisced, and measured seed number per fruit. Additionally, for maternal plants that produced fruits from both cross types, (10 and 8 maternal families for greenhouse and field crosses, respectively) I weighed individual seeds to the nearest hundredth milligram. I analyzed the effect of cross type on the number of seeds per fruit for these 18 maternal plants using paired t-tests separately for each cross-location. I analyzed seed size with a mixed model ANOVA (Proc MIXED, SAS Institute); cross-location, cross type, and their interaction were treated as fixed effects, and maternal plant nested within cross-location, and maternal plant by cross type nested within cross-location were treated as random effects. A significant maternal plant by cross type interaction for log-transformed data may indicate differences in inbreeding depression among the maternal lines (Johnston and Schoen 1994).

Comparison of early offspring fitness

In May 2002, I germinated the individually weighed seeds from the greenhouse and field crosses. I randomized the position of seeds in wells of sand filled flats in a seed germinator set at 10-hour days with a day/night temperature of 20.5° and 18.5° Celsius respectively. When the cotyledons emerged, I scored germination and recorded the date. I then transplanted the seedlings into flats of field-collected soil in the greenhouse. I analyzed the effect of cross-location (greenhouse or field), maternal plant, cross type (self or outcross), and all interactions on probability of germination using a categorical model (Proc CATMOD, SAS Institute). To compare field germination rates between cross types, I conducted an additional set of germination trials using collections of selfed and outcrossed seed from maternal plants that produced seeds of only one type (35 maternal plants). I conducted these at Saint Marks NWR in

12 December 2002. I randomly selected 180 outcross and 360 selfed seeds, and sowed 60 seeds in each of 0.25 m2 plots (1 plot for outcrossed seeds, 2 for self) in each of 3 different blocks. I tested the effects of block, cross type, and their interaction on probability of germination using a categorical model (Proc CATMOD, SAS institute).

Comparison of survival and reproductive fitness components

To measure survival and reproductive fitness components, in October 2002 I transplanted the seedlings grown in the greenhouse into two environments: one in the greenhouse, and one in the field. I also scored survival to transplant for all seedlings. In the greenhouse, I transplanted 65 total seedlings from 4 maternal families (2-29 seedlings of each cross type from each maternal plant) into 4-inch pots filled with field-collected soil. All 4 maternal plants were from the greenhouse crosses. In the field, I used 360 total seedlings from 14 maternal families (8 from the field and 6 from the greenhouse crosses). All 14 families grown in the field are unique from the greenhouse families. I transplanted 1-8 seedlings of each cross type from each maternal plant randomly into 4 blocks at Saint Marks NWR (90 seedlings per block). For each plant in each environment, I measured: survival to each flowering season, as well as CL seed number, CH ovule number, and CH pollen viability, for two CL and CH flowering seasons. From these data, I calculated total CL seed and CH ovule production over the 2-year period, and a total reproductive fitness index as the sum of CL seed CH ovule production.

Rep. fit. index = CL seed number + CH ovule number (1)

I was only able to count CH ovule number and not seed number because the sampling for pollen was destructive. Pollen viability was estimated using the aniline blue staining technique outlined by Kearns and Inouye (1987). This measure of male fitness is one component of male fitness, and does not

13 incorporate pollen dispersal or pollen competitive ability, but is often the most feasible to obtain. I use CH ovule production as my measure of female fitness. For the plants grown in the field, I estimated total plant biomass in June of 2004 by measuring the width of the largest leaf, which is significantly correlated (R2 = 0.85) with plant biomass for this species (Winn, unpub. data). All data were analyzed using SAS version 8.0 (SAS institute). When possible, continuous variables, such as ovule and seed number, were analyzed using mixed-model ANOVA (Proc MIXED), with the restricted maximum likelihood (REML) method. This method is generally preferable to the type-3 method for unbalanced data (SAS users manual, SAS institute). I tested the significance of the random effects in the model using a Chi-square test on the difference in the - 2 log likelihood score between a model with random effect(s), and the model with no random effects included. The degrees of freedom for these tests are the differences in the number of random terms in the model. All data analyzed using ANOVA were natural log (x+1) transformed to meet the assumptions of the model. When data could not be transformed to meet the assumption of the model, the effect of cross type was tested using paired t-tests on family means. I analyzed discrete data such as probability of survival using a categorical model (Proc CATMOD, SAS institute).

Calculation of cumulative fitness and inbreeding depression

I calculated cumulative fitness for each cross type for each maternal plant using the sum of the total reproductive fitness index (Eq. 1) for i total offspring of a cross type, divided by the number of seeds produced from the initial crosses for each cross type.

W family = (Σi Rep. fit. index) / Initial seed number (2)

I chose this measure because it generates an average fitness for each cross type for each family, which is less influenced by the unbalanced design than is total fitness per family. I excluded fruit set because the small number of flowers each parent produced made it imprudent to interpret family mean fruit

14 set, and because I found evidence that the emasculation regime of the controlled outcrosses may have reduced fruit set (see results). I also calculated a pooled, population level cumulative fitness for each cross type in the field using a multiplicative measure, substituting the field germination rates from the pooled bulk seed collections for the actual observed germination rates of the experimental plants.

W multiplicative population field = (Mean seed number * probability of field germination * probability of survival * mean rep. fit. index) (3)

This incorporates the effect of field germination rates into the overall estimate of fitness, but should be interpreted cautiously, as it is based on a pooled sample of seeds from a different set of maternal plants from the field and greenhouse crosses. Because of low rates of field germination, it would have been impossible to conduct the field experiment starting with germination. I calculated family level inbreeding depression using the cumulative fitness measures (Eq. 2) for cross and self-derived plants of each maternal parent as:

σ family = (W family, outcross – W family, self) / the larger of the two (4)

Calculated in this way inbreeding depression is bounded by –1 and 1, with positive values indicating inbreeding depression, and negative values indicating outbreeding depression (Ågren and Schemske 1993). While this method of calculation has the problem of different estimates being divided by different denominators, I used it because it scales negative inbreeding depression (which can approach negative infinity) and because of some instances of complete negative inbreeding depression, which are undefined using the traditional measure of (outcross fitness – self fitness)/outcross fitness (see results). For both the greenhouse and field plants, I calculated mean population inbreeding depression as the mean of family level inbreeding depression (Eq. 4), and constructed 95% confidence intervals around those estimates using both normal theory, and jackknife re-sampling of families (Sokal and Rohlf 2000). For the field plants, I also calculated a pooled population level estimate of inbreeding

15 depression using the multiplicative pooled field measure of fitness (Eq. 3), which incorporates field germination rates.

σ pooled field = (W multiplicative outcross – W multiplicative self) / the larger of the two (5)

Although, this measure is somewhat contrived (see above), it gives a rough estimate of the expected cumulative inbreeding depression that might arise from field germinated plants. Finally, I examined the relationship between family level inbreeding depression (equation 4) and individual selfing rate using a Spearman correlation of the ranked values.

Results

Autogamy and effects of emasculation

Of the 62 control emasculations conducted, none set fruit, thus I can be confident that my outcross pollinations were not the result of accidental selfing. Eleven of 35 (33.3%) of the early emasculation treatment (used for outcross pollinations) crosses set fruit, compared to 21 of 41 (51%) of the late emasculation treatment (used for self pollinations). The effect of the emasculation treatment was not significant (χ2 = 2.38, d.f. = 1, p = 0.12), but the difference in fruit set is large enough to warrant omitting this stage from my measure of cumulative fitness (see below). Of the 92 flowers bagged to estimate autogamous selfing, only 1 set fruit, indicating that there is virtually no CH autogamy in this species. For the estimation of individual selfing rates of the field maternal plants, 25 field maternal plants produced flowers over the two-year period. The mean selfing rate was 0.83, with a standard deviation of 0.28 (Figure 1). Mean (std. dev.) selfing rate of 6 maternal plants that had produced both self and outcross seeds in the controlled pollinations was 0.69 (0.23).

16 Fruit and seed set on maternal plants

Of the 26 and 78 initial maternal plants pollinated in the greenhouse and field respectively, 19 and 41 successfully produced seeds from at least one cross type. At the population level, there were no significant effects of cross-location, cross type, or their interaction on fruit set (results not shown). The seeds from 18 maternal plants that produced seeds of both types were used for the remaining measures of components of relative fitness (except for field germination). The majority of maternal plants produced only 1 fruit from each cross type. In cases where a maternal plant produced more than one fruit per cross type, I averaged (across fruits to get) seed number per fruit, and pooled across fruits for subsequent measures. Seed number per fruit was somewhat greater for outcrosses than for selfs, especially for the greenhouse pollinations (Table 1.1). However, the effect was not significant for either the field, (paired t = 0.3, d.f. = 7, p < 0.79) or the greenhouse (paired t = 1.0, d.f. = 9, p < 0.35). Because most maternal plants produced only 1 fruit per cross type, I was unable to test for a maternal plant*type interaction for this stage. For average individual seed weight, there were no significant effects of cross type, or the cross type by cross-location interaction, but there were significant effects of cross-location, maternal plant nested within location, and the maternal plant by cross type interaction nested within location (Table 1.2). Outcross derived seeds were larger than self derived seeds for the greenhouse pollinations, but the opposite pattern is shown for the field pollinations, and overall the greenhouse pollinations yielded larger seeds than did the field pollinations (Table 1.1, Figure 1.2). For germination in the lab, outcrossed seeds had slightly higher germination than selfed seeds (Table 1.1), but this difference was not significant (χ2 = 0.86, d.f. = 1, p = 0.354). There was a highly significant maternal plant by cross type interaction (χ2 = 55.6, d.f. = 17, p < 0.001), indicating differences among maternal plants in the degree of inbreeding depression. This result was

17 for all maternal families pooled across the two cross-locations, but the results obtained separately by location are qualitatively the same. For the field germination trials of seed from maternal plants that only produced seeds from 1 cross type, selfed germination was 33% (std. dev. 33%) compared to 18% (47%) for the outcross seeds. This difference was highly significant (χ2 = 19.52, d.f. = 1, p < 0.001). Significant effects of block (χ2 = 74.62, d.f. = 2, p = 0.001) and block*type interaction (χ2 = 94.52, d.f. = 2, p < 0.001) suggest variation among microenvironments in suitability for germination. Outcross-derived progeny had somewhat greater survival to transplant (Table1), however, this effect was not significant (χ2 = 0.03, d.f. = 1, p = 0.85). There was no significant effect of maternal plant by cross type nested within cross-location (not shown).

Greenhouse fitness

There were no detectable differences in survival between selfed and outcrossed progeny raised in the greenhouse (only 1 out of 63 plants failed to survive). Of the different experiments and years, CH production in the greenhouse in 2003 was the only case for which there was uniform enough flowering (self and outcross plants from the same maternal family) to compare inbreeding depression for male and female fitness components (Table 1.3). In that year, there was no effect of cross type on family mean CH ovule production (paired t = 0.734, d.f. = 3, p = 0.516), but self-derived progeny had marginally greater family mean pollen viability (paired t = -2.48, d.f. = 3, p = 0.09) than outcross-derived progeny. Self-derived plants had on average higher total CL seed production over the two-year period than did outcross plants (Table 1.3), but this difference was not significant (Table 1.4). There were significant effects of maternal plant, and maternal plant by cross type interaction on CL seed production (Table 1.4). For family mean total CH ovule production over the two-year period, outcross derived progeny produced on average, more ovules than did progeny derived from selfing (Table 1.3), but the effect was not significant (paired t = 0.402, d.f. = 3, p =

18 0.72). There was no significant effect of cross type on total reproductive fitness index (the sum of CL seed and CH ovule production), though outcross-derived plants had on average higher reproductive fitness than self derived plants (Table 1.3 and 1.5). There were significant effects of maternal plant, and a significant maternal plant by type interaction on total reproductive fitness (Table 1.5, Figure 1.3).

Field fitness

In the field, only 25% of the 360 offspring derived from controlled pollinations produced any flowers; consequently, I present measures of total reproductive fitness components summed over the two-year experiment. One family from the greenhouse crosses was omitted from the analyses of reproductive fitness components because neither the cross nor self-derived plants produced flowers of any kind; however this family was included in the cumulative survival and biomass analyses and in the calculation of the means of family means (Table 1.6). There was no difference between self and outcross plants (χ2 = 0.94, d.f. = 1, p =0.33) for cumulative survival in the field (Table 1.6). There was no significant effect of maternal plant by cross type nested within location (not shown). This analysis was from data pooled across blocks, due to the low number of plants per cross type per family in each block. Overall, few plants in the field produced flowers, necessitating the comparison of total reproductive fitness using family means for each cross type within a block, averaged across blocks. Stage specific measures for CL seed, CH ovule and CH pollen viability will not be presented. Overall, the field reproductive fitness data are well summarized by the mean reproductive fitness index, which was 100% (plants from field pollinations) and 40% (plants from greenhouse pollinations) higher for outcross-derived than for self-derived plants (Table 1.6, Figure 1.3), but these differences were not significant (paired t = 1.108, d.f. = 13, p = 0.288). For the estimated family mean biomass, outcross derived plants were significantly larger than self derived plants (paired t = -2.462, d.f. = 13, p =

19 0.029). For plants from the field pollinations, outcross-derived plants were on average 15% larger than the self-derived plants, and this difference was even greater (27%) for the plants from the greenhouse pollinations (Table 1.6, Figure 1.4).

Inbreeding depression

Mean inbreeding depression for the 4 maternal families (from Eq. 4) in the greenhouse (Table 1.7), was 0.33 (std. dev. 0.34). However, the 95% confidence interval for this value is large, –0.21 to 0.88, and so inbreeding depression is not significantly different from zero. Confidence intervals based on jackknife re- sampling, were smaller, but qualitatively similar. Mean inbreeding depression for the 14 maternal families (from Eq. 4) in the field (Table 1.7), was 0.11 (std. dev. 0.77). The 95% confidence interval for this value was also large, –0.34 to 0.55, and inbreeding depression is not significantly different from zero. Outcross relative to self-fitness varied considerably among maternal families (Figure 1.5), ranging from cases of complete inbreeding depression (cross fitness > 0, self fitness = 0), to complete negative inbreeding depression (cross fitness = 0, self fitness > 0). The multiplicative pooled “population” level inbreeding depression (from Eq. 5) for the field plants, using field germination, was –0.14. I was able to estimate maternal family level inbreeding depression and individual selfing rates for 5 of the field maternal plants. There is a slight positive, but non-significant relationship between individual selfing rate and inbreeding depression (rs = 0.33, p = 0.55), though more data are needed to draw any reliable inference.

20 Discussion

I found neither strong, nor consistent evidence for inbreeding depression at the population level in Viola septemloba. Measures of mean cumulative inbreeding depression were 33% for the greenhouse grown plants, and 11% for the field grown plants. When field germination rates were used to calculate overall pooled inbreeding depression, an estimate of –14% was obtained. This indicates that although outcross plants had higher reproductive fitness on average, this effect may be far outweighed by the apparent superiority of selfed seeds for germination. The inbreeding depression I found for plants grown under field conditions, is therefore well below the 50% threshold required by simple models (Lande and Schemske 1985) to prevent the evolution of complete selfing. My measure however, could be an underestimate if inbreeding depression for survival and reproduction increases over the life of the plants. My estimates of low inbreeding depression are in general agreement with other studies of cumulative inbreeding depression in cleistogamous species (Waller 1984, Schmitt and Gamble 1990, Clay and Antonovics 1985, Lu 2002) though these studies compared the fitness of CH and CL offspring, and therefore may confound differences in fitness due to flower type (e.g. differences in the mass of seeds produced by the two flower types) with differences due to inbreeding depression. For another perennial violet, Culley (2000) reported negative inbreeding depression (–54% recalculated following Ågren and Schemske 1993), though this estimate was based on a greenhouse study of only 4 maternal families. Only 3 other studies report measures of cumulative inbreeding depression for perennials grown in the field. Schemske (1983) found total inbreeding depression of 29-56% in 3 species of Costus. Similarly, Johnston (1992) found total inbreeding depression of 7-73% in 2 species of Lobelia. These species are predominantly outcrossing, and are therefore expected to carry higher levels of inbreeding load than V. septemloba. Clay and Antonovics (1985) found total

21 inbreeding depression in Danthonia spicata, a cleistogamous (and thus regularly selfing) perennial, to be less than 6%. It is expected that inbreeding depression for perennials and highly selfing species should be expressed later in the life cycle, such as in survival and reproduction (Husband and Schemske 1996). The only stage where I found significant inbreeding depression was for estimated biomass. This may be considered a component of later inbreeding depression, and therefore, is consistent with the expectations of Husband and Schemske (1996). Though inbreeding depression for male fitness (not significant) in the greenhouse was estimated to be negative, inbreeding depression for female fitness (not significant) was estimated to be considerably less than the 50% required for differential inbreeding depression in male and female function to promote stable mixed mating (Rausher and Chang 1999). Despite the lack of significant inbreeding depression at the population level, there is considerable evidence that maternal lines express different levels of inbreeding depression (Tables 1.2, 1.4, 1.5, Figures 1.2-1.5). Variation among maternal lines in their degree of inbreeding depression has also been found in many studies (Schemske 1983, Dudash 1990, Ågren and Schemske 1993, Norman et al. 1995, Carr et. al. 1997, Dudash et. al 1997, Mutikainen et al. 1998, Rankin et al. 2002, Pico et al. 2003, but see Johnston 1992, Chang and Rausher 1999). Despite the small number and unbalanced nature of seed production from the initial crosses, which made it impossible for me to test for a maternal plant by cross type interaction at many stages, there is a consistent trend of differential inbreeding depression among lineages. In 4 of 6 of the stages for which I was able to test for a maternal plant by type interaction (seed size from the initial crosses, lab germination, CL seed production in the greenhouse experiment, total reproductive fitness index for the greenhouse experiment), I found it to be highly significant (Tables 1.2, 1.4, and 1.5). Although direct comparisons between the magnitude of inbreeding depression in the field and the greenhouse are difficult because the plants were derived from different maternal families, and because of the small sample of

22 maternal families in the greenhouse, there are some obvious, if not significant differences. There were no differences between self and outcross seeds for probability of germination in the greenhouse, but there was significant negative inbreeding depression for field-germinated seeds (Figure 1.3). Adult plants grown in the greenhouse had nearly 100% survival, compared to an overall average of about 75% for the field experiment. Proportion of plants flowering was also greater and much more uniform in the greenhouse experiment (Tables 1.3 and 1.6, Figure 1.3); plants grown in the greenhouse produced on average, 2 orders of magnitude more seeds and ovules than those grown in the field. Cumulative inbreeding depression in the greenhouse (33%) was larger than that in the field (11%), although there was considerable variation around these estimates. This result contrasts with Dudash (1990), who found greater inbreeding depression in the field than in the greenhouse (see also Husband and Schemske1996). It may be that decreased environmental variance in the greenhouse increased the likelihood of observing inbreeding depression (Schemske 1983). This may simply be a statistical issue, but could also reflect the differential expression of genes in the field plants due to small-scale environmental heterogeneity. If there were few suitable sites in the field, then the low probability of an outcrossed progeny being planted in such a site would result in much larger variance in inbreeding depression compared to the more uniform greenhouse environment. This reinforces the importance of measuring inbreeding depression under natural conditions. However, it may also be that the sample of maternal lines is too small in the greenhouse to make any general inferences about the differences in magnitude of inbreeding depression between adult plants in the field and the greenhouse. Furthermore, the majority of the non-flowering plants in the field have not yet reached reproductive maturity, and some opportunities to observe inbreeding depression have not yet arisen. The explanation for the differences in magnitude of inbreeding depression between the field and greenhouse is probably a combination of the above factors. In this study, where the crosses were conducted (i.e. the maternal environment) often played as great a role as where the experiment was

23 conducted (offspring environment) in determining the magnitude (and even direction) of inbreeding depression (Tables 1.1, 1.6), although conclusive evidence would require that plants of the same genotype be crossed in both environments, and their offspring be grown in both environments. Not enough of the maternal lines that produced both self and outcrossed progeny went on to produce flowers in subsequent years to permit reliable insight into the genetic basis of inbreeding depression (based on the relationship between family level inbreeding depression and individual selfing rate) in this species. From the 5 maternal lines that I examined, there was no apparent relationship between individual selfing rate and inbreeding depression, which provides no support that overdominance for fitness is responsible for inbreeding depression. This result is consistent with the findings of other studies in which no relationship between inbreeding depression and selfing history was found (Carr et al. 1997, Mutikainen 1998, Rankin et al. 2002). Studies that have found a relationship between selfing history and inbreeding depression, have found it to be negative. This suggests purging, and that deleterious recessive alleles rather than overdominance, is the genetic basis for inbreeding depression (Chang and Rausher1999, Stone and Motten 2002). My measure of individual selfing rate does not consider within flower CH selfing facilitated by pollinators, and so may underestimates the actual individual selfing rate, though morphological features of the CH flowers make this type of self-fertilization difficult (Beattie 1974). It should also be noted that the ratio of CH/CL flower production can be highly influenced by environment (Schemske 1978, Waller 1980, Clay 1982), and that there is only scant evidence that the proportion of total flowers that are CL is under genetic control (Clay 1982). Therefore, my estimate of individual selfing rate is only a rough indicator of possible genetically controlled differences in selfing rates between maternal plants. One possible explanation for differences in inbreeding depression among maternal lines in this study is differences in the history of selfing of the maternal parents. Maternal plants that are the result of successive generations of selfing

24 may have purged deleterious recessive alleles and therefore produce selfed offspring that are equally fit as outcrossed offspring (Dudash et al. 1997, Charlesworth and Charlesworth 1999, Carr and Dudash 2003). The obligately selfing nature of CL flowers produced in this species seems especially conducive to serial selfing (Lu 2002). Maternal plants that are the result of more recent outcrossing events would be expected to carry a larger genetic load, and thus, produce selfed offspring that express higher levels of inbreeding depression. A more unusual result, and one that is more difficult to interpret, is the abundance of negative inbreeding depression. Stone and Motten (2002) also found negative inbreeding depression, and suggest positive epistasis in plants that have purged their genetic load through serial selfing as a possible explanation (see also Allard 1996). Although the data in this study are spare, they suggest that there is little inbreeding depression in V. septemloba. This further suggests that this inbreeding depression is the result of deleterious recessive alleles, which have been purged from lines with more recent histories of selfing. Additional work with a larger sample of maternal plants would be needed to definitively test the relationship between inbreeding depression and individual selfing rate, though such data may prove extremely difficult to obtain for V septemloba. What stabilizes mixed-mating in V. septemloba? Given the lack of substantial inbreeding depression, coupled with the probable low cost of CL flowers, why doesn’t this species evolve complete selfing? It may be that cleistogamy is maintained as a bet-hedging strategy, in which regular cleistogamous selfing acts to purge the genetic load (Lu 2002), and provides a way by which to cheaply produce offspring with low variance in fitness (Waller 1980), while temporal environmental variation favors occasional fitness gains through CH outcrossing.

25 Table 1.1 Family means (std. dev.) for early life history stages by cross- location (field or greenhouse), and cross type (self or outcross).

Stage Location Cross Type n Mean (Std. Dev.) Seed Number Field Outcross 8 16.3 (9.8) Per Fruit Self 15.1 (7.5)

Greenhouse Outcross 10 13.2 (9.5) Self 9.7 (4.4) Seed Weight Field Outcross 8 1.59 (0.20) Self 1.66 (0.23)

Greenhouse Outcross 10 1.98 (0.28) Self 1.89 (0.25) Germination Field Outcross 8 87% (18) Self 91% (14)

Greenhouse Outcross 10 87% (18) Self 80% (24) Survival to Field Outcross 8 95% (11) Transplant Self 96% (4)

Greenhouse Outcross 10 95% (11) Self 86% (17)

26 Table 1.2 Results of mixed model ANOVA on individual seed weight (natural log transformed) by cross-location (field or greenhouse), cross type (outcross or self), maternal plant nested within cross-location, and interactions. Note the necessary difference in appearance of this and subsequent tables from those of traditional mixed model ANOVAs, due to different methods used to test the random factors using the REML method (see materials and methods).

Random Effects χ2 df p Maternal Plant (Location) 5.9 1 <0.025 Maternal Plant*Type (Location) 36.4 2 <0.001

Fixed Effects F value df p Location 8.46 1 0.011 Type 0.03 1 0.860 Location*Type 2.92 1 0.108

27 Table 1.3 Greenhouse environment: Family means (std. dev.) for reproductive fitness components by cross type.

Stage Cross Type n Mean (std. dev.)

CH Ovule Number 2003 Cross 4 263.9 (104.4) Self 206.2 (115.5)

CH Pollen Viability 2003 Cross 4 59.7% (3.9) Self 68.7% (6.9)

Total CL Seed Number Cross 4 215.0 (65.1) Self 269.0 (19.8)

Total CH Ovule Number Cross 4 276.5 (101.8) Self 246.3 (128.1)

Total Rep. Fit. Index Cross 4 491.5 (133.6) (Ch Ovules + CL Seeds) Self 515.4 (143.6)

Table 1.4 Greenhouse environment: Results of mixed model ANOVA on total CL seed production (natural log transformed) by cross type, maternal plant and interaction.

Random Effects χ2 df p Maternal plant 6.0 1 < 0.025 Maternal plant*Type 9.5 2 < 0.01

Fixed Effect F value df p Type 1.61 1 0.302

28 Table 1.5 Greenhouse environment: Results of mixed model ANOVA on total reproductive fitness index (CH ovules + CL seeds):(natural log transformed) by cross type, maternal plant and interaction.

Random Effects χ2 df p Maternal plant 7.9 1 <0.005 Maternal plant*Type 11.6 2 <0.005

Fixed Effect F value df p Type 0.77 1 0.452

Table 1.6 Field environment: Family means, averaged over blocks, for fitness components by cross-location (field or greenhouse), and cross type.

Stage Cross Location Cross Type n Mean(Std. Dev.) Cumulative Field Outcross 8 77.0% (15.9) Survival Self 8 73.3% (13.2)

Greenhouse Outcross 6 70.4% (12.1) Self 6 71.5% (14.8)

Total Fitness Field Outcross 8 9.72 (13.4) Index (CL Seeds Self 8 4.72 (4.28) Plus CH Ovules) Greenhouse Outcross 6 7.07 (4.80) Self 6 5.09 (5.91)

Estimated Dry Field Outcross 8 139.3 (26.2) Biomass (mg) Self 8 119.8 (19.5)

Greenhouse Outcross 6 134.6 (29.1) Self 6 99.3 (28.0)

29

Table 1.7 Summary estimates of cumulative inbreeding depression (δ) by cross-location (parental environment) and where the plants were grown (offspring environment). Fitness measured as family total reproductive fitness index by cross type/seed number from initial crosses.

Cross-Location Environment Maternal Plant δ

Greenhouse Greenhouse 34 -0.15 37 0.41 33 0.43 29 0.64

Greenhouse Field 27 -0.51 28 -0.42 1 0.00 31 0.35 32 0.94 30 1.00

Field Field 14 -1.00 19 -1.00 21 -0.74 11 -0.34 18 0.42 13 0.80 5 1.00 20 1.00

30

80 70 60 50 40 30 % of Plants % of 20 10 0 0-20% 20-40% 40-60% 60-80% 80-100% Selfing Rate

Figure 1.1 Mean selfing rates over a two-year period for all maternal field plants that flowered (n = 25).

2.6

2.2

1.8

Mean Seed Wt. (mg) Mean Seed Wt. 1.4

1 Cross Self

Figure 1.2 Family mean individual seed weight (mg) by cross type. Solid lines indicate crosses conducted in the field (n = 10), dashed lines indicate crosses conducted in the greenhouse (n = 8).

31

Greenhouse

800

700

600 x

500

400

300 Total Rep. Fit. Inde 200

100

0 Tot Rep Fit Cross Tot Rep Fit Self

Field

40

35

30 x

25

20

15 Total Rep. Fit. Inde 10

5

0 Tot Rep Fit Cross Tot Rep Fit Self

Figure 1.3 Total reproductive fitness index (total CL seeds + CH ovules) for the greenhouse (above) and field (below). Solid lines represent maternal lines from the field crosses (n = 8). Dashed lines represent maternal lines from the greenhouse crosses (n = 10).

32

200

180 160

140 120

100

80 60

Estimated Biomass (mg) Biomass Estimated 40 20

0 Cross Self

Figure 1.4 Field environment: Family mean estimated biomass. Solid lines represent maternal lines from the field crosses (n = 8). Dashed lines represent maternal lines from the greenhouse crosses (n = 6).

33 Field and Greenhouse

700 600 500 400 300 200

Total Self Fitness 100 0 0 100 200 300 400 500 600 700 Total Outcross Fitness

Field Only

40 35 30 25 20 15 10

Fitness Self Total 5 0 0 5 10 15 20 25 30 35 40

Total Outcross Fitness

Figure 1.5 Distribution of family mean relative reproductive fitness index for the greenhouse and field combined (above), and for the field only (below). The diagonal line is where inbreeding depression equals 0 (outcross fitness equals self fitness). Points below the line indicate positive inbreeding depression, while points above the line indicate negative inbreeding depression.

34

CHAPTER 2

THE MANTINANCE OF CLEISTOGAMY AS A MIXED-MATING SYSTEM

Introduction

Mating systems have long been of interest to evolutionary biologists because they dictate the genetic structure of populations, having profound effects on genetic variability, and therefore the potential response to selection. Plant mating systems are of particular interest because of the wide variety of strategies that plants employ (Jain 1976). Plant mating system theory predicts that a selfing genotype has a 50% fitness advantage (automatic transmission advantage) over an obligate outcrossing genotype, contributing pollen and ovules to selfing, as well as contributing pollen to outcrossing (Fisher 1941, Jain 1976). Inbreeding depression is thought to counter the automatic transmission advantage, and inbreeding depression greater than 0.5 will result in complete outcrossing. Thus, complete selfing or complete outcrossing are the only predicted outcomes (Lande and Schemske 1985). That many plant species exhibit intermediate levels of selfing and outcrossing therefore remains a great puzzle (reviewed in Schemske and Lande 1985, Barrett and Eckert 1990, Vogler and Kalisz 2001). Cleistogamous species stand in stark contrast to the predictions of mating system theory. Though few estimates of selfing rates are available for cleistogamous species, cleistogamy is a system of dimorphic flowering that inherently facilitates mixed selfing and outcrossing. Individuals produce open

35 (chasmogamous), often showy flowers available for outcrossing, as well as highly reduced (cleistogamous) flowers that obligately self in the bud. Cleistogamy is a widespread phenomenon in flowering plants, having been reported in 114 genera in 56 different families (Lord 1981, Plitmann 1995). The appearance of cleistogamy in many widely divergent families, each of which include non-cleistogamous species, suggests that it has evolved independently multiple times, and that selective regimes favoring cleistogamy are not uncommon. Cleistogamy is frequent among wind-pollinated monocots, but is also common in dicot families with floral morphologies specifically adapted to animal pollination, such as the Acanthaceae, Fabaceae, Scrophulariaceae, and Violaceae (Lord 1981, Plitmann 1995). As is the general trend with partially selfing species (reviewed in Barrett and Eckhardt 1990), most cleistogamous (about 80%) species are herbaceous annuals or perennials (Plitmann 1995). Among other systems of mixed-mating, cleistogamy is unique in two key ways. The CL flowers obligately self in the bud, and cannot donate any pollen to outcrossing (complete pollen discounting), which eliminates the automatic transmission advantage of selfing for the cleistogamous flowers. The CL flowers also require much less energy to produce, and therefore may provide a cheap method of reproductive assurance (Schemske 1978, Waller 1979, Schoen and Lloyd 1984). Reproductive assurance, the ability to guarantee and even increase reproductive output when conditions for outcrossing are unfavorable, can be favored even in the face of strong inbreeding depression. There is a diversity of flowering strategies under the broad banner of cleistogamy. Some species position their cleistogamous flowers such that CL seeds experience reduced dispersal, the most extreme case being geocarpy, where the CL flowers are produced underground (Lord 1981, Plitmann 1995). There is also variation in the relative phenology of the two flower types. Many species produce CH and CL flowers in an overlapping fashion, which is common among annuals such as species of Impatiens (Schemske 1978, Waller 1980). Other species produce CH and CL flowers in temporally distinct seasons,

36 common among perennials as in the genera Oxalis and Viola (Berg and Redbo- Torstensson 1999, Berg 2003). Given the energetic economy of CL flowers, along with the potential for reproductive assurance, it is easy to see how an initial mutation creating a CL flower would be selected for. The interesting question becomes, what prevents the fixation of CL flower production once it arises? The prevalence and diversity of cleistogamous systems in plants make this question all the more intriguing, and indicate that there may be more than one explanation depending upon the species under consideration. Here I review the literature on the adaptive significance of cleistogamy, including the current theoretical framework for the evolutionary stability of mixed CH/CL. I then review empirical studies that measure at least some of the relevant variables, paying special attention to comprehensive measurements of all parameters in a particular system. I then point out areas where adequate data are lacking, or need further consideration. I also suggest some new approaches to forward our understanding of this mixed-mating system.

Theoretical Review

Although there is a large body of theory on the evolution of mating systems in plants, both genetic (Lande and Schemske 1985, Campbell 1986, Uyenoyama 1986, Charlesworth et. al. 1990, 1991, Uyenoyama et al. 1991, 1993), and ecological (Lloyd 1979, 1992, Holsinger 1991, Johnston 1998), there is relatively little theory that takes into account the special features of cleistogamy. Schoen and Lloyd (1984) and Masuda et al. (2001) present the only mathematical models describing conditions that can favor mixed CH/CL. Schoen and Lloyd (1984) present three phenotypic optimization models for the evolution of cleistogamy that incorporate relative costs of reproduction by the two flower types. They also consider relative fitness of progeny derived from the two flower types, including the effects of flower type per se (i.e. viability due to differential allocation to seed size) and inbreeding depression. The first model

37 compares the fitness of two phenotypes that differ only in their degree of cleistogamy (proportion of total flower number that are CL) in a homogeneous environment. Fitness is assigned as the sum of gametic contributions: Pollen and ovules via CL and CH selfing, CH outcrossed ovules fertilized, and CH pollen donation (assumed to be equal to CH ovule fertilization). The result of this model is analogous to models of mixed mating in species with monomorphic flowers (e.g. Lande and Schemske 1985), and predicts that either complete cleistogamous selfing, or complete chasmogamous outcrossing should be favored depending on the relative costs of CH and CL, the selfing rate of CH flowers, and the relative viabilities of CH and CL offspring. This model cannot explain the stable coexistence of mixed CH/CL. The second model incorporates temporal environmental variation at the level of reproducing parental plants into the above framework. This model assumes that during a portion of the flowering season, some conditions will favor CH reproduction. For the remainder of flowering season, CH fertility and pollen donation is reduced, and CL reproduction will be selected for. The model compares two phenotypes that differ only in their ability to respond to a reliable environmental cue by producing the most appropriate flower type. One phenotype is able to respond perfectly; the other is able to respond, but to a lesser extent. Not surprisingly, the result is that the phenotype that is able to respond best to the environment is always favored, and temporally separated mixed CH/CL production is stable. However, if a plant is unable to detect and respond to the cue, then it must produce the flower type that is best most of the time. Without 2 different temporal environments, each favoring a flower type, and without a reliable cue to predict temporal changes in the environment, the mixed production of CH/CL is not stable. This model does not explicitly state what the advantage of CH outcrossing may be, and must be invoking an advantage to outcrossing per se. The only way reproduction by CH could be favored over CL, is if fitness through CH reproduction is enough to outweigh the inevitably cheaper production cost of CL (see summary of empirical data). The authors suggest that seasonally lower

38 pollinator activity creates conditions where CH flowers are not favored, but do not explain why CH flowers would be favored over CL flowers, which don’t require pollinators and are cheaper to produce in the first place. The third model of Schoen and Lloyd (1984) is an ESS model that applies more restrictively to cases in which the seeds of the two flower types have different dispersal abilities. This model assumes that CL seeds are cheaper to produce, but do not disperse as far as CH seeds, and therefore experience density dependent competition for establishment sites that CH seeds do not. It also assumes no CH selfing; that CH pollen export is large and beyond the seed shadow of the maternal plant, and that none of the half-sibs derived from CH matings experience density dependent competition for establishment sites. The prediction is that, for a narrow range of relative establishment probabilities for the two seed types, an ESS of mixed CH/CL can exist. The conditions of this model may however be, so restrictive as to be unlikely, even for the subset of species with differential dispersal capabilities. Masuda et al. (2001) address the persistence of mixed CH/CL with two phenotypic optimization models that also incorporate differential production costs of the two flower types. The first model, comparing two phenotypes that differ only in their degree of cleistogamy, predicts that with moderate inbreeding depression and large seasonal variation in CH fertility, mixed CH and CL could persist if the two flower types were produced in temporally distinct seasons and selection favored each flower type in a different season. This model is similar to Schoen and Lloyd’s (1984) second model. The second model of Masuda et al. (2001) incorporates an increase in the level of geitonogamous CH selfing with increased CH flower production. This model implicitly requires that conditions favor CH reproduction, and asks if an increase in geitonogamy with increased CH flowering can stabilize mixed CH/CL. This model predicts that, given a low level of inbreeding depression, mixed CH/CL can occur in an overlapping fashion. Without evidence for conditions that favor CH reproduction however, this is just a theoretical exercise. The important question that needs to be addressed is not what maintains CL flowering, the

39 benefits of which are easily explained, but what maintains CH reproduction. With the exception of the differential dispersal model of Schoen and Lloyd (1984), both mathematical models predicting stable mixed CH/CL (Schoen and Lloyd, model 2, 1984, Masuda et al., model 2, 2001) implicitly require that CH reproduction must be favored due to an advantage of outcrossing per se. This idea of an inherent advantage to outcrossing is analogous to arguments invoked for the evolution and maintenance of (Williams 1975, Maynard Smith 1978, Kondrashov 1993), in which outcrossing is proposed to be favored because of it’s ability to break up linkage disequilibrium, allowing the combination of newly arisen beneficial alleles and the elimination of detrimental alleles, as well as providing genetic variation needed to adapt to changing environments. Verbal models based on an explicit advantage of CH outcrossing have been proposed to explain maintenance of mixed CH/CL (Waller 1980), and suggest that mixed CH and CL may be a bet-hedging strategy. There are several verbal models of the maintenance of cleistogamy that invoke bet-hedging (Zeide 1978, Waller 1980). Under most conditions, CL reproduction is advantageous due to its relative energetic economy, greater fertilization efficiency, and reduced variance among offspring fitness. Occasionally however, unpredictable, temporal (cross-generational) environmental variation may provide a potentially large, fitness advantage to CH reproduction. This could result in an increase in the geometric mean fitness of a mixed CH/CL flowering system over that which would be achieved by a purely CL system, thus stabilizing mixed CH/CL. A variety of mechanisms have been proposed for such a CH advantage. One mechanism proposed to provide a CH advantage is decreased sibling competition among CH progeny relative to CL progeny (Waller 1980). These models are analogous to sib-competition models for the maintenance of sexual reproduction (Williams 1975, Maynard Smith 1978, Young 1981). Half-sibs (the result of CH outcrossing) are genetically more dissimilar than full-sibs (the result of CL selfing), and therefore experience less inter-sibling competition than do CL offspring (Cheplick 1993). In this scenario, mixed CH/CL would be stabilized by

40 temporal variation in the degree to which siblings experience competition. Frequency-dependent competition among CL progeny in a constant environment may also promote CH reproduction and stabilize mixed CH/CL production (Waller 1980, Price and Waser 1982). With the exception of the differential dispersal model of Schoen and Lloyd (1984), which is only questionably applicable to amphicarpic CL species, the mathematical models that predict stable mixed CH/CL production assume that some conditions favor CH over CL reproduction, but provide no mechanism by which this occurs. The verbal bet-hedging models, which have considered the biological conditions that might favor CH reproduction, are based on temporal variation in the relative fitness of CH and CL offspring. The data most useful for evaluating the potential for temporal variation in the inherent advantage of outcrossing to lead to stable mixed CH/CL, would therefore be factors influencing the relative overall reproductive success via CH and CL, and the degree to which this balance varies over time. Factors influencing relative reproductive success of each flower type include costs (per seed) of reproduction by each type, and the fitness of offspring produced by each type. Knowledge of the cost per seed for the two flower types would permit comparisons within a species, and provide a standardized measure to make comparisons across species. Cost per seed, for each flower type, is determined by the fertility of the flower (probability of successful fruit set), the cost of the individual structures (flower, fruit and seeds), and the number of seeds per fruit. The lower the fertility of a given flower type, for example lower fertility of CH flowers due to pollinator failure, the higher the cost per seed because of the energy wasted on flowers that produced no seeds. The costs of producing the individual structures will directly increase the cost per seed. Finally, the more seeds produced per fruit, the lower the cost per seed. The most efficient flower type would have high relative fertility and lower relative flower and fruit production costs. Differences in fitness between CH and CL progeny may be due to flower type per se, as in cases where seed size differs between the flower types

41 (McNamara and Quinn 1977, Zeide 1978), or may be due to inbreeding depression in the CL progeny. Because CH flowers are often capable of self- fertilization, the selfing rate of CH flowers, and inbreeding depression in the progeny resulting from such selfing must also be considered. Ultimately what matters is the overall relative fitness of progeny from the two flower types, if CH selfing rate is low, then relative fitness of CH and CL progeny is all we require. If CH selfing rate is high, or highly variable, then using such an approximation may obscure our understanding of the relationship between CH and CL fitness. Understanding this balance is critical to gauging the magnitude of the advantage of outcrossing required for the maintenance of CH/CL. Therefore, in cases where CH selfing is moderate to high, it would be useful to separate fitness differences due to flower type and those due to inbreeding depression.

Summary of Empirical Data

I found estimates in the literature for cost per seed and two of its major components: relative fertility (the probability of a flower successfully setting fruit), and relative flower costs (Table 2.1). I omit studies that report only a small portion of the data needed to calculate cost per seed (i.e. those that present only relative seed number per fruit). Schemske (1978) reported that for two species of Impatiens, CH flower costs were 100 times greater than CL (including nectar production costs), CH flowers were 20-60% less likely to successfully produce fruits than CL flowers, and that overall, cost per seed was 2-3 times higher for CH reproduction than for CL reproduction (Table 2.1). Waller (1980) found a similar relative cost per seed for Impatiens capensis (Table 2.1). Other studies reported only some components of relative cost per seed, with the most common being relative fertility (probability that a flower will successfully set fruit). Relative fertilities of CL vs. CH flowers ((mean 3.2, std. dev. 5.22); Table 2.1) were reported in 12 studies, and varied widely from 0.43 and 0.99 in two species of Viola (Culley 2002, Berg 2003), to 19.2 in Scutellaria indica (Sun 1999). Although complete data are limited, there is a consistent trend that CL flowers are less

42 costly, and have higher probability of being fertilized, which indicates a lower cost per seed for CL reproduction (Table 2.1). Measurements of inbreeding depression were reported in 19 studies; here I present only the results of studies considering cumulative fitness, fecundity and/or biomass. To the best of my knowledge, Culley (2000) is the only study to report relative fitness due to flower type, as well as inbreeding depression, for more than one life history stage (Table 2.2). She reports a cumulative relative

fitness (W) for CL derived progeny of 1.56 (WCL/ WCH selfed), as well as inbreeding

depression of –0.49 (recalculated here as (W outcrossed CH – W selfed CH)/W selfed CH). The cumulative fitness measure used included germination, survival, and vegetative biomass measured in the greenhouse from 4 maternal families. While interpretation of these results has obvious limitations, contrary to expectations, CL and inbred CH fitness are overwhelmingly larger than outcrossed CH fitness. Other studies estimated inbreeding depression by comparing open- pollinated CH and CL progeny. This method of measuring inbreeding depression may underestimate the actual level of inbreeding depression when CL seeds are inherently larger than CH seeds, or when CH selfing is common. In two studies of Impatiens (Table 2.2), the ratio of average cumulative fitness of CL/CH derived progeny (relative fitness) was about 0.65, corresponding to inbreeding depression of 35% (Waller 1984, Schmitt and Gamble 1990). In Danthonia spicata, Clay and Antonovics (1985), report a cumulative relative fitness of CL progeny to be 0.94, corresponding to inbreeding depression of 6%. Additional reports of individual stage specific relative CL fitness corroborate the above trend, that inbreeding depression is low or in some cases negative (Table 2.2). Selfing rates of CH flowers have been reported in 10 studies, 5 of which are for species of Impatiens (Table 2.3). The overall average was 66% (std. dev. 26%), and ranged from 24% in Impatiens (Mitchell-Olds and Waller 1985) to greater than 96% in Lespedeza capitata (Cole and Biesbor 1992). These high levels of CH selfing suggest that the above measures of inbreeding depression using open pollinated CH vs. CL seeds may be underestimates.

43 No direct-test of the general bet-hedging hypothesis could be found for cleistogamous species. Because few studies compare fitness for more than one year, there is little information on temporal variation in inbreeding depression (or outcrossing advantage per se.). Berg and Redbo-Torstensson (1998) found that in Oxalis, CH, but not CL, flower fertilization success varied temporally. They suggest that this is evidence for bet hedging, but do not consider offspring fitness. Many studies have reported that CL flowers are produced until a certain size threshold is reached, after which, CH flowers are produced (Waller 1980, Cheplick and Quinn 1982, Schnee and Waller 1986, Kaul et al. 2002). This is consistent with, but not conclusive evidence for, a conservative bet-hedging strategy in which small plants decrease variance in offspring fitness through CL selfing, while larger plants are able to “bet” resources on the riskier possibility of large fitness gains through CH outcrossing (Waller 1980). Most empirical tests of the sibling competition hypothesis in cleistogamous species do not support this mechanism for promoting mixed CH/CL: CH derived plants (half-sibs) do not outperform CL derived plants (full-sibs) when grown in competition (Schmitt et. al. 1985, Schmitt and Erhardt 1987, Wilson et. al. 1986, McCall et. al. 1989, Berg and Redbo-Torstensson 1999, 2000). Cheplick (1993) found that CH progeny of Sporobolus vaginiflorus suffered less from competition than did CL progeny, but this was likely explained by greater dispersal of CH progeny (i.e. model 3, Schoen and Lloyd 1984) rather than a release from competition due to increased genetic variability in the CH progeny. In a review of 22 other studies comparing plants and insects of similar and dissimilar genotypes grown in competition, Price and Waser (1982) report, a weak (3%) average advantage of dissimilar genotypes compared to similar genotypes. Other studies find the opposite trend, siblings grown in competition outperformed non-siblings grown in competition (Tonsor 1989, Donohue 2003), or no detectable difference (Kelley 1989b, Argyres and Schmitt 1992, Burt and Bell 1992, but see Schmitt and Antonovics 1986, Kelley 1989a). This suggests more generally that sibling competition is an unlikely explanation for the maintenance of sexual reproduction

44 (Kondrashov 1993), and by analogy, an unlikely mechanism for the maintenance of CH (outcrossing) in cleistogamous species.

Implications and Conclusions

Mixed CH/CL is a widespread phenomenon among flowering plants, having evolved independently multiple times (Plitmann 1995), and therefore may be the result of a variety of different selective scenarios. In all cases however, cleistogamy appears to facilitate adaptive mixed mating, contrary to predictions of general plant mating system theory (Lande and Schemske 1985, Charlesworth et al. 1990), which predicts either complete selfing or complete outcrossing depending on the level of inbreeding depression. Mathematical models developed to explain the persistence of mixed CH/CL (Schoen and Lloyd 1984, Masuda et al. 2001) require, contrary to the empirical evidence, a strong, short-term advantage of CH reproduction; or are restricted to amphicarpic species (Schoen and Lloyd 1984, model 3). Another limitation of current theory is that all of the mathematical models imply an annual life history. Morgan et al. (1997) illustrated differences between annuals and perennials with respect to the evolution of selfing, and noted a relative dearth of selfing among long-lived woody perennials (Barrett and Eckhardt 1990). They offer 2 possible non-exclusive explanations. They point out that for a given effect of inbreeding depression on survival to reproduction, a perennial life history would experience greater lifetime inbreeding depression than would an annual life history. This cumulative inbreeding depression of the perennial life history would more strongly counter the automatic transmission advantage, making the evolution of selfing more difficult. This is especially true for long-lived perennials, in which each year permits another opportunity for the expression of inbreeding depression on survival (Morgan et al 1997). They further point out that perennials may experience a between-year seed discount (Lloyd 1992). Whenever selfing increases overall seed set, it will increase fitness via reproductive assurance. But in a perennial, increased reproduction via selfing

45 in a given year may come at the cost of resources for future survival and future outcross reproduction. In general, the cost of increased reproduction on future survival and reproduction in perennials is well documented (Zimmerman and Aide 1989, Calvo and Horvitz 1990, Primack and Hall 1990). Cumulative inbreeding depression (Morgan et al. 1997) may also explain why there are so few long-lived woody perennials that are cleistogamous (Plitmann 1995), and a between year seed discount (Lloyd 1992, Morgan et al. 1997) would diminish the advantages of CL selfing even in short -lived perennials. One goal then, should be to develop mathematical models that incorporate both the unique features of cleistogamy and iteroparity, to permit examination of the conditions that promote cleistogamy in a large class of organisms that have been implicitly ignored by current theory. Bet-hedging models (Zeide 1978, Waller 1980) provide a more general framework for assessing the evolutionary stability of mixed CH/CL than do available mathematical models, but complete data on the net relative fitness of reproduction via the two flower types is lacking, and data on the temporal variation of this balance is non-existent. Of the 41 studies reviewed, none provided comprehensive data on relative costs and fitness of the two flower types. Relative cost per seed of the two types of reproduction has only been reported for 2 species of Impatiens (Schemske 1978, Waller 1979). Cumulative measures of relative fitness from CL and CH offspring have only been reported in 4 studies (Clay and Antonovics 1985, Waller 1984, Schmitt and Gamble 1990, and Culley 2000), and only 1 of these reported differences in fitness due to inbreeding depression separate from differences due to flower type (Culley 2000). Of the species for which we have measures of relative fitness, there are only estimates of CH selfing rates for two species, Impatiens capensis (Mitchell-Olds and Waller 1985, McCall et al. 1989, Waller and Knight 1989, Lu 2002) and Viola pubescens (Culley 2002). The literature reviewed here indicates that CL flowers are generally cheaper to produce, have higher fertilization probabilities, and produce progeny that experience relatively low levels of inbreeding depression. However, a

46 complete data set for variables required to calculate the overall relative fitness of reproduction via the two flower types is available for only 1 species. Comprehensive measurement of these variables in a broader diversity of cleistogamous species is needed both to begin to make generalizations about the magnitude and frequency of CH advantage needed to stabilize CH/CL (Lloyd 1992, Morgan et al. 1997). Data needed to assess the relative fitness derived from CL and CH reproduction in a single generation would include relative costs of reproduction for both flower types, independent measures of differential fitness of progeny due to flower type and inbreeding depression, and the selfing rates of CH flowers. Measures of fitness should ideally include all stages of the life cycle, and be obtained under natural conditions. To assess the role of bet-hedging we need measures of these quantities over a number of years. From this information we could begin to determine if the magnitude and frequency of temporal variation in relative fitness via each flower type in nature is enough to stabilize mixed CH/CL as proposed by bet-hedging models. High geometric mean fitness of a mixed CH/CL system may also be due to variance in CH male outcrossing success, an aspect often ignored. While at the population level, average male outcrossing success equals average female outcrossing success (the number of fertilizations is constrained by the number of available ovules); there may be considerable variation among individuals in CH outcrossing success. Temporal variation in male outcrossing success, such that there are occasional large fitness payoffs through outcrossing, may be another mechanism that promotes a bet-hedging strategy of mixed CH and CL production; therefore data on variation in the proportion of total gametic contributions, of individuals, attributable to outcross pollen donation would also be relevant. Our ability to measure individual male outcrossing success in natural populations is very limited. I found no reports of estimates of individual male outcrossing success for cleistogamous species. There are some studies of non- cleistogamous species that have provided estimates of individual male

47 outcrossing success. He and Smouse (2002) have shown that in Ophiopogon xylorrhizus, outcross pollen donation can account for greater than 20% of an individual’s reproductive success. Vassiliadis et al. (2002) have shown an average male outcrossing success of 4% in Phillyrea angustifolia, a wind pollinated species. Fishman (2000) has shown experimentally, that levels of outcrossing pollen donation may vary widely depending on the history of selfing of the donor plant and its neighbors. No studies have presented data on temporal variation in male outcrossing success. Future empirical and theoretical efforts should be aimed at assessing the magnitude and frequency of a short-term advantage to CH reproduction. Unpredictable temporal environmental variation in fitness via CH reproduction is the most common and most generally plausible mechanism proposed for maintaining mixed CH/CL production. Theoretical work should be directed at predicting a range of magnitudes and frequencies of selection for CH reproduction that can lead to stable mixed production over time. While difficult to obtain, data on temporal fluctuations in CH advantage, and male outcrossing success over time are necessary to understand the maintenance of mixed CH/CL production. A very different empirical approach that may also prove useful is comparisons of closely related species that differ in their degree of chasmogamy (ratio of CH flowers/total flower number). By examining a number of species pairs, closely related but differing in degree of chasmogamy, we can identify environments in which a higher degree of chasmogamy may be favored With theory in place to describe the different situations under which CH and CL reproduction are favored, and comprehensive data for species for each type of situation, we will begin to make progress on understanding the mechanism(s) that underlies persistence of mixed CH/CL. Better understanding of this facultative and adaptive system of mixed mating may furthermore give us greater insight into the evolutionary maintenance of mixed mating systems in general.

48 Table 2.1 Estimated relative cost per seed and two of its components: Relative fertility, and relative flower cost. All values are the ratio of CL/CH.

Species Fertility Flower Cost Cost Per Seed Reference A. purshii 4.89 McNamarra and Quinn 1977 C. benghalensis 1.11 Kaul 2002 C. grandiflora 0.9 Wilken 1982 C. melitensis 1.02 Porras and M. A. 1999 I. biflora 1.40 0.01 0.46 Schemske 1978 I. capensis 0.67 Waller 1979 I. pallida 1.77 0.01 0.38 Schemske 1978 M. polynoda 4.26 0.78 Schoen 1984 O. acetosella 1.25 Berg 2003 S. indica 19.2 0.15 Sun 1999 V. hirta 1.01 Berg 2003 V. mirabilis 1.08 Berg 2003 V. pubescens 0.43 Culley 2002 V. riviniana 0.99 Berg 2003

49

Table 2.2 Estimated relative fitness of CL and CH progeny, as a ratio of CL/CH fitness for cumulative fitness, fecundity, and biomass. In cases of multiple years or treatments, I averaged across years and/or treatments for simplicity of presenting the data. Only 1 study presented true measures of inbreeding depression separate from flower type, these measures are reported in bold. The where column refers to where the study was conducted (GAR = garden, GH = greenhouse, F = Field).

Reference Where Species Biomass Fecundity Cumulative Wilken 1982 GH C. grandiflora 1 0.95 Clay and Antonovics 1985a F D. spicata 0.97 0.94 Waller 1984 GH I. capensis 0.57 0.67 Schmitt and Gamble 1990 F I. capensis 0.8 0.64 Schmitt and Erhardt 1987 GH I. capensis 1.03 0.99 Berg and R-T 2000 F O. acetosella 1.35 Berg and R-T 2000 GAR O. acetosella 1.23 Culley 2000 GH V. canadensis 0.94 1.16 1.56 -0.14 -0.09 -0.49 Berg and R-T 1999 GAR V. hirta 0.89 Berg and R-T 1999 GAR V. mirabilis 1.1

Table 2.3 Estimated CH flower selfing rates.

Species Selfing Rate Reference Centaurea melitensis 92% Porras & Munoz Alvarez 1999

Impatiens capensis 24-70% Mitchell-Olds & Waller 1985 McCall et al. 1989 Waller & Knight 1989 Lu 2002

Lespedeza capitata 97% Cole & Biesbor 1992

Triodanis perfoliata 89% Gara & Muenchow 1990

Viola pubescens 36% Culley 2002

50

CONCLUSION

The evolution of mating systems has been the subject of interest to evolutionary biologists for some time, yet data needed to evaluate the unique conditions required by models predicting stable mixed mating are lacking, and general information common to many models, such as estimates of inbreeding depression, have important limitations (Husband and Schemske 1996). I found little evidence that overall inbreeding depression is responsible for maintaining outcrossing in this species. Contrary to expectations (Husband and Schemske 1996), the two stages for which I found significant inbreeding depression were early life history stages (seed weight and early seedling survival). I found inbreeding depression for greenhouse grown plants to be greater (and more uniformly so) than that of field grown plants, which suggests that the results of previous greenhouse inbreeding depression studies should be interpreted with caution. I found no support for models predicting stable mixed mating based on the relationship between inbreeding depression for male and female fitness components (Rausher and Chang 1999). Although I found consistent evidence for variation among maternal plants in the degree to which their offspring experience inbreeding depression, I was unable to accurately asses the covariance of inbreeding depression with selfing rate of the maternal plants, and so am unable to make a reliable inference about the genetic basis of inbreeding depression (reviewed in Uyenoyama et al. 1993). The few other studies that have looked for this relationship have found no pattern, or have found the opposite pattern, indicating purging of the genetic load. Without an overdominant genetic basis, inbreeding depression alone is inadequate to explain the persistence of mixed mating.

51 Cleistogamy is one of the most interesting contexts in which to study evolution of mating systems because it is likely the most clear cut system for facilitation of mixed mating. Existing mathematical theory is not adequate to explain the persistence of mixed CH/CL in any general sense because models assume an advantage to CH reproduction that contradicts the available empirical data (Schoen and Lloyd 1984, Masuda et al. 2001). The literature reviewed here indicates that CL flowers are generally cheaper to produce, have higher fertilities, and produce progeny that experience relatively low levels of inbreeding depression. Verbal bet-hedging models may provide a more plausible and general explanation for the maintenance of mixed CH/CL (Zeide 1978, Waller 1980); unpredictable temporal variation in selection for genetically variable progeny, or male outcrossing success could stabilize mixed CH/CL. Future theoretical and empirical work should be directed at addressing the magnitude and frequency of CH advantage required to stabilize mixed CH/CL production. Because of important differences between annual and perennial life history strategies with respect to the evolution of the selfing rate (Morgan et al. 1997), future theoretical work should be extended to accommodate features of a perennial life history. The widespread nature, and the multiple independent evolutionary origins of cleistogamy suggest that conditions favoring this facilitated mixed mating system are not uncommon, making this an appealing syndrome in which to study the evolution of mating systems (Lord 1980, Plitmann 1995). Also intriguing is the very pronounced advantage to selfing that the inexpensive CL flowers provide in addition to other advantages such as reproductive assurance and the potential for efficient purging through serial selfing (Lu 2002). Using cleistogamous species a model system in which to study how a presumed advantage of outcrossing maintains mixed CH/CL production can give us greater insight into the question of how mixed-mating systems persist, and perhaps even a better understanding of how the advantages of genetically variable offspring maintain sexual reproduction.

52

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BIOGRAPHICAL SKETCH

Christopher G. Oakley was born September 2, 1974 in Torrance, CA. He received an A.A. Degree in Liberal Arts from Sierra College in Rocklin, CA. He then received his B. S. degree in Botany from the University of Washington in 1998.

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