Crocanthemum Canadense (L.) Britt

MIXED MATING AND ITS ECOLOGICAL IMPACT ON AN ENDANGERED

PLANT: CROCANTHEMUM CANADENSE (L.) BRITT.

Emily Evans

Thesis submitted in partial fulfillment of the

requirements for the Degree of

Bachelor of Science with

Honours in Biology

Acadia University

April, 2017

This thesis by Emily Evans

is accepted in its present form by the

Department of Biology

as satisfying the thesis requirements for the degree of

Bachelor of Science with Honours

Approved by the Thesis Supervisors

______

Dr. Rodger Evans Date

______

Dr. Kirk Hillier Date

Approved by the Head of the Department

______

Dr. Brian Wilson Date

Approved by the Honours Committee

______

Dr. Jun Yang Date

I, Emily Evans, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit

basis. I, however, retain the copyright in my thesis.

______

Signature of Author

______

Date

iii

Acknowledgements

I would like to thank my supervisors, Dr. Rodger Evans and Dr. Kirk Hillier, for providing me with support and guidance throughout my research experience. Through their passion for their work, both were huge motivators throughout the summer and academic year.

Rodger’s positivity and sense of humour always brightened the lab space, while Kirk’s curiosity greatly inspired me to discuss and pursue questions of my own. A big thanks to Victoria Brown for lending a helping hand during the many hours we spent in the field, baking in the hot sun.

Also, thank you Erica Gillis for your support and for introducing your awesome music to our lab space. In the K.C. Irving Environmental Science Centre, Phyllis Essex-Fraser, Dr. David Kristie, and Dr. Robin Browne provided critical guidance and feedback on my project, especially regarding germination procedures and statistical analyses. Phyllis, your dedication to helping students and hilarious sense of humour was a highlight of my honours experience! Finally, I would like to thank my fellow honours students and the faculty in Acadia’s Biology Department for providing such a supportive and welcoming environment, especially during the summer months.

This project was made possible by the cooperation of the Canadian Forces Base 14 Wing

Greenwood and on-site support by Alan Ng and Alyssa Walker, who helped locate plant populations. Funding from both the Arthur Irving Academy Foundation and NSERC through an

Undergraduate Student Research Award was critical for the progression of the project.

Table of Contents

Acknowledgements ...... iv

List of Tables ...... vii

List of Appendices ...... x

Abstract ...... xi

1. Introduction ...... 1

1.1 Studying Endangered Plants ...... 1

1.2 Crocanthemum canadense in Nova Scotia ...... 1

1.3 Reproductive Biology ...... 3

1.4 Florivory by Mompha capella ...... 7

1.5 Objective ...... 8

2. Materials and Methods ...... 11

2.1 Sample Collections of C. canadense ...... 11

2.2 Differentiating Between Flower Types ...... 11

2.3 Quantifying Floral Characteristics ...... 12

2.3.1 Dissection ...... 12

2.3.2 Pollen Counts and Viability Testing ...... 13

2.4 Floral Morphology ...... 14

2.4.1 Scanning Electron Microscopy ...... 14

2.4.2 Spurr’s Resin Embedding ...... 15

2.5 Pollinator Exclusion Trial ...... 16

2.6 Seed Studies ...... 17

2.6.1 Germination Trial ...... 17

2.6.2 Viability Testing ...... 18

2.7 Infestation Rate by Mompha capella ...... 19

3. Results ...... 21

3.1 Quantifying Floral Characteristics ...... 21

3.2 Floral Morphology ...... 24

3.3 Pollinator Exclusion Trial ...... 29

3.4 Seed Studies ...... 30

3.5 Infestation Rates by Mompha capella ...... 31

4. Discussion ...... 35

4.1 Seasonal Transition in Floral Morphology and Mating Strategy ...... 35

4.2 Floral Contributions to Genetic Diversity and Fitness ...... 37

4.3 Implications of Mompha capella on Future Populations ...... 38

5. References ...... 41

Appendix A ...... 45

Appendix B ...... 49

List of Tables

Table 1. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the mean number of stamens between C. canadense flower types ...... 45

Table 2. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the mean number of pollen grains per anther between C. canadense flower

types ...... 45

Table 3. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the estimated mean number of pollen grains per flower between C. canadense

flower types...... 46

Table 4. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the mean number of ovules between C. canadense flower types ...... 46

Table 5. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the mean number of seeds between C. canadense flower types ...... 47

Table 6. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in the estimated mean inviable-to-total pollen grains per anther of seasonal flower

types of C. canadense ...... 47

Table 7. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess

differences in seed production between flower types of C. canadense treated under open (un-

bagged) and closed (bagged) pollinator conditions ...... 48

Table 8. Data under comparison in a post hock Tukey’s multiple comparisons test to assess

differences in seed germination percentage between flower types of C. canadense after a

two-week incubation period ...... 48

Table 9. Summary of estimated contribution per plant to the genetic diversity of subsequent

generations based on mean number of flowers per plant, seeds produced per flower, and

germination % of seeds for each flower type ...... 49

vii

List of Figures

Figure 1. Distribution of C. canadense across North America according to USDA National

Resources Conservation Service (2017) ...... 2

Figure 2. Crocanthemum canadense plant and flower morphology ...... 4

Figure 3. Scanning electron micrographs illustrating chasmogamous flowers of C. canadense pre-

anthesis while infested by larvae of M. capella...... 8

Figure 4. Flower classification system based on the degree of branching relative to the stem and

temporal separation within the season ...... 12

Figure 5. Bright field microscope images of C. canadense pollen grains in Alexander stain on a

gridded microscope slide ...... 14

Figure 6. A quantitative comparison between the reproductive traits of C. canadense flower types

based on degree of branching from the main stem ...... 22

Figure 7. A comparison between the inviable-total pollen grains per anther of seasonal flower

types of C. canadense...... 23

Figure 8. A comparison between the viable pollen-ovule ratios of the seasonal flower types of C.

canadense ...... 24

Figure 9. Scanning electron micrographs illustrating the reproductive structures across flower

types of C. canadense prior to pollen dehiscence...... 26

Figure 10. Scanning electron micrographs illustrating the reproductive structures between select

flower types of C. canadense during or after pollen dehiscence...... 27

Figure 11. Light micrographs of cross-sectioned anthers of C. canadense flower types. Circular

shapes with dark blue staining are pollen grains ...... 28

Figure 12. Comparison between seed production of open flower types of C. canadense under open

pollination (un-bagged) and excluded (bagged) insect pollinator conditions...... 29

viii

Figure 13. Relationship between the number of open C. canadense flowers producing fruits and

late season lateral branch characteristics during an in-field exclusion trial ...... 30

Figure 14. Germination percent of seeds from various C. canadense flower types after a two-week

incubation period at 15OC ...... 31

Figure 15. Scanning electron micrographs of unidentified larvae infesting closed flowers of C.

canadense ...... 33

List of Appendices

Appendix A. Data from multiple post hoc statistical comparisons tests to assess differences in

several reproductive traits ...... 45

Appendix B. Summary of estimated contribution per plant to the genetic diversity of subsequent

generations based on mean number of flowers per plant, seeds produced per flower, and

germination % of seeds for each flower type ...... 49

Abstract

Reproductive biology has important implications for the genetic makeup and demography of a population. By playing a major role in controlling genetic diversity, the mating strategy utilized by a species can help predict the adaptability and future viability of populations. The objective of this study was to provide a comprehensive understanding of the unique mixed mating system utilized by an endangered plant, illuminating potential related ecological repercussions.

Crocanthemum canadense (L.) Britt. (Rockrose or Canada Frostweed) is a small perennial herb listed as critically imperiled under the Nova Scotia Endangered Species Act. Rockrose exhibits a unique mixed mating system called dimorphic cleistogamy, whereby a single plant produces both cross-pollinating open flowers and self-pollinating closed flowers temporally separated within a single season. The seasonal transition from one floral type to another was assessed by comparing several floral characteristics using various microscopic analyses. Moreover, seed production in a field-based exclusion trial and pollen-ovule ratios of flower types were compared to determine the dominant mating strategy of the plants. Seed viability characteristics were also compared between flower types to determine if a difference in fitness existed between progeny from crossed versus self-pollinated flowers. Results indicate that flowers gradually change in morphology and reproductive strategy within a season, with transitional states between dominantly cross- pollinating and obligate self-pollinating flowers. Seeds from late-season closed flowers had a lower percentage germination success than open flowers, revealing a possible decrease in fitness of progeny from closed flowers. An increase in the infestation rate of open flowers by larvae of

Mompha capella in comparison to previous records in 2014 was also documented. By consuming seeds from open flowers predispersal, the florivorous moth larva further exaggerates the number of inbred seeds produced per plant relative to outcrossed seeds, decreasing a plant’s contribution to the genetic diversity of subsequent generations and ultimately posing risks for the adaptability and success of future Rockrose populations.

1. Introduction

1.1 Studying Endangered Plants

In recent years, the study of rare plant species has sparked renewed interest within the scientific community, with increased effort devoted to understanding patterns associated with endangerment and improving conservation methods (Peńas et al. 2016). Both intrinsic factors related to the biology of a species and extrinsic environmental factors play a role in species endangerment and population viability. Through its links to demography and population genetics, understanding the reproductive biology of an endangered plant may provide critical insight into the future viability of populations and appropriate conservation strategies (Carlsen et al. 2001).

1.2 Crocanthemum canadense in Nova Scotia

Crocanthemum canadense (L.) Britt. (Rockrose or Canada Frostweed) is a small perennial herb of the family Cistaceae that inhabits eastern Canada and the United States (Yorke 2007). In

Nova Scotia, the species is confined to dry, sandy barren ecosystems in Queens and Kings

Counties, with the largest documented populations in Annapolis Valley scattered between the closely associated communities of Kingston and Greenwood (Figure 1). Crocanthemum canadense prefers to inhabit areas bordering mixed woods and ditches, especially in disturbed sites provided by all-terrain-vehicle traffic, highway maintenance, and nearby runway developments at the Canadian Forces Base in Greenwood (Newell 2007). Over the past few decades, a gradual decline in C. canadense abundance and area of occupancy throughout Nova

Scotia has led to its listing as critically imperilled under the Nova Scotia Endangered Species Act

(Newell 2007; NatureServe 2015). Specifically, surveys conducted by Newell (2007) during the

2006 growing season identified between 5000 and 5500 plants remaining in wild populations and confirmed extirpation of a historic population in Halifax County. With no recovery plan established to save diminishing populations in Nova Scotia, C. canadense is at risk of progressive population decline. The reduction in provincial populations of C. canadense is mainly attributed to

the widespread destruction of the sand barren habitat throughout the province, reduced to as low as 3% of its original area (White 2016). Increased anthropogenic disturbances, including agricultural development, housing and road construction, and off-road vehicle traffic, are some of the largest contributors to habitat loss (Newell 2007). As a poor competitor, C. canadense relies on physical disturbances to create open areas with limited vegetation to reduce competition from neighbouring plants. Historically, wild fires and the grazing of extirpated woodland caribou

(Rangifer tarandus caribou) reduced the growth of woody species in sand barrens that would outcompete C. canadense for light and nutrient resources (White 2015). The invasion of Nova

Scotian sand barrens by the European Scots Pine, Pinus sylvestris (L.), has drastically reduced the provincial populations of C. canadense (Catling and Carbyn 2005; Newell 2007). Moreover, mechanical scarification and thermal pre-treatment promote seed germination of C. canadense in vitro, demonstrating further importance of grazing and wildfires for the germination success of the plant in its natural habitat (Newell 2007).

Figure 1. Distribution of C. canadense across eastern North America according to USDA National Resources Conservation Service (2017). Magnification of Nova Scotia illustrates the location of three provincial populations, including A (Kingston, Kings County); B (Greenwood, Kings County); and C (Greenfield, Queens County). Material throughout the study was collected from populations A and B.

Genetic analyses conducted by Yorke et al. (2011) determined that Nova Scotian populations of C. canadense in Kings and Queens Counties are genetically distinct from one another and from their next closest neighbouring populations in Southern Quebec and Southern

Maine. The lack of gene flow between provincial populations is likely due to geographical barriers between the two populations, such as the distance and difference in elevation which would prevent insect pollinators from facilitating interbreeding between individuals (White 2015). Moreover,

Nova Scotia C. canadense populations have the lowest levels of population-level genetic variation compared to other populations investigated in Maine, New Hampshire, and Quebec. A low genetic diversity is associated with a decrease in the ability of a species to adapt to changing environmental conditions, increasing the risk of extirpation greatly amongst the small C. canadense populations left in Nova Scotia (Dostálek et al. 2009; Yorke et al. 2011). By analyzing the reproductive biology of C. canadense, the pattern of genetic transmission and its direct effect on genetic diversity can be better understood (Kearns and Inouye 1993; Rodríguez-Pérez 2005).

1.3 Reproductive Biology

Crocanthemum canadense exhibits a mixed mating strategy referred to as dimorphic cleistogamy or true cleistogamy, whereby a single plant produces two distinct flower types that are temporally separated within a single growing season (Lord 1981; Newell 2007). More than

500 species of flowering plants from over 30 families exhibit this same mating strategy, including other species from the Cistaceae, Fabaceae, Violaceae, and Poaceae families (Culley and Klooster

2007). Chasmogamous flowers are the first flowers to appear in early June and continue to bloom until early July, opening on sunny days to display five yellow petals that attract insect pollinators

(Figure 2A). Open flowers promote outcrossing between individuals, increasing the genetic diversity of offspring by means of sexual recombination (Cortés-Palomec et al. 2006). Dispersal of genetically diverse seeds into the plant population reduces likeliness of inbreeding depression: the decrease in fitness of a species because of continuous breeding with related individuals

(Dostálek et al. 2009). Fitness can be described as the success of a species in its ability to survive, grow, and reproduce, and is compared between plants on the bases of various vegetative and reproductive measurements including plant size, flower size, pollen output, and ovule numbers

(Primack and Kang 1989).

Figure 2. Crocanthemum canadense plant and flower morphology. A. Chasmogamous flower during anthesis with anthers splayed beside the pistil. B. Late season branches baring many cleistogamous fruits of C. canadense. Dehiscing fruit of a chasmogamous flower is circled. C. Dehiscing chasmogamous fruit with seeds visible. A= anther; C= petal; Fr= fruit valve; F= insect frass; K=sepal; S=seeds. Scale bars = 15 mm.

At the beginning of the growing season, C. canadense plants have limited branching and chasmogamous flowers develop on the terminal ends of the main stem, and on one or two short lateral branches immediately below the terminal flower. Later in the season, approaching August,

C. canadense plants become highly branched to support the growth of numerous cleistogamous flowers (Figure 2B); (White 2015). This flower type is structurally distinct from chasmogamous flowers in that they are significantly smaller in size, are apetalous, and remain unopened throughout their reproductive development (Lord 1981). By remaining sealed, cleistogamous flowers are inaccessible to insect pollinators and have developed a unique mechanism of autogamous self-pollination, whereby stamens remain in close physical contact with the pistil

during floral development and pollen tube growth is initiated upon contact with the receptive stigma (White 2015). Due to this efficient pollen transfer mechanism, cleistogamous flowers develop markedly fewer stamens and ovules than their chasmogamous counterpart (Cruden 1977;

Lord 1981). Without nectar or elaborate petals, development of cleistogamous flowers is much less energetically expensive than chasmogamous flowers, and thus cleistogamous flowers are favourable in ecologically marginal habitats or when pollinators are limited (Winn and Moriuchi

2009). Several studies have provided evidence that true cleistogamous plants can demonstrate phenotypic plasticity in the ratios of chasmogamous and cleistogamous flowers grown in a single season as an adaptive response to variation in external environmental factors (Lord 1980; Lu

2002; Winn and Moriuchi 2009). Thus, C. canadense may be able to balance the advantages of adaptive trait combinations from outcrossing of chasmogamous flowers and reproductive assurance from cleistogamous flowers in variable environmental conditions (Ruan and da Silva

2012).

In their studies of 96 species of plants from various families, Cruden (1977) found that a flower’s pollen-ovule ratio is correlated with its breeding system and developed a classification system of flowers based on these different ratios. Highest ratios occur in outcrossing flowers that require large pollen output to maximize the probability of pollen transfer between plants, whereas selfing flowers have more efficient pollen transfer mechanisms and do not require excess pollen grains for successful fertilization of ovules. Lord (1981) reported that many true cleistogamous species produce an intermediate flower type in the transition between chasmogamous and cleistogamous flower display. Specifically, pollen-ovule ratios on true cleistogamous plants vary on a continuum rather than two distinct ratios, as would be expected if only purely chasmogamous and cleistogamous flowers existed (Lord 1980). Termed ‘pseudocleistogamous’ flowers, these intermediates may be morphologically indistinguishable from chasmogamous flowers, but differ in the fact that they do not open and may possess the ability to self-pollinate within the developing bud (Lord 1981). The growth of intermediate flowers with smaller petals and fewer stamens and

ovules than chasmogamous flowers has been reported in local C. canadense populations in previous years (R. Evans, personal communication 2016). As no studies have been conducted to examine the reproductive biology of these intermediate flowers in C. canadense, it remains unclear if they open to participate in cross pollination, self-pollinate, or are even reproductively viable. Studying the reproductive biology of these transition flowers will illuminate how they contribute to the population dynamics and genetic diversity of C. canadense populations.

Fruits from chasmogamous and cleistogamous flowers take between three to four weeks to mature, eventually forming a dehiscent fruit capsule housing 30-45 seeds in chasmogamous fruits and 5-10 seeds in cleistogamous fruits (Figure 2C) (Newell 2007). In addition to the ratio of chasmogamous to cleistogamous seed production per plant, the viability of each seed type is a critical determinant of the number of progeny arising from each flower type in future seasons.

Though the study of plant fitness has traditionally focused on seed production, plants that produce fewer seeds may have seeds of higher quality than those that produce many seeds, allowing them to contribute more to subsequent generations (Carlsen et al. 2001; Primack and Kang 1989).

Correspondingly, there is some evidence suggesting that seeds from chasmogamous fruits may be more fit than from cleistogamous fruits in species such as Microstegium vimineum (Trin.) from the

Poaceae family. This may confer a fitness advantage for chasmogamous seeds in plants where total seed production per plant is dominated by cleistogamous flowers (Huebner 2011). Simple germination trials and seed viability tests can be used to quantify seed viability and vigor, providing an indication of seedling fitness between different flower types (Verma and Majee

2013; Kearns and Inouye 1993). Therefore, any factor that limits the seed output of a plant or reduces the viability of its seeds will directly reduce its reproductive potential and population growth rate.

1.4 Florivory by Mompha capella

In 2013, insect larvae were documented consuming developing seeds of chasmogamous fruits predispersal (R. Evans, personal communication 2016). In 2014, the infestation rate in chasmogamous flowers was as high as 50% at the Canadian Forces base in Greenwood (White

2015). After rearing larvae to adulthood, White (2016) used DNA barcoding techniques to identify the larvae as Mompha capella (Busck) moth (Lepidoptera; Momphidae), a small florivorous moth previously undocumented in the Maritimes provinces. Dissecting infested chasmogamous flowers revealed a novel mechanism by which larvae could induce self-pollination and subsequent seed production in a chasmogamous flower (R. Evans, personal communication 2016). Without documentation of M. capella infesting cleistogamous flowers, larger chasmogamous flowers may be more heavily parasitized due to the larger buds providing more resources to support larval development (Dart and Eckert 2014).

Prior to anthesis, larvae enter developing chasmogamous flowers between the sepals and petals. Larvae then chew at the base of growing petals, causing the wilting petals and stamens to aggregate into a cone shape on the upper portion of the pistil and press the anthers against the receptive stigma (Figures 3A and 3B). As the anthers dry out and dehisce, the pollen falls on the stigma and fertilize ovules within the ovary, thereby initiating seed development. Larvae then chew a hole into the side of the ovary and can feed on the nutrient-rich seeds as they develop within the closed fruit (Figure 3C); (R. Evans, personal communication 2016). Although florivory is common in nature, the effect of pre-dispersal seed predation on perennial plant species has been understudied, making the fate of C. canadense populations in Nova Scotia uncertain (McCall and

Irwin 2006).

Moreover, the relationship between floral antagonists and variation in floral morphology and plant mating systems has rarely been studied (Penet et al. 2008). Florivorous insects may be a potent selective agent in the expression of different flower types in a single plant species, as florivores may select for smaller, less conspicuous flowers, thus indirectly promoting the increase

in cleistogamous flower development (Dart and Eckert 2014; Penet et al. 2008). By reducing the seed output from outcrossing chasmogamous flowers, M. capella directly reduces female reproductive fitness and may further reduce the genetic diversity within affected populations, triggering a potential increase in inbreeding depression (Rodríguez-Pérez 2005). With C. canadense populations facing habitat destruction, insect predation risks additional loss of individuals for populations already in decline.

Figure 3. Scanning electron micrographs illustrating chasmogamous flowers of C. canadense pre- anthesis while infested by larvae of M. capella. A. Damage to petals by a larva is visible, initiating the wilting of the petals around the intact developing stamens. B. Larval activity within developing flower. Pollen tubes are visible surrounding the stigma as self-pollination is induced. C. Larva within a fertilized ovary consuming developing seeds. A= anther; C= petal; F= insect frass; K=sepal; L=larva; O=ovary; Ov=ovules. Scale bars = 1 mm.

1.5 Objective

The overall objective of this study is to provide a comprehensive understanding of the unique mixed mating system utilized by Crocanthemum canadense, illustrating the potential ecological repercussions that may be associated. To clarify the seasonal transition in floral morphology and dominant mating strategy utilized by the plant, floral characteristics were compared both quantitatively and qualitatively using dissection, pollen staining, and microscopic analyses. Moreover, in-field exclusion trials provided additional insight into the reproductive strategies utilized by different flower types, specifically whether open chasmogamous have an

autonomous self-pollination mechanism to ensure seed set in the absence of pollinators, and if the number of cleistogamous flowers produced later in the season is a plastic response to reproductive success of chasmogamous flowers. As indicators of plant fitness, differences between the germination characteristics and viability of seeds produced from various uninfested and infested flower types were determined using a germination trial and seed viability test. Finally, the infestation rate and intensity of open flowers by M. capella was documented across three populations in Nova Scotia to assess potential changes since the most recent measurements in

2014 (White 2015). Results will ultimately elucidate how the mating system of C. canadense may affect the adaptability and future viability of populations in Nova Scotia and how the prevalence of M. capella may be further exaggerating these effects in affected populations.

2. Materials and Methods

2.1 Sample Collections of C. canadense

To obtain a representative sample of floral material at various stages of development, flowers were collected on a weekly basis through June 5, 2016 to September 8, 2016 from three sites in King’s county. These sites included a road-side site along the treeline at the western end of the 14 Wing Greenwood Canadian Forces Base runway (UTM: 347081.00 mE, 4982629.00 mN), a walking trail through the sand barrens west of the Greenwood Military Aviation Museum

(UTM: 347429.00 mE, 4982172.00 mN), and along the Kings County Rail Trail adjacent to the

Highway 201 (UTM: 350041.00 mE, 4985489.00 mN). Early in the season, while chasmogamous flowers were developing, samples of the top few centimeters of stem tissue were collected with the attached flower buds to preserve the arrangement of the developing flowers on the stem. By mid-July, when plants began to develop cleistogamous flowers later in the season, entire branches were removed at the basal intersection with the stem. Plants were sampled using an opportunistic method, whereby collections took place every 5-10m while walking through each population.

Samples were immediately placed in vials containing 50% Formalin Acetic Alcohol Fixative

(5:5:90 by volume; Formalin: Acetic Acid: 50% Ethanol). After 48 hours, material was transferred to 70% ethanol prior to dissection and microscopic analyses.

2.2 Differentiating Between Flower Types

To assess changes in floral reproductive traits during a single growing season, flowers were distinguished from one another based upon their degree of branching relative to the stem and temporal separation within the season. Terminal flowers were the first flowers to open, with a single flower grown at the apex of the stem. Later, two branches often developed at the intersection of the stem and the terminal flower. The flower occupying the end of these branches was termed the primary flower, which typically reached anthesis after the terminal flower. From the same branches, 1-2 secondary flowers occasionally developed from axillary nodes after the

primary flower was initiated. After these early season flowers reached maturity, alternating branches grew off the stem below the terminal flower. The apical ends of the branches were occupied by flowers referred to as terminal cleistogamous flowers, while flowers growing along the branches were termed axillary cleistogamous flowers (Figure 4).

Figure 4. Flower classification system based on the degree of branching relative to the stem and temporal separation within the season. T = terminal flower; 1 = primary flower; 2 = secondary flower; TCL = terminal cleistogamous flower, CL = axillary cleistogamous flower.

2.3 Quantifying Floral Characteristics

2.3.1 Dissection

Floral material was dissected in a Petri dish containing 70% ethanol and viewed with an

Nikon SMZ-1000 stereo zoom macroscope (Nikon Corporation, Tokyo, Japan). The quantified reproductive traits included the number of ovules, seeds, aborted ovules, and stamens in each flower type. One-way ANOVAs and Tukey’s multiple comparison tests (a=0.05) were used to assess if significant differences in these traits existed between flower types. All statistical analyses and figure constructions were completed using GraphPad Prism version 7.00 for Mac, (GraphPad

Software, La Jolla California USA).

2.3.2 Pollen Counts and Viability Testing

Mature stamens were collected from dissected, preanthetic flower buds and placed in vials containing 70% ethanol. To ensure that the pollen counts were an accurate representation of the number of pollen grains within each anther, mature anthers were distinguished from immature or post-dehiscent anthers based on their bright yellow colouration, visibility of individual pollen grains through the anther tissue, and lack of anther sac perforations. Stamens were selected from a pooled vial of stamens taken from at least 10 individuals during the floral dissection process. Each anther was separated from the filament using forceps and placed on the centre of an uncoated, gridded microscope slide for ease of counting the pollen grains. One drop of Alexander Stain

(Alexander 1969) was applied to the centre of the slide. Forceps were used to gently tease apart the anther, freeing pollen from anther sacs and creating a suspension of free pollen grains in the drop of stain. After applying a coverslip, the slide was left to sit for approximately 5 minutes at room temperature. Stained pollen grains were viewed and photographed with a Nikon Eclipse 50i bright field microscope and Nikon Fi1 digital camera (Nikon Corporation, Tokyo, Japan) (Figure

5A). Viable pollen grains, with protoplasm staining bright pink (Figure 5B), and inviable pollen grains with green stain only in the pollen wall (Figure 5C), were counted and recorded for 10 anthers of each flower type.

To estimate total amount of viable pollen produced by a single flower, number of viable grains in a single anther was multiplied by mean number of stamens in the associated flower type determined from analysis of dissection data. To estimate pollen-ovule ratios as an indication of the mating system utilized by the flower types, estimated number of viable pollen grains per flower was then divided by mean number of ovules in that flower type determined from dissections. One- way ANOVAs and Tukey’s multiple comparisons tests (a=0.05) were used to determine if a significant difference existed between flower types in terms of their total number of pollen grains per stamen, ratio between aborted pollen grains and total number of pollen grains per stamen, total number of pollen grains per flower, and total pollen-ovule ratio of the individual flowers. The

ratio between aborted and total pollen grains was transformed using an arc-sine function and the viable pollen-ovule ratios were transformed using a log function in order to meet the normality requirements for ANOVA. Though the number of pollen grains per flower was log-transformed to display data in graph format, the ANOVA was completed on untransformed data. All statistical analyses and figure constructions were completed using GraphPad Prism.

Figure 5. Bright field microscope images of C. canadense pollen grains in Alexander stain on a gridded microscope slide. A. Pollen grains at low magnification at which viable and inviable grains were counted across the entire slide. Scale bar = 1 mm. B. Viable pollen grains with protoplasm stained pink. Scale bar = 100 µm. C. Inviable pollen grains with pollen wall stained green and no protoplasm staining. Scale bar = 100 µm.

2.4 Floral Morphology

2.4.1 Scanning Electron Microscopy

Material was dissected in a Petri dish containing 70% ethanol and separated into glass vials. A graded ethanol series was used to dehydrate the samples, which involved 24-hour transitions between 80%, 95% with dye, and three consecutive changes of 100% ethanol.

Afterwards, the material underwent critical point drying using a Polaron E3000 Series Critical

Point Drier (Quorum Technologies, East Sussex, UK). The samples were then mounted on aluminum stubs and coated with gold-palladium using a SC7640 Sputter Coater (Quorum

Technologies, East Sussex, UK). A JEOL JSM-5900LV scanning electron microscope (JEOL,

Peabody, MA, USA) was used to view and capture images of the samples. Preview (Apple Inc,

California, USA) was used to edit all photos.

2.4.2 Spurr’s Resin Embedding

Mature stamens were collected from dissected flower buds preanthesis. With stamens from different flower types separated into vials, samples underwent a graded ethanol series to dehydrate tissue involving 24-hour transitions through 80%, 95% with dye, and two rounds of

100% ethanol. Dehydration was followed by 24-hour transitions through the following solutions in series: 2 parts 100% ethanol to 1 part 100% acetone, 1 part 100% ethanol to 1 part 100% acetone, 1 part 100% ethanol to 2 parts 100% acetone, and two changes of 100% acetone. The samples were then submerged in vials containing 15 parts 100% acetone to 2 parts Spurr resin for

12 hours, 7 parts 100% acetone to 1 part Spurr resin for 8 hours, 3 parts 100% acetone to 1 part

Spurr resin for 12 hours, and three changes of pure Spurr resin for two hours each (Spurr 1969).

The samples were then embedded in moulds using fresh Spurr resin and left overnight in a 70°C oven. The blocks were removed from the moulds and trimmed prior to sectioning with a Leica

RM 2065 histological microtome fit with a diamond blade. Sections were cut at 1.5 µm and placed floating on slides coated with distilled water. A hot plate set at 60°C was used to dry the slides prior to storage. Slides were stained by heating them on a 60°C hot plate for 10 minutes and applying a solution of 0.5% Toluidine Blue O (TBO) in 0.1% sodium carbonate with a filtered syringe. The stain was left on the slides for 30 seconds prior to rinsing with distilled water. Excess water was removed using blotting paper and the slides were dried for 24 hours in a slide box.

Three drops of Resolve™ oil were placed on the slides and coverslips were carefully mounted.

After drying the slides overnight, they were viewed and photographed with a Nikon Eclipse 50i bright field microscope and Nikon Fi1 digital camera. Preview (Apple Inc.) was used to edit all photos.

2.5 Pollinator Exclusion Trial

On June 9 at the Greenwood Military Aviation Museum site, a field experiment was initiated to evaluate the necessity of pollinators for seed production in open flower types of C. canadense, and to assess the plasticity of chasmogamous-cleistogamous flower ratios of plants in response to changes in the reproductive success of chasmogamous flowers. Twenty-four patches of plants were opportunistically selected for the trial by walking throughout the field site and marking clusters of plants at least two metres from one another. To exclude airborne and terrestrial insect pollinators, one plant bearing immature chasmogamous flowers from each of the targeted areas was covered loosely with a breathable 1 mm plastic mesh bag propped upwards by a flag and pinned to the soil by two metal fasteners. A separate, uncovered plant in each of the clusters was tagged by wrapping a segment of plastic straw around its stem. The plant pairs were monitored on a weekly basis and evidence of developing chasmogamous flowers was recorded.

Due to the public access of the site, some bags were torn off throughout the course of the trial.

Bags were only replaced early during the course of the experiment before chasmogamous flowers began to open. Mature chasmogamous fruits and aborted flowers were removed from the plants to quantify seed production under both pollinator conditions throughout the month of July. Most fruits were collected before seed dehiscence, allowing an accurate quantification of mature seeds and aborted ovules. Some flowers abscised and were lost before they could be collected, excluding them from seed counts.

Through August 16 to September 8, late-season branches baring developing cleistogamous flowers were collected. Providing an accurate quantification of the total number of cleistogamous fruits produced per plant posed a challenge, as mature fruits near the end of the branches began to abscise before flowers initiated development closer to the stem. As an alternative to counting the total number of cleistogamous flowers produced per plant, branches were harvested at the same developmental stage when terminal cleistogamous fruits were mature and dehiscing. Visible cleistogamous flowers and scars left from abscised flowers were summed

together to provide comparisons between cleistogamous flower counts between plants, under an assumption that no additional cleistogamous flowers would be produced. Length of each branch and number of nodes on each branch were also recorded.

To compare seed production of open flower types under the two pollinator conditions, seed-ovule ratios of collected flowers were arc-sine transformed and subjected to two-way

ANOVA and Tukey’s multiple comparisons tests (a=0.05). For assessment of plasticity in cleistogamous flower development depending on reproductive success of earlier open flowers, the number of fruits from open flowers reaching maturity per plant was considered a categorical variable since it took on a limited number of values (range: 0-2). For the dependent variables, number of visible axillary cleistogamous flowers summed with scars from abscised flowers, number of lateral branches, total length of lateral branches, and total number of axillary nodes took on a broader range of values and were treated as continuous. One-way ANOVAs and

Tukey’s multiple comparisons tests (a=0.05) were used to assess whether each of these measurements differed depending on the number of open flowers developing into fruits earlier during the season. All statistical analyses and figure constructions were completed using

GraphPad Prism.

2.6 Seed Studies

2.6.1 Germination Trial

Ripened, open fruit capsules were collected throughout the season from various flower types at the Greenwood Military Aviation Museum site. Seeds were removed from the fruits and stored at 4 oC for 3-4 months. Optimal scarification and germination conditions were selected based on a previous study of the germination of five species of Helianthemum (Crocantheumum) by Pérez-García and González-Benito (2006). Fine sandpaper was used to gently scarify the seeds in circular motions until the seed coat was noticeably damaged. Scarified seeds were plated on

Petri dishes lined with two sheets of filter paper moistened with 5mL of distilled water. Each

flower type was represented by three replicates consisting of a plate of 20 seeds each. Plates were covered and sealed within Ziploc bags. The seeds were incubated at 15oC for a 12-hour photoperiod at low level light with an irradiance of 50 µmol m−2 s−1. Germinated seeds, with an emerged radicle, were counted and removed every 12-24 hours over a 2-week incubation period.

Seeds with evidence of bacterial or fungal contamination were counted, removed from their plate, and re-plated together on a separate, labelled Petri dish lined with moistened filter paper.

To calculate final germination percent of each plate after the two-week incubation period, the number of unimbibed seeds was subtracted from the total number of seeds for each plate. A one-way ANOVA and Tukey’s multiple comparisons test (a=0.05) was used to assess if a significant difference existed between the final germination percent of seeds from each flower type. All statistical analysis was completed using GraphPad Prism.

2.6.2 Viability Testing

As a follow-up test to study seed types that had not reached near 100% germination, seeds from terminal cleistogamous flowers and axillary cleistogamous flowers were subjected to a tetrazolium chloride test to assess for viability of seed embryos. Three replicates of 20 seeds for each flower type were manually scarified with fine sandpaper. Each replicate was plated in a closed Petri dish lined with 2 sheets of filter paper soaked with 5mL of distilled water. Seeds were imbibed at room temperature (~20oC) for 24 hours. After imbibition period, seeds were transferred to separate glass shell vials and immersed in 1% tetrazolium chloride solution (Verma and Majee

2013) and sealed with parafilm. After incubation in darkness at 35oC for 6 hours, seeds were rinsed well in distilled water and subsequently immersed in 2mL of distilled water with 3 drops of clearing solution (1:1:1 by volume of distilled water, lactic acid, glycerin) for 3 minutes. Seed lots were then rinsed and viewed under a stereomicroscope. Viable seeds stained bright pink while inviable seeds had no staining and remained a brownish-yellow colour. To compare the embryo viability between the seeds from different flower types, the number of viable seeds was divided by

the total number of seeds in each lot and the groups were subjected to an unpaired two-tailed t-test with a Welch’s correction (a=0.05) using GraphPad Prism.

2.7 Infestation Rate by Mompha capella

To quantify infestation rate of open flowers by M. capella across different sampling sites, unopened flowers were collected weekly between June 28 and July 20, 2016. Due to the small population size at the Highway 201 site, only 15 flowers were collected weekly during this period, while ~30 flowers from the CFB Greenwood and Greenwood Military Aviation Museum sites were collected due to the large population sizes. Developing flowers were dissected and infestation was recorded if larvae or frass were present within the closed flowers. For fruits that were closed and had frass but no larva present, the number of remaining intact seeds after infestation was also recorded. The percentage of flowers infested from each sample collection at each site was calculated and arcsine transformed to meet the normality requirements of ANOVA.

A one-way ANOVA and Tukey’s multiple comparisons test (a=0.05) completed on GraphPad

Prism were used to assess for significant differences in the infestation rates of open flowers between the populations studied.

3. Results

3.1 Quantifying Floral Characteristics

Comparison of reproductive traits of different flower types revealed significant differences between the mean number of stamens (One-way ANOVA: F4,480 =405.9, p<0.0001)

(Figure 6A), pollen grains per anther (One-way ANOVA: F4,45 =68.5, p<0.0001) (Figure 6B), pollen grains per flower (One-way ANOVA: F4,45 =119.1, p<0.0001) (Figure 6C), ovules (One- way ANOVA: F4,597 =405.9, p<0.0001) (Figure 6D), and seeds (One-way ANOVA: F4,352 =171.4, p<0.0001) (Figure 6E) between the various flower types. Though some flower types closest in their degree of branching and temporal separation had no significant differences in some reproductive traits, reproductive traits generally decreased in number gradually across flower types from terminal chasmogamous to axillary cleistogamous.

A B

C D

Figure 6. A quantitative comparison between the reproductive traits of C. canadense flower types based on degree of branching from the main stem. Traits including number of stamens (A), number of pollen grains per anther (B), number of pollen grains per flower (C) number of ovules (D), and number of seeds (E) were compared using one-way ANOVAs and Tukey’s multiple comparisons tests between flower types. The bars represent the mean while the error bars represent the standard error of the mean. Significant differences between bars are denoted with different letters. Sample sizes were variable between flower types and between quantified traits. T= terminal flower; 1= primary flower; 2= secondary flower; TCL= terminal cleistogamous flower; CL= axillary cleistogamous flower (Appendix A).

Comparing the percentage of inviable pollen grains from the total pollen produced per anther of each flower type revealed no significant differences (One-way ANOVA: F4,45 =1.331, p=0.27) in

the relative viability of pollen between flower types. Secondary, terminal cleistogamous, and axillary cleistogamous flowers had notably larger standard error of the mean than earlier terminal and primary flowers (Figure 7).

Figure 7. A comparison between the inviable-total pollen grains per anther of seasonal flower types of C. canadense. Values were arcsine-transformed for comparison using one-way ANOVA. The bars represent the mean while the error bars represent the standard error of the mean. No significant differences were found between flower types. N=10 for each flower type. T= terminal flower; 1= primary flower; 2= secondary flower; TCL= terminal cleistogamous flower; CL= axillary cleistogamous flower.

The estimated viable pollen-ovule ratios showed a similar trend as the reproductive traits compared between flower types, with significant differences between flower types (One-way

ANOVA: F4,45 =66.7, p<0.0001). As the branching degree increased throughout the season, the pollen-ovule ratio decreased as well. No significant differences were found between the pollen- ovule ratios of early season terminal and primary flowers and late season terminal cleistogamous and axillary cleistogamous flowers (Figure 8).

Figure 8. A comparison between the viable pollen-ovule ratios of the seasonal flower types of C. canadense. Values were log-transformed and compared using one-way ANOVAs and Tukey’s multiple comparisons tests. The bars represent the mean while the error bars represent the standard error of the mean. Comparisons between bars are significant when denoted by different letters. N=10 for each flower type. T= terminal flower; 1= primary flower; 2= secondary flower; TCL= terminal cleistogamous flower; CL= axillary cleistogamous flower (Appendix A).

3.2 Floral Morphology

Noteworthy differences were found in stamen morphology across flower types. The first flowers to develop early in the season and later in the season on lateral branches had straight stamens that grew parallel with the pistil as they approached maturity (Figures 9A, 9B, and 9D).

As the degree of branching increased, coiling of the stamens of secondary and axillary cleistogamous flowers brought the anthers into closer association with the stigma (Figure 9C and

9E). All early season flowers bared petals, while axillary cleistogamous flowers developing last within the season were never recorded with petals. Developing terminal cleistogamous flowers were documented both with and without petals during dissections and microscopic analysis.

Figure 9. Scanning electron micrographs illustrating the reproductive structures across flower types of C. canadense prior to pollen dehiscence. A. Partially dissected (forward-facing sepals, petals, stamens, and ovary wall removed) pre-anthesis terminal flower with erect stamens, straight and parallel with the pistil. Scale bar = 1 mm. B. Partially dissected (forward-facing sepals, petals, stamens, and ovary wall removed) pre-anthesis primary flower with similar stamen morphology as terminal flower. Scale bar = 1 mm. C. Partially dissected (sepals and petals removed) secondary flower pre-anthesis. Coiling of filaments creates a non-parallel alignment of stamens with the pistil. Scale bar = 1 mm. D. Partially dissected (sepal and anthers removed) terminal cleistogamous flower. Stamens are erect and parallel with ovary. Scale bar = 500 µm E. Axillary cleistogamous flower partially dissected (sepals removed) showing a high degree of coiling of the stamens around the ovary. Scale bar = 500 µm. A=anther; C = petal; K= sepal; O= ovary Ov= ovule; S= stigma.

At maturity when anthers began to dehisce, flower types differed in their pollen dispersal mechanism. Early season (terminal, primary, and secondary) flowers opened to pollinators, causing straight stamens to fall against the petals (Figure 2A). Sepals closed after pollen dehiscence, re-erecting stamens beside the pistil (Figure 10A). The last flowers to develop within the season (axillary cleistogamous flowers) remained closed during pollination, as the pollen tubes grow from within the anther sacs. The coiling of the filaments and close proximity between the anthers and stigma enabled the anthers to tightly press against the stigma during self-pollination

(Figure 10C). Intermediate to these flower types, terminal cleistogamous flowers displayed a unique mechanism of autonomous self-pollination whereby straightened stamens permitted self- pollination regardless of a greater spatial separation between the stamens and the pistil than axillary cleistogamous flowers (Figure 10B). Pollination occurs as pollen tubes are initiated within the anthers without pressing tightly against the stigma as seen in axillary cleistogamous flowers.

Figure 10. Scanning electron micrographs illustrating the reproductive structures between select flower types of C. canadense during or after pollen dehiscence. A. Partially dissected (sepals and stamens removed) post-anthesis terminal flower. Stamens have re-erected after anthesis as sepals closed inwards. Anther sacs have dehisced and remnant pollen is visible within anthers. Scale bar = 1mm. B. Partially dissected terminal cleistogamous flower (petals and sepals removed) during autonomous self-pollination event. Filaments remain straighten and spatially separated from ovary wall. Pollen tubes are visible in contact with the stigma. Scale bar = 500 µm. C. Partially dissected (with forward-facing sepals removed) axillary cleistogamous flower during self- pollination. Stamens are coiled and in close association with the ovary. Anthers are pressed tightly against the stigma and pollen tubes are visible in contact with the stigma. Scale bar = 500 µm. A=anther; K = sepal; O= ovary Ov= ovule; S= stigma.

Cross sections of anthers revealed a transition in the anther morphology from tetrasporangiate with extrorse dehiscence in early season flowers to bisporangiate with a paddle- like shape and introrse dehiscence in late season flowers. Specifically, terminal and primary flower anthers had four uniform anther sacs (Figures 11A and 11B), while secondary flowers had some disfiguration in the anther wall and a reduction in size of the introrse anther sacs relative to the extrorse sacs (Figure 11C). Anthers of terminal cleistogamous flowers often had a reduction in anther sac number all together, as one introse locule was often absent or reduced. Extrorse anther sacs also became oriented lateral to connective filament tissue rather than above, as in the cases of the earlier flower types (Figure 11D). Latest in the season, anthers of axillary cleistogamous flowers took on a paddle-like shape as the anther sacs reduced to two in number and became

oriented below connective filament tissue. As a result, the anther was curved toward the interior of the developing flower (Figures 10C, 11E).

Figure 11. Light micrographs of cross-sectioned anthers of C. canadense flower types. Circular shapes with dark blue staining are pollen grains. A. Terminal flower with four uniform anther sacs. B. Primary flower with four uniform anther sacs. C. Secondary flower. Introrse anther sacs are reduced and anther wall tissue is not uniform. D. Terminal cleistogamous flower. Only one introrse locule is present and the orientation of extrorse anther sacs is lateral to the filament connective tissue. E. Axillary cleistogamous flower. Anther sacs reduce to two in number and are positioned below filament connective tissue. Paddle-like shape of anther is curved towards the interior of the flower. Scale bar = 100 µm.

3.3 Pollinator Exclusion Trial

Un-bagged plants that were monitored over the course of the season grew an average of

1.00 ± 0.00 terminal flowers (N=24), 0.71 ± 0.142 primary flowers (N=24), 0.04 ± 0.040 secondary flowers (N=24), 4.95 ± 0.358 terminal cleistogamous flowers (N=20), and 61.4 ± 7.25 axillary cleistogamous flowers (N=19) per plant. In comparing seed production between open- pollinated and excluded flowers, terminal and primary flowers showed a significant reduction in the number of seeds produced when excluded (Two-way ANOVA: F2,81 =55.3, p<0.0001), while there was no significant difference in seed production between open and closed pollinator conditions for terminal cleistogamous flowers (p=0.99) (Figure 12).

Figure 12. Comparison between seed production of open flower types of C. canadense under open pollination (un-bagged) and excluded (bagged) insect pollinator conditions. Bars represent the arcsine of the mean seed-ovule ratio of the flowers while the error bars represent the standard error of the mean. A significant difference (a=0.05) in seed production between treatments for each flower type is denoted by “*”, while no significant difference is denoted by “ns”. Sample sizes are variable between individual groups. T= terminal flower; 1= primary flower; TCL= terminal cleistogamous flower (Appendix A).

Moreover, the number of open flowers that developed into fruits did not have a significant effect on the number of axillary cleistogamous flowers (One-way ANOVA: F2,28 =0.29, p=0.75) (Figure

13A), number of lateral branches (One-way ANOVA: F2,28 =0.19, p=0.83) (Figure 13B), total length of lateral branches (One-way ANOVA: F2,28 =0.60, p=0.56) (Figure 13C), or total number

of axillary nodes (One-way ANOVA: F2,28 =0.01, p=0.99) (Figure 13D) that developed later during the season.

A B

C D

Figure 13. Relationship between the number of open C. canadense flowers producing fruits and late season lateral branch characteristics during an in-field exclusion trial. Response variables included the number of closed flowers produced on lateral branches (A), the number of lateral branches developed (B), the sum of the length of lateral branches (C), and the total number of axillary nodes on lateral branches (D). The bars represent the mean while the error bars represent the standard error of the mean. All comparisons between bars on the same axis are insignificant. Sample size for zero fruits was 10 plants, one fruit was 11 plants, and two fruits was 10 plants.

3.4 Seed Studies

Following a two-week incubation period, final germination percentages of seeds from the various flower types were significantly different (One-way ANOVA: F4,10 =21.6, p<0.0001).

Terminal, infested terminal, and primary flowers had no significant difference in final germination percent, with germination percentages as high as 94.3±3.48, 95.0±2.89, and 98.3±1.67, respectively. Late season terminal cleistogamous and axillary cleistogamous flowers had

significantly lower final germination percentages than early season flowers, at 18.3±4.41 and

61.7±9.28 percent, respectively. The germination percentages of seeds from late season flowers were not significantly different from one another (Figure 14).

Figure 14. Germination percent of seeds from various C. canadense flower types after a two-week incubation period at 15OC. Bars represent arcsine of mean germination percentages and error bars represent standard error of the mean. Comparisons between bars using a Tukey test (a=0.05) are significantly different if labelled with different letters (Appendix A). Sample size was three plates with 20 seeds per plate for each flower type. T= terminal flower; 1= primary flower; TCL= terminal cleistogamous flower; CL= axillary cleistogamous flower

Of the imbibed seeds that had not germinated after the two-week incubation period, 61.1±5.55 percent of terminal cleistogamous seeds and 61.9±9.21 percent of axillary cleistogamous seeds had fungal contamination. Comparative analysis of the viability of terminal cleistogamous and axillary cleistogamous seeds following a tetrazolium chloride test revealed that seed embryos were 33.3±4.41 and 45.0±7.64 percent viable, respectively. The viabilities of the two seed types were not significantly different from one another (Unpaired t-test: df=2, p=0.27).

3.5 Infestation Rates by Mompha capella

Each of the sub-populations studied had a significantly different infestation rate of chasmogamous flowers (One-way ANOVA: F2,9 =18.5, p<0.001). Infestation at the CFB

Greenwood site was highest at 54.1±4.032 percent, followed by the Greenwood Military Aviation

Museum at 25.6±10.12 percent. No infested open flowers were recorded at the Highway 201 site.

Of fruits previously infested by M. capella, an average of 1.8±0.662 intact seeds remained after infestation (N=15).

Dissection of cleistogamous flowers revealed small unidentified larvae within developing flower buds (Figure 15A). Larvae were documented consuming both unfertilized ovules and developing seeds within the ovary of both terminal cleistogamous and axillary cleistogamous flowers (Figure 15B), though no petal cone formation was recorded in terminal cleistogamous flowers. Larvae then proceeded to consume all ovules and eventually ovary tissue (Figure 15C), chewing through the receptacle tissue to exit the empty flower bud (Figure 15D). Oftentimes, neighbouring axillary cleistogamous flowers along a branch were noted with signs of larval damage, but only the lowest flower on the stem housed a larva. Multiple larvae were never documented on the same branch and were found within cleistogamous flowers at all sampling sites.

Figure 15. Scanning electron micrographs of unidentified larvae infesting closed flowers of C. canadense. A. Partially dissected (forward-facing sepals, petals, and stamens removed) terminal cleistogamous flower with larvae present within closed bud. No damage to petals is evident. B. Larva consuming developing seeds within an axillary cleistogamous flower ovary. C. Larval growth surpassing ovary size. All developing seeds were consumed and ovary tissue was partially consumed. D. Larva exiting empty cleistogamous flower bud through the base of the closed flower. A = anther; C = petal; F = insect frass; K = sepal; L = larva; O = ovary; Ov = ovule; S = stigma. Scale bar = 500 µm.

4. Discussion

4.1 Seasonal Transition in Floral Morphology and Mating Strategy

While previous studies have characterized flower types of C. canadense into only two categories, quantitative and morphological comparisons of flower types in this study suggest a continuum of floral morphs exist between the first chasmogamous flower and obligate cleistogamous flowers that grow in a single season on each plant. This clarifies an earlier finding by White (2015), where variances in ovule and stamen number were high in chasmogamous flowers generalized into a single category on the basis of having petals. Here, I found quantitative distinctions between the reproductive characteristics of several open and closed flower types, as the number of stamens per flower, pollen grains per anther, total pollen per flower, and ovules per flower, gradually decreased in subsequent flower types over the course of the season (Figure 6). A flower’s degree of branching relative to the main stem may have an effect on the morphology and quantity of floral structures due to the architectural effect, whereby resource allocation to floral development differs based on a bud’s position relative to the axis of the plant (Ortiz et al. 2009).

Thus, a reduction in floral traits of closely associated flowers may be a result of diluted resource allocation to axillary flowers relative to flowers found at the terminus of either the stem, or lateral branches. Though not statistically significant, a trend of increased inviable pollen per anther in subsequent flower types was observed, as would be expected if fewer resources were allocated to flower development in later season flowers.

Moreover, the reduction in number of reproductive traits between the flower types was accompanied by a reduction in pollen-ovule ratios over the course of the season. This provided evidence that transitional flower types demonstrate a steady shift in mating strategy. Based on the mating system classifications outlined by Cruden (1977), the terminal and primary flowers fell within the range of other facultative xenogamous flowers; self-compatible yet primarily outcrossing. This conclusion was further supported in the field-based exclusion trial, where

terminal and primary flowers had a significant reduction in seed production when excluded from pollinators (Figure 12). Secondary flowers overlapped with typical pollen-ovule ratios classified for facultative autogamous flowers, which are predominantly self-pollinating prior to, or during, flower opening. Though morphologically similar to the first chasmogamous flower to bloom in a season, inasmuch as they may produce petals (Figure 15A), the pollen-ovule ratios of terminal cleistogamous flowers were not significantly different from obligate autogamous axillary cleistogamous flowers, indicating that they too are likely predominately selfing flowers.

Moreover, gradual changes in floral morphology coincided with the quantitative changes in reproductive traits as the mating strategy of flowers transitioned towards obligate autogamy.

Over the course of the season, filaments supporting the anthers became increasingly coiled and anthers assumed a paddle-like shape as flowers grew axillary relative to the stem and lateral branches. The close proximity of the anthers to the stigma during floral development increases the likeliness of autonomous self-pollination. Interestingly, the presence of petals and parallel orientation of the anthers with the stigma in terminal cleistogamous flowers did not correlate with the assumed autogamous mating strategy of the flower based on its pollen-ovule ratio.

Microscopic images provided evidence that terminal cleistogamous flowers can self-pollinate prior to and after anthesis and fall under the classification of pseudocleistogamous flowers described by Lord (1981). This classification was further supported in the field-based exclusion trial, where no significant difference in seed production occurred in terminal cleistogamous flowers when excluded from pollinators due to a reliable selfing mechanism.

Within the Cistaceae family, the majority of species develop only xenogamous flowers.

However, species within the Tuberaria, Helianthemum, and Lechea genera possess the ability to self-pollinate (Culley and Klooster 2007). Few studies have provided evidence that plants from the Cistaceae family that use dimorphic cleistogamy as a mating system have transitional flower types. Interestingly, Tuberaria guttata (L.) displays only one type of flower, which may represent a transition towards cleistogamy within this species. After anthesis, the open flowers are able to

self-pollinate as the sepals fold over the gynoecium once the petals are shed. Similar to the terminal cleistogamous flowers observed in the exclusion trial within this study, open flowers of

T. guttata reliably produce seeds in the absence of pollinators under a similar exclusion experiment (Herrera 1991).

4.2 Floral Contributions to Genetic Diversity and Fitness

Clarifying floral traits and dominant mating strategy of each flower type was a critical stepping stone for determining how each flower type may contributes to the genetic diversity of subsequent generations of plants. Based on the number of flowers produced per plant, the number of seeds produced per flower, and the germination percentage of seeds from each flower type, an estimate of the total number of genetically-recombined and inbred seeds produced per plant in a single season was calculated (Appendix B). With a three-fold increase, the sum of progeny derived from autogamous fertilization per plant greatly exceeded that from outcrossing flowers.

As relatively more inbred progeny enter subsequent generations, a reduction in genetic variability and an increase in inbreeding depression over time become possible threats to future C. canadense populations.

To quantify generational changes in plant vigor and survivability as a result of inbreeding, fitness measurements must be compared between plants derived from both outcrossed and inbred seeds. Lower seed germination percent and viability of seeds derived from self-fertilization compared to outcrossing provide preliminary evidence suggesting that seedling fitness may also be reduced. The percentage of terminal cleistogamous seeds that were viable based on the TTZ test was considerably higher than the percentage of seeds of the same flower type that germinated after two weeks. This discrepancy may be an indication that these seeds have a physiological dormancy mechanism or reduced vigor compared to earlier season seeds. In seedling studies of other dimorphic cleistogamous plant species such as Impatiens capensis (L.) Meerb. and

Triodanis perfoliata (L.) Nieuwl., germination success and seedling vigor were related and

reduced in cleistogamous seed compared to chasmogamous seed (Waller 1984; Gara and

Muenchow 1990). Future studies should focus on comparing measurements of vegetative and reproductive fitness between plants produced from outcrossing and selfing pollination strategies using standardized greenhouse trials. Recent initiatives to propagate plants in greenhouse conditions have not been successful (R. Browne, personal communication 2016), so attention should be directed towards improving ex situ growth techniques so that measurements of plants with a known pedigree can be conducted in the future.

4.3 Implications of Mompha capella on Future Populations

In addition to the innate ecological threat that the mating strategy of C. canadense poses on future populations, M. capella may be further exaggerating this effect. By consuming 98% of seeds produced in chasmogamous flowers, insect predation by M. capella increases the relative contribution of inbreed seeds to future generations in highly infested populations. Since 2014, the infestation rate of open flowers at the Canadian Forces Greenwood site has increased slightly from

50% to 54%, effectively doubling the number of inbred progeny relative to outcrossed in this population (White 2015). Though larva-induced seed production may be beneficial for terminal flowers in the absence of pollinators, the plant-insect interaction is unlikely to develop into a mutualism since few seeds are left unconsumed. Since infestation of open flowers was not recorded at the Highway 201 site, quantifying pollinator activity between sites may reveal the extent of gene flow between populations, helping to boost genetic diversity in infested populations. In addition, infestation by M. capella in other C. canadense populations within

Canada and the United States should be explored, as the distribution and widespread impact of M. capella is currently unknown.

The field-based exclusion trial was designed as a preliminary test to assess how pollinators or M. capella activity may indirectly affect the subsequent development of cleistogamous flowers later in the season. If low reproductive success of plants earlier during the

season increases their cleistogamous fruit output later in the season as a mechanism of reproductive assurance, it was hypothesized that infestation of flowers by M. capella would increase the relative contribution of inbred seeds to the population beyond simply consuming outcrossed seeds. Based on the homogeneity of cleistogamous flower counts and lateral branch measurements between plants with differing reproductive success of early season chasmogamous flowers, within-season phenotypic plasticity in cleistogamous flower production was not evident.

Due to the public access of the site where the exclusion trial took place, multiple bags were torn from plants and the final sample size was reduced by about 30%. Moreover, considerable variation in plant size and environmental conditions made comparisons between the small groups of plants non-representative. A controlled experiment with a greater degree of replication in a more private field site could illuminate potential differences in cleistogamous flower production in plants with developing chasmogamous flowers removed, left open to pollinators, or glued to simulate infestation by M. capella.

Infestation of cleistogamous flowers by larvae of an unidentified species at all study sites raises the question of whether M. capella infests both flower types within a single season. If this is the case, later season larvae appear to be reduced in size compared to larvae infesting open flower types, enabling them to fit within the smaller ovary. Moreover, the infestation of cleistogamous flowers by M. capella would indicate that larvae assume a different feeding behaviours between the different flower types. With no petals in axillary cleistogamous flowers to feed on, larvae bore into the ovary before or after self-pollination. Damage to multiple axillary cleistogamous flowers along a single branch suggests that larvae exit consumed flower buds and infest multiple adjacent flowers prior to pupating, contrary to what is documented in larger chasmogamous flowers where a single flower is parasitized per larva. In future studies, larvae should be collected from cleistogamous flowers and DNA barcoded to form a foundation for the study of M. capella larval morphs and lifecycle with C. canadense as a host. By improving our understanding of this plant- insect interaction, a novel insect specialist paradigm in pollination biology can be established.

5. References

Alexander, M.P. 1969. Differential staining of aborted and non-aborted pollen. Stain Technol.

44(3): 117-122.

Carlsen, T.M., Espeland, E.K., and Pavlik, B. 2001. Reproductive ecology and persistence of an

endangered plant. Biodivers. Conserv. 11(7): 1247-1268. doi:

10.1023/A:1016066618824

Catling, P.M. and Carbyn S. 2005. Invasive Scots Pine, Pinus sylvestris, replacing Corema,

Corema conradii, heathland in Annapolis Valley, Nova Scotia. Can. Field Nat. 119(2):

237-244. doi: 10.22621/cfn.v119i2.112

Cortés-Palomec, A.C., McCauley, R.A., and Ballard, H.E. 2006. Population genetic structure in

temperate and tropical species of Viola (Violaceae) with a mixed breeding system. Int. J.

Plant Sci. 167(3): 503-512. doi: 10.1086/502665

Cruden, R.W. 1977. Pollen-ovule ratios: a conservative indicator of breeding systems in flowering

plants. Evol. 31(1): 32-46. doi: 10.2307/2407542

Culley, T.M. and Klooster, M.R. 2007. The cleistogamous breeding system: a review of its

frequency, evolution, and ecology in angiosperms. Bot. Rev. 73(1): 1-30. doi:

10.1663/0006-8101(2007)73[1:TCBSAR]2.0.CO;2

Dart, S., and Eckert, C.G. 2014. Variation in pollen limitation and floral parasitism across a

mating system transition in a Pacific coastal dune plant: evolutionary causes or ecological

consequences. Ann. Bot. 115(2): 315-326. doi: 10.1093/aob/mcu243

Dostálek, T., Münzbergová, Z., and Plačková, I. 2009. Genetic diversity and its effects on the

fitness in an endangered plant species, Dracocephalum austriacum L. Conserv. Genet.

11(3): 773-783. doi: 10.1007/s10592-009-9879-z

Gara, B. and Muenchow, G. 1990. Chasmogamy/cleistogamy in Triodanis perfoliata

(Campanulaceae): some CH/CL comparisons in fitness parameters. Am. J. Bot. 77(1): 1-6.

doi: 10.2307/2444786

Herrera, J. 1991. Flower variation and breeding systems in the Cistaceae. Pl. Syst. Evol. 179(3):

245-255

Huebner, C.D. 2011. Seed mass, viability, and germination of Japanese stiltgrass (Microstegium

vimineum) under variable light and moisture conditions. Invas. Plant Sci. Mana. 4(1): 274-

283. doi: 10.1614/IPSM-D-10-00090.1

Kearns, C. A., and Inouye, D. W. 1993. Techniques for pollination biologists. University Press of

Colorado.

Lord, E.M. 1980. Intra-inflorescence variability in pollen/ovule ratios in the cleistogamous species

Lamium amplexicaule (Labiatae). Am. J. Bot. 67(4): 529-533

Lord, E.M. 1981. Cleistogamy: a tool for the study of floral morphogenesis, function and

evolution. Bot. Rev. 47(4): 421-449. doi: 10.1007/BF0286053

Lu, Y. 2002. Why is cleistogamy a selected reproductive strategy in Impatiens capensis

(Balsaminaceae)? Biol. J. Linnean. Soc. 75(4) 543-553. doi: 10.1046/j.1095-

8312.2002.00039.x

McCall, A.C. and Irwin, R.E. 2006. Florivory: the intersection of pollination and herbivory. Ecol.

Lett. 9(12): 1351-1365. doi: 10.1111/j.1461-0248.2006.00975.x

NatureServe. 2015. Helianthemum canadense [online]. Available from

http://www.natureserve.org [accessed June 8, 2016]

Newell, R. 2007. Nova Scotia provincial status report on Rockrose (Canada Frostweed)

Helianthemum canadense (L.) Michx, Wolfville N.S. Publ.

Ortiz, P.L., Berjaro, R., Talavera, M., and Arista, M. 2009. The role of resources and architecture

in modeling floral variability for the monoecious amphicarpic Emex spinosa

(Polygonaceae). Am. J. Bot. 96(11): 2062-2073. doi: 10.3732/ajb.0800417

Peñas, J., Barrios, S., Bobo-Pinilla, J., Lorite, J., Martínez-Ortega, M.M. 2016. Designing

conservation strategies to preserve the genetic diversity of Astragalus edulis Bunge, an

endangered species from western Mediterranean region. PeerJ 4(1): e1474. doi:

10.7717/peerj.1474

Penet, L., Collin, C.L., and Ashman, T.L. 2008. Florivory increases selfing: an experimental study

in the wild strawberry, Fragaria virginiana. Plant Biol. 11(1): 38-45. doi: 10.1111/j.1438-

8677.2008.00141.x

Pérez-García, F. and González-Benito, M.E. 2006. Seed germination of five Helianthemum

species: effect of temperature and presowing treatments. J. Arid Environ. 65(4): 688-693.

doi: 10.1016/j.jaridenv.2005.10.008.

Primack, R.B. and Kang, H. 1989. Measuring fitness and natural selection in wild plant

populations. Annu. Rev. Ecol. Evol. Syst. 20: 367-396. doi:

10.1146/annurev.es.20.110189.002055

Rodríguez-Pérez, J. 2005. Breeding system, flower visitors and seedling survival of two

endangered species of Helianthemum (Cistaceae). Ann. Bot. 95(7): 1229-1236. doi:

10.1093/aob/mci137

Ruan, C. and da Silva, J.A.T. 2012. Evolutionary assurance vs mixed mating. Crit. Rev. Plant Sci.

31(4): 290-302. doi: 10.1080/07352689.2011.645442

Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J.

Ultrastruct. Res. 26(1): 31-43. doi: 10.1016/S0022-5320(69)90033-1

USDA National Resources Conservation Service. 2017. The PLANTS Database. National Plant

Data Team, Greensboro, NC. Available from

https://plants.usda.gov/core/profile?symbol=HECA3 [accessed January 2017]

Verma, P. and Majee, M. 2013. Seed germination and viability test in tetrazolium (TZ) assay. Bio-

protoc. 3(17): e884. doi: 10.21769/BioProtoc.884

Waller, D. 1984. Differences in fitness between seedlings derived from cleistogamous and

chasmogamous flowers in Impatiens capensis. Evol. 38(2): 427-440.

White, S.N. 2015. A tale of two flowers: a comparative analysis of chasmogamous and

cleistogamous flower development in Helianthemum canadense (L.) Michx. B.Sc.H.

thesis, Department of Biology, Acadia University, Wolfville, N.S.

White., S.N., Stewart, D.T., Hillier, N.K., and Evans, R.C. 2016. Identification of Mompha

capella Busck, a microlepidopteran predator of an endangered plant, Crocanthemum

canadense (L.) Britton, in Nova Scotia. Northeast. Nat. 23(2):211-218. doi:

http://dx.doi.org/10.1656/045.023.0203

Winn, A.A., and Moriuchi, K.S. 2009. The maintenance of mixed mating by cleistogamy in the

perennial violet Viola septemloba (Violaceae). Am. J. Bot. 96(11): 2074-2079. doi:

10.3732/ajb.0900048

Yorke, A. 2007. Genetic variation and phytogeography of Nova Scotia’s isolated populations of

Canada Frostweed (Helianthemum canadense (L.) Michx.) as revealed with AFLP

Markers. B.Sc.H. thesis, Department of Biology, Acadia University, Wolfville, N.S.

Yorke, A.F., Mockford, S., and Evans, R. 2011. Canada frostweed (Helianthemum canadense (L.)

Michx.; Cistaceae) at the northeastern limit of its range: implications for conservation.

Bot. 89(2): 83-89. doi: 10.1139/B10-08

Appendix A

Table 1. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the mean number of stamens between C. canadense flower types at a=0.05.

Flowers compared Mean 1 Mean 2 N1 N2 P Value T vs. 1 32.8 22.93 94 97 <0.0001 T vs. 2 32.8 12.65 94 43 <0.0001 T vs. TCL 32.8 9.852 94 88 <0.0001 T vs. CL 32.8 3.877 94 163 <0.0001 1 vs. 2 22.93 12.65 97 43 <0.0001 1 vs. TCL 22.93 9.852 97 88 <0.0001 1 vs. CL 22.93 3.877 97 163 <0.0001 2 vs. TCL 12.65 9.852 43 88 0.0914 2 vs. CL 12.65 3.877 43 163 <0.0001 TCL vs. CL 9.852 3.877 88 163 <0.0001

Table 2. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the mean number of pollen grains per anther between C. canadense flower types at a=0.05. N=10

Flowers compared Mean 1 Mean 2 P Value T vs. 1 738.3 737.4 >0.9999 T vs. 2 738.3 528.5 0.0035 T vs. TCL 738.3 115.8 <0.0001 T vs. CL 738.3 90.9 <0.0001

1 vs. 2 737.4 528.5 0.0037 1 vs. TCL 737.4 115.8 <0.0001 1 vs. CL 737.4 90.9 <0.0001 2 vs. TCL 528.5 115.8 <0.0001 2 vs. CL 528.5 90.9 <0.0001 TCL vs. CL 115.8 90.9 0.9909

Table 3. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the estimated mean number of pollen grains per flower between C. canadense flower types at a=0.05. N=10.

Flowers compared Mean 1 Mean 2 P Value T vs. 1 24216 16909 <0.0001 T vs. 2 24216 6686 <0.0001 T vs. TCL 24216 1141 <0.0001 T vs. CL 24216 351.8 <0.0001

1 vs. 2 16909 6686 <0.0001 1 vs. TCL 16909 1141 <0.0001 1 vs. CL 16909 351.8 <0.0001 2 vs. TCL 6686 1141 <0.01 2 vs. CL 6686 351.8 <0.001 TCL vs. CL 1141 351.8 0.98

Table 4. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the mean number of ovules between C. canadense flower types at a=0.05.

Flowers compared Mean 1 Mean 2 N1 N2 P Value T vs. 1 53.19 40.85 178 125 <0.0001 T vs. 2 53.19 34.44 178 36 <0.0001 T vs. TCL 53.19 20.12 178 97 <0.0001 T vs. CL 53.19 6.916 178 166 <0.0001

1 vs. 2 40.85 34.44 125 36 <0.05 1 vs. TCL 40.85 20.12 125 97 <0.0001 1 vs. CL 40.85 6.916 125 166 <0.0001 2 vs. TCL 34.44 20.12 36 97 <0.0001 2 vs. CL 34.44 6.916 36 166 <0.0001 TCL vs. CL 20.12 6.916 97 166 <0.0001

Table 5. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the mean number of seeds between C. canadense flower types at a=0.05.

Flowers compared Mean 1 Mean 2 n1 n2 P Value T vs. 1 40.84 28 99 64 <0.0001 T vs. 2 40.84 20.69 99 13 <0.0001 T vs. TCL 40.84 16.37 99 68 <0.0001 T vs. CL 40.84 4.566 99 113 <0.0001 1 vs. 2 28 20.69 64 13 0.14 1 vs. TCL 28 16.37 64 68 <0.0001

1 vs. CL 28 4.566 64 113 <0.0001 2 vs. TCL 20.69 16.37 13 68 0.64 2 vs. CL 20.69 4.566 13 113 <0.0001 TCL vs. CL 16.37 4.566 68 113 <0.0001

Table 6. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in the estimated mean inviable-total pollen grains per anther of seasonal flower types of C. canadense. The means displayed arc-sine transformed inviable- total pollen grain ratios as analyzed in a one-way ANOVA. a=0.05. N=10

Flowers compared Mean 1 Mean 2 P Value T vs. 1 0.02253 0.02788 >0.9999 T vs. 2 0.02253 0.07526 0.81 T vs. TCL 0.02253 0.08033 0.76 T vs. CL 0.02253 0.1174 0.30

1 vs. 2 0.02788 0.07526 0.86 1 vs. TCL 0.02788 0.08033 0.82 1 vs. CL 0.02788 0.1174 0.36 2 vs. TCL 0.07526 0.08033 >0.9999 2 vs. CL 0.07526 0.1174 0.91 TCL vs. CL 0.08033 0.1174 0.94

Table 7. Data under comparison in a post hoc Tukey’s multiple comparisons test to assess differences in seed production between flower types of C. canadense treated under open (un-bagged) and closed (bagged) pollinator conditions. Means displayed are arcsine- transformed seed-ovule ratio per flower as analyzed in a two-way ANOVA at a=0.05.

Flowers compared Mean 1 Mean 2 n1 n2 P Value Un-bagged:T vs. Un-bagged:1 0.8685 1.1 15 12 0.39 Un-bagged:T vs. Un-bagged:TCL 0.8685 1.228 15 11 <0.05 Un-bagged:T vs. Bagged:T 0.8685 0.05734 15 17 <0.0001 Un-bagged:T vs. Bagged:1 0.8685 0.3342 15 15 <0.001 Un-bagged:T vs. Bagged:TCL 0.8685 1.308 15 17 <0.01 Un-bagged:1 vs. Un-bagged:TCL 1.1 1.228 12 11 0.92 Un-bagged:1 vs. Bagged:T 1.1 0.05734 12 17 <0.0001 Un-bagged:1 vs. Bagged:1 1.1 0.3342 12 15 <0.0001 Un-bagged:1 vs. Bagged:TCL 1.1 1.308 12 17 0.48 Un-bagged:TCL vs. Bagged:T 1.228 0.05734 11 17 <0.0001 Un-bagged:TCL vs. Bagged:1 1.228 0.3342 11 15 <0.0001 Un-bagged:TCL vs. Bagged:TCL 1.228 1.308 11 17 0.99 Bagged:T vs. Bagged:1 0.05734 0.3342 17 15 0.13 Bagged:T vs. Bagged:TCL 0.05734 1.308 17 17 <0.0001 Bagged:1 vs. Bagged:TCL 0.3342 1.308 15 17 <0.0001

Table 8. Data under comparison in a post hock Tukey’s multiple comparisons test at a=0.05 to assess differences in seed germination percentage between flower types of C. canadense after a two-week incubation period. N=3.

Flowers compared Mean 1 Mean 2 P Value T vs. T infested 1.3 1.315 >0.9999 T vs. 1 1.3 1.465 0.85 T vs. TCL 1.3 0.1847 <0.001 T vs. CL 1.3 0.6778 <0.05 T infested vs. 1 1.315 1.465 0.89 T infested vs. TCL 1.315 0.1847 <0.001 T infested vs. CL 1.315 0.6778 <0.05 1 vs. TCL 1.465 0.1847 <0.0001 1 vs. CL 1.465 0.6778 <0.01 TCL vs. CL 0.1847 0.6778 0.080

Appendix B

Table 9. Summary of estimated contributions of flowers per plant to the genetic diversity of subsequent generations based on mean number of flowers per plant, seeds produced per flower, and germination % of seeds for each flower type.

C Estimated Dominant A B Mean ± SEM Reproductive Total Mating Flower Mean ± SEM Mean ± SEM % Germination Potential Reproductive Strategy Type Flowers/Plant Seeds/Flower (N=3) (A x B x C) Potential

T 1.0±0.00 (N=24) 40.8±1.21(N=99) 94.3±3.48 38.5 Outcrossing 1 0.7±0.14 (N=24) 28.0±1.85 (N=64) 98.3±1.67 19.3 58.6 2 0.04±0.041 (N=24) 20.7±4.62 (N=13) N.A.* 0.8

TCL 5.0±0.36 (N=20) 16.4±1.14 (N=68) 18.3±4.41 15.0 Inbreeding 189.3 CL 61.4±7.26 (N=19) 4.6±0.23 (N=113) 61.7±9.28 174.3

* No seeds were collected in the field to quantify germination percentage.