Abstract an Anatomical Comparison of Wild Type

ABSTRACT

AN ANATOMICAL COMPARISON OF WILD TYPE AND HOMEOTIC MUTANT FLOWERS OF CLARKIA TEMBLORIENSIS

by Chelsea Elizabeth Obrebski

Flowers consist of four whorls of organs (sepals, petals, stamens and carpels), each expressing unique characteristics. In floral homeotic mutants, organs develop abnormally, and floral organs of one whorl express characteristics of another whorl. In the homeotic mutant crinkle petal (cp) of Clarkia tembloriensis, previous investigations demonstrated that cp petal is a hybrid organ combining characteristics of sepals and petals. This investigation extends those studies with a comparative anatomical study of trichomes, stomata, and marginal cells in sepals, WT petals, and cp petals. The aim was to determine if cp petals exhibit a hybrid anatomy of sepals and WT petals. Floral spikes (containing buds 0.6mm, 1.0mm, 1.6mm, and 3.0mm in length), buds at pre-anthesis, and flowers 1-day post anthesis were collected and prepared for SEM or light microscopy. Trichomes and stomata were counted and marginal cells were observed and described in flowers harvested one day post-anthesis. Developmental observations of trichomes, guard cells, and marginal cells were also made in all pre-anthesis bud sizes. The results of this anatomical study demonstrated that cp petals of C. tembloriensis exhibit characteristics of both sepals and WT petals. This provides further support for the hybrid nature of cp petals in Clarkia tembloriensis.

AN ANATOMICAL COMPARISON OF WILD TYPE AND HOMEOTIC MUTANT FLOWERS OF CLARKIA TEMBLORIENSIS

A Thesis

Submitted to the

Faculty of Miami University

in partial fulfillment of

the requirements for the degree of

Master of Science

Chelsea E. Obrebski

Miami University

Oxford, Ohio

2019

Advisor: Dr. Nancy Smith-Huerta

Reader: Dr. Alfredo Huerta

Reader: Dr. Carolyn Keiffer

This Thesis titled

AN ANATOMICAL COMPARISON OF WILD TYPE AND HOMEOTIC MUTANT FLOWERS OF CLARKIA TEMBLORIENSIS

Chelsea E. Obrebski

has been approved for publication by

The College of Arts and Science

and

Department of Biology

______Dr. Nancy Smith-Huerta

______Dr. Alfredo Huerta

______Dr. Carolyn Keiffer

Table of Contents Introduction ...... 1 Subject of This Investigation ...... 1 General Models of Floral Anatomy and Development ...... 2 Homeotic Mutants That Do Not Fit Current Flower Development Models ...... 3 Origins of Homeotic Mutants ...... 3 Anatomical Features of Floral Organs...... 4 Goals of the Current Study...... 5 Materials and Methods ...... 6 Sample Seed Collection and Growth Chamber Culture ...... 6 Sample Preparation and Viewing ...... 6 Light Microscopy ...... 6 Scanning Electron Microscopy ...... 6 Analysis ...... 7 Results ...... 8 Trichomes on Sepals, WT petal, and cp Petals...... 8 Guard Cells on sepals, WT petals, and cp petals ...... 10 Organ Margins, Redifferentiated Epidermal Cells and Postgenital Fusion in Sepals, WT Petals, and cp Petals ...... 11 Mature sepals, WT petals and cp petals...... 11 sepals...... 11 WT petal...... 11 cp petals...... 12 Organ Development ...... 13 Sepal...... 13 WT petal and cp petal development...... 14 Discussion...... 16 The Hybrid Nature of the cp Organ ...... 16 Epidermal cells investigated in this study ...... 16 Trichomes...... 16 Guard cells...... 18 Redifferentiated epidermal cells (REC) in the marginal region of floral organs and their role in post genital fusion (PGF)...... 19

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Other Single Whorl B-type Mutants in the Onagraceae ...... 23 The Future of C. tembloriensis in the Wake of Climate Change ...... 23 Homeotic Mutants as Model Systems to Study Floral Evolution...... 25 Future Studies ...... 25 Conclusions ...... 27 Reference ...... 59 Appendix 1 ...... 65 Trichome Elemental Analysis ...... 65

List of Tables Table 1 Average Count of Trichomes on the Adaxial and Abaxial Side of Mature Sepal, WT Petal, and cp Petal at the Apex, Middle of the Sepal/Base of the Limb in Petals, and Base of Sepal/Claw of Petals

Table 2 Average Total Counts of Stomata on the Adaxial and Abaxial Sides of the Sepal, WT Petal, and cp Petal on the Apex, Middle/Base Limb, and Base/Claw Positions of the Organs

Table 3 Spot Elemental Analysis of the Surface of the Falcate Trichome

List of Figures Figure 1. Drawing of C. tembloriensis sepal, WT petal, and cp petal. Figure 2. SEM images of floral organ trichomes. Figure 3. Sepals of developing floral bud prior to PGF fusion. Figure 4. Trichomes on WT petal and cp petal in 3.0mm buds. Figure 5. Image of a stomata surrounded by specialized epidermal cells called guard cells on a C. tembloriensis sepal. Figure 6. Light micrographs continuing basipetally through the bud with a partially fused set of sepals. Figure 7. Light sections of sepal margins in mature buds 1-day pre- anthesis. Figure 8. Side view of mature sepal tip after anthesis. Figure 9. SEM images of sepal edge after anthesis. Figure 10. Margins and edge of WT petal after anthesis. Figure 11. SEM images of WT petal edges post anthesis at the apex. Figure 12. Tip of anthesis WT petal lobe with stomata on the margin. Figure 13. SEM images of WT petal margin after anthesis progressing acropetally. Figure 14. Light images of one day pre-anthesis cp petal edges and PGF. Figure 15. Various SEM images of cp petal post anthesis at the apex. Figure 16. SEM image of cp petal lobe post anthesis near the apex. Figure 17. Light image of cp petal lobe post anthesis near the apex. Figure 18. SEM images of cp petal edges post anthesis progressing acropetally. Figure 19. Floral meristem showing the progression of REC on the developing sepals. Figure 20. Closing floral buds. Figure 21. Light section of the apical region of floral bud. Figure 22. Light sections through a single bud with a partially fused set of sepals. Figure 23. SEM images of young petals in 1.0mm floral buds. Figure 24. SEM image of petals in 1.6mm floral buds. Figure 25. Light sections of petals in 1.6mm floral buds.

Figure 26. Light sections of petals in 1.0mm floral buds. Figure 27. Light sections of petals in 3.0mm floral buds. Figure 28. SEM image of petals in 3.0mm floral buds. Figure 29. cp petal flowers during anthesis illustrating the different degrees of obstruction by the sepal hood. Figure 30. Conical and falcate trichome elemental analysis.

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Acknowledgements Dr. Nancy Smith-Huerta, Dr. Alfredo Huerta, Dr. Carolyn Keiffer, Dr. Dan Gladish, Richard Edelmann, Matt Duley, Mary Carol Johantgen (deceased), Miami University Biology Department, and Miami University CAMI facility.

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Introduction

The wildflower Clarkia tembloriensis grows in discrete populations in the arid foothills of the Temblor range of California (Vasek, 1976). The flowers of the majority of the plants growing in these populations display a form and function typical of plants in the family Onagraceae. The flowers are 4-merous, (have sepals petals stamens and gynoecium) with an inferior ovary. In certain populations some of the plants deviate from this normal condition, and the petals are replaced with organs that resemble sepals (Vasek, 1966 and Smith-Huerta, 1992). Previous investigations of these abnormal flowers have demonstrated that the trait is determined by a single recessive gene named CRINKLE PETAL (cp) (Vasek, 1966 and Vasek & Harding, 1976). In addition, previous developmental studies have demonstrated that the cp organ is not a simple replacement of a petal with a sepal, but instead the cp organ combines characteristics of WT petals and sepals (Smith-Huerta, 1996). The current study further investigates the nature of this hybrid cp organ, with an anatomical comparison of wild-type (WT) and cp mutant flowers of Clarkia tembloriensis. More specifically the study focuses on trichomes, guard cells, and the marginal cells of sepals, WT petals and cp organs. Subject of This Investigation The subject of this investigation is Clarkia tembloriensis, a flowering plant in the family Onagraceae and one of 44 species in the genus Clarkia (Lewis and Lewis 1955, Sytsma & Smith, 1992, and Vasek, 2008). A native wildflower from California, this species inhabits arid grassland and shrub woodland in regions of the Inner Coastal Range (Vasek, 1977, 2008). Clarkia tembloriensis ranges in California from Alameda county in the North, to Kern county and San Luis Obispo in the south (Vasek, 1977, 2008). The species is divided into two subspecies based on flower size. Clarkia tembloriensis subsp. longistyla has relatively large flowers and has a primarily outcrossing breeding system (Vasek, 2008). Clarkia tembloriensis subsp. tembloriensis has small flowers, some expressing outcrossing and others a selfing breeding system (Vasek, 2008). Within certain populations of the small selfing C. tembloriensis subsp. tembloriensis, plants may express one of two flower phenotypes. Some plants have normal flowers (WT) and other plants have flowers with small crinkled petal (cp) flowers (Vasek, 1964, 2008). Clarkia flowers are 4-merous, with 4 sepals, 4 petals, 8 stamens (4 long and 4 short) and a gynoecium of 4 fused carpels. The ovary is inferior with a short hypanthium (Lewis and Lewis 1955, Sherry & Lord, 1996, 2000 and Smith-Huerta, 1992). The sepals of normal WT flowers of C. tembloriensis are elongate in shape and are greenish- pink in color (Smith-Huerta, 1992). There is a small maroon spot on the sepal at the point of attachment to the floral tube (Smith-Huerta, 1992). At anthesis, the sepals are recurved and reflex back, away from the rest of the floral whorls (Leong et al., 2001). The petals of WT flowers consist of an expanded deltoid-shaped limb, attached to an elongated slender claw (Leong et al., 2001). The claw attaches to the floral tube and the petal is mostly flat along its entirety with some undulations along the perimeter of the limb (Smith-Huerta, 1992). The background color of WT petals is pink. Petals are polymorphic for the presence of maroon spots occurring at the base of the limb and the claw (Smith-Huerta, 1992). More specifically, the flowers of some plants possess spots while the flowers of other plants do not.

The stamens of Clarkia flowers consist of long stamens alternating with the petals and short stamens opposite the petals (Sherry & Lord, 2000). The anthers of the large stamens are lavender to red colored, while the anthers of small stamens are similar in color, but much paler (Lewis, 2012). Carpels have a long style with four stigma lobes (Sherry & Lord, 1996). Ovaries in the smaller selfing flowers of C. tembloriensis are slightly longer than the ovaries of the larger flowers of outcrossers (Sherry & Lord, 1996). These ovaries have eight grooves and are pubescent similar to the sepals (Lewis, 2012). The styles of the small selfing flowers are shorter than the styles of the larger outcrossing flowers (Sherry & Lord, 1996). The cp mutant flowers that occur in certain natural populations, have normal sepals, stamens, and carpels similar to those of WT flowers (Smith-Huerta,1992, 1996). The cp flower differs from the WT flower in the shape and coloration of the petals (Leong et al. 2001 and Smith- Huerta,1992, 1996). Instead of the elongated claw and expanded limb of WT flowers, the petals of cp flowers resemble sepals in both shape and coloration (Vasek, 1966 and Smith-Huerta, 1992, 1996). Although cp petals are sometimes referred to as sepaloid (Smith-Huerta,1992, 1996 and Vasek, 2008), past research revealed that the cp petal expresses characteristics of both the WT petals and WT sepals (Leong et al., 2001 and Smith-Huerta,1992, 1996). This implies that the cp petal is a hybrid between two organs rather than a complete conversion from one organ to another (Ford & Gottlieb, 1992 and Smith-Huerta,1992, 1996). Evidence for the hybrid nature of the cp petal comes from the appearance of the cp petal, which resembles elongated, linear- lanceolate, greenish pink sepals with a small maroon spot present or absent at the point of attachment (Leong et al., 2001 and Smith-Huerta, 1992). The cp petal characteristics are similar to WT petals in that they remain erect rather that reflexed back at anthesis, and the time and place of organ initiation within the flower (Leong et al., 2001 and Smith-Huerta, 1992). One characteristic unique to cp petals is their wrinkled appearance at anthesis (Leong et al. 2001 and Smith-Huerta, 1992). General Models of Floral Anatomy and Development Flowers are typically composed of four whorls, each consisting of a single floral organ (Theißen & Saedler, 2001). Whorl one organs are sepals, whorl two are petals, three are stamens, and four are carpels (Hintz et al., 2006 and Theißen & Saedler, 2001). Not all plants and their flowers develop normally. Mutations may occur that disrupt normal reproductive or vegetative organ development (Hintz et al., 2006). Some of these mutations cause an organ or tissue to develop partial (Halfter et al., 1994 and Krizek et al., 1996) or complete characteristics of another type of tissue or organ (Benedito et al., na., and Theißen et al., 2016, Smith-Huerta, 1992,). This type of mutation is called a homeotic mutation and the most well-known homeotic mutations in plants are those found in flowers (Hintz et al., 2006). The study of these, and other types of mutants, has led to the elaboration of models to explain how normal flowers develop (Aceto & Gaudio, 2011, Halfter et al., 1994, Hintz et al., 2006, and Zahn et al., 2005). One current floral developmental model is the ABCDE model, which states that the identity of organs in each whorl is controlled by different overlapping factors, or groups of homeotic genes (Benedito et al., 2003 and Murai, 2013). According to the model, each factor denoted as A, B, C, D, or E, acts alone or in conjunction with other overlapping factors to produce a certain floral organ type (Benedito et al., 2003, Halfter et al., 1994, Leong et al., 2001, and Zahn et al., 2003). For example, the expression of factor A+E results in the development of sepals, A+B+E results in petals, B+C+E generates stamens, C+E generates carpels, and lastly

C+D+E produces ovules (Leong et al., 2001, Murai, 2013, Theißen & Saedler, 2001, Theißen et al., 2016, and Zahn et al., 2005). Expression of factor E throughout the floral apex in conjunction with the other factors is required for the floral organs to develop correctly (Aceto & Gaudio, 2011, Hintz et al., 2006, and Murai, 2013). When floral organ mutations occur, they can be categorized into A-type, B-type, and C-type, depending upon what organs are affected (Hintz et al., 2006, Theißen & Saedler, 2001, and Theißen et al., 2016). With the recent growth of molecular analysis, discovery of the MADS-box gene family allowed for the development of the Quartet model of floral development (Zahn et al., 2005). This model was built on, and extended the ABCDE model to include recent advances in molecular analysis of floral development (Aceto & Gaudio, 2011, Hintz et al., 2006, Theißen et al., 2016, and Zahn et al., 2005). The quartet model explains that the identity of the four floral organs is determined by four quartets of MADS-box proteins that work together as transition factors to regulate the specific downstream gene patterns for the initiation and development of the floral organs (Aceto & Gaudio, 2011, Hase et al., 2000, Theißen, & Saedler, 2001, Theißen et al., 2016, and Zahn et al., 2005). The study of homeotic mutants, in general, has been invaluable for understanding the evolution and development of morphologic diversity in flowers, as these mutations allow for diversity to develop through the modification of existing MADS-box genes and floral structures (Hintz et al., 2006, Murai, 2013, and Smith-Huerta, 1992). Although extensive studies have been conducted on this topic, many questions still remain to be answered concerning the molecular mechanisms that drive floral development (Theißen et al., 2016). In addition, there are floral homeotic mutants that do not entirely follow current models and these mutants have not been extensively studied, such as the fbp7/11 petunia mutant (Mach, 2012), gp petunia mutant (van der Krol & Chua, 1993), and bicalyx mutant of C. concinna (Ford & Gottlieb, 1992 and Hintz et al., 2006). Homeotic Mutants That Do Not Fit Current Flower Development Models The cp petal mutant of C. tembloriensis and the bicalyx mutant of Clarkia concinna are two of several unique homeotic mutations that do not fit completely with current floral development models in that only one whorl (petals) is affected by the mutation (Ford & Gottlieb, 1992, and Smith-Huerta, 1992). Most other mutants, like those in Arabidopsis and Antirrhinum, exhibit two affected whorls (Coen & Meyerowitz, 1991, van der Krol & Chua, 1993 and Yanofsky et al., 1990). Typically, in B-type mutants, both petals and stamens are affected, resulting in infertility of the stamens as they either become replaced by another organ (Tsuchimoto et al., 2000), are absent, or fused to the gynoecium (Smith-Huerta, 1992). The cp petal and the bicalyx mutant stamens, however, maintain fertility (Ford & Gottlieb, 1992 and Smith-Huerta & Huerta, 2015). This fact allows mutant plants to grow and persist in natural populations (Ford & Gottlieb, 1992 and Smith-Huerta & Huerta, 2015). Though there is evidence that the cp petal has reduced pollen production, compared to WT petal, in conjunction with lower outcrossing rates (0.03), this does not lead to reduced seed production, as flowers with cp petals have slightly higher seed set than flowers with WT petals (Smith-Huerta & Huerta, 2015 and Vasek, 2008). The petunia gp mutant was also observed by van der Krol and Chua (1993) to maintain its fertility, despite some homeotic effects on the stamens including petaloid cells found growing on the filaments of the stamens. Origins of Homeotic Mutants

Most of the homeotic mutants used to develop models of floral development were selectively bred or created in the laboratory using mutagenic treatments, such as ethyl methane sulfonate (Yanofsky et al., 1990), x-ray, γ-ray, heavy-ion beams with high-linear energy transfer radiation (Hase et al., 2000), the activation of transposons, (Carpenter, R., & Coen, 1990, van der Krol & Chua, 1993), or by T-DNA insertion mutation (Yanofsky et al., 1990). Some laboratory- induced homeotic mutations are temperature sensitive and will decrease in their degree of expression or completely loose expression and appear more like WT flowers under a different temperature regime. For example, Gómez et al. (1999) demonstrated that development of sl/sl mutant tomato flowers showed a complete reversal in mutant petal phenotype expression and more than 15% reduction in expression at temperatures lower (17 C day/ 7 C night) than standard temperature (26 C day/ 20 C night) and optimum greenhouse environmental conditions (spring 28 C max. day/ 12 C night min., winter 18 C max. day/ 6 C night min.). The subject of this study (cp mutant of C. tembloriensis) was not created in a laboratory but was discovered by Vasek (1964 and 1966) growing naturally in California populations. This discovery spurred other studies including the significance of the cp mutant to reproduction in natural populations (Smith-Huerta and Huerta 2015), development of the cp mutant (Smith- Huerta, 1992, 1996), and a molecular genetic linkage map of the cp trait (Leong and Smith- Huerta 2001). The cp mutant is one of a few naturally occurring homeotic mutants found sustained in nature, sometimes at high frequencies, and even has been found genetically fixed in one population around Cholame California (Smith-Huerta, 1992, 1996 and Vasek, 2008). Another natural mutant of Clarkia concinna (bicalyx) was found to be stable over a four-year observation period in California population (Ford & Gottlieb, 1992 and Hintz et al., 2006). The genus Clarkia is not unique in the possession of naturally occurring B-type floral homeotic mutants in the family. Homeotic sepaloid petal mutants have been observed in two other genera within the Onagraceae, Epilobium and Oenothera (Ford & Gottlieb, 1992, Leong et al., 2001, and Renner, 1959). The mutation in these genera within the family Onagraceae is conditioned by a single recessive gene (Ford & Gottlieb, 1992, Renner, 1959, and Vasek, 1966). Anatomical Features of Floral Organs. The general anatomy of sepals and petals has been described in several investigations (Endress, 1996 and Weberling, 1989). In general, sepals possess thick spongy or palisade parenchyma with large rounded cells with thickened cell walls and many chloroplasts (Endress, 1996 and van der Krol & Chua, 1993). However, despite their leafy appearance, sepals are reduced in size compared to leaves (Endress, 1996). Compared to petals, sepals have smaller intercellular air spaces (Endress, 1996 and van der Krol & Chua, 1993). Petals, on the other hand, are thinner than sepals with simple small mesophyll cells that are either rounded or branched irregularly (Endress, 1996 and van der Krol & Chua, 1993, and Weberling, 1989). Most petal cells also have large extensive intercellular air spaces that lack chloroplasts (Endress, 1996, van der Krol & Chua, 1993, and Weberling, 1989). The epidermis of floral organs consists of specific cell types including trichomes, guard cells, and pavement epidermal cells (Glover, 2000). Pavement epidermal cells are jigsaw puzzle shaped epidermal cells that are morphologically unspecialized and make up most of the epidermal layer of plants (Glover, 2000). They do not perform any gas exchange or have any protrusions on their surface and mainly serves the purpose of ensuring correct spacing out of

more morphologically specialized cells and protecting the subepidermal tissue layers (Glover, 2000). The presence or absence, and structural characteristics of these cell types can vary considerably between floral organs, as Cildir et al., (2012) has demonstrated in the petals and sepals of several different species in the Lathyrus taxa. For example, sepals typically are characterized as having numerous trichomes on both the adaxial and abaxial surfaces (Endress, 1996, Gómez et al. 1999, and van der Krol and Chua, 1993), while petals typically are characterized as having few to no trichomes on either surface (van der Krol & Chua, 1993 and Weberling, 1989). Guard cells are another epidermal cell type that can vary between sepals and petals. Sepals can have numerous guard cells on both the adaxial and abaxial side (Endress, 1996, Gómez et al. 1999, and van der Krol & Chua, 1993). However, petals can have few or no guard cells (van der Krol & Chua, 1993 and Weberling, 1989). Epidermal cells may also vary between floral organs. For example, epidermal cells on the margins of a floral organ have the potential to redifferentiate and fuse with other redifferentiated cells on adjacent organs as demonstrated in Catharanthus roseus by Lolle & Pruitt.(1999) In this mechanism termed post genital fusion (I will refer to as PGF), organs may partially or completely merge with one another by means of reciprocal epidermal recognition and interaction (Lolle & Pruitt, 1999 and Pruitt, 2000). The anatomical features of PGF have been described in detail in Catharanthus roseus by Verbeke (1992), beginning with initial contact between two interacting organs. Prefusion epidermal cells were described as rectangular in shape, with dense cytoplasm, a thin cuticle, and divided anticlinally (Verbeke 1992). The convex epidermal cells of the organs at initial contact became flattened and the cuticle, if present, disintegrated. When epidermal cells made contact, they redifferentiated to isodiametric parenchyma cells with large vacuoles, and proceeded to divide periclinally. During the process, the cells that had made contact interlocked and finally the epidermal and subepidermal cells decreased in size (Verbeke, 1992). The overall result was a suturing of organs that had begun their development at inception as separate entities (Endress, 1996). Goals of the Current Study As described above, the cp petal of Clarkia tembloriensis has been the subject of morphological, developmental, genetic, and ecological investigation, however, no studies to date have focused on an anatomical comparison of WT and cp flowers in C. tembloriensis. This project seeks to fill that gap with an anatomical comparison of WT and cp petal flowers using SEM and light microscopy. More specifically, this study focused on epidermal cells including trichomes, guard cell, and marginal epidermal cells, of C. tembloriensis sepals, WT petals, and cp petals. The previous morphological and developmental studies of Clarkia tembloriensis flowers revealed that the cp petal is a unique organ which combines the characteristics of both sepals and WT petals. For this reason, I hypothesize that the cp petal will exhibit characteristics of both sepals and WT petal for the features observed in this study. This study will contribute to current understanding of the development of floral organs, and current models of floral development. In addition, this study will advance knowledge on the model organism C. tembloriensis and its homeotic mutant crinkle petal.

Materials and Methods

Sample Seed Collection and Growth Chamber Culture Seeds of Clarkia tembloriensis subsp. tembloriensis, collected from the Crocker Canyon population in southern California, USA were used for these investigations (Smith-Huerta & Huerta, 2015). Plants growing in this population are polymorphic for the cp trait, with approximately 50% of individuals expressing the cp trait, the other 50% expressing WT (Smith- Huerta & Huerta, 2015). Seeds were sown in pots of vermiculite and placed into a growth chamber at a photoperiod of 12hr light/12hr dark and a constant temperature of 20 C. After the seedlings had germinated and started to produce their first true leaves, plants were transplanted to 6-inch pots containing Fafard 3B potting soil (Sun Gro® Horticulture). Plants reached maturity at approximately 5 months after the seeds were sown and consisted of a main central spike bearing flowers with approximately 5 lateral branches which also produced flowers. Sample Preparation and Viewing The apical region of the main floral spike (containing floral buds in different stages of development), a closed bud (1-day pre-anthesis), and a flower 1-day post-anthesis were collected from 10 different plants of each floral type and placed into formalin-acetic acid-alcohol (FAA) (recipe from Ruzin, 1999) and fixed for a minimum of 24hrs. In order to allow for better penetration of the FAA solution into the tissue, one sepal was removed from each of the apical sections being soaked. After fixation with FAA, the smaller floral buds at 0.6mm, 1.0mm, 1.6mm, and 3.0mm in length were collected from the spike using a dissecting microscope. The fixed petals and sepals were also collected from the mature flowers that had been fixed one day post anthesis. These fixed samples were then prepared for use in either light or scanning electron microscopy observations. All samples were put through an ethanol dehydration series at 95% and 100%. Samples for light section microscopy 3.0mm buds and one day pre-anthesis buds were additionally put through a 50/50% mix of 100% ethanol and 100% acetone and then a 100% acetone soak. Light Microscopy After sample preparation, light microscopy (LM) samples were put through an ethanol and Spurrs’ resin exchange series. The 3.0mm bud and one day pre-anthesis buds, although, used acetone instead of ethanol. Exchange ratios were 3:1, 1:1, 1:3, and 100% resin. Samples in resin were then placed into molds and cured in the oven (60 C). Blocks were then sectioned to a thickness of 1µm with a Reichert-Jung Ultracut E Ultramicrotome and a Diatome diamond knife and the resulting sections placed onto glass slides on a heated table. Samples were stained with 1% toluidine blue, covered with a cover slip, and then viewed and photographed using an Olympus AX-70 Microscope. Scanning Electron Microscopy For SEM, previously fixed samples (as described above) were critical point dried (CPD) after the ethanol/acetone dehydration series, mounted onto SEM specimen stubs, and sputter coated with 20nms of gold. Samples were then viewed using a JEOL JSM-840A SEM using the appropriate settings. Trichome SEM samples were also prepared for viewing and EDS (Energy- dispersive X-ray spectroscopy) elemental analysis using a mature one-day post-anthesis sepal,

WT petal, and cp petal. In addition, samples were CPD, mounted onto stubs, coated with carbon, and viewed with a Zeiss Supra 35 VP FEG SEM using the appropriate settings. Analysis LM and SEM images were magnified and viewed using the Microsoft Window Paint program. Trichomes were named based on the published images of Maheshwari (1963). Trichomes and stomata were counted in a 0.55mm2 area on five different SEM images of floral organs at one day post anthesis. Counts of trichomes and stomata were made in sepals near the apex, at the base, and near the mid-point (figure 1.A). Counts in WT petals were made near the apex of the limb and the base of the limb and at the middle of the claw (figure 1.A). Trichome and stomata counts in the cp petals were made near the apex, near the base, and at the middle (figure 1.A). Average counts of trichomes and stomata were calculated for each region stated for the organs. The cells in the marginal regions of the mature organs harvested one day post-anthesis were imaged using SEM and sampled as described above in the areas indicated in figure 1B. Some epidermal cell striations and cell types were characterized based on their similarity to the cells observed and characterized in the study by Ojeda et al. (2009) and Bailes & Glover, (2018). Their two main categories were based off the cells general perimeter shape which were tabular, which can have rectangular like or jigsaw shaped perimeters, and papillose conical cells, which have oval or circular shaped perimeters Bailes & Glover, (2018). The tabular cells are further subdivided based on their morphology in being either flat or rugose, which are cells that are ridged or raised Bailes & Glover, (2018). Cell surface micromorphology presence was then determined, whether cells had striations or not (Bailes & Glover, 2018) and Ojeda et al., 2009).

Results Trichomes on Sepals, WT petal, and cp Petals Four types of trichomes were identified in SEM images: conical, clavate, falcate, and glandular trichomes (figure 2). The conical trichomes were elongated and needle-shaped with multiple verrucate projections on their surfaces (figure 2A). Clavate trichomes were club-shaped with smooth surfaces (figure 2A). Falcate trichomes were also needle-shaped, however, they were shorter, thicker, and more curved towards the tip than the needle-shaped conical trichomes (figure 2B). These falcate trichomes also had verrucate projections that were even more dense than those of conical trichomes (figure 2A and B). The glandular trichomes were much shorter than the other trichomes and had a rounded multi-celled head on top of a slender stalk (figure 2C). Elemental and spot analysis of these trichomes revealed no difference in elemental composition, however, the falcate trichomes did show a higher relative abundance of calcium on their verrucate projections, which likely was in the form of calcium carbonate. Elemental analysis results can be found in appendix 1. Trichome numbers in mature floral organs varied between organ types (Table 1). Overall, mature sepals had the greatest number of total trichomes of all the organs and mature WT petals had the least, while the mature cp petals possessed a number intermediate to sepals and WT petals (Table 1). Sepals had a greater overall average total number of trichomes on their abaxial surface and WT petal the most on the adaxial surface (Table 1). The cp petals were similar to sepals in that they had the highest overall average total trichome count on the abaxial surface, however unlike sepals, they had a relatively larger number of trichomes on the adaxial surface, which was more like WT petals (Table 1). Both the overall average total adaxial and abaxial trichome counts for cp petals were intermediate between the sepal and WT petal. Falcate trichomes were the most abundant type of trichome in sepals, especially at the apex of the abaxial surface. Second most abundant was the glandular trichome, which were mostly on the abaxial surface. Conical trichomes were absent on the sepal. Clavate trichomes were the most abundant type in WT petals, especially on the adaxial surface, while falcate trichomes were absent on the organ (Table 1). Conical trichomes were only found on the WT petal adaxial side. Clavate trichomes were the most abundant on cp petals similar to WT petals, and the presence of falcate trichomes was similar to sepals. Presence of conical cells only on the cp petal adaxial surface was also similar to the WT petal. Trichomes were also observed and photographed in sepals, WT petals, and cp petals in early stages of floral organ development (Fig 3, 4). Trichomes developed on very young sepals, first appearing in buds that were about 150µm in length (Figure 3). These first trichomes observed resembled the clavate trichomes of mature sepals. Trichomes appeared on WT petals and cp petals in buds around 3.0mm in length (figure 4). WT petals possessed very few trichomes early in development (figure 4A and C). The petals pictured in figure 4.A, C each had only a single trichome visible. The cp petals of young buds (0.9mm) possessed numerous well developed trichomes on the adaxial and abaxial surfaces, as shown on the abaxial side in the SEM and on both surfaces in the light sections (figure 4.B, D). The trichomes pictured on cp petals in figure 4 had matured sufficiently to make the presence of clavate, falcate, and glandular trichomes recognizable. This presence of trichomes on the cp petal was similar to young sepals as shown in figure 3.

Guard Cells on sepals, WT petals, and cp petals Stomata, consisting of a stomatal pore surrounded by 2 guard cells, were observed on the adaxial and abaxial surfaces of sepals, WT petals, and cp petals (figure 5). Average numbers of stomata counted and averaged on the mature sepals, WT petals, and cp petals are summarized in table 2. Overall, sepals had the greatest number of stomata, while WT petals had the least. The cp petals were intermediate between the sepals and WT petals in overall average numbers of stomata and average stomata on the adaxial and abaxial surface (Table 2). Sepals had many more stomata on the abaxial surface than the adaxial surface while petals had the most stomata on the adaxial surface and fewer on the abaxial surface (Table 2). The cp petals were more similar to the sepals in surface distribution of stomata with more stomata occurring on the abaxial surface than the adaxial surface (Table 2). On the adaxial surface of the organs, sepals showed a relatively even distribution of stomata, while WT petals had a greater abundance of stomata in the middle of the organ. The cp petals were unique with the greatest number of stomata on the apical area of the adaxial surface (Table 2). On the abaxial surfaces of the WT petal and cp petal, stomata had normal distribution from apex to base (Table 2). The patterns of quantity and distribution of stomata were similar to the patterns observed in trichome distribution (Table 1 and Table 2). One exception to this was in the abaxial surface of the cp petal in which trichomes were more abundant at the apex of the organ, while stomata were not present at a higher density in this area (Table 1 and 2).

Organ Margins, Redifferentiated Epidermal Cells and Postgenital Fusion in Sepals, WT Petals, and cp Petals The presence or absence of redifferentiated epidermal cells (REC) and postgenital fusion (PGF) was documented in the marginal regions of mature sepals, WT petals and cp petals using SEM and LM. Mature sepals, WT petals and cp petals. sepals. The marginal epidermal cells of sepals harvested one day pre-anthesis were viewed with LM near the apex, in the mid region, and at the base. The cells near the apex and middle section had narrow and acuminate papilliform REC (Figure 6 A and B), while towards the base there were fewer of these cells (Figure 6C and D). Small, fused REC were observed in the marginal regions of mature sepals (harvested one day pre anthesis) (figure 7.A). These cells were located at the junction of adjacent sepals and were papilliform with an outward facing end and an acuminate cuticular projection (figure 7.B). The cells interlaced with similar neighboring sepal cells, resembling interlocking teeth of a closed zipper (figure 7.B). Surrounding these cells were larger epidermal cells (figure 7.A). Observation made with SEM added further details to the nature of the marginal epidermal cells in mature sepals. Cells in several different stages of development were observed at the apex of mature sepals harvested one day post anthesis (figure 8). The flat epidermal cell consisted of pavement cells with developed striations (red arrow) (figure 8). Flat cells were also observed to have partially undergone redifferentiation and produced a rounded extension on their surfaces. However, these cells ceased redifferentiation mid-way and deposited striations (purple arrow), leaving them with the rounded extension. Some parenchyma REC developed striations while still being rounded (yellow arrow). The cells that had previously fused to adjacent sepals were narrow elongated papilliform REC that projected out with acuminate cuticular tips and had no striations (green arrow) (figure 8). Adjacent to the papilliform REC were rounded REC with no striations (gray arrow). These cells have the potential to go through an elongation and flattening process similar to those cells designated by the green arrow (figure 8). Lastly, large rounded REC cells were seen on the adaxial side at the top of the sepal (blue arrow). Some of these cells had an impression in them most likely formed from being pressed up against the adjacent sepal cells. This shows that the region of redifferentiation was not limited entirely to the edge of the sepal. All along the edge of the WT flower sepal, after anthesis, elongated non-striated papilliform REC were observed with SEM (figure 9). These cells were elongated on the long axis of the sepal. Lining both sides of the papilliform cells were non-striated narrow elongated previously REC that did not take on a papilliform shape (figure 9). The apex (figure 9.A) and the middle section (figure 9.B) had more acuminate papilliform REC than the cells at the base of the sepal (figure 9.C). WT petal. The marginal epidermal cells of WT petals harvested one day post anthesis and viewed with LM were flat to rounded in shape and possessed thin striations on both adaxial and abaxial

surfaces (figure 10.A). Trichomes were found occasionally on the edges of the organs (figure 10.B). Observation made with SEM added further details to the nature of the marginal epidermal cells in mature WT petals. The cells in the margins at the tip of the mature WT petals viewed with SEM (figure 11) were similar to those cells and striation observed with the light microscope (figure 10). Along the mid and upper parts of the WT petal limb the overall margins of the organ were wavy and occasionally small lobes were present. Stomata were sometimes observed at the edge of those lobes (figure 12). These stomata were observed only in SEM, and not in light micrographs. Along the claw of the mature WT petal (harvested one day post-anthesis), elongated and a few short tabular flat cells with longitudinal striations (TFS) (cellular description from Ojeda et al., 2009) were present. These cells were thick and extremely wavy. Striations perpendicular to the long axis of the cells were also present (figure 13.A). Some of these cells had rounded uplifted portions, similar to those cells with partially rounded extension on their surfaces observed in the sepal apex. Those cells had begun to dedifferentiate into REC, but then ceased part way and developed striations (figure 8). Cells were slightly larger and more elongated in shape at the base of the limb portion of the petal (figure 13.B). However, compared to the claw, the striations were thinner and perpendicular striations were lacking. Also, at this position at the base of the limb, trichomes appeared on the edge, but rounded uplifted cells were not observed. The middle portion of the limb (figure 13.C) appeared similar to the previous section except for a few more shorter cells and the striations being slightly thinner. The section near the apex portion of the petal (figure 13.D) appeared similar to the previous section. cp petals. Marginal cells of cp petals include REC slightly reduced in size and lacking striations (figure 14.A and B), similar to the cells observed in the sepal margins. The epidermal cells further from the margin on the adaxial and abaxial surface of the lamina were striated (figure 14.A and B). In the cp petal, the edge of the cp petal towards the apex became fairly close in proximity to the sepal as the cp petal margins curved backwards towards the sepal (figures 14.C and D). The fact that cp petals have REC similar to sepals suggests that cp petals have the potential to undergo PGF similar to that observed in sepals. No complete fusions were observed, but the potential for fusion was evident in that REC cells with cuticular projections, were present in both sepals and cp petals (figure 14.B) When the margins of mature cp petals came into contact with the adaxial surface of a sepal within the bud, the REC potentially interacted with the sepal cells (figure 14.D). This interaction consisted of a weak interdigitation of the epidermal cells. Although this weak interaction was observed, the cp petals and sepals were never observed fusing completely with one another, to the stamens, or to the gynoecium. Observations with SEM provided additional details of cp petal marginal cells. The cells at the apex of the cp petal were variable and similar to the cells at the apex of mature sepals and WT petals (Figure 15). The cells were short or elongated, with striations (figure 15.A). Trichomes were also observed, resembling those of mature WT petals (figure 15.A). In some cp petals cells at multiple stages of development were observed (figure 15.B) and in others multiple striated RECs were observed similar to mature sepals (figure 15.C).

The marginal region just below the apex of the cp petal varied between individual organs. The margin may be straight, similar to the sepal, or may have a short lobe, similar to WT petals (figure 16). The lobes had elongated TFS epidermal cells with extremely thick wavy striations and striations that were oriented perpendicular to the long axis of the cp petal (figure 16). These cells were similar to the cells of WT petals; however, the types and patterns of striations were different in the two organ types. Short cells were also observed on the lobe (figure 16) as well as a stoma at the tip of the lobe (figure 16 and in the light section in figure 17), similar to the lobe in the WT petal (figure 12). The cells along the edge of the lobes had epidermal cells with striations, however, further from the lobe, the cells were elongate REC with no striations, a condition similar to that found in sepals (figure 16). Along the claw of the cp petal (figure 18. A) there were elongated non papilliform REC with no striations. The base of the limb section edge (figure 18. B) looked similar to the claw section. At the middle limb (figure 18. C), the cells were elongated REC, but some had endings that rounded up and projected outwards. Further up the organ, near the curled over part, were some short and slightly wider cells. The section near the apex (figure 18.D) was similar to the middle limb section, having elongated REC and elongated REC with endings that were round and projected outwards. However, the elongated cells with endings that were round and projected outwards were more frequently observed in this section than the middle limb. Organ Development Sepal. The sepals were initiated in opposite pairs, one pair initiating slightly after the other, so that one pair was slightly ahead of the other in development (Figure 19). Sepals produced REC very early in their development, soon after they were initiated. The REC first appeared on the sepal apex (figure 19). These cells observed were rounded, somewhat toothed shaped and lacked striations. Large, older sepals possessed REC that were larger and more completely developed (figure19). As the sepals grew even larger, REC began to develop along the margins, maturing from the apex basipetally (figure 20.A). The margins of the sepals sutured together as the apices approached one another (figure 20.B). As the apices of the oldest (opposite) sepals contacted each other, the cells extended outwards to the opposite sepal (figure 20.C). This pattern of REC development in young buds was further observed and documented with LM (figure 21, A-D). Near the apices of the sepals, just before the junction of all four sepals (figure 21.A), the papilliform REC elongated towards the adjacent sepals. This was particularly evident in the larger older sepals (Figure 21 A). In the middle region of the junction between the sepals (figure 21.B), many small REC were observed, and these REC interlocked extensively with one another. In regions of the buds still further from the apex, the four individual sepals were evident surrounding a central area (figure 21.C). The REC lining the adaxial surface of the sepals in the central bud area were enlarged and projected inward. However, small REC also remained on the margins of the sepals. Further from the sepal apex junction (figure 21.D) four sepals were evident, surrounding a hollow central chamber. The epidermal cells in this chamber were reduced in size and were rounded along the adaxial surface of the sepals (figure 21.D). At the edge junctions, some REC extended out into the chamber,

while those suturing the sepals together were small and interwoven (figure 21.D). It was clear from these images that PGF begins between sepals very early during bud development. Further details of the process of PGF were documented in LM presented in figure 22. The images were produced by photographing sections of a young bud made sequentially from its apex to its base. Since the cells at the margins of the sepals of these buds were in different stages of fusion, these sections provide an excellent illustration of the process of PGF of sepal margins. The process of PGF in the sepals began with the anticlinal division of ground tissue cells near the sepal’s apex, leading to enlargement. This enlargement brought adjacent sepals closer to one another (figure 23.A, B). As the ground tissue cells in this region increased in number, the REC at the sepal’s margins appeared rounded and extended outward (figure 22.A, B). As the sepals continued to enlarge, the margins of adjacent sepals became closer to one another, and the cells appeared acuminate and possessed a cuticular projection (figure 22.B). In the region still further from the apex, periclinal division of the ground tissue cells had occurred, resulting in thicker sepals in this region (figure 22.C, D). Continued growth of the sepals brought the REC of the adjacent sepals into closer contact, and the process of PGF began interlocking the adjacent sepals together (figure 22.C, D). Near the base of the sepals, the continued periclinal cell divisions in this area resulted in increasing the number of REC which eventually participated in PGF (figure 22.D-E). As PGF proceeded, the REC appeared smaller (figure 22.F). WT petal and cp petal development. Both WT petals and cp petals were initiated in buds approximately 0.6 mm in length and visible differences between them could be recognized in buds as small as 1.0 mm (figure 23) At this very early stage of development REC were not observed on the margins of WT petals, however, cp petals were similar to sepals in the presence of REC (figure 23). The REC were first observed on the cp petal towards the apex (figure 23. B). These cells projected outwards from the organ, giving it a bumpy appearance along its margins. In comparison, the WT petal appeared smooth along its margins (figure 23. A). In slightly larger buds, (1.6 mm and 3.0 mm in length) WT petals retained smooth margins (figure 24. A and figure 25. A) and the cp petal showed its REC. As the cp petal grew larger, the marginal areas covered by the REC grew larger (figure 24. B and figure 25. B). This also demonstrates that the cp petal is similar to the sepal in that the REC develop basipetally. The LM images of figures 26, 25 and 27 further document the differences between developing WT and cp petals and the presence or absence of REC. The WT petal and cp petal continued to develop in the same way in the 3.0mm bud (figure 28. A, B and figure 27. A, B). At this stage, the REC in the cp petal covered significantly more area than that of the previous sizes (figure 28. B).

The observation described above correlates with observations made at the macroscopic level during anthesis. During anthesis of WT flowers, the sepals separated completely along their margins. However, during anthesis of some cp petal flowers, the sepals did not separate completely from one another (figure 29). In some cases, the narrow cp petals escaped through the sepals towards the base where the sepal junctions had a weaker PGF connection, as was illustrated in the light microscopy sections (figure 6.C, D). However, toward the apex of the bud, the sepals were often found remaining fused together, likely due to a greater number of REC interlaced toward the tips. In these cases, when the sepals were still intact, they acted like a hood and wrapped over some of the petals (figure 29.B) or stamens (figure 29.A), preventing them from being exposed. The flowers with WT petals always opened fully. The broad deltoid blades of WT petals, had a larger surface area than cp petals and exerted more force during anthesis, thus resulting in the differences in sepal separation during anthesis.

Discussion The naturally occurring homeotic mutant of floral development crinkle petal (cp) of Clarkia tembloriensis has been the subject of ecological, genetic, and developmental studies (Leong et al, 2001, Sherry & Lord, 2000, Smith-Huerta, 1992, Smith-Huerta, 1996 Smith-Huerta & Huerta, 2015, Vasek, 1966, and Vasek & Harding, 1976). The previous developmental investigations revealed that the cp petal can be considered a hybrid organ, combining characteristics of sepals and WT petals. This investigation extends those studies with a comparative anatomical study of trichomes, stomata, and marginal cells in sepals, WT petals, and cp petals. The results, discussed below, further support the hybrid nature of the cp petals of Clarkia tembloriensis. The Hybrid Nature of the cp Organ The work of Smith-Huerta (1992, 1996) established the hybrid nature of the cp organ. Her work is summarized briefly here as an introduction to the topic of this research. Beginning at the inception of the floral meristems, no differences were observed between WT and cp plants (Smith-Huerta, 1992). Sepals were initiated first and developed trichomes very early in development. Both cp petals and WT petals were initiated later and began their development at the same time and location in the bud, however, the two different organ types assumed slightly different shapes shortly after inception (Smith-Huerta, 1992). For both WT and cp petals, this pattern of initiation followed the usual petal development pattern in Clarkia in which petals are initiated almost simultaneously to one another and after the sepals (Endress, 1996). As the WT and cp petals continued their early development in the bud, they maintained a similar shape, however in buds of approximately 3 mm in length the cp petals developed trichomes, and the WT petals did not. An analysis of organ growth from inception to maturity also revealed that the sepal and cp petal have similar allometric growth patterns (both different from WT petals), resulting in similar shapes at maturity (Smith-Huerta, 1992). During the process of anthesis, the WT petals expanded more rapidly than both the sepals and cp petals. (Smith-Huerta, 1992). Vascular system development was also studied in the three organ types by Smith-Huerta (1996). Sepals were found to have three primary veins and the tracheary elements of these organs developed rapidly in a basipetal pattern. WT petals had a single primary vein and the tracheary elements of WT petals developed relatively slowly and in an acropetal pattern. The cp petals combined characteristics of sepals and WT petals exhibiting a pattern of maturation similar to sepals and an overall vein architecture similar to WT petals (Smith-Huerta, 1996). It can be concluded from these observations that cp petals were similar to WT petals with respect to initiation, early development and vascular architecture, and were similar to sepals in allometric growth, the early development of trichomes and patterns of tracheary element maturation. The cp petal was unique in its final size and crinkled appearance (Smith-Huerta 1992). Epidermal cells investigated in this study Trichomes.

All floral organs had trichomes on both surfaces and four different types of trichomes were identified. The cp petals were most like sepals with respect to having the greatest number of trichomes on the abaxial surface, presence of falcate on the abaxial surface, and early development soon after organ initiation. The cp petals were most similar to WT petals in that clavate trichomes were the most abundant type present in both organs, conical trichomes were present on the adaxial surface, and relatively high presence of trichomes on the adaxial surface. Finally, the cp petals were intermediate to sepals and WT in overall numbers of trichomes present and overall average total on the adaxial and abaxial surface. Below is a detailed discussion of these results in consideration of general trichome characteristics of flowers. Trichomes are epidermal cells that occur on the surface of some plant shoot organs (Callow et al., 2000), and they may be unicellular or multicellular. The trichomes that occur on the sepal and WT petals of C. tembloriensis followed a pattern of trichome number and distribution typical of some other species. In general, sepals typically have numerous trichomes on both the adaxial and abaxial surfaces, for example, Lycopersicon esculentum, Petunia hybrida, and (Endress, 1996, Gómez et al. 1999, and van der Krol & Chua, 1993) while petals have few to no trichomes on either surface (van der Krol & Chua, 1993 and Weberling, 1989). This pattern was observed in the present study for C. tembloriensis with sepals possessing more than twice as many trichomes as WT petals. In the present study, the sepals had more trichomes on the abaxial side than the adaxial side, while the species in previous studies possessed an equal abundance of trichomes on both surfaces (Endress, 1996, Gómez et al. 1999, and van der Krol and Chua, 1993). The cp petals were similar to sepals in that they both had a greater density of trichomes on the abaxial surface and were intermediate to sepals and WT petals in overall numbers of trichomes. The present study also confirms and extends the observations of Smith- Huerta (1992), who observed that mature sepals were covered with trichomes, and that these trichomes appeared very early in development. The WT petals, on the other hand, had a smooth texture (Smith-Huerta, 1992). The mature cp petals were also covered in trichomes, like the sepals. However, unlike sepals, trichomes began to develop on cp later in development (Smith- Huerta, 1992) (cp petal about 910 µm tall in a 3.0mm bud size). Clarkia concinna is another species that has a naturally occurring B-type mutant called bicalyx (Ford and Gottlieb 1992). The bicalyx petals were described by Ford and Gottlieb (1992) as being intermediate between sepals and WT petals with respect to the presence of trichomes. However, that study did not provide a detailed description and analysis as the present study did. Previous investigations, including one of C. tembloriensis, have found trichomes to be generally lacking on petals (van der Krol & Chua, 1993 and Weberling, 1989 Smith-Huerta 1992). However, this conclusion could be the result of how trichomes were visualized. The mature WT petals were not observed with SEM by Smith-Huerta (1992). When observing floral organs of C. tembloriensis with the unaided eye, it is possible that the appearance of trichomes could go undetected and the WT petals could be described as smooth in texture. This is supported by the fact that WT petals had the lowest overall total number of trichomes of the three floral organs studied. The tomato mutant sl/sl, is another b-type homeotic mutant with petals resembling sepals (Gómez et al., 1999). A study of the sl/sl mutant found the presence of trichomes on the entire surface of both sides of the sepaloid organ similar to that of cp petals. In addition, the greatest number of trichomes occurred at the apex of the organs and on the abaxial side, similar to cp petals (Gomez et al 1999). The sl/sl mutant petal had foot-cell trichomes on its abaxial surface

similar to sepals. The study of sl/sl tomato petal mutant compared trichomes between sepals and the mutant petal and did not include an analysis of a WT petal (Gómez et al. 1999). For this reason, it is not known whether the mutant organ had any trichome characteristics similar to WT petals. The trichome characters described by Keating (1982) on the leaves of members of the plant family Onagraceae provide useful information for this study since Clarkia is a member of the Onagraceae. The characteristics of trichomes observed in the present study were very similar to those observed by Keating (1982). Trichomes of Onagraceae leaves were described as either smooth or textured, with no regular pattern of distribution. The trichomes were mostly unicellular, simple, elongated, and tapered to a tip, with constricted or slightly expanded bases (Keating, 1982). Some trichomes were also described as being slightly recurved, sharply tipped, short, and very broad, while others were long and straight (Keating, 1982). Overall, these trichomes are very similar to the falcate and conical trichomes observed on the floral organs of this study. Additionally, in all Clarkia species observed by Keating (1982) the trichome surfaces possessed fine striate-papillate or tuberculate surfaces. The falcate and conical trichomes observed in this study of C. tembloriensis possessed similar surfaces. In the present study there were no trichomes observed on developing cp petals of either 0.2 or 0.4 mm in length. By the time cp petals were 0.9 mm in length, trichomes were visible on the surface. From this observation, it can be concluded that trichomes developed on cp petals sometime between 0.4 and 0.9 mm in length. This observation was similar to what was observed by Smith-Huerta (1992). In her study, trichomes were visible in cp petals that were 0.5 mm in length, a value which falls between the 0.4 mm and 0.9 mm range as observed in the present study The presence of trichomes on the abaxial surface of cp petals might be an advantage to the flower since it is known that many trichomes on the sepal’s surface likely acted as a protective layer against irradiation by the sun (Endress, 1996). Presence of trichomes in the cp petal petals could also act as an advantage in increasing the boundary layer thickness of the organs, thus slightly decreasing transpiration. Guard cells. All three floral organs had guard cells on both adaxial and abaxial surfaces, with sepals possessing the greatest number. The cp petals were most like sepals in their overall distribution patterns with more guard cells occurring on the abaxial than the adaxial surface. The cp petals were most like the WT petal in having a normal distribution pattern on the abaxial surface. The cp petals were intermediate to sepals and WT petals in the overall average number of stomata per organ. The cp petals were also intermediate to sepals and WT petals for average stomatal numbers on both the adaxial and abaxial surface. Guard cells are a type of epidermal cell found in plants (Glover, 2000). The guard cells occur in pairs and surround a stomatal pore on the epidermis. The guard cells, function to regulate gas exchange by controlling the size of the stomatal pore (Roberts, 2007). Sepals usually have numerous guard cells on both the adaxial and abaxial side (Endress, 1996, Gómez et al. 1999, and van der Krol & Chua, 1993). However, petals usually have few or no guard cells (van der Krol & Chua, 1993 and Weberling, 1989).

The sepals of C. tembloriensis possessed numerous guard cells similar to other flowers such as Petunia hybrida (Endress, 1996 and van der Krol & Chua, 1993). The mature WT petals in this study possessed guard cells, which is not the usual condition. The petals of many plants do not possess guard cells (van der Krol & Chua, 1993 and Weberling, 1989), although exceptions exist as in the case for Gerbera hybrid, flowers (Kotilainen et al. 2000). In Gerbera petals, stomata form on the abaxial surface (Kotilainen et al. 2000). The bicalyx mutant of C. concinna was similar to C. tembloriensis, possessing guard cells on all three of the mature organ types (Ford and Gottlieb 1992). Total numbers of guard cells in the area used for counting on the bicalyx petals were intermediate in numbers between the sepals and WT petals (Ford and Gottlieb 1992). In the homeotic (sl/sl) mutant of tomatoes sepals, WT petals and sepaloid petals all possessed guard cells (Gomez et al 1999). Numbers of guard cells on the sepaloid petals were similar to numbers on the sepals, and the WT petals had fewer guard cells than the other two organs (Gómez, et al. 1999). Redifferentiated epidermal cells (REC) in the marginal region of floral organs and their role in post genital fusion (PGF). In the present study the marginal cells of the sepals had REC, and these cells differentiated early in the development of the organ. The WT petals never developed REC. The cp petals were similar to sepals in the early development of REC at their margins. The presence of REC in sepals allowed the process of PGF to occur between the margins of adjacent sepals. The presence of REC in cp petals did not lead to the process of PGF, most probably due to the fact that the marginal REC of the cells of adjacent cp petals did not come into contact with one another during development in the bud. In rare instances, the REC of cp margins came in contact with the adaxial surface of a sepal in the bud. When this occurred, a weak interaction occurred between the REC of the cp margins and the adaxial surface of the sepal. Below is a detailed discussion of these results in consideration of general REC characteristics of flowers and their role in PGF. Redifferentiated epidermal cells are parenchyma cells that have thin cuticles, are rectangular in cross-section, have dense cytoplasm, and divide anticlinally (Verbeke, 1992). These REC’s play a role in PGF in plants. During PGF distinct organs may fuse during development through the contact of the REC’s on adjacent organs. Soon after RECs come into contact with other RECs they change shape from rectangular to isodiametric, become highly vacuolate, and begin to divide periclinally (Verbeke, 1992). As periclinal divisions proceed, the cells become papilliform and pointed and the REC’s from adjacent organs become interlaced (Weberling, 1989). This results in the PGF of the cells of the adjacent organs, described in greater detail below. Each perianth whorls within flower buds may assume a variety of positions depending on the species. Floral organs may overlap (imbricate), the margins may touch end to end, but not overlap (valvate), or not overlap or touch (apart/open) (Endress, 1994, 2011). The arrangement of the perianth organs in regards to one another in the floral bud are referred to as the aestivation pattern, which may have implications for PGF potential (Endress, 1996). These patterns are usually displayed as a cross section through the bud. The seven classes of aestivation include quincuncial, contort, ascending, descending, apart/open, imbricate, and valvate (Endress, 1994, 2011). In pentamerous flowers, an imbricate pattern with two perianth completely inside two

surrounding perianth and the fifth perianth is positioned in-between the inside and outside sets is referred to as quincuncial (Endress, 1994). A second imbricate pattern, called contort, has all the perianth having one of their margins covered by a different organ’s margin and its other margin covering a different organ’s margin (Endress, 1994). The two other imbricates are cochlear descending, where four organs are contort except one that is outside the others, and cochlear ascending, which is similar to descending except the one organ is inside the contort organs (Endress, 1994). In this study, observations of C. tembloriensis sepals in pre-anthesis buds in LM sections indicate that they have a valvate aestivation pattern. As suggested above, the aestivation pattern of a flower influences how the floral organs potentially interact with one another. One way that perianth parts may interact is through a process called congenital fusion, were organs are united during initiation in the meristem (Lolle & Pruitt, 1999). Another way that perianth parts may interact is through PGF (Lolle & Pruitt, 1999). In this mechanism, organs may partially or completely merge with one another by means of reciprocal epidermal recognition and interaction by direct contact (Lolle & Pruitt, 1999 and Pruitt, 2000). The overall result of PGF is a suturing of organs together, via cell wall adhesion vs cytoplasmic union, though the cells that are making contact, even though they had originated as separate entities at inception (Endress, 1996, Lolle et al., 1992, and Verbeke, 1992). This contrasts with congenital fusion where the organs are united at inception (Endress, 1996). This mechanism can occur in almost all floral organs (Lolle et al., 1992 and Verbeke, 1992) and even in vegetative tissue, as was the case in the fiddlehead mutant of Arabidopsis thaliana (fdh) (Lolle et al., 1992). The majority of research on PGF conducted to date seeks to explain carpel fusion (Verbeke, 1992 and Lolle & Pruitt, 1999). Emphasis on this organ harkens to its importance in floral evolution and angiosperm reproductive success in that it protects the ovules from exposure to the outside environment, allows for species to adapt diverse modifications to seed dispersal, and enables multi carpel pistils to allow ovules of more than one carpel to be fertilized (Lolle & Pruitt, 1999). Gynoecium fusion events have been described in Apocynaceae (Lolle & Pruitt, 1999) and by Bessey in members of Rosaceae, Alismaceae, and Ranunculaceae (Verbeke, 1992). The process of PGF has been documented very rarely in the corolla (Yang et al., 2017) and calyx (Verbeke, 1992). Few exceptions are noted in the Apocynaceae (Verbeke, 1992) such as in the genus Vinca (Yang et al., 2017). In contrast, PGF events have been documented to be common in the androecium of several species (Tragopogon pratensis, Tagetes patula, Solanum dulcamara, members of Asteraceae and some Campanulaceae) and in the gynoecium of Catharanthus roseus (Verbeke, 1992 and Yang et al., 2017). Extensive morphological studies on carpel fusion and the required cellular signals have been conducted in Catharanthus roseus (Lolle et al., 1992 and Lolle & Pruitt, 1999). The sequence of events taken during PGF seem relatively similar between species like C. roseus, Arabidopsis thaliana fdh mutant, and other plants capable of PGF (Lolle et al., 1992, Lolle & Pruitt, 1999). The process of PGF requires that the cells of different organs be in direct contact with one another and have reciprocal recognition in localized areas (Lolle et al., 1992, Lolle & Pruitt, 1999, Verbeke, 1992). The initiation of PGF requires that organs grow and come into contact with one another (Lolle et al., 1992 and Verbeke, 1992) and that morphogenetic factors be exchanged between cells in direct contact. Morphogenetic factors are small, fairly stable, water soluble, and rapidly diffusible molecules for which the molecular basis has not been determined (Lolle & Pruitt,1999 and Verbeke, 1992). These epidermal cells dedifferentiate and then redifferentiated into isodiametric convex parenchyma-cells that contain many vacuoles

(Lolle & Pruitt, 1999 and Verbeke, 1992). The contacting areas of the epidermal cell walls then flatten, interlock, and often undergo periclinal divisions (Verbeke, 1992). At this point, there is a divergence on what then occurs next. Verbeke (1992) documented the loss of the cuticle in the REC that had undergone PGF and also a decline in cell size. Lolle & Pruitt (1999), on the other hand, described that fragments of cuticle were present in much of the sutured tissue, with the fewest cuticle fragments in areas where fusion had occurred earlier. In a third study Baum (1948) observed that PGF of organs occurred before cuticle formation. Lolle & Pruitt (1999) later showed that epidermal cells late in making contact were found not to redifferentiate, but retained epidermal cell characters and adhered only by the cell wall. The above studies demonstrate that PGF can vary in the order of fusion events and can result in varying degrees of fusion (Verbeke, 1992). Another variation to PGF is when the interacting cells become cytoplasmically connected. This contrasts with the definition of postgenital fusion, which states that the involved cells do not become connected through the symplasm, but rather adhere to one another via the cell wall (Baum 1948, Cusick 1966; Verbeke, 1992, and Walker 1975a, b). However, van der Schoot et al. (1995) found secondary plasmodesmata forming in low frequencies in the epidermal cell walls of fusing carpels of Catharanthus roseus. This process was seen incurring before the dedifferentiating and redifferentiating stage that results in the formation of parenchyma cells (Lolle & Pruitt, 1999). The development of cytoplasmic connections between fused carpels in C. roseus is believed to be one of the necessary elements required for the creation of a compitum in the gynoecium, which allows pollen tubes to travel between different carpels to different ovules (Vialette‐Guiraud, & Scutt, 2018). Exceptions like C. roseus bring question to the long-held definition of PGF (van der Schoot et al.,1995). As stated above, sepals of C. tembloriensis possessed REC very early in their development. These RECs participated in PGF of adjoining sepals during floral bud closure. In the early stages of C. tembloriensis bud development, the sepal marginal cells dedifferentiated and then redifferentiated, and this occurred even before they made actual contact. Also, not all the cell walls flattened after the process of PGF was complete. In fact, most cells developed an acuminate cuticular projection, which is where the cuticle develops into a long tapering projection from the cell surface. This type of PGF seen in the sepal margins in the developing bud of C. tembloriensis, is called dentonection. Dentonection is an interlocking mechanism for epidermal adhesion by which papilliform cells develop pointed ends with a cuticular projection and then interlace together (Weberling, 1989). This mechanism occurs in C. tembloriensis due to the valvate aestivation pattern of the sepals in the bud (Endress, 1996). Dentonection is also the mechanism observed in joining the keel petals in Papilionaceae and the anthers and stylar channel in Campanula species and Oenothera biennis (Weberling, 1989). A characteristic commonly observed in sepals is organ fusion at the tips, or upper portions, while remaining separated at the base (Weberling, 1989). This pattern was also observed in the present study of C. tembloriensis. More specifically, acuminate cells were observed towards the tips of the sepals that had undergone anthesis, while cells at the base were flattened. These acuminate cells had been fused with cells of adjacent sepals before anthesis while cells at the base were not. The time of PGF initiation (from tip to base) led to the cells at the tip completing PGF while those at the base did not. Baum, who studied PGF in 140 allegedly apocarpous (distinct un-joined carpels) species, also found that the degree of fusion between organs depended on how early the PGF event occurred during ontogeny (Baum, 1948a, Baum,

1948b, and Verbeke, 1992). The degree of fusion relates back to the degree of suturing between the cells, for example, Helleborus was found to undergo PGF very early in development in the carpels, and possessed highly notched REC. In contrast, Aconitum was shown to undergo PGF in the carpels late in development and the REC in these plants were flat (Baum, 1948b and Verbeke, 1992). As stated above, the WT petals on C. tembloriensis did not have REC and did not undergo PGF. The majority of cells in the WT petals margins appeared similar to the epidermal cells in the petal lamina. These were tabular cells with striations on their surface as described by Bailes & Glover, (2018) and Ojeda et al (2009). This differs from the petals of other plants in which papillose conical cells, which have oval or circular shaped perimeters and usually possessing steep high anticlinal cell walls, make up the epidermal regions of the petal lamina (Bailes & Glover, 2018, Ojeda et al., 2009, and Whitney et al., 2011). However, in some plants, such as the Fabaceae, petal lamina may possess both tabular and papillose conical cells within the same flower or the same petal (Ojeda et al., 2009). The marginal cells of various species have been observed to be either tabular as in Cassia emarginata or papillose conical as in Cassia roxburghii (Ojeda et al.,2009). Though almost all of the marginal cells of WT C. tembloriensis petals resembled tabular cells, a portion of the lower petal claw had striated cells with rounded raised regions that resembled parenchyma cells. These might have been REC cells adjacent to the area of PGF that formed the short basal floral tube. Many Onagraceae members have petaloid floral tubes, including those in the genus Clarkia (Beidleman et al., 2014). Beidleman et al. (2014) characterized C. tembloriensis as having a floral tube length of 1-5mm above the ovary. As described above, some cp petals of C. tembloriensis were similar to sepals in the presence of REC at their margins, however the expression of REC was incomplete when compared to sepals. More specifically, the claw, bottom limb, and middle limb margins of the cp petals posed REC similar to those observed in the sepals while the margins at the apex varied in the expression of REC depending on the sample. Other cp petals were similar to the WT petal with the occurrence of a short lobe on the marginal section just below the apex. This mixed expression of sepal and WT petal characteristics in the cp petal was also observed in the petaloid sepals of Arabidopsis p35S-PI (Krizek & Meyerowitz, 1996) and in the sepaloid petals of gp Petunia (Halfter et al., 1994). The variability in the apex and region just below the apex of the cp petal supports the suggestion of Vasek (2008) that flower development in Clarkia exhibits a degree of plasticity. In addition, Halfter et al. (1994), observed different levels of gp gene expression in Petunia which resulted in different degrees of sepal conversion into petal. Initiation of the REC cells on the cp petal occurred early in development, similar to sepals. Furthermore, the marginal epidermal cells of the mature sepals and cp petals (observed post anthesis) possessed similar numbers of REC. However, the REC appeared different in structure between mature sepals and cp petals. The sepals had been previously fused together by PGF and had their REC were cuticular tip papilliform cells while the cp petal REC did not exhibit this morphology. Additionally, on either side of these papilliform cells in the sepal, there were REC cells that had not gone through PGF and remained elongated and non-striated. Lolle et al. (1992) also observed this loss of topology in the cells surrounding the cells involved in the primary fusion of fdh Arabidopsis mutant sepals and petals.

Although the distribution of REC on the cp petal margins was similar to those on the sepals, fusion between adjacent cp petals did not occur like fusion between the sepals. This was likely due to the cp petals open aestivation pattern and to the narrow widths of the organs which prevented physical contact. This was in contrast to the petals of Vinca rosea which are wide enough to have adequate marginal contact for PGF (Boke, 1948). Previous investigations have identified the need for the diffusion of small molecules between adjacent organs to elicit PGF, and the presence of a cuticle which prevents molecule exchange between the organs (Pruitt et al., 2000). Arabidopsis plants have specific lipids found on their organ surfaces which modify the cuticle properties, allowing for the exchange of molecules and thus promoting cell-cell interaction (Pruitt et al, 2000). Since the cp petals were observed to interact only weakly with adjacent sepals, recognition chemicals may not have been produced by the marginal cells of the cp petal. This observation suggests that the recognition factors are specific to the sepals. Again, the degree of homeotic gene expression coding for sepaloid characteristics in the cp petal might dictate the degree to which these organs are able to fuse to other organs in the flower (Halfter et al., 1994). This study has provided evidence that the REC of cp petals may have the potential to fuse with other floral organs, as evidenced by the interactions observed between the margins of cp petals and the adaxial surface of sepals in the bud. However, this occurrence is probably rare, since few sections in this study documented this phenomenon. It is interesting to note that Lolle et al. (1992), documented that all the floral organs of the fdh Arabidopsis homeotic mutant were able to fuse with any other floral organ including fusion between sepals and petals. However, Lolle et al. (1992) did observe areas on petals that were not fused with the overlying sepals, but did show that the petals were reacting to the contact (smooth areas on petals). This was probably due to brief contact between the organs (Lolle & Pruitt, 1999), which might be the case for the limited evidence of petal-sepal fusion seen in C. tembloriensis. Other Single Whorl B-type Mutants in the Onagraceae Naturally occurring sepaloid homeotic plants have also been identified in populations of Clarkia concinna as described above (Ford and Gottlieb, 1992) in Clarkia exilis (Vasek, 1966) and in the genera Epilobium and Oenothera of the Onagraceae (Renner 1959, Ford & Gottlieb, 1992 and Leong et al., 2001). The expression of the sepaloid petal trait in all of these mutants in the Onagraceae is determined by a single recessive gene, similar to the cp mutant of C. tembloriensis (Ford & Gottlieb, 1992, Renner, 1959, and Vasek, 1966). The Future of C. tembloriensis in the Wake of Climate Change As described previously, C. tembloriensis inhabits the coast ranges of California. Annual rainfall varies across the range of species from an average of 23 cm at the core of the range to 12 cm at its margins (Smith-Huerta and Huerta 2015). The Crocker canyon population was the origin of the seeds used in this study, and this population occurs at the margins of the species range. The persistence of C. tembloriensis in marginal regions of its range could be in question if climate change brings increased temperatures and less rainfall to these marginal areas. At present, several populations of C. tembloriensis are polymorphic for the cp trait, with approximately fifty percent of individuals expressing the trait (Smith-Huerta and Huerta 2015). If the cp trait provides an adaptive survival advantage under increasing temperature and drought conditions, it is possible that the frequency of the cp trait could increase in populations. Below is a discussion of the characteristics of cp petals that might increase survival under such a scenario.

The presence of trichomes on the cp petals abaxial surface might be an advantage to the flower since it is known that many trichomes on the sepal’s surface likely acted as a protective layer against irradiation by the sun (Endress, 1996). Presence of trichomes in the cp petals could also provide an advantage by increasing the thickness of the boundary layer of the organs, thus decreasing transpiration. A reduction in petal size, like that found in the cp petal, may act as an advantage in reducing the need for water and metabolic resources (Smith-Huerta and Huerta 2015). In habitats with limited water, flowers with large colorful petals, like that in WT petal, would require more resources and, despite the advantage of attracting pollinators and increasing outcrossing, it may limit the availability of energy and resources for seed production, as proposed by Smith-Huerta and Huerta (2015). Other species that inhabit arid environments have also developed modifications that preserve resources, like cacti that have evolved morphological and physiological traits that result in the water conservation. Examples of these traits include extremely thick stem tissue composed of water storing parenchyma, a thickened cuticle that prevents water loss through the epidermis, and a photosynthetic adaptation called Crassulacean acid metabolism (CAM), which minimizes water loss by opening the stomata at night when its cooler and the evaporative demand is lower (Gibson, 1998). Another advantage for cp petal might be its green coloration. The green pigment of the organ indicates the presence of chlorophyll with the potential for photosynthesis. This would provide an advantage in the form of added resources to cp petal flowers, especially for seed set. The WT petals are not photosynthetic and thus do not contribute positively to the carbon balance of the flower. Although it has not been investigated, the adaptive advantages of trichomes to reduce water loss and presence of chlorophyll with the potential for photosynthesis might help to explain the persistence and high frequency of cp petal plants in the lower moisture marginal habitats (Smith-Huerta & Huerta, 2015). The cp petal flowers are less conspicuous to pollinators compared to WT petal flowers, and this may result in fewer pollinator visits to cp plants. However, the normal mating system in populations where plants are present is self-pollination (Smith-Huerta & Huerta, 2015). Pollinators are not required for full seed set in these marginal populations. It is common for selfing populations of plants to be found near the margins of their range (Smith-Huerta and Huerta, 2015). Many species have already been impacted by changes in the climate, especially in arid regions of the planet (Archer & Predick, 2008 and Miranda et al, 2011). For example, it is known that many species of bees are experiencing declining populations, and this could be due in part to changes in climate (González-Varo et al, 2013 and Le Conte & Navajas, 2008) This change in regional pollinator population size might also influence the success of outcrossing species since fewer pollinators would potentially be available to effect cross pollination. In this case plants with a selfing habit would have a survival advantage. The highly selfing population of C. tembloriensis, which contain cp plants, are able to set seed without pollinator visits giving them a survival advantage where pollinators are rare or absent. Considering the above, further studies of the cp petal of C. tembloriensis could lead to an increased understanding of how plant species might adapt to a changing environment through changes in breeding system and floral morphology.

Homeotic Mutants as Model Systems to Study Floral Evolution In a recent study of a naturally occurring homeotic mutant of Capsella bursa-pastoris, Nutt (2008) explored the criteria necessary to make homeotic mutants good model systems for the study of floral evolution. Nutt (2008) suggested the following characteristics are necessary to make a naturally occurring homeotic mutant a good candidate for a model to study floral evolution: 1. The mutation causing the trait is simple and heritable. 2. The mutant plants are reproductively fit, producing comparable seed numbers to WT plants. 3. The plants have short generation times in populations, allowing rapid observations of fitness through time. 4. The plants are amenable to molecular genetic analysis. 5. Plants occur in sufficient numbers for continued study through numerous generations. The bicalyx homeotic mutant of C. concinna is one species considered by Nutt (2008) for model status since it was naturally occurring and had remained stable in the population for four years (Ford and Gottlieb, 1992) Nutt disqualified C concinna as a model plant for the following reasons. First, mutant plants occur in very low numbers in nature, making up only 20-30% of a single small population occurring near Point Reyes, CA. Second, a molecular characterization of the bicalyx gene had not been undertaken and third, the relative reproductive fitness of mutant plants compared to WT plants had not been measured (Hintz et al., 2006 and Nutt, 2008). Unlike the bicalyx mutant, the cp petal homeotic mutant of C. tembloriensis should probably be explored further as a model system to study floral evolution. As described above, it occurs in relatively high frequency in a number of natural populations. It is an annual plant, completing its life cycle in a single year. In addition, Smith-Huerta and Huerta (2015) established the reproductive fitness characteristics of WT vs cp plants. The only criterion not fully met for suitability as a model system is a full molecular characterization of the mutation. It has been established that the mutation is controlled by a single recessive gene (Vasek, 1966, Vasek and Harding, 1976) and several markers bordering the CRINKLED PETAL gene have been identified in a linkage map produced by Leong et al., (2001). However, additional molecular analysis is necessary to meet the criterion of molecular characterization required for model development (Nutt, 2008). The likely reasons for the limited descriptions of naturally occurring homeotic mutants such as cp of C. tembloriensis, are that they are generally rare and occur in low frequencies in natural populations. They have often been viewed by modern researchers as having little evolutionary significance and have thus have been simply ignored by researchers (Hintz et al., 2006). Continued study of this species would be highly valuable to further the understanding of floral development, evolution, the interaction of the environment on the species in the face of climate change, and of this species as a whole.

Future Studies Genetic analysis should be the next step in evaluating this species to better determine the mechanism behind this one whorled homeotic mutation. It has been suggested, that alleles of the homeotic genes that express weakly could be responsible for producing single whorl homeotic mutations (van der Krol & Chua, 1993). It has been seen in Antirrhinum that differing mutant alleles of the DEFICIENS-A (DEFA) B-type homeotic gene produce individuals that develop varying second and third whorls (Schwarz-Sommer et al., 1990). A null allele could also be a

possibility as was the case for a line of gp, evaluated by van der Krol & Chua (1993), after a chromosomal deletion. Exploration into pMADS1, a petunia specific MADS box gene that has a role in 2nd and 3rd whorl development (van der Krol et al., 1993), might reveal more about the timing of development of the cp petal and be able to lead to an explanation on some of the cp petal hybrid characteristics and why they all are highly plastid. Experiments by van der Krol et al. (1993) on petunia with pMADS1 restoration and suppression produced multiple types of flowers with mutant petals containing differing degrees of sepal and petal characteristics. Under normal petunia WT petal development, the early and continued differentiation of cells characteristic to petals will hinder cells differentiating into ones characteristic of sepals, however, if petal destoned cells are delayed in development in localized areas of the organ, this will then result in cells developing more sepaloid characteristics in those areas (van der Krol & Chua, 1993). Ultimately, the time of activation of the petal differentiation pathway determines how far cells complete differentiation into a petaloid cell (van der Krol et al., 1993). In the transgenic pMADS1 petunias, the petal differentiation process was delayed more than that in the WT petal, which resulted in more sepaloid characteristic development and a sepaloid phenotypic appearance (van der Krol & Chua, 1993).

Conclusions The results of this anatomical study, demonstrate that the cp petals of Clarkia tembloriensis exhibit characteristics of both sepals and WT petals. The hypothesis proposed in this investigation, that cp petals are unique hybrid organs, was supported by the results of this study. Furthermore, this study complements and extends the previous investigations conducted by Smith-Huerta (1992, 1996) who first documented the hybrid nature of the cp petal mutant.

A B Figure 1. Drawing of C. tembloriensis sepal, WT petal, and cp petal. Blue squares indicate areas viewed and surveyed for A) trichome, stomata (blue box) and B) margin characteristics (blue box). Bars = 2mm. Modified from (Smith-Huerta, 1992).

A B C Figure 2. SEM images of floral organ trichomes. A) WT petal adaxial surface showing a conical (red arrow) and clavate trichome (blue arrow). B) Sepal abaxial surface showing a falcate trichome (yellow arrow). C) cp petal adaxial surface showing a glandular trichome with a multicellular head (green arrow). Bars = 10μm.

Table 1 Average Count of Trichomes on the Adaxial and Abaxial Side of Mature Sepal, WT Petal, and cp Petal at the Apex, Middle of the Sepal/Base of the Limb in Petals, and

Base of Sepal/Claw of Petals Adaxial Surface Floral organ

Sepal WT petal cp petal

Trichome

Falcate Conical Glandular Clavate Falcate Conical Glandular Clavate Falcate Conical Glandular type Clavate Total/sec Total/sec Total/sec Apex 1.6 0.2 0.0 0.4 2.2 12.0 0.0 0.6 0.6 13.2 8.2 0.0 7.0 1.6 16.8 Middle/ Base Limb 1.2 0.0 0.0 0.0 1.2 13.8 0.0 1.2 2.6 17.6 2.8 0.0 2.4 1.4 6.6 Base/ Claw 0.6 0.0 0.0 0.6 1.2 10.6 0.0 1.2 1.8 13.6 2.2 0.0 1.8 0.8 4.8 Ave Total/ Trichome 3.4 0.2 0.0 1.0 33.6 0.0 3.0 5.0 13.2 0.0 11.2 3.8 Overall Ave Total 4.6 44.4 28.2 Abaxial Surface

Floral organ Sepal WT Petal cp Petal

Trichome

Falcate Conical Glandular Clavate Falcate Conical Glandular Clavate Falcate Conical Glandular type Clavate Total/sec Total/sec Total/sec Apex 9.8 47.0 0.0 11.6 68.4 0.0 0.0 0.0 0.0 0.0 21.0 21.0 0.0 1.8 43.8 Middle/ Base 7.2 22.0 0.0 11.6 40.8 3.2 0.0 0.0 0.0 3.2 13.6 13.6 0.0 1.4 28.6 Limb Base/ Claw 6.8 18.4 0.0 13.6 38.8 14.2 0.0 0.0 0.0 14.2 7.2 7.2 0.0 0.8 15.2

Ave Total/ 23.8 87.4 0.0 36.8 17.4 0.0 0.0 0.0 41.8 29.2 0.0 4.0 Trichome Overall Ave Total 148.0 17.4 75.0 Total Trichome Type /organ 27.2 87.6 0.0 37.8 51.0 0.0 3.0 5.0 55.0 29.2 11.2 7.8 Total Trichomes of organ 152.6 61.8 103.2 Notes. Surveys were over a 0.55mm2 area. Middle and base refer to positions on the sepal and base if the limb and claw refer to positions on the petals, which are equivalent to one another. n=5.

Figure 3. Sepals of developing floral bud prior to postgenital fusion. Developing trichomes (orange arrow). Cells that will undergo postgenital fusion are indicated with a purple arrow. Bar = 100μm.

A B

Adaxia l

Abaxia l C D Figure 4. Trichomes on WT petal and cp petal in 3.0mm buds. A) SEM (abaxial side seen) of WT petal and B) cp petal, C) Light micrograph of WT petal and D) cp petal. Trichomes (orange arrows). Clavate trichome (blue circle), falcate trichome (yellow circle), glandular trichome (green circle). Bars = 100μm.

Figure 5. Image of a stomata surrounded by specialized epidermal cells called guard cells on a C. tembloriensis sepal. Bar = 50µm.

Table 2 Average Total Counts of Stomata on the Adaxial and Abaxial Sides of the Sepal, WT Petal, and cp Petal on the Apex, Middle/Base Limb, and Base/Claw Positions of the Organs Adaxial Surface Average Total/sect. Sepal WT Petal cp Petal Apex 4.8 6.0 11.8 Middle/Base Limb 4.0 16.6 5.8 Base/Claw 4.6 6.2 7.8 Average Total Adaxial 13.4 28.8 25.4 Abaxial Surface Average Total/sect. Sepal WT Petal cp Petal Apex 53.2 1.6 16.4 Middle/ Base Limb 40.4 4.4 21.6 Base/Claw 36.2 3.6 15.8 Average Total Abaxial 129.8 9.6 53.8 Average Total 143.2 38.4 79.2 Notes. Middle and base refer to positions on the sepal and base of the limb and claw refer to positions on the petals, which are morphologically equivalent to one another. n=5.

A B

C D Figure 6. Light micrographs continuing basipetally through the bud with a partially fused set of sepals. A) Fused sepals near the apex, B-C) junction between sepals at the mid position of the bud, D) base near the hypanthium. Bars = 50 μm.

B Figure 7. Light micrographs of sepal margins in mature buds 1-day pre anthesis. A) Separated sepal showing REC (white arrow) and surrounding epidermal cells (green arrow), B) higher magnification of REC (yellow arrow). Bars = 50µm.

Abaxial

Adaxial

Figure 8. Side view of mature sepal tip after anthesis. Flat epidermal cell consisting of pavement cells with developed striations (red arrow), partially flat cell that was partially dedifferentiating into a REC, but then stopped part way and developed striations (purple arrow), round previously parenchyma REC that developed striations (yellow arrow), narrow elongated papilliform PGF cell with pointed cuticular tips and no striations (green arrow), rounded previously parenchyma REC with no striations (gray arrow), large rounded cell were some had an indent formed from the adjacent sepal cells (blue arrow). Bar = 10 μm.

A B C Figure 9. SEM images of sepal edge after anthesis. A) near the apex, B) midsection, and C) base of sepal. Elongated non-striated papilliform REC (green arrow). Narrow elongated dedifferentiated PGF cell not in papilliform shape (gray arrow). Bars = 10 μm.

A B Figure 10. Margins and edge of WT petal after anthesis. A) Fat and rounded cells with thin striations, B) edge with trichome. Trichome (orange arrow). Bars = 50µm.

Figure 11. SEM images of WT petal edges post anthesis at the apex. Bar = 10 μm.

Figure 12. Tip of anthesis WT petal lobe with stomata on the margin. Bar = 100 μm.

A B C D Figure 13. SEM images of WT petal margin after anthesis progressing acropetally. A) claw, B) base of the limb C) middle limb, D) Near the apex. Cells with a rounded up lifted portion (orange arrow). Bars = 10 μm.

A B

C D Figure 14. Light micrograph images of one day pre anthesis cp petal edges and PGF. A) Typical cp petal edge, B) papilliform cell with cuticular projection, C) potentially reacting epidermal cells on side of sepal to cp petal edge, D) cp petal potentially fusing with sepal side (image by Smith-Huerta). REC at edge (blue arrow), non-PGF epidermal cells with stations (red arrow), papilliform cells (yellow arrow), sepal epidermal cells potentially reacting to the REC in adjacent cp petal (orange cells). Bars = 100µm.

A B

C Figure 15. Various SEM images of cp petal post anthesis at the apex. A) Apex with striated elongated and short cells and trichome, B) apex with cells in multiple stages of development, C) apex with striated previously REC. Short striated epidermal cell (red arrow), un-striated REC cell (blue arrow), dedifferentiated parially striated epidermal cell (purple arrow), REC that became striated epidermal cell (yellow arrow), trichomes (orange arrow), unstriated elongate PGF cell (gray arrow). Bar = 50 μm.

Figure 16. SEM image of cp petal lobe post anthesis near the apex. Elongated TFS epidermal cells with thick wavy striations (green arrow), stomata (pink arrow), short cells (dark gray arrow), elongate REC (orange arrow). Bar = 100 μm.

Figure 17. Light micrograph image of cp petal lobe post anthesis near the apex. Elongated TFS epidermal cells with thick wavy striations (green arrow), stomata (pink arrow). Bar = 100 μm.

A B C D Figure 18. SEM images of cp petal edges post anthesis progressing acropetally. A) Claw, B) base of the limb, C) middle limb, D) near the apex. Elongated non-papilliform REC with no striations (gray arrow), elongated REC with rounded and outward projecting end (green arrow), short and slightly wider cells (dark gray arrow). Bars = 10 μm.

Figure 19. Floral meristem showing the progression of REC on the developing sepals. Red brackets show two of the oldest sepals (#1, #2) with REC just starting to develop. In a slightly older floral apical meristem the two youngest sepals (#3, 4) shown in red brackets, are beginning to develop REC. Bar = 100 μm.

A B

C Figure 20. Closing floral buds. A) REC developing basipetally along the edge of the sepal (yellow bracket), B) REC suturing along the edge of the sepals in an older bud (yellow bracket), C) cells extending out to the adjacent sepal (grey arrow). Bars = 100µm.

A B

C D Figure 21. Light micrograph section of the apical region of floral bud. A) Position just above sepal junction (purple arrow- papilliform REC), B) middle of junction, C) starting to open up into bud chamber (blue arrow), D) past sepal tip junction (yellow box- fused REC only along edges of sepals at junctions, purple arrow- projecting REC). Samples prepared by Mary Carol Johantgen. Bars = 100 μm.

A D

B E

C F Figure 22. Light micrograph sections through a single bud with a partially fused set of sepals. A-F) Shows the progression of sepal fusion from the side of a partially fussed bud progressing basipetally. These images illustrate how the sepals merged together during growth. Anticlinal directed cell division (red arrows), rounded and extended out epidermal cell (blue arrow), formation of a cuticular projection on the rounded and extended out epidermal cell (green arrow), periclinal-directed cell division in mesophyll (yellow arrows), periclinal-directed cell division in epidermis (purple arrow). Bars = 100 μm.

A B Figure 23. SEM images of young petals in 1.0mm floral buds. A) WT petal, B) cp petal. Comparing edges on petals (purple arrow), area REC seen on edge of cp petal (orange bracket area estimation based on light microscopy section observations). Bars = 50 µm.

A B Figure 24. SEM image of petals in 1.6mm floral buds. A) WT petal, B) cp petal. Edges of petals (purple arrow), area REC seen on edge of cp petal (orange bracket area estimation based on light microscopy section observations). Bars = 100 µm.

A B Figure 25. Light microscopy sections of petals in 1.6mm floral buds. A) WT petal with smooth edges, B) cp petal with bumpy edge from REC. Compare cells on edges of petals (purple arrow). Bars = 100 µm.

A B Figure 26. Light microscopy sections of petals in 1.0mm floral buds. A) WT petal with smooth edges, B) cp petal with bumpy edge from REC. Compare cells on the edges of petals (purple arrow). Bars = 100 µm.

A B Figure 27. Light microscopy sections of petals in 3.0mm floral buds. A) WT petal with smooth edges, B) cp petal with bumpy edges from REC. Compare cells on edges of petals (purple arrow). Bars = 100 µm.

A B Figure 28. SEM image of petals in 3.0mm floral buds. A) WT petal, B) cp petal. Comparing cells on edges on petals (purple arrow), area REC seen on edge of cp petal (orange bracket area estimation based on light microscopy section observations). Bars = 100 µm.

A B Figure 29. cp petal flowers during anthesis illustrating the different degrees of obstruction by the sepal hood. A) Petals penetrate though bud base to be exposed, but the sepal hood remains covering the flower face and some stamens, B) floral face is exposed, but the sepal hood is wrapped around and obstructing the petals exposure. Bars = 15mm.

Reference

Aceto, S., & Gaudio, L. (2011). The MADS and the beauty: genes involved in the development of orchid flowers. Current genomics, 12(5), 342-356.

Archer, S. R., & Predick, K. I. (2008). Climate change and ecosystems of the southwestern United States. Rangelands, 30(3), 23-29.

Bailes, E. J., & Glover, B. J. (2018). Intraspecific variation in the petal epidermal cell morphology of Vicia faba L. (Fabaceae). Flora, 244, 29-36.

Baum, H. (1948a). Postgenitale Ver· wachsung in und zwischen Karpel!· und Staubblattkreisen. Sitzungsber. Oesterr. Akad. Wiss. Math.·Naturwiss. Kl. Abt. II 1 57:17-38 (In German).

Baum, H. (1948b). Uber die postgenitale Verwachsung in Karpellen. Osterr. Bot. z. 95:86-94 (In German).

Beidleman, L. H., & Kozloff, E. N. (2014). Plants of the San Francisco Bay Region: Mendocino to Monterey. Univ of California Press.

Benedito, V. A., Angenent, G. C., Van Tuyl, J. M., & Krens, F. A. (2003). Lilium longiflorum and molecular floral development: the ABCDE model. In XXI International Eucarpia Symposium on Classical versus Molecular Breeding of Ornamentals-Part II 651 (pp. 83-89).

Benedito, V. A., Angenent, G. C., & Krens, F., (n.a.), A. FLORAL HOMEOTIC MUTANTS IN LILY.

Boke, N. H. (1948). Development of the perianth in Vinca rosea L. American Journal of Botany, 413-423.

Callow, J. A., Hallahan, D. L., & Gray, J. C. (Eds.). (2000). Plant trichomes (Vol. 31). Elsevier.

Carpenter, R., & Coen, E. S. (1990). Floral homeotic mutations produced by transposon- mutagenesis in Antirrhinum majus. Genes & Development, 4(9), 1483-1493.

Cildir, H., Kahraman, A., & Dogan, M. (2012). Petal and sepal epidermal micromorphology of six Lathyrus taxa (Fabaceae) and their systematic value. Notulae Botanicae Horti Agrobo tanici Cluj-Napoca, 40(1), 35-41.

Coen, E. S., & Meyerowitz, E. M. (1991). The war of the whorls: genetic interactions controlling flower development. Nature, 353(6339), 31.

Cusick, F. (1966) On phylogenetic and ontogenetic fusions. In: Trends in plant morphogenesis, pp. 170-183, Cutter, E.G., ed. Longmans, Green and Co., London

Endress, P. K. (1996). Diversity and evolutionary biology of tropical flowers. Cambridge University Press.

Ford, V. S., & Gottlieb, L. D. (1992). Bicalyx is a natural homeotic floral variant. Nature, 358(6388), 671-673.

Gibson, A. C. (1998). Photosynthetic organs of desert plants. Bioscience, 48(11), 911-920.

Glover, B. J. (2000). Differentiation in plant epidermal cells. Journal of Experimental Botany, 51(344), 497-505.

Gómez, P., Jamilena, M., Capel, J., Zurita, S., Angosto, T., & Lozano, R. (1999). Stamenless, a tomato mutant with homeotic conversions in petals and stamens. Planta, 209(2), 172-179.

González-Varo, J. P., Biesmeijer, J. C., Bommarco, R., Potts, S. G., Schweiger, O., Smith, H. G., ... & Vilà, M. (2013). Combined effects of global change pressures on animal-mediated pollination. Trends in ecology & evolution, 28(9), 524-530.

Halfter, U., Ali, N. A. Z. E. E. M., Stockhaus, J., Ren, L., & Chua, N. H. (1994). Ectopic expression of a single homeotic gene, the Petunia gene green petal, is sufficient to convert sepals to petaloid organs. The EMBO journal, 13(6), 1443-1449.

Hase, Y., Tanaka, A., Baba, T., & Watanabe, H. (2000). FRL1 is required for petal and sepal development in Arabidopsis. The Plant Journal, 24(1), 21-32.

Hintz, M., Bartholmes, C., Nutt, P., Ziermann, J., Hameister, S., Neuffer, B., & Theissen, G. (2006). Catching a ‘hopeful monster’: shepherd's purse (Capsella bursa-pastoris) as a model system to study the evolution of flower development. Journal of Experimental Botany, 57(13), 3531-3542.

Keating, R. C. (1982). The evolution and systematics of Onagraceae: leaf anatomy. Annals of the Missouri Botanical Garden, 770-803.

Kotilainen, M., Elomaa, P., Uimari, A., Albert, V. A., Yu, D., & Teeri, T. H. (2000). GRCD1, an AGL2-like MADS box gene, participates in the C function during stamen development in Gerbera hybrida. The Plant Cell, 12(10), 1893-1902.

Krizek, B. A., & Meyerowitz, E. M. (1996). The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development, 122(1), 11-22.

Le Conte, Y., & Navajas, M. (2008). Climate change: impact on honey bee populations and diseases. Revue Scientifique et Technique-Office International des Epizooties, 27(2), 499- 510.

Leong, L. L. Y., Wilson, K. G., & Smith-Huerta, N. L. (2001). A Linkage Map for CRINKLED PETAL: A Homeotic Gene of Clarkia tembloriensis (Onagraceae). Journal of Heredity, 92(1), 78-81.

Lewis, H. (1953). The mechanism of evolution in the genus Clarkia. Evolution, 7(1), 1-20.

Lewis, H. and Margaret E. Lewis. (1955). The genus Clarkia. Univ. Calif. Publ. Bot., 20: 241-392.

Lewis, H. (2012), Clarkia tembloriensis subsp. tembloriensis, in Jepson Flora Project (eds.) Jepson eFlora, http://ucjeps.berkeley.edu/eflora/eflora_display.php?tid=49915, accessed on August 09, 2018.

Lolle, S. J., Cheung, A. Y., & Sussex, I. M. (1992). Fiddlehead: an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells. Developmental biology, 152(2), 383-392.

Lolle, S. J., & Pruitt, R. E. (1999). Epidermal cell interactions: a case for local talk. Trends in plant science, 4(1), 14-20.

Mach, J. (2012). A Petunia Twist on the ABC Model of Floral Organ Specification. Plant Cell, 24(6), 2237.

Maheshwari, P. (1963). Embryology in relation to taxonomy. Recent researches in plant taxonomy, 55-97.

Miranda, J. D. D., Armas, C., Padilla, F. M., & Pugnaire, F. I. (2011). Climatic change and rainfall patterns: effects on semi-arid plant communities of the Iberian Southeast. Journal of Arid Environments, 75(12), 1302-1309.

Murai, K. (2013). Homeotic genes and the ABCDE model for floral organ formation in wheat. Plants, 2(3), 379-395.

Nutt, P. (2008). Characterisation of the natural homeotic variety Stamenoid petals (Spe) in the Sherpherd'Purse (Capsella bursa-pastoris): establishment of a new model system (Doctoral dissertation, Jena, Univ., Diss., 2008).

Ojeda, I., Francisco-Ortega, J., & Cronk, Q. C. (2009). Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity. Annals of botany, 104(6), 1099-1110.

Pruitt, R. E., Vielle-Calzada, J. P., Ploense, S. E., Grossniklaus, U., & Lolle, S. J. (2000).

FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proceedings of the National Academy of Sciences, 97(3), 1311-1316.

Renner, O. (1959). Somatic conversion in the heredity of the cruciata character in Oenothera. Heredity, 13, 283-288.

Roberts, K. (Ed.). (2007). Handbook of plant science (Vol. 1). John Wiley & Sons.

Ruzin, S. E. (1999). Plant microtechnique and microscopy (Vol. 198). New York: Oxford University Press.

Schwarz-Sommer, Z., Huijser, P., Nacken, W., Saedler, H., & Sommer, H. (1990). Genetic control of flower development by homeotic genes in Antirrhinum majus. Science, 250(4983), 931.

Sherry, R. A., & Lord, E. M. (1996). Developmental stability in flowers of Clarkia tembloriensis (Onagraceae). Journal of Evolutionary Biology, 9(6), 911-930.

Sherry, R. A., & Lord, E. M. (2000). A comparative developmental study of the selfing and outcrossing flowers of Clarkia tembloriensis (Onagraceae). International Journal of Plant Sciences, 161(4), 563-574.

Smith-Huerta, N. L. (1992). A comparison of floral development in wild type and a homeotic sepaloid petal mutant of Clarkia tembloriensis (Onagraceae). American journal of botany, 1423-1430.

Smith-Huerta, N. L. (1996). A comparison of venation pattern development in wild type and a homeotic sepaloid petal mutant of Clarkia tembloriensis (Onagraceae). American journal of botany, 712-716.

Smith-Huerta, N. L., & Huerta, A. J. (2015). Floral biology and the evolution of selfing in natural populations of Clarkia tembloriensis Vasek (Onagraceae). Journal of the Torrey Botanical Society, 142(3), 240-248.

Sytsma, K. J. & Smith, J. F. (1992). Molecular systematics of Onagraceae: examples from Clarkia and Fuchsia. In: Molecular systematics of plants (P.S. Soltis, D.E. Soltis & J.J. Doyle, eds), pp. 245. Springer US, New York.

Theißen, G., & Saedler, H. (2001). Plant biology: floral quartets. Nature,409(6819), 469-471.

Theißen, G., Melzer, R., & Rümpler, F. (2016). MADS-domain transcription factors and the floral quartet model of flower development: linking plant development and evolution. Development, 143(18), 3259-3271.

Tsuchimoto, S., Mayama, T., van der Krol, A., & Ohtsubo, E. (2000). The whorl‐specific action of a petunia class B floral homeotic gene. Genes to Cells, 5(2), 89-99. van der Krol, A. R., Brunelle, A., Tsuchimoto, S., & Chua, N. H. (1993). Functional analysis of petunia floral homeotic MADS box gene pMADS1. Genes & Development, 7(7a), 1214- 1228. van der Krol, A. R., & Chua, N. H. (1993). Flower Development in Petunia. The Plant Cell, 5(10), 1195. van der Schoot, C., Dietrich, M. A., Storms, M., Verbeke, J. A., & Lucas, W. J. (1995). Establishment of a cell-to-cell communication pathway between separate carpels during gynoecium development. Planta, 195(3), 450-455.

Vasek, F. C. (1964). The evolution of Clarkia unguiculata derivatives adapted to relatively xeric environments. Evolution, 18(1), 26-42.

Vasek, F. C. (1966). A case of gene homology in Clarkia. Evolution, 20(2), 243-244.

Vasek, F. C., & Harding, J. (1976). Outcrossing in natural populations. V. Analysis of outcrossing, inbreeding, and selection in Clarkia exilis and Clarkia tembloriensis. Evolution, 30(3), 403-411.

Vasek, F. C. (1977). Phenotypic variation and adaptation in Clarkia section Phaeostoma. Systematic Botany, 251-279.

Vasek, F. C. (2008). A taxonomic reassessment of Clarkia calientensis and Clarkia tembloriensis. Madroño, 55(4), 297-302.

Verbeke, J. A. (1992). Fusion events during floral morphogenesis. Annual review of plant biology, 43(1), 583-598.

Vialette‐Guiraud, A. C., & Scutt, C. P. (2018). Carpel evolution. Annual Plant Reviews online, 1- 34.

Walker, D.B. (1975a) Postgenital carpel fusion in Catharanthus roseus (Apocynaceae). I. Light and scanning electron microscopic study of gynoecial ontogeny. Am. J. Bot. 62, 457-467

Walker, D.B. (1975b) Postgenital carpel fusion in Catharanthus roseus (Apocynaceae). III. Fine structure of the epidermis during and after fusion. Protoplasma 86, 43-63

Weberling, F. (1989). Morphology of flowers and inflorescences. [Transl. by RJ Pankhurst.]. Cambridge Univ. Press: Cambridge, 405, 236-7.

Whitney, H. M., Bennett, K. V., Dorling, M., Sandbach, L., Prince, D., Chittka, L., & Glover, B. J. (2011). Why do so many petals have conical epidermal cells?. Annals of botany, 108(4), 609-616.

Yanofsky, M. F., Ma, H., Bowman, J. L., Drews, G. N., Feldmann, K. A., & Meyerowitz, E. M. (1990). The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature, 346(6279), 35.

Yang, S. J., Ohno, S., Deguchi, A., Sato, M., Goto, M., Doi, M., ... & Hosokawa, M. (2017). The histological study in sympetalous corolla development of pinwheel-type flowers of Saintpaulia. Scientia Horticulturae, 223, 10-18.

Zahn, L. M., Leebens-Mack, J., DePamphilis, C. W., Ma, H., & Theissen, G. (2005). To B or not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms. Journal of Heredity, 96(3), 225

Appendix 1 Trichome Elemental Analysis Each of the trichome types were subject to elemental analysis. No difference was found in elemental composition between individual trichome, however, the falcate trichome’s surface did reveal a higher relative abundance of calcium. Spot analysis of the falcate surface indicated that this higher concentration of calcium was located on their verrucate projections and was most likely in the form of calcium carbonate, which made up about 37.67% of the mass analyzed (Table 3). The conical trichome elemental analysis (figure 30.A) was used as a representative for the other trichome, since none of them differed much from one another, to compare to the falcate trichome (figure 30.B). In the conical trichome, Ca was at a much smaller relative abundance, as demonstrated by the conical trichome graph requiring the enlargement of the Y-axis to see the Ca peak (blue arrows, figure 30.A). In contrast, the falcate trichome had a larger relative abundance of Ca, as demonstrated by the graph, which did not require enlarging as much to see (blue arrows, figure 30.B). Additionally, within the falcate trichome graph, one could see that the Ca peak was similar in height to the Na and Mg peaks (yellow circle), but that the K peak was too small to see (green circle). In contrast, the conical trichome graph showed the Ca peak similar to the K peak in height and both smaller than the Na and Mg peaks.

Table 3 Spot Elemental Analysis of the Surface of the Falcate Trichome

Notes. Yellow underline highlights potential calcium source and mass norm.

35.0 1.0

N Mg K Ca Na M Ca a g

A B Figure 30. Conical and falcate trichome elemental analysis. (X-axis= energy (keV), Y-axis = relative abundance). Conical trichome graph requires enlargement to see the Ca peak, (blue arrows) relative to falcate trichome. A) Conical: Ca peak is shown as similar in height to K (green circle) and smaller than Na and Mg peaks (yellow circle). B) Falcate: K peak does not appear and Ca appears similar in height to Na and Mg peak.