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 by Chelsea E. Obrebski Miami University Oxford, Ohio 2019 Advisor: Dr. Nancy Smith-Huerta Reader: Dr. Alfredo Huerta Reader: Dr. Carolyn Keiffer ©2019 Chelsea E. Obrebski This Thesis titled AN ANATOMICAL COMPARISON OF WILD TYPE AND HOMEOTIC MUTANT FLOWERS OF CLARKIA TEMBLORIENSIS by 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 iii 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 iv 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 v 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