East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 12-2007 Evidence of Ecological Speciation in Phacelia. Pamela Michele Glass East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Botany Commons Recommended Citation Glass, Pamela Michele, "Evidence of Ecological Speciation in Phacelia." (2007). Electronic Theses and Dissertations. Paper 2143. https://dc.etsu.edu/etd/2143 This Thesis - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. Evidence of Ecological Speciation in Phacelia A thesis presented to the faculty Department of Biological Sciences East Tennessee State University In partial fulfillment of the requirements for the degree Masters of Science in Biological Sciences by Pamela Michele Glass December 2007 Foster Levy, Ph.D. - Chair Dhirendra Kumar, Ph.D. Timothy McDowell, Ph.D. Keywords: Phacelia, ecological speciation, internal transcribed spacer (ITS), reproductive isolation, seed germination, pollen tubes ABSTRACT Evidence of Ecological Speciation in Phacelia by Pamela Michele Glass Phacelia purshii Buckley and P. fimbriata Micheaux are two species that are nearly morphologically indistinguishable. Seed germination experiments showed that the high elevation endemic, P. fimbriata requires lower temperatures to trigger germination. Following interspecific crosses, pollen tubes enter ovules and maternal tissue of the gynoecium matures but hybrid diploid and triploid organs fail to develop. DNA sequences from the ribosomal DNA internal transcribed region showed that P. fimbriata and P. purshii comprise a monophyletic clade but that P. fimbriata is more differentiated from related species. In contrast, P. purshii supported significantly higher levels of intraspecific polymorphism. Phacelia fimbriata and P. purshii are sister species with similar morphology but they are unable to hybridize, they are differentiated in physiological characteristics related to environment, and they inhabit different elevations. This pattern of relationship and differentiation suggests P. fimbriata may be the product of ecological speciation. 2 CONTENTS Page ABSTRACT .......................................................................................................................... 2 LIST OF TABLES ................................................................................................................ 5 LIST OF FIGURES .............................................................................................................. 7 Chapter 1. INTRODUCTION ........................................................................................................ 9 2. METHODS ................................................................................................................... 19 Ecological Analysis................................................................................................... 19 Hybridization Experiment.......................................................................................... 22 Pollen Tube Visualization ......................................................................................... 24 Phylogenetic Analysis................................................................................................ 26 3. RESULTS ..................................................................................................................... 29 Germination Experiments .......................................................................................... 29 Hybridization Experiment.......................................................................................... 33 Pollen Tube Experiments .......................................................................................... 41 Molecular Evidence of Phylogenetic Relationships and Differentiation................... 45 4. DISCUSSION ............................................................................................................... 54 Germination Analysis................................................................................................ 54 Hybridization Analysis.............................................................................................. 55 Pollen Tube Analysis ................................................................................................ 56 Phylogenetic Analysis................................................................................................ 56 Genetic Diversity....................................................................................................... 57 3 Chapter Page Systematic Implications ............................................................................................ 57 REFERENCES ...................................................................................................................... 67 APPENDICES ....................................................................................................................... 71 Appendix A: Phacelia purshii and P. fimbriata Field Germination Data Collected During Fall 2005 ....................................................................................................... 71 Appendix B: Phacelia purshii and P. fimbriata Laboratory Germination Data Collected Fall 2005 ................................................................................................................... 72 Appendix C: Phacelia fimbriata and P. purshii Pollen Tube Data .......................... 75 Appendix D: DNA Sequences Aligned Using Multalin (Corpet 1988) ................... 77 VITA ..................................................................................................................................... 78 4 LIST OF TABLES Table Page 1. Phacelia fimbriata and P. purshii Populations used for the Germination and Hybridization Studies ....................................................................................................................... 20 2. Crossing Design for P. purshii, P. fimbriata, and P. dubia Hybridizations ...................... 23 3. Crossing Design for P. purshii ♀ x P. fimbriata ♂ Interspecific Crosses ........................ 24 4. Crossing Design for P. fimbriata ♀ x P. purshii ♂ Interspecific Crosses ....................... 24 5. Crossing Design for P. fimbriata and P. purshii Pollen Tube Assay ................................ 25 6. Results of Nested ANOVA Comparing Numbers of Field-Germinated Seeds Between Species and Populations of P. fimbriata and P. purshii ............................................ 31 7. Results of One-Way ANOVAs Comparing Numbers of Field-Germinated Seeds Among Populations Within Species....................................................................................... 31 8. Nested ANOVA Comparison of Numbers of Laboratory-Germinated Seeds Between Species and Populations of P. fimbriata and P. purshii ........................................... 33 9. Interspecific and Intraspecific Crossing Combinations Sorted by Species and Population 34 10. Qualitative Summary of Results from Controlled Crosses.............................................. 35 11. Test for Heterogeneity Among Researchers in Percentage of Swollen and Not Swollen Ovaries ....................................................................................................................... 39 12. Analysis of Frequencies for Intra- and Interspecific Crosses Comparing Swollen and Not Swollen Ovaries ................................................................................................. 40 13. Comparison of Frequencies of Fertilization in Crosses Preserved at 4-Hours and 24-Hours Using Fisher’s Exact Test.......................................................................... 42 14. Potential Reproductive Barriers and Evidence for Their Applicability to Hybridization of P. fimbriata and P. purshii ................................................................................... 44 5 Table Page 15. Summary of the P. fimbriata and P. purshii Sequences Used in the DNA Phylogenetic Analysis...................................................................................................................... 45 16. A List of the Sequences Downloaded from NCBI Database........................................... 45 17. Analysis of Frequencies for ITS DNA Sequence Similarities for P. fimbriata and P. purshii.................................................................................................................... 46 18. Summary of All Variable Sites (Excluding Insertions and Deletions) in Phacelia Subgenus Cosmanthus .............................................................................................. 47 19. Point Mutations for Phacelia Subgenus Cosmanthus When Compared to Western Phacelia Species (See List From Table 16) .............................................................. 49 20. Synapomorphies for P. fimbriata and P. purshii ............................................................ 49 21. Point Mutations Unique to P. fimbriata .......................................................................... 49 22. Point Mutations Unique to P. dubia With the Exception of Position 676 Shared with P. ranunculacea