TAXONOMIC AND PHYLOGENETIC EVALUATION OF STYLISMA (CONVOLVULACEAE) Sondi Jones Hoffman A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Department of Biology and Marine Biology University of North Carolina Wilmington 2009 Approved by Advisory Committee _ __Dr. Ami Wilbur______ _Dr. Michael Durako ______ ______Dr. Gregory Chandler Chair Accepted by Dean, Graduate School TABLE OF CONTENTS ABSTRACT.........................................................................................................................v ACKNOWLEDGMENTS ................................................................................................ vi DEDICATION ................................................................................................................. vii LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix CHAPTER 1. GENERAL INTRODUCTION .................................................................. 1 Stylisma....................................................................................................................1 Review of taxa and taxonomic history ....................................................................2 Species concepts ......................................................................................................9 Current taxonomy ..................................................................................................11 Conservation ..........................................................................................................11 Objectives ..............................................................................................................12 Hypothesis..............................................................................................................12 Methodology..........................................................................................................14 Morphometric vs. molecular techniques................................................................14 Introduction to morphological methodology .........................................................14 Ordination ..............................................................................................................15 Introduction to molecular methodology.................................................................16 The chloroplast genome.........................................................................................15 CHAPTER 2. MATERIALS AND METHODS ..............................................................20 Morphological methods .........................................................................................20 Taxon sampling......................................................................................................20 iii Morphological measurements................................................................................20 Morphological data analysis ..................................................................................23 Molecular methods.................................................................................................26 Taxon sampling......................................................................................................26 DNA extraction, amplification, and sequencing....................................................26 Sequence data analysis...........................................................................................31 CHAPTER 3 RESULTS....................................................................................................32 Morphological results ............................................................................................32 Molecular results....................................................................................................49 Sequence characteristics ........................................................................................49 Phylogenetic reconstruction...................................................................................52 Major clades...........................................................................................................54 CHAPTER 4. DISCUSSION............................................................................................55 Morphology............................................................................................................55 Molecular genetics.................................................................................................58 Conclusions............................................................................................................60 CHAPTER 5. DESCRIPTION OF PROPOSED TAXA..................................................65 Stylisma abdita.......................................................................................................65 Stylisma pickeringii................................................................................................65 Stylisma paten ........................................................................................................66 LITERATURE CITED ......................................................................................................68 iv ABSTRACT Stylisma (Convolvulaceae) is a small genus of dawnflowers found in the sandhills of southeastern United States. The genus is in need of a taxonomic revision because the last revision was long ago, it did not appropriately resolve the relationships within the genus, and there are several threatened or endangered taxa in the genus. A phylogenetic study of the genus is presented here, using two chloroplast markers, the trnT-L intergeneric spacer and the rps16 intron. A phenetic study of the genus is also presented examining 40 characters in 430 specimens and analyzing the resulting matrix with ordination (PCA) methods. Both data sets result in three clades of similar topology. The molecular data combines Stylisma patens and Stylisma humistrata into a single clade, Stylisma pickeringii into a monospecific clade, and Stylisma villosa, Stylisma abdita, and Stylisma aquatica into a separate clade. The morphological data agree with the molecular data as to the placement of S. pickeringii into its own clade, but separates S. abdita into a second clade and lumps the remainder of the genus into a third clade. The combined morphological and molecular results suggest Stylisma to be a complex group that requires more work to determine the spurious species boundaries. v ACKNOWLEDGMENTS This thesis would not have been possible without the help of my advisor, Gregory Chandler, who was available to me day and night throughout this process. I must also thank my committee, Ami Wilbur and Mike Durako. A special thanks goes out to Steven Brewer, who was such a great help, even from Belize. Thanks to Marcel van Tuinen for the use of lab space. Thanks to David Webster and Craig Bailey for all of their support. I would like to thank Florida State University, University of South Florida, Rutgers University, University of North Carolina, and Kansas State University for providing herbarium loans. I would like to extend thanks to my field crew, Dallas, Justin, and Erich. Amy Cherry Millis was invaluable, as she provided a sounding board for the entire project. A huge thanks is in order for the biology office staff, especially Tracie and Lori, without who I would surely have failed. vi DEDICATION I would like to dedicate this thesis to my husband, Dallas, whose continued support and encouragement have been invaluable. vii LIST OF TABLES Table Page 1. Stearn’s leaf terms...........................................................................................................5 2. Total specimens studied and their locations (county and state)....................................21 3. Morphological characters measured ............................................................................22 4. Numbers assigned to qualitative characters in matrix ..................................................25 5. Key to abbreviations used in ordination .......................................................................27 6. Genebank and voucher information..............................................................................28 7. Primer list......................................................................................................................30 8. Characters used in ordination matrices.........................................................................33 9. The variance and eigenvalues .......................................................................................34 10. Correlations of the first six eigenvectors ....................................................................48 11. Sequence characteristics .............................................................................................51 viii LIST OF FIGURES Figure Page 1. Phenogram representing current infrageneric relationships .........................................13 2. Diagram of the trnT-L region and the rps16 intron ......................................................19 3. Representative flower and leaf for measurements........................................................24 4. Visual output of PCA analysis axes 2 v.1.....................................................................36