The University of Southern Mississippi The Aquila Digital Community Honors Theses Honors College Spring 5-2017 Phylogenetic Relationships of Salicaceae Based on Analyses of Nuclear DNA Data John Marshall Diffey University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/honors_theses Part of the Botany Commons Recommended Citation Diffey, John Marshall, "Phylogenetic Relationships of Salicaceae Based on Analyses of Nuclear DNA Data" (2017). Honors Theses. 521. https://aquila.usm.edu/honors_theses/521 This Honors College Thesis is brought to you for free and open access by the Honors College at The Aquila Digital Community. It has been accepted for inclusion in Honors Theses by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi Phylogenetic Relationships of Salicaceae Based on Analyses of Nuclear DNA Data by John Marshall Diffey A Thesis Submitted to the Honors College of the University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science in the Department of Biological Sciences May 2017 ii Approved by ____________________________________ Mac H. Alford, Ph.D., Thesis Adviser Associate Professor of Biological Sciences ____________________________________ Janet Donaldson, Ph.D., Chair Department of Biological Sciences ____________________________________ Ellen Weinauer, Ph.D., Dean Honors College iii Abstract Salicaceae are an economically and ecologically important family of flowering plants. The family includes willows and cottonwoods and was recently enlarged to include a large number of tropical species formerly placed in the family Flacourtiaceae. Relationships of these tropical relatives to willows and cottonwoods have been explored at a basic level using morphology and plastid DNA data, but to date no molecular phylogenies have been constructed with significant sampling of nuclear DNA, which sometimes results in a different picture of relationships because of its biparental inheritance. For this project, I sampled one region of nuclear DNA (GBSSI) across the family to infer relationships among the genera of Salicaceae. These results were mostly congruent with previous analyses, although sequences from some key species closely related to Salix and Populus were not obtained, possibly due to multiple copies of the gene. Key Terms: Flacourtiaceae, nuclear GBSSI, phylogenetic systematics, Salicaceae iv Acknowledgments I would like to thank Dr. Mac H. Alford for mentoring me in this project and Corey Pagart for partnering with me in the laboratory work. Some DNA samples were provided by the Royal Botanic Gardens, Kew, and the Missouri Botanical Garden. The Drapeau Center for Undergraduate Research at the University of Mississippi provided funds for the project through their Eagle SPUR grant program. Finally, I wish to thank the Honors College, which has provided me many opportunities for intellectual growth. v Table of Contents List of Figures ................................................................................................................... vii List of Tables ................................................................................................................... viii Chapter 1: Introduction ....................................................................................................... 1 Chapter 2: Literature Review .............................................................................................. 4 Chapter 3: Materials and Methods ...................................................................................... 8 Chapter 4: Results ............................................................................................................. 13 Chapter 5: Discussion ...................................................................................................... 16 Literature Cited ................................................................................................................. 18 vi List of Figures Figure 1: Strict consensus of 8 most parsimonious trees recovered in phylogenetic analysis of morphology and plastid DNA (trnL-F and ndhF), based on a subset of Alford (2005). There were 219 potentially parsimony informative characters in a matrix of 4429 aligned characters. L=379, RI=0.74. ........................................................ 14 Figure 2: Strict consensus of 66 most parsimonious trees recovered in phylogenetic analysis of nuclear DNA (GBSSI) (this study). There were 212 potentially parsimony informative characters in a matrix of 849 aligned characters. L=577, RI=0.75. .............. 15 vii List of Tables Table 1: Samples utilized for DNA work ................................................................... 11–12 viii 1 Chapter 1: Introduction Plants are vitally important to life on Earth; humans, too, could not survive without them. They provide us with oxygen that we need to breathe and carbohydrates that we need to eat. Many of the medications that we use to treat diseases are produced by plants. Plants also provide us with building materials for shelter and furniture. Beyond benefits to our species, plants are important to the ecosystems in which they reside in many important ways beyond just oxygen and chemical energy, including soil conservation. Without plants, the soil would simply wash away. In order to study plants, it is necessary to have a system that enables us to classify them and communicate unambiguously about them. Taxonomy is the scientific field associated with classification of organisms. Providing informative taxonomy involves determining relationships between organisms, which are inferred from examining and analyzing morphological, anatomical, chemical, genetic, and fossil evidence. Knowing which organisms are most closely related and how organisms (and their features) evolved provides a plethora of useful information. For one, scientists use phylogenetic relationships to analyze factors that influence evolution of traits through time. This information also enables scientists to determine if morphological characteristics were passed through a lineage from a common ancestor or if they developed independently in different branches of an evolutionary tree. Understanding these relationships allows scientists to make predictions about inadequately studied 2 characteristics that can potentially apply to all plants within a family as these characteristics are discovered. Flacourtiaceae were a plant family that included 80–95 genera and 800–1000 species of woody, pantropical plants that were difficult to identify (Chase et al., 2002). Due to new understanding of their relationships after phylogenetic analysis of DNA sequence data, the family was divided into several groups and sometimes fused with other plant families, primarily Salicaceae and Achariaceae (Chase et al., 2002), or even more finely into additional families like Samydaceae and Scyphostegiaceae (Alford, 2005). Traditionally, Salicaceae included two genera, Salix and Populus (Cronquist, 1981; Leskinen & Alstrom-Rapaport, 1998), which have highly reduced flowers with no obvious sepals and petals and have hairy, wind- and water-dispersed seeds. While classifications within Populus have remained relatively stable, Salix has undergone several revisions due to its complexity (Leskinen & Alstrom-Rapaport, 1998). The big surprise, though, was the addition of new genera to this family from tropical Flacourtiaceae, a family with mostly “normal” flowers. This result from genetic data (Chase et al., 2002; Alford, 2005) led to the development of new questions regarding the relationships within Salicaceae. Answering these questions is the interest of a small group of botanists, but while the number of researchers in this field is quite small, the need is great. An accurate phylogeny can greatly decrease the time involved in finding new compounds that can be used to synthesize medications. Several medications have been synthesized from extracts of species within both Salix and Populus. Perhaps the most widely known of these 3 medications is aspirin, which is currently one of, if not the most, widely used drugs in the world (Rainsford, 2004). In the eighteenth century, salicin, extracted from willow bark (Salix), was found to have antipyretic and analgesic effects. Salicylic acid was found to be the ingredient responsible for these medicinal properties. In 1897, Bayer synthesized acetylsalicylic acid from salicylic acid in 1897 and named his synthesized product Aspirin in 1899 (Glaser, 2000). Platelet aggregate inhibitors (anti-clotting agents) have also been extracted from Populus sieboldii (Kagawa et al., 1992). Given their close relationship, other medications with similar properties could possibly be found among species of Salicaceae, which increases the need for an accurate phylogeny of this family. In fact, antimicrobial properties have been found in Oncoba spinosa, a member of Salicaceae that was once a member of Flacourtiaceae (Djouossi et al., 2015). Another use is phytoremediation. Willows (Salix) have been used for taking up heavy metals from contaminated soils, and not surprisingly, three of the suspected relatives in the old Flacourtiaceae, Homalium, Lasiochlamys, and Xylosma from New Caledonia, have high recorded levels of nickel uptake (Jaffré et al., 1979). In order to address some of the questions about taxonomy of Salicaceae, the old Salicaceae and new members