ABSTRACT A MORPHOLOGICAL AND MOLECULAR TAXONOMIC REVIEW OF THE TRIFOLIUM DEPAUPERATUM (FABACEAE) SPECIES COMPLEX by Lydia Grace Tressel The amphitropical Trifolium depauperatum Nutt. species complex (Fabaceae) is a group of closely related clovers with a disjunct native distribution from Chile and western North America. Based on the most recent treatment by Vincent and Isely in 2012, the species complex includes two species, one of which contains three varieties. Historically, the taxonomy of this species complex has been unstable, so a morphological and molecular analysis was conducted from herbarium specimens and fresh tissue. Multivariate and cluster analyses of morphological data support the recognition of four taxa: T. depauperatum var. depauperatum Desv., T. depauperatum var. amplectens Torr. & Gray, T. depauperatum var. truncatum (Greene) Martin ex Isely, and T. hydrophilum Greene. Each taxon is typified, synonymy is provided, and descriptions and distributional information is given. Molecular analysis was inconclusive; however, a strategy for using molecular analysis in future investigations of these taxon boundaries is discussed. A MORPHOLOGICAL AND MOLECULAR TAXONOMIC REVIEW OF THE TRIFOLIUM DEPAUPERATUM (FABACEAE) SPECIES COMPLEX A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science by Lydia Grace Tressel Miami University Oxford, Ohio 2020 Advisor: Michael A. Vincent Advisor: Richard C. Moore Reader: Eric J. Tepe ©2020 Lydia Grace Tressel This Thesis titled A MORPHOLOGICAL AND MOLECULAR TAXONOMIC REVIEW OF THE TRIFOLIUM DEPAUPERATUM (FABACEAE) SPECIES COMPLEX by Lydia Grace Tressel has been approved for publication by The College of Arts and Science and Department of Biology ____________________________________________________ Michael A. Vincent ______________________________________________________ Richard C. Moore _______________________________________________________ Eric J. Tepe Table of Contents Chapter 1. A morphological review of the Trifolium depauperatum species complex. Introduction………………………………………………………………………..1 Materials and Methods…………………………………………………………….5 Results……………………………………………………………………………..6 Discussion……………………….……………………………………………….10 Chapter 2. A molecular review of the Trifolium depauperatum species complex. Introduction………………………………………………………………………39 Materials and Methods…………………………………………………………...40 Results……………………………………………………………………………42 Discussion………………………………………………………………………..42 Conclusion……………………………………………………………………….43 iii List of Tables Chapter 1 Table 1: Names published in Trifolium depauperatum species complex…………3 Table 2. Key morphological differences in floral and involucre characters that allow for practical distinctions of taxa in the field………………………………..9 iv List of Figures Chapter 1 Figure 1: Principal components analysis projection of entire T. depauperatum species complex………………………………………………………...7 Figure 2: Phenogram produced from a Ward’s method cluster analysis of all specimens……………………………………………………………….8 Figure 3: McDermott (1910) specimen drawing of Trifolium hydrophilum……..15 Figure 4: Map of T. hydrophilum species distribution…………………………...16 Figure 5. McDermott (1910) specimen drawing of Trifolium depauperatum var. amplectens……………………………………………………………..19 Figure 6. Map of T. depauperatum var. amplectens species distribution………..20 Figure 7. McDermott (1910) specimen drawing of Trifolium depauperatum var. truncatum………………………………………………………………25 Figure 8. Map of T. depauperatum var. truncatum species distribution…………26 Figure 9. McDermott (1910) specimen drawing of Trifolium depauperatum var. depauperatum………………………………………………………….30 Figure 10. Map of T. depauperatum var. depauperatum species distribution…...31 Chapter 2 Figure 1: Bayesian phylogenetic tree of the data set of ITS region……………...44 v Dedication This work is dedicated to my fiancé Grant Taylor Bowling. Thank you so much for your patience, love, and support through this process. vi Acknowledgements First, I owe a special thanks to my family: my grandmother Nancy for being a master gardener and first teaching me about plants, my mother Susan for always encouraging me to explore outside, my father Timothy for encouraging me to pursue botany at Miami University as an undergraduate, and my sister and her fiancé, Leah and Antonio for always being supportive of me in my academic career. You guys have always believed in me even when I didn’t believe in myself. I also thank my dear friends and lab-mates Lauren Rogers, Brody Betsch, Patrick Garrett, Hannah Scheppler, and Evan Gallagher for always loving and supporting me through the stress of graduate school. Extra thanks go to Patrick for spending countless hours helping me in the lab. Without his guidance, chapter 2 of this thesis would not be possible. I owe a great deal of thanks to my fiancé Grant Bowling for being the foundation of love and support in my life. I also want to thank Lisa and Mike Mandelert for their love and support as my Miami parents. I could not have gotten through this process without each of you. Thank you to all of the staff at the Channel Islands National Park and Catalina Island Conservancy for allowing Lauren Rogers and I to collect specimens from the Channel Islands. I would also like to thank the W.S. Turrell Herbarium (MU) Fund for supporting the monetary portion of this project. Finally, I would like to thank my committee members including my co-advisors Drs. Michael Vincent and Richard Moore, as well as Dr. Eric Tepe. Thank you for all of your advice, criticism, and support in the lab and in life. Your dedication to me as a student is greatly appreciated. vii Chapter 1: A MORPHOLOGICAL REVIEW OF THE TRIFOLIUM DEPAUPERATUM SPECIES COMPLEX Introduction The economically and ecologically important family Fabaceae includes many useful plants such as crops, timber, ornamentals, herbivorous insect food and medicinal plants (Yahara et al. 2013). Fabaceae is the third largest family of flowering plants, with about 770 genera and 19,500 species (LPWG 2017). Fabaceae is phylogenetically placed in the order Fabales (Stevens 2001) and contains six subfamilies: Duparquetioideae, Cercidoideae, Detarioideae, Dialioideae, Caesalpiniodeae, and Papilionoideae (LPWG 2017). The Papilionoideae contains approximately 14,000 species including many important crop species such as the cultivated soybean (Glycine max) and common bean (Phaseolus vulgaris L.) (LPWG 2017; Yahara et al. 2013). Members of the Papilionoideae have papilionoid flowers, which are characterized by imbricate petals with the upper petal (banner) outermost, the wings lateral, and two basal petals (keel) connate or coherent at the apex (Judd et al. 2008). The plant family Fabaceae contains the Papilionoideae genus Trifolium. Trifolium L. is a widely distributed genus of plants known for their mostly 3-parted leaves commonly known as clovers. Individuals in this genus are annual, biennial, or perennial herbaceous plants with often trifoliolate leaves, papilionoid flowers that are found in racemose- umbellate heads to head-like inflorescences, with diadelphous stamens and persistent corollas. The genus includes at least 240 species distributed worldwide across temperate and subtropical regions of the Northern and Southern Hemispheres (Ellison et al. 2006; Zohary & Heller 1984). Native clovers are absent from Southeast Asia and Australia, where they have been introduced for agricultural purposes (Ellison et al. 2006). The greatest species diversity is found in the Mediterranean region, western North America, and eastern Africa (Ellison et al. 2006; Zohary & Heller 1984). A phylogenetic study of the genus was conducted using ITS and chloroplast trnL intron sequences by Ellison et al. (2006) which confirmed the monophyly of the group. In the most recent monograph of the genus, that of Zohary and Heller (1984), recognized eight sections in Trifolium. All native North American species were included in two sections, Lotoidea Crantz and Involucrarium Hooker. Twenty-five species having heads subtended by an involucre of fused bracts were placed in sect. Involucrarium. The remainder of the North American species, those without an obvious involucre, were placed in sect. Lotoidea. 1 Ellison et al. (2006) placed all North American clovers into a more broadly defined sect. Involucrarium as a result of their phylogenetic analyses. A portion of sect. Involucrarium, including T. depauperatum Desv., T. fucatum Lindley, and T. hydrophilum Greene, can be separated from the rest of the species by characteristic traits such as fused involucral bracts that form a cup or bowl at the base of the inflorescence, glabrous calyces, banner petals that inflate when in fruit, and an annual lifecycle. Part of this group is being treated here as the T. depauperatum species complex. The T. depauperatum species complex is a group of taxa native to western North and South America, and is known from British Columbia, Washington, Oregon, Idaho, California, Mexico, and Chile. In total, 31 names have been published for taxa within this species complex (Table 1; Vincent & Isely 2012). In general, plants in this group are annual, decumbent to erect herbs with leaflets that are 0.5-0.2 cm long and oblong to obovate, and inflorescences that are head-like, 0.5-1 cm wide, with 3 to several flowers. The corollas are 4.5-9 mm long and the banners inflate when in fruit. The most recently published treatment of the T. depauperatum species complex (Vincent and Isely 2012) recognized the