ABSTRACT A MORPHOLOGICAL ANALYSIS OF THE TRIFOLIUM AMABILE KUNTH SPECIES COMPLEX IN NORTH AMERICA by Tia Ahlquist The Trifolium amabile Kunth species complex (Fabaceae) is a group of New World of clovers ranging from the mountains of southern Arizona of the United Sates, throughout the mountainous regions of Central America, and continuing southward to northern-central Argentina. As currently circumscribed in Zohary and Heller’s monograph (1984), this group is comprised of a singular species, T. amabile, and five varieties. Specimens from herbaria worldwide have been utilized for a morphometric examination to determine specific boundaries for the North American members of this complex. In this paper nine species, including four new species, are recognized as distinct and described. These species include: Trifolium amabile, T. blanquitum sp. nov., T. cognatum, T. goniocarpum, T. hickeyi sp. nov., T. laciae sp. nov., T. lozani, T. mexicanum, and T. sonoranensis sp. nov. A MORPHOLOGICAL ANALYSIS OF THE TRIFOLIUM AMABILE KUNTH SPECIES COMPLEX IN NORTH AMERICA A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Botany by Tia Kay Ahlquist Miami University Oxford, Ohio 2012 Advisor _________________________________ Michael A. Vincent Reader__________________________________ R. James Hickey Reader__________________________________ Richard C. Moore © Tia K. Ahlquist 2012 Table of Contents Introduction……………………………………………………………………………………1 Materials and Methods………………………………………………………………………6 Results…………………………………………………………………………………………14 Discussion…………………………………………………………………………………….47 Taxonomic Treatment………………………………………………………………………48 Literature Cited………………………………………………………………………………99 Appendix 1………………………………………………………………………………….101 Appendix 2………………………………………………………………………………….104 Appendix 3………………………………………………………………………………….110 Appendix 4………………………………………………………………………………….128 Appendix 5………………………………………………………………………………….141 Appendix 6………………………………………………………………………………….153 iii List of Tables 1. List of published names……………………………………………………………..5 2. List of herbaria………………………………………………………………………..8 3. P values resulting from a Student’s t-Test for T. amabile………………….26 4. P values resulting from a Student’s t-Test for T. blanquitum……………..28 5. P values resulting from a Student’s t-Test for T. cognatum………………..30 6. P values resulting from a Student’s t-Test for T. goniocarpum……………32 7. P values resulting from a Student’s t-Test for T. hickeyi………………..…34 8. P values resulting from a Student’s t-Test for T. laciae…………………….36 9. P values resulting from a Student’s t-Test for T. lozani………….…………38 10. P values resulting from a Student’s t-Test for T. mexicanum……………..40 11. P values resulting from a Student’s t-Test for T. sonoranensis…………..42 iv List of Figures 1. Illustration of Trifolium amabile………………………………………………..….4 2. Diagram of selected vegetative characters………………………………………9 3. Diagram of selected inflorescence characters…………………………………10 4. Diagram of selected floral characters…………………………………………..11 5. Diagram of selected fruit characters……………………………………………12 6. Diagram of pubescence characters……………………………………………..13 7. Preliminary results from NTSYSpc analyses………………………………….16 8. Results from a PCA and SAHN clustering analysis of the largest group….. …………………………………………………………………………………………..17 9. Box and whisker plot of peduncle length………………………………………19 10. Box and whisker plot of wing petal lamina width (widest)…………………20 11. Box and whisker plot of the characters used for the fifth couplet……….21 12. Box and whisker plot of the characters used for the sixth couplet………22 13. Box and whisker plot of the characters used for the seventh couplet…..23 14. Box and whisker plot of the characters used for the eighth couplet…….24 15. Differences in terminal leaflet shapes and sizes……………………………..44 16. Line diagram comparing the mean petal shapes and sizes………………..45 17. Photograph of the Trifolium amabile type specimen…………………………57 18. Trifolium amabile…………………………………………………………………….58 19. Distribution of Trifolium amabile………………………………………………...59 20. Trifolium blanquitum……………………………………………………...............63 21. Distribution of Trifolium blanquitum, T. cognatum, and T. lozani…………64 22. Trifolium cognatum………………………………………………………………….68 23. Trifolium goniocarpum………………………………………………………………73 24. Distribution of Trifolium goniocarpum…………………………………………..74 25. Trifolium hickeyi………………………………………………………………….....79 26. Distribution of Trifolium hickeyi and T. mexicanum…………………………80 27. Trifolium laciae……………………………………………………………………….84 28. Distribution of Trifolium laciae and T. sonoranensis………………………..85 v 29. Trifolium lozani…………………………………………………………….........….89 30. Trifolium mexicanum………………………………………………………………..94 31. Trifolium sonoranensis…………………………………………………...............98 vi Dedication I would like to dedicate this thesis to my mother for all of her love and support. vii Acknowledgements First I would like to thank my family for all of their support. I would also like to thank my advisor and committee for all of their help and advice they gave me. Finally I would like to thank my friends and fellow graduate students for their advice and support. viii Introduction The legume family, Fabaceae, is the third largest family of angiosperms after the Orchidaceae and Asteraceae, with more than 727 genera and 19,000 species (Lewis et al., 2005), and second in terms of economic importance (Judd et al., 2008), after the Poaceae. Despite variation in floral structure, Fabaceae is held together by the presence of its unique fruit, the legume (Lewis et al., 2005). Other unifying features include a unilocular superior ovary, parietal placentation, compound leaves (or simple leaves derived from ancestrally compound leaves), pulvini, and stipules (Lewis et al., 2005; Wojciechowski et al., 2006). Traditionally there have been three subfamilies within the Fabaceae, the Caesalpinioideae, the Mimosoideae, and the Papilionoideae, the latter being by far the largest of the three with 476 genera and over 13,800 species (Wojciechowski, 2006). However, recent molecular phylogenetic analyses have shown that while the Fabaceae is a monophyletic unit, the caesalpinioids are paraphyletic, and the mimosoids (excluding the genus Dinizia) and papilionoids are each monophyletic (Wojciechowski et al., 2004). The Papilionoideae are characterized by having a papilionaceous flower, that is with a banner petal, two wing petals, and two keel petals, in which the banner petal is the outermost in bud (Judd et al., 2008). Within this subfamily are many agriculturally important taxa including Medicago (alfalfa), Glycine (soybean), Arachis (peanut), and sixteen or more species of Trifolium (clover), which are important food and fodder crops (Ellison et al., 2006; Lewis et al., 2005). There are 36 tribes within the family, with 28 in the Papilionoideae (Lewis et al., 2005). The tribe Trifolieae contains six genera and over 480 species, more than half of which (about 250 species) belong to the genus Trifolium (Zohary and Heller, 1984; Lewis et al., 2005); some of these have been recognized since the times of Theophrastus, Dioscorides, and Pliny (Zohary and Heller, 1984). Other genera in this tribe include Parochetus, Ononis, Melilotus, Trigonella, and Medicago (Lewis et al., 2005). There are three centers of 1 diversity of Trifolium, two in the Old World and one in the New World (Ellison et al., 2006). The diversity in the Old World is concentrated around the Mediterranean basin and the eastern African highlands, and diversity in the New World is centered in western North America (Ellison et al., 2006). Trifolium species are mostly sun-tolerant and occur in a variety of different habitats from prairies and meadows to alpine peaks (Ellison et al., 2006). Despite its large size and economic importance, relatively little comprehensive work has been done on the genus, especially the New World taxa (Zohary and Heller, 1984). According to Zohary and Heller (1984), the genus Trifolium is divisible into eight sections, but most of these have been shown not to be monophyletic (Ellison, et al., 2006). Only two of Zohary and Heller’s sections, Lotoidea and Involucrarium, occur natively in the New World. Recent molecular analyses of cpDNA loci matK and trnL, and nrDNA locus ITS have shown that Zohary and Heller’s (1984) section Lotoidea is polyphyletic (Ellison, et al., 2006). All New World Trifolium formerly placed in section Lotoidea are now included in a more broadly circumscribed, monophyletic, section Involucrarium (Steele and Wojciechowski, 2003; Ellison et al., 2006). Section Trifoliastrum, containing species such as Trifolium repens L., has been shown to be sister to section Involucrarium (Steele and Wojciechowski, 2003; Ellison et al., 2006). Within section Involucrarium, Trifolium amabile Kunth is sister to the rest of the section excluding T. breweri S. Watson (Ellison et al., 2006). The Trifolium amabile species complex is a very widespread group, ranging from the mountains of southern Arizona, through the mountainous regions of Central America, and continuing southward to the province of Córdoba in north-central Argentina. In addition to its extensive geographical distribution, members of this species complex vary greatly in their size, habit, and morphological features. Trifolium amabile (Fig. 1) was described by Kunth in 1824, from material collected in Mexico by Humboldt and Bonpland. Since then, over thirty names have been published for members of this complex (Table 1).