ABSTRACT A MORPHOLOGICAL ANALYSIS OF THE TRIFOLIUM AMABILE KUNTH SPECIES COMPLEX IN SOUTH AMERICA by Justin Hendy The Trifolium amabile Kunth species complex (Fabaceae) is a group of New World clovers found in mountainous regions ranging from southern Arizona in the United Sates, through Central America, where it occurs in Mexico, Guatemala, and Costa Rica, and south along the Andes Mountains, from Colombia, Ecuador, Peru, and Bolivia, to Argentina. Zohary and Heller (1984) circumscribe this group as a single species, T. amabile, with five varieties. Ahlquist (2012) examined material from North and Central America, and recognized nine species in that geographical range. This study focuses on the South American range of the species complex. Species boundaries were determined using morphometric analyses of herbarium specimens, including PCA and cluster analysis, using NTSYS-pc, Student’s t-tests, and box and whisker plots. Six taxa, including four new species, are recognized as distinct and described. These species include: Trifolium amabile, T. andesicola sp. nov., T. australe sp. nov., T. cochabambense sp. nov., T. peruvianum, and T. tafiense sp. nov. A MORPHOLOGICAL ANALYSIS OF THE TRIFOLIUM AMABILE KUNTH SPECIES COMPLEX IN SOUTH 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 Justin Hendy Miami University Oxford, Ohio 2013 Advisor _________________________________ Michael A. Vincent Reader__________________________________ Richard C. Moore Reader__________________________________ M. Henry H. Stevens © Justin Hendy 2013 Table of Contents Introduction……………………………………………………………………….………………………………………………………1 Materials and Methods………………………………………………………………………………………………………………6 Results……………………….………………………………………………………………………………………………………………8 Discussion…………………………………………………………………………………..…………..……………………………….26 Taxonomic Treatment…………………………………………………………………………………..…………………………27 Conclusion……………………………………………………………………………………………………………………………….59 Literature Cited………………………………………………………………………………..………………………………………61 Appendix 1……………………………………………………………………………………………………………..……………….63 Appendix 2………………………………………………………………………………………………………..…………………….66 Appendix 3…………………………………………………………………………………………………………………..………….71 Appendix 4………………………………………………………………………………………………………………..…………….83 Appendix 5…………………………………………………………………………………………………………………………….107 iii List of Tables 1. List of published names………………………………………………………………………………………………………….5 2. P-values resulting from student’s t-tests of each species against each other species…………..13 iv List of Figures 1. PCA of all 109 specimens…………………………………………………………………………………………………..…10 2. Dendrogram of all 109 specimens………………………………………………………………………..…….………..11 3. Box and whisker plot of characters used for the first couplet……………………………………….………14 4. Box and whisker plot of characters used for the second couplet………………………………….………17 5. Box and whisker plot of the characters used for the third couplet……………………………………....19 6. Box and whisker plot of the characters used for the fourth couplet…………………………………….21 7. Box and whisker plot of the characters used for the fifth couplet…………………………………….….23 8. Trifolium amabile……………………………………………………………………………………………………….………..33 9. Geographical range of T. amabile………………………………………………………………………………….……..34 10. Trifolium peruvianum……………………………….…………………………………………………………….………….39 11. Geographical range of T. peruvianum………………………………………………………………………..……….40 12. Trifolium tafiense………………………………………………………………………………………………………..……..43 13. Geographical range of T. tafiense……………………………………………………………………………….……..44 14. Trifolium andesicola…………………………………………………………………………………………..………………48 15. Geographical range of T. andesicola…………………………………………………………………………..………49 16. Trifolium australe……………………………………………………………………………………………………………...53 17. Geographical range of T. australe…………………………………………………………………………..………….54 18. Trifolium cochabambense…………………………………………………………………………………………….…….57 19. Geographical range of T. cochabambense………………………………………………………………………….58 v Acknowledgements First and foremost, I would like to thank my wife for all of her encouragement and support. Without her and my family none of this would have been possible. I would also like to thank my advisor, Dr. Mike Vincent, without whom I would have not accomplished something so great. I would also like to thank my thesis committee members, Drs. Hickey, Moore, and Stevens for all of their advice and help. Finally I would like to thank all of the other graduate students and my lab-mates for offering support, advice and keeping things fun. vi Introduction The genus Trifolium L. (clovers; Fabaceae, Faboideae, Trifolieae) consists of approximately 250 species (Ellison et al. 2006, Zohary & Heller 1984). The name Trifolium derives from tri, three, and folium, leaf, referring to the trifoliate leaf found commonly in the genus. Trifolium species, which are annual, biennial, or perennial herbs, occur in temperate regions, with a few species in subtropical areas. The range of the genus is North America, Central America, and South America, Europe, Africa, and temperate Asia; no native clovers are found in tropical Asia, Australia, or the Pacific Ocean islands. Three major centers for speciation in the genus are the Mediterranean region, the California region, and the eastern African highlands (Zohary & Heller 1984). Within Fabaceae, Trifolium is placed in the tribe Trifolieae. Lewis et al. (2005) defined this tribe as consisting of six closely related genera: Ononis, Parochetus, Trigonella, Medicago, Melilotus, and Trifolium. Trigonella, Medicago, Parochetus, and Melilotus are Old World genera, but some species have been cultivated and have spread from cultivation outside their native ranges. Ononis (another Old World genus) can be distinguished from Trifolium by its alternately dorsifixed and basifixed dimorphic anthers and its usually glandular and spiny nature (Wei & Vincent 2010). Trifolium can be separated from Parochetus, Melilotus, Medicago, and Trigonella by its persistent corolla after anthesis, a legume that is included in or slightly exerted from the persistent corolla, and filaments that are dilated distally (Gleason & Cronquist 1991, Wei & Vincent 2010). Clovers are important for livestock feed, and at least 16 species are actively cultivated (Ellison et al. 2006). Trifolium repens L., white clover, and T. pratense L., red clover, are the most widely cultivated. Clovers have been cultivated for several reasons. They produce a plentiful crop that will continue to grow after heavy grazing; they are nutritious for livestock; and they can grow in many climates due to their cold hardiness; they grow in a wide range of soil types (Taylor 1985). Due to these favorable properties T. pratense (red clover) has been cultivated since the 4th century A.D. in Europe (Zohary & Heller 1984). 1 Pre-Linnean botanists made preliminary attempts to classify Trifolium into subgeneric groups, but were only aware of a very small number of species. Linnaeus (1753) divided his 41 species of Trifolium into five units; Meliloti, Lotoidea, Lagopoda, Vesicaria, and Lupulina (Zohary & Heller 1984). Not all of his species are now placed in Trifolium, with some instead assigned to Trigonella and Melilotus. After Linnaeus, many scientists studied clovers in their own regions, but no world-wide studies were conducted until Seringe (1825) completed the first revisions of the entire genus, including 150 species from both the Old and New Worlds. Presl (1831) split Trifolium into nine genera: Paramesus, Amarenus, Lupinaster, Amoria, Micrantheum, Trifolium, Mistyllus, Galearia, and Calycomorphum. His work was not followed by others; however, most of his genera were later accepted as sections within Trifolium. Celakovsky (1874) examined the sections of Trifolium and attempted to describe their phyletic relationships. The last major work on the entirety of Trifolium before Zohary and Heller was that of Bobrov (1967). He organized eleven sections of Trifolium into two tribes, Trifolieae and Lupineae. In the most recent monograph, Zohary and Heller (1984) recognized 237 species, and arranged them into eight sections: Lotiodea, Paramesus, Mistyllus, Vesicaria, Chronosemium, Trifolium, Trichocephalum, and Involucrarium. The New World species were included within Sections Lotoidea (containing both New and Old World species) and Involucrarium (containing only New World species). Ellison et al. (2006) conducted a molecular phylogenetic analysis of Trifolium, including 255 species covering the geographical and morphological range of the genus. As a result of these analyses, they recognized two subgenera, Chronosemium and Trifolium, with the latter encompassing eight sections. Ellison et al. (2006) place all of New World Trifolum in a newly defined Section Involucrarium of subgenus Trifolium. Trifolium breweri S. Wats. (a Californian species) is basal within the Involucrarium clade, with T. amabile Kunth + T. peruvianum Vogel as sister to the remaining 65 species (Ellison et al. 2006). Trifolium amabile was described by Kunth (in Humboldt et al. 1824) from material from Mexico collected by Humboldt and Bonpland. As circumscribed by Zohary and Heller (1984), T. amabile is a highly variable taxon, in which five varieties were recognized; T. amabile Kunth var. 2 amabile, T. amabile Kunth var. hemsleyi (Lojac.),Heller & Zoh., T. amabile Kunth var. pentlandii Ball, T. amabile var. mexicanum (Hemsl.) Heller & Zoh., T. amabile Kunth var. longifoliolum Hemsl.,