Abstract a Morphological and Molecular Taxonomic

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

Lydia Grace Tressel

Miami University

Oxford, Ohio

2020

Advisor: Michael A. Vincent

Advisor: Richard C. Moore

Reader: Eric J. Tepe

This Thesis titled

A MORPHOLOGICAL AND MOLECULAR TAXONOMIC REVIEW OF THE TRIFOLIUM DEPAUPERATUM (FABACEAE) SPECIES COMPLEX

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

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

Dedication

This work is dedicated to my fiancé Grant Taylor Bowling. Thank you so much for your patience, love, and support through this process.

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.

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 varieties T. depauperatum var. depauperatum, T. depauperatum var. amplectens (Torr. & A. Gray) Rattan, T. depauperatum var. truncatum (Greene) Martin ex Isely, and the species T. hydrophilum. Vincent and Isely covered only material from California, and material from the rest of the native range remained largely unexamined, especially material from Mexico and South America. In addition to the traits discussed above, I identify and describe traits that may be used to separate these taxonomic entities. In my study, I conducted analyses of morphological data from specimens in the T. depauperatum species complex to reexamine the taxonomy of the species complex. The objective of this chapter was to determine the most appropriate taxon delimitations within the species complex by testing the hypothesis that Vincent & Isely’s (2012) treatment is the most appropriate.

Table 1. Names published in the Trifolium depauperatum species complex. Names in bold are recognized by Vincent & Isely (2012). Name Publication Author Lupinaster depauperatus Symb. Bot. 1(3): 50. 1831. Presl, Carl Bořivoj Trifolium amplectens Fl. N. Amer. 1(2): 319–320. Torrey, John 1838. Gray, Asa Trifolium amplectens var. Man. Fl. Pl. Calif. 537. 1925. Jepson, Willis Linn diversifolium Trifolium amplectens var. Fl. W. Calif. 311. 1901. Jepson, Willis Linn hydrophilum Trifolium amplectens var. Man. Fl. Pl. Calif. 537. 1925. Jepson, Willis Linn stenophyllum Trifolium amplectens var. Man. Fl. Pl. Calif. 537. 1925. Jepson, Willis Linn truncatum Trifolium anodon Pittonia 5(27): 107. 1903. Greene, Edward Lee Trifolium brachyodon Pittonia 5(27): 107. 1903. Greene, Edward Lee Trifolium decodon Pittonia 5(27A): 108. 1903. Greene, Edward Lee Trifolium depauperatum fo. Ill. Key Amer. Trifolium 140. McDermott, Laura franciscanum 1910. Frances Trifolium depauperatum fo. Ill. Key Amer. Trifolium 133. McDermott, Laura laciniatum 1910. Frances Trifolium depauperatum fo. Ill. Key Amer. Trifolium 144. McDermott, Laura truncatum 1910. Frances Trifolium depauperatum var. Anal. Key West Coast Bot. 43. Rattan, Volney amplectens 1887. Trifolium depauperatum var. Fl. W. Calif. 311. 1901. Jepson, Willis Linn angustatum Trifolium depauperatum var. Journal de Botanique, Appliquée Desvaux, Nicaise depauperatum à l'Agriculture, à la Pharmacie, à Auguste la Médecine et aux Arts 4: 69–70, pl. 32, f. 2. 1814. Trifolium depauperatum var. Ill. Key Amer. Trifolium 135. McDermott, Laura diversifolium 1910. Frances Trifolium depauperatum var. Brittonia 32(1): 55. 1980. Martin, James hydrophilum Stillman Trifolium depauperatum var. Fl. W. Calif. 311. 1901. Jepson, Willis Linn laciniatum Trifolium depauperatum var. Ill. Key Amer. Trifolium 137. McDermott, Laura stenophyllum 1910. Frances

Table 1 cont. Trifolium depauperatum var. Brittonia 32(1): 56. 1980. Martin, James truncatum Stillman Trifolium diversifolium Proc. Acad. Nat. Sci. Nuttall, Thomas Philadelphia 1: 152. 1847. Trifolium franciscanum Man. Bot. San Francisco Bay Greene, Edward Lee 100. 1894. Trifolium franciscanum var. Man. Bot. San Francisco Bay Greene, Edward Lee truncatum 100. 1894. Trifolium hydrophilum Man. Bot. San Francisco Bay Greene, Edward Lee 100. 1894. Trifolium laciniatum Pittonia 1(1): 7–8. 1887. Greene, Edward Lee Trifolium laciniatum var. Man. Bot. San Francisco Bay Greene, Edward Lee angustatum 101. 1894. Trifolium minutiflorum Pittonia 3(17B): 215. 1897. Greene, Edward Lee Trifolium quercetorum Pittonia 1(11): 172–173. 1888. Greene, Edward Lee Trifolium stenophyllum Proc. Acad. Nat. Sci. Nuttall, Thomas Philadelphia 1: 151. 1848. Trifolium truncatum Proc. Acad. Nat. Sci. Greene, Edward Lee Philadelphia 47(3): 546. 1895[1896].

Materials and Methods

Specimens were obtained from the following herbaria: B, CAS, E, ECON, GH, JEPS, K, MSC, MU, NDG, NY, P, PENN, PH, SBBG, SD, UC, US, W and WS (acronyms from Thiers [continuously updated]). From among those specimens, 146 specimens representing the entire geographical range of the T. depauperatum species complex were utilized for morphological examination. Macroscopic characters were measured using a Westcott flexible stainless-steel ruler. Characters from specimens less than 1 cm were considered microscopic and measured using a dissecting microscope. Microscopic characters were measured using a dissecting microscope with an ocular micrometer with 10x magnification measured to the nearest 0.1 mm. Flowers were rehydrated using 1% Aerosol OT in order to obtain accurate measurements of floral parts (Ahlquist 2012; Ayensu 1967; Foster 2015). Flowers were then dissected in order to yield measurements for the calyx, corolla, stamen, and pistil characters. Measurements from 35 characters (Appendix 1) were used in the statistical analyses: peduncle length, stem diameter, leaflet length, leaflet width, calyx circumference, calyx tube length, calyx lobe lengths, banner petal length, banner width, wing petal width, wing petal length, wing petal claw length, keel petal width, keel petal length, keel petal claw length, single stamen length, stamen cluster length, stamen tube length, ovary width, ovary length, pistil length, style length, ovule number, pistil stipe length, stipule length, stipule width, involucral bract length, involucral bract width, involucral lobe length, involucral lobe width at widest part, involucral lobe apex angle, floral bract length, rachis length, pedicel length, and number of whorls of flowers. Principal component analysis (PCA; Pearson 1901, Hotelling 1933) was carried out using R (Appendix 2; R Core Team, R Foundation for Statistical Computing, Vienna, Austria). PCA was conducted using the princomp function and projected into R. Only variable characters were used in the analysis, as invariant characters were removed (Appendix 1; Sneath and Sokal 1973). Maps of specimens used in statistical analyses were produced in R (R Core Team 2017) using the packages ggplot2 (Wickham 2016), sf (Pebesma 2018), and rnaturalearth (South 2017). The phenogram was produced from a Ward’s method cluster analysis (CA) of all specimens using

SAS software (Ward 1963; JMP® 2019).

Results Multivariate analyses indicate the presence of two groups (Fig. 1). The specimens of T. depauperatum var. depauperatum (black circles, Fig 1.), T. depauperatum var. amplectens (green pluses, Fig 1.), and T. depauperatum var. truncatum (blue crosses, Fig 1.) are intermixed with one another in the projection of principle component (PC) 1 and PC 2. There is overlap of T. depauperatum var. depauperatum with both vars. amplectens and truncatum, but the remainder of the specimens separate in distinct clusters. T. hydrophilum specimens (red triangles, Fig. 1) occupy a discrete cluster in the PCA plot space. The phenogram produced by the Ward’s method cluster analysis (Fig. 2) contains five clusters that supports the previously published taxonomic boundaries (Vincent & Isely 2012). McDermott’s (1910) sketches of involucral characters are mapped onto the clusters that correspond to each taxon. All T. depauperatum var. truncatum are clustered on the same two branches of the tree, and the T. depauperatum var. truncatum branch was the first branch to split from the rest of the phenogram. Specimens of T. depauperatum var. amplectens, T. hydrophilum, and T. depauperatum var. depauperatum group in species-specific clusters. The topography of this cluster analysis indicates that T. depauperatum var. amplectens is more morphologically similar to T. hydrophilum than any other taxon. The involucre, calyx, corolla, and stem length characters were the most distinct characters among taxa (Table 2). Involucre shape is most variable in T. depauperatum var. amplectens with 4-5 bracts of varying shape. The calyx length varies between 3-4 mm and the corolla length varies between 5-6 mm. T. hydrophilum has basally fused involucral bracts and a corolla that tends to be longer and thinner (6.5-9mm), almost fusiform, than other taxa in this species complex. T. depauperatum var. depauperatum has a ring-like involucre lacking bracts. T. depauperatum var. truncatum has an involucre with free bracts and membranous margins. The high average stem length of 32 ± 16 cm of T. hydrophilum distinguishes it from other taxa, which have average stem lengths of 10 cm to 17 cm (Table 2).

Figure 1. Two-dimensional principal components analysis projection of all T. depauperatum species complex. Colors and shapes correspond as follows: T. depauperatum var. depauperatum (black circle), T. hydrophilum (red triangle), T. depauperatum var. amplectens (green plus), and T. depauperatum var. truncatum (blue cross). Principle Component 1 (Comp. 1) explains 28.3% of variation, Principle Component 2 (Comp. 2) 14.7%.

Figure 2. Phenogram produced from cluster analysis of all specimens using Ward’s method. Colors correspond as follows: T. depauperatum var. depauperatum (black), T. hydrophilum (red), T. depauperatum var. amplectens (green), and T. depauperatum var. truncatum (blue). McDermott’s (1910) sketches of involucral characters are mapped onto the cluster that corresponds to each taxon.

Table 2. Key morphological differences in floral, involucral, and stem characters that allow for practical distinctions of taxa in the field.

Character T. T. hydrophilum T. depauperatum T. depauperatum depauperatum var. depauperatum var. truncatum var. amplectens Involucre 4-5 involucral Involucre bracts Involucre ring-like Involucre bracts description bracts, with basally fused. free, margins margins that are membranous toothed at the tip and variable in shape. Involucre Length 1-4 mm <1 mm 0.1 mm 2-2.5 mm Calyx Length 3-4 mm 2.5-5 mm 2.5-3.5 mm 2.5-3 mm Corolla Length 5-6 mm 6.5-9 mm 6-9 mm 4.5-7.5 mm Stem Length 10-24 cm 16-48 cm 5-15 cm 7-27 cm

Discussion

The results of the PCA and CA support the recognition of four taxa within the Trifolium depauperatum species complex: T. hydrophilum, T. depauperatum var. amplectens, T. depauperatum var. truncatum, and T. depauperatum var. depauperatum. These results support the previous taxonomic treatment and taxonomic designations of Vincent & Isely (2012). Significant quantitative and qualitative morphological gaps between clusters were used to identify and describe these taxa. Trifolium hydrophilum is supported as a unique species compared to the T. depauperatum sub-taxa, even though it is clustered within them in the phenogram. T. hydrophilum is morphologically separated from the other sub-taxa in the PCA. In particular, the stem length of T. hydrophilum is considerably longer than the other sub-taxa. There is also a clear ecological disjunction of T. hydrophilum since it grows in marshes and open areas in alkaline soils, while all other sub-taxa persist in in grasslands and coastal woodlands. Therefore, T. hydrophilum is supported as a separate species if we apply both the ecological and morphological species concepts (Queiroz 2007). T. depauperatum var. amplectens, T. depauperatum var. truncatum, and T. depauperatum var. depauperatum are supported as separate taxa based on the CA phenogram. The PCA suggests the morphological distinctions of the specimens are not as delimited as T. hydrophilum, therefore, rendering it unjustifiable to designate these as separate species. The taxonomic boundaries can be used in the future for necessary conservation decisions. The delineation of species is extremely important taxonomically as well as for the implication in conservation. When a taxonomist is making a decision, they need to take into account the potential problems of inappropriate designation of species for conservation purposes (Frankham et al. 2012). The only taxon within this species complex of any conservation concern is T. hydrophilum. According to the California Native Plant Society (CNPS; 2020), T. hydrophilum is listed as rare or endangered in California and imperiled. In the following taxonomic treatment, species delineations are defined and described in order to inform future conservation decisions about this species complex.

Taxonomic Treatment

Key to the species of the Trifolium depauperatum species complex 1. Involucral bracts <1 mm, not reduced to an unlobed ring, fused at base; inflorescence 1- 1.5 cm wide; corolla 6.5-9 mm long; found in salt marshes and open areas in alkaline soils…………….1. T. hydrophilum 1.’ Involucral bracts 2-4 mm, free or partially fused, or reduced to an unlobed ring; inflorescence 0.5-1 cm wide; corolla 4.5-9 mm; found on grassy flats, disturbed slopes, openings in woodlands……………………………………………………………………2 2. Fruit 3-4 mm, oblong, involucral bracts free or partially fused, margins widely scarious; corolla 5-6 mm……………………………2. T. depauperatum var. amplectens 2’. Fruit 2-3 mm, ovate or obovate; involucral bracts free, margins membranous, or reduced to an unlobed ring; corolla 4.5-9 mm…………………………………………...3 3. Leaflet tip generally truncate, toothed; involucral bracts 2-3 mm, margins membranous…………………………………3. T. depauperatum var. truncatum 3.’ Leaflet tip notched or lobed; involucre reduced to an unlobed ring………………………………………4. T. depauperatum var. depauperatum

1. Trifolium hydrophilum Greene Trifolium hydrophilum Greene, Man. Bot. San Francisco Bay 100, 1894. T. amplectens Torr. & A. Gray var. hydrophilum Greene (Jeps.), Fl. W. Calif. 311, 1901. T. depauperatum Desv. var. hydrophilum, (Greene) J.S. Martin ex Isely, Brittonia 32: 55, 1980. TYPE: USA. California: Belmont, 9 May 1886, E. L. Greene s.n. (lectotype, designated by Gillett (1966): NDG 36031(=25355); syntype: NDG 36030 (=25483). (Fig. 3).

Plants herbaceous, glabrous, annual; stems erect, branching, 26-49 cm long, 1-3.9 mm diameter mid-stem; stipules adnate to petioles, sheathing, entire, 6-12x2-6 mm; petioles 35- 83x0.5-0.7 mm; leaves trifoliolate; leaflets linear or oblong, 12-29.5x3-10 mm, dentate or somewhat serrulate, obtuse or truncate; inflorescences racemes, axillary, umbelliform, 1-1.5 cm wide, rachises 0.5-1.5 mm long, glabrous, involucral bracts fused basally, typically half the length of calyces; pedicels 0.2-0.5 mm long, glabrous; calyces 1.3-8.5 mm long, tube 0.3-2.5 mm long, broadly campanulate, membranous or scarious, throat oblique, lobes unequal, subulate, typically as long or longer than tube, longest 1-6 mm; banner petals pink-purple, brown with age, claw absent, blade 6.5-9.5x1.2-3.8 mm, striate, broadly ovate, strongly inflated at maturity; wing petals purple, brown with age, asymmetrically clawed, claws 0.3-0.9 mm long, blade 5.4- 8.7 mm long, laminae elliptic-orbicular; keel petals purple, darker at edges, brown with age, asymmetrically clawed, claws 0.2-0.6 mm long, blade 5.3-8.6 mm long, laminae elliptic-ovate; stamens diadelphous, connate filaments 4.2-6.7 mm long, free filaments 4.4-7.3 mm long, anthers 0.2 mm; pistils stipitate, stipes 0.4-0.8 mm, ovaries 1.7-5.7x0.6-2.4 mm, glabrous, styles 0.8-5.9 long, ovules 1-2; legumes inflated, glabrous; seeds 1-2, globular, 1.3x1.8x0.6 mm, brown, seed coat smooth, shiny. Trifolium hydrophilum is endemic to California, where it ranges from San Luis Obispo County, north through Yolo County (Fig. 4). It grows in marshes and open areas in alkaline soils. Collections have been made from elevations ranging from 0-150m. Trifolium hydrophilum flowers during the months of April, May, and June. Trifolium hydrophilum has been considered a synonym of T. diversifolium Nutt. in some previously published taxonomic and floristic treatments (McDermott 1910). Examination of the

12 type shows that T. diversifolium is a synonym of T. truncatum and T. stenophyllum. The only difference McDermott (1910) saw between T. depauperatum and T. depauperatum var. diversifolium was that T. depauperatum var. diversifolium was much taller than the other specimens. McDermott (1910) found specimens were abundant in the salt marshes in the sea- coast counties of California. Jepson (1901) recombined T. hydrophilum as a variety of T. amplectens. Martin (1943) considered T. hydrophilum to be a variety of T. depauperatum, while Munz and Keck (1959) placed T. hydrophilum as a variety of T. amplectens. In the most recent monograph, Zohary and Heller (1984) considered T. hydrophilum equivalent to T. depauperatum var. diversifolium. Greene (1894), in his original publication, describes Trifolium hydrophilum in this way: “Diffuse, glabrous, the branches flaccid though not very slender, 1-2 ft. long: stipules ovate, entire, subulate-pointed; leaflets linear or oblong, obtuse or truncate, repandly dentate or somewhat serrulate, 1 in. long: peduncles slender, little exceeding the leaves: heads 8-15 flowered: involucre of about 5 small ovate or oblong bracts: calyx-teeth very long, subulate-aristiform: corolla in age oblong, slightly inflated and about equally so from end to end, conspicuously striate: pod 2-seeded: seed transversely oblong, sinuous- rugose. In low moist lands along the seaboard, preferring the vicinity of the salt marches; but also around ponds among the hills, and even on subaline plains of the lower Sacramento. A most distinct species every way, and one which, having its lowest leaves narrowest and its uppermost and later ones broadest, reverses that order of leaf-widening which is otherwise universal in Californian clovers. It is T. diversifolium of the Flora Franciscana; but Nuttall’s species cannot be identified by his meagre description, and it is hardly probable that he had this plant in view.”

Representative Specimens USA. California. Alameda County: May 1891, C.H. Michener s.n. (MU); Sacramento

County: 16th St. Station, Oakland, marsh, 20 April 1888, E.L. Greene (ND-G, UC); San Benito County: wet field along south side of Rt. 25, 0.5 miles west of Shore Road, northwest of Hillside, 9 April 1998, M. A. Vincent et al. 8188 (BRY, CAN, ISC, MU, RSA, WSU); San Francisco County: in low fields near Stege, 24 March 1900, J. P. Tracy 629 (K, UC, US); San Luis Obispo County: beside railroad, Mrs. R. W. Summers s.n. (GH, UC); Santa Clara County: pond adjacent

13 to railroad tracks and on west side US 101, 1 mile N of the Pajaro River, 28 April 1998, D. W. Taylor 16357 (MU); ibid.: San Jose, 01 June 1903, A. D. E. Elmer 4816 (B, COLO, G, E, MO, ORE, OSC, UC, W, WIS); Solano County: foothill valley, 5 miles northeast of Fairfield, 06 May 1954, B. Crampton 1835 (MU); ibid.: rich meadow near Vallejo, 20 April 1874, E.L. Greene 107 (GH, ISC); ibid.: Sulfur Springs Ridge, Vallejo, North Columbus Parkway, 13 April 2009, D. G. Kelch 9.077 (MU).

Figure 3. Trifolium hydrophilum from McDermott (1910), as T. depauperatum var. diversifolium. O: the ovary, w: the wing petal, v: the banner petal, i: the involucre, k: the keel petal, l: the leaf, h: the inflorescence, and c: the calyx.

Figure 4. Map of T. hydrophilum species distribution. Collections are mapped according to information taken from label data. Mapped specimens include those used in statistical analyses.

2. Trifolium depauperatum var. amplectens (Torr. & A. Gray) Rattan, Anal. Key West Coast Bot. 43, 1887 (as “amplectans”). Trifolium amplectens Torr. & Gray, Fl. N. Am. 1: 319. 1838. TYPE: USA. California, 1833, D. Douglas s.n. (LECTOTYPE (here designated) K 001051250; isolectotypes CAS, E, GH, K, US (photo)). (Fig. 5).

Trifolium quercetorum, Greene, Pittonia 1: 172. 1888. TYPE: USA: California, Oakland Hills, between Oakland and the Moraga Valley, towards Mt. Diablo, April 1888, V.K. Chesnut and A.B. Simonds s.n. (not located). NEOTYPE (here designated): USA. California: Alameda, Livermore, 14 April 1904, A. A. Heller 7319 (US).

Plants herbaceous, glabrous, annual; stems decumbent to erect, branching, 9-29.6 cm long, 0.3-1.7 mm diameter mid-stem; stipules adnate to petioles, sheathing, entire, 4.6-10.4x1.6- 4.3 mm; petioles 17-44x0.3-0.5 mm; leaves trifoliolate; leaflets linear or oblong, 6.5-13.2x2.2- 5.7 mm, dentate or somewhat serrulate, cuneate or decurrent; inflorescences racemes, axillary, umbelliform, 0.9-1.2 cm wide, rachises 0.4-2.1 mm long, glabrous, involucral bracts broad, irregular with wide, hyaline margins; pedicels 0.2-0.8 mm long, glabrous; calyces 0.9-4.4 mm long, tube 0.4-2.2 mm long, broadly campanulate, membranous or scarious, throat oblique, lobes unequal, subulate, typically as long or longer than tube, longest 1.9-4.9 mm; banner petals pink- purple, brown with age, claw absent, blade 5.8-10.7x2.3-4.6 mm, striate, broadly ovate, strongly inflated at maturity; wing petals purple, brown with age, asymmetrically clawed, claws 0.4-0.7 mm long, blade 4.7-9.3 mm long, laminae elliptic-orbicular; keel petals purple, darker at edges, brown with age, asymmetrically clawed, claws 0.2-0.5 mm long, blade 4.2-8.3 mm long, laminae elliptic-ovate; stamens diadelphous, connate filaments 4.2-5.9 mm long, free filaments 3.2-6.3 mm long, anthers 0.2 mm; pistils stipitate, stipes 0.3-2.3 mm, ovaries 1.9-5.3x0.8-2.5 mm, glabrous, styles 0.3-2.4 long, ovules 2-6; legumes inflated, glabrous; seeds 1-5, globular, 1.7x2.3x1.1 mm, brown, seed coat smooth, shiny. Specimens have been collected in California and Mexico (Fig. 6). The first specimen of Trifolium amplectens was collected in California. Trifolium depauperatum var. amplectens grows in grasslands and coastal woodlands, and flowers during the months of April, May and June. McDermott (1910) found that specimens from San Diego county were identical to those in

Santa Clara county, making its distribution almost as extensive as that of T. depauperatum in her study of this species complex. Torrey and Gray (1838) describe Trifolium amplectens as follows: “Glabrous, erect, branching: leaflets obovate-cuneiform, mucronately denticulate; stipules ovate, scarious, entire, aristate-mucronate; peduncles shorter than the leaves; involucre about half the length of the 5-6 flowered head, 4-5 parted; the segments somewhat lobed, obtuse; calyx much shorter than the corolla, cleft almost to the base; the teeth subulate, very unequal; vexillum free; covering the wings; legume sessile, 6-seeded. California, Douglas!—Plant 4-6 inches high. Leaflets very small, on slender petioles. Peduncles axillary. Heads less than half an inch diameter. Involucre scarious. Upper teeth of the calyx very short, about one-third the length of the others.”

Representative Specimens USA. California: Calaveras County: 1 mile east of Red House Ranch, open grassland in foothills, 9 May 1967, Beecher Crampton 7900 (MU); San Diego County: San Diego, April 1905, J.S. Brandegee (US); Santa Clara County: serpentine hills at Edenvale, about south-east of San Jose, 22 March 1961, J. H. Thomas 8999 (MU); Tehama County: on the north side of Dye Creek, about 1/2 mile east of the Dye Creek buildings, Dye Creek Preserve, about 6 miles (air) northeast of Los Molinos, 17 April 2003, L. Ahart, J. Dittes, Daphne C. & H. Stevens 10106 (MU); Ventura County: Diamond Ranch, 01 May 1998, R. Burgess, T. Austin, G. Austin & P. Munro 2844 (SBBG). Mexico. Baja California: Guadalupe Island, along trail from northwest anchorage to first large valley on route to springs and Cypress Grove, 25 April 1958, Ira L. Wiggins & W.R. Ernst 92 (GH); ibid.: 19 April 1925, H. L. Mason (GH); ibid.: common about northeast spring, 25 April 1958, Reid Moran 6656 (SD); ibid.: just above barracks at NE anchorage, 28 March 1988, S.A. Junak 3471 (MU); ibid.: Laguna Hanson, Constitucion National Park, Sierra de Juarrez, near west margin of Laguna Hanson, 28 May 1983, R. F. Thorne, W. Wisura & W. Steinmetz 55872 (SD).

Figure 5. Trifolium depauperatum var. amplectens from McDermott (1910). O: the ovary, w: the wing petal, v: the banner petal, i: the involucre, k: the keel petal, l: the leaf, h: the inflorescence, and c: the calyx.

Figure 6. Map of T. depauperatum var. amplectens species distribution. Collections are mapped according to information taken from label data. Mapped specimens include those used in statistical analyses.

3. Trifolium depauperatum var. truncatum (Greene) Martin ex Isely, Brittonia 32: 56 (1980). T. franciscanum Greene var. truncatum Greene, Man. Bot. Reg. San Franc. Bay 100 (1894). T. truncatum (Greene) Greene, Proc. Acad. Philad. 1895: 564 (1896); T. amplectens Torr. & Gray var. truncatum (Greene) Jeps., Man. Fl. Pl. Calif. 537 (1925); T. depauperatum Desv. var. amplectens (Torr. & Gray) Wats. f. truncatum (Greene) McDerm., N. Am. Sp. Trif. 144. (1910). LECTOTYPE (designated by Gillett 1966): USA: California, Livermore Valley, E.L. Greene s.n., 3 April 1895 (NDG 36019). (Fig. 7).

T. anodon Greene, Pittonia (27): 107 (1903). LECTOTYPE (designated by Gillett 1966): USA. California, San Diego, T.S. Brandegee s.n., April 1902 (distributed by Baker as no. 282) (NDG 36029) [photo DAO]; isolectotype: B, ECON, G, K, MSC, NY, US [photo DAO], W). SYNTYPE: ibid, T.S. Brandegee s.n., April 1903 (distributed by Baker as no. 3422) (B, ECON, G, K, NDG (2), UC, UC (photo), W).

T. brachyodon Greene (non Celak. 1881), Pittonia 5: 107 (1903). LECTOTYPE (designated by Gillett 1966): USA. California, Santa Catalina Island, common on dry peaks, Mrs. Trask s.n., March 1901 (NDG 36036, lower specimen). SYNTYPE: USA. California, Santa Catalina Island, Mrs. Trask s.n., March 1901 (NDG 36038, upper specimen).

T. decodon Greene, Pittonia 5(27A): 108 (1903). LECTOTYPE (designated by Gillett 1966): USA. California, San Diego, T. S. Brandegee s.n., 20 May 1903 (distributed by Baker as no. 3371) (NDG 1328 [photo DAO]); isolectoypes B (2), CAS, G, GH, K, MSC, NDG, NY, UC (3), UC (drawing), US, W).

T. franciscanum Greene, Man. Bot. Reg. San Franc. Bay 100 (1894). T. depauperatum Desv. var. stenophyllum (Nutt.) McDerm. f. fransciscanum (Greene) McDerm., Ill. Key Amer. Trifolium 140 (1910). LECTOTYPE (designated by Gillett 1966): USA. California, San Francisco, E.L. Greene s.n., 22 Apr 1891 (NDG 36019 [photo DAO]; isolectotype JEPS).

T. minutiflorum Greene, Pittonia 3 : 215 (1897). LECTOTYPE (designated by Gillett 1966): USA. California, San Diego, C.R. Orcutt s.n., 24 Mar 1884 (NDG 36009).

T. stenophyllum Nutt., Proc. Acad. Nat. Sci. Philadelphia 4(1): 8 (1848). T. amplectens Torr. & Gray var. stenophyllum (Nutt.) Jeps., Man. Fl. Pl. Calif. 537 (1925). T. depauperatum Desv. var. stenophyllum (Nutt.) McDerm., N. Am. Sp. Trif. 137 (1910). LECTOTYPE (designated here): USA. California, island of Santa Catalina, Gambel s.n., s.d. (GH; likely isolectotype GH, K).

Plants herbaceous, glabrous, annual; stems decumbent to erect, branching, 5.2-39.6 cm long, 0.4-1.8 mm diameter mid-stem; stipules adnate to petioles, sheathing, entire, 4.6-10.4x1-5 mm; petioles 20-41x0.2-0.6 mm; leaves trifoliolate; leaflets linear or oblong, 5.4-17.7x1-5.3 mm, dentate or somewhat serrulate, cuneate or decurrent; inflorescences racemes, axillary, umbelliform, 0.7-1.4 cm wide, rachises 0.2-1.9 mm long, glabrous, involucral bracts as long as the calyx and free; pedicels 0.1-0.4 mm long, glabrous; calyces 0.8-4.2 mm long, tube 0.9-1.8 mm long, broadly campanulate, membranous or scarious, throat oblique, lobes unequal, subulate, typically as long or longer than tube, longest 0.6-3.8 mm; banner petals pink-purple, brown with age, claw absent, blade 3.2-7.2x1.8-4.5 mm, striate, broadly ovate, strongly inflated at maturity; wing petals purple, brown with age, asymmetrically clawed, claws 0.3-1.1 mm long, blade 2.4-6.4 mm long, laminae elliptic-orbicular; keel petals purple, darker at edges, brown with age, asymmetrically clawed, claws 0.2-0.7 mm long, blade 3.1-7.1 mm long, laminae elliptic-ovate; stamens diadelphous, connate filaments 2.3-5.9 mm long, free filaments 2.2-4.9 mm long, anthers 0.2 mm; pistils stipitate, stipes 0.1-0.6 mm, ovaries 1.2-3.8x0.4-2.5 mm, glabrous, styles 0.2-2.6 long, ovules 1-2; legumes inflated, glabrous; seeds 1-2, globular, 1.6x2.2x1.4 mm, brown, seed coat smooth, shiny. Trifolium depauperatum var. truncatum is found in the high coastal ranges of California to Baja California, Mexico (Fig. 8). No type was designated when the name was published, so Gillet lectotypified Trifolium depauperatum var. truncatum in 1966. Trifolium depauperatum var. truncatum is the only taxon of this species complex that is found on the Channel Islands. McDermott (1910) equated T. depauperatum var. truncatum with the name T. depauperatum var. stenophyllum. If T. depauperatum var. truncatum were to be recognized as a distinct species, the name that would correspond to this taxon is T. stenophyllum Nuttall (1848). Nuttall (1848), originally described Trifolium stenophyllum as follows: “Annual, branching from the base; leaves ternate, smooth and linear, distantly serrulate; stipules

subulate, sparingly denticulate; peduncles elongated, filiform. Heads small and nearly round, the vecillum, at length, forming a membranous inflated sac of equal breadth throughout, embracing the small wings and small carina, which is monopetalous, but with one broad claw attached to the vexillum. With all the aspect of the involucrate clovers. Flowers brownish, the keel deeper colored. Stamens diadelphous. Pod stipitate, flat, two- seeded, seeds obcordate. Stigma small, capitate. About four to six inches high: leaves about one or two lines wide. Hab. The island of Santa Catalina, and San Pedro, Upper California. Flowering in February.”

Representative Specimens USA. California. Alameda County: growing wild on the University campus, Berkeley, 12 April 1907, Miss H.A. Walker 551 (MU, US); Butte County: Chico-Centerville road 3 miles from Chico at the lower limit of the digger pine belt, in low ground in adobe, 20 April 1915, A. A. Heller 11851 (GH); ibid: near Clear Creek, 15 April 1897, H. E. Brown (US); Colusa County: along Bear Valley Rd., 2.5 miles N jct. Wilbur Springs Rd, 30 March 1996, M. A. Vincent & R. Rhode 7289 (MU); Contra Costa County: Mount Diablo state park, shallow soil on outcrops of "China Wall" formation, N-facing grassland, 15 April 2001, Barbara Ertter & Daniel Norris 17611 (MU); ibid: Byron, 27 April 1903, C. F. Baker 2863 (US); Fresno County, Little Panoche Road, ca. 2 miles west of Hwy 5, 26 March 1998, Randall Morgan (MU); Glenn County: Elk Creek-Stonyford Rd. at the salt springs 0.2 mi N of Salt Creek, 3.2 mi N of the Colusa County line, 6 April 1994, V. H. Oswald & Lowell Ahart 6054 (MU); Kern County: near Caliente, 07 April 1905, A. A. Heller 7620 (MO); Los Angeles County: San Clemente Island: central upland portion of the island, SW of 'Ledge', 15 April 1992, T. Ross and A. Kendall 6192 (MU); ibid: Santa Catalina Island, dry grassy W-facing slope, Blue Cavern Point, NE of Isthmus Cove, 4 April 1966, Robert F. Thorne 35772 (SD); ibid: Los Angeles, 24 March 1889, J. Q. A. Fritchey (MO); ibid: San Clemente Island, E side of island, along N side of Horton Canyon Road, ca. 0.6 miles E of San Clemente Island Ridge Road, 22 April 1997, Steven A. Junak SCI-848 (SBBG); ibid: Santa Catalina Island, ridge on W side of Cape Canyon, 21 March 1998, Steven A. Junak et al. SCa-493 (SBBG); Monterey County: Pacific Grove in pine woods, 16 May 1903, A. A. Heller 6734 (US); Orange County: Newport Beach, edge of dry mesa facing Newport Bay and the ocean, along main coast highway, 14 April 1935, Donald G. Nelson 287 (SD); Riverside County:

NW Palomar Mountains, Agua Tibia Mountains: Pechanga Indian Reservation, 29 March 1997, Darin L. Banks 1605 (MU); ibid: Santa Rosa Plateau, open desiccated mud at margin of vernal marsh, Mesa de Colorado, 18 April 1968, O. Clark & S. Carlquist 37438 (SD); San Benito County: wet field along south side of Rt. 25, 0.5 miles west of Shore Road, northwest of Hillside, 9 April 1998, M. A. Vincent, E. H. Freid, R. Morgan 8192 (MU); San Bernardino County: Red Hill, near Upland, 8 April 1917, Ivan Johnston 1192 (MU); San Diego County: north slope of San Miguel Mt., 28 March 1935, Frank F. Gander (SD); ibid: National Ranch, 01 April 1882, E. F. G. (SD); ibid: San Diego, 23 April 1888, Daniel Cleveland (SD); ibid: Stonewall mine, Cuyamaca Mts., 05 June 1897, S. B. Parish 4417 (MO); San Francisco County: San Francisco, May 1891, C. H. Michener (MU); Santa Barbara County: Santa Cruz Island, along W. side of Centinela Connector Road, 15 April 1992, Steven A. Junak SC-3178 (SBBG); ibid: San Miguel Island, SW side of island, 16 April 1998, Steven A. Junak 6361 (SBBG); ibid: Santa Rosa Island, Canada Verde & Dry Canyon, 12 April 1993, S.A. Junak SR-574 (SBBG). Santa Clara County: near Frenchman's Lake, 14 March 1900, W. F. Wright 134 (US); Santa Cruz County: Scotts Valley, grassland north of Casa Way, 10 April 1989, Randall Morgan 1463 (MU); ibid: Scotts Valley, grassland north of Casa Way, 10 April 1989, Randall Morgan 1463 (MU); ibid: grassland west of Meder Street and south of UCSC campus, 29 March 1989, Randall Morgan 1391 (MU); Solano County: S.S.E. Davis, Tremont Rd. 1/4 mile east of its junction with Bulkley, agricultural area, 9 April 1979, Beecher Crampton 9676 (MU); ibid: Cordelia, vernal meadow (dry), adobe soil, 26 April 1966, Beecher Crampton 7673 (MU); ibid: Vallejo, 16 March 1914, W. W. Jones 188 (GH); Sonoma County: Kenwood, May 1893, Michener & Bioletti (MU); Ventura County: San Nicolas Island, mesa, just N of airfield, E of N runway access road, across airfield from control tower, 10 April 2001, Steven A. Junak SN-1671 (SBBG); ibid: San Nicolas Island, NE portion of mesa, N of Beach Road at NW end of airfield, 09 April 2001, Steven A. Junak SN-1650 (SBBG); Mexico. Baja California: scarce on dry grassy slope, 2 km SW of Redondo Station, 27 May 1978, Reid Moran 26045 (SD); ibid., common in marshy ground, 2 km north of Las Juntas, 07 June 1980, Reid Moran 28727 (SD); ibid., common in muddy bottom, south of Agua Amarga, 5 km north of Laguna Hanson, 28 May 1979, Reid Moran 27530 (SD).

Figure 7. Trifolium depauperatum var. truncatum from McDermott (1910), as T. depauperatum var. stenophyllum. O: the ovary, w: the wing petal, v: the banner petal, i: the involucre, k: the keel petal, l: the leaf, h: the inflorescence, and c: the calyx.

Figure 8. Map of T. depauperatum var. truncatum species distribution. Collections are mapped according to information taken from label data. Mapped specimens include those used in statistical analyses.

4. Trifoliumn depauperatum var. depauperatum Trifoliumn depauperatum Desv., Journal de Botanique, Appliquée à l'Agriculture, à la Pharmacie, à la Médecine et aux Arts 4: 69–70, pl. 32, f. 2, 1814. Lupinaster depauperatus (Desv.) Presl, Symb. Bot. 1:50, 1831. LECTOTYPE (here designated): Journal de Botanique, Appliquée à l'Agriculture, à la Pharmacie, à la Médecine et aux Arts 4: pl. 32, f. 2; EPITYPE: Chile. Perou, Dombey s.n. (P02733815).

Trifolium laciniatum Greene, Pittonia 1(1): 7-8, 1887. T. depauperatum Desv. var. laciniatum (Greene) Jeps., Fl. W. Mid. Calif. 311, 1901. T. depauperatum Desv. fo. laciniatum (Greene) McDerm., N. Am. Sp. Trif. 133, 1910. TYPE: USA. California: Byron Springs, April 1884, E. L. Greene NDG 36005 (isolectotype, designated by Gillett (1966) NDG 36007). (Fig. 9). Trifolium laciniatum Greene var. angustatum Greene, Man. Bot. Reg. San Franc. Bay 101, 1894. TYPE: USA. California: Byron Springs, 24 March 1889, E. L. Greene (lectotype, designated by Gillett (1966): NDG 36006.

Plants herbaceous, glabrous, annual; stems decumbent to erect, branching, 5-21 cm long, 0.4-1.5 mm diameter mid-stem; stipules adnate to petioles, sheathing, entire, 4.9-9x1-3.4 mm; petioles 21-33x0.4-0.6 mm; leaves trifoliolate; leaflets linear or oblong, 4.9-14.8x0.9-4.7 mm, dentate or somewhat serrulate, cuneate or decurrent; inflorescences racemes, axillary, umbelliform, 0.9-1.3 cm wide, rachises 0.1-1.7 mm long, glabrous, involucral bract cup-like; pedicels 0-0.4 mm long, glabrous; calyces 1.2-6.2 mm long, tube 0.3-2.2 mm long, broadly campanulate, membranous or scarious, throat oblique, lobes unequal, subulate, typically as long or longer than tube, longest 1.1-4 mm; banner petals pink-purple, brown with age, claw absent, blade 5.2-9.2x2.7-4.9 mm, striate, broadly ovate, strongly inflated at maturity; wing petals purple, brown with age, asymmetrically clawed, claws 0.3-1 mm long, blade 3.7-7.9 mm long, laminae elliptic-orbicular; keel petals purple, darker at edges, brown with age, asymmetrically clawed, claws 0.2-0.6 mm long, blade 4.4-7.3 mm long, laminae elliptic-ovate; stamens diadelphous, connate filaments 3.8-7.1 mm long, free filaments 3.3-6.3 mm long, anthers 0.2 mm; pistils stipitate, stipes 0.2-0.7 mm, ovaries 1.2-4.2x0.7-2.7 mm, glabrous, styles 0.7-3.4 long, ovules 2-4; legumes inflated, glabrous; seeds 1-2, globular, 0.5x0.6x0.5 mm, brown, seed coat smooth, shiny. 2n= 16 (Ellison et al. 2006; Zohary & Heller 1984). Ellison (2006) and

Zohary & Heller 1984 have a chromosome count of Trifolium depauperatum, however, no variety was designated. Specimens have been collected in British Columbia on Vancouver Island, Washington, Oregon, California, and southern Chile. The first specimen of Trifolium depauperatum was collected in Santiago Chile (Fig. 10). Trifolium depauperatum is the sub-taxon that exhibits the amphitropical disjunct distribution in this species complex. Carlquist (1983) hypothesized that this distribution was due to the migratory patterns of shore birds. Trifolium depauperatum flowers during the months of March, April, and May. Desvaux (1814) originally described T. depauperatum as follows: “Only a few inches high, branched from the base; flacid, decumbent, glabrous, few leaved: leaflets one half in. long, cuneate-oblong, obtuse or emarginate, denticulate, head long, stalked, few- flowered; involucre greatly reduced with truncate short lobes, corolla longer than in the last, less inflated pod one or two seeded; seeds a little broader than long, rather angular, tuberculate-rugose. Less common than the last (T. stenophyllum) and a small rather obscure species seldom collected. It appears to be one of the few plants common to the Wester coasts of both North and South America.”

Representative Specimens USA. California: Butte County: 28 March 2001, R. R. Halse 5975 (MU); Colusa County: along Bear Valey Rd., 2.5 mi. N. jct. Wilbur Springs Rd., 20 March 1996, M. A. Vincent and Robert Rhode 7290 (MU); Contra Costa County: Byron Springs, 23 March 1889, E. L. Greene (OSC); Fresno County: Little Panoche Road, ca. 2 miles west of Hwy 5, 26 March 1998, R. Morgan (MU); Placer County: 2.5 miles of Lincoln, 14 April 1966, Beecher Crampton 7648 (MU); San Diego County: mountains east of San Diego, California, 1923, M. F. Spencer (MU); Santa Cruz County: Scotts Valley grassland north of Casa Way, 5 April 1989, R. Morgan 1457 (MU); Sonoma County: Kenwood, 1893, Michener & Bioletti (MU); Stanislaus County: 10 miles south of Modesto, 12 April 1940, R. F. Hoover 4330 (US); Oregon: Jackson County: Rogue Valley, on top of table Rock, 16 April 1887, T. Howell 638 (GH); Curry County: bays of the Chetco River, 12 May 1929, L. F. Henderson 10118 (PH); Jackson County: Agate Desert, near Medford, 4 December 1940, L. E. Detling 3952 (WS); Canada. British Columbia: Vancouver Island: two miles ESE of Langford, just NW of Victoria, 12 May 1957, J. A. Calder, D. B. O.

Saville, and R. L. Taylor 20787 (US); Vancouver Island, Cedar Hill, 14 May 1887, J. Macoun (US); ibid: Mt. Finlayson a few miles north of Victoria, 23 May 1961, J. A. Calder and K. T. Mackay 29436 (GH); Chile. Santiago: Limache, Fundo Pangal, 19 September 1921, S. Looser and R. A. Philippi 957 (GH).

Figure 9. Trifolium depauperatum var. depauperatum from McDermott (1910). O: the ovary, w: the wing petal, v: the banner petal, i: the involucre, k: the keel petal, l: the leaf, h: the inflorescence, and c: the calyx.

Figure 10. Map of T. depauperatum var. depauperatum species distribution. Collections are mapped according to information taken from label data. Mapped specimens include those used in statistical analyses.

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Appendix 1. List of quantitative and qualitative characters measured. Character Type of Character Peduncle Length Continuous Quantitative Stem Diameter Continuous Quantitative Stem Length Continuous Quantitative Length of Leaflet Continuous Quantitative Width of Leaflet Continuous Quantitative Calyx Circumference Continuous Quantitative Calyx Tube Length Continuous Quantitative Calyx Lobe Length Continuous Quantitative Banner Petal Width Continuous Quantitative Banner Petal Length Continuous Quantitative Wing Petal Width Continuous Quantitative Wing Petal Length Continuous Quantitative Wing Petal Claw Length Continuous Quantitative Keel Petal Claw Length Continuous Quantitative Keel Petal Width Continuous Quantitative Keel Petal Length Continuous Quantitative Single Stamen Length Continuous Quantitative Stamen Tube Length Continuous Quantitative Ovary Width Continuous Quantitative Ovary Length Continuous Quantitative Style Length Continuous Quantitative Number of Ovules Discontinuous Quantitative Pistil Stipe Length Continuous Quantitative Stipule Length Continuous Quantitative Stipule Width Continuous Quantitative Involucre Bract’s Length Continuous Quantitative Involucre Bract’s Width Continuous Quantitative Involucre Lobe Length Continuous Quantitative Involucre Lobe Width at Widest Part Continuous Quantitative Involucre Lobe Apex Angle Discontinuous Quantitative Involucre Margin Hyaline Description Binary Qualitative Floral Bract Length Continuous Quantitative Rachis Length Continuous Quantitative Pedicel Length Continuous Quantitative How many whorls of flowers in inflorescence Semiquantitative *Seed Number Discontinuous Quantitative *Seed Width Continuous Quantitative *Seed Length Continuous Quantitative *Seed Thickness Continuous Quantitative *Seed Coat Texture Multistate Qualitative *Seed Coat Texture Description Multistate Qualitative *Seed Coat Color Multistate Qualitative

*Non-variable characters.

Appendix 2. R code for selected statistical analyses. #Standardizing data <- read.csv("Master Spreadsheet.csv") rownames(data)<-data[,1] data_stand <- scale(data[,3:37], center=TRUE, scale=TRUE)

#PCA data <- read.csv("Master Spreadsheet.csv") PC<-princomp(data[,c(3:37)], cor=TRUE) lambda <- PC$sdev*sqrt(nrow(PC$scores)) plot (t(t(PC$scores)/lambda), pch=as.numeric(data[,2]), col=as.numeric(data[,2]), xlab="Comp. 1", ylab="Comp. 2") legend("topleft",c("T. depauperatum var. depauperatum","T. depauperatum var. truncatum","T. hydrophilum"), pch = c(1,2,3),col=c("black","red","green","blue"), horiz = FALSE, cex = 0.3)

#Map Plotting install.packages('maps') install.packages('mapdata') library('maps') library('mapdata')

#Map of each taxon Lydia <- read.csv("Book1.csv") View(Lydia) points(Lydia$Long, Lydia$Lat, pch=19, col="blue", cex=0.3) install.packages(c("cowplot", "googleway", "ggplot2", "ggrepel", + "ggspatial", "libwgeom", "sf", "rnaturalearth", "rnaturalearthdata")) library("ggplot2") theme_set(theme_bw()) library("sf") library("rnaturalearth") library("rnaturalearthdata") world <- ne_countries(scale = "medium", returnclass = "sf") install.packages("rgeos") ggplot(data = world) + + geom_sf() ggplot(data = world) + geom_sf() + xlab("Longitude") + ylab("Latitude") + ggtitle("World map", subtitle = paste0("(", length(unique(world$NAME)), " countries)")) ggplot(data = world) + geom_sf(color = "black", fill = "lightgreen")

ggplot(data = world) + geom_sf() + coord_sf(xlim = c(-125.15, -50.12), ylim = c(-35, 50.12), expand = TRUE)

# Map of Trifolium depauperatum var. depauperatum ggplot(data = world) + geom_sf() + coord_sf(xlim = c(-125.15, -50.12), ylim = c(-35, 50.15), expand = TRUE)

(sites <- data.frame(longitude = c(-121.6885, -122.3216, -122.3216, -116.61, -122.3216, -122.38, -120.64, -122.01, -122.05, -121.57, -120.04432, -120.85987, -121.55, -121.293, -122.05827, - 122.06377, -122.00107, -121.54495, -122.93, -71.26, -70.67, -70.97, -122.98, -124.07, -122.76, - 123.49563, -123.34531, -123.53877, -123.46376, -72.23743, -122.89837), latitude = c(39.5855, 38.212, 38.212, 32.6, 38.212, 40.59, 37.98, 37.05, 36.99, 39.59, 38.46835, 36.7076, 39.55638, 38.89156, 36.99158, 36.98993, 37.06753, 39.58926, 42.44, -33.01, -33.45, -33.97, 42.49, 42.18, 42.33, 48.44746, 48.44205, 48.48149, 48.46477, -37.71788, 42.42901))) ggplot(data = world) + geom_sf() + geom_point(data = sites, aes(x = longitude, y = latitude), size = 1, shape = 25, fill = "blue") + coord_sf(xlim = c(-125.15, -50.15), ylim = c(-35, 50.15), expand = TRUE)

# Map of Trifolium depauperatum var. amplectens ggplot(data = world) + geom_sf() + coord_sf(xlim = c(-125.15, -50.12), ylim = c(-35, 50.12), expand = TRUE)

(sites <- data.frame(longitude = c(-122.44, -122.01551, -121.495, -120.68, -118.26, -118.15, - 118.15, -118.16, -118.1925, -121.768, -117.16108, -118.27605, -115.90688), latitude = c(37.67, 40.06306, 37.165, 38.2, 29, 29.03, 28.58, 28.59, 27.075, 37.68187, 32.71573, 29.05252, 32.04456))) ggplot(data = world) + geom_sf() + geom_point(data = sites, aes(x = longitude, y = latitude), size = 1, shape = 22, fill = "red") + coord_sf(xlim = c(-125.15, -100.15), ylim = c(20, 50.15), expand = TRUE)

# Map of Trifolium depauperatum var. truncatum ggplot(data = world) + geom_sf() + coord_sf(xlim = c(-125.15, -50.12), ylim = c(-35, 50.15), expand = TRUE)

(sites <- data.frame(longitude = c(-121.96, -118.25, -122.55, -122.02, -122.54, -122.43, -121.75, -118.33, -117.79, -121.92, -122.13, -122.06, -122.07, -122.03, -116.47, -116.235, -115.555, - 116.938533, -117.153839, -116.993508, -117.382, -122.02, -120.8556, -116.9819, -116.6442, - 116.6442, -122.01468, -117.54254, -118.49807, -122.01031, -121.82374, -122.25853, - 122.55184, -120.04432, -121.83747, -122.25663, -121.91662, -117.24299, -117.92984, - 117.11218, -116.93701, -117.16108, -121.64541, -120.1849, -122.25853, -116.58236, - 121.91662, -117.62172, -117.18519, -121.638, -118.24368, -116.56918, -118.62786, - 122.46619, -122.1347, -122.2333, -120.99687, -120.3976, -118.47169, -118.41596, -120.05434, -118.32822, -118.4773), latitude = c(38.04, 33.19, 38.41, 37.05, 38.44, 37.76, 38.5, 33.34, 33.76, 37.88, 38.21, 36.97, 36.97, 37.05, 32.305, 32.3175, 32.065, 32.704611, 32.672969, 32.8063, 33.193022, 37.05, 36.6925, 33.40972, 32.8825, 32.8826, 37.05105, 33.75001, 32.90288, 37.06696, 38.27535, 37.87189, 39.39569, 38.46835, 39.72849, 38.10408, 36.61773, 33.53159, 33.61888, 32.90473, 32.69737, 32.71573, 39.67416, 39.90814, 37.87189, 32.98887, 36.61773, 34.11389, 32.76845, 37.86714, 34.05223, 32.98282, 35.29107, 37.79887, 37.25, 37.40139, 37.63909, 37.18536, 33.38572, 33.34891, 34.0099, 33.34281, 33.44808))) ggplot(data = world) + geom_sf() + geom_point(data = sites, aes(x = longitude, y = latitude), size = 1, shape = 21, fill = "green") + coord_sf(xlim = c(-125.15, -100.15), ylim = c(20, 50.15), expand = TRUE)

# Map of Trifolium hydrophilum ggplot(data = world) + geom_sf() + coord_sf(xlim = c(-125.15, -50.12), ylim = c(-35, 50.12), expand = TRUE)

(sites <- data.frame(longitude = c(-122.1208, -121.72, -121.87, -122.04, -121.71, -122.2194, - 121.4464, -121.7589, -122.25663, -122.15857, -120.65961, -121.48901, -121.48866, -122.2913, -122.24163, -122.25379, -121.88632, -122.32719, -121.88632), latitude = c(38.084, 37.6, 37.66, 38.25, 36.88, 38.15694, 36.94222, 38.33278, 38.10408, 38.04936, 35.28275, 38.56977, 38.56989, 37.82717, 37.7652, 37.7718, 37.3382, 37.91659, 37.3382))) ggplot(data = world) + geom_sf() + geom_point(data = sites, aes(x = longitude, y = latitude), size = 1, shape = 25, fill = "pink") + coord_sf(xlim = c(-125.15, -100.15), ylim = c(20, 50.15), expand = TRUE)

Chapter 2: A PRELIMINARY MOLECULAR ANALYSIS OF THE TRIFOLIUM DEPAUPERATUM SPECIES COMPLEX Introduction DNA-based markers in phylogenetic reconstructions provide researchers with the ability to study biological diversity and evolution from the population level up to the comparison of families and kingdoms (Moritz & Hillis 1996). Molecular sequences have contributed most significantly in areas where morphologies were inconclusive or poorly analyzed (Patterson et al. 1993). Since molecular systematics was developed, many studies have included both molecular and morphological datasets in analyses to support phylogenetic hypotheses (Denk et al. 2005; Doyle 2005; Kron et al. 2002). Through the use of polymerase chain reactions (PCR), analysis of minute pieces of DNA is made possible, particularly the investigation of specimens in museums and herbaria (Lookerman & Jansen 1996; Pääbo 1990). A successful amplification from museum or herbarium specimens depends mostly on the preservation of the DNA and the purification of the DNA extracted (Blattner 1999). Generally, the unfavorable slow drying when the specimen was collected initially and storage under unfavorable conditions of biological material can result in severely degraded DNA, rendering the amplification of DNA fragments in the range of 150-400 bp extremely improbable (Golenburg et al. 1996; Höss et al. 1996). Analyses of DNA from older samples is often only possible through stepwise amplification and the sequencing of short overlapping sequences of DNA (Blattner 1999). Internal transcribed spacers (ITS) of the nuclear ribosomal DNA are important molecular markers in phylogenetic studies (Blattner 1999). Nuclear ribosomal DNA (nrDNA) consists of the coding subunits 18S-, 5.8S- and 26S- which are separated by ITS-1 and ITS-2 (White et al. 1990). These ITS regions occur in high copy numbers in the genome. The ITS regions make up the transcriptional unit but are not included in the final ribosomal RNA (Blattner 1999). Key parts of the spacer sequences are essential for rRNA secondary structure and are evolutionarily conserved among plants (Hershkovitz & Zimmer 1996; Liu & Schardl 1994; Mai & Coleman 1997). The other components of the spacers are variable between species. This variation makes the ITS marker a useful tool in resolving evolutionary relationships, even those phylogenetic relationships between closely related individuals in a species complex. Conserved flanking regions of the two spacers have also allowed for the construction of universal primers suitable

39 for PCR amplification making ITS an attractive nuclear marker for phylogenetic analysis for species-level questions (Baldwin 1992; Baldwin et al. 1995; Blattner 1999). Noncoding regions of cpDNA, which are presumably under less functional constraint and thus evolve more rapidly, also provide useful phylogenetic information at the lower taxonomical levels, including within species complexes (Clegg et al. 1994; Gielly & Taberlet 1994; Sang et al. 1997). The two intergenic spacers of chloroplast DNA (cpDNA), psbA-trnH and trnL are often used to inform phylogenetic reconstructions besides nuclear ribosomal DNA (Sang et al. 1997; Bruyns et al. 2006; Miller et al. 2003). For the current study, six individuals from the Trifolium depauperatum species complex were sequenced and analyzed using the internal transcribed spacers region. The objective of this chapter was to infer phylogenetic relationships of taxa within the Trifolium depauperatum species complex in order to inform taxonomic delineations.

Materials and Methods Leaf tissue from eight herbarium specimens was removed and used for DNA extraction with permission of herbaria from which specimens were obtained (MU, OSC, WS). Specimens sampled included two specimens of T. depauperatum var. depauperatum collected in California, two specimens of T. depauperatum var. truncatum collected in California, one specimen of T. depauperatum var. amplectens collected in California, one specimen of T. hydrophilum collected in California, one specimen of T. depauperatum var. depauperatum collected in Chile, and one specimen of T. repens collected in Ohio which was included as an outgroup. Nuclear and chloroplast genomic DNA was extracted from dried leaves using the DNeasy Plant Mini Kit (QIAGEN, Inc.). Most of the material from this study was collected from herbarium specimens with severely degraded DNA. In order to ensure successful amplification, two separate amplification reactions were conducted in order to cover the entire region. Through the combination of the external and internal primers (Blattner 1999; Sang et al. 1997) one complementary strand was created. The ITS region, psbA-trnH intergenic spacer, and trnL intergenic spacer was PCR- amplified using the primers ITS-a and ITS-b (Blattner 1999), psbA forward and trnH reverse (Sang et al. 1997), and TAB-e and TAB-f (Taberlet et al. 1991) in a 10 L reaction volume. The

PCR conditions were carried out in the SimpliAmpTM Thermal Cycler (Thermo Fisher Scientific

Inc., Carlsbad, CA, USA) as follows. An initial denaturation at 95C for 5 minutes was followed by 2 cycles of annealing at 57C for 15 s, extension at 72C for 30 s, and denaturation at 95C for 15 s; followed by changing annealing temperatures to decrease from 56C to 51C over 10 total cycles (1 degree per 2 cycles); followed by 15 cycles of annealing at 50C for 15 s, extension at 72C for 30 s, and denaturation at 95C for 15 s; with a final extension at 72C for 7 minutes. Amplification was carried out with 7.5L of GoTaq Green Master Mix (Promega, Madison, WS, USA), 1 L of DNA, 0.6 L of each primer, and 5.3 L of water. DNA quantity and quality were checked on a 1% agarose gel with approximately 5L of PCR product. After checking by gel, the PCR products were cleaned with the Wizard SV PCR Clean-Up System (Promega, Madison, WS, USA). 48 purified PCR products, 8 for each primer, were then sent to Eton Bioscience and sequenced using Sanger sequencing. A few sequences were low quality, so amplification was run a second time at the facility. After the second amplification, most of the sequences received from Eton Bioscience were successfully amplified and analyzed. Alignment and consensus sequence generation was conducted using the program Geneious Prime (Geneious Prime 2020.1.1). The ITS, psbA-trnH intergenic spacer, and trnL intergenic spacer were aligned and corrected manually. The GTR model of DNA substitution was selected for the Bayesian phylogenetic analysis and run using BEAST 2.5 (Bouckaert et al. 2019). A consensus tree was then generated based on these results (Fig. 1).

Results

The psbA-trnH intergenic spacer alignment represented 8 taxa and was made up of 495 base pairs. The trnL intergenic spacer alignment represented 8 taxa and was made up of 212 base pairs. The psbA-trnH intergenic spacer and trnL intergenic spacer showed no variable characters in the consensus sequences, so these markers were not included in the analysis. Of the eight specimens processed, only seven samples had amplifiable products for ITS. Amplification failed for DNA isolated from the T. depauperatum var. depauperatum specimen from Chile that was collected in the 1800s. The ITS alignment represented 7 taxa and was made up of 674 base pairs. In the ITS alignment there were twelve informative variable sites among the taxa in this group. The

41 consensus tree generated from Bayesian phylogenetic analysis using ITS alignment yielded a statistically supported tree based on the posterior probabilities. T. repens was the outgroup chosen for this analysis because of its old-world origin (Ellison et al. 2006). T. depauperatum var. amplectens was sister to the branch with T. depauperatum var. truncatum specimens. T. hydrophilum was nested within a clade with both T. depauperatum var. depauperatum specimens.

Discussion These results support a close phylogenetic relationship among these taxa. There are clear differences between the morphology-based clustering and the phylogeny of this group, however. The preliminary molecular data suggest that molecularly, Trifolium depauperatum var. amplectens and Trifolium depauperatum var. truncatum are more closely related to one another, whereas Trifolium hydrophilum in nested within Trifolium depauperatum var. depauperatum. According to the phenogram, T. depauperatum var. amplectens is more morphologically similar to T. hydrophilum than any other taxon. Oftentimes morphology overcomes weak molecular evidence in cases where molecular and morphological trees conflict (Doyle & Endress 2000). That the cpDNA and ITS regions these taxa have not diverged appreciably genetically, suggests a close evolutionary relationship among these taxa. This lack of variation is consistent with the designation of these taxa at the varietal level, except for T. hydrophilum which has a divergent ecological habitat. A more robust dataset using more specimens should be generated before this analysis can inform any decisive taxonomic conclusions. At the moment, the current molecular analysis does not support or refute the morphological data set, nor does it inform us as to whether this species complex is made up of one species with four taxa or four completely separate species. Morphological and molecular data do not always agree with one another, and this conflict can be due to differences in assumptions between analyses (Hillis 1987). Change within monophyletic groups, whether morphological or molecular, is positively correlated with time. However, the degree of constancy of change between data sets is a point of considerable debate (Britten 1986; Hillis 1987; Negrón-Ortiz & Watson 2002). I hypothesize that members of the Trifolium depauperatum species complex have radiated rapidly morphologically relatively recently across

42 its geographic distribution, at least in a time frame that prevents the substantial accruement of sequence differences in the ITS, psbA-trnH, and trnL regions.

Future Directions Due to the COVID-19 pandemic, I was unable to have access to a lab in order to complete a more robust molecular dataset. The data presented in this chapter is not enough to make any taxonomic conclusions that add to the morphological study of the Trifolium depauperatum species complex. However, it is clear that this group has recently diverged, and more analysis is necessary. As of now, PCR amplification has been conducted for 44 additional specimens in the Trifolium depauperatum species complex. They are ready to be sent to Eton Bioscience to be sequenced. Once the results of the sequencing have been sent, both data sets will be combined and analyzed using the same protocol lined out in the methods portion of this chapter. A more thorough comparison can then be made between each data set. With a more robust data set, I hope to understand how the taxonomy of the Trifolium depauperatum species complex can best reflect the insights provided my multiple lines of evidence.

Figure 1. Bayesian phylogenetic tree of the data set of ITS region. Numbers above branches correspond to Bayesian posterior probabilities.

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