A New Species of Bidens (Asteraceae: Heliantheae) from Starbuck Island Provides Evidence for a Second Colonization of Pacific Islands by the Genus

A new species of Bidens (Asteraceae: Heliantheae) from Starbuck Island provides evidence for a second colonization of Pacific islands by the genus

by Cynthia Sayre BSc, (1994), University of British Columbia

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

in

THE FACULTY OF GRADUATE STUDIES Department of Botany

We accept this thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA July 2001 ©Cynthia Sayre, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department

The University of British Columbia Vancouver, Canada ABSTRACT

An unknown Bidens was recently discovered on Starbuck Island, a small, remote coral atoll in the South

Pacific and one of the least visited islands in the world. I determined the identity and evolutionary origin of this Bidens using both morphological analyses and phylogenetic relationships in Bidens (Asteraceae;

Heliantheae) and Coreopsis (Asteraceae; Heliantheae) using variation in nuclear rDNA internal transcribed spacer (ITS) sequences. The ITS analyses include all species of Bidens sequenced to date, including five from this study and sequences from Ganders et al. (2000) and a selection of Coreopsis sequences from Kim et al. (1999). Using morphological and molecular data 1 tested the following hypotheses: (1) Starbuck Island Bidens is an endemic species that evolved from a second colonization of Pacific Islands that has not led to further speciation or adaptive radiation, independent of the colonization that radiated in the Hawaiian Islands and French Polynesia; (2) Starbuck Island Bidens is B. aurea or B. serrulata; (3) Starbuck Island Bidens recently evolved from either B. aurea or B. serrulata. Based on ITS sequences, B. aurea and B. serrulata are rejected as being the same as or even closely related to Starbuck

Island Bidens. Surprisingly, Bidens cynapiifolia, a weedy species that occurs from Mexico to Brazil and is widely introduced elsewhere, appears as a very close sister species to Starbuck Island Bidens.

However, the two taxa are distinct morphologically. Starbuck Island Bidens is described as a new species, endemic to Starbuck Island. Also revealed in the ITS phylogeny are new sectional and generic relationships. Two species of Cosmos (Asteraceae; Heliantheae), originally included as outgroup species, appear nested within Coreopsis and are most closely related to section Pseudoagarista from Mexico.

Bidens mitis (section Meduseae) is nested within section Bidens, indicating that there is no support for the recognition of section Meduseae. Bidens section Psilocarpaea remains polyphyletic, now composed of four distinct clades. Both Bidens laevis (section Bidens) and Bidens squarrosa (section Greenmania) cluster with the other species in their respective sections. My data indicate that section Greenmania is monophyletic and section Bidens is monophyletic if section Meduseae is included in it. It is now known

ii that one of the South Pacific Bidens species, Starbuck Island Bidens, is not closely related to the Hawaiian and Marquesan species of Bidens. To fully understand the evolutionary relationships among indigenous Pacific island Bidens a complete ITS phylogeny should be constructed that includes the remaining South Pacific Bidens species not yet sequenced.

iii TABLE OF CONTENTS

ABSTRACT ii

LIST OF TABLES : vi

LIST OF FIGURES vii

ACKNOWLEDGEMENTS ix

Chapter 1 GENERAL INTRODUCTION 1 1.1 Starbuck Island Bidens 1 1.1.1 Possible origin of Starbuck Island Bidens 4 1.1.2 Description of Starbuck Island 4 1.1.3 Flora of Starbuck Island 8 1.1.4 Bidens present on the Line Islands 8 1.2 The genus Bidens 9 1.2.1 General biology and classification 9 1.2.2 Pacific Island Bidens 10 1.3 Characteristics of island taxa 10 1.3.1 Long distance dispersal 10 1.3.2 Evolutionary change in dispersal characters 11 1.3.3 Adaptive radiation and island endemism 12 1.4 ITS phylogeny of Bidens : 12 1.5 Hypotheses 13

Chapter 2 METHODS 16 2.1 ITS sequencing • 16 2.1.1 Specimen sources 16 2.1.2 DNA Extraction and amplification 16 2.1.3 Sequencing reaction 18 2.1.4 Data analysis 18 2.2 Morphological studies 23 2.2.1 Seed source '• 23 2.2.2 Growth environment 23 2.2.3 Traits measured 24 2.2.4 Chromosome counts 24

Chapter 3 RESULTS , 26 3.1 ITS Sequence Phylogeny 26 3.1.1 Species for which ITS sequences were obtained 26 3.1.2 ITS sequence phylogeny of Bidens and Coreopsis 26 3.1.3 Continental sister species of Starbuck Island Bidens 27 3.1.4 A new continental sister species of the Hawaiian and Marquesan Bidens 27 3.1.5 Sectional relationships revealed within Bidens 27 3.1.6 Relationship of Cosmos to Bidens and Coreopsis 31 3.2 Morphology of Starbuck Island Bidens 31 3.2.1 Floral and achene characters 32 3.2.2 Vegetative characters 34

Chapter 4 DISCUSSION 36 4.1 Starbuck Island Bidens: a new endemic Pacific island species 36 4.1.1 Evolutionary origin of Starbuck Island Bidens 36 4.1.2 Possible autopolyploid origin 37 4.1.3 Evidence for evolutionary adaptations to island life 38 4.1.4 The future of Starbuck Island Bidens 39 4.2 Sectional relationships within Bidens 40

iv 4.3 Generic relationships between Bidens, Coreopsis and Cosmos 41

4.4 Implications for future research 41

Chapter 5 TAXONOMIC TREATMENT 43

Chapter 6 REFERENCES 46 APPENDIX 50 Appendix A: Alignment of ITS 1 and ITS 2 sequences used in phylogenetic analyses. Alignment is also available in TreeBASE 50

V LIST OF TABLES

Table 1. Information on plant species (Garnett, 1983; IUCN, 1991; Kepler and Kepler, 1994), protection status (Dahl, 1986; IUCN, 1991) and human activity (Garnett, 1983; UNEP/IUCN, 1988) in the Line Islands. Islands are listed in order from north to south 9 Table 2. Specimen sources for Bidens species sequenced. Herbaria are identified by the standard international abbreviation as listed in the Index Herbariorum (Holmgren et al, 1981) 17 Table 3. Sequences and sources used in phylogenetic analysis. Sequences are deposited in the National Center for Biotechnology Information (GenBank) or the National Center for Genome Resources (GSBO). Sequences for newly sequenced species will be available in GenBank July 31, 2002 19 Table 4. Mean, median, minimum and maximum values for floral, achene and vegetative traits of Starbuck Island Bidens 31

vi LIST OF FIGURES

Figure 1. Starbuck Island Bidens on Starbuck Island. Photograph used with permission, by A. K. Kepler, 1995 2 Figure 2. Starbuck Island Bidens on Starbuck Island, detail of leaves and flower head. Photograph used with permission, by A. Whistler, 1995 3 Figure 3. Map of the Pacific Ocean and location of Starbuck Island 6 Figure 4. Map of the Line Islands and the location of Starbuck Island within the archipelago. The eleven islands, from north to south, are: Kingman Reef, Palmyra, Teraina (formerly Washington), Tabuaeran (formerly Fanning), Kiritimati (formerly Christmas), Jarvis, Maiden, Starbuck, Caroline (now Millennium), Vostok and Flint 7 Figure 5. Aerial view of Starbuck Island. Photograph used with permission, by A. K. Kepler, 1995 7 Figure 6. ITS phylogeny by Ganders et al. (2000). Percentages represent parsimony bootstrap values, while whole numbers represent the minimum number of base-pair changes required for each step 14 Figure 7. Diagram of internal transcribed spacers (ITS) and the 5.8s gene of ribosomal DNA, including primer locations and orientation 18 Figure 8. One of 10 trees with the highest maximum likelihood score from 44 equally most parsimonious trees for 53 taxa. Tree length 768. Numbers shown are bootstrap percentages from the parsimony analysis. Branch lengths are proportional to the number of substitutions. Clades are identified by capital letters. Underlined species were sequenced in this study 28 Figure 9. Strict consensus of 44 equally parsimonious trees of length 768, for 53 taxa. Clades are identified by capital letters 29 Figure 10. Maximum likelihood tree for 45 taxa with 8 duplicate taxa omitted. The analysis used optimized parameters with 7% of sites invariable, a transition/trans version ratio of 1.80 and a gamma shape parameter of 0.93. Branch lengths are proportional to the estimated number of substitutions. Clades are identified by capital letters 30 Figure 11. Photographs showing floral characteristics of Starbuck Island Bidens. Plants grown at the University of British Columbia from seed collected by A. Whistler on Starbuck Island. (A) Heads, side view. (B) Heads, face view. (C) Ray florets from three plants. (D) Head with rays removed. (E) Ray floret with sterile stamens. (F) Disc florets. (G) Inner involucral bracts. (H) Outer involucral bracts. (I) Chaffy bracts 33 Figure 12. Photograph of Starbuck Island Bidens achenes. Achenes represent variation among three plants grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island 34 Figure 13. Photograph of leaves from a single plant of Starbuck Island Bidens. Plant was grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island 34 Figure 14. Photograph of leaf variation in Starbuck Island Bidens. Leaves are from fourteen plants grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island 35 Figure 15. Bidens cynapiifolia. Illustration adapted from Sheriffs monograph on Bidens (Sheriff, 1937). (a) Portion of fruiting specimen (x 0.85). (b) Upper leaves and (c) lower leaves (x 0.85). (d) Outer involucral bract, (e) inner involucral bract and (f) chaffy bract (x 6.82). (g) Disc floret (x 6.82). (h) Outer achene and (i) inner achene (x 3.41) 38

Vll Figure 16. Illustration of Starbuck Island Bidens. (a) General growth habit (x 0.4). (b, c, d) Detail of three leaf types (x 0.4). (e) Head with rays removed (x l.l).(f) Head (x 1.1). (g, h, i) Achenes with one, two and three awns respectively (x 1.1). (j) Outer involucral bract (x 2.75). (k) Inner involucral bract (x 2.75). (1) Chaffy bract (x 2.75). (m) Disc floret (x 1.1). (n) Ray floret (x 1.1) 45

viii ACKNOWLEDGEMENTS

Special thanks to my supervisor, Dr. Fred Ganders, for his generous support and direction, and without whom I could not have carried out this research. I am also indebted to Dr. Arthur Whistler of the University of Hawaii, for providing the first specimen, achenes, photographs and information that planted the seed for this project, and to Dr. Kay Kepler for patiently answering my questions and generously sharing her rare photographs and information. The DNA sequencing portion of my research would not have been possible without the helpful guidance and lab facilities offered by Dr. Jeannette Whitton, Dr. Mary Berbee, and the respective members of their labs. Thanks also to Dr. Daniel Crawford of Ohio State University for sharing his ITS sequence data and to Dr. Bruce Bohm and Dr. Helen Kennedy for their words of wisdom and advice. Elaine Simons and the administrative staff of the Botany Department at UBC are recognized for the outstanding job that they do, for genuinely caring about the students and making a difference. A special thanks to Chris Sayre, Ross Kubb, Eric Sayre, Diana Dobson, Carolyn Jones, Tia Ramos and my family for their assistance and constant support. Partial financial support was provided by the Frances Chave Memorial Graduate Scholarship in Botany.

ix Chapter 1 GENERAL INTRODUCTION

1.1 Starbuck Island Bidens The genus Bidens provides a classic example of colonization of oceanic islands by long distance dispersal and subsequent speciation, adaptive radiation and evolutionary change in dispersal mechanisms (Ganders, 1989; Carlquist, 1974, 1980). It is one of the most thoroughly studied taxonomic groups in the Pacific island flora. Until recently, extensive research using chromosomes (Gillet and Lim, 1970), crossing experiments (Gillet and Lim, 1970; Gillet, 1975; Ganders and Nagata, 1983,1984), isozymes (Helenurm and Ganders, 1985), polyacetylenes (Marchant et al, 1984), flavonoids (Ganders et al, 1990) and ITS sequences (Ganders et al, 2000) indicated that all known indigenous Pacific island Bidens evolved from a single ancestral species that colonized the Pacific from the Americas. However, a recently discovered Bidens on Starbuck Island, in the Line Islands of the South Pacific provides new evidence that Pacific island Bidens evolved from at least two independent colonization events.

In 1995 during a brief visit, Dr. Arthur Whistler of the University of Hawaii discovered the unknown Bidens (Figure 1 and Figure 2) on Starbuck Island. This flat, uninhabited coral atoll is extremely small, with a total land area of just over 16 square kilometers. It supports only thirteen species of vascular plants, all but one non-weedy species found throughout the Pacific, and this Bidens is one of the most common on the island (Whistler, personal communication, 1998).

A recent study by Ganders et al. (2000) using sequences of the internal transcribed spacers (ITS) of ribosomal DNA showed that the unidentified Starbuck Island Bidens is not closely related to any of the known indigenous Pacific island Bidens. Instead, it is most closely related to section Greenmania, a group of woody vines found from Mexico to South America.

The purpose of this study is to determine the identity of the Starbuck Island Bidens and its evolutionary origin through the use of ITS sequences, cytological and morphological data. Similar studies that combine several forms of experimental analysis have proven extremely useful in resolving complex evolutionary relationships among taxa of vascular plants. Studies combining morphological, chemical and genetic techniques have been conducted on the systematics of the Bidens pilosa complex in North and Central America (Ballard, 1986), the Bidens ferulifolia

1 Figure 1. Starbuck Island Bidens on Starbuck Island. Photograph used with permission, by A. K. Kepler, 1995.

2 Figure 2. Starbuck Island Bidens on Starbuck Island, detail of leaves and flower head. Photograph used with permission, by A. Whistler, 1995. complex (Hart, 1979), Bidens section Greenmania (Roseman, 1986) and Hawaiian Bidens (Ganders, 1989).

1.1.1 Possible origin of Starbuck Island Bidens Based on morphology, Starbuck Island Bidens appears most similar to Bidens aurea or Bidens serrulata.

Bidens aurea occurs from southern Arizona, through most of Mexico and Guatemala and it has become naturalized in France and Italy (Sherff, 1937). Starbuck Island Bidens is similar to Bidens aurea but the two species differ in ray floret number, ray length and achene length. Most significantly, Starbuck Island Bidens achenes are larger than those of B. aurea. It is a common evolutionary pattern for island species to produce larger fruit and seeds than their mainland relatives (Carlquist, 1967, 1974) so Starbuck Island Bidens could have evolved from B. aurea. In addition, most botanical treatments of B. aurea are based on the description in Sherff (1937), so achene size in B. aurea may actually have a broader range than is currently recorded, and Starbuck Island Bidens could be within that range. Another difference between achenes of the two taxa is the position of hairs on the achene body, with B. aurea exhibiting hairs only along the margins, and Starbuck Island Bidens with hairs covering the upper third of the achene body. All morphological comparisons are based on the description of B. aurea in Sherff (1937).

Bidens serrulata occurs from Mexico to Guatemala (Sherff, 1937). Like B. aurea, B. serrulata also differs from Starbuck Island Bidens mainly in achene characters. Most importantly, B. serrulata has dimorphic achenes while Starbuck Island Bidens does not. However, while achenes of Starbuck Island Bidens are completely unlike the outer achenes of B. serrulata, they are similar to the description of the inner achenes of B. serrulata (Sherff, 1937). According to Sherff s description of B. serrulata (Sherff, 1937), achenes of Starbuck Island Bidens are similar to the inner achenes of B. serrulata in overall shape, length, awn number, the presence of retrorse barbs on the awns and the position and location of hairs on the achene body.

Bidens aurea and Bidens serrulata appear to be the most similar species to Starbuck Island Bidens and they occur within a geographical range that could reasonably have been the origin of a long-distance dispersal event to Starbuck Island. On the other hand, the Bidens found on Starbuck Island has not been reported on any other Pacific island. The fact that this plant is common, on an isolated, relatively undisturbed island which contains mostly non-weedy species (Whistler, personal communication, 1998), suggests that it is also possible that the Starbuck Island Bidens is an endemic species.

1.1.2 Description of Starbuck Island One of the least visited islands in the world, Starbuck Island is also one of the least understood in terms of its flora and fauna. This low lying, nearly filled-in coral atoll is located just south of the equator in the South Pacific, at 5°37'S 155°53'W, as shown in Figure 3. Starbuck Island is small, only 16.2 square kilometers, and extremely isolated. Its nearest neighbor, Maiden Island, is 258 kilometers to the northeast. Figure 4 shows the position of Starbuck Island within the Line Islands chain, one of the most remote island groups in the world with the closest continent, South America, nearly 9000 kilometers away. Figure 5 shows an aerial view of Starbuck Island, the center of which is dominated by dry salt flats and a salt lagoon. The island reaches a maximum elevation of 5 meters at the beach crest (Kepler and Kepler, 1994).

4 In 1819, Starbuck Island was first discovered by Captain J. Henderson of the ship Hercules. In 1823 it was again visited by Captain Valentine Starbuck, for whom it is now named, of the English whaling ship L'Aigle. The island was initially claimed by the British and the United States, but it is now one of 33 islands belonging to the Republic of Kiribati (pronounced Kiri- bas). Other, more descriptive names by which the island has been known include Low, Starve, Hero, Barren, Coral Queen and Volunteer Island (Garnett, 1983).

Starbuck Island is one of the Line Islands, formed during the mid Cretaceous to the late Eocene as the result of ridge building volcanic activity involving four hot spots (Haggerty et al, 1982). In the southern Line Islands, including Starbuck Island, evidence points to two main periods of recurrent volcanism: first from 95 to 72 million years ago, and again from 45 to 39 million years ago (Haggerty et al, 1982; Sager and Keating, 1984; Schlanger et al, 1984). The precise geological age of Starbuck Island has not been determined because of the complex manner in which the Line Islands chain has evolved.

The climate on Starbuck Island is dry, with a mean annual rainfall of 800 mm and intermittent drought (Wester et al, 1992; Janowiak et al, 1995). There is no other source of freshwater on the island. Winds are strong, fed by the prevailing easterly trade winds, and severe storms, typhoons and hurricanes are not uncommon. The mean annual temperature in Kiribati is 27°C. Soil quality is very poor with the soil composed mostly of coral sand and fragments of rock. Vegetation on the island consists mainly of a low scrub mixed with a few herb and grass species. The fauna is limited to at least 11 species of sea birds including a large breeding colony of several million sooty terns (Sterna fuscata), green turtles (Chelonia mydas), a single gecko species (Lepidodactylus lugubris), feral cats (Felis cattus) and Polynesian rats (Rattus exulans) which presumably live on seabirds and their eggs (Garnett, 1983). There is no published information on the invertebrate fauna of Starbuck Island.

Starbuck Island remains relatively undisturbed, with human activity limited to a brief period, from 1870 to 1893, in which the island was mined for guano. Guano, composed of bird droppings, is extremely rich in soluble phosphate and its abundance on many Pacific islands has spurred mining over the past century, mainly for use in fertilizers. Since the short duration in which the island was inhabited by miners, Starbuck has received only the occasional visitor. Limited access due to a dangerous fringing coral reef, which has caused numerous shipwrecks since the island's discovery, and the relatively isolated and barren state of the island help ward off casual visitors. Starbuck Island is now classified as a Wildlife Sanctuary and a Closed Area by the government of Kiribati (IUCN, 1991).

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Figure 4. Map of the Line Islands and the location of Starbuck Island within the archipelago. The eleven islands, from north to south, are: Kingman Reef, Palmyra, Teraina (formerly Washington), Tabuaeran (formerly Fanning), Kiritimati (formerly Christmas), Jarvis, Maiden, Starbuck, Caroline (now Millennium), Vostok and Flint.

Figure 5. Aerial view of Starbuck Island. Photograph used with permission, by A. K. Kepler, 1995.

7 1.1.3 Flora of Starbuck Island While published floras or species lists exist for some of the northern Line Islands (Rock, 1916; Fosberg, 1937, 1943, 1953; St. John and Fosberg 1937; Chock and Hamilton, 1962; St. John, 1974; Wester, 1985; Wester et al, 1992; Kepler and Kepler, 1994), there is no published flora for Starbuck Island. However, personal communications with Dr. A. Whistler (1998) and Dr. A. K. Kepler (1998) revealed that there are currently at least 13 species of vascular plants on the island. Almost all of these are non-weedy species that are found throughout the Pacific and all but Boerhavia albiflora are found on other islands in the Line Islands chain (Rock, 1916; Fosberg, 1937; St. John and Fosberg, 1937; Dawson, 1959; Chock and Hamilton, 1962; Garnett, 1983; Wester, 1985; Wester et al, 1992; Kepler and Kepler, 1994). The vegetation is typical of dry, low coral atolls in the Pacific (Van Balgooy, 1960). The checklist of plants that A. Whistler found on Starbuck Island includes all of the following species except for Cordia subcordata which was noted by A. K. Kepler during the same visit: Bidens sp. (Asteraceae) Boerhavia albiflora Fosb. (Nyctaginaceae) Boerhavia repens L. (Nyctaginaceae) Cocos nucifera L. (Arecaceae) Cordia subcordata Lam. (Boraginaceae) Digitaria pacifica Stapf. (Poaceae) Ipomoea macrantha R. & S. (Convolvulaceae) Ipomoea pes-caprae (L.) R. Br. (Convolvulaceae) Lepturus repens (Forst. f.) R. Br. (Poaceae) Pisonia grandis R. Br. (Nyctaginaceae) Portulaca lutea Soland. ex Forst. f. (Portulacaceae) Sida fallax Walp. (Malvaceae) Tribulus cystoides L. (Zygophyllaceae) A single clump of coconut palm (Cocos nucifera), planted at the landing site along the shore, is the only tree on the island except for a few stunted Cordia subcordata. The palm tree serves as the only reminder of an attempt to start a coconut plantation during the guano mining era. Most of the island outside the salt flats is covered with a stunted Sida fallax scrub, herbs, grasses and a few shrubs (Garnett, 1983).

1.1.4 Bidens present on the Line Islands Bidens pilosa has been found as an introduced weed on Kiritimati (formerly Christmas), Tabuaeran (formerly Fanning), Teraina (formerly Washington), Maiden and Flint Islands in the Line Islands (Garnett, 1983; Wester, 1985). No Bidens have been reported on any of the Phoenix islands, east of the Line Islands in Kiribati (Garnett, 1983).

Information on the number of plant species, protection status and human activity for each of the Line Islands is listed in Table 1.

8 Table 1. Information on plant species (Garnett, 1983; IUCN, 1991; Kepler and Kepler, 1994), protection status (Dahl, 1986; IUCN, 1991) and human activity (Garnett, 1983; UNEP/IUCN, 1988) in the Line Islands. Islands are listed in order from north to south.

Island Bidens Number of Total Land Protection Status Human Activity pilosa Indigenous Estimated Area present Plant No. of Plant (km2) 1 Species Species Kingman No 0 0 1 US National Now uninhabited; Reef Marine Wildlife US naval station in 1930s Refuge Palmyra No 21 58 12 US National Now uninhabited; Marine Wildlife U.S. military air base from Refuge 1940-61 Teraina Yes 25 91 14 Settled in 1800s (Washing• ton) Tabuaeran Yes 23 123 35 Settled in 1800s (Fanning) Kiritimati Yes 19 69 321 Cook Islet is a Settled in 1800s; (Christmas) Wildlife Sanctuary Atomic bomb testing in and Closed Area 1950s Jarvis No 8 N/A 4.5 US National Now uninhabited; Wildlife Refuge Settled in 1935 Maiden Yes 9 9 39 Wildlife Sanctuary; Now uninhabited; Closed Area Mined 1860-1930; Atomic bomb testing in 1950s Starbuck No 13 13 16 Wildlife Sanctuary; Now uninhabited; Closed Area Mined 1870-1893 Vostok No 3 3 0.24 Wildlife Sanctuary Uninhabited; No human activity

Flint Yes 18 43 3.2 • Now uninhabited; Mined 1800s Millennium No 23 or 24 27 0.23 Wildlife Sanctuary Now uninhabited; (Caroline) Settled in 1800s

1.2 The genus Bidens 1.2.1 General biology and classification In his monograph on Bidens Sherff (1937) describes over 230 species, including annual and perennial herbs, shrubs and vines found throughout the world. However, there are currently fewer species recognized due to subsequent taxonomic revisions within the genus (Ganders and Nagata, 1990). The name Bidens is derived from the Latin words for 'two' (bi) and 'teeth' (dens), and refers to the two characteristic 'tooth-like' pappus awns (Ganders and Nagata, 1990). Most of the species are native to the Americas, Africa, and Polynesia, with some in Asia and Europe. In addition, weedy species have become naturalized all over the world.

Of the 14 sections of Bidens described by Sherff (1937), there are five major sections that are based on morphological and ecological distinctions. These include sections Campylotheca, Greenmania, Meduseae, Bidens, and Steppia. Section Psilocarpaea includes more than half of

9 the Bidens species, and Sherff acknowledges that this section will likely need to be split into smaller groups. Bidens aurea is the type for this section. Seven of the remaining 8 sections are monotypic, and section Heterodonta, which is morphologically very similar to section Bidens, includes only two species (Sherff, 1937).

Members of Bidens can easily be confused with species of Coreopsis (Asteraceae; Heliantheae) and the distinction between these two genera is uncertain (Ganders and Nagata, 1990). Several recent taxonomic revisions have involved transfer of species between the two genera (Rayner, 1992a; Rayner, 1992b; Ryding and Bremer, 1992). All of the African taxa formerly assigned to Coreopsis were recently transferred to Bidens (Tadesse, 1993).

A recent study by Kim et al. (1999) revealed the complexity of the relationship between Bidens and Coreopsis. Using ITS sequences, the study found that neither genus is monophyletic as currently described, with Bidens appearing as two distinct clades nested within Coreopsis. Therefore Bidens, Coreopsis or both will have to be redefined.

1.2.2 Pacific Island Bidens Endemic species of Bidens occur on many of the Pacific islands, including the Society Islands, Marquesas Islands, Pitcairn, Oeno, and Henderson Island in the South Pacific, and the Hawaiian Islands in the North Pacific Ocean (Figure 1). All are presently classified in section Campylotheca (Ganders and Nagata, 1990), although Sherff (1937) had segregated B. cosmoides in the monotypic section Degenaria.

A few non-endemic species of Bidens are also found throughout the Pacific. The widespread Bidens pilosa is present as an introduced weed on many populated islands in the western Pacific. Bidens alba and B. cynapiifolia are also introduced weeds and are present on most of the Hawaiian Islands. Bidens gardneri has only been reported on the Hawaiian island of Molokai, where it is likely an escaped garden plant (Ganders, personal communication 1999).

1.3 Characteristics of island taxa Oceanic islands provide a unique opportunity to observe the evolutionary patterns of flowering plants because they are so isolated from the Earth's continental land masses. It is well documented that oceanic islands, including the volcanic islands of the Pacific, have acquired their floras and faunas through long-distance dispersal (Carlquist, 1967, 1974,1980). Following successful colonization, isolation from the parent population and new habitats provide the setting for speciation and the evolution of new endemic species. The genus Bidens provides important examples of island evolution and endemism in flowering plants, including adaptive shifts in habit, dispersal and mating characteristics (Ganders, 1989).

1.3.1 Long distance dispersal Geological evidence has shown that many of the Pacific islands, including the Line Islands chain, have never been connected to a land mass and have only been in existence for a short period of time compared to continental land masses. Therefore plants have had to cross great distances within a geologically small window of time, 5-10 million years, in order to colonize these islands. In some cases, especially in the Hawaiian Islands chain, older islands may have served as intermediate 'stepping stones' for plants to disperse to younger islands where they are found today (Carlquist, 1980).

10 Despite the evolution of elaborate dispersal mechanisms in many plants, relatively few species have succeeded in colonizing Pacific islands. Many of the large taxonomic groups in continental floras are entirely absent from the Pacific. Some island colonists are the result of chance dispersal events while others have benefited from their specialized adaptations to long-distance dispersal (Carlquist, 1980). Examples of the most common natural vectors of long-distance dispersal in plants include flotationvi a oceanic currents, wind and birds. Today, additional species, some of which would never have reached Pacific islands on their own, have been introduced by humans, sometimes intentionally. Humans are now very important vectors in long-distance dispersal and virtually any plant that can tolerate island conditions can potentially become an island dweller.

In Bidens long-distance dispersal across oceanic distances is likely achieved primarily by attachment to bird feathers. Achenes (dry, indehiscent, single-seeded fruit) of Bidens have evolved adaptations for dispersal by mammals, but they are equally suited to dispersal by birds (Carlquist, 1980). The needle-like achenes, equipped with one or more barbed awns, are easily caught in fur, the fibers of clothing, or in bird feathers. Barbs, and bristly hairs on the achene body, ensure that the fruit is unlikely to fall off once it has become attached (Carlquist, 1980). Dispersal by mechanical attachment to birds probably accounts for the majority of colonists to Pacific islands. For example, in the Hawaiian Islands, 39% of the 255 hypothetical immigrant Hawaiian flowering plants are hypothesized to have arrived in this manner (Carlquist, 1980).

1.3.2 Evolutionary change in dispersal characters Pacific Bidens species have been interpreted as an example of evolution toward a 'loss of dispersibility,' a common evolutionary pattern among island plants (Carlquist, 1980). While a plant may have used a particular long-distance dispersal mechanism in order to reach an island, it may no longer be useful and may even be detrimental, once the plant has arrived. Like Bidens, plants that arrived through attachment to bird feathers may lose their ability to adhere to feathers and fur if birds and mammals are much less common or are entirely lacking in the new environment.

Barbed awns, characteristic of continental Bidens, are reduced or absent in achenes of most Hawaiian species which Carlquist (1974, 1980) interprets as a loss of dispersibility. However, such changes in achene morphology may also represent an adaptive shift to a new type of dispersal mechanism where the old one is no longer functional (Ganders, 1989). Some Hawaiian Bidens species have evolved flattened, sometimes winged or helical achenes, which seem to be better adapted to dispersal by wind in an environment where birds and terrestrial mammals are much less common (Ganders, 1989).

Evolutionary loss of attachment characteristics in Bidens can include one or more of the following changes (Carlquist, 1980): 1. Awns become shorter 2. Awns move closer together (are less spreading) 3. Awns lose their retrorse barbs 4. Awns are lost entirely 5. Upward-pointing hairs on the achene body are diminished or lost 6. Achenes increase in size 7. Achene shape changes from narrow and needle-like to various shapes, some twisted or contorted

11 1.3.3 Adaptive radiation and island endemism Endemism on oceanic islands is extremely high, with over 90% of the Hawaiian flowering plant species endemic to Hawaii (Wagner et al, 1990). Bidens species endemic to Pacific islands are the result of long-distance dispersal followed by adaptive radiation and speciation.

Hawaiian Bidens is one of the best, most thoroughly studied examples of adaptive radiation and speciation in vascular plants (Ganders, 1989). The most recent revision of Hawaiian Bidens recognizes 19 native species and 8 subspecies, which have been classified based on morphological traits and ecogeographical factors (Ganders and Nagata, 1990). Among these species, the range of morphological and ecological diversity is greater than that within the rest of the genus (Ganders and Nagata, 1990).

While Hawaiian Bidens species are all interfertile, they do not normally hybridize in the wild. Natural hybridization is mostly prevented by geographical isolation or allopatry. Ecological isolation, differences in pollinators and seasonal isolation are also important in preventing hybridization (Ganders and Nagata, 1984). Speciation in Hawaiian Bidens has been the result of gradual genetic divergence of these allopatric populations (Ganders, 1989). 1.4 ITS phylogeny of Bidens Recent work suggests that analysis of variation in sequences of ribosomal spacer regions, such as ITS, is a useful means of constructing phylogenies and identifying continental sister groups of island taxa. This has been particularly well demonstrated in Bidens (Ganders et al, 2000) and the Hawaiian tarweeds (Baldwin, 1992; Baldwin and Wessa, 2000).

In flowering plants, phylogenetic reconstruction using ITS sequences is also a useful method for resolving relationships at the generic and species level. Since the ITS region is not expressed phenotypically, mutations accumulate with relatively little selection pressure. Assuming a constant rate of mutation, genetic differences in ITS sequences provide a relatively unbiased reflection of phylogeny within a group of closely related organisms, through which one can infer evolutionary relationships.

A phylogeny generated from differences in the ITS sequences of ribosomal genes, rather than morphology, is also a more accurate reflection of the evolutionary relationships between taxa where convergence or rapid divergence of morphological characters has occurred, as commonly occurs in island taxa. In these cases, the magnitude of morphological differentiation among taxa may not reflect the magnitude of genetic change. For example, the Hawaiian species have identical ITS sequences, reflecting how recently they evolved from a single common ancestor. However, Hawaiian species of Bidens exhibit an enormous amount of morphological variation which, when compared with the rest of the genus, might lead one to conclude that the Hawaiian species are genetically much more distantly related from one another than they really are (Ganders et al, 2000). The magnitude of morphological variation in the Hawaiian Bidens is partly due to the fact that many morphological traits in Bidens are controlled by only one or two loci (Ganders, 1989).

The recent Bidens ITS phylogeny by Ganders et al. (2000) revealed important information about sectional and ecogeographical relationships in Bidens, as well as the position of Starbuck Island Bidens within the Bidens ITS phylogeny. This was the first evidence that the Bidens from Starbuck Island had a different evolutionary origin than the rest of the Pacific Bidens. Figure 6

12 illustrates the maximum likelihood tree generated from that study. The tree divides Bidens into two major groups. The first is composed of six north temperate species from wet habitats, and the second of southern, tropical and subtropical species from well-drained habitats (Ganders et al, 2000). It is the latter that includes all of the Pacific Island Bidens and the Bidens from Starbuck Island.

In Figure 6 all of the known Pacific island species form a monophyletic group, with the Marquesan species appearing as sister to the Hawaiian species. It is hypothesized that the endemic Bidens species from southern Polynesia are a more recent radiation from the Hawaiian species. The known Pacific island Bidens species evolved from a single colonist from the Americas which underwent speciation and adaptive radiation after colonizing the Pacific (Ganders et al, 2000). Previous biosystematic studies of Hawaiian Bidens have included studies of chromosomes (Gillet and Lim, 1970), isozymes (Helenurm and Ganders, 1985), polyacetylenes (Marchant et al, 1984), flavonoids (Ganders et al, 1990) and breeding experiments (Gillet and Lim, 1970; Gillet, 1975; Ganders and Nagata, 1983,1984). All of these studies, along with Ganders et al (2000), support the conclusion that the members of Hawaiian Bidens form a monophyletic group that evolved from a single ancestor that colonized the Pacific.

Surprisingly, as shown in Figure 6, the Bidens from Starbuck Island is not closely related to the other Pacific Bidens species. It is most closely related to the woody vines of section Greenmania, while the continental sister group to the known Pacific Bidens is a group of Latin American herbs in section Psilocarpaea. However, the authors acknowledge that the identity of the Starbuck Island Bidens is undetermined and that it may simply be a recently introduced continental species that is not indigenous to the Pacific (Ganders et al, 2000). Also this study did not include very many species.

1.5 Hypotheses The purpose of this study was to determine the identity and evolutionary significance of the Starbuck Island Bidens by testing the following hypotheses:

1) Starbuck Island Bidens is endemic to the Line Islands, representing a second, independent colonization of a Pacific island that has not led to further speciation or adaptive radiation. 2) Starbuck Island Bidens is Bidens aurea or Bidens serrulata and is not indigenous, and the fruit morphology is within the range of the species in North America. 3) Starbuck Island Bidens is Bidens aurea or Bidens serrulata that has evolved morphologically different fruit following its introduction (natural or by humans) to the island.

These hypotheses were tested by phylogenetic reconstruction using ITS sequences from Bidens aurea and Bidens serrulata, the best candidates for the nearest relative of Starbuck Island Bidens based on morphology and geographical origin, combined with morphological and cytological studies. ITS sequences were also obtained for a selection of other Bidens species to possibly reveal a closer continental relative to the Starbuck Island Bidens and to expand the ITS phylogenies of Bidens and Coreopsis by Ganders et al. (2000) and Kim et al. (1999). The species chosen for sequencing represent a selection of Bidens sections and a range of morphologically distinctive types previously not sequenced. They are Bidens cynapiifolia, B. laevis, B. mitis, B. ostruthioides and B. urophylla.

13 Calycadenia """ pauciflora Outgroups, Arnica mollis Mexico and Cosmos bipinnatus N. America

Bidens sp. 1 94 Section B. Sp. Tahiala' Campylotheca rl1B. sp.2 South Pacific, B. mauiensis Marquesas Islands B. conjuncta 92 B. sandvicensis Section Campylotheca Hawaii B. cosmoides B. hillebrandiana 65 100 B. hillebrandiana X B. cosmoides B. populifolia 97 X B. conjuncta |99j- B. pilosa Section Psilocarpaea B. alba Mexico and Caribbean 100 B. ferulaefolia Section Psilocarpaea 100 B. andicola Mexico and S. America 7g r— B. reptans Section Greenmania TH m B. boquetiensis Central, S. America, 100 Caribbean j 94 I— B. segetum

87 B. Sp. 'Starbuck Island' South Pacific B. frondosa 66 Section Bidens 3 B. cf. amplissima 03 LH

Figure 6. ITS phylogeny by Ganders et al. (2000). Percentages represent parsimony bootstrap values, while whole numbers represent the minimum number of base-pair changes required for each step

14 Bidens cynapiifolia (section Psilocarpaed) has achenes different from any Bidens sequenced so far. It is a weedy selfing species that occurs from Mexico to Brazil and it is widely introduced elsewhere, including the Pacific where it is a common weed on the Hawaiian Islands.

Bidens laevis (section Bidens) is an aquatic species found in sloughs, rivers and shallow lakes, from southern USA to South America. Several authors have questioned whether this species is specifically distinct from B. cernua. Including the ITS sequences of B. laevis in the ITS phylogeny may reveal whether the two species are in fact distinct fromon e another and whether section Bidens is really monophyletic.

Bidens mitis (section Meduseae) is one of only five species in its section. None of the species in Meduseae were sequenced until now and having a representative helps to complete the Bidens ITS phylogeny and shows whether Starbuck Island Bidens is closer to section Meduseae than to section Greenmania.

Bidens ostruthioides including var. costaricensis (section Psilocarpaed) is not similar to Starbuck Island Bidens. Its placement within Bidens has been questioned. It is one of only two species currently included in Bidens that have pistillate rather than sterile ray florets. It was chosen for sequencing to possibly determine whether there is support for its inclusion in Bidens and to further reveal the phylogenetic structure of the present section Psilocarpaea.

Bidens urophylla (section Greenmania) differs from the rest of its section in achene morphology. It was chosen for sequencing to determine whether it belongs to section Greenmania and to possibly reveal a closer sister taxon to Starbuck Island Bidens within section Greenmania.

15 Chapter 2 METHODS

2.1 ITS sequencing

2.1.1 Specimen sources Sequencing reactions were performed on DNA extracted from herbarium specimens, with two exceptions. Bidens cynapiifolia and Bidens squarrosa specimens were grown from open- pollinated, wild collected seed. In addition, the sequence for Starbuck Island Bidens obtained by Ganders et al. (2000) was confirmed. ITS sequences were obtained for all of the species listed in Table 2.

I thank curators of the following herbaria for loans of specimens: Ann Arbor (MICH), Herbarium of the University of Michigan; Chicago (F), Field Museum of Natural History; Honolulu (BISH), Bishop Museum; New York (NY), The New York Botanical Garden; Saint Louis (MO), Missouri Botanical Garden; Tempe (ASU), Arizona State University; Tucson (ARIZ), Herbarium of the University of Arizona; Washington (US), United States National Herbarium, Smithsonian Institution.

2.1.2 DNA Extraction and amplification Genomic DNA of Starbuck Island Bidens was isolated from a small portion of a single young leaf using a standard CTAB lysis buffer. For Bidens aurea, Bidens serrulata, and all other species sequenced, genomic DNA was isolated with QIAGEN DNeasy™ Plant Mini Kit, following their recommended protocol for isolation of DNA from plant tissue.

In most cases, genomic extractions were run on an agarose gel to estimate concentration for polymerase chain reaction (PCR). With some specimens, where it was consistently difficult to amplify the ITS region, fluorometer readings of genomic elutions were taken to get more accurate estimates of DNA concentration.

16 Table 2. Specimen sources for Bidens species sequenced. Herbaria are identified by the standard international abbreviation as listed in the Index Herbariorum (Holmgren et al, 1981).

Taxon Speci- Geographical source Collection number Herbarium men where deposited

Bidens aurea (Ait.) Sherff 1 Mexico: Municipio de T.R. Van Devender ASU 344035 Yecora Sonora 97-1254A

Bidens aurea (Ait.) Sherff 2 Arizona: Santa Rita Mts., S. P. McLaughlin ASU 264262 Pima County 4057

Bidens cynapiifolia 1 Hawaii: Kona Sayre 201 UBC H.B.K.

Bidens cynapiifolia 2 Hawaii: Kona Sayre 202 UBC H.B.K.

Bidens laevis (L.) B.S.P. 1 USA: Florida Ganders & Kennedy UBC 97-20

Bidens laevis (L.) B.S.P. 2 USA: Florida Ganders & Kennedy UBC 97-21

Bidens mitis (Michx.) 1 USA: Florida, Marion S. L. Orzell & E. L. NY Sherff County Bridges 15734 2 USA: Florida, Osceola S. L. Orzell & E. L. NY Bidens mitis (Michx.) County Bridges 16356 Sherff

Bidens serrulata (Poir.) 1 Mexico: Mts. SE of T. Reeves 6220 ASU 97521 Desf. Texcoco

Bidens serrulata (Poir.) 2 Mexico: NW of San Luis Nestor Naranjo J. 73 ASU110530 Desf. Ayucan

Bidens squarrosa H.B.K. 1 Costa Rica: Guanacaste Ganders s.n. (grown UBC Province from seed collected in Costa Rica)

Bidens squarrosa H.B.K. 2 Costa Rica: Guanacaste Ganders s.n. (grown UBC Province from seed collected in Costa Rica)

Bidens sp. (Starbuck 1 Kiribati: Starbuck Island A. Whistler 9879 BISH Island)

Primers ITS 2 and ITS 5 with ITS 3 and ITS 4, or primers ITS 4 and ITS 5 were used at a concentration of lOuM to amplify the ITS 1 and ITS 2 regions (and the 5.8s gene where ITS 4 and ITS 5 were used) from genomic DNA. Primer positions relative to the ITS of nuclear ribosomal DNA are shown in Figure 7, and primer sequences are published in White et al. (1990).

17 Figure 7. Diagram of internal transcribed spacers (ITS) and the 5.8s gene of ribosomal DNA, including primer locations and orientation.

PCR was carried out using Amersham Pharmacia Biotech Ready-To-Go® PCR Beads. Tubes were placed in a PTC-100™ Programmable Thermal Controller (MJ Research) for an initial 5 minutes at 95°, followed by 29 cycles of 1 minute at 95°, 1 minute at 48°, and 45 seconds (plus 4 seconds per cycle) at 72°. The reaction was completed by a final incubation time of 7 minutes at 72°, followed by storage at 4°.

The double-stranded PCR product was purified, prior to sequencing, using QIAGEN QIAquick™ PCR Purification Kit. Purified DNA was pelleted and stored at -4°C for later sequencing.

2.1.3 Sequencing reaction Sequencing reactions using primers ITS 4, ITS 5 and, in some cases, ITS 2 and ITS 3 were performed to obtain sequences for both complementary strands of the complete ITS 1 and ITS 2 regions, and the 5.8S gene (except where only ITS 2 and ITS 3 were used) of nuclear ribosomal DNA. Sequencing was completed in accordance with the protocol in the Applied Biosystems PRISM™ TaqDyeDeoxy™ Terminator cycle sequencing kit.

To verify sequences, DNA was again extracted, amplified and sequenced, from a different herbarium specimen where possible, for each taxon. Where two different specimens of the same taxon differed in their sequences, both were included in phylogenetic analyses.

2.1.4 Data analysis ITS sequences were assembled in AutoAssembler and a consensus sequence built for each taxon. Consensus sequences were then imported into SeqApp (Gilbert, 1993) for manual alignment with the addition of new sequences to the existing alignments by Kim et al. (1999) and Ganders et al. (2000). The result was a single alignment including all of the existing Bidens and Coreopsis ITS sequences. Both alignments, from Kim et al. (1999) and Ganders et al. (2000), were modified as necessary with the addition of new taxa. The 5.8s data was unavailable for Fitchia speciosa, Dahlia coccinea, Dahlia macdougallii and the Coreopsis taxa so the 5.8s data was excluded from the alignment for all analyses.

To keep the total number of taxa within a reasonable size for analysis only a selection of the taxa from Kim et al. (1999) were retained for the final alignment. Some of the Coreopsis species were

18 excluded, but at least one taxon from every major branch in the Kim et al. phylogeny was kept. The resulting final alignment includes the ITS 1 and ITS 2 sequences for all of the Bidens and a representative selection of the Coreopsis species, listed in Table 3, sequenced so far. The final alignment is included in the appendix and can be obtained from TreeBASE at http://www.herbaria.harvard.edu/treebase.

In all phylogenetic analyses Dahlia macdougallii, Dahlia coccinea and Fitchia speciosa were defined as outgroup taxa. Like Bidens and Coreopsis, both Dahlia and Fitchia belong to tribe Heliantheae in the sunflower family (Asteraceae).

Initially, Cosmos bipinnatus, also in tribe Heliantheae, was included with the outgroup but preliminary analyses showed it to be nested within Coreopsis and Bidens in the ingroup. This prompted the addition of another Cosmos sequence to determine if the position of Cosmos bipinnatus within the ingroup was representative of other Cosmos species. ITS sequences for Cosmos atrosanguineus were available in GenBank so that species was included in the final alignment.

Table 3. Sequences and sources used in phylogenetic analysis. Sequences are deposited in the National Center for Biotechnology Information (GenBank) or the National Center for Genome Resources (GSBO). Sequences for newly sequenced species will be available in GenBank July 31, 2002.

Taxon Geographical source Collection number Accession number Bidens L.

Section Bidens L. (formerly Section Platycarpaea DC.)

B. amplissima E. Greene Canada: British Columbia Kennedy & Ganders 5044 U67095

B. cernua L. Canada: British Columbia Goward 81-742 U67098

B. frondosa L. Canada: British Columbia Kennedy & Ganders 5042 U67094

B. laevis (L.) B.S.P.: sequence USA: Florida Ganders & Kennedy 97- AY046937 replicate 1 20

B. laevis (L.) B.S.P.: sequence USA: Florida Ganders & Kennedy 97- AY046938 replicate 2 21

B. tripartita L. Canada: British Columbia Kennedy & Ganders 5043 U67097

Section Campylotheca (Cass.) Nutt. (including section Degeneria Sherff)

B. conjuncta Sherff Hawaii: Maui Ganders s.n. U67099

B. cosmoides (A. Gray) Sherff Hawaii: Kauai C. Guppy 85-29 U67104

B. hillebrandiana (Drake) Hawaii: Maui Ganders 81-14 U67105 Degener ssp. polycephala Nagata & Ganders

19 Table 3. Continued.

Taxon Geographical source Collection number Accession number

Section Campylotheca cont.

B. mauiensis (A. Gray) Sherff Hawaii: Maui Ganders 81-16 U67101

B. sandvicensis Less. ssp. Hawaii: Kauai Ganders s.n. U67102 sandvicensis

FI hybrid of B. hillebrandiana Ganders s.n. U67103 (Drake) Degener ssp. polycephala Nagata & Ganders X B. cosmoides (A. Gray) Sherff

FI hybrid of B. populifolia Sherff Hawaii: Oahu Ganders s.n. U67100 X B. conjuncta Sherff

B. (Tahiata) Marquesas Islands: Tahiata , Perlman 14917 AF078927

B. sp. 1 (Marquesas Islands) Marquesas Islands: Fatu Hiva Perlman 14948 sequence same as AF078927

B. sp. 2 (Marquesas Islands) Marquesas Islands: Fatu Hiva Perlman 14996 AF078928

Section Greenmania Sherff

B. boquetiensis (Roseman) Costa Rica Kennedy 4566 U67111

B. reptans (L.) G. Don Puerto Rico Kennedy, G. & M. U67110 Breckon 4784C

B. segetum Mart, ex Colla Brazil Ganders s.n. U67112

B. squarrosa H.B.K. Grown at UBC from seed Ganders s.n. (collected AY046934 collected in Costa Rica seed)

Section Hydrocarpaea A. Gray

B. beckii Torr. Canada: Ontario C. E. Garton 20535 U67096

Section Meduseae (Nutt.) Sherff

B. mitis (Michx.) Sherff: USA: Florida S. L. Orzell & E. L. AY046935 sequence replicate 1 Bridges 15734

B. mitis (Michx.) Sherff: USA: Florida S. L. Orzell & E. L. AY046936 sequence replicate 2 Bridges 16356

Section Psilocarpaea DC.

B. andicola (A. Gray) Sherff Peru C. & E. Franquemont 343 U67109

B. alba (L.) DC var. radiata USA: Florida Ganders s.n. U67107 (Schultz-Bip.) Ballard ex Melchert

B. aurea (Ait.) Sherff: sequence Mexico T.R. Van Devender AY046932 replicate 1 97-1254A

B. aurea (Ait.) Sherff: sequence Arizona: Santa Rita Mts., S. P. McLaughlin 4057 AY046933 replicate 2 Pima County

20 Table 3. Continued.

Taxon Geographical source Collection Accession number number

Section Psilocarpaea cont.

B. cynapiifolia H.B.K.: sequence Grown at UBC from seed Sayre 201 AY046941 replicate 1 collected in Kona, Hawaii

B. cynapiifolia H.B.K.: sequence Grown at UBC from seed Sayre 202 AY046942 replicate 2 collected in Kona, Hawaii

B. ferulifolia (A. Gray) Sherff Mexico Kennedy & U67108 cv. Gold Marie Ganders 5401

B. pilosa L. USA: Florida Ganders s.n. U67106

B. schimperi Sch. Bip. ex Walp. Ethiopia Mesfin s.n. GSDB:S:1386341, GSDB:S:1386384

B. serrulata (Poir.) Desf.: sequence Mexico T. Reeves 6220 AY046939 replicate 1

B. serrulata (Poir.) Desf.: sequence Mexico Nestor Naranjo J. AY046940 replicate 2 73

Section unassigned

B. macroptera (Sch. Bip. ex Ethiopia Mesfin s.n. GSDB:S: 1386342, Chiov.) Mesfin GSDB:S: 1386385

B. pachyloma (Oliv. & Hiern) Ethiopia Mesfin s.n. GSDB:S:1386344, Cufod. GSDB:S: 1386387

B. prestinaria (Sch. Bip.) Cufod. Ethiopia Mesfin. s.n. GSDB:S:1386343, GSDB:S:1386386

Bidens sp. (Starbuck Island) Kiribati: Starbuck Island A. Whistler 9879 U67113 Coreopsis L.

C. californica (Nutt.) USA: California Crawford 1475 GSDB:S:1386355, var. californica Sharsmith GSDB:S:1386398

C. delphinifolia Lam. USA: South Carolina Crawford & Lewis GSDB:S: 1386370, 1461 GSDB:S:1386413

C. laevenworthii T. & G. USA: Florida Crawford 1441 GSDB:S:1386374, GSDB:S: 1386417

C. latifolia Michx. USA: North Carolina Crawford & Lewis GSDB:S: 1386365, 1466 GSDB:S: 1386408

C. lopez-mirandae Sagast. Peru Stuessy et al. GSDB:S: 1386352, 12,573B GSDB:S:1386395

C. maritima (Nutt.) Hook. f. USA: California Elisens s.n. GSDB:S:1386364, GSDB:S: 1386407

C. mutica DC. var. leptomera Mexico Spooner 2211 GSDB:S:1386357, Sherff GSDB:S:1386400

C. petrophila A. Gray Mexico Crawford et al. GSDB:S: 1386345, 1389 GSDB:S: 1386388

21 Table 3. Continued.

Taxon Geographical source Collection number Accession number Coreopsis cont.

C. pubescens Ell. USA: Alabama Smith 3739 GSDB:S:1386381, GSDB:S:1386424

C. pulchra Boynton in Small . USA: Alabama Crawford 1456 GSDB:S:1386366, GSDB:S:1386409

C. rhyacophila Greenm. Mexico Yahara 255 GSDB:S: 1386346, GSDB:S:1386389

C. tripteris L. USA: Ohio Crawford s.n. GSDB:S:1386368, GSDB:S:1386411

C. woytkowskii Sherff Peru Stuessy et al. 12,553 GSDB:S:1386351, GSDB:S: 1386394

Outgroups

Cosmos atrosanguineus (Hook.) unavailable unavailable AF165847 Voss

Cosmos bipinnatus (A. Gray) Canada: British Columbia Ganders s.n. U67114 Sherff

Dahlia coccinea Cav. Mexico Saar 784 GSDB:S:2668016, GSDB:S:2668017

Dahlia macdougallii Sherff Mexico Saar 779 GSDB:S:2668018, GSDB:S:2668019

Fitchia speciosa Cheeseman Hawaii Motley s.n. GSDB:S:2668014, GSDB:S:2668015

A heuristic parsimony search was completed for the entire data set, using the default settings in PAUP, and the results were compared with those of omitting 8 duplicate taxa from the same analysis. The excluded taxa were B. conjuncta, B. sandvicensis ssp. sandvicensis, B. hillebrandiana ssp. polycephala, B. hillebrandiana ssp. polycephala X B. cosmoides, B. cosmoides, B. sp. (Tahiata), B. cf. amplissima and B. tripartita. Maximum likelihood scores were calculated for the set of 44 equally parsimonious trees obtained from the analysis of the complete data set. For 500 parsimony bootstrap replicates, I used 50 replicates of multiple random addition of taxa for each bootstrap replicate, the 'heuristics' option and the default settings in PAUP.

To test whether regions of uncertain alignment affected the results of phylogenetic analyses 13 sites near gaps were omitted from the original alignment of 519 sites. Excluded were sites 53, 129, 130, 131, 148, 149, 306, 479, 480, 491, 492, 502 and 503 from the alignment in Appendix A. A heuristic parsimony search was completed for both the original and the reduced data sets. Results from these analyses were compared to determine whether these areas of uncertain alignment changed the results. Bootstrap values were calculated with 500 parsimony bootstrap replicates, 50 replicates of multiple random addition of taxa for each bootstrap replicate, the 'heuristics' option and the default settings in PAUP

22 A maximum likelihood analysis was performed that allowed for a model of evolution with different rates of evolution at different sites within the ITS 1 and ITS 2 regions. Omitting the 8 duplicate taxa listed above, a maximum likelihood tree was calculated with 500 replicated heuristic searches and the default settings in PAUP, with equal nucleotide frequencies and equal probability of substitution at all sites. The resulting maximum likelihood tree was used to optimize the likelihood of the data, with the highest likelihood when 7% of the sites were in the invariable category, with zero probability of change. The estimated transition/transversion (Ti/Tv) ratio was 1.80 and the estimated gamma shape parameter was 0.93. These parameter estimates were then used to search for a new maximum likelihood tree, with 10 replicated heuristic searches and random addition of taxa, omitting the 8 duplicate taxa listed above.

To determine if the default evolutionary model changed the results, four different distance corrections were used to calculate Neighbor Joining trees for the complete data set (not excluding duplicate taxa) with 1000 bootstrap replicates. Four trees were calculated with the following corrections: Jukes-Cantor, Kimura 2-parameter, the General Time Reversible model (GTR) and the Maximum Likelihood correction for multiple hits, with the estimated proportion of invariable sites, Ti/Tv ratio and gamma shape parameter from above. The results were compared to the parsimony bootstrap and maximum likelihood trees to determine whether the ITS phylogeny is dependent upon inherent evolutionary assumptions in the analyses.

2.2 Morphological studies 2.2.1 Seed source Achenes of Starbuck Island Bidens were collected from one or a few plants on Starbuck Island in June, 1995 by Dr. A. Whistler of the University of Hawaii. From these achenes, plants were grown for use in subsequent morphological studies examining vegetative, floral and achene characteristics. 2.2.2 Growth environment Achenes were surface-sterilized in a 2% bleach solution followed by several rinses in distilled water. Then the tip of the achene, just below the awns, was cut and removed to allow for immediate germination, bypassing the need for the embryo to break through the seed coat. Achenes were placed on moist filter paper, in a sterile petri dish in the dark. Seedlings emerged two to three days following treatment, and were then placed in the light and grown for three to four more days before transplanting to vermiculite in 2 inch pots.

Young seedlings were raised within a growth chamber at the following settings: light period from 6:00 AM to 8:00 PM at 25°C; dark period from 8:00 PM to 6:00 AM at 18°C. Relative humidity was kept constant at 50%. During the light period, full spectrum lights operated at 67% of full capacity, with only 8 of the 12 available lights operating at any given time.

A few weeks after transplanting, a slow-release Osmocote fertilizer was added to the vermiculite. When the young plants grew large enough they were transferred to 4" pots (and later 6" pots) containing Sunshine Mix, a standard potting mix of peat and pearlite, with Osmocote fertilizer. Plants were watered every few days as needed, allowing the soil surface to dry out between waterings.

23 2.2.3 Traits measured Vegetative, floral and achene characteristics were included in the morphological studies. Measured vegetative traits included leaf length (including petiole), leaf width (widest point), number of leaflets, leaflet length, leaflet width (widest point) and leaf serration (total number of teeth per leaflet edge, and number of teeth per cm of leaflet edge). These were measured and recorded for the largest and smallest leaf, and in some cases an intermediate leaf, for each of 18 plants raised from seed in the growth chamber. Also observed and recorded were plant height, growth habit, shape of the stem cross section, leaf pubescence and range of leaf variation.

Floral and inflorescence traits measured included inner and outer involucral bract length, number of inner and outer involucral bracts, number of chaffy receptacular bracts, number of disc florets, number of ray florets, ray floret length, ray floret width and peduncle length (longest). These characters were measured on 70 flower heads from 7 different plants. The number of heads measured per plant ranged from 2 to 20. At least 10 heads per plant were measured if available, but some plants died or stopped flowering before this number was reached. Whether heads were determinate or indeterminate, inflorescence type, number of heads per inflorescence and peduncle pubescence were also recorded.

Measured achene characters included achene length (not including awns), achene width (at the widest point), number of awns, and length of the longest awn. Furthermore, non-quantitative characters, such as color, location of hairs (if any), presence or absence of barbs on the awns, and the position of grooves and other identifying features, were recorded. These traits were measured on each of the 72 achenes collected on Starbuck Island. Also measured were 142 achenes collected from 3 different plants grown from seed within the growth chamber, after flowering and seed set. These achenes were most likely the result of selfing, although cross-fertilization cannot be ruled out. Pollen transfer may have occurred via air currents in the growth chamber's circulation system or by greenhouse whitefly (Trialeurodes vaporariorum) moving from plant to plant. White fly, a common greenhouse pest that is partial to Bidens, was present throughout the life of these plants. Awn number was measured on the above achenes as well as on 1619 unfertilized ovaries from flower heads, so misleading measurements involving awns that broke off during handling would be less likely.

Thirty-five millimeter slides were made to document the range of variation in size and morphology of achenes, leaves and floral characters, including disc florets, ray florets, inner and outer involucral bracts and chaffy bracts. Results from the morphological studies were incorporated into a description and diagnosis of Starbuck Island Bidens.

2.2.4 Chromosome counts An attempt was made to obtain the chromosome number of Starbuck Island Bidens to reveal whether Starbuck Island Bidens is likely to be derived from any of the Bidens for which chromosome counts are known, including Bidens aurea and Bidens serrulata.

Tissue for cell squashes was obtained from the growing tips of young roots from mature plants. Root tips were hydrolyzed in a solution of one part HCI to one part 95% ethanol for 30-90 seconds, followed by several rinses in distilled water. The tissue was then transferred to a glass slide, where the distal meristematic region was removed and the rest discarded. A drop of acetocarmine was added to the meristematic tissue to stain the chromosomes for increased visibility under the microscope. After letting the stain absorb for one minute and gently

24 macerating the tissue, a drop of Hoyer's Mounting Medium (Hansen et al, 1978) was added and mixed evenly with the stained tissue. A cover slip was placed over the mixture and pressure was applied evenly to flatten the cells and make a semi-permanent slide. No cell fixative was used.

Initial cell squashes were inconclusive, with the quality too poor to get an exact chromosome count. This was due to the high number and small size of chromosomes, weak staining, and the fact that few cells were at the appropriate stage of mitosis, with visibly condensed chromosomes. Crude counts indicate that the chromosome number of Starbuck Island Bidens is greater than 2N=70, but to determine the exact number would require alteration of the methods used in the initial trials, with the addition of a cell fixative and a different stain.

25 Chapter 3 RESULTS

3.1 ITS Sequence Phylogeny 3.1.1 Species for which ITS sequences were obtained ITS Sequences were obtained for Bidens aurea, B. serrulata, B. cynapiifolia, B. laevis, B. mitis and B. squarrosa. The sequence of Starbuck Island Bidens obtained by Ganders et al. (2000) was confirmed.

Unfortunately, usable sequences could not be obtained for Bidens ostruthioides and Bidens urophylla. This was due to the highly degraded state of the DNA from the available herbarium specimens and consequent difficulty in amplifying the ITS region. A complete ITS sequence was obtained for one of eight herbarium specimens of B. ostruthioides but the sequence obtained from a second isolation of the same specimen conflicted with the original sequence. Two of five specimens of B. urophylla yielded incomplete ITS sequences but inconsistency in the results suggested contamination of the genomic DNA rendering those sequences unusable as well.

3.1.2 ITS sequence phylogeny of Bidens and Coreopsis There is high congruency among the trees generated from parsimony, maximum likelihood and neighbor joining analyses. Figure 7 shows one of 10 trees with the highest maximum likelihood score, of 44 equally parsimonious trees with length 768. Figure 8 is the strict consensus of those 10 trees. The results of parsimony analyses were identical with or without omitting the 8 duplicate taxa. The maximum likelihood tree with optimized parameters, omitting 8 duplicate taxa is shown in Figure 9. All trees are rooted with outgroup species Fitchia speciosa, Dahlia coccinea and Dahlia macdougallii.

Differences in branching order between the parsimony, maximum likelihood and neighbor joining trees (not shown) involve only weakly-supported branches with less than 55 percent bootstrap support in the parsimony tree. These differences include branch E which is collapsed in the likelihood tree, branch I which is collapsed in all neighbor joining trees, and the unresolved positions of Bidens beckii, Coreopsis latifolia, C tripteris, C. pulchra and C. delphinifolia within clade J. The lack of resolution within clade J is consistent wjth the findings of Kim et al.

26 (1999) and the addition of taxa from this study to their alignment did not resolve the relationships within that clade. Neighbor joining trees with distance corrections retained all branches with more than 55 percent bootstrap support in the parsimony tree.

Branching order within clade K, which includes Starbuck Island Bidens, is identical among parsimony, maximum likelihood and neighbor joining trees with Jukes-Cantor, Kimura 2- parameter, GTR and the maximum likelihood corrections. The only exception is that identical taxa were rearranged randomly among the various trees.

Omitting 13 sites near gaps from the original alignment changed the position of only one taxon compared to the maximum parsimony tree from the complete data set. With the reduced data set, the position of Bidens beckii within clade J is unresolved, while the tree from the complete data set places B. beckii as sister to the rest of the north temperate Bidens with 54 percent parsimony bootstrap support. This is consistent with the findings of Ganders et al. (2000)

3.1.3 Continental sister species off Starbuck Island Bidens The most surprising and significant result is that Bidens cynapiifolia appears as a very close sister species to Starbuck Island Bidens in the ITS phylogeny. The two taxa differ by only 12 base pairs or 2% of their ITS sequences and their relationship as close sister species, with 100 percent parsimony bootstrap support, is confirmed in the parsimony, maximum likelihood and all neighbor joining trees. In the ITS phylogeny Starbuck Island Bidens and B. cynapiifolia together are sister group to the species in section Greenmania. Neither Bidens aurea nor Bidens serrulata has ITS sequences the same as or similar to those of Starbuck Island Bidens.

3.1.4 A new continental sister species off the Hawaiian and Marquesan Bidens While Bidens aurea is not closely related to Starbuck Island Bidens, it turns out to be a close relative of the Hawaiian and Marquesan species. In clade T, Bidens aurea is basal to B. pilosa and B. alba var. radiata. Together these three species are the most closely related continental species to the Polynesian clade. Furthermore, the results show that B. serrulata is not closely related to either Starbuck Island Bidens or the Hawaiian and Marquesan species.

3.1.5 Sectional relationships revealed within Bidens Neither Bidens aurea nor Bidens serrulata is closely related to Starbuck Island Bidens according to ITS sequences. Bidens aurea and B. serrulata appear within two distinct clades with other species in section Psilocarpaea. In clade R, Bidens serrulata is basal to B. ferulifolia and B. andicola with 100 percent parsimony bootstrap support. Bidens aurea, in clade T, is basal to B. pilosa and B. alba var. radiata, with 79 percent parsimony bootstrap support. These findings confirm the non-monophyletic nature of section Psilocarpaea shown by Ganders et al. (2000) and Kim et. al (1999). Section Psilocarpaea is now known to be composed of at least four separate clades: clade R, clade T, Bidens schimperi in clade N and B. cynapiifolia in clade P.

27 - Fitchia speciosa 100 i— Dahlia coccinea Outgroups L Dahlia macdougallii ] — C. mutica var. leptomera Sect. Electra Mexico D 1001 C. maritima Sect. Tuckermannia O California u 77 ' C. californica var. californica Sect. Leptosyne o F 100 i C. petrophila ] Sect. Pseudoagarista — Mexico U A C. rhyacophila 89 H 100 p—— Cosmos bipinnatus ] Mexico Cosmos atrosanguineus II C Sect. Silphidium 80 5li C. latifolia '— C. tripteris

r—• C. pulchra Sect. Palmatae O C. delphinifolia J 99 i

Figure 8. One of 10 trees with the highest maximum likelihood score from 44 equally most parsimonious trees for 53 taxa. Tree length 768. Numbers shown are bootstrap percentages from the parsimony analysis. Branch lengths are proportional to the number of substitutions. Clades are identified by capital letters. Underlined species were sequenced in this study.

28 Fitchia speciosa Dahlia coccinea Dahlia macdougallii C. mutica var. leptomera D C. maritima C. californica var. californica C. petrophila C. rhyacophila H Cosmos bipinnatus Cosmos atrosanguineus C. latifolia C. tripteris C. pulchra C. delphinifolia C. leavenworthii C. pubescens B. beckii B. frondosa B. mitis 1 B. mitis 2 B. laevis B. amplissima B. cf. amplissima F| B. tripartita B. cernua C. lopez-mirandae C. woytkowskii B. schimperi B. pachyloma B. macroptera B. prestinaria B. sp. (Starbuck Island) B. cynapiifolia B. reptans B. squarrosa B. boquetiensis B. segetum B. serrulata B. ferulifolia B. andicola j B. aurea I I B. pilosa I B. alba var. radiata B. populifolia X B. conjuncta B. mauiensis B. conjuncta B. sandvicensis ssp. sandvicensis B. hillebrandiana ssp. polycephala B. hillebrandiana ssp. polycephala X B. cosmoides B. cosmoides B. sp.2 B. sp. 1 B. sp.(Tahiata)

Figure 9. Strict consensus of 44 equally parsimonious trees of length 768, for 53 taxa. Clades identified by capital letters.

29 • Fitchia speciosa r— Dahlia coccinea L Dahlia macdougallii — C. mutica var. leptomera D maritima — C. californica var. californica C. petrophila I C. rhyacophila j Cosmos bipinnatus ' Cosmos atrosanguineus C. latifolia C. tripteris M— C. pulchra C. delphinifolia - B. beckii — C. leavenworthii C. pubescens I— B. frondosa [j B. mitis 1 " B. mitis 2 B. laevis B. amplissima B. cernua _r C. lopez-mirandae '— C. woytkowskii B. schimperi — B. pachyloma j—B. macroptera "1—pB . prestinaria -B. sp. (Starbuck Island) K B. cynapiifolia I B. reptans B. squarrosa boquetiensis M B. segetum r—B. serrulata jrB. ferulifolia ^B. andicola O i—B. aurea Lr B. pilosa LB. alba var. radiata B. populifolia X B. conjuncta l_ B. mauiensis r-B. sp. 2 '—B. sp.(Tahiata)

Figure 10. Maximum likelihood tree for 45 taxa with 8 duplicate taxa omitted. The analysis used optimized parameters with 7% of sites invariable, a transition/transversion ratio of 1.80 and a gamma shape parameter of 0.93. Branch lengths are proportional to the estimated number of substitutions. Clades are identified by capital letters.

30 The only representative from section Meduseae, Bidens mitis, appears nested within section Bidens in trees generated from all analyses. The two specimens of Bidens mitis that were sequenced differ by four base pairs in their ITS sequences, including two ambiguous nucleotides, so both sequences were included in the phylogenetic analyses. They are labeled as B. mitis 1 and B. mitis 2 and they are sister taxa in 98 percent of parsimony bootstrap replicates, in the parsimony tree, maximum likelihood and all neighbor joining trees. The two specimens of B. mitis cluster with B. frondosa in the parsimony tree with 100 percent bootstrap support, as well as in the maximum likelihood and neighbor joining trees.

Classified in section Bidens, B. laevis appears with the other species in its section. In 91 percent of parsimony bootstrap replicates it appears as sister species to the four north temperate Bidens with identical ITS sequences: B. amplissima, B. cf. amplissima, B. tripartita and B. cernua. This relationship is confirmed in parsimony, maximum likelihood and neighbor joining trees. Section Bidens is now paraphyletic only because B. mitis is nested within that clade.

Bidens squarrosa appears in clade P with the other species in section Greenmania. Bidens squarrosa is only weakly basal to B. boquetiensis and B. segetum but it clusters with the latter two species in 95 percent of parsimony bootstrap replicates. With the addition of B. squarrosa to the ITS phylogeny, section Greenmania remains monophyletic.

3.1.6 Relationship off Cosmos to Bidens and Coreopsis Cosmos, a genus of approximately 26 annual and perennial herbs and subshrubs is currently represented by only two species in the Bidens I Coreopsis ITS phylogeny. The two representatives of Cosmos form a monophyletic clade nested within Coreopsis with 100 percent parsimony bootstrap support. The exact position of Cosmos is unresolved, with section Pseudoagarista of Coreopsis either basal to or sister group to Cosmos. Some species of Cosmos have been placed in both Bidens and Dahlia previously. 3.2 Morphology of Starbuck Island Bidens A summary of the morphological analysis of Starbuck Island Bidens is shown in Table 4. Included are the mean, median, minimum and maximum values for all measured morphological traits. This information is incorporated into the species diagnosis and description in Chapter 5.

Table 4. Mean, median, minimum and maximum values for floral, achene and vegetative traits of Starbuck Island Bidens.

Trait measured Floral and Inflorescence traits N Median Mean Min. Max. S.D. S.E. Peduncle length (mm) 69 51.23 58.32 13.12 119.71 26.34 3.17 Disc radius including rays (mm) 17 21.68 21.85 15.32 28.39 3.70 0.90 Disc radius with rays flat (mm) 17 25 25.51 21.19 29.77 2.70 0.66 Disc radius without rays (mm) 10 9.65 8.97 6.23 10.42 1.53 0.48 Number of outer involucral bracts 68 9 9.07 5 13 2.10 0.25 Number of inner involucral bracts 68 8 7.32 5 10 1.27 0.15 Number of ray florets 69 8 7.25 5 12 1.40 0.17

31 Table 4. Continued. Trait measured Number of disc florets 69 24 24.42 7 41 7.27 0.88 Number of chaffy bracts 67 25 24.52 8 37 6.98 0.85 Outer involucral bract length (mm) 621 4.8 4.97 2.8 13.07 1.36 0.05 Inner involucral bract length (mm) 498 5.3 5.32 3.82 7.39 0.56 0.03 Ray floret length (flat) (mm) 493 10.92 10.84 2.69 16.22 1.93 0.09 Ray floret width (flat) (mm) 127 7.65 7.52 4.18 9.38 1.03 0.09 Extra ray ligule length (flat) (mm) 27 9.22 9.14 2.19 13.89 2.86 0.55 Achene traits Achene length, not incl. awns (mm) 214 9.67 9.84 4.73 15.46 1.89 0.13 Achene width: widest point (mm) 214 1.03 1.04 0.51 1.52 0.17 0.01 Achene width: narrowest point (mm) 214 0.18 0.18 0.03 0.33 0.05 0.00 Number of awns 214 2 1.67 0 3 0.74 0.05 Length of longest awn (mm) 180 1.26 1.26 0.57 1.96 0.25 0.02 Vegetative traits Plant height (cm) 8 68.5 67.88 54 78 8.41 2.97 Leaf length (cm) 41 9.02 9.24 2.73 19.84 4.37 0.68 Leaf width (cm) 41 6.63 6.96 1.45 16.1 3.29 0.51 Number of leaflets 40 5 4.45 3 5 0.90 0.14 Leaflet length (cm) 183 3.95 4.16 1.05 9.74 1.88 0.14 Leaflet width at widest point (cm) 183 1.72 1.85 0.39 4.71 0.87 0.06 Average number of teeth per cm 183 2.5 2.58 0 8 1.17 0.09 Total number of teeth per leaflet side 183 6 6.45 0 19 4.43 0.33

3.2.1 Floral and achene characters Figure 11 shows variation in floral traits of Starbuck Island Bidens. Unique characteristics of this species, some of which may suggest a high degree of inbreeding, can be seen here as well. For example, curled ray florets (Figure 11 A) are typical of Starbuck Island Bidens, while ray florets with sterile stamens and an underdeveloped style (Figure 11 C and 11 E) are atypical but not uncommon. The latter sometimes appears to be a sort of modified disc floret, occasionally with an extra corolla ligule or blade, as shown in Figure 11 C. Of the ray florets measured 4% had an extra corolla ligule. There was no evidence that the sterile stamens of ray florets produced any pollen. A typical ray floret of Starbuck Island Bidens is completely sterile with a strap-shaped corolla and no evidence of stamens or style and no pappus.

Achenes of Starbuck Island Bidens are quite variable in form, from straight and needle-like to curved or twisted, as shown in Figure 12. In comparison, Bidens cynapiifolia (Figure 15), the closest continental relative of Starbuck Island Bidens, has straight achenes with no evidence of curving or twisting. Awn number also differs between Starbuck Island Bidens and B. cynapiifolia, the former with typically two, occasionally one or three, retrorsely-barbed awns compared to 4-6 barbed awns in the latter. Achene length overlaps considerably between the two taxa with achenes of Starbuck Island Bidens, from 5-15 mm, somewhat shorter than those of B. cynapiifolia which range from 8-17 mm. Achene characters are important in distinguishing Starbuck Island Bidens from Bidens cynapiifolia as well as from the rest of the genus.

32 Figure 11. Photographs showing floral characteristics of Starbuck Island Bidens. Plants grown at the University of British Columbia from seed collected by A. Whistler on Starbuck Island. (A) Heads, side view. (B) Heads, face view. (C) Ray florets from three plants. (D) Head with rays removed. (E) Ray floret with sterile stamens. (F) Disc florets. (G) Inner involucral bracts. (H) Outer involucral bracts. (I) Chaffy bracts.

33 0 0.5 cm 1 i ... I Figure 12. Photograph of Starbuck Island Bidens achenes. Achenes represent variation among three plants grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island.

3.2.2 Vegetative characters Starbuck Island Bidens exhibits wide variation in leaf morphology which is common within species in the genus Bidens. Although its leaves are quite similar to those of B. cynapiifolia, leaf characters are not the most taxonomically significant character in distinguishing Starbuck Island Bidens from other Bidens species. Figure 13 shows the variation in leaf size and shape within a single plant while Figure 14 shows the variation in leaf morphology among 14 different plants.

Figure 13. Photograph of leaves from a single plant of Starbuck Island Bidens. Plant was grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island

34 0 5 cm i ill

Figure 14. Photograph of leaf variation in Starbuck Island Bidens. Leaves are from fourteen plants grown at the University of British Columbia, from seed collected by A. Whistler on Starbuck Island Chapter 4 DISCUSSION

4.1 Starbuck Island Bidens: a new endemic Pacific island species Results from sequencing the ITS of ribosomal genes for both Bidens aurea and Bidens serrulata have disproved hypotheses 2 and 3. Both species appear in clades with other species in section Psilocarpaea and are not closely related to Starbuck Island Bidens. Evidence from ITS sequences and morphological studies support hypothesis 1, that Starbuck Island Bidens is a new species that is endemic to the Line Islands and it represents a second, independent colonization of a Pacific island that has not led to further speciation or adaptive radiation, unless it is the last survivor of a radiation in which all other species have gone extinct. 4.1.1 Evolutionary origin of Starbuck Island Bidens According to Sherff s monograph of Bidens (1937), Starbuck Island Bidens appears most similar to B. aurea and B. serrulata. However, both species can now be distinguished from Starbuck Island Bidens through ITS sequences, floral and achene characters.

The most surprising result is the discovery that Bidens cynapiifolia, not B. aurea or B. serrulata, is the most closely related to Starbuck Island Bidens. As suggested by Ganders et al. (2000) further sequencing in the diverse and poorly sampled section Psilocarpaea did indeed reveal a closer relative to the Starbuck Island Bidens. While ITS sequences indicate that Starbuck Island Bidens is most closely related to Bidens cynapiifolia, shown in Figure 15, morphology indicates that they are clearly not the same species. They differ considerably in growth habit, floral and achene characters. However, leaves of Bidens cynapiifolia are within the range of those of Starbuck Island Bidens, both in size and form, and similarities in certain achene characters suggest that it is not unlikely that the two share a recent common ancestor or that Starbuck Island Bidens has evolved from Bidens cynapiifolia following its introduction to Starbuck Island.

That the closest known relative to Starbuck Island Bidens is not very similar morphologically is not that surprising. Studies of island floras, especially Hawaiian Bidens, have shown that morphological similarity is not a good indicator of overall relatedness. In the Hawaiian Bidens there is more morphological variation than within the rest of the entire genus, yet all of the

36 Hawaiian Bidens share identical ITS sequences and are relatively recently derived from a single common ancestor (Ganders, 1989; Ganders et al, 2000). There is evidence that many of the morphological traits that distinguish Hawaiian Bidens species from one another are determined by only one or two gene loci and are therefore not useful as indicators of relatedness (Ganders, 1989).

Achenes of Starbuck Island Bidens exhibit some of the unique characteristics of Pacific Island Bidens. Like many Hawaiian Bidens species (Ganders, 1989; Carlquist, 1974), Starbuck Island Bidens shows evidence of a recent or ongoing adaptive shift in dispersal mechanism. For instance, Figure 12 shows how achene shape ranges from straight and needle-like to curved or twisted, which has been hypothesized to represent an adaptation to dispersal by wind in Hawaiian Bidens (Ganders, 1989). In comparison, Bidens cynapiifolia (Figure 15), the closest continental relative of Starbuck Island Bidens, has straight achenes with no evidence of curving or twisting. Awn number also differs between Starbuck Island Bidens and B. cynapiifolia, the former with typically two, occasionally one or three, retrorsely-barbed awns compared to 4-6 retrorsely barbed awns in the latter. Achene length overlaps considerably between the two taxa with achenes of Starbuck Island Bidens, from 5-15 mm, somewhat shorter than those of B. cynapiifolia which range from 8-17 mm.

Starbuck Island Bidens is distinguished from the rest of the genus by a unique combination of morphological characteristics, listed in the species diagnosis below. Furthermore, Starbuck Island Bidens possesses many of the traits associated with island endemism in flowering plants, including extreme sensitivity to growth environment, a tendency toward a woody growth form compared to its closest continental relative, change in seed dispersal characteristics, including twisted achenes and odd physical mutations that may reflect a high degree of inbreeding. All of this points to Starbuck Island Bidens having evolved on the island after a direct colonization event from Latin America. The evidence indicates that Starbuck Island Bidens is derived from a different ancestral colonist than the rest of the Pacific Island Bidens species.

4.1.2 Possible allopolyploid origin The chromosome number of B. cynapiifolia is 2N = 24 (Ganders and Nagata, 1990), while that of Starbuck Island Bidens is 2N = ca. 70 or higher. Therefore if Starbuck Island Bidens evolved directly from B. cynapiifolia, it could be an autopolyploid. However, not many chromosome counts are available for B. cynapiifolia and it is possible that polyploid races could also exist within that species. It is extremely unlikely that a hybridization event involving two different Bidens species occurred on Starbuck Island. Bidens pilosa has chromosome numbers of 2N = 24, 36, 46,48, 72 and ca. 76 (Ganders and Nagata, 1990), it is the only other Bidens commonly found throughout the Pacific as an introduced weed and it is not closely related to Starbuck Island Bidens in the ITS phylogeny. The possible existence of a closer continental relative to Starbuck Island Bidens seems equally unlikely. However, to rule out this possibility the ITS phylogeny would have to be completed for the entire genus Bidens.

37 h i E. E. Sherff Del. f Bidens cynapiifolia H.B.K.

Figure 15. Bidens cynapiifolia. Illustration adapted from Sherff s monograph on Bidens (Sherff, 1937). (a) Portion of fruiting specimen (x 0.85). (b) Upper leaves and (c) lower leaves (x 0.85). (d) Outer involucral bract, (e) inner involucral bract and (f) chaffy bract (x 6.82). (g) Disc floret (x 6.82). (h) Outer achene and (i) inner achene (x 3.41).

4.1.3 Evidence for evolutionary adaptations to island life While atoll floras are typically low in species diversity, the flora of the Southern Line islands is especially impoverished. Several features contribute to this, including an isolated location, low island profile, homogenous topography, low habitat diversity, little rainfall and nutrient poor soils (Kepler and Kepler, 1994). For islands like these long distance dispersal and plant hardiness are important factors in establishing a flora but, once a plant species becomes established, the dispersal characteristics that helped it to reach the island may no longer be advantageous.

38 A loss or change in dispersal characteristics is just one aspect of the major evolutionary changes, especially in gross morphology, that island immigrants typically undergo. Endemic flowering plant species in the Hawaiian and other Pacific floras show a tendency toward a loss of dispersibility by animals compared to their mainland counterparts (Carlquist, 1967). This loss of dispersibility is characterized by a marked increase in seed size, often accompanied by lowered seed production (Lord et al. 1997).

In Pacific Island Bidens evolutionary change in dispersal characters has been very well documented, particularly in species of the Hawaiian Islands, where there is a wide range in achene morphology (Carlquist, 1980; Ganders, 1989). Among the South Pacific Bidens, only a few species have largely retained their dispersal characteristics. These include the three most southeastern species, B. hendersonensis, B. mathewsii and B. saintjohniana, and the Hawaiian species B. hillebrandiana. All have barbed awns and have maintained the presence of hairs on both dorsoventral and lateral margins (Carlquist, 1974). While Starbuck Island Bidens does not have larger achenes than those of Bidens cynapiifolia it does have markedly fewer and shorter awns which can be interpreted as a loss of dispersibility.

Carlquist proposes several possible explanations for a change in dispersal characters. Loss of dispersibility may be due to an ecological shift as a weedy pioneering immigrant species shifts to a more stable forest existence. Precinctiveness may also play a role, particularly in species that have become highly specialized to a particular ecological niche. Precinctiveness is the phenomenon whereby a plant sheds its propagules within a limited zone of stable conditions to which the species is highly ecologically adapted. In Bidens, Carlquist proposes that the original dispersal mechanism that brought Bidens to the islands was no longer adaptive after the original immigrant species radiated into the new ecological niches. In particular, in the new habitat, the dispersal vector may have been absent. Pacific insular species of Bidens that have best maintained adaptations to dispersal by birds are found on cliffs where sea birds nest. But Bidens species that have evolved into higher-elevation, forested sites have either lost their dispersal mechanisms, evolved new ones or both. Where dispersal mechanisms are simply lost it may be due to evolutionary loss of a structure that no longer serves a specific function. Carlquist specifically chose Asteraceae to demonstrate loss of dispersal characteristics in a diverse family that is well represented in the Pacific (Carlquist, 1966).

4.1.4 The future of Starbuck Island Bidens Starbuck Island Bidens is vulnerable to extinction in the near future. Its existence depends entirely on the protection of the 16 km island that it inhabits. In 1975 Starbuck Island was classified as a Wildlife Sanctuary and Closed Area but there is no specific measure to protect the vegetation of Starbuck Island. However, Kiribati is party to the International Agreement on Endangered Species and Biodiversity (IUCN, 1991) and in the future may develop legislation to provide specific protection for plants that are considered rare and endangered within its territory.

Pacific island endemics are especially vulnerable to extinction. They have often lost the dispersal mechanisms that allowed them to reach their island and that would enable them to expand their range to other locations. Also, many island species have become highly specialized to an ecogeographical niche and they are no longer able to adapt well to new environments. Currently the most persistent threats to Pacific island endemics include human disturbance and the introduction of exotic plants and animals. These factors have had a devastating impact on the

39 plant communities of many Pacific islands, including the loss of some rare and endemic species. Even Bidens has been affected. A recent inventory of the 16 vascular plant species on Oeno atoll failed to turn up the island's only endemic, Bidens hendersonensis var. oenoensis (Florence et al., 1995) .

For now, Starbuck Island Bidens appears to be thriving on the tiny island that remains unaffected by the typical destruction of native plants caused by humans. Even the most sparsely populated of the Line Islands have faced alteration of their native floras through the development of urban areas and villages, clearing of land for agriculture and coconut plantations and the introduction of exotic species (IUCN, 1991). Of the Line Islands chain, Starbuck and Vostok are the only two that remain relatively undisturbed. Probably the biggest threat to Starbuck Island is rising sea levels due to global warming. Over the last 20 years the climate in the Pacific has changed significantly. Between 1977 and 1994 the mean annual temperature in Kiribati rose 0.3°C and rainfall increased by 30% compared to averages before 1977 (Salinger, 1995; Salinger et al., 1996) .

Regardless of human effort to protect this species eventually Starbuck Island Bidens will face extinction as the island completes its cycle as a coral atoll and becomes completely submerged. For now, the island's remote location and the lack of visitors favor the survival of Starbuck Island Bidens within its rare and fragile ecosystem.

4.2 Sectional relationships within Bidens The data from this study confirms the polyphyletic nature of section Psilocarpaea revealed in Ganders et al. (2000) and Kim et al. (1999). Clearly section Psilocarpaea must be divided into several separate sections. Unfortunately, ITS sequences are not known for most of the 124 species currently assigned to this section. Furthermore, no morphological distinguishing characters or synapomorphies have been discovered for the clades revealed by ITS sequences. Therefore sectional reassignment of Bidens species within Clade K (Figure 8) will require that many more species are sequenced.

ITS sequence differences between the two specimens of B. mitis may represent sequence divergence between the two populations fromwhic h they were collected. Two of the four nucleotide differences involve an ambiguous nucleotide in one of the specimens that may or may not represent a real difference between the two sequences. However, the other two sites appear to be real differences. The first specimen, B. mitis 1, was collected in Marion County, Florida at 29° 11'55"N, 81°39'04"W in a white cedar-palm-bay seepage spring swamp forest. The second specimen, B. mitis 2, was collected in Osceola County, Florida at 28°12'53"N, 81°17'29"W in a disturbed, seepy flatwoods-savanna and cypress slough. The collection sites are separated by a distance of ca. 35 km.

Although ITS sequence data should be obtained for all of the species in section Meduseae, it appears that section Meduseae should not be recognized as separate fromsectio n Bidens.

Together, Bidens aurea, B. pilosa and B. alba var. radiata are sister to the Hawaiian and Marquesan species in section Campylotheca. Bidens pilosa, which is rayless, and B. alba, which has white ray florets, would seem to be less likely than B. aurea, which has yellow ray florets, as the ancestral species to the Polynesian Bidens, which also have yellow ray florets. This discovery narrows down the continental ancestor to the Hawaiian and Marquesan Bidens from Ganders et

40 al. (2000) and also indicates that B. ferulifolia, B. andicola and B. serrulata, in clade R, are not as closely related to section Campylotheca as was previously thought. 4.3 Generic relationships between Bidens, Coreopsis and Cosmos Recently, ITS sequences have proven useful in resolving evolutionary relationships within groups of closely related genera in tribe Heliantheae, including subtribe Engelmanniinae (Clevinger and Panero, 2000), the Encelia alliance of genera (Clark, 1995), subtribe Zinniinae (Urbatsch et al, 1996) and the tarweed-silversword subtribe Madiinae (Baldwin and Wessa, 2000).

ITS sequences show that neither Bidens nor Coreopsis is a monophyletic genus. My ITS phylogeny also shows that Cosmos is nested within Coreopsis, which further complicates this already complex taxonomic problem. With only two representative species though, the complete relationship of Cosmos to Bidens and Coreopsis cannot be determined. This lack of resolution is consistent with the results of a study by Ryding and Bremer (1992) which found no resolution between Bidens, Coreopsis and Cosmos based on a data matrix of 37 morphological characters. Further sequencing within Cosmos will be necessary to determine if Cosmos is a monophyletic genus, as it is currently circumscribed, and to reveal its precise relationship to Coreopsis and Bidens. It is not surprising that Cosmos is so closely related to Bidens. In recent taxonomic revisions two species, B. ocellatus and B. acrifolia var. langlassei, were transferred from Cosmos to Bidens (Melchert, 1990; Melchert and Turner, 1990).

Ultimately several more genera will have to be recognized. The basal Clades B, D and F (see Figure 8) will have to be recognized at the generic level. Most already have, at one time or another, and there are good morphological characters separating them. For example, species of Clade D have alternate leaves and pistilate ray florets, while most species of Bidens and Coreopsis in Clades J and K have opposite leaves and sterile ray florets. Cosmos (Clade H) and Clade L should be recognized at the generic level, allowing the two largest Clades, J and K, to be recognized as separate genera.

At present, the type species of both Coreopsis and Bidens are in Clade J. Perhaps the best proposal would be to conserve the genus Bidens with a new type in Clade K, leaving Clade J as Coreopsis. Although this would mean that the familiar northern North American and European species of Bidens would become Coreopsis, many have been named in Coreopsis already. This proposal would require the fewest new combinations and it would preserve the currently used names for all of the commonly cultivated species of both genera. 4.4 Implications for future research Starbuck Island Bidens is a rare species, endemic to one of the most isolated coral atolls in the Pacific, and it provides a remarkable example of a second Pacific island colonization that has apparently not diversified evolutionarily. Its discovery is a significant contribution to our understanding of island biology in general and more specifically, the biogebgraphy of the genus Bidens.

Future research should include crossing experiments between Starbuck Island Bidens and B. cynapiifolia to determine if the two species are interfertile. If they are interfertile, interspecific hybrids should be tested to estimate hybrid fertility and the closeness of the relationship between

41 the two species. If Starbuck Island Bidens and B. cynapiifolia are capable of producing fully fertile hybrids then the species barrier between them is primarily one of geographical isolation and ecology.

To fully understand the evolutionary relationships among Pacific island Bidens an effort should be made to obtain specimens and ITS sequences for all of the endemic Pacific island Bidens that have not yet been sequenced. These include the remaining six of nine endemic species described from the Marquesas Islands, as well as a number of undescribed species from the Marquesas, seven endemic species from the Society islands, B. hendersoniensis from Henderson Island, B. hendersoniensis var. oenoensis fromOen o Island, B. mathewsii fromPitcair n and B. saintjohniana from Marotiri, near Rapa. The recent work on this genus has shown that morphology alone is not informative when it comes to estimating phylogeny. The discovery of Starbuck Island Bidens has shown us that there is much left to learn about the biogeography of this important genus. Only by obtaining a complete ITS data set for the genus Bidens will we begin to fully understand the unique and complex relationships of the Pacific island Bidens that we see today.

42 Chapter 5 TAXONOMIC TREATMENT

Bidens kiribatiensis Sayre & Ganders sp. nov. Figure 16

Differt a B. cynapiifolia flosculis radiatis (5-) 8 (-10), flavis, 3-16 mm longis et 4-9 mm latis. Differt a B. aurea flosculis radiatis (5-) 8 (-10) versus 5-6, achaeneis 5-15 mm longis versus 4- 7 mm longis. Differt a B. serrulata flosculis radiatis (5-) 8 (-10) versus (4-) 5 (-7), achaeneis non dimorphis.

Plants erect, perennial subshrubs from 40 to 68 cm. Stems branched, green, maturing to reddish or reddish-brown, square in cross-section, glabrous. Leaves opposite, medium green, 3-20 cm long including petiole, 1.5-16 cm wide, once-pinnately compound to bipinnate-pinnatifid with 3 or 5 (-9) leaflets. Leaflets acute to acuminate at apex, 1-10 cm long, 0.5-5 cm wide, serrate (rarely entire); (0) 1-4 (-8) teeth per cm along leaflet margin, sparsely to moderately pubescent on both surfaces. Inflorescence with mostly 3-7 heads in simple or compound cymes. Heads 6- 10 mm wide without rays, 2.1-3.0 cm with rays extended. Pedunclel-12 cm long, medium green. Involucre basally hispid. Involucral bracts in two series; outer involucral bracts 5-13, linear-spathulate, 3-7 (-13) mm long, medium to dark green, pubescent on both surfaces and along margins; inner involucral bracts 5-12, linear-lanceolate, 4-7 mm long, medium to light green, adaxial surface glabrous, abaxial surface pubescent at tip, margins hyaline. Chaffy bracts of receptacle linear-lanceolate, pale green, hyaline. Ray florets yellow, sterile, 8 (5-10) per head; ligule obovate, often curled under, 3-16 mm long and 4-9 mm wide measured flat; secondary ligule occasionally present, 2-14 mm long excluding claw, sometimes accompanied by sterile, partially-formed stamens; disc florets perfect, (7) 11-37 (41) per head, disc corollas yellow. Achenes dark brown to black, linear, straight or somewhat curved or twisted, basically quadrangular with four longitudinal grooves, sparsely hairy mostly within the upper third of the achene body. Achenes 5-15 mm long, 0.5-1.5 mm wide at the widest point. Pappus of mostly 2 awns (one or a few achenes per head may have 0,1 or 3 awns), parallel to spreading or divergent, dark brown, 0.5-2 mm long, retrorsely barbed.

Type: KIRIBATI. Line Islands: Starbuck Island, June 1995, A Whistler 9879 (Holotype: BISH).

43 Specimens examined: Cultivated at the University of British Columbia from seed collected by A. Whistler on Starbuck Island, Kiribati, C. Sayre 99-1., 17 Dec.1999 (UBC!) (Voucher for species illustration); C. Sayre 99-2., 17 Dec.1999 (UBC!); C. Sayre 99-3., 17 Dec.1999 (UBC!).

Distribution: Starbuck Island, Kiribati, endemic.

ITS sequences and morphological data indicate that the Bidens on Starbuck Island is a new species. As indicated by ITS sequences, its closest relative is Bidens cynapiifolia but the two are clearly not the same species. They are easily distinguished from one another based on floral characteristics, such as the presence or absence of ray florets, the number of ray florets and the number of pappus awns. Starbuck Island Bidens has radiate heads with (5-) 8 (-10) ray florets, 3- 16 mm long, while B. cynapiifolia has only 3-4 ray florets, 5-7.5 mm long, or none at all. The pappus of Starbuck Island Bidens never exceeds 3 awns while the pappus of B. cynapiifolia is of 4— 6 barbed awns.

When keyed out in Sherff s monograph on Bidens (Sherff, 1937), Starbuck Island Bidens appears to be morphologically most similar to Bidens aurea and Bidens serrulata. However, B. aurea and B. serrulata are distinguished from Starbuck Island Bidens by ITS sequences, floral and achene characters.

Starbuck Island Bidens is distinguished from Bidens aurea by the former having more ray florets, shorter rays and longer achenes than the latter. While heads of Starbuck Island Bidens typically have 8 (5-10) rays, from 0.3-1.6 cm long, B. aurea has only 5 or 6 rays per head, ranging in length from 1-3 cm. Achene length in Starbuck Island Bidens is 5-15 mm versus 4-7 mm in B. aurea.

Starbuck Island Bidens is easily distinguished from Bidens serrulata by the presence of dimorphic achenes in B. serrulata and monomorphic achenes in Starbuck Island Bidens. Other differences between the two species include the presence of hairs on outer involucral bracts, inner involucral bract length, ray number and ray length. Starbuck Island Bidens has pubescent outer involucral bracts and inner involucral bracts ranging in length from 4-7 mm versus glabrous outer involucral bracts and inner involucral bracts from 7-11 mm long in B. serrulata. Heads of Starbuck Island Bidens have (5-) 8 (-10) ray florets from 0.3-1.6 cm long versus (4-) 5 (-7) rays from1.5- 3 cm long in Bidens serrulata.

A unique combination of morphological characteristics, listed in the species diagnosis, distinguishes Starbuck Island Bidens from the rest of the genus.

Starbuck Island Bidens has not been assigned to a section within Bidens. Its closest relative, Bidens cynapiifolia, is currently assigned to section Psilocarpaea which, as previously mentioned, is polyphyletic. The next most closely related group to Starbuck Island Bidens is the clade containing species within section Greenmania. There is no point in assigning Starbuck Island Bidens to a section until the sectional taxonomy of Bidens and Coreopsis are revised.

The specific epithet commemorates the country Kiribati to which Bidens kiribatiensis is endemic. The epithet starbuckensis was not used because I did not want to imply any connection with or support from Starbucks Coffee.

44 d j k I m n

C. M. Sayre

Figure 16. Illustration of Starbuck Island Bidens. (a) General growth habit (x 0.4). (b, c, d) Detail of three leaf types (x 0.4). (e) Head with rays removed (x l.l).(f) Head (x 1.1). (g, h, i) Achenes with one, two and three awns respectively (x 1.1). (j) Outer involucral bract (x 2.75). (k) Inner involucral bract (x 2.75). (1) Chaffy bract (x 2.75). (m) Disc floret (x 1.1). (n) Ray floret (x 1.1).

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