Sondi Jones Hoffman a Thesis Submitted to the University of N

TAXONOMIC AND PHYLOGENETIC EVALUATION OF STYLISMA (CONVOLVULACEAE)

Sondi Jones Hoffman

A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science

Department of Biology and Marine Biology

University of North Carolina Wilmington

2009

Approved by

Advisory Committee

_ __Dr. Ami Wilbur______Dr. Michael Durako ______

______Dr. Gregory Chandler Chair

Accepted by

Dean, Graduate School

TABLE OF CONTENTS

ABSTRACT...... v

ACKNOWLEDGMENTS ...... vi

DEDICATION ...... vii

LIST OF TABLES...... viii

LIST OF FIGURES ...... ix

CHAPTER 1. GENERAL INTRODUCTION ...... 1

Stylisma...... 1

Review of taxa and taxonomic history ...... 2

Species concepts ...... 9

Current taxonomy ...... 11

Conservation ...... 11

Objectives ...... 12

Hypothesis...... 12

Methodology...... 14

Morphometric vs. molecular techniques...... 14

Introduction to morphological methodology ...... 14

Ordination ...... 15

Introduction to molecular methodology...... 16

The chloroplast genome...... 15

CHAPTER 2. MATERIALS AND METHODS ...... 20

Morphological methods ...... 20

Taxon sampling...... 20

iii Morphological measurements...... 20

Morphological data analysis ...... 23

Molecular methods...... 26

Taxon sampling...... 26

DNA extraction, amplification, and sequencing...... 26

Sequence data analysis...... 31

CHAPTER 3 RESULTS...... 32

Morphological results ...... 32

Molecular results...... 49

Sequence characteristics ...... 49

Phylogenetic reconstruction...... 52

Major clades...... 54

CHAPTER 4. DISCUSSION...... 55

Morphology...... 55

Molecular genetics...... 58

Conclusions...... 60

CHAPTER 5. DESCRIPTION OF PROPOSED TAXA...... 65

Stylisma abdita...... 65

Stylisma pickeringii...... 65

Stylisma paten ...... 66

LITERATURE CITED ...... 68

iv ABSTRACT

Stylisma (Convolvulaceae) is a small genus of dawnflowers found in the sandhills of southeastern United States. The genus is in need of a taxonomic revision because the last revision was long ago, it did not appropriately resolve the relationships within the genus, and there are several threatened or endangered taxa in the genus. A phylogenetic study of the genus is presented here, using two chloroplast markers, the trnT-L intergeneric spacer and the rps16 intron. A phenetic study of the genus is also presented examining 40 characters in 430 specimens and analyzing the resulting matrix with ordination (PCA) methods. Both data sets result in three clades of similar topology. The molecular data combines Stylisma patens and

Stylisma humistrata into a single clade, Stylisma pickeringii into a monospecific clade, and

Stylisma villosa, Stylisma abdita, and Stylisma aquatica into a separate clade. The morphological data agree with the molecular data as to the placement of S. pickeringii into its own clade, but separates S. abdita into a second clade and lumps the remainder of the genus into a third clade. The combined morphological and molecular results suggest Stylisma to be a complex group that requires more work to determine the spurious species boundaries.

v ACKNOWLEDGMENTS

This thesis would not have been possible without the help of my advisor, Gregory

Chandler, who was available to me day and night throughout this process. I must also thank my committee, Ami Wilbur and Mike Durako. A special thanks goes out to Steven Brewer, who was such a great help, even from Belize. Thanks to Marcel van Tuinen for the use of lab space.

Thanks to David Webster and Craig Bailey for all of their support. I would like to thank Florida

State University, University of South Florida, Rutgers University, University of North Carolina, and Kansas State University for providing herbarium loans.

I would like to extend thanks to my field crew, Dallas, Justin, and Erich. Amy Cherry

Millis was invaluable, as she provided a sounding board for the entire project. A huge thanks is in order for the biology office staff, especially Tracie and Lori, without who I would surely have failed.

DEDICATION

I would like to dedicate this thesis to my husband, Dallas, whose continued support and encouragement have been invaluable.

vii

LIST OF TABLES

Table Page

1. Stearn’s leaf terms...... 5

2. Total specimens studied and their locations (county and state)...... 21

3. Morphological characters measured ...... 22

4. Numbers assigned to qualitative characters in matrix ...... 25

5. Key to abbreviations used in ordination ...... 27

6. Genebank and voucher information...... 28

7. Primer list...... 30

8. Characters used in ordination matrices...... 33

9. The variance and eigenvalues ...... 34

10. Correlations of the first six eigenvectors ...... 48

11. Sequence characteristics ...... 51

viii LIST OF FIGURES

Figure Page

1. Phenogram representing current infrageneric relationships ...... 13

2. Diagram of the trnT-L region and the rps16 intron ...... 19

3. Representative flower and leaf for measurements...... 24

4. Visual output of PCA analysis axes 2 v.1...... 36

5. 3D output of PCA analysis axes 1 v.2 v. 3 ...... 37

6. Visual output of PCA analysis axes 1 v.3...... 38

7. 3D output of PCA analysis axes 1 v.2 v. 4 ...... 39

8. Visual output of PCA analysis axes 1 v.4...... 40

9. Visual output of PCA analysis axes 2 v.4...... 41

10. Visual output of PCA analysis axes 3 v.4...... 42

11. Visual output of PCA analysis axes 2 v.3...... 43

12. Visual output of PCA of Group III axes 1 v.2 ...... 44

13. 3D output of PCA of Group III axes 1 v.2 v. 3...... 45

14. Visual output of PCA of Group III axes 1 v.3 ...... 46

15. 3D output of PCA of Group III axes 2 v.3 v. 4...... 47

16. The character vectors from the PCA...... 50

17. Strict consensus tree of molecular data...... 53

18. 3D output of PCA including molecular ...... 61

19. Distribution maps of Stylisma...... 67

ix CHAPTER 1. GENERAL INTRODUCTION

Stylisma Raf.

Stylisma, commonly known as the dawnflower, is a member of Convolvulaceae, the morning glory family, tribe Cresseae (Weakly 2007). Convolvulaceae is sister to Solanaceae within

Solanales and sits firmly within the Eurastid I clade (Olmstead et al. 1993; APG II 2003).

Defining characters of Eurastid I include fused carpels, epipetaly, and an equal or smaller number of stamens as lobes on the corolla. Members of Solanales have flowers that are radially symmetrical with plicate, sympetalous corollas and leaves that are alternate, simple, and exstipulate. Convolvulaceae are usually twining or trailing herbs with rhizomes, lactifers, hairs, determinate inflorescences, bisexual flowers (pentamerous) with superior ovaries, and capsular fruits. Cresseae contains plants that are herbs to shrubs, often lianas, and their leaf bases are not cordate, unlike other tribes (Stefanovic et al. 2003).

Convolvulaceae comprises 1600-1700 species distributed among 55-60 genera. Most genera within Convolvulaceae are large, with Ipomoea L. (~650 species) being the largest, and most exhibit tropical or subtropical distributions, with just a few representatives from temperate regions (Stefanovic et al. 2003). Stylisma, however, is a small genus that is restricted primarily to temperate southeastern North America, with one species being endemic to Florida (Stylisma abdita Myint). Stylisma consists of only six species, two of which are separated into subspecific taxa ranging from New Jersey, Illinois, and Iowa, south into Florida and Texas (Weakly 2007).

Stylisma are perennial, vining, herbaceous plants that are often slender, weak, or somewhat twining. The leaves are quite thin, being linear, narrowly elliptic, narrowly oblong, or narrowly lanceolate. Unlike many other genera of Convolvulaceae, the leaves are not cordate. The flowers are long-pedunculate except in S. abdita, and are generally arranged in loose cymes of a few flowers, or they are solitary. The calyx can be pubescent or glabrous, with small, thin, acute, acuminate, or rarely obtuse sepals. A small, inconspicuous, five-angled corolla with white petals

[except S. aquatica (Walter) Raf., which is pink or purple] is standard. The corolla has a unique pattern of trichomes with pubescence along the margins and in strips where the petals are fused to one another. Stylisma stamens are included or partially exerted, with weakly sagittate anthers and filiform filaments, while the ovary is bicarpellate and four-ovuled with bifid style topped with two capitate stigmas. The capsules are one or two seeded (rarely three or four), with glabrous or minutely pubescent seeds and linear, deeply bifurcated cotyledons (House 1907;

Myint 1966).

Review of taxa and taxonomic history

Stylisma abdita, also known as the hidden dawnflower, was described by Myint (1966).

They are perennial, prostrate vines with densely pubescent stems. The leaves are narrowly linear-elliptic, sessile with an acute or obtuse apex and are densely villous with silvery grey or brown trichomes, with indistinct secondary veins. Stylisma abdita has solitary flowers on short peduncles with a densely pubescent calyx and an unusually large ratio of calyx to corolla length

(~1:2) being the major distinguishing feature commonly used in keys. These plants flower from

April to November, and are a rare endemic found in xeric sand hill and scrub habitats in peninsular Florida. They often occur in open sunny areas dominated by Ceratiola ericoides

Michx. or Quercus geminata Small, with plants seldom occurring in densely vegetated areas

(Austin & Burch 1992). The individuals of this species are small and inconspicuous, often almost hidden by grasses, other small plants, and even fallen leaves, hence the specific epithet, which means “hidden.” This species is currently listed as endangered in Florida (Myint 1966).

2 Stylisma aquatica is commonly known as the water dawnflower. They have prostrate or twining, thick, hairy stems with narrowly oblong or narrowly oblong-elliptic leaves, which sit on short petioles and have obtuse-mucronate apexes. The leaf surface is tomentose with distinctly thick veins, and the inflorescences are axillary cymes of 1-3 flowers. The sepals are short with silvery grey hairs. This is the only species in the genus with petals that are not white, rather they are lavender, pink, or red. Stylisma aquatica flowers from Late May to August (Myint 1966), and is often found in clay-based Carolina bays and wet savannas. As the specific epithet implies, they are found in wetter habitats than the rest of the genus. They range from

Southeastern North Carolina south to the Florida panhandle and west to Southeastern Arkansas and Eastern Texas, and are on the NC rare list (Weakly 2007).

Stylisma humistrata (Walter) Chapman is commonly referred to as the southern dawnflower.

It has prostrate, twining, and glabrous to villous stems, the leaves have petioles that range from

1-12 mm long, and blades that are narrowly oblong to oblong with apices that are obtuse, acute or obtuse-mucronate. The inflorescences are axillary cymes of 3-7 flowers, though they can rarely be solitary through the abortion of lateral buds. The calyx is glabrous and has ciliate margins. Stylisma humistrata flowers from May through August (Myint 1966), and can be found on sand hills or other dry woodlands (Weakly 2007). Of all of the taxa in the genus, S. humistrata has the broadest distribution (Bates & Lewis 1984) and is found from southeastern

Virginia south to northern Florida, west to Arkansas and eastern Texas, north in the interior to northern Alabama and western Tennessee. This species is on the Virginia watch list (Weakly

2007).

Stylisma villosa (Nash) House is commonly known as the hairy dawnflower. The stems are twining or prostrate and thick, and villous with grey or brown hairs. The wooly-tomentose

3 leaves are mostly obtuse with petioles 3-10 mm long, and the apex is obtuse, acute or rarely obtuse-mucronate. Inflorescences are solitary or cymes of 3-7 flowers. The sepals are larger and densely villous with grey or brownish hair. Stylisma villosa is commonly mistaken for S. aquatica when not in flower. This species flowers from April to August (Myint 1966) and can be found in the dry sandy soils of sand hills, pinewoods, high pinelands, and occasionally on calcareous lands. They range from southern Georgia south to southern Florida west to eastern

Texas (Weakly 2007).

Stylisma patens (Desr.) Myint is one of two species complexes in this genus, and is commonly confused with others within the genus because of its morphological variability.

Plants of this species vary greatly in many characters, namely the size and shape of the leaves and the pubescence of the sepals. At the time of the last taxonomic revision there was little herbarium material available, so the morphological bounds of the species are currently unclear

(Myint 1966). Myint (1966) divided this species into two subspecies: S. patens subsp. patens and subsp. angustifolia based on sepal pubescence (villous or glabarous) and leaf length to width

(greater or less than six times the width).

Stylisma patens subsp. patens, the common dawnflower, has stems that are prostrate, rarely twining, and range from nearly glabrous to moderately and densely pubescent with grey-silver trichomes. The leaves are narrowly elliptical, which is defined by Stearn (1995) as having a 6:1 length to width ratio and being acute at each end with the sides curved equally from the middle

(Table 1). Inflorescences are axillary cymes of 1-5 flowers and the sepals are moderately to densely villous. They flower from May to September, and are the most common taxon in the group.

4 Table 1. Length to width ratios of commonly used leaf terms from Stearn (1995).

Elliptic series Length: width

Very narrowly elliptic 6:1

Narrowly elliptic 3:1

Elliptic 2:1

Broadly elliptic 3:2

Rotund 6:5

Circular 6:6

Oblong series

Linear 12 or more:1

Cultrate 6:1

Narrowly oblong 3:1

Oblong 2:1

Broadly oblong 3:2

Very broadly oblong 6:5

5 They are usually encountered on dry sandy soils and occur in sand hills, pine lands, oak-pine woods, and rarely in lowlands, ranging from eastern North Carolina south to northern Florida and west to southern Mississippi (Myint 1966; Weakly 2007).

Stylisma patens subsp. angustifolia (Nash) Myint is the narrow leaf dawnflower. The stems are prostrate, thick, and sparsely pubescent with linear leaves (Stearn 1995). The flowers are solitary, or very rarely in simple cymes (an unusual condition, as most cymes are compound).

The sepals are glabrous or ciliate rarely with a few scattered hairs. This species flowers from

May to July and can be found in dry sandy soils in pine lands, sand-hill scrubs, oak-pine flatwoods, and coastal plains (Myint 1966), ranging from northern to central Florida with spotty occurrence in north to southeastern North Carolina. This species is on the North Carolina watch list (Weakly 2007).

Stylisma pickeringii (Torr. ex M. A. Curtis) A. Gray was first split into four varieties, though

Myint (1966) found that there were no characters to adequately distinguish the initial number of subspecific taxa, though he did split the species into two varieties because the eastern group has longer stylar branches than the Midwestern groups. The two varieties also have different sepal shapes, with the eastern variety possessing obtuse sepals while the western variety has acute sepals. The morphological differences are correlated with geographical distribution (Lammers

1984).

Stylisma pickeringii var. pickeringii is known as Pickering’s dawnflower. The stems are prostrate, or trailing, and slightly pubescent. The leaves are sessile, linear, and moderately pubescent, with the veins obscure except for the midrib. The inflorescences are cymes of 2-5 flowers with the central flower sessile, or solitary. The sepals are obtuse with fulvous pubescence. This group can easily be recognized by its growth pattern as it has numerous stems

6 arising from a central point, forming a mound. Flowering occurs from May to August (Myint

1966), and they occur in the driest, most barren sand hills, and can occasionally be found on disturbed sandy roadsides. They range from southern North Carolina south to Alabama and west to Mississippi. There is also a disjunct population in New Jersey. This taxon is labeled as threatened in Georgia, rare in South Carolina, and endangered in North Carolina (Weakly 2007).

Stylisma pickeringii var. pattersonii (Fern.& Schub.) Myint only differs from var. pickeringii by its stylar branches and sepals. The sepals in this variety are acute with closer (hoary) pubescence and the stylar branches are shorter. This variety is found in well drained soils, sandy open woods, and occasionally in the moist sandy soils of inland plains (Myint 1966) and ranges from Illinois and Indiana south through Kansas and Oklahoma to eastern Texas, western

Louisiana and western Mississippi.

During the course of a book review of Elliott’s A Sketch of the Botany of South Carolina and

Georgia Rafinesque (1818) first proposed the name Stylisma based on the cleft style of the species now known as Stylisma humistrata (Walt.) Chapm. (Myint 1966). Stylisma is closely related to Bonamia DuPetit-Thouars., a moderately sized genus of about 45 species, primarily found in tropical and southern temperate regions. Another close relative, Breweria R. Br., is in a stage of taxonomic flux and has anywhere between five and 45 species that are primarily herbaceous vines found throughout Australia and Africa. These genera have been taxonomically merged and split multiple times primarily based on their shared floristic features (Lewis 1971).

Choisy, in De Candolle’s (1845) Prodromus Systematis Naturalis Regni Vegetabilis, treated these as three distinct genera. Asa Gray was the first to question the separation of the genera.

He first adopted Stylisma for the North American plants in his Manual of the Botany of the

Northern United States (Gray 1856), but then in a later edition of this publication (Gray 1863) he

7 included them in Bonamia.. Bentham and Hooker (1873) placed the North American species within Breweria, and Gray (1878) agreed with this placement, keeping Bonamia a monotypic genus. Hallier (1893) combined Bonamia and Breweria as Bonamia, but separated Stylisma into a separate genus. Peter (1897) produced a classification of this group including Stylisma as a subgenus of Breweria and separating two species out as Bonamia, while House (1907) chose to accept Hallier’s previous description of Stylisma separate and the rest of the species lumped into

Bonamia. Fernald and Schubert (1949) treated Bonamia as a separate genus and included

Stylisma under Breweria, then Wilson (1960) moved Stylisma back into Bonamia. Myint (1966) most recently recognized Stylisma as a separate genus, the major distinguishing character being the difference in cotyledon shape. Forty of the 45 species of Bonamia examined by Austin and

Staples (1985) had ovate-oblong to bilobed and emarginate, broad cotyledons, while the other five species, which belong to Stylisma, possess deeply bifid cotyledons with long slender and filiform lobes. Lewis (1971) also found clear differences between the pollen of Bonamia and

Stylisma. Using light and scanning electron microscopy he showed that Stylisma pollen is characterized by an advanced three-aggrecolpate aperture totaling 12 or 15 colpi. In contrast,

Bonamia pollen is typically a three-colpate type, which is more primitive (Lewis 1971). In addition to these embryological and microscopic characters, there are a number of gross morphological differences between the two genera. Bonamia generally consists of woody vines or shrubby plants, with ovate, obvate, cordate, thick and leathery leaves. The flowers are shortly pedunculate in dense cymes of many flowers, in contrast to the loose cymes of Stylisma. The sepals are generally large as are the corollas with the calyx ranging from 3-10 mm and the corolla ranging from 5-22mm. The fruits are mostly two or four seeded, rarely one-seeded

(Myint 1966).

8 Species concepts

Although it has been the subject of much attention the question “what is a species?” has yet to be resolved. Reflecting this confusion, there are many definitions of species in scientific literature. These concepts include the biological species, the phylogenetic species, and the phenetic species (for examples, Cracraft 2000; Templeton 1989). Each concept relies on definitions that are often as difficult to define as the species itself, such as population (Nelson &

Platnick 1981).

The Biological Species Concept (BSC) was introduced by Mayr (1982) and is widely known (Balakrishnan 2005). In its simplest form, the BSC is based on reproductive isolation, or a lack of gene flow, between naturally occurring populations (Coyne 1994). A biological species must form a reproductive community, an ecological unit, and a genetic unit (Mayr 2000). Many botanical systematists have largely abandoned this concept for a multitude of reasons.

Specifically in plants, hybridization, selfing, and asexual reproduction are all common and prevent them from fitting into the BSC. The application of this concept would lead to a vast number of clones and selfing individuals being recognized as different species making the species virtually impossible to identify in many cases. Specifically, this concept would prove impractical for delineating plant species because each study would have to include pollination experiments to test for reproductive isolation (Coyne 1994; Balakrishnan 2005). Another issue with the BSC is the assumption that gene flow represents relatedness. Reproductive compatibility is the primitive state of a lineage in plants, so only some species of that lineage may have lost the ability to interbreed with other members (Rosen, 1978). Often times in plants other characters diverge first, leaving more distantly related organisms with the ability to hybridize. Based on these, biological species are inappropriate to botanical phylogenetic

9 systematics, where lineages are being studied.

Another competing hypothesis is the Phylogenetic Species Concept (PSC). This concept defines a species as a group of organisms that share at least one derived character (Agapow et al.

2004). The phylogenetic species concept focuses upon the historical relatedness of organisms and the distribution of characters (Baum 1992; Velasco 2009). Under this concept, only monophyletic groups, consisting of an ancestor and all its descendants, can be considered taxa.

If a group subjected to cladistic analysis can be placed on a phylogenetic tree properly, then it has a unique evolutionary history. This history is what the BSC and many other concepts may lack (Velasco 2009). A number of different versions of PSC have been proposed over the years.

In the Autapomorphic Species Concept species are defined by at least one autapomorphy, or unique diagnostic character (Donoghue 1985; Mishler 1985; Mishler & Brandon 1987; de

Queiroz & Donoghue 1988). Diagnostability Species Concept defines a species as the smallest aggregation of populations or lineages diagnosable by a unique combination of character states in comparable individuals (Nixon & Wheeler 1990), a polytypic species concept as opposed to the monotypic concept of the Autapomorphic Species Concept. The Genealogical Species

Concept defines species by basal exclusivity; individuals of a species are more closely related to one another than to any individuals outside the group (Baum and Shaw 1995), though this can be difficult and laborious to demonstrate, while the Internodal Species Concept is based on the notion that species exist between two branching points in a lineage (Kornet 1993).

The Phenetic Species Concept defines taxa as categories of clusters that are outwardly discrete, with a species being the smallest group that is consistently distinct (Andersson 1990).

More commonly known as the Morphological Species Concept, or Type Species Concept, and it is the most common basis for a practical taxonomy. This concept is purely operational, basing

10 taxa entirely on empirically demonstrable facts (Andersson 1990), which can then be studied using more elaborate techniques to show various degrees of relatedness.

In practice, a species can be defined as samples that two biologists can distinguish and agree upon and communicate to others how they can be diagnosed (Nelson & Platnick, 1981). In this study, a species will be recognized as a discrete unit in phenetic space separate from all other units in that space, based on the data set.

Current taxonomy (Myint 1966)

Myint (1966) presented the most recent taxonomic study of the genus Stylisma. He looked at 14 characters he thought showed differences among species; habit, stem, leaf width, leaf tip, villosity, length of peduncle, inflorescence, villosity of sepal, tip of sepal, margin of corolla, villosity of filament, fusion of stylar branches, length of stylar ranches, and fusion of cotyledonary petioles. He gave each character a value of 0, 0.5, or 1, with what he considered the least specialized condition represented by a 0. These are what he called his specialization values, but how he determined which character was more or less specialized is impossible to determine based on the information presented in his paper, that is, it is not falsifiable. These values were then transferred to a graph (Figure 1) which he stated indicated a probable evolutionary pattern for the genus, but again how the data produced the graph is difficult to determine (Myint 1966). The lack of molecular data and the lack of clarity in Myint’s study drove me to begin a study that looks more closely at this genus.

Conservation

Many of the taxa in this genus are considered rare, threatened or endangered. Stylisma abdita is endangered, S. aquatica is rare in North Carolina, S. humistrata is on the Tennessee threatened list and the Virginia watch list, S. patens ssp. angustifolia is on the North Carolina

11 watch list, S. pickeringii var. pattersonii is endangered in Illinois, and S. pickeringii var. pickeringii is threatened in Georgia, endangered in North Carolina and rare in South Carolina. A taxonomic revision could change the conservation status of members of this genus and help determine if the rare taxa are actually rare.

Objectives

The objectives of this study are to provide a taxonomic revision and phylogeny of Stylisma, using morphological and molecular characters. The genus was last revised by Myint (1966), before the advent of cladistics and phylogenetic theory. A lack of phylogenetically informative characters has plagued morphological phylogenetics, particularly in angiosperm systematics, with unresolved trees the norm when large numbers of terminal taxa are examined (Chandler and

Crisp 1998). Molecular systematics has helped mitigate this dilemma, by providing relatively large numbers of informative characters. Both morphological and molecular characters will be used to assess the taxa within Stylisma, as well as to examine the somewhat spurious species boundaries among several of its members.

Hypothesis

Species are scientific hypotheses regarding the existence of novel biological and evolutionary entities (Mayden 2002). They are presented on the basis of evidence that leads to theories that some populations are unique and form independent lineages relative to other populations (de

Queiroz 2005). The current taxonomy presented by Myint (1966) provides a testable hypothesis, with his phylogeny shown in Fig. 1. I present a null hypothesis that there will be no difference between the new phylogeny and the existing one; with the H1 being that there the new phylogeny will be vastly different from the original.

Figure 1. Proposed relationship scheme of Stylisma based on a distance matrix of specialization indices (Myint 1966). The abbreviations on each node correspond with the characters examined. Relevance of uppercase vs. lowercase was not presented clearly in the paper. A-habit, B-stem length, C-length to width ratio of the leaves, D-leaf tip, E-villosity of leaves, F-length of peduncle, G-Inflorescence, H-villosity of sepal, I-tip of sepal, J-margin of corolla, K-villosity of filament, L-fusion of stylar branches, M-length of stylar branches, N-fusion of cotyledonary petioles

13 There are many uncertainties in the current phylogeny and taxonomy, including that it was based on just a few characters, many of which are ambiguous, none of them were molecular, and all the characters are continuous variables, making the phylogeny phenetic rather than cladistic.

The origins of the specialization indices used to create the scheme of relationships in the Myint

(1966) study were not clearly defined leaving questions as to the validity of the results and making reproduction of the study impossible.

Methodology

Morphometric vs. molecular techniques

There is an ongoing controversy in plant systematics as to whether molecular or morphological data are better sources of phylogenetic information (Patterson et al. 1993; Baker et al. 1998; Bateman et al 2006). Even in the most recent journals you can find a variety of both molecular and morphological papers. I thought it would be most beneficial to the genus to look at both types of data and compare them.

The two data sets will not be analyzed together, rather, they will be compared, because in this instance the data are different, the molecular data are cladistic and more species level whereas the morphological data are phenetic and more population level.

Introduction to morphological methodology

The Linnaean system that is the basis for our current system of taxonomy was based largely on morphological features and has served taxonomy well for more than 300 years.

Classifications based on morphological characters are the most practical for most users who would not be able to identify species based solely on molecular techniques (Dunn 2003).

Phenetic characters have the disadvantage of being subject to both genetic and environmental factors, but the advantage of being readily recordable (Thorpe 1983).

14 Phenetic analysis, also known as numerical taxonomy, attempts to classify organisms based on morphological similarity. Phenetic analysis of the morphological characters was used to determine the distinctness of taxa within Stylisma. Phenetic techniques can be used to describe the pattern of relationships among taxa by ordination or cluster analysis (James and McCulloch

1990). Both ordination and clustering begin with the same data matrix of distances, but differ in their graphical output.

Ordination

Ordination is the reduction of a matrix of distances or similarities among the attributes or among the objects to one or a few dimensions (James and McCulloch 1990). These techniques combine large amounts of information into a few dimensions and are frequently used in taxonomy (Pimentel 1981; Chandler and Crisp 1998). Ordination is advantageous because it can identify several overlapping patterns, and does not apply hierarchical structure to the data

(Chandler and Crisp 1998). It can also independently determine whether variation is discreet or continuous, and objects are displayed in graphical space with the axes being gradients of combinations of the attributes (James and McCulloch 1990).

Principle component analysis (PCA) is one of the more widely used ordination methods because it makes use of all of the data from the matrix to determine the axes (Austin 1985). PCA is used wherever large and complicated multivariate data sets have to be reduced to a simpler form and has two primary aims: to reduce many variables to a smaller number of variables and to reveal patterns in the data. This technique is generally accurate for higher order groups, familial and above, though it is widely used at the species level (Chandler and Crisp 1998).

Multidimensional scaling (MDS) is a multivariate statistical technique that can be employed to visually represent similarities and hidden structure among data. MDS produces a spatial

15 representation using a configuration of points that correspond to each of the specimens (Stevens et al. 2006). There are three commonly used forms of MDS, metric (MMDS), non-metric

(NMDS), and hybrid (HMDS). MMDS is restricted by assuming a linear relationship, whereas

NMSD circumvents the linearity assumption because it is based on the rankings of distances between points, and is the less restrictive of the two models, while HMDS is a combination of the two models and is used for ecological rather than taxonomic purposes (Chandler and Crisp

1998). NMDS is considered a robust ordination method (Saldarriaga-Córdoba et al. 2009;

Pimentel 1981; Minchin 1987; Chandler and Crisp 1998).

Introduction to molecular methodology

Molecular phylogenetics is a field that expanded rapidly throughout the 1990’s giving systematists many tools to uncover patterns of relatedness, including microsatellites, restriction fragment length polymorphism (RFLP), and rapidly amplified polymorphic DNAs (RAPD), among many others (Jordan et al. 1996). Direct DNA sequencing is currently being widely used for phylogenetic reconstruction in botany, using mainly noncoding regions of chloroplast DNA and nuclear DNA for reconstructing phylogenies at lower taxonomic levels (Chandler et al.

2001). The polymerase chain reaction (PCR) is a commonly used tool to rapidly amplify the genomic region of choice and it provides major advantages, including the ability to select specific areas of the genome for analysis (Jordan et al. 1996). PCR produces large amounts of template DNA, usually negating the need for cloning (Semagn et al. 2006). When using PCR, different regions of the sequence can be selected based on the taxonomic level to be examined.

For example, coding regions like rbcL with relatively low variation are more useful when examining higher taxonomic levels, such as familial or ordinal ranks, while regions that are more

16 rapidly evolving, like trnT-K or rps16, are more useful at lower taxonomic levels, including intrafamilial relationships (Small et al. 1998).

The chloroplast genome

The chloroplast genome is a circular molecule ranging from c. 71 kb (kilo-bases) to c. 220 kb in higher plants (Palmer 1987). The smallest known chloroplast genome belongs to a non- photosynthetic root parasite in Orbanchaceae, Epifagus virginiana Barl (DePamphilis and

Palmer 1990), while the largest known chloroplast genome belongs to the green alga

Chlamydomonas moewusii Gerloff (Sugiura 1992). Many papers have been published with complete chloroplast genomes from various plant groups (e.g., Nicotiana tabacum L.; Sugiura

1992; Oryza sativa L.;Hiratsuka et al. 1989; Solanum bulbocastanum Dunal and S. lycopersicum

L.; Daniell et al. 2006; Pinus thunbergii Franco; Wakasugi et al. 1994; Zea mays L.;Maier et al.

1995; Marchantia polymorpha L.; Ohyama et al. 1986). This availability of information has made it easier to design primers to target specific regions of the genome and has lead to an abundance of commonly used regions and “universal” plant-specific primers, which presents the modern systematists with a dilemma: which region to use? I used data from Shaw et al. (2005) to choose the two regions used in this study. They performed an extensive study on 21 typically used chloroplast DNA regions, with Solanum L. used to represent Solanales. The trnT-K region, which includes the trnT-L intergenic spacer, was part of the most variable regions studied and rps16 also showed high variability.

For this reason I chose those two noncoding regions of chloroplast DNA, the trnT-L intergeneric spacer and the rps16 intron (Figure 2). Noncoding regions tend to be used in studies at lower taxanomic levels since they are under fewer functional constraints than coding regions and are able to evolve more rapidly than coding regions (Shaw et al. 2005). Taberlet (1991)

17 found the trnT-L region to be especially suitable for PCR amplification due to the highly conserved nature of transfer RNA genes and the large (several hundred base pairs) noncoding region. The rps16 intron is a group II intron flanked by the two rps16 exons and varies from 790 bp to 887 bp (Oxelman et al. 1997). Oxelman et al. (1997) found this intron to be useful at the family level and below. Group II introns direct and catalyze the splicing of the flanking exons

(Michel and Feral 1995).

I attempted to include ITS, a nuclear marker commonly used in genus level taxonomic studies, but I could not get it to amplify and was forced to abandon the work due to financial constraints.

18 trnL trnL trnT trnF 5′ Exon 3′ Exon

Spacer Intron Spacer

A C E B D F a

Figure 2. a.) The trnT-trnF region of the chloroplast DNA, including the trnT-L intergeneric. b.) The rps16 intron on the chloroplast DNA.

19 CHAPTER 2 MATERIALS AND METHODS

Morphological methods

Taxon sampling

Specimens were sampled and pressed in the field, with flowering material preserved in water, ethanol, and glycerol mixture to preserve features, according to the method of Chandler and

Crisp (1998). Thirty-two specimens were collected at the locations listed in Table 2. No measurements were taken until the specimens were dried to maintain consistency of measurements.

Vouchers of all specimens collected are housed in the David Siren Herbarium (WNC) with any replicates distributed to NCU. The relatively small number of field collected specimens was offset by a large number of herbarium specimens, allowing for a more accurate presentation of the variation of each taxon, as well as a larger geographic range. Each of the herbarium specimens, approximately 400, was rekeyed using both Myint’s key and the more recent key presented by Weakley (Myint 1966; Weakley 2007).

Morphological measurements

The study used the morphology of herbarium specimens and preserved flowers. The number of specimens assessed depended on the availability of material and the extent of the morphological variation of each species. Table 2 lists the specimens studied and collection sites.

Table 3 shows the character states that were measured for the flowering and herbarium specimens. The herbarium specimens were on loan from Florida State University (FLAS),

University of Southern Florida (USF), Rutgers University (CHRB), Kansas State University

(KANU), University of North Carolina (UNC), and The David J. Siren Herbarium (WNC).

20 Table 2. Specimens of Stylisma studied and the states and counties where they were found. Locations I collected from are marked with an asterisk.

Taxa Total number Location S. abdita 49 specimens FL: Clay, Marion, Lake, Citrus, Orange Polk, Highlands*, Collier S. aquatica 35 specimens FL: Santa Rosa, Washington, Jackson, Calhoun NC: Robeson, Scotland*, Moore AL: Mobile, Baldwin MS: Jackson, Perry LA: Union, Morehouse, Allen, Beauregard, Calcasieu SC: Clarendon, Williamsburg, Orangeburg S. humistrata 86 specimens Fl: Walton, Jackson, Gulf, Franklin, Madison, Lafayette, Dixie, Union, Hernando VA: Southampton, Isle of Wight, New Kent, Nansemond LA: Claiborne, Union, Ouachita, Winn*, Sabine, Grant*, Rapides, Tangipahoa GA : Decatur, Lincoln, Screven AK : Ashley, Union, Nevada, Bradley, Ouachita MS: Clarke, Forrest, Stone, Harrison NC: Brunswick*, New Hanover*, Pender*, Duplin, Sampson, Robeson*, Hoke, Richmond, Cleveland, Wayne, Greene, Pitt, Pamlico, Edgecomb, Gates, Northampton AL: Marengo, Clarke, Escambia, Geneva, Lee SC: Jasper, Beaufort, Charleston, Berkeley, Georgetown, Abbeville, Anderson, Cherokee, York S. patens var. 48 specimens FL: Okaloosa*, Calhoun, Liberty, Gadsden, Jefferson, Lafayette, Polk, patens Highlands*, Walton* SC: Aiken, Georgetown MS: Harrison NC: Brunswick*, New Hanover* S. patens 78 specimens NC : Scotland*, Carteret, Bladen, Harnett, Hoke, Moore, Richmond, var.angustifolia Brunswick*, Gadston, New Hanover GA: Brooks, Chatham, Bulloch MS: Forrest SC: Lee, Kershaw, Edgefield, Clarendon, Lexington FL: Gilcrist, Pinellas, Hernando, Sumter, Polk, Osceola, Leon, Tayler, Hillsborough, Lake, Citrus, Marion*, Putnam, Clay*, Duval S. pickeringii 28 specimens NC : New Hanover, Bladen, Scotland*, Cumberland ssp. pickeringii NJ: Atlantic, Burlington, Camden, Ocean TX: Angelina S. pickeringii 35 specimens LA : Bienville, Winn, Natchitoches, Grant ssp. patersonii KS : Rice, Stafford, Reno AR : Ouachita IL: Cass OK: Grant, Kingfisher S. villosa 71 specimens FL : Escambia, Okaloosa, Walton, Bay, Gulf, Suwanwee, Nassau, Alachua, Marion, Lake, Orange, Hillsborough, Polk*, Osceola, Brevard, Manatee, Desoto, Highlands*, Martin, Broward, Miami-Dade Total studied: 430 specimens

21 Table 3. Morphological characters of Stylisma spp. measured from field and herbarium specimens, characters with an asterisk were also measured during flower dissections. Character states: Leaf length Bracteole length Leaf width Bracteole width Leaf shape (ratio) Bracteole shape (ratio) Leaf thickness Length of sepals* Leaf pubescence (villous, glabarous) Sepal tip* Leaf tip (acute, acuminate, obtuse) Sepal pubescence* Leaf base (cordate, cuneate) Length of corolla* Mucro Corolla color (white, pink)* Petiole Corolla pubescence* Stem width Corolla margin* Stem pubescence Lobe depth* Stem habit Pubescence of filament* Internode Anther length* Type of fruit (berry capsule) Fusion of stylar branches* Length of fruit Length of stylar branches* Width of fruit Length of style* Number of seeds Pedicel length Bract length Peduncle length Bract width Type of inflorescence Bract shape (ratio) Number of flowers per inflorescence

22 I examined 433 pressed specimens and dissected 33 flowers from across the geographic distribution of the taxa. Each specimen was keyed prior to making measurements because members of this genus are often incorrectly keyed. I re-determined approximately 40 specimens and there were 20-30 specimens that could not be identified based on the information provided in the specimens. The characters used to key Stylisma are mostly continuous variables that could be determined incorrectly by a botanist that is inexperienced or in a hurry (you really have to look at multiple areas of the specimen to accurately determine the identification).

The quantitative character states were measured to 0.1 mm using a vernier caliper. The leaf, bract, and bracteole lengths were measured from the base of the leaf to the tip. When applicable they included the mucro, but not the petiole as these were measured separately (Fig. 3b). Leaf width was measured at the widest point as was the leaf thickness. The stem width and internode length were measured near the middle of the plant. The corolla was measured from the end of the pedicel to the tip of the corolla (Fig. 3a). The qualitative character states examined were assigned a number from 0 to 7 so they could be analyzed in a statistical matrix (Table 4). These numbers were not assigned based on any evolutionary assumptions, i.e., they are unweighted and unordered.

Morphological data analysis

Phenetic analysis of the morphological characters was used to analyze relationships within the genus. The data were analyzed using PC-ORD, 5.16 (McCune and Mefford 2006).

Both PCA and NMDS were used with a variety of different matrices. NMDS is thought to be a more robust technique for infrageneric studies (see Chandler and Crisp 1998 for a discussion), but in this case did not provide as much information as PCA.

Figure 3. Stylisma flower and leaf. A) This figure shows the unique pattern of pubescence on the corolla. The vertical line represents the points of measurement of the corolla. B) The vertical line represents the point measurements for leaves, bracts, and bractioles.

24 Table 4. The qualitative characters and numbers assigned to the character states of Stylisma specimens. Qualitative Characters character states assigned value

Shape (leaf, bract, bractiole) very narrowly elliptic 0 narrowly elliptic 1 elliptic 2 pubesence glabarous 0 sparsely villous 1 villous 2 wooly 3 tomentose 4 velutinous 5 ciliate 6 pilose 7 leaf tip acute 0 emarginate 1 apiculate 2 obtuse 3 obcordate 4 accuminate 5 leaf apex cuneate 0 slightly cordate 1 rounded 2 habit trailing 0 prostrate 1 twining 2 color white 0 pink/purple 1 corolla margin lobed 0 entire 1 filament pubesence glabarous 0 at base 1 1/2 villous 2 3/4 villous 3 all villous 4 Inflorescence solitary 0 cyme 1 dichasial cyme 2 Presence yes 0 no 1

25 PCA also provided more information in the form of character vectors. Four matrices were examined, looking at different characters and axes. Table 5 is a key to the character abbreviations used. The data were first split into three different matrices to account for missing data. There was missing data because specimens were in different stages of reproduction, damaged, or badly mounted.

Molecular methods

Taxon sampling

Specimens were sampled from across their geographic range (Table 2). All specimens were pressed in the field and young leaf material was preserved in silica gel for molecular studies. No

S. pickeringii var. pattersonii were obtained in the field, so it was necessary to obtain the molecular data from herbarium material. Leaf material was taken from the most recently collected specimens from Kansas State University (KANU), and University of North Carolina

(NCU), these being collected in 1997, 1998, and 2001. Vouchers of all specimens collected are housed in the David Siren Herbarium (WNC) with any replicates distributed to NCU.

DNA extraction, amplification, and sequencing

Total DNA was isolated from silica dried leaves or herbarium specimens using the DNeasy

Plant Mini Kit (Qiagen, Valencia, California). DNA was extracted from 35 specimens; the locations for those that were sequenced are presented in Table 6. The DNeasy protocol was followed except the plant tissue was broken down using liquid nitrogen prior to the DNA extraction. Both the trnT-L and the rps16 region of the chloroplast genome were then amplified by polymerase chain reaction (PCR) using Taq DNA polymerase. The PCR samples were heated to 94°C for 3 minutes prior to the addition of DNA polymerase to denature unwanted proteases and nucleases.

26 Table 5. Key to the morphological abbreviations used in the PCA.

Character Abbreviation

Leaf length LL Leaf width LW Leaf ratio LR Petiole length PL Stem Width SW internode INT Bractiole length BL Length of sepals LS Peduncle length PL # of flowers per inflorescence FLS leaf shape LfS leaf pubescence LfP leaf tip LfT leaf base LfB Mucro MU Stem pubescence SP Sepal tip ST Sepal pubescence STP Bractiole shape BS Bractiole width BW Bractiole ratio BR corolla color CC length of corolla LC corolla pubescence CP Length of SB LSB Length of style LST Fusion of stylar branches FUS Pubescence of filament PF Anther length LA

Table 6. The Genbank accession number and voucher information for the specimens sequenced. Sequences from Ipomoea purpurea were obtained from a whole chloroplast genome. Taxon Voucher trnT-L Rps16 Location information

Ipomoea purpurea - EU118126 EU118126 -

Stylisma abdita SJH 107 WNC TBA TBA Fl: Highlands Co.

Stylisma abdita SJH 108 WNC TBA TBA Fl: Highlands Co.

Stylisma aquatica SJH 128a WNC TBA TBA NC: Scotland Co.

Stylisma aquatica SJH 128d WNC TBA TBA NC: Scotland Co.

Stylisma villosa SJH 105 WNC TBA TBA Fl: Highlands Co.

Stylisma villosa SJH 109 WNC TBA TBA Fl: Polk Co.

Stylisma patens ssp SJH 121 WNC TBA TBA NC: Brunswick Co. angustifolia Stylisma patens ssp SJH 102 WNC TBA TBA FL; Marion Co. angustifolia Stylisma patens ssp SJH 132 WNC TBA TBA NC: Scotland Co. angustifolia Stylisma patens SJH 112 WNC TBA TBA FL: Walton Co.

Stylisma patens SJH 127a WNC TBA TBA NC: Robeson Co.

Stylisma patens SJH 123 WNC TBA TBA NC: Brunswick Co.

Stylisma pickeringii KANU TBA TBA KS: Reno Co. var. pattersonii 00336255

Stylisma pickeringii NCU TBA TBA MS: Perry Co. var. pattersonii 325953

Stylisma pickeringii SJH 133 WNC TBA TBA NC: Scotland Co.

28 The double-stranded PCR products were produced via 30 cycles of denaturation (94°C for 1 minute), primer annealing (48°C for 1 minute), and extension (72°C for 1 minute). A seven minute final extension cycle at 72°C followed the 30th cycle to ensure the completion of all strands.

Taberlet et al.’s (1991) forward a and reverse b primers were used to both amplify and sequence the trnT-L region, while the rps16 intron was amplified using the rps16 forward and rps162 reverse primers (Table 7).

The PCR reaction mixture consisted of 33.75µl of sterile water, 5µl of 10x reaction buffer,

3µl of 40 mmol dNTP solution, 2µl of each primer, 4µl of template DNA, and 0.25µl of Taq polymerase for a total volume of 50µl.

PCR products were sequenced by Macrogen Inc. (South Korea). Due to funding constraints I was only able to send 22 specimens to be sequenced. GenBank numbers for all taxa used in the molecular portion of this study and voucher specimen locations are in Table 7. Ipomoea purpurea sequences were obtained from Genbank (Table 7) and were used as a root for the phylogenetic trees.

Sequences were assembled using Sequencher™ 3.0 (Gene Codes Corporation, Ann Arbor

Michigan, USA), transferred to Clustal W (Larkin et al. 2007), then manually adjusted by eye to check for errors.

This was completed following the principles of noncoding sequence alignment presented by

Kelchner and Clark (1997) to allow for secondary structure in the DNA strand. Of the 22 specimens sequenced, four were not useable because of sequence noise or a lack of usable information.

29 Table 7. This is a list of primers and their references used in this study. Primer Primer sequence Reference rps16 (F) GTG GTA GAA AGC AAC GTG CGA CTT Oxelman et al. (1997) Rps162 (R) TCG GGA TCG AAC ATC AAT TGC AAC Oxelman et al. (1997) trnT-Fa (F) ACA AAT GCG ATG CTC TAA CC Talberlet et al. (1991) trnT-Fb (R) TCT ACC GAT TTC GCC ATA TC Talberlet et al. (1991)

Sequence data analysis

Sequence data were analyzed using parsimony and maximum likelihood in PAUP 4.0b10

(Swofford, 2002), on a Macintosh OS 9.2 computer. The data matrix contained 19 total taxa, 18 representing the ingroup and one outgroup. Phylogenetic construction was performed on unweighted characters by a heuristic maximum parsimony search with the maximum number of trees set at 1000 and by maximum likelihood analysis (ML). The initial tree was created by random edition sequence with 1000 random initial starts. The large number of maximum trees was to avoid the island problem, however, I found only one island consisting of three equally parsimonious trees. The two regions were analyzed separately and together. The resulting trees were tested using bootstrapping with 1000 replicates. ModelTest (Posada and Crandall 1998) was used to fit the best model of DNA evolution for ML analysis. To assess confidence of the clades, bootstrap analysis (BS; Felsenstein 1985) was performed using 1000 replicates of full heuristic searches.

CHAPTER 3 RESULTS

Morphological results

Although PCA is generally less useful for more closely related taxa it proved to be the best option for the data I collected, because NMDS did not provide clear separation of my samples.

Four matrices were examined, each looking at different characters and axes. The data were first split into three different matrices to account for missing data. There was missing data because specimens were in different stages of reproduction, damaged, or badly mounted. The first matrix consisted of 304 samples and 24 characters, the second consisted of 160 samples and 25 characters and the third consisted of 67 samples and 27 characters (Table 8). A fourth matrix was created after conducting a multivariate regression to account for missing data, that included both categorical and continuous data combined (Table 8). The last matrix showed the highest resolution and used the most characters, so it is the one I chose to analyze. A graphical interpretation of the PCA results can be seen in Figure 4. There are three significant axes and three major groupings (Group I, II, and III). The first three axes account for 50% of the variance in the PCA with values of 24.7%, 14% and 11.2%. Table 9 shows the eigenvalues for 10 of the principal components along with individual and cumulative percent of the variance explained.

Group I, the ‘abdita’ group, is the most distinct from the rest of the data and contains all of the S. abdita samples. Group II, the ‘pickeringii’ group, consists of S. pickeringii var. pickeringii and

S. pickeringii var. pattersonii, while Group III, the ‘patens’ group, contains S. patens ssp. patens,

S. patens ssp. angustifolia, S. aquatica, S. humistrata, and S. villosa. Group III is cluttered and complex, with no species boundaries discernable from the data.

Table 8. The characters used in the four PCA ordination matrices and the number of samples in each matrix. Number of Matrix Number of characters Characters samples

LL, LW, LR, PL, SW, INT, BL, 1 304 24 LS, PL, FLS, LfS, LfP, LfT, LfB, MU, SP, ST, STP, BS, BW, BR, CC,LC, CP

2 160 25 Above characters and FUS

3 67 27 Above characters, PF, and LA

4 304 27 Matrix 1, LSB, LST, and LA

Table 9. The variance and eigenvalues for the first 10 principal component axes, which account for 80% of the total morphological variation. Cumulative % Axis Eigenvalue % variance variance

1 6.434 24.744 24.744 2 3.639 13.996 38.741 3 2.895 11.135 49.875 4 1.810 6.963 56.839 5 1.614 6.208 63.047 6 1.160 4.463 67.510 7 0.966 3.717 71.227 8 0.875 3.366 74.593 9 0.759 2.920 77.512 10 0.743 2.859 80.371

Based on the indistinct clustering (Figure 4) I decided to look more in depth at the PCA output. I plotted each axis (up to number six) against each other axis and looked at 3D representations of the first six axes. The graphs that show notable variation are presented here

(Fig. 5-15) while the remainder are presented as Appendix A.

Figure 4 shows the main three groups. Figure 5 is a three dimensional representation of the 3 strongest axes. It shows that S. abdita is by far the most distinct group. Figure 6 shows axes 1 v

3. In this graph S. patens spp. angustifolia and S. humistrata seem to be very distinct, but there is still a central area with some overlap. Figures 7, 8, 9, and 10 show a separation of S. aquatica from the rest of Group III. Figure 11 shows axis 2 and 3. In this figure Group II is combined, but Group I is still distinct. Figures 12-15 show Group III with a variety of different axes, as an attempt to tease out the taxa within the group.

The axes represent a combination of traits (see Table 10 for the correlations with the axes).

Axis one is strongly correlated with leaf width, leaf ratio (negative correlation), bracteole length

(negative correlation), and the length of stylar branches. Axis two is strongly correlated with leaf length, stem width, bracteole ratio, and length of anther. Axis three is correlated with the number of flowers per inflorescence, length of corolla (negative correlation), and the length of sepals (negative correlation).

Axis four is correlated with sepal tip, corolla color, and bracteole shape (negative correlation). Axis five is correlated with leaf base, length of sepal, and corolla pubescence, all of which are negative correlations. Axis six is correlated with sepal tip (negative correlation), corolla pubescence (negative correlation), and leaf tip.

Figure 4. The visual output of the PCA analysis showing axis 2 vs. 1. Group I is depicted in the red circle, Group II is in the green circle, and Group III is in the blue circle. The colored circles were hand drawn. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica- yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

Figure 5. The 3D output of the PCA analysis showing axis 1 v 2 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii- pink, S. pickeringii var. pattersonii-khaki.

Figure 6. The visual output of the PCA analysis showing axis 1 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

Figure 7. The 3D output of the PCA analysis showing axis 1 v 2 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii- pink, S. pickeringii var. pattersonii-khaki.

Figure 8. The visual output of the PCA analysis showing axis 1 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

Figure 9. The visual output of the PCA analysis showing axis 2 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

Figure 10. The visual output of the PCA analysis showing axis 3 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii- pink, S. pickeringii var. pattersonii-khaki.

Figure 11. The visual output of the PCA analysis showing axis 2 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii- pink, S. pickeringii var. pattersonii-khaki.

Figure 12. The visual output of the PCA analysis of Group III showing axis 1 v 2. Each taxa is represented by a color: S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green.

Figure 13. The3D output of the PCA analysis of Group III showing axis 1 v 2 v 3. Each taxa is represented by a color: S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green.

Figure 14. The visual output of the PCA analysis of Group III showing axis 1 v 3. Each taxa is represented by a color: S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green.

Figure 15. The 3D output of the PCA analysis of Group III showing axis 2 v 3 v 4. Each taxa is represented by a color: S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green.

Table 10. The first six eigenvectors, scaled to unit length. The strongest correlations are highlighted in bold and italicized. Eigenvectors Traits 1 2 3 4 5 6 LL 0.0040 0.4225 -0.0803 -0.0309 0.1541 -0.0098 LW 0.3036 0.0905 0.2072 -0.0586 0.2274 0.1747 LR -0.2717 0.2316 -0.2305 0.0411 0.0371 0.0272 PL 0.2607 0.2011 0.1337 -0.1114 0.1235 0.2166 SW -0.0703 0.3628 0.2023 -0.1249 0.0574 -0.1260 INT 0.2083 0.2010 0.0940 0.1202 0.0481 0.1113 BL -0.2740 0.2617 0.1108 -0.1697 -0.0760 -0.0952 LS 0.2214 -0.0093 -0.2717 -0.0636 -0.3289 -0.0689 FLS 0.0891 0.2207 0.3016 -0.0371 0.2523 0.0461 BW -0.1857 0.0033 0.1318 -0.4261 -0.0423 -0.2749 BR -0.1928 0.3043 0.0202 0.2048 -0.2046 0.2158 LC 0.2439 0.1134 -0.3421 -0.0178 -0.1426 -0.0469 LSB 0.3378 -0.0149 -0.1765 0.1513 0.0147 0.0627 LS 0.2576 0.2389 -0.2021 0.0061 -0.1430 -0.0511 L 0.0982 0.3240 -0.2049 -0.0603 0.1587 -0.1390 Lf 0.2655 -0.1114 0.2696 -0.0472 0.1626 0.1680 Lf -0.1793 0.1574 0.1797 0.2425 -0.0885 0.0867 LfT 0.1422 0.1189 0.1609 -0.0898 -0.2507 -0.5148 LfB 0.2326 -0.0716 0.1214 -0.1005 -0.3367 0.0275 MU -0.2387 -0.1105 -0.2578 0.0317 0.1957 0.2847 SP -0.0409 -0.0912 0.2431 0.2782 -0.1527 -0.1917 ST 0.1514 0.0279 -0.0578 0.3058 0.2481 -0.3397 STP 0.0017 -0.0170 -0.2476 0.0882 0.1913 -0.1876 BS 0.0560 -0.2786 0.0318 -0.3280 0.2633 -0.1425 CC 0.0098 -0.0706 0.2410 0.5193 -0.0636 -0.1410 CP 0.0511 0.0395 0.0948 -0.1753 -0.4139 0.3423

Figure 16 shows the character vectors from the PCA. Stem width is the longest vector and is strongly correlated with leaf length; this is logical because a larger stem can support a larger leaf.

Number of flowers per inflorescence, peduncle length, internode, leaf tip and leaf width are all correlated with each other and negatively correlated with mucro. Bractiole length, width and ratio are all correlated with each other and leaf pubescence, stem pubescence, and leaf ratio while they are negatively correlated with sepal tip, leaf shape, length of corolla, leaf base, and sepal length. Sepal tip, leaf shape, leaf base, and length of sepal are all positively correlated.

Leaf length is not correlated with leaf width and bracteole width. Leaf length is negatively correlated with bracteole shape and stem pubesence. Leaf tip, internode length, length of style, and peduncle length are all positively correlated, uncorrelated with mucro and stem pubescence, and not correlated with bracteole length, bracteole ratio, leaf ratio, and leaf pubescence.

Ideally, I would have also run a PCA that included only the characters that Myint (1966) used, but was unable to because of the large amount of missing data (even the regression technique mentioned above could not account for the amount of missing data). Once all of the missing data were excluded there was nothing left to analyze. Many of the characters used by

Myint were very tiny and required destructive sampling, which I was not able to do on a large scale, as many specimens had only one flower and it is unethical to disrupt such samples as it leaves nothing for future scientists to examine.

Molecular results

Sequence characteristics

Table 11 shows the statistics, including length variations, G/C content, and the number of informative characters.

Figure 16. The character vectors from the PCA. The key to the abbreviations can be seen in

Table 5.

Table 11. Sequence characteristics of the rps16 intron and the trnT-L intergeneric spacer sequenced in this study.

Sequence characteristics rps16 trnT-F Combined Length range (bp) 659-769 758-784 1506-1559

Length mean (bp) 754.29 765 1521.26

Aligned length (bp) 762 907 1667

G+C content 36% 25% 31%

Number of constant sites 744/762 (98%) 782/907 (86%) 1486/1667 (89%)

Number of variable sites 19/762 (2%) 99/907 (11%) 149/1667 (9%)

Number of parsimony informative sites 6/762 (1%) 26/907 (3%) 32/1667 (2%)

Number of indels 1 10 11

Combined sequence lengths varied from 1506 bp in Ipomoea purpurea to 1559 bp in S. abdita. The trnT-L spacer sequences ranged from 758 bp in S. villosa and S. aquatica to 784 bp in S. abdita and the rps16 intron ranged from 659 bp in S. pickeringii var. pattersonii to 769 bp in S. abdita.

G/C content in the combined matrix was 31% for all sequences, except S. pickeringii where it was 30%. In the trnT-L spacer G/C content was 25% in all sequences, except S. villosa and S. humistrata where it was 26%. In the rps16 intron G/C content ranged from 35% in S. pickeringii to 37% in S. pickeringii var. pattersonii. The transition to transversion ratio is 13:27. This ratio is within the expected range of 1:2.

There were a variety of indels in the sequence ranging from 2 to 28 bp. Most of these were synapomorphic among all Stylisma excluding I. purpurea. Stylisma villosa had an autapomorphic indel as did S. abdita and S. aquatica (see Table 11).

Phylogenetic reconstruction

A parsimony heuristic search of all nucleotide characters revealed three trees of 186 steps, with a consistency index (CI) of 1.00, a retention index (RI) of 1.00, and a rescaled consistency index (RC) of 1.00. Identical sequences were excluded with no change in CI or RI values. A strict consensus tree including the bootstrap values as well as the number of synapmorphies supporting each branch is presented as Figure 17. ModelTest chose the GTR + I model as the best fit for my data [rates (among site variation) = equal; base frequences = estimate; rate matrix

= estimate]. The same tree topology was found among all analyses, so only the maximum parsimony tree is presented.

Figure 17. Strict consensus tree of three trees. Tree length is 186 steps. CI=1.00, RI=1.00. The major clades are marked A-C. Bootstrap values are above the line and the number of synapomorphies are below in italics.

Major clades

Molecular data ascertained the three major clades shown in Figure 17, which are described below, all of which are strongly supported by bootstrap analysis.

Clade A, the ‘patens’ group contains S. patens, S. patens ssp. angustifolia, and S. humistrata

(BS = 96; 3 synapomorphies), though relationships among the members of this group remain unclear.

Clade B, the ‘villosa’ clade, contains S. villosa, S. aquatica, and S. abdita (BS = 74; 3 synapomorphies), though this clade also contains one of the S. pickeringii var. pattersonii specimens. This clade forms a polytomy, so that the relationships within the clade are not fully resolved.

Clade C, the ‘pickeringii’ clade contains S. pickeringii and S. pickeringii var. pattersonii (BS

= 79; 8 synapomorphies). This appears to be the most distinct clade with the two taxa separated by 13 synapomorphies

CHAPTER 4 DISCUSSION

Morphology

Ordination of principal component analysis results showed three distinct morphological groups within the genus Stylisma. Group I (the 'abdita' group) seems to be a distant outlier from the other two groups. The central part of group II (the 'pickeringii' group) is dissimilar to group

III (the 'patens' group), but there is some overlap in the outliers. Group I is strongly correlated with PCA axes one and two, Group II is correlated with axis two and Group III is not closely correlated with either axis. Group I is the most distant in multivariate space from the other two groups (see Figures 4 and 5). Figure 6 combines Groups I and II based on axis one and three, which is likely due to the lack of influence of bracteole characters that are key to discerning S. pickeringii from other members of the genus. The relationships of the taxa within Group III are much more difficult to discern. In Figures 5-15 the Group III cluster is explored further with a variety of axes and 3D images, but regardless of how it is presented there is no clear definition of taxa within this group. Figures 7-10 show S. aquatica being separated from the rest of Group III though there is still some overlap with the rest of the group. This only happens when axis four is used, with its strongest character being corolla color, which seems to be the driving force behind this separation. Color is a very unreliable character in plants; in fact only one or two genes may be responsible for determining flower color and shape (Gottleib 1984; Coen 1991; Coen and

Meyerowitz 1991), such that little genetic variation would be required to drastically alter floral morphology, undermining the conclusive separation of S. aquatica as a distinct species. Even though it is a categorical variable, it is weak in this analysis, further undermining S. aquatica.

Combining this with the fact that in each PCA there was still some overlap between S. aquatica

55 and the remainder of Group III leads me to believe that there should be no separation of S. aquatica.

In an attempt to further resolve Group III, Groups I and II were removed from the PCA. This removed variation attributable to Groups I and II, giving the taxa in Group III more room to separate on the ordinations, which should resolve any groupings that were being hidden by the tight cluster formed when all of the data were included in the PCA. The same six axes were plotted and the most informative ordinations were presented here (Figs. 12-15) and the rest appear in Appendix I. Figures 12-15 show that there are no distinct clusters within Group III.

Some of the taxa clustered together (for example, S. patens ssp. angustifolia ands. humistrata), but there is always a large, central region of overlap in the group.

Most of the character vectors are correlated with PCA axis two, with leaf length, stem width, anther length and bracteole shape being the most correlated. Length of style, length of stylar branch, leaf width, mucro, bracteole width, and length of corolla are all correlated with axis one.

Much of the variation within Stylisma is based on vegetative characters rather than reproductive characters, as seen in the line vectors (Fig.16). Corolla color, corolla pubescence, and stem pubescence were very short vectors and explained little of the variation in the data.

Most of the characters with a strong effect were not the characters essential in species identification (Weakley 2007; Myint 1966). Sepal pubescence, corolla length, and corolla color were three of the key characters in both Myint and Weakley’s keys, but they were not exceptionally strong or highly correlated with any PCA axes. Bractiole length is one of the main characters in both keys and is one of the strongest characters used based on the size of the character vector. The reproductive characters are not the strongest, but they are all correlated

56 with each other, while the vegetative characters are the strongest, but are not necessarily correlated with each other. The characters that account for the most variation within the genus are: leaf length, stem width, bracteole length and ratio (shape), leaf ratio (shape), length of anther, length of style, peduncle length, length of stylar branches, and leaf base. Vegetative characters are generally less reliable in phylogenetic studies, because they are more variable based on environmental factors (Chandler and Crisp 1998); this is evident if we examine any plant species that display clinal variation along an altitudinal gradient.

Group I is the most divergent, but tightly clustered and contains only S. abdita, supporting this taxon as a distinct species. Group II contains S. pickeringii var. pickeringii and S. pickeringii var. pattersonii uniting them as a species, but within the clade there is little resolution which suggests that there is one variable species, rather than a species with subspecific taxa.

Group III shows that all of the taxa within the group are likely to represent one large, variable species.

The current taxonomy of Stylisma is based on obscure and difficult to discern morphological characters. As a result, identifications are often erroneous, as can be seen on many of the herbarium specimens that I examined. The lack of reliability of the vegetative characters that play such a strong role in the variation of this genus is reflected in its cryptic nature. Many of the specimens I used were keyed incorrectly and even more could fall into multiple species depending on how the key was interpreted. The PCA output shows many of these characters to be of little importance in explaining variation among the taxa, again pointing to the likely potential of there being fewer, more variable, species within the genus Stylisma than are currently recognized.

Molecular genetics

Clade A represents includes two species that show little to no variation between them in most of the specimens suggesting that S. patens and S. humsitrata are one species. There are between zero and two synapomorphies among the specimens in this group. This clade is strongly supported by a bootstrap value of 96.

Clade B is a polytomy, so the relationships among the taxa within this clade cannot be resolved, but the 6 synapomorphies separating S. abdita hints to the possibility of it being a different species. The clade is moderately supported by a bootstrap value of 74. Stylisma villosa is weakly supported as a clade, based on a bootstrap value of 67. The S. pickeringii var. pattersonii was sister to the S. villosa and it is discordant with the rest of the tree, and upon further examination of the specimen the identification is questionable. The specimen is a herbarium specimen not in flower, so many of the defining characters are missing. The main character that sets S. pickeringii apart from the rest of the genus is the size of the bractioles, with

S. pickeringii having bractioles longer than 1 cm and the rest of the group having bractioles shorter than 1 cm. This particular specimen had bractioles between 0.8 cm and 1.1 cm. Based on the lack of informative characters and the ambiguity of the characters that are present I suggest this specimen is either a hybrid or incorrectly identified; field identification of plants from that particular population is needed in the future. The specimen was collected from

Louisiana (Winn Parish) which is an area of geographical overlap for the genus; S. pickeringii var. pattersonii, S. humistrata, S. patens ssp. patens, S. villosa, and S. aquatica are all found in this area.

Clade C is the most differentiated clade within the genus, and definitively separates S. pickeringii from the rest of the group. The clade is supported by a bootstrap value of 79 and eight synapomorphies. The 13 synapomorphies separating the 2 varieties of S. pickeringii points to them possibly being two different species.

There were only 32 parsimony informative sites in the combined sequence data. This lack of informative characters is likely the cause for the lack of resolution in the phylogeny. The two regions of chloroplast DNA used have provided both informative and less informative results in other similar studies. The trnT-L intergenic spacer has proven to be a useful region for genus level studies (Nishikawa et al 2002; Hamzeh and Dayanandan 2004), but there has also been other instances where it has provided limited informative characters (Baumel et al. 2002; Helsen et al. 2009). The rps16 intron was successfully used in a generic level study by Wanntorp et al in 2002 and Haston et al. (2005). The rps16 region has been used much more frequently at the family and tribe level (Urophylleae, Smedmark et al 2008; Burseraceae, Clarkson et al 2002;

Rubioideae, Andersson and Rova 1999).

Although there are relatively few informative characters in the data set, the data I have are robust. Both the retention index and the consistency index resulted in values of 1.00 for all of the data, with 1.00 being the best possible value. Further analysis with other molecular markers needs to be undertaken to fully resolve the relationships within this genus. I suggest using the trnL-F chloroplast region as well as a nuclear marker, possibly ITS from nuclear DNA. It would be beneficial to expand the study to include Bonamia to determine the monophyly of Stylisma.

The tribe Cresseae that contains Stylisma is currently paraphyletic and could benefit from further

59 study as well. I suggest using the rps16 intron in the tribe level study; perhaps it would prove to be a better marker at that taxonomic level.

Conclusions

Stylisma is a small genus, within a family of large genera, that has been largely ignored by modern systematists. This study set out to increase our knowledge of the genus and its infrageneric taxa. The results indicate that Stylisma is a complex genus, and I was unable to discern all of the species boundaries set out by Myint (1966) by either phonetic or molecular analyses.

The morphological and molecular data agree on the placement of some taxa, specifically the separation of S. pickeringii from the remainder of the genus. The large number of synapomorphies separating S. abdita from the rest of Clade B and the morphological separation of the species into Group I is another point of agreement.

There is some disagreement between the two data sets, however. The morphological data show S. humistrata and S. patens to be in Group III, but forming separate clusters with some overlap, whereas the molecular data do not show any variation between S. humistrata and S. patens. The molecular data separates S. villosa and S. aquatica into a trichotomy with S. abdita in Clade B, whereas the morphological data includes them into Group III with S. patens and S. humistrata. These contradictory data could be due to lack of informative characters in the molecular data. A more informative molecular site could provide some differentiation between

S. humistrata and S. patens. The morphological data placed most of the specimens into Group

III, and despite looking at multiple topologies, no separation of the taxa in the group could be found.

Figure 18. The 3D visual output of the PCA analysis showing axis 1 v 2. The + symbols represent specimens that were used in the molecular study as well. Each taxa is represented by a color: S. abdita-khaki, S. aquatica-green, S. humistrata-pink, S. villosa- magenta, S. patens ssp. patens-red, S. patens ssp. angustifolia-sky blue, S. pickeringii var. pickeringii- purple, S. pickeringii var. pattersonii-yellow

Figure 18 shows the specimens that were used for both the morphological and molecular data on the PCA ordination. This figure shows that specimens used for the molecular analyses did not generally represent morphological extremes, although several of them are on the fringe of their color groups.

The lack of strength influence of the reproductive characters in the morphological data presents evidence that variation among the vegetative characters is most important in determining the species boundaries of Stylisma. These characters are not nearly as reliable as as reproductive characters, as they are more subject to variation in response to environmental factors (Chandler and Crisp 1998; Baker and Desalle 1998). The evolutionary pattern presented by Myint (1966; Fig. 2) has similarities to and differences from the results of this study (Fig. 3 and 5). In both studies, S. pickeringii is the most distantly related to the rest of the group.

Myint’s specialization indices placed S. pickeringii four to five steps away from any other species while parsimony placed it eight steps away, though how Myint’s “steps” compare to those of parsimony is unknown. Infraspecific data within S. pickeringii taxa are variable across each method. Myint (1966) had both subspecies being closely related (one step apart), but parsimony separates them by 13 steps, while PCA lumps them together with no discernable differences. Thus, there is a clear need for further molecular study, likely a population level molecular study, as the subspecies could represent cryptic morphological species that are genetically quite distantly related.

Myint (1966) suggested that S. abdita and S. aquatica are sister to each other, and that S. humistrata and S. villosa are also sister taxa. Phylogenetic analysis of the molecular data showed a polytomy between S. villosa, S. aquatica, and S. abdita, and it grouped S. humistrata

62 with S. patens. PCA separated S. abdita from the rest of the genus morphologically. Myint separated S. patens into infraspecific taxa based on two steps, as does parsimony analysis. In other words, Group III is particularly complex and may contain cryptic species. The presence of cryptic species presents the problem of proper identification which would have to be addressed for each species, but I suggest only separating species that can be field-identified.

This study provided new information on the relationships within the genus Stylisma, but without further study that includes Bonamia the monophyly of the group cannot be ascertained. I was unable to include Bonamia due to lack of funding and the distribution of this genus.

Bonamia is primarily found in tropical and south temperate regions, with only four species occurring in three states (Florida, Hawaii, and Texas), making sampling this genus impossible for this project. Currently the two genera are separated based on strong morphological and molecular data (House 1907; Lewis 1971; Myint 1966).

I do not feel that there are enough data presented in this study to make final taxonomic conclusions because of the lack of clear molecular data and disparities between the morphological and molecular analyses. Based on my data and analyses, I suggest that S. abdita and S. pickeringii remain separate species. I recommend combining S. pickeringii var. pickeringii and S. pickeringii var. pattersonii, based on the distinct cluster formed by these two varieties that was separated from the other taxa using the morphological data. The remaining taxa in the genus should be lumped together into one species, based on the lack of clear distinctions in their morphologies. Tentative descriptions of these taxa derived from the data collected in this study follow.

This change in taxonomy would result in some changes in the conservation status of the genus. Stylisma abdita would remain on the Florida endangered species list. There would also not be a change to the S. pickeringii taxa, they would remain on the Illinois endangered species list, South Carolina rare list, Georgia threatened list, and North Carolina endangered list. The combination of taxa into S. patens would remove S. patens spp. angustifolia from the North

Carolina watch list and S. aquatica from the North Carolina rare list. The resulting S. patens would be on the Virginia watch list and the Tennessee threatened list.

While I cannot ascertain how Myint (1966) calculated his specialization indices to develop his phylogeny, there are some similarities to the current molecular data as well as some strong disagreements. This study rejects Myint’s hypothesis of relationships among the taxa of

Stylisma, tentatively replacing it with the phylogeny uncovered using molecular data and the taxonomy presented here, which is based on morphological and molecular data sets. It is clear that further study is needed to fully resolve the phylogeny of Stylisma.

CHAPTER 5. DESCRIPTION OF PROPOSED TAXA

Stylisma abdita

Perennial, prostrate vines with densely pubescent stems. Internodes are short (2.4-9.4mm)

Leaves are narrowly linear-elliptic, sessile with an acute or obtuse apex and are densely villous with silvery grey or brown trichomes, with indistinct secondary veins and no mucro. Leaf length ranges from 5.7-12.5mm and width ranges from 0.9-2.8mm. Solitary flowers on short peduncles with a densely pubescent calyx and an unusually large ratio of calyx to corolla length (~1:2).

Corolla white, sepal woolly or densely villous.

Vernacular name: Hidden Dawnflower

Flowering period: April to November

Distribution: Figure 19a

Habitat: Xeric sand hill and scrub habitats

Affinity: This species is the most easily recognizable species in the genus, it can be distinguished from the rest by its small size and compact internodes.

Stylisma pickeringii

Prostrate, or trailing, and slightly pubescent. The leaves are sessile or nearly sessile (petiole

0-2.8mm) and linear and villous along the midrib which is the only discernible vein. Mucro is absent or tiny. Leaves are 5.4-65mm long and 0.8-3.5mm wide. Bractioles are long ranging from 4.5-24.5mm long. The inflorescences are cymes of 2-7 flowers with the central flower sessile, or solitary. The sepals generally are acute, occasionally obtuse and wooly to villous, the corollas are white. Styles are fused with branches ranging from 0.1-2.4mm.

Vernacular name: Pickering’s dawnflower

6565

Flowering period: May to August

Distribution: Figure 19b

Habitat: Dry barren sand hill and scrub habitats

Affinity: This group can easily be recognized by its growth pattern, it has numerous stems arising from a central point, forming a mound and often times by the length of bractioles, they have much longer leaf like bractioles than the rest of the genus.

Stylisma patens

The stems are twining or prostrate and thick, and glabrous to villous. The villous to wooly leaves are mostly elliptic to narrowly elliptic with petioles 0.8-7.8mm and range from 12-52mm long and 4-23mm wide. The leaves have a mucro that is often large. Inflorescences are solitary or cymes of 2-6 flowers Calyx is villous, glabrous, or ciliate, corolla is white or pink-purple.

Filament is villous at the base, entirely villous, or glabrous and styles are free or fused with the branches ranging from 3-11.7mm.

Vernacular name: Common dawnflower

Flowering period: April to September

Distribution: Figure 19c

Habitat: Dry sandy soils of sand hills, pinewoods, high pinelands, and occasionally on calcareous lands, Carolina Bays

Affinity: This is the most diverse Stylisma, having a wide range of morphological traits.

a b

Figure 19. Distribution of a.) S. abdita b.) S. pickeringii c.) S. patens

LITERATURE CITED Agapow P., O. R. P. Bininda-Emonds, K. A. Crandall, J. L. Gittleman, G. M. Mace, J. C. Marshall, A. Purvis. 2004. The impact of species concept on biodiversity studies. The Quarterly Review of Biology 79(2): 161-179. Andersson, L. 1990. The Driving Force: Species Concepts and Ecology. Taxon 39(3): 375-382. Andersson L. J. H. E. Rova 1999 The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Plant Systematics and Evolution 214: 161-186. Angiosperm Phylogeny Group. 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society 141: 399–436 Austin D. F. and J. N. Burch. 1992. Status of Stylisma abdita (Convolvulaceae) in Southwestern Florida. Florida Scientist 55(2): 99-102. Austin D.F. and G. W. Staples. 1985. Petrogenia as a synonym of Bonamia (Convolvulaceae), with comments on allied species. Brittonia 37(3): 310-316. Austin M. P. 1985. Continuum concept: ordination methods and niche theory. Annual Review of Ecology and Systematics 16: 113-148. Baker R. H., X. Yu, and R. DeSalle. 1998. Assessing the relative contribution of molecular and morphological characters in simultaneous analysis trees. Molecular Phylogenetics and Evolution 9(3): 427-436. Balakrishnan R. 2005. Species concepts, species boundaries and species identification: a view from the tropics. Systematic Biology 54(4): 689-693. Bates V. and P. Lewis. 1984. Rediscovery of Stylisma humistrata (Convolvulaceae) in Tennessee. Rhodora 86: 393-394. Baum B.R. 1992. Combining trees as a way of combining data sets for phylogenetic inference, and the desirability of combining gene trees. Taxon 41: 3-10. Baum, D. A. & K. L. Shaw. 1995. Genealogical perspectives on the species problem. Pp. 289- 303 in P. C. Hoch & A. G. Stephenson, eds. Molecular and experimental approaches to plant biosystematics. Missouri Botanical Garden, St. Louis. Bentham G. and J. D. Hooker. 1873. Genera Plantarum. , Lowell Reeve & Co, Williams & Norgate, London. Bock H. H. 1996. Probabilistic models in cluster analysis. Computational Statistics & Data Analysis 23: 5-28. Carcraft J. 2000. Species concepts in theoretical and applied biology: A systematic debate with consequences. In Species concepts and Phylogenetic theory. Q. D. Wheeler and R. Meiser, eds. Columbia University Press. Chandler G. T. and M. D. Crisp. 1998. Morphometric and phylogenetic analysis of the Daviesia ulicifolia complex (Fabaceae, Mirbelieae). Plant Systematics and Evolution 209: 93- 122. Chandler G. T., R. J. Bayer, and M. J. Crisp. 2001. A molecular phylogent of the endemic Australian genus Gastrolobium (Fabaceae: Mirbelieae) and allied genera using chloroplast and nuclear markers. American Journal of Botany 88(9): 1675-1687. Clarks, J. J., M. W. Chase, and M. M. Harley. 2002. Phylogenetic Relationships in Burseraceae Based on Plastid rps16 Intron Sequences. Kew Bulletin. 57(1): 183-193.

Coen, E. S. (1991). The role of homeotic genes in flower development and evolution. Annual Review of Ecology and Systematics 42: 241-279. Coen, E. S., and E. M. Meyerowitz. 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353: 31–37. Coyne J. A. 1994. Ernst Mayr and the Origin of Species. Evolution 48(1): 19-30. Daniell, H., S. Lee, J. Grevich, C. Saski, T. Quesada-Vargas, C. Guda, J. Tomknis, R. K. Jansen. 2006. Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes. Theory Applied Genetics 112: 1503-1518. de Queiroz, K., and M. J. Donoghue. 1988. Phylogenetic systematics and the species problem. Cladistics 4(4):317-338. De Candolle, A. P. 1845. Prodromus systematis naturalis regni vegetabilis, sive Enumeratio contracta ordinum generum Vol. 9. Sumptibus Sociorum Treuttel et Würtz. Donoghue, M. J. 1985. A Critique of the Biological Species Concept and Recommendations for a Phylogenetic Alternative. Bryologist 88(3): 172-181. Dunn C. P. 2003. Keeping taxonomy based in morphology. Trends in Ecology & Evolution 18(6): 270-271. Feliner G. N. and J. A. Rossello. 2006. Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants. Molecular Phylogenetics and Evolution 44: 911-919. Felsenstein J. 1985. Confidence limits on Phylogenies: an approach using the bootstrap. Evolution 39: 783-791. Fernald M. L. and B. G. Schubert. 1949. Some identities in Breweria. Rhodora 51: 35-43. Gottlieb, L. D. (1984). Genetics and morphological evolution in plants. American Naturalist 123: 681-709. Gray, A. 1856. Manual of the botany of the northern United States. Second ed. New York: Ivison & Phinney. Gray A. 1863. Manual of the botany of the northern United States. Fourth ed. New York: Ivison & Phinney. Gray A. 1878. Synoptic Flora of North America. New York: American Book Company. Hallier H. 1893. Versuch einer naturlichen Gliederung der Convolulaceen auf morphologischer und anatomischer Grundlage. Botanische Jahrbücher für Systematik 16: 453-591. Hamzeh, M. and S. Dayanandan. 2004. Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast TRNT-TRNF region and nuclear rDNA. American Journal of Botany. 91: 1398-1408. Haston, E. M., G. P. Lewis, J. A. Hawkins. 2005. A phylogenetic reappraisal of the Peltophorum group (Caesalpinieae: Leguminosae) based on the chloroplast trnL-F, rbcL and rps16 sequence data. American Journal of Botany. 92: 1359-1371. Helsen, P, R. A. Bowne, D. J. Anderson, P. Verdyck, and S. V. Dongen. 2009. Galápagos’ Opuntia (prickly pear) cacti: extensive morphological diversity, low genetic variability. Biological Journal of the Linnean Society. 96: 451–461.

Hiratsuka J., H. Shimada, R. F. Whittier, T. Ishibashi, M. Sakamoto, M. Mori, C. Kondo, Y. Honji, C. R. Sun, B. Y. Meng, Y. Li, A. Kanno, Y. Nishizawa, A. Hirai, K. Shinozaki, and M. Sugiura. 1989. The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Molecular and General Genetics 217: 185-194. House H. D. 1907. Studies in the North American Convolvulaceae. III. Calycobolus, Bonamia and Stylisma. Bulletin of the Torrey Botanical Club 34: 143-149. James F. C. and C. E. McCulloch. 1990. Multivariate analysis in ecology and systematics: panacea or Pandora’s box? Annual Review of Ecology and Systematics 21: 129-166. Jordan W. C., M. W. Courtney, and J. E. Neigel. 1996. Low levels of intraspecific genetic variation at rapidly evolving chloroplast DNA locus in North American duckweeds (Lemnaceae). American Journal of Botany 83: 430-439. Kelchner S. A. and L. G. Clark. 1997. Molecular evolution and phylogenetic utility of the chloroplast rpl16 intron in Chusquea and the Bambusoideae (Poaceae). Molecular Phylogenetics and Evolution 8 (3): 385-397. Kornet, D. 1993. Internodal species concept. Journal of Theorlogical Biology 104: 407-435. Lammars T. G. 1984. Stylisma pickeringii var. pattersonii (Fern &Schub.) Myint (Convolvulaceae) rediscovered in Iowa. Sida 10(3): 256-257. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. 2007. Clustal W and Clustal X version 2.0. Bioinformatics, 23: 2947-2948. Lewis W. H. 1971. Pollen differences between Stylisma and Bonamia (Convolvulaceae). Brittonia 23(4): 331-334. Maier, R. M., K. Neckermann, G. L. Iglio, and H. Kössel. 1995. Complete sequence of the maize chloroplast genome: gene content, hotspots of divergence and fine tuning of genetic information by transcript editing. Journal of Molecular Biology 251: 614-628. Mayden R. L. 2002. On biological species, species concepts and individuation in the natural world. Fish and Fisheries 3: 171-196. Mayr E. 2000. A critique from the Biological Species Concept perspective: What is a species and what is not. In Species concepts and Phylogenetic theory. Q. D. Wheeler and R. Meiser, eds. Columbia University Press. McCune, B. and M. J. Mefford. 2006. PC-ORD: Multivariate analysis of Ecological Data. Version 5 MjM Software. Gleneden Beach, OR, US Michel F. and J. Feral. 1995. Structure and activities of group II introns. Annual Review of Biochemistry 64: 435-461. Minchin P. R. 1987. An evaluation of the relative robustness of techniques of ordination. Vegetatio 69: 89-107. Mishler, B. D. 1985. The morphological, developmental, and phylogenetic basis of species concepts in bryophytes. Bryologist 88: 207-2l4.

Mishler, B. D., R. N. Brandon. 1987. Individuality, pluralism, and the phylogenetic species concept. Biology and Philosophy 2: 397-414. Myint T. 1966. Revision of the genus Stylisma (Convolvulaceae). Brittonia 18: 97-117.

Nelson G. and N. Platnick. Systematics and biogeography: cladistics and vicariance. New York: Columbia University Press, 1981. Nishikawa, T., K. Okazaki, and T. Nagamine. 2002. Phylogenetic Relationships among Lilium auratum Lindley, L. auratum var. platyphyllum Baker and L. rubellum Baker Based on Three Spacer Regions in Chloroplast DNA. Breeding Science. 52: 207-213. Nixon, K. and Q. Wheeler. 1990. An amplification of the phylogenetic species concept. Cladistics 6:211–223. Ohyama K., H. Fukuzawa, T. Kohchi, H. Shiria, T. Sano, S. Sano, K. Umesono, Y. Shiki, M. Takeuchi, Z. Chang, S. I. Aota, H. Inokuchi, and H. Ozwki. 1986.Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572-574. Olmstead R. G., B. Bremer, K. M. Scott, and J. D. Palmer. 1993. A parsimony of the Asteridae sensu Lato based on rbcL sequences. Annuals of the Missouri Botanical Garden press 80 (3):700-722. Oxelman B., M. Lidén, and D. Berglund. 1997. Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Systematics and Evolution 206: 393-410. Palmer J. D. 1987. Chloroplast DNA evolution and biosystematic uses of chloroplast DNA variation. American Naturalist 130: 6-29. Patterson C., D. M. Williams, and C. J. Humphries. 1993. Congruence between molecular and morphological phylogenies. Annual Review of Ecology and Systematics 24: 153-188. Peter A. 1897. Convolvulaceae. In: Natürlichen Pflanzenfamilien IVa:25~31 ed.by A. Engler & Prantl, K. Pimentel R. A. 1981. A comparative study of data and ordination techniques based on a hybrid swarm of sand Verbenas (Abronia Juss.). Systematic Zoology 30: 250-267. Posada D. and K. A. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics (Oxford, England) 14: 817-818. Queiroz K. D. 2005. Different species problems and their resolution. Bioessays 27: 1263-1269. Rosen D. E. 1978. Vicariant Patterns and Historical Explanation in Biogeography Systematic Zoology. 27(2):159-188. Salatino A., M. L. F. Salatino, R. D. Mello-Silva, M. V. Sluys, D. E. Giannasi, and R. A. Price. 2001. Phylogenetic inference in Velloziaceae using chloroplast TrnL-F sequences. Systematic Botany 26(1): 92-103. Saldarriaga-Córdoba M. M., M. Sasa. R. Pardo. M. A. Méndez. Phenotypic differences in a cryptic predator: Factors influencing morphological variation in the terciopelo Bothrops asper (Garman, 1884; Serpentes: Viperidae). Toxicon 54: 923-937. Shaw J., E. B. Lickey, J. T. Beck, S. B. Farmer, W. Liu, J. Miller, K. C. Siripun, C. T. Winder, E. E. Schilling, and R. L. Small. 2005. The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. American Journal of Botany 92 (1): 142-166. Semagn K., A. Bjørnstad, and M. N. Ndjiondjop. 2006. An overview of molecular marker methods for plants. African Journal of Biotechnology 5(25): 2540-2568.

Small R. L., J. A. Ryburn, R. C. Cronn, T. Seelanan, and J. F. Wendel. 1998. The tortoise and the hare: choosing between noncoding plastome and nuclear ADH sequences for phylogeny reconstruction in a recently diverged plant group. American Journal of Botany 85: 1301-1315. Smedmark, J. E., E. Rydin, C. Razafimandimbison, S. G. Khan, S. A. Liede-Schumann, S. Bremer. 2008. A phylogeny of Urophylleae (Rubiaceae) based on rps16 intron data Taxon. 57(1): 24-32. Soltis D. E., E. V. Marvrodiev, J. J. Doyle, J. Rauscher, and P. S. Soltis. 2008. ITS and ETS sequence data and phylogeny reconstruction in allopolyploids and hybrids. Systematic Botany 33(1): 7-20. Stearn W. T. Botanical Latin. Fourth Edition. Oregon: Timber Press, Inc., 1995. Stefanovic S., D. Austin, and R. Olmstead. 2003. Classification of Convolvulaceae: a phylogenetic approach. Systematic Botany 28(4):791-806. Stevens D. A., R. F. Smith, H. T. Lawless. 2006. Multidimensional scaling of ferrous sulfate and basic tastes. Physiology & Behavior 87: 272-279. Sugiura M. 1992. The Chloroplast Genome. Plant Molecular Biology 19: 149-168. Swofford D. 2002. PAUP: phylogenetic analysis using parsimony, pre-release version 4.0b10. Laboratory of Molecular Systematics, Smithsonian Institution, Washington, D.C., and Sinauer, Sunderland, Massachusetts, USA. Taberlet P., L.Gielly, G. Pautou, and J. Bouvet. 1991. Universal primers for amplification of free noncoding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109. Templeton A. R. 1989. The meaning of species and speciation: a genetic perspective. 3–27 in D. Otte and J. A. Endler, eds. Speciation and its consequences. Sinauer Associates, Sunderland, MA. Thorpe R. S. 1983. A review of numerical methods for recognizing and analyzing racial differentiation. In Felsenstein, J., (Eds): Numerical taxonomy, pp. 404-423. Berlin, Heidelberg, New York: Springer. Velasco J. D. 2009. When monophyly is not enough: exclusivity as the key to defining a phylogenetic species concept. Biology and Philosophy 24: 473-486. Wakasugi T., J. Tsudzuki, S. Ito, K. Nakashima, T. Tsudzuki, and M. Sugiura. 1994. Loss of all ndh genes as determined by sequencing the entire chloroplast genome of the black pine Pinus thunbergii. Proceedings of the National Academy of Sciences of the USA 91: 9794-9798. Wanntorp, L., H. E. Wanntorp, and M. Kallersjo. 2002. Phylogenetic relationships of Gunnera based on nuclear ribosomal DNA ITS region, rbcL and rps16 Intron. Systematic Botany. 27(3): 512-521. Weakley A. S. 2007. Flora of the Carolina’s, Virginia, Georgia, and Surrounding Areas. UNC Press, Chapel Hill. Wilson K. A. 1960. The genera of Convolvulaceae in the Southeastern United States. Journal of the Arnold Arboretum 41: 298-317.

72 APPENDIX A

The 3D output of the PCA analysis showing axis 1 v 2 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 1 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 1 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 2 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 2 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 3 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 3 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 4 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 4 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis showing axis 5 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The 3D output of the PCA analysis of Group III showing axis 1 v 2 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The 3D output of the PCA analysis of Group III showing axis 1 v 2 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 1 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 1 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 1 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 2 v 3. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 2 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 2 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 2 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 3 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The 3D output of the PCA analysis of Group III showing axis 2 v 3 v 4. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 3 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 3 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 4 v 5. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 4 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.

The visual output of the PCA analysis of Group III showing axis 5 v 6. Each taxa is represented by a color: Stylisma abdita-dark blue, S. aquatica-yellow, S. humistrata-sky blue, S. villosa- purple, S. patens ssp. patens-red, S. patens ssp. angustifolia-green, S. pickeringii var. pickeringii-pink, S. pickeringii var. pattersonii-khaki.