Molecular Phylogenetics and Breeding System Evolution of the Turneraceae

Paul D.J. Chafe

A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO

NOVEMBER 2009

Abstract

This study examined the phylogenetic relationships among the species of the heterostylous plant family the Turneraceae, with a focus on a single genus, Piriqueta. It includes the first phylogenetic analysis of species from Old World species in the Turneraceae. Using combined ITS and ndhF sequence data several phylogenies were constructed. The sequence data generated resolved the family quite well, and was successful even at the sub generic level.

The family was found to be composed of three or four clades, depending on the sequence data included and the type of analysis performed. These clades are the genus Turnera, the genus

Piriqueta, a clade of mainland African species, and a clade composed of disjunct island endemics and Erblichia odorata, a Central American species. The results of this study indicate that there have been numerous breakdowns of heterostyly in the Turneraceae. Finally, I discuss intercontinental disjunctions and identify species whose historical taxonomic treatment requires revision based on the phylogenies constructed.

Acknowledgments I would like to take this opportunity to thank Dr. Joel Shore for allowing me the opportunity to further my education. This work would not have been completed without his guidance, patience, and advice. I would also like to thank my parents, brothers, and girlfriend for their support and for pushing me to complete this work. To my lab mates Jonathan, Darya, and Henry, thank you for the interesting discussions on all topics scientific or otherwise. Thanks also to my fellow graduate students for help in coursework, lab work, and life. Finally, thanks to Dr. Laurence Packer for his help in my yearly progress reports.

Table of Contents:

Abstract ...... iv

Acknowledgments...... v

List of Tables ...... ix

List of Figures ...... x

List of Abbreviations ...... xi

List of Appendices ...... xii

Glossary ...... xiii

1.0 Introduction ...... 1

1.1 Historical Context ...... 2

1.2 The Turneraceae ...... 8

1.2.1 Historical Classification of the Turneraceae ...... 9

1.3 Intercontinental Disjunctions...... 14

1.4 Molecular Phylogenetics ...... 15

1.4.1 Maximum likelihood ...... 17

1.4.2 Parsimony ...... 18

1.4.4 New Technology ...... 20

1.5 The nuclear ribosomal intergenic spacer (ITS) ...... 21

1.6 The cpDNA ndhF gene ...... 25

1.7 Molecular Phylogenetics in heterostylous families ...... 27

1.8 Objectives of the Thesis ...... 32

2.0 Materials and Methods: ...... 33

2.1 Plant Material ...... 33

2.2 DNA Extraction ...... 33

2.3 Primer Design ...... 35

2.4 Polymerase Chain Reaction ...... 35

2.5 Agarose Gel Electrophoresis ...... 36

2.6 Molecular Cloning ...... 37

2.6.1 Cloning of PCR Product ...... 37

2.6.2 Plasmid Isolation ...... 37

2.7 DNA Sequencing ...... 39

2.8 DNA Sequence Analysis...... 39

2.9 Phylogenetic Analyses ...... 40

2.10 Mapping Breeding System ...... 41

3.0 Results ...... 42

3.1 Amplification of ndhF and ITS ...... 49

3.2 Sequence Variation ...... 55

3.3 Phylogenetic Analyses ...... 55

3.31 Piriqueta ...... 56

3.32 All Genera of Turneraceae ...... 61

3.4 Breeding System Evolution ...... 64

4.0 Discussion...... 78

4.1 Phylogenetic Analyses ...... 79

4.12 Evolutionary relationships among genera ...... 79

4.13 Evolution of African Lineage ...... 80

4.16 Evolution of Island Endemics ...... 84

4.2 South American and African Disjunctions ...... 86

vii

4.4 Evolution of Breeding System ...... 89

4.4 Conclusions ...... 92

References: ...... 95

Appendices:...... I

viii

List of Tables

1. The distribution of breeding system in the Turneraceae. 43 2. List of taxa, collection number, and DNA sequences 44 successfully amplified 3. Species name, breeding system, and tissue DNA was 47 extracted from, for all species in the current study 4. Oligonucleotide primers designed to amplify the cpDNA 50 ndhF gene in the Turneraceae 5. Taxa in the current study and their native range 51

List of Figures 1. A graphical representation of distyly 7 2. Structural composition of the transcriptional unit of 24 nuclear ribosomal DNA 3. Structural composition of the cpDNA ndhF gene 26 4. Example of .7% Agarose gels containing PCR amplified 54 product of both ndhF and ITS 5. Results of a 1000 replicate parsimony analysis of 66 Piriqueta based on ndhF. 6. Results of a 1000 replicate parsimony analysis of 67 Piriqueta based on ITS 7. Results of a 1000 replicate parsimony analysis of 68 Piriqueta based on combined ndhF and ITS sequence data. 8. Results of a Bayesian analysis of Piriqueta based on 69 ndhF and ITS sequence data 9. A 100 bootstrap replicate maximum likelihood 70 bootstrap analysis of Piriqueta based on combined ndhF and ITS sequence data 10. A 1000 bootstrap replicate parsimony analysis of the 71 Turneraceae based on ndhF sequence data. 11. 1000 replicate parsimony analysis of the Turneraceae 72 based on ITS sequence data 12. Results of a 1000 replicate Parsimony analysis of the 73 Turneraceae based on combined ITS and ndhF sequence data 13. Bayesian phylogeny of the Turneraceae based on ITS 74 data 14. Results of a 100 bootstrap replicate maximum 75 likelihood analysis of Turneraceae 15. Breeding system mapped on family wide ITS 76 parsimony analysis 16. Maximum likehood analysis of ITS with branch lengths 77 indicated

List of Abbreviations CTAB- hexadecyltrimethylammonium bromide NaCl- Sodium Chloride NaOH- Sodium hydroxide DNA- Deoxyribonucleic acid ITS- Nuclear ribosomal internal transcribed spacer PCR- Polymerase Chain Reaction SDS- Sodium dodecyl sulfate v/v- volume/volume w/v- weight/volume g- gram ml- milliliter ng- nanogram ʅ>- Microlitre pmole- picomole

List of Appendices Appendix A. Comprehensive List of all Species in the I Turneraceae. Appendix B. Current taxonomic treatment of the VI Turneraceae Appendix C. Molecular phylogenetic treatment of Turneraceae and related families based on ITS sequence VII data Appendix D. Molecular phylogenetic treatment of the VIII Turneraceae based on ndhF and ITS sequence data. Erblichia bernariana is included but only has ndhF sequence available.

xii

Glossary

Ancestral State- Character state present in the ancestor of a particular lineage

Anther- The pollen containing male floral structure

Apomorphy- A derived character state

Approach Herkogamy- A floral morphology in which the style extends beyond the anthers

Autapomorphy- A uniquely derived character state

Bifurcating- A node that leads to two branches is bifurcating

Consensus Tree- A compromise among equally parsimonious trees. The two most common types are strict and majority rule.

Clade- A monophyletic group

Character- An observable feature of an organism which may take on different character states in different taxa. In the case of DNA, a nucleotide position can have the states A, G, C, or T.

Derived state- Character state that evolved from the ancestral state

Distyly- A form of heterostyly in which there are two floral morphologies: a long styles morph and a short styles morph.

Heterostyly- The coexistence of genetically controlled hermaphrodite floral types with different style lengths.

Homoplasy- A similarity in character state among taxa that is not homologous. It is the result of parallel or convergent evolution, or reversal.

Ingroup- The group being studied. The clade whose members are being subjected to phylogenetic analysis.

Multifurcation- A node that leads to three or more branches is multifurcating

Monophyletic group- A group that contains a common ancestor and all of its decedents

Node- A branching point on a tree. In regard to a species tree, a node represents a speciation event.

xiii

Outgroup- A group, or species, used in an analysis that is not included in the ingroup. These are used to assess the polarity of character state changes within the ingroup, and represent the ancestral state.

Paraphyletic- A group that contains a common ancestor but not its decendants

Pleisomorhpy- The ancestral character state

Pollen- Male gametophyte that germinates to form pollen tubes.

Polytomy- A node that leads to three or more branches on a tree. Such a group is considered unresolved.

Sister group- Two monophyletic groups who are also their own closest relatives.

Stigma- The floral structure that receives pollen grains.

Synapomorphy- A shared derived character state

Symplesiomorphy- An ancestral character state shared by two or more taxa

Taxon- A named group of organisms of any rank (species, family, etc.)

Topology- The branching pattern of a tree.

Tristyly- A form of heterostyly in which there are three morphs, short, mid, and long styled.

Unweighted- Each character is given an equal weight.

Weighting- An analysis where certain characters are given a higher value based on their particularly high evolutionary importance.

xiv

1.0 Introduction

Heterostyly is a breeding system found in 28 Angiosperm families, in which populations contain two (distyly) or three (tristyly) distinct floral morphs. This condition has evolved on numerous occasions in various angiosperm families (Barrett and Shore 2008). The morphs vary in the lengths of both stamens and pistils (figure 1). In distyly, one morph, the long-styled or pin, has short stamens and long pistils, while the other morph, the short-styled or thrum, contains long stamens and short pistils (Darwin, 1877). Occasionally this condition has broken-down into a breeding system with only one morph that is strongly self-compatible. This breeding system is defined as homostyly (Truyens et al. 2005). Plants that possess the monomorphic condition of floral form will be referred to as homostylous, following Barrett and Shore (1987).

Beginning with the investigations of Hildebrand and Darwin, distyly has been an important model system in plant genetics and breeding system evolution (Darwin 1877; Ganders

1976; Barrett 1992; Ornduff 1992). Several hypotheses on the genetic architecture, evolution, development, and pollen competition systems have been proposed and tested, based primarily on observations and experiments on Primula species. Over the past century much has been accomplished with regard to the study of the evolution and breakdown of distyly, however, the majority of the work has focused at or below the species level (Shore et al. 2006). To better understand the evolutionary pathways of heterostyly, molecular phylogenetic analysis of heterostylous families is necessary. Several recent studies have explored various hypotheses for breeding system evolution (Kohn et al., 1996; Schoen et al, 1997; Graham et al., 1998; Conti et al., 2000; Huelsenbeck et al., 2003; Graham and Barrett, 2004; Truyens et al., 2005). The work of

Truyens et al (2005) focused on a single genus, Turnera (Turneraceae), with 135 species, subspecies, and taxonomic varieties. The genus Turnera contains many unresolved species relationships as well as both distylous and homostylous species. Truyens et al. (2005) found that there were no primary homostylous species (i.e. the ancestral state in Turnera appears to be distyly) within the genus. It is hoped that through a phylogenetic study of the entire family,

Turneraceae, any species which diverged prior to the evolution of distyly can be identified, if indeed there were any.

1.1 Historical Context

The term heterostyly was coined by Hildebrand (1866) in reference to the distinct morphological traits he observed in Primula flowers. Charles Darwin greatly expanded upon on

ƚŚĞǁŽƌŬŽĨ,ŝůĚĞďƌĂŶĚĂŶĚŝƐǁŝĚĞůǇƌĞŐĂƌĚĞĚĂƐƚŚĞĨĂƚŚĞƌŽĨƚŚĞƐƚƵĚǇŽĨŚĞƚĞƌŽƐƚǇůǇ͘ĂƌǁŝŶ͛Ɛ

ďŽŽŬ͕͞dŚĞĚŝĨĨĞƌĞŶƚĨŽƌŵƐŽĨĨůŽǁĞƌƐŽŶƉůĂŶƚƐŽĨƚŚĞƐĂŵĞƐƉĞĐŝĞƐ͟;ϭϴϳϳͿ͕ŝƐƚŚĞĨŽƵŶĚĂƚŝŽŶĂů text in the study of heterostyly. Darwin was very fond of his work on Primula. On his work with

Primula Darwin wrote͗͞no little discovery of mine ever gave me so much pleasure as the making out the meaning of heterostyled flowers͟(P. 136, 1902), which speaks volumes for the work on heterostyly coming from the major founder of the theory of evolution by natural selection. Darwin also studied heterostyly in organisms other than Primula. In fact, in 1865

Darwin wrote of Lythrum ƐƉĞĐŝĞƐ͞/ŶƚŚĞŝƌŵĂƚƚĞƌŽĨĨĞƌƚŝůŝǌĂƚŝŽŶƚŚĞƐĞƉůĂŶƚƐŽĨĨĞƌĂŵŽƌĞ

ƌĞŵĂƌŬĂďůĞĐĂƐĞƚŚĂŶĐĂŶďĞĨŽƵŶĚŝŶĂŶǇŽƚŚĞƌƉůĂŶƚŽƌĂŶŝŵĂů͘͟ĂƌǁŝŶǁĂƐƚŚĞĨŝƌƐƚƚŽŶŽƚĞ the reciprocal-herkogamy, the reverse orientations of styles and stamens in the morphs, and self-incompatibility of heterostylous plants (Figure 1).

With his work on Primula Darwin laid the foundation for subsequent investigations. The origins of heterostyly, the selective forces that maintain it, and why it often breaks down into other breeding systems, are questions that are often investigated by researchers focusing on heterostylous groups (Charlesworth 1978; Schoen et al. 1997). The large volume of research undertaken on heterostyly, given its relative rarity as a breeding system, has to do with several outstanding characteristics that have made it a model system for addressing many questions in evolutionary biology (Barrett 1992; Barrett and Shore 2008). The reciprocal-herkogamy, self- incompatibility, pollen size dimorphisms, as well as readily identifiable morphs, made distylous

Primula an obvious choice for pioneering studies. The number and quality of the studies on

Primula (due to the factors outlined above), made it a model system. It was one of the first plants used to demonstrate Mendelian genetics, showing a two-allele one-locus system which is still often cited in a majority of genetics textbooks (Barrett 1992).

Since Darwin, work on heterostyly has been undertaken in two major periods with different research emphases. The first, until around 1960, concerned genetic and biosystematic studies largely by European workers on a few herbaceous taxa such as Primula, Lythrum, and

Oxalis (Barrett 1992)͘^ŝŶĐĞƚŚĞϭϵϲϬ͛Ɛ͕ƚŚĞŐƌŽǁƚŚŽĨƉŽƉƵůĂƚŝŽŶďŝŽůŽŐǇĐŽŵďŝŶĞĚǁŝƚŚƚŚĞ relative ease of field study in tropical areas, has led to an increase in work on reproductive ecology, population genetics, and the evolution and breakdown of heterostyly (Barrett 1992).

More recently, the use of molecular phylogenetics has gained a foothold, as has molecular genetics in an effort to find the genes responsible for heterostyly (Barrett and Shore 2008).

Early post-Darwinian research on distyly was largely genetic in nature, concerned with determining the inheritance of the polymorphism (Barrett 1992). Such notable scientists as

Bateson, Fisher, Haldane, Ernst, and Mather, completed studies on topics as diverse as inheritance, linkage, recombination, supergenes and polymorphic equilibria (reviewed in Lewis and Jones 1992). At the time, these studies were part of the mainstream of genetics, and aided in the development of theories concerned with the regulation of recombination and evolution of genetic systems (Barrett 1992).

The inheritance of heterostyly has now been determined for at least 23 species in 11 families, although the condition occurs in numerous species in 28 families (Lewis and Jones

1992; Barrett 1992; Barrett and Shore 2008). The dialleic locus, S,s in distyly, and the dominance of the short-styled morph (S), are almost uniform in distribution among the distylous species studied to date. Interestingly, only three (Limonium, Hypercium and Oxalis) of the taxa studied to date vary from the normal dominance of the short-styled morph (Lewis and Jones 1992).

Despite the defining characters of heterostyly being morphological, studies focusing on the structural and developmental aspects of heterostyly are few in number (Barrett 1992).

Dulberger (1992) pointed out that far more studies have devoted time to the adaptive significance of reciprocal herkogamy than to function of the structural differences between pollen grains and stigmas of the floral morphs. She also argues that while the morphological aspects of heterostyly promote out-crossing, their primary function is to prevent inbreeding through incompatibility. However, not everyone working in the field would agree with this, for example Lloyd and Webb (1992) argue the morphology is there to enhance male fitness by getting pollen to the compatible stigmas.

Heterostyly has evolved many times in different Angiosperm families. However, understanding how this breeding system evolves and breaks down is among the least

4 understood aspects in the field (Barrett 1992). Clearly, there have been multiple independent evolutionary events in the establishment and breakdown of heterostyly. However, more work, in the form of phylogenetic studies of plant families containing heterostyly are necessary to help clarify the evolutionary pathways involved (Barrett 1992; Truyens et al. 2005).

Darwin (1877) postulated that distylous taxa first evolved the system of reciprocal herkogamy which arose prior to self-incompatibility, as a means of efficient pollen transfer, and this view was shared by Lloyd and Webb (1992). However, Baker (1966) suggested that, in the

Plumbaginaceae at least, heterostyly arose in several steps, beginning with dialleic- incompatibility.

Several genera, notably Amsinckia (Ray and Chisaki 1957), Cryptantha (Casper 1985),

Eichhornia (Barrett 1988), Melochia (Martin 1967), and Nivenia (Goldblatt and Bernhardt 1990), contain the morphological aspects of heterostyly (i.e. reciprocal herkogamy) but are thoroughly self-compatible (Barrett 1992). Recent work on Amsinckia (Schoen et al. 1997), however, determined that heterostyly is the ancestral condition in the genus. It seems that the existence of self-compatible species would be consistent with the notion that self-compatibility is associated with the breakdown of the breeding system, and that the species lacking self- incompatibility may be in the process of loss of dimorphism.

Perhaps the best known theoretical work on the evolution of distyly was written by

Charlesworth and Charlesworth (1979). In this paper, convincing arguments and a population genetics model are given for the evolution of self-incompatibility prior to the system of reciprocal-herkogamy. They argue that distyly arose through a complex series of mutations, morphological and physiological, resulting in two tightly linked gene complexes, with one

5 dominant over the other (Charlesworth and Charlesworth 1979). Lloyd and Webb (1992) used phenotypic models and argued the reverse. They argued that reciprocal-herkogamy likely appeared first as a means of increasing male fitness through precision of pollination, followed by evolution of incompatibility. Both points of view are feasible, therefore, further study is necessary to determine how heterostyly first appeared.

Figure 1: Diagram of distyly, compatible pollinations are shown using solid arrows, other pollinations (self, short x short, long x long) are incompatible (after Barrett 1992).

1.2 The Turneraceae

The Turneraceae is largely a tropical family of flowering plants (Malpighiales; eurosid I;

APG 2003) composed of approximately 226 species in 10 genera. The largest genus in the family is Turnera, with 135 specific and subspecific taxa (Truyens et al. 2005; Shore et al. 2006). The family has two areas of high species richness, one in southeastern Africa ( Hyalocalyx, Loewia,

Mathurina, Stapfiella, Tricliceras) and another in central South America (Turnera, Piriqueta), especially in Brazil.

Seven of the ten genera within the Turneraceae contain distylous species, and those lacking distylous species are either monotypic or contain few species (Shore et al. 2006; Table

1). In all, there are 168 distylous species, 40 homostylous species and 10 species with populations containing either homostylous or distylous plants (Shore et al 2006; table 1). The abundance of homostylous and distylous species as well as species containing both breeding systems makes the Turneraceae ideal for investigations on the evolution and breakdown of heterostyly. Recent phylogenetic analysis of the Primulaceae has indicated that all homostylous

Primula are secondarily homostylous (i.e. have lost distyly) (Mast et al. 2006). Which homostylous species within Turneraceae represent primary homostyles (i.e. these homostyles possess the breeding system of the ancestral Turneraceae species), if any exist, or the breakdown of distyly, requires an extensive phylogenetic study (Shore et al. 2006). An initial study by Truyens et al. (2005) indicated that homostyly evolved from distyly within the genus

Turnera on at least 3 separate occasions.

Truyens et al. (2005) completed the first phylogenetic analysis of Turneraceae. The focus of the phylogenetic analysis included 40 species of Turnera. The species of Turnera are

8 largely native to South and Central America, although there are two species native to southern

Africa (Arbo 1997; Arbo 2000). Truyens et al. (2005) used sequence data of the internal transcribed spacer region (ITS) of the nuclear ribosomal genes, to address questions concerning both chromosome and breeding system evolution. This work found that distyly is the ancestral breeding system in Turnera.

The current study will build on the phylogenetic work of Truyens et al (2005). The aforementioned study used ITS sequences to resolve the phylogeny within Turnera. The ITS sequences generated by Truyens et al. (2005) did not yield a fully resolved tree. The current study will employ a cpDNA encoded gene, ndhF, in addition to ITS, in an attempt to obtain a more robust and resolved phylogeny. The rapidly evolving chloroplast DNA sequence ndhF is known to be a good candidate gene for constructing phylogenies (Graham and Barrett 2004), and ITS is the most widely used sequence in phylogenetic studies of plants (Alvarez and Wendel

2003). This study should provide novel insights into the evolution and breakdown of distyly within the Turneraceae and provide evidence for generic relationships.

1.2.1 Historical Classification of the Turneraceae

The Turneraceae, as a family, was described by Dr. I. Urban in 1883 as containing 88 species in 5 genera (Wormskjioldia(now Tricliceras) (7 species), Streptopetalum (2 species),

Piriqueta (22 species in 2 sections), Mathurina (1 species), and Turnera (54 species). Several species that Urban (1883) described have since been revised, and many new genera and species have been described.

Of particular interest to the current study are the taxonomic positioning of disjunct old world species of primarily new world genera in both Piriqueta and Turnera; the treatment of

Erblichia, with one species in the Central America, and four endemic to Madagascar; the treatment of all old world genera within the family, and the allied families of the Malpighiales

(eurosid I; APG 2003). Erblichia, Mathurina, and Adenoa are large growing, homostylous species, features that make them unique among the homostylous species of the Turneraceae. Appendix

A provides a complete current list of all taxa in the Turneraceae, while appendix B contains the current taxonomic classification of the Turneraceae, based on morphological and cytogenetic traits (from Arbo 1995).

The issue of how to treat species within the same genus but on opposite sides of the

Atlantic has been problematic historically (Gillett 1980). However, through the use of molecular phylogenetics it is hoped that some insight may be gained. There has been some disagreement on how to treat the African members of the genus Turnera, Turnera thomasii and Turnera oculata. Turnera thomasii was originally described as a member of the genus Turnera by Urban, subsequently moved to the genus Loewia by J. Lewis (1971), due to its African origin, and then moved back to Turnera (Gillett 1980). This study will identify the likely affiliation of both African members of the genus Turnera. Also, within the genus Turnera, Truyens et al. (2005) found that

Turnera capitata (series Capitatae) was the basal lineage of Turnera, using Piriqueta as an outgroup. The relationship of the members of Turnera series Capitatae to Turnera and the rest of the family will be addressed given the inclusion of additional genera of the Turneraceae in this study.

The genus Piriqueta was first described in 1775 by Aublet, but, it was not until 1883 that

Urban provided the first monograph of the family. Urban (1883) described Piriqueta as containing 2 sections: section Eupiriqueta, which contained most of the modern American species; and section Erblichia, which contained species of the modern genus Erblichia as well as

Piriqueta capensis, the sole African species retained in the genus. The current taxonomic treatment has 2 sections in Piriqueta: section Piriqueta contains all of the American species, and all but one species in the genus; section Africana contains Piriqueta capensis (Gonazlez and Arbo

2003). Section Africana can be differentiated using a few taxonomic characters including a lack of glandular hairs (simple trichomes) (Gonzalez and Arbo 2003), and the lack of an obtuse chalaza in the seed (Arbo 1995). The current study will address the placement of Piriqueta capensis, and whether its inclusion in Piriqueta is warranted.

In Erblichia, the sole American species, Erblichia odorata, has been classified alongside

4 species from Madagascar (Arbo 1979; Shore et al. 2006). These are all trees ranging from 4 to

27 meters, and they are homostylous. Where available, this study will present an initial attempt to treat these taxa using phylogenetic information. Erblichia odorata was the first species described in the genus, in 1853 by Seeman and was followed by E. bernariana (as Turnera bernariana) (Tulanse) Arbo. E. madagascariensis Hoffman., E. integrifolia (as Paropsia integrifolia, Passifloraceae) (Claverie) Arbo., and E. antsingyae (as Piriqueta antsingyae)

(Capuron) Arbo. (Arbo 1979). Urban (1883) treated Erblichia as a section of the genus Piriqueta, and grouped the known species of Erblichia with Piriqueta capensis. The genus was first treated in its current form in 1979 by Arbo, who moved several species into the genus based on morphological similarities. In addition to the morphological affinities, Arbo (1979) suggested

11 that the pollen of the species of Erblichia were similar, with minor differences among the species. Moreover, Arbo found that Erblichia is closely related, not only to Piriqueta, but to

Mathurina, Adenoa, and Stapfiella. The classical taxonomic breakdown of the genera in the

Turneraceae is given in appendix B, adapted from Arbo (1996). E. odorata is perhaps the only

New World Turneraceae known to be pollinated by hummingbirds (Arbo 2006).

The first molecular phylogenetic treatment of the other African genera, including

Mathurina, Stapfiella, Streptopetalum, Loewia, Tricliceras, and Hyalocalyx, will be undertaken.

This will allow the relationships among them to be clarified. Shore et al. (2006) note that in four of the African genera (Hyalocalyx, Loewia, Streptopetalum, and Tricliceras) flowers possess stamens of different lengths, mostly two shorter and three longer within a flower, in both heterostylous and homostylous species. Unfortunately, the presence/absence of an incompatibility system is unknown. If detailed studies were to be conducted this could lead to interesting insights into breeding system evolution in the Turneraceae.

Mathurina is a monotypic genus represented only by the species Mathurina penduliflora, an endemic to Rodriques Island. This genus and species was first described by

Balfour (1876) as a member of the Turneraceae. The species is a small tree, found primarily at higher elevations, and is noted by Balfour as being closest in relation to Erblichia odorata, the only known Erblichia at the time. This genus is also homostylous, however the incompatibility systems have yet to be studied. Interestingly, Arbo (2006) notes that the seeds of Mathurina are wind dispersed, with the aril modified into slender long filaments, a feature unique in the family.

Most species of Turneraceae possess a fleshy aril and are ant dispersed (Shore et al. 2006).

Given the closer proximity to Madagascar, and the majority of the species in the genus Erblichia, a close relationship between Mathurina and the Madagascarean Erblichia is a possibility.

Stapfiella is a genus composed of shrubs up to 3 meters in height confined to tropical

Africa. The genus contains 7 species and subspecific taxa. The breeding system of the majority of these taxa is unclear, but of those that have been classified, 2 are known to be homostylous and

1 is thought to be distylous (Shore et al. 2006). Loewia is composed of 3 species found in East

Africa, including Kenya, Ethiopia and Somalia (Arbo 2006). One species is homostylous, L. microphylla, while the others are distylous, L. glutinosa and L. tanaensis (Shore et al. 2006).

The largest African genus, in terms of number of species, is Tricliceras. This genus contains 19 taxa (16 species and 3 subspecific taxa), distributed throughout Central and East

Africa (Arbo 2006). Although the breeding systems of these species have been incompletely studied, it appears that distyly occurs in at least 12 of the taxa, homostyly in at least 3 taxa, and one species, T. glanduliferum, has populations of both distylous and homostylous populations

(Shore et al. 2006).

Monotypic Hyalocalyx setiferus is an annual herb growing up to 30cm in height and distributed in Madagascar and south-eastern Africa. It has both heterostylous and homostylous populations. Streptopetalum is another of the African genera composed of perennial or annual herbs, distributed in East Tropical and Southern Africa (Arbo 2006). There are 6 species, 5 are distylous, and 1 is homostylous.

Adenoa is a monotypic genus endemic to serpentine soils on the island of Cuba (Arbo

1977). Adenoa cubensis is a homostylous shrub growing up to 3 meters in height. It is known to

13 be a nickel hyperaccumilator (Reeves et al. 1999). The genus was described in 1977 for the monotypic A. cubensis, which had formerly been classified as a Piriqueta, but was moved due to it having exclusive characters which separate it from the rest of the family (Arbo 1977).

1.3 Intercontinental Disjunctions

As described above, the family, Turneraceae, contains many genera with disjunct species. Erblichia contains 4 species in Madagascar and 1 in Central America (Arbo 1979; Shore et al. 2006). Piriqueta contains 44 species in the New World and 1 in Southern Africa. Turnera has around 128 species, two of which are distributed in Africa (Shore et al. 2006). It has long been a challenge to properly treat intercontinental disjunctions within genera. For example, within Turnera, T. thomasii was moved to the genus Loewia by J. Lewis (1971) simply because it is disjunct in Africa and not distributed in the New World.

Several hypotheses have been proposed for the origins of disjunct populations, and these vary according to the relatedness between genera within families (Renner 2004; Davis et al. 2004). The dispersal events between South America and Africa often invoke either

Gondwanan dispersal (the last connection between America and Africa was around 105 million years ago), or long-distance dispersal (Davis et al. 2004). Other hypotheses have been presented to explain recent dispersal events. The Boreo-tropical theory postulates that tropical families moved north in warm periods and were able to spread across to the Old World via land bridges, then were able to colonize southward into Africa at various intervals when land conditions were conducive to migration (Davis et al. 2004). More recent, anthropogenically facilitated disjunctions can be seen in many weedy plant species. For example, Backer (1951) describes 3 species of Turneraceae that have become established in Malaysia, Piriqueta racemosa, Turnera

14 subulata, and Turnera ulmifolia. All of these species originated in the New World and were brought by humans to Malaysia. Furthermore, Renner (2004) notes that trans-Atlantic disjunctions at the genus-level are likely due to long distance dispersal, while species-level disjunctions would most likely be anthropogenic. Family level disjunctions would likely date back to the breakup of Africa and South America. Givnish et al. (2004) describe intercontinental disjunctions in the Raptaceae and Bromeliaceae in West Africa by discussing the possibility of long distance dispersal, facilitated by winds. The relationships of the disjunct taxa in the

Turneraceae to their current genera, and their retention in these genera will be investigated.

1.4 Molecular Phylogenetics

Molecular phylogenetics is the study of evolutionary relationships among organisms through the use of DNA and protein sequences, in addition to other molecular markers (Graur and Li 2000). Molecular data lends itself readily to phylogenetic studies as the characters used are likely to be independent of the morphological trait changes, including those associated with breeding system evolution (Barrett 1992). High levels of homoplasy may occur for floral characters, thus complicating phylogenetic analysis based upon those characters (Graham et al.

1998; Truyens et al. 2005). Sequence data is unambiguous in the sense that it is not altered by the environment (Graur and Li 2000). Additionally, assessment of homology is often easy with molecular data. Molecular data, especially protein and DNA sequences, can be used to assess relationships among distantly related organisms (Graur and Li 2000). When trying to determine the origin of homologous loci in allopolyploids, molecular data is necessary (Wendel 2000). In the identification of cryptic species, those species which look similar and may even have

15 traditionally been lumped into one species, morphological data is also ineffective. For example, through the use of DNA barcoding, Hebert et al (2004) found that the Neotropical skipper butterfly, Astraptes fulgerator, was actually a cryptic species complex of ten different species.

However, it should be noted that it is still necessary to describe new species based on morphological traits, and Hebert et al. (2004) did recommend the description of the ten cryptic species based on slight morphological differences among the species, as well as their reproductive isolation.

Morphological data do have some additional merit, other than use in describing and identifying species. In plants there are often subtle morphological data that differentiate species with similar, or even identical DNA sequences, and are reproductively isolated. For example, species of Trachycarpus palm from the Himalayas, which are reproductively isolated but which show little to no variation in their ITS sequence, have been classified according to their morphological traits because it is difficult, if not impossible, to separate them using molecular data obtained thus far (Stürk 2006).

Molecular data sets are, however, liable to problems similar to those of morphological data. dŚĞůŝŬĞůŝŚŽŽĚŽĨĂĐŚĂŶŐĞŝŶƚŚĞƐƚĂƚĞŽĨĂŶƵĐůĞŽƚŝĚĞ;Ğ͘Ő͘͟͞ƚŽ͞'͟Ϳ͕ĂŶĚƚŚĞ͚ǀĂůƵĞ͛ŽĨ such a change is difficult to measure (Wenzel 1997). Resolutions of morphological homoplasy are also more logical than are those in DNA data sets (Wenzel 1997). Finally, interpretation of aligned DNA sequences might be difficult both due to the presence of gaps and multiple substitutions at the same site (Graur and Li 2000).

The construction of a phylogenetic tree is a representation of our best estimate of evolutionary history (Swofford et al. 1996). This is true both of trees generated using morphological or molecular data, or both simultaneously. Evolutionary events are inferred from

16 extant species and molecules (or morphology), and some species will have been lost to extinction or may not have been included in the study, which can lead to an incomplete phylogeny.

When aligned DNA sequence data are used in tree reconstruction, the sequence is broken into individual sites (i.e. with individual nucleotides as character states). Each of these sites is a treated as a multistate character, which can have one of 4 states: A, T, G, or C. DNA data is usually treated as unordered, which means that it is equally likely that a transition or transversion of any nucleotide may occur (Graur and Li 2000). In a parsimony analysis ancestral character states are plesiomorphic, while derived character states are termed apomorphic.

Derived character states shared by two or more taxa are termed synapomorphies, and are phylogenetically informative, while shared ancestral character states called symplesiomorphies, are not phylogenetically informative. An autopomorphy is a derived character state that is unique to a particular taxon (Graur and Li 2000).

The major methods of phylogenetic reconstruction have historically been: Maximum likelihood, Parsimony, and distance. Recently, another type of reconstruction has gained popularity: Bayesian inference. Further advances have been made involving the algorithms employed to obtain the most parsimonious trees. One such advance is the New Technology of

Golobof et al. (2008). Maximum likelihood, Parsimony, and Bayesian inference will be employed in the present study and will be discussed in turn.

1.4.1 Maximum likelihood

Maximum likelihood methods infer tree topology by determining the probability of observing the data (i.e. nucleotide sequence) under a proposed model of evolution (Hall 2001).

This method assumes that the evolutionary history with the highest probability of leading to the present state is most likely to be correct (Swofford et al. 2006). There are many model options available in maximum likelihood analyses. These models vary in assumptions on processes of nucleotide substitution. The fact that maximum likelihood tests different models of evolution is one of the major benefits of this method of assessing phylogeny. Branch length is considered when evaluating a tree via maximum likelihood analysis, with evolutionary changes being more likely to occur over long branches than short ones (Swofford et al. 1996). Maximum likelihood is known to outperform other methods of phylogenetic analysis under a range of different conditions (Huelsenbeck et al. 2002).

Maximum likelihood is, however, not without criticism. It has been found to take much longer to infer a phylogeny, up to months for large data sets. Second, this method of analysis leads to only one tree so comparison of trees is not possible (Hall 2001). Finally, a phylogeny created using maximum likelihood is only valid if evolution proceeds according to the evolutionary model being used in an analysis (Swofford et al. 1996).

1.4.2 Parsimony

Parsimony is perhaps the easiest phylogenetic analysis to comprehend. It assumes that the answer that requires the smallest number of steps is the correct one(s) (Hall 2001). Using maximum parsimony, the tree structure (s) that requires the smallest number of evolutionary changes to explain the relationships among the taxa, is chosen as the best estimate of phylogeny (Graur and Li 2000). Often, when running a parsimony analysis a number of equally parsimonious trees may be found, although they can not all be the correct phylogenetic tree.

Parsimony assumes that taxa which share a character state do so because they inherited this character state mutually from a common ancestor. When the assumptions above are violated, it can be explained by: a reversal (a changed character state reverted back to its original state), convergence (the same character evolves separately in two unrelated taxa), or parallelism (different taxa may be predisposed to developing the same character state) (Hall

2001). Homoplasy is the term for these three phenomena collectively. The mathematical algorithm of parsimony seeks to select the tree (or trees) that require the least number of steps to explain the data, therefore homoplasy must be minimized as it involves extra steps to explain the data (Hall 2001).

Methods of measuring the amount of consistency within a data set have been developed. The CI (consistency index) measures the amount of homoplasy contained within a data set (Schuh 2000). The CI always falls between 0 and 1, and the closer the number is to 1, the less homoplasy that is contained within the data. The RI (retention index) measures the fraction of synapomorphy retained as synapomorphy on a phylogenetic tree (Shuch 2000). A character that is consistent fully with a phylogenetic tree has a RI of 1, while a character with no synapomorphic content would have an RI of 0 (Shuch 2000).

Swofford et al. (1996) criticized parsimony because it does not require a model of evolutionary change. Another criticism is that parsimony implies an efficiency to evolution that may not be true (Hall 2001). Lastly, using computer simulations where the true tree is known, parsimony has been known to produce incorrect tree topologies particularly involving long branch attraction (Swofford et al. 1996). Recently, new methods for rapidly finding the most parsimonious tree have been developed. These searches use newly developed algorithms to rapidly search phylogenetic data, and will be discussed in section 1.4.4 (Golobof et al. 2008).

1.4.3 Bayesian Inference

More recently, Bayesian inference of phylogeny has gained popularity. Bayesian inference is based on the notion of posterior probabilities, which are estimated after we already know something about the data (Hall 2001). Like maximum likelihood, Bayesian inference is based on the likelihood function (Huelsenbeck et al. 2002). Because Bayesian inference is based on the likelihood function it inherits many of the positive statistical properties that maximum likelihood is known to possess. In addition, Bayesian inference also has the added benefit of allowing the researcher to include prior information about phylogeny through the specification of prior probabilities (Huelsenbeck et al. 2002).

Bayesian inference is not without questions, and critics. First, Bayesian posterior probabilities tend to give higher values than either parsimony or maximum likelihood bootstrap values (Huelsenbeck et al. 2002). Huelsenbeck et al. (2002) offer as an explanation the fundamental differences between a bootstrap analysis (based on a resampling of the data from the original data matrix) and a Bayesian posterior probability (which explicitly measures uncertainty based on the specified evolutionary model). Another drawback of the Bayesian method is that it is also dependant on the choice of evolutionary model, similar to maximum likelihood (Huelsenbeck et al. 2002). Furthermore, the ͞prior information͟ used in the analyses is commonly unknown.

1.4.4 New Technology

Recently, new computer algorithms have been developed for phylogenetic analysis.

With the introduction of TNT (Golobof et al. 2008), a method of evaluating large data sets quickly, has arisen. The introduction of new technology algorithms with cyclic perturbation and search phases, including ratchet and tree-drifting, allows large data sets to be evaluated quickly

(Golobof et al. 2008). Tree-fusing allows for sub-tree exchanges between trees, affecting those that improve the tree score. Sectorial search creates reduced data sets and submits this data to

ĂƐĞĂƌĐŚĂůŐŽƌŝƚŚŵ͘ůůĨŽƵƌŽĨƚŚĞƐĞƐĞĂƌĐŚŵĞƚŚŽĚƐĐĂŶďĞĞŵƉůŽǇĞĚŝŶĂƐŝŶŐůĞ͚ƐĞĂƌĐŚ͛ƋƵŝĐŬůǇ resulting in a tree that can be subjected to bootstrapping, jackknifing, and Bremner supports

(Golobof et al. 2008). The goal is to discover all the most parsimonious trees more quickly than using older software.

1.5 The nuclear ribosomal intergenic spacer (ITS)

Structurally, the internal transcribed spacer (ITS) region is contained within the nuclear ribosomal DNA (nrDNA; Figure 2)(Baldwin 1993). This cistron is comprised of three genes that encode the 18S, 5.8S, and 26S ribosomal subunits. ITS 1 separates the 18S and 5.8S subunits, while ITS 2 separates the 5.8S and 26S subunits (figure 2). Thousands of copies of this repeat can exist in the nuclear genome, which each cistron being separated by an intergenic spacer

(Baldwin et al. 1995).

Although ITS 1 and 2 are transcribed, they are not included in mature ribosomes. It has been hypothesized that these spacers serve a function in the maturation of nrRNAs, resulting in

21 some evolutionary constraint in both structure and function (Baldwin et al. 1995). Studies using yeast have shows that deletions in ITS 1 can inhibit production of mature rRNAs, and point mutations in ITS 2 have been found to reduce processing of large subunit RNAs (van der Selde et al. 1992).

ITS is, however, the most common DNA sequence used in phylogenetic studies of plants.

According to Alvarez and Wendel (2003) as of 2003, of the 244 phylogenetic papers published in the preceding five years, 66% included ITS sequence data, and 34% of all published phylogenies were based exclusively on ITS sequence data. The popularity of this sequence can be accounted for by the numerous advantages that it possesses over other regions. Among the advantages of the ITS region are, firstly that Hamby and Zimmerman (1992) found that members of the ITS gene family had undergone intraspecific homogenization (concerted evolution), resulting in intergenomic homogeneity among the repeated units. Secondly, the coding regions are highly conserved, even though the spacers are sharply divergent, even within genera. This allows the design of universal primers that will amplify phylogenetically informative gene sequence from most plant and fungal phyla (White 1990). Thirdly, as a nuclear sequence, ITS is biparentally inherited, unlike many cpDNA and mtDNA genes, which are usually maternally inherited

(Baldwin 1992, Baldwin et al. 1995). Lastly, since ITS regions are under reduced evolutionary constraint, ITS provides a multitude of nucleotides that may be neutral in their evolution (van der Selde et al. 1992).

Recently, there has been some criticism over the widespread use of ITS in phylogenetic studies (Alvarez and Wendel 2003). The authors cite a number of potential pitfalls to the sole use of ITS in molecular phylogenetic studies. The presence of paralogous copies of ITS and ITS

22 pseudogenes could cause confusion over proper sequencing and phylogenetic relationships, a lack of concerted evolution in the genome, and the accuracy of the alignment using ITS sequences. The most pressing issue when working with ITS of divergent taxa can be the alignment of multiple sequences. Using a protein encoding gene, the alignment issue is less problematic, since there is a built in check system, every three nucleotides. No such check exists for ITS since it is non-coding, but the inclusion of slowly evolving nrDNA subunits gives areas of consensus in ITS sequence data (Alvarez and Wendel 2003). In ITS, the alignment problem is magnified since the region has a tendency to accumulate indels (insertions/deletions). Since ITS is GC rich it tends to cause an increase of indel formation, which is not the case for most nuclear introns, which tend to be AT rich (Alvarez and Wendel 2003). The rapid evolution of ITS can lead to problems aligning ingroup and outgroup sequences. The inclusion of the 5.8s nrDNA gives an

͚ĂŶĐŚŽƌ͛ƐĞƋƵĞŶĐĞƚŽŚĞůƉŐƵŝĚĞĂůŝŐŶŵĞŶƚ͘

Alvarez and Wendel (2003) recommend the use of multiple single-copy nuclear genes as opposed to ITS. In an analysis of potential barcoding genes, Fazekas et al. (2008) found that multiple rapidly evolving cpDNA genes peaked at a maximal resolution of around 71% of species, where the greatest resolution given with a single gene, the rapidly evolving match gene was

56%. Though cpDNA sequences are usually not biparentally inherited (although in Turnera ulmifolia Shore and Triassi (1998) found paternal bias in cpDNA inheritance), it has been shown that cpDNA more readily amplifies in PCR reactions than do single-copy nuclear DNA in aged samples (Drabkova et al. 2002). For this reason, the current study also employs the rapidly evolving cpDNA gene, ndhF, in addition to ITS.

Figure 2: Structural composition of the transcriptional unit of nuclear ribosomal DNA. The genes that encode the 18S, 5.8S, and 26S ribosomal subunits are separated by two internal transcribed spacers. The location and direction (indicated by arrows) of primers ITS2, ITS3, ITS4, and ITS5 are given at the bottom of the figure (From Baldwin 1993).

1.6 The cpDNA ndhF gene

The plastid ndhF gene encodes subunit F of the multisubunit NADH: plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa

(Schluchter et al. 1993). This gene has been used in a number of phylogenetic studies, either on its own, or in multigene analyses (e.g. Kim et al. 2001; Backlund et al. 2000; Steinman and Porter

2002; Graham and Barrett 2003; Olmstead et al. 2000; Li 2008).

Since ndhF is protein-encoding, it lacks a number of the problematic alignment issues present with ITS sequence. The gene, or part thereof, is known to have good resolving power in some families, and has enough parsimony informative characters for some sub-generic resolution (Olmstead et al 2000). Figure 3 shows a diagrammatic representation of the ndhF gene. The length of the gene illustrated is based on the cpDNA genome of Nicotiana tabacum, and is 2223 bp long (Shinozaki et al. 1986). The location of primers that were designed for the current study is indicated and arrows giving their orientation are shown.

Figure 3: Diagrammatic representation of the ndhF gene with the primers designed in this study and their direction noted. The gene is 2223 bp long based on the Nicotiana tabacum genome

(Shinozaki et al. 1986).

1.7 Molecular Phylogenetics in heterostylous families

Many of the phylogenetic analyses of distylous taxa have focused on the evolution and breakdown of distyly, most of these at or below the species level. Phylogenetic analysis is necessary for the evolutionary pathways of distylous groups to be tested (Donoghue 1989;

Weller and Sakai 1999). Recent phylogenetic research based on DNA sequences and character mapping in the Pontederiaceae (Kohn et al. 1996; Graham et al. 1998; Huelsenbeck et al. 2003),

Amsinckia (Schoen et al. 1997), Primula (Conti et al. 2000; Mast et al. 2004), Houstonia (Church

2003), Narcissus (Graham and Barrett 2004), Linum (Armbruster et al. 2006; McDill et al. 2009),

Turnera (Truyens et al. 2005), and Lythraceae (Morris 2007) have investigated hypotheses for breeding system evolution in heterostylous taxa.

Schoen et al. (1997) and Graham and Barrett (2004) investigated the evolutionary origins of distyly in Amsinckia and Narcissus, respectively. Kohn et al. (1996) explored the evolutionary origins and breakdown of tristyly in the Pontederiaceae. Ancestral state reconstruction of heterostyly in Linum and Linacaeae was recently undertaken by McDill et al.

(2009). Finally, there have been attempts to map breeding system evolution in the Primulaceae

(Conti et al. 2000; Mast et al. 2006). The methods and findings of these studies will be discussed below.

Schoen et al. (1997) investigated the evolution and breakdown of distyly in Amsinckia, a genus composed of about 20 species, 5 of which are distylous. Restriction fragment length polymorphism (RFLP) of the chloroplast genome was used infer the phylogeny of the family.

They investigated differential weighting for the gain and loss of distyly. To avoid sampling error

27 the authors analyzed the data under different assumptions, in separate analyses both distyly and homostyly were considered to be ancestral. The authors used the outgroup, Cryptantha flava, only to root the phylogeny. This species was not used to infer the ancestral condition in character mapping because the authors did not want to bias their results. In character mapping analyses, the authors treated the gain and loss of distyly as a weighted or unweighted character.

Using differential weighting and both distyly and homostyly as the ancestral breeding system, the authors mapped breeding system on the phylogeny (Schoen et al. 1997). When the authors treated distyly as the ancestral condition, or when the loss of distyly was easier than its gain, the results indicated that self-fertilizing taxa (homostyles) are of recent origin from outcrossing relatives. The authors conclude that it is likely that distyly is the ancestral state in Amsinckia due to the difficulty of its evolution and ease of breakdown, as discussed below.

There are cases in which heterostyly appears to have arisen independently several times within the same genus. Using DNA sequence data from both ndhF and the trnL-trnF (both trnL- trnF), Graham and Barrett (2004) found that heterostyly had evolved on two separate occasions

(distyly once, and tristyly once) in Narcissus. They also found several independent evolutionary events have led to dimorphic stigma height. This condition is rare in angiosperms and is hypothesized (Lloyd and Webb 1992) to be the immediate precursor to the evolution of heterostyly. In this model the immediate ancestors of distylous taxa were monomorphic for style length and possessed approach herkogamy, with long styles and stamens below the stigma. In Narcissus, the immediate ancestor of the lone distylous species possesses stigma- height dimorphism. The authors concluded that the ancestral condition in the genus appears to be stylar monomorphism and approach herkogamy (Graham and Barrett 2004).

Kohn et al. (1996) attempted to explore the evolution of SI (self-incompatibility) and heterostyly in different lineages of the Pontederiaceae. Restriction fragments of cpDNA were used to create a phylogeny. Using this phylogeny they analyzed two different weighting schemes for shifts in the evolution and breakdown of breeding system and incompatibility. They had some difficulty in reconstructing the primitive floral form in the family due to some topological uncertainty. The authors came to a few broad conclusions: Tristyly originated in the

Pontederiaceae on either one or two occasions; The breeding system has repeatedly broken down leading to selfing; Homostylous taxa, and self-incompatibility, probably arose after the origin of tristyly. The last finding contradicts the evolutionary model of Charlesworth and

Charlesworth (1979). Their model predicted that self-incompatibility would evolve prior to the evolution of heterostyly, counter to the last finding in the study of the Pontederiaceae (Kohn et al. 1996).

The evolution of breeding systems has been mapped in the Primulaceae by Conti et al.

(2000) and again by Mast et al. (2006). Conti et al. (2000) used phylogenies generated using ITS sequence data to map the breeding system, chromosome number, and biogeographic features for 21 species of Primula (Primulaceae). The study found that homostyly is a derived breeding system state. Additionally, they found a strong link between hybridization, allopolyploidy and derived homostyly.

Mast et al. (2006) used cpDNA sequence data to infer the phylogeny of the family and to map the ancestral breeding system state within the family. The authors used differential weighting schemes to determine the ancestral condition in the family. Various weightings were explored, and it was found that with a weighting of 5:1 (gain: loss of distyly) one origin of distyly

29 was inferred for the family. When the gain to loss was weighted at 1:1, the phylogeny suggested there might be up to 4 separate origins of distyly in the family. Given the evolutionary models of both Charlesworth and Charlesworth (1979) and Lloyd and Webb (1992) it is far more likely that the gain of distyly is more difficult evolutionarily than the loss, indicating that the results from the 5:1 weighting is likely to be correct. In Turnera, Tamari et al. (2004) found that spontaneous mutant homostyles arise in hybrid plants of Turnera subulata x T. krapovickasii and in tetraploid

T. scabra supporting the idea that derived homostyly can have a simple genetic basis. The simple genetic basis and widespread occurrence of derived homostyly supports the evolutionary models of both Charlesworth and Charlesworth (1979) and Lloyd and Webb (1992). As mentioned above, these models suggest that the breakdown of heterostyly is less complex than its evolution.

In a recent study focusing on Linum and Linaceae, McDill et al. (2009), used nuclear DNA sequence data from ITS and cpDNA sequences from ndhF, rbcL, trnL-F, and the trnK ϯ͛ŝŶƚƌŽŶ, to infer a phylogeny. The authors used this DNA sequence data to work out the systematics, explore the biogeography of the genus and family, and to address questions of the evolution of heterostyly. The authors found that they were unable to determine the ancestral state conclusively in Linum. However, they do note that with complete taxon sampling some of the ambiguity surrounding the ancestral state could be eliminated. They also found breeding system transitions on three separate occasions in the family. One is a gain of heterostyly in a species whose ancestors are unequivocally homostylous, as well as two independent losses of heterostyly.

Schoen et al. (1997) found that homostylous taxa in two of the lineages of Amsinckia are polyploid, possibly having arisen from past hybridization of separate species and consequent doubling of chromosomes. In Primula it was found that in some lineages glacial retreats allowed for expansion in the geographic ranges of some species (Conti et al. 2000). The expansion and subsequent hybridization events led to the creation of homostylous allopolyploid taxa. In

Turnera homostyles tend to be higher polyploids (Barrett and Shore 1987, Truyens et al. 2005), but not always (Shore et al. 2006).

The identification of species that are primary homostyles, species that have diverged prior to the evolution of distyly or tristyly, if they exist, is important to infer the ancestral breeding system condition in a lineage. The determination of their Self-Incompatibility (SI) or

Self-Compatibility (SC) is also of great importance in inferring the evolutionary history of heterostyly and testing the evolutionary models (Charlesworth and Charlesworth 1979; Lloyd and Webb 1992). Primary homostylous taxa identified in phylogenetic studies will allow testing of the evolutionary models of distyly. These taxa will also be very useful when distyly genes are discovered, as a means of determining the changes required for the evolution of the breeding system. Given the range of families that contain heterostylous taxa it is most likely that the evolutionary pathway of heterostyly will vary from family to family.

1.8 Objectives of the Thesis

The current study sets out to resolve the phylogeny of the Turneraceae using the rapidly evolving nrDNA intergenic spacers, ITS 1 and ITS 2, as well as a portion of the cpDNA ndhF gene.

Using this data the evolutionary relationships among the species in the Turneraceae will be investigated. The correct phylogenetic placement of disjunct species of Turnera, Piriqueta, and

Erblichia will be investigated. Finally, the evolution and/or breakdown of distyly within the family will be analyzed.

2.0 Materials and Methods:

2.1 Plant Material

A complete list of the plant material employed in the current study can be found in

Table 2. Taxa representative of most of the genera of the Turneraceae were used as well as sequences from outgroups (for rooting purposes), including Passiflora (Passifloraceae), Populus

(Salicaceae), and Ricinus (Euphorbiaceae), all of which are in the order Malpighiales (APG 2003).

Attempts were made to sample as many of the genera of the Turneraceae as possible.

The majority of the samples in this study were extracted from preserved dry-leaf tissue samples taken from variously aged herbarium specimens provided by a colleague, Maria Arbo

(Universidad Nacional del Nodeste). Additionally, where available, DNA was extracted from fresh tissue of plants grown in the glasshouse at York University. One dry leaf sample of Erblichia odorata was sent from Costa Rica by Dr. Daniel Janzen (University of Pennsylvania).

2.2 DNA Extraction

DNA extractions of both fresh and dry leaf samples were performed using two different protocols: 1) the FastDNA® kit and the FastPrep® instrument following the manufacturer's protocol (Qbiogene inc., CA) ; 2) Doyle and Doyle (1987) CTAB method. In protocol 1, 100-200mg of tissue was ƐƵƐƉĞŶĚĞĚŝŶϴϬϬʅůŽĨƚŚĞƉƌŽǀŝĚĞĚ>^-s&ƐŽůƵƚŝŽŶĂŶĚϮϬϬʅůŽĨWW^͘dŚŝs mixture was then placed in the FastPrep® machine and homogenized for 40 seconds at a setting of 6.

This homogenized mixture was then centrifuged for 10 minutes at 14,000 x g and the supernatant was transferred into a clean microcentrifuge tube and an equĂůǀŽůƵŵĞ;Đ͘ϲϬϬʅůͿŽĨ binding matrix. Next, this solution was gently agitated for 5 minutes at room temperature

33 before being centrifuged at 14,000 x g for 5 minutes to pellet the DNA and binding matrix. The pellet was then gently resuspended using the foƌĐĞŽĨƚŚĞϱϬϬʅůŽĨůŝƋƵŝĚ^t^-M from the pipette tip. This was again centrifuged at 14,000 x g for 1 minute, and the supernatant was

ƌĞŵŽǀĞĚ͘dŚĞEǁĂƐƚŚĞŶĞůƵƚĞĚďǇƌĞƐƵƐƉĞŶĚŝŶŐƚŚĞƉĞůůĞƚŝŶϭϬϬʅůŽĨ^ĂŶĚŝŶĐƵďĂƚĞĚ

ĨŽƌϱŵŝŶƵƚĞƐĂƚϱϱȗ͘dŚĞƐŽůƵtion was then centrifuged at 14,000 x g, and the eluted DNA was transferred to a clean microcentrifuge tube and stored at -ϮϬȗ͘

For Doyle and Doyle extractions, approximately 0.5g of fresh flower buds, flowers, or leaf tissue, or 0.2g of dry leaf tissue, were used. CTAB buffer, 5 ml (2% w/v CTAB, 1.4M NaCl,

0.2% v/v mercaptoethanol, 20mM EDTA, 100mM Tris-HCl ph 8.0) ǁĂƐŚĞĂƚĞĚƚŽϲϬȗŝŶĂϭϱŵů polypropylene tube. The tissue was then ground in the buffer using a mortar and pestle. The resultant slurry was then incubated for 60-90minutes with occasional mixing. Next, a single extraction with 24:1 chloroform:isoamyl alcohol (v/v) was completed. Samples were then centrifuged for 10 minutes at room temperature at 5,400 rpm in a Megafuge 1.0R (Heraeus

Instruments). The aqueous phase was then transferred to a clean 15 ml polypropylene tube to which an equal volume of cold isopropanol was added and the sample was then inverted a few times to mix.

DNA was then pelleted with 10 minutes centrifugation as above. The resultant pellet was transferred to a 1.5 ml microfuge tube and then washed with 1ml of wash buffer (76%v/v ethanol, 10mM ammonium acetate) in wash buffer for 1 hour then was again pelleted by centrifugation at 13,000rpm for 5 minutes. The wash buffer was then poured off and the pellet was allowed to air dry over night. The dried pellet was then resuspended in 200-ϰϬϬʅůŽĨd buffer (10mM Tris-,ů;ƉŚϴ͘ϬͿ͕ϭŵDdͿ͘ůůƐĂŵƉůĞƐǁĞƌĞƚŚĞŶƚƌĞĂƚĞĚǁŝƚŚϭʅůZEĂƐĞ

;ϮϱŵŐͬŵůͿĂŶĚŝŶĐƵďĂƚĞĚĂƚϯϳȗĨŽƌϯϬŵŝŶutes to remove RNA. DNA was then re-precipated by adding 1/5 a volume of 10M ammonium acetate and two volumes of 95% ethanol. DNA was pelleted by centrifugation as above. The pellet was then washed with 1 ml of 70% ethanol, centrifuged and, then air dried. The final pellet was resuspended in an appropriate volume (30-

ϮϬϬʅůͿŽĨϬ͘ϭydďƵĨĨĞƌ͘

2.3 Primer Design

Appropriate primers for the ndhF gene were necessary to amplify the gene across the

Turneraceae. Therefore, family-specific primers were designed using sequences available online from taxa in families known to be related to the Turneraceae, as listed above. These sequences were then aligned using CLUSTALX (Thompson et al. 1997). Conserved regions of the gene sequence were then selected for initial primer design and then selected primer regions were checked for potential problems (e.g. primer dimers, etc) using gene Runner version 3.05

(Hastings Software) and the selected primers (Table 2; Figure 3) were then synthesized

(Invitrogen Life Technologies). In addition to the ndhF primers described above, ITS primers: ITS

2, ITS 3, ITS 4, Baldwin (1993), and ITS 5 (Figure 4), Sang et al. (1995), were also synthesized

(Invitrogen Life Technologies).

2.4 Polymerase Chain Reaction

All PCR reactions were conducted using an Eppendorf mastercycler thermal cycler

(ƉƉĞŶĚŽƌĨĂŶĂĚĂ͕DŝƐƐŝƐƐĂƵŐĂ͕KŶƚĂƌŝŽͿ͘ZĞĂĐƚŝŽŶƐĐŽŶƚĂŝŶĞĚϭϬʅůŽĨZdĂƋZĞĂĚǇDŝǆWZ reaction mix (Sigma). Each sample contained 1-Ϯʅů;Đ͘ϮϬ-ϱϬŶŐͿŽĨƚĞŵƉůĂƚĞE͕ϰʅůŽĨƚŚĞ

35 appropriate primer pair ;ϭϬƉŵŽůͬʅůͿ͕ĂŶĚϰ-ϱʅůŽĨĚĞŝŽŶŝǌĞĚĚŝƐƚŝůůĞĚǁĂƚĞƌƚŽĂĨŝŶĂůǀŽůƵŵĞŽĨ

ϮϬʅ>͘

ĨƚĞƌĂŶŝŶŝƚŝĂůŚŽƚƐƚĂƌƚĂƚϵϰȗĨŽƌϮ͗ϬϬŵŝŶƵƚĞƐ͕WZ͛ƐǁĞƌĞĐĂƌƌŝĞĚŽƵƚŝŶŽŶĞŽĨƚǁŽ ways:

1) For ndhF ĂŵƉůŝĨŝĐĂƚŝŽŶƐƚŚĞƌĞǁĞƌĞϯϴĐǇĐůĞƐŽĨϬ͗ϱϬƐĞĐĂƚϵϰȗ͕Ϭ͗ϯϱƐĞĐĂƚϱϮȗ͕ϭ͗ϬϬ

ŵŝŶĂƚϳϮȗ͕ĨŽůůŽǁĞĚďǇĂĨŝŶĂůĞǆƚĞŶƐŝŽŶŽĨϮ͗ϬϬŵŝŶĂƚϳϮȗĂŶĚƐƵďƐĞƋƵĞŶƚĐŽŽůŝŶŐƚŽ

ϰȗĂƚƚŚĞĞŶĚŽĨƚŚĞƌƵŶ͘

2) /d^ĂŵƉůŝĨŝĐĂƚŝŽŶƐŚĂĚϯϱĐǇĐůĞƐŽĨϭ͗ϬϬŵŝŶĂƚϵϰȗ͕Ϭ͗ϯϬƐĞĐĂƚϱϱȗ͕ĂŶĚϭ͗ϯϬŵŝŶĂƚ

ϳϮȗ͕ĨŽůůŽǁĞĚďǇĂĨŝŶĂůĞǆƚĞŶƐŝŽŶĂƚϳϮĨŽƌϮ͗ϬϬŵŝŶĂŶĚƐƵďƐĞƋƵĞŶƚĐŽŽůŝŶŐƚŽϰȗĂƚ

the end of the run.

2.5 Agarose Gel Electrophoresis

Agarose gels (0.7%) were prepared with molecular biology grade agarose and 1X TAE

buffer (0.04M Tris-Acetate, 0.001M EDTA). All gels were stained with ethidium bromide

;ϭʅŐͬŵůͿĂŶĚǁĞƌĞƌƵŶĂƚϱϬ-80V. Agarose gels were visualized with UV to confirm adequate

ŵŝŐƌĂƚŝŽŶŽĨE͕ĂŶĚƚŚĞŶƉŚŽƚŽŐƌĂƉŚĞĚƵƐŝŶŐƚŚĞůƉŚĂŝŵĂŐĞƌΡĞůĞĐƚƌŽŶŝĐƵůƚƌĂ-violet

photographic equipment and software present in the York University biology department.

DNA bands corresponding to the expected size of PCR-amplified DNA were excised

under UV light using a clean razor blade and then extracted using the QIAQuick Gel

ǆƚƌĂĐƚŝŽŶ<ŝƚΞ;YŝĂŐĞŶͿĨŽůůŽǁŝŶŐƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛprotocol. The purified PCR product (2

ʅ>ͿǁĂƐƚŚĞŶƌĞ-run on an agarose gel as above, to verify that there was a sufficient yield of

DNA for subsequent DNA sequencing (see below).

2.6 Molecular Cloning

2.6.1 Cloning of PCR Product

If there was insufficient DNA yield for direct sequencing, the PCR product was concentrated into a smaller volume using DNA precipitation with Pellet Paint© following the

ŵĂŶƵĨĂĐƚƵƌĞƌ͛Ɛprotocol (Novagen).

Cloning was done using the pT7-Blue-3 Perfectly Blunt Cloning Kit© (Novagen) following

ƚŚĞŵĂŶƵĨĂĐƚƵƌĞƌ͛ƐƉƌŽƚŽĐŽů͘ŵŝŶŝŵƵŵŽĨϮϬŶŐŽĨWZƉƌŽĚƵĐƚǁĂƐƵƐĞĚŝŶĞĂĐŚĞŶĚ conversion reaction to ensure an optimal insert to vector molar ratio of 1:1 to 2.5:1. All ligation reactions were incubated for 2 hours in an attempt to maximize transformation

ĞĨĨŝĐŝĞŶĐǇ͘ŽƚŚϮϬʅůĂŶĚϱϬʅůŽĨƚƌĂŶƐĨŽƌŵĂŶƚƐǁĞƌĞƉůĂƚĞĚŽƵƚƚŽŝŶĐƌĞĂƐĞƚŚĞůŝŬĞůŝŚŽŽĚ that successfully transformed colonies would appear on each plate.

X-gal plates (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) ĐŽŶƚĂŝŶŝŶŐϯϬʅŐͬŵů of amƉŝĐŝůůŝŶĂŶĚϯϬʅŐͬŵů/Wd';/ƐŽƉƌŽƉǇůɴ-D-1 thiogalactopyranoside) were used to screen transformants for the presence of an insert. Plates were incubated for 12-ϭϱŚŽƵƌƐĂƚϯϳȗ

ƚŽĂůůŽǁĨŽƌĐŽůŽŶǇŐƌŽǁƚŚ͘dŚĞƉůĂƚĞƐǁĞƌĞƚƌĂŶƐĨĞƌƌĞĚƚŽĂϰȗƌĞĨƌŝŐĞƌĂƚŽƌĨor 3 hours to allow for color development. Using this method darkly stained colonies do not possess an insert.

2.6.2 Plasmid Isolation

Successful transformants were selected from the plated colonies and harvested for growth in 16x125 mm glass tubes containing 3ml of LB broth (0.03g tryptone, 0.015g yeast

ĞǆƚƌĂĐƚ͕Ϭ͘ϬϬϯŐEĂů͕Ɖ,ϳ͘ϱͿĐŽŶƚĂŝŶŝŶŐϯϬʅŐͬŵůĂŵƉŝĐŝůůŝŶ͘dŚĞĐƵůƚƵƌĞƐǁĞƌĞƚŚĞŶ grown

ŽǀĞƌŶŝŐŚƚ;ΕϭϮŚŽƵƌƐͿĂƚϯϳȗǁŝƚŚǀŝŐŽƌŽƵƐƐŚĂŬŝŶŐ͘

Plasmids were isolated from overnight cultures using the alkali lysis miniprep method of

Maniatis et al (1989). The bacteria were first pelleted by centrifugation at 13,000rpm for one minute, and the LB medium was removed by aspiration. The resultant pellet was

ƌĞƐƵƐƉĞŶĚĞĚŝŶϭϬϬʅůŽĨ^ŽůƵƚŝŽŶ/;ϱϬŵDŐůƵĐŽƐĞ͕ϭϬŵDd͕ϮϱŵDdƌŝƐ-HCl pH 8.0) and incubated at room temperature for 5 minutes. Then, ϮϬϬʅůŽĨ^ŽůƵƚŝŽŶ//;ĨƌĞƐŚ͘ϮEEĂK, and 1% SDS) was added to each sample, and the contents gently mixed and stored on ice for

ϱŵŝŶƵƚĞƐ͘EĞǆƚ͕ϭϱϬʅůŽĨƐŽůƵƚŝŽŶ///;ϯDƉŽƚĂƐƐŝƵŵϱDĂĐĞƚĂƚĞ͕Ɖ,ϰ͘ϴͿǁĂƐĂĚĚĞĚĂŶĚĂůů samples were gently vortexed in an inverted position to mix the contents thoroughly, and then stored on ice for 5 minutes. The samples were then centrifuged at 13,000rpm for 5 minutes and the supernatant was transferred to a fresh 1.5ml microcentrifuge tube.

A single phenol/chloroform extraction was performed followed by centrifugation at

13,000rpm for 2 minutes. The supernatant was transferred to a clean 1.5ml microcentrifuge tube. Two volumes of 95% ethanol was then added, the samples were gently vortexed and spun (13,000rpm) for 5 minutes in a microcentrfuge. The supernatant was discarded, the pellet rinsed with 70% ethanol and then centrifuged for 2 minutes at 13,000rpm. The supernatant was again discarded and the pellets were left to air dry. Once dry, each pellet

ǁĂƐƌĞƐƵƐƉĞŶĚĞĚŝŶϱϬʅůŽĨϬ͘ϭydĐŽŶƚĂŝŶŝŶŐϭʅůŽĨϮϱŵŐͬŵůZEĂƐĞĂŶĚŝŶĐubated at

ϯϳȗĨŽƌŽŶĞŚŽƵƌ͘

A sample of the plasmid DNA was then digested with the ECORI (New England Biolabs)

restriction enzyme to confirm that it contained a DNA band of the appropriate size. The

isolated plasmid DNA ;ϮʅůͿ was added to an appropriate amount of ECORI RXN buffer and

ECORI restriction enzyme and ddH2Ϭ͕ĂŶĚŝŶĐƵďĂƚĞĚĂƚϯϳȗĨŽƌŽŶĞŚŽƵƌ (the reaction was

ŐĞŶĞƌĂůůǇĐĂƌƌŝĞĚŽƵƚŝŶĂĨŝŶĂůǀŽůƵŵĞŽĨϮϱʅů͕ĐŽŶƚĂŝŶŝŶŐϮ͘ϱʅůZyEďƵĨĨĞƌ͕ϱƵŶŝƚƐŽĨ

enzyme and the remainder as ddH20). The sample was then run on an agarose gel (as

above). If the size of the insert was correct, the sample was sent to the Molecular Core

Facility (York University) for DNA sequencing, using universal plasmid primers.

2.7 DNA Sequencing

Automated DNA sequencing was completed in the Molecular Core Facility at York

University, using BigDye Terminator chemistry on the Applied Biosystems 3130xL DNA

Sequencer. Sequencing runs were performed using primers ITS 4 and ITS 5, for the ITS

amplifications (sequencing both strands), or ndhF-F4 and ndhF-R7 (sequencing both strands)

for the ndhF amplifications. If identical sequences were not obtained in the first runs

additional sequences of either ITS 4 or ITS 5, for ITS sequences, or ndhF-R7 were run to

obtain a consensus. When a plasmid insert was sequenced, universal plasmid primers were

used.

2.8 DNA Sequence Analysis

DNA sequences were aligned using CLUSTALX (Thompson et al. 1997). The alignment

parameters were set according to those recommended in Hall (2001). For the pairwise

alignment parameters, gap opening was set to 10.00 and gap extension to 0.20, and delay

divergent sequences was set to 25%. The resultant alignment was then further adjusted

manually using Mesquite (Maddison and Maddison 2009). All sequence gaps were treated

as missing data in phylogenetic analyses, unless otherwise indicated.

2.9 Phylogenetic Analyses

Phylogenetic analyses were undertaken using a number of different programs and

methods. MrBayes (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003), was

used for Bayesian inference of phylogeny. Parsimony analyses were undertaken using TNT

(Golobof et al. 2008). Maximum likelihood and parsimony analyses were undertaken using

PAUP 4.0b (Swofford 2001).

Analyses using MrBayes were completed using files prepared using alignments exported

from mesquite (Maddison and Maddison 2009). These files were adjusted to allow the

specification of an outgroup prior to completing the Bayesian phylogenetic analyses. The

evolutionary model was set to GTR (general time reversible) with gamma-distributed rate

variation across sites. The program was run with 10000000 generations with a burnin

frequency of 25% of the total generations, ƚŚĞ͚ĐŚĂŝŶƐ͛ǁĞƌĞƐĂŵƉůĞĚĞǀĞƌǇϭϬϬϬ

generations.

Several methods of analyzing the alignments were completed using TNT. Prior to

completing any analysis an appropriate outgroup was defined for the analysis being

undertaken. Parsimony analyses were undertaken using equal character weights and

random addition sequences branch swapping (each with TBR). TNT allows for the use of

highly effective algorithms capable of finding minimum length trees extremely quickly.

These algorithms were employed in this study to quickly find the minimum length of trees in

each data set. New technology analyses were undertaken with all options checked. Set

minimum length was set to 1000 (or more); the perturbation phases were set to 5 and 20

iterations in the ratchet algorithm; drift was set to 20 cycles. Additionally, trees were

resampled using bootstrapping and jackknifing to calculate uncertainty values around a

most parsimonious tree.

For maximum likelihood analyses, a parsimony analysis was first completed to provide

the program with trees to use as a starting point in seeking the most likely tree. A general

time reversible model (GTR+G) and six substitution types was used for additional branch

swapping. A bootstrap procedure with 1000 replicates and ten random sequence additions

per replicate was performed to investigate internal clade support.

2.10 Mapping Breeding System

Breeding system was mapped on phylogenies using MacClade 4.05 (Maddison and

Maddison 2000), following the methodology of Xiang et al. (1998). A data matrix of breeding system states was created. ǇƐĞůĞĐƚŝŶŐ͞ƚƌĞĞĚŝƐƉůĂǇǁŝŶĚŽǁ͟ƵŶĚĞƌ͞display͟ in MacClade, a random tree was generated. The topology of this tree was then manually modified to match the topology of the consensus tree generated by either TnT (Golobof et al. 2008), PAUP (Swofford

2001), or MrBayes (Huelsenbeck 2003). For the purposes of this thesis the loss of distyly was treated as equal to the gain, since it is unclear whether differential weighting is appropriate.

3.0 Results

DNA extractions from fresh tissue were the most successful, consistently yielding a large quantity of high quality of DNA . The majority of the DNA, however, was extracted from dried leaf tissue extracted from herbarium specimens, and the success of PCR amplification appeared to be related to the age and method of drying, or poisoning, to which each sample was subjected (Tables 1 and 2). Dried leaf samples that were extremely old or had been heated or treated with pesticides, yielded DNA of inferior quality (highly sheared) or no DNA at all.

Table 1: Summary of the number of species/subspecific taxa with distyly, homostyly or both breeding systems in all genera of the Turneraceae. The 12 taxa with ambiguous or unknown breeding systems have not been included. Number of Number of Species Number of Total number of species/subspecific with ndhF Species with ITS Distyly and species/subspecific taxa in our successfully successfully Genus Distyly Homostyly Homostyly taxa collection sequenced sequenced See Truyens et al. Turnera 109 21 5 135 50 5 2005 Piriqueta 38 6 4 48 30 28 29 Adenoa 0 1 0 1 1 1 0 Erblichia 0 5 0 5 3 2 1 Hyalocalyx 1 0 0 1 1 0 0 Loewia 2 1 0 3 2 0 0 Mathurina 0 1 0 1 1 1 0 Stapfiella 1 2 0 7 4 1 1 Streptopetalum 5 3 0 6 3 0 1 Tricliceras 12 3 1 19 15 2 1 Family Wide 168 42 10 226 101 40 33

Table 2: Taxa, collection number, and DNA sequence successfully amplified and sequenced in this study.

Species Collection Number ndhF ITS Adenoa cubensis Zuloaga 9600 Y Y Erblichia bernariana Madagascar Brulfert & Bardot-Vaucoulois 2712 Y N Erblichia odorata Janzen 2008 Y Y Erblichia odorata Morales 6055 N Y Mathurina penduliflora DFHJJ 2 Y Y Piriqueta abariana Arbo - Nunes 112 Bahia Y N Piriqueta abariana Giulietti et al. 2192 Rio de Contas. Bahia, Brazil Y Y Piriqueta asperfolia Unknown Y Y Piriqueta aurea Arbo et al. 3849 Y Y Piriqueta australis Unknown Y Y Piriqueta breviseminata Arbo et al. 3526 Y Y Piriqueta capensis Codd 3619 Y Y Piriqueta cistoides ssp. caroliniana Unknown, Florida, USA Y Y Piriqueta carnea Tourinho et al. 34. Gentio do Ouro, Bahia, Brazil Y Y Piriqueta carnea Arbo et al. 5330 Y N Piriqueta constellata Passos et al. PCD 4849 Y N Piriqueta constellata Harley & Giulietti 54638. Mucuge, Bahia, Brasil Y Y Piriqueta densiflora Arbo et al. 7490 Y N Piriqueta densiflora Guedes et al. PCD 3088. Y N Piriqueta dentata Arbo et al. 5417 Y N Piriqueta douradinha Borba 1872 Y Y Piriqueta duarteana Arbo et al. 7613 Y Y Piriqueta duarteana var. duarteana Harley & Maycoorm PCD 3771 Y N Piriqueta duarteana var. ulei Goneicoo 1588 Y Y

Piriqueta grandifolia Solís Neffa et al. 1924 Y Y Piriqueta guianensis - subsp. elongata Araujo F. S. 1349. Crateus, Ceara, Brazil Y Y Piriqueta morongii Panseri et al. 31/2 Y Y Piriqueta mortonii Harting 11 Y Y Piriqueta nanuzae Arbo et al. 5767 Y Y Piriqueta ochroleuca Gonzalez et al. 17 Y Y Piriqueta racemosa Arbo et al 5503 Y Y Piriqueta revoluta Giulietti et. al. 2385. Rio de Contas, Bahia, Brazil Y Y Piriqueta rosea Dematteis et al. 2907 Y Y Piriqueta sarae Queiroz 7143 Y Y Piriqueta sidifolia var. multiflora Arbo et al. 3863 Y Y Piriqueta sidifolia var. multiflora Souza et al. 28286 Y N Piriqueta sidifolia var. sidifolia Arbo et al. 3802 Y Y Piriqueta tamberlikii Arbo et al. 3789 Y Y Piriqueta taubatensis Unknown N Y Piriqueta viscosa Arbo Y Y Stapfiella usambarica Borhidi 85625 Y Y Streptopetalum serratum Friis I et al 9002 N Y Tricliceras brevicaule Napper 2187 N Y Tricliceras lobatum Bidgood et al. 1211 N Y Tricliceras tancetifolium Onderstall 1060 N Y Tricliceras longipedunculatum BGZ 8747 Y Y Tricliceras longipedunculatum var. longipedunculatum Balkwill & Cadman 2494 Y Y Turnera oculata Mendes 120 Y Y Turnera oculata var. paucipilosa Giess 9377 Y Y Turnera oculata var. paucipilosa Kotze 118 Namibia Y Y Turnera thomassii Gillett and Newbould 19205 Kenya Y Y

Turnera capitata Cordeiro 2750 Y Y Turnera diffusa Ricters Herbs Y Y Turnera panamensis Unknown Y Y Turnera subulata(BRY) Barrett and Shore 1374 Y Y *- Incomplete sequence Y= Successfully Sequenced. N= Not successfully Sequenced

Table 3: Species included in the present study, their breeding system and the tissue from which DNA was isolated. Species Breeding System Tissue Adenoa cubensis Homostylous Fresh Material Erblichia bernariana Homostylous Dried Leaf Erblichia odorata Homostylous Dried Leaf Erblichia odorata Homostylous Dried Leaf Mathurina penduliflora Homostylous Dried Leaf Piriqueta abariana Distylous Dried Leaf Piriqueta abariana Distylous Dried Leaf Piriqueta asperfolia Distylous Dried Leaf Piriqueta aurea Distylous Dried Leaf Piriqueta australis Distylous Dried Leaf Piriqueta breviseminata Distylous Dried Leaf Piriqueta capensis Homostylous/Distylous Dried Leaf Piriqueta cistoides ssp. caroliniana Distylous Fresh Material Piriqueta carnea Distylous Dried Leaf Piriqueta carnea Distylous Dried Leaf Piriqueta constellata Distylous Dried Leaf Piriqueta constellata Distylous Dried Leaf Piriqueta densiflora Distylous Dried Leaf Piriqueta densiflora Distylous Dried Leaf Piriqueta dentata Distylous Dried Leaf Piriqueta douradinha Distylous Dried Leaf Piriqueta duarteana Distylous Fresh Material Piriqueta duarteana var. duarteana Distylous Dried Leaf Piriqueta duarteana var. ulei Distylous Dried Leaf Piriqueta grandifolia Distylous Dried Leaf Piriqueta guianensis ssp. elongata Distylous Dried Leaf Piriqueta morongii Homostylous/Distylous Dried Leaf Piriqueta mortonii Homostylous Dried Leaf Piriqueta nanuzae Distylous Dried Leaf Piriqueta ochroleuca Distylous Dried Leaf Piriqueta racemosa Homostylous/Distylous Dried Leaf Piriqueta revoluta Distylous Dried Leaf Piriqueta rosea Distylous Dried Leaf Piriqueta sarae Distylous Dried Leaf Piriqueta sidifolia var. multiflora Distylous Dried Leaf Piriqueta sidifolia var. multiflora Distylous Dried Leaf

Piriqueta sidifolia var. sidifolia Distylous Dried Leaf Piriqueta tamberlikii Distylous Dried Leaf Piriqueta taubatensis Distylous Dried Leaf Piriqueta viscosa Homostylous Fresh material Stapfiella usambarica Distylous Dried Leaf Streptopetalum serratum Homostylous Dried Leaf Tricliceras brevicaule Distylous Dried Leaf Tricliceras lobatum Homostylous Dried Leaf Tricliceras tancetifolium Distylous Dried Leaf Tricliceras longipedunculatum Distylous Dried Leaf Tricliceras longipedunculatum var. longipedunculatum Distylous Dried Leaf Turnera oculata Homostylous Dried Leaf Turnera oculata var. paucipilosa Homostylous Dried Leaf Turnera oculata var. paucipilosa Homostylous Dried Leaf Turnera thomassii Homostylous Dried Leaf Turnera capitata Distylous Dried Leaf Turnera diffusa Distylous Fresh material Turnera panamensis Distylous Fresh material Turnera subulata Distylous Fresh material

3.1 Amplification of ndhF and ITS

Universal oligonucleotide primers for the ndhF gene of the Turneraceae were designed and successfully synthesized (Table 2; Figure 4). In agarose gels (figure 4) the upper bands were cut, cleaned as described in the methods, and sequenced. The lower band is self-annealing primer. The primers ndhF-f4 and ndhF-r7 are able to amplify throughout the family, but were most effective within the genera Piriqueta and Turnera. For other genera, the primer pair ndhF- f5 and ndhF-r4, produced more consistent results (table 2). Using different combinations of these primers (ndhF-f7, ndhF-r7 or ndhF-f5, ndhF-r7) I was able to amplify DNA for 48 species as indicated in table 3. The remainder of the species in the list (Table 2) were either amplified but failed to sequence properly, were shown to be contaminants, or samples had insufficient DNA to perform successful PCR.

For ITS, the primers ITS-4 and ITS-5 were successful in amplifying DNA throughout the family (Table 3). Of the 48 samples which had ndhF sequenced, 40 also had ITS successfully sequenced as well. An additional 6 samples failed to have ndhF sequence but had ITS successfully sequenced. There were DNA samples sequenced for ndhF but unsuccessful for ITS.

These were either purposely not sequenced since they had been previously, not sequenced since samples of the same species had been sequenced, had insufficient DNA, or sequencing was attempted but failed. The internal primers ITS 2 and ITS 3 were tested, and worked well, but since the exterior primers worked well they were not used (Figure 4).

Table 4: Conserved oligonucleotide primers for amplification of the ndhF gene. These were designed to universally amplify a portion of the ndhF gene in the Turneraceae. Primer Name Sequence ndhF-f4 CCG CAG TAA TTT TGA AAA TGC ndhF-r4 GCA TTT TCA AAA TTA CTG CGG ndhF-f5 GTA ACC ACG ATT ATA GGA CCA ATC ndhF-r5 GGA ACA TAC ATA TCA ATA TTT ATG G ndhF-r6 GGG AAT TAG TTG GAA TGT GCT C ndhF-r7 GCT CGA CTT CTT CCT CTT TTC ndhF-f7 GAA AAG AGG AAG AAG TCG AGC

Table 5: Taxa included in this study and their native range. New World/ Species Old World Native Range Adenoa cubensis New World Cuba (endemic) Erblichia bernariana Old World Madagascar (endemic) Erblichia odorata New World Central America Mathurina penduliflora Old World Rodriguez Island (endemic) Piriqueta abariana New World South America (Brazil) Piriqueta asperfolia New World South America (Brazil) Piriqueta aurea New World South America (Brazil) Piriqueta australis New World South America (Argentina, Paraguay) Piriqueta breviseminata New World South America (Brazil) Piriqueta capensis Old World Southern Africa (South Africa, Zimbabwe, Swaziland, Mozambique) Piriqueta cistoides ssp. caroliniana New World North America, Caribbean, Central America Piriqueta carnea New World South America (Brazil) Piriqueta constellata New World South America (Brazil) Piriqueta densiflora New World South America (Brazil) Piriqueta dentata New World South America (Brazil) Piriqueta douradinha New World South America (Brazil) Piriqueta duarteana New World South America (Argentina, Bolivia, Brazil) Piriqueta duarteana var. duarteana New World South America (Argentina, Bolivia, Brazil) Piriqueta duarteana var. ulei New World South America (Brazil) Piriqueta grandifolia New World South America (Argentina, Bolivia, Paraguay) Piriqueta guianensis - subsp. Elongata New World South America (Brazil) Piriqueta morongii New World South America (Paraguay)

Piriqueta mortonii New World Central America (Mexico) Piriqueta nanuzae New World South America (Brazil) Piriqueta ochroleuca New World South America (Paraguay) Piriqueta racemosa New World South America (Brazil, Columbia, Bolivia) Piriqueta revoluta New World South America (Brazil) Piriqueta rosea New World South America (Brazil, Paraguay) Piriqueta sarae New World South America (Brazil) Piriqueta sidifolia var. multiflora New World South America (Brazil) Piriqueta sidifolia var. sidifolia New World South America (Brazil) Piriqueta tamberlikii New World South America (Brazil) Piriqueta taubatensis New World South America (Argentina, Brazil, Paraguay, Uruguay) Piriqueta viscosa New World North America, Central America, Caribbean, South America Stapfiella usambarica Old World Southeast Africa (Tanzania) Streptopetalum serratum Old World Eastern Africa (Eritrea, Ethiopia, Somalia, South Africa, Tanzania) Tricliceras brevicaule Old World Southern Africa Tricliceras lobatum Old World Southern Africa (Angola, Tanzania, Zambia) Tricliceras tancetifolium Old World Southeast Africa (Mozambique) Tricliceras longipedunculatum Old World Africa (Botswana, Malawi, South Africa, Tanzania, Zambia) Tricliceras longipedunculatum var. longipedunculatum Old World Southeastern Africa (South Africa, Swaziland, Mozambique) Turnera oculata Old World Southwest Africa (Namibia, endemic) Turnera oculata var. paucipilosa Old World Southwest Africa (Namibia, endemic) Turnera thomasii Old World Southeastern Africa (Kenya, endemic) Turnera capitata New World South America (Brazil) Turnera diffusa New World Americas (Widespread)

Central America (Belize, Costa Rica, El Salvador, Guatemala, Turnera panamensis New World Honduras, Nicaragua, Panama) Turnera subulata New World Caribbean, Central America, South America

Figure 4: Photo of agarose gels showing successful amplification of both ITS and ndhF in a range of species. Lanes 1, 3, 5, 7, and 11 are of the 1kb plus ladder (New England Biolabs). Lanes 2, 4,

6, and 8-10 are amplifications of a portion of the ndhF gene in the following species: lane 2:

Turnera subulata; lane 4: Piriqueta cistoides ssp. caroliniana; lane 6: Adenoa cubensis; Lane 8:

Piriqueta dentata; Lane 9: Piriqueta viscosa; Lane 10: Piriqueta morongii. Lanes 12-18 are amplifications of the nrDNA ITS region in the following species Piriqueta racemosa (lane 12),

Piriqueta viscosa (lane 13), Piriqueta breviseminata (lane 14), Piriqueta abariana (lane 15),

Piriqueta rosea (lane 16), Piriqueta (lane 17), and Piriqueta capensis (lane 18). Upper bands represent either the successfully amplified ndhF or ITS DNA sequence. The lower band is most likely primer dimer or self annealing primer.

3.2 Sequence Variation

The ndhF sequence employed in this study was a consistent 471bp in all species. ITS sequences ranged from a short length of 584bp in species of Piriqueta to over 638 bp in

Stapfiella usambarica. In species of Passifloraceae the homologous ITS sequences ranged from

639bp to 660bp, and were always longer than the sequences from the Turneraceae.

3.3 Phylogenetic Analyses

Here I present the results of the phylogenetic analyses generated in this study. There are 10 figures of phylogeny based on ndhF, ITS, or combined ndhF and ITS sequence data. In addition to the data, these figures also differ in phylogenetic methodology. Parsimony, maximum likelihood, and Bayesian inference were all used to analyze the data. Due to the complex nature of these results I will proceed methodically through the results displayed in these figures. Below, I first describe the phylogenies displayed in figures 5 through 15. I then give the results of the phylogenetic analyses that pertain to Piriqueta, followed by a general discussion of the remainder of the Turneraceae.

The first figures (Figure 5 through 9) focus on the data for Piriqueta species. A parsimony analysis of the ndhF gene is displayed in figure 5. Next, we see a parsimony analysis of the ITS sequence data (Figure 6). The following phylogeny (figure 7) is a parsimony analysis of combined ndhF and ITS sequence data. A Bayesian analysis based on ITS and ndhF data is shown in figure 8. Finally, the final analysis focusing on Piriqueta is a maximum likelihood analysis of the combined ITS and ndhF sequence data.

The remaining figures (10 through 14) are family-wide analyses. Figure 10 is a parsimony analysis of species from throughout the Turneraceae based on ndhF sequence data. A larger sampling of species is seen in figure 11, a parsimony analysis of ITS sequence. A family wide parsimony analysis of the combined ndhF and ITS sequence is next (Figure 12). Figure 13 is a

Bayesian analysis of ITS data using the same large data set found in figure 11. A 100 replicate maximum likelihood analysis of the ndhF and ITS sequence data is found in figure 14. This figure also indicates the corresponding Bayesian probabilities for the same dataset. Figure 16 is an equally weighted character reconstruction of breeding system in the Turneraceae.

3.31 Piriqueta

No phylogenetic study has been undertaken for Piriqueta, the second largest genus in the Turneraceae. Below, I give the phylogenetic results pertaining to Piriqueta. I first give the results for the parsimony analyses figures 5, 6, and 7, and identify well supported clades in

Piriqueta. I then give results for the Bayesian analysis (Figure 8), and maximum likelihood (Figure

9). Next, I identify the phylogenetic differences among the analysis types, and the general organization of the genus. The geographic origin of each species of Piriqueta can be found in table 5.

Figures 5, 6, and 7 are all 1000 replicate parsimony bootstrap analyses of Piriqueta generated in TnT. Figure 5 displays a phylogeny of the genus, with Passiflora suberosa as an outgroup, based on a section of the cpDNA ndhF gene. Figures 6 is based on ITS sequence data and figure 7 is based on combined ndhF and ITS data. Several clades, varying only slightly in support and species composition are identified in the analyses. In all analyses there are 4

56 separate, well supported, clades identified in Piriqueta. Figure 8 is a Bayesian phylogeny of

Piriqueta based on the combined ndhF and ITS dataset. Figure 9 is a maximum likelihood analysis of Piriqueta based on the same dataset.

The genus Piriqueta has been separated into a number of clades common among the different data sets and phylogenetic methodologies. Considering figure 5 first, the clades have been identified by an assigned letter. The outgroup in this figure is Passiflora suberosa, and several species of Turnera have been included for context. The genus Piriqueta is split into three major clades, labeled here as clade B, clade C, and clade D. There are also two clades nested within clade B, identified as clade A and clade H. All of these clades are consistent throughout the phylogenetic analyses. Clade A, with 78% bootstrapping support, is nested within clade B and is composed of the following species: Piriqueta aurea, Pi. tamberkilii, Pi. australis, Pi. morongii, Pi. grandiflora, Pi. rosea and Pi. ochroleuca. The interior branching of this clade has Pi. aurea and Pi. tamberkilii emerging from a polytomy. Also branching from the same polytomy are the sister taxa Pi. australis and Pi. morongii, and another clade with Pi. grandiflora diverging before the sister taxa Pi. rosea and Pi. ochroleuca. Clade B, with bootstrap support of 73%, consists of clade A as well as Pi. dentata, Pi. constellata, Pi. guiaensis, Pi. mortonii, Pi. viscosa, Pi. breviseminata, Pi. duarteana, Pi. carnea and Pi. nanuzae. Clade H, nested within clade B, has

98% bootstrap support and contains Pi. duarteana, Pi. carnea, and Pi. nanuzae. Clade C is poorly resolved in Figure 5, but has higher support in subsequent analyses, it includes Pi. revoluta, Pi. dourdiana, Pi. sarae, Pi. sidifolia, Pi. abariana, Pi. densiflora and Pi. asperfolia. The interior structure, though poorly resolved, is evident. Piriqueta revoluta is basal, and is followed by the divergence of Pi. dourdinha, Pi. sarae, and then a polytomy. The interior structure of this

57 polytomy has Pi. sidifolia, Pi. abariana, and the sister taxa Pi. densiflora and Pi. asperfolia. Clade

D has 81% bootstrap support and contains only Pi. cistoides ssp. caroliniana and Pi. racemosa. In this analysis the basal clade of the genus is clade D with clade B and clade C being sister clades.

Piriqueta capensis, the lone African member of the genus, diverges prior to the rest of the genus.

Next, we have a parsimony analysis of the ITS sequence data (Figure 6). Unlike the previous figure (figure 5), the basal lineage of the genus is unclear due to polytomy. It is uncertain whether Clade B, Clade C, or the species Piriqueta constellata is basal in this analysis.

The clades discussed above are similar with the following exceptions, Pi. nanuzae is moved from clade B to clade C as the sister taxon to Pi. sarae. Piriqueta constellata is moved from clade B to a polytomy at the base of the genus. Clade A, with bootstrap support of 77, is nested within clade B, which has bootstrap support of 64. Similarly clade H, which is also nested in clade B has

57% bootstrap support. Clade C, with bootstrap support of 99%, emerges at a polytomy with clade D, 91% support, and the species Pi. taubatensis. Once again, Pi. capensis falls outside the rest of the genus.

A parsimony analysis of Piriqueta based on the combined ndhF and ITS dataset is next

(Figure 7). The base of the genus is a polytomy involving clade B, Clade C and clade D. Clade A has 91% bootstrap support, and is nested within clade B, which has 68% bootstrap support.

Clade H is also within clade B and has 99% bootstrap support. Pi. constellata is, once again, basal in clade B. Clade C has 95% bootstrap support and contains Pi. nanuzae in this analysis. Clade D has bootstrap support of 95 and emerges at the polytomy at the base of the genus. Pi. capensis,

58 though clearly belonging in the Turneraceae, is only distantly related to the remainder of the genus.

A Bayesian phylogeny of the genus Piriqueta is next (figure 8). In this figure the monophyly of Piriqueta is clearly supported with a posterior probability of 100. The base of

Piriqueta is a polytomy with clade B, clade C, clade D, and Pi. taubatensis. Clade B, posterior probability of 89, has a polytomy at its base with Pi. constellata, Pi. dentata, and the rest of the clade emerging from the trifurcation. Pi. mortonii diverges next, followed by a polytomy with Pi. viscosa, Pi. guianensis, clade A and clade H. Clade A has a posterior probability of 100, clade H has a posterior probability of 94. Clade A also forms a basal polytomy with Pi. tamberkilii, Pi. aurea, and two clades with strong support. The first clade is composed of the sister taxa Pi. morongii and Pi. australis, with a probability of 100. Clade C has a posterior probability of 99. Pi. nanuzae is basal in this clade. Interior to this there is a polytomy with Pi. abariana, Pi. sidifolia var. sidifolia and two clades containing 3 species each. The first of these clades, with a weak support, has Pi. sidifolia var. multiflora diverging prior to the well supported sister taxa Pi. asperfolia and Pi. densiflora, posterior probability of 99. The other clade has Pi. sarae diverging prior to the sister taxa Pi. dourdinha and Pi. revoluta, with a probability of 77. Finally clade D emerges from the basal polytomy and has a posterior probability of 100.

In a 100 replicate maximum likelihood bootstrap analysis based on the combined ndhF and ITS sequence data (Figure 9)the tree structure is similar to figure 8, but with increased polytomy and lower support values. Pi. taubatensis as well as clades B, C, and D, emerge from a basal polytomy.

To summarize the above findings, the well supported clades A and H are nested within the larger clade B. Clade A is composed of Piriqueta tamberkilii, Pi. aurea, Pi. morongii, Pi. australis, Pi. rosea, Pi. grandiflora, and Pi. ochroleuca. This clade is well supported in all analyses, with bootstrap support of 91 in figure 7 and Bayesian posterior probability of 100 in Figure 8.

Clade H contains Pi. breviseminata, and the sister taxa Pi. carnea and Pi. duarteana. It is strongly supported in all analyses.

Clade B contains clade A, above, as well as clade H. In addition to the species in the nested clades A and H, clade B contains Piriqueta dentata, Pi. constellata, Pi.guianensis, Pi. mortonii, Pi. viscosa, and Pi. breviseminata. In figures 8 and 9, Pi. breviseminata is included in clade H with strong support. Clade B is less well supported than clades A or H, but is present in all phylogenies. The support values range between 68% bootstrap support in figure 7 and a posterior probability of 89 in figure 8.

Clade C is separate from both A and B above. Clade C is composed of Piriqueta nanuzae,

Pi. revoluta, Pi. dourdinha, Pi. sarae, Pi. abariana, Pi. sidifolia, and Pi. asperfolia. This clade is weakly supported in figure 5, with 37% bootstrap support. In other figures, including the more analyses shown in figures 7 and 8, this clade has 95% bootstrap support (Figure 7) and a posterior probability of 99 (Figure 8).

One additional common clade, clade D, is identified in the genus Piriqueta. This small clade is composed of Piriqueta cistoides ssp. caroliniana and Pi. racemosa. This clade is well supported in all analyses with bootstrap support of 95% (figure 7) and a posterior probability of

100 (figure 8).

The monophyly of the genus, with the exception of Piriqueta capensis, is strongly supported in all phylogenetic figures. There is bootstrap support of 100 (figure 7), and a posterior probability of 100 (figure 8). In all analyses Piriqueta section Africana (Piriqueta capensis) does not belong in the genus Piriqueta. Its inclusion in Piriqueta makes the genus paraphyletic.

Piriqueta taubatensis appears to be basal in the genus (Figure 6, figure 7, figure 8, and figure 9), with clade D branching next (Figure 5, 6 and 7). The branching within Piriqueta is uncertain in figures 8 and 9, even with the more robust dataset.

3.32 All Genera of Turneraceae

No phylogenetic analysis has yet been undertaken at the family level in the

Turneraceae. Figures 10 through 14 represent a preliminary attempt to construct a phylogeny and determine the evolutionary history of the family. Figure 15 is a maximum likelihood analysis of the Turneraceae with the branch lengths indicated as number of substitutions per 100bp. For reference, the current taxonomic treatment of the family can be seen in Appendix B, and the geographic origin of each species in this study is found in table 5.

Figure 10 is the result of a 1000 replicate parsimony bootstrap based on ndhF sequence data. Next is a 1000 replicate parsimony bootstrap using the ITS sequence data (Figure 11).

There is a much larger sampling of species in this and subsequent ITS analyses since this includes the sequence obtained by Truyens et al. (2005) as well as additional species sampled in this study. A combined ndhF and ITS 1000 replicate parsimony bootstrap is next (Figure 12).

Following that is a Bayesian inferred phylogeny using the large ITS sequence dataset (Figure 13).

The final phylogeny is a likelihood analysis of the Turneraceae based on the combined ndhF and

ITS dataset (Figure 14). I now proceed through these figures in greater detail identifying major clades and differences among the analyses.

Figure 10 displays a phylogeny of the Turneraceae based on a section of the ndhF gene, bootstrapping support above 50% is noted beside the nodes. This analysis has a polytomy at the base of the family. This polytomy has Piriqueta capensis, the combined clades E and F, and a clade containing both Piriqueta and Turnera, emerging from it. Clade E, which is composed of

Mathurina penduliflora, Erblichia odorata, and Adenoa cubensis in all other analyses, is here joined with clade F, which is composed of the African Turneraceae. Adenoa cubensis and

Turnera capitata are basal to the remainder of the family, but without significant bootstrap support. Turnera then splits from Piriqueta, with 81% bootstrap support. The monophyly of

Piriqueta section Piriqueta is supported( 96% bootstrap support).

Figure 11 includes the sequence data set containing most of the Turnera species analyzed by Truyens et al. (2005). It also contains additional species of Turnera sequenced since that publication, many species of Piriqueta, and the representatives from genera within the

Turneraceae. This figure is based on ITS sequence data, numbers beside the nodes indicated bootstrapping support. Ricinus communis is the outgroup for this analysis. The family is divided into 2 poorly supported lineages in this phylogeny. One, labeled clade F, contains most of the

African genera, including Piriqueta capensis, Stapfiella, Streptopetalum, and Tricliceras. The other is the rest of the family. This is divided between the Piriqueta/Turnera lineage and clade E, made up of island endemics and the sole American member of the genus Erblichia, E. odorata.

In this instance Turnera section Capitatae is nested within the genus Turnera, but basal to rest of the genus, as Truyens et al. (2005) found. The African Turnera, Tu. thomasii and Tu. oculata, are within Turnera series Turnera. Also in Turnera, series Leiocarpe is paraphyletic, with the Tu. sidoides species complex falling outside the remainder of the series. One final note about

Turnera is that there is a large polytomy in the genus with Tu. panamensis, Tu. ignota, and Tu. weddeliana, all species from different series, emerging from the polytomy along with other, well supported, clades. The monophyly of Piriqueta is strongly support.

In figure 12 we see the sole tree found in a 1000 replicate new technology search based on the combined ndhF and ITS sequence data. The monophyly of Piriqueta has 100% bootstrap support. While Turnera, excluding series capitatae, has 89% bootstrap support. Turnera series capitatae is given 70% bootstrap support for inclusion in Piriqueta, although it is basal to the genus. The family is divided into two lineages. One of these lineages houses clades E, 77% bootstrap support, and clade F, 55% bootstrap support. The other lineage of the family contains both Piriqueta and Turnera.

Figure 13 is a Bayesian inference of the phylogeny of the Turneraceae, based on ITS sequence data. This phylogeny is very similar to that seen in figure 11, with the exception that the clades are better supported in the Bayesian analysis. This phylogeny has the Turneraceae forming a trifurcation with 3 separate clades, one with Turnera (with a posterior probability of

73), one with Piriqueta (posterior probability 100), and another composed of clades E and F

(posterior probability of 80). Turnera once again has Tu. thomasii and Tu. oculata nested within

Turnera series Turnera. Turnera series Capitatae is basal to the genus, with Tu. capitata and Tu. maracasana forming a well resolved clade with a probability of 99. Series Leiocarpe is, again,

63 paraphyletic. An additional clade, clade G, is identified in this phylogeny. This clade consists of species from a diverse number of series in the current taxonomic treatment of Turnera. The monophyly of Turnera series Turnera is strongly supported with a posterior probability of 100.

Similarly, the monophyly of Piriqueta section Piriqueta is well supported. A third lineage, with a posterior probability of 80, contains clades E and F. Clade E contains only monomorphic species, it has a probability of 97. Clade F, with a posterior probability of 100, contains the African

Turneraceae, and has at its base Pi. capensis, the only African member of the genus Piriqueta.

Figure 14 shows a 100 replicate maximum likelihood analysis of the Turneraceae based on combined ITS and ndhF sequence data. Bootstrap/posterior probabilities are indicated next to the nodes. A dash (-) indicates support of less than 50%, and polytomy in the maximum likelihood analysis. The sister clades E and F emerge outside the rest of the family with 60% bootstrap support. Clade E has a posterior probability of 94 and 76% bootstrap support. Clade F is well supported with 89% bootstrap support and a posterior probability of 95. The remainder of the family, Turnera and Piriqueta, emerge from a polytomy with poor support. The monophyly of Turnera is strongly supported with 99% bootstrap support and a posterior probability of 98. One caveat to this is that Turnera capitata is basal in Piriqueta, with 87% bootstrap support and a posterior probability of 99. The monophyly of Piriqueta, is strongly supported with both bootstrapping and posterior probability of 100. Within Piriqueta the clades identified earlier are once again strongly supported.

3.4 Breeding System Evolution

Figure 16 shows an equally weighted (loss/gain of distyly) character map of the evolution of breeding systems in the Turneraceae. This figure is based on the consensus tree from the ITS sequence data, since that analysis had the largest number of taxa represented. A separate matrix containing one character was created using MacClade 4.05 (Maddison and

Maddison 2000) and then a random tree was generated. The topology of this tree was then changed to match that of the consensus tree and breeding system was mapped. The outgroups,

Ricinus and Passiflora are from the Euphorbiaceae and Passifloraceae and are not distylous.

Based on character mapping (Figure 16) it appears that prior to the divergence of the

Turneraceae the ancestral breeding system is homostyly. If distyly is ancestral then it has arisen only once in the family, and subsequently broken down on several occasions. If, however, the ancestral character state is homostyly, then there are one or possibly two separate episodes of the evolution of distyly in the Turneraceae. There is one appearance of distyly in the African taxa, and another in the New World taxa.

In Piriqueta, it seems that secondary homostyly has arisen independently within the genus at least 1 occasion, and this number grows to 4 times when species with both homostylous and distylous populations are included. In Turnera homostyly has arisen on at least three separate occasions. In the African lineage, clade F, the ancestral state is equivocal meaning that it is unclear on how many occasions homostyly has arisen independently. In clade

E the ancestral state is equivocal, but the entire clade is homostylous. If the ancestral state of this clade is distyly then it has broken-down on only one occasion. If, however, homostyly is ancestral, then this clade could represent remnants of the ancestral breeding system condition in the Turneraceae.

Figure 5: Unweighted Parsimony analysis of primarily Piriqueta species completed using a 1000 replicate new technology search in TNT. This is based on ndhF sequence data and only one tree that was found, tree length is 243, and where branch length is 0 the tree has been collapsed.

Bootstrap support values are indicated beside the nodes. CI= 0.764, RI= 0.778.

Figure 6: Parsimony analysis of ITS sequence data from Piriqueta. Completed using a 1000 replicate new technology search in TNT, tree shown is the strict consensus of the six trees found. Tree length is 490 and nodes have been collapsed where branch length is 0. Numbers beside the nodes indicate bootstrap support values. CI= 0.754, RI= 0.729.

Figure 7: A parsimony analysis of combined ndhF and ITS sequence data of Piriqueta. Tree length is 622 and is the strict consensus of the 27 trees found during a 1000 replicate new technology search. Trees have been collapsed where branch length = 0. Numbers beside the nodes indicate the bootstrap support values. CI= 0.751, RI= 0.736.

Figure 8: Bayesian Phylogeny of Piriqueta based on the combined ITS and ndhF dataset.

Numbers beside the nodes represent the posterior probabilities for each clade.

Figure 9: A 100 bootstrap replicate maximum likelihood analysis of Piriqueta based on combined

ITS and ndhF DNA sequence data. A 50% majority consensus tree was generated using the GTR

+G model of sequence evolution. The numbers beside the nodes indicate bootstrap support values.

Figure 10: Unweighted parsimony analysis of ndhF from genera within the Turneraceae.

Consensus of 5 trees found, the best score was 198, generated using TnT (Golobof et al. 2008).

Bootstrap support values are found beside the nodes. CI= 0.757, RI= 0.871.

Figure 11: Consensus of a family wide sample of ITS sequence data, generated via a 1000 replicate new technology search in TnT (Golobof et al. 2008). In the analysis 72 trees were found and the best tree length was 1831, all taxa sampled in this study and by Truyens et al. (2005) have been included. CI= 0.454, RI= 0.712.

Figure 12: Results of a 1000 replicate new technology search, generated using TnT (Golobof et al. 2008) based on combined ndhF and ITS sequence data. Genera from throughout the

Turneraceae have been included. The best score was 1767 and only one tree was retained.

Numbers beside nodes indicate the bootstrap support. CI= 0.593, RI=0.710.

Figure 13: Bayesian Inference of the phylogeny of the Turneraceae based on ITS sequence data, all taxa sampled in this study as well as those sampled by Truyens et al. (2005) have been included. Clade posterior probability support values are indicated beside the nodes.

Figure 14: Results of a maximum likelihood bootstrap analysis of the Turneraceae based on combined ITS and ndhF DNA sequence data. A 50% majority consensus tree was generated by

100 bootstraps using the GTR +G model of sequence evolution. The first number beside the nodes indicate bootstrap support values, the second number is the posterior probability. A slash

(-) indicates a support value of less than 50%.

Figure 15: Maximum Likelihood branch lengths in the Turneraceae. The branch lengths are given as number of substitutions per 100 base pairs.

Figure 16: ITS analysis of the entire family, from figure 11, with breeding system mapped using MacClade (Maddison and Maddison 2000), following the method of Xiang et al. (1998).

4.0 Discussion

The Turneraceae is composed of 10 genera and 226 species and subspecific taxa. There are two regions of high diversity, one in central South America and the other in tropical East

Africa. The majority of the species in the family are in two genera Turnera (c. 128 species) and

Piriqueta (45 species). The rest of the species are distributed among the other 8 genera in the family. Adenoa is a monotypic genus endemic to Cuba. Erblichia contains 1 species in Central

America and 4 in Madagascar. Mathurina is monotypic and endemic to Rodriques Island.

Hyalocalyx is monotypic and native to Mozambique. Loewia contains 3 species distributed in

Eastern Africa. Stapfiella has 7 species and Streptopetalum has 6 species, and both are from eastern Africa. Tricliceras is the most species rich of the African genera with 19 species and subspecific taxa. The phylogenetic relationships of all of the genera and taxa in the Turneraceae are currently unresolved, with the exception of a portion of the genus Turnera, studied by

Truyens et al. (2005). The current taxonomic relationships within the family are shown in appendix B (Arbo 1995).

Sequences of both ITS and the cpDNA ndhF gene were used to infer a preliminary phylogeny of the family. There are few old world species included in this study because samples of high quality are difficult to obtain. The majority of those I attempted to PCR amplify were from very old herbarium specimens. Here, I consider the findings of this work and state any conclusions that can be drawn from it. I begin with a discussion of the results using different phylogenetic analyses and any differences found using the different methods or DNA sequence data. Next, I discuss the evolutionary relationships between the genera in the Turneraceae using ndhF, ITS, and combined DNA sequence data. I then discuss how the results relate to the

78 intercontinental disjunctions in the family. Next, I discuss the evolutionary history of breeding system in the family, and finish with the conclusions that can be made as well as future directions for this and related work.

4.1 Phylogenetic Analyses

The majority of the phylogenies generated during this study were created using parsimony in TnT (Golobof et al. 2008). Additional phylogenies were created using MrBayes

(Huelsenbeck and Ronquist 2001), for Bayesian inference, and PAUP 4.0b (Swofford 2001) for maximum likelihood. All types of analysis produced similar results and major clades both within genera and more generally were identified. These clades were consistent and, usually, well supported in all analyses. I now discuss the overall evolutionary relationships among the genera in the Turneraceae, followed by an analysis of the identified clades and their impact on the taxonomic classification of the Turneraceae.

4.12 Evolutionary relationships among genera

All phylogenies generated for the current study used outgroups from either the

Passifloriaceae (Passiflora) or the Euphorbiaceae (Ricinus). Both families are in the same order as the Turneraceae, the Malpighiales (APG II 2003; Appendix C). These outgroups are appropriate since these families are known to be closely related to the Turneraceae (APG II

2003; Appendix C). The use of Ricinus as the outgroup in some analyses is pertinent because it has been proposed that the Turneraceae are nested within the Passifloraceae. However,

79 analyses in the current study have found that the Passifloriaceae have remained sister to the

Turneraceae (Appendix C).

All genera in the Turneraceae, excluding Hyalocalyx and Loewia, are represented in the phylogenies discussed here. Attempts to extract and amplify DNA from dried leaf samples of these genera were unsuccessful.

The family is divided into three or four major clades, depending on the data and analysis. One contains the genus Turnera. One contains Piriqueta section Piriqueta. One is composed of the majority of the mainland African species, Clade F. The last is composed of island endemics and Erblichia odorata, clade E. The latter clade, however, may not contain all taxa from the genus Erblichia because E. bernariana, from Madagascar, had ndhF successfully amplified and was not sister to E. odorata, suggesting they may not comprise a monophyletic group (Figure 10; Appendix D).

4.13 Evolution of African Lineage

This study represents the first time that the African Turneraceae have been analyzed phylogenetically. The geographic origin of all species sequenced in this study can be found in

Table 5. In all phylogenetic analyses, excluding the family-wide ndhF analysis (Figure 10), the mainland African Turneraceae form a clade of their own, identified in figures 10 through 14 as clade F. This clade has GC support value of 57 in Figure 11, 66 in figure 12, and a posterior probability of 100 in the Bayesian phylogeny (figure 13). In figures 13 and 14, clade F diverges with clade E, from a trifurcation at the base of the family. Clade F contains members of the genera Stapfiella, Streptopetalum, Tricliceras, and Piriqueta capensis (Figure 13). These genera

80 represent the bulk of the species found in the east African hub of species diversity. These species represent diverse growth forms ranging from sub-shrubs, through herbaceous perennials, to annuals. Their breeding systems also are diverse, including species that are homostylous, distylous, or have separate populations that are either homostylous or distylous.

Unfortunately, compatibility, breeding system, and inheritance have not been studied in this group so few inferences about these traits can be made. The branch lengths in these clades are also generally longer than they are in the New World species, even within genera (e.g.

Tricliceras, Figure 15).

The taxonomic treatment of African species has been sparsely updated, usually in the form of the addition of a new ƐƉĞĐŝĞƐ͕ƐŝŶĐĞƚŚĞϭϵϱϬ͛Ɛ;>ĞǁŝƐϭϵϱϰ͖ĂůƚŚŽƵŐŚƐĞĞƌďŽϭϵϳϵͿ͘

The scope of the current study does not allow for in-depth analysis of the evolutionary relationships in this clade. However, the branching structure within clade F, in the ITS Bayesian analysis has strong posterior probabilities (Figure 13). Along with the ITS parsimony analysis

(Figure 11), it appears that Pi. capensis is basal in the clade. Stapfiella diverges next, followed by the sister genera Streptopetalum and Tricliceras. The internal structure of Tricliceras is poorly supported, with a posterior probability of 56, and bootstrapping support of 49%. Any inferences on the evolution of breeding system in this group will be made in section 4.3.

4.14 Evolution of Piriqueta

In every phylogeny generated during this study Piriqueta is polyphyletic. Piriqueta section Piriqueta and section Africana form two distinct lineages. Section Africana contains only

Piriqueta capensis, which always falls into a clade F with most of the mainland African species

(table 5). Based on the data presented here, it would be prudent to move Piriqueta capensis to a different genus, one of the existing African genera, or to create a new monotypic genus for this taxon.

In the phylogenetic analyses presented here, Piriqueta has been broken down into clades that consistently group together. These clades have been labeled Clade A, Clade B, Clade

C, Clade D, and Clade H, based on the order in which they were identified. Three of these clades,

Clade B, Clade C and clade D are very consistent across all analyses and are likely representative of the different lineages in Piriqueta. The current taxonomic treatment of the genus (Arbo 1995) divides Piriqueta into only two sections, Piriqueta and Africana. I propose that morphological characters uniting these species should be explored, while section Africana is transferred to a different genus.

4.15 Evolution of Turnera

Evolutionary events in Turnera have been discussed by Truyens et al. (2005), so, only new information will be discussed. The African Tu. thomasii and Tu. oculata are nested within series Turnera, as their classical treatment predicted (Arbo 2005). This was unexpected, since it was assumed that the similarities between the African and American Turnera might be due to homoplasy, rather than shared common ancestry. To ensure that this result was not due to DNA contamination, each species was sequenced three times. The sequence similarities between the

American and African Turnera indicates one of two scenarios. The first is that the proliferation of

Turnera species in the New World is due to rapid speciation by an invading African lineage.

Second, is that these species represent a recent invasion of Africa by species of Turnera series

Turnera. The DNA sequence data, short branch lengths (Figure 15), secondarily homostylous characteristics, and large number of related species present in America make the second option more likely. The branch lengths shown on Figure 15, showing much shorter branch lengths in

Turnera and Piriqueta than in the African lineages also supports this option.

The basal lineage of Turnera is series Capitatae, represented in this study by Tu. capitata and Tu. maracasana, as indicated by Truyens et al. (2005). The family-wide ITS phylogenies, however, allows for a broader placement of this series, than did Truyens et al. (2005). The current study includes more species of Turneraceae and the outgroups are more distantly related. In the ndhF analysis (Figure 10) Turnera series Capitatae forms a trifurcation with

Turnera and Piriqueta. In figures 12 and 13, the ITS analyses, this series falls outside Turnera, appearing as a basal lineage of Piriqueta. The same scenario is found in the robust combined ndhF and ITS analyses (Figure 14) with strong support (posterior probability of 99, bootstrap support of 87) for the inclusion of Turnera capitata in Piriqueta. The larger sampling of both

Turnera and other genera, in the ITS analysis allows for a more accurate phylogenetic placement of series Capitatae then was provided in Truyens et al. (2005). The above data certainly indicate that Turnera capitata might not properly included in the genus Turnera. However, additional genetic data and increased taxon sampling from series capitatae is necessary to determine if the series, and all species in this series, have been treated properly classified.

In common with Truyens et al. (2005) the ITS data suggests that several of the series of

Turnera are polyphyletic. Series Leiocarpe is represented by both the Tu. siddoides species complex, as well as a second clade that includes Tu. hasslariana and Tu. pumilea (Figure 11;

Figure 13). Both of these well-supported lineages emerge from a polytomy in Figure 11. In the

Bayesian analysis (Figure 13), the clade containing Tu. hassleriana and Tu. pumilea is sister to

Turnera series Turnera. An additional clade, labeled clade G (Figure 11; Figure 13), is identified in the phylogenies presented here. This clade consists of species from a number of different

Turnera series. This clade is well supported (figure 13), with a posterior probability of 79, and the internal supports are even higher. Species not included in this clade in figure 11, Tu. panamensis, and Tu. ignota, are included at the base of this clade (figure 13). Tu. panamensis and Tu. ignota, as well as a third species, Tu. weddeliana, are in Turnera series salicifolia. In figure 11 these species all arise at a polytomy. However, in figure 13 Tu. weddeliana is basal to the family, diverging immediately after series capitatae, while Tu. ignota and Tu. pananmensis are basal in clade G. The other species in clade G are classified in different series: Tu. diffusa and Tu. calyptrocarpa in series microphyllae, Tu. cearensis and Tu. bahiensis in series anomalae, and Tu. chamaedrifolia in series papilliferae. Unfortunately, the species classified in the same series are not sister in figures 11 and 13. In these figures Tu. diffusa is sister to Tu. chamaedrifolia, with strong bootstrap support of 94%, and a Bayesian probability of 100. The other lineage in clade G, with 94% bootstrap support (Figure 11), has Tu. bahiensis diverging prior to the sister taxa Tu. cearensis and Tu. calyptrocarpa. Additional DNA sequence data is needed before revision of the series, using morphological characters, can be undertaken.

4.16 Evolution of Island Endemics

Perhaps the most intriguing clade in terms of evolutionary origin, labeled clade E in analyses, contains Mathurina, Adenoa, and the Central American Erblichia odorata (table 5). All

84 of these species are homostylous. This group, labeled clade E in the analyses above, is represented by two island endemic species, Mathurina penduliflora, from Rodriquez Island, and

Adenoa cubensis, from Cuba. The lone American species of the genus Erblichia, E. odorata, from

Central America is also in this clade. This clade is well resolved in Figures 11, 12, 13 and 14.

However, in ndhF analyses it is included with the African lineage, clade F (Figure 10). In the combined ndhF and ITS analyses this clade is well supported (Figure 12; Figure 14), as it is in the

Bayesian ITS analysis (Figure 13). The consensus seems to be that this clade is most closely related to clade F, the mainland African species.

The intriguing aspect of this clade is that the representatives are not species that fit the description of traditional secondarily-homostylous species. Schoen et al. (1997) described derived homostylous members of the partially distylous genus Amsinckia (Boraginaceae) as colonizing plants of roadsides, pastures, and agricultural fields (i.e. disturbed sites). Similar traits are associated with derived homostylous Turneraceae, though large flowers, mixed mating, and residual dialleic incompatibility systems have been noted in Turnera (Belaoussoff and Shore

1995; Tamari et al. 2001). These plants still tend to be small plants primarily of disturbed sites.

Adenoa, Mathurina, and Erblichia odorata are all trees or large shrubs. E. odorata is a shrub or tree growing up to 27 meters in height. Mathurina is a tree growing to 12 meters. Adenoa is a shrub growing to 3 meters in height (Arbo 2007). These species all also have large flowers, limited distribution, and do not rapidly spread to disturbed habitats. The incompatibility systems of these species have not been tested, so it is unclear if they are predominately outcrossing.

Erblichia odorata is known to be pollinated by hummingbirds, suggesting that this species is outcrossing (Arbo 2006). Given the habit, attributes, and stature of these taxa, it seems unlikely

85 that these plants represent a breakdown product of heterostyly. It is possible that these represent the ancestral condition of the family, and are primary homostyles. However, the character mapping analysis (Figure 16) indicates that the base of the family is equivocal, meaning that it is unclear whether the families last common ancestor was distylous or homostylous. It is possible that they diverged from the rest of the Turneraceae prior to the establishment of heterostyly in the family, or that this clade failed to develop heterostyly while the others did. Certainly this possibility is worthy of further study.

The only Erblichia species that had both ITS and ndhF successfully sequenced was E. odorata. However, E. bernariana did have ndhF successfully sequenced (Table 2; Figure 11;

Appendix D) and in it diverges after E. odorata. In addition a parsimony analysis was done on the combined ndhF and ITS sequence data including all species with at least one gene sequenced

(appendix D). In this analysis E. bernariana is most closely related to clade E (Appendix D). Also in clade E in this analysis one Tricliceras, Tr. Longipedunculatum, with only ndhF sequence data also fell out with clade E, and not with the remainder of Tricliceras. However, before any conclusions can be drawn from this placement, additional data is needed for a more robust analysis.

4.2 South American and African Disjunctions

The Malpighiales (APG II 2003) have been deduced to be no older than 125 million years

(Davis et al. 2005). There has been great diversification of the families within Malpighiales since that time, and we can assume that the Turneraceae diverged more recently than this date.

Africa and South America were last connected approximately 105 million years ago (Davis et al.

2004). It has been hypothesized that the Passifloriaceae clade (including both the

Malesherbiaceae and the Turneraceae) diverged from the remainder of the Malpighiales around

100 million years ago. The Malesherbiaceae split from the lineage around 70 million years ago, then Turneraceae split from the Passifloraceae around 60 million years ago(Davis et al. 2005).

Nowak (2002) argued that the age of the Malesherbiaceae is closer to 40 million years based on climatic, genetic, and distribution data. She also hypothesized that the Malesherbiaceae were most closely related to the Turneraceae, which is unlikely given the analysis of Davis et al.

(2005). However, in preliminary investigations using ITS sequence data, the current study has found some support for EŽǁĂŬ͛Ɛ theory (Appendix C). Again, this general phylogeny could benefit from the sequencing of additional genes, to make it more robust. The Turneraceae have no fossil record, making it difficult to estimate a time frame of divergence. The 60 million year estimate, based on genetic divergence and fossil evidence, given by Davis et al. (2005) will be used as a starting point for discussion of the disjunctions.

Three genera within the Turneraceae, Erblichia, Piriqueta, and Turnera, contain disjunct species between South America and Africa. Erblichia has 4 species in Madagascar and 1 in

Central America. Piriqueta has 44 species in the New World and 1 in the Southern Africa.

Turnera has c. 128 species in the New World and 2 in Southern Africa (Shore et al. 2006; table

5). The disjunct Piriqueta capensis seems to be most closely related to other mainland African genera. The genetic data indicates that this species is not properly placed with the remainder of the American Piriqueta section Piriqueta. Figures 9, 10, 11, 12, 13, and 14 indicate that Pi. capensis is most closely related to the African species of Turneraceae. These figures also indicate

87 that the African lineages are basal lineages in the family. It is unclear whether this lineage is more closely related to the outgroup due to a more recent shared common ancestor, or due to earlier divergence. If the latter is true, it would indicate that the ancestral homeland of the

Turneraceae is in Southern Africa, and that a later invasion of the New World by the family allowed for rapid speciation and accounts for the large number of species in Turnera and

Piriqueta. The divergence among the majority of the Old and New World taxa suggests that these lineages have been separated for a long time. The species in clade E may represent remnants of previous expansions by the family. Looking at the branch lengths (Figure 15), the species in Turnera and Piriqueta do seem to be more closely related than those in the African lineage. The long branch lengths in clades E and F, both within and among genera, indicate that these clades have been separated longer than have the New World taxa.

The phylogenetic placement of Mathurina, Erblichia odorata, and Adenoa cubensis, in figures 9, 10, 11, 12, 13 and 14 gives credit to the notion that these taxa are remnants of the spread of the Turneraceae. These taxa are geographically isolated from each other, yet retain many similarities. In the case of Erblichia these similarities have placed species in the same genus even though they are found on opposite sides of the world. In addition, Balfour, in his description of Mathurina, noted that its closest living relative was the, at that time, monotypic

Erblichia odorata of Central America (Balfour 1876). This suggests that clade E possibly consists of remnants of the invasion event or events that resulted in the family spreading from continent to continent. The long branch lengths in this clade support the notion that these species are remnants of past expansion, since we expect that nucleotide substitutions would increase with

88 time since divergence (Figure 15). The identification of fossil evidence of the Turneraceae would greatly help in inferences on the origin and ramification of the family.

The phylogenetic position of the African members of the genus Turnera, Tu. thomasii and Tu. oculata, is confirmed in every phylogeny in which these species were included. It is known that when the same species occupies several continents, its proliferation has usually been anthropogenically introduced (Renner 2004). However, it is not often that species within the same genus, let alone the same series of a genus, are found on opposite sides of an ocean.

Renner (2004), attributes within genus disjunction to recent invasion events, and given the extremely low number of substitutions found between Tu. oculata, Tu. thomasii, and related

New World taxa; we found 10 nucleotide differences between the African Turnera and Tu. subulata in c. 500 bp. of ndhF, 5 of which are shared between the African taxa. The ITS sequences are also very similar with 15-20 nucleotide differences and deletions among the species. Looking at the branch lengths (Figure 15) it is clear that these taxa have recently diverged from the rest of Turnera series Turnera. It is likely that these species represent a very recent invasion of the African continent by a largely American lineage. It is unclear what sort of time frame this number of nucleotide substitutions represents, but it is clear that Tu. thomasii and Tu. oculata represent the most recent, and perhaps only, non-anthropogenic reinvasion of

Africa by New World Turneraceae.

4.4 Evolution of Breeding System

Based on character mapping (Figure 16) it appears that prior to the divergence of the

Turneraceae the breeding system is homostylous. However, the selection of outgroup, taxon

89 sampling, and character weighting have guided this decision. For the Turneraceae the ancestral condition is equivocal, meaning that it is unclear given the taxon sampling and distribution of the distylous species among the clades. In both Turnera and Piriqueta section Piriqueta, it appears that distyly is the ancestral condition, and that all homostyles in these genera are the result of the breakdown of distyly. Ancestral breeding system in the African lineage, clade F, and the homostylous clade E, is equivocal with equally weighted gain: loss of breeding system.

In Piriqueta section Piriqueta, distyly is the ancestral condition and appears to have broken down on at least 3 separate occasions within the genus, and two of these breakdown events have occurred within species (Figure 16). The occurrence of homostyly in Pi. viscosa and

Pi. mortonii may have been from the same evolutionary event since these species are sister taxa

(Figure 16). The homostylous populations of Pi. morongii and Pi. racemosa likely arose in independent events. The addition of extra homostylous taxa: Pi. cistoides ssp. cistoides, Pi. hapala, Pi. mexicana, and Pi. venezuelana, as well as species with both distylous and homostylous populations, such as Pi. assuruensis, to this analysis would allow better insight into the number of separate origins of homostyly in the genus.

Evolutionary events in the genus Turnera have been discussed by Truyens et al. (2005), however, the addition of extra taxa here allows for further insight into the genus. The ancestral state for the genus is distyly (Truyens et al. 2005; Figure 16), with homostyly being derived on at least 5 separate occasions with 2 separate origins of homostyly within species, and the remaining 3 in separate taxa. The scenario displayed in Figure 16, in series Turnera in particular, is unlikely to be the true scenario, because it shows polyploids giving rise to diploid species.

Within series Turnera, it appears that distyly has broken down, then reappeared later. The most

90 likely scenario is that homostyly has appeared one or more times in the polyploid taxa, while the diploid taxa retained distyly, since there is no known mechanism for polyploids to revert to the diploid state.

The appearance of homostyly in Tu. pumilea and Tu. candida, in separate lineages are almost certainly derived homostyly, as are the occurrences of homostylous populations of both

Tu. chamaederfolia and in the Tu. sidoides complex. This study agrees with Truyens et al. (2005) that distyly in the ancestral condition within the genus.

It is possible that distyly arose independently in both the Old World and New World. The differences in the distylous breeding system between Turnera and Piriqueta section Piriqueta, and that of the distylous African lineage may represent convergent evolution, and, with equal weighting, this is a possibility, since the base of the family is equivocal (Figure 16). However, as mentioned in the introduction the loss of distyly is considered more difficult evolutionarily than the gain.

If it is more difficult to gain distyly than to lose it (Schoen et al. 1997), then the most likely scenario is that distyly arose once in the Turneraceae. This would have had to occur before the family was split between the New and Old World. However, the question of the homostylous clade E remains. It is know that these plants are homostylous (Arbo 1977; Arbo

1979; Arbo 2006), and that they do not fit into the description of derived homostylous species

(Schoen et al. 1997), the ancestral state of these species remains in question. Are they representative of the ancestral breeding system? The character reconstruction with equal weighting indicates that the lineage containing this clade is equivocal, meaning the ancestral

91 condition is unclear (Figure 16). However, if the weighting is changed to 2:1, or if Piriqueta capensis is treated as distylous (it contains different populations that are either homostylous or distylous) then the ancestral breeding system condition for this clade is distyly. As mentioned earlier, however, the size, probable outcrossing, and unclear incompatibility systems indicate that these species are more similar to primary homostyles then secondary homostyles. To clarify this situation, it is recommended that the incompatibility systems and additional genetic data be obtained for further study. If it can be clearly shown that the identified putative primary homostyles are representative of the ancestral condition then testing of the hypotheses put forth by Charlesworth and Charlesworth (1979) and Lloyd and Webb (1992) can be undertaken.

4.4 Conclusions

This study represents the first attempt to classify the family Turneraceae phylogenetically. It is also one of the few studies to look at evolution and breakdown of distyly using phylogenetic techniques on a family-wide scale. Analysis of DNA sequence data from ITS and ndhF have led to the identification of species that potentially represent the ancestral breeding system condition in the family. The phylogenies in this study include representative taxa from 8 of the 10 genera within the Turneraceae. Future studies should attempt to include both Hyalocalyx and Loewia. The phylogenies generated were used to address breeding system history, family history, and disjunction both within genera and in the Turneraceae in general.

Detailed analyses of the genus Piriqueta led to the conclusion that, at least in section

Piriqueta, the ancestral condition is distyly and that homostyles are representative of the breakdown of this breeding system. In addition it was discovered that one member of the genus

Piriqueta, Piriqueta capensis, has been improperly placed in the genus, and should be either moved to another genus or a new genus. Turnera capitata often is located at the base of the genus, including in the robust dual gene analyses. It is unclear whether Turnera series capitatae should be moved to Piriqueta, retained in Turnera, or moved to a new genus.

Analysis of the genus Turnera indicates that series Capitatae is the basal clade. However, this series often does not always form a strong clade with the remainder of the genus. The two

African members of the genus Turnera were found to have been properly treated taxonomically, as they were found to be most closely related to series Turnera. The genetic information indicates that these species represent a recent reinvasion of Africa by the New World genus

Turnera. The most likely scenario is that these species represent a very recent introduction of

Turnera into Africa from South America. The current taxonomic treatment of Turnera, is not supported by the ITS sequence data. It is recommended that this genus be revised taxonomically, and that additional DNA sequence be obtained for phylogenetic analysis. A more robust data set would help with any ambiguity in the ITS analyses in the current study, and in

Truyens et al. (2005).

The phylogenetic placement of several isolated taxa, mostly island endemics, Adenoa cubensis, Erblichia odorata, and Mathurina penduliflora, indicate that these could represent a remnant of the ancestral condition on the Turneraceae. These potential primary homostyles should be studied in more detail, and whether they possess an incompatibility system should be established as a means of testing the hypotheses of the evolution of distyly.

There is limited information to support the break up of the genus Erblichia. This genus, with one species in Central America and 4 in Madagascar, is homostylous, and there is a possibility that all of these species belong in the clade discussed above. However, more phylogenetic information on the genus is necessary before any conclusions can be drawn.

Further resolution of the origin and breakdown of distyly can be accomplished using more thorough taxon sampling, inclusion of other rapidly evolving genes or intergenic spacers, and single or low copy genes. Distyly has broken down in Piriqueta on at least three occasions, in

Turnera on at least three occasions, and on at least one occasion in the African lineage, clade F.

Prior to the divergence of the Turneraceae the breeding system was homostylous, based on the outgroups, but the breeding system of the most recent common ancestor is equivocal. Since the divergence distyly has arisen one, or more, times. The ancestral breeding system condition of clade E, could help to determine whether homostyly is ancestral or derived in this clade.

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Appendices: Appendix A: Comprehensive List of all taxa within the Turneraceae (Shore et al. 2006):

Adenoa Arbo Erblichia Seeman A. cubensis (Britton & Wilson) Arbo E. antsingyae (Capuron) Arbo E. bernieriana (Tulasne) Arbo Piriqueta E. integrifolia (Claverie) Arbo Piriqueta Section Piriqueta Arbo E. madagascariensis O. Hoffman Pi. abairana Arbo E. odorata Seeman. Pi. araguaiana Arbo E. odorata var. mollis (Standley and Steyermark) L.O. Williams Pi. asperfolia Arbo Pi. assuruensis Urb. Mathurina Balf. Pi. aurea (St.-Hillaire, Jussieu & Cambess.) Urb. M. penduliflora Balf. Pi. australis (Urb.) Arbo Pi. breviseminata Arbo Hyalocalyx Rolfe Pi. caiapoensisArbo H. setiferus Rolfe Pi. carnea Urb. Pi. cistoides (L.) Grisebach subsp. caroliniana (Walter) Arbo Loewia Urb. Pi. cistoides (L.) Grisebach subsp. cistoides Arbo L. glutinosa Urb. Pi. constellata Arbo L. microphylla (Chiov.) Roti-Mich Pi.corumbensisMoura L. tanaensis Urb. Pi. cristobaliae Arbo Pi. dentata Arbo Pi. densiflora Urb. Stapfiella Gilg Pi. densiflora var. goiasenis Arbo Sta. claoxyloides Gilg Pi. douradina Arbo Sta. lucida var. lucida Robyns Pi. duarteana var. duarteana (St.-Hillaire, Jussieu & Cambess.) Urb. Sta. lucida var. pubescens B. Verdcourt Pi. duarteana var. ulei Urb. Sta. muricata Staner

Pi. emasensis Arbo Sta. ulugurica Mildbr. Pi. flammea Arbo Sta. usambarica J. Lewis Pi. grandifolia (Urb.) Arbo Sta. zambesiensisR. Fernandes Pi. guianensis subsp. Guianensis N.E. Brown Pi. guianensis subsp. elongata (Urb. And Rolfe) Arbo Pi. hapala Arbo Pi. lourteigiae Arbo Streptopetalum Hochst. Pi. mesoamericana Arbo Str. arenarium Thulin Pi. mexicana Fryxell & Koch Str. graminifolium Urb. Pi. morongii Rolfe Str. hildebrandtii Urb. Pi. mortonii Koch & Fryxell Str. luteoglandulosum R. Fernandes Pi. nanuzae Arbo Str. serratum Hochst. Pi. nitida Urb. Str. wittei Staner Pi. ochroleuca Urb. Pi. plicata Urb. Tricliceras Thonn ex DC. Pi. racemosa (Jacq.) Sweet Tr. auriculatum(A & R Fernandes) R. Fernandes Pi. revoluta Arbo Tr. bivinianum (Tul.) R. Fernandes Pi. rosea (St.-Hillaire, Jussieu & Cambess.) Urb. Tr. brevicaule var. brevicaule R. Fernandes Pi. sarae var. saraeArbo Tr. brevicaule var. rosulatum (Urb.) R. Fernandes Pi. sarae var. glabrescens (Urb.) Arbo Tr. elatum(A & R Fernandes) R. Fernandes Pi. scabrida Urb. Tr. glanduliferum(Klotzsch) R. Fernandes Pi. sidifolia var. multiflora Urb. Tr. hirsutum(A & R Fernandes) R. Fernandes Pi. sidifolia var. sidifolia (St.-Hilaire, Jussieu & Cambess.)Urb. Tr. lacerata (Oberm.) Oberm. Pi. suborbicularis (St.-Hillaire & Naudin) Arbo Tr. lanceolatum(A & R Fernandes) R. Fernandes Pi. subsessilis Urb. Tr. lobatum(Urb.) R. Fernandes Pi. sulfurea Urb. & Rolfe Tr. longipedunculatum (Mast) R. Fernandes Pi. tamberlikii subsp. rotundiflora (St-Hillaire, Jussieu & Cambess) Arbo Tr. longipedunculatum var. eratense R. Fernandes Pi. tamberlikii subsp.. tamberlikii Urb. Tr. mossambicense (A & R Fernandes) R. Fernandes

Pi. taubatensis (Urb.) Arbo Tr. pilosum (Willd.) R. Fernandes Pi. undulataUrb. Tr. prittwitzii (Urb.) R. Fernandes Pi. venezuelana Arbo Tr. schinzii var. schinzii (Urb.) R. Fernandes Pi. viscosa Grisebach subsp. viscosa Urb. Tr. schinzii var. juttae (Urb.) R. Fernandes Pi. viscosa subsp. tovarensis Urb. Tr. tancetifolium (Klotzsch) R. Fernandes Tr. xylorhizum B. Verdcourt Piriqueta Section Africana Arbo Series Stenodictyae Pi. capensis (Harv.) Urb. Tu. acuta Willd. Ex Schult Tu. annectens Arbo Turnera Tu. aurantica Benth. Turnera Series Salicifoliae Tu. benthamiana M.R. Schomb. Tu. amapensis R.S. Cowan Tu. castilloi Arbo Tu. brasiliensis Wiild ex. Schult Tu. cicatricose Arbo Tu. clausseniana Urb. Tu.longipes Urb. Tu. glaziovii Urb. Tu. macrophyllae Urb. Tu. hindsiana subsp. hindsiana Benth Tu. urbanii Arbo Tu. hindsiana subsp. brachyantha Arbo Tu. ignota Arbo Series Annulares Tu. panamensis Urb. Tu. annularis Urb. Tu. rupestris var. rupestris Aubl. Tu. aromatica Arbo Tu. rupestris var. frutescens Urb. Tu. breviflora Moura Tu. serrata var. serrata Vell. Tu. odorata Richard Tu. serrata var. brevifolia Urb. Tu. serrata var. latifolia Urb. Series Leiocarpe Tu. steyermarkii Arbo Tu. acaulis Griseb. Tu. venosa Urb. Tu. argentea Arbo Tu. weddeliana Urb. & Rolfe Tu. callosa Urb. Tu. cipoensis Arbo

III

Series Capitatae Tu. crulsii Urb. Tu. albicans Urb. Tu. curassavica Urb. Tu. capitata Cambess. Tu. dasytricha Pilger Tu. dasystyla Urb. Tu. dichotoma Gardner Tu. hatschbachii var. hatschbachii Arbo Tu. dolichostigma Urb. Tu. hatschbachii var. miniata Arbo Tu. elliptica Urb. Tu. maracasana Arbo Tu. foliosa Urb. Tu. marmorata Urb. Tu. genistoides Cambress. Tu. pernambucensis Arbo Tu. goyazensis Urb. Tu. princeps Arbo Tu. guianensis Aublet Tu. schomburgkiana Urb. Tu. harleyii Arbo Tu. waltheriodes Urb. Tu. hassleriana Urb. Tu. hilaireanab Urb. Tu.huberi Arbo Tu. incana Cambress Series Microphyllae Tu. lamiifolia Cambress Tu. asymmetrica Arbo Tu. lanceolata Cambress Tu. calyptrocarpa Urb. Tu. lineata Urb. Tu. collotricha Arbo Tu. longiflora Cambress Tu. diffusa var. diffusa Willd ex. Schult. Tu. luetzelburghii Sleumer Tu. diffusa var. aphrodisiaca Urb. Tu. melanorhiza Urb. Tu. hebepetala Urb. Tu. melochia Triana Tu. melochioides Cambress Tu. nana Cambress Series Papilliferae Tu. nervosa Urb. Tu. caatingana Arbo Tu. oblongifolia Cambress Tu. chamaedrifolia Cambress Tu. opifera Mart. Series Turnera Tu. pinifolia Cambress

Tu. arcuata Urb. Tu. parauana Arbo Tu. aurelii Arbo Tu. pohliana Urb. Tu. caerulea subsp. Caerulea DC. Tu. prancei Arbo Tu. caerulea subsp. surinamensis Obermeyer Tu. pumilea var. pumilea L. Tu. campaniflora Arbo Tu. pumilea var. pumilea Urb. Tu. candida Arbo Tu. revoluta Urb. Tu. concinna Arbo Tu. sidoides subsp. sidoides L. Tu. coriacea Urb. Tu. sidoides subsp. carnea Arbo Tu. cuneiformis Poir. Tu. sidoides subsp. holosericea Arbo Tu. grandidentata Arbo Tu. sidoides subsp. integrifolia Arbo Tu. grandiflora Arbo Tu. sidoides subsp. pinnatifida Arbo Tu. hermanniodes Cambress. Tu. stachydifolia Urb. & Rolfe Tu. joelii Arbo Tu. subnuda Urb. Tu. krapovickasii Arbo Tu. tenuicaulis Urb. Tu. leptosperma Urb. Tu. trigona Urb. Tu. lucida Urb. Tu. uleana Urb. Tu. oculata Story Tu. rubrobracteata Arbo Tu. oculata var. paucipilosa Arbo & Fernandez Tu. orientalis Arbo Series Annomalae Tu. purpurascens Arbo Tu. bahiensis Urb. Tu. scabra Millspaugh Tu. blanchetiana var blanchetiana Urb. Tu. simulans Arbo Tu. blanchetiana var. subspicata Urb. Tu. stenophylla Urb. Tu. cearensis Urb. Tu. subulata Smith Tu. chrysocephala Urb. Tu. thomasii Story Tu. gardneriana Arbo Tu. ulmifolia var. ulmifolia L. Tu. stipularis Urb. Tu. ulmifolia var. acuta Urb. Tu. tapajoensis moura Tu. velutina Presl.

Appendix B: Current taxonomic treatment of the Turneraceae modified from Arbo (1996):

Appendix C: Relationship of the Turneraceae to related families based on ITS sequence data. Parsimony Bootstrap generated using TnT, support values are indicated at the nodes.

Hollrungia Passiflora suberosa 100 Passiflora candida Paropsia madagascariensis Malesherbia linearifolia 99 86 100 Malesherbia turbinea Malesherbia weberbaueri 98 Piriqueta capensis Stapfiella usambarica 100 97 Streptopetalum serratum 92 Tricliceras brevicaule 99 80 Tricliceras tanacetifolium Tricliceras lobatum Adenoa cubensis 72 Mathurina penduliflora 100 83 Erblichia odorata 97 Erblichia odorata 100 Piriqueta aurea 100 Piriqueta viscosa 88 Turnera subulata Turnera panamensis

VII

Appendix D: Complete analysis of all species with at least one gene sequenced. 1000 replicate New Technology bootstrap. Clade supports over 50% are shown beside the nodes.

P.suberosa T.pananmensis T.ignota T.weddelliana T.sidoides ssp. integrifolia 99 T.sidoides T.sidoides ssp. pinnatifida 85 T.sidoides ssp. carnea T.hassleriana_ 80 T.pumilea 54 T.nervosa T.melochioides 88 T.opifera T.diffusa 99 T.chamaedrifolia 74 T.bahiensis 98 T.calyptocarpa 100 T.cearensis T.hermaniodes T.stenophylla T.joelii T.coerulea T.coerulea var. surinamensis 100 T.candida_ T.fernandezi_ 96 61 T.grandiflora T.velutina 100 T.thomasii T.scabra_ T.oculata T.subulata 79 Ζd͘ƐƵďƵůĂƚĂ͚Zz͛ 74 T.occidentalis 90 T.orientalis T.campaniflora 98 T.ulmifolia T.krapovickasii 69 T.cocinna 96 T.grandidentata T.maracasana 95 T.capitata A.cubensis E. .odorat ͚Janzen' 85 82 E.odorata 76 E.bernieriana 72 Tr.longpedunculatum 55 89 M.penduliflora Sta.usambarica P.capensis 65 Str.serratum 97 Tr.brevicaule 80 Tr.lobatum 58 Tr.tanacetifolium P.dentata P.sidifolia_var. sidifolia P.densiflora P.taubatensis P.asperfolia P.sidifolia var. multiflora P.abairana P.nanuzae P.sarae P.dourdinha P.revoluta P.mortonii 99 P.constellata P.viscosa P.breviseminata P.guianensis P.racemosa 96 P.caroliniana P.duarteana var. ulei 70 P.duarteana P.carnea P.tamberlikii P.aurea P.grandifolia 55 P.rosea 90 P.ochroleuca P.morongii 91 P.australis

VIII

Figure 1: The genetic polymorphisms of distyly. The compatible pollinations are shown using solid arrows, other pollinations (self, short x short, long x long) are incompatible (after Barrett 1992).

Figure 3: Diagrammatic representation of the ndhF gene with the primers designed in this study and their direction noted. The gene is 2223 bp long based on the Nicotiana tabacum genome (Shinozaki et al. 1986).

Figure 4: Photo of gels showing successful amplification of both ITS and ndhF in a range of species. Lanes 1, 3, 5, 7, and 11 are of the 1kb plus ladder (New England Biolabs). Lanes 2, 4, 6, and 8-10 are amplifications of a portion of the ndhF gene in the following species: lane 2: Turnera subulata; lane 4: Piriqueta cistoides ssp. caroliniana; lane 6: Adenoa cubensis; Lane 8: Piriqueta dentata; Lane 9: Piriqueta viscosa; Lane 10: Piriqueta morongii. Lanes 12-18 are amplifications of the nrDNA ITS region in the following species Piriqueta racemosa (lane 12), Piriqueta viscosa (lane 13), Piriqueta breviseminata(lane 14), Piriqueta abariana (lane 15), Piriqueta rosea(lane 16), Piriqueta (lane 17), and Piriqueta capensis(lane 18).

Figure 5: Unweighted Parsimony analysis of primarily Piriqueta species, completed using a 1000 replicate new technology search in TNT. This is based on ndhF sequence data and only one tree that was found, tree length is 243, and where branch length is 0 the tree has been collapsed. Passiflora suberosa Turnera panamensis T.diffusa T.subulata 100 59 T.oculata 94 86 T.thomasii 100 Piriqueta capensis Turnera capitata P.racemosa 75 81 P. cistoides ssp. caroliniana P.revoluta 51 P.dourdiana 37 P.sarae 35 P.sidifolia var. sidifolia 99 P.sidifolia var. multiflora 26 P.abariana 12 85 P.abariana P.densifolia 62 P.asperfolia 79 P.dentata 87 P.dentata P.constellata P.guiaensis 86 P.constellata P.mortonii 73 Pviscosa P.breviseminata P.duarteana P.carnea 51 P.duarteana 98 P.carnea 43 P.duarteana var. duartena P.nanuzae P.aurea P.tamberkilii P.australis 78 81 P.morongii P.grandifolia P.rosea 64 62 P.ochroleuca Figure 6: Parsimony analysis of ITS sequence data from Piriqueta. Completed using a 1000 replicate new technology search in TNT, tree shown is the strict consensus of the six trees found. Tree length is 490 and nodes have been collapsed where branch length is 0. P.suberosa P.capensis T.subulata P.constellata 100 P.taubatensis P.racemosa 91 P.caroliniana 76 55 P.sidifolia var. multiflora P.asperifolia 99 P.abairana P.dourchiana 96 56 P.revoluta P.sarae 51 P.nanuzae P.mortonii P.viscosa P.breviseminata P.guianensis P.duarteana var. ulei 64 57 P.duarteana P.carnea P.tamberkilli P.grandifolia P.aurea 77 P.rosea 81 P.ochroleuca P.australis 61 P.morongii Figure 7: A parsimony analysis of combined ndhF and ITS sequence data of Piriqueta. Tree length is 622 and is the strict consensus of the 27 trees found during a 1000 replicate new technology search. Trees have been collapsed where branch length = 0. P.suberosa P.capensis T.subulata P.racemosa 100 95 P.caroliniana P.nanuzae 59 P.revoluta P.dourchiana 95 P.sarae 100 P.abariana P.sidifolia var. multiflora 55 P.asperifolia P.constellata P.guianensis p.mortonii 68 P.viscosa P.breviseminata 59 P.duarteana 56 99 P.carnea P. aurea P. tamberkillii P. grandifolia 91 P.rosea 94 P.ochroleuca P.australis 98 P.morongii Fig 8: Figure 8: Bayesian Phylogeny of Piriqueta based on the combined ITS and ndhF dataset. P suberosa T subulata P capensis P taubatensis P nanuzae P abariana 99 P sidifolia var sidifolia P sidifolia var. multiflora 58 50 99 P asperifolia P densiflora P sarae 79 7 P dourdinha 7 P revoluta P constellata P dentata 89 P mortonii 100 P viscosa P guianensis 78 P tamberkillii P aurea 100 100 P morongii 50 P australis

98 P grandifolia 100P ochroleuca P rosea

94 P breviseminata 100P carnea P duarteana 100 P caroliniana P racemosa Figure 9: A 100 bootstrap replicate maximum likelihood analysis of Piriqueta based on combined ITS and ndhF DNA sequence data. A 50% majority consensus tree ws generated using the GTR +G model of sequence evolution. The numbers beside the nodes indicate bootstrap support values. P.suberosa P.capensis T.subulata P.taubatensis P.sidifolia var. multiflora P.abariana P.sarae P.dourdinha 91 P.revoluta 55 P.sidifolia var. sidifolia P.nanuzae

59 P.asperifolia P.densiflora P.viscosa p.mortonii P.guianensis 100 P.constellata P.dentata P. tamberkillii 91 P.aurea 95 100 P.morongii P.australis P.grandifolia 70 96 P.ochroleuca P.rosea P.breviseminata 71 100 P.carnea P.duarteana

100 P.caroliniana P.racemosa

ITS Figure 10: Unweighted parsimony analysis of ndhF from genera within the Turneraceae. Consensus of 5 trees found, the best score was 198, generated using TnT (Golobof et al. 2008).

Populus alba Populus trichocarpa Passiflora biflora 100 Adenoa cubensis 100 Erblichia odorata 17 Stapfiella usambarica Erblichia bernieriana 94 7 31 Tricliceras longepedunculatum 88 Mathurina penduliflora 24 Tricliceras longepedunculatum Piriqueta capensis T.capitata Piriqueta cistoides ssp. caroliniana 13 4 98 P.morongii 54 P.asperfolia 29 48 P.sarae Turnera panimensis 92 T.diffusa 54 T. subulata 98 T.oculata 83 T.thomasii Figure 11: Consensus of a family wide sample of ITS sequence data, generated via a 1000 replicate new technology search in TnT (Golobof et al.

2008). In the analysis 72 trees were found and the best tree length was 1831, all taxa sampled in this study and by Truyens et al. (2005) have been included. Ricinus communis Passiflora.suberosa PiriquetaStapfiella capensis usambarica 56 Streptopetalum serratum 16 Tricliceras brevicaule 95 Tricliceras lobatum 100 70 Tricliceras tanacetifolium Adenoa49 cubensis Mathurina pendulifora 57 54 Erblichia odorata 100 83 Erblichia odorata 91 P.cistoidesP.racemosa ssp. caroliniana 6 P.taubatensisP.asperifolia 11 33 P.sidifolia var. multiflora P.abairana 99 P.nanuzae 36 P.sarae 97 40 P.dourchiana 2 P.revoluta P.constellata61 P.mortonii P.viscosa 31 P.breviseminata P.guianensis 47 P.duarteana var. ulei 34 P.duarteana 54 61 P.carnea P.tamberkilli P.aurea 23 P.grandifolia P.rosea 41 P.ochroleuca 65 P.morongii 89 P.australis T.maracasana 96 T.capitata T.pananmensisT.ignota T.weddelliana_ T.sidoides ssp. integrifolia 11 99 T.sidoides 31 T.sidoides ssp. pinnatifidacarnea 79 T.diffusa T.chamaedrifolia 98 T.bahiensis 63 42 T.calyptocarpa 94 99 T.cearensis T.hassleriana T.pumilea 69 18 T.nervosa T.melochioides 39 T.opifera 81 T.candida 1 T.coerulea T.coerulea var. surinamensis 18 99 T.fernandezii 61 84 T.grandiflora 90 T.stenophylla T.joelii 20 T.hermaniodes T.campaniflora 6 95 T.ulmifolia 8 T.orientalisT.occidentalis 83 T.orientalis 61 T.velutina

T.oculataT.thomasii 17 Turneraceae T.subulata 1 62 T.scabra T.subulataT.scabra 6 T.krapovickasii T.cocinna 67 91 T.grandidentata Figure 12: Results of a 1000 replicate new technology search, generated using TnT (Golobof et al. 2008) based on combined ndhF and ITS sequence data. Genera from throughout the Turneraceae have been included. The best score obtained was 1767 and only one tree was retained. Passiflora suberosa Piriqueta capensis Stapfiella usambarica 100 Adenoa cubensis 51 Erblichia odorata 72 Mathurina pendulifera 66 Turnera capitata Piriqueta morongii 79 100 P.cistoides ssp. caroliniana 84 P.asperfolia 99 P.sarae Turnera panamensis 94 T.diffusa 89 T.subulata 100 T.oculata T.thomasii Figure 13: Bayesian Inference of the phylogeny of the Turneraceae based on ITS sequence data, all taxa sampled in this study as well as those sampled by Truyens et al. (2005) have been included. Generated using MrBayes (Huelsenbeck and Ronquist 2001). Ricinus P suberosa T opifera 100 T melochioides 89 T nervosa 100 95 T pumilea T hassleriana T candida 61 T coeruleavsurinamensis 67 100 T grandiflora 100 94 T grandiflora T fernandezii T coerulea T joelii 100 T stenophylla T hermaniodes T ulmifolia 100 T campaniflora 60 T orientalis 100 T orientalis T occidentalis T subulata 100 T subulata 98 100 T subulata 58 62 T scabra T scabra 54 T velutina 100 T grandidentata 56 100 T cocinna T krapovickasii 99 T thomasii Toculata 55 100 T cearensis 100 T calyptocarpa T bahiensis 99 T chamaedrifolia 74 1.00 T diffusa T ignota 99 T pananmensis T sidoidessspcarnea 79 100 T sidoides ssp pinnatifida 73 100 T sidoides ssp integrifolia T sidoides T weddelliana T capitata 99 T maracasana P carnea 100 P duarteana P duarteanaulei 97 P guianensis P breviseminata P australis 100 P morongii 99 P grandifolia 100 52 P ochroleuca 100 P rosea 98 P aurea P tamberkilli 91 P viscosa 84 P mortonii P constellata P revoluta 96 P dourchiana 100 98 P sarae 59 P nanuzae 100 P abairana P sidifoliamultiflora 84 60 P asperifolia 85 P taubatensis P caroliniana 100 P racemosa P capensis Streptopetalumserratum 100 100 56 Tricliceras tanacetifolium 100 Triclicerasbrevicaule 69 Tricliceras lobatum 80 Stapfiella usambarica 100 Erblichiaodorata 100 Erblichia odorata 97 Mathurina penduliflora Adenoa cubensis Figure 14: Results of a maximum likelihood bootstrap analysis of the Turneraceae based on combined ITS and ndhF DNA sequence data. A 50% majority consensus tree was generated by 100 bootstraps using the GTR +G model of sequence evolution. The numbers beside the nodes indicate bootstrap support values. Passiflora suberosa

T.capitata

T.panamensis

98 T. diffusa

96 T.subulata 80 100 T. thomasii 75 T. oculata

P.morongii

100 70 P.cistoides ssp. caroliniana 81 P. sarae 100 P. asperfolia

A. cubensis 85 M. pendulifera 99 E. odorata

P. capensis 59 S. usambarica Figure 15: ITS analysis of the entire family, from figure 11, with breeding system mapped using MacClade (Maddison and Maddison 2000), following the method of Xiang et al. (1998). T.cocinna 8900 T.grandidentata 6109 T.krapovickasii 46355 T.subulata 1374 T.scabra 689 T.scabra 308 T.subulata 298 T.subulata T.thomasii Toculata86concensus T.velutina 309 T.orientalis 312 T.occidentalis T.orientalis 1538 T.ulmifolia Mad009 T.campaniflora 1337 T.hermaniodes 5680 T.joelii 1373 T.stenophylla 979 T.grandiflora 19260 T.grandiflora T.fernandezii 5286 T.coeruleavsurinamensis T.coerulea 1789 T.candida 2588 T.melochioides 1734 T.opifera 4325 T.nervosa 2076 T.pumilea 38624 T.hassleriana 8897 T.diffusa 310 T.chamaedrifolia 3175 T.bahiensis 57 T.calyptocarpa 1479 T.cearensis 53491 T.sidoidessspcarnea 21711 T.sidoides T.sidoidessspintegrifolia 2 T.sidoidesssppinnatifida T.weddelliana 8845 T.ignota 4111 T.pananmensis T.maracasana 224 T.capitata 2750 P.constellata P.mortonii P.viscosa P.guianensis P.breviseminata P.duarteana P.duarteanaulei P.carnea P.ochroleuca P.rosea P.morongii P.australis P.aurea P.tamberkilli P.grandifolia P.dourchiana P.revoluta P.nanuzae P.sarae P.abairana P.sidifoliamultiflora P.asperifolia P.taubatensis P.caroliniana P.racemosa Adenoacubensis Mathurina Eodorata Erblichiaodorata Tricliceraslobatum Triclicerastanacetifolium Triclicerasbrevicaule Streptopetalumserratum Stapfiellausambarica P.capensis P.suberosa Ricinus