Quick viewing(Text Mode)

Phylogenetic Analysis of Vitaceae Based on Plastid Sequence Data

Phylogenetic Analysis of Vitaceae Based on Plastid Sequence Data

PHYLOGENETIC ANALYSIS OF BASED ON PLASTID SEQUENCE DATA

by

PAUL NAUDE

Dissertation submitted in fulfilment of the requirements for the degree

MAGISTER SCIENTAE

in

BOTANY

in the

FACULTY OF SCIENCE

at the

UNIVERSITY OF JOHANNESBURG

SUPERVISOR: DR. M. VAN DER BANK December 2005 I declare that this dissertation has been composed by myself and the work contained within, unless otherwise stated, is my own

Paul Naude (December 2005) TABLE OF CONTENTS

Table of Contents Abstract iii Index of Figures iv Index of Tables vii Author Abbreviations viii Acknowledgements ix

CHAPTER 1 GENERAL INTRODUCTION 1 1.1 Vitaceae 1 1.2 Genera of Vitaceae 6 1.2.1 6 1.2.2 7 1.2.3 8 1.2.4 9 1.2.5 Clematocissus 9 1.2.6 10 1.2.7 11 1.2.8 11 1.2.9 12 1.2.10 13 1.3 The 13 1.4 Previous taxonomic studies on Vitaceae 14 1.5 Main objectives 18

CHAPTER 2 MATERIALS AND METHODS 21 2.1 DNA extraction and purification 21 2.2 Primer trail 21 2.3 PCR amplification 21 2.4 Cycle sequencing 22 2.5 Sequence alignment 22 2.6 Sequencing analysis 23 TABLE OF CONTENTS

CHAPTER 3 RESULTS 32 3.1 Results from primer trail 32 3.2 Statistical results 32 3.3 Plastid region results 34 3.3.1 rpL 16 34 3.3.2 accD-psa1 34 3.3.3 rbcL 34 3.3.4 trnL-F 34 3.3.5 Combined data 34

CHAPTER 4 DISCUSSION AND CONCLUSIONS 42 4.1 Molecular evolution 42 4.2 Morphological characters 42 4.3 Previous taxonomic studies 45 4.4 Conclusions 46

CHAPTER 5 REFERENCES 48

APPENDIX STATISTICAL ANALYSIS OF DATA 59

ii ABSTRACT

Five plastid regions as source for phylogenetic information were used to investigate the relationships among ten genera of Vitaceae. These comprised the tmL intron, trnL-F intergenic spacer, rpL16 intron, rbcL gene and accD- psa/ spacer. Congruent results were obtained between separate, combined and Bayesian analysis with all four major clades being shared among . All bootstrap consensus trees obtained from single sequences or combined analysis suggest that Vitaceae is a monophyletic group with Leea weakly supported as sister to Vitaceae. The results presented provide novel insights into the relationships within ten Vitaceae genera and suggest direction for further studies.

iii INDEX OF FIGURES

Figure 3.5 One of the 8180 most parsimonious trees (TL = 1491, CI 40 = 0.73, RI = 0.67) based on the combined plastid data for 40 of Vitaceae and outgroups. Numbers above the branches indicate Fitch lengths (DELTRAN optimasition) and those below the branches indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree Figure 3.6 Bayesian analysis of the combined plastid data set. One 41 of the 10001 majority rule consensus trees with PP shown above the branches

CHAPTER 4 DISCUSSION AND CONCLUSIONS 42 Figure 4.1 Combined tree indicating the color groups and the 44 morphological characters shared

APPENDIX STATISTICAL ANALYSIS OF DATA 59 Figure A-1 An illustration of the search tree for the branch-and- 66 bound algorithm (Swofford, 1993) Figure A-2 The process of Nearest Neighbour Interchange (NNI) 69 where an interior branch is dissolved and the four subtrees connected to it are isolated. These can then be reconnected in two other ways (after Felsenstein, 2004) Figure A-3 An example of subtree pruning and regrafting (after 70 Felsenstein, 2004) Figure A-4 An example of rearrangement via bisection and (after 71 Felsenstein, 2004) Figure A-5 Three trees (1, 2, and 3) and their strict consensus (4), 74 majority rule consensus (5), and Adams consensus (6) trees (after Felsenstein, 2004)

vi

INDEX OF FIGURES

CHAPTER 3 RESULTS 32 Figure 3.1 One of the 2365 equally parsimonious trees (TL = 290, 36 CI = 0.84, RI = 0.81) found from the analysis of rpL16 sequences for 26 species of Vitaceae and outgroups. Numbers above the branches are Fitch lengths (DELTRAN optimisation), and those below are Fitch bootstrap percentages above 50%. Branches not recovered in the strict consensus are indicated with solid arrows Figure 3.2 One of the 377 most parsimonious trees (TL = 364, CI = 37 0.81, RI = 0.63) based on the analysis of accD psa1 for 22 species of Vitaceae and Leea. Numbers above the branches indicate Fitch lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not recovered in the strict consensus tree Figure 3.3 One of the 414 most parsimonious trees (TL = 545, CI = 38 0.64, RI = 0.63) found from the analysis of rbcL for 38 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not present in the strict consensus tree Figure 3.4 One of the 4560 most parsimonious trees (TL = 346, CI 39 = 0.86, RI = 0.82) from the analysis of trnL-F for 37 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimasation) and the numbers below indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree INDEX OF FIGURES

CHAPTER 1 GENERAL INTRODUCTION 1 Figure 1.1 Taxonomic placement of Vitaceae (Soltis et al., 2003) 2 Figure 1.2 The worldwide distribution of Vitaceae (Heywood, 1993) 2 Figure 1.3 The defining features of the family Vitaceae. Illustrations 5 A-R are of : (A) portion of a flowering ; (B) staminate with dropped off petals; (C) inflorescence opposite a ; (D) flower bud; (E) bisexual flower bud opening with the petals forming a cap; (F) opened bud with pseudoconnate petals falling of; (G) longitudinal section of the ; (H) four seeded , cross section; (I) branch with tendril opposite and an infruitescence; (J) cross section of ; (K) seed; (L) abaxial surface of seed; (M,N) adaxial surfaces of seed; (0) cross section of seed; (P) seed in longitudinal section; (Q, R) embryo. S-W illustrations of V. vulpine: (S) portion of vine with leaf and opposite infruitescence; (T-V) seed abaxial surface; (W) seed adaxial surface (Judd et al., 1999) Figure 1.4 (www. -pictures. corn) 7 Figure 1.5 (www. plant—pictures. corn) 8 Figure 1.6 (www.plant-pictures.com ) 10 Figure 1.7 Parthenocissus quinquefolia (www. plant-pictures. corn) 12 Figure 1.8 Tetrastigma voinierianum (www.plant-picture.corn) 14 Figure 1.9 One of the shortest trees found with successive 17 weighting (SVV). Fitch branch lengths are shown above the branches and Fitch bootstrap percentages are shown below the branches. Solid arrowheads indicate groups not found in the strict consensus tree of the Fitch analysis and open arrowheads indicate groups not found in either the Fitch or the SW consensus tree (Ingrouille et al., 2002)

iv

INDEX OF TABLES

CHAPTER 1 GENERAL INTRODUCTION 1 Table 1.1 A summary of the morphological character diversity in 19 Vitaceae, X — indicates morphological characters that are present (Ingrouille et at., 2002)

CHAPTER 2 MATERIAL AND METHODS 21 Table 2.1 List if taxa with voucher information and GenBank 25 accession numbers ('Albert et al., 1992; 2Ingrouille et al., 2002; 3Savolainen et al., 2000a; 4Savolainen et al., 2000b; 5sequences from this study; *sequences from this study submitted to GenBank but still awaiting accession numbers) Table 2.2 The various denaturing and annealing temperatures with 29 the number of cycles used for each gene in the primer trail Table 2.3 Regions studied and PCR primer sequences 30 Table 2.4 PCR protocols used for the different genes 31 Table 2.5 Unamplified taxa 31

CHAPTER 3 RESULTS 32 Table 3.1 Plastid and nuclear regions tested and their success 32 rates Table 3.2 Statistics from PAUP analysis of separate and combined 33 molecular data matrices

vii AUTHOR ABBREVIATION

Blume Carl(Karl) Ludwig von Blume 1796-1862 Desc. Bernard M. Descoings 1931- DieIs Friedrich Ludwig Emil Diels 1874-1945 Domin Karel Domin 1882-1953 Engelm. Georg (George) Engelmann 1809-1884 Gagnep. Francois Gagnepain 1866-1952 G. Don George Don 1798-1856 Griff. William Griffith 1810-1845 Jackes Betsy Rivers Jackes 1935- J.Kern. Johann Simon von Kerner 1755-1830 J.VahI Jens Laurentius(Lorenz) Moestue Vahl 1796-1854 L. 1707-1778 Lam. Jean Baptiste Antoine Pierre de Monnet de Lamarck 1744-1829 Michx. Andre Michaux 1746-1803 Planch. Jules Emile Planchon 1823-1888 Rupr. Franz Josef (Ivanovich) Ruprecht 1814-1870 Trautv. Ernst Rudolf von Trautvetter 1809-1889 Vahl Martin (Henrichsen) Vahl 1749-1804

viii ACKNOWLEDGEMENTS

I would like to thank Dr. M. Van der Bank for her support and guidance throughout this study. I would also like to thank Dr. V. Savolainen and the personnel at the Jodrell Laboratory at the Royal Botanic Garden Kew, London for their support and guidance. The financial support by the National Research Foundation and the Thuthuka Program is also greatly appreciated. Lastly I would like to thank my wife, Karlien, for her support and patience.

ix CHAPTER 1

GENERAL INTRODUCTION CHAPTER 1 GENERAL INTRODUCTION

The intrageneric relationships within Vitaceae have been uncertain, although many authors have attempted to resolve it (Hooker, 1862; Planchon, 1887; Gagnepain, 1911; Latiff, 1982; Watson and Dallwitz, 1992; Ingrouille et al, 2002; Rossetto et al., 2002). This is mainly due to the lack of floral characters as the are small and very inconspicuous. Morphological characters used at present to distinguish between genera include the structure of the nectar disk, configuration of the endosperm in cross section, and length of the style (Judd et al., 1999). Some highly variable characteristics are used as well such as leaf form, inflorescence structure, and tendril arrangement (Ingrouille et a!, 2002). Rosetto et al., 2002, and Ingrouille et al., 2002, used molecular data from various plastid and nuclear genes to resolve the generic relationships, but still some genera remained unresolved. This chapter describes the morphological traits of the family, as well as the distinguishing morphological characters of each genus. Previous taxonomic studies will also be mentioned.

1.1 Vitaceae The of representatives of Vitaceae is still poorly understood and uncertain. It is often associated with , since both possess opposite the petals, and Takhtajan (1997) placed Vitaceae near Protenae, in . Molecular data do not link it unambiguously with any other core , although they are definitely basal in the group. Soltis et al. (2000) placed it sister to , but only with moderate support, and even this moderate support has weakened in a subsequent four-gene analysis (Soltis et al. 2003). Figure 1.1 illustrates the current placement of the family (Soltis et al., 2003), where Vitaceae is of the order , which is of the class Magnoliopsida, and the division Magnoliophyta.

Distribution — Vitaceae has a widespread distribution, occurring in warmer climates and in some temperate regions (Figure 1.2; Heywood, 1993). The grapevine (Vitis vinifera) is cultivated worldwide, even as far north as the lower Rhineland, Germany (Wild and Drummond, 1963).

1

CHAPTER 1 GENERAL INTRODUCTION

Gunnera CL Myrothamnus

Santales

Saxifragales

Berberidopsidales

- Vitaceae

Rosids

Dilleniaceae

-

Asterids

Figure 1.1 Taxonomic placement of Vitaceae (Soltis et a/., 2003).

Figure 1.2 The worldwide distribution of Vitaceae (Heywood, 1993).

2 CHAPTER 1 GENERAL INTRODUCTION

Morphological features — The defining features (Figure 1.3) of the family are that they are mostly and are easily recognised by their distinctive leaf morphology with the tendrils opposite them (Wild and Drummond, 1963). The tendrils are either modified shoots or inflorescences and may end in disk like suckers (Heywood, 1993). In the case of Vitis (the grapevine) the tendril is negative phototropic, which thus forces it to grow away into crevices and other similar places (Heywood, 1993). In these dark places, the tendrils expand into large tissue balls, which in turn become sticky and mucilaginous and helps the vine to firmly adhere itself to its support (Heywood, 1993). The Vitaceae habit includes twining vines, or small trees (Wild and Drummond, 1963). The (Figure 1.3) are alternate and simple, often palmately lobed and stipulate. It may also be opposite, spiral or two-ranked and palmately or pinnately veined (Figure 1.3). Vitaceae may thus be recognised by their caducously stipulate leaves with palmately compound, -lobed or -veined blades and often rather coarse teeth (Wild and Drummond, 1963). Flowers (Figure 1.3) are bisexual or unisexual and actinomorphic; the inflorescences may be a raceme, panicle, or cyme (Wild and Drummond, 1963). Stamens are opposite the petals and the anthers are dorsifixed, with a narrow transition to the filament and a thin connective (Wild and Drummond, 1963). The gynoecium, as can be seen in Figure 1.3, is syncarpous and consists of two carpels; the ovary is usually superior with one or two per locule (Wild and Drummond, 1963). The is a berry and the berry often has ruminated seeds (Wild and Drummond, 1963). The chromosome number (n) of the family varies between 11-16, 19 and 20 (Verdcourt, 1993).

Economic uses — Vitaceae is known worldwide thanks to its economic uses. This is because of its extreme importance as the source of table , raisins and grapes (Vitis vinifera; Zohary, 1982). Wine is produced by the action of yeast on fructose found in grapes. Grapes have all three because yeast naturally occurs on the skins. It is one of the oldest cultivated , and was grown by the Egyptians 6000 years ago and highly developed by the Greeks and Romans (Hill, 1937). The grapevine originates from the Orient and North West (Heywood, 1993). More than 25 million tons of

3 CHAPTER 1 GENERAL INTRODUCTION wine is produced annually from this species and has become a scientific study (Heywood, 1993). The are also dried resulting in various delicacies such as, raisins or sultanas, if the grape is of a seedless variety (Heywood, 1993).

Vitis vinifera is very susceptible to various fungus, insects and pests particularly root louse (; Hill, 1937). Fortunately the American grapes are less susceptible and are now used as stock on which the European varieties are grafted (Hill, 1937). These American grapes include, , V. rotundifolia, V. vulpina and V. aestivalis (Harrison et al., 1985).

Vitaceae is also commonly cultivated for the climbing ornamentals Virginia creeper and Boston ivy, both from the genus Parthenocissus (Hill, 1937). Various members of Vitaceae are also used medicinally, for instance: Vitis leaf tea for diarrhoea and stomach-aches, and can be applied externally for aches and fevers; Parthenocissus root teas are used for various ailments but may be toxic and leaves are toxic, causing dermatitis in autumn (Altschul, 1973).

4 CHAPTER 1 GENERAL INTRODUCTION

Figure 1.3 The defining features of the family Vitaceae. Illustrations A-R are of Vitis rotundifolia: (A) portion of a flowering vine; (B) staminate flower with dropped off petals; (C) inflorescence opposite a petiole; (D) flower bud; (E) bisexual flower bud opening with the petals forming a cap; (F) opened bud with pseudoconnate petals falling of; (G) longitudinal section of the gynoecium; (H) four seeded ovary, cross section; (I) branch with tendril opposite leaf and an infruitescence; (J) cross section of berry; (K) seed; (L) abaxial surface of seed; (M,N) adaxial surfaces of seed; (0) cross section of seed; (P) seed in longitudinal section; (Q, R) embryo. S-W illustrations of V. vulpine: (S) portion of vine with leaf and opposite infruitescence; (T-V) seed abaxial surface; (W) seed adaxial surface (Judd et a/., 1999).

5 CHAPTER 1 GENERAL INTRODUCTION

1.2 Genera of Vitaceae There are fifteen genera and about 1000 species worldwide with 5 genera and 53 species occurring in southern . The fifteen genera of Vitaceae include: Acareosperma Gagnepain, Ampelocissus Planchon, Ampelopsis Michx, Cayratia Planchon, Cissus Planchon, Planchon, Cyphostemma Planchon, Nothocissus Latiff, Parthenocissus (creeper) Planchon, Pterisanthes Blume, Pterocissus Urb and Eckman, Rhoicissus Planchon, Tetrastigma Planchon, Vitis (grape) L, and C. L. Li. The largest genus is Cissus, with the number of named species depending on the researcher, from 200 (Mabberley 1995), to 250 (Lavie, 1990), to 350 (Galet, 1967). The genera found in include Ampelocissus, Cayratia, Cissus, Cyphostemma and Rhoicissus (Merxmuller and Schreiber, 1969). Genera cultivated in southern Africa include: Ampelopsis, Parthenocissus and Vitis. Table 1.1 is a short summary of the various genera in Vitaceae and their morphological features. The genus Leea is also included into this table as it is closely related to Vitaceae and often included into the family.

1.2.1 Vitis Vitis (grapevine) with 65 species are found in the north temperate regions. Most species are woody, deciduous tendril climbers, but some can be - like (Merxmuller and Schreiber, 1969). Vitis exhibits a scandent habit (Figure 1.4). The genus consists of various leaf arrangements, as can be seen in Table 1.1 (Ingrouille et W., 2002). The flower bud is globose and develops into a cupulate-lobed calyx with an expanding corolla (Table 1.1). The flower disk is cupulate and the flowers are hermaphroditic. The fruit are berries, which are used commercially in the wine industry. Species included in the present study are: V. aestivalis Michx., V. amurensis Rupr., V. arizonica Engelm., V. berlandieri Planch., V. coignetiae Diels., V. lincecumii Michx., V. riparia Michx., and V. rotundifolia Michx.

6 CHAPTER 1 GENERAL INTRODUCTION

vies vim rent OT om ats. St:hoe:0,Q

Figure 1.4 Vitis vinifera (www.plant-pictures.com )

1.2.2 Cayratia Cayratia (50 species) is found in tropical regions of the Old World, normally in drier rainforests and along stream banks. It is also known as the Hairy water vine (Jackes, 1989b). It is an herbaceous deciduous vine with a woody . Cayratia has compound leaves with three cordate to ovate coarsely toothed leaflets (Jackes, 1989b). Each leaflet is about 15cm long and 8cm wide that is softly hairy (Table 1.1). New growth is covered with soft white hairs. Branched tendrils oppose each leaf (Jackes, 1989b). Green to yellow flowers in axillary cymes occurs in early spring and black globular fruits appear thereafter (Jackes, 1989b). Cayratia acris L., which is included into this study, is often used as an ornamental plant as the species has attractive leaves (Jackes, 1989b). The other two species included are C. mollisimo Gagnep. and C. trifoliate L.

7 CHAPTER 1 GENERAL INTRODUCTION

1.2.3 Cissus Cissus, the largest genus in the grape family, with about 300 species, are found mainly in tropical and subtropical regions, with eight species occurring in southern Africa (Dunaiski Junior, 1992). In the 80's the genus was split according to some details of the flower, and the large caudiciform species were moved to the genus Cyphostemma (Jackes, 1988). The remaining species are mostly vining species native of tropical and subtropical areas. The flowers, typical of the grape family, are green and insignificant (Lombardi, 2000). They produce fruit shaped as small grapes with many of the species possessing poisonous fruit (Lombardi, 2000). I have included five representatives of the genus namely; C. aratifolia, C. discolor Blume, C. penninervis Planch., and C. reniformis Domin and C. quadrangularis L.

Figure 1.5 Cissus quadrangularis (www.plant—pictures.com )

8 CHAPTER 1 GENERAL INTRODUCTION

1.2.4 Cyphostemma Cyphostemma (250 species) is found in tropical and subtropical regions with 34 species found in southern Africa (Barkhuizen, 1987). As mentioned previously it consists of caudiciform species that used to belong to the genus Cissus. They originated in Africa and Madagascar and vary in size, from small shrub to tree-like (Joffe, 1993). It contains both twinning inflorescences and tendrils (Table 1.1) for adhesive support to structures. As can be seen in Table 1.1, the inflorescence may be either a cyme or an umbel (Barkhuizen, 1987). The globose flower bud develops into a cupulate calyx and an expanding corolla, with a cupulate disk (Table 1.1). Cyphostemma consists of both monoecious and hermaphroditic flowers. Propagation is from seeds that form grape like, poisonous fruit in late summer (Barkhuizen, 1987).

Cyphostemma juttae Desc., also known as Basterkobas or Bastard Cobas, is the only species included in this study. It originated in Namibia and South Africa (Joffe, 1993). It is a caudiciform tree which grown up to 6 feet tall (1.8 m). The basterkobas is a very unusual and attractive tree with red grape-like toxic fruits (Joffe, 1993).

1.2.5 Clematocissus Clematocissus is a monospecific genus native to Western Australia (Jackes, 1989a). Clematocissus angustissima Planch. is a sprawling, deciduous tuber forming vine (Jackes, 1989a). The cymose inflorescences terminate one or both branches of the tendrils (Ingrouille et a/, 2002). The corolla has five free lobes that reflex at anthesis but are soon lost, and the disk is copular and adnate to the ovary (Ingrouille et a/, 2002). This feature and the relatively weak specialisation of the tendril/inflorescences indicate an intermediate degree of specialisation within Vitaceae. Rossetto et aL, (2002) found that Clematocissus was related to some Australian representatives of Cissus.

9

CHAPTER 1 GENERAL INTRODUCTION

,G7 -77-•

1-61 •;* 411 • 41- . •

ai

, CA )hostel ii-14 *a liti) • . et _,T, ,.-, .. • -.4 1, .c.•.: S■ . , ' hert, -

Figure 1.6 Cyphostemma juttae (www.plant-pictures.corn)

1.2.6 Ampelopsis Ampelopsis has about 25 species native to ( and ) and (Lodhi and Reisch, 1995). Most are woody, deciduous shrubs but some grow as vines, they are mostly grown as garden omamentals. This deciduous vine with tendrils grows up to fifteen feet tall (Rose, 1998). Ampelopsis brevipendiculata Trautv. has an alternate leaf arrangement, with its leaves being simple and deciduous (Table 1.1). The leaves may grow up to 10cm long and are lobed with a cordate leaf base and a serrated leaf edge. The leaves are dark green in colour (Lombardi, 2000). The inflorescence may be a cyme, thyrse, or an umbel (Table 1.1). The flowers have no ornamental value and are green. The fruit are a metallic blue berry and very showy. A. brevipendiculata is used commercially for its fruit or as a cover for fences (Rose, 1998). Three more species are included in this study; A. megalophylla Diels, A. cordata Michx. and A. arborea L.

10 CHAPTER 1 GENERAL INTRODUCTION

1.2.7 Ampelocissus Ampelocissus, with about 100 species worldwide with two species found in southern Africa: (Lour) Merr. is found in northern Namibia, and Ampelocissus obtusata Planch. is found in Namibia, and South Africa, in the Limpopo province (Verdcourt, 1993). Ampelocissus commonly has thyrsoid or pendant spicate inflorescences that bear tendrils (Ingrouille et al., 2002). The primary branch of the inflorescence commonly forms a branched tendril, and there are leaf opposed tendrils and inflorescences present as well (Lombardi, 2000). It has a globose and sometimes rather elongated bud, and the petals are cucullate, relatively thick and spreading, but are soon lost (Lombardi, 2000). indicate that Ampelocissus is an ancient taxon, which was once more widely distributed (Cevallos-Ferriz and Stockey, 1990). Ampelocissus thyrsifolia L. is the only species included into this study.

1.2.8 Parthenocissus Parthenocissus is a genus of about 10 species of deciduous tendril climbers found in the Himalayas, East Asia, and North America (Wilson and Posluszny 2003). They are grown as garden ornamentals. This study deals with four species of Parthenocissus, Parthenocissus quinquefolia (L.) Planch., P. himalayana Planch. P. tricuspidata Planch. and P. inserta Kern. I will only discuss the morphological characters of two of these species. P. quinquefolia (Virginia creeper) is also known as: American ivy, five-fingered ivy, five- fingered poison ivy, five-leafed ivy, Virginia creeper or woodbine (Gerrath et at., 1989). It is a fast-growing, high-climbing vine that attaches itself with tendrils, which expand, disk-like, on their tips (Gupta, 1978). The leaves are palmately compound with (typically) five leaflets radiating outward from a central petiole like spokes on a wheel (Table 1.1). Each leaflet is about six to 14cm long and is two to five cm wide (Gerrath et al., 1989). The leaves turn fiery red in fall and are very showy. The individual flowers are tiny and inconspicuous, and arranged in elaborate long-stemmed clusters, with each flower at the tip of its own peduncle; such an inflorescence is called a cyme (Table 1.1). The whole inflorescence is about eight to 12cm across (Gerrath et

11 CHAPTER 1 GENERAL INTRODUCTION al., 1989). The berries are blue-black, less than a cm across and much relished by birds. P. quinquefolia is native to eastern North America, from Quebec to , and west to . Hardy throughout, the Virginia creeper occurs in all kinds of and in clearings and on hedgerows.

Figure 1.7 Parthenocissus quinquefolia (www.plant-pictures.com )

Parthenocissus himalayana Planch. is a new climber from the western hills of Dali in China. A vigorous hardy vine that climbs by tendrils not suckers. The new spring growth is a beautiful coppery green, which as the summer progresses turns to a dark green (Gupta, 1978). By autumn the leaves have turned through bronze to a rich red, making a spectacular show. The winter vine is a trellis of fine stems that lace together.

1.2.9 Rhoicissus Rhoicissus with ten species are all native to Africa with seven of them occurring in southern Africa (Merxmuller and Schreiber, 1969). Planch. is widely distributed throughout South Africa. It grows fast, has beautiful leaves and is not capricious. It has also been recorded from the lower Eocene of England (Poole and Wilkinson, 2000). They are generally slender plants and have cymose inflorescences, which often bear tendrils, and could be confused with the genus Ampelopsis (Ingrouille et al., 2002). The

12 CHAPTER 1 GENERAL INTRODUCTION leaf-opposed tendrils are sometimes branched. It has a globose bud shape as Ampelopsis with thick spreading petals, but the petals are less likely to be deciduous and sometimes whiter while still attached (Table 1.1). The disk is cupulate but not always as strongly developed as Ampelopsis (lngrouille et al., 2002). The leaves are commonly trifoliate or simply or palmately lobed (Table 1.1; Ingrouille et al., 2002). Rhoicissus rhomboidea Planch., R. tomentosa Lam., and L. are the three species of this genus included into this study.

1.2.10 Tetrastigma Tetrastigma is better known as the 'Chestnut vine' or the 'Vietnamese Grapevine' (Tetrastigma voinierianum) (Li, 1997). It is a giant, rapidly growing vine, which is endemic to Vietnam. The leaves are about 30cm long and of deep green colour (Yamasaki, 1994) with a silver green hairy covering on the underside. The stems are woody and covered in the same hair as the leaves (Yamasaki, 1994). The tendrils sprout out from the base of the leaf stalks. Four species of the genus are represented in this study namely: T. crenatum Jackes, T. hookeri L., T. trifoliate L. and T. obovatum L.

1.3 The genus Leea Leea is also included into this study as it is closely related to Vitaceae and often included into the family (Soltis et al., 2000). Its habit ranges from herbs to trees with raphides barbed. Leaf edges are teethed with a small glandular apex and one lateral vein continues its course above the tooth. Stipules are borne along the petiole margin and the cymose inflorescence is terminal (Soltis et al., 2000; Table 1.1). The calyx is cupulate and the corolla spreading with the flower disk being annular and the stigmas minute (Table 1.1). Flowers may be either monoecious or hermaphroditic (Table 1.1). Flowers are in much-branched cymes and are up to 18cm across. Individual flowers are two cm across. Leea guineensis G.Don, included in this study, is an evergreen shrub or small tree native to tropical Africa.

13 CHAPTER 1 GENERAL INTRODUCTION

Figure 1.8 Tetrastigma voinierianum (www.plant-picture.com )

1.4 Previous taxonomic studies on Vitaceae The range in variation in the reproductive characteristics of Vitaceae is so little that originally Hooker (1862) recognised only three genera in the family Leea, Vitis, and Pterisanthes. In 1887, Planchon, recommended the placing of Leea into its own family, Leeaceae, and recognised 10 genera in the family Vitaceae. Planchon (1887) also recognised three sections in the genera Cissus which are now regarded as three separate genera; Cissus, Cyphostemma and Cayratia (Gagnepain, 1911). The distinguishing characteristics, between the three genera are described in detail by Descoings (1960), and are mainly based on seed morphology and the nature of the endosperm (Rossetto et al., 2002). As mentioned before, Cissus is characterised by simple leaves and one seeded fruits, Cyphostemma by compound leaves and one seeded fruits and Cayratia by compound leaves

14 CHAPTER 1 GENERAL INTRODUCTION and multiple-seeded fruits. Watson and Dallwitz (1992) followed on Latiff's work (1982) and recognised 15 genera, excluding Leea namely; Ampelocissus, Ampelopsis, Cayratia, Cissus, Cyphostemma, Parthenocissus, Pterisanthes, Rhoicissus, Tetrastigma, Vitis and Yua, and four monotypic genera, Pterocissus, Clematocissus, Acareosperma and Nothocissus (Ingrouille et al., 2002).

Floral and especially vegetative differences have historically been used to establish generic limits in Vitaceae and to separate Leea into its own family, Leeaceae. These generic borders have become increasingly difficult to establish as more and more species have been described and other species re-examined. A good example would be the Australian Vitaceae representatives. The differences given by Gagnepain (1911) and Latiff (1981) between Cayratia and Cissus are that Cayratia has compound leaves, a one to four seeded berry, and Cissus has simple leaves and a one seeded berry. This, however, is not true of all Australian representatives of these genera (Jackes, 1987a). The small flowers and a lack of floral characters have led to highly variable characters within the genera such as, the form of the leaf, branching and tendril arrangement, and inflorescence structure (Ingrouille et al., 2002). Therefore, Ingrouille et al., (2002), reported the results of a cladistic analysis of rbcL DNA sequence data for most of the genera of Vitaceae and reinterpreted the morphological data in the light of this analysis.

Ingrouille et at., (2002), found that the phylogenetic relationships revealed by the cladistic analysis of rbcL DNA sequence data did not correlate perfectly with the morphological patterns that they assessed. This could partly be because several of the genera studied could be paraphyletic or polyphyletic, but the data was too limited to properly investigate it. The distribution of the morphological characters over the various genera, as illustrated in Table 1.1, also disrupted the correlation. As is clear from Table 1.1, various characters are restricted to one genus and many are highly variable within the genera. The rbcL tree (Figure 1.4) is correlated with a regular pattern of floral ontogenetic change, with Leea and Ampelopsis most ancestral, Cissus and

15 CHAPTER 1 GENERAL INTRODUCTION

Parthenocissus intermediate and the most derived Vitis. The development of petals and stamens varies from strongly centripetal in Leea to simultaneous in some Vitis species (Ingrouille et al., 2002). The most derived flowers are also in some respect the simplest, with thin membranous petals and weakly developed disks (Ingrouille et al., 2002). Leea has the largest and most robust flowers, with a thick and complex corolla and in Ampelopsis and Ampelocissus the corolla is also thick and there is a well-developed cupular disk.

Another evolutionary trend correlated with the Ingrouille et al., 2002, rbcL tree is an increasing frequency of unisexual flowers. Leea and Ampelopsis have hermaphroditic flowers (Watson and Dallwitz, 1992) and in other genera there is a tendency towards monoecy. Some species of Cissus are polygamous, and in Vitis some species are hermaphroditic and some species are functionally dioecious, and Tetrastigma is dioecious.

Habit is another correlating feature found on the rbcL tree. Leea, Ampelopsis and Ampelocissus have the least specified habit, and the most modified in Parthenocissus, Cyphostemma and some species of Cissus. However, there has been considerable parallel evolution in some of the vegetative features such as leaf shape, and tendril and inflorescence structure (Ingrouille et al., 2002). Nevertheless, there is a clear trend. Leea lacks tendrils and has pinnate to multiply compound leaves, Ampelopsis is weakly tendrillate but has a tendrillate inflorescence and multiply compound leaves, other genera are more regularly ternate or have simple leaves and clearly distinct tendrils and inflorescences (Ingrouille et al., 2002). However, even in some of these genera, a tendrillate inflorescence may be observed. Other findings by Ingrouille et al, 2002, where that the family Vitaceae was monophyletic but only with a weak bootstrap support (BP) of 53%, with Vitis beeing paraphyletic. Rhoicissus and Cissus were the only two genera found to be monophyletic with BP of 97% and 100% respectively.

16

CHAPTER 1 GENERAL INTRODUCTION

14 Phoradendr 1 I 50 Schoepfk 25 Osyrk Outgroups: Tetracen Santales & 9 72 10 Schumaceri 10C 96 15 10 19 77 Pachynem; 51 14 22 CuratelL 2A 97 Cayratia trifoli 11 Tetrastigma hook( A Tetrastigma trifoliate 82 4 Tetrastigma obovati. 4 Vitis rotundifol 2 13 59 Vitis vinifer 21 90 Cyphostemma jut; 7 Parthenociccus quinquef(

Parthenocissus himalaya Vitis aestival 12 Ampelocissus thyrsifl 16 19 Cissus quandragula 10C 19 Cissus disco! Clematocissus angustiss Rhoicissus rhomboic 7 9 97 4 Rhoicissus digfta 53 95 L, Rhoicissus tomento 12 23 Ampelopsis brevipendicul 17 10C Ampelopsis megaloph. 14 Leea guineens.

Figure 1.9 One of the shortest trees found with successive weighting (SW). Fitch branch lengths are shown above the branches and Fitch bootstrap percentages are shown below the branches. Solid arrowheads indicate groups not found in the strict consensus tree of the Fitch analysis and open arrowheads indicate groups not found in either the Fitch or the SW consensus tree (Ingrouille et aL, 2002).

17 CHAPTER 1 GENERAL INTRODUCTION

1.5 Main objectives Because of the difficulty in identifying unequivocal morphological synapomorphies within Vitaceae, this study aimed to use sequence information to resolve relationships across a selection of taxa. A number of issues that could not be clarified by the analysis of morphological characteristics alone gave rise to two main questions. What are the relationships between the Vitaceae genera? Should Leea be a separate family or included in Vitaceae since it share many characteristics with Vitaceae but lacks tendrils? Thus the aim of the study was to answer these and other questions by investigating selected regions of the plastid genome namely the trnL intron, trnL-F intergenic spacer, rpL16 intron, rbcL gene and accD-psal spacer.

18

a) a) r-- X XX 5 XXX XXX X XXX X 2 ow X X X 0) C E. a) 4.) U) x x X X x x x 2 0 X X X X a. 2 1a)12 ‘t a) xx :::L x xxx

ir 2 N _c ..) xx XXX X XXX XXX X xx X 0) N2 E O O ii

O E a) 3

X X xx xx X xx X X X a) a) ad . as xx xx xx XXX X xx X

U) I a) xx X xx V xx xx

2 a)

X xxxx xx 80) 0 O_c 2- O E x xxxxxxxx x a) ..c

O I I a" xx xx x x E X xxx E z D.

U)

S..- I

m

t mm t

so e t

te E te m 0 .0 te ire 0 co m = te te t a ec a 131 an ■ a -0 ,., .- 5 la la 2 11 .E... E 0 ...

en lm den 16 a

er io an inn a) . inn l lio >, a.. 0. r 2 a E .. r- 01! O an a:C C' 0 a cr fo . fo ... A a' 5

bi• _ .. 2 E La 1- 0 s 3- Z .... a) sc o ,... CNI . C j O 0 ve 1 - c _a -0. a 3 a) c in Le "--- . I as 1

19 X X X

X X

X

X

X X x X X

X

x X X X

X X X

X X x x X

X X X X

'0 co co vt to co r.— a) 03. a' 7 0 V -IZ -E g.E., 2 7 •— to 2 -0 0) = = 0 c= TY4) in0 0 0 -0 C 6 5 c v — 5 5 2 0 0 2 co '-' X c c .2 E C03 "T Z 0 9 -C as 2 a) = al co • a = a) 0 ca LI — co co 2 . 2 , U l0 x cs To R 0 .c CD 0 — C E G) -5 co o AL X '5 4, 01 E zr, ho 41 .c =c., 0 co

20 CHAPTER 2

MATERIAL AND METHODS CHAPTER 2 MATERIAL AND METHODS

2.1 DNA extraction and purification No fresh material was used in this study, as there was already total DNA samples present in the DNA Bank of the Royal Botanical Gardens, Kew. The necessary samples were obtained from the DNA Bank in 100p1 aliquots. The Voucher information, GenBank accession numbers, and literature citations are listed in Table 2.1. Samples were purified and concentrated using the QlAquick PCR Purification Kit according to the manufacturer's protocol.

2.2 Primer trail Although in plants numerous loci from all three genomes have been successfully targeted to infer relationships (summarised in Soltis and Soltis, 1998), it is extremely difficult to judge a priori rates of sequence divergence for specific groups. The rate of sequence variation for both coding and non- coding regions varies widely across groups even among closely related taxa. Previous studies (Ingrouille et al., 2002) indicates very low sequence divergence within Vitaceae and it was therefore necessary to survey several DNA regions (rps16, rp116, accD-psa1, psbC-trnS, ITS, trnV-rbcL) to assess their potential for reconstructing relationships within Vitaceae. Five species (Cissus discolor, Vitis rotundifolia, Cayratia acris, Leea guineensis and Parthenocissus himalayana) representing Viataceae were included in this trail. In Table 2.2 the denaturing and annealing temperatures used for the various genes are summarised.

2.3 PCR amplification The chloroplast genes, accD psal, rpl16, rbcL and trnL-F (see Table 2.3 for list of PCR primer sequences and references) were amplified from purified DNA using the polymerase chain reaction, in 50p.I reactions contained 50units/m1 of Taq DNA polymerase supplied in a proprietary reaction buffer (pH 8.5), 40011M dATP, 400p.M dGTP, 400p.M dCTP, 400p.M dTTP, 3mM MgCl2 and DNA template. Bovine serum albumin (BSA) was added to the reactions to stabilise enzymes, reduce secondary structure problems and favour precise annealing (Palumbi, 1996).

21 CHAPTER 2 MATERIAL AND METHODS

PCR reactions were subjected to three steps, namely denaturation, annealing and extention. The different plastid regions used different programs for amplification (see Table 2.4). Amplified double stranded DNA fragments were purified using QlAquick PCR purification kit in accordance with manufacturer's instructions. For all regions, amplification primers were then used as sequencing primers.

2.4 Cycle sequencing The protocol used for cycle sequencing is a modification of the protocol described in Perkin Elmar's ABI PRISM TM Terminator Cycle Sequencing Ready Reaction Kit manual. For each reaction, the following was added: 35ng PCR product, 1pl Terminator Ready Reaction Mix (Perkin Elmar; dNTPs, ABI PRISMTM, Dye labelled ddNTPs terminators and AmpliTag R DNA polymerase FS), 0.3p1 amplification primer, and sterile distilled water ending with a final volume of 10p1. This was subjected to a cycle sequencing thermal profile consisting of 26 cycles of 10sec denaturation at 96°C, 5sec annealing at 50°C and 4min extention at 60°C. Samples were purified using ethanol precipitation to remove any excess of dye terminators and then run on a Spectrumedix SCE 2410 Genetic Analysis system. The software BaseSpectrum from Spectrumedix was used to analyse the raw data.

2.5 Sequence alignment Sequencer (Gene Codes Corporation) was used for assembly and editing of the complimentary strands. DNA sequences from the genes rbcL, accD, rpL16 and the trnL-F region were aligned manually by sequential pair wise comparison. Alignment required interpretation of gaps, which appeared in all the sequences of all the studied taxa. There are two ways into which indels (insertions and deletions of nucleotides) can be incorporated in the phylogenetic analysis of a group of taxa (Wojciechowski et al., 1993). Each gap position can either be treated as a missing data item, or alternatively as a new character. Treating gaps as missing data allows information to be retained on base substitutions occurring in those taxa within the indel region. But, this will exclude information regarding the evolutionary events or

22 CHAPTER 2 MATERIAL AND METHODS transformation involved in the insertion or deletion of bases. Secondly indels can be scored as separate characters. This, however, will increase the risk of overweighing indels in the analysis, if adjacent gaps are non independent due to erroneous decisions made during alignment. In this study indels were scored as missing data.

2.6 Sequence analysis Maximum parsimony (MP) The data matrices of the aligned plastid genes used in the study were analysed using the software package PAUP for Macintosh version 4.01b (Swofford, 1993) on a Macintosh G5. The data matrices of all five genes as well as the combined matrix were analysed using a heuristic search with 1000 random sequence additions, TBR (tree bisection reconnection) branch swapping, and all character transformations treated as equally likely (Fitch parsimony; Fitch, 1971). Deceleration of transformations (DELTRAN) character optimisation was used to illustrate branch lengths throughout the analysis (due to reported errors with acceleration of transformation (ACCTRAN) optimisation in PAUP version 4.0b1). Internal support was accessed with 1000 bootstrap replicates (Felsenstein, 1985) with Fitch weights using TBR swapping, but holding only ten trees per replicate. Only groups of greater than 50% frequency were reported.

Congruence of the separate data sets was assessed by visual inspection of the individual bootstrap consensus trees. The bootstrap trees were considered incongruent only if they displayed "hard" (i.e., with high bootstrap support) rather than "soft" (with low bootstrap support) incongruence (Seelanan et al., 1999; Wiens, 1998). An arbitrary scale was applied for describing support percentages: 50-74% low; 75-84% moderate; 85-100% high. The statistical analysis methods are described in more detail in the Appendix.

Bayesian analysis Bayesian analysis was performed using MrBayes (Huelsenbeck and Ronquist, 2001). An HKY85 model was specified in which all transitions and

23 CHAPTER 2 MATERIAL AND METHODS transversions have potentially different rates. The analysis was performed using four chains, run for a total of 10 000000 generations, with trees sampled for every 10 generations. Resulting trees were plotted against their likelihood to determine the point where the likelihoods converged on a maximum value, and all trees before this convergence were discarded as the `burn-in' phase. All the remaining trees were imported into PAUP* 4.0b1 and a majority rule consensus tree was produced showing the frequencies (i.e., posterior probabilities) of all observed bi-partitions.

Choice of outgroups and were selected as outgroups on the bases of a previous molecular study (Ingrouille et al., 2002). Not all of the regions could be amplified for all taxa, and thus the matrices do not contain identical sets of taxa (Table 2.5).

24 Table 2. 1 List if taxa with voucher information and Gen Ba n k accession -NC w. .0 1E Cu C C 0 0 U) ti) to N E 4 i G) is O O 0 O 5; E 'Es tn O = C (:) O O rn Cu C C co )7)

/) tQ

3Savolaine n et but still awaiting access ion numbers).

GenBank accession numbers CO O .

Dilleniaceae Cs1 0)

CurateIla americana L. Chase 973 (K) 0-4 ) Tetracera asiatica (Lou r.) HooglandC hase 1238( K) Phoradendron Nickrent 2077 (ILL) 25

Ampelocissus 4 A. thyrsifolia Planch. Chase 1386 (K) 4 A. brevipenduculata Maxim. Chase 848 (K) 4 megalophylla Diels and Gielg Chase 849 (K) A cordata Michx. Chase 1 0225 (K) u c . ) Ni A. arborea L. 4 Cayratia mollisimo Gagnep. Chase 14162( K) 4 (s 4 i "") 'Kt t_ Cayratia trifoliate Domin Chase 1387 (K) Table 2. 1 Continued.

GenBa nk access ion numbers J me. Co 4 u.. 0 11. a) In a. a) U

, it I1 CO CV LO Nr < Cayratia acris L. it K it < C. aratifolia Blume Chase 14160 (K) CA it %-. ..-- CV 101.0 0) 03C is- l— it C. discolor Blu me Chase 853( K) ei r— a K C. quadrangularis L. Chase 852( K) tO CV ul Nr i 1 O CD 26 v C. penninervis Planch. B.Jackes 9850 (K) 7 CV it 0 C. reniformis Domin. B.Jackes 1.2. 86 (K) it Zlach s. n. (K) Clematocissus Ci * C •i- r"-- CV * < D C. angustissima (F. Mue ll) Pla nch. Chase 2202( K) ("4 * 0) r•-• * < --, C. juttae (Dienter an d Gie lg) Desc. Chase 535 (K) Parthenocissus Cs1 _ * 0) CV CO in < — •i- 0 CV 03 I's 0) it 3 Chase 850 (K) V' K — it P. quinquefolia Planch. Chase 967 (K) 3 • it P. tricuspidata Planch. Chase 8468 (K) i t P. inserta Kern. Chase 38 1 (K)

4'

s ber

co num n

io Q. it it it it 4' k acc ess n

Ba L Gen it it #

N.4. N 1r) M It) ILO CNI CN1 O 0) rn vt 0 < < Q s < < * * it #

JCT) (

K) K)

( ( K) K) K) K) K) ( ( ( ( K) 6 K) 962 ( (K) ( (K) 1 8 9

14 s 579 ( 891 51 54 0228 47 469 470 138 1 8 8 1249 5 6

226 8 8 ke 8 8 e e e e c se se se se s se Ja ha hase has hase ha ha has hase Cha C Chas C C C B. Cha C C Cha C C

w

N

ch. h. d.

n

r. lm.

E ls. h. e hx. Pla c lanc ie inue

t P Pup D a n

M ic i Eng

1p r

is

CD L. Plan ide ta s is ie O tiae d

E ica ta ta al 1 Co ta tum bo C .0) en ne issus lia tiv on ita lan na ur

2.

U s

O ig den iz ic i le ifo hom oE O dig tr b ber tr

r

ho ae am ar co cre O. ID T. Ta R. T. V. V. R V. V. R. V. R. a 1--

27 Table 2. 1 Continued.

GenBa nk access ion numbers CO a) a) C.) O.

V. lincecumi Michx. V. palmata Michx. Chase 10230 (K) V. riparia M ichx. Chase & Fay 14588( K) (-8 r-- < v- 0

V. rotundifolia Michx. Chase 966 (K) V. vinifera L. Chase 10231 (K) a) a) L() r— co M < L. guineensis G. Don Chase 71 2( K) • as a) z 0 a) a a) co a) a) co Co e- 28

Table 2.2 The various denaturing and annealing temperatures with the number of cycles used for each gene in the primer I t _

Temperature (°C) 4 fa a_ CD C C N R C ea I- N a) C 0 E w ,..

. Number of Cycles

Plastid regions CO 01Cf)CV 0 LO U)IC)AtLe)1.0 A- 1---0304CN1Is- 01 0)a) Act V-CI01Ad"A:1- 0 CO

29 psb C (ps i, 44kd protein) /tmS( tRNA-Ser UGA) 0 tpS 16 1 F / rpS 16 2 R CO rpL 1671F / rpL 16 1 661R rpL 16 internal F /rpL 16 internal R

Nuclea r region CO 0 LC, 01

Table 2. 3 Regions studied and PCR primer sequences

0 Primer sequence (5'-3') (.3 1- 0 1- 1- 0 1- 0 I- 1- I- 0 I- (.0 0) I's la C.) RI Q. 998 et al., 1 Small accD-psal spacer < ,-, 00 ‹ 0 / < 0 F F 0 < < 0 < ti V) CO _ ._ Z rn rn - .0 11) a a 0 < ° 0 < 0 < I- 0

Ct. l., < Small eta E3

0 0 8 0 CO CD

03 ac z 03 Cl) 0 Q (.1 ... o .-4...0 as c c 0 E ..c E 't a) a) co a. .ivc.-. t•-• a) ccccc as (13 sz .-co E EEEEE Q. Q. MT- T- T- U . Ca ,....0.... .-0,- z O e 16 co E . 4.2 o (0 E •:t Y- CV CV 0 z J- a) CD CD 1.0 h- h- CI e i-- 0 a) .c :. co (.$

ccccc IE. Q. Z Co E EEEEE co a)> -.. 0 .0,0 4:1 0 2 o 0. 1-...: .in- 0 F., 43- (13 co

ta 09 CNI 0 co 0 ...so'e CD CD 1.0 N. h- CO U y inser in- O- i...:

P. .E (,) E (71C O-...- ccccc ta; .as -6 as L E EEEEE ida E c

sp ......

icu as s. tr 0 Q c-ss E s ne 0

e as " ed

issu ,.. g assQ) e ifi

t .4- .4- OD CV CV CD l c 0

n CD CD V- N. h- CO 1-0

amp heno as as- t iffere re .-as d

Par 0) fl ii

he a) d we ta; t E E as co de

C.) da -Q (4,0 for r lu 0 y 0

o 0 d 1••- c inc 0

se a is F2 s s

x CO ie u Et U) Q c ls lop

e o id e OD CV CV OD d ta a) 't co CD CDCD '1 N. h■ CV

to CL w

lifie E m E o

o c All sp Amp

r ¢ a ¢ .-

C o amp wa) PCR p

c Un in CD C CD 4

2. 5: C

2. U.. (0 a) .a 76. w 0 le E cw ca) 49 co ° a) Q ble b 112 a) c ">< 0 .4 C.) Ta Ta a_a

31 CHAPTER 3

RESULTS CHAPTER 3 RESULTS

3.1 Results from primer trail As can be seen from Table 3.1, accD-psal (using primer pair 769F and 75R), rpL16 and rps16 successfully amplified representatives of Vitaceae. I however decided only to use accD-psal and rpL16 together with rbcL and trnL-F, since they were more variable than rps16.

Table 3.1 Plastid and nuclear regions tested and their success rates.

Primer Result accD psal 769F / accD psal 75R Positve accD psa1 769F / accD psal 2640R Negative psbC (psll 44kd protein) / trnS (tRNA-Ser UGA) Negative rpS16 1F / rpS16 2R Positive rpL16 71F / rpL16 1661R Positive rpL16 internal F / rpL16 internal R Negative tm V / rbcLr Negative ITS 4 / 5 Negative

3.2 Statistical results A summary of the statistics for each analysis is provided in Tables 3.2

32

Table 3. 2 Statistics from PAUP ana lys is of separate and combined molecu lar data matrices. -5 14. U 4:1 "c E c tv z .0 N e--- CO (3) .-- CO I's-- ..-- 'Cr ,co0, O ‘-Nt.‘71-V'LO co a....- e-- CO0) (.0 e-- 0) EEEEEE If) LOr•---CDe-- , coCD10CO co ..-....-, = 0

included characters zz -0 0) Cr).- CY) e-a) CO '- N- 1.0 U)CDLOV' CI = CD 0 constant characters .0 CNI ...... , co --.. 0"<" ..-, Irs- 0 .--.. e--- co Csl 0 e- ,-- <0. c* c`' ...-..., Lti ii .....- e- CD•CrV)cicNi 03 a- 0) o'L' LO CO CNI = 0 v. . c? .."*. ., ) :' ■

u n informative sites z -0 N- 10 LC) 8". CO C0c;c:ic.; NI- 0-CO ...., — -;rcoci oi co /1 0) 0 N: 0.-... co I's = CD 0 1 --. 5 0 ° e ■ ." .... . "

parsimony informative s ites

z -0 1 CO CV CNI 0- ..t'—0, CO CK)%.' 0) O = 0 trees saved .0 CD z -0 0 0) a) = 0 5 CO 00 6 cz;c.i co a CO CO CsI00 c=; oi Is.-- CO( Cr) GI - CO '62 - 6 r---- 33 e- 0-; Cel N O

Average number of changes per variable site (no. of steps / no. of varia ble sites)

CHAPTER 3 RESULTS

3.3 Plastid region results 3.3.1 rpL16 Of the 1219 included characters 132 (10.8%) were uninformative and 91 (7.5%) potentially parsimony informative. The analysis resulted in 2365 equally most parsimonious trees with 290 steps with a CI of 0.84 and a RI of 0.81. One of these trees is illustrated in Figure 3.1.

3.3.2 accD psal In this analysis 874 characters were included with 176 (20.1%) sites uninformative and 84 (9.6%) potentially informative. Fitch analysis produced 377 shortest trees with a tree length of 364 (CI = 0.81, RI = 0.63). Figure 3.2 illustrates on of these shortest trees.

3.3.3 rbcL The 1408 characters included into this analysis had 164 (11.6%) uninformative sites and 134 (9.5%) potentially informative sites. The analysis produced 414 shortest trees (TL = 545, CI = 0.64. RI = 0.63). One of the shortest trees is shown in Figure 3.3.

3.3.4 trnL-F Of the 1016 aligned positions, 159 (15.6%) were uninformative and 99 (9.7%) potentially parsimony informative. The Fitch analysis produced 4560 most parsimonious trees with a tree length of 1491 (CI = 0.86. RI = 0.82). One of these trees is illustrated in Figure 3.4.

3.3.5 Combined data No strongly supported incongruent patterns were visible after visual inspection and thus the plastid data was combined. There were 4516 included characters of which 570 (12.4%) were uninformative and 395 (8.7%) potentially informative. The analysis yielded 8180 most parsimonious trees (Figure 3.5) with 1491 steps (CI = 0.73, RI = 0.67). The Bayesian results can be seen in Figure 3.6.

34 CHAPTER 3 RESULTS

The family Vitaceae was found to be monophyletic in all the analysis (MP=67BP and ML=PP0.84). Leea is sister to Vitaceae (PP1.0). As indicated in Figures 4.5 and 4.6 Vitaceae can be divided into four informal groupings. These groups are indicated in color to aid with the interpretation thereof.

The first group, purple, comprised of the genera Parthenocissus and Cissus. This was strongly supported in the ML analysis PP0.9 but received less than 50% support in the MP analysis. Four of the Cissus species (C. disco/or, C. quadrangularis, C. reniformis, C. aratifolia) are grouped together with a 100BP support in the combined analysis, and a Posterior Probability of 1.0 in the Bayesian analysis. The fifth Cissus species, C. penninerfis, was grouped together with Parthenocissus quiquefolia. The second group, green, consisted of the genus Vitis. It was found to be monophyletic (PP0.84). Although only weakly supported (PP0.53) Ampelocissus thysifolia is sister to groupings one and two.

Ampelopsis, Rhoicissus, and Clematocissus made up the third (blue) group. Rhoicissus was found to be monophyletic in the combined results MP (90BP) and ML (PP0.97) analysis. In the ML analysis Amelopsis was found to be monophyletic (PP0.99) with Clematocissus angustissima sister to Ampelopsis and Rhoicissus.

Three genera made up the fourth group (red). Cayratia, Tetrastigma and Cyphostemma were grouped together with strong support (BP82, PP1.0). Tetrastigma was found to be monophyletic (100BP; PP1.0) with Cayratia paraphyletic in all analyses.

35 CHAPTER 3 RESULTS

10 Ampelopsis brevipedunculata

.1 Parthenocissus quinqeufolia

Ampelopsis megalophylla

Parthenocissus himalayana 3 10 63 100 11 Cissus quadrangularis 99 10 22 Cissus aratifolia 68 100 20 Cissus discolor

10 Rhoicissus rhomboidea

Clematocissus angustissima

Parthenocissus tricuspidata

6 52 1 4 Vitis atizonica 61 1 Vitis lincecumii

2 Vitis tiparie 684 2 27 78 4 88 1 Vitis rotundifolia

Ampelocisus thyrsifolia 6 90 25 Cayratia molissimo

28 Cyphostemma juttae 9 99 32 Cayratia trifoliate 1 Tetrastigma hooked 7 54 Tetrastigma crenatum

11 Tetrastigma trifoliate

Leea sp.

Figure 3.1 One of the 2365 equally parsimonious trees (TL = 290, CI = 0.84, RI = 0.81) found from the analysis of rpL16 sequences for 26 species of Vitaceae and outgroups. Numbers above the branches are Fitch lengths (DELTRAN optimisation), and those below are Fitch bootstrap percentages above 50%. Branches not recovered in the strict consensus are indicated with solid arrows.

36 CHAPTER 3 RESULTS

Ampelocissus thyrsifolia

Ampelopsis brevipendiculate

Rhoicissus rhomboidea

56 15

Clematocissus angustissima

14 Cayratia acris 4 55 19 Cyphostemma juttae

15 Cissus discolor 21 10 100 Cissus aratifolia 17 100 Cissus quedrangularis

Parthenocissus himalayana

Parthenocissus tricuspidata

8 7- Parthenocissus quingeufolia

Vitis coignetiae

Vitis lincecumii

--2-- Vitis amurensis

Vitis rotundifolia

13 Ampelopsis megalophylla

114 Cissus sp.

33 8 50 7 Tetrastigma hookeri

Leea sp.

Figure 3.2 One of the 377 most parsimonious trees (TL = 364, CI = 0.81, RI = 0.63) based on the analysis of accD psal for 22 species of Vitaceae and Leea. Numbers above the branches indicate Fitch lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not recovered in the strict consensus tree.

37 CHAPTER 3 RESULTS

27 Amplocissus thysiforia Ampelopsis brevipedunculata

10 J142 Ampelopsis megalophylla 68 F Ampelopsis arborea 2 Rhoicissus tomentosa 7 Rhoicissus digitata 90 7 Rhoicissus rhomboidea 4 Clematocissus angustissima 31 27 Cayratia trifolia 55 18 Cyphostemma juttae

10 a— Cayratia mollisimo 100 2 Cayratia acris 7 Tetrastigma crenatum 73 Tetrastigma hooked Tetrastigma obovatum 6 79 Tetrastigma trifoliata 19 Cissus discolor ...1L_ Cissus quadrangularis 100 11 84 Cissus reniformis 98 11 Cissus aratifolia ii 2 Cissus penninervis 72 100 4/1-41— Parthenocissus quinquefolia Parthenocissus himalayana Vitis aestivalis Vitis bedandieri Vitis lincecumii Vitis coignetiae 20 Vitis vinifera lel—fi__ Vitis rotundifolia Vitis amurensis Vitis palmata

42 Leea sp 100 Leea guineensis 103 Tetracera asiatica Phoradendron

Figure 3.3 One of the 414 most parsimonious trees (TL = 545, CI = 0.64, RI = 0.63) found from the analysis of rbcL for 38 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimisation) and the numbers below indicate Fitch bootstrap percentages over 50%. Solid arrows indicate branches not present in the strict consensus tree.

38 CHAPTER 3 RESULTS

8 Ampelocissus thysifolia Cissus penninervis Vitis aestivalis Vitis bertandieri 341-1- 4 Vitis lincecumii I Vitis coignetiae Vitis riparia Vitis amurensis Vitis vinifera Vitis arizonica Vitis rotundifolia 34 Cayratia trifolia Tetrastigma trifoliate 72 Tetrastigma obovatum 8 691-10 Tetrastigma hooked 100 4 Tetrastigma crenatum 31 8 10 Cayratia acris 72 73 13 Cayratia mollisimo 13 23 Cyphostemma juttae 67 8 Cissus discolor 20 13 2 Cissus quadrangularis 100 7 76 4 Cissus reniformis 99 3 Cissus aratifolia Parthenocissus himalayana 7 Parthenocissus quiquefolia 5 Parthenocissus bicuspidate 9 Ampelopsis arborea Ampelopsis megalophylla Rhocissus rhomboidea 3 2 Rhocissus tomentosa 85 6 3 5 41 Rhocissus digitata 67 6 Rhocissus tridentate 2 Ampelopsis brevipedunculata 100 6 96 Ampelopsis cordate 34 Clematocissus angustissima 2 Leea sp. 11 Leea guineensis

Figure 3.4 One of the 4560 most parsimonious trees (TL = 346, CI = 0.86, RI = 0.82) from the analysis of trnL-F for 37 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimasation) and the numbers below indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree.

39 CHAPTER 3 RESULTS

36 Ampelocissus thysifolia Parthenocissus inserta 241-- Vitis coignetiae 8 Vitis amurensis 92 Vitis vinifera 1 Vitis palmata 10 Vitis aestivalis Vitis lincecumii 6 Vitis riparia 54 le Vitis berlandieri Vitis arizonica 19 54 16

73 Cissus discolor 71 20 Cissus quadrangularis 100 " I 13 33 35 90 Cissus reniformis 29 58 100 Cissus aratifolia 23 Parthenocissus himalayana 18 19 Parthenocissus tricuspidata 1 16 19 Cissus penninervis 10 Parthenocissus quiquefolia 111 Cayratia trifolia 23 Tetrastigma trifoliata 89 Tetrastigma obovatum 100 Tetrastigma hookeri Tetrastigma crenatum 49 86 82 Cyphostemma juttae 34 45 Cayratia acris 100 Cayratia mollisimo 8 32 I Ampelopsis brevipedunculata 89 19 9 Rhocissus rhomboidea 67 2ie r3— Rhocissus tomentosa 12 90 22 6 Rhocissus tridentata 75 Rhocissus digitata 31 Clematocissus angustissima

63 Ampelopsis megalophylla 71 24 Ampelopsis arborea 30 Phoradendron 99 F-7216– Tetracera asiatica 47 Leea sp. 100 it Lees guineensis Figure 3.5 One of the 8180 most parsimonious trees (TL = 1491, CI = 0.73, RI = 0.67) based on the combined plastid data for 40 species of Vitaceae and outgroups. Numbers above the branches indicate Fitch tree lengths (DELTRAN optimasition) and those below the branches indicate Fitch bootstrap percentages above 50%. Arrows indicate branches not present in the strict consensus tree.

40 CHAPTER 3 RESULTS

Ampelocissus thysifolia _ Cissus discolor 1.1L 1 0 Cissus quadrangularis Cissus reniformis 0 90 10 Cissus aratifolia 0 28 053. Parthenocissus himalayana 0 90 Parthenocissus tricuspidata 0 44 Cissus penninervis Parthenocissus quiquefolia Vitis aestivalis 0 38 097[ Vitis berlandieri 10 Vitis arizonica

0 79 Vitis lincecumii 10 Vitis riparia Vitis coignetiae .2.95 0 731 Vitis amurensis 041 Vitis palmata Vitis vinifera Vitis rotundifolia 1 0 Ampelopsis brevipedunculata

0 99 Ampelopsis cordata 1 0 Ampelopsis megalophylla 9.6 Ampelopsis arborea Rhocissus rhomboidea 12Z Rhocissus tomentosa 0 98 0 73 87 Rhocissus digitata Rhocissus tridenteta Clematocissus angustissima Cayratia trifolia 10 1 0 Tetrastigma trifoliata 10 10 Tetrastigma obovatum 0 50 0 — Tetrastigma hookeri cop Tetrastigma crenatum 10 Cyphostemma juttae

1 0 Cayratia acris Cayratia mollisimo

1 0 Leea sp Leea guineensis Phoradendron Tetracera asiatica

Figure 3.6 Bayesian analysis of the combined plastid data set. One of the 10001 majority rule consensus trees with PP shown above the branches.

41 CHAPTER 4

DISCUSSION AND CONCLUSIONS CHAPTER 4 DISCUSSION AND CONCLUSIONS 4.1 Molecular evolution Of the genes used, accD, had a significantly higher amount of variable sites (20.1%; Table 3.1) than trnL-F (15.6%), rbcL (11.6%), rpL16 (10.8%). The number of parsimony informative characters was the highest in non-coding gene trnL-F (9.7%), followed by accD (9.6%), rbcL (9.5%), rpL16 (7.5%). The difference between the non-coding and coding regions in parsimony informative characters were insignificant in this study. The variable sites in rbcL (3.32 steps/site) evolved faster than those of rpL16 (2.19 steps/site), trnL-F (2.18 steps/site) and accD (2.07 steps/site). The genes accD and rbcL performed equally (RI = 0.63) as did the genes rpL16 and trnL-F (RI = 0.82).

4.2 Morphological characters Shared morphological traits within clades are illustrated in Figure 4.1.

The green group (Ampelocissus and Vitis) share various morphological traits. Firstly, both have a scandent habit. The leaves are entire, palmate, digitate or three foliolate. The flowers on the inflorescence are opposite, and the inflorescence is used to twine. The bud is globose. Ampelopsis is the only genus which has dioecious flowers.

Leaf characteristics, inflorescence type and stigma are the three morphological traits which are shared by the purple group (Cissus and Parthenocissus). The leaves are palmate and digitate, and the inflorescence a cyme. The stigma is minute. Parthenocissus is the only genus that does not twine, a trait shared by the Leea.

The red group (Cayratia, Cyphostemma, Tetrastigma) all share palmate leaves. Cayratia and Cyphostemma share three or five foliolate leaves. The flowers are all opposite on the inflorescence and Tetrastigma has a peculiar inflorescence. This peculiar inflorescence supports the placing of Tetrastigma as a monophyletic group. The inflorescence is also used to twine. The bud is globose and the calyx cupulate. Cyphostemma is the only genus with a decumbent habit.

42 CHAPTER 4 DISCUSSION AND CONCLUSIONS The blue group (Ampelopsis, Rhoicissus, Clematocissus) all have root tubers and a scandent habit. The shared leave characteristic is the three foliolate leaves and tendrils are used to twine. The inflorescence is a cyme and all species are hermaphroditic. The genera Rhoicissus and Ampelopsis share the morphological trait of monoecious flowers. Rhoicissus also has two distinct morphological characters namely trichasium inflorescence and glandular disk.

The basal genus Ampelopsis is also supported in this position as it has various morphological characters in common with the outgroup Leea. They share a scandent habit with root tubers. The leaves are entire, palmate and three or five foliolate. They use tendrils for twinning and the cymose inflorescence has opposite flowers. The bud is globose. The corolla is spreading with an annular disk and a minute stigma. The genera have both monoecious and hermaphroditic flowers.

A general floral ontogenetic change was found, with Leea and Ampelopsis (Leea has large robust flowers with a thick complex corolla, and Ampelopsis has a thick corolla and well developed disk) most basal, Cissus and Parthenocissus intermediate and Vitis the most derived (thin membranous petals and weakly developed disks). An increase in the frequency of unisexual flowers was also found with Leea, Ampelopsis and Rhoicissus being monoecious and hermaphroditic, and Ampelocissus dioecious and hermaphroditic. A parallel evolution in some other vegetative features was also noticed. Leea lacks tendrils and has multiple compound leaves. Ampelopsis is weakly tendrilate and has multiple compound leaves. The other genera have regular ternate or simple leaves, distinct inflorescences and are distinctly tendrillate.

43 CHAPTER 4 DISCUSSION AND CONCLUSIONS

Ampelocissus thysifolia Parthenocissus inserta Vitis coignetiae

Vitis amurensis Leaves entire palmate digitate 1-3 foliolate 99 Vitis vinifera Habit ova ndent Vitis palmata in Vitis aestivalis Inflorescence opposite flowers Vitis lincecumii Bud. globose Vitis riparia 5"4 -w Twinnmq inflorescence 4 Vitis berlandieri Vitis arizonica 19 54 1A Vitis rotundifolia 71 Cissus discolor 90 _7_1_ Cissus quadrangularis Leaves: palmate / digitate 100 lc 90 1 11 Cissus reniformis 33 Inflorescence: cyrnose 58 0 100 ..2c) Cissus aratifolia Stigma : minute 9'1 Parthenocissus himalayana 1f1 12 Parthenocissus tricuspidata lA 19 Cissus penninervis in Parthenocissus quiquefolia 1 Cayratia trifolia Leaves: pall ate 3-5 folo ate 23 19 Tetrastigma trifoliata 89 90 6 obovatum :nflo re see nce opposite A flowers 100 1/1 Tetrastigma hookeri 14 Calyx: cupiiiate Tetrastigma crenatum 49 RR globose Cyphostemma juttae 82 %Inning: I ntorescente 45 Cayratia acris 100 Cayratia molfisimo 32 R Ampelopsis brevipedunculata Habit scandent 89 Ampelopsis cordata Root tubers 19 Rhocissus rhomboidea Leaves: 3 foliolate 67 Rhocissus tomentosa Twinning: tendrils 1 9 90 22 Rhocissus tridentata Inflorescence: cymose 75 Rhocissus digitata Bud: globose 31 Clematocissus angustissima 14 Sexuality: morroecious / Al Ampelopsis megalophylla hermaphroditic 24 71 Ampelopsis arborea 77 in Phoradendron 9R 99 Tetracera asiatica

47 Leea sp. 100 Leea guineensis

Figure 4.1 Combined tree indicating the color groups and the morphological characters shared.

44 CHAPTER 4 DISCUSSION AND CONCLUSIONS 4.3 Previous taxonomic studies Originally Hooker (1862) recognised only three genera in the family: Leea, Vitis, Pterisanthes. In 1887, Planchon, recommended the placing of Leea into its own family, Leeaceae, and recognised ten genera in the family Vitaceae. Planchon (1887) also recognised three sections in the genera Cissus which are now regarded as three genera; Cissus, Cyphostemma and Cayratia (Gagnepain, 1911). The distinguishing characteristics, between the three genera are described in detail by Descoings (1960), and are mainly seed morphology and the nature of the endosperm (Rossetto et a/., 2002). Watson and Dallwitz (1992) followed on Latiff's work (1982) and recognised fifteen genera, excluding Leea namely; Ampelocissus, Ampelopsis, Cayratia, Cissus, Cyphostemma, Parthenocissus, Pterisanthes, Rhoicissus, Tetrastigma, Vitis and Yua, and four monotypic genera, Pterocissus, Clematocissus, Acareosperma and Nothocissus.

Floral and especially vegetative differences have historically been used to establish generic limits in Vitaceae and to separate Leea into its own family. These generic borders have become increasingly difficult to establish as more and more species have been described and other species re-examined. The small flowers and a lack of floral characters have led to highly variable characters within the genera such as, the form of the leaf, branching and tendril arrangement, and inflorescence structure (Ingrouille et al., 2002). Therefore, Ingrouille et al., (2002), reported the results of a cladistic analysis of rbcL DNA sequence data for most of the genera of Vitaceae and reinterpreted the morphological data in the light of their analysis.

The results of the present study agree with several of the findings of Ingrouille et al., (2002). Vitaceae was once again found to be monophyletic, but with an increased BP of 67 (previously 53BP). Rhoicissus was found monophyletic with a strong BP support of 90BP. Ampelopsis was once again the most basal genus. Several findings of this study also disagreed with the phylogeny proposed by Ingrouille et al. (2002). Vitis was found as monophyletic in all four genealogical analyses with its strongest BP support in rpL16 (67BP). Cissus

45 CHAPTER 4 DISCUSSION AND CONCLUSIONS was found as paraphyletic as Cissus penninerfis was grouped together with Parthenocissus in the combined results.

4.4 Conclusions A phylogenetic tree for Vitaceae was produced using DNA sequenced data from plastid regions to resolve relationships within the family and also to evaluate the position of the genus Leea. Combining the different datasets provided resolution within groupings that were largely in agreement with the current taxonomy. Vitaceae was found to be monophyletic with a moderate BP and PP support with four informal groupings described in this study. All analyses suggest that Leea is sister to Vitaceae and should not be included in the family. The results of this study agreed with several of the findings of Ingrouille et al., (2002) namely the monophyly of the family and the basal position of Ampelopsis. Several findings of this study also disagreed with the phylogeny proposed by Ingrouille et al. (2002). In this study Vitis was found as monophyletic in all four genealogical methods. Cissus was found as paraphyletic, which agrees with Rossetto et al. (2002), as Cissus penninerfis was grouped together with Parthenocissus in the combined results.

A general floral ontogenetic change was found, with Leea and Ampelopsis most basal, Cissus and Parthenocissus intermediate and Vitis the most derived. An increase in the frequency of unisexual flowers was also found with Leea, Ampelopsis and Rhoicissus being monoecious and hermaphroditic, and Ampelocissus dioecious and hermaphroditic. A parallel evolution in some other vegetative features was also noticed. Leea lacks tendrils and has multiple compound leaves. Ampelopsis is weakly tendrillate and has multiple compound leaves. The other genera have regular ternate or simple leaves, distinct inflorescences and are distinctly tendrillate.

Overall, the results presented provided new insights into the relationships within a number of Vitaceae genera. Several genera where found to be monophyletic and the monophyly of Vitaceae is also strongly supported. Although the intrageneric relationships between Cayratia, Tetrastigma and

46 CHAPTER 4 DISCUSSION AND CONCLUSIONS Cyphostemma as well as Cissus and Parthenocissus remained unresolved, it is evident that strong relations exist within these two groups. Nevertheless, it is premature to propose a phylogenetic-based nomenclature of all the genera of Vitaceae, as in most cases the type species for the genera were not included in the analysis. Overall the analysis among the four clades could not be resolved in this study. This is not an uncommon occurrence at this taxonomic level. As a result, further sequencing and a greater number of species (including the type species of the genera investigated) will be needed to increase the resolving power of future studies.

47 CHAPTER 5

REFERENCES CHAPTER 5 REFERENCES A

Adams, E.N. 1972. Consensus techniques and the comparison of taxonomic trees. Systematic Zoology 21: 390-397.

Albert, V.A., Williams, S. E, and Chase, M.W. 1992. Carnivorous plants: phylogeny and structural evolution. Science 257: 1491 — 1495.

Altschul, S. von R. 1973. Drugs and Foods from Little-Known Plants. Cambridge, Mass.: Harvard Univ. Press.

B

Barkhuizen, B.P. 1987. Succulents of southern Africa. Purnell, Cape Town.

C

Camin, J.H. and Sokal, R.R. 1965. A method for deducing branching sequences in phylogeny. Evolution 19: 311-326.

Cevallos-Ferris, S.R.S. and Stockey, R.A. 1990. Permineralised fruits and seeds from the Princeton chert Middle Eocene of British Columbia, Canada, Vitaceae. Canadian Journal of Botany 68: 288-295.

D

Davies, J.I., Simmons, M.P., Stevenson, D.W. and Wendell, J.F. 1998. Data decisiveness, data quality, and incongruence in phylogenetic analysis: An example from using mitochondria! atpA sequences. Systematic Biology 47(2): 282-310.

48 CHAPTER 5 REFERENCES Descoings, B. 1960. Un genre meconnu de Vitacees: comprehensior et distinction des genres Cissus et Cyphostemma (Planch.) Alston. Notu/ae Systematicae 16: 113-125.

Dollo, L. 1893. Les lois de revolution. Bulletin de la Societe Belge de Geologie, de Paleontologie et d'Hydrologie 7: 164-166.

Dunaiski Junior, A. 1992. Two species of Vitaceae: Cissus cervii sp. N. and Cissus marcocarpa sp. N. Acta Biolkgica Paranaeanse 21: 135 — 141.

E

Eck, R.V. and Dayhoff, M.O. 1966. Atlas of Protein Sequences and Structure. National Biomedical Research Foundation, Silver Spring, Maryland.

Edwards, A.W.F. and Cavalli-Sforza, L.L. 1964. The reconstruction of evolution. Annals of Human Genetics 27: 105.

F

Farris, J.S. 1969. A successive approximations approach to character weighting. Systematic Zoology 18: 374-385.

Farris, J.S. 1972. Estimating Phylogenetic Trees. American Nature 106: 645- 668.

Farris, J.S. 1989. The retention index and rescaled consistency index. Cladistics 5: 417-419.

Felsenstein, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Systematic Zoology 27: 401-410.

49 CHAPTER 5 REFERENCES Felsenstein, J. 1981. Evolutionary trees from DNA sequences: a maximum likelihood approach. Journal of Molecular Evolution 17: 368-376.

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using bootstrap. Evolution 39: 783-791.

Felsenstein, J. 1988. Phylogenies from molecular sequences: inference and reliability. Annual Revision in Genetics 22: 521-565.

Felsenstein, J. 2004. Inferring phylogenies. Sinnauer Associates, Inc. Publishers Sunderland, Massachusetts.

Fitch, W.M. 1971. Toward defining the course of evolution: minimum change for a specified tree topology. Systematic Zoology 20: 406-416.

Fitch, W.M. 1981. A non-sequential method for constructing trees and hierarchical classifications. Journal of Molecular Evolution 18: 30-37.

Fitch, W.M. 1984. Cladistic and other problems: Problems, pitfalls, and potentials. In. Duncan, T. and Stuessey, T.G. (eds.), Cladistic perspectives on the reconstruction of evolutionary history, pp. 221-252. Columbia University Press, New York.

Fitch, M.W. and Margoliash, E. 1967. Construction of phylogenetic trees. Science 155: 279-293.

G

Gagnepain, F. 1911. Classification des Cissus et Cayratia. In: Lecomte, ed Notulae Systematicae. Vol. 1. Paris. 339-343.

50 CHAPTER 5 REFERENCES Galet, P. 1967. Recherches sur les methods d'indentification et de classification des Vitaceae des zones temperees. PhD Thesis: University of Montpellier, France.

Gerrath, J.M., Posluzny, U. and Dengler, N.G. 1989. Primary vascular patterns in the Vitaceae. International Journal of Plant Science 162: 729-745.

Goloboff, P.A. 1991. Homoplasy and the choice among cladograms. Cladistics 7: 215-232.

Greybeal, A. 1998. Is it better to add taxa or characters to a difficult phylogenetic problem? Systematic Biology-47: 9-17.

Gupta, P. 1978. Floral anatomy of Parthenocissus Planchon Journal of the Indian Botanical Society. 57: 29-30.

H

Harrison, S.G., Masfield, G.B. and Wallis, M. 1985. The Oxford book of food plants. Oxford University Press, London.

Heywood, V.H. 1993. Flowering plants of the world. B.T. Batsford. London.

Hill, A.F. 1937. Economic Botany: A textbook of useful plants and plant products. McGraw-Hill Book Company. London.

Hillis, D.M. 1998. Taxonomic sampling, phylogenetic accuracy, and investigator bias. Systematic Zoology 47: 3-8.

Hooker, J.D. 1862. Ampelideae. In: Bentham, G. and Hooker, J.D. eds. Genera Plantarum. London: Reeve, 386-388.

51 CHAPTER 5 REFERENCES Huelsenbeck, J.P. and Ronquist, F. 2001. MRBAYES. Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755.

Huelsenbeck, J.P., Ronquist, F., Nielsen, R., Bollback, J.P. 2001. Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294: 2310-2314.

I

Ingrouille, M.J., Chase, M.W., Fay, M.F., Bowman, D., van der Bank, M., and Bruijn, A.D.E. 2002. Systematics of Vitaceae from the viewpoint of plastid rbcL DNA sequence data. Botanical Journal of the Linnean Society 138: 421-432.

J

Jackes, B.R. 1987a. Revision of Australian Vitaceae 2. Cayratia Juss. Austrobaileya 2: 365-379.

Jackes, B.R. 1988. Revision of Australian Vitaceae 3. Cissus L. Austrobaileya 2: 481-505.

Jackes, B.R. 1989a. Revision of Australian Vitaceae 4. Clematocissus Planch. Austrobaileya. Austrobaileya 3: 11-19.

Jackes, B.R. 1989b. Revision of Australian Vitaceae 5. Tetrastigma (Miq) Planchon. Austrobaileya 3: 149-158.

Joffe, P. 1993. The gardeners guide to South African plants. Tafelberg, Cape Town.

Judd, W.S., Campbell, C.S., Kellogg, E.A., and Stevens, P.F. 1999. Plant Systematics: a phylogenetic approach. Sinauer associates. USA.

52 CHAPTER 5 REFERENCES K

Kitching, I.J. 1992. Tree builiding techniques. In. Forey, P.L., Humphries, C.J., Kitching, I.L., Scotland, R.W., Siebert, D.J. and Williams, D.M. (eds.), Cladistics: A pratical course in systematics, pp. 44-71. Oxford Science Publication, London.

Kluge, A.G. and Farris, J.S. 1969. Quantitative phyletics and the evolution of anurans. Systematics of Zoology 18: 1-32.

L

Latiff, A. 1981. Studies in Malaysian Vitaceae V. The genus Cayratia in the Malay Peninsula. Sains Malaysiana 10: 129-139.

Latiff, A. 1982. Studies in Malaysian Vitaceae II. Nothocissus a new Malaysian genus. Federation Museums Journal, Perak 27: 70-74.

Lavie, P. 1990. Vitaceae de l'ouest Africain. Especes senegalaises. Senegal: Ministere de la cooperation et du development.

Li, C. 1997. New taxa of Vitaceae from . Acta Botanica Yunnanica China, 19 (3) 217-220,

Lodhi, M.A. and Reisch, B.I. 1995. Nuclear content of Vitis species, and other genera of Vitaceae. Theoretical and Applied Genetics 90: 11-16.

Lombardi, J.A. 2000. Vitaceae: generos Ampelocissus, Ampelopsis, Cissus. Neotropica Monograph 80.

53 CHAPTER 5 REFERENCES M

Mabberley, D.J. 1995. Vitaceae. Pp 446 — 482 in A revised handbook to the flora of Ceylon, ed. M.D. Dassanayake. Amerind Publishing Company.

Maddison, W.P. and Maddison, D.R. 1992. MacClade, version 3.0. Sinauer, Sunderland, Massachusetts.

MerxmCiller, H. and Schreiber, A. 1969. Vitaceae. Prodromus einer Flora von SOdwestafrika 80: 1-8.

P

Page, R.D.M. and Holmes, E.C. 1998. Molecular evolution: a Phylogenetic approach. University Press, Cambridge, UK.

Palumbi, S.R. 1996. Nucleic acids 2: The polymerase chain reaction. In Hillis, D.M., Moritz, C. and Mable. B.K. (eds.), Molecular Systematics. 206- 247. Sinauer, Sunderland, Massachusetts, USA.

Planchon, J.E. 1887. Vitaceae. In: De Candolle, A. and De Candolle, C. eds. Monographiae Phaneroamarum 5: 2.

Poole, I. and Wilkinson, H.P. 2000. Two early Eocene vines from southeast England. Botanical Journal of the Linnaean Society 133: 1-26.

R

Richardson, J.E. 1999. Molecular systematics of the genus L. with an emphasis on the island species. Ph. D. thesis. University of Edinburgh, UK.

Rose, N. 1998. Field Notes: Ampelopsis brevipedunculata 'Elegans'. American Nurseryman.

54 CHAPTER 5 REFERENCES Rossetto, M., Jackes, B.R., Scott, K.D. and Henry, R.J. 2002. Is the genus Cissus monophyletic? Evidence from plastid and nuclear ribosomal DNA. Systematic Botany 27(3): 522-533.

S

Saitou, N. and Nei, M. 1987. The neighbor joining method: a new method for reconstructing phylogenetic tree. Molecular Biology and Evolution 4: 406-425.

Savolainen, V., Chase, M.W., Hoot, S.B., Morton, C.M., Soltis, D.E., Bayer, C., Fay, M.F., de Bruijn, A.Y., Sullivan, S., Qui, Y-L. 2000a. Phylogenetics of flowering plants based upon a combined analysis of plastid atpB and rbcL gene sequences. Systematic Biology 49: 306 — 362.

Savolainen, V., Fay, M.F., Albach, D.C., Backlund, A., van der Bank, M., Cameron, KM., Johnson, S.A., Liedo, M.D., Pintaud, J-C., Powell, M., Sheahan, M.C., Soltis D.E., Soltis, P.S., Weston, P., Whitten, W.M., Wurdack, K.J., Chase, M.W. 2000b. Phylogeny of the eudicots: a nearly complete familial analysis based on rbcL gene sequences. Kew Bulletin 55: 257 — 309.

Seelanan, T., C.L. Brubaker, J. McD. Stewart, L.A. Craven, and J.F. Wendel. 1999. Molecular Systematics of Australian Gossypium section Grandicalyx (Malvaceae). Systematic Botany 24:183-208

Siddal, M.E. 1998. Prior agreement: Arbitration or arbitrary? Success of parsimony in the four taxon case: Long branch repulsion by likelihood in the Farris zone. Cladistics 14: 209-220.

Siddal, M.E. and Kluge, A.G. 1997. Probabilism and phylogenetic inference. Cladistics 13: 313-336.

55 CHAPTER 5 REFERENCES Siebert, D.J. 1992. Tree statistics, trees and confidence; consensus trees; alternatives to parsimony; character weighting ,character conflicting and its resolution. In. Forey, P.L., Humphries, C.J., Kitching, I.L., Scotland, R.W., Siebert, D.J. and Williams, D.M. (eds.), Cladistics: A pratical course in systematics, pp. 72-88. Oxford Science Publication, London.

Small, R.L., Rybum, J.A., Cronn, R.C., Seelanan, T. and Wendel, J.F. 1998. The tortoise and the hare: choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group. American Journal of Botany 85: 1301-1315.

Sokal, R.R. and Rohlf, F.J. 1981. Biometry. 2nd edition. Freeman, NY

Soltis, D.E. and Soltis, P.S. 1998. Choosing an approach and an appropriate gene for phylogenetic analysis. In Sotis, D.E., Soltis, P.S. and Doyle, J.J. (eds.), Molecular systematics of plants H. DNA sequencing. Kluwer Academy Publishers Boston.

Soltis, D.E., Soltis, P.S., Chase, M.W., Mort, M.E., Albach, D.C., Zanis, M., Savolainin, V., Hahn, W.H., Hoot, S.B., Fay, M.F., Axtell, M., Swensen, S.M., Price, L.M., Kress, W.J., Nixon, K.C. and Farris, J.S. 2000. Angiosperm phylogeny inferred from 18S rDNA, rbcL and atpB sequences. Botanical Journal of the Linnean Society 133: 381-461.

Soltis, D. E., Senters, A.E., Zanis, M.J., Kim, S., Thompson, J.D., Soltis, P.S., Ronse De Craene, L.P., Endress, P.K. and Farris, J.S., 2003. Gunnerales are sister to other core eudicots: implications for the evolution of pentamery. American Journal of Botany 90(3): 461-470.

Swofford, D.L. 1991. On the utility of the distance Wagner procedure. In. Fund, V.A. and Brooks, D.R. (eds.), Advance in cladistics ,pp. 25-43. New York Botanical Garden, New York.

56 CHAPTER 5 REFERENCES Swofford, D.L. 1993. PAUP: Phylogenetic Analysis Using Parsimony, version 3.1. Illinois Natural History Survey, Champaign, Illinois.

Swofford, D.L., Olsen, P.J., Waddell, P.J. and Hillis, D.M. 1996. Phylogenetic inference. In. Hillis, D.M., Moritz, C. and Marble, B.K. (eds.), Molecular systematics, pp. 407-514. Sinauer Associates, Sunderland, M.A.

T

Taberlet, P., Gielly, L., Pautou, G. and Bouvet, J. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology 17: 1105-1109.

Takhtajan, A. 1997. Diversity and classification of flowering plants. Columbia University Press, New York

V

Verdcourt, B. 1993. Flora of Tropical East Africa. Vitaceae: 1-149.

W

Wagner, W.H. 1961. Problems in the classification of ferns. Recent Advances in Botany 1: 841-844.

Watson, L. and Dallwitz, M.J. 1992. The families of Flowering plants: descriptions, illustrations, identification, and information retrieval, Version 16. http://www. kei I. ukans. edu/delta/.

Wiens, J. J. 1998. The accuracy of methods for coding and sampling higher- level taxa for phylogenetic analysis: A simulation study. Systematic Biology 47:381-397.

57 CHAPTER 5 REFERENCES Wild, H. and Drummond, R.B. 1963. Vitaceae. Flora zambesiaca 2: 439-492.

Wilson, T. and Posluszny, U. 2003. Novel variation in the floral development of two species of Parthenocissus. Canadian Journal of Botonay. 81(7): 738- 748 (2003)

Wiley, E.O., Siegal-Causey, D., Brooks, D.R. and Funk, V.A. 1991. The complete cladist: A primer of phylogenetic procedures. The University of Kansas, Lawrence, Kansas.

Williams, D.M. 1992. DNA analysis: methods. In. Forey, P.L., Humphries, C.J., Kitching, LL., Scotland, R.W., Siebert, D.J. and Williams, D.M. (eds), Cladistics: a practical course in systematics, pp. 102-103. Oxford Science Publications, London.

Wojciechowski, M.F., Sanderson, M.J., Baldwin, B.G. and Donoghue, M.J. 1993. Monophyly of aneuploid Astergalus (): Evidence from nuclear ribosomal DNA internal transcriber spacer sequence. American Journal of Botany 74: 766.

Y

Yamasaki, T. 1994. A new species of Tetrastigma (Vitaceae) from the Ryukyus. Journal of Japanese Botany 69(3): 176 — 178.

Z

Zohary, M. 1982. Plants of the Bible. Cambridge University Press. London.

58 APPENDIX

STATISTICAL ANALYSIS OF DATA APPENDIX STATISTICAL ANALYSIS OF DATA The task of molecular phylogenetics is to convert information on the sequences into an evolutionary tree for those sequences. There are several methods used for inferring phylogenetics and this number is ever increasing. Data obtained from DNA sequences can be categorised as either discrete or distance characters. Distance methods first convert aligned sequences into a pairwise distance matrix, and then input that matrix into a tree building method, whereas discrete methods consider each nucleotide site directly.

1 Distance data Distance measures were first used to reconstruct phylogenetic trees on the amino acid sequences of cytochrome c (Fitch and Margoliash, 1967). Williams, 1992, defined distance data as the degree of dissimilarity between two taxa or genes. Thus, two taxa would be identical if zero distance (100% similarity) separates them. In DNA sequences, this would mean that the level of dissimilarity between two sequences in a pairwise comparison, is equal to the total number of aligned sequence positions with non identical basis divided by the number of sequence positions compared (Swofford et al, 1996). For tree construction, distance data may be divided into two methods, additive distance data and ultrametric distance data.

1.1 Additive distance data Additive distance data trees are trees where the evolutionary distance between any pair of taxa would be equal to the sum of the length of the branches connecting them (Swofford et al., 1996). However, the true topology of an additive tree can only be obtained if no character changes its state more than once, i.e. no homoplasy (Fitch, 1981, 1984). Two examples of additive distance methods include: Neighbour joining method (Saitou and Nei, 1987) and Distance Wagner method (Farris, 1972).

1.2 Ultrametric distance data Ultrametric data trees are trees in which the distance between any two taxa is equal to the sum of the branches joining them. This tree may be rooted, so that all the taxa are the same distance from the root. Thus, assuming that a

59 APPENDIX STATISTICAL ANALYSIS OF DATA molecular clock is operating at the same rate in all lineages, a tree is constructed by connecting the least distant pair of taxa then adding the successively more distant taxa until all taxa have been joined into the tree (Swofford et al., 1996). A common method used to construct trees from ultrametric data is the UPGMA (Unweighted Pair Group Method with Arithmetic mean) procedure. This is the simplest method of tree construction, and was originally developed for constructing phenograms. Because this method does not take into account heterogeneity, it can produce erroneous tree if some lineages evolved faster than others did.

2 Discrete data The use of sequence data for phylogenetic analysis is straightforward. For a set of DNA sequences, the character is represented by a corresponding position in the sequence, while the nucleotide observed, at such a position represents the character state. Character distinction is dependent on two assumptions (Swofford et al., 1996). The first is positional homology or identity, which is the nucleotide observed at a given position in the taxa under study is said to have derived from a single ancestral nucleotide for a particular position. Positional homology is established by aligning sequences, so as to minimise mismatches. Accurate alignment of indels must be conducted (by inserting gaps) to attain positional homoplasy (Swofford et at., 1996). The second assumption is that characters are assumed to be independent variables, whose possible values are drawn from a collection of exclusive character states. The assumption of independence among characters is common to most character-based methods of phylogenetic analysis. A loss of information can occur when converting characters into distances. There are two ways of using discrete characters, maximum likelihood and maximum parsimony.

2.1 Maximum likelihood The principal of maximum likelihood suggests that the explanation that makes the observed outcome the most likely, i.e. the most probable. Mathematically this would mean, LD = Pr (D/H), which is the probability of obtaining D given H

60 APPENDIX STATISTICAL ANALYSIS OF DATA (Page and Holmes, 1998). In DNA sequence data, the nucleotides observed at each nucleotide site are considered separately and the log likelihood for having these nucleotides are computed for a given topology by using a particular probability model. This log likelihood is added for all the nucleotides sites and the sum of the log likelihood is maximized to estimate the branch- length of a tree. This procedure is repeated for all possible topologies and the topology that shows the highest likelihood is chosen as the optimal one (Richardson, 1999).

Edwards and Cavalli-Sforza (1964) were the first to apply maximum likelihood to the estimation of phylogenies using gene frequency data. Then Felsenstein (1981) gave methods for computing the likelihood of a tree with an arbitrary number of species and of finding branch-lengths that maximizes the likelihood. Problems with maximum likelihood arise due to the computational intensity (matrices containing more than 40 taxa can not be analysed) and because there is empirical evidence refuting all molecular models (Siddall and Kluge, 1997). Further, maximum likelihood methods are based on explicit models of evolutionary change. They make more complete use of all available information, i.e. all sites are informative and they are supposedly more consistent and efficient than parsimony (Felsenstein, 1988). However, Siddal (1998) demonstrated cases in which maximum likelihood was inconsistent and inaccurate. Maximum likelihood requires an explicit model of evolutionary change and the methods are therefore more 'assumption laden' than parsimony. There is also a lack of empirical to support proposed models of evolutionary change. In addition, these methods are relatively slow in computing time.

2.1.1 Bayesian analysis

Bayesian analysis considers the many possible histories of substitution weighted by their probability of occurring in a specific model of evolution (Huelsenbeck et al., 2001) and was performed using MrBayes (Huelsenbeck and Ronquist, 2001). An HKY85 model was specified in which all transitions and transversions have potentially different rates. The analysis was

61 APPENDIX STATISTICAL ANALYSIS OF DATA performed using four chains, run for a total of 10 000 000 generations, with trees sampled for every ten generations. Resulting trees were plotted against their likelihoods to determine the point where the likelihoods converged on a maximum value, and all trees before this convergence were discarded as the turn—in' phase. All the remaining trees were imported into PAUP 4.0b1 and a majority rule consensus tree was produced showing the frequencies (i.e., posterior probabilities) of all observed bi-partitions.

2.2 Maximum parsimony Maximum parsimony chooses the tree, or trees, that require the lowest number of evolutionary changes. The data for maximum parsimony comprise of individual nucleotide sites. For each nucleotide site, the goal is to reconstruct the evolution of that site on a tree subject the constraint of fewest possible evolutionary changes. Parsimony maximizes the amount of evolutionary similarity that can be explained as homologous similarity, due to common ancestry (Richardson, 1999). Parsimony has a long history, Edwards and Cavalli-Sforza (1964) first introduced the concept of 'method of minimum evolution', then Camin and Sokal (1965) introduced the term parsimony, and the first application of the principles of parsimony on the molecular evolution of sequences was done by Eck and Dayhoff (1966).

Parsimony's advantages are that it is relatively straightforward to understand, it makes a few assumptions about the evolutionary processes, it has been studied extensively, and there are powerful software implementations available. A problem with parsimony includes the fact that it does not use all the available characters (i.e. ignores the parsimony uninformative sites) and it is supposedly inconsistent (i.e. when rates of change are unequal, it does not always converge on the correct answer as more data is added) (Felsenstein, 1978). However, Greybeal (1998) has demonstrated that the accuracy of reconstruction of a four taxon tree improved dramatically with the addition of more taxa, and also improved with the addition of more characters. Parsimony is also supposedly only reliable when rates of change are low. However, Hillis (1998) simulated an increase in the expected amount of change along all

62 APPENDIX STATISTICAL ANALYSIS OF DATA braches of a particular tree and demonstrated that various methods for inferring phylogeny, including parsimony, preformed better when the rates of change were, in fact, higher.

There are three steps to finding the most parsimonious tree: 1) determining the optimality criterion to infer the tree that specifies the restrictions imposed on character state changes; 2) specifying the algorithm that is used to search for optimal trees under the conditions imposed by the optimality criterion and 3) the measures used to evaluate the result.

3 Choice of parsimony optimality criterion The following optimality criterion has been described: Wagner Parsimony (Wagner, 1961). For a binary character, a change from state 0 to state 1 is given the same weight as a state change from 1 to 0. A consequence of this reversibility is that the length of a tree is independent of the position of the root (Kitching, 1992). This means that an unrooted tree can be rooted at any point, without changing the length of the tree.

Fitch parsimony (Fitch, 1971). Characters with three or more states are unrooted, i.e. they can be transformed into any other state directly. This criterion was formulated for DNA as sequences as it has four character states (A,G,T,C).

Wagner and Fitch parsimony criteria are applicable whenever the probabilities of any character state change are unknown or where they are symmetrical (i.e. change from 0 to 1 has the same probability as a change from 1 to 0). Only the Fitch criterion is appropriate for DNA sequences.

Dollo parsimony (Dollo, 1893). This is appropriate when a reverse change probability (i.e. 1 to 0) is zero. In other words, character polarity is specified. Every derived character state is uniquely defined (parallel

63 APPENDIX STATISTICAL ANALYSIS OF DATA gains of the derived condition are not allowed). Reversal and not parallelism must account for all homoplasy.

4. Camin-Sokal parsimony (Camin and Sokal, 1965). Character evolution is irreversible (equivalent to ordered but not reversible). Under this criterion all homoplasy must be accounted for by parallel or convergent change.

Characters optimised under Dollo or Camin-Sokal parsimony criteria are examples of directed characters.

4 Algorithm used to search for optimal trees A frequently used computer package for inferring phylogenies from distance character data under the principal of maximum parsimony is PAUP (Phylogenetic Analysis Using Parsimony). PAUP provides two basic classes of methods for searching for optimal trees, exact methods and heuristic methods. Exact methods guarantee to find the optimal tree but may require extensive computer time for medium to large seized data sets. Heuristics do not guarantee optimality but generally require far less computer time.

4.1 Exact Methods Exact methods comprise of mainly two searching procedures, the exhaustive search and the branch-and-bound algorithm.

4.1.1 Exhaustive search The conceptually simplest approach to search for the optimal tree is simply to test every possible tree. Assuming that exact methods exists for evaluating the length of any particular tree, an algorithm that generates all possible tree topologies, and evaluates them, is guaranteed to find the optimal trees. Initially the first three taxa in the data set are connected to form the only possible unrooted tree for these taxa. Then, the next taxon is added and evaluated in all topologies (three different possible positions). Then each additional taxon is added and every single tree topology is evaluated as the

64 APPENDIX STATISTICAL ANALYSIS OF DATA subsequent taxa are added at each step. The problem with this method is that the number of trees, to be evaluated, increases dramatically with the addition of more taxa (Swofford et al., 1996).

4.1.2 Branch-and-Bound algorithm This method closely resembles the exhaustive search algorithm described previously. This method employs a search procedure, which has provision for discarding tree without evaluating them in detail. In the first step of tree building (Figure A-1, Swofford, 1993) three taxa (A, B, C) are used to obtain the first tree A. then another taxon is added to this tree resulting in three possible tree topologies (B1, B2, B3). The fifth taxon may then be added to these three tree possibilities, in five different places, resulting in fifteen tree topologies. However, if an upper bound (with regards to tree length) was incorporated into the search, then all of the trees exceeding this upper bound in length will be disregarded. In practice, when the upper bound is exceeded the branch will be cut off and no evaluation of trees with additional branches connected to this branch will be done. Thus it is possible to backtrack down the search tree and proceed up another branch to determine if this will produce a tree with a length less than the upper bound (Swofford et al., 1996).

65 APPENDIX STATISTICAL ANALYSIS OF DATA

Figure A-1: An illustration of the search tree for the branch-and-bound algorithm (Swofford, 1993).

Several factors affect the running time of the branch-and-bound algorithm (Swofford, 1993). The quality of the data is perhaps the most important, large data sets with little homoplasy will run quickly as most paths of the search tree are terminated early. Another important factor is the speed at which the length of each tree can be evaluated. This is a function of character types (Swofford, 1993). Undirected character types run faster than directed character types because certain methods, for rapidly computing tree lengths, can be applied when the tree lengths does not depend on the position of the root. Ordered characters are much faster than unordered characters for the same reasons as above (Swofford, 1993). Then the final factor affecting run times would be the computer speed available on which the program is being run (Swofford, 1993).

66 APPENDIX STATISTICAL ANALYSIS OF DATA 4.2 Heuristic Methods When a data set is too large to permit the use of exact methods, heuristic methods are used which sacrifice the guarantee of optimality in favor of reduced computing time (Swofford et al., 1996). The search begins by building a single tree, which may or may not be the optimal tree in regard to its topology. The manner to which each taxon is added to the tree is constrained. This results in a tree, which is optimal for the constraints given to it. In other words, taxa are added and rearranged to improve the tree topology, when no more improvements can be done, the optimal tree was found. This method is analogous to proceeding along a reasonable path for obtaining this tree (which may or may not be the best tree) without the option of backtracking to explore other possibilities (Wiley et at., 1991). The heuristic procedure may be improved in several ways so that the tree with the local optimum may approach the global optimum with regard to its topology. Two procedures are used in PAUP to achieve this: stepwise addition and branch swapping.

4.2.1 Stepwise addition Stepwise addition operates by connecting taxa, one at a time, to a developing tree until all taxa have been placed. First, three taxa are chosen for the initial tree. Next, one of the remaining unplaced taxa is selected for addition to the tree. Each of the resulting trees, from placing the unplaced taxon in one of the tree possible branches, is evaluated and the one whose length is most optimal is saved for the next round. Now, yet another unplaced taxon is selected for addition to one of the five possible branches on the saved tree from the previous round. Once again all the trees are evaluated and the tree with the most optimal topology is saved for the next round. This process terminates when all taxa have been joined to the tree.

The problem with stepwise addition is determining which three taxa should be joined initially and thereafter which taxon should added to the tree next. PAUP provides four options for specifying the addition sequence.

67 APPENDIX STATISTICAL ANALYSIS OF DATA As is. The taxa are simply added in the same order in which they are presented in the data matrix, starting with the first three and sequentially adding the rest. This method is usually not very effective.

Closest. Initially the length of all possible three taxon trees, formed by joining a triplet of terminal taxa to a single internal node, are evaluated. The three taxa yielding compose the starting tree. At each successive step the remaining taxa are considered for connection to every branch on the tree, and the taxon-branch combination that requires the smallest increase in tree length is chosen. This method requires a lot more computer time than does As is.

Simple. This option corresponds to the order in which taxa are connected in the 'simple algorithm' of Farris (1969). The distance between each taxon and a reference taxon is calculated, Farris called this distance an 'advancement index'. The taxa are then added in order of increasing 'advancement'. So, the reference taxon and the two closest taxa to it, form the starting tree, there after the remaining taxa are joined in the order given by their advancement index.

Random. A pseudorandom number generator is used to obtain a permutation of the taxa to be used as the addition sequence.

4.2.2 Branch swapping Branch swapping is conducted by performing a set of predefined rearrangement of branches within trees. These rearrangements are preformed in the hope to obtain a better tree. If a new tree is found, a new rearrangement is initiated on this tree and the process is continued to come closer to a topology approaching the global optimum. PAUP uses three branch-swapping algorithms (Swofford et al., 1996). In order of increasing effectiveness, they are: (1) nearest neighbor interchanges (NNI), (2) subtree pruning-regrafting (SPR), and (3) tree bisection-reconnection (TBR).

68 APPENDIX STATISTICAL ANALYSIS OF DATA In NNI swapping, each internal branch of the tree defines a local region of four subtrees connected by the internal branch (Figure A-2, Felsenstein, 2004). Interchanging a subtree on one side of the branch with one from the other constitutes a NNI. Two such arrangements are available for each internal branch, as shown in Figure A-2.

A subtree is rearranged by dissolving the connections to an interior branch

and reforming them in one of the two possible alternative ways:

Figure A-2: The process of Nearest Neighbour Interchange (NNI) where an interior branch is dissolved and the four subtrees connected to it are isolated. These can then be reconnected in two other ways (after Felsenstein, 2004).

In subtree pruning and regrafting (SPR), a subtree is pruned from the tree [e.g. the subtree containing terminal nodes A and B, Figure A-3 (after Felsenstein, 2004). The subtree is then regrafted to a different location on the tree. All possible subtree removals and reattachment points are evaluated.

69

APPENDIX STATISTICAL ANALYSIS OF DATA

Break a branch, separate the subtiees

A

Connect a branch of one to a branch of the other Here is the result:

H

Figure A-3: An example of subtree pruning and regrafting (after Felsenstein, 2004).

In tree bisection and reconnection, the tree is bisected along a branch yielding two disjoint subtrees (Figure A-4, Felsenstein, 2004). Joining a pair of branches, one from each subtree, then reconnects the subtrees. All possible bisections and reconnections are evaluated.

70 APPENDIX STATISTICAL ANALYSIS OF DATA

Break a branch, remove a subtree

B

Add it in, attaching it to one (*) of the other branches Here is the result:

E— K Figure A-4: An example of rearrangement via bisection and (after Felsenstein, 2004).

5 Measures used to evaluate results There are several tests to evaluate the resulting trees for robustness, fitness and reliability. These are discussed below.

5.1 Measures of robustness of a cladogram A number of indices are used to test the robustness of a cladogram. Firstly, the consistency index (CI), introduced by Kluge and Farris (1969), and is still the most widely used today, is a measure of how the transformation series and the entire data matrix fit into a tree's topology. If the characters in the data set are perfectly congruent with each other and the tree is thence without homoplasy, then the observed number of steps will equal the minimum, and the CI will equal 1, which is the highest possible value. On the other hand, the greater the chaos in the data and the tree, the greater the homolpasy and the

71 APPENDIX STATISTICAL ANALYSIS OF DATA greater the number of observed steps, and the more the CI shrinks (0 being the lowest possible value).

Farris (1989) introduced another index namely the retention index (RI). RI is used to express the amount of synapomorphies in a data set by examining the actual amount of homoplasy as a function of the maximum number of possible homoplasy. RI can be regarded as the proportion of similarities in a tree due to synapomorphies (Swofford et al., 1996). Thus when a character fits a tree as poorly as possible the retention index will be 0.

When CI is multiplied by RI the resulting answer will be the rescaled consistency index RC. RC ranges from 0 to 1, with higher RC values indicating that characters in the data set are more congruent with each other and the tree (Maddison and Maddison, 1992).

5.2 Reliability of inferred trees Finally the confidence, which can be placed in the topology of a tree, has to be tested. This is a difficult test as one needs to distinguish between psychological and statistical confidence and in the case of a branching diagram, the topology either correctly reflects the historical relationship, or it does not (Siebert, 1992). There are several methods used to attach confidence parameters on branches of trees, these include; the bootstrap method, the jack-knife method, and data decisiveness.

The Bootstrap method was introduced by Felsenstein (1985) and involves random sampling with replacement (compared to random sampling without replacement of the Jack-knife method) of either character rows or columns in a data set to build many bootstrap data sets of the same size as the original data set. Each bootstrap data set is then analysed using a heuristic or branch- and-bound search to give a tree or a set of trees. This procedure of random sampling and tree generation is repeated at least 100 times and a percentage of occurrences of a specific group or component that appears among the

72 APPENDIX STATISTICAL ANALYSIS OF DATA bootstrap trees can be considered as an index of support of that group, although it is not a true statistical confidence support (Siebert, 1992).

The Jack-knife method is also a numerical resampling program. It resamples without replacement and data sets are simulated by systematically leaving characters, or taxa, out of the simulated data sets (Siebert, 1992).

Data decisiveness (DD) is based on the observation with a lot of homoplasy (low CI or RI) are not necessarily uninformative (Goloboff, 1991). Data are considered informative insofar that some cladograms are more efficient summaries thereof than others. This is measured by cladogram length. For any data matrix, DD = S — s / S — M, where S is the mean length of all possible cladograms, s is the length of the most parsimonious cladogram and M is the observed variation in the matrix (the sum of the minimum number of steps for each character). When the length of the most parsimonious cladogram approaches the observed variation in the matrix, DD approaches 1. The matrix is thus consistent with relatively few trees and is decisive. On the other hand, when the DD index approaches 0, the mean length of all possible cladograms equals the length of the most parsimonious tree. The matrix discriminates only weakly among possible trees and it is indecisive (Davies et al., 1998). DD does not capture all relevant attributes of data quality, but it is an informative index of the overall robustness of the support for relationships.

7. Consensus trees Consensus trees are hierarchical summaries of the information common to a set of 'rival' trees. PAUP provides the following consensus tree methods: strict, semistrict, majority rule, and Adams (Swofford, 1993). These methods vary in the criteria used for retaining groups of taxa in the consensus. A consensus tree for a group of minimal trees, will in general, be longer than any of those trees (Swofford, 1993). This is because consensus trees are usually less resolved than any of the rival trees. This means that some characters will have to change more times on the unresolved consensus tree than on the more resolved rival trees. Thus, the consensus tree is not the optimal tree for

73 APPENDIX STATISTICAL ANALYSIS OF DATA a particular data set; it is only a summary of the most parsimonious trees found for that particular data set.

GACB E D F GACF B E D

3

GACF B E D

Figure A-5. Three trees (1, 2, and 3) and their strict consensus (4), majority rule consensus (5), and Adams consensus (6) trees (after Felsenstein, 2004).

7.1 Strict consensus A strict consensus tree contains only those groups that appear in all of the rival trees (Sokal and Rohlf, 1981). This can be considered to be the most conservative estimate of consensus and is the simplest to interpret (Swofford, 1993). In Figure A-5 (Felsenstein, 2004) tree (4) is the strict consensus tree of trees (1), (2) and (3). It preserves only the monophyletic groups that are totally unambiguous.

7.2 Majority rule consensus This method retains groups that appear on a certain pre-specified percentage of the rival trees (Swofford, 1993). Thus a group may appear in the consensus tree, even if there are some conflicting groups on other rival trees (Swofford, 1991). In Figure A-5, the Majority rule consensus tree is represented by (5).

74 APPENDIX STATISTICAL ANALYSIS OF DATA 7.3 Adams consensus These trees (Adams, 1972) are designed to find the maximum number of components for a given set of cladograms by placing conflicting taxa at the most resolved node common to all trees. The Adams consensus shows `nestings' shared among trees instead of monophyletic groups (Swofford, 1993). A group is said to 'nest' within a larger group if the most recent common ancestor of the smaller group is a descendant of the most recent common ancestor of a larger group, which need not require monophyly of either group (Swofford, 1993).

8. Outgroups The inclusion of an outgroup is an important criterion in any phylogenetic analysis. An outgroup may be defined as any taxon used in a phylogenetic analysis that is assumed to be phylogenetically outside the group of taxa under study (Swofford, 1993). It is used for comparative purposes, usually determining character polarity and assigning the direction of change of character transformation (Swofford et aL, 1996). It is also used for determining the root of a phylogenetic tree (Swofford et al., 1996). Outgroups are often chosen as a sister group in the sense that it is a taxon that is genealogically most closely related to the ingroup, but must not be the ancestor of the ingroup.

75 UNIVERSITY OF JOHANNESBURG UNivEnSiT+ .104AaESDA'.' LIBRARY SERVICES

DIGITAL THESIS AND DISSERTATIONS HANDING-IN FORM

Please indicate the handing-in formats of the electronic theses and dissertations, to enable the University of Johannesburg Library to make the theses and dissertations available electronically.

CONTACT DETAILS

Student Number.: 9:4 005 el- Name: Pti u, Nrkt.4 of -t Title of thesis/dissertation: pirl c 5 ear c aN6 Iti N 5 ex/ V faca_cot 13c4Ted O1,, Degree: MSC SC9( er dcairci ,,p ( Department 806 CHNI9 I afr■Ad 610 &ea" ivoiC/51 Contact numbers: Tel.: Cell: Fax: Postal address: E-mail: Date: a 4 19- 1 20c

COPIES

Number of unbound paper copies handed in 1

3. SAVING FORMAT FORMAT QUANTITY

E.g. 1 CD-ROM or 2 Stiffies. Stiffy * CD-ROM ">< I * Other: Specify: FILE FORMAT

* MS * WordPerfect I * PostScript I * PDF I * Other acceptable and approved format

Specify: FILE NAMES

Digital files may be handed in as single files or they may be divided into several files. Should one stiffy not accommodate a dissertation, the document may be divided into several files (and stiffies). FILE NAME LOGICAL DESCRIPTION e,t,t NictA.,aa fdl Sc. A-p e ei,c1 i g

rin,ii-ci- I — 5 T, f-Ve t extrcis

6. ADDITIONAL DOCUMENTS

Please give a complete description below in cases where separate graphs, photo's, figures. etc. in another format and/or separate from the text are handed in.

NAME LOGICAL DESCRIPTION

IM . SIGNATURE: STUDENT SIGNATURE: HEAD: FACULTY ADMINISTRATION

DATE

DECLARATION BY STUDENT

1. CONTACT DETAILS

Student Number.: 9 00 54-(o Name: Paul 11 ct,ii.dc, Title of thesis/dissertation: piij i,yi,v2.4-ic i4 of- 1/44cacte &lad plohd kl uaPtce Degree: Pi5c.

2. DECLARATION

I, the undersigned, hereby declare that the electronic format of the dissertation/thesis is a true replica of the examined dissertation/thesis.

I am also aware of the fact that I will be subject to criminal prosecution, should this declaration be false or contai misrepresentation.

SIGNATURE: STUDENT

6/3/2002

Top View