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Winter 1997

Systematics of the Hamamelidaceae based on morphological and molecular evidence

Jianhua Li University of New Hampshire, Durham

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SYSTEMATICS OF THE HAMAMELIDACEAE BASED ON MORPHOLOGICAL AND MOLECULAR EVIDENCE

BY

JIANHUA LI B.S. Henan Normal University, Xinxiang, China, 1984 M.S. Central China Normal University, Wuhan, China, 1987

DISSERTATION

Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Plant Biology

December, 1997

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SYSTEMATICS OF THE HAMAMELIDACEAE BASED ON MORPHOLOGICAL AND MOLECULAR EVIDENCE

BY

JIANHUA LI B.S. Henan Normal University, Xinxiang, China, 1984 M.S. Central China Normal University, Wuhan, China, 1987

DISSERTATION

Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Plant Biology

December, 1997

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9819680

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation has been examined and approved.

Dissertation Director, Dr. A. Linn Bogle Professor of Plant Biology

Dr.//Garrett E s c r o w Professor of Plant Biology

/

Dr. Anita^?. Klein Associate Professor of Biochemistry and Molecular Biology

Dr. Thomas D. Lee Associate Professor of Plant Biology

Dr. Leland S J J&hnke Associate Professor of Plant Biology

'9 Dati

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THIS DISSERTATION IS DEDICATED TO

my parents and parents-in-laws

Jinsheng and Yuolian Li

&

Jiabao and Yuozhao Zhang

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

I owe great indebtedness to my advisor, Professor Dr. A.

Linn Bogle. Without his continuing encouragement, unreserved

support, great understanding and consideration, I would not

be able to finish this project.

Dr. Anita S. Klein has been very supportive and generous

to me technically and sometimes financially. I am indebted

to her generosity and guidance.

I would like to extend my sincere thanks to the other

members of my dissertation committee, Dr. Garrett Crow, Dr.

Tom Lee, and Dr. Lee Jahnke, who have made the course of my

doctorate education a rich and rewarding experience.

Thanks are extended to Dr. Tao Sang, Michigan State

University, East Lansing, MI for providing matK primers; Dr.

Bruce Baldwin for technical advice; Donald Padgett for the

conversations and friendship we shared as fellow graduate

students in Plant Systematics; Joselle Germano and Danielle

Friel for sharing lab space and technical assistance.

The following people and their institutions deserve

special thanks for helping me collect materials or sending me

DNA for this study: Patricia Grady and Vinnie Simeone of the

Planting Field Arboretum at Oyster Bay, Long Island, New

York; Dr. Niangjun Chung, Forest Experimental Station,

National University of Taiwan; Dr. Shumiaw Chaw, Botanical

iv

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Institute, Academia Sinica, Taiwan; Dr. Tracy D. Omar,

Washington University Arboretum, Seattle, WA; Chris Strand,

Arnold Arboretum of Harvard University, Boston, MA; Dr. Rob

Nicholson, Smith College, MA; Zhongchun Luo, Forest Bureau of

Xinning County, Hunan, China; Dr. Peter K. Endress, Institute

of Systematic Botany, University of Zurich, Switzerland; Dr.

Yinlong Qiu, Indiana University; Dr. Leng Guan Saw, Forest

Research Institute Malaysia, Kepong, Malaysia; Dr. Richard

Saunders, University of Hong Kong; Dr. Armand Randrianasolo,

Missouri Botanical Garden, St. Louis, MI; Longwood Gardens,

PA; Etienne A. Rakotobe, National Center for Pharmaceutical

Research, Madagascar; Division of State Park, Hawaii.

My sincere thanks also extend to the following Herbaria

for allowing me to access specimens of the Hamamelidaceae, or

loaning me specimens of the Hamamelidaceae for this study:

Harvard University Herbaria, Missouri Botanical Garden,

National Herbarium at the Smithsonian Institution, New York

Botanical Garden, and Hong Kong Herbarium.

My special thanks go to the Department of Plant Biology

and the Graduate School of the University of New Hampshire

for awarding me financial support in the form of Teaching and

Research Assistantships, Travel Funds, Graduate School Summer

Teaching Assistant Fellowships, Graduate Student Research

Enhancement Funds, and a Dissertation Fellowship. Financial

support for research from the Powers Fund is also greatly

appreciated.

v

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. My heartfelt thanks are extended to my wife Yuling and

daughter Ran for their support, encouragement, and great

companionship.

I would also like to thank Mrs. M. Bogle for her

hospitality and consideration to me and my family. She made

us feel at home and warmly-loved.

Finally, thanks are extended to many unnamed people who

have offered me help in different ways and enriched my life

in the University of New Hampshire.

vi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

DEDICATION ...... iii

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES ...... ix

LIST OF FIGURES ...... X

LIST OF APPENDICES ...... xiv

ABSTRACT ...... xv

INTRODUCTION

i-1. General Information of the Hamamelidaceae...... 1

i-2. Brief History of Systematics of the

Hamamelidaceae ...... 5

i-3. Systematics Problems of the Hamamelidaceae...... 9

i-4. Parsimony Analysis and Molecular Systematics ....10

i-5. Objectives ...... 11

CHAPTER I. A PHYLOGENETIC ANALYSIS OF THE HAMAMELIDACEAE BASED ON MORPHOLOGICAL DATA

1.1. Introduction ...... 24

1.2. Materials and Methods...... 25

1.3. Results ...... 28

1.4. Discussion ...... 48

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II. PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE INFERRED FROM NUCLEOTIDE SEQUENCES OF THE PLASTID GENE MATK

2.1. Introduction...... 83

2.2. Materials and Methods ...... 87

2.3. Results ...... 91

2.4. Discussion ...... 92

CHAPTER III. PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE INFERRED FROM SEQUENCES OF INTERNAL TRANSCRIBED SPACERS OF NUCLEAR RIBOSOMAL DNA

3.1. Introduction...... 149

3.2. Materials and Methods ...... 152

3.3. Results ...... 158

3.4. Discussion ...... 161

CHAPTER IV. PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE: EVIDENCE FROM MORPHOLOGY AND NUCLEOTIDE SEQUENCES OF ITS AND MATK GENE

4.1. Introduction...... 199

4.2. Materials and Methods ...... 201

4.3. Results ...... 204

4.4. Discussion ...... 205

CHAPTER V. TRANSLATING PHYLOGENY OF THE HAMAMELIDACEAE INTO A CLASSIFICATION SYSTEM

5.1. Introduction...... 251

5.2. Materials and Methods ...... 252

5.3. Results ...... 253

5.4. Discussion ...... 253

CHAPTER VI. GENERAL CONCLUSIONS ...... 271

LITERATURE CITED ...... 273

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

TABLE 1.1, Morphological character codings in the Hamamelidaceae ...... 63

TABLE 2.1. Sources and vouchers of the species sequenced for matK ...... 104

TABLE 2.2. Location and base composition of amplification and sequencing primers ...... 106

TABLE 2.3. Sequence characteristics of the matK gene in the Hamamelidaceae ...... 107

TABLE 2.4. Codon usage of the matK gene in the Hamamelidaceae ...... 110

TABLE 3.1. Species sequenced for nrDNA ITS ...... 171

TABLE 3.2. Sequence divergence of ITS-1 and ITS-2 (below and above diagonal) in the Hamamelidaceae, calculated in PAUP* (mean distance x 100) ...... 173

TABLE 3.3. Characteristics of the most parsimonious trees obtained by PAUP heuristic searches of ITS-1 sequences aligned using different combination of gap and gap length penalties ..176

TABLE 3.4. Characteristics of the most parsimonious trees obtained by PAUP heuristic searches of ITS-2 sequences aligned using different combination of gap and gap length penalties ..177

TABLE 3.5. Sequence characteristics of the two internal transcribed spacers, separated and combined, in 32 species of the Hamamelidaceae...... 178

ix

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

FIG.i.l. Geographic distribution of the Hamamelidaceae...... 12

FIG.i.2. Classification system of the Hamamelidaceae proposed by Reinsch (1889) formatted into a c l a d o g r a m ...... 14

FIG.i-3. Generic relationships of the Hamamelidaceae depicted by Tong (1930), formatted into a c l a d o g r a m ...... 16

FIG.i.4. Classification system of the Hamamelidaceae proposed by Harms (1930), formatted into a c l a d o g r a m ...... 18

FIG.i.5. Classification system of the Hamamelidaceae (non-Chinese genera not included, Chang, 1973) formatted into a cladogram ...... 20

FIG.i.6. Classification system of the Hamamelidaceae proposed by Endress (1989b, c), formatted into a cladogram...... 22

FIG.1.1. Stomatal apparatus in the sampled species of the Hamamelidaceae ...... 64

FIG.1.2. Strict consensus of the 12 shortest trees of 219 steps based on morphological data of the Hamamelidaceae ...... 66

FIG. 1.3. 50% majority consensus of the 12 shortest trees based on morphological data of the Hamamelidaceae ...... 68

FIG. 1.4. 50% majority consensus tree of 12 shortest trees based on morphological data, showing the distribution of numbers of characters that change unambiguously on branch ...... 70

FIG.2.1. Approximate relative locations of matK primers ...... 113

FIG.2.2. The Strict consensus of the six most parsimonious trees of 582 steps long based on sequences of the matK gene ...... 115

x

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIG.2.3. One of the six most parsimonious trees of 582 steps long based on sequences of the matK gene, showing the number of unambiguous substitutions along the branches ...... 117

FIG.3.1. The nrDNA ITS region ...... 179

FIG.3.2. UPGMA phenogram of the Hamamelidaceae generated by analysis of nrDNA sequences aligned using the Optimality method. Jukes-Cantor's one parameter model and gamma rate (1.0) were used in the analysis ..... 181

FIG.3.3. Strict consensus of 90 shortest trees of 1041 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method ...... 183

FIG.3.4. 50% Majority consensus of 90 shortest trees of 1041 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method ...... 185

FIG.3.5. Strict consensus of 12 shortest trees of 6747 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned with the Elision method ...... 187

FIG.3.6. One of the shortest tree of 1041 steps using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method, showing the number of unambiguous character state changes of ITS sequences ...... 189

FIG.4.1. Strict consensus of 203 most parsimonious trees of 185 steps based on morphology of the Hamamelidaceae ...... 217

FIG.4.2. Strict consensus of six most parsimonious trees of 381 steps based on sequences of the matK gene in the Hamamelidaceae ...... 219

FIG.4.3. Strict consensus of 16 most parsimonious trees of 6952 steps based on the ITS grand data set of the Hamamelidaceae ...... 221

FIG.4.4. Strict consensus of morphology and ITS of the Hamamelidaceae obtained using the Consensus approach ...... 223

xi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIG.4.5. Strict consensus of morphology and matK of the Hamamelidaceae obtained using the Consensus approach ...... 225

FIG.4.6. Strict consensus of ITS and matK of the Hamamelidaceae obtained using the Consensus approach ...... 227

FIG.4.7. Strict consensus of morphology, ITS and matK of the Hamamelidaceae obtained using the Consensus approach ...... 229

FIG.4.8. Strict consensus of two shortest tree of 7161 steps based on the combined data set of morphology and ITS of the Hamamelidaceae ....231

FIG.4.9. Strict consensus of eight shortest trees of 590 steps based on the combined data set of morphology and matK of the Hamamelidaceae ...... 233

FIG.4.10. Single shortest tree of 7338 steps based on the combined data set of ITS and matK of the Hamamelidaceae ...... 235

FIG.4.11. Single shortest tree of 7546 steps based on the combined data set of morphology, ITS and matK of the Hamamelidaceae ...... 237

FIG.4.12. Single most parsimonious tree found in the analysis of the combined data of morphology, ITS and matK. showing distribution of character states of leaf form in the Hamamelidaceae ...... 239

FIG.4.13. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of the number of ovules per carpel in the Hamamelidaceae ...... 241

FIG.4.14. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of the character states of petals in the Hamamelidaceae ...... 243

xii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FIG.4.15. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of pollination types in the Hamamelidaceae...... 245

FIG.4.16. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of character states of sexuality in the Hamamelidaceae...... 247

FIG.4.17. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of character states of chromosome base numbers in the Hamamelidaceae ...... 249

xiii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF APPENDICES

APPENDIX 1.1. Herbarium and chemically fixed specimens observed for this s t u d y ...... 72

APPENDIX 2.1. Aligned sequences of the chloroplast gene matK in the Hamamelidaceae ...... 119

APPENDIX 2.2. Amino acid sequences translated from the matK gene sequences ...... 140

APPENDIX 3.1. Aligned nrDNA ITS sequences in the Hamamelidaceae...... 191

xiv

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

SYSTEMATICS OF THE HAMAMELIDACEAE BASED ON

MORPHOLOGICAL AND MOLECULAR EVIDENCE

by

Jianhua Li University of New Hampshire, December, 1997

The Hamamelidaceae are a plant family of 31 genera and

more than 140 species distributed in both the Old and New

worlds. There has long been debate about the subfamilial,

tribal, as well as generic, relationships within the family.

Parsimony analysis was performed to examine the

relationships in the Hamamelidaceae based on morphology, and

DNA sequences of the Internal Transcribed Spacers (ITS) of

nuclear ribosomal DNA, and the chloroplast matK gene. The

morphology-based phylogeny shows weak support for all clades

except for the Altingioideae, and differs greatly from the

molecular phylogenies. Phylogenies based on the matK and ITS

data are basically congruent. The consensus of the most

parsimonious trees generated from separate analyses of the

morphology, ITS and matK data sets resolves few

relationships. However, the analysis using the combined data

set of morphology, ITS and matK gene data resolves all

suprageneric relationships of the Hamamelidaceae.

xv

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In the combined phylogeny, the first clade containing

Altinqia and t,iqiHriainhar is sister to the second clade

including the other genera of the Hamamelidaceae, supporting

the recognition of the Altingioideae. Exbucklandia and

Rhodoleia form a clade, suggesting a close relationship

between the two genera. Mytilaria forms its own clade, as

does Disanthus. Paraphyly of Disanthus, Mytilaria and

Exbucklandia does not support the combination of these genera

in one subfamily. Disanthus is the immediate sister taxon to

the clade of the Hamamelidoideae.

A phylogenetically annotated classification system is

presented here that reflects natural relationships of the

genera, facilitates communication of taxon relationships, and

minimizes nomenclatural modifications. This classification

system recognizes six subfamilies, and six tribes in the

subfamily Hamamelidoideae: 1) Altingioideae,

2) Rhodoleioideae, 3) Exbucklandioideae, 4) Mytilarioideae,

5) Disanthoideae, and 6) Hamamelidoideae. Tribes are

1) Corylopsideae, 2) Loropetaleae Trib. Nov.,

3) Hamamelideae, 4) Fothergilleae, 5) Eustimgateae, and

6) Dicorypheae Trib. Nov.

The combined phylogeny manifests that parallel evolution

has occurred for several important morphological characters

that have been used to classify the family, including loss of

petals, wind pollination, and monoecy.

xvi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION

General Information of the Hamamelidaceae

Diagnostic Features

The Hamamelidaceae (Witch-hazel) are a family of plants

in the subclass Hamamelidae (Cronquist, 1981). Plants of

this family can be identified by the following morphological

characteristics. Trees or shrubs. Leaves persistent or

deciduous, generally alternate or rarely opposite, stipulate,

simple or palmately lobed; venation pinnate or palmate;

epidermal trichomes simple, stellate or peltate scales.

Inflorescences racemose, spicate or capitate, sometimes

subtended by showy bracts. Flower morphology very variable,

ranging from complete to incomplete, from bisexual to

andromonoecious, and to monoecious; calyx composed of four or

five united sepals, variable or lacking (absent in Distylium

S.et Z. and Distvliopsis Endress); corolla of four or five

distinct petals, reduced or lacking (absent in Liquidambar

L ., Parrotiopsis Schneider, Fotherqilla Murray,

Semiliquidambar Chang, Distylium. Matudaea Lundell,

Molinadendron Endress, Parrotia C.A.Mey, Sycopsis Oliv. and

Altinqia Nor.); stamens (2)-4, 5, 10-(>20), anthers

tetrasporangiate (bisporangiate in Exbucklandia R.W.Br. and

Hamamelis L.); ovaries 2-carpellate, superior, semi-inferior,

1

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or inferior, each locule with one or more ovules. Fruit, a

capsule, with woody exocarp and horny endocarp. Seeds one to

several per carpel, winged or unwinged; seed coat bony,

embryo straight, and endosperm albuminous.

Monophyly of the Hamamelidaceae

The Hamamelidaceae have long been recognized as a

natural group (Bentham and Hook, 1865; Reinsch, 1889;

Niedenzu, 1891; Harms, 1930; Bogle and Philbrick, 1980;

Endress, 1989c). Moreover, the monophyly of this family has

been supported by phylogenetic analyses based on both

morphological (Hufford, 1992) and molecular data (Hoot and

Crane, 1996; Hoot et al., 1997; Li et al., unpublished).

Floristics and Geographic Distribution

There are 31 genera in the Hamamelidaceae as of this

date (Zhang and Lu, 1995). The hamamelidaceous plants are

distributed in every Continent (Fig. i.l ). Whereas most of

the genera are distributed in the Northern Hemisphere,

several genera occur in the Southern Hemisphere, including

Trichocladus Pers., Ostrearia Baill., Neostrearia L.S.Smith,

Noahdendron Endress, Hyland et Tracey, and Dicoryphe Du

Petit-Thouars. Many genera in the Hamamelidaceae are

narrowly endemic to one island or a small geographic area.

For instance, Trichocladus is endemic to southeastern Africa;

Ostrearia. Neostrearia and Noahdendron cure distributed only

2

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in Queensland, Australia; Mainqaya Oliv. is endemic to

Malaysia, Dicoryphe to Madagascar, Chunia Chang to Hainan,

while Fortunearia Rehd.et Wils., Sinowilsonia Hemsl.,

SemilicpTi-riamhar. and Shaniodendron Deng, Wei et Wang can be

found only in China. However, there are two genera in the

family whose species are widely and disjunctly distributed in

several continents. One of them is Liquidambar. which can be

found in central Asia, southeastern Asia, southeastern North

America and Central America (Bogle, 1968, 1986); the other is

Hamamelis, which contains species distributed in southeastern

Asia, southeastern North America and Central America (Mione

and Bogle, 1990).

There are about 140 species in the Hamamelidaceae (Zhang

and Lu, 1995). However, more than half of the genera in the

Hamamelidaceae are either monotypic (single species) or

oligotypic (several species). Only a few genera contain more

than ten species, e.g., Corylopsis S.et Z. (7-36), Dicoryphe

(10-20), and Distylium (10-18). However, the number of

species in most of the large genera is uncertain, and all

groups need revision.

Zhang and Lu (1995) analyzed the ecogeographical pattern

of the genera in the Hamamelidaceae and recognized two types

and nine subtypes of distribution: I. Tropical distributions

(18 genera), including: 1) tropical Asia, 2) tropical Central

America, 3) tropical Africa, and 4) tropical Australia; and

II. Temperate distributions (13 genera), including: 1) Asian,

3

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2) western Asian, 3) western Asian-eastern Asian-southeastern

North American, 4) eastern Asian-North American disjuncts,

and 5) southeastern North American. Zhang and Lu (1995) also

made a statistical analysis of the genera and species

distributed in different floristic regions recognized by

Takhtajan (1969). These included Eastern Asiatic Region,

81/16 (species/genera); North-American Atlantic Region, 6/3;

Iran-Turanian Region, 3/3; Sudano-Zambezian Region, 3/1;

Madagascan Region, 13/1; Indo-Chinese Region, 40/12; Malesian

Region, 6/4; Caribbean Region, 8/4; Cape Region, 2/1; and

Northeast Australian Region, 3/3. Several genera are

distributed in more than one region.

Economic Significance

Many species in the Hamamelidaceae are large trees whose

wood has long been used as lumber for different kinds of

construction and furniture making (Chang, 1979). The best

known ones are r.igm'danihar and Altinaia. Several tree

species have long been planted as street trees in many

cities, such as Liouidambar, while many shrubby species are

widely cultivated as ornamentals, including Corylopsis.

Fotherailla. Hamamelis. and Loropetalum R.Br.ex Reichb. A

fragrant gum (storax) used in perfumery, as an expectorant,

inhalant and as a fumigant in treatment of skin diseases is

derived from Liouidambar styraciflua and lu orientalis

(western Asia). Liquidambar species are also frequently

4

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. grown for their colorful autumn foliage. Hamamelis

virainiana yields the widely used astringent and soothing

lotion for cuts and bruises known as 'Witch Hazel'. Water

diviners ('dowsers') favor Witch hazel twigs for their

dowsing (Mione, 1987).

Brief History of Systematics of the Hamamelidaceae

The name Hamamelidaceae dates back to 1818 when Brown

recognized it as a natural group, including the four genera

Hamamelis L., Dicoryphe Thouars, Dahlia Thunb.

(= Trichocladus Pers.), and Fotherailla L. f. Since then

more genera have been continually added to the Hamamdlidaceae

(See Bogle, 1968). As a result, many botanists have studied

the natural relationships of the taxa in the Hamamelidaceae.

De Candolle (1830) recognized two tribes within the

family: the tribe Hamamelideae (Hamamelis, Dicoryphe. and

Trichocladus) and the tribe Fothergilleae (Fotherqilla).

Gardner (1849) proposed a broad concept of the

Hamamelidaceae, including the segregate families

Altingiaceae, Bruniaceae and Helwingiaceae.

Bindley (1853) divided all the known genera of the

Hamamelidaceae into two groups: Group I, characterized by

solitary ovules, included Dicoryphe. Corylopsis.

Trichocladus. Hamamelis. Loropetalum. Parrotia, Fotherqilla.

and Distylium. Group II, characterized by several ovules in

each locule, consisted of Bucklandia (=Exbucklandia).

5

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sedaewickia (= Altinqia). Rhodoleia Champ.ex Hook, Eustiama

Gardn.et Champ, and Tetracrypta (= Anisophyllea.

Anisophylleaceae).

Oliver (1862) arranged the uniovulate genera of the

Hamamelidaceae into three groups, based on the absence or

presence, and on the forms of petals. The apetalous group

consisted of Parrotia. Fotherqilla. Distylium. and Sycopsis.

The second group, with linear-elongate, lanceolate to

spathulate petals, was composed of Corvlopsis. Dicoryphe.

Hamamelis. Loropetalum. and Trichocladus. The third group,

with squamiform petals, was made up of Tetrathvrium Benth.

and Eustioma.

Bentham and Hooker (1865) proposed a system of the

Hamamelidaceae which contained 15 genera. They firstly

divided the family into two groups based on the number of

ovules per locule, a uniovulate-locule group and a

multiovulate group. The first group was the same as those

three groups of Oliver (1862, see preceding paragraph), while

the second included Rhodoleia. Bucklandia (= Exbucklandia).

Altinqia. and Liquidambar.

Reinsch (1889) was the first botanist to combine data

from both morphology and anatomy, and to provide a natural

classification system for the family with a hierarchy of

subfamilies. In this system, he recognized three

subfamilies: the Altingioideae, Bucklandioideae, and

Hamamelidoideae (Fig. i.2).

6

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Niedenzu (1891) revised Reinsch's system by: 1) reducing

subfamilies Altingioideae and Bucklandioideae into the rank

of tribe under the subfamily Bucklandioideae; 2) removing

Mvrothamnus (= Myrothamnaceae) from the family, and 3)

recognizing two tribes in the Hamamelidoideae, Parrotieae,

which included Distylium. Parrotia, Fotherqilla. and

Corvlopsis. and Hamamelideae, which contained Tetrathyrium.

Mainqava. Loropetalum. Eustioma. Sycopsis. Hamamelis.

Dicoryphe. Trichocladus. and Franchetia in the

Hamamelidoideae.

Tong (1930) was the first to devise a classification

system of the Hamamelidaceae explicitly on the basis of

floral evolution. After analyzing the possible evolutionary

course of floral characters, he proposed several criteria for

determining primitive flowers in the Hamamelidaceae: 1)

flowers hermaphroditic, 2) floral organs in definite numbers,

3) sepals only slightly connate, 4) petals well developed, 5)

stamens variable, and 6) ovules in each locule numerous.

Consequently, he revised Niedenzu's system to reflect his

hypothesis of floral character evolution (Fig. i.3).

Harms (1930) made a considerable revision of Niedenzu's

system. In fact, his revised classification scheme had been

considered as the most comprehensive until a more recent one

was proposed by Endress (1989b). In his system of the

Hamamelidaceae, shown in Fig. i.4, Harms (1930) established

five subfamilies, the largest of which, the Hamamelidoideae,

7

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was further subdivided into five tribes. However, he did not

place the two, then little-known genera, Mytilaria Lecomte

and Ostrearia.

Chang (1948, 1973, 1979) basically adopted Harms's

system, but he erected a new subfamily Mytilarioideae to

include Mytilaria and his new genus Chunia (Fig. i.5).

Schulze-Menz (1964) transferred Sinowilsonia from the

Tribe Distylieae into the Corylopsideae, which consisted of

Corvlopsis and Fortunearia.

More recently, Endress (1989a) reviewed the previous

systems, especially those of Harms and Chang. He recognized

four subfamilies for the Hamamelidaceae, including

Altingioideae, Exbucklandioideae, Rhodoleioideae and

Hamamelidoideae (Fig. i.6). His Exbucklandioideae is a

combination of Exbucklandioideae, Disanthoideae, and the

Mytilarioideae. For the largest subfamily Hamamelidoideae,

Endress (1989b, c) combined two tribes, Fothergilleae and

Distylieae, forming the tribe Fothergilleae sensu lato. He

not only recognized the replacement of Sinowilsonia by

Schulze-Menz but also united Fortunearia and Sinowilsonia

with Eustigma, thus establishing the Eustigmateae sensu lato.

Further, he separated the five genera of the Southern

Hemisphere (Ostrearia, Neostrearia. Noahdendron. Dicoryphe,

and Trichocladus1 from the rest as a subtribe Dicoryphinae in

the tribe Hamamelideae.

Deng et al. (1992a) described a segregate genus

8

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Shaniodendron and placed it in the Fothergilleae.

Systematic Problems of the Hamamelidaceae

There is no doubt that the systematics of the

Hamamelidaceae has evolved to a better understanding of the

relationships of the taxa due to the accumulation of more

data from studies in a number of different disciplines,

including morphology, anatomy, embryology, floral ontogeny,

and cytology (Baillon, 1871, 1871-1873; Harms, 1930; Tong,

1930; Chang, 1948, 1962, 1973, 1979; Endress, 1967, 1976,

1989a, b, c; Bogle, 1968, 1970, 1986, 1987a, b, 1989, 1990,

1991; Melikian, 1973a, b, 1975; Rao, 1974; Goldblatt and

Endress, 1977; Bogle and Philbrick, 1980; Wisniewski and

Bogle, 1982; Mione and Bogle, 1990; Pan et al., 1991).

However, the Hamamelidaceae are so widely distributed in

several continents of the World that it is almost impossible

to access every genus for many studies, especially for those,

such as embryology and floral ontogeny that require serial

collections of flowers and fruits once every three to four

days for at least one growing season. Both field study and

laboratory research are costly, while financial assistance,

on the other hand, is hard to acquire for systematics

projects. Encouragingly, as sciences and technologies that

are related to systematics develop, systematists get more and

more new characters that might be used to better estimate

phylogenies, thus providing a basis for revisions of previous

9

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. classification schemes. Therefore, every system reflects

what has been understood up to that time. This can be

attested by the differences among the several suprageneric

systems of the Hamamelidaceae (Harms, 1930; Tong, 1930;

Chang, 1973, 1979; Endress, 1989b, c).

As can be summarized from the controversies concerning

suprageneric relationships of the Hamamelidaceae, several

problems need to be addressed: 1) How many subfamilies should

be recognized in the Hamamelidaceae so that each subfamily

represents a monophyletic group? 2) What are the

relationships among the subfamilies? 3) Within the

Hamamelidoideae, how many monophyletic tribes can be

recognized? 4) Are the defining features in the

Hamamelidaceae derived from one ancestral form? In other

words, how many times have some of the morphological

characters in the family evolved?

Parsimony Analysis and Molecular Systematics

Given the assumption that evolution takes place in a

minimum number of steps, maximum parsimony analysis has been

created to search for the most parsimonious, i.e., the

shortest, phylogenetic trees that should reflect the

evolutionary relationships of organisms. This type of

analysis has been used extensively and proven to be effective

in resolving relationships at various levels in many groups

of organisms (Olmstead and Palmer, 1994).

10

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Molecular data, especially DNA sequences, have been

widely favored for studying natural relationships among

organisms because DNA sequence differences are free of non-

heritable environmental perturbations that obscure true

genetic relationships (Doyle, 1993).

Objectives

The objectives of this study include the following: 1)

to conduct parsimony analyses using both morphological and

molecular data, thus being able to assess the suprageneric

systems previously proposed for the Hamamelidaceae; 2) to

understand the possible evolutionary directionalities of

important characters in the family; 3) to provide a revised

classification system for the family so that only

monophyletic groups will be recognized.

11

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. i . 1. Geographic distribution of the Hamamelidaceae (Drawn based on Elias, 1980; Heywood et al., 1993; Zhang and Lu, 1995; and Rakotobe, 1996).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.i.2. Classification system of the Hamamelidaceae proposed by Reinsch (1889) formatted into a cladogram.

14

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altinqia Altingioideae Myrothamnus Liquidambar Bucklandia Bucklandioideae Disanthus Rhodoleia Eustigma Maingaya Fothergilla (D M H- Loropetalum CL 0 Hamamelis H- CL (D Tetrathyriurn 0) (D Dicoryphe Franchetia Corylopsis Parrotia Trichocladus Distylium Sycopsis

15

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.i.3. Generic relationships of the Hamamelidaceae depicted by Tong (1930), formatted into a cladogram. Arrows indicate the proposed ancestral taxa in the original diagram.

16

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bucklandia Rhodoleia

Disanthus

Altingia •Liquidambar

Mytilaria

Distylium

Sycopsis

Parrotia

Parrotiopsis

Fotherqilla

Sinowilsonia

Eustigma

.Corylopsis

Fortunearia

Maingaya

Loropetalum

Hamamelis

Trichocladus

Dicoryphe

Franchetia

17

i

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.i.4. Classification system of the Hamamelidaceae proposed by Harms (1930), formatted into a cladogram.

18

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Disanthoideae Disanthus Altingioideae Liquidambar Altingia

Disylium — Distylieae Sycopsis Sinowilsonia Parrotia Fothergilleae Parrotiopsis Fothergilla Hamamelis — Loropetalum

19

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. i.5. Classification system of the Hamamelidaceae (non- Chinese genera not included, Chang, 1973), formatted into a cladogram.

20

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Disanthoideae Disanthus

Altingia Altingioideae Semiliquidambar

Liquidambar Rhodoleioideae Rhodoleia Exbucklandioideae Exbucklandia

Mytilarioideae Chunia c Mytilaria

Distylium Distylieae Sycopsis

Sinowilsonia

Parrotia Fothergilleae Parrotiopsis

Fothergilla Hamamelidoideae Corylops ideae Corylopsis c Fortunearia Eustigmateae Eustigma

Hamamelis

Embolanthera Hamamelideae Tetrathyrium

Loropetalum

21

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. i.6. Classification system of the Hamamelidaceae proposed by Endress (1989b, c), formatted into a cladogram.

22

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Altingioideae Semiliquidambar Liquidambar Rhodoleioideae Rhodoleia Mytilaria Exbucklandioideae Chunia Exbucklandia Disanthus Distylium Matudaea Distyliopsis Sycopsis Parrotia Fothergilleae Fothergilla Parrotiopsis Molinadendron Eustigmateae Eustigma Fortuneeuria Sinowilsonia Hamamelidoideae Corylopsis Hamamelis Hamamelidinae Embolanthera Maingaya Tetrathyriurn ■ Loropetalum J

.Dicoryphe Loropetalinae Trichocladus Dicoryphinae Ostrearia 'Neostrearia • Noahdendron

23

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

A PHYLOGENETIC ANALYSIS OF THE HAMAMELIDACEAE

BASED ON MORPHOLOGICAL DATA / INTRODUCTION

Morphology, or the external form of an organism, has

been and still is the type of data used most in plant

classification (Davis & Heywood, 1963).

Today, even though more efforts and financial support

are directed to using molecular markers to study natural

relationships of plants, non-molecular data, including

morphology, anatomy, embryology and floral ontogeny, etc.,

are still invaluable systematically. They produce

phylogenetically informative data for resolving issues such

as homology, and for assessing relationships among taxa at

different levels. This is attested by the fact that in many

taxa whose systematics has been studied on the basis of both

non-molecular and molecular data, the phylogenies produced

from the two sources are to some extent congruent (Donoghue

and Sanderson, 1992).

In the Hamamelidaceae, three major classification

systems have previously been proposed in recent decades

(Harms, 1930; Chang, 1973; Endress, 1989a). It can be seen

from Figs. i.4-i.6 that there are many differences among the

24

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. three systems. Endress (1989b) also published a polytomous

tree showing his view of the phylogenetic relationships in

the subfamily Hamamelidoideae. However, until the present a

comprehensive phylogenetic analysis based on morphological

data has not been conducted for the family. Therefore, the

objectives of this study are dual: 1) to conduct a parsimony

analysis of the Hamamelidaceae using morphological

characters; and 2) to evaluate the existing systems based

upon the analysis.

MATERIALS AND METHODS

There are 31 genera in the Hamamelidaceae. In this

analysis, however, 30 genera were used. The one excluded

genus is Semiliquidambar. which is morphologically similar to

Licruidambar. and is possibly a hybrid between the latter and

Altinaia (Chang, 1948; Bogle, 1986).

Data collection was carried out mostly by using

literature information, as many observations have been made

by various researchers in the last 250 years (Murray, 1774;

Noronha, 1790; Persoon, 1807; Du Petit-Thouars, 1804; Brown,

1818; Candolle, 1830; Meyer, 1831; Siebold and Zuccarini,

1835; Gardner and Champion, 1849; Bentham, 1861, 1865;

Maximowicz, 1866; Baillon, 1871, 1871-1873; Reinsch, 1889;

Oliver, 1860, 1862, 1872, 1890; Niedenzu, 1891; Schneider,

1905; Hemsley, 1906; Merrill, 1909; Rehder and Wilson, 1913;

Lecomte, 1924; Harms, 1930; Lundell, 1940; Brown, 1946;

25

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Smith, 1958; Bogle, 1968, 1987; Chang, 1948, 1973, 1979;

Endress, 1969, 1970, 1977, 1978a, 1993; Melikian, 1973a, b;

Endress et al., 1985; Deng et al., 1992a, b; Zhang and Lu,

1995). I also did my own first-hand investigations on some

of the characters, either to test previous observations or to

fill the previously missing data for some taxa. For such,

herbarium specimens were borrowed from Missouri Botanical

Garden (MO), United States National Herbarium at Smithsonian

Institute (US), Hong Kong Herbarium (HK), and Harvard

University Herbaria (GH).

I also dissected chemically fixed specimens of most of

the genera in the family using a Wild Heerbrugg M-5

stereoscope with multiple magnifications. The materials

observed are listed in Appendix 1.1.

To study the leaf epidermis of a dozen genera that had

not previously been observed, leaves from either herbarium or

F.A.A. (formalin: glacial acetic acid: 70% alcoho1=90:5:5)

fixed specimens were bleached with 5% sodium hypochlorite

(Chlorox) for one to two days depending on leaf thickness.

Then, both lower and upper epidermis layers were cleaned with

a pen-brush, stained with safranin, dehydrated via alcohol

series, and mounted with Permount mounting medium (Fisher

Scientific Co., NJ).

Parsimony analyses were conducted using PAUP 3.1.1

(Swofford, 1993) with a Powermac 7200/120 computer.

Cercidiphvllum ~i aponicum was chosen as the outgroup since

26

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. recent molecular studies (Chase et al., 1993; Hibsch-Jetter

and Soltis, 1996; Hoot and Crane, 1996; Qiu et al.,

unpublished) have all suggested that this species was a

sister group to the Hamamelidaceae.

Given the limit of computer memory and the size of the

data set, the heuristic tree search option was used with the

TBR (Tree Branch Reconnection) branch swapping strategy,

coupled with steepest descent off and multipars on. The

support for each branch of the cladogram was first assessed

by calculating all cladograms one, two, etc., steps longer

than the most parsimonious ones. The corresponding strict

consensus trees show the stability of groups when longer

trees were allowed. This was originally proposed by Farii et

al. (1982) for distance analysis, and by Bremer (1988) for

parsimony analysis (see Gustafsson and Bremer, 1995). This

support was called "Bremer support" or "decay index"

(Donoghue et al., 1992). Bootstrap analysis was also applied

to show the relative strength of branches (Felsenstein,

1985).

Owing to a large number of trees obtained upon the

relaxation of parsimony and the memory capability of the

computer, bootstrap analysis could not get to 100 replicates.

Alternatively, bootstrap analysis was conducted for 1000

replicates with multipars off, and 10 trees were saved in

each bootstrap replicate. To avoid any a priori assumptions,

all characters and their states were unweighted, and every

27

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. multistate transformation was treated as unordered.

RESULTS

Characters and their states

A total of 70 characters were collected. However, only

52 of these were used in this analysis because either the

information for the other 18 characters was missing for most

of the taxa in the Hamamelidaceae, or the characters were

invariable throughout the family. Table 1.1 lists the

characters and their state coding used for this analysis.

The character numbers are as those used in the following

character descriptions. The delimitations of subfamilies,

tribes, and subtribes for the character discussion followed

the system of Endress (1989c), unless otherwise indicated.

1. Habit - evergreen (0), semi-evergreen (1), deciduous (2 ).

In the Hamamelidaceae, nine genera are deciduous and

distributed in Temperate regions of the Northern Hemisphere,

including Disanthus Maxim., Fortunearia. Fothergilla.

Hamamelis. Liquidambar, Parrotia. Parrotiopsis.

Shaniodendron. and Sinowilsonia (Bogle, 1968, 1970; Endress,

1993; Zhang and Lu, 1995). In Coryloosis. while most species

are deciduous, a couple of species are semi-evergreen, such

as CL multiflora Hance. In contrast, the rest of the genera

are evergreen and mainly distributed in subtropical or

tropical areas (Zhang and Lu, 1995).

28

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 . Leaf venation - pinnate (0), intermediate (1), palmate

(2 ).

Most of the genera in Hamamelidaceae possess pinnate

venation (e.g., Sycopsis. Distylium. and Distylioosis. while

some other genera have palmate venation, including

Licruidambar. Exbucklandia. Chunia, Mytilaria, and Disanthus.

Intermediate venation pattern (pinnate venation with basal

crowding of secondary veins), however, is found in several

genera, such as Corylopsis. Fotherailla. Hamamelis, Parrotia

Parrotiopsis. and Shaniodendron (Harms, 1930; Endress, 1993;

Zhang and Lu, 1995).

3. Number of prophylls - one (0), two (1), three (2).

In most of the genera of the Hamamelidaceae there is a

solitary prophyll, while two prophylls are found in Matudaea

Molinadendron. Fortunearia. and Sinowilsonia. Corylopsis is

the only genus with three prophylls on the branch (Hufford

and Crane, 1989; Endress, 1993).

4 . Phyllotaxy - alternate (0), spiral (1), opposite (2).

Leaves of the genera of the Hamamelidaceae are

alternately arranged with the exception of Altingioideae,

where a spiral pattern can be observed, and of Trichocladus

and Dicoryphe. where occasionally subopposite or opposite

leaf arrangement occurs (Harms, 1930; Endress, 1993; Zhang

and Lu, 1995).

5. Peltate leaf blade - absent (0), present (1).

Leaves in the Hamamelidaceae are petiolate, and in most

29

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genera petioles are basifixed. However, in Molinadendron,

Dicoryphe. and Trichocladus. petioles tend to be peltate

(Baillon, 1871, 1871-1873; Endress, 1969, 1989b, 1993).

6 . Leaf margins - entire (0 ), crenate (1), half serrate

(2), serrate (3), lobed (4), lobes serrate (5).

Three or five-lobed leaves are predominant in

Altingioideae (Altiniaa. Liquidambar) and Exbucklandioideae

(Exbucklandia and Mytilaria). However, in Altingia,

Exbucklandia. and Mytilaria. both simple and lobed leaves can

be found on an individual tree (Harms, 1930; Hutchinson,

1967; Bogle, 1968; Chang, 1979; Endress, 1993; Zhang and Lu,

1995), showing leaf dimorphism in these genera. In

Liquidambar. leaf lobes are serrate. Simple leaves are

typical of the rest of the genera in the Hamamelidaceae, the

evergreen genera of which have entire leaves, but several

taxa have half-serrate leaves, or leaves serrate in the upper

half to 1/3, including Distylium. Distyliopsis. Eustiama.

Svcopsis. and Tetrathyrium. Whereas most of the deciduous

taxa have toothed leaf margins, the leaves of Hamamelis are

somewhat crenate (Bradford and Marsh, 1977).

7. Modal Anatomy - trilacular (0), multilacunar (1).

The genera of the family typically have

trilacunar-three-trace nodes (Sinnott and Bailey, 1914;

Metcalfe and Chalk, 1979; Li et al., 1993) with the exception

of Chunia and Mytilaria. where multilacunar nodal anatomy has

been reported (Bogle, 1990, 1991).

30

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8. Secretory canals (gum ducts) - absent (0), present

(1).

These structures occur in the stems and petioles of the

Altingioideae, Chunia, Mytilaria, and Ostrearia (Baillon,

1871; Harms, 1930; Huang and Lee, 1982; Huang, 1986).

9. Leaf tooth type - absent (0), Altingioid (1),

Fothergilloid (2).

The Altingioid type of leaf tooth occurs exclusively in

the Altingioideae of the Hamamelidaceae, while the

Fothergilloid type is represented in the rest of the

hamamelidaceous genera (Li and Hickey, 1988).

10. Stomatal apparatus - Paracytic (0), Stephanocytic (1),

Cyclocytic (2), Anomocytic (3).

The Paracytic type of stomatal apparatus prevails in

the Hamamelidaceae (this study, Fig. l.la-1; Metcalfe and

Chalk, 1979; Pan et al., 1990). The Stephanocytic type

occurs only in Mvtilaria and Tetrathyrium. the Cyclocytic

type is found in Exbucklandia and Rhodoleia (Pan et al.,

1990). The Anomocytic type occurs with the Paracytic type in

Corylopsis. Hamamelis. Fotheroilla. and Sinowilsonia.

11. Leaf trichomes - simple (0), glandular (1), stellate

(2), scale (3).

Simple or 2-branched trichomes occur in Altingioideae

(Liquidambar and Altingia) and Exbucklandioideae

(Exbucklandia. none were found in Chunia, and Disanthus)

(Warren Davis, University of New Hampshire, unpublished).

31

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stellate trichomes are represented in most of the genera of

the Hamamelidoideae with the exception of Distylium.

Distyliopsis, Tteostraari a. Noahdendron. Ostrearia, Sycoosis.

and Rhodoleia. where the scale type of trichome is found (Li

and Hickey, 1988; Fang and Fan, 1993; Warren Davis,

unpublished). Multicellular glandular hairs are found on

petioles and primary veins of several species of Corylopsis.

blade margins of the primary leaves of Matudaea, and stipules

or bracts of Matudaea, Embolanthera Merr., and Tetrathyrium

(Endress, 1989b).

12. Foliar sclereids - absent (0), columnar (1), fusiform

(2), libriform (3), and idiofibrosclereids (4).

Four types of foliar sclereids were defined by Li and

Bogle (1995) in the Hamamelidaceae. No sclereids were found

in Altingioideae, Eustigmateae (excl. Eusticnna). and

Corylopsideae, while the columnar and fusiform types occur in

Hamamelis. the Exbucklandioideae and the Fothergilleae sensu

stricto. Libriform and idiofibrosclereids exist in the

Loropetalinae and Dicoryphinae.

13. Stipule shape - absent (0), linear (1), leafy but not

appressed (2), leafy and appressed (3).

Leaves of the Hamamelidaceae are stipulate, but in

Rhodoleia stipules are not formed in all leaves of a shoot

(Endress, 1978b). Stipules are small and linear in the

following genera: Disanthus. Distylium. Distyliopsis.

Fortunearia. Sinowilsonia. Eusticnna. Trichocladus. Among the

32

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genera containing leafy stipules, Chunia, Exbucklandia. and

Mytilari a have stipules that are paired up and appressed to

each other (Endress, 1969, 1989b, c, 1993; Bogle, 1986).

14. Stipule habit - persistent (0), deciduous (1).

Stipules are deciduous in most of the genera of the

Hamamelidaceae, but in Chunia. Dicoryphe. Exbucklandia,

Mytilaria. and Noahdendron stipules are persistent (Chang,

1948, 1973, 1979; Endress, 1989a).

15. Ovary pubescence - absent (0), present (1).

Glabrous ovaries are found in the Altingioideae,

Exbucklandioideae, Rhodoleia, Eustigma, Fortunearia. and a

couple of species of Corylopsis (Baillon, 1871; Harms, 1930;

Tong, 1930; Hutchinson, 1967; Endress, 1967, 1976, 1989a, b,

1993; Bogle, 1968, 1986).

16. Ovary lenticels - absent (0), present (1).

Eustiama and Fortunearia are the only two genera in the

Hamamelidaceae that have lenticels on the ovaries (Chang,

1979; Endress, 1989b, c, 1993).

17. Number of flowers per inflorescence - two (0), more

than two (1).

The number of flowers per inflorescence varies

considerably in the Hamamelidaceae, or even within a genus,

such as from 5 to more than 30 in Corylopsis (Li et al.,

1993). However, two flowers per inflorescence are found

almost constantly in Disanthus (Maximowicz, 1866; Pan et al.,

1991).

33

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18. Flower parts - five (0)f four (1), variable (2).

The number of flower parts, especially stamens, is 5-

merous in most of the petaliferous genera of the

Hamamelidaceae, while Hamamelis is constantly 4-merous and

several genera have both 5- and 4-merous flowers, such as

Loropetalum. Dicoryphe. and Trichocladus (Baillon, 1871-1873;

Harms, 1930; Tong, 1930; Mione and Bogle, 1990). In

contrast, in most of the apetalous genera, the number of

stamens is variable, such as 15-45 in Shaniodendron (Deng et

al., 1992a, b; Hao et al., 1996), and 21-25 in Parrotiopsis

(Bogle, 1970; Endress, 1976, 1993).

19. Sexuality - bisexual (0), andromonoecious (1),

monoecious (2).

Flowers are both morphologically and functionally

bisexual in petaliferous genera and several apetalous members

of the Hamamelidaceae. However, in several apetalous genera,

both stamen and pistil are present in a flower, but one is

not functional, thus the flower is functionally unisexual.

In this case, both pistillate and staminate flowers are

generally on the same individual, thus giving rise to

monoecy. These taxa include the Altingioideae and

Sinowilsonia (Harms, 1930; Hutchinson, 1967; Bogle, 1968;

Bogle and Philbrick, 1980; Endress, 1993). Andromonoecy

(both bisexual and staminate flowers on the same individual)

occurs in some taxa, including the Fothergilleae (except for

Matudaea and Molinadendron) and Exbucklandia (Bogle, 1970;

34

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chang, 1979; Endress, 1993).

Fortunearia has long been believed to be monoecious

because the pollen grains were thought to be non-functional

in pistillate flowers (Chang, 1979; Endress, 1993). I

collected fresh pollen grains from a small tree of

Fortunearia cultivated in the greenhouse of the University of

New Hampshire, and conducted a pollen viability test on the

pollen from staminate and pistillate flowers using the

Aniline Blue method (Bradford et al., 1974). More than 500

pollen grains were counted and the results showed that about

84% of the pollen grains were viable in both staminate and

"pistillate" flowers. Therefore, this genus is

andromonoecious instead of being monoecious.

20. Pollination - bird (0), insect (1), wind (2).

Rhodoleia is the only genus that is pollinated by birds

in the Hamamelidaceae (Harms, 1930; Bogle, 1989; Endress,

1989b). Although species with showy petals are insect

pollinated, several hamamelidaceous species that have lost

their petals in the course of evolution continue to have

insect pollination because of the specialization, or

modification of other parts, either floral or vegetative, to

attract insects, such as the whitish, showy stamens in

Fotherqilla. and the large, white inflorescence bracts in

Parrotiopsis (Harms, 1930; Bogle, 1968, 1970; Endress,

1989b). Both Eustiama and Fortunearia have rather reduced

petals, but they share purplish and largely expanded stigmas,

35

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. especially in Eustiama. It is likely that the showy

stigmatic surfaces have a function as insect optical

attractants.

21. Flower symmetry - symmetric (0), asymmetric (1).

All of the genera in the Hamamelidaceae have symmetric

flowers except for Rhodoleia. where asymmetric flowers are

found. That is, petals are not initiated on the adaxial side

of a flower due to flower crowding and fusion by condensation

of the inflorescence into a capitulum in the course of

evolution (Harms, 1930; Bogle, 1968, 1989; Endress, 1989a).

22. Sepals - absent (0), present (1).

All of the genera in the Hamamelidaceae possess sepals

except for several Altinaia species, Distylium, Distyliopsis.

Matudaea, and Liquidambar acalycina (Endress, 1989a, 1993).

23. Fusion of sepals into a tubular structure - absent

(0), present (1).

Fusion of sepals into a tubular structure that ruptures

at anthesis and/or falls off by means of circumscissile

dehiscence occurs in Mainaaya and Embolanthera of the

Loropetalinae, in Neostrearia. Dicoryphe. Trichocladus

grandiflorus. and 1\. Goetzei of the Dicoryphinae. Sepals are

partly fused in the Fothergilleae, while in Parrotia persica

differences between individuals in the amount of fusion are

considerable (Harms, 1930; Hutchinson, 1967; Endress, 1967,

1976, 1989a, b, 1993; Bogle, 1968, 1970, 1986).

36

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24. Showy bracts - absent (0), present (1).

This character is present exclusively in Parrotiopsis

(Harms, 1930).

25. Fimbriate glandular hairs - absent (0), present (1).

Bracts with fimbriate glandular hairs are found only in

Matudaea and Embolanthera (Bogle, 1968; Endress, 1989a, 1993;

Zhang and Lu, 1995).

26. Bract fusion - absent (0), present (1).

Fusion of bracts to form a calyx-like structure occurs

in the American genus Matudaea (Bogle, 1970; Endress, 1989b,

c ) .

27. Petals - geniculate with two lateral auricles (0),

geniculate with two dorsal swellings (1), orbicular (2),

spathulate (3), ribbon-like and coiled circinately in the bud

(4), ribbon-like, but not coiled circinately in the bud (5),

ribbon-like and fleshy (6), ribbon-like with expanded broad

base and tapering tip (7), reduced (8), and absent (9).

Petal morphology is fairly diverse in the

Hamamelidaceae. Corylopsis is the only genus having

orbicular petals (Morley and Chao, 1977; Li et al., 1993).

Rhodoleia and Exbucklandia share spathulate petals. Many

genera in the Hamamelidaceae have ribbon-like petals, with

petals coiled circinately in the bud, including Hamamelis.

Loropetalum. Mainaaya. Neostrearia. Noahdendron. Ostrearia,

Tetrathyrium. and Trichocladus. However, Dicoryphe is an

exception, where petals are strap-shaped, but they are not

37

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. coiled circinately in the bud (Endress, 1989a, b ) . Mytilaria

has ribbon-like petals, but the petals are thick and fleshy.

Petals in Disanthus are broadly lanceolate and glandular at

the base (Maximowicz, 1866; Pan et al., 1991). Both

Fortunearia and Sinowilsonia have reduced petals (Rehder and

Wilson, 1913; Bogle, 1968; Chang, 1979; Endress, 1993). In

Embolanthera and Eustiama. petals have auricles, but the

auricles are lateral and more distinct in the former and

dorsal and minute in the latter. No petals are present in

the Altingioideae, Chunia and the Fothergilleae.

28. Fusion of stamen to petal - absent (0), present (1).

Fusion of petals to stamens to form a tube occurs in

some members of the Hamamelidaceae such as Embolanthera.

especially in E^. qlabrescens (Tardieu-Blot, 1965), and in

Dicoryphe. especially in D^. stipulacea. but not in D.

viticoides (Endress, 1989b).

29. Stamen organization - cyclic (0), centripetal (1),

centrifugal (2).

In most of the genera in the Hamamelidaceae, regardless

of how many stamens occur in a flower, stamens cure cyclically

initiated, however, the centripetal pattern of initiation was

found in Matudaea, while the centrifugal initiation pattern

was seen in Fotherailla (Endress, 1976).

30. Anther orientation - Introrse (0), latrorse (1),

extrorse (2).

Latrorse anthers are prevalent in the Hamamelidaceae.

38

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, in Disanthus, extrorse anthers occur (Baillon,

1871). Gardner and Champion (1849) described the anthers in

Eusticnna as extrorse. However, later observations

(Hutchinson, 1967, and this study) indicated that the anthers

are latrorse instead. Introrse anthers are found in the

Dicoryphinae, Hamamelis and Exbucklandia (Endress, 1989b, c).

31. Showy stamens - absent (0), present (1).

In Fotherailla. the stamen filaments are elongate,

clavate, whitish and showy at anthesis, whereas all the other

members of the Hamamelidaceae have simple filaments (Bogle,

1970; Endress, 1989b).

32. Filament length - equal to or longer than the anther

(0), shorter than the anther (1).

Filament lengths vary greatly in the Hamamelidaceae.

Long filaments can be found in several Corylopsis species,

Chunia, Fothergilleae (excl. Molinadendron). Rhodoleia. and

Hamamelis. while short filaments can be seen in the

Eustigmateae, the Loropetalinae, the Dicoryphinae, Disanthus,

Exbucklandia. and Mytilaria (Bogle, 1968, 1970; Chang, 1979;

Endress, 1989b).

33. Elongate anthers - absent (0), present (1).

Oblong and elongate anthers are found in the

Altingioideae, Exbucklandia. and several genera of the

Fothergilleae (Distyliopsis. Matudaea, Parrotia.

Shaniodendron. and Svcopsis) (Bogle, 1970; Endress, 1989b).

39

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34. Anther connective protrusion - adaxial (0), axial

(1), absent (2).

The length of the anther connective protrusion is

variable in the Hamamelidaceae (Baillon, 1871; Bogle, 1968,

1970; Chang, 1979; Endress, 1989b). Long protrusions are

found in the Loropetalinae (excl. Embolanthera 1, and in the

Dicoryphinae (excl. Neostrearia\. where protrusions become

highly adaxial, covering the inner gynoecium completely

before anthesis. In several genera, however, protrusions are

long and erect, or axial, including Exbucklandia. Eustigma,

Chunia. Corylopsis. and the Fothergilleae (excl. Parrotiopsis

and Fotherqillal. Anthers show little connective protrusion

in the rest of the Hamamelidaceae, including the

Altingioideae, Disanthus, Mytilaria, Hamamelis, Embolanthera.

Fortunearia. Sinowilsonia. and Neostrearia (Bogle, 1968).

35. Staminodial phyllomes - more than one whorls (0), one

whorl (1), absent (2).

There is a great diversity of sterile phyllomes in the

Hamamelidaceae (Bogle, 1968, 1970, 1986, 1989; Chang, 1979;

Wisniewski and Bogle, 1982; Endress, 1989b). Sterile

structures in most of the genera in the Hamamelidaceae are of

staminodial origin, while those of the Altingioideae possibly

have carpellary characteristic (Wisniewski and Bogle, 1982).

More than two whorls of the staminodial phyllomes can be

found in Corylopsis. Loropetalum. Mainaaya. and Mytilaria.

whereas one whorl has been observed in Exbucklandia.

40

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hamamelis. Tetrathyrium. Ostrearia. Rhodoleia, and Disanthus.

However, there is no staminodial phyllomes in Chunia,

Embolanthera. the Eustigmateae, the Dicoryphinae (excl.

several Dicoryphe species, and Ostrearia), and the

Fothergilleae.

36. Pollen exine sculpture - coarsely reticulate (0),

finely reticulate (1), foveolate (2), scrobiculate (3).

Coarsely reticulate pollen exines are most prevalent in

the Hamamelidaceae (Bogle and Philbrick, 1980). Finely

reticulate exines are found in Chunia, Distylium and

Parrotiopsis. Rhodoleia is the only genus in the

Hamamelidaceae that has scrobiculate pollen grains, while the

Altingioideae and Matudaea have foveolate pollen exines.

37. Pollen aperture - tricolpate (0), tricolporate (1),

rugate (2), polyporate (3).

Pollen aperture is rather diverse in the Hamamelidaceae.

Tricolpate pollen is the basic type in this family, while

rugate type occurs in several genera, including Chunia,

Matudaea. Molinadendron. and Sycopsis. Rhodoleia is reported

to have tricolporate pollen grains, while polyporate pollen

type is found in the Altingioideae. However, more than one

aperture pattern can be found within a genus. For example,

both tricolpate and tetracolpate pollen grains are reported

in Corylopsis and Hamamelis (Bogle and Philbrick, 1980; Zhang

and Zhang, 1991).

41

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38. Pollen exine - non-verrucate (0), verrucate (1).

Pollen exines of most of the genera in the

Hamamelidaceae are non-verrucate, while in several apetalous

genera or genera with reduced petals, the pollen exine is

verrucate, including the Altingioideae, Fortunearia.

Parrotiopsis. Parrotia, Sycopsis. Distylium, and

Mo1inadendron (Bogle and Philbrick, 1980).

39. Pollen aperture margins - ragged (0), smooth (1).

Smooth pollen aperture margins are found in several

genera including Dicoryphe. Fortunearia. Sinowilsonia.

Embolanthera. Parrotia. Sycopsis. Molinadendron. and

Distylium. while the ragged aperture margin occurs in the

rest of the genera in the Hamamelidaceae (Bogle and

Philbrick, 1980).

40. Pollen aperture membranes - fine (0), coarse (1).

Coarse pollen aperture membranes are the basic type in

the Hamamelidaceae, while fine pollen aperture membranes

occur in Hamamelis. Fothercrilla. Embolanthera. Mainoaya. and

Mytilaria (Bogle and Philbrick, 1980).

41. Anther dehiscence - four valves for four sacs (0), two

valves for four sacs (1), two valves for two sacs (2), simple

slits (3).

Anthers of the Hamamelidaceae are tetrasporangiate with

the exception of Exbucklandia and Hamamelis. where anthers

are bisporangiate. Anthers in several genera dehisce by

simple slits (Disanthus, Parrotia, Shaniorienrirnn. and

42

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sycopsis). while in most genera, anthers dehisce by valves

(Baillon, 1871; Bogle, 1968, 1970; Endress, 1989a). There

are three types of anthers with valvate dehiscence (Endress,

1989a). The first type occurs most commonly, where each

theca has two pollen sacs and opens with two valves. In the

second type, each theca has one pollen sac and opens with one

valve. This dehiscence pattern occurs in only two genera:

Hamamelis and Exbucklandia. The third anther dehiscence type

is most unusual and systematically most interesting. It is

somewhat between the two valvate types above. Although each

theca has two pollen sacs, it has only one valve serving both

pollen sacs. The valve opens toward the floral center as in

the second type. This type is known only from the subtribe

Dicoryphinae, whose genera are confined to the southern

Hemisphere, including southeastern Africa (Trichocladus).

Madagascar (Dicoryphe), and northeastern Australia

(Ostrearia, Neostrearia. and Noahdendron).

42. Inflorescences - raceme (0), spike (1), spadix (2),

capitulum (3), complex capitulum (4).

Flowers in the Hamamelidaceae are clustered into

inflorescences even when there are only a couple of flowers

in an inflorescence (such as in Disanthus). These small to

middle-sized flowers, mostly sessile, and rarely with a short

pedicel, are mostly in dense spikes or capitula. The raceme

occurs in Eusticnna. Fortunearj a. Loropetalum. and in some

species of Corylopsis. Dicoryphe. Madudaea. Molinadendron and

43

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sycopsis. The capitulum type of inflorescence is found in

many taxa such as the Altingioideae, Exbucklandia,

Disanthus, and Trichocladus. among which the Altingioideae

has complex capitulum (Harms, 1930; Tong, 1930; Hutchinson,

1967; Endress, 1967, 1976, 1989a, b, 1993; Bogle, 1968,

1986). Mytilaria and Chunia share the spadix type of

inflorescence (Chang, 1979).

43. Hypanthium tube - Absent (0), present (1).

Hypanthium tubes are formed in Distyliopsis. Sycopsis

and Sinowilsonia enclosing ovaries completely at anthesis

(Harms, 1930; Bogle, 1968; Chang, 1979; Endress, 1989a, b).

However, it is absent in all of the other genera of the

Hamamelidaceae.

44. Ovary position - inferior (0), semi-inferior (1),

superior (2).

The position of the ovary in the Hamamelidaceae ranges

from superior (Sycopsis. Distylium. and Matudaea) to

different levels of fusion of ovary with the hypanthium.

This character is stable within a genus with the exception of

Corylopsis and Fothergilla. where superior to semi-inferior

ovaries occur (Harms, 1930; Bogle, 1968; Chang, 1979;

Endress, 1989b, c). There are several genera possessing

inferior ovaries, including the Altingioideae, Eustigma,

Embolanthera. Loropetalum. and Tetrathyrium (Endress, 1993).

45 . Endocarp - Papery (0), thick but inseparable from

exocarp (1), thick but not ballistic (2), thick and ballistic

44

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (3).

Some fruits in the Hamamelidaceae show an explosive seed

discharge mechanism (Endress, 1989a). This mechanism is

caused by the ballistic activities of the thick and bony

endocarps. It is restricted to one-seeded carpels of the

Hamamelidoideae. In the Altingioideae, the endocarps are

papery and thus are not ballistic. Thick endocarps are

present in the fruits of Rhodoleia and Exbucklandia. but they

are not separable from the exocarp, thus do not discharge

seeds explosively. The endocarps in Disanthus and Mytilaria

are similar to those of the Hamamelidoideae, but they are not

ballistic (Endress, 1989a).

46. Styles - equal to or longer than the stamens (0),

shorter thcin the stamens (1).

In most of the members of the Hamamelidaceae, styles are

equal to or longer than the stamens, however, the styles are

much shorter than the stamens in several genera, including

Ostrearia. Neostrearia. Noahdendron. and Loropetalinae

(Chang, 1979; Endress, 1989b).

47. Stigmas - unexpanded (0), greatly expanded (1).

Both Eustiama and Fortunearia have expanded purplish

stigmas. However, this characteristic does not occur in any

other members of the Hamamelidaceae (Harms, 1930; Chang,

1979; Endress, 1989b, 1993).

48. Decurrent stigmas - absent (0), present (1).

In both long- and short-styled carpels, the stigma is

45

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. often decurrent, though in some entomophilous genera it is

more restricted to the apical region, including Rhodoleia

(Bogle, 1986), Corylopsis, and Hamamelis (Endress, 1989b).

49. Humber of ovules - three or more (0), two to three

(1), one (2).

In the Hamamelidoideae, generally, there is only one

seed in each locule of the bicarpellate ovary, although there

are several genera in this subfamily having more than one

ovule in each locule (Bogle, 1968; Chang, 1979; Wisniewski

and Bogle, 1982; Mione, 1987; Endress, 1989a, b; Pan et al.,

1991), including Corylopsis. Dicoryphe. Hamamelis.

Neostrearia. and Noahdendron. In contrast, there are more

than three ovules in each locule in the other subfamilies.

However, in most of these multiovulate subfamilies

(Rhodoleioideae, Exbucklandioideae, Liquidambaroideae), only

one or two ovules enlarge, reach maturity and become seeds,

while the other ovules remain much smaller and sterile. The

only exception is the Disanthoideae, where there are two or

more fertile seeds per carpel.

50. Seed appendage - unwinged (0), winged (1)

Most of the genera in the Hamamelidaceae do not have a

seed appendage, or seed wing. Winged seeds are found in the

Altingioideae, Chunia, and Rhodoleia (Chang, 1948, 1979;

Endress, 1989a; Zhang and Wen, 1996).

51. Capsule dehiscence - ventricidal (0), loculicidal (1),

septicidal (2).

46

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The capsules are ventricidal in Licruidambar and

septicidal in Disanthus. while the other genera of the

Hamamelidaceae show both ventricidal and septicidal dehiscing

patterns (Baillon, 1871; Chang, 1979).

52. Chromosome numbers (n) - 8 (0), 12 (1), 13 (2), 21

(3).

The chromosome number n=12 is typical of the

Hamamelidoideae and Rhodoleia. Mytilaria has a chromosome

number of n=13 (Pan and Yang, 1994), while n=8 is reported in

the rest of the genera, including Disanthus (2n=16),

Exbucklandia (2n=32), and the Altingioideae (2n=32)

(Goldblatt and Endress, 1977; Morawetz and Samuel, 1989;

Oginuma and Tobe, 1991; Pan and Yang, 1994).

Phylogenetic trees

The parsimony analysis generated 12 shortest trees of

219 steps, and the indices of the trees were 0.45, 0.55, 0.59

and 0.27 for consistency index (Cl), homoplasy index (HI),

retention index (RI), and rescaled consistency index (RC)

respectively. In the strict consensus tree (Fig.1.2),

Altinoia and Liquidambar formed the basal clade with a

bootstrap of 76% and a decay index of one, followed by the

clade of Chunia. Each of the following genera, Exbucklandia.

Mytilaria. Disanthus and Rhodoleia, formed its own clade and

showed paraphyletic relationships consecutively. However,

the bootstrap percentages were less than 50% and the decay

47

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. indices were one step for each of the successive clades.

The genera of the Hamamelidoideae formed a single clade

in both the strict consensus and the majority consensus trees

(Figs.1.2, 1.3). Within the Hamamelidoideae (Fig.1.3), the

five genera that are distributed exclusively in the Southern

Hemisphere formed a monophyletic group, in which Ostrearia

was the basal taxon followed by Noahdendron and Neostrearia.

and Trichocladus and Dicoryphe were allied in a clade.

Mainqaya, Loropetalum. Tetrathyrium. and Embolanthera were

paraphyletic to one another in the most parsimonious trees

(Figs.1.2, 1.3). The rest of the genera of the

Hamamelidoideae formed a clade, with the three genera of the

Eustigmateae being the basal branch. Sister to the

Eustigmateae was the clade consisting of the Fothergilleae

and Corylopsis-Hamamelis.

The bootstrap values were less than 50%, and decay

indices were only one step for all the clades except for a

few, including Altinqia-Liauidambar; Corylopsis-Hamamelis;

Sinowilsonia- Fortunearia; and ((Distylium. Distyliopsis)-

Sycopsis1 (Fig.1.2).

DISCUSSION

The phylogenetic analysis in this study provides pros

and cons for the hypotheses regarding intergeneric

relationships in the Hamamelidaceae suggested by the three

48

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. major classification systems (Harms, 1930; Chang, 1979;

Endress, 1989b, c). I will examine the suprageneric

relationships in each of the subfamilies, tribes, and

subtribes of the system of Endress (1989c).

Altingioideae

This subfamily includes three genera: Altingia,

Liquidambar and Semiliquidambar (Chang, 1962, 1979; Bogle,

1968; Wisniewski and Bogle, 1982; Zhang and Lu, 1995).

Morphologically, Semiliquidambar stands as intermediate

between the other two genera of the Altingioideae.

Therefore, it has been considered either as a possible hybrid

(Chang, 1962; Bogle, 1968; Wisniewski and Bogle, 1982), or a

doubtful genus (Endress, 1989c, 1993). Although it has been

widely recognized that Altingioideae are a monophyletic group

(Bogle, 1968; Endress, 1989a, c), it has long been

controversial concerning whether this subfamily should be

raised to familial status. Nakai (1943) proposed that each

of the five subfamilies of Harms (1930) be raised to family

rank, including the Altingioideae. Family recognition of the

Altingioideae was further supported by palynological studies

(Wang, 1992), wood anatomy (Huang 1986), and seed coat

anatomy (Melikian, 1973a, b). However, Pan et al. (1990)

preferred to keep Altingia and Liquidambar in the

Hamamelidaceae based on their study of leaf epidermis in the

family. This view is still reflected in a recent

49

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. classification system of the Hamamelidaceae (Endress, 1989c).

Takhtajan (1997), however, recognized the family Altingiaceae

and considered it to be closely related to the

Hamamelidaceae.

It should be noticed that most of the characters that

researchers have used to support the separation of the

Altingioideae from the other members of the Hamamelidaceae

are also found in one or more genera in the other

subfamilies. For example, Wang (1992) studied pollen grain

types in the Hamamelidaceae and concluded that the

Altingioideae should be separated from the Hamamelidaceae

proper because of the occurrence of polyporate pollen in the

three genera of the Altingioideae. However, similar types of

pollen grains have been reported in several other genera of

the Hamamelidaceae, including Chunia, Matudaea. Sycopsis and

Distylium (Bogle and Philbrick, 1980). Therefore, using this

one piece of evidence to determine the systematic rank of a

taxon is not convincing, but rather inappropriate, even

though it provides insights for better understanding the

phylogenetic relationships.

The present phylogenetic analysis indicates that the

Altingioideae was supported by five character transformations

(Fig.1.4), these being winged seed, chloroanthoid leaf tooth

type, absence of foliar sclereids, monoecy, and polyporate

pollen grains. This clearly supports the monophyly of the

Altingioideae. However, most of these synapomorphies can

50

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. also be found in other hamamelidaceous genera.

Recent studies (Hoot and Crane, 1996; Hoot et al., 1997;

Li et al., unpublished) based on both nuclear and chloroplast

gene sequences have also shown that the Hamamelidaceae sensu

lato are monophyletic. Therefore, I prefer to retain these

genera in the Hamamelidaceae as the subfamily Altingioideae.

Exbucklandioideae

This subfamily includes four genera, Exbucklandia

(= Symincrtonia 1. Disanthus, Mvtilaria and Chunia (Endress,

1989c).

Brown (1946) was the first to substitute the name

Exbucklandia for Bucklandia since Bucklandia had been

previously used for a fossil gymnosperm by Presley (See van

Steenis, 1952) and must therefore be rejected as a later

homonym. Later van Steenis (1952) also proposed another

name, Syminqtonia. for Bucklandia, apparently unaware that it

was six years after Brown's Exbucklandia; the latter has

nomenclatural priority and is the valid name for the genus

(Kaul and Kapil, 1974).

Exbucklandia has, in the past, been placed close to

Liquidambar and Altingia (Lindley, 1846, 1853; Gardner and

Champion, 1849; Bentham and Hooker, 1865; Baillon, 1871-

1873), or allied with Rhodoleia (Reinsch, 1889; Niedenzu,

1891). Later, Harms (1930) established the subfamily

Exbucklandioideae, which included only a single genus

51

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Exbucklandia.

Disanthus was first described by Maximowicz (1866), and

later was placed into the Bucklandioideae (Reinsch, 1889;

Niedenzu, 1891), or treated as a monogeneric subfamily

(Harms, 1930; Pan et al., 1991).

Mytilaria was described by Lecomte (1924) and was placed

by Tong (1930) in the tribe Altingieae. However, Harms

(1930) did not include consideration of this little known

genus. Later, Chang (1948) described Chunia, a relative of

Mytilaria. and erected a new subfamily Mytilarioideae for the

two genera, implying a close relationship between Chunia and

Mytilaria. Bogle (1990, 1991) reported that both Mytilaria

and Chunia have multilacunar nodal anatomy, which is not

found in any other members of the Hamamelidaceae, supporting

the close relationship of the two genera. However, the

evident differences of Chunia and Mytilaria in petiolar

anatomy, floral and pollen morphology led Bogle (1991) to the

suggest that these two genera be placed in the subfamily

Exbucklandioideae, but treated as a distinct tribe, the

Mytilareae.

A cytological study (Pan and Yang, 1994) pointed out the

karyomorphological differences between Mytilaria and the

other members of the Exbucklandioideae and supported the

recognition of the subfamily Mytilarioideae (Karyomorphology

of Chunia was not studied in that study).

Endress (1989c) took a broader view of the subfamily and

52

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. put all four genera together in his concept of the

Exbucklandioideae. The characters he claimed to define the

subfamily are 5-8 ovules per carpel, simple or tricuspidate

leaves, actinodromous venation, large and persistent

stipules, x=8 for Disanthus and Exbucklandia, and seemingly

bisexual flowers. Most recently, however, Takhtajan (1997)

has reclaimed the subfamily Disanthoideae from the

Exbucklandioideae.

In the present phylogenetic analysis, the four genera

each formed their own clade and were paraphyletic to one

another. This strongly suggests that they do not form a

natural group (Figs.1.2, 1.3). Furthermore, Chunia is very

isolated from the others in the cladogram, as supported by a

group of distinct characteristics, including lack of petals,

winged seeds, and wind pollination.

Rhodoleioideae

Rhodoleia was first described by Hooker (1850). Before

being recognized as belonging to a separate monogeneric

subfamily (Harms, 1930), Rhodoleia was consistently

associated with Exbucklandia and Liquidambar (Reinsch, 1889;

Niedenzu, 1891). Nakai (1943) elevated the Rhodoleioideae to

familial status, which found support from Shaw (1966) and

Wolfe (1973). Nevertheless, this view has not been widely

adopted by others.

Wood anatomy led Tang (1943) to the conclusion that

53

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rhodoleia is distinct from Exbucklandia and Disanthus. and

should be a separate subfamily. Skvortsova (1960a, b, c)

found Rhodoleia to be comparable to a number of genera of the

Hamamelidoideae in petiole vasculature, leaf venation and

stomatal type, but distinct from Exbucklandia, Mytilaria.

Disanthus, Altinaia. and Liquidambar. Melikian (1971, 1972,

1973a, b, 1975) found the seed coat anatomy of Rhodoleia to

be similar to that of Exbucklandia and Chunia, and concluded

that the three genera could be included in a single

subfamily, Rhodoleioideae, perhaps as tribes Exbucklandieae

and Rhodoleieae. A similar conclusion was reached by Rao

(1974), who studied seed anatomy of the Hamamelidaceae.

In the strict consensus tree generated in this study

(Fig.1.2), Rhodoleia formed its own clade and was sister to

the Hamamelidoideae. The most important characteristic that

allies Rhodoleia and the Hamamelidoideae is the chromosome

number n=12, which is different from any of the other

subfamilies of the Hamamelidaceae, where n=8 or 13 (Goldblatt

and Endress, 1977; Morawetz and Samuel, 1989; Oginuma and

Tobe, 1991; Pan and Yang, 1994). Therefore, this

phylogenetic analysis supports the most widely recognized

treatment, that is, to keep Rhodoleia as a monogeneric

subfamily within the Hamamelidaceae. The Rhodoleioideae are

supported by a combination of several morphological

characteristics, including cyclocytic stomatal apparatus,

tricolporate and scrobiculate pollen grains, asymmetric

54

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. flowers, long spathulate petals, long stamen filaments, and

presence of staminodial phyllomes.

Hamamelidoideae

The hamamelidaceous taxa had long been divided into a

minimum of two groups (Gardner and Champion, 1849; Lindley,

1853; Bentham and Hooker, 1865; Baillon, 1871-1873), the

largest of which is the Hamamelidoideae, characterized by

uniovulate locules, and the remaining genera, which are

multiovulate. In the phylogeny (Figs.1.2, 1.3), the genera

of the Hamamelidoideae formed a well-defined clade,

suggestive of the monophyly of this taxon. This result

agrees with previous morphological studies (Bogle, 1968;

Bogle and Philbrick, 1980; Endress, 1989b, c; Hufford and

Crane, 1989), and further supported by recent DNA sequence

analyses of the rbcL gene (Qiu et al., unpublished); ITS and

matK (Li et al., 1997a). There are several synapomorphies

defining this natural subfamily, such as the ballistic

mechanism of seed dispersal, several to one ovule per locule,

and the chromosome base number of n=12.

There are about 23 genera in the Hamamelidoideae,

accounting for more than 75% of the genera in the family. A

great morphological diversity can be seen among the genera.

As a result, there has been disagreement about the

intergeneric relationships within the Hamamelidoideae.

55

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Corylops ide ae

The circumscription of this tribe varies, depending on

the author. In Harms's system (1930), the Corylopsideae

included two genera, Corvlopsis and Fortunearia. which he

believed to be similar in leaf morphology. Schulze-Menz

(1964), however, recognized the similarity of Sinowilsonia

with Fortunearia in both floral structures and leaf

morphology, and thus transferred Sinowilsonia from the

Distylieae into the Corylopsideae, resulting in a three-genus

tribe. Corvlopsis has two unique characteristics that can

not be found anywhere else in the Hamamelidaceae, or even the

subclass Hamamelidae. They are orbicular petals and the

horizontally reflexed styles. It is these peculiarities that

led Endress (1989b, c) to the conclusion that Corylopsis

should constitute a monogeneric tribe.

In the phylogenetic tree produced in this study

(Fig. 1.2), Corylopsis was grouped with Hamamelis and the

clade was supported by four morphological states, including

presence of staminodes, non-decurrent stigma, 2-3 ovules per

carpel, and the semi-palmate venation. The clade of

Corylopsis and Hamamelis, however, did not share immediate

ancestry with the tribe Eustigmateae sensu latp, thus

offering support for a distant relationship between

Corvlopsis and the genera Fortunearia and Sinowilsonia. as

proposed by Endress (1989b, c).

56

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Eustiamateae

Eustiama was first described by Gardner and Champion

(1849), who noticed the peculiarities of this plant.

However, it was Harms (1930) who recognized Eustiama as

forming a monogeneric tribe because of the its small, bilobed

petals, and its greatly expanded purplish stigmas. The close

relationship between Fortunearia and Sinowilsonia has been

proposed and widely recognized (Schulze-Menz, 1964; Bogle,

1968; Endress, 1969), but the association of Eustiama with

Fortunearia and Sinowilsonia was only recently put forward

(Endress, 1989b,c). The present study offers support for

that proposition (Figs.1.2, 1.3). The unambiguous

morphological characters supporting the relationship include

expanded purplish stigmas (personal observations), and ovary

lenticels (Endress, 1989b).

Distylieae and Fotherai1leae

The tribes Distylieae and Fothergilleae combined include

eight genera that have been treated taxonomically in a number

of ways by various authors (Candolle, 1830; Hallier, 1903;

Harms, 1930; Endress, 1989c).

The Distylieae originally included Distylium and

Svcopsis (Hallier, 1903), to which was added Sinowilsonia

(Harms, 1930), while the Fothergilleae (Candolle, 1830)

contained only Fotherailla. Harms's (1930) Fothergilleae,

however, included Fotherqilla. Parrotia, and Parrotiopsis.

57

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. As described by Harms (1930), the major differences

between the two tribes are: 1) flowers are bisexual in the

Fothergilleae and andromonoecious in Distylieae; 2) leaves

are deciduous in the Fothergilleae, but persistent in

Distylieae.

Lundell (1940) described Matudaea. but it was Walker

(1944) who placed Matudaea in the Distylieae. Walker (1944)

also recognized several Central American species of

Distvlium. Endress (1969), however, separated the Central

American species of Distvlium (D. cruatemalense. D.

hondurense, and D^. sinaloense) from the east Asian species of

Distylium as a new genus, Molinadendron. He also suggested

that Mo1inadendron was closer either to Fotheroilla and

Parrotiopsis. or to Fortunearia and Sinowilsonia. rather than

to the Distylieae. Endress (1970) separated some species of

Svcopsis. e.g., S^. dunni. S. tutcheri, as a new genus,

Distyliopsis. and suggested that Distyliopsis is more closely

related to Distylium than to Sycopsis.

Shaniodendron. the most recently described segregate

genus (Deng et al., 1992a) was originally identified as a

species of Hamamelis (Chang, 1962, 1979). Deng et al.

(1992a, b) placed Shaniodendron in the Fothergilleae.

In the present phylogeny (Fig.1.2), Molinadendron.

Matudaea, Distylium. Distyliopsis. and Svcopsis comprised a

monophyletic clade, while the Fothergilleae did not form a

clade. Svcopsis was sister to the clade of Distvlium-

58

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Distyliopsis. supporting the close relationship of

Distyliopsis and Distylium. as suggested by Endress (1969).

The tribes Distylieae and Fothergilleae altogether formed a

clade (Fig.1.2), indicating that it is reasonable to unite

the two tribes as the Fothergilleae sensu lato (Endress,

1989b, c). Endress (1989b, c) united the two tribes on the

basis of a fertile, spontaneous intergeneric hybrid between

Svcopsis and Parrotia, which Endress and Anliker (1968)

described as XSycoparrotia.

My observations of floral structures indicate that the

members of the Fothergilleae not only lack petals, as widely

recognized previously (Baillon, 1871; Harms, 1930; Bogle,

1968; Endress, 1993) but also have andromonoecy (except for

Mo1inadendron and Matudaea), as implied by Bogle (1970).

Hamamelideae

The concept of the Hamamelideae has not been changed

much since the establishment of the tribe (Harms, 1930;

Chang, 1979; Endress, 1989b, c), except for the addition of a

few new genera (Smith, 1958; Endress, 1969; Endress et al.,

1985). This tribe is characterized by strap-shaped petals

and 4- or 5-merous flower parts. Endress (1989b, c) did a

revision of the tribe and recognized three subtribes,

Hamamelidinae, Loropetalinae, and Dicoryphinae. The

Hamamelidinae included only one genus, Hamamelis.

characterized by 4-merous flowers and deciduous leaves.

59

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. However, the parsimony analysis placed Hamamelis in a

position close to Corvlopsis (Fig.1.2), which in turn was

sister to the Fothergilleae.

The Loropetalinae contained four genera, including

Tetrathyrium. Mainaaya. Loropetalum, and Embolanthera.

Tetrathyrium and Loropetalum are so closely related that they

might be congeneric species (Endress, 1993). However,

Loropetalum is rather unique in the family by its very small

leaves. Maincraya is endemic to the northwestern part of the

Malay Peninsula (Parak and Penang). It has more than two

whorls of staminodial phyllomes, and the calyx tube falls off

after anthesis, leaving a ring-like scar on the hypanthium.

Embolanthera is distributed in the Philippines, the southern

tip of China, and adjacent Vietnam. Its petals are small and

geniculate with two lateral lobes. Petals and stamens are

fused at the base. The disjunct distributions and

morphological uniqueness of each of the four genera seems to

dictate a 'not-very-closely-related' assessment, even though

they share the character of long, horn-like connective

protrusions in their anther. In Figs.1.2, and 1.3, these

four genera did not form a clade, suggestive of their 'loose'

relationships.

The third subtribe, Dicoryphinae, is composed of five

genera that are exclusively distributed in the Southern

Hemisphere. These genera share a unique anther dehiscence

pattern, i.e., dehiscing by two valves serving four pollen

60

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sacs. The majority consensus tree (Fig. 1.3) showed that

Dicoryphinae was a monophyletic group, as implied by Endress,

(1989b), even though the strict consensus tree did not

resolve the relationships. However, this suggestion does not

provide support for the attempt to recognize the five genera

as a separate family, as suggested by Zhang and Lu (1995).

In summary, the present phylogenetic analysis recognizes

the monophyly of the subfamilies: Altingioideae,

Exbucklandioideae sensu Harms (1930), Disanthoideae,

Rhodoleioideae, and Hamamelidoideae. Also recognized by the

phylogeny are the following tribes and subtribes: the

Eustigmateae sensu Endress (1989c), Fothergilleae sensu Harms

(1989c), and the Dicoryphinae. Polyphyletic taxa suggested

by the present phylogenetic analysis include

Exbucklandioideae sensu Endress (1989c), Mytilarioideae,

Hamamelideae, and Loropetalinae.

Noticeably, both supporting indices (bootstrap

percentage and decay index) were small for all the clades

except for Licruidambar-Altinqia and Corylopsis-Hamamelis.

Also, the consistency indices were low (CI=0.46, RC=0.27),

and the gl value (skewness test) was only -0.42. These low

indices indicate that there are high homoplasies in this

morphological data set. Therefore, the relationships

obtained in this study should be considered with caution.

That is, some characteristics could be the result of parallel

or reverse evolution. Therefore, other lines of evidence are

61

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. needed to further evaluate the relationships within the

Hamamelidaceae.

62

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1.1. Morphological character codings in the Hamamelidaceae. Cereidiphy 1 lum is the outgroup. (Characters and their states are given in the text). a=01, b=02, c=12, d=34, e=03, f=23, g=24, h=14, i=124, j=13, k=39, m=04, ?=missing data.

Character Genus 10 20 30 40 52

Altingia OOOlOdOOlO 1011001222 0000009001 0112221101 040000010110 Chunia 02?00mll00 0d30001002 0100009001 0001212101 0200200101c? Corylopsis 112003002e f021a01001 0100002001 OaOlOOOOOl 0a0c300010cl Dicoryphe OOOblOOOOO 3120101a01 0110005a01 OlOOaOOOll Ia0c30011011 Disanthus 2200000000 0111000001 0100007002 0102100001 330120010020 Distylium 0000020020 3d21101212 0000009001 0all21f111 3102300120cl Distyliopsis 0000020020 3d21101212 0000009001 0all21f111 3112300120C1 Embolanthera 0020030020 3h21101001 0110100101 0102200010 0100310120c? Eustigma 0020020020 3gll011001 0100001001 01012f0001 0000301120c? Exbucklandia 02000m0002 lg30001212 OlOOOOkOOl 0111100001 2301100100C0 Fortunearia 2010030020 3011011011 0100008001 0102200111 0001301120C1 Fothergila 210003002e 3011101211 01a0009021 1002200000 0101300120C1 Hamamelis 210001002e 3a21101101 0100004000 0102100000 2 j01300010cl Liquidambar 2200050110 1011001222 0000009001 0112223101 040000010100 Loropetalum 0020000000 3d21101a01 0100004001 0100100001 0000310120C1 Maingaya 0020000000 3dlll01001 0110004001 0100000000 0 j01310120c? Matudaea 0010000000 £111101202 0000119011 0011222001 0a023001201? Molinadendron 0010100000 3dlll01202 0100009001 0101202111 0a01300120cl Mytilaria 02200mll01 3g30001001 0100006001 0102000000 0201200100C2 Neostrearia 0020000000 4d21101001 0110004000 0102200001 1101310110c? Noahdendron 0020000000 4220101001 0100004000 0100200001 1101310110C1 Ostrearia 0020000100 4d21101001 0100004000 0100100001 1201310120cl Parrotia 2100030020 3221101212 01a0009001 001120blll 3101300120C1 Parrotiopsis 2100030020 3221101211 0101009001 01a2210101 0101300120C1 Rhodoleia 0000000002 42bl001200 1100003001 0002131001 0301100001C1 Shaniodendron 2100030020 3221101212 0100009001 0011200001 3101300120c? Sinowilsonia 201003002e 3011101022 0100008001 0102200011 0111300120C1 Sycopsis OOOOObOObO 4d21101212 0100009001 0all20f111 0al2300120cl Tetrathyrium 0020020021 3hlll01001 0100004001 0100100001 0j00310120c? Trichocladus OOOblOOOOO 3d20101a01 01a0004000 0100200001 1301300120cl Cere idiphy Hu m 2c020c0003 0011001232 0000009001 0011210111 330270010103

63

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.1.1. Stomatal apparatus in the sampled species of the Hamamelidaceae. All lower epidermis and 200X unless otherwise indicated, a. Dicoryphe stipulacea. Randrianasolo 543; b. Dicoryphe stipulacea (upper epidermis); c. Embolanthera spicata. Soefarto and Fernando 7345 (GH); d. Mainqava malayana (no collection data); e. Matudaea hirsuta. Bogle 848; f. Matudaea hirsuta (upper epidermis); g. Molinadendron sinaloense. Bogle 864; h. Neostrearia fleckeri. Endress s.n. (400X) i. Noahdendron nicholasii. Endress s.n.: j. Ostrearia australiana. Irvine 2081; k. Parrotia persica. Bogle 884; 1. Trichocladus crinitus. Bogle, 1545.

64

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a

j■

65

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.1.2. Strict consensus of the 12 shortest trees of 219 steps based on morphological data of the Hamamelidaceae. Branches have a decay value of one and a bootstrap percentage of less than 50% unless otherwise indicated. Numbers below and above the branches are decay indices and bootstrap percentages. CI=0.45, RC=0.27.

66

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 76 Altingia Liquidambar Chunia 66 Corylopsis Hamamelis 67 Distylium Distyliopsis Matudaea Molinadendron -tE Sycopsis Parrotia Shaniodendron Fothergila Parrotiopsis Eustigma Fortunearia £ Sinowilsonia Embolanthera Tetrathyriurn Loropetalum Dicoryphe Trichocladus Maingaya Neostrearia Noahdendron Ostrearia Rhodoleia Disanthus Mytilaria Exbucklandia Cereidiphy Hu m (Outgroup)

67

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 1.3. 50% majority consensus of the 12 shortest trees based on morphological data of the Hamamelidaceae. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

68

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. -| Altingia -i A^oi^ae Liquidambar -I “ “ Chunia — EXBoideae r— Corylopsis — cOReae— Hamamelis — HAMinae (HAMeae) Distylium j— c Distyliopsis Matudaea 0) Molinadendron (0 Cercidiphyllum (Outgroup)

69

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 1.4. 50% majority consensus tree of 12 shortest trees based on morphological data, showing the distribution of numbers of characters that change unambiguously on branch.

70

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. i Altingia

i Liquidambar i Chunia

i Corylopsis

Hamamelis D isty liu m Distyliopsis Matudaea Molinadendron Sycopsis P a rro tia Shaniodendron Fothergilla Parrotiopsis Eustigma Fortunearia Sinowilsonia

Embolanthera Tetrathyrium Loropetalum

Maingaya Dicoryphe Trichocladus

t Chars, that change Neostrearia Unambiguously on branch Noahdendron CZ3 o Ostrearia UZ3 1 Rhodoleia ES3 2 Disanthus 3 4 M y tila ria 5 Exbucklandia 6 Cercidiphyllum 7 8 9 or more

71

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX 1.1

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix.1.1. Herbarium and chemically fixed specimens observed for this study. GH, Harvard University Herbaria; MO, Missouri Botanical Garden; HK, Hong Kong Herbarium; US, U.S. National Herbarium at Smithsonian Institution.

HERBARIUM SPECIMENS

Corvlopsis aotoana Makino

JAPAN. Honshu: Mt. Aterayama, Murata and Iwatsuki 908

(MO).

C . glaucescens Hand.-Mazz.

CHINA. Tibet: Rock 11226 (US).

£_«_ himalavaoa Griff.

INDIA. Khasia and Jynteah Hills, Gallatly 627 (US).

Sirhoi: Kinqdon-Ward 17205 (MO).

£_*_ multiflora Hance

CHINA. Guangxi: Yuan Tung Shan, Chinq 5722. 6038 (US).

Guizhou: Fanjing Shan, Jiangkou Xian, Sino-American Botanical

Expedition 113 (MO).

C . pauciflora S.et Z.

JAPAN. Honshu: Sakiya 78 (MO).

SLi. Platvoetala R.et W.

CHINA. Sichuan: Dujiangyan, Boufford and Bartholomew

24584 (MO).

C . sinensis Hemsl.

CHINA. Anhui: Jinzhai, Yao 8899 (MO). Hunan: Xinning, Li

et al. 1823 (MO). Jiangxi: Lu Shan, Steward 2473 (MO); Chiao

18588 (US).

73

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C . soicata S.et Z.

JAPAN. Honshu: Sakiya 81 (MO). Shikoku: Osaka-toge,

Nangoku-shi, Kochi-ken, June 20, 1973, Ohashi s.n.(US

willmottiae R.et W.

CHINA. Sichuan: Guan Xian, Wane 870075 (MO).

wilsonii Hemsl.

CHINA. Guangdong: Le Chang Xiang, Tsanq 20757 (MO). C.

vunnanensis Diels. CHINA. Yunnan: Chienchuan, Mekong divide,

Forrest 23588 (MO); Tsangshan Range, west of Talifu, Rock

6376 (US).

P\fftVij.9Pgiff A-flvr-i.f9.lAa (Hemsl.) Endress

CHINA. Yunnan: Lunan Xian, Sino-American Botanical

Expedition 1584 (GH ).

PiStyiiMffi bnxifolium (Hance) Merr.

CHINA. Hubei: Western Hupeh, Wilson 115 (GH); A. Henry,

3626 (GH). Zhejiang: Barchet 170 (US). IK gracile Nakai.

CHINA. Taiwan: Taipeh, Botanical Garden (cultivated), Keng

K1047 (GH); Wilson 11107 (US).

L . Nakai

JAPAN. Tokyo: Ogasawara Isis, Fuiita and Shimizu 72

(MO).

raggmggwm s.et z.

CHINA. Taiwan: Botel Tobago, Taitung county, Chang 2793

(MO); Tienchih, Huang 9378 (MO).

JAPAN. Hondo: Waterfall of Ozaka, Pref. Nara, Faurie

74

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12077 (MO). Honshu: Pref. Yamaguchi, Kasayama, Hagi-shi,

Murata 22143 (MO). Kagoshima: Kyushu, Mitsuta. Doei and

Naaamasu 53 (GH); Wilson 6039 (MO). Komaba: Ohashi and

Tateishi 10949 (GH). Kosugidani: Arakawa Dam, Yahara.

Murakami, and Watano 7633 (GH). Linkiu Islands: Wilson 8070

(GH). Okinawa: Isl. Iriomote, along Yutsun river, Taketomi-

cho, Okada. Takahashi and Naaamasu 358 (GH).

Embolanthera spicata Merr.

PHILIPPINES. Palawan: Panacan, Aborlan, Dulit 3725 (GH).

Eustiama oblongifolium Gardn.et Champ.

CHINA. Guangdong: Sin-fung District, Taam 389 (MO);

Luofu Xiang, Chun 40455 (MO). Guangxi: Yao Shan, Wang 39379

(GH); Shang-sze district, Tsanq 22213 (GH). Hainan: Chim Fung

Mt. Kan-en District, Lau 5678 (GH); Wright 35219 (US). Hong

Kong: Peel Rise, Choi 403 (HK); Mt. Gough, Victoria, Hu and

But 21997 (GH); Choi and Hu 12739. 13251 (GH); Wright 186

(US ). Taiwan: Wilson 9959 (GH ).

§_«_ balansae Oliv.

CHINA. Guangdong: Ding-hu Shan, Ting 1569 (GH). Guangxi:

Steward and Cheo 696 (GH).

SsfrttsJslanflia pypvLagfl (R.Br.) R.w.Br.

CHINA. Hong Kong: Sunset Peak, Li 73 (GH). Yunnan: Henry

11068 (GH); Hsien and Wang 72631 (GH); Between Kambaiti and

Tengyueh, Rock 7574 (GH).

75

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 1.1. Continued

Fortunearia sinensis R.et W .

CHINA. Anhui: Huangshan, Chow 101 (GH, MO); IP 6339

(US). Jiangsu: I-hinq and Fana 7902 (MO); Bau Hwa Shan, Tsu

1680 (US). Zhejiang: Tihtaishan, Chinq 1550 (US).

P.9tllgr.q341a qardenii Murr.

U.S.A. North Carolina: Scotland Co., just south of the

bridge over Lumber Creek (Hoke-Scotland Co. Line) on US 15-

501, Weaver Jr. 350 (GH); Onslow county, May 11, 1948, Boyce

and Moreland s.n. (GH). South Carolina: Horry county, Griscorn

16547 (GH).

F . maior (Sims) Loddiges

U.S.A. North Carolina: Burke Co., Bell 6564 (GH); Rable

Rock Mountain, Stone and Family 1889 (GH); Burke Co. Shortoff

Mountain, Wilbur 7012 (GH); Polk County, Weaver. Jr. 1333

(GH).

Liquidambar formosana Hance

CHINA. Guangdong: Manchi Shan (Wan Zhi Shan), Jen-hwa

District, Tsanq 26304 (GH); Loh Hoh Tsuen, Ling Yun Ksien,

Steward and Cheo 442 (GH). Guangxi: Liang 69991 (GH).

Guizhou: Ta-ho-Yen, Fan Ching Shan, Steward. Chiao and Cheo

716 (GH). Hong Kong: Hu and But 20064 (GH). Hubei: western

Appendix 1.1. Continued

Hupeh, Wilson 513. 5218 (GH).

JAPAN. Kyoto: Yase, Muroi 4987 (GH).

76

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kj_ styraciflua L.

U.S.A. Alabama: Tuscaloosa County, Ingram and Findley 66

(GH). Arkansas: Drew county, Demaree 16661 (GH); Saline

County, Demaree 8517 (GH). Delaware: Newcastle county, Long

32326 (GH).

frgrgpetalEfl chinense (R.Br) Oliv.

CHINA. Anhwei: Ching 3284 (GH). Fukien: Chung 1013. 8548

(GH). Guangxi: Ling-chuan District (Ling Chuan County), Tsang

27845 (GH). Hong Kong: Hu and But 20118 (GH). Jiangxi:

Lushan, Cheng 74 (GH).

INDIA: Khasia and Jaintia Hills, Ruse 47 (GH).

JAPAN. Honshu: Taoda 3680 (GH).

Maingava malavana Oliv.

MALAYSIA. Kepong: Frim Arboretum, van Balgooy 5755 (GH).

Penang: Penang Hill, Viaduct Rd., Lesmv 33667 (GH).

Matudaea triuervia Lundell

COSTA RICA. Hammel 17420 (GH).

HONDURAS. Esperanza and Intibuca: Standley 25512 (GH).

MEXICO. Chiapas: Matuda 3984 (GH). Jalisco: south of

Talpa de Allende, McVauoh 23317A (GH).

Molinadendron guatemalense (Harms) Endress

GUATEMALA. Alta Verapaz: Williams. Terua. Williams, and

Molina 40373 (GH); von Turckheim 187 (US).

77

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M . sinaloense (Standi.& Gentry) Endress

MEXICO. Sinaloa: Sierra Surotato, Gentry 7254. (Isotype)

(US).

M. hondurense (Standi.) Endress

HONDURAS. La Mision: Allen 6188 (GH); Molina 7226 (GH).

Mvtilaria laosensis H.Lee

CHINA. Guangxi: Shap Man Taai Shan, Shang-sze District,

Tsana 2498 (GH).

Neostrearia fleckeri L.S.Smith

AUSTRALIA. Queensland: Mosspanan River Gorge, Brass 2140

(GH).

Ostrearia australiana Baill.

AUSTRALIA. Queensland: Upper Parrot Creek, Annan River,

Cape York Peninsula, Brass 20266 (GH); Cook District, Smith

11209 (GH); Sanderson 43 (GH).

PgHT9ti9Pgjg iacquemontiana Schneider

INDIA. Kashmir: Bhadraloah, Rao 9064 (GH). Jhelum

Valley: Stewart 12088 (GH). Punjab: Kabl, Chamba, Stewart

2331 (GH).

PAKISTAN. Swat District: Showa Khuar Kalam, Shah and

Javed 231 (GH).

Sinowilsonia henrvi Hemsl.

CHINA. Hubei: Shennongjia Forest District, Sino-American

Botanical Expedition 1483 (GH); Wilson 584 (GH, US); Henry

6559 (Isotype) (US).

78

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Sv c o p s x s sinensis Oliv.

CHINA. Taiwan: Hwalien, Kao 9842 (MO).

CHEMICALLY FIXED SPECIMENS

(Stored in the Laboratory of Plant Morphology and

Anatomy at the University of New Hampshire).

Alttflqia excelsa Nor.

CHINA. Hong Kong: Hong Kong Botanical Garden, Bogle 583.

MALAYSIA. Cameron Highlands: Mentigi F. R., Bogle 313.

C M l U a friLS.fr IflAfliaAdSfi Chang

CHINA. Hainan, Liang 64169.

Corvlopsis veitchiana Bean

U.S.A. Massachusetts: Arnold Arboretum (cultivated),

Li 02.

Picorvphe so.

Madagascar: Dorr 3982.

Disanthus cercidifolius Maxim.

JAPAN. Tokyo: Tokyo University Botanical Garden,

Bogle 1268.

U.S.A. Pennsylvania: Morris Arboretum (cultivated),

Bogle 1474. Washington: University of Washington Arboretum

(cultivated), Bogle 1343.

Pistvlium racemosun S.et Z.

U.S.A. New Hampshire: University of New Hampshire

greenhouse (cultivated), Bogle 1459. Washington: University

79

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 1.1. Continued

of Washington Arboretum (cultivated), Boole 1361. 1385.

E.ygt-i.qffifl oblonqifolium Gardn.et Champ.

CHINA. Hong Kong: Aberdeen Rd., ALB 589. Taiwan: Chung

s.n., serial collections every three days for two years,

1996-1997.

Exbucklaodia populnea (R.Br.) R.W.Br.

MALAYSIA. Cameron Highlands: Tanah Rata Vill., Boole

314.

Fortunearia sinensis R.et W.

U.S.A. New Hampshire: University of New Hampshire

greenhouse (cultivated), Boole 1458.

Hamamelis macrophvlla Pursh

U.S.A. Washington: University of Washington Arboretum,

Boole 749.

H^_ mollis Oliv.

U.S.A. New Hampshire: University of New Hampshire campus

(cultivated), Boole 1352.

kjttvteflamfear. gtYracifiua l . U.S.A. Massachusetts: Arnold Arboretum (cultivated), M.

Wisniewski, Oct. 5, and 25, 1979. Pennsylvania: Service St.,

Philadelphia, Boole 605. 1267.

Loropetalnn chinense (R.Br.) Oliv.

JAPAN. Tokyo University Botanical Garden, Boole 719.

U.S.A. New Hampshire: University of New Hampshire

greenhouse (cultivated), ALB 1294.

80

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 1.1. Continued

qai-Bgaza malavana Oliv.

MALAYSIA. Kepong: Boole 1314. Kepono Research Institute

25856.

Matudaea ££..

MEXICO. Behucos-Nanchititea: Boole 848.

Mvtilarira laosensis Lee.

CHINA. Guangxi, Liano 6980.

Qgtrear4-a australiana Baill.

AUSTRALIA. Queens land: Gray 1036.

Parrotia persica C.A.Mey

U.S.A. Massachusetts: Harvard University campus

(cultivated), Boole 1488. New Hampshire: University of New

Hampshire greenhouse. Jan. 14, 17 1994, Li s.n.

Parrotiopsis iacouemontana Schneider

U.S.A. Massachusetts: Harvard University campus

(cultivated), Boole 1482. 1498. Washington: University of

Washington Arboretum (cultivated), Boole 1327.

Rhodoleia championii Hook. f.

MALAYSIA. Cameron Highlands: Tanah Rata Vill., ALB 316;

Kuala Lumper: Klang Gates, Boole 276.

Sinowilsonia henrvi Hemsl.

U.S.A. New York: Oyster Bay Planting Field Arboretum,

Long Island (cultivated), Boole 1518.

ffYSAPgiS sinensis Oliv.

U.S.A. Washington: University of Washington Arboretum

81

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Appendix 1.1. Continued

(cultivated), Bogle 1315. 1367.

Tetrathvriuro subcordatum Benth.

CHINA. Hong Kong: Bowen Rd., Bogle 586.

Trichocladus crinitus Pers.

U.S.A. Pennsylvania: Longwood Gardens (cultivated),

Bogle 1513. 1545.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II

PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE

INFERRED FROM NUCLEOTIDE SEQUENCES OF THE PLASTID GENE MATK

INTRODUCTION

The Hamamelidaceae, a family of 30-31 widely recognized

genera and about 140 species, are distributed in the

tropical, subtropical and temperate areas in both the Old and

New Worlds (Endress, 1993; Zhang and Lu, 1995). The uniform

characters in the Hamamelidaceae include woody habit,

2-carpelled pistils and multicellular stigmatic papillae

(Endress, 1989a). Other characters are highly diverse. For

example, leaves are stipulate, persistent or deciduous,

simple and pinnately veined or palmately lobed and veined.

Most species are bisexual, but some are andromonoecious (both

staminate and bisexual flowers found in an individual), still

others are monoecious (staminate and pistillate flowers

develop on an individual). Flowers are complete and 5-merous

in most genera, 4-merous in several genera and variable in

others; a few genera have an incomplete perianth or are

naked.

Well-known examples of the Hamamelidaceae are

Witch-hazel (Hamamelis virqinianal, which yields the widely

used astringent and soothing lotion for cuts and bruises;

83

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Winter-hazel (Corylopsis), which has long been cultivated as

an ornamental; and Sweetgum (Licruidambar). which produces a

fragrant gum (storax) used in perfumery, as an inhalant, and

as a fumigant in treatment of skin diseases.

Both morphological (Hufford, 1992) and molecular (Hoot

et al., 1997; Hoot and Crane, 1996) studies have demonstrated

that the Hamamelidaceae are monophyletic. As for the

systematic position of the Hamamelidaceae, most of the

traditional systems place this family in the "Lower"

Hamamelidae that include Cercidiphyllaceae, Tetracentraceae,

Trochodendraceae, Daphniphyllaceae, Platanaceae,

Myrothamnaceae and Eupteleaceae (Takhtajan, 1980; Cronquist,

1988). Endress (1977) suggested this family to be a

connecting taxon between the "Lower" and the "Higher"

Hamamelidae (e.g. Betulaceae, Fagaceae, and Juglandaceae).

Recently, some members of the Hamamelidaceae have been

considered as linking taxa between the "Lower" hamamelids and

some basal elements of rosids and asterids (Hufford, 1992;

Chase et al., 1993; Endress, 1993; Morgan and Soltis, 1993).

Apparently, a more comprehensive study (more taxa and more

sources of data) is needed to further assess the systematic

position of the Hamamelidaceae.

The phylogeny of the Hamamelidaceae remains unresolved

at both subfamilial and tribal levels. At the end of the

19th century, Reinsch (1889) proposed a three-subfamily

scheme of the Hamamelidaceae [Altingioideae, Bucklandioideae

84

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (= Exbucklandioideae) and Hamamelidoideae]. Niedenzu (1891),

however, suggested a two-subfamily system [Bucklandioideae

(= Exbucklandioideae) and Hamamelidoideae]. In this century,

the first comprehensive classification system was proposed by

Harms (1930) (Fig.i.4). He recognized five subfamilies

[Disanthoideae, Hamamelidoideae, Rhodoleioideae,

Bucklandioideae (= Exbucklandioideae), and

Liquidambaroideae]. Chang (1973, 1979) reviewed the

hamamelidaceous flora of China, recognizing the five

subfamilies of Harms but also erecting a new subfamily for

Mytilaria and his own new genus Chunia (Fig.i.5). Most

recently, Endress (1989c) provided a suprageneric scheme for

the Hamamelidaceae (Fig.i.6). In this system he combined the

three subfamilies, Disanthoideae, Mytilarioideae and

Exbucklandioideae, thus recognizing four subfamilies in the

Hamamelidaceae (Altingioideae, Rhodoleioideae,

Exbucklandioideae, and Hamamelidoideae).

The controversy concerning tribal or subtribal

delimitations focuses on the Hamamelidoideae since the other

subfamilies have only one or a few genera, and further

subdivision does not seem to be justified. Harms (1930)

divided the Hamamelidoideae into five tribes, including

Cory lops ideae, Distylieae, Fothergilleae, Hamamelideae, and

Eustigmateae. Endress (1989c) revised Harms's (1930) system

and recognized four tribes in the Hamamelidoideae by

including the Distylieae in the Fothergilleae. In that

85

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. system, he also made some generic rearrangements.

Nucleotide sequences of chloroplast genes have proven to

be very promising for resolving phylogenetic relationships in

angiosperms (Olmstead and Palmer, 1994). However, genes may

have different substitution rates, suggesting that to resolve

phylogeny at different levels, gene(s) of appropriate

evolutionary substitution rates should be employed. The most

widely used and so-called "gene of choice" the rbcL gene that

encodes the large subunit of ribulose-l,5-bisphosphate

carboxylase/oxygenase has been found to be informative mostly

at the familial or higher levels (Chase et al., 1993).

Nevertheless, in the rbcL phylogenies (Chase et al., 1993),

where the Hamamelidaceae were poorly represented, the clade

containing both hamamelidaceous genera and others was not

resolved. Compared to the rbcL, the matK gene, which encodes

a chloroplast maturase and is located in the trnK intron, has

a relatively higher substitution rate (Olmstead and Palmer,

1994), indicating that it may be more appropriate for

systematic studies at lower levels. This proposition has

been confirmed by several cladistic analyses (Johnson and

Soltis, 1994; Steele and Vilgalys, 1994), including a

phylogenetic study of the "Higher" Hamamelidae (Manos and

Steele, 1997).

Therefore, in this study, I chose to use DNA sequences

of the matK gene to investigate phylogenetic relationships

within the Hamamelidaceae and to evaluate the existing

86

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. classification systems of the family.

MATERIALS AND METHODS

Plant Materials

Thirty hamamelidaceous species from 27 genera were

sampled, representing all of the subfamilies, tribes and

subtribes previously proposed by Harms (1930), Chang (1979),

and Endress (1989c). Sources, vouchers, and sequence GenBank

accession numbers are listed in Table 2.1. The aligned matK

sequences are given in Appendix 2.1.

Molecular Techniques

Total genomic DNAs were extracted from fresh or silica

gel dried leaves using the standard DNA extraction procedures

of Doyle and Doyle (1987). Polymerase chain reaction (PCR™)

amplification was conducted using forward primer matKFl and

reverse primer matKRl kindly provided by Tao Sang (Michigan

State University, East Lansing, MI). The amplification

reaction included 50-100 ng of genomic DNA, 3-4 units of Tag

(Promega, Madison, WI), 3-4 units of Tag Extender

(Stratagene, La Jolla, CA), IX Tag Extender buffer, 2.5 mM

dNTP, and 20 gM primers. Amplifications were conducted in

thin-walled tubes using a MJ thermocycler (Watertown, MA)

following the PCR program of Johnson and Soltis (1994).

The PCR amplified products were purified on 0.8% low

87

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. melting point agarose gels in IX TBE buffer (pH8.0). The

matK bands identified by lambda HindiII DNA size markers were

excised and agarase-digested for 30 minutes. The purified

PCR products were then used as templates for direct

double-stranded sequencing using dyedideoxynucleotide

terminator cycle sequencing on an Automated Sequencer 373A

(Applied Biosystems, Foster City, CA). The procedures were

carried out according to the manufacturer's instructions at

the University of New Hampshire Sequencing Facility Center.

Sequencing primers, FI, F2, F3 (forward) and Rl, R2 and R3

(reverse) were generously provided by Tao Sang. In addition,

I designed forward primers matKF4. matKF4-2 (for Maingaya),

matKF5, matKF6 (for Liquidambar). and reverse primer matKR2-2

(for Liquidambar) in order to get complete matK gene

sequences. Figure 2.1 shows the relative positions of these

primers; base composition of the primers is listed in Table

2 .2 .

Sequence Analysis

Sequence chromatograms were analyzed using the SEQED

program (Applied Biosystems, Foster City, CA), and the

overlap option was employed to assure correct base-calling.

The analyzed sequences were then exported as EDITSEQ files.

Base composition, translated amino acid sequences, and codon

usage were obtained using the EDITSEQ program. The sequences

were aligned using the MEGALIGN program. Both EDITSEQ and

88

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MEGALIGN are programs in the DNASTAR software package

(version 3.72, Madison, WI).

Phylogenetic Analyses

Parsimony analyses were conducted using the test version

4.0d55 of PAUP* written by David L. Swofford (Smithsonian

Institution). Given the limit of computer memory and the

sample size, heuristic searches were performed with the other

options of TBR swapping, multipars on, and steepest descent

off. I have tried a 1:1.3 weighting scheme for transition

(Ts) and transversion (Tv) according to the estimated

frequencies of character changes using the MacClade program

(Maddison and Maddison, 1992), but did not find any effect on

the topology of phylogenetic trees. Therefore, I report only

the results of the analyses using unweighted characters and

unordered transformations. Indels in the data matrix were

coded as missing data. However, some indels are

phylogenetically informative and will be mentioned in the

Discussion section of this Chapter.

The trees were rooted by outgroup comparison using

Sullivantia sullivantii (GenBank accession #20130) and

Saxifraqa inteqrifolia (GenBank accession #20131)

(Saxifragaceae) as outgroups. These taxa were two of only a

few taxa whose complete matK sequences were available in the

GenBank. In addition, the possible relationship of the

Hamamelidaceae and Saxifragaceae has been previously proposed

89

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Hufford, 1992; Chase et al., 1993; Endress, 1993; Morgan and

Soltis, 1993).

Bootstrap analysis (Felsenstein, 1985) of 100 replicates

and the Constraint decay analysis (Bremer, 1988; Morgan,

1997) were performed to obtain the indices of relative

support for individual clades.

Skewness of tree length distributions has been proposed

to be a level indicator of phylogenetic information contained

in a data matrix (Kallersjo, 1982; Huelsenbeck, 1991). The

skewness test was implemented using the Random Tree option of

PAUP* and 10,000 random trees were examined. Another test of

phylogenetic information of a data set is the randomization

test, which produces the permutation tail probability (PTP)

statistics. Data sets with values of PTP<0.01 are considered

to be considerably different from randomized data (Faith and

Cranston, 1991; Plunkett et al., 1997). The permutation test

was performed using the Permutation option of PAUP* with 100

replicates and heuristic searches.

The aligned sequences were imported into the MacClade

computer program, version 3.03 (Maddison and Maddison, 1992)

to estimate transition and transversion ratio, and character

changes in the first, second and third codon using one of the

most parsimonious tree, and to compare the competing

hypotheses concerning generic relationships of the

Hamamelidaceae. The MacClade program was also used to

estimate the number of unambiguous changes along branches.

90

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS

Sequence Characteristics

The sequence length of the matK gene in the

Hamamelidaceae ranged from 1506 bases in Pisan thus to 1521

bases in Molinadendron. In most of the other genera, this

gene was 1515 bases long, while in Altinaia and Liquidambar

(Altingioideae), Exbucklandia. Rhodoleia. and Sinowilsonia,

the matK gene was 1512 bases in length. The number of amino

acids ranged from 502 to 507 (Appendix 2.2). In terms of

nucleotide composition, the matK gene was AT rich in the

Hamamelidaceae; the GC contents were ca. 34% (Table 2.3).

Pairwise sequence divergence basically corresponded to

taxonomic levels: 0.5-1% within Corvlopsis. Hamamelis, and

Liquidambar: 2-3% at generic levels, and 4-6% at the

subfamilial level (Table 2.3). Alignment of the sequences in

the Hamamelidaceae required five deletions, two of which were

three bases long, while the other three were two, six, and

seven bases in length (See Appendix 2.1).

The ratio of transitional and transversional changes

obtained using the MacClade program was about 1.3 for the

matK gene sequences in the Hamamelidaceae, and the relative

percentages of character state changes were 32%, 25%, and 43%

for the first, second, and third codon positions

respectively. Codon usage of matK gene in the Hamamelidaceae

91

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was far from balanced among the codons encoding the same

amino acid (Table 2.4).

Phylogenetic Trees

The parsimony analysis resulted in six equally shortest

trees with a length of 582 steps and a consistency index (Cl)

of 0.85 (strict consensus tree shown in Fig.2.2). Altinqia

and Liquidambar formed the basal clade supported by a

bootstrap percentage (BP) of 100% and a decay index (DI) of

eight. The next clade contained Exbucklandia and Rhodoleia

and the supporting indices were 100% BP and DI of seven.

Mvtilaria formed its own clade followed by the clade of

Disanthus« which was in turn sister to the trichotomous clade

of the Hamamelidoideae. The first clade in the

Hamamelidoideae included Corvloosis and the branch of

Maingava. Loropetalum. and Matudaea, while the second one

contained two groups: Dicoryphinae Endress and the

Eustigmateae sensu Endress (1989c) (incl. Molinadendron).

The third clade was composed of the Fothergilleae sensu

Endress (1989c), but excluding the New World Matudaea and

Molinadendron. and including Hamamelis).

DISCUSSION

MatK Sequence Variation

The matK gene is approximately 1500 bases long (Johnson

92

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and Soltis, 1995). So far, only a portion of the matK gene

has been sequenced for a variety of plants (Johnson and

Soltis, 1994, 1995; Steele and Vilgalys, 1994; Soltis et al.,

1996b; Hilu and Liang, 1997; Manos and Steele, 1997; Plunkett

et al., 1997), thus it is premature to give an accurate range

of the length variation of the matK gene in the angiosperms.

Nevertheless, on the basis of the available matK sequence in

the Hamamelidaceae (this study), Saxifragaceae (Johnson and

Soltis, 1994), Poaceae (Hilu and Liang, 1997), Nymphaeaceae

(Padgett, 1997), and Brassicaceae (GenBank X04826), the matK

gene in angiosperms is approximately 1500-1600 bases in

length.

Plunkett et al. (1997) studied the relationship between

Apiaceae and Araliaceae using DNA sequences of both the rbcL

gene and two thirds of the matK gene. They found that the

ratio of transition (Ts) to transversion (Tv) was 1.13 for

the matK gene, and codon position substitution ratios were

1.2:1:1.68 for the first, second and third codon

respectively. The matK gene in the Hamamelidaceae shows

similar ratios both for Ts/Tv (1.3:1), and for codon

substitutions (1.3:1:1.74). It is noticeable that more

variation occurs at the third codon position for Apiales

(0.43) (Plunkett et al., 1997), for Polemoniaceae (0.38-0.5)

(Steele and Vilgalys, 1994), and for the Hamamelidaceae

(0.43) (this study).

93

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Phvloaenetic Usefulness of matK Gene

The matK gene has been increasingly applied in molecular

systematics of plants because of its several characteristics.

First, there is a certain degree of evolutionary constraint

imposed by the function of the maturase it encodes (Neuhaus

and Link, 1987; Wolfe et al., 1992). This property

facilitates sequence alignment. In this study, the

divergence of the matK gene between the hamamelidaceous

genera and the outgroup genera from the Saxifragaceae is 8-

11%, and the sequences are unambiguously alignable (See

Appendix 2.1). This suggests that the matK DNA sequences are

likely to be useful in resolving deep relationships at family

or even order levels. This agrees with recent studies

(Soltis et al., 1996b; Plunkett et al., 1997). Second, the

matK gene is more variable than the rbcL gene that is

informative in resolving relationships at the family or

higher taxonomic levels (Chase et al., 1993). Thus, the matK

gene may be more informative at lower levels. Sequence

divergence within a genus is rather small, ca. 1%, providing

a limited number of informative sites. However, the sites

have been proved very useful in resolving interspecific

relationships in some genera such as T.iguidambar (Li et al.,

1997b). In contrast, informative sites are about 8.6% in the

Hamamelidaceae, giving rise to about 130 informative

characters. As a result, phylogenetic analyses using the

matK data set resolved most of the intergeneric relationships

94

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the Hamamelidaceae. In addition, a higher percentage of

phylogenetically-informative sites has been reported in other

families (Plunkett et al., 1997). Therefore, as pointed out

by Hilu and Liang (1997), the matK gene is informative in

reconstructing phylogenetic relationships at different

taxonomic levels and is a promising source of data in

studying the molecular systematics of plants.

Both gi (tree length skewness) statistics and the PTP

(permutation tail probability) test have shown that matK data

matrices contain a large amount of phylogenetic structure in

the Apiales (gi=-0.51, P<0.01, Plunkett et al., 1997) and the

Hamamelidaceae (gi=-1.89, P<0.01, this study), thus

quantitatively indicating the usefulness of matK gene in

these phylogenetic studies.

Phyloaenv of the Altinaioideae

The Altingioideae includes three genera, Altinaia.

Liquidambar. and Semilicruidambar (Chang, 1973, 1979; Bogle,

1986; Endress, 1989c). Semiliquidambar is morphologically

intermediate, and thus has been considered to be a hybrid

between the other two genera (Chang, 1962; Bogle, 1968,

1986). Altinqia is very similar to the species of

Liquidambar in many morphological structures (Harms, 1930;

Tong, 1930; Bogle, 1968, 1986; Melikian, 1973a, b; Rao, 1974;

Chang, 1979; Bogle and Philbrick, 1980; Wang, 1992). The

95

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phylogenetic analysis reported here supports a close

relationship between Altinoia and Liquidambar (Fig.2.2 ). As

can be seen in Figure 2.3, 35 unambiguous nucleotide changes

support this clade. Also, the species in the Altingioideae

share a three-base deletion and several synapomorphic amino

acid substitutions (Appendix 2.2). Interestingly, the

sampled species of Altinaia is located within the clade of

the two Liquidambar species, indicating that either one of

the two genera is derived from the other, or both genera are

not monophyletic. Unfortunately, it is impossible to

determine which of the two hypotheses is correct because this

study sampled only one Altinqia species. Therefore, a more

inclusive study of the two genera is needed using sequences

of matK gene or a piece of DNA of higher variation.

The long-standing controversy regarding the

Altingioideae has been whether this group should be

considered as a separate family (Endress, 1989c; Pan et al.,

1991; Wang, 1992; Takhtajan, 1997). I prefer to keep this

clade in the Hamamelidaceae because of the monophyly of the

Hamamelidaceae supported by several recent molecular studies

(Hibsch-Jetter and Soltis, 1996; Hoot and Crane, 1996; Hoot

et al., 1997; Li et al., unpublished). Furthermore, many

morphological characters that have been used to support the

separation of the Altingioideae as a family occur in both the

Altingioideae and some other members of the Hamamelidaceae.

96

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Polyphyly of the Exbucklandioideae

This subfamily, as recently prescribed by Endress

(1989c), consists of four genera, Disanthus, Exbucklandia.

Mvtilaria. and Chunia, the latter was not available for this

analysis. However, each of the other three genera has been

treated as representing a separate subfamily based on

morphological characters, which suggests their distant

relationships (Harms, 1930; Chang, 1948, 1979). The fact

that these genera share several morphological

characteristics, such as palmately veined leaves; large

persistent stipules; and 5-8 ovules in each locule, led

Endress (1989c) to propose a unification of the genera.

Takhtajan (1997), however, moved Disanthus out of the

Exbucklandioideae and treated the genus as representing the

subfamily Disanthoideae.

The matK phylogeny suggests a polyphyletic relationship

among these genera (Fig.2.2). Disanthus and Mytilaria each

forms a monogeneric clade with 35 and 25 unambiguous

character state changes respectively (Fig.2.3), while

Exbucklandia and Rhodoleia are nested with strong support

from both BP and DI (Fig.2.2). Forcing Mytilaria. Disanthus,

and Exbucklandia into a monophyletic clade required 17 more

steps than the most parsimonious trees. Disanthus is the

most closely related genus to the Hamamelidoideae, which is

concordant with previous studies (Hufford and Crane, 1989;

Pan et al., 1991).

97

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rhodoleioideae

Rhodoleia has long been treated as a monotypic subfamily

(Harms, 1930; Endress, 1989c; Takhtajan, 1997). However, in

the matK-based phylogeny, Rhodoleia is allied with

Exbucklandia in a well-supported clade, suggesting a close

relationship of the two genera. Ten unambiguous character

changes occur along the branch supporting the relationship.

This cladistic pattern agrees with the phylogeny based on

rbcL data (Chase et al., 1993; Qiu et al., unpublished).

Furthermore, splitting Exbucklandia from Rhodoleia required

nine steps more than the minimum character changes.

Interestingly, Reinsch (1889) recognized the Bucklandieae

including Bucklandia (= Exbucklandia) and Rhodoleia based on

his comparative study of floral morphology. The clade of

Rhodoleia and Exbucklandia is located in the phylogenetic

tree (Fig.2.2) as the taxa linking the Altingioideae and the

clades of Mytilaria. Disanthus and the Hamamelidoideae. This

agrees with the implications from floral ontogenetic studies

(Bogle, 1986, 1989).

Tribal and subtribal relationships in the Hamamelidoideae

There is no doubt that the Hamamelidoideae are a

monophyletic group, as shown by previous studies (Bogle and

Philbrick, 1980; Endress, 1989a, b; Hufford and Crane, 1989)

and by this analysis. In the matK-based phylogeny, the

Hamamelidoideae clade is well supported by both BP and DI,

98

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and by five unambiguous character changes (Fig.2.2).

However, intergeneric relationships within the

Hamamelidoideae have long been debated (Harms, 1930; Schulze-

Menz, 1964; Chang, 1979; Endress, 1989c).

Three clades constitute the Hamamelidoideae (Fig.2.2).

The first clade includes species of Corylopsis and the branch

containing Mainaaya. Loropetalum. and Matudaea. The

relationships of Corylopsis and Mainaaya-Loropetalum-Matudaea

are strongly supported by BP and DI, and characterized by 10

unambiguous substitutions. The fact that Corylopsis was

phylogenetically far from Fortunearia and Sinowilsonia

supports the separation of this genus from the latter two

genera, as hypothesized by Endress (1989c). The well-

supported relationship between Mainaaya. Loropetalum, and

Matudaea is a new and unexpected pattern. Interestingly,

these genera share several morphological characteristics such

as bisexual flowers, long anther connective protrusions, and

valvate anther dehiscence. However, Matudaea differs

strongly from the other two genera in its irregular calyx,

lacking petals and large, variable number of stamens.

Loropetalum and Matudaea form a clade, but the supporting

indices are small (Fig.2.2). In addition, the other two

morphologically similar taxa (Embolanthera and Tetrathyrium)

were not available for this study. Therefore, the result is

not very conclusive.

The second clade in the Hamamelidoideae associates the

99

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. subtribe Dicoryphinae with the tribe Eustigmateae (incl.

Molinadendron). The Dicoryphinae, which includes the genera

exclusively distributed in the Southern Hemisphere remnants

of the former Gondwanaland, forms a monophyletic clade. This

result agrees with the unique anther dehiscence pattern the

five genera share (Endress, 1989a). Considering the

exclusive distribution in the Southern Hemisphere and the

unique pattern of anther dehiscence, Zhang and Lu (1995)

suggested family status for the five genera. However, the

matK-based phylogeny does not support this proposition. In

contrast, the Dicoryphinae is closely nested with the

Eustigmateae and their clade is moderately supported by BP

and DI (Fig.2.2). As described above, Endress (1989c) placed

both Fortunearia and Sinowilsonia in the Eustigmateae, which

originally included one genus, Eusticrma (Harms, 1930).

Fortunearia and Sinowilsonia were put together based on their

similarities in leaf morphology and reduced petals (Schulze-

Menz, 1964), while the association of Fortunearia and

Eustiqma involves several common characteristics including

pedicellate flowers, covering sepals, small petals, sessile

anthers, large lenticellate fruits, and phloem in the

inflorescence axis containing libriform fibre groups

(Endress, 1989b). At the nucleotide level, two character

changes occur along the branch of the four genera (Fig.2.3).

Molinadendron is put in a clade with Sinowilsonia in the

matK-based phylogeny, which agrees with the implication that

100

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Molinadendron is closer to Fortunearia and Sinowilsonia. or

the Fothergilleae group than to Distylium (Endress, 1967).

My close examination of specimens of Sinowilsonia and

Molinadendron found that the two genera, along with Eustiama

and Fortunearia, share several characteristics including

linear stipules, covering sepals, naked floral buds, and two

prophylls flanking each lateral bud.

The third clade in the Hamamelidoideae is basically

genera of the tribe Fothergilleae, plus Hamamelis (Fig.2.2).

In Harms's system (1930), two tribes were recognized for the

apetalous Hamamelidoideae, one being Distylieae (Distylium

and Svcopsis) and the other the Fothergilleae (Fotherailla.

Parrotia, and Parrotioosis). However, Endress (1989c)

combined the two tribes and recognized the Fothergilleae

sensu lato because of the inherent connection between the two

tribes suggested by the production of a spontaneous hybrid

(XSycoparrotia) between Sycopsis and Parrotia (Endress and

Anliker, 1968). The matK data did not put genera of the

Distylieae and the Fothergilleae sensu stricto into

individual monophyletic clades, but clustered them into one

clade, thus supporting the tribal unification proposed by

Endress (1989c). My observations revealed that flowers are

not strictly bisexual, but andromonoecious in the

Fothergilleae sensu stricto. This further diminishes the

proposed distinctness of separate tribes of Harms (1930), the

Distylieae and the Fothergilleae, and supports the union of

101

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the genera into one tribe, the Fothergilleae.

As has been pointed out above, Mo1inadendron and

Matudaea do not sort out as members of the Fothergilleae.

The former falls into the clade of the tribe Eustigmateae and

the latter into the Cory lops is -Loropetalum group. These two

genera are apetalous, but bisexual, thus supporting their

separation from the andromonoecious Fothergilleae.

Furthermore, attempting to place Matudaea and Mo 1 inadendron

into the Fothergilleae clade entailed 38 more steps than the

minimum length.

The most interesting genus is Hamamelis. This analysis

placed this genus within the clade of the tribe Fothergilleae

(Fig.2.2). However, Endress (1989c) treated it as a

monogeneric subtribe of the tribe Hamamelideae. Hamamelis is

characterized by strictly 4-merous flowers and bisporangiate

anthers (Endress, 1989b; Mione and Bogle, 1990). The

parsimony analysis with Hamamelis removed from the data set

did not change the DI, but increased BP from 54 to 69 for the

clade of Fothergilleae (tree not shown). This implies a weak

relationship between Hamamelis and the other genera of the

Fothergilleae, and a stronger relationship within the

Fothergilleae.

The only obvious similarity between Hamamelis and the

Fothergilleae (Parrotia. Parrotiopsis. Shaniodendron) is the

presence of the semicraspedodromous venation in both groups.

However, the recent discovery of a hamamelidaceous fossil

102

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. flower in Upper Cretaceous deposits in Sweden might suggest

an ancient relationship among these genera since this fossil

flower had bisporangiate anthers, as in extant Hamamelis, and

a variable number of stamens, as in the Fothergilleae

(Endress and Friis, 1991).

Loropetalum. Hamamelis and genera of the subtribe

Dicoryphinae were previously grouped in the tribe

Hamamelideae (Harms, 1930; Chang, 1979; Endress, 1989c).

Forcing these taxa into a monophyletic group in the matK-

based phylogeny, However, required 21 steps more than the

minimum character changes.

Parrotia is morphologically similar to Shaniodendron and

Svcopsis (Bogle, 1968, 1970; Endress, 1970, 1993), and a

phylogenetic analysis of the Fothergilleae sensu Endress

(1989c) using nuclear DNA sequences supports the close

relationship of the three genera (Li et al., 1997d).

However, in the matK phylogeny, Parrotia is phylogenetically

far distinct from the other two genera (Fig.2.2). It is

likely that lineage sorting has occurred in the evolutionary

processes of the Fothergilleae. This will be discussed in

detail in Chapter IV.

103

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U77092 AF013045 AF013042 AFO13043 GenBank AF013038 AF013039 AF015650 AF015651 AF013037

Hawaii Univ. of New Hampshire campusHampshire New of Univ. AF013046 MA.Arboretum, Arnold Hampshire New of Univ. Greenhouse Arboretum Washington of Univ. AF013047 SourceTaiwan number Tulear, MadagascarTulear, SC. Inc. Woodlanders, AF013040 AF013041 MA.Arboretum, Arnold AF013044 Gard. Bot. Missouri AF013059 Manuka State Roadside Park, Roadside State Manuka China MA. Arboretum, Arnold SC.Inc. Woodlanders, SC. Inc. Woodlanders, U77091 BOGLE QIU 03LI MA. Arboretum, Arnold LI 02 LI J.-H. LI J.-H. LI y.-L. J.-H. A. L. A. A. L.A. BOGLE A. L.A. BOGLE L.A. BOGLE A. L.A. BOGLE A. Randrianasolo 543 Randrianasolo A. Voucher HVI HVE DIR L. A. BOGLE DIC DIS FORFOT J.-H. 04 LI J.-H. LI campus Hampshire New of Univ. EUS N.-J. CHUNG EXB LFO LOR D. T. Omar CSP LSI Taxon Collector and CSI J.-H. accession ALT tutcheri DOT used in this analysis. Hamamelis Hamamelis virginiana Corylopais sinensis Corylopais Distyliopsis Distylium racemosum Corylopsis Corylopsis spicata Dicoryphe stipulacea Fortunearia sinensis Hamamelis vernalis Loropetalum sinense Species Disanthus cercidifolius abbr. majorFothergilla Exbucklandia populnea Exbucklandia Liquidambar formosana Liquidambar orientalis Eustigma Eustigma oblongifolium Altingia excelsa Table 2.1. Sources and vouchers of the species sequenced for matK gene and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Harvard Univ. campus campus Univ. Harvard Hawaii AF013054 PA. Gardens, Longwood AFO13058 Forest Research Institute, Institute, Research Forest Malaysia Kepong, AF025393 Garden Zurich. Botanical of AFO13048 Garden Zurich. Botanical of AF013049 Garden Zurich. Botanical of AF013050 Garden Zurich. Botanical of AF013051 Garden Zurich. Botanical of AF013052 Hampshire New of Univ. Greenhouse AF013053 Honolulu, Arboretum, Lyon U77094 China Jiangsu, MA Arboretum, Arnold AFO13056 AF013055 Guangxi, China Guangxi, U77093 SC. Inc. Woodlanders, AFO13057

BOGLE SAW ENDRESS ENDRESS ENDRESS BOGLE 05 LI BOGLE LUO ENDRESS ENDRESS BOGLE BOGLE QIU P. P. P. J L. A A A. SHASIN Y SYSTRI A PAR PPSRHO A MAT OST MAI MOL MYTNEO Z. NOA P. P. Trichocladus Trichocladus crinitus Ostrearia Ostrearia australiana Sinowilsonia Sinowilsonia henryi Sycopsis sinensis Parrotia persica Parrotia Parrotiopsis jacquemontiana Rhodoleia championi Shaniodendron Shaniodendron subaequale Table 2.1 Continued Neostrearia fleckeri Noahdendron nicholasii Maingaya malayana Matudaea trinervia Molinadendron guatemale Mytilaria laosensis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.2. Locations and base compositions of amplification and sequencing primers used in this study. * this primer was synthesized with equal parts of "C" and "T" at base position 6.

Designed Primer 5' sequence 3' by Forward matKFl ACT GTA TCG CAC TAT GTA TCA T. Sang matKF2 GTT CACTAATTGTGAAACGT T. Sang matKFA ACC CCA CCC CAT CCA TCT J. Li matKFA-2 TGG TTC AAA CCC TTC GCT ACT J. Li matKF5 TGG AGYCCTTCTTGAGCGA* J. Li matKF6 TCA GTG GTA CGG AAT CAA ATG C J. Li Reverse matKRl GAA CTA GTC GGA TGG AGT AG T. Sang matKR2 TTC ATG ATT GGCCAG ATCA T. Sang matKR2-2 ACG GGGCCATAAGAA AGT CG J. Li matKR3 GAT CCG CTG TGATAA TGA GA T. Sang

106

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.3. Sequence characteristics of the matK gene (Taxon abbreviations as in Table 2.1). Divergence Species Length G+C(%) ALTCSICSPDIC DIS DIRDOT EUS EXB

ALT 1512 35 -

CSI 1515 34 6.1 -

CSP 1515 34 6.1 0.5 -

DIC 1515 34 4.7 2.2 2 -

DIS 1506 34 6.1 4.4 5 3.1 -

DIR 1515 34 5.2 2.7 2 1.3 4 -

DOT 1515 34 5 2.4 2 1 4 0.3 -

EUS 1515 34 5.3 3 3 1.1 4 2.1 1.8 -

EXB 1512 35 5.4 4.8 5 3.3 5 3.8 3.6 4.1 - FOR 1515 34 5.8 3.2 3 1.5 4 2.2 1.9 1.5 4.5 FOT 1515 34 5 2.3 2 0.9 3 1 0.7 1.5 3.6 HVI 1515 34 4.9 2 2 0.7 3 0.9 0.6 1.5 3.4 HVE 1515 34 5.3 2.4 2 1.1 4 1.3 1 1.8 3.8 LFO 1512 35 0.5 5.9 6 4.6 6 5 4.8 5.2 5.3 LOR 1512 35 0.9 5.8 6 4.4 6 4.9 4.6 5 5 LSI 1515 34 5.8 2.1 2 2 4 2.6 2.3 2.8 4.5 MAI 1515 34 5.8 2 2 2 4 2.1 2.2 2.8 4.5 MAT 1515 34 5.9 2.2 2 2.2 5 2.8 2.5 3 4.9 MOL 1521 34 5.2 2.6 3 0.7 4 1.8 1.5 1.3 3.7 MYT 1515 34 5.7 4.1 4 2.4 4 3.2 2.8 3.2 3.9 NEO 1515 34 5.2 2.6 3 0.5 4 1.7 1.4 1.3 3.7 NOA 1515 34 5 2.4 3 0.4 3 1.6 1.3 1.3 3.6 OST 1515 34 5.3 2.7 3 0.6 4 1.8 1.5 1.6 3.8 PAR 1515 34 5.5 3.4 4 1.8 4 2.2 1.9 2.6 4 PPS 1515 34 4.8 2.1 2 0.6 3 0.7 0.4 1.4 3.4 RHO 1512 34 5.2 4.7 5 3 5 3.7 3.4 3.8 2.4 SHA 1515 34 5 2.4 2 0.9 4 0.6 0.3 1.7 3.5 SIN 1515 35 5.6 2.8 3 1.2 4 1.9 1.5 1.7 4.1 SYS 1515 34 5.2 2.5 2 1.1 4 0.3 0.1 1.9 3.8 TRI 1515 34 5.4 3 3 1.1 4 2.2 1.9 2 4.2 SAX 1518 32 12 12 12 11 . 11 11 11 11 12 SUL 1521 34 8.9 9.1 9 7.5 8 8.1 7.8 8.1 8.3

107

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.3 Continued

Divergence Species FOR FOT HVI HVE LFO LOR LSI MAI MAT MOL MYT NEO NOA OST ALT CSI CSP DIC DIS DIR DOT EUS EXB FOR

FOT 1.9 -

HVI 1.9 0.5 -

HVE 2.2 0.9 0.4 - LFO 5.6 4.8 4.7 5.1 -

LOR 5.5 4.7 4.6 5 0.7 -

LSI 3.1 2.2 1.8 2.2 5.6 5.4 -

MAI 3.1 2.2 1.8 2.2 5.6 5.5 1.5 -

MAT 3.2 2.4 2.1 2.5 5.7 5.6 1.7 1.8 - MOL 1.7 1.3 1.1 1.5 5 4.8 2.4 2.4 2.6 -

MYT 3.5 2.8 2.6 3 5.4 5.2 3.6 3.6 3.9 3 -

NEO 1.9 1.3 1.1 1.5 5 4.8 2.4 2.4 2.6 1 2.8 -

NOA 1.7 1.1 0.9 1.3 4.8 4.7 2.2 2.2 2.5 1 2.7 0.5 - o 00

OST 2 1.4 1.2 1.6 5.1 5 2.5 2.5 2.8 1 2.9 • 0.7 - PAR 3 1.9 1.7 2 5.3 5.2 3.2 3 3.5 2 3.6 2.2 2 2.3 PPS 1.8 0.3 0.2 0.6 4.6 4.5 1.9 1.9 2.2 1 2.5 1 0.9 1.1 RHO 4.2 3.4 3.2 3.6 5 4.7 4.2 4.4 4.6 3 3.8 3.4 3.3 3.6 SHA 2 0.5 0.5 0.9 4.8 4.6 2.2 2 2.4 1 2.8 1.3 1.1 1.4 SIN 1.6 1.7 1.5 1.9 5.4 5.3 2.8 2.8 3 1 3.2 1.6 1.5 1.7 SYS 2 0.8 0.7 1.1 5 4.8 2.4 2.3 2.6 2 3 1.5 1.4 1.7 TRI 2.4 1.8 1.6 1.9 5.2 5.1 2.9 2.9 3.2 2 3.4 1.3 1.3 1.5 SAX 12 11 10 11 12 12 11 11 12 11 12 11 11 11 SUL 8.7 7.8 7.6 8 8.6 8.6 8.6 8.5 9.1 8 8.2 8 7.8 7.9

108

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE 2.3 Continued

Divergence Species PAR PPS RHO SHA SIN SYS TRX SAX SUL ALT CSI CSP DIC DIS DIR DOT EUS EXB FOR FOT HVI HVE LFO LOR LSI MAI MAT MOL MYT NEO NOA OST PAR

PPS 1.6 -

RHO 4 3.1 -

SHA 1.7 0.3 3.4 - SIN 2.7 1.4 3.8 1.7 -

SYS 2 0.5 3.6 0.4 1.7 - TRI 2.7 1.5 4 1.8 2.1 2 SAX 11 10 11 10 11 11 11 - SUL 8.4 7.6 8.1 7.8 8.4 8 8.2 8.4

109

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 8 8 5 8 8 6 6 6 8 8 8 8 7 gag 15 15 7 15 16 16 17 17 16 7 15 7 16 7 15 7 15 8 15 8 19 16 7 16 17 16 7 15 16 6 16 7 15 15 15 16 16 7 16 7 15 8 Glu GAA 3 15 8 4 4 4 16 7 4 4 16 8 4 ______16 14 4 15 5 15 3 15 15 16 13 16 3 16 Gin caacag

7 55 17 3 4 15 3 5 5 5 15 4 7 5 5 15 4 ______1 5 14 4 1 5 15 3 1 4 14 3 1 0 5 15 3 0 0 0 5 15 4 1 5 15 3 0 0 5 15 3 0 6 14 3 0 0 00 5 6 16 4 15 3 0 6 15 4 0 0 00 50 5 5 16 3 16 3 15 3 0 50 15 4 Cys ugcugu

14 16 14 0 6 15 3 1414 1 4 13 5 ______5 14 5 15 5 16 2 5 4 15 4 14 0 5 16 4 5 14 5 144 0 13 5 15 5 13 4 14 5 144 0 5 5 15 5 14 5 14 0 5 5 14 5 14 1 4 5 15 5 6 15 5 14 5 14 5 14 Asp gacgau

23 5 12 26 21 21 23 23 23 23 25 23 24 23 ______7 5 24 5 14 5 23 5 24 5 24 4 16 8 6 23 5 5 23 5 23 5 5 21 35 16 23 5 5 23 5 5 23 5 14 5 5 23 6 6 22 5 13 4 8 205 5 23 5 5 Asn aacaau

4 3 5 5 5 7 5 4 5 5 8 5 5 cgu

5 5 2 2 3 3 8 46 5 4 5 4 8 8 21 5 5 5 4 4 5 5 6 cgg

1 3 4 6 22 1 1 1 3 5 6 23 cgc

______9 1 9 3 8 2 9 1 9 9 10 1 4 10 10 10 1 4 10 1 10 10 1 4 10 10 1 10 1 4 Arg cga

4 8 4 6 5 25 5 4 4 4 3 5 4 9 1 5 5 5 10 0 4 4 9 4 agg

6 6 4 9 1 4 5 5 6 6 6 4 9 5 7 6 4 10 1 4 5 6 4 9 1 5 5 6 5 8 aga 9 7 9 9 9 9 7 8 11 11 10 10 7 10 1111 6 6 5 10 10 6 4 10 5 4 11 1 5 6 10 6 4 9 1 5 5 1 9 9 6 1 1 10 6 4 8 1 4 1 11 2 9 9 3 1 11 210 5310145 524 2 9 6 4 9 1 5 2 10 6 4 2 10 6 3 3 10 6 4 2 2 11 6 4 10 1 3 ______5 2 10 6 4 9 1 5 5 5 2 10 6 4 5 2 5 1 5 1 6 3 55 2 1 5 2 5 2 5 2 10 6 5 2 10 6 4 9 1 Ala 1 1 1 1 1 4 1 1 2 3 2 1 5 2 (Taxon abbr. as in Table 2.1). SAX 4 2 SUL 3 4 SHA 1 SIN SYS TRI 0 5 3 FOR 5 FOT HVI 1 5 PAR 1 4 PPS RHO 3 CSI DISDOT 4EXB 5 1 1 3 HVE 1 5 LOR 1 LSIMAI 2 2 5 NEO 1 5 OST 2 4 DIR CSPDIC 1 3 2 ALT 1 6 EUS 1 5 2 LFO 1MAT 6MOL 1 2 4 MYT 2 5 NOA Species Species GCAGCCGCGGCU Table 2.4. Codon usage of the matK gene in the Hamamelidaceae

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Table 2.4 Continued •H JS rH rH J (3 X cu j E ■p 04 M m a) >1 (0 0) 0) u u 0 >i 9 9 U u o 3 3 < 3 3 3 CD a CD CD CD 3 3 3 o < 3 < 3 3 3 a 3 CD 3 3 3 D D < D 3 CD 3 2 < S a < 3 3 3 3 < 3 3 3 < a u 3 3 U O D 3 < < 3 CJ < 3 3 3 3 < 3 n i n t n i n n t f i n t n ^ ^ i n i n i o r o ^ ^ m i n i n t n t n i n t n M O i n i n i n i n t o n ^ H HCMNNHOHMOOMMHHNfOnNHHCnnMfOtMHCMOHM'Ofn nnnnnfnn^^rnnnfOonnconfnNnfn^fnn^nnnmnn cococororocorocMrococMcococococococorococoeocororococoTpcorocNTp on rH rH - r CO rH r** CMCMCMCMCMCMCMCNCMCMCMCMCMCMCMCMCNCMCMCMCMCMPMCMCMCMCMCMCMCMCMCM in^o,00000p-p-000000000000000r*-000r*-00 oDp-0p-0p-p-*p**p-p'p-r-p-p-p-p**p-cop-*p'r-p'*p»p-p-0p-GOp-co0CDOjHHHHHHHHHHHHMHHHHHHHHHHHHHHHONn TP rH rH GO 0 TP aN0p-oDaNr'*oocop-cooooooooNOocococooocNp**p*coaNOoaococooDCOTpp«. ,*»0000

MCM CM P- rH m TP rH 0C 010 10 CO 10 10 HrH rH rH m m m m o o MC CM CM CM on Hr HrH rH rH rH IT CM CM 10 OTP CO H rH 00 10 OC CO CO CO CM rH P* HrH rH CM CO HCM rH o o in rH H H NONON m ON rH HH rH o o o CM in ON HrH rH m rH HH rH o o rH MC CM CM CM MCMCM m CM CM 00 TP oo rH m rH a HH rH H p- m o H nm in H CO HrH rH rH o CM CM 10 PTf TP rH 00 an in rH p- CM CM CM CM -p* p- CO HH rH HrH rH in an ON H rH H *P ONON rH HrH rH nin in o o ON rH CM OTP CO H rH rH PTP TP TP 00 rH P- MCMCM CM in rH V0 NONON rH rH CM rH rH Hr rH rH rH GO rH 0 rH o CM nm in m o HrH rH 111 rH rH CM VO CM CM CO rH rH in p- rH NO TP rH o rH CM CM o CM p- HrH rH rH PTP TP ON 0p» V0 rH r-* H m rH CM CM TP p* rH H 01 rH NP - 0 P-* P* ON H GO rH p* rH 00 HrH rH rH V0 CM CM P** CO CM H no in rH P*» rH CO in Hr Hr HrH rH rH rH rH rH in o o H TP rH rH rH CM CM 10 in CM CM CM rH CO 10 O t - ( O O O O O m O O O O t H O V0 rH rH rH nm in o MC MCM CM CM CM in HrH rH rH 11 CM TP rH P* HrH rH rH CO m rH 0 0 rH o o O 0 0 0 0 0 0 •O' P^ ON ON MCMCM MCM CM rH f T an HrH rH TP rH 0GO 00 0 rH O H HHr rH rH H rH CM CM CM H H H H HHr HrH rH rH H rH ON H CO TP O0 CO 0 0 0 0 0 0 0 0 0 0 0 0 rH rH o o rH CM 0 an 0 0 0 rH rH TP 0 0 0 O O an CM CM CM CM rH H m P-* rH TP rH ON HH rH o MCMCM MCMCM MCM CM 0 CM CM an -ON p- rH TP Hr rH rH rH rH o H CO O' rH rH OC CO CO CO H -0 000 00 00 P- rH O ON © rH MCMCM CM H rH rH TP CM -P* P- H Tp rH ON rH O O CM Hr HrH rH rH rH H 00 H CM rH •H -0C 0 0 CO 0 P- rH an r-» rH rH P- rH co o o o o rH MC CM CM CM CM ^4 rH *O in O' M* 0 rH HrH rH OCO CO HrH rH HrH rH 0 o ON 0 HrH rH CM on rH 0 0 HrH rH ON rH H P- 0 0 O an rH H CM N0CO 0 ON CO 0 0 ON 0 Hr rH rH rH rH HrH rH rH O MCMCM MC CM CM CM CM iH HHrH H rH CM P«» PTP TP rH TP TP rH co o o rH 10 CO CM 00 ON rH TP rH CM 0 H t P CM CM GO 0 ON 0 0 rH rH rH TP rH rH 0 0 rH CO CM TP Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without prohibited reproduction Further owner. copyright the of permission with Reproduced

Table 2.4 Continued j CO > E E a> 0) <0 G 1 > u U U U U Q< h h ^tNCMnfnnn^cNinnnn^inoiNCMnnnnn'fnfnfninnfn^cN VO H ( N ( S C M f S N ( N M H i nn ( M m ( M i M n H H i ( N f ( t N N ^ ( m M H i ( S n N i H ( f M ( l N « O N C M N D f i M O invovovoinvomvoTi* n { N t n ,3'inin*3, i o v n m n n in i m o n v i o v n in ' O in o w v ^ in i o n v m i o n v t o v f o l v i in n r-» i o n v >\o o i n i n i n < o 2 r ' 2 « « o o i ^ c ^ r ^ r ^ © r ^ c o © f l o 2 H ® r ‘ 'C O I ^r ^r v ®®r ^®®p ‘c ^v 0 0Q 'C \ 0 0 O ' \ Q \ C Pioinvoinn-tninininvotnvo\ovovoiov©i©vointninmHrinv©ininintnvoin f " « - O \ g \ C P q v < O 0 0 Q P O \ O \ O \ Q 0 ^ Q P O \ O \ 0 0 G 0 C 0 0 D 0 D t , - * 0 > C 0 O V t * - » V O 0 D N(Nf ' H M M ( f') N N ( N M ( N fN N ( fN N H N M H M C m N M C N N J r**» O M H M J O H M C N M S ( O f H f M r H O \ r H r H O N O O r H r H O O O O N r H r H Ooooooooooooooooooooooooooooooooo N ' - H ' ‘ H 'oooooooooooooooooooooooooooooooo - H r H O r H r H r H O r H O O O O N M ( Nn M n f f o M n ^ M ^ ^ M N c ( » N ) { { N ^ N ' N i n ( ( N n f C M ( M N i C n i N n N ^ ' N t f H ^ M < n N o j C ^ M ,n ^ ( ( n i n M ^ ^ ' { n i M n ^ ' f > l < N N N N N N f M ( N M ( > i ( N r*» rH r t H H H H H H H H N H H H r t H H H H H M N H H H H H r t H H H N n t ~ r ~ r ^ [ ~ r ' v o r ~ r ~ r ~ i o t ^ r ~ c ~ r ~ v o t ~ r ~ r ~ t ~ - r ^ r ~ r ~ r ~ c o [ ~ t ~ t ~ r ~ r ~ i — a > 2 13 00 on o 13 OO o 13 10 on o CO 15 CM - r 0OO 00 rH 14 CO *H o o o 14 10 m on 000 00 14 VO 14 10 00 Hf 000 00 13 VO o o 12 11 non onon CO 16 000 00 HI* © 16 112 o 16 VO VO 00 CM 13 VO r* r-* H 13 000 00 CO o o 15 10 00 16 00 HI* rH 15 10 ON in 00 o 15 VO CM 14 10 ON 000 00 o o 15 on 15 000 00 CO o 14 10 o nM M* M* in 16 10 ON 00 o 16 r*- r— rH 13 as o m 00 16 m ON o o o 14 i i m CO 14 10 ON in 00 17 in rH m * c—C*- HrHrH ON VO rH Fig. 2.1. Approximate relative locations of matK primers (base compositions are listed in Table 2.1, shaded areas are introns).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ______5]______3' trnK j j matK ^ trnK

matK FI F2 F4 / F4-2 F5 R3 F6 R2/R2-2 R1 > > > > <><<

114

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.2.2. The Strict consensus of the six most parsimonious trees of 582 steps long based on sequences of the matK gene. CI=0.85, RI=0.82. Numbers above and below branches are decay indices and bootstrap percentages; boxed numbers denote the three major clades in the Hamamelidoideae. Taxon abbreviations as in Table 2.1. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

115

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I 100L LFO 100 I------LOR- 7 I---- CSI=T 100 I. CSP J 100 l*SI-|LORinae 61 (HAMeae) FOTeae 96 MAI J DIC NEO NOA 58 71 H OST TRI- 1 81 EUS-i •H EUSeae 69 FOR- 85 MOL FOTeae 62 SIN-I DIR-1 70 SYS 92 DOT 91 SHA 66 FOT PPS <50 HVI ea) d

72 DIS-i MYT EXB-1 RHO-RHOideae SAX (Outgroup) SUL

116

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.2.3. One of the six most parsimonious trees of 582 steps long based on sequences of the matK gene, showing the number of unambiguous substitutions along the branches. Taxon abbreviations as in Table 2.1.

117

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT 35 LFO LOR 14 CSI CSP LSI MAT 11 MAI DIC NEO NOA OST 14 TRI EUS FOR MOL SIN DIR SYS DOT SHA FOT PPS HVI — HVE —21 PAR 35 DIS 25 MYT 10 EXB 14 RHO SAX SUL (Outgroups)

118

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDICES 2.1-2.2

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80 80

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 [1120 C C [1120 ...... A A ...... A A ...... G ...... A A A A A A ..C A. A A A A A A A A...C A A A A A A A A A A...C T A T T A T T A T ...... C C G.R ...... T T A C.. A ...... G G G G G G G G G G G G G G G G G G G G G A .G A .G ...... G ...... DIS DIS ...C CSI CSI CSP CSP DIC DIR DOT S. .G ALT AGTGGTACGG AATCAAATGC TAGAAAATTC GTTTATAATA GATAATGCTA TTAAGAAGTT CGATATCATA GTTCCAATTA GTTCCAATTA CGATATCATA ALT TTAAGAAGTT GATAATGCTA [1120 GTTTATAATA TAGAAAATTC AATCAAATGC AGTGGTACGG EUS EUS EXB G FOR C... .G G C... .G Appendix 2.1. Continued PAR PAR SAX SUL ..C SHA SHA SIN SYS C... .G FOT FOT HVI HVE OST OST PPS RHO LFO LFO LOR MAT MAT MOL MYT NEO NOA C... .G TRI U> U> MAI £ LSI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 [1200 A [1200 .... T ...... T T TT....T... [1200 T T [1200 ...... T T ...... C C C C C C C C C C C C C C C C C C C C C C C C C C T T... c C ---- G....A T. .T G ...... T T T T ...... T ...... C G ...... G C..G ...... G ...... T T ...... T A ...... A.. CA A.. A ...... DIC DIC T.A DIS DIS DIR DOT T.A T.A HVI HVI T.A SUL SUL .A A.. CSP CSP T.A SHA SHA SYS T.A T.A PPS PPS SIN T.A SAX T.A CSI CSI T.A Appendix 2.1. Continued EUS EUS EXB FOR ..G.G FOT T.A HVE T.A T.A T.A NEO NEO T.A TRI T.A PAR PAR RHO T G ALT TTCCTCTGAT TGGATCATTG GCTAAAGCGA AATTTTGTAA CGTATTAGGG CATCCTATTA GTAAGCCGGC CCGGGCCGAT CCGGGCCGAT GTAAGCCGGC ALT CATCCTATTA CGTATTAGGG [1200 AATTTTGTAA GCTAAAGCGA TGGATCATTG TTCCTCTGAT MAT MAT MOL T.A T.A LOR LOR LSI MAI T.A C . MYT T.A. NOA OST A.A T.A T.A LFO LFO

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [1280 [1280 [1280 G. G. [1280 ..... A A [1280 A [1280 A A [1280 A [1280 A [1280 [1280 A [1280 A [1280 A [1280 . . . . [1280 CC A [1280 A A [1280 A [1280 A [1280 A C C [1280 C C A [1280 [1280 [1280 [1280 C...CC [1280 A C C C C [1280 [1280 [1280 A A [1280 [1280 A [1280 C C [1280 _____ ...... T T T T T..G T T T T T T T T T T T T T T T T T T T ---- ______---- T T T T T T T ...... G ...... A. A. ..A A ...... G A ...... A ...... T T T T T T T T T T T T T T T T T T T T..W T T T AT ...... C C G ...... A T G...G ...... A ...... A T ...... T T...A ...... G ...... T G G G G ...... A ...... A ...... SAX G SUL SHA SIN SYS TRI DIS DOT DIR EXB FOR MAT NOA PAR PPS C CSI DIC FOT LOR MOL RHO EUS HVI HVE LFO MYT OST C CSP NEO Appendix 2.1. Continued ALT TCATCAGATT CTGATATTAT CGACCGATTT GTGCGTATAT GCAGAAATCT TTCTCATTAT CACAGCGGAT CCTCGAAAAA [1280 ^ MAI i_i i_i LSI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 [1360 ...... A [1360 A A A A A A A A A A A A A A A [1360 A A A A A [1360 A A A A [1360 A..T... [1360 CA C. C. ...A C. ...A ...... T ...... G ...... A ...... C C C G ...... G ...... G ...... TG ...... SHA SIN SYS SAX ...CT SUL PPS TRI PAR RHO OST ...A DIS DIR HVI HVE MYT NOA ...A ALT CSI AAAGAGTTTG TATCGAATAA .M AGTATATACT TCGACTTTCT TGTGCTAGAA CTTTGGCTCG TAAACACAAA AGTCCTGTAC [1360 CSP DIC DOT EXB FOR FOT LSI MOL LFO MAI NEO ...A Appendix 2.1. Continued LOR MAT EUS

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[1440 [1440 [1440 [1440 1440 [ [ 1440 [ [1440 [1440 [1440 [1440 [1440 [1440 1440 [ 1440 [ [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 [1440 T T T. T. T. T. T...... TTTGATCGTC ...... C G. G. G...... GAGGAAGAACAAGTTCTTTC ATTCCTTACG TATTGGAAGA .G. . .G. .G. . .G. .G. . .G. • G. . G. • . G. • . .G. ....T.. .G. GGTTCGGAAT c ...... C..C GTGCTTTTTT GAAAAGATTA

DIC DIS ALT CSP HVE DOT EXB FOR CSI DIR FOT SAX SUL Appendix 2.1. Continued HVI SHA SIN SYS TRI EUS LOR PAR OST PPS RHO MAT MOL LFO MYT NOA NEO LSI m CO -o MAI

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 [1520 T. T. [1520 ...... T T T T T T T T T T T T T T T T T T T T T T T .T T . .T. . [1520 T. T...T...T [1520 T T. T. .T ...... C C C C C C C C C C C C C C C C C C C ...... G..C G C...C ...... T T T ...... C T ...... C T T T T T T T T K T T T T T C. C. ...T C. C. ...T ...... G...G G A G G G G G G G GG G G G G ...... T G ...... T G G G G G G G ...... T G ...... T G G A .G ...... C C ...... C C ...... A ...... T A. .G A C ...... C.A C . A C . A C . C .A C.A T .T G. .T G. AA. .G ..... C...T .T ...... G T ...... GCTT CTTCTACTTC GCGGAGGTTA TATAGAGGGC GTATTTGGTA TTTGGATATT ATTTGTATCA ACGATCTGGC [1520 ...... T ...... --- .... DIC ...AAA DIR ...AAA DIS ...AAA ALT CSI CCA ...AAA CSP ...AAA DOT EUS ...AAA EXB ...AAA FOR ...AGAFOT ...AAA HVI ...AAA HVE ...AAA ...AAA Appendix 2.1. Continued SUL ...AGA PAR PPS ...AAA ...AAA SHA SIN ...AAA SYS ...AAA ...AAA SAX .AGAA . . LFO LOR ...... MAT MOL ...AAAMYT ...AAA NEO ...AAANOA ...AAA OST ...AAA ...AAA RHO ...AGA TRI ...AAA M LSI ...AAA oo oo MAI ...AAA

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[60 [60 [60 [60 [60 [60 [60 [60 [60 [60 [60 [60 KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL [60 KSSSLIVKRL [60 KSSSLIVKRL KSSSLIVKRL KSSSLIVKRL [60 KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL [60 KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL KSSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL [60 NSSSLIVKRL [60 KfSSLIVKRL [60 KfSSLIVKRLKSSSLIVKRL [60 KSSSLIVKRL [60 KSSSLIVKRL [60 KfSSLIVKRL [60 ILLENVGYDN ILLENVCYDN ILLENVGYDN ILLENVGYDN ILLENVGYEN ILLENVGYDN ILLENVGYDN ILLENLGfiDN LAHDHGLNRS LAHDHGLNRS ILLENVGYDN LAHDHGLNRS ILLENLGfiDN LAHDHGLNRS y PLIFQEYIYA LAHDHGLNRS PLIFQEYIYA PLIFQEYIYA LAHDHGLNRS PLLFQEYIYA LAHDHGLNRS ILLENVGYDN PLIFQEYIY PLIFQEYIYA DKSRQHDFLY DKSRQHDFLY PLIFQEYIYA DKSRQHDFLY DKSGQHDFLY DKSRQPDFLY PLIFQEYIYA LAHDHGLNKS ILLENLGYDN HKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVRYDN 10 10 20 30 40 50 60 MEEFQGYLEL MEEFQGYLEL MEEFQGYLEL MEEfiQGYLEL MEEFQGYLEL MEEFQGYLEL DKSRQHDFLY MEEFQGYLEL MEEFQGYLEL DKSRQHDFLY MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN (Taxon abbreviations as in Table 2.1). Note the underlined lower case letters. DIC DIS FORSIN MEEFQGYLELFOT DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS MEEFQGYLEL ILLENVGYDN PPS DQSRQHDFLY PLIFQEYIYA LAHDHGLNRS MEEFQGYLEL ILLENVGYDN DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRSSYS ILLENVGYDN MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRSHVI ILLENVGYDN HVE MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN SHA MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRSDIR ILLENVGYDN MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN OST LFOLOR DKSRQHDFLY LAHDHGLNRS MEEfiQGYLEL PLIFQEYIYfi EXBRHO MEEFQGYLEL DKSRQHYFLY PLIFQEYIYA LAHDHGLNRS MEKFQGYLEL ILLENVGYDN DKSRQYDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN ALT LAHDHGLNRS DKSRQHDFLY MEEfiQGYLEL PLIFQEYIYjf ILLENLGfiDN CSP MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN PAP MKEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRSDOT ILLENVGYDN CSI MEEFQGYLELLSI DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN EUS NOA MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN MYT MEEFQGYLEL DKSRQHYFLY Appendix 2.2. Amino acid sequences translated from the matK gene sequences MATMAI MEEFQGYLEL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS MEEFQGYLELMOL DKSRQHDFLY PLIFQEYIYA LAHDHGLNRS ILLENVGYDN MEEFQGYLEL DQSRQHDFLY PLIFQEYIYA LAHDHGLNRS NEO

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 [120 120 [ 120 [ VSSLEGKEIV GSSLEGKEIV VSSLE£KEIV VSSLErKEIV VSSLE£KEIV VSSLEGKERV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV VSSLEGKEIV IVEIPFSLRL IVEIPFfiLRL IVEIPFSLRL IVEIPFSLRL IVEIPFSLRL IVEIPFjaLRL IVEIPFjaLRL IVEIPFSLRS IVEIPFSLRS IVEIPFSLRS IVEIPFSLRS IVEIPFSLRL IVEIPFSLRS IVEIPFSLRL IVEIPFSLRS IVEIPFSLRS IVEIPFSLRS IVEIPFSLRS IVEIPFSLRL IVEIPFSLRL IVEIPFSLRL IVEIPFSLRL IVEIPFSLRL SQMISEGFAV SQMISQGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQMISEGFAV SQIISEGFAV IFLGYNKNLY PFLGHNKNLY PFLGHNNNLYSQIISEGFAI PFLGHNKNLYSQMISEGFAIIVEIPFSLRL TFLGLNKNLYSQMISEGFAIIVEIPFSLQL PFLGHNKNLYSQMISEGFAV PFFGHNKNLYFQMISEGFAIIVEIPFSLRL PFLGHNKNLF PFLGHNKNLY PFLGHNKNLY PFLGHNKNLY PFLGHNKNLYSQMISEGFAV PFLGHNNNLYSQIISEGFAIIVEIPFSLRL PFLGHNKNLF PFLGHNKNLYSQMISEGFAV PFLGHNKNLY PFLGHNKNLY PFLGHNKNLYSQMISEGFAV PFLGHNKNLY PFLGHNKNLYSQMISEGFAV IISANDSNQYPFLGHNKNLY IISANDSNQNPFLGHNKNLY IISVNDSNQN IISANDSNQNPFLGHNKNLYSQMISEGFAVIISVNDFNQN IISANDSNQNPFLGHNKNLY IISVNDSNQN IISVNDFNQN IISVNDSNQNPFLGHNKNLYSQMISEGFAIIVEIPFSLRLIISANDSNQNPFLGHNKNLYSQMISEGFAVIISANDSNQN IISVNDSNQN IISVNDSNQN IISANDSNQN IISANDSNQN IISANDSNQNPFLGHNKNLYSQMISEGFAVIISANDSNQNPFLGHNKNLY IISANDSNQNPFLGHNKNLYSQMISEGFAVIVEIPFSLRLIISANDSNQN IISANDSNQN IISANDSNQN IISANDSNQN IISANDSNQN IISVNDSNQN IISANDSNQN IISANDSNQN IISANDSNQN NISANDSNQN 70 70 80 90 100 110 120 ITRMYQQNHL ITRMYQQ-NL ITRMYQQNQL ITRLYQQNQL ITRMYLQNHL ITRMYQQNHL ITRMYQQNHL ITRMYQQNQL ITRMYQQNQL ITRMYQQNHL DIS Appendix Continued2.2 ALTITRMYQQNRL LORITRMYQQNRLEXBITRMYQQNHLSISANDSNQDRHOITRMYQQNHL FORITRMYQQNQLSIN PPSSHAITRMYQQNHL ITRMYQQNHL LFOITRMYQQNRL MYTIIRMYQQNHLCSICSPITRMYQQNHL ITRMYQQNHL HVE ITRMYQQNHL DIRITRMYQQNHLDOT HVI DIC PAPSYS ITRMYQQNHL LSIMATITRMYQQNHL ITRMYQQNHL FOT MAI EUS ITRMYQQNQL NEOITRMYQQNQLNOA OST MOLITRMYQQNQL

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [178 [180 [178 [178 [178 [178 DASSLDLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF [178 DASSLDLLRF DASSLHLLRF DASSLHLLRF [178 DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF [178 DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRFDASSLHLLRF [178 DASSLHLLRF [178 DASCLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRF DASSLHLLRFDASSLHLLRF [178 LVQTLRYWVK LVQTLRYWVK LVQTLRYWVK LVQTLRYWVK LVQVLRYWVK LVQTLRYWVK LIPHPIHLEI LIPHPIHLEI LIPHPIHLEIWDQTLRYWVK LIPHPIHLEILVQTLRYWVK LIPHPIHLEILVQTLRYWVK LIPHPIHLEILVQTLRYWGK LIPHPSHLEILVQTLRYWVK LIPHPIHLEILVQTLRYWVK LIPHPIHLEILVQTLRYWVK LIPHPTHLEILVQTLRYWVK LIPHPIHLEILVQTLRYWVK LIPHPIHLEI LIPHPIHLEILVQTLRYWVK LIPHPTHLEILVQILRYWVK LIPHPIHLEIWQTLRYWVKLIPHPIHLEI LIPHPIHLEI FLHLNYVSDI FLHLNYVSDI FLHLNYVSDILIPHPIHLEILVQTLRYWLK FLHLNYVSDI FLHLNYVSDI FLHLNYVSDI FLHLNYVSDI FLHLNYVSDILIPHPIHLEILVQTLRYWVK FLHLNYVSDI LLHLNYVSDILIPYPIHLEILVQTLRYWVK FLHLNYVSDI LLHLNYVSDI VLHLNYMSDI HSIFPFLEDK HSPFPFLEDKLLHLNYVSDI HSIFPFLEDK HSIFPFLEDRFLHLNSVSDI HAIFPFLEDN HSIFPFLEDKLLHLNYVSDILIPHPIHLEILVQTLRYWVKHSIFPFLEDKLLHLNYVSDI HSIFPFLEDK HSIFPFLEDK HSIFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVKHSIFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVKHSIFPFFEDK HSIFPFFEAK HSIFPFLEDK HSIFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVK HSVFPFPEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVK HSVFPFLEDK HSIFPFLEDK HSIFPFLEDKFLHLNYVSDI HSIFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVK HSIFPFFEDKFLHLNYVSDILIPHPIHLEI HSIFPFLEDK HSLFPFLEDKFLHLNYVSDI HSIFPFLEDK HSIFPFLEDK HSIFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVK HSVFPFLEDKFLHLNYVSDILIPHPIHLEILVQTLRYWVK 130 130 140 150 160 170 180 QSH—NLRSI QSH—NLRSI QSH—NFRSI QSH— NLRSI QSH—NLRSIHSIFPFLEDKFLHLNYVSDIQSH— NLRSI QSH— NLRSI QSH—NLRSI QSH—NLRSI QSH— NLRSI QSH—NLRSI QSH— NLRSI QSH— NLRSI HAIFPFLEDK FLHLNYVSDI QSHTHNLRSI QSH— NLRSI KSH— NLRSI icSH— NLRSI SYS SHA QSH— NLRSI DIR HVE QSH— NLRSI DIC OST DIS QSH— NLRSI PAP RHOMYT CSI QSH— NLRSI DOTHVI QSH— NLRSI QSH— NLRSI NOA QSH— NLRSI EXB MAT FOR SINQSH—NLRSIPPS QSH— NLRSI CSP EUS MAI QSH— NLRSI LSI FOT NEO QSH— NLRSI Appendix 2.2 Continued ALT LFO LOR JtSH— NLRSI MOL N)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [238 [238 [238 [240 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [238 [235 [238 [238 [238 SSYLRSTSSG [238 SSYLRSTSSG [238 SSYLRSTSSG SYHLRSTSSG [237 SSYLRSTSSG SSHLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQ ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQ ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFVFLRNQ EYIFLFFRNQSSYLRSTSSG ESIFVFLRNQSSHLRSTSSG EYIFLFFRNQSSYLRSTSSG ESIFFFFCNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSGESIFLFFRNQSSYLRSTSSG ESIFLFFRNQ ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQ ESIFLFFRNQSSYLRSTSSG ESIFLFFRNQSSYLRSTSSG ESIFVFLRNQ SSHLRSTSSG [237 ESyFVFLRNQ ESyFVFLRNQ SSHLRSTSSG ESvFVFLRNQ SSHLRSTSSG FLYNSHVCEY FLYNSHVCEY F L Y N S H V y E Y FLYNSHVCEY FLYNSHVCEY FLYNSHVCEY FSKRNQRLFLFLYNSHVCEY FSKRNQRLFLFLYNSHVCEY FSKRNQRLFLFLYNSHVCEY FSKRNQRLFLFLYNSHVCEY FSKRNQRLFL FSKRNQRLFLFLYNSHVCEY FSKRNQRLFLFLYNSHVCDYESIFVFLRNQSSYLRSTSSG FSKRNQRLFL FLYNSHVyEY FSKRNQRLFL FSKRNQRLFL FLYNSHVyEY FSKRNQRLFLFLYNSHVCEY FSKRNQRLFLFLYNSHVCEY L-KRNQRLFLFLYNSHVCEY L-KRNQRLFL ; Y --- LITPKKSISI LITPKKSISI LITPKKSISI LITPKKSISIFSKRNKRLFLFLYNSHVCEY LITPKKSISIFSKRNQRLFLFLYNSHVCEYLITPKKSISIFSKRNKRLFLFLYNSHVCEY LITPKKSISIFSKRNKRLFL LITPKKSISIFSKRNKRLFLFLYNSHVCEY LITPKKSISI LITPKKSISI LITPKKSISI LITPKKPISI LITPKKPISI l i n p k k s i fl y i n p k k s i fLINPKKSIFy y LITPKKSISI LITPKKSISIFSKRNQRLFL LITPKKSISI 190 190 200 210 220 230 240 FFYEYYNWNSLITPKKSISIFSKRNQRLFLFLYNSHVCEY FFYEYHNWNS FFYEYHNWNS FFYEYYNWNS FFYEYYNWNS FFYEYYNWNSLITPKKSISIFSKRNKRLFSFLYNSHVCEY FFYEYHNWNSLITPKKSISLFSKGNQRLFLFLYNSHVCEY FFYEYHNWNSLITPKKSISLFSKRNQRLFLFLYNSHVCEY FFYEYYNWNS FFYEYYNWNS FFYEYHNWNSLITPKKSISIFSKRNQRLFLFLYNSHVCEY FLYEYHNWNS FLYEYRNWNS FFYEYYNWNSLITPKKSISLFSKRNQRLFLFLYNSHVCEY FFYEYYNWNS FLYEYRNWNS FLYEYRNWNS FFYEYRNWNSLITPKK FFYEYHNWNS FFYEYYNWNS FFYEYYNWNSLITPKKSISIFSKRNQRLFLFLYNSHVCEY LFYEYYNWNSLITPKKSISIFSKRNQRLFLFLYNSHVCEY HVI FFYEYYNWNS SHA DIR DOT HVE DIC OST FOT CSI LSI LFO DIS FOR SIN PAP EUS PPS FFYEYYNWNS SYS NEOFFYEYHNWNS NOAFFYEYHNWNS EXB RHOFLYEYRNWNS MYT CSP MAT MOLFFYEYHNWNSLITPKKSIPIFSKRNQRLFL MAIFFYEYYNWNS Appendix 2.2 Continued ALT LOR CO

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [298 [295 [297 [297 [298 [298 [297 [298 [298 [298 [298 TPLLMNKWKY TPTLMNKWKY LPLLMNKWKY [298 TPLLMNKWKY TPLLMNKWKY [298 QGKSILASKGTPLLMNKWKY QGKSILASKGTPLLMNKWKY QGKSILASKGTPLLMNKWKY QGKAILASKG QGKSILASKG QGKSILASKG TPLLMNKWKYQGKSLLASKG [298 QGKSILASKGTPLLMNKWKY QGKSILASKG TPLLMNKWKY [298 QGKSILASKG TPLLMNKWRY [298 QGKSILASKGTPLLMNKWKY QGKAILASKG APLLMNKWKY [298 QGKSILASKG TPLLMNKWKY [300 QGKSILASKGTPLLMNKWKY QGKSILASKGTPLLMNKWKY QGKSILASKG TPLLMNKWKY [298 QGKSILASKG TPLLMNKWKYQGKSILASKG [298 TPLLMNKWKY [298 KDPFVHYVRY KDPFMHYVRY KDPFMHYVRYQGKSILASKGTPTLMNKWKYKDPFMHYVRYQGKSILASKG KDPFVHYVRY QGKSILASKG TPLLMNKWKY [298 KDPFVHYVRY KDPFVHYVRY KDPFMHYVRY KDPFMHYVRY QGKSILASKG TPLLMNKWKY [298 KDPFMHYVRY QGKSILASKG TPLLMNKWKY [298 KDPFMHYVRY KDPFMHYVRY KDPFMHYVRYQGKSILASKG NDFQASPWLF NDFQAILWLF NDFQAILWLFKDPFMHYVRY NDFQAILWLF NDFQAILWLF NDFQATLWLF NDFQAILWLFKDPFMHYVRY NDFQAIPWLF NDFQAILWLF KDPFVHYVRYNDFRAILWLFKDPFMHYVRY QGKSILASKGNDFQAILWLFKDPFMHYVRY TPLLMNKWKY [298 NDFRPS-YGV NDFQAILWLFKDPFMHYVRY NDFQAILWLF KDPFMHYVRY QGKSILASKGNDFQAILWLFKDPFMHYVRY TPLLMNKWKY [298 KIKHLVEAFA KIKHFVEVFV KIKHLVEVFV KIKHLVEVWV KIKHLVEVFV NDFQAILWLF KDPFMHYVRY QGKSILASKG TPLLMNKWKY [298 KIKHLVEVFVNDFQAILWLF KIKHLVEVFV KIKHLVEVFV KIKHLVEVFVNDFQAILWLF KIKNLVEVFVNDFQAILWLFKDPFMHYVRY KIKNLVEVFVNDFQAILWLF KIKHLVEVFV-DFQAILWLF KIKHLVEVFVNDFQAILWLFKDHFMHYVRY KIKHLVEVFVNDFQAILWLF KIKHLVEVFV NDFQAILWLFKIKHLVEVFVNDFQAILWLF KDPFVHYVRY QGKSILASKGKIKHLVEVFVNDFQAILWLFKDTFVHYVRY TPLLMNKWKY [298 KIKHLVEVFVNDFQPILWLFKDPFMHYVRY KIKHLVEVFVNDFQAILWLFKDPFMHYVRY KIKHLVEVFV NDFQAILWLF KDPFVHYVRY QGKSILASKG TPLLMNKWKY [298 250 250 260 270 280 290 300 ALLERIYFYG ALLERIYFYG ALLERIYFYGKIKHLVEVFA ALLERIYFYGKIKHLVEVFA ALLERIYFYGKIKHLVEAFA ALLERIYFYGKIKHLIEVFA ALLERIYFYGKIKHLVEVFA ALLERIYFYG ALLERIFFYG ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERIFFYGKIKHLVEVFV ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERIYFYG ALLERICFYG ALLERIYFYG ALLERIYFYG ALLERIYFYGKIKNLVEVFV ALLERIYFYGKIKNLVEVFVALLERIYFYG ALLERIYFYGKIKHLVEVFV DIS EXB FOR SIN CSI RHO MYT EUS FOT PPS SHA SYS HVE DIC ALLERIYFYG CSP LFO LOR Appendix 2.2 Continued ALT MAI MOL PAPDIR ALLERIYFYG DOT HVI NOA LSI MAT OST NEO

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [358 [358 [355 [357 [357 [358 [358 [358 [358 [358 [358 [358 [358 [358 [358 [358 [358 [358 [358 [357 [358 ENSFIIDNAI ENSFIIDNAI ENSFIIDNAI ENSFIIDNAI ENSFIIDNDI ENLFIIDNAI ENSFIIDNAI [358 ENSFIIDNAI ANSFLIDNAI NPSWRSQMLENSFLIDNAI NPSWRSQMLENSFIIDNAI NPSWRnQML NPSWRSQML-NSFIIDNDINPSWRSQML NPSWRSQMLENSFIIDNAI NPSWRSQMLENSFIIDNAI NPSWRSQMLENSFIIDNAI NPSWRSQMLNPSWRSQMLANSFLIDNAI ENSFIIDNAI [360 NPSWRSQMLENSFIIDNAI NPSWRSQML ENSFIIDNAINPSWRSQMLENLFIIDNAI [358 NPSWRSQMLENSFIIDNAI NPSWRSQMLENSFIIDNAINPSWRSQMLENSFIIDNAINPSWRSQMLENSFIIDNAI FLGYLSSVGL FLGYLSSVGL FLGYLSSVGL NPSWRnQML ENSFIIDNAIFLGYISSVGL [358 FLGYLSSVGLNPSWRSQML FLGYLSSVGL FLGYLSSVGL FLGYLSSVGL FLGYLSSVGL FLGYLSSVGL FLGYLSSVGLNPSWRSQMLFLGYLSSVGL NPSW-SQML ENLFIIDNAIFVGNLSSVGL [358 d S-D -- INQLSNHSF^ INQLSNHSL INQLSNHSLGFLGYLSSVGL INQLSNHSLD INQLSNHSFDFLGYLSSVGL INQLSNHSFD INQLSNHSFD FLGYLSSVGL NPSWRnQML ENSFIIDNAI [358 INQLSNHSFDFLGYLSSVGLNPSWRSQMLINQLSNHSFD INQLSNHSFDFLGYLSSVGL INQLSNYSFDFLGYLSSVGL INQLSNHSFD FLGYLSSVGL NPSWRSQML ENSFIIDNAI [358 INQLSNHSFDFLGYLSSVGL INQLSNHSFD INQLSNHSFDFLGYLSSVGL INQLSNHSFDFLGYLSSVGLNPSWRSQMLINQLSNHSFD INQLSNHSFD INQLSNHSFDFLGYLSSVGL LNQLSNHSFDFLGYLSSVGLNPSWRSQML INQLSNHSFDFLGYLSSVGL INQLSNHSFD FLGYLSSVGL NPSWRSQML ENSFIIDNAIINQLSNHSFDFLGYLSSVGLNPSWRSQIL [358 YVWSQPGRIYINQLSNHSLD YVWSQPVRIY YVWSQPVRIY YVWSQPVRIY YVWSQPGRIY YVWSQPGRIH YVWSQPGRVY YVWSQPGRIYINQLSNHSFD YVWSQPGRIY YVWSQPGRIYRNQLSNHSFD YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIY YVWSQPGRIYINQL YVWSQPGRIY YVW-QPGRIYINQLSNHSFD YVWSQPGRIY 310 310 320 330 340 350 360 YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF YLVNFWQCHF DIS LORYLVNFWQCHFEXBYLVYFWQCHFRHO MYTYLVNFWQCYFCSI LSI YLVNFWQCHF LFOYLVNFWQCHF FORYLVNF-QCHFSIN YLVNFWQCHF ALTYLVNFWQCHF CSPYLVNFWQCHFMATYLVNFWQCHFMAIEUS YLVNFWQCNF FOT YLVNFWQCHF YVWSQPGRIY SHAYLVNFWQCHFPAPSYS YLVNFWQCHF YLVNFWQCHF YVWSQPGRIY MOLYLVNFWQCHF DIR DOT YLVNFWQCHF DIC YLVNFWQCHF YVWSQPGRIY PPS HVE YLVNLWQCHF Appendix Continued2.2 HVI NEO NOAYLVNFWQCHFYVWSQPGRIY OSTYLVNFWQCHFYVWSQPGRIYINQLSNHSFDFLGYLSSVGLNPSWRSQML

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [417 [418 [418 [418 [418 [420 [417 [418 [418 [418 [418 [418 RNLSHYHSGSRNLSHYHSGS [417 RNLSHYYSGS RNLSHYHSGSRNLSHYHSGS [418 RNLSHYHSGS [418 RNLSHYHSGS [418 RNLSHYHSGSRNLSHYHSGS [418 [418 RNLSHYHSGS RNLSHYHSGS -NLSHYHSGS [418 RNLSHYYSGS [418 KNLSHYHSGS RNLSHYYSGSRNLSHYYSGS [418 RNLSHYYSGS [418 RNLSHCHSGS RNLSHYHSGSRNLSHYHSGS [418 RNISHYHSGS [415 RNLSHYHSGS RNLSHYYSGS RNLSHYYSGS [418 RNLSHYHSGS RNLSHYHSGSRNLSHYHSGS [418 [418 RNLSHYHSGS [418 RNLSHYHSGS [418 DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDQFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIY DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC EIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFGRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC DIIDRFVRIC KPARADSSDS KPARADSSDYDIIDRFVRIC KPARADSSDS KPARADSSES FCNVLGHPISKPARADSSDS FCNVLGHPISKPARADSSDS FCNVLGHPISKPARADSSDS FCNVLGHPISKPARADSSDS FCNVLGHPIS FCNVLGHPISNPARADSSDS FCNVFGHPISKPARADSSDS FCNVFGHPISKPARADSSDS PLIGSLAKAKFCNVLGHPISKPARADSSDS PIIGSFAKAK PLIGSLAKAKFCNVLGHPISKPARSDSSDSPLIGSLAKAK PLIGSLAKAK PLIGSLAKAK PLIGSLAKAKFCNVLGHPISKPARADSSDS ALIGSLAKAR 370 370 380 390 400 410 420 KKFDIIVPII KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPIS KKFDIIVPII KKFDIIVPIIPLIGSLAKAKFCNGLGHPISKPARADSSDSKKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKLARADSSDS KKFDIIVPII KKFDIIVPII KKFDIIVPIIPLIGSLAKAKFCNVLGHPIS KKFDIIVPII KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVSGHPISKPVRADSSDSKKFDIIVPII KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAK KKFDIIVPIIPLIGSLAKAK KKFDIIVPIIPLIGSLAKAK KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS KKFDIIVPIIPLIGSLAKAKFCNVLGHPISKPARADSSDS SYS DOT RHO CSI LSI SIN KKFDIIVPII PLIGSLAKAK FCNVLGHPIS KPARADSSDS SHAKKFDIIVPIIPAP DIR HVI HVE NOAKKFDIIVPIIPLIGSLAKAKFCNVFGHPISKPARADSSDSDIC OST LOR EXB MAT MAI DIS MYT CSP EUS FOR FOT PPS NEOKKFDIIVPIIPLIGSLAKAKFCNVFGHPIS LFO Appendix 2.2 Continued ALTKKFDIIVPII MOL

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [478 [477 [477 [478 [478 [478 [478 [478 [478 [477 [477 VLSLIFPKAS [478 VLSLIFPKAS VLSLIFPKAS [478 VLSLIFPKAS [478 VLSLIFPKAS [478 VLSLIFPKAS VLSLIFPKASVLSLIFPKAS [478 [478 VLSLIFPKAS [478 VLSLIFPKAS [478 VLSLIFPKAS VLSLIFPKAS [478 ALSLIFPKAS [478 VLSLIVP-AS LEEFLTEEEQ LEEFLTEEEQ VLSLIFPKAS [480 LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ VLSLIFPKAS [478 LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ LEEFLTEEEQ VLSLIVP-AS [477 LEEFLTEEEQ LEEFLTEDEQ VLSLIFPKAS [475 LEEFLTEEEQ AFLKRFGSGL AFLKRLGSGL AFLKRLGSGLLEEFLTEEEQVLSLIFPKAS AFLKRFGSGL AFLKRLGSgL LEEFLMEEEQ VLSLIFPRAS AFLKRLGSGL AFLKRLGSGL AFLKRLGSGL AFLKRLGSGLLEEFLTEEEQVLSLIFPKASAFLKRLGSGL AFLKRLGLGL AFLKRLGSELLEEFFTEEEQVLSLIFPKASAFLKRLGLGL LEEFLTEEEQ VLSLIFPKAS [478 AFLKRLGSGL AFLKRFGSGL AFLKRLGLGL LEEFLTEEEQAFLKRLGSGL VLSLIFPKAS [478 AFLKR-GLGL AFLKRLGSgL AFLKRLGSgL AFLKRFGSGLLEEFLTEEEQ AFLKRLGLGL LEEFLTEEEQ VLSLIFPKASAFLKRLGSGLLEEFLTEEEQ [478 AFLKRLGSGL AFLKRLGSGLLEEFLTEEEQVLSLIFPKAS AFLKRLGSgL LEEFLTEEEQ VLSLIVP-AS AFLKRLGSGLLEEFLTEEEQVLSLIFPKAS AFLKRLGSGL LARKHKSTVR LARKHKSTVR LARKHKSTVR AFLKRLGSGLLARKHKSTVR LEEFLMEEEQ VLSLIFPRAS [477 LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSPVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHTSTVR LARKHKSTVR LARKHKSTVR LARKHTSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSTVR LARKHKSPVR LARKHKSPVR LARKHKSTVR LARKHKSTLR YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART YILRLSCART 430 430 440 450 460 470 480 SKKKSLYRIK SKKKSLYRIK SKKKSLYRIK SKKKSLYRIKYILRLSCART SKEKSLYRIK SKKKSLYRIKYILRLSCART SQKKSLYRIKYILRLSCART SKKKSLYRIKYILRLSCART SKKKSLYRIK SKKKSLYRIR SKP-SLYRIK SKKKSLYRIKYILRLSCART SKKKSLYRIK SKKKSLYRIK SKKKSLYRIK SIN SQKKSLYRIK YILRLSCART EUS FOR FOTSHASKKKSLYRIKYILRLSCART SKKKSLYRIK PAP PPS SKKKSLYRIK SYS SQKKSLYRIK HVI SKKKSLYRIK YILRLSCART DIS DIRSQKKSLYRIK RHO LSI HVE SKKKSLYRIK DIC SKKKSLYRIK DOT SQKKSLYRIK CSP SQKKSLYRIK LOR Appendix 2.2 Continued LFO MAI MAT MOL MYT EXB CSI NEOSKKKSLYRIKYILRLSCARTOSTSKKKSLYRIK ALT NOASKKKSLYRIK

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DLANHE. [504 IWYLDIVCIN 490 490 500 DIC STSRRLYRGR IWYFDIISINDLANHE. [505 OST STSRRLYRGR IWYFDIISINDLANHE. [505 SIN STSRRLYRGR IWYFDISSINDLANHE. SHA [504 PAP STSRRLYRGR SYS IWYFDIISINDLANHE. STSRRLYRGR IWYFDIISINDLANHE. STSRRLYRGR [505 IWYFDIISINDLANHE. [505 HVI [505 HVE STSRRLYRGR IWYFDIISINDLANYE. STSRRLYRGR IWYFDIISINDLANYE. [505 [505 EUS STSRRLYRGR FOR IWYFDIISINDLANHE. [505 STSRRLYRGR FOT IWYFDILSINDLANHE. PPS [505 STSRRLYRGR IWYFDIISINDLANHE. STSRRLYRGR IWYFDIISINDLANHE. [505 [505 DIR DOT STSRRLYRGR IGYLDIISINDLANHE. STSRRLYRGR IWYFDIISINDLANHE. [505 NEO [505 NOA STSRRFYRGR IWYFDIISISDLANHE. STSRRLYRGR IWYFDIISINDLANHE. [505 [505 DIS SSLRRLYRER IWYFDIICINDLANHE. [502 CSP STSQRLYRGR IWHFDIISINDLANQE. [505 LFO STSRRLYRGR IWYLDIICINDLANHE. [504 RHO STSRRLYRGR CSI STSQRLYRGR LSI IWHFDIISINDLANQE. MAT [505 FTSQRLYRGR MAI IWY-DIISINDLANQE. STSQRLYRGR IWYFDIISINDLANQE. [505 STSQRLYRGR MOL IWYFDIISINDLANQE. [505 [504 STSRRLYRGR IWYFDIISINDLANHE. [507 EXB STSRRLYRGR IWYLDIICINDLSNHE. [504 ALT STSRRLYRGR LOR IWYLDIICINDLANHE. [504 STSRRLYRGR ILYLDIICINDLANHE. [504 MYT STSRRLYRGR IWYFDIICINDLANHE. [505 Appendix 2.2 Continued

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III

PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE INFERRED

FROM SEQUENCES OF INTERNAL TRANSCRIBED SPACERS

OF NUCLEAR RIBOSOMAL DNA

INTRODUCTION

Using sequence variation between homologous genes has

become an increasingly routine method of molecular

systematics to reconstruct the phylogenetic relationships of

a taxon, although it is still controversial concerning

whether the phylogenetic trees obtained via cladistic

analysis are species trees or just gene trees (Doyle, 1993).

Many studies have been conducted on phylogenies of organisms

at various hierarchical levels, from populations to

divisions, using single gene sequences (Chase et al., 1993).

Most studies suggested similar relationships to those using

other lines of evidence such as morphology and anatomy

(Donoghue and Sanderson, 1992; Chase et al. 1993). As

expected, discrepancies exist as well, indicating where

future research should be concentrated.

It has been well known that different genes evolve at

different rates (Gaut et al., 1992). Thus, finding

appropriate genes with meaningful substitution rates for

assessing phylogenetic relationships at various levels has

149

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. been one of the frontiers in molecular systematics. It is

often the first step for a thorough study of phylogenetic

relationships of a group of organisms.

One of the widely used segments of DNA sequence is the

Internal Transcribed Spacer (ITS) region of 18S-26S nuclear

ribosomal DNA (nrDNA) (Fig.3.1). This region includes three

components: two spacers designated ITS-1 and ITS-2 and the

5.8S subunit intercalated between the two spacers. The ITS

region is part of the transcriptional unit of nr DNA, but the

spacers of the transcript are not incorporated into mature

ribosomes. However, the spacers probably function in the

maturation of nrRNAs (Musters et al., 1990; van der Sande et

al., 1992; van Nues et al., 1994). Therefore, ITS-1 and

ITS-2 are under some evolutionary constraints in structure

and sequence (Baldwin et al., 1995). Lower length variation

and a relatively high base substitution rate indicate that

DNA sequences of these spacers might be readily alignable

across closely related taxa, yet be sufficiently variable to

allow resolution of lower-level phylogenetic questions in

angiosperms (Sytsma and Schaal, 1985, 1990; Jorgensen and

Cluster, 1988; Yokota et al., 1989; Kim and Mabry, 1991;

Baldwin, 1992).

There are three advantages for using the ITS region in

phylogenetic analyses of angiosperms. First, along with

other components of the nrDNA multigene family, the ITS

region is highly repeated in the plant nuclear genome. The

150

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. entire nrDNA repeat unit is present in up to many thousands

of copies at a chromosomal locus or at multiple loci (Rogers

and Bendich, 1987; Hamby and Zimmer, 1992). The high copy

number promotes detection amplification, cloning and

sequencing of nrDNA (Baldwin et al., 1995). Second, albeit

many-copied, the sequences of the spacers do not vary much

due to intraspecific homogenization by virtue of concerted

evolution (including unequal crossing over and gene

conversion) (Zimmer et al., 1980; Dover, 1982; Hillis et al.,

1991). Homogenization can even affect sequences of nrDNA of

non-homologous chromosomes (Arnheim et al., 1980; Wendel et

al., 1995). This property systematically promotes accurate

reconstruction of species relationships from the sequences

(Hamby and Zimmer, 1992; Sanderson and Doyle, 1992). Third,

due to the small size of the ITS region (<700 bp in

angiosperms) and the presence of highly conserved sequence

flanking each of the two spacers, it is relatively easy to

amplify the region using universal eukaryotic primers

designed by White et al. (1990) (Fig.3.1).

Although this ITS region is mostly used for

phylogenetic study of interspecific relationships, many

studies have also suggested its informativeness for resolving

intergeneric relationships of closely related taxa (Baldwin

et al., 1995; Bogler and Simpson, 1996; Downie and Katz-

Downie, 1996; Schilling and Panero, 1996; Soltis et al.,

1996; Kron and King, 1996).

151

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. As mentioned in the Introduction Chapter, the

relationships among the genera of the Hamamelidaceae have

been controversial for more than a century (Figs.i.2-i.6).

The objective of the research reported here, therefore,

is to study the phylogenetic relationships among the

hamamelidaceous genera using DNA sequences of the ITS region.

MATERIALS AND METHODS

Plant Material

Material of 32 species was used in this study (Table

3.1), representing 28 of the total 31 genera in the

Hamamelidaceae. Unfortunately, three genera were not

available for analysis: Chunia, Semiliouidambar. and

Embolanthera. Vouchers for the sampled species are deposited

in the Hodgdon Herbarium of the University of New Hampshire

(NHA). Sequences of both ITS-1 and ITS-2 have been submitted

to the GenBank and the accession numbers are given in Table

3.1. The aligned ITS sequences are shown in Appendix 3.1.

Molecular Techniques

Total genomic DNAs were extracted from fresh or silica

gel dried leaves or buds following the protocol of Doyle and

Doyle (1987). Polymerase Chain Reaction11*1 (PCR) was conducted

in 0.2 ml thin-walled microcentrifuge tubes. Each 50 ^1

reaction included 50-100ng DNA, 5pl of Tag extender buffer

152

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Stratagene, CA), 4 fjl of 2.5 jjm dNTP, 1-4 units of Tag

extender (Stratagene, La Jolla, CA) and Tag polymerase

(Promega, WI), 1 gl of 20pm primers, and an appropriate

amount of UV-treated deionized water.

The PCR reaction was conducted with a three-minute 'hot-

start' (D'aquila et al., 1991). Tag polymerase and Tag

extender were added into the reaction tube at the end of the

hot-start, when the temperature was stabilized at 80°C. Then,

30 cycles of PCR reaction were started following the

thermocycler program of 105 seconds at 45°C, 115 seconds at

72°C and 30 seconds at 94°C. The last cycle ended at 72°C for

15 minutes of extension. The PCR primers were universal ITS4

and ITS5 of White et al. (1990).

The PCR products were loaded on 0.8% LMP (Low Melting

Point) agarose gel along with 0X174 Haelll DNA size markers

and separated for 2-3 hours at 40 volts in 0.5X TBE buffer.

The band identified by comparison to the markers was then

excised from the gel, liquified at 650C, and treated with

agarase (Sigma Chemical Co., St. Louis, MO) for 30 minutes at

37°C. This gel-purified PCR product was used directly as a

sequencing template. Sequencing reactions were carried out

using Cycle Sequencing Kit, FS (catalog#: 402119) and

following the manufacturer’s protocols (Applied Biosystems,

Foster City, CA).

The sequencing primers were ITS2, ITS3, ITS4 and ITS5 of

White et al. (1990). The cycle sequencing products were then

153

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. separated on 6% polyacrylamide gel using an Automated

Sequencer 373A (Applied Biosystems, Foster City, CA) in the

Sequencing Facility Center of the University of New Hampshire

(UNH).

For Tetrathyrium and Maingaya, 0.8% DMSO

(Dimethylsulfoxide) was added to both the PCR and cycle

sequencing reactions.

For Liquidambar formpsana, when the PCR product was used

directly as a template for sequencing, I could not acquire

reliable sequences. Therefore, the standard pBiuescript gene

clone technique (Promega, Madison, WI) was used to clone the

PCR product of the ITS region. The PCR primers are ITS4 and

ITS5 of White et al. (1990), with BamHl and EcoRl restriction

sites added to their 5' ends respectively. Cycle sequencing

procedures sure as mentioned above. Sequence from a single

clone of the ITS region was used in this analysis.

The chromatograms were analyzed using the SEOED program

(Applied Biosystems, Foster City, CA). Also, in order to

assure correct base-calling, I overlapped sequences generated

from adjacent primers of either the same or opposite

directions. The boundaries of ITS-1 and ITS-2 were

determined by comparing sequences of the 3 ’ 18S, 5.8S and the

5 1 end of the 26S ribosomal genes of Canella winterana

(GenBank accession number L03844)

154

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequence Alignment.

The ITS sequences within a genus and among closely

related genera were easily aligned by sight. However, ITS

sequences among genera of different subfamilies were not

readily alignable, thus resulting in some ambiguous regions.

According to some authors, these ambiguous sites, as

identified by alignability by eye, were eliminated from the

data matrix before a phylogenetic analysis was conducted

(Downie and Katz-Downie, 1996). This "culled" method tends

to create clades where internal relationships are not well

resolved (Wheeler et al., 1995; Soltis et al., 1996a).

Wheeler et al. (1995) proposed a method called the

"Elision" approach for the alignment of ambiguous DNA

sequences. Alignments change with gap penalty (GP) and gap

length penalty (GLP), thus, setting different GPs and GLPs

produces various alignments. The resulting aligned sequences

using different penalties are then strung together, giving

rise to a combined, grand data set. Elision is essentially a

fine character weighting scheme based on character

consistency in the sets of alignments (Wheeler et al., 1995).

Another way of dealing with alignments with ambiguous

sites is to select a so-called optimal alignment. This

approach involves consistency indices of the aligned

sequences and the phylogenetic trees produced based on the

data. Bogler and Simpson (1996) used this "optimality"

method to analyze the phylogenetic relationships of the

155

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Agavaceae. Many indices can be used to assess the optimality

of sequence alignment, including the number of trees

generated, the Consistency Index (Cl), Retention Index (RI),

and Rescaled Consistency Index (RC). Bogler and Simpson

(1996), however, implied that the higher the RC was, the

better the alignment. This seems to be reasonable because

the RC index excludes characters that do little to the 'fit'

of the tree but inflate the Cl (Wiley et al., 1991).

In this study, both the Elision and Optimality methods

were employed to generate data matrix of the ITS sequences of

the Hamamelidaceae. For the Optimality method, the alignment

that created trees with the highest RC index was considered

as optimal for both ITS-1 and ITS-2. When two or more

alignments having equally highest RC were encountered, the

one with the highest Cl was chosen. For the Elision method

the procedures were carried out following Wheeler et al.

(1995), and Soltis et al. (1996a). Individual alignments

were conducted using the CLUSTAL option of the MEGALIGN

program of DNA* software package (DNA* Inc., CA).

Phylogenetic Analysis

The resulting data matrices from both alignments

(Optimality and Elision) were imported into the beta-test

version of PAUP* 4.0d57 computer program, written by David L.

Swofford (1997) at the Smithsonian Institution, for

phylogenetic analyses.

156

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Indels were treated as missing data because this coding

strategy retains information about substitutions that occur

in other taxa in the indel region. However, it does not

convey the information regarding the evolutionary event

involved in the insertion or deletion, and thus can introduce

ambiguities (Platnick et al., 1991; Wojciechowski et al.,

1993). To compensate for the disadvantage, attention was

paid to the evolutionary significance of some unambiguous

indels.

All characters and their states were equally weighted in

both parsimony and distance analyses. Sequence divergence

was analyzed using the pairwise difference obtained from

PAUP*.

Due to the size of the data set and the limitation of

computer memory, the heuristic search option was used to find

the shortest trees with TBR (Tree Bisection and Reconnection)

branch swapping, mulpars on, and steepest descent off.

In the parsimony analysis, Altinoia was used as the

outgroup because: 1) both sequence divergence (Table 3.2) and

cluster analysis of the ITS sequences (UPGMA, Fig.3.2)

indicated that Altinoia. along with Liquidambar. was rather

distinct from other Hamamelids; 2) the fossil record has

shown that Altinoia is the most ancient member in the

Hamamelidaceae (Zhang and Lu, 1995); and 3) both morphology

and matK gene phylogenies (See Chapter I and II) suggested

that Altinoia. together with Liquidambar. is the basal taxon

157

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the Hamamelidaceae.

Both bootstrap and decay analyses were conducted using

the PAUP program to test the relative strength of putative

clades (Felsenstein, 1985; Bremer, 1988; Donoghue et al.,

1992).

MacClade 3.03 (Maddison and Maddison, 1992) was used to

trace unambiguous changes along branches and to compare

competing hypotheses concerning the relationships.

The skewness test (Huelsenbeck, 1991) was implemented

using the Random tree option of the PAUP*, and 10,000 random

trees were examined to evaluate the phylogenetic information

contained in ITS data matrix. Also, the permutation test

(Faith and Cranston, 1991; Plunkett et al., 1997) was

performed using the Permutation option of the PAUP* with 100

replicates and heuristic searches.

RESULTS

Sequence Alignment

Among the seven pairs of alignment parameters, GPs/GLPs,

including 8/8, 8/15, 10/20, 10/10, 15/8, 20/8, and 20/20, the

pair of 8/8 resulted in the alignment of the highest RC index

for ITS-1 (Table 3.3) and that of 20/20 produced the optimal

alignment for ITS-2 (Table 3.4).

158

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■Sequence Characteristics

The lengths (bp) of spacers 1 and 2 range from 236 to

277 and from 224 to 241 respectively in the Hamamelidaceae

(Table 3.5), and the means are 267 bases for ITS-1 and 236

for ITS-2. Several genera have roughly equal length of ITS-1

and ITS-2 including Fortunearia. Eustioma. Molinadendron. and

Sinowilsonia. GC content ranged from 58% to 67% with an

average of 63% in ITS-1, and from 61% to 68% with an average

of 65% in ITS-2. Statistical analysis (Chi-square test)

showed that the differences of nucleotide composition in both

ITS-1 and ITS-2 across taxa, and between the two spacers were

not significant with P>0.9.

Pair-wise sequence divergence in ITS-1 was generally

higher than that in ITS-2, with averages of 18.1% and 16.5%

respectively. Pair-wise divergence between the two genera of

Liquidambaroideae and the other member of the Hamamelidaceae

was higher than 30% for both spacers. The percentages of

potentially informative sites were 52% and 51% for ITS-1 and

ITS-2 respectively (Table 3.5).

The alignment of the sequences required 45 and 31 indels

in ITS-1 and ITS-2 respectively (Table 3.5). in both

spacers, about 70% of the indels were a single base in

length. There were three indels of 10 or more bases in the

ITS region: two indels (12 and 26 bases) were in ITS-1, and

one (10 bases) in ITS-2.

The parsimony analysis using the Optimality data of ITS-

159

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 and ITS-2 yielded 90 shortest trees of 1041 steps, with a

consistency index of 0.59. Figure 3.3 shows the strict

consensus tree. Liquidambar is the first taxon to branch

following the outgroup Altincria. Mytilaria. Exbucklandia.

and Rhodoleia form a clade, while Disanthus is sister to the

clade containing the sampled genera of the Hamamelidoideae.

In the Hamamelidoideae, three major clades are recognized:

the Corylopsis clade, Hamamelis clade, and the Trichocladus

clade, among which the Corylopsis clade is basal. Both

Corylopsis and Hamamelis clades are strongly supported,

whereas the Trichocladus clade is weakly supported (Fig.3.3).

The Corylopsis clade consists of two subclades, the

first of which is comprised of three species of Corylopsis:

the second includes Loropetalum, Tetrathyrium. Maingaya, and

Matudaea. Both subclades receive strong support of a 100%

bootstrap and a decay index of more than five steps.

In the Trichocladus clade, four branches are recognized:

Dicoryphe. Trichocladus. the Eustigmateae sensu Endress

(1989b) (incl. Molinadendron). and the three Australian

genera. However, in the 50% majority consensus tree, the

five Southern Hemisphere (Gondwanaland) genera (Dicoryphe.

Trichocladus. Ostrearia. Neostrearia. and Noahdendron) form a

single clade (Fig.3.4). Among the three Australian genera,

Neostrearia is basally sister to the latter two genera

(Figs.3.3, 3.4).

In Fig.3.3, Molinadendron and Sinowilsonia are bound

160

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with a bootstrap value of 84% and a decay index of three

steps.

In both Fig.3.3 and 3.4 the Hamamelis clade is

essentially the Fothergilleae sensu Endress (1989c), plus

Hamamelis. In Fig.3.3 there are four clades whose

relationships are not well resolved, including Hamamelis,

Fotherqilla, Parrotiopsis. and the well-supported Distylium

group consisting of Distylium. Distyliopsis. Parrotia,

Shaniodendron. and Sycopsis. However, in Fig.3.4 Fotherqilla

is the basal clade, followed by the clades of Hamamelis

species, Parrotiopsis. and the Distvlium group.

Twelve most parsimonious trees of 6747 steps were

generated based on the grand data set from the Elision

alignments. The strict consensus tree (Fig.3.5, CI=0.578) is

largely congruent with the phylogeny that was generated using

the Optimality alignment data. However, the subtribe

Dicoryphinae of Endress (1989c) forms a clade and so does

Mytilaria in the Elision phylogeny.

DISCUSSION

Sequence Characteristics

In angiosperms ITS-1 and ITS-2 are each less than 300

bases long: ITS-1 ranges from 187 to 298bp and ITS-2 from 187

to 252bp (Baldwin et al., 1995; Johnson and Soltis, 1995;

Downie and Katz-Downie, 1996; Padgett, 1997). The length of

161

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the two spacers in the Hamamelidaceae falls within the above

range, but ITS-1 sequences in most of the species sampled are

in the high end of the range, except for the four genera

(Eustigma, Fortunearia. Molinadendron. and Sinowilsonia) that

share two unique deletions (12 and 26 bases). The ITS-2 in

the Hamamelidaceae is relatively less variable in length,

mostly in the range of 224 to 241, which agrees with a

generalization that ITS-2 is more conservative than ITS-1

(Hershkovitz and Lewis, 1996; Liston et al., 1996).

A permutation test for the data sets from both the

Elision and the Optimality alignments resulted in a

probability of 0.01, indicating that the ITS data are

significantly phylogenetic, and supporting the

appropriateness of both alignments. This is consistent with

the skewness of tree length distribution (Table 3.5).

Monophvly of the Altinaioideae

When Altinoia was used as an outgroup, the first taxon

following Altinoia was Liquidambar. indicating a close

relationship between the two genera. In the UPGMA tree

(Fig.3.2), Altinoia and Liquidambar formed a clade sister to

a clade consisting of all the other taxa of the

Hamamelidaceae. Additionally, there were some 80

substitutions between the Altingioideae and the other

hamamelidaceous taxa (Fig.3.6). Therefore, this study

strongly suggests that the Altingioideae are monophyletic.

162

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It has been proposed that the Altingioideae be raised to

the family status (Melikian, 1973afb; Huang and Li, 1982;

Wang, 1992; Takhtajan, 1997). However, recent molecular

studies support inclusion of the Altingioideae in the

Hamamelidaceae (Hoot and Crane, 1996; Hibsch-Jetter and

Soltis, 1996; Hoot et al., 1997). Moreover, many

morphologically diagnostic apomorphies found in the

Altingioideae are also represented in other hamamelidaceous

genera (Pan et al., 1990; Zhang and Lu, 1995). Thus, the

Altingioideae are retained in the Hamamelidaceae as a

distinct subfamily.

Polvphvletic Exbucklandioideae sensu Endress

The Exbucklandioideae sensu Endress (1989c) includes

four genera, Chunia, Disanthus, Exbucklandia. and Mytilaria.

The morphological synapomorphies of this subfamily are large,

persistent stipules, palmate venation, and 4-6 ovules per

carpel. However, the phylogenetic analysis based on the ITS

DNA sequences provided a different picture of this taxon

(Chunia was not available for this study). Exbucklandia was

grouped with Rhodoleia, and Disanthus forming its own clade,

While Mytilaria was either allied with the clade of

Exbucklandia and Rhodoleia (Fig.3.3), or constituted its own

clade. This indicates that the Exbucklandioideae sensu

Endress (1989c) are not monophyletic. Furthermore, when the

three genera (Disanthus, Exbucklandia. and Mytilaria) were

163

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. forced into a monophyletic group, 34 more steps were

required.

Disanthus and Mytilaria have been previously recognized

as subfamilies Disanthoideae and Mytilarioideae, respectively

(Harms, 1930; Chang, 1948, 1973, 1979). Mytilaria formed its

own clade in the Elision phylogeny (Fig.3.5, but not in the

Optimality phylogeny (Figs.3.3, 3.4). However, the alliance

of this genus with the clade of Exbucklandia-Rhodoleia in the

Optimality tree was not well-supported. Moreover, separating

Mytilaria from the Exbucklandia-Rhodoleia group required only

one more step, while putting Mytilaria into the clade of the

Exbucklandia-Rhodoleia group entailed 13 more steps than the

shortest length. Thus, isolation of Mytilaria from

Exbucklandia and Rhodoleia is more parsimonious. Therefore,

this study recognizes the isolated position of both Disanthus

and Mytilaria in the Hamamelidaceae as individual

subfamilies.

Disanthus is the immediate sister taxon to the

Hamamelidoideae in the phylogenetic trees (Figs.3.3, 3.4,

3.5), reminiscent of the conclusions from a previous

morphological analysis (Hufford and Crane, 1989; Pan et al.,

1991). However, the intermediate systematic position of

Disanthus between the Exbucklandia-Rhodoleia clade and the

Hamamelidoideae in the ITS-based tree did not provide

evidence for the hypothesis that this genus is most primitive

in the Hamamelidaceae, as has been suggested by some authors

164

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Reinsch, 1889; Tong, 1930; Cronquist, 1981; Pan et al.,

1991; Takhtajan, 1997).

Exbucklandia-Rhodoleia Clade

Exbucklandia has been placed in its own subfamily

(Harms, 1930; Chang, 1979), or in a subfamily that includes

Chunia, Disanthus, and Mytilaria (Endress, 1989c). However,

the present phylogenetic analysis strongly supports a close

relationship of Exbucklandia with Rhodoleia. These two

genera also share 22 unambiguous base substitutions

(Fig.3.6). Interestingly, this pattern agrees with an

earlier taxonomic treatment (Reinsch, 1889). Nevertheless,

these two genera are rather different in both vegetative and

reproductive structures, suggestive of an ancient divergence.

Therefore, it seems reasonable to continue to treat them as

belonging to individual subfamilies (Harms, 1930; Chang,

1973, 1979; Endress, 1989b, c).

Monophyly of the Hamamelidoideae

It has been widely recognized that the Hamamelidoideae

are a monophyletic group (Bogle, 1968; Endress, 1989a, b, c;

Hufford and Crane, 1989). The present analysis adds strong

evidence to this hypothesis because the clade of this

subfamily receives a 97% bootstrap value and a decay index of

more than six steps, and is further supported by 17

unambiguous base substitutions (Fig.3.6).

165

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The intergeneric relationships within the

Hamamelidoideae, however, have been contentious (Harms, 1930;

Schulze-Menz, 1964; Endress, 1989b, c). The relationships

among the five tribes recognized by Harms (1930) and more

recently revised by Endress (1989c) and others, are discussed

below.

Corvlops ideae

The concept of the tribe Corylopsideae has been revised

several times in the past decades. Harms (1930) delimited

Corylopsideae as including Corylopsis and Fortunearia. while

Schulze-Menz (1964) added Sinowilsonia to this tribe.

However, Endress (1989b,c) considered Corylopsideae as

containing a single genus, Corylopsis.

In the ITS-based phylogenies (Figs.3.3, 3.5), the three

species of Corylopsis sampled formed a well-supported clade

and were sister to the branch comprised of Loropetalum,

Tetrathyrium. Mainaaya. and Matudaea, while Fortunearia and

Sinowilsonia are phylogenetically distant from Corylopsis.

Therefore, Corylopsis is not closely related to either

Fortunearia or Sinowilsonia.

Hamamelideae

In Harms's system (1930), all of the genera in the

Hamamelidoideae that have strap-shaped petals were placed in

the tribe Hamamelideae and no further divisions were

166

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. attempted. Endress (1989c), however, recognized three

subtribes in the Hamamelideae, including the Hamamelidinae,

Loropetalinae, and the Dicoryphinae. The Hamamelidinae

contained a single genus, Hamamelis: the Dicoryphinae

included the five Southern Hemisphere (Gondwanaland) genera

(Dicoryphe. Trichocladus. Ostrearia, Neostrearia. and

Noahdendron): while the Loropetalinae contained the remaining

four genera (Loropetalum. Embolanthera. Maingaya, and

Tetrathyrium).

The analysis of the ITS data placed the genera of the

Hamamelideae into three different clades (Figs.3.3, 3.5),

suggesting that the tribe is polyphyletic. Nevertheless, the

Dicoryphinae was suggested to be monophyletic in both the

majority consensus tree of the Optimality analysis and the

strict consensus tree of the Elision analysis. A

monophyletic origin agrees with the exclusive Gondwanaland

distribution and the unique pattern of anther dehiscence of

these five genera (Endress, 1989a, b, c).

Eustiqmateae

The delimitation of the Eustigmateae has expanded from

monogeneric (Harms, 1930) to trigeneric (Endress, 1989c).

The small, auriculate petals and greatly enlarged stigmatic

surfaces earned Eustiama a tribal status (Harms, 1930).

However, the similarities of Eustiama with Fortunearia in

other morphological characters such as pedicellate,

167

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lenticellate fruits, and covering sepals led Endress (1989c)

to the belief that Eustigmateae should include Fortunearia

and Sinowilsonia.

The ITS-based trees (Figs.3.3, 3.5) offer strong support

for Endress's hypothesis, and further corroborates the

implication (Endress, 1967) that Molinadendron and

Sinowilsonia are closely related. Interestingly, these four

genera share four unambiguous base substitutions (Fig.3.6)

and two unique deletions totaling 36 bases (Li et al.,

1997c).

Combination of Distylieae and Fotherqilleae

In the ITS-based phylogeny, one of the three major

lineages in the Hamamelidoideae includes the clade of

Hamamelis and the branch containing the Distylieae

(Distylium. Distyliopsis. and Svcoosis) and Fothergilleae

sensu Harms (1930) (Fotherqilla. Parrotia, and Parrotiopsis)

(Figs.3.3, 3.5). Neither Distylieae nor Fothergilleae forms

a monophyletic clade. Instead, a close but unresolved

relationship is discerned among Sycopsis. Parrotia, and

Shaniodendron. This agrees with the finding that Svcopsis

and Parrotia are interfertile, giving rise to the hybrid

taxon, XSvcoparrotia (Endress and Anliker, 1968). Thus, the

Fothergilleae sensu Endress (1989c) is recognized.

The apetalous genera of the Hamamelidoideae have long

been considered as monophyletic (Harms, 1930; Endress,

168

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1989c), but the ITS-phylogenies (Figs.3.3, 3.5) suggest that

loss of petals in this subfamily has evolved at least three

times independently: Matudaea in the Corylopsis clade,

Mo1inadendron in the Trichocladus clade, and the

Fothergilleae in the Hamamelis clade. Therefore, the

apetalous genera do not belong to a single monophyletic

group, and apetaly is homoplasious in this family.

Fotherqilla and Parrotiopsis each form their own clades

in the ITS phylogenies (Figs.3.3, 3.5). This is consistent

with the showy specialized structures they have evolved for

insect pollination: long, white, and clavate stamen filaments

in Fotherqilla. and large, white subfloral bracts in

Parrotiopsis (Bogle, 1970; Endress, 1989a). These

specializations do not occur in any other members of the

Fothergilleae sensu Endress (1989c) or in the Hamamelidaceae.

It is a novel relationship that Hamamelis is grouped

with Fothergilleae sensu Endress (excl. Matudaea and

Molinadendron). Interestingly, this pattern finds support

from leaf venation (Harms, 1930; Chang, 1979; Li and Hickey,

1988). The systematic position of Hamamelis is not well

resolved in the ITS phylogenies (Figs.3.3, 3.5), however,

this genus differs greatly from the Fothergilleae in a group

of floral characters, including showy, strap-shaped petals,

bisexuality, nectariferous phyllomes, and insect pollination

(Endress, 1989a). Therefore, it should be isolated from the

Fothergilleae in taxonomic treatments.

169

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In summary, at the subfamily level, this study

recognizes the monophyly of Liquidambaroideae,

Exbucklandioideae, Mytilarioideae, Rhodoleioideae,

Disanthoideae, and Hamamelidoideae. Furthermore, it suggests

a close relationship of Exbucklandia with Rhodoleia, and the

paraphyly of Exbucklandioideae sensu Endress (1989c). At the

tribal and subtribal levels, the monophyletic groups are the

Corylopsideae sensu Endress (1989c), Eustigmateae sensu

Endress (1989c, incl. Molinadendron). Fothergilleae sensu

Endress (1989c, incl. Hamamelis), Dicoryphinae, and

Loropetalinae (incl. Matudaea). The Hamamelideae, however,

is polyphyletic. The ITS phylogeny further suggests that

some morphological characteristics commonly utilized to

classify the Hamamelidoideae have originated several times

independently. These characteristics include strap-shaped

petals, apetaly, monoecy, and wind pollination. Therefore,

whenever a classification system is attempted, this homoplasy

should be taken into account so that natural relationships

are reflected.

170

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U65464 U65465 AF015425-26 U65462 AF015423-24 AF015656-57 U65463 AF015436

campus Park, Hawaii Park, Hampshire New of Univ. campus Hampshire New of Univ. Taiwan U65466 Tulear, MadagascarTulear, AF015419-20 China AF015417-18 greenhouse. Gard. Bot. Missouri AF015427-28 Woodlanders, Inc. SC.Inc. Woodlanders, AF015421-22 Roadside State Manuka MA.Arboretum, Arnold U65467 Arnold Arboretum, MA. Arboretum, Arnold MA.Arboretum, Arnold AF015655 BOGLE SC. Inc. Woodlanders, BOGLE BOGLE 04 LI BOGLE SC. Inc. Woodlanders, BOGLE LI 01LI MA. Arboretum, Arnold QIU 03 LI BOGLE Hampshire New of Univ. LI LI 02 MA.Arboretum, Arnold U65461 J.-H. LI J.-H. LI J.-H. L. A. A. L. A. A. L. A. FOT J.-H.HVI LI DIR FOR J.-H. EXB DIC 543 Randrianasolo A. DOTDIM L. A. BOGLE L. A. SC.Inc. Woodlanders, AF019231-2 HVE DIS Taxon Taxon CPA J.-H. GenBank EUS N.-J. CHUNG CPA J.-H. CSI LSI L. A. LFO L. A. ALT Y.-L. Fothergilla majorFothergilla Hamamelis virginiana Corylopsis pauciflora Corylopsis Corylopsis Corylopsis sinensis Distyliumracemosum Fortunearia sinensis Distyliopsis Distyliopsis tutcheri myricoides Distylium Disanthus Disanthus cercidifolius Species Species Corylopsis spicata Abbr. Voucher and Collector Source populnea Exbucklandia Accessions Dicoryphe stipulacea Eustigma oblongifolium Hamamelis vernalis Liquidambar formosana Loropetalum sinense Altingia excelsa Table 3.1. Species sequenced for nrDNA ITS for this analysis.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

U65469 AP015439 AF015441 AFO15443 AF015442 AP015429-30 U65468 AF015438 AP015440 AF015431-32 AF015433-34 AF019233 AF022241 AF015437 AF022242 AF015435

Zurich, Switzerland Zurich, Zurich, Switzerland Zurich, Switzerland Zurich, Zurich, Switzerland Zurich, greenhouse. Zurich, Switzerland Zurich, Botanical Garden of Garden Botanical Univ. of New Hampshire New of Univ. Harvard Univ. campus Univ. Harvard Botanical Garden of Garden Botanical Botanical Garden of Garden Botanical Jiangsu, China Jiangsu, Kong Hong Kepong, Malaysia Kepong, Garden, Botanical China Guangxi, of Garden Botanical Lyon Arboretum, Hawaii Arboretum, Lyon MA Arboretum, Arnold SC. Inc. Woodlanders, PA. Gardens, Longwood

Z.-C. LUO Z.-C. P. K. ENDRESS K. P. ENDRESS K. P. ENDRESS K. P. ENDRESS K. P. QIU Y.-L. LI 05 J.-H. P. K. ENDRESS K. P. BOGLE L. A. BOGLE L. A. Saunders R. L. G. SAW G. L. BOGLE L. A. BOGLE L. A. BOGLE L. A.

PPS MOL TET SHA

NOA OST TRI NEO MYT RHO SIN MAT SYS MAI PAR Tetrathyrium subcordatum Tetrathyrium crinitus Trichocladus Shaniodendron Shaniodendron subaequale henryi Sinowilsonia Sycopsis sinensis Ostrearia Ostrearia australiana Molinadendron Molinadendron guatemalense Neostrearia fleckeri Noahdendron nicholasii Parrotia persica Parrotia Parrotiopsis Parrotiopsis jacquemontiana Rhodoleia championi Matudaea Matudaea trinervia Mytilaria laosensis Table 3.1. Continued malayanaMaingaya - j N)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2. Sequence divergence of ITS-1 and ITS-2 (below and above diagonal) in the Hamamelidaceae. The values are derived from pairwise distances, calculated in PAUP* (mean distance x 100). Taxon abbreviations as in Table 3.1.

Taxon CPA CSI CSPDIS DICDIMDIRDOTEUSEXBFOR CPA - 3 3.9 24.9 16.7 15.4 15.8 15.1 15.1 19.5 14.1 CSI 4.1 - 0.9 23.5 15.7 14 14 13.3 14.1 18.5 13.6 CSP 3.7 2.2 - 24 16.6 14.9 14.9 14.1 15 19 14.4 DIS 24.4 26.3 26.2 - 25.1 23.4 23.3 22.5 25.3 28.1 25.2

DIC 14.9 16.8 17.5 24.3 - 11.6 10.7 9.8 8.1 22.9 5.9 DIM 15.3 17.3 17.2 23.6 11.2 - 1.7 2.6 9.9 19.4 8.2

DIR 15.3 17.2 17.2 24 12 1.1 - 1.7 9.1 19.7 8.6 DOT 15.6 17.6 17.5 23.7 12 0.7 0.4 - 8.3 20.3 8.6 EUS 10.9 13.7 13.6 23.2 10.8 8 8.5 8.4 - 21.1 6 EXB 30.6 32.8 32.7 31.9 30.1 30.4 30.6 30.8 28.6 - 20.6 FOR 12.9 14.9 14.9 22.7 9.4 8 8.4 8.4 3.4 28.1 - FOT 14 16.4 16 22.9 11.9 5.3 5.7 5.7 7.6 30.4 8.4 HVE 14.5 16.5 16.4 22.1 11.1 3.7 4.1 4.1 7.7 30.5 7.7 HVI 15.6 17.9 17.9 23.6 11.9 4.5 4.9 4.9 8.5 30.9 8.5 LSI 11.5 12.5 12.8 26.4 18 17.7 17.7 17.7 15.7 30.7 16.8 MAI 15.2 15.8 15 27.1 17.4 18.3 18.6 18.7 17.5 31.7 16.8 MAT 13.7 13.5 13.5 26 18.7 19.6 19.6 19.6 17 30.8 17.3 MOL 12.2 15 14.9 24.4 10.2 8.4 8.4 8.4 3.4 27.7 5.5 MYT 30 32.2 32.1 29.6 28.8 28.5 27.9 28.2 26 25.9 23.8 NEO 13.8 17.2 16.8 25.1 13.1 11.9 12.3 12.3 6.4 31 7.1 NOA 12.9 15.7 15.3 24.8 10.7 11.2 11.5 11.5 7.3 30.9 8 OST 11.6 14.9 14.5 25.1 10.4 10.4 10.8 10.8 7.7 32.5 8.5 PAR 16.8 18.7 18.6 24.8 12 1.9 3 2.6 8.6 29.7 8.4 PPS 16 17.9 17.9 23.6 13 6.3 7.1 7.1 8.5 29 9.3 RHO 28.7 30.8 31.5 28.8 28.2 30.1 29.4 29.7 27.2 19.1 25.7 SHA 16 18.3 18.3 23.6 10.8 2.2 3.3 3 9.4 30.8 8.5 SIN 13 15.9 15.8 26.1 10.7 8.8 9.2 9.2 3.8 30.9 5.1 SYS 17.5 19.4 19.3 26.5 13.5 3.4 4.1 3.7 9.8 31.5 9.8 TET 11.4 13.2 12.8 25.7 16.7 16.9 17.2 17.2 15.4 27.9 16.9 TRI 15.6 16.4 16.4 25.1 11.5 11.6 11.6 11.6 9.4 32.2 9.8 LFO 32.5 34 34.3 34.8 36.2 32.1 31.3 32.1 28.2 38.9 29.1 ALT 35.5 37.4 36.9 36 35.6 32.7 32 32.8 31.4 39.3 30.5

173

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FOT HVE HVILSI MAIMAT MOLMYT NEO NOA 0ST PAR PPS 15 12 13.3 19 11.2 12.1 15.3 23.1 17.6 14 15 14.6 13.6 14 11.5 12.8 18 9.1 10.8 13.5 23.9 16.6 13 14.4 12.7 12.7 14.9 12.4 13.7 18.9 9.9 11.7 14.4 23.4 17.5 13.9 15.3 13.6 13.5 22.9 23.4 23.6 25.2 25.4 24.2 24.1 31.3 23.5 24.1 25.1 22 23.4 10.7 10.3 10 17.2 15.1 14.8 8.9 26 9.4 8.5 8.1 10.3 9.6 6.4 4.3 4.7 19.1 16.9 17.9 10.3 25.7 13.4 11.6 11.1 2.1 4.8 5.5 5.2 4.7 18.1 15.6 17 9.8 25.2 12.5 10.7 10.2 1.3 3.9 4.7 4.3 3.9 16.7 15.6 17.1 10.8 24.8 12.5 9.9 9.4 1.3 3 8.2 8.6 8.2 17.5 15.6 16.5 8.5 25.8 11.2 7.7 7.7 8.6 8.4 20.7 20.3 20.8 27.5 21.3 23.8 19.7 26.8 24.5 24.5 21.9 19.3 20.6 9.4 8.1 7.8 15.9 14.2 18.2 5.9 26.9 9.8 6.8 8.1 9 7.9 - 4.7 4.3 20.2 16.7 17.4 11.5 25.1 12.4 10.2 9.8 4.3 3.9 3.7 - 2.1 19.5 16.4 17.1 12 25.5 12.9 10.3 9.8 3.9 3.5 4.5 1.5 - 19.1 16.1 16.8 11.6 25.8 13 10.4 9.5 3.4 3 17.5 17.2 18.3 - 19.9 11.2 15.8 31.9 17.3 14.5 17.6 17.6 18.2 18.2 18.2 19.1 11.6 - 14.7 14.5 24.9 16.9 14 15.5 15.1 14.6 19 18.4 19.6 9.7 11.2 - 14.7 27.6 17.5 14.7 15.1 16.6 15.4 7.6 8.1 8.9 16.9 18.2 17.8 - 27.9 12 10.2 9.7 10.3 11.3 28.9 27.1 26.7 30.5 32.7 31 25.6 - 26.8 26.7 26.7 25.1 24.6 11.2 10.5 11.9 19.2 21 21.1 6.8 30 - 9.4 8.1 12.9 12.7 9.6 9.6 10.3 18.2 18.9 20.1 7.6 30.5 6.7 - 5.5 10.3 9.6 9.2 9.2 10 17.6 18.3 19.2 8.1 30.8 7.3 5 - 9.8 10 5.7 3.4 4.1 18.4 19.5 20 8.9 27.5 12 11.9 11.2 - 2.6 6.4 5.6 6.3 18.7 20.5 20.3 8.9 28.8 12.2 12.5 11.5 5.2 - 28.8 28.2 28.6 29.3 29.5 28.3 25.8 23.8 29.1 30.3 28.6 29.1 27 6.1 3 4.5 18 18.3 19.2 9.8 27.9 12.3 12.3 11.6 1.8 6.3 9.3 8.5 9.3 17.4 18.7 19.2 4.7 26.8 8 9 9.4 9.3 10.1 7.5 5.2 5.6 18.7 19.5 21.1 9.4 29.1 14.3 13 12.7 3.4 7.9 17.1 17.2 18.4 5.6 11.2 10.5 17 28.7 19.9 18.9 18.3 16.9 18.3 11.1 10.7 11.5 18.3 18.5 19.1 8.9 30.4 11.5 9.2 9.6 13.1 12.2 33.1 33 33 36.2 36.5 35.9 29 33.4 35.1 33.3 32.5 33.1 33.5 34 32.9 32.9 37 36.5 36.3 31.3 36.8 36.1 33.8 34.3 32.9 34

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RHOSHA SIN SYSTETTRI LFOALT 21.9 15.7 18.2 15.9 12.5 14.4 39.7 38.8 20 13.9 16.4 14.1 12 14.8 40 39.1 20.4 14.8 17.3 15 12.9 15.7 40.5 39.6 29.7 22.5 26.2 23.3 22.6 23.9 41.5 39.1 24.4 9.9 9.3 10.7 16.8 9.3 37.7 37.9 23.9 2.6 12.9 3.4 18.6 11.2 37.5 36.7 23 1.7 12.9 2.5 17.2 11.1 36 35.2 23.1 1.7 12.1 0.4 17.3 10.3 35.8 35.9 23.6 9.1 9.4 9.1 17.7 10.3 38.2 38.8 15.9 20.5 24.7 20.7 22.4 23.7 37.5 37.8 23 9.4 7.6 9.4 17.5 10.2 37.6 37.8 24.4 5.5 11.9 5.1 18.5 11.6 37.4 37.9 24 5.2 12 5.2 17.8 9.9 37.8 38.3 23.7 4.7 12.1 4.7 17.4 10.3 38.1 38.5 28 17.1 19.6 17.5 14.1 19 35.3 35 22.7 16.3 18.8 16.4 15.1 17.5 40.4 37.8 23.7 17 17.2 17.9 9.4 17.7 37.4 35.6 23 10.7 7.6 11.6 16.7 11 38.5 36.9 30.1 24.5 29.2 25.7 25.4 25.5 39.4 39.3 24.4 12.5 13.6 13.3 15.5 12.4 38.4 38.5 24.3 10.7 10.6 10.7 14.4 11 38.5 38.6 23.4 10.2 10.6 10.2 15 10.2 37.6 37.8 22.6 1.3 12.4 2.1 16.8 10.7 36.5 35.7 23.7 3.9 11.7 3.9 16.8 10.9 36.6 36.7

- 23.3 24.9 23.9 22.8 22.7 39.4 40.2 29.1 - 12.8 2.5 16.2 11.1 35.5 34.7 29.6 10.2 - 12.9 20.5 11.9 40.4 40.5

30.5 3.7 10.6 - 18.1 11.1 36.5 36.7 28.5 16.5 17.9 18.4 - 18.4 38.5 37.1 30.5 13.5 11.1 13.4 18.2 - 38.4 38.5 35 33.4 30.5 34.5 35.8 33.9 - 6.3 36.7 33.7 31.8 34.7 37 35.1 14.6 -

175

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.3. Characteristics of the most parsimonious trees obtained by PAUP heuristic searches of ITS-1 sequences aligned using different combination of gap and gap length penalties. Abbreviations: Consistency Index (Cl), Retention Index (RI), Rescaled Consistency Index (RC), number of steps (Steps), number of trees found (Trees), Number of characters in the matrix (Nchar), the aligned considered optimal is highlighted.

Penalty (Gap/gap length) Steps Trees Nchar Cl RI RC 10/10 581 90 291 0.613 0.69 0.423 15/8 582 402 291 0.612 0.695 0.425 20/20 665 1716 284 0.561 0.662 0.371 20/8 586 492 289 0.600 0.689 0.414 8/15 576 234 289 0.611 0.696 0.425 8/20 583 858 288 0.602 0.696 0.419 8/8 548 210 294 0 . 620 0.696 0.432

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.4. Characteristics of the most parsimonious trees obtained by PAUP heuristic searches of ITS-2 sequences aligned using different combination of gap and gap length penalties. Abbreviations: Consistency Index (Cl), Retention Index (RI), Rescaled Consistency Index (RC), number of steps (Steps), number of trees found (Trees), Number of characters in the matrix (Nchar), the aligned considered optimal is highlighted.

Penalty (Gap/gap length) Steps Trees Nchar Cl RI RC 10/10 435 753 260 0.556 0.623 0.347 15/8 440 180 259 0.566 0.633 0.358 20/20 480 54 251 0. 577 0.635 0.366 20/8 450 162 258 0.573 0.639 0.366 8/15 453 138 255 0.559 0.625 0.349 8/20 463 252 252 0.572 0.634 0.363 8/8 413 81 262 0.557 0.606 0.337

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64 61.5-67 471-522 1.4-38.2 Combined 168 168 (30.8) 377 377 (69.2) (ITS-1 (ITS-1 and ITS-2) 156121 365 244 235 501.8 -1.2 -1.0 ITS-2 168 168 (66.9) 45 30 75 294 251 545 266.9 ITS-1 236-277 229-241 1.5-39.3 1.7-40.5 57.5-66.6 61.3-68.2

and combined, in 32 species of the Hamamelidaceae. (gi value (gi for 10,000 random trees) -0.8 Skewness of tree length distribution Transversions (minimum)Transition/transversion 118 1.68 1.29 1.5 Sequence divergence (%) Number of potentially informative sitesNumber of constant sitesTransitions (minimum) 153 (52) 128 (51) 281 (51.5) 85 (28.9) 83 (33.1) 198 Number of variable sites 209 (71.1) G+C content mean (%) 63 64.9 Length range (bp) G+C content range (%) Number of indels Lengthmean (bp) Aligned length (bp) Table 3.5. Sequence characteristics of the two internal transcribed spacers, separated

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 3.1. The ITS region. Arrows indicate orientation and approximate positions of primer sites. Primer names and sequences as in White et al. (1990).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ITS5 ITS3

— > — > 5.8S 18S nrDNA ITS-1 nrDNA ITS-2 26S nrDNA

< - - < - ITS2 ITS4

ITS Region

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3.2. UPGMA phenogram of the Hamamelidaceae generated by analysis of nrDNA sequences aligned using the Optimality method. Jukes-Cantor's one parameter model and gamma rate (1.0) were used in the analysis. Taxon abbreviations as in Table 3.1.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT LFO CPA CSI CSP MAI MAT LSI TET DIC EUS FOR MOL SIN NEO NOA OST DIM DIR DOT PAR SHA SYS FOT HVE HVI PPS TRI DIS EXB RHO MYT

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3.3. Strict consensus of 90 shortest trees of 1041 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method. Taxon abbreviations as in Table 3.1. Numbers above and below branches are bootstrap percentages and decay values respectively. CI=0.59, and RC=0.39. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT — ALToideae CPA-1 100 aat CSI a> Corylopsis dadi >5 99 os CJo 100 \ >5 c s p -j >5 97 LSI-i LORinae 95 >5 TET (HAMeae) >5 62 MAI J 1 MAT— FOTeae DIC — DXCinae (HAMeae) EUS-i 97 80 FOR BUSeae Trichocladus dade >5 84 MOL —FOTeae 72 ^ 4 SIN-1 1 NEO-| J3JL NOA

73 DXCinae HAMoideae OST (HAMeae) TRI-1 66 62 DIM “I 80 <50 DIR DOT (0v PAR 5 / FOT 100 S «> 97 HVE 2 to >5 Hamamelis dade •S $ HVI □ PPS DISn 99 I-----' EXB EXBoideae 70 I >5 I----- RHO RHOideae •MYT- 1LFO— ALToideae

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3.4. 50% Majority consensus of 90 shortest trees of 1041 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method. Taxon abbreviations as in Table 3.1. Numbers are clade frequencies. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT — ALToideae CPA 1 100 100 CSI 100 CSP J LSI 1 100 LORinae 100 TET (HAMeae) 100 MAI -J MAT— FOTeae

60 NEO OlCinae 100 (HAMeae) 100 NOA 100 OST 100 TRI —1

100 EUS EUSeae FOR (HAMeae) 60 HAMoideae 100 MOL FOTeae SIN-1 DIM - 100 100 100 DIR 100 DOT 100 PAR SHA SYS 67 PPS 100 100 100 HVE HVI POT -I DIS— 100 EXB EXBoideae 100 RHO RHOideae MYT. LFO— ALToideae

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3.5. Strict consensus of 12 shortest trees of 6747 steps, generated using nrDNA ITS sequences of the Hamamelidaceae, aligned with the Elision method. Taxon abbreviations as in Table 3.1. Numbers above and below branches are bootstrap percentages and decay values respectively. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DlCinae=Dicoryphinae.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT — ALToideae CPA -i 1001 >6 I 1001 CSI >6 I 100 CSP J >6 99 LSI-1 LORinae 100 >6 TET (HAMeae) >6 56 MAI-I MAT — FOTeae <50 DIC-I DICinae <50 TRI (HAMeae) 100 NEO 100 >6 NOA 100 >6 I 93 >6 OST — 'EUS— | EUSeae 100 , FOR (HAMeae) >6 HAMoideae 1 liooj MOL FOTeae SIN-I 100 DIM 91 >6 DIR 100 >6 DOT 100 PAR >6 SHA SYS 100 FOT 91 >6 >6 100 HVE •H >6 HVI PPS 100 >6 DIS—I EXBoideae 100 EXB >6 RHI RHOideae MYT— LFO— ALToideae

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.3.6. One of the shortest tree of 1041 steps using nrDNA ITS sequences of the Hamamelidaceae, aligned using the Optimality method, showing the number of unambiguous character state changes of ITS sequences. Taxon abbreviations as in Table 3.1.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ALT CPA CSI CSP 28 T

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX 3.1

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20 20 40 60 as as in Table 3.1. represents gaps. SHA SIN TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TGC-GGGG-GCGAGGGG-AGGCATGC-CCCSYS TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATGC-TAT-GGGG-GCGAGGGG-AGGCATGC-CCC TCGAAACCT-GCCCAGBAGAACGACCCGCGAACACGT-NT-CAATGC-TAC-GGGG-GCGAGGGG-AGGCATGC-CCC Teuton Teuton |ITS-1 -> CPA CSI TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AAAAA-TACAAGT-GGGG-GCGGGGGGGGCSP TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AAAAAATACAAGT-GGGG-GGGGGGGGGCA-TTCGTGCTCDIC TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AAAAAATACAAGT-GGGG-GCGGGGGGGCAATCCGTGCTC TCGAAACCT-GCCCAGCAGAACGACCCGCGAACGAGT-AA-GAAAGC-TAC-GGGG-GGGAGGGG-AGGCATGC-CCC CATGCTC FOT HVE TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TAT-GGGG-GCGAGGGGTGGGCATGC-CCC TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TGT-GGGG-GCGAGGGG-AGGCATGC-CCC PAR PPS TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TAC-GGGG-GCGAGGGG-AGGCATGC-CCCRHO TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TAC-GGGG-GCGGGGGG-AGGCATGC-CCC TCGAAACCT-GCACAGCAGAACGACCCGCGAACCGGTAAAC— ACGT TCGGGGGGGAGCGGG-GGGTGCAAGCCC ALT TCGATGCCCCGCAAACAAAACCAACCCGCACACATATTATTCA CATATCGGGGGGCGGGGGG— GGCGAAACCCC DIS DIM TCGAAACCT-GCCCAGCAGAACGACCCGTGAACATGT-ATA-AACTCCCGTGGGGGAGCGGAGGGGGGGCATGC-CTCDIR TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TAC-GGGG-GCGAGGGG-AGGCATGC-CCCDOT TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGCATAA-GGGG-GCGAGGGG-AGGCATGC-CCCEUS TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AT-CAATGC-TAA-GGGG-GCGAGGGG-AGGCATGC-CCCEXB TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATGC-CAT-GGGG-GCGAGGGG-AGGCATGC-CCC TCGAAACCT-GCACAGCAGAACGACCCGCGAACCGGTTACA— ACGT HVI CCGGGGGGAGGGGTGTGGGTGCAAGCCC TCGAAACCT-GCCCAGTAGAACGACCCGCGAACACGT-AT-CAATGA-TAT-GGGG-GCGAGGGG-AGGCATTC-CCCMAI TCGAAACCT-GCACAGCAGAACGACCCGCGAACATGT-AAAAA-TACTGCCCGGTG-GCGAAGGGGCATTCC CCC TRI TCGAAACCT-GCTCAGCAGAACGACCCGCGAACACGT-AA-CAATGC-TAT-GGGG-GTGAGGGG-AGGCATGC-CCT Appendix 3.1. Aligned nrDNA ITS sequences in the Hamamelidaceae. Taxon Abbreviations MYT TCGAAACCT-GCCCAGTAGAACGACCCGCGAATACGTAACACAACGT TTGGGGGGCGAGGGG— GGCGCAGGCCC TET TCGAAACCT-GCACAGCAGAACGACCCGCGAACACGT-AAAAA-CACAAGCCGGGG-GCGG-GGGGGATTCC CCT NEO TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATAC-TGT-GGGG-GCGAGGGG-AGGCATGC-CCCOST TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-TAATAC-TAT-GGGG-GCGAGGGG-AGGCATGC-CCC FOR TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATKC-CAT-GGGG-GCGAGGGG-AGGCATGC-CCC MAT TCGAAACCT-GCACAGCAGAACGACCCGCGAACATGT-AAATA-CACAAGTTGGGG-G-AG-GGGGCATTCC ACC MOL TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATGC-TAT-GGGG-GCGAGGAG-AGGCATGC-CCC NOA TCGAAACCT-GCCCAGCAGAACGACCCGCGAACACGT-AA-CAATAC-TAT-GGGG-GCGAGGGG-AGGCATGC-CCC M fo LFO LSI TCGATGCCC-GCAAAGCAGAACGACCCGCGAACACAT-ACGAA TCGAAACCT-GCACAGCAGAACGACCCGCGAACATGT-GAAAA-CACAAGTCGGTG-GCGG-GGGG-ATTTC AACATCGGGGGGGCGGGGG— GGCACGAGCCC CCC

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ------CGGCAA CGGCAA CGGCAA CGGCAA GGGGC-AAGCTTC ------GTCGGGACGCGTCCGGTGCTT GTCGAGACGCGTCCGGTGCTT GTCGAGACGCGTCCGGTGCTT GTCGGGACGCGCTCGGTGCTCGGTCCGCGACCCGTGC-CAC-GTGCTCGG-GAAGCCCCGTCGGGACACGCTCGGTCCCCGGCCCGCATCCCGTGC-CGC-GTGCTCGG-GAAGCCCC ------GTCGAGACGCGTCCGGTGCTC ----- • • • • • MAI CTCGC-CCCC— AGA SIN CTCTCACCCCC-AAA SHA CTCTCTCCCCCCAAA GTCGGGACGCGCTCGGTGCTCGATCCGCCGACCGTGC-CAC-GTGCGCGG-GAAGCACC PAR CTCTCTCCCCCCAAA GTCGGGACGCGCTCGGTGCTCGATCCGCCAACCGCGC-CAC-GTGCGCGG-GAGGCACC SYS TET CTCTCTCCCCC-AGA CTCGC-CCCC— AAA GCCGGGACGCGCTCGGTGCTCGATCCGCCAACCGTGC-CAC-GTGCCCGG-GAAACCCC GTCGGGACGCGCTCGGCGCTCGGTCCGCAGCCCGCGC-CAC-GTGCTCGG-GAAGCCCC HVI CTCTCTCCCCC-AAA GTCGGGACGCGCTCGGTGCCCGATCCGCCAACCGTGC-C-CCGTGCGCGG-GAAGCATC DIC DIS CCCTCGCCCCC-AAG CTCTCTCTCCTCTACCC—DIR GTCGAGACGCGCTCGGAGCTCGATCCGCAGCCCGTGC-C-CCGTGCGCGG-GAAGCTTC ATCGGGATGCGCTTGGCGTCCGACCCGCGACCCGTGT-C— CTCTCTCCCCC-ATA C-TGCACGG-AGAGCATC GTCGGGACGCGCTCGGTGCTCGATCCGCCAACCGTGC-C-CCGTGCACGG-GAAGCACC PPS RHO CTCTCTCCCCC-AAA -CTC-TCTTCC— TCCCGTGTCGGGACGCGCTCGGCAC-CGCCCCGTGAC— GTCGGGACGCGCTCGGTGCTCGACTCGCCAAACGTGC-C-CCGCGCGCGG-GAGGCATC CTGCGCGATGCGTGCAGGGAGCCGGC TRI CTCTCAACCCC-AGA GTCGGGACACGCTCGGTGCACGTTTCGCCACCCGTGC-C-CCGTGTACGG-GAAGCTTC EXB FOR TCTCCTCCTCCACCCCCAAGTCGGGATGCGCATGGCTT-CGCACCGCGCC—FOT CTCTCACCCCC-AAC HVE CCGTGCCCAGTGTGCGGGGAGC— CTCTCTCCCCC-AAA GT CTCTCTCCCCC-AAA GTCGGGACGCGCTCGGTGCTCGATCCGCCAACCGTGC-C-CCGTGCGCGG-GAAGCATC GTCGGGACGCGCTCGGTGCCCGATCCGCCAACCGTGC-C-CCGTGCGCGG-GAAGCATC Taxon 80 CPA CSI CTCTCACCCCC-AAA CSP CTCTCACCCCC-AAA CTCGAGACGCGCTCGGTGCTCGATCAGCAACGCGTGC-CTC-GCGCACGG-GAAGCTTC CTCTCACCCCC-AAA ATCGGGACGCGCTCGGTGCTCGATTAGAAACGCGTGC-CTC-GTGCACGG-GAAGCTTCDIM ATCGGGACGCGCTCGGTGCTCGATTAGCAACGCGTGC-CTC-GTGAACGG-GAAGCTTC CTCTCTCCCCC-AAA 100 DOT GTCGGGACGCGCTCGGTGCTCGATCCGCCAACCGTGC-C-CCGTGCACGG-GAAGCACC CTCTCTCCCCC-ATA GTCGGGACGCGCTCGGTGCTCGATCCGCCAACCGTGC-CACCGTGCACGG-GAAGCACC 120 140 LFO CTTCTCCCTTCC CC— GTCGAGAAGCGGCCAGTGCT-AGCGTGCCCCTCGACCACGG-GT-TAGGGGAAAGCGTC MYT -CTCTCCTTCCCWTC OST GTCGGGATGCGCTCGGTGC-TGCTCCGCACCTCCCATGCCCTGTGCACGGGAAGC— CTCTCACCCCC-AAA GT GTCGAGACACGCTCGGTGCTCGATCCGCCACCCGTGC ALT CTTCTCCCCTCT TC— GTCGGRAAGCGGCCAGTGCG-GGCGT-CCCCTCGCCCACGG-GT-TGGGGGAAAGCGTC EUS CTCTCACCCCT-AAA NEO NOA CTCTCACCCCC-AAA CTCTCACCCCC-AAA GTCGAGACGCGCTATGTGCTCGATCTGCCACCCGTGC-C-ACGCGTGCGGCAAGCAAAA GTCGAGACACGCTCGGTGCTCGATCTGCCACCCGTGC-C-CCGTGTGCGGCAAAGCTTC MAT MOL CTCAC-CCCCA-AAA CCCTCACCCCC-AGA GTCGGGACGCGCTCGGTCTCCGGTTCGCGACCCGGGC-CAC-GTGCTCGG-GAAGCCCC Appendix 3.1 Continued 3.1 Appendix co £ LSI CTCGC-CCCCC-AAA

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CGCCGTGCTCTCGACA—TCACAACGAACCCCGACGCAAAACGCGTCAAGGAAA-CGCAATGCAAGAGCATG CGGCGTGCTCCCGGTA—TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAATGCAAGAGCATG CGCCGTGCTCTCGATA—TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAATGCAAGAGCATG CGTCGTGCTCTCGATA— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAATGCAAGAGCATG • • • • • ------DIS GGCT-CCAACGCATTCCCGATC— ACACAACGAACCCCGGCGCAAAACGCGCCAAGGAA-CTTTAACGCAAGAGCATGDOT GGCA-CCGGCGTGCGCTCGGCA— TCACAACGAACCCCGGCGCAAATCGCGTCAAGGAAA-CGAAACGCAAGAGCTTG FOT HVE GGCA-CCGGCGTGCGCTCGATA—HVI GGCA-CCGGCGTGCGCTCGGCA— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAACGTAAGAGTCTG GGCA-CCGGCGTGCGCTCGGCA— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAACGCAAGAGCCCG TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAACGCAAGAGCCCG SYS GGGAACCGGCGTGGGCTCGGCA—TRI TCACAACGAACCCCGGCGCAAATCGCGTCAAGGAAA-CGAAACGCAAGAGCCCG GGCG-CCAGCATGCTCTCGATA— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAC-CGCAATGCAAGAGCGTG PAR GGCA-CCGGCGTGCGCTCGGCA— TCACAACGAACCCCGGCGCAAATCGCGTCAAGGAAA-CGAAACGCAAGAGCCCGSHA SIN GGCA-CCGGCGTGCGCCCGGCA— TCACAACGAACCCCGGCGCAAATCGCGTCAAGGAAA-CGAAACGCAAGAGCCCG Taxon 160 CSI GGTG-CCGTCGTGCTCTCGATC—DIC TCACAACGAACCCCGGCCCAAAACGCGTCAAGGAATCTAAAATGCAAGAGCGTG GGCG-CCGGCGTGCTCCCGACA—DIM TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAC-CGTAATGCAAGAGCGTGDIR GGCA-CCGGCGTGCGCTCGGCA— 180 GGCA-CCGGCGTGCGCTCGGCA— TCACAACGAACCCCGGCGCAAATCGCGTCAAGGAAAACGAAACGCAAGAGCTTG BUS TCACAACGAACCCCGGCGCAAACCGCGTCAAGGAAA-CGAAACGCAAGAGCTTGFOR 200 220 LFO GGTGCTTGTCGTGTTCTTAGCCC—MAI CATAACGAACCCCAGAGCAAACCGCGCCAAGGAAACT-CAACTCGAGAGCATGMAT GGCG-CCGTCGTGCTCCCGATC— GGCG-CCGTCGTGCTCCCGATC— ACACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTCAA-CGTAAGAGCGTG TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTCAA-TGCAAGAGCGTG TET GTCC-CCGTCGCGCTCTCGATC— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTCAA-CGCAAGAGCGTG MYT NEO CTGTGCCGGTGCGCTTCTCG-ACCTCACAACGAACCCCGGCGCAAAACGCGCCAAGGAA-CTTGAACSCAAGAGAGCWNOA GGCA-CCGGCATGCTCTCGATA—OST GGCA-CTGGGGTGCTCTCGATA— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAC-CGCAATGCAAGAGCGTG GGCA-TCGGCGTGCCCTCGATA— TCACAATGAACCCCGGCGCAACACGCGTCAAGGAAC-CGCAATGCAAGAGCGTGPPS TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAC-CACAATGCAAGAGCGTGRHO GGCA-CCGGCGTGCGCTCGATA— GAGTGTCGGTGCGCTCCCGATACACAACAACGAACCCCGGCGCAAAACGCGCCAAGGAA-CTCTAACGCAAGAGCACG TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAAA-CGCAACGCAAGAGCCCG ALT CPA GGTGCTTGTCGTGTTCTCGGCCC— GGTG-CCGTCGTGCTCTCGATC— CATAACGAACCCCGGCGCAAACCGCGCCAAGGAAATT-TAACTCGAGAGCATGCSP TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTAAA-TGCAAGAGCGTG GGTG-CCGTCGTGCTCTCGATC— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTAAAATGAAAGAGCGTG EXB TCGTGCCGG— CGCTCTCGAT-C-TAACAATAATCCCCGGCGCAAGACGCGCCAAGGAA-CTCTAACGCAAGAGCATG Appendix 3.1 Continued 3.1 Appendix MOL £ LSI GTCC-CCGTCGTGCTCTCGATC— TCACAACGAACCCCGGCGCAAAACGCGTCAAGGAATCTCAA-TACAAGAGCGTG

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. jlTS-2. DIS DIS CGTATTGCGTTGCCCCCC AGCCCCGTG— CCCCGTTCTCGGGACGCCCGGGGGGTAGTGCAATCTTCGATAT-ATC-T SHA SHA CGTATCGCGTCGCCACCC C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTAT-AC Taxon Taxon 240 CPA CGTATCGCGTCGCCCCCC CSI C-TCCCATC— CGTATCGCGTCGCCCCCC CCCCGGTCACGGGTTGTGGTGGGGGCAATGTCATCTTCGATATTATT-C CSP C-TCCCATC— CGTATCGCGTCGCCCCCC GCCCGGTCACGGGTTTTGGTGGGGGCAATGTCATCTTCGATATTATT-C DIC C-TCCCATC— CGTATCGCGTCGCCCCCA GCCCGGTCACGGGTTTTGGTGGGGGCAATGTCATCTTCGATATTATT-C C-TCCCATC— CCCCGGTCCCGGGCCGCGGTGGGGGCAATGGCATCTTCGATATTATT-C DIM CGTATCGCGTCGCCACCC DIR C-TCCCATC— CGTACCGCGTCGCCACCC CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTAT-AC DOT C-TCCCATC— CGTATCGCGTCGCCACCC CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTAT-AC 260 EUS C-TCCCATC— CGTATCTCGTCGCCCCCA CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTAT-AC EXB C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCAATGGCATCTTCGATATTATT-C CGTATCACGTCCTCCCCC CCCCTGCTCTGCCCCGGTCTCGGGGCGTGTGGCGGGCGATGTCATCTCTGAAA-TCT HVE CGTATCGCGTCGCCACCC C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCAATGGCATCTTCGATATTATT-C 280 |-> 300 RHO CCCCCTTGCTGCCCCGATCTCGGGGCGTGCGGGCGGCTGTGTTATCTTCGAAA-TCT RHO CGTATCACGTCGCCCCCA CCCCCTTGCTGCCCCGATCTCGGGGCGTGCGGGCGGCTGTGTTATCTTCGAAA-TCT SIN CGTATCGCGTCGCCCCCA SYS T-TCCCATC— CGTATCGCGTCGCCACCC CCCCGGTCCCGGGTCGTGGTGGGGGCAACAGCATCTTCGATATTATT-C C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTATT-C TRI CGTATCGCGTCGCCCCCA C-TCCCATC— CCCCGGTCTCGGGACGCGGTGGGGGCAATGGCATCTTCTATATTATT-C Appendix 3.1 Continued 3.1 Appendix OST CGTATCGCGTCGCCCCCA PAR C-TCCCACC— CGTATCGCGTCGCCACCC CCCCGGTCTCGGGCCGTGGCGGGG-CAATGTCATCTTCGATATTATT-C PPS C-TCCCATC— CGTATCGCGTCGCCACCC CCCCGGTCTCGGGTCGTGGTGGGGGCGATGGCATCTTCGATATTAT-AC C-TCCCATC— CCCCGGTCTCGGGTCGCGTTGGGGGCGATGGCATCTTCGATACTATTAC TET CGCATCGCGTCGCCCCCC C-TCCCATC— CCCCGGTCACGGGTGCCGGTGGGGGCAATGGCTTCTTCGATATTATT-C ALT CGCGCCCGTCGTCCCGGTCCCGGGGCGTGCGGGAGGCGGTGCGATCTTGTATGTATACAT CGTCACGCATCGCGTCGC ALT CGCGCCCGTCGTCCCGGTCCCGGGGCGTGCGGGAGGCGGTGCGATCTTGTATGTATACAT FOR CGTATCGCGTCGCCCCCA FOT C-TCCCATC— CGTATTGCGTCGCCACCC CCCCGGTCCCGGGCCGTGGTGGGGGCAATGGCATCTTCGATATTATT-C C-TCCCATC— CCCCGGTCTCGGGTTGTGGTGGGGGCATTGGCATCTTCGATATTATT-C HVI CGTATCGCGTCGCCACCC C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCAATGGCATCTTCGATATTATT-C LFO CGCGCCCGCCGCCTCGGTCTCGGGGTGTGCGGGAAGCGGTGCGATCTTGTATGTATACAC CG LFO LSI CGCGCCCGCCGCCTCGGTCTCGGGGTGTGCGGGAAGCGGTGCGATCTTGTATGTATACAC CGCATCGCGTCGCCCCCC CAACCCATCGC C-TCCCATC— CCCCGGTCACGGGTGCCGATGGGGGCAATCGCTTCTTCGATATTATT-C MAT CGTATCGCGTTGCCCCCC MOL C-TCCCATC— CGTATCGCGTCGCCCCCA CCCCGGTCCCGGGTGCTGGTGGGGGCAGTGGCATCTTCGATACTAAT-C C-TCCCATC— CCCCGGTCTCGGGTCGTGGTGGGGGCAGTGGCATCTTCGATATTATT-C NEO CGTATCGCGTCGCCCCCA NOA C-TCCCATC— CGTATCGCGTCGCCCCCA CCCCGGTCTCGGGCCGTGGTGGGGGYAATGTCATCTTCGATATTATTA- C-TCCCATC— CCCCGGTCTCGGGCCGTGGTGGGGGCAATGTCATCTTCGATATTATTCT MYT TGCACCCGCTGCCTCGGTCTCGGGGCCCGCGGGAGSCAGTSCCATCTTCGATACTCT MYT CGTATTGCGTCGCACCCC TGCACCCGCTGCCTCGGTCTCGGGGCCCGCGGGAGSCAGTSCCATCTTCGATACTCT U* U* MAI CGTATCGCGTCGCCCCCC C-TCCCATC— CCCCGGTCCCGGGCGTTGGTGGGGGCAACGGCATCTTCGATATTATT-C vo

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T— GCG -- .... 320 340 360 380 SIN SIN GCG CAAACCCTTTCCCACGTATATCG-TGGCTGGTGGCATTATATGTGGGGGCGGAGATTGGCCTCCCGTGCCC-C— SHA SHA TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— CAAACCCCCGCCCACGCATGCCG-TGATGCGGGGGGTCCT— Taxon Taxon DIM TCG GTGGGGGCGGATATTGGCCTCCCGTGCGC-T— DIR CAA-CCCCCGCACACGCATGCCG-TGATGCGGGGCGTACT— TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— DOT CAA-CCCCCGCCCACGCATGCCG-TGATGCGGGGCGTCCT— TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— CAA-CCCCCGCCCACGTATGCCG-TGATGCGGGGCGTCCT— EXB FOR — GCG ACG AAACCCTGTCCACGG-TGCCCGTGGTGAGGTGCTTTGGGCG GAGCGGATGTTGGCCTCCCGTGCGT-C— FOT CAA— TCG CCCCGCACACGTACACCG-TGGAGAGGGGCATTATATGTGGGGGCGGAGATTGGCCTCCCGTGCAC-C— GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— HVE CAA-CCCCCGTCCACGTATATCG-TGCTGCGGGGCGTCAT— CAA TCG GTGGGGGCGGATATTGGCCTCCCGTGCGCGT— TCCGCACACGTATACCG-TGCTGCGGGGCATCAT— PPS TCG — GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— AACCCCCGCCCACGTACACCG-TGCCGCGGGGCGTCTT— SYS TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— TET CAA-CCCCCGGCCACGTATGCCG-TGATGCGGGGCGTCCT— GCG C— AACCCTGCCCACGCA-AGCCGTGGTGCGGGGCATCGTGCG-GGGG-CGGATACTGGCCTCCCGTGCAC-C— CSP CSP DIC GCG GCG DIS AACCTCGCCCACATCCGACG-TGGTGCGGGGCATTATGCG-GGGG-CGGATATTGGCCTCCCGTGCGA-T— CAA— CCCCGCCCACGTATACCG-TGGCATGGGGCACCATGTGTGGGGGCGGAGATTGGCCTCCCGTGCAC-C— CAA-TCCCCGCCCACGGATCCCG-TGGTGCGTGGCCTT-TCTGTGGGGGCGGATACTGGCCTCCCGTTC EUS EUS GCG CAA— CCCCGCCCACGTATATCG-TGGAGAGGGGCA-CACAGGTGGGGGCGGATATTGGCCTCCCGTGCAC-T— HVI CAA TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— CCCGCACACGTATACCG-TGCTGCGGG-CATCAT— OST GAG CAA— CACCGCCCACGTATATCG-TGGTGCGGGTCATCGTGCGTGGGGGCGGAGATTGGCCTCCCGTGCAC-C— RHO CCG GAGCGGATATTGGTCTCCCGTGCAT-C— CAAAACCTCACCCACGT-TGCTCGTGGTGCGGGGATTTGAGCG TRI CTG CAA— CCCCTCCCACGTATACCG-TGGTGCGGGGCATCATGTGTTGGGGCGGAGATTGGCCTCCCGTGTGC-T— Appendix 3.1 Continued CPA CSI GTG GCG AACCCCGCTCACGTCCAACG-TGGTGCGGGGCATTATGCG-GGGA-TGGATATTGGCCTCCCGTGCGC-T— AACCTCGCCCACGTCCGACG-TGGTGCGGGGCATTATGCG-GGGG-CGGATATTGGCCTCCCGTGCGC-T— ALT ALT CCCCCCGAACCCCGC-CATCCTTT-GGTGGCGTGGGGCTTCGCGGGGAGCGGAGATTGGCCTCCCGTGAACCACGGTG LFO LFO LSI CCCCCCGAACCCCGC-TATCCTTTTGGTGGCGTGGGGCTTTGCAGGGAGCGGAGATTGTCCTCCCGTGAACCATGGTG MAI GCG MAT CCAATCCGGGGGCGGAGATTGCCCTCCCGTGCAC-C— GCG AACCCTTCCCACGAACCCCG-TGGTGATGGGC— GCG MOL AACCCCGCCCGCGTTCGACGTTGGGCCGGGGCACCGTGTG-GGGGGCGGATATTGGCCTCCCGTGCAC-C— CCAAACCCCGCCCGCGCACAGCGTTGGCCGGGGGCATTGTGAG-GGGG-CGGAGATTGGCCTCCCGTGCAC-C— GCG MYT CAA— CCCTTCCCACGCATGCCG-TGGCGAGGGGCATTATATGTGGGGGCGGAGATTGGCCTCCCGTGCAC-C— — TTC GACCCCCGCCCA-ATGTCATCTTGGCGCGGGGCCTCGCGTG GAGCGGATACTGGCCTCCCGTG-GGAT— NOA GCG CAG— CACCGCCCACGTACATCG-TGGTGCGGGGCATCGTATGTGGGGGCGGAGATTGGCCTCCCGTGCAC-T— PAR TCG GTGGGGGCGGATATTGGCCTCCCGTGCAC-T— CAA-CCCCCGCCCACGCATGCCG-TGATGCGGGGCGTCCT— NEO NEO GCG CAA— CGCCGCCCACGTGAATCG-TGGTGCGGGGCTTCATGTGT-GGGGCGGAGATTGGCCTCCCGTGCAC-C—

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ---- CGGACGTCACGATAAGTGGTGGTTTTTAAGCCGCA CAGAACGTCACGATAAGTGGTGGTCTACAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGACCGT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT TAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT TCGAACGTCACGACAAGTGGTGGTTGTTAA-CACCGACCTT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT CGGAACGTCACGACAAGTGGTGGTTGTTAGGCCCCGGCCTC CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCGT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT CAGAACGTCACGATAAGTGGTGGGTGATAAGCCCCGGCCCT CAGAACGTCACGATAAGTGGTGGTTTATAAGCCCCGACCCT CACAACGTCACGATAAGTGGTGGTTTACAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT CG-AACGTCACGACAAGTGGTGGTTGACAGGCCTCGGCCTC CAGAACGTCACGATAAGTGGTGGTTGATAAGCCTCGGCCCT CAGAACGTCACGATAAGTGGTGGTTTATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTGATAGGCCCCGACCCA CAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT CAGAACGTCACGATAAGTGGTGGT-GATAAGCCCCGGCCCT CAGAACGTCACGATAAGTGGTGGTTTATAAGCCCCGACCCT CAGAACGTCACGATAAGTGGTGGTTTATAAGCCCCGACCCT ------A— AGAACGTCACGATAAGTGGTGGTTAACAACCCCCGATCCG ------ACCAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT ------ACCAGAACGTCACGATAAGTGGTGGTTGATAAGCCCCGGCCCT ------.... 400 400 420 440 460 SHA SHA SIN CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA SYS CAGCGGTTGGCCTAAAAGAGAGCCCCGGGCGA CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCCG TRI TRI CCGCGGTTGGCCTAAAAGTGAGCCCCGGGCGA PPS PPS CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA TET TET CTGCGGTTGGCCTAAAAACGAGCCCCGGGCGA DIS DIS DIM GTGCGGTTGGCTTAAAAACGAGCCCCGAGCG DIR CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA DOT CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA CCGCGGTTGGCCTAAAAGCGAGCCCCGGGC-G FOT CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA HVI CCGCGGGTGGCCTAAAAGCGAGCCCCGGGCGA PAR CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA RHO TTGCGGTTGGCCTAAAAATCGGTCCCGAGCGA CPA CPA CTGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA CSP DIC CTGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA EXB EXB CCGCGGTTGGCTTAAAAGCGAGCCCCGGGCGA OST CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA NEO NEO CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA CSI CSI CTGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA FOR CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA HVE CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA LFO TCGCGGTTGGCTTAAAAGCGTGCCCCAGGCGATGAACGCCAACGTCTCAAGCAGTGGTGGTTACCAAACCCCGGCATC MAT MOL TTGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA NOA CTGCGGTTGGCCTAAAAACGAGCCCCGGGCGA Appendix 3.1 Continued 3.1 Appendix Taxon EUS EUS CCGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA MYT MYT CCGCGGCTGGCCTAAATGCGAGCCCCGAGCGA ALT ALT TCGCGGTTGGCTTAAAAGCGTGCCCCGGGCGACGAACGCCAACGTCTCAAGCAGTGGTGGTTACCAAACCCCGGCATC LSI MAI CTGCGGTTGGCCTAAAAACGAGCCCCGGGCGG TTGCGGTTGGCCTAAAAGCGAGCCCCGGGCGA I

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GGCACAGAGACCCCCGAACGCGTCGCACACG-CGATGT GGCACAGAGACCCCCGAACGCGTCGCATATG-CGGCGT -- -- .... 480 480 500 520 540 TGCGGTGTCGTGCGTTGTCCCGTCGCTCGCT-GGTGCTGCGTGACCCCGATGCTCCGTCGAATGGCGGTGC ------SHA SIN TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCCCGCT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGCSYS TCAA-GTGTCGAGTCGTGCGTTGCGCTGTCGCCTGTT-GGAGCTACGAGACCCC-GATGTGTCGTCCC-GACGATGCTET TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCCCGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGC CTCA-GTGTCGTGTCGTGCGTTGCACTGTCGCCCGCTGGAGCCA-CGAGACCCC-GATGCACCGCA-ACCGCGGTGC DIM TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCCCGTCCGGAGCTACGAGACCC— GATGCGTCGTCCC-GACGATGC PPS TCAA-GTGTCATGTCGTGCGTTGCC-TGTCGCTCGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGC Taxon FOR TCAA-GTGTCGTGTCGTGCGTTGCGCTGTCGCCTGTC-GGAGCCACGAGACCCC-GATGCGTCGTCCC-GACGATGC HVI TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCTCGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCCCGACGATGC DIC DIS TCGA-GTGTCGTGTCGTGCGTTGTGCTGTCGCCTTCT-GGAGCCACGAGACCC— GATGCGTCGTCCC-CACGATGC RHO AGCG-GTATCGTGTCGTGCGTTTTTCCGTCGCTCGTCGGAGCGAACGAGACCCC-GATGCGTCGC-CCCGACGATGC TRI TCGA-GTGTCGTGTCGTGCGTTTTGCTGTCGCCCGTT-GGAGCTACGAGACCCCCGATGCGTCGTACG-GACGATGC DIR TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCCCGTC-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGC NOA OST TAAA-GTGTCGTGTCGTGCGCTGCGCTGTCGCCTGTT-GGAGCCACGAGACCCC-GATGCGTCGTCCC-CACGATGC AAAA-ATGTCGTGTCGTGCGTTGCGCTGTCGCCTGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-CACGATGC DOT EUS TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCCCGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGCEXB TCAA-GTGTCGTGTCGTGCGTTGCGCTGTCGCCTGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGC AGCG-GTATCGTGTCGTGCGTTGCCCCGTCGCTCGTCGGAGCCAACGAGACCCT-GACGCGTCGT-CCCGACGACGCFOT HVE TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCTTGTT-GGAGCTACGAGACCCC-GATGCGTCGTTCC-GACGATGC TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCTCGTT-GGAGCTATGAGACCCC-GATGCGTCGTCCCCGACGATGC CPA CSI TGCA-GTGTCGTGTCGTGCGTTGCACTGTCGCTCGTTGGAGCTA-TGAGACCCC-GATGCGCCGTA-CCGACGGTGCCSP TGCA-GTGTCGTGTCGTGCGTTGCACTGTCGCTCGTTGGAGCTAATGAGACCCC-GATGCGCCGTG-CCGACGGTGC TGCA-GTGTCGTGTCGTGCGTTGCACTGTCGCTCGTTGGAGCTAATGAGACCCC-GATGCGCCGTG-CCGACGGTGC PAR TCAA-GTGTCATGTCGTGCGTTGCCCTGTCGCTCGTT-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGATGC LSI MAI GAAACGTGTCGTGTCGTGCGTTGCACTGTCGCCCGCT-GGAGCCACGAGACCCC-GATGCGCCCCAACC-ACGGTGC TTCA-GTGTCGTGTCGTGCGTCGCGCTGTCGCTCGTCGGAGCTA-CGAGACCCC-GATGCGTCTTG-CTGA-GGCGCMOL MYT TGCA-GTGTCGAGTCGTGCGTTGCGCTGTCGCCTGTC-GGAGCTACGAGACCCC-GATGCGTCGTCCC-GACGGTGCNEO CAAA-GCGTCGTGTCGTGCGTTGTACTGTCGCCTGCC-GGAGCTACGAGACCCC— GTGTCGTATCGTGCGTTGC-CCGTCGCCAACGGGAGCCACACAATACCCCCGTGCGTCGTCATCG-CGACGC GTGCATCTCCCC-CACGATGC Appendix 3.1 Continued 3.1 Appendix LFO GATGATGTTTGCCTTGTGCGTTGCCACGTCGCCCGC ALT GATGATGTTTGCCTTGTGCGTTGCCTCGTCGCCCGC MAT TCCA-GTGTCGTGTCGTGCGTTGCACTGTCGCCCGTTGGAGCCA-CGAGACCCC-GATGCGCCGCG— CCGCGGTGC

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

PHYLOGENETIC RELATIONSHIPS OF THE HAMAMELIDACEAE: EVIDENCE

FROM MORPHOLOGY AND NUCLEOTIDE SEQUENCES OF ITS AND MATK GENE

INTRODUCTION

In the Hamamelidaceae, there are 31 genera and more than

140 species (Zhang and Luf 1995). These taxa are rather

diverse in morphology. For example, some are trees, while

others are shrubs; some species are deciduous, others are

evergreen; some are bisexual, others are either

andromonoecious or monoecious; some are wind pollinated,

while others are insect or bird pollinated. With respect to

biogeographic distribution, the hamamelidaceous plants are

distributed in southeastern Africa, Madagascar, northeastern

Australia, western-, central-, and southeastern Asia, eastern

North America, Central America and even northern South

America. The fossil record of several hamamelidaceous taxa

dates back to the Upper Cretaceous, indicating the long

evolutionary history of this family (Endress and Friis, 1991;

Zhang and Lu, 1995).

Given the ancient diversification of the Hamamelidaceae

in both morphology and biogeography, it is not surprising

that there has been a great deal of disagreement concerning

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the natural classification of the family, especially, since

various authors have placed an emphasis on different

characters in ascertaining relationships among certain groups

of species. Consequently, two, three, four, five, and even

six subfamilies have been recognized in the Hamamelidaceae

(Reinsch, 1889; Niedenzu, 1891; Harms, 1930; Chang, 1962,

1973; Endress, 1989c). Also controversial are the

intergeneric relationships within the largest subfamily, the

Hamamelidoideae. The result has been the division of the

subfamily into five or four tribes (Harms, 1930; Endress,

1989c).

Evidence from several sources, including morphology,

palynology, and DNA sequences, etc., contributes to a better

understanding of the natural relationships of a particular

taxon, but phylogenetic information contained in each data

set may differ, thus giving rise to various hypotheses about

relationships. Unfortunately, evolutionary history can only

recognize one correct phylogeny. Therefore, many relevant

data should be taken into account when proposing phylogenetic

hypotheses (Tehler, 1995). This is especially important for

molecular data, since the phylogeny obtained based on the

nucleotide sequences in a single segment of DNA is probably

not an organismic tree but a gene tree (Doyle, 1993). In

addition, some other biological processes (such as lineage

sorting and introgression) may get involved in shaping

genetic history, and thus distort some relationships (Ritland

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and Eckenwalder, 1992). In contrast, because of different

inheritance patterns in nuclear and chloroplast genes,

comparison of phylogenies from both data sources may provide

insights on the origin of some taxa (Soltis et al., 1996).

In this Chapter, therefore, I present a phylogenetic

analysis of the Hamamelidaceae using data from morphology,

and nucleotide sequences of the ITS (Internal Transcribed

Spacers) of nuclear ribosomal DNA, and of the plastid matK

gene.

MATERIALS AND METHODS

Data Sources

The morphological characters used in this analysis are

those described in Chapter I, and the ITS and matK sequences

are those presented in Chapters II and III, respectively.

Phylogenetic Analysis

There have been great debates over the approaches that

should be used in phylogenetic analyses when multiple data

sets are available for a taxon (Penny et al., 1982; Cracraft

and Mindell, 1989; Kluge, 1989; de Queiroz, 1993; Tehler,

1995). The advocates for the consensus approach argue that

this means of analysis gives equal weight to each data set,

thus reducing the potential for data sets with relatively

large numbers of characters to swamp data with fewer

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. characters (Kluge, 1983), and that a conservative estimate

should be generated using the consensus method (Hillis,

1987). However, proponents of the combined approach counter

that equally weighting data sets of different numbers of

characters results in an arbitrary weighting of characters

(Cracraft and Mindell, 1989). Consensus trees do not

necessarily indicate the most parsimonious pattern of

character change (Miyamoto, 1985), and consensus trees can

actually contradict combined trees, thus not necessarily

showing a conservative pattern (Barrett et al., 1991).

However, as noted by de Queiroz (1993), each method is

preferred under different circumstances. When different data

sets give conflicting trees that cure strongly supported,

consensus is preferred over a combined approach. When non­

independence of characters is suspected within (but not

among) data sets, but conflicts among the trees resulting

from individual analyses are not strongly supported,

consensus should be used in conjunction with a combined

analysis. But, when there is not obvious non-independence of

characters within data sets, a combined approach is preferred

over consensus. As far as defining reasonable limits of

support, Kellogg et al. (1996) treated clades as "poorly

supported" when their bootstrap values are less than 70%, and

corresponding decay indices are one or two.

In this study, I applied both the consensus and combined

approaches to the data sets. The consensus approach was

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. carried out by reconstructing phylogeny from each data set

and then determining the consensus tree from all estimates,

while the combined method was performed by combining the data

sets from the outset.

Because the individual data sets of morphology, ITS and

matK do not contain exactly the same taxa, I had to reduce

the number of taxa so that a common set of taxa is included

in the combined data set. Both the morphological analysis

using Cercidiphyllum as the outgroup, and the matK gene

sequence analysis using two genera of the Saxifragaceae as

the outgroups, placed Altinqia in the basal clade of the

Hamamelidaceae (See Chapters I and II). In addition, neither

the ITS nor the matK sequences of Cercidiphyllum were

available for this study. Furthermore, the distinct

morphological differences between either Cercidiphyllum or

the Saxifragaceae and the Hamamelidaceae cast doubt on the

alignability of the ITS sequences between these taxa.

Therefore, Altincria was chosen as the outgroup in the

combined analysis.

Phylogenetic analyses were conducted using the PAUP*

test versions 4.0d57-d59 computer program, written by David

Swofford of the Smithsonian Institution. The options were

Heuristic search, TBR branch swapping, Mulpars on, and

Steepest descent off. All characters were unweighted, and

all state transformations were unordered. Both bootstrap

statistics of 100 replicates (Felsenstein, 1985) and the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Constraint decay analysis (Morgan, 1997) were performed for

all data sets to indicate relative supports for individual

clades.

RESULTS

Consensus Approach

Strict consensus trees based on each data set are

presented in Figs.4.1, 4.2, and 4.3 for morphology, matK, and

ITS respectively. When the Consensus approach was applied,

the consensus tree of neither morphology+ITS nor

morphology+matK resolved many relationships (Figs.4.4, 4.5);

however, ITS+matK consensus resolved most of the intergeneric

relationships of the Hamamelidaceae (Fig.4.6). There is

little resolution in the consensus of morphology+matK+ITS

(Fig.4.7).

Combined Analysis

When the Combined approach was used, the most

parsimonious phylogenies of morphology+ITS and

morphology+ITS+matK resolved all the intergeneric

relationships (Figs.4.8, 4.11), while those of

morphology+matK and ITS+matK showed slightly less resolution

for intergeneric relationships (Figs.4.9, 4.10). Figure 4.11

is the single tree generated in the combined analysis of

morphology+ITS+matK . In this phylogeny, Liquidambar is the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. first clade following the outgroup Altinqia. Exbucklandia

and Rhodoleia are clustered in one clade, followed by two

paraphyletic clades of Mvtilaria and Disanthus. Within the

Hamamelidoideae, the basal clade is Corylopsis-Loropetalum-

Mainqaya-Matudaea. followed by a dichotomy of Hamamelis-

Fothergilleae and Eustigmateae-Dicoryphinae. The

Dicoryphinae Endress forms a monophyletic group, within which

Dicorvphe and Trichocladus are of a clade, and its sister

clade contains the three Australian genera (Neostrearia,

Ostrearia. and Noahdendron). The Eustigmateae sensu Endress

(1989c) is a clade, with the inclusion of Molinadendron.

Hamamelis is basal in the clade of Hamamelis-Fothergilleae.

within which Fotherqilla. Parrotiopsis. Parrotia, and

Shaniodendron form a consecutive paraphyletic pattern, while

Distylium. Distyliopsis. and Sycopsis are located on the top

of the clade.

DISCUSSION

Consensus Approach Reduces Resolution

The trees based on the two DNA sequence data sets (ITS

and matK) are mostly congruent to each other (Figs.4.2, 4.3),

thus producing a consensus that resolves some relationships

of the Hamamelidaceae (Fig.4.6). However, the tree

topological patterns based on either of the two DNA sequence

data sets rarely agree with those generated using

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. morphological data, resulting in a consensus tree of little

resolution (Figs.4.4, 4.5, 4.7). This is a drawback of the

consensus approach for the data sets.

If bootstrap values less than 70% are considered as weak

supports (Kellogg et al., 1996), the clades in the

morphology-based phylogeny are not well supported except for

several closely related genus-pairs (Fig.4.1). As a result,

when applied in the consensus approach, which does not

differentiate the well-supported and poorly-supported

topologies, morphological data offset the resolving power

from the other data sets, resulting in a poor overall

resolution for the Hamamelidaceae. Thus, the combined

approach should be preferred in this situation, as

recommended by de Queiroz (1993) and Kellogg et al. (1996).

However, some relationships are recognizable when the

phylogenetic estimates from each data set are compared. For

instance, Eustiama. Fortunearia. and Sinowilsonia are

clustered in one group in all three trees (Figs.4.1, 4.2,

4.3), supporting the recognition of the Eustigmateae sensu

Endress (1989c).

The Combined Approach Increases Resolution

Parsimony analyses were conducted for different

combinations of the three data sets, including ITS-morphology

(Fig.4.8), matK-morpholooy (Fig.4.9), ITS-matK (Fig.4.10),

and ITS-matK-morphology (Fig.4.11). It is readily noticeable

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that well-supported relationships in one data set overperform

the weakly-supported ones in another. For example, the

weakly supported relationships produced by morphological

data, such as Corylopsis-Hamamelis. and Matudaea-

Molinadendron. are rarely shown when the morphology data set

is combined with either ITS or matK data, both of which have

demonstrated much stronger clade supports.

Interestingly, the morphological data set produces few

well-supported clades, but it increases resolution for some

clades when combined with either the ITS or matK data. For

example, in the ITS tree (Fig.4.3), the relationships of

Fothergilla. Hamamelis, and Parrotiopsis remain unclear,

while the analysis using the combined data set of ITS and

morphology resolves the relationships of the genera

(Fig.4.8).

It seems to be appropriate to combine data sets when

there are no conflicting hypotheses that are well-supported

by different, independent data sets (Kellogg et al., 1996).

However, I believe that performing both the consensus and

combined analyses has several advantages. First, the

consensus approach often produces highly unresolved

phylogeny, but when it does resolve a relationship, greater

confidence should be placed in the reality of this pattern,

especially when the analysis involves data from multiple

independent sources. Second, the combined analysis generally

produces a phylogeny with a high resolution when data sets

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. are not obviously conflicting to one another. Third, when

individual analyses are conducted, and the phylogenetic trees

are compared, the discrepancies may shed some new light on

biological processes leading to the differentiated pattern,

such as lineage sorting and chloroplast capture (Soltis et

al., 1996a).

Implications of the topological differences between

phyloqenies based on nuclear and chloroplast DNA sequences

In the ITS-based phylogeny, Parrotia is located next to

Sycopsis. suggesting a close relationship (Fig.4.3); however,

in the matK tree, Parrotia is the basal taxon of the

Fothergilleae, but Sycopsis is at the top of the clade, far

away from Parrotia (Fig.4.2). These two genera are known to

be interfertile, producing a fertile hybrid (=XSycoparrotia.

Endress and Anliker, 1968). This is consistent with their

close relationship, as shown in the ITS phylogeny. Parrotia

and Svcopsis also share some morphological characteristics,

especially floral structures (Bogle, 1968; Endress and

Anliker, 1968). Since morphology is not consistent with the

chloroplast gene-based tree, and there is a strong

differentiation of the matK gene between Parrotia and

Sycopsis. it does not make sense to use chloroplast capture

to explain the differences in the phylogenetic patterns of

ITS and matK for the two genera (Soltis et al., 1996a).

Alternatively, lineage sorting may be the cause of the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pattern (Rieseberg and Brunsfeld, 1992; Soltis and Soltis,

1995; Soltis et al., 1996a). High polymorphism of the

chloroplast genome may have developed in the common ancestor

of these two genera, and their geographical separation since

the Late Miocene (Deng et al., 1992b; Zhang and Lu, 1995) may

have provided an opportunity for further accumulation of base

substitutions.

Shaniodendron (Chang) Deng, Wei & Wang is the most

recently described segregate genus in the Hamamelidaceae

(Deng et al,. 1992a). It is endemic to the eastern ranges of

Dabieshan mountains and the northern parts of the Tianmushan

mountains of eastern China (Deng et al., 1992a, b; Hao et

al., 1996). Because of its rarity, Shaniodenriron has been

added to the list of the most endangered plant species in

China (Deng et al., 1992a, b). Deng et al. (1992a) suggested

that this genus was more closely related to Fothergilleae

(incl. Fotherailla. Parrotia. Parrotiopsis) than to

Distylieae (incl. Distylium. Matudaea. Molinadendron. and

Sycopsis) (See Harms, 1930; Endress, 1989a, b ) . Recently-

conducted comparative studies of floral morphology (Hao et

al., 1996) and wood anatomy (Fang and Deng, 1996) support

Deng et al.'s placement (1992a) of Shaniodendron in the

Fothergilleae. Furthermore, the study of floral morphology

led Hao et al. (1996) to the conclusion that Shaniodendron is

most closely related to Parrotia.

In the ITS phylogeny (Fig.4.3), Shaniodendron is placed

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. between Parrotia and Sycopsis, while in the matK tree

(Fig.4.2), it is closely allied within the clade consisting

of Sycopsis and Distylium. but separated from Parrotia.

Shaniodendron shares morphological characters with both

Sycopsis and Parrotia. but especially with Parrotia.

including deciduous leaves, andromonoecy, and elongated

stamens. Therefore, the morphological data support the

relationships among the three genera shown in the ITS-based

phylogeny. As stated above, Sycopsis and Parrotia are able

to hybridize, producing a hybrid, XSvcoparrotia.

Interestingly, Shaniodendron shows many somewhat intermediate

morphological characteristics between Parrotia and Sycopsis,

as in parallel with the hybrid genus XSycoparrotia, such as

middle-sized lanceolate stipules, slightly elongated anthers,

shallow hypanthium tubes, and semi-inferior ovaries (See Deng

et al., 1992a, b; Fang and Deng, 1996; Hao et al., 1996).

Therefore, it is possible that Shaniodendron could be derived

from an intergeneric hybridization event between species of

Parrotia and Sycopsis. Furthermore, in the maternally

inherited matK gene phylogeny, Shaniodendron is closely

related to Sycopsis. but distant from Parrotia. One could

interpret this as an indication that Sycopsis might be the

maternal parent for this putative hybrid.

Parrotia is a monotypic genus endemic to the Caspian Sea

region of central Asia (Zhang and Lu, 1995). Shaniodendron

is also a monotypic taxon, but narrowly distributed in

210

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eastern China (Deng et al., 1992a, b; Zhang and Lu, 1995).

In contrast, Sycopsis is widely distributed in southeastern

Asia from southern Vietnam to central areas of China (Chang

1979; Zhang and Lu, 1995). Therefore, Parrotia is disjunct

from both Shaniodendron and Sycopsis. Moreover, the

divergence time of Parrotia and Shaniodendron was estimated

to be in the late Tertiary (Li et al., 1997d). Thus, the

hypothesized hybrid origin of Shaniodendron. if true, must

be an ancient event.

Phylogenetic relationships in the Hamamelidaceae

Most of the relationships suggested by this combined

analysis are similar to the ones shown in previous analyses

using individual data sets of the matK gene (Fig.2.2, Chapter

II) and ITS (Figs.3.3, 3.4, 3.5, 3.6, Chapter III). These

relationships are as follows: 1) the monophyly of several

groups including the Altingioideae, Exbucklandia-Rhodoleia.

and the Hamamelidoideae; 2) polyphyly of Exbucklandioideae

sensu Endress (1989c); 3) the Hamamelidoideae containing

three major branches: Corylopsis-Loropetalum-Mainqava-

Matudaea: Dicoryphinae-Eustigmateae sensu Endress (1989c),

but including Molinadendron: and Fothergilleae sensu Endress

(1989c), but including Hamamelis. In the combined analysis

Hamamelis was placed in the position sister to the

Fothergilleae clade. This relationship is morphologically

reasonable.

211

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Evolution of Some Morphological Characters

The Hamamelidaceae are rather diverse morphologically,

thus resulting in controversies about a correct natural

classification of the family. A phylogenetic analysis based

only on morphological data produced trees with a small

consistency index (Cl) of 0.45 (Chapter I), indicating high

homoplasy of morphological characters in the Hamamelidaceae.

Therefore, a combined analysis of DNA sequence data and

morphological data may shed some light on the evolution of

some morphological characters, especially the ones that are

commonly used in defining groups in previous classification

systems.

Palmately lobed leaves occur in several paraphyletic

clades of the tree based on the combined data set (Fig.4.12),

including Liquidambar. Mvtilaria. and Exbucklandia.

Therefore, the most parsimonious interpretation of the

distribution of the character is that it represents a

plesiomorphic feature.

In the Hamamelidoideae, most of the genera have only one

ovule in each locule of the two-carpelled pistil, while in

several genera two to three ovules have been observed per

locule, yet only one is fertile and becomes seed. The one-

seeded condition is functionally correlated with explosive

seed dispersal (Endress 1989b). But in other taxa, such as

Exbucklandia. Rhodoleia, Mytilaria. and Disanthus, there are

several to many ovules in each locule. Accordingly, these

212

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genera do not have the ballistic seed discharge mechanism in

the fruit. Endress (1989b) described this mechanism in

detail and concluded that a one-seeded pistil with explosive

seed discharge is more advanced. The combined phylogeny

(Fig.4.13) shows this directionality of ovule evolution in

the Hamamelidaceae, and that the one-seeded carpel is a

synapomorphy of the Hamamelidoideae.

The tendency toward wind pollination has been proposed

to be a driving force of character evolution in the

Hamamelidaceae, such as loss of petals and change of

sexuality from bisexual via andromonoecious to monoecious

(Tong, 1930; Bogle and Philbrick, 1980). The phylogenetic

analysis (Fig.4.14) reveals that loss of petals is

distributed in at least five clades in the Hamamelidaceae,

including the Altingioideae, Exbucklandia. Fothergilleae,

Matudaea. and Molinadendron. Forcing the character to become

a single synapomorphy increases the number of tree steps

greatly compared to the most parsimonious trees. Therefore,

parallel evolution may have occurred in this character state.

The same appears to be true for wind pollination,

andromonoecy and monoecy (Figs.4.15, 4.16).

Within the Hamamelidoideae, three lineages can be

recognized (Fig.4.11). Interestingly, each lineage exhibits

similar evolutionary trends in certain floral characters

adapting to wind pollination. In the first lineage, the

bisexual flower is a synplesiomorphy, while petals have

213

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. evolved in the direction of loss from Loropetalum to Matudaea

(Fig. 4.14). In the second lineage, flowers change from

bisexual in Eustiama and Molinadendron. to andromonoecious in

Fortunearia. and monoecious in Sinowilsonia (Fig.4.16). In

the third lineage, flowers evolve from insect pollination in

Parrotionsis and Fotheroilla to wind pollination in Parrotia,

Shaniodendron. Distylium. Distyliopsis. and Svcopsis

(Fig.4.15).

Possession of showy structures other than petals

(stamens in Fotheroilla and inflorescence bracts in

Parrotiopsis) has been considered as secondarily evolved in

the course of evolution (Endress, 1989b). However,

Fotheroilla and Parrotiopsis are located in the basal

position of the clade that also includes Distyliopsis,

Distylium. Sycopsis. and Shaniodendron. Therefore, these

characteristics may be secondarily evolved but in conjunction

with the plesiomorphic insect pollination.

The haploid chromosome numbers (n) in the Hamamelidaceae

exhibit two basic series: 8, 16, 32, and 12, 24, and 36

(Santmour, 1965, 1972; Goldblatt and Endress, 1977; Morawetz

and Samuel, 1989; Oginuma and Tobe, 1991). Goldblatt and

Endress (1977) suggested that x=7 was the basic chromosome

number in the Hamamelidaceae, and that later descending

aneuploidy resulted in x=6, which, via polyploidy, gave rise

to n=12 group (Rhodoleia and Hamamelidoideae), whereas later

ascending aneuploidy produced x=8, which also via subsequent

214

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. polyploidy led to the n=8 alliance (Altingioideae and

Exbucklandioideae). However, they admitted that it is also

possible that either x=8 or x=6 is the basic chromosome

number. Morawetz and Samuel (1989) considered x=8 as basic

in the Hamamelidaceae, and suggested that 2n=24 was of

triploid origin from x=8 via anorthoploidization. Oginuma

and Tobe (1991) agreed with both Goldblatt and Endress (1977)

and Morawetz and Samuel (1989), and suggested that x=8 is

basic to an evolutionary line of Exbucklandioideae and

Altingioideae, while x=6 is basic in the Hamamelidoideae and

Rhodoleioideae. Interestingly, a new base number, x=13

(2n=26), was first reported in Liquidambar acalycina (Huang

et al., 1985) [no photographs were provided in the paper,

thus it needs to be confirmed]. This number (2n=26) was also

recently reported in Mytilaria (Pan and Yang, 1994), an

isolated genus in the Exbucklandioideae (Endress, 1989c).

In the phylogeny based on the combined data set

(Fig.4.17), x=8 occurs in the most basal clade, the

Altingioideae, followed by the clade of Exbucklandia (x=8)

and Rhodoleia (x=12). In the succeeding clades, Mytilaria

has x=13, which probably is derived from x=12 via ascending

aneuploidy, while Disanthus exhibits the base number of x=8.

However, the Hamamelidoideae share x=12 with Rhodoleia. This

phylogeny does not support the two evolutionary-line

hypothesis (Oginuma and Tobe, 1991). In the evolutionarily

more recent clade, the Hamamelidoideae, x is invariably equal

215

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to 12. Therefore, x=8 is probably the ancestral base number,

while x=12 is a base number of possibly ancient polyploid

origin. The close relationship between Exbucklandia (x=8)

and Rhodoleia (x=12) in the phylogenetic analysis seems to

support the suggestion that x=12 is of triploid origin of x=8

via anorthoploidization (Morawetz and Samuel, 1989), since it

is difficult to believe that a series of cytological events

has taken place, including descending and ascending

aneuploidy, and polyploidization, in this strongly supported

clade. The further ploidy levels, n=24 and 36, may be

explained as having been derived directly from the ancient

base number, x=12, via polyploidy.

216

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.1. Strict consensus of 203 most parsimonious trees of 185 steps based on morphology of the Hamamelidaceae. CI=0.5, RC=0.31. Branches have bootstrap percentage of less than 50% and decay values of one unless otherwise indicated above and below the branches respectively.

217

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis Hamamelis Dicoryphe Trichocladus Loropetalum Maingaya 2 Neostrearia Noahdendron Ostrearia Disanthus Distylium Distyliopsis Eustigma { Fortunearia Sinowilsonia Exbucklandia Fothergilla Matudaea t Molinadendron Mytilaria Parrotia Parrotiopsis Rhodoleia Shaniodendron Sycopsis Liquidambar

218

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.2. Strict consensus of six most parsimonious trees of 381 steps based on sequences of the matK gene in the Hamamelidaceae. CI=0.87, RC=0.72. Numbers above and below branches are bootstrap percentages and decay indices.

219

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis 100 611---- Loropetalum 96 Matudaea 2 i Maingaya Dicoryphe Neostrearia 58 71 Noahdendron 1 1 ---- Ostrearia 81 Trichocladus 69J— Eustigma 89 5 | 85 Fortunearia 2 62 r “ Molinadendron 2 1--- Sinowilsonia 70 1---- Distylium 92 1 1____ Sycopsis 1 4 62 Distyliopsis Shaniodendron Fothergilla <50 Parrotiopsis 95 Hamamelis Parrotia 100 Disanthus Mytilaria

100 Exbucklandia ■ c Rhodoleia Liquidambar

220

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.3. Strict consensus of 16 most parsimonious trees of 6952 steps based on the ITS grand data set of the Hamamelidaceae. CI=0.6, RC=0.36. Numbers above and below branches are bootstrap percentages and decay indices.

221

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis 100 Loropetalum 17 100 “l2\ 82 Maingaya Matudaea

<50 Dicoryphe

55 Trichocladus Neostrearia 100 100 Noahdendron 100 Ostrearia Eustigma 100 Fortunearla 14 10 Molinadendron Sinowilsonia 100 99 Distylium 96 11 Distyliopsis 100 Parrotia 15 51 Shaniodendron Sycopsis 64 M Hamamelis 100 Parrotiopsis Fothergilia 100 20 Disanthus Mytilaria

100 Exbucklandia Rhodoleia Liquidambar

222

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.4. Strict consensus of morphology and ITS of the Hamamelidaceae obtained using the Consensus approach.

223

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis Dicoryphe Disanthus Distylium Distyliopsls Eustigma Fortunearia Exbucklandia Fothergilla Hamamelis Loropetalum Maingaya Matudaea Molinadendron Mytilaria Neostrearia Noahdendron Ostrearia Parrotia Parrotiopsis Rhodoleia Shaniodendron Sinowilsonia Sycopsis Trichocladus Liquidambar

224

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.5. Strict consensus of morphology and matK of the Hamamelidaceae obtained using the Consensus approach.

225

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis Dicoryphe Disanthus Distylium Distyliopsis Eustigma Fortunearia Exbucklandia Fothergilla Hamamelis Loropetalum Maingaya Matudaea Molinadendron Mytilaria Neostrearia Noahdendron Ostrearia Parrotia Parrotiopsis Rhodoleia Shaniodendron Sinowilsonia Sycopsis Trichocladus Liquidambar

226

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.6. Strict consensus of ITS and matK of the Hamamelidaceae obtained using the Consensus approach.

227

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■■ ■■ Altingia — — Corylopsis — —— Loropetalum — ~ Maingaya — — Matudaea ■■ Dicoryphe — Neostrearia — — Noahdendron

11 11 ■ Ostrearia — — Trichocladus — Eustigma

11 Fortunearia — — Molinadendron — — Sinowilsonia —— Distylium ■— — Distyliopsis

— 1— Fothergilla — — Hamamelis " Parrotia — — Parrotiopsis — — Shaniodendron ■ Sycopsis

1 Disanthus — — Mytilaria | Exbucklandia ' Rhodoleia — Liquidambar

228

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 4.7. Strict consensus of morphology, ITS and matK of the Hamamelidaceae obtained using the Consensus approach.

229

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia Corylopsis Dicoryphe Disanthus Distylium Distyliopsis Eustigma Exbucklandia Fortunearia Fothergilla Hamamelis Loropetalum Maingaya Matudaea Molinadendron Mytllaria Neostrearia Noahdendron Ostrearia Parrotia Parrotiopsis Rhodoleia Shaniodendron Sinowilsonia Sycopsis Trichocladns Liquidambar

230

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.8. Strict consensus of two shortest tree of 7161 steps based on the combined data set of morphology and ITS of the Hamamelidaceae. CI=0.6, RC=0.36. Numbers above and below branches are bootstrap percentages and decay indices. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

231

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia — ALToideae Corylopsis _ C0Reae Loropetalum LORinae (HAMeae) 14 75 Maingaya '] Mafcuda&a — FOTeae Dicoiryphe — Trxchocladus DICinae Neostrearla (HAMeae) Noahdendron Ostrearia _ (Q

232

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.9. Strict consensus of eight shortest trees of 590 steps based on the combined data set of morphology and matK of the Hamamelidaceae. CI=0.72, RC=0.49. Numbers above and below branches are bootstrap percentages and decay indices. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

233

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 Altingia — ALToideae Corylopsis — coReae 90 Loropetalum -j Maingaya _1 (HAMeae) Matudaea — FOTeae Dicoryphe

Neostrearia DICinae 83 77 Noahdendron (HAMeae) Ostrearia Trichocladus— Distylium — 100 Distyliopsis 8| 57 Sycopsis 1 78 Shaniodendron 6 <50 Parrotia 1 52 Parrotiopsis 1 84 Fothergilla— 70 3 Hamamelis — •HAMinae (HAMeae) Eustigma EUSeae Foxrtunearia 95 60 d8 0) •Mytilaria •w•o o o X 93 Exbucklandia— w c Rhodoleia — RHOideae 1 Liguidantbar — ALToideae

234

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.10. Single shortest tree of 7338 steps based on the combined data set of ITS and matK of the Hamamelidaceae. CI=0.62, RC=0.38. Numbers above and below branches are bootstrap percentages and decay indices. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae , EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamame1idoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

235

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Altingia — ALToideae

Corylopsis — coReae Loropetalum L0Rinae Maingaya J (HAMeae)

Matuda&a — FOTeae Dicoryphe — Trichocladus DICinae Neostrearia (HAMeae) Noahdendron Ostrearia _ Eustigma Fortunearia EUSeae

Molinadendron -FOTeae Sinowilsonia _ Distylivm — Distyliopsis

Fothergilla OJ 0) 5C « a) Hamamelis — Parrotiopsls —

Disanthus (d0) d) Mytilaria •HTJ O CQ Exbucklandia —I w* Rhodoleia RHOideae

Llquldambar — ALToideae

236

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.11. Single shortest tree of 7546 steps based on the combined data set of morphology, ITS and matK of the Hamamelidaceae. CI=0.61, RC=0.37. Numbers above and below branches are bootstrap percentages and decay indices. Groupings on the right side follow Endress (1989c). ALToideae=Altingioideae, EXBoideae=Exbucklandioideae, RHOideae=Rhodoleioideae, HAMoideae=Hamamelidoideae, COReae=Corylopsideae, HAMeae=Hamamelideae, HAMinae=Hamamelidinae, FOTeae=Fothergilleae, EUSeae=Eustigmateae, LORinae=Loropetalinae, DICinae=Dicoryphinae.

237

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 'Alti.ngi.6i — ALToideae

Cory lops is — coReae

I O r ° P e t a I u m -|L0R i„ . e Maingaya -I

Matudaea — FOTeae Dicoryphe — Trichocladus DXCinae Neostrearia (HAMeae) Noahdendron Ostrearia Eustigma id(U Fortunearia a) •H•a Molinadendron o Sinowilsonia— Distylium — Distyliopsis (00) Sycopsis e o Shaniodendron fa Parrotia

Paurrotiopsis v d) Fothergilla - •h s Hamamelis -- a> Disanthus •a Mytilaria •H O CD X Exbucklandia —I w Rhodoleia - RHOideae 1 Liquidambar — ALToideae

238

I

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.12. Single most parsimonious tree found in the analysis of the combined data of morphology, ITS and matK, showing distribution of character states of leaf form in the Hamamelidaceae.

239

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Liquidambar Shaniodendron Hamamel is Hamamel Trichocladus Parrotiopsis Ostrearia Fothergilla Eustigma Sinowilsonia Dicoryphe Noahdendron Molinadendron Neostrearia is Corylops Loropetalum Maingaya Mytilaria iExbucklandia \Disanthus i nDistylium uDistyliopsis nFortunearia a mParrotia a a o aMatudaea in in jaAltingia ■■ Sycopsis in in in mRhodoleia □a □n l=in jpzin in "I*— _r— in [fE * cm K> O

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.13. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morophology, ITS and matK. showing distribution of the number of ovules per carpel in the Hamamelidaceae.

241

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Sycopsis Shaniodendron Liquidambar Parrotiopsis Trichocladus Parrotia Hamamelis Sinowilsonia Eustigma Fortunearia Dicoryphe Ostrearia Molinadendron Neostrearia Noahdendron Corylopsis Loropetalum Disanthus Maingaya Exbucklandia Mytilaria Rhodoleia Matudaea \Distylium iDistyliopsis i i \Fothergilla aAltingia i i to to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.14. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK, showing distribution of the character states of petals in the Hamamelidaceae.

243

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. q q tn O 01 O (Q 0} q Vh q <0 M *H 3 q •H <0 01 XJ oi rH -H XJ t? XJ •H XI § Q< q Q, -h oi ^ q o oi q Ih q q q q O oi qqo-H-wqqqoi •q m q -H q q q H ^ *H -H XI tn «s XJ q (h & XJ M M 01 o U 01 1 § <0 'H q q q q q -H ft "H 0) E q -q in Oi XI +J +J o C •H O OII O -w O12. 01 01 o (0 q O *H —I -H •H 'H S, .q q *0 a -h -h w q q Q Q tQ to S! to Q Eh BBBBBBBBQBIBBBQBBBB

T Petal unordered lyl&yl auriculate dorsaly orbicular long clawed orbicular ribbon-like coiled circinately ribbon-like but not coiled ribbon-like and fleshy ■ ■ ribbon-like with broad base ■ ■ reduced ■ ■ absent fevAvt uncertain I i equivocal

244

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.15. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of pollination types in the Hamamelidaceae.

245

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sycopsis Distylium Diatyliopsis Shaniodendron Trichocladus Parrotia Parrotiopsis Fothergilla Hamamelis Sinowilsonia Altingia Liquidambar Dicoryphe Ostrearia Neostrearia Noahdendron Corylopsis Molinadendron Loropetalum Disanthus Exbucklandia Rhodoleia Maingaya Matudaea Mytilaria \Eustigma iFortunearia

I I bird P.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.16. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK. showing distribution of character states of sexuality in the Hamamelidaceae.

247

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

a lari Shaniodendron Distyliopsis Parrotia Altingia s i Hamamel Trichocladus Parrotiopsis Fothergilla Sinowilsonia Dicoryphe Ostrearia Eusti gma Eusti Fortunearia Neostrearia Molinadendron Corylopsis Noahdendron Disanthus Loropetalum Maingaya Matudaea Rhodoleia

Myt i i Myt i nDistylium aSycopsis mLiquidambar in in i o o a in in v d m in L = . r— in l UzzaaExbucklandia j = i n ~~|Lmn PM L

h to a> c oi to ss a p> a a> H- H H- 2 H cr (0 a> 3 3 h H- 0 3 (0 2! o O 3 n> o 1 d o 3 0 30 a r H- o e(0 O K> 00

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig.4.17. Strict consensus of the two most parsimonious trees found in the analysis of the combined data of morphology, ITS and matK, showing distribution of character states of chromosome base numbers in the Hamamelidaceae.

249

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

Sycopsis Shaniodendron Liquidambar Hamamelis Altingia Trichoc ladusTrichoc Parrotia Fothergilla Dicoryphe Fortunearia Parrotiopsis Neostrearia Corylopsis Eustigma Noahdendron Mol i nadendron i Mol Maingaya Matudaea Exbucklandia Mytilaria Rhodoleia i i aSinowilsonia aOstrearia nDistylium a uDisanthus i i \nLoropetalum •saa •saa — in hzaaDistyliopsis Rssa Rssa razan razan fclrmn fclrmn V j ■'■ -jtri ■'■ j :|tinna :|tinna L L nw

unordered rsj cn o

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V

TRANSLATING PHYLOGENY OF THE HAMAMELIDACEAE INTO

A CLASSIFICATION SYSTEM

INTRODUCTION

Since the publication of The Origin of Species by

Charles Darwin in 1859, taxonomists have been pursuing

classifications that reflect the natural history (phylogeny)

of organisms. In recent decades, the development of computer

technology and molecular systematics, together with improved

techniques for classical studies of morphology, embryology

and floral ontogeny, etc. has rejuvenized the field of plant

systematics. A number of explicit phylogenetic hypotheses

for some plant groups have been proposed to be translated

into classification systems (Cantino et al., 1995; Donoghue,

1995; Morgan, 1995; Olmstead, 1995). The exercise of making

the necessary taxonomic and nomenclatural changes has been

urged for systematists conducting phylogenetic analyses

(Judd, 1995). For the Hamamelidaceae, the most recently

proposed classification system has largely improved the

previous systems in terms of recognizing monophyletic groups

(Endress, 1989c); however, my phylogenetic analyses (Chapter

IV) based on both morphological and molecular data have shown

that some groups are still polyphyletic. In order to reflect

251

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. new understandings of the natural relationships in the

Hamamelidaceae, I present in this chapter a new

classification system for the Hamamelidaceae.

MATERIALS AND METHODS

The combined analysis using data from morphology, ITS

and matK DNA sequences was conducted as in Chapter IV.

The recognition of only monophyletic groups as taxonomic

units is widely accepted by systematists; however, there are

alternative ways to formally name these groups (Kron, 1995).

One approach is the sequencing convention (Wiley et al.,

1991), in which sister groups are considered "equivalent" and

given the same ending, thus allowing familiar names and

endings to be applied. However, the proliferation of names

and the possible "loss" of some well-known names becomes a

concern, particularly in regions of the tree that are highly

asymmetric (pectinate). According to de Queiroz and Gauthier

(1995), a phylogenetic systematic approach names clades

without the restrictions imposed by the Linnaean hierarchical

approach. However, abandonment of the Linnaean hierarchy

obscures the inherently hierarchical nature of genealogy,

thus creating difficulties in communicating the known

phylogenetic relationships (Kron, 1995). Therefore,

following the belief that the goal of an evolutionary

taxonomist is to develop a classification system that

252

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. recognizes monophyletic groups, but disrupts the present

classification as little as possible (Funk, 1985), I

translate the combined phylogeny (Fig.4.11) into an ordered

and phylogenetically annotated classification system for the

family, recognizing monophyletic clades, and keeping well-

known names intact whenever it is reasonable to do so. The

classification system starts from the basal clade up, with

each taxon followed by a brief note indicating its

phylogenetic position.

RESULTS

The phylogeny produced in Chapter IV, based on the

combined data set, resolved the intergeneric relationships of

the Hamamelidaceae (Fig.4.11). Six monophyletic groups,

herein treated as subfamilies, are recognized, including

Altingioideae, Rhodoleioideae, Exbucklandioideae,

Mytilarioideae, Disanthoideae, and Hamame lido ide ae. Within

the Hamamelidoideae, six tribes are recognized. Tribe 1.

Corylopsideae, Tribe 2. Loropetaleae, Tribe 3. Eustigmateae,

Tribe 4. Hamamelideae, Tribe 5. Fothergilleae, and Tribe 6.

Dicorypheae.

DISCUSSION

It is rarely disputable that defining monophyletic

groups should be the mission of taxonomy, and that

253

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phylogenetic patterns should be embodied in the

classification system. In the Hamamelidaceae, several

monophyletic groups are recognized in the present study,

including Altinaia-Semiliquidambar-Liquidambar. Exbucklandia

Rhodoleia, Mytilaria. Disanthus. and the subfamily

Hamamelidoideae. The latter group, the Hamamelidoideae,

contains three major clades: 11 Corvlopsis-Loropetalum-

Maingaya-Matudaea. 2) Eustiama-Fortunearia-Mo1inadendron-

Sinowilsonia-Dicoryphinae. and 3) Hamamelis-Fotherqilleae.

Some of the monophyletic groups share one or several

morphological character states, while others do not. When a

clade does not have a common morphological characteristic(s )

treating the major clade as a taxon of whatever rank is not

reasonable in terms of communicating phylogeny among

systematists. Therefore, it may be preferable to split a

monophyletic group so that a practicable definition can be

made (Bremer, 1978). Accordingly, I divided certain clades

into two (or more) equally-ranked taxonomic units;

furthermore, their close relationships are indicated in the

classification system by explicitly stating their sister

relationships.

Altinqia-Semilicruidambar-Liauidambar

These three genera are distinct from the rest of the

members of the Hamamelidaceae in many morphological

characters and nucleotide substitutions (See Chapter I, II,

254

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ill), thus there is no doubt that they are a monophyletic

group. However, the controversy has been whether they should

be separated from the rest of the Hamamelidaceae and be

recognized as an individual family (Nakai, 1943; Melikian,

1973; Huang and Lee, 1982; Pan et al., 1990; Wang, 1992;

Zhang and Lu, 1995; Takhtajan, 1997). Molecular studies have

shown that Liquidambar and Altingia are closely knit with the

other Hamamelidaceae (Hibsch-Jetter, 1996, and pers. comm.;

Hoot and Crane, 1996; Hoot et al., 1997). Therefore, these

three genera can be treated either as a subfamily

Altingioideae, or as a separate family Altingiaceae closely

related to the Hamamelidaceae. However, I prefer to retain

them in the Hamamelidaceae because many of the charateristics

that have been used to define them as a family Altingiaceae

can also be found in some genera of the Hamamelidaceae, such

as monoecy, polyporate pollen grains, and apetaly.

Exbucklandioideae and Rhodoleioideae

Exbucklandia and Rhodoleia have long been treated as

monogeneric subfamilies (Harms, 1930; Chang, 1948, 1979).

However, Endress (1989c) expanded the Exbucklandioideae to

include not only Exbucklandia. but Disanthus, Mytilaria. and

Chunia as well, based on their having 5-8 ovules per carpel

and their possession of persistent stipules. In contrast,

the present phylogeny associates Exbucklandia with Rhodoleia,

supporting the treatment of putting the two genera into one

255

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. subfamily proposed by Reinsch (1889). However, these two

genera are rather different morphologically, even though both

have complex capitulate inflorescences. Leaves are palmate-

veined in Exbucklandia. but pinnatly-veined in Rhodoleia:

flowers are andromonoecious, rarely have petals, are

symmetric, and possibly wind-pollinated in Exbucklandia.

while in Rhodoleia. flowers are bisexual, have asymmetrically

distributed showy petals (usually on the abaxial side only),

and are bird pollinated. In addition, the chromosome base

number is x=8 in Exbucklandia and x=12 in Rhodoleia.

Therefore, Exbucklandia and Rhodoleia are treated herein as

closely related individual subfamilies.

Mytilarioideae

Mytilaria was considered as a little known genus by

Harms (1930), and not included in his system. Chang (1948)

described the genus Chunia, and erected a subfamily,

Mytilarioideae, for Mytilaria and Chunia. These two genera

are rather similar in leaf morphology, spadix inflorescence

(Chang, 1979), presence of resin canals (Huang, 1986), and

multilacunar nodal anatomy (Bogle, 1990, 1991).

Unfortunately, material of Chunia was not available for

molecular study (the genus is rare, and endemic to Hainan).

Mytilaria. Chunia, Disanthus. and Exbucklandia have recently

been combined into a subfamily Exbucklandioideae by Endress,

(1989c). However, the paraphyletic relationships among the

256

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genera do not support this union of the four genera.

Instead, Mytilaria forms its own clade. Therefore, the

Mytilarioideae should be recognized as a subfamily.

Interestingly, karyotype study has shown that Mytilaria has a

chromosome base number of x=13 (the chromosome number in

Chunia is unknown), which is different from any other

Hamamelidaceae (x=13 has also been reported in Liquidambar

(Huang et al., 1985, but no photograph was provided, and the

report could be spurious), thus supporting the recognition of

this genus as a subfamily. The systematic position of Chunia

needs further study, but I place this genus in the subfamily

Mytilarioideae for the time being, based on their

morphological similarities.

Disanthoideae

The paraphyly of Disanthus with Mytilaria and

Exbucklandia does not provide support for the expanded

Exbucklandioideae sensu Endress (1989c). Instead, Disanthus

forms its own clade. Disanthus is specialized in many

morphological characters, including rounded palmately-veined

leaves, strap-shaped petals with broad and glandular bases,

capitulate inflorescence with two flowers fused back to back,

and two or more seeds in each carpel locule. Therefore, I

treat Disanthus as representing a monogeneric subfamily

Disanthoideae. This treatment agrees with several studies

(Harms, 1930; Chang, 1979; Takhtajan, 1997). Disanthoideae

257

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. have been proposed to be the most primitive taxon in the

Hamamelidaceae (Harms, 1930; Pan et al., 1991; Takhtajan,

1997). However, it is intercalated between Mvtilaria and the

Hamamelidoideae in the phylogenetic tree, suggesting that it

is evolutionarily intermediate in the Hamamelidaceae.

Obviously, Disanthoideae are a transitional taxon between the

Hamamelidoideae and other more primitive subfamilies.

Hamamelidoideae

There is no doubt that the Hamamelidoideae are a

monophyletic group (Bogle and Philbrick, 1980; Hufford and

Crane, 1989; Endress, 1989a, b, c). However, morphologies

are so diverse in this subfamily that controversies

concerning tribal and subtribal relationships of the

Hamamelidaceae exist only for this subfamily.

Corylops ideae

The tribe Corylopsideae sensu Harms (1930) included

Corylopsis and Fortunearia. while Schulze-Menz (1964)

transferred Sinowilsonia to this tribe due to his recognition

of the similarity of Sinowilsonia and Fortunearia. Recently,

Endress (1989c) separated Corylopsis from Fortunearia and

Sinowilsonia and recognized it as a monogeneric tribe. The

present phylogenetic analysis recommends the monophyletic

group of Corylopsis-Loropetalum-Mainqaya-Matudaea (Fig.4.11).

However, Corylopsis, as pointed out by Endress (1989b,c), is

258

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the only genus in the family, even the entire subclass

Hamamelidae, that has clawed, orbicular petals. This unique

characteristic, together with several other attributes, such

as presence of seed caruncle and polyembryogeny (Li and

Bogle, unpublished), led me to recognize Corylopsis as a

monogeneric tribe following Endress (1989c). This tribe is

closely related to Loropetalum-Mainqaya-Matudaea.

Loropetaleae

Loropetalum. Maingaya, and Tetrathyrium. along with a

fourth genus, Embol anther a. which was not available for this

study, but may soon become available, have been placed in the

tribe Hamamelideae (Harms 1930) and subtribe Loropetalinae of

the Hamamelideae (Endress, 1989c), while Matudaea is placed

in the apetalous group of the Hamamelidoideae, namely, the

tribe Fothergilleae (Endress, 1989c). However, the present

phylogenetic analysis puts these four genera in a well-

supported clade (Figs.2.2, 3.5, 4.11). There cure two

morphological characters shared by these four genera,

including distinct adaxial anther connective protrusions and

bisexual flowers. Nevertheless, Matudaea is apetalous, and

its ovaries are almost superior, while Loropetalum. Maingaya,

and Tetrathyrium have showy strap-shaped petals, and almost

inferior ovaries. Therefore, different subtribes could be

recognized. However, I have not done that in this study for

several reasons. First, in the phylogeny (Fig.4.11),

259

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Loropetalum is sister to the clade of Maingaya and Matudaea.

Thus, if Matudaea is treated as a subtribe, the other genera

should not be recognized as the other subtribe because they

do not belong to a single clade, and thus are not

monophyletic. Second, Embolanthera was not available for

this study, and it is possible that inclusion of Embolanthera

in a future analysis may change the intergeneric

relationships of these genera.

Hamamelideae

The definition of the tribe Hamamelideae has not been

changed since Harms (1930), although Endress (1989c) revised

its infratribal relationships. The floral specializations of

Hamamelis earned it monogeneric subtribal status (Endress,

1989c). In the present phylogenetic tree, this genus is in a

separate clade from the other members of the Hamamelideae,

thus recognizing its isolated systematic position. Instead,

Hamamelis has a sister relationship with the Fothergilleae

sensu Endress (1989c) (excl. Matudaea and Molinadendron).

However, while Hamamelis is similar in leaf morphology with

several genera of the Fothergilleae sensu Endress (1989c),

including Fotherailla. Parrotia, Parrotiopsis. and Parrotia,

it is rather different from the Fothergilleae in floral

structure. For example, in Hamamelis. flowers are strictly

4-merous, have showy petals, pre-carpel initiation of

staminodial nectars, and are bisexual (Mione and Bogle,

260

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1990), while in the Fothergilleae sensu Endress (1989c),

flowers are apetalous, andromonoecious, and number of stamen

is variable (Bogle, 1970). Therefore, I prefer to recognize

two tribes in the Fothergilleae clade (Fig.4.11), one being

the Hamamelideae (Hamamelis), and the other the Fothergilleae

(Fothercrilla. Parrotiopsis. Parrotia, Shaniodendron,

Sycopsis. Distyliopsis, and Distylium).

Eusticmateae

Eustigmateae sensu Harms (1930) is a monogeneric tribe,

while that sensu Endress (1989c) includes three genera,

Eustiqma. Fortunearia. and Sinowilsonia. Based on the

present phylogeny, Molinadendron also belongs to the

Eustigmateae and the grouping is strongly supported

(Fig.4.11). Therefore, this study recognizes the

Eustigmateae sensu Endress (1989c), but with the addition of

Molinadendron. This tribe is supported by a couple of

synapomorphies, such as linear stipules and two prophylls on

a branch. Within this tribe, Eustiama and Fortunearia are

closely related to each other and this pattern is supported

by several synapomorphies, including pedicellate and

lenticellate capsules, expanded purplish stigmatic surfaces,

and reduced petals. On the other hand, Sinowilsonia and

Molinadendron are allied in a clade, suggesting a close

relationship. The synapomorphies supporting the clade of

Molinadendron-Sinowilsonia include wind pollination and great

261

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduction or absence of petals (Bogle, 1970; Endress, 1969).

Dicorypheae

It is Endress (1989b,c) who first recognized the

possible monophyly of the five genera endemic to the

Gondwanaland Continents of the Southern Hemisphere (Africa,

Madagascar, Australia). These genera, Dicoryphe.

Trichocladus. Ostrearia, Noahdendron. and Neostrearia. share

this geographic distribution and a special pattern of anther

dehiscence in which anthers are tetrasporangiate and

bithecate, but dehisce via one valve per theca. This

characteristic does not occur in any other members of the

Hamamelidaceae (Endress, 1989a). In the present phylogeny

(Fig.4.11), these five genera form a strongly supported

clade, within which Dicoryphe and Trichoc ladus. and the three

Australian genera form their own clades respectively. Some

species of the former two genera tend to have oppositely

arranged, peltate leaves, persistent stipules, and stellate

foliar trichomes. The three Australian genera are very

closely related and share a couple of synapomorphies,

including scale trichomes (Warren Davis, per. comm.) and

short styles (Endress, 1989a, b).

An annotated classification system for the Hamamelidaceae

As discussed above, I propose below a detailed,

phylogenetically annotated classification system for the

262

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Hamamelidaceae R.Br.

Subfam.I. Altingioideae Reinsch (= Liquidambaroideae

Harms) - basal clade in the Hamamelidaceae and paraphyletic

to Exbucklandioideae-Rhodoleioideae.

Liqniriamhar L.

Semiliquidambar Chang

Altinqia Nor.

Sub£am.II. Exbucklandioideae Reinsch (= Bucklandioideae,

Symingtonioideae) - sister to Rhodoleioideae.

Exbucklandia R.W.Br. (= Bucklandia Griffith, Svmincrtonia

Steenis).

Subfam.IIl. Rhodoleioideae Harms

Rhodoleia Champ.ex Hook.

Subfam.IV. Mytilarioideae Chang - paraphyletic to

Disanthoideae

Mytilaria Lecomte

Chunia Chang

Subfam.V. Disanthoideae Harms - sister to Hamamelidoideae.

Disanthus Maxim.

Subfam. VI. Hamamelidoideae Reinsch - tribes I and II as

one basal clade.

Tribe I. Corylopsideae Harms - sister to

Loropetaleae.

Corylopsis Sieb. et Zucc.

263

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tribe II. Loropetaleae Li. Trib. Nov.

Loropetalum R.Br.ex Reichb.

Tetrathyrium Benth.

Maingaya Oliv.

Embolanthera Merr.

Matudaea Lundell

Tribe III. Hamamelideae A. DC. - sister to

Fothergilleae.

Hamamelis L.

Tribe IV. Fothergilleae Harms

Fotherqilla Murray

Parrotiopsis Schneider

Parrotia Mey.

Shaniodendron Deng, Wei, et Wang

Svcopsis Oliv.

Distvlium Sieb. et Zucc.

Distvliopsis Endress

Tribe V. Eustigmateae Harms - sister to Dicorypheae.

Eustiqma Gardn. et Champ.

Fortunearia Rehd. et Wils.

Sinowilsonia Hemsl.

Molinadendron Endress

Tribe VI. Dicorypheae Li. Trib. nov.

Dicoryphe Du Petit-Thouars.

Trichocladus Pers.

Neostrearia Smith

264

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Ostrearia Baill.

Synopsis of the Hamamelidaceae

Family Hamamelidaceae R.Brown, Abel.Narr. Journey China:

374 (1818) ("Hamamelideae"). (Harms, 1930; Chang, 1979;

Endress, 1989c; Zhang and Lu, 1995).

Trees or shrubs. Leaves persistent or deciduous,

generally alternate or rarely opposite, stipulate, simple or

palmately lobed; venation pinnate or palmate; epidermal

trichomes simple, stellate or peltate scales. Inflorescences

racemose, spicate or capitate, sometimes subtended by showy

bracts. Flower morphology very variable, ranging from

complete to incomplete, from bisexual to andromonoecious, and

to monoecious; calyx composed of four or five united sepals,

variable or lacking; corolla of four or five distinct petals,

reduced or lacking; stamens (2)-4, 5, 10-(>20), anthers

tetrasporangiate or bisporangiate; ovaries 2-carpellate,

superior, semi-inferior, or inferior, each locule with one or

more ovules. Fruit, a capsule, with woody exocarp and horny

endocarp. Seeds one to several per carpel, winged or

unwinged; seed coat bony, embryo straight, and endosperm

albuminous.

Subfamily I. Altingioideae Reinsch, Bot. Jahrb. Syst. 11:

389, 1889 (Harms, 1930; Bogle, 1968, 1970; Chang, 1979;

265

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Endress, 1989c; Zhang and Lu, 1995).

Evergreen or deciduous trees. Leaves ovate, simple to

tricuspidate or palmately lobed, pinnately or palmately

veined, margins toothed or rarely entire. Stipules linear

and caducous. Flowers unisexual, sessile, aggregated into

dense capitula (or spikes). Perianth lacking. Stamens

numerous, anthers tetrasporangiate and bithecate. Ovaries

bicarpellate, semi-inferior, each carpel with numerous

ovules. Seeds, winged, not ejected ballistically.

Chromosome base number x=8. Three genera and about 20

species in eastern and western Asia, Central and North

America.

Subfamily II. Exbucklandioideae Reinsch

(= Bucklandioideae), Bot. Jahrb. Syst. 11: 389, 1890 (Harms,

1930; Bogle, 1968; Chang, 1979; Zhang and Lu, 1995).

Evergreen trees. Leaves simple, broadly ovate to 3-

lobed, palmately veined, margins entire. Stipules large,

appressed, elliptic, and persistent. Flowers

andromonoecious, aggregated as dense capitula. Petals

reduced or lacking. Stamens numerous. Anthers monothecate.

Ovaries semi-inferior. Ovules numerous. Seeds winged. One

genus and about four species, distributed in southeastern

Asia.

266

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subfamily III. Rhodoleioideae Harms in Engler et Prantl,

Nat. Pflanzenfam., 2 ed., 18a: 335, 1930 (Bogle, 1968, 1986;

Chang, 1979; Bogle and Philbrick, 1980; Endress, 1978b,

1989c, 1993; Zhang and Lu, 1995).

Evergreen trees or shrubs. Leaves simple, ovate, rarely

cordate, pinnately veined, margins entire. Stipules mostly

lacking. Flowers bisexual and zygomorphic, aggregated into a

pseudanthium, surrounded by an involucre of broad bracts, and

bird-pollinated. Sepals and petals present. Stamens 6-8,

anthers bithecate. Ovaries semi-inferior, bicarpellate, each

carpel with 10-20 ovules. Seeds, winged, not ejected out of

the capsule. Chromosome base number x=12. One genus and

about 10 species in eastern Asia and Malaysia.

Subfamily IV. Mytilarioideae Chang, Sunyatsen Univ. Bull.

1: 57, 1973 (Chang, 1948, 1979; Bogle, 1968; Pan and Yang,

1994; Zhang and Lu, 1995).

Evergreen tree. Leaves broadly ovate to 3-lobed,

palmately veined, margins entire. Stipules large, appressed

and persistent. Flowers bisexual, spirally arranged on a

spadix. Sepals present or absent, petals strap-like or

absent. Stamens 8-13. Ovaries inferior, sunken in spadix.

Carpels with 6 ovules. Chromosome base number x=13 (unknown

for Chunia). Two genera and 2 species in southern China,

Hainan, and northern Laos and northern Vietnam.

267

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Subfamily V. Disanthoideae Harms in Engler et Prantl. Nat.

Pflanzenfam. 2 ed., 18a: 316, 1930 (Bogle, 1968; Chang, 1979;

Pan et al., 1991; Zhang and Lu, 1995).

Deciduous shrubs or small trees. Leaves simple, broadly

ovate to orbicular, margins entire, palmately veined.

Inflorescence capitulate, two-flowered. Flowers bisexual, 5-

merous. Sepals present, petals strap-like, circinately

coiled. Ovaries semi-inferior. Carpels with 4-6 ovules.

Chromosome base number x=8. One genus and one species in

southern Japan and several provinces of eastern and central

China.

Subfamily VI. Hamamelidoideae Reinsch, Bot. Jahrb. Syst.

11: 389, 1890 (Endress, 1967, 1970, 1989a, b,c, 1993; Bogle,

1968, 1970; Chang, 1979; Zhang and Lu, 1995).

Trees or shrubs, evergreen or deciduous. Leaves simple,

pinnately veined. Flowers, bisexual, andromonoecious, or

monoecious. Inflorescence a spike, raceme, or capitulum.

Perianth present or lacking. Ovaries superior to inferior.

Carpels mostly with one ovule (rarely one fertile and two

sterile). Seed ejected at maturity. Chromosome base number

x=12). Twenty three genera and about 100 species widely

distributed in Asia, North, Central, and South America,

Australia, Africa, and Madagascar.

268

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tribe I. Corylopsideae Harms in Engler et Prantl, Nat.

Pflanzenfam., 2 ed., 18a: 325, 1930 (Bogle, 1968; Morley and

Chao, 1977; Endress, 1989b, c; Li et al., 1993).

Deciduous or rarely semi-evergreen shrubs, or small

trees. Flowers 5-merous, bisexual. Sepals present. Petals

yellow, orbicular, and clawed. Carpels with 2-3 ovules (one

fertile and two sterile). Capsules woody, with curved beaks.

One genus and 11 species in southeastern Asia.

GENUS TYPICUM: Corylopsis Sieb.et Zucc.

Tribe II. Loropetaleae Li. Trib. Nov.

Flores bisexuales. Petala lineari-eloncata v. nulla.

Antherae basifixae. connectivo producto acuminato.

Four genera about 5 species in southeastern Asia.

GENUS TYPICUM: Loropetalum R.Br.ex Reichb.

Tribe III. Dicorypheae Li. Trib. Nov.

Flores bisexuales. Petala lineari-elonaata. Antherae

bithecae. valva unica dehiscens. Five genera and about 20

species in eastern and southern Africa, northeastern

Australia, Madagascar, and Comoros Islands).

GENUS TYPICUM: Dicoryphe Du Petit-Thouars.

Tribe IV. Eustigmateae Harms in Engler et Prantl, Nat.

Pflanzenfam., 2 ed., 18a: 324, 1930 (Endress, 1989c).

Buds naked. Branch prophylls 2. Flowers bisexual or

269

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Stamens 5 or variable. Four genera and about eight species

distributed in eastern Asia and central America.

GENUS TYPICUM: Eustiqma Gardn. et Champ.

Tribe V. Hamamelideae A. DC. Prodr. Syst. Nat. 4: 268,

1830 (as Hamameleae) (Mione and Bogle, 1990).

Flowers 4-merous, bisexual, with ribbon-like, showy

petals. Carpels with 2-3 ovules (one fertile and one or two

sterile). One genus and about 5 species in eastern Asia,

Central and North America.

GENUS TYPICUM: Hamamelis L.

Tribe VI. Fothergilleae A. DC., Prodr. Syst. Nat. 4: 269,

1830 (incl. Distylieae Harms in Engler et Prantl, Nat.

Pf lanzenf am., 2 ed., 18a: 331, 1930) (Endress, 1989c, excl.

Molinadendron and Matudaea).

Flowers andromonoecious, apetalous (in a few genera also

asepalous), Sepals and stamens highly variable. Eight genera

and about 20 species in southeastern and western Asia, and

eastern North America.

GENUS TYPICUM: Fotherqilla Murray

270

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GENERAL CONCLUSIONS

The classification system of Endress (1989c) recognized

four subfamilies in the Hamamelidaceae, four tribes in the

subfamily Hamamelidoideae, and three subtribes in the tribe

Hamamelideae. In the present study, parsimony analyses of

the Hamamelidaceae were conducted based on both morphological

and molecular data to examine the naturalness of the groups

in the Endress classification system.

The phylogeny based on sequences of the Internal

Transcribed Spacers (ITS) of nuclear ribosomal DNA is

basically congruent with that based on nucleotide sequences

of the chloroplast gene matK. In contrast, the phylogeny

based solely on morphology hardly agrees with the molecular

phylogenies.

A combined analysis using the pooled data of morphology

and DNA sequences (ITS and matK) resolves all of the

intergeneric relationships in the Hamamelidaceae. The

resulting phylogeny recognizes the naturalness of several

suprageneric taxa in Endress's system (1989c), including the

subfamilies Altingioideae, Rhodoleioideae, and

Hamamelidoideae. In the Hamamelidoideae, the tribes

Corylopsideae, Eustigmateae, and Fothergilleae, and the

271

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. subtribes Dicoryphinae, Hamamelidinae, and Loropetalinae are

also natural. However, several taxa are suggested to be

polyphyletic, including the subfamily Exbucklandioideae and

the tribe Hamamelideae. The combined phylogeny also

indicates that the apetalous, wind-pollinated genera in the

Hamamelidoideae do not form a single clade, but are

distributed in three separate clades, suggesting that

parallel evolution has occurred in the family.

A revision of the current classification system of the

Hamamelidaceae is carried out to reflect the phylogenetic

relationships suggested by the present analysis, resulting in

a phylogenetically annotated suprageneric classification

system. This new classification system recognizes six

subfamilies, and six tribes in the subfamily Hamamelidoideae.

At the subfamily level, this new classification system agrees

with the system of Chang (1979), while at the tribal level,

this system recognizes the four tribes in the system of

Endress (1989c), with two new tribes Loropetaleae and

Dicorypheae. The six subfamilies include the Altingioideae

(basal), Exbucklandioideae (sister to Rhodoleioideae),

Rhodoleioideae, Mytilarioideae (paraphyletic to

Disanthoideae), Disanthoideae (sister to the Hamamelidoi­

deae), and Hamamelidoideae. The six tribes are Corylopsideae

(sister to Loropetaleae), Loropetaleae, Eustigmateae (sister

to the Dicorypheae), Dicorypheae, Hamamelideae (sister to the

Fothergilleae), and Fothergilleae.

272

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LITERATURE CITED

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Arnheim, N., M. Krystal, G. Wilson, 0. Ryder, and E. A. Zimmer. 1980. Molecular evidence for genetic exchange among ribosomal genes on non-homologous chromosomes in man and apes. Proc. Natl. Acad. U.S.A. 77: 7323-7327.

Baillon, H. 1871. Saxifragacdes, Hist. PI. 3: 325-464. rHamamelis series, 389-397; 414-415; Hamamelideae, 456- 461. Liquidambar series, 397-400; 415; Liquidambareae, 461-462.]

______. 1871-1873. Nouvelles notes sur les Hamamdlidees. Adansonia 10: 120-137.

Baldwin, B. G. 1992. Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the Compositae. Mol. Phylogenetics Evol. 1: 3-16.

______, M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, C. S. Campbell, and M. J. Donoghue. 1995. The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247-277.

Barrett, M . , M. J. Donoghue, and E. Sober. 1991. Against consensus. Syst. Zool. 40: 486-493.

Bentham, G. 1861. Flora Hongkongensis. L. Reeve, London.

______and J. D. Hooker. 1865. Hamamelideae. Gen. PI. 1: 664-669.

Bogle, A. L. 1968. Floral vascular anatomy and the nature of the hamamelidaceous flower. Ph.D. thesis, University of Minnesota.

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