Systematics and Evolution Of The Palm Genus Areca

SYSTEMATICS AND EVOLUTION OF THE PALM GENUS

ARECA

BY

CHARLIE DANNY HEATUBUN

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

2009

I, Charlie Danny Heatubun, certify that this dissertation is my own original work and has not been submitted in a previous application for a higher degree.

June, 25th 2009

ABSTRACT

CHARLIE DANNY HEATUBUN, Systematics and Evolution of Palm Genus Areca _L. supervised by SRI S. TJITROSOEDIRDJO, JOHANIS P.MOGEA, WILLIAM J. BAKER, and MIEN A. RIFAI.

Areca L. (Arecoideae: Areceae: Arecinae) is treated in this study as a genus of South Asian–West Pacific palm and best known for its type species Areca catechu, the betel nut palm. This genus comprises 41 species and included into two subgenera; subgenus Areca

and subgenus Beccarioareca. Seven species are newly recognized (A. bakeri Heatubun, A. churchii Heatubun, A. dransfieldii Heatubun, A. gurita Heatubun, A. mogeana Heatubun,

A. riparia Heatubun, and A. triginticollina Heatubun). Eight previously recognized species (A. jobiensis Becc., A. multifida Burret, A. nannospadix Burret, A. nigasolu Becc., A. rechingeriana Becc., A. rostrata Burret, A. torulo Becc. and A. warburgiana Becc.) are reduced to synonymy with A. macrocalyx Zipp. ex Blume; two species (A. guppyana Becc. and A. salomonensis Burret) are also reduced to A. novohibernica (Lauterb.) Becc. and one species (A. celebica Burret) to A. oxycarpa Miq.; and also one species A. macrocarpa

Becc. is also synonym to A. catechu L. While, three species (A. chaiana J. Dransf., A. congesta Becc., and A. ledermanniana Becc.) are considered to species incertae sedis (doubtful or uncertain species). A determination key is presented to subgenera and all easts of Wallace’s line taxa, including their detailed descriptions and for new species. A phylogenetic analysis of certain species in the genus was performed based on DNA sequences from two low-copy nuclear genes, phosphoribulokinase (PRK) and the second largest subunit of RNA polymerase II (RPB2). The monophyly of Areca as genus is reconfirmed together with its two lineages are also recovered. Subgenera and sections in the genus were assessed based on phylogenetic relationship together with their biogeography explanation. Natural history observations, including uses and conservation status are also presented in this study.

Key words: palms, Arecaceae, Areca, systematics, morphology, DNA, phylogeny, classification, species.

ABSTRAK

CHARLIE DANNY HEATUBUN. Sistematika dan Evolusi Marga Palem Areca _L. Dibimbing oleh SRI S. TJITROSOEDIRDJO, JOHANIS P. MOGEA, WILLIAM J. BAKER dan MIEN A. RIFAI.

Areca L. (Arecoideae: Areceae: Arecinae) adalah marga palem yang distribusinya dari Asia Selatan sampai Pasifik Barat dan sangat terkenal dengan jenis tipenya Areca catechu, pinang sirih. Marga ini terdiri dari 41 jenis yang tergabung dalam dua anak marga; anak marga Areca dan anak marga Beccarioareca. Tujuh jenis adalah jenis baru (A. bakeri

Heatubun, A. churchii Heatubun, A. dransfieldii Heatubun, A. gurita Heatubun, A. mogeana Heatubun, A. riparia Heatubun, and A. triginticollina Heatubun). Delapan jenis yang sebelumnya dikenal (A. jobiensis Becc., A. multifida Burret, A. nannospadix Burret,

A. nigasolu Becc., A. rechingeriana Becc., A. rostrata Burret, A. torulo Becc. and A. warburgiana Becc.) merupakan sinonim baru untuk A. macrocalyx Zipp. ex Blume; dua jenis (A. guppyana Becc. and A. salomonensis Burret) juga merupakan sinonim A. novohibernica (Lauterb.) Becc. dan satu jenis (A. celebica Burret) untuk A. oxycarpa Miq.; demikian pula A. macrocarpa Becc. merupakan sinonim untuk A. catechu L. Sementera tiga jenis (A. chaiana J. Dransf., A. congesta Becc., and A. ledermanniana Becc.) dipertimbangkan sebagai jenis incertae sedis (meragukan atau jenis yang tidak pasti). Kunci determinasi ditampilkan untuk anak marga dan jenis-jenis yang berasal dari sebelah Timur Garis Wallace, demikian pula deskripsi lengkapnya dan juga untuk jenis baru. Analisis filogenetika untuk beberapa jenis pada marga didasarkan pada sekuens DNA dari dua low-copy gen inti; phosphoribulokinase (PRK) dan the second largest subunit of RNA polymerase II (RPB2). Monofiletik Areca sebagai marga kembali dikonfirmasi bersama dengan penemuan dua jalur evolusi di dalamnya. Anak marga dan seksi-seksi pada marga ini dikaji berdasarkan hubungan kekerabatan (filogeni) dan juga penjelasan tentang biogeografinya. Pengamatan tentang peri kehidupan alam termasuk kegunaan dan status konservasi juga ditampilkan dalam penelitian ini.

Kata kunci: palem, Arecaceae, Areca, sistematika, morfologi, DNA, filogeni, klasifikasi, jenis.

SUMMARY

Areca L. was the first palm genus was described by Linneaus in Species Plantarum in 1753 based on Rumphius’ Pinanga published in Herbarium Amboinense in 1741 (see Corner, 1966; Moore & Dransfield, 1979). It is also the type genus of the family name

Arecaceae Bromhead. This genus is best known for its type species, Areca catechu – the widely cultivated betel nut palm, the seed of which is chewed with the leaf or inflorescence of Piper betle L. (Piperaceae), lime and sometimes tobacco and spices, principally as a mild stimulant. Betel nut is consumed by an estimated 200–400 million people world-wide (Gupta & Warnakulasuriya, 2002; Loo et al., 2006; Dransfield et al., 2008) – extensively in the Asia-Pacific region and expatriate Asian communities – making betel nut the fourth most widely “abused” substance after nicotine, alcohol and caffeine (Norton, 1998; Loo, et al. 2006).

The genus Areca has approximately 47 species (Dransfield et al., 2008), and is distributed from India and south China through Malesia to New Guinea and the Solomon Islands (Dransfield, 1984; Uhl & Dransfield, 1987; Dransfield et al., 2008).

The objective of this research is to provide a modern taxonomic treatment of Areca

including studies of its phylogenetic relationships, natural history, uses and conservation status. The work will be based on exhaustive studies of existing literature, a thorough examination of herbarium materials in international herbaria, extensive fieldwork and laboratory-based molecular systematic research. The project consists of four main subprojects: 1). Taxonomic revision of the genus Areca; 2). Species level phylogeny estimation; 3). Comparative morphological and molecular study of the genus Areca with aim to understand the morphological changes that occurred during their evolution; 4). and if possible to reconstruct the historical biogeography and origin of A. catechu L.

Taxon sampling and observations for the morphology and distribution of the species were based on herbarium samples or specimens (dried and spirit-preserved materials) deposited at international herbaria, namely A, AAU, B, BH, BO, BRI, FI, K, KEP, L, LAE, MAN, PNH, SAN, SAR, and SING (herbarium acronyms follow Holmgren et al., 1990), as well as the newly established small herbarium in Balai Penelitian Kehutanan

(Forestry Research Institute) in Manokwari, West Papua, Indonesia. Measurements were taken from spirit-preserved material and dried herbarium specimens and from living collections. Floral parts were measured from spirit-preserved material or dried specimens and rehydrated by boiling. Basic morphological characters such as habit, stem, leaves, inflorescence, staminate flower, pistillate flower, fruit, seed and their details were used to describe and recognize taxa; all morphological data was used for producing the descriptions of each taxon, while the key to species was constructed from the diagnostic characters only. The morphological species concept or taxonomic species concept was applied as a framework to define taxa, and assessed later with a phylogenetic species concept (Davies & Heywood, 1963; McDade, 1995; Gornall, 1997; Mayden, 1997; Dransfield, 1999), especially based on the result of molecular phylogenetics analysis of the genus Areca (Heatubun et al., in prep.). The conservation status of each species of the genus Areca in east of Wallace’s line was assessed based on the IUCN red list categories and criteria version 3.1 (IUCN, 2001).

DNA-materials were collected directly in the fields or obtained from botanical gardens. Also were using collections of DNA bank of Jodrell Laboratory, Royal Botanic Gardens Kew, UK. All specimens will be fully vouchered with specimens, they were deposited in appropriate herbaria, and their identities confirmed. All available sequences of species of the genus Areca were included in the taxon sampling, with species of Nenga

and Pinanga (Loo et al. 2006), Bentinckia condapanna (Arecoideae: Areceae: unplaced),

Clinostigma savoryanum (Arecoideae: Areceae: unplaced), Cyrtostachys renda

(Arecoideae: Areceae: unplaced), Cyphokentia macrostachya (Arecoideae: Areceae:

Clinospermatinae) and Tectiphiala ferox (Arecoideae: Areceae: Oncospermatinae) as outgroup (Norup et al., 2006; Dransfield et al., 2008; Baker et al., 2009). For DNA extraction, total genomic DNA extracted from silica gel-dried leaf materials (Chase & Hills, 1991) using the 2×CTAB method of Doyle & Doyle (1987). DNA were precipitated with 100% ethanol at –20°C, purified by cesium chloride/ethidium bromide gradients (1.55 g/mL) followed by dialysis and removal of ethidium bromide using butanol. Primer sequences for PRK and RPB2 were obtained from published sources (Lewis & Martinez, 2000; Lewis & Doyle, 2002; Roncal et al. 2005; Loo et al. 2006; Norup et al. 2006; Trenel

et al., 2007; Cuenca et al., 2007). Sequence alignment follow Loo et al. (2006) and Norup

et al. (2006) which every base position in the reverse and forward sequences were check and assembled using Sequencher 4.1 (Gene Codes Corp, Ann Arbor, Michigan, USA) and will be deposited in GenBank. The sequences then enter and aligned manually into data matrices in PAUP* version 4b10 (Swofford, 2002) for Macintosh computer. Phylogenetic analyses in this study were using parsimony analysis and Bayesian analysis. the standard procedures as follow: congruence between the PRK and RPB2 datasets will evaluated using the incongruence length difference (ILD) test of Farris et al. (1994) as implemented in PAUP* (Swofford 2002) and Mr. Test in MrBayes version 3.0b4 (Huelsenbeck & Ronquist, 2001). In parsimony analyses, uninformative characters and ambiguously aligned regions were excluded. All included characters were unordered and equally weighted. Initial analyses employed 1000 heuristic searches, each with starting trees obtained by random taxon addition, tree-bisection-reconnection (TBR) swapping, and keeping multiple trees per step (MulTrees on). Only groups that were found in the strict consensus tree and 50% or more of the replicates were recorded. Bayesian analyses were carried out using the program MrBayes version 3.0b4 (Huelsenbeck & Ronquist, 2001). Models of sequence evolution that the best fit the individual datasets were determined using Mr. Test. The models were evaluated by the Akaike information criterion (Akaike, 1973) implemented in the program. Parameters based on patterns in the data matrices. For all three datasets, four incrementally heated Markov chains were used in an analysis that was run for 100,000 generations initially with trees saved at every 10th generations. Trees produced prior to stationarity were discarded as the burn-in.

From this study, Areca is interesting palm genus not only for the species diversity but also for its natural history. This genus comprises 41 species and included into two subgenera; subgenus Areca and subgenus Beccarioareca. Seven species are newly recognized (A. bakeri Heatubun, A. churchii Heatubun, A. dransfieldii Heatubun, A. gurita

Heatubun, A. mogeana Heatubun, A. riparia Heatubun, and A. triginticollina Heatubun). Eight previously recognized species (A. jobiensis Becc., A. multifida Burret, A. nannospadix Burret, A. nigasolu Becc., A. rechingeriana Becc., A. rostrata Burret, A. torulo Becc. and A. warburgiana Becc.) are reduced to synonymy with A. macrocalyx

Zipp. ex Blume; two species (A. guppyana Becc. and A. salomonensis Burret) are also reduced to A. novohibernica (Lauterb.) Becc. and one species (A. celebica Burret) to A. oxycarpa Miq.; and also one species A. macrocarpa Becc. is also synonym to A. catechu L. While, three species (A. chaiana J. Dransf., A. congesta Becc., and A. ledermanniana

Becc.) are considered to species incertae sedis (doubtful or uncertain species). A determination key is presented to subgenera and all easts of Wallace’s line taxa, including their detailed descriptions and for new species.

A phylogenetic analysis of certain species in the genus was performed based on DNA sequences from two low-copy nuclear genes, phosphoribulokinase (PRK) and the

second largest subunit of RNA polymerase II (RPB2). The monophyly of Areca as genus is reconfirmed together with its two lineages are also discovered. Subgenera and sections in the genus were assessed based on phylogenetic relationship together with their biogeography explanation. The morphological characters are still relevant and applicable to apply species concept in the genus Areca, however those characters are independently each others and evolved several times in this palm genus, so is needed a depth phylogeny interpretation to understand relationship and changes during the evolution. Dispersal is the main biogeography factor to reflect the recent distribution pattern of the genus Areca. The origin of the cultivated wide-spread species – Areca catechu L. is still unresolved from this study. Natural history observations, including uses and conservation status are also presented in this study.

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SYSTEMATICS AND EVOLUTION OF THE PALM GENUS

ARECA

BY

CHARLIE DANNY HEATUBUN

G 361060021

As partial requirement fulfilment for the Doctoral Degree

in Plant Systematics

DEPARTMENT OF BIOLOGY

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

2009

External Examiners

Examination I : Dr. Dra. Rugayah, M.Sc.

Herbarium Bogoriense, Puslitbang Biologi, Indonesia Institute of Sciences (LIPI), Cibinong, Bogor, Indonesia.

Dr. Dedy Duryadi Solihin, DEA

Head of Biology Study Programme, Faculty of Mathematics and Natural Science, Bogor Agricultural University,

Darmaga, Bogor, Indonesia.

Examination II : Dr. Timothy M. A. Utteridge

South-East Asia Section, Herbarium, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, United Kingdom.

Dr. Kuswata Kartawinata

Herbarium Bogoriense, Puslitbang Biologi, Indonesia Institute of Sciences (LIPI), Bogor, Indonesia; UNESCO office Jakarta, Indonesia and Department of Botany, Field Museum, Chicago, Illinois 60605-2496, USA.

Dissertation Title : Systematics and Evolution of the Palm Genus Areca

Name of Student : Charlie Danny Heatubun

Register Number : G361060021

Study Programme : Biology

Sub Study Programme : Plant Taxonomy

Approved by 1. Advisory Committee

(Dr. Sri S. Tjitrosoedirdjo, M.Sc) (Prof. Dr. Johanis P. Mogea) (Chairman) (Member)

(William J. Baker, M.Sc, Ph.D, FLS) (Prof. Dr. Mien A. Rifai) (Member) (Member)

2. The Biology Study Programme 3. The Graduate Schools

(Dr. Ir. Dedy Duryadi Solihin, DEA) (Prof. Dr. Ir. Khairil A. Notodiputro, MS) (Head) (Dean)

ACKNOWLEDGEMENTS

Many individuals and institutions contributed to the success of this work and I am grateful to them all. I would like to thank my advisors Dr. Sri S. Tjitrosoedirdjo, Professor Johanis P. Mogea, Dr. William J. Baker, Professor Mien A. Rifai and Dr. John Dransfield, whose always guided my work and gave me countless and useful recommendations to finished this dissertation. Professor Edwino S. Fernando is gratefully acknowledged for sharing his expertise on the Philippine’s Areca. Drs. Felix Forest, James J. Clackson and Professor Mark W. Chase to let me in to the lab, introduced me to DNA and Molecular Systematics, and guided me with the palm molecular phylogeny in the Jodrell Laboratory of Royal Botanic Gardens Kew. Drs. Christine D. Bacon and Raymond Baker from the Lyon Arboretum provided me with DNA materials of Areca from their garden collections in Hawaii. Dr. Tom D. Evans sent his collection of a new species of Areca from Cambodia. Professors Anders Barfod, Finn Borschenius and Henrik Balsev to invite me, arranged my travel and allowed me working with Areca collections in University of Aarhus and Copenhagen. Drs. Piero Cuccuini and Chiara Nepi also arranged my visit to Florence and allowed me working with the type specimens from Beccari’s historical collections. Ms. Julia Anak Sang and Mr. Shahbuddin Moh. Shabki are thanked for assistance with permits and all things regarding my fieldtrips to Sarawak. Dr. George Argent is responsible to my visiting in Edinburgh and Dr. Jef Veldkamp for my Leiden trip. Dr. Benito Tan and Ms. Serena Lee for accessing herbarium data base and type specimens of Areca kept in the Singapore Botanic Garden. I am grateful to the Keepers and/or curators of herbaria A, AAU, B, BH, BO, BRI, FI, K, KEP, L, LAE, MAN, PNH, SAN, SAR, SING and BPK Manokwari for access to their specimens, data bases, and loan materials for study. I also thank to Lucy T. Smith for preparing the wonderful plates for my new species. Drs. Rugayah, Dedy Duryadi Solihin, Timothy M. A. Utteridge and Kuswata Kartawinata are acknowledged for their willingness being examiners in my final examination.

I am grateful to Professor Eko B. Waludjo, Drs. Dedy Duryadi Solihin, Drajat Martianto, Dedy Jusadi, Marimin, Nunik Sri Aryanti, Harry Wiriadinata, Yohanes Purwanto, Dedy Darnaedi, Rugayah, Elisabeth Widjaja, Teguh Triono, and Ary Keim, and also Himmah Rustiami, Rani Asmarayani, Arief Hidayat from BO and IPB; Hans Arwam, Rudi

Maturbongs, Marthinus Iwanggin, Elieser Sirami dan Pilep Mambor from UNIPA; Daud Leppe, Herman Remetwa, Thomas Nifinruri and Krisma Lekitoo from BPK Manokwari; Drs. Rogier de Kok, Tim Utteridge, Aaron Davis and Soraya Villalba, and Melinda Trudgen, Edith Kapinos, Lzaslo Cziba, Steve Graham and Anne Morley-Smith from K; Hermus Indou, Herkilaus Rumaikewi, Tobias Paiki, Maikel Simbiak and Piter Matani supported my research in various way. My colleagues, friends and my Papuan families in Bogor always offered good suggestion and words of hope during the hard times of my study.

Rector and the Dean of Fakultas Kehutanan Universitas Papua (UNIPA) Manokwari, Rector and the Dean of Sekolah Pascasarjana Institut Pertanian Bogor (IPB), they allow me to do my PhD. Financial supports came from BPPS Dikti Depdiknas and Royal Botanic Gardens Kew, UK for the PhD scholarships. The Royal Botanic Gardens Kew and Royal Botanic Gardens Edinburgh were funded my fieldtrips to Tanah Papua and North Sulawesi. My fieldtrips to Sarawak was granted by International Palm Society (IPS Endowment Fund 2007); visit to Nationaal Herbarium of Nederland, Leiden Branch by Flora Malesiana Foundation (Kostermann Funds) and Beasiswa Unggulan Dikti Depdiknas; visit to Royal Botanic Gardens Edinburgh, Scotland by the Sibbald Trust; travel to herbarium of University Aarhus and Monocot IV Symposium in Copenhagen, Denmark by the university of Aarhus; visit to Xishuangbanna Tropical Botanical Garden, Yunnan, China by Centre for Tropical Forest Science, Harvard University and China Academy of Sciences; and visit to herbarium Sezione Botanico, Meseo di Storia Naturale, Università degli Studi di Firenze, Florence, Italy by Royal Botanic Gardens Kew. The plates were funded by Pacific Biological Foundation and the Royal Botanic Gardens Kew.

Finally, to my wife Oktarina Simanjuntak – Heatubun, my son Edward Glorious Excelsa Heatubun and my daughter Narcissa Elegantia Heatubun for continuous supports during my PhD studies, especially for their patience while I was away far from home.

CURRICULUM VITAE

Charlie Danny Heatubun is a son of Clemens Yoseph Heatubun (†) and Selfina Endemina Hursepuny (†). He was born on December 6th, 1974 in Manokwari, West Papua. In 1985 he was graduated from primary school, and secondary school in 1988 and also high school 1991, all in Manokwari, West Papua. He was continued his study at Department of Forestry, Faculty of Agriculture, Cenderawasih University in Manokwari and graduated in 1997.

Since 1997, he has been enrolled as academic staff in Forestry Department, Faculty of Agriculture; Cenderawasih University (now is Faculty of Forestry, University of Papua). In 2006, at the same year after finished his Master degree (M.Si) on Plant Taxonomy in Biology Department, the Graduate Schools of Bogor Agricultural University (SPs–IPB); he was continued his study to doctoral level in the same department of the same university with the scholarships from BPPS Dikti Depdiknas and the Royal Botanic Gardens Kew, UK. He was married Oktarina S. Simanjuntak and has a son Edward Glorious Excelsa Heatubun and a daughter Narcissa Elegantia Heatubun.

He is Fellow of the Linnean Society of London (FLS) and joint the International Association of Plant Taxonomy (IAPT), the Systematics Association (SYSTASS), the International Palm Society (IPS), the Biological Society of New Guinea, Indonesian Association of Plant Taxonomy (PTTI), and the IUCN Species Survival Commission (SSC) Palm Specialist Group (PSG) as member. He is the founder of Herbarium Papuaense of Forestry Research Institute in Manokwari (Balai Penelitian Kehutanan Manokwari) and he was the visiting fellow on several herbaria and botanic gardens in ASEAN, China, Australia and Europe. He is also co-authors of the books about tree species of Gunung Meja in Manokwari, West Papua and the edible fruits from the same area. His articles related with plant taxonomy and palm systematics were appeared in the several journals, including peer review international journals such as Systematic Botany, Kew Bulletin, Blumea, Flora Malesiana Bulletin and Folia Malaysiana.

CONTENTS

Page

LIST OF TABLES... xvii

LIST OF FIGURES... xviii

LIST OF APPENDIXES... xix

GENERAL INTRODUCTION Background... 1

Objectives... 2

Literature Cited... 2

PHYLOGENY OF ARECA (ARECACEAE) BASED ON DNA Abstract... 6

Introduction... 7

Materials and Methods... 8

Results... 12

PRK Analysis... 12

RPB2 Analysis... 13

Combined Analysis... 15

Discussion... 20

Phylogenetics Value of PRK and RPB2... 20

Morphology... 21

Systematics Implications... 22

Biogeography... 22

Acknoledgements... 24

A CONSPECTUS OF GENUS ARECA L. (ARECACEAE)

Summary... 30

Introduction... 31

Materials and Methods... 32

Taxonomic Treatment... 35

Genus Description... 35

Infrageneric Classification of the Genus Areca... 36

Key to Subgenus of Areca... 36

I. Subgenus Areca... 37

II. Subgenus Beccarioareca... 54

Excluded and Uncertain Names... 72

Acknowledgements... 81

Literature Cited... 82

A MONOGRAPH OF ARECA OF EAST OF WALLACE’S LINE Summary... 88

Introduction... 89

Materials and Methods... 92

Results and Discussion... 93

Morphology... 93

Habit... 93

Stems... 94

Leaves... 94

Indumentum... 96

Inflorescences... 96

Fruits and Seeds... 100

Natural History and Conservation... 101

Habitat and Ecology... 101

Pollination, Seeds Predation and Seeds Dispersal... 102

Biogeography... 104

Conservation Status... 105

Uses... 105

Taxonomic Treatment... 106

Key to Species of Areca East of Wallace’s Line... 106

Species Description of Areca East of Wallace’s Line... 107

1. Areca catechu L... 107

2. Areca macrocalyx Zipp. ex Blume... 113

3. Areca mandacanii Heatubun... 121

4. Areca novohibernica (Lauterb.) Becc... 123

5. Areca oxycarpa Miq... 127

6. Areca vestiaria Giseke... 129

Doubful and Uncertain Species... 132

Acknowledgements... 132

References... 133

Appendix: List of Specimens Examined and Identified... 139

GENERAL DISCUSSION Taxonomic Revision of the Genus Areca... 142

Phylogeny of the Genus Areca... 143

Literature Cited... 144

LIST OF TABLES

Page

2.1 Statistics calculated from parsimony analyses of PRK, RPB2 and combined

datasets.….…...11

3.1 Comparison of previously recognised taxa and infrageneric classification in Areca

LIST OF FIGURES

Page

1.1 The 50% majority rule consensus tree of the Bayesian inference analysis of PRK

dataset...16

1.2 The 50% majority rule consensus tree of the Bayesian inference analysis of RPB2 dataset...17

1.3 The 50% majority rule consensus tree of the Bayesian inference analysis of combined (PRK and RPB2) dataset...18

1.4 Areca bakeri Heatubun...40

1.5 Areca dransfieldii Heatubun...45

2.1 Areca riparia Heatubun...52

2.1 Areca churchii Heatubun...57

2.3 Areca gurita Heatubun...61

3.1 Areca mogeana Heatubun...66

3.2 Areca triginticollina Heatubun...70

3.3 Distribustion map of the species Areca which native to east of Wallace’s line...91

3.4. The inflorescence (infructescence) and the pistillate flowers morphology of Areca macrocalyx...97

3.5 Reproductive organ of Areca vestiaria and Areca oxycarpa...100

3.6 Areca macrocalyx Zipp. Ex Blume...116

LIST OF APPENDIXES

I. Papers have been published during PhD study (2006–2009) in the Graduate Schools of Institut Pertanian Bogor (part of the dissertation and/or related to the Palms Systematics in general):

1. Dransfieldia (Arecaceae) – A New Palm Genus from Western New Guinea. Systematic Botany 31: 61–69 (2006).

2. Apakah Jenis Itu? (Sebuah Pelajaran Penerapan Konsep Jenis Pada Suku Palem-Paleman Di New Guinea). Rampak Serantau 14: 190–199 (2007).

3. Two Species of Licuala (Arecaceae; Coryphoideae) from Western New Guinea. Blumea 53: 429–434 (2008).

4. A new Areca from Western New Guinea. Palms 52: 198–202 (2008)

GENERAL INTRODUCTION

Background

Areca L. was the first palm genus was described by Linneaus in Species Plantarum in 1753 based on Rumphius’ Pinanga published in Herbarium Amboinense in 1741 (see Corner, 1966; Moore & Dransfield, 1979). It is also the type genus of the family name Arecaceae

Bromhead. This genus is best known for its type species, Areca catechu – the widely cultivated betel nut palm, the seed of which is chewed with the leaf or inflorescence of Piper betle L. (Piperaceae), lime and sometimes tobacco and spices, principally as a mild stimulant. Betel nut is consumed by an estimated 200–400 million people world-wide (Gupta & Warnakulasuriya, 2002; Loo et al., 2006; Dransfield et al., 2008) – extensively in the Asia-Pacific region and expatriate Asian communities – making betel nut the fourth most widely “abused” substance after nicotine, alcohol and caffeine (Norton, 1998; Loo, et al. 2006). The genus Areca also contains species of horticultural and agro-forestry significance (Ray & Reddy, 2001) and recent work has revealed considerable potential for therapeutic drugs to be extracted from some species, including cholesterol reducing agents (Byun et al., 2001) and neurological and dermatological treatments (Lee & Choi, 1999; Sullivan et al., 2000).

The genus Areca has approximately 47 species (Dransfield et al., 2008), and is distributed from India and south China through Malesia to New Guinea and the Solomon Islands (Dransfield, 1984; Uhl & Dransfield, 1987; Dransfield et al., 2008). Although varying in size from undergrowth palmlets to moderately tall tree palms, the genus is well defined in its traditional concept by the presence of a crownshaft, infrafoliar inflorescences, the single inflorescence bract (prophyll), complete floral triads confined to the basal part of the branched inflorescence or its main axis, the symmetrical fruits with basally attached seed, and the ruminate endosperm with basal embryo (Dransfield, 1984). However, Areca displays perplexing variation in inflorescence structure and flower presentation that is in need of further analysis, particularly in view of its wide distribution throughout Southeast Asia, straddling Wallace’s line (Dransfield, 1995). In the recent phylogenetic classification of the palm family, the genus Areca is placed together with Nenga and Pinanga in subtribe

Arecinae; tribe Areceae; subfamily Arecoideae (Dransfield et al., 2005, 2008; Loo et al., 2006); differing from the previous classification which also included Gronophyllum, Gulubia,

Hydriastele,Siphokentia (these four now reduced to a single genus Hydriastele – see Baker & Loo, 2004) and Loxococcus (Dransfield & Uhl, 1986, 1998; Uhl & Dransfield, 1987, 1999).

2 Current phylogenetic evidence indicates that Areca is sister to a clade comprising Nenga and

Pinanga (Loo et al., 2006; Norup et al., 2006; Baker et al., 2009).

The last infrageneric classification of the genus Areca was proposed by Furtado (1933) comprising two subgenera and five sections: subgenus Blumeoareca (sections Arecella,

Oeotheanthe and Axonianthe); and subgenus Beccarioareca (sections Microareca and

Mischophloeus). However, these relationships within the genus are based on morphological affinities alone (Furtado, 1933; Dransfield, 1984; Harley & Dransfield, 2003), and it has been suggested that they need to be reassessed using modern methods (Dransfield, 1984). Moreover, the geographic origin of economic species A. catechu is still uncertain, with the Philippines (Beccari, 1919; Furtado, 1933), Malaysia (Corner, 1966; Jones, 1995) or Celebes (Corner 1966) currently hypothesised to be the locality of the species’ origin.

Objectives

The general aim of this PhD project is to provide a modern taxonomic treatment of Areca

including studies of its phylogenetic relationships, natural history, uses and conservation status. The work will be based on exhaustive studies of existing literature, a thorough examination of herbarium materials in international herbaria, extensive fieldwork and laboratory-based molecular systematic research. The project consists of four main subprojects: 1). Taxonomic revision of the genus Areca; 2). Species level phylogeny estimation; 3). Comparative morphological and molecular study of the genus Areca with aim to understand the morphological changes that occurred during their evolution; 4). And if possible to reconstruct the historical biogeography and origin of A. catechu L. The main subprojects would be accommodating in the next following papers or chapters.

Literature cited

Asmussen, C. B. and M. W. Chase. 2001. Coding and non-coding plastid DNA in palm systematics. Amer. J. Bot.88: 1103−1117.

Baker, W. J. and A. H. B. Loo. 2004. A synopsis of the genus Hydriastele (Arecaceae).

Kew Bull.59: 61−68.

Baker, WJ, Asmussen CB, Chase MW, Dransfield J, Forest F, Harley MM, Savolainen V, Uhl NW Wilkinson M. 2009. Complete Generic Level Phylogenetic Analyses of Palms (Arecaceae) with Comparisons of Supertree and Supermatrix Approaches. Syst. Biol. doi: 10.1093/sysbio/syp021

3

Beccari, O. 1919. The palms of Philippines Islands. Phillipp. J. Sci.14: 295−362.

Byun, S. J., H. S. Kim, S. M. Jeon, Y. B. Park, and S. M. Choi. 2001. Supplementation of

Areca catechu L. extract alters triglyceride absorption and cholesterol metabolism in rats. Ann. Nutri. Metabol.45: 279−284.

Corner, E. J. H. 1966. The natural history of palms. London: Weidenfeld and Nicolson.

Dransfield, J. 1984. The genus Areca (Palmae: Arecoideae) in Borneo. Kew Bull.39: 1−22.

Dransfield, J. and N.W. Uhl. 1986. An outline of classification of palms. Principes 30:

3−11.

Dransfield, J. and N. W. Uhl. 1998. Palmae. In: Kubitzki, K (Ed.), the families and genera of vascular plants, vol. IV. pp. 306−389, Berlin: Springer.

Dransfield, J., N. W. Uhl, C. B. Asmussen, W. J. Baker, M. M. Harley and C. E. Lewis. 2005. A new phylogenetic classification of the palm family, Arecaceae. Kew Bull.60:

559−569.

Dransfield, J., N. W. Uhl, C. B. Asmussen-Lange, W. J. Baker, M. M. Harley and C. E. Lewis. 2008. Genera Palmarum: The Evolution and Classification of Palms. Kew: Royal Botanic Gardens Kew.

Flynn, T. 2004. Morphological variation and species limits in the genus Areca (Palmae) in New Guinea and the Solomon Islands. Unpublished Master thesis, University of Wales, Bangor.

Furtado, F. X. 1933. The limits of the genus Areca L. and its sections. Repert. Spec. Nov. Regni Veg.33: 217−239.

Govaerts, R., and J. Dransfield. 2005. World checklist of palms. Kew: Royal Botanic Gardens Kew.

Gupta, P. C., and S. Warnakulasuriya. 2002. Global epidemiology of Areca nut usage.

Addict. Biol.7: 77−83.

Hahn, W. J. 2002a. A molecular phylogenetic study of the Palmae (Arecaceae) based on

atpB, rbcL and 18S nrDNA sequences. Syst. Biol.51: 92−112.

Hahn, W. J. 2002b. A phylogenetic analysis of the Arecoid line of palms based on plastid DNA sequence data. Mol. Phylogenet. Evol23: 189−204.

Harley, M. M. and J. Dransfield. 2003. Triporate pollen in the Arecaceae. Grana 41:

3−19.

Jones, D. L. 1995. Palms throughout the world. Sidney: Reed Books.

Lee, K. K., and J. D. Choi. 1999. Areca catechu L. extract. I. effect on elastase and aging.

4

Lewis, C. E., and J. J. Doyle. 2002. A phylogenetic analysis of tribe Areceae (Arecaceae) using two low-copy nuclear genes. Plant Syst. Evol.236: 1−17.

Loo, A. H. B., J. Dransfield, M. W. Chase and W. J. Baker. 2006. Low copy nuclear DNA, phylogeny and the evolution of dichogamy in the betel nut palms and their relatives (Arecinae; Arecaceae). Mol. Phylogenet. Evol.39: 598−618.

Moore H. E., Jr. and J. Dransfield. 1979. Typification of Linnean palms. Taxon 28:

59−70.

Norton, S. A. 1998. Betel: consumption and consequences. J. Am. Acad. Dermatol. 38:

81−88.

Ray, A. K., and D. V. S. Reddy. 2001. Performance of Areca-based high density multi species cropping system under different level of fertilizer. Tropical Agriculture 78:

152−155.

Sullivan, R. J., J. S. Allen, C. Otto, J. Tiobech, K. Nero. 2000. Effects of chewing betel nut (Areca catechu) on the symptoms of people with schizophrenia in Palau, Micronesia. British Journal of Psychiatry177: 174−178.

Uhl, N. W. and J. Dransfield. 1987. Genera Palmarum: A Classification of Palms based on the work of Harold E. Moore Jr. Lawrance: L. H. Bailey Hortorium and International Palm Society.

Uhl, N. W. and J. Dransfield. 1999. Genera Palmarum after ten years. Mem. New York Bot. Gard.83: 245−253.

PHYLOGENY OF

ARECA

₍

ARECACEAE

_{) BASED ON DNA}

CHARLIE D.HEATUBUN1,2,3,4,WILLIAM J.BAKER3,JOHN DRANSFIELD3,JIM J.CLARKSON3, AND FELIX FOREST3

1

Fakultas Kehutanan, Universitas Papua, Jl. Gunung Salju, Amban, Manokwari 98314, Papua Barat, Indonesia;

2

Departemen Biologi, Sekolah Pascasarja Institut Pertanian Bogor, Kampus Darmaga, Bogor 16680, Jawa Barat, Indonesia;

3

Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK. 4

Author for correspondence (charlie_deheatboen@yahoo.com)

ABSTRACT. Phylogenenetics analysis of the palm genus Areca was performed based on the sequences of two low-copy nuclear genes phosphoribukilonase (PRK) and RNA-polymerase II subunit B (RPB2). Two lineages of the genus Areca are discovered and the monophyly of Areca as a genus is also reconfirmed. The previous infrageneric classification within Areca (subgenera and sections) were assess based on the phylogenetic tree and together with their biogeography explanation. Dispersal is more likely the main factor to reflect the present distribution of the genus Areca. And the origin of the wide spread cultivated species Areca catechu still unresolved from this study.

HEATUBUN ET AL.: PHYLOGENY OF ARECA

I

NTRODUCTION

Areca is the type genus of the palm family Arecaceae and it distribute in the old world tropics from India, Srilanka, south of China to Malesian region to Solomon Island in the Pacific (Dransfield et al. 2008). The genus is well known from its type species, Areca catechu L., the betel nut palm or pinang. The betel nut palm is important commodity and has been developed to the large-scaled plantations for supplying the nuts demanding. Traditionally, the seeds are chewing for a mild stimulant (Loo et al., 2006; Dransfield et al., 2008), but in the recent years, the betel nut palm and other species of Areca have been used widely in therapeutic drugs; including cholesterol reducing agents (Byun et al., 2001), and neurogical and dermalogical treatments (Lee & Choi, 1999; Sullivan et al., 2000); horticulture and agroforestry significance (Ray & Reddy, 2001); and even further to maintain the biodiversity and conservation (The Economist, 8th November 2008: 100).

The genus Areca has approximately before 47 species by Dransfield et al. (2008) and reduces to 41 species in the recent revision (Heatubun et al., in prep.). Morphologically, the development of generic concept of Areca has been discussed in details by previous authors (Furtado, 1933; Dransfield, 1984; Uhl & Dransfield, 1987; Dransfield et al., 2008), including its relationships with other palm genera from morphology point of view (Harley & Dransfield, 2003; Baker et al., 2009).

In the recent phylogenetic classification of the palm family, the genus Areca is placed together with Nenga and Pinanga in subtribe Arecinae; tribe Areceae; subfamily Arecoideae

(Dransfield et al., 2005, 2008; Loo et al., 2006); differing from the previous classification which also included Gronophyllum, Gulubia, Hydriastele, Siphokentia (these four now reduced to a single genus Hydriastele – see Baker & Loo, 2004)and Loxococcus (Dransfield & Uhl, 1986, 1998; Uhl & Dransfield, 1987, 1999). While, the current phylogenetic evidence indicates that Areca is sister to a clade comprising Nenga and Pinanga (Loo et al., 2006; Norup et al., 2006; Baker et al., 2009).

The last infrageneric classification of the genus Areca was proposed by Furtado (1933) comprising two subgenera and five sections: subgenus Blumeoareca (sections Arecella,

Oeotheanthe and Axonianthe); and subgenus Beccarioareca (sections Microareca and

Mischophloeus). However, these relationships within the genus are based on morphological affinities alone (Furtado, 1933; Dransfield, 1984; Harley & Dransfield, 2003), and it has been suggested that they need to be reassessed using modern methods (Dransfield, 1984).

HEATUBUN ET AL.: PHYLOGENY OF ARECA

Moreover, the geographic origin of economic species A. catechu is still uncertain, with the Philippines (Beccari, 1919; Furtado, 1933), Malaysia (Corner, 1966; Jones, 1995) or Celebes (Corner, 1966) and western New Guinea (Heatubun, 2008) currently hypothesised to be the locality of the species’ origin.

The application of phylogenetics analysis to construct classification system in the palm family is widely accepted, and the sequences of specific DNA region also proved as powerful tools to solved taxonomic problem in palms. From DNA data used in the palms systematics, data from nuclear regions more variable and appear to evolve more rapidly (Dransfield et al., 2008), and this very useful at species level phylogeny analysis, especially low-copy nuclear gene (Lewis & Doyle, 2002). Recently, sequences of two low-copy nuclear genes have been shown to provide satisfactory phylogenetics information at lower taxonomic levels in the palm family (Bayton 2005; Lewis & Martinez 2000; Lewis et al. unpublished; Norup 2004; Roncal et al. 2005; Thomas et al. 2006), especially in subtribe Arecinae (Loo et al. 2006): nrDNA PRK (phosphoribolukinase, a Calvin cycle enzyme) intron 4 (Lewis & Doyle 2001, 2002) and RPB2 (RNA polymerase II) intron 23 (Roncal et al. 2005). And also have been successful in molecular dated phylogeny analysis to construct biogeography history and dispersal events in palms (Gunn, 2004; Norup et al., 2006; Trenel et al., 2007; Cuenca et al.,

2007).

Based on an existing preliminary dataset of subtribe Arecinae (Loo et al., 2006), it is already known that these regions are useful for resolving relationships among certain species in subtribe Arecinae, including Areca. Thus, sequences from PRK and RPB2 were used in this study; to evaluating monophyly and relationships within the genus Areca and testing subgeneric and sectional grouping proposed by Furtado (1933), and if possible to reconstruct biogeographic history and origin of the betel nut palm Areca catechu.

M

ATERIALS AND

M

ETHODS

DNA-materials were collected directly in the fields by first author in western New Guinea (the Indonesian provinces of Papua and West Papua), North Sulawesi and Sarawak or obtained from botanical gardens, especially provided by Christine Bacon and Raymond Baker from Lyon Arboretum, Hawaii and Carl Lewis from Fairchild Tropical Botanical Garden Miami, Florida, USA. Also were using collections of DNA bank of Jodrell Laboratory, Royal

HEATUBUN ET AL.: PHYLOGENY OF ARECA

Botanic Gardens Kew. All specimens will be fully vouchered with specimens, they were deposited in appropriate herbaria, and their identities confirmed.

All available sequences of species of the genus Areca were included in the taxon sampling, with species of Nenga and Pinanga (Loo et al. 2006), Bentinckia condapanna

(Arecoideae: Areceae: unplaced), Clinostigma savoryanum (Arecoideae: Areceae: unplaced),

Cyrtostachys renda (Arecoideae: Areceae: unplaced), Cyphokentia macrostachya

(Arecoideae: Areceae: Clinospermatinae) and Tectiphiala ferox (Arecoideae: Areceae:

Oncospermatinae) as outgroup (Norup et al., 2006; Dransfield et al., 2008; Baker et al., 2009).

For DNA extraction, total genomic DNA extracted from silica gel-dried leaf materials (Chase & Hills, 1991) using the 2×CTAB method of Doyle & Doyle (1987). DNA were precipitated with 100% ethanol at –20°C, purified by cesium chloride/ethidium bromide gradients (1.55 g/mL) followed by dialysis and removal of ethidium bromide using butanol.

Primer sequences for PRK and RPB2 were obtained from published sources (Lewis & Martinez, 2000; Lewis & Doyle, 2002; Roncal et al. 2005; Loo et al. 2006; Norup et al. 2006; Trenel et al., 2007; Cuenca et al., 2007). The primers for PRK (prk717f: 5’-GTG ATA TGG AAG AAC GTG G-3’, prk969r: 5’-ATT CCA GGG TAT GAG CAG C-3’) have been designed to be specific to the smaller of the two paralogues known in the palms (Loo et al., 2006) The primers for RPB2 (RPB2-PALM-INT23F: 5’-CAA CTT ATT GAG TGC ATC ATG G-3’, RPB2-PALM-INT23R: 5’-CCA CGC ATC TGA TAT CCA C-3’) are specific to palms. Each PCR product was amplified using a 25 µl reaction mix consisting of 1.1×ReddyMix PCR Master Mix (2.5mM MgCl2, ABgene), 0.3 µl of each primer (10mM, final concentration), 1–5µl of template DNA, 0.9 µl bovine serum albumin (BSA) and optional dimethyl sulfoxide (DMSO) at 1% for recalcitrant amplifications of PRK and RPB2. Reaction mixtures will be subjected to the following temperature profile: initial denaturation at 95 °C for 5 min, 38 cycles of 96°C at 1 min each, 50°C for 1 min and 72°C for 1 min, and a final extension at 72 °C for 5 min. Amplification products were clean using the QIAquick PCR Purification Kit. The cleaned PCR products were cycle sequenced using the PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems: ABI) following the manufacturer’s protocols. Amplification primers will use as sequencing primers. Subsequent cycle sequencing protocols and purification of cycle sequencing

HEATUBUN ET AL.: PHYLOGENY OF ARECA

10 products follow that of Asmussen and Chase (2001) and Salazar et al. (2003). The sequences were run on an automated sequencer (ABI).

Sequence alignment follow Loo et al. (2006) and Norup et al. (2006) which every base position in the reverse and forward sequences were check and assembled using Sequencher 4.1 (Gene Codes Corp, Ann Arbor, Michigan, USA) and will be deposited in GenBank. The sequences then enter and aligned manually into data matrices in PAUP* version 4b10 (Swofford, 2002) for Macintosh computer. Variable positions in the alignment will verified against the raw data to ensure that these will not a result of base-calling errors.

Phylogenetic analyses in this study were using parsimony analysis and Bayesian analysis. the standard procedures as follow: congruence between the PRK and RPB2 datasets will evaluated using the incongruence length difference (ILD) test of Farris et al. (1994) as implemented in PAUP* (Swofford 2002) and Mr. Test in MrBayes version 3.0b4 (Huelsenbeck & Ronquist, 2001). Character partitions were designated in the combined dataset and analyzed in 10000 replicates, each with a single heuristic search, saving trees with each replicate.

The two dataset partitions were analyzed separately and simultaneously. Taxon sampling varied between the PRK and RPB2 datasets, and therefore the combined dataset included only taxa for which both gene sequences were available.

For parsimony analyses, uninformative characters and ambiguously aligned regions were excluded. All included characters were unordered and equally weighted. Initial analyses employed 1000 heuristic searches, each with starting trees obtained by random taxon addition, tree-bisection-reconnection (TBR) swapping, and keeping multiple trees per step (MulTrees on). Only groups that were found in the strict consensus tree and 50% or more of the replicates were recorded.

Bayesian analyses were carried out using the program MrBayes version 3.0b4 (Huelsenbeck & Ronquist, 2001). Models of sequence evolution that the best fit the individual datasets were determined using Mr. Test. The models were evaluated by the Akaike information criterion (Akaike, 1973) implemented in the program. Parameters based on patterns in the data matrices. For all three datasets, four incrementally heated Markov chains were used in an analysis that was run for 100,000 generations initially with trees saved at every 10th generations. Trees produced prior to stationarity were discarded as the burn-in.

HEATUBUN ET AL.: PHYLOGENY OF ARECA

11 Table 1. Statistics calculated from pasimony analyses of PRK, RPB2 and combined datasets.

Analysis Number of taxa

Alignment length

Ambigously aligned characters

Parsimony Informative Characters

MPTs Tree length

CI RI RC Nodes in

strict consensus

Nodes with >50% bootstrap support

Nodes with >90% bootstrap support

PRK 54 627 102 63 300 91 0.7473 0.9358 0.6992 23 23 4

RPB2 45 776 - 73 3021 106 0.8302 0.9572 0.7947 19 19 11

Combined 41 1403 102 127 (PRK:

55; RPB2: 72)

HEATUBUN ET AL.: PHYLOGENY OF ARECA

R

ESULTS

Results of the phylogenetics analysis of the genus Areca based on the molecular data nuclear DNA; PRK, RPB2 and combined as shown in the Table 1 and Figs 1–3.

PRK Analysis

In the PRK dataset, there were 54 sequences of 36 taxa, including 34 sequences from 20 species of Areca available. The average length of the genes is about 700 bp in the Areca

and the final length from data matrices is 627 bp and consisting of 23 bp exon 4, all of intron 4 and 130 bp exon 5, with 102 ambiguous characters and 63 parsimonious informative characters (Table 1). In the 5 samples, including A. chaiana was unsuccessful amplified and/or recovered apparently the existence of non-orthologous copies or pseudo-gene, thus all these genes were excluded from the analyses. Overall, parsimony and Bayesian inference analysis of PRK produced well-resolved and strongly supported topologies (Fig. 1). The parsimony bootstrap analysis resulted 300 the most parsimonious trees (MPT) with a length of 91 trees (CI = 0.7473, RI = 0.9538) and recovered 23 nodes over 50% bootstraps supports (one not shown in Fig. 1) including 4 nodes over 90% of bootstraps supported. As previously expected, that the Bayesian inference analysis will be generating more strong supported and more resolved in the PRK analysis than the bootstrap parsimony analysis. Of 25 resolved nodes in the Bayesian majority-rule tree, 21 nodes has value above 90% Bayesian posterior probability support and only 2 nodes below 75%.

The Arecinae is strongly resolved (100 Bayesian posterior probability, BPS and 90 bootstraps percentage, BP), whereas the outgroups is unresolved with polytomies (Bentinckia condapanna, Clinostigma savoryanum and Cyphokentia macrostachya) and poorly resolved in Cyrtostachys renda and Tectiphiala ferox (55 BPS and less than 50 BP). The topology supports the monophyly of Arecinae.

The monophyly of subtribe Arecinae which contain genera Areca, Nenga and Pinanga

is reconfirmed and Areca as sister to Nenga and Pinanga is resolved with strongly supports (100 BPS and 90 BP). And also Nenga is sister to Pinanga resolved with strongly supports in Bayesian (95 BPS) and moderate support in parsimony analysis (70 BP).

Areca is strongly supported (100 BPS, 91 BP) with two lineages to define two different clades. The discovery of two lineages within Areca with highly resolution (100 BPS in both, 89 and 55 BP) is indicate the infrageneric classification as proposed before by

HEATUBUN ET AL.: PHYLOGENY OF ARECA

Furtado (1933), two subgenera (subgenus Areca and Beccarioareca). However, the species are included in these two subgenera still need to re-arrangements based on the result of this study.

The subgenus Areca is strongly supports in Bayesian (100 BPS) and weak in parsimony analysis (55 BP). In general, the topology is low resolution with polytomies and the relationships among the species still unclear, except to A. triandra clade (A. triandra and

A. concinna) with strongly supports (100 BPS, 97 BP), the Philippines congested inflorescence species clade (A. caliso, A. camarinensis and A. ipot) also with strongly supports (100 BPS, 96 BP) and A. oxycarpa (Heatubun 977 and Asmarayani 461).

The resolution in subgenus Beccarioareca is better than subgenus Areca, the node is highly resolved (100 BPS, 89 BP) and the relationships among the species in the subgenus more resolved. In this clade is also revealed that Areca furcata is sister to all species in subgenus Beccarioareca with relatively moderate supports (75 BPS, 69 BP).

The clade consist A. minuta, A. jugahpunya and A. ridleyana is resolved with high resolution in Bayesian analysis (100 BPS) and moderate in parsimony bootstrap analysis (71 BP). Also within the clade, the relationships between one accession A. minuta (Heatubun 892) and A. ridleyana recovered with strongly supports (100 BPS, 84 BP), whereas another accession of A. minuta (s.n.) and A. jugahpunya still unclear. While, two accession of A. minuta (Heatubun 887 and 888) are not clearly resolved as polytomies together with A. tunku.

Areca guppyana (= A. novohibernica) and A. vestiaria forming the Mischophloeus clade (94 BPS, 62 BP) and indicates A. novohibernica is sister to A. vestiaria. This clade is similar to section Mischophloeus of subgenus Beccarioareca in Furtado’s infrageneric classification (1933).

The other node is resolved with low supports (50 BPS, less than 50 BP) and expressed the relationships between A. insignis var. moorei and A. subacaulis. And this splits from other accession of A. insignis.

RPB2 Analysis

In RPB2 dataset, 54 sequences were in total from 36 taxa, including 30 sequences of 18 species of Areca were available. The length of the genes around 850 bp in the Areca and the final length from data matrices is only 776 bp of intron 23 with 73 parsimony informative characters. Similarly, about 7 samples unsuccessful, thus those sequences excluded from

HEATUBUN ET AL.: PHYLOGENY OF ARECA

RPB2 analysis and also in the combined analysis. The tree topologies in RPB2, nodes support and resolution performed by the parsimony bootstrap analysis lower than the results of Bayesian analysis. In the analysis produced 3021 most parsimonious trees (MPT) with a tree length 106 (CI = 0.8302, RI = 0.9572). Of 19 nodes resolved in majority rule tree 11 nodes over 90% BP (Fig. 2). Whereas in the Bayesian inference analysis, from 21 nodes resolved in the majority rule tree, 18 nodes exceeding 90% BPS.

The monophyly of Arecinae is reconfirmed with strongly supports in Bayesian analyisis (100 BPS) and moderate in parsimony bootstrap analysis (78 BP). From outgroups, only Clinostigma savoyarum and Cyrtostachys renda is resolved with strongly supports (100 BPS, 92 BP), while Bentinckia condapanna, Cyphokentia macrostachya and Tectiphiala ferox

are not clearly resolved their relationships.

Three lineages have been discovered from Arecinae in this analysis; two lineages (evolutionary lines) are also recovered in PRK analysis which contains Areca line and Nenga-Pinanga line and the third is Areca chaiana line. The topology is supports exclusion of Areca chaiana from Areca and as sister to all genera of Arecinae.

Areca is monophyletic and sister to Nenga and Pinanga are reconfirmed with strongly supports (100 BPS, 100 BP). Nenga is sister to Pinanga also reconfirmed from this analysis (90 BPS, 60 BP) and supports the monophyly of each genus in subtribe Arecinae.

Two lineages within Areca are also discovered with highly resolution in strongly supports in both analyses Bayesian and parsimony (100 BPS, 99 BP and 100 BPS, 100 BP). Two clades defined as clade of subgenus Areca and clade of Beccarioareca recovered with different resolution in tree topology. The relationships among species within subgenus in

Areca are more resolved than Beccarioareca.

Subgenus Areca is composed by nine resolved nodes with different resolutions. From those resolved nodes, three major clades have been identified as the congested inflorescence species clade, Areca catechu clade and Areca triandra clade. The congested inflorescence species clade is containing species accession of Areca from The Philippines, Sulawesi and New Guinea. This clade is strong supports in Bayesian analysis (95 BPS) and relatively weak supports in parsimony bootstrap analysis (62 BP), and from topology supports A. ipot as sister to all congested inflorescence species (A. camarinensis, A. caliso, A. macrocalyx and A. oxycarpa). Three accessions of Areca macrocalyx from New Guinea (Baker 1317, Heatubun 787 and 876) defined a resolved clade with strongly supports (100 BPS, 95 BP) and two of

HABITAT. This palm grows in primary or secondary forest in the lowlands or hill forest at an altitude of 30– 400 m above sea level.

VERNACULAR NAMES. Papua Indonesian: tnang nyi

(Sentani, Jayapura);gap(Marap, Tami R.); terep/terrip

(Yei/Je, Merauke); Nibung (Indonesian dialect in Papua, also used for other tree palms). Papua New Guinea: yomberi (Timbunke, Sepik); yowoh (Waskuk, Sepik);hek/he-ek(Amele, Madang);terep(Jal, Madang);

apaku (Mekeo, Maipa); flim (Mianmin); lobu (Wapi, Marok); mun (Orme, Walwali); wai’eba (Kutubu);

toono-i (Bougainville Island); a ikul (New Ireland Island). Solomon Islands:kwara’ae(Aatarae).

USES.Cyrtostachys loriaeis one of the more useful species in the genus from an ethnobotanical point of view. It has been used traditionally by people native in both New Guinea and the Solomon Islands. The stems and leaves are used as building materials for traditional houses, e.g. piles,flooring, water pipes, thatch and mattresses. The palm heart or“cabbage”is also eaten fresh or cooked. CONSERVATION STATUS. Least Concern (LC).Cyrtostachys loriaeis widespread in the Papuasian region, and as yet the conservation status of this species seems not to be a cause for concern. However, land conversion for oil palm plantations or other purposes, including illegal logging activities in West New Guinea (Indonesian Provinces of Irian Jaya Barat and Papua) could have a severe effect on the populations of the palm. Detailed population studies are still needed to assess its conserva-tion status more precisely.

NOTES. Cyrtostachys loriae was the first species of the genus to be published from the Papuasian region (Beccari1905). This palm is easily distinguished by its solitary and robust habit, spherical crown, pendulous leaflets, very short (– 10 cm long) or missing petiole and an inflorescence more robust than in other species, branched to 3 orders, with robust rachillae bearing large and deep pits.

Re-examination of the type specimens ofCyrtostachys brassii,C. kisu,C. loriae, C. microcarpa,C. peekelianaandC. phanerolepis revealed no significant differences among them except those caused by differences in develop-mental stages, despite the inadequate nature of the specimens. Morphological variation among them is con-tinuous, especially after comparison with more adequate specimens from recent collections. No disjunctions in variation occur that would allow the consistent separation of six species as recognised by previous authors. The narrow species concept used in the past reflects limited information obtained from single collections.

Moore (1966) pointed out his suspicions that the five taxa above might not be distinct; they all have a solitary habit and 12 stamens except for Cyrtostachys phanerolepis — six stamens, pits in 9 series and larger fruits (Burret 1936). Burret did not realise that the specimenClemens1353 (the type ofC.phanerolepis) had been mixed with some maleflowers ofLicuala, and he

described C. phanerolepis with staminate flowers from

Licuala.

Cyrtostachys loriaeis the most widespread species of the genus in the Papuasian region, and also occupies a wide range of ecological conditions from swampy areas in the lowlands to heath forest in lower montane vegetation, from evergreen rain forest to dry areas in savannah lands and from the main island of New Guinea to small off-shore islands and the Solomons. The adaptation to various habitats is reflected in the very variable appearance, a plasticity that occurs not only in size and shape, but also the number of certain organs, such as number of stamens. In some speci-mens, different numbers of stamens can be found within one inflorescence or in different collections from the same locality.

There are two collections from savannah areas in Merauke, Indonesian Province of Papua, Maturbongs

654 andvan Royen 4734, which look quite distinct in the appearance of their leaves. The petiole and rachis are covered by scaly indumentum, and the leaflets are slender with discolorous surfaces (glaucous adaxially, purplish-brown abaxially). Other characters such as habit, stem, inflorescence andflowers, fruits and seeds fit withCyrtostachys loriae. The variation in leaf charac-ter may reflect different ecological conditions in savannah areas.

7. Cyrtostachys renda Blume (1838: 66, 1843: 101).

Bentinckia renda (Blume) Mart. (1853: 316). Type: Indonesia, Sumatra, East Sumatra, around Indrapura,

Korthals s.n. (lectotype L!, designated here; isolecto-type K!).

Areca erythropoda Miq. (1861a: 6). Type: Indonesia, Sumatra, Bangka Island, Djebus, Teisjmann s.n. (holotype BO!).

Cyrtostachys lakka Becc. (1885: 141). Type: Malaysia, Borneo, Sarawak, Kuching, Nov. 1866, Beccari PB 2674 (lectotype FI!,designated here).

Cyrtostachys lakka var. singaporensis Becc. (1885: 141). Type: Singapore,”cultivato nel giardino del Sig. Wham-poa”, Anon. (holotype FI!)

Invalid names:

Pinanga purpureaMiq. (1861b: 590), nom. inval., in synon.

Ptychosperma coccineaTeijsm. & Binn. (1866: 69); nom. nud.

Areca erythrocarpa H. Wendl. in Kerch. (1878: 231), nom. nud.

Pinanga rubricaulisLinden (1885: 61), nom. nud. Slender, clustering tree palm with up to c. 3 or more adult stems up to 15 (–20) m high.Stemc. 6–10 cm diam., green with greyish stripes or yellow with somewhat greenish and purplish stripes, internodes 15 – 24 cm long, crown appearing shuttle-cock

shaped.Leaves7–10 in crown, erect, stiff, to 150 cm long; sheath tubular, c. 100 cm long, forming distinct crownshaft, scarlet to bright red, with scattered black thick scales; petiole elongate, 5 – 50 cm long, 1.5 –

2.5 cm wide and 1–2 cm thick at the base, channelled adaxially, rounded abaxially, red, indumentum as sheath; leaflets regularly arranged, leathery, 26 – 40 leaflets on each side, 56 – 107 × 3–6 cm at middle portion, apical leaflets 10 – 20 × 1 – 2 cm, briefly pointed with long tip and sometimes notched at apices, green, discolorous when dried, glaucous adax-ially, waxy white abaxadax-ially, mid-vein with discontinuous membranous brown scales. Inflorescencestrongly divar-icate, to 90 cm long, branched to 2 (possibly 3) orders, creamy, green to dark purplish-red; peduncle 5–8 cm long; rachilla 27–73.5 cm long and 4–6 mm diam., calyx persistent on rachillae when fruits fallen off; pits 2 –5 mm diam., 5– 7 pits per 1 cm rachilla length.

Staminate flowers 2 – 2.5 × 2 – 3 mm, asymmetrical; sepals 1.8 – 2 × 2 mm, imbricate, rounded, strongly keeled; petals 1–_{2 × 1}–1.8 mm, triangular, brown at apex and base; stamens 12 – 15; filaments 0.7 – _{1 ×}

0.2–0.3 mm; anthers 1–1.5 × 0.5–0.8 mm; pollen size, long axis 36–43 µm, short axis 27–33 µm, proximal wall thickness 1.5 – 2 µm, distal wall thickness not observed, tectum surface microfossulate-rugulate, trichotomosulcate grains present; pistillode 0.7 – _{1 ×}

0.2–0.5 mm, trifid.Pistillate flowers4– _{5 × 3}– 4 mm; sepals 3 – 4 × 2 – 3 mm, imbricate, strongly keeled, dark brown to black; petals 3 – 3.5 × 2 – 2.5 mm; gynoecium 3.5 × 1.5 mm (including three recurved stigma 0.5– 1 mm); staminodes circular, 0.5– 1 mm height.Fruits7–_{10 × 4}–7 mm, ellipsoid to ovoid, light green becoming black when ripe.Seeds4–5 × 3–5 × 3–5, ellipsoid to ovoid. (Fig.3J–M).

DISTRIBUTION. This is the only species found to the west of Wallace’s Line, occurring in the southern part of Thailand, Malay Peninsula, Sumatra and Borneo. SPECIMENS EXAMINED. THAILAND. Narathiwat Province: Tak Ban, Phru Kok Daam, March 1985,Niyondham852 (BKF, K!); Tho daeng, c. 30 km SE of Narathiwat, 50–

100 m a.s.l., Nov. 1990, Barfod & Ueachirakan 41772 (AAU, BKF, K!, PSU); to Daeng, 75 m a.s.l., Oct. 1996,

Barfod et al.43888 (AAU!, BKF, PSU). N of Sritamerat, Ta Samet, c. 50 m a.s.l., Jan. 1928, Kerr 14332 (K!). SUMATRA. Aceh, Asdat 171 (BO!); Asahan, Polak s.n. (BO!); Bangka Island, Djebus Teysmann s.n. (BO!); Bengkalis, Selat panjang, 3 m a.s.l., Nov. 1919,Bequin

457 (L!, BO!); near Indrapura,Korthalss.n. (L!, K!; the type); Riau, Widyatmoko 399 (BO!); Widyatmoko 400 (BO!); South Sumatra, Dransfield JD 1252 (BO!); Natuna Island,Mogea 2990 (BO!). MALAY PENINSULA. Selangor: Telok swamp forest, Klang, March 1968,

Dransfield713 (K!).SINGAPORE.”cultivato nel giardino del Sig. Whampoa”, Anon. (holotype FI!).BORNEO. SABAH: Kudat distr., Pulau Balembangan, NE inner side Telok

Lung, 10 m a.s.l.,BCS-EFA-LM et. al. SAN 86702 (K!, KEP, L!, SAR, SING); Sandakan, Jan. 1921,Wood1111 (A!, PNH, SAN). SARAWAK: Kuching, Nov. 1866,

Beccari PB 2674 (FI!); 1865 – 1869, Beccari PB 3438 (K!); Bintulu, Sept. 1867,BeccariPB 4038 (FI!); 1929,

Clemens 21377 (A!, BO!, K!); Miri distr., Rian road, 50 m a.s.l., April 1959, Saleh 1214 (K!, L!, S, SAR). BRUNEI DARUSSALAM: Belait, Labi, km 20 Labi road, burnt over white sand forest, level land, 50 m a.s.l., March 1992, Dransfield JD 7279 (K!); Bukit Bakong, Oct. 1992,Bernstein278 (K!); Maruntungan, May 1932,

Keith2491 (K!). CULTIVATED. Indonesia: North Suma-tra, Sibolangit Botanic Garden, 500 m a.s.l., Sept. 1927,

Lörzing12083 (L!); West Java, Bogor Botanic Garden, origin from Banka Island, loc. V. K. 37, April – May 1936,FurtadoSFN 3/1/68 (BO!, K!, L!, SING); loc. V. G. no. 4., May 1903, Schoute s.n. (L!); Jan. 1906,n.n.

(L!); n.n. (BO!). Malaysia: Penang Botanic Garden, Sept. 1900, Curtis 3527 (K!). UK: Royal Botanic Gardens Kew, Palm House, May 1998, 1982-5882 (K!). Seychelles: Victoria, Mali, Dec. 1971, Elizabeth

111 (K!). Singapore: Singapore Botanic Garden, Lawn K, Sept. 1929, Furtado s.n. (K!, SING). Thailand: Peninsular Botanic Garden Khao Chong, Trang,

Barfods.n. (AAU (photo)!).

HABITAT. Cyrtostachys renda grows in lowland peat swamp forest, especially in coastal areas, but more rarely occurs in peat swamps in uplands from 0 –

500 m above sea level.

VERNACULAR NAMES. Thailand: kap daeng, mark-dang

(Thai). Malay Peninsula:pinang rajah (Malay). Suma-tra: pinang renda or rende’ (Indrapura);pinang rimbou

(Sibolga); pinang lempiauw or pinang lepiaw (Bangka island). Borneo: malawaring, raring (Brunei). Trade names:pinang merah,palem merah,(Malay/Indonesia);

sealing wax palm, lipstick palm(English);hsing hsing yeh tzu(Chinese);rode palm(Dutch).

USES. This palm has limited traditional uses; stems are used forflooring and leaves for thatch. It is, however, a highly desirable and widely cultivated ornamental for tropical regions.

CONSERVATION STATUS. Vulnerable (VU). See Drans-field & Johnson (1991), Kiew (1991), and Mogea (1991) for conservation status assessment.

NOTES.Cyrtostachys rendadiffers from all other species in the bright red crownshaft and leaf sheath, the lowest number of leaflets (26–40 on each side), the leaflets being waxy white abaxially, the inflorescence branched mostly to 2 orders (possibly up to 3), the tectum surface of pollen rugulate, and its preferred habitat in lowland peat swamp forest in southern Thailand, Malay Peninsula, Sumatra and Borneo.

In the protologue ofCyrtostachys renda, no material is cited although a reference is made to Blume’s account in Rumphia, published some years later, in which the Korthals specimen is mentioned (Blume 1838, 1843). We formally designate this specimen as

lectotype. In the case ofC. lakka, Beccari (1885) cited two specimens, Beccari PB 2674 and 4038, both of which are extant at FI. Harold E. Moore annotated

Beccari PB 2674 (FI) as lectotype in 1956, but to our knowledge did not publish this lectotypification. We have formalised this typification here. The transfer of

Cyrtostachys lakka to a synonym ofC. renda has already been made by Whitmore (1982).

The bright green stems and brilliant red to orange crownshafts make Cyrtostachys rendaa highly desirable and widely cultivated ornamental. Infraspecific taxa have been described from cultivation and the number of these could increase in parallel to horticultural demand. Ellison & Ellison (2001) introduced two cultivars, C. renda ‘Apple’ and C. renda ‘Orange Crownshaft’, followed by Waddel (2002) with his C. renda‘Theodora Buhler’. Before them, Tucker (1992) reported, that in Singapore Botanic Garden grew C. renda‘Ruby’and that it was one of the most treasured specimens of all, and he also discussed a strange brown crownshafted form of C. renda in Florida. All the cultivars of C. renda were described based on different stem and crownshaft colours.

Specimens of Uncertain Affinity Heatubun679 & 680 (Cyrtostachyssp.).

This specimen was collected from a plant cultivated at the Kebun Raya Bogor (Bogor Botanic Garden) in the lawn XIB. XX, close to the nursery and plant conservation building. In its reddish sheaths this palm resemblesCyrtostachys rendasomewhat but is nevertheless quite distinct. It is a large, clustering tree palm growing to 20 m high with a stem of more than 10 cm diam. with elongate internodes. The crown is hemispherical in outline and the leaves bear pendulous leaflets, a short petiole and a greenish-red to yellowish-red sheath and petiole with a few green stripes. The inflorescence is large, branched to 3 orders (never 2-branched). Al-though the clustering habit and red leaf sheath suggest characters of C. renda, it is far too robust to match that species. The taxon could well be a result of hybridisation between two different species (C. elegans

and C. renda) that have been planted together in the garden (Dransfield1999). Further studies are required before a new hybrid species can be formally described.

Excluded and Uncertain Names

Cyrtostachys ceramica (Miq.) H. Wendl. in Kerch. (1878: 242) = Rhopaloblaste ceramica (Miq.) Burret (1928: 288).

Heterospathe compsoclada(Burret)Heatubuncomb. nov.

Cyrtostachys compsocladaBurret,Notizb. Bot. Gart. Berlin-Dahlem 13 (118): 325 (1936). Type: Papua New

Guinea, Central Province, Boridi, Sept. 1935, Carr

13136 (holotype B†; isotypes K!, L!).

After re-examination of the type of Cyrtostachys comp-socladaBurret, no characters diagnostic ofCyrtostachys

were found, while all the characters fit with Hetero-spathe. There are no pits, and the rachillae are covered in thick brown indumentum. In the staminateflowers, petals and filaments are free (connate or united in

Cyrtostachys), and stamens 6 in number (8 – 15 in

Cyrtostachys). Leaf and leaflets are very small for an adult palm inCyrtostachysand theflower colour is said to be purple — colourful inflorescences and flowers are known inHeterospathe. Moreover, the cited altitude, 5000 feet (1500 m) a.s.l., is higher than usual for

Cyrtostachys. The highest recorded altitude for Cyrtos-tachys being 900 m a.s.l. forC. barbata, whereas Hetero-spathe is most frequently found at submontane to montane elevations.

Cyrtostachys ledermannianaBecc. (1923: 450). See notes underCyrtostachys barbatafor the discussion.

Acknowledgements

Many individuals and institutes contributed to the completion of this work. We would like to thank the curators and staff of herbaria A, AAU, BH, BO, BRI, FI, K, KEP, L, LAE, MAN, PNH, SAN and SING for access to their specimens, databases and for providing loans in many cases. Dr Hannah Banks is thanked for assistance with pollen laboratory work and microscopy. Dr Sander van der Kaars provided fossil pollen information. Lucy T. Smith prepared the elegant plates. This research was conducted while CDH did his Master’s degree in Institut Pertanian Bogor. CDH would like to thank the Dransfields, the Bakers and the staff of the Herbarium, Royal Botanic Gardens, Kew for their support and hospitality during his recent visits. Special thanks to Marthinus Iwanggin, Piter Gusbager, Herkilaus Rumakewi, Tobias Paiki, Krisma Lekitoo, Piter Matani, Maikel Simbiak, Hermus Indou for help during fieldtrips to Tanah Papua. Financial support came from BPPS DIKTI Depdiknas, local government of Provinsi Irian Jaya Barat and local government of Kabupaten Manokwari, the BAT Bio-diversity Partnership to the PONG project of the Royal Botanic Gardens, Kew, the Pacific Biological Foundation, the Sibbald Trust, and the Ethnobotanical Conservation Organisation for South East Asia (ECO-SEA).

List of Specimens Examined and Identified Species numbers are given in brackets and in bold after collection numbers.

Key: (1)C. bakeri, (2)C. barbata, (3) C. elegans, (4)C. excelsa, (5)C. glauca, (6)C. loriae, (7)C. renda.

Anon (Cult. Singapore)s.n. (7)

Baker et al.1110 (6), 1138 (1);Barfod478 (6), 482 (6);

Barfods.n. (7);Barfod & Uechirakan41772 (7);Barfod et al.395 (6), 454 (5), 463 (6), 467 (6), 43888 (7); BCS-EFA-LM et al. SAN 86702 (7); Beccari PB2674 (7), PB3438 (7), PB4038 (7);Bernstein278 (7);Bequin457 (7);Brass7162 (6), 7757 (6), 13707 (2), 13807 (6)

Carr12253 (6);Clemens1353 (6), 21377 (7);Croft et al.

LAE 68805 (5);Curtis3527 (7)

Desianto 01 (6); Derbyshire 867 (6); Derbyshire & Hoog-land 8020 (6); Dransfield JD713 (7), JD1252 (7), JD7279 (7)

Elizabeth111 (7);Essig & KatikLAE 55009 (5);Essig & YoungLAE 74052 (6)

Furtado SFN 3/1/06 (3), 3/1/28 (3), 3/1/68 (7);

Furtados.n. (7)

Guppy235 (6);Gusbager23 (6)

Heatubun et al.194 (3), 208 (6), 279 (6), 330 (4), 341 (3), 527 (6), 532 (6), 533 (6), 546 (6), 547 (6);

Hoogland & Craven10114 (6)

Kajewski 2220 (6); Kanehira & Hatusima 12747 (3), 12851 (3);Katik LAE 62219 (5); Keith2491 (7);Kerr

14332 (7); Kjaer & Magun512 (5); Korthals s.n. (7);

Kostermanss.n. (6)

Lörzing12083 (7)

Maturbongs 654 (6); Meijer Dress 501 (6); Mogea 2990 (7);Moore9272 (5);Morren & Frodin3189 (6)

Niyondham852 (7)

Peekel444 (6);Polaks.n. (7);Pullen1692 (6), 7305 (6), 8198 (5), 8212 (5), 8231 (5), 8409 (5)

Saleh1214 (7);Schoode2248 (6);Schoutes.n. (7)

Takeuchi8770 (5);Takeuchi et al.13217 (5);Teysmanns. n. (7)

van Royen4734 (6)

WhitmoreBSIP 3945 (6), 4210 (6);Widyatmoko399 (7), 400 (7);Wood1111 (7)

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