Biosystematics of pandanaceae in Java

BIOSYSTEMATICS OF

PANDANACEAE

IN JAVA

SRI ENDARTI RAHAYU

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

STATEMENT OF RESEARCH ORIGINALITY AND

INFORMATION SOURCE

This is to certify that my dissertation entitled Biosystematics of Pandanaceaein Java is my own work and never been submitted to any institution before. All the incorporated data and information are valid and stated clearly in the text and listed in the references.

Bogor, Januari 2011

Sri Endarti Rahayu NRP G361040061

ABSTRACT

SRI ENDARTI RAHAYU. Biosystematics of Pandanaceae in Java. Supervised by ALEX HARTANA, TATIK CHIKMAWATI, KUSWATA KARTAWINATA, and MIEN A. RIFAI.

Pandan family (Pandanaceae) is represented in Java by two genera: Freycinetia Gaud. and Pandanus Parkins. Since the studies by Backer and Bakhuizen van den Brink in 1968 and Stone in 1972, there were no further exploration on the pandan flora of the island have been made, thus the pandan flora remains largely unknown. Taxonomical problem as far as the Java pandans concerned are centered not only on species status ofP. odoratissimus L.f and P. tecroriusvar.littoraliswhich are regarded as synonym, but also the species status which are given as synonym ofP. furcatus Roxb. by Backer and Bakhuizen van den Brink. Backer and Bakhuizen van den Brink included of P. bantamensis Koord., P. oviger Martelli, P. pseudolais Warb. and P. scabrifolius Martelli as synonym to P. furcatus Roxb. This Backer and Bakhuizen van den Brink’s classification is in contrast with Stone who stated that these species were regarded as four different species. For the reason of this taxonomical problem, therefore an effort has been made to redescribe these species in detail, using morphological, anatomical and molecular data such as sequence data of atpB-rbcL IGS, since a satisfactory classification depends upon the interpretation of many characters as possible. In this study we only found three of four species mentioned above, viz. P. bantamensis Koord., P. pseudolais Warb., and P. scabrifolius Martelli. The result showed that P. bantamensis Koord., P. pseudolais Warb, and P. scabrifolius Martelli are treated as three different species, and P. odoratissimus L.f and P. tectorius var.littoralis are treated as two different species. The ISSR marker showed that six species of Freycinetia and thirteen species of Pandanus from Java have high genetic diversity, although Freycinetia has a bit lower than Pandanus. This research showed seven species of Freycinetia could be recognized, viz. F. angustifolia Bl., F. funicularis Merr., .F. imbricata Bl., F. insignisBl.,F. javanicaBl.,F. scandensGaud., andF. sumatranaHemsl.; sixteen species of Pandanus, viz. P. amaryllifolius Roxb., P. bantamensis Koord., P. bidur Jungh., P. dubius Spreng., P. faviger Backer, P. kurzii Merr., P. labyrinthicus Kurz, P. multifurcatus Fagerl., P. nitidus Kurz, P. odoratissimus L.f., P. polycephalus Lam., P. pseudolais Warb., P. scabrifolius Martelli, P. spinistigmaticusFagerl., P. tectorius Parkins. with two varieties, viz. P. tectorius var. littoralis, P. tectorius var. samak, one cultivar, i.e Pandanus tectorius cv. Sanderi; P. utilis Bory; one variety P. leram Jones var. andamanensium (Kurz) Stone, and one cultivar,P. spuriusMiq. cv. Putat.Pandanaceaein Java are most diverse in lowland rain forest and hill forest.

ABSTRAK

SRI ENDARTI RAHAYU. Biosystematics of Pandanaceae in Java. Di bawah bimbingan ALEX HARTANA, TATIK CHIKMAWATI, KUSWATA KARTAWINATA dan MIEN A. RIFAI.

Pandanaceae di Jawa diwakili oleh dua marga, yaitu Freycinetia Gaud. danPandanusParkins. Sejak penelitian oleh Backer dan Bakhuizen van den Brink pada tahun 1968 dan Stone pada tahun 1972, belum pernah dilakukan eksplorasi lebih jauh tentang flora pandan di Jawa sehingga sampai saat ini banyak hal yang belum diketahui tentang flora pandan tersebut. Masalah taksonomi tentang Pandanaceae yang ada di Jawa, tidak hanya tentang status spesies dari P. odoratissimus L.f dan P. tectorius var. littoralis yang dianggap sinonim, tetapi juga status spesies yang merupakan sinonim dari P. furcatus Roxb, yaitu P. bantamensisKoord.,P. oviger Martelli,P. pseudolais Warb., dan P. scabrifolius Martelli. Backer dan Bakhuizen van den Brink memasukkan P. bantamensis Koord., P. oviger Martelli, P. pseudolais Warb., dan P. scabrifolius Martelli ke dalam satu spesies, yaituP. furcatusRoxb., sedangkan menurut Stone ke empat spesies tersebut adalah empat spesies yang berbeda. Berdasarkan latar belakang permasalahan tersebut perlu dilakukan penelitian untuk dapat mendeskripsikan kembali spesies-spesies tersebut dengan menggunakan data morfologi, anatomi dan molekular, seperti sekuen antar genatpB-rbcL, karena suatu klasifikasi yang paling memuaskan tergantung pada interpretasi dari sebanyak mungkin karakter. Dalam penelitian ini hanya ditemukan tiga dari empat spesies tersebut di atas, yaitu P. bantamensisKoord., P. pseudolais Warb., dan P. scabrifolius Martelli. Hasil penelitian menunjukkan bahwa P. bantamensis Koord., P. pseudolais Warb., dan P. scabrifolius Martelli diperlakukan sebagai tiga spesies yang berbeda, dan P. odoratissimus L.f dan P. tecrorius var. littoralis diperlakukan sebagai dua spesies yang berbeda. Penelitian ISSR menunjukkan bahwa ke enam spesies Freycinetia dan 13 spesies Pandanus di Jawa memiliki keanekaragaman genetik yang tinggi, walaupun keanekaragaman genetik Freycinetia sedikit lebih rendah bila dibandingkan denganPandanus.Hasil penelitian menunjukkan bahwa di Jawa dapat dikenali tujuh spesies Freycinetia dan 16 spesies Pandanus. Ketujuh spesiesFreycinetia tersebut mencakupF. angustifoliaBl.,F. funicularis Merr., F. imbricata Bl., F. insignis Bl., F. javanicaBl., F. scandensGaud., dan F. sumatrana Hemsl. Keenambelas spesies Pandanus tersebut terdiri dari P. amaryllifolius Roxb.,P. bantamensis Koord.,P. bidur Jungh.,P. dubius Spreng, P. favigerBacker,P. kurziiMerr.,P. labyrinthicusKurz, P. multifurcatusFagerl., P. nitidus Kurz, P. odoratissimus L.f., P. polycephalus Lam., P. pseudolais Warb., P. scabrifoliusMartelli, P. spinistigmaticus Fagerl.,P. tectorius Parkins, dengan dua varietas, yakniP. tectoriusvar.littoralis, P. tectoriusvar.samak, satu kultivar, yaitu Pandanus tectoriuscv. Sanderi, P. utilisBory; satu varietas, P. leramJones var.andamanensium( Kurz) Stone, dan satu kultivar,P. spuriusMiq. cv. Putat. Di Jawa, keanekaragaman Pandanaceae yang paling tinggi terdapat di hutan pegunungan bawah dan bukit pegunungan.

SUMMARY

SRI ENDARTI RAHAYU. Biosystematics ofPandanaceaein Java. Supervised by ALEX HARTANA, TATIK CHIKMAWATI, KUSWATA KARTAWINATA, and MIEN A. RIFAI.

Pandan family (Pandanaceae) is represented in Java by two genera, Freycinetia Gaud. and Pandanus Parkins. Since the studies by Backer and Bakhuizen van den Brink in 1968 and Stone in 1972, there were no further exploration on the pandan flora of the island have been made, thus the pandan flora remains largely unknown. Taxonomical problem as far as the Java pandans concerned, are centered not only on the species status ofPandanus odoratissimus L.f andP. tecrorius var.littoralis which are considered as synonym, but also the species status which are given as synonym ofPandanus furcatusRoxb. by Backer and Bakhuizen van den Brink. Under the latter they included of P. bantamensis Koord.,P. oviger Martelli,P. pseudolaisWarb., and P. scabrifoliusMartelli into one species ofP. furcatusRoxb. This classification is in contrast with Stone who stated that these species were regarded as four different species. Therefore an effort has been made to redescribe these species in detail, using morphological, anatomical and molecular data such as sequence data ofatpB-rbcL IGS to provide better understanding of morphological, anatomical and molecular characters in supporting taxa delimitation and its distribution in Java.

This study was based mainly on available specimens at the Herbarium Bogoriemse (BO), National Herbarium Netherlands, Leiden (L) and Herbarium of the Royal Botanical Gardens Kew (K) and new collection specimens obtained from field work in different location in Java. In addition, living plants grown in botanical garden were also studied. Five species that was planted in Bogor Botanical Garden, viz. Pandanus kurzii, P. labyrinthicus, P. multifurcatus, P. polycephalusandP. spinistigmaticuswere also examined.

Characters of leaf shape, leaf apex, the morphology of leaf auricles and type of pistillate inflorecence were found useful in delimitation and identification of Javanese Freycinetia, while character of habit, the surface of stem, present or absent of prop root, the surface of prop root, the leaf shape, leaf apex, the armature of leaf margins and midrib, the colour of leaf margin and midrib teeth, the distinctness or indistinctness of tertiary cross vein, present or absent of apical ventral pleats, the texture of leaves in dry state, phalange shapes, the position of infructescence, position of seed chamber and stigma shape are proved useful for distinguishing species of JavanesePandanus.

Besides gross morphological characters, anatomical characters of leaf are used as well. They have proved useful especially for distinguishing between closely related species, because anatomical characters can provide information and strengthening conclusion based on morphological characters. Stomata in Javanese Freycinetia is relatively uniform, the degree might be in the qualitative manner, except in Freycinetia sumatrana stomata are arranged in neat longitudinal rows which are alternating with each row of stomata is one row of cubical crystals, while stomata in Pandanus were variable, and the variation largely involved papillae developed on subsidiary and neighbouring cells. The range of variation in

the epidermal tissue including the stomata proves to be a great value in identification ofPandanusspecies in Java.

Morphological, anatomical and comparative sequence data of atpB-rbcL IGS were able to solve the taxonomical problem of Pandanus furcatus complex and P. tectorius complex and as a result P. bantamensis Koord, P. pseudolais Warb. and P. scabrifolius Martelli treated as three different species, and P. odoratissimusL.f andP. tectoriusvar.littoralis treated as two different species.

ISSR (Inter Simple Sequence Repeat) study showed that six species of Freycinetia and thirteen species of Pandanus from Java have high genetic diversity, although Freycinetia has a bit lower of genetic diversity than Pandanus, while Principal component analysis (PCA) for Javanese thirteen Pandanusand six JavaneseFreycinetiashowed a bit different in clustering pattern and species relationships compared to the cluster analysis.

This study showed that in Java there are seven species of Freycinetia, viz. F. angustifolia Bl., F. funicularis Merr., F. imbricata Bl., F. insignis Bl., F. javanica Bl.,F. scandensGaud., andF. sumatranaHemsl.; and sixteen species of Pandanus, viz.P. amaryllifoliusRoxb., P. bantamensisKoord., P. bidur Jungh., P. dubius Spreng., P. faviger Backer, P. kurzii Merr., P. labyrinthicusKurz, P. multifurcatus Fagerl., P. nitidus Kurz, P. odoratissimus L.f., P. polycephalus Lam., P. pseudolais Warb., P. scabrifolius Martelli., P. spinistigmaticus Fagerl., P. tectoriusParkins, with two varieties, viz.P.tectoriusvar.littoralis,P. tectorius var. samak, one cultivar, i.e Pandanus tectorius cv. Sanderi, P. utilis Bory; one variety P. leram Jones var. andamanensium (Kurz) Stone, and one cultivar, P. spuriusMiq. cv. Putat.

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BIOSYSTEMATICS OF

PANDANACEAE

IN JAVA

SRI ENDARTI RAHAYU

Dissertation

Submitted as a part of the requirement for the fulfillment of Doctor degree in Biology

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

Examiners in comprehensive examination: Dr. Rugayah MSc

Researcher In Herbarium Bogoriense, LIPI Cibinong Dr. Rita Megia, DEA

Lecturer in Biology Department, Faculty of Mathematics and natural Sciences, Bogor Agricultural University, Bogor

Examiners in final examination:

Dr. Sri Soedarmiyati Tirtosoedirdjo MSc

Lecturer in Biology Department, Faculty of Mathematics and natural Sciences, Bogor Agricultural University, Bogor

Dr. Ruliyana Susanti MSi

ACKNOWLEDGMENT

This work could not have been accomplished without the kind assistance of many persons and institutions. I would like to thank to my advisors Prof. Dr. Ir. Alex Hartana MSc (chairman of the advisory committee), Dr. Ir. Tatik Chikmawati MSi, Dr. Kuswata Kartawinata and Prof. Dr. Mien A. Rifai (members of the advisory committee) for their advices, guidance, encouragement and valuable discussion throughout this study. Prof. dr. Peter C van Welzen of the National Herbarium Netherlands – Leiden University are gratefully acknowledged for his kind assistance in offering permits and providing facilitation for my study at Leiden, Dr. Jef Veldkamp for helping to translate German literatures, Dr. Barbara Gravendeel and MCM Eurlings for allowing me to work in the laboratory, introducing me with PCR and guiding me how to determine DNA sequence with sequencer in Van der Klauw Laboratory, Leiden University, Dr. William J. Baker, Dr. Timothy M.A Ulteridge arranged my visit to Kew Herbarium and allowing me working withPandanaceaecollection, Dr. Rogier de Kok from Kew Herbarium for helping me translate German literature, Dr. Tim Fulcher to let me work with sequencer in the Jodrell Laboratory of Royal Botanic Gardens Kew. I sincerely thank to Dr. Ir. Dedy Duryadi Solihin, DEA for his advice, Dr. Eko Baroto Walujo for giving me opportunity to studyPandanaceae specimens and conduct anatomical study at the Laboratory of Anatomy, Morphology and Cytology, Herbarium Bogoriense (BO), Cibinong. I Also would like to thank the Directors and Curators of National Herbarium of Netherland (L) and Herbarium of Kew Botanical Garden (K) for giving me an opportuninty to study Pandanaceae specimen kept at L and K, Dr. Irawati for giving me opportunity to study living collection of Pandanaceae in Bogor Botanical Garden.

I am grateful to Dr. Rugayah MSc, Dr. Rita Megia, DEA, Dr. Sri Sudarmiyati Tjitrosoedirdjo, MSc and Dr. Ruliyana Susanti MSi are acknowledged for their willingness being examiners in my final examination. Many people have helped me through good advice, suggestions and supports. In particular. I would like to thank to Dr. Sunaryo, Drs. Erlin Rachman, Eka Fatmawati T, S.Si, Ujang Hapid, Widoyanti and Dr. Dorly for their help and

technical supports during my anatomical study, Dr. Elizabeth A. Widjaja, for her kindness in giving the specimens of Freycinetia and giving references of Pandanaceae, Dr. Arry P. Keim, for his valuable discussion and critics on Pandanaceae., Dra, Tutie Djarwaningsih, MSi, for her help for publication in Floribunda, Dr. Fitmawati, Dr. Soaloan Sinaga, Sulassih SP for their help and technical supports during ISSR study and Dr. Nunik Sri Ariyanti for her help in making keys.

Rector and the Dean of Fakultas Biologi Universitas Nasional, Jakarta, Koordinator Kopertis Wilayah III, Rector and Dean of Sekolah Pascasarjana Institut Pertanian Bogor (IPB) for the opportunity given to me to undertake this study. Financial support came from BPPS Dikti Depdiknas, Universitas Nasional, Fundamental research grant from DP2M-Dikti Depdiknas, International Publication grant from Dikti Depdiknas, Sandwich program from Dikti Depdiknas, and from Yayasan Damandiri.

I sincerely thank to all staff at Herbarium Bogoriense for sharing their experties in different fields, all my colleagues in Fakultas Biologi Universitas Nasional, and all post graduate student of Botany – Taxonomny IPB, Dr. Nurhaidah Iriany Sinaga MSi, Dr. Nursahara Pasaribu MSc., Dr. Pudji Widodo, Dr. Iris Rengganis and Dra. Ratna Siahaan MSi who always offered good suggestion and words of hope during the hard times of my study.

Finally, my deepest appreciation is also sent to my parents, my brothers and sisters, my brothers and sisters in law, especially my husband Drs. Darmanto and my daughters Dea Anggraini and Della Anggiani for their deep understanding and great patient and for giving me their moral support.

Bogor, January 2011 Sri Endarti Rahayu

CURRICULUM VITAE

Sri Endarti Rahayu was born on 25 August, 1958 in Bandung, West Java. She spent her childhood with her parents in Bandung until she graduated from the Padjadjaran University in 1984.

Since 1988 she has been working at Biology Faculty at National University, Jakarta. In September 1991, she had an opportunity to continue study at IPB sponsored by TMPD Dikti Depdiknas., and admitted to the degree of MSi in October 1995.

In September 2004 she had an opportunity to continue study at IPB for her PhD sponsored by BPPS Dikti Depdiknas. In 2008, she had the opportunity to follow sandwich program, sponsored by Directorate General of Higher Education (DGHE) of Indonesia to study Biosystematics of Pandanaceae in Java for three months at Nationaal Herbarium of The Netherlands (NHN), Leiden University and one month at Kew Herbarium of Royal Botanic Garden, Kew, UK.

A part of dissertation has been published under two different title articles : (1) Genetic Diversity of Pandanus and Freycinetia from Java based on ISSR markers, Floribunda 3(4) : 95 – 103. 2007; (2) A taxonomic study of the Pandanus furcatus andPandanus tectorius complexes with special emphasis on plants from Java submitted to the 8th International Flora Malesiana Symposium in Singapore Botanic Garden, 23–27 August 2010.

TABLES OF CONTENTS

Page

LIST OF TABLES ... ix

LIST OF FIGURES ... x

GENERAL INTRODUCTION Taxonomical Aspects ofPandanaceaein Java and Its Systematic Problems ... 1

Objectives ... 3

MORPHOLOGY OF JAVANESE SPECIES OFPANDANACEAE Introduction ... 5

Materials and Methods ... 6

Results and Discussion ... 6

Morphology of JavaneseFreycinetia ... 6

Morphology of JavanesePandanus ... 12

LEAF ANATOMICAL FEATURES INPANDANUS (PANDANACEAE) FROM JAVA Introduction ... 23

Materials and Methods ... 24

Results and Discussion ... 25

A TAXONOMIC STUDY OF PANDANUS FURCATUSCOMPLEX AND PANDANUS TECTORIUSCOMPLEX WITH SPECIAL EMPHASIS ON PLANTS FROM JAVA Introduction ... 39

Materials and Methods ... 40

Results and Discussion ... 41

GENETIC DIVERSITY OF JAVANESEPANDANUSAND FREYCINETIA BASED ON ISSR MARKER Introduction ... 51

Materials and Methods ... 53

Results and Discussion ... 56

THE DISTRIBUTION AND ECOLOGY OFPANDANACEAEIN JAVA Introduction ... 67

Materials and Methods ... 67

Page SPECIFIC PART :

A. BIOLOGICAL FLORA OF JAVA :Pandanus tectoriusParkinson 75

B TAXONOMIC TREATMENT ... 87

GENERAL DISCUSSION ... 137

CONCLUSION ... 143

LIST OF TABLES

Page 4.1 Morphological characters of species recognized within

Pandanus furcatuscomplex ... 41 4.2 Summary of contrasting characters of species recognized within

Pandanus furcatuscomplex. Leaf base colour, scale bar = 5 cm; Peduncle, scale bar = 13 cm; Cephalia shape, scale bar = 10 cm;

Style shape, scale bar = 1 cm ... 42 4.3 Morphological characters of species recognized within

Pandanus tectoriuscomplex ... 43 4.4 Summary of contrasting characters of species recognized within

Pandanus tectoriuscomplex; Prickles on leaf apex, scale bar = 2 cm; Cephalium shape, scale bar = 10 cm ; Phalange apex,

scale bar = 1 cm ... 44 4.5 Polymorphic sites inatpB-rbcL IGS sequence of

Pandanus furcatuscomplex ... 45 4. 6 Polymorphic sites inatpB-rbcL IGS sequence of

Pandanus tectoriuscomplex ... 46 5.1 Thirteen species ofPandanusof Java used in this study ... 54 5.2 Six species ofFreycinetiaof Java used in this study ... 55 5.3 ISSR primers sequence and amplified results on thirteen species of

Pandanusof Java ... 58 5.4 ISSR primers sequence and amplified results on six species of

Freycinetiaof Java ... 58 5.5 Nei and Li’s similaritymatrix of thirteenPandanusspecies of Java

by using ISSR primers ... 59 5.6 Nei and Li’s similarity matrix of sixFreycinetia species of Java

by using ISSR primers ... 60

LIST OF FIGURES

Page 2.1 Habit ofFreycinetiaspp.: A.F. javanica, scale bar = 15 cm;

B. F. sumatrana, scale bar = 2 m. ... 7 2.2 Auricles type ofFreycinetiaspp.: A. Membranous (F. javanica)

B. Coriaceous (F. insignis).Auricles was shown with arrow. Scale bar = 2 cm ... 9 2.3 Inflorescence type ofFreycinetiaspp.: A. Umbel (F. javanica),

B. Racemiform (F. angustifolia), C. Lateral (F. funicularis).

Scale bar = 1 cm ... 11 2.4 Berries type ofFreycinetiaspp.: A. Obconic (F. angustifolia)

B. Lageniform (F. insignis), scale bar = 25 mm ... 12 2.5 Habit ofPandanusspp.: A. Shrub(P. amaryllifolius), scale bar = 10

cm; B. Tree (P. odoratissimus.), scale bar = 1 m ... 13 2.6 Prop root type ofPandanusspp.: A. Slender (P. spuriuscv. Putat):

B. Stout (P. odoratissimus); C. No prop root (P. kurzii);

D. Muricate in longitudinal line (P. utilis). Scale bar for A = 75 cm, Scale bar for B, C and D = 5 cm ... 14 2.7 Prickles type ofPandanus leaves: A. White prickles (P. tectorius

var. littoralis); B. Red prickles (P. utilis), scale bar = 1 cm ... 15 2.8 Inflorescence type ofPandanusspp.: A. Staminate inflorescence erect

(P. labyrinthicus); B. Long staminate inflorencence (P. utilis); C. Pistillate inflorescence in spike (P. odoratissimus); D. Raceme of spikes (P. polycephalus). Scale bar for A and B = 10 cm, scale bar for C and D = 2 cm ... 18 2.9 Stamen types ofPandanusspp.: A. Sessile(P. tectoriusvar. littoralis)

B. Pedicellate (P. utilis), scale bar = 2 cm ... 18 2.10 Stigma type ofPandanusspp.: A. Sessile (P. utilis), B. On style

(P. scabrifolius), scale bar = 1 cm ... 19 2.11 Cephalia type ofPandanusspp.: A. Cephalia consist of numerous

drupes (P. bantamensis); B. Drupe (P. bantamensis); C. Cephalia consist of numerous phalanges (P. utilis); D. Phalange (P. utilis); E. Cephalia covered by flat scales (P. kurzii); F. Apical phalange with cracks on the centre of apical sinuses (P.tectoriusvar.littoralis). Scale bar for A and C = 20 cm.; scale for B, D, E and F = 1 cm. 21

Page 3.1 Light micrographs of leaves: (A)- transverse leaf section ofP.

multifurcatusFagerl.; (B)- elongated abaxial epidermal cells of P. bantamensisKoord.; (C)- undulate adaxial anticlinal cell walls ofP. kurziiMerr ; (D)- adaxial epidermis cells with cubical crystals ofP. bantamensisKoord.;(E)- adaxial stomata with lobed papillae ofP. utilisBory; (F)- costal zone in the abaxial epidermis of P. amaryllifoliusRoxb. Scale bar for A & F = 100 µm.; Scale bar

for B, C and D = 50 µm; scale bar for E = 20 µm ... 26 3.2 Light micrographs of leaves: (A)- abaxial epidermal cells of

P. dubiusSpreng.; (B)- abaxial papillae in lateral subsidiary cells of P. tectoriusvar.littoralis; (C)- abaxial papillae on epidermal cells of P. spuriusMiq. cv Putat; (D)- hypodermis ofP. labyrinthicusKurz; (E)- crystal cells in outermost hypodermis ofP. pseudolaisWarb.; (F)- circular palisade cells ofP. amaryllifoliusRoxb. Scale bar for

A, D, E. & F = 50 µm.; scale bar for B & C = 20 µm ... 29 3.3 Light micrographs of leaves: (A)- sclerenchyma in spongy tissue of

P. tectoriusParkinson cv.Sanderi, (B)- raphide sacs in internal palisade ofP. amaryllifoliusRoxb.; (C)- unspecialized abaxial stomata ofP. tectoriusParkinson cv. Sanderi ; (D)- abaxial papillose lateral subsidiary cells ofP. odoratissimusL.f ; (E)- abaxial papillose terminal and lateral subsidiary cells ofP. scabrifoliusMartelli; (F)- abaxial papillose neighbouring and subsidiary cells ofP. kurzii Merr.; (G)- abaxial, overarching, lobed or dendritic papillae ofP. utilis Bory. Scale bar for A & B = 100 µm, scale bar for C, D, E, F &

G = 20 µm ... 31 5.1 ISSR profile ofPandanusspp and Freycinetiaspp of Java using

primer ISSR4;M : Marker, S1:P. bantamensis, S2:P. tectoriusvar. littoralis, S5:P. odoratissimus, S6:F .javanica, S7:F. angustifolia, S14:P. nitidus,S15:F. angustifolia, S20:F. sumatrana,

S22:F. imbricata, S24:P. kurzii, ... 57 5.2 ISSR profile of Pandanusspp and Freycinetiaspp of Java

using primer ISSR7.; M: Marker, S3:P. bidur,

S4:P. spinistigmaticus,S8:P. pseudolais, S13:P. multifurcatus S14: P. nitidus, S21:P. amaryllifolius, S23:P. polycephalus,

S24:P.kurzii, S25:P. scabrifoliusand S26: P.dubius... 57 5.3 UPGMA dendrogram based on ISSR analysis of thirteen

Pandanusspecies of Java ... 60 5.4 UPGMA dendrogram based on ISSR analysis of sixFreycinetia

Page 5.5. Principal component analysis for thirteen JavanesePandanus

based on ISSR data ... 62 5.6. Principal component analysis for six JavaneseFreycinetiabased

on ISSR data ... 63 6.1 Diversity ofPandanaceaein different habitat in Java, BF (Beach

Formation); LRF (Lowland Rain Forest); HF (Hill Forest); LMF

GENERAL INTRODUCTION

Taxonomical Aspects ofPandanaceaein Java and Its Systematic Problems

Pandanaceae was first recognized by Robert Brown in 1810. The Pandanaceae, the sole representative of the Pandanales, is arborescent or scandent monocotyledons confined to the Old World tropics and subtropics (Cox 1990). Pandanaceae is a large family, consist of 4 genera: Freycinetia Gaud., Pandanus Parkins., Sararanga Hemsl., and Martellidendron Callm. & Chassot (Callmanderet al. 2003).Pandanaceaecommonly known as screw pine or screw palm has a unique growing form. The large serrated leaves up the trunk are formed in a circular motion, giving it the screw like look. The family is important in several regions, wherein it has contributed to the fundamental structure and physiognomy of the vegetation (Stone 1983b).

Pandan family (Pandanaceae) is represented in Java by two genera: Freycinetia Gaud. andPandanus Parkins. (Backer and Bakhuizen van den Brink 1968; Stone 1972). The genus Freycinetia was described in 1824 by the French botanist Gaudichaudii (Stone 1970), and may be called climbing pandans. This genus has been known to occur in Java for a long time, since most of the oldest taxa nomenclaturally speaking are typified by specimens from Java (Stone 1968), and according to Stone (1970) the real home ofFreycinetiaamong others is Java. The name Pandanus originates from the Malay word pandan, it was latinized and first published by George Eberhard Rumpf (Rumphius) whose Herbarium Amboinensis of 1743 contains the description of eleven species of which eight are illustrated (Stone 1965). Java harbours a large number of species of Pandanus,not less than fifteen species of Pandanus are mentioned in the key given by Backer and Bakhuizen van den Brink (1968). It becomes evident that Pandanusin Java is rich in species, considering the widely variable morphology of this genus and of the species already known from this island.

The last attempt at a comprehensive treatment of Pandanaceae of Java was Backer and Bakhuizen van den Brink (1968) in their “Flora of Java” in which they recognized seven species of Freycinetia and fifteen species of Pandanus. Since a short visit to Hortus Bogorienses by Stone (1972), there were no further exploration on the pandan flora of the island have been made, thus the pandan flora remains largely unknown.

Since a large number of morphological characters are now known for Freycinetia and Pandanus species, it appears useful to consider their use in identifyingPandanaceaeof Java, because morphological data are regarded as the most appropriate and the most rapid mean for identification and for constructing map of diversity of plant (Davis & Heywood 1963). Morphological characters have the great advantage over other characters that we can see the plant variability easier. Unfortunately complete material is not always available for the various numbers of a group being studied, and sometimes we need to identify incomplete material or small fragment of material. In this case we use anatomical characters. It is now realized that anatomical characters is as valuable as morphological ones (Stace 1989). Some of these anatomical characters are so diagnostic that they are now commonly used in identification or identification of fragment of plants (incomplete material). Valuable taxonomic evidence has been obtained from the study of leaf epidermis and stomata ofPandanus, and the appearance of particular anatomical characters seems sufficiently constant. In this study we are able to propose anatomical key to species forPandanusin Java. Taxonomical problem as far as the Java pandans concerned, are centered not only on the species status ofP. odoratissimusandP. tecroriusvar.littoralis, which are considered as a synonym, but also the species status which are given as synonyms of Pandanus furcatus Roxb. Backer and Bakhuizen van den Brink (1968) included P. bantamensis Koord.,P. oviger Martelli,P. pseudolaisWarb., and P. scabrifoliusMartelli into one species ofP. furcatusRob. This classification is in contrast with Stone (1972) who stated that these species were regarded as four different species. In this study we only found three of four species mentioned above, viz.P. bantamensisKoord.,

P. pseudolaisWarb, P. scabrifoliusMartelli. Therefore an effort has been made to redescribe these species in detail, using morphological, anatomical and molecular data, i.e sequence data of atpB-rbcL IGS since a satisfactory classification depends upon the interpretation of as many characters as possible (Davis & Heywood 1963). Molecular data can be useful in solving different kinds of taxonomic problem, and choloroplast marker such as atpB-rbcL IGS can be particularly valuable at lower taxonomical level (Soltis & Soltis 1998).

The pandans in general tend to be horticulturally interesting and often curious or unusually elegant, and tend to cause considerable public interest. The species that are already completely well known in cultivation are few, viz. P. dubius, P. spurius cv. Putat, P. tectorius cv. Sanderi and P. utilis. In view of its importance and considering the large number of wild species germsplasm available for Freycinetia and Pandanus in Java, the genetic analysis through molecular marker is a prerequisite to have a deep insight of the genome organization in the wild species. It is imperative, threfore to establish strategies for the preservation of Freycinetia and Pandanus germplasm. In this study, genetic diversity have been assessed using ISSR marker. This analysis is a preliminary step to ensure the conservation and the development of genetic resources, and to know the most important location to conserve Pandanaceae in Java, we studied the distribution and ecology ofPandanaceaein Java.

Every chapter was written in different style, following the format of the journal we are going to publish these articles, e,g. the article of Biological Flora of Java:Pandanus tectoriusParkinson is in preparation for publication in Journal of Ecology.

The objectives of the study

Our objective were to unravel species which is having taxonomical problem. A combined morphological, anatomical and molecular marker was undertaken to evaluate the suitability of the species concept; to determine genetic diversity, and to know the distribution and ecology ofPandanaceaein Java.

MORPHOLOGY OF JAVANESE SPECIES OF

PANDANACEAE

Introduction

The last attempt at a comprehensive treatment of Pandanaceae of Java was Backer and Bakhuizen van den Brink (1968) in their “Flora of Java” in which they recognized seven species of Freycinetia and fifteen species of Pandanus. Since a short visit to Hortus Bogorienses by Stone (1972), there were no further exploration on the pandan flora of the island have been made, thus the pandan flora remains largely unknown.

Since a large number of characters are now known for Freycinetia and Pandanusspecies, it appears useful to consider their use in identifying plants from Java. The species are invariably classified by the feature of the female for several reasons (Kam 1971). First, the male flowers are very short-lived, generally they bloom and decay within 2 or 3 days. Furthermore, there seem to be a higher percentage of females in any given population. Thirdly, the fruit offers a greater number of useful diagnostic features. The male inflorescences are generally strongly scented, and sometimes offer powering fragrance. The male flowers themselves are simply masses of stamens gathered into units representing branching systems of various ranks. Fieldwork carried out for this study has provided stronger foundation for understanding morphological variation within the species.

Characters of leaf shape, leaf apex, the morphology of leaf auricles and type of pistillate inflorescence were found useful in delimitation and identification of Javanese Freycinetia, while characters of habit, the surface of stem, present or absent of prop root, the surface of prop root, the leaf shape, leaf apex, the armature of leaf margins and midrib, the colour of leaf margin and midrib teeth, the distinctness or indistinctness of tertiary cross vein, present or absent of apical ventral pleats, phalange shapes, the position of infructescence, position of seed chamber and stigma shape are proved useful for distinguishing among species of JavanesePandanus.

This study was undertaken to have a better understanding on the morphology of the family in order to make a better species delimitation, particularly for species found in Java.

Materials and Methods

Morphological study on Javanese species of Pandanaceae has been conducted. This study was based mainly on available specimens at the BO, K and L and new collection specimens obtained from field work in different location in Java. In addition, living plants grown in botanical garden were also studied. Five species that was planted in Bogor Botanical Garden, viz. Pandanus kurzii, P. labyrinthicus, P. multifurcatus,P. polycephalusandP. spinistigmaticus were also examined. The process of undertaking in this study followed the methods described by Rifai (1976) and Vogel (1987).

Basic morphological characters such as habit, stem, leaves, inflorescence, staminate flower, pistillate flower, fruit and their details were used to describe and recognize taxa; all morphological data was used for producing the description of each taxon, while the key to species was constructed from the diagnostic characters only.

The morphological species concept was applied as a framework to define taxa, in which distinction is based on perceived discontinuities in morphological variation (Davis & Heywood 1963).

Results and Discussion

Morphology of JavaneseFreycinetia Habit

All species of Freycinetia found in Java are climber with different size. Three species of Freycinetia (F. imbricata, F. javanica and F. scandens) are smallish to medium climber, while F. insignis and F. sumatrana are medium climbers, and the smallish climber is found inF. angustifolia.

The stems are found hanging on trees and branch richly, such as found in F. angustifoliaandF. javanica(Fig. 2.1 A); while inF. imbricatathe main stem remain adherent to tree trunk, and in some cases makes a complete cover which

makes the trunk of the host tree invisible, such as found in F. insignis and F. sumatrana(Fig.2.1 B).

Figure 2.1 Habit ofFreycinetiaspp.: A.F. javanica, scale bar = 15 cm B. F. sumatrana, scale bar = 2 m.

Stem

Stems of JavaneseFreycinetiavary in size, nodes and colour. In term of size, F. angustifolia, F. imbricata, F. javanica and F. scandens are included in slender species, whileF. funicularis, F. insignisandF. sumatranaare included in robust species. Variation is observed in stem shape. This study showed that F. angustifolia, F. imbricata,F. insignis, F.scandens, andF. sumatranahad terete stem, whereasF. javanicahad subterete to terete stem.

Variation is also observed in internode shape. This study showed that F. angustifolia, F. imbricata, F. insignis, F. javanica, and F. sumatranahad terete internode, whereas F. scandens had subterete to terete internode. The surface of the stem can be sulcate or sulcate to canaliculate. The colour also varies from yellowish, greenish brown to reddish brown.

Leaves

The leaves ofFreycinetiaare usually green to dark green coloured on the upper surface, but paler green on the lower surface. In Java F. angustifolia possesses the smallest and most slender leaves, whereas F. sumatrana has the most robust leaves. The leaves arrangement in species of Freycinetia found in Java is alternate and imbricate. Four species of Freycinetia (F. angustifolia, F.

A A

funicularis,F. javanicaandF. scandens) have alternate leaves, and the other three species (F. imbricata,F. insignisand F. sumatrana) had imbricate leaves.

Leaves are simple blade and usually linear-lanceolate in outline, but variation do exist in some species.F. scandenshave variation in leaf shape, from elliptic, oblanceolate to lanceolate. Leaf apex can be abruptly attenuate below the apex to gradually attenuate toward the subulate apex, whereas in F. sumatrana it is long tapering to a slender subulate tip. The margin usually was armed in basal part, apical part and upper midrib of its leaves with serrate prickles. The basal part ofF. insignisandF. scandens are denticulate, and basal part of F. sumatrana is dentate. The colour of the prickles may be wholly straw colour or with brown tipped as in F. angustifolia. The laminar part can be chartaceous, subcoriaceous to coriaceous.

Auricles

The auricle is an organ of flange-like extensions found on the leaf-sheath, and can be easily seen on the young leaf. Auricles are usually membranous (Fig. 2.2 A), transparent, fragile or coriaceous (Fig.2.2 B). Auricles can be regarded as a good identification character of Freycinetia in the field, because in herbarium specimen they are hardly seen in fine condition. The shape, size, texture, nature of margin, nerves and colour of auricle vary between species. Freycinetia angustifolia possess the smallest auricle, while F. sumatrana possess the biggest auricle. Colour of auricles also varies from pale green in F. insignisto brownish green–brownis yellow in F. javanica.

InFreycinetia, they presented in varying from tapered or rounded to the apex or adnate to the apex. The margin is entire or denticulate to spinulose at apex (F. insignis), or armed almost to the base (F. imbricata). The lamina is usually membranous inF. javanica(Fig. 2.2 A) or coriaceous inF. insignis(Fig. 2.2 B), some of them fragmenting transversally in F. sumatrana or fragmenting laterally in F. scandens, with 4 widely spaced nerved in F. insignis or 1-2 septate in F. javanica.

Figure 2.2 Auricles type ofFreycinetiaspp.: A. Membranous (F. javanica) B. Coriaceous (F. insignis).Auricles was shown with arrow. Scale bar = 2 cm

Bracts

Bracts are persistence or caduceus, located on the peduncle. Bracts vary in shapes and sizes. Bracts are usually ovate, cymbiform to lanceolate, with entire margin to slightly armed with prickles. The sizes of bracts are in concurrent with their habits. Slender species like F. angustifolia, F. imbricata and F. javanica possess minute bracts, while robust species such as F. insignisandF.sumatrana have robust bracts as well. Their apex have various shapes, varying according to the species from acute to acuminate in F. imbricata, acute to cuspidate in F. javanica) mucronate to aristate in F. angustifolia, and aristate with aculeate prickles inF. sumatrana.

Peduncle and pedicel

The peduncle usually is straight and short. The peduncle may be slender or robust, densely or sparsely pubescent or rarely glabrous, only in F. insignis the peduncle had bract scar. Usually the pedicell is longer than peduncle. The pedicel shape usually is half-terete, slender (F. scandens), usually as thick as peduncle, or only somewhat stouter than the peduncle with 0.2-1.5 cm diameter (F. insignis). The surface of pedicels can be glabrous or covered with indumentum. F. angutifolia, F. javanica and F. scandens had glabrous pedicel, while F. imbricataandF. insignishad puberolous to hirsute pedicell.

Inflorescentia

The inflorescence in most ofFreycinetiafound in Java are usually located on terminal part of the stems (i.e terminal inflorescence). Lateral inflorescence are less often seen. This study showed that there is one species that possess such feature, viz.F. funicularis. In lateral inflorescence, not only bract that is present, but also prophyll, that is located in the upper part of bracts.

Freycinetia is mostly dioecious, which means that male and female flowers are formed on different plant. Staminate inflorescence is invariably terminal on a normal leafy shoot, usually a raceme of spikes, each of the three or four spikes. Staminate inflorescence are more rarely seen than that of pistillate inflorescence, due to the anthesis which takes only one to three days (Stone 1983b). Similar to other genera of Pandanaceae, Freycinetia identification is therefore mainly based on the structure of female inflorescence and infruitescence. In Freycinetia, the spikes (both male and female) tend to be closely adjacent and often ternate, so that the open ripe inflorescence seems to be an umbel like in F. javanica (Fig. 2.3 A) or pseudoumbel. In F. angustifolia, the inflorescence is racemiform (Fig. 2.3 B), whereas in F. funicularis, the inflorescence is lateral (Fig. 2.3 C).

Male flowers (Stamen)

The stamens are simple, without branched filaments, and each terminate is in a small rather short anther or sessile stamens, and staminate pedicel is absent.

Female flowers (Pistil)

The pistil consists of multiovulate carpel with carpels separated to base or carpel united. Each carpel tipped by a stigma, and the stigma is sessile. The stigmas vary in number and shape, from 2 to 5 in number, and from ovate (F. javanica), depressed ovate-suborbicular (F. angustifolia, F. imbricata, and F. insignis), pentagonal-suborbicular (F. scandens) or protude (F. funicularis) in shape. The position of the stigma varies from horizontal to sunken in position.

Fig.2.3 Inflorescence type ofFreycinetiaspp.: A. Umbel (F. javanica), B. Racemiform (F. angustifolia), C. Lateral (F. funicularis). Scale bar = 1 cm

Cephalia

Cephalium (plural: cephalia) is the complex fruit in Pandanaceae. Some of the important morphological character inFreycinetiaare found in the cephalia. In the Javanese species of Freycinetia cephalia vary in shapes, size and colours. Four species (F. funicularis F. insignis, F. javanica, and F. sumatrana) have cylindric cephalia. F. angustifolia have cylindric to oblanceolate cephalia, F. scandens have cylindric to oblong cephalia, and F. imbricata have narrowly elliptic to broadly oblong cephalia. The number of cephalia per inflorescence is usually three, two or four.

A B

Berries

Berry is the simple fleshy fruit ofFreycinetia. InFreycinetia a cephalium consisted of numerous berries. A berry contain many fused ovules, thus a multiovulate fruit, while inPandanusthe simple fruit is always uniovulate. In this circumstances a cephalium can be regarded as a complex fruit. The shape of berries vary from obconic like in F. angustifolia (Fig. 2.4 A) obovate (F. funicularis), oblong (F. javanica) and subpyramidal such as in F. sumatrana. Three species, viz. F. imbricata, F. insignis(Fig. 2.4 B) andF. scandens are the species observed with various shapes of berry from pentagonal to lageniform, pentagonal, lageniform to oblong, and pentagonal, lageniform, ovate to oblong respectively. The apical part of a berry is usually harder and stiffer, while the basal part is usually fleshy.

Fig.2.4 Berries type ofFreycinetiaspp.: A. Obconic (F. angustifolia) B. Lageniform (F. insignis), scale bar = 25 mm

Morphology of JavanesePandanus

Habit

UnlikeFreycinetiathat have climbing habit,Pandanusspecies habit found in Java vary from shrub to tree. Shrub species are P. amaryllifolius(Fig. 2.5 A), P. kurzii, P. nitidusandP. polycephalus. Tree species include P. bantamensis,P. bidur, P. dubius, P. faviger. P. labyrinthicus, P. leram var. andamanensium, P.

multifurcatus, P. odoratissimus) (Figure 2.4 B),P. pseudolais, P. scabrifolius, P. spinistigmaticus,P. spuriuscv Putat,P. tectoriusandP. utilis.

Figure 2.5 Habit ofPandanusspp.: A. Shrub(P. amaryllifolius), scale bar = 10 cm ; B. Tree (P. odoratissimus.), scale bar = 1 m

Stems

Stems of Javanese Pandanus vary in size, colour and branch. Slender stem is found in P. amaryllifolius or short stem in P. kurzii. Erect stem and unbranched are found in P. bantamensis,P. dubius andP. pseudolais, while P. labyrinthicus, P. multifurcatus, P. nitidus, P. odoratissimus and P. spinistigmaticus had spreading branches, whereas P. faviger, P. tectorius var. littoralis and P. utilis had dichotomous branching. Surface of stem can be smooth likeP. kurzii, sulcate inP. amaryllifolius, abundant rootlet to thorny in P. bidur, P. dubius, P. labyrinthicus, P. multifurcatus, P. nitidus, P. odoratissimus, P. spinistigmaticus, and P. tectorius var. littoralis or ringed by leaf scars in P. polycephalusandP. utilis. Colour of stem also varies from grey to green or brownish.

Prop root

A prop root is a root formed from the stem, usually close to the ground which helps hold the stem erect and anchor the plant. There are variation in prop root sizes, number and surfaces. Prop root can be small and few in P. amaryllifolius, small and abundant in P. labyrinthicus, P. odoratissimus and P. tectoriusvar.samak, slender in P. spurius cv Putat (Fig. 2.6 A), some arise from

lateral branches inP.nitidusandP. spuriuscv Putat, long and stout as inP. bidur, P. dubius, P. faviger, P. leram var. andamanensium, P. multifurcatus, P. odoratissimus(Fig. 2.6 B) andP. spinistigmaticus or absent in P. kurzii(Fig. 2.6 C). Surface of the prop root can be smooth in P. scabrifolius, muricate in longitudinal line such as in P. utilis (Fig. 2.6 D), P. tectorius var. littoralis, P. pseudolais, andP. nitidus or armed with prickles or spine inP. spinistigmaticus, P. polycephalusand P. multifurcatus.

Figure 2.6 Prop root type ofPandanusspp.: A. Slender (P. spuriuscv. Putat): B. Stout (P. odoratissimus) ; C. No prop root (P. kurzii) :

D. Muricate in longitudinal line (P. utilis). Scale bar for A = 75 cm, Scale bar for B, C and D = 5 cm.

Marginal Spine

Leaves usually set with prickles along its length or part of it. The prickles decrease in size nearing the leaf apex (P. utilis, P. bantamensis, P. pseudolais, P. tectoriusvar. littoralis, andP. nitidus), always antrorse (ascending) but those near leaf base sometimes retrorse, or absent. Their margin are usually prickly,

A B

occasionally entire (P. spuriuscv. Putat), or prickly very near and at apex only (P. amaryllifolius), or occasionally smaller at leaf base, larger at midsection and decreasing in size nearing the leaf apex (P. bidur, P. dubius, P. odoratissimus, andP. scabrifolius). Twin lateral pleats are often well distinguished, smooth (P. bantamensis, P. nitidus, P. pseudolais, and P. scabrifolius), or in some species prickly serrate or absent (P. odoratissimus, P. spinistigmaticus, P. spurius cv. Putat, and P. tectorius var. littoralis). Some species have white spine in P. tectorius var. littoralis (Fig. 2.7 A), green prickles with brown tipped (P. bantamensis, P. pseudolais, andP. scabrifolius) or yellowish green (P.dubius) or red prickles inP. utilis(Fig. 2.7 B).

Figure 2.7 Prickles type ofPandanus leaves: A. White prickles (P. tectorius var. littoralis) ; B. Red prickles (P. utilis), scale bar = 1 cm. Leaves

The leaves of Pandanus are usually dark green coloured, glossy and glaucous on upper (adaxial) surface, but paler green on the lower surface, whereas in P. tectoriuscv. Sanderi have longitudinal of white to pale yellow band from centre to leaf margin. Compared with the other members ofPandanaceae, the size of leaf in Pandanus is noticeably longer and bigger. In Java P. amaryllifolius has the smallest and most slender leaves, whereas P. pseudolais possess the longest leaves (299-574.5 cm long). The leave arrangement in all species of Pandanus found in Java is spiral. The leaves are simple and usually ensiform to linear in shapes. However variation do exist as in some species, the leaves are observed in ligulate such as found inP.labyrinthicus, P. multifurcatus, P. odoratissimus and P. spurius cv. Putat. The apical part can be abruptly

terminating in a point to gradually long tapering to subulate apex. Caudate apices are observed only in one species, i.eP. dubius. The margin can be entire as inP. spuriuscv. Putat or noticeably armed with spines throughout the length, except in P. amaryllifoliuspossess prickles only in the apical part of its leaves. The laminar part can be chartaceous, thin coriaceous, coriaceous to thick coriaceous. Longitudinal lines are more prominent abaxially, with tertiary cross vein on both surfaces. In P. amaryllifolius, P. bantamensis, P. bidur, P. dubius, P. kurzii, P. pseudolais, and P. scabrifolius tertiary cross vein form a network of meshes, oblong or rhombic meshes, whereas in P. leram var. andamanensium tertiary cross vein makes tesselate appearance. The basal part of leaves of P. kurzii, P. polycephalus and P. scabrifolius are whitish, reddish in P. utilis, or reddish brown inP. bantamensis, and P. pseudolais.

Bracts

Bract are covering inflorescence and usually formed by the three levels of trichiously arranged bracts. The interior bract covers the inflorescence, while the other two namely exterior and middle bracts protect the interior bract and the inflorescence inside.

Bracts vary in shapes and sizes. Bracts are usually lanceolate in shape, with margins armed with prickles to spines. The sizes of bracts are in concurrent with their habits. Robust species such as P. bantamensis, P. pseudolais and P. scabrifolius have robust bracts as well, while slender species like P. nitidus possess slender and minute bracts. The exterior bracts are leaf-like in forms, except inP. kurziithe exterior bracts are cymbiform in shape. The inner bracts are shorter and lighter in colour.

The colour of bracts also varies. The colour of exterior and interior bracts can be different. The differences can be seen within the same bract. The colour of the apical part, middle and basal parts may not be uniform such as in P. odoratissimus, and in few species the colour of bracts are uniform such as inP. polycephalus (white) and P. utilis (green). The apex of bracts are observed abruptly acute inP. utilis, gradually tapering to a subulate tip inP. labyrinthicus, P. odoratissimusandP. tectorius.

Inflorescentia

Pandanus are dioecious, which means that male and female flowers are produced on separate plants. The inflorescence in all species ofPandanusin Java is usually found in apical part of the stem (i.e terminal inflorescence). Staminate inflorescences are fragrant and usually pendent, except in P. labyrinthicus staminate inflorescence is erect (Fig. 2.8 A). Staminate inflorescence is also varies in sizes. In P. odoratissimusflowering part of spike is 21.3-33 cm long and bear 9-18 lateral racemes, whereas inP. utilisflowering part of spike is 41-43 cm long and bear 20 lateral racemes (Fig. 2.8 B). Staminate inflorescence are more rarely seen than that pistillate inflorescences, thereforePandanusidentification is mainly based on the structure of female inflorescences and infructescence.

Pistillate inflorescences may be represented by a single cephalium (head) as in P. odoratissimus (Fig. 2.8 C) or by s spike of several cephalia. In P. favigerthe pistillate are observed spike of 3 cephalia, whereas inP. polycephalus pistillate inflorescence is raceme of spike (Fig. 2.8 D).

Peduncle

The peduncle is stout and glabrous, generally straight, whereas in P. bantamensis,P. tectoriusvar. littoralis, andP. utilisthey are slightly curved at the end; in P. pseudolais it is curved at the end. Their shapes are usually 3-sided, whereas in P. polycephalus it is obtusely trigonous. Their size is larger at apex and decrease nearing the base. Their colour is varied according to the species from whitish green (P. bantamensis) to green (P. pseudolais, andP. utilis).

Male Flower (Staminate)

The stamens are to be borne in small or large clusters on short or long axes (P. labyrinthicus, P. odoratissimus, P. spurius cv. Putat, P. tectorius var. littoralis, andP. utilis). Most of the male flowers are sessile (Fig. 2.9 A), whereas inP. utilisit is pedicellate ( Fig. 2.9 B).

Figure 2.8 Inflorescence type ofPandanusspp.: A. Staminate inflorescence erect (P. labyrinthicus); B. Long staminate inflorencence (P. utilis); C. Pistillate inflorescence in spike (P. odoratissimus); D. Raceme of spikes (P. polycephalus). Scale bar for A and B = 10 cm, scale bar for C and D = 2 cm.

Figure 2.9 Stamen types ofPandanusspp.: A. Sessile(P. tectoriusvar. littoralis) B. Pedicellate (P. utilis), scale bar = 2 cm

C D

A B

Female Flower (Pistillate)

Female flowers consist of a single uniovulate carpel with a single stigma. The stigma may be sessile as in P. bidur, P. dubius. P. odoratissimus, P. polycephalus, P. tectorius var. littoralis, and P. utilis (Fig. 2.10 A) or borne on style in P. bantamensis, P. kurzii, P. nitidus, P. pseudolais, and P. scabrifolius (Fig. 2.10 B). The stigmas have various shapes, varying according to the species from linear (P. kurzii) or rounded (P. polycephalus), cordate, elliptic, lanceolate, lip-like (P. bidur) or separating in the forkes (P. bantamensiis, and P. scabrifolius).

Figure 2.10 Stigma type ofPandanusspp.: A. Sessile (P. utilis), B. On style (P. scabrifolius), scale bar = 1 cm

Cephalia

Some of the important morphological character for species identification in Pandanus are in their cephalia (singular, cephalium). In Javanese species of Pandanuscepalia vary in shapes, sizes and colours. Five species, i.eP. kurzii, P. odoratissimus, P. scabrifolius, P. tectorius var. littoralis and P. utilis have

subglobose cephalia, while the other species such as inP. bantamensis,P. nitidus, P. polycephalus and P. pseudolais possess ellipsoid to oblong cephalia. The number of cephalia per infruitescence is usually one, other numbers such as three, five or six are also present but less seen.

In Pandanus, a cephalium can be consisted of numerous drupes or phalanges. Drupes is composed of free, a single carpel while phalanges (polydrupes) are carpels that are permanently fused into compound structure. Drupes or phalanges are of great importance in Pandanus classification. The

result of this study showed that seven species. i.e P. bantamensis (Fig. 2. 11 A dan B), P. faviger, P. kurzii. P. nitidus, P. polycephalus, P. pseudolais and P. scabrifolius have cephalium consisted of numerous drupes, while the other species such as in P. bidur, P. dubius, P. leram var. andamanensium, P. odoratissimus, P. tectorius var.littoralis andP. utilis (Fig. 2.11 C and D) have cephalia consisted of numerous phalanges. Drupe varies in shape from conical, cuneate to oblanceolate, while phalange shapes vary from clavate, cuneate, obovate to oblong.

Drupes usually have one stigma while phalanges (polydrupes) have more than one stigma. Some species have sessile stigma, while in other species stigma is on style. The surfaces of cephalium can be smooth, such as in P. utilis or covered by flat scales like inP. kurzii (Fig. 2.11 E), while in the surface of the apical phalanges of Pandanus usually have no cracks on centre apical sinuses, except in Pandanus tectorius var. littoralis, there are cracks on the centre of apical sinuses on its the surface apical of the phalange (Fig. 2.11F ). The apical part of drupe of phalange is usually harder, and the basal part is usually fibrous and fleshy.

Figure 2.11 Cephalia type ofPandanusspp.: A. Cephalia consist of numerous drupes (P. bantamensis); B. Drupe (P. bantamensis); C. Cephalia consist of numerous phalanges (P. utilis); D. Phalange (P. utilis); E. Cephalia covered by flat scales (P. kurzii); F. Apical phalange with cracks on the centre of apical sinuses (P.tectoriusvar. littoralis). Scale bar for A and C = 20 cm.; scale for B, D, E and F = 1 cm.

A B

C _D

Conclusion

In this study, based on gross morphological characters, seven species of Freycinetia can be recognized, viz. F. angustifolia Bl., F. funicularis Merr., F. imbricataBl., F. insignis Bl., F. javanica Bl., F. scandensGaud., F. sumatrana Hemsl; and sixteen species of Pandanus, viz. P. amaryllifolius Roxb., P. bantamensis Koord., P. bidur Jungh., P. dubius Spreng., P. faviger Backer, P. kurziiMerr., P. labyrinthicus Kurz, P. multifurcatus Fagerl., P. nitidus Kurz,P. odoratissimus L.f., P. polycephalus Lam., P. pseudolais Warb., P. scabrifolius Martelli.,P. spinistigmaticusFagerl., P. tectoriusParkins, with two varieties, viz. P. tectorius var. littoralis, P. tectorius var. samak, one cultivar, i.e Pandanus tectorius cv. Sanderi, P. utilis Bory; one variety P. leram Jones var. andamanensium (Kurz) Stone, and one cultivar, P. spurius Miq. cv. Putat are reported from Java.

LEAF ANATOMICAL FEATURES IN

PANDANUS

(PANDANACEAE) FROM JAVA

Introduction

Pandanus Parkinson is characterized by a number of very obvious features, forming a unique combination of characters. These distinctive characters are generally an erect stem (sometimes sprawling), basally supported by many long stilt and prop roots, branching more or less dichotomously, usually rather prickly because of short, blunt or sharp specialized adventitious roots; leaves in 3 regular, close-set spirals, on the rounded or slightly 3-angled trunks, leaves entire, long, narrow, deeply channelled along the midrib and pleated once on each side, the tip, margin, midrib (below) and the pleats (above, sometimes) set with stout or fine prickles (Stone 1965).

Pandanus, with three other genera,FreycinetiaGaud., SararangaHemsl., and Martellidendron Callm. & Chassot form the Pandanaceae. Pandanus contains more than 600 species which are distributed throughout the tropics of the Old World and this large genus is internally very diverse (Kam 1971). The last revisions of thePandanaceaeof Java were made by Backer & Bakhuizen van den Brink (1968) and Stone (1972). They recognized 16 species and produced some classifications, which invariably used morphological characters of the pistillate flowers and fruits as diagnostic features. It is, therefore virtually impossible to identify sterile or staminate plants (Kam 1971).

Edeoga and Ikem (2001) showed that leaf epidermal features are as useful in systematic botany as for instance DNA sequences or chemical compounds. The taxonomic value of leaf anatomical features was considered in some detail by Stace (1965). Examining the shape of epidermal, guard and subsidiary cells of stomata may prove useful for the identification of selected plant species (Jakubska 2007). Stone (1976) constantly reaffirmed that the variation in the epidermal tissue (including stomata) is of great value in systematic botany. Tomlinson (1965) founded a classification of stomatal types based on progressive complexity, and this system was used by Kam (1971) to demonstrate that there is a corelation between stomatal characters and the sectional delimitation within

Pandanus.The aims of the present study are to describe the epidermal variation in 15 Javanese species ofPandanusand to evaluate the usefulness of the characters for identification and classification purposes.

Materials and Methods

The survey was largely based on fresh material collected from the field, from plants cultivated at the Bogor Botanical Garden and from herbarium material supplied by the Herbarium Bogoriense, Bogor. A full list of names and sources is provided in the appendix 1.

Leaves from living and dried specimens were used. Dried leaves were boiled in water for a few minutes to soften the leaf until they became unfolded and were ready for epidermal scrapping. Fresh leaves were fixed in 70% FAA. Leaf samples were prepared according to the modified method of Johansen (1940). The fresh or dried leaves were placed in a tube with 10% nitric acid and kept in boiling water for about 10-15 minutes. Nitric acid softens the leaf tissue and facilitates peeling of the epidermis.

Both epidermal layers were stripped off gently by brushing away unwanted tissue with the help of a pointed needle and forceps, after which the epidermis was stained with safranin. An excess of safranin was washed off and the epidermis was temporarely mounted in an aquaous glycerol solution.

For sectioning, a rotary microtome was used to make 15-20 µm thick sections. The ribbons were placed on clean slides smeared with a thin film of Haupt’s albumen and allowed to dry, after which a drop of water was added prior to mounting. The slides were placed on a hot plate at 40oC for a few minutes to let the ribbons expanding and they were stored overnight. In the next day the slides were immersed for 2-5 minutes in a solution of xylene and absolute alcohol (1:1 ratio v/v). The slides were then transferred to another solution of xylene and alcohol in the ratio 1:3 (v/v) for few minutes, after which they were washed in a series of 95%, 90%, 70% and 50% alcohol. The slides were stained with a few drops of fast green and counterstained with safranin for two minutes, then dehydrated in a series of 50%, 70%, 80%, 90% xylene/alcohol solution and mounted in Canada balsam. The slides were dried on a hot plate at 30o C

(Johansen 1940). The slides were examined and photographed with a Nikon Eclipse 80i digital microscope.

The voucher specimens are deposited in the Herbarium of the Biology Faculty of the National University in Jakarta (Indonesia).

Results and Discussion Results

Leaf anatomy

The transverse sections of all species are very uniform. All leaves have the same basic construction,i.e., the leaf is isolateral (Fig. 3.1A).

Epidermis

A distinct cuticle is sometimes developed. Pandanus labyrinthicus Kurz has a thick cuticle (4.96µ), while the rest of the species have a thin cuticle (<.2.48µ). The adaxial epidermis is never differentiated into costal and intercostal regions. The shape of the adaxial and abaxial epidermis cells is similar in P. amaryllifolius Roxb., P. multifurcatus Fagerl., P. nitidus Kurz, P. tectorius var. littoralis andP. tectorius cv. Sanderi, whereas the adaxial epidermal cells have a very different shape than the abaxial cells in P. bantamensis Koord., P. dubius Spreng., P. kurzii Merr., P. labyrinthicus Kurz, P. odoratissimus L.f., P. polycephalusLam.,P. pseudolaisWarb.,P. scabrifoliusMartelli,P. spuriusMiq. cv. Putat andP. utilisBory.

Six types of epidermal cells could be adaxially distinguished. Each species always shows only a single type, thus the shape of the adaxial epidermal cells is constant at the species level. The six types of the epidermal cells are: (1) rectangular adaxial epidermal cells are found only inP. kurziiand restricted to the adaxial surface, (2) isodiametric epidermal cells occur in P. pseudolais and P. scabrifolius, (3) elongated adaxial epidermal cells were observed in P. bantamensis (Fig. 3.1B), (4) polygonal epidermal cells were found in P. dubius, and P. spurius cv. Putat, (5) slightly extended epidermal cells arranged in longitudinal rows appear inP. amaryllifolius, and (6) four layers of longitudinally extended epidermal cells alternating with rows of smaller and thick walled cells

1

Figure 3.1 Light micrographs of leaves: (A)- transverse leaf section of P. multifurcatus Fagerl.; (B)- elongated abaxial epidermal cells of P. bantamensis Koord.; (C)- undulate adaxial anticlinal cell walls ofP. kurziiMerr.; (D)- adaxial epidermis cells with cubical crystals of P. bantamensis Koord.; E)- adaxial stomata with lobed papillae of P. utilis Bory; (F)- costal zone in the abaxial epidermis ofP. amaryllifoliusRoxb. Scale bar for A & F = 100 µm.; Scale bar for B, C and D = 50 µm; scale bar for E = 20 µm

were recorded in P. labyrinthicus, P. multifurcatus, P. nitidus, P. odoratissimus, P. polycephalus,P .tectoriusvar. littoralis, P. tectoriuscv. Sanderi and P. utilis.

The anticlinal walls of the adaxial epidermal cells are straight or undulate. The type of anticlinal walls is constant on the species level. Pandanus kurzii

A B

C D

shows adaxially undulate anticlinal walls (Fig. 3.1C), while the rest of the species have straight anticlinal walls.

The distribution of cubical crystals in the fifteen species is variable. Cubical crystals are absent in P. amaryllifolius, P. dubius, P. kurzii, P. labyrinthicus, P. tectorius var. littoralis, andP. bantamensis (Fig. 3.1D); present in both epidermis inP. multifurcatusandP. nitidus; only abaxially presents inP. odoratissimus, P. polycephalus, P. pseudolais, P. scabrifolius, P. spurius cv. Putat, and P. tectorius cv. Sanderi; and finally, only adaxially presents in P. utilis.

Stomata occur in the adaxial and abaxial epidermis, but the stomata are always more abundant in the abaxial than in the adaxial epidermis, and the polar subsidiary cells of the latter are slightly larger than those of the abaxial stomata. All species have the tetracytic type of stomata. There are four subsidiary cells adjacent to the guard cells, the two are called terminal (or polar) subsidiary cells, situated at the end of the guard cells and smaller in size than the other two, the lateral subsidiary cells.

In most species studied the adaxial epidermis has simple stomata without papillae, except inP. utiliswith stomata with lobed papillae (Fig. 3.1E).

The abaxial epidermis is variable throughout the species studied. It may or may not be differentiated into costal and intercostal regions. The demarcation into zones is very clear-cut in P. amaryllifolius (Fig. 3.1F), P. dubius, P. kurzii, P. multifurcatus, P. nitidus, P. odoratissimus, P. polycephalus, P. spuriuscv. Putat, P. tectorius var. littoralis, P. tectoriuscv. Sanderi, and P. utilis. However, inP. labyrinthicusthe demarcation is not so clear, and inP. bantamensis,P. pseudolais andP. scabrifoliusthe abaxial epidermis shown no division into regions.

The shape of the abaxial epidermal cells can only be divided into three classes, i.e. (1) epidermal cells often divided into 4 or 5 layers of longitudinally elongated cells with thin walls, alternating with a single layer of thick walled cells of smaller size are found inP. dubius(Fig. 3.2A), P. multifurcatus, P. nitidus,P. pseudolais, and P. tectorius cv. Sanderi, (2) longitudinally elongated abaxial epidermal cells occur in P. amaryllifolius, P. bantamensis, P. labyrinthicus, P.

kurzii,P. odoratissimus,P. scabrifolius,P. spuriuscv. Putat, andP. utilis; and (3) polygonal epidermal cells inP. polycephalus.

In most of species, the abaxial epidermis is frequently associated with papillae. The distribution, size and shape of the papillae is highly variable throughout all species studied and can be used with caution for systematic purposes.

Papillae, when present may occur in all normal epidermal cells or only certain cells. Lateral and terminal subsidiary cells of the stomatal apparatus are often associated with papillae. Papillae which occur in lateral subsidiary cells are always arranged in a linear row of three to four as found in P. tectorius var. littoralis (Fig. 3.2B), and four to five in P. amaryllifolius, P. scabrifolius andP. spurius cv. Putat. The papillae vary from simple, globose to elaborately lobed ones, while those present in terminal subsidiary cells vary from simple, forked to dendritic.

The number of papillae of one epidermal cell varies from one to several. One to three globose papillae are found in each epidermal cell of P. spurius cv. Putat (Fig. 3.2C). InP. labyrinthicus, P. tectoriusvar.littoralisandP. utilis only one papilla is found in each epidermal cell, but the papillae are elaboratly lobed at the distal end.P. kurziiandP. polycephalushave dendritic papillae on the abaxial epidermal cells, while inP. scabrifolius andP. dubiuspapillae are absent on the abaxial epidermis.

Hypodermis

A hypodermis is always present and consists of at least two layers of colourless cells as in P. amaryllifolius, P. multifurcatus, P. pseudolais and P. tectorius cv. Sanderi; three layers of colourless cells are usually present in P. labyrinthicus(Fig. 3.2D).

The adaxial hypodermis is more uniform than the abaxial hypodermis, because it is not interrupted by many stomatal chambers. The adaxial hypodermal layers are usually somewhat thicker than the abaxial ones. The outermost hypodermal layers are sclerotic, while the inner cells are larger and isodiametric and remain thin walled. Hypodermal cell rows do not coincide with the epidermal

Figure 3.2 Light micrographs of leaves: (A)- abaxial epidermal cells ofP. dubius Spreng.; (B)- abaxial papillae in lateral subsidiary cells of P. tectorius var. littoralis; (C)- abaxial papillae on epidermal cells of P. spurius Miq. cv. Putat; (D)- hypodermis of P. labyrinthicus Kurz; (E)- crystal cells in outermost hypodermis of P. pseudolais Warb.; (F)- circular palisade cells of P. amaryllifoliusRoxb. Scale bar for A, D, E. & F = 50 µm.; scale bar for B & C = 20 µm

rows. The number of epidermal cells above one of the outermost hypodermal cells in transverse direction can be used as a diagnostic feature for certain species. For example, in P. labyrinthicus the cells of the first hypoderrmal layer are long and correspond to 4 or 5 epidermal cells, 5 or 6 cells inP. amaryllifolius, and 7 or 8 epidermal cells inP. tectoriuscv. Sanderi.

A B

C D

Crystal cells are of a cuboid, and they are found in some of the species studied, viz. P. amaryllifolius, P. bidur, P. labyrinthicus, P. multifurcatus, P. pseudolais, P. tectorius var.littoralis andP. tectoriuscv. Sanderi. Crystal cells are generally found in the outermost hypodermis. The crystal cells are distributed uniformly, either solitary or in pairs. In P. amaryllifolius and P. pseudolais the large cuboid crystal cells occurs in the outermost layer of the hypodermis (Fig. 3.2E).

Mesophyll

The leaves of all species are isolateral. The mesophyll comprises parallel veins separated by large colourless cells, which collapse in mature leaves, resulting in the formation of pseudo-lacunae. In mature leaves there is a wide transverse air lacuna between each adjacent pair of veins. The adaxial chlorenchyma may be a two-layered palisade as in P. pseudolais or even four-layered palisade in P. amaryllifolius; abaxially the palisade is always 1 (or 2) layered. The shape of the palisade tissue is variable. Most species show columnar and compactly arranged palisade cells (P. multifurcatus, P. pseudolais and P. tectorius cv. Sanderi); the only exception is P. amaryllifolius with circular palisade cells (Fig. 3.2F).

Sclerenchyma strands are variable, and occur solitarily or in groups of 2 or 3 cells. The cells are hexagonal, rectangular or triangular and present next to the adaxial and abaxial epidermis. They are sometimes found within the hypodermis and even within the palisade tissue inP. amaryllifolius; in the spongy tissue inP. tectorius cv. Sanderi (Fig. 3.3A), or in the palisade and spongy tissue as in P. labyrinthicus. The cell is characterized by concentric layering with narrow, circular to oval laminae and in some of the cells also have cone-shaped silica bodies that project into the lamina.

Raphides

Idioblast as raphide sacs are present in the palisade of P. amaryllifolius (Fig. 3.3B),P. multifurcatusandP. pseudolais; in the spongy tissue inP. tectorius

Figure 3.3 Light micrographs of leaves: (A)- sclerenchyma in spongy tissue ofP. tectoriusParkinson cv.Sanderi, (B)- raphide sacs in internal palisade ofP. amaryllifoliusRoxb.; (C)- unspecialized abaxial stomata ofP. tectoriusParkinson cv. Sanderi; (D)- abaxial papillose lateral subsidiary cells ofP. odoratissimusL.f; (E)- abaxial papillose terminal and lateral subsidiary cells ofP. scabrifolius Martelli; (F)- abaxial papillose neighbouring and subsidiary cells ofP. kurzii Merr; (G)- abaxial, overarching, lobed or dendritic papillae ofP. utilisBory. Scale bar for A & B = 100 µm; scale bar for C, D, E, F & G = 20 µm.

A B

C D

E _F

Upper epidermis

cv. Sanderi or in the palisade and spongy tissue inP. labyrinthicus. The raphides are as pencil shaped ,i.e.flat at one end and pointed at the other.

Stomata

Tomlinson (1965) recorded a considerable range in stomatal structures found in 30 Pandanus species, and he provided a general description of the stomatal apparatus. The variation in the stomatal structure depends on the number of papillae that develop on the subsidiary and neighbouring cells. Tomlinson (1965) and Kam (1971) considered stomata without associated papillae to represent the unspecialized condition, and they grouped such stomata into Class 1. A series of stomatal types, which become increasingly more papillate, can be recognized, whereby the most complex class of stomata has guard cells that are completely obscured by overarching papillae. Tomlinson (1965) and Kam (1971) recognized five arbitrary classes, which are applied in this study.

Class 1. Unspecialized stomata. Each stoma has two guard cells, with each guard cell more or less symmetric in transverse view. The lateral subsidiary cell are thin-walled, and conspicously different from normal epidermal cells. Terminal subsidiary cells are short, but otherwise less well distinct from normal epidermal cells. The subsidiary and neighbouring cells lack papillae. This type of stomata is found inP. dubius,P. multifurcatus,P. nitidus,P. pseudolais, and P. tectoriuscv. Sanderi (Fig. 3.3C).

Class 2. Papillose lateral subsidiary cells. The structure of the guard cells and subsidiary cells is similar to that of class 1 except for the addition of a row of four to six papillae on the outer surface of each lateral subsidiary cell. There is no other stomatal outgrowth, except for a tendency for the terminal subsidiary cells to overarch the lateral subsidiary and guard cells to a greater extent than in class 1. Class 2 stomata are observed together with intermediate stomata, in which papillae occured on one of the two lateral subsidiary cells belonging to a single stoma. At least one full row of papillae is always developed. Class 2 stomata have been observed inP. bantamensis, P. odoratissimus(Fig. 3.3D) andP. spurius cv. Putat.

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