Biosystematic Study Of The Fern Genus Diplazium In West Malesia

BIOSYSTEMATIC STUDY OF THE FERN GENUS

DIPLAZIUM IN WEST MALESIA

TITIEN NGATINEM PRAPTOSUWIRYO

DEPARTMENT OF BIOLOGY

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

STATEMENT OF RESEARCH ORIGINALITY AND

INFORMATION SOURCE

This is to verify that my dissertation entitled: Biosystematic Study of the Fern GenusDiplazium in West Malesia is my own work and never been submitted to any institution before. All of the incorporated data and information are valid and stated clearly in the text, and listed in the references.

Bogor, September 2008 Titien Ngatinem Praptosuwiryo NIM P176 00005

ABSTRACT

TITIEN NGATINEM PRAPTOSUWIRYO. Biosystematic Study of the Fern GenusDiplazium in West Malesia. (Under the supervision of Prof. Dr. Ir. Edi Guhardja, M.Sc., Prof. Dr. Mien A. Rifai, M.Sc., Prof. Dr. Masahiro Kato, M.Sci., and Dr. Dedy Darnaedi, M.Sc. )

Diplazium is a large genus consisting of about 400 species occur mainly in the tropics, sparingly in the subtropic and only locally extending into temperate. It was estimated that 300 species of the records were occurred in Malesia. Taxonomically,Diplazium is very difficult and quite insufficiently known. Therefore a comprehensive study onDiplazium in West Malesia was conducted by using morphological, ecological, geographic distribution, anatomical, palinological, cytological, as well as DNA analysis to understand the diversity and relationship among species.

Based on gross morphological study on 1051 collection number of specimens as well as living collections, it was concluded that West MalesianDiplazium comprises of 69 species with 14 varieties. Thirteen species of them are proposed and described as new species, namelyDiplazium asymmetricum, D. batuayauense, D. crameri, D. densisquamatum, D. halimunense, D. loerzingii, D. megasegmentum, D. megasimplicifolium, D. meijeri, D. parallelivenium, D. profluens, D. subalternisegmentum,andD. subvirescens. Two new varieties are poposed, namelyD. accedens var. spinosum and D. silvaticum var. pinnae-ellipticum. D. pallidum var. montanum and D. accedensvar.ridleyi are proposed as new status.

Based on their main habitats,Diplaziumcan be classified into three major groups, viz. dryland (dominant), riparian and rheophytic species. Species diversity was culminated at 1000-1500 m above sea level. The individuals with different genetic load in the same species sometimes grow in the different habitat gradients. Based on the range of the geographical distribution, West Malesian species can be divided into three types: (1) very wide species (19 species), (2) Malesian species (27 species), and (3) locally endemic species (23 species).

Anatomical study on the transversal section of stipe of 27 species showed that the vascular bundle shape is varying among species. Therefore the leaf-trace shapes are important diagnostic features which support species delimitation in Diplazium. Spore morphology study showed that perine ornamentations support in delimitating species in Diplazium. However the phylogenetic analysis using parsimony revealed that morphological variation of spore is inadequate to depict natural relationship amongDiplazium species.

Cytological study on 117 collection number from 54 localities included in 31 species found that West MalesianDiplazium has six ploidy levels with x = 41 (diploid, triploid, tetraploid, pentaploid, hexaploid, and octoploid). New cytological information for science on 19 species are recorded. They areD. aequibasale(2n = 164),D. angustipinna (2n = 123).D. asymmetricum(2n = 123),D. batuayauense(2n = 164, 205),D. crenatoserratum(2n = 123, 164),D. halimunense(2n = 123),D. hewittii(2n = 123),D. profluens(2n = 164),D. loerzingii(2n = 82, 123),D. pallidum (2n = 82),D. petiolare(2n = 82),D. porphyrorachis (2n = 164),D. riparium(2n = 82, 123),D. spiniferum (2n = 82), D. subserratum (2n = 82, 123, 164), D. subvirescens (2n = 123), D. tomentosum(2n = 82, 205),D. xiphophyllum(2n = 82, 246), andD. wahauense(2n=164).

Phylogenetic analysis on morphological data sets of 69 species using parsimony revealed that the phylogenetic relationship among species in the genus Diplazium was very difficult to explain due to the lack of or weak support Bootstrap value. However the lack of or weak support for a phylogenetic tree does not strictly indicate that the pattern observed is incorrect but it does limit the amount of confidence that can be placed in the relationships between taxa.and the conclusions can be drawn from them. This study showed that some terminal clades formed are consisting of species that presumed to be closely related by formerly authors.

DNA analysis resulted new generbcL sequences data on 25 species. GenerbcL sequence is very well in supporting species delimitation and revealing the intraspecific diversity within species ofDiplazium. Phylogenetic analysis on 29 species from West Malesia and 9 references species outside Malesia using parsimony revealed that gene rbcL is more informative than morphological data in inferring phylogeny of Diplazium and showed that West Malesian Diplazium is monophyletic. The position of D. porphyrorachis at the basal clade of the morphological tree is supported by the phylogenetic tree generated from molecular data (gene rbcLsequence). This study also showed the congruence between the clade of riparium group drawn by generbcL tree and the clade of imparipinnate frond group drawn by morphological tree.

RINGKASAN

TITIEN NGATINEM PRAPTOSUWIRYO. Biosystematic Study of the Fern GenusDiplazium in West Malesia. (dibimbing oleh Prof. Dr. Ir. Edi Guhardja, M.Sc., Prof. Dr. Mien A. Rifai, M.Sc., Prof. Dr. Masahiro Kato, M.Sci. dan Dr. Dedy Darnaedi, M.Sc.).

Diplazium merupakan marga besar tumbuhan paku yang beranggotakan lebih kurang 400 jenis yang sebagian besar ditemukan di daerah tropis, sedikit di daerah sub tropis dan hanya secara lokal meluas ke daerah beriklim sedang. Dari jumlah tersebut, diperkirakan 300 jenis terdapat di kawasan Malesia. Marga tumbuhan paku terestrial ini mempunyai ciri-ciri diagnosa sebagai berikut: Alur tangkai daun dan tulang daun utama terbuka dan alur ini diteruskan sampai tulang anak daun berikutnya; alur daun berbentuk U dengan dasar pipih pada sebagian besar jenis; anak daun basal yang mengarah ke rembang (acroscopic side) seimbang atau lebih kecil, pinggir lembaran daun tidak menulang; sori menggaris, ganda (diplazioid) atau tunggal (asplenoid), yang tunggal membuka ke arah urat-urat daun utama atau urat-urat-urat-urat daun pusat dari cuping utama, yang ganda membuka dengan arah bertolak belakang.

Secara taksonomi, Diplazium sangat sulit dan kurang dipahami. Tumbuhan muda mungkin saja subur dan sulit untuk dikenali sebagai suatu jenis. Banyak takson memiliki variasi morfologi. Adanya poliploidi, apomiksis dan hibrid dalam marga paku ini menambah sulit dalam membuat pembatasan jenis. Pengelompokan anak marga dari marga ini secara alami belum pernah dilakukan walaupun variasi morfologinya sangat luas. Pembatasan marga Diplazium juga masih meragukan.

Di Malesia pada umumnya dan Malesia Barat pada khususnya, penelitian sistematika Diplazium dengan menggunakan pendekatan biologi secara menyeluruh belum pernah dilakukan untuk seluruh kawasan. Oleh karena itu penelitian biosistematikaDiplazium dengan menggunakan pendekatan morfologi, ekologi, distribusi geografi, anatomi, palinologi, sitologi dan juga analisa DNA dilakukan untuk memahami keanekaragaman jenis dan hubungan kekerabatannya.

Berdasarkan pengamatan morfologi pada 1051 nomor koleksi specimen dan juga koleksi hidup, disimpulkan bahwa DiplaziumMalesia Barat terdiri dari 69 jenis dan 14 varitas. Tiga belas jenis diantaranya diusulkan sebagai jenis baru, yaitu Diplazium asymmetricum, D. batuayauense, D. crameri, D. densisquamatum, D. halimunense, D. loerzingii, D. megasegmentum, D. megasimplicifolium, D. meijeri, D. parallelivenium, D. profluens, D. subalternisegmentum dan D. subvirescens. Dua varitas baru diusulkan, yaitu D. accedens var. spinosumdanD. silvaticum var. pinnae-ellipticum. D. pallidumvar. montanumdanD. accedensvar.ridleyidiusulkan sebagai status baru.

Berdasarkan habitat utamanya, Diplazium dapat dikelompokkan dalam tiga group utama, yaitu jenis lahan kering (dryland species, 64 species), jenis riparian (5 jenis) dan reofit (2 jenis). Keanekaragaman jenis memuncak pada ketinggian 1000-1500 m dpl. Individu-individu dengan tingkat ploidi berbeda pada jenis yang sama seringkali menempati habitat berbeda berdasarkan ketinggian. Berdasarkan jangkauan distribusi geografinya, jenis Diplazium Malesia Barat dapat dibagi dalam tiga tipe: 1) jenis tersebar luas (19 jenis), 2) jenis Malesia (27 jenis) dan 3) jenis endemik setempat (23 jenis).

Penelitian anatomi irisan melintang tangkai daun pada 27 jenis Diplazium memperlihatkan bahwa bentuk pembuluh vaskular bervariasi diantara jenis dan penting untuk menyokong pembatasan jenis. Pengamatan morfologi spora pada 46 nomor koleksi yang tercakup dalam 26 jenis memperlihatkan bahwa hiasan perine menyokong pembatasan jenis Diplazium. Walaupun demikian analisa filogeni dengan menggunakan parsimoni menunjukkan bahwa variasi perine tidak cukup untuk menggambarkan hubungan kekerabatan alami diantara jenisDiplazium.

Penelitian sitologi pada 117 nomor koleksi dari 54 lokasi yang mencakup 31 jenis menemukan bahwa Diplazium Malesia Barat mempunyai enam tingkat ploidi dengan jumlah kromosom dasar x = 41 (diploid, triploid, tetraploid, pentaploid, heksaploid dan oktoploid). Informasi sitologi baru bagi dunia ilmu pengetahuan dilaporkan untuk 19 jenis, yaitu D. aequibasale (2n = 164), D. angustipinna (2n = 123). D. asymmetricum (2n = 123), D. batuayauense (2n = 164, 205), D. crenatoserratum (2n = 123, 164), D. halimunense (2n = 123), D. hewittii (2n = 123), D. profluens (2n = 164), D. loerzingii (2n = 82, 123), D.

pallidum (2n = 82), D. petiolare (2n = 82), D. porphyrorachis (2n = 164), D. riparium (2n = 82, 123),D. spiniferum(2n = 82), D. subserratum(2n = 82, 123, 164),D. subvirescens(2n = 123), D. tomentosum(2n = 82, 205),D. xiphophyllum (2n = 82, 246) danD. wahauense(2n=164).

Analisa filogeni pada seri data morfologi dari 69 jenis dengan menggunakan parsimoni menunjukkan bahwa hubungan kekerabatan filogeni diantara jenis Diplazium sangat sulit dijelaskan karena tidak adanya atau lemahnya nilai Bootstrap. Bagaimanapun, tidak adanya atau lemahnya penyokong statistik bagi pohon filogeni tidaklah menandakan bahwa pola-pola yang diamati tidak benar namun ini hanya membatasi tingkat kepercayaan yang dapat ditempatkan pada hubungan kekerabatan diantara takson dan dari nilai-nilai tersebut kesimpulan dapat ditarik. Beberapa cabang ujung pohon morfologi tersusun dari jenis-jenis yang diduga berkerabat dekat oleh para peneliti sebelumnya.

Analisa DNA menghasilkan data baru sekuensi gene rbcL dari 25 jenis Diplazium. Sekuensi generbcLsangat bagus untuk menyokong pembatasan jenis, mengungkap keanekaragaman genetik dalam jenis dan juga lebih banyak memberikan informasi untuk menduga filogeni Diplazium dibanding data morfologi. Berdasarkan analisa filogeni pada 29 jenis Malesia Barat dan 9 jenis referensi dari luar Malesia, Diplazium terbukti monofiletik berdasarkan gene rbcL. Kedudukan group porphyrorachis pada cabang pangkal pohon filogeni morfologi disokong oleh pohon filogeni generbcL. Cabang group daun menyirip gasal pada pohon morfologi selaras dengan cabang group riparium pada pohon generbcL.

Penelitian ini tidak mengusulkan suatu kerangka sistematika di dalam marga Diplazium, sebab: (1) Pohon hipotesa filogeni yang dihasilkan dari data morfologi tidak didukung oleh alasan-alasan statistik yang obyektif; (2) Pohon hipotesa filogeni yang dihasilkan dari sekuensi gene rbcL tidak kokoh karena sebagian besar jenis kunci yang diduga dari pohon filogeni morfologi tidak dimasukkan dalam analisa disebabkan ketidaktersediaan sampel material segar pada jenis-jenis tersebut; (3) Analisa filogeni pada seri kombinasi data molekuler dan non molekuler untuk menduga filogeniDiplazium belum dilakukan.

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BIOSYSTEMATIC STUDY OF THE FERN GENUS

DIPLAZIUM IN WEST MALESIA

TITIEN NGATINEM PRAPTOSUWIRYO

A dissertation submitted to fulfill one of the requirements for the

Doctorate Degree at the Study Program of Biology,

Graduate School, Bogor Agricultural University

DEPARTMENT OF BIOLOGY

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

Examiner of first examination:

Dr. Tatik Chikmawati, M.Si. Department of Biology, Faculty of Mathematic and Natural Sciences, Bogor Agricultural University, Bogor Examiners of second examination:

1. Dr. Sri Sudarmiyati Tjitrosoedirdjo, M.Sc. Department of Biology, Faculty of

Mathematics and Natural Sciences, Bogor Agricultural University, Bogor 2. Dr. Rugayah, M.Sc.

Herbarium Bogoriense, Botany Division, Research Center for Biology – Indonesian Institute of Sciences, Cibinong Sciences Center, Cibinong

Dissertation Title : Biosystematic Study of the Fern GenusDiplazium in West Malesia

Name of student : Titien Ngatinem Praptosuwiryo Number of student : P176 00005

Study Program : Biology

Certified by Supervisor Committee

Prof. Dr. Ir. Edi Guhardja, M.Sc. Prof. Dr. Mien A. Rifai, M.Sc (Chairman) (Member)

Prof. Dr. Masahiro Kato, M.Sci. Dr. Dedy Darnaedi, M.Sc. (Member) (Member)

The Biology Study Program Graduate School

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

PREFACE

Research on the West Malesian Diplazium under the tittle Biosystematic Study of the Fern GenusDiplazium in West Malesia was conducted from 2001-2007. Field study was carried out at 54 localities in the primary and secondary forest of Jawa, Sumatra and Kalimantan. Specimens examination was conducted at Herbarium Bogoriense (BO) and Herbarium of Singapore Botanic Gardens (SING). Study on the anatomy, cytology, and mode of reproduction type were conducted at the Anatomical and Cytological Laboratory of Herbarium Bogoriense. Observation on spore morphology by using Scanning Electron Microscope and DNA squencing (gene rbcL squencing) were conducted at the Laboratory of Department of Chemical and Biological Sciences, Faculty of Science, Japan Women s University and the Laboratory of Molecular Systematic of Department of Biological Science, University of Tokyo.

A part of this dissertation (Chapter 5) has been published under two different tittles articles: (1) Cytological Study of Some Species of Ferns Genus Diplazium in Java I. that published inFloribunda 2 (5): 128-137 (2004) and (2) Cytological Study of Some Species of Ferns Genus Diplazium in Java II. that published inFloribunda2 (8): 209-221 (2005).

I greatly appreciate to my supervisors: Prof. Dr. Ir. Edi Guhardja, Prof. Dr. Mien A. Rifai, Prof. Dr. Masahiro Kato and Dr. Dedy Darnaedi, for their advice, guidance and encouragements throughout this study. I sincerely thank Dr. Irawati and Dr. Eko Baroto Walujo for their advices and technical support. I am indebted to Prof. Dr. Ryoko Imaichi, Dr. Chie Tsutsumi, Dr. Y. Kita, Dr. Mami Konomi, Dr. Mamiko Sato, and Mie Hashino for their helpful advices and technical supports. I would like express my appreciation to Dr. Chin See Chung and Ms. Serena Lee M. L. for giving me an opportunity to studyDiplazium specimens at the Singapore Botanic Gardens Herbarium. I am grateful to Dr. R.J. Johns for fruitful discussion and his guidance when I was studying Diplazium specimens in Singapore Botanic Gardens. I would like to sincerely thank Dr. Teguh Triono, Dr. Titik Rugayah, and Dr. Joko Ridho Witono for the fruitful discussion, criticism, and technical supports. I also thank Dr. Sri Sudarmiyati Tjitrosoedirdjo and Dr. Tatik Chikmawati for their suggestions and corrections.

I sincerely thank Dr. Rudy Lukman for his helpful advices and soft literatures. I thank Abdulrokhman Kartonegoro, S.Si. and Dr. Nunik S. Ariyanti for taking some pictures of specimens type and literatures from Leiden. I would like to thank Prof. Dr. Hiroshi Okada and Dr. Hirokazu Tsukaya for involving me in the field study in Java and Central Kalimantan. I also thank to Dra. Esti Munawaroh, Wita Wardani, S.Si., Arief Hidayat, S.Si., Bp. Bambang Purwadi, Bp. Ujang Hapid and Sri Wahyuni, S.P. for sampling and fieldtrips. I am also grateful to Bp. Endjum and Ibu Ahati for maintaining the living collections.

I thank the following institute in which I used their facilities: a) Herbarium Bogoriense, Botany Division, Research Center for Biology Indonesian Institute of Science (Lembaga Ilmu Pengetahuan Indonesia), b) Center for Plant Conservation, Bogor Botanic Gardens LIPI, c) Department of Chemical and Biological Sciences, Faculty of Science, Japan Women s University, d) Department of Biological Science, University of Tokyo, Japan.

Finally, my grateful goes to my dear father (Bp. Ng. Praptosuwiryo who has passed away on 25th February 2006) and mother (Ibu Kardiyem) my sisters and brother (Yu Ni, Dik Tum, and Dimas Bas) for their deep understanding and moral supports.

This research was partly supported by Proyek Kompetitif Pengembangan IPTEK LIPI TA 2004 , Prof. Dr. Masahiro Kato (Department of Botany, National Museum of Nature and Science, Amakubo, Tsukuba, Japan), Prof. Dr. Ryoko Imaichi (Department of Chemical and Biological Sciences, Faculty of Science, Japan Women s University, Japan) and The Singapore Botanic Gardens Research Fellowship 2005.

CURRICULUM VITAE

Titien Ngatinem Praptosuwiryo was born on 27 March 1969 in Boyolali, Central Java, the second daughter from four children from father the late Ngadimin Praptosuwiryo and mother Kardiyem. She was graduated from Bogor Agricultural University (Institut Pertanian Bogor) in 1994. In 1994 to 1996, she worked at Herbarium Bogoriense, Puslitbang Biologi LIPI, Bogor as an honorary researcher in ferns. In September 1996, she had an opportunity to continue study at IPB sponsored by Prof. Dr. Masahiro Kato, the University of Tokyo, Japan and admitted to the degree of M.Sc. in August 1999. In 1999 to 2000 she continued working at Herbarium Bogoriense.

In September 2000, she had an opportunity to continue study at IPB for her PhD. This study was partly supported by MENRISTEK and LIPI. Since 2001 she has been working at the Center for Plant Conservation-Bogor Botanic Gardens, LIPI, Bogor as a researcher in plant systematic. Her research area is biosystematic study on ferns.

TABLE OF CONTENTS

Page

LIST OF TABLES ……….. iv

LIST OF FIGURES ………..………. v

LIST OF APPENDIXES ……… vi

1 INTRODUCTION Taxonomical Aspects ofDiplazium and Its Systematic Problems ………... 1

The Diversity ofDiplazium in Malesia ... 7

The Biological Aspects on Systematics Study of Ferns …... 8

Morphological and Anatomical Evidences in Taxonomy ………... 8

The Constribution of Palynology to Systematics: Spore Morphology Evidence in Pteridophytes ... 9

Cytological Evidence in the Revealing Taxonomic Problems onDiplaziumand Its Closely Related Genera ... 10

The Utility of Molecular Techniques for phylogenetic studies of pteridophytes: Generbcl Sequences ... 11

Objectives ……….………. 12

2 DIVERSITY AND ECOLOGY OF DIPLAZIUM Introduction ……… 13

Materials and Methods ………... 14

Results and Discussion ………... 14

Ecology .………... 14

RheophyticDiplazium ………... 15

RiparianDiplazium ………... 16

Diplaziumin Dryland ……..………... 16

Diversity ofDiplaziumBased on Elevation …... 19

Conclusions ………... 22

3 THE DISTRIBUTION OF WEST MALESIAN DIPLAZIUM INSIDE AND OUTSIDE MALESIA Introduction ……… 23

Materials and Methods ………... 24

Results and Discussion ……….. 25

Very Wide Distribution Species ………... 26

Endemic Species to Island in West Malesia ………... 27

Conclusions ………... 40

4 THE STELAR ANATOMY OF STIPE AND ITS TAXONOMIC SIGNIFICANT IN DIPLAZIUM Introduction ……….. 41

Material and Methods ………... 42

Results and Discussion ………. 42

Conclusions ……… 46

5 CYTOLOGICAL AND REPRODUCTIVE STUDIES ON DIPLAZIUM IN WEST MALESIA Introduction ……….. 47

Materials and Methods ………. 49

Results and Discussion ………. 50

Chromosome Number Variations and Mode Reproduction Types onDiplazium ………... 50

The Relationship between Ploidy Level and Morphological Variation within Species and Closely Related Species ofDiplazium ... 66

Relationship between ploidy level and habitat gradient ... 71

Correlations between reproductive mode and habitat ... 72

Conclusions ... 81

6 PHYLOGENETIC STUDIES OF DIPLAZIUM FROM WEST MALESIA: EVIDENCE FROM MORPHOLOGY Introduction ………... 83

Character Selection and Construction ………. 85

Character Selection ……… 85

Character Type ……….. 85

Character Coding ……… 86

Character Variation within West MalesianDiplazium ……….. 87

Materials and Methods ……….... 100

Taxon Sampling ……….... 100

Character Examination ofDiplazium ………... 100

Phylogenetic Analysis ……… 101

Results and Discussion ……… 110

Conclusions ………... 117

7 SPECIFIC DELIMITATION AND RELATIONSHIP AMONG SPECIES OF DIPLAZIUM BASED ON SPORE MORPHOLOGY Introduction ... 118

Materials and Methods ... 120

Spore Characters ofDiplaziumand Its Use in

Supporting Species delimitation and Identification …... 122

Phylogenetic Analysis ………... 137

Conclusions ... 143

8 MOLECULAR SYSTEMATIC OF DIPLAZIUM FROM WEST MALESIA Introduction ……….. 146

Materials and Methods ………. 148

DNA Analysis ……… 149

Phylogenetic analysis ………. 161

Results and Discussion ……… 161

Infraspecific Genetic Diversity inDiplazium ……… 161

Species Delimitation inDiplaziumbased on Gene rbcL Sequence ………... 163

Informative Characters of GenerbcL Sequences for Inferring Phylogenetic Hypothesis ofDiplazium ………. 165

Phylogenetic Analysis ……… 166

The Monophyly ofDiplazium ……….. 166

Relationships among species withinDiplazium ….…….. 165

Conclusions ………... 175

9 TAXONOMIC STUDY OF THE FERN GENUS DIPLAZIUM IN WEST MALESIA Introduction ……….. 176

Materials and Methods ………. 176

Taxonomic Treatment ……….. 177

10 GENERAL DISCUSSION Synthesis ……….. 283

General Discussion ……….. 284

Systematic Implications for the GenusDiplazium ………... 285

11 CONCLUSIONS ………... 187

LITERATURES ………... 292

LIST OF TABLES Table 2.1. Classification ofDiplaziumbased on their main habitat …… 18

Table 3.1. Distribution of West MalesianDiplazium inside and

outside Malesia ………..…. 28 Table 3.2. Endemic Species ofDiplaziumin West Malesia ……… 39 Table 3.3. Species diversity and endemism of Diplaziumin

four mainlands of West Malesia ……… 40 Table 5.1. Somatic Chromosome Numbers, Ploidy Level and

Mode Reproduction Type of Diplaziumfrom West Malesia ... 52 Table 5.2. Polyploid series ofDiplaziumin West Malesia Based on

Present Study ... 73 Table 6.1. Characters, character states, and coding for 88 characters

utilized in construction of morphological data set ofDiplazium 102 Table 7.1. Spores Description of Diplazium in West Malesia ... 124 Table 7.2. Characters, character states, and coding for 17 characters

utilized in construction of spore morphology

dataset ofDiplazium ……… 143 Table 7.3. Coding for 17 characters utilized in construction of

spore morphology data set ofDiplazium ……… 144 Table 8.1. List of Taxa Used in This Study ……… 152 Table 8.2. Primers Used for Amplifying and Sequencing DNA from

Diplazium (Hasebeet al, 1994) ……… 160 Table 8.3. Infraspecific Genetic Variatons ofDiplazium based

on GenerbcL Sequences ………. 162 Table 8.4. Interspecific Genetic Variatons ofDiplazium based

on GenerbcL Sequences ………. 164

LIST OF FIGURES

Figure 2.1. a-b. Light shade-ferns ofDiplazium ………. 21 Figure 2.2. Elevational distribution ofDiplazium species in

West Malesia ………. 21 Figure 4.1. Vascular structure of the leaf axis ………. 43 Figure 4.2. Leaf-trace shapes inDiplazium ………... 44

Figure 4.3. Leaf-trace shapes inDiplazium. ... 45

Figure 5.1. Somatic chromosome of Diplazium. ……… 74

Figure 5.2. Somatic chromosome ofDiplazium ………... 75

Figure 5.3. Somatic chromosomes ofDiplazium ……...……….. 76

Figure 5.4. Somatic chromosomes of Diplazium ……… 77

Figure 5.5. Somatic chromosomes ofDiplazium cordifolium ……… 78

Figure 5.6. Somatic chromosomes ofDiplazium ... 79

Figue 6.1. Rhizome appeareance ofDiplazium ………. 89

Figure 6.2. The variation of scale shapes inDiplazium ……… 91

Figure 6.3. Margin of scales ……….……… 92

Figure 6.4. Stipes appearances of Diplazium ……….………. 93

Figure 6.5. Frond architectures ofDiplazium ……… 95

Figure 6.6. Venation types ofDiplazium ………. 98

Figure 6.7. Sori variation in Diplazium .………...…... 99

Figure 6.8. Strict consensus of 8 trees of length 1366 from unweighted morphological dataset comprises 88 morphological caharcters. ... 111

Figure 7. 1. Group I. a and b.D. accedens; c and d.D. bantamense; e.D. lobbianum; f and g.D. pallidum; h-j. D. procumbens; k-l. D. sorzogonense ... 133

Figure 7.2. Group II. a.D. subserratum; b-c.D. vestitum; d-e. D. vestitumvar.borneense; Group III. f-g. D. crenatoserratum; h-i. D. prescottianum; Group IV. j-l.D. silvaticum ... 134

Figure 7.3.Group V. a-c D. pallidum; Group VI. d-e.D. cordifolium; g-i.D. tomentosum; j.. D. malaccense; k-l.D. megasegmentum; m -o.D. simplicivenium ……… 135

Figure 7.4. Group VII. a-c. D. profluens; Group VIII. d. D. spiniferum; Group IX. e-f. D. subvirescens ………... 136

Figure 7.5. Tree number 1 of 100 the most parsimonius trees ………… 141

Figure 7.6. The strict consensus of 100 the most parsimonious trees ……….. 142

Figure 8.1. Strict consensus of the 200 equally most-parsimonious trees obtained in maximum parsimony analysis of therbcL sequence data. …………..……….. 169

LIST OF APPENDIXES Appendix 1. Matrix of 88 Morphological Characters for Maximum Parsimony ………. 309

Appendix 2. GenerbcL Sequence Data ……….. 321

Plate 1. Diplazium asymmetricum Praptosuwiryo ………. 351

Plate 2. Diplazium batuayauense Praptosuwiryo ………... 352

Plate 3. Diplazium crameri Praptosuwiryo ... 353

Plate 4. Diplazium densisquamatum Praptosuwiryo ... 354

Plate 5. Diplazium halimunensePraptosuwiryo ... 355

Plate 6. Diplazium loerzingii Praptosuwiryo ……….. 356

Plate 7. Diplazium megasegmentum Praptosuwiryo ……… 357

Plate 8. Diplazium megasimplicifolium Praptosuwiryo ……… 358

Plate 9. Diplazium meijerii Praptosuwiryo ……….. 359

Plate 10. Diplazium parallelivenium Praptosuwiryo ... 360

Plate 11. Diplazum profluens Praptosuwiryo ... 361

Plate 12. Diplazium subalternisegmentum Praptosuwiryo ... 362

Plate 13. Diplazium subvirescens Praptosuwiryo ... 363

CHAPTER 1

GENERAL INTRODUCTION

1.1. Taxonomical Aspects ofDiplazium and Its Systematic Problems

Diplazium was established by Swartz (1801) and typified by Asplenium plantaginifolium L. (Diplazium plantaginifolium Sw.). Etymologically Diplazium is formed from the Greek diplazios which means double, because indusia lie on both sides of the vein.

Diplazium is a large genus consisting of about 400 species occur mainly in the tropics (Ching 1964a; Copeland 1947; Tagawa & Iwatsuki,1988), sparingly in the sub tropic and only locally extending into temperate (Krameret al 1990). This genus member has diagnostic characters as follow: Groove of frond axis open to admit the groove of axis of lower order; frond axes U-shaped with a flat base in most species; acroscopic basal pinnules equal or smaller, laminar margin not cartilaginous; sori linear, double (diplazioid) or single, the single ones opening toward the main veins or the central veins of the ultimate segments, the double ones in opposite directions (van Alderwerelt van Rosenburgh 1908; Holttum 1966; Kato 1977; Tagawa & Iwatsuki 1988; Krameret al 1990).

a. Species Delimitation.

Taxonomically,Diplazium is very difficult and quite insufficiently known. It is in great need of monographic study. The young plants may be fertile and difficult to assign to a species (Kramer et al, 1990). Many taxa are considerably morphologically diversified. Their morphological variations are continuous through apparently intermediate forms, which are commonly regarded as putative hybrids (Takamiyaet al 1999).

b. Polyploidy and Polymorphic Species Complex

Many cytological complexities including polyploidy from 4x to 8x, hybridity, apomixis, have been reported in ca. 20% of cytological investigation of Diplazium taxa (Lovis 1977). Recent studies on JapaneseDiplazium revealed that one of the reason for the taxonomic complexity of the Diplazium species group with bi- to tripinnate leaves is apomictic reproduction and most of the group’s members have been found to be triploid apomics (2n=3x=123). Many polymorphic Diplazium taxa having large evergreen bi-to tripinnate leaves occur under evergreen broad-leaved forest from the Ryukyus Islands to southwestern Japan. Several putative hybrids are also known within Diplazium, thus it is increasing the taxonomical dispute (Takamiya et al 1999). Cytological observation ofDiplazium from Java (Praptosuwiryo & Darnaedi 1994, 2004) and Lesser Sunda Island (Praptosuwiryo 2003) also showed many polyploid types in which several species were apomix. Therefore, polyploidy, apomixis and hybrid in diplazioid ferns creates difficulty in species delimitation.

c. Subdivision ofDiplazium.

Natural subdivision of the genus has not been assigned (Kramer et al 1990). Van Alderwerelt van Rosenburgh (1908) divided this genus artificially into two sections based on its venation, viz. Eudiplazium andAnisogonium. The first section includes species with free veins such as Diplazium bantamense, D. crenatoserratum and D. porphyrorachis; while the second section includes species with anastomosing veins, such as D. cordifolium, D. esculentum, and D. fraxinifolium. Copeland (1908), in his revision on the Philippine species of Athyrium, merged Diplazium into Athyrium and recognized 13 non formal group ofAthyrium, namely drynarioid species (A. hyalostegium,A. loheri),A. japonicum group (A. japonicum, A. grammitoides, A. acrotis), A. filix-femina group (A. drepanopteron), A. macrocarpum group (A. halconense, A. anisopteron), A. nigripes group (Diplazium aristulatum, D. aristulatum var. sphanicolum, Athyrium philippinense, A. brevipinnulum, A. nigripes var. mearnsianum, A. elmeri, A. stramineum, A. platyphyllum), A. cyatheaefolium group (A. cyatheaefolium=Diplazium ebenum, A. atratum=D. atratum, A. oligosorum=D. oligosorum), A. silvaticumgroup (A. silvaticum, A. blumei=D. polypodioides, A.

fructuosum= D. fructuosum, A. dolichosorum=D. dolichosorum, A. maximum, A. vestitum= D. vestitum, A. davaoense= D. davaoense, A. esculentum= D. esculentum), A. umbrosum group (A. meyenianum= D. caudatum, A. sorzogonense= D. sorzogonense= D. woodii, D. brachysoroides= D. brachysoroides),A. williamsigroup (A. deltoideum, A. whitfordii=D. whitfordii, A. bolsteri= D. bolsteri, D. geophilum, A. williamsi= D. williamsi), A. pinnatum group (A. pinnatum= D. petiolare, A. cultratum, A. crenato-serratum= D. crenatoserratum, D. inconspicuum, A. pallidum=D. pallidum),A. accedensgroup (A. accedens= D. accedens), A. fraxinifolium group (A. fraxinifolium=D. bantamense,A. cumingii=D. cumingii, A. tabacinum = D. tabacinum, A. pariens, A. cordifolium= D. cordifolium), and A. porphyrorachis group (A. merrillii= D. merrillii). However he did not explain characters delimiting his classification.

Based on the characters such as scales, stipe, lamina and venations, Kato (1977) recognized genus Diplazium that consisting of five groups, namely: (1) Diplazium dilatatum group that includes member with groove generally U-shaped with a flat base, acroscopic basal pinnules or segments equal to or smaller than the basiscopic or subsequent ones, scales entire or toothed, but not clathrate. The representative species are D. dilatatum, D. esculentum, D. donianum, D. mettenianum, D. subsinuatum, D. hachijoense, D nipponicum, D. sibiricum, D. squamigerum, D. tomitaroanum, D. pullingeri, D. lobatum, D. yaoshanense, and D. kawakami; (2) Diplazium wichurae group that includes species member with groove U-shaped, acroscopic base of pinna auricled, adaxial surface of lamina concave along veins, scales entire, sometimes subclathrate. The representatives are D. wichurae, D. okudairae, and D. pin-faense; (3) Diplazium mesosorum group that having characters groove U-shaped with a flat base, acroscopic basal pinnules or segments equal to or slightly larger than the basiscopic or subsequent ones, scales entire and subclathrate and only represented by D. mesosorum; (4) Diplazium javanicum group in which includes species with groove V-shaped, frond pinnate or imparipinnate, laminar margin entire or undulate, veins sagenoid-reticulate and scales entire. This group represented by D. heterophlebium, D. javanicum, D. cavalerianum, andD. marginatum; and (5)Diplazium longicarpun group. This group includes species with frond pinnate, acroscopic base of pinna

truncate, basiscopic cuneate, adaxial surface of lamina not concave along veins, scales entire. This group represented only by one species,D. longicarpum.

d. The status ofDiplazium a separates genus.

The delimitation of Diplazium is still in doubt. Some taxonomist merged Diplazium into Athyrium, while others maintained them as separate genera. Beddome (1883) separated Diplazium from Athyrium based on the difference of sorus shape. Athyrium species have special reniform or round sori, meanwhile Diplazium has elongated sori. Copeland (1908) transferred Diplazium in Philippine intoAthyrium. In accord with his previous papers, Copeland (1929) has described a considerable number of diplazioid ferns as species of Athyrium, merged the whole species of Diplazium. Furthermore Copeland (1947) united Diplazium into Athyrium because he believed that Athyrium s.str. (Euathyrium) andDiplaziumas a whole is almost surely a phyletic entity. He was unable to find any line where the genus can be divided to produce distinguishable natural groups. Ching (1964a, 1964b) splits Diplazium into several genera, e.g., Allantoidea, Callipteris, Diplaziopsis, Monomelangium, and Diplazium sensu stricto. Ching (1964a) delimited Diplazium Swartz as represented by D. plantaginifolium (Linn) Urban of Tropical America, D. bantamense Blume of Java and D. donianum (Mett.) Tard.-Blot of the Orient which characterized by imparipinnate (sometimes simple or trifoliolate) fronds of firm texture with large similar pinnae (2-6 pairs or rarely more), in similar way the simple frond, the upper side of pinna-costa has a very shallow or even obsolete longitudinal canal provided with low rounded edges on each side and not open to the rachis-groove at point of insertion and by very long, linear and more often double sori extending from the costa to near the leaf margin. Diplazium sensu stricto is a pantropical genus of medium-size (Ching 1964a). On the other hand Allantoidia sensu Ching (1964a) included the bulk of the species of Diplazium of Christensen’s Index Filicum asrepresented by Diplazium dilatatum Blume, D. polypodioidesBlume, and their allies. Their short-linear sori and indusia being asplenioid or sometimes diplazioid but not allantoid. Ching (1964a) delimited Allantoidea R. Brown as follow: fronds vary from simply to 2-3-pinnate with lateral pinnae gradually becoming shorter upwards and finally merged into a deltoid, acuminate pinnatifid

apical part, with the pinna-rachis or costa or costule of pinnules (in the compound-leaved species) deeply grooved above and raised (becoming flat upon drying) knife-edge margins on each side, which are decurrent along the rachis or costa of pinnae or costule of pinnule, and the grooves of rachis. Pinna-rachis or of costa and costule are open to each other at the point of insertion, the leaf-texture is herbaceous or rarely chartaceous and the sori are thick, short-linear or ovoid-oblong only with the anterior basal one usually diplazioid. It is a large genus of about 350 species mostly in tropical and subtropical Asia with a few extending to the temperate region in the Northern Hemisphere.

However, recent molecular phylogenetic study on the ferns species included inAthyriaceae sensu Ching (1964a) by using evidence from chloroplast TrnL-F region sequences revealed that Diplaziopsis C.Chr.is included in the Diplazium Sw. clade (Wang et al 2003). Wang et al (2003) also gave evidence thatAllantoidea R. Br. andCallipteris Bory should be included inDiplazium Sw.

Christensen & Holttum (1934) separated Diplazium from Athyrium because they thought that merging the whole mass of species of Diplazium in Athyrium would result too unnatural grouping. Holttum (1940) originally showed that in Malaya Diplazium and Athyrium are quite distinct, so that he recognized the genus Diplazium. Holttum pointed out that Diplazium had pinnae and pinnules of pinnate frond with subequal base, venation never anadromous, sori elangated along the veins with lateral indusium, the diplazioid sori usually with two quite separate indusia, near grading into horse-shoe shape, with indusium continuous all around, while Athyrium had pinnules often with equal bases, an anadromous venation and division, and sori are always short and broad, the diplazioid ones often of a horse-shoe shape, the two sides of the sorus being connected. In contrast to his 1940 treatment, Holttum (1947, 1955) recognized the close relationship of Diplazium with Athyrium and hence associated the two genera because the distinction between them were no longer distinct to justify their separation. Inconsistently, Holttum (1966) without doubt stated that genus Diplazium should be maintained because cytologically the two groups were constantly different. According to Holttum (1966) Athyrium has x = 40 and Diplazium x = 41. Despite Holttum (1966) others taxonomist such as, Ching

(1964a), Pichi-Sermolli (1977), Kato (1977; 1994) and Tagawa & Iwatsuki (1988) maintainedDiplaziumseparated fromAthyrium.

Kato (1977) differentiated between the two genera as follows. Athyrium had stipe bases on ascending to erect rhizomes swollen with pneumatophores, frond axes V-shaped in transaction, acroscopic basal pinnules larger than others, laminar margin cartilaginous or not, spines present adaxially at the junction of costules or not, sori horse-shoe- or J-shaped, or linear, scales entire. Meanwhile Diplazium had stipe bases neither swollen nor bearing pneumatophores, frond axes U-shaped with a flat base in most species, acroscopic basal pinnules equal or smaller, laminar margin not cartilaginous, spines absent, sori linear, scales toothed or entire. Furthermore, Kato (1977) merged Callipteris Bory, Allantoidea R. Brown,Hemidictyum Presl,Diplaziopsis C. Christensen,Monomelangium Hayata, Dictyodroma Ching and Rhachidosorus Ching into Diplazium. In accord with Kato (1977), recent molecular phylogenetic studies included Monomelangium in the monophyleticDiplaziumclade; Rhacidosorusis separated from monoplyletic Diplazium clade and not closely related to eitherAthyriumandDiplazium(Sanoet al 2000a, 2000b).

e. Classification ofDiplazium.

Despite the recognition ofDiplaziumas genus, the higher classification of Diplazium (family level) is still unclear. Tardieu-Blot (1932) placed this genus in the tribeAsplenieae of the familyPolypodiaceae. Alston (1956), Ching (1964a). Pichi-Sermilli (1977) and Tagawa & Iwatsuki (1988) placed the genus in Athyriaceae, the family that proposed for the first time by Ching (1954) and validated by Alston (1956). Holttum (1966) placed the genus inDenstaedtiaceae, the largest family of modern pteridophytessensu Holttum. Tryon & Stolze (1991) also placed this genus in Dryopteridaceae. Brummit (1992) and Kato (1994) included Diplazium in Woodsiaceae, a family previously established by Herter (1949). Nayar (1970) included Diplazium in Athyrioideae, a subfamily of Dryopteridaceae. Kramer et al (1990) placedDiplazium in Physematieae, a tribe ofAthyrioideae,a subfamily ofDryopteridaceae.

Based phylogenetic trees generated from chloroplast TrnL-F region sequences that inferred using the neighbor-joining and maximum-parsimony methods, Wang et al (2003) to include Diplazium into subfamilyDiplazioideae and proposed to divide Athyriaceae into five subfamilies : Cystopterioideae, Athyrioideae, Deparioideae, DiplazioideaeandRachidosorioideae.

1.2. The Diversity ofDiplazium in Malesia

Malesia is one of the center megadiversity of tropical plants. With ca. 40,000 species of vascular plants, the Malesian region is among the most species-rich areas world-wide (Roos 1993).

It is firstly recognized by Swiss botanist, Heinrich Zolliger, in 1858, that Malesion region is different from the Asia and Australia due to a vast diversity of vascular plants. It is predicted that not less than 40.000 species of vascular plants occur in the region (from John 1995). This region extends from Malay Peninsula to New Guinea and cover more than 3,000,000 km2 at 0-5000 a.s.l. The region included seven countries, namely Brunai Darrusalam, Indonesia, Malaysia, Timor Leste, Papua New Guinea, Philippines and Singapore. Phytogegraphically, this region is divided into three subregion, viz. West Malesia, Central Malesia and East Malesia. West Malesia, known as Sunda Shelf, cover Malay Peninsula, Sumatra, Borneo, Palawan, Jawa and Bali. Central Malesia is subregion covering Philippina, Sulawesi, Moluccas and Lesser Sunda. East Malesia, recognized as Sahul Shelf or the Papuasia, is a subregion included Irian Jaya and Papua New Guinea (John 1995).

Malesia is also a species-rich region for Pteridopyta. It is estimated that 4,400 fern species of ca. 12,000 fern species known in the world are distributed in this region (Roos 1993). New Guunea is the world’s most species-rich island subcontinent with ca. 2,000 species, Borneo and the Philippines each have 1,000 species, and Java, sumatra and Celebes each have 500 species. Seram Island, a small island of 18,000 km2 in the Moluccas, is quite rich and is known to have about 700 species (Kato 1992). Parris et al (1992) reported that more than 600 fern species occur in Mt. Kinabalu (400 km2). Because of the species richness, Malesia ferns are very suitable for studying their biodiversity and evolution.

Malesian region is the center of Diplazium diversity. It is predicted that 75% of species in the world (ca.300 species) are existing in this region (Roos, 1995). However the revision of Diplazium for the Malesian region has not yet been done. However, some short studies based on local area has been conducted for Diplazium. Holttum (1940) described 25 species of Diplazium from Malay Peninsula and recognized 29 species on his monumental book ‘Ferns Flora of Malaya (Holttum 1966).

Mitsuta (1985) listed 11 species from West Sumatra. In Java, Backer and Posthumus (1939) described 17 species. Sixty years after that Praptosuwiryo (1999) recognized 22 species and 4 varieties of Javan Diplazium. Tagawa (1972) listed 15 species of Diplazium of Borneo based on ferns collected by M. Hirano and M. Hotta. Iwatsuki & Kato (1984) reported 15 species of diplazioid ferns of East Kalimantan, moreover Parris et al (1992) listed 31 species from Mt. Kinabalu. In addition to the records, Kato (1994) reported 32 species of Diplazium from Ambon and Seram (Moluccas).

1.3. The Biological Aspects on Systematics Study of Ferns 1.3.1 Morphological and Anatomical Evidence in Taxonomy

Morphological characters are very important in systematics, although many biological approaches are applied in constructing classification system, such as anatomy, palinology, cytology, and molecular analysis. The morphological data are used for grouping, identifying, studying the relationship of plants (Davis & Heywood 1963).

Fern systematists have employed various species concept. Some systematists employed morphological species concept, using morphology as primary criterion in recognizing species and formulating initial hyphotheses about new lineages. In addition, modern floristic surveys are also based primarily on morphology and provide the first clues to identify certain individuals or populations as potentially as potentionally unique, and deserving of further scrutiny (Haufleret al 2000).

The comparative study of plant structure, morphology and anatomy, has always been the backbone of plant systematics to elucide plant diversity,

phylogeny and evolution. The second half of the 20th century has been a fascinating period in which systematics and structural studies greatly profited from new techniques and methods. The advancement of new techniques and methods such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), sinematografi, cladistics, evolutionary paleobotany, and molecular systematics and molecular developmental genetics are just exactly supporting morphological data (Endresset al 2000).

Although morphological data series has revealed more homoplasy than those from molecular, however combination of morphology and molecular data might produced more robust phylogeny. Thus, in reality the two data are mutually complementary. Even, there is an amazing fact that the succes of molecular phylogenetic studies are predicted based on previous the morphological data (Endresset al, 2000).

1.3.2. The Role of Palynology in Systematics: Spore Morphology Evidence in Pteridopytes

Palynological characters have now been used in plant systematics for almost two centuries. Studies of a various taxa employing transmission electron microscopy (TEM) and or SEM (Scanning Electron Microscopy) have elucidated the complex nature of both exorspore and perispore development (e.g. Mitsui 1986; Tryon 1986). These investigations suggest that fine-scaled comparisons of spores among taxa may be necessary to distinguish superficial and gross similarity from true synapomorphy.

Measurement of spores serves as useful probes for establishing hyphothesis of evolutionary relatioships within polyploid complexes. Spore size of polyploid is dependent upon two factors: size of cells in diploid progenitors and ploidy level. Both factors may be used to predict cell size of missing members of polyploid complexes from cell-size means of the known members, as long as environmental variation does not compromise the analysis (Barringtonet al1986)

1.3.3. Cytological Evidence in revealing taxonomic problems on Diplaziumand Its Closely Related Genera

Once a species has been diagnosed, it represents a working hyphothesis that can be tested through the application of new evidence or analytical methods (Haufler et al 2000). The first auxiliary tool that had a major impact on species concept on ferns was the development of techniques for determining chromosomes numbers and studying meiotic behaviour (Manton 1950). The new perspectives that were obtained through chromosomal studied led to a revolution in he recognition of species boundaries in many groups. Polyploids discovery in taxa that had been considered simply as ‘polymorphic assemblages’, Manton demonstrated that evolutionary mechanism in ferns were more complex than previously appreciated and that hybridization between distinct species was an important component of the history of fern lineages (Manton 1950). This new approach results a new perception of fern species. Manton (1950) and her followers analysed meiosis in artificial hybrids to characterize the limits of fern lineages as reproductively isolated units, and to identify genetic similarities between genomes. These studies showed that interactions among ‘primary’ species involving hybridization and polyploidy generated ‘secondary’ species (terminology sensu Grant 1981), and resulted in reticulate species complexes whose intricacies challenged subsequent systematists.

As reported by Löve et al (1977) cytological research on Diplazium was firstly conducted by Manton (1953) for Ceylon species. From 1953 until 1977, only about 15.5% of ca. 400 species were recognized for its chromosome number.

Intensive studies on cytology of Diplazium in Japan (Ohta & Takamiya 1999; Takamiyaet al 1999, 2000, 2001; Sanoet al 2000) revealed that cytological approach through chromosomal information, is very helpful in analysing polymorphic species. However, few cytological investigations on Malesian Diplazium have been conducted. Manton (1954) initiated cytological examination on this genus for Malayan species, moreover Holttum & Roy (1965) conducted similar study for New Guinean species.

Preliminary cytological observation onDiplazium in Java was reported by Darnaedi (1992) for tetraploid D. cordifolium and diploid D. esculentum. In

addition, Praptosuwiryo and Darnaedi (1994) reported cytological research on six species ofDiplazium from Gede-Pangrango National Park, West Java (one species of its, viz.D. opacum, have been treated asCornopteris opaca)

Praptosuwiryo (2003) and Praptosuwiryo & Darnaedi (2004) reported recent cytological infromation of Diplazium from Malesian region. The first covered two species of Diplazium from Lombok Island, namely D. malaccense and D. pallidum, while the second report 43 collection numbers from 10 localities of Java included 10 species.

1.3.4. The Utility of Molecular Techniques for phylogenetic studies of pteridophytes: Generbcl Sequences

The use of morphology in reconstructing phylogeny of ferns is often complicated due to the lack of phylogenetically informative characters (Haufler & Rangker 1995). For example, the simplicity of foliar morphology of Ophioglossaceae has limited number of characters available for reconstructing classifications and understanding relationships (Hauket al 2003).

The lack of informative morphological characters in ferns led to the search for new sources of characters in molecular data, including restriction site and nucleotide sequence data, to infer phylogenetic relationships (Eastwood et al 2004).

Nucleic acid sequencing is a relatively new approach in plant systematic, however the power of the techniques and the data generated have made it become one of the most utilized for inferring phylogenetic history. DNA sequence data are the most informative tool in molecular systematics. Comparative analysis of DNA sequences is becoming increasingly important and so valuable in plant systematics. The major reasons of its valuable area: (1) the characters (nucleotides) are the basic units of information encoded in organism and (2) the potential sizes of informative data sets are immense. Systematic informative variation is essentially inexhaustible and different genes or parts of the genome might evolve at different rates. Therefore, questions at different taxonomic level can be addresed using different genes or different region of a gene (Soltis & Soltis 1998)

Molecular systematic studies of the pteridophytes have generated robust phylogenies at all taxonomic scales. In many cases, molecular phylogeny are highly congruent with morphological-based hyphothesis (Wolf & Conant 1994). The use of molecular data to infer phylogeny has yielded valuable insight into the relationships and evolution of ferns, some with taxonomic implications (Hasebeet al 1995, Gastony & Ungerer 1997, Murakamiet al 1999).

1.4. Objectives

The objectives of the research are:

(1) To obtain delimitation of the genus and species concepts

(2) To provide better data on distribution of species in the West Malesia region

(3) To provide an identification keys to the species and infra species. (4) To collect data on species diversity.

(5) To understand the anatomical and palinological data in supporting the species delimitation.

(6) To recognize ecological aspect of species.

(7) To provide cytological map of species in its distribution site. (8) To unravel species which is having taxonomical problems. (9) To obtain phylogenetic hypotheses forDiplaziumbased on morphological data and generbcL sequence.

CHAPTER 2

DIVERSITY AND ECOLOGY OF DIPLAZIUM

2.1. Introduction

The number of organisms in a particular area is determined by speciation and extinction, and by immigration and local loss. The relative importance of these processes depends upon the scale of investigation. Ecological processes are crucial to many theories of speciation (Godfray & Lawton 2001).

Delimitation of species is, of course, performed by choice of a certain combination of critical characters on comparative-morphological basis. Each Linnaean species is essentially a self-perpetuating population in the genetic sense, i.e. a mixture of individual which are to a larger or smaller degree genetically different. The genetically defined characters do not manifest themselves in exactly the same way in different individuals, as their manifestation in influenced during the stages of their ontogenetical development by the environmental condition (Steenis 1957).

Many environmental factors can affect various ferns, including the present of other plants, the activities of animals (amongst which man now plays a predominant role in many areas), and a whole range of physical and climatic factors (Edie 1978). For the sake of simplicity, most authors on fern taxonomy usually explained the main factors which act on ferns growing under or less natural conditions. These factors include the substratum where the fern grows, type of soil, rock, ect., surrounding vegetation, exposure to light and climatic conditions, microclimate which surrounds the fern itself (temperatures, humidity, etc.), and availability of water.

In order to grasp a better understanding in species delimitation on Diplazium and also to recognize the correlation between ecology and species richness, ecological studies of this genus from Western Malesia are conducted by doing field studies, specimens examination, and literatures study. The aims of this study are: (1) to study the ecology ofDiplazium species and (2) to recognize the diversity ofDiplazium species in the different habitat gradient.

2.2. Materials and Methods

Ecological and distribution data were collected directly in the field in Java, Sumatra and Borneo and information obtained from the specimens examined. Specimens vouchers are housed at Herbarium of Bogor Botanic Gardens (BOHB) and will be distributed to some herbaria, including Herbarium Bogoriense (BO). In addition, some literatures are cited for comparison.

2.3. Results and Discussion

All species of Diplazium grow terrestrially, except D. lomariaceum. It grows both terrestrially and epipetric on wet rocks, in disturbed or secondary forest and primary forest. Most of species are mountain ferns. They distribute from 10 m – to 3400 m above sea levels. The ecology of West Malesian Diplazium for each species is summarized in Table 2.1. The ecology and the distribution of all species are discussed below.

2.3.1. Ecology

Some authors used habit and habitats for classifying ferns. Holttum (1966) classified ferns into 8 groups based on its habit and habitats, namely: terrestrial sun-ferns, terrestrial shade-ferns, climbing ferns, epiphytes of sheltered places, epiphytes of exposed places, rock-ferns and river-bank-ferns, aquatic ferns, and mountain ferns. Edie (1978) classifies the characteristics of ferns from each type of habitat in general: terrestrial shade ferns, terrestrial sun ferns, epiphytes, rock ferns and aquatic ferns. Parris et al(1992) explained the ecological characteristic of ferns growing in Mt. Kinabalu based on its habit and habitats: roadside and thicket-ferns at lower elevations, high-elevation thicket-ferns, tree ferns, ferns of cultivated areas, shade ferns of forest, ferns of mountain ridges, ferns high elevations, epiphytic ferns, filmy ferns, high-elevation and epiphytes. Chin (1997) described habitat of tropical ferns and its diversity and then he recognized six habitats of fern, viz. lowland rainforest, mountain forest, secondary forest, agricultural areas, swamps and open waters, and urban areas.

Based on their main habitat, Diplazium can be included into three major groups: dryland, riparian and rheophytic. Rheophytes are plant species which inhabit the beds of swift-running streams and rivers and grows up to flood-level, but not beyond the reach of regularly occurring flash floods (van Steenis, 1981). The rheophytic plants are characterized by having particular morphological characters as follow: a) narrow lanceolate leves or leaflets; b) mattet root systems; c) short erect, ascending, or creeping rhizomes tightly attached to streambed substrates; and flexible stems and petioles. Dryland species generally do not occur in the flooded zone where rheophytes occur, while rheophytes do not occur in dryland habitat where dryland plants thrive (Imaichi & Kato 1997). As pointed out by Imaichi and Kato (1997), there is distinct habitat segregation between rheophyte and dryland species, especially in the humid tropics. Following the definition described by Lincolnet al (1982), riparian means living or situated on the banks of rivers and streams, whereas terrestrial is living habitually on the land or ground surface.

2.3.1.1. Rheophytic ofDiplazium

Rheophytic of Diplazium are smallest group of Diplazium in West Malesia. There are only two species reported in this study, viz. D. aequibasale andD. wahauense (Table 2.1. and 2.2.). Katoet al (1991) reported three species, namely D. aequibasale, D. wahaunse and D. subsinuatum. The last species however has been moved into Deparia and treated as Deparia lancea (Thunb.) Fraser-Jenk based on morphology, cytology and molecular characters (Sano et al 2000). In this paperD. subsinuatumis excluded. In Sumatra and Borneo, the first species are usually growing on lowland clayey stream-bank, whereas the second species, that is only found in Borneo, growing at streambed in flood zone in lowland.

Van Steenis (1981) classifies rheophytes into obligate and facultative, according to preference for rheophytic habitats. In Borneo, many plants of D. esculentum are growing well on river banks in the flood zone, for example at Sungai Joloi (track to Batikap, Central Kalimantan). This species may be included in facultative rheophytes. But anatomical observations show that leaves

of D. esculentumhave poorly developed intercellular spaces of rheophytes (Kato & Imaichi unpublished), there is no distinct morphological difference between the reophytic and dryland plants. Kato (1991) regarded such species as evolutionarily incipient rheophytes.

Kato et al (1991) assumed that most rheophytes are products of primary speciation from dryland mother species. Diplazium wahauense presumably derived from D. riparium, Deparia biserialis and De. confluens from D. petersenii, Phronephrium hosei fromP. rhombeum orP. menisciicarpon.

2.3.1.2. RiparianDiplazium

Only few species occur on river banks or stream. They are D. aequibasale,D. riparium,D. fuliginosum, D. lomariaceumandD. porphyrorachis (Table 2.2.). However these species do not strictly grow at this habitat. The last three species are more usual growing in dryland of shady ravine in the forest. In Borneo, much of individual plants ofD. ripariumare also growing well in shady dryland. All species mentioned above, are usually found on lowland mountain forest.

Environmental condition seems very important component of speciation in Diplazium although this of course depend upon of how one of species response the ecological change. Diplazium riparium and its closely related species (D. aequibasale andD. wahauense) may be a good example for it. Katoet al (1991) presumed that D. wahauense may have been derived from D. riparium which occurs in riparian and dryland forest of Borneo. As they explained and supported by this studies (see Chapter 8), these two species share black scales, somewhat crisped, entire scales, blackish stipes, dark brown, naked rachises, and imparipinnate leaves with 4 pairs of entire lateral pinnae. Diplazium wahauense differs from D. riparium mainly in its narrow pinnae, which are characteristic of rheophytes.

2.3.1.3.Diplaziumin Dryland

Most of Diplazium species are growing terrestrially in dryland forest. (Table 2.1. and 2.2.). According to the light intensity, the drylandDiplazium can

be divided into three groups: a) opened area, b) light shady ferns, and c) deep shady ferns. The species that adapted to the light intensity are discussed below.

a. Opened area ferns

D. esculentum usually grows on opened areas. This species grows well at the dump soil of opened areas both in the forest and at the farming areas. Therefore sometimes this species often form small population at the edges of rivers or stream.

b. Light shady ferns

There are great number of species growing in the light shady area. Some species adapted to very humid conditions and living near streams, such as D. accedens, D. kunstlerii, D. procumbens, D. profluens, D. squarrosum, D. spiniferum, and D. vestitum. The humidity of the air in primary forest near streams is always high, even in the places well away from streams its average is much above that of the open air outsite the forest.

Many species are growing well on dry areas, tolerating drier soil and air. They are found further from streams on forested hill slopes, viz. D. asymmetricum; D. atrosquamosum D. bantamense, D. barbatum, D. batuayauense, D. betimusense, D. cordifolium, D. crenatoserratum, D. densisquamatum, D. dilatatum, D. dolichosorum, D. donianum , D. fraxinifolium, D. halimunense, D. hewittii, D. hottae, D. latisquamatum, D. lobbianum, D. loerzingii, D. malaccense, D. megasegmentum, D . meijerii, D. pallidum, D. parallelivenium, D. petiolare, D. poiense, D. polypodioides, D. sorzogonense, D. speciosum,andD. simplicivenium.

Shady ferns usually grow more slowly than sun-ferns. The weak light prevents plants to make its carbohydrates fast and the high humidity may make less loss of water and less root activity. However this condition does not prevent many species of Diplazium from attaining a immense size, such as D. accedens, D. dilatatum, D. polypodioides, D. subpolypodioides, D. megasegmentum, andD. sorzogonense.

Table 2.1. Classification ofDiplaziumBased on their main habitat

Major Group

Species Number of

Species Rheophytic D. aequibasale, D. wahauense 2 Riparian D. aequibasale,D. riparium,D. fuliginosum, D.

lomariaceum, D. porphyrorachis

5

Dryland D. accedens,D. albidosquamatum,D. angustipinna,D.

asymmetricum,D. atrosquamosum,D. bantamense,D. barbatum, D. batuayauense,D. beamanii, D. betimusense,D. christii ,D. cordifolium,D. crameri,D. crenatoserratum,D. crinitum,D. cumingii D. densisquamatum,D. dilatatum,D. dolichosorum,D. donianum,D. esculentum ,D. fraxinifolium,D. halimunense,D. hewittii,D. hottae,D. insigne,D. kunstlerii,D. laevipes,D. latisquamatum,D. lobbianum,D. loerzingii,D. malaccense,D. megasegmentum,D. megasimplicifolium, D. melanolepis,D. meijerii,D. moultonii,D. pallidum,D. parallelivenium,D. petiolare,D. poiense,D. polypodioides,D. prescottianum,D. procumbens,D. profluens,D. riparium, D. silvaticum,D.

simplicivenium,D. sorzogonense,D. speciosum,D. spiniferum,D. squarrasum,D. subintegrum, D.subalternisegmentum,D. subserratum,D. subvirescens,D. tomentosum,D. tricholepis,D. umbrosum,D. velutinum,D. vestitum,D. xiphophyllum

64

c. Deep shady ferns.

Many species are adapted to deep shady areas, such as D. albidosquamatum, D. angustipinna, D. beamanii, D. christii, D. cumingii, D. fuliginosum, D. laevipes, D. lomariaceum, D. moultonii, D. poiense, D. tomentsum, and D. umbrosum. Therefore they are nearly all have much thinner fronds than the light shady ferns. The deep shady ferns species do not need to be tough to avoid being shriveled by the sun’s heat. These species do not need to store water as much as some light shade ferns do. The deep shady ferns usually also show more dark green or blue metallic fronds than the light shade ferns (Figure 2.1.).

This classification is not strict as some species are also grow well in opened and light shady areas. D. polypodioides are seen in the gap areas of thick forest and also in the margin forest. There are several species found growing in both light shady and deep shady areas. D. bantamense, D. batuayauense, D. cordifolium, D. crinitum, D. crameri, D. dilatatum, D. donianum, D. fraxinifolium, D. lobbianum, D. loerzingii, D. malaccense, D. melanolepis, D. riparium,andD. sorzogonense can be found in light shady and deep shady areas.

2.3.2. Diversity ofDiplaziumBased on Elevation

Table 2.3.. and Figure 2.2. show the diversity of Diplazium based on elevation. The species number ofDiplazium culminate at 1000-1500 m. Amount of 19 species ofDiplazium distribute at the elevation. Some studies on diversity across elevation (Shmida & Wilson 1985; Gentry & Dodson 1987; Colwell & Hurtt 1994) also reveal that peak diversity occur at intermediate elevations. Lomolino (2001) predicted that species-density should peak at an intermediate elevation and the peak should occur at transition zone between the two species-rich, juxtaposed communities. Because detailed information on climatic gradients, dispersal, population persistent and anthropogenic disturbance are generally unavailable, it is difficult to evaluate critically some of the predictions associated with causal explanation for peaks in diversity at intermediate elevations (Lomolino 2001).

Some species ofDiplazium, such asD. atrosquamosum, D. beamanii,and D. squarrosum, are only occurring at upper elevation zones (Table 2.3.). Above 3000 m, there was only foundD. moultonii. Some scientists (Kikkawa & Wlliams 1971; Gentry & Dodson 1987; McCoy 1990; Rahbek 1995) reported that speciation and endemicity peak at the intermediate to high elevations. Rahbek (1995) presumed that upper elevation zones may provide the geographical isolation required for speciation. If the montane zones are both isolated and large enough to allow population persistence and divergence over evolutionary time, they may represent hotspots of speciation and endemicity.

Some species that presumed to be closely related have different in the range of distribution based on altitude. D. lobbianum and D. bantamense are presumed closely related. The two species share character combination as follow: scales toothed; fronds imparipinnate; pinnae oblong-ovate; vein free, forked, texture chartaceous. EcologicallyD. lobbianum is found at 1500-1800 m, whereas D. bantamense usually below 1500 m.

Table 2.2. Diversity ofDiplaziumbased on Elevation Elevation m Species

20- 500 D. accedens, D. acuminatum, D. aequibasale, D. angustipinna, D. bantamense, D. batuayauense, D. crenatoserratum, D. crinitum, D. hewittii, D. lomariaceum, D. pallidum, D. petiolare, D. polypodioides, D. porphyrorachys, D. riparium , D. tomentosum, D. wahauense, D. xiphophyllum

500-1000 D. accedens, D. acuminatum, D. bantamense, D.. cordifolium, D. crenatoserratum, D. cumingii, D. hewittii, D. hottae, D. laevipes, D. lomariaceum, D. pallidum, D. petiolare, D. poiense, D. polypodioides, D. simplicivenium, D. sorzogonense, D. tomentosum, D. umbrosum, D. xiphophyllum

1000-1500 D. accedens, D. asymmetricum, D. barbatum, D. cordifolium, D. crenatoserratum, D. cumingii, D. dilatatum, D. halimunense, D. hewittii, D. laevipes, D.

lomariaceum, D. latisquamatum, D. moultonii, D. pallidum, D. petiolare, D. poiense, D. polypodioides; D. sorzogonense; D. tomentosum, D. tricholepis, D. umbrosum, D. velutinum, D. xiphophyllum.

1500-2000 D. atrosquamosum, D. barbatum, D. cordifolium, D. fuliginosum, D.

latisquamatum, D. lomariaceum, D. moultonii, D. poiense, D. polypodioides, D. sorzogonense, D. speciosum, D. tricholepis, D. umbrosum, D. velutinum 2000-2500 D. atrosquamosum, D. barbatum, D. cordifolium, D. fuliginosum, D.

latisquamatum, D. moultonii, D. sorzogonense, D. speciosum, D. tricholepis 2500-3000 D. cordifolium, D. fuliginosum, D. latisquamatum, D. moultonii, D. speciosum 3000-3400 D. moultonii

The individuals with different ploidy level in the same species sometimes grow in the different altitude (See Chapter 5). Diploid D. pallidum from Java, Borneo, and Sumatra were found at 200 m, 240 m and 30 to 85 m, respectively. Whereas the tetraploid ones were found at 1000 – 13000 m.

Some closely related species may occur in overlapped distribution. D. insigne is evidently closely allied toD. accedens (Holttum 1940). The two species share in characters: dull brown toothed scales with narrow thickening black strands, the deltoid deeply lobed apex of fronds and copiously goniopterid venation. The first species distributes from 600 m to 1200 m and the second species from 80 m to 1400 m.

Figure 2.1. a-b. Light shade-ferns ofDiplazium. a. D. hottae; b.D. loerzingii; c-d. Deep shade-ferns. c.D. cordifolium; d.D. tomentosum.

0 5 10 15 20 25

0-500 501-1000 1001-1500 1501-2000 2001-2500 2501-3000 3000-3500

Elevation Range (meter)

N

umber

of Spec

ies

2.4. Conclusions

Based on their main habitat, Diplazium can be divided into three major groups: dryland, riparian and rheophytic. Most of species are terrestrial dryland ferns and found at 20 – 3400 m above sea level in the primary and secondary forest on moist humus-rich soil in light and deep shady places (64 species). There are only found five of the riparian species, namelyD. aequibasale, D. riparium, D. fuliginosum, D. lomariaceum, and D. porphyrorachis. The rheohytic species are found inD. aequibasaleand D. wahauense.

The most species number ofDiplazium were culminated at 1000-1500 m. The lowest number of species occurred at 2500-3400 m. The individuals with different ploidy level in the same species sometimes grow in the different gradient habitats.

CHAPTER 3

THE DISTRIBUTION OF WEST MALESIAN DIPLAZIUM INSIDE AND OUTSIDE MALESIA

3.1. Introduction

The fern genusDiplazium is an important component of tropical rainforest of the Old World and the New World. It is a terrestrial ferns which is commonly found in the moist ground at the humus rich soil both in the primary and secondary forest at 20-3400 m altitude. Most of species grow in shadowed place and fond of moist humus rich soil. Some species are locally abundant by stream in the mountains. Only a few species are found in limestone areas and in rheophitic areas (See Chapter 2).

Since 1801 Diplazium have been the subject of numerous taxonomic studies conducted, including morphological, anatomical, cytological and molecular investigations. It is estimated that the genus consisting of about 400 species (Ching 1964a, Copeland 1947). Roos (1995) estimated that 300 species of its are found in Malesian region. Few species are found in continental Africa (Kramer et al 1990). In the Neotropics there are nearly 100 species (Pacheco 2004).

Distributional data are important in answering many questions about polyploidy and speciation (Baack 2004). Usually, increasing in ploidy level is associated with the origin of novel adaptations (Levin 2002). Polyploid often occupy different habitats from those of their diploid parents (Soltis & Soltis 2000).

Study of the biogeographical distribution of organisme are also very useful in inferring the monophyly of a taxon. The elementary questions of historical biogeography concern areas of endemism and their relationships (Nelson & Platnick 1981). By analogy to phylogenic systematics, where species or higher taxa are grouped, in cladistic biogeopgraphy the units grouped are areas of endemism (Linder 2001). For example, study on molecular phylogenetic and historical biogeography of Hawaiian Dryopteris(Dryopteridaceae) (Geiger & Ranker 2005) indicate that Hawaiian Dryopteris is not monophyletic, and there

were at least five separate colonizations of the Hawaiian Islands by different species of dryopterioid ferns, with most of the five groups having closest relatives in SE Asia.

This chapter presents a preliminary compilation of Western Malesian Diplazium distribution and their inferred center of diversity in Malesia based on specimens examined and direct observation in the field and their distribution outside West Malesia that obtained from literature studies. In the light of current concerns on the loss biodiversity it is also useful to highlight the areas with hight species numbers and endemicity. The aims of the research are map the distribution of Western MalesianDiplazium and determine the endemic species.

3.2. Materials and Methods

The determination of species distribution patterns was studied by examining specimens deposited at BO and SING, conducting field work in Java, Sumatra, and Borneo. Beside that many literature were also studied for determining the range area distribution of each species.

Following those conducted by Parris (2003) in presenting the distribution of Grammitidaceae in the world, for the purposing of examining the distribution of Western Malesian Diplazium, it is convenient to divide the world into five regions: (1) New World (North, Central and South America, the West Indies and the islands of the Galapagos, Falklands and South Georgia); (2) Africa (continental Africa, Madagascar and the islands of the Azores, Canaries, Ascension, St. Helena, Tristan da Cunha group, Seychelles, Comoros, Mauritius, Reunion, Marion, Crozets and Kerguelen); (3) Asia excluding Malesia (India,Sri Langka, Nepal, Thailand, Cambodia, Laos, Vitenam, China, Taiwan and Japan); (4) Malesia (Malaysia, Singapore, Brunei Darussalam, Indonesia, Philippines and Papua New Guinea), and (5) Pacific (Australia, New Zealand and the islands of Micronesia, Melanesia and Polynesia).

3.3. Results and Discussion

A large number of Diplazium specimens (1051 collections number ) deposited at BO and SING and new collections obtained from the field have been examined. Sixty nine species of Diplazium are recorded within West Malesian region. Diplazium of Malay Peninsula, Borneo, Sumatra and Java (including Bali) comprises of 28, 40, 29, and 30 species, respectively (Table 3.2. ). The distribution for each species inside and outside West Malesia are presented in Table 3.1.

Revision on Bornean and Sumatran species has not been conducted since van Alderwereld van Rosenburgh (1908). For Bornean species, formerly author who ported the check list of this genus locally were Iwatsuki & Kato (1984), Tagawa (1972), Katoet al (1991), and Parriset al 1992). For Sumatran species, the check list provided is only those reported by Mitsuta (1985) of West Sumatra. Thus study was the first account of Diplazium throughout Borneo and Sumatra. Beside that three and four species are now recognized as new species for Borneo and Sumatra, respectively (See Chapter 9).

For Javanese species the recent account after Backer & Posthumus (1939) was Praptosuwiryo (1999). The first author described 17 species, three species of them have been included in other genera. Meanwhile second author described 22 species and 4 varieties. Now, eight species were added for Java and seven species of them are proposed as new species (See Chapter 9).

Holttum (1940, 1966) reported 27 species of Malay Peninsula that include D. heterophlebium, D. curtisii and D. amplissimum. As stated in Chapter 9, the first two species are included in the doubtful names. The latest species was included in Cornopteris as C. atroviridis (v.A.v.R.) M. Kato (Kato 1979). D. aequibasale , D. christii , andD. fraxinifolium are new record for Malay Peninsula (See Chapter 9).

Based on the range of its geographical distribution, the West Malesian species can be divided into three types: (1) very wide distribution species, (2) Malesian species, and (3) endemic species to island in West Malesia. The three types of the geographical distribution are discussed below.

Plate 10.

Diplazium parallelivenium

Praptosuwiryo,

Holotype

Plate 11.

Diplazum profluens

Praptosuwiryo.

Holotype (

T.Ng. Praptosuwiryo

1820,

BO). a. Pinna; b. Pinnulae. c. Middle part of pinnule with detail venation

and sori

Plate 13.

Diplazium subvirescens

Praptosuwiryo.

Paratype (

T.Ng.

Praptosuwiryo 1013

, BO).

a. Bipinnate frond; b. A part of punnule bearing sori.

c. Long creeping rhizome with base of stipes bearing brown scales.

ABSTRACT

TITIEN NGATINEM PRAPTOSUWIRYO. Biosystematic Study of the Fern GenusDiplazium in West Malesia. (Under the supervision of Prof. Dr. Ir. Edi Guhardja, M.Sc., Prof. Dr. Mien A. Rifai, M.Sc., Prof. Dr. Masahiro Kato, M.Sci., and Dr. Dedy Darnaedi, M.Sc. )

Diplazium is a large genus consisting of about 400 species occur mainly in the tropics, sparingly in the subtropic and only locally extending into temperate. It was estimated that 300 species of the records were occurred in Malesia. Taxonomically,Diplazium is very difficult and quite insufficiently known. Therefore a comprehensive study onDiplazium in West Malesia was conducted by using morphological, ecological, geographic distribution, anatomical, palinological, cytological, as well as DNA analysis to understand the diversity and relationship among species.

Based on gross morphological study on 1051 collection number of specimens as well as living collections, it was concluded that West MalesianDiplazium comprises of 69 species with 14 varieties. Thirteen species of them are proposed and described as new species, namelyDiplazium asymmetricum, D. batuayauense, D. crameri, D. densisquamatum, D. halimunense, D. loerzingii, D. megasegmentum, D. megasimplicifolium, D. meijeri, D. parallelivenium, D. profluens, D. subalternisegmentum,andD. subvirescens. Two new varieties are poposed, namelyD. accedens var. spinosum and D. silvaticum var. pinnae-ellipticum. D. pallidum var. montanum and D. accedensvar.ridleyi are proposed as new status.

Based on their main habitats,Diplaziumcan be classified into three major groups, viz. dryland (dominant), riparian and rheophytic species. Species diversity was culminated at 1000-1500 m above sea level. The individuals with different genetic load in the same species sometimes grow in the different habitat gradients. Based on the range of the geographical distribution, West Malesian species can be divided into three types: (1) very wide species (19 species), (2) Malesian species (27 species), and (3) locally endemic species (23 species).

Anatomical study on the transversal section of stipe of 27 species showed that the vascular bundle shape is varying among species. Therefore the leaf-trace shapes are important diagnostic features which support species delimitation in Diplazium. Spore morphology study showed that perine ornamentations support in delimitating species in Diplazium. However the phylogenetic analysis using parsimony revealed that morphological variation of spore is inadequate to depict natural relationship amongDiplazium species.

Cytological study on 117 collection number from 54 localities included in 31 species found that West MalesianDiplazium has six ploidy levels with x = 41 (diploid, triploid, tetraploid, pentaploid, hexaploid, and octoploid). New cytological information for science on 19 species are recorded. They areD. aequibasale(2n = 164),D. angustipinna (2n = 123).D. asymmetricum(2n = 123),D. batuayauense(2n = 164, 205),D. crenatoserratum(2n = 123, 164),D. halimunense(2n = 123),D. hewittii(2n = 123),D. profluens(2n = 164),D. loerzingii(2n = 82, 123),D. pallidum

(2n = 82),D. petiolare(2n = 82),D. porphyrorachis (2n = 164),D. riparium(2n = 82, 123),D. spiniferum (2n = 82), D. subserratum (2n = 82, 123, 164), D. subvirescens (2n = 123), D. tomentosum(2n = 82, 205),D. xiphophyllum(2n = 82, 246), andD. wahauense(2n=164).

Phylogenetic analysis on morphological data sets of 69 species using parsimony revealed that the phylogenetic relationship among species in the genus Diplazium was very difficult to explain due to the lack of or weak support Bootstrap value. However the lack of or weak support for a phylogenetic tree does not strictly indicate that the pattern observed is incorrect but it does limit the amount of confidence that can be placed in the relationships between taxa.and the conclusions can be drawn from them. This study showed that some terminal clades formed are consisting of species that presumed to be closely related by formerly authors.

DNA analysis resulted new generbcL sequences data on 25 species. GenerbcL sequence is very well in supporting species delimitation and revealing the intraspecific diversity within species ofDiplazium. Phylogenetic analysis on 29 species from West Malesia and 9 references species outside Malesia using parsimony revealed that gene rbcL is more informative than morphological data in inferring phylogeny of Diplazium and showed that West Malesian Diplazium is monophyletic. The position of D. porphyrorachis at the basal clade of the morphological tree is supported by the phylogenetic tree generated from molecular data (gene

rbcLsequence). This study also showed the congruence between the clade of riparium group drawn by generbcL tree and the clade of imparipinnate frond group drawn by morphological tree.