BIOSYSTEMATIC STUDY OF THE FERN GENUS DIPLAZIUM … · ekologi, distribusi geografi, anatomi, palinologi, sitologi dan juga analisa DNA dilakukan untuk memahami keanekaragaman jenis

BIOSYSTEMATIC STUDY OF THE FERN GENUSDIPLAZIUM IN WEST MALESIA

TITIEN NGATINEM PRAPTOSUWIRYO

DEPARTMENT OF BIOLOGYTHE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITYBOGOR

2008

STATEMENT OF RESEARCH ORIGINALITY ANDINFORMATION SOURCE

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

Bogor, September 2008

Titien Ngatinem PraptosuwiryoNIM P176 00005

ABSTRACT

TITIEN NGATINEM PRAPTOSUWIRYO. Biosystematic Study of the Fern Genus Diplazium inWest 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 300species of the records were occurred in Malesia. Taxonomically, Diplazium is very difficult andquite insufficiently known. Therefore a comprehensive study on Diplazium in West Malesia wasconducted 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 asliving collections, it was concluded that West Malesian Diplazium comprises of 69 species with 14varieties. Thirteen species of them are proposed and described as new species, namely Diplaziumasymmetricum, D. batuayauense, D. crameri, D. densisquamatum, D. halimunense, D. loerzingii,D. megasegmentum, D. megasimplicifolium, D. meijeri, D. parallelivenium, D. profluens, D.subalternisegmentum, and D. subvirescens. Two new varieties are poposed, namely D. accedensvar. spinosum and D. silvaticum var. pinnae-ellipticum. D. pallidum var. montanum and D.accedens var. ridleyi are proposed as new status.

Based on their main habitats, Diplazium can 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 speciessometimes grow in the different habitat gradients. Based on the range of the geographicaldistribution, West Malesian species can be divided into three types: (1) very wide species (19species), (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 thevascular bundle shape is varying among species. Therefore the leaf-trace shapes are importantdiagnostic features which support species delimitation in Diplazium. Spore morphology studyshowed that perine ornamentations support in delimitating species in Diplazium. However thephylogenetic analysis using parsimony revealed that morphological variation of spore isinadequate to depict natural relationship among Diplazium species.

Cytological study on 117 collection number from 54 localities included in 31 speciesfound that West Malesian Diplazium has six ploidy levels with x = 41 (diploid, triploid, tetraploid,pentaploid, hexaploid, and octoploid). New cytological information for science on 19 species arerecorded. They are 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), and D. wahauense (2n=164).

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

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

RINGKASAN

TITIEN NGATINEM PRAPTOSUWIRYO. Biosystematic Study of the Fern

Genus Diplazium 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 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 biosistematika Diplazium 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 Diplazium Malesia 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. spinosum dan D. silvaticum var. pinnae-ellipticum. D. pallidum var.

montanum dan D. accedens var. ridleyi diusulkan 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 jenis Diplazium.

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) dan D. 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 gene rbcL sangat 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 gene rbcL. Cabang group daun menyirip

gasal pada pohon morfologi selaras dengan cabang group riparium pada pohon

gene rbcL.

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 filogeni Diplazium belum dilakukan.

Copyright @ 2008, Bogor Agricultural University

All Right Reserved

1. It is prohibited to cite all or part of this dissertation without referring to

and mentioning the source.

a. Citation only permitted for the sake of education, research,

scientific writing, report writing, critical writing or reviewing

scientific problems.

b. Citation does not inflict the name and honor of Bogor Agricultural

University.

2. It is prohibited to republish and reproduce all part of this dissertation

without copyright permission from Bogor Agricultural University.

BIOSYSTEMATIC STUDY OF THE FERN GENUSDIPLAZIUM IN WEST MALESIA

TITIEN NGATINEM PRAPTOSUWIRYO

A dissertation submitted to fulfill one of the requirements for theDoctorate Degree at the Study Program of Biology,

Graduate School, Bogor Agricultural University

DEPARTMENT OF BIOLOGYTHE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITYBOGOR

2008

Examiner of first examination:

Dr. Tatik Chikmawati, M.Si.Department of Biology, Faculty ofMathematic 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 Genus Diplazium 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)

Examination Date: 25 March 2008 Graduation Date:

PREFACE

Research on the West Malesian Diplazium under the tittle Biosystematic

Study of the Fern Genus Diplazium 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 in Floribunda 2 (5): 128-137 (2004) and (2)

Cytological Study of Some Species of Ferns Genus Diplazium in Java II. that

published in Floribunda 2 (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 study Diplazium

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.

i

TABLE OF CONTENTS

Page

LIST OF TABLES …………………………………………………….. iv

LIST OF FIGURES ………..……………………………………………. v

LIST OF APPENDIXES ………………………………………………… vi

1 INTRODUCTION

Taxonomical Aspects of Diplazium and Its Systematic Problems …………………………………………………............ 1

The Diversity of Diplazium in Malesia .......................................... 7The 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 on Diplazium and Its Closely Related Genera ........................................................................ 10

The Utility of Molecular Techniques for phylogeneticstudies of pteridophytes: Gene rbcl Sequences ....................... 11

Objectives ……………………………………….………………. 12

2 DIVERSITY AND ECOLOGY OF DIPLAZIUM

Introduction ……………………………………………………… 13Materials and Methods ……………………………………......... 14Results and Discussion ……………………………………......... 14

Ecology .………………………………………….................. 14Rheophytic Diplazium ……………………...................... 15Riparian Diplazium ………………………....................... 16Diplazium in Dryland ……..……………........................ 16Diversity of Diplazium Based on Elevation …............... 19

Conclusions …………………………………………….............. 22

3 THE DISTRIBUTION OF WEST MALESIAN DIPLAZIUMINSIDE AND OUTSIDE MALESIA

Introduction ……………………………………………………… 23Materials and Methods …………………………………………... 24Results and Discussion ………………………………………….. 25

Very Wide Distribution Species ………………….................. 26Malesian Species …………………………………................. 26

ii

Endemic Species to Island in West Malesia ………................. 27Conclusions ………………………………………………........... 40

4 THE STELAR ANATOMY OF STIPE AND ITS TAXONOMIC SIGNIFICANT IN DIPLAZIUM

Introduction …………………………………………………….. 41Material and Methods …………………………………………... 42Results and Discussion …………………………………………. 42Conclusions …………………………………………………………… 46

5 CYTOLOGICAL AND REPRODUCTIVE STUDIES ON DIPLAZIUM IN WEST MALESIA

Introduction …………………………………………………….. 47Materials and Methods …………………………………………. 49Results and Discussion …………………………………………. 50

Chromosome Number Variations and Mode ReproductionTypes on Diplazium ………………………………………... 50The Relationship between Ploidy Level andMorphological Variation within Species and CloselyRelated Species of Diplazium ............................................... 66Relationship between ploidy level and habitat gradient ....... 71Correlations between reproductive mode and habitat ........... 72

Conclusions ................................................................................ 81

6 PHYLOGENETIC STUDIES OF DIPLAZIUM FROM WEST MALESIA: EVIDENCE FROM MORPHOLOGY

Introduction …………………………………………………...... 83Character Selection and Construction …………………………. 85

Character Selection ………………………………………… 85Character Type …………………………………………….. 85Character Coding …………………………………………… 86

Character Variation within West Malesian Diplazium ……….. 87Materials and Methods ……………………………………….... 100

Taxon Sampling ………………………………………….... 100Character Examination of Diplazium …………………....... 100Phylogenetic Analysis ……………………………………… 101

Results and Discussion ………………………………………… 110Conclusions …………………………………………………..... 117

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

Introduction .................................................................................. 118Materials and Methods ................................................................. 120Results and Discussion ................................................................. 122

iii

Spore Characters of Diplazium and Its Use in Supporting Species delimitation and Identification …........... 122

Phylogenetic Analysis ……………………………………... 137Conclusions ................................................................................. 143

8 MOLECULAR SYSTEMATIC OF DIPLAZIUM FROM WEST MALESIA

Introduction …………………………………………………….. 146Materials and Methods …………………………………………. 148

DNA Analysis ……………………………………………… 149Phylogenetic analysis ………………………………………. 161

Results and Discussion ………………………………………… 161Infraspecific Genetic Diversity in Diplazium ……………… 161Species Delimitation in Diplazium based on GenerbcL Sequence ……………………………………………... 163

Informative Characters of Gene rbcL Sequencesfor Inferring Phylogenetic Hypothesis of Diplazium ………. 165Phylogenetic Analysis ……………………………………… 166

The Monophyly of Diplazium ………………………….. 166Relationships among species within Diplazium ….…….. 165

Conclusions ……………………………………………………... 175

9 TAXONOMIC STUDY OF THE FERN GENUS DIPLAZIUMIN WEST MALESIA

Introduction …………………………………………………….. 176Materials and Methods …………………………………………. 176Taxonomic Treatment ………………………………………….. 177

10 GENERAL DISCUSSION

Synthesis ……………………………………………………….. 283 General Discussion …………………………………………….. 284 Systematic Implications for the Genus Diplazium ……………... 285

11 CONCLUSIONS …………………………………………………... 187

LITERATURES ……………………………………………………....... 292

LIST OF TABLES

Table 2.1. Classification of Diplazium based on their main habitat …… 18

Table 2.2. Diversity of Diplazium based on elevation …………………. 20

iv

Table 3.1. Distribution of West Malesian Diplazium inside and outside Malesia …………………………………………..…. 28

Table 3.2. Endemic Species of Diplazium in West Malesia …………… 39

Table 3.3. Species diversity and endemism of Diplazium in four mainlands of West Malesia …………………………… 40

Table 5.1. Somatic Chromosome Numbers, Ploidy Level and Mode Reproduction Type of Diplazium from West Malesia ... 52

Table 5.2. Polyploid series of Diplazium in 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 of Diplazium 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 of Diplazium ………………………………………… 143

Table 7.3. Coding for 17 characters utilized in construction of spore morphology data set of Diplazium …………………… 144

Table 8.1. List of Taxa Used in This Study …………………………… 152

Table 8.2. Primers Used for Amplifying and Sequencing DNA fromDiplazium (Hasebe et al, 1994) …………………………… 160

Table 8.3. Infraspecific Genetic Variatons of Diplazium based on Gene rbcL Sequences …………………………………. 162

Table 8.4. Interspecific Genetic Variatons of Diplazium based on Gene rbcL Sequences …………………………………. 164

LIST OF FIGURES

Figure 2.1. a-b. Light shade-ferns of Diplazium ………………………. 21

Figure 2.2. Elevational distribution of Diplazium species in West Malesia ……………………………………………………. 21

Figure 4.1. Vascular structure of the leaf axis …………………………. 43

Figure 4.2. Leaf-trace shapes in Diplazium ………………………....... 44

v

Figure 4.3. Leaf-trace shapes in Diplazium. ............................................. 45

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

Figure 5.2. Somatic chromosome of Diplazium ………………………... 75

Figure 5.3. Somatic chromosomes of Diplazium ……...……………….. 76

Figure 5.4. Somatic chromosomes of Diplazium ……………………… 77

Figure 5.5. Somatic chromosomes of Diplazium cordifolium …………… 78

Figure 5.6. Somatic chromosomes of Diplazium ...................................... 79

Figue 6.1. Rhizome appeareance of Diplazium …………………………. 89

Figure 6.2. The variation of scale shapes in Diplazium ………………… 91

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

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

Figure 6.5. Frond architectures of Diplazium …………………………… 95

Figure 6.6. Venation types of Diplazium ………………………………. 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. vestitum var. 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

vi

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 the rbcL sequence data. …………..………………………………….. 169

LIST OF APPENDIXES

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

Appendix 2. Gene rbcL 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 halimunense Praptosuwiryo ..................................... 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

Plate 14. Diplazium subvirescens Praptosuwiryo ………………………. 364

1

CHAPTER 1

GENERAL INTRODUCTION

1.1. Taxonomical Aspects of Diplazium 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 (Kramer et 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; Kramer et 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 (Takamiya et al 1999).

2

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 Japanese Diplazium 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 of Diplazium 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 of Diplazium.

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 and Anisogonium. 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

of Athyrium, 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. silvaticum group (A. silvaticum, A. blumei= D. polypodioides, A.

3

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. williamsi group (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. accedens group

(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, and D. marginatum; and (5) Diplazium longicarpun

group. This group includes species with frond pinnate, acroscopic base of pinna

4

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 of Diplazium 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 into Athyrium. 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)

and Diplazium as 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 as represented by Diplazium dilatatum Blume, D. polypodioides Blume,

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

5

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 in Athyriaceae 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

that Allantoidea R. Br. and Callipteris Bory should be included in Diplazium 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

6

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

maintained Diplazium separated from Athyrium.

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 monophyletic Diplazium clade; Rhacidosorus is separated from monoplyletic

Diplazium clade and not closely related to either Athyrium and Diplazium (Sano et

al 2000a, 2000b).

e. Classification of Diplazium.

Despite the recognition of Diplazium as genus, the higher classification of

Diplazium (family level) is still unclear. Tardieu-Blot (1932) placed this genus in

the tribe Asplenieae of the family Polypodiaceae. 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 in Denstaedtiaceae,

the largest family of modern pteridophytes sensu 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) placed Diplazium in Physematieae, a tribe

of Athyrioideae, a subfamily of Dryopteridaceae.

7

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 subfamily Diplazioideae

and proposed to divide Athyriaceae into five subfamilies : Cystopterioideae,

Athyrioideae, Deparioideae, Diplazioideae and Rachidosorioideae.

1.2. The Diversity of Diplazium 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.

8

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 (Haufler et al 2000).

The comparative study of plant structure, morphology and anatomy, has

always been the backbone of plant systematics to elucide plant diversity,

9

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 (Endress et 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 (Endress et al, 2000).

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

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 (Barrington et al 1986)

10

1.3.3. Cytological Evidence in revealing taxonomic problems onDiplazium and 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; Takamiya et al 1999, 2000, 2001; Sano et 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 on Diplazium in Java was reported by

Darnaedi (1992) for tetraploid D. cordifolium and diploid D. esculentum. In

11

addition, Praptosuwiryo and Darnaedi (1994) reported cytological research on six

species of Diplazium from Gede-Pangrango National Park, West Java (one species

of its, viz. D. opacum, have been treated as Cornopteris 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 ofpteridophytes: Gene rbcl 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 (Hauk et 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)

12

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 (Hasebe et

al 1995, Gastony & Ungerer 1997, Murakami et 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 for Diplazium based on

morphological data and gene rbcL sequence.

13

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 of Diplazium species and (2) to recognize

the diversity of Diplazium species in the different habitat gradient.

14

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.

15

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 Lincoln et 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 of Diplazium

Rheophytic of Diplazium are smallest group of Diplazium in West

Malesia. There are only two species reported in this study, viz. D. aequibasale

and D. wahauense (Table 2.1. and 2.2.). Kato et 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 paper D. subsinuatum is 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

16

of D. esculentum have 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 from P. rhombeum or P. menisciicarpon.

2.3.1.2. Riparian Diplazium

Only few species occur on river banks or stream. They are D.

aequibasale, D. riparium, D. fuliginosum, D. lomariaceum and D. 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 of D. riparium are 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 and D. wahauense) may be a good example for it. Kato et 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. Diplazium in Dryland

Most of Diplazium species are growing terrestrially in dryland forest.

(Table 2.1. and 2.2.). According to the light intensity, the dryland Diplazium can

17

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, and D. 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, and D.

sorzogonense.

18

Table 2.1. Classification of Diplazium Based on their main habitat

MajorGroup

Species Number ofSpecies

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

lomariaceum, D. porphyrorachis5

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, and D. sorzogonense can be found in light shady and deep shady areas.

19

2.3.2. Diversity of Diplazium Based on Elevation

Table 2.3.. and Figure 2.2. show the diversity of Diplazium based on

elevation. The species number of Diplazium culminate at 1000-1500 m. Amount

of 19 species of Diplazium 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 of Diplazium, such as D. atrosquamosum, D. beamanii, and

D. squarrosum, are only occurring at upper elevation zones (Table 2.3.). Above

3000 m, there was only found D. 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. Ecologically D. lobbianum is found at 1500-1800 m, whereas

D. bantamense usually below 1500 m.

20

Table 2.2. Diversity of Diplazium based 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 to D. 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.

21

Figure 2.1. a-b. Light shade-ferns of Diplazium. a. D. hottae; b. D. loerzingii; c-d. Deepshade-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)

Num

ber

of S

peci

es

Fig. 2.2. Elevational distribution of Diplazium species in West Malesia

22

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, namely D. aequibasale, D. riparium,

D. fuliginosum, D. lomariaceum, and D. porphyrorachis. The rheohytic species

are found in D. aequibasale and D. wahauense.

The most species number of Diplazium 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.

23

CHAPTER 3

THE DISTRIBUTION OF WEST MALESIAN DIPLAZIUM INSIDE ANDOUTSIDE MALESIA

3.1. Introduction

The fern genus Diplazium 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

24

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 Malesian Diplazium 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).

25

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), Kato et al (1991), and Parris et 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 , and D. 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.

26

3.3.1. Very Wide Distribution Species

A conception of very wide species adopted here is referred for species that

is distributed at least two region or more of regional division following those

adopted by Paris (2003). Of the sixty nine species of Diplazium from West

Malesia, 19 of them distribute very wide (Table 3.1.), in the New World. They

are D. accedens, D. bantamense, D. cordifolium, D. crenatoserratum, D.

dilatatum, D. donianum, D. esculentum, D. fuliginosum, D. malaccense, D.

pallidum, D. polypodioides, D. prescottianum, D. riparium, D. silvaticum, D.

simplicivenium, D. sorzogonense, D. subserratum, D. tomentosum and D.

xiphophyllum . Three species of them are distributing across four main region

(Africa, Asia, Malesia, Pacific), viz. D. accedens, D. dilatatum, and D.

esculentum.

Some species that widely distributed are varying in morphology and

genetic (see Chapter 2 and 9). Therefore some varieties are found in these

species. Kato (1995) recorded 2 varieties in D. donianum and D. dilatatum. In

this study (see Chapter 9), D. accedens, and D. cordifolium are rezognized to

have three varieties. Meanwhile in D. pallidum and D. silvaticum are recorded

two varieties.

The very wide species in general are also showing the long distance

gradient distribution. They are commonly growing in lowlands or medium

altitude of mountain forest. For example, D. accedens, D. polypodioides, and D.

pallidum are found at 80-1400 m, 200-1900 m, and 600-1500 m, respectively (See

Chapter 2).

3.3.2. Malesian Species

Malesian species denote for species that having wide distribution range in

Malesia region only. These species may be only found in West Malesia or

distributed throughout Malesia. Most of species are distributed widely in

Malesian region. Some species, such as D. aequibasale, D. angustipinna, D.

kunstlerii, D. latisquamatum, D. procumbens, D. subintegrum and D.

subpolypodioides are only found in West Malesia.

27

3.3.3. Endemic Species to Island in West Malesia

Endemic species concept adopted here refer to a species that restricted to a

particular local or island. List of endemic species of Diplazium are showed in

Table 3.3 Endemic species criteria was determined by inspection of distribution

maps cited from literatures. The total number of local endemic species of West

Malesian species recorded in this study are 23 species. This number are high

enough. Tryon (1970), Smith (1972) and Wagner (1972) have discussed the high

proportion of pteridophytes on oceanic islands. Smith (1972) compiled data from

several islands and showed that ferns have much lower of endemism than

angiosperm at both the genus and species levels. On Hawaii, about 16% of

angiosperms genera are endemic vs. 6.7% of pteridopnytes genera. Ranker et al

(1994) give interpretation for this fact in two fold: first, ferns are more capable of

long-distance dispersal and establishment, second, continued gene flow from

mainland sources may slow speciation rates for ferns, thereby constraining the

evolution of endemic taxa.

Most of endemic species are found mountain forest at 900-1600 m above

sea level. It is evidence that the lower and intermediate elevation of mountain

forest is also the center of endemism areas.

Some endemic species have a relative strict distribution based on

elevation. D. betimusense was found at 300-400 m. D. atrosquamsosum and D.

lobbianum were only found at 1500-2100 and 1600-1800 m, respectively. One

species that has longer distant gradient distribution is D. tricholepis, 100-2100 m.

Some endemic species are found in certain habitats. D. wahauense only

grows at the reophytic areas of Borneo. D. albidosquamatum and D. crinitum

grow in shady places of limestone area. D. betimusense and D. squarrosum were

found growing at shady forest near streams.

This endemic statements, however, can change in accordance with the

science advances. The new exploration and the change of the taxon delimitation

would give new data for a taxon. Consequently taxa that in the past stated as

endemic species would be non endemic species. For example, D. atrosquamoum,

D. barbatum, D. laevipes, and D. vestitum are not endemic to Mount Kinabalu

now (Parris et al 1992).

28

Table 3.1. Distribution of West Malesian Diplazium Inside and Outside Malesia

No. Species Inside MalesiaPM

B S Pa J B Ph C M L S I I J PNGOutsideMalesia

Literatures GeographicalDistributionRange

1. D. accedens Blume V V V V V V V V V V V V North-easternQueensland;Africa,islands ofIndianOcean,Somoa,Thailand.

Present StudyAndrews(1990)Holttum (1940)Tagawa &Iwatsuki(1988)Kato (1992)Mitsuta (1985)

Very wide

2. D. acuminatum Blume V Present study Endemic

3. D. aequibasale (Baker)C.Chr.

V V V V Present studyIwatsuki &Kato (1984)

Malesia

4. D. albidosquamatum Alderw. V Present study Endemic

5. D. angustipinna Holtt.(Holtt.)

Present studyHolttum (1966)

Malesian

6. D. asymmetricumPraptosuwiryo, sp. nov.

V Present study Endemic

7. D. atrosquamosum Copel.)C.Chr. & Holtt.

V Present study Endemic

Note: PM = Peninsular Malaysia, B = Borneo, S = Sumatra, Pa = Palawan, J = Java, B = Bali, Ph = Philippines, C = Celebes, M = Moluccas, LSI = Lesser Sunda Islands, IJ = Irian Jaya, PNG = Papua New Guinea.

29

Table 3.1. Continued

No. Species Inside MalesiaPM

B S Pa J B Ph C M L S I I J PNGOutsideMalesia

Literatures GeographicalDistributionRange

8. D. bantamense Blume V V V V V V Thailand Present studyHolttum (1940)Iwatsuki &Kato (1984)Tagawa &Iwatsuki(1988)Mitsuta (1985)

Very wide

9. D. barbatum C.Chr. inC.Chr. & Holtt.

V Present study Endemic

10. D. batuayauensePraptosuwiryo, sp. nov.

V Present study Endemic

11. D. beamanii M.G. Price V Endemic

12. D.betimusense Alderw. V Present study Endemic13. D. christii C. Chr. V Present study Endemic14. D. cordifolium Blume V V V V V V V V V V V India to

SolomonIsland,Thailand

Present studyHolttum (1940)Tagawa (1972)Tagawa &Iwatsuki(1988)Kato (1992)Mitsuta (1985)

Very wide

30

Table 3.1. Continued

No. Species Inside MalesiaPM

B S Pa J B Ph C M L S I I J PNGOutsideMalesia

Literatures GeographicalDistributionRange

15. D. crameri Praptosuwiryo,sp. nov.

V Present study Endemic

16. D. crenatoserratum (Blume)Moore

V V V V V Thailand Present studyHolttum (1940)Tagawa (1972)Tagawa &Iwatsuki (1988)Kato (1992)Mitsuta (1985)

Very wide

17. D. crinitum (Baker) C..Chr. V Present studyTagawa (1972)Iwatsuki &Kato (1984)

Endemic

18. D. cumingii (Presl) C.Chr. V V Present studyTagawa (1972)Iwatsuki &Kato (1984)

Malesia

19. D.densisquamatumPraptosuwiryo, sp. nov.

V Present study Endemic

31

Table 3.1. Continued

No. Species Inside MalesiaPM

B S Pa J B Ph C M L S I I J PNGOutsideMalesia

Literatures GeographicalDistributionRange

20. D. dilatatum Blume V V V V V V V V V V Thailand,India,Burma, S.China,Taiwan,Ryuku, S.Japan,Indochina,N.Australia.

Present studyHolttum (1940)Tagawa &Iwatsuki(1988)

Very wide

21. D. dolichosorum Copel. V V Present study Malesia

22. D. donianum (Mett.) Tardieu V V V Japan,Taiwan, S.China,Indochina,Thailandand India

Present studyTagawa &Iwatsuki(1988)

Malesia

23. D. esculentum (Retz.)Swartz

V V V V V V V V V V V V Tropic ofAsia toOceania

Present studyHolttum (1940)Tagawa (1972)Tagawa &Iwatsuki(1988)Kato (1992)

Very wide

24. D. fraxinifolium Presl V V V V Present study Malesia

32

Table 3.1. Continued

No. Species Inside MalesiaPM

B S Pa J B Ph C M L S I I J PNGOutsideMalesia

Literatures GeographicalDistributionRange

25. D. fuliginosum (Hook.) M.G.Price

V V V BismarckArch. (New

Ireland)

Present study Malesia

26. D. halimunensePraptosuwiryo, sp. nov.

V Present study Endemic

27. D. hewittii (Copel.) C.Chr V Present studyIwatsuki & Kato(1984)

Endemic

28. D. hottae Tagawa V V Present studyTagawa (1972)Iwatsuki & Kato(1984)

Malesia

29. D. insigne Holtt. V Present studyHolttum (1966)

Endemic

30. D. kunstlerii Holtt. V V V Present studyHolttum (1966)Mitsuta (1985)

Malesia

31. D. laevipes C.Chr. in C.Chr.& Holtt.

V Present studyIwatsuki & Kato(1984)

Endemic

32. D. latisquamatum Holtt. V V V Present studyHolttum (1966)

Malesia

33. D. lobbianum Moore V V V V Present studyVan Alderweldvan Rosenburgh(1908)

Malesia

33

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

34. D. lomariaceum (Christ.)Price

V V V V V V Present studyIwatsuki &Kato (1984)Kato (1992)

Malesia

35. D. loerzingii Praptosuwiryo,sp. nov.

V V Present study Malesia

36. D. malaccense Presl. V V V V Thailand,Indochina

Present studyHolttum (1940)Tagawa &Iwatsuki(1988)Mitsuta (1985)

Malesia

35. D. loerzingii Praptosuwiryo,sp. nov.

V V Present study Malesia

36. D. malaccense Presl. V V V V Thailand,Indochina

Present studyHolttum (1940)Tagawa &Iwatsuki(1988)Mitsuta (1985)

Malesia

37. D.megasegmentumPraptosuwiryo, sp. nov.

V Present study Endemic

38. D.megasimplicifoliumPraptosuwiryo, sp. vov.

V Present study Endemic

39. Diplazium meijeriiPraptosuwiryo

V Present study Endemic

34

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

40. D. melanolepis Alderw. V Present study Endemic41. D. moultonii (Copel.) Tagawa V Present study

Tagawa (1972)Endemic

42. D. pallidum (Blume) Moore V V V V Queensland Present studyHolttum (1966)Kato (1992)Mitsuta (1985)

Very wide

43. D. paralleliveniumPraptosuwiryo, sp. nov.

V Present study Endemic

44. D. petiolare C. Presl. V Present study Endemic45. D. poiense C. Chr. in C.Chr. &

Holtt. V Present study

Iwatsuki & Kato(1984)

Endemic

46. D. polypodioides Blume V V V V V V V V V V V V Thailand,Sri Langka,S. India,Himalaya,Indochina,Taiwan.

Present studyHolttum (1940)Tagawa &Iwatsuki (1988)Mitsuta (1985)

Very wide

47. D. porphyrorachis (Baker)Diels

V Present studyTagawa (1972)

Endemic

48. D. prescottianum (Wall.) Moore V Thailand Present studyHolttum (1940)Tagawa &Iwatsuki (1988)

Endemic

35

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

49. D. procumbens Holtt. V V V Present studyHolttum (1940)

Malesia

50 D. profluens Praptosuwiryo, sp.nov.

V Present study Endemic

51. D. riparium Holtt. V V V V V V Thailand Present studyHolttum (1940)Tagawa (1972)Tagawa &Iwatsuki (1988)Kato (1992)

Malesia

52. D. silvaticum (Bory) Swartz V V V V V Thailand;Mauritus,India,Burma

Present studyHolttum (1940)Iwatsuki & Kato(1984)Tagawa &Iwatsuki (1988)

Malesia

36

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

53. D. simplicivenium Holtt. V V Thailand Present studyHolttum (1940)Tagawa &Iwatsuki (1988)

Malesia

54. D. sorzogonense C. Presl. V V V V Thailand,Vietnam,Indo-China

Present studyHolttum (1940)Iwatsuki & Kato(1984)Tagawa &Iwatsuki (1988)Kato (1992)Mitsuta (1985

Malesia

55. D. speciosum Blume V V V V V Present studyHolttum (1940)

Tagawa (1972)Iwatsuki & Kato(1984)Mitsuta (1985)

Malesia

56. D. spiniferum Alderw. V Present studyTagawa (1972)

Endemic

57. D. squarrasum K. Iwats. & MKato

V Present studyIwatsuki & Kato(1984)

Endemic

37

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

58. D. subintegrum Holtt. V V Thailand Present studyHolttum (1940)Tagawa &Iwatsuki (1988)

Malesia

50. D.subalternisegmentumPraptosuwiryo, sp. nov.

V Present study Endemic

60. D. subpolypodioides Alderw. V V Present study Malesia

61. D. subserratum (Blume) Moore V V V Thailand Present studyHolttum (1940)Tagawa &Iwatsuki (1988)Mitsuta (1985)

Malesia

62. D. subvirescens Praptosuwiryo,sp. nov.

V Present study Endemic

63. D. tomentosum Blume V V V V V Thailand,Burma,Vietnam

Present studyHolttum (1940)Tagawa (1972)TagawaIwatsuki (1988)Kato (1992)Mitsuta (1985)

Very wide

64. D. tricholepis C. Chr. V Present studyTagawa (1972)Iwatsuki & Kato(1984)

Endemic

38

Table 3.1. Continued

No. Species Inside MalesiaPM B S Pa J B Ph C M L S I I J PNG

OutsideMalesia

Literatures GeographicalDistributionRange

65. D. umbrosum (Smith) Beddome V Present study Endemic66. D. velutinum Holtt. V Present study

Holttum (1940)Endemic

67. D. vestitum C. Presl. V V V Present studyKato (1992)

Malesia

68. D. wahauense Kato, Darnaedi etK. Iwatsuki

V Present study Endemic

69. D. xiphophyllum (Baker) C.Chr.

V V V V V Thailand Present StudyHolttum (1940)Tagawa (1972)Tagawa &Iwatsuki (1988)Kato (1992)

Very wide

39

Table 3.2. Endemic Species of Diplazium in West Malesia

No. Species Locality

1. D. albidosquamatum Sumatra: Bengkulu (Lebong Tandai)2. D. asymmetricum

Praptosuwiryo, sp. nov.Java: Mt. Gede, Mt. Halimun (West Java)

3. D. atrosquamosum Borneo: Mt Kinabalu, Tenompok-Humu-humu, Southof Panataran River.

4. D. barbatum Borneo: Mt. Kinabalu (North Borneo), Mt. Besar(South Kalimantan)

5. D. batuayauensePraptosuwiryo, sp. nov.

Borneo: Batu Ayau (Muller Range)

6. D. beamanii Borneo: Mt. Kinabalu7. D. betimusense Sumatra: Betimus River (Sibolangit)8. D. christii Malay Peninsula: G. Muntahak, (Johore)9. D. crameri,

Praptosuwiryo, sp. nov.Sumatra: Kenali (Sukaraja)

10. D. crinitum Borneo: G. Njapa and Long Keluh, Berau (CentralKalimantan)

11. D. densisquamatumPraptosuwiryo, sp. nov.

Sumatra: Bukit Tapan (Jambi)

12. D. halimunensePraptosuwiryo, sp. nov.

Java: Mt. Halimun (West Java)

13. D.megasegmentumPraptosuwiryo, sp. nov.

Java: Mt. Salak (West Java)

14. D.megasimplicifoliumPraptosuwiryo, sp. nov.

Borneo: Bukit Raya

15. D. meijeriiPraptosuwiryo, sp. nov.

Sumatra: Mt. Sago (Payakumbuh)

16. D. melanolepis Sumatra: G. Singgalang17. D. parallelivenium

Praptosuwiryo, sp. nov.Java: Mt. Halimun (West Java)

18. D. profluensPraptosuwiryo, sp. nov.

Java: Mt. Halimun (West Java)

19. D. squarrosum Borneo: Mt. Besar (South Kalimantan)20. D. subalternisegmentum

Praptosuwiryo, sp.nov.Mount Kinabalu, Borneo

21. D. subvirescensPraptosuwiryo, sp. nov.

Java: Mt. Gede and Mt. Halimun (West Java)

22. D. tricholepis Mount Kinabalu, Borneo23. D. velutinum Malay Peninsula: Cameron Highlands24. D. wahauense Borneo: Along Jenta River, Miau Baru, north of

Muara Wahau (Central-East Kalimantan), BelatungRiver, Muller Range (Central Kalimantan)

40

Table 3.3. Species diversity and endemism of Diplazium in four mainlands of West Malesia

Island Account of Species Number Endemics FoundJava 30 6Sumatra 29 6Malay Peninsula 28 2Borneo 40 9

3.4. Conclusions

Sixty nine species of Diplazium are distributed in West Malesia. The total

number of species for each main island are 40, 30, 29, 28 for Borneo, Java,

Sumatra, and Malay Peninsula, respectively. 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) locally

endemic species.

Most species are only found in Malesia. Nineteen species of West Malesia

distribute very wide. They are D. accedens, D. bantamense, D. cordifolium, D.

crenatoserratum, D. dilatatum, D. donianum, D. esculentum, D. fuliginosum, D.

malaccense, D. pallidum, D. polypodioides, D. prescottianum, D. riparium, D.

silvaticum, D. simplicivenium, D. sorzogonense, D. subserratum, D. tomentosum

and D. xiphophyllum.

Twenty four species are presumed as endemic in the islands of West

Malesia. The highest number of endemic species is occurring in Borneo (9

species): D. atrosquamosum, D. barbatum, D. batuayauense, D. beamanii, D.

crinitum, D. squarrosum, D. subalternisegmentum, D. tricholepis and D.

wahauense. Whereas the lowest number is in Malaya Peninsula (2 species), viz D.

christii and D. velutinum. Six species are endemic to Java and Sumatra. The six

endemic species of Sumatra are D. albidosquamatum, D. betimusense, D. crameri,

D. densisquamatum, D. meijerii, and D. melanolepis, and six species endemic to

Java are D. asymmetricum, D. halimunense, D. megasegmentum, D.

parallelivenium, D. profluens, and D. subvirescens.

41

CHAPTER 4

THE STELAR ANATOMY OF STIPE AND ITS TAXONOMICSIGNIFICANT IN DIPLAZIUM

4.1. Introduction

Anatomical studies of the ferns have had a long and historically

significant place in the professional literatures (e.g. Bower 1912, 1913). These

anatomical studies in particular, played a large part in the conclusions concerning

fern systematic relationships and evolution drawn by Bower. Between the years

of 1960 – 2000, anatomical data have emerged once again as important to an

accurate understanding of relationships and evolution among ferns (e.g. Holttum

& Sen 1961; Bir 1969; Kato 1972; Tryon 1970; Nishida & Nishida 1982; White &

Weidlich 1995; Qiu et al 1995).

Detailed anatomical investigation can serve several useful purposes

including the addition of new knowledge about particular taxa and the useful

application of these kind data on the problems of relationships among fern taxa

(White 1974). Broad and detailed comparative anatomical studies are important

in problems solving of fern systematic and evolution.

One of the kinds of comparative studies usually used in the anatomical

studies on the ferns is vascular (stelar) pattern (White 1974). The importance of

the vascular tissue as a unified tissue system (the stele) was recognized in the late

19th century (e.g. Tiegham & Douliot 1886). Reviewing from the many cases

studies on vascular pattern of ferns, White (1974) came in a concussion that stelar

anatomy is a potentially powerful tool in systematic although the vascular tissue

data alone do not provide the basis on which systematic (or taxonomic)

conclusions are based.

The stelar anatomy of Athyrioid ferns and their relatives has been studied

in detail by some pteridologist (e.g. Tardieu-Blot 1932, Bir 1969, Kato 1972). All

these studies came to the concussion that anatomical evidence is important to

support taxa delimitation. This chapter presents the anatomical data of stipe on 27

species of West Malesian Diplazium. The aim of this study was to collect stelar

anatomical data to support species delimitation in Diplazium.

42

4.2. Materials and Methods

Transection of frond axis, stipe near lamina (upper portion of stipe), were

studied on 27 species collected from West Malesia . A pieces of stipe near blade

of fresh material were hand sectioned with a sharp razor blade. The sections were

embedded in glycerine jelly after staining them with 0.5% methyl green to obtain

semi permanent slides.

4.3. Results and Discussion

As also reported by Praptosuwiryo (1999), this study resulted that all

species under observation have strand of vascular tissue termed amphicribal

vascular bundles. This structure consist of a central strip of xylem completely

surrounded by phloem. Foster & Gifford (1959) termed meristele for this such

concentric strand of vascular tissue. The meristele in the stipe is embedded within

a conjunctive parenchyma which posses large intercellular spaces. Between the

cortical parenchyma and the single layered cuticularized epidermis is a band of

hypodermal schlerenchyma whose fiber are heavily lignified. Meristele structure

in the rhizome and leaf strands is the usual type for Diplazium, Athyrium,

Diplaziopsis, etc. (Tardieu-Blot 1932, Bir 1969). Therefore this structure is not an

important for diagnostic feature which supports the taxonomic separation among

the species of Diplazium.

Leaf-trace are binary. As showed in Figure 4.1. the xylem of a leaf-trace

of most species are same hippocampus-shaped bundle in transaction. The shape

and position of this vascular bundle are varying among species. The similar

anatomy is illustrated by Tardieu-Blot (1932) and Kato (1977). In D.

polypodioides and D. subpolypodioides, the xylem of the leaf-trace develop ridges

and grooves become somewhat W-shaped. This shape is similar with D.

latifolium illustrated by Bir (1969).

The leaf-trace shapes of the stipe, as a whole, are varying among species

and constant among the adult individuals in a species (Figure 4.2. and 4.3.). The

shapes are comprised of five types: (1) uninterrupted V-shaped (Figure 4.2.a-c,

e.), (2) interrupted V-shaped (Figure 4.2.g.), (3) uninterrupted U-shaped (Figure

4.2.i. ), (4) interrupted U-shaped (Figure 4.2.h & k), and (5) W-shaped (Figure

43

4.3.i.). These types similar those illustrated by Tardieu-Blot (1932), Kato (1977)

and Bir (1969).

Figure 4.1. Vascular structure of the leaf axis. a. D. cordifolium; b. D. esculentum;c. D. umbrosum; d. D. procumbens. e. D. polypodioides. Bar = 1 mm for b, c, d, and e. Bar = 0.4mm for a.

The uninterrupted V-shaped leaf-trace is seen in D. tomentosum, D.

angustipinna, and D. cordifolium. Interrupted V-shaped is shown in D.

silvaticum. Interrupted U-shaped leaf-trace is seen in D. accedens var. spinosum,

D. batuayauense and D. D. subserratum.. Most species are possessing

44

uninterrupted U-shaped leaf trace, such D. halimunense, D. procumbens, D.

sorzogonense, D. speciosum, D. subserratum, D. umbrosum and D. vestitum. As

mentioned above, W-shaped leaf trace is found in D. subpolypodioides and D.

polypodioides. Some species may have intermediate shape between two types. For

example, D. riparium and D. crenatoserratum have the intermediate shape between type

one and four.

Figure 4.2. Leaf-trace shapes in Diplazium. Groove U-shaped with flat base. a. D. tomentosum;b. D. cordifolium (simple frond); c. D. angustipinna; d. D. silvaticum var. silvaticum;e. D. riparium; f. D. xiphophyllum; g. D. crenatoserratum h. D. subserratum;i. D. subvirescens; j. D. accedens var. accedens; k. D. accedens var. spinosum. Bar = 2 mm fora, c, d, g, and h. Bar = 1.6 mm for b and e. Bar = mm for f and d. Bar = 2.5 mm for j and k.

45

Figure 4.3. Leaf-trace shapes in Diplazium. a. D. halimunense; b. D. donianum;c. D. simplicivenium; d. D. sorzogonense; e. D. procumbens; f. D. vestitum; g. D. speciosum;h. D. subpolypodioides; i. D. polypodioides; j. D. umbrosum; k. D. spiniferum;l. D. batuayauense. Bar = 1.5 mm for a, b, d and g. Bar = 1 mm for l. Bar = 2 mm for c, e, f, h,i, j, and k.

46

Each type of the leaf traces or the vascular bundle may diversify into the

derivative forms. These character variations can be used to determine a species

among closely related species. D. speciosum and D. sorzogonense are

morphologically very similar. Anatomically, the two species share characters:

vascular bundle form uninterrupted U-shaped, flat base on the two directions,

inward and outward (Figure 4.3.). But, the U-shaped vascular bundle of first

species is with an angle 90º and end slightly ridge, whereas the second species

with an angle 110º and end almost simple. Even, the vascular bundle type of D.

acedens var. accedens (Figure 4.2.j.) is different from D. accedens var. spinosum

(Figure 4.2.k.). Var accedens has an uninterrupted U-shaped vascular bundle

with ridges that formed at the outward of lower base, angles, and ends.

Meanwhile var. spinosum has an interrupted U-shaped with more blunt ridges on

the angle and end. Therefore the leaf-trace shapes are important diagnostic

features that support species delimitation in Diplazium.

4.4. Conclusions

The leaf-trace shape of Diplazium stipe is varying among species and

constant among the adult individuals in a species. Based on the result of this

study and also previous authors (Tardieu-Blot 1932, Bir 1969, Kato 1977) it is

concluded that the leaf-trace shapes of Diplazium can be classified into five main

types: (1) uninterrupted V-shaped, (2) interrupted V-shaped, (3) uninterrupted U-

shaped, (4) interrupted U-shaped, and (5) W-shaped. Each type seems to vary among

the species. Each type may diversify into some different derivative forms that enables to

determine a species among closely related species. Therefore the leaf-trace shapes are

important diagnostic features which support species delimitation in Diplazium.

47

CHAPTER 5

CYTOLOGICAL AND REPRODUCTIVE STUDIES ON DIPLAZIUM INWEST MALESIA

5.1. Introduction

Cytological studies of ferns have given a big influence on the classification

on ferns and ferns allies dating from the publication of Manton (1950), ‘Problems

of Cytology and Evolution in the Pteridophyta’, in which a new simple techniques

was introduced. Her book inspired a great upsurge in chromosomes work on the

ferns and fern allies not only in Europe but also by authors in North America,

India, Japan and New Zealand (Tindale & Roy, 2002). However, by 1977 it was

estimated by Love et. al. that less than 20% of the Pteridophyta had been

examined cytologically, although few genera have escaped attention since that

date. Love et. al. (1977) stated that there are about 3.200 species referred to 446

genera and 65 families on the basis of Pichi Sermolli’s classification (1977).

As reported by Löve et al (1977), cytological observations of Ceylon

Diplazium by Manton (1953) was preliminary cytological research of Diplazium.

From 1953 to 1977 were only about 15.5% of ca. 400 species of Diplazium in the

world known their chromosomal information. In Malesia, it is predicted that

cytological information since Manton (1954) to Praptosuwiryo & Darnaedi (2004)

were only 6% of ca.300 species of Diplazium.

Polyploidization and mode reproduction types has played an important

role in the geographical distribution and evolution of Diplazium. Polyploids,

which contain more than two sets of chromosomes per cell, have important in

plant evolution (Stebbins 1971; Lewis 1980). Up to 95% of pteridophytes may be

polyploid (Grant 1981) and recent polyploids may comprise 45% of extant

homosporous fern (Vida 1976; Haufler & Soltis 1986). Polyploidy may lead to

significant reproductive isolation (Ramsay & Schemske, 1998), and change in

gene expression (Adams et al 2003), and ecological interactions (Segraves et al

1999).

48

Cytological observation have clearly disclosed many apomictic species

within Diplazium (Lovis, 1977). Apomixis is an asexual reproduction via

chromosomal unreduction of spores and gametophytes, and the subsequent

apogamous reproduction (in a strict sense), i.e., asexual reproduction of a

sporophyte from somatic cells of the gametophyte. About 10% of fern species in

world are apomictic (Lovis 1977). About 15% of Japanese ptridophytes are

apomictic (Takamiya 1996, 1997). How many percent of apomictic species of

Malesian pteridophytes are not known because the cytological observations of

peridophytes have not been done completely.

Since the late 20th, cytological and reproductive studies on Japanese

Diplazium have been doing intensively. Takamiya et al (1999) examined 162

plants representing 16 species of Japanese Diplazium, including polymorphic

terrestrial with evergreen bi- to tripinnate leaves. Their studies clearly give

clarification the morphological complexities on some species, moreover would be

a good evident in clarifying its taxomical status. Mitotic and meiotic

chromosomes studies on 80 plants representing seven species with summer-green

bi- to tripinnate leaves were also unraveled many cases of the morphological

complex in Japanese Diplazium (Takamiya et al. 2000; 2001).

Cytological researches on Diplazium in Malesian region are not so many.

Cytological information on Diplazium were firstly reported by Manton (1954) and

Holttum & Roy (1965). The first reported 11 species of Diplazium of Malaya,

and the second observed 4 species of Papua New Guinea. In Java, preliminary

cytological observation of Javanese Diplazium was conducted by Darnaedi (1992)

in which he found tetraploid D. cordifolium and diploid D. esculentum.

Furthermore, Praptosuwiryo and Darnaedi (1994) reported chromosome numbers

of 6 species of Diplazium (one species of its, viz. D. opacum has been changed to

Cornopteris opaca) of Gede-Pangrango National Park. Recent cytological

informations for Malesian region were reported by Praptosuwiryo (2003),

Praptosuwiryo & Darnaedi (2004). The first reported two species tetraploid

Diplazium of Lombok Island, viz. D. malaccense and D. pallidum. While the

second reported cytological research of 10 species of Javanese Diplazium

included 43 collection number of 10 localities.

49

This chapter reports cytological and mode reproduction type studies of

Diplazium from West Malesia included 31 species. This aims of this studies are;

(1) to examine somatic chromosomes number of Diplazium; (2) to determine the

mode reproduction type of Diplazium; (3) to recognize the correlation between

ploidy level and morphological variation in the Diplazium species; 4) to recognize

the correlation between ploidy levels and its habitats.

5.2. Materials and Methods

Living plants of Diplazium included in 33 species and 117 collection

number were collected from 54 localities in West Malesia (Table 5.1.). Living

materials were planted in the green house at Bogor botanic Gardens. Now, the

voucher specimens are deposited in the herbarium of Bogor Botanic Gardens

(BOHB) and its duplicates will be distributed to Herbarium Bogoriense (BO).

Cytological investigations were carried out by observing the somatic

chromosomes at the root tips following a procedure developed by Manton (1950)

and modified by Darnaedi (1991). Root tips pretreated with 0.002 M 8-

hydroxyquinolin for 4-6 hrs at about 18-20°C, and then fixed with 45% acetic acid

for 10 minutes. The fixed roots were macerated with a mixture of 45% acetic acid

and 1 N HCl (1:3) for 3-4 minutes at 60°C, and then stained with 1% aceto-orcein

solution. Chromosomes counting were carried out during observation under a

light microscope with 100x objective lens, and photographs were taken by Nikon

Camera in Herbarium Bogoriense.

Mode reproduction types were determined by counting spores per

sporangium. Sporangium with 64 normal spores treated as sexual reproduction,

while the sporangium with 32 spores treated as apogamous one.

50

5.3. Results and Discussion

Somatic chromosome account and mode reproductive type of Diplazium

from West Malesia included 33 species and 117 collections number collected

from 54 localities are reported (Table 5.1.). All species examined revealed basic

chromosome number x = 41. These are consistent with those reported by many

recently researchers on cytology of Diplazium, such as Ohta & Takamiya (1999)

and Takamiya et al. (1999, 2000, 2001). The 33 species of Diplazium successfully

examined its chromosomes number and mode reproduction type are discussed as

follow.

5.3.1. Chromosome Number Variations and Mode Reproduction Types onDiplazium

Diplazium accedens Blume. Eight individuals of D. accedens revealed

2n = 82 (diploid), three individuals from East Kalimantan and five individuals

from five localities in East Java and West Java.. Somatic chromosomes number of

D. accedens from Kalimantan is firstly report in this study. Eighteen individuals

of plant from four localities in East Java and West Java reported by Praptosuwiryo

& Darnaedi (1994 and 2004) also showed diploid level. Total of 23 plants are

consistently diploid sexual. Manton (1954) in Malaya also reported diploid level

only. Since we understand that ploidy is derived from the diploid one, we

presumed that D. eccedens is a good species or probably native to Java.

Diplazium aequibasale (Baker) C.Chr. Only one individual of D.

aequibasale succeded to be examined and showed tetraploid (2n=164). It is the

first cytological report for this species. There is not a cytological observation

reported yet except this study.

Diplazium angustipinna Holttum. There are two levels ploidy found in D.

angustipinna, viz. triploid (2n=123) and tetraploid (2n=164) from Borneo. The

two cytotypes are the first cytological record for Bornean Diplazium, and the

triploid one is also the first record for science. Manton (1954) reported only n =

82 for Malayan plant. However it is not recognized whether this plant is sexual

tetraploid or apogamous diploid because its somatic chromosome number was not

51

reported. Two specimens types from two different ploidy levels, 2n=123 dan

2n=164, can not differentiated.

Diplazium asymmetricum Prtaptosuwiryo, sp. nov. Four individuals (TNgP

1094, TNgP 1334, and TNgP 1732 ) of this species has been observed

cytologically and showed 2n = 123 (triploid). The small frond of Diplazium

asymmetricum is in a glance similar to those of D. procumbens. However D.

asymmetricum can be differentiated from D. procumbens by the following

characters combination: rhizome short, erect; lamina more incised (up to

tripinnate) basiscopic pinnulae or segments and lobes are larger than the

acroscopic ones (acroscopic and basiscopic pinnuale or segments and lobes

asymmetric); indusia thicker, margin entire. While D. procumbens has rhizome

long creeping; acroscopic and basiscopic pinnulae and lobes almost symmetric;

indusia thin and lacerate at margin.

Diplazium bantamense Blume. Four individuals of D. bantamense

collected from Cangkuang Forest, Mt. Salak, West Java, found to be tetraploid

race. Two individuals are successfully examined their reproduction types. TNgP

1212 showed 32 spores per sporangium presumed as apogamous type and TNgP

1454 showed 64 spores per sporangium as sexual type. The others two plants

have not been recognized their reproduction type. Most of individuals

successfully examined were tetraploid sexual (Praptosuwiryo & Darnaedi, 1994).

As pointed out by Kato (1992), at diploid gametophytes level, sexual species

(tetraploid in sporophyte) are more abundant than apogamosporous ones. Kato

(1992) also explained the hypothetical origins of tetraploid agamosporous type.

Tetraploid agamosporous type may be derived from a crossing between diploid

sexual species and triploid agamosporous. While, triploid agamosporous are

derived from diploid and tetraploid sexual species, as a demonstrated by

Dryopteris sparsa compelx (Darnaedi et al 1989).

Based on this analysis, it is presumed that in Java, diploid sexual and

triploid apogamous of D. bantamense may exist in nature. However up to now

the information concerning the existence of both diploid sexual and triploid

apogamous are not exist. Further cytological survey of this species is urgently

needed to confirm whether the diploid ancestral is still exist or not.

52

Table 5.1. Somatic Chromosomes numbers, Ploidy Level and Mode Reproduction Type of Diplazium from West Malesia

Species ChromosomesNumbers (2n)/ PloidyLevel/ ReproductiveType

VoucherSpecimens

Locality

D. accedens 82/diploid/82/diploid/82/diploid/82/diploid/

82/diploid/

82/diploid/82/diploid/seksual82/diploid/seksual83/diploid/seksual

TNgP1001TNgP1447TNgP1786TNgP1399

TNgP1649

TNgP1211TR 53.1TR 53.2TR 53.3

Gede-Pangrango National ParkG. Salak, West Java. Ca. 1500 s.l.G. Halimun, West Java. Ca. 950 m s.l.Cidaon-Cibunar, Ujung Kulon NationalPark, West Java.Gondang Forest, G. Wilis Utara, Kec.Kare, Kab. Madiun, East JavaG.Salak, West JavaEast KalimantanEast KalimantanEast Kalimantan

D. aequibasale 164/tetraploid/ TNgP2026 Bank of Sungai Abang Ai, TNBD, JambiD. angustipinna 123/triploid/

ca.164/tetraploid/

TNgP1904

TNgP1905b

Kobet Forest, near S. Kobet, trek to BatuAyau, Peg. Muller, KALTENG. 440 m.Kobet Forest, near S. Kobet, trek to BatuAyau, Peg. Muller, KALTENG. 440 m.

D. asymmetricumPraptosuwiryo

123/triploid/apogamous

123/triploid/apogamous

123/triploid/apogamous

TNgP 1094

T.Ng.P 1334

TNgP 1732

Petak 4 Desa Kemutuk Lor Wana WisataBaturraden, G. Slamet, Central Java. 970-1000 m. a.s.l.Cibodas Forest, behind Cibodas BotanicGardens, Mt. Gede, Gede-PangrangoNational Park, West Java. ca. 1450 mTrack Cikaniki – Citalahap, HalimunNational Park, West Java. Ca. 1000 m.

D. bantamense 164/tetraploid/apoga-mous164/tetraploid/

164/tetraploid/Sexual

164/tetraploid/

-/-/Sexual

-/-/Sexual

-/-/Apogamous

TNgP1212

TNgP1321

TNgP1454

TNgP1383

TNgP1516

TNgP1517

TNgP1707

Cangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeG. Slamet, Track to Sang Hyang Ropoh,Central Java. 1060 m s.l.G. Slamet, Track to Sang Hyang Ropoh,Central Java. 1060 m s.l.Cikaniki Forest, Halimun National park,West Java

D. batuayauensePraptosuwiryo

164/tetraploid/-

Ca. 205/pentaploid/-

TNgP1927c

TNgP1909

Above Sungai Talikot Puhung Kucan,Batu Ayau, Muller Range, CentralKalimnatan, Borneo. 450 m.Kobet Forest, near S. Kobet, track to BatuAyau, Muller Range, Central Kalimantan,Borneo. 440 m s.l.

53

Table 5.1. Table continued

Species ChromosomesNumbers (2n)/PloidyLevel/Reproductive Type

VoucherSpecimens

Locality

D. cordifolium“pinnate fronds”“simple fronds”

“pinnate fornds”

“pinnate fronds”

“pinnate fornds”

“simple fronds”

“simple frond”

“pinnate fronds”

“pinnate fronds”

“pinnate fronds”

“pinnate fronds”

“pinnate fronds”

“simple fronds”

‘pinnate frond’

“pinnate frond”

“pinnate frond”

“pinnate frond”“simple fronds”

“simple fronds”

“simple fronds”

D. cordifolium‘pinnate fronds’D. cordifolium‘simple fronds’

164/tetraploid/

Ca.246/hexaploid/

Ca.205/pentaploid/

164/tetraploid/

Ca.205/pentaploid/

Ca.205/pentaploid/

Ca.164/tetraploid/

164/tetraploid/

ca205/pentaploid/

-/-/Sexual

-/-/Sexual

-/-/Sexual

164/tetraploid/

164/tetraploid/

164/tetraploid/

ca.205/pentaploid/

164/tetraploid/205/pentaploid/

164/tetraploid/

164/tetraploid/

ca. 164/tetraploid/ .

328/octoploid/

TNgP1194

TNgP1201

TNgP1307

T NgP1375

TNgP1355

TNgP1204

TNgP1203

TNgP1442

TNgP1441

TNgP1456

TNgP1457

TNgP1460

TNgP1458

TNgP1735

TNgP1774

TNgP1808

TNgP1736TNgP1813

TNgP2284

TNgP2281

TNgP1910

TNgP 1926b

Cangkuang Forest, G. Salak-West Java,Southern SlopeCangkuang Forest, G. Salak-West Java,Southern SlopeCangkuang Forest, G. Salak-West Java,Southern SlopeCangkuang Forest, G. Salak-West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java.Southern SlopeCangkuang Forest, G. Salak-West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak-West Java,Southern SlopeCurug Macan Trail, Cikaniki Forest, G.Halimun, West JavaOwa Trail, Cikaniki Forest, G. Halimun, WestJavaPlot II/C6, Cikaniki Forest, G. Halimun, WestJava. Ca. 950 m.Loop Trail, G. Halimun, West Java.Plot I, near Tea Plantation, Cikaniki ResearchStation, G. Halimun, West Java. Ca. 950 m.Track to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West JavaTrack to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West Java.Near Sungai Anak Kobet, track to Batu Ayau,Batu Ayau. 440 m s.l.Near Sungai Anak Kobet, track to Batu Ayau,Mts. Muller, Desa Tumbang Topus, Kec.Tumbang Konyi, Kab. Murung Raya, CentralKalimantan. ca. 450 m.

54

Table 5.1. Table continued

Species ChromosomesNumbers (2n)/ PloidyLevel/ Reproductive Type

VoucherSpecimens

Locality

D. cordifolium‘pinnate fronds’

164/tetraploid/ ...

ca. 164/tetraploid/ ...

164/tetraploid

TNgP2128

TNgP2129

TNgP2342b

Track S. Sopan-Bukit Batikap, between PonotPenanginan-Camp Lapangan Heliped II, ca.6 msebelah Timur S. Joloi, near Tanjakan NikkiArdilla, Mts. Muller, Central Kalimantan. 240 mTrek S. Sopan-Bukit Batikap, between PonotPenanginan-Camp Lapangan Heliped II, ca.6 mthe eastern of Sungai Joloi, near Tanjakan NikkiArdilla, Peg.Muller, Central Kalimantan. 240 mCikuda Paeh, G. Halimun, West Java

D. crenatoseratum 164/tetraploid/

164/ tetraploid/

164/ tetraploid/

164/tetraploid/

ca. 164/ tetraploid/

ca. 123/triploid/apogami

TNgP2067a

TNgP2026a

TNgP2026c

TNgP2044

TNgP1971

TNgP2075

Bukit Pal, Taman Nasional Bukit Dua Belas,Jambi, Sumatra. ca. 70 m.Bank of Abang Ai, Taman Nasional Bukit DuaBelas, Jambi, Sumatra. 20 m.Bank of Abang Ai, Taman Nasional Bukit DuaBelas, Jambi, Sumatra. 20 m.Bukit Air Keruh, Taman Nasional Bukit DuaBelas, Jambi, Sumatra. 50 m.Air Terjun Tomoroh, S. Belatung, Peg. Muller,KALTENG. 150 m.Bukit Suban Punai Banyak, Taman Nasional BukitDua Belas, Jambi, Sumatra. Ca. 55m.

D. dilatatum 123/triploid/apogamous

123/triploid/apogamous

123/triploid/apogamous

123/triploid/apogamous

123/triploid/apogamous

123/triploid/apogamous

TNgP1073

TNgP1339

TNgP1343

TNgP1011b

TNgP1025

TNgP1526

Petak 55, Desa Karang Mangu, Wana WisataBaturraden, G. Slamet, Central JavaG. Gede, Gede-Pangrango National Park, WestJavaG. Gede, Gede-Pangrango National Park, WestJavaG. Gede, Gede-Pangrango National Park, WestJavaG. Gede, Gede-Pangrango National Park, WestJavaG. Pangrango, 1925 m dpl, Gede-PangrangoNational Park

D. donianum 164/tetraploid/ XIX.C.III.65 Bukit Ubar, Sumatra. Cultivated in Bogor BotanicGardens.

D. esculentum 82/diploid/-

82/diploid/-82/diploid/-

TNgP1291

TNgP1784TNgP2094

Pasir Buntu, Geger BentangG. Pangrango, West JavaCikuda Paeh Trail, Cikaniki Forest, G. HalimunHutan Sungai Air Keruh, TNBD, Jambi

D. profluensPraptosuwiryo

164/tetraploid/ TNgP1798 Canopy Bridge Trail, Cikaniki Forest, G. Halimun

D. halimunensePraptosuwiryo

123/triploid/apogami TNgP 2341b Cikuda Paeh, G. Halimun, West Java

D. hewittii 123/triploid/apogami TNgP1913b Hutan Batu Ayau, Peg.Muller, KALTENGD. loerzingii 82/diploid/-

123/triploid/apogamiTNgP 2339cTNgP 2339d

Cikuda Paeh, G. Halimun, West JavaCikuda Paeh, G. Halimun, West Java

D. megasegmentumPraptosuwiryo

-/-/ Apogamous-/-/ Apogamous

TNgP1451TNgP1452

Cangkuang Forest, G. Salak, West Java, SouthernSlopeCangkuang Forest, G. Salak, West Java, SouthernSlope

55

Table 5.1. Table continued

Species ChromosomesNumbers (2n)/ PloidyLevel/ Reproductive Type

VoucherSpecimens

Locality

D. petiolare 82/diploid/-

82/diploid/-

TT993.2

TT993.3

Cagar Alam Rimbo Panti, Kec. Panti, Kab.Pasaman, West Sumatra. 240-900 m dpl.Cagar Alam Rimbo Panti, Kec. Panti, Kab.Pasaman, West Sumatra. 240-900 m dpl.

D. pallidum 164/tetraplioid

164/tetraploid

164/tetraploid164/tetraploid82/diploid/82/diploid/ -ca.82/diploid82/diploid82/diploid

TNgP1377

TNgP1764

TNgP 1151TNgP1172TNgP1406TNgP2088TNgP2036cTNgP2045TNgP2192b

Cangkuang Forest, G. Salak, Southern slope, WestJava. Ca. 1250 m s.l.Jalur G. Andam, Cikaniki Forest, HalimunNational Park. Ca. 1200 m s.l.Cibodas, G. Gede, JABAR. ca. 1300 m.Cibodas, G. Gede, JABAR. ca. 1300 m.G. Payung, T.N. Ujung Kulon, JABAR. 200 mBukit Lubuk Semak, TNBD, Jambi, 85 mHutan Sukodibeyung, TNBD, Jambi. ±30 m.Hutan Sukodibeyung, TNBD, Jambi. 45 m.S. Joloi, Peg. Muller, KALTENG. ±240 m.

D. polypodioides 82/diploid/82/diploid/

82/diploid/

82/diploid

TNgP1647TNgP1604

TNgP1812

TNgP2285

Gondang Forest, 1000 m s.l., G. Wilis, East JavaBurning Forest of Ngliman, Pace Subdistrict, G.Wilis, East JavaPlot I, near Nirmala Plantation, Cikaniki, G.Halimun, West JavaTrack to Curug Seribu, Taman Wisata AlamGubung Salak Indah, G. Salak, West Java

D. procumbens 123/triploid/Apogamous

123/triploid/Apogamous

-/-Apogamous

-/-/Apogamous

-/-/Apogamous

-/-/Apogamous

TNgP1312

TNgP1348

TNgP1173

TNgP1384

TNgP1453

TNgP1693

G.Gede, Gede-Pangrango National Park, WestJavaCangkuang Forest, Southern Slope of G. Salak,West JavaRawa Denok II, Gede-Pangrango National Park,West JavaCangkuang Forest, Southern Slope of G. Salak,West JavaCangkuang Forest, Southern Slope of G. Salak,West JavaTrack to Wilis Water Fall, Southern Slope of G.Wilis, Dusun Turi, Kec. Geger, Kec. Sendang,Kab. Tulung Agung, East Java

D. porphyrorachis ca. 164/tetraploid TNgP1885 S. Ruhai, G. Pumpung Sapat, Batu Ayau, Peg.Muller, KALTENG. 260 m dpl.

D. riparium ca. 82/diploid

123/triploid

TNgP2147

TNgP 1847

Tract to Sungai Sopan-Bukit Batikap, betweenCamp. Lapangan Heliped II – Sungai Sopan, ca.14 km from Camp. Lapangan Heliped II, ca. 150m to the East of Sungai Joloi, Mt. Muller ProtectedArea. Ca.240 m s.l.Track to Batu Ayau, Muller-Range, CentralKalimantan. 280 m

56

Table 5.1. Continued

Species ChromosomesNumbers (2n)/ PloidyLevel/ Reproductive Type

VoucherSpecimens

Locality

D. simplicivenium 123/triploid/apogamous

123/triploid/apogamous123/triploid/apogamous

123/triploid/apogamous-/-/apogamous

-/-/apogmous

TNgP1229

TNgP1386*TNgP1343

TNgP1371TNgP1179

TNgP 1523

Track Honje Warak-Cibatu Lawang, Gede-Pangrango National Park, West Java. 1545 mCangkuang forest, G. Salak, West JavaCibodas, G. Gede, Gede-Pangrango NationalPark, West Java. 1500 m s.l.Cangkuang Forest, G. Salak, Southern SlopeCibodas Forest, Mt. Gede, Gede-PangrangoNational Park, West JavaCibodas, G. Gede, Gede Pangrango NationalPark, West Java. 1520 m s.l.

D. silvaticum var. silvaticum

ca.164/tetraploid/-ca.164/tetraploid/Sexualca.164/tetraploid/-ca.164/tetraploid/-164/tetraploid/-

164/tetraploid/-

TNgP1300TNgP1301TNgP1302TNgP1303TNgPs.n.21May04TNgPs.n.11May04

Wild Ferns of Bogor Botanic GardensWild Ferns of Bogor Botanic GardensWild Ferns of Bogor Botanic GardensWild Ferns of Bogor Botanic GardensWild Ferns of Bogor Botanic Gardens

Wild Ferns of Bogor Botanic Gardens

D. silvaticumvar.pinnae-ellipticum

123/triploid/apogamous-/-/apogamous123/triploid/apogamous123/triploid/apogamous123/triploid/apogamous-/-/apogamous-/-/apogamous

TNgP2001aTNgP 2007bTNgP2085dTNgP2119cTNgP2019aTNgP 2016aTNgP 2028c

Bukit Lubuk Semak, TNBD, Jambi. 70 m.TNBD JambiBukit Berumbung, TNBD, Jambi. 25 m.Bukit Berumbung, TNBD, Jambi. 25 m.Pematang Berumbung, TNBD, Jambi, 15 m.Pematang Berumbung, TNBD, Jambi, 15 m.Pematang Berumbung, TNBD, Jambi, 15 m.

D. speciosum var.speciosumvar. speciosum

var. speciosum

var. majorvar. speciosumvar. major

82/Diploid/Sexual

82/diploid/Sexual

82/diploid/Sexual

82/diploid/82/diploid/82/diploid/

*TNgP1363

*TNgP1366

TNgP1803

TNgP1727TNgP1745TNgP1758

Cangkuang Forest, G. Salak Nature Reserve,Southern Slope, West Java. Ca. 1600 m s.l.Cangkuang Forest, G. Salak Nature Reserve,Southern Slope, West Java. Ca. 1600 m s.l.Jalur Plot II, Cikaniki Forest, G. Halimun,West JavaCikaniki-Citalahap, G. Halimun, West JavaJalur Owa, Cikaniki Forest, West JavaJalur G. Andam, G. Halimun, West Java

D. procumbens 123/triploid TNgP1344 G.Gede, Gede-Pangrango National Park,West Java

D. speciosum 82/diploid/Sexual82/diploid/Sexual

TNgP1359TNgP1362

Hutan Cangkuang, G. Salak, JABAR. 1600 m.Hutan Cangkuang, G. Salak, JABAR. 1600 m.

D. sorzogonense 82/diploid/Sexual82/diploid/-82/diploid/-82/diploid/-

TNgP1803TNgP1727TNgP1745TNgP1758

Jalur Plot II, Cikaniki , G. Halimun, JABARJalur Cikaniki-Citalahap, G. Halimun,JABARJalur Owa, Cikaniki, JABARJalur G. Andam, G. Halimun, JABAR

57

Table 5.1. Continued

Species ChromosomesNumbers (2n)/ PloidyLevel/ Reproductive Type

VoucherSpecimens

Locality

D. spiniferum(indiv.)(27)

82/diploid/-82/diploid/seksual82/diploid/

TNgP1896aTNgP1896cTNgP1896b

G. Pumpung Sapat, Peg. Muller, KALTENGG. Pumpung Sapat, Peg. Muller, KALTENGG. Pumpung Sapat, Peg. Muller, KALTENG

D. subpolypodioi- des

82/diploid/

-/-/sexual

82/diploid/sexual

-/-/sexual

TNgP1825

TNgP 2285

TNgP2292

TNgP 2303

Track to Perkebunan P.T. Gayatri , G. PasirPacet-G. Galunggung, Lingamulya, Kec.Leuwisari, Kab. Tasikmalaya, West Java.Curug Seribu, Gunung Salak Indah, G.Salak, West Java. 940 mTrack to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, WestJava.940 m.Track to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West Java.940 m.

D. subserratum ca.123/triploid/apogamous

-/-/Sexual

-/-/sexual

-/-/sexual

-/-/sexual

82/diploid/ Sexual

164/tetraploid/

164/tetraploid/

82/diploid/

82/diploid/

123/triploid/

-/-/sexual

-/-/sexual

-/-/sexual

-/-/sexual

-/-/sexual

-/-/sexual

TNgP1072

TNgP1439

TNgP1458

TNgP1459

TNgP1462

TNgP1463

TNgP1379

TNgP1380

TNgP2282

TNgP2283

TNgP2287

TNgP1444

TNgP1705

TNgP1706

TNgP 2282

TNgP 2287

TNgP 2289

Secondary foreest, Petak 55, Desa KarangMangu, Mt. Slamet, Wana WisataBaturaden, Central JavaCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, West Java,Southern SlopeCangkuang Forest, G. Salak, Southern slope,West JavaCangkuang Forest, G. Salak, Southern slope,West Ja va,Cangkuang Forest, G. Salak, Southern slope,West JavaTrack to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West JavaTrack to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West JavaTrack to Curug Seribu, Taman Wisata AlamGunung Salak Indah, G. Salak, West JavaCangkuang Forest, G. Salak, West Java,Southern SlopeCikaniki Forest, Halimun National park,West Java. 1000 m.Cikaniki Forest, Halimun National park,West Java. 1000 m.Curug Seribu, Gunung Salak Indah, G salak,West Java. 940 mCurug Seribu, Gunung Salak Indah, G salak,West Java. 940 mCurug Seribu, Gunung Salak Indah, G salak,West Java. 940 m

58

Table 5.1. continued

Species ChromosomesNumbers (2n)/ PloidyLevel/ Reproductive Type

VoucherSpecimens

Locality

D. subserratum -/-/sexual

-/-/sexual

TNgP 2290

TNgP 2294

Curug Seribu, Gunung Salak Indah, G salak,West Java. 940 mCurug Seribu, Gunung Salak Indah, G salak,West Java. 940 m

D.subvirescensPraptosuwiryo

123/triploid/apogamous

123/ triploid.apogamous

TNgP 1177

TNgP 1013

Cibodas forest, behind Cibodas BotanicGardens, Mt. Gede, Gede-PangrangoNational Park, West Java.Cibodas forest, behind Cibodas BotanicGardens, Mt. Gede, Gede-PangrangoNational Park, West Java.

D. tomentosum 82/diploid/

164/teraploid

205/pentaploid/

TNgP2336b

TNgP2066

TNgP1722

Cikuda-Paeh, Mt. Halimun, West Java. Ca.1300 m.Near Kali Talun – Air Terjun talun, BukitPal, Taman Nasional Bukit Dua Belas.Ca.70 m.Loop Trail, Cikaniki, Mt. Halimun, WestJava. Ca. 1000 m.

D. umbrosum 82/diploid/ TNgP1348 Cibodas forest, behind Cibodas BotanicGardens, Mt. Gede, Gede-PangrangoNational Park, West Java. ±1400 m

D. xiphophyllum 82/diploid/-

±164/tetraploid/-

246/hexaploid/-

164/tetraploid/-

164/teraploid/-

TNgP 1841

TNgP2040b

TNgP1190

RI 867

TD902

Track to Batu Ayau, Peg. Muller, CentralKalimantan. Ca. 280 mRawa Edam, Sukodibeyung SecondaryForest, in TNBD Sumatra.Hutan Cangkuang, G. Salak, West Java. Ca.1200 m.Malampah Nature Reserve, Pasaman Barat,West Sumatra. 950 m dpl.East Kalimantan

D. wahauense 164/tetraploid/ 1972c S. Belitung, Peg. Muller, KALTENG. 150 m.

Diplazium batuayauense Praptosuwiryo, sp. nov. D. batuayauense has

two ploidy levels, tetraploid (2n=164) and pentaploid (2n = ca. 205 ). The two

cytotypes were found on the relatively same habitat. They were growing on

humus rich soil and on rather shady places between the altitudes 440-450 m.

Diplazium cordifolium Blume. There are two kind of morpholodical

types in D. cordifolium, viz. simple frond and pinnate frond. Four ploidy levels

are found in the simple fronds of D. cordifolium. They are tetraploid, pentaploid,

hexaploid (TNgP 1201), and octoploid (TNgP1926b). The tetraploid (TNgP 1203)

was found in Mt. Salak (Ca.1200 m). Two individual pentaploid were found in

Mt. Salak at ca. 1200 m (TNgP 1204) and Mt. Halimun at ca. 950 m (TNgP

1808). The hexaploid and octoploid were growing at Mt. Salak (Ca. 1200 m) and

59

Mts. Muller (Ca.450 m), respectively. While, in the imparipinnate fronds are

found to have tetraploid (TNgP 1194, 1375, 1376) and pentaploid (TNgP 1307,

1355, 1442) from Mount Salak. From Mount Halimun, imparipinnate fronds are

also found to be tetraploid (TNgP 1735, 1736, 1774) and pentaploid (TNgP 1813).

Cytological observation of this species from Malaya reported by Manton (1954)

revealed 2n= >200, might be pentaploid and was not known whether ‘simple’ or

‘imparipinnate’ fronds. Cytological report from Java by Praptosuwiryo &

Darnaedi (2004) gave information concerning existence of the ‘imparipinnate’

tetraploid of this species in G. Patuha and those ‘simple’ pentaploid in Selabintana

G. Gede-Pangrango. The occurance of different morphological characters of

(simple and imparipinnate at the same ploidy level) bring us to the questions

whether they have different ancestor diploid races. Further cytological study is

strongly recommended.

Diplazium crenatoserratum (Blume) Moore. There are two levels ploidy

reported in this study, triploid (2n = 123) and tetraploid (2n=164). The triploid

plant was from Bukit Suban Punai Banyak, Bukit Dua Belas National Park

(Sumatra), minewhile the tetraploid plants were collected from the forest near

Tomoron Water Fall (Muller Range of Central Kalimantan) and Bukit Dua Belas

National Park (Sumatra). The all cytological records of this species are new

information for science .

Diplazium dilatatum Blume. Six individuals of Diplazium dilatatum

collected from West Java and Central Java showed apogamous triploid.

Cytological observations of this species from Gede-Pangrango National Park Wes

Java by Praptosuwiryo and Darnaedi (1994) found some apogamous triploid and

one individual tetraploid. In Japan, two varieties of D. dilatatum has been found,

viz. D. dilatatum Bl var. dilatatum and D. dilatatum Bl var. heterolepis Seriz.

The first variety was reported to have diploid and triploid, while the second

variety is only have triploid race (Takamiya et. al. 1999).

Diplazium donianum (Mett.) Tardieu. Only one plant of D. donianum

successfully examined its chromosome number. This plant is cultivated at Bogor

Botanic Gardens (collected from Bukit Ubar, Sumatra) and showed tetraploid (2n

= 164). Apogamous tetraploid was reported from Japan (Kato 1995). In Japan,

60

Kato (1995) reported D. donianum var. aphanoneuron (Ohwi) Tagawa with

chromosome number 2n = 123 (triploid). This variety is differ from var.

donianum in: lamina thicker and veins invisible beneath. West Malesian D.

donianum has characters: thin lamina and vein visible beneath (Figure 6.6.c.,

Chapter 6). Thus West Malesain plants are D. donianum var. donianum.

Diplazium esculentum (Retz.) Swartz This species usually grows in

opened areas with wet soils and distribute from Tropics of Asia to Oceania.

Mature plants are found to be pinnate and bipinnate. Veins anastomousing with a

few veins from adjacent veins joining into excurrent ones below sinuses between

lobes. Two individuals collected from two localities (Mt. Halimun and Mt. Gede)

showed 2n=82, diploid. Cytological information of this species from eight

localities (Malaya, Ceylon, North and South India) from 1954 to 1970 revealed

diploid without giving type of reproduction information (Love et. al., 1977).

Takamiya et. al. (1999) was also reported sexual diploid D. esculentum of

Shigetomi, Japan. Tetraploid D. esculentum is only found in India (Bhavanandan

& Amal, 1991). It is indicate that diploid D. esculentum is distributed in broad

range.

Diplazium halimunense Praptosuwiryo, sp. nov. There is only one

individual of D. halimunense succesfully examined (TNgP 2341b) and showed 2n

= 123 (triploid). This species is very rare, therefore only two individuals that can

be collected in Mt. Halimun. This species was found growing among the small

populations of D. bantamense and D. donianum. Morphologically, Diplazium

halimunense seems to be intermediate of D. bantamense and D. donianum.

Diplazium hewittii (Copel.) C.Chr. Onle one ploidy level from one plant

succesfully examined, triploid (2n = 123, TNgP 1913b), from Muller Range,

Central Kalimantan. There is no cytological report of this species up to this

present study. This psecies has a wide range of frond morphology, from bipinnate

to tripinnate. Therefore further cytological examination and mode reproduction

type study of this species are needed.

Diplazium profluens Praptosuwiryo, sp. nov. One individual of D.

profluens showed tetraploid (2n=164). The recent survey (in September 2003),

around Cikaniki Station Research, Halimun National Park, West Java, succeed to

61

find this species in shadowed place in the small river bank at track Macan and

track Canopy Bridge.

Diplazium loerzingii Praptosuwiryo, sp. nov. The recent living plants of

this species are found in Mt. Halimun (West Java) and Bukit Tapan (Jambi,

Sumatra). Two individuals from Java showed diploid (TNgP 2339c) and triploid

(TNgP 2339d). This species may have affinity to D. malaccense. This species

differs from D. malaccense in the following characters: lower base of 1-2 pairs

basal pinnae less cut, base of lower pinnae almost equally truncate, texture thicker

or subcoriaceous, upper rachis much gemmiferous, lobes truncate, sori medial on

veinlets or close to margin, attachments sides of indusia darker.

Diplazium pallidum (Blume) Moore. Three individuals of D. pallidum

observed from three different localities from Java showed two ploidy levels. Two

individuals which collected from mountain rainforest above the elevation 1100 m

showed tetraploid level, whereas one individual from lowland rainforest, viz. Mt.

Payung, Ujung Kulon National Park, with the elevation ca.200 m showed diploid

level. The diploid race is very scarcely occurred. Most of individuals of D.

pallidum collected from mountain forest above the elevation 1,200 m from three

localities in Java showed tetraploid level (Praptosuwiryo & Darnaedi, 1994;

Praptosuwiryo & Darnaedi, 2004).

Cytological examination of D. pallidum from Taman Nasional Bukit Dua

Belas (TNBD) in Sumatra and Bukit Batikap, Muller Range Central Kalimantan

has diploid level. These plants grow at 70 – 90 m in TNBD Sumatra and at 200 m

in Muller Range These facts support the hypothesis explained that diploid level of

D. pallidum habits at lowland under 200 m alt.

Diplazium petiolare C. Presl. Two individuals of Diplazium petiolare

collected from Rimbo Panti Nature Reserve, West Sumatra, showed diploid (2n =

82). This cytological account is a new record for Malesian ferns.

Diplazium porphyrorachis (Baker) Diels. Only one individual of D.

porphyrorachis succed its chromosomes counting, viz. tetraploid (2n=164). This

plant (TNgP 1885) was found in Gunung Pumpung Sapat, Muller Range (Central

Kalimantan). This is a new cytological account for Malesia. There is no

cytological report up to this present research.

62

Diplazium procumbens Holttum. Two individuals from two different

localities in, G. Salak and G. Gede, showed triploid only (TNgP 1348 and TNgP

1312). Formerly report by Praptosuwiryo & Darnaedi (2004) from four

localities in West Java and Eas Java also showed 2n=123, triploid. There is no

report of diploid D. procumbens. Cytological observation of this species from

Malaya (Manton 1954) and Ceylon (Manton & Sledge 1954) were also revealed

2n = 123, triploid apogamous.

Diplazium riparium Holttum. Two cytotypes of this species were found,

namely diploid (2n=82) and triploid (2n=123). The two cytotypes were collected

from the same island (Borneo) and in different locality. The diploid one was

collected from tract to Sungai Sopan-Bukit Batikap (Mts. Muller) at ca. 240 m

a.s.l. whereas the triploid from track Batu Ayau (Mts. Muller) at ca. 280 m a.s.l.

Since described by Holttum (1940), there is no chromosome observation for this

species. Thus, the two cytotypes are first record for science.

Diplazium silvaticum (Bory) Swartz. There are two varieties in D.

silvaticum, var. silvaticum that have lanceolate pinnae and var. pinnae-ellipticum

(new variety proposed in Chapter 9) those have elliptical pinnae. The first variety

revealed tetraploid and found in ‘nature habitat’ of the Bogor Botanic Gardens.

Whereas the second variety is triploid and collected from the lowland forest of

Sumatra (ca. 20 m). Manton & Sledge (1954), reported this species from Ceylon

to have ca. 200, while Manton (1953) also reported 2n= ca. 205 from Ceylon.

Abraham et. al. (1962) gave information 2n=205 from South India. Thus,

triploid cytotype was first record for science.

Diplazium simplicivenium Holtt. D. simplicivenium, in a glance, similar to

D. dilalatum. As stated by Holttum (1940) this species differs from D. dilatatum

in the following combination characters: scales wider and longer of coarser

texture; veins all single (a few may be forked on transition pinnae near apex of

fronds but not on the typical larger pinnae, even of large frond); usually not more

than 5 pairs of veins; sori occupying ¾ or longer of the length of veins. Three

individual of D. simplicivenium from three localities proved to be 2n=123,

triploid. Cytological observations of the species outside of Java were also showed

63

triploid, viz. In Malaya (Manton, 1954), Ceylon (Manton & Seldge 1954), and

New Delhi (Bir, 1969).

The rough morphological similarity between D. simplicivenium and D.

dilatatum are sometimes cause difficulties to diagnose the two species fast in the

field. Connecting to the same of chromosome numbers, ploidy level, and the

similarity of morphological appearances, the two species presumable involve in

the complexity of their species relationship.

Diplazium sorzogonense Presl. All plants examined from Mt. Salak and

Mt. Halimun revealed diploid. Two plants from Mt. Salak and Mt. Halimun were

sexual diploid. Morphological examination of both herbarium specimens

deposited at BO and living plants growing in Mt. Salak and Mt. Halimun revealed

that this species is varying in size and in the degree of lobing of its pinnae.

Diplazium speciosum Blume . One of the interested matter of D.

speciosum is its morphological variation of fronds. Therefore, two varieties of this

species have been described, viz. D. speciosum var. speciosum and D. speciosum

var. major. The two varieties can be recognized by the differences of its lobes and

venations. D. speciosum var. speciosum has lobes moderately toothed, end

rounded; veinlets 7-9 pairs, simple, While, D. speciosum var. major has lobes

strongly toothed, end mostly acute; veinlets 10-16 pairs, mostly forked.

However we do not find the differences of the two varieties based on

choromosome numbers. All individuls succesfully examined, include the two

varieties, revealed 2n=82, diploid. Three individuals belonging to D. speciosum

var. speciosum showed sexual type. While two individuals included in D.

speciosum var. major were not recognized its reproduction type.

Diplazium spiniferum Alderw. All three plants collected from G.

Pumpung Sabat (Muller Range, Central Kalimnatan) examined are diploid (2n =

82) and one of them showed sexual. There are no cytological record and mode

type reproduction information for D. spiniferum until this present observation. D.

spiniferum is one species of Diplazium that can grow on limestones area of

mountain forest at altitude 100-1300 m above sea level of Borneo. Stipe dark

green when living with sharply spine and rounded scales are characters that

64

differentiate this species from other bipinnate species of Bornean Diplazium.

The fronds are in varying and usually from bipinnatifid to bipinnate.

Diplazium subserratum (Blume) Moore. Previous cytological reports of

D. subserratum is not known until this research conducted. Four individulas of D.

subserratum from two localities of Mt. Slamet and Mt. Salak showed three ploidy

level: diploid, triploid and tetraploid. Diploid revealed sexual, while those

tetraploid are not known, and those probably sexual of triploid race presumed to

be apogamous.

Diplazium subvirescens Praptosuwiryo, sp.nov. Two indvidual plants

collected from Mt. Gede showed triploid (2n = 123, TNgP 1177 and TNgP 1013).

This species may closely related to D. virescens that distributed in Japan, Korea,

Taiwan, China and Indochina.

Diplazium tomentosum Blume. Three levels ploidy obtained from

cytological observation, viz. Diploid (2n = 82, Mt. Halimun, Java), tetraploid (2n

= 164, Bukit Pal Taman Nasional Bukit Dua Belas, Sumatra) and pentaploid

(2n=205, Mt. Halimun, West Java). Diploid and pentaploid race are new

cytological information for science. Cytological observation of this species from

Ceylon (Manton & Sledge 1954) showed only tetraploid and those from Fraser’s

Hill Malaya (Manton 1954) reported n = 82. The existence of diploid type and

also the higher ploidy level in this species presumed that Malesia, especially Java,

is the center origin for D. tomentosum.

Diplazium umbrosum (Smith) Bedd. Diplazium umbrosum is belonging to

the Diplazium species group with bi-tripinnate leaves. Its segments mostly 3.5-5

mm wide, one basal basiscopic lobes the largest, to 10 by 6 mm, apex blunt or

truncate, margin crenate or lobed 1/3 way to costulet of segments; texture

herbaceous; vein pinnate in each segments 4-6 pairs, mostly forked once in each

crenations, on larger crenation pinnate 2-3 pairs of second veinlets. Sori

elongated from near costulet of segments or on middle veinlets. Cytological

investigation revealed that there is no differences with the former information

from Java by Praptosuwiryo & Darnaedi (2004) collected from Mt. Welirang and

Mt. Patuha), in this recent paper one individual of D. umbrosum from Cibodas,

Mt. Gede, showed 2n=82, diploid. Other cytological observation from Europe by

65

Manton in Jermy (1964) gave 2n=ca.82, diploid also, under the name D.

caudatum (Cav.) Jermy sensu stricto (synonim of D. umbrosum).

Diplazium xiphophyllum (Bak.) C.Chr. Diplazium xiphophyllum is pinnate

species group. Its pinnae 9 pairs, terminal one like the rest; texture thin, drying

light brownish. Pinnae elliptical to 26 cm long, 4 cm wide, narrowed gradually to

slightly unequal cuneate base, ubrubtly to acuminate-caudate apex, margin entire

to irregularly toothed throughout. Veins in small group, at about 55o to costa,

commonly of one basal pairs and one central veins which is forked 1-3 times,

scarcely 4 times, sometimes acroscopic basal veins anastomousing with

basiscopic basal veins of the nearest vein group or with the nearest branch of

central vein near margin.

Three level ploidy for D. xiphophyllum are reported (diploid, tetraploid

and hexaploid). The finding of diploid and hexaploid race from Borneo (Batu

Ayau of Muller Range, Central Kalimantan) and Jawa (Mt. Salak), respectively,

are new record for science. Meanwhile the tetraploid race is new record for Java

and Sumatra. Formerly research was reported tetraploid race from Taiping Hill

(Manton 1954). As stated by Holttum (1966) and this study (Chapter 2 and 9), D.

xiphophyllum commonly grows in lowland forest and valley forest of moderate

mountains. The hexaploid race was found at 1200 m a.s.l. While, in Malaya D.

xiphophyllum was recorded at 914 m s.l. Two individuals tetraploid plant from

Sumatra, TNgP2040b and RI 867, were collected from Jambi at 55 m and from

West Sumatra at 950 m, respectively. It appeared that diploid and tetraploid race

of D. xiphophyllum exist at lower altitude than the hexaploid. This cytological

study on D. xiphophyllum showed a common phenomena that ploidy levels has

any correlation to habitat gradient. .

Diplazium wahauense Kato, Darnaedi et K. Iwatsuki. Diplazium

wahauense was firstly described by Kato et al. (1991) based on specimen

collected from along Jenta River, north of Muara Wahau, East Kalimantan. Since

that time, there is no cytological record for this species up to this present

research.. Due to the difficulties on maintaining the living collections of this

species, of the four collections number collected from Muller Range (Central

66

Kalimantan), there was only one collection that was successfully examined its

chromosome number, viz. TNgP 1972c. It was tetraploid (2n = 164).

5.3.2. The Relationship between Ploidy level and Morphological Variation within Species and Closely Related Species of Diplazium

Summary of the ploidy levels of Diplazium from West Malesia is

presented in Table 5.2. This study showed that intraspecific diversity on West

Malesian Diplazium are high enough. Twelve species of the 31 species

successfully examined are having series ploidy. Thirteen species showed only

polyploid race, from triploid and tetraploid. Whereas nine species revealed only

diploid race. The relationship between ploidy level and morphological variation

within species and closely related species are discussed below.

Diplazium accedens. All species of D. accedens examined are showing

diploid. Nevertheles, morphological variation exist. Three individual of

collections number TR 53 from Sumatra are showing very different appearances:

Roots are not bearing buds, stipes sharply spiny, vascular bundles of stipes

transversal sections near blade showing interupted U shape, rachis not

gemmiferous, basal pinnae much reduced. While collection number TNgP 1001,

1211, 1399, 1447, 1649, and 1786, from Java bearing buds both on roots and

rachise, stipes bearing green protuberances after falling scales, vascular bundles

of stipes transversal sections near blade showing continued U-shape, basal pinnae

slightly reduced.

Diplazium accedens is distributed throughout Malesia and grows well on

moist ground by stream or in humid in evergreen forest, by preferences more or

less shadowed localties, at 60-1550 m altitude. In very large fronds there are extra

areoles between the normal groups adjacent to the main lateral veins; a specimens

having this character formed the basis of Athyrium ridleyi Copeland. Having

opportunity to examine the type specimen of A. ridleyi deposited at SING (Ridley

13970) I agree with Holttum (1966) who also included it in the present species. In

my present field work in 2006 at Bukit Tapan, in Kerinci Seblat National Park

Sumatra, I found the plants similar to the type specimen of A. ridleyi.

67

This species is closely related to D. proliferum (Lam.) Thouars, therefore

the two should perhaps be united (Holttum, 1940). Andrews (1990) placed this

species in the genus Callipteris as Callipteris prolifera (Lam.) Bory and in his

synonymy includes D. proliferum (Lam.) Kaulf., Asplenium decussatum Sw., D.

accedens Bl., and D. proliferum var. accedens (Bl.) v.A.v.R.

As stated by Holttum (1940), the name D. accedens is a little complicated.

The three names of D. accedens, D. repandum, and D. Swartzii, all published in

the same book by Blume (1828), are to be regarded as synonymous. Later authors

have regarded all as synonyms of Lamarck’s earlier name, or of Asplenium

decussatum Sw.; the names Swartzii and accedens have been taken up and used

in the genera Callipteris, Asplenium and Athyrium, but the name D. repandum

appears to have been almost or entirely ignored. Backer & Posthumus (1939),

however, have revived the name D. repandum, apparently on grounds of page

priority (though they do not state this) which is not admitted by Rules. In their

synonymy, however, they include Diplazium proliferum Thouars, an older name;

their use of the name D. repandum is therefore contrary to the rules.

Diplazium angustipinna. Two cytotypes of D. angustipinna (triploid and

tetraploid) do not show any morphological differents. It is presumed that the

mechanism conctrolled is autopoliploidi.

Diplazium bantamense. Diplazium bantamense is usually found in moist

shady forest, chiefly in the hills, sometimes near streams in lowland forest. All

individuals examined here cytologically were collected from highland forest.

Assuming that most of diploid are generally living in tropical area diploid race

probably occur in the lowland. Examinations of many living collection grown in

Bogor Botanic Gardens nursery and dried specimens housed at BO revealed that

fronds of this species have enough variations on both leaf shape and size of

pinnae. Praptosuwiryo & Darnaedi (1994) reported four individuals of the

tetraploid sexual and two octoploid sexual of D. bantamense from Gunung Gede-

Pangrango National Park, West Java. However the features distinguishing the

tetraploid and octoploid type are unclear. Further examination on chromosome

number and its mode reproduction type from all of the range of its habitats,

including from lowland, would clarify their variations.

68

Diplazium cordifolium. Species concept of D. cordifolium stated by

Tagawa dan Iwatsuki (1988) covers individuals having simple frond narrowly

oblong-subdeltoid, cordate at broadest base, narrowing upwards towards

acuminate apex, subentire to undulate at margin and individuals having

imparipinnate fronds with a few pairs of lateral pinnae becoming smaller

upwards, lateral pinnae sessile, bearing gemmae at junction between rachis and

costa. Tagawa & Iwatsuki (1988) treated all morphological variations, the simple

fronds (D. cordifolium Blume) and the pinnate fronds (D. integrifolium Blume),

as one entity, viz. D. cordifolium Blume. The cytological evidence of this

research supports in differentiating D. cordifolium Blume from D. integrifolium

Blume. The simple fronds plants have one serial ploidy from tetraploid,

pentaploid and hexaploid, meanwhile those simply pinnate fronds plants reveal

tetraploid and pentaploid. Observation on living plants revealed that those simple

fronds never change into the simply pinnate fronds. However some characters of

the two groups, such as scales, lamina texture, and venation, are similar.

Mitsuta (1985) recognized two varieties of Sumatran D. cordifolium, var.

integrifolium (Bl.) Mitsuta and var. pariens (Copel.) C.Chr. The two varieties are

differentiated with characters as follow. var. integrifolium has 2-3 pairs of lateral

pinnae, and base of terminal pinnae sessile, while var. pariens with 4-6 pairs of

lateral pinnae, and base of terminal pinna usually wide cuneate.

Cytological observation showed that the simple fronds revealed a series

ploidy (2n=4x, 5x, dan 6x) and those imparipinnate aslo have a series ploidy

(2n=4x dan 5x). Morpholigical observation in the living collections revelaed that

the plant with simple fronds did not change into the pinnate fronds. Further

morphological differences are provided in the identification key in the Chapter 9.

This study showed that genetic variation within D. cordifolium are varying and

has brougth out into the morphological variation. The molecular evidence from

gene rbcL sequence also supported this results (Chapter 8).

Diplazium crenatoserratum. As also reported by Holttum (1940), D.

crenatoserratum is a very common fern of lowland forest. This species shows

much variation in size and in the degree of lobing of its pinnae. Small plants with

blunt entire pinnae may be fertile. The tetraploid type of Tomoroh Water Fall

69

(Muller Range, Central Kalimantan) has small size with blunt entire pinnae while

the plants with larger size and pinnae more incision, margin lobed ¼-1/2 way

towards costa, revealed triploid and tetraploid. Therefore it is presumed that there

are no morphological differences between triploid and tetraploi. However further

cytological observation of this species for all its range distribution are needed to

clarify this preliminary study.

Diplazium dilatatum. In Japan, two variaties of D. dilatatum have been

recognished, viz. D. dilatatum Blume var. dilatatum dan D. dilatatum Blume var.

heterolepis Seriz. (Kato, 1995). The first variety are diploid and triploid, while the

second variety is only triploid (Takamiya et. al. 1999). The triploid race of

Javanese species presumed as var dilatatum, due to morphological similarity with

those of Japanese species. Kato (1995), stated that D. dilatatum var. dilatatum

differs from D. dilatatum var. heterolepis in the scales cahacters. D. dilatatum

var. heterolepis has scales at the stipe base lanceolate, to 20 mm long, black at

margin. While D. dilatatum var. heterolepis has scales at stipe broadly lanceolate,

10-15 mm long, 1-3 mm broad, hardly black at margin. Javanese plants match to

the D. dilatatum var. dilatatum, because its scales characters similar to this

variety, viz. liniery lanceolate, 15 mm long or more, 1 mm wide, yellowish brown

at middle, black and sharply toothed at margin. One individual of the tetraploid

and thirteen individuals of triploid of D. dilatatum from West Java were reported

by Praptosuwiryo & Darnaedi (1994). But, morphological differences between the

two cytotypes are not significant.

Diplazium pallidum. Morphologically, the appearances of the two ploidi

level, diploid and triploid, showed much differences. The tetraploid race showed

following character spots: terminal pinna deltoid and deeply lobed; upper base of

lateral pinnae broadly truncate, lower base narrowly rounded. Whereas the

diploid race has following characters spots: terminal pinna conform to lateral with

one or two lobes; upper base of lateral pinnae rounded, lower base cuneate.

Further examination to see the conssitence of the morphological

differences between the two race of D. pallidum (diploid dan tetraploid) is needed

by examining much more sample from different altitude. In addition, examination

70

of reproduction types, spore mophology, anatomy of the fornds, and isozyme or

DNA analysis would give clarification of the taxonomical status of the two race.

Diplazium silvaticum. There are morphological distinct among the triploid

types and tetraploid types. Tetraploid plants have pinnae 8-13 pairs, lower pinnae

lanceolate, 7-15 x 1.6-3 cm, upper base subtruncate, lower base cuneate.

Meanwhile the triploid ones have pinnae 2-5 pairs, lower pinnae elliptic, 3.3 -5.4

x 2.0-2.7 cm, upper base subrounded, lower base cuneate. Those tetraploid are

sexual, while the triploid are apogamous. It is suggested that the triploid was

originally hybrid. Further studies are needed.

Diplazium subserratum. The differrences of ploidy level is presumed to

be correlated to its morphological variations. The results of the morphological

comparison between diploid (TNgP 1463) and tetraploid (TNgP 1379) is showed

in the following characters: The diploid has stipe 1.5 mm diam., length 15 cm;

lamina 34 cm length, 3.1 cm wide (in the middle), margin entire; veins forked 2-3

times. While those tetraploid has stipe 1.5-2 mm diam., 13.0-16.5 cm length;

lamina 15-29 cm, 2.0-4 cm wide (in the middle); margin 1/6-2/3 portion of base

entire and waved 1/3 – 5/6 portion upper; veins forked 2-4 times. The correlation

between the ploidy level, type of reproduction, and morphological appearance of

this species seem very interesting from the point view of speciation in Diplazium.

Therefore more cytological observations of this species over its range areas of

distribution are needed to verify the correlation between ploidy levels and

morphological differences.

Diplazium tomentosum. 82/diploid/ (TNgP 2336b) ; 164/teraploid (TNgP

2066); 205/pentaploid/ (TNgP1722). There are not any significant qualitative

characters differences among the three cytotypes of D. tomentosum. However

they have several differences in quantitative. The diploid type has lamina up to

21.5 cm by 10 cm, pinnae up to 14 pairs; veinlets on the largest lobe of basal

pinna up to 5 pairs, mostly simple. Tetraploid type has lamina up to 24 cm by 15

cm, pinnae up to 17 pairs; veinlets up to 6 pairs, mostly simple. Whereas the

pentaploid type has lamina up to 32 cm by 17 cm, pinnae to 19 pairs; veinlets on

the largest lobe of basal pinna up to 8 pairs, mostly once forked. These results

indicate that the ploidy seri in this species is autoploid.

71

Diplazium xiphophyllum. Three voucher spesimens of the three cytotypes

(diploid, tetraploid and hexaploid) could not be made comparison because they

are in different stage of growth. However qualitatvely the three cytotypes are not

different. Therefore it is presumed that they are autoploid.

Diplazium aequibasale, D. riparium and D. wahauense. Diplazium

aequibasale (2n=164), D. riparium (2n=82) and D. wahauense (2n=164) almost

have similar morphological appearances. Diplazium aequibasale have allied form

between D. riparium and D. wahauense. D. wahauense is closely related to D.

riparium. Kato et.al. (1991) presumed that D. wahauense derived from D.

riparium which occurs in riparian and dryland forest in Borneo. The two species

share many character, viz. black, somewhat crisped, entire scales, blackish stipes,

dark-brown, naked rachis, and imparipinnate leaves with 3-4 pairs of entire lateral

pinnae. D. wahauense differs from D. riparium mainly in its narrow pinnae,

which are characteristic of rheophytes.

5.3.3. Relationship between ploidy level and habitat gradient

Although data are available for fern floras from different parts of the

wolrd, there is not simple relationship between ploidy level and habitat (Walker

1979). Some studies have revealed that lower-ploids occur in warmer habitats

than higher-ploid, for example Lepisorus thunbergianus (diploid vs. Tetraploid)

(Mitui et al 1987), Woodwardia orientalis (diploid vs. tetraploid) (Mitui 1968),

Dryopteris erythrosora (diploid vs triploid) ( Hyrabayashi 1974), Pteris dispar

(diplaid vs tetraploid) (Nakato 1981), and Diplazium nipponicum (triploid vs

tetraploid) (Takamiya et al 2000). However, many parts of Europe all three

cytotypes (2x, 3x, 4x) of the Polypodium vulgare complex were found in close

proximity (Shivas 1961). Matsumoto (2003) did not find a significant relationship

between latitude and ploidy level in the Cyrtomium falcatum complex. However

there was an association between ploidy level and habitat: diploid grew on sea

cliffs or in dry forests, triploid grew at forest edges, and tetraploid grew in wet

forests. In the study of the Pteris fauriei complex in Taiwan, Huang et al. (2007)

recorded any correlation between habitats, latitude, and elevation of P. fauriei.

72

The three parameters are related to temperatures. In general, triploid plants grow

in low temperature sites than diploid plants.

In many species of Diplazium from West Malesia, however, there are not

any strick correlation between ploidy level and habitat gradient. All species with

only diploid type, viz. D. accedens, D. petiolare, D. polypodioides, D, speciosum,

D. sorzogonense, D. spiniferum and D. umbrosum, are growing from 420 m to

1500 m. Species with two different ploidy levels are growing on almost the same

elevation. The tetraploid D. crenatoserratum were collected from 20 – 150 m sea

level where the triploid one was also found in the range, 55 m sea level. Triploid

and tetraploid D. angustipinna were found at the same altidude, 440 m. In

contrary, pentaploid D. batuayauense was growing at 440 m, whereas the

tetraploid plant was found at 450 m. Overlap distribution was seen in D.

cordifolium. Tetraploid D. cordifolium was growing from 240 m to 1200 m,

whereas pentaploid plants were occuring from 1000 m to 1200 m. As has been

mentioned above, there are only few species that has a correlation between ploidy

levels and habitat gradient, such is D. riparium and D. xiphophyllum.

Some species of West Malesian Diplazium showed a correlation between

ploidy level and altitute gradient. Diploid D. riparium was found at 150 m,

whereas the triploid plant was at 280 m. Tetraploid plants of D. pallidum were

only found above 1000 m, whereas those diploid were growing from 30 m to 250

m. Triploid and tetraploid D. silvaticum are growing at ca. 20 m and ca. 280 m,

respectively. The hexaploid D. xiphophyllum was found at higher altitude than

those of tetraploid and diploid.

5.3.4. Correlations between reproductive mode and habitat

Some studies have found distinct correlation between reproductive mode

and habitat (Holbrook-Walker & Lloyd 1973; Lloyd 1974). Sexual reproduction

is most frequent in moist habitats, whereas apomictic reproduction is more

common in ferns growing in xeric habitats (Tryon 1968). However, some

apomictic species do not live in xeric habitats, for example some Japanese

Diplazium (Takamiya et al 1999) and Cornopteris christenseniana (Park & Kato

2003).

73

Table 5.2. Polyploid series of Diplazium in West Malesia Based on Present Study

Ploidy Level

Species

2X 3X 4X 5X 6X 8X

D. accedensD. aquibasaleD. asymmetricumD. bantamenseD. batuayauenseD. cordifoliumD. crenatoseratumD. dilatatumD. donianumD. esculentumD. halimunenseD. hewittiiD. loerzingiiD. pallidumD. petiolareD. polypodioidesD. procumbensD. profluensD. porphyrorachisD. ripariumD. simpliciveniumD. silvaticumD. speciosumD. procumbensD. sorzogonenseD. subpolypodioidesD. spiniferumD. subserratumD. subvirescensD. tomentosumD. umbrosumD. xiphophyllumD. wahauense

V-------

-V - -VVVV---V--V-VVVV-VVV-

--V---VV

--

V V

V---V--VVV-V---VV----

-V-VVVVV*

V-

- -

-V---VV--V-----V-V-VV

----VV----

- -

-----------------V---

-----V--

--

- -

-------------------V-

---

V*-V--

--

- -

---------------------

TOTAL 15 13 15 3 2 2

Notes:

V Present study

V* Praptosuwiryo & Darnaedi (1994)

74

Figure 5.1. Somatic chromosome of Diplazium. a. D. accedens var. accedens , 2n = 82 ;b. D. accedens var. spinosum, 2n = 82; c. D. angustipinna, 2n = 164 (TNgP 1906b); d. D.angustipinna, 2n = 123; e. D. spiniferum (TNgP 1896a), 2n = 82; f. D. halimuense, 2n=123(TNgP 2341b). Bar = 3 m.

75

Figure 5.2. Somatic chromosome Diplazium. a. D. umbrosum, 2n = 82 (TNgP1348);b. D. petiolare 2n = 82 (TT993-3), c. D. tomentosum, 2n = 82 (TNgP 2336b);d. D. tomentosum, 2n = 205 (TNgP1722); e. D. tomentosum, 2n = 164 (TNgP2066);f. D. asymmetricum, 2n=123 (TNgP 1094). Bar = 3 m.

76

Figure 5.3. Somatic chromosomes of Diplazium. a. D. xiphophyllum (TNgP), 2n = 246; b. D.xiphophyllum (TD902), 2n = 164; c. D. xiphopyllum (TNgP1841 ), 2n = 82; d. D. batuayauense(TNgP 1927), 2n = 123; e. D. porphyrorachis (TNgP1885), 2n = 164. Bar = 3 m.

77

Figure 5.4. Somatic chromosomes of Diplazium. a. D. subserratum, 2n = 82 (TNgP1463),b. 2n = 123 (TNgP2287), c. 2n = 164 (TNgP1379); d. D. subpolypodioides, 2n = 82 (TNgP2292);e. D. procumbens, 2n = 123 (TNgP1348). Bar = 3 m.

78

Figure 5.5. Somatic chromosomes of Diplazium cordifolium. a. 2n = 164 (TNgP1735);b. 2n = 205 (TNgP1204); c. 2n = 246 (TNgP1201); d. 2n = 328 (TNgP 1926b). Bar = 3 m.

79

Figure 5.6. Somatic chromosomes of Diplazium. a. D. pallidum, 2n = 82 (TNgP1406);b. D. pallidum 2n = 164 (TNgP1764); c. D. riparium, 2n = 123 (TNgP 1847);d. D. polypodioides, 2n = 82 (TNgP2285); e. D. dilatatum, 2n = 123 (TNgP1073); f. D. speciosum,2n=82 (TNgP1359). Bar = 3 m.

80

Reproductive studies on Diplazium from West Malesia showed that both

the sexual and apomictic species can occur in the moist habitats. The apogamous

triploid D. procumbens and D. simplicivenium are found in dryland areas of the

slopes on Mt. Gede-Pangrangrango and Mt. Salak in which have the relatively

high amount of rainfall (ca. 200 mm per month in October - May). The

apogamous pentaploid D. batuayauense was also growing at the moist soil in

shady place in the forest. The sexual and apogamous tetraploid of D. bantamense

are found at the relatively same habitats condition. The two race are found at the

moist humus-rich soil of the dryland areas of Cangkuang forest in Mt. Salak.

These results revealed that there are not distinct correlation betweed the

reproductive mode and habitats in West Malesian Diplazium.

In many ferns reproductive barriers are likely to exist between polyploid

and their ancestors. This increases the potential for the development of their

spatial segregation (Vogel et al 1999). Some researchs demonstrate that

microenvironment (habitat) selection is partially responsible for maintaining the

separation of different cytotypes in ferns and flowering plants (Kumaret et al

1987). In West Malesian Diplazium the separation between polyploid and their

ancestors have not been seen clearly. Beacuse most of species with seri ploidy

have not been examined well their mode of reproductive by sampling many

individual plants. Moreover, the ancestors of the many polyploid species

observed have not been found, their ancestors (diploid types) have not been found

yet. The diploid of D. bantamense, D. simplicivenium, D. procumbens, and D.

asymmetricum, for example, have not been recognized their existence. Therefore

further study on cytology and the mode reproductive of West Malesian Diplazium

to understand its speciation mechanism are needed.

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5.4. Conclussions

All West Malesian species of Diplazium examined showed basic

chromosome number x = 41. New cytological information for science on 19

species are recorded. They are D. aequibasale (tetraploid), D. angustipinna

(triploid), D. asymmetricum (triploid), D. batuayauense (tetraploid and

pentaploid), D. crenatoserratum (triploid and tetraploid), D. halimunense

(triploid), D. hewittii (triploid), D. profluens (tetraploid), D. loerzingii (diploid

and triploid), D. pallidum (diploid), D. petiolare (diploid), D. porphyrorachis

(tetraploid), D. riparium (diploid and triploid), D. spiniferum (diploid), D.

subserratum (diploid, triploid, tetraploi), triploid D. subvirescens, D. tomentosum

(diploid and pentaploid), D. xiphophyllum (diploid, hexaploid), and Diplazium

wahauense (2n=164).

This study showed that intraspecific diversity on West Malesian Diplazium

is high enough. Twelve species of the 31 species successfully examined their

somatic chromosome numbers are having ploidy level series: D. angustipinna

(triploid and tetraploid), D. bantamense (traploid and oktoploid). D. cordifolium

(tetraploid, pentaploid, dan hexaploid, octoploid), D. pallidum (diploid and

tetraploid), D. silvaticum (triploid and tetraploid), D. tomentosum (diploid,

tetraploid, pentaploid), D. subserratum (diploid, triploid, and tetraploid), D.

xiphophyllum (diploid, tetraploid and hexaploid). Thirteen species showed only

polyploid race: D. aequibasale (tetraploid), D. profluens (tetraploid), D.

porphyrorachis (tetraploid), D. procumbens (triploid), D. simplicivenium

(triploid). Whereas nine species revealed only diploid race (D. accedens, D.

esculentum, D. malaccense, D. petiolare, D. polypodioides, D. speciosum, D.

spiniferum, D. sorzogonense, D. subpolypodioides, and D. umbrosum).

Study on the relationships between ploidy level and morphological

variation in some species, such as D. batuayauense, D. crenatoserratum, D.

loerzingii, D. riparium, D. tomentosum, and D. xiphophyllum, suggested that they

are autoploid. Triploid D. silvaticum was presumed alloploid. However further

cytological study are needed to verify the polyploidy mechanisme involved in the

species mentioned above and the others.

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Study on the relationship between ploidy level and habitat gradient

showed that generally West Malesian Diplazium species are showing no any

strick correlation between ploidy level and altitude, but D. pallidum, D. riparium,

D. silvaticum and D. xiphophyllum. Reproductive studies revealed that many

species are apogamous triploid and both the sexual and apomictic species can

occur in the relatively moist habitats.

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CHAPTER 6

PHYLOGENETIC STUDIES OF DIPLAZIUM FROM WEST MALESIA:EVIDENCE FROM MORPHOLOGY

6.1. Introduction

Most of pteridologist thought that Diplazium are quite distinct and should

be separated from Athyrium (See Ching 1940; van Alderwerelt van Rosenburgh

1908; Alston 1956; Sledge 1962; Holttum 1940, 1966; Kato 1977, 1995; Edie

1978; Tagawa & Iwatsuki 1988; Andrews 1990; Kramer et al 1990). Strictly

Kato (1977) showed the differences between Diplazium and Athyrium. In

Athyrium, the stipes bases on ascending to erect rhizome 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 horseshoe- or J-shaped, or linear, scales entire.

Whereas in Diplazium, the stipes bases neither swollen nor bearing

pneumatophores, frond axes U-shaped with flat base in most species, acroscopic

basal pinnules equal or smaller, laminar margin not cartilaginous, spine absent,

sori linear, scales toothed or entire. Moreover the separation of Diplazium from

Athyrium has been supported by both cytological evidence and molecular data.

The different in the basic chromosome number of Athyrium (x= 40) and

Diplazium (x=41) is useful diagnostic character (Tryon & Tryon 1982).

Preliminary phylogenetic study of Diplazium conducted by Sano et al (2000)

based on chloroplast rbcL gene sequences showed monophyletic of this genus.

By using evidence from chloroplast trnL-F region sequences Wang et al (2003)

also supported the monophyletic of Diplazium clade that include Callipteris

Bory, Allantoidea R. Br. Emend. Ching, and Diplaziopsis C. Chr.

Morphological variation among species of Diplazium are very diverse.

But, a natural subdivision of this genus has not been given yet. Van Alderwereld

van Rosenburgh (1908) tried to divide Malayan Diplazium, include those of the

Malay Peninsula,The Philippines and New Guinea, into two sections based on

only the venation type: (1) Eudiplazium for Diplazium species having free veins

and (2) Anisogonium for Diplazium species that possess anastomousing veins.

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Based on the characters such as scales, stipe, lamina and venations, Kato (1977)

recognized Japanese 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; (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; (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; (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; and (5) Diplazium

longicarpun group that includes species with frond pinnate, acroscopic base of

pinna truncate, basiscopic cuneate, adaxial surface of lamina not concave along

veins, scales entire. However the naturalness of the subdivision of Diplazium

circumcribed by van Alderwereld van Rosenburgh (1908) and Kato (1977) have

never been tested and analyzed phylogenetically using morphological datasets.

Therefore phylogenetic studies on Diplazium using morphological datasets

should be introduced.

Most grouping on the tree of life were first inferred using morphological

characters, which have been widely used since Darwin’s time. Most of the

million-plus described species are know only from morphology, and their inferred

phylogenetic positions are based on these characters; with between 90 and 99

percent of life still to be formally recognized, usually initially from morphological

features alone (Lee 2004). Although the pool of morphological characters is

much smaller than molecular characters (See Chapter 8), and often insufficient

for robust phylogentic resolution, is a common view (e.g. Hillis 1987; Givnish &

Sytsma 1997), however morphological datasets often contain as much relevant

phylogenetic signal as typical molecular datasets that have orders of magnitude

more characters (Lee 2004).

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This chapter presents phylogenetic study of Malesian Diplazium by using

morphological datasets generated from sixty nine species from West Malesia. All

data on the characters analyzed here are cited from this present work (Chapter 9).

This study was conduct to address question of phylogenetic relationships among

species within Diplazium. The objectives of this study were to: (1) to reconstruct

the phylogenetic relationships among the species in the genus Diplazium; (2)

identify monophyletic species groups within Diplazium, (3) establish sister-group

relationships among these monophyletic groups.

6.2. Character Selection and Construction

6.2.1. Character Selection

Cladistic analysis is the most common method currently used to

reconstruct phylogenetic trees, even it is the best method (See Bremer &

Wanntorp 1978, Estabrook 1978, Wiley 1980). For generating a set of trees, it

involves two basis phase-exploration of characters (including selection and

examination), followed by analysis of the data (Thiele 1993). Thiele (1993)

described an ideal morphological character as one in which the character states

vary between terminal units in the analysis but not in other members of the unit

(e.g. conspecific individuals) being represented.

6.2.2. Character Type

In the morphological systematic studies, there are two types of characters

used: qualitative and quantitative. Qualitative characters are mostly obtained by

examination without measurement. These characters can be divided into binary

characters and multistate characters. Binary characters comprises two character

states, such as scales margin (without thickening black strands or with thickening

black strands, in character 14, Table 6.1.). Multistate characters consist of more

than two character states such as lamina division that comprise 7 character states

(simple, pinnatifid, imparipinnate, bipinnatifid, bipinnate, tripinnatified, and

tripinnate, in character 23, Table 6.1.). Generally, qualitative characters are more

86

acceptable and seem to be unambiguous in cladistic analysis because their states

are considered to be clearly defined and no overlapping (Kitching et al 1998).

Quantitative characters are obtained by measurement. Generally

quantitative characters are continuously variable. Continuously variable should

only be exclude if the cladistic analysis cannot handle such data or if it can be

shown empirically that those characters convey no information of phylogenetic

signal relative to other characters in the data matrix (Kitching et al. 1998). In this

study quantitative characters are included in the analysis because they convey

information of phylogenetic signal relative to other characters in data matrix.

Another argument for inclusion of quantitative characters is that some qualitative

characters may be a collection or transformation of quantitative characters. For

example. Leaf shape (a qualitative characters) can be defined by ration of leaf

length to leaf width, which is quantitative caharcters (Thiele 1993.). Characters

number 7, 8, 16, 17, 25, 26, 37, 38, 49, 50, and 56 in the Table 6.1. are

quantitative characters that are important and having tight correlation with the

qualitative characters examined. Excluding these characters would give illogical

relationships among the species. Therefore these characters were included in

analyis.

6.2.3. Character Coding

The crucial point in the phylogenetic analysis using morphological

datasets is how features might be usefully coded so as to reflect accurately our

observations for particular scale problem (Kiching et al 1998). Character coding

is the link between observation and explanation (Strong & Lipscomb 1999).

Therefore, ability, or inability, of a coding method to reflect the evidential

significant of observations should be the primary concern in considering

alternative methods of coding.

Basing on the philosophy above, in cladistic analysis of morphological

characters in Diplazium, a conservative approach of ‘composite characters coding’

(Strong & Lipscomb 1999) was employed. Composite coding refers to the

creation of single multistate character from several potentially dependent

character (method ‘A’ in Kitching et al 1998) that designed to minimize the effect

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of character linkage. Kitching et al (1998) stated that the more that characters

become linked, i.e. dependent on other characters, the greater is the departure

from independence and consequently the risk that one false homology can obscure

the topologies of true homologies decreased.

Composite coding applied in this study was also designed to reduce the

error that extensive amounts of missing data (a character state scored as ‘?’) can

create in a parsimony analysis (Maddison 1993). The fronds architecture of

Diplazium are varying, from simple to quadripinnate. Consequently taxa with

simple, pinnate, bipinnate fronds do not posses any organ that possessed by those

with pinnate, bipinnate and tripinnate fronds, respectively.

Character 36. (Table 6.1) is an example of composite characters in this

study. It was created from the combination of two characters: 1) Lateral pinnae

exist/not exist and 2) Shape of lateral pinnae oblong / oblong lanceolate /

elliptical / liniery triangular / oblong subtringular / lanceolate / ovate. These two

characters in which the latter is dependent on the former, were combined into a

single character with eight character states (lateral pinna oblong / oblong

lanceolate / elliptical / liniery triangular / oblong subtringular / lanceolate / ovate/

organ not exist). This method reduced the numbers of linked characters and

limited the influence of multiple missing characters states on the analysis for

previously coded characters ‘lateral pinnae oblong / oblong lanceolate / elliptical /

liniery triangular / oblong subtringular / lanceolate / ovate’ in those taxa which

have no lateral pinnae. Maddison (1993) stated that missing character states can

cause difficulties for parsimony analysis if the missing state for the character

occurs in more than one clade.

6.3. Character Variation within West Malesian Diplazium

Previous pteridologist worked on Diplazium have summarized the

principal characters of the genus Diplazium and identified the important

morphological features in identification and classification. Characters those

particularly variable across the taxa and which may be significant in an

evolutionary sense are discussed as follow.

88

a. Rhizome

Most of West Malesian species posses erect rhizome. The appearances of

the erect rhizome are varying. The very short and small rhizome are seen in D.

crenatoserratum and D. tomentosum. The stout and medium rhizome are found in

D. dilatatum, D.kunstlerii, D. megasegmentum, D. polypodioides, and D.

subpolypodioides. The stout long rhizome is present in D. esculentum. Some

species have sreeping rhizome, such as D. donianum, D. procumbens, and D.

subvirescens Mickel (1974) presumed that the main lines of the ferns begun with

probably creeping rhizome. Thus the erect rhizome is regarded as derived. Figure

6.1. showed the rhizome patterns of Diplazium.

b. Scales and Hairs

Scales in Diplazium are varying. As showed by Holttum (1940, 1966),

Kato (1977, 1995), and Tagawa & Iwatsuki (1988), scales are very important

characters in determining species in Diplazium (Figure 6.2.). The scales mainly

attach on upper of the rhizome and stipes. On some species scales are also

present on the rachis or the costa, but in smaller size than those on the rhizome

and stipes. Scales are generally linear lanceolate to obong lanceolate. Rounded or

ovate scales are present on the projections of a muricate stipe such as in D.

megasegmentum and D. profluens (new species proposed, See Chapter 9), D.

kunstlerii, and D. latisquamatum. Rounded or ovate scales which is initially

having a function as an covered organ of spines are seen on the stipe of D.

spiniferum.

Scales are generally attached with a basal point. Some species with

rounded or ovate scales, such as in D. kunstlerii, D. latisquamatum, D.

megasegmentum and D. spiniferum, have scales with a subbasal point attachment

(Figure 6.2.g. in this Chapter; Chapter 9). Scales with a subbasal point attachment

may be derived. Dickason (1946) presumed that a scales with a basal point of

attachment may be more easily derived from a hair than one with broad base or

one peltately attached.

Diplazium scales are entire or toothed, with tooth consisting of two

upturned ends of adjacent marginal cells. The marginal may be with thickening

brown-black strand or not. Toothed scales may show sharp and blunt teeth,

89

regular or irregular teeth construction, and forked or not forked teeth. The

existence of glandular cell at the marginal scales are important in differentiating

among species with entire scales or with blunt toothed scales, such as in D.

tomentosum, D. pallidum and D. spiniferum. Scales with entire margins may be

simpler than those with toothed margins. Because evolution never starts with

complex constructions (Schölch 2000), the toothed margin scales may be derived.

Figure 6.1. Rhizome appearance of Diplazium. a. short-creeping rhizome of D.donianum; b. medium-creeping rhizome of D. subvirescens; c. medium-erect rhizome ofD. accedens; d. short-erect rhizome of D. halimunense; e. stout-erect rhizome of D.megasegmentum.

90

Scale colour are also important in species identification. Scales colour

may be yellowish, pale brown, brown, dark brown to black. For example, black

scales of D. meijerii will be differentiate fast this species from its related species,

D. atrosquamatum, that has brown scales.

The taxonomical evaluation of the articulated hairs (trichomes) in the

Athyriaceae has been conducted by Kato (1973). He summarised that the features

of the articulated hairs are important in the classification of Athyriaceae. In the

genus Diplazium, Kato (1972) showed two kinds of articulated hairs, viz. hairs

with glandular cells and without glandular cells in Monomelangium pullingeri

(that has been proposed to be placed in Diplazium and become D. pullingeri) and

D. tomentosum, respectively. There are only several West Malesian species that

posses hairs. The hairs are without glandular cells. D. squarrosum posseses

stellate hair. D. tomentosum shows simple articulate hairs densely covered stipe,

rachis and costa. D. crenatoserratum posseses simple articulated hair sparsely on

stipes when living and then it will be scarsely found on dried material.

c. Stipe

Diplazium stipe is not swollen and becomes gradually thicker downwards.

The stipe is generally green. The stipe anatomy of some species of Diplazium

has been studied by Tardieu-Blot (1932), Bir (1962, 1969), and Kato (1972). Leaf

traces are binary. The xylem of a leaf-trace is the same hippocampus-shaped

bundle in transaction (See Chapter 4). In this study the anatomical characters of

Diplazium were no used in phylogenetic analysis, because most of species

examined were based on only herbarium specimens.

Stipe characters used in this study were only those from the gross

morphology, such as the size, colour, and the existence of multicellular hairs,

scales, protuberances and spine (Figure 6.4.). Most of Diplazium posses scales

only. Multicellular hairs and scales are seen in D. crenatoserratum, D. silvaticum

D. tomentosum. The multicellular hairs in D. crenatoserratum and D.silvaticum

are usually only seen in living plants, they are fallen when dry. Whereas in D.

tomentosum the multicelullar hairs can be seen both in living plants and dried

materials. The existence of stellate hairs and scales on the stipes and rarchis of

D. squarrosum make this species is very distinct among the West Malesian

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Diplazium. Green protuberances are seen in D. accedens var accedens and D.

accedens var. swartzii. Spines are present in D. accedens var. spinosum and D.

spiniferum.

Figure 6.2. The variation of scale shapes in Diplazium. a. D. halimunense; b. D.tomentosum; c. D.. silvaticum var. silvaticum; d.. D. esculentum; e. D. petiolare; f. D.batuayauense; g. D. spiniferum; h. D. sorzogonense; i. D. malaccense; j. D. donianum; k.D. silvaticum var. pinnae-ellipticum; l. D. angustipinna.

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Figure 6.3. Margin of scales. a. entire with glandular cells and irregular thickeningblack strands (D. atrossuamatum); b. margin entire without glandular cells andthickening black strands (D. cumingi); c and d. margin sharply toothed with regularthickening black strands (c. D. subalternisegmentum , d. D. simplicivenium); e. marginentire with glandular cells and thickening dark brown strands (D. sorzogonense); f.margin sharply toothed irregularly with irregularly thickening black strands (D.halimunense); g. Margin with densely glandular cells without thickening black strands(D. batuayauense); h. margin entire without thickening brown or black strands (D.malaccense); i. margine toothed without thickening black strands D. petiolare.

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When old or in dried specimens, stipes may glabrous, sparsely or densely

scales throughout its length. Chistensen (1911) said that the best and most

constant specific characters is to be found in the dermal appendages, hairs and

scales. A glabrous shoot may be thought ofas more primitive than one having a

potentiality for the developmental of dermal outgrowth (Dickason 2007).

Figure 6.4. Stipes appearances of Diplazium. a. Stipes densely scales (D.simplicivenium); b. stipes dark green, covered sparsely by rounded-ovate scales, scalesfallen when dry (D. spiniferum); c. Stipes covered by green protuberances (D.accedensvar. swartzii) ; d. stipes spiny, scales fallen when old (D. accedens var. spinosum)

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d. Rachis and costa

As showed by Kato (1977), the rachis and costa groove is usually open to

admit the groove of leaf axis of lower order. D. accedens, D. crenatoserratum, D.

subserratum, D. tomentosum, D. xiphophyllum have rachis that prominently raised

above with shallow grooves and their grooves are U-shaped with flat base

(Chapter 4., Figure 4.2.). The appearance of rachis groove would give more

informative parsimony characters. The cross section of rachis are generally

similar to the cross section of stipes near lamina. Unfortunately, in this study,

anatomical data of the cross section of stipes near lamina were only obtained

from 27 species due to the lack of the living collections in most species.

Therefore grooves characters on rachis or stipes did not used in this analysis,

because they would cause many missing data on many species.

Generally, rachis are glabrous. Some species has fibrillose or sparsely

minutely scales or spiny. Fibrillose rachis seen in D. sorzogonense and D.

loerzingii. Spines are present in D. accedens var spinosum and D. spiniferum.

Because the existence of fibrillose scales or spine are only in few species, in this

study the rachis and costa characters were not used.

e. Fronds architecture

Fronds architectures in Diplazium are varying, they are simple to

quadripinnate. Most of West Malesian species have pinnate to bipinnate fronds.

Simple fronds is seen in D. subserratum, but in very young fronds are pinnate

(Figure 6.5.). Deeply pinnatifid fronds, sometimes with one pair of free to

numerous segments, are seen in D. fuliginosum, D. lomariaceum and D.

porphyrophyllum. D. cordifolium has both simple and imparipinnate fronds.

Imparipinate fronds are in D. aequibasale, D. bantamense, D. lobbianum D.

donianum, D. halimunense (a new species proposed in Chapter 10), D. riparium,

D. xiphophyllum, and D. wahauense. The intermediate fronds between pinnate

and imparipinnate is seen in D. pallidum. Bipinnate-tripinnatifid fronds are

present in D. umbrosum. Tripinnatifid fronds are seen in D. megasegmentum, D.

melanolepis and D. subpolypodioides. In D. subalternisegmentum fronds are

tripinnate. Tripinnatifid to quadripinnate frond is seen in D. moultonii.

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Figure 6.5. Frond architectures of Diplazium. a. & b. D. subserratum, a. juvenile fronds, b. adultfronds; c. pinnate frond of D. silvaticum var. pinnae-ellipticum; d. imparipinnate frond of D.donianum; e. bipinnatifid frond of D. spiniferum; f. bipinnate frond of D. esculentum.

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e. Venation types

There are two kind of venation, viz. free venation and anastomosing

venation (Figure 6.6.). Free venation type may be forked or pinnate in the lobes.

Generally forked veins are present in the species with simple to simply pinnate

fronds or in species that have intermediate fronds between simpli pinnate-pinnate

such as D. subserratum, D. aequibasale, D. bantamense, D. lobbianum, D.

riparium and D. pallidum. Veins may forked once or more. Whereas pinnate

veins are seen in the species with pinnate to quadripinnate such as D. petiolare, D.

silvaticum, D. malaccense, D. dilatatum, D. polypodioides and so on.

Reticulate venations are seen in D. accedens, D. angustipinna, D.

esculentum, D. cordifolium, D. cumingi, D. insigne, D. megasimplifolium (a new

species proposed, See Chapter 9), and D.squarrosum. There are some species that

generally have free venation, but occasionally show veins uniting at margin such

as in D. fraxinifolium, D. riparium and D. xiphophyllum.

Kato (1977) showed two types of reticulate venation in Diplazium,

goniopterid venation and sagenoid venation. In West Malesian Diplazium,

goniopterid venation are seen in D. esculentum, D. accedens, D. insigne and D.

squarrosum.. Meanwhile the sagenoid venation are present in D. angustipinna,

D. cordifolium, D. cumingii and D.megasimplifolium.

Anastomosing venation in Ophioglossum is believed advanced, free

venation in other genera as primitive (Kato 1987). However the development of

anastomousing venation from free venation may be parallel, it may evolved in

different phyletic lines.

f. Sori

Diplazium sori are dorsal on the vein and linear, elongated along veins

with lateral indusium (Figure 6.6.). They are either single (Asplenoid) or double

(Diplazioid). Asplenoid sori generally occur along the acroscopic side of a vein

and Diplazioid sori are beared on both sides of the basal acroscopic vein. As

stated by Bower (1928), Holttum (1947), and Kato (1977), Asplenioid sori are

produced by abortion of the shorter arm of J-shaped sori along the basiscopic side

of a vein, in view of all intermediate forms. Bower and Holttum interpreted that

Diplazioid sori are produced by interruption of horseshoe- or J-shaped sori at the

97

distal end. Kato (1977) added that the interpretation of Bower and Holttum may

lead to the possibility of Diplazioid sori on higher veins in addition to the basal

acroscopic vein. Diplazioid sori bifurcate along the bifurcating basal acroscopic

vein, as in Athyrium and Deparia. As pointed out by Kato (1977) Diplazioid sori

are not regarded as a morphological unit, but each one of a pair as a mere single

Asplenioid sorus facing either the costule or costa.

The position of sori in Diplazium is important in species determination.

Mainly, there are two type of sori position, viz. subcostular sori and medial sori.

The subcostular sori can be divided into two group, viz. that running from until

touching midveins and those reaching at proximal end. The medial sori may not

touching midvein (inframedial) or almost perfectly medial. Most of West

Malesian Diplazium posses subcostular sori.

Generally sori are not impressed, but some species show impressed sori.

The impressed are present in D.poiense, D. sorzogonense, and D. subserratum.

The existence of impressed sori on D. sorzogonense becomes one of the

diagnostic characters to differentiate this species from D. speciosum.

In the past, a number of phylogenetic schemes presented for ferns was

based great stress on the position of the sorus (marginal vs. dorsal) with only rare

instances of changing from one to another, as in Bower’s “phyletic slide” of

Bower’s scheme (1923-28). Now, this do not faithfully represent all the diversity

within the ferns, the shift from marginal to dorsal sori occurring several times

(Mickel 1974). In the case of soral position on Diplazium, It is not known

whether those subcostular sori are advance or primitive.

g. Indusia

Most species have firm and persistent indusia. Some species posses very

thin indusia and fragile, such as D. procumbens.

Generally, the indusia is concolour, there is not color difference between

attachment side and margin. But some species posses distinctive indusia, marginal

indusia are paler than those at the attachment side, e.g. in D. cumingii and D.

loerzingii (new species proposed, see Chapter 10).

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Figure 6.6. Venation types of Diplazium. a-c. Free venation, a. vein pinnate in thelobus, veinlets simple, D. batuayaense; b. vein once forked, D. pallidum var. montanum;c. vein forked 3-4 times, D. donianum; d-e. vein anastomousing, d. goniopteris venation,D.accedens; e. sagenoid venation, D. megasimplifolium.

99

Figure 6.7. Sori variation in Diplazium. a. D. densisquamatum; b. D. cordifolium; c. D.accedens; d. D. xiphophyllum; e. D. megasegmentum; f. D. subvirescens; g. D.asymmetricum; h. D. esculentum.

100

Indusia may be entire or not. It would be one of the important characters

in species determination. Most of Diplazium have entire indusia. Generally,

entire indusia are seen in species with simple to pinnate fronds, such as D.

suberratum, D bantamense, D. cordifolium, D. lobbianum, D. pallidum, D.

riparium, D. silvaticum, and D. wahauense. Whereas non entire indusia (toothed

or fringed or lacerate) are found in species with bipinnate fronds, such as D.

atrosquamosum, D. beamanii, D. betimusense, D. esculentum, D. Kunstlerii, D.

laevipes, and D. meijerii.

6.4. Materials and Methods

6.4.1. Taxon Sampling

All species of Diplazium described in the Chapter 9 (69 species) were

included in this study (Table 6.1.). Athyrium anisopterum was chosen as

outgroup. The description of A. anisopterum taken from Holttum (1966).

Athyrium was chosen as an outgroup because this genus was considered as the

closest relatives of the ingroup (See Kato 1977).

6.4.2. Character Examination of Diplazium

Morphological characters used in this investigation were obtained mainly

from the observations of dried specimens. All characters used by Holttum (1940,

1966), Kato (1995) and Tagawa & Iwatsuki (1988) were assessed. Table 6.1.

showed characters, character states, and coding for 88 character utilized in

construction of morphological dataset of Diplazium. A list of taxa studied and the

scores for characters states are provided in Appendix 1.

The morphological datasets matrix was constructed by scoring character

states of the characters chosen from the species descriptions (Chapter 10). All

qualitative characters were coded as binary and multistate. When characters states

could not be defined due to incomplete specimens examined, it was noted as ‘?’.

Missing character states were marked as ‘-‘.

Quantitative characters are coded by using gap-weigting method (Thiele

1993). The steps are as follows: (1) The raw data are initially ranked as an

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ordered set of states, arranged according to the values of the means; (2) The data

are then range standardized:

xs = (x- min) / max – min) n

where, x = the raw datum, xs = the standardized datum, n = the maximum

number ordered states allowed by the cladistic computer program, in this study n

= 9; (3) The value is coded as the rounded integer of the standardized values; (4)

The characters are treated as an ordered multistate for analysis.

6.4.3. Phylogenetic Analysis

The data matrix was run using PAUP Version 4.0 beta (Swofford, 1998).

Due to the complexity of the dataset, Heuristic searching was employed. All

characters are of type unordered and have equal weight. Gaps are treated as

"missing”. Multistate taxa interpreted as uncertainty. Starting tree(s) obtained via

stepwise addition. Number of trees held at each step during stepwise addition = 1.

Branch-swapping algorithm used tree-bisection-reconnection (TBR). Initial

'MaxTrees' setting = 100. The number of rearrangements tried were unlimited.

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Table 6.1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium.

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

1. Rhizome Length Short Long

2.Rhizome habit Erect Suberect Creeping

3.Rhizome appearance

Slender Medium Stout

4. Occurrence ofscales on stipes

Sparsely atbase

Sparselythroughoutits length

Densely atbase

Densely atbase tomiddle

Denselythroughoutits length

5. Attachment side ofscales

Subbasal Basal

6. Scales shape Linear Oblongsubtriangular

Lanceolate Narrowlytriangular

Subulate Oblongovate

Ovate Rounded

7. Scales Length (mm) <2.50 2.50 x<5.75 5.75 x<7.5 7.5 x <9.75 9.75x<11.75

11.75 x<13.75) 13.75 x<16.75 16.75 x<18.75 8.75 x<20.75 75 x

8. Scales Width (mm) <0.6 0.6 x <1.25 1.25 x<2.00

2.00 x<2.75

2.75 x<3.30 3.3 x<3.75 3.75 x <4.75 4.75 x <5.75 5.75 x <6 6

9. Scales colour Yellowish Light brown Palebrown

Brown Darkbrown

Black

10. Scales thickeness Thin Thick11. Existence of teethat scales margin

entire bluntlytoohed

sharplytoodhed

12. Type of teeth onscale marginal

teeth simple(not forked

teeth forked

13. Scales (existence ofmargin glands)

Withoutglandularscells

Withglandulars cell

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Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium.

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

14. Scales marginthickness

Withoutthickeningblack strands

Withthickeningblack strands

15. Stipe appearance Slender Medium Stout

16. Stipe (length, mm) <12.25 12.25 x<28.25

28.25 x<36.75

36.75 x<50.75

50.75 x<64.75

64.75 x<78.25

78.25 x<92.75

92.75 x <106.25

106.25 x <120.75

120.75

17. Stipe thickness (nearbase, mm)

<1.25 1.25 x<2.75

2.75 x < 4 x < 5.25 5.25 x<6.75

6.75 x <7.75

7.75 x < 8.75 8.75 x<10.25

10.25 x11.75

11.75

18. Stipe surface Smooth Protuberance Prickly

19. Hair existence onstipe

Glabrous Glabrescent Tomentose

20. Existence ofscales on upper partof stipe

Glabrescent Scales

21. Existence ofscales on stipe base

Glabrescent Sparsely scales Denselyscales

22. Stramineous darkstramineous

paleolivaceous

yellowish palebrown

darkbrown

.black

23. Lamina (division) Simple Pinnatifid Imparipinnate Pinnate Bipinnatifid Bipinnate Tripinnatified Tripinnate

24. Lamina shape Oblong Oblonglanceolate

Oblongsubtriangular

Oblongovate

Oblongsubdeltoid

Oblongovoid

Elliptic Lanceolate Ovatesubdeltoid

Deltoid

25. Lamina length (cm) 13.50< 13.50 x<30.75

30.75 x<47.25

47.25 x<62.75

62.75 x<77.25

77.25 x<94.75

94.74 x<111.25

111.25 x<128.75

128.75 x<145.25

145.25

26. Lamina width(cm)

<10.75 10.75 x<17.25

17.25 x<27.75

27.75 x<38.25

38.25 x<49.75

49.75 x<57.75

57.75 x<68.25

68.25 x<78.75

78.75 x<88.25

88.25 <

27. Lamina incision Entire Undulate Crenate Divided

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Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

28. Lamina base Cuneate Subcordate Cordate29. Lamina apex Acuminate Attenuate Caudate30. Number of lateralpinnae

Absent less than 5 pair 5-10 pairs 11-16 pairs 17-22 pairs 23 – 28 29 or more

31. Position of mostlateral pinnae to therachise

Absent Patent Ascending

32. Deflection oflowest pinnae

Absent Not deflexed Quitedistinctlydeflexed

33. Reduction oflowest pinnae

not reduced slightly reduced muchreduced

34. Stalk existence oflower lateral pinnae

Absent Sessile Adnate Stalked

35. Stalk existence oflower lateral pinnae

Absent Sessile Adnate Stalked

36. Shape of lateralpinnae

Absent Oblong Oblonglanceolate

Elliptical Linierytriangular

Oblongsubtringular

Lanceolate Ovate

37. Length of the largestlateral pinnae (mm)

(<8) x<12.75 12.75 x<20.25 20.25 x<29.1 29.1x<36.90)

36.90 x<43.1)

43.1 x<50.9)

50.9 x<57.1

57.1 x<64.9

64.9 < x

38. Width of the largestlateral pinnae (cm)

<2.75 2.75 x <6.25 6.25 x <10.25 10.25 x<14.75

14.75 x<18.25

18.25 x<22.75

22.75 x<26.25

26.25 x<30.75

30.75 x<34.25

34.25

39. Upper base oflower lateral pinnae

Absent Truncate Subtruncate Cuneate

40. Lower base oflower lateral pinnae

Absent Truncate Subtruncate Cuneate Subcordate Cordate Rounded Auricled

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Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

41. Margin of lowerlateral pinnae

Absent Entire Subentire Undulate Crenulate Crenate Serrate Toothed Lobed ordivided

42. Margin of upperlateral pinnae

Absent Entire Serrate Toothed Slightlycrenate

Lobed

43. Incision of lateralpinnae

Absent Less than ¼way to costa

1/4 – ½ wayto costa

2/3 - ¾ wayto costa

4/5 – 5/6way to costa

6/7 way tocosta– towithin 1mm ofcosta

divided

44. Apex of lateralpinnae

Absent Rounded Acute Acuminate Caudate Attenuate

45. Pinnulae number Absent less than 10pairs

10 -20 pairs more than 20pairs

46. The existence ofstalk on lowerpinnuae

Absent Shortly stalked Subsessile Sessile

47. The existence ofstalk on upperpinnulae

Absent Shortly stalked Subsessile Sessile

48. Pinnulae shape Absent Lineary Oblong Oblongsubtriangular

Oblonglanceolate

Oblongelliptical

Oblongsubdeltoid

Hastate

49. Length of largestpinnule (cm)

<2.25 2.25 x <3.50 3.50 x <5.75 5.75 x <7.75 7.75 x <9.75

9.75 x<11.75

11.75 x<12.25

12.25 x<15.75

15.75 x<17.25

17.25

50. Width of largestpinnule (cm)

(<0.7 0.7 x < 1.3 1.3 x <1.7 1.7 x<2.4 2.4 x <3.0) 3.0 x <3.7 3.7 x < 4.1 4.1 x < 4.5 4.5 x < 4.9 4.9 )

51. Pinnule base Absent Truncate Subtruncate Broadlycuneate

Subcordate

52. Pinnule margin Absent Entire Serrate Crenulate Lobed

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Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium.

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

53. Pinnule incision Absent less than ¼way to costa

1/4 – ½ wayto costa

2/3 - ¾ wayto costa

4/5 – 5/6way to costa

6/7 way tocosta– towithin 1mm of costa

divided = 7(formingsegments

54. Pinnule (apex) Absent rounded acute acuminate attenuate

55. Shape balanceof pinnulae orsegment lobes

Absent not oblique slightlyoblique

56. The width oflobes

Absent less than 5mm wide

5 mm or morewide

57. Form of lobesapice

Absent Truncate Obtuse Rounded Acute

58. Margine oflobes

Absent Entire Subentire Slightlyserrate

Serrate Slightlytoodhed

Crenate Lobed

59. Form ofterminal pinnae

Absent Similar tolateral ones

Deltoid withlobes at base

Withoutdistinctterminalpinna

60. Texture oflamina or pinnae orpinnulae

Herbaceous Subherbaceous Subcoriaceous Coriaceous Shoftlychartaceous

Chartaceous Papyraceous

61. Surfacedepression

Surface notdepressedbelow sori

Surfacedepressedbelow sori

62. Rachis glabrous minutely hairy minutelyscaly

densely hairy denselyscales

107

Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium.

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

63. Rachise(viviparous or not)

Notgemmiferous

Gemmiferous

64. Costae Glabrous Tomentose/hairy

Sparselyminutescales

Denselyscales

65. Vein reticulationtype

Free Anastomousing

66. Vein branchingtype

Pinnate Forked

67. Veinlet number onpinnated vein

less than 4pairs

4- 6 pairs 7 pairs ormore

68. Veinlet branchingon pinnated veins)

Simple Forked 1 – 3times

Forkedmore than3 times

69. Vein (uniting withthe opposite onesforming excurrentveinlets or not)

uniting withoppositeones

not unitingwith oppositeones

70. Vein (forking type) Forked 1-2times

Forked morethen 2 times

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Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

71. Vein (part ofanastomousing)

occasionallyanastomousingnear margim but nevercopiouslyanastomousing

anastomousing¼ from themargin

anastomousing1/3 from themargin

anastomousing1/2 from themargin

anastomousing2/3 from themargin

anastomousingmore than43/4 fromthe margin

72. Veins in small groups at anangle about .... to themidrib or costa

about 45º about 50º about 60ºormore

73. Sori position near basal ofveins

middle ofveins or

from nearbasal of veinsor veinletalmost to reach margin

74. Sori covering on the veins or veinlets

Less than 1/3 ofveinlets length

1/3 of vein orveinlet length

1/2 of vein orveinlet length

2/3 – ¾ Nearly thewhole lengthof veinlets

75. sori existence on veinlets

On part ofveinlets

On all veinlets

76. Sori number on each vein group

1 2-4 5 or more

77. Sori (on basal acroscopic vein)

sometimesdiplazioid

usuallydiplazioid

alwaysdiplazioid

78. Indusia form reniform/hooked crescentic linear79. Indusia Width Narrow Broad

109

Table 1. Characters, character states, and coding for 88 characters utilized in construction of morphological dataset of Diplazium.

Characters (0) (1) (2) (3) (4) (5) (6) (7) (8) (9)

80. Indusia appearance Hardlyevident inmaturesori

Evident inmatrure sori

81. Indusia opening openingbeforemature

openingwhenmature

82. Indusia curling Not rolledback whenold

Rolled backwhen old

83. Indusia colour palebrown

brown darkbrown

84. Colour continuity ofindusia

Concolour Attachmentsside darker

85. Indusia margin entirewhenopening

subentire toothed orfringed orlacerate orcrisped

86. Indusia existence Notpersistent

Persistent

87. Indusiastrengtheness

Fragile Robust

88. Spines at thejunction of costules

Absent Present

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6.5. Results and Discussion

The eighty eight morphological characters (Table 6.1. and Appendix 1.)

were analysed to determine relationship among the 69 species of West Malesian

Diplazium. The eighty eight morphological characters comprises eighty five

parsimony-informative characters, one constant character, and two parsimony-

uninformative characters. The eight equally most parsimonious trees of 1366 steps

produce a Consistency Index (CI) = 0.24, Retention Index (RI) = 0.48, RC = 0.12,

and Homoplasy Index (HI) = 0.75. Figure 6.8. showed the topology of a strict

consensus tree of the eight equally most parsimonious trees of 1366 steps without

the high level of Bootstrap value.

Due to the low level of Bootstrap support, it is very difficult to state any

statement with confidence about the relationships within West Malesian

Diplazium generated from morphological date derived from unweighted

maximum parsimony analysis. The out group (Athyrium anisopterum) is separated

from the in group without Bootstrap value. The relationships within Diplazium

were also unresolved, without high Bootstrap values but clade that comprised D.

silvaticum and D. petiolare and clade that include D. lomariaceum, D.

megasimplifolium, D. subserratum and D. prophyrorachis with Bootstrap support

65% and 82%, repectively.

The lack of support of the monophyly of the genus Diplazium due to the

high proportion of homoplastic characters (HI = 0.75). Homoplasy – the

independent origin or loss of one or more traits in different organism – can distort

the inference of phylogenetic relationship, tying together similar but unrelated

taxa (Givnish & Sytsma 1997).

Although the phylogenetic tree derived from the maximum parsimony was

lack of or weak support, however, it does not indicate that the pattern observed is

incorrect. Triono (2006) stated that the lack of or weak support for a phylogeny

does limit the amount of confidence that can be placed in the relationships

between taxa and also the conclusions that can be drawn from the inferred

phylogeny. Therefore some clades generated from the phylogenetic analysis of

Diplazium that seems formed from the closely related taxa were discussed below.

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Figure 6.8. Strict concensus of 8 trees of length 1366 from unweighted morphologicaldataset comprises 88 morphological characters. Bootstrap support values between 50-90 are givenabove line. Only support value above 50% are shown.

112

Two major clades were identified in the morphological parsimony analysis

without well-supported Bootstrap value: Clade I and Clade II (Figure 6.8.).

Clade I consist of D. fuliginosum, D. lomariceum, D. porphyrorachis, D.

megasimplifolium and D. subserratum in which D. megasimplifolium and D.

subserratum are the most closely related. In this clade D. fuliginosum is at the

base clade and diverse into four species, viz. D. lomariceum, D. porphyrorachis,

D. megasimplifolum and D. subserratum, respectively. Clade II included most of

West Malesian species and splited into two clades with very low-supported

Bootstrap value, viz. D. tomentosum alone (Clade II.1.) and all the remaining

species (Clade II.2.) . In the Clade II.2. D. crenatoserratum is positioned at the

basal clade and diverse into two subclade, the small clade in which composed of

D. velutinum, D.silvaticum and D. petiolare (Clade II.2.A.) and the large clade

(Clade II.2.B.)

The affinity of D. fuliginosum, D. lamariceum and D. porphyrorachis. In

the topological tree (Figure 8.6.) D. fuliginosum, D. lomariceeum, D.

megasimplifolium, D. suseratum and D. porphyrorachis form a separated clade

from the other West Malesian species and the wo species, D. subserratum and D.

megasimplifolium (a new species proposed, see Chapter 9), are the most closely

related. The affinity of the two species will be discussed separately below. The

affinity of D. fuliginosum, D. lomariceum and D. porphyrorachis are supported by

the share characters as follow: rhizome short-erect, wiry black roots; fronds

narrowly elliptic, deeply pinnatifid or pinnate, apex coadnate, segments numerous,

usually very dark green when living; scales abundant on stipe and rachis, narrow,

usually dark and shiny; veins free. Price (1983) stated that D. fuliginosum is one

of the most unusual of all diplazia, was not transferred to the genus Diplazium

until recently. This species is strickingly peculiar by the smooth rachis channel

without raised sides, uninterrupted by the insertion of pinna-costa. In small

fronds, and distally on large fronds, the rachis is almost flat above. Other features

unusual to Diplazium are sori informally extending from costa to margin, and thin

translucent pinna margin. D. lomariaceum is very closely related to D.

porphyrorachis and until Price (1983) distinguished it from D. porphyrorachis,

the name seems to have been ignored since Christ himself who described is as

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Asplenium lamoariaceum, reduced lomariaceum to porphyrorachis in Ann. Jard.

Bot. Buitenz. 15 (1898, p.119). Examining many specimens from K, L, MICH,

NY, P, PNH, UC, and US, Price (1983) showed the signs of apparent gene

interchange between D. porphyrorachis and D. lomariceium. In relation of the

two species with D. fuliginosum, Price (1983) believed that rather than to D.

lomariceum and D. porphyrorachis, the closest relationship of D. fuliginosum is to

D. cumingii (Presl.) C.Chr., with which it agrees in dark frond colour and black

axis aging to greeyish; axes without cartilaginous ridges; scales on stipe abundant,

dark, entire; and indusia black with very narrow pale brown margin, curling back

at maturity; even though D. cumingii is very different in its conform frond apex

and broadly elliptic pinnae. Price (1983) added that almost exactly the same frond

form (and even margin structure) as D. fuliginosum was independently evolved in

the Central American D. harrisonii (Bak.) C. Chr. which otherwise differs

markdedly in scales and the architecture of the axes.

The affinity of D. megasimplifolium and D. subseratum. In the Clade I the

closely related between D. megasimplifolium and D. subseratum is not supported

by well-Bootstrap value. The two species possess similarities on characters as

follow: lamina simple with cuneate bases, margin entire on lower part, apex

acuminate. However the two species are very different. The lamina of D.

megasimplifolium is elliptic, much wider (up to 10.5 cm wide), margin always

entire; veins copiously anastomousing up to 4/5 way of margin. D. subserratum

possess lamina lanceolate, less than 4 cm wide, margin entire to irregularly

crenate; veins free. In juvenile stage lamina of D. subserratum is pinnate.

Holttum (1940) presumed that D.subserratum is probably allied to D. lanceum of

India and China but is larger, and a shorter rootstock which is more or less with

tufted fronds; the scales also appear to be smaller.

The relationship among D. malaccense, D. sorzogonense and D

tricholepis. Of three species, D. malaccense is closest related to D. sorzognense.

Ecologically the two species also grow in similar localities, species of lowland

and mid mountain forest in West Malesia. As also stated by Holttum (1940), D.

malaccense is differing from D. sorzogonense in glabrescent stipe and rachis.less

deeply lobed pinnae, and sori not at all impressed. Whereas, D. sorzogonense

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seems to have affinity with D. tricholepis, mainly its fibrillose rachis, in deeply

lobed of lanceolate pinnae, oblong lobes and veinlet number in each lobe.

‘Imparipinnate frond group’. ‘Imparipinnate frond group’ refer to the

clade in the Figure 8.6. that comprises D. aequibasale, D. angustipinna, D.

donianum, D. cordifolium, D. halimunense, D. cumingii, D. subintegrum, D.

bantamense, D. lobbianum, D. fraxinifolium, D. xiphophyllum, D. hottae, D.

crameri, D. riparium, and D. wahauense. The affinity of these species seems to

be natural. The affinity among these species are discussed below.

The affinity of Clade D. cumingii. Clade D. cumingii consists of D.

cumingii, D. halimunense, D. cordifolium, D. donianum, D. angustipinna, and D.

aequibasale. In this clade, two pair of species, D. aequibasale and D.

angustipinna, D. cordifolium and D. halimunense, are the closest related, D.

donianum to be the sister clade of D. aequibasale and D. angustipinna, and D.

cumingii at the basal clade. The relationship patterns among the species of Clade

D. cumingii seems unnatural. The similarities on many quantitative characters

among these species that seems not to be correlated with their qualitative resulted

the unnatural patterns. The closest relationship of D. aequibasale is to D. riparium

or D. wahauense. Morphologically, D. aequibasale is intermediate between D.

riparium and D. wahauense. The three species share in characters such as

lanceolate dark brown entire scales and oblong lateral pinnae with cuneate base

and margin entire. D. angustipinna and D. halimunense should to be closely

related to D. cordifolium and D. donianum, respectively. D. angustipinna and D.

codifolium share light brown scales, margin entire with irregularly thickening

black strands, ovate-lanceolate lateral pinna, and copiously anastomousing veins.

While D. donianum and D. bantamense share in irregular sharp toothed scales and

ovate-lanceolate pinnae..

The relationship among D. bantamense, D. lobbinaum, D. subintegrum, D.

fraxinifolium, D. xiphophyllum ,D. hottae. D. crameri. The closest relation of D.

lobbianum and D. bantamense and also D. xiphophyllum and D. fraxinifolium are

very reasonable and natural. The two first species share in the following character

combination: dark brown toothed scales; pinnae ovate-lanceolate with rounded

base, margin entire or serrate near apex; vein free and forked several times.

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Meanwhile D. fraxinifolum and D. xiphophylum are in the collection often mixed

due to the misidentification. D. fraxinifolium differs from D. xiphophyllum in

lineary lanceolate scales (less that 10 mm long), waved-crenate lateral pinnae,

forking type of veins (5-7 times). D. hottae seems to have close relation to D.

xiphophyllum and D. subintegrum. Tagawa (1972) stated that D. hottae is alled to

D. subitegrum. D. hottae differs in: terminal pinna not lobed at the base and

similar to upper lateral ones, pinnae apparently entire, venation obscure, sori

narrower. D. crameri is more close to D xiphophyllum, and differs from this

species mainly in characters: scales dark brown, pinnae stalked to 1.5 cm long,

ovate-lanceolate, all veins free (D. xiphophyllum is occasinolly showing the

uniting veins near margin) and forked to 2 times.

The affinity of D. riparium and D. wahauense. As explained in Chapter 2,

5 and 9 D. riparium and D. wahauense are morphologically very similar and

closely related (Kato et al 1991). Topological tree showed in Figure 8.6. also

revealed that the two species are closely related. Thus the statement of Kato et

al (1991) has been verified in this study.

The affinity of D. pallidum and D. prescottianum. The morphological

characters between the two species are very similar. Holttum (1940) presumed

that D. prescottianum has close relation to D. pallidum. D. prescottianum differs

from D. pallidum in large brown scales. More lobed edges to pinnae, and many

more soriferous veins in each group.

Relationship among D. velutinum, D. petiolare and D. silvaticum. In the

topological tree inferred from morphological data the relationshop among D.

velutinum, D. petiolare and D. silvaticum are not resolved because the Bootstrap

value is very low (<50%), but the closest related of D. petiolare and D. sivaticum

are supported (Bootstrap value 65%). Morphologically, the two species are very

similar (See Chapter 9). D. petiolare differ from D. silvaticum in characters

combination as follow: D. petiolare has scales linear with distantly teeth and

thickening black strand; upper surface of lamina light green when living, pinnae

lobed ¾ way toward costa, upper base not auricle. Whereas D. silvaticum has

lanceolate scales with closely teeth without thickening black strands; upper

surface of lamina dark green, pinnae lobed ¼-1/2 toward costa

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This result also showed that this phylogenetic tree generated from

morphological data did not support the taxonomical treatment of van Alderwerelt

van Rosenburgh 1908). Treating Diplazium into two sections based on the

venation type, Eudiplazium for those having free veins and Anisogonium in which

composed of species having anastomousing veins (van Alderwerelt van

Rosenburgh 1908), is not natural. In the topology of the phylogenetic tree

generated from morphological data in this study showed that the species members

of the two sections are mixed and did not form into two distinct lines. D.

megasimplifolium (Anisogonium) is included in Clade I in which also composed

of the other species with free veins (Eudiplazium). In the Clade II the species

with copiously anastomousing veins, D. accedens, D. squarrosum, D.

angustipinna, and D.cordifolium are nested within the free veins species and also

do not fall in one terminal clade. These results suggests that the anastomousing

venation pattern in Diplazium may develop in different line. In evolution this

character occurred parallel. Dickason (1946) stated that free and reticulate

venation are not two fundamentally different things, but rather that reticulate

venation may develop from the open type wherever a diverging meristimatic

center on the margin of the blade primordium meets and merges with an adjacent

center.

Comparison between the phylogenetic tree resulted in this study and the

classification of Kato (1977) could not be done proportionally due to the lack

living collection in many species Nevertheless, all the 27 species of Diplazium

that successfully examined their groove and vascular bundles characters (Chapter

4) could not also be referred to the five Diplazium group of Kato (1977). There

are only two group, D. dilatatum group and D. javanicum group, that could be

reffered to West Malesian species. This topological tree showed that the member

of D. dilatatum group (D. batuayauense, D. dilatatum, D. donianum, D.

polpodioides, D. sorzogonense, D. subpolypodioides, D. speciosum, D.

spiniferum, D. subserratum, D. umbrosum, and D. xiphophyllum) distributed in

many different terminal clades of the main clade (Figure 6.8.). It is indicate that

Kato’s classification could nod be applied on West Malesian Diplazium.

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6.6. Conclusions

The phylogenetic relationship among species in the genus Diplazium was

very difficult to explain due to the lack of or weak support Bootstrap value.

Therefore the monophyletic species groups in this genus could not be identified

confidently. Consequently, sister-group relationships among these species also

could not be established.

The high homoplastic on morphological characters in Diplazium distort the

inference of phylogenetic relationship among species. Moreover parallel evolution

seems occurring.

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 and

congruence with the some terminal clades of gene rbcL tree, such as the affinity

of D. porphyrorachis group and ‘imparipinnate frond group’ {((D. subintegrum

(D. bantamense, D. lobbianum)) ((D. fraxinifolium, D. xiphophyllum) D. hottae))

D. crameri) (D. riparium, D. wahauense)} This study also revealed that the

classification of van Alderwereld van Rosenburgh (1908) in dividing Diplazium

into two sections (Eudiplazium and Anisogonium) is not natural. Moreover, this

study gave indication that Kato’s classification (1977) could not be applied on

West Malesian Diplazium. The lack of phylogenetic signal in morphological

datasets shows the need for other more informative data, such as molecular data,

for inferring phylogentic relationships.

Cytological data in the Chapter 5 showed that Diplazium is very

complicated. It indicates that cytological information is very important in

detecting the evolution and speciation in the Diplazium. Therefore in the future

cytological data, including both somatic and gametic chromosome, the

chromosomal behavior in meiosis, and chromosomal karyotype, should be

incorporated in the phylogenetic analysis of Diplazium.

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CHAPTER 7

SPECIFIC DELIMITATION AND RELATIONSHIP AMONG SPECIESOF DIPLAZIUM BASED ON SPORE MORPHOLOGY

7.1. Introduction

Ferns spores display remarkable variation and have been extremely heplful

in systematic studies. In many cases particular spore morphology are distinctive

for families or genera, or in some instance individual species (Taylor & Mickel

1974). Extensive studies on the spore of Pteridophyta showed that spore

morphology can be used as one of the taxonomic evidence for delimiting taxa.

Ferrarini et al. (1986) reported the illustration, by scanning electron microscope,

of the spores of the Pteridophyta native in Italy amount to 124 specific and

intraspecific taxa. They showed that spore morphology provide supporting

taxonomic evidence for delimiting both generic and species level in the

Pteridophyta. Even they recognized some infraspecific of Asplenium, such. A.

trichomanes, A. ruta-muraria, A. seelosii and A. officinarum, based on its spore

morphological characters. Harris (1955) described spores from 170 taxa of New

Zealand fern based on samples taken from herbarium specimens. Forty six years

after Large & Braggins (1991) revised this work and provided full spores

description of 211 species from New Zealand based on samples taken from fresh

material. These works also reveled that spore morphology among the genera in a

family are diversified so that a tentative general key to the genera of pteridophytes

from New Zealand is given. However they did not discuss dealing the correlation

between palynological characters and its taxomical treatments. Ohta & Takamiya

(1999) used spore morphology characters that observed by using SEM to

distinguish Diplazium griffithii from other species in the Diplazium mettenianum

complex.

Palynological characters may also provide evidence to illustrate the pattern

of relationship. Blackmore (2000) showed the the use of pollen morphology, in

isolation, as a means of explicitly illutrating the relationship pattern of species

included in subtribe Scorzonerinae (Asteraceae). The direction of the

transformation was determined by method of outgroup comparison, because it is

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very difficult to determine the transformation of characters on the basis of the

evolutionary trend approach. Even it is also difficult to polarize characters on the

basis ontogeny. Therefore one solution to the issue of character polarization is to

analyze the characters as unordered, or unpolarized, unless development evidence

for the transformation is available, and allow parsimony to determine the direction

of change.

In effort to develope new circumscriptions of genera in the Vittariaceae,

with the goal of orgnizing the species into genera that are srictly monophyletic,

Crane (1997) mapped the states for four morphological characters, including

spores type, onto the tree generated from phylogenetic analysis using rbcL gene

sequences data (Crane et al 1995).

The exospore is considered to be extremely important for establishing

evolutionary relationship (van Konijnenberg-van Citteret 1999). Exospore

architecture is very diverse. The detailed exospore structure revealed by light and

electron microscopy was first studied by Pettit (1966). Since then, many

pteridologists have dealt with the subject and its culminated with the work of

Tryon and Lugardon (1991) which describes pteridophytes spores from all over

the world using SEM (Scanning Electron Microscope) and TEM (Transmission

Electron Microscope).

Any determination of what constitutes a primitive (ancestral) or derived

state in the structure of fern spores must be based on homologies. Connecting this

subject, Van Konijnenburg-van Cittert (1999) discussed the evolution of various

characters of spore shape and wall structure based on fossil evidence. The

characters discussed include number of spores per sporangium, spore size, overall

spore shape in connection with laesura, thickness and sculpture of exospore,

absence or presence of a perispore, and its sculpture. The evolutionary trend in

fossil fern spores are summarized as follow: (1) A number of 256 spores per

sporangium is primitive, while 128 or less spores per sporangium is derived.

There is a trend of reduction of the number of spores per sporangium; (2)

Standard spore diameter is between 30 and 60 um. Spore diameter over 60 um is

derived. Spore diameter under 30 um may derived, but is probably the most

primitive stage; (3) Tetrahedral, trilete spores are primitive. Bilateral, monolete

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spores are derived. The evolution from trilete to monolete spores are seen in

several fern families. Trilete and monolete spores may even occur in one

sporangium; intermediates have been recorded; (4) The most primitive overall

spore shape in trilete spores is probably globose spores, while (rounded) triangular

spores are derived; (5) Exospores between 1 and 2 um are usually primitive, but

thin and smooth exospores correlated with a thick and elaborate parispore may be

derived. Exospores over 3 um and certainly over 6 um, are derived; (6) Smooth or

fine exospore ornamentation is possibly primitive, but in the Schizaeceae the

oldest recorded spores appear only late during evolution; (7) Exospores

sculptures that are certainly derived are: bisculpate spores; strongly raised

laesurae; laesurae bordered by margo; valvate spores; and elaboration of the

equatorial region; (8) Absence of perispore is primitive; presence of perispore is

derived; (9) Thin, smooth or finely ornamented perispores consisting usually of

more than one layer and having an elaborate ornamentation are derived.

This chapter present spore morphology of Diplazium from West Malesia

observed by using both Light Microscope (LM) and Scanning Electron

Microscope (SEM. Due to the available of spores collection, only 27 species

were observed of 69 species recorded in West Malesia. The aims of the research

were to: (1) get supporting data for species delimitation on Diplazium; (2) provide

a tentative key for species based on spore morphology; and (3) infer phylogenetic

relationship between species in the genus Diplazium based on spore characters.

7.2. Materials and Methods

Spores of twenty six species of 46 collection numbers of Diplazium were

observed by using scanning electron microscopy (SEM) for getting detail of its

perine ornamentation. The list of collection number examined are presented on

Table 1. These species were chosen based the spore sample availability.

However the 26 species sampled have represented two main clades and eight

subclade of the topological tree generated from morphological data in Chapter 6.

Spore sample were collected both from fresh material collecting from the

field and herbarium material housed at Herbarium Bogoriense (BO). For

determining its size, both polar (P) and equatorial (E) measurements, and

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morphological outline, 65 collections number were observed by using light

microcopy (LM).

a. Preparation for light microscopy (LM)

Spore sample, both obtained from fresh spores (air drying without

alcoholic treatments) and from herbarium specimens, were directly transfered on

slides and mounted with glycerine jelly for study with light microscopy. Spore

size, polar (P) and equatorial (E) measurements, were taken for each spores. Each

individual plant was taken 20 spores randomly. Each species was represented by

one to five collection numbers.

b. Preparation for scanning electron microscopy (SEM)

Separation and Attaching Spores on the Holder. Spores sample used were

fresh spores (air drying without alcoholic treatments) and spore of herbarium

specimens housed at BO. Spore sample were separated from the indusium and

sporangium fragment of indusium. Separation was conducted manually. One

spatula of spora sample were spread over on waxpaper 4.5 x 4.5 cm. Waxpaper

with spores were shaked by hand. Spores will separate from the fragments.

Evaporation and Platinum Coating. Spores were spread over and attached

on the holder. Spores samples on the holders were evaporated by using Ion

Sputtering for 5-7 minutes. After that spores sample were coated with Platinum

Carbon by using HITACHI HCP-2 for 10 minutes by using Electron Beam

Coating Method.

Observation and Taking Pictures. Spore sample on the holder coated by

Platinum Carbon were observed by using scanning electron microscope (SEM) S-

800 HITACHI. Spore pictures were taken on the longitudinal ekuator, transversal

ekuator, dan polar views.

c. Description format and taxonomic characters

Description are given in full for each taxon. Terminology used to describe

spores morphology following those used by Harris (1955) and Large & Braggins

(1991).

d. Phylogenetic Analysis

Data matric (Table 5.2.) was analyzed using maximum parsimony, PAUP

Version 4. (Swofford 1998), with the Heuristic search settings. All characters are

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of type unorder and have equal weight. Gaps are treated as "missing". Starting

tree(s) obtained via stepwise addition. Athyrium filix-femine was used as

reference taxon, out group. Number of trees held at each step during stepwise

addition = 1. Branch-swapping algorithm was runned by using tree-bisection-

reconnection (TBR).

7.3. Results and Discussion

7.3.1. Spore Characters of Diplazium and Its Use in Supporting Species Delimitation and Identification

All Diplazium species have spores morphological characteristic

similarities as follow: golden yellow to brown when released, monolete,

bilaterally symmetrical, and heteropolar; polar outline elliptical, sides convex;

equatorial longitudinal view concave-convex to plano convex; equatorial

transverse view, proximal face planar to concave, distal face convex to

hemispherical. SEM observations revealed that a thick perine is present; laesura

is often concealed by perine. Perine is alate to costate alate, wing-like folds. The

description of each species is presented on Table 7.1.

Character used to describe Diplazium spores are as follow: 1) Pattern: The

general pattern of the perine, may appear, (a) folded, or b) spinulose; 2)

Fenestration: perine may be, (a) fenestrate throughout, (b) fenestrate in the

lacunae of any reticulation, or (c) without fenestrae; 3) Ornamentation: Overall

ornamentation is (a) smooth, (b) veined or wrinckled, or (c) raised ribs within the

lacunae. Large & Braggins (1991) mentioned one more character state, viz. (c)

tuberculate with knob-like elements, however this character state is not found for

Diplazium of Western Part malesian Region. 4) Pattern density: Density classes

are (a) sparse and (b) dense; 5) Perine fold type: Perine folds may vary, general

forms are (a) winged (alate) or (b) ridged (costate); ridges may be rounded or

slightly crested; 6) Fold margins: The edges of the perine folds may (a) extend

into serrate to echinate projections, (b) bear minute teeth, papillae, or (c) be

smooth.

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Kramer et al (1990) described general morphological chraracteristic for

spores Diplazium, viz. with long, usually prominent, wing-like folds, often with

echinate borders, and sometimes cristate or echinate, but without giving further

explanation for the existence of the grouping based on spores characters. Roubik

(2003) showed alate spores of D. grandifolium of Barro Colorado.

Spores of West Malesian Diplazium fall into nine main pattern groups

based on the perine ornamentation. The nine group are discussed as follows.

(1) Group I. The spore form most common among the diplazioid ferns of

Western Part of Malesian region are costate, non-fenestrate, and ornamented with

a wing-like pattern raised to form a pattern of slightly rounded ridges. D.

accedens, D. bantamense, D. lobbianum, D. polypodioides, D. pallidum, D.

procumbens, D. sorzogonense, D. speciosum, D. subpolypodioides, D. dilatatum,

and D. xiphopyllum are included in this group. The existence of additional

ornaments, however, would differentiate among the species. Reticulation type of

wing-like muri or wing-like costae, terminating margin of wing-like muri, and

surface of perine (both on lacunae and wing-like muri) of these members are in

varying character states. Therefore these characters can be used to support species

delimitation. Spores of D. accedens, D. bantamense, and D. polypodioides are

similar appearance. They are seen incomplete reticulation of wing-like muri or

costae often incomplete with terminating margins entire or smooth. D. accedens

and D. polypodioides differ with D. bantamense in the projection of wing-like

muri or costae. The two first species showed wing-like muri or costae project to 7

m, while the third species has wing-like muri or costae project to 13 m. D.

accedens and D. polypodioides would differ in the size of their irregular polygons

lacunae, the first species is with 10 m or more across while the second species 9

m or less across.

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Table 7.1. Spore Description of Diplazium in West Malesia

No. Species Spores Description & Specimen Examined

1. D. accedens Blume Monolete, bilaterally symmetrical, heteropolar; polar outlineelliptical, sides convex; equatorial longitudinal view concave-convex to plano-convex; equatorial transverse view, proximal faceplanar to concave, distal face hemispherical; perinate. E:25.34(31.47)36.02±2.54; P: 15.32(19.51)23.59±2.82. Laesure:concealed by perine ridge. Perine: costate-alate, reticulation oftenincomplete; lacunae shallow, project 10-17 m ;wing-like muri orcostae project c.0.5-6 m, terminating margins entire. Exine: oftenvisible through perine, smooth under LM, smooth under SEM.

Specimens Examined: Java: West Java, Mt. Gede, T.Ng.Praptosuwiryo s.n.

2. D. bantamenseBlume

Monolete, bilaterally symmetrical, heteropolar; polar outlineelliptical, sides convex; equatorial longitudinal view concave-convex; equatorial transverse view, proximal face concave, distalface hemispherical; perinate. E: 42.81(53.88)66.05±5.69; P:22.64(31.48)37.47±3.38 Laesure: concealed by perine ridge.Perine: costate-alate, loosely reticulate irregular envelope, costaeform a large reticulation; separated from the spores; reticulationoften incomplete; lacunae large irregular polygons 11-25 macross; costae or alate project 3-13 m, terminating marginsentire; surface of perine smooth. Exine: often visible throughperine, smooth under LM.

Specimens examined: Java: West Java, T.Ng. Praptosuwiryo1707; 1766.

3. D. cordifolium Blume Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex; equatorial longitudinal view (excludingperine) plano-convex; equatorial transverse view, proximal faceplanar, distal face convex; perinate. E: 37.02(42.15)51.62 ±5.09P: 20.23(26.56)31.21±2.97. Laesura: concealed by perine wing.Perine: alat to costate-alate, loose reticulate; irregular envelopeseparated from exine surrounds the spore in continuousanastomosing wings, forming a loose reticulation; lacunae largeirregular polygons 15-20 µm across; thin wing-like muri project2-8 µm, terminating margins are often echinate; surface of perinesparsely echinate or ciliate; echinae project 0.5-0.8 µm. Exine:visible through perine, smooth under SEM.

Specimens examined: Java, West Java, Mt. Salak, T.Ng.Praptosuwiryo 1367, 1457; Mt. Halimun, T.Ng. Praptosuwiryo1708; 1709; 1775.

125

Table 7.1. Continued

No. Species Morphological characteristic of Spores

4. D. crenatoserratum(Blume) Moore

Monolete, bilaterally summetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transversely elliptical, sidesconvex; equatorial longitudinal view (excluding perine) concave-convex;equatorial tranverse view, proximal face concave, distal face convex;perinate. E: 29.86(38.19)45.74±4.95, P: 17.92(23.48)27.36±2.94.Laesura: concealed by thin wing-like muri. Perine: alate, looselyreticulate; irregular envelope, separated from exine, surrounds the spore inanastomosing wings, forming loose reticulation, reticulation irregular andoften incomplete; lacunae irregular polygons, 8-16 µm across, irregularholes and small papillae within; holes caused by the fallen papillae; murithin, wing-like, projected c. 1-8 µm, terminating margin ciliate; surface ofperine fibrous-like and holed, holes irregular and formed by fallen ciliae.

5. D. dilatatumBlume

Monolete, bilaterally symatrical (made asymmetric by prine), heteropolar,polar outline (excluding perine) elliptical, sides convex; equatoriallongitudinal view (excluding perine) concave-convex; equatorialtransverse view, proximal face concave, distal face convex; perinate. E:31.85(45.69)49.56±4.78; P: 19.84(28.55)34.62±3.89. Laesura: concealedby perine. Perine: alate-to costate-alate, irregular envelope separated fromexine surrounds the spore; wing-like muri projected c.6 -15 µm µm,terminating margin are often ciliate; surface of perine smooth. Exine:visible through perine, smooth under SEM. Specimens examined: J.J.Afriastini Bl-170

6. D. esculentum(Retz.) Swartz

Monolete, bilaterally symmetrical, heteropolar; polar outline elliptical,sides convex; equatorial longitudinal view concave-convex to plano-convex; equatorial transverse view, proximal face planar to concave, distalface hemispherical; perinate. E: 31.72(38.39)43.09±3.35P: 22.74(26.37)29.12±1.39. Laesure: not visible, concealed by perine.Perine: smooth under LM, micro rugulate under SEM. Exine: Exine: oftenvisible through perine, granulate under LM.Specimens examined: T.Ng. Praptosuwiryo 637

7. D. lobbianumMoore

Monolete, bilaterally symmetrical (made asymmetrical by perine),heteropolar; polar outline elliptical, sides convex; equatorial longitudinalview concave-convex to plano-convex; equatorial transverse view,proximal face planar to concave, distal face hemispherical; perinate. E:38.20(46.10)51.87±3.22; P: 20.47(26.28)32.81±3.73. Laesure:concealed by perine ridge or wing-like muri. Perine: costate-alate; wing-like muri surrounds the spore without forming reticulation or with formingloose reticulatons, terminating margins ciliate; surface of perine smooth toscatterly ciliate or pappilate. Exine: often visible through perine under LM,smooth under SEM. Specimens examined: T.Ng. Praptosuwiryo 1340

8. D. lomariaceum(Christ) Price

Monolete, bilaterraly symmetrical (made asummetric by perine),heteropolar; polar outline (excludong perine) elliptical; equatoriallongitudinal view (excluding perine) biconvex; equatorial tranverse view,proximal face convex, distal view hemispherical; perinate. E:35.03(39.41)54.55±4.20; P: 19.55(24.57)30.13±3.46. Laesura: concealedby wing-like costae. Perine: alate to costate-alate, alae forming loosereticulation; irregular envelope separated from exine surrounds the spore inwing-like muri reticulation, lacunae large, 13-21µm; wing-like muri thin,project 3-10 µm, terminating margin waved or almost entire. Exine: visiblethrough perine, rough rugulate under SEM. Specimens examined: M.Kato, Gen Murata & JP MogeaB7407

126

Table 7.1. Continued

No. Species Morphological characteristic of Spores

9. D. malaccense Monolete, bilaterally summetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) elliptical; equatoriallongitudinal view planar convex to concave-convex; equatorialtransverse view, proximal face concave, distal view convex.Laesura: concealed by wing-like muri. E:37.73(34.24)27.53±3.03; P: 26.01(20.80)13.33±2.82. Perine:alate to costate-alate, often loosely reticulate; irregular envelopeseparated from exine surrounds the spore in continuousanastomosing wings, forming a loose reticulation; wing-like muriproject 1.5-8 µm, terminating margins sparsely ciliate. Exine:visible though perine, smooth granulate under SEM. Specimensexamined: T.Ng. Praptosuwiryo 831, 944-3

10. D. megasegmentumPraptosuwiryo

Monolete, bilaterally symmetrical (mades asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex; equatorial longitudinal view (excludingperine) concave-convex; equatorial transverse view, proximalface convex, distal face convex; perinate. E:39.61(51.42)57.01±4.28P: 18.51(31.49)35.85±3.59. Laesura: concealed by perine.Perine: alate; alae sometimes loosely reticulate; wing-like muriproject 4-13 m, terminating margins almost entire. Specimens examined : T.Ng. Praptosuwiryo 1451.

11. D. pallidum (Blume)Moore

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical; equatorial longitudinal view (excluding perine) plano-convex; equatorial view tranverse view proximal face convex,distal face hemispherical; perinate. Size: E:32.65(47.96)65.02±8.98; P: 17.08(27.09)34.12±5.12Laesura: concealed by perine. Perine: alate under LM, costateunder SEM, irregular envelope separated from exine surrounds thespore in irregular, wing-like costae, often anastomosing to formloose reticulation, lacunae shallow irregular polygons 15-18 µmwide, muri 0.8 – 5 µm wide, surface of muri smooth and lacunaesmoothly granulate. Exine: visible through perine, granulateunder LM.Specimens examined: T.Ng. Praptosuwiyo 1688, 1759; HolttumSFN 31350; J. v. Borsum 2787.

12. D. polypodioidesBlume

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropalar; polar ouline (excluding perine) transversely elliptical,sides convex; equatorial longitudinal view plano-convex toconcave-convex; equatorial tranverse view, proximal view planarto concave, distal view convex; perinate. Size: E:34.36(39.63)43.17± 2.33, P: 16.31(22.92)27.43±2.97Laesura: concealed by wing- like perine. Perine: costate-alate,loosely reticulate; irregular envelope separated from exinesurround the spore with costae forming loose irregular reticulationor wing-like muri, reticulation often incomplete, lacunae largeirregular polygons, 3-9 µm across; wing-like muri project 1-7µm,terminating margins entire; surface or perine smooth under SEM.Specimens examined: T.Ng. Praptosuwiryo 1582, 1604, 1612,1651.

127

Table 7.1. Continued

No. Species Morphological characteristic of Spores

13. D. porphyrorachis(Baker) Diels

Monolete, bilaterally summetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex; equatorial longitudinal view (excludingperine) plano-convex, ; equatorial tranverse view, proximal,proximal face planar, distal convex; perinate. E:39.89(46.39)50.98±3.53, P: 21.74(27.57)30.49±2.87Laesura: concealed by perine. Perine: alate to costate-alate, alaesometimes anastomosing forming loose reticulation; irregularenvelope, separated from exine, surrounds the spore in looseanastomosing wing, forming a very loose reticulation, reticulationoften incomplete; lacunae very irregular polygons, 10-16 macross; muri thin, wing like muri project 1.5-9.0 m; terminatingmargin ciliate or papillate; surface of perine holed, holes irregularand formed by fallen papillae. Specimens examined: W. Meijer872, T.Ng. Praptosuwiryo 930

14 D. prescottianum(Wall.) Moore

Monolete, bilaterraly symmetrical (made asymmetric by perine),heteropolar, polar outline (excluding perine) transverselyelliptical; equatorial longitudinal view plano-convex; equatorialtransverse view, proximal face planar, distal face concave;perinate.E: 32.72(39.11)51.44±5.16, P: 20.23(24.79)33.56±3.63Laesura: concealed by perine. Perine: alate, costate-alate, looselyreticulate; irregular envelope separated from exine surrounds thespore in continuous anastomosing wings, forming a loosereticulation; lacunae large, irregular polygons 5-8 µm; thin wing-like muri project 4-9 µm , terminating margins ciliate or echinate.Exine: visible through perine, smooth-granulate under SEM.Specimen examined: J.v. Borssum W. (17-6-1953)

15. D. procumbens Holtt. Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical; equatorial longitudinal view (excluding perine) plano-convex; equatorial transverse view, proximal face convex, distalface hemispherical; perinate. Size: E: 43.03(53.78)63.20±4.72,P: 29.52(33.22)37.31±2.39Laesura: concealed by perine. Perine: alate under LM, costateunder SEM, irregular envelope separated from exine surrounds thespore in irregular, wing like costae, often anastomosing to form aloose reticalution, lacunae shallow irregular polygons 8-22 µm,muri 1.6-6.6 µm, surface of muri and lacunae smooth. Specimensexamined: R.E. Holttum 36503; T.Ng. Praptosuwiryo 1455.

16. D. profluensPraptosuwiryo

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline elliptical, sides convex; equatoriallongitudinal view concave-convex to plano-convex; equatorialtransverse view, proximal face planar to concave, distal facehemispherical; perinate. E: 36.44(44.06)63.70±8.92; P:23.89(29.04)43.07±5.73. Laesure: concealed by perine ridge.Perine: micro costate, costae broken, reticulate irregularly, denselyechinate under SEM, echinae single elements irregularly apart andprojecting 1.1-2.1 µm. Exine: often visible through perine,smooth. Specimen examined: T.Ng. Praptosuwiryo 1798

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Table 7.1. Continued

Species Morphological characteristic of Spores

17. D. simpliciveniumHoltt.

Monolete, bilaterally symmetrical, heteropolar; polar outlineelliptical, sides convex; equatorial longitudinal view plano-convexto concave-convex to plano-convex; equatorial transverse view,proximal face planar to concave, distal face hemispherical;perinate.E: 43.99(50.61)54.60±3.36, P: 23.31(29.42)33.78)±2.79Laesure: concealed by perine ridge. Perine: alate to costae alate,irregular envelope separated from exine surrounds the spore inirregular and in continuous or discontinuous anastomosing wing,forming a loose reticulation; lacunae large irregular polygons,occasionally with granulate deposite within; thin wingilike muriprojected 3-8µm, terminating margins are almost entire. Exine:smooth regulate under SEM. Specimens examined: T.Ng.Praptosuwiryo 1250

18. D. silvaticum(Bory) Sw.

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar, polar outline (excluding perine) transverselyelliptical; equatorial longitudinal view (excluding perine) concave-convex; equatorial transverse view, proximal face concave, distalface hemispherical; perinate. E: 31.59(41.31)49.30±3.82, P:18.64(23.56)26.41±2.46Laesura: concealed by perine. Perine: alate under LM, costate-alate under SEM, irregular envelope separated from exine; alaeform loose reticulation; lacunae irregular polygons c. 6.6-20.0 µmacross, interior of lacunae showing micro irregularreticulate/fenestreta under SEM; muri very thin, wing-like,terminating margin irregularly echinate. Exine: smooth-

19. D. speciosumBlume

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex-straight; equatorial longitudinal view(excluding perine) concave-convex; equatorial tranverse view,proximal face convex, distal face biconvex; perinate. E:29.98(39.29)47.25±5.53; P:17.37(21.69)25.83±2.56. Laesura:concealed by perine ridge. Perine: alate, costate-alate, irregularenvelope separated from exine; alae occasionally form looseretication; lacunae irregular polygons c. 6.6-10.8 µm across,interior of lacunae smooth under SEM; terminating margins ofwing-like muri entire. Exine: rugulate, rugulae large, shallow,irregular anastomousing under SEM after separating from perine.Specimens examined: T.Ng. Praptosuwiryo 1242; 1805

20. D. spiniferumAlderw.

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excludng perine) transversely elliptical,side convex; equatorial longitudinal view concave-convex toplano-convex; equatorial transverse view, proximal face planar toconvex, distal face hemispherical; perinate. E:31.96(39.52)47.77±4.18, P: 14.87(23.92)29.21±4.14. Laesura:concealed by perine. Perine: micro-costate, densely echinateunder SEM, costae broken, densely micro reticulate, coralline;irregular envelope, separated from exine, surrounds the spore indensely coralline with enchinae project 1.5-2.3 µm; lacunae arevery small, irregular polygons less than 0.5-1.5 µm across; heavilyfenestrate throughout under SEM, giving coralline appearance.Exine: visible through perine. Specimens examined: M.Kato, M.Okamoto & EB Walujo B10800

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Table 7.1. Continued

Species Morphological characteristic of Spores

21. D. sorzogonensePresl

Monolete, bilaterally summetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex; equatorial longitudinal view (excludingperine) plano-convex; equatorial transverse view, proximal faceplanar to convex; distal face hemispherical; perinate. E:27.80(33.03)37.90±3.91, P: 13.95(20.92)26.39±3.82. Laesura:concealetd by perine. Perine: alate, costate-alate, irregularenvelope separated from exine; lacunae irregular polygons c. 8.3-16.6 m accross; terminating margins of costae or wing-like murientire. Exine: smooth under SEM.Specimens examined: T.Ng. Praptosuwiryo 1713.

22. D.subpolypodioidesBlume

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar; polar outline (excluding perine) transverselyelliptical, sides convex-straight; equatorial longitudinal view(excluding perine) concave-convex; equatorial tranverse view,proximal face convex, distal face biconvex; perinate. E:32.49(40.29)47.17±4.08, P: 17.15(21.54)28.28±2.90Laesura: concealed by perine ridge. Perine: alate, costate-alate,irregular envelope, reparated from exine; costae or alae sometimesform loose reticulation; lacunae irregular polygons c. 3.3—12.5µm, interior of lacunae smooth under SEM; muri, wing-like,smooth, terminal margin entire. Exine: smooth under SEM.Specimens examined: T.Ng. Praptosuwiryo 1335, 1605

23. D. subserratum(Blume) Moore

Monolete, bilaterally symmetrical, heteropolar; polar outlineelliptical, sides convex; equatorial longitudinal view concave-convex to plano-convex; equatorial transverse view, proximal faceplanar to concave, distal face hemispherical; perinate. Size:E:27.88(41.19)45.49±5.03, P: 16.54 (27.13)20±5.03. Laesure:concealed by perine ridge. Perine: costate-alate, irregularenvelope, separated from exine; costae form irregular loosereticulation; lacunae irregular polygons c.to 23 µm across; interiorof lacunae and costae smooth. Exine: visible throught perine,smoothly granulate under LM. Specimens examined: T.Ng.Praptosuwiryo 1705, 1704

24. D. subvirescensPraptosuwiryo

Monolete, bilaterally symmetrical (made asymmetric by perine),heteropalar; polar outline (excluding perine) transversely elliptical,sides convex; equatorial longitudinal view (excluding perine)plano convex, equatorial transverse view, proximal face planar,distal view convex; perinate. Laesura: concealed by wing-likealae spinous coralline perine. Perine: alate to costate alate, alae orcostae forming irregular reticulation, reticulation often incomplete;lacunae with echinae and coralline within, wing-like muri project3-11 µm, terminating margins pappilate or echinate; pappilaeproject c.0.7-1.5 µm; surface of perine echinae – coralline withdensely irregular hole, windows-like. Exine: not visible throughperine under SEM. Specimens examined: T. Ng. Praptosuwiryo1178.

130

Table 7.1. Continued

Species Morphological characteristic of Spores

25. D. tomentosumBlume

Monolete, bilaterally summetrical, heteropolar; polar outlineelliptical, sides convex, equatorial longitudinal view plano-concex-concave convex, equatorial transverse view, proximal faceplanar to concave, distal face convex; perinate. E:34.64(41.06)47.19±3.88, P: 23.14(26.77)28.67±2.13. Laesura:concealed by perine. Perine: alate to costate-alate, looselyreticulate; irregular envelope separated from exine surrounds thespore in continuous anastomosing wings, forming a loosereticulation, occasionally wing-like alae only form 3 reticulationsurraound the spore; lacunae large irregular polygons, papillaewithin; thin wing–like muri projected 3-12 µm, terminatingmargin are often ciliate, ciliae projected to ca. 0.5 µm. Exine:visible through perine, granulate under LM. Specimens examined:T.Ng. Praptosuwiryo 827, 829, 911.

26. D. vestitum Presl Monolete, bilaterally symmetrical (made asymmetric by perine),heteropolar, polar outline (excluding perine) transverselyelliptical, sides vonvex; equatorial longitudinal view (excludingperine) concave-convex; equatorial tranverse view, proximal faceconvex, distal face convec to hemispherical; perinate. E:28.21(32.58)35.33±2.26, P: 16.55(19.94)22.73±1.64. Laesura:concelated by perine. Perine: costate-alate, almost no reticulationirregular envelop, separated from exine, surrounds the spore withalae-costae ridge (occasionally) projected 2-5 µm; surface ofperine smooth. Exine: visible through perine, smooth under SEM.Specimens examined: M.Kato & H. Wiriadinata B 6097

Spores of West Malesian Diplazium fall into nine main pattern groups

based on the perine ornamentation. The nine group are discussed as follows.

(1) Group I. The spore form most common among the diplazioid ferns of

Western Part of Malesian region are costate, non-fenestrate, and ornamented with

a wing-like pattern raised to form a pattern of slightly rounded ridges. D.

accedens, D. bantamense, D. lobbianum, D. polypodioides, D. pallidum, D.

procumbens, D. sorzogonense, D. speciosum, D. subpolypodioides, D. dilatatum,

and D. xiphopyllum are included in this group. The existence of additional

ornaments, however, would differentiate among the species. Reticulation type of

wing-like muri or wing-like costae, terminating margin of wing-like muri, and

surface of perine (both on lacunae and wing-like muri) of these members are in

varying character states. Therefore these characters can be used to support species

delimitation. Spores of D. accedens, D. bantamense, and D. polypodioides are

similar appearance. They are seen incomplete reticulation of wing-like muri or

131

costae often incomplete with terminating margins entire or smooth. D. accedens

and D. polypodioides differ with D. bantamense in the projection of wing-like

muri or costae. The two first species showed wing-like muri or costae project to 7

m, while the third species has wing-like muri or costae project to 13 m. D.

accedens and D. polypodioides would differ in the size of their irregular polygons

lacunae, the first species is with 10 m or more across while the second species 9

m or less across.

(2) Group II. Second group included D. subserratum and D. vestitum are

revealed spores with costate-alate and sparsely fenestrate perine, micro holes are

irregularly scattered. The existence and its density of the pappilae on the wing-

like muri would be important in differentiating taxa in this group. D.

subbserratum showed entire terminating margin of wing-like muri, while D.

vestitum have ciliate wing-like of muri.

(3) Group III. The third group included members D. crenatoserratum and

D. prescottianum have alate and sparsely fenestrate perine spores. Additional

decoration of perine on both lacunae and wing-like muri add the variation of spore

sculpture of this group.

(4) Group IV. Spores of D. silvaticum are very distinct among the

diplazioid species of Western Part of Malesian region. Its wings-like muri and

lacunae are coralline and fenestrate, irregular windows or holes large (ca. 0.5-1.5

m across) and decorated with irregular dense echinae on terminating margins of

wing-like muri.

(5) Group V. One species of Diplazium showed a rather simple

ornamentation is D. esculentum. Its perine is micro rugulate without wing-like

muri or costae. Spores with monolete, bilaterally symmetrical, elliptical, sides

convex and regulate perine is also found in other genera, such as Athyrium. Using

LM, Tseng-Chieng (1981) observed spores of this species under the name

Anisogonium esculentum and described that its perine are reticulate with scabrate

processes. As reported by Large & Braggins (1991), spores with micro-rugulate

perine is showed in Athyrium filix-femina. However this species is decorated with

costae sparsely that formed shallow and very large lacunae. A. erythropodum and

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A. vidalii of Thailand observed by Tseng-Chieng (1981) were also revealed

rugulate spores.

(6) Group VI. Alate non-fenestrate perine sculpture can be seen in D.

cordifolium, D. tomentosum, D. simplicivenium, D. megasegmentum, and D.

malaccense. The variation of wing-like muri ornamentation, such as its

reticulation, lacunae formed, projection, and terminating margins, differentiate

among species. Spores of D. tomentosum and D. malaccense are similar. The

two species would differ in the projection of wing-like muri. The first species is

with wing-like muri project to 14 m, and the second with projection of wing-like

muri to 8 m. D. cordifolium differ from other species of this group in its

incomplete reticulation and large lacunae (to 16 m or more across). Spores of D.

simplicivenium differ from those of D. megasegmentum in the existence of

granulate deposit in lacunae. The first have granulate deposit within lacunae,

while the second is without granulate deposit in the lacunae.

(7) Group VII. Spores that are decorated in echinate perine and non-

fenestrate are found in D. profluens. Echinae is only a single elements and its

projection size is diverse. Density and size projection of echinae would be very

useful for differentiating species in this group. Echinate spores under LM were

also showed in D. subsinuatum and D. virescens of Thailand (Tseng-Chieng

1981). The projection of echinae was also useful to differentiate the two species,

echinae of D. subsinuatum spores were far longer than of those D. virescens.

(8) Group VIII. Diplazium spiniferum, D. lomariaceum, D.

porphyrorhachys are have spores with coralline, echinate and fenestrate

sculptures. Three species can be differentiated by its more detail perine

sculptures. D. spiniferum shows densely corraline with echinae project 1.5-2.3

m with very small lacunae (0.5-1.5 m). D. lomariceum has large lacunae (13-

21 m) with wing-like muri project 3-10 m. The projections of wing-like muri

of D. porphyrorachis (1.5-9.0 m) and its across size of irregular lacunae

polygons (10-16 m) are overlap with those of D. lomariceum. Morphologically,

the two last species are very similar.

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Fig. 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. Bar = 15 m for a, b, j, ; Bar = 30 m for c,d, e, g, h, and k. Bar = 3 m for i and j.

134

Figure 7.2. Group II. a. D. subserratum; b-c. D. vestitum; d-e. D. vestitum var.borneense; Group III. f-g. D. crenatoserratum; h-i. D. prescottianum; Group IV. j-l. D.silvaticum. Bar = 30 m for a, b, d, f, h, j, and i. Bar = 3 m for c, e, g, i, and k.

135

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. Bar =30 m for a, c, d, e, f, g, h, i, k, and m. Bar = 3 m for b, h, l, n, and o.

136

Figure 7.4. Group VII. a-c. D. profluens; Group VIII. d. D. spiniferum; Group IX.e-f. D. subvirescens. Bar = 30 m for a, b, and e. Bar = 3 m for c, d, and f.

(9) Group IX. Spores that have form alate-echinate and fenestrate are

found in D. subvirescens. In a glance, this spore type is similar to those of D.

profluens. However D. subvirescens spores would be differentiated from D.

profluens spores with its micro lacunae sculpture. The lacunae decorated with

coralline and densely irregular windows.

Diplazium spores of Thailand observed by Tseng-Chieng (1981) using LM

revealed alate to costate-alate sculpture in eight species, viz. D. dilatatum, D.

squamigerum, D. wichurae, D. megaphyllum, D. mettenianum, D. petri, D.

phaeolepis and D. doederleinii. More detail of the its perine surface, however,

can not be seen so that the comparison between these costate-alate species and

costate-alate species of Malesia could no be done. Because Tseng-Chieng (1981)

did not provide the spores figures from SEM.

137

This study indicates that perine characteristics are very important in

supporting delimitation of species Diplazium observed. So that a tentative key for

species of Diplazium observed is successfully given.

Key to the Species of Diplazium based on Spore Morphological Characters Observed byUsing SEM

1. Perine alate to costate-alate

2. Perine coralline, fenestrate on wing-like muri ………………………………….......... D. silvaticum

2. Perine not coralline, not fenestrate on wing-like muri

3. Terminating margins of wing-like muri or costae almost entire

4. Reticulation often incomplete

5. The mean of equatorial size over 40 m or more

6. Wing-like costae project up to 13 m; exine smooth under LM D. bantamense

6. Wing-like costae project up to 5 m; exine visible granulate under LM ................................................................... D. pallidum

5. The mean of equatorial size less than 40 m

6. Lacunae (irregular polygons) 10 m or more across …........... D. accedens

6. Lacunae (irregular polygons) 9 m or less across ……............. D. polypodioides

4. Reticulation complete

5. Perine alate

6. Lacunae with granulate deposit within ….................. D. simplicivenium

6. Lacunae without granulate deposit within

7. Exine regulate ……………………………............ D. lomariceum

7. Exine smooth

8. Wing-like muri project up to c. 7 m ........... D. procumbens

8. Wing-like muri project up to c. 13 m .. D. megasegmentum

5. Perine costate-alate

6. Surface of perine (lacunae) smooth granulate . ..... D. subpolypodioides

6. Surface of perine (lacunae) smooth irregular fibrous

7. Wing-like muri project up to c. 4 m ...………… D. speciosum

7. Wing-like muri project up to c.6 m ………….. D. sorzogonense

138

3. Terminating margins of wing-like muri or costae echinate or ciliate

4. Reticulation often incomplete

5. Lacunae to 12 m across

6. Surface of perine smooth or scarcely micro papillate; not coralline ………………………………………………… D. xiphophyllum

6. Surface of perine densely sharp echinae or spine; coralline .… D. subvirescens

5. Lacunae to 16 m or more across

6. Perine not forming holes from fallen ciliae ……………..… D. cordifolium

6. Perine forming holes from fallen ciliae

8. Like-fiber materials on muri not forming continuous lines …………………………… D. porphyrorachis

8. Like-fiber materials on muri forming continuous lines …………………………... D. crenatoserratum

4. Reticulation complete

5. Terminating margins of wing-like muri almost entire or smooth …. D. subserratum

5. Terminating margins of wing-like muri ciliate

6. Costae or alae usually not forming reticulations ……………… D. vestitum

6. Costae or alae forming many reticulations

7. Perine alate

8. Wing-like muri project to 9 m

9. Wing-like muri with irregular holes ………. D. prescotianum

9. Wing-like muri without irregular holes

10. Wing-like muri project up to 8 m …….. D. malaccense

10. Wing-like muri project up to 15 m ….…. D. lobbianum

8. Wing-like muri project to 14 m ………….………. D. tomentosum

7. Perine costate-alate

8. Lacunae without irregular holes within ………...…. D. dilatatum

1. Perine not alate to costate-alate

2. Perine rugulate ………………………………….……………………………… D. esculentum

2. Perine ciliate to coralline-echinate

3. Not fibrous-coralline, not fenestrate . ……………………………………….. D. profluens

3. Thick fibrous-coralline, fenestrate .............................................................. .............. D. spiniferum

139

7.3.2. Phylogenetic Analysis

The evolutionary trends presented by Van Konijnenburg-van Cittert

(1999) can not be applied in plylogenetic relationship in Diplazium because they

appear suitable to be implicated to genus level and above. All derived spore

characters states summed up by Konijnenburg-van Cittert (1999) are exist in

Diplazium (see Table 1.) All species of Diplazium usually reveal 32 and 64 spores

per sporangium for apogamous and sexual, respectively. Therefore the direction

of the characters state transformation was determined by method of outgroup

comparison. Table 5.2. showing the characters and character states utilized in

phylogenetic analysis of Diplazium. The rooting of the inferred phylogenetic

relationship revealed in Figure 1. was conducted by selecting spore description of

Athyrium filix-femina cited from Large & Braggins (1991).

The 17 spore morphological characters (Table 7.2. and 7.3.) were

analysed to determine relationship among the 27 species of West Malesian

Diplazium. The 100 equally most parsimonious trees of 131 steps produce a

consistency index (CI) = 0.53 and retention index (RI) = 0.50.

Figure 7.5. showed tree number 1 of 100 the most parsimonius trees.

However the strict consensus of 100 the most parsimonious trees resulted a

polytomy tree with very low supported Boostrap value (Fig. 7.6.). Separation

between the ingroup and outgroup was also without a high supported Bootsrap

value.

The results showed that there is an incongruence between the tree

generated from gross morphological data and those generated from spore

morphology (See Chapter 6). As showed in Figure 7.6., the concensus tree of

100 most parsimonius tree, relationship among the species of Diplazium based

on spore morphology are polytomy and elusive. Although topological of tree

number 1 of 100 most parsimonious trees showed differentiation among 27

species analyzed (figure 7.5.), however, it did not give any logical relationships

between the two closely related species.

Figure 7.5. showed that both the main clades and sub clades are formed by

the grouping of species that are not closely related species based on the gross

morphological evidences. For example, D. porphyrorachis and D. lomariaceium

140

are closely related (See Chapter 5). In the clade II the two species were separated,

the first species was in the same dichotomous branch with D. cordifolium and the

second species was forming dichotomous branch with D. prescottianum. In the

morphological tree, D. porphyrorachis and D. lomariacium were uniting,

meanwhile D. prescottianum formed a dichotomous branch with D. pallidum. As

explained in the Chapter 6, the reasonable affinity of D. porphyrorachis should

be with D. lomariaceum, and D. pallidum with D. prescottianum.

Incongruence between two independent or more data in phylogenetic

analysis of ferns occasionally occurs. Dubuisson et al (1998) has evaluated the

interaction between three independent data sets (anatomy/morphology, cytology

and molecules) within the ferns genus Trichomanes (Hymenophyllaceae). It was

revealed that the data sets showed high and significant level on incongruence.

The illogical relationship of the spore topological tree result in this study

also indicate that superficial similarity of mature perispores may not be the best

indicator of systematic relationship among species. This indication was also

showed by Rangker (1989) in the study on spore morphology of new aorld

Hemionitis, Gymnopteris, and Bommeria (Adiantaceae). Ranker (1989)

presumed that similar surface patterns of spore may be derived through different

development pathways.

141

Figure 7.5. The cladogram of tree number 1 of 100 the most parsimonius trees

142

Figure 7.6. The strict consensus of 100 the most parsimonious trees. The numberabove line showed supported by the Bootstrap value.

143

Table 7.2. Characters, character states, and coding for 17 characters utilized in construction of spore morphology dataset of Diplazium

(0) (1) (2) (3) (4) (5) (6) (7) (8) (9)1. Equatorial longitudinalview

concave-convex

plano-convex

biconvex {01}

2. Proximal face concave planar convex {01} {12}3. Distal face hemispherical convex concave biconvex4. The Equatorial sizemean

31.47-32.37 32.58-34.24

34.25-38.18 38.19-39.63

40.29-42.15

2. 44.06 45.69-47.39

47.96 50.61-51.42

53.78-53.88

5. The Polar size mean(um)

19.51-19.94 20.80-2154 21.69-22.92 23.48 -24.57

24.88-26.28

26.37-27.57

28.55-29.04

29.42 31.48-33.22

6. The existence ofperine fold

no fold with fold

7. Perine fold type costate costate-alate

alate echinate {12}

8. End of ridge rounded slightlycrested

{01}

9. The existence ofcoralline

not exist exist

10. Type of perine foldreticulation

incompletereticulate

completereticulate

{01}

11. The widest lacunae 1.50 – 2.50 2.60-4.60 4.70-6.70 6.80-10.7 10.8-12.5 10.90-18.5 18.6-20.3 20.4-23.7 23.8-26.112. Fold of CrestMargine

Smooth/entire Waved Papillate Ciliate Echinate {01} {23} {24} {34}

13. The longestprojection of wing likemuri or costae

1-2.1 2.9-4 5 6-7 8 9 10-11 12 13 15

14. Perine surface Smooth Fibrous-like

Ciliate/papilate Holed Echinate Smallgranulate

{05}

15. Existence ofmicroscopic holes onperine

Absent Present

16. Exine appearanceunder SEM

Smooth Granulate Rugulate {01}

17. Exine appearanceunder LM

Smooth Granulate Rugulate {01}

144

Table 7.3. Coding for 17 characters utilized in construction of spore morphology dataset of Diplazium

Species Characters and Characters States

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Athyrium filix-femina 3 3 0 2 4 1 0 0 0 0 0 0 0 5 0 2 0D. accedens 3 3 0 0 0 1 1 0 0 0 0 0 3 0 0 0 0D. bantamense 0 0 0 9 8 1 1 1 0 0 9 0 8 0 0 0 0D. cordifolium 1 1 1 4 5 1 3 2 0 1 7 4 4 0 0 0 0D. crenatoserratum 0 0 1 3 3 1 2 1 0 0 6 2 4 1 1 0 0D. dilatatum 0 0 1 6 6 1 3 1 0 0 0 3 9 0 0 0 0D. esculentum 3 3 0 3 5 0 - 0 - - 0 - 0 0 0 1 2D. lobbianum 3 3 0 6 4 1 1 1 0 0 0 6 - 2 0 0 0D. lomariaceum 2 1 0 3 3 1 3 1 0 - 0 5 6 0 0 2 2D. malaccense 3 0 1 1 1 1 3 2 0 1 0 3 4 0 0 0 1D. megasegmentum 0 2 1 8 8 1 2 1 0 0 0 0 8 0 0 - -D. pallidum 1 2 0 7 8 1 0 0 0 0 6 0 2 0 0 1 0D. polypodioides 3 3 1 3 2 1 1 2 0 0 3 0 3 0 0 1 0D. porphyrorachis 1 1 1 6 5 1 3 1 0 0 7 0 5 0 0 0 0D. precottianum 1 1 2 3 3 1 3 1 0 1 3 8 4 0 0 0 3D. procumbens 1 2 0 9 9 1 2 2 1 0 8 5 3 0 0 0 0D. profluens 3 3 0 5 6 1 0 1 0 0 0 4 0 4 0 0 0D. simplicivenium 3 3 0 8 7 1 3 1 0 2 0 0 4 6 0 3 0D. silvaticum 0 0 0 4 3 1 2 1 0 0 7 4 - 0 0 3 0D. speciosum 0 2 3 3 1 1 3 1 0 0 4 0 - 0 0 2 0D. spiniferum 3 4 0 3 3 0 1 1 0 0 0 - 0 0 0 0 0D. sorzogonense 1 4 0 1 1 1 3 2 0 0 6 0 - 0 0 0 0D. subpolypodioides 0 2 3 4 1 1 3 2 0 0 4 0 - 0 0 0 0D. subserratum 3 3 0 4 1 1 1 2 0 1 8 0 - 0 0 - 0D. subvirescens 1 1 1 - - 1 3 1 1 0 0 7 - 4 1 - -D. tomentosum 3 3 1 4 5 1 3 2 0 1 0 3 7 0 0 1 0D. vestitum 0 2 3 1 0 1 1 0 0 0 0 0 2 0 0 0 0D. xiphophyllum 3 4 1 4 4 1 3 2 0 0 3 2 6 0 0 0 0

145

7.4. Conclusions

All species Diplazium have monolete, bilateraly symmetrical, heteropolar,

polar outline elliptical, perinates with laesura concealed by its perine. Perine

ornamentation varied from alate to costate alate and rugulate with variation of

additional decoration such as ciliae, coralline, echinae, and fenestrae. Perine

ornamentations support in delimitating Diplazium species. Nevertheless

morphological variation of spore is inadequate to depict natural relationship

among Diplazium species. Superficial similarity of mature perispores would not

indicate on systematic relationship among species as similar surface pattern it

derived through different development pathways.

146

CHAPTER 8

MOLECULAR SYSTEMATIC OF DIPLAZIUM FROM WEST MALESIA

8.1. Introduction

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). Phylogenetic analysis on Diplazium based on morphological characters

(Chapter 6) revealed that Diplazium has only a little informative morphological

characters and a hight homoplasy (HI=0.76). The lack of the informative

morphological characters and the high of homoplasy suggests that additonal

caharcters are required to asses the relationship within this genus.

Advances in molecular biology have provided systematists with a valuable

source of characters. Correlating in inferring phylogeny of the vascular plants,

plant sistematists are depending upon chloroplast genome. Most phylogenetic

reconstructions in plant systematics conducted so far is based on molecular data

from cpDNA genes. The chloroplast genome is well suited for evolutionary and

phylogenetic studies, because: (1) the chloroplast genome is small (typically

between 120 and 200 kb), making it relatively easy to examine the entire genome

via ristriction site analysis; (2) it contains primarily single copy genes; (3) has a

conservative rate of nucleotide subtitution; and (4) extensive background for

molecular information on the chloroplast genome is available (Soltis & Soltis

1998).

The most common gene used to provide sequence data for plant

phylogenetic analysis is the plastid-encoded rbcL gene (Chase et al 1993). It is

located in the large single-copy region of the chloroplast genome and encodes the

large subunit or ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO).

This single copy gene is aproximately 1430 base pairs in length; insertions or

deletions (indels) are extremely rare.

147

The use of gene rbcL sequences for phylogenetic analysis has been

reviewed in Palmer et al (1988). On family level and above, rbcL is preferred for

inferring phylogeny. rbcL are not only widely used in the analyzing the extant

taxa, but also found in the leaves fossil samples from Miocene, ca. 17-20 millions

ago (Golenberg et al. 1990, Soltis et al, 1992; Manen et al, 1995). This gene have

proved useful in elucidating higher-order phylogenetic relationships in

angiosperms (Chase et al, 1993; Duvall et. al. 1993).

Recent use of rbcL sequence data in constructing pteridophyte phylogeny

showed that interspecific as well as higher order relationship could be resolved

(Hasebe et al. 1994; Wolf et al. 1994; Hennequin et al 2006). rbcL also could

circumscribe the genera of the fern family (Crane 1997; Smith et.al. 2006).

Phylogenetic trees based on rbcL sequences support the current classification that

the Matoniaceae and its genera, Matonia and Phanerosorus, each are treated as

monophyletic groups (Kato & Setoguchi 1999). Moreover a large number of rbcL

sequence variation has been reported in various species such as Asplenium nidus

(Yatabe et al 2001), Hymenasplenium obliquissimum (Murakami et al 1998),

Stegnogramma pozoi (Thelypteridaceae) (Yatabe et al 1998), Osmunda

cinnamomea, O. claytonia, and O. regalis (Osmundaceae) (Yatabe et al 1999) and

Cheiropleuria bicupsis (Dipteridaceae) (Kato et al 2001). The substitutions rates

of rbcL in Osmundaceae were estimated to be one nucleotide substitution

between two rbcL sequences occurs only one in 5 million years on average

(Yatabe et al 1999). Considering this slow evolutionary rate for rbcL, the large

amount of rbcL sequence variation within a single morphological species may

suggest that these species contain several cryptic species that are reproductively

isolated from each other.

rbcL gene sequence has been used as a tool in unraveling the taxonomic

problems in ferns. Based on rbcL gene sequence which are also supported by

morphological and cytological characters, Sano et al (2000a) moved D.

subsinuatum and D. tomitaroanum into Deparia lancea and Di. tomitaroana,

respectively. The phylogenetic analysis on nucleotide sequences of the

chloroplast-encoded rbcL gene that included Loxoscaphe thecifera (Aspleniaceae)

and Actiniopteris radiata (Pteridaceae) results robust clades and consequently the

148

two species should be included into Asplenium and Onychium, respectively

(Gastony & Johnson 2001). Moreover characters of rbcL sequences information

are useful in the discovery of cryptic species in ferns, e.g. Asplenium nidus

(Yatabe et al 2001).

rbcL gene sequence has been applied in studying the phylogeny of the

Lady fern group, tribe Physematieae (Dryopteridaceae) (Sano et al 2000a). Based

on the chloroplast rbcL gene sequence included 42 species of tribe Physematieae,

including Diplazium, Sano et al (2000a) showed that: (1) the monophyletic

Diplazium clade included Monomelangium; (2) Athyrium, Cornopteris,

Pseudocystopteris, and Anisochampium form a monophyletic clade and Athyrium

is polyphyletic. However the result of Sano et al (2000a) is preliminary because

they examined only few (10 species) of the ca. 400 species of Diplazium. In their

preliminary inferred phylogeny also showed that Diplazium wichurae from

eastern Asia and D. lonchophyllum from central America form a clade in the rbcL

trees. They suggested that more detailed studies including species from wider

areas should be useful for biogeographic studies of Diplazium.

In this present study, gene rbcL sequences from 54 collections number

representing 29 species of West Malesian Diplazium and 9 collections number of

9 references species outside Malesia were analyzed. The objectives of this were

five-fold: 1) to recognize genetic diversity within species; 2) to obtain supporting

evidences in species delimitation; 3) to obtain more informative characters for

inferring phylogenetic hypothesis of Diplazium; 4) to evaluate the monophyly of

Diplazium, and 5) to investigate species-level relationships within the genus.

8.2. Materials and Methods

DNA analysis was carried out at the Laboratory of Molecular Systematic

of Department of Biological Science, University of Tokyo, from 2002 to 2003

and 2006-2007. Materials were collected from 25 localities of Java, Sumatra, and

Borneo (Table 8.1.).

149

8.2.1. DNA Analysis

a. Plant materials.

Sample used were obtained from fresh dried material (kept in Silica Gell)

collected both from the field and plant growing at green houese of Bogor Botanic

Gardens (Table 8.1.). Voucher specimens were deposited in Herbarium

Bogoriense (BO) and Herbarium of Bogor Botanic Gardens(BOHB).

b. DNA Extraction.

DNA was extracted by a modified CTAB method of Doyle and Doyle

(1978) or using the Dneasy Plant Mini Kit (Qiagen, Inc., Tokyo) following the

manufacturer’s intruction. (1) 500 um CTAB was poured in the 1.5 ml tube and it

was incubated on 60ºC. (2) A piece of lamina (± 4 cm2) was ground on the bowl

by using liquit nitrogen until forming smooth powder . (3) The smooth powder

was poured into the tube containing 500 um CTAB and shaked for 1 minute and

then incubated in 60ºC for 30-60 minutes.

c. Separating DNA from Protein.

(1) 500 um of cloroform and isoamil alcohol mixture was poured into the

tube containing sample and 500 um CTAB and shaked by hand for 10 minutes. (2)

The tube was centrifuged on 20ºC for 20 minutes at 15000 rpm. (3) The top layer

(containing water with DNA) was poured into the 1.5 um tube. Step no.1 – no.3

were repeated once more.

d. DNA Purification.

(1) 400 ul Isoamilalcohol was added into the tube containing DNA liquit

and than kept in the freezer for15 minutes or longer. (2) The tubes were

centrifuged at 0ºC for 15 minutes at 15000rpm. (3) The liquid was disposed of

(the residu was DNA). (4) 300 ul 70% Ethanol was added into the tube and

shaked by using vortex and centrifuged at 20ºC for 10 minutes 15000rpm. (5)

The ethanol was evaporated by using aspirator. (6) Tubes containing DNA was

dried in the desicator for10 minutes. (7) The DNA was dissolved in 100um TE

and than shaked well.

150

e. rbcL Multiplication by Using PCR.

PCR was performed using a DNA thermal cycler (Perkin-Elmer 9700),

Applied Bosystems, CA) with Ex Taq DNA polymerase (TaKaRa Biomedical,

Tokyo). Some samples were amplified with Amp direct (Shimazu, Kyoto), whike

most others were amplified with Ex buffer. (1) 10xPCR Buffer, dNTPmix, 10 pM

primer x 2, MgCl2, DNA template, Taq polymerase and DW (Destilate Water)

were poured into 0.2 ml tubes. Taq must be kept at 0ºC (in the ice box) before

PCR was begun to run. (2) PCR was runned at 94ºC for 3 minutes and turned on

35 cycles at 94ºC for 30 seconds, at 48ºC for 40 second, at 72ºC for 90 seconds,

and at 72ºC for 7 minutes and than the tubes were kept at 4ºC.

f. Checking PCR Product by Using Electrophoretic

(1) making gel agarose LE 1%. (2) 3.5 ul PCR product was mixed with

dye in the parafilm and than poured into sample holes (3) Elektroforesi was

runned (4) When blue colour reaching the forth line electrophoretic was stopped.

Gel was moved and pu t into etidiumbromid for 20 minutes. (5) Gel was

illuminated by using UV beam; if bars pictures with proportion sizes were seen it

was concluded that PCR product have been multiplied well.

g. PCR Product Purification

PCR products were purified with GFX DNA and GEL purification Kit

(Amarsham Pharmacia Biotech, Piscataway, NJ) or with ExoSAP-IT (USB

corporation, Ohio) following the manufacture’s intruction. a. Autocycle. (1) 3

ul premix, 0.5 ul sequence buffer, 0.5 ul MgCl2, 1.6 ul 2pm primer, and 4.4 ul

DNA were put into the 0.2 ml tubes. The premix was containing of dNTP,

ddNTP, buffer and taq polymerase. Taq polymerase must be kept at 0ºC before

autocyle. (2) PCR was begun at 96ºC for 90 second and than runned at 30 cycles

96ºC for 10 second, at 50ºC for 15 second and at 60ºC for 4 minutes and than the

tubes were kept at 4ºC. b. Purifying Autocycle Product. (1) 12.5ul 100 Etanol

and 0.5um 3M sodium asetat were put into 1.5 ml tubes (2) Autocycle product

were added into the tubes and shaked by using vortex. (3) The tubes were kept in

the ice box for 20 minutes. (4) The tubes were centrifuged for 20 minutes at 20ºC

15000rpm and than their liquit were run off. (5) 250ul etanol 70% was added into

151

the tubes and than shaked by using vortex. (6) The tubes were centrifuged at

20ºC for 10 minutes at 15000rpm speed and than ethanol was evaporated by

using aspirator. (7) Autocycle product in the tubes were dried in the desiccator

for 15 minutes.

h. Gel Squencing Preparation

(1) Glass plate for gel was washed off with water and rinsed off with

miliQ and isoprophanol, and than dried at room temperature. (2) preparing gel.

18g urea was mixed with 5 ml 10x TBE buffer and amount of water and than

stirred by using magnetic stirrer for 10 minutes (3) The mixture was added with

water till 48 ml volume. (4) The mixture was evaporated by using aspirator and

than kept in the refrigerator (5) The mixture was evaporated by using dessicator

for 5 minutes. (6) 10 % APS was made in the tube. (7) Glass plate was lay out

on the flat tabel, tweezers were tightened (8) TEMED 30ul and 270ul APS 10%

were poured into the mixture and shaked manually (9) The Gel was poured into

the glass plate and than chomb was installed. (10) After 2 hours, manicure was

smeared for preventing buffer spilled out.

i. Preparing Samples for Squencing

(1) Formamide and blue dextran (5:1) mixtured was made. 3ul DNA

sample for every coll. number were also prpepared. All samples and blue dextran

must alwasy be kept in the ice box and blue dextran. (2) The tubes containing

samples were shaked by using shaker for 5 minutes. (3) The tubes cantaining

were incubated at 98ºC for 2 minutes and than kept in the ice box..

j. Run Sequencer

(1) Chomb was removed from gel and than changed with the chomb with

36 holes. (2) Glass plate gel was washed and rinsed off with isopropanol and

mili-Q till the white line not seen. (3) Glass plate gel was installed on the

chamber sequencer and than cassette recorder was also installed on the

Sequencer. (4) Checking glass plate gell (if the noise voice was heared from the

machine we must clean again the plate. (5) Chamber was installed on the

sequencer and than 1x TBE buffer was poured into the chamber. (6) All of the

bubbles attaching the holes chomb must be removed by using injector.

152

Table 8.1. List of Taxa Used in This Study

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Outgroup

Athyrium distentifolium Unknown AB059577 Yatabe andMurakami(2001)

Athyrium filix-femina (L.) Roth exMeretens

AY818676 Skog et al (2004)

Athyrium niponicum (Mett.) Hance Tokyo, Japan CT1005 TI AB232413 Tsutsumi and Kato(2006)

Athyrium vidalii (Miq.) Koidz. Chiba, Japan Sano 25 CBM D43894 Sano et al (2000b)Athyrium sheareri Aichi, Japan Sano 41 CBM D43892 Sano et al (2000b)Athyrium yokoscense (Fr. Et Sav.)Christ

Chiba, Japan Sano 22 CBM D43893 Sano et al (2000b)

Ingroup

Diplazium angustipinna Holttum (

Near Sungai Kobet, Track to BatuAyau, Muller Range, CentralKalimantan, Borneo. 440 m.

T.Ng. Praptosuwiryo1905

BO Present Study

Diplazium asymmetricum PraptosuwiryoSp. Nov.

Petak 4 Desa Kemutuk Lor, Convertedforest-Natural forest boundary,Wanawisata Baturraden, Mt. Slamet,Cetral Java. 970-1000 m.

T.Ng. praptosuwiryo1094

BO Present Study

Diplazium asymmetricum PraptosuwiryoSp. Nov. (2=1)

Loop Trail, Cikaniki Forest Research,Mt. Halimun, West Java. Ca. 1000 m

T.Ng. praptosuwiryo1728

BO Present Study

Diplazium cordifolium Blume ‘simplefronds’

Cangkuang Forest, Southern Slopes ofMt. Salak, West Java

T.Ng. Praptosuwiryo1202

BO Present Study

153

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium cordifolium Blume‘Pinnate fronds’

Cangkuang Forest, Southern Slopes ofMt. Salak, West Java

T.Ng. Praptosuwiryo1237

BO Present Study

Diplazium cordifolium Blume‘Pinnate fronds’

Cangkuang Forest, Southern Slopes ofMt. Salak, West Java

T.Ng. Praptosuwiryo1238

BO Present Study

Diplazium cf cordifolium Blume Primary forest between S. Anak Kobet-S. Kobet, track to Batu Ayau, CentralKalimantan, Borneo.

T.Ng. Praptosuwiryo1912

BO Present Study

Diplazium accedens Blume Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1001

BO Present Study

Diplazium accedens Blume Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1161

BO Present Study

Diplazium accedens Blume Cangkuang Forest, Southern Slopes ofMt. Salak, West Java.

T.Ng. Praptosuwiryo1211

BO Present Study

Diplazium bantamense Blume Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park,West Java

T.Ng. Praptosuwiryo1160

BO Present Study

Diplazium batuayanense Praptosuwiryo Above Sungai Kobet, Track to BatuAyau, Muller Range, CentralKalimantan, Borneo. 440 m.

T.Ng. Praptosuwiryo1909

BO Present Study

D_cavalerianum (Christ.) C.Chr. Chiba, Japan Sano 11 CBM D43909 Sano et al (2000b)D. chinense (Bak.) C.Chr. Kumamoto, Japan Ohta &Takamiya 739 KUMA AB021718 Sano et al (2000b)

154

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium dilatatum Blume Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1011

BO Present Study

Diplazium simplicivenium Holttum Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1025

BO Present Study

Diplazium simplicivenium Holtum Converted Forest, Petak 55, DesaKarang Mangu, Wana WisataBaturraden, G. Slamet, Baturraden,Central Java. 860 m.

T.Ng. Praptosuwiryo1073

BO Present Study

Diplazium dilatatum Blume Kagoshima, Japan Ohta & Takamiya602

KUMA AB021719 Sano et al (2000b)

Diplazium donianum (Mett.) Tard. Kagoshima, Japan Sano 29 CBM D43911 Sano et al (2000b)Diplazium esculentum (Retz.) Sw. Situ Patengan, Mt. Patuha, Bandung,

West Java.T.Ng. Praptosuwiryo730

BO Present Study

Diplazium esculentum (Retz.) Sw. Gede-Pangrango National Park, WestJava

T.Ng. Praptosuwiryo1227

BO Present Study

Diplazium esculentum (Retz.) Sw. Kagoshima, Japan Hasebe 276001 TI U05619 Hasebe et al (1994)Diplazium hottae Tagawa Near Sungai Anak Kobet, Track to

Batu Ayau, Muller Range, CentralKalimantan, Borneo. 450 m.

T.Ng. Praptosuwiryo1911

BO Present Study

Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1007

BO Present Study

155

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1031

BO Present Study

Diplazium lobbianum Moore Cibodas Forest, Behind CibodasBotanic Gardens, Track I Mt. Gede,Gede-Pangrango National Park, WestJava. 1500 m

T.Ng. Praptosuwiryo1181

BO Present Study

Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park,West Java

T.Ng. Praptosuwiryo1008

BO Present Study

Diplazium lonchophyllum Kunze Unknown U05920 Wolf et al (1994)Diplazium megasegmentumPraptosuwiryo Sp.Nov.

Cangkuang Forest, Southern Slope, Mt.Salak, West Java.

T.Ng. Praptosuwiryo1450

BO Present Study

Diplazium mesosorum Tochigi, Japan Sano 44 CBM D43910 Sano et al (2000)Diplazium okudairaekamiDiplazium pallidum (Blume) Moore Near Telaga Warna, Gede-Pangrango

National Park, West JavaT.Ng. Praptosuwiryo1172

BO Present Study

Diplazium pallidum (Blume) Moore Mt. Payung, Ujung Kulon NationalPark, West Java

T.Ng. Praptosuwiryo1406

BO Present Study

Diplazium poiense C.Chr. in C.Chr. &Holttum

Unknown TML1

Diplazium polypodioides Blume Cangar Nature Reserve, Mt. Welirang,Batu, East Java. 1370-1400 m.

T.Ng. Praptosuwiryo667

BO Present Study

Diplazium polypodioides Blume Cibodas Forest, Mt. Gede, Gede-Pangrango National Park, West Java

T.Ng. Praptosuwiryo1185

BO Present Study

156

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium porphyrorachis Unknown TML2Diplazium procumbens Holttum Cibodas Forest, Track I of Mt. Gede,

behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1015

BO Present Study

Diplazium procumbens Holttum Cibodas Forest, Track I of Mt. Gede,Behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1047

BO Present Study

Diplazium procumbens Holttum Cibodas Forest, Behind CibodasBotanic Gardens, Track I Mt. Gede,Gede-Pangrango National Park, WestJava

T.Ng. Praptosuwiryo1163

BO Present Study

Diplazium procumbens Holttum Converveted Forest-Natural ForestBoundary, Petak 4 Desa Kemutuk LorWana Wisata Baturraden, Mt. Slamet,Central Java. 970-1000 m.

T.Ng. Praptosuwiryo1094

BO Present Study

Diplazium procumbens Holttum Cimisblung, Mt. Masigit, Mt.Pangrango, Gede-Pangrango NationalPark. 1300 m.

T.Ng. Praptosuwiryo1281

BO Present Study

Diplazium procumbens Holttum Cangkuang Forest, Souther Slopes ofMt. Salak, West Java.

T.Ng. Praptosuwiryo1216

BO Present Study

Diplazium rhachidosorus Unknown TML3Diplazium riparium Holttum Unknown TML4

157

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium sibiricum var. sibiricum Akita, Japan Ohta & Takamiya695

KUMA AB021720 Sano et al (2000b)

Diplazium silvaticum (Bory) Sw. Wild fern of Bogor Botanic Gardens,Java

T.Ng. Praptosuwiryo1300

BO Present Study

Diplazium silvaticum (Bory) Sw. Wild fern of Bogor Botanic Gardens,Java

T.Ng. Praptosuwiryo1302

BO Present Study

Diplazium silvaticum (Bory) Sw. Wild fern of Bogor Botanic Gardens,Java

T.Ng. Praptosuwiryo1303

BO Present Study

Diplazium simplicivenium Holttum Track to Cibeureum, Mt. Gede, Gede-Pangrango National Park, West Java

T.Ng. Praptosuwiryo1141

BO Present Study

Diplazium simplicivenium Holttum Rawa Denok I, Gede-PangrangoNational Park, West Java

T.Ng. Praptosuwiryo1171

BO Present Study

Diplazium simplicivenium Holttum Near Batu Kukusan I, Gede-PangrangoNational Park, West Java

T.Ng. Praptosuwiryo1175

BO Present Study

Diplazium simplicivenium Holttum Cibodas Forest, Mt. Gede, Gede-Pangrango National Park, West Java

T.Ng. Praptosuwiryo1179

BO Present Study

Diplazium sorzogonense C. Presl. Loop Trail , Cikaniki Research Center,Mt. Halimun, West Java. Ca. 1000 m.

T.Ng. Praptosuwiryo1720

BO Present Study

Diplazium sorzogonense C. Presl. Loop Trail , Cikaniki Research Center,Mt. Halimun, Halimun National Park,West Java. Ca. 1000 m.

T.Ng. Praptosuwiryo1725

BO Present Study

Diplazium sorzogonense C. Presl. Loop Trail, Cikaniki Forest ResearchCenter, Mt. Halimun, HalimunNational Park, West Java. Ca. 950 m.

T.Ng. Praptosuwiryo1731

BO Present Study

Diplazium sorzogonense C. Presl. Jalur Owa, Cikaniki Reseacrh Center,Mt. Halimun, West Java. Ca. 975 m.

T.Ng. Praptosuwiryo1744

BO Present Study

158

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium speciosum Blume Geger Bentang, Mt. Pangrango, Gede-Pangrango National Park, West Java.1960 m.

T.Ng. Praptosuwiryo1243

BO Present Study

Diplazium squamigerum (Mett.)Matsum.

Kumamoto, Japan Ohta & Takamiya893

KUMA Sano et al (2000b)

Diplazium subpolypodioides (Alderw.)Alderw.

Cibodas Forest, Mt. Gede, Gede-Pangrango National Park, West Java.

T.Ng. Praptosuwiryo1184

BO Present Study

Diplazium subserratum (Blume) Moore. Converted Forest, Petak 55, DesaKarang Mangu, Wana WisataBaturraden, G. Slamet, Baturraden,Central Java. 860 m.

T.Ng. Praptosuwiryo1070

BO Present Study

Diplazium subserratum (Blume) Moore. Converted Forest, Petak 55, DesaKarang Mangu, Wana WisataBaturraden, G. Slamet, Baturraden,Central Java. 860 m.

T.Ng. Praptosuwiryo1072

BO Present Study

Diplazium subvirescens Praptosuwiryo Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park1500 m.

T.Ng. Praptosuwiryo1178

BO Present Study

Diplazium subvirescens Praptosuwiryo Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National

T.Ng. Praptosuwiryo1012

BO Present Study

Diplazium tomentosum Blume Loop Trail, Cikaniki Forest Research,Mt. Halimun, West Java. Ca. 1000 m

T.Ng. Praptosuwiryo1722

BO Present Study

Diplazium umbrosum (Smith) Bedd. Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1002

BO Present Study

159

Table 8.1. Continued

Species Locality Collectorand Number

HerbariumHoldingVoucher

GenebankAccessionNo.

References

Diplazium umbrosum (Smith) Bedd. Cibodas Forest, Track I of Mt. Gede,behind Cibodas Botanic Gardens GuestHouse, Gede-Pangrango National Park

T.Ng. Praptosuwiryo1050

BO Present Study

Diplazium wichurae (Mett.) Diels Shizuoka, Japan Sano 13 CBM D43915 Sano et al (2000b)Diplazium xiphophyllum (Baker) C.Chr. Eas Kalimantan, Borneo TD 902 Living Coll.

at Nuserryof BBG

Present Study

Diplazium xiphophyllum (Baker) C.Chr. Loop Trail, Cikaniki Forest Research,Mt. Halimun, West Java. Ca. 950 m.

T.Ng. Praptosuwiryo1717

BO Present Study

Diplazium xiphophyllum (Baker) C.Chr. Loop Trail, Cikaniki Forest Research,Mt. Halimun, West Java. Ca. 950 m.

T.Ng. Praptosuwiryo1719

BO Present Study

Diplazium xiphophyllum (Baker) C.Chr. HM 39 Cikuda Paeh, Cikaniki ForestResearch, Mt. Halimun, West Java.

T.Ng. Praptosuwiryo1791

BO Present Study

160

(7) Prerun for 5 minutes. (8) Stopping for a moment and than samples with odd

number collectons were injected into the odd holes. (9) Resume for 5 minutes.

(10) Stop prerun and than the sample with the even collentions number were

injected into the even holes and than the chambercover was installed (11)

Running.

k. Gene rbcL sequence alignment.

The PCR product were sequenced with the Big Dye Terminator Cycle

Squencing Kit Ver. II or Ver. III (Applied Biosystems, CA) following

manufacturer’s intructions with amplification primers of Hasebe et al (Tabel 1.).

l. Homology searching on the data base by internet (using a reprsentative

database of nucleotide sequence, DNA DataBase of Japan (DDBJ,

http://www.ddbj.nig.ac.jp/) to compare predicted gene sequence with the

registered sequence in the database.

Table 8.2. Primers Used for Amplifying and Sequencing DNA from Diplazium(Hasebe et al, 1994)

Primer Sequence (5’ to 3’) Position

rbcL-aF

rbcL-bF

rbcL-sR

rbcL-aR

rbcL-cF

rbcL-sF

rbcL-cR

rbcL-bR

ATGTCACCACAAACAGAGACTAAAGC

TATCCCCTGGATTTATTTGAGGAAGGTTC

GAACCTTCCTCAAATAAATCCAGGGGATA

CTTCTGCTACAAATAAGAATCGATCTCTCCA

TGAAAACGTCGTGAATTCCCAACCGTTTATGCG

ACTGTAGTGGGCAAATTGGAAGGCGAACG

GCAGCAGCTAGTTCCGGGCTCCA

CGTTCGCCTTCCAATTTGCCCACTACAGT

1-26

307-335

335-307

670-640

609-638

988-1016

1373-1351

1016-988

161

8.2.2. Phylogenetic analysis

DNA sequences were edited and aligned by using Clustal W. All

characters were treated as equally weighted and unordered. Maximum

Parsimony (MP) analysis was performed with PAUP 4.0b10 (Swofford 1998)

using the heuristic search option with 100 random replicates of stepwise data

addition with TBR swapping and Multrees set to 200 trees limit. Boostrap analysis

(Felsenstein 1985) was performed with 200 replicates to evaluate internal support.

Athyrium filix-femina, A. distentifolium, A. niponicum, A. sheareri. A.vidalii and

A. yokoscence were selected as outgroup taxa.

8.3. Results and Discussion

Chloroplast rbcL gene sequences of 25 species of Diplazium from West

Malesia were newly generated (Table 1). Length of the sequences of the 29

species are varying, from 640 to 1445 base pair (bp). There are many missing

data on gene between pair number 722 – 1445. Therefore 721-bp region from the

pair number 1 to 721 were used to make comparison between two cytotypes in a

species or among the closely related species and used for phylogenetic analysis.

However the 721-pb region possess many informative characters, viz. 544 (75%).

8.3.1. Infraspecific Genetic Diversity in Diplazium

Intraspecific nucleotides differences in Diplazium are showed in Table

8.3. The differences among individuals in the same species are 0 – 5 nucleotides,

but in D. dilatatum, D. procumbens and D. subvirescens. There are not any

differences on gene rbcL squences in D. esculentum, D. polypodioides, D.

subserratum, D.simplicivenium, D. silvaticum, and D. xiphophyllum (TNgP1717 -

1719). As revealed in Chapter 5, the two first species are diploid. D. subserratum

showed diploid, triploid and tetraploid. D. simplicivenium and D. silvaticum are

triploid and tetraploid, respectively. Whereas the voucher specimen of the last

species was not know its chromosomes number. These results indicate that these

species are autoploid.

162

The diploid and tetraploid of D. pallidum differ from each other by five

nucleotides between pair number 74 – 721. There are missing data on the diploid

(TNgP 1406) on pair number 1-73. Takamiya et al (2001) reported genetic

comparison between triploid and tetraploid of Diplazium doederleinii. The two

cytotypes differ from each other by only two nucleotides in the rbcL sequences,

and there are no variations in cytotype except for a single plant of the triploid.

Tabel 8.3. Infraspecific Genetic Variaton of Diplazium based on Gene rbcL Sequences

Species Number ofNucleotidesDifference

Notes

D. esculentum TNgP 730 – TNgP 1227 0D. esculentum (Kagoshima-Japan) – Java (TNgP1227)

3

D. accedens TNgP 1001 – TNg 1161 4 In the same locality, G. Gede-G.Pangrango National Park

D. accedens TNgP 1161 (G. Gede-Pangrango) –TNgP 1211 (G. Salak)

1 On nucleotide number 649, inwhich TNgP 1161 has Thymineand TNgP 1211 has Cytosine

D. cordifolium ‘simple’ TNgP 1202 – D.cordifolium ‘pinnate’ (TNgP 1237)

1 On nucleotide number 340, inTNgP 1202 Cytosine pyrimidinebase and TNgP 1237 has Adeninpurine base

D. cordifolium TNgP1238 - 1237 10D. cordifolium TNgP 1912 (berbulu) – D.cordifolium TNgP 1237

8

D. xiphophyllum TD902 – TNgP1717 1D. xiphophyllum TNgP1717 - 1719 0D. polypodioides TNgP 677, TNgP 1184, 1185 0D. procumbens 1015 - 1047 5D. procumbens 1015-1216 7D. procumbens 1047-1216 6 Missing data on nucleotide

number 1-4 and 649 to 721 inTNgP 1216

D. pallidum tetraploid ( 1172)– diploid (TNgP1406)

5 Missing data on TNgP 1406 in1-73

D. subserratum TNgP 1070 - 1072 0D. lobbianum TNgP 1031-1181 5 Missing data on TNgP 1181 in

nucleotide number 1-3D. lobbianum TNgP 1181 - 1008 4D. lobbianum TNgP 1031 - 1008 4D. silvaticum TNgP 1300 – 1302 0D.simplicivenium TNgP 1025, 1073, 1141,1171, 1175, 1179

0

D. dilatatum TNgP 1011 (G. Gede Java –ABO21719 (Japan)

16

D. subvirescens TNgP 1012 – D. subvirescensTNgP 1178

22

163

The high number of nucleotides differences of D. dilatatum, D.

procumbens and D. subvirescens are presumed to due they are hybrid. In Java,

the three species mentioned above are triploid apogamous. However their putative

parents have not been found. Further study are needed to prove this suggestion.

8.3.2. Species Delimitation in Diplazium based on Gene rbcL Sequence

These results revealed that rbcL nucleotides variations in Diplazium are

large enough, but between the two pair of the closely related species D.

sorzogonense TNgP 1744 – D. batuayauense and D. xiphophyllum TNgP 1791 -

D. hottae TNgP 1911. Among species are differentiated from each other by 4-37

nucleotides between pair number 1– 721 (Table 8.4.). The more closely related

species the lower the differences number of nucleotides. Morphological character

of D. cordifoliuman is very similar to D. angustipinna. The two species are

sharing in: imparipinnate fronds with copiously anastomosing veins; scales light

brown, lanceolate, margin entire. The pinnate fronds plant of D. cordifolium

differs from D. angustipinna by 11 nucleotides between pair number 103-363.

While the simple fronds plant of D. cordifolium differs from D. angustipinna by 4

nucleotides between pair number 63-717. The distantly related West Malesian

species of D. megasegmentum and D. porphyrorachis are difference each other in

19 nucleotides. The first species is a huge plant with tripinnate fronds whereas the

second species is a small plant with pinnatifid fronds. These results revealed that

gene rbcL sequences are well supporting in delimiting species in Diplazium.

Recently comparison of rbcL sequences among species on ferns showed

that five or more nucleotides differences are enough to differentiate species. In

genus Diplazium (Sano et al. 2000a, 2000b) also showed that rbcL nucleotide

variation are great, more than four nucleotides, between different species, while

there are no or only two sequence changes within species. The intraspecific

variation study at the molecular level (using rbcL sequencing) in the Japanese

Asplenium of sect. Thamnopteris showed that there are as many as 14-23

differences in the rbcL sequences between Asplenium australasicum (J. Sm.) and

A. setoi N. Murak. et Seriz. (Murakami et al. 1999b).

164

Tabel 8.4. Interspecific Genetic Variaton of Diplazium based on Gene rbcL Sequences

Species Number ofNucleotidesDifference

Notes

D. donianum – D. bantamense 18D. bantamense – D. lobbianum TNgP 1031 13D. cordifolium (TNgP 1737) – D. riparium(TML4)

16

D.esculentum – D. cavalerianum 37D. esculentum – D. chinense 16D. wichurae – D. okudairaekami 5D. wichurae – D.lonchophyllum 17D. squamigerum – D. sibiricum 6D. mesososrum – D. rhachidosorus 12D. cavalerianum – D. okudairaekami 36D. pallidum = D. procumbens TNgP 1163D. cordifolium ‘simple’ – D. angustipinnaTNgP 1905

4

D. cordifolium TNgP 1238 – D.angustipinna TNgP 1905

11

D. xiphophyllum TNgP 1791 – D. hottaeTNgP 1911

1

D. speciosum TNgP 1243– D. batuayauenseTNgP 1909

15

D. xiphophyllum TNgP 1791 – D. ripariumTML4

7

D. dilatatum TNgP 1011 – D.simplicivenium TNgP 1025

16 Missing data in 13 sites

D. dilatatum TNgP 1011 – D. procumbensTNgP 1015

12

D. bantamense TNgP 1160 – D. lobbianumTNgP 1181

11

D. sorzogonense TNgP 1720- 1725 -1731 –1744 – D. batuayauense

0

D. simplicivenium TNgP 1179 – D.dilatatum TNgP 1011

18 Missing data on manyplace, mainly in nucleotidenumber 711-721

D.megasegmentum (TNgP 1450) – D.prophyrorachis

19 TNgP 1450 (nucleotidenumber 63-76 is in N)

D. porphyrorachis(missing data onnucleotides number 699 –721)

D. poiense – D. porphyrorachis 14 D. poiense (missing dataon nucleotide number 649-721)

165

The lack or low of nucleotides difference between D. sorzogonense TNgP

1744 – D. batuayauense and D. xiphophyllum TNgP 1791 - D. hottae TNgP 1911

should be re-checked again in the future studies as the length of the gene rbcL

compared in this study are only 721-bp region from the pair number 1 to 721. The

nucleotides differences between the two pair of these species may be existed

between the pair number 722 – 1445. Because morphological studies (See

Chapter 9) showed D. batuayauense and D. sorzogonense are having many

differences. D. batuayauense differs from D. sorzogonense in the following

characters: Scales sharply toothed with thickening dark brown stand irregularly,

fronds are much smaller, never reaching 1 m, lacks the fibrillose scales on stipe

and rachis, pinnae lobed only to ½ way to costa, sori not impressed. Therefore D.

batuayauense is propsed as new species. The morphogical differences between D.

hottae and D. xiphophyllum can be seen in the Chapter 9.

8.3.3. Informative Characters of Gene rbcL Sequences for Inferring Phylogenetic Hypothesis of Diplazium

In the molecular phylogenetic relationships studies on ferns, generally

rbcL shows a high parsimony informative characters. Wolf et al. (1994) reported

465 phylogenetic informative characters of the 1320 nucleotide sites of

Dennstaedtioid ferns. In the molecular phylogenetic relationships study in the

heterosporous fern genus Marsilea Nagalingum et al (2007) obtained 58

parsimony informative characters from 1233-pb. Sano et al. (2000a) reported 342

parsimony informative characters in the phylogenetic analysis of 68 taxa of ferns

in inferring phylogenetic relationship of the Lady fern group, tribe Physematieae,

including Diplazium, by using 1206 bp region between 73-1278. Lu et al. (2007)

showed 144 parsimony informative characters from 1320-necleotide segment of

the rbcL gene in studying the phylogeny of the Polystichoid ferns in Asia. Hauk et

al (2003), in the phylogenetic studies of Ophioglossaceae, the adder’s tongue

ferns that has limited the number of characters available for understanding

relationships, got 341 potentially parsimony-informative characters of the 1321-

bp.

The DNA sequence of West Malesian Diplazium that only included 721

base pair (bp) contained a high parsimony informative characters, 544 (75%) of

166

parsimony-informative characters. This result showed that gene rbcL sequence is

a good enough marker for inferring phylogenetic hiphothesis of Diplazium.

8.3.4. Phylogenetic Analysis

The 721-bp region from the pair number 1 to 721, numbered from the

initial methionin codon of Nicotiana tabacum was used for analysis. As a

references, species outsite of Malesia are included in the analysis: D.

lonchophyllum from Central America and D. cavalerianum, D. mesosorum, D.

rhachidosorus, D. sibiricum, D. squamigerum and D. wichurae from Japan and

(Table 8.1.).

The aligned matrix of the 77- taxon contained 544 (75%) of parsimony-

informative characters, 35 (5%) variable of parsimony-uninformative characters

and 142 (20%) constant characters. The consistency index (CI) and retention

index (RI; Farris 1989) are 0.79 and 0.94, respectively. The topology of Most

Parsimony (MP) of 8 trees and those strict concensus trees are generally similar

(Figure 8.1.).

8.3.4.1. The Monophyly of Diplazium

Recent molecular phylogenetic analysis of Diplazium and closely related

genera revealed that Diplazium is monophyletic. Preliminary phylogenetic study

of Diplazium conducted by Sano et al (2000a) based on chloroplast rbcL gene

sequences showed the monophyletic of Diplazium clade supported by Bootstrap

value 86%. This clade includes Monomelangium pullingeri (Bak.) Tagawa. By

using evidence from chloroplast trnL-F region sequences Wang et al (2003)

showed the monophyletic of Diplazium clade that include Callipteris Bory,

Allantoidea R. Br. Emend. Ching, and Diplaziopsis C. Chr. with supported

Bootstrap value 98%.

All species studied in this study form a monophyletic group. The

monophyly of Diplazium is strongly support in the tree with 100% bootstrap

value. It is revealed that separation of Diplazium from Athyrium are strongly

resolved. Thus, the monophyly of West Malesian Diplazium has been verified

based on this molecular study.

167

Morphologically, Diplazium can be distinguished from other close related

genera (including from Athyrium) by characters combination as follow (Kato,

1977): (1) stipe bases neither swollen nor bearing pneumatophores; (2) Frond

axes U-shaped with flat base in most species; (3) Acroscopic basal pinnules equal

or smaller; lamina margin not cartilaginous, spines absent; (4) Vein free, or

goniopetrid or sagenoid – anstomousing, ending near the margin; (5) Sori dorsal

on the vein and linear, either single (Asplenoid) or double (Diplazioid); generally

Asplenoid sori along the acroscopic side of a vein and Diplazioid sori are confined

to both sides of the basal acroscopic; both parts of double sori of about equal

length; (6) Scales entire or toothed with consisting of two upturned ends of

adjacent marginal cells. This morphological study (see Chapter 9) supported the

previous workers (Ching 1940; van Alderwerelt van Rosenburgh 1908; Alston

1956; Sledge 1962; Holttum 1940, 1966; Kato 1977, 1995; Edie 1978; Tagawa &

Iwatsuki 1988; Andrews 1990; Kramer et al. 1990).) in separating Diplazium from

Athyrium.

8.3.4.2. Relationships among species within Diplazium

In the monophyletic of Diplazium, D. porphyrorachis form a clade alone

and diverges earliest (Clade I), it is the most basally positioned in all studied taxa

(Clade II). But the divergency of D. porphyrorachis from the all taxa studied is

without well-supported Bootstrap value. Subsequently Clade II diverges into two

main clades without well-supported Boostrap value, viz. Clade II.1. that contains

only D. subvirescens (TNgP 1012) and Clade II.2. that diverges again into four

main clades, one main well-supported group, Clade A, with Bootstraping value

100% and three main weakly-supported group, Clade B, C, and D. The

relationships inferred among the species included in these cades are discussed

below. Before discussing the relationship inferred among the species, the

polytomy occurrence in the topological tree generated is discussed first.

In ferns, polytomy relationship patterns among species on the evolutionary

relationship inferred that generated from molecular data are usual. As showed in

the Figure 8.1., Clade A2 diverges into six subclades. Subsequently in the

terminal clade, Diplazium from Japan, Eastern Asia and Central America are also

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forming a polytomy. The polytomy pattern is also showed in the sister clade of D.

riparium that comprised D. hottae, D. xiphophyllum, D. cordifolium, and D.

angutipinna. Dubuisson et al (1998) showed polytomy relationship pattern of the

sister clade of Trichomanes thysanostomum that consisted of T. minutum, T.

bipunctatum, T. speciosum, and D. diaphanum. In the monophyly of the section

Lepidoglosa (Elaphoglossaceae), Elaphoglossum asterolepis, E. splendens and E.

rufidulum formed a polytomy (Rouhan et al. 2004). Hauk et al (1993) showed the

polytomy pattern in the sister clade of Ophioglossum pusillum that included O.

reticulatum, O. vulgatum, and O. richardsiae. The polytomy patterns indicate that

more data, including more taxa and more informative characters, are needed in

studying the relationships of the taxa in the future.

D. porphyrorachis. D. porphyrorachis is the sister clade of the main

clade (Clade II). Morphologically, D. prophyrorachis is very distict among the

West Malesian Diplazium. D. porphyrorachis posses a suite of morphological

characters distinguishing it from other West Malesian species. As presented in

Chapter 6 and 9 this species is having diagnostic characters as follow: Lamina

pinnatifid lanceolate apex lobed or toothed, the lower 2/3–6/7 deeply pinnatifid

into many or numerous, close, spreading, subfalcate, linear oblong, blunt, slightly

crenate, serrate or toothed segments 8-15 mm broad, which have not seldom the

sides entire and only the apex serrate; lower segments gradually growing smaller,

the 1-4 lowest free and deflexed. The position of D. prophyrorachis at the basal

tree of the rbcL tree is also supported by the topology of the phylogenetic tree

generated from morphological data. In the tree generated from morphological

data, D. porphyrorachis is forming a clade that include its closely related species,

viz. D. fuliginosum and D. lomariaceum and also at the basal clade of the

monophyly of West Malesian Diplazium. As stated by Price (1983) D.

prophyrorachis and its closely related species, D. lomariceum and D. fuliginosum,

constitute unusual Malesian Diplazium that agree in the following characters:

rhizome short-erect, with thick wiry black roots; fronds narrowly elliptic, pinnate

of subpinnate, apex coadunate, segments numerous, usually dark; scales abundant

on stipe and rachis, narrow, usually dark and shiny; veins free.

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Figure 8.1. Strict consensus of the 200 equally most-parsimonious trees obtained inmaximum parsimony analysis of the rbcL sequence data. Bootstrap values are indicatedabove the branches occurring in more than 50% of 100 bootstrap replicates.

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Clade C. Clade C without well Bootstrap support comprised D.

procumbens 1047, D. asymmetricum 1094, D. subvirescens 1178, D.

asymmetricum 1728, D. procumbens 1281. Due the very low level of Boostrap

value, it is difficult to state the relationship among them. Therefore each species

is discussed separately below.

D. asymmetricum. This species in a glance is very similar to D.

procumbens as the two species are having affinity in characters combination:

scales on stipes fallen when mature, dark brown, ovate-lanceolate, margin with

blunt teeth; lamina oblong; lobes oblong with blunt or truncate apex; veinlets

forked. Diplazium assymetricum differs from D. procumbens in the following

characters combination: rhizome short, erect; lamina more incised (to tripinnate)

basiscopic pinnulae or segments and lobes are larger than the acroscopic ones;

indusia thicker, margin entire. Therefore it is possible that the two species are

closest related in the phylogenetic tree inferred based on gene rbcL sequence.

Diplazium subvirescens. In the topology of rbcL tree, Diplazium

subvirescens (TNgP 1012) constitute the sister clade of Clade II in which this

speices is also included in unresolved Clade II.2. (TNgP 1178). Cytological

observations on two individuals of this species (Chapter 5) showed only triploid

apogamous and it is presumed hybrid. Suggestion that this species is originally

hybrids may be support by the evidence from the gene rbcl sequence. Two plants

successfully sequenced (TNgP 1012 and TNgP 1178) showed differences each

other in 22 nucleotides. This difference number was high enough although

morphologically the two collection number are very similar and should be treated

as one species. Morphologically, D. subvirescens is closely related to D.

virescens. As mentioned in the Chapter 9., D. subvirescens has affinity to D.

virescens in the characters combination as follows: rhizome long creeping, black

with densely scales on younger part; scales on stipes lineary lanceolate, polish

dark brown with toothed margin; lamina deltoid, deep green, firm herbaceous with

veinlets prominent on both surface; sori oblong to linear, medial to supramedial;

indusia thin-membranaceous, laciniate at margin, irregurarly broken at maturity.

Two collections number that successfully examined their chromosomes number

(TNg 1013 and TNgp 1177) (See Chapter 5) showed triploid and the two

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collentions number are having similar morphological characters with TNgP 1012

and TNgP 1178. Therefore, based on these cytological and rbcL evidence it is

suggested that this species are hybrids.

Diplazium procumbens. Diplazium procumbens distributes on four

different clades in rbcL Most Parsimonious tree. Firstly, this species is sister

species of D. pallidum that has subcoriaceous pinnate fronds with lineary

lanceolate sharply acuminate toothed pinnae (up to 27 pairs) less than 3 cm wide

with veins forked 1-3 times and soriferous on upper simple branch, with

Bootstrap value 62%. Secondly, D. procumbens form a clade with D. dilatatum

(TNgP 1011) without supporting Bootstrap value. Thirdly, D. procumbes (TNgP

1047; 1281) form a clade with D. asymmetricum and D subvirescens (TNgP 1177)

without supporting Bootstrap value. The latter, D. procumbens (TNgP 1216)

form a clade alone and become the sister clade of well resolved of Clade A and

weakly supported of Clade B dan C.

D. procumbens is bipinnate diplazioid fern with rhizome long creeping,

very narrow slightly toothed scales, veins copiously forked in the oblong lobes

and sori with thin indusia that break down in the middle. Therefore the

relationship between D. pallidum and D. procumbens in the topological tree

generated from gene rbcL is not logical. In the tree generated from morphology,

D. pallidum is closely related to D. prescottianum (See Chapter 6) and this

relation is more logic.

Cytological observations on eight individuals of D. procumbens from five

localities in Java (See Chapter 5., Praptosuwiryo & Darnaedi 2004, 2005) showed

triploid apagamous. DNA rcbL sequence showed that nucleotides differences

within D. procumbens are high enough, viz. from 1 – 10. As mentioned above,

whereas, interspecific nucleotides differences in Diplazium are 4 – 37 (Table 8.4.).

It is indicate that D. procumbens may be polyphyletic and originally hybirids. In

Malesia, D. procumbens is distributed in Malay Peninsula (Holttum 1940),

Sumatra (present study) and Jawa (Praptosuwiryo 1999).

The position of D. procumbens and D. subvirescens in two or more

different clades may be correlated with the hybrid occurrence in these species.

Funk (1985) stated that if hybridization has occurred among the species of a taxon

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under cladistic analysis the results are varied but always present additional

difficulties. Hybridization results in incongruent intersecting data that obscure the

underlying hierarchy.

Clade B. Clade B consisted of D. donianum, D. tomentosum, D.dilatatum

and D. simplicivenium without Boostrap support. It is difficult to make a

statement about the relationship among these species due to the very low level of

the Bootstrap value, but between D. simplicivenium and D. dilatatum, D.

simplicivenium may has affinity to D. dilatatum. The two species

morphologically very similar (see Chapter 9). Whereas the relationship between

D. donianum and D. tomentosum is difficult to explain and not logic. The two

species are morphologically very different (See Chapter 9). The affinity of D.

donianum shoud be with D. halimunese (not included in rbcL analysis, See

Chapter 9), and D. maoenense Ching. Moore et al (2002) revealed that D.

donianum and D. maoenense are closely related species. D. donianum is different

from D. maoenense in the following characters: D. maonense has lobed terminal

pinna wuth free lobes in lower part, pinnae crenate at margin and serrate near apex

whereas D. donianum has unlobed terminal pinna, pinnae entire at margin and

slightly serrate near apex.

Clade A. Clade A with well supported Bootstrap values level (100%)

included 30 species in which species from Japan (D. chinense, D. rhachidosorus,

D. mesosorum, D. cavalerianum, D. sibiricum, D. squamigerum and D.

okudairaekami), Eastern Asia (D. wichurae) Central America (D. lonchophyllum)

nested. This Clade diverged into two clade, viz A1 that only consisted D.

megasegmentum and Clade A2 that include 29 species. Subsequently A2 diverged

into five branches that are polytomy: (1) A2.1 had one species only (D. poiense);

(2) well supported clade of A2.2. (65%) that comprised D. bantamense, D.

subserratum, D. speciosum, D. lobbianum, and D. silvaticum; (3) Clade A2.3. that

include two individual of D. umbrosum with strong Bootstrap support; (4) Clade

A2.4 that comprised D. accedens, D. polypodioides and D. subpolypodioides

without Bootstrap value support; (5) Clade A2.5. that included D. pallidum, D.

procumbens TNgP 1163, D. esculentum and species from Japan, Eastern Asia and

Central America; and Clade A2.6. Subsequently, Clade A2.6. diverges into two

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monophyletic group with well-supported Bootstrap value, ‘sorzogonense’ Group

(Clade A2.6.1) and ‘riparium’ Group (Clade A2.6.2). The first group with well-

supported Bootstrap value (96%) includes two species, D. sorzogonense and D.

batuayauense, meanwhile the second group with Bootstrap value 51% includes

D. cordifolium, D. angustipinna, D. xiphophyllum, D. hotta and D riparium. The

relationships among species within these clades are dicussed below.

Clade A.2.6.2 (=‘riparium Group’). This study showed that the closely

related species (D. angustipinna and D. cordifolium; D. xiphophyllum and D.

hottae inferred from phylogenetic analysis using morphology are supported by

gene rbcL sequence data. The monophyletic of ‘riparium Group’ is characterized

by the entire scales and simple pinnate frond with terminal distinct terminal

pinnae, terminal pinnae conform to lateral pinnae. In the monophyletic of

‘riparium Group’ D. riparium is at basal clade and diverge earliest. Of all species

within ‘riparium group’, but D.riparium, are dryland species. D. riparium grows

on both riparian and dryland areas. In this group D. cordifolium and D

angustipinna are the most closely related and become the sister group of D.

xiphophyllum and D. hottae. D. cordifolium and D. angustipinna have similar

morphological characters in glossy light brown lanceolate scales on stipes and

copiously anastomosing veins up to 1/3-1/2 way of margin. In the topology of

rbcL tree, the relationship of D. xiphophyllum and D. hottae are not resolved.

Tagawa (1972) stated that D. hottae whic is occurring on Malay Peninsula and in

Northern Sumatra is allied to D. subintegrum Holtt.

Affinity between D. dilatatum and D. simplicivenium. D. dilatatum and D.

simplicivenium form a clade without supporting Bootstrap value (Clade B2). The

closest related of the two species in rbcL tree is supported by the similarity in

morphological appearance such as the lineary lanceolate of black-margined

toothed scales, the tuft of gigantic bipinnate fronds, and oblong subtriangular

pinnulae. However, in the morphological tree (Sees Chapter 6) these species are

separated into two different terminal clades, but still in the same middle clade.

Relationships among non West Malesian Species. Clade A2.5.2 without

high Bootstraping value, include D. rachidosorus, D. mesosorum, D.

cavalerianum, D. squamigerum, D. wichurae, D. okudairaekami, D.

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lonchophyllum and D. esculentum. As revealed also by Sano et al (2000), in the

rbcL tree in this study, D. wichurae from eastern Asia and D. lonchophyllum from

Central America consistently form a clade in the rbcL trees and nested among

species from Japan (D. chinense, D. rachidosorus, D. mesosorum, D.

cavalerianum, D. squamigerum, D. okudairaekam ) and West Malesian species.

It is indicate that more detailed molecular phylogenetic studies that include

species from wider areas should be for phylogeographic studies of Diplazium.

Phylogenetic relationships of D. dilatatum group and the others groups of

Kato (1977). The member of D. dilataum group are spread into many different

terminal clades and also positioned at the subbasal clades. Whereas D.

mesosorum dan D. javanicum group are also mixed in the clade in which the

member of D. dilatatum group forming a calde. It is indicate that Diplazium

groups established by Kato (1977) was not monophyletic. Therefore the all of

West Malesian Diplazium can not be referred to the Kato’s Diplazium groups.

Phylogenetic relationships of the section Anisogonium and Eudiplazium

(van Alderwerelt van Rosenburgh 1908). The rbcL tree is congruent with the

phylogenetic tree generated from morphological data in drawing the polyphyletic

of the section Anisogonium and Eudiplazium. Both the member of the section

Anisogonium and Eudiplazium are mixed, the species of Anisogonium nested in

the many places in which the member of Eudiplazium forming terminal clades.

This study represents the first attempt to explore the phylogeny of West

Malesian Diplazium based on gene rbcL sequences. These results showed that

some clades generating from this sequence are congruence with the clades of

phylogenetic tree generated from morphological data. However this result is

preliminary because this study examined only 29 species from West Malesia.

Molecular phylogenetic study that includes all species of Diplazium from West

Malesia and also use more molecular marker would give the more robust or well

resolved phylogenetic hyphothesis tree.

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8.4. Conclusions

Gene rbcL sequence is very well in supporting species delimitation among

species within the genus Diplazium. Even this study also showed most of West

Malesian species are showing genetic diversity, mainly those having polyploidy

types or seri ploidy. Therefore the division of D. pallidum into two varieties is

supported with this gene rbcL sequences. This study give an indication that

species in which have both morphological difference and seri ploidy, such D.

accedens, D. cordifolium, D. crenatoserratum, D. bantamens, D. subserratum and

D. tomentosum, should be more studied by encompassing much more sample from

their range of distribution.

This study revealed that gene rbcL is more informative in inferring

phylogeny of the genus Diplazium in West Malesia. Therefore the phylogenetic

analysis of gene rbcL sequences found that Diplazium in West Malesia is

monophyletic.

The position of D. porphyrorachis at the basal clade of the philogenetic

tree generated from morphological data and separated from other species is

supported by the phylogenetic tree generated from molecular data (gene rbcL

sequence). This result is in accordance with the statement of Price (1983) that D.

porphyrorachis and close related species are belonging to the different lines

within the genus Diplazium. This study also showed the congruence between the

clade of ‘riparium Group’ drawn by rbcL tree and morphological tree.

The result of this study also showed that the classification of van

Alderwerelt van Rosenburgh (1908) is not monophyletic. The classification of

Kato (1977) in dividing Japanese Diplazium into six group can not be referred to

West Malesian Diplazium. Moreover the Diplazium groups of Kato (1977) are

also not monophyletic.

This study indicates that some uncertain relationships require further

analysis in the future. More data, including both more taxa that include all West

Malesian species and from wider areas and more molecular data, are needed

before a well resolved phylogenetic hypothesis for West Malesian Diplazium can

be offered.

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CHAPTER 9

TAXONOMIC STUDY OF THE FERN GENUS DIPLAZIUMIN WEST MALESIA

9.1. Introduction

The genus Diplazium was established by Swartz (1801) and typified by

Asplenium plantaginifolium L (Diplazium plantaginifolium Sw.). Since then, a

total of 1401 of binomials have been published by various authors for Diplazium

in the world (IPNI, 2006). Hassler & Swale (2002) listed 474 species and 8

hybrids of Diplazium in the world.

Malesian region is the center of Diplazium diversity as it was predicted

that 75% of species in world (ca.300 species) are existing in this region (Roos,

1995). However, since van Alderwereld van Rosenburgh (1908), there has not

been a comprehenship study, including a revision, on Malesian Diplazium, yet.

Previous workers only studied Diplazium based local area. Holttum (1940, 1966)

in his revision on ferns of Malaya reported 27 species of Malay Peninsula.

Tagawa (1972) listed 15 species of Diplazium from Borneo. Iwatsuki & Kato

(1984) reported 15 species of diplazioid ferns of East Kalimantan. Parris et al

(1992) listed 31 species from Mt. Kinabalu. Mitsuta (1985) listed 11 species from

West Sumatra. In Java, Backer and Posthumus (1939) decribed 17 species. Sixty

years after that Praptosuwiryo (1999) recognized 22 species and 4 varieties of

Java. Kato (1994) reported 32 species of Diplazium from Ambon and Seram

(Moluccas).

This chapter presented the account of West Malesian Diplazium. The aims

of this study are: (1) to revise the genus Diplazium in West Malesia; (2) to

provide the delimitation of the genus and species, and (3) to provide an

identification keys to the species and infra species.

9.2. Materials and Methods

Morphological study on Diplazium covered West Malesian species have

been conducted. This study is based mainly on available specimens housed at the

BO and SING and new collection specimens obtained from field work study in

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Java, Sumatra and Kalimantan from 1992 to 2006. The supplemented with images

of type specimens from the data bases/websites of the National Herbarium of the

Netherlands, University of Leiden Branch (L), the United States National

Herbarium in the National Museum of Natural History at the Smithsonian

Institution (US), and Herbarium Universitatis Mosquensis (MW) were also used.

The total number of 1051 collections have been examined. Living plants

in the field were studied in Java, Sumatra and Borneo. In addition, the living

plants grown in botanical gardens and cultivated plants in the green-houses of

Bogor Botanic Gardens were also studied. Three species growing naturally in

Bogor Botanic Gardens, viz. D. esculentum, D. silvaticum, and D.

subpolypodioides, were also examined. The process of undertaking this study

follow the steps those described by Davis & Heywood (1963), Rifai (1976), Vogel

(1987) and Maxted (1992).

9.3. Taxonomic Treatment

Diplazium

Diplazium Sw., Schrad. J. Bot. 1800 (2): 61. 1801. –Type: Diplazium

plantaginifolium Sw.; Backer & Posth., Varenfl. Java: 123. 1939; Holttum, Gard.

Bull. S.S. 11: 77. 1940; Tagawa & K. Iwats., Fl. Thailand 3: 449. 1988.

Callipteris Bory, Voy. 1: 282. 1804. –Type: Callipteris prolifera (Lam.)

Bory; Copel., Gen. Fil.: 152. 1947.

Rhizome creeping to erect, scaly. Stipe well-developed, often stout,

occasionally muricate or spiny, rarely persistently scaly, adaxially sulcate; scales

orbicular to linear, margin entire or toothed; teeth composing two adjacent cells.

Lamina simple to pinnately compound; rachis distinctly grooved, occasionally

bearing adventitious buds; adaxial groove of rachis usually with flat bottom,

glabrous; no spine-like appendages at bases of bases of costae or costules; costa

and costules adaxially grooved. Ultimate divisons costate, costae anadromously

pinnate, veins simple or forked, free or anastomousing, without free included

veinlets. Sori elongate along one or both sides of veins; indusium lateral; double

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sori with two quite separate indusia. Spores monolete, bilaterally symmetrical,

plano-convex or concave-convex, usually prominent, wing-like folds, often with

echinate border, sometimes cristate or echinate, occasionally rugullate.

CHROMOSOME NUMBERS. X=41.

TYPE SPECIES. Diplazium plantaginifolium (L.) Urban.

DISTRIBUTION. Tropical of both hemispheres. Throughout Malesia.

ECOLOGY. Terrestrially, usually on moist humus-rich soil of mountain

slopes of primary and secondary forest of shadowed places. Only a few species

found in the opened areas and grow on the riparian and rheophytic habitats.

Elevation: 20-3400 m.

NOTES. (1) Doubful Species. Seven species of Diplazium published in

some literatures that I doubt and could not found its specimens as follows:

Diplazium allantoideum M.G. Price, D. curtisii Holttum, D. heterophlebium

(Mett.) Diels, D. mesocarpum Alderw., D. falcinellum C.Chr. in C. Chr. &

Holttum, D. megistophyllum (Copel.) Tagawa, D. tabacinum Copel.

(2) Excluded Species. There some species of West Malesian Diplazium

that excluded from Diplazium and then have been included in the other genera,

namely D. chrysocarpum Alderw., D. grammitoides Presl, D. japonicum Bedd.,

D. subsinuatum (Wall. ex Hook ) et Grev.) Tagawa and D. amplissimum. D.

chrysocarpum Alderw was included as Athyrium stramineum Copel.). D.

grammitoides Presl, D. japonicum Bedd., D. subsinuatum (Wall. ex Hook ) et

Grev.) Tagawa were included in Deparia and treated as Deparia confluens

(Kunze) M. Kato, De japonica (Thunb.) M. Kato (Kato 1984), and De lancea

(Thunb.) Fraser-Jenk (1997), respectively. While was D. amplissimum included in

Cornopteris as C. atroviridis (v.A.v.R.) M. Kato (Kato 1979).

(3) Now, West Malesian Diplazium is reported comprising 69 species

with 15 varities. Thirteen new species and two new varieties are described. The

thirteen new species described in this chapter are: D. asymmetricum, D.

batuayauense, D. crameri, D. densisquamatum, D. halimunense, D. loerzingii, D.

megasegmentum, D. megasimplicifolium, D. meijerii, D. parallelivenium, D.

profluens, D. subalternisegmentum, and D. subvirescens. While the two new

varieties descrided are: D. accedens var. spinosum and D. silvaticum var. pinnae-

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ellipticum. Two new status of Diplazium are proposed: D. pallidum var.

montanum and D. accedens var. ridleyi.

Key to Species of Diplazium in Western Malesia

1. Veins free

2. Lamina simple

3. Rhizome creeping; scales lanceolate, margin entire ………........... D. subsinuatum

3. Rhizome erect or suberect; scales oblong subtriangular, margin shortly toothed. ……………………................................. D. subserratum

2. Lamina simply or compoundly pinnate

3. Lamina simply pinnate to bipinnatifid

4. Terminal pinnae conform to lateral pinnae or deltoid with deeply lobed

5. Scales margin toothed

6. Rhizome short, erect; lateral pinnae commonly 3-18 pairs; terminal pinna larger or deltoid with deeply lobe at base

7. Lateral pinnae commonly 3-5 pairs, 4 cm or more wide; texture chartaceous or thinly papyrceous

8. Rachise non gemmiferae; base of lateral pinnae cuneate …………………………… D. halimunense

8. Rachise gemmiferae; base of lateral pinnae rounded ………………………….. D. bantamense

7. Lateral pinnae commonly 7-18 pairs, 3 cm or less wide; exture thinly coriaceous …………………….. D. lobbianum

6. Rhizome medium, creeping; lateral pinnae commonly to 3 pairs; terminal pinna conform to lateral ones …… D. donianum

5. Scales margin entire

6. Rheophytic or riparian; scales on stipe black; lateral pinnae to 4 pairs

7. Rheophytic; Stipe more than 20 cm long;lateral pinnae shortly stalked 4.5 mm long, lanceolate,

more than 12 cm long, 2 cm broad ……… D. aequibasale

7. Riparian; Stipe less than 20 cm long; lateral pinnae adnate, lineary oblong, usually less than 8 cm long, 1.5 cm broad …. D. wahauense

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6. Terrestrial; scales on stipe pale to dark brown; lateral pinnae 5 or more pairs

7. Scales pale or light brown to brown;

8. Lateral pinnae ovate-lanceolate, larger ones to 3 wide

9. Scales brown; pinnae to 5 pairs; veins group formingangle 50-60º to costa …………… D. crameri

9. Scales light brown; pinnae to 20 more pairs; veinsgroup forming angle ca. 45º

to costa ….………………………. D. hottae

8. Lateral pinnae elliptical, larger ones 4 cm or more wide ......……. D. xiphophyllum

7. Scales dark brown

8. Lateral pinnae to 10 pairs; texture thinly or softly chartaceous

9. Veins forked to 3 times; pinnae crenate or shallowly lobed,shortly stalked ……. D. prescottianum

9. Veins forked to 4 times; pinnae subentire or toothed near apex, distinctly stalked .... D. subintegrum

8. Lateral pinnae to 27 pairs; texture subcoriaceous …………………………….…. D. pallidum

4. Terrninal pinnae absent, upper pinnae gradually reduced

5. Scales abundant on stipe, rachis, costae, and vein beneath; Lamina nearlyelliptic- lanceolate, to 13 cm broad

6. Lower lamina fully pinnate to 2/3 part; free pinnae to 32 or more pairs; veins forked to 3 times ………..…………………… D. fuliginosum

6. Lower lamina deeply pinnatifid to 5/6; sometimes with one pair of reduced free basal pinnae; veins mostly once forked

7. Lamina elliptic; lobes to 10 mm broad, oblong-lanceolate, narrowing towards apex, subentire ……… D. lomariaceum

7. Lamina lanceolate; lobes to 15 mm broad, liniery oblong, slightly crenate, or toothed and not seldom the sides entire only the apex serrate ………………… D. porphyrorachis

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5. Scales usually abundant on lower stipe only; lamina oblong subtriangular – oblong lanceolate, to 30 cm broad

6. Scales entire

7. Lamina to about 15 cm wide, subtriangular, costae hairy beneath …………………… D. tomentosum

7. Lamina usually wider, oblong-lanceolate, costae not hairy beneath

8. Margin of pinnae shortly toothed or almost entire; veins forked once to twice, usually soriferous on basal acroscopic side …………………… D. pallidum

8. Margin of pinnae deeply lobed; veins pinnate in the lobes; usually soriferous on all veinlets

9. Rachise fibrillose; margin of pinnaelobed ¾ -11/12 way to costa …. D. sorzogonense

9. Rachise glabrous; margin of pinnae lobed 1/3-2/3 way to costa

10. Scales light brown without thickening blackish strandmargin; lower base of lower pinnae not cut away; soricover veinlets to 2/3 of their lengt …..… D. christii

10. Scales dark brown with thickening blackish strandsmargin; lower base of lower pinnae often much cutaway; sori cover veinlets to ¾ of their length or almostreaching margin

11. Rachise not gemmiferous; lobus rounded; texture papyraceous; sori elongate from near base of veins to near margin of lobus; indusia medium brown, concolour ……….……………… D. malaccense

11. Rachise gemmiferous; lobus truncate; texture subcoriaceous; sori medial or close to margin; indusia dark brown, attachment side darker ....... D. loerzingii

6. Scales toothed

7. Stipe, rachis and costa tomentose ….…………… D. tomentosum

182

7. Stipe, rachis and costa not tomentose

8. Lateral pinnae to 4 cm or more broad, margin lobed to 5/6 way to costa …..………………….. D. speciosum

8. Lateral pinnae less than 4 cm broad, margin lobed to ¾ way to costa

9. Pinnae lobed ¼ - ½ towards costa

10. Scales dark brown or nearly black, without thickening black strand; sori subcostular, touching midveins or nearly so at proximal end

11. Pinnae chartaceous, often auriculate below;scales irregularly andshortly toothed ……… D. crenatoserratum

11. Pinnae thin in texture, not auriculate below;scales strongly toothed …… D. silvaticum

10. Scales brown, with thickening dark brown strand; sori medial or supramedial (not touching midveins) …….... D. batuayauense

9. Pinnae lobed ¾ way towards costa

10. Pinnae lanceolate, texture herbaceous; Veins forming an angle about 60º to costa ……... D. petiolare

10. Pinnae lineary subtriangular, texture thicker; Veins forming an angle 50-55º to costa … D. acuminatum

3. Lamina bipinnate to tripinnate

4. Rhizome erect

5. Lamina bipinnate

6. Scales rounded

7. Pinnulae lobed more than ¾ way to costa

8. Costae bearing scattered small ovate-rounded scales;pinnulae subtriangular, lower stalked to 1 mm long,apex of lobus truncate; sori usually diplazioid onbasal acroscopic veinlets only ………… D. latisquamatum

8. Costae glabrous; pinnulae lanceolate, lower stalkedto 2.5 mm long, apex of lobus rounded-acute;sori diplazioid both on basal and medianacroscopic veinlets ……………………. D. profluens

183

7. Pinnulae lobed ¾ way or less to costa

8. Stipes prickly

9. Stipe muricate, dark brown when dry; lower pinnulaeshortly stalked to 1.5 mm long, margin lobed to1/2 way to costa ………………… D. betimusense

9. Stipe spiny, rather stramineous when dry; lower pinnulaeadnate, margin lobed less than ¼ way to costaor almost entire …………………. D. spiniferum

8. Stipes not prickly

9. Lower pinnulae stalked to 2 mm long; Sori on medialon veins ……………………………………. D. kunstlerii

9. Lower pinnulae stalked less than 1 mm long oralmost adnate; Sori on basal veins ................ D. laevipes

6. Scales ovate-liniery lanceolate

7. Scales toothed

8. Pinnulae lobed 1/3 way or less to costa or almost entire

9. Stipes scales throughout

10. Pinnulae lobed less than 1/4 way to costa (crenulate);veinlets to 9 pairs ….……….……… D. barbatum

10. Pinnulae lobed to 1/3 way to costa; veinlets to 6 pairs

11. Pinnulae to 20 pairs, less than 12 mm broad;veins forked 1-3 time …………. D. melanolepis

11. Pinnulae to 12 pairs, 15 mm or more broad; veinspinnate to 6 pairs of veinlets

12. Pinnulae elliptical, less than 5 cm long, 2 cmbroad; apex of vein whitish-limy(dot like) …………….. D. albido-squamatum

12. Pinnulae oblong, to 6 cm long, 1.5 cm broad,base broadly cuneate; apex of vein not whitish-limy ……………………………. D. crinitum

9. Stipes scales at base only

10. Stipe spiny; pinnulae to 12 pairs, lower stalked to3 mm long; veins to 8 pairs, once forkedon lower lobes …………………..……. D. vestitum

10. Stipe not spiny; pinnulae to 16 pairs, lower stalkedto 1.5 mm long; veins to 5 pairs,commonly 4 pairs, all simple …… D. simplicivenium

184

8. Pinnulae lobed ½ or more to costa

9. Lamina less than 100 cm long; scales light brown, withoutthickening black strand; texture herbaceous … D. umbrosum

9. Lamina more than 150 cm long; scales dark brown, teeth mostlyforked, with thickening black strand; texture papyraceous

10. Stipe prickly; lobes oblong to subquadrangular;sori less than 2.5 mm long …………. D. polypodioides

10. Stipe not prickly; lobes subdeltoid to semiorbicular;sori more than 3 mm long .…………… D. dilatatum

7. Scales entire

8. Stipes not not muricate at base

9. Stipe and rachise not clothed with scattered multicellular brownhairs;

Sori on medial veins, impressed ………….. D. poiense

9. Stipe and rachise clothed with scattered muslticellular hairs; Sori on basal veins, not impressed …………. D. velutinum

8. Stipes muricate at base

9. Stipes densely scales throughout; free pinnulaeto 18 pairs .……………. ………….. D. densisquamatum

9. Stipes densely scales only at base; free pinnulae to 13 pairs

10. Lower pinnulae shortly stalked to 1.5 mm long,lanceolate, more than 6 cm long, lobed to 4/5 way tocosta …………………………… D. atrosquamosum

10. Lower pinnulae adnate – sessile, nearly hastate, less than 5cm long, crenate ….…………...………… D. hewittii

5. Lamina tripinnatifid – tripinnate

6. Pinnulae lobed less than ¾ way to costa

7. Pinnulae to 16 pairs; larger pinnule stalked to 5 mm long,oblong subtriangular, to 18 cm long, 4 cm broad,apex attenuate, margin lobed ½ way to costa ……. D. dilatatum

7. Pinnulae to 20 pairs; larger pinnule adnate, oblong-lanceolate, to 5.5 cm long, 1 cm broad, commonlyless than 4 cm long, 8 mm broad, apex rounded-acute, margin lobed to 1/3 way to costa ……... D. melanolepis

185

6. Pinnulae lobed ¾ way or more to costa

7. Pinnulae lobed to within one mm or less of costule, forming segments

8. Lower costule of larger pinnulae winged; free segment notpresent

9. Scales lineary triangular-lanceolate; Indusia opening whensori mature ………………………. D. subpolypodioides

9. Scales rounded; Indusia opening beforesori mature ………………............ D. megasegmentum

8. Lower costule of larger pinnulae not winged; free segmentspresent

9. Pinnulae 17 pairs or more; larger pinnulae oblong- subtriangular; sori brown when dry

10. Segments crenate or lobed 1/3 way to costulet; sori bearing near basal or on middle veinlets cover ½ of their length; indusia pale brown, attachments side more dark, margin entire ………….. D. umbrosum

10. Segments crenulate, apex rounded; sori bearing on basal costulet cover veinlets 1/3 of their length; indusia brown, concolour, margin fringed ………………….…... D. moultonii

9. Pinnulae less than 16 pairs; larger pinnulae subtriangularlanceolate; sori nearly golden

yellow when dry …………………….. D. chrysocarpum

7. Pinnulae lobed to within more than 2 mm of costa, not formingsegments

8. Scales narrowly linier, margin toothed with thickening blackstrands; Stipes densely scaly near base, surface prickly;

Sori occupying from the base half-way or more to the edge ……............................... D. polypodioides

8. Scales rounded, margin entire; Stipes scales throughout, surfacenot prickly; Sori occupying only the lower half or lessof the veins ………………………… D. latisquamatum

4. Rhizome creeping

5. Margin of pinnae lobed to ¾ towards costa; Sori elongated from basal veinlets …………………………………………………. D. procumbens

5. Margin of pinnae lobed to 2/3 towards costa; Sori elongated on medial or submedial veinlets ……………………………. D. subvirescens

186

1. Veins occasionally uniting at margin or copiously anastomousing

2. Veins rarely anastomousing, occasionally uniting at margin

3. Lateral pinnae 6-9 pairs, elliptical; veins occasionally anatomousing; gemmae present at the junction between rachise and costa .…. D. xiphophyllum

3. Lateral pinnae 2-4 pairs, oblong; veins free or very rarely anastomousing; gemmae absent ………..……………………………………….. D. riparium

2. Veins copiously anastomosing

3. Lamina simply or simply pinnate

4. Lamina simply

5 . Lamina subdeltoid, base cordate, veins anastomousing 1/3-1/2 ………………………... D. cordifolium

5. Lamina elliptical, base subequally cuneate, veinsanastomousing 2/3-4/5 way of margin ….... D. megasimplicifolium

4. Lamina simply pinnate

5. Scales dense on stipe, rachise& costa; lower surface of laminabearing stelate trichome scatterly ………………………. D. squarrasum

5. Scales dense on base of stipes only; lower surface occasionally bearinguniseriat trichome with glandular cells

6. Terminal pinnae differ to lateral ones, deltoid ……. D. accedens

6. Terminal pinnae conform to lateral ones

7. Scales on stipes dark brown nearly black, lateral pinnae oblong, base cuneate; veins anastomousing

1/3 way of margin ……………………………... D. angustipinna

7. Scales on stipes light brown, lateral pinnae lanceolate, lower base rounded to auricled; veins anastomousing¼ way of margin ………………………………. D. cumingii

3. Lamina bipinnate

4. Scales dull brown; stipe spiny toward the base; Pinnulae fully adnate, texturefirmly herbaceous; vein anastomousing like D. accedens …..... D. insigne

4. Scales dark brown; stipe smooth; Pinnulae shortly stalked, texturepapyraceous; Vein in pinnate group in the lobes, 8-10 pairs of side veins, thelower 2-3 pairs of adjacent group anastomousing, forming irregularintermediate excurrent vein leading towards a sinus between

adjacent lobes …………………………………………... … D. esculentum

187

1. Diplazium accedens Blume

Diplazium accedens Blume., En. Pl. Jav.: 192. 1828; Holttum, Gard. Bull.

S.S. 11: 81. 1940; Tagawa & K. Iwatsuki, Fl. Thailand 3: 451. 1988. – Athyrium

accedens (Blume) Milde, Holttum, Rev. fl. Malaya 2: 558. f. 329. 1966.

Diplazium repandum Blume, En. Pl. Jav.: 191. 1828; Backer & Posth.,

Varenfl. Java: 125. 1939. TYPE: Blume s.n. (?, L!).

Diplazium swartzii Blume, En. Pl. Jav.: 191. 1828; Alderw. Ferns. Suppl.

1: 275. 1916.

Callipteris prolifera (Lam.) Bory; Copel., Fern Fl. Philip. 3: 419. 1960;

Andrews, Ferns of Queensland: 72. f. 5.1.C. 1990. TYPE: Callipteris prolifera

(Lam.) Bory.

Asplenium proliferum Lam. in Lam. & Poiret, Encycl. 2: 307. 1786

Athyrium ridleyi Copel., Phil. Journ. Sci. XI (1) : 39. 1908. TYPE:

Ridley 13970 (Isotype, SING!).

Key to the Variety

Rhizome bearing buds. Stipe protuberances, vascular bundles of its transversal sections nearblade continuously U shaped. Rachise not spiny, always gemmiferous.

Stipe sparsely protuberances; protuberance light green. Veinlets pale green when living;extra areola between adjacent normal group of veins few, the line of series extra areola up to1/3 way to costa ................................... ..…………………………………... var. accedens.

Stipe densely protuberances; protuberance deep green. Veinlets light green when living;extra areola between adjacent normal group of veins copious, the line of series extra areola upto 5/6 way to costa ......................................................................................... var. ridleyi.

Rhizome not bearing buds. Stipe sharply spinuous, vascular bundles of its tranversal sections nearblade interrupted U shaped. Rachise bluntly spiny, not gemmiferous ……… var. spinosum.

a. var. accedens

Rhizome stout, short, erect, bearing buds. Stipe stout, green, clothed with

green protuberances toward base, up to 80 cm long, 1-1.2 cm thick, scales at base;

scales linier, 10-13 mm long, 1.2-1.5 mm wide, dull brown, thick in texture with

narrow black strand toothed at margin. Lamina simply pinnate, oblong with

188

acuminate apex in outline, to 110 cm long or more, 40 cm wide, pinnae to 18

pairs; lower pinnae stalked to 7 mm long, sessile in upper ones, basal pinnae or

subbasal pinnae the largest, about 30 cm long, 7 cm wide, oblong with acuminate

apex, base broadly cuneate to subtruncate, margin entire or undulate, or double

crenate; terminal deltoid with 1 or 2 deeply lobed at base; papyraceous, glabrous

above; rachis grooved above, gemmiferous; costa prominent below, grooved

above with distinct ridge; veins pinnate, veinlets 8-10 pairs, veinlets adjacent

groups anastomousing producing a series of parallelogram-shape areolus. Sori

elongate along veinlets, often throughout their length forming areoles; indusia

very thin, fragile, margin entire.

SPORES. Monolete, bilaterally symmetrical, heteropolar; polar outline

elliptical, sides convex; equatorial longitudinal view concave-convex to plano-

convex; equatorial transverse view, proximal face planar to concave, distal face

hemispherical; perinate. E: 25.34(31.47)36.02±2.54; P: 15.32(19.51)23.59±2.82.

Laesure: concealed by perine ridge. Perine: costate-alate, reticulation often

incomplete; lacunae shallow, project 10-17 m ;wing-like muri or costae project

c.0.5-6 m, terminating margins entire. Exine: often visible through perine,

smooth under LM, smooth under SEM.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an uninterrupted U-shaped with an angle about 90º, the outward of the

lower base, angle, and end ridge. Stomata: polocytic and copolocytic.

CHROMOSOMES. 2n = 82 (Cytotype: TNgP1447, BOHB).

DISTRIBUTION. Peninsular Thailand, Malesia, Pacific.

ECOLOGY. Terrestrial, spread throughout Western Part Malesia Region

at 400 – 1550 m sea level in moist soil, by preferences more or less shadowed

localities, in primaryforest, secondary forest in ravine, forest borders, and

meadows.

VERNACULAR NAMES. Paku buwah, paku careham (Sundanese),

pakis angkrik (Javanese).

USE. The yound fronds and bulbis in the axils of leaflets are eaten after

cooking.

189

SPECIMENS EXAMINED. --- JAVA: Arsin s.n.; Backer s.n., 6307,

10891, 16200, 23754; Backer & Posthumus 201; Bakhuizen v/d Brink 2393;

Beguin 83; Brinkman 445; Burk s.n.; Coert 910; Danser 6783; Hallier 201;

Heurn 201, 202, 203; Kooders 373*, 8778, 10891, 21179B, 23356B, 23725B,

41459B, 41508B; Lörzing 933, 1745; Meijer 31, 172; Pleyte 162; Raciborski

109; Sciffner s.n..; Winckel 1279B, 1419B; Zippelius s.n.. ---- MALAY

PENINSULA: Md Nur 32894, s.n. (28-8-1923); M.R. Henderson s.n. (28-8-

1923); Br. G. Allan 3477, s.n. (30-6-1957); Wale 9111. --- SUMATRA: J.A.

Lörzing 5577, 12808, 5562;.Bünnenmejer 4316; D. Darnaedi 107; J.A. Lörzing

5562; C.G.G.J. van Steenis 3470; J.A. Lörzing 12809, 7001; Dr. Cramer no. 25;

J.A. Lörzing 4226; Karta 21; Jacobson 1451, 2498; K. Iwatsuki, Gen Murata, J.

Dransfield & D. Saerudin S- 1542; Bünnenmeyer 4314a; M.Hotta 26131;

W.J.J.O. de Wilde & B.E.E. de Wilde-Duyfjes 12583; H. Surbeck 81; Dr. O.

Posthumus 1105. ---- BALI: O. Posthumus 3722; C.G.G.J. van Steenis 8017; W.

Meijer 10546; W. Meijer 10453.

b. var. spinosum Praptosuwiryo, var. nov.

TYPE: Tahrodin TR53 (holotype, BOHB), East Kalimantan

Stipe sharply spiny, pale green on upper surface when living, black on

lower surface. Pinnae up to 7 pairs; lower pinnae stalked to 3 mm long, basal

pinnae reduce to 6 cm long, 2.3 cm broad, margin undulate; rachise not

gemmiferous.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form interrupted U-shaped with an angle about 90º, angle and end bluntly ridge.

CHROMOSOMES. 2n = 82 (Cytotype: TR 53, BOHB)

SPECIMENS EXAMINED: --- BORNEO: TR 53; YN 04. ---

SUMATRA: H.A.B. Bünnenmeijer 8665.

190

c. var. ridleyi (Copel.) Praptosuwiryo, stat. nov.

Athyrium ridleyi Copel., Phil. Journ. Sci. XI (1) : 39. 1908. TYPE:

Ridley 13970 (Isotype, SING!).

Stipe deep green when living, bearing densely deep green protuberances,

minutely pilosa. Lateral pinnae up to 7 pairs; larger ones up to 10.8 cm broad,

apex caudate, margin entire or crenate. Veinlets very distict, light green. Extra

areola between adjacent normal group of veins copious, the line of series extra

areola up to 5/6 way to costa.

DISTRIBUTION. Malay Peninsula, Suamtra.

SPECIMENS EXAMINED: --- MALAY PENINSULA: Ridley 13970. ---

SUMATRA: T.Ng. Praptosuwiryo 2525a.

2. Diplazium acuminatum Blume

Diplazium acuminatum Blume, En. Pl. Jav.: 193. 1828; Alderw., Mal.

Ferns: 409. 1908.

Rhizome short, erect-suberect. Stipe 34-36 cm long, 2 mm thick when

dry, brown, black and scales at base, glabrous upward; scales lineary triangular, 5-

10 mm long, 0.5-mm broad, dark brown, margin sharply toothed. Lamina

pinnate, subtriangular- lanceolate in outline, 45-50 cm long, 15-23 cm broad, free

pinnae 15-21 pairs below deeply lobed acuminate apex of lamina; lower pinnae

shortly stalked to 6 mm long, upper ones adnate–sessile, lineary subtriangular,

larger pinnae 12-12.5 cm long, 2-2.2 cm broad, base unequal, lower base cuneate,

upper truncate, apex acuminate, margin lobed ½-3/4 way to costa; lobus oblong,

basal acroscopic the largest, 3.5-5.5 mm wide, ends truncate, entire or slightly

toothed; texture firm; rachise gemmiferous on upper part; veins free, forming

angle about 50-55° to costa, pinnate in the lobes, veinlets 5-7 pairs, all simple,

forming angle about 15-20º to main veins. Sori elongate from near costule covers

1/3-2/3 way of their length or almost reaching the margin, basal acroscopic

diplazioid; indusia brown, rolled back, margin entire, firm.

DISTRIBUTION. Sumatra.

ECOLOGY. In the mountain forest at 400-1000 m dpl.

191

SPECIMENS EXAMINED: --- SUMATRA: J.A. Lörzing 14051, 6311; J.

Buumee A. 841.

NOTES. Kato (1994) states that this species is similar and perhaps related

to D. megaphyllum (Baker) Chist from E. Myamma, SW China, Taiwan, Vietnam

and Thailand, in general habit and leaf morphology, but from it in the regularly

anastomosing veins. D. megaphyllum generally has free veins.

3. Diplazium aequibasale (Baker) C.Chr.Diplazium aequibasale (Baker) C.Chr.; Ind. 227. 1905; Alderw., Mal.

Ferns: 404. 1908. J. Fac. Sci. Univ. Tokyo 3 (15): 101. 1991.Rhizome short, erect. Stipe 21-28 cm long, pale brown when dry, black

and scales at base; scales ovate-lanceolate, 3-5 mm long, 1-1.5 mm broad, fragile,

margin entire. Lamina simply pinnate, lateral pinnae 3-4 pairs, terminal pinnae

like the laterals, the largest; lower pinnae shortly stalked to 3-4.5 mm long, upper

adnate, lanceolate, to 14 cm long, 2.5 cm broad, base cuneate, margin entire or

crenate near apex, suddently narrowed near apex, acuminate; texture rather thin,

surface naked; costa rounded beneath, grooved on upper surface; veins group

forming angle about 65-70° to costa, each group of 3 veins, middle vein forked 1-

3 times, occasionally the outer of vein group uniting with the inner close to the

margin. Sori bearing at 1-3 veins in each vein group, those on the outer vein of

the group extending from the costa to margin, rest shorter, acroscopic outer veins

usually diplazioid; indusia brown, narrow, margin entire, persistent, fragile,

opening when mature.

CHROMOSOMES. 2n = 164 (Cytotype: T.Ng. Praptosuwiryo 2026, BO)

DISTRIBUTION. Java, Sumatra, Malay Peninsula, Borneo.

ECOLOGY. It is rheophytic species and usually grown at lowland clay

stream-bank. 20- 400 m.

SPECIMENS EXAMINED. --- JAVA: Scheffer 16584. --- BORNEO:

A. Kostermans 10448, T.Ng. Praptosuwiryo 2148. --- SUMATRA: O.

Posthumus 695; T.Ng. Praptosuwiryo 2026a, 2026b, 2157. --- MALAY

PENINSULA: R.E. Holttum SFN 24780.

192

4. Diplazium albido-squamatum Alderw.

Diplazium albido-squamatum Alderw., Bull. Buiten. II no. 23. 9. 1916. --

-TYPE: C.J. Brook 272/S. (holotype, BO!), Soelit, Lebong, Bengkulu, Sumatra;

C.J. Brook s.n. (isotype, L!), Lebong Tandai, Bengkulu, Sumatra.

Stipes 38 cm long, 6 mm thick, grooved above, dark brown-blackish,

scales throughout; scales lineary lanceolate or subulate, to 1 cm long, 1 mm wide,

dark brown, margin toothed with thickening black strand. Lamina bipinnate, ca.

70 cm long, (?) cm broad, lanceolate in outline, pinnated pinnae 5 pairs below ca.

5 pairs of pinnatifid pinnae, apical lamina (?); lower pinnae stalked, stalked to 17

mm long, 8-10 cm apart, ovate-lanceolate, 15-24.5 cm long, 8.5-9 cm broad,

pinnulae 5-8 pairs; lower pinnulae shortly stalked to 1 mm long or adnate, 2.5-3

apart, upper pinnulae sessile with subequally broadly cuneate; larger pinnulae

oblong-elliptical, 4.8 cm long, 1.9 cm broad, base subequally subtruncates, apex

acute, margin lobed ¼-1/3 way to costule; lobus 4-6.5 mm wide, ends rounded-

subtruncate; texture subherbaceous, upper surface glabrous, to apex vein

whitish-limy, dot-like; rachise obscurely dark brown, minutely scales, at length

base persistent scales fallen away rugged; veins free, pinnate, mid-veins forming

angle ca. 60º to costule; veinlets 4-6 pairs, length base persistent scales fallen

away rugged all simple, forming angle about 20-25° to costule. Sori elongate

along veinlets, cover ½-2/3 of its length, acroscopic basal diplazioid, others

asplenoid; ndusia dark brown, stiff, margin entire, opening when mature.

DISTRIBUTION. Sumatra.

ECOLOGY. Grown in limestone.

SPECIMENS EXAMINED. --- SUMATRA: C.J. Brooks 272/S.

NOTES. young plants simply pinnate, with the fronds similars to the

largest pinnae of the adult ones but less copiously soriferous.

193

5. Diplazium angustipinna (Holttum) Holttum

Diplazium angustipinna (Holttum) Holttum, Rev. Fl. Malaya 2. Appendix

II: 637. 1966. Athyrium angustipinna Holttum, Gard. Bull. S.S. 14: 8. 1953;

Rev. Fl. Malaya 2: 549. 1966. --- TYPE: R.E. Holttum SFN 39416 (holotype,

SING!), Frasser’s Hill, Pahang, Malay Peninsula.

Rhizome short, erect. Stipe to 36 cm long, 3 mm thick, scales at base;

scales narrow, to 8 mm long, 1 mm broad, medium margin entire, some larger

ones with black firm edges. Lamina simply pinnate, fertile ones to 41 cm long,

lateral pinnae 4-6 pairs below a terminal leaflet of similar shape; largest fertile

pinnae on different fronds 10-18 cm long, 1.6-2.8 cm broad, widest 1/3 from the

base, tapered evenly to the narrow apex and to the base which is truncate on a

stalk 1 mm. long, margin slightly and irregularly sinuous; frond and rachis

glabrous except for very short hairs in groove of upper surface of rachis and small

scales on bases of costae; rachise not proliferous; veins anastomousing freely in

outer 1/3 of each half of the lamina, forking 2 or 3 times before anastomousing.

Sori long, diplazioid on acroscopic branch of each first forking, reaching from

costa 2/3 towards the margin, also 1-3 short sori on distal anastomousing vein-

branches; indusia narrow, firm, perisistent, margin entire.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form uninterrupted V-shaped, with an angle 70º, ends simple, not forming a ridge.

CHROMOSOMES. 2n = 123 (Cytotype: T.Ng. Praptosuwiryo 1904, BO),

164 (T.Ng. Praptosuwiryo 1905b, BO).

DISTRIBUTION --- Malay Peninsula, Borneo.

ECOLOGY --- Terrestrially on humus rich soil or rock soil in dense

jungle. 400-900 m.

SPECIMENS EXAMINED --- BORNEO: T.Ng. Praptosuwiryo 1904,

1905b. --- MALAY PENINSULA: R.E. Holttum SFN 3946; King’s 8026.

194

6. Diplazium asymmetricum Praptosuwiryo, sp. nov. Plate 1.TYPE. Java. West Java, Mt. Halimun, track Cikaniki-Citalahap, ca. 1000

m., 27 September 2003, T.Ng. Praptosuwiryo 1728 (holotype, BO).

Rhizoma breve erectum. Stipites ad 40 cm longi, basi squamis subulatis

vel oblongis ovatis ad 4.5 mm longis 1.5 mm latis margine dentatis vestitae.

Lamina deltoideus, 46-50 longae, 46-50 latae, bipinnata-tripinnatifida, pinnate

pinnae 3-4 jugatae infra pinnatifis pinnae, apice deltodeus peniti-lobus. Pinnae

infimus maximae, stipipatae ad 4 cm longae, ubtriangulare, libere pinnulae 6-10-

jugatae infra apice deltoideus pinnatifidus; pinnulae infimus basiscopicus

maximae, stipitae ad 1.5 mm longae, subtrianglare, asymmetricus, base

subcordatus vel truncates, apice acuminatae, cetera margine lobatae ad 4/5 costam

versus; lobi angulum plus minusve 90° cm costa formantes, ad 8 mm latis supra

basi, margine leviter denticulatus, apice subtruncatus vel obliquely subrotundatus.

Vennae in uno lobo ad 6-jugatae, plerumque furcatae. Sori basaliter, aliquando

medianus, in 1/3-1/2 longitudine venularum sedentes, in basi venulae

acroscopicus diplazioideus. Indusia badius, fragilis, maturis aperiens.

Rhizome short, erect. Stipe pale green when living, scales at black base,

glabrous upwards, 30-101 cm long, 5-8 mm diam. near base; scales dark brown,

subulate-oblong ovate, to 4.5 mm long, 1.5 mm wide, margin toothed. Lamina

bipinnate-tripinnate, deltoid in outline, 46-50 cm long, 46-50 cm broad, pinnate

pinnae 3-4 pairs below pinnatifid pinnae, apex deltoid with deeply lobed. Basal

pinnae the largest, stalked to 7 cm long, subtriangular in outline, pinnule 6-10

pairs below acuminate deeply lobed deltoid apex of pinnae, basiscopic pinnules

the larger than acroscopic ones; pinnulae shortly stalked to 4 mm long,

subtriangular, asimetrical, base subcordate-truncate, apex sharply toothed

attenuate or caudate, margin lobed 4/5-7/8 way to costule (on bipinnate lamina),

or forming segments (on tripinnate lamina); basiscopic segments larger than

acroscopic ones, to 4 cm long, 1.8 cm broad, base truncate or broadly cuneate,

apex acute, margin lobed to 1/3 way to costulet; lobus almost at right angle, to 4

mm broad (on tripinnate lamina) or to 8 mm broad above base (on bipinnate

leaves), margin slightly toothed, ends subtruncate-obliquelly subrounded. Veins

pinnate in the lobes, veinlets to 6 pairs, usually once forked. Sori from near

195

costule, sometimes medial, cover 1/3-1/2 of veinlets, diplaziod on basal

acroscopic veinlets. Indusia pale brown, fragile, margin entire, opening when

maturity, rolled back.

PARATYPE. JAVA. West Java: Mt.Gede, Cibodas Forest, behind

Cibodas Botanic Gardens, ca. 1450 m, 19 August 2002, T.Ng. Praptosuwiryo

1334 (BO); Mt. Salak, Southern Slope, Cangkuang Forest, 18 December 2002,

T.Ng. Praptosuwiryo1365 (BOHB ); Mt. Halimun, Track Cikaniki-Cikuda Paeh,

ca. 1250 m, 29 September 2003 T.Ng. Praptosuwiryo 1780 (BO).

CHROMOSOMES. 2n = 123.

DISTRIBUTION. Only found from Java, from Mt. Gede, Mt. Salak and

Mt. Halimun.

ECOLOGY. Growing on moist humus-rich soil, shadowed places of

mountain forest at altitude 1000-1500 above sea level.

ETYMOLOGY. This species is named in relation to the specific

characters of its pinnae and pinnulae. Pinnae and pinnulae are usually

asymmetric, the acroscopic pinnulae and segments or lobes are larger than the

basiscopic.

SPECIMENS EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 1728, 1334,

1365, 1780.

NOTES. Diplazium asymmetricum is similar to D. procumbens The two

species share in scales on stipes fallen when mature, ovate-lanceolate dark brown

scales with blunt teeth, deltoid lamina, oblong lobes with blunt or truncate apex,

and forked veinlets. Diplazium assymetricum differs from D. procumbens in the

following characters combination: rhizome short, erect; lamina more incised (to

tripinnate) basiscopic pinnulae or segments and lobes are larger than the

acroscopic ones; indusia thicker, margin entire.

196

7. Diplazium atrosquamosum (Copel.) C.Chr. & Holtt.

Diplazium atrosquamosum (Copel.) C.Chr. & Holttum, Gard. Bull. S.S. 7:

274. 1934. --- Athyrium atrosquamosum Copel., Phillip. J. Sci. 12c: 59. 1917.

--- TYPE: Clemens 11051 (holotype PNH?†; isotype MICH, s.n.; photo of isotype

at UC, K, s.n.).

Rhizome short, erect. Stipe light brown, 65 cm long, 7 mm thick.,

muricate and scales fallen. Lamina bipinnate, subdeltoid, 56 cm long, 40 cm

broad, pinnated pinnae to 4 pairs; pinnae stalked to 5.5 cm long, lanceolate, 32 cm

long, 14 cm broad, free pinnulae 12 pairs below pinnnatifid apex of pinnae, basal

acroscopic slightly reduced; lower pinnulae shortly stalked to 2 mm long, upper

andate or sessile, lanceolate, to 9.2 cm long, 2 cm broad, apex sharply acuminate,

margin lobed ¾- 4/5 way to the costule; lobes oblong, 2-5-3.5 mm wide above

base, ends truncate, slightly toothed; rachis gemmiferous at the adjacent to the

costae at the apex of lamina, glabrous; costae and costule sometimes scales,

mainly at the adjacent costae and costule or costule and costulets; veins black,

mainvein forming angle about 70º to costa, free, pinnate in the lobes; veinlets 5-6

pairs, simple, forming an angle about 35º to the costulet. Sori on basal veinlets,

elongate from near costulet cover 1/3-1/2 way to margin, indusia pale brown, thin,

broad, persistent, margin lacerate when opening.

DISTRIBUTION. Borneo.

ECOLOGY. Terrestrial in mountain forest. Elevation: 1500-2700 m.

SPECIMENS EXAMINED. --- BORNEO: R.E. Holttum SFN 25429; J.

M.S. Clemens 29716, 28103, 32558.

8. Diplazium bantamense Blume

Diplazium bantamense Blume, En. Pl. Jav.: 191. 1828; Copel., Polypod.

Philipp.: 72. 1905; Backer & Posth., Varenfl. Java: 128. 1939; Holttum, Gard.

Bull. S.S. 11: 82. 1940; Tagawa & K. Iwats., Fl. Thailand 3: 455. 1988. –

Athyrium bantamense (Blume) Milde, Bot. Zeit.: 353. 1870: Holttum, Rev. Fl.

Malaya 2: 558. f. 330. 1966.

Rhizome short, erect, suberect. Stipe up to 90 cm long, 4-6 mm thick near

base, glabrescent, clothed with scales at base; scales narrow, to 15 mm long, 2

197

mm wide at base, dark brown, margin toothed. Lamina simple pinnate, oblong in

outline, up to 50 cm long, 26 cm wide; lateral pinnae 2-5 pairs, ascending, shortly

stalked, upper ones sessile, oblong, rounded at base, widest at a little above the

base, gradually narrowing towards acuminate apex, margin subentire or serrate

near apex, up to 25 by 5.5 cm; terminal pinnae similar to lateral ones or

occasipnally with large lobe at base; texture chartaceous, glabrescent; rachis

grooved above, often gemmiferous at the junction with costa of upper pinnae;

costa raised below, grooved above; veins free, 2-3 times forked. Sori elongate

along veinlets, usually diplazioid. Indusia brown, fragile, margin entire.

SPORES. Monolete, bilaterally symmetrical, heteropolar; polar outline

elliptical, sides convex; equatorial longitudinal view concave-convex; equatorial

transverse view, proximal face concave, distal face hemispherical; perinate. E:

42.81(53.88)66.05±5.69; P: 22.64(31.48)37.47±3.38. Laesure: concealed by

perine ridge. Perine: costate-alate, loosely reticulate irregular envelope, costae

form a large reticulation; separated from the spores; reticulation often incomplete;

lacunae large irregular polygons 11-25 m across; costae or alate project 3-13

m, terminating margins entire; surface of perine smooth. Exine: often visible

through perine, smooth under LM.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form uninterrupted U-shaped with an angle about 120º, base almost flat, end

ridges on both dirrection, outward and inward, bend outward to form an angle

150º. Stomata: polocytic and copolocytic.

CHROMOSOMES. 2n = 164 (Cytotype: T.Ng. Praptosuwiryo 1454,

BOHB), 328 (Cytotype: Tin 1-3, BOHB).

DISTRIBUTION. Malesia throughout.

ECOLOGY. Terrestrial in lowland and lower montane forest. Occurs in

shady places in hills and occasionally by streams in the lowlands, but not

common. 20-1600 m.

SPECIMENS EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 1366, 1369,

1373, 1383, 1212, 1454, 1510, 1511, 1516, 1517, 1752. ---BORNEO: M. Kato,

M.Okamoto, K.Ueda, D. Darnaedi & E.B. Walujo B-8355; M. Kato, M. Okamoto

& E.B. Walujo B-10609; M. Kato, G. Murata, Y.P. Mogea B-3773; A.A. Church,

198

A.C. 319. --- SUMATRA: Dr. Cramer 40; W.J.J.O. de Wilde and B.E.E. de

Wilde-Dyfjes 12793; O. Posthumus s.n. (27 July 1925); J.A. Lörzing 14730; J.A.

Lörzing 5342a; J. v. Borssum W. 2767; J.J. Afriastini 613; J.A. Lörzing 6894. ---

MALAY PENINSULA: G.H. Addison SFN 37159.

9. Diplazium barbatum Christensen

Diplazium barbatum C.Chr. in C.Chr. & Holttum, Gard. Bull. S.S. 7: 272.

pl. 59. 1934. --- TYPE: Borneo, Mt. Kinabalu, Holttum SFN 25386 (Holotype,

BM n.v.; isotype, US!; K n.v., SING n.v.).

Rhizome short, erect. Stipe ca. to 30 cm long, light brown throughout,

densely scales toward base; scales light brown, linear lanceolate, 3-1.4 mm long,

0,25-1 mm broad, concolours, without thickening black strands, margin toothed,

teeth distinctly forked. Lamina broad lanceolate, ca. to 65 cm long, 19 cm broad,

pinnae 10 pairs; lower shortly stalked to 3 mm long, pinnae pinnate, ovate

lanceolate, to about 12 cm long, 5.3 cm broad, free pinnulae 2-4 pairs below

pinnatifid apex of pinnae; pinnulae sessile, almost at right angle to costa, oblong,

margin entire, ends acute-rounded, slightly toothed; texture papyraceous, rachis

and costa densely minute scales; veins free, pinnate in the pinnulae; veinlets to 9

pairs, once forked. Sori elongate from near costule continuing to acroscopic

branch of veinlets covers 2/3-3/4 of their length, indusia pale brown, margin

entire, persistent.

DISTRIBUTION. Borneo.

ECOLOGY. Terrestrial in mountain forest. Elevation 1400-2100 m.

SPECIMENS EXAMINED. --- BORNEO: J. & M.S. Clemens 32607;

R.E. Holttum 25386; M. Kato, G. Murata & Y.P.Mogea.

NOTES. As notified by Christensen & Holttum (1934), D. barbatum is

somewhat resembling D. speciosum in general habit, colour and texture, but very

distinct by its densely squamose stipe, rachis and costae..

199

10. Diplazium batuayauense Praptosuwiryo, sp. nov.

TYPE: Borneo, Central Kalimantan, Mts. Muller, Batau Ayau, Above S.

Talikot Puhung Kucan, ca. 450 m, 14 June 2004, T.Ng. Praptosuwiryo 1927

(holotype, BO).

Rhizoma breve erectum. Stipites gracilis, 19-5-23 cm longae, 3 mm

crassa, squamis caducus brunneus, linear-lanceolatus, 4-10 mm longae, 0.5 mm

latis, margine irregularis dentibus furcatis cum filum niger spissescens. Lamina

pinnata, lanceolatis, 41 cm longis, 20 cm latis, pinnae 15-jugatae infra apice

pinnatifidus; pinnae inferiora stipitae ad 3.5 mm longis, superiora adnatus,

lanceolatae, 11.2 cm longis, 2 cm latis, basi inaequaliter truncatae ad cuneatae, e

margine 1/3-1/2 costam lobatae; lobi 4-5.5 mm latae supra basim, truncatae,

leviter crenati. Rachides non gemmiferae, glabrae. Venulae distinctus in

superficiebus ambabus, liberis, pinnatus in uno lobo, venae principalis angulum

55º cum costa formantes, venulae 4-5-jugatae, simpliciter. Sori medial or close to

margin of lobes, cover 1/3-1/2 of veinlets length (3 mm length). Indusiis

brunneis, concolour, persistens, ineteger.

Rhizome short, erect. Stipe slender, 19.5 -23 cm long, 3 mm thick, fallen

scales; scales brown, lineary lanceolate, 4-10 mm long, 0.5 mm broad, margin

toothed, teeth apart, with thickening dark brown strand irregularly, glandular cells

present,. Lamina pinnate, lanceolate in outline, 41 cm long, 20 cm broad, pinnae

15 pairs below pinnatifid apex of lamina; lower pinnae stalked 3.5 mm long,

upper pinnae adnate, one pair basal pinnae bending downward with cut away

base; pinnae lanceolate, widest at 1/3 part from basal, 11..2 cm long, 2 cm broad,

base unequally truncate on lower pinnae, cuneate at upper ones, apex acuminate,

margin lobed to within 4 mm of costa (or 1/3-1/2 way to costa); lobus widest at

base, 4-5.5 mm wide above base, ends truncate, slightly crenate. Rachise not

gemmiferous, glabrous. Texture thin. Veins distinct on both surface, free, pinnate

in the lobus, main vein forming angle 55° to costa, veinlets 4-5 pairs, all simple,

reaching margin. Sori medial or close to margin of lobes, cover 1/3-1/2 of

veinlets length (3 mm length). Indusia brown, persistent, margin entire.

200

PARATYPE. BORNEO: Central Kalimantan, Mts. Muller, Above S.

Talikot Puhung Kucan, track to Batu Ayau, 450 m, 14 June 2004, T.Ng.

Praptosuwiryo 1927a, 1927b, 1927c, 1927d, 1927e.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form interrupted U-shaped, with an angle 115º, angle slightly ridges, end almost

simple.

CHROMOSOMES. 2n = 164 (Cytotype: T.Ng. Praptosuwiryo 1927c,

BOHB), 205 (Cytotype: T.Ng. Praptosuwiryo 1909, BOHB).

DISTRIBUTION. Borneo

ECOLOGY. Terrestrial in lower montane forest. It is Grows on humus

rich soil in shady places.

NOTES. This species closely related to D. sorzogonense. Diplazium

batuayauense differs from D. sorzogonense in the following characters: Scales

sharply toothed with thickening dark brown stand irregularly, fronds are much

smaller, never reaching 1 m, lacks the fibrillose scales on stipe and rachis, pinnae

lobed only to ½ way to costa, sori not impressed. D. batuayauense is also similar

to D. malaccense.

ETYMOLOGY. The specific epithet is from the locality where this

species found, Batu Ayau, the mountain forest of Mts. Muller in Central

Kalimantan, Borneo.

11. Diplazium beamanii M.G. Price

Diplazium beamanii M.G. Price, Contr. Univ. Mich. Herb. 16: 193, f. 1-3.

1987. Type: Borneo: Pinosuk Plateau, 1400 m, Beaman 10724 (Holotype,

MICH!; Isotype , K!., MSC n.v.).

Rhizome erect. Stipe stramineus, 66 cm long, 7 mm thick, densely scales

at base; scales pale brown, ovate, 10 mm long, 4.5 mm broad, margin entire.

Lamina ovate, 60 cm long, bipinnate to deeply tripinnatifid at base, pinnae (?)

pairs; pinnae stalked to 2.5 cm long, subbasal the largest, to 37 cm long, ca. 17 cm

broad, pinnulae to 13 pairs, basal acroscopic slightly reduced; pinnulae stalked to

ca. 1 mm long, 2 cm distance, subtriangular-lanceolate, to 8 cm long, 2 cm broad,

base truncate, apex acuminate, margin lobed to 5/6 way to costa (within 2 mm of

201

costa); segments to ca. 5 mm wide, ends subtruncate, margin slightly denticulate-

crenulate; veins free, pinnate, veinlets to 5 pairs, usually simple. Sori from near

costule cover 1/3 way of veinlets (1.0-2.5 mm long), basal acroscopic often

diplazioid; indusia broad, margin irregularly fringed, persistent.

DISTRIBUTION. Borneo.

ECOLOGY. Terrestrial. Lower montane forest by stream. Elevation:

1400 m. Endemic to Mount Kinabalu.

SPECIMEN EXAMINED. --- BORNEO: Beaman 10724.

12. Diplazium betimusense Alderw.

Diplazium betimusense Alderw., Bull. Buit. III. 2. 142. 1920. ---TYPE:

J.A. Lörzing 5718 (Holotype, BO!; Isotype, L!), Sumatra, Sibolangit, Betimus

River.

Rhizome short, erect, 17 mm thick. Stipe dark brown, 48 cm long,

sparingly muricate, scales towards the base, scales on stipes dark brown, rounded-

ovate, margin entire, deciduous, leaving the stipes roughish by their persistent

bases, 3.5 mm long, 3 mm broad. Lamina bipinnate, (?) cm long, (?) cm broad,

pinnae (?) pairs; lower pinnae stalked to 5.3 cm long, oblong subtringular, 40 cm

long, 18 cm broad, pinnulae 6-9 pairs below pinnatifid deltoid apex of pinnae;

lower pinnulae shortly stalked to 1.5 mm long, upper ones adnate-sessile,

lanceolate, 8-10.5 cm long, 2-3 cm broad, base subequally truncate-cuneate,apex

acuminate-caudate, sharply toothed, margin lobed to within 7 mm of costa (or 1/3-

1/2 to cosule; lobus more or less widest at base, 5-6.5 mm broad, ends truncate,

slightly toothed; rachise dark brow, glabrous; texture firm; veins free, pinnate in

the lobus, main veins forming angle 50-55° to costule, veinlets 4-5 pairs, forming

angle about 10-15 º to main veins, distinct on both surface, simple, all reaching

the margin. Sori subbasal or medial, covers to 1/3 of their length, sometimes

diplazioid on basal acroscopic veinlets; indusia narrow.

DISTRIBUTION. Sumatra.

ECOLOGY. Growing on shade part of forest, near river at ca. 400 m sea

level.

202

SPECIMENS EXAMINED. --- SUMATRA: J.A. Lörzing 5718

13. Diplazium christii C.Chr.

Diplazium christii C.Chr., Index Fil.: 229. 1905; C.Chr. & Holttum,

Gard. Bull. S.S. 7: 270. 1934.

Rhizome (?). Stipe 35 cm long, 4 mm diam., scales at base , dark brown;

scales on stipes light brown, concolours, shining, 3-7 mm long, 0.5-1.5 mm diam.

near base, margin entire, without thickening black strands. Lamina pinnate,

lanceolate, 65 cm long, 18 cm broad, pinnae 19 pairs below deeply deltoid apex of

lamina (?); pinnae shortly stalked to 3 mm long, lanceolate 15 cm long, 1.8 cm

broad, base cuneate, apex acuminate, margin lobed ½ way to costa; lobus oblique,

widest at base, to 6 mm above base, ends subtruncate, slightly toothed; veins free,

pinnate in the lobes; veinlets usually 5-6 pairs, simple. Sori elongate at middle

veinlets covers 2/3 of their length; indusia light brown, broad, margin entire,

opening when mature.

DISTRIBUTION. Malay peninsula.

ECOLOGY. In dense forest, near summit 600 m.

SPECIMENS EXAMINED. --- MALAY PENINSULA: R.E. Holttum

SFN 19912.

14. Diplazium cordifolium Blume

Diplazium cordifolium Blume, En. Pl. Jav.: 190. 1828; Holttum, Gard.

Bull. S.S. 11: 83. 1940. – Anisogonium cordifolium (Blume) Bedd., Ferns Br.

Ind.: t. 331. 1870; Hand. Ferns Br. Ind.: 191. f. 92. 1883. -- Athyrium cordifolium

(Bl.) Copel., Holttum, Rev. Fl. Malaya 2: 548. f. 322. 1966. TYPE: C.J.A. van

Hasselt s.n. (?, L!), Kapala Tjibarran, Java.

Diplazium integrifolium Blume, En. Pl. Jav.: 190. 1828. TYPE: Blume

s.n. (?, L!), Bogor, Java.

Callipteris cordifolia (Blume) J. Sm., Copel., Polypod. Philipp.: 70.

1905.

Athyrium pariens Copel., Phil. Journ. Sci. IIIc : 299. 1908.

203

Key to the varieties

1. Lamina simple, oblong subdeltoid, base cordate, apex acuminate; veins forked to 5 times ………………………………………………... var. cordifolium

1. Lamina simply pinnate

2. Terminal pinnae usually the largest, oblong subdeltoid; lateral pinnae oblong, base cordate, apex acuminate; texture coriaceous …………………... var. integrifolium

2. Terminal pinnae usually similar shape to lateral ones, lanceolate; lateral pinnae lanceolate, base rounded to moderately auricled at basiscopic and truncate to cunetae at acroscopic, apex attenuate; texture subcoriaceous ……. var. pariens

Rhizome erect, suberect, scales on younger part. Stipe pale brown, 3-4

mm diam., 30-55 cm long, grooved above, brown, nearly black and scales toward

base; scales oblong ovate with acuminate apex, 6-9 mm long, 1-2 mm wide near

base, shining dark brown, margin entire with black thickening above base to tip

when old, occasionally with glandular cells. Lamina of two kinds, simple and

imparipinnate; simple ones oblong subdeltoid, 23-30 cm long, 8-13 cm wide

above base, widest 1/3 from base, base cordate, apex acuminate, margin entire;

imparipinnate ones nearly oblong deltoid in outline, 28-45 cm long, 24 cm wide;

lateral pinnae 1-6 pairs, upper smaller, usually terminal ones largest, oblong

subdeltoid, to 13-18 by 4-8 cm; upper pinnae sessile, broadly cuneate at base,

lower pinnae shortly stalked, the largest 16 by 5.5 cm, cordate at base, apex

acuminate, margin entire, rachis distincty beneath, gemmiferous at the junction

with costa; texture coriaceous; veins at about 45-70°to costa on pinnate ones, 50-

80º to midrib on simple ones, forked close to midrib, lower branch forked again 2-

5 times, anastomousing irregularly about 1/3-1/2 from margin. Sori elongate

along veinlets on both side or on acroscopic ones; indusia thin, persistent, margin

entire.

SPORES. Monolete, bilaterally symmetrical (made asymmetric by

perine), heteropolar; polar outline (excluding perine) transversely elliptical, sides

convex; equatorial longitudinal view (excluding perine) plano-convex; equatorial

transverse view, proximal face planar, distal face convex; perinate. E:

37.02(42.15)51.62 ±5.09 P: 20.23(26.56)31.21±2.97. Laesura: concealed by

perine wing. Perine: alat to costate-alate, loose reticulate; irregular envelope

separated from exine surrounds the spore in continuous anastomosing wings,

204

forming a loose reticulation; lacunae large irregular polygons 15-20 µm across;

thin wing-like muri project 2-8 µm, terminating margins are often echinate;

surface of perine sparsely echinate or ciliate; echinae project 0.5-0.8 µm. Exine:

visible through perine, smooth under SEM.

SPECIMENS EXAMINED. MALAY PENINSULA. --- SINGAPORE:

H.N. Ridley 5867. --- BORNEO: Turaya 1894.

DISTRIBUTION. Malesia throughout, eastward to Solomon Islands.

a. var cordifolium

Diplazium cordifolium Bl., En. Pl. Jav.: 190. 1828. ---TYPE: J.C.A. van

Hasselt s.n. (L!, No.Reg. L 0051534), Kapala Cibaran, Java.

Lamina simple, about 27 cm long, 13 cm broad; veins branching to 8

times, anastomousing 1/3 from margin to the costa; veins group forming an angle

about 70ºto costa (at the middle), soriferous on outer veins, inner also, diplazioid

on basal acroscopic.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an uninterrupted V-shaped, with an angle 65º, end simple, not forming a

ridge.

CHROMOSOMES. 2n = 164 (Cytotype: T.Ng. Praptosuwiryo 1203, BO),

205 (Cytotype: T.Ng. Praptosuwiryo 1204, BO), 246 (T.Ng. Praptosuwiryo 1201,

BO), 328 (Cytotype: T.Ng. Praptosuwiryo 1926b, BO).

DISTRIBUTION. Sumatra, Malay Peninsula, Java, Borneo.

ECOLOGY. Occurs on moist sandy soil or humus rich soil of mountain

slopes in dense forest at low or medium altitudes. 400 – 2000 m.

SPECIMENS EXAMINED. ---- JAVA. West Java: A. Hidayat & H.

Wiriadinata AH 501; T.Ng.Praptosuwiryo 1201, 1202, 1203, 1204, 1206, 1207,

1208, 1456, 1460, 1461, 1737, 1768, 1807, 1813, 1710, 1768, 1807, 1808. ---

BORNEO: T.Ng. Praptosuwiryo 1910, 1926a, 1926b, 2128a, 2128c, 2128e,

2194a, 2194c, 2128b, 2194d, 2194e; Amdjah 271; J. & M.S. Clemens 26898;

Amdjah 273; J. & M.S. Clemens 33810; Amdjah 718; Kunio Iwatsuki, M. Kato,

Gen Murata & Y.P Mogea B-779; K. Iwatsuki, M. Kato, G. Murata & Y.P.

Mogea B-1935; A.Kostermans 8130; Amdjah 718; Kostermans 9051; Teysman

s.n.; M.Kato, M. Okamoto & E.B. Walujo B-10079; M. Kato & H. Wiriadinata B-

205

6362; M. Kato & H. Wiriadinata B-4727; M. Kato & H. Wiriadinata B-7098; M.

Kato & H. Wiriadinata B-6900; Kunio Iwatsuki, M. Kato, Gen Murata & Y.P.

Mogea B-2961; K. Iwatsuki, M. Kato, Gen Murata & Y.P. Mogea B-2271. ---

MALAY PENINSULA: King 6274; M.A. Donk no B.T. 10; R.E. Holttum 9898;

R.E. Holttum 31219; R.E. Holttum 23340; R.E. Holttum 10799; Md Nur SFN

19773. --- SUMATRA: S. Prawiroatmodjo 2576; W.J.J.O. de Wilde & B.E.E. de

Wilde-DUyfjes 19946; v. Steenis 3792; D. Darnaedi 81; L. Ajoeb 90; L. Ajoeb

96; J. v. Borssum W. 2788.

b. var. integrifolum

Diplazium integrifolium Blume, En. Pl. Jav.: 190. 1828. ---TYPE: Blumes.n. (L!, No.Reg. L 0051533 ), Java. Diplazium cordifolium Blume var.integrifolium (Blume) Mitsuta, Acta Phytotax Geobot. 36 (1-3): 78. 1985.

Lamina imparipinnate. Terminal pinnae usually the largest, oblong

subdeltoid; lateral pinnae 1-6 pairs, the largest 16 by 5.5 cm, oblong, base cordate,

apex acuminate; texture coriaceous; veins 1-3 forked, anastomousing in the

marginal 1/3-1/2.

SPORES. Monolete, bilaterally symmetrical (made asymmetric by

perine), heteropolar; polar outline (excluding perine) transversely elliptical, sides

convex; equatorial longitudinal view (excluding perine) plano-convex; equatorial

transverse view, proximal face planar, distal face convex; perinate. E:

37.02(42.15)51.62 ±5.09 P: 20.23(26.56)31.21±2.97. Laesura: concealed by

perine wing. Perine: alat to costate-alate, loose reticulate; irregular envelope

separated from exine surrounds the spore in continuous anastomosing wings,

forming a loose reticulation; lacunae large irregular polygons 15-20 µm across;

thin wing-like muri project 2-8 µm, terminating margins are often echinate;

surface of perine sparsely echinate or ciliate; echinae project 0.5-0.8 µm. Exine:

visible through perine, smooth under SEM.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an uninterrupted U-shaped with an angle about 110º, base flat on inward and

outward direction, end ridges equally on both both directions.

CHROMOSOMES. 2n = 164 (T.Ng. Praptosuwiryo 2128, BO).

DISTRIBUTION. Sumatra, Java, Malay Peninsula, Borneo.

206

SPECIMENS EXAMINED. --- JAVA: TNgPraptosuwiryo 1192, 1205,

1206, 1207, 1208, 1305, 1367, 1368, 1375, 1374, 1457, 1706, 1708, 1709, 1729,

1735, 1736, 1742, 1774, 1775, 2280, 2281, 2284, 2288, 2291, 2293, 2297, 2298,

2300, . --- SUMATRA: de Vogel & Vermeulen 7504.; Kerling s.n., T.Ng.

Praptosuwiryo 2524a, 2524b, 2539. --- BORNEO: M. Kato, M. Okamoto, K. Ueda

& E.B. Walujo B-7397; M. Kato, M. Okamoto, K. Ueda & E.B. Walujo B-7386;

Masahiro Kato & H. Wiriadinata B-4984; M. Kato & H.Wiriadinata B-5243;

Halllier 1727; M. Kato, M. Okamoto & E.B. Walujo B-10236; M. Kato, M.

Okamoto & E.B. Walujo B- 10580; M. Kato, M. Okamoto & E.B. Walujo B-

9622; M. Kato, M. Okamoto, E.B. Walujo B-10847.

c. var. pariens (Copel.) C. Chr., Gard. Bull. S.S. 7 (3): 274. 1934; Ind.

Fil. Suppl. III: 75. 1934; Mitsuta, Acta Phytotax. Geobot. 36 (1-3): 78. 1985.

Lamina pinnate, pinnae 4-8 pairs; lower lateral pinnae shortly stalked to 3

mm, upper ones sessile, lanceolate, gradually reduce in size upwards, larger ones

12 cm long, 2.8 cm wide, attenuate at apex, rounded to moderately auricled at

basiscopic and truncate to cunetae at acroscopic bases, margin entire, terminal

pinnae similar to the lateral ones; texture subcoriaceous, rachis gemmiferous at

the adjacent to costa; veins 1-3 forked, anastomousing in the marginal 1/3-1/2.

SPECIMENS EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 1192, 1205,

1305, 1367, 1368, 1369, 1374, 1375, 1457, 1708.

NOTE: Mitsuta (1985) also recognized two varieties of D. cordifolium

of Sumatra, var. integrifolium (Blume) Mitsuta and D. pariens. The two varieties

are differentiated with characters as follow: Var. integrifolium has 2-3 pairs of

lateral pinnae and base of terminal pinna sessile, while var. pariens with 4-6 pairs

of lateral pinnae and base of terminal pinna usually wide cuneate.

15. Diplazium crameri Praptosuwiryo, sp. nov.

TYPE: Sumatra, Sukaraja, Kenali, 27 August 1915, DR. Cramer 41

(Holotype, BO).

Rhizoma erect (?). Stipites dilutus brunneus nitidus, 38 cm longis, 4 mm

crassis, basi piceus squamis caducus lanceolatus, c.7 mm longis, 2 mm latis,

integris, brunneis. Lamina simpliciter imparipinnatae, oblongae, 25.5 cm longae,

207

21 cm latae; pinnae 5-jugatae, stipitae ad 1.5 cm longis, 3.5-4 cm seorsum, ovate-

lanceolate, gradatin decrescente in statura, pinnae infernus ad 12.5 cm longis, 3

cm latis, basi cuneatae, marginae subintegrae, versus apice leviter serratus, apice

acuminatae, glabrae, in sicco superne atrobrunneae, inferne brunneae, pinnae

terminalis ad lateralis conformes; rhachides non gemmiferae, glabrae, costae

inferne prominens; vennae distingubilis, libere, angulum 50-60° cum costa

formantes, propinquus costae furcated, ramus superis simpliciter et soriferus,

ramus infernis simpliciter vel furcatis, plerumque furcatis. Sori e propinquus

costae elongati ad 7/9 venulae occupants, soris infimus acroscopicus

diplazioideus. Indusiis latis, brunneis, margine leviter crispatus, persistens.

Rhizome erect (?). Stipe pale brown, glossy, 38 cm long, 4 mm thick,

black at base, fallen scales; scales lanceolate, ca. 7 mm long, 2 mm broad, margin

entire, dark brown. Lamina simply pinnate, oblong, 25.5 cm long, 21 cm broad,

pinnae 5 pairs, terminal pinnae conform the lateral ones; lateral pinnae stalked to

1.5 cm long, 3.5-4 cm apart, below apical pinnae adnate, ovate-lanceolate,

gradually decrising in size, lower pinnae to 12.5 cm long, 3 cm broad, margin

entire, slightly serrate towards apex, base equally cuneate; rachise not

gemmiferous, costa raised below, glabrous; texture very firm. Veins free, distinct

on both surface, forming angle 50-60 to costa, forked near costa, upper branch

simple and soriferous, lower branch simple-once forked, commonly once forked.

Sori elongate from near basal veinlets (0.5-3.5 mm distance from costa) cover to

7/9 of lengt veinlets (irregularly), acroscopic basal diplazioid, other asplenoid.

Indusia broad, brown, margin slightly crisped, persistent.

DISTRIBUTION. Sumatra.

ECOLOGY. This is a dry land terrestrial fern that grows in shady places

of primary forest .

NOTES. Diplazium crameri may closely related to D. xiphophyllum.

The two species share stramineous stipe, lanceolate entire concolour scales,

simply pinnate oblong lamina, base of pinnae cuneate and texture subcoriaceous.

Diplazium crameri differs from D. xiphophyllum in its scales dark brown, pinnae

stalked to 1.5 cm long, ovate-lanceolate, all veins free and forked to 2 times.

Meanwhile D. xiphophyllum has pinnae elliptical, lower pinnae shortly stalked,

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veins often anastomousing near margin and forked to 5 times. There is one

specimen only found among assemblage of specimens at BO that collected from

Sumatra. (22 Sept. 2007).

ETYMOLOGY. This species is named after DR. Cramer, the first

collector of this species.

SPECIMENS EXAMINED. --- SUMATRA: DR. Cramer 41.

16. Diplazium crenatoserratum (Blume) Moore

Diplazium crenatoserratum (Blume) Moore, Ind. Fil.: 121. 1859; Bedd.,

Hend. Ferns Br. Ind.: 177. 1883; Backer & Posth., Varenfl. Java: 130. 1939;

Holttum, Gard. Bull. S.S. 11: 99. 1940. –Asplenium crenatoserratum Blume,

Enum. Pl. Jav.: 177. 1828. –Athyrium crenatoserratum (Blume) Milde, Bot. Zeit.:

353. 1870; Holttum, Rev. Fl. Malaya 2: 561. f. 322. 1966.

Rhizome short, erect. Stipe 30-50 cm long, glabrescent, pale brown, black

and scales at base, distinctly grooved above; scales narrowly oblong subtriangular,

about 5 by 1 mm, concolour, dark brown, margin toothed irregularly, teeth not

forked. Lamina pinnate without distinct terminal pinnae of the fornd, oblong

triangular in outline with attenuate apex, widest at base, 33-70 cm long, 14-20 cm

or more wide; rachis grooved above; pinnae 9-22 pairs, stalked 3.5-5 mm long,

liniery oblong, 7-11 cm long, 1.4-2.7 cm wide, upper ones sessile with cuneate

lower base, lower ones stalked, upper base strongly auricled, less lower base or

slightly rounded; margin lobed ¼- ½ to costa or almost entire, lobes irregularly in

size; texture softly chartaceous; veins pinnate in lobes to 3 pairs or once-twice

forked in subentire pinnae. Sori alongate from nearly costa almost reach margin

of pinna, single or occasionally diplazioid on acroscopic veinlets of forked veins,

usually diplazioid on acroscopic of basal pinnate veins; indusia thin brown,

fragile, persistent, margin entire.

SPORES. Monolete, bilaterally summetrical (made asymmetric by

perine), heteropolar; polar outline (excluding perine) transversely elliptical, sides

convex; equatorial longitudinal view (excluding perine) concave-convex;

equatorial tranverse view, proximal face concave, distal face convex; perinate. E:

29.86(38.19)45.74±4.95, P: 17.92(23.48)27.36±2.94. Laesura: concealed by thin

209

wing-like muri. Perine: alate, loosely reticulate; irregular envelope, separated

from exine, surrounds the spore in anastomosing wings, forming loose

reticulation, reticulation irregular and often incomplete; lacunae irregular

polygons, 8-16 µm across, irregular holes and small papillae within; holes caused

by the fallen papillae; muri thin, wing-like, projected c. 1-8 µm, terminating

margin ciliate; surface of perine fibrous-like and holed, holes irregular and formed

by fallen ciliae.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an interrupted V-shaped formed from two long oval leaf traces, end simple

without ridges at both inward and outward directions.

CHROMOSOMES. 2n = 123 (Cytotype: T.Ng. Praptosuwiryo 2075, BO),

164 (Cytotype: T.Ng. Praptosuwiryo 2067, BO).

DISTRIBUTION. Peninsular, Malaysia, Sumatra, Java, Borneo,

Moluccas, Thailand.

ECOLOGY. Terrestrial in primary and secondary forest, dry land, shaded

in slope and rigde of lowlands and montane forest. It can grow on various soil

from yellow-red loamy, red clay, and on humus rich soil. 20 – 1000 m.

SPECIMENS EXAMINED. --- JAVA: Backer 10644, 9927, 9999,

25555; Bakhuizen v/d Brink 3330, 3416, 6403, 7282. --- MALAY PENINSULA:

H.N. Ridley 3068, S.Z. Lin S2 2002-65; Sidek & Rozali Jaman RJ 1783; V.L.

Gurung 2. --- BORNEO: A. Kostermans 9747, 4145, 4139; K. Iwatsuki, M.

Kato, G. Murata & YP. Mogea, B-2273; B-2274; K. Iwatsuki, M.Kato, G. Murata

& Y.P.Mogea B-2958; K Iwatsuki, M. Kato, G. Murata, Y.P. Mogea B-717; B-

736; F.H. Endert 2796; Amdjah 723. BANGKA: Anta 728; Huguenin s.n., A.C.

Church, U.W. Mahyar, A.Ruskandi, Nurdin -192; A.C. Church, U.W. Mahyar,

A.Ruskandi, Nurdin -442; Sibatak Luang S 24733. --- MENTAWAI ISLANDS:

Iboet 557; Amdjah 186. --- SUMATRA: W.J.J.O de Wilde & B.E.E. de Wilde-

Duyfjes 19733; W. Meijer 15818; O. Posthumus 664; T.Ng. Praptosuwiryo

2011a, 2011b, 2011c, 2142a, 2142c, 2044, 2067a, 2067b, 2073a, 2073b, 2074a,

2074b, 2074c, 2074d, 2075, 2075c, 2075d, 2084a, 2085a, 2085b; 2208, 2210.

210

17. Diplazium crinitum (Baker) C.Chr.

Diplazium crinitum (Baker) C.Chr.; Ind. Fil. 230. 1905. ---Athyrium

crinitum (Bak.) Copel. Brittonia 1: 73. 1931. ---Athyrium vestitum of Copel.

Sarawak Mus. Journ. 2: 377. 1917.

Rhizome (?). Stipe 25-45 cm long, 2.5-7 mm diam. near base, light

brown, densely scales throughout; scales on stipes yellowish, shining, lanceolate,

4-7 mm long, 0.5-1 mm broad, margin toothed with thickening black strands

irregularly, fragile; teeth forked irregularly. Lamina bippinate, ovate in outline,

(?) cm long, 24 cm broad, pinnae (?) pairs. Pinnae shortly stalked to 6 mm long,

oblong in outline, 25 cm long, 10 cm broad, pinnulae to 11 pairs; lower pinnulae

adnate, upper sessile below deeply deltoid apex of pinnae; pinnulae oblong, larger

ones to 6 cm long, 1.5 cm broad, base broadly cuneate, apex acuminate, margin

lobed 1/3 way to costule; lobes oblique, widest at base, ends truncate or slightly

rounded, subentire; rachise dark brown, grooved on upper surface, densely minute

toothed scales; costa and costule also densely scales as like as rachise; texture

herbaceous-memranaceos; veins free, pinnate in lobes, veinlets 4-6 pairs, simple,

soriferous 1-4 pairs from basal. Sori elongate from near cosulet covers 1/4-2/3 of

their length, diplazioid on basal acroscopic; indusia light brown, attachment side

darker, margin entire, fragile.

DISTRIBUTION. Borneo.

ECOLOGY. Limestone areas. Terrestrial on mountain slope in deep

shade. 50-100 m.

SPECIMENS EXAMINED. --- BORNEO: M. Kato & H. Wiriadinata B-

5953; B-5955; B-5956; M. Kato, H. Wiriadinata B-5414; M. Kato & H.

Wiriadinata B-5953; B-5935.

18. Diplazum cumingii (Presl) C. Chr.

Diplazum cumingii (Presl.) C. Chr. Ind. Fil. 230. 1905. --- Athyrium

cumingii (Presl) Milde. --- Ochlogramma cumingii Presl. --- Calliptiteris

alismifolia J. Smith (nomina nudum). --- TYPE: Cuming 116 (Isotype, MW!).

Rhizome short, erect. Stipe dark brown or nearly black, 25.5-39 cm long,

3-4 mm diam. near base, blackish, fallen scales at base. Scales on stipe dark

211

brown, blackish, concolour, subulate, 4-7 mm long, 0.5-1.0 mm broad, margin

entire. Lamina imparipinate, oblong, 37-45 cm long, 19-32 cm broad, lateral

pinnae 2-3 pairs, terminal pinnae like the rest, the largest. Pinnae subopposite,

shortly stalked 2.5-3 mm long, oblong, base cuneate, apexcaudate, margin entire,

to 19 cm long, 4.7 cm broad; terminal pinnae 21.5-26 cm long, 6.8-7.0 cm broad.

Coatae distinctly raised below, dark brown when dry, minutely scales on lower

surface. Veins in small group of 2-4 veins, forming an angle about … ºto costa,

veilets forked 2-3 times again, outer and inner veins uniting with the nearest veins

forming areoles 1 mm wide, or nastomousing 1/7-¼ way from margin to costa.

Sori elongate from costa continuously to near margin, forming areoles near

margin, outer acroscopic diplazioid, inner also. Indusia black, margin entire, firm,

persistent, opening when old, margin of indusia leaving line scar between veins

like false veins.

DISTRIBUTION. Borneo

ECOLOGY. Terrestrial on stream bank in deep shade, lowlands ever

green rain forest, on humus rich slope. 270 – 1500 m.

SPECIMENS EXAMINED. ---BORNEO: Kato & H. Wiriadinata B-

4729; B-5060; M Okamoto & E.B. Walujo B-11491; M.Kato, M. Okamoto &

E.B. Walujo B-11227; M. Kato, M. Okamoto, K. ueda, D. Darnaedi & E.B.

Walujo B-8353; M. Kato, M. Okamoto & E.B. Walujo B-9882; M. Kato, Gen

Murata, & Y.P. Mogea B-3775.

NOTE: The sori and the indusia of this species are very distinctive.

Indusia are rolled back when old, always leaving a distinct ridge indicating the

original position of their outer edges.

DISTRIBUTION. Borneo, Philippines.

19. Diplazium densisquamatum Praptosuwiryo, sp. nov. Plate 4.

Type: SUMATRA. Jambi, Kerinci Seblat National Park, Sungai Penuh,

Bukit Tapan, secondary forest, 1290-1300 m, 5 September 2006, T.Ng.

Praptosuwiryo 2491 (holotype, BO).

Rhizoma breve erecta. Stipites ad 47.5 cm longae, 0.6 cm cassi fere basi,

squamis brunneis nitidus integris ovatis-lanceolatis 7-17 mm longae 0.5-2.5 mm

latae densus penitus vestitae. Lamina ad 95 cm longae, 65 latae, bipinnata

212

deltoideus; pinnate pinnae ad 6 jugatae, pinnatida pinnae ad 7 jugatae infra apice

pinnatifidus. Infimus pinnae stipes ad 1 cm longae, 38-41 cm longae 10.5-13.0 cm

latae, lanceolate, libere pinnulae 13-18-jugatae; pinnulae infimus acroscopicus

leviter dimidia ad 2.8 cm longis, 1 cm latis; pinnulae maximae stipitae ad 1 mm

longis, superiora adnatus-sessilis, oblong subtriangulare, ad 6.8 cm longiae, 1.9

cm latae, basi truncatae, apice acuminatae, e margine ¾ costam versus lobatae;

lobi oblongis, basi basiscopicus maximae, ad 4 mm latae, plerumque 3 mm latae,

truncati, leviter dentatis. Venulae libere, pinnatae in lobo, 5-6-jugatae,

plerumque 5-jugatae, simplices vel in lobo infimus basiscopicus furcatis Rachise

et costae, minute squamis, non gemmiferae. Sori e basi venarum ½, ad 1.5 mm

longae, soris infimus acroscopicus diplazioideus. Indusiis latis, brunneus,

concolorus, marginem lacerus, persistens.

Rhizome short, stout, erect, scales densely on younger part. Stipes dark

brown, black toward base, 47.5 cm long, 0.6 cm cm thick, scales densely

throughout; scales dark brown, ovate-lanceolate, 7-17 mm long, 0.5-2.5 mm

broad, margin entire, without thickening black strands, glandular cell present.

Lamina bipinnate, deltoid, to 95 cm long. 65 cm broad; pinnate pinnae 6 pairs,

pinnatifid pinnae 7 pairs below pinnatifid apex of lamina; lower pinnae stalked to

1 cm long, lanceolate, 38-41 cm long, 10.5-13.0 cm broad, free pinnulae 13-18

pairs; basal acroscopic pinnulae a little reduced to 2.8 long, 1 cm broad; larger

pinnae short stalked to 0.5-1 mm long, upper adnate-sessile, oblong subtriangular,

5.6-6.8 cm long, 1.7-1.9 cm broad, base truncate, apex acuminate, margin lobed ¾

way to costa; lobes oblong, basal basiscopic the widest, 2-4 mm wide, commonly

3 mm wide, ends truncate, slight toothed. Veins free, pinnate in the lobes, veinlets

5-6 pairs, commonly 5 pairs, commonly simple, once forked on basal basiscopic

lobes. Rachise and costa minutely scales, not gemmiferous. Texture

subpapyraceous. Sori elongate from basal covers ½ of its length, 1.5 mm long,

basal acroscopic diplazioid. Indusia broad, dark brown, concolour, margin

lacerate, persistent.

DISTRIBUTION. This species has hitherto only been found in shady

forest of Bukit Tapan, Kerinci Seblat National Park, Sumatra.

ECOLOGY. In shady place of dry land in secondary forest. 1000-1200 m.

213

SPECIMENS EXAMINED. SUMATRA: T.Ng. Praptosuwiryo 2491.

NOTES. Diplazium desisquamatum is in a glance similar to the small

plants of D. polypodioides. However the dense scales throughout the stipes with

ovate –lanceolate and entire margin will differentiate from D. polypodioides

fastly. The stipes of D. polypodioides are densely covered by lineary lanceolate

sharply toothed scales at base only. Moreover D. desisquamatum is differ from D.

.polypodioides in characters combination as follow: larger pinnulae lobed to ¾

way to costa and veinlets 6 pair or less, while D. polypodiodes has larger pinnulae

lobed more deep close to costules and veinlets to 11 pairs.

ETYMOLOGY. The species epithet is from the Latin densus and

squamatus meaning scales are dense in illustrating the densely scales throughout

the stipes and rachis.

20. Diplazium dilatatum Blume

Diplazium dilatatum Blume, En. Pl. jav.: 194. 1828; C.Chr. Holttum,

Gard. Bull. 7: 273. 1934. Backer & Posth., Varenfl. Java: 132. 1939; Holttum,

Gard. Bull. S.S. 11: 85. 1940.

Rhizome stout, short, erect. Stipe to 80 cm or larger, 8 mm or more thick

near base, dark green when living, black and very densely scales at base; scales

narrowly linier, to 15 mm long, 1 mm wide, yellowish brown at middle, blackish

brown and sharply toothed at margin. Lamina bipinnate-tripinnatifid, about 90 cm

long, 70 cm wide, variable in size; pinnae oblong, narrowing toward acuminate

apex, to 58 cm long, 70 cm wide, pinnules to about 16 pairs below deltoid lobed

apex of pinna; basal pinnules usually a little reduce; larger pinnules stalked to 5

mm long, to 18 long, 4 cm broad, oblong subtriangular with attenuate apex, basal

lobes a little reduce, base truncate-cordate on stalked ones, lobed ½ way to costa

or a little more; lobes slightly subdeltoid-semiorbicular, apex rounded, margin

subentire or serrate, 15 by 9 mm, commonly less; rachis glabrescent, costa with

scattered narrow brown scales to 20 by 3 mm; veins in lobes pinnate with 5-7

pairs of simple or forked veinlets. Sori elongate along veinlets from near base to

2/3 length

214

SPORE. Monolete, bilaterally symatrical (made asymmetric by prine),

heteropolar, polar outline (excluding perine) elliptical, sides convex; equatorial

longitudinal view (excluding perine) concave-convex; equatorial transverse view,

proximal face concave, distal face convex; perinate. E: 31.85(45.69)49.56±4.78;

P: 19.84(28.55)34.62±3.89. Laesura: concealed by perine. Perine: alate-to

costate-alate, irregular envelope separated from exine surrounds the spore; wing-

like muri projected c.6 -15 µm µm, terminating margin are often ciliate; surface of

perine smooth. Exine: visible through perine, smooth under SEM.

ANATOMY. Stomata: Polocytic, copolocytic and seppolocytic.

CHROMOSOMES. 2n= 123 (Cytotype: T.Ng. Praptosuwiryo 1073)

DISTRIBUTION. India, Burma, S. China, Taiwan, Ryuku, S. Japan,

Indochina, Malesia throughout to N. Australia.

ECOLOGY. Terrestrial. On humus rich soil of mountain slopes primary

forest or mixed forest at 100-1800 m sea level.

VERNACULAR NAMES. Pakis layung (Javanese), paku beunyeur

(Sundanese).

USE. The young fronds can be eaten as vegetable after cooking.

SPECIMENS EXAMINED. --- JAVA: Adelbert 142; Alston 12771;

Dillewgn 706; Donk s.n., 603; Lefebu 113; Matthew 610; Mogea 2344, 2345;

Mousset 50, 760; Popta 225; Posthumus 3584, 3768, 3939; Sapiin 2660, 2713,

2717; Zippelius 239. --- BORNEO: M. Kato, G. Murata & Y.P. Mogea B-3873;

R.E. Holttum SFN 25555; M.Kato, G.Murata & Y.P. Mogea B-3738; M. Kato,

M. Okamoto, & E.B. Walujo B-10036.

NOTES. Kato (1995) recognized two varieties D.dilatatum in Japan, viz.

var. dilatatum and var. heterolepis. The first variety has scales on stipe base

lanceolate, to 20 mm long, black at margin, while the second variety has scales on

stipe broadly lanceolate, 10-15 mm long, 1-3 mm broad, hardly black at margin.

215

21. Diplazium dolichosorum Copel.

Diplazium dolichosorum Copel., Philip. J. Sci. 1 Suppl.: 151. 1906; M.G.

Price, Contr. Univ. Mich. Herb. 16: 195. 1987. --- TYPE: Philippines,

Mindanau, Zamboanga, San Ramon, 800 m, Copeland 1716 (Holotype, s.n.)

Rhizome stout; scales lineary, 1 cm long, black-dark brown. Stipe

crowded, ca. 35 cm long, 8 mm thick, base densely scales, muricate, upper;

subglabrous; scales dark, lineary, 4-8 mm long; lamina ovate, 1-1.5 mm long,

bipinnate, apex pinnatifid; larger pinnae 45 cm long, 20 cm broad, lower smaller;

pinnulae shortly stalked, subfalcate, to ca. 10 cm long, 2 cm broad, base truncate,

apex serrate, margin lobed to 1/3 way to costa; lobus truncate, toothed; rhachis

glabrescent, grooved on upper surface; texture papyraceous, glabrous, upper

surface shiny, lower surface pale; veins free, pinnate in the lobus, veinlet to 6

pairs, simple, lower curved. Sori lineary, to 8 mm long; indusia broad, brown.

DISTRIBUTION. Borneo, Philippines

ECOLOGY. Terrestrial. Hill dipterocarp forest. Elevation: 1000 m.

SPECIMEN EXAMINED: Borneo: Beaman 10641 (K).

NOTES. This species is described based on photograph of specimen

deposited at Rijks Herbarium, Kew.

22. Diplazium donianum (Mett.) Tardieu

Diplazium donianum (Mett.) Tardieu, Asplén. Tonkin: 58, t 5 1 & 2.

1932; Tagawa & K. Iwats., Fl. Thailand 3 (3): 455. 1988. Asplenium donianum

Mett., Farngatt. 6: 177. 1859.

Rhizome creeping, 4-5 mm thick, blackish, with leaves 1-2 cm apart,

scales on younger part; scales brown shining, ovate-narrowly lanceolate, 2.5-9

mm long, 1-3 mm broad, apex acuminate, brown, margin toohed irregularly with

thickening black strand. Stipe longer than lamina, 28-57 cm long, 3-4.5 mm

thick, dark brown and scales at base, upper pale green when living. Lamina

simply pinnate, oblong in outline, 30-42 cm long, 15-19 cm broad, lateral pinnae

subalternate, to 4 pairs, terminal pinnae conform to lateral ones; pinnae stalked 3-

216

6 mm long, ovate-lanceolate to narrowly oblong, to 16.2 cm long, 3-4.5 cm broad,

base cuneate, apex acuminate, margin entire, slightly toothed; rachise glabrous;

texture firm-papyraceous, upper surface rather light green, glossy, lower pale

green; slightly serrate near apex; veins group forming angle 55-60º to costa, free,

forked near costa, upper branch simple or one forked and soriferous, lower forked

1-4 times again, outer usually also soriferous but shorter. Sori bearing on outer

veins, sometimes also on inner ones, basal acroscopic usually the longest,

diplazioid, elongate from near costa almost reaching the margin; indusia

concolour, brown, rolled back, margin subentire, persistent, opening when mature.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an uninterrupted U-shaped, with an angle 110, flat base both inward and

outward, angles not forming ridges, ends slightly ridges outward.

CHROMOSOMES. 2n = 164 (Cytotype: XIX.C.III.65, BOHB, living

plant is cultivated at Bogor Botanic Gardens).

DISTRIBUTION. Japan, Taiwan, S. China, Indochina, Thailand, India,

Sumatra, Java, New Guinea.

ECOLOGY. On mountain slopes in light shade or in dense primary forest,

at low elevations lower than 1300 m.

SPECIMENS EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 2340. ---

SUMATRA: XIX.C.III. (Cultivated at Bogor Botanic Gardens).

NOTES. This species is new record for Java and Sumatra. Kato (1995)

also reported chromosomes number ’n’= 164 (apogamous).

23. Diplazium esculentum (Retz.) Sw.

Diplazium esculentum (Retz.) Sw., Scrad. Jour. Bot. 1801 92): 312. 1803;

Syn. Fil. : 92. 1806; Holttum, Gard. Bull. S.S. 11: 86. 1940.

Rhizome erect. Stipe 5-10 mm diam., 14-80 cm long, brown, glabrescent,

black and scales toward base; scales narrowly linier, 7-13 mm long, 1-1.5 mm

wide, concolour, dark brown, margin toothed with thickening black strand.

Lamina pinnate-bipinnate, large, various in size; lower one-two pairs of pinnae

usually reduced; larger pinnae 27-47 cm long, 12-25 cm wide, bearing numerous

217

of pinnules; pinnules oblong, narrowing towards acuminate or attenuate apex,

varying much in size, larger ones 8-13 cm long, 1.5-2.8 cm wide, lower shortly

stalked about 1 mm long, the rest sessile truncate-subcordate or broadly cuneate,

auricled one or both side at base, margin crenate or lobed about 2 mm or less from

margin to ¼ way to costa; lobes or crenation truncate or rounded, and serrate at

apex; texture papyraceous, sometimes fibrillose hairy beneath; rachis glabrescent

or occasionally bearing fibrillose hairs beneath; costa or costae grooved with

distinct ridge above, bearing scattered minutely scales; vein in pinnate group in

the lobes, 8-10 pairs of side veins, the lower 2-3 pairs of adjacent group

anastomousing, forming irregular intermediate eccurrent vein leading towards a

sinus between adjacent lobes. Sori occupying almost the whole length of the

veins, often also on part of the joint excurent vein, 1-4 pairs from basal sometimes

diplazioid; indusia not so thin, dark brown, persistent, margin toothed when

opening.

SPORES. Monolete, bilaterally symmetrical, heteropolar; polar outline

elliptical, sides convex; equatorial longitudinal view concave-convex to plano-

convex; equatorial transverse view, proximal face planar to concave, distal face

hemispherical; perinate. E: 31.72(38.39)43.09±3.35; P: 22.74(26.37)

29.12±1.39. Laesure: not visible, concealed by perine. Perine: smooth under

LM, micro rugulate under SEM. Exine: Exine: often visible through perine,

granulate under LM.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an interrupted U-shaped with angle about 100º, base flat, end ridges outward

to form an angle about 140º. Stomata: polocytic and seppolocytic.

CHROMOSOMES. 2n= 82 (Cytotype: T.Ng. Praptosuwiryo 1784, BO)

DISTRIBUTION. India to Fiji, throughout Malesia, north to Japan.

ECOLOGY. On moist soil in light shade, ravine, swampy ground,

grassland (not no dry), hedges, dith banks, roadside, riverside in the forest at

elevation 5-1600 m sea level.

USE. Young fronds of this species is sold in the local marked as

vegetables. It is eaten after cooking.

218

SPECIMENS EXAMINED. SPECIMENS EXAMINED --- JAVA:

Adelbert 435; Arsin 19455; Backer 4479, 7855, 12004, 12574, 17745, 18638;

Backer & Posthumus 4, 108, 162, 308, 400, 419, 601; Bakhuizen v/d Brink 1440,

2401, 3715, 5508; Beguin 76; Dorgello 1938; Hallier 678b; v. Heurn s.n.

(Pujon); Kooders 22754B; Lörzing 698, 699; Oosten a.17; Polak s.n. (Rawa

Lakbok); Posthumus 1455a; T.Ng. Praptosuwiryo 630, 635, 637, 638, 1784,

1785; Raciborski s.n. (Kota Batu); Rumka 3; Rutten 323; Zippelius 178. ---

MALAY PENINSULA: Newton, Z. Teruya 2343. --- BORNEO: K. Iwatsuki M.

Kato, Gen Murata & Y.P.Mogea B-194; M. Kato & Y.P.Mogea B-224;

K.Iwatsuki, M. Kato, M. Okamoto, K. Ueda & E.B. Walujo B-7190; M. Kato &

H. Wiriadinata B-6868; O. Posthumus 2065. ---SUMATRA: CH Lamourex

5652. --- BORNEO: T.Ng. Praptosuwiryo 2017, 2080, 2094b, 2094c, 2161b,

2161c, 2248a, 2248b.

24. Diplazium fraxinifolium Presl

Diplazium fraxinifolium Presl, Rel. Haenk 1: 49. 1825.

Athyrium fraxinifolium (Presl) Milde, Bot. Zeit. 28: 353. 1870.

Rhizome erect, 12 mm thick. Stipe 44.5-67 vm long, 4-7 mm thick, fallen

or sparsely scales at base; scales on stipe 2.5-8 mm long, 1-1.5 mm broad, margin

entire, brown, concolour. Lamina simply pinnate, 37.5-46 cm long 22-44 cm

broad, lateral pinnae 4-5 pairs, terminal pinna conform to the rest; pinnae 4-8 cm

distance, lower stalked 6-7 mm long, upper adnate, elliptical, 21.5-26 cm long,

4.5-6 cm broad, base cuneate, margin waved- crenate entirely or only toward

apex, or lobed to 1/6 way to costa, apex acuminate or caudate; veins group

forming angle 50-65º, each crenation for one vein group; vein forked 5-7 times,

outer veinlets in each veinlets group uniting with outer adjacent veinlets group at

1/5-1/4 way from margin or less. Sori almost on each veinlet, cover ½-5/6 of

veinlet length (2.5-17 mm long), diplazioid on basal acroscopic, others asplenoid

and opening toward acroscopic, except subbasal acroscopic ones opening towards

basiscopic; indusia pale brown, margin entire and darker, persistent, opening

when mature.

DISTRIBUTION. Malaya, Borneo, New Guinea, Philippines.

219

ECOLOGY. Terrestrial on slope in light-deep shade, lowland rain forest

at 20-750 m s.l.

SPECIMEN EXAMINED. --- Borneo: C. Boden Kloss 19028 (BO,

SING); M. Kato & H. Wiriadinata B-5568; B-4728; M.Kato, M. Okamoto & K.

Ueda B-11615, B-11659.

25. Diplazium fuliginosum (Hook.) M.G. Price

Diplazium fuliginosum (Hook.) M.G. Price, Brit. Fern Gaz. 10: 260. 1973;

Gard. Bull. S.S. 36 (1): 26. 1983. Asplenium fuliginosum Hook. Sp. Fil. 3: 120.

1859; C.Chr. Gard. Bull. S.S. 7: 280. 1934; Athyrium fuliginosum (Hook.) Copel.

Philip. J. Sci. 56: 476. 1935; Asplenium lugubre Hook. Second Cent. Ferns

(1861) t.3 (non Liebm. 1849). Type: North Norneo, Mt. Kinabalu, H. Low (K).

Athyrium longissimum Copel. Philip. J. Sci. 38: 139. 1929; Fern Fl.

Philip. 3: 411. 1961; Diplazium longissimum (Copel.) C.Chr. Ind. Fil. Suppl. 3:

74. 1934. Type: Philippines, Leyte, Dagami, Ramos BS 15269, Aug. 1912

(MICH).

Rhizome short, erect. Scales on stipes blackish, shining, margin entire, 6

mm cm long, 1.5 mm broad. Stipe 2.5-7 cm long, 3-3.5 mm diam, blackish when

dry, densely scales. Lamina very narrowly elliptic, 31-79 cm long, 9-13 cm broad

at about 1/3 of upper part, lower 2/3 part fully pinnate, upper 1/3 part pinnatifid,

free pinnae 15-32 pairs, lower pinnae 17 pairs gradually reduced downward;

pinnae subfacate, sessile, lanceolate, to 4.8 -6.7 cm long, 1.2-1.7 cm broad, base

truncate, apex sharply acute, margin subentire; texture very thin; rachis densely

scales beneath; veins free, 1-3 forked, soriferous on acroscopic branch. Sori

elongate along veinlets from basal almost reaching the margin; indusia dark

brown, margin entire, opening when mature.

DISTRIBUTION. Bismarck Arch. (New Ireland), New Guinea

(widespread), North Borneo (Mt. Kinabalu, common), Philippines (Leyte, one

collection).

ECOLOGY. Occurring in shaded moist ravines in montane forests, c.

1000-3000 m.

220

SPECIMENS EXAMINED --- BORNEO: R.E. Holttum SFN 25529;

J.M.S. Clemens 33723; J. & M.S. Clemens 31797.

26. Diplazium halimunense Praptosuwiryo, sp. Nov. Plate 5.

TYPE: Java: G. Halimun, Cikuda Paeh-Cikaniki, ca. 1300 m, 25 February

2006, T.Ng. Praptosuwiryo 2341 (holotype, BO).

Rhizoma breve erectum. Stipites 31-43 cm longae, in sicco stramineus,

basi brunneus squamatuss deciduis; squama linearis lanceolatis, ad 7 mm longae,

1 mm latae, margine irregularis dentatus cum filum niger spissescens. Lamina

pinnata, oblongus, pinnae laterales ad 3-6-jugatae, pinna terminalis ceteris similis,

rachis non prolifera; pinnae stipitatae ad 6 mm longae vel adnatus, ovate-

lanceolate, 15-21 cm longae, 3.5-5.5 cm latae, basi subequaliter cuneate, margine

fere integrae, apicem acuminatus leviter serratus. Venae libere, angulum 60-70°

cum costa formantes, furcatis Sori basalis vel medius, 1/8-4/5 venulae

occupantes. Indusia brunneus, marginem subintegris, persistens.

Rhizome short, erect, scales on younger part. Stipe 31.5-43 cm long,

stramineus when dry, dark brown and fallen scales at base; scales lineary

lanceolate, 5-7 mm long, 1 mm broad, margin with thickening black strand,

irregularly toothed. Lamina simply pinnate, oblong in outline, lateral pinnae 3-6

pairs, terminal pinnae conform to lateral ones; pinnae ovate-lanceolate, 15-21 cm

long, 3.5-5.5 cm broad, base cuneate, margin entire, slightly serrate near apex,

apex acuminate; rachis non gemmiferae, texture thinly papyraceous, light green

when living. Veins free, veins group forming angle 65-70º, forked near costa,

upper branch simple and soriferous, lower branch 2-3 times forked again, outer

basiscopic also soriferous but shorther and asplenoid. Sori linear from near costa

or submedial, cover 1/8 – 4/5 of their length, basal acroscopic the longer and

diplazioid. Indusia brown, margin subentire, opening when matur.

PARATYPE. JAVA, West Java, Bandung, Mt. Patuha, Situ Patengan, ca.

1300-1400 m, 26 Dec. 1997, T.Ng. Praptosuwiryo 749 (BO).

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an interrupted U-shaped with angle about 110º, flat base on both inward and

outward directions, end bluntly ridges to form an angle 140º.

221

CHROMOSOMES. 2n = 123 (Cytotype: T.Ng. Praptosuwiryo 2341, BO)

DISTRIBUTION. Java.

ECOLOGY. This species is growing on moist humus-rich soil of shady

places in mountain forest. In the Mt. Halimun, D. halimunense was found

growing among D. donianum and D. bantamense. Ca. 1300 m.

SPECIMEN EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 749, 2341.

NOTES. D. halimunense may be closely related to D. bantamense, but the

last two species appears to differ from the first species. Diplazium halimunese

has irregularly sharp toothed scales with thickening black strands, while the

margin scales of D. bantamense are minutely and regularly toothed; Pinnae of D.

halimunense are cuneate at base with margin entire, while pinnae of D.

bantamense are round at base with subentire or serrate at posterior portion. It also

resembles D. donianum, but D. halimunense has erect rhizome, while D.

donianum with creeping rhizome. The two species have similarities in irregular

sharp toothed scales and ovate-lanceolate pinnae.

Cytological observation of one individual of D. halimunense (TNgP

2341b) showed 2n = 123 (triploid). Whereas most indivual of D. bantamense

from Java are tetraploid and only some individual oktoploid. One collection

number of D. donianum planted in Bogor Botanic Gardens (from Sumatra) is

tetraploid (See Chapter 5).

ETYMOLOGY. The specific epithet is formed from name of the locality

where this species is found, Mt. Halimun, Halimun National Park, West Java.

27. Diplazium hewittii (Copel.) C.Chr

Diplazium hewittii (Copel.) C.Chr., Index Fil. Suppl. 1: 26. 1913.; C.Chr.

& Holttum, Gard. Bull. S.S. 7: 273. 1934.

Athyrium paripinnatum Copel., Philip. J. 11 (2): 147. 1915.

Diplazium paripinnatum Alderw., Handb. Suppl. 1 : 266. 1916.

Rhizome short, erect. Scales on stipes concolour, dark brown-blackish,

shiny, ovate, to 2.5 mm long, 2 mm broad, margin entire, glandular cell on tip

when young. Stipe 25-67 cm long, 2.5-3.5 mm diam. near base, light brown,

slightly muricate, black at base. Lamina pinnate – bipinnate, subtrangular in

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ouline, 30 – 70 cm long, 20-27 cm broad. Pinnae opposite-subopposite; lower

pinnae stalked to 1.5 cm long, lanceolate, 22 cm long, 5.7 cm broad, pinnate, apex

acumimate; lower pinnulae adnate – subsessile, upper pinnule sessile below

deeply lobed apex of pinnae; larger pinnulae nearly hastate, 1.8-3.3 cm long, 0.4 –

1.0 cm broad, base broadly cuneate, upper base distinctly auricle, lower base less,

apex acute-acuminate, margin crenate; middle pinnae with pinnulae sessile; upper

pinnae gradually less pinnate-pinnatifid; texture subcoriaceous; surface naked,

lower pale, upper dark green when living; rhacise naked; veins free, forked in

each crenatiron, oblique. Sori elongated along veinlet from near costule covers

5/6 way to margin, acroscopic branch diplazioid; indusia brown, fragile,

persistent, margin entire.

CHROMOSOMES. 2n = 123 (Cytotype: T.Ng. Praptosuwiryo 1913b,

BO).

DISTRIBUTION. Borneo

ECOLOGY. Growing on humus rich soil, low sandy ridges, on shaded

places in the forest at 400-450 m.

SPECIMENS EXAMINED. --- BORNEO: M. Kato, M. Okamoto, K.

Ueda & E.B. Walujo B-7383; M. Kato, M. Okamoto, K. Ueda & E.B. Walojo B-

7902; B-7903. K. Ueda & D. Darnaedi B-8835; T. Ng. Praptosuwiryo 1913a;

1913b; 1913c; 1908a; 1908b; 1908c; 1942; 2171; 2172a; 2172b, 2174; 2177;

2178; 2179; 2180.

NOTES. Christensen & Holttum (1935) stated that a series of specimens

collected by Mjoberd in Sarawak has shown that Athyrium Hewittii, A.

sarawakense, and A. paripinnatum of Copeland probably all forms of the same

species, different in size but otherwise scarcely distinguishable.

My collections from Muller Range, Borneo, showed that this species has

variation in morphology from a young to adult. Young plants in which spores

have not been produced are showing pinnate fronds while adult plants having

fronds with pinnate-bipinnate.

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28. Diplazium hottae Tagawa

Diplazium hottae Tagawa, Acta Phytotax. Geobot. 25 (2-3): 65. 1972. ---

TYPE: Hotta 15185 (Holotype, KYO, n.v.), Gunung Mulu, Sarawak.; Kokawa &

Hotta 2364, 2415 (Paratype, KYO, n.v.).

Rhizome shorth, stout, erect. Scales on stipes light brown, shining,

concolours, 9-15 mm long, 1-2 mm broad.margin entire, Stipe 56-67.5 cm long, 3-

6 mm diam. at base stramineous, deeply grooved upper surface, base dark brown,

scales, upward glabrous. Lamina oblong, lanceolate, ca. 85 cm long, 36 cm broad,

pinnae 11-21 pairs; lower pinnae stalked to 5 mm long, lanceolate, larger pinnae

28 cm long, 2.5 cm broad, base narrowly cuneate, apex acuminate and slightly

crenate, margin entire; rachis glabrous, gemmiferous at the adjacent between

rachise and costa near terminal pinna; veins group forming an angle about 45º to

costa; veins forked near costa, upper branch simple, lower branch forked again

once-twice; Sori elongate along acroscopic branch, mostly not so close to costa,

almost reaching the margin; indusia light brown, shining, margin entire, rolling

back, not so broad.

DISTRIBUTION. Sumatra, Borneo,

ECOLOGY. Terestrial on rather dry mountain in light shade. 300 –

1350 m.

VERNACULAR NAMES. Paku rahang (Dayak).

USE. Dayak use this plant for medicine. The very young fronds are

crushed for sore poultice.

SPECIMENS EXAMINED. --- BORNEO: M. Kato, M. Okamoto, K.

Ueda 7 E.B. Walujo B-7428; M. Kato, M. Okamoto, K. Ueda & E.B. Walujo B-

7675; K. Ueda & D. Darnaedi B-11571; M. Kato, M. Okamoto, K. Ueda & E.B.

Walujo B-7879; T.Ng.Praptosuwiryo 1911. --- SUMATRA: C.J. Brooks 330 S.,

T.Ng. Praptosuwiryo 2536a.

NOTES. Tagawa (1972) stated that D. hottae is allied to D. subintegrum

Holtt. Occurring on Malay Peninsula and in Northern Sumatra. This species

differs with D. subintegrum constantly in: (1) terminal not lobed at base and

similar to apper laterals ones in shape and size, (2) uppermost 1 or 2 lateral pinnae

gemmiferous at the base on the rachis, (3) pinnae apparently entire, but in reality

224

provided with minute and remote incision on margin, (4) venation obscure,

acroscopic veinlet of group simple and soriferous, basiscopic one once forked and

sterile, (5) sori all asplenioid, narrower, very unequal in length, their anterior ends

arranged in an intramarginal uneven line, (6) scales at the base of stipe much

longer.

29. Diplazium insigne HolttumDiplazium insigne Holttum, Gard. Bull., S.S. 9: 123. 1937; Holttum,

Gard. Bull. S.S. 11: 87. 1940. --- TYPE: R.E. Holttum SFN 21635 (holotype,

SING!), Trans Valley, Frasser’s Hill, Pahang, Malay Peninsula.

Rhizome stout, short, erect. Stipe stout, c.1 m long, spiny toward the base,

the spines 2 mm long, each at first bearing a scale; scales dull brown 1.5 cm long

by 1.5 cm wide at base, with a narrow black toothed edge, deciduous. Lamina to

1.5 m long, bipinnate; lowest pinnae about 28 cm long and 6.5 cm wide, narrowed

and stalked at the base, the margins lobed half-way to the costa, the apex

acuminate; middle pinnae largest, to 60 cm long and 16 cm wide, pinnate;

pinnules slightly oblique, adnate to the rachis (the lowest ones narrowly, the upper

fully adnate and grading into the lobed apical lamina of the pinna), to 9 cm long

and 2.3 cm wide, cunete at the base at an angle of about 45o on each side,

narrowed gradually from the base and then suddenly at 1.5 – 2 cm, from the apex,

margins slightly serrate; texture firmly herbaceous; veins anastomousing as in D.

accedens. Sori few or copious, the lowest acroscopic sorus in each vein-group

usually diplazioid, and occasionally a few others also.

ECOLOGY. In moist shady valley forest. 600-1200 m

DISTRIBUTION. Malay Peninsula.

SPECIMENS EXAMINED. --- MALAY PENINSULA: R.E. Holttum

SFN 21635.

NOTES. As stated by Holttum (1940) this species is evidently closely

allied to D. accedens and the apex of afrond might pass for that species, but its

copious bipinnate form marks it as a quite distinct species. Holttum (1940) added

that this species is also similar to D. Smithianum from Ceylon, but appears to be

225

much larger; probably both D. Smithianum and D. insigne are local derivatives of

D. accedens (or D. proliferum).

30. Diplazium kunstlerii Holttum

Diplazium Kunstlerii Holttum, Gard. Bull. S.S. 11: 88. f. 2. 1940. –

Athyrium kunstlerii (Holtt.) Holttum, Rev. Fl. Malaya 2: 564. f. 335. 1966. ---

TYPE: R.E. Holttum SFN 31194 (holotype, US!).

Rhizome suberect, about 2 cm diam., scales on younger part. Stipe 7 mm

diam at base, 70 cm long, glabrescent, dark brown, nearly black and scales at

base; scales rounded. Lamina bipinnate, about 107 cm long, 60 cm wide; pinnae

about 11 pairs; larger pinnae 52 cm long, about 16 cm wide, on stalk to 11 cm

long, upper pinnae with shorter stalk; lowest pinnules to about 5 cm apart, largest

ones 9.2 cm long, 2.7 cm broad, on stalk to 2 mm long, base broadly cuneate to

truncate, margin lobed to 2/3 way toward costa; lobes slightly oblique, 5-7 mm

wide, base dilated, margin slightly toothed, apex rounded; texture thicker than in

allied species; rachis gemmiferous at upper portion; veins in each lobe 6-9 pairs,

oblique, mostly simple, distinct but hardly prominent on both surface. Sori at

middle part of the veins or nearly close the costules, basal acroscopic veinlets

usually diplazioid; indusia narrow, hardly evident in mature sori, very dark brown.

DISTRIBUTION. Malaya, Java.

ECOLOGY. Locally abundant near streams in shady forest in the

foothills. Ca. 900-1000 m.

SPECIMENS EXAMINED --- MALAY PENINSULA: R.E. Holttum

SFN 31194. --- JAVA: Winckel 1556B.

31. Diplazium laevipes C.Chr. in C.Chr. & Holttum

Diplazium laevipes C.Chr. in C.Chr. & Holttum, Gard. Bull. S.S. 7: 271.

Pl. 58. 1934. TYPE: Borneo, Mt. Kinabalu, H. 25259 (Holotype, SING!).

Stipe 55-75 cm long, ca. 3-8 cm diam.near base, glabrescent, dark brown

and scales towards base. Scales on stipes rounded, to 3.5 mm diam., dark brown,

margin entire, more thick toward central. Lamina subdeltoid, bipinnatifid-

226

bipinnate, pinnae numerous; pinnae shortly stalked 5-15 mm long, oblong

subtriangular-ovate outline, basal pinnae smallest, oblong lanceolate, base

subcordate-subttruncate, subbasal pinnae 32-37 cm long, 8-19 cm wide, pinnulae

to 10 pairs below pinnatifid or deltoid lobed apex of pinnae, acroscopic basal

basal pinnulae a little reduced; pinnulae almost at right angle to costa, lower

shortly stalked less than 1 mm long, oblong subtriangular, 10 cm long, 3.5 cm

broad, base subtruncate, apex acuminate with slightly toothed, margin lobed 1/3-

2/3 way to costule; lobus oblong, slightly oblique, to 4 mm above base, acroscopic

basal a little reduce, ends truncate, entire-slightly toothed; texture stiff , blackish

on upper surface and dark brown beneath when dry; rachis and costa deeply

grooved and rather tomentose above, gemmiferous, gemmae bearing at adjacent

between upper part of rachis and costa; veins pinnate in each lobe, free, not so

distinct on both surface, forming an angle about 65-80º to costule; veinlets simple,

to 9 pairs, occasionally once forked on one-two pair basal lobus, forming an angle

about 25-35º to costulet. Sori elongate from basal veinlet cover ¼-2/3 their

length, diplazioid on basal acroscopic; indusia dark brown, fragile.

DISTRIBUTION. Borneo.

ECOLOGY. Occurring on forest floor in deep shade near river or ravine

at 900-1300 m.

SPECIMENS EXAMINED. --- BORNEO. Kalimantan Timur: M. Kato,

M. Okamoto, E.B. Walujo B-11261, B-9596, B-10748, B-11486, B-9625, B-

7399; M. Kato, M. Okamoto, K. Ueda, D. Darnaedi & E.B. Walujo B-8345; M.

Kato, M. Okamoto, K. Ueda & E.B. Walujo B-7399.

NOTES. As pointed out by Christensen & Holttum (1934), D. laevipes is

closely related to D. spiniferum. The two species are quite similar in size, colour

and division, but the step of D. spiniferum is prckly, the segments all entire, the

sori far from the costule, and the frond coriaceous.

227

32. Diplazium latisquamatum Holttum

Diplazium latisquamatum Holttum, Gard. Bull. S.S. 9 : 124. 1937. Fig. 3.

Gard. Bull. S.S. 11: 90. 1940. Fig. 3; --- TYPE: R.E. Holttum SFN 31311

(holotype, SING!), S. Terla, Ulu Telom, Pahang, Malay Peninsula. –Athyrium

latisquamatum (Holtt.) Holttum, Rev. Fl. Malaya 2: 563. f. 334. 1966.

Rhizome short, erect. Stipe 52-100 cm long, 5-9 mm thick, dark brown,

nearly black at base, scales throughout, more dense at base; scales rounded-ovate,

dark brown, center thicker, margin entire, 4-8 mm long, 2.5-4 mm broad,

thickening black strand irregularly near apex. Lamina bipinnate-tripinnatifid, 50-

125 cm long, about 40-80 cm wide; pinnae stalked 2.5-5.5 cm long, lanceolate,

larger pinnae 26-54 cm long, 15-18 cm wide, pinnulae 10-13 pairs below

pinnnatifid apex of pinnae, basal pinnules a little reduce, smaller on acroscopic

than on basiscopic side; pinnulae sessile on upper ones, shortly staked on lower

ones, the larger 6.5-11 cm long, 1.8-3 cm wide, base broadly cuneate to

subtruncate, gradually narrowed to acuminate apex, margin lobed to within 1.5-2

mm of the costa (3/4 way to costa), lobes slightly oblique, 2.5-6 mm wide,

commonly 5 mm wide, apex truncate, margin entire or toothed near apex; texture

thin, firm; rachis occasionally gemmiferous at the adjacent to the costae at the

apex of lamina, glabrous; costae bearing scattered small rounded-ovate scales

beneath; veins pinnate in lobes, veinlets 5-7 pairs, simple or forked. Sori covering

¼-1/2 way of veinlet, acroscopic basal veinlets usually diplazioid. Indusia thin,

brown, broad, torn from margin to the base when opening, persistent.

DISTRIBUTION. Malay Peninsula, Java, Borneo.

ECOLOGY. In moist shady valley of montane forest. These plants

often grow in very wet ground. Elevation: 1200-2700 m.

SPECIMENS EXAMINED --- JAVA: Matthew 609; Meijer 1453, 1818;

Popta 203; Raciborski 108. --- MALAY PENINSULA: R.E. Holttum SFN 31311

(holotype, SING!). --- BORNEO: J. & M.S. Clemens 27122, 27951, 28391,

29716, 32516, 32952.

228

33. Diplazium lobbianum Moore

Diplazium lobbianum Moore, Ind. Fil.: 331. 1861; Alderw., Malay. Ferns

Hand.: 408. Asplenium lobbianum Hook., 2nd Cent. of Ferns, t. 17. 1861.

Rhizome short, erect. Stipes up to 70 cm or larger. 7 mm diam. when

dry, glabrescent, dark brown, black and scales toward base, distinctly grooved on

upper surface; scales subulate, to 15 mm long, 5 mm wide at base, brown, toothed

with thickening black strand at margin. Lamina simple pinnate, oblong-ovate in

outline, up to 53 cm long, 27 cm wide near subbasal, pinnae to 18 pairs below

deltoid acuminate with deeply lobed of terminal frond; pinnae subhorizontal,

ascending, oblong lanceolate to linier-oblong, lower ones on stalk to 3 mm long,

basal pinnae up to about 10-14 cm long, 2-2.8 cm wide or larger, subbasal pinnae

14-15.5 cm long, 2.3 cm wide, upper base subtruncate, lower base slightly

rounded, upper pinnae sessile with obliquely rounded-cuneate at base; margin

entire on lower part, toothed near apex, apex acuminate, subfalcate; texture thinly

coriaceous, vein-group at an angle 50º to costa, forked near costa, upper branch

simple, soriferous, lower branch forked 1-2 times again, scarcely 3 times,

basiscopic and also one pair subbasal soriferous. Sori on acroscopic veinlets

elongate nearly from the costa almost reaching the margin or more far (covers ½-

3/4 way to wargin), usually diplazioid, basiscopic veinlets soriferous also, shorter,

mostly simple; ndusia frim, fragile, brown, margin entire, persistent.

SPORES. Monolete, bilaterally symmetrical (made asymmetrical by

perine), heteropolar; polar outline elliptical, sides convex; equatorial longitudinal

view concave-convex to plano-convex; equatorial transverse view, proximal face

planar to concave, distal face hemispherical; perinate. E: 38.20(46.10) 51.87

±3.22; P: 20.47(26.28)32.81±3.73. Laesure: concealed by perine ridge or wing-

like muri. Perine: costate-alate; wing-like muri surrounds the spore without

forming reticulation or with forming loose reticulatons, terminating margins

ciliate; surface of perine smooth to scatterly ciliate or pappilate. Exine: often

visible through perine under LM, smooth under SEM.

ANATOMY. Tranverse section of stipe near lamina: vascular bundle

form an anterrupted U-shaped with angle about 115º, base flat on inward and

229

outward directions, angle and end not forming ridges. Stomata: polocytic and

seppolocytic.

CHROMOSOMES. 2n=164 (Cytotype: T.Ng. Praptosuwiryo 720, BO).

DISTRIBUTION. Java, Philippines, New Guinea.

ECOLOGY. On moist ground or humus-rich soil in mountain slopes

secondary and primary forest in altitude beween 1500-1800 m sea level.

SPECIMENS EXAMINED. --- JAVA: Backer 14716, 15910; Donk 612,

632, 771; Popta 204; Posthumus 204; T.Ng. Praptosuwiryo 720, 722, 723, 1190,

1240, 1239, 1245, 1246, 1251, 1346, 1487 (G. Slamet), 1488 (G. Slamet);

Raciborski 52.

34. Diplazium lomariaceum (Christ) Price

Diplazium lomariaceum (Christ) Price, Gard. Bull. S.S. 36 (1): 27. 1983.

Asplenium lomariaceum, Verh. Naturf. Ges. Basel 11: 229. 1895. Type: central

Celebes, Takalekadjo, F.& P. Sarasin 994, 8 Feb. 1895 (P, lectotype, n.v.)

Diplazium merrillii Copel. Philip. J. Sci. 2C: 128., t.2A. 1907; Hieron.

Bot. Jahrb. 56: 134. 1920; Athyrium merrillii (Copel.) Copel. Philip. J. Sci. 3C:

300. 1908; 56: 476. 1935; Fern Fl. Philip. 3: 411. 1961. Type: Philippines,

Mindoro, Mt. Halcon., Merrill 5914, Nov. 1906 (Lectotype MICH; US, n.v.)

Diplazium porphyrolepium v.A.v.R. , Bull. Jard. Bot. Buitenz. II, 20: 11.

1915. Type: Celebes, Soemalilah, Capt. Van Vuuren’s Explor. Comm., Rachmat

418 (BO?; L).

Diplazium porphyrophyllum v.A.v.R., Bull. Jard. Buitenz. II, 28: 18.

1918. Lectotype: Ceram, Wai Lantabi, L. Rutten’s Explor. Comm., Kornassi

1240, 4 May 1918 (L!).

Athyrium altum Copel. Philip. J. Sci. 38: 138. 1929; Fern Fl. Philip. 3:

411. 1961; Diplazium altum (Copel.) C.Chr. Ind. Fil. Suppl. 3: 72. 1934. Type:

Philippines, Mindanau, agusan, Mt. Urdenta, Elmer 14081, Oct. 1912 (MICH; L,

NY, US, n.v.) .

230

Scales blackish, shiny, entire, to 9 by 1 mm, gradually narrowed to a hair-

tip, abundant and persistent on stipe, rachis, costae, and vein beneath, Stipe of

fertile fronds to 15 cm long, of sterile tp 9 cm. Lamina narrowly elliptic, to 50 by

9.5 cm, deeply pinnatifid, one pair of reduced basal pinnae sometimes free and

sessile; lobes to 4.5 by 1 cm, oblong-lanceolate, narrowing towards apex,

subentire, blunt. Colour dark greenish-brown, more or less shiny below when dry,

dark bluish-green when living; rachises above with a channel formed by raised

cartilaginous sides, either continuous or interrupted at each junction with a middle

of a lobe, and paleate at that point whether or notinterrupted; indusia brown,

margin erose. Spore brown, with irregular short wing.

SPORES. Monolete, bilaterraly symmetrical (made asummetric by

perine), heteropolar; polar outline (excludong perine) elliptical; equatorial

longitudinal view (excluding perine) biconvex; equatorial tranverse view,

proximal face convex, distal view hemispherical; perinate. E:

35.03(39.41)54.55±4.20; P: 19.55(24.57)30.13±3.46. Laesura: concealed by

wing-like costae. Perine: alate to costate-alate, alae forming loose reticulation;

irregular envelope separated from exine surrounds the spore in wing-like muri

reticulation, lacunae large, 13-21µm; wing-like muri thin, project 3-10 µm,

terminating margin waved or almost entire. Exine: visible through perine, rough

rugulate under SEM.

DISTRIBUTION. Philippines, Borneo, Sumatra, Celebes, Ceram, New

Guinea.

ECOLOGY. This species usually grows in moist montane forest, 400-

2000 m.

SPECIMENS EXAMINED --- BORNEO: Garry Shea SHEA 23160;

Maskuri 845; Veldkamp 7874. -- SUMATRA: CJ Brooks 322S.

NOTE: Price (1983) state that the holotype was destroyed in 1945 at the

PNH so he designated the MICH specimen as lectotype. D. porphyrolepium and

D. porphyrophyllum are not excep not exceptional in any way. The latter had a

syntype purportedly from Sumatra, Brooks 322S. Price (1983) designated the

specimen from Ceram at L as lectotype. Athyrium altum is a form with narrow

fronds not otherwise distinguishable, Three specimens from eastern Kalimantan

231

(Kortermans 9089, Meijer 577, 872 – all L) differ by having fewer and brownish

paleae but agree in distribution of paleae, and in frond form and colour.

Diplazium lomariaceum is very closely related to D. pophyrorachis and until now

the name sees to have been ignored since Christ himself reduced lomariaceum to

porphyrorachis in Ann. Jard. Bot. Buitenz. 15 (1989, p. 119).

35. Diplazium loerzingii Praptosuwiryo, sp. nov.

TYPE: SUMATRA. North Sumatra, E. Mount Sibajak, Upper Petani

Valley, primary forest, 1250-1350 m, 5 Feb. 1929, J.A. Lörzing 15124 (holotype,

BO).

Rhizoma breve erectum. Stipites ad 45 cm longi, in sicco brunnei, basin

versus aquamis brunneis nitidis lanceolatis, ad 13 mm longis 1.5 mm latis integris

acuminatis vestiti. Lamina pinnata lanceolatis, ad 75 cm longa et 32 cm supra

basin lata, pinnae ad 24-jugatae; rachis supra sulcata, infra acem gemmifera.

Pinnae subbasales maximae (1-3 jugatae deflexae), petiolulatae ad 5 mm longae,

lanceolatae, ad 17.5 cm longae, 2.8 cm latae, basi truncatae, apice attenuatae,

margine ½-2/3 costam lobatae; lobi maximi ad basim, 5-9 mm lati, apice truncati,

margine apices leviter dentati. Rachis supra sulcata, glabrae, infra apicem

gemmifera. Textura subcoriacea vel plus solid; in vivo colorae supra atroviridis,

subtus pallidus. Venae perspicuus in uno lobo 3-5 jugatae, plerumque 5 jugatae,

omnes simplices. Sori medius in 1/3-3/4 longitudine venularum. Indusia

brunneus, in affixus atro brunneus, marginem integrum, non revolutus, persistens.

Rhizome short, erect, scales densely on younger part; scales lanceolate, 7-

13 mm long, 0.5-1.5 cm broad, dark brown-blackish, with thickening black

strands, shining, margin entire. Stipe 33-45 cm long, 4-5 mm thick, pale brown

when dry, black at base, fallen scales. Lamina lanceolate, pinnate, 61-75 cm long,

24-32 cm broad, pinnae 22-24 pairs below pinnatifid triangular apex of lamina.

Lower pinnae stalked to 3-5 mm long, 2.3-3.6 cm apart, oblong-lanceolate, larger

ones 10.3-21.3 cm long, 1.8-3.2 cm broad, base of 1-3 pairs basal pinnae cut away

lower, base truncate, margin lobed to ½-2/3 way to costa, apex attenuate, toothed;

upper ones adnate-sessile; lobus widest at base, larger ones 5-9 mm broad, ends

truncate, slightly toothed. Rachise gemmiferous, bearing buds at the adjacent

232

between rachis and costa, glabrous, costa glabrous. Texture subcoriaceous or

firmer, dark green upper surface, pale green lower when living. Veins free,

pinnate in the lobus, mid-veins of lower lobus forming angle 70-75 ° to costa,

veinlets 3-5 pairs, commonly 5 pairs, distinct, simple, all reaching margin,

forming angle 25-30º to midveins. Sori medial, (leaving sterile part of veinlets

1.5-2.5 mm) or close to margin of lobus, elongate cover 1/3-3/4 of veinlet length,

basal acroscopic diplazioid. Indusia broad, brown, attachment side darker, margin

entire, opening when mature, not rolled back, persistent.

PARATYPE. JAVA. West Java, Mt. Halimun, track Cikuda Paeh-

Cikaniki, ca. 1300 m, 25 February 2006, T.Ng. Praptosuwiryo 2339 (BO).

SUMATRA. Sibolangit Nature Reserve, 400-500 m., 10 December-1928, J.

Buumee A. 841; Jambi, Kerinci Seblat National Park, Sungai Penuh, Bukit

Tapan, secondary forest Pal 823-829, 1360 m, 9 Sept. 2006, T.Ng. Praptosuwiryo

2519, 2520, 2521 (BO).

CHROMOSOMES. 2n = 82 (Cytotype: T.Ng. Praptosuwiryo 2339c , 123

(T.Ng. Praptosuwiryo 2339d).

DISTRIBUTION. Java and Sumatra.

ECOLOGY. Secondary and primay forest, light-deep shady places, on

moist and humus rich soil. 1250-1400 m.

NOTES. Diplazium loerzingii is closely related to Diplazium malaccense.

This species differs from D. malaccense in the following characters: lower base

of 1-2 pairs basal pinnae less cut, base of lower pinnae almost equally truncate,

texture thicker or subcoriaceous, upper rachis much gemmiferous, lobes truncate,

sori medial on veinlets or close to margin, attachments sides of indusia darker.

ETYMOLOGY. This species is named after J.A. Lörzing who collected

this plant for the first time. He collected D. loerzingii in 1920 at Mt.Sibajak

Sumatra. Nine year after that he collected this plant again in the same place. C.J.

Brook also found this species at Mt. Dempo (Sumatra) in 1923. I found this plant

at Mt. Halimun (Java) and Bukit Tapan (Kerinci Seblat National Park, Sumatra)

respectively in Februari and September 2006.

233

36. Diplazium malaccense Presl.,

Diplazium malaccense Presl, Epim.: 86. 1849; Tard. & C.Chr. in Fl. Gén.

I.-C. 7(2): 258. 1940; Tagawa & K. Iwats., Southeast As. St. 5 : 104. 1967; Acta

Phytotax. Geobot. 23 : 56. 1968. -- Athyrium malaccense (Presl.) Hottum, rev. Fl.

Malaya 2 : 525. 1955.

Rhizome short, ca. 1.5-1.8 cm diam, erect, densely scales on young part.

Stipe 39- 48.5 cm long, 0.3-0.5 cm diam near base, densely scales at base; scales

10-12 mm long, 1 mm broad, lineary triangular, brown. Lamina pinnate, oblong

elliptical –subtriangular in outline, 33-64.5 cm long, 15-30 cm broad, pinnae 16-

19 pairs below lobed apex of lamina. Lower pinnae stalked 2-5 mm long, 1-2

pairs basal pinnae bending downward, lineary- subtriangular, 9-19.5 cm long, 1.1-

2.8 cm broad, base very unequal, lower base cut away, upper truncate, upper

pinnae with base subequally truncate or cuneate, Pinnae thin in texture, drying

rather light green, margin lobed 1/3-2/3 way to the costa, apex acuminate; lobes

about 4-7 mm wide at base, oblique, apex rounded to subtruncate, slightly

toothed. Veins 4-7 pairs in each lobe, all simple. Sori from near base of veins to

near margin of lamina, acroscopic basal sorus usually diplazioid. Indusia medium

brown, thin but firm, narrow, margin entire.

SPORES. Monolete, bilaterally summetrical (made asymmetric by

perine), heteropolar; polar outline (excluding perine) elliptical; equatorial

longitudinal view planar convex to concave-convex; equatorial transverse view,

proximal face concave, distal view convex. Laesura: concealed by wing-like

muri. E: 37.73(34.24)27.53±3.03; P: 26.01(20.80)13.33±2.82. Perine: alate to

costate-alate, often loosely reticulate; irregular envelope separated from exine

surrounds the spore in continuous anastomosing wings, forming a loose

reticulation; wing-like muri project 1.5-8 µm, terminating margins sparsely ciliate.

Exine: visible though perine, smooth granulate under SEM.

ECOLOGY. In evergreen forest, terrestrial on rather moderate slope, in

light shade, on humus-rich soil ground. 20 – 1000 m.

SPECIMENS EXAMINED. --- MALAY PENINSULA. Pahang: M.R.

Henderson 11235; Md Nur 10508; R.E. Holttum 20788; A.B. Murdok s.n. (12

June 1913); H.N. Ridley s.n. (1891). Negeri Sembilan: R.E. Holttum 9565; Md

234

Nur s.n. (29 Nov. 1923). Melaka: A. Derry 14. Penang: R.E. Holttum SFN

31196. --- BORNEO. Kalimantan Timur: K. Ueda & D. Darnaedi B-8982; M.

Kato, M. Okamoto & E.B. Walujo B-9070; M. Kato, M. Okamoto, K. Ueda &

E.B. Walujo B-7398; M. Kato, M . Okamoto & E.B. Walujo B-10909; M. Kato,

M. Okamoto, K. Ueda, E.B. Walujo B-7871; K. Iwatsuki, M. Kato, G. Murata &

Y.P. Mogea B-2472. Kalimantan Selatan: M. Kato, G. Murata & Y.P. Mogea B-

4155; 4175. --- SUMATRA: W.J.J.O. de Wilde & B.E.E. de Wilde – Duyfjes

12560; W.J.J.O. de Wilde & B.E.E. de Wilde – Duyfjes 12920; T.Ng.

Praptosuwiryo 2218, 2220, 2209, 2217, T.Ng. Praptosuwiryo 2223, 2224, 2225,

2226, 2229, 2230, 2233, 2235, 2236, 2237, 2238.

37. Diplazium megasegmentum Praptosuwiryo, sp. nov. Plate 7.

TYPE. JAVA. West Java, Mt. Salak, Southern Slope, Cangkuang Forest,

19 December 2002, T.Ng. Praptosuwiryo 1382 (foto) (holotype, BO).

Rhizoma breve erectum, crassum. Stipites ad 122 cm longis, prope basin

10 mm crassi, in sicco brunneis, basin versus squamis deciduis nigris rotundis vel

ovatis peltatis integris imbricates vestitae. Lamina ad 100 cm longae, 80 cm latae,

subdeltoideus, tripinnatifidus, pinnae ad 14-jugatae; pinnae inferus bipinnatidus,

stipites ad 3.5 cm longae, lanceolatus, 50 cm longae 26 cm latae, libere pinnulae

ad 16-jugatae, apice pinnatifidus; pinnulae inferus subalternatus, 2.5-3.5 cm

seorsum, stitipatae ad 3 mm longae, oblongus-lanceolatus, ad 13 cm longae 3.5

cm latae, basi truncatae, apice acumitae, margine lobatae segmenta formantes,

segmenta inferiora plus minusve opposite, 2.5-5.0 mm seorsum, sessilis;

segmenta superus subalternate; segmenta maximae 2.4 cm longae et 8 mm latae,

apice rotundatus-acutus, margine 1/3-1/2 versus venae principalis; pinnae medius

pinnatus, pinnae superus pinnatifidus. Venae liberis, in segmento pinnatus,

venulae ad 6-8-jugatae, in lobo furcatis vel bifurcate. Sori e basi venulae ramis

acroscopicus 1/3-1/2 extensi, plerumque in basi venulae acroscopicus

diplazioideus. Indusiis brunneus, margine laciniatus, ante sporangiis maturis

aperiens, persistens.

235

Rhizome erect, stout. Stipes dark brown, 122 cm long, 10 mm diam. near

base, fallen scales; scales rounded, ca. 6 mm long and broad. Lamina bipinnate-

tripinnatifid, subdeltoid in outline, 100 cm long, 80 cm broad, pinnae to 14 pairs,

lower pinnae bipinnatifid, middle pinnae pinnate, upper pinnae pinnatifid. Larger

pinnae stalked to 3.5 cm long, lanceolate, 50 cm long, 26 cm broad, free pinnulae

16 pairs below pinnatifid apex of pinnae. Lower pinnulae subalternate, 2.5-3.5

cm apart, shortly stalked to 3 mm long, oblong-lanceolate, 13 cm long, 3.5 cm

broad, base truncate, apex acuminate, margin lobed deeply to whitin 1 mm of

costa forming segments; lower segments opposite, 2.5-5.0 mm distance, sessile;

upper segments subalternate below deeply lobed acuminate apex of pinnulae;

larger segments oblong, 2.4 cm long, 8 mm broad, apex rounded-acute, margin

lobed 1/3-1/2 way to main vein. Veins free, pinnate in the segments, to 6-8 pairs

Veinlets once-twice forked in lobes. Sori bearing on basal acroscopic branch of

veinlets, elongate from basal covers 1/3 -1/2 of their length, diplazioid on basal

acroscopic veinlets. Indusia brown, persistent, opening when sori young, margin

laciniate.

PARATYPE. Java: West Java, Mt. Salak, Southern Slope, Cangkuang

Forest, 28 December 2002, T.Ng. Praptosuwiryo 1443, 1450, 1451, 1452.

SPORES. Monolete, bilaterally symmetrical (mades asymmetric by

perine), heteropolar; polar outline (excluding perine) transversely elliptical, sides

convex; equatorial longitudinal view (excluding perine) concave-convex;

equatorial transverse view, proximal face convex, distal face convex; perinate. E:

39.61(51.42)57.01±4.28; P: 18.51(31.49)35.85±3.59. Laesura: concealed by

perine. Perine: alate; alae sometimes loosely reticulate; wing-like muri project 4-

13 m, terminating margins almost entire.

DISTRIBUTION. Java.

ECOLOGY. Diplazium megasegmentum is growing well on moist humus-

rich soil in shady places.

NOTES. Diplazium megasegmentum is very distinct among Javanese

species Diplazium in the combination of the following characters: pinnules cut to

within 1 mm of costa to form a big oblong segments to 8 mm broad that lobed

again to ½ way to main vein; basalis sori which not more than half away to the

236

main vein. It is grows on moist humus-rich soil in shady forest in southern slope

of Mt. Salak West Java.

ECOLOGY. The species epithet is from the Latin mega and segmentum

meaning the large segment in illustrating the large size of its large lobed

segments.

38. Diplazium megasimplicifolium Praptosuwiryo, sp. nov. Plate 8.

TYPE: Borneo: Bukit Raya, Tumbang Tubus, Veldkamp 7998, 150 m, 5

May 1983 (holotype, BO).

Rhizoma breve erectum, gracilis. Stipites ad 15 cm longae, in sicco

brunneus dilutus, glabratus, basi deciduis squamatus; squamis (?). Lamina

simpliter, ellipticus, ad 40 cm longae, 7.5 – 10.6 cm latae, angustatus versus

apicem acuminatus, gradatin decrescens versus basin qui subito contractus e

latitude 1 cm vel minus, basi cuneatus, margine integrae; subcartaceus; rhachides

prominens; venae in parvus caterva in angulum 65-70º cum costa formantes, ad

5-7 mm seorsum, copiosus anastomantes (e margine 2/3-4/5 versus costae). Sori

in venae lateralis elongate, venae interiora et venae exteriora areolae c.1.5 mm

latis formantes, ramis basalis acrocopicus et venulae anastomosis diplazioideus.

Indusiis dilutis brunneis, integris, persistens.

Rhizome short, erect, slender. Stipe 14-15 cm long, pale brown, glabrous,

fallen scales at base. Scales on stipes (?). Lamina simple, elliptical, to about 40

cm long, 7.5 – 10.6 cm wide, narrowed toward acuminate apex, narrowed

gradually to the base which is suddenly contracted from a wide of 1 cm or less,

base subequally cuneate, margin entire. Texture thin, subcartaceous pale green on

upper surface when dry, paler beneath. Rachis distinct on both surface; veins in

small group at an angle about 65-70º to the costa, the group about 5-7 mm apart,

each vein group forked at the costa, lower branch forked 5-7 times, lateral

members of the vein group copiously anastomousing (2/3-4/5 way from margin to

costa). Sori elongate along lateral veins, both outer and inner vein group forming

areoles about 1.5 mm wide, basal acroscopic branch and also those anastomousing

veinlets diplazioid. Indusia thin, pale brown, margin entire, persistent.

DISTRIBUTION. Borneo.

237

ECOLOGY. This species grows terrestrially on lowland mountain forest

at 150 m. Hitherto it has only been found in Bukit Raya.

NOTES. This species is very distinct among Malesian Diplazium in

characters combination as follow: Lamina simple elliptical with base subequaly

cuneate, veins copiously anastomousing to 4/5 way of margin to form areoles

about 1.5 mm wide on both ounter and inner veins group. This species similar to

simple frond of D. cordifolium in its copiously anasomousing veins so that the

specimen was formerly identified as D. cordifolium by de Joncheere.

Unfortunately, scales that is the important characteristic for identifying Diplazium

is fallen, not found on the specimens.

ETYMOLOGY. The species epithet is chosen in illustrating the large

simple frond.

39. Diplazium meijerii Praptosuwiryo, sp. nov. Plate 9.

TYPE: Sumatra: Central Sumatra, Payakumbuh, Northern slope of Mt.

Sago, 900-1200 m, 21 July 1955, W. Meijer 3772 (Holotype, BO).

Rhizome subprocumbent. Stipites ad 62 cm longis, prope basin 4 mm

crassi, in sicco dilutus brunneus, basi nigris squamis; squama nitidus nigris,

linearis lanceolatis, ad 9 mm longis 1 mm latis, margine regularis dentatus cum

filum niger spissescens, dentatis furcatis. Lamina ad 50 cm longis et latis,

bipinnata deltoideus; pinnae inferiora stipitae ad 6.5 mm latis, ad 9 cm seorsum,

ascendens, oblongis subtriangularis, ad 28 cm longis 12 cm latis, punnulae ad 13

jugatae; pinnulae inferiora stipitae at 2 mm longis, superiora adnatae vel sessile,

lanceolatis, ad 7.5 cm longis, 2 cm latis, basi truncates, apice acuminatus, margine

2/3-3/4 costam lobatae; lobus basalis acroscopicus multus deminutus ad 2 latis,

lobi magniora ad 6 mm latae, obiquus, basin latissimus, apice truncates vel to

roundrotundatus, integer; venae liberis, in lobo pinnatus, venulae ad 6-8-jugatae,

simplices vel furcatis. Textura tenuis papyraceous. Sori medius in 1/4-3/4

longitudine venularum.. Indusiis tenuis, paleis brunneis, persistens, margine

laciniatus.

238

Rhizome subcreeping (?), densely scales on younger part. Stipe to 62 cm

long, 4 mm thick, light brown when dry, black and scales at base; scales lineary

lanceolate, to ca. 9 mm long, 1 mm broad, black, shiny, margin toothed, with

regularly thickening black strands, teeth forked at tip. Lamina bipinate, deltoid,

ca. 50 cm long and broad; lower pinnae stalked to 6.5 cm long, to 9 cm apart,

ascending, oblong subtringular, to 28 cm long, 12 cm broad, pinnulae to 13 pairs;

lower pinnulae stalked to 2 mm long, upper adnate to sessile, lanceolate, to 7.5 cm

long, 2 cm broad, base truncate, apex sharply acuminate, margin lobed 2/3-3/4

way to costa; basal acroscopic lobes much reduce to 2 mm broad, larger lobes to 6

mm broad, oblique, broadest at base, apex truncate to rounded, entire; rachise not

gemmiferouse; veins free, pinnate in the lobes, veinlets 6-7 pairs, commonly 7

pairs, simple or once forked. Texture thinly papyraceous. Sori medial, cover ¼-

3/4 of veinlets length; indusia thin, pale brown, persistent, margin laciniate.

DISTRIBUTION. Sumatra.

ECOLOGY. Terrestrially, in primary forest at 900-1200 m.

NOTES. Diplazium meijeri may closely related to D. latisquamatum. D.

meijeri differs from D. latisquamatum in its black lineary toothed scales with

regular thickening black strands; sori medial. D. latisquamatum has dark brown

round-ovate entire scales with irregularly thickening black strand; sori basalis.

ETYMOLOGY. This species is named after W. Meijer, the collector of

this species.

40. Diplazium melanolepis Alderw.Diplazium melanolepis Alderw., Bull. Jard. Bot. Buit. II nr. XI. 8. 1913. -

--TYPE: Sumatra, G. Singgalang, C.G. Matthew 507 (holotype, BO!).

Rhizome (?). Scales on stipes dark brown, concolour, lanceolate, 5-12 mm

long, 1-2 mm broad, margin toothed, Stipe 56 cm long, 7 mm thick,blackish when

dry, scales throughout, dense toward base. Lamina bipinnate-bipinnatifid, pinnae

(?) pairs; lower pinnae stalked to 13 mm long, 7.5 cm apart, almost at right angle,

lanceolate, widest one third from the base, 34.5 cm long, 8 cm broad, pinnulae 20

pairs below pinnatifid apex of pinna; pinnulae adnate-sessile, oblong-lanceolate,

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one pair basal reduced to 12 mm long, 3.5 mm broad, larger pinnulae to 5.2 cm

long, 0.9 cm broad, commonly less than 4 cm long, 0.8 cm broad, apex rounded-

acute, margin lobed to 1/3 way to costule; lobus 2-2.5 mm broad, oblique, ends

subtruncate, slightly toothed. Rachise and costae scales. Texture firm. Veins

forming angle 40-50º to costule, free, forked at costule, upper branch simplend

soriferous, usually diplazioid, lower brach simple or forked once-twice again,

basiscopic branch usually also soriferous, asplenoid. Sori from basal cover 2/3-3/4

of their length. Indusia dark brown, usually rolled back, persistent, margin entire.

DISTRIBUTION. Sumatra.

ECOLOGY. Occurs in light shade, on mountain forest at ca. 1900 m

above sea level.

SPECIMENS EXAMINED. --- SUMATRA: C.G. Matthew 507.

41. Diplazium moultonii (Copel.) Tagawa

Diplazium moultonii (Copel.) Tagawa, Acta Phyotax. Geobot. 25: 67.

1972. --- Athyrium moultonii Copel. Journ. Str. Br. Asiatic Soc. No. 63. 71.

1912; C.Chr. Gard. Bull. S.S. 7: 267. 1934.

Lamina tripinnatifid, (?) cm long, 25 cm broad; pinnae numerous, (?)

pairs. Pinnae alternate, about 3-4 cm apart, costa forming an angle about 70-80º

to rachis, stalked to about 8 mm long, oblong subtriangular, to about 28 cm long,

8 cm broad, pinnulae to 24 pairs below pinnatifid apex of pinna, gradually

decrease in size towards apex of pinna; pinnulae almost at right angle to costa,

shortly stalked to 33 mm long; lower pinnule oblong subtriangular, to about 6 cm

long, 1.6 cm wide, base subequally truncate, lower segmented, segments 1-4 pairs,

upper deeply lobed to 4/5 – 6/7 way to costulet (deeply lobed to within 1 mm of

costule), apex acuminate; segments adnate to sessile, forming an angle about 60º

to costule, oblong, to about 9 mm by 4 mm, apex rounded, crenulate. Veins

pinnate in each segment, free, to 4 pairs, veinlet simple in each crenation, distinct,

black when dry. Sori from basal costulet cover veinlets 1/3 of their length.

Indusia brown, thin, broad, persistent, margin tearing of.

DISTRIBUTION. Borneo.

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ECOLOGY. Growing on shade parts of ridge mountain forest at 1100-

1850 m sea level.

SPECIEMNS EXAMINED: BORNEO: Kalimantan Timur: M Kato, M.

Okamoto & E.B. Walujo B-9806; M. Kato, M. Okamoto & E.B. Walujo B10018;

M. Kato, M. Okamoto & E.B. Walujo B10925; M. Kato, M. Okamoto & E.B.

Walujo B-9566.

42. Diplazium pallidum (Blume) Moore

Diplazium pallidum (Blume) Moore, Ind.: 333. 1861; Bedd., Hand. Ferns

Br. Ind: 175. 1883; Copel., Polypod. Philipp.: 73. 1905. Backer & Posth.,

Varenfl. Java: 129. 1939; Holttum, Rev. Fl. Malaya Appendix II : 637. 1966. –

Asplenium pallidum Blume, En. Pl. Jav.: 177. 1828. -- Diplazium montanum

Alderw., Bull. Jard. Bot. Buitenz. II Ser., 28: 19. 1918. – Athyrium montanum

(Alderw.) Holttum, Rev. Fl. Malaya 2: 555. 1966.

Key to the varieties

Terminal pinna deltoid and deeply lobed; upper base of lateral pinnae broadly truncate, lower basenarrowly rounded ……………………….…………………………………… var. pallidum

Terminal pinna conform to lateral or with one or two lobes; upper base of lateral pinnae rounded,lower base cuneate …………………………………………………………… var. montanum

a. var. pallidum

Rhizome erect, suberect. Stipe 17-57 cm long, black and scales densely at

base; scales dark brown, nearly black, concolours, 7-13 mm long, 1-1.8 mm wide.

Lamina nearly oblong, pinnae 11-27 pairs; rachis grooved on upper surface;

pinnae 7.8-23.5 cm long, 1.2-2.6 cm wide; lower pinnae on stalk up to 5 mm long,

upper pinnae sessile, apical lamina of the frond deltoid and deeply lobed; upper

pinnae with cuneate base, lower pinnae with base broadly truncate, lower base

narrowly rounded; margin of pinnae toothed, apex toothed acuminate;texture

subcoriaceous; veins free, forked near the costa, upper branch simple and

soriferous, lower branch forked again 1-3 times. Sori on acroscopic veinlet

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simple, sometimes double, reaching from the costa almost to margin of pinnae.

Indusia firm, persistent, dark brown, margin entire.

SPORES. Monolete, bilaterally symmetrical (made asymmetric by

perine), heteropolar; polar outline (excluding perine) transversely elliptical;

equatorial longitudinal view (excluding perine) plano-convex; equatorial view

tranverse view proximal face convex, distal face hemispherical; perinate. Size: E:

32.65(47.96)65.02±8.98; P: 17.08(27.09)34.12±5.12. Laesura: concealed by

perine. Perine: alate under LM, costate under SEM, irregular envelope separated

from exine surrounds the spore in irregular, wing-like costae, often anastomosing

to form loose reticulation, lacunae shallow irregular polygons 15-18 µm wide,

muri 0.8 – 5 µm wide, surface of muri smooth and lacunae smoothly granulate.

Exine: visible through perine, granulate under LM.

ANATOMY. Transverse section of stipe near lamina: Vascular bundle

form an uninterrupted U-shaped with an angle 105º, base flat, ends bluntly ridge

both inward and outward.

CHROMOSOMES. 2n = 164 (Cytotype: T.Ng. Praptosuwiryo 1151,

BO).

DISTRIBUTION. Malay Peninsula, Sumatra, Java.

ECOLOGY. Grows in shady forest in the hills and mountains, 1000 –1600 m.

SPECIMENS EXAMINED. --- JAVA: T.Ng. Praptosuwiryo 1151, 1237,

1335, 1377, 1484, 1505, 1630, 1739, 1759, 1764; W.S. Hoover, J.M. Hunter, H.

Wiriadinata, D.Girmansyah & A. Ruskandi ARs 29; A. Hidayat & H. Wiriadinata

AH 558. SUMATRA: T.Ng. Praptosuwiryo 2246.

b. var. montanum (Alderw.) Praprosuwiryo, com. nov.Diplazium montanum (Alderw.), Bull. Buitenz. II Ser. 28:19. 1918; Holtt.,

Gard. Bull. S.S. 11: 92. 1940. –Athyrium montanum (Alderw.) Holttum, Rev. Fl.

Malaya 2: 555. f. 327. 1966.

Pinnae to 16 pairs, 8.5-21 cm long, 1.3-2.3 cm wide; lower pinnae on stalk

2-4 mm long, upper base rounded, lower base cuneate; upper pinnae sessile with

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broadly cuneate base; terminal pinane like others, occasionally with one-two

rounded lobes; margin of pinnae shortly toothed or almost entire.

CHROMOSOMES. 2n = 82 (Cytotype: T.Ng.Praptosuwiryo 1406, BO).

DISTRIBUTION. Sumatra, Java.

ECOLOGY. Terrestrial. Dense jungle, rich soil on rock, wet ground.

Elevation: 20 – 400 m.

SPECIMENS EXAMINED. --- SUMATRA: T.Ng. Praptosuwiryo 2244,

2537; J. v. Borssum W. 2787; J. v. Borssum W. 2334; J. v. Borssum W. 2338, ---

MALAY PENINSULA. Pahang: L.B. Molesworth – Allen 4247; M.R.

Henderson 18583; Haniff 4047; L. Wray 3617; King 10959; H.N. Ridley 14209;