Biosystematics of pandanaceae in Java

BIOSYSTEMATICS OF

PANDANACEAE

IN JAVA

SRI ENDARTI RAHAYU

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

STATEMENT OF RESEARCH ORIGINALITY AND

INFORMATION SOURCE

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

Bogor, Januari 2011

Sri Endarti Rahayu NRP G361040061

BIOSYSTEMATICS OF

PANDANACEAE

IN JAVA

SRI ENDARTI RAHAYU

THE GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY

BOGOR

STATEMENT OF RESEARCH ORIGINALITY AND

INFORMATION SOURCE

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

Bogor, Januari 2011

Sri Endarti Rahayu NRP G361040061

ABSTRAK

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

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

ABSTRACT

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

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

SUMMARY

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

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

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

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

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

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

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

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

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

GENERAL INTRODUCTION

Taxonomical Aspects ofPandanaceaein Java and Its Systematic Problems

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

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

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

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

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

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

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

The objectives of the study

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

A TAXONOMIC STUDY OF

PANDANUS FURCATUS

AND

PANDANUS TECTORIUS

COMPLEXES WITH SPECIAL

EMPHASIS ON PLANTS FROM JAVA

Introduction

The term species complex is used to describe species aggregation sharing the specific morphological and molecular features (Juddet al. 1999). Within this complex a complicated morphological overlap, without any discontinuities, has led to taxonomic difficulty (Pak and Kawano 1990). Although their taxonomic affinity may be difficult to determine, some form of taxonomic resolution is desirable.

According to Stone (1972) Pandanus in Java contains many rather problematical species. He suggested that detailed studies were required to get more refined taxonomic scheme. The main problem as far as the Java plant are concerned appears to be the status species which are given as synonyms of P. furcatus Roxb. by Backer and Bakhuizen van den Brink (1968). According to Backer and Bakhuizen van den Brink,P. bantamensisKoord.,P. ovigerMartelli, P. pseudolais Warb., and P. scabrifolius Martelli; and P. odoratissimus L.f are regarded as synonymous with P. tectorius var. littoralis. Stone (1972) did short revision on Pandanaceae in Java based on specimen herbarium kept in BO and living plant cultivated in Hortus Bogoriensis and tried to develop a more stable species concept forPandanus furcatusRoxb. In Stone’s opinion, Backer’s species concept for Pandanus furcatus is far too comphrehensive that required certain readjusment because some species omitted or reduced to be lumped toPandanus furcatus Roxb., and by Stone these species were regarded as four different species, viz.P. bantamensisKoord.,P. pseudolaisWarb.,P. scabrifoliusMartelli, and P. oviger Martell, while the status of P. tectorius var. littoralis and P. odoratissimus L.f. is still more or less in question. Therefore those circumscription should now be reviewed in light of our recent fieldwork in Java with these taxa. A practical difficulty here is that almost all morphological characters and/or character states used for evaluating species complex are only slightly differentiated from one another and usually show considerable overlap. Therefore the identification taxa within species complex usually requires

combination of several characters. The aims of the study was to provide taxonomic resolution of the P. furcatus and P. tectorius complexes based on morphology, anatomy and molecular approach, i.e comparative sequence ofatp B-rbcL IGS.

Materials and Methods

Studies of herbarium specimens were conducted in Herbarium Bogoriemse (BO), Herbarium of the Royal Botanical Gardens Kew (K) and National Herbarium Netherlands, Leiden (L). Observations on living plants and anatomical were undertaken in Herbarium Bogoriense, while the molecular data was analyses in Van der Klauw Laboratory, Leiden.

Morphology— Data on morphology were collected from specimens and field collections. The procedure for morphological variation followed those described by Rifai (1976) and Vogel (1987). Measurement were taken from spirit-preserved material and dried herbarium specimen and from living collection. Floral parts were measured from spirit-preserved material, dried specimens and rehydrated by boiling.

Anatomy — Leaf anatomy was investigated by first fixing the leaves (small part of the middle base) in FAA. Paradermal sections were taken from the upper and lower surfaces of leaves, then stained with safranin 1% in water and then mounted in glycerin (Johansen 1940).

atpB-rbcL IGS sequences — Genomic DNA of the silica gel dried leaf were extracted according to the protocol described by Doyle and Doyle (1987). Double stranded DNA was directly amplified by PCR. Reaction volumes were 25 µl and contained 2.5 µl PCR buffer, 2.5 µl dNTPs, 1 µl each of the 5 mM primers, 0.3 µl Taq Pol and 12.7 µl ddH2O. Approximately 5 µl genomic DNAs were

added to the PCR mixture. PCR was performed 3 min at 94oC for the activation of the polymerase, followed by 35 cycles of 49 sec at 94oC, 45 sec at 55oC, 2 min at 72oC, with a final extension period of 10 min at 72oC. The primers used in this study for atpB-rbcL intergenic spacers are forward 5’

-GAAGTAGTAGGATTGATTCTC- 3’ and reverse 5’

-TACAGTTGTCCATGTACC AG-3’. The PCR product was checked on 1% agarose gel, and purified using a purification kit of Wizard SV Gel and PCR

clean up system (PROMEGA) following the manufacturer’s protocol prior to sequencing. The DNA concentration was measured with the nanodrop. Cycle sequencing was performed by Macrogen Korea. The sequences were edited using sequencher 4.6 and MEGA 3.0 (Kumaret al. 2004).

Result and Discussion Result

Morphological Characters ofPandanus furcatuscomplex

In this study, we only found three of four species that recognized withinP. furcatuscomplex. i.e P. bantamensis,P. pseudolaisand P. scabrifolius.

Concerning the prop root characters of Pandanus furcatus complex, prickles in prop root did not display consistence differentiation among the taxa analysed, except for P. scabrifolius where it was no prickles on the prop root. Leaf base colour divided the taxa into two groups which one of it was overlapping group. P. bantamensisandP. pseudolaiswere taxa with reddish brown leaf base, andP. scabrifolius displayed yellowish white leaf base. The leaf dimensions of Pandanus furcatus complex were highly variable. Pandanus pseudolais were characterized by bigger and longer leaf, whereas the leaf of P. scabrifolius were clearly smaller and shorter.P. bantamensiswas characterized by intermediate leaf dimension. Number of drupe per cephalium of P. pseudolais were higher compared with the other taxa. Drupe length divided the taxa into two group which one of it was overlapping group. P. scabrifolius showed longer drupe compare with the other taxa (Table 4.1).

Table 4.1 Morphological characters of species recognized within Pandanus furcatuscomplex

Species Characters

Leaf length (cm)

Prickles on prop root

Number of drupes

Drupe length (cm) P. bantamensis 216-441 muricate in

lines

475-795 2.7–4.5 P. pseudolais 299.4-574.5 muricate in

lines

724-1053 2.6–4.5

Among all morphological characters of species recognized within P. furcatus complex, there were four characters, viz. leaf base colour, peduncle shapes, fruit shapes and style shapes could be used to discriminate among species recognized withinP. furcatuscomplex. The summary of contrasting characters of species recognized withinPandanus furcatuscomplex is shown in Table 4.2.

Table 4.2 Summary of contrasting characters of species recognized within Pandanus furcatuscomplex. Leaf base colour, scale bar = 5 cm; Peduncle, scale bar = 13 cm; Cephalia shape, scale bar = 10 cm; Style shape, scale bar = 1 cm

Character P. bantamensis P. pseudolais P. scabrifolius

Leaf base colour Reddish brown Reddish brown Yellowish white

Peduncle shape Bit curved at the end

Curved at the end Straight

cephalia shape Cylindric-suboblong Oblong-ellipsoid Subglobose

Style shape Bifurcate nearly half way

Morphological Characters ofPandanus tectoriuscomplex

Leaf dimension, leaf shape, cephalium size, cephalium shape and number of phalanges per cephalium did not display consistence differentiation between the taxa analysed, but united taxa into overlapping group (Table 4.3). Among all morphological characters of species recognized within P. tectorius complex, there were three characters, viz. prickles on leaf apex, phalange shapes, and phalange apexes could be used to discriminate among species recognized within P. tectorius complex. The summary of contrasting characters of species recognized withinPandanus tectoriuscomplex is shown in Table 4.4.

Table 4.3 Morphological characters of species recognized within Pandanus tectoriuscomplex

Species

Characters Leaf

length (cm)

Leaf shape

Cephalium size (cm)

Cephalium shape

Number of phalanges

P. tectoriusvar. littoralis

112-199 ligulate or sword

26 x 25 subglobose 79-83 P. odoratissimus 98-126 ligulate

or sword

22 x 25 subglobose-globose

Table 4.4 Summary of contrasting characters of species recognized within Pandanus tectoriuscomplex ; Prickles on leaf apex, scale bar = 2 cm; Cephalium shape, scale bar = 10 cm; Phalange apex, scale bar = 1 cm Character P. tectoriusvar.littoralis P. odoratissimus Prickles on

leaf apex

on one side of leaf prickly, another side smooth

prickly on both sides of leaves

Cephalium shape Oblong Obovate

Phalange apex Carpel tips flat Grooved between carpel tips

Anatomical Characters

Anatomical characters are just as valuable as morphological characters and could be used to delimit species. These characters, qualitative characters were observed directly on paradermal surfaces, viz. occurence of papillae on stomata. Four types of stomata were found in Pandanus tectoriusand Pandanus furcatus complexes. The variation on stomatal structures in Pandanus depends on the number of papillae that develops on the subsidiary and neighbouring cells (Tomlinson 1965; Kam 1971).

The species recognized within Pandanus furcatus complex had different stomatal type. P. bantamensis, P. pseudolais, and P. scabrifolius had stomatal

type 2, type 1 and type 3 respectively; while the species recognized within Pandanus tectorius complex had also different stomatal type. P. tectorius var. littoralisandP. odoratissimushad stomatal type 4 and type 2 respectively.

Molecular Characters

atpB-rbcL IGS sequence was a good marker for delimiting the different species in Pandanus furcatus complex and Pandanus tectorius complex. Polymorphic site in atpB-rbcL IGS sequence of Pandanus furcatus complex is shown in Table 4.5.

Table 4.5 Polymorphic sites inatpB-rbcL IGS sequence ofP. furcatuscomplex

Species

Nucleotide at polymorphic site at indicated position

4 6 16 17 18 12

1 1 2 3 1 4 9 1 5 0 1 6 6 1 6 7 1 6 8 2 4 9 2 7 1 2 7 2 2 7 3 2 9 2

P .bantamensis A G A A A C C G A A G T T A A T T

P.. pseudolais T T A C C A T A A A A T A G G A A

P. scabrifolius T G C C T T C A G G T G T G A G T

Table 4.5 Continued

Species

Nucleotide at polymorphic site at indicated position 2 9 3 2 9 6 2 9 7 4 4 6 4 4 7 4 4 8 5 2 0 5 2 1 5 2 2 5 5 0 5 5 1 5 5 2

P. bantamensis T A G T A G A C C T C A

P.. pseudolais T A T G G G A T A T T A

P. scabrifolius A T A T A A T A C A T C

Polymorphic site inatpB-rbcL sequence ofPandanus tectoriuscomplex is shown in Table 4.6.

Table 4.6 Polymorphic sites inatpB-rbcL sequence IGS ofP. tectoriuscomplex

Species

Nucleotide at polymorphic site at indicated position

3

1 ₃2 ₃3 ₆1 2_{6 6}3 ₈5 ₈6 ₈7 51_{1 1}17 ₁19 02_{1 1}99 ₂00 ₂01

P. tectorius var. littoralis

A G T T C T T A A G T T C C T C

P. odoratissimus G T A G A C G T T A A A G T G A

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

2 0 4 2 2 9 2 3 1 2 3 2 2 3 3 2 4 1 2 4 2 2 6 0 1 2 6 9 3 3 4 3 3 5 3 3 6 3 8 0 4 0 5 4 3 8 4 4 1

P. tectorius var. littoralis

T A G G A A T T T C A T A G C G

P. odoratissimus A G T T C T A A A T T G T A A C

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

4 6 4 4 6 5 4 7 6 4 7 7 5 0 7 5 2 0 5 2 1 5 7 1 5 7 3 5 7 8 5 7 9 5 8 6 5 8 9 5 9 0 5 9 1 6 0 8

P. tectoriusvar. littoralis

A G A A G T C A A A C G A A T T

P. odoratissimus G A T T C A A T T G G C C C A G

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

6 0 9 6 1 3 6 1 4 6 2 3 7 3 9 7 4 0 7 4 8 7 7 0 7 7 1 7 7 4 7 7 5 7 8 9 8 0 9 8 1 0 8 5 6 8 5 7

P. tectorius var. littoralis

G A C T C A T C A A C T G T A G

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position 8 5 8 8 9 5 8 9 6 8 9 7 9 0 1 9 0 2 9 0 3

P. tectorius var. littoralis

A A A A G G A

P. odoratissimus C G T C T C C

Discussion

Pandanus furcatuscomplex

In the species recognized within Pandanus furcatus complex, they were sharing some morphological features, viz. leaf length, prickles on prop root, number of drupes, and drupe length. Beside those characters, these species also had four contrasting morphological characters that could be used to separate each species, viz. leaf base colour, peduncle shape, fruit shape and style shape. P. bantamensishad reddish brown leaf base, peduncle with a bit curved at the end, cylindric-suboblong fruit shape and style that bifurcate nearly half way. P. pseudolaiswere characterized by reddish brown leaf base, peduncle that curved at the end, oblong-ellipsoid fruit shape and hornlike style; whereas P. scabrifolius had yellowish white leaf base, straight peduncle, subglobose fruit shape and style that was bifurcate third way.

These species also had different anatomical characters especially on stomatal type. The difference was on the occurence of papillae, whether occured in lateral subsidiary cells as in P. bantamensis, in terminal and lateral subsidiary cells as in P. scabrifolius, or papillae absent in stomata that was called unspecilaized stomata as inP. pseudolais.

Molecular variation of the chloroplast noncoding region between atp B-rbcL genes amongP. bantamensis,P. pseudolais, andP. scabrifoliusshowed that most variation were contributed by nucleotide substitution, 19, 24 and 22 nucleotide substitution were found betweenP. bantamensis andP. pseudolais,P. pseudolais and P. scabrifolius, and P. bantamensis and P. scabrifolius respectively.

Pandanus tectoriuscomplex

AlthoughPandanus tectoriusvar.littoralis andP. odoratissimusare very closely related, they could be distinguished (Stone 1994). Stone have proposed delimitingP. odoratissimusby just two characters, fleshy shoulders on phalanges and large white spines on the leaves (Stone 1967). In our study we found that P. odoratissimushas large white spines, the same asP. tectoriusvar.littoralis,but it does not have fleshy shoulders on the phalanges. It is supported by John (1979) who stated that P.odoratissimus has large white spines, but does not have fleshy shoulders. Morphological observation revealed that large white spine on the leave character are not good characters for separating the two species because the two species have those characters on their leaves.

In this study, we found three contrasting morphological characters that could be used to discriminate each species recognized within P. tectorius complex, i.e prickles on leaf apex, phalange shape and phalange apex.P. tectorius var.littoraliswas characterized by leaf apex that had not prickly on both sides of leaves, oblong phalange shape, and carpels tip flat; whereas P. odoratissimushad leaf apex that is prickly on both sides of leaves with obovate phalange shape, and grooved between carpel tips.

These species also had different anatomical characters, especially on stomatal type. The stomata was differed on the occurence of papillae on stomata. InP. odoratissimusthe papillae only occured on lateral subsidiary cells, while in P. tectorius var. littoralis papillae was occured also in neighbouring subsidiary cells.

Molecular variation of atpB-rbcL intergenic spacer in both P. tectorius var. littoralis and P. odoratissimus showed that most variation were contributed by 71 substitution.

Conclusion

Pandanus furcatuscomplex

Based on morphological, anatomical and comparative sequence of atp B-rbcL IGS, we concluded that Pandanus bantamensis Koord., Pandanus pseudolais Warb., dan Pandanus scabrifolius Martelli are treated as three

different species. Our study confirm the opinion of Stone (1972) that three species should recognized in Java, viz. Pandanus bantamensisKoord.,Pandanus pseudolaisWarb., andPandanus scabrifoliusMartelli.

Key to species of thePandanus furcatuscomplex

1a. Leaf base reddish brown ... 2 1b. Leaf base yellowish white ... P. scabrifolius 2a. Peduncle a bit curved at the end, fruit shape cylindric–suboblong, style

bifurcate nearly halfway, stomata with papillae on lateral

subsidiary cells ... P. bantamensis 2b. Peduncle curved at the end, fruit shape oblong–ellipsoid, style hornlike,

stomata without papillae ... P. pseudolais

Pandanus tectoriuscomplex

Based on morphological, anatomical and comparative sequence of atp B-rbcL IGS, P. tectorius var.littoralis should be recognized as a separate species and distinct from P. odoratissimusL.f.

Key to species of thePandanus tectoriuscomplex

One side of the leaf apex smooth, phalange oblong, phalange apex flat, stomata with papillae on neighbouring epidermal and subsidiary cells

... P. tectorius var.littoralis Both sides of the leaf apex prickly, phalange obovate, phalange apex grooved between carpel tips, stomata with papillae on lateral

A TAXONOMIC STUDY OF

PANDANUS FURCATUS

AND

PANDANUS TECTORIUS

COMPLEXES WITH SPECIAL

EMPHASIS ON PLANTS FROM JAVA

Introduction

The term species complex is used to describe species aggregation sharing the specific morphological and molecular features (Juddet al. 1999). Within this complex a complicated morphological overlap, without any discontinuities, has led to taxonomic difficulty (Pak and Kawano 1990). Although their taxonomic affinity may be difficult to determine, some form of taxonomic resolution is desirable.

According to Stone (1972) Pandanus in Java contains many rather problematical species. He suggested that detailed studies were required to get more refined taxonomic scheme. The main problem as far as the Java plant are concerned appears to be the status species which are given as synonyms of P. furcatus Roxb. by Backer and Bakhuizen van den Brink (1968). According to Backer and Bakhuizen van den Brink,P. bantamensisKoord.,P. ovigerMartelli, P. pseudolais Warb., and P. scabrifolius Martelli; and P. odoratissimus L.f are regarded as synonymous with P. tectorius var. littoralis. Stone (1972) did short revision on Pandanaceae in Java based on specimen herbarium kept in BO and living plant cultivated in Hortus Bogoriensis and tried to develop a more stable species concept forPandanus furcatusRoxb. In Stone’s opinion, Backer’s species concept for Pandanus furcatus is far too comphrehensive that required certain readjusment because some species omitted or reduced to be lumped toPandanus furcatus Roxb., and by Stone these species were regarded as four different species, viz.P. bantamensisKoord.,P. pseudolaisWarb.,P. scabrifoliusMartelli, and P. oviger Martell, while the status of P. tectorius var. littoralis and P. odoratissimus L.f. is still more or less in question. Therefore those circumscription should now be reviewed in light of our recent fieldwork in Java with these taxa. A practical difficulty here is that almost all morphological characters and/or character states used for evaluating species complex are only slightly differentiated from one another and usually show considerable overlap. Therefore the identification taxa within species complex usually requires

combination of several characters. The aims of the study was to provide taxonomic resolution of the P. furcatus and P. tectorius complexes based on morphology, anatomy and molecular approach, i.e comparative sequence ofatp B-rbcL IGS.

Materials and Methods

Studies of herbarium specimens were conducted in Herbarium Bogoriemse (BO), Herbarium of the Royal Botanical Gardens Kew (K) and National Herbarium Netherlands, Leiden (L). Observations on living plants and anatomical were undertaken in Herbarium Bogoriense, while the molecular data was analyses in Van der Klauw Laboratory, Leiden.

Morphology— Data on morphology were collected from specimens and field collections. The procedure for morphological variation followed those described by Rifai (1976) and Vogel (1987). Measurement were taken from spirit-preserved material and dried herbarium specimen and from living collection. Floral parts were measured from spirit-preserved material, dried specimens and rehydrated by boiling.

Anatomy — Leaf anatomy was investigated by first fixing the leaves (small part of the middle base) in FAA. Paradermal sections were taken from the upper and lower surfaces of leaves, then stained with safranin 1% in water and then mounted in glycerin (Johansen 1940).

atpB-rbcL IGS sequences — Genomic DNA of the silica gel dried leaf were extracted according to the protocol described by Doyle and Doyle (1987). Double stranded DNA was directly amplified by PCR. Reaction volumes were 25 µl and contained 2.5 µl PCR buffer, 2.5 µl dNTPs, 1 µl each of the 5 mM primers, 0.3 µl Taq Pol and 12.7 µl ddH2O. Approximately 5 µl genomic DNAs were

added to the PCR mixture. PCR was performed 3 min at 94oC for the activation of the polymerase, followed by 35 cycles of 49 sec at 94oC, 45 sec at 55oC, 2 min at 72oC, with a final extension period of 10 min at 72oC. The primers used in this study for atpB-rbcL intergenic spacers are forward 5’

-GAAGTAGTAGGATTGATTCTC- 3’ and reverse 5’

-TACAGTTGTCCATGTACC AG-3’. The PCR product was checked on 1% agarose gel, and purified using a purification kit of Wizard SV Gel and PCR

clean up system (PROMEGA) following the manufacturer’s protocol prior to sequencing. The DNA concentration was measured with the nanodrop. Cycle sequencing was performed by Macrogen Korea. The sequences were edited using sequencher 4.6 and MEGA 3.0 (Kumaret al. 2004).

Result and Discussion Result

Morphological Characters ofPandanus furcatuscomplex

In this study, we only found three of four species that recognized withinP. furcatuscomplex. i.e P. bantamensis,P. pseudolaisand P. scabrifolius.

Concerning the prop root characters of Pandanus furcatus complex, prickles in prop root did not display consistence differentiation among the taxa analysed, except for P. scabrifolius where it was no prickles on the prop root. Leaf base colour divided the taxa into two groups which one of it was overlapping group. P. bantamensisandP. pseudolaiswere taxa with reddish brown leaf base, andP. scabrifolius displayed yellowish white leaf base. The leaf dimensions of Pandanus furcatus complex were highly variable. Pandanus pseudolais were characterized by bigger and longer leaf, whereas the leaf of P. scabrifolius were clearly smaller and shorter.P. bantamensiswas characterized by intermediate leaf dimension. Number of drupe per cephalium of P. pseudolais were higher compared with the other taxa. Drupe length divided the taxa into two group which one of it was overlapping group. P. scabrifolius showed longer drupe compare with the other taxa (Table 4.1).

Table 4.1 Morphological characters of species recognized within Pandanus furcatuscomplex

Species Characters

Leaf length (cm)

Prickles on prop root

Number of drupes

Drupe length (cm) P. bantamensis 216-441 muricate in

lines

475-795 2.7–4.5 P. pseudolais 299.4-574.5 muricate in

lines

724-1053 2.6–4.5

Among all morphological characters of species recognized within P. furcatus complex, there were four characters, viz. leaf base colour, peduncle shapes, fruit shapes and style shapes could be used to discriminate among species recognized withinP. furcatuscomplex. The summary of contrasting characters of species recognized withinPandanus furcatuscomplex is shown in Table 4.2.

Table 4.2 Summary of contrasting characters of species recognized within Pandanus furcatuscomplex. Leaf base colour, scale bar = 5 cm; Peduncle, scale bar = 13 cm; Cephalia shape, scale bar = 10 cm; Style shape, scale bar = 1 cm

Character P. bantamensis P. pseudolais P. scabrifolius

Leaf base colour Reddish brown Reddish brown Yellowish white

Peduncle shape Bit curved at the end

Curved at the end Straight

cephalia shape Cylindric-suboblong Oblong-ellipsoid Subglobose

Style shape Bifurcate nearly half way

Morphological Characters ofPandanus tectoriuscomplex

Leaf dimension, leaf shape, cephalium size, cephalium shape and number of phalanges per cephalium did not display consistence differentiation between the taxa analysed, but united taxa into overlapping group (Table 4.3). Among all morphological characters of species recognized within P. tectorius complex, there were three characters, viz. prickles on leaf apex, phalange shapes, and phalange apexes could be used to discriminate among species recognized within P. tectorius complex. The summary of contrasting characters of species recognized withinPandanus tectoriuscomplex is shown in Table 4.4.

Table 4.3 Morphological characters of species recognized within Pandanus tectoriuscomplex

Species

Characters Leaf

length (cm)

Leaf shape

Cephalium size (cm)

Cephalium shape

Number of phalanges

P. tectoriusvar. littoralis

112-199 ligulate or sword

26 x 25 subglobose 79-83 P. odoratissimus 98-126 ligulate

or sword

22 x 25 subglobose-globose

Table 4.4 Summary of contrasting characters of species recognized within Pandanus tectoriuscomplex ; Prickles on leaf apex, scale bar = 2 cm; Cephalium shape, scale bar = 10 cm; Phalange apex, scale bar = 1 cm Character P. tectoriusvar.littoralis P. odoratissimus Prickles on

leaf apex

on one side of leaf prickly, another side smooth

prickly on both sides of leaves

Cephalium shape Oblong Obovate

Phalange apex Carpel tips flat Grooved between carpel tips

Anatomical Characters

Anatomical characters are just as valuable as morphological characters and could be used to delimit species. These characters, qualitative characters were observed directly on paradermal surfaces, viz. occurence of papillae on stomata. Four types of stomata were found in Pandanus tectoriusand Pandanus furcatus complexes. The variation on stomatal structures in Pandanus depends on the number of papillae that develops on the subsidiary and neighbouring cells (Tomlinson 1965; Kam 1971).

The species recognized within Pandanus furcatus complex had different stomatal type. P. bantamensis, P. pseudolais, and P. scabrifolius had stomatal

type 2, type 1 and type 3 respectively; while the species recognized within Pandanus tectorius complex had also different stomatal type. P. tectorius var. littoralisandP. odoratissimushad stomatal type 4 and type 2 respectively.

Molecular Characters

atpB-rbcL IGS sequence was a good marker for delimiting the different species in Pandanus furcatus complex and Pandanus tectorius complex. Polymorphic site in atpB-rbcL IGS sequence of Pandanus furcatus complex is shown in Table 4.5.

Table 4.5 Polymorphic sites inatpB-rbcL IGS sequence ofP. furcatuscomplex

Species

Nucleotide at polymorphic site at indicated position

4 6 16 17 18 12

1 1 2 3 1 4 9 1 5 0 1 6 6 1 6 7 1 6 8 2 4 9 2 7 1 2 7 2 2 7 3 2 9 2

P .bantamensis A G A A A C C G A A G T T A A T T

P.. pseudolais T T A C C A T A A A A T A G G A A

P. scabrifolius T G C C T T C A G G T G T G A G T

Table 4.5 Continued

Species

Nucleotide at polymorphic site at indicated position 2 9 3 2 9 6 2 9 7 4 4 6 4 4 7 4 4 8 5 2 0 5 2 1 5 2 2 5 5 0 5 5 1 5 5 2

P. bantamensis T A G T A G A C C T C A

P.. pseudolais T A T G G G A T A T T A

P. scabrifolius A T A T A A T A C A T C

Polymorphic site inatpB-rbcL sequence ofPandanus tectoriuscomplex is shown in Table 4.6.

Table 4.6 Polymorphic sites inatpB-rbcL sequence IGS ofP. tectoriuscomplex

Species

Nucleotide at polymorphic site at indicated position

3

1 ₃2 ₃3 ₆1 2_{6 6}3 ₈5 ₈6 ₈7 51_{1 1}17 ₁19 02_{1 1}99 ₂00 ₂01

P. tectorius var. littoralis

A G T T C T T A A G T T C C T C

P. odoratissimus G T A G A C G T T A A A G T G A

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

2 0 4 2 2 9 2 3 1 2 3 2 2 3 3 2 4 1 2 4 2 2 6 0 1 2 6 9 3 3 4 3 3 5 3 3 6 3 8 0 4 0 5 4 3 8 4 4 1

P. tectorius var. littoralis

T A G G A A T T T C A T A G C G

P. odoratissimus A G T T C T A A A T T G T A A C

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

4 6 4 4 6 5 4 7 6 4 7 7 5 0 7 5 2 0 5 2 1 5 7 1 5 7 3 5 7 8 5 7 9 5 8 6 5 8 9 5 9 0 5 9 1 6 0 8

P. tectoriusvar. littoralis

A G A A G T C A A A C G A A T T

P. odoratissimus G A T T C A A T T G G C C C A G

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position

6 0 9 6 1 3 6 1 4 6 2 3 7 3 9 7 4 0 7 4 8 7 7 0 7 7 1 7 7 4 7 7 5 7 8 9 8 0 9 8 1 0 8 5 6 8 5 7

P. tectorius var. littoralis

G A C T C A T C A A C T G T A G

Table 4.6 Continued

Species

Nucleotide at polymorphic site at indicated position 8 5 8 8 9 5 8 9 6 8 9 7 9 0 1 9 0 2 9 0 3

P. tectorius var. littoralis

A A A A G G A

P. odoratissimus C G T C T C C

Discussion

Pandanus furcatuscomplex

In the species recognized within Pandanus furcatus complex, they were sharing some morphological features, viz. leaf length, prickles on prop root, number of drupes, and drupe length. Beside those characters, these species also had four contrasting morphological characters that could be used to separate each species, viz. leaf base colour, peduncle shape, fruit shape and style shape. P. bantamensishad reddish brown leaf base, peduncle with a bit curved at the end, cylindric-suboblong fruit shape and style that bifurcate nearly half way. P. pseudolaiswere characterized by reddish brown leaf base, peduncle that curved at the end, oblong-ellipsoid fruit shape and hornlike style; whereas P. scabrifolius had yellowish white leaf base, straight peduncle, subglobose fruit shape and style that was bifurcate third way.

These species also had different anatomical characters especially on stomatal type. The difference was on the occurence of papillae, whether occured in lateral subsidiary cells as in P. bantamensis, in terminal and lateral subsidiary cells as in P. scabrifolius, or papillae absent in stomata that was called unspecilaized stomata as inP. pseudolais.

Molecular variation of the chloroplast noncoding region between atp B-rbcL genes amongP. bantamensis,P. pseudolais, andP. scabrifoliusshowed that most variation were contributed by nucleotide substitution, 19, 24 and 22 nucleotide substitution were found betweenP. bantamensis andP. pseudolais,P. pseudolais and P. scabrifolius, and P. bantamensis and P. scabrifolius respectively.

Pandanus tectoriuscomplex

AlthoughPandanus tectoriusvar.littoralis andP. odoratissimusare very closely related, they could be distinguished (Stone 1994). Stone have proposed delimitingP. odoratissimusby just two characters, fleshy shoulders on phalanges and large white spines on the leaves (Stone 1967). In our study we found that P. odoratissimushas large white spines, the same asP. tectoriusvar.littoralis,but it does not have fleshy shoulders on the phalanges. It is supported by John (1979) who stated that P.odoratissimus has large white spines, but does not have fleshy shoulders. Morphological observation revealed that large white spine on the leave character are not good characters for separating the two species because the two species have those characters on their leaves.

In this study, we found three contrasting morphological characters that could be used to discriminate each species recognized within P. tectorius complex, i.e prickles on leaf apex, phalange shape and phalange apex.P. tectorius var.littoraliswas characterized by leaf apex that had not prickly on both sides of leaves, oblong phalange shape, and carpels tip flat; whereas P. odoratissimushad leaf apex that is prickly on both sides of leaves with obovate phalange shape, and grooved between carpel tips.

These species also had different anatomical characters, especially on stomatal type. The stomata was differed on the occurence of papillae on stomata. InP. odoratissimusthe papillae only occured on lateral subsidiary cells, while in P. tectorius var. littoralis papillae was occured also in neighbouring subsidiary cells.

Molecular variation of atpB-rbcL intergenic spacer in both P. tectorius var. littoralis and P. odoratissimus showed that most variation were contributed by 71 substitution.

Conclusion

Pandanus furcatuscomplex

Based on morphological, anatomical and comparative sequence of atp B-rbcL IGS, we concluded that Pandanus bantamensis Koord., Pandanus pseudolais Warb., dan Pandanus scabrifolius Martelli are treated as three

different species. Our study confirm the opinion of Stone (1972) that three species should recognized in Java, viz. Pandanus bantamensisKoord.,Pandanus pseudolaisWarb., andPandanus scabrifoliusMartelli.

Key to species of thePandanus furcatuscomplex

1a. Leaf base reddish brown ... 2 1b. Leaf base yellowish white ... P. scabrifolius 2a. Peduncle a bit curved at the end, fruit shape cylindric–suboblong, style

bifurcate nearly halfway, stomata with papillae on lateral

subsidiary cells ... P. bantamensis 2b. Peduncle curved at the end, fruit shape oblong–ellipsoid, style hornlike,

stomata without papillae ... P. pseudolais

Pandanus tectoriuscomplex

Based on morphological, anatomical and comparative sequence of atp B-rbcL IGS, P. tectorius var.littoralis should be recognized as a separate species and distinct from P. odoratissimusL.f.

Key to species of thePandanus tectoriuscomplex

One side of the leaf apex smooth, phalange oblong, phalange apex flat, stomata with papillae on neighbouring epidermal and subsidiary cells

... P. tectorius var.littoralis Both sides of the leaf apex prickly, phalange obovate, phalange apex grooved between carpel tips, stomata with papillae on lateral

GENETIC DIVERSITY OF JAVANESE

PANDANUS

AND

FREYCINETIA

BASED ON ISSR MARKER

Introduction

The pandan family (Pandanaceae) of the monocotyledonous Angiosperm or flowering plants is represented in Java by two genera, Pandanus and Freycinetia(Stone 1970). The genusPandanuscontain about 500-600 species of trees and shrubs of the Old World tropics, distributed from East Africa westward through Indomalaysia to remote island of Polynesia, it extends south to tropical Australia (but not to New Zealand), north to Ryukyus, Bonins, Taiwan, and to Hawaiian island (Stone 1965), whileFreycinetiacontains about 180-200 species of root climbing lianas or low growing shrubs occur from Sri Lanka throughout South-Eastern Asia to Northern Australia, Polynesia and New Zealand (Coxet al. 1995). The habitat of Pandanus include nearly all possible habitats available to plants of comparable size, from sea level to the highest peaks (Stone 1966), whereas Freycinetia are usually lacking in open environment because they are dependent on forest environment (Stone 1982).

Pandanus species specialize in wind pollination. This anemophily, coupled with syncarpus dispersal unit (the pistillate phalange) and also the presence of facultative apomixis (Cox 1990) allowing Pandanus species with these features have considerable ability to colonize new areas. Immediate genetic isolation as well as exposuring to new habitats without regard to pollinator availability may account for the extreme richness of species inPandanusas well its very wide distribution (Cox et al. 1995), whereas Freycinetia has been believed to be strictly dioecious (Dahlgren et al. 1985), but recent fieldwork has indicated that a variety of breeding systems exist inFreycinetia(Coxet al. 1984). Some individuals of Freycinetia imbricata in Sumatra produce staminate and pistillate shoots on the same plant, such divergences from a dioecious breeding system may be important in island colonization (Baker & Cox 1984) particularly since monoecious individuals of Freycinetia scandens in Philippine have been found to be self compatible (Cox et al. 1984). Freycinetia lack facultative

apomixis and water dispersal. However, their attractiveness to a wide variety of vertebrate pollination and disperses, as well as infrequent leaky dioecy would assure them a large range and high speciation rate (Cox 1990).

Many species ofPandanushave been used by Indonesian people for daily purposes, i.e. as the raw materials for mats, and other handicraft such as hat and bag, but not all species of Pandanus are suitable as raw materials for mat, the most suitable species are P. dubius, P. furcatus,and P. tectorius. All along the leaves border of the three species are thorny, but their texture are flexible, unbreakable (Purwanto 2007). Pandanus amaryllifolius is rare in the wild, but cultivated and widely used as flavouring in cooking (Ng & Yap 2003). Some species are cultivated as ornamentals, i.e. P. dubius, P. spurius cv. Putat, P. tectorius cv. Sanderi, andP. utilis, (Thomsonet al. 2006), whereas some species ofFreycinetiaare important plants in the Pacific as an emergency food and for the construction of fish traps (Brown 1931). In Indonesia, utilization of the Freycinetiais still minimum, but looking at its elegant figure, Freycinetiahas the potential as an ornamental plant (Purwanto 2007).

In view of its importance and considering the large number of wild species germplasm available for Pandanus andFreycinetia in Java, the genetic analysis through molecular marker is a prerequisite to have a deep insight of the genome organization in the wild species. Therefore it is imperative to establish strategies for the preservation of Pandanus and Freycinetia germplasm. This analysis is a preliminary step to ensure the conservation and the development of genetic resources.

In the past, genetic diversity in species has typically been assessed using morphological, physiological and biochemical traits. Since morphological and physiological traits are subject to environmental influences, emphasis has shifted to biochemical studies (Moodieet al. 1997). In particular, allozyme analysis has been used to document genetic diversity in a range of different species. However, allozymes may underestimate genetic diversity (Esselmanet al. 1999). Recently more sensitive DNA based-techniques have been developed to detect the genetic diversity in different plant groups. The commonly used polymerase chain reaction (PCR)-based DNA marker system are random amplified polymorphic

DNA (RAPD), amplified fragment length polymorphism (AFLP), and more recently simple sequence repeat (SSRs) or microsatelites (Staubet al. 1996). The major limitation of these methods are low reproducibility of RAPD, high cost of AFLP and the need to know the flanking sequence to develop species specific primers for SSR polymorphism. ISSR-PCR is a technique that overcome most of these limitations (Zietkiewiczet al. 1994).

Inter simple sequence repeats (ISSR) exhibit a few advantages over other markers. ISSR primer anneal to simple sequence repeats that are abundant through the eukaryotic genome and evolve rapidly, and hence may reveal a high level of polymorphism (Zietkiewiczet al.1994). In addition, ISSR may produce more reliable and reproducible bands than RAPD because of the higher annealing temperature and longer primer sequences (Wei et al. 2001), have proved their usefulness to detect genetic diversity in Ficusspecies (Rout & Asparajita 2009) andMorusspecies (Awasthiet al. 2004).

There is no information regarding the genetic diversity of Pandanus and Freycinetia from Java. Our objective was to obtain information based on ISSR marker ofPandanusandFreycinetiaspecies sampled.

Materials and Methods Plant materials

Totally 19 samples of species ofPandanusandFreycinetiawere collected from various places in Java. They consisted of 13 species of Pandanus and 6 species of Freycinetia. All samples were identified to species based on morphological characteristic following the method of Stone (1983b) (Table 5.1 and Table 5.2). One sample represented a species. Two or three leaves were collected from each species and stored in silica gel in the field. In the laboratory, samples were maintained at–5oC until DNA extraction could be performed.

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

DNA extraction

Genomic DNA of the silica gel dried leaf samples were extracted according to the protocol described by Doyle and Doyle (1987) with minor modification mainly aimed to minimize the presence of phenolic compounds. For each sample, 100 mg of leaf were ground to fine powder, followed by the addition of 1 ml preheated (65oC) extraction buffer constitute 3% (w/v) CTAB, 1.4 MnCl, 0.2% (v/v) β-mercaptoethanol, 20mM EDTA, 100mM Tris-HCl (pH 8.0) and 1% (w/v) PVP-40. The homogenous was incubated at 65oC for 30 min and extracted two times with a phenol chloroform: isoamyl alcohol (25:24:1) solution, and phenol was eliminated by chloroform: isoamyl alcohol (24: 1) solution. Then DNA was precipitated in cold isopropanol and treated with Rnase A (37oC) for 60 min. After electrophoresis with a standard DNA on 1% agarose gels, stained with ethidium bromide, DNA concentration were determined by comparison against the standard of DNA with known concentration. The DNA was suspended to a final concentration of 10 ng/µl in 0.5 x TE and stored at 4oC.

Table 5.1 Thirteen species ofPandanusof Java used in this study Sample Code. Species Location S1 S2 S3 S4 S5 S8 S13 S14 S21. S23 S24 S25 S26

Pandanus bantamensisKoord. Pandanus tectoriusvar.littoralis Pandanus bidurJungh.

Pandanus spinistigmaticusFagerlind Pandanus odoratissimusL.f

Pandanus pseudolaisWarb. Pandanus multifurcatusFagerlind Pandanus nitidusKurz

Pandanus amarillyfoliusRoxb Pandanus polycephalusLamk Pandanus kurziiMerr.

Pandanus scabrifoliusMartelli Pandanus dubiusSprengel

Jampang Kulon Ujung Genteng Ujung Kulon

Botanical Garden (BO) Ujung Kulon

Bodogol

Bogor Botanical (BO) Jampang Kulon Depok

Bogor Botanical (BO) Bogor Botanical (BO) Cibodas

Table 5.2 Six species ofFreycinetiaof Java used in this study Sample

Code.

Species Location

S6 S7 S12 S19 S20 S22

Freycinetia javanicaBlume Freycinetia angustifoliaBlume Freycinetia scandensGaud. Freycinetia insignisBlume Freycinetia sumatranaHemsl. Freycinetia imbricataBlume

Ujung Kulon Halimun Cibodas Cibodas

Jampang Kulon Bodogol

DNA amplification

Total of 12 ISSR primers (Fermentas GmbH - Germany) were used for screening the amplification of unambiguously visible and polymorphic ISSR bands. A final set of 6 ISSR primers (Table 5.3) which produced unambiguously visible and polymorphic bands across the 19 samples were chosen for further analysis.

PCR Conditions

PCR was performed in a total volume of 25 µl containing 1X reaction buffer, 50 ng genomic DNA, 3.0 mM of MgCl2, Taq polymerase (2.5 units), 0.4

mMdNTPs and 10 µM primer ISSRs were amplified using GeneAmp PCR System 2400 Perkin Elmer. The amplification was programmed for 1 cycle in 2 min at 94oC, and 35 cycles in 1 min at 94oC, 1 min at 53oC, and 1 min at 72oC, followed by a final extension in 10 min at 72oC. PCR products were run at 1% (w/v) agarose gel in 1X TAE buffer. Gels were run for 1 to 2 h at 90 V, with a 100 bp ladder (Promega – USA) as the size standard. Gels was stained with ethidium bromide (10 µg/ml) and visualized under UV light and photograph with a digital camera.

Data Analyses

The numerical taxonomy and Multivariate Analyses System (NTSYS-pc) were used in this study. The presence of band was scored from the photograph. Only clear and reproducible bands were considered. These bands were considered

a polymorphic when they were absent in some samples in frequency a greater than 1% (Jorde 1995). Change in band intensity was not considered as a polymorphism. Clear band were scored as present (1) or absent (0) at particular position or distance migrate on the gel. The data matrix of 1’s and 0’s has been prepared from the scorable bands and entered into the data analysis package (Amstronget al. 1994). The indices similarity was calculated across all possible pair wise comparisons of species following the method of Nei and Li (1979). Cluster analysis of the data was carried out using UPGMA (Sneath and Sokal 1973) method. The bootstrap values were calculated using WINBOOT (Yap and Nelson 1996). Principal component analysis (PCA) was performed in order to make more effective view of the clustering pattern of the taxa included in this study.

Results and Discussion Result

Primer Selection

Six primers were applied on thirteen species of Pandanusand six species of Freycinetia from Java. The results showed that different primers generated various numbers of fragments with different length of amplified products as shown in Table 5.3 and Table 5.4. The six primers amplified 50 band position in Pandanus, and 32 band position in Freycinetia. The number of amplification bands per primer ranged from 6 to10 inPandanusand from 2 to 8 inFreycinetia. The primer ISSR4 produced more bands in Pandanus and primer ISSR7 produced more bands in PandanusandFreycinetiathan the other primers (Table 5.3 and Table 5.4), probably because ISSR4 and ISSR7 are greater abundance in PandanusandFreycinetiagenome. The repeats (GA)n were most abundant in rice (Nagarajuet al. 2002) and date palm (Trifiet al. 2000) genome. This data might indicate that dinucleotide-based ISSR-PCR marker could provide potential markers in thePandanusandFreycinetiagenome. A representative amplification pattern obtained by using primer ISSR4 and ISSR7 are shown in Figure 5.1 and Figure 5.2 respectively.

M s1 s2 s5 s6 s7 s14 s15 s20 s22 s24

1500 1250 1000 750 500

250

ISSR4

Figure 5.1 ISSR profile ofPandanusspp and

Freycinetiaspp of Java using primer ISSR4.

M : Marker, S1:P. bantamensis,

S2:P. tectoriusvar.littoralis,; S5:P. odoratissimus S6:F. javanica; S7:F. angustifolia, S14:P. nitidus, S15:F. angustifolia, S20:F. sumatrana,

S22:F. imbricata, S24:P. kurzii,

Kb s3 s4 s8 s13 s14 s21 s23 s24 s25 s26

.

1000 750 500 250

ISSR7

Figure 5.2 ISSR profile of Pandanusspp and

Freycinetiaspp of Java using primer ISSR7.

M: Marker, S3:P. bidur; S4:P. spinistigmaticus S8:P. pseudolais, S13:P. multifurcatus

S14: P. nitidus, S21:P. amaryllifolius S23:P. polycephalus, S24:P. kurzii S25:P. scabrifoliusand S26: P. dubius

Table 5.3 ISSR primers sequence and amplified results on thirteen species of Pandanusof Java

Primer Primer sequence

(5’ – 3’)

Size (bp) No.of bands scored No. of polymorphic bands Percentage of polymorphism (%) ISSR2 ISSR3 ISSR4 ISSR5 ISSR6 ISSR7 (AC)8TT (AG)8T (AG)8AA (AG)8TA (AG)8TT (GA)9A

250 - 1000 250 - 1000 200 - 1200 200 - 700 250 - 1000 200 - 800

7 9 10 8 6 10 7 9 8 7 4 9 100 100 80 87.5 67 90

Total 50 44

Average 8.3 87.4

Table 5.4. ISSR primers sequence and amplified results on six species of Freycinetiaof Java

Primer Primer sequence

(5’ – 3’)

Size (bp) No. of bands scored No. of polymorphic bands Percentage of polymorphism (%) ISSR2 ISSR3 ISSR4 ISSR5 ISSR6 ISSR7 (AC)8TT (AG)8T (AG)8AA (AG)8TA (AG)8TT (GA)9A

300 - 750 250 - 1000 200 - 1000 500 - 700 250 - 500 200 - 750

5 8 6 4 2 7 5 7 4 4 2 5 100 87.5 67 100 100 71

Total 32 27

Average 5.3 87.5

ISSR Survey

A total of 50 ISSR fragments were generated by six primers from thirteen species of Pandanus, and 32 ISSR fragments were generated in six species of Freycinetia. The highest number of fragments in Pandanus was detected in Pandanus amaryllifolius (34), P. spinistigmaticus (29) and P. pseudolais (28) respectively, whereas the least number was found in P. multifurcatus containing six fragments. In other species the number of fragments ranged from 12 in P.bantamensis and P. tectorius var. littoralis to 22 in P. bidur, while within Freycinetia, the highest number of fragments was detected inF. javanica andF. imbricatacontaining 21 fragments respectively, while the least number was found inF. scandenscontaining 11 fragments. In other species, the number of fragment

ranged from 15 in F. angustifoliato 18 in F. sumatrana. The average number of fragments generated by all analyzed primer was 8.3 in Pandanus and 5.3 in Freycinetia.The choice of primers used in amplification is critical to demonstrate high polymorphism. The results showed that samples had different banding pattern. Primers had several characteristic that can affect the number and quality of DNA fragment amplified during PCR (Hassel and Gunnarsson 2003). Among all the species subjected in this research, no species specific band was detected.

Genetic similarity and species relationships

Genetic similarity can reflect the difference among species in certain extend. The average similarity index for Pandanus was 0.250 to 0.889. The similarity indices among species ofPandanusare represented in Table 5.5. High genetic similarity was observed between P. nitidus and P. scabrifolius (0.889), whileP.amaryllifoliuswere distantly related toP. multifurcatus(0.250). Genetic similarity in other species varied from 0.333 (P. bantamensis and P. multifurcatus) to 0.789 (P. kurziiandP.odoratissimus).

Table 5.5.Nei and Li’s similaritymatrix of thirteenPandanusspecies of Java by using ISSR primers

S1 S2 S3 S4 S5 S8 S13 S14 S21 S23 S24 S25 S26

S1 1.000 S2 0.750 1.000 S3 0.529 0.412 1.000 S4 0.488 0.488 0.745 1.000 S5 0.667 0.733 0.450 0.511 1.000 S8 0.550 0.500 0.480 0.702 0.565 1.000 S13 0.333 0.444 0.286 0.286 0.333 0.353 1.000 S14 0.600 0.733 0.350 0.426 0.778 0.565 0.333 1.000 S21 0.522 0.478 0.464 0.635 0.538 0.774 0.250 0.577 1.000 S23 0.563 0.563 0.714 0.694 0.526 0.625 0.462 0.526 0.519 1.000 S24 0.625 0.688 0.429 0.490 0.789 0.667 0.308 0.842 0.593 0.550 1.000 S25 0.667 0.733 0.350 0.468 0.833 0.565 0.333 0.889 0.577 0.526 0.842 1.000 S26 0.545 0.545 0.558 0.760 0.513 0.735 0.444 0.462 0.618 0.732 0.537 0.513 1.000

In Freycinetia, the average similarity index was 0.396 to 0.923. The similarity indices among species ofFreycinetiaare represented in Table 5.6. High

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