SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS IN PARASHOREA (DIPTEROCARPACEAE) OF MALAYSIA

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1 Malays. Appl. SYSTEMATIC Biol. (2007) 36(2): SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS 47 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS IN PARASHOREA (DIPTEROCARPACEAE) OF MALAYSIA NORAINI TALIP 1 * and DAVID CUTLER 2 1 School of Environmental and Natural Resource Sciences Faculty of Science and Technology, Universiti Kebangsaan Malaysia Bangi, Selangor, Malaysia 2 Jodrell Laboratory, Royal Botanic Garderns, Kew, Richmond Surrey TW9 AB, ntalip@ukm.my ABSTRACT The petiole and midrib anatomy of genus Parashorea from Malaysia were investigated. In this study eight species representative of genus Parashorea were selected. Results from this study showed that the midrib outline can be used as a diagnostic character for P. tomentella. The presence of glandular and simple unicelullar hairs is proved to be characteristic for Parashorea species. The presence of resin canals adjacent to central vascular bundles in the petioles and midribs can be used to distinguish species such as P. densiflora and P. smythiesii. Results from this study have also shown that the number of resin canals adjacent to the adaxial side of the peripheral vascular bundles in the petiole and midrib and those related to the central vascular bundles in the petiole and midrib has taxonomic value for each species of this genus. ABSTRAK Kajian anatomi petiol dan tulang daun telah dijalankan ke atas genus Parashorea di Malaysia. Dalam kajian ini lapan spesies yang mewakili genus Parashorea telah dipilih. Hasil kajian menunjukkan bentuk garis luar tulang daun boleh digunakan sebagai ciri diagnosis P. tomentella. Kehadiran trikom ringkas unisel dan trikom berkelenjar adalah ciri untuk spesies dalam genus Parashorea. Kehadiran saluran rembes pada bahagian berkas vaskular petiol dan tulang daun boleh digunakan untuk membezakan antara spesies tertentu seperti P. densiflora dan P. smythiesii. Hasil kajian juga menunjukkan bilangan saluran rembes yang hadir pada bahagian adaksial berkas vaskular luar dan berkas vaskular tengah petiol dan tulang daun mempunyai nilai taksonomi bagi setiap spesies yang dikaji dalam genus ini. Keywords: Dipterocarpaceae, Parashorea, anatomy, micromorphology INTRODUCTION The Dipterocarpaceae is a well-known plant family with approximately species (Ashton, 1982; Maury-Lechon and Curtet, 1998; Ashton, 1998). The family is placed in Malvales (APG, 1998; APG II, 2003), and generally regarded as allied to some families of the order. The Asiatic dipterocarps are divided into two tribes, Dipterocarpeae and Shoreae. Dipterocarpeae has valvate fruit calyx lobes, scattered * To whom correspondence should be addressed. resin canals, and a basal chromosome number of x = 11. Members of this tribe are Vateria, Vateriopsis, Stemonoporus, Vatica, Cotylelobium, Upuna, Anisoptera and Dipterocarpus. The other is tribe Shoreae which has imbricate fruit calyx lobes, resin canals in tangential bands and a basal chromosome number of x = 7. The members of this tribe are Dryobalanops, Parashorea, Hopea, Neobalanocarpus and Shorea. Most species of Hopea, Shorea, Parashorea and Neobalanocarpus are of economic value either for their timber products, non-timber products or regarded as a part of dynamic biodiversity and ecosystem in the

2 48 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS tropical rain forest. Parashorea Kurz. is a small but widely distributed genus which occurs from Myanmar, Southern China, through the Malay Peninsula, Sumatra and Borneo and to the Philippines. It consists of about 13 species and three species are restricted to Peninsular Malaysia which are P. densiflora Sloot et Sym., P. globosa Sym. and P. stellata Kurz. As with species in many other angiosperm plant families, species of Dipterocarpaceae are fairly difficult to identify in the absence of fruits or flowers. Flowering in this family is generally considered to be inconsistent, at irregular intervals of 2 to 10 years (Burgess, 1972) with a few species have specific flowering patterns (Sasaki et al,. 1979; Sakai, 2002). Field characters such as bole, buttress, bark and cut (blaze) also have their limitations in that they are more variable within the species, and less reliable for specific identification, than are the characters of flowers and fruits (Rath et al., 1998). Moreover, most forest characters vary with the age of the tree and sometimes with the habitat (Symington, 1974). Problems in identifying Dipterocarpaceae species may result in the wrong identification of sterile material such as in herbarium specimens, hence there is a great need for the use of anatomical evidence in addition to leaf morphological characteristics for species identification. MATERIALS AND METHODS s used for this work were obtained from the arboretum at the Forest Research Institute, Kepong, Malaysia and the Pasoh Forest Reserve, Negeri Sembilan, Malaysia. Herbarium specimens were obtained from the Herbarium, Royal Botanic Gardens, Kew, Richmond, United Kingdom. Eight species with several replicates of Malaysian Parashorea were used in this study. Voucher specimens are kept at Royal Botanic Gardens, Kew, and Universiti Kebangsaan Malaysia Herbarium. A full list of species and the localities from which they were collected is given in Appendix 1. Fixation, embedding and sectioning follow the method described by Johansen (1940) and Sass (1958) with suitable modifications. Fresh material was fixed in A:A (Acid acetic: Alcohol). While dried herbarium material was boiled followed by fixation. Sections from the middle parts of the petioles and midribs were obtained by sectioning using a sliding microtome, stained in safranin and alcian green, dehydrated then mounted in Euparal. Photographs of sections were taken using Leitz Diaphlan polarizing microscope or Reichert Polyvar 2 Microscope and a Olympus digital camera. Images were processed using Analysis Docu Software (soft-imaging system) and Adobe Photoshop software. All the slides were deposited in the anatomy section at Jodrell Laboratory, Royal Botanic Gardens, Kew, United Kingdom and Microtechnique Laboratory, Universiti Kebangsaan Malaysia, Malaysia. RESULTS Midrib transverse sections Outline: adaxial slightly concave, abaxial prominent U-shaped or slightly rounded (Fig. 1 a - h) except in P. tomentella (Fig. 1 h), where adaxial slightly convex, abaxial U-shaped. Trichomes: Simple, tufted (Fig. 1 j), glandular. Vascular bundles: upright semicircular abaxially or outline incomplete circle, inner circle or ring present; sclerenchyma between phloem in the central vascular bundles (Fig. 1 i). Sclerenchyma: completely ensheathing vascular bundles. Resin canals: 2 13 adjacent to the adaxial side of peripheral vascular bundles, most of species has 5 8 resin canals, 1-8 canals related to central vascular bundles. Petiole transverse section Outline: adaxial slightly concave to straight, abaxial surface rounded, circle or U-shape in P. densiflora (Fig. 2 a), P. malaanonan (Fig. 2 d), P. parvifolia (Fig. 2 e) and P. tomentella (Fig. 2 h); adaxial flat and abaxial three quarter of circle or U-shape in P. globosa (Fig. 2 b), P. lucida (Fig. 2 c), P. smythiesii (Fig. 2 f) and P. stellata (Fig. 2 g). Epidermal cells: ± isodiametric. Trichomes: Simple, tufted (Fig. 2 i), glandular. Vascular bundles: opened vascular bundles abaxially; complete ring vascular bundles in the medullary. Sclerenchyma: sparsely scattered fibres around peripheral vascular bundles (Fig. 2 j) and present as phloem sclerenchyma between phloem in central vascular bundles. Resin canals: 5 11 adjacent to the adaxial side of peripheral vascular bundles and 1 7 related to central vascular bundles. Cell inclusions: druses densely scattered in parenchyma cells. Outer parenchyma: c layers of parenchyma cells. DISCUSSION AND TAXONOMIC IMPLICATIONS Trichomes The occurrence of trichomes in the species studied in the midrib and petiole is summarized in Table 1. The indumentum in Dipterocarpaceae is reported to consist of simple unicellular hairs,

3 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS 49 a b c d e f g h Tufted trichome Phloem sclerenchyma i j Fig. 1. Transverse sections of midribs of Parashorea species. a) P. densiflora, b) P. globosa, c) P. lucida, d) P. malaanonan, e) P. parvifolia, f) P. smythiesii, g) P. stellata, h) P. tomentella, i) Phloem sclerenchyma in the midrib vascular bundles, j) Tufted trichome. Scale bar: 200 μm.

4 50 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS a b c d e f g h Tufted trichome i j Scattered fibres Fig. 2. Transverse sections of petioles of Parashorea species. a) P. densiflora, b) P. globosa, c) P. lucida, d) P. malaanonan, e) P. parvifolia, f) P. smythiesii, g) P. stellata, h) P. tomentella, i) Tufted trichome, j) Scattered fibres in the petiole vascular bundles. Scale bar: 500 μm

5 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS 51 Table 1: The occurrence of trichomes in the midrib and petiole Midrib Petiole Species Simple Compound Simple Compound Unicellular Lobed Tufted Glandular Unicellular Lobes Tufted Glandular (lobes) (arms) (cells) (lobes) P. densiflora + _ c _ + + P. globosa + _ c _ + + P. lucida + _ c _ + + P. malaanonan + _ c _ + + P. parvifolia + _ c _ + + P. smythiesii + _ c _ + + P. stellata + _ c _ + + P. tomentella + _ c _ + + Table 2: The occurrence of resin canals in the midrib and petiole Vascular bundles in petiole Vascular bundles in midrib Species Adjacent to Adjacent to adaxial Adjacent to Adjacent to adaxial central vascular side of peripheral central vascular side of peripheral bundles vascular bundles bundles vascular bundles P. densiflora 2(5)5 7(9)9 1(1)2 6(6)6 P. globosa 2(2)3 8(8)8 2(2)3 7(7)7 P. lucida 0(0)0 5(5)5 0(0)0 5(5)5 P. malaanonan 0(0)0 6(8)8 0(0)0 6(6)6 P. parvifolia 0(0)0 5(5)5 0(0)0 3(3)5 P. smythiesii 2(3)3 5(7)7 2(2)2 5(5)7 P. stellata 0(0)0 7(7)7 0(0)0 5(5)5 P. tomentella 0(0)0 13(13)13 6(6)6 7(7)7 Note: The number of resin canals present adjacent to peripheral and central vascular bundles, measured by taking the minimum, maximum and common number is written as follows: minimum number (in brackets is the most common number) maximum number. For instance 3(4)6, 3 is the minimum number of resin canals, 4 is the most common number of resin canals present, 6 is the maximum number of resin canals present. +: present, : absent tufted hairs and various glandular hairs (Solereder, 1908; Metcalfe and Chalk, 1950). However there is no specific information on the distribution of hair type within species or genera. In this present study glandular hairs with 2 16 cells were found to be present in all Parashorea species indicating that this trichome type is common to species belonging to the Parashorea. As noted by Fritsch (1903), the constancy of a certain type of hair throughout an entire order is believed to be inherited from its ancestors and the presence and structure of glandular hairs characterizes families. Dickison (2000) in another statement stated that the occurrence of glandular hairs, especially with knob-like secretory apical swelling, is particularly important taxonomically. Therefore in this case the presence of glandular hairs is proven to be characteristic for the Parashorea species and definitely of taxonomic value. Simple unicellular hairs are common and present in all species of Parashorea. This hair type can be long or short, and has thick walls and narrow lumina or less thickened walls and wider lumina. Tufted hairs are present in almost all Parashorea species (Fig. 1 j and Fig. 2 i) except in P. lucida and P. parvifolia. The presence of tufted or tufted hairs has been reported by Solereder (1908) and Metcalfe and Chalk (1950). Resin canals A summary of the occurrence of resin canals adjacent to the vascular bundles in the petiole and midrib is presented in Table 2. The most outstanding anatomical characteristic is the occurrence throughout the genus of a branched system of resin canals which are usually lined with

6 52 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS a delicate epithelium (Metcalfe and Chalk, 1950; Solereder, 1908). Metcalfe and Chalk (1950) suggested their size, number and distribution show a considerable range in different genera and species, and are thus of taxonomic value. Resin canals occur in all the species studied, adjacent to the adaxial side of peripheral vascular bundles in the petiole, midrib and as well as in primary vascular bundles in the lamina and can be used as a common character for species belonging to Parashorea. Some of the species studied do not have resin canals adjacent to the central vascular bundles either in the petiole or midrib, namely P. lucida, P. malaanonan, P. parvifolia and P. stellata. Some species studied have resin canals adjacent to the adaxial side of the peripheral as well as to the central vascular bundles such as in P. densiflora, P. globosa and P. smythiesii. The presence of resin canals adjacent to the central vascular bundles in the petioles and midribs therefore, are diagnostic for individual species. Results in this present study have also shown an amazingly unexpected constant number of resin canals adjacent to the adaxial side of peripheral vascular bundles in the petiole and midrib or related to the central vascular bundles such as in P. globosa, P. lucida, P. malaanonan, P. stellata and P. tomentella. Therefore the number of resin canals could be of taxonomic value for each species of this genus. However more taxa and specimens need to be examined to confirm the consistency of number of resin canals present in Parashorea. Midrib transverse sections Midrib transverse sections are not as commonly used as petiole transverse sections in anatomical studies but this character could be of taxonomic value and very useful for differentiating species (Noraini et al., 2005). In the Dipterocarpaceae there are no previous reports on midrib anatomy. According to Metcalfe and Chalk (1950), the midrib vascular structure is always complex and exhibits a range of structures which might prove to be of considerable diagnostic value. Members of the Dipterocarpaceae can be differentiated from those of others plant families by the complex structure of the vascular bundles in the midrib. In this study, the midrib vascular systems are classified using two main types of components. The main vascular strand is closed with bundles arranged in a circular or lenticular configuration, either in as groups or in one continuous ring. The second component is the central vascular bundles, an open system, either joined or grouped in a U or V shape. The vascular bundles systems are complex and may incorporate one or both of these components. There are also a number of resin canals accompanying the vascular strands. The number, configuration and distribution of the central vascular bundles in the central vascular bundles, varies between species but does not provide much information that can be useful in classification. Sclerenchyma cells either completely or incompletely ensheathing the vascular bundles and phloem sclerenchyma in the midrib are common in all species belonging to the Parashorea studied. Dickison (2000) noted that the abundance and distributional patterns of phloem sclerenchyma or fibres are characteristic for some families or even for lower taxa. All the species examined have sclerenchyma cells completely ensheathing the vascular bundles in the. In addition to this all species are characterized by the presence of phloem sclerenchyma in the midrib medullary vascular bundles. The outline of the midribs in transverse sections does not provide much support for classification. This character shows a potentially close interrelationship between species in Parashorea. Petiole transverse sections According to Solereder (1908) a closed ring of vascular bundles enclosing a medullary vascular bundle system is always present in the petiole. This present study has shown it is not necessary to have a closed ring of vascular bundles in the petiole, in fact there are many species which have no closed ring of vascular bundles. The petiolar vascular structure is always complex and exhibits a range of structure which might prove to be of considerable diagnostic value. Solereder (1908), Metcalfe and Chalk (1950), Ashton (1982), Rojo (1987) and Munawirah et al. (1991) also reported that the members of the Dipterocarpaceae can be differentiated from others families by the complex characteristics of the vascular bundles in the petiole. The petiole vascular systems is similar to that of the midrib by having two main components, that is, an outer main strand consisting of a closed ring of bundles arranged in a circular or lenticular configuration, either in groups of bundles or in one continuous ring, and a second type in the medullary region, with an open system of vascular bundles joined or grouped together in a U or V shape. This system is complex and may incorporate one or both of these components. There are also a number of resin canals accompanying the vascular strands. Although the number, configuration and distribution of central vascular bundles in the medullary region, varies within species, they do not provide much information that can be useful in classification or identification. The difficulty of

7 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS 53 selecting comparable positions along the petiole for sectioning may contribute to this problem. Rojo (1987) reported that three Philippine Shorea species can be identified with certainty using petiole in conjuction with other anatomical features. According to Rudall (1992), the presence of sclereids and and fibres in the leaves of vascular plants is often with taxonomic applications. Sclerenchyma cells or fibres scattered around peripheral vascular bundles in the petiole were found in all species investigated and also can be a very good indicator for Parashorea species (Fig. 2 j). All species examined also have phloem sclerenchyma in the vascular bundle system in the petiole. It is suggested that these two anatomical characters are useful as diagnostic characters for the Parashorea. Petiole transverse sections have shown very little interspecific variation but give much support for identification of particular species and could be useful in classification. The petiole outline in this study is being classified based on its abaxial and adaxial shape. Despite the variability in vascular structure, the combination of petiole outline with type of vascular bundle arrangement could be very useful in the classification of Parashorea. Cells inclusions The presence of mucilaginous cells was reported by Solereder (1908) and Metcalfe and Chalk (1950). According to Metcalfe and Chalk (1950), mucilage cells are sometimes found in rows situated in the ground tissue of the petiole and midrib in certain species of Hopea and Shorea and in the lateral veins of Neobalanocarpus heimii. However no mention is made for Parashorea. Mucilaginous idioblasts occur in all species examined and vary from being very sparsely scattered to very dense, and grouped in the ground tissue of the petiole and midrib. They are most frequently recorded in the ground tissue of the midrib and petiole, and in the upper leaf epidermis and more rarely in the lower epidermis or mesophyll (chlorenchyma or bundle sheath), and this corresponds with Gregory and Baas (1989) on the distribution on mucilage cells within the plant. As a conclusion, results of the study reveal a number of interesting features with some characters which could be of taxonomic and diagnostic value. ACKNOWLEDGEMENTS We wish to thank Dr. Khatijah Haji Hussin for her comments on the manuscript. Deepest thanks to Miss Tivvy Harvey for her guidance in the herbarium of Royal Botanic Gardens, Kew,. Special thanks to Dr. Richard Chung and Abu Hussin for their technical help in the field and at the FRIM Herbarium, Kepong, Malaysia. REFERENCES APG (Angiosperm Phylogeny Group) An ordinal classification for the families of flowering plants. Annals of Missouri Botanical Garden, 85: APG II (An update of the angiosperm phylogeny group classification for the orders and families of flowering plants) The angiosperm phylogeny group. Botanical Journal of the Linnean Society, 141: Ashton, P.S Dipterocarpaceae. Series 1 Spermatophyta. In: Van Steenis, C. G. G. J. (Ed.) Flora Malesiana, 1(9): Ashton, P.S Dipterocarpaceae. In: Kubitzki, K. and Bayer, C. (Eds.). The Families and Genera of Vascular Plants. Springer-Verlag, Berlin. pp Burgess, P.F Studies on the regeneration of the hill forests of the Malay Peninsula. Malayan Forester, 35: Dickison, W.C Integrative Plant Anatomy. Harcourt Academic Press, San Diego. 530 pp. Fritsch, F.C The use of anatomical characters for systematic purposes. New Phytologist, 2: Gregory, M. and Baas, P A survey of mucilage cells in vegetative organs of the dicotyledons. Israel Journal of Botany, 38: Johansen, D.A Plant Microtechnique. McGraw-Hill, New York and London. 97 pp. Khatijah, H., Halijah, I., Dayangku, N.H.A., Gen, W.Q. and Ping, L.J. 2000a. Anatomical variations in leaves of Boesenbergia O. Kuntz. and Kaempferia, L. species (Zingiberaceae). Journal of Tropical and Subtropical Botany, 9(1): Khatijah. H., Chua, T.S., Halijah, I., Wu, Q.G., Liao, J.P. and Liu N. 2000b. Comparative leaf anatomy of Alpinia Roxb. species (Zingiberaceae) from China. Botanical Journal of the Linnean Society, 133(20): Maury-Lechon, G. and Curtet, L Biogeography and evolutionary systematics of Dipterocarpaceae. In: Appanah, S. and Turnbull, J.M. (eds.), A Review of Dipterocarps: Taxonomy, Ecology and Silviculture. Center for International Forestry Research, Bogor, Indonesia.

8 54 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS Metcalfe, C.R. and Chalk, L Anatomy of the Dicotyledons: Leaves, Stem and Wood in Relation to Taxonomy with Notes on Economic Uses. Vol. I. The Clarendon Press, Oxford. 276 pp. Munawirah, P., Khatijah, H. and Latiff, A Anatomi petiol beberapa spesies daripada famili Dipterocarpaceae Malaysia. Prioriti Penyelidikan IRPA, pp. Noraini, T., Khatijah, H. and Halijah, H Comparative leaf anatomy of Alpinia species (Zingiberaceae). Nordic Journal of Botany, 23: Rath, P., Rajasegar, G., Chong, J.G. and Kumar, P.P Phylogenetic analysis of Dipterocarps using random amplified polymorphic DNA markers. Annals of Botany, 82: Rojo, J Petiole anatomy and infrageneric interspecific relationship of Philippine Shorea (Dipterocarpaceae). In: Kostermans, A. J. G. H. (ed.), Proceedings of the Third Round Table Conference on Dipterocarps, Biotrop, Bogor, Indonesia. Rudall, P Anatomy of Flowering Plants: an Introduction to Structure and Development. 2nd ed. Cambridge University Press, Cambridge. 60 pp. Saas, J.E Botanical Microtechnique. 3 rd ed. Iowa State University, Ames. 78 pp. Sakai, S General flowering in lowland mixed dipterocarps. Biological Journal of the Linnean Society, 75(2): Sasaki, S., Tan, C.H. and Zulfatah, H.A.R Some observations on unusual flowering and fruiting of dipterocarps. The Malaysian Forester, 42(1): Solereder, H Systematic Anatomy of the Dicotyledons: A Handbook for Laboratories of Pure and Applied Botany. Volume I and II. The Clarendon Press, Oxford. Symington, C.F Foresters Manual of Dipterocarps. Malayan Forest Records, no nd ed. Penerbit University Malaya, Kuala Lumpur.

9 SYSTEMATIC SIGNIFICANCE OF PETIOLE AND MIDRIB ANATOMICAL CHARACTERS 55 Appendix 1: Specimens examined, code, locality, source, collectors and year of collection No Species Code Locality Source Collectors and date of collection 1. P. densiflora v. Sloot. & Symington NN18 Pasoh Forest Reserve, Negeri SembilanPasoh NN24 Forest Reserve, Negeri Sembilan NN54 FRIM Kepong Arboretum, Selangor FMS32387 Kali Forest Reserve, Mersing, Johore. Symington P. globosa Symington Kledang Sayong, Chepun Valley, Perak Symington April 1931 BB32754 Panngabean, Sumatera, Indonesia Unknown P. lucida Kurz SAN95283 Belurun, Sabah Amin, Gambio P. malaanonan Merr St. Lucia, Pinayas, Tawau, Sabah Unknown Bengkoa Forest Reserve, Sabah Sheat, Minjulu K50456 Pangi Tenom, Sandakan, Sabah Sadaui NN47 FRIM Kepong Arboretum, Selangor P. parvifolia Wyatt-Sm. ex P. S. Ashton Mount Templer Forest Reserve, Sabah Ulu Limbang, Bt. Antu, Ulu Sebayang, Sarawak G. Shea, P. S. Ashton WKM1614 Lamunin, Tutong, Brunei Rosli, Hj. Jili P. smythiesii Wyatt-Sm. ex. P. S. Ashton BRUN3169 BRUN472 Bt. Biang, Brunei Unknown P. S. Ashton P. stellata Kurz 5611 Larut, Perak Slooten Larut, Perak King 1885 NN55 FRIM Kepong Arboretum, Selangor P. tomentella (Symington) Meijer SAN21532 Sepilok Forest Reserve, Balau Compt. Sandakan, Sabah Meijer SAN96625 Kg. Kapuakan, Labuk, Sugut, Sabah Aban, Dewol Key to table: P = Parashorea, RBG = Royal Botanic Garden. FRIM = Forest Research Institute of Malaysia