Anatomy and ultra-structures of leaf of Vatica L. (Dipterocarpaceae) in Peninsular Malaysia

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1 1 Anatomy and ultra-structures of leaf of Vatica L. (Dipterocarpaceae) in Peninsular Malaysia HOUSSEIN M. ALI EL-TAGURI & A. LATIFF Faculty of Science, Benghazi University, Benghazi, Libya. Faculty of Science & Technology, Universiti Kebangsaan Malaysia, Bangi,Selangor, Malaysia Abstract: anatomical leaf of 28 species of Vatica including: petiole outline, shape of vascular bundles, fibre distribution, sclereid number, latex canals number and distribution; midrib outlines, shape of vascular bundles, fibre distribution, sclereid number, latex canals number and distribution; leaf margin in cross-section outline; adaxial, abaxial epidermal cells size, the type of vascular bundle fibre and thickness of lamina; leaf surfaces cell wall pattern of adaxial, abaxial epiderm, stomata and trichomes; venation have done. Most of anatomical characters can be using to identification the specie and some are helpful in distinguishing some species. Key words : Vatica, Leaf anatomy, Dipterocarpaceae,Peninsular Malaysia INTRODUCTION The genus Vatica L. (Dipterocarpaceae) or locally known as resak is a small- to medium-sized trees and is among the important producers of semi-durable hard woods for house posts and other minor construction materials. In the world it is represented by about 67 species distributed from Sri Lanka, southern and eastern India, Myanmar, Indochina, southern China, Thailand and throughout Malesia. In Malaysia it is usually an understorey and main canopy trees. In the Malay Peninsula, Ridley (1922) had produced the earliest and most comprehensive account of the genus and later Symington (1943, revised 2004) had comprehensively treated the genus for the Malay Peninsula and he recognized a total of 23 species. He had also given ecological and geographical distribution maps. Ashton (1982) in his treatment of the Dipterocarpaceae for Flora Malesiana listed and described a total of 20 species of Vatica for Peninsular Malaysia, 11 in sect. Vatica and 9 in sect. Sunaptea. Recently, two species, namely V. sarawakensis Heim and V. pedicellata Brandis were reported to occur in Peninsular Malaysia by El-Taguri and Latiff (2010). Subsequently, two new species of Vatica from Peninsular Malaysia, V. spatulata (Sect. Sunaptea) andv. cuneata (Sect. Vatica) and two new records, namelyv. rassak (Korth.) Blume and V.acrocarpa Sloot. (Sect. Vatica) are reported for Peninsular Malaysia (El-Taguri & Latiff, 2012). Many species of the families are incorrectly or misidentified. Previously, phylogenetic relationships within the dipterocarps have been addressed using

2 2 morphological data, pollen analysis, fossil records and anatomy (Ashton, 1982). In particular, anatomical data has provided valuable taxonomic information at the subfamily, tribe, generic and species levels for different groups of plants (Stuessy, 1990), including the delimitation of species in the Dipterocarpaceae. Heim (1892) and other authors have used the petiole anatomy of the Dipterocarpaceae, and particularly the arrangement of the vascular bundles as seen in transverse section at the distal end as a guide to classification and species determination. Recently, Nor Nafizah (2006) has described many Vatica species useing anatomical characters such as leaf surface (epidermal cells, trichomes, venation), leaf transverse section (outline, epidermal cells, trichomes, resin canals and crystals) and petiole transverse section (outline, epidermal cells, trichomes, sclerenchyma, resin canals and crystals) but she has not used these characters to classify the genus. This study attempts to use leaf anatomical characters for the identification of Vatica species. MATERIALS AND METHODS Fresh and dried leaves were used their specimens in the herbaria KEP, SING, UKMB, KLU, UPMF and SFK (Herbarium, School of Forestry, Kepong, Malaysia).The fresh leaves were fixed in three parts of alcohol and one part of acetic acid. The dried leaves from herbarium collections were boiled in water for one hour or until the leaves sank to the bottom and later fixed in similar fixative. Polystyrene cork was used to support petioles and leaves before sectioned using Reichert sliding microtome. Sections were then bleached with Clorox, rinsed and stained in safranin and alcian blue. Subsequently, sections were dehydrated in alcohol in an ascending series and mounted on a slide using euparal (Johansen, 1940; Sass, 1951). The slides were dried in an oven at 60o for two weeks. The slides were viewed under Zeiss microscope which was attached to a computer with image analysis packages, high resolution colour printer 1400dpi, National video camera and adaptor. Magnification of 4 x 10 was used to better view the sections. For large sections, IP photo deluxe assistant image editor was used. For epidermal preparations, the adaxial surface of the leaf was scraped gently to remove the epidermal tissue. Later, the leaf of ca. 1 cm2 was soaked in Jeffrey s solution for an hour until a thin layer of the abaxial epidermis remained. The epidermis was rinsed and stained with alcian green. Then, the epidermis was dehydrated in alcohol in an ascending series, mounted on a slide using euparal and viewed under Zeiss light microscope. Scanning electron microscope (SEM) was used to observe the leaf epidermis and type of indumentum present in specimens. The dry specimens were cut into c. 1 mm2 piece. Two parts were required, close to midrib adaxial and abaxial. The pieces of fresh samples were dehydrated in an ascending series of alcohol of 30%,

3 3 50%, 75% and 95% for 30 minutes and 100% three times at one hour interval, followed by three times 100% acetone at 10 minutes intervals before CO2 critical point drying. Fully dried samples were later mounted on SEM stub with doublesided tape and coated with gold. Then the samples were examined on Philips XL30 scanning electron operated at 3 to 10 Kv to magnification requirement of 50 to The trichomes, stomata types and distribution patterns from the epidermis were identified, measured and photographed. Three readings of sizes and density of the stomata were taken and photographed. The pattern of distribution for stomata was observed under low magnification of 50. RESULTS AND DISCUSSION The results of petiole anatomy obtained in this study is discussed based on petiole outlines, shape of vascular bundles, fibre distribution, sclereid number, latex canals number and distribution; midrib outlines, shape of vascular bundles, fibre distribution, sclereid number, latex canals number and distribution; margin in cross-section outline; adaxial, abaxial epidermal cells size, the type of vascular bundle fibre and thickness of lamina; leaf surfaces includes the study of anticlinal cell wall pattern of adaxial, abaxial epiderm, stomata and trichomes; venation: a- Petiole outline in cross-section usually is not so different within the same genus for example Cotylelobium species have petiole outline oblong to oval laterally and Dryobalanops species have petiole outline adaxially strongly grooved or slightly grooved (Nor Nafizah 2006). In this study the petiole outline of Vatica is classified into six different types as follows: Type I = subcircular. Type II = oblong or ovate, laterally flat. Type III = oblate or ovate, dorsi-ventrally flat. Type IV = adaxial side more or less concave. Type V = adaxial side more or less V -shape groove. Type VI = more or less hexagonal. (Figure 1) Type I is found in many species (e.g. V. bella, V. cuspidata, V. hullettii, V. lowii, V. maingayi, V. nitens, V. pauciflora, V. rassak, V. scortechinii, V. stapfiana, V. umbonata, V. venulosa and V. yeechongii), several species have type II (e.g. V. cinerea, V. havilandii, V. heteroptera, V. mangachapoi, V. odorata, V. pallida, V. pedicellata, V. ridleyana and V. stapfiana), both V. sarawakensis and V. spatulata have type III, only V. lobata has type IV, both V. acrocarpa and V. cuneata have type V and both V. flavida and V. perakensis have type VI. The outline of petiole is useful character in identification some species for example we can identify V. perakensis by more or less hexagonal petiole outline. Also we can identify V.

4 4 lobata by oblong shape and adaxial side more or less concave. (Figure 1). Nor Nafizah (2006) and Noraini (2006) have used the outline of the petiole in their anatomical study of Dipterocarpaceae. They classified the species depending on the outline of the petiole in most genera by the same way in this study and then used this character in their keys to identification the species. a. b. c. d.

5 5 e. f. Figure 1 Type of petiole outline. a: Type I in V. pauciflora, b: Type II in V. ridleyana, c: Type III in V. spatulata, d: Type IV in V. lobata, e: Type V in V. cuneata and f: Type VI in V. perakensis. b- Petiole vascular bundle arrangement is one of the important anatomical characters which can be used to diagnose the genus, and to identify the species. In this study it is classified into six different types: Type I = outer continuous ring; inner continuous ring; centre continuous ring or continuous inverted arc or continuous arc. Type II = outer continuous ring or interrupted ring; inner continuous arc or continuous crescent or continuous inverted arc; centre continuous ring or continuous inverted arc or continuous arc. Type III = outer interrupted ring or interrupted crescent or continuous crescent; inner interrupted ring or continuous ring or continuous ring at top; centre continuous inverted arc or continuous arc. Type IV = outer interrupted ring; inner continuous ring; centre two continuous arcs in an upright position. Type V = outer interrupted crescent; inner continuous ring; centre many continuous inverted arcs in the distribution of random. Type VI = outer continuous ring; inner continuous crescent; centre two continuous rings in by side position. (Figure 2). Type I is observed in V. cuneata, V. cuspidata, V. hullettii, V. lobata, V. lowii, V. nitens, V. odorata and V. umbonata, type II in V. flavida, V. maingayi, V. pallida, V. perakensis, V. ridleyana, V. sarawakensis and V. spatulata, type III in V. acrocarpa, V. bella, V. cinerea, V. havilandii, V. heteroptera, V. pauciflora, V. scortechinii, V. stapfiana and V. venulosa, type IV in V. rassak and V. yeechongii, type V in V. pedicellata and type VI in V. mangachapoi. (Figure 2).

6 6 All of the species exhibit three groups of vascular bundles outer, inner and centre but the pattern may be further divided into six types based on interrupted or continuous, ring, crescent or arc shape and arrangement of bundles. The majority of outer vascular bundles occurred in Vatica were ring shape. The vascular bundle of petiole is useful character in identification some species for example we can identify V. pedicellata by outer interrupted crescent, inner continuous ring and centre many continuous inverted arcs in the distribution of random while V. mangachapoi by outer continuous ring, inner continuous crescent and centre two continuous rings in by side position and V. cuspidate by outer continuous ring, inner continuous ring centre continuous arc. (Figure 2). Nor Nafizah (2006) and Noraini (2006) have used the vascular bundle of petiole in their anatomical study of Dipterocarpaceae. They classified the species depending on the vascular bundle of petiole in most genera by the same way in this study. Only Noraini used this character in her key to identification the species. a b

7 7 c d e f Figure 2 Type of petiole vascular bundle arrangement. V. cuspidata (a), V. flavida (b), V. heteroptera (c), V. rassak (d), V. pedicellata (e) and V. mangachapoi (f) c- Study of Petiole Sclerenchyma showed that there are a few different arrangements of fibres in the petiole in this study as shown in Table 1. Most species with fibres interrupted around outer ring and scattered around inner ring. (Figures 1 and 2). Nor Nafizah (2006) described sclernchyma in Vatica petiole as scattered around vascular bundle complex almost completely ensheathing the vascular bundle complex while Srinual & Thammathaworn (2008) pointed out that the vascular bundles of Vatica are surrounded by sclerenchyma cells. In Vatica no study referred to sclereids before. Brachysclereid is present within the cortex of the petiole. The sclereid number is different in some species so it can be useful in identifying for example no sclereid in V. mangachapoi (Figures 2 f). Big number of

8 8 big size sclereid appeared in V. rassak (Figures 2 d). Different numbers of sclereid studied were showed in Table 1. Table 1 Sclerenchyma in petiole. Species Petiole fibres arrangement Sclereid No. in one petiole cross-section Mean sclereid No. in 1 mm² of petiole crosssection V. acrocarpa interrupted around outer ring and scattered around inner ring V. bella semi continuous around outer ring and scattered around inner ring V. cinerea semi continuous around outer ring and scattered around inner ring V. cuneata scattered around outer ring and scattered around inner ring V. cuspidata continuous around outer ring and interrupted around inner ring V. flavida interrupted around outer ring and interrupted around inner ring V. havilandii semi continuous around outer ring and scattered around inner ring ± ± ± ± ± ± ±14.3 V. heteroptera make caps at outer ring's bundles and interrupted around inner ring 0 0 V. hullettii semi continuous around outer ring and interrupted around inner ring V. lobata interrupted around outer ring and interrupted around inner ring V. lowii semi continuous around outer ring and semi continuous around ± ± ±17.9

9 9 inner ring V. maingayi continuous around outer ring and scattered around inner ring 5-60 ±13.0 V. mangachapoi scattered around outer ring and scattered around inner ring 8-12 ±7.8 V. nitens interrupted around outer ring, interrupted sheath around some outer ring's bundles and scattered around inner ring V. odorata interrupted around outer ring and scattered around inner ring V. pallida semi continuous around outer ring and scattered around inner ring V. pauciflora interrupted around outer ring and scattered around inner ring ± ± ± ±12.0 V. pedicellata V. perakensis interrupted sheath around some outer ring's bundles and scattered around inner ring interrupted around outer ring and scattered around inner ring ± ±13.0 V. rassak interrupted around outer ring and scattered around inner ring V. ridleyana interrupted around outer ring and scattered around inner ring ± ±6.8 V. sarawakensis V. scortechinii scattered around outer ring and scattered around inner ring scattered around outer ring and scattered around inner ring ± ±16.7 V. spatulata interrupted around outer ring and scattered around inner ring V. stapfiana make caps at outer ring's bundles and scattered around inner ring V. umbonata semi continuous around outer ring and interrupted around inner ring ± ± ±18.1

10 10 V. venulosa interrupted around outer ring and scattered around inner ring 1-3 ±0.7 V. yeechongii interrupted around outer ring and interrupted around inner ring ±2.6 d- The number and distribution of resin canals in petiole can be helpful to identify species e.g. V. rassak and V. yeechongii have canals in outer ring and 1-3 canals at centre of petiole while V. cinerea and V. mangachapoi have 3 canals in outer ring and 0-1 canals at centre of petiole. Nor Nafizah (2006) and Noraini (2006) have used the number and distribution of resin canals of the petiole in their anatomical study of Dipterocarpaceae. They used these characters in their keys to identification the species. Different number and distribution of resin canals studied were showed in Table 2, Figure 3. Table 2 Resin canals number in petiole. Species Number of resin canals in outer ring Number of resin canals at centre of petiole V. acrocarpa 5 1 V. bella V. cinerea V. cuneata 7 1 V. cuspidata V. flavida 8 0 V. havilandii 5 1 V. heteroptera V. hullettii 5 1 V. lobata 7 1 V. lowii 5 1 V. maingayi 5 1 V. mangachapoi 3 0

11 11 V. nitens 7 1 V. odorata V. pallida 3 2 V. pauciflora V. pedicellata 7 1 V. perakensis 7 0 V. rassak 11 1 V. ridleyana 5 1 V. sarawakensis V. scortechinii V. spatulata 7 1 V. stapfiana 7 1 V. umbonata V. venulosa V. yeechongii a) b) Figure 3 Number and distribution of resin canals in petiole. V. yeechongii (a), V. cinerea (b) (R = resin canals)

12 12 e- In this study the midrib outline of Vatica is classified into five different types: Type I = Midrib adaxial convex or moderately convex and midrib abaxial V -shaped or V -shaped to semi-circular. Type II = Midrib adaxial more or less prominent ridge and midrib abaxial three quarters of a circle to U -shaped, slightly narrowed toward the adaxial side. Type III = Midrib adaxial more or less prominent ridge and midrib abaxial U -shaped or semi-circular to U -shaped. Type IV = Midrib adaxial convex or moderately convex and midrib abaxial semicircular or semi-circular to U -shaped. Type V = Midrib adaxial slightly concave and midrib abaxial U -shaped. Figure 4. Vatica acrocarpa, V. pallida, and V. cinerea have type I, both V. nitens and V. yeechongii have type II, several species have type III (V. bella, V. havilandii, V. heteroptera, V. odorata, V. perakensis, V. sarawakensis, V. scortechinii, V. spatulata and V. stapfiana), many have type IV (e.g. V. cuneata, V. flavida, V. hullettii, V. lobata, V. lowii, V. mangachapoi, V. pauciflora, V. pedicellata, V. rassak, V. ridleyana, V. umbonata and V. venulosa) and only V. cuspidata has type V. The outline of midrib is useful character in identification some species e.g. V. cuspidata can be identified by midrib adaxial slightly concave and midrib abaxial U -shaped while V. nitens and V. yeechongii midrib adaxial more or less prominent ridge and midrib abaxial three quarters of a circle to U -shaped, slightly narrowed toward the adaxial side. (Figure 2). Nor Nafizah (2006) and Noraini (2006) have used the outline of the midrib in their anatomical study of Dipterocarpaceae. They classified the species depending on the outline of the midrib in most genera by the same way in this study and then used this character in their keys to identification of the species. a. b.

13 13 c. d. e. Figure 4 Type of midrib outline. a: Type I in V. acrocarpa, b: Type II in V. nitens, c: Type III in V. bella, d: Type IV in V. pauciflora and e: Type V in V. cuspidata f- The vascular bundle arrangement in midrib can be of help in identifing the species e.g. V. nitens (Figure 4 b), V. rassak (Figure 5 b), V. sarawakensis and V. yeechongii can be identifing by the outer vascular bundle arrangement in continuous ring and inner two continuous arcs in an upright position. In this study it classified into two different types: I = outer continuous or interrupted ring and inner continuous arc or fused with 2-3 arcs. II = outer continuous ring and inner two continuous arcs in an upright position. Type I is found in most species (e.g. V. acrocarpa Figure 4 a, V. bella, V. cinerea, V. cuneata, V. cuspidata, V. havilandii, V. heteroptera, V. hullettii, V. lobata, V. lowii, V. maingayi, V. mangachapoi, V. odorata, V. pallida, V. pauciflora Figure 4 d, V. pedicellata Figure 5 a, V. perakensis, V. ridleyana, V. scortechinii, V.

14 14 spatulata, V. stapfiana, V. umbonata and V. venulosa) and type II in some species (e.g. V. flavida, V. nitens, V. rassak Figure 5 b, V. sarawakensis and V. yeechongii). (Figure 4). Nor Nafizah (2006) and Noraini (2006) have used the vascular bundle of midrib in their anatomical study of Dipterocarpacea. They classified the species depending on the vascular bundle of midrib in most genera by the same way in this study. Only Nor Nafizah used this character in her key to identification the species. Srinual & Thammathaworn (2008) pointed out that the vascular bundles of Vatica exhibit a very complex structure with the distal end as a closed or very slightly open ring, surrounding a central medullary region. a. b. Figure 5 Type of midrib vascular arrangement. a: Type I in V. pedicellata and b: Type II in V. rassak (I = inner vascular bundles, O = outer vascular bundles, F = fibres, S = sclereids) g- The appearance of fibres arrangement in the midrib is not really different in this study as showen in Table 3, Figure 4 and 5. Brachysclereids is present within the parnchyma of midrib. The sclereid number is present in some species with a few number so it can be useful in identifying some species for example V. pedicellata (Figure 5 a), V. scortechinii, V. stapfiana and V. yeechongii can be identify by 20 sometimes 40 sclereid number in one cross-section of midrib. Different numbers of sclereid studied were showed in Table 3. Srinual & Thammathaworn (2008) pointed out that the vascular bundles of Vatica are surrounded by sclerenchyma cells. Sharma (1970) concluded that the differences in the sclereid features in stem and leaves are helpful in distinguishing Capparis and Crataeva (Capparidaceae). In Vatica no study referred to sclereids before. Table 3 Sclerenchyma in midrib. Species Midrib fibre Sclereid number in one cross-section of midrib

15 15 V. acrocarpa continuous around outer ring and V. bella continuous around outer ring and V. cinerea continuous around outer ring 0 V. cuneata continuous around outer ring and V. cuspidata continuous around outer ring and V. flavida continuous around outer ring and continuous under each of inner arcs V. havilandii continuous around outer ring and continuous under inner arc V. heteroptera continuous around outer ring and V. hullettii continuous around outer ring and V. lobata continuous around outer ring and V. lowii continuous around outer ring and V. maingayi continuous around outer ring and V. mangachapoi continuous around outer ring and V. nitens continuous around outer ring and continuous under each of inner arcs V. odorata continuous around outer ring and continuous under inner arc V. pallida continuous around outer ring and V. pauciflora continuous around outer ring and V. pedicellata continuous around outer ring and

16 16 V. perakensis continuous around outer ring and V. rassak continuous around outer ring and interrupted under each of inner arcs V. ridleyana continuous around outer ring and V. sarawakensis continuous around outer ring and continuous under each of inner arcs V. scortechinii continuous around outer ring and V. spatulata continuous around outer ring and V. stapfiana continuous around outer ring and V. umbonata continuous around outer ring and V. venulosa continuous around outer ring and V. yeechongii continuous around outer ring and continuous under each of inner arcs h- The number and distribution of resin canals in midrib can be helpful to identify species for example we can identify V. rassak, V. sarawakensis (Figure 6 a) and V. yeechongii by more than ten resin canals in the outer ring of midrib vascular bundle.while the others have less than ten resin canals in the outer ring of midrib vascular bundle for example V. spatulata (Figure 6 b). Nor Nafizah (2006) and Noraini (2006) have used the number and distribution of resin canals of the midrib in their anatomical study of Dipterocarpaceae. They used these characters in their keys to identification the species.the different number and distribution of resin canals studied were shown in Table 4.

17 17 a) b) Figure 6 The number and distribution of resin canals in midrib. a: more than ten in V. sarawakensis and b: less than ten in V. spatulata (R = resin canals) Table 4 Resin canals number in midrib. Species Resin canals in outer ring Resin canals at centre V. acrocarpa 5 1 V. bella V. cinerea 3 0 V. cuneata 5 0 V. cuspidata V. flavida 7 5 V. havilandii 3 1 V. heteroptera 5 0 V. hullettii 3 0 V. lobata V. lowii 3 1 V. maingayi V. mangachapoi 1 0 V. nitens V. odorata

18 18 V. pallida 3 0 V. pauciflora V. pedicellata 3 1 V. perakensis V. rassak V. ridleyana 3 1 V. sarawakensis V. scortechinii V. spatulata 5 1 V. stapfiana V. umbonata V. venulosa 3 1 V. yeechongii i- The margin in cross-section outline appeared obtuse in most species (e.g. V. acrocarpa, V. bella, V. cinerea, V. flavida, V. havilandii, V. heteroptera, V. lobata, V. lowii, V. maingayi, V. mangachapoi, V. nitens, V. odorata, V. pauciflora, V. pedicellata, V. perakensis, V. rassak, V. spatulata, V. stapfiana and V. venulosa), in some species it is obtuse to slightly acute (e.g. V. cuneata, V. cuspidata, V. ridleyana, V. sarawakensis, V. umbonata and V. yeechongii) and three species have acute shape (V. hullettii, V. pallida and V. scortechinii) (Figure 7). a.

19 19 b. c. Figure 7 Margin cross section outline. a: obtuse in V. mangachapoi, b: slightly acute in V. umbonata and c: acute in V. hullettii Many species have slightly curved margin (Figure 8 a) (V. acrocarpa, V. bella, V. cinerea, V. cuneata, V. cuspidata, V. flavida, V. havilandii, V. hullettii, V. lobata, V. lowii, V. maingayi, V. mangachapoi, V. odorata, V. pauciflora, V. rassak, V. scortechinii and V. umbonata), some species have curved margin (Figure 8 b) (e.g. V. nitens, V. pallida, V. pedicellata, V. ridleyana, V. stapfiana and V. yeechongii) and some straight margin (Figure 8 c) (e.g. V. heteroptera, V. perakensis, V. sarawakensis, V. spatulata and V. venulosa). Schlerenchyma is present at margin in all species.

20 20 a. b. c. Figure 8 Margin type. a: slightly curved margin in V. acrocarpa, b: curved margin in V. stapfiana and c: straight margin in V. sarawakensis j- The epidermal cell is one of the characters which can help to distinguish between species for example we can distinguish V. yeechongii by oblong adaxial epidermal cells the height about duple times the width, while most of Vatica species have oblate-quadrate adaxial epidermal cells the height about same to less than the width. The proportion of length to width in this case is useful for comparison (Cutler et al. 2008). In this study of the adaxial epidermal cells some species appeared with height more than width up to 1.9:1 in V. yeechongii (Figure 9 a) Some species the height of adaxial epidermal cells quite same as width for example V. maingayi (Figure 9 b), several species the height is less than width for example V. bella (Figure 9 c), while the abaxial epidermal cells is mostly height less than width, some species height quite the same as width. Nor Nafizah (2006) and Noraini (2006) have used the height and width of adaxial epidermal cells in their anatomical study of Dipterocarpaceae. They haven t used this character in their keys to identification of the species.the height and width of leaf epidermal cells are shown in Table 5.

21 21 a b c Figure 9 Adaxial epidermal cells a: oblong adaxial epidermal cells in V. yeechongii, height of adaxial epidermal cells quite same as width in V. maingayi and b: height is less than width in V. bella

22 22 Table 5 Leaf epidermal cells size and height and width rate. Species adaxial epidermal cells HxW µm adaxial epidermal cells H:W ratio abaxial epidermal cells HxW µm abaxial epidermal cells H:W ratio V. acrocarpa 13.6 x :1 7.4 x :1 V. bella 10.6 x : x :1 V. cinerea 25.8 x : x :1 V. cuneata 5.4 x :1 7.8 x :1 V. cuspidata 15.4 x : x :1 V. flavida 12.7 x :1 6.5x :1 V. havilandii 10.9 x :1 7.9 x :1 V. heteroptera 10.8 x :1 7.5 x :1 V. hullettii 5.3 x :1 5.3 x :1 V. lobata 9.5 x :1 6.8 x :1 V. lowii 6 x :1 6 x :1 V. maingayi 18.1 x : x :1 V. mangachapoi 16.1 x :1 8.1 x :1 V. nitens 10 x :1 7.8 x :1 V. odorata 12.5 x :1 8.8 x :1 V. pallida 10.5 x :1 6.6 x :1 V. pauciflora 11.4 x :1 9.5 x :1 V. pedicellata V. perakensis 15 x :1 8.3 x :1 7.2 x :1 7.2 x :1 V. rassak 8.6 x :1 7.5 x :1 V. ridleyana 16.5 x :1 9.6 x :1

23 23 V. sarawakensis V. scortechinii 7.4 x :1 8.1 x :1 10 x :1 7.3 x :1 V. spatulata 10.1 x :1 7.2 x 8 0.9:1 V. stapfiana 20.3 x :1 8.1 x :1 V. umbonata 9.9 x :1 9.2 x 9.2 1:1 V. venulosa 8.8 x :1 7 x :1 V. yeechongii 19.2 x :1 9 x :1 H = height and W = width k- The types of vascular bundle fibre in lamina are found on the upper side only (e.g. V. bella, V. hullettii, V. maingayi, V. nitens, V. perakensis and V. umbonata), on the upper and lower sides (e.g. V. cinerea, V. cuneata, V. cuspidata, V. flavida, V. havilandii, V. heteroptera, V. lobata, V. lowii, V. mangachapoi, V. odorata, V. pallida, V. pauciflora, V. pedicellata, V. rassak, V. ridleyana, V. spatulata, V. stapfiana, V. umbonata and V. venulosa) and without fibre (e.g. V. acrocarpa, V. sarawakensis, V. scortechinii and V. yeechongii). The type of vascular bundle fibre in lamina can be used to distinguish related species for example V. acrocarpa and V. umbonata as well as some others. Nor Nafizah (2006) pointed out that the bundle sheaths are secondary, tertiary and some smaller bundles with inner sclerenchyma sheath and outer sheaht. Inner sheath mostly completely ensheating vascular bundles or sometimes forming fibre caps at xylem and phloem pole as seen in V. mangachapoi. l- The thickness of lamina can be useful in identify species. In this study some species have less than 150 µm thick (e.g. V. pallida, V. odorata, V. perakensis, V. venulosa, V. havilandii, V. heteroptera and V. nitens), several species have 150 to 200 µm (e.g. V. mangachapoi, V. spatulata, V. bella, V. maingayi, V. cuneata, V. sarawakensis, V. rassak, V. lowii, V. lobata and V. cuspidata), some species have 201 to 250 µm (e.g. V. yeechongii, V. flavida, V. hullettii, V. scortechinii, V. pauciflora, V. umbonata, V. cinerea and V. acrocarpa) and a few species have more than 250 µm (e.g. V. ridleyana, V. pedicellata and V. stapfiana). m- The anticlinal cell wall pattern can be used to identify species. The anticlinal cell wall pattern of adaxial epiderm appeared straight (e.g. V. acrocarpa, V. cinerea, V. cuneata, V. flavida, V. heteroptera, V. hullettii, V. lobata, V. pallida, V. pedicellata, V. perakensis and V. spatulata), curved to undulate (e.g. V. bella,

24 24 V. maingayi, V. yeechongii, V. rassak, V. odorata, V. umbonata, V. sarawakensis and V. scortechinii) and straight to curved (e.g. V. cuspidata, V. havilandii, V. lowii, V. mangachapoi, V. nitens, V. pauciflora, V. ridleyana, V. stapfiana and V. venulosa). The anticlinal cell wall pattern of abaxial epiderm appeared straight (e.g. V. cinerea, V. cuneata, V. flavida and V. perakensis), curved to undulate (e.g. V. acrocarpa, V. lowii, V. maingayi, V. rassak, V. ridleyana, V. bella, V. heteroptera, V. lobata, V. mangachapoi, V. odorata, V. pallida, V. pauciflora, V. pedicellata, V. umbonata, V. venulosa, V. yeechongii, V. sarawakensis and V. scortechinii) and straight to curved (e.g. V. cuspidata, V. havilandii, V. hullettii, V. nitens, V. spatulata and V. stapfiana). (Figure 10) a. b. c. d. e. f. Figure 10 The anticlinal cell wall pattern of adaxial epiderm. a: straight in V. acrocarpa, b: curved to undulate in V. bella, c. straight to curved in V. havilandii; the anticlinal cell wall pattern of abaxial epiderm d:

25 25 straight in V. cinerea, e: curved to undulate in V. sarawakensis and f: straight to curved in V. spatulata n- Examination of the epidermal surface view showed that there were not much variations in stomata type. The stomata type is staurocytic with 4 to 6 subsidiary cells. (Figure 11). The size of stomata is from 14 µm to 31 µm (Table 6). The frequency of stomata is high when the size of stomata is small. It is from 7 stomata to 40 stomata in 0.4 mm 2. The stomata are present on abaxial epidermis only. (Table 6, Figure 11). Tewary and Sarkar (1985) studied the leaf epidermis of Dipterocarpaceae in Indian forests and recognized two stomatal types in Vatica: cyclocytic and tetracytic stomata. Srinual and Thammathaworn (2008) pointed that most Vatica species in Thailand have cyclocytic stomata, except V. bella Slooten, V. harmandiana Pierre and V. mangachapoi ssp. obtusifolius (Elmer) P.S. Ashton which present both cyclocytic and actinocytic stomata but they haven t study size and density. While Nor Nafizah (2006) pointed out that Vatica stomata are staurocytic with 4-7 subsidiary cells also she hasn t studied size and density. The size and density of stomata in Vatica species not so different. Only two species V. maingayi and V. stapfiana have stomata bigger than 22 µm. V. pedicellata has the much density of stomata that has stomata in 0.4 mm 2 while other species less than 30 stomata in 0.4 mm 2. Petronela & Nevena (2010) have used stomata density to distinguish Sesleria heufleriana from Sesleria uliginosa that stated it is more numerous in S. heufleriana. a. b.

26 26 c. d. e. f. Figure 11 Staurocytic stomata with 4 to 6 cells. a: V. bella and b. V. scortechinii; stomata size c: small 16 to 18 µm in V. heteroptera and d: big 25 to 31 µm in V. stapfiana; stomata frequency in 0.37 mm 2 e: V. havilandii and f: V. pedicellata Table 6 Stomata size and frequency. Species Stomata size in µm number of stomata in 0.4 mm 2 V. acrocarpa V. bella V. cinerea V. cuneata V. cuspidata V. flavida V. havilandii V. heteroptera

27 27 V. hullettii V. lobata V. lowii V. maingayi V. mangachapoi V. nitens V. odorata V. pallida V. pauciflora V. pedicellata V. perakensis V. rassak V. ridleyana V. sarawakensis V. scortechinii V. spatulata V. stapfiana V. umbonata V. venulosa V. yeechongii o- There are two main types of trichomes, glandular and non glandular trichomes. The glandular trichomes usually are present on abaxial epidermis, consist of a single cell representing a foot and eight cells that make oblate head (Figure 12).

28 28 a. b. c. d. Figure 12 Glandular trichomes on abaxial epidermis. a: lamina cross section of V. umbonata (LM), b: surface view of V. cuspidata (LM), c and d: surface view of V. scortechinii (SEM) The non-glandular trichomes are mostly stellate, sometimes simple or with two to five arms and rarely dendritic (Figure 13). These trichomes can be found on adaxial and abaxial lamina and midrib epidermis as well as petioles. Table 7 shows size and distribution of different shape of these trichomes. The trichome characters are useful on identify species for example big stellate trichomes more than 300 µm can be found only on leaves of V. stapfiana, while V. cinerea and V. lowii don t have trichomes. Nor Nafizah (2006) pointed out that Vatica trichomes are simple, dendritic and glandular.

29 29 a. b. c. d. Figure 13 Non glandular trichomes on abaxial epidermis. a: stellate of V. umbonata (SEM), b and c: simple or with two to five arms of V. pallida (SEM), d: dendritic of V. hullettii (LM) Table 7 Size and distribution of trichomes. Species Petiole trichomes in µm Midrib and lamina trichomes in µm G. ST. S. D. G. ST. S. T. ST.1 V. acrocarpa V. bella V. cinerea V. cuneata V. cuspidata V. flavida

30 30 V. havilandii V. heteroptera V. hullettii V. lobata V. lowii V. maingayi V. mangachapoi V. nitens V. odorata V. pallida V. pauciflora V. pedicellata V. perakensis V. rassak V. ridleyana V. sarawakensis V. scortechinii V. spatulata V. stapfiana V. umbonata

31 V. venulosa V. yeechongii G. = glandular trichomes, ST. = stellate trichomes, S. = simple trichomes, D. = dendritic trichomes, T. = two to five arms trichomes and ST.1 = stellate trichomes with one arm long p- The examination of the venation showed that there is not much variation in marginal ultimate venation types as well as areolation shape and veinlets. The marginal ultimate venation types are looped in all species. The areolation shapes are mostly quadranglar to polygonal some species appeared polygonal (e.g. V. acrocarpa, V. cinerea and V. cuspidata). The veinlets are simple curved and branched once or twice in many species (e.g. V. acrocarpa, V. bella, V. cuneata, V. flavida, V. havilandii, V. heteroptera, V. hullettii, V. lobata, V. lowii, V. pallida, V. pedicellata, V. rassak, V. ridleyana, V. sarawakensis, V. scortechinii and V. spatulata) and several species have 3-branched (e.g. V. cinerea, V. cuspidata, V. maingayi, V. mangachapoi, V. nitens, V. odorata, V. pauciflora, V. perakensis, V. stapfiana, V. umbonata, V. venulosa and V. yeechongii). Figure 14 show venation in two species. Nor Nafizah (2006) pointed out that Vatica venation are same in all species that without a maginal vein. Primary veins joining together towards the leaf tips sometimes with loops and there is space between them and margin. a. b. Figure 14 Venation a: V. bella and b: V. perakensis ACKNOWLEDGMENTS

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