Reinwardtia a Journal on Taxonomic Botany, Plant Sociology and Ecology

REINWARDTIA A JOURNAL ON TAXONOMIC BOTANY, PLANT SOCIOLOGY AND ECOLOGY

ISSN 0034 – 365 X | E-ISSN 2337 − 8824 | Accredited 10/E/KPT/2019

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2019 18 (2)

REINWARDTIA

A JOURNAL ON TAXONOMIC BOTANY, PLANT SOCIOLOGY AND ECOLOGY

Vol. 18 (2): 51 – 133, December 10, 2019

Chief Editor Kartini Kramadibrata (Mycologist, Herbarium Bogoriense, Indonesia)

Editors Dedy Darnaedi (Taxonomist, Herbarium Bogoriense, Indonesia) Tukirin Partomihardjo (Ecologist, Herbarium Bogoriense, Indonesia) Joeni Setijo Rahajoe (Ecologist, Herbarium Bogoriense, Indonesia) Marlina Ardiyani (Taxonomist, Herbarium Bogoriense, Indonesia) Himmah Rustiami (Taxonomist, Herbarium Bogoriense, Indonesia) Lulut Dwi Sulistyaningsih (Taxonomist, Herbarium Bogoriense, Indonesia) Eka Fatmawati Tihurua (Morphologist, Herbarium Bogoriense, Indonesia) Topik Hidayat (Taxonomist, Indonesia University of Education, Indonesia) Eizi Suzuki (Ecologist, Kagoshima University, Japan) Jun Wen (Taxonomist, Smithsonian Natural History Museum, USA) Barry J. Conn (Taxonomist, School of Life and Environmental Sciences, The University of Sydney, Australia) David G. Frodin (Taxonomist, Royal Botanic Gardens, Kew, United Kingdom) Graham Eagleton (Wagstaffe, NSW, Australia)

Secretary Ruslan Bukhori

Layout Liana Astuti

Illustrators Wahyudi Santoso Anne Kusumawaty

Correspondence on editorial matters and subscriptions for Reinwardtia should be addressed to: HERBARIUM BOGORIENSE, BOTANY DIVISION, RESEARCH CENTER FOR BIOLOGY– INDONESIAN INSTITUTE OF SCIENCES CIBINONG SCIENCE CENTER, JLN. RAYA JAKARTA – BOGOR KM 46, CIBINONG 16911, P.O. Box 25 CIBINONG INDONESIA PHONE (+62) 21 8765066; Fax (+62) 21 8765062 E-MAIL: [email protected] http://e-journal.biologi.lipi.go.id/index.php/reinwardtia

Cover images: Dinochloa glabra Widjaja & Ervianti, spec. nov. A. Culm sheath. B. Leaves. C. Leaf sheath. D. Inflorescence (1. Floret. 2. Palea. 3. Lemma. 4. Glume (a, b, c). 5. Lodicule (a, b, c). 6. Anthers. 7. Stigma. 8. Fruit). From Widjaja EAW 8864 (BO), drawing by Wahyudi Santoso (BO).

The Editors would like to thank all reviewers of volume 18(2):

Abdul Latiff Mohamad, Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor, Malaysia Andrew Powling, School of Biological Sciences, University of Portsmouth, United Kingdom Barry J. Conn, School of Life and Environmental Sciences, The University of Sydney, Australia Hans Joachim Esser, Botanische Staatssammlung München, Germany Martin Dancak, Faculty of Science Palacky University, Czech Republic Sumitra Salam, Nambol L. Sanoi College, Bishnupur, Manipur, India Wong Khoon Meng, Herbarium Singapore, Singapore Botanic Gardens, 1 Cluny Road, Singapore

80 REIN WARD TIA [VOL.18 REINWARDTIA Vol. 18. No. 2. pp: 81−96 DOI: 10.14203/reinwardtia.v18i2.3753 D., VUKOJIČIĆ, V., STOJANOVIĆ, V., studies. Environmental Publication, Karad, LAZAREVIĆ, M. & STEVANOVIĆ, V. India. Pp. 1‒79. THE SYSTEMATIC SIGNIFICANCE OF LEAF EPIDERMAL MICRO- 2009. Threat status revision of some taxa from ZAMAN, M., NADERUZZAMAN, A. T. M., HA- MORPHOLOGY OF TEN NEPENTHES SPECIES (NEPENTHACEAE) “The Red Data Book of Flora of Serbia 1”. Bo- SAN, M. & NAZ, S. 2011. Ecology, FROM PENINSULAR MALAYSIA tanica Serbica 33(1): 33‒43. morphology and anatomy of Aldrovanda TRIVEDI, R. K. & GOEL, P. K. 1984. Chemical vesiculosa L. (Droseraceae) from Bangladesh. Received August 20, 2019; accepted October 26, 2019 and biological methods for water pollution Bangladesh Journal of Botany 40(1): 85‒91. MOHD NORFAIZAL GHAZALLI Resource Utilisation and Agrobiodiversity Conservation Programme (BE2), Agrobiodiversity and Environment Research Centre, MARDI Headquarters, 43400 Serdang, Selangor, Malaysia. Email: [email protected]

AMIN ASYRAF TAMIZI Agri-Omics and Bioinformatics Programme (BT1), Biotechnology and Nanotechnology Research Centre, MARDI Headquarters, 43400 Serdang, Selangor, Malaysia. Email: [email protected]

MUHAMAD IKHWANUDDIN MAT ESA Department of Biology, Faculty of Science, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia. Email: [email protected]

EDWARD ENTALAI BESI Department of Biology, Faculty of Science, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia. Email: [email protected]

DOME NIKONG Digital Dome Photography, 21500 Permaisuri, Terengganu, Malaysia. Email: [email protected]

ANUAR RASYIDI MOHD NORDIN Resource Utilisation and Agrobiodiversity Conservation Programme (BE2), Agrobiodiversity and Environment Research Centre, MARDI Headquarters, 43400 Serdang, Selangor, Malaysia. Email: [email protected]

AHMAD ZAKI ZAINI Electron Microscopy Unit, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM), 43600 Bangi Selangor, Malaysia. Email: [email protected]

ABSTRACT GHAZALLI, M. N., TAMIZI, A. A., ESA, M. I. M., BESI, E. E., NIKONG, D., NORDIN, A. R. M. & ZAINI, A. Z. 2019. The systematic significance of leaf epidermal micromorphology of ten Nepenthes species (Nepenthaceae) from Peninsular Malaysia. Reinwardtia 18(2): 81−96. — The pitcher plants of Malaysia belong to the genus Nepenthes and can be found thriving in swampy areas, along the roadside, on hillslopes and in mountainous terrains depending on species and their ecological preferences. In this study, cuticle micromorphology of ten species of Nepenthes (Nepenthaceae) collected from Peninsular Malaysia was intensively studied through scanning electron microscopy (SEM) to characterise and investigate diagnostic characters of cuticle micromorphology that can be useful in Nepenthes classification. A total of eleven characters from the inner and outer cuticles were enumerated in details and these characters have a value either for infrageneric classification or for diagnostic identification of the species. Characters observed and analysed were related to the epidermal cells, subsidiary cells, stomatal complex i.e type of waxes on both epidermal surfaces, abaxial and adaxial cuticular ornamentation, stomata characteristics, stomata formation, stomata frequency, cuticular ornamentation on stomata, shape of the stomata, stomata size, trichome existence and type of trichomes. Nepenthes ampullaria is clearly distinguished from the other species by markedly different types of tufted and multicellular trichomes of the epidermal cells on both leaf epidermal surfaces. For N. alba, its cuticular feature showed groovy cuticular pattern on the abaxial and adaxial surface, hence, can serve as a diagnostic cuticular pattern for this species. From these findings, the species delimitation based on cuticular features show a clear resolution, however some species might be individually distinct based on the combination of characters examined.

Key Words: Characters, classification, identifications, Nepenthes, Peninsular Malaysia,

ABSTRAK GHAZALLI, M. N., TAMIZI, A. A., ESA, M. I. M., BESI, E. E., NIKONG, D., NORDIN, A. R. M. & ZAINI, A. Z. 2019. Signifikansi sistematik mikromorfologi epidermis daun dari sepuluh jenis Nepenthes (Nepenthaceae) dari Semenanjung Malaysia. Reinwardtia 18(2): 81−96. — Kantong semar di Malaysia dimasukkan ke dalam marga Nepenthes dan dapat ditemukan tumbuh di rawa-rawa, tepian jalanan, tebing bukit dan tebing gunung tergantung jenis dan keperluan ekologinya. Mikromorfologi kutikula sepuluh jenis Nepenthes (Nepenthaceae) yang diperoleh dari Semenanjung Malaysia telah diteliti secara intensif dengan menggunakan ‘scanning microscope electron ’ (SEM).

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Sebelas karakter kutikula bagian luar dan dalam telah diuraikan secara terperinci. Karakter-karakter ini mempunyai nilai untuk klasifikasi infragenerik dan identifikasi diagnostik jenis. Karakter yang diobservasi dan dianalisis berkaitan dengan sel epidermis, sel tetangga, kompleks stomata yaitu tipe lilin pada kedua permukaan epidermis, ornamentasi kutikula di abaksial dan adaksial, karakter, formasi dan frekuensi stomata, ornamentasi kutikula pada stomata, bentuk dan ukuran stomata, keberadaan dan tipe trikoma. Nepenthes ampullaria secara jelas berbeda dengan jenis-jenis Nepenthes yang lain dengan kehadiran variasi trikoma multiseluler dan trikoma berumpun pada kedua permukaan epidermis. Karakter kutikula yang bergerigi pada kedua permukaan abaksial dan adaksial pada Nepenthes alba dapat dijadikan panduan untuk ciri identifikasi diagnostik untuk jenis ini. Hasil penelitian ini menunjukkan pembatasan jenis berdasarkan ciri kutikula jelas menunjukkan resolusi yang bermakna, namun demikian sebagian jenis mempunyai kemungkinan untuk dibedakan secara individu berdasarkan kombinasi karakter-karakter yang diuji.

Kata Kunci: Identifikasi, karakter, klasifikasi, Nepenthes, Semenanjung Malaysia.

82 REINWARDTIA [VOL.18 2019] GHA ZALL I et al. : The sy stematic sign ificance of lea f epider mal mic romor pholog y 83

Kata Kunci: Identifikasi, karakter, klasifikasi, Nepenthes, Semenanjung Malaysia.

INTRODUCTION cies enumerations and has gained more attention with the advent of SEM (Cantrill, 1989; Dilcher, Nepenthes is one of the most abundant and 1974; Herbin & Sharma, 1969; Hill & Carpenter, diversified carnivorous plant groups. There are 1991; Martin & Juniper, 1970; Marques, 2000; about 160 species recorded worldwide that Stockey & Ko, 1986; Wells & Hill, 1989). As an distributed widely in the wet Old World from example, recent microstructural research on the Madagascar, the Seychelles, Sri Lanka, India, cuticular characteristics in leaf micromorphology across to China, the Philippines, New Guinea, of multiple plants including Lepisanthes (Sapinda- Australia and New Caledonia (Adam, 1992; ceae), Melastoma (Melastomaceae) and Parashorea Alastair et al., 2019; McPherson, 2009; Phillipps (Dipterocarpaceae) species in Malaysia (Mohd et al., 2008; Shivas,1984). In Malaysia, the genus Norfaizal et al., 2018; Noorma Wati et al., 2016; is represented by 46 species; thriving in lowlands Noraini & Cutler, 2009) had revealed relationship and mountain forests, also on disturbed land between cuticle characters and the currently including the roadsides and old mining sites accepted taxonomy classification, shedding light (Phillipps & Lamb, 1996; Saibeh et al., 2016). into the potential of utilizing these characters for To date, there are 11 species can be found in taxonomic classification. Peninsular Malaysia (N. alba Ridl., N. albomar- In this study, we investigated the usefulness of ginata T. Lobb. ex Lindl., N. gracillima Ridl., N. leaf cuticle micromorphology of ten out of eleven ampullaria Jack, N. benstonei C. Clarke, N. gra- Nepenthes species in Peninsular Malaysia in order Fig. 1. Studied species of Nepenthes. A. Nepenthes alba. B. N. albomarginata. C. N. ampullaria. D. N. ben- cilis Korth., N. macfarlanei Hemsl., N. rafflesiana to add the different cuticular characters for stonei. E. N. gracilis. F. N. ramispina. G. N. macfarlanei,.H. N. mirabilis. I. N. sanguinea. J. N. rafflesiana. Jack., N. mirabilis (Lour.), Druce, N. ramispina infrageneric classification of pitcher plants. This (Photos by: Amin Asyraf & Mohd Norfaizal, 2019). Ridl. and N. sanguinea Lindl.) (Fig. 1); while the approach of using cuticular characters has not been rest of 35 species are distributed randomly enumerated and explored for Nepenthes species throughout Malaysian Borneo (Clarke, 2001; distinction; hence, it is thought this effort will be Clarke, 2002). As suggested by their vernacular worth to be tested. The data obtained will also organic material except the cuticle was dissolved. RESULTS AND DISCUSSION name (pitcher plant or monkey cup), all Nepenthes serve as reference material useful in identifying Related SEM preparation procedures were species produce morphological variation of vegetative specimens of Nepenthes remains and conducted following Alvin & Boulter (1974); This study deals with Nepenthes cuticle micro- jug-shaped pitchers that function to attract, trap affix more systematic evidences in Nepenthes Halbritter (1997); Yoshie & Sakai (1985). Isolated morphology and has documented a new character and digest small animals specifically the insect genus for the identification purpose. Nepenthes epidermal cuticles were then rinsed in set which will be useful in identification and groups. However, some species have preferred distilled water. Targeted leaves cuticles were classifying different Nepenthes species. Eleven dead leaves, and even animal excretions, over mounted on aluminium stubs with double-sided important ultrastructural characters from both the insects for nutrients as can be observed in MATERIALS AND METHODS adhesive tape and proceeded for air drying. The inner and outer cuticular surfaces have been N. ampullaria and a Bornean species N. lowii stubs were then sputter-coated with pure gold to a described, representing the first report of such (Clarke, 1997 & 2001). Nepenthes leaves used in this study were maximum thickness of 15 nm and examined with characters for the Peninsular Malaysian The plant cuticle is a technically important obtained from field collection and their LEO (Model 1450 SEM)-Field-Emission Scanning Nepenthes. Enumeration of the Nepenthes character for plants that basically functions as a identification were conducted in line with Electron Microscope (FESEM). The remaining micromorphological features of both abaxial and protective layer for the plant and it imprints the available herbarium specimens that are available at cuticles were soaked in 5% ammonia and mounted adaxial surfaces under scanning electron sculptural layer of the epidermis (Barranova, 1992; KEP; MDI and UKMB collections (see Table 1 for on microscope slides in Canada balsam. All the microscope are shown in Figs. 2–6. Examination Hu, 1986 & Fahn, 1990). Related research voucher details). In total, ten species with one to stubs and slides prepared are housed in the SEM of the leaves sculpturing, stomata and epidermal invol-ving the study of cuticular layer of plants three replications of Nepenthes originating in Collection of Plant Anatomy and Palynology characters of the species showed interesting were initiated by several research projects that Peninsular Malaysia were examined. Epidermis Laboratory, Agrobiodiversity and Environment information-of which some of them can serve as investigated the potential of epidermis cuticular preparative fragments about 3 mm long were Research Centre, MARDI Serdang, Selangor, diagnostic characteristics-specifically on the study of the fossil conifers and several other spe- excised from the middle portion of approximately Malaysia, while the SEM analysis was conducted stomata features, stomata formation, trichome cies, which has sparked interests among plant tax- three mature leaves of each species. Cuticles part at Microscopy Electron Unit, Faculty of Science distribution and type of trichomes. The leaves of onomists due to the stable chemical nature of the were prepared by soaking leaf blade fragments in and Technology, Universiti Kebangsaan Malaysia, all species examined consisted of either plant cuticle. This character is then utilized in spe- 5–10% aqueous chromium trioxide until all Bangi Campus, Selangor, Malaysia. hipostomatic or amphistomatic, the stomata were dispersed randomly all over the whole abaxial/

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Table 1. Nepenthes species examined, their sources and voucher details. Species Localities

Nepenthes alba Malaysia, Pahang, Gunung Tahan. Muhammad Ikhwanuddin M.E. MDI 12346, 8 February 2019 (MDI) N. albomarginata Malaysia, Kedah, Gunung Jerai FR, Compartment 20-23. Kamarul Hisham, M., Noorsiha, A. & Kueh, H.L, FRI 52039, 10 June 2006 (FRI) Malaysia, Pulau Pinang, Bukit Bendera, Mohd Norfaizal, G., MDI 12337, 16 December 2018 (MDI) Malaysia, Johor, Gunung Ledang. Ruth Kiew, RK1677, 8 May 1985 (FRI) N. ampullaria Malaysia, Johor, Mersing. Rohana M.S. RMS 081, 28 November 1989 (FRI) Malaysia, Terengganu, Sungai Tong. MDI 12341, Mohd. Norfaizal, G. Amin Asyraf, T., Dome, N. 31 January 2019 (MDI) Malaysia, Selangor, Sungai Karang FR, Tanjung Karang. FRI 38751. Lilian, C. 28 April 1993 (FRI) N. benstonei Malaysia, Kelantan, Machang, Bukit Bakar. FRI 53169. Yao, T.L. Duistermart, H. Kiew, R. Kueh, H.L. 27 September 2006 (FRI) Malaysia, Kelantan, Machang, Bukit Bakar Summit. MDI 12333. Mohd Norfaizal, G. & Salmaniza, S. 5 March 2019 (MDI) Malaysia, Terengganu, Gunung Tebu. FRI 13147. Mohd Shah, Ahmad Shukor, Mahmud Awang. 31 May 1974 (FRI) N. gracilis Malaysia, Pahang, Temerloh, Tasik Bera. FRI52720. Rafidah, A.R., Mohd. Nazri, A., Kueh, H.L. 10 July 2007 (FRI) Malaysia, Bangi, UKM Bangi. MDI 12336. Mohd. Norfaizal, G. & Amin Asyraf, T. 15 December 2018 (MDI) Malaysia, Kedah, Pulau Langkawi, FRI63422. Beentje, H.J., Rosdi, M., Angan. 18 February 2009 (FRI) N. macfarlanei Malaysia, Pahang, Bentong, Gunung Ulu Kali. FRI 52572. Nor Ezzawanis, A.T. 16 February 2007 (FRI) Malaysia, Pahang, Summit Ridge of Gunung Ulu Kali to Gunung Rajah. Lilian Chua. FRI 40516. 16 January 1995 (FRI) Malaysia, Pahang, Pathway to Telekom Telecommunication Tower, Genting Highlands. MDI 12331. Mohd Norfaizal, G. & Amin Asyraf, T. 9 December 2018 (MDI) N. mirabilis Malaysia, Selangor, Hulu Selangor, Bukit Tunggal FR. FRI 73129. Chew, M.Y., Mohd Hairul, M.A. & Mohd Afiq, K. 24 May 2016 (FRI) Malaysia, Selangor, Puncak Alam. MDI 12416. Mohd Norfaizal, G. Anuar Rasyidi, M.N., Ahmad Syahman, M.D.& Muhammad Syakir. 30 December 2018 (MDI) N. ramispina Malaysia, Selangor, Gunung Bunga Buah. FRI65498. Yao, T.L. Kiew, R. & Jun, W. 31 October 2010 (FRI) Malaysia, Pahang, Pat hway to Telekom Telecommunication Tower, Genting Highlands. MDI 12332. Mohd Norfaizal, G. & Amin Asyraf, T. 9 December 2018 (MDI) N. sanguinea Malaysia, Terengganu, Hulu Terengganu, Gunung Padang. FRI70887. Mohd Hairul, M.A., Imin, K., Rafidah, A.R. Ummul Nazrah, A.R. Kueh, H.L. 21 March 2010 (FRI) Malaysia, Selangor, Gunung Ulu Semangkok. MDI 12418. Mohd Norfaizal, G. Anuar Rasyidi, M.N., Ahmad Syahman, M.D.& Muhammad Syakir.13 January 2019 (MDI) Malaysia, Perak, Gunung Hijau summit plateau, FRI 40475. 6 September 1994. Lilian Chua (FRI) N. rafflesiana Malaysia, Johor, Kluang, Kluang FR, Gunung Belumut. FRI60328. Chew, M.Y. & Teo, Y.L. 10 August 2009 (FRI) Malaysia, Johor, Gunung Ledang, FRI33511. Lilian Chua. 4 June 1993 (FRI)

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Table 1. Nepenthes species examined, their sources and voucher details. adaxial and abaxial surfaces, and the stomata were compound trichome observed on adaxial surface. all of either slightly raised above, same level with Species Localities the epidermis or sunken below the epidermis. 1.3. N. AMPULLARIA Jack Nepenthes alba Malaysia, Pahang, Gunung Tahan. Muhammad Ikhwanuddin M.E. MDI 12346, Details on the species micromorphological features Type of waxes: film layer observed on the 8 February 2019 (MDI) are described as below: abaxial epidermal surface; film layer and flakes observed on the adaxial epidermal surface. Abaxial N. albomarginata Malaysia, Kedah, Gunung Jerai FR, Compartment 20-23. Kamarul Hisham, M., 1.1. NEPENTHES ALBA Ridl. cuticular ornamentation: Type I, clear – anticlinal Noorsiha, A. & Kueh, H.L, FRI 52039, 10 June 2006 (FRI) Type of waxes: film layer and crustose observed wall clear and distinct while periclinal wall sunken. Malaysia, Pulau Pinang, Bukit Bendera, Mohd Norfaizal, G., MDI 12337, 16 on the adaxial epidermal surface; crustose No cuticular striation. Adaxial cuticular December 2018 (MDI) observed on the abaxial epidermal surface. Abaxial ornamentation: Type I, clear – anticlinal wall clear Malaysia, Johor, Gunung Ledang. Ruth Kiew, RK1677, 8 May 1985 (FRI) cuticular ornamentation: Type III, unclear – anti- and distinct while periclinal wall sunken. No N. ampullaria Malaysia, Johor, Mersing. Rohana M.S. RMS 081, 28 November 1989 (FRI) clinal wall unclear and periclinal wall sunked. cuticular striation. Stomata characteristics: Malaysia, Terengganu, Sungai Tong. MDI 12341, Mohd. Norfaizal, G. Amin Adaxial cuticular ornamentation: Type I, clear – hipostomatic. Stomata formation: superficial, Asyraf, T., Dome, N. 31 January 2019 (MDI) anticlinal wall can be observed clearly, periclinal raised from the epidermal wall. Stomata frequency: Malaysia, Selangor, Sungai Karang FR, Tanjung Karang. FRI 38751. Lilian, C. wall sunked. Cuticular striation exists on the very dense population on the abaxial surface. 28 April 1993 (FRI) adaxial surface. Stomata characteristics: hiposto- Cuticular ornamentation on stomata: stomatal rim matic. Stomata formation: superficial. Stomata raised and clearly noticeable. Stomatal rim and N. benstonei Malaysia, Kelantan, Machang, Bukit Bakar. FRI 53169. Yao, T.L. Duistermart, frequency: exist with dense population on the frame unclear. Epidermal cells do not wrap the H. Kiew, R. Kueh, H.L. 27 September 2006 (FRI) abaxial surface. Cuticular ornamentation on stoma- stomatal surface. Shape: broad ellipse. Subsidiary Malaysia, Kelantan, Machang, Bukit Bakar Summit. MDI 12333. Mohd Norfaizal, G. ta: stomatal rim raised and clearly noticeable. cell and epidermal can be differentiated easily. & Salmaniza, S. 5 March 2019 (MDI) Shape: ellipse. Epidermal cell do not wrap the Stomata semi-raised. Stomatal cuticular striation: Malaysia, Terengganu, Gunung Tebu. FRI 13147. Mohd Shah, Ahmad Shukor, stomatal rim surface. Subsidiary and epidermal absent. Stomatal size: L (11.48–16.12 µm) × W Mahmud Awang. 31 May 1974 (FRI) cells can be differentiated easily. Stomatal (9.01‒10.02 µm). Trichome distribution: can be N. gracilis Malaysia, Pahang, Temerloh, Tasik Bera. FRI52720. Rafidah, A.R., Mohd. cuticular striation: absent. Stomatal size: L (13.50– observed on the adaxial and adaxial epidermis Nazri, A., Kueh, H.L. 10 July 2007 (FRI) 16.89 µm) × W (8.01‒9.07 µm). Trichome surfaces. Trichome type: peltate glandular distribution: can be observed on the adaxial and trichome (unicellular terminal) can be observed on Malaysia, Bangi, UKM Bangi. MDI 12336. Mohd. Norfaizal, G. & Amin Asyraf, adaxial epidermis surfaces. Trichome type: simple, the abaxial surface. Stellate rotate trichome can be T. 15 December 2018 (MDI) unicellular trichome (short, bended) can be observed on the adaxial surface. Peltate glandular Malaysia, Kedah, Pulau Langkawi, FRI63422. Beentje, H.J., Rosdi, M., Angan. observed on the adaxial surface. Capitate glandular trichome (unicellular; sessile). Simple, unicellular 18 February 2009 (FRI) trichome (sessile) can be observed on the adaxial trichome (unicellular, bended, smooth surface) and N. macfarlanei Malaysia, Pahang, Bentong, Gunung Ulu Kali. FRI 52572. Nor Ezzawanis, A.T. surface. Peltate glandular trichome observed with be observed on abaxial surface. Scale trichome 16 February 2007 (FRI) random formation on the abaxial surface. observed on the adaxial surface. Papillae Malaysia, Pahang, Summit Ridge of Gunung Ulu Kali to Gunung Rajah. Lilian ornamentation also can be seen covering the Chua. FRI 40516. 16 January 1995 (FRI) 1.2. N. ALBOMARGINATA W.Lobb ex Lindl. abaxial epidermal surface.

Malaysia, Pahang, Pathway to Telekom Telecommunication Tower, Genting Type of waxes: film layer observed on the adaxial epidermal surface; film layer and granule 1.4. N. BENSTONEI C.Clarke Highlands. MDI 12331. Mohd Norfaizal, G. & Amin Asyraf, T. 9 December Type of waxes: film layer observed on the 2018 (MDI) observed on the abaxial epidermal surface. Abaxial cuticular ornamentation: Type III, unclear – anti- abaxial epidermal surface; film layer, crustose, N. mirabilis Malaysia, Selangor, Hulu Selangor, Bukit Tunggal FR. FRI 73129. Chew, M.Y., clinal wall unclear and periclinal wall sunken. observed on the adaxial epidermal surface. Abaxial Mohd Hairul, M.A. & Mohd Afiq, K. 24 May 2016 (FRI) Adaxial cuticular ornamentation: Type III, unclear cuticular ornamentation: Type I, clear – anticlinal Malaysia, Selangor, Puncak Alam. MDI 12416. Mohd Norfaizal, G. Anuar Rasyidi, – anticlinal wall unclear and periclinal wall wall clear while periclinal wall sunken. with M.N., Ahmad Syahman, M.D.& Muhammad Syakir. 30 December 2018 (MDI) sunken. Cuticular striation observed clearly on the cuticular striation. Adaxial cuticular ornament- N. ramispina Malaysia, Selangor, Gunung Bunga Buah. FRI65498. Yao, T.L. Kiew, R. & Jun, adaxial surface. Stomata characteristics: amphisto- tation: Type II, unclear – anticlinal wall W. 31 October 2010 (FRI) matic. Stomata formation: same level with the epi- semi-raised while periclinal wall sunken. Cuticular Malaysia, Pahang, Pat hway to Telekom Telecommunication Tower, Genting dermal wall. Stomata frequency: exist with dense striation exists on the adaxial surface. Stomata Highlands. MDI 12332. Mohd Norfaizal, G. & Amin Asyraf, T. 9 December population on the abaxial surface. Cuticular characteristics: hipostomatic. Stomata formation: 2018 (MDI) ornamentation on stomata: stomatal rim raised and paraficial, semi-raised from the epidermal wall. Stomata frequency: dense population on the N. sanguinea Malaysia, Terengganu, Hulu Terengganu, Gunung Padang. FRI70887. Mohd clearly noticeable. Shape: ellipse. Epidermal cell abaxial surface. Cuticular ornamentation on Hairul, M.A., Imin, K., Rafidah, A.R. Ummul Nazrah, A.R. Kueh, H.L. 21 do not wrap the stomatal rim surface. Subsidiary stomata: stomatal rim not clearly noticeable. March 2010 (FRI) and epidermal cells can be differentiated easily. Stomatal rim and its frame unclear. Epidermal cells Malaysia, Selangor, Gunung Ulu Semangkok. MDI 12418. Mohd Norfaizal, G. Stomatal cuticular striation: absent. Stomatal size: do not wrap the stomatal surface. Shape: rounded. Anuar Rasyidi, M.N., Ahmad Syahman, M.D.& Muhammad Syakir.13 January L (14.29–18.12 µm) × W (7.01‒10.07 µm). Subsidiary cell and epidermal can be differentiated 2019 (MDI) Trichome distribution: can be observed on the easily. Stomata semi-raised. Stomatal cuticular Malaysia, Perak, Gunung Hijau summit plateau, FRI 40475. 6 September 1994. adaxial and adaxial epidermis surfaces. Trichome striation: absent. Stomatal size: L (10.61–13.17 Lilian Chua (FRI) type: simple, unicellular trichome (short, bended, smooth) can be observed on the abaxial surface. µm) × W (12.89–14.63 µm). Trichome distribu- N. rafflesiana Malaysia, Johor, Kluang, Kluang FR, Gunung Belumut. FRI60328. Chew, M.Y. Capitate glandular trichome (sessile) can be tion: can be observed on the adaxial and adaxial & Teo, Y.L. 10 August 2009 (FRI) observed on the adaxial surface. Peltate glandular epidermis surfaces. Trichome type: peltate Malaysia, Johor, Gunung Ledang, FRI33511. Lilian Chua. 4 June 1993 (FRI) trichome (unicellular terminal) observed with glandular trichome (multicellular terminal, sessile) random formation on the abaxial surface. Stellate can be observed with scattered formation on the

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Fig. 2. SEMs of adaxial surfaces of leaf showing the surface sculpturing of A. Nepenthes alba. B. N. albomarginata. C. N. ampullaria. D. N. benstonei. E. N. gracilis. F. N. macfarlanei. G. N. mirabilis. H. N. rafflesiana. I. N. ramispina. J. N. sanguinea. (Magnification A-J: 100×). (Photos by: Mohd Norfaizal & Ahmad Zaki).

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abaxial surface. Scale trichome observed on the 1.7. N. MIRABILIS (Lour.)Druce adaxial surface. Type of waxes: film layer, sheet and granule observed on the abaxial epidermal surface; film layer and sheet observed on the adaxial epidermal 1.5. N. GRACILIS Korth. surface. Abaxial cuticular ornamentation: Type II, Type of waxes: film layer and granule observed unclear – anticlinal wall semi-raised while on the abaxial epidermal surface; film layer and periclinal wall sunken. Cuticular striation flakes observed on the adaxial epidermal surface. observed on the abaxial surface. Adaxial cuticular Abaxial cuticular ornamentation: Type III, unclear ornamentation: Type III, unclear – anticlinal wall – anticlinal wall unclear while periclinal wall unclear while periclinal wall sunken. Cuticular sunked. No cuticular striation. Adaxial cuticular striation exists on the adaxial surface. Stomata ornamentation: Type II, unclear – anticlinal wall characteristics: hipostomatic. Stomata formation: semi-raised while periclinal wall sunken. No paraficial, in random formation, sunken in the cuticular striation exists on the adaxial surface. epidermal wall. Stomata frequency: very dense Stomata characteristics: hipostomatic. Stomata population on the abaxial surface. Cuticular formation: superficial, in parallel formation, raised ornamentation on stomata: stomatal rim clearly from the epidermal wall. Stomata frequency: very noticeable. Stomatal rim and its frame clear. dense population on the abaxial surface. Cuticular Epidermal cells wrapping the stomatal surface. ornamentation on stomata: stomatal rim not clearly Shape: ellipse. Subsidiary cell and epidermal not noticeable. Stomatal rim and its frame unclear. distinguishable. Stomata raised on the epidermal Epidermal cells do not wrap the stomatal surface. surface. Stomatal cuticular striation: present. Shape: ellipse. Subsidiary cell and epidermal not Stomatal size: L (15.74–16.00 µm) × W (10.00 distinguishable. Stomata semi-raised. Stomatal 12.37 µm). Trichome distribution: can be cuticular striation: absent. Stomatal size: L (12.74– observed on the abaxial and adaxial epidermis 14.00 µm) × W (8.00–9.50 µm). Trichome surfaces. Trichome type: peltate glandular distribution: can be observed on the adaxial trichome (multicellular terminal) can be observed epidermis surfaces. Trichome type: peltate on the abaxial and adaxial surfaces. Peltate glandular trichome (multicellular terminal) can be glandular trichome (unicellular terminal, short- observed on the adaxial surface. stalked) observed on the abaxial epidermal surface.

1.6. N. MACFARLANEI Hemsl. 1.8. N. RAMISPINA Ridl. Type of waxes: film layer observed on the Type of waxes: film layer and granule observed abaxial epidermal surface; film layer and flakes on the abaxial and adaxial epidermal surfaces. observed on the adaxial epidermal surface. Abaxial Abaxial cuticular ornamentation: Type I, clear – cuticular ornamentation: Type II, unclear – anticlinal wall raised while periclinal wall sunken. anticlinal wall semi-raised while periclinal wall No cuticular striation observed on the abaxial sunken. Cuticular striation observed on the abaxial surface. Adaxial cuticular ornamentation: Type I, surface. Adaxial cuticular ornamentation: Type II, clear – anticlinal wall arise while periclinal wall unclear – anticlinal wall semi-raised while sunken. Cuticular striation exists on the adaxial periclinal wall sunken. No cuticular striation exists surface. Stomata characteristics: hipostomatic. on the adaxial surface. Stomata characteristics: Stomata formation: superficial, in random hipostomatic. Stomata formation: superficial, in formation, raised on the epidermal wall. Stomata tangential parallel formation, raised from the frequency: very dense population on the abaxial epidermal wall. Stomata frequency: very dense surface. Cuticular ornamentation on stomata: population on the abaxial surface. Cuticular stomatal rim clearly noticeable. Stomatal rim and ornamentation on stomata: stomatal rim clearly its frame clear. Epidermal cells do not wrap the noticeable. Stomatal rim and its frame clear. stomatal surface. Shape: ellipse. Subsidiary and Epidermal cells do not wrap the stomatal surface. epidermal cells not distinguishable. Stomata raised Shape: ellipse. Subsidiary cell and epidermal not on the epidermal surface. Stomatal cuticular distinguishable. Stomata raised on the epidermal striation: absent. Stomatal size: L (16.74–17.00 surface. Stomatal cuticular striation: absent. µm) × W (14.02–15.00 µm). Trichome distri- Fig. 2. SEMs of adaxial surfaces of leaf showing the surface sculpturing of A. Nepenthes alba. Stomatal size: L (15.74–16.00 µm) × W (8.00– bution: can be observed on the abaxial and adaxial B. N. albomarginata. C. N. ampullaria. D. N. benstonei. E. N. gracilis. F. N. macfarlanei. G. N. mirabilis. 9.37 µm). Trichome distribution: can be observed epidermis surfaces. Trichome type: peltate H. N. rafflesiana. I. N. ramispina. J. N. sanguinea. (Magnification A-J: 100×). (Photos by: Mohd Norfaizal on the abaxial and adaxial epidermis surfaces. glandular trichome (multicellular terminal) can be & Ahmad Zaki). Trichome type: peltate glandular trichome observed on the abaxial surface. Peltate glandular (multicellular terminal) can be observed on the trichome (unicellular terminal, short-stalked) abaxial surface. Simple, unicellular (smooth observed on the abaxial epidermal surface. su rface, pointed tip) can be observed randomly on Simple, unicellular (pointed tip) can be observed the adaxial epidermal surface. on the abaxial surface. Papillae ornamentation can

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Fig. 3. SEMs of abaxial surfaces of leaf showing the surface sculpturing of A. Nepenthes alba. B. N. albomarginata. C. N. ampullaria. D. N. benstonei. E. N. gracilis. F. N. macfarlanei. G. N. mirabilis. H. N. rafflesiana. I. N. ramispina. J. N. sanguinea. (Magnification A-J: 100×). (Photos: Mohd Norfaizal & Ahmad Zaki).

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Fig. 4. Plate of diagnostic stomata formation in Nepenthes; A. Nepenthes alba, sunked stomata without striation. B. N. albomarginata, same level with the epidermal surface formation without striation, and C. N. macfarlanei, raised formation stomata as compared with the epidermal surface, with stomatal-rim formation. (Magnification A-C: 100×). (Photos: Mohd Norfaizal & Ahmad Zaki).

Fig. 5. A-E. Variation of peltate glandular trichomes (unicellular and multicellular) and F. scale trichome observed in Nepenthes genus (Magnification A-F: 1000×). (Photos: Mohd Norfaizal & Ahmad Zaki).

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Fig. 6. A-E. Variation of non-glandular trichomes observed in Nepenthes genus. A. Simple, unicellular branched trichome. B. Tufted unicellular trichome. C. Simple, unicellular trichome with scales on the surface. D. Stellate rotate trichome. E. Simple, unicellular trichome, unbranched, smooth surface. (Magnification A-E: 1000×). (Photos: Mohd Norfaizal & Ahmad Zaki). be observed on the abaxial surface. Peltate on the abaxial surface. Peltate glandular trichome glandular trichome (multicellular terminal, (multicellular terminal, short-stalked) observed on short-stalked) observed on the abaxial epidermal the abaxial epidermal surface. Simple, unicellular surface. (pointed tip) can be observed on the adaxial surface. 1.9. N. RAFFLESIANA Jack Type of waxes: film layer observed on the 1.10. N. SANGUINEA Lindl. abaxial epidermal surface; film layer and granule Type of waxes: film layer and granule observed on the adaxial epidermal surface. observed on the abaxial epidermal surface; film Abaxial cuticular ornamentation: Type II, unclear layer observed on the adaxial epidermal surface. – anticlinal wall semi-arise while periclinal wall Abaxial cuticular ornamentation: Type I, clear – sunken. Cuticular striation observed on the abaxial anticlinal wall raised while periclinal wall sunken. surface. Adaxial cuticular ornamentation: Type Cuticular striation observed on the abaxial surface. III, unclear – anticlinal wall unclear while Adaxial cuticular ornamentation: Type I, clear – periclinal wall sunken. Cuticular striation exists anticlinal wall arise while periclinal wall sunked. on the adaxial surface. Stomata characteristics: No cuticular striation exists on the adaxial surface. hipostomatic. Stomata formation: paraficial, in Stomata characteristics: hipostomatic. Stomata random formation, raised on the epidermal wall. formation: paraficial, in random formation, raised Stomata frequency: dense population on the on the epidermal wall. Stomata frequency: sparse abaxial surface. Cuticular ornamentation on population on the abaxial surface. Cuticular stomata: stomatal rim clearly noticeable. Stomatal ornamentation on stomata: stomatal rim clearly rim and its frame clear. Epidermal cells wrapping noticeable. Stomatal rim and its frame clear. the stomatal surface. Shape: ellipse. Subsidiary Epidermal cells wrapping the stomatal surface. cell and epidermal not distinguishable. Stomata Shape: ellipse. Subsidiary and epidermal cells not raised on the epidermal surface. Stomatal cuticular distinguishable. Stomata sunken in the epidermal striation: absent. Stomatal size: L (11.93–13.05 surface. Stomatal cuticular striation: absent. µm) × W (9.00–10.05 µm). Trichome distribution: Stomatal size: L (14.74–16.00 µm) × W (7.00‒8.50 can be observed on the abaxial and adaxial µm). Trichome existence: can be observed on the epidermis surfaces. Trichome type: peltate abaxial and adaxial epidermis surfaces. Trichome glandular trichome (multicellular terminal) can be type: peltate glandular trichome (multicellular observed on the abaxial surface. Peltate glandular terminal) can be observed on the abaxial surface. trichome (unicellular terminal, short-stalked) Peltate glandular trichome (multicellular terminal, observed on the abaxial epidermal surface. short-stalked) observed on the abaxial and abaxial Simple, unicellular (pointed tip) can be observed epidermal surfaces.

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The lamina surface has been the subject of stomata is usually an ecological adaptation related investigations compared other plant micro- to control of water loss under direct exposure to morphology characteristics that often have wind and solar radiation. In the case of sunken taxonomic significance (Stace, 1969; Rudall, stomata, guard cells are placed at the base of a 1992). Through observation of studied Nepenthes stomatal pit and this arrangement prevents water lamina surfaces, the anticlinal wall of the adaxial loss through the stoma (Das & Ghose 1997; and abaxial epidermal cells was either straight- Mbagwu & Edeoga 2006). Nevertheless, our study wavy, straight or groovy which therefore appears indicates the correlation between sunken, to be a common character for this genus. paraficial and superficial stomata and their According to Stace (1965), Wilkinson (1979) and potential taxonomic and ecological significance Wu et al. (2005), cuticular sculpturing may be a for Peninsular Malaysian Nepenthes still needs to diagnostic character of some species. In fact, be further assessed and established in order to see several related study of the character of cuticular the physiological adaptation factors that sculpturing in selected genera e.g. Parnassia and influenced these characters. All of the studied Pinus could serve as a diagnosis note to species level Nepenthes species also possessed guard cell pairs (Price 1998; Wu et al., 2005; Yoshie & Sakai 1985). with either an elliptical, broadly-elliptical or In this study, the Nepenthes epidermal surfaces rounded outline. revealed a number of important micro- Photomicrographs of stomata also have morphological characters and these characters revealed an interesting key feature that may be of Fig. 6. A-E. Variation of non-glandular trichomes observed in Nepenthes genus. A. Simple, unicellular exhibited interesting interspecific variations that diagnostic value (Fig. 3). From series of branched trichome. B. Tufted unicellular trichome. C. Simple, unicellular trichome with scales on the are of significance for species identification. observations, stomatal features as seen under the surface. D. Stellate rotate trichome. E. Simple, unicellular trichome, unbranched, smooth surface. Further observation under SEM of the adaxial SEM could be useful for identification especially (Magnification A-E: 1000×). (Photos: Mohd Norfaizal & Ahmad Zaki). lamina surfaces showed a few taxonomically at the species level as observed in Nepenthes alba significant characteristics, i.e. the abaxial and that posseses sunken stomata without striation, adaxial surface being groovy (Nepenthes alba), N. albomarginata with its stomata same level with be observed on the abaxial surface. Peltate on the abaxial surface. Peltate glandular trichome possibly owing to the thick cuticle, lack of stomata the epidermal surface formation without striation, glandular trichome (multicellular terminal, (multicellular terminal, short-stalked) observed on on the surfaces and type of trichomes. On the other and N. macfarlanei with raised stomata from the short-stalked) observed on the abaxial epidermal the abaxial epidermal surface. Simple, unicellular hand, when the leaf abaxial surface was viewed epidermal surface together with stomatal-rim surface. (pointed tip) can be observed on the adaxial under the SEM at low magnification, a high formation (Fig. 4). Detailed observation of the surface. diversity in the ‘cellular patterns’ and the characters from stomatal microstructure and type 1.9. N. RAFFLESIANA Jack distribution of idioblastic elements such as of trichome also has succeeded in adding three Type of waxes: film layer observed on the 1.10. N. SANGUINEA Lindl. trichomes were readily recognised. The appearance more diagnostic characters (stomatal formations, abaxial epidermal surface; film layer and granule Type of waxes: film layer and granule of the cuticular wax - film layer, granular or trichome distribution and observation of the observed on the adaxial epidermal surface. observed on the abaxial epidermal surface; film crustose - provides much information for epidermal surface) that are unique to certain taxa Abaxial cuticular ornamentation: Type II, unclear layer observed on the adaxial epidermal surface. classification and observation that may be useful as thus becoming important for systematic – anticlinal wall semi-arise while periclinal wall Abaxial cuticular ornamentation: Type I, clear – an additional systematic evidence together with significance of Nepenthes species. Based on sunken. Cuticular striation observed on the abaxial anticlinal wall raised while periclinal wall sunken. morphological and anatomical observations. stomatal formations – that can be classified as surface. Adaxial cuticular ornamentation: Type Cuticular striation observed on the abaxial surface. All of the species examined showed superficial, same level or paraficial – III, unclear – anticlinal wall unclear while Adaxial cuticular ornamentation: Type I, clear – characteristics of amphistomatic and hipostomatic, N. albomarginata herein could be clearly periclinal wall sunken. Cuticular striation exists anticlinal wall arise while periclinal wall sunked. agreeable with preliminary study of Nepenthaceae separated from the rest of the species for having on the adaxial surface. Stomata characteristics: No cuticular striation exists on the adaxial surface. characters by Metcalfe and Chalk (1950). In this same-level-with-the-epidermal surface stomatal hipostomatic. Stomata formation: paraficial, in Stomata characteristics: hipostomatic. Stomata study, amphistomatic stomata features could be feature. Based on trichome distribution on the random formation, raised on the epidermal wall. formation: paraficial, in random formation, raised only observed in N. albomarginata, while the other epidermal surfaces, N. gracilis it the only species Stomata frequency: dense population on the on the epidermal wall. Stomata frequency: sparse nine Nepenthes species showed hipostomatic showing trichome distribution on the adaxial abaxial surface. Cuticular ornamentation on population on the abaxial surface. Cuticular stomata feature; hence such a unique trait can give epidermal surface while the other nine species stomata: stomatal rim clearly noticeable. Stomatal ornamentation on stomata: stomatal rim clearly an affirmative diagnostic feature for possess trichome distribution on both sides of rim and its frame clear. Epidermal cells wrapping noticeable. Stomatal rim and its frame clear. N. albomarginata. Metcalfe and Chalk (1950) also epidermal. This finding is coherent with a the stomatal surface. Shape: ellipse. Subsidiary Epidermal cells wrapping the stomatal surface. reported the type of stomata in Nepenthaceae as previous study by Xiang et al. (2010) that reported cell and epidermal not distinguishable. Stomata Shape: ellipse. Subsidiary and epidermal cells not being of ranunculaceous. However, our finding the morphology and distribution of trichomes have raised on the epidermal surface. Stomatal cuticular distinguishable. Stomata sunken in the epidermal concludes Nepenthes from Peninsular Malaysia are valuable systematics significance at species level striation: absent. Stomatal size: L (11.93–13.05 surface. Stomatal cuticular striation: absent. characterized by not only ranunculaceous but also in Chelonopsis (Lamiaceae). Factor that µm) × W (9.00–10.05 µm). Trichome distribution: Stomatal size: L (14.74–16.00 µm) × W (7.00‒8.50 paracytic stomatal type, adding a new delimiting trichome distribution on the adaxial can be observed on the abaxial and adaxial µm). Trichome existence: can be observed on the information that is useful in defining Nepenthes surface can be compensated by thick epicuticular epidermis surfaces. Trichome type: peltate abaxial and adaxial epidermis surfaces. Trichome species. Further observation also showed that waxes (film layer and flakes) on the adaxial leaf glandular trichome (multicellular terminal) can be type: peltate glandular trichome (multicellular stomata in Nepenthes were either raised or same surface and delimiting the species description in observed on the abaxial surface. Peltate glandular terminal) can be observed on the abaxial surface. level, and sunken in some species (Fig. 4). In the genus Nepenthes. However, further evaluation trichome (unicellular terminal, short-stalked) Peltate glandular trichome (multicellular terminal, general, sunken and raised stomata are related to is relevantly recommended to determine whether observed on the abaxial epidermal surface. short-stalked) observed on the abaxial and abaxial habitat preference of the species and environ- these features are influenced by environmental Simple, unicellular (pointed tip) can be observed epidermal surfaces. mental adaptations. The presence of sunken factors due to its habitat preferences.

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Fig. 7. UPGMA analysis of micromorphology characteristics of Peninsular Malaysia Nepenthes using Gower General Similarity Coefficient (HL: highland species; LL: lowland species); (Yellow and Purple Lines: Cluster 1; Blue Line: Cluster II; Red Line: Cluster III).

Through this study, it is also clearly N. mirabilis), they are considered as monophyletic indicated that N. ampullaria can be differentiated due to their lowland habitat preference but poorly from other studied Nepenthes species with resolved in this analysis as cuticular study does not variation in the types of trichomes for having support that, due to only one systematic evidence combination of simple, glandular and scale analysis conducted in this study. It is suggested trichome, and we have recorded six types i.e. that other systematic evidences – using thorough peltate glandular (unicellular terminal, short- morphological, anatomical and molecular analyses stalked), stellate rotate (on adaxial surface), – should be conducted and incorporated in future peltate glandular (unicellular, sessile), simple research to complement this microstructural unicellular trichome (smooth surface), scale section analysis. trichome and combination of papillae formation. All features of the Nepenthes leaf epidermal This observation of trichomes are agreeable with micromorphology analysed in this study are related research involving different taxa by Chen produced in Appendix I and II. et al. (2013) and Guimares et al. (1999) that refer and employed the trichome characteristics in CONCLUSION systematic studies of Melastomaceae family. Finally, we incorporated the features of the Leaf micromorphology analyses have shown a Nepenthes leaf epidermal micromorphology to significant taxonomic value in the genus infer the relationships among the ten studied Nepenthes (Nepenthaceae) through the surface species. In this study, N. ampullaria, N. alba, N. sculpturing ornamentation, trichome morphology gracilis and N. albomarginata are clearly and its distribution as well as the stomatal feature. distinguished from the other species with the It is concluded that this study has added new observation of the epidermal ornamentation, systematics evidences that are useful in defining stomata features as well as trichome morphology. the Nepenthes species, hence it can be postulated UPGMA (unweighted pair group method with that the micromorphology of the leaf surfaces arithmetic mean) groupings analysis using varies greatly between species. cuticular micromorphology, however, gave poor resolution. As illustrated, the typical highland ACKNOWLEDGEMENTS Nepenthes species namely N. sanguinea, N. macfarlanei, N. ramispina, N. alba and N. We thank the curators of KEP, UKMB, MDI benstonei are not clustered as monophyletic, while deposition collections specifically to Dr Richard subsection III occupying the basal position and Chung (Forest Research Institute Malaysia, FRIM) comprised of N. ampullaria and N. albomarginata and Assoc. Prof Dr. Noraini Talip (UKMB are not related to each other in terms of ecology Herbarium) for help in obtaining permission and morphology (Fig. 7). For the remaining involving the herbarium specimens. Special thank Nepenthes species (N. gracilis, N. rafflesiana and also due to Mdm. Salmaniza Salleh, Miss

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Nurshahidah Mohd Rusli (Plant Anatomy and Biology and Technology 42: 485−493. Palynology Laboratory, MARDI), Mohamad Alias HALBRITTER, H. 1997. Preparing material for Shakri and Muhammad Ikhwan Afandi Md Daud Scanning Electron Microscope using 2,2- for their assistance in field guide and specimen dimethoxypropane (DMP) and critical point preparation. drying. Biotechnic and Histochemistry 73: 137−143. REFERENCES HERBIN, G. A. & SHARMA, K. 1969. Studies on plant cuticular waxes: the wax coatings of ADAM, J. H., WILCOCK, C. C. & SWAINE, M. pine needles: a taxonomic survey. Phyto- D. 1992. The ecology and distribution of chemistry 8: 151–160. Bornean Nepenthes. Journal of Tropical Forest HILL, R. S. & CARPENTER, R. J. 1991. Evolu- Science 5: 13−25. tion of Acmopyle and Dacrycarpus ALASTAIR, S. R., MICHAL, R. G., MARC, B., (Podocarpaceae) foliage as inferred from YOSUKE, S., JENNIFER, J. F., DIEGO, G., macrofossils in south-eastern Australia. Aus- CHANDLER, N. G., ADI, O. L., NICK, W.R. tralian Systematic Botany 4: 449–479. M., STEWART, R. M., GREGORY, J. P., HU, Y. 1986. SEM observation of the inner IVAN, P., BRIAN, D. Q. & JEFF, S. 2019. surface structure of needle cuticles in Pinus. Revisions in Nepenthes following explorations Acta Phytotaxonomica Sinica 24: 464–468. of the Kemul Massif and the surrounding MCPHERSON, S. R. 2009. Pitcher Plants of the Fig. 7. UPGMA analysis of micromorphology characteristics of Peninsular Malaysia Nepenthes using region in north-central Kalimantan, Borneo. Old World Vol. II. Poole: Redfern Natural Gower General Similarity Coefficient (HL: highland species; LL: lowland species); (Yellow & Purple Lines: Phytotaxa 392(2): 97−126. History Productions. Borneo. Cluster 1; Blue Line: Cluster II; Red Line: Cluster III). ALVIN, K. L. & BOULTER, M. C. 1974. A MARQUES, A. R., GARCIA, Q. S., REZZENDE, controlled method of comparative study for J. L. P. & FERNANDES, G. W. 2000. Varia- taxodiaceous leaf cuticles. Botanical Journal of tions in leaf characteristics of two species of Through this study, it is also clearly N. mirabilis), they are considered as monophyletic the Linnean Society 69: 277–286. Miconia in the Brazilian cerrado under differ- indicated that N. ampullaria can be differentiated due to their lowland habitat preference but poorly BARRANOVA, M. 1992. Principles of ent light intensities. Tropical Ecology 41: 47– from other studied Nepenthes species with resolved in this analysis as cuticular study does not comparative stomatographic studies of 60. variation in the types of trichomes for having support that, due to only one systematic evidence flowering plants. The Botanical Review MARTIN, J. T. & JUNIPER, B. E. 1970. The combination of simple, glandular and scale analysis conducted in this study. It is suggested 58: 49−99. Cuticles of Plants. London: Edward Arnold. trichome, and we have recorded six types i.e. that other systematic evidences – using thorough CANTRILL, D. J. 1989. An Albian coniferous flo- MBAGWU, F. N. & EDEOGAGA, H. O. 2006. peltate glandular (unicellular terminal, short- morphological, anatomical and molecular analyses ra from the Otway Basin, Victoria; taxonomy, Leaf anatomy of some Nigerian species of stalked), stellate rotate (on adaxial surface), – should be conducted and incorporated in future palaeocology and palaeoclimatology. PhD Vigna savi (Leguminosae−Papilionoideae). peltate glandular (unicellular, sessile), simple research to complement this microstructural Dissertation, University of Melbourne, Mel- Agricultural Journal 1: 5−7. unicellular trichome (smooth surface), scale section analysis. bourne, Australia. METCALFE, C. R. & CHALK, L. 1950. Anato- trichome and combination of papillae formation. All features of the Nepenthes leaf epidermal CHEN, J. L., LAN, S. R., WU, S. S., YANG, H., my of the Dicotyledons. Vol 1. Oxford: This observation of trichomes are agreeable with micromorphology analysed in this study are WU, H. C. & PENG, D. H. 2013. Leaf surface Clarendron Press. related research involving different taxa by Chen produced in Appendix I and II . characteristics of 6 species of Melastoma L. MOHD NORFAIZAL, G., NORAINI, T. & et al. (2013) and Guimares et al. (1999) that refer and their taxonomic significance. Journal of LATTIFF, A. 2018. Leaf micromorphology of and employed the trichome characteristics in CONCLUSION Fujian College of Forestry 2: 106−112. Lepisanthes Blume (Sapindaceae) in Peninsu- systematic studies of Melastomaceae family. CLARKE, C. M. 1997. Nepenthes of Borneo. lar Malaysia. American Institute of Physics Finally, we incorporated the features of the Leaf micromorphology analyses have shown a Natural History Publications (Borneo), Kota Conference Proceedings 1940, 020038; Nepenthes leaf epidermal micromorphology to significant taxonomic value in the genus Kinabalu. https://doi.org/10.1063/1.5027953. infer the relationships among the ten studied Nepenthes (Nepenthaceae) through the surface CLARKE, C. M. 2001. Nepenthes of Sumatra and NOORMA WATI, H., NORDIYANAH, A. & species. In this study, N. ampullaria, N. alba, N. sculpturing ornamentation, trichome morphology Peninsular Malaysia. Natural History RUBASHINY, V. 2015. The taxonomic gracilis and N. albomarginata are clearly and its distribution as well as the stomatal feature. Publications (Borneo), Kota Kinabalu. significance of leaf micromorphology in the distinguished from the other species with the It is concluded that this study has added new CLARKE C. M. 2002 Nepenthes of Borneo. genus Melastoma L. (Melastomataceae). Sains observation of the epidermal ornamentation, systematics evidences that are useful in defining Victorian Carnivorous Plant Society Journal Malaysiana 44(5): 643–650. stomata features as well as trichome morphology. the Nepenthes species, hence it can be postulated 65:19. NORAINI, T. & CUTLER, D. F. 2009. Leaf ana- UPGMA (unweighted pair group method with that the micromorphology of the leaf surfaces DAS, S. & GHOSE, M. 1997. Development of tomical and micromorphological characters of arithmetic mean) groupings analysis using varies greatly between species. stomata and leaf hairs in some mangroves. some Malaysian Parashorea (Dipterocarpa- cuticular micromorphology, however, gave poor Phytomorphology 47: 389−394. ceae). Journal of Tropical Forest Science 21 resolution. As illustrated, the typical highland ACKNOWLEDGEMENTS DILCHER, D. L. 1974. Approaches to the identifi- (2): 1−7. Nepenthes species namely N. sanguinea, N. cation of angiosperm leaf remains. The Botani- PHILLIPS, A. & LAMB, A. 1996. Pitcher Plants macfarlanei, N. ramispina, N. alba and N. We thank the curators of KEP, UKMB, MDI cal Review 40: 1−157. of Borneo. Natural History Publications benstonei are not clustered as monophyletic, while deposition collections specifically to Dr. Richard FAHN, A. 1990. Plant Anatomy 4th Ed. Pergamon (Borneo). Kota Kinabalu. subsection III occupying the basal position and Chung (Forest Research Institute Malaysia, FRIM) Press. PHILLIPS, A., LAMB, A. & LEE, C. C. 2008. comprised of N. ampullaria and N. albomarginata and Assoc. Prof Dr. Noraini Talip (UKMB GUIMARAES, P. J. F., RANA, N. T. & MAR- Pitcher Plants of Borneo 2nd ed. Natural are not related to each other in terms of ecology Herbarium) for help in obtaining permission TINS, A. B. 1999. Morphology of trichomes in History Publications (Borneo). Kota Kinabalu. and morphology (Fig. 7). For the remaining involving the herbarium specimens. Special thank Tibouchina section Pleroma (D.Don) Cogn. PRICE, R. A, LISTON, A. & STRAUSS, S. H. Nepenthes species (N. gracilis, N. rafflesiana and also due to Mdm. Salmaniza Salleh, Miss (Melastomataceae). Brazilian Archives of 1998. Phylogeny and systematics of Pinus. In:

94 REIN WARD TIA [VOL.18

D. M. RICHARDSON (Ed.) Ecology and micromorphology of Araucariana de Jussieu. Bio- geography of Pinus. Cambridge: Cam- Botanical Gazette 147: 508–548. bridge University Press. 49–68. WELLS, P. M. & HILL, R. S. 1989. Leaf RUDALL, P. 1992. Anatomy of Flowering morphology of the imbricate–leaved Podocar- Plants: An Introduction to Structure and paceae. Australian Systematic Botany 2: 369– Development. 2nd ed. Cambridge University 386. Press, Cambridge. WILKINSON, H. P. 1979. The plant surface SAIBEH, K., SUGAU, J., REPIN, R. 2016. The (mainly leaf). Part 1: Stomata. In: C. R. checklist of plants occurring at the METCALFE & L. CHALK (Eds.) Anatomy of abandoned Mamut Copper Mine, Sabah, the Dicotyledons. 2nd ed. The Clarendon Press, Malaysia. Journal of Tropical Biology and Oxford. pp. 97−167. Conservation 13: 71−84. https:// WU, D., WANG, H., LU, J. M. & LI, D. Z. 2005. www.ums.edu.my/ibtpv2/files/06-JTBC13- Comparative morphology of leaf epidermis in 004-914.pdf. Parnassia (Parnassiaceae) from China. Acta SHIVAS, R. G. 1984. Pitcher Plants of Phytotaxonomica Sinica 43: 210−224. Peninsular Malaysia and Singapore. XIANG, C. L., DONG, Z. H., PENG, H. & LIU, Z. Maruzen Asia. Singapore. W. 2010. Trichome micromorphology of the STACE, C. A. 1965. Cuticular studies as an aid East Asiatic genus Chelonopsis (Lamiaceae) to plant taxonomy. Bulletin of the British and its systematic implications. Flora 205: Museum Natural History Botany 4: 3−78. 434−441. STACE, C. A. 1969. The use of indumentum YOSHIE, F. & SAKAI, A. 1985. Types of florin characters in taxonomic studies. Botanical rings, distributional patterns of epicuticular Journal of Linnean Society 62: 131−168. wax, and their relationships in the genus Pinus. STOCKEY, R. & KO, H. 1986. Cuticle Canadian Journal of Botany 63: 2150–2158.

94 REIN WARD TIA [VOL.18 2019] GHA ZALL I et al. : The sy stematic sign ificance of lea f epider mal mic romor pholog y 95

D. M. RICHARDSON (Ed.) Ecology and micromorphology of Araucariana de Jussieu.

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96 REIN WARD TIA [VOL.18

Appendix II. Summary of Nepenthes leaf epidermal micromorphology characteristics (Trichome)

Species Frequency of Frequency Frequency Frequency Occurrence Occurrence Bodystalk of simple trichome of tufted of stellate of cushioned of glandular of glandular glandular trichome trichome stellate trichome on trichome on trichome trichome abax adax Nepenthes Medium- High Medium High Medium Low Sessile ampullaria frequency

N. alba Low- frequency No No Low Medium High Sessile N. albomarginata Low- frequency Medium High Low Medium Low Shortly-stalked N. benstonei Low- frequency No No No Medium Low Shortly-stalked N. gracilis No No No No No Low Shortly-stalked N. macfarlanei Low- frequency No No No Low Low Shortly-stalked

N. mirabilis Low- frequency No No No Low High Shortly-stalked N. ramispina Low- frequency No No No Medium High Shortly-stalked

N. rafflesiana Low- frequency No No No Low High Shortly-stalked N. sanguinea No No No No High Low Shortly-stalked

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Journal : KRAENZLIN, F. 1913. Cyrtandraceae novae Philippinenses I. Philipp. J. Sci. 8: 163–179. MAYER, V., MOLLER, M., PERRET, M. & WEBER, A. 2003. Phylogenetic position and generic differentiation of Epithemateae (Gesneriaceae) inferred from plastid DNA sequence data. American J. Bot. 90: 321–329. Proceedings : TEMU, S. T. 1995. Peranan tumbuhan dan ternak dalam upacara adat “Djoka Dju” pada suku Lio, Ende, Flores, Nusa Tenggara Timur. In: NASUTION, E. (Ed.). Prosiding Seminar dan Lokakarya Nasional Etnobotani II. LIPI & Perpustakaan Nasional. Pp. 263–268. (In Indonesian). SIMBOLON, H. & MIRMANTO, E. 2000. Checklist of plant species in the peat swamp forests of Central Kalimantan, Indonesia. In: IWAKUMA, T., INOUE, T., KOHYAMA, T., OSAKI, M., SIMBOLON, H., TACHIBANA, H., TAKAHASHI, H., TANAKA, N., YABE, K. (Eds.). Proceedings of the International Symposium on: Tropical Peatlands. Pp. 179 ‒ 190. Book : RIDLEY, H. N. 1923. Flora of the Malay Peninsula 2. L. Reeve & Co. Ltd, London. Part of Book : BENTHAM, G. 1876. Gesneriaceae. In: BENTHAM, G. & HOOKER, J. D. (Eds.). Genera Plantarum 2. Lovell Reeve & Co., London. Pp. 990–1025. Thesis : BAIRD, L. 2002. A Grammar of Kéo: An Austronesian Language of East Nusantara. Australian National University, Canberra. [PhD. Thesis]. Website : http://www.nationaalherbarium.nl/fmcollectors/k/KostermansAJGH.html. (Accessed 15 February 2012).