Malays. Appl. Biol. (2018) 47(5): 289–293

PRELIMINARY ANATOMICAL STUDY ON LEAF SURFACES OF BORNEAN (TRIBE ALPINIEAE) FROM NORTH EAST SARAWAK

SALASIAH, M.* and MEEKIONG, K.

Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia *E-mail: [email protected]

Accepted 2 October 2018, Published online 30 November 2018

ABSTRACT

Among the family Zingiberaceae, the biggest subfamily in Borneo is Alpinioideae. The highest diversity is recorded for tribe Alpinieae, with nine genera from Borneo. Tribe Alpinieae is, in fact, the most challenging that need further analysis for recircumscription of many of the genera. The study reported in this paper was carried out to investigate if the anatomy of leaf surfaces particularly epidermal cells, stomata, and trichomes are applicable as supportive data in finding the intergeneric variations within the tribe Alpinieae. The leaf surfaces of selected species from six genera of tribe Alpinieae (viz. Alpinia, Amomum, Elettaria, Etlingera, Geocharis, and Plagiostachys) from the north east of Sarawak was preliminarily studied. The epidermal cells of the adaxial surface are hexagonal or more or less polygonal in shapes in all selected species with straight anticlinal walls at both surfaces. The cells of the abaxial epidermis are much more variable and irregular in shapes and sizes. Different sizes of epidermal cells are also shown in all species. In addition, crystals are observed in epidermal cells of E. longipilosa only. Meanwhile, the type of stomata observed is of tetracytic form in all species. All species except for Alpinia aquatica possessed simple and unicellular trichomes. Study on leaf surface anatomy alone may only provide a baseline data to facilitate in taxonomical classification and a thorough leaf anatomical study on the lamina, leaf margin, midrib and petiole involving more species in each genus would provide more insightful information.

Key words: Alpinieae, Zingiberaceae, leaf anatomy, leaf surface, Borneo

INTRODUCTION Tribe Alpinieae is grouped under subfamily Alpinioideae together with Riedelieae. The The pantropical Zingiberaceae is one of the subfamily is morphologically defined by the plane important herbaceous of tropical forests and of distichous leaves perpendicular to rhizome and the largest family among seven others of order the reduction or absence of the two lateral . There are at least 60 genera and 1,500 staminodes (Smith, 1985; Kress et al., 2002). There species in the family of which 1,000 species occur are currently nine genera in the tribe Alpinieae that in tropical Asia (Kress, 1990; Larsen et al., 1999). commonly occur in Borneo namely Alpinia Roxb., In Borneo, 21 genera with nearly 250 named taxa Amomum Roxb., Elettaria Maton, Elettariopsis have been reported of which five genera namely Bak., Etlingera Giseke, Geocharis (K. Schum.) Ridl., Haniffia, Burbidgea, Haplochorema, Myxochlamys Geostachys (Bak.) Ridl., Hornstedtia Retz. and and Tamijia are endemic to the island (Poulsen, Plagiostachys Ridl (Lamb et al., 2013). Tribe 2006; Lamb et al., 2013; Wong et al., 2014). Many Alpinieae is characterised by having fleshy or of the Bornean species are only determined to genus indehiscent fruit and lacking extrafloral nectaries level such as Amomum, Hornstedtia, Plagiostachys, (Smith, 1985; Kress et al., 2002). Genus Etlingera Zingiber and Boesenbergia and many other species represents the highest species number of Bornean remain undescribed and are yet to be documented gingers with 42 known species followed by (Takano et al., 2004; Julius et al., 2010; Lamb et Amomum with 36 named taxa (Lamb et al., 2013). al., 2013). Lowland forests up to 600 m altitudes are the richest zone for family Zingiberaceae and species richness * To whom correspondence should be addressed. drops at higher altitude. However, the species of 290 ANATOMICAL STUDY ON LEAF SURFACES OF BORNEAN ZINGIBERACEAE (TRIBE ALPINIEAE)

Alpinia, Amomum, and Hornstedtia can still be MATERIALS AND METHODS found around the lower montane forest from 1400 – 2300 m compared to other species in the family Six species from different genera in tribe Alpinieae (Lamb et al., 2013). Species in the genera Etlingera, from Similajau National Park, Bintulu, Sarawak Hornstedtia, and Plagiostachys predominate in the were used in the study. The specimens were disturbed areas which have been logged or have identified based on published materials and been affected by fires during droughts as the matched herbarium specimens (from herbaria) and habitats provide more sunlight (Julius et al., 2010; identified as Alpinia aquatica (Retz.) Roscoe, Lamb et al., 2013). Amomum xanthophlebium Bak., Etlingera nasuta Alpinia is the only genus in Alpinieae that has (K. Schum.) R.M. Sm., Elettaria longipilosa S. terminal inflorescences on the leafy shoot (or near Sakai & Nagam., Geocharis sp., and Plagiostachys the top of the leafy shoots, for example, A. hansenii cf. megacarpa A. Julius & A. Takano. Fresh leaf and A. havilandii) and mostly have long petioles specimens were collected from the field and (Smith, 1985; Kress et al., 2002). Meanwhile, for epidermal peels were prepared by scraping. Plagiostachys, the inflorescence is also terminal but Specimens were washed with sodium hypochlorite breaking through the side of the leaf sheath and and stained with safranin. The samples were then appearing lateral on the leafy shoot (Smith, 1985; rinsed in series of ethyl alcohol (50%, 70%, 90% Kress et al., 2002; Julius, 2006). The other seven and 100%) to remove excess water. All slides were genera are commonly having radical inflorescence mounted in Canada Balsam after dehydration. Images separate from the leafy shoot (Smith, 1985; Kress were then captured using a light microscope and et al., 2002). Systematics study on Bornean examined. Zingiberaceae especially on subfamily Alpinioideae by Sakai and Nagamasu (1998, 2001, 2003) from Lambir Hills National Park has provided valuable RESULTS AND DISCUSSION taxonomic data which primarily based on morphological features of species. Epidermal cells A thorough analysis of the anatomy of the The anticlinal walls are straight in cells of both family Zingiberaceae was first performed by adaxial and abaxial surfaces. The epidermal cells of Solereder and Meyer (1930) (in Tomlinson, 1956) the adaxial surface are hexagonal or more or less on ca. 70 species. Followed by Tomlinson (1956) polygonal in shape with a longer axis perpendicular on selected species in the family. Tomlinson (1956, to the longitudinal veins. In this case, polygonal 1960) who carried out a more comprehensive study shape refers to the two-dimensional shape with of the family, suggested a possibility of certain straight lines which consist of rectangular, anatomical features of the leaf being of diagnostic pentagonal, hexagonal, heptagonal and octagonal. significance and there is a great need to study Meanwhile, the abaxial epidermis is more irregular interspecific and intergeneric variation within the in shape and arrangement and generally slightly family. Interspecific anatomical variations in leaf transversely stretched. Different species present structure have been reported for Alpinia (Hussin et different epidermal cell sizes. The size of the al., 2000; Talip et al., 2005), Zingiber (Hussin & epidermis of P. cf. megacarpa is rather large Ibrahim, 1989), Boesenbergia and Kaempferia compared to other species with measurement ranging (Hussin et al., 2001), and Curcuma (Nonkratok et 20.68 – 23.14 µm long and 9.60 – 11.77 µm wide al., 2012). Study on intergeneric variation within on adaxial surface and 26.81 – 30.17 µm long and Zingiberaceae based on leaf anatomy is still lacking. 12.26 – 13.46 µm wide on the abaxial epidermis. In addition, the identification of Zingiberaceae Ergastic substances particularly silica bodies can be species especially tribe Alpinieae based on fresh observed in all species whereas calcium oxalate materials or herbarium specimens alone may crystals is clearly recorded in E. longipilosa only. sometimes result in wrong identification. Therefore, Figure 1 (a) to (f) illustrate the leaf epidermis of there is a need to utilize anatomical evidence in several specimens examined. addition to leaf morphological characteristics in species identification. The aim of this study was to Stomata preliminarily investigate whether or not leaf surface Generally, the stomata are amphistomatic but anatomy could be of taxonomic value in systematic occur much more frequent on the abaxial than and diagnostic investigations to differentiate the adaxial surface. The stomata are randomly between genera particularly in tribe Alpinieae. distributed in most species. Whereas in E. ANATOMICAL STUDY ON LEAF SURFACES OF BORNEAN ZINGIBERACEAE (TRIBE ALPINIEAE) 291 longipilosa, they are distributed in rows near veins Trichomes with fewer stomata randomly distributed in between. The types of trichomes that occur in the five The sizes of the stomata are different in all species species are of simple, unicellular, projecting and lie but constant on both surfaces. The guard cells lie at scattered on the leaf surface especially on the the same level as other epidermal cells and are of abaxial epidermis. Alpinia aquatica did not show the normal type with the long axis of the pore any presence of trichomes on both surfaces. No parallel to the veins. The stomata of all species trichomes are observed on its leaf sheath, ligule and studied are of tetracytic form which referring to four petiole as well. Trichomes on E. longipilosa, E. subsidiary cells that are constantly present around nasuta and P. cf. megacarpa are long and dense each of the stomata i.e. two on each lateral and especially on the abaxial surface while trichomes terminal, sometimes with slight irregularities. on A. xanthophlebium are short and rarer on the Stomata cells of P. cf. megacarpa are also larger abaxial surface. Figure 1 (e) and (f) present the type compared to other species measuring 21.15 – 23.57 of trichomes in selected species examined. Table 1 µm long and 13.07 – 13.93 µm wide. Figure 1 (c) presents the comparable leaf surface features of all and (d) show the types of stomata on abaxial the six species. epidermis in species examined.

Fig. 1. Leaf epidermis of studied specimens. (a) The adaxial epidermis of E. longipilosa with calcium oxalate crystals in each cell. (b) The adaxial epidermis of Geocharis sp. (c) Abaxial epidermis showing stomata of tetracytic type in E. nasuta. (d) Abaxial epidermis showing stomata of tetracytic form in P. cf. megacarpa. (e) – (f) Trichomes of simple, unicellular and long in E. longipilosa and P. cf. megacarpa. Scale bar = 200 µm. 292 ANATOMICAL STUDY ON LEAF SURFACES OF BORNEAN ZINGIBERACEAE (TRIBE ALPINIEAE)

Table 1. Leaf surface anatomical characters of specimens examined

Characters Alpinia Amomum Elettaria Etlingera Geocharis Plagiostachys Species aquatica xanthophlebium longipilosa nasuta sp. cf. megacarpa

Shape of Hexagonal or more or less polygonal epidermis

Presence of Silica bodies ergastic Silica bodies Silica bodies Silica bodies Silica bodies Silica bodies and crystals substances

Size of adaxial 13.18–23.04 × 16.63–17.68 × 14.70–20.95 × 15.34–20.34 × 17.05–20.46 × 20.68–23.14 × epidermis 5.40–6.96 µm 4.25–8.39 µm 5.69–7.08 µm 4.45–6.85 µm 4.26 – 6.75 µm 9.60–11.77 µm

Size of abaxial 9.29–16.74 × 14.83–16.76 × 11.03–15.64 × 12.35–17.74 × 11.29–13.16 × 26.81–30.17 × epidermis 3.61–6.68 µm 4.76–7.64 µm 4.56–6 µm 4.8–6.5 µm 4.39–6.45 µm 12.26–13.46 µm

Type of stomata Tetracytic

Size of 11.08–22 × 16.46–20.05 × 14.69–16.32 × 13.96–18.32 × 10.83–13.01 × 21.15–23.57 × stomata 8.04–10.45 µm 10.22–12.26 µm 8.55–9.70 µm 8.32–11.67 µm 7.93–8.85 µm 13.07–13.93 µm

The leaf surface of selected species studied ACKNOWLEDGEMENTS possessed characters as reported for the family Zingiberaceae by Tomlinson (1956, 1960). The We thank our colleagues and staff of Faculty of straight epidermal anticlinal walls are common in Resource Science and Technology, Universiti all species (Tomlinson, 1956). Whilst, simple and Malaysia Sarawak, Forest Department Sarawak for unicellular trichomes observed in most species are Research Permit (Permit No.WL20/2018) and also recorded as the only type for Zingiberaceae Sarawak Forestry Corporation for their great family. The absence of trichomes in leaf surface of assistance and useful suggestions in completing this A. aquatica was also recorded by Talip et al. (2005). study. The presence of hairs on the vegetative part of the is considered as distinct characters for the species. Meanwhile, the presence of crystals in REFERENCES epidermal cells of E. longipilosa may be the useful characteristic in differentiating the species or the Hussin, K., Ibrahim, H., Aminah, D. & Ali, H. genus. 2001. Anatomical variations in leaf of Study of leaf surface anatomy alone provided Boesenbergia O. Kuntze and Kaempferia L. limited information in order to learn variation species (Zingiberaceae). Journal of Tropical and between different genera in tribe Alpinieae. A detail Subtropical Botany, 9(1): 49-54. study of leaf anatomy should cover the appearance Hussin, K.H. & Ibrahim, H. 1989. Taxonomic of the midrib, petiole, and sheath in the transverse implications of several Zingiber species section which somewhat constant and characteristic (Zingiberaceae) based on morphological and for the genus. Besides, the constancy of these anatomical characteristics. Malaysian Applied diagnostic features can only be determined by Biology, 18(2): 155-161. investigating a much larger number of species in Hussin, K.H., Chua, T.S., Ibrahim, I., Wu, Q.G., Ping, each genus in the tribe. On the other hand, data on L.J. & Liu, N. (eds). 2000. Comparative leaf the size of epidermal cells and stomata, presence of anatomy of AIpinia Roxb. species (Zingi- crystals in epidermis, as well as the presence, length beraceae) from China. Botonical Journal of and density of trichomes may still be useful as Linnaen Society, 133: 161-180. supplementary characters for identification of the species. ANATOMICAL STUDY ON LEAF SURFACES OF BORNEAN ZINGIBERACEAE (TRIBE ALPINIEAE) 293

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