Comparative leaf anatomy of ten Nepenthes L. species (Nepenthaceae) from Peninsular Malaysia

The genus Nepenthes (Nepenthaceae) in Peninsular Malaysia includes fourteen species that can be found from the sea-level to over 1000 m in the mountains. Our observation indicated that Nepenthes can be found in five major habitat types: specifically in tropical lowland evergreen rain forest, heath forest, peat swamp forest, montane forest, and limestone forest. Their leaves have several anatomical characters that remain underexplored. There were specific differences between species that could be potential identification characters. The following anatomical characteristics were explored for their diagnostic value and comprised of ten distinct anatomical characteristics in Nepenthes, viz., density and stomatal index (SI), hypodermis cell – cell layers, occurrence of fiber groups mixed with the hypodermis cells, adaxial cuticle thickness, vascular bundle arrangement, midrib outline shape, venation plasticity, druses appearance and appendage/trichome type.


Introducti Introducti Introducti Introduction on on on
There are fourteen species of Nepenthes L. recorded in Peninsular Malaysia and mainly distributed in heath forests, lowland area, including montane species up to 1000 m altitude and above (Table 1)  . The primary centers of diversity of Nepenthes are in Borneo, the Philippines and Sumatra, with updated enumeration of 170 species already recorded. It is also important to include their secondary centers of diversity that covers Peninsular Malaysia, Sulawesi and New Guinea (Jebb and Cheek, 1997;Cheek and Jebb, 2001;Christenhusz, 2016;Clarke, 2001 and2002;McPherson, 2009;Robinson et al., 2019). Out of the fourteen Nepenthes species recorded in Peninsular Malaysia, ten are considered endemic, one -N. gracillima seems to need to be further study and confirmation, and the other three annotated species are noted as common species with wider distribution records. Upper hill forest habitat (800 -1000 m) 11. 11. 11. 11.

N. sanguinea
Lower and upper montane forests (300-1800 m) Pitcher's shape in morphological observation is the main character used for systematic identification of Nepenthes (Ridley, 1924;Corner, 1988). Nepenthes produces leaves that form into the shape of a jug with watery fluid inside intended to absorb and digest nutrients from the insects, organic matter or dead leaves trapped into the pitcher fluid, which are later transported to other parts of Nepenthes that promote growth and survival needs (Clarke, 2001 and2002). Pitcher morphology is very important for the identification process as there is confusion due to the wider morphological variation between true species and their natural hybrids (Danser, 1928;Beveridge et al., 2013). In view of this, is important to study other systematic characters of Nepenthes to assist alternative identification for this genus. Difficulties in identification of sterile Nepenthes specimen without lower or upper pitchers may also result in wrong identification and interpretation of the species.
Investigation of leaf anatomy provides as much information as alternative identification from the leaf lamina, venation and stomatal type, petiole, margin, appendages variation and vascular bundle patterns. de Candolle (1879) conducted a comprehensive study on petiole anatomy that enabled the description of several fundamental concepts of vascular bundles that can be implemented in other genus or family descriptions. Metcalfe and Chalk (1950) pioneered the study of Nepenthes anatomical characteristics using the slides collection obtained from Kew Botanic Gardens and successfully enumerated the basic qualitative observations from its basic anatomical description. Several other studies were conducted by Toma et al. (2002), Pavlovic et al. (2007), Biati (2012) and Paluvi et al. (2015) using cultivated Nepenthes specimens of N. maxima, N. mirabilis, N. alata and N. gracilis, but the studies are still incomplete due to the specimen collection restrictions and lack of wild specimen enumeration.
Limited knowledge and information on the anatomical descriptions of Nepenthes species, specifically the Peninsular Malaysia collections, prompted further evaluation and characterisation, complementing the existing type specimens in safekeeping. Thus, the aim of this research is to analyse the anatomical description of Nepenthes and its systematic information that can be utilised for alternative identification of the Nepenthes species studied.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Nepenthes specimen authentication and observation were conducted based on the available specimens from two herbaria (UKMB and KEP). All the ten Nepenthes species were collected in the field following the methods described by Martins and Filgueiras (2010). All examined specimens are listed in Table 2. Fresh collected specimens were removed from the middle third of a leaf segment, located in the median leaf region. For some species which required herbarium material observation, the sample was rehydrated in 50% (v/v) aqueous glycerine in a stove at c. 60 °C for 48 h, dehydrated in an ethanolic gradient and conserved in 70% ethanol. We analysed at least three individuals per species. Anatomical study Cross-sections were made with a sliding microtome (Leica SM2500). Paradermal sections were made using acid dissociation (Krauss and Arduin, 1997). Standard plant anatomy procedures were used to make slides (Johansen 1940). At least three replicates were used per individual. The sections were analysed using a Zeiss optical microscope and the images obtained with a Leica photomicroscope associated with the computer, using the LASEZ image capture system.
Our study shows that there is significant anatomical variation among the ten species of Nepenthes studied. Some anatomical leaf characters in the Nepenthes species could be generally influenced by environmental conditions; however, some characters are stable and have taxonomic significance i.e. venation pattern plasticity, druse observation, midrib outlines, vascular bundle arrangements and also leaf margin outlines. In some species, these characters could be further analysed due to the uniqueness of the characteristics that can serve as diagnostic characteristics specifically involving taxa having leaf anatomical characters influenced by environmental conditions such as N. ampullaria, N. macfarlanei and N. ramispina in which the analysed features of appendages type, layer of hypodermis are varied. Figure 10. Figure 10. Figure 10. Five types of vascular bundle arrangements combined with four types of midrib outer shape were identified (Figures 11, 12, 13). The vascular bundle is arranged in collateral, which too had been explained by Metcalfe and Chalk (1965). The results have shown that all the Nepenthes species studied have a complex vascular structure with closed or open ring system and consist of additional and medullary vascular bundles. This result supported the findings by Metcalfe and Chalk (1979). The complex vascular structure present in all the species studied consists of outer and medullary vascular bundles with closed or open ring system. Specifically, this is the first effort in determining and describing the complex structure of plant vascular tissues in Nepenthes , observation of Type 1 (adaxial VB: five VB in stacks; medullary VB: absent, additional VB: present as several smaller size VB near abaxial epidermis); Type 2A (outer ring present: circular in shape; medullary VB present: one to three scattered VB; additional VB: absent); Type 2B (outer ring present: triangular in shape; central or medullary VB present: one to three scattered VB; additional VB: present -several smaller size VB near abaxial epidermis); Type 3 (outer ring present (rectangular in shape); medullary VB: absent; additional VB present: several smaller size VB near epidermis abaxial and two VB present on the adaxial side of outer ring VB at the left and right side); Type 4 (outer ring present: triangular in shape; medullary VB: one to three scattered VB; additional VB: present as several smaller size VB near abaxial and adaxial epidermis) and Type 5 (outer ring present: triangular, elliptic or rectangular in shape; medullary VB: one to three scattered VB; additional VB: several smaller size VB near abaxial epidermis and two VB present on the adaxial side of outer ring VB on the left and right side). Type 1 was present in Nepenthes macfarlanei,Type 2A in N. ramispina,Type 2B in N. alba and N. ampullaria,Type 3 in N. rafflesiana,Type 4 in N. gracilis and Type 5 in N. benstonei,N. mirabilis,N. sanguinea and N. albomarginata (Figure 11). Figure 11. Figure 11. Figure 11.   Based on the vascular bundle arrangement in the petiole, it is clearly shown that this character can be useful in species differentiation, especially for the ten studied species. It is also clearly shown that the types of vascular bundle systems can be used for species grouping in the genus Nepenthes. These findings agree with the study of Mohamad Ruzi (2007), who found that the types of vascular bundle based on the arrangement and system in the vascular bundle strands can be used for grouping of species in Dipterocarpus and help in species identification. As Metcalfe (1942) stated, the anatomy of both leaf and stem gives numerous characteristics, which in combination are reliable for diagnostic purposes; some species can be identified with certainty based on the vascular bundle arrangement (Figure 11). Therefore, anatomical characteristics could be valuable for taxa identification and differentiation.
For outer shape of the midrib, four outline types were characterised in this study, which is Type 1 (Abaxial: prominent UV-shaped; Adaxial: very slightly concave) found in Nepenthes benstonei, N. mirabilis, N. sanguinea, N. albomarginata and N. ramispina; Type 2 (Abaxial: arch shaped; Adaxial: slightly flattened) characterized in N. gracilis and N. ampullaria, Type 3 (Abaxial: prominent UV-shaped; Adaxial: prominent Ushaped) in N. rafflesiana and Type 4 (Abaxial: prominent V-shaped; Adaxial: prominent wide V-shaped) which is showed in N. macfarlanei & N. alba). This characters clearly distinguished N. rafflesiana from other Nepenthes taxa and can be used in diagnostic charaterisation (Figures 12 and 13). Selected anatomical characters observed in the Nepenthes species studied are stomatal density and length, trichome types and distribution, cuticle thickness, thickness of adaxial hypodermis, and the number of adaxial hypodermis layers (Table 3). Some characters are present in all the species studied and considered common characters that enumerate this carnivorous plant genus; for example, all the species studied have a single layer of palisade cells with variable height. Druses are present in enlarged idioblast cells in palisade layers of most of the studied species. Other anatomical leaf features are also fairly uniform. Two to four palisade layers are found with extracellular spaces in spongy cell layers. Sessile glands tend to be orbicular and flower-like in shape, spread on adaxial and abaxial surfaces, and the base is buried up to the hypodermal layer. In N. alba and N. albomarginata, druses are not only confined to the idioblast cells, but also occur in the palisade and spongy cell layers and in the vascular tissue, that can be observed in the parenchyma cells of the midrib, and in the phloem cells. However, most of the species showed the occurrence of solitary crystals in the palisade and spongy mesophyll cells, also in the marginal and in the leaf lamina. Anomocytic stomata were present in all species, which is considered as characteristic for Nepenthaceae (Metcalfe and Chalk, 1950). Anomocytic stomata are also known as ranunculaceous type of stomata (Metcalfe and Chalk, 1965). This feature also in line with the finding involving new taxa of N. domei, N. latiffiana which also characterized the same anomocytic stomata features .
Stomata anatomical characteristics can be used in species identification and also in dividing the species based on its ecological habitat.  (2012) noted that stomatal density can be affected by environmental and climatic conditions such as drought and will influence stomatal count in plant species. Tomlinson et al. (2011) also stated that stomata in pitcher plants do not necessarily occur in continuous regular longitudinal rows. Hypodermis cells are the cells like those found in Poaceae that have the same function as bulliform cells (adaxially occurring in grasses species leaf lamina) (Turpe, 1967;Oliveira et al. (2015); Reis et al. 2015). The hypodermis cells in Nepenthes are present on both abaxial and adaxial surfaces. Variations between species in number of cell layers and the occurrence of fibre groups mixed with the hypodermis cells were significant for species differentiation in Nepenthes. Hypodermal cells layer on the adaxial side of Nepenthes are varied, compared to the abaxial side. This study supported the previous study by Biati (2012) which found N. gracilis and N. rafflesiana having a single layer of hypodermal cells, while typical highland species such as N. gymnamphora have between 3 and 4 variable layers. It demonstrates that hypodermal layers and their thickness will increase according to the altitude or elevation of the natural habitat (Toma et al., 2002). Analysis showed that adaxial hypodermal layer varies within species -N. alba, N. sanguinea, N. mirabilis,N. rafflesiana, N. ampullaria and N. albomarginata with single layer, N. gracilis and N. benstonei with 1-2 layers, N. macfarlanei with 1-4 layers) and N. ramispina with 1-3 layers (Table 3).
In this study, it is noted that the adaxial cuticle thickness varies from 0.8-12.5 μm, tending to be thicker than the cuticle on the abaxial side. However, at the base of N. mirabilis the leaf blade occasionally has some part not covered by cuticle. Riederer (2006) and Javelle (2010) stated that trichome and cuticle function against abiotics response were not much different, such as control transpiration, temperature, gas exchange and sunlight intensity. Lowland associated species such as N. ampullaria and N. mirabilis have adaxial cuticle to 5.9μm thick (Table 3). Typical highland species such as N. albomarginata, N. macfarlanei, N. ramispina, N. sanguinea and N. alba have adaxial cuticle to 12.5μm thick. The differences may be due to the population of these species, which grow in colder climate and nearby water resources. Therefore, there is less effort to stabilize transpiration on the leaves, considering that cuticles and trichome are modifications from epidermis (Haron, 2008) and both of those modifications appear due to the influence of environmental stress (Esau, 1965). Table 3. Table 3. Table 3. Prominent leaf characters play an important role in supporting identification and classification; for example, the number of nerves and secretory cells are usually diagnostic, especially when complete specimens are available for observation (Dickinson, 2000). Combinations of marginal and areolar venation patterns for certain taxon are unique, the veinlet endings vary greatly among them, namely uni-, simple and linear, bi-, with their ultimate marginal venation complete or incomplete (Table 4). The abundance of druses on the lamina surface of N. alba and N. ramispina can be a diagnostic character for them (Figure 8 H, I, Figure 10 H, I). The presence of swollen veinlets or swollen tracheids can also be used as additional characters for differentiation.
For example, swollen veinlets are present in N. ampullaria ( Figure 2M) and N. macfarlanei (Figure 5 L). Three veinlet patterns were observed in this study, namely simple and linear veinlets, uni-veinlets, bi-veinlets. In particular, N. alba showed no ending veinlets and this is diagnostic for this taxon. Others showed either uni-, or simple and linear, or bi-veinlets and these can be the diagnostic characters for taxa identification. Only one type of ultimate marginal venation was observed in this study, which is closed ultimate marginal venation, which refers to higher vein order fused into a vein running just inside the margin. Another systematic evidence is the variation in the appendages between Nepenthes taxa ( Table 5). Combination of tufted and simple unicellular trichome on the abaxial leaf blade surface can be observed in N. ampullaria and simple unicellular trichome on the abaxial leaf blade surface in N. albomarginata, N. benstonei, N. mirabilis, N. rafflesiana and N. ramispina, while both abaxial and adaxial parts of the leaf surface were demonstrated in N. macfarlanei and N. alba, which is supported by the studies of Metcalfe and Chalk (1950, 1965and 1979 and Farishy (2017) on the variation of appendage type and dispersion on the leaf surfaces of Nepenthes.

Conclusions Conclusions Conclusions Conclusions
There are ten distinguishing anatomical characteristics of Nepenthes that combine the features of density and stomatal index (SI), hypodermis cell -cell layers, occurrence of fibre groups mixed with the hypodermis cells, adaxial cuticle thickness, vascular bundle arrangement, midrib outline shape, venation plasticity, druses appearance and appendage/ or trichomes type. Some of these characters such as stomatal density, hypodermis cell analysis and cuticle thickness can be influenced by environmental conditions specifically in altitudinal changes but some characters are worth interpretated in taxonomic significance that involves venation pattern plasticity, druse observation, midrib outlines, vascular bundle arrangements and also leaf margin outlines. These characters are important and can serve as systematically related features or diagnostic characters for Peninsular Malaysia's Nepenthes species interpretation, as well as an additional identification evidence in supporting morphological observation.