Nepenthes latiffiana and N. domei (Nepenthaceae), two new species of pitcher plants from Terengganu, Peninsular Malaysia

Two new species of Nepenthes from Terengganu, Peninsular Malaysia, N. latiffiana M. N. Faizal, A. Amin & N. Dome and N. domei M. N. Faizal, A. Amin & A. Latiff, are described and illustrated.


INTRODUCTION
In Peninsular Malaysia, a total of eleven species of pitcher plants ( Nepenthes) or locally known as "periuk kera" have been recorded (Jebb and Cheek 1997;Cheek and Jebb 2001;Clarke 2001;McPherson 2009). The first taxonomic account of the genus in Peninsular Malaysia was given by Ridley (1924) who recorded a total of ten species, namely N. ampullaria Jack., N. albomarginata Lobb., N. gracillima Ridl., N. alba Ridl., N. ramispina Ridl., N. sanguinea Lindl., N. macfarlanei Hemsl., N. gracilis Korth., N. rafflesiana Jack and N. phyllamphora Willd. (= N. mirabilis (Lour.) Druce). After Ridley's treatment, there have been some further researches on the genus by Holttum (1940), Kiew (1990), Turner (1995), Clarke (2002), Adam et al. (2005) Latiff et al. (2011), Clarke and Lee (2012) and Latiff and Norsiah (2016) regarding the diversity, distribution, and ecological study of Nepenthes species and natural hybrids. Another taxonomic study of the Peninsular Malaysian taxa was carried out by Rohana in 1988 that covers basic anatomical features and systematic study of the species and hybrids. Turner (1995) listed ten species, which instead included two natural hybrids, namely N. × hookeriana and N. × trichocarpa, with N. alba and N. ramispina being excluded. Clarke (1999) added N. benstonei, a species then known from Bukit Bakar, Kelantan, to the list and the latest was Adam and Hamid (2007) who described N. sharifah-hafsahii which happens to be a natural hybrid between N. gracilis and N. mirabilis rather than a true species (McPherson 2009).
The most comprehensive and well accepted taxonomic enumeration works of the genus were given by Jebb and Cheek (1997), Cheek and Jebb (2001) and Clarke (2001) who recognised eleven species. In the course of continuous field trips to the state of Terengganu, the authors had encountered several populations of pitcher plants in Setiu whose features did not match the currently described taxa of Peninsular Malaysia. From this finding, we further analysed the morphological, anatomical and micromorphological characteristics and have discovered two distinct new species (as described in this article). The descriptions of N. domei and N. latiffiana herein can be considered as a comprehensive work in the taxa identification as the sheet contains the sterile/fertile parts and pitchers, anatomical and micromorphological evidences, and enumeration of prominent plant characteristics. We also conducted a comparative molecular study on N. domei using internal transcribed spacer (ITS) DNA region to further differentiate it from a closely related species.

MATERIALS AND METHODS
Analyses of morphological characteristics on new Nepenthes specimens were based on living plants observed in situ and also from herbarium type specimens (MDI12424 and MDI12423) deposited at MARDI Herbarium (MyGenebank™ Complex MARDI Serdang).
For leaf anatomy analysis, collected and described Nepenthes leaves were fixed in AA solution (1 Acetic acid : 3 Alcohol). Investigated leaf parts including midrib, lamina and margin were sectioned in a range of thickness from 15-30 μm using sliding microtome. The leaves then were cleared using bleaching agent, washed in various alcohol series and stained in Safranin and Alcian blue for 5 minutes each before proceeded for dehydration stage through an ascending alcohol series (50-100%). During the final dehydration stage, the leaf specimens were differentiated in 70% alcohol with a drop of hydrochloric acid (HCL) before mounted in Euparal. Specimen slides were kept in the oven for two weeks at about 60-70°C. Photographs of the investigated sections were taken using Olympus SZH40 microscope and the images were processed using CellSens Image Analysis software.
For micromorphology study, Nepenthes epidermis preparative fragments about 3 mm long were excised from the middle portion of approximately three mature leaves of each species. Cuticle parts were prepared by soaking leaf blade fragments in 5-10% aqueous chromium trioxide until all organic material except the cuticle was dissolved. Targeted leaf cuticles were mounted on aluminium stubs with double-sided adhesive tape and proceeded for air drying. The stubs were then sputtercoated with pure gold to a maximum thickness of 15 nm and examined with LEO (Model 1450 SEM) -Field-Emission Scanning Electron Microscope (FESEM). The remaining cuticles were soaked in 5% ammonia and mounted on microscope slides in Canada balsam.
Results of the adaxial and abaxial epidermal layers under scanning electron microscope (SEM) and leaf anatomical study of N. domei, N. latiffiana, N. benstonei and N. sanguinea are shown in Figure 5 to 12 and summarised in Table 2 and 3. These results are combined with transverse section of the leaf lamina and margin showing epidermal cells, mucilaginous idioblast, glandular and simple trichomes, solitary crystals observation on the parenchyma and along a vein, and significant venation characters. The systematic significant of leaf anatomy and micromorphology evidences differentiating these investigated Nepenthes species are presented in Table 2 and 3. Finally, for molecular study, all genomic DNA purifications were done according to DNeasy Plant Mini Kit (Qiagen) manufacturer's protocol and matured leaves (100 mg per sample) from N. domei sp. nov. (Terengganu), N. benstonei (Bukit Bakar, Kelantan) and N. sanguinea (Fraser's Hill, Pahang) were used. The internal transcribed spacer (ITS) partial gene (~610bp) was PCRamplified from Nepenthes DNA using universal ITS1-2 primers (ITS_F: 5'-AGGAGAAGTCGTAACAAGGTT; ITS_R: 5' GATGCAACCTTGGCCTT) and Q5 High-Fidelity DNA polymerase (New England Biolabs, UK). The parameters for thermocycler were set as follows: initial denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 61°C for 15 s, extension at 72°C for 20 s, and final extension at 72°C for 2 min. The amplicons were sent for Sanger sequencing and the sequencing data was trimmed and aligned using BioEdit Sequence Alignment Editor. Phylogenetic inference of nine peninsular Nepenthes species (including N. domei sp. nov.) was reconstructed in Molecular Evolutionary Genetics Analysis software (MEGA5) using Neighbor-Joining (NJ) method (1000 bootstraps) based on the ITS partial sequences isolated in this study as well as sequences obtained from the NCBI database (Bunawan et al. 2017;Alamsyah and Ito 2013;Renner and Specht 2011

Diagnosis
Nepenthes latiffiana differs from N. sanguinea in peristome morphology which is considerably developed, loosely cylindrical, with expanded outer margin part towards both sides of the mouth forming flap-like structure which is especially prominent in upper pitcher (vs. simple, expanded, outer margin usually markedly sinuate where the peristome is widest); climbing stem with simple hairs, cross section cylindrical (vs. glabrous, sharply angular) and lid margin slight wavy to wavy that retains its morphology in dried and wet preserved specimens (vs. flat).

Description
Terrestrial climber to 0.5-1.5 m tall. Climbing stems cylindrical to rounded, particularly towards the leaf nodes, 0.8-1.2 cm in diameter. Internodes 2.2-3.5 cm long. Leaves coriaceous, sessile to less amplexicaul, linear-lanceolate, apex obtuse to emarginated, 15-20 cm long, 5-6 cm wide; base clasping stem for about ½ of its circumference; longitudinal veins 1-3 on each side of the midrib, pennate nerves conspicuous; tendril with slender formation in the middle, 10-14 cm long, with minute hairs. Lower pitchers up to 18-26.5 cm long, 4.9-8 cm wide, fleshy coriaceous texture, arising abruptly from the tendril; broad cylindrical at the lower part of the pitcher, ½ upper of the pitcher slightly swollen with hip formation, 9 cm length, up to 7 cm above of the hip till nearly closer to the mouth; cylindrical above, with slight widening towards the mouth, surface of the hip smooth; two fringed wings up to 0.9-1.0 cm wide, runs along the pitcher and widest at the mouth, with multicellular hair elements up to 0.3-0.6 cm long; spur with branched form, reaching 1.0 cm long; mouth rounded; peristome loosely cylindrical but widening or expanded towards lateral sides of the pitcher mouth (ca. 1.5 cm wide each side), outer surface smooth, reaching 1.1 cm wide (in front of the mouth), ribs (at the part of the peristome) reaching 1.6 cm wide; lid rounded, up to 6.5 cm long and 6 cm wide, base retuse, without appendages or trichomes on the lower surface of the lid, ~105-126 dotted nectar glands superfluous and dense at the center and base of the lid. Upper pitchers up to 20-23.5 cm long, 5-7.5 cm wide, coriaceous texture, arising slight abruptly from the tendril; broad cylindrical at the ¼ lower part of the pitcher, ¾ upper of the pitcher cylindrical and slight narrowing towards the mouth; with minute wings; mouth ovoid to sub ovoid, peristome cylindrical but with expanded lateral sides (1.3-1.4 cm wide each side) and flattened towards the front of the mouth part, outer surface smooth, reaching 9mm wide, ribs reaching 1.3 mm wide; lid rounded, up to 4.8-5.2 cm long and 5.5 cm wide, base cordate, without appendages or trichomes on the lower surface of the lid. Male inflorescence a raceme, peduncle up to 16 cm, rachis up to 40 cm, partial peduncles single-flowered, bracts absent, pedicel 10-13 mm long, tepal ovate, up to 2-5 mm, staminal column 2.5-6 mm long, anther head 2-3.75 mm. Colour of the living plants: Lower pitcher combination of green-yellowishbrown hues with several red blotches formation on the 1/3 of the upper part of the pitcher body. Upper pitcher light green with fade specks. Pitcher interior greenyellowish to reddish. Leaves green. Stem green to dark green. Colour of the dried specimen light brown -pale brown (lamina, stem and pitcher).

Etymology
Named in honour of Emeritus Professor Dato' Dr. A. Latiff Mohamad, a prominent figure in Malaysian Botany Field from Universiti Kebangsaan Malaysia, Bangi Campus, Malaysia.

Distribution and habitat
Nepenthes latiffiana is currently known only from the restricted area at the type locality in Setiu, Terengganu.

Ecology
Nepenthes latiffiana occurs as terrestrial climbers on hilly terrains with slight shaded, upper hill forest habitats, at an altitude of c. 1000-1100 m.

Diagnosis
Nepenthes domei differs from N. benstonei in several lower pitcher characteristics that include thick-leathery coriaceous texture (vs. coriaceous texture), broad rounded formation in the lower 1/3 or 1/2 part of the lower pitcher (vs. broad ovoid -ovoid in the lower part), two fringed wings 5.5-7 mm wide narrowest at the base and widest at the mouth (vs. less than 4mm runs the whole length of the pitcher body), multicellular fringe elements up to 2-4 mm long (vs. overally shorter, 1-3 mm long). The lower pitchers of the new species frequently burrow into humus, a feature that has not observed in N. benstonei from Bukit Bakar. The lower pitchers exhibit whitish red to dark ruby red colouration with the majority of individuals sparsely overlain with or entirely absent of specks on the inner and outer wall (vs. brownish red-dark red, inner wall heavily overlain with specks). The inflorescence rachis length 28-35 cm (vs. less than 30-31 cm). Leaves and stems without waxy cuticle (vs. covered with waxy cuticle).

Description
Terrestrial climber to 3.5 m tall. Stem and climbing stems slight rounded in cross section, up to 0.7-0.9 cm in diameter. Internodes 4-8 cm on climbing stems and 1.3-2.5 cm on rosette stems. Leaves of lower stem coriaceous, lanceolate, sessile, 12-13 cm long and 4-5 cm wide, sheathing the stem up to ¼ of its circumference. Leaf blades of the climbing stems narrowly linear-lanceolate, sessile, 25-32 cm long, 5-8 cm wide, base contracted into the stem up to ½-¾ of the stem, apex narrow acuminate, tendril insertion simple. Longitudinal nerve 1-2 on each side of the midrib, pennate nerves spreading towards the leaf margins. Leaves of climbing stems with slight differentiation to those of the lower stems which is bigger in size. Tendrils coiled, up to 13-37 cm long. Rosette/ lower pitchers up to 15-17 cm long, 4.4-6.5 cm wide; thick-leathery coriaceous texture; arising slight gradually from the tendril; broad rounded formation at the lower part ca. 1/3 with slight pronounced hip, straight cylindrical towards above, widening slightly towards the mouth. Inner surface of the portion below the lid throughout surfaces above the hip smooth and waxiness, light green in colour; two fringed wings up to 7 mm wide (narrowest at the base and widest at the mouth), run from the top of the pitcher to the bottom of the pitcher, consist of multicellular fringe elements up to 2-4 mm long; mouth ovoid-rounded and oblique, concave, rising at the rear of the pitcher towards the lid; peristome slight cylindrical, front up to 6 mm wide, sides up to 4 mm, up to 1.2 cm wide at the rear; outer surface with minute unicellular trichomes, lid ovate, up to 2.7 cm long and 2.4-2.5 cm wide, base generally rounded (not cordate), no hairs or appendages observed on the lower surface. ~60-87 dotted -rounded nectar glands scattered on the lower part of the lid, and concentrated at the base of the lid. Upper pitchers 10-12 cm long, 1.5-4 cm wide, thin coriaceous; arising gradually from the tendril; slightly infundibular to cylindrical in the lower part, upper part cylindrical and slight widening towards the mouth; peristome similar to the lower pitchers, differs in mouth shape (wide and length of the mouth and peristome); lid narrow ovate, up to 1.8 cm long and 2.3 cm wide, base rounded (not cordate), no hairs or appendages on the lower surface of the lid; spur simple, unbranched less than 0.5-0.6 cm. Male inflorescence a raceme, peduncle up to 13 cm, rachis up to 35 cm, partial peduncles single-flowered, bracts absent, pedicel 11-14 mm long, tepal wide ovate, up to 4-6 mm, staminal column 3-4 mm long, anther head 2-2.5 mm. Colour of living plants lower pitcher whitish red to dark ruby red throughout or green-reddish, sometimes with a few red-purple blotches on the inner surfaces and lid, peristome dark red to green with varying degrees of green and red. Leaves and stems without waxy cuticle. Stems reddish-green to green. Leaves dark green. Colour of dried specimens dark brown (lamina, stem and pitcher).

Etymology
Named in honour of the first person who discovered the population, Mr. Dome Nikong.

Distribution
Nepenthes domei is currently known only from the restricted area at the type locality in Setiu, Terengganu.

Ecology
Terrestrial climber in humus rich soil, immature and mature lower/rosette pitchers tend to burrow in the soil rich with humus up to 1/3 -1/2 of the pitcher. Upper hill forest habitats, at an altitude of c. 850-1000 m.

Conservation notes
Nepenthes domei and N. latiffiana are terrestrial climbers; with N. latiffiana observed growing as understorey  plants on a hill slope with rocky substratum at an elevation about c. 1100 m. The vegetation of the locality consisted of various montane species dominated by Dicranopteris linearis (Gleicheniaceae), Dipteris conjugata (Dipteridaceae) and Machaerina maingayi (Cyperaceae), thriving together with rattans, Pandanus sp. and Leptospermum flavescens. Some climbing stems of N. domei were also observed scrambling over Pandanus tree. Based on our observation, more than five individuals of N. domei were seen at three scattered locations, while only one to two individuals of N. latiffiana were recorded at a single location. Both species are known to be restricted in a single mountain since it was discovered. Thus, we assign N. domei and N. latiffiana in this article as Vulnerable under criterion -D2 of IUCN (2001). The habitat had experienced logging in the past; hence, this could explain the limited number of the individuals discovered in the area.  Upper pitchers Up to 10-12 cm high × 1.5-4 cm wide.
Less than 15 cm high × and 3 cm wide.

Anticlinal walls of lamina epidermal cells under light microsope (LM)
According to Stace (1969), the lamina surface has been the focal subject of investigations (as compared with other plant surfaces) that some of studied taxa showed consistent characteristics which often serve as a taxonomic application. From observations of the lamina surface in studied Nepenthes, the anticlinal wall of N. domei is straight to slight curve for both abaxial and adaxial epidermis surfaces while N. benstonei showed straight to wavy adaxial epidermis surface and sinuous abaxial epidermis surface. On the other hand, N. latiffiana showed straight to straight curve epidermis wall for both of its surfaces as compared to N. sanguinea which possessed straight -slightly curve and straight for its abaxial epidermis. Therefore, these characters appear to be diagnostic for those two new species, respectively.

Margin and Midrib Transverse Section (TS)
Studied Nepenthes species showed interspecies variations in lamina margin transverse section (TS) which can serve as a useful characteristic for species identification. The outline of the TS of the margin is bluntly rounded (pointed 75-80°) for N. domei, sharply pointed downwards that reached 85° for N. latiffiana, sharply pointed with 20-25° curve for N. benstonei, while N. sanguinea having up to 80° and rough creased abaxial and adaxial surfaces. Midrib outline for N. domei, N. sanguinea and N. benstonei is characterised as Type I (abaxial with prominent U-V shape and adaxial with very Table 3. Comparison of leaf anatomical features between Nepenthes domei, N. latiffiana, N. benstonei and N. sanguinea (Figure 9-12 slight concave shape) while N. latiffiana having Type IV (abaxial with prominent V shape and adaxial with prominent wide V shape) midrib outline. For midrib TS, the variations are as follows: N. domei is having Type I (adaxial vascular bundle in stacks, medullary vascular bundle absent, additional vascular bundle present in several smaller size nearby the abaxial epidermis) vascular bundle arrangement, while N. latiffiana, N. benstonei and N. sanguinea are having Type VI vascular bundle arrangement (outer ring present, elliptic; central medullary vascular bundle present in scattered formation; additional vascular bundle present in several smaller size nearby the adaxial side of outer ring at the left and right side). These observations (disparity in vascular bundle arrangement combined with midrib outline) agree with some studies conducted by Metcalfe and Chalk (1950) who demonstrated the two characteristics could greatly serve for diagnosis in certain species, as recorded and showed in some Parashorea species.

Marginal and areolar venation
The variation of leaf venation patterns of angiosperms was extensively studied and classified by Hickey (1979) and Sun et al. (1997) and this further addresses the importance of comprehensive treatment in taxon differentiation. Leaf architecture is primarily used for classification which includes the leaf shape, leaf margin structure and other possible characters. Indeed, the characterisation of an angiosperm leaf venation pattern starts by observing the primary veins and then proceeded to the branching secondary veins. Combination of marginal venation and areolar venation patterns for every taxon are unique, even for those taxa classified under the same genus (Inamdar et al. 1983). In studied Nepenthes species, marginal and lamina/areolar venation features showed diagnostic characteristics that are useful in species differentiation and evaluation. Nepenthes domei and N. sanguinea showed incomplete univeinlet, while N. benstonei and N. latiffiana are characterised with closed type, with none veinlet. Another feature analysed is the areolar / lamina venation, in which all taxa showed incomplete areolar venation but varied in type of venation ending as follows: N. domei showed simple veinlet with uni-veinlet (linear to curved), while N. latiffiana and N. benstonei shared the same feature of uni-bi veinlet (curved) that slightly differs in N. benstonei venation ending with linear-curved (1-3 branched) and N. sanguinea characterised with uni-veinlet (slight curved). These variable patterns of leaf venation are taxonomically significant in these pitcher plants species as they can be used as additional evidences for species differentiation.
Cuticular wax and epidermal sculpturing under scanning electron microscopy (SEM) Cuticular sculpturing also holds considerable diagnostic values as reported by Wilkinson (1979) and Wu et al. (2005). In this study, the epidermal surfaces revealed a number of important micromorphological characters, exhibited interesting interspecies variations that are significant for species identification. Leaf epidermis surfaces (abaxial and adaxial) of the investigated Nepenthes species that were viewed under the SEM (low to high magnifications) gave certain features -distribution of idioblastic elements such as trichomes, glands and stomata, combined with the appearance of epicuticular wax. In this study, the appearance of epicuticular wax on both of the leaf surfaces could be tubular granular or flaked, or a combination of different types of wax. Nepenthes domei differed from others for having granular wax on both abaxial and adaxial epidermis surfaces, and tubular wax only on abaxial surface ( Figure  5). Meanwhile its closely related species, N. benstonei, showed numerous wax flakes (Figure 7) for its adaxial epidermis surface and granules for its abaxial surface. It seems the presence of these numerous flakes supports Clarke's (2001) observation saying that the leaves and stems of N. benstonei were covered with thick waxy cuticle. Nepenthes latiffiana showed similar features as that of N. benstonei, while N. sanguinea had wax flakes on both abaxial and adaxial surfaces. Stomata features also implicate a significant diagnostic value for our species of interest. Nepenthes domei and N. sanguinea leaves are amphistomatous (stomata on both surfaces); however N. benstonei and N. latiffiana are hypostomatous (stomata only on the lower surface). While hypostomatous stomata feature is common for the majority of species for monocotyledons and dicotyledons (Meidner and Mansfield 1968), amphostomatous feature is much rare (Drake et al., 2018). Drake et al. (2018) further points out that amphistomaty may help in better CO2 absorption but with higher rate of transpiration and this discovery of two different groups of leaves (hypostomatous vs. amphostomatous) within the same genus is truly intriguing. The indumentum feature in dicotyledons was reported to consist of simple unicellular and various glandular trichomes (Metcalfe and Chalk, 1950). In this study, glandular trichomes was present in all species but N. domei showed diagnostic character with the observation of scale-type trichomes, known as peltate, on its abaxial epidermis surface that did not exist in other investigated species. Simple unicellular trichomes are common and present in all species of studied Nepenthes except in N sanguinea that also can be postulated as a diagnostic characteristic that differentiates N. latiffiana from N. sanguinea. This trichome type can be long or short, and has either thick wall ornamentation or smooth.
Identification key based on anatomy and micromorphology characters

3) Molecular analysis of ITS sequence to infer the relationship between N. benstonei and other species
The ITS is a nuclear DNA region which is highly conserved and it has been used successfully to discriminate different plant species including Nepenthes (Giudicelli et al. 2015;Gogoi and Bhau 2018). Based on nucleotide search on the public NCBI database (BLASTn), we noted partial ITS region (~610bp) is more than 95% conserved among the majority species in the Nepenthes genus and the ITS may not be sensitive to differentiate sub-populations under the same species. However, we tested the degree of ITS sequence conservation from a few different populations of N. sanguinea in Peninsular Malaysia and it is proven the partial ITS sequence is 100% conserved within this particular species (data not shown), saying that ITS intraspecific variation of a closed population should be close to 0.
In order to further establish N. domei as a separate taxon from its most closely related species N. benstonei, we conducted a molecular analysis by comparing the partial ITS sequence from both plants ( Figure 13) and then inferred phylogenetic relationship (NJ method) of the new species with nine peninsular Nepenthes species (Figure 14). Based on sequence alignment, N. domei sp. nov shares up to 97.2% ITS nucleotide identity to N. benstonei, and the 2.8% nucleotide dissimilarity -a figure significant enough to differentiate Nepenthes species in this study -is due to nucleotide polymorphisms that occur at 15 different positions, denoted as non-coloured letters (Figure 13). In addition to this, the phylogenetic inferring has positioned N. domei on a separate branch from N. benstonei in the highland/intermediate highland clade (Figure 14). This molecular scrutiny -on top of morphology, leaf anatomy and micromorphology -serves as an addition evidence that has positioned N. domei as a distinct species from N. benstonei.

CONCLUSION
The combination approach of morphology-anatomy-micromorphology analyses used in this comparison study is useful in providing as much aspects as possible in distinguishing plant characteristics of N. domei and N. latiffiana against their congener species, N. benstonei and N. sanguinea. As a whole, pitcher morphology of both new species combined with detailed descriptions of the midrib, lamina and epidermis have addressed comprehensive taxonomic resolution in defining these new Nepenthes species. Also, the phylogenetic inference has provided an additional supportive evidence for the placement of N. domei as a new species. Hence, N. domei and N. latiffiana described in this paper are confidently positioned as two new species discovered from eastern part of Peninsular Malaysia.          Phylogenetic tree (NJ method) reconstructed using ITS sequences obtained from the public database (NCBI) and ITS isolated in this study. Bootstrap values are as indicated above relevant branches and an American carnivorous pitcher plant (Sarracenia flava) serves as an outgroup.