Leaf Epidermal Comparison of Phasey Bean ( Macroptilium lathyroides ) and Siratro ( Macroptilium atropurpureum )

. Phasey bean ( Macroptilium lathyroides ) and siratro ( Macroptilium atropurpureum ) are introduced legumes that become a common species in pastureland. The nutritional content of these legumes has been explored, but on the contrary for the anatomical study. The anatomical traits, especially leaf epidermal, have been used to increase the understanding of taxonomical study. This study aimed to investigate the leaf epidermal variability among phasey bean and siratro. The method used longitudinal section for upper and lower epidermal, then stained in safranin 0,1%. The results show that the upper epidermal in phasey bean has a polygonal epidermal cell, while the lower part and both parts of siratro have irregular-shaped. The type of stomata in the upper epidermal of phasey bean is paracytic and the lower epidermal is paracytic and anomocytic. Both epidermal sides in siratro have paracytic and anomocytic stomata. The index of stomata in phasey bean is higher than siratro, but the index of trichomes in phasey bean is lower than siratro. The trichomes were only absent in the upper epidermal of the phasey bean. Both plants have similar types of trichomes: capitate glandular trichomes and linear non-glandular trichomes.


Introduction
Phasey bean (Macroptilium lathyroides (L.) Urb.) and siratro (Macroptilium atropurpureum (DC.) Urb.) were the two common legumes that were used as a pasture plant. In Indonesia, these two species were introduced from Central and South America [1], and over a long period have been naturalized in Indonesia. These species have high adaptabilities in the tropical region, such as habitat variability and altitudinal distribution from the sea level up to 2,000 m a.s.l. [1,2].
Phasey bean is an erect legume with a stem up to 1.5 m. Phasey bean is an annual plant (rarely biennial); the stem is woody at the base; leaf pinnately trifoliated, leaflet oval -rhomboid, 3-8 x 1-3.5 cm, margin entire, acute at the base, acute-obtuse at the apex; inflorescence in a raceme, length 15-25 cm (rarely reach 50 cm); flower typical papilionoid, colored in red to red-purple, 1-1.5 cm in diameter; pods in a linear shape, 5.5-12 cm in length, contains 20-30 seeds [1].
In the botanical study, anatomical observation was widely used to increase the understanding of physiological processes and also in the taxonomical study. The anatomical approach is more convenient to distinguish species with similar morphological traits [3] or to complement the macromorphological traits that might be plesiomorphic or by-products of parallelism [4]. Some of the anatomical traits also show an evolutionary geometric relationship among characters [5], so it can be used to produce a more robust phylogenetic tree.
Leaf was one of the organs that have many variations in anatomical structures that have been considered as important taxonomical data [3,4,6], especially the epidermal tissue [7]. The epidermal tissue in the plant is the outermost tissue that plays a major role as a protective barrier to biotic and abiotic threats, while also maintaining the gas and water exchanges [8]. These multiple roles of epidermal tissue are supported by epidermal cells and some epidermal cells that developed certain structures, known as derivates of epidermal.
The derivate of epidermal can be observed in form of stomata and trichomes. These two derivates are frequently used for biosystematics studies because of their structure was influenced by the environment [3,9,10]. The prior research, trichomes can be used as a diagnostic character in Lamiaceae [11] and tribe Trifolieae of Leguminosae [12], and to provide the taxonomical feature in Caryophyllaceae [7], Piper [13], and Polygonatum [10]. Trichomes also can be combined with stomata, crystal, and oil cell distribution as complementary data in the systematic study of the genus Zingiber [3]. In further research, those epidermal structures, particularly stomata, potentially can be used as a major tool to construct paleoclimatic because of their correlation to functional ecology [14].
Not only the derivates, the epidermal cell also has been studied in many approaches. The pavement of the epidermal cell was correlated with genetic traits that later affect the mechanical process in the formation of the epidermal cell wall [15]. The shape and pavement of epidermal cells can be used to connect the plant evolutionary from fern to eudicots [16]. The epidermal cell also has clade-specific traits such as in Asian Cinnamomum [4]. Despite many benefits from the anatomical trait, the anatomical data of the phasey bean and siratro is still insufficient. Then, this leaf epidermal study in phasey bean and siratro aimed to investigate the anatomical characteristic among those plants. In the long term, it could provide the anatomical spectrum of epidermal in the genus Macroptilium.

Leaf epidermal observation
The leaf epidermal observation used a longitudinal section for upper and lower epidermal tissue. Preparat were made by using hand-section from the prepared sample in alcohol 70%, then stained in 0,1% safranin for 10 minutes and soaked in the aquades for 2 minutes. Preparate observed in a microscope equipped with Optilab (with Optilab viewer 2.2) to capture the images. The cell measurement using Image Raster 3. For a more representative epidermal structure, the observation was added with the illustration. The data collected in the observation was the structure of the epidermal cell, subsidiary cell, type of stomata, and type and structure of trichomes.

Data analysis
The obtained data were analyzed by descriptive and by index. The descriptive analysis by describing the anatomical structure of the epidermal tissue. The index analysis uses the index of stomata and the index of trichomes with the formula below. S = the number of stomata E = the number of epidermal cells T = the number of trichomes E = the number of epidermal cells

Results and Discussion
The epidermal component that can be observed in phasey bean and siratro consists of epidermal cells, stomata, and trichomes. The summary of the epidermal variation is shown in Table 1, while the epidermal illustration for the phasey bean is shown in Figure 2 and for the siratro in Figure 3. In siratro, both upper and lower epidermal cells are irregular-shaped with more grooves than lower epidermal cells of phasey bean. The irregular-shaped epidermal cell showed an identical shape to the typical epidermal cell in Solanum [17]. The shape and size of the epidermal cell can be affected by environmental conditions, such as temperature, soil, nutrient, and humidity [8]. The shape of epidermal cells in the upper and lower epidermal can have a similar shape or might be different. In phasey bean, the lower epidermal have an irregular-shaped epidermal cell, while the upper epidermal have polygonal-shaped epidermal cell. In the different shapes of the epidermal cell, the irregular-shaped (or also known as undulatedshaped) epidermal cell was more often found in the lower part of the leaf [16].
The undulations have increased the research curiosity because of the pattern in higher taxa. The epidermal cell in the fern has a strongly undulating margin, monocots have weakly undulating margins, and eudicots have no particular degree of undulation [16]. The high undulation epidermal cell is commonly found in drought-tolerant species [11]. There is some hypothesis about the undulation structure, which might be to increase the cells' contact to improve the efficiency of   The most interesting part of the phasey bean and siratro is the stomata. Stomata is a derivate of epidermis that plays a major role in water and gas exchange between the plant and the atmosphere [14,19]. The functionality of stomata is related to the two guard cells that are responsible for pore formation. In the majority of the plant, there is also an integral part of stomata called subsidiary cells that flanked those guard cells. The subsidiary cells are defined as cells that are adjacent to guard cells and are distinct (have a unique morphology or molecular signature) from other epidermal cells [20]. In some plants, such as grasses (Poaceae), the stomata consist of two dumbbell-shaped guard cells with parallel subsidiary cells associated with faster movement to increase the efficiency of water and gas exchange [21]. That typical characteristic of stomata in grasses is known as the Halter type.
Phasey bean and siratro have stomata in the upper and lower epidermal, also known as amphistomatic. The amphistomatic leaves are associated with the fast-growing species that have high adaptability in the dry environment [22]. The main concern of those stomata is the variation in http://www.eksakta.ppj.unp.ac.id/index.php/eksakta I Made Saka Wijaya, Eniek Kriswiyanti subsidiary cells that led to different types of stomata. In phasey bean, the upper epidermal has paracytic-type stomata that are indicated by two subsidiary cells that paralleled with the guard cells. The size of subsidiary cells was smaller than the epidermal cells. The subsidiary cells also have different sizes among them, the smaller ones and the larger ones.
The lower epidermal in phasey bean has two types of stomata, the paracytic and the anomocytic. The anomocytic-type of stomata is characterized by the undifferentiated shape of the subsidiary cells, so it looks like did not have subsidiary cells. In Fig. 2b, the top sequence showed the anomocytic-type of stomata. The cells that flanked around guard cells vary in number, ranging from 2 to 4 cells. In the middle sequence of Fig. 2b, the parasitic-type stomata also have different shapes of subsidiary cells, the near leaf nerve has polygonal-shaped subsidiary cells, while the leaf blade has irregular-shaped subsidiary cells.
In siratro, the upper and lower epidermal show the same structure of stomata. Both sides have parasitic and anomocytic stomata and irregular-shaped subsidiary cells. The epidermal that has more than one type of stomata was rarely reported. Some species of wild tomato (Solanum spp.) have two types of stomata, anisocytic and anomocytic [17]. In bananas (Musa acuminata), the type of stomata can be classified as paracytic and hexacytic [20].
The type of stomata is defined by the shape and formation of subsidiary cells, so it can be used as a unique morphological trait in the taxonomical study. In Pteridaceae, the shape of the subsidiary cell has the highest value to distinguish among clades [6]. As the morphological structure reflect the functional system, the different type of subsidiary cells might be correlated with the environment. Subsidiary cells support the function of the guard cell in water and ions regulation or enhance the particular morphology that affects the gas exchange [20]. Although the specific water and gas regulation in every type of stomata is unclear, the occurrence of two types of stomata in phasey bean and siratro can be observed in the elaborated study.
The stomata that occurred in the upper and lower epidermis have a different index of stomata (SI). In the upper epidermis, the SI value for phasey bean is 17.55  3.50 %, while the lower epidermis has a higher SI value, 20.15  3.29 %. In siratro, the SI value is lower than phasey bean, but has the same trend, which is the upper epidermal tends to have lower SI than the lower epidermal. In siratro, the SI value for the upper epidermal is 11.08  3.15 %, while for the lower epidermal is 17.73  0.59 %. The SI value correlates with the plant adaptation to drought. The drought-tolerant have more SI values, such as phasey bean and siratro. For a plant that needs high humidity, such as Zingiber, the SI value of the upper epidermal was less than 3 %, while for the lower epidermal was less than 10 % [3].
The trichomes in phasey bean are only found in the lower epidermal. The glandular trichomes are shaped in capitate, while the non-glandular trichomes have a linear shape. The length of nonglandular trichomes is 1124.65  338.88 µm, while the size of non-glandular trichomes is 154.27  21.44 × 77.41  12.00 µm. Siratro has glandular and non-glandular trichomes in both upper and lower epidermal. The glandular trichomes are shaped in capitate, the same shape as the phasey bean. Trichomes might be the derivate of epidermal that have specialization against biotic and abiotic threats [23]. The glandular and non-glandular trichomes have different roles in plant protection. The glandular trichomes secret exudates as protection for herbivory or pathogen [24], while the nonglandular trichomes tend to deal with abiotic stress [10,23]. The high amount of non-glandular trichomes indicated adaptability in the xeromorphic environment [7,11]. In the early stage of development, the non-glandular trichomes have produced phenolic compounds that are similar to glandular trichomes, but then diffused to the cell wall at the maturation [23]. So, the non-glandular trichomes also have a role in UV protection, especially against UV-B radiation damage [23].
The non-glandular trichomes in phasey bean were only found in the lower epidermal, while in siratro were found on both sides of the leaf. These differences might be implied that siratro has more adaptability in drought environments, or it might be a form of compensation for the broader leaf. In drought stress or low water potential, plants tend to produce reduced-size of leaf to maintain the stomatal conductance [19], or increase the density of non-glandular trichomes [10]. Siratro has a broader leaf than phasey bean which caused the higher rate of evapotranspiration, but it is also in accordance with the higher IT value.
The leaf anatomical traits were widely used as supporting data in plant taxonomy. In this study, the leaf epidermal traits in phasey bean and siratro provided nine characters, and some of them (such as the type of stomata, shape of the epidermal cell, and trichomes distribution) were the most varied characters. Based on the epidermal characteristics, both phasey bean and siratro show the drought adaptation, especially in form of amphistomatic leaf irregular-shaped epidermal cells with high curved, and the presence of non-glandular trichomes. Trichomes and stomata have been known as valuable taxonomical traits [10], but in this study, the shape of the upper epidermal cell was more informative to distinguish the leaf between phasey bean and siratro. This study only used epidermal traits, so for further research, the use of anatomical traits in stem and root nodules can be considered as the main variables.

Conclusion
In conclusion, the lower epidermal of phasey bean and siratro have relatively similar traits, such as the shape of the epidermal cell, type of stomata, and type of trichomes. The quantitative measurement of size and index (stomata and trichomes) was varied. The upper epidermal provides more distinct characters. The upper epidermal in phasey bean only have anomocytic stomata, polygonal-shaped epidermal cells, and without any trichomes, while siratro have paracytic and anomocytic stomata, irregular-shaped epidermal cells, and have glandular and non-glandular trichomes.