Introduction
The Verbenaceae family comprises a wide range of flowering plants with considerable significant morphological differences that challenge established taxonomic classifications. In recent years, researchers have focused on the morphological aspects of leaves, especially the characteristics of the leaf epidermis, to better understand the systematic relationships and ecological adaptations of the different Verbenaceae species [1]. Verbenaceae, commonly known as Verbena or Vervain family, is the largest group of angiosperms distributed in a wide range of habitats. It belongs to the order Lamiales and comprises mainly tropical flowering plants [2, 3]. It was established in 1805 by J. St. Hil. The family is mainly distributed in tropical and subtropical regions and comprises 34 genera and 1,200 species [4, 5]. About 17 genera and 35 species have been recorded in Pakistan [6]. However, the geographical distribution of species and phylogenetic studies show that the Verbenaceae comprises 32 genera and 800 species [7, 8]. Recently, a taxonomic and phylogenetic study of specific groups within the Verbenaceae proposed that the subfamily Verbenoideae comprises eight tribes [2, 9-11]. Molecular and phylogenetic studies on the Verbenaceae have resolved many problems concerning the major lineages and genetic relationships in most parts of this group [8]. The species of the Verbenaceae family are mostly found in tropical and subtropical regions, with a few species in temperate regions, commonly distributed in Africa, Europe, Southeastern Asia, South America, and Pakistan [12]. The species of the family are well represented in arid and semi-arid regions [13], but only a few species have been found in moist to dry tropical forests, cloud forests, and grasslands of the high Andean [14]. Members of the Verbenaceae are closely related to the family Lamiaceae (Labaiateae), which is characterized by the presence of a bilabiate corolla and a persistent calyx [15]. The Verbenaceae are also related to the Bignoniaceae and Martyniaceae due to the nature of the calyx, inflorescence, and fruit [16]. In addition to teak, which is very highly valuable because of its wood, Verbenaceae also includes many medicinal and ornamental plants [17].
The distribution of stomata is an important characteristic of the foliar epidermis [18, 19]. Differences in stomatal density and arrangement provide useful information for the identification of closely related species and the understanding of their physiological adaptations to different environmental situations [20]. Trichomes, hair-like structures on the leaf surface, are another important component of the foliar epidermis [21]. Trichome density, form, and the presence of glandular structures have been shown to be effective in distinguishing species and can be used as reliable taxonomic identifiers [22, 23].
The study of anatomical characteristics is a useful tool for the identification and systematic modification of taxa [24, 25]. It enables the correct identification of herbal medicines [26]. Morphological and anatomical characteristics of a leaf are used as markers to assist in the correct recognition of the plant species. The role of anatomical data of leaves has long been recognized in traditional taxonomy, as anatomical features have been used to reflect the variations within species, genera, or a family. Leaf anatomical features such as trichomes, types of stomata, and other features are useful anatomical tools [27, 28]. Although many studies have been conducted on the wood anatomy and gross morphology of plants, they have proven to be valuable for plant identification and differentiation because identification criteria would be incomplete without considering the epidermal morphological features of leaves [29, 30]. Scientists particularly focused on the anatomical features to identify small scraps of plant material. The epidermal cells usually show variations in their configuration. The epidermal features of leaves, including cuticular characters, trichomes, epidermis, subsidiary cells, and stomatal types, have been proven to be systematically valuable [31]. Most anatomical studies of the Verbenaceae genera such as Lippia [32-34], Aloysia [35], Acantholippia [36], Glandularia [37], and Junellia [38]. The anatomical revisions of Verbenaceae were comprehensively performed by [39]. A study on the ontogeny of stomata and leaf epidermal was described by [40]. The most frequently found stomata patterns are paracytic, anomcytic, and diacytic. The trichomes were found to be uniseriate glandular capitate, multicellular glandular capitate, and non-glandular uniseriate filiform [41]. The most common stomata types are diacytic as found in Gmelina arborea Roxb. and occasionally anomocytic in Duranta erecta L. [42]. In Pakistan, the epidermal anatomy of the Verbenaceae family leaves has been least studied. The current study aims to investigate the distinguishing features of the leaf epidermis to prove its major role in establishing the taxonomic position of five species of the Verbenaceae family. Several research papers have focused on the morphology of epidermal cells, such as their shape, size, and arrangement. This study explores comprehensive insights from foliar characteristics using Light Microscopy (LM) and Scanning Electron Microscopy (SEM) for selected Verbenaceae taxa from Pakistan. The investigation explores the taxonomic significance of these features, contributing valuable knowledge to the understanding of Verbenaceae taxa.
Materials and Methods
Site Description and Climate
The district of Peshawar covers an area of 1257 square kilometers and lies at approximately 34°15 north latitude and 71°42 east longitude, with an elevation of about 1173 feet above sea level [43]. In the west, it borders Mohmand and Khyber Agency, while the northern border is shared with the Charsadda district [44]. In the east, it is bordered by the Nowshera district, and the southeastern parts are connected to tribal areas linking Kohat and Peshawar districts [44, 45]. The climate in the Peshawar district is semi-arid and is characterized by hot summers and mild winters. The average temperature fluctuates between 5ºC and 39ºC in the months of January to February and June to July respectively, accompanied by an annual rainfall of approximately 513 mm. July and August are the months with the highest rainfall [46].
Floristic Studies
The plants were collected from various territories of Peshawar University and surrounding areas. Information about the plants was collected, and specimens were dried in newspapers and identified with the help of (http://www.wfoplantlist.org/) and Flora of Pakistan [47, 48]. They were labeled with voucher numbers and deposited in the herbarium of the Department of Botany, University of Peshawar.
Light Microscopy
The modified technique of [36, 49] was used for epidermal studies. A leaf sample was collected in a test tube and mixed with 2 g potassium chloride and 1 mL of distilled water. The test tube was poured with 4 mL of concentrated nitric acid and the mixture was then carefully boiled according to the method of [50]. After a few minutes, the epidermal peels were washed with water after separation and then placed on a slide, to which one to two drops of bleach were added for 30 seconds to remove chloroplasts. After bleaching, the strips were washed again with distilled water and then treated with one to two drops of lactic acid to cover the entire mixture with a coverslip. Peeling the epidermis by hand is the most conventional method for peeling a large number of specimens. Prepare the abaxial surface by placing the leaf blade on a cutting plate or tile so that the adaxial surface is facing upwards. Using a sharp razor, carefully scrape the adaxial surface until the abaxial surface remains, and then use a camel hair brush hairbrush to remove all the above materials following the procedure of [51]. The epidermal peels were treated with one to two drops of bleach to remove additional chloroplast material and washed again with water. The leaf epidermis materials treated with lactic acid drops were placed on a clean glass, covered with slip, and observed under a light microscope.
Scanning Electron Microscopy
The dried leaves of the samples were used for scanning electron microscopy (SEM). For the examination, a mature, undamaged leaf section was cut from the upper middle part. The epicuticular wax on the upper and lower surface of the epidermis was removed by soaking the material in xylene for 12 to 24 h [50, 51]. Two leaf pieces were removed and then mounted on stumps with double-coated adhesive tape. One leaf piece with the lower surface was mounted to expose the upper surface and the other piece was taped to stumps from the upper side to expose the lower surface in stumps [25, 45]. The gold-palladium samples were coated by sputtering and then observed under a scanning electron microscope at the Central Resource Library (CRL) of the University of Peshawar.
Results
In the present study, the foliar anatomical features of five species of the Verbenaceae were investigated using light and scanning electron microscopy. The features recorded were based on the qualitative and quantitative characteristics shown in Tables 1 and 2. Fig. 1 and 2 show statistical graphs generated through foliar quantitative characters, whereas Fig. 3 and 4 depict foliar epidermal features through LM and SEM. These features were used as taxonomic tools for the identification of complex taxa.
Leaf Epidermal Morphology and Epidermal Cell Size
The leaf epidermis of five species of the Verbenaceae has various shapes and sizes. The shape of the epidermal cells is pentagonal, rectangular, square, and polygonal on both leaf surfaces of the examined representatives of the Verbenaceae. However, the hexagonal and oval shape of the epidermis was observed in V. negundo. Polygonal shapes with thick epidermal walls were observed in most species except P. nodiflora, which has smooth epidermal walls on both adaxial and abaxial surfaces (Table 1). In P. nodiflora, the unicellular trichomes are trailed by a layer of palisade cells with smooth edges.
On the abaxial surface of V. negundo, tomentose and glandular hairs were observed in enormous numbers as well as microtrichomes. Glandular hairs were observed in L. indica, V. negundo and V. officinale. The trichomes were divided and branched in V. negundo. In this study, the size of epidermal cells was found to vary in both length and width on both leaf surfaces. The mean length of epidermal cells was higher on the upper epidermis cell than on the lower surface. The highest epidermal length (32-40 μm) on the adaxial surface was observed in V. officinalis, followed by P. nodiflora (24- 28 μm), D. repens (24-27 μm), and L. indica (22-24 μm). The maximum value (17.9-20 μm) of the leaf epidermal width on the upper surface of the leaf was found in V. officinalis. The length of the epidermis on the abaxial leaf surface was greatest (42-47.5 μm) in V. officinalis among the Verbenaceae members compared to the other species. However, the width of the epidermis ranged from 22.5-25 to 8-13 μm on the lower epidermis.
Stomata Type and Size
The leaves of L. indica, V. officinalis, V. negundo and P. nodiflora, were classified as amphistomatic, having stomata on both the upper and lower epidermis. In D. repens, however, the stomata were absent on the adaxial surface. Normally, the stomata are anisocytic and animocytic and were observed on both leaf surfaces in most species. In V. negundo, the stomata were not visible because the stomata were covered with an enormous number of glandular hairs. V. officinalis with diacytic stomata on the upper leaf surface. The stomata on the lower epidermis were smaller and anomocytic and paracytic. In P. nodiflora, the stomata are located more on the adaxial than on the abaxial surface and were anisocytic, anomocytic, and diacytic. The lower stomata are diacytic with some guard cells and two subsidiary cells (Table 1). In this study, we also measured the size of the stomata on both leaf surfaces. The largest length (28-31 μm) of stomata on the adaxial surface was found in P. nodiflora, followed by L. indica (13-17 μm) and V. officinalis (8-13 μm). The highest length (30-33 μm) of stomata was found on the abaxial surface of P. nodiflora, whereas the lowest length (6-12.5 μm) of stomata was recorded in L. indica.
Table 1.
Qualitative leaf epidermal characters of family Verbenaceae based on LM.
Table 2.
Quantitative leaf epidermal characters of family Verbenaceae under light microscopy.
The width of stomata on both leaf surfaces ranged from 3-8 to 22.1-25 μm. The length was quite variable from species to species. In this study, the length of the stomatal pore was larger on the adaxial surface than on the abaxial surface. Larger (39.9-44 μm) stomata were observed in V. officinalis and smaller (24-26 μm) stomata in P. nodiflora. The long width of stomata varied from a maximum (72.9-74 μm) in P. nodiflora to a minimum (3-9 μm) in V. officinalis on the upper surface and from a maximum of 71-77 μm (V. officinalis) to a minimum of 3.9-5 μm (L. indica) on the lower surface.
Subsidiary Cell Size and Stomatal Index
The size of the subsidiary cells was quite variant in various species. A long subsidiary cell was found in V. officinalis and a small subsidiary cell in P. nodiflora on the upper surface, while a large subsidiary cell in P. nodiflora and a small one in L. indica on the lower surface. It was investigated that the maximum length of the subsidiary cell is 55-61 μm and the minimum length of a subsidiary cell is 18-21.1 μm on the upper surface.
On the other hand, the maximum length of the subsidiary cell in P. nodiflora is 50.9-52 μm, while the minimum length of the subsidiary cell in L. indica is 14.6-16 μm on the lower surface. The width of the subsidiary cell ranged from 35-43.6 μm to 22.9-24 μm on the upper surface and from 39.9-42 μm to 15.9-17 μm on the lower surface of a leaf. In this study, the stomatal index is an important tool to differentiate the complex taxa. The stomatal index shows different variations, with the largest stomatal index observed in D. repens and the smallest in V. officinalis on both surfaces of the epidermis.
Fig. 3.
Light micrograph of foliar epidermal characteristics of Verbanaceae taxa, A) D. repens (upper epidermis), B) D. repens (lower epidermis), C) L. indica (upper epidermis), D) L. indica (upper epidermis), E) L. indica (lower epidermis), F) L. indica (lower epidermis), G) P. nodiflora (upper epidermis), H) P. nodiflora. (upper epidermis), I) P. nodiflora (lower epidermis), J) P. nodiflora (lower epidermis), K) V. officinalis (lower epidermis), L) V. officinalis (lower epidermis), M) V. officinalis (upper epidermis), N) V. officinalis (lower epidermis), O) V. negundo (upper epidermis), P) V. negundo (upper epidermis).
Discussion
The leaf prepares the food for plant growth and development of the plant through the process of photosynthesis and respiration, and is therefore considered an important organ of the plant [52, 53]. Although it is the site where carbohydrates are formed, it is also the source of important features that have been used as the basis for the correct classification of important complicated plant taxa [53, 54]. Leaf anatomical studies are now considered an important aspect of plant systematics, and the epidermal features of leaves have been studied in many angiosperm families [30, 54-56]. Leaf anatomical features are important for the delimitation of various plant species [28]. The use of foliar epidermal characters is helpful in the identification and delimitation of taxa identified by previous studies [57, 58]. Leaf anatomical variations are important tools in the classification and differentiation of complex taxa [59]. Foliar anatomical characters provide a basis for the delimitation of complex taxa of the family Verbenacaeae, which have been described by many authors [42, 60-63]. The use of light and scanning electron microscopy can play an important role in the study of anatomical features of leaves, and these epidermal features of leaves are important for the characterization of groups abroad within subgroups and tribes [45, 51].
Fig. 4.
Electron micrographs of Verbancaeae taxa, A-D) D. repens; E-H) L. indica; I-L) P. nodiflora; M-P) V. officinalis;Q-T) V. negundo.
The present study shows that the five species of the Verbenaceae family exhibit marked differences in the size of the leaf epidermal cells, the size and shape of the stomata, the subsidiary cells, and the stomatal index. Two types of trichomes, glandular and non-glandular trichomes, were observed in the members of Verbenaceae as documented by [60, 61, 64]. Many studies have reported that the presence of non-glandular trichomes in Verbenaceae species shows considerable differences in their chemical composition and morphological features [65, 66]. [32] reported at least five species of glandular trichomes and one species of non-glandular trichomes in the leaves of Lippia citriodora L. Uniseriate and tomentosa, non-glandular trichomes were observed on the leaves of Lippia javanica (Burm.f.) Spreng. [67]. However, the number and shape of trichomes vary from species to species [33] in contrast to the study of [33], in which six types of glandular trichomes ranging from unicellular to tetracellular trichomes and three types of tectorial trichomes were observed in the leaves of Lippia species, [68] observed only two types of glandular trichomes in L. scaberrima Sond. The presence of trichomes in Verbenaceae species is a characteristic feature of this family [60, 68, 69]. Identification on the basis of trichome characteristics has a high taxonomic value and their classification can even be made to the specific level [70, 71]. Features of the epidermal wall and ontogeny of stomata are important taxonomic characters to distinguish between advanced and primitive taxa and are used for morphological and ontogenetic classification [61, 72, 73]. In this study, the leaf epidermal cells of five different species of Verbenaceae show variation in their morphology. Polygonal with thick epidermal walls and straight edges were the common features of most species. The results are in agreement with those of [61, 74-76]. However, rectangular, sporadic, square, pentagonal, and hexagonal epidermal shapes with smooth edges were also observed in the studied species. Stomata are small pores on the adaxial and abaxial surfaces of leaves and plants that play an important role in plant identification and classification and also have a significant role in plant physiological processes [77]. The anisocytic, anomocytic, and diacytic stomata were commonly observed on both leaf surfaces in the Verbenaceae family [67]. Our results were in agreement with the findings of [75], who also reported the same types of stomata in the Verbenaceae in Egypt. However, [74] reported tetracytic types of stomata in Clearodendrum species. In our result, the leaves of L.indica, V. officinalis, V. negundo, and P. nodiflora, were classified as amphistomatic, with stomata on both the upper and lower epidermis- a result similar to that of [67, 78]. In our finding, the stomata were not seen on both surfaces of the epidermal cells of V. negundo due to the presence of an enormous number of tomentose and glandular hairs. This result was in agreement with [79]. The leaf anatomical features of the leaves provided information on the fossil phylogeny and revealed taxonomic relationships [80]. The larger epidermal cell was observed on the adaxial surface in V. officinalis with 32-40 μm and smaller epidermal cells on the adaxial surface were found in L. indica with 22-24 μm. On the abaxial surface, the large stomata were observed in Phyla nodiflora with a size of 30-33μm, while smaller stomata were observed in L. indica with a size of 6-12.5 μm. In P. nodiflora, the stomata are located more on the adaxial than on the abaxial surface and were anisocytic, anomocytic, and diacytic. This finding differs from the results of [73, 81], who reported that the stomata are larger on the abaxial surface than on the adaxial surface.
The subsidiary cells have a disjunct role on the upper surface. It is investigated that the maximum length of a subsidiary cell is 55-61 μm and the minimum length of a subsidiary cell is 18-21.1 μm on the upper surface. A long subsidiary cell was found in V. officinalis and a small subsidiary cell in P. nodiflora on the upper surface. On the other hand, the large subsidiary cell in P. nodiflora and the small one in L. indica are on the lower surface of a leaf. The stomatal index shows different variations, with the largest stomatal index observed in D. repens and the smallest observed in V. officinalis on both surfaces of the epidermis. The stomatal index can affect plant-environment interactions such as assimilation and transpiration [50, 82]. Overall, this study is important for the delimitation of Verbenaceous species based on leaf anatomical features and provides us with important information on the relevance and importance of foliar epidermal features in solving many problems related to the classification of complex taxa [30, 83, 84].
Conclusion
The above study suggests that examination of the leaf epidermis can be helpful in identifying and differentiating different Verbenaceous species. Various characteristics of the leaf epidermis such as the absence or presence of stomata, size, and shape of epidermal cells, the absence or presence of trichomes, and the types of trichomes are preliminary steps to delimit different taxa at generic and specific levels. Finally, the current work deals with the complex architecture and morphology of the leaf epidermis in Verbenaceae species. Through a thorough examination of the leaf epidermis of different species of this plant family, we gained important insights into the unique adaptations and features that these structures exhibit. The different epidermal characters discovered in this study may be useful diagnostic features for taxonomists and botanists attempting to classify the Verbenaceae species. It is crucial to highlight that, while this work provides insight into the epidermal anatomy and morphology of the leaves of the Verbenaceae taxa, further research is needed to investigate the functional significance of these morphological differences.
Future Perspectives
As we continue to explore the complex field of Verbenaceae foliar epidermis, future research should attempt to fill information gaps and uncover the larger ecological consequences of the observed diversity. Understanding the adaptive importance of different epidermal characteristics could shed light on the dynamic interactions between Verbenaceae taxa and their habitats. Investigating the relationship between certain epidermal features and environmental conditions, such as climate, soil composition, or altitude, could shed light on the adaptive methods of Verbenaceae species. Deciphering these relationships could help us to understand plant ecology and improve conservation measures. Exploring potential applications of the foliar diversity of Verbenaceae in industries such as medicine, agriculture, and industry could be a promising direction. Certain features of the epidermis could have medicinal properties, and the research could inspire improvements in biomimicry for agricultural practices or materials science. In summary, research into the leaf epidermis of Verbenaceae is not a goal in itself, but rather a step towards a more comprehensive understanding of plant biology and its practical applications. The combination of ecological, genetic, and evolutionary perspectives promises a fertile ground for future research, providing both theoretical insights and potential applications in practice.
