Foliar epidermal and trichome micromorphological diversity among poisonous plants and their taxonomic significance

During the current study, 25 poisonous plant species were collected from various locations in the lower Himalayas between March and September 2021. The collected species were dried, pressed, mounted and labelled. Plant species were identified using herbarium specimens from the Herbarium of Pakistan (ISL) QAU, Islamabad, as well as information from the flora of Pakistan (http://www.eflora.org). The Plant List (TPL) (http://www.theplantlist.org) and the International Plant Names Index (http://www.ipni.org) were used to confirm the plant species. Table 1 lists the names of plant species, voucher number, collector, location and altitude.

Using a light microscope, fresh leaf samples of 25 poisonous plants were investigated in which trichomes were examined in nine species following the method described by Raza et al. (2020). To keep the leaves from drying out, they were picked from actively developing plants and dipped in water for some time. Then, 1 or 2 leaves were placed in a test tube and dipped in 70% lactic acid and 30% nitric acid for 2 min or until the leaves became translucent. The leaves were then transferred into a cell culture dish, and the translucent sections were rinsed 2–3 times with water. Through a camel hair brush and sharp needle, the epidermal portions from the abaxial and adaxial sides of the sample were meticulously detached. The isolated epidermis was processed with a droplet of lactic acid to clean the section before being placed on a slide by coverslips. To make permanent slides, the margins of coverslips on slides were covered with translucent nail polish. For each plant species, six or seven samples of the abaxial and adaxial surfaces were prepared. A Nikon Microscope with Plan-40X/0.65 lens was used to examine the set slides. Using an XSP-45LCD microscope, the characteristics of the leaf epidermis were photographed. The following characteristics were observed under microscopy: width and length of epidermal cells, stomatal apparatus, trichomes and morphology of the epidermal cell, stomatal complex, the pattern of anticlinal walls (AW), and types of trichomes. Table 2 and Table 3 summarise the qualitative and quantitative characteristics, respectively. Mean (minimum–maximum) ± standard error SE (e.g., 56.7–160 = 89.6 ± 9.5) are used to express quantitative properties. For each abaxial and adaxial surface, five readings of each characteristic were recorded. The quantitative data were analysed using SPSS software IBM, Chicago, USA to calculate the values of mean, maximum, minimum and standard error. These data are extremely useful in identifying species and various epidermal features. These indices include information of length and width of the epidermal cell, subsidiary cells, stomatal complex and trichomes.

For SEM investigation, dried and mature leaves were washed with ethanol to expel the flotsam and jetsam. For slide preparation, the leaf cuttings were placed on stubs with a twofold covered scotch tape. The samples were super-coated with gold-palladium and examined by SEM (Show JEOL-5910, USA) introduced in the Central Library Office of Material Science College of Peshawar. A Polaroid P/N 665 film was used to take pictures. The samples were analysed beneath the magnifying lens and scrutinised the different micromorphological highlights (epidermal cells, trichomes, stomata) of the leaf (Gul et al., 2019).

Leaf epidermal morphology plays a crucial role in plant taxonomy and systematics. The quantitative attributes of leaf epidermal cells were measured in terms of their width and length on both the abaxial and adaxial surfaces. The maximum length of epidermal cells was observed on the lower surface, while the maximum width was observed on the upper surface. For example, E. helioscopia had the maximum length of epidermal cells (99.50 ± 9.02 μm) on the adaxial surface, whereas I. carnea had the lowest length (29.25 ± 1.31 μm). Similarly, the maximum width of epidermal cells was observed in S. nigra (45.05 ± 1.9 μm), while the lowest width was observed in S. incanum (12.25 ± 0.35 μm). Subsidiary cells, which are present alongside epidermal cells, also exhibited variations in width and length on both surfaces. S. halepense had the maximum length of subsidiary cells (61.9 ± 2.46 μm) on the adaxial surface, while R. communis had the lowest length (19.85 ± 1.2 μm). The maximum width of subsidiary cells was observed in Ranunculus sceleratus (32.40 ± 2.4 μm), while the minimum width was observed in E. helioscopia (7.9 ± 0.36 μm). On the abaxial surface, A. mexicana had the highest length of subsidiary cells (60.85 ± 4.02 μm), while I. carnea had the shortest length (23.40 ± 0.40 μm). Similarly, the highest width of subsidiary cells was observed in A. mexicana (37.95 ± 1.27 μm), while the lowest width was observed in E. helioscopia (9.50 ± 0.50 μm).

Variations in the width and length of stomatal pore and guard cells on the abaxial and adaxial sides were also observed in this study. On the adaxial surface, Alocasia macrorrhizos had the maximum length of stomata (34.30 ± 0.69 μm), while the minimum length of stomata was observed in E. Helioscopia (16.50 ± 1.0 μm). The highest width of stomatal length was observed in E. Royleana (29.85 ± 0.69 μm) and the lowest in E. Helioscopia (2.75 ± 0.14 μm). On the abaxial side, S. Nigra (46.95 ± 1.15 μm) had the maximum length of stomata, while the minimum length of stomata was examined in the following three plants: S. incanum (14.15 ± 0.4 μm), I. carnea (14.15 ± 0.36 μm) and B. monosperma (14.15 ± 0.36 μm). The highest width of stomata was observed in S. nigra (34.55 ± 0.64 μm) and the lowest in E. helioscopia (2.75 ± 0.14 μm). On the upper side, the highest length of guard cell was detected in A. macrorrhizos (34.60 ± 0.64 μm) and B. monosperma (14.15 ± 0.36 μm). On the other hand, the highest width of guard cell was found in E. Helioscopia (19.50 ± 0.50 μm) and the lowest in T. repens (5.20 ± 0.30 μm). On the lower surface, the extreme length of guard cells was examined in S. Nigra (42.95 ± 0.60 μm), while the lowest length of guard cells was observed in B. monosperma (14.15 ± 0.36 μm), I. carnea (14.15 ± 0.36 μm) and S. incanum (14.15 ± 0.43 μm). Similarly, the highest width of guard cell was observed in E. helioscopia (20.25 ± 0.61 μm), while the lowest was found in T. repens (3.75 ± 0.28 μm).

Trichomes also vary in length and width. On the adaxial side, the highest length of trichome was found in E. helioscopia (895.2 ± 70 μm) and I. carnea (39.55 ± 0.60 μm). On the other hand, the highest width of trichomes was observed in B. versicolor (46.40 ± 1.93 μm), while C. tinctoria (10.45 ± 1.05 μm) was found to have the lowest width of trichomes. On an abaxial surface, the maximum length of trichomes was found in Brugmansia versicolor (307.35 ± 2.62 μm), while the minimum was identified in Chenopodium ambrosioides (57.70 ± 2.28 μm). The highest width of trichomes was observed in S. nigrum (39.85 ± 2.47 μm), while the lowest was found in C. tinctoria (9.95 ± 0.40 μm).

The qualitative attributes of foliar epidermal anatomy showed significant variations among the studied plant species. Epidermal cells exhibited different shapes, including irregular, isodiametric polygonal, curvy and rectangular. Rectangular-shaped epidermal cells were present on both surfaces of S. halepense and on the adaxial surface of S. incanum. The pattern of AW also varied, ranging from deeply sinuous, wavy to sinuous, undulate, straight and angular. Thick sinuous walls were observed only in S. halepense. Stomata, which are crucial for gas exchange in plants, were observed on both surfaces of the leaves. The upper surface generally had a higher number of stomata than the lower surface. Different types of stomata were identified in the present study, including anisocytic, anomocytic, paracytic and cyclocytic. Cyclocytic stomata were found only in I. carnea. Anisocytic stomata have been observed in members of the Solanaceae family. Some species exhibited elliptical and wide elliptical stomatal pores. Dumbbell-shaped guard cells were observed only in S. halepense. Trichomes, or plant hairs, are another important aspect of leaf anatomy that can vary significantly among different plant species. Various types of trichomes were observed, including glandular trichomes and non-glandular trichomes. Glandular trichomes were found in Datura, which secrete toxic substances. Non-glandular trichomes, which do not produce any secretions, were observed in plants such as R. communis and S. nigrum. The density of trichomes also varied, with some plants having a sparse distribution, while others had a dense coverage of trichomes on leaf surfaces. For example, Datura innoxia exhibited a high density of glandular trichomes, giving the leaves a rough texture, while S. nigrum had a lower density of non-glandular trichomes, and thus a smoother leaf surface. The identified anatomical characteristics play a significant role in distinguishing and identifying poisonous species. The consistent presence or absence of specific characteristics in our study can serve as valuable diagnostic traits for species identification. For example, in our study, we consistently observed irregular epidermal cells with undulate AW, anomocytic stomata and glandular multicellular trichomes in the species of Solanaceae. This combination of features may serve as a distinguishing characteristic for species within this family. By highlighting these consistent features, we provide taxonomists with specific anatomical traits that can be used as diagnostic tools. These traits can be incorporated into identification keys or guides, enabling accurate species identification based on the microscopic examination of leaf epidermal characteristics.

The present study aimed to explore and compare the foliar anatomy of selected species, shedding light on the structural variations and adaptations present in their leaves. Based on the examination of the abaxial side, irregular epidermal cells with undulate AW and anomocytic stomata were observed in Solanaceae species, which is consistent with the findings of Adedeji et al. (2007). Ibrahim et al. (2016) reported rectangular-shaped epidermal cells with undulate cell walls in D. innoxia on both surfaces, whereas the present study revealed irregular and polygonal-shaped epidermal cells. The presence of anomocytic and anisocytic stomata was consistent with the findings of Ibrahim et al. (2016), and multicellular non-glandular trichomes were also observed. In S. nigrum, polygonal epidermal cells with a wavy pattern of AW on the upper surface and irregular cells with sinuous patterns on the lower surface were observed, which aligns with the findings of previous studies except for the undulate AW on the lower surface. The presence of anisocytic and anomocytic stomata, along with multicellular non-glandular trichomes, was consistent with the findings of previous research. Zahra et al. (2014) studied various Euphorbiaceous species and observed irregular epidermal cells with undulating walls and anisocytic stomata in E. pulcherrima, which is similar to the findings of the present study. Multicellular non-glandular trichomes with bulbous bases were also observed, consistent with the findings of previous work. In E. royleana, the observed shape of epidermal cells was polygonal, and paracytic stomata with straight AW were found only on the abaxial side. These results deviate from those of previous studies, which reported different-shaped epidermal cells with diacytic stomata. Najmaddin (2020) reported E. helioscopia with tetracytic and anomocytic stomata on the abaxial surface, while the current research identified polygonal and irregular epidermal cells with undulating and entire walls, and retained anomocytic stomata. Tyagi et al. (2013) examined the transverse section of R. communis, observing collenchyma, microcrystals, oil glands, parenchyma, palisade layers, prismatic crystals and rosette crystals, but there has been no additional foliar anatomical work on R. communis. The present study examined polygonal epidermal cells with straight AW and paracytic stomata on both surfaces.

Ramzan et al. (2019) previously described the anatomical characteristics of B. monosperma and found the absence of stomata on the adaxial surface and paracytic stomata on the abaxial surface, which is consistent with the current findings. Glandular, unicellular trichomes with pointed tips were also observed, which were not reported by the earlier work. However, the present research identified irregular and polygonal epidermal cells, which deviates from the previous study. Gostin (2009) observed foliar cross-sections of T. repens, noting palisade and parenchyma cells, but unfortunately, there is no epidermal anatomical study found in the literature. The current study revealed irregular and polygonal epidermal cells with anomocytic and anisocytic stomata on both the adaxial and abaxial surfaces, along with glandular multicellular trichomes.

In Ranunculus sceleratus, irregular epidermal cells with sinuous AW and anomocytic stomata were observed on both the abaxial and adaxial surfaces, with no trichomes recorded, which is consistent with the results of Salim et al. (2016). Bashir et al. (2020) examined irregular and polygonal epidermal cells with wavy to straight AW and paracytic stomata in T. peruviana, while the present study identified anisocytic stomata, which was not reported in the literature. Trichomes were not found in both the current and recent research. Arogundade and Adedeji (2019) recently investigated polygonal epidermal cells with straight walls, while brachyparacytic stomata were observed in Alocasia macrorrhiza in previous work, whereas the present study found paracytic stomata. In S. halepense, sinuous walls of rectangular epidermal cells with paracytic stomata were noted, which is consistent with the finding of Chaudhari et al. (2014). Only S. halepense exhibited dumbbell-shaped guard cells in all the mentioned varieties. The abaxial surface of Duranta erecta exhibited polygonal-shaped epidermal cells, which was also observed by Shekhawat and Manokari (2017). Gaafar (2019) observed wavy polygonal and irregular epidermal cells with only anisocytic stomata in Chenopodium ambrosioides, while the present results showed anisocytic stomata and a wide elliptical shape of the stomatal pore. Al-Mousawi et al. (2019) inspected thick anticlinal-walled epidermal cells and anisocytic stomata in A. mexicana, whereas the present study showed polygonal epidermal cells with thin AW and anomocytic stomata.

This research exhibited irregular or rectangular epidermal cells with slightly sinuous AW, along with cyclocytic stomata, in I. carnea. Both glandular capitate and non-glandular unicellular conical-shaped trichomes were also observed. These outcomes are consistent with the previous work of Ashfaq et al. (2019). Koyuncu et al. (2008) recorded polygonal epidermal cells with anisocytic stomata in P. harmala, while the present study described anomocytic stomata, showing slight deviations from prior work. Amini et al. (2019) examined the foliar cross-section of S. nigra and observed parenchyma cells and vascular bundles. Atkinson and Atkinson (2002) examined the high density of stomata on the abaxial surface and the absence of stomata on the adaxial surface. In contrast, the present study revealed polygonal epidermal cells with angular walls, exhibiting anomocytic stomata and multicellular trichomes, which were not mentioned in the literature.

It is important to explore why there are variations in the anatomical features observed in different studies. Factors such as genetic variability, environmental conditions, geographic location and different plant developmental stages could contribute to the observed differences. By focussing on these anatomical characteristics, our research contributes to the understanding of plant diversity and species identification. The consistent traits can be incorporated into taxonomic keys, guides and databases, enabling accurate and efficient identification of poisonous species. Additional research and comparative studies are needed to validate the diagnostic value of these anatomical characteristics. However, our findings lay the foundation for future investigations and provide a starting point for researchers and taxonomists interested in the identification and classification of poisonous species based on leaf anatomical features.

Overall, understanding the foliar anatomy of poisonous plants provides valuable information for their identification and classification. By recognising specific morphological features, dermal structures, glandular structures, secondary metabolites and leaf colouration, botanists, toxicologists and other experts can effectively distinguish poisonous plants from non-toxic species, contributing to public safety and environmental management.