INTRODUCTION
The genus Ficus is among the premier genera of shrubs and trees, which are known for its medicinal, socioeconomic and religious importance. As regards its medicinal importance, the Ficus spp. are known to biosynthesize bioactive compounds including phenolics, flavonoids, terpenes etc., which are used to treat the ailments such as diabetes, toothache, gum infection, piles etc. (Salehi et al. 2021; Devi et al. 2022). Interestingly all parts of plant including bark, roots, leaves and fruits are used in the treatment of various ailments (Murugesu et al. 2021).
Morphological features of plants have close association with the biochemical pathways operative in leaves and other parts (Klem et al. 2019). Photosynthesis, which depends upon the stomatal functions in the exchange of water and gases, is one of the fundamental processes involved in the production of primary and secondary metabolites. Hence, the stomatal density and function in the leaves may directly influence the synthesis of primary metabolites and indirectly the secondary metabolites (Ördög et al. 2013).
Plants have historically been categorised according to the appearance of their fruits and blooms. In order to accurately identify and classify closely related taxa, plant scientists have been using leaf epidermal morphology (Abdulrahaman and Oladele 2010; Qiu et al. 2023; Hussain et al. 2025). Moreover, to differentiate between various taxonomic ranks, such as families, tribes, genera, and species, morphological traits are essential (Alaida and Aldhebiani 2022). However, several species of flowering plants have similar morphological attributes, so it might be difficult to differentiate them based only on morphological traits (Nazir et al. 2013). Determining the taxonomic placements of these species thus requires an awareness of both the similarities and differences within a sub-genus. Numerous crucial micromorphological characteristics were identified through the examination of Ficus epidermal surfaces; some of these features exhibit noteworthy interspecific variations that are significant from a taxonomical standpoint (Mubo et al. 2004).
Essential diagnostic features of the epidermis that offer helpful suggestions for identification include size, stomata orientation, stomata form, guard cell shape, and structural features of epidermal cells (Munir et al. 2011; Trofimov and Rohwer 2018). In a related study, it was shown that trichome size and shape, stomata size and form, and the presence or absence of stomatal clusters were useful diagnostic traits for differentiating distinct fern species (Rahman et al. 2017).
Although it is now thought that epidermal traits are important for taxonomy, there is little information on the epidermal morphology of Ficus (Hussain et al. 2025). Notable characteristics such as epidermal cell structure, shape, orientation, stomatal complex size, and trichome types have major taxonomic implications (Rahman et al. 2017).
Leaf is the source tissue where the biosynthesis of majority of medicinally and economically important compounds takes place. Ficus is an important genus known for the synthesis of quite a few metabolites of medicinal value (Devi et al. 2022), which may be associated to the metabolite profiles. Thus, the current study was conducted to gain a comprehensive understanding of the importance of leaf epidermal structure in the identification of F. elastica, F. elastica and F. natalensis. This study will add to the existing literature on the use of leaf epidermal architecture in plant identification and provide insights for researchers and practitioners in plant taxonomy and categorisation.
MATERIALS AND METHODS
An experiment was conducted in order to explore stomatal modifications in lower leaf lamina, petiole length and leaf surface area of different Ficus species in November, December and January. The leaves were collected from the New Botanical Garden, University of Agriculture, Faisalabad. Samples were preserved in FAA (formaldehyde-acetone-alcohol) fixative for 24 hours and then shifted to 70% ethanol for preservation. The hand sectioning of the leaf lamina was performed. The leaf lamina was peeled off from the selected leaf samples and immediately placed in 30% ethanol for 10–15 minutes. These tissues were shifted to 50% ethanol, followed by 70% ethanol for 10–15 minutes, respectively. After 70% ethanol treatment, a few drops of safranin were added for 5 minutes. Safranin with 90% ethanol was used for 5 minutes. Then samples were washed 2–3 times with 100% ethanol. The samples were treated with 25%, 50% and 100% xylene for clearing them. Each section was permanently preserved in a drop of Canada balsam. The images of the samples were taken under an electron microscope.
RESULTS
Length of petiole
The comparison of different Ficus species petiole length recorded at different intervals (November, December and January) showed significant variations. Data showed a gradual increase in the petiole length of F. elastica over time. In contrast, F. natalensis consistently exhibited the minimum petiole length throughout all the experimental months, whereas other Ficus species showed no specific pattern in increase or decrease in length (Fig. 1A).
Leaf area
Results revealed that F. elastica var. varigata leaf area was decreased from November to December and again increased from December to January. The F. natalensis had the smallest leaf area but remained constant throughout all the experimental months (Fig. 1B).
Number of stomata
The comparison of Ficus species leaves epidermis collected at different time intervals (dawn, dusk, mid-day) in winter months revealed that there’s a subsequent increase in the opening of stomata during mid-day. Among the species studied, F. natalensis had the maximum no. of stomata in November and December, but in January, F. elastica var. varigata had the maximum no. of stomata. Moreover, F. elastica ‘Burgundy’ had the least no. of stomata in almost all data taking months (Fig. 1C).
Number of subsidiary cells
Data showed that F. natalensis showed the maximum no. of stomata and F. hawai and F. macrophylla had the least no. of subsidiary cells (Fig. 1D).
Stomatal pore area
Results showed
that the stomatal pore size of F. macrophylla and F. elastica ‘Burgundy’
gradually increased across the observation periods, while F. natalensis
pore size increased from November to %2024010105%20Ok_files/image001.png)
December
and then decreased from December to January (Fig. 2A–4).
%2024010105%20Ok_files/image002.png)
Length of guard cells
The comparison of different Ficus species based on the length of guard cells recorded at different intervals in winter months (November, December and January) showed subsequent variations (Fig. 3–4). Results showed that the length of the guard cells of F. elastica gradually decreased over time, while the length of the guard cells of F. hawai increased across the observation periods (Fig. 2B).
Width of guard cell
The results for guard cell width showed a gradual increase over time in F. macrophylla, whereas in F. elastica, guard cell width decreased with the passage of time (Fig. 7).
DISCUSSION
The genus Ficus, commonly referred to as fig, belongs to the family Musaceae. Ficus is one of the largest plant genera, with more than 750 described species distributed worldwide, mainly in tropical countries. Fig trees are often ecologically important ‘Keystone’ components of tropical forests, because of the large number of vertebrates that feed on their figs, more than any other group of plants (Hussain et al. 2025).
A study was conducted to compare different
morphological and anatomical characteristics of Ficus species. The
result revealed that F. elastica had the maximum number of stomata, and the
least were found in F. natalensis. The petiole length of F. elastica
var. varigata increased over time. Over time, plants evolved different
mechanisms to %2024010105%20Ok_files/image003.png)
optimise their stomatal density in response to
environmental cues to enhance their survival and fitness (Hou et al.
2023) (Fig. 1–2). Moreover, F. natalensis had shorter petiole length and
showed no specific pattern in the increase and decrease in length during the
observation period. The leaf area of F. elastica var. varigata decreased
from November to December. and again, increased from December to January. In
contrast, Shrestha et al. 2025) reported non-significant changes in the leaf
area of F. religiosa during the winter season. In contrast, F.
natalensis had the smallest leaf area, and no variation was marked from November
to January. Furthermore, F. natalensis had the maximum number of stomata, and F.
hawai and F. macrophylla had the least number of subsidiary cells. Stomatal
pore area of F. macrophylla and F. elastica ‘Burgundy’ gradually
increased with the passage of time, while F. natalensis pore area
increased from November to December and then decreased from December to January
(Fig. 3–4). Stomatal pore size or movement follows circadian rhythm, which is
influenced by light exposure and the plants internal clock (Hou et al.
2023).
The
length of the guard cell of F. elastica gradually decreased over time, while
in F. hawai it increased from November till January. In addition, the width
of the guard cells of F. macrophylla gradually increased across the
observation period, and in F. elastica the guard cell width decreased,
respectively (Fig 1–4). Plants are affected by the environmental conditions
during all phases of growth and %2024010105%20Ok_files/image004.png)
development. Especially, stomatal number reportedly
changes when plants are grown in different seasons. Although the measurements were
done on an area basis, this approach does not account for possible anatomical changes
during different environmental conditions. It seems likely that the stomatal
pattern may operate and respond to a range of conditions that can be explored
in further studies. Stomata frequency declined in response to increasing CO2
and may have occurred over geological time (Hofmann et al. 2025).
CONCLUSIONS
changes in the number of epidermal cells, such as the number of stomata and subsidiary cells of Ficus species, were greatly affected by the environmental and climatic changes. These differences highlight the adaptive responses of each species to seasonal and diurnal changes. This study highlighted the taxonomic importance of leaf epidermal features, emphasising their effectiveness as diagnostic traits for distinguishing among Ficus species. Future research should focus on the molecular and physiological mechanisms underlying stomatal regulation and anatomical adaptations in Ficus species under varying environmental conditions. Long-term field studies and genetic analyses could provide deeper insights into their adaptive strategies.
ACKNOWLEDGMENTS
The author expresses his sincere gratitude to the Department of Botany, University of Agriculture, Faisalabad, for providing facilities for this work.
AUTHOR CONTRIBUTIONS
Muhammad Zubair Akram: Topic decision in publication, journal approach, publication process, data defining, writing and data collection. Ali Nawaz: Data collection, writing of article
CONFLICTS OF INTEREST
The authors affirm that they possess no conflicts of interest.
DATA AVAILABILITY
The data will be made available on a fair request.
ETHICS APPROVAL
Not applicable to this paper.
FUNDING SOURCE
This project is not funded by any agency.
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