Grewia tembensis Fresen and Grewia trichocarpa Hochst. ex A.Rich. (Grewioideae Hochr; Malvaceae Juss.) Micromorphological Study and Comparison via Electron Microscopy

Abstract

Grewia tembensis and Grewia trichocarpa inhabit dry tropical zones and arid environments, adapting to extreme climatic conditions and limited moisture supplies. Overall, Grewia L. possesses a significant variety of bioactive chemical constituents of great therapeutic importance. Indeed, for these species, precise morphological analyses are poor, and their detailed characterization is almost non-existent. This research attempts to investigate and compare the micromorphological traits of G. tembensis and G. trichocarpa species through scanning electron microscopy (SEM). Micromorphological characteristics of the leaf and fructiferous structures turned out to be highly effective in separating the two species, G. tembensis and G. trichocarpa, especially regarding the type, density, and distribution of trichomes on the lower and upper surfaces of the leaves, along with the stomatal and trichome types on the surfaces of the fruits. Statistical analyses using principal component analysis, t-tests, and hierarchical clustering conducted on micromorphological data of the leaves, flowers, and fruits showed considerable variation within samples of G. tembensis and samples of G. trichocarpa. On the basis of their morphological assessment characteristics, the samples of both species were distinct and clustered into separate groups. This study emphasizes the necessity of performing detailed morphological studies of species by means of an electron microscope and proves that the leaf features are important for separating species. Such morphological traits of trichomes would offer an efficient tool to distinguish the species. Within the findings, this suggests that such diagnostics are likely to be highly useful for species identification in Grewia, especially in cases where there are no fruits available.

1. Introduction

Order Malvales Juss. ex Bercht. and J. Presl contains four families: Tiliaceae (Juss.), Sterculiaceae (Vent.), Bombacaceae (Kunth.), and Malvaceae (Juss.) [1]. It is worth mentioning that the classification in this order is facing several challenges, as numerous genera have been reassigned among families. Initially, the genus Grewia L. was classified under the family Tiliaceae; however, it has been moved to the family Malvaceae according to the results of phylogenetic analyses, and the Tiliaceae has become a subfamily of Malvaceae (Tilioideae) [2,3]. Within Malvaceae, there are currently nine subfamilies: Bombacoideae (Burnett), Brownlowioideae (Burret), Byttnerioideae (Burnett), Dombeyoideae (Beilschm.), Grewioideae (Hochr.), Helicteroideae (Meisn.), Malvoideae (Burnett), Sterculioideae (Beilschm.), and Tilioideae (Arnott) [2]. The genus Grewia L. belongs to the subfamily Grewioideae [4,5,6]. The Grewia genus comprises 280–300 species, as mentioned by Al-Hawshabi [7]. Currently, 274 species have been accepted within the Grewia genus, as recorded in data from the Royal Botanic Gardens, (Kew) & Plants of the World Online [8]. Grewia species are distributed primarily in tropical Africa, Madagascar, the Arabian Peninsula, the Himalayas, Pakistan, India, China, Myanmar, Thailand, Indo-China, Malesia, the Pacific Islands (e.g., Tonga and Samoa), and northern Australia [5,9].

In the genus Grewia, several taxonomic studies have highlighted the value of morphological and anatomical characters. Hashmi and Qaiser [10] looked at two species of Grewia (G. erythraea and G. tenxa) from Pakistan and indicated that these species can be distinguished from each other based on these morphological features. The investigation by Chung [9] explored several species of Grewia in Malaysia; G. multiflora, G. leavigata, G. polygama, and G. huluperakensis. It mentioned that these species can be distinguished from one another through a range of morphological characteristics. In addition, Nuha and Fatima [11] studied Grewia spp. in Sudan and noted that the species G. tenax is identifiable by its flower’s colors. More recently, Hawshabi [7] examined the vegetative and reproductive phenotypic traits of seven Grewia spp. in Yemen and reported that the identified characteristics have significant taxonomic relevance.

Regarding molecular and phylogenetic studies, there are some works on Grewia species that published the complete chloroplast genome, such as Grewia biloba Xu [12] and Grewia biloba var. parviflora reported by Hou [13]. Dorr and Wurdack [14] reconstructed the Grewia phylogenetic tree involving several species from genera found in the subfamily Grewioideae, using ITS sequences. The interrelationships within Grewia suggest a biogeographical scenario of complexity.

The medicinal significance of Grewia species has been reported. These constituents include quercetin, β-sitosterol, lupeol, stigmasterol, triterpenoids, lipid compounds, flavonoids, steroids, saponins, and tannins [15,16]. It is used in traditional medicine to treat ailments in Africa, such as coughs. It is used to treat microbial infections, typhoid fever, gastroesophageal reflux, hypochondriasis, decreased appetite, and a distended diaphragm [17].

The flora of Saudi Arabia is one of the most diverse ecological regions in the Arabian Peninsula and contains important genetic resources of crops and medicinal plants. The components of this flora are an illustration of a mixture of effects coming from Asia, together with African assets, as well as the Mediterranean region [18]. Chaudhary, Khalik, and Al-Ruzayza [19,20] recorded 13 genera and 54 species of Malvaceae in Saudi Arabia, including cultivated varieties. Currently, the Flora of Saudi Arabia list (latest updated on 1 September 2024 [21]) includes about 16 genera and 64 species distributed in three subfamilies, namely: Dombeyoideae, Grewioideae, and Malvoideae. A total of nine species of Grewia have been reported in the Kingdom of Saudi Arabia, namely, Grewia arborea (Forssk.) Lam., Grewia erythraea Schweinf., Grewia gillettii Sebsebe and B. Mathew, Grewia mollis Juss., Grewia tenax (Forssk.) Fiori, Grewia tembensis Fresen., Grewia trichocarpa Hochst. ex A. Rich., Grewia velutina (Forssk.) Lam., and Grewia villosa Willd. [21]. However, these species lack full taxonomic studies, and detailed descriptions are needed to determine their taxonomic positions.

It grows as a shrub in a desert biome and dry shrubland biome. Morphological characters of G. tembensis and G. trichocarpa are similar, and divergence is often based on flowering and fruiting. Microscopic examinations of micromorphological features play an essential role in the processes of distinction and identification of samples to species and sex at the taxonomic level. However, few studies have evaluated Grewia with scanning electron microscopy (SEM), such as in Grewia pindanica R.L. Barrett [22]. This work aims to study and compare the morphological features of “G. tembensis and G. trichocarpa” by scanning electron microscopy.

2. Materials and Methods

2.1. Plant Specimen Collection and Study Area

For the purpose of morphological evaluations, specimens of foliage, flowers, and fruits from the species Grewia tembensis and Grewia trichocarpa were collected from the Faifa Mountains in the Jazan region of southwestern Saudi Arabia (17°14′45.1′′ N 43°05′27.2′′ E), which is situated in the southwestern segment of the Arabian Peninsula as well. This area holds significant importance from both a floristic and phytogeographical standpoint, serving as a biogeographical connection between the continents of Asia and Africa [23,24]. The Faifa Mountains constitute a pivotal hotspot for botanical diversity in Saudi Arabia. This region is characterized by a complex array of environments and varied habitats, which collectively foster a rich and diverse floral assemblage [25]. The heightened levels of plant species diversity in the southwestern region of Saudi Arabia can be attributed to substantial annual precipitation exceeding 300 mm and an altitudinal gradient that ascends from sea level to 3100 m. The climatic conditions in the Faifa Mountains remain temperate throughout the year; however, temperatures are prone to decline during winter at higher elevations and increase during summer at lower altitudes. Specifically, summer temperatures in these mountains range from approximately 16 to 28 degrees Celsius, whereas winter temperatures vary between 3 to 25 degrees Celsius.

This collection occurred from September to November 2024. Five individuals (trees) of the species were studied. All study samples were collected from Jabal Fifa and lived in the same environmental conditions. The identification of the samples was validated by specialists, who utilized both herbarium specimens and morphological characteristics documented in relevant literature. Voucher specimens were meticulously prepared, and thereafter, these specimens were archived within the Herbaria of the Biology Department at the Faculty of Science, Umm Al-Qura University, Makkah, Saudi Arabia (Table S1), showing information about the vouchers.

2.2. Examination of Micromorphological Characteristics by Scanning Electron Microscopy

The samples were dried, and the dry flowers and fruit specimens, as well as the abaxial (AB) and adaxial (AD) surfaces of the leaves of the species studied in this investigation, were fixed to stubs by double-sided adhesive tape. The samples were then platinum-coated for 2 minutes prior in a Cressington 108 coating unit (Micro to Nano/Haarlem, The Netherlands). Observation and photography were conducted using an LTD JSM7610F, with a magnification power of 1,000,000, contrast power of 1 NM, and 15 kV (JEOL/Peabody, MA, USA) at the Central Research Laboratory of King Saud University. The qualitative features that were visible on either side of the leaves (AB and AD), as well as the general surface patterns related to flowers and fruits fruits were systematically recorded. The complex relief of the leaf surface and trichomes was described using the previously specified terminology in Chung [26]. The outer stomatal ledge and outer stomatal aperture were described using terminology in Pautov et al. [27]. The photomicrographs were taken in the same position for all samples when measuring. A total of 31 qualitative characters (for leaves, flowers, and fruits) were defined to be recorded, along with 21 quantitative characters measured (Table S2 and Table S4). Measurements of length, width, and area for leaves, fruits, petals, and ovaries are shown in the schematic drawing provided in the Supplementary File (Figure S1). The measurements were conducted using ImageJ v. 1.53t analysis software [28]. The processes of measurement using ImageJ software involved uploading sample photos into ImageJ, setting a scale, preparing the image for measurement, and using Adjust Jope in the menu. Then, image analysis for estimating the area was performed by choosing the submenu Tools-ROI Manager, followed by exporting the ImageJ data file for further analysis [29].

2.3. Statistical Analysis

The quantitative micromorphological features (Table S4) were prepared for immediate application, and the morphometric dataset was used in the PCA analysis. This PCA was conducted with XLSTAT version 2024.2.1 [30]. The PCA helps us examine how well the selected traits (variables) can cluster samples based on their resemblances. In addition, the collection of summary statistics concerning morphological traits and graphical representations and assay the association of the phenotypic traits to observe the correlation between them and the association according to the species.

The correlation coefficients between the different morphological characters were determined in XLSTAT version 2024.2.1 [30], with Bootstrap observations chart: Number of samples: 50, Seed (random numbers).

It was applied box plot and t-test analysis (GraphPad Software version 10.4.1 for Windows, [31]) to compare the means in this respect for two species, Grewia tembensis and Grewia trichocarpa. Statistical significance was tested using 5 replicates per sample and 95 permutations; a t-test p-value < 0.05 was considered significant, while p-value > 0.05 indicated an insignificant result.

Where R-Squared (R² or the coefficient of determination) was calculated; R-squared (R² or the coefficient of determination) is a statistical measure of how close the data are to the fitted regression line. So, R-squared is essentially what percentage of how well the data fits the regression itself. Using the R-squared, which ranges between 0 and 1, describes the degree to which the model fits the data: 1 = perfect fit of the model to the data; 0 = the model explains none of the variance in your dependent variable. As a rule of thumb, in academic research, one can expect R-squared to be statistically significant somewhere between 0.50 and 0.99.

It was used the Heatmapper web server [32] to generate a heatmap of variation and hierarchical clustering based on the G. tembensis and G. trichocarpa morphological data. Additionally, a morphological dendrogram grouping was built through the ‘factoextra’ package Version 1.0.7 following [33] and RStudio software Version 2.0 [34]. Consequently, it enhances the potential of exploring the relations between two species, Grewia tembensis and Grewia trichocarpa, of morphological traits. Hierarchical representation and clustering: Samples were clustered (branch and subbranch formation) based on dissimilarity.

3. Results

3.1. Morphological and Micromorphological Characteristics of the Leaf Epidermis, Stomata, Ovaries, and Fruits

Characteristics of the Grewia species, G. tembensis and G. trichocarpa, via SEM are presented in Table S2 and Figure 1, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6. The G. tembensis species is distinguished by long cuticular striations on the (AB and AD) leaf surfaces, whereas they are short and less frequent on the AD leaf surface of G. trichocarpa.

In this study, we observed the presence of several types of glandular and nonglandular trichomes on the leaf surfaces in both species (Table S2). a. Nonglandular trichomes are as follows: (1) simple long trichomes that are tapered at the tip (Figure 1b,g; (2) 2, 3, or 4-armed tufted trichomes on the (AB) and (AD) leaf surfaces, which are observed only in G. tembensis (Figure 1c–e,h); (3) stellate trichomes without a central cushion that are present on both the (AB) and (AD) leaf surfaces; these trichomes had more arms (3–10 arms) and were more highly dense on the AB leaf surface in G. trichocarpa (Figure 2a–d) than in G. tembensis (5–8 arms/Figure 1f), and they had a twisted appearance and were swollen at the tips of G. trichocarpa on both the AB and AD leaf surfaces (Figure 3a–c); and (4) stellate trichomes with a central cushion present only on the leaf surfaces of G. trichocarpa, which were 6–8-armed, long, twisted, and swollen at the tips of both the (AB) and (AD) leaf surfaces (Figure 3a,d). b. Glandular trichomes were observed on the AB and AD leaf surfaces of both G. tembensis and G. trichocarpa, with short stalks and elliptical, broadly elliptical, or rounded heads (Figure 4c,f).

Stomata were present only on the AB leaf surfaces of G. tembensis and G. trichocarpa; they were elliptical (G. tembensis) to broadly elliptical (G. trichocarpa) and were elevated slightly above the surrounding epidermal cells. The stomatal shape was anomocytic in G. tembensis (Figure 4a,b), and it was paracytic in G. trichocarpa; however, the high density of stellate trichomes on the AB surface of G. trichocarpa obscures the clear visibility of the stomata (Figure 4d,e).

The petals and stamen of G. tembensis were yellow in color. The petal color of G. trichocarpa was light orange, whereas the stamen color was orange. Different types of tri-chomes were observed as well on the ovary surface (Table S2) as follows: (1) simple, long trichomes tapered at the tip, present on the ovary and highly dense at the base of the ovaries of G. tembensis and G. trichocarpa (Figure 5b,d,h); (2) tufted trichomes with 2 arms, present only on the surface of the ovary and base of the ovary in G. tembensis (Figure 5a); (3) stellate trichomes without a central cushion, which are 8- to 12-armed and present on the floral stalks of G. trichocarpa (Figure 5g), but long and 6- to 8-armed in G. tembensis (Figure 5c); and (4) 5- to 8-armed stellate trichomes with a central cushion, which have only been found on the floral stalks of G. tembensis (Figure 5c). Fruit colors are orange in both species, with 4 fruit lobes in G. tembensis and 2 in G. trichocarpa. The fruit surface of G. tembensis is distinct due to nonglandular trichomes that are simple, long, and tapered at the tip, and 2-armed tufted trichomes, which are present only in G. tembensis (Figure 6b–d). Stellate trichomes without a central cushion are observed only on the fruit surface of G. trichocarpa (Figure 6g,h).

3.2. Outcomes of Data Analysis

The PCA outcomes demonstrated significant variability in the morpho- and micromorphological attributes of the leaves, flowers, and fruits of G. tembensis and G. trichocarpa. This phenomenon was evident in the allocation of the specimens within the two main groups on the basis of the PCA results.

Table S3 and Figure S2 in the Supplementary Materials show the eigenvalues and variability of the principal component analysis (PCA) axes, where Axes 1, 2, and 3 represent the highest values (38.383, 14.550, and 13.114, respectively). The characteristics that exhibited the most substantial positive and negative loadings on Axis 1, 2, and 3 are shown in Table S4. The blade width, fruit lobe length, fruit lobe width, and fruit area characteristics exhibited the most significant positive contributions on Axis 1 of the PCA (0.849, 0.727, 0.866, and 0.822, respectively). In contrast, the blade length, area of the outer stomatal aperture, outer ledge on the abaxial (AB) surface of the leaf, and ovary area exhibited the most significant negative contributions on Axis 1 of the PCA (−0.749, −0.808, −0.864, and −0.817, respectively). The outer stomatal ledges length on the abaxial (AB) leaf surface and the ovary width characteristics presented the most significant positive and negative loadings on Axis 2 of the PCA (0.869 and −0.581, respectively). The petal length and petal area characteristics had the highest positive loadings (0.710 and 0.619, respectively) on Axis 3 of the PCA, whereas the blade area had the highest negative loadings (−0.502).

Figure 7 and Figure S3 in the Supplementary Materials show the loading plots and cluster loading distributions of samples on Axes 1 and 2 of the principal component analysis. The samples of G. tembensis and G. trichocarpa are clearly distributed within two groups according to the species. The PCA results show that the species under study differ along axis 1, and that axis 2 variables contribute to intraspecific variability. A similar effect of micromorphological characteristics on sample distribution was also observed for (Figure S4 and Figure S6 in the Supplementary Materials) Axes 1 and 3 of the PCA, as well as Axes 2 and 3 of the PCA (Figure S5 and Figure S7 in the Supplementary Materials). They obtained similar results from grouping the study samples into two separate groups according to species in heatmaps of the variation and hierarchical clustering analysis and cluster dendrogram, as shown in Figure S8 and Figure S9 in the Supplementary Materials.

Table S5 represents the correlation matrix among the micro-morphological features of the leaves, flowers, and fruits of G. tembensis and G. trichocarpa. A robust positive correlation was observed between the length of the flower pedicel and blade pedicel length characteristics (0.888). However, the most negative correlation was between the fruit lobe width and outer stomatal ledges area (−0.806).

The results of the T-test for the two species, namely, G. tembensis and G. trichocarpa (Table S6 and Figure 8), revealed that some micromorphological traits exhibited by the leaves, flowers, and fruit structures employed in this investigation significantly differed between the study samples. Specifically, the blade length, blade width, outer stomatal aperture area on the abaxial (AB) leaf surface, outer stomatal ledges area on the abaxial (AB) leaf surface, ovary width, ovary area, fruit lobe length, and fruit lobe width were significantly different between the two species, with p-values < 0.05 and R-squared values ranging between 0.537 and 0.804. The G. trichocarpa samples presented the largest values recorded for the blade length, blade width, outer stomatal aperture area AB, outer stomatal ledges area AB, ovary width, and ovary area in the box plot (Figure 8a,c–f), with means of 7.498, 16.163, 183.390, 4.650, and 8.031, respectively (Table S6 in the Supplementary Materials). The G. tembensis samples presented the greatest variation in the blade width, fruit lobe length, and fruit lobe width characteristics in the boxplot (Figure 8b,g,h), with means of 4.471, 0.699, and 0.519, respectively (Table S6).

Notably, these characteristics also reflect high loadings on the PCA axes, as shown in Table S6, and play a role in distinguishing the G. tembensis and G. trichocarpa samples (Figure 8).

4. Discussion

Micromorphological properties studied in this research strongly reflect that G. tembensis and G. trichocarpa share no close similarity. In fact, differences between these two species can be found in the abundance and types of trichomes on AB and AD leaf surfaces, ovary surfaces, and stalks, as well as fruit surfaces. The results of this study further suggest that the morphological characteristics of stomatal structures are important for the taxonomy of Grewia species. These findings are consistent with previous studies, which highlighted the importance of morphological characteristics in the classification of this genus. Other characters based on petiole length, leaf blade morphology, texture of blade surfaces, flower peduncle, ovary surface, and fruit morphology were also elucidated by Hashmi and Qaiser [10] in an investigation of Grewia species. In addition, Chung [9] showed that Grewia species can be differentiated from each other along several morphological parameters, such as leaf margin types, inflorescence types and their number, flower sexuality, surface texture of petals, and number of fruit lobes.

In a micromorphological study on Grewia L. in Peninsular Malaysia and Borneo, [26] reported four general categories and nine subcategories of nonglandular trichomes, as follows: (1) Simple trichomes, characterized by their long, tapered apex. (2) Tufted trichomes: likely classified into 2- to 4-armed. (3) Stellate trichomes, in 4- to 6-armed formations without a central cushion. (4) Cushioned stellate trichomes in 4- to 8-armed and with a central cushion. In the current analysis of samples from G. tembensis and G. trichocarpa, these basic trichome types were observed. However, there are differences in the shape, number of arms, density, and distribution of the trichomes.

In our investigation, we observed a distinction in the morphology of cuticular striation on the epidermis between the two species, characterized by elongated and pronounced cuticular striation on the leaf surfaces of G. tembensis, contrasted with shorter and less frequently occurring cuticular striation on the leaf surfaces of G. trichocarpa. This observation aligns with the findings presented by Chung [26], who noted that cuticular striation in Grewia species can be categorized as either elongated, infrequent, or shortened in nature.

The micromorphological characteristics of the leaves, flowers, and fruits tested in this study showed significant differences between G. tembensis and G. trichocarpa based on differences identified using principal component analysis, t-tests, and hierarchical clustering.

Specifically, blade length, blade width, outer stomatal aperture area on the abaxial surface of leaves, outer stomatal ledges area on the abaxial surface of leaves, ovary width, ovary area, fruit lobe length, and fruit lobe width were determined to be effective in classifying the species samples into two classes.

The fine morphological features of the foliage, including the type, size, and abundance of trichomes on the abaxial and adaxial surfaces and types of stomata, were scrutinized in this study and were found to differ measurably between the two species (G. tembensis and G. trichocarpa); hence, these features provided the most complete separation of the two taxa. Identification of these species is also critical, owing to their importance in traditional medicines in many parts of Asia and Africa, as well as their value as a source of high-quality timber and as feed for herbivorous livestock. Given that most species identification methods rely heavily on the descriptive and morphological features of fruits and flowers, structures that are not always available throughout the year or are limited to certain seasons; it proposes that such leaf features may provide an important contribution to the taxonomy of G. tembensis and G. trichocarpa when only vegetative material is available and species identification must rely solely on foliar material.

Intensive studies [35,36] confirmed that the micromorphological features of the stem and leaf were valid features to differentiate genera and species in the order Malvales. In addition, ref. [26] emphasized the significance of the unique glandular trichomes in species under the Malvaceae family as an important prerequisite for species identification and reliable authentication of herbal medicine.

Karakish et al. [37] investigated the micromorphological traits of 17 species from within the Malvaceae family of Saudi Arabia. Notable characteristics of the stem, petiole, lamina, trichomes, crystals, ducts, and star-shaped idioblasts were significant; they used these characteristics for the development of an artificial key.

In general, many recently published studies from various families support the idea that the analysis of micromorphological traits of leaves, trichomes, or stomatal complexes could be useful taxonomic tools to separate specimens at the genus or even species level, as mentioned before in the Scrophulariaceae family [38]. The study of relations of Silene leucophylla Boiss. and Silene schimperiana Boiss. demonstrated by [39]. Additionally, the study of micromorphological characteristics for species from the family Loranthaceae Juss [40] confirmed the importance of micromorphological characteristics.

5. Conclusions

The features of the qualitative and quantitative micromorphology analyzed throughout this study show the differences among the specimens of both species (G. tembensis and G. trichocarpa).

It observed fine variations between G. tembensis and G. trichocarpa samples in trichome features with respect to: the number of arms, trichome morphology, density, and distribution in leaves, ovaries, and fruits, in addition to variation in stomatal shape.

The results of the comparative analysis found differences between the microcharacters observed in: leaves, flowers, and fruits of both species. In particular, blade length, blade width, outer stomatal aperture area on the abaxial leaf, outer stomatal ledges area on the abaxial leaf, ovary width, ovary area, length of the fruit lobes, and fruit lobes width.

Also, encourage future studies to investigate the Grewia species through SEM methods, pursuing this powerful methodology to explore the highly diverse structural potential that exists within this genus.