Plant taxonomy plays a critical role in plant diversity assessment, conservation, phytogeographic deductions, optimum utilization, and inferences (Mukherjee 2014). As such, the events of plant species misidentification could lead to detrimental results. The genus Prunus (Family Rosaceae) comprises over 400 plant species and displays vast biodiversity globally, although only approximately 98 species of the genus are of great importance, including P. domestica (Linn.), P. persica (L.) Batsch, P. amygdalus (L.) Batsch, P. cerasoides (D.) Don, P. armeniaca (Linn.), and P. africana (Hook f.) Kalkman (Biswajit et al., 2011). African cherry, also known as Red stinkwood or African almond (P. africana, synonym: Pygeum africanum Hook. F) is an evergreen tree species that occurs within the sub-Saharan countries of Africa including Angola, Burundi, Cameroon, the Democratic Republic of Congo, Equatorial Guinea, Ethiopia, Kenya, Lesotho, Madagascar, Malawi, Mozambique, Nigeria, Rwanda, Sao Tome, South Africa, South Sudan, Swaziland, Tanzania, Uganda, Zambia, and Zimbabwe (Komakech and Kang 2019). The genus name Prunus derives from the Latin word that refers to the plum family and the binomial Latin name P. africana indicates the African origin of the species (Komakech et al. 2017). P. africana is a highland forest tree, growing in the humid and semi-humid highlands and humid midlands. The wild tree species is found mainly in tropical forests at altitudes of approximately 900-3,400 m above the sea level, with a mean annual rainfall and temperature of 890-2,600 mm and 18-26°C, respectively (Komakech et al. 2017). The mature P. africana tree is approximately 10-25 m high with open branches (Fig. 1a). The outer part of the stem bark is corrugated or rough and black to brown in color (Fig. 1b). The leaves are alternate and simple, approximately 8-20 cm in length, dark green on the top, and pale green at the bottom with mildly serrated margins (Fig. 1c). The flowers are small, white or greenish, hairy, and borne in bunches. The fruits are spherical, purplish-brown, and bilobed, with a thin tough pericarp (Fig. 1d). The seeds are yellowish-brown and oval in shape (Fig. 1e) (Komakech et al. 2017).

Figure 1. Botany of Prunus africana. a. P. africana tree. b. P. africana stem with part of its bark harvested for medicinal purposes. c. P. africana leaf. d. P. africana fruit. e, f. P. africana seeds

Prunus africana has been used in the treatment and management of several diseases including benign prostatic hyperplasia, prostate cancer, gonorrhea, chest pain, fevers, gastrointestinal conditions, urinary disorders, malaria, diabetes, obesity, mental illness, hypertension, infertility, and kidney disease (Jimu 2011; Komakech and Kang 2019; Steenkamp 2003). Owing to its medicinal importance, a micropropagation protocol for P. africana was recently developed to meet the ever-increasing demand for it (Komakech et al. 2020). However, as an important medicinal plant, providing features that enhances its accurate taxonomic identification and authentication is pivotal (De Souza et al. 2018). Microscopic observation is one of the important approaches to identify characteristic features that could be used to standardize medicinal plant characterization. Although a study focusing on the anatomy of P. africana bark and wood structure has been published (Kotina et al. 2016), there is limited information regarding its leaf, fruit, and seed anatomy. This study thus attempted to provide anatomical characteristics of P. africana leaf, seed, fruit, and stem which could be important additional features for its accurate plant identification, quality control, and phylogenetic studies in the future.

The Natural Chemotherapeutics Research Institute, Ministry of Health, Uganda provided the P. africana sample for the purpose of this study. The voucher specimen number KIOM201901022377 was deposited in the Korean Herbarium of Standard Herbal Resources (Index Herbarium code: KIOM) at the Korea Institute of Oriental Medicine (KIOM), South Korea. The dried leaves and seeds for the anatomy study were stored in water for 24 h, followed by maceration in boiling water for 10 min. An ascending ethanol series (50-100%) was used to dehydrate the tissues for 1 h at each concentration. The samples were then embedded using Technovit^® 7100 (Heraeus-Kulzer, German), based on a previously described protocol (Yeung and Chan 2015). After complete polymerization, 10 µm sections of the resulting resin blocks were prepared using a microtome (SM 2010R; Leica, Wetzlar, Germany) with a tungsten carbide knife. The resin films containing the tissue sections were attached to the glass slides using warm water, followed by staining with Toluidine Blue O before the mounting using Permount^® (Fisher Science, Hampton, USA). The slides of the studied material were observed under a microscope (BX53; Olympus, Tokyo, Japan) and photographed using a digital camera (DP-51; Olympus, Tokyo, Japan).

For the leaf cuticle morphological observations, living material samples were stored using 70% ethanol, then cut into small pieces (1.0 cm × 1.0 cm). For the light microscopic observations, the samples were dipped in 6% sodium hypochlorite for 8 h. The samples were then thoroughly washed in distilled water. The epidermis of both surfaces of the leaves was peeled off using a single-edge blade (DN-52, Dorco, Seoul, Korea), colored in 1% safranin-50% ethanol for 3 min and mounted in Canada balsam. The mounted slides were examined under a light microscope (Olympus BX-53, Olympus, Tokyo, Japan), and captured using a digital camera (Olympus DP21, Olympus, Tokyo, Japan). The distribution of the epidermal types and stomata density were recorded and compared from the central part of the leaves. The cuticle morphological terminology used to describe the leaves in this study followed previously published indications (Evert 2006; Wilkinson 1979).

Leaf morphology plays an integral role in plant taxonomy, identification, and systematics (Vincenzo and Cardini 2011). In this study, the leaves of P. africana were observed to exhibit a smooth surface and hypostomatic nature with stomata size ranging 18.90-(22.34)-26.90 × 15.41-(18.40)- 21.22 μm (Table 1). The leaves were also observed to display an isodiametric or irregular cell arrangement (Table 2). Tetracytic and anisocytic stomata complexes and three anticlinal cell wall types (straight, undulated, and straight/ curved) were observed on the leaf surfaces (Fig. 2A and B). These stomatal complex types are important factors in accurate plant classification (Abdulrahaman et al. 2009), determination of plant origin, evolution, and phylogenetic relationships (Hong et al. 2018).

Table 1 . Overview of representative stomatal characteristics of Prunus africana.

Prunus species	Stomatal complex			Size of stomata (μm)
	Position		Type	Length	Width
Prunus africana	HP	AB	Ani, Tet	18.90-(22.34)-26.90	15.41-(18.40)-21.22

HP - hypostomatic; AB - abaxial surface; Act - actinocytic; Ani - anisocytic; Ano - anomocytic; Tet - tetracytic..

Table 2 . Overview of representative leaf epidermal surface characteristics of Prunus africana.

Prunus species		Primary sculpture			Crystals		Trichomes
		Outline	Anticlinal wall	Periclinal wall	DR	ST	SS	LS	GT
Prunus africana	AD	iso	und	ft	−	++	−	+	−
	AB	iso	cur/st, und	ft	−	++	−	−	−

AD - adaxial surface; AB - abaxial surface; iso - isodiametric; st - straight; cur - curved; und - undulate; ft - flat; DR - druse-shaped crystal; ST - star-shaped crystal; SS - short simple trichomes; LS - long simple trichomes; GT - glandular trichomes. −, absent; +, present; ++, dominant..

Figure 2. Light microscopic micrographs revealing leaf characteristics of Prunus africana. A. Leaf adaxial surface. B. Leaf abaxial surface. C. Druse-shaped crystals. D, E: Transverse sections of leaf petiole. F, H: Transverse sections of leaf blade. C, E, H: The observation was performed using the differential interference contrast (DIC) mode of a light microscope. ep: epidermis, ics: intercellular space, pa: palisade parenchyma, spo: spongy parenchyma, vb: vascular bundle, xy: xylem, arrows: druse crystals. Scale bars for A, B: 50 μm, C: 20 μm

In addition to morphology, leaf anatomy has gained importance as a key tool in plant taxonomy over the years (Araújo et al. 2010; Kolb et al. 2020). In this study, the transverse-section through the leaf mid-vein and petiole of P. africana showed the presence of characteristic druse crystals (Fig. 2C, E, and H), calcium oxalate crystals distributed in all plants and known to protect plants against herbivores (Franceschi and Nakata 2005). The shape of these crystals is reportedly genetically controlled which explains their consistency in each plant species (Ilarslan et al. 2001). The presence of these druse crystals might thus play an important role in plant taxonomy since they occur in various morphological shapes as per given plant species, including druses, prisms, styloids, raphides, and crystal sand that vary from one plant species to another (Konyar et al. 2014). Previous studies also reported the presence of druse crystals in the leaves of other prunus species including P. serotina (Lersten and Horner 2006) and P. virginiana (Lersten and Horner 2004).

Our observation showed that the transverse-section of the P. africana petiole contained vesicular bundles with prominent xylem vessels arranged in a circular pattern (Fig. 2D and E). The P. africana mid-leaf transverse-section revealed the presence of upper and lower epidermal layers with cuticle and a single layer of end-to-end densely packed epidermal cells with large vacuoles (Fig. 2F and G). It is an important structure that protects the plant against moisture loss, microbial, and physical harm (Crang et al. 2018). The palisade layer located on the adaxial side just beneath the upper leaf epidermis was made of closely packed cylindrical cells, and a spongy layer located in the abaxial side of the leaf with loosely arranged irregular shaped cells and wide intercellular space (Fig. 2G and H).

The young stems have green and glabrous surface (Fig. 3A). The anatomical section of the stem showed the presence of a layer of isodiametric cells covered by a smooth cuticle. The cortical parenchyma inner regions contain the intercellular spaces. Sclerenchyma cells were absent. Primary vascular bundles xylem and phloem vessels were arranged in the shape of a ring (Fig. 3B). The pith was centrally located, consisting of spongy parenchyma cells (Fig. 3B-D). The cortex consisted of isodiametric, thin-walled parenchyma cell layers containing druse crystals (Fig. 3D). Druse crystals were observed in the cortex region of the stem. The mature bark is corrugated or rough and black to brown in color. It contains brown dots and/or patches of lenticels and adherent scales (Fig. 3E). The microscopic examination of the stem bark of mature P. africana (Fig. 3F) showed mainly the presence of rhytidome-secondary phloem and the oldest periderm densely arranged in multiple layers (Fig. 3G). Furthermore, a cluster of stone cells was observed scattered among the rhytidomes. Sclerenchyma was observed within the phelloderm of mature stems (Fig. 3H).

Figure 3. Stem morphology and anatomy of Prunus africana. A. Young stem morphology. B: Transverse section of young stem. C, D: Longitudinal sections of young stem. E. Mature stem bark morphology. F. Dried stem bark. G, H. Rhytidome. The observation was performed using the DIC mode of a light microscope. cor: cortex, ep: epidermis, pf: phloem fiber, ph: phloem, xy: xylem, arrows: druse crystals, per: periderm, sc: sclerenchyma. Scale bars for G, H: 200 μm

The structures observed in P. africana stem bark including the periderm in multiple layers, intercellular spaces, secondary phloem, the sclerenchyma tissues, occurrence of druse crystals, almost exclusively simple perforation plates, and stone cells are shared by other prunus species that were previously studied including P. serotina, P. Avium, P. Pennsylvanica, and P. Pennsylvanica (Bastin 1895); indicated that they all belong to the genus Prunus.

The anatomy of the P. africana pericarp showed the presence of epicarp, mesocarp, and endocarp with a thickness of approximately 350-400, 300-350, and 30-50 μm, respectively, and vascular bundles (Fig. 4 A, B, C).

Figure 4. Anatomy of fruit and seed of Prunus africana. A, B, C: Transverse section of pericarp. D: Transverse section of seed. epc: epicarp, mec: mesocarp, end: endocarp, vb: vascular bundle, cot: cotyledon, pl: plumule, ts: testa, arrows: sclerenchyma

Seed anatomy plays a vital role in the taxonomy of plants (Vaughan 2009). In this study, the transverse section through the seed of P. africana showed the presence of testa with a thickness of approximately 50-60 μm, plumule, and sclenchyma tissue.

The internal structures of the plants play critical roles in the understanding of the relationships between the taxa. Consequently, the results obtained in this study will play a crucial role in the taxonomy of P. africana.

This research was funded by the framework of International Cooperation Program (Korea-South Africa Cooperative Research Project for Excavation of Candidate Resources of Complementary and Alternative Medicine) managed by National Research Foundation of Korea (grant no. 2017093655 and KIOM: D17470). Additionally, this work was also supported by Development of Foundational Techniques for the Domestic Production of Herbal Medicines (K18405), Development of Sustainable Application for Standard Herbal Resources (KSN2013320), Korea Institute of Oriental Medicine through the Ministry of Science and ICT, Republic of Korea.

The authors declare no conflict of interest.