Anatomical characterization of Semi-arid Bignoniaceae using light and scanning electron microscopy

Abstract

Background

The present research work was done to evaluate the anatomical differences among selected species of the family Bignoniaceae, as limited anatomical data is available for this family in Pakistan. Bignoniaceae is a remarkable family for its various medicinal properties and anatomical characterization is an important feature for the identification and classification of plants.

Methodology

: In this study, several anatomical structures were examined, including stomata type and shape, leaf epidermis shape, epidermal cell size, and the presence or absence of trichomes and crystals (e.g., prisms, raphides, and druses). Three statistical tools—heat map analysis, correlation analysis, and principal component analysis (PCA)—were used to highlight distinctions and similarities among the species.

Results

On both the upper and lower leaf surfaces, polygonal, irregular, and hexagonal epidermal cells with thick cell walls were observed. Three patterns of anticlinal cell walls were detected: curved, straight, and sinuous. Distinct stomatal types were also identified across the different species. For instance, sunken stomata were observed in Kigelia africana and Jacaranda mimosaefolia, while anomocytic stomata were found in Oroxylum indicum, Pyrostegia venusta, Tecoma stans, Tecomella undulata, Mansoa alliacea, Heterophragma adenophylla, Handroanthus impetiginosus, Campsis radicans, and Anemopaegma chamberlaynii. Paracytic stomata were examined in B. callistegioides and Dolichandra unguis-cati. Tabebuia aurea was the only species with Tetracytic stomata. A contiguous type of stomata was only observed in Millingtonia hortensis. This family contained three types of trichomes. Glandular peltate trichomes contained a basal epidermal cell, a very small monocellular stalk and a circular or round multicellular head containing 12 cells arranged in a single circle. Non-glandular trichomes had a thin apex without a head and a pointed end. Branched trichomes contained several arms arising from a common base.

Conclusion

This anatomical examination, using advanced microscopic techniques, is the first to classify several species that are not listed in the e-flora of Pakistan. Leaf anatomical research has proven valuable in resolving challenging taxonomic issues.

Background

The Bignoniaceae family contains about 120 genera and 800 species worldwide. It is characterized in Pakistan by three native genera and eighteen commonly cultivated species (http://www.efloras.org/flora). The plant species of this family are located globally. Nevertheless, some are found in subtropical and tropical countries. However, some of the temperate species are found in East Asia and North America. Tropical rainforests, savannas, and dry forests are the preferred habitats for the Bignoniaceae family plant species [1].

The taxonomic classification of the Bignoniaceae is defined by Gentry [2, 3]. Traditionally, the categorization of the Bignonieae had contained some large and small genera through several difficult generic delimitations. In the most current angiosperm categorizations, Bignoniaceae was placed within Lamiales order [4]. Fisher [5] has done the current categorization of the Bignoniaceae, and he documented eight tribes: Crescentieae, Bignonieae, Oroxyleae, Eccremocarpeae, Coleeae, Tourrettieae, Tecomeae and Schlegelieae.

Members of the Bignoniaceae family primarily consist of trees, shrubs, climbers, and herbs. Morphologically, they typically exhibit opposite, simple, or compound leaves that may transform into tendrillar organs and lack stipules. Their flowers are irregular, bisexual, and hypogynous, featuring a 5-lobed calyx, tubular or campanulate corolla, and 4 to 5 epipetalous, didynamous stamens. Staminodes may be present, and the anthers are bilocular with longitudinal dehiscence. The floral disk may or may not be present, and the ovary demonstrates axile placentation with multiple ovules. Distinctive wood features of Bignoniaceae include septate fibers, absence of vasicentric tracheids, and minimal paratracheal parenchyma, setting them apart from lianas of other families. Additionally, this family contains numerous crystal-like structures confined to ray cells [6].

The species of Bignoniaceae are economically important in agriculture as well as horticulture, while aboriginal individuals utilize some plants for various reasons (food, timber, therapeutic and ritual purposes). Some species are also used as an ornamental plant [7]. The different species of this family have relatively enthusiastic and colourful flowers, which provide ornamental significance. Furthermore, the timber of this family contains a high marketable significance, which is a commercial purpose for afforestation [8].

In addition to their ornamental value, members of the Bignoniaceae family play a significant role in managing and treating various diseases affecting humans. The effectiveness of different plant parts varies among communities and nationalities. For instance, in Tobago and Trinidad, the leaves are utilized to help control high blood pressure. In Mexico, particularly in the Antilles and Yucatan, the fruit pulp is employed to treat respiratory ailments and internal abscesses. In Colombia, the unripe fruit is used for treating snakebites, while in the Philippines, it is applied in the treatment of diarrhea, inflammation, and hypertension [9].

Although the Bignoniaceae family is included in the flora of Pakistan, there is a notable deficiency in anatomical information regarding many of its species. Specifically, several important taxa, such as Tabebuia aurea Benth. & Hook.f. ex S.Moore, Mansoa alliacea (Lam.) A.H. Gentry, Bignonia callistegioides Cham., Handroanthus impetiginosus (Mart. ex DC.) Mattos, and Anemopaegma chamberlaynii (Sims) Bureau & K.Schum, have not been documented in the available flora literature. This lack of information poses a significant challenge for taxonomists and botanists seeking to accurately identify and classify these species based on their anatomical features.

To address this gap, the present research undertakes a comprehensive anatomical analysis of the aforementioned species, in conjunction with other members of the Bignoniaceae family. By employing advanced microscopic techniques and thorough examination methods, this study aims to elucidate the anatomical characteristics of these underrepresented species. The analysis includes the evaluation of key features such as leaf structure, epidermal cell morphology, stomatal arrangement, trichome types, and the presence of specialized structures like crystals.

This research not only seeks to fill the existing knowledge void but also aims to provide valuable insights into the anatomical diversity within the Bignoniaceae family in Pakistan. By documenting and analyzing these anatomical features, the study contributes to a deeper understanding of the taxonomic relationships among these species, thereby facilitating accurate identification and classification. Ultimately, the findings are expected to enhance the overall botanical knowledge of the Bignoniaceae family in Pakistan and serve as a reference for future research and conservation efforts related to this important group of plants. Anatomical features are valuable tools for plant identification and differentiation, particularly in clarifying taxonomic relationships. Researchers have recognized the importance of leaf anatomy in this context, as it showcases variations in epidermal characteristics across 16 taxa within the Bignoniaceae family, thereby aiding species identification and classification [10, 11]. All these clarifications provide immense value in the botanical documentation and calibration of the drug in crude form [12].

This research provides a detailed study of the leaf anatomy and in silico approach of the Bignoniaceae family. This information would be helpful for taxonomists in correctly identifying species of the family Bignoniaceae.

Materials and methods

This research was done to evaluate the important anatomical features of some members of the family Bignoniaceae. The whole experimental work was performed in the Taxonomy Lab of Lahore College for Women University, Lahore.

Plant species collection and identification

A variety of Bignoniaceae family plant species were collected from District Lahore (Lahore College for Women University, Bagh-e-Jinnah, Government College University, Canal Road, Lahore Nursery, and Jilani Park). The collection of samples was conducted during the peak growing season, from April to August, in the years 2022–2023. About 15 different plant samples were collected and separately placed in plastic zipper bags with tags indicating their common and botanical names. The species were then properly identified by consulting different floras (http://www.efloras.org), after which voucher numbers were assigned and used for taxonomic studies.

Morphological characterization

Vegetative features, e.g. colour and height of stem; type of root (tap root or adventitious root); leaf (colour, venation, type, length, width and margin), were examined and documented. Two types of texture were observed such as Glabrous and Coriaceous. Some reproductive characteristics such as stamens, flowers, inflorescence, and the characteristics of floral whorls will be observed using a stereomicroscope and magnifying glass [13].

Anatomical characterization

Light Microscopy

The fresh plant samples were used to study the anatomy of the leaf epidermis. Then, a solution of fluid comprising 70% concentration of Lactic acid and 30% concentration of nitric acid was prepared. Then, the plant leaf samples were bubbled in a water bath for around 15–20 min, and the clearance of chlorophyll lactic acid was extinguished, which activated the discolouration of leaves. After boiling, the mixture was allowed to cool down. The unwinding of the leaf was achieved through the effortless peeling off the epidermis. For anatomical analysis, both the upper and lower surfaces of leaves were assembled.

For the formation of abaxial surface slides, the adaxial surface of leaves was deposited on the top side of the tile. By using a blade and camel hairbrush, the adaxial surface of the leaves was peeled off. The leaves were washed away continuously within a solution of 70% cold lactic acid so that the upper surface of the leaves was detached and the adaxial surface of the leaves was left on the tile. With the help of a small drop of lactic acid, the lower part of the leaf was shifted on the glass slide. Then, it was observed through a light microscope (SZF model Kyowa, Japan). Some important anatomical features of the leaf epidermis were observed, such as the number and shape of stomata shape and the size of epidermal cells, e.g. irregular, polygonal, hexagonal, trichomes, etc. The same process was followed for the preparation of the adaxial surface slide [13].

Scanning electron microscopy

For SEM analysis, sections of leaves were taken from the leaf margins and the middle. These sections were cut into sizes suitable for the stubs of the scanning electron microscope. The samples were attached with adhesive tape and coated with gold-palladium before observation under an EVO/LS10 model scanning electron microscope. Microphotographs were taken at magnifications ranging from 1500× to 10,000×. This protocol follows the established methods described by Kedar et al. (2018) with minor modifications to adapt to our specific samples and equipment [14].

Statistical analysis

Mean, Stranded Deviation and Stranded Error were calculated for descriptive analysis for each sample. The mean value was taken for further evaluation. TB tool (toolkit for Biologistics) was used for heat map analysis. R software (R-4.4.1) was used for Principal Component Analysis and Correlation analysis. The analysis included variables such as epidermal cell length and width, length and width of stomatal cells, guard cells, subsidiary cells, and trichomes.

Results

The taxonomic diversity of different plants of the Bignoniaceae family is studied through light and scanning electron microscope (Table 1a). In this research, we investigated various qualitative and quantitative characteristics of leaves, which are explained below (Fig. 1).

Table 2.

B: qualitative data of adaxial epidermis of selected species of family Bignoniaceae

No of Plan ts	Plant names	Leaf epidermis cells	Epidermis cell margins		Type of Stomata	Guard cells	Trichomes	Crystals
1	Kigelia Africana (Lam.) Benth	Irregular or sinuous shape of epidermal cells	curved to sinuate and thick anticlinal cell wall		Sunken stomata, open and closed stomata	Kidney shaped	Absent	Prismatic crystals
2	Oroxylum indicum L.	Polygonal, irregular, sinuous or smooth shape of epidermal cells	curved to wavy and thick anticlinal cell wall		Absent	Absent	Glandular peltate and multicellular	Absent
3	Dolichandra unguis-cati L.	Irregular or sinuous shape of epidermal cells	Curved to wavy and thick anticlinal cell wall		Paracytic stomata, open stomata	Kidney shaped	Absent	Present
4	Pyrostegia venusta Ker Gawl.	Polygonal or smooth shape of epidermal cells		thick anticlinal cell wall	Absent	Absent	Absent	Raphides
5	Tecoma stans L.	Irregular or sinuous shape of epidermal cells		wavy to undulate and thick anticlinal cell wall	Absent	Absent	Glandular peltate and multicellular	Prismatic crystals, druses
6	Tecomella undulata Sm.	Irregular or sinuous shape of epidermal cells		curved to undulate and thick anticlinal cell wall	Anomocytic and open stomata	Kidney shaped	Glandular peltate and multicellular	Prismatic crystals
7	Tabebuia aurea Benth & Hook.f.ex.moore	Hexagonal shape of epidermal cells		thick and undulate anticlinal cell wall	Tetracytic, open and closed stomata	Kidney shaped.	Glandular peltate and multicellular	Prismatic crystals
8	Millingtonia hortensis L.f	Irregular, sinuous or polygonal shape of epidermal cells		curved, smooth or straight anticlinal cell wall	Anomocytic stomata, contiguous stomata and open stomata	Kidney shaped.	Glandular peltate, multicellular and non-glandular	Prismatic crystals, druses
9	Mansoa alliacea Lam.	Irregular or sinuous shape of epidermal cells		thick and wavy anticlinal cell wall	Absent	Absent	Glandular peltate and multicellular	Druses
10	Jacaranda mimosifolia D.Don	Irregular, sinuous or polygonal shape of epidermal cells		thick, smooth and curved anticlinal cell wall	Absent	Absent	Glandular peltate, multicellular and non-glandular	Prismatic crystals, druses
11	Fernandoa adenophylla Wall. ex G.Don	Polygonal or hexagonal shape of epidermal cells		straight, smooth and thick anticlinal wall	Absent	Absent	Branched, Glandular peltate and multicellular	Raphides, druses
12	Bignonia callistegioides Chem.	Polygonal shape of epidermal cells		curved, thick and smooth anticlinal cell wall	Absent	Absent	Absent	Prismatic crystals, druses
13	Handroanthus impetiginosus Mart. ex DC	Irregular, sinuous or polygonal shape of epidermal cells		straight, curved, smooth and thick anticlinal cell wall	Absent	Absent	Glandular peltate and multicellular	Prismatic crystals, druses
14	Campsis radicans L.	Irregular or sinuous shape of epidermal cells		curved to undulate anticlinal cell wall	Absent	Absent	Absent	Prismatic crystals, druses
15	Anemopaegma chamberlaynii Sims.	Irregular, sinuous or polygonal shape of epidermal cells		thick, smooth and curved anticlinal cell wall	Absent	Absent	Glandular, peltate and non-glandular with pointed ends	Druses

Fig. 1.

Studies members of Bignoniaceae A Kigelia africana B Oroxylum indicum C Dolichandra unguis-cati D Pyrostegia venusta E Tecoma stans F Tecomella undulata. G Tabebuia aurea H Millingtonia hortensis I Mansoa alliacea J Jacaranda mimosifolia K Fernandoa adenophylla L Bignonia callistegioides Cham. M Handroanthus impetiginosus N Campsis radicans Seem. O Anemopaegma chamberlaynii

Epidermal cells

Epidermal cells play a crucial role in plant identification due to their structural diversity, adaptations, and unique features in different plant species, families, and genera. These cells form the outermost layer of plant organs such as leaves, stems, and roots, and they exhibit a range of characteristics that can be used for taxonomic purposes. in the present studies, some variations were observed in the epidermal cell shapes of Bignoniaceae studied members. In upper and lower leaf surfaces, polygonal, irregular and hexagonal shapes of epidermal cells with thick cell walls were observed. It showed all three patterns of anticlinal cell wall, e.g. curved, straight and sinuous (Fig. 2). Polygonal shapes of epidermal cells with thick cell walls were observed in Oroxylum indicum(L.) Benth. ex Kurz, Pyrostegia venusta (Ker Gawl.) Miers, Fernandoa adenophylla (Wall. ex G. Don) Steenis, Bignonia callistegioides Cham. Hexagonal shaped of epidermal cells were observed in Tabebuia aurea Hook. f. ex S. Moore. Irregular shapes of epidermal cells were observed in Kigelia africana (Lam.) Benth, Dolichandra unguis-cati L., Tecoma stans L., Tecomella undulata (Sm.) Seem, Millingtonia hortensis L.f., Mansoa alliacea (Lam.), Handroanthus impetiginosus L., Campsis radicans L., Anemopaegma chamberlaynii L. and Jacaranda mimosaefolia D. Don. The length of epidermis cells ranged from 37.8 μm to 209.3 μm (Fig. 3). K. africana had the largest length, and J. mimosaefolia had the smallest length. The width of epidermis cells ranged from 18.1 μm to 189.1 μm. K. africana had the largest width, and J. mimosaefolia had the smallest width (Tables 1 and 2). According to [15], the anticlinal cell walls were mainly sinuous in this family.

Fig. 2.

Light microscopy of epidermal cells of studied species of Bignoniaceae (EC = epidermis cell, S = stomata A Kigelia africana showing irregular shape of epidermal cells. B and C Oroxylum indicum showing irregular and polygonal shape of epidermal cells. D Dolichandra unguis-cati showing an irregular shape of epidermal cells. E and F Pyrostegia venusta M E) showing an irregular and polygonal shape of epidermal cells. G Tecoma stans (L.) showing an irregular shape of epidermal cells. H) Tecomella undulata showing an irregular shape of epidermal cells. I Tabebuia aurea showing a hexagonal shape of epidermal cells. J and K Millingtonia hortensis L.f. shows irregular and polygonal shapes of epidermal cells. L Mansoa alliacea A.H.Gentry is showing an irregular shape of epidermal cells. M and N Jacaranda mimosifolia showing an irregular and polygonal shape of epidermal cells. O Fernandoa adenophylla Steenis showing a polygonal or hexagonal shape of epidermal cells. P Bignonia callistegioides showing a polygonal shape of epidermal cells. Q Handroanthus impetiginosus showing irregular and polygonal shape of epidermal cells. R Campsis radicans Seem. showing irregular shapes of epidermal cells. S Anemopaegma chamberlaynii showing an irregular shape of epidermal cells

Fig. 3.

Scanning electron microscopy of epidermal cells of studied species of Bignoniaceae A Kigelia africana showing irregular shape of epidermal cells. B and C Oroxylum indicum showing irregular and polygonal shape of epidermal cells. D Dolichandra unguis-cati (L.) L. G. Lohmann showing irregular shape of epidermal cells. E and F Pyrostegia venusta (E) showing the irregular and polygonal shape of epidermal cells. G Tecoma stans showing irregular shape of epidermal cells. H Tecomella undulata G showing irregular shape of epidermal cells. I Tabebuia aurea showing the hexagonal shape of epidermal cells. J Millingtonia hortensis L.f. shows irregular and polygonal shapes of epidermal cells. K Mansoa alliacea showing irregular shape of epidermal cells. L Jacaranda mimosifolia showing the irregular and polygonal shape of epidermal cells. M Fernandoa adenophylla Steenis showing polygonal or hexagonal shape of epidermal cells. N Bignonia callistegioides showing polygonal shape of epidermal cells. O Handroanthus impetiginosus showing irregular and polygonal shape of epidermal cells. P Campsis radicans Seem. showing the irregular shape of epidermal cells. Q Anemopaegma chamberlaynii showing irregular shape of epidermal cells

Table 1.

A: qualitative data of abaxial epidermis of selected species of family Bignoniaceae

Sr no.	Plant names	Leaf epidermis cells	Epidermal cell margins	Type of Stomata	Guard cells	Trichomes	Crystals
1	Kigelia africana (Lam.) Benth	Irregular or sinuous shape of epidermal cells	Curved to undulate and thick cell wall	Anomocytic, sunken stomata, open and closed stomata	Kidney shaped	Glandular peltate and Multicellular	Absent
2	Oroxylum indicum L.	Irregular or sinuous shape of epidermal cells	curved to undulate or wavy anticlinal cell wall	Anomocytic stomata. open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Prismatic crystals
3	Dolichandra unguis-cati L.	Irregular or sinuous shape of epidermal cells	curved to wavy and thick anticlinal cell wall	Paracytic stomata open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Druses
4	Pyrostegia venusta Ker Gawl .	Irregular or sinuous shape of epidermal cells	anticlinal cell wall	Anomocytic stomata open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Raphides
5	Tecoma stans L.	Irregular or sinuous shape of epidermal cells	undulate striation and wavy anticlinal cell wall	Anomocytic stomata and open stomata.	Kidney-shaped and elliptic with wide opening	Glandular peltate and multicellular	Absent
6	Tecomella undulata Sm.	Irregular or sinuous shape of epidermal cells	curved to undulate thick anticlinal cell wall	Anomocytic raised stomata and open stomata	Kidney shaped	Glandular peltate and multicellular	Prismatic crystals
7	Tabebuia aurea Benth. & Hook.f.ex moore	Hexagonal shape of epidermal cells	thick and undulate anticlinal cell wall	Tetracytic stomata open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Absent
8	Millingtonia hortensis L.f	Irregular or sinuous shape of epidermal cells	curved or undulated thick anticlinal cell wall	Anomocytic stomata and open stomata	Kidney shaped	Glandular, peltate, multicellular and non-glandular	Prismatic crystals, druses
9	Mansoa alliaceaLam.	Irregular or sinuous shape of epidermal cells	undulate striation or wavy anticlinal cell wall	Anomocytic or Anisocytic stomata. Open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Druses
10	Jacaranda mimosifolia D.Don	Irregular or sinuous shape of epidermal cells	wavy anticlinal cell wall	Sunken stomata, anomocytic and open stomata	Absent	Glandular peltate, multicellular and non-glandular trichomes	Prismatic crystals or druses
11	Fernandoa adenophylla Wall. ex G. Don	Polygonal or hexagonal shape of epidermal cells	straight, smooth and thick anticlinal cell wall	Anomocytic stomata, open and closed stomata	Kidney shaped	Branched, glandular peltate and multicellular	Prismatic crystals and druses
12	Bignonia callistegioides Cham.	Polygonal shape of epidermal cells	curved, thick and smooth anticlinal cell wall	Paracytic stomata, open and closed stomata	Kidney shaped	Absent	Prismatic crystals, raphides, druses
13	Handroanthus impetiginosus Mart. ex DC	Irregular, sinuous or polygonal shape of epidermal cells	straight, curved, smooth and thick anticlinal cell wall	Anomocytic stomata, open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Prismatic crystals, druses
14	Campsis radicans L.	Irregular or sinuous shape of epidermal cells	curved to undulate and thick anticlinal cell wall	Anomocytic stomata, open and closed stomata	Kidney shaped	Glandular peltate and multicellular	Prismatic crystals, raphides, druses
15	Anemopaegma chamberlaynii sims.	Irregular, sinuous or polygonal shape of epidermal cells	curved and smooth anticlinal cell wall and striations	Anomocytic and Aniosocytic stomata, open and closed stomata	Kidney shaped	Glandular peltate, multicellular and non-glandular with pointed ends	Druses, raphides

Stomata

Stomata play a crucial role in plant identification, particularly when distinguishing between species or even genera, because they exhibit variations in structure, size, arrangement, and density, which are often species-specific. In the current investigation, distinct stomatal types were observed in the leaves of different Bignoniaceae species. In K. africana and J. mimosaefolia, sunken stomata were observed. In contrast, anomocytic stomata were found in O.indicum, P.venusta, T. stans, T. undulata, M. alliacea, F. adenophylla, H. impetiginosus, C. radicans, and A. chamberlaynii. Anisocytic stomata were also observed in M. alliacea and (A) chamberlaynii. Paracytic stomata were examined in (B) callistegioides and Dolichandra unguis-cati. T. aurea was the only species with Tetracytic stomata. A contiguous type of stomata was observed only in M. hortensis. Many variations were found in the length and width of stomata size. The average length of stomata ranges from 57.1 μm to 104.6 μm. T.stans had the largest length, and M. hortensis had the smallest length (Table 3). The average width of stomata ranges from 39.2 μm to 78.4 μm. Dolichandra unguis-cati had the largest width, and the M. hortensis had the smallest width (Fig. 4).

Table 3.

A: quantitative data of abaxial epidermis of selected species of family Bignoniaceae

No of Plan ts	Plant names	Leaf epidermis cells				Total number of Stomata		Stomatal cavity						Guard cells				Subsidiary cells				Trichomes							No of Trichomes
		Length µm		Width µm		Open	closed	Length µm	Width µm					Length µm	Width µm			Length µm			Width µm	Length µm		Width µm
1	Kigelia africana	206.1 (209.3 −210.3)		189.1 (183.8–195.9)		22	8	78.2 (51.5–92.4)	56.9 (49.5–68.2)					22.4 (22.0–23.0)	8.1 (7.0–9.0)			158.9 (70.4–173.0).			80.6 (55.6–99.0)	80.9 (77.4–83.9)		78.9 (70.7–82.5)					3
2	Oroxylum indicum	124.8 (104.2–136.5)			68.8(49.8–80.4)	16	5	73.8 (65.8–70.0).	52.5 (51.6–53.0)					65.7 (60.6–71.0)	26.5 (21.1–30.0)			71.1 (57.3–94.0)			30.6 (25.2–36.8 )	179.4 (173.8- 184.8)		158.3(141.8–175.3)					3
3	Dolichandra unguis-cati	204.7 (160–268.4)			139.6 (133.8–145)	10	5	104.3 (102.0- 108.0)	78.4 (68.9–85.0)					100.3 (91.2–108.0)	24.1 (21.2–28.3)			108.5 (104.0- 109.0)			75.4 (65.7–89.0)	148.9 (134.3–150.0)	122.9 (116.8- 127.9)						4
4	Pyrostegia venusta	156.1 (105.7–192.2)		75.7(66.3–88.9)		19	1	101.1 (97.9–106.8)			66.7 (58.1–74.3)			100.1µ (93.4–107.0)		25.7(20.0–29.4)		108.5 (104. 109.0)	75.4 (65.7–89.0)			269.3 (234.8- 313.5)				219.4 (195.0- 250.1)			3
5	Tecoma stans	104.3 (91.1–115.8)		40.5 (28.1–50.2)		9	0	104.6 (98.3–116.4)		58.9 (51.2–73.8)				86.3 (77.8–97.1)		21.2 (19.8–23.2)		92.8 (85.6–76.7)	61.6 (51.5–44.6)			145.0 (104.4– 182.3)				93.3 (84.3– 105.2)			10
6	Tecomella undulata	52.6 (41.1–67.9)		21.9 (18.4–24.8)		19	0	72.4 (66.9–75.7)				59.2 (52.9–67.1)		63.5 (61.9–65.5)			22.5 (18.9–24.8)	100.3 (78.8–108.0)	44.7 (39.3–54.1)			106.0 (98.1–117.0)						108.3 (97.0–117.0)	9
7	Tabebuia aurea	55.3 (45.8–66.0)		38.4 (31.2–45.9)		13	2	77.4 (72.3–82.5)			61.6 (58.6–64.0)			74.5 (68.1–79.7)			24.4 (23.0- 25.6)	69.9 (62.9–71)	22.2 (17.0–27.0)			76.2 (73.4–78.4)						64.9 (55.6–76)	3
8	Millingtonia hortensis	61.5 (57.3–64.8)		22.3(20.0–35.0)		0	0	57.1 (49.0–65.8)			39.2 (36.7–42.6)			70.2 (62.6–78.0)			17.2 (15.1–21.0)	64.3 (60.9–67.2)	34.1 (24.8–42.1)			151.8 (114.6–174.0)						158.8(117.0–181.3)	12
9	Mansoa alliacea	140.6 (127.0- 149.5)	61.8 (50.0–69.8)			4	16	89.6 (83.4–95.6)					70.5 (54.6–80.4)	81.6 (72.1–89.9)			30.5 (29.5–31.8)	94.8 (76.8–111.0)		45.6 (31.1–69.6)		179.3 (151.3– 205.8)					178.4 (154.3– 216.7)		11
10	Jacaranda mimosifolia	37.8 (35.1–40.5)	18.1 (13.0–21.4)			20	0	58.7 (56.1–62.0)					52.0 (44.1– 56.6)	57.3 (51.0–62.0)			13.3 (11.3– 17.0)	55.7 (50–60.1 )		42.0 (44.0–50.0)		161.2 (155.3 − 165.1)					156.6 (145.2–162.7)		5
11	Fernandoa adenophylla	73.5 (63.5–88.1)	41.3 (34.0–49.0)			1	2	69.8 (55.8–96.1)					50.5 (38.9– 67.7)	77.9 (55.0–89.8)			18.7 (11.0–25.1)	94.7 (90.8–99.0)		64.3 (55.4–77.0)		204.5 ( 201.1–206-0)				195.7 (192.2–197.5)			1
12	Bignonia callistegioides	130.0 (111.4– 157.0)	51.3 (46.8–57.0)			22	8	84.7 (79.8–87.6)					76.4 (73.3–79.6 )	68.5 (56.6 − 76.8 )			28.3 (24.0–32.0)	95.7 (83.1–103.4)		17.9 (16.4–21.0 )		0							0
13	Handroanthus impetiginosus	88.0 (81.9–94.1 )	42.7 (37.4–46.0)			2	3	84.7 (74.3–94.5)				42.3 (31.1–51.9 )		75.0 (62.2–86.0 )			23.7 (12.8–29.4 )	73.4 (60.2–95.7)		43.7 (32.6–51.0 )		195.9 (188.0–200.1)				210.8 (209.5–212.1)			3
14	Campsis radicans	88.7 (78.6–95.2)	34 (28.4–41.0)			2	17	96.5 (96.0–97.7)			68.9 (62.0– 75.1)			84.0 (79.0–90.1 )			24.7 (22.0–29.0 )	71.8 (62.6–78.3)		36.1 (28.0–42.2)		144.1 (138.3–148.1)				142.9 (134.0–155.5)			9
15	Anemopaegma chamberlaynii	85.4 (80.4–91.3)	33.3 (26.9–41.1)			5	20	70 (60.8– 87.0)			52.0 (41.0– 64.1)			79.7 (75.2–83.1).			25.7 (23.8– 28.4)	89.0 (60.4– 132.7)		24.7 (21.0–32.0)		180.9 (154.2- 200.1)			39.7 (34.2–45.3)				28

Fig. 4.

Light microscopy of stomata and guard cells of studied species of Bignoniaceae (EC = epidermis cell, T = trichome, GC = guard cell) A Kigelia africana (showing anomocytic and sunken stomata. B Oroxylum indicum (L.) showing anomocytic stomata. C Dolichandra unguis-cati (L.) L. G. Lohmann showing paracytic stomata. D) Pyrostegia venusta E showing anomocytic stomata. E Tecoma stans showing anomocytic stomata. F Tecomella undulata (Sm.) Seem. showing anomocytic stomata. G Tabebuia aurea showing tetracytic stomata. H) Millingtonia hortensis L.f. showing contiguous stomata. I Mansoa alliacea (showing anaomocytic and anisocytic stomata. J Jacaranda mimosifolia D showing anomocytic and sunken stomata K Fernandoa adenophylla Steenis showing anomocytic stomata. L Bignonia callistegioides showing paracytic stomata. M Handroanthus impetiginosus N showing anomocytic stomata. Campsis radicans Seem. showing anomocytic stomata O Anemopaegma chamberlaynii. showing anaomocytic and anisocytic stomata

Guard cells

Guard cells, the specialized cells that surround and control the opening and closing of stomata, also play an important role in plant identification. Their shape, arrangement and size play a significant role in identification. Guard cells observed in Bignoniaceae were all kidney-shaped. The average length of guard cells ranged from 22.4 μm to 100.3 μm. Dolichandra unguis-cati had the largest length, and the K. africana had the smallest length. The average width of guard cells ranged from 8.1 μm to 30.5 μm. M. alliacea had the largest width, and the K. africana had the smallest width. Most of the stomata of selected members of Bignoniaceae were anomocytic, in which stomata are surrounded by subsidiary cells that have the same shape, arrangement and size as the rest of the epidermal cells (Table 4). Only one species possessed tetracytic stomata, which contained four subsidiary cells. Two species possessed paracytic stomata that contained one or more pairs of subsidiary cells parallel to the guard cells. Only a few species possessed anisocytic stomata, which contained one smaller subsidiary cell and two larger subsidiary cells. Only one species possessed contiguous stomata, and stomata occurred in the pair form (Fig. 5).

Table 4.

B: quantitative data of adaxial epidermis of selected species of family Bignoniaceae

No of Plan ts	Plant names	Leaf epidermis cells		Total number of Stomata		Stomatal Cavity		Guard cells			Subsidiary cells						Trichomes		No of Trichomes
		Length µm	Width µm	Open	Closed	Length µm	Width µm	Length µm	Width µm		Length µm					Width µm	Length µm	Width µm
1	Kigelia africana	209.3 (188.1- 230.8)	159.3 (148.4- 199.7)	18	7	78.4 (51.6–92.9)	56.9 (49.0–68.9)	22.8 (22.9–24.0)	8.2 (7.1–9.1)		158.9 (70.3– 168.5.0)					80.6 (55.9–97.0)	0	0	0
2	Oroxylum indicum	148.3 (134.2- 171.2)	62.3 (42.4–74.7)	0	0	0	0	0	0		0					0	210.3 (181.8–232.8)	188.3 (156.2–214.6)	3
3	Dolichandra unguis-cati	183.1 (159.4–216.5)	105.0 (88.6–135.6)	4	0	102.9 (90.0–117.3)	65.0 (49.6–82.2)	95.9 (73.5–122.5 )	21.5 (19.1–24.1)		104.8 (95.0- 103.0)	35.5 (25.5–34.0)					0	0	0
4	Pyrostegia venusta	147.4 (123.8–169.3)	79.5 (76.1–85.9)	0	0	0	0	0	0		0	0					0	0	0
5	Tecoma stans	93.3 (84.3–105.2)	42.9 (33.5–48.4)	0	0	0	0	0	0		0	0					152.0 ( 148.6–153.1)	150.2 (148.3–152.4)	1
6	Tecomella undulata	65.0 (57.2–75.0)	36.8 (34.0–39.8)	18	0	83.6 (71.3–93.4)	59.7 (54.9–69.3)	56.5 (48.4–64.0)	16.3 (11.4–23.3)		106.5 (101.1–116.4 )	42.5 (32.6–54.1)					108.1 (94.4- 116.1)	118.9 (106.6–134.0)	7
7	Tabebuia aurea	49.1 (42.7–53.6)	31.2 (28.3–34.2)	1	2	73.8 (68.6–83.7)	55.7 (52.6–58.1)	74.1 (70.1–79.8)	18.2 (17.5–19.1)		63.7 (57.9–66.9)	22.9 (16.3–29.1)					101.9 (80.7–113.7)	96.4 (76.1- 108.7)	10
8	Millingtonia hortensis	61.5 (57.6–65.0)	25.7 (18.8–35.3)	0	9	78 (70.2–82.9 )	58.1 (50.2–64.6)	64.9 (55.3–71.2)	15.2 (11.4–17.3)		49.1 (45.0–55.4 )	34 (28.0–38.5)					182.3 (121.0–214.0)	175 (132.0- 199.0)	7
9	Mansoa alliacea	84.9 (76.2–97.3)	57.8 (54.2–62.6)	0	0	0	0	0	0		0	0					199.4 (175.1- 220.5)	193.4 (170.8–208.5 )	14
10	Jacaranda mimosifolia	53.7 (48.0–57.3)	23.1 (19.1–27.7)	0	0	0	0	0	0		0		0				292.6 (260.3- 334.7)	54.9 (50.0- 59.8)	21
11	Fernandoa adenophylla	92.8 (91.4–95.2)	49.5 (41.1–60.4 )	0	0	0	0	0	0		0		0				165 (163–166.4)	53.4 (51.2–54.8)	1
12	Bignonia callistegioides	126.3(111.1- 150.3)	77 (68.6–91.0).	0	0	0	0	0	0		0			0			0	0	0
13	Handroanthus impetiginosus	63.9 (63.5–64.7 )	36.7 (33.9–39.4 )	0	0	0	0	0	0		0			0			207.3 (200.2- 213.7)	213.5 (208.7–217.1)	3
14	Campsis radicans	126.7 (110.6–147.1)	57.5 (48.3–66.7)	0	0	0	0	0	0		0					0	0	0	0
15	Anemopaegma chamberlaynii	90.4 (88.7–103.9)	58.1 (50.1–67.8)	0	0	0	0	0		0	0				0		167.8 (131.1–193.8)	35.4 (27.5–40.5)	35

Fig. 5.

Scanning microscopy of stomata of studied species of Bignoniaceae A Kigelia africana showing anomocytic and sunken stomata. B Oroxylum indicum showing anomocytic stomata. C Dolichandra unguis-cati showing paracytic stomata. D Pyrostegia venusta showing anomocytic stomata. E Tecoma stans showing anomocytic stomata. F Tecomella undulata showing anomocytic stomata. G Tabebuia aurea showing tetracytic stomata. H Millingtonia hortensis showing anomocytic stomata. I Mansoa alliacea showing anaomocytic and anisocytic stomata. J Jacaranda mimosifolia showing anomocytic and sunken stomata K Fernandoa adenophylla showing anomocytic stomata. L Bignonia callistegioides. showing paracytic stomata. M Handroanthus impetiginosus showing anomocytic stomata. N Campsis radicans showing anomocytic stomata O Anemopaegma chamberlaynii. showing anaomocytic and anisocytic stomata

Subsidiary cells

Subsidiary cells, which are specialized epidermal cells surrounding guard cells in the stomata, play a significant role in plant identification. Their number, shape, arrangement, and relationship to guard cells can be highly diagnostic. Various variations were observed in the size of subsidiary cells. Most of the stomata of selected members of Bignoniaceae were normocytic, in which stomata are surrounded by subsidiary cells that have the same shape, arrangement and size as the rest of the epidermal cells. Only one species possessed tetracytic stomata, which contained four subsidiary cells. Two species possessed paracytic stomata that contained one or more pairs of subsidiary cells parallel to the guard cells. Only a few species possessed anisocytic stomata, which contained one smaller subsidiary cell and two larger subsidiary cells. On the adaxial side, only one species possessed contiguous stomata in which stomata occurred in the pair form.

The average length of subsidiary cells reached from 55.7 (50- 60.1) µm to 158.9 (70.4–173.0) µm. The largest length of subsidiary cells was observed in K africana, and the smallest length of subsidiary cells was observed in J. mimosifolia. The average width of subsidiary cells reached from 17.9 (16.4–21.0) µm to 80.6 (55.6–99.0) µm. The largest width of subsidiary cells was observed in K. africana, and the smallest width of subsidiary cells was observed in B. callistegioides.

Trichomes

Trichomes, the small hair-like structures found on the surface of plants, are important for plant identification due to their wide variety in form, function, and distribution. Trichomes can vary between species, genera, and families, making them a valuable trait in taxonomy. This family contained three types of trichomes. Glandular peltate trichomes contained a basal epidermal cell, a very small monocellular stalk and a circular or round multicellular head containing 12 cells arranged in a single circle. Non-glandular trichomes had a thin apex without a head and a pointed end. Branched trichomes contained several arms arising from a common base (Fig. 6). Souza [16] stated that glandular trichomes have been reasonably well documented in some Bignoniaceae species. Glandular peltate trichomes were observed in K. africana, O. indicum, Dolichandra unguis-cati, P. venusta, T. stans, T. undulata, T. aurea, M. alliacea, F. adenophylla, H. impetiginosus and C. radicans. M. hortensis, J. mimosifolia and A. chamberlaynii had non-glandular trichomes. Only Fernandoa adenophylla possessed branched trichomes. The average length of trichomes ranged from 76.2 μm to 292.6 μm. J. mimosifoia had the largest length, and the T. aurea had the smallest length. The average width of trichomes ranged from 39.7 μm to 219.4 μm. P. venusta had the largest width, and the A. chamberlaynii had the smallest width (Figure 7).

Fig. 6.

Light microscopy of trichomes of studied species of Bignoniaceae (EC = epidermis cell, T = trichome) A Kigelia africana showing glandular peltate trichomes. B Oroxylum indicum. glandular peltate trichomes. C Dolichandra unguis-cati showing glandular peltate trichomes. D Pyrostegia venusta showing glandular peltate trichomes. E Tecoma stans showing glandular peltate trichomes. F Tecomella undulata showing glandular peltate trichomes. G Tabebuia aurea showing glandular peltate trichomes. H and I Millingtonia hortensis L.f. showing glandular peltate and non-glandular trichomes. J Mansoa alliacea showing glandular peltate trichomes. K and L Jacaranda mimosifolia showing glandular peltate and non-glandular trichomes. M Fernandoa adenophylla showing glandular peltate trichomes. N Handroanthus impetiginosus(Mart. ex DC.) Mattos showing glandular peltate trichomes. O Campsis radicans Seem. glandular peltate trichomes. P and Q Anemopaegma chamberlaynii showing glandular peltate and non-glandular trichomes

Fig. 7.

Scanning electron microscopy of trichomes of studied species of Bignoniaceae (A) Kigelia africana (Lam.) Benth. showing glandular peltate trichomes. B Oroxylum indicum. showing glandular peltate trichomes. C Dolichandra unguis-cati (L.) showing glandular peltate trichomes. D Pyrostegia venusta (showing glandular peltate trichomes. E Tecoma stans (L.) showing glandular peltate trichomes. F Tecomella undulata (Sm.) Seem. showing glandular peltate trichomes. G Tabebuia aurea showing glandular peltate trichomes. H and I Millingtonia hortensis L.f. showing glandular peltate and non-glandular trichomes. J Mansoa alliacea showing glandular peltate trichomes. K Jacaranda mimosifolia showing glandular peltate and non-glandular trichomes. L Fernandoa adenophylla Steenis is showing glandular peltate and branches of trichomes. M Handroanthus impetiginosus showing glandular peltate trichomes. N Campsis radicans Seem. showing glandular peltate trichomes. O and P Anemopaegma chamberlaynii showing glandular peltate and non-glandular trichomes

Crystals

Crystals in plants, often composed of calcium oxalate or calcium carbonate, are important for plant identification because their presence, structure, and distribution are often species- or family-specific. These crystals can be found in various plant tissues such as leaves, stems, roots, and fruits, and their characteristics are used by botanists for taxonomic purposes. Some types of crystals are involved in protection against herbivores such as prismatic, Druses and raphides. Different types of crystals were observed in the leaves of different Bignoniaceae species. Prismatic crystals were observed in K. africana, O. indicum, T. undulata, M. hortensis, J. mimosifolia, B. callistegioides, F. adenophylla, H. impetiginosus (Fig. 8). Raphides were observed in (A) chamberlaynii, P. venusta, (B) callistegioides and (C) radicans. Druses were observed in (A) chamberlaynii, C. radicans, H. impetiginosus, (B) callistegioides, F. adenophylla, J. mimosifolia, Dolichandra unguis-cati, M. alliacea and M. hortensis [16]. stated that raphides were only observed in the Amphilophium crucigerum.

Fig. 8.

Light microscopy of crystals of studied species of Bignoniaceae (EC = epidermis cell, C = crystal, T = trichomes) (A) Kigelia africana (Lam.) Benth. showing prismatic crystals. B Oroxylum indicum showing prismatic crystals. C Dolichandra unguis-cati (L.). showing druses. D Pyrostegia venusta showing raphides. E Tecoma stans (L.) showing prismatic crystals. F Tecomella undulata (Sm.) Seem. showing prismatic crystals. G Tabebuia aurea showing prismatic crystals. H Millingtonia hortensis L.f. showing prismatic crystals or druses. I Mansoa alliacea showing druses. J Jacaranda mimosifolia showing prismatic crystals or druses. K Fernandoa adenophylla) Steenis showing raphides. L Bignonia callistegioides showing druses. M Handroanthus impetiginosus showing prismatic crystals and druses. N Campsis radicans Seem. showing raphides. O Anemopaegma chamberlaynii showing raphide

Taxonomic key

1. Irregular or sinus epidermal cell shape……………………..……………………(2)

1. Hexagonal epidermal cell shape……………………………………Tabebuia aurea

2. Paracytic type of stomata ……………….……..…………….…………………...(3)

2. Anomocytic or anisocytic type of stomata ………………….…………………..(4)

3. Trichomes are present in epidermal cell……. ……………Dolichandra unguis-cati

3. Trichomes are absent in epidermal cells……………………Bignonia callistegioides

4. Branched grandular multicellular trichomes are present...… Fernandoa adenophylla

4. Unbranched grandular multicellular trichomes are absent……………………….(5)

5. Crystals are present in epidermis cells ……..………….…………………………(6)

5. Crystals are absent in epidermis cells………………..……………………………(7)

6. Smooth anticlinal cell walls of epidermal cells…….. Anemopaegma chamberlaynii

6. Wavy anticlinal cell walls of epidermal cells…………………………………….(8)

8. Prismatic and druses crystals types are present in epidermal cells………………..(9)

8. Raphides crystals type are present in epidermal cells…………... Pyrostegia venusta

9. Only Anomocytic type of stomata is observed ...…………….…………………..(10)

9. Both Anomocytic and anisocytic types of stomata were observed……Mansoa alliacea

10. Length of epidermal cells is greater than 100 µm ……………..…. Oroxylum indicum

10. Length of epidermal cells is lesser than 100 µm …………………………………(11)

11. Length of trichome cells is greater than 150µm………………..…………………(12)

11. Length of epidermal cells is lesser than 150 µm …………………………………(13)

12. More than 10 trichomes seen in epidermis…………………... Millingtonia hortensis

12. Less than 10 trichomes seen in epidermis………………………………………..(14)

14. Length of epidermal cells is less than 50 µm ………………Jacaranda mimosifolia

14. Length of trichome cells is greater than 50µm…………Handroanthus impetiginosus

13. Prismatic crystals type is present in epidermal cells…………… Tecomella undulata

13. Prismatic, raphides and druses crystals type are present in epidermal cells…………………..…………………………………………………….. Campsis radicans

7. Nearly 30 no. of stomatas are observed……………………………. Kigelia Africana

7. Nearly 9 no. of stomatas are observed ………………….……………. Tecoma stans

Analysis of anatomical trait relationships

The analysis of anatomical traits on the adaxial surface utilized heatmaps combined with hierarchical clustering to reveal patterns and relationships within the data. Hierarchical clustering, represented by dendrograms on both axes, grouped plant species and tissue types based on their similarity (Fig. 9). The dendrogram on the left identified clusters of species and tissue types with closely related anatomical profiles, while the top dendrogram categorized samples based on their overall trait expression. Certain species exhibited distinct patterns in trait values. For instance, Campsis radicans consistently displayed lower anatomical values across multiple conditions, while Kigelia africana showed relatively higher values. These groupings suggest a potential alignment of species within clusters based on shared anatomical adaptations or ecological strategies. The clustering patterns indicate that species within the same cluster exhibit closer similarities in their anatomical traits compared to those in separate clusters.

Fig. 9.

Heatmap visualization and hierarchical clustering of anatomical traits on the adaxial surface. The dendrogram on the left groups plant species and tissue types based on anatomical similarity, while the dendrogram at the top clusters samples by overall trait expression

The PCA biplot provided a visual representation of principal component analysis, combining score plots (samples) and loading plots (variables) PC1 indicates 54.01% of the total variance, whereas PC2 explains 26.26% of the total variance. Together, PC1 and PC2 explained 80.27% of the variance, capturing a significant portion of data variability. PCA can be used as a tool to identify patterns of variations among different species (Fig. 10).

Fig. 10.

Principal component analysis: The x-axis represents the first principle component, the y-axis represents the second principal component, Red lines show the variables’ contributions, the same direction shows a positive correlation, the oppositive direction shows a negative correlation and blue labels show similarities in variables

Correlation analysis was performed to know the relationship between examined parameters. A Pearson analysis was performed to observe the relation between different leaf anatomical features. Length of stomata was observed to be positively correlated with the width of stomata (R2 = 0.97) followed by the length of guard cells (R2 = 0.94), the width of guard cells (R2 = 0.91), and length of subsidiary cells (R² = 0.91) (Fig. 11). The length of subsidiary cells was observed to be significantly correlated with the width of stomata [R² = 0.92], the length of trichomes was observed to have a significant negative correlation with the width of the epidermis (R²=−0.4) and the length of the epidermis (R² = 0.39) (Fig. 12).

Fig. 11.

Correlation analysis of the Adaxial side of species

Fig. 12.

Correlation analysis of Abaxial side of species

Discussion

The present research aimed to investigate the anatomical diversity of 15 Bignoniaceae species collected from semi-arid regions in Lahore, Pakistan, using light and scanning electron microscopy. The study focused on key anatomical traits, such as epidermal cells, stomata, trichomes, subsidiary cells, guard cells, and crystal structures, to explore their role in species differentiation and adaptation to environmental stress. The findings provide a comprehensive understanding of the taxonomic, ecological, and evolutionary significance of these traits in the family Bignoniaceae. The observed diversity in epidermal cell morphology and arrangement highlights their taxonomic value in distinguishing Bignoniaceae species. For example, hexagonal cells were a unique feature of Tabebuia aurea, while irregular cells were predominant in species such as Kigelia africana and Jacaranda mimosifolia [16]. The differences in cell dimensions, with Kigelia africana showing the largest epidermal cells and Jacaranda mimosifolia the smallest, underscore the utility of these measurements in taxonomic classification [17]. Similarly, the variation in anticlinal cell wall patterns, from wavy and curved to straight, provides additional diagnostic tools for species identification [18].

Stomatal morphology further contributes to species differentiation. Sunken stomata in Kigelia africana and Jacaranda mimosifolia, anomocytic stomata in most studied species, and tetracytic stomata in Tabebuia aurea highlight distinct taxonomic markers within the family [19]. These findings align with prior studies emphasizing the taxonomic significance of stomatal diversity in angiosperms [20]. The use of subsidiary cell structure and arrangement as additional distinguishing features strengthens the anatomical framework for identifying Bignoniaceae species, especially in regions with limited floristic documentation [21]. The presence of glandular and non-glandular trichomes further enriches the taxonomic understanding of the family. While glandular peltate trichomes were common across most species, branched trichomes were exclusive to Fernandoa adenophylla, underscoring the specificity of trichome types to certain taxa [22]. Similarly, crystal structures such as raphides, druses, and prismatic crystals provide supplementary taxonomic markers, reflecting evolutionary divergence among species [23, 24].

The anatomical traits observed in this study reflect adaptations to the semi-arid environments where these species thrive. Semi-arid ecosystems are characterized by high temperatures, intense solar radiation, limited rainfall, and significant seasonal variability, which exert selective pressure on plant morphology and physiology [25]. The observed anatomical features suggest evolutionary strategies that enhance water-use efficiency, thermal regulation, and overall resilience in challenging environments. Several species displayed traits indicative of xerophytic adaptations. For instance, Kigelia africana and Jacaranda mimosifolia exhibited large, thick-walled epidermal cells and sunken stomata, adaptations known to reduce water loss by minimizing transpiration [26, 27]. Sunken stomata, in particular, shield the stomatal pores from direct exposure to air currents and sunlight, thereby maintaining higher humidity in the stomatal chamber and reducing evaporative water loss [28]. Such adaptations are critical in regions with prolonged dry spells and high evapotranspiration rates [29].

The predominance of anomocytic stomata in species like Tecoma stans and Tecomella undulata reflects an adaptation that allows for efficient gas exchange under fluctuating moisture conditions [30, 31]. The lack of specialized subsidiary cells in anomocytic stomata may confer flexibility in stomatal function, enabling these species to optimize water use during periods of drought [32]. Conversely, the tetracytic stomata observed in Tabebuia aurea suggest an alternative strategy for regulating gas exchange and water loss during environmental stress [12]. The widespread occurrence of glandular and non-glandular trichomes across the studied species suggests their importance in mitigating environmental stress. Glandular peltate trichomes, observed in species such as Mansoa alliacea and Pyrostegia venusta, likely serve dual roles in reducing leaf temperature and preventing water loss by creating a localized microclimate on the leaf surface [15]. Non-glandular trichomes, as seen in Millingtonia hortensis and Jacaranda mimosifolia, further enhance water-use efficiency by reflecting sunlight and reducing direct exposure to solar radiation [19]. The presence of branched trichomes in Fernandoa adenophylla may indicate an additional adaptation for herbivore deterrence, highlighting the multifunctional nature of trichomes in plant survival [23].

Crystals, including raphides, druses, and prismatic crystals, were observed in various species, with prismatic crystals being particularly abundant in Kigelia africana and Millingtonia hortensis. These structures may play roles in mineral storage, structural support, and defense against herbivores [33]. In semi-arid soils that are often nutrient-poor or saline, the ability to store minerals such as calcium in the form of crystals could confer a significant advantage [8]. The diversity in stomatal size, density, and arrangement among the studied species reflects varied strategies for optimizing water-use efficiency. Species such as Oroxylum indicum and Dolichandra unguis-cati with paracytic stomata may have specialized pathways for controlling gas exchange under low moisture availability [34]. Additionally, the larger stomata and subsidiary cells observed in Dolichandra unguis-cati suggest adaptations that balance the need for carbon dioxide uptake with the imperative to conserve water [35, 36].

Broader implications and applications

The anatomical diversity documented in this study has broader implications for taxonomy, ecology, and conservation. The findings contribute to the growing body of knowledge on the Bignoniaceae family, providing a reference point for identifying species in poorly documented regions. This is particularly important in Pakistan, where several species are not included in existing flora records. The observed adaptations underscore the resilience of Bignoniaceae species to semi-arid conditions, making them valuable candidates for ecological restoration and afforestation projects in water-scarce regions. The morphological features associated with water conservation, mineral storage, and thermal regulation offer potential insights into the evolutionary pressures shaping xerophytic plant lineages. The study emphasizes the importance of anatomical markers in understanding plant responses to environmental stress. By linking anatomical traits to ecological functions, this research highlights the evolutionary ingenuity of Bignoniaceae species in surviving under semi-arid conditions. Future studies could build on these findings by exploring the genetic basis of these adaptations and assessing their functional roles in greater detail.

Conclusion

Anatomy serves as a crucial taxonomic tool for taxonomists to identify and differentiate closely related taxa. This study highlights the significance of leaf anatomical research in addressing complex taxonomic issues, particularly as it provides insights into species not documented in the e-flora of Pakistan. Utilizing microscopic techniques, this pioneering anatomical examination focused on both the abaxial and adaxial sides of leaves, encompassing both qualitative and quantitative analyses. Various characteristics, including the type, shape, and size of epidermal cells, stomata, trichomes, and crystals, were meticulously observed. Future research could expand on this foundational study by incorporating molecular techniques alongside anatomical analyses to further elucidate the evolutionary relationships among species in the Bignoniaceae family. Additionally, exploring the ecological implications of the observed anatomical features could provide insights into how these traits contribute to adaptation in specific environments. Moreover, establishing a comprehensive database of anatomical characteristics could aid in the identification and classification of new species, enhancing our understanding of biodiversity within the region.

Authors’ contributions

Author Contributions: Conceptualization, R.S. and S.S.; Methodology, R.S; software, M.W. and S.S.; validation, S.M.H. and S.I.; formal analysis, S.M.H; investigation, M.W.; resources, M.M. and M.W.; Data curation, R.S.; writing—original draft preparation, R.S., S.S; writing—review and editing, M.W., M.M., A.H., R.S., S.I, A.H., E.F.A., and S.A.S.; visualization, M.W.; supervision, S.S. and M.W.; project administration, S.S.; funding acquisition E.F.A., A.H. All authors having substantial contributions in research, read and agreed to the published version of the manuscript.

Funding

The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2025R356), King Saud University, Riyadh, Saudi Arabia.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable—this manuscript has no personal data from the authors.

Competing interests

The authors declare no competing interests.

Competing of interest

The authors declare no conflicts of interest.