Tendril Anatomy: A Tool for Correct Identification among Cucurbitaceous Taxa

This research examined the histological micro-structure of tendril vasculature in cucurbitaceous taxa. In this research, the tendril anatomy of 17 taxa of Cucurbitaceae categorized into seven genera, including Cucumis (five species), Cucurbita and Luffa (three species each), Citrullus and Momordica (two species each) while Lagenaria and Praecitrullus (one species each), collected from different areas of the Thal desert were examined via microscopic imaging to explore its taxonomic significance. Tendril transverse sections were cut with a Shandon Microtome to prepare slides. The distinctive characteristics of taxonomic value (qualitative and quantitative) include tendril and vascular bundle shape, variation in the number of vascular bundles, tendril diameter length, layers of sclerenchyma, and shape of collenchyma and epidermal cells. Tendril shapes observed are irregular, slightly oval-shaped, slightly C shaped, angular (4-angled, 6-angled, or polygonal), and star shaped. Quantitative measurements were taken to analyze the data statistically using SPSS software. Cucurbita pepo had a maximum tendril diameter length of 656.1 µm and a minimum in Momordica balsamina of 123.05 µm. The highest number of vascular bundles (12) were noticed in Luffa acutangula var.amara. Angular type was prominent in collenchyma, and irregular shape was dominant in sclerenchyma cells. A maximum of seven to nine sclerenchyma layers were present in Lagenaria siceraria and a minimum of two or three layers in Cucumis melo subsp. agrestis, Cucumis melo var. flexuosus, and Cucumis melo var.cantalupensis. Epidermis cells also show great variations with a rectangular shape being dominant. Statistical UPGMA dendrogram clustering of tendril vasculature traits shows that histological sections studied with microscopic techniques can be used to identify species and will play a vital role in future taxonomic and phylogenic linkages.

The UPGMA clustering dendrogram for Cucurbitaceous taxa is presented in Figure  10. Seventeen taxa of Cucurbitaceae fall into two major clusters based on the difference in qualitative features. Similarity relationships among different Cucurbitaceous species were explored using UPGMA clustering using tendril anatomical characters. The UPGMA phenogram shows two main clusters, C1 and C2. The first principle cluster, C1, represents sections C. maxima and C. pepo. The second cluster C2 further divided into two sub-clusters comprising C2a1 of 5 species in which C. melo and C. balsamina were closely related based on Euclidean distance mapping. The second sub-cluster, C2a2, represents ten species, among which, based on Euclidean distance L. cylindrica and M. charantia was placed at the minimum distance in this sub-cluster, showing the similarity in tendril qualitative features.  Seventeen taxa of Cucurbitaceae fall into two major clusters based on the difference in qualitative features. Similarity relationships among different Cucurbitaceous species were explored using UPGMA clustering using tendril anatomical characters. The UPGMA phenogram shows two main clusters, C1 and C2. The first principle cluster, C1, represents sections C. maxima and C. pepo. The second cluster C2 further divided into two sub-clusters comprising C2a1 of 5 species in which C. melo and C. balsamina were closely related based on Euclidean distance mapping. The second sub-cluster, C2a2, represents ten species, among which, based on Euclidean distance L. cylindrica and M. charantia was placed at the minimum distance in this sub-cluster, showing the similarity in tendril qualitative features.

Discussion
Various studies described morpho-anatomical features to classify Cucurbitaceous species [13,14]. Different researchers have carried out the tendril anatomical investigation of a few species of Cucurbitaceae [20] and studied the anatomical characteristics of some species of the genus cucurbita, like flower stalk, petiole, and stem, and examined some tendril characteristics. Leaf anatomy of Cucurbitaceous species has been briefly mentioned by [21], but no information is available on the tendril anatomy of Cucurbitaceous taxa, so this study elaborates tendril histology of 12 Cucurbitaceous taxa to find out taxonomic markers for their correct identification. The tendril's shape was four and five an-

Discussion
Various studies described morpho-anatomical features to classify Cucurbitaceous species [13,14]. Different researchers have carried out the tendril anatomical investigation of a few species of Cucurbitaceae [20] and studied the anatomical characteristics of some species of the genus cucurbita, like flower stalk, petiole, and stem, and examined some tendril characteristics. Leaf anatomy of Cucurbitaceous species has been briefly mentioned by [21], but no information is available on the tendril anatomy of Cucurbitaceous taxa, so this study elaborates tendril histology of 12 Cucurbitaceous taxa to find out taxonomic markers for their correct identification. The tendril's shape was four and five angled furrows in Momordica charantia and Cucumis sativa [22], which shows similarities with current results. While current studies show tendrils outlined in transverse view in different cucurbitaceous taxa are mostly irregular, slightly oval-shaped, slightly C shaped, angular (four-angled, six-angled, or polygonal), and star-shaped (Table 1). The maximum tendril length was observed in Cucurbita pepo (656.1 µm) and minimum was observed in Momordica balsamina (123.05 µm) as shown in Figure 11. Whereas the maximum width of tendril was noted in Cucurbita maxima (489.6 µm) and minimum in Momordica balsamina (112.95 µm). There was a single-layered epidermis present in the tendril histological section and it was predominantly irregular in shape while oval-shaped cells were recorded [22]. However, [23] elaborates on rectangular cells whereas our findings show square, oval, isodiametric, irregular, pentagonal, hexagonal, and polygonal types. Significant variation in the tendril micromorphology of Cucurbitaceous taxa was observed on both the adaxial and abaxial epidermal sides. There was variation in the epidermal cell length and width of the studied species (Table 3). The largest cell lengthwise was noted on the adaxial side in Praecitrullus fistulosus (28 µm) and the smallest in Luffa cylindrica (13 µm). The maximum epidermal cell width was noted in Cucumis melo var.cantalupensis (18.2 µm) and minimum in Luffa acutangula (8.05 µm) as shown in Figure 12. There was a single-layered epidermis present in the tendril histological section and it was predominantly irregular in shape while oval-shaped cells were recorded [22]. However, [23] elaborates on rectangular cells whereas our findings show square, oval, isodiametric, irregular, pentagonal, hexagonal, and polygonal types. Significant variation in the tendril micromorphology of Cucurbitaceous taxa was observed on both the adaxial and abaxial epidermal sides. There was variation in the epidermal cell length and width of the studied species (Table 3). The largest cell lengthwise was noted on the adaxial side in Praecitrullus fistulosus (28 µm) and the smallest in Luffa cylindrica (13 µm). The maximum epidermal cell width was noted in Cucumis melo var.cantalupensis (18.2 µm) and minimum in Luffa acutangula (8.05 µm) as shown in Figure 12. rectangular cells whereas our findings show square, oval, isodiametric, irregular, pentagonal, hexagonal, and polygonal types. Significant variation in the tendril micromorphology of Cucurbitaceous taxa was observed on both the adaxial and abaxial epidermal sides. There was variation in the epidermal cell length and width of the studied species (Table 3). The largest cell lengthwise was noted on the adaxial side in Praecitrullus fistulosus (28 µm) and the smallest in Luffa cylindrica (13 µm). The maximum epidermal cell width was noted in Cucumis melo var.cantalupensis (18.2 µm) and minimum in Luffa acutangula (8.05 µm) as shown in Figure 12.  Correspondingly, the largest cell length was calculated along the abaxial in Praecitrullus fistulosus (27.65 µm) and the shortest in Cucumis melo subsp. agrestis (12.85 µm). The cell width was observed to be maximum on the abaxial surface in Cucurbita pepo var. cylindrica (18 µm) and minimum in Luffa acutangula var. amara (4.1 µm). Layers of sclerenchyma and chlorenchyma (both one to eight layers) and collenchyma cells (two to six layers). However, [22] mentioned some variations in sclerenchyma two to nine, chlorenchyma one to three and collenchyma two to six as mentioned in Figure 13.
Angular collenchyma cells were observed from Iraq by [23] among Cucurbitaceae species from Iraq, while angular lamellar types were examined in this study (Table 1). In previous studies, two to four layers of collenchyma were present [22], while present measurements revealed a distinct continuous layering of cells below the epidermis with two to six layers of collenchyma ( Table 2). The maximum layers were present in Cucurbita maxima and Luffa acutangula var.amara both having six layers, while the minimum number of layers was present in Citrullus lanatus, with two or three layers. Collenchymatous cell size showing maximum length in in Cucumis sativus (27.85 µm) while minimum length was in Cucurbita pepo (12.85 µm). Whereas the largest width was calculated for Cucumis sativus (17.7 µm) the lowest width was for Cucumis melo var. flexuosus (8.55 µm) as illustrated in Figure 14.
There were mostly two or three layers of chlorenchyma in tendrils of Cucurbitaceae species [22], while in recent studies, a single layer of chlorenchyma cells lies beneath collenchyma but in some species, more than single layers noticed two or two to three layers ( Table 2). Maximum chlorenchymateous layers were present in Lagenaria siceraria in two to three layers. Different shapes of chlorenchyma cells were inspected, such as rectangular, irregular, pentagonal, hexagonal, and polygonal (Table 1). The chlorenchymateous cell size range from largest lengthwise was measured in Cucumis sativus (44.25 µm) while the shortest was in Cucumis melo subsp. agrestis (20.6 µm). Likewise, the largest cell widthwise was seen in Cucumis melo var. cantalupensis (27.45 µm), and the smallest cell widthwise was observed in Cucumis melo subsp. agrestis (8.9 µm) Figure 15. lus fistulosus (27.65 µm) and the shortest in Cucumis melo subsp. agrestis (12.85 µm). The cell width was observed to be maximum on the abaxial surface in Cucurbita pepo var. cylindrica (18 µm) and minimum in Luffa acutangula var. amara (4.1 µm). Layers of sclerenchyma and chlorenchyma (both one to eight layers) and collenchyma cells (two to six layers). However, [22] mentioned some variations in sclerenchyma two to nine, chlorenchyma one to three and collenchyma two to six as mentioned in Figure 13. Angular collenchyma cells were observed from Iraq by [23] among Cucurbitaceae species from Iraq, while angular lamellar types were examined in this study (Table 1). In previous studies, two to four layers of collenchyma were present [22], while present measurements revealed a distinct continuous layering of cells below the epidermis with two to six layers of collenchyma ( Table 2). The maximum layers were present in Cucurbita maxima and Luffa acutangula var.amara both having six layers, while the minimum number of layers was present in Citrullus lanatus, with two or three layers. Collenchymatous cell size showing maximum length in in Cucumis sativus (27.85 µm) while minimum length was in Cucurbita pepo (12.85 µm). Whereas the largest width was calculated for Cucumis sativus (17.7 µm) the lowest width was for Cucumis melo var. flexuosus (8.55 µm) as illustrated in Figure 14. There were mostly two or three layers of chlorenchyma in tendrils of Cucurbitaceae species [22], while in recent studies, a single layer of chlorenchyma cells lies beneath collenchyma but in some species, more than single layers noticed two or two to three layers ( Table 2). Maximum chlorenchymateous layers were present in Lagenaria siceraria in two to three layers. Different shapes of chlorenchyma cells were inspected, such as rectangular, irregular, pentagonal, hexagonal, and polygonal (Table 1). The chlorenchymateous to three layers. Different shapes of chlorenchyma cells were inspected, such as rectangular, irregular, pentagonal, hexagonal, and polygonal (Table 1). The chlorenchymateous cell size range from largest lengthwise was measured in Cucumis sativus (44.25 µm) while the shortest was in Cucumis melo subsp. agrestis (20.6 µm). Likewise, the largest cell widthwise was seen in Cucumis melo var. cantalupensis (27.45 µm), and the smallest cell widthwise was observed in Cucumis melo subsp. agrestis (8.9 µm) Figure 15. Species like Citrullus colocynthis and Citrullus lanatus have continuous sclerenchyma cells, similar to our findings. All the studied species illustrated the continuous sclerenchymatous cells except Cucumis melo var catanlupensus, Cucurbita pepo var cylindrica, and Momordica charantia, discontinuous sclerenchymatous cells were recorded in these species. The continuous layer of sclerenchymatous cells that makes up the tendril acts as an anchor and requires it to be robust enough to hold the weight of the plant and its fruits, especially when it is rising [22]. The most prominent and darkly stained layers of sclernchyma cells were just below the chlorenchyma cell layers. Sclerenchyma cell layers range from a minimum of two to three layers in three species of Cucumis melo subsp. agrestis, Cucumis melo var. flexuosus, and Cucumis melo var. cantalupensis, while the maximum number of layers was noticed in Lagenaria siceraria seven to nine layers, Table 2. Shapes of sclerenchyma cells are dissimilar in tendrils of studied plant species. Mainly, sclerenchyma was perceived as irregular, trigonal, tetragonal, hexagonal, and polygonal shaped, Table 1. There were variations in sclerenchyma cell size ranges, maximum cell sizes lengthwise were seen in Luffa acutangula var. amara (38.05 µm) compared to minimum cell size lengthwise in Cucumis melo subsp. agrestis (13.55 µm). The maximum width of sclerenchyma cells was noted in Luffa acutangula var. amara (17.95 µm), while minimum was observed in Cucumis melo subsp. agrestis (9 µm) Figure 16.
In earlier studies, different parenchyma cells were irregular, pentagonal, and polygonal (Table 1), butt angular parenchyma cells were also recorded [23]. In the current studies, parenchymatous cells were present in all species, mostly occupying the pith region in tendrils. The number of parenchyma cell layers differed in all studied species in Table 2. The maximum parenchyma layers were present in Cucumis sativus six-layers, whereas the minimum was observed in Luffa acutangula var. amara of two-layers. Parenchyma cells were the largest cells in size present in tendrils. The largest cell lengthwise is Praecitrullus fistulosus (91.55 µm), while mini cells were present in Cucumis melo (29 µm). The sizeable cells, widthwise, are present in Citrullus colocynthis (63.35 µm), and compact cells widthwise were existingin Luffa acutangula (13.5 µm) Figure 17. ceived as irregular, trigonal, tetragonal, hexagonal, and polygonal shaped, Table 1. There were variations in sclerenchyma cell size ranges, maximum cell sizes lengthwise were seen in Luffa acutangula var. amara (38.05 µm) compared to minimum cell size lengthwise in Cucumis melo subsp. agrestis (13.55 µm). The maximum width of sclerenchyma cells was noted in Luffa acutangula var. amara (17.95 µm), while minimum was observed in Cucumis melo subsp. agrestis (9 µm) Figure 16. In earlier studies, different parenchyma cells were irregular, pentagonal, and polygonal (Table 1), butt angular parenchyma cells were also recorded [23]. In the current studies, parenchymatous cells were present in all species, mostly occupying the pith region in tendrils. The number of parenchyma cell layers differed in all studied species in Table 2. The maximum parenchyma layers were present in Cucumis sativus six-layers, whereas the minimum was observed in Luffa acutangula var. amara of two-layers. Parenchyma cells were the largest cells in size present in tendrils. The largest cell lengthwise is Praecitrullus fistulosus (91.55 µm), while mini cells were present in Cucumis melo (29 µm). The sizeable  Vascular bundles were mainly arranged in a subsidiary manner, and few were centrally arranged in the case of Cucumis melo var. cantalupensis and Momordica charantia, Table 1. Bicollateral-types of vascular bundles were present in tendrils. Various writers have reported this feature in the Cucurbitaceae, which is constant across the analyzed taxa [3,4,14,24]. Their shapes vary from oval, elliptical, rounded, irregular, and dumbbell, Table 1. Each studied species has a different number of vascular bundles ( Table 2). The max- Vascular bundles were mainly arranged in a subsidiary manner, and few were centrally arranged in the case of Cucumis melo var. cantalupensis and Momordica charantia, Table 1.
Bicollateral-types of vascular bundles were present in tendrils. Various writers have reported this feature in the Cucurbitaceae, which is constant across the analyzed taxa [3,4,14,24]. Their shapes vary from oval, elliptical, rounded, irregular, and dumbbell, Table 1. Each studied species has a different number of vascular bundles ( Table 2). The maximum number of vascular bundles was detected in Luffa acutangula var. amara, having 12 vascular bundles, while the minimum number of vascular bundles was in Momordica balsamina, having three vascular bundles. Vascular bundles also vary in size. The largest vascular bundle lengthwise was present in Cucumis sativus (224.25 µm), while the smallest was observed in Cucurbita pepo var. cylindrica (26.1 µm). The largest vascular bundle widthwise was observed in Cucumis sativus (125.3 µm), and the smallest vascular bundle was present in Cucurbita pepo var. cylindrica (19.35 µm) Figure 18. There was no record found about the vessel elements of the vascular bundle previously, while the current study showed a distinct number of vessel elements in the xylem of the studied species' tendrils. Their numbers and size vary from species to species. The highest number of vessel elements were present in Cucumis sativus, of about 15, and the lowest number of vessel elements existed in Cucumis melo var. cantalupensis, at around four elements ( Table 2). The biggest vessel element lengthwise was found in Cucumis sativus (42.55 µm), while the smallest vessel element lengthwise was present in Momordica balsamina (9.5 µm). The largest vessel element widthwise was observed in Cucumis melo var. cantalupensis (30.45 µm); meanwhile, the shortest was analyzed in Citrullus colocynthis (7.82 µm) Figure 19. There was no record found about the vessel elements of the vascular bundle previously, while the current study showed a distinct number of vessel elements in the xylem of the studied species' tendrils. Their numbers and size vary from species to species. The highest number of vessel elements were present in Cucumis sativus, of about 15, and the lowest number of vessel elements existed in Cucumis melo var. cantalupensis, at around four elements ( Table 2). The biggest vessel element lengthwise was found in Cucumis sativus (42.55 µm), while the smallest vessel element lengthwise was present in Momordica balsamina (9.5 µm). The largest vessel element widthwise was observed in Cucumis melo var. cantalupensis (30.45 µm); meanwhile, the shortest was analyzed in Citrullus colocynthis (7.82 µm) Figure 19.
No tendril anatomical data were found about Luffa aegyptiaca. However, the species was used against hydrocarbon-contaminated soil through rhizoremediation, and chemical analysis of this species was also carried out in research papers [25]. In Western Africa, three genera of the Cucurbitaceae family, e.g., Momordica, Luffa, and Trichosanthes, were studied for their foliar epidermis and tendril morphology. The significant differences in their leaf and tendril morphology provided additional data for classifying three genera in separate tribes [26]. Furthermore, many authors studied praecitrullus fistulosus for its medicinal, anthelmintic, and anticancer activities [27][28][29]. Among the tendril anatomical characters presented in the study, only a few discussed characters have been studied earlier for selected species [20,22,23], while other species investigated in the current project have not been investigated. A detailed review of the literature revealed that there is no comprehensive study regarding the tendril anatomical features of these plants. No tendril anatomical data were found about Luffa aegyptiaca. However, the species was used against hydrocarbon-contaminated soil through rhizoremediation, and chemical analysis of this species was also carried out in research papers [25]. In Western Africa, three genera of the Cucurbitaceae family, e.g., Momordica, Luffa, and Trichosanthes, were studied for their foliar epidermis and tendril morphology. The significant differences in their leaf and tendril morphology provided additional data for classifying three genera in separate tribes [26]. Furthermore, many authors studied praecitrullus fistulosus for its medicinal, anthelmintic, and anticancer activities [27][28][29]. Among the tendril anatomical characters presented in the study, only a few discussed characters have been studied earlier for selected species [20,22,23], while other species investigated in the current project have not been investigated. A detailed review of the literature revealed that there is no comprehensive study regarding the tendril anatomical features of these plants.

Study Site and Selected Species of Family Cucurbitaceae
This research was conducted in the desert areas of districts of Bhakkar and Layyah in Punjab province. During March to July 2022, 17 Cucurbitaceous species each with 5 specimens were collected during field trips. Cucurbitaceous species sampling sites were georeferenced using a GPS device (German eTrex Venture) ( Table 5).

Study Site and Selected Species of Family Cucurbitaceae
This research was conducted in the desert areas of districts of Bhakkar and Layyah in Punjab province. During March to July 2022, 17 Cucurbitaceous species each with 5 specimens were collected during field trips. Cucurbitaceous species sampling sites were georeferenced using a GPS device (German eTrex Venture) ( Table 5).  The whole specimen was collected, including the tendrils, roots, stems, petioles, leaves, and flowers. Cucurbitaceous species were grown in cultivated field crops and wild places of the studied region. Plant specimens were pressed and dried in newspapers, identified and authenticated from the Herbarium of Pakistan (ISL). Cucurbitaceous species names were verified from the International Plant Name Index (www.ipni.org accessed on 9 October 2020) and Flora of Pakistan (www.eflora.org accessed on 9 October 2020). After preservation with ethanol and mercuric chloride solution the Cucurbitaceous herbarium specimens were deposited in the ISL herbarium.

Tendril Fixation
A solution of FAA (one part of 40% formaldehyde, 18 parts of 70% ethanol, and one part of glacial acetic acid) was used to fix mature tendrils for anatomical study for 12 h. They were moved for 2 h in 50% ethanol and then dipped into 70% ethanol for 2 h. Afterward, put in absolute ethanol at room temperature [22].

Histological Sectioning
Successive fixation of tendrils were sectioned using the standard technique outlined by [30] with several changes. Tendril sections were cleaned for about a minute, and any extra water was then removed by treating the sections with a series of alcohols ranging from 70% to 100%. The dehydrated pieces were then submerged in xylol for one hour to permeate the wax. At 60 • C, molten wax was used to preserve the tissues. Sections were transferred to cast using forceps and needles, and a cast was used for this. This wax-filled cast and its sections were chilled with cold water. The cast was afterward lifted out and processed for microtomes. A piece of around 15-20 µm was cut from half of the length from the base with the aid of a Shandon Microtome (Finesse 325). On a glass slide, these sections were moved, and egg albumen was scattered all over the slide. The slide was moved onto a hot plate and placed in an oven at 60 • C, where the wax expanded. The tissues were extracted from the wax using xylol for five minutes. Sections were washed thoroughly before being successively dehydrated with alcohol at concentrations of 100%, 90%, 80%, and 70%. For staining, fast green stain and Safranin O were applied. Slides were stained for 15 to 20 min before being cleaned with distilled water. Rehydration using a sequence of alcohol concentrations of 70%, 80%, 90%, and 100%, respectively was then performed. To make the slide better visible, xylol was employed. DPX mountant was placed on the slide for mounting, and the area received a cover slip. Each slide had a proper label and was dried [31].

Tendril Micromorphology
Slides were examined using a 40x-objective LM (OPTIKA Microscope, Italy). Digital cameras were used to capture photos of each sample under the 4, 10, and 40 objective lenses.

Light Microscopy
A reading sheet was used to record quantitative data. 10 to 15 readings of each species were taken under the Meiji (MT 4300H) LM at a magnification of 40×. For each species, minimum, maximum, mean and standard deviation values of various microanatomical parameters, including the length and width of the tendrils, the vascular bundles, collenchymatous cells, sclerenchyma cells, chlorenchyma cells and parenchyma cells, abaxial and adaxial epidermal cells, and the vessel elements, were calculated [32].

Statistical Analysis
The statistical SPSS 16.0 tool was used to analyze the corresponding average data for the measured values of tendril anatomical traits [33]. The effectiveness of the quantitative and qualitative features were evaluated using the UPGMA clustering analysis based on the Euclidean distance coefficient using PAST 4.03 software [34].

Conclusions
Through microscopic magnifications, the anatomical morphometry of tendril micromorphological features in Cucurbitaceous taxa exhibited variations. The resulting tendril micromorphology provides trustworthy traits that help identify different species. Accurate taxonomy will be achieved through the analysis of tendril characteristics. For taxonomic examination, it is crucial to consider the shape of the tendril outline, vascular bundle arrangements, sclerenchyma layers, and the morphology of the collenchyma and epidermal cells. The largest collenchyma cell was found in Cucumis sativus (27.85 µm), whereas the longest sclerenchyma cell was found in Luffa acutangula var. amara (38.05 µm). The findings show that the taxonomic identification of these species and their relationships will benefit from quantitative anatomical tendril features through clustering (UPGMA) analysis

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.