Biosystematics and molecular characterization of three biotypes of Convolvulus arvensis L. (convolvolaceae) in Marsa Matrouh, Egypt

Abstract

The notable characteristic of phenoplasticity in Convolvulus species is flower color polymorphism, with blooms ranging from white to pink. This variation is due to various factors, including genetic drift and natural selection. The primary objective is to conduct a study on the morphoplasticity of eight accessions collected from diverse locations and categorized into three distinct biotypes based on floral pigmentation: Biotype 1 (White; accessions 1, 4, and 7), Biotype 2 (Pale Pink; accessions 2, 5, and 8), and Biotype 3 (Pink; accessions 3 and 6) of Convolvulus arvensis L. Based on the aforementioned characteristics—flower colour, stem outline, leaf shape, pollen features, shape, colpus and pori shapes, epidermal cell shape, and the number of vascular bundles in both stem and petiole—the study employed morphological and anatomical studies, as well as CDDP markers, to evaluate differences among accessions. Leaf tissues are parenchyma above the main vascular bundle, with an angle between the midrib and the wing region. Three distinct biotypes (B1, B2, and B3) have been defined. CDDP marker analysis revealed high polymorphism (74.36%) and Rp (9.50). Samples 7 and 8 (location 3) showed the most informative polymorphism of flower color (white/pale pink/pink). Genetic distances clustered samples primarily by location rather than by color, underscoring the dominant role of location in structuring populations. The interaction between genotype and ecological conditions led to the genetic variation in flower color in C. arvensis.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1186/s12896-026-01202-5.

Introduction

The Convolvulaceae family, commonly known as bindweeds or morning glories, holds significant economic, cultural, and ecological importance. Its uses span from ornamental plants to sources of food and medicine, with some species even used as recreational drugs [1, 2]. Geographically, this diverse family is widespread across temperate, tropical, and subtropical regions [3]. The family is structured into 6 subfamilies and 12 tribes, encompassing 57 genera and 1,955 accepted species [4].

In Egypt, the family is represented by 47 species across 10 genera and 6 tribes. A systematic study by Abdel Khalik [5] in Egypt utilized numerical taxonomy based on vegetative characteristics, pollen grains, and seed morphology to organize the family’s genera. This research identified five dominant groups: Convolvuleae, Cuscuteae, Cresseae, Dichondreae, and a mixed group from Poor and Merremieae.

Within the Convolvulaceae family, the genus Convolvulus is the largest, with 203 species [4]. It is the namesake for the tribe Convolvuleae and subfamily Convolvuloideae. Convolvulus species are found worldwide in temperate zones, with significant diversity in the Mediterranean and Western Asia. In Egypt, the genus is represented by 20 species [6]. The classification of Convolvulus has evolved over time. Boissier [7] divided the genus into 10 unranked groups based on habit, the presence of spines, and hairiness of the ovary and capsule. Sáad [8] revised species in the Mediterranean and Western Asia, classifying them into three sections and 12 subsections. Wood et al. [9] assigned species in a comprehensive monograph to seven morphological and geographical units, aligning with a phylogenetic framework.

C. arvensis L. Sp. Pl. 1:153. 1753 ([10] p.153), a morpho-plastic perennial herb, is native to Europe but has spread globally [11]. Its notable characteristic is flower color polymorphism, with blooms ranging from pink to white [12, 13]. This variability has been linked to natural selection [14]. The species’ morphoplasticity has been the subject of several studies [15–17]. Research on biotypes, ecotypes, and varieties of C. arvensis suggests that much of its phenotypic variation is inherited and clone-specific [18]. Sixty varieties have been identified in Europe, and classifying them formally has been challenging. In Egypt, while no varieties of C. arvensis have been recorded [19, 20], recent molecular and morphological studies provide more detailed insights. Soliman et al. [21] used RAPD to differentiate and establish taxonomic relationships among three varieties.

More recently, Hamed et al. [22] identified five distinct genotypes from populations in the same habitat in Egypt. These genotypes showed differences in leaf and flower morphology, as well as chromosomal variations. Crucially, the study concluded that the observed phenoplasticity is genetically controlled, rather than being driven by ecological factors, since all populations were collected from the same environment.

Pollen morphology has been exceptionally important in classifying the Convolvulaceae family. Early work by Hallier [23] was foundational, dividing the family into two groups based on whether the pollen exine was echinate (spiny) or psilate (smooth). This initial classification, known as Echinoceniae and Psiloconiae, highlights the early recognition of pollen’s taxonomic value. More recent and detailed studies have confirmed and expanded on this. Telleria and Daners [24], identified three main pollen types with multiple subtypes across 75 species, while Saensouk and Saensouk [25] classified pollen from 45 taxa into six types based on their apertures using a scanning electron microscope (SEM). Localized studies, such as those in Egypt by Osman and Abdel Khalik [26] and Abd El-Raouf et al. [27], have also shown that pollen characteristics can effectively differentiate between species and genera. Notably, the study by Abd El-Raouf et al. [27] demonstrated a high correlation between pollen taxonomy and DNA barcoding, suggesting that micro-morphological and molecular approaches can be effectively integrated for plant identification.

Beyond pollen, seed structure and other anatomical features also provide critical taxonomic data. Takhtajan [28] notably placed Convolvulaceae into its own order, Convolvulales, largely because of its unique seed structure and pollen morphology, which are not shared with other families in the order Solanales. Research on seed coat anatomy and morphology, such as the work by Sampathkumar and Ayyangar [29], and Abdel Khalik and Osman [30], has also proven valuable for separating genera, even leading to the separation of Cuscuta from the rest of the family. Anatomically, the family is characterized by intra-xylary phloem and successive cambia, which are rare among angiosperms. The presence of articulated, non-anastomosing laticifers (latex-producing cells) is another distinguishing feature [31].

Foliar (leaf) anatomical features, particularly trichomes (hairs), are also vital for correct identification and classification. Specific trichome types can define genera, species, and even families [32]. Ashfaq et al. [33] demonstrated the importance of leaf epidermal micromorphology for Convolvulaceae systematics. Revisions of genera, such as Convolvulus, have used trichome type as a key diagnostic character at the species level, as seen in the work by Aykurt and Sümbül [34]. Detailed studies by Roudi et al. [35] confirmed the systematic significance of trichome micromorphology, showing its effectiveness in distinguishing sections, subsections, and species within the genus Convolvulus. These findings highlight the potential for integrating trichome studies with both molecular and traditional classification methods. The taxonomy of Convolvulaceae relies heavily on combining micro- and macro-morphological characteristics, including pollen, seed structure, and foliar features, such as trichomes, which provide robust evidence of phylogenetic relationships) [36].

DNA-based markers are valuable tools for identifying and distinguishing various forms of materials within and between species. The genetic assembly within species populations reflects the integration of various processes, including mutation, gene flow, mating system, genetic drift, and selection [37]. Recent DNA-based markers grant botanists and geneticists a wider array of genetic tools for studying genetic variation than was previously available [38]. Many genetic techniques and markers, including random amplified polymorphic DNA (RAPD), start codon target (SCoT), inter-simple sequence repeats (ISSR), simple sequence repeats (SSR), and single-nucleotide polymorphism (SNP) markers, have been used recently. These have become a means of investigating genetic diversity within and between populations and are widely used to survey population genetic structure [39].

Repeated surveys and collection missions were conducted in Marsa Matrouh governorate, Egypt, under the authority of the Botany and Microbiology Department, Faculty of Science, Menoufia University, Egypt. It was observed in three phytogeographical localities in the same habitat that a wide variation exists. Flower color is the most important attribute of phenoplasticity in C. arvensis L., as mentioned above. Three biotypes were collected from three localities in Marsa Matrouh governorate, Egypt. Eight accessions were collected according to the morphoplasticity attribute of flower color: (1) white flower (biotype 1), (2) pale pink flower (biotype 2), and (3) pink flower (biotype 3), as mentioned in Table 1. This morphoplasticity, including the primary morphoplasticity of flower color (white, pale pink, and pink), was investigated under ecological conditions and genetic structure by identifying and characterizing these biotypes based on macro- and micro-morphological attributes, as well as molecular characterization.

Table 1.

The phyto-geographical locations of the studied convolvulus populations

Localities	Accessions	GPS coordinates		Height (m)
		N	E
1	1, 2, 3	31° 21’ 658”	0 27° 09’ 661”	21
2	4, 5, 6	31° 23’ 013”	0 27° 04’ 474”	8
3	7, 8	31° 26’ 662”	0 26° 49’ 322”	8

Materials and methods

Plant materials and collection

Eight accessions of C. arvensis L. were collected during spring 2024–2025 from three distinct localities in the same habitat- road sides. It was conducted during the annual flora excursion of the Botany and Microbiology Department, Faculty of Science, Menoufia University, Egypt. Three biotypes were collected from three localities across Marsa Matrouh governorate, Egypt. Eight accessions were collected according to the morphoplasticity attribute of flower color: (1) white flower (biotype 1), (2) pale pink flower (biotype 2), and (3) pink flower (biotype 3) (Table 1). The identification of specimens was carried out by Prof. Dr. Zaki Abd El-Hamied Turki, Professor of Plant Taxonomy and Flora, Botany and Microbiology Department, Faculty of Science, Menoufia University, with the aid of local flora [6, 19, 20]. Representative plant samples were arranged flat in newspaper and dried in a plant press. Specimens are then mounted on acid-free paper and labeled with detailed collection data. Voucher specimens are deposited in Cairo University Herbarium (CAI); their numbers are 88.377.1117.207, 88.377.1117.208, 88.377.1117.209.

Macro- and micro-morphological investigations

Plant phenotyping was conducted with hand-held instruments to measure all growth stages of the idiotypes that differed phenologically. A single plant for each was chosen visually to perform more detailed measurements. Morphological description was taken according to the terminology of Stearn [40]. Descriptors follow Täckholm [19] and Boulos [6, 20].

Fresh plants of each were directly used for studying different macro- and micro-morphological attributes, and photographed with a digital camera.

For micro-morphological investigation, specimens were fixed in the FAA mixture that consists of formalin: glacial acetic acid: alcohol 70% (5:5:90/100 ml) for 24 h at room temperature. Then it was dehydrated and processed using the schedule of the paraffin method according to Johansen [41]. Sections of stem-and leaf specimens (i.e., petioles and blades) of 20–30 µm in sections were made using a LEICA rotary microtome. Sections were stained with safranin-fast green solution [41]. Anatomical examination and measurements were achieved using a Leica light Research Microscope model of a Zeiss light stereomicroscope, and photographed with a digital camera (OPTIKA). The terminology follows Pandey [42], and Abd El-Gawad et al. [43].

Palynological and seed surface electron Microscopy

For each biotype, Pollen grains and seeds were mounted onto stubs with double sided adhesive tape, and then these stubs were sputter-coated with gold [44]. After coating, they were examined and photographed using a scanning electron microscope (Model JEOL JSM-IT 200 Series) installed in the Faculty of Science, Alexandria University, Egypt. The measurements of pollen grains and seeds were based on at least 15–20 pollen grains or seeds for each specimen.

The pollen terminology was followed by that recommended by Punt et al. [45]. Seed terminology was followed as given by Barthlott [46, 47] and Abdel Khalik and Van Der Maesen [48].

Genomic DNA extraction and CDDP-PCR amplification

Genomic DNA was isolated from fresh plant tissues according to Mahdy [49] and quantified using 0.8% agarose gel electrophoresis. All samples were adjusted to a concentration of 10ng/μl for subsequent analyses. Ten CDDP primers (Table 2) were synthesized according to [50]. The PCR was conducted in a 25 μl reaction mixture, containing 25 ng DNA, 0.2 μM dNTPs, 1 μM primer, 1.5 mM MgCl₂, 1X PCR buffer, and 1 U Go-Taq Flexi polymerase (Promega). PCR amplification was programmed with an initial denaturation cycle at 94ºC/5 min, followed by 35 cycles comprising 94ºC/1 min, 50ºC/1 min, and 72ºC/90s, with a final extension at 72ºC/7 min.

Table 2.

Sequences of CDDP primers used

Primer name	Sequence
MADS-1	ATGGGCCGSGGCAAGGTGC
Myb1	GGCAAGGGCTGCCGC
ERF2	GCSGAGATCCGSGACCC
WRKY-R3B	CCGCTCGTGTGSACG
WRKY-R1	GTGGTTGTGCTTGCC
WRKY-R3	GCASGTGTGCTCGCC
KNOX-2	CACTGGTGGGAGCTSCAC
WRKY-F1	TGGCGSAAGTACGGCCAG
ABP1-2	ACSCCSATCCACCGC
Myb2	GGCAAGGGCTGCCGG

Gel electrophoresis and band detection

PCR products (10 μl) were mixed with 3 μl of 6x gel loading buffer and loaded on 1.2% w/v of agarose gel electrophoresis in 0.5x of Tris-Borate-EDTA (TBE) buffer at 70 V/30 min, stained with 2 μl ethidium bromide. The amplified products were detected using the Bio Doc Image Analysis System with Uvisoft analysis package (Cambridge Electronic Design Ltd, Cambridge, UK).

Data integration and statistical coding

The molecular profile, consisting of CDDP markers recorded as binary presence/absence (0/1) data, was integrated with macro- and micro-morphological characters scored as multi-state variables (1, 2, and 3). To ensure a representative analysis of the three biotypes; Biotype 1 (White; accessions 1, 4, 7), Biotype 2 (Pale Pink; accessions 2, 5, 8), and Biotype 3 (Pink; accessions 3, 6), mean values for each biotype were calculated. For the binary CDDP data, row means were rounded to the nearest integer to maintain the 0/1 biological significance.

Clustering and Visualization Hierarchical Cluster Analysis (HCA) was performed using the Ward. D2 linkage method, which minimizes total within-cluster variance. Genetic and morphological distances were calculated using the Euclidean distance metric. The integrated relationships were visualized using the pheatmap and factoextra packages in R (version 4.5.3). Essential packages including poppr, polysat, ggplot2, heatmap, and cluster for metrics, distances, and visualization were added to R software (version 4.5.3) [51].

The morphological data were averaged for all attributes and fed into the XLSTAT 2018 software to compute all attributes (Addinsoft 2022). The individuals’ coordinates were used to cluster biotypes by Hierarchical Ascending Classification [52]. A heat map was plotted according to [53]. Molecular analysis begins by loading binary marker data into R software, where rows represent primers and columns represent eight Convolvulus arvensis accessions scored as absent (0) or present (1) bands [51].

Results

Convolvulus arvensis L. (Sp. Pl. 1:153. 1753 ([10] p.153).

Morphology

The morphological characterization of plant species of vegetative and reproductive organs is essential for distinguishing and understanding the variation between the studied accessions. The following description provides a comprehensive anatomical analysis of the vegetative structures. Both SEM of seed and pollen features are studied giving valuable characterizations.

Figure 1 and Table 3 provide a detailed description of the characteristics that differentiate between the studied biotypes. Biotypes are perennial prostrate herbs, with stems are angular-cylindrical with ridges, sparsely-moderately hairy, twining or trailing. Leaves alternate, simple, glabrous-pubescent, with variable shape; hastate- sub sagittate, sagittate with auricle-triangular base, with entire margin and apiculate- acute apices (Fig. 1; 4–6). Flowers are with 1–2 elliptic -oblong bract(s) that are borne alone or in pairs on peduncles. Peduncle cylindrical with ridges, divided into two parts; 5–17 mm long below the bract, thin and 2-5 mm long upper bract and thick. Bract 2-4×0.4–1 mm, elliptic -oblong, with entire margins and acute apex, hairy at margins only (Fig. 1; 7–9). Flowers are bisexual; sepals are five, unequal, 3–6 mm long, imbricate, leathery, green, glabrous- spreading hairy, with entire margin and emarginate-obtuse apex. Corolla 5–15(−25) mm long, pubescent; white, pale pink-pink, contorted funnel shaped with greenish violet-greenish white dorsal midpetaline area, light violet-white where the petals unite internally (Fig. 1; 1–3). Five epipetalous unequal stamens; filament 1- 4(−6) mm long, smooth with papillate base, anther 2-4×0.8–1.7 mm, white-dark pink, sagittate (Fig. 1; 13–18). Gynoecium is carried on a 0.2–0.5 mm thick annular disc; ovary 0.5–4.5×0.4–1 mm, ovate and smooth, stigma 0.6–2(−2.5) mm long, bird head-like in closed flower, while oblong in shaped when fully opened and smooth (Fig. 1; 19–24). Fruit is 5.5-9×4-8 mm, subglobose with persisting calyx, smooth, contains 2–4 seeds (Fig. 1; 25–30). Seed is 3.8–4.4×2.1–3.2 mm, dull, brownish black to black, sectoroid with three faces; two flattened sides and one rounded and rough.

Fig. 1.

Field photographs showing the morphological characters of Convolvulus arvensisbiotypes; biotype 1; 1,4,7,10,13,16,19,22,25,28, biotype 2;2,5,8,11,14,17,20,23,26,29, biotype 3; 3,6,9, 12,15, 18,21, 24,27, 30. 1–3 flowers, 4–6 Leaves, 7–9 bracts, 10– 12 sepals, 13–15 base of filaments, 16–18 stamens, 19–21 stigma shapes, 22–24 ovaries, 25–27 fruits, 28–30 seeds

Table 3.

Morphological characters within the studied convolvulus arvensis biotypes (significant differences are in italics)

Characters\Studied taxa		C. arvensis biotype 1	C. arvensis biotype 2	C. arvensis biotype 3
Macro-morphological characters
Habit		Prostrate	Prostrate	Prostrate
Internode length (cm)		2.5–4	1.5–3.5(−4)	1.5–5
outline		Cylindrical with Ridges	Angular	Angular
Hairness		Moderately hairy	Sparsely hairy	Moderately hairy
Leaves
Length ×width (cm)		(3.5-)4-5 × 1.2–2	(2.2-)3.3–3.7x (0.8-)1.8–2	3–4.5 × 1.7–2
Length\width ratio		3–5	1.8–2.2	1.7–2.2
Shape		Sagittate	Sub-sagittate	Hastate
Apex		Apiculate -Acute	Acute	Apiculate
Base		Ear like- auricle	Triangular	Triangular
Petiole length(cm)		0.5–2(−2.5)	0.6–2.3(−3)	1–4-2(−3)
Flower
Bract	length ×width (mm)	2.6–2.8 × 0.5-1hairy	2.2–2.6(−3.5) x0.7–1	3-4 × 0.6
	shape	Elliptic	Elliptic	Elliptic -oblong
	Hairness	Hairy at margins	Hairy at margins	Hairy at margins
Pedicel length (mm)		(9-)12–13(−22) hairy	11–14	7-11hairy
Flower number		1	1	1
Color		White	Pale pink	Pink
Flower length (mm)		10–11	6–14	6–9
Calyx	length (mm)	4–4.2(−5)x(2.5-)3.2–3.4(−4)	3–4(−6)x2.5–3	3.5–4.5(−6)x2-3(−3.5)
	Shape	Obovate-cordate	Obovate-cordate	Obovate-cordate
	Apex	Emarginate	Obtuse-emarginate	Obtuse-emarginate
	Hairness	Hairy at margins	Sparsely hairy at margins	Hairy on margins
Corolla	length (mm)	10–11.5(−25)	6–6.5(−25)	6–10(−25)
	Shape	Funnel -Contorted	Funnel -Contorted	Funnel -Contorted
	Hairness	Hairy at apex	Hairy at apex	Hairy at apex
Androecium Length (mm)		6.5–9	(2.6-)3–6.8(8)	3–5.8 (−7)
Anther	length ×width (mm)	2.5-3 × 1	2–2.8(−3.8) x0.7–0.8	2–3(−4)x0.7–1.7
	shape	Sagittate	Sagittate	Sagittate
	Colour after ripen	White	pink	White - pink
Filament	length (mm)	4–6	1–4	1–2.6(−3)
	base	Papillate	Papillate	Papillate
Gynoecium	Disc length (mm)	(0.2-)0.4–0.5	0.2–0.4 × 0.9	0.2–0.4
	length (mm)	(3.6-)4.2–9.5(−1.5)	4.3–7.1(−13.5)	6.7–8.5(−12.4)
	Ovary L x W (mm)	0.8–2(−4)x0.6–0.8	0.5–1(−4.5) x0.4–0.6	1–1.1(−3.5) x0.9–1
	Ovary shape	Ovate	Ovate	Ovate
	Style length (mm)	2–4(−6)	1.5–3.5(−6)	4–5(−6) angular
	Stigma shape	Bird head like-Oblong-shaped	Bird head like -Oblong-shaped	Bird head like Oblong-shaped
	stigma LxW(mm)	(0.6-)1–2.5	1.5–1.6(−2)	1.5–2(−2.5) smooth
Fruit
peduncle length (cm)		3	1.5	3.2
Number of fruits		1	1	1
L x W (mm)		6-8 × 6-7	5.5-7 × 4-5	8-9 × 7-8
Fruiting calyx length (mm)		3	4–4.5	2.5 hairy
Shape		Subglobose	Subglobose	Subglobose
Number of seeds per fruits		2–3	2–3	4
Seed	Shape	Sectoroid	Sectoroid	Sectoroid
	Color	Black	Black	Black
	Length ×width (mm)	3.8-4 × 2.1–3	4–4.4 × 3-3.2	4–4.4 × 2.5–3.2
	Surface	Rough	Rough	Rough

Pollen

Pollen grains were symmetrical, isopolar, monad, oblate, subprolate-perprolate according to polar: equatorial ratio, oblong, spheroidal according to equatorial view (Fig. 2; 1–6, Table 4).

Fig. 2.

SEM photomicrographs of pollen grains of Convolvulus arvensis biotypes; biotype 1; 1,4,7,10, biotype 2;2,5,8,11, biotype 3; 3,6,9, 12. 1–3; equatorial view, 4–6; polar view,7–9; aperture, 10–12; exine ornamentation

Table 4.

SEM of pollen characteristics of Convolvulus arvensis biotypes (significant differences are in italics)

Characters\Studied taxa		C. arvensis biotype 1	C. arvensis biotype 2	C. arvensis biotype 3
Pollen shape		Oblate	Subprolate-Perprolate	Oblate
Equatorial view	shape	Oblong	Spheroidal	Oblong
	Lxw(µm)	65 × 40	47.86 × 57.9	65 × 44.29
Polar view	shape	spheroidal	spheroidal	spheroidal
	Lxw(µm)	25 × 25	85.56 × 85.56	52 × 52
Polar⁄ equatorial ratio		0.385	1.79	0.8
Aperture	Class	Trizonocolpate	Trizonocolpate	Trizonocolpate
	Length ×width(µm)	52.14 × 0.4	45.71 × 14.13	57.14 × 0.42
	L:w ratio	130.35	3.234	136.05
Exine surface		Papillate, Scabrate - punctate	Papillate, Scabrate - punctate	Papillate, Scabrate – punctate
Number of papillae in 5 × 5 µm		25–27	18–20	37–39
Endoapreture sculpture		Hollow with solitary papillae	Irregular papillate solid bulk	Hollow with tufts of papillae

Trizonocolpate apertures, papillate; papillae with obtuse and blunt apices, scabrate–punctate sculpture, pores less than 1 µm diameter, irregularly distributed. Number of papillae in 5 × 5 µm: 19–38. Endoapertual sculpture varied from a hollow, narrow papillate slit to an irregular papillate and granulate solid bulk area (Fig. 2; 7–12, Table 4).

The studied Convolvulus arvensis taxa show extinct variation in many aspects; oblate in biotypes 1 and 3, and subprolate-perprolate in biotype 2, according to polar⁄equatorial ratio; while oblong in biotypes 1 and 3, and spheroidal in biotype 2, according to equatorial view. Number of papillate in 5 × 5 µm: 18–20 in biotype 2, 25–27 in biotype1, 37–39 in biotype 3. Hollow endoapreture with solitary papillae in biotype 1, hollow with tufts of papillae in biotype 3, and irregular papillate and granulate solid bulk in biotype 2 (Fig. 2; 7–12, Table 4).

Seed

Examined seed are sectoroid-spheroid, with a length-to-width ratio ranging between 1.36 and 1.48 (Fig. 3; 1–3) and (Table 5). The hilum is apical, transverse, and oblong, with a length-to-width ratio ranging from 0.49–0.55. The gross seed coat sculpture is divided into two levels: the top is continuous or dissected wrinkled tubercles, and the bottom is reticulated. The epidermal cell shape is polygonal, with 5–6 Gonal cells. These findings are consistent with Abdel Khalik and Osman [30]. Anticlinal cell wall borders undulate, with increased relief at the cell edges, which conflicts with Abdel Khalik and Osman [30]. The curvature of the outer periclinal wall is concave (Fig. 3; 10–15). The relief of the cell wall boundaries is raised. The lateral wall ornamentation of epidermal cells ranged from striated to wrinkled: striated in biotype 1, and wrinkled in biotypes 2 and 3 (Fig. 3; 13–15, Table 5).

Fig. 3.

SEM photomicrographs of the seeds of Convolvulus arvensis taxa;. biotypes; biotype 1; 1,4,7,10,13, biotype 2;2,5,8,11,14, biotype 3; 3,6,9, 12,15. 1–6 seed shape, 7–10 hilum shape; 11–15 magnified spermoderm surface

Table 5.

SEM of seed of Convolvulus arvensis biotypes. (significant differences are in italics)

Characters\Studied taxa		C. arvensis biotype 1	C. arvensis biotype 2	C. arvensis biotype 3
Seed	Shape	Sectoroid, spheroid	Sectoroid, spheroid	Sectoroid, spheroid
	Length (µm)	3700-3750	388-3857.143	3918-3928.57
	Width (µm)	2500-2607.14	2811-2821.43	2809-2821.43
	L/W ratio	1.43-1.48	1.367-1,38	1.392-1.394
Hilum	Shape	Transverse oblong	Transverse oblong	Transverse oblong
	position	Apical	Apical	Apical
	Length (µm)	285.71-292.3	285.71 -292.3	271.428-280.4
	Width(µm)	542.857 -546.7	571.43-546.7	500 -508.3
	L/W ratio	0.526-0.535	0.49-0.535	0.543-0.552
Gross seed coat sculpture		Wrinkled tubercled, reticulate	Wrinkled tubercled, reticulate	Wrinkled tubercled, reticulate
epidermal cell	Shape	6-7 gonal	6-7 gonal	6-7 gonal
	diameter (μm)	8-22x8-22	7-18x7-20	10-20x7-20
anticlinal cell wall	Shape	± undulate	± undulate	± undulate
	relief of cell boundaries	Raised	Raised	Raised
distal appendage
Curvature of outer periclinal wall		Concave	Concave	Concave
Lateral wall ornamentation		Striated	Wrinkled	Wrinkled

Anatomy

Stem anatomy

In cross‒sections, the outline of the stems is ridged-circular to flattened, with sparse hairs (Fig. 4; 1–3). The epidermis is composed of a single layer of isodiametric‒tangentially elongated cells covered by a very thin, wavy cuticle. Cortex differentiated into 2‒3 outer palisade tissue composed of isodiametric-radially elongated cells, followed by 4–8 layers of inner parenchymatous tissue with tangentially elongated-angular cells. The number of big polygonal parenchyma cells varied from 6 to 8 in parenchymatous tissue (Fig. 4; 4–6). Vascular bundles are 8–16 collateral bundles; 3–6 layers of phloem. Xylem with 2‒5 vessels, mainly in radial groups of 3‒6 arches. A large central pith, composed of 11‒18 layers with Isodiametric-polygonal parenchymatous cells present in the center, occasionally sand crystals are present (Fig. 4; 4–6, Table 6).

Fig. 4.

Transverse section of stem and petiole ofConvolvulus arvensisbiotypes; biotype 1; 1,4,7,10,13, biotype 2;2,5,8,11,14, biotype 3; 3,6,9, 12,15. 1–6. Stem; 1–3 whole stem, 4–6 cross section.7–15. petiole; 7–9. Whole petiole, 10–12. Cross sections of petiole showing epidermis and collenchyma tissue in ear zone, 13–15. Main vascular bundle in petiole

Table 6.

Micromorphological features of stem, petiole and leaf of convolvolus arvensis biotypes

Characters\taxa			C. arvensis biotype 1	C. arvensis biotype 2	C. arvensis biotype 3
Stem
Diameter(µm)			804.92–882.72	919.56–1368.51	712.59–805.52 × 596.85
Outline			3 ridged- circular	2-ridged -circular	3-ridged-flattened
Epidermis	Thickness(µm)		18–20.9	10.6–15.9	18.67–20.75
	Cell shape		Tangentially elongated -isodiametric	Tangentially elongated -isodiametric	Isodiametric-tangentially elongated
	Cutin Thickness(µm)		4–5.3	5–6	3.84–5.12
Cortex	Thickness(µm)		148.04–153.4	90.45–105.52	100.5–109.45
	Palisade tissue	Thickness(µm)	23.04–26.2	40.2–50.25	30.15–35
		Number of layers	2–3	2–3	2–3
		Cell shape	Isodiametric-radially elongated	Isodiametric-radially elongated	Isodiametric-radially elongated
	Parenchyma	Thickness(µm)	125–127.2	50.25–55.27	70.35–74.45
		Number of layers	7–8	5–6(−8)	4–6
		Cell shape	Tangentially elongated-tubular- angular	Tangentially elongated	Isodiametric-tangentially elongated
	Shape of polygonal parenchyma cells		7–8	6	Tetragonal-tangentially elongated
Vascular bundles	Number		8–9	12–13	15–16
	Thickness(µm)		207.29–235.57	177.85–190	122.73–152.86
	Phloem	Thickness(µm)	20.9–26.2	30.15–32	41.88–52.36
		Number of layers	3–4	3–4	5–6
	Cambium	Thickness(µm)	8.38–10.47	7–10	10.5–20.1
		Number of layers	1–2	1–2	1–2
	Xylem	Thickness(µm)	178.01–198.9	140.7–148	70.35–80.4
		Number of arches	3–6	4–6	3–6
		Number of vessels	3–5	3–5	2–5
Pith	Thickness(µm)		50.26–52.38	351.76–733.67	221.11–230.16
	Number of layers		14–18	11–15	13–18
	Cell shape		Isodiametric-angular, 4–5 gonal	Isodiametric-polygonal	Isodiametric-polygonal
Petiole
Thickness ×width (µm)			(±)571.06–618.42 × 793.97.8	(±)590.6–656.2522.6 × 703.72–713.56	(±)533.64–587.51 × 793.97
Outline			Horse shoe	Ovate	Horse shoe
Epidermis	Thickness(µm)		20.9–23.04	31.4–36.65	15.07–17
	Cell shape		Tubular(tetragonal)-isodiametric	Radially elongated-polygonal	Tangentially elongated-Tetragonal
	Cutin thickness(µm)		15.08–18.1	10–15.07	3–5.03
Parenchyma	Thickness(µm)		160.8–180	209.4–219.9	100.5–110
	Number of layers		9	7–9	4–5
	Cell shape		Isodiametric	Radially elongated	Isodiametric-radially elongated
Vascular bundle	Thickness(µm)		126.8–132.8 × 180.9–221.106	110.6–130.65 × 221.11–231.16	150.75–172.86
	Number of Subsidiary vascular bundles		2	2	2
	Phloem	Thickness(µm)	18.2–20.3	30.15–40.2	40.2–60.3
		Number of layers	4–5	3–4	4–6
	Xylem	Thickness(µm)	108.6–112.5	80.4–90.45	110.55–112.56
		Number of arches	12–15	13–15	12–13
		Number of vessels	1–4	1–4	1–6
Parenchyma	Thickness(µm)		188.48–198.95	157–167.5	211.06–221.11
	Number of layers		9–10	7–8	9–11
	Cell shape		5–7 gonal, Tangentially-radially elongated	Isodiametric-angular	Isodiametric
	Big cell number		10–13	12	12–15
Collenchyma	Thickness(µm)		38–40.2	50.25–60.3	35.18–40.2
	Number of layers		2–3	3	2
	Cell shape		tangentially elongated	Radially-isodiametric	Isodiametric- tangentially elongated
Lower Epidermis	Thickness(µm)		18–20.1	18.9–20.9	15.08–16.08
	Cell shape		Tetragonal-Isodiametric	Tangentially elongated-isodiametric	Tangentially elongated-isodiametric
	Cutin Thickness(µm)		3–5.23	3–5.23	3–5.23
Leaf
Midrib region
Outline			U-shaped	U-shaped	U-shaped
Thickness(µm)			(±)344.875- 402.82 × 552.8–562.8	(±)404.155–464.38 × 603.02–613.07	(±)313.4–344.6 × 462.3–472.4
Upper Epidermis	Thickness(µm)		15.07–18.09	24.13–26	18–21.1
	Cell shape		Isodiametric-tangentially elongated	Isodiametric-radially elongated	Radially elongated
	Cutin thickness(µm)		5.03–7.2	5.025–10.05	7–10.05
Collenchyma	Thickness(µm)		15.075–17	60.3–70.4	40.2–50.3
	Number of layers		2	3–4	3–4
	Cell shape		Tangentially elongated	Isodiametric-radially elongated	Isodiametric-tangentially elongated
Parenchyma	Thickness(µm)		50.3–54	20.1–25	-
	Number of layers		3–4	1	-
	Cell shape		Isodiametric-angular	Isodiametric-radially elongated	-
Vascular Bundle	Thickness(µm)		110.7–130.6 × 211.06–221.1	110.6–130.7 × 271	90.5–102.4 × 201-211.06
Xylem	Thickness(µm)		80.4 -90.4	80.4–90.5	70.4–80.4
	Number of arches		12–13	14–16	13–15
	Number of vessels		2–5	2–4	2–5
Phloem	Thickness(µm)		30.3–40.2	30.2–40.2	20.1–22
	Number of layers		3–4	3–4	3–4
Parenchyma	Thickness(µm)		110.6–120.6	130.7–132	90.5–100.5
	Number of layers		5–7	4–5	5–7
	Cell shape		Angular-irregular isodiametric-radially elongated	Angular-isodiametric	Isodiametric-radially elongated
	Number of big cells		5-(4–6-gonal)	5-(4-6gonal)	5- Pentagonal
Collenchyma	Thickness(µm)		20.1–30.2	30.2–40.2	20.1–30.1
	Number of layers		2–3	1–3	1–2
	Cell shape		Isodiametric-tangentially elongated	Isodiametric	Tangentially elongated
Lower Epidermis	Thickness(µm)		15–20.1	20.1–25	19–20.1
	Cell shape		Isodiametric-radially elongated	Tetragonal	Isodiametric-radially elongated
	Cutin thickness(µm)		3–5.03	3–5.03	8–10.05
Wings region
Wing	Thickness(µm)		(±)216.4–254.65	(±)180.17–213.2	(±)172.48–192.86
Upper Epidermis	Thickness(µm)		15.2–20.1	10.05–15.08	15.07–20.01
	Cell shape		Tangentially elongated-isodiametric	Tangentially elongated Isodiametric	Tangentially elongated-Isodiametric
	Cutin thickness(µm)		5–6	5–6	4.2–5.23
Mesophyll	Thickness(µm)		178.01–209.42	157.07–178.01	136.13–146.59
	Number of layers		5–7	4–8	7–8
	Cell shape		Radially elongated	Radially elongated	Radially elongated
Lower Epidermis	Thickness(µm)		15.2–20.1	10.05–15.08	14.08–16
	Cell shape		Radially elongated- isodiametric	Tangentially elongated-isodiametric	Tangentially elongated
	Cutin thickness(µm)		3–5.03 wavy	3–5.03	3–5.03

Petiole anatomy

The outline is a horseshoe-shaped oval with a small depression in the adaxial part. The epidermis is tangentially elongated (tetragonal)-radially elongated cells that are coated in an undulating cuticle and multicellular hairs (Fig. 4; 7–9).

The cortex is 5–9 layers of isodiametric, radially elongated parenchymatous cells. The vascular system consists of a core elongated, arc‒shaped collateral vascular bundle, in which 3–6 layers of phloem, the xylem arches are 12–15, each with 1‒6 vessels, and 2 accessory vascular bundles at each upper end of the main vascular bundle (Fig. 4; 7–9, 13–15).

Below: the main Vascular bundles, 7–11 layers of isodiametric-angular, radially elongated parenchymatous tissue, followed by collenchyma represented by 2–3 layers of isodiametric, tangentially, radially elongated cells, lower epidermis is tangentially elongated, isodiametric cells covered by an undulating cuticle layer (Fig. 4; 7–9, Table 6).

Leaf anatomy

Leaf blade has a U-shaped midrib region. Upper epidermal cells are isodiametric, radially elongated, covered with a wavy cuticle and multicellular trichomes (Fig. 5; 7–12). Collenchyma is 2–4 layers with isodiametric, tangentially, radially elongated cells. Parenchyma tissue may be present or absent, has 1–4 layers, and isodiametric, radially elongated cells. The main vascular bundle is collateral; a broad boat‒shaped strand of xylem band inside with 12–16 arches, each with 2–5 vessels, followed by 3–4 layers of phloem inside (Fig. 5; 7–12).

Fig. 5.

Transverse sections of petiole and leaf of Convolvulus arvensis biotypes; biotype 1; 1,4,7,10,13, biotype 2;2,5,8,11,14, biotype 3; 3,6,9, 12,15. 1–6. Petiole; 1–3. angular parenchyma cells, 4–6. Epidermal cells. 7–15. Leaf, 7–10. Whole leaf, 8–12. Midrib region, 13–15 wing region

Below the main vascular bundles, there are 4–7 layers of parenchymatous tissue with angular-irregular, isodiametric, radially elongated cells. Followed by parenchyma tissue, 1‒3 collenchyma layers with isodiametric-tangentially elongated cells. Lower epidermal tissue with isodiametric, radially elongated cells covered with wavy cuticle. In the wing’s region, the mesophyll consists of one type of cells; 5–8 layers with radially elongated cells (Fig. 5; 13–15 Table 6).

Artificial key of C. arvensis L. biotypes based on morphological, anatomical, seed and pollen characters

1a- Leaves sagittate, cylindrical with ridges stem outline, greenish white dorsal midpetaline area … … … … … … … … … … … … … … … … … … … … … … … … … … … ,,,,,,,,,,,,, … . biotype 1

1b- Leaves subsagittate-hastate, angular stem outline, dark violet dorsal midpetaline area … … … … … … … … … … … … … … . … … … … … … … … … … … … … … … . … … … … … 0.2

2a- Leaves subsagittate, subprolate-perprolate pollen grains, polar⁄ equatorial ratio up to 1.79, irregular papillate solid bulk endoapreture sculpture. In leaf; 3–4 parenchyma layers upper main vascular bundle … … … … … … … … … … . … … … … … … … … … … … … … … … … … biotype 2

2b- Leaves hastate, oblate pollen grains, polar⁄ equatorial ratio up to 0.8, hollow with tufts of papillae endoapertural sculpture. In leaf; absence of parenchyma tissue upper main vascular bundle … … … … … … … … … … … … … … … … … … … … … … … … … … … … … . … … biotype 3

3.4. Genetic parameters

Seven markers out of 10 CDDPs were successfully amplified and distinguished between accessions (Fig. 6). These markers were used for subsequent biostatistical analyses, including clustering, genetic variance, and relationships. These estimates were to differentiate between biotypes in three localities. The evaluation of CDDP marker efficiency across the eight accessions revealed a significant level of genetic informativeness and discriminating capacity, as summarized in the table below. The total number of bands (T. Bands) per sample ranged from 24 to 29, with accessions 2, 7, and 8 exhibiting the highest primer binding stability. Genetic diversity analysis indicated that accession 7 possessed the highest levels of heterozygosity and polymorphism information content (H_Index = 0.1497; PIC_Index = 0.1154), followed closely by accession 8. Conversely, accession 5 showed the lowest genetic contribution, recording a minimum Resolving Power (Rp_Score) of 5.75. The observed correlation between high Rp_Score and elevated PIC values in accessions 7 and 8 underscores their importance as genetically distinct individuals within the population. These indices collectively suggest that the CDDP markers employed are highly effective for biotype identification and for establishing unique DNA fingerprints, with accessions 7 and 8 serving as the most informative for conservation strategies (Table 7).

Fig. 6.

Profile of CDDP markers. a)C6, b) WRKY-R3BC5, c)c 8,d) ERF2–C2, e) MADS1–C1,and f) Myb1 and g)KNOX-2 S13

Table 7.

H Index: expected heterozygosity (genetic Diversity); PIC Index: polymorphism information Content; Rp Score: resolving Power (ability of the primer to distinguish genotypes)

#	T. Bands	1	0	Poly%	H	PIC	Rp
1	39	28	11	71.79	0.1201	0.0937	7.50
2	39	29	9	74.36	0.1382	0.1082	8.50
3	39	26	12	66.67	0.1168	0.0900	7.50
4	39	26	12	66.67	0.1102	0.0857	7.00
5	39	24	14	61.54	0.0880	0.0682	5.75
6	39	26	12	66.67	0.1102	0.0857	7.00
7	39	29	9	74.36	0.1497	0.1154	9.50
8	39	29	9	74.36	0.1472	0.1140	9.25

The pairwise genetic distance heatmap effectively partitioned the strains into two distinct phylogenetic clusters, identifying accessions 7 and 8 as the most closely related pair with a minimum distance of 0.06. The maximum divergence (0.46) was observed between accession 2 and accessions 7 and 8. These findings suggest that accessions 7 and 8 are the most informative for establishing DNA fingerprints, and the high genetic distance between them and accession 2 presents a strategic opportunity for maximizing genetic recombination in future breeding programs (Fig. 7).

Fig. 7.

The pairwise genetic distance heatmap by CDDP marker. The genetic divergence among the samples solely based on molecular (CDDP) markers, identifying specific genetic fingerprints eight samples

The CDDP marker analysis of eight revealed significant genetic diversity and structured relationships influenced by flower color and geographic location. Accessions 7 (white) and 8 (pale pink flowers), from the third location, emerged as the most genetically informative, exhibiting the highest PIC indices (0.1154 and 0.1140) and Resolving Power (9.50 and 9.25), indicating their high utility for DNA fingerprinting. The genetic distance heatmap effectively clustered the samples into two major groups: the first comprising samples from the first location (accessions 1, 2, 3) along with accession 4, and the second grouping samples from the second (accessions 5 and 6) and third locations (accessions 7 and 8). Notably, the lowest genetic distance (0.06) was found between accessions 7 and 8, suggesting that geographic proximity in the third location has a stronger influence on genetic similarity than flower color. Conversely, the highest divergence (0.46) was observed between accession 2 (pale pink, location 1) and the cluster containing accessions 7 and 8, highlighting a clear genetic separation driven by both geographic distance and environmental factors across the three collection sites.

CDDP marker demonstrated robust genetic diversity detection, revealing high polymorphism (74.36%) across primers and resolving powers (Rp) peaking at 9.50 for sample 7. Genetic distances in the heatmap range from 0.00 (likely within biotypes 7–8) to 0.46, forming two clusters primarily aligned with locations rather than flower colors. This structure reflects conserved DNA-derived polymorphism (CDDP) in targeting gene regions for reproducible diversity assessment, consistent with studies in related plants. Accessions 1, 4, and 7 (white flowers) show variable bands (26–29), polymorphisms (71.79–74.36%), PIC (0.115–0.147), and Rp (8.70–9.50), with accession 7 excelling. Pale pink accessions 2, 5, and 8 exhibit similar ranges (26–29 bands, 66.67–74.36% poly), but accession 8 has high PIC (0.114) and Rp (9.25). Pink accessions 3 and 6 have 26 bands each, 66.67% polymorphism, and a moderate PIC/Rp (0.110/9.10), indicating lower informativeness. Table 7 and Fig. 7.

Location 1 (accessions 1–3: white/pale pink/pink) clusters tightly, suggesting shared local adaptation despite color variation. Location 2 (accessions 4–6: white/pale pink/pink) partially overlaps but diverges more, while location 3 (accessions 7–8: white/pale pink) shows the tightest similarity (distance ~0.06), prioritizing geography over color. Heatmap gradients confirm location-driven structure, with maximal distances (0.4–0.46) between locations 1 and 3.

The integrated study of eight accessions categorized into three biotypes (Biotype 1 (White), Biotype 2 (Pale Pink), and Biotype 3 (Pink)) reveals a significant correlation between genetic fingerprints and internal structures. Hierarchical clustering demonstrates that Biotypes 1 and 2 share a high degree of similarity and cluster closely together. In contrast, Biotype 3 is clearly defined as a distinct accession, characterized by unique CDDP molecular markers and macro- and micro-morphological characters Fig. 8.

Fig. 8.

The pairwise genetic distance heatmap with genetic marker, macro and micromorphological characters: this matrix quantifies the total biological distance between biotypes

The pairwise genetic and morphological distance matrix quantifies the biological dissimilarity between the groups. The lowest distance value (12.37) recorded between Biotype 1 and Biotype 2, confirming they are very close. Biotype 3 showed the highest degree of divergence, with 13.30 from Biotype 1 and 12.49 from Biotype 2. The matrix confirms consistent dissimilarity values, reinforcing the reliability of the integrated 0/1 (molecular) and 1/2/3 (morphological) data scaling.

The transition in floral pigmentation from white to pink is accompanied by measurable changes in 66 macro and micromorphological characters Fig. 9, including vascular bundle thickness and seed parameters, which are intrinsically linked to specific CDDP genomic markers. This confirms that the observed biotypes are distinct biological entities with unique internal and genetic profiles.

Fig. 9.

The distance heatmap with CDDP marker, macro and micromorphological characters with different biotypes: this visualization demonstrates cluster into three distinct groups (B1: white, B2: pale pink, and B3: pink) based on a combination of genetic and structural traits

Discussion

Macro-morphological variations

Robinson and Fernald [54], recognized C. arvensis var. obtusifolia Choisy as a variety in North America. Gray [55] introduces C. arvensis f. cardifolius with cordate leaves and broad basal lobes, and C. arvensis f. auriculatus Descre. with linear-oblong or lanceolate blades with acute ear-shaped lobes. In Europe, sixty or more C. arvensis varieties have been identified because of variation in environmental factors [56]. In North America, plants with intermediate features are common. Gleason [57] grouped C. ambigens with C. arvensis.

Whitesides [58], reported the establishment of three ecotypes of C. arvensis from Oregon that varied in length and number of vines, number of leaves and flowers, and seed output per plant when cultivated under similar conditions.

In Egypt, there is no previous records on biotypes of C. arvensis until Täckholm [19] and Boulos [20]. Soliman et al. [21] differentiated between three varieties of C. arvensis var. arvensis, C. arvensis var. linearfolious and C. arvensis var. nov using RAPD. Hamed et al. [22] reported notable morphological diversity of five distinct accessions of C. arvensis, with two ploidy levels, and unrelated to environmental factors.

The studied accessions in the present study showed considerable morphological variation although specimens collected from the same habitat had an angular stem in biotypes 2 and 3, and a cylindrical stem with ridges in biotype 1. The sagittate leaf shape in biotype 1, subsagittate in biotype 2 and hastate in biotype 3. White flower in biotype 1, pale pink in biotype 2, and pink in biotype 3. Dorsal midpetaline is greenish white in biotype 1, pink in biotypes 2 and 3. White radiating colour where the petals unite internally in biotype 3, pale pink in biotype 2.

Palynological and seed micro-morphological

Telleria and Daners [24], describe a highly diverse pollen morphology in the Convolvulaceae family, which has unique taxonomic significance. Wide variation in pollen descriptions in the previous work on Convolvulus species; prolate-spheroidal to oblate-spheroidal, and rarely prolate [59], and suboblate, oblate-spheroidal, prolate-spheroidal, and prolate [60]. In the current study; the shape of biotype 2 pollen grains is prolate and perforate [61]. Ashfaq et al. [33] reported similar results. Sculptures are micro-echinates or spines with obtuse and blunt apices that have been irregularly distributed [60, 62], which agrees with the present results. Pori are characterized by rounded or elongate tectal perforations, less than 1 μm in length or diameter. Tricolporate pollen type which is a primitive type according to Ashfaq et al. [33].

Seeds have a limited role in differentiation between the studied taxa in the majority of features, except for the lateral wall ornamentation of epidermal cells, which is striated in biotype 1 and wrinkled in biotypes 2 and 3.

Anatomical features

In stem, circular outline with ridges in biotypes 1 and 3; tri-angled in biotype 1 (agreed with 63), di-angled in biotype 2 while ridged and flattened in biotype 3. Number of vascular bundles up to 9 in biotype 1, 12–13 in biotype 2, 15–16 in biotype 3. Cortex has palisade tissue; the stem is solid; pith is present (as mentioned by 63).

In leaf, the cuticular layer on the upper epidermis is relatively thin, as mentioned by Farid [63]. In petiole; horseshoe outline in biotypes 1 and 3, and ovate in biotype 2. Number of parenchyma layers: 4–5 in biotype 3, up to 9 layers in biotypes 1 and 2. Epidermal cells radially in biotype 2, tubular (tetragonal)-isodiametric in biotype 1, and tangentially elongated-tetragonal in biotype 3. In midrib: The upper epidermis was concave in surface; these results are in harmony with Farid [63]. The cuticular layer in the leaves of the studied taxa differs with that of Malik and Trapathi [64], Madani and Majbour [65], and Farid [63]. Collenchyma below the upper epidermis has 2–4 layers and up to 3 layers above the lower epidermis; the main vascular bundle is collateral [66]. In the midrib, there is no parenchyma tissue above the main vascular bundle in biotype 3. 1 layer of isodiametric-radially elongated cells in biotype 2 and 3–4 layers of isodiametric-angular in biotype 2. Upper epidermis in the midrib forms nearly right angle with the epidermis of the wing in biotype 1, while an obtuse angle is formed in both biotype 2 and 3; bigger than 150 in biotype 2 and between 120 and 150 in biotype 3.

Among the angiosperms, laticifers in different parts and tissues (stem, midrib, phloem, cortex and pith) have been reported [67, 68], including the Convolvulaceae [68–70].

CDDP analysis marker and genetic diversity metrices

CDDP marker analysis of eight Convolvulus arvensis L. accessions from three locations reveals robust genetic diversity, with polymorphism levels reaching 74.36% and resolving power (Rp) peaking at 9.50 for accession 7 (white flower, location 3)

The genetic distance heatmap shows two main clusters aligned primarily by location: location 1 (accessions 1–3: white/pale pink/pink) groups tightly, location 2 (accessions 4–6: white/pale pink/pink) partially overlaps, and location 3 (accessions 7–8: white/pale pink) exhibits the lowest distance (~0.06), indicating geography overrides flower color effects [71, 72].

White flower accessions (1, 4, and 7) display 26–29 bands, 71.79–74.36% polymorphism, PIC values of 0.115–0.147, and Rp up to 9.50, with sample 7 most informative. Pale pink accessions (2, 5, and 8) show comparable ranges (66.67–74.36% poly), while pink accessions (3 and 6) have moderate PIC (0.110) and Rp (9.10). These CDDP metrics confirm marker reproducibility for fingerprinting in weeds, such asfield bindweed [50].

Locality-driven structure dominates, as in location 3, contrasting with higher distances (0.4–0.46) between locations 1 and 3. Flower color shows limited influence, consistent with prior C. arvensis studies using CDDP and RAPD markers [73].

Flower color showed a weak correlation with clustering, as white/pale pink pairs per location group closely with prior RAPD/CDDP findings on C. arvensis, where geography overtakes minor traits. CDDP’s high reproducibility suits C. arvensis fingerprinting for weed management or breeding, outperforming random markers like RAPD. Environmental factors likely amplify location effects in this perennial weed. [74].

Integrative ApproachesTwo dominant groups were identified using the CDDP markers. The first main group includes the accessions 1, 2, 3, and 4. The second main group is divided into two subgroups; the first subgroup consists of accessions 5 and 7, while the second one includes accessions 6 and 8. These results differ from those derived from morphological, anatomical, and SEM of Seed and pollen attributes (Tables 2, 3, 4, 5 and 6), as well as collection cores of localities (Table 1). Various factors influence the morphoplasticity and the phenology of biotypes, including the distribution and spread of accessions across three localities. These include ecological mechanisms, biosystematics, the number of individuals in a population, the number of localities, the distance between populations, dominant pollinators in a locality, and the methods of collection [17, 75, 76]. The variance between clusters and ideotypes occurs gradually. This confirms the set of C. arvensis into three biotypes based on the ecological mechanisms, and the collection method plays a primary role in classifying the biotypes. This may be the primary role of environmental mechanisms, and spreads across localities. GPS coordinates of the first (31° 21’ 658” and 27° 09’ 661”) and second (31° 23’ 013” and 27° 04’ 474”) localities are very close to each other. The first idiotype collected is likely the 4 before others in the same locality; therefore, it clusters with the first localities (including accessions 1, 2, and 3). The distance between the two localities is approximately 2 km (Table 1). The effect of geographical distribution on genetic variance between two date palm accessions in two arid regions was studied using CDDP and SCoT markers [77]. They investigated the polymorphism of both markers as a tool to identify the genetic similarity and correlation of two populations. Mahdy and Rizk [78], highlighted additional insights into gene pools of three Calligonum polygonoides populations using random amplified polymorphic DNA (RAPD). These gene pools are valuable reservoirs that enrich genetic variation and provide the basic information for biodiversity and conservation, which can be exploited under the same environmental conditions.

We estimated some parameters and non-parameters to differentiate whether two gene pools (populations) are different or not. Our results are in agreement with previous studies [75, 79, 80]. The obtained results showed a variance between the two gene pools. These may be due to the impact of environmental conditions or the genetic makeup of the collection. The genetic variation within accessions of a species can lead to the emergence of new traits and improved morphological characteristics that enhance plant performance. These may lead plant life to face significant challenges, including mating systems in Convolvulus, which is open-pollinated by insects. Gene flow plays a significant role in the rearrangement of a plant’s genetic variation across phytogeographical locations and time [81], and is one of the evolutionary tools to adopt for conservation [82]. The reproduction of species depends on the interactions between genotypes and the environment that drive development, evolution, and diversity [83]. Attitudinal changes to local native seed for ecological restoration have been studied in shifting seed sourcing [84]. Hufford et al. [85] defined the genetic determination of local seed collection zones along a climate gradient. This revealed that the obtained results were between those from morphological and molecular parameters. In fact, the obtained contradiction reveals that morphoplasticity is due to one or more factors for plant life to face ecological stresses [86]. Therefore, it needs further precise clarification, such as SNP and DNA barcoding [76]. It can be said that accurate identification must be based on various approaches [79].

While both morphological and molecular data are valuable, their combined use can provide a more comprehensive understanding of relationships. The conflict often arises due to differences in the inherent challenges of both data types. Additionally, the inherent genealogical deficit of comparative morphology and genetic signal erosion can exacerbate these conflicts, making it crucial to use genetically validated characters for accurate classification. Cohen [87] suggested that between taxon description and clades, logical circularity must be avoided in cladistic analysis, and follows rules away from PhyloCode-like approaches to systematics if they allow the output of cladistic analysis to serve as the input to further such analyses. A simple method for identifying and removing convergence-prone characters improves phylogenetic accuracy, potentially enabling, when necessary, the inclusion of morphologies and hence fossils for reliable tree inference [88]. de Almeida et al. [89] recommended the pivotal need to observe plant morphology with molecular phylogenetic results, particularly when new hypotheses disagree with the existing classification, is to avoid taxonomic mistakes. Keating et al. [90] advised that the congruence between morphological and molecular data partitions needs to be considered in combined analyses. Their results revealed that morphology and molecules must be combined in order to best estimate history and reveal hidden support for novel relationships. Studies that analyse only phenomic or genomic data in isolation are unlikely to provide the full picture [90]. Understanding these conflicts is essential for accurately interpreting relationships and avoiding misclassification in taxonomic studies. Morphological data can experience more presence than molecular data, but this does not necessarily mean that data is less reliable; it can be more informative for resolving relationships among closely related species. Addressing these conflicts requires careful consideration of the specific characteristics used in the analysis.

Conclusion

It can be said that the method of collection, with the distribution and spread across environmental conditions, influenced the clustering of the accessions into three groups by CDDPs. Additionally, the CDDP markers were separated into biotypes based on the color of flowers. Although the mechanisms of patterns remain largely unresolved, this study showed contrasting patterns in the native ranges of C. arvensis. Detailed research, including population genetics, phylogeography, or investigation of pollinators, may help reveal the factors shaping the observed patterns. Additionally, this study highlights the role of citizen science platforms. It can be concluded that studies analyzing only phenological or genetic data in isolation are unlikely to provide a full picture of how ecological stresses and genotype structure influence the morphoplasticity of flower color in Convolvulus arvensis biotypes.

Electronic supplementary material

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Author contributions

FS, and MH designed the research. FS, MH, ME and EM conducted experiments. ME& MH finalized the manuscript. All authors read, edited and approved the final version of the manuscript.

Funding

No funding was received for conducting this study.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files or available upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.