Received: April 11, 2025; Accepted: October 5, 2025; Published: December 24, 2025

Taxonomic evaluation of three Egyptian Solanum species based on morphology, DNA sequences, and chromosome analysis

Amany S. Abdo^1,* , Rim S. Hamdy^2,3 , Ibrahim A. El Garf¹ , Mona E. Abd El Gawad⁴

¹ Department of Botany and Microbiology, Faculty of Science, Cairo University, Egypt

² Biological science department, Faculty of Science, Galala University, Suez, Egypt

³ Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza, Egypt

⁴ National Gene Bank, Agriculture Research Center (ARC), Egypt

*Corresponding author. Email: amanysallam88@gmail.com

Abstract. We investigated the intra-specific relationships between three Solanum species in Egypt: Solanum nigrum, Solanum villosum, and Solanum sinaicum, in addition to the inter-specific relationships among the populations of the three studied species. These species’ taxonomic difficulty is primarily due to its inconsistent infra-specific treatments in various floras. The morphological studies revealed distinguishing characteristics for the three investigated Solanum species mainly fruit and flower characteristics. The morphological differences allowed S. villosum to be divided into two subspecies: subsp. villosum, and subsp. miniatum. The genetic variation between the three Solanum species was clarified using AFLP technique. The ribosomal DNA ITS1-5.8S-ITS2 region of the three studied species was sequenced using the universal primers ITS4 and ITS5. The DNA sequences of Solanum species were counted. The karyotypes of the species under examination were established using the chromosome number and genome size acquired from mitotic chromosomal preparations.

Keywords: AFLP, karyotype, Solanum, S. nigrum, S. sinaicum, S. villosum.

Contents

Introduction

Materials and methods

Morphological study

Data analysis

Cytogenetics study

Chromosomes number and Karyotype formulae

Chromosome preparations

Microscopic examination karyotyping, idiograming and signals imaging

Molecular study

DNA extraction

PCR reactions and data analysis

Results morphological diversity

Key to studied species of the genus solanum in egypt

Morphological correlation between the three Solanum species

Cytogenetics study

Chromosomes number and Karyotype Formula

Molecular analysis

Genetic correlation between the studied Solanum species

Discussion

Acknowledgements

References

Introduction

Solanaceae is one of the largest and most essential families of flowering plants. This family serves as a significant source for medicine, food, and spice (Afroz et al. 2020). It is extensively dispersed in both tropical and temperate regions; it has 100 genera and over 2500 species thriving in a variety of environments and ecologies and exhibits different morphologies (Nderitu et al. 2023). Solanaceae is represented in Egypt by eight genera including 30 species (Boulos 2002). Solanum L. is the family’s largest genus (El-Shaboury et al. 2020). There are several species of Solanum that are used in traditional medicine, and some of these species are employed as sources of drugs in pharmacology (Jainu and Devi 2005). Due to its morphological plasticity and infraspecific genetic variation, Solanum possesses a taxonomic challenge (Jennifer and James 1997), also because of the 6,931 names that have been published, many of which are connected to the cultivated and widely distributed species of the genus (Särkinen et al. 2018).

Solanum is represented in Egypt by 9 species (Boulos 2002). However, Täckholm (1974) recorded 10 species. Zohary (1966) recorded two subspecies of Solanum villosum: subsp. villosum and subsp. puniceum. Boulos (2002) recorded that Solanum villosum Mill. is represented in Egypt with two subspecies: subsp. villosum and subsp. miniatum. The name Solanum sinaicum is considered a synonym of Solanum villosum (Särkinen et al. 2018; powo.science.kew.org/) but the name is accepted by Khafagi et al. (2018) and (www.solanaceaesource.org).

Low taxonomic level phylogenetic relationships have been successfully solved using AFLP (Despres et al. 2003; Koopman 2005; Meudt and Clarke 2007). The use of AFLP offers numerous advantages. It generates data that is highly reproducible (Jones et al. 1997), and doesn’t require a priori sequence knowledge. Previous attempts using AFLP markers have proved effective in resolving taxonomic issues and illuminating relationships among species of Solanum (Kardolus et al. 1998; Mace et al. 1999a, 1999b; Coulibaly et al. 2002; Jacoby et al. 2003; Dehmer and Hammer 2004; Olet 2004).

The objectives of this study are to investigate the morphological and genetic diversity found in the populations of the three selected Solanum species: S. nigrum, S. villosum and S. sinaicum, to solve the taxonomic problem of these species in Egypt due to the confused infra-specific groupings.

Materials and methods

Morphological study

This study was based on the examination of specimens deposited at Cairo University Herbarium (CAI), in addition to the authentic type specimens preserved in virtual herbaria that are accessible online (the JSTOR Global Plants database). Acronyms follow Index Herbariorum (http://sweetgum.nybg.org/ih/). In addition to the examination of fresh representative specimens of each of the three species collected between 2022–2024. These specimens belonging to 5 different localities from different phytogeographical regions of Egypt. The coordinates of these localities: 30°49’56’’ & 29°34’49’’; 30°00’18’’ & 31°12’45’’; 30°00’49’’ & 31°12’04’’; 29°17’23’’& 30°51’11’’; and 28°33’38’’ & 33°58’20’’. All specimens were examined for morphological variations in all distribution localities. There were 44 different morphological features analyzed, including those for the stem, leaves, flower, and fruit. Scientific names for the species follow IPNI (http://www.ipni.org/). Vouchers Samples were placed in (CAI).

Data analysis

44 morphological characteristics were analyzed in order to establish a correlation between the samples of the three Solanum species. All samples had their characteristics measured and documented, and the software (R-4.3.1 for Windows) was used to analyze the data and create the heat map. A heatmap is a visual representation of numerical data where each value is represented as a square, with lighter squares representing smaller numerical values and darker squares representing larger values (Tiessen et al. 2017).

Cytogenetics study

Chromosomes number and Karyotype formulae

Chromosome preparations

Between 2022 and 2024, plant material was collected for cytogenetic analysis. A total of thirty specimens were sampled, comprising ten individuals from each of the three Solanum species. Seeds were extracted from the specimens and soaked in distilled water for 1 h prior to germination at room temperature. Root tips approximately 1 cm in length were excised and pretreated with 0.025% colchicine (C₂₂H₂₅NO₆) for 2 h at room temperature to arrest cells at metaphase, followed by rinsing with distilled water. The treated samples were subsequently fixed in a 3:1 (v/v) ethanol:glacial acetic acid solution.After thorough washing with distilled water, the root tips were hydrolyzed in 1 N HCl at 64 °C for 5 min. For slide preparation, the root tips were squashed in 45% acetic acid and stained with acetoorcein solution to visualize chromosomes. The procedure followed previously described protocols (Ibrahim et al., 2019; Elsayed et al., 2024).

Microscopic examination karyotyping, idiograming and signals imaging

Chromosomes examination was done via a vertical fluorescence microscope (Leica DM2500) equipped with a cooled monochrome digital camera (Leica DFC340FX). Twenty cells with clearly observed and well spread chromosomes were checked and photographed at 100X magnification under oil immersion. Chromosome counting and karyotype has performed via the automated Karyotype and FISH software processing (Leica CW4000) system. Ideograms were constructed from complete chromosomes which showed the greatest possible banding pattern in at least ten different metaphase plates. (Ibrahim et al. 2019; Abdo et al. 2023).

Molecular study

DNA extraction

For the chosen samples of Solanum nigrum, Solanum villosum and Solanum sinaicum, genomic DNA was isolated from one gram of juvenile leaves using the CTAB (Cetyl-trimethyl ammonium bromide) extraction buffer approach as described by Doyle and Doyle (1990) and modified by Allen et al. (2006).

PCR reactions and data analysis

PCR amplification was carried according to Williams et al. (1990) with some modification. The reaction volume of 25 μl containing 12.5 μl Dream Taq Green PCR Master Mix (2X), 1 μl Forward primer, 1 μl Reverse primer, 2 μl Template DNA and completed to 25 μl with water (nuclease-free) was placed in a thin-walled PCR tube on ice, then gently vortex the samples and spin down. PCR reaction was performed using the recommended thermal cycling conditions: One cycle of initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 45 seconds followed by annealing at 57°C for 45 seconds, extension at 72°C for 60 seconds, and one cycle of final extension at 72°C for 10 minutes.

The reaction products were separated by electrophoresis on a 1.6% agarose gel in a 1x TBE buffer, run in the same buffer at 100 V for an hour, and then visualized by staining with 0.5 g/ml of ethidium bromide and being photographed under UV light.

To elucidate the genetic variation and construct the phylogeny of the studied Solanum species, the ribosomal DNA ITS1-5.8S-ITS2 region of the three species was sequenced using ABI377 DNA sequencer (ABI, USA). DNA was amplified using the universal primers ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) and ITS5 (5´-GGAAGTAAAAGTCGTAACAAGG-3´), these primers described by White et al. (1990). Then, BLAST program was employed to look for sequence similarity in DNA databases. Multiple sequence alignment and the determination of genetic distances among the analyzed species were performed using MEGA5 software. Neighbour joining dendrogram was created to highlight the genetic links between the three species. The retrieved sequences were registered on the ncbi under accession numbers PP701899, PP707086, PP707087, and PP707088. The reference sequences from other countries of Solanum nigrum and Solanum villosum used to construct the heat map are available online in the Gene bank

Results
morphological diversity

Old herbarium specimens and 150 newly collected specimens of the three studied Solanum species were the subject of morphological studies and taxonomy revision based on 44 morphological traits, including plant height, leaf features, as well as inflorescence and fruit characters. The flower, fruit and leaf characters were the most distinctive characters between the species, and according to the variation in morphological characters we considered the two different forms of Solanum villosum as two different subspecies Solanum villosum subsp. villosum and Solanum villosum subsp. miniatum (Table 1, Figs 1, 2, 3, 4).

Solanum nigrum L., Sp. Pl., ed. 1, 219 (1753).
Common name: Black nightshade

Annual erect herb; stem glabrous to pubescent, green, angular, woody, branched, up to 70 cm tall; leaves simple, alternate, petiolate, ovate or deltoid-rhomboid, entire or irregularly dentate, acute, base cuneate to truncate, 2-7 x 1.5-3.5 cm, petiole up to 2 cm, stipules absent; inflorescences unbranched cymes, number of flowers per inflorescence 5-10, peduncle length 1-1.5 cm; Flowers pentamerous, hermaphrodite; pedicel 3-10 mm; calyx lobes triangular with acute or rounded apex, green, (1.5-2.5 x 1 mm); corolla white with yellow midrib, oblong or ovate to lanceolate, 4-5 x 2-2.5 mm, acute; stamen filament 1.5-2 mm, anthers yellow, oblong, 1.5-2.5 x1 mm; ovary globose, 0.5-1 mm in diameter, style 2-3.5 mm long, stigma is small; fruit berry, globose, black, 6-7 mm in diameter, fruiting calyx lobes spreading to reflexed 2-3 x 2 mm; seeds ovoid, 1.5–2 mm in diameter; hairs non glandular unicellular papillose, or non-glandular basal cell with long narrow apical cell, or non-glandular bicellular uniseriate with hooked or obtuse apical cell.

Solanum villosum subsp. miniatum (Bernh. ex Willd.) J.M.Edmonds, Bot. J. Linn. Soc. 89: 166 (1984).
Common name: Woolly nightshade, Red-berried nightshade

Annual to perennial erect herb to small shrub; stem pubescent, green, angular, woody, branched, up to 70 cm tall; leaves simple, alternate, petiolate, broadly to narrowly ovate to elliptic or deltoid, entire or irregularly sinuate-dentate, acute to acuminate or obtuse, base cuneate to truncate or cordate, 2-6 x 1.5-5 cm, petiole up to 2 cm, stipules absent; inflorescences unbranched cymes, number of flowers per inflorescence 2-5, peduncle length 0.5-2.5 cm; flowers pentamerous, hermaphrodite; pedicel 7-11 mm; calyx lobes rounded, green, (2.5 x 1 mm); corolla white with black midrib, oblong or ovate to lanceolate, 8 x 4 mm, acute; stamen filament 2 mm, anthers yellow, oblong, 2.5 x1 mm; ovary globose, 1 mm in diameter, style 5 mm long, stigma is small; fruit berry, globose, bright red, 6-9 mm in diameter, fruiting calyx lobes spreading to reflexed 2-3 x 1-2 mm; seeds 1.5–2.5 mm long, pale yellow; hairs non glandular bicellular uniseriate with hooked or obtuse apical cell.

Solanum villosum subsp. villosum Miller, Gard. Dict. 8th edn, no. 2 (1768)

It differs from subsp. miniatum in having acute leaves, base cuneate to truncate, cordate or hastate, 2-8.5 × 1.5-6.5; number of flowers per inflorescence 3-6 flowers, peduncle length 1-1.5 cm; pedicel 9-12 mm; calyx lobes 2 x 1 mm; corolla white with yellow midrib, 6-7 x 3 mm; stamen filament 1.5 mm, anthers 3 x1.5 mm; fruit orange, 3-4 x 1.5-2 mm.

Solanum sinaicum Boiss., Diagn. Pl. Orient. 11: 135 (1849)
Common name: Sinai Nightshade

perennial erect herb to small shrub; stem sparsely pubescent, green, angular, woody, branched, up to 60 cm tall; leaves simple, alternate, petiolate, oblong-rhombic to oblong-ovate, sinuate-dentate rarely entire, acute, base cuneate to truncate, 2-6 x 1-3 cm, petiole up to 3 cm, stipules absent; inflorescences unbranched cymes, number of flowers per inflorescence 4-8 mostly 7, peduncle length 1.5-2.5 cm; flowers pentamerous, hermaphrodite; pedicel 8-11 mm; calyx lobes linear-oblong with acute or rounded apex, green, (1-2 x 1-2 mm); corolla white with yellow midrib, petals strongly recurved, 7-10 x 3-5 mm, acute; stamen filament 2 mm, anthers yellow, oblong, 2-3 x1-1.5 mm; ovary globose, 1 mm in diameter, style 5 mm long, stigma is small; fruit berry, globose, orange, 7-9 mm in diameter, fruiting calyx lobes spreading, 4 x 2 mm; seeds 1.5–2.5 mm long; hairs glandular, bicellular uniseriate stalk, with unicellular head, and glandular multicellular uniseriate stalk, unicellular head.

Key to studied species of the genus solanum in egypt

1Fruit berry black in color; calyx lobes triangular with acute or rounded apexS. nigrum

–Fruit berry bright red or orange in color; calyx lobes rounded or linear-oblong2

2Fruit berry bright red in color; calyx lobes rounded; corolla white with black midribS. villosum subsp. miniatum

–Fruit berry orange in color; calyx lobes rounded or linear-oblong with acute or rounded apex; corolla white with yellow midrib3

3Leaves 2-8.5 × 1.5-6.5 cm, broadly to narrowly ovate to elliptic or deltoid; calyx lobes rounded; filament length 1.5 mm longS. villosum subsp. villosum

–Leaves 2-6 × 1-3 cm, oblong-rhombic to oblong-ovate; calyx lobes linear-oblong with acute or rounded apex; filament length 2 mm longS. sinaicum

Morphological correlation between the three Solanum species

Based on the 44 morphological characters that were investigated, the correlation between S. nigrum, S. sinaicum, S. villosum subsp. miniatum, and S. villosum subsp. villosum was constructed (Figure 5, Table 2). A clustering of the heat map identified two groups. S. villosum subsp. miniatum and S. villosum subsp. villosum together constituted the first group. S. nigrum and S. sinaicum were included in the second group. The heat map revealed that S. nigrum and S. sinaicum had the highest correlation (0.429), S. villosum subsp. miniatum and S. villosum subsp. villosum also have a high correlation of 0.415, followed by S. sinaicum and S. villosum subsp. villosum with correlation of 0.314, and there was a negative correlation (-0.127) between S. villosum subsp. miniatum and S. nigrum.

Cytogenetics study

Chromosomes number and Karyotype Formula

karyotype formula (Deanna et al., 2022), determined the fraction of m chromosomes for each of the three Solanum species (S. nigrum, S. sinaicum and two different subspecies of S. villosum) (Figure 6, Table 3) recorded highly variation. Chromosome counts from 10-20 well-scattered metaphase plates of each population of each species revealed interspecific differences in the diploid chromosome number. S. nigrum showed 2n = 72 chromosomes, while S. sinaicum 2n= 24 chromosomes. The two different subspecies of S. villosum subsp. villosum and S. villosum subsp. miniatum showed 2n=24 and 2n= 48 chromosomes respectively. Accordingly, the x number of genome of the three Solanum species has been determined (Table 3). Both S. nigrum (6x) and S. villosum subsp. miniatum (4x) showed polyploidy, while S. sinaicum and S. villosum subsp. villosum showed diploid number (2x). (Figure 6, 7).

Molecular analysis

The PCR amplification products of the four investigated Solanum specimens produced four distinct, high-quality bands during gel electrophoresis. The DNA sequences ITS1-5.8S-ITS2 of 4 Solanum specimens that were the subject of molecular study have been registered on the National Center for Biotechnology Information (NCBI), and the following accession numbers PP701899, PP707086, PP707087, and PP707088 were given to these sequences.

Genetic correlation between the studied Solanum species

The phylogenetic dendrogram based on the ribosomal DNA ITS1-5.8S-ITS2 sequence of the three studied species from Egypt (Figure 8), showed that these sequences generate three clusters. The first cluster included the two subspecies of S. villosum that showed close affinity with low divergence distance of 0.02. The second cluster included S. nigrum that showed high divergence distance from both S. villosum subspecies especially S. villosum subsp. miniatum and this result is consistent with the morphological correlations across these taxa. The third cluster included S. sinaicum which showed moderate genetic distance from S. nigrum and both S. villosum subspecies.

The genetic correlation between S. nigrum and S. villosum worldwide as shown in Figure 9 was indicated based on the constructed heat map using the ribosomal DNA ITS1-5.8S-ITS2 sequence and the genetic distance value between each two pairs, from the first sample to the last sample, is represented by each little square in the IBS distance matrix. The larger values of genetic distance between two specimens are closer to red, and smaller values are closer to blue. The heat map clustering separated two groups. The first group included specimens of S. nigrum, specimens 7 from UK and 11 from India have no genetic divergence, while specimens 9, 10 from India and specimen 13 from UK have very low genetic distance less 0.05, While other specimens of S. nigrum (Specimen 5 from UK, specimen 6 from Indonesia, specimens 6, 8 from India, specimen 1 from Egypt) have genetic distance from 0.05 to 0.2. S. villosum specimens comprised the second group, sp. 2 of S. villosum subsp. miniatum, sp. 3 of S. villosum subsp. villosum, and sp. 4 of S. sinaicum from Egypt comprised one subgroup with very low genetic distance less than 0.05, in addition the remaining specimens of S. villosum (sp. 17, 18, 19, 20 from India, sp. 15 from France, sp. 14 from South Korea, sp. 16 from UK, and sp. 21 from USA) also have low genetic distance less than 0.1.

Discussion

Morphological identification might not be a trustworthy way to identify species in the Solanaceae because their members are cryptic and share phenotypic features that have been altered by genetic and environmental factors (Nderitu et al. 2023). However, Pojarkova (1997) and Yousaf et al. (2010) confirmed that an essential source for the classification of Solanum species was provided by morphological features. According to Knapp et al. 2019, Solanum L. possesses challenges for identification due to the lack of group-specific regional keys. There are 17 identified sections within the Solanum subgenus (Manoko 2007). About 50 global species make up the section Solanum (Child and Lester 2001), the Solanum nigrum complex is another name for the section Solanum. Since Linnaeus first described S. nigrum in 1753, the species in this section have undergone numerous reclassifications up till the present. The ploidy levels of the species, which range from diploid to hexaploid, the genetic variation between populations of the same species, and finally their naturally occurring inter-specific hybridization all contribute to the taxonomic complexity of this section (Edmonds and Chweya 1997).

Morphological studies of freshly collected species, in addition to the herbarium specimens of S. nigrum, S. villosum, and S. sinaicum based on 44 morphological characters (Table 1) showed that flower, fruit, and leaf characters were the most distinctive characters among the studied species. The examined S. villosum populations in Egypt exhibited inter-specific variations in morphology. These morphological differences allowed S. villosum to be divided into two subspecies: subsp. villosum has fruit with orange color, and white corolla with yellow midrib, while subsp. miniatum has fruit with a bright red color and white corolla with black midrib. These results agree with Boulos (2002). The morphological correlation of the three studied species as shown in Figure 5, Table 2 detected that S. nigrum and S. sinaicum had the highest correlation (0.429), followed by S. villosum subsp. miniatum and S. villosum subsp. villosum with a correlation of 0.415.

For each of the three Solanum species, we found the x number of genomes (Table 3). Polyploidy was demonstrated in S. nigrum (6x), This result agrees with Melo et al. (2011), Sultana and Alam (2007), and Särkinen et al. (2018), S. sinaicum and S. villosum subsp. villosum displayed diploid number (2x), however S. villosum subsp. miniatum displayed polyploidy (4x), this agrees with Sultana and Alam 2007 and Särkinen et al. 2018.

It is possible to determine the genetic diversity of individuals or populations by using morphological and molecular markers. Given that environmental factors and the stage of plant development determine morphological traits (El-Domyati et al. 2011). Previous research studies showed that ITS2 provides more accurate results on interspecific variation than intraspecific variation compared to RBCL (Duan et al. 2019). In this investigation, the ribosomal DNA ITS1-5.8S-ITS2 sequences of the studied species were used to examine the interspecific similarities showed the creation of three clusters (Figure 8), the two S. villosum subspecies that exhibited strong affinity and a low divergence distance of 0.02 were included in the first cluster. S. nigrum in the second cluster, had a high divergence distance from both S. villosum subspecies, particularly S. villosum subsp. miniatum. These findings were confirmed with the morphological correlation seen among these taxa Figure (5). S. sinaicum shows a moderate genetic divergence from S. nigrum, and both S. villosum subspecies were separated in the third cluster.

The heat map clustering based on the ribosomal DNA ITS1-5.8S-ITS2 sequence from worldwide specimens separated two groups (Figure 9). The first group included specimens of S. nigrum from Egypt and worldwide, the second group included specimens of S. sinaicum, S. villosum subsp. miniatum, and S. villosum subsp. villosum from Egypt in addition to worldwide specimens of S. villosum.

Acknowledgements

We are grateful to all members of Taxonomy and Flora section in Cairo University Herbarium (CAI) for their assistance and providing all possible facilities.

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