Biosystematic Study of the Egyptian Datura Stramonium (Solanaceae)

Phytotaxa 408 (3): 178–194 ISSN 1179-3155 (print edition) https://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2019 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.408.3.3

Biosystematic study of the Egyptian Datura stramonium (Solanaceae)

RANIA A. HASSAN* & WAFAA M. AMER Botany and Microbiology Department, Faculty of Science, Cairo University, Giza 12613, Egypt *Author for correspondence: Email: [email protected] / [email protected]

Abstract

Datura stramonium (Solanaceae) is an annual weed found in most temperate and subtropical regions of the world. The taxonomic identity of this species is still under debate since Linnaeus first published the name. Early on, many varieties and forms were recognized. In Egypt, D. stramonium presents in two forms: the white-flowered ‘stramonium’ form and violet- flowered ‘tatula’ form. Some authors treated D. stramonium and D. tatula as two distinct species, while others included D. tatula within D. stramonium as either a variety or forma. The present study aimed to elucidate the taxonomic identity of both the white ‘stramonium’ and the violet ‘tatula’ forms based on morphological, palynological and cytogenetic studies extended to karyotyping. A taxonomic study of these forms was carried out using 75 morphological and pollen characters. Significant morphological differences were observed; the most important ones were flower and stem colour, in addition to flower and fruit dimensions. The anatomical examination of juvenile-fruit in acropetal transverse sections, revealed the gradual displacement of parietal placentation at the fruit base to axile at the fruit apex. Pollen of both forms showed no significant differences. Cytogenetic results revealed the presence of a diploid chromosome number (2n=2x=24) in both forms, with minor aneuploidy in the ‘tatula’ form. According to the karyotyping, notable differences were found between the two studied forms, including the centromeric index, total genomic length, and intrachromosomal asymmetry index. Morphological and cytogenetic data revealed that the two forms are different enough to be treated taxonomically as two distinct varieties, namely D. stramonium var. stramonium and D. stramonium var. tatula.

Keywords: cytogenetics, flower colour, phenoplasticity, placentation, taxonomy

Introduction

Datura Linnaeus (1753: 179) (Datureae, Solanoideae, Solanaceae) is a genus of 12 (Dupin et al. 2017) or 14 species (Jiao et al. 2002), comprising of annual or perennial herbs, and rarely shrubs or trees. Datura stramonium is a cosmopolitan weed of cultivated fields, gardens, waste places, barnyards, and other disturbed habitats. It is characterized by its narcotic, hallucinogenic, and medicinal properties, as well as its effects in human poisonings (Mikolich et al. 1975, Hightower 1979, Maldonado & Lott 1991, Dupin & Smith 2018). It has been used in both human and veterinary medicine (Kingsbury 1964), and in folk medicine (Hightower 1979). Tribe Datureae originated in Andean regions and subsequently expanded its range to North America and non- Andean regions. Most Datura species are native to tropical dry forests in Mexico, and arid lands in northern Mexico and southwest United States of America (Symon & Haegi 1991, Shonle & Bergelson 2000, Dupin et al. 2017, Dupin & Smith 2018). Linnaeus (1753) described D. stramonium as American, but Fernald (1950) declared it to be from Asia. The taxonomic identity of D. stramonium is still under debate since Linnaeus (1753). He used the name ‘Tatula’ in the first edition of Species Plantarum as a synonym of D. stramonium. However, in the second edition of Species Plantarum, D. tatula Linnaeus (1762: 256) was separated from D. stramonium, and described as being of larger size, with pale blue flowers, and purple-tinged branches. Some authors maintained this treatment, while others included D. tatula within D. stramonium as a variety or forma (Haegi 1976). Many varieties and forms of D. stramonium were recognized early on (https://www.tropicos.org), namely: D. stramonium var. canescens Wall. in Roxburg (1824: 239); D. stramonium var. tatula (L.) Torrey (1824: 232); D. stramonium var. chalybaea Koch (1837: 510); D. stramonium var. inermis (Juss. ex Jacq.) Lundström (1914: 84); D. stramonium f. inermis (Juss. ex Jacq.) Hupke (1938: 135); D. stramonium var. godronii Danert (1954: 351); D.

178 Accepted by Ronell Klopper: 19 Jun. 2019; published: 1 Jul. 2019 stramonium f. bernhardii (C.E. Lundstr.) Danert in Mansfield (1959: 385); D. stramonium f. tatula (L.) Danert (1959: 385); and D. stramonium f. labilis Hammer (1983: 42). only three variants of D. stramonium were early represented in the Egyptian Flora. Täckholm (1974) reported the white-flowered D. stramonium with two varieties: D. stramonium var. stramonium L., which possessed spiny capsules; and D. stramonium var. inermis Safford (1921: 175) with smooth capsules. She treated the violet-flowered form as a separate species, namely D. tatula with two varieties: D. tatula var. tatula with spiny capsules, and D. tatula var. inermis Timmerman (1927: 574) with smooth capsules. Later, Hepper (1998) and Boulos (2002) treated D. tatula as a synonym of D. stramonium based on capsule shape and leaf indumentum, without reference to the petal and stem colour. Both treatments lack infraspecific details. In spite of the medicinal importance of this species, its taxonomic identity is still uncertain. The use of karyological data in taxonomy, traditionally referred to cytotaxonomy or karyosystematics (Greilhuber & Ehrendorfer 1988), contributes to the evaluation of genetic relationship among species or populations, and helps to understand the way they diverged from each other (Guerra 2008). There is huge genetic diversity within the genus Datura, which Gaire (2008) ascribed to mutation. The most common chromosome number in D. stramonium is 2n=24 (Satina 1959, Fedorov 1969, Tutin et al. 1976, Goldblatt 1981), while Blakeslee (1921) described naturally occurring variants with 2n=12, 25, 26, 36 and 48 chromosomes in the United States. In addition, Spurna et al. (1981) in Czechoslovakia described a population with 2n=21–25. The aim of the present study was to elucidate the taxonomic identity of the two forms of D. stramonium currently present in Egypt, namely—D. stramonium f. tatula (violet-flowered) and D. stramonium f. stramonium (white- flowered). Morphological, palynological and cytogenetic studies (extended to karyotyping) were used to obtain conclusive evidence regarding the taxonomic treatment of these forms as one species with infraspecific taxa, or as different species.

Materials and Methods

The present study was based on examination of D. stramonium specimens kept in the major Egyptian herbaria [Cairo University (CAI), Agricultural Museum (CAIM), National Research Centre (CAIRC)] to define the geographic distribution of this species in Egypt, and to estimate the phenotypic variability of the studied forms. In addition, specimens housed at the herbarium of the Royal Botanic Garden, Kew (K), and other virtual herbaria available on-line [New York Botanical Garden (NYBG) (https://www.nybg.org/), the JSTOR Global Plants database (http://plants.jstor. org), Harvard University Herbaria & Libraries (HUH) (https://huh.harvard.edu/), the herbarium of the Botanic Garden and Botanical Museum Berlin (B) (https://www.bgbm.org/en)] were analyzed. Acronyms follow Thiers (2019). Field studies were conducted between 2016 and 2017 in the Pharmacy Faculty experimental field in Giza, Egypt, where the two forms of D. stramonium (f. tatula and f. stramonium) were cultivated, to exclude environmental variability, for subsequent comparative morphological and genetic analyses. A total of 20 populations for each form were selected. From each population, ten individuals were used in the taxonomic investigation of 75 morphological traits (including stem, leaf, inflorescence, fruit, and pollen characters; see Table 1). Ten fresh samples per form were selected for anatomical examination using a light microscope (AmScope M150C-I 40X-1000X). Voucher specimens of the studied forms were deposited in CAI. Morphology:—The epidermal system of the stem and leaf was examined using a light microscope (AmScope M150C-I 40X-1000X). Anatomical examination of juvenile fruit was performed using nine transverse sections in acropetal succession. Data analysis:—All morphological character states (Table 1) were converted into binary code (0 or 1). Differences between the studied forms were evaluated by a simple matching measure to compare the similarity of binary data using SPSS (version 20.0, 2011). A similarity matrix was obtained from the morphological data analysis of the studied forms (Table 1) using simple matching measure. Sample preparation for scanning electron microscope (SEM):—Fresh anthers were collected from floral buds of the studied D. stramonium forms. Pollen samples were prepared and scanned on a Joel 1200 EX ІІ SEM at 20 kv. Size measurements were obtained from the average of 25 randomly selected grains from 25 individuals per form. Pollen terminology used in this study followed Erdtman (1952) and Punt et al. (1994).

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 179 Chromosome counting:—Actively growing root tips were cut from ten plants from different populations—for each form, pretreated with colchicine (0.25%) for 2 h and fixed in Carnoy solution (3 ethanol:1 acetic acid) for 4 h at room temperature. Thereafter, samples were washed thoroughly with distilled water and macerated with enzymatic mixture (4% cellulose, 1% pectinase, 75 Mm KCl and 7.5 Mm EDTA) on glass slides in a moisture chamber at 37°C for 40 min before staining with 1% Aceto orcein (Lobal Chemie, Mumbai, India) according to the methodology of Fukui & Nakayama (1996). Karyotyping:—For each studied form, analysis of metaphase spreads was performed to produce their karyotypes. Chromosome counting was performed on mitotic metaphase cells under a light microscope (Leica DM 2500, Wetzlar, Germany). For each studied form, ten clearly observable and well-spread metaphases of ten individuals from different populations were selected and photographed, using Image Processing Analysis System Standard and high-resolution automated karyotyping software processing (Leica CW4000). Metaphase chromosomes were arranged in a decreasing order of size and keeping in view the centromere position that constitutes a karyotype. Classification of Levan et al. (1964) was used for each chromosome pair. Mean length of short arm (s) and long arm (l) was measured and the mean total length of each chromosome (c) was calculated (c=s+l). The mean relative length (RL) of each chromosome pair was calculated by (c/sum c) × 100. Mean centromeric index (ci) was calculated by (s/c) × 100 for each chromosome pair to determine the position of the centromere. The chromosome was considered metacentric when the (ci) value ranged between 37.5–50.0, submetacentric when the (ci) value ranged between 25.0–37.5, subtelocentric when the (ci) value ranged between 12.5–25.0, acrocentric when the (ci) value ranged between 0–12.5, and telocentric when this value was zero. Karyotype asymmetry was estimated for the relations between the chromosome arms in each studied form using the Romero Zarco equation (1986) for the intrachromosomal asymmetry (A1) as follows:

A1 = 1-

where n is the number of homologous chromosome pairs; bi is the average length for short arms in every homologous chromosome pair; Bi is the average length for long arms in every homologous chromosome pair. In addition, we used the interchromosomal asymmetry (A2) to estimate the variation in chromosome length using the Romero Zarco (1986) index based on Pearson’s dispersion coefficient as follows:

A2 = where S is the standard deviation; X̅ is the mean of chromosome length for each sample.

Results

Taxonomic treatment

Datura stramonium Linnaeus (1753: 179) Type:—Herb. Clifford 55, Datura I, fol. 1 (lectotype BM), lectotype designated by Hepper (1987: 406).

Herbaceous annual; root thick, shallow. Stems stout, hollow, erect, up to 1 m tall, pseudo-dichotomously branched, puberulent, yellowish green or purplish green. Leaves simple, ovate-elliptic, coarsely-sinuate dentate, apex acuminate, 50–150 × 20–120 mm, petiolate; petioles 5–60 mm long; hairs dense on adaxial surface, sparse abaxially, simple multicellular, eglandular or glandular. Flowers actinomorphic, solitary, axillary in fork of branching stem; pedicel short, 2–10 mm long, densely hairy; calyx 5 or 6, tubular, light green or purplish green, 30–50 × 13– 15 mm, strongly prismatic, unequal teeth 3–8 mm; corolla 5 or 6 , white or violet (Figs. 1–2), tubular infundibular, 50–100 mm long, 5- or 6-lobed, lobes cuspidate 13–20 mm long; stamens 5 or 6, whitish brown or purple (Figs. 1–2), equal, 40–60 mm, inserted near base of corolla; stigma 2-lobed. Capsule ovoid or ovoid–ellipsoid, erect, 30–55 × 25–35 mm, dehisced regularly by 4 valves in basipetal order; false septum at ovary base divides 2 carpels into 4 locules. Fruit pericarp densely covered with unequal spines; spines up to 10 mm, rarely smooth. Seeds dark brown- black, flat, reniform, surface alveolate, 2 –3 × 2.0–2.5 mm.

180 • Phytotaxa 408 (3) © 2019 Magnolia Press hassan & amer FIGURE 1. Morphological features of Datura stramonium var. stramonium. A. Leaves and white flowers with 5 petals. B. Leaves and white flower with 6 petals. C. Flower with 5 stamens. D. Flower with 6 stamens. E. Immature and mature capsules. F. Seed. (photos taken by Rania A. Hassan).

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 181 FIGURE 2. Morphological features of Datura stramonium var. tatula. A. Leaves and purplish flowers. B. Flower with 5 petals. C. Nearly- smooth capsules (red arrows) and spiny capsules (in top of picture). D. Flower with 5 stamens. E. Immature and mature spiny capsules. F. Seed. (photos taken by Rania A. Hassan).

Character ‘stramonium’ form ‘tatula’ form

Stem characters 1. height up to 1 m less than 1 m 2. habit erect erect 3. branching pseudo-dichotomously branched pseudo-dichotomously branched 4. surface smooth abaxially, hairy adaxially smooth abaxially, hairy adaxially 5. colour yellowish green purplish green Leaf characters 6. shape ovate-elliptic elliptic-ovate 7. composition simple simple 8. margin coarsely sinuate-dentate coarsely sinuate-dentate 9. apex acuminate acuminate 10. hairs on adaxial surface dense dense 11. hairs on abaxial surface sparse sparse 12. hair type on leaf blade eglandular & glandular eglandular & glandular 13. base asymmetric asymmetric 14. length up to 150 mm less than 150 mm 15. width up to 120 mm less than 120 mm 16. petiole length ≤ 60 mm ≤ 60 mm 17. petiole indumentum densely hairy densely hairy 18. hair type on petiole eglandular & glandular eglandular & glandular Flower characters 19. insertion in fork of branches in fork of branches 20. number of fl. / infl. solitary solitary 21. pedicel length < 10 mm ≤ 10 mm 22. pedicel indumentum densely hairy densely hairy 23. calyx length ≤ 40 mm ≤ 50 mm 24. calyx width 15 mm ≤ 15 mm 25. calyx indumentum densely hairy densely hairy 26. calyx colour light green purplish green 27. calyx teeth length ≤ 6 mm ≤ 8 mm 28. calyx teeth shape unequal unequal 29. calyx teeth number 5 or 6 5 30. calyx apex acuminate acuminate 31. corolla shape infundibular infundibular 32. corolla length < 100 mm ≤ 100 mm 33. corolla width ≤ 30 mm > 30 mm 34. corolla tube up to 35 mm up to 45 mm 35. corolla funnel ≤ 30 mm > 30 mm 36. corolla lobes length up to 15 mm up to 20 mm 37. corolla lobes apex cuspidate cuspidate 38. corolla lobes number 5 or 6 5 39. corolla colour white violet 40. calyx / corolla ratio < 0.5 ≤ 0.5 ...... continued on the next page

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 183 TABLE 1 (Continued) Character ‘stramonium’ form ‘tatula’ form 41. stamen number 5 or 6 5 42. filament length < 60 mm up to 60 mm 43. filament surface hairy at adnate part hairy at adnate part 44. anther length < 5 mm up to 5 mm 45. anther width < 2 mm up to 2 mm 46. anther colour yellowish brown purple 47. anther indumentum sparsely hairy sparsely hairy 48. ovary shape ovate ovate 49. carpel number 2 2 50. ovary length up to 5 mm < 5 mm 51. ovary width up to 5 mm < 5 mm 52. style length up to 55 mm up to 50 mm 53. stigma length up to 4 mm up to 4 mm 54. stigma width ≤ 2 mm up to 25 mm Capsule characters 55. capsule length ≤ 50 mm < 50 mm 56. capsule width up to 35 mm up to 35 mm 57. capsule L / W ratio 1.0–1.4 1.1–1.3 58. capsule shape ovoid-ellipsoid ovoid-ellipsoid 59. capsule habit erect erect 60. capsule surface spiny spiny, rarely smooth 61. spine length up to 10 mm < 10 mm 62. spine number > 50 / valve ≤ 50 / valve 63. ratio of spines ≤ 10 mm > 50 % < 50 % 64. peduncle length ≤ 10 mm ≤ 10 mm 65. seed length up to 3 mm up to 25 mm 66. seed width up to 25 mm up to 2 mm 67. seed shape reniform reniform 68. mature seeds colour dark brown-black dark brown-black 69. seed ornamentation alveolate alveolate Pollen grain characters 70. polar axis (P) 23.26–34.88 (29.07) μm 26.67–31.40 (29.03) μm 71. equatorial axis (E) 23.26–33.64 (28.45) μm 25.00–27.91 (26.46) μm 72. P/E ratio 1.02 1.1 73. shape prolate-spheroidal prolate-spheroidal 74. colpus length 11.63–15.00 (13.32) μm 11.67–23.26 (17.47) μm 75. colpus width 4.65–7.5 (6.08) μm 3.33–4.65 (3.99) μm

The ‘stramonium’ form:—Stem yellowish green; calyx 5 or 6, light green; corolla 5 or 6, white; stamens 5 or 6, anthers yellowish brown; capsule spiny (Fig. 1). The ‘tatula’ form:—Stem purplish green; calyx 5, purplish green; corolla 5, violet; stamens 5, anthers purple; capsule spiny, rarely smooth (Fig. 2). Sophisticated morphological variations among the two studied forms are outlined in Table 1. Global distribution:—Cosmopolitan, introduced from America and naturalized in warm temperate regions. Distribution in Egypt:—Found as a weed of cultivation along the Nile Delta and Valley; the two studied forms have a sympatric distribution with some overlap. Flowering period:—June–October.

Calyx and receptacle collar:—The 5-lobed calyx forms a clearly prismatic 5-angled tube. After fertilization, the receptacle collar extends just above the fruit base and becomes thicker. Then, the calyx separates transversally at a disjunction ring and falls down leaving the persistent receptacle collar, which hardens forming the fruit collar (Fig. 3).

FIGURE 3. Development of the receptacle collar in Datura stramonium, showing the falling calyx and disjunction ring in mature flowers, and the receptacle collar in the mature fruit.

The transverse section in the calyx, made to examine its epidermal system, showed the presence of a thin-walled epidermal layer with abundant stomata, supported with a thin cuticle layer and covered with eglandular and glandular trichomes. Mesophyll tissue consists of 5–6 layers of chlorenchyma cells with clearly observed intercellular spaces and druses crystals. Examination of the receptacle collar showed the presence of a thin-walled epidermal layer covered with a thick cuticle. Stomata were inconspicuous and the two observed types of trichomes were present. The mesophyll composed of 10–15 layers of compact chlorophyllous cells with few druses crystals. Vascular strands of the receptacle collar expanded to the calyx with the same pattern. Trichomes and stomata:—Glandular and eglandular trichomes were present in both studied forms. Trichomes were abundant on the adaxial leaf surface, but less frequent on the stem and abaxial leaf surface. Eglandular trichomes (Fig. 4) were simple, uniseriate, conical, and consisted of 3–4 cells with thin warty walls. Glandular trichomes extended their presence to the sepals and fruit pericarp. They consisted of a short-stalk of 1–2 cells, with a unicellular or multicellular, ovoid-pyriform head (Fig. 4). Anomocytic stomata were observed on the adaxial and abaxial leaf surfaces of both studied forms.

FIGURE 4. Trichomes observed on both ‘stramonium’ and ‘tatula’ forms. A. Eglandular trichome. B. Glandular trichome with unicellular head. C. Glandular trichome with multicellular head.

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 185 Carpels:—Morphologically, the 2-carpeled ovary of the two studied forms showed four clear longitudinal lines that lack epidermal spines. After fertilization, these lines denote the position of fruit dehiscence, and form regular basipetal dehiscence lines of the 4-valved Datura capsule. Anatomical examination of juvenile fruit, using transverse sections in an acropetal succession, showed four locules at the base (Fig. 5), with four protruding placentae and numerous ovules, while two locules appeared at the fruit apex. Ovary axes (true and false) showed no considerable anatomical differences. Each axis consisted of an epidermal layer from both sides enclosing 4–6 layers of compact rectangular parenchyma cells, followed by rounded parenchyma with narrow intercellular spaces. The vascular system of these axes consisted of a median dorsal well-developed amphicribral (phloem surrounds xylem) vascular bundle and two placental bundles at the four placental positions. Four similar bundles observed in the fruit pericarp corresponded to the four dehiscence lines. Morphological and anatomical data revealed that the D. stramonium fruit pericarp is composed of four units that later developed, forming the four regular valves in the mature fruit.

FIGURE 5. Diagram showing placentation displacement from parietal (capsule base) to axile placentation (capsule apex). A. Transverse sections in a juvenile capsule. B. Anatomical examination of the juvenile capsule in an acropetal succession from 1–9. 1. Parietal. 2–3. st st st nd nd st nd Parietal-axile on 1 axis. 4–6. Axile on 1 axis. 7. Transitional between 1 & 2 axis. 8–9. Axile on 2 axis. (1 axis = false axis; 2 axis = true axis) (Photographs and line drawings by Rania A. Hassan).

Placentation displacement:—At the fruit base (see Fig. 5A–5B, section 1 of nine transverse sections shown), ovules are on four parietal placentae close to the first axis (false septum that divides the two carpels into four locules). These placentae moved towards the ovary center and appeared as parietal–axile in sections 2 and 3. In section 4, placentae appeared nearly axile on the first axis, whereas in sections 5 and 6 they appeared perfectly axile on the same axis. Apically, the placentae were still moving to the center, part of the first axis (false septum) disintegrated and the four placentae fused into three (section 7). Finally, sections 8 and 9 showed the complete disintegration of the false septum and formation of two locules with two axile placentae on the second axis (true). In the mature opened capsule, we observed the disintegration of both axes at the capsule apex, fusion of placentae and arrangement of ovules on the placental column.

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 187 Pollen features:—Pollen grains of the ‘stramonium’ and ‘tatula’ forms were isopolar, radially-symmetrical, tri- zonocolporate, with rugulate–reticulate exine ornamentation (Fig. 6). No significant variation was traced between the two forms. In terms of size, pollen grains of the two studied forms belonged to the medium-sized class with a mean polar axis of 29.03–29.07 µm (Table 1). Equatorial diameter in the ‘stramonium’ form was larger than that of the ‘tatula’ form (28.45 and 26.46 respectively). Pollen grain shape was prolate–spherodial in both forms according to the P/E ratio (1.03–1.14, Table 1). In terms of apertures, both forms have tri-zonocolporate pollen grains. Colpi were wider at the equator and gradually narrowing towards the poles. In the ‘tatula’ form colpi were sunken, while deeply-sunken colpi were found in the ‘stramonium’ form (Fig. 6). Colpi dimensions varied in both forms. The longest (17.47 µm) and narrowest colpus (3.99 µm) was found in the ‘tatula’ form, whereas the shortest (13.32 µm) and widest (6.08 µm) one was found in the ‘stramonium’ form (Table 1). Exine structure in both forms was semitectate, columellate with irregular rugulate-reticulate exine ornamentation (Fig. 6). Similarity index:—In the similarity matrix obtained from the morphological data analysis (Table 1), a similarity percentage of 68 % was estimated among the two studied forms (dissimilarity percentage of 32 %).

FIGURE 6. Scanning electron micrographs of pollen grains of Datura stramonium forms. A. ‘stramonium’ form. B. ‘tatula’ form.

Cytogenetic results

Chromosome number:—Somatic chromosome number 2n=2x=24 was recorded in both studied forms of D. stramonium. Minor aneuploidy was recorded in the ‘tatula’ form by addition of an extra copy to chromosome number 8 and 9 (2n=2x+2=26, Figs. 7–8). Karyotype analysis:—In metaphase spreads, differences were observed in chromosome morphology among the karyotypes of each studied form (Table 2 and Figs. 8–9). According to the centromeric index (ci) of the twelve chromosome pairs, four groups were recorded namely: metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (t). The ‘stramonium’ form followed the diploid karyotypic formula 16m+4sm+2st+2t, while the ‘tatula’ form followed the karyotypic formula 13m+9sm+2st. Chromosome sets of twelve (x=12) were highly variable in their lengths among the two studied forms (Table 2). The first chromosome pair of the ‘tatula’ form was longer than that of the ‘stramonium’ form (with lengths of 8.81 μm and 6.74 μm respectively). Moreover, the last chromosome pair of the ‘tatula’ form was longer than that of the ‘stramonium’ form (3.32 μm and 2.63 μm respectively). Mean relative lengths (RL) of the largest and smallest chromosomes in the ‘stramonium’ form were 13.01 and 5.02 respectively. In the ‘tatula’ form, the RL of the largest chromosome was 12.88 and of the smallest 4.85. Total genomic length in the ‘stramonium’ form was shorter (51.8 μm) than that of the ‘tatula’ form (68.4 μm). According to asymmetry indices, the higher intrachromosomal asymmetry (A1) was recorded in the ‘tatula’ form (0.35), while the interchromosomal asymmetry (A2) was similar (0.02) in both forms (Table 2).

188 • Phytotaxa 408 (3) © 2019 Magnolia Press hassan & amer FIGURE 7. Representative photomicrographs of well-spread mitotic metaphase indicating number of chromosomes in the Datura stramonium forms. A. ‘stramonium’ form (2n = 24). B. ‘tatula’ form (2n = 24 or 26).

FIGURE 8. Karyotypes of Datura stramonium forms showing the presence of diploid chromosome number (2n=24) in ‘stramonium’ (top) and ‘tatula’ (bottom) forms, with minor aneuploidy in chromosomes 8 and 9 in the ‘tatula’ form (2n+2=26).

FIGURE 9. Mean relative length (RL) and centromeric index (ci) percentage of the twelve chromosome pairs in the studied forms (st = ‘stramonium’ form; ta = ‘tatula’ form).

Biosystematic study of Datura stramonium Phytotaxa 408 (3) © 2019 Magnolia Press • 189 TABLE 2. Karyological data on homologous chromosome pairs (1–12) in the complement of Datura stramonium forms (2n=24 in ‘stramonium’ form; 2n=24 and 26 in ‘tatula’ form; s = short arm mean length; l = long arm mean length; RL = mean relative length of each chromosome pair; ci = mean centromeric index; m = metacentric; sm = submetacentric; st = subtelocentria, t = acrocentric; A1 = intrachromosomal asymmetry; A2 = interchromosomal asymmetry). ‘stramonium’ form ‘tatula’ form Chromosome pair s l s+l s l s+l RL ci RL ci (μm) (μm) (μm) (μm) (μm) (μm) 3.35 ± 3.39 ± 6.74 ± 49.7 4.19 ± 4.62 ± 8.81 ± 47.56 1 13.01 12.88 1.84 1.85 3.69 m 0.67 1.04 1.70 m 2.88 ± 3.03 ± 5.91 ± 48.73 3.48 ± 4.21 ± 7.69 ± 45.25 2 11.41 11.24 1.42 1.62 3.03 m 0.59 1.24 1.78 m 2.7 ± 2.76 ± 5.46 ± 49.45 2.76 ± 4.58 ± 7.34 ± 3 10.54 10.73 37.5 m 1.43 1.45 2.87 m 0.47 1.97 1.54 2.39 ± 2.46 ± 4.85 ± 43.15 2.76 ± 3.95 ± 6.71 ± 41.13 4 9.36 9.81 1.17 1.18 2.22 m 0.51 1.17 0.86 m 2.08 ± 2.74 ± 4.82 ± 49.27 2.88 ± 3.37 ± 6.25 ± 46.08 5 9.30 9.14 1.08 1.08 2.16 m 0.45 0.56 0.93 m 1.72 ± 2.44 ± 4.16 ± 2.53 ± 3.05 ± 5.58 ± 45.34 6 8.03 30.53 sm 8.16 0.59 1.22 1.79 0.24 0.60 0.83 m 1.83 ± 2.00 ± 3.83 ± 47.78 2.48 ± 2.83 ± 5.31 ± 7 7.39 7.76 46.7 m 1.00 0.75 1.75 m 0.42 0.40 0.76 1.67 ± 1.95 ± 3.62 ± 46.13 1.69 ± 3.28 ± 4.97 ± 34.00 8 6.99 7.27 0.71 0.97 1.70 m 1.03 0.79 0.77 sm 1.67 ± 1.88 ± 3.55 ± 47.04 1.54 ± 3.00 ± 4.54 ± 33.92 9 6.85 6.64 0.81 0.92 1.72 m 0.31 0.23 0.75 sm 0.39 ± 2.95 ± 3.34 ± 11.68 1.5 ± 2.65 ± 4.15 ± 36.14 10 6.45 6.07 0.35 1.55 1.73 t 0.57 1.10 0.63 sm 0.49 ± 2.41 ± 2.90 ± 16.90 0.87 ± 2.86 3.73 ± 11 5.60 5.45 23.32 st 0.45 1.07 1.32 st 0.23 ±0.59 0.41 0.71 ± 1.92 ± 2.63 ± 0.89 ± 2.43 ± 3.32 ± 26.81 12 5.02 27.00 sm 4.85 0.25 1.07 1.29 0.20 0.73 0.59 sm Total genome length = 51.8 ± 1.26 μm Total genome length = 68.4 ± 1.70 μm

A1 = 0.28 A1 = 0.35

A2 = 0.02 A2 = 0.02

Discussion

Datura stramonium is a cosmopolitan weed showing phenotypic plasticity in terms of flower colour (white or violet) and capsule surface (spiny or smooth). Blumer (1996) reported that variation within a species is very common over a geographic range, and may be a product of genetic differences between the subpopulations at different sites. The taxonomic identity of D. stramonium is still under debate worldwide. Also, Egyptian taxonomists did not determine the actual taxonomic status of this species in Egypt. Of the many recognised varieties and forms of D. stramonium only two forms are currently grown in Egypt: the ‘stramonium’ form with white flowers, and the ‘tatula’ form with violet flowers. Fernald (1950) and Satina & Avery (1959) recognized var. inermis (with smooth capsules) as a third variant of D. stramonium, together with var. tatula and var. stramonium. Our field observations revealed the rare presence of smooth capsules along with spiny ones on the same plant in the ‘tatula’ form (Fig. 2). Also, Safford (1921) and Satina & Avery (1959) reported the presence of smooth capsules in both white and violet-flowered daturas. In addition, Blakeslee (1921) and Weaver & Warwick (1984) stated that the smooth capsule was merely a difference in a single gene subjected to Mendelian segregation. Accordingly, D. stramonium var. inermis does not exist in Egypt. Therefore, this study was carried out to elucidate the taxonomic identity of the only two forms of D. stramonium present in Egypt and their taxonomic relationship.

190 • Phytotaxa 408 (3) © 2019 Magnolia Press hassan & amer The taxonomic assessment was based on 75 macro- and micromorphological characters (Table1). The most important morphological characters making the two forms easily distinguishable were flower and stem colour (Figs. 1–2), in addition to flower and fruit dimensions (Table 1). Morphological analysis of spine length on capsules of both forms revealed the presence of dense and unequal spines in the capsules of both forms (Figs. 1–2). These data agree with that of Avery (1959), who claimed that these features were present in both ‘stramonium’ and ‘tatula’ forms. Our results revealed the presence of anisocytic stomata, eglandular and glandular trichomes in both forms (Fig. 4), thus supporting results by Ibrahim et al. (2016), whereas Dave et al. (1980) claimed that D. stramonium had only multicellular, eglandular trichomes. Although palynological criteria have been used to solve taxonomic problems in various taxa of the family Solanaceae (Bernardello & Lujan 1997, Knapp et al. 1998, Al-Quran 2004), in this study pollen morphological diversity showed no great significance between the two studied forms. Minor differences were traced, such as the deeply-sunken colpi in the ‘stramonium’ form (Fig. 6), in addition to the colpi dimensions that varied among the two forms (Table 1). This study is the first to report the placentation displacement in D. stramonium ovaries from parietal–axile on the first axis (false), to axile on the second axis (true) (Fig. 5A–B). These observations, in addition to the morphological and anatomical features of the four dehiscence lines on the ovary, and the presence of four well-developed amphicribral vascular bundles in the fruit pericarp corresponding to the four dehiscence lines, likely indicate that the 4-loculed ovary in D. stramonium is derived from fusion of four carpels (this assumption needs future study). Similar findings were given by Dave et al. (1980), who reported that D. innoxia Miller (1768: n.5) had four placentae at the ovary base and two placentae at the apex. Additionally, Simpson (2010) claimed that in most Solanaceae, the ovary is synascidiate up to the apex of the placental region and symplicate in the apical part. Cytogenetic investigations carried out on the Egyptian D. stramonium forms revealed the presence of a diploid chromosome number (2n=2x=24) in both forms. Similar results were achieved earlier by Bonicke (1911), who reported that the haploid chromosome number in both D. stramonium and D. tatula is 12. Other studies, such as Satina (1959), Fedorov (1969), Tutin et al. (1976) and Goldblatt (1981), also reported the same results. Chromosomes of the ‘tatula’ form showed presence of minor aneuploidy in chromosome number 8 and 9 (2n=2x+2=26, Fig. 8). Similar data were reported earlier by Blakeslee (1921) in the United States. According to the karyotyping data (Table 2, Fig. 8), differences were observed in chromosome morphology of the two studied forms. No notable differences were found among the studied forms regarding their relative chromosome lengths (Table 2, Fig. 9). However, differences in centromeric index (ci) led to different karyotypic formulas recorded for the two forms (‘stramonium’ form = 16m+4sm+2st+2t; ‘tatula’ form = 13m+9sm+2st (Table 2, Fig. 7). Total genomic length in the ‘stramonium’ form was shorter (51.8 μm) than in the ‘tatula’ form (68.4 μm, Table 2). Intrachromosomal asymmetry index (A1) varied between the two forms with a higher value in the ‘tatula’ form (0.35), which had more asymmetric chromosomes. Interchromosomal asymmetry index (A2) was very low (0.02) and similar in both forms. The latter index reflects a conservation among chromosome size in the karyotype, whereas both indices reflect variation between the two studied taxa.

Conclusion

Our morphological and palynological data revealed that the two studied forms of Datura stramonium in Egypt are closely related taxa with a significant similarity (68 %), but show some conspicuous differences (dissimilarity 32%). According to the definitions of infraspecific taxa of Stuessy (1990), we prefer to treat these entities at the level of variety of the same species as follows: Datura stramonium var. stramonium Datura stramonium var. tatula (L.) Torrey (1824: 232) Basionym:—Datura tatula Linnaeus (1762: 256) Homotypic synonym:—Datura stramonium f. tatula (L.) Danert (1959: 385) Type:—Herb. Linn. No. 243.2 (lectotype, LINN), lectotype designated by Hadkins et al. (1997: 305). our assumption is supported by their sympatric distribution, similar ploidy level, and different karyotypes. In this study, the two Egyptian forms are growing as mixed populations in the same locality, and accordingly, the environmental factors show no influence on their phenoplasticity. Further studies are required to elucidate the origin of the D. stramonium gynoecium, referring to the misperception of morphological and anatomical features.

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