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