© 2020 The Japan Mendel Society Cytologia 85(2): 163–168 Karyological Investigation on Three Zephyranthes Species and Its Taxonomic Significance Chandan Kumar Dash, Md. Oliur Rahman and Syeda Sharmeen Sultana* Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh Received February 2, 2020; accepted February 21, 2020 Summary Three species of Zephyranthes Herb. viz. Z. candida (Lindl.) Herb., Z. carinata Herb. and Z. tubis- patha Herb. were cytotaxonomically studied to characterize and elucidate probable evolutionary relationship among them. These species were found to possess different chromosome number and karyotype formula such as 2n=38=32m+6sm in Z. candida, 2n=24=14m+10sm in Z. carinata and 2n=48=16m+32sm in Z. tubispatha. A pair of satellites was observed only in Z. tubispatha. Despite three Zephyratnes species showed close relationship with reference to morphological characters, however, they displayed notable differences in the karyological study. The present cytogenetical and taxonomical findings indicated that Z. tubispatha was relatively advanced and Z. carinata was primitive from the evolutionary point of view. Keywords Asymmetric index, Cytotaxonomy, Karyotype, Zephyranthes. The genus Zephyranthes belongs to Amaryllidaceae, also reported in Z. carinata where the somatic chromo- comprises about 70 species with neotropical distribution some number was found to be 2n=38, 42, 46 and 48 (Chowdhury and Hubstenberger 2006, Spurrier et al. (Singh and Roy 1973, Mehra and Sachdeva 1976, Kumar 2015). In Bangladesh, four species of Zephyranthes viz. and Subramaniam 1987). Z. tubispatha is less studied so Z. atamasco (L.) Herb., Z. candida (Lindl.) Herb., Z. far and reported to have 2n=24 chromosomes (Lakshmi carinata Herb. and Z. tubispatha Herb. are available 1980, Joshi and Ranjekar 1982). No previous cytogeneti- (Afroz et al. 2018). This genus has several medicinal cal records regarding their karyotype are known for Z. uses and reported to have anticancer, antifungal and an- tubispatha. tibacterial activities (Katoch and Singh 2015). Besides, In this research, three Zephyranthes species, viz. Z. many of them are popular as ornamentals and cultivated candida, Z. carinata, and Z. tubispatha were analyzed widely (Lorenzi and Souza 1999). cytogenetically aiming to characterize these species Taxonomically, Zephyranthes is a very multifarious with karyomorphological data and comparing the chro- genus, with poorly distinct and closely morphological mosomal data of previous information to describe a nu- margins among species. The existing chromosome data- merical and structural modification of karyotype. base of about 50 species of Zephyranthes represented it as evolutionarily vibrant genus with variability in basic Materials and methods and somatic chromosome numbers and ranging from 2n=10 in Z. seubertii (Daviña 2001) to 2n=200 in a Three Zephyranthes species, namely Z. candida, horticultural hybrid (Flory and Smith 1980, Greizerstein Z. carinata and Z. tubispatha were collected from the and Naranjo 1987, Oliveira 2006). Reports on variations compound of University of Dhaka as well as different in chromosome number and the reports of B chromo- nurseries of Dhaka city and maintained in the Botanic somes in Z. mesochloa, Z. brasiliensis, Z. grandiflora, Z. Garden, Department of Botany, University of Dhaka, chlorozolen, Z. candida, and Z. sylvatica indicated the Bangladesh. The plant specimens were critically studied chromosomal and karyological diversity of this genus morphologically and identified with special reference (Raina and Khoshoo 1971, Bhattacharyya 1972, Greiz- to taxonomic values (Utech 2002, Siddiqui et al. 2007, erstein and Naranjo 1987, Felix et al. 2007, 2008). Afroz et al. 2018). Healthy roots of at least 50 individu- Z. candida is a species with variable chromosome als were collected and pre-treated with 2 mM 8-hydroxy- numbers of 2n=19, 24, 28, 32, 36, 38, 42, 49 and 50 quinoline for 3 h 30 min at 18°C followed by fixation in (Kumar and Subramaniam 1987, Zhu et al. 1991, Devi Carnoy’s fixative (ethanol : acetic acid=3 : 1) and pre- and Borua 1996). Chromosome number variability was served at 4°C. Roots were then hydrolyzed in a mixture of 1M HCl and 45% acetic acid (2 : 1) at 65°C for 4 min. The root tips were stained and squashed in 1% aceto- * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.85.163 orcein for 2 h. Then these slides were observed under a With Supplement files of Tables S1–S3 microscope (Nikon eclipse 100). To get an accurate mea- 164 C. K. Dash et al. Cytologia 85(2) surement of lengths, chromosomes from five metaphase carinata and Z. tubispatha were presented in Figs. 6–8 plates were measured for each case. The idiograms were and Table 2. In Z. candida, 2n=38 chromosomes were made based on chromosome size in decreasing order. A observed (Fig. 6, Tables 2, S1). The total length of the procedure proposed by Levan et al. (1964) was followed chromosome complements (TCL) was 457.01±7.04 µm, for determining the centromeric type of chromosomes. average chromosome length (ACL) was found to be Various karyomorphological parameters including sym- 12.03 µm, and the relative length (RL) of chromosome metry and asymmetry indices were calculated with such ranged from 1.74–3.88%. The karyotype formula (KF) as the total form percent (TF%) (Huziwara 1962), karyo- of this species was 32m+6sm. The longest chromo- type asymmetry index (As K%) (Arano 1963), the index some length (17.71±1.25 µm) was more than double of karyotype symmetry (Syi%) (Greilhuber and Speta than the shortest chromosome (7.94±0.47 µm). The As 1976), the index of chromosomal size resemblance (Rec K%, TF%, Syi% and Rec index were 55.36%, 44.64%, index) (Greilhuber and Speta 1976), Intrachromosomal 80.64% and 67.93%, respectively. The other karyomor- and interchromosomal asymmetry index (Zarco 1986), phological parameters such as A1, A2, A, and AI values degree of asymmetry of karyotypes (A) (Watanabe et al. were 0.18, 0.24, 0.11 and 2.82, respectively. No satellite 1999), the asymmetry index (AI) (Paszko 2006) and was observed. The karyotype was quite symmetric and Stebbins’s classification (Stebbins 1971). corresponded to the 2B category of Stebbins (1971). Z. carinata was found to possess 2n=24 chromo- Results somes (Fig. 7, Table S2). TCL was 268.76±4.71 µm which was the shortest among three species. This spe- The present study revealed that Zephyranthes candida cies possessed 14 metacentric and 10 submetacentric possessed ovoid bulb, white flowers, ovate to lanceolate chromosomes. The range of individual chromosomal perianth, oblong anthers, slender style, yellowish-green length was from 8.40±0.78 to 16.22±1.02 µm and RL fruits, and angular seeds, and the ovary was covered ranged from 3.12–6.03%. The length of longest chromo- by a spathe. Z. carinata was characterized by having some was almost double to that of the shortest one. With a tunicated bulb, rose to pink flowers, sub-elliptic to 17.51 µm average chromosome length; 59.00% As K%, oblong-lanceolate perianth, narrowly linear anthers, fili- 41.00% TF%, 69.49% Syi% and 69.06% Rec index were form style, dark green fruits, and angular seeds, and the also calculated for this species. The A1, A2, A and AI ovary was not encircled by a spathe. Presence of tuni- values were 0.28, 0.20, 0.18 and 2.63, respectively (Table cated bulbs, linear leaves, yellow flowers, funnel-shaped 2). Z. carinata was included in the 2A category of Steb- perianth, linear anthers, filiform styles, yellowish-green bins (1971). fruits, and oblong seeds were the key characters as ob- Somatic cells with 2n=48 and karyotype with 16 served in Z. tubispatha. The ovary was not enclosed by metacentric and 32 submetacentric chromosomes were spathe in Z. tubispatha (Figs. 1–5, Table 1). observed in Z. tubispatha (Fig. 8, Table S3). The species Results of karyological investigation on Z. candida, Z. presented 821.09±9.97 µm TCL with 17.11 µm ACL. Figs. 1–5. Morphology of three Zephyranthes species. (1) Plant specimens, (2) flowers, (3) L. S. of flowers, (4) anthers and (5) stigmas; a=Z. candida, b=Z. carinata and c=Z. tubispatha. 2020 Cytotaxonomy of Zephyranthes 165 Table 1. A comparative account of three Zephyranthes species based on morphological characters. Characters Z. candida Z. carinata Z. tubispatha Bulb Tunicated, ovoid, c. 2.5 cm in diameter Tunicated, up to 2 cm in diameter Tunicated, c. 1.0 cm in diameter Leaves Simple, terete, linear, up to Simple, flat, linear, up to 35.0×0.8 cm, Simple, flat, linear, c. 30×0.3 mm, 35.0×0.5 cm, hollow, obtuse obtuse, appearing with flowers obtuse, appearing with flowers Spathe Covers the ovary Does not cover the ovary Does not cover the ovary Flowers White Rose or pink Yellow Perianth Segments 6, ovate-lanceolate, free, Segments 6, rarely up to 8, sub-elliptic Segments 6, funnel-shaped, c. 3.7 cm c. 3.7 cm long to oblong–lanceolate, 2–4 cm long long Stamens 6, free, about half as long as the 6, sometimes 7–8, adnate to the throat 6, adnate to the throat of the perianth perianth of the perianth Filament Yellowish-white, c. 1.0 cm long White, up to 2 cm long Yellowish-white, c. 1.4 cm long Anthers Oblong, dorsifixed, c. 0.9 cm long, Linear, narrow, dorsifixed, c. 0.8 cm Linear, dorsifixed, c. 0.7 cm long, yellow long, yellow orange Style Slender, c. 1.7 cm long Filiform, c. 3 cm long Filiform, c. 2 cm long Stigma 3–lobed Deeply 3–4 lobed 3–lobed Placentation Axile Axile Axile Fruit c. 0.8×1.2 cm, yellowish-green, sub- c. 0.5×0.5 cm, dark green, 6–10 seeded c.