© 2020 The Japan Mendel Society Cytologia 85(2): 163–168

Karyological Investigation on Three 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 Zephyranthes belongs to , 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 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 cies possessed 14 metacentric and 10 submetacentric possessed ovoid , 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 , 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 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 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. 0.8×1.0 cm, subglobose, loculicid- globose, 16–25 seeded ally 3-valved, yellowish-green, 16–20 seeded Seeds Angular Angular Oblong, angled

Figs. 6–8. Orcein-stained mitotic metaphase chromosomes and haploid idiograms of three Zephyranthes species. (6a) Meta- phase and (6b) idiogram of Z. candida; (7a) metaphase and (7b) idiogram of Z. carinata; (8a) metaphase and (8b) idio- gram of Z. tubispatha. Bars=10 µm. 166 C. K. Dash et al. Cytologia 85(2)

Table 2. Comparative karyomorphological features of three Zephyranthes species.

Features Z. candida Z. carinata Z. tubispatha

2n 38 24 48 Satellite ̶ ̶ 2 KF 32m+6sm 14m+10sm 16m+32sm TCL (µm) 457.01±7.04 268.76±4.71 821.09±9.97 RCL (µm) 7.94±0.47–17.71±1.25 8.40±0.78–16.22±1.02 10.83±0.65–28.79±2.07 RL % 1.74–3.88 3.12–6.03 1.32–3.51 ACL (µm) 12.03 11.20 17.11 AsK % 55.36 59.00 61.61 TF % 44.64 41.00 38.39 Syi % 80.64 69.49 62.32 Rec index 67.93 69.06 59.43

A1 0.18 0.28 0.37 A2 0.24 0.20 0.24 A 0.11 0.18 0.23 AI 2.82 2.63 3.31 Stebbins’s classification 2B 2A 2B

The RL of the individual chromosomes was ranging observed for this species which correlates with reports from 1.32–3.51%. The individual chromosome length of Sveshnikova and Zemskova (1988). ranged from 10.83±0.65 to 28.79±2.07 µm in which the Previously, several authors (Raina and Khoshoo 1971, length of the largest chromosome was about three times Felix et al. 2011b) have concluded that the basic chro- to that of the smallest one. As K%, TF%, Syi%, and mosome number of the genus Zephyranthes is x=6 and Rec index were found to be 61.61%, 38.39%, 62.32%, it is also claimed that this basic chromosome number and 59.43%, respectively (Table 2). The A1, A2, A and maintained for the entire tribe Zephyrantheae. Besides, AI values were 0.37, 0.24, 0.23 and 3.31, respectively, there were other basic numbers suggested for this genus is included in the 2B category of Stebbins (1971). In Z. viz. x=5, 7, 9, 11, 13, and 15 (Naranjo 1969, Daviña and tubispatha, a pair of satellites (2.93 µm) was found at the Fernández 1989), however, those numbers were found to short arms of chromosome pair 21 (Table S3). exist in very few cases. Felix et al. (2011b) reported Z. robusta and Z. sylvatica as diploid, Z. rosea, Z. brachy- Discussion andra and Z. grandiflora (=Z. carinata) as tetraploid considering x=6. In this study, 2n=4x=24 in Z. carinata Somatic and basic chromosome number (tetraploid) and 2n=8x=48 in Z. tubispatha (octaploid) Zephyranthes is one of the evolutionarily dynamic represents the base number x=6 that supports the pre- genera of Amaryllidaceae which displays and retains an vious findings. However, 2n=38 in Z. candida could array of chromosome numbers. In this study, three Zeph- occur as hexaploid (2n=6x=36) followed by secondary yranthes species were found to possess different chro- modification (hyper-aneuploidy), similarly reported by mosome number such as 2n=38 in Z. candida, 2n=24 in Felix et al. (2011b). Because of the most frequent occur- Z. carinata and 2n=48 in Z. tubispatha (Figs. 6–8, Table rence, it is to be expected that variation in base number 2). Several researchers reported 2n=38 chromosomes might be originated from the normal ancestor having in Z. candida earlier (Kumar and Subramaniam 1987, x=6. Based on this base number and available data from Zhu et al. 1991, Devi and Borua 1996, Ma et al. 1997) this experiment and previous studies, it can be suggested which correlates to that of present observation. Different that a polyploid series is available for this studied genus, chromosome number for this species were also available from 2n=2x=12 and extended to 2n=16x=96 (Raina and such as 2n=19, 24, 28, 32, 36, 42, 49 and 50 (Kumar and Khoshoo 1971, Felix et al. 2011a, b). Some aneuploid Subramaniam 1987, Zhu et al. 1991, Devi and Borua reports were also available for different species of this 1996). The chromosome numbers of 2n=38, 42, 46 and genus (Raina and Khoshoo 1971, Felix et al. 2011b). In- 48 were reported for Z. carinata (Singh and Roy 1973, traspecific polymorphisms regarding chromosome num- Mehra and Sachdeva 1976, Kumar and Subramaniam ber for this genus indicated that it could be changed due 1987). Felix et al. (2011a) reported 2n=24 in Z. grandi- to existing polyploid series of cytotypes and possible flora Lindl. (synonym of Z. carinata). The present study secondary modifications among them which ultimately only correlates with the reports of Felix et al. (2011a). Z. showed multiple basic numbers. tubispatha are less studied so far and reported to have 2n=24 and 48 chromosomes (Fedorov 1974, Lakshmi Karyotype analysis 1980, Joshi and Ranjekar 1982, Kumar and Subrama- Three Zephyranthes species employed in the present niam 1987, Sveshnikova and Zemskova 1988). In this in- investigation showed karyotypes formed by metacentric vestigation, 2n=48 chromosomes for Z. tubispatha were to submetacentric chromosomes with gradual reduc- 2020 Cytotaxonomy of Zephyranthes 167 tion in chromosome size. However, these species had number of seeds per fruit is another important character distinct KF viz. 32m+6sm in Z. candida, 14m+10sm in where species with relatively a smaller number of seeds Z. carinata and 16m+32sm in Z. tubispatha. However, per fruit show primitiveness over other species that have all the investigated individuals under each species had a higher number of seeds per fruit (Sambamurty 2005). same karyotype and did not show any significant varia- Z. carinata produces six to ten seeds per fruit which is tion. Previously, Felix et al. (2011a, b) reported 2n=38 lower than those of Z. candida (16–25 seeded) and Z. chromosomes in Z. candida with KF 18m+20sm and tubispatha (16–20 seeded). The above facts suggest that 18 m+10sm+10a (a=acrocentric) from Campina Grande, Z. carinata is more primitive than Z. candida and Z. PB, , and 2n=24 chromosomes in Z. grandi- tubispatha. This hypothesis also supports the compara- flora (=Z. carinata) from São Paulo, SP, Brazil with KF tive karyotype asymmetry i.e., Z. carinata have low- 4m+10sm+10a. The chromosome number 2n=24 in Z. est AI value (2.63) relating to Z. candida (2.82) and Z. tubispatha was reported by Lakshmi (1980) with KF tubispatha (3.31). 6m+10sm+8st (st=subtelocentric) from . If the KF is taken into consideration, the present findings are not Acknowledgement in agreement with the previous reports. The variation in KF of these species indicates the modification or evolu- This research is dedicated by the authors to Late tion of karyotype as a distinct lineage due to having dif- Professor Dr. Sheikh Shamimul Alam for his eminent ferent geographical and environmental factors. contribution in Cytogenetics Laboratory, University of The average chromosome length was found signifi- Dhaka, Bangladesh. cantly higher in Z. tubispatha (17.11 µm) and the remain- ing two species had almost similar average chromosome References length i.e., 12.03 µm in Z. candida and 11.20 µm in Z. carinata. The variation in TF%, Syi%, As K%, Rec Afroz, S., Rahman, M. O. and Hassan, M. A. 2018. and index, A , A , AI and A had a perfect or almost posi- reproductive biology of the genus Zephyranthes Herb. (Liliaceae) 1 2 in Bangladesh. Bangladesh J. Plant Taxon. 25: 57–69. tive or negative correlation with each other. As K% had Arano, H. 1963. Cytological studies in subfamily Carduoideae (Com- a perfect negative correlation with two indices: TF% positae) of Japan. IX. The karyotype analysis and phylogenetic and Syi% (Table 2). The AI was developed recently to considerations on Pertya and Ainsliaea. Bot. Mag. Tokyo 76: generate a single value that evaluated karyotype asym- 32–39. metry where higher AI was considered as higher levels Bhattacharyya, N. K. 1972. Chromosome inconstancy in Zephyran- thes mesochloa Baker. Cytologia 37: 423–433. of karyotypic heterogeneity (Paszko 2006). Among the Chowdhury, M. R. and Hubstenberger, J. 2006. Evaluation of cross three studied species, Z. tubispatha showed higher AI pollination of Zephyranthes and species and hybrids. values (3.31) which indicated that this species possessed J. Arkansas Acad. Sci. 60: 113–118. more heterogenous karyotype than others, and Z. cari- Daviña, J. R. 2001. Estudios citogeneticos en algunos generos argen- nata had relatively symmetric karyotype, which was tinos de Amaryllidaceae. Ph. D. Thesis, National University of reflected with AI and A values. This observation also Cordoba, Cordoba. 2 Daviña, J. R. and Fernández, A. 1989. Karyotype and meiotic be- suggested that Z. tubispatha was advanced from the evo- havior in Zephyranthes (Amaryllidaceae) from . lutionary point of view (Stebbins 1971). Cytologia 54: 269–274. Several taxonomic characters have been analyzed to Devi, T. T. and Borua, P. K. 1996. Karyotype analysis of three species separate three Zephyranthes species from one another of Zephyranthes (Amaryllidaceae). J. Cytol. Genet. 31: 15–22. including morphology of bulbs, leaves, spathe, flower, Fedorov, A. N. A. 1974. Chromosome Numbers of Flowering . Otto Koettz Science Publishers, Königstein. perianth, , filament, anther, style, stigma, pla- Felix, W. J. P., Almeida, A., Melo, N. F. and Felix, L. P. 2007. Cito- centa, fruit and seeds (Figs. 1–5, Table 1). However, genética de duas espécies de Zephyranthes Herb. (Amaryllida- based on these taxonomical characters these three spe- ceae–Hipeastreae) cultivadas. Rev. Bras. Bioci. 5: 294–296. cies were found to be closely allied, but they showed Felix, W. J. P., Dutilh, J. H. A., Melo, N. F., Almeida, A. and Felix, complete divergence in cytogenetical view. Floral mor- L. P. 2008. Intrapopulational chromosome number variation in Zephyranthes sylvatica Baker (Amaryllidaceae: ) phology, for examples, number and size of floral parts from northeast Brazil. Rev. Bras. Bot. Braz. J. Bot. 32: 371–375. can be employed for constructing evolutionary relation- Felix, W. J. P., Felix, L. P., Melo, N. F., Dutilh, J. H. A. and Carvalho, ship among closely related taxa (Moyroud and Glover R. 2011a. Cytogenetics of Amaryllidaceae species: Heterochro- 2017). The species with many numbers of floral parts matin evolution in different ploidy levels. Plant Syst. Evol. 292: can be considered are primitive in nature as compared 215–221. Felix, W. J. P., Felix, L. P., Melo, N. F., Oliveira, M. B. M., Dutilh, J. to the species with relatively few numbers of floral parts H. A. and Carvalho, R. 2011b. Karyotype variability in species (Stebbins 1974, Sambamurty 2005). Among the studied of the genus Zephyranthes Herb. (Amaryllidaceae–Hippeast- species, Z. carinata displayed infrequent occurrence of reae). Plant Syst. Evol. 294: 263–271. up to eight perianth and stamens (generally six) in com- Flory, W. S. and Smith, G. L. 1980. High chromosome number in sev- parison with Z. candida and Z. tubispatha, those have eral Zephyrantheae taxa. Plant Life Stanford Conn. 36: 63–72. strictly six perianth and stamens. On the other hand, a Greilhuber, J. and Speta, F. 1976. C-banded karyotypes in the Scilla 168 C. K. Dash et al. Cytologia 85(2)

hohenackeri group, S. persica and Puschkinia (Liliaceae). Plant asymmetry indices. Plant Syst. Evol. 258: 39–48. Syst. Evol. 126: 149–188. Raina, S. N. and Khoshoo, T. N. 1971. Cytogenetics of tropical bul- Greizerstein, E. J. and Naranjo, C. A. 1987. Estudios cromosomicos bous ornamentals VI: Chromosomal polymorphism in cultivated en especies de Zephyranthes (Amaryllidaceae). Darwinian 29: Zephyranthes. Caryologia 24: 217–227. 169–186. Sambamurty, A. V. S. S. 2005. Taxonomy of Angiosperms, I. K. Inter- Huziwara, Y. 1962. Karyotype analysis in some genera of Compositae national Pvt Ltd. New Delhi, India. VIII. Further studies on the chromosome of Aster. Am. J. Bot. Siddiqui, K. U., Islam, M. A., Ahmed, Z. U., Begum, Z. N. T., Hassan, 49: 116–119. M. A., Khondker, M., Rahman, M. M., Kabir, S. M. H., Ahmad, Joshi, C. P. and Ranjekar, P. K. 1982. Visualization and distribution A. T. A., Rahman, A. K. A. and Haque, E. U. 2007. Encyclope- of heterochromatin in interphase nuclei of several plant species dia of Flora and Fauna of Bangladesh. Vol. 11, Angiosperms: as revealed by a new Giemsa banding technique. Cytologia 47: (Agavaceae–Najadaceae). Asiatic Society of 471–480. Bangladesh, Dhaka. Katoch, D. and Singh, B. 2015. Phytochemistry and pharmacology of Singh, V. K. and Roy, S. K. 1973. Somatic chromosomes of Zephyran- genus Zephyranthes. Med. Aromat. Plants 4: 1000212. thes. Herb. Rev. Biol. 9: 141–145. Kumar, V. and Subramaniam, B. 1987. Chromosome Atlas of - Spurrier, M. A., Smith, G. L., Flagg, R. O. and Serna, A. E. 2015. A ing Plants of the : Dicotyledons. Botanical new species of Zephyranthes (Amaryllidaceae) from . Survey of India, Kolkata. Novon 24: 289–295. Lakshmi, N. 1980. Cytotaxonomical studies in eight genera of Ama- Stebbins, G. L. 1971. Chromosomal Evolution in Higher Plants. Uni- ryllidaceae. Cytologia 45: 663–673. versity Park Press, Baltimore. Levan, A., Fredga, K. and Sandberg, A. A. 1964. Nomenclature for Stebbins, G. L. 1974. Plant Species: Evolution above the Species Lev- centromeric position on chromosomes. Hereditas 52: 201–220. el. The Belknap Press of Harvard University Press, Cambridge. Lorenzi, H. and Souza, H. M. 1999. Plantas Ornamentais no Brasil: Sveshnikova, L. I. and Zemskova, E. A. 1988. Chromosome numbers Arbustivas, Herbáceas e Trepadeiras. Instituto Plantarum, São in some members of the Amaryllidaceae. Bot. Žhurn. Engelmann Paulo. Online 73: 1207–1208. Ma, Y., Cai, K. X. and Cui, M. Z. 1997. Karyotype analysis of Zephy- Utech, F. H. 2002. Flora of North America: North of Mexico, Vol. 26. ranthes candida. Acta Agric. Boreal. Sin. 12: 77–80. Flora of North America Editorial Committee. Oxford University Mehra, P. N. and Sachdeva, S. K. 1976. Cytological observations on Press, New York. some west Himalayan monocots. IV. Cytologia 41: 31–53. Watanabe, K., Yahara, T., Denda, T. and Kosuge, K. 1999. Chromo- Moyroud, E. and Glover, B. J. 2017. The evolution of diverse floral somal evolution in the genus Brachyscome (, As- morphologies. Curr. Biol. 27: R941–R951. tereae): Statistical tests regarding correlation between changes in Naranjo, C. A. 1969. Cariotipos de nueve especies de Rho- karyotype and habit using phylogenetic information. J. Plant Res. dophiala, , Zephyranthes y Habranthus (Amaryl- 112: 145–161. lidaceae). Kurtziana 5: 67–87. Zarco, C. R. 1986. A new method for estimating karyotype asymme- Oliveira, R. S. 2006. Flora da Cadeia do Espinhaco: Zephyranthes try. Taxon 35: 526–530. Herb. & Habranthus Herb. (Amaryllidaceae). Ph. D. Thesis, In- Zhu, J., Xie, Z. W. and Wu, K. F. 1991. Isozymes and karyotype anal- stituto de Biociências, São Paulo. ysis of Zephyranthes candida (Lindl.). Herb. J. Shanghai Agric. Paszko, B. 2006. A critical review and a new proposal of karyotype Coll. 9: 290–296.