8 KAWAKAMI ET AL

Chromosome Botany (2019) 13 (2): 61-67 ©Copyright 2019 by the International Society of Chromosome Botany

A Karyotype analysis in revoluta (Hyacinthaceae) with a new Cytotype

Priyanka T. Giranje1, Mayur D. Nandikar2* and Shrirang R. Yadav3

1,2 Naoroji Godrej Centre for Research, 431, Lawkim Campus, Shindewadi, Shirwal, Dist. Satara, , 412 801. 3Professor and BSR Faculty Fellow, Department of Botany, Shivaji University, Kolhapur, Maharashtra, India 416 004.

*Author for correspondence ([email protected]) Received November 21, 2018; accepted December 21, 2018

ABSTRACT: Ledebouria Roth is a complex and it shows a wide range of chromosome counts. Present paper reports chromosome number 2n (6X) = 90 in L. revoluta (L.f.) Jessop a number that is not known so far in the entire genus Ledebouria which exhibits highly asymmetrical (2C) karyotype. The karyotype symmetry of the chromosomes and extreme level of polyploidy indicates that the L. revoluta is actively evolving in varied climatic and geographical regions.

KEYWORDS: Karyotype, Ledebouria, Hyacinthaceae, new cytotype, India

The genus Ledebouria Roth (Hyacinthaceae) consists of Rahangdale 2016) were collected from the hill slopes of ca. 61 species and 2 infraspecific species in the world Junnar, district, Maharashtra (Fig. 1A). The (WCSP 2018). Although, Ledebouria was described as for the cytological study were grown in Naoroji Godrej early as 1821 by Roth, it has been treated as a synonym of Centre for Plant Research (NGCPR) Botanical garden, in most Indian literature (Giranje and Nandikar Shirwal, Satara, Maharashtra. The voucher specimens 2016). The genus has very few qualitative characters of (M.D. Nandikar 1403, M.D. Nandikar 1418 and P.T. diagnostic value in determination of the species. Although, Giranje 1434) have been deposited at NGCPR. the characters such as blotches on the leaf, size, shape and orientation of the leaves (erect, sub–erect or prostrate), Sampling of karyotype: The were kept for rooting. inflorescence size, number, etc have been used for Water grown healthy root tips (0.5 cm long) were pre– identification of the species, however, these characters are treated with a saturated solution of para–dichlorobenzene varies greatly with reference to their climatic and (pDB) at 10 ±2°C for 3–4 hrs. Root tips were hydrolyzed geographical conditions. in 1 N HCL and then stained and squashed with 2 % In India the genus is represented by Ledebouria aceto–orcein. revoluta Roth and other three ill–defined species viz. L. The well separated somatic plates were photographed viridis S. Dutta and P. Harvey ex M. R. Almeida, L. by OPTSCOPES IS–300 camera under LEICA DM 500 karnatakensis Punekar and Lakshmin., and L. microscope at 100x magnification. Five well separated hyderabadensis M. V. Ramana, Prasanna and Venu. somatic chromosome plates were selected for karyotype Ledebouria revoluta is wide spread from Africa to analysis. Chromosomes were sorted into different peninsular India and shows great diversity both in categories on the basis of type of chromosome and arm morphological and chromosome number in different ratio by following Levan et al. (1964). Evaluation of habitats. Leaf tip bulbils is a predominant mode of Karyotype symmetry has been determined by using vegetative reproduction in majority of the populations categories of Stebbins (1971). which is subjected to cytological variations (Jessop 1973; Dixit et al. 1989; Jehan et al. 2014). The chromosome RESULTS counts reported so far in Ledebouria revoluta shows 10 The karyotype analysis of Ledebouria revoluta has been cytotypes (2n = 28, 30, 44, 45, 46, 58, 60, 64, 68, and 70: given in Table 2. Different karyotypic parameters such as Table 1) indicating active cytological differentiation. The a total chromosome length of haploid compliment (THCL), present report (2n= 90) confirms hexa–ploidy (6X) nature total chromosome length percent (TCL %), total from of populations, which is first time recorded not only in percent (TF %), symmetric index (SI), Gradient Index Ledebouria revoluta but also in entire genus Ledebouria. (GI) Coefficient of variation indices (CVcl and CVci) (Paszko 2006), and asymmetry indices (Ai, A1, A2) MATERIALS AND METHODS (Zarco 1986) were examined for L. revoluta, and are Plant material: Plant material of Ledebouria revoluta tabulated in Table 3. (= described as L. junnarensis by Rahangdale and 62 GIRANJE ET AL.

Table 1. A detailed account of previous chromosome counts in Ledebouria revoluta (L.f.) Jessop Reported as taxa n (2n) Studied by Country/Provision Ledebouria revoluta (L. f.) Jessop 15 30 Haque and Ghosh (2015) India (West Bengal, Kolkata, Rahara) Ledebouria revoluta (L.f.) Jessop 64, 68 Sheeba and Vijayavalli (1998) (=Scilla indica (Wight) Baker) Ledebouria revoluta (L.f.) Jessop 22 Johnson and Brandham (1997) South Africa (=Scilla revoluta (L.) Baker) Ledebouria revoluta (L.f.) Jessop 28 Stedje (1996) East Africa, Kenya, Machakos, between Koma Rocks Ledebouria revoluta (L.f.) Jessop 30 Dixit et al. (1989) India (Maharashtra, Mumbai, Kanheri caves) (=Scilla hyacinthina (Roth) J. F. Macbr) 45 India (Maharashtra, Satara, Kas plateau) 60 India (Maharashtra, Pune, Dive ghat) Ledebouria revoluta (L.f.) Jessop 30 Jha and Sen (1980), India (Maharashtra, Satara, Mahabaleshwar) (=Scilla hyacinthina (Roth) J. F. Macbr.) 60 Nair (1989) India (Maharashtra, Mumbai) Ledebouria revoluta (L.f.) Jessop 45 Sheriff and Rao (1981) India (Tamil Nadu, Chennai, Adyar & Saidapet) (=Scilla indica Roxb.) Ledebouria revoluta (L.f.) Jessop 20,22, Subramanian (1981) (=Scilla indica Baker ) 44,45, 46,58 Ledebouria revoluta (L.f.) Jessop 10 Sarkar et al. (1980) India (Maharashtra, Uklapani) (=Scilla hyacinthina (Roth) J. F. Macbr.) Ledebouria revoluta (L.f.) Jessop 30 Sen (1973), Western Coastal belts of India (=Scilla indica (Wight) Baker ) Chakravarty and Sen (1992) Ledebouria revoluta (L.f.) Jessop 9 Jessop (1973) South Africa (Rhodes University garden; Henderson's 10 Farm; 34 miles west of Potgietersrust; 11 5 miles west of Colenso; Kroomie, Fort Beaufort; 2 miles 12 north-west of Grahamstown; Willowmore; 13 SWAZILAND;34 miles west of Potgietersrust). 15 16 17 22

A KARYOTYPE ANALYSIS IN LEDEBOURIA REVOLUTA (HYACINTHACEAE) WITH A NEW CYTOTYPE 63

Table 1. Continued Reported as taxa n (2n) Studied by Country/Provision Ledebouria revoluta (L.f.) Jessop 15 Jessop (1970) South Africa (Eastern Cape,Grahamstown 20 miles N. of Pretoria Rhodes Botany Dept. Garden) Ledebouria revoluta (L.f.) Jessop 30 Rao (1956) India (Maharashtra, Mumbai; Karnataka, Dharwad, (=Scilla indica (Wight) Baker ) 44 Mysore; Tamil Nadu, Tiruchirapalli, Chennai; 45 Andhra Pradesh, Krishna, Machilipatnam; Madhya 46 Pradesh, Sagar; Maharashtra, Nagpur) 58 60 Ledebouria revoluta (L.f.) Jessop 44, 45, Darlington and Wylie (1955) (= Scilla indica (Wight) Baker ) 46, 58 Ledebouria revoluta (L.f.) Jessop (=Scilla 30, 44, Rao (1953) indica (Wight) Baker ) 45, 46, 58 Ledebouria revoluta (L.f.) Jessop 30 Sheriff and Murthy (1946) India (Karnataka, Kolar hills) (=Scilla indica (Wight) Baker ) Ledebouria revoluta (L.f.) Jessop 44 Raghavan and Venkatasubban (1939) India (Tamil Nadu, Chennai, Adyar) (=Scilla indica Roxb.) 46 India (Tamil Nadu, Chennai, Saidapet) 45 India (Tamil Nadu, Chennai, Soundarya Nursery)

64 GIRANJE ET AL.

Fig. 1: Ledebouria revoluta A. habitat, B. habit, C. chromosome count (2n =90), D. idiogram.

A KARYOTYPE ANALYSIS IN LEDEBOURIA REVOLUTA (HYACINTHACEAE) WITH A NEW CYTOTYPE 65

Table 2. Karyotype details of Ledebouria revoluta Chromosome Length of Length of Total length d=l-s Arm ratio Centromeric Centromeric pairs long arm (l) short arm (s) c=l+s (µm) r=l/s index Position (µm) (µm) i=s/c*100 1 4.30 ±1.08 2.03 ±0.48 6.33±1.53 2.27 2.12 32.05 sm 2 3.96±0.87 1.83±0.50 5.79±1.32 2.13 2.16 31.63 sm 3 3.38±0.91 1.67±0.60 5.05±1.47 1.71 2.02 33.08 sm 4 2.61±0.61 1.55±0.47 4.16±1.05 1.06 1.68 37.28 sm 5 2.46±0.41 1.28±0.36 3.74±0.76 1.18 1.92 34.21 sm 6 1.91±0.56 1.19±0.06 3.10±0.58 0.72 1.60 38.46 m 7 1.53±0.43 1.20±0.17 2.73±0.60 0.33 1.27 44.03 m 8 1.45±0.31 0.99±0.14 2.44±0.38 0.46 1.47 40.51 m 9 1.42±0.25 0.96±0.11 2.38±0.34 0.45 1.47 40.47 m 10 1.32±0.21 0.95±0.14 2.27±0.32 0.37 1.38 41.95 m 11 1.22±0.22 0.95±0.07 2.17±0.28 0.28 1.29 43.62 m 12 1.12±0.14 0.96±0.14 2.07±0.27 0.16 1.16 46.22 m 13 1.26±0.18 0.81±0.13 2.07±0.24 0.46 1.57 38.93 m 14 1.18±0.18 0.77±0.16 1.96±0.26 0.41 1.53 39.49 m 15 1.11±0.20 0.80±0.12 1.91±0.25 0.31 1.39 41.90 m 16 1.22±0.24 0.65±0.20 1.87±0.22 0.57 1.87 34.87 sm 17 1.00±0.10 0.81±0.15 1.81±0.22 0.19 1.23 44.82 m 18 1.05±0.14 0.71±0.10 1.76±0.21 0.34 1.48 40.32 m 19 0.98±0.17 0.72±0.05 1.70±0.20 0.26 1.36 42.34 m 20 0.99±0.13 0.66±0.14 1.65±0.21 0.32 1.48 40.24 m 21 1.07±0.09 0.55±0.24 1.63±0.18 0.52 1.95 33.94 sm 22 0.96±0.16 0.66±0.07 1.62±0.18 0.30 1.46 40.71 m 23 1.06±0.06 0.60±0.19 1.66±0.18 0.46 1.76 36.17 sm 24 0.94±0.09 0.63±0.08 1.57±0.16 0.30 1.48 40.32 sm 25 0.91±0.08 0.63±0.21 1.54±0.15 0.29 1.46 40.73 sm 26 1.03±0.14 0.51±0.14 1.54±0.15 0.52 2.00 33.31 sm 27 0.98±0.18 0.55±0.15 1.52±0.16 0.43 1.79 35.82 sm 28 0.94±0.22 0.55±0.17 1.49±0.18 0.39 1.70 37.01 sm 29 0.89±0.08 0.60±0.20 1.49±0.18 0.29 1.48 40.39 m 30 0.82±0.07 0.63±0.11 1.45±0.17 0.19 1.31 43.35 m 31 1.08±0.18 0.37±0.25 1.44±0.16 0.71 2.92 25.51 sm 32 0.93±0.14 0.49±0.29 1.42±0.18 0.44 1.90 34.49 sm 33 0.90±0.15 0.44±0.25 1.35±0.30 0.46 2.04 32.84 sm 34 0.95±0.20 0.42±0.29 1.37±0.19 0.52 2.24 30.84 sm 35 0.98±0.26 0.36±0.23 1.34±0.18 0.63 2.74 26.72 sm 36 0.87±0.16 0.44±0.19 1.30±0.18 0.43 1.98 33.51 sm 37 0.99±0.21 0.30±0.33 1.29±0.17 0.68 3.25 23.54 st 38 0.89±0.20 0.38±0.25 1.27±0.15 0.52 2.37 29.66 sm 39 0.84±0.15 0.40±0.19 1.24±0.15 0.44 2.11 32.12 sm 40 0.81±0.21 0.38±0.14 1.19±0.15 0.42 2.10 32.21 sm 41 0.82±0.21 0.33±0.21 1.15±0.13 0.50 2.52 28.43 sm 42 0.95±0.23 0.14±0.21 1.08±0.14 0.81 6.88 12.69 st 43 0.74±0.17 0.20±0.12 0.94±0.10 0.54 3.69 21.30 st 44 0.90±0.22 0.07±0.16 0.97±0.10 0.83 12.87 20.21 st 45 0.82±0.08 0.04±0.08 0.86±0.11 0.78 21.91 14.37 st

The karyotype analysis of Ledebouria revoluta can be µm. Based on the centromeric position (Table 2) somatic summarized as follows: haploid chromosome number chromosome complement can be broadly classified into 3 n=15; hexaploid chromosome number 2n (6x) =90 (Fig. distinct chromosome groups: 1) 17 pairs of median 1C); total haploid chromosome length (THCL) = 90.72 chromosome, 2) 23 pairs of sub–median chromosome, 3) 66 GIRANJE ET AL.

Table 3. Different karyotypic parameters of Ledebouria revoluta Parameters Result THCL 90.72 TF% 35.47 SI 54.95 GI 13.56 CVcl 60.03 CVci 26.73 AI 16.05 A1 0.45 A2 0.60 Karyotype formula (K) 17m ± 23sm ± 5st Classification as per Stebbins (1971) 2C

5 pairs of sub–terminal chromosome. The karyotype the species is highly polyomorphic with three cytotypes formula can be expressed as 17m ± 23sm ± 5st and (2n=30, 45 and 60: Dixit et al. 1989), the present reports idiogram is given in Figure 1D. The karyotype is adds one more cytotype (2n=90). These records also moderately asymmetrical and falls into "2C" category of evidenced by Dixit et al. (1989) that adaptation of the taxa Stebbins (1971). The chromosome length ranged from to the different eco–climatic regions: diploids (2n=30) are 6.33 to 0.86 µm. The intra–chromosomal difference in usually found in plains and reproduce sexually, triploids long arm and short arm (d value) ranged from 0.16 to 2.27. (2n=45) and present hexaploids (2n=90) are so far known The arm ratio (r value) ranged from 1.16 to 21.91. The from higher altitudes in the Western Ghats adapted to high Centromere index (I value) ranged from 4.37 to 46.22. rainfall zone and usually reproduce vegetatively by The somatic chromosome count 2n = 90 with total haploid formation of bulbils at leaf tip. In addition to these, some chromatin length =90.72 µm, symmetric index (SI) =54.95, of the triploids also shares the habitat with tetraploids in and TF% is 35.47 shows highly asymmetrical gradient drier parts of peninsular India. However, all above index (GI) =13.56. cytotypes when grown under humid conditions, have tendency to produce bulbils at leaf tip. DISCUSSION Polyploidy played an important role in evolution of The superfluous and illegitimate name Ledebouria angiosperm. Cytological data provides important junnarensis Rahangdale and Rahangdale (2016) refers to evidences to evaluate the impact of genetic divergence L. revoluta (Giranje and Nandikar 2016) and there among species and population, and it has been estimated comparison with Ledebouria viridis is erroneous. that more than 15% flowering plants have impacted by Ledebouria viridis (Blatt. and Hallb.) S. Dutta and P. polyploidy (Guerra 2008; Wood et al. 2009). In many Harvey ex M. R. Almeida (2009) was described as a instances polyploidy causes changes in shape and texture distinct species based on its hysteranthous habit, less than of plant organs, like the leaves and petals are usually 40 flowered racemes and pinkish–purple to green thicker and firmer, lowering of fertility and seed perianth, whereas L. junnarensis is synanthous and production, which helps in the process of stabilization and typically agrees with L. revoluta. We studied the cytology establishment of new habitats (Stebbins 1971). The of L. junnarensis from its type locality which revealed present studied population of L. revoluta shows diploid chromosome number 2n= 90, which does not morphological variations such as linear to linear– match with the number (i.e. 2n= 70) previously reported lanceolate, faintly blotched leaves with fleshy, bulbils at by Rahangdale and Rahangdale (2016). The present report apices, axillary, solitary flowering scape with 25–30 confirms hexa–ploid (6X) nature of populations, which flowers, 10–15 mm long pedicels, and mauve perianth being first time recorded in the genus Ledebouria. (Fig. 1B) as compared to the other adjoin populations of L. In asymmetrical karyotype centromere position shifts revoluta. Our study indicates that there is much towards the median to subterminal, or their relative size evolutionary advancements in L. revoluta is possibly has too differences to make karyotype more favoured due to the polyploidy and highly asymmetrical heterogeneous (Stebbins 1971). The previous studies on karyotype. Therefore, such populations should be morphology and cytology in L. revoluta have revealed that A KARYOTYPE ANALYSIS IN LEDEBOURIA REVOLUTA (HYACINTHACEAE) WITH A NEW CYTOTYPE 67 considered as one of the races instead of a new taxon, as it Nair, A. S. 1989. Micropropagation of Scilla hyacinthia (Roth) merely trying to establish or adapt to the new habitat. Macbride. Proc. Indian Natl. Sci. Acad., B 55(2): 121– 124. Paszko, B. 2006. A critical review and a new proposal of ACKNOWLEDGEMENT Authors are thankful to Mr. Vijay karyotype asymmetry indices. Plant Syst. Evol. 258: 39–48. Crishna, Director Naoroji Godrej Centre for Plant Research for Raghavan, T. S. and Venkatasubban, K. 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