© 2012 The Japan Mendel Society Cytologia 77(1): 113–119 Comparative Karyotype Analysis of Delphinium malabaricum var. malabaricum (Huth) Munz. and Delphinium malabaricum var. ghaticum Billore. Firdose R. Kolar*, Kumar Vinod Chhotupuri Gosavi, Arun Nivrutti Chandore, Shrirang Ramchandra Yadav and Ghansham Balbhima Dixit Department of Botany, Shivaji University, Kolhapur-416004 (M.S.), India Received August 25, 2011; accepted February 1, 2012 Summary Delphinium malabaricum (Huth) Munz. and its variety D. malabaricum var. ghaticum Billore (Ranunculaceae) were cytologically examined. Both taxa were found to possess 2n=16 dip- loid chromosome number. The 8 pairs of homologous chromosomes were characterized into 1 meta- centric, 1 submetacentric and 6 subtelocentric chromosomes. The total length of diploid comple- ments and range of chromosome length were more or less the same in the species and its variety. The length of metaphase chromosomes ranged from 1.42 to 5.35 μm in D. malabaricum var. malabaricum and from 1.77 to 5.30 μm in D. malabaricum var. ghaticum with an average length of 2.88 μm and 2.92 μm, respectively. The total chromosome length (TCL) of the former was 23.05 μm and that of the latter was 23.38 μm. The prevalence of chromosomes with subtelocentric centromeres indicated a tendency towards asymmetric karyotype, which revealed an advanced feature. Hence both the taxa belong to Stebbinsʼs 2B type karyotypic symmetry class. The r esults revealed that D. malabaricum var. malabaricum and D. malabaricum var. ghaticum have a similar karyotype pattern, despite differing morphologically. Thus, it may be concluded that the external morphological varia- tion occurred independently of the chromosomal variation, which may have played a prominent role in the intraspecific differentiation of D. malabaricum. Key words Delphinium malabaricum, Ranunculaceae, Endemic, Karyotype symmetry, Western Ghats, India. The genus Delphinium is one of the most important genera of the family Ranunculaceae, which represents a group of very beautiful annuals, rarely biennials and perennial plants, com- monly called Larkspurs. The diversity of colors and shape of their flowers bestows this genus a very interesting ornamental potential. The genus has about 370 species distributed throughout the north temperate regions of the world (Blanché 1991). According to Rau (1993) Delphinium L. is represented in India by 24 species, almost all confined to Himalayan regions except D. malabaricum, which is an endemic perennial herb restricted to only a few locations in Western Ghats of India and is considered as a rare species (Billore 1973). This is the only species of the genus growing naturally in Western India. It is represented by its 1 species and 1 variety D. malabaricum var. malabaricum (Figs. 1a, 1c) and D. malabaricum var. ghaticum (Figs. 1b, 1d). D. malabaricum var. malabaricum is stated vulnerable and D. malabaricum var. ghaticum as critically endangered due to its small fragmented population and distribution (Mishra and Singh 2001). The existence of previous chromosome counts for the studied species of the genus Delphinium has been checked in the indices of plant chromosome numbers (Mehra and Ramanandan 1972, Langlet 1927, Lewitzky 1931, Lawrence 1936, Gregory 1941, Lewis et al. 1951, Al-Kelidar and Richards 1981, Love 1981, 1984, and Subramanian 1985) and revealed the presence of diploid * Corresponding author, e-mail: fi[email protected] 114 F. Kolar et al. Cytologia 77(1) Fig. 1. Delphinium malabaricum var. malabaricum (Huth) Munz.: a: Infloresence, c: Side view of flower showing spur, e: Meocyte showing haploid chromosome number n=8, g: Somatic chromosome number 2n=16, i: Karyogram and Delphinium malabaricum var. ghaticum Billore.: b: Infloresence, d: Side view of flower showing spur, f: Meocyte showing haploid chromosome number n=8, h: Somatic chromosome number, 2n=16, j: Karyogram. (2n=16), triploid (2n=24), tetraploid (2n=32) and hexaploid (2n=48) levels for the genus (Table 1). The basic chromosome number of the genus is reported to be x=8 (Darlington and Janaki-Ammal 1945). Cytological data is of great importance for systematic and evolutionary studies of plants and have been widely used to elucidate intraspecific and/or interspecific relationships and delimit spe- cific or generic circumscriptions (Stebbins 1971, Raven 1975, Hong 1990). D. malabaricum var. malabaricum has been previously investigated cytologically in which 2n=16 chromosome number was reported (Pai et al. 2007). However, D. malabaricum var. ghaticum has not been explored cy- tologically. The present study is intended to re-examine the chromosome numbers and karyomor- 2012 Karyotype Comparison in 2 Varieties of Delphinium malabaricum (Huth) Munz. 115 Table 1. Previous reports of somatic chromosome number in Delphinium species Chromosome number Taxa Author and year n 2n D. ajacis L. 8 16 Langlet 1927, Gregory 1941 D. andersoni Gray 8 16 Lewis et al. 1951 D. altissimum Wallich. 8 16 Mehra and Kaur 1963 D. belladonna (hort) 16, 24 24, 48 Langlet 1927, Subramanian 1985 D. brunonianum Royle. 8, 16 16, 32 Lewitzky 1931, Al-Kelidar and Richards 1981 D. californicum T. & G. 8 16 Lewis et al. 1951 D. candelabrum Ostenf. var. monanthum 8 16 Yang and Wu 1993 D. cardeopetallum DC. 8 16 Lewitzky 1931 D. cardinale Hook. 8 16 Mehlquist et al. 1943 D. carolinianum Walt 16 32 Gregory 1941 D. cashmerianum Royle. 8, 16 16, 32 Love 1981, Al-Kelidar and Richards 1981 D. decorum Fisch. & Mey. ssp. tracyi Ewan 8 16 Lewis et al. 1951 D. denudatum Wallich. 8, 10, 16 16, 20, 32 Me hra and Ramanandan 1972, Sarkar et al. 1982, Al-Kelidar and Richards 1981 D. elatum L. 16 32 Lawrence 1936 D. formosum Boiss. & Huet 16 32 Gregory 1941 D. glaucum Wats. 8 16 Lewis et al. 1951 D. grandiflorum L. 8 16 Propach 1940 D. gypsophilum Ewan 8, 16 16, 32 Lewis et al. 1951 D. glaucum Wats. 8 16 Lewis et al. 1951 D. gracilentum Greene 8 16 Lewis et al. 1951 D. hanseni Greene 8, 16 16, 32 Lewis et al. 1951 D. hesperium Gray 8 16 Lewis et al. 1951 D. hesperium var. cuyamacae (Abrams) Jeps. 8 16 Le wis et al. 1951 D. malabaricum var. malabaricum (Huth) Munz. 8 16 Pai et al. 2007, present communication. D. malabaricum var. ghaticum Billore 8 16 present communication. D. nudicaule T. & G. 8 16 Gregory 1941 D. scabriflorum D. 8 16 Love 1984 D. variegatum T. & G. 8, 16 16, 32 Lewis et al. 1951 D. vestitum Wallich. 8 16 Mehra and Kaur 1963 phology of D. malabaricum var. malabaricum and compare it with the respective karyomorphology of D. malabaricum var. ghaticum, seeking chromosomal characters potentially important for under- standing the taxonomy of these varieties. Materials and methods In order to study the karyotypic characteristics, seeds of D. malabaricum var. malabaricum (Huth) Munz and D. malabaricum var. ghaticum Billore were collected from the localities Ajinkyatara in the Satara district and Harhargadh in the Nasik district of Maharashtra, respectively. The seeds were germinated on sterile moist filter paper in petri dishes at 25°C. The root tips were excised and pretreated with aqueous saturated solution of para-dichlorobenzene (pDB) for 4 h at 8–10°C. Then they were hydrolyzed with 1N HCl at 60°C and stained with 2% aceto-orcein. Slides were observed using a Carl Zeiss microscope and were randomly photographed (Nikon Coolpix 4500 digital camera) to obtain 10 appropriate metaphase plates. The plates were used for the analy- sis of chromosome complements. The lengths of long and short arms were measured and these measurements were used for further calculations of arm ratio, centromeric index and percentage of relative chromosomal length (RL%). The chromosomes were classified according to their arm ra- tios into metacentric, submetacentric and subtelocentric (Levan et al. 1964). For the estimation of 116 F. Kolar et al. Cytologia 77(1) karyotype asymmetry, 3 numerical parameters, namely intrachromosomal asymmetry index (A1), interchromosomal asymmetry index (A2) and asymmetry index (AI), were used (Romero Zarco, 1986 and Paszko, 2006). To analyze the variability of the karyotypes among these 2 taxa, the asym- metry index, mean RL% of haploid chromosomes and mean arm ratio were compared by analysis of variance (ANOVA) using Graphpad instat software. Chromosome pairing was also studied in pollen mother cells (PMCs) of D. malabaricum. Anthers were fixed in Carnoyʼs fluid, and after hydrolysis were stained with 2% aceto-orcein. The slides were examined under a Carl Zeiss microscope and Chromosome pairing was determined from cells at diakinesis or metaphase I. Results The present study demonstrated that D. malabaricum var. malabaricum and D. malabaricum var. ghaticum were diploids 2n=16 chromosome complement (Fig. 1g, 1h). The individual chromo- some length ranged from 1.42 μm to 5.35 μm in D. malabaricum var. malabaricum and from Table 2. Karyological features of Delphinium malabaricum var. malabaricum Chromosome Long arm Short arm Total length ʻdʼ value ʻrʼ value ʻiʼ value Relative Centromeric Type pair L (μm) S (μm) C=L+S (μm) L–S (μm) L/S (μm) S/CX100 length (%) position I 3.17∓0.21 2.18∓0.39 5.35∓0.57 0.99 1.45 40.75 23.24 m A II 2.93∓0.34 1.58∓0.21 4.52∓0.36 1.34 1.85 35.08 19.61 sm B III 2.26∓0.07 0.43∓0.93 2.69∓0.07 1.82 5.18 16.17 11.70 st C IV 1.98∓0.07 0.47∓0.07 2.46∓0.13 1.50 4.16 19.35 10.67 st C V 1.90∓1.57 0.39∓0.12 2.30∓0.15 1.50 4.80 17.24 09.98 st C VI 1.82∓0.07 0.39∓0.07 2.22∓0.13 1.42 4.60 17.85 09.63 st C VII 1.82∓0.07 0.23∓1.96 2.06∓0.23 1.58 7.66 11.53 08.94 st D VIII 1.19∓0.00 0.23∓1.43 1.42∓0.15 0.95 5.00 16.66 06.19 st E Trait TCL A1 A2 AI TF% L/S 23.05 0.69 0.46 20.96 25.82 3.76 TCL: Total haploid chromosome length, A1: intrachromosomal asymmetry index, A2: interchromosomal asymmetry index, AI: Asymmetry index, TF%: Total form percent, L/S: Longest chromosome/Shortest chromosome.