Comparative Karyotype Analysis of Delphinium Malabaricum Var

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 diploid chromosome number. The 8 pairs of homologous chromosomes were characterized into 1 metacentric, 1 submetacentric and 6 subtelocentric chromosomes. The total length of diploid complements 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 results 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 variation 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 cytologically. 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 analysis 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 ratios 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 asymmetry 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 chromosome 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.

Table 3. Karyological features of Delphinium malabaricum var. ghaticum

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 3.13∓0.14 2.16∓0.19 5.30∓0.11 0.97 1.45 40.80 22.68 m A II 2.66∓0.11 1.54∓0.13 4.21∓0.18 1.12 1.72 36.67 18.00 sm B III 2.11∓0.19 0.55∓0.10 2.67∓0.12 1.55 3.78 20.90 11.44 st C IV 2.11∓0.01 0.53∓0.45 2.65∓0.01 1.58 3.94 20.22 11.35 st C V 1.96∓0.03 0.50∓0.11 2.46∓0.06 1.46 3.92 20.30 10.53 st C VI 1.85∓0.11 0.40∓0.31 2.25∓0.24 1.44 4.54 18.03 09.66 st C VII 1.66∓0.25 0.37∓0.24 2.03∓0.14 1.29 4.50 18.18 08.71 st D VIII 1.40∓0.17 0.37∓0.11 1.77∓0.19 1.03 3.80 20.83 07.60 st E

Trait TCL A1 A2 AI TF% L/S

23.38 0.65 0.41 15.01 27.59 2.99

Table 4. Types of chromosomes in D. malabaricum var. malabaricum and var. ghaticum.

Types of chromosomes Depiction

Type ʻAʼ (Chromosome I): A pair of longest chromosomes (D. malabaricum var. malabaricum: 5.35 μm, D. malabaricum var. ghaticum: 5.30 μm) with median centromere (m). Type ʻBʼ (Chromosome II): A pair of long chromosomes (D. malabaricum var. malabaricum: 4.52 μm, D. malabaricum var. ghaticum: 4.21 μm) with sub-median centromere (sm). Ty pe ʻCʼ (Chromosomes III, IV, V, VI): 4 pairs of short chromosomes (D. malabaricum var. malabaricum: 2.69–2.46– 2.30–2.22 μm, D. malabaricum var. ghaticum: 2.67–2.65–2.46–2.25 μm) with sub-terminal centromere (st). Type ʻDʼ (Chromosome VII): A pair of short chromosomes (D. malabaricum var. malabaricum: 2.06 μm, D. malabaricum var. ghaticum 2.03 μm) with sub-terminal centromere (st). Type ʻEʼ (Chromosome VIII): A pair of shortest chromosomes (D. malabaricum var. malabaricum: 1.42 μm, D. malabaricum var. ghaticum 1.77 μm) with sub-terminal centromere (st).

1.77 μm to 5.30 μm in D. malabaricum var.ghaticum. Arm ratios were between 1.45 and 7.66 in D. malabaricum var. malabaricum and between 1.45 and 4.54 in D. malabaricum var. ghaticum with an average length of 2.88 and 2.92 μm, respectively. The total chromosomal length of the haploid set of former was 23.05 μm and that of later was 23.38 μm. The average RL% of the longest haploid chromosomes in D. malabaricum var. malabaricum and D. malabaricum var. ghaticum was 23.24 and 22.68 and the average RL% of the shortest haploid chromosome was 6.19 and 7.60, respectively (Tables 2, 3). The chromosome complements of both the varieties possess one pair of chromosomes with median-region centromere and one pair with submedian-region centromeres. The remaining 6 pairs have subterminal-region centromeres (Table 4). The karyotypes of these varieties were formulated as K: 2n: 16: 2Am+2Bsm+8Cst+2Dst+2Est. With regard to karyotype asymmetry, the karyotype was classiﬁed into category 2B (Stebbins 1971). The analysis of the karyotype asymmetry indices showed values 0.69, 0.46 and 20.96 in D. malabaricum var. malabaricum and 0.65, 0.41 and 15.01 in D. malabaricum var. ghaticum for A1, A2 and AI, respectively which con- ﬁrmed that the karyotype was asymmetric. The meiotic observations of microspore mother cells revealed that both the taxa of D. malabaricum viz. var. malabaricum and D. malabaricum var. ghaticum form 8 bivalents at diakinesis (Figs. 1e, 1f).

Discussion

The diploid chromosome number 2n=16 has been observed in most of the Delphinium species such as D. ajacis (Langlet 1927), D.cardinale (Mehlquist et al. 1943), D. grandiflorum (Propach 1940), D. altissimum (Mehra and Kaur 1963), D. nudicaule (Gregory 1941), D. zalil (Gage 1953), and D. malabaricum var. malabaricum (Pai et al. 2007). In the genus Delphinium diploid species are by far the most numerous, tetraploids are occasional and hexaploids are known only in cultivated forms (Gage 1953). The karyotype analysis of D. malabaricum var. malabaricum (Fig. 1g) and D. malabaricum var. ghaticum (Fig. 1h) showed the presence of diploid chromosome number 2n=16 like the most other species of the genus indicating that in respect of centromeric position they are symmetrical (Figs. 1i, 1j). The total length of 2n chromosome complement and range of individual chromosome length were more or less the same in these taxa (Tables 2, 3). Based on the morphological features of the chromosomes, all 8 chromosomes can be grouped into 2 long chromosomes (1 with an almost median, the other with a submedian centromere); 4 of intermediate length (with subtelocentric centromeres) and 2 of a relatively short length (with subtelocentric centromeres). The genome of the various Delphinium species are morphologically very similar, a fact 118 F. Kolar et al. Cytologia 77(1) that has also been observed by several other investigators (Hocquette 1922, Tjebbes 1927, Lawrence 1936, Propach 1939, Gregory 1941, Mehlquist et al. 1943 and Lewis et al. 1947 and 1951). The karyotype in the studied taxa mostly comprised of metacentric, submetacentric and subtelocentric chromosomes as indicated by their mean arm ratio. The predominance of chromosomes with centromeres in a telomeric position indicated that the intrachromosomal symmetry in D. malabaricum and its variety were very low and the karyotype of both taxa were asymmetric. Karyotype evolution is one of the most important aspects of the whole evolutionary processes (Imai et al. 1986) and considered as an isolating mechanism in speciation and has its own evolutionary trends independent of genetic evolution (Imai et al. 2001). Stebbins (1971) pointed out that the pattern of karyotype evolution is generally from symmetry to asymmetry in higher plants. Accordingly the karyotype of Delphinium malabaricum var. malabaricum and D. malabaricum var. ghaticum is considered an advanced karyotype. Stebbins (1971) explained the mechanism governing this karyotype asymmetry as the result of pericentric inversions and unequal translocations of fragments of chromosome arms, which occur without any change in the number of centromeres or of independent chromosomes. Karyotypes are one of the parameters by which authentic identification of a specimen is possi- ble, since varieties usually possess the same chromosome numbers and major chromosomal differences do not exist among different varieties of a species. The karyotype of D. malabaricum var. ghaticum when compared with the karyotype of D. malabaricum var. malabaricum was found to be similar. In addition to similarity in the karyotype, RL% and arm ratio values of each chromosome pair were not significantly different (p＜0.05) between the taxa, confirming that they belong to the same species and have a close relationship. Although on the basis of morphological criteria, D. malabaricum var. ghaticum can be distinguished from D. malabaricum var. malabaricum, at the chromosomal level both remain undistinguished. These facts indicate that external morphological variations in this species occurred independently of the chromosome variations. Thus, it is assumed that there is no or little reproductive isolation between D. malabaricum var. malabaricum and D. malabaricum var. ghaticum with the same karyotype, and that the morphological differences between taxa are caused by several genes which do not play a significant role in the intraspecific differentiation of D. malabaricum. In other words, the morphological classification of both taxa are not biologically significant. These findings further indicate that both taxa differ morphologically but cytotaxonomically they are same. This conclusion may be further supported by molecular in- vestigations which will reveal the similarity and differences if any between these taxa.

Acknowledgements

The ﬁrst author wishes to express her special thanks to Dr. Swaroopa R. Ghatge and Dr. Sandeep R. Pai for the helpful discussions and review of the manuscript. The author is also in- debted to Dr. Nilesh V. Malpure for providing the photographs of the variety ghaticum.

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