Abstract: In order to investigate the karyotype of Dianthus, a study was carried out on nine populations of Dianthus crinitus, D. binaludensis (an endemic species in Iran) and D. polylepis in the North-East of Iran. For present research, 8- Hydroxy quinolein 0.0002 M and aceto-orcein were used as pretreatment solution and staining. The basic chromosome number of Dianthus was X= 15. D. binaludensis and D. polylepis were diploid, while D. crinitus was found to be tetraploid. In addition, for the first time the karyotypes of the three species of Dianthus were prepared and compared with their morphological characters. D. binaludensis and D. polylepis were found to be morphologically close, with the same ploidy level but D. crinitus differs from the other species by in that it is tetraploid suffruticous in habit, has white petals and a membranous herbaceous stigma with nerves.
INTRODUCTION
Dianthus L. belongs to the Silenoideae tribe of the Caryophyllaceae family (Engler, 1887). Over 300 Dianthus species have been identified in Europe, Asia, North America, north Africa (Groshkova, 1970; Davis, 1965-85; Punt and Hoen, 1995), On the basis of Flora Iranica report, Dianthus has six sections, 30 and 12 species and subspecies respectively. Just, four species belong to sect. Fimbriati grow in the province of Khorasan-Razavi (northeastern of Iran). These species are: D. crinitus Sm. p.p, D. binaludensis Rech. f, D. polylepis Bienert ex Boiss. and D. orientalis Adams. p.p. (Rechinger, 1988). This genus is important by having medicinal characters, aromatic materials and polymorphism in morphology, genetic and hybridization (McGeorge and Hammett, 2002; Facciola, 1990; Hughes, 1993; Lee et al., 2005; Su Yeons, 2002). The differences of morphology among the populations prevent to identify species easily. On the other hand, the recognizing of distinct species is difficult because of similarity among the species. Previous morphological studies of this genus confirmed the variation in the amount of indument on the stem and leaves and in the size, shape and color of the leaves, petals and the calyx (Carine and Shykoff, 2003). The aim of present research was to identify chromosome number and find relationship between variation of karyotype and morphology. According to previous karyological reports, this genus was diploid and seldom triploid in some hybrid like D. caryophyllus x D. isensis (Nimura et al., 2004, 2006; Nimura, 2008; Goralski et al., 2009). There also have been some karyological studies on Dianthus sect. Plumaria, Dianthus nardiformis Joe ka. and Dianthus spiculifolius Schur (Weiss et al., 2002; Ioni et al., 2003). Karyological reports cited by Weiss et al. (2002) were shown in Table 1. Likewise, for the first time in Iran, this study investigated the karyological characteristics of Dianthus growing in northeastern Iran for identifying any relationship between their morphological characters and the cytological data.
MATERIALS AND METHODS
The materials were collected from Northeastern Iran (Table 2) during May to June 2008. Voucher specimens were deposited in the herbariums of IAUM and FUMH. For karyotype preparing, a pretreatment was performed at room temperature for three hours before root tip fixation of the three species of Dianthus in 0.002 M 8-hydroxyquinoline. Then following fixation in a cold mixture of ethanol and acetic acid (3:1), the next steps were maceration in 1N HCl at 60°C for 5-8 min, washing in water, cutting off the meristems and squashing them in a drop of 45% acetic acid (Krahulkova, 2003). Next, the chromosomes were described according to Levan’s terminology (Levan et al., 1964). Then, the karyotypes were compared using total form percentage (Forni-Martins et al. 1994). Finally, the ratios of the longest to the shortest chromosomes were calculated (Verma, 1980).
| Table 1: | Previous reports of somatic chromosomes number in Dianthus |
| Table 2: | The locations of the studied Dianthus populations |
Symmetry karyotypes were determined using Stebbins' two-way system that is based on percent of r-value (Stebbins, 1971). Present study was conducted in plant research lab of Islamic Azad university, Mashhad branch.
RESULTS
The number of somatic chromosomes along with the details of the karyotypes of the studied species of Dianthus is shown in Table 3. The results indicated that D. binaludensis (Moghan population) is diploid (2n = 30) and has the karyotype formula (11M+1m+2sm+1st) with 76.65% metacenteric chromosomes (Fig. 1a, 4a). The Boujan population of this species had the same ploidy level (2n = 30) but with the karyotype formula (12M+1m+1sm+1st) and 50% metacenteric chromosomes (Figs. 1b, 4b). Also, the Zoshk population was diploid with the karyotype formula (12M+1m+2sm) and 80% metacenteric chromosomes that were found to be more symmetric than the population in Boujan (Fig. 1c, 4c). Dianthus polylepis (Kardeh population) was found to be diploid with the karyotype formula (10M+1m+4sm) and 66% metacenteric chromosomes (Fig. 2a, 5a) that were more asymmetrical than the other populations of this species. The Torbat-e Heydariye population was also found to be diploid with the karyotype formula (11M+2m+2sm) and 73% metacenteric chromosomes (Fig. 2b, 5b).
| Fig. 1: | D. binaludensis (a) Moghan population, (b) Boujan population and (c) Zoshk population |
| Table 3: | Karyotypic details of the studied Dianthus populations |
| TL: Total chromosome length, S: Shortest chromosome, L: Longest chromosome, L/S: Longest/shortest, TF%: Total karyotype form percentage, T.V: Total volume, D.R.L: Difference of range of relative length, S%: Bazzichelli index D. binaludensis: (1) (Moghan population). 2) Boujan population. 3) Zoshk population D. polylepis: 4) Kardeh population. 5) Torbat Heidariye population. 6) Fariman population D. crinitus subsp. Turcominicus: 7) Sarrud population. 8) Moghan population. 9) Ardak population | |
| Fig. 2: | D. polylepis (a) Karde population (b) Torbat Heydariye population and (c) Fariman |
| Fig. 3: | D. crinitus (a) Sarrud population (b) Moghan population and (c) Ardak population |
| Fig. 4: | Ideogram
of D. binaludensis (a) Moghan population (b) Boujan opulation and
(c) Zoshk population |
| Fig. 5: | Ideogram
of D. polylepis (a) Kardeh population (b) Torbate Heydariyeh population
and (c) Fariman population |
| Fig. 6: | Ideogram of D. crinitus (a) Ortokand population (b) Moghan population and (c) Ardak population |
The Fariman population diploid with the karyotype formula (11M+1m+3sm) and 73% metacenteric chromosomes (Fig. 2c, 5c). All populations of D. crinitus were found to be tetraploid (2n = 60). In the Sarrud population the karyotype formula was determined as (25M+3m+1sm+1st). Of all the chromosomes identified, 83% were metacenteric (Fig. 3a, 6a). The karyotype formula of the populations of Moghan and Ardak were (23M+6m+1sm) with 76% etacenteric chromosomes and (20M+8m+2sm) with 66% metacenteric chromosomes, respectively (Fig. 3b, c, 6b, c). All populations fell into class 2B. Also, the findings with regard to total chromatin length and the size of the longest and shortest chromosomes are shown in Table 2. In this regard, the Ardak population of D. crinitus had the highest chromatin length, with a total chromatin length of 183.72 μM, while the Fariman population of D. polylepis had the lowest with 71.68 μM.
DISCUSSION
The basic number of chromosomes in Dianthus is X = 15. Among the previous studies on Dianthus sect. Plumaria, Weiss identified tetraploid and hexaploid cytotypes for D. nardiformis Jan ka (Weiss et al., 2002). Also Ioni reported diploid and tetraploid cytotypes for D. spiculifolius, Schur (Ioni et al., 2003). Diploid was reported in D. cyprius by Yildiz and Gucel (2006). According to interesting report, different cytotype of D. broteri had 2n = 45, 90 and 180 (Balao et al., 2009). But usually diploid were identified (Carolin, 1957). According to the karyological results in the present study, the whole populations of D. binaludensis and D. polylepis were found to be diploid, while the populations of D. crinitus were identified as tetraploid. Also, the karyotypes of the above mentioned populations was relatively symmetric. Thus, on the basis of Stebbins' two- way table, all of the populations in this study fall into class 2B. The populations of D. polylepis in Fariman and Torbat-e Heydarieh were the most asymmetric ones. On the other hand, previous morphological studies of this genus confirmed the variation in the amount of indument on the stem and leaves and in the size, shape and color of the leaves, petals and the calyx. In other words, the characteristics of this species vary from one population to another (Carine and Shykoff, 2003). In the present study, however, D. binaludensis and D. polylepis were found to be morphologically close, with the same ploidy level, habit and flowers of similar color, size and shape. They are caespitose-suffruticous and have similarities like white to purple petals, scaly stigma without nerves and scaly bracts. However, they differ in that D. polylepis has lanceolate bracts while D. binaludensis has ovate bracts. Both of them grow on northern slopes of stony mountains and at the same altitude. D. crinitus differs from the other species by in that it is tetraploid suffruticous in habit, has white petals and a membranous herbaceous stigma with nerves. This species is divided into varieties. In spite of the variation in morphological characteristics, the types of chromosomes are almost homogeneous. There was no relationship between the type of chromosomes and the morphological finding.