Karyotype analysis of four species of Dianthus section Fimbriati (Caryophyllaceae, Sileneae)

Abstract

Karyotypic characters, mitotic metaphase chromosomes, monoploid ideograms and karyograms of Dianthus L. section Fimbriati Boiss., D. tabrisianus Bien.ex Boiss., D. sessiliflorus Boiss., D. stramineus Boiss. & Heldr., and D. erythrocoleus Boiss. were investigated for the first time. Analysis of somatic metaphases showed that the chromosome numbers and the karyotype formula of these taxa were 2n = 30 = 22m + 6sm + 2st for D. tabrisianus, 2n = 30 = 14m + 8sm + 8st for D. sessiliflorus, 2n = 30 = 20m + 4sm + 6st for D. stramineus and 2n = 30 = 18m + 8sm + 4st D. erythrocoleus. No satellites were observed in the karyotypes of these species. The interchromosomal and intrachromosomal karyotype asymmetries were estimated with coefficient of variation of chromosome length (CV_CL) and mean centromeric asymmetry (M_CA).

Keywords:

• Dianthus
• ideogram
• karyotype
• symmetry/asymmetry index

Introduction

Dianthus L. is placed in the subfamily Sileneae DC. in the family Caryophyllaceae. It is a large genus, consisting of approximately 300 species found in Europe, Asia, Africa and North America (Bittrich 1993; Vaezi et al. 2014). The genus was represented by 121 species in Flora Europaea (Tutin 1964), 78 species in Flora of the USRR (Shishkin 1995), 49 species in Flora Iranica (Rechinger 1988), 19 species in Flora of Syria, Palestine and Sinai (Post 1932), eight species in Flora Palestina (Zohary 1966), three species in Flora of Iraq (Rechinger 1964), and three species in Flora of Cyprus (Meikle 1977). The most comprehensive taxonomic study of Turkish Dianthus was carried out by Reeve (1967). In the light of the latest data, the genus includes 74 species, two subspecies and 12 varieties found in five sections in the flora of Turkey (Davis et al. 1988; Güner 2000; Menemen and Hamzaoğlu 2000; Aytaç and Duman 2004; Vural 2008; Yılmaz et al. 2011; İlçim et al. 2013). The taxa of section Fimbriati are distinguished from other taxa with fimbriate petals (Boissier 1867; Reeve 1967). The section Fimbriati includes nine species in the Flora of Turkey (Reeve 1967). Petal surface with glabrous or barbulate is one of the most important distinctive characters among species. D. tabrisianus, D. sessiliflorus, D. stramineus, D. erythrocoleus and D. crinitus Sm. have glabrous petals (Reeve 1967).

The chromosomal measurements and karyotype symmetry/asymmetry are the most important taxonomic characters, together with morphological characters (Eroğlu et al. 2013; Peruzzi and Eroğlu 2013), as the chromosome numbers of organisms are highly variable (Peruzzi and Bedini 2014). There are different karyotype symmetry/asymmetry formulae in higher plants (Paszko 2006; Peruzzi and Eroğlu 2013), animals and humans (Eroğlu 2015). Different chromosome counts have been reported in the genus Dianthus. However, the most frequent chromosome number in the genus Dianthus is diploid 2n = 30. Other counts such as 2n = 60, 90 and 120 are also known (Güner 2000).

Although there are several chromosome reports on Dianthus (Güner 2000), no information on chromosome numbers and karyotype analysis of the investigated species was found in the literature. The aim of this study is to investigate the chromosome number, karyotype, idiogram and other detailed measurements within four species of Dianthus section Fimbriati. D. sessiliflorus and D. stramineus of these species are narrow endemic in Turkey. D. tabrisianus and D. erythrocoleus grow wild in Turkey and Iran (Reeve 1967; Rechinger 1988).

Material and methods

The plant materials were collected from natural habitats in different localities (Table 1). Ergin Hamzaoğlu and Murat Koç authenticated the plant material. The voucher specimens have been deposited in the Herbarium of Gazi University (GAZI). The seeds were germinated between moist Whatman papers in Petri dishes. The root tips were fixed in Carnoy’s fixative (3:1 absolute alcohol: glacial acetic acid) at +4°C overnight after pre-treatment in colchicine (0.2% w/v) for 2 h. Root tips were stained with acetocarmine. Preparations were made using the squash method. At least 10 metaphase cells were used to determine chromosome numbers. The metaphase chromosomes were photographed by Olympus BX53 digital camera (Olympus Corporation, Tokyo, Japan) and measured using the Bs200ProP image processing and analysis system (BAB Image Analysing Systems, Ankara, Turkey).

Table 1. Localities, coordinates, collector name and numbers of studied species.

Species	Locality	Coordinate	Collectors and herbarium
D. tabrisianus	Kars: Digor, between Digor and Iğdır, 27 August 2012, 1375 m	40° 10' N 43° 30' E	Hamzaoğlu 6657 & Koç (GAZI)
D. sessiliflorus	Muş: Varto, Bingöl Mountain, 25 July 2013, 3120 m	39° 20' N 41° 20' E	Hamzaoğlu 6935 & Koç (GAZI)
D. stramineus	Konya: Akören, between Belkuyu and Dutlu, 18 June 2013, 1200 m	40° 40' N 42° 10' E	Hamzaoğlu 6767 & Koç (GAZI)
D. erythrocoleus	Van: Bahçesaray, around Karapet Pass, 24 July 2013, 3100 m	38° 00' N 42° 50' E	Hamzaoğlu 6911 & Koç (GAZI)

Karyotype formula was determined by chromosome morphology based on centromere position according to the classification of Levan et al. (1964). The following parameters were estimated to characterize the karyotypes numerically: long arm length (LAL), short arm length (SAL), total chromosome length (TCL) = [LAL + SAL], arm ratio (AR) = [LAL/SAL] and centromeric index (CI) = [SAL / (LAL + SAL) × 100]. For each species, karyograms were drawn based on length of chromosome size (arranged large to small).

The interchromosomal and intrachromosomal karyotype asymmetries were estimated with coefficient of variation of chromosome length (CV_CL) (Paszko 2006) and mean centromeric asymmetry (M_CA) (Peruzzi and Eroğlu 2013). Asymmetry indexes were compared with Pearson correlation.

Results

Dianthus tabrisianus

Mitotic metaphase chromosomes, karyotype and monoploid ideogram of D. tabrisianus are given in Figures 1–3, respectively. The measurement data of these chromosomes are given in Table 2. Analysis of somatic metaphases showed that the chromosome number of the species is 2n = 30. The formula is 22m + 6sm +2st. No satellite was observed in the chromosomes. The length of chromosomes varied from 1.09 to 2.68 μm, and the total haploid length was 28.36 μm. The average length of chromosomes was 1.89 μm. The CI varied from 25.00 to 50.00 μm. The analysis of the interchromosomal and intrachromosomal karyotype asymmetry indexes showed values of 24.17 and 15.81 for CV_CL and M_CA, respectively.

Figure 1. Somatic metaphase chromosomes of four species of Dianthus section Fimbriati. A: D. tabrisianus; B: D. sessiliflorus; C: D. stramineus; D: D. erythrocoleus. All scale bars = 10 μm.

Figure 2. Karyograms of four species of Dianthus section Fimbriati. A: D. tabrisianus; B: D. sessiliflorus; C: D. stramineus; D: D. erythrocoleus. Scale bar = 5 μm.

Figure 3. Ideograms of four species of Dianthus section Fimbriati. A: D. tabrisianus; B: D. sessiliflorus; C: D. stramineus; D: D. erythrocoleus.

Table 2. The measurement data of chromosomes of species of Dianthus section Fimbriati.

Species	Chromosome pair	TCL(μm)	LAL(μm)	SAL(μm)	AR	Chromosome type	CI
D. tabrisianus	1	2.68	1.42	1.26	1.127	m	47.01
D. sessiliflorus	1	2.63	1.36	1.27	1.071	m	48.29
D. stramineus	1	2.33	1.30	1.03	1.262	m	44.21
D. erythrocoleus	1	2.57	1.70	0.87	1.954	sm	33.85
D. tabrisianus	2	2.53	1.39	1.14	1.219	m	45.06
D. sessiliflorus	2	2.48	1.25	1.23	1.016	m	49.60
D. stramineus	2	2.18	1.09	1.09	1.000	m	50.00
D. erythrocoleus	2	2.32	1.19	1.13	1.053	m	48.71
D. tabrisianus	3	2.48	1.24	1.24	1.000	m	50.00
D. sessiliflorus	3	2.16	1.20	0.96	1.250	m	44.44
D. stramineus	3	1.97	1.00	0.97	1.031	m	49.24
D. erythrocoleus	3	2.05	1.31	0.74	1.770	sm	36.10
D. tabrisianus	4	2.27	1.19	1.08	1.102	m	47.58
D. sessiliflorus	4	1.84	0.97	0.87	1.115	m	47.28
D. stramineus	4	1.71	0.88	0.83	1.060	m	48.54
D. erythrocoleus	4	2.00	1.03	0.97	1.062	m	48.50
D. tabrisianus	5	2.03	1.07	0.96	1.115	m	47.29
D. sessiliflorus	5	1.75	0.91	0.84	1.083	m	48.00
D. stramineus	5	1.70	1.02	0.68	1.500	m	40.00
D. erythrocoleus	5	1.79	0.96	0.83	1.157	m	46.37
D. tabrisianus	6	1.92	1.06	0.86	1.233	m	44.79
D. sessiliflorus	6	1.73	0.91	0.82	1.110	m	47.40
D. stramineus	6	1.63	0.98	0.65	1.508	m	39.88
D. erythrocoleus	6	1.72	1.10	0.62	1.774	sm	36.05
D. tabrisianus	7	1.91	1.06	0.85	1.247	m	44.50
D. sessiliflorus	7	1.61	0.90	0.71	1.268	m	44.10
D. stramineus	7	1.53	1.05	0.48	2.188	sm	31.37
D. erythrocoleus	7	1.59	0.81	0.78	1.038	m	49.06
D. tabrisianus	8	1.88	0.95	0.93	1.022	m	49.47
D. sessiliflorus	8	1.42	0.90	0.52	1.731	sm	36.62
D. stramineus	8	1.47	0.82	0.65	1.262	m	44.22
D. erythrocoleus	8	1.43	0.78	0.65	1.200	m	45.45
D. tabrisianus	9	1.82	1.19	0.63	1.889	sm	34.62
D. sessiliflorus	9	1.39	0.88	0.51	1.725	sm	36.69
D. stramineus	9	1.39	0.79	0.60	1.317	m	43.17
D. erythrocoleus	9	1.26	0.82	0.44	1.864	sm	34.92
D. tabrisianus	10	1.78	1.07	0.71	1.507	m	39.89
D. sessiliflorus	10	1.24	0.82	0.42	1.952	sm	33.87
D. stramineus	10	1.38	0.81	0.57	1.421	m	41.30
D. erythrocoleus	10	1.21	0.61	0.60	1.017	m	49.59
D. tabrisianus	11	1.66	1.03	0.63	1.635	m	37.95
D. sessiliflorus	11	1.15	0.73	0.42	1.738	sm	36.52
D. stramineus	11	1.29	0.76	0.53	1.434	m	41.09
D. erythrocoleus	11	1.12	0.69	0.43	1.605	m	38.39
D. tabrisianus	12	1.60	1.03	0.57	1.807	sm	35.63
D. sessiliflorus	12	0.93	0.71	0.22	3.227	st	23.66
D. stramineus	12	1.16	0.88	0.28	3.143	st	24.14
D. erythrocoleus	12	1.02	0.52	0.50	1.040	m	49.02
D. tabrisianus	13	1.40	1.05	0.35	3.000	st	25.00
D. sessiliflorus	13	0.86	0.67	0.19	3.526	st	22.09
D. stramineus	13	1.12	0.85	0.27	3.148	st	24.11
D. erythrocoleus	13	0.93	0.56	0.37	1.514	m	39.78
D. tabrisianus	14	1.31	0.66	0.65	1.015	m	49.62
D. sessiliflorus	14	0.77	0.58	0.19	3.053	st	24.68
D. stramineus	14	1.10	0.79	0.31	2.548	sm	28.18
D. erythrocoleus	14	0.68	0.52	0.16	3.250	st	23.53
D. tabrisianus	15	1.09	0.73	0.36	2.028	sm	33.03
D. sessiliflorus	15	0.63	0.49	0.14	3.500	st	22.22
D. stramineus	15	1.09	0.85	0.24	3.542	st	22.02
D. erythrocoleus	15	0.64	0.52	0.12	4.333	st	18.75

Abbreviations: TCL: total chromosome length; LAL: long arm length; SAL: short arm length; AR: arm ratio; CI: centromeric index; m: median; sm: sub-median; st: sub-terminal.

Dianthus sessiliflorus

Mitotic metaphase chromosomes, karyotype and monoploid ideogram of D. sessiliflorus are given in Figures 1–3, respectively. The measurement data of these chromosomes are given in Table 2. Analysis of somatic metaphases showed that the chromosome number of the species is 2n = 30. The formula is 14m + 8sm + 8st. No satellite was observed in the chromosomes. The length of chromosomes varied from 0.63 to 2.63 μm, and the total haploid length was 22.59 μm. The average length of chromosomes was 1.51 μm. The CI varied from 22.09 to 49.60 μm. The analysis of the interchromosomal and intrachromosomal karyotype asymmetry indexes showed values of 40.24 and 24.61 for CV_CL and M_CA, respectively.

Dianthus stramineus

Mitotic metaphase chromosomes, karyotype and monoploid ideogram of D. stramineus are given in Figures 1–3, respectively. The measurement data of these chromosomes are given in Table 2. Analysis of somatic metaphases showed that the chromosome number of the species is 2n = 30. The formula is 20m + 4sm + 6st. No satellite was observed in the chromosomes. The length of chromosomes varied from 1.09 to 2.33 μm, and the total haploid length was 23.05 μm. The average length of chromosomes was 1.54 μm. The CI varied from 22.02 to 50.00 μm. The analysis of the interchromosomal and intrachromosomal karyotype asymmetry indexes showed values of 25.31 and 23.81 for CV_CL and M_CA, respectively.

Dianthus erythrocoleus

Mitotic metaphase chromosomes, karyotype and monoploid ideogram of D. erythrocoleus are given in Figures 1–3, respectively. The measurement data of these chromosomes are given in Table 2. Analysis of somatic metaphases showed that the chromosome number of the species is 2n = 30. The formula is 18m + 8sm + 4st. No satellite was observed in the chromosomes. The length of chromosomes varied from 0.64 to 2.57 μm, and the total haploid length was 22.33 μm. The average length of chromosomes was 1.49 μm. The CI varied from 18.75 to 49.59 μm. The analysis of the interchromosomal and intrachromosomal karyotype asymmetry indexes showed values of 39.22 and 20.26 for CV_CL and M_CA, respectively.

Discussion

Cytogenetic characters, especially chromosome number, chromosome size and chromosome asymmetry, are preferred characters in plant taxonomy. These data are also important to elucidate the origin, speciation and phylogenetic relationships of plants (Eroğlu et al. 2013). The chromosome numbers of Dianthus tabrisianus, D. sessiliflorus, D. stramineus and D. erythrocoleus are 2n = 30. All species have small chromosomes between 0.63 and 2.68 μm. The chromosomes were showed little variation in size within the complement (Table 2).

There are nine species and 12 taxa in section Fimbriati (Reeve 1967). In section Fimbriati it is reported that the chromosome numbers of Dianthus crinitus var. crinitus (Khatoon and Ali 1993), D. orientalis Donn subsp. orientalis (Gadnidze et al. 1998) and D. orientalis Donn subsp. nassireddini (Stapf) Rech.f. (Ghaffari 2006) were 2n = 30. The chromosome numbers of three populations of D. crinitus were 2n = 60 (Jafari and Behroozian 2010). The predominant diploid number of chromosomes in Dianthus is 2n = 30. Other common chromosome numbers are 2n = 60, 90 and 120 (Khatoon and Ali 1993; Gadnidze et al. 1998; Ghaffari 2006; Güner 2000). In section Fimbriati the diploid number of chromosomes is 2n = 30.

The CV_CL value is perfectly suited for the measure of interchromosomal asymmetry (Paszko 2006). It varies between 0 (no variation) and 100. The CV_CL values of Dianthus tabrisianus, D. sessiliflorus, D. stramineus and D. erythrocoleus are 24.17, 40.24, 25.31 and 39.22, respectively. The M_CA value is perfectly suited for the measure of intrachromosomal asymmetry (Peruzzi and Eroğlu 2013). It varies between 0 (perfectly symmetric) and 100 (perfectly asymmetric). The M_CA values of D. tabrisianus, D. sessiliflorus, D. stramineus and D. erythrocoleus are 15.81, 24.61, 23.81 and 20.26, respectively. The values of CV_CL and M_CA increase with increasing asymmetry. The symmetrical karyotype is defined with median and sub-median chromosomes. The location of centromere position changes in intrachromosomal karyotype asymmetry. In addition, the sizes of large and small chromosomes are quite different in interchromosomal karyotype asymmetry (Peruzzi et al. 2009). According to the CV_CL and M_CA values, D. tabrisianus is the most symmetrical karyotype, while D. sessiliflorus is the most asymmetrical karyotype. According to the CV_CL value, D. stramineus is more symmetrical than D. erythrocoleus, but D. erythrocoleus is more symmetrical than D. stramineus in M_CA value. Weak positive correlation was observed between CV_CL and M_CA (r = 0.441) (Figure 4). The weak positive correlation is a result of a change in place of symmetry/asymmetry position between D. stramineus and D. erythrocoleus. This is due to the CV_CL parameters. The CV_CL provides the most reliable data when used together with M_CA. For instance, the karyotype could not be discriminated by CV_CL, if there is not variation in chromosome size (Peruzzi and Eroğlu 2013).

Figure 4. Scatter diagram between CV_CL and M_CA: A: D. tabrisianus; B: D. sessiliflorus; C: D. stramineus; D: D. erythrocoleus.

Dianthus tabrisianus, D. sessiliflorus, D. stramineus and D. erythrocoleus are quite different species morphologically. A key to closely relate to the taxonomic status of these species is given below. The key was made from plant samples collected for cytogenetic analysis.

1.	Stems 2–12 cm, 1–3-noded; epicalyx scales 4(–6), indistinctly 1‒3-veined at apex .............................................................................................2
–	Stems 15–45 cm, 5–11-noded; epicalyx scales (4–)6–10(–12), distinctly 3‒5-veined or indistinctly 5–9-veined at apex .............................................................................................3
2.	Calyx 20–26(–30) mm; teeth 5–7.5 mm long; petal 23–28(–32) mm, limb 9–15-toothed .............................................................................................D. erythrocoleus
–	Calyx 14–18 mm; teeth 3–5 mm long; petal 18–21 mm, limb 5–9-toothed .............................................................................................D. sessiliflorus
3.	Calyx teeth narrowly triangular, 2.2–5 mm long; inner epicalyx scales 1/2–3/5 as long as calyx, with scarious (0.4‒0.6 mm) margins; petal limb 7–9-toothed, claw 14–19 mm long, collar as wide as claw .............................................................................................D. tabrisianus
–	Calyx teeth linear-oblong, 6.5–8.5 mm long; inner epicalyx scales 1/3–2/5 as long as calyx, with scarious (0.2‒0.3 mm) margins; petal limb 11–15-toothed, claw 20–26 mm long, collar 1/2 as wide as claw ……………........………………...…. D. stramineus

There are differences among species in karyotype formulae and karyotype asymmetry indexes. These differences seemed to support morphological differences. Although karyotype formula and karyotype asymmetry are not distinctive characters for all Dianthus taxa, there are similarities between morphological differences and cytogenetic differences of studied species. Seijo and Fernandez (2003) reported that the differences found among species in asymmetry indexes and karyotype formulae may contribute to the diversification of the genus. For example, D. sessiliflorus and D. erythrocoleus are different from D. tabrisianus and D. stramineus morphologically and grow in alpine regions and high altitudes (Table 1). Also, D. sessiliflorus and D. erythrocoleus are different from D. tabrisianus and D. stramineus in the following ways: (i) lower number of median chromosomes; and (ii) more asymmetric karyotype. D. sessiliflorus and D. erythrocoleus show different locations in Figure 4.

In this study, the chromosome numbers, karyotypes, ideograms and karyotype asymmetry degrees of four species of section Fimbriati were determined. The chromosome numbers of the studied species were firstly reported. In all species the same chromosome number (2n = 30) was found. The karyotypes of Dianthus tabrisianus, D. sessiliflorus, D. stramineus and D. erythrocoleus were quite symmetric, because they have a large number of median chromosomes, a small number of sub-median chromosomes and several sub-terminal chromosomes.

Disclosure statement

No potential conflict of interest was reported by the author.

Funding information

This work was financially supported by TUBITAK [project number: KBAG–111T873] and Scientific Research Projects Fund of Bozok University [project number: 2015FBE/T183 and 184].