Abstract
The somatic chromosome numbers and detailed morphometric properties of 18 specimens belonging to eight taxa (six species) of genus Carduus L. distributed in Turkey were examined. The somatic chromosome numbers were determined as 2n = 2x = 22 in C. olympicus Boiss. subsp. hypoleucus (Bornm.) P. H. Davis, C. tmoleus Boiss. subsp. tmoleus and C. acanthoides L. s.l.; 2n = 2x = 18 in C. candicans Waldst. & Kit. s.l. and 2n = 2x = 16 in C. nutans L. subsp. falcato-incurvus P. H. Davis (2798). In addition, tetraploid chromosome numbers (2n = 4x = 32) were also observed in C. nutans subsp. falcato-incurvus (2737) and C. onopordioides Fisch. ex M. Bieb. subsp. turcicus (Kazmi) P. H. Davis. Karyotype analysis indicated that chromosomes of Carduus taxa generally have median region (m) and submedian region (sm) karyotypes. A median point (M) karyotypes is found in C. acanthoides subsp. acanthoides (2728). Secondary structures and one to three pairs of satellite chromosomes (sat-chromosome) were observed in some of the examined specimens. The findings for each of the analysed taxa are compared with the results of previous studies. The chromosome number and morphology of C. olympicus subsp. hypoleucus, C. nutans subsp. falcato-incurvus, C. onopordioides subsp. turcicus, C. candicans subsp. globifer and C. acanthoides subsp. sintenisii are reported here for the first time.
Introduction
Carduus L. is spiny member of Asteraceae (Cardueae), including c.90 species distributed in Eurasia (mainly in the Mediterranean region), Northern Africa, West Asia and introduced in America and Australia (Tamamschian Citation1963; Davis Citation1975; Kadereit and Jeffrey Citation2007). The genus was represented by 17 species (28 taxa) in Turkey (Arabacı Citation2012, Köstekci and Arabacı Citation2013).
The chromosome numbers of some Carduus taxa have been previously given. According to these studies, the genus has 2n = 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 30, 32, 34, 36, 40, 50, 54, 60, 62, 64, 80 and n = 12, 13, 14, 31 chromosome numbers with the basic chromosome numbers of x = 8, 9, 10, 11 (Arano Citation1957; Kazmi Citation1964; Mehra et al. Citation1965; Favarger Citation1969; Borgen Citation1974; Van Loon and De Jong Citation1978; Corrias and Villa Citation1980; Devesa Citation1980; Devesa and Talavesa Citation1981; Gremaud Citation1981; Zemskova and Ciklauri Citation1987; Ghaffari Citation1989; Kuzmanov et al. Citation1991; Vogt and Aparicio Citation1999, Kadereit and Jeffrey Citation2007; Gedik et al. Citation2014).
The chromosome numbers of Turkish Carduus species are currently being studied in order to clarify its taxonomy and make contributions to other multidisciplinary studies of the genus. Eight annual taxa of the genus were previously examined by the authors (Gedik et al. Citation2014). This study presents karyological analyses of 18 specimens of another eight taxa (six species) of the Turkish Carduus species: C. olympicus Boiss. subsp. hypoleucus (Bornm.) P. H. Davis; C. tmoleus Boiss. subsp. tmoleus; C. nutans L. subsp. falcato-incurvus P. H. Davis; C. onopordioides Fisch. ex M. Bieb. subsp. turcicus (Kazmi) P. H. Davis; C. candicans Waldst. & Kit. subsp. candicans; C. candicans Waldst. & Kit. subsp. globifer (Velen.) Kazmi; C. acanthoides L. subsp. acanthoides; and C. acanthoides L. subsp. sintenisii Kazmi.
Materials and methods
Plant materials were collected from field studies during the fruiting season in 2008 and 2013 as a part of a taxonomic revision of the genus Carduus in Turkey. The voucher specimens were deposited at the İnönü University Herbarium (INU). The list of the examined specimens, localities and voucher numbers are given in Table . Meristematic cells of root tips are used for the karyological studies. The seeds were germinated on moist filter paper in Petri dishes at 25°C. The actively growing root tips were pretreated with aqueous paradichlorobenzene for 4 h at room temperature, aqueous colchicine (0.05%) for 2 h at room temperature and α-mono-bromonaphthalene for at least 12 h at 4°C in a refrigerator. Then, the root tips were fixed with Farmer (1:3 glacial acetic acid–absolute ethanol) for at least 24 h at 4°C, hydrolysed in 1 N HCl at 60°C for 15 min, and subsequently rinsed in tap water for 3–5 min. Then they were stained in Feulgen for 1 h and mounted in 45% acetic acid (Gedik et al. Citation2014). Digital microphotographs from at least three well-spread metaphase plates were taken using an Olympus BX51 microscope (Olympus Optical Co. Ltd, Tokyo, Japan), and were recorded with an Olympus Camedia C-4000 digital camera (Olympus Optical Co. Ltd) (Figure ). The short arm, long arm and total lengths of each chromosome were measured and the relative lengths (RL), arm ratios (AR), and centromeric indices (CI) were determined from images of selected cells. Levan et al. (Citation1964) was used for the classification of chromosomes. The intrachromosomal asymmetry index (A1) and the interchromosomal asymmetry index (A2) are given according to Romero-Zarco (Citation1986).
Table 1. Localities and voucher numbers of Carduus taxa investigated.
Figure 1. Somatic metaphases in Carduus taxa. (a) C. olympicus subsp. hypoleucus (2n = 22); (b) C. tmoleus subsp. tmoleus (2795) (2n = 22); (c) C. tmoleus subsp. tmoleus (2803) (2n = 22); (d) C. tmoleus subsp. tmoleus (2804) (2n = 22); (e) C. tmoleus subsp. tmoleus (2817) (2n = 22); (f) C. tmoleus subsp. tmoleus (2818) (2n = 22); (g) C. tmoleus subsp. tmoleus (2820) (2n = 22); (h) C. nutans subsp. falcato-incurvus (2737) (2n = 32); (i) C. nutans subsp. falcato-incurvus (2798) (2n = 16); (j) C. onopordioides subsp. turcicus (2n = 32); (k) C. candicans subsp. candicans (2n = 18); (l) C. candicans subsp. globifer (2n = 18); (m) C. acanthoides subsp. acanthoides (2728) (2n = 22); (n) C. acanthoides subsp. acanthoides (2811) (2n = 22); (o) C. acanthoides subsp. acanthoides (2812) (2n = 22); (p) C. acanthoides subsp. acanthoides (2813) (2n = 22); (q) C. acanthoides subsp. acanthoides (2814) (2n = 22); (r) C. acanthoides subsp. sintenisii (2n = 22). Scale bars: 10 μm. Arrows indicate the satellite chromosomes.
The chromosome numbers of the species were identified and checked using the relevant literature (Tamamschian Citation1963; Kazmi Citation1964; Davis Citation1975) and the online chromosome number databases (Goldblatt and Johnson Citation1979; Watanabe Citation2014).
Results
The number of somatic chromosomes, ploidy level, karyotype formula, morphometric parameters, A1 and A2 values were determined for each taxa (Table ). Idiograms of haploid chromosomes were drawn (Figure ). The examined taxa and characteristics of somatic chromosomes are given below.
Table 2. Somatic chromosome number (2n), ploidy level, karyotype formula, ranges of chromosome length, total karyotype length (TKL), relative length (RL), arm ratio (AR), centromeric index (CI) (100S/TL) and asymmetry indexes (A1, A2) of Romero Zarco (1986) of the studied Carduus taxa.
Figure 2. Haploid idiograms. (a) C. olympicus subsp. hypoleucus (2n = 22); (b) C. tmoleus subsp. tmoleus (2795) (2n = 22); (c) C. tmoleus subsp. tmoleus (2803) (2n = 22); (d) C. tmoleus subsp. tmoleus (2804) (2n = 22); (e) C. tmoleus subsp. tmoleus (2817) (2n = 22); (f) C. tmoleus subsp. tmoleus (2818) (2n = 22); (g) C. tmoleus subsp. tmoleus (2820) (2n = 22); (h) C. nutans subsp. falcato-incurvus (2737) (2n = 32); (i) C. nutans subsp. falcato-incurvus (2798) (2n = 16); (j) C. onopordioides subsp. turcicus (2n = 32); (k) C. candicans subsp. candicans (2n = 18); (l) C. candicans subsp. globifer (2n = 18); (m) C. acanthoides subsp. acanthoides (2728) (2n = 22); (n) C. acanthoides subsp. acanthoides (2813) (2n = 22); (o) C. acanthoides subsp. acanthoides (2811) (2n = 22); (p) C. acanthoides subsp. acanthoides (2812) (2n = 22); (q) C. acanthoides subsp. acanthoides (2814) (2n = 22); (r) C. acanthoides subsp. sintenisii (2n = 22). Scale bars: 1 μm.
C. olympicus Boiss. subsp. hypoleucus (Bornm.) P. H. Davis
An endemic taxon growing on rocky slopes and Pinus nigra forest openings between altitudes of 1400 and 1875 m asl (Davis Citation1975). The somatic chromosome number of this species was found to be 2n = 22. The chromosome length is 1.81–3.69 μm, and the ratio of the longest to shortest chromosome is 2:1. It consists of six median region (m) and five submedian region (sm) chromosomes. Chromosome arm ratios are 1.26–2.17 μm. Centromeric index is 31.53–44.14 μm, and relative lengths are 7.03–14.28 μm. As far as we know, this is the first chromosome number and morphology report for this species (Table , Figures. a, a).
C. tmoleus Boiss. subsp. tmoleus
A many-stemmed perennial taxon growing in mountain woods, forest openings, and stream and field sides between 525 and 1900 m asl (Davis Citation1975). Previous studies reported chromosome counts for this taxon as 2n = 22 (Gremaud Citation1979; Van Loon and Snelders Citation1979; Kuzmanov et al. Citation1983). In this study, six specimens of C. tmoleus subsp. tmoleus collected from different localities were investigated (Table ). There are some differences in karyotype analyses, but the chromosome numbers are the same with 2n = 22 in the six examined specimens. The specimen labelled “Arabacı 2795” has metaphase chromosome lengths of 1.80–3.36 μm, longest to shortest chromosome ratio of 1.8:1, a karyotype comprising seven m and four sm chromosomes, chromosome arm ratios of 1.17–2.21 μm, centromeric index of 31.14–46.07 μm, and relative lengths of 6.54–12.22 μm (Table , Figures b, b). The specimen labelled “Arabacı 2803” has six m and five sm chromosomes, metaphase chromosome length of 2.09–3.51 μm, longest to shortest chromosome ratio of 1.6:1, chromosome arm ratios of 1.38–2.03 μm, centromeric index of 32.97–41.86 μm, and relative lengths of 6.85–11.49 μm (Table , Figures c, c). The specimen labelled “Arabacı 2804” has seven m and four sm chromosomes, metaphase chromosome length of 1.62 to 3.25 μm, longest to shortest chromosome ratio of 1.2:1, chromosome arm ratios of 1.22–2.19 μm, centromeric index of 31.26–45.00 μm, and relative lengths of 5.74–11.50 μm (Table , Figures d, d). The specimen labelled “Arabacı 2817” has seven m and four sm chromosomes, metaphase chromosome length of 2.06–3.81 μm, longest to shortest chromosome ratio of 1.8:1, chromosome arm ratios of 1.23–2.09 μm, centromeric index of 32.32–44.66 μm, and relative lengths of 6.10–11.31 μm (Table , Figures e, e). The specimen labelled “Arabacı 2818” has 10 m chromosomes and one sm chromosome, metaphase chromosome length of 2.06–3.79 μm, longest to shortest chromosome ratio of 1.8:1, chromosome arm ratios of 1.02–2.01 μm, centromeric index of 33.14–49.42 μm, and relative lengths of 6.31–11.60 μm (Table , Figures f, f). The specimen labelled “Arabacı 2820” has seven m and four sm chromosomes, metaphase chromosome length of 2.18–3.97 μm, longest to shortest chromosome ratio of 1.8:1, chromosome arm ratios of 1.27–2.05 μm, centromeric index of 32.78–43.86 μm and relative lengths of 6.52–11.89 μm. Two pairs of satellites are observed in specimen “Arabacı 2820” (Table , Figures g, g).
C. nutans L. subsp. falcato-incurvus P. H. Davis
This endemic taxon is distributed on limestone and chalky slopes and mountains, c.800 m asl (Davis Citation1975). Specimens collected from two different localities are investigated (Table ). The specimen labelled “Arabacı 2737” has a chromosome number of 2n = 32, and it consists of 11 m and five sm chromosomes. The metaphase chromosome length is 1.46–3.06 μm and longest to shortest chromosome ratio is 2.0:1. Chromosome arm ratios are 1.28–2.27 μm, the centromeric index is 30.55–43.83 μm, and relative lengths are 4.20–8.78 μm (Table , Figures h, h). The specimen labelled Arabacı 2798 has 2n = 16 chromosome number and consists of seven m chromosomes and one sm chromosome, has a metaphase chromosome length of 3.33–4.74 μm and longest to shortest chromosome ratio of 1.4:1, chromosome arm ratios of 1.96–2.76 μm, centromeric index of 32.58–44.33 μm, and relative lengths of 10.49–14.94 μm. A pair of satellite chromosomes (sat-chromosome) and secondary structures was observed in this specimen (Table , Figures i, i). According to our knowledge, this is the first chromosome number and morphology report for this taxon.
C. onopordioides Fisch. ex M. Bieb. subsp. turcicus (Kazmi) P. H. Davis
An endemic taxon growing on fallow fields between 1300 and 1500 m asl (Davis Citation1975). Somatic chromosome number of this species was observed as 2n = 32. It consists of 10 m and six sm chromosomes. The chromosome length is 1.83–3.37 μm, the ratio of the longest to shortest chromosome is 1.8:1, chromosome arm ratios are 1.23–2.23 μm, the centromeric index is 30.93–44.67 μm, and relative lengths are 4.64–8.56 μm. As far as we know, this is the first chromosome number and morphology report for this species (Table , Figures j, j).
C. candicans Waldst. & Kit. subsp. candicans
This taxon was collected from field margins at 150 m asl (Table ). A haploid chromosome number count (n = 8–9) was given in previous studies (Gremaud Citation1979). According to our studies, this taxon has 2n = 18 chromosome numbers with a karyotype consisting of seven m and two sm chromosomes. The chromosome length is 2.32–4.28 μm and longest to shortest chromosome ratio is 1.8:1. Chromosome arm ratios are 1.02–2.06 μm. Centromeric index is 32.57–49.26 μm and the relative lengths are 8.15–15.01 μm. Two pairs of satellite chromosomes (sat-chromosome) were observed (Table , Figures k, k).
C. candicans Waldst. & Kit. subsp. globifer (Velen.) Kazmi
This taxon was collected from field margins at 80 m asl (Table ). The chromosome number is determined as 2n = 18 for this taxon. The karyotype consists of five m and four sm chromosomes, the chromosome length is 2.80–4.34 μm, longest to shortest chromosome ratio is 1.5:1, chromosome arm ratios are 1.30–2.14 μm, the centromeric index is 31.84–43.39 μm and the relative lengths are 8.86–13.70 μm. Three pairs of satellite chromosomes (sat-chromosome) are observed. A diploid chromosome number count (2n = 30) were given in previous studies for C. onopordioides subsp. onopordioides (Zemskova and Ciklauri Citation1987). But, according to our knowledge, this is the first study which presents the chromosome number and morphology of C. onopordioides subsp. turcicus (Table , Figures l, l).
C. acanthoides L. subsp. acanthoides
Five specimens of this taxon were collected from field margins and roadsides between 560 and 1410 m asl. Previous studies reported chromosome counts for C. acanthoides subsp. acanthoides as 2n = 16 (Wentworth et al. Citation1991) and 2n = 22 (Harriman Citation1975; Kuzmanov and Georgieva 1976; Vachova Citation1976; Murin and Vachova Citation1978; Van Loon and Kieft Citation1980; Kuzmanov et al. Citation1991; Albers and Pröbsting Citation1998; Lövkvist and Hultgård Citation1999). In this study, the chromosome numbers of examined specimens are found as 2n = 22 (Table ). The specimen labelled “Arabacı 2728” has metaphase chromosome length of 1.96–3.66 μm, and a longest to shortest chromosome ratio of 1.8:1; the karyotype comprises one median point (M), eight m and two sm chromosomes. Chromosome arm ratios are 1.00–2.05 μm, centromeric index is 32.70–50.00 μm, and relative lengths are 6.13–11.47 μm (Table , Figures m, m). In the specimen labelled “Arabacı 2811”, the karyotype comprises five m and six sm chromosomes with the metaphase chromosome length of 2.35–3.45 μm and longest to shortest chromosome ratio of 1.4:1. Chromosome arm ratios are 1.35–2.06 μm, centromeric index is 32.60–42.50 μm and relative lengths are 7.33–10.76 μm. Three pairs of satellites are observed in these specimens (Table , Figures n, o). The specimen labelled “Arabacı 2812” has six m and five sm chromosomes. Metaphase chromosome length is 1.99–3.86 μm, and the longest to shortest chromosome ratio is 1.9:1. Chromosome arm ratios are 1.30–2.19 μm, centromeric index is 31.33–43.42 μm and relative lengths are 6.24–12.11 μm. A pair of satellites is also observed (Table , Figures o, p). The specimen labelled “Arabacı 2813” has five m and six sm chromosomes, metaphase chromosome length of 1.76–2.97 μm and longest to shortest chromosome ratio of 1.6:1. Chromosome arm ratios are 1.42–2.08 μm, centromeric index is 32.43–41.28 μm, and relative lengths are 6.68–11.29 μm (Table , Figures p, n). In the specimen labelled “Arabacı 2814”, the karyotype comprises seven m and four sm chromosomes. Metaphase chromosome length is 2.02–3.83 μm and longest to shortest chromosome ratio is 1.8:1. Chromosome arm ratios are 1.21–2.25 μm. Centromeric index is 30.72–44.66 μm, and relative lengths are 6.29–11.89 μm (Table , Figures q, Fig q).
As seen from Figures and , chromosome lengths vary between 1.76 and 3.86 μm in the specimens. Two of the five specimens of C. acanthoides subsp. acanthoides have pairs of satellites. The specimen labelled “Arabacı 2811” has three pairs of satellites and the specimen labelled “Arabacı 2812” has one pair of satellites.
C. acanthoides L. subsp. sintenisii Kazmi
An endemic species growing on igneous banks and field margins between 960 and 2100 m asl (Davis Citation1975) (Table ). The somatic chromosome number of this species is 2n = 22, metaphase chromosome length is 1.81–2.33 μm, and the longest to shortest chromosome ratio is 1.2:1. The karyotype comprises seven m and four sm chromosomes, chromosome arm ratios of 1.32–1.96 μm, centromeric index of 33.70–42.92 μm, and relative lengths of 7.83–10.10 μm. As far as we know, this is the first chromosome number and morphology report for this species (Table , Figures r, Fig r).
Discussion
The studied taxa are the biennial or perennial members of Turkish Carduus genus. These taxa are morphologically characterized by hemispherical to globose involucres, and zygomorphic corollas (Davis Citation1975).
Karyotype analysis of the examined Carduus taxa showed that the taxa have x = 8, 9 and 11 basic chromosome number and all of them are diploid except for C. nutans subsp. falcato-incurvus (2737) and C. onopordioides subsp. turcicus (2766). Analysis of the karyotype formulae indicated that chromosomes of eight studied Carduus taxa generally have m and sm karyotypes. An M karyotype is also observed. The mean chromosome length is 1.46–4.74 μm. The smallest chromosome length is in tetraploid specimens of C. nutans subsp. falcato-incurvus (1.46–3.06 μm) while the longest chromosome length was seen in its diploid specimens (3.33–4.74 μm). Centromeric index ranges from 30.55 to 50.00 μm, and relative lengths from 4.20 to 15.01 μm. The intrachromosomal asymmetry index (A1) is ranges from 0.24 to 0.40 μm. Interchromosomal asymmetry index (A2) varies between 0.08 and 0.22 μm.
The somatic chromosome numbers were determined as 2n = 2x = 16 in the specimens of C. nutans subsp. falcato-incurvus labelled “Arabacı 2798”. In addition, a tetraploid chromosome number (2n = 4x = 32) were observed in C. nutans subsp. falcato-incurvus labelled “Arabacı 2737”. Yüksel et al. (Citation2013) reported that specimens collected from various geographical regions can have different chromosome numbers and this is related with their reproductive biology, such as self- and cross-pollination.
Three pairs of satellite chromosomes (sat-chromosomes) were observed in C. acanthoides subsp. acanthoides (2811) and C. candicans subsp. globifer, two pairs of satellite chromosomes are seen in C. tmoleus subsp. tmoleus (2820) and C. candicans subsp. candicans, and one pair of satellite chromosomes was observed in C. acanthoides subsp. acanthoides (2812) and C. acanthoides subsp. sintenisii. In addition, one pair of satellite chromosomes and secondary structures was seen in C. nutans subsp. falcato-incurvus (2798) (Figures , 2).
In this study, chromosome numbers of eight Turkish Carduus taxa were established. Numerous counts were reported for C. candicans s.l., C. onopordioides s.l., C. tmoleus s.l. and C. acanthoides s.l in previous studies. But, as far as we know, the chromosome number and morphology of the taxa C. olympicus subsp. hypoleucus, C. nutans subsp. falcato-incurvus, C. onopordioides subsp. turcicus, C. candicans subsp. globifer and C. acanthoides subsp. sintenisii are newly reported here. This study will contribute to further studies on the karyotype properties and implications on systematic of genus Carduus from Turkey.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding information
The authors want to thank İnönü University Scientific Projects Research Fund (İÜBAPB) [project no. 2011/39] for financial support.
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