BIHAREAN BIOLOGIST 13 (2): 66-70 ©Biharean Biologist, Oradea, Romania, 2019 Article No.: e191102 http://biozoojournals.ro/bihbiol/index.html

Karyological study of six Echinops L. (: Cardueae) from Iran

Behnaz ALIJANPOOR1, Hejraneh AZIZI2*, Seyamak MASHAYEKHI3, Leila FALLAH-HOSSEINI4 and Hamed KHODAYARI5

1. Researcher, Research and Education Agriculture and Natural Research of Tehran Province Agricultural Research, Training and promotion Organization, Tehran, Iran. E-mail: [email protected]. 2. Faculty of Biological Sciences, Shahid Beheshti University, Tehran, Iran. E-mail: [email protected], H_ [email protected]. 3. Agriculture Economic, Research Center of Agriculture and Natural Resource of Tehran Provinc. E- mail: [email protected]. 4. Phytology, Research Institute of Forests and Rangelands, Tehran, Iran. E-mail: [email protected]. 5. Department of Biology, Faculty of Science, Lorestan University, Khorramabad, Iran. E- mail: [email protected], [email protected]. * Corresponding author, H. Azizi, E-mail: [email protected]

Received: 15. October 2018 / Accepted: 04. February 2019 / Available online: 19. February 2019 / Printed: December 2019

Abstract. Echinops is a of Asteraeceae that is represented in Iran by 72 species Six species of the genus (E. robustus, E. polygamous, E. orientalis, E. ilicifolius, E.pungens, E. chardinii) from twelve different Iranian populations, belonging to three sections (Oligolepis, Ritropsis, Echinops), are studied cytologically. Somatic chromosome numbers of four species are reported for the first time. Chromosome number 2n=2x= 34 is reported for the first time in section Oligolepis. The chromosome numbers of the studied species and populations were different among members of sections (2n=2x=30, 32 and 34). The chromosomes were mainly metacentric or sub-metacentric. The species differed significantly in the total chromosome length and Mean chromosome length indicating the role of quantitative genomic changes in the Echinops species diversification. The Echinops species were placed in 3A and 3B classes of Stebbins karyotype symmetry. Cluster analysis of karyotype features indicated that cytological studies did not support sectional classification.

Key word: Karyotype, Echinops, sectional classification, chromosome number.

Introduction from Sect. Oligolepis to Sect. Ritropsis is suggested. In the cur- rent study we included 12 Populations of six species distrib- The genus Echinops L. ( Lam. & DC., family As- uted in five provinces to investigate the relationships and teraceae Bercht. & J. Presl) is a large genus containing about test the sectional classification among these species and 120-130 species. The species of this genus are distributed populations. mostly in the Northern hemisphere (tropical Africa, the Mediterranean Basin and temperate region of Eurasia) (Jäger 1987, Petit 1988). The key taxonomic characteristics of the Material and methods

Cynareae, such as the pappus the type and density of the in- material dumentum on the stems, leaves and phyllaries, cannot easily karyological study was performed in 12 populations (plant seeds of be used to distinguish between Echinops species (Mozaf- The gene bank of Research institute of forests and Rangelands, Te- farian 2006, Sánchez-Jiménez et al. 2010). Sectional classifica- hran, Iran) of six Echinops (E. robustus, E. polygamous, E. orientalis, E. tion of Echinops is districted to only a few Flora such as: ilicifolius, E. pungens, E. chardinii) species from Iran (Tables 1, 2). Flora of USSR (Shishkin 1997) and Flora Iranica (Rechinger 1979). In Flora of USSR, the species are organized in eight Table1: Echinops species and locality studied. sections: Echinops, Chamaechinops, Nanecinops, Oligolepis, Pli- Species Locality acmes, Ritro, Ritrodes and Terma in Flora Iranica 54 species are E.robustus Bunge Isfahan: Golpaigan included in five sections: Oligolepis, Ritropsis, echinops, E.polygamus Bunge Semnan: Semnan Hololeuce and Nanechinops Subsequently, 18 new species of E.orientalis Trautv Qom: Qom, Khaljestan, Anjileh village this genus have been introduced in Iran (Mozaffarian & E. ilicifolius Bunge Yazd: Taft Ghahreman 2002, Mozaffarian 2006) species from East Africa E. ilicifolius Bunge Fars: Khoram bid, Khorami village are placed in four sections (Fries 1923). Therefore sectional E. ilicifolius Bunge Yazd: Ardakan classification of Echinops is difficult due to great morphologi- E.ilicifolius Bunge Yazd: Mehriz cal uniformity. Four species of Echinops are spread through- E.ilicifolius Bunge Yazd: Bafgh out Iran (Rechinger 1979). Molecular studies as well as mor- E.pungens Boiss Markazi: Komijan phological studies emphasize that this genus has a taxo- E.pungens Boiss Markazi: Tafresh nomic problem. (Garnatje et al. 2005, Sánchez-Jiménez et al. E. chardinii Boiss &Bushe. West Azerbaijan: Urmia 2010). The most common chromosome numbers in Echinops E. chardinii Boiss &Bushe. West Azerbaijan: Naghadeh are 2n= 28, 30, 32 and less common is 2n= 34 (Sheidai et al. 2000). Karyotype studies of Echinops members are recorded Cytological studies in Balkan region (Sanchez-Jimenez et al. 2012), world (Ga- For Karyological study freshly grown root tips were collected from rantje et al. 2004a, b, Goldblatt & Johnson 1983, Strid & Fran- the seeds of at least ten randomly selected in each species, zen 1981, Moore 1982) and in Iran by Ghaffari (1982) and pretreated with 0.002 mol/L 8-hydroxyquinolin and fixed in abso- Sheidai et al (2000). There are B chromosomes of the Echinops lute ethanol: glacial acetic acid (3:1) for 24 hrs. The fixed tips were grijsii Hance (Peng 1986). Sheidai et al. (2000) Using somatic then washed thoroughly in distilled water and hydrolyzed in 40°C chromosome number as a criterion for section delimitation, 1N HCl for about 18 min. Squash technique was used the root tips the transfer of E. gedrosiacus, E. cyanocephalus and E. sojakii which were stained with 2% aqueous aceto-orcein. The somatic Karyological study of six Echinops L. species from Iran 67 chromosome number and karyotype details were studied in at least 5 chromosome length occurred in Anjileh population of E. ori- well-prepared metaphase plates. The chromosomes were photo- entalis (2.98μm), while the highest value occurred in E. ilici- graphed by digital camera and measured. The chromosomes were folius (Bafgh) (Table 2). The highest value of Coefficient of identified according to Levan et al. (1964) , karyotype symmetry was Variation (CV= 30) for chromosome size was recorded in E. determined according to Stebbins (1971), while other karyotype pa- rameters like total form percentage (TF %), coefficient of variation robustus, indicating the highest degree of size variation (CV) of the chromosome size as well as A1 and A2 indices according among its chromosomes, while the least CV=18 value was to Romero-Zarco (1986) Total chromosome length (TL), Size of the registered in E. orientalis (Anjileh). The total form percentage longest chromosome pair(L), Size of the shortest chromosome value (TF%) varied from 41% in E. robustus (Golpaighan), E. pair(S), Mean chromosome length(X), Stebbins symmetry class (ST) ilicifolius (Ardakan and Bafgh) to 44% in E. orientalis (An- and Karyotype formulae(KF) were determined. Grouping of the taxa jileh), E. pungens (Tafresh) and E. chardinii (Naghadeh) a was performed using clustering methods (single linkage and higher value of TF% indicates the presence of a relatively WARD) and ordination of taxa on the first two principal components more symmetrical karyotype. The chromosomes of the stud- axes (PCA) (Sheidai & Jalilian 2006). Pearson correlation among karyotype characters and PCA were ied species were mostly categorized in m (metacentric) and performed with PAST software (Hamer 2012). sm (sub-metacentric) chromosome types (Table 2). Also, Karyotype formulae of studied species are different (Table 2). According to Stebbins’ karyotype classification, these Results species fall into 3A and 3B classes among species that are placed in class 3B E. ilicifolius shows a higher value of A1 in- Karyotype analysis dex (0.29) of Romero Zarco. In Table 2, the chromosome numbers and Karyotype fea- A PCA analysis of the karyotype characters revealed that tures of the studied Echinops species are presented. Figs 1 & the two PCA components comprised about 80 % of the total 2 show Idiograms and chromosome morphology of studied variation. The TL (total chromosome length) (>0.9) and x species. E. ilicifolius (Taft, Khorami, Ardakan, Mehriz, (Mean chromosome length) characters are the first PCA axis Bafgh), E. punges (Kamijan, Tafresh), E. orientalis (Anjileh) component, L (Size of the longest chromosome pair) and L/S have chromosome numbers 2n=2x=30, E. robustus (Golpai- (Size of the longest chromosome pair/Size of the shortest gan), E. chardinii (Urmia, Naghadeh) 2n=2x=34 and E. po- chromosome pair) characters are the second PCA axis com- lygamous (Semnan) 2n=2x=32. The lowest haploid and mean ponent. Therefore, these characters are the most variable

Table 2: Karyotype features of the Echinops species and population studied.

Species Locality 2n TL L S L/S X ST A1 A2 CV TF KF (µm) (µm) (µm) (µm) E. robustus Bunge Golpaigan 34 79.27 7.38 2.68 2.75 4.66 3B 0.28 0.30 30 41 17m E. polygamus Ban. Semnan 32 66.3 6.20 1.72 3.60 4.15 3B 0.26 0.29 29 42 14m+2sm E. orientalis Trautv Anjileh 30 44.72 3.88 2.03 1.91 2.97 3A 0.21 0.18 18 44 15m E. ilicifolius Bunge Taft 30 53.6 5.45 1.73 3.16 3.57 3B 0.24 0.27 27 42 15m E. ilicifolius Bunge Khorami 30 56.42 5 2.55 1.96 3.76 3A 0.24 0.20 20 42 15m E. ilicifolius Bunge Ardakan 30 63.7 6.12 2.65 2.31 4.25 3B 0.29 0.23 23 41 1sm+14m E. ilicifolius Bunge Mehriz 30 63.11 6 2.6 2.31 4.21 3B 0.23 0.23 23 43 1sm+14m E. ilicifoliusB unge Bafgh 30 83.62 8.38 3.4 2.47 5.57 3B 0.28 0.25 25 41 1sm+14m E. pungensBoiss Kamijan 30 59.64 6.1 2.25 2.71 3.98 3B 0.24 0.27 27 42 15m E. pungensBoiss Tafresh 30 51.51 5.02 2.30 2.18 3.43 3B 0.21 0.24 24 44 15m E. chardinii Boiss &Bushe. Urmia 30 63.7 5.23 2.02 2.59 3.41 3B 0.17 0.27 27 43 17m E. chardinii Boiss&Bushe. Naghadeh 34 52.48 4.41 1.81 2.43 3.09 3B 0.19 0.27 27 44 17m

Abbreviations: TL = Total chromosome length, L = Size of the longest chromosome pair, S = Size of the shortest chromosome pair, X = Mean chromo- some length, ST =Stebbins, symmetry class, A1 & A2 = Romero- Zarco indices, CV =coefficient of variation, TF% = total form percentage, KF = Karyotype formulae.

Figure 1/a. Karyotypes of E. robustus (2n = 34), E. polygamous (2n = 32), E. orientalis (2n = 30). Scale bar = 10 μm. 68 B. Alijanpoor et al.

Figure 1/b. Karyotypes of E. ilicifolius (2n = 30), E. chardini (2n =34), E. pungens. (2n=30) Scale bar = 10 μm.

Figure 2. Idiograms of E. robustus, E. polygamous, E. orientalis, E. ilicifolius, E. pungens, E. chardini Karyological study of six Echinops L. species from Iran 69

Figure 3. Ward dendrogram of Echinops species based on karyotype data. Abbreviations: 1: E. robustus, 2: E. polygamous, 3: E. orientalis, 4-8: E. ilicifolius, 9, 10: E. pungens, 11, 12: E. chardinii . sec.Olig: section Oligo- lepis, sec. Rit: section Ritropsis, sec. Ech: section Echinops.

karyotype features. Pearson correlation determined among 2n=2x=30 was recorded for E. pungens (syn. E. persicus, E. the karyotype features showed a positive significant correla- adenocaulis) which is in accordance with a previous report. tion between the TL and S (size of the shortest chromosome (Garnatje et al. 2004a) pair) (r >0.68, p<0.05). Therefore, the significant quantitative In the present study, the chromosome count reported for change in the chromatin material has occurred in the size of E. orientalis, E. pungens that agrees with previous report only short chromosome arm during the species diversifica- (Garnatje et al. 2004a) While chromosome number of E. ro- tion. Also, total chromosome length and total form percent- bustus, E. polygamous, E. ilicifolius, E. chardinii are reported for age had negative correlation (r<0.76, P<0.01). Different clus- the first time. Also 2n=2x=34 is reported for the first time in tering methods and ordination plot of the Echinops species sec. Oligolepis whereas 2n= 28, 30 were previously recorded based on karyotype data were similar. Therefore, Ward den- for sec. Oligolepis (Sheidai et al. 2000, Garnatje et al. 2004a). drogram is discussed here (Fig. 3). In general, two major Due to structural changes of chromosomes during Echinops clusters are formed; (1) The first cluster includes sec. species diversification, variation occurs in the size of chro- Oligolepisis (2) The second cluster consists of two sub- mosomes, type of chromosomes, TF%, CV and Stebbins clusters that sec. Ritropsis, sec. Echinops and sec. Oligolepisis symmetry class. Pearson correlation among the karyotype are located in different locations in dendrogram. The species features shows that increase in somatic chromosome number of sections did not locate together in the one clade for is accompanied to increasing in degree of asymmetry and example sec. Oligolepisis seen in various situations of chromosomes size changes. The mean chromosome size de- dendrogram so sec. Ritropsis and sec. Echinops are located in creases significantly which is due to loss of chromatin. On two different locations in dendrogram. Cluster analysis of the basis of sheidai et al. (2000) variation observed in basic karyotype features indicated that cytological studies did not chromosome number indicates the possible role played by support sectional classification. (Fig. 3). centric fusion/fission during species diversification and it is not possible to indicate which number is the primitive one. According to Stebbins (1971), symmetrical karyotypes are Discussion more primitive than asymmetrical ones and usually chroma- tin loss provides changes in symmetry. Most of the taxa Sheidai et al. (2000) reported 2n=2x=28 for E. orientalis in studied of this genus are 3B Class that it is asymmetrice, the populations of Fars province that’s one of the southeast area occurrence of asymmetric karyotypes is probably because of of country while we are reported 2n=2x=30 in populations of Consequence chromosomal structural changes, especially Qom province that’s located in the central area of Iran. Shei- centric fusion/fission. Sheidai et al. 2000 proposed that dai et al. (2000), also, reported TL: 29, L: 2.8, S: 1.6, X: 2, TF: changes in chromosome number in Iranian Echinops have oc- 44.2, ST: 2A the same species (E. orientalis) while we are re- curred via centric fusion or fission. Most of the species in the ported TL: 44.72 L: 3.88, S: 2.03, X: 2.98, TF: 44, ST: 3A in Asteraceae family are recorded to have the basic chromo- populations of Qom province. Garnatje et al. (2004b) re- some number x = 9. Garnatje et al. (2004b) suggested x = 8 as ported 2n=2x=30 for population of Poland E. orientalis indi- the ancestral basic chromosome number. On the other hand, cating the role of aneuploidy and geographical distance in during polyploidization, genome downsizing often occurs cytological evolution of Echinops. In the present study, (Semple & Watanabe 2009, Leitch et al. 2008). Sapci et al. 2n=2x=32 was found for E. polygamous, 2n=2x=34 for E. char- (2015) reported 2n = 34 in E. emiliae, as well as 2n = 36 in E. dini, 2n=2x=30 for E. ilicifolius and 2n=2x=34 was reported transcaucasicus Iljin (Sánchez-Jiménez et al. 2010) that would for E. robustus for the first time. The chromosome number rather support an ancient basic chromosome number of x =

70 B. Alijanpoor et al.

Figure 4. PCA analysis based on Karyotype features (10 quantitative and 2 qualitative) Echinops robustus, E. polygamous, E. orientalis, E. ilicifolius, E. pungens, E. chardini.

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