DOI: http://dx.doi.org/10.22092/ijb.2014.6154 New CHROMOSOME COUNTS (ASTERACEAE) IN IRAN OF SIX TAXA OF CARDUEAE M. R. Parishani, M. R. Rahiminejad, M. Mirtadzadini & H. Saeidi Received 2013. 12. 11. Accepted for publication 2014.03.01 Parishani, M. R., Rahiminejad, M. R., Mirtadzadini, M. & Saeidi, H. 2014. 06. 31: New chromosome counts of six taxa of Cardueae ( Asteraceae) in Iran.-Iran. J. Bot. 20 (1): 29-35. Tehran. In this study, the chromosome numbers of six Iranian taxa belonging to tribe Cardueae were counted. The chromosome numbers in Jurinea cartilaginea Mozaff. 2n=30, J. carduiformis (Juab. & Spach) Boiss. 2n=34, Aegopordon berardioides Boiss. 2n=34, Nikitinia leptoclada (Bornm. & Sint.) Iljin 2n=30, Centaurea amadanensis Schultz-Bip. var. gymnoclada (Jaub. & Spach) Wagenitz 2n=18 and Cousinia argentea Mehregan & Assadi 2n=26 were determined. Of the chromosome counts reported four are new to the world. Based on this study, three taxonomic changes are confirmed and a new suggestion is presented here. Mohammad Reza Parishani (correspondence <mrr@sci.ui.ac.ir >), Mohammad Reza Rahiminejad, & Hojatollah Saeidi, Department of Biology, Faculty of Science, University of Isfahan, Isfahan, Iran. - Mansour Mirtadzadini, Department of Biology, Faculty of Science, University of Shahid Bahonar, Kerman, Iran. Key words: Cardueae; chromosome number; Asteraceae; Iran ‫ )ﺗﻴﺮه ﻛﺎﺳﻨﻲ( در اﻳﺮان‬Cardueae ‫ﺷﻤﺎرش ﻫﺎي ﻛﺮوﻣﻮزوﻣﻲ ﺟﺪﻳﺪ ﺑﺮاي ﺷﺶ آراﻳﻪ از ﻃﺎﻳﻔﻪ‬ .‫ داﻧﺸﻜﺪه ﻋﻠﻮم داﻧﺸﮕﺎه اﺻﻔﻬﺎن‬،‫ ﮔﺮوه زﻳﺴﺖ ﺷﻨﺎﺳﻲ‬،‫ داﻧﺸﺠﻮي دﻛﺘﺮي‬،‫ﻣﺤﻤﺪ رﺿﺎ ﭘﺮﻳﺸﺎﻧﻲ‬ .‫ داﻧﺸﻜﺪه ﻋﻠﻮم داﻧﺸﮕﺎه اﺻﻔﻬﺎن‬،‫ اﺳﺘﺎد ﮔﺮوه زﻳﺴﺖ ﺷﻨﺎﺳﻲ‬،‫ﻣﺤﻤﺪ رﺿﺎ رﺣﻴﻤﻲ ﻧﮋاد‬ .‫ داﻧﺸﻜﺪه ﻋﻠﻮم داﻧﺸﮕﺎه ﻛﺮﻣﺎن‬،‫ اﺳﺘﺎدﻳﺎر ﮔﺮوه زﻳﺴﺖ ﺷﻨﺎﺳﻲ‬،‫ﻣﻨﺼﻮر ﻣﻴﺮﺗﺎج اﻟﺪﻳﻨﻲ‬ .‫ داﻧﺸﻜﺪه ﻋﻠﻮم داﻧﺸﮕﺎه اﺻﻔﻬﺎن‬،‫ داﻧﺸﻴﺎر ﮔﺮوه زﻳﺴﺖ ﺷﻨﺎﺳﻲ‬،‫ ﺳﻌﻴﺪي‬...‫ﺣﺠﺖ ا‬ Jurinea cartilaginea ‫ اﻋﺪاد ﻛﺮوﻣﻮزوﻣﻲ ﺑﺮاي‬.‫ در اﻳﺮان ﺷﻤﺎرش ﮔﺮدﻳﺪ‬Cardueae ‫در اﻳﻦ ﭘﮋوﻫﺶ ﻋﺪد ﻛﺮوﻣﻮزوﻣﻲ ﺷﺶ آراﻳﻪ از ﻃﺎﻳﻔﻪ‬ ‫ ؛‬Aegopordon berardioides Boiss. 2n = 34 ‫ ؛‬J. carduiformis (Juab. & Spach) Boiss. 2n=34 ‫ ؛‬Mozaff. 2n=30 Centaurea amadanensis Schultz-Bip. var. gymnoclada (Jaub. & ‫ ؛‬Nikitinia leptoclada (Bornm. & Sint.) Iljin 2n=30 ‫ ﭼﻬﺎر ﻣﻮرد از اﻋﺪاد ﻛﺮوﻣﻮزوﻣﻲ‬.‫ ﺑﻪ دﺳﺖ آﻣﺪ‬Cousinia argentea Mehregan & Assadi 2n = 26 ‫ و‬Spach) Wagenitz 2n=18 ‫ ﺑﺮ اﺳﺎس اﻳﻦ ﻣﻄﺎﻟﻌﻪ ﺳﻪ ﺟﺎﺑﻪﺟﺎﻳﻲ ﻣﻮﻗﻌﻴﺖ ﺗﺎﻛﺴﻮﻧﻮﻣﻲ ﺗﺄﻳﻴﺪ و ﻳﻚ ﺗﻐﻴﻴﺮ‬.‫ ﮔﺰارشﻫﺎي ﻛﺮوﻣﻮزوﻣﻲ ﺟﺪﻳﺪي ﺑﺮاي دﻧﻴﺎ ﻣﻲ ﺑﺎﺷﻨﺪ‬،‫اراﻳﻪ ﺷﺪه‬ .‫ﻣﻮﻗﻌﻴﺖ ﺳﻴﺴﺘﻤﺎﺗﻴﻚ ﭘﻴﺸﻨﻬﺎد ﻣﻲﺷﻮد‬ INTRODUCTION The rediscovery of Mendel’s Laws in the year 1900 caused the formulation of ‘chromosome theory of inheritance’ and the foundation of cytogenetic. In the last few decades of the last century, the interest in plant cytogenetic that combined study and use of structural and numerical changes of chromosomes largely declined. However, with reviving the interests in chromosomal data in a new molecular form, plant cytogenetic had a re-birth (Gupta 2006). Chromosomal data such as number, morphology, staining characteristics and behavior of chromosomes profoundly affected the evaluation of taxonomic investigations (Stace 2000). The Asteraceae contain the largest number of described species of any plant family, 24,000-30,000, distributed in 1600-1700 genera occurring on all continents except Antarctica. In Asteraceae (Compositae), chromosome 30 IRAN. J. BOT. 20 (1), 2014 Table 1. The list of studied taxa in present survey (All samples were collected by Parishani and Mirtadzadini). Genera and species are arranged in alphabetical order. No Taxon Collection site & altitude (meter) Coordinates Herbarium vouchers 1 Aegopordon berardioides Boiss. Isfahan, Khomeinishahr, Ladour fountain, N 32º42‫׳‬35‫״‬ 18096 HUI 1981 m E 51º26‫׳‬53‫״‬ 2 Centaurea amadanensis Schultz-Bip. Kordestan, Saqqez to Marivan, after Soote N 35º59'42" 18097 HUI var. gymnoclada (Jaub. & Spach) village, 1747 m E 46º25'56" Wagenitz 3 Cousinia argentea Mehregan & Khorasan, Torbat-Heydariyeh to N 34º52'51" 18098 HUI Assadi Mashhad, near to Robot-Sefid, 1100 m E 47º23'31" 4 Jurinea carduiformis (Jaub. & Spach) Isfahan, University of Isfahan, 1743 m N 32º35‫׳‬45‫״‬ 18101 HUI Boiss. E 51º39‫׳‬32‫״‬ Kerman, East Kerman, 1220m N 30º17‫׳‬30‫״‬ 18102 HUI E 57º01‫׳‬59‫״‬ 5 J. cartilaginea Mozaff. Khuzestan, Behbahan to Ramhormoz, N 31º00‫׳‬14‫״‬ 18099 HUI Maghar strait, 944 m E 50 º06‫׳‬20‫״‬ 6 Nikitinia leptoclada (Bornm. & Sint.) Khorasan, north west Bojnord, between N 37º44'51" 18103 HUI Iljin Pish Ghalae and Tanghe Torkaman, 808 E 56º56'28" m number range changes from n=2 to high level of polyploidy with n=114 (Funk et al. 2005). This family includes 43 tribes that Cardueae is one of them. The Cardueae is monophyletic and based on the molecular survey, includes five sub-tribes (Carlininae, Echinopsinae, Carduinae, Centaurinae, and Cardopatiinae) (Susanna and Garcia-Jacas 2007, Funk et al. 2009). Jurinea Cass. and Aegopordon Boiss. from JurineaSaussurea group of Carduinae, Cousinia Cass. from the Arctium-Cousinia group of Carduinae and, Centaurea L. and Nikitinia Iljin form the Centaurinae belong to the tribe Cardueae (Häffner 2000, Susanna et al. 2003, Funk et al. 2009). Although, until render the article of Susanna et al. (2002), Nikitinia Iljin was known one Carduinae, but then it was recognized as one Centaureinae. Based on the latest molecular investigation (Susanna et al. 2006), Outreya carduiformis Juab. & Spach is transferred to Jurinea and also Jurinea pungens Boiss. was transferred to Cousinia on the basis of the study of Mehregan and Assadi (2009). Jurinea cartilaginea was newly added to the Iranian list of this genus (Mozaffarian 1988). The several reports of cytological studies on Cardueae in Iran have been investigated for different taxa that in some of these cases, only a simple chromosome counts has been studied (Afzal-Rafii 1980, Amirimoqadam et al. 2011, Aryavand 1975 and 1977, Bakhshi-Khanaki, 1995a,1995b and 1996, Djavadi 2005, 2007 and 2012, Djavadi & Attar 2010, Djavadi et al. 2007, Djavadi & Ghaffari 1999, Estilai 1977, Ghaffari 1984, 1986, 1987a,b, 1988, 1989, 1999a, 1999b and 2006, Ghaffari & Chariat-Panahi 1985, Ghaffari & Djavadi 1998, Ghaffari et al. 2000, Ghaffari et al. 2006, Ghaffari & Kelich 2006, Ghaffari & Shahraki 2001, Ghaffari & Tajik 2007, Garcia-Jacas et al. 1998, Garcia-Jacas et al. 2009, Garnatje et al. 2004, Garnatje et al. 2006, Hellwig 1994, Hidalgo et al. 2007, Khidir & Knowles 1970, Lopez-Vinyallonga et al. 2009, Nouroozi et al. 2010, Ranjbar & Negaresh 2013, Romaschenko et al. 2004, Schank & Knowles 1964, Sheidai et al. 2000, Sheidai et al. 2006, Sheidai et al. 2009, Sheidai et al. 2012a &b, Sheidai et al. 2013, Susanna et al. 2003b, Vilatersana et al. 2000, Yazdani et al. 2013). The purpose of this investigation is to give information concerning the chromosome counts of angiosperm taxa of the Iranian flora and help to complete index of plant chromosome number. MATERIAL AND METHODS The chromosome counts were obtained from mitotic cells following Sharma and Sharma (2002). Root meristems from germinating cypsela collected in the wild were used. The germination duration was between 5 days to 2 weeks, depending on the species. Samples were pretreated with 0.002 M 8-hydroxyquinoline at 4◦C for 8 h. The material was fixed with Carnoy for 24 h at low temperature. Before staining, the material was hydrolyzed with 1 N HCl for 20 minutes at room temperature. It was stained with 1% aceto-orcein and mounted in 45% acetic acid (Sharma & Sharma 2002, Singh 2002). For all the counts, at least three plates from different individuals were examined. The best metaphase plates were photographed using a BX40 microscope (Olympus, Tokyo, Japan) with digital 31 camera attachment. The examined plant materials are presented in Table 1 that they were collected from different localities in Iran, in 2010-2013. The herbarium vouchers are preserved in the herbarium of Department of Biology at the University of Isfahan (HUI). IRAN. J. BOT. 20 (1), 2014 It exhibited 2n=30 (Table 1, Fig. 6). The monotypic genus is endemic to the Kopet Dag mountains in south of Turkmenistan and north east of Iran (Rechinger 1979, Häffner 2000). This is the first chromosome count for this species. DISCUSSION RESULTS The provided results of each species are given below: Jurinea cartilaginea Mozaff. This taxon is endemic to south west of Iran and is found in a limited area of north east of Khuzestan province (Mozaffarian 1988). The chromosome number in this species showed 2n=30 (Table 1, Fig. 1). According to our data, this is the first chromosome count for this species. J. carduiformis (Jaub. & Spach) Boiss. J. carduiformis is an endemic taxon in Flora Iranica range (Rechinger 1979). We studied two populations of this taxon, one from a population of Kerman province, and another from a population of Isfahan province. It exhibited 2n = 34 (Table 1, Fig. 2) in both of the mentioned populations. This is the third count for this species. Our results did not agree with the previous reports of n = 16 by Ghaffari (1986 and 1988). This is a new different chromosome number for this taxon. Aegopordon berardioides Boiss. This taxon is endemic to Iran, Afghanistan and Pakistan (Rechinger 1979). It exhibited 2n=34 (Table 1, Fig. 3). In here mixoploidy is shown in the same preparations. Where diploid chromosomes (2n=2x=34) were counted, tetraploid cells (2n=4x=68) were observed also (Fig. 7). With this study, the chromosome number of the taxon is given for the first time. Cousinia argentea Mehregan & Assadi It is determined that the somatic chromosome number is 2n = 26 (Table 1, Fig. 4). This taxon is endemic to north east of Iran (Rechinger 1979, Mehregan & Assadi 2009). According to our data, the chromosome number of the taxon is reported here for the first time. Centaurea amadanensis Schultz-Bip. var. gymnoclada (Jaub. & Spach) Wagenitz . As far as we reviewed, this is the third count for this species, and also this is the first count for this variety. This variety is endemic to west of Iran according to Flora Iranica (Wagenitz 1980). In our new count, chromosome number observed is 2n = 18 (Table 1, Fig. 5) that is in accordance with the previous reports (Garcia-Jacas et al. 1998b, Ghaffari & Shahraki 2001). In this taxon, mixoploidy is observed in the same preparations. Where diploid chromosomes (2n=2x=18) were counted, tetraploid cells (2n=4x=36) were determined also (Fig. 8). Nikitinia leptoclada (Bornm. & Sint.) Iljin Variation in chromosome number is probably through hybridization, euploidy, and aneuploidy in Asteraceae (Funk et al. 2009, Häffner 2000). It may be suggested based on the data that the tribe Cardueae could be subdivided into two main phyletic groups which one with x = 9 and 8 and the second with x = 6, 5 and 4 (Missouri Botanical Garden, http://www.tropicos.org/ Project/IPCN; Watanabe 2013, http://www.lib.kobeu.ac.jp/products/asteraceae/e-index.html). Nevertheless, the chromosome numbers differ considerably among different genera of the tribe and the differences are correlated with appreciable morphological variability and these characters will render them useful in construction of general classification. Chromosomes data are capable to provide information to confirm natural boundaries for generic delimitations. Akhtar (2005) argued that chromosomal features are basic to an understanding of relationships in Cardueae and it is more useful at generic level. Based on the chromosome numbers reported of the genus Jurinea (Missouri Botanical Garden, http://www.tropicos.org/Project/IPCN; Watanabe 2013, http://www.lib.kobe-u.ac.jp/products/asteraceae/eindex.html), 2n=30 in Jurinea cartilaginea is not farfetched. Nonetheless, the suggestion of changing systematic position of this species and the possibility of its separation from Jurinea or at least changing its section, can be taken into account on the bases of the basic chromosome number proposed for Jurinea (n=x=17). The above suggestion can be strengthened based on the fact that J. cartilaginea is unique for some of its cypsela features such as the mucilage coat of cypsela during soaking (which does not happen in the other Iranian Jurinea species), the inner papus of 5-7 long bristles with a broader base than the base of the other bristles, and pentagonal structure which are unique among the other Jurinea species. On the other hand, this reduction in basic chromosome number (n=x=17 compare with n=x=15) may be related to climatic and ecosystem conditions of the habitat of this taxon. Because this plant lives in a semi-arid environment, very warm (at summer) and partly mountainous; likely this descending dysploidy could be correlated with taxon adaptation to the warm and xeric conditions (the same reports in Selvi & Bigazzi 2002, Watanabe et al. 1999). IRAN. J. BOT. 20 (1), 2014 32 Figs. 1-6. Somatic metaphases: 1- Jurinea cartilaginea 2n=30; 2- J. carduiformis 2n=34; 3-Aegopordon berardioides 2n=34; 4- Cousinia argentea 2n=26; 5-Centaurea amadanensis var. gymnoclada 2n=18; 6- Nikitinia leptoclada 2n=30. Figs. 7-8. Polyploidy in Somatic metaphases: 7- Aegopordon berardioides 2n=4x=68; 8- Centaurea amadanensis var. gymnoclada 2n=4x=36. Susanna et al. (2006) transferred Outreya carduiformis to Jurinea carduiformis on the bases of molecular similarity. Based on the chromosome numbers reported for Jurinea which mostly are 2n=34 (Missouri Botanical Garden, http://www.tropicos.org/Project/ IPCN; Watanabe 2013, http://www.lib.kobeu.ac.jp/products/asteraceae/e-index. html), the exhibited chromosome number in our study for this species (2n=34) can confirm this changing. The obtained chromosome number for Aegopordon berardioides in the present study (2n = 34) shows more similarities between the two genera Aegopordon and Jurinea, which can be taken as evidence for a systematic change of Aegopordon berardioides to Jurinea berardioides, a suggestion provided by Susanna et al. (2006) on the basis of a molecular investigation (of course, it was not indexed in IPNI yet). It was mentioned in the introduction that Mehregan and Assadi (2009) transferred Jurinea pungens to their nomen novum Cousinia argentea. According to the chromosome number given for Cousinia argentea in the present study, we can confirm their decision. This is in accordance with 2n=26 and n = 13 for Cousinia reported by: Afzal-Rafii, (1980), Djavadi (2005, 2007, 2012), Djavadi & Attar (2010), Djavadi & Ghaffari (1999), Ghaffari (1984, 1986a), Ghaffari & Chariat- 33 Panahi (1985), Ghaffari & Djavadi (1998), Ghaffari et al. (2006), Lopez-Vinyallonga et al. (2009a), Sheidai et al. (2006), Sheidai et al. (2012) and Susanna et al. (2003b). Considering the chromosome numbers reported for the section Paraphysis and the other related sections of the genus Centaurea (Bakhshi-Khanaki 1995a,1995b and 1996, Ghaffari 1986, 1988 and 1989, Ghaffari & Shahraki 2001, Garcia-Jacas et al. 1998, Ranjbar & Negaresh 2013), we concluded that the common basic chromosome number in these sections can be x=9. Also, the chromosome number obtained for Centaurea amadanensis var. gymnoclada in this research supports this issue. At 1960, Iljin changed Jurinea leptoclada to Nikitinia leptoclada based on the different morphological characters such as receptacle subcylindrical, warty filaments and multi-seriate pappus. Then Susanna et al. (2002) did another systematic change in the position of the subtribe of this taxon. They transferred it from Carduinae to Centaureinae based on a study of pappus characters. The diploid number (2n=30) along with the considerably small size of the chromosomes comparing to that of the Jurineas’ confirm the changes offered above, surely. Based on our study we suggest that the dysploidy and polyploidy can be regarded as the main mechanisms of chromosomal evolution in Cardueae. Indisputable, detailed karyotype analyses combined with molecular studies can help to overcome the unresolved taxonomic confusions of Cardueae plants. 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