Contribution to Cytotaxonomy of Iranian Cirsium (Asteraceae)

Maryam Nouroozi1, Masoud Sheidai1,*, Farideh Attar2 and Zahra Noormohammadi3

1 Shahid Beheshti University, GC., Faculty of Biological Sciences, Tehran, Iran 2 School of Biological Sciences, University of Tehran, Tehran, Iran 3 Biology Department, School of Basic Sciences, Science and Research Branch, Islamic Azad University (SRBIAU), Poonak, Tehran, Iran

Received February 5, 2010; accepted February 19, 2010

Summary A meiotic study was performed on 21 populations of 17 Cirsium species growing in Iran. The species of C. spectabile, C. congestum, C. strigosum, C. ciliatum, C. osseticum, C. aduncum, C. haussknechtii, C. turkestanicum, C. echinus, C. obvallatum, C. libanoticum, C. alatum, C. echinus, C. obvallatum, C. libanoticum, C. hygrophilum and C. arvense had 2nϭ2xϭ34 chromosome number, while the species of C. lappaceum, C. vulgare and C. elodes showed 2nϭ4xϭ68. The chromosome numbers of C. spectabile, C. strigosum, C. haussknechtii, C. lappaceum, C. turkestanicum and C. libanoticum are new to science and a new polyploidy level (4x) has been reported for C. elodes. The ANOVA test revealed significant differences for chiasma frequency and chromosome pairing among the species studied and the Pearson coefficient of correlation determined showed significant positive correlation between intercalary chiasmata and the mean number of quadrivalents. Although diploid species and populations are expected to form only bivalents in metaphase I, quadrivalents were formed in C. arvense, C. ciliatum, C. congestum, C. echinus, C. strigosum and C. libanoticum possibly due to the occurrence of heterozygote translocations. Interestingly enough, tetraploid species of C. lappaceum did not form any quadrivalents showing diplontic behavior. The occurrence of large pollen grains (possibly 2n pollen grains) was observed along with smaller (normal) pollen grains in species and populations showing tripolar cells. Cytological characteristics may be used in species delimitation in Cirsium but may not be used in sectional delimitation of the genus.

Key words Cirsium, Heterozygote translocation, Polyploidy.

The genus Cirsium Mill. (Asteracea) contains about 250 perennial, biennial or rarely annual and spiny species (Zomlefer 1994, Bures et al. 2004, Segarra-Moragues et al. 2007). Cirsium species mainly grow in the Northern hemisphere, occupying various habitats, but are also distribu- ted in Europe, North Africa, Siberia, Central Asia, West and East Africa, and Central America. The genus is considered to be taxonomically complex due to the variability and intergradation of diagnostic characters among taxa (Dabydeen 1980). The complex nature of the genus may be partly due to high degree of hybridization occurring among different species and also because of highly variable germplasm interacting with different environmental conditions. Several hybrids have been described from Caucasus and adjoining regions of Asia minor (Ownbey 1951, Moore and Frankton 1974, Charadze 1998, Bures et al. 2004). About 44 Cirsium species have been reported in Flora Iranica (Rechınger 1979), which have been classified in 5 sections. Cirsium is a typical example of a genus with a high affinity to form natural interspecific hybrids (Wagenitz 1987). Hybrid plants persist primarily through vegetative growth, forming clusters of flowering shoots often connected by rhizomes. Because hybrids are usually fertile, they

* Corresponding author, e-mail: [email protected]

119_127.pdf 1 10.5.26 9:11:23 AM 120 M. Nouroozi et al. Cytologia 75(1)

can often produce introgressive hybrids with the parental species or triple hybrids with other taxa (Wagenitz 1987). Cytotaxonomical studies have been considered useful in showing species relationships, taxonomic delimitation and genetic differences in several plant groups including grass genera of Aegilops (Sheidai et al. 1999b, 2000, 2002), Bromus (Sheidai and Fadaei 2005, Sheidai and Nouroozi 2005, Sheidai and Nouroozi 2006. Sheidai et al. 2008), Stipa (Sheidai and Attaei 2005, Sheidai et al. 2006), Festuca (Sheidai and Bagheri-Shabestarei 2007), and Hordeum (Sheidai and Rashid 2007), as well as in other plant groups like Asparagus (Sheidai and Inamdar 1997), Hyoscyamus (Sheidai et al. 1999a) and Echinops (Sheidai et al. 2000). Available literature shows that limited cytological studies have been performed on the genus Cirsium, (Frankton and Moore 1961, Tonian 1981a, b, 1982, Dempsey 1994, Ghaffari 1999, Ozcan et al. 2008) and similar studies are almost entirely lacking from Iran. Therefore, the present study considers meiotic analysis in 21 populations of 11 Cirsium species of Iran considering chiasma frequency, chromosome pairing and meiotic abnormalities leading to unreduced gamete formation.

Materials and methods Plant material Meiotic studies were performed in 21 populations of 11 Cirsium species belonging to 4 sections (Table 1), all growing in Iran, namely: 1-Sect. Psudoepitrachys Petrak, Cirsium spectabile DC. E., C. congestum Fitch & C.A. Mey, C. congestum Fitch and C.A. Mey, 2-Sect. Epitrachys DC., C. strigosum (M.B) M.B., C. ciliatum (Murray) Moench, C. osseticum (Adams ) Petrak, C. aduncum Fitch and C.A. Mey, C. haussknechtii Boiss., C. lappaceum M.B., C. turkestanicum (Regel) Petrak, C. vulgare (Savi) Ten., 3-Sect. Echenais (Cass.) Petrak, C. echinus (M.B.) Hand. Mzt., 4-Sect. Cirsium Petrak, C. obvallatum (M.B.) M.B., C. libanoticum DC., C. alatum (S.G. Gmelin) Bobrov, C. elodes M.B., C. hygrophilum Boiss., 5-Sect. Cephalonoplos (Necker) DC., C. arvense (L.) Scop. The voucher specimens are deposited in the Herbarium of Shahid Beheshti University (HSBU) and the Herbarium of Tehran University (HTU).

Cytological studies Meiotic studies were performed on young ﬂower buds collected using minimum 100 metaphase/diakinesis pollen mother cells (PMCs) and 500 anaphase and telophase cells for data collection (Sheidai and Rashid 2007). Pollen satiability as a measure of fertility was determined by staining minimum 1000 pollen grains with 2% acetocarmine: 50% glycerin (1 : 1) for about 0.5 h. Round complete pollens which were stained were taken as fertile, while incomplete, shrunken pollens with no stain were considered infertile (Sheidai and Rashid 2007).

Statistical analyses In order to study the species relationship based on cytogenetic similarities, relative data (each cytogenetic parameter divided by the chromosome number) was used. Such relative data makes possible the study of the species having different chromosome number (Sheidai et al. 2006). In order to reveal signiﬁcant difference in chaisma frequency and chromosome pairing data, the analysis of variance (ANOVA) followed by the least signiﬁcant difference test (LSD) were performed among the species and populations studied (Sheidai et al. 2006). Cytological distinctness of the species studied was checked by using different clustering methods of ordination plotting of principal components analysis (PCA) (Sheidai et al. 2006). A consensus tree was obtained from the different clustering analyses performed. Statistical analyses used SPSS ver. 9 (1998) and DARwin ver. 5.0.155 (2006) software.

119_127.pdf 2 10.5.26 9:11:24 AM 2010 Contribution to Cytotaxonomy of Iranian Cirsium 121

Fig. 1. Representative meiotic cells in Cirsium species studied. AϭDiakinesis cell in Asbdavani population of C. ehinus showing ring and rod bivalents. BϭMetaphase I cell in Gachsar population of C. congestum showing ring and rod bivalents. CϭMetaphase I cell in Givi population of C. ciliatum showing bivalents and univalents. DϭDiakinesis cell in Shabil population of C. obvallatum showing bivalents and univalents. EϭMetaphase I cell in Gachsar population of C. congestum showing univalents. FϭMetaphase I cell in Meshkinshahr population of C. lappaceum showing bivalents. GϭAnaphase I cell in Asbdavani population of C. ehinus showing laggard chromosomes. HϭTripolar cell in Asbdavani population of C. ehinus. IϭTripolar cell in Mishodagh population of C. libanoticum. JϭTripolar cell in Manjil population of C. congestum. K, LϭUnreduced pollen grains (bigger size) in Rasem population of C. elodes and Manjil population of C. congestum. Scale barϭ10 mm.

Results The species of Cirsium spectabile, C. congestum, C. strigosum, C. ciliatum, C. osseticum, C. aduncum, C. haussknechtii, C. turkestanicum, C. echinus, C. obvallatum, C. libanoticum, C. alatum, C. echinus, C. obvallatum, C. libanoticum, C. hygrophilum and C. arvense had 2nϭ2xϭ34 chromosome number while the species of C. lappaceum, C. vulgare and C. elodes showed 2nϭ4xϭ68 (Fig. 1A–F), supporting the earlier reports on C. congestum (Tonian 1982), C. ciliatum (Ozcan et al. 2008), C. osseticum (Tonian 1982), C. aduncum (Tonian 1982), C. vulgare (Dempsey 1994), C. echinus (Tonian 1981a), C. obvallatum (Tonian 1981b), C. alatum (Ghaffari 1999), C. elodes (Tonian 1982), C. hygrophilum (Ghaffari 1999) and C. arvense (Ozcan et al. 2008). According to our knowledge, the chromosome number reports for C. spectabile, C. strigosum,

119_127.pdf 3 10.5.26 9:11:24 AM 122 M. Nouroozi et al. Cytologia 75(1)

Fig. 2. Consensus tree of Cirsium species based on relative cytological data. Species abbreviations: aduncϭC. aduncum, alatϭC. alatum, arvϭC. arvense, ciliaϭC. ciliatum, congϭC. congestum, echiϭC. ehinus, eldϭC. elodes, huaskϭC. haussknechtii, hygrϭC. hygrophilum, lapaϭC. lappaceum, libonatϭC. libanoticum, obaϭC. obvallatum, ossetϭC. osseticum, spectϭC. spectabile, strigϭC. strigosum, turkϭC. turkestanicum and vulgareϭC. vulgare.

Table 1. Cirsium species and populations studied and their localities in Iran

Species Voucher No. Localities

Cirsium spectabile 883000 Kashan, Ghohrood, 2403 m C. congestum 883001 Ghazvin-Rasht Road, 30 km Lowshan, 1300 m C. congestum 883002 Karaj-Chaloos road, Gachsar 2800 m C. strigosum 883003 Firoozkuh C. strigosum 883004 Zanjan, Gheidar 2091 m C. ciliatum 883005 Ardabil, Heiran, 1547 m C. ciliatum 883006 Khalkhal, Givi, 1836 m C. osseticum 883007 Firoozkuh, Gaduk, 2348 m C. aduncum 883008 Meshkinshahr, 1169 m C. haussknechtii 883008 Tabriz, Kandovan, Hilevar village, 1300 m C. lappaceum 883009 Meshkinshahr, 169 m C. turkestanicum 883010 Semnan C. vulgare 883011 Rasht, 25 m C. echinus 883012 Asalem to Khalkhal, Asbdavani, 1950 m C. obvallatum 883013 Sabalan, Shabil C. libanoticum 883014 Tabriz, Mishodagh, 1900 m C. alatum 883015 Kiamakidagh road, 736 m C. elodes 883016 Road of Haraz, 5 km Lasem, 1700 m C. hygrophilum 883017 Damavand, 1900 m C. arvense 883018 Tabriz, 1470 m C. arvense 883019 Arak, 1750 m

119_127.pdf 4 10.5.26 9:11:25 AM 2010 Contribution to Cytotaxonomy of Iranian Cirsium 123 Ring ϭ Terminal chiasmata per Terminal ϭ Total chiasmata, RIIN Total ϭ Terminal chiasmata, TOX Terminal ϭ Intercalary chiasmata per chromosome, TXN ϭ species studied Intercalary chiasmata, TX Cirsium ϭ Quadrivalent per cell, IXN Quadrivalent ϭ Quadrivalent, IX Quadrivalent, ϭ Meiotic characteristics of Univalent, IV Univalent, Table 2. ϭ Univalent per cell, IVN Univalent ϭ RII RD I IV IX TX TOX RIIN RDN IN IVN IXN TXN TOXN Rod bivalent, I Rod bivalent, ϭ n Rod bivalent per cell, IN Rod bivalent ϭ Total chiasmata per chromosome. Total Ring bivalent, RD Ring bivalent, ϭ ϭ ArakTabrizGiviHeiranManjil 34 34GachsarAsbdavani 13.88 34 34 12.03Firoozkooh 34Gheidar 3.02 34 34 3.77 5.67Gaduk 4.87 34 10.03Mishodagh 0.19 1.47 11.71 3.91 9.27 4.65Ghohrud 34 3.10 7.5Meshkinshahr 0.00 12.37 34 11.67 0.20 0.42 1.80 34Hilevar 13.7 34 2.77Shabil 0.50 0.12 0.51 34 0.40 0.20 0.13 0.49 2.12Kiamakidagh 5.03 14.23 30.64 0.40 1.82 0.84Damavand 28.20 0.37 0.24 14.55 0.50 2.51 0.95 34 34 11.97Semnan 31.14 0.00 14.64 0.00 28.4 4.25 21.50 0.51Meshkinshahr 34 13.63 21.70 0.18 0.29 34 0.00 0.82Rasht 2.23 3.04 0.00 1.00 1.00 25.41 22.00 20.54 68 22.60 21.96 0.71 1.48Rasem 0.061 2.35 34 14.77 0.00 13.83 0.18 19.10 29.69 1.67 21.02 0.00 0.00 22.13 0.28 17.33 0.33 0.10 0.22 14.7 0.00 20.10 0.00 15.21 0.00 0.25 19.83 0.11 0.59 4.13 0.23 16.52 0.40 0.27 19.00 0.69 68 0.54 0.08 0.49 68 22.07 19.83 0.00 0.17 0.00 12.87 0.25 0.00 18.16 19.10 0.00 0.44 0.73 0.00 0.68 0.02 17.43 18.03 0.11 22.07 0.012 0.16 0.00 18.20 0.81 0.00 0.008.34 0.00 0.00 0.13 0.03 0.01 16.3 0.03 18.49 0.00 0.02 0.01 0.008 0.029 0.29 19.23 24.86 19.92 0.84 0.05 0.11 0.03 0.00 0.00 0.85 1.80 0.049 0.03 0.022 0.15 5.67 0.05 0.014 1.66 0.00 19.23 0.70 19.92 19.3 0.11 18.5 0.00 0.86 0.00 0.25 0.00 0.03 50.17 0.01 0.02 1.83 1.26 0.80 1.28 1.67 0.13 0.13 0.00 0.18 19.35 21.130.23 18.50 0.00 55.79 0.06 0.04 1.49 1.21 1.29 0.00 1.29 0.08 1.33 0.73 0.87 21.13 0.00 0.81 0.14 0.23 0.10 0.00 0.51 0.00 1.02 1.75 1.27 1.30 0.00 51.13 0.00 0.00 0.2442.34 0.00 0.01 0.86 0.89 1.66 0.48 0.02 1.16 51.73 1.12 0.03 42.57 0.00 0.38 1.29 0.00 0.00 0.01 1.16 0.00 1.07 0.51 1.12 1.06 0.24 0.00 0.00 1.29 0.00 0.00 0.00 0.00 1.07 0.48 1.09 0.73 1.13 0.00 1.17 0.00 0.00 0.16 0.00 0.00 1.13 0.00 1.17 1.13 1.09 1.47 0.00 0.00 1.24 1.14 1.09 1.64 0.02 0.01 1.24 1.50 2.49 1.52 2.50 Species Locality 2 chromosome, TOXN bivalent per cell, RDN bivalent Abbereviations: RII Abbereviations: C. arvense C. arvense C. ciliatum C. ciliatum C. congestun C. congestum C. ehinus C. strigosum C. strigosum C. osseticum C. libanoticum C. spectabile C. aduncum C. haussknechti C. obvallatum C. alatum C. hygrophilum C. turkestanicum C. lappaceum C. vulgare C. elodes

119_127.pdf 5 10.5.26 9:11:26 AM 124 M. Nouroozi et al. Cytologia 75(1)

Table 3. ANOVA of meiotic characteristics

Character Sum of Squares df Mean Square F Sig.

Ring bivalent Between Groups 3484.53 20 174.22 26.09 0.01 Within Groups 527.46 79 6.67 Total 4012 99 Rod bivalent Between Groups 1617.53 20 80.87 11.97 0.01 Within Groups 533.46 79 6.75 Total 2151 99 Univalent Between Groups 67.84 20 3.39 2.149 0.01 Within Groups 124.80 79 1.57 Total 192.64 99 Quadrivalent I Between Groups 6.19 20 0.30 1.79 0.01 Within Groups 13.60 79 0.17 Total 19.79 99 Intercalary chiasmata Between Groups 1352.14 20 67.60 52.17 0.01 Within Groups 102.36 79 1.29 Total 1454.51 99 Terminal chiasmata Between Groups 9747.69 20 487.38 64.33 0.01 Within Groups 598.50 79 7.57 Total 10346.19 99 Total chiasmata Between Groups 15079.44 20 753.97 79.98 0.01 Within Groups 744.66 79 9.42 Total 15824.11 99

C. haussknechtii, C. turkestanicum, C. lappaceum and C. libanoticum are new to science. Tonian (1982) reported 2nϭ2xϭ34 for C. elodes, while we obtained 2nϭ4xϭ68 for this species; therefore we report a new polyploidy level for this species. Among those species with 2nϭ34 chromosome number, the highest value of mean total and terminal chiasmata occurred in the Arak population of C. arvense (31.14 and 30.64 respectively, Table 2), while the lowest value of the same occurred in the Meshkinshahr population of C. aduncum (18.20 and 18.16, respectively). Similarly, the highest value of intercalary chiasmta was observed in the Manjil population of C. congestum (4.25). Among the species showing 2nϭ68 chromosome number, the highest value of mean total and intercalary chiasmata occurred in the Meshkinshahr population of C. lappaceum (55.79 and 5.67, respectively, Table 2), while the lowest value of the same occurred in the Lasem population of C. elodes (42.57 and 0.23, respectively). Similarly, the highest value of terminal chiasmta was observed in the Rasht population of C. vulgare (51.13) while the lowest values occurred in the Lasem population of C. elodes (42.43). Diploid species studied formed mainly bivalents in metaphase of meiosis I, however, quadrivalents were formed in C. arvense, C. ciliatum, C. congestum, C. echinus, C. strigosum and C. libanoticum. Interestingly enough, tetraploid species of C. lappaceum did not form any quadrivalents showing diplontic behavior. Univalents were usually formed in most of the species studied, ranging from one to many (Fig. 1C–E). Such univalents may lead to laggard chromosome formation observed in anaphase stages (Fig. 1G). The ANOVA and LSD tests revealed significant differences (pϽ0.01) for chiasma frequency and chromosome pairing among the species and populations studied (Table 3). The pearson coefficient of correlation determined among cytological characteristics showed significant positive correlation between the mean number of ring bivalents and terminal, intercalary and total chiasmata (pϽ0.01). Similarly, the mean number of intercalary chiasmata was positively correlated with total chiasmata and the mean number of quadrivalents (pϽ0.01).

119_127.pdf 6 10.5.26 9:11:26 AM 2010 Contribution to Cytotaxonomy of Iranian Cirsium 125

Along with the laggard chromosomes observed in anaphase I and II, tripolar cells were observed in some of the species (Fig. 1H–J). The occurrence of large pollen grains (possibly 2n pollen grains) was observed along with smaller (normal) pollen grains in species and populations showing tripolar cells (Fig. 1K, L). The large pollen grains comprised about 1% of pollen grains in these populations. The grouping of the species based on relative meiotic characteristics by different clustering methods showed almost similar results with the highest cophenetic correlation for NJ and UPGMA methods (rϭ0.98). A consensus tree of these methods is presented in Fig. 2 As the results show, the species of the sections studied are not completely separated from each other; however, the species of C. hygrophilum, C. libanoticum, C. obvallatum and C. alatum from the sect. Cirsium show cytological similarity and have been place a close to each other in a single cluster. C. elodes of the same section, however, has been placed far from these species. The species of C. haussknechtii, C. aduncum, C. strigosum, C. ciliatum, C. turkestanicum, C. osseticum, C. strigosum, C. vulgare and C. lappaceum from the sect. Epitrachys although show some degree of cytological similarity to each other but have been placed in different clusters. Two species of C. spectabile and C. congestum from the sect. Pseudopitrachys differ in their cytological characteristics and have been placed far from each other. Populations of C. congestum, C. strigosum and C. ciliatum stand far from each other showing meiotic variation, while populations of C. arvense are placed close to each other, also supported by the ANOVA test results.

Discussion The occurrence of different ploidy levels in Cirsium species and populations indicates the role played by polyploidy in the species diversification. It has been suggested that 2nϭ34 based on nϭ17 is a primitive and ancestral chromosome number in the genus Cirsium (Moore and Frankton 1963, Dabeydeen 1980). Most of the taxa investigated in present study have a diploid chromosome number, but tetraploid chromosome numbers were observed in C. vulgare. C. lappaceum and C. elodes. The most frequent chromosome number within the genus Cirsium is diploid (2nϭ34), reported for approx. 69% of the species; tetraploid (2nϭ68) is also relatively common, occurring in approximately 10% of species. The less frequent chromosome numbers reported are 2nϭ30 and 2nϭ32 (both in approximately 5% of the species; Bures et al. 2004). Significant differences obtained for meiotic features which are under genetic control (Quicke 1993), partly indicates the genomic distinctness of the species studied. Variation in chiasma frequency and localization observed among the species and populations studied is suggested to be genetically controlled (Quicke 1993) and has been reported in populations of different species (Rees and Jones 1977). Such a variation in species and populations with the same chromosome number is considered a means for generating new forms of recombination, influencing the variability within natural populations in an adaptive way (Rees and Jones 1977). Quadrivalent formation in metaphase of meiosis-I in diploid species may indicate the, occurrence of heterozygote translocations between 2 pairs of chromosomes. Such chromosomes structural changes may increase the amount of genetic variability in the gametes by forming new genetic linkage groups, which may be used for adaptation to adverse environmental conditions. The significant correlation obtained between quadrivalent formation and intercalary chiasmata supports such a suggestion, as an increase in the number of intercalary chiasmata also increases genetic recombination by involving the genes present in the middle part of chromosomes in recombination; therefore both heterozygote translocation and an increase in intercalary chiasmata add to the genetic diversity of the next generation gametes. Ownbey et al. (1975) and Dabeydeen (1980)

119_127.pdf 7 10.5.26 9:11:26 AM 126 M. Nouroozi et al. Cytologia 75(1)

suggested that reduction in chromosome number accompanied by translocation between non- homologous chromosomes has played a signiﬁcant role in Cirsium speciation. The results of cluster analyses showed that cytological data does not separate species of each section from each other and that such data is not useful for sectional delimitation in Cirsium. However, different populations of C. congestum, C. strigosum and C. ciliatum stand far from each other, showing meiotic variation. If such cytological differences are accompanied by morphological variation (under investigation), we may consider these populations as a new variety or subsp. Therefore cytological characteristics may be of use in species delimitation. The presence of giant pollen grains has been used as an indication of the production of 2n pollen (Vorsa and Bingham 1979, Bretagnolle and Thompson 1995). Unreduced gametes are known to produce individuals with higher ploidy level through a process known as sexual polyploidization (Villeux 1985), which has been considered as the major route to the formation of naturally occurring polyploids. The occurrence of multipolar cells might be considered as the possible mechanisms of unreduced meiocytes and pollen grain formation in Cirsium.

References

Bretagnolle, F. and Thompson, J. D. 1995. Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol. 129: 1–22. Bures, P., Wang, Y., Horova, L. and Suda, J. 2004. Genome size variation in central European species of Cirsium (Compositae) and their natural hybrids. Ann. Bot. 94: 353–363. Dempsey, R. E., Gornall, R. J. and Bailey, J. P. 1994. Contributions to a cytological catalogue of the British and Irish ﬂora. Watsonia 20: 63–66. Charadze, A. L. 1998. Cirsium Mill. emend. Scop. In: Bobrov E. G. and Cherepanov S. K. (eds.), Flora of the USSR. Bishen Singh Mahendra Pal Singh and Koeltz Scientic Books 28: 52–214. Dabeydeen, S. 1980. Cytotaxonomy of the Genus Cirsium Mill. (Compositae) in Nebraska. University of Nebraska, Lincoln. Frankton, C. and Moore, R. J. 1961. Cytotaxonomy, phylogeny and Canadian distribution of Cirsium undulatum and Cirsium ﬂodmanii. Can. J. Bot. 39: 21–33. Ghaffari, S. M. 1999. Chromosome studies in the Iranian Asteraceae II. Iran. J. Bot. 8: 91–104. Moore, R. J. and Frankton, C. 1963. Cytotaxonomic notes on some Cirsium species of the Western United States. Can. J. Bot. 41: 1553–1567. — and —. 1974. The Cirsium arizonum complex of the southwestern United States. Can. J. Bot. 52: 543–551. Ownbey, G. B. 1951. Natural hybridization in the Cirsium I. C. discolor (Muhl. ex. Willd) Spreng. X C. munitum Michx. Bul. Torrey Bot. Club. 18: 233–253. —, Raven, P. H. and Kyhos, D. W. 1975. Chromosome numbers in some American species of the genus Cirsium. III. Western United States, Mexico and Guatemala. Brittonia 27: 297–304. Ozcan, M., Hayirlioglu-Ayaz, S. and Inceer, H. 2008. Chromosome counts of some Cirsium (Asteraceae, Cardueae) taxa from Turkey. Caryologia 61: 375–382. Quicke, D. L. J. 1993. Principles and Techniques of Cotemporary Taxonomy. Blackie Academic & Professional, Glasgow. pp. 298. Rechinger, K. H. 1979. Cirsium Adans. In: Rechinger K. H. (ed.). Flora Iranica, Tomus 139a. Compositae III-Cynareae. Akademishe Druck-und-Verlansanstalt, Graz. pp. 231–280. Rees, H. A. and Jones, R. N. 1977. Chromosome Genetics. Edward Arnold, London. pp. 160. Segarra-Moragues, J. G., Villar, L., Lopez, J., Perez-Collazos, E. and Catalan, P. 2007. A new Pyrenean hybrid Cirsium (Asteraceae) as revealed by morphological and molecular analyses. Bot. J. Linn. Soc. 154: 421–434. Sheidai, M. and Inamdar, A. C. 1997. Cytomorphology of Asparagus taxa using multivariate statistical analysis. Nucleus 40: 7–12. —, Mosalanejad, M. and Khatamsaz, M. 1999a. Karyological studies in Hyoscyamus species of Iran. Nord. J. Bot. 19: 369–373. —, Saeed, A. M. and Zehzad, B. 1999b. Meiotic studies of some Aegilops (Poaceae) species and populations in Iran. Edinb. J. Bot. 56: 405–419. —, Arman, M., Mohamadi-Saeed, A. and Zehzad, B. 2000. Notes on cytology and seed protein characteristics of Aegilops species in Iran. Nucleus 43: 118–128. —, Nasirzadeh, A. and Kheradnam, M. 2000. Karyotypic study of Echinops (Asteraceae) in Fars Province of Iran. Bot. J.

119_127.pdf 8 10.5.26 9:11:26 AM 2010 Contribution to Cytotaxonomy of Iranian Cirsium 127

Lin. Soc. 134: 453–463. —, Arman, M. and Zehzad, B. 2002. Chromosome pairing and B-chromosomes in some Aegilops species and populations of Iran. Caryologia 55: 261–271. — and Attaei S. 2005. Meiotic studies of some Stipa (Poaceae) species and populations in Iran. Cytologia 70: 23–31. — and Fadaei, F. 2005. Cytogenetic studies in some Bromus L. species sec. Genea. J. Genet. 84: 189–194. — and Nouroozi, M. 2005. Cytological studies on some species of Bromus sect. Bromus. Bot. Lithuanica 11: 141–150. —, Attaei, S. and Khosravi-Reineh, M. 2006. Cytology of some Iranian Stipa (Poaceae) species and populations. Acta. Bot. Croat. 65: 1–11. — and Nouroozi, M. 2006. B-chromosomes, diffuse stage and seed proteins in Bromus L. species sect. Bromus. Nucleus 49: 26–34. — and Bagheri-Shabestarei, E.-S. 2007. Cytotaxonomy of some Festuca species and populations in Iran. Acta Bot. Croat. 66: 143–151. — and Rashid, S. 2007. Cytogenetic study of some Hordeum L. species in Iran. Acta Biol. Szeged. 51: 107–112. —, Saeidi, S., Nourozi, M and Fadaei, F. 2008. Karyotype Analysis in Fourteen Species and Varieties of the Genus Bromus L. (Poaceae). Cytologia 73: 453–461. Tonian, T. R. 1981a. Kryological data on species of Cirsium Mill. growing in Armenia. Biol. Zhu. Arm. 34: 769–772. — 1981b. New chromosome numbers of species of Cirsium Mill. from Armenia. Biol. Zhu. Arm. 34: 641–645. — 1982. New chromosome numbers of the species of Cirsium in Armenia. Ucen. Zap. Erevan Univ. 3: 115–120. Villeux, R. 1985. Diploid and polyploid gametes in crop plants: mechanisms of formation and utilization in plant breeding. Plant Breed. Rev. 3: 253–288. Vorsa, N. and Bingham, E. 1979. Cytology of 2n pollen formation in diploid alfalfa, Medicago sativa. Can. J. Genet. Cyto. 21: 525–530. Wagenitz, G. 1987. Cirsium Mill. em. Scop. In: Hegi G. (ed.). Illustrierte Flora von Mitteleuropa, 2nd edition. Vol. 6/4. Paul Parey Verlag, Berlin et Hamburg, pp. 866–916. Zomlefer, W. 1994. Guide to Flowering Plant Families. University of North Carolina Press, Chapel Hill.

119_127.pdf 9 10.5.26 9:11:26 AM