Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(4): 123-131, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1678 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Seyed Mahmood Ghaffari¹, Seyed Mohsen Hesamzadeh Hejazi² (2022). Cytogenetic studies in the Cen- taurea aucheri group (sect. Phaeo- pappus). Caryologia 75(4): 123-131. doi: 10.36253/caryologia-1678 Received: June 06,2022 Accepted: December 06, 2022 Published: April 28, 2023 Copyright: © 2022 Seyed Mahmood Ghaffari¹, Seyed Mohsen Hesamza- deh Hejazi². This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID SMG: 0000-0002-6777-6992 Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) Seyed Mahmood Ghaffari¹,*, Seyed Mohsen Hesamzadeh Hejazi² ¹ Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran ² Research Education and Extension Organization (AREEO), Tehran, Iran *Corresponding author. E-mail: mghaffary@ut.ac.ir Abstract. In this study, it was aimed to evaluation of the revision in Centaurea aucheri group by the cytogenetical studies and determines the chromosome number for C. albonitens species, to determine chromosome morphology and karyotype analysis. Results of meiotic behavior and karyomorphologycal parameters of Centaurea aucheri group indicated that raised this group in five distinct species are correct. Somatic and gametic chromosome numbers indicated that C. albonitens, C. assadii, C. farsestanica, C. indistincta and C. phaeopappa are diploid with n=9 and 2n=18 and C. aucheri is tetraploid with n=18 and 2n= 4x= 36+0-2B. Gametic chromosome number and karyo- morphology of C. aucheri and C. indistincta species is reported here for the first time. Also, meiotic behavior and karyomorphology parameters of C. albonites, C.assadi , C. farsestanica and C. phaeopappa are newly reported here. Analysis of karyotype and behavior of meiosis indicated that C. aucheri is a natural allotetraploid species. Kary- otype symmetry parameters showed that all the studied species were classified in the class 2A except of C. aucheri which was located in class 2B. Keyword: Allotetraploid, Centaurea aucheri, Cytogenetic, Karyomorphology, Meiotic behavior. INTRODUCTION Centaurea, as the fourth largest genus among the genera in Asteraceae and also the second largest genus in the tribe Cardueae consist of 200–250 species placed in 40 sections (Wagenitz and Hellwig 1997; López et al. 2011 Hilpold et al. 2014 ). The present classification of Centaurea in 40 sections is highly problematic (Wagenitz 1975) and does not take into account all of the pollen, karyological and carpological evidence (Susanna et al. 1995). In Flora Iranica 88 species belong to 28 sections are reported (Wagenitz 1980). Centaurea section Phaeopappus (DC.) O. Hoffm.is one of the section com- prising of five species in Flora Iranica. Of these, 3 taxa viz: C. albonitens, C. aucheri and C. spectabilis are distributed in Iran (Wagenitz 1980). Ranjbar and Heydari (2016) elevated this section to 14 species in the world, which 12 are belongs to Iran (10 species are endemic to the country). In flora of Iran 5 subspecies has been cited for C. aucheri viz: subsp. aucheri, subsp. elbur- 124 Seyed Mahmood Ghaffari, Seyed Mohsen Hesamzadeh Hejazi sensis, subsp. farsistanica, subsp. indistincta and sub- sp. szowitsii (Wagenitz 1980; Mozafarian 2018). Ran- jbar and Negaresh (2013) raised these five subspecies to the C. aucheri (DC.) Wagenitz, C. assadii Ranjbar and Negaresh, C. farsistanica (Wagenitz) Ranjbar & Negaresh, C. indisticta (Wagenitz) and C. phaeopappa (DC.) Schultz Bipontinus respectively based on the mor- phological characteristics. The aims of this present paper is evaluation of the revision in Centaurea aucheri group by the cytogenetical studies. MATERIALS AND METHODS The origin of the plant material studied here is shown in Table 1. For meiotic studies floral buds of plants found in nature were collected and immediately fixed in Piennr’s fluid containing ethanol 96%, chloro- form, propionic acid, 6:3:2 (v/v/v)for 24 hours at room temperature. Anthers dissected out from the buds were squashed and stained with 2% acetocarmine. Chromo- some counts obtained from a minimum of 50 pollen Table 1. The species and origin of the material examined. Taxon Origin Altitude(m) Longitude and Latitude H. Code Centaurea albonitens Turrill Azerbaijan: Tabriz, 20 Km towards Marand 1360 E:46°03’57”; N:38°15’48” 109080 Azerbaijan: between Khoy and Salmas 1530 E: 44°54’15”; N: 38°24’39” 109081 Zanjan, 10 Km towards Miyaneh 1500 E: 47°27’35”; N: 37°12’36” 109101 Zanjan, 30 Km towards Miyaneh 1600 E: 47°30’53”; N: 37°14’41” 109102 Centaurea aucheri Kordestan:Saqqez to Divandareh 2200 E: 46°54’15”; N: 36°24’39” 109091 Kordestan: W Saqqez 2160 E: 46°16’13”; N: 36°15’39” 109092 Qazvin: Avaj, 10 Km. to Hamadan 2400 E: 49°09’38”; N: 35°32’17” 109201 Qazvin: Neck of Avaj 2320 E: 49°12’05”; N: 35°35’35” 109202 Qazvin: Abgarm to Avaj 2300 E: 49°14’07”; N: 35°42’58” 109203 Hamadan: 90 Km. to Qazvin 2070 E: 49°02’19”; N: 35°27’54” 109301 Lorestan: Doroud, Gahar, Saravand 2110 E: 49°10’03”; N: 33°22’26” 109421 Markazi: Tafresh, Noghre-kamar 2080 E: 50°08’13”; N: 34°41’53” 109501 Centaurea assadii Azerbaijan:Miyaneh to Bostanabad 1720 E: 47°18’27”; N: 37°32’05” 109087 Tehran: Kouhdashteh 1800 E: 51°20’13”; N: 35°44’23” 109701 Tehran: Kouhdashteh 1950 E: 51°23’50”; N: 35°44’29” 109702 Tehran: Sorkheh hesar 1500 E: 51°36’03”; N: 35°43’12” 109714 Tahran: Sorkheh hesar 1700 E: 51°35’46”; N: 35°43’09” 109712 Tehran: Jajroud 2200 E: 51°29’45”; N: 35°42’39” 109754 Tehran: Abali 2400 E: 51°57’55”; N: 35°45’54” 109718 Centaurea farsistanica Shiraz: Bamoo park 1750 E: 52°36’52”; N: 29°42’48” 109614 Shiraz: Bamoo park 1820 E: 52°37’15”; N: 29°43’11” 109615 Shiraz: Bamoo park 2000 E: 52°36’33”; N: 29°12’41” 109617 Fars: Dasht arjan, Khers dareh 2400 E: 51°58’56”; N: 29°39’47” 109687 Yasouj, Kakan 2300 E: 51°35’57”; N: 30°38’18” 109813 Yasouj: Kamehr 2400 E: 51°35’38”; N: 30°39’55” 109815 Centaurea indistincta Tehran: Kouhdashteh 1800 E: 51°20’13”; N: 35°44’23” 109706 Tehran: Kouhdashteh 2300 E: 51°23’50”; N: 35°44’30” 109709 Lorestan: Doroud, Gahar 2100 E: 49°10’03”; N: 33°22’25” 109432 Zanjan 1630 E: 47°33’53”; N: 37°14’41” 109132 Centaurea phaeopappa Karaj, Eshtehard, Dakin 1550 E: 50°27’36”; N: 35°37’18” 109745 Qazvin: Abgarm to Avaj 2300 E: 49°14’07”; N: 35°42’58” 109208 Tahran: Kohdashteh 1800 E: 51°20’13”; N: 35°44’24” 109784 Karaj, Ziyaran 2000 E: 50°31’43”; N: 36°07’06” 109764 Azerbaijan: Oroumiyeh to Mahabad 1330 E: 45°19’22”; N: 37°13’52” 109075 Azarbaijan: Salmas to Oroumiyeh 1375 E: 45°36’08”; N: 37°34’46” 109094 Azerbaijan: Mishodagh Mt. 2040 E: 45°38’23”; N: 38°17’50” 109049 Qazvin: Takestan to Hamadan 2350 E: 49°01’44”; N: 35°20’46” 109257 125Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) mother cells within each collection. Actively growing root tips were used for mitotic analysis. Roots were pre- treated with 0.002M, 8-hydroxyquinoline at 20˚C for 3 hr, and then fi xed in 3:1 (ethanol: acetic acid). Staining was carried out with the Feulgen reaction enhanced by squashing in 2% acetocarmine. Nomenclature adopted by Levan et al. (1964) was followed for recognizing chro- mosome types. Both mitotic and meiotic slides were made permanent by the venetain turpentine (Wilsom 1945). Voucher specimens are preserved in the Her- barium of Research Institute of Forests and Rangelands (RIFR). RESULTS Centaurea albonitens Turrill Centaurea albonitens is widely distributed in west- ern Iran, especially Zanjan, west and east Azerbaijan Provinces. Five samples of this taxon showed chromo- some numbers of n=9 and 2n=18 in both meiosis and mitotic respectively. Meiosis in this taxon showed 9 bivalents at diakinesis which ring tetravalent in some cells were observed (Figure 1A). Also, two bivalents were associated with the nucleolus at diakinesis, which is con- fi rmed to presence of two satellite chromosomes in this species. First metaphase indicated 9 bivalents, which most of them were in rod shape (Figure 1B). Anaphase I showed (9-9) chromosomes segregation (Figure 1C). Somatic chromosome counts in 50 root tips disclosed a chromosome number of 2n=18 (Figure 1D, E). Th e rep- resentatives of chromosome set at mitotic metaphase are shown in Figure 1F and Table 2. Th e karyotype of C. albonitens consists of one pair of metacentric chromo- some and 8 pairs of sub-metacentric chromosomes. Th e total length of the chromosomes varied from 5.18 µm to 2.96 µm (Table 2). Th is count (2n=18) agrees with pre- vious reports by Garcia-Jacas et al. (1998) and Ranjbar and Negaresh (2013). Karyotype analysis and meiotic count for this taxon is reported here for the fi rst time. C. aucheri (DC.) Wagenitz Eight samples of this taxon showed chromosome numbers of n=18 and 2n=36 in both meiosis and mitotic respectively. Meiosis showed some clamping of chromo- somes at fi rst metaphase. Th e bivalents at metaphase I were usually in the shape of rod with one terminal chi- asmata (Figure 2A). Many cells were observed in order to ascertain the correct chromosome count. Th e number C F Figure 1. Meiosis and mitotic micrographs of Centaurea albonitens. A; Diakinesis, showing ring tetravalent (arrow). B: Metaphase I (n=9). C: Anaphase I (9-9). D: Late prophase (2n=18). E: Metaphase (2n=18). F: Karyotype showing nine pairs of chromosomes. Scale= 5μm. 126 Seyed Mahmood Ghaffari, Seyed Mohsen Hesamzadeh Hejazi n=18 observed at diakinesis (Figure 2B) and (18-18) seg- regation at first anaphase (Figure 2C). In the first meiosis stage, we did not observe any tetravalents at metaphase I and diakinesis. Also we did not found any abnormal- ity at first and second anaphase of meiosis, which is prevalent in autopolyploids species. These results indi- cated that this taxon is natural allotetraploid species. Somatic chromosome counts of 50 root tips disclosed a chromosome number of 2n= 36+0-2B (Figure 2D,E). These B-chromosomes were not found at meiosis stage. The karyotype consisted of 15 metacentric pairs, and 3 submetacentric pairs (Figure 3A). The total length of the chromosomes varied from 6.11 µm to 2.85 µm (Table 3). Different chromosome number of this taxon (2n=36) with others subspecies (2n=18) and allotetraploid behav- Table 2. Measurement of somatic chromosomes in a diploid C. albonitens (Obtained from 50 cells). No. of chromosome Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 5.18 2.59 2.59 1 2 4.44 2.59 1.85 1.4 3 3.89 2.22 1.67 1.33 4 3.33 2.22 1.11 2 5 3.33 2.04 1.29 1.85 6 3.33 2.04 1.29 1.85 7 3.14 1.85 1.29 1.43 8 2.96 1.85 1.11 1.67 9 2.96 1.85 1.11 1.67 Figure 2. Meiosis and mitotic micrographs of Centaurea aucheri group A- E. C. aucheri, A: metaphase I. B: diakinesis. C: anaphase I. D: mitotic metaphase, showing B-chromosome (arrow). E: mitotic metaphase, showing 2B-chromosomes (arrows). Scale = 5μm. 127Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) ior indicated that upgrade to species rank (C. aucheri (DC.) Wagenitz ) by Ranjbar and Negaresh (2013) is cor- rect. In addition, we found some specimens of this taxon together with C. aucheri subsp. szowitsii (C. phaeopappa) in one place (Abgarm to Avaj, Table 1), that chromo- some examination showed independence in chromo- some number for each species ( subsp. aucheri , n=18, 2n=36: subsp. szowitsii n=9, 2n=18) and did not show any hybrid adjective between this two taxon. C. assadii Ranjbar & Negaresh Wagenitz (1980) is introduced the C. aucheri sub- sp. elbursensis (C. assadii) as a new endemic subspecies to Iran for the first time. Previous chromosome num- ber report for this taxon is n=9 by Ghaffari (1986). The results obtained in this study showed nine bivalents in pollen mother cells at diakinesis (Figure 2F). Another stages of meiosis showed chromosome segregation (9-9) at anaphase I and 9 chromatid segregation at anaphase II (Figure 2G,H). Chromosome complement at metaphase of mitotic was 2n=18 (Figure 2I). Karyotype consisted of seven pairs of metacentric and two pairs of submeta- centric chromosomes (Figure 3B). The total length of the chromosomes varied from 4.74 µm to 2.79 µm (Table 4). This taxon distributed in Azerbaijan, Mazandaran, Qaz- vin, and Tehran provinces. We found overlapping of this species with C. indistincta and C. phaeopappa in one place (Tehran, kouhdashteh, see Table 1) and did not see any hybrid between them. Lack of geographical distribu- tion independence, joint with other subspecies, morpho- Figure 3. A: Karyotype of C. aucheri. B: Karyotype of C. assadii. C: Karyotype of C. farsestanica. D: Karyotype of C. indistincta. E: Karyo- type of C. phaeopappa Scale bar = 10 μm. Table 3. Measurement of somatic chromosomes in a tetraploid C. aucheri (Obtained from 12 cells). No. of chromosome Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 6.11 3.67 2.44 1.50 2 5.61 3.35 2.26 1.48 3 5.11 3.02 2.08 1.45 4 4.76 2.49 2.26 1.10 5 4.41 2.49 1.91 1.30 6 4.38 2.47 1.91 1.29 7 4.23 2.44 1.79 1.36 8 4.20 2.61 1.58 1.64 9 4.16 2.84 1.32 2.14 10 4.06 2.44 1.61 1.51 11 3.91 1.97 1.94 1.01 12 3.55 2.14 1.41 1.52 13 3.52 2.23 1.29 1.72 14 3.50 2.17 1.32 1.64 15 3.32 2.11 1.20 1.75 16 3.23 1.82 1.41 1.29 17 2.91 1.76 1.14 1.53 18 2.85 1.61 1.23 1.30 128 Seyed Mahmood Ghaffari, Seyed Mohsen Hesamzadeh Hejazi logical characteristics and difference in chromosomal characteristics with C. aucheri, indicated that this taxon is a distinct species. C. farsistanica (Wagenitz) Ranjbar & Negaresh Meiosis in this taxon was regular and showed nine bivalents at metaphase I which more of them were in rod shape (Figure 2J). Anaphase I indicated (9-9) chro- mosomes segregation (Figure 2K). Mitotic study showed 2n=18 chromosomes at metaphase (Figure 2L). This dip- loid species has a symmetrical karyotype with six pairs of metacentric and three pairs of submetacentric chro- mosomes (Figure 3C). The total length of the chromo- somes varied from 3.98 µm to 2.25 µm (Table 5). This taxon is completely different with others subspecies in morphological characteristic and pattern of distribution (Ranjbar and Negaresh 2013), which are introduced by Wagenitz (1980) and Mozaffarian (2018). Centaurea indistincta (Wagenitz) Ranjbar & Negaresh This taxon is reported by Wagenitz (1980) as a new endemic subspecies species (C. aucheri subsp. indistinc- ta) for flora of Iran, which was introduced by Ranjbar and Negaresh (2013) as a distinct species. Meiosis in this species showed nine bivalents at metaphase I and (9-9) univalents segregation at first anaphase (Figure 2M,N). Chromosome complement in this species was 2n=18 (Figure 2O). Karyotype consisted of five pairs of meta- centric and four pairs of submetacentric chromosomes (Figure 3E). The total length of the chromosomes varied from 4.08 µm to 2.52 µm (Table 6). Centaurea phaeopappa (DC.) Schulpz & Bipontinus Meiotic and mitotic divisions were examined on eight samples of this species (Table 1). Meiosis showed nine bivalents at diakinesis and metaphase I (Figure 2P,Q). Nondisjunction of (8-10) segregation at anaphase I was observed (Figure 2R). Also, in some cells laggard chromosomes at anaphase II were observed (Figure 2S). Mitotic stages in this species indicated chromosome complement of 2n= 18 (Figure 2T) which is agrees with the previous report by Ghaffari (1988). The karyotype consisted of six pairs of metacentric and three pairs of submetacentric chromosomes (Figure 3F). The total length of the chromosomes varied from 4.21 µm to 2.70 µm (Table 7). DISCUSSION The study has detected the somatic and gamet- ic chromosome number and karyomorphology of C. Table 4. Measurement of somatic chromosomes in a diploid C. assadii (Obtained from 9 cells). Chromosome No Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 4.74 2.43 2.30 1.05 2 4.67 2.51 2.15 1.16 3 4.10 2.45 1.65 1.48 4 3.87 2.43 1.43 1.69 5 3.43 1.94 1.48 1.31 6 3.23 2.28 0.95 2.40 7 3.19 1.84 1.35 1.36 8 3.02 1.80 1.22 1.47 9 2.79 1.54 1.24 1.23 Table 5. Measurement of somatic chromosomes in a diploid C. far- sistanica (obtained from 14 cells). Chromosome No Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 3.08 2.24 1.73 1.29 2 3.34 1.95 1.39 1.39 3 3.09 1.82 1.27 1.43 4 2.71 1.90 0.80 2.36 5 2.54 1.31 1.23 1.06 6 2.46 1.39 1.06 1.31 7 2.38 1.27 1.11 1.14 8 2.29 1.44 0.85 1.69 9 2.25 1.49 0.76 1.94 Table 6. Measurement of somatic chromosomes in a diploid C. indistincta (obtained from 8 cells). Chromosome No Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 4.08 2.21 1.86 1.19 2 3.72 2.30 1.42 1.62 3 3.15 1.95 1.37 1.41 4 2.92 2.08 1.06 1.96 5 2.92 1.59 1.33 1.20 6 2.75 1.55 1.37 1.12 7 2.57 1.73 1.02 1.69 8 2.13 1.64 0.93 1.76 9 2.52 1.59 0.93 1.71 129Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) aucheri and C. indistincta species for the first time. Also, meiotic behavior and karyomorpholog y parameters of C. albonites, C.assadi and C. phaeopappa are newly reported here. C. albonitens, C. assadii, C. indistincta , C. farsestanica and C. phaeopappa are diploid with n= 9, 2n=2x=18 and C. aucheri is tetraploid with 2n=4x=36+0- 2B. All taxa had the basic chromosome number of x= 9. By definition, a subspecies designation is applied to a plant that is geographically isolated from other mem- bers of its species in habitat and therefore does not inter- breed for this reason. Five subspecies (C. aucheri subsp. aucheri, C. aucheri subsp. elbursensis, C. aucheri subsp. farsestanica, C.aucheri subsp. indistincta, C. aucheri subsp. szowitsii) which is introduced by Wagenitz (1980) and Mozafarian (2018) for flora of Iran, were often adja- cent to each other [see Table 1 and pattern of distribu- tion of these subspecies in the research article by Ran- jbar and Negaresh (2013)]. Therefore, they cannot be considered as subspecies. Eight samples of the C. aucheri showed chromosome numbers of n=18 and 2n=36 in both meiosis and mitotic respectively. Meiosis in pollen mother cells in this taxon showed that the most com- mon chromosome configurations were bivalents at diak- inesis and first metaphase. Analysis of karyotype and behavior of meiosis indicated that this taxon is a natural allotetraploid species. Therefore, the results of meiotic behavior and karyomorphologycal parameters of five subspecies which are introduced by Wagenitz (1980) and Mozaffarian (2018) are not correct and revision of them by Ranjbar and Negaresh (2013) in five independence species are correct. In this study, most of the chromosomes of the eva luated species were metacent ric (m) or sub- metacentric(sm). Kar yot y pe sy mmetr y parameters showed that all the studied species were classified in the class 2A of the category of Stebbins (1971), except of C. aucheri which was located in class 2B (Table 8). Total form percentage (TF%) for C. indistincta and C. phaeopappa were 40.41 and 40.02 respectively, that indicated similarity between them. Also, this TF% similarity can be seen between C. assadii and C. far- sistanica with 41.75 and 40.82 respectively. According to the data obtained from the five species (C. aucheri, C. assadii , C. farsistanica, C. indisticta and C. phaeo- pappa), the karyotype formulas were different between them (Table 8). Also, the inter and intrachromosomal asymmetry index (A1 and A2) for five taxa were differ- ent (Table 8). In this study, C. farsistanica had a higher DI value, which is associated with an enhanced order of karyotypic specialization. C. aucheri had the highest A2 values therefore its karyotype was more asymmet- ric than the other species. To analyze the variability of the karyotypes among species, all karyotype char- acteristics of Centaurea species (Table 8) were com- pared by one-way design. Using principal components analysis (PCA), the first two independent components accounted for about 81.68% of total variation (Table 9). The first component indicated that TL, LA, SA, CI, TF% and A2 were important characters for classifica- tion of species with about 61% of total variation. AR, LA%, SA%, DRL, A1 and DI were important traits in the second component (21%) (Table 9). Table 7. Measurement of somatic chromosomes in a diploid C. phaeopappa (obtained from 23 cells). Chromosome No Total length (µm) Long arm (µm) Short arm (µm) Arm ratio L/S = r 1 4.21 2.13 2.08 1.02 2 3.72 2.21 1.50 1.47 3 3.59 2.04 1.55 1.31 4 3.55 2.21 1.33 1.66 5 3.51 2.21 1.29 1.71 6 3.46 2.17 1.28 1.68 7 2.88 1.68 1.19 1.40 8 2.75 1.86 0.88 2.10 9 2.70 1.68 1.02 1.65 Table 8. Karyotype parameters of Centaurea taxa; Total Length of chromosome(TL), Long Arm (LA), Short Arm (SA), Arm Ratio(AR), Centromeric Index(CI ), Long Arm percent(LA % ), Short Arm percent(SA %); Total Form percent (TF % ), Difference of Relative Length (DRL), intrachromosome asymmetry index(A1 ), interchromosome asymmetry index(A2), Dispersion Index(DI), symmetry classes of Stebbins(SC), Haploid Karyotype Formula(H.K.F.) (m: metacentric , sm: submetacentric). species TL LA SA AR CI %LA %SA %TF DRL A1 A2 DI SC H.K.F C. assadi 3.68 2.14 1.54 1.47 0.41 6.47 4.64 41.75 5.89 0.28 0.19 8.11 2A 7m+2sm C. aucheri 4.11 2.43 1.68 1.48 0.41 3.29 2.27 40.83 4.41 0.31 0.22 8.41 2B 15m+3sm C. farsistanica 2.79 1.65 1.14 1.52 0.41 6.58 4.54 40.82 6.87 0.30 0.21 9.12 2A 6m+3sm C. indistincta 3.11 1.85 1.26 1.52 0.40 6.62 4.49 40.41 5.55 0.32 0.17 7.65 2A 5m+4sm C. phaeopappa 3.38 2.03 1.35 1.56 0.40 6.66 4.45 40.02 4.96 0.33 0.15 5.47 2A 6m+3sm 130 Seyed Mahmood Ghaff ari, Seyed Mohsen Hesamzadeh Hejazi Th e tree phylogeny (Figure 4) of the fi ve species indi- cated two major clades. Th e fi rst major clade consists of two species (C. indistincta and C. phaeopappa) showed a degree of affi nity and were placed close to each other, while, C. aucheri joined the other species at a great dis- tance. Th e second clade contained C. assadii and C. fars- estanica. 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Parameters First component Second component TL 0.328 -0.054 LA 0.336 -0.050 SA 0.316 -0.061 AR 0.294 0.308 CI -0.320 -0.206 LA% -0.275 0.381 SA% -0.304 0.316 TF% -0.326 -0.169 DRL -0.204 0.528 A1 0.133 -0.176 A2 0.332 0.218 DI 0.215 0.471 Eigen value 7.3257 2.4761 Percentage of Variance 61.0479 20.6346 Cum percentage of variance 61.0479 81.6824 C. phaeopappa Figure 4. Dendrogram of cluster analysis (Ward) of Centaurea species based on karyotype characteristics. Cophenetic correlation r=0.94. -3 -1 1 C. assadiis C. aucheri C. farsistanica C. indistincta C.phaeopappa Figure 5. Scatter plot of Centaurea species for the fi rst two princi- pal components. 131Cytogenetic studies in the Centaurea aucheri group (sect. Phaeopappus) Stebbins G.L. 1971. Chromosomal evolution in higher plsnts. Edward Arnold Ltd. London. Susanna A, Garcia-Jacas N, Soltis DE, Soltis P.S. 1995. Phylogenetic relationship in tribe Carduae (Aster- aceae) based on ITS sequence. American Journal of Botany 82: 1056-1068. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 75, Issue 4 - 2022 Firenze University Press Avicennia genus molecular phylogeny and barcoding: A multiple approach Laleh Malekmohammadi1, Masoud Sheidai1,*, Farrokh Ghahremaninejad2, Afshin Danehkar3, Fahimeh Koohdar1 Studying some morphological responses of stevia (Stevia rebaudiana Bertoni) to some elicitors under water deficiency Basoz Sadiq Muhealdin1, Sahar Hussein Hamarashid1,*, Fairuz Ibrahim Ali1, Nakhshin Omer Abdulla1, Syamand Ahmad Qadir2 Morphological and cytogenetic characterization in experimental hybrid Aloe jucunda Reyn. x Aloe vera (L.) Burm. f. 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