Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(1): 35-46, 2023 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1988 Citation: Fatemeh Nezhadi, Farzad Fayaz, Ezzat Karami, Hooshmand Safari, Abdol Rahman Rahimi (2023). A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran. Caryologia 76(1): 35-46. doi: 10.36253/caryologia-1988 Received: January 17, 2023 Accepted: June 10, 2023 Published: September, 19, 2023 Copyright: © 2023 Fatemeh Nezhadi, Far- zad Fayaz, Ezzat Karami, Hooshmand Safari, Abdol Rahman Rahimi. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided 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. A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran Fatemeh Nezhadi1, Farzad Fayaz2,*, Ezzat Karami2, Hooshmand Safa- ri3, Abdol Rahman Rahimi2 1 Department of Agronomy and Plant breeding, Sanandaj Branch, Islamic Azad Univer- sity, Sanandaj, Iran 2 Faculty member of Department of Agronomy and Plant Breeding, Sanandaj Branch, Islamic Azad University, Sanandaj, Iran 3 Faculty member of Forest and Rangeland Research Department, Kermanshah Agri- culture and Natural Resources Research and Education and Extension Organization (AREEO), Kermanshah, Iran *Corresponding author. E-mail: far.fayaz@gmail.com Abstract. We conducted the present study on seven important medicinal species of Achillea (in a total of 28 populations) in their natural habitats. The results indicated that the populations had a base number (x= 9) and the diploid, tetraploid, and hexa- ploidy levels were observed. In addition to the inter-species diversity, there was the intra-species genetic diversity as A. millefolium (4x, 6x), A. vermicularis (2x, 4x), A. tenuifolia (2x, 4x), A. Aleppica (2x), A. talagonica (2x), A. biebersteinii, and A. wilhelm- sii (4x). Furthermore, studies also indicated that 11 out of 28 populations had 1A sym- metry, 15 populations had 2B symmetry, a population had 2A, and another population had 2B. Cluster analysis of cytogenetic variables could differentiate only the species of A. aleppica, A. talagonica and A. wilhelmsii but others could not separate differences between species appropriately, probably due to the superiority of intra-species diver- sity of populations to inter-species diversity. Furthermore, we found %TF and DRL are useful parameters for differentiating intrachromosomal variation of species. Keywords: Achillea, cytogenetic, cytomorphology, cluster analysis, chromosome struc- ture. INTRODUCTION Achillea genus of family Asteraceae is of about 130 species that are dis- tributed from southeastern Europe to southeastern Asia and has spread to North America through Eurasia. Different species of this genus have shown significant adaptation to different environmental conditions and have spread from deserts and coastal areas to rocky regions. The plants of this genus are perennial, allogamous and they are pollinated by insects (Mozaffaria, V. 2003). There are 19 herbaceous species of this genus available in Iran. Other species of this genus also grow in Anatolia, Syria, Caucasus, Lebanon, Pal- estine, Central Russia, Transcaucasia, Turkmenistan, Afghanistan, Southwest https://doi.org/10.36253/caryologia-1988 https://doi.org/10.36253/caryologia-1988 36 Fatemeh Nezhadi et al. Asia, and Central Asia in addition to Iran (Ghahreman 1984). Yarrow is a popular medicinal herb that is widely used in traditional medicine to treat diseases, particular- ly burns and scars (Muẓaffarīyān 1996). Cytogenetics is study of relationship of chromosome structure with cellular function. Karyotype, the highest level of functional and structural organization of nucle- ar genome, that is essential for studying chromosomal characterization of plant species (Altınordu et al. 2016). Comparative chromosomal taxon has been provid- ing useful knowledge about patterns and evolutionary mechanisms in speciation (Flavell 2021). Chromosomal features, such as chromosome length, centromere index, number of chromosomes are crucial variables for inves- tigating interrelationships and intrarelationships of taxa. However, in addition of chromosomal morphotype, pop- ulation geographical origin is an important parameter in interpreting taxon’s diversity (Ramsey 2011) . The basic chromosome number x = 9 is commonly reported for Achillea but variation in chromosome num- bers and different ploidy levels are frequently occurring in this genus. Unfortunately, only few cytological studies have been published concerning karyological aspects on this genus in Iran. Ploidy and number of chromosomes in A. aleppica DC. species has been varied from 2x to 8x and with high symmetrical characteristic karyotype (2A), however in some population asymmetrical chromosomes (2B) have been reported (Rad and Javaheri 2014). A. ver- micularis, A. wilhelmsii and A. millefolium species have shown different levels of ploidies 2x, 4x, 6x and 8x with 2A symmetrical Stebbins’s index (Afshari et al. 2013). Mean- while, study of nine populations of A. biebersteinii species demonstrated a diploid (2x); however, chromosomal inter- species variation has been observed and there has been a symmetrical Stebbins’s index 1A and 2A (Chehregani Rad et al. 2017). The results of an another study, that was con- ducted on 14 populations of 8 Achillea species showed A. talagonica and A. berbersteinii species have been 2x ploidy, meanwhile vermicularis and wilhelmsii showed 4x, 6x ploidy levels respectively (Sheidai et al., 2009). Unfortu- nately, we have few studies on different species of Achillea particularly in Iran. Therefore, to fill this gap, we try to conducted this study on 28 population of seven species of Achillea in their natural habitats in Sanandaj find out the cytogenetical characteristics features. MATERIALS AND METHODS Plant materials All of the 28 samples in this study, including seven species of Achillea (A. millefolium, A. vermicularis, A. tenuifolia, A. aelppica, A. biebersteinii, A. wilhelmsii, and A. talagonica), with four replicates in each species, were collected in west of Iran, Kurdistan, Sanandaj. This region is located at a longitude of 46° 59’ 45” E and lati- tude of 35° 19’ 00” N. To identify every species, a sam- ple was collected from each point. Figs 1, 2 represent the exact position of each location and morphological popu- lation features. Furthermore, we recorded the geographi- cal position of each location using the GPS. Table (1) presents the latitude and longitude of each site. Cytogenetical study of species The seeds obtained from every point were disin- fected employing the solution of the Sodium hypochlo- rite 2%, under sterile conditions, inside a Petri dish, and on the filter paper. Afterwards, the seeds germi- nated at room temperature. Following two to five days, Table 1. Details on population sites including geographical coordi- nates, altitude and origin of samples. Population Longitude Latitude Altitude/m Origin W13 46.71 35.29 2,208 Klatei W12 46.88 35.36 1,974 Arandan W11 47.00 35.51 1,999 Sofla Mamox W14 46.79 35.51 2,026 Gav Dareh TA1 46.94 35.47 1,628 Chrandoo TA2 46.98 35.58 1,980 Biaenchob TA4 46.99 35.51 2,145 Chrandoo TA3 46.98 35.49 1,919 Sofla Mamox TE4 46.96 35.46 1,577 Sarab Ghamish TE2 46.96 35.51 1,841 Chrandoo TE1 46.99 35.49 1,866 Mamox TE3 47.02 35.57 1,934 Bazi Rabab AL3 46.94 35.28 1,829 Hassan Abad AL4 46.58 35.32 1,607 Goyran AL1 46.98 35.30 1,653 Pakr kodak AL2 47.13 35.29 2,095 Salvat Abad BI3 46.58 35.30 1,436 Danikesh BI2 46.60 35.32 1,436 Pichon BI1 46.89 35.13 1,334 Savarian BI4 46.93 35.46 1,593 Chelgazii VE4 46.97 35.30 1,838 Dole Rahman VE1 46.92 35.55 2,334 Ghalvazei VE3 46.92 35.58 2,152 Sangi Sefied VE2 46.99 35.51 2,145 Sofla Mamox MI2 47.07 35.36 1,498 Babareiz MI1 47.15 35.25 1,985 Salvat Abad MI4 47.12 35.54 1,642 Jebreillian MI3 47.11 35.49 1,993 Dolbandi 37A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran their roots reached the proper size for sampling (roots with a length of 0.5-1 cm are appropriate for sampling). After applying the pre-treatment, the root samples were exposed to a 0.5% α-bromonaphthalene solution for 4 h, and running water for 30 min to remove the remains of the solution. Subsequently, we performed the fixation. Thus, we used Levitsky solution as a suit- able fixator for karyotypic studies, and the samples were in the for 16 h (Levitsky 1931, Levitus et al. 2010). Following the fixation, the samples were rinsed with running water for 3 h to eliminate the residuals of the fix. Then we used squash at an optimal level to sepa- rate the cells and put them at the same level, and make staining better. To this end, we removed the roots from 70% ethyl alcohol, rinsed them with running water for 30 minutes, put them in a hydrolyzer (1 M NaOH), and placed them in the oven at 60 °C for 8 min. After hydrolysis, the samples were dried with filter paper and placed in hematoxylin for 3-4 h, to stain the chromo- somes (Abbaszade et al. 2017). Chromosomal images were transferred to the monitor and saved with a digi- tal color CCD camera mounted on a light microscope. The chromosomes of each cell were cut in Photoshop and arranged in a separate file. Using Micro Measure software and specifying the beginning to end of chro- mosomes and their centromere locations, certain char- acteristics such as short and long arm length, the total chromosome length, and relative chromosome length were calculated. The results were stored in Excel. In the present study, five cells (replications) were selected and evaluated from each slide to measure chromo- somal parameters. The parameters calculated for the karyotypes were as follows: Short arm relative length percentage (SA%), Long arm relative length percent- age (LA%), total length (TL), Relative length percentage (RL%), Arm ratio (AR), Total form percentage (TF%), Centromere index (CI), Difference of the range of rela- tive length (DRL), Value of relative chromatin (VRC), Intrachromosomal asymmetry Index (A1) and Inter- chromosomal asymmetry Index (A2) (Altınordu et al. 2016). According to the number of the replications, we calculated the standard deviation for the traits and the confidence interval for some of them. The chromosome form was determined using a method by Levan (Levan 1964). After measuring the chromosomes, we drew the ideogram associated with the karyotype of the popula- tions based on the lengths of short and long arms, in which the order of chromosomes was considered based on the length of the short arm (from large to small). We utilized the Stebbins method for comparing the karyo- typic symmetry in the species (Stebbins 1971). Statistical data analysis We performed all the statistical analyses by employ- ing R software. The cluster analysis was performed for cytogenetic data series using the statistical packages, fac- toextra, FactoMineR, and devtools (Kassambara 2017). To map extract geological we used raster and MapTool packages by using R software. (Bivand and Lewin-Koh 2013) (Fig. 1). RESULTS Comparison of cytogenetic parameters between species Mitotic metaphase chromosomes, ideograms, and morphological diversity of chromosomes were showed in Fig. 3, 4. The comparison of the results of karyotypic characteristics in the populations indicated that the base chromosome number was x=9 in all the populations and there were hexa-, tetra- and diploid levels for the popu- lations. Regarding the ploidy level, there was diversity not only among the species, but also among the popu- lations of the three species, A. millefolium (tetra- and hexa-ploidy), A. vermicularis (tetra and diploidy), and A. tenuifolia (tetra and diploidy). A. alepine and A. tala- gonica species were diploid and A. biebersteinii and A. willhelmsii species were tetraploid (Table 2). Karyotype formulas of inter-species and intra-species populations were different and all the chromosomes were metacen- tric only in populations AL2, TA1, VE1, TE1, and TE4; the karyotype consisted of a large number of metacen- tric chromosomes and a small number of chromosomes were submetacentric in other populations. According to the Stebbins’ s index, most of the populations were in 1A and 1B, only population BI4 was in 2B, and MI1 in the 2A (Table 2). Therefore, a symmetrical karyotype was observed for the species of this genus. The highest rela- tive amount of chromatin belonged to population AL3 with an average of 4.15 μm whereas the lowest relative amount of chromatin belonged to population TA2 with an average of 2.55. Except for population AL3, the rela- tive chromatin levels of the populations were less than 4 and more than 2 μm. Since the relative difference in the lengths of chromosomes had an inverse relationship with intra-species ploidy levels, the most asymmetric chromosomes among the hexaploidy populations, based on DRL index, belonged to population MI4 with an average of 38.3% (Tables 2, 3). For diploid populations, BI1 population had the highest rate of chromosomal asymmetry with the highest DRL (5.32 %). Among the diploid populations, VE1 population and four popula- tions of A. talagonica species had the highest rate of 38 Fatemeh Nezhadi et al. DRL, and the most asymmetric chromosomes. The per- centage of overall chromosomes form ranged from 37.6 to 46.11, and the highest percentage of overall chromo- some form belonged to populations TE4, TE1, and TA1; thus, they had a more symmetrical karyotype compared to the other populations. On the contrary, AL3, MI2, MI4, and WI4 had the lowest percentage of overall chro- mosome form; therefore, they had the most asymmet- ric karyotypes (Tables 2, 3). The lowest A1 belonged to populations TE4, TE1, and TA1; consequently, they had more symmetrical karyotypes than the other popula- tions. Based on index A1, AL3 and Wl4 had the high- est chromosomal asymmetry. Hence, it was found that the intra-species diversity was high for A1 and TF%, and the species were indistinguishable based on the param- eters. For the A2, the intra-species diversity was some- what lower, and the species could be divided into three categories; the species of the first class included A. alep- pica and A. millefolium, whose populations had an A2 of less than 0.2 and symmetrical chromosomes based on the index. On the contrary, the populations of two spe- cies, A. talagonica and A. vermicularis, had an index A2 of over 0.2 and asymmetric chromosomes based on the index. However, the populations of other species had higher intra-species diversity compared to the above- mentioned four species populations and also had popu- lations with low inter-chromosomal asymmetry and high A2 (Table 2). In terms of the CI (Table 3), populations TE4, MI1, TE1, VE3, and TA1 had a centromeric index between 0.42 and 0.46 and they had symmetrical chromosomes based on the index. Meanwhile, populations AL3, BI3, MI2, MI4, WI2, and WI4 with a CI of 0.38 had the most asymmetric chromosomes based on the index. The low- est ratio of long to short arm belonged to TE4, TE1, and TA1 populations with average values of 1.19, 1.37, and 1.4, respectively, and had symmetrical chromosomes based on the index. On the other hand, the highest value for the index with long to short arm ratio between 1.6 and 1.69 belonged to populations AL3, BI3, MI2, MI4, WI4, TE3, and WI2. Thus, they had asymmetric chro- mosomes. The highest average total chromosome length belonged to population AL3 with an average of 4.15 µm, and other populations had an average total chromosome length between 2.55 and 3.64 μm, among which popu- lations TA2, TE1, TA4, TE4, BI2, VE2, MI1, VE1, BI4, Figure 1. Positions the population samples were collected on the map. 39A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran Figure 2. Shows different morphological attributes of seven species of Achillea L. in west of Iran, Kurdistan in their natural habitats. (a) A. tenifolia (b) A. vermicularis (c) A. allepica (d) A. biebesteinii (e) A. wilhielmsii (f) A. millefolium (g) A. talagonica. 40 Fatemeh Nezhadi et al. Figure 3. Haploid ideogram of seven species population samples. (Red (SA): Relative length of short arm, Blue (LA): Relative length of long arm, Black (Sat): satellite chromosome, Scale bar = 5 μm, X-axis express No. of chromosome, Y-axis express Relative of long and short arm scale bar = 5 μm). 41A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran Figure 4. The morphological diversity metaphase chromosomes between and within species (Scale bar = 5μm). 42 Fatemeh Nezhadi et al. and BI1 had an average total chromosome length of less than 3 μm. The other populations had an average total chromosome length between 3 and 3.64 µm. Therefore, it was found that intra-species diversity was high for AR, CI, and TL indices (Table 3). Based on the parameters, the species were indistinguishable. The range of the total chromosome length varied widely from a minimum range of 1.59 µm in the population BI2 to a maximum of 9.85 µm in the population VE1; hence, the longest chromosome was 6.19 times higher than the shortest chromosome. Results of analysis of cytogenetic variables Fig. 5 depicts the results of cluster heatmap (based on Euclidian distance and ward method) analysis for cytogenetic variables of different species. A total of 10 attributes were included in the analysis. The results exhibited that the accessions were assigned two main distinct groups. According to the figure 5, in the first cluster A. talgonica (2x) species was separated from others. The second group consists of all other species with different ploidy levels, but interestingly in con- trary side of first group (maximum distance from first group) a diploid species A. aleppica (2x) was located. In other words, two diploid species were assigned to different sides of clustering. Obviously, cluster analy- sis could not detect differences between interspecies, however three species including A. talagonica, A. alep- pica and A. willhelmsii were clearly discriminated from others. It can be noted that cluster analysis could not detect ploidy pattens among the Achillea populations (annotation group in cluster analysis) (Fig.5). Among the attributes a few variables showed the highest inter- relationship variation population accessions which are including %TF, LA and DRL. Table 2. Cytogenetic indices data A2, A1, % TF, DRL, VRC, SC, and KF. Species KF 2n SC VRC DRL %TF A1 A2 Population A. aleppica 8m+1sm 2x=18 1A 3.49 5.90 39.97 0.334 0.170 AL1 9m 2x=18 1A 3.27 5.67 39.78 0.335 0.162 AL 2 6m+3sm 2x=18 1A 4.15 5.90 37.60 0.395 0.169 AL 3 8m+1sm 2x=18 1A 3.30 5.92 39.27 0.351 0.166 AL 4 A. biebersteinii 14m+4sm 4x=36 1B 2.98 5.32 38.42 0.356 0.228 BI1 15m+3sm 4x=36 1B 2.76 4.43 39.43 0.343 0.190 BI 2 14m+4sm 4x=36 1B 3.62 4.39 38.44 0.376 0.195 BI 3 14m+4sm 4x=36 2B 2.97 4.39 38.62 0.343 0.217 BI 4 A. millefolium 20m+7sm 6x=54 2A 3.02 2.54 38.80 0.355 0.153 MI1 17m+10sm 6x=54 1B 2.79 2.94 37.89 0.378 0.167 MI2 15m+3sm 4x=36 1A 3.06 3.67 39.29 0.337 0.183 MI3 20m+7sm 6x=54 1B 3.16 3.38 37.85 0.375 0.194 MI4 A. talagonica 9m 2x=18 1B 3.53 8.80 42.02 0.277 0.236 TA1 7m+2sm 2x=18 1B 2.55 8.07 40.37 0.311 0.224 TA 2 7m+2sm 2x=18 1B 3.38 8.05 40.68 0.315 0.223 TA 3 6m+3sm 2x=18 1A 2.62 7.34 38.86 0.363 0.209 TA 4 A. vermicularis 9m 2x=18 1A 2.85 7.53 39.74 0.339 0.210 VE1 15m+3sm 4x=36 1B 2.76 4.59 39.84 0.321 0.218 VE 2 17m+1sm 4x=36 1B 3.19 4.94 41.20 0.281 0.224 VE 3 17m+1sm 4x=36 1B 3.64 4.49 40.52 0.317 0.218 VE 4 A. tenuifolia 18m 4x=36 1A 2.62 3.04 42.50 0.257 0.159 TE1 14m+4sm 4x=36 1B 2.82 4.50 39.36 0.347 0.223 TE 2 11m+7sm 4x=36 1B 3.70 3.96 38.56 0.369 0.199 TE 3 9m 2x=18 1A 2.69 5.23 46.11 0.141 0.147 TE 4 A .wilhelmsii 15m+3sm 4x=36 1B 3.41 4.17 39.00 0.346 0.190 WI1 14m+4sm 4x=36 1B 3.08 4.61 38.13 0.371 0.205 WI2 16m+2sm 4x=36 1A 3.35 3.95 39.13 0.342 0.168 WI3 14m+4sm 4x=36 1A 3.28 4.04 37.75 0.380 0.183 WI4 43A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran DISCUSSION There are high diversity and differences in chro- mosomal length characteristics of the inter and intra- species of this genus Fig. 3, 4. Given that the existence of diversity and difference in chromosome length indi- cates an advanced karyotype and has chromosomes in different sizes (Afshari et al. 2013), the species of this genus have advanced karyotypes. The existence of x=9 as the base chromosome number on the yarrow genus has been proven in several reports, yet the number of chromosomes and ploidy levels vary among different species of this genus, which could range from 2n=2x= 18 to 2n=8x=72 even though most species are Dip- loid( Guo et al. 2005, Baltisberger and Widmer 2016). In addition to inter-species diversity in ploidy levels, there are numerous reports of ploidy level diversity in populations within a species. In other words, different ploidy levels are reported for populations of a species ( Hoshi et al. 2010, Ebrahim et al. 2012). Accordingly, a range between diploid to hexaploidy has been reported for A. aleppica species (Rad and Javaheri 2014); how- ever, all the accessions of the species were diploid in the present study. The tetraploid level was reported for A. bieberestini species (Afshari et al. 2013), which was consistent with the present result. Afshari reported diploid and tetraploid levels for A. millefolium spe- cies. In another study, hexa and octa-ploidy levels were reported for the species (Ebrahim et al. 2012). The two reports were consistent with the present study in terms of A. millefolium species. For four populations of A. talagonica species, the diploid level was in accordance with results of studies by Sahin et al. (2006). Finally, the results obtained for ploidy levels of two species, A. vermicularis and A. tenuifolia, were in agreement with other reports (Afshari et al. 2013, Rad and Javaheri Table 3. Karyotypes and chromosomal parameters in this study for each species. Population CI AR %RL TL %SA %LA Chromosome range length (µm) AL1 0.4 ± 0.02 1.52 ± 0.1 11.11 ± 1.23 3.49 ± 0.39 4.44 ± 0.59 6.67 ± 0.71 2.63 – 4.48 AL2 0.4 ± 0.01 1.52 ± 0.05 11.11 ± 1.17 3.27 ± 0.34 4.42 ± 0.41 6.69 ± 0.77 2.53 – 4.19 AL3 0.38 ± 0.01 1.69 ± 0.09 11.11 ± 1.23 4.15 ± 0.46 4.18 ± 0.44 6.93 ± 0.81 3.18 – 5.39 AL4 0.39 ± 0.01 1.56 ± 0.07 11.11 ± 1.2 3.3 ± 0.36 4.36 ± 0.46 6.75 ± 0.76 2.58 – 4.33 BI1 0.39 ± 0.01 1.59 ± 0.07 5.59 ± 0.58 2.98 ± 0.31 2.14 ± 0.16 3.36 ± 0.36 1.95 – 4.81 BI2 0.4 ± 0.01 1.56 ± 0.08 5.56 ± 0.49 2.76 ± 0.24 2.19 ± 0.19 3.37 ± 0.31 1.59 – 3.79 BI3 0.38 ± 0.01 1.63 ± 0.06 5.56 ± 0.5 3.62 ± 0.33 2.14 ± 0.21 3.42 ± 0.3 2.52 – 5.38 BI4 0.39 ± 0.02 1.56 ± 0.1 5.56 ± 0.56 2.97 ± 0.3 2.15 ± 0.15 3.35 ± 0.37 2.01 – 4.35 Ml1 0.44 ± 0.01 1.59 ± 0.07 3.7 ± 0.21 3.02 ± 0.17 1.44 ± 0.08 2.24 ± 0.13 2.22 – 4.29 MI2 0.38 ± 0.01 1.63 ± 0.05 3.7 ± 0.23 2.79 ± 0.18 1.4 ± 0.07 2.28 ± 0.14 2.02 – 4.24 Ml3 0.4 ± 0.01 1.53 ± 0.06 5.56 ± 0.47 3.06 ± 0.26 2.18 ± 0.16 3.32 ± 0.28 2.15 – 4.18 Ml4 0.38 ± 0.01 1.63 ± 0.05 3.7 ± 0.27 3.16 ± 0.23 1.4 ± 0.09 2.25 ± 0.14 2.24 – 5.12 TA1 0.42 ± 0.01 1.4 ± 0.05 11.11 ± 1.71 3.53 ± 0.54 4.67 ± 0.75 6.44 ± 0.97 2.34 – 5.14 TA2 0.41 ± 0.02 1.49 ± 0.14 11.11 ± 1.63 2.55 ± 0.37 4.49 ± 0.58 6.63 ± 1.09 1.83 – 3.68 TA3 0.41 ± 0.01 1.49 ± 0.09 11.11 ± 1.62 3.38 ± 0.49 4.52 ± 0.72 6.59 ± 0.92 2.35 – 4.8 TA4 0.39 ± 0.01 1.59 ± 0.07 11.11 ± 1.52 2.62 ± 0.36 4.32 ± 0.61 6.79 ± 0.92 1.8 – 3.54 VE1 0.4 ± 0.01 1.53 ± 0.06 11.11 ± 1.52 2.85 ± 0.39 4.41 ± 0.59 6.7 ± 0.95 4.92 – 9.85 VE2 0.4 ± 0.01 1.52 ± 0.08 5.56 ± 0.56 2.76 ± 0.28 2.21 ± 0.19 3.29 ± 0.33 2.35 – 3.29 VE3 0.42 ± 0.01 1.42 ± 0.07 5.56 ± 0.58 3.19 ± 0.33 2.29 ± 0.19 3.23 ± 0.36 2.11 – 4.95 VE4 0.41 ± 0.01 1.49 ± 0.06 5.56 ± 0.56 3.64 ± 0.37 2.25 ± 0.23 3.3 ± 0.34 2.36 – 5.30 TE1 0.43 ± 0.01 1.37 ± 0.05 5.56 ± 0.41 2.62 ± 0.19 2.36 ± 0.17 3.19 ± 0.25 1.94 – 3.37 TE2 0.39 ± 0.01 1.56 ± 0.07 5.41 ± 0.61 2.75 ± 0.31 2.19 ± 0.23 3.37 ± 0.34 1.82 – 4.1 TE3 0.39 ± 0.01 1.61 ± 0.06 5.39 ± 0.58 3.59 ± 0.39 2.14 ± 0.2 3.41 ± 0.31 1.6 – 4.93 TE4 0.46 ± 0.01 1.19 ± 0.05 11.11 ± 1.06 2.69 ± 0.26 5.12 ± 0.47 5.99 ± 0.61 2.16 – 3.43 WI1 0.4 ± 0.1 1.56 ± 0.06 5.56 ± 0.49 3.41 ± 0.3 2.17 ± 0.16 3.34 ± 0.3 2.25- 4.85 WI2 0.38 ± 0.01 1.6 ± 0.05 5.56 ± 0.53 3.08 ± 0.29 2.12 ± 0.17 3.39 ± 0.31 2.1 – 4.65 WI3 0.39 ± 0.01 1.54 ± 0.05 5.56 ± 0.43 3.35 ± 0.26 2.17 ± 0.13 3.33 ± 0.25 2.46 – 4.84 WI4 0.38 ± 0.01 1.63 ± 0.05 5.56 ± 0.47 3.28 ± 0.28 2.1 ± 0.14 3.4 ± 0.27 2.45 – 4.84 44 Fatemeh Nezhadi et al. 2014). Therefore, no new reports were found for the ploidy levels of the species. The karyotypic formulas of all the species consisted of a large number of metacentric chromosomes and a small number of sub-metacentric chromosomes (Table 2). On this basis, the populations of the species of this genus had symmetrical karyotypes, and there were diverse karyotypic formulas for both species and intra- species populations. In several reports on the cytogenet- ic analysis of species of the yarrow genus, more metacen- tric chromosomes and less submetacentric chromosomes have been reported ( Sahin et al. 2006, Afshari et al. 2013, Rad and Javaheri 2014). Moreover, there were some reports on the subtelocentric chromosomes (Baltisberger and Widmer 2016). However, there were almost sym- metrical chromosomes for species of the genus. Accord- ing to the Stebbins table regarding 28 populations, 11 populations had A1 symmetry, 15 had B1, a population had A2, and a population had B2 symmetry (Table 2); hence, there were more symmetrical chromosomes in the present research than other reports since the karyo- type A2 was mostly reported in other reports, and few- er cases had A1 and B1 symmetries (Kiran et al. 2012, Sahin et al. 2006). Accordingly, no obvious differences were reported in karyotype asymmetry between yarrow species; all the species had symmetrical karyotype struc- tures because most chromosomes were metacentric and sub-metacentric (Kiran et al. 2012). Satellites were observed more in populations with tetra- and hexaploidy levels and on chromosome 1 (Fig. 3). No satellites were observed in diploid populations, and there was only a satellite for each population. Our results were consistent with those of a report by Sahin et al. ( 2006). On the contrary, no satellites were reported in certain studies ( Hoshi et al. 2010, Kiran et al. 2012, Afshari et al. 2013, Rad and Javaheri 2014) whereas one Figure 5. Depicts the results of cluster heatmap (based on Euclidian distance and ward method analysis for cytogenetic parameters). 45A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran to three satellites have been reported in some other researches (Afshari et al. 2013). Additionally, more sat- ellites were observed in submetacentric chromosomes and the results were consistent with those of the present study (Hoshi et al. 2010). There were chromosomes B in two populations of A. tenuifolium species (Table 2). A chromosome B was also reported for the species in some populations (Chehrega- ni Rad et al. 2017), and there were some reports on the existence of B chromosome in other species on the genus (Baltisberger and Widmer 2016). Nevertheless, there was no B chromosome in some reports (Kiran et al. 2012). There was no inter-species diversity for the chroma- tin content, arm length, and chromosome length. Fur- thermore, the intra-species populations showed more diversity (Table 3), but there was inter-species diversity for ratios to arms (and large to small); however, the intra- species populations had diversity. Therefore, the evolution and speciation of the genus was through A1 rather than increasing or decreasing the chromatin content and chro- mosome length. The average length of each chromosome ranged from 2.93 to 3.55 mμ for the species, which was consistent with other reports (Sahin et al. 2006 Afshari et al. 2013) . Meanwhile, the chromosome length range was higher in certain reports than that in the results of the present study, and longer chromosomes were reported for the species (Aksu et al. 2013). Based on the karyotypic characteristics, the A. aleppica had more karyotypic evo- lution in terms of chromatin content, and three species, A. biebersteinii, A. wilhemsii, and A. millefolium, had more complete karyotypes due to the A1 and a higher evolution in terms of chromosome length characteris- tics and chromatin content. A. talangonica, A. tenifolia, and A. vermicularis had karyotypic evolution due to the chromosomal asymmetry; thus, A. biebersteinii, A. wil- hemsii and A. millefolium had more evolved karyotypes than the other species. According to the results, the karyotypic characteristics could not separate the popula- tions of yarrow species due to the intra-species diversity, and the populations of different species were in the same group in several cases. 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