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Cytological investigations and new chromosome number reports in yarrow (Achillea millefolium Linnaeus, 1753) accessions from Iran

Abstract In this study, a new chromosome number for Iranian yarrow (Achillea millefolium L.) accessions was reported. Cytological analyses on four Achillea millefolium accessions, indicated that two accessions were diploids (2n=2x=18) and two tetraploids (2n=4x=36). Cluster analysis based on chromosomal characteristics and karyotype asymmetry, categorized the four accessions separated into two groups. In terms of the Stebbins’ system, the karyotype of diploid accessions grouped in 2A class. The average value of the total form percentage (TF%) in the group one (diploid accessions) and two (tetraploid accessions) were 40.85 and 41.15, respectively. The group one had the highest mean value for the symmetry index (S%=57.5). Consequently, it can be inferred that diploids belonging to the group one are the earlier evolutionary forms.


Introduction
Achillea is one of the most recent genera of the Asteraceae family which exists throughout the world (Rechinger 1963). More than 100 species have been identified in this genus. Many of those who used these plants reported properties such as anti-inflammatory, anti-rheumatic, antiseptic, antispasmodic, analgesic, astringent, carminative, diaphoretic, digestive, expectorant, hypotensive, stomachic and etc. (Balbir et al. 2012). These plants are native to Europe and Western Asia but are also found in Australia, New Zealand, and North America (Rechinger 1963).
Achillea millefolium has a high genetic and phenotypic variation in Iran . The basic chromosome number is often reported in different species of Achillea is x = 9; however, the diversity in chromosome numbers and ploidy levels are frequently occurring in the genus (Ebrahim et al. 2012). Polyploid taxa have originated in many clades including 4x, 6x and 8x species, and as a result, several Achillea species show high morphological variability (Sheidai et al. 2009). Biste (1987) explained worthy diversity in leaf width, height, shoot number, and stomata length in different populations of the same species.
Achillea millefolium has been cytologically analyzed extensively in different regions of the world (Felfoldy 1947, Mizianty and Frey 1973, Pireh and Tyrl 1980, Lavrenko et al. 1991, Guo et al. 2012, Bala and Gupta 2013. Three cytological studies have been reported in Iran and showed the following ploidy levels: tetraploid 2n = 4x = 36, hexaploid 2n = 6x = 54 and octoploid 2n = 8x = 72 (Farsi et al. 2000, Ebrahim et al. 2012, Sheidai et al. 2009). The aims of this study were (1) to determine the chromosome numbers of four A. millefolium accessions and (2) to find any relationship between the karyotype characteristics and asymmetrical index with ploidy levels.

Material and methods
The aerial parts of the four Achillea millefolium accessions were collected from three provinces in north, west and south of Iran (Table 1). Voucher samples were deposited at the herbarium of Research Institute of Forests and Rangeland (RIFR) of Tehran, Iran. Seeds were germinated on moist filter paper in Petri dishes. Actively growing root tips, 1 to 2 cm length were cut from the germinating seeds and pretreated with 8-hydroxyquinoline (0.002M) for 2 to 4 hours and fixed in Farmer (1:3, glacial acetic acid : ethanol 95%) for 24 hours at 4° C. Thereafter, the root tips were hydrolyzed in 1 N NaOH at 60° C for 5-10 minutes, stained for 45 minutes in esterase stain at 30° C, and squashed in 45% glacial acetic acid. Finally, the chromosome images were obtained with photomicroscope.
Karyotypec characteristics such as differences of range relative length (DRL), mean chromosome length (MCL), and mean arm ratio (MAR) were calculated using MI-CROMEASURE (Version 3.3) Software (Reeves 2001). Stebbin's classification was calculated (Stebbins 1971). Cluster analysis was performed to differentiate the accessions according to the Ward's method SPSS software for Windows 20.0 (SPSS Inc., Chicago, IL, USA).
Karyotypic analysis indicated asymmetrical pattern in the four accessions of A. millefolium (Table 2). Mitotic metaphases and karyograms of the four accessions are shown in Figure 1. The highest TCL value was found in Am3 (60.9 µm) and the lowest was found in Am2 (24.5 µm) ( Table 3). The lowest and the highest DRL values were found in Am3 and Am2 accessions, respectively (Table 3). High DRL value leads to more changes in the construction of chromosomes. DRL values in the two diploid accessions were higher than the tetraploid ones; it can be a relationship between ploidy level and DRL value. The tetraploid accessions had the most symmetric karyotypes.
Other parameters that indicate karyotype asymmetry are total form percentage (TF %; Huziwara 1962) and symmetrical index (S% or S/L%; Battaglia 1955) ( Table 2). Group one (Am1 and Am2) had the highest mean value for the symmetrical index (S%=57.5) than the group two (S%= 50) ( Table 2). It can be inferred that the group one, as diploids, are the earlier evolutionary form. The average value of the total form percentage (TF%) in the group one and two were 40.85 and 41.15, respectively. The TF% index has frequently been used to explain karyotype asymmetry (Mercado-Ruaro and Delgado-Salinas 1998, Ruas et al. 2000). In terms of the Stebbins' system, the karyotype of Am1 and Am2 grouped in 2A class, and it can be ancient evolutionary origin of A. millefolium species.

Cluster Analysis
Cluster analysis was done based on karyotypic characteristics (TCL, MCL, MAR and DRL) and karyotype asymmetry (TF% and S%) ( Figure 2) and agrees with Ebrahim   (2012). The results of cluster analyses divided the four accessions in two groups ( Figure 2); based on ploidy levels. The first group included diploid accessions (Am1 and Am2), while the second group comprised tetraploid accessions (Am3 and Am4). In the dendrogram, distance between diploid accessions is lower than tetraploid accessions that confirm the result of Stebbins' system that both of diploid accessions grouped in 2A class.

Conclusion
The results of the present study illustrated a new ploidy level (2n = 2x = 18) in Iranian Achillea millefolium accessions. Cluster analysis indicated that accessions can be classified based on ploidy levels.