of Achillea sipikorensis Hausskn. and Bornm. and Achillea sintenisii Hub.-Mor. (Asteraceae)

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1 CARYOLOGIA Vol. 57, no. 3: , 2004 numbers, karyotypes and 4C DNA contents of Achillea sipikorensis Hausskn. and Bornm. and Achillea sintenisii Hub.-Mor. (Asteraceae) SÈifa Turkoglu* and H. Askin Akpulat Cumhuriyet University, Faculty of Art and Science, Department of Biology, 58140, Sivas, TURKEY Abstract The cytological features including chromosome numbers, detailed chromosome measurements and 4C DNA values were reported in Achillea sipikorensis and A. sintenisii from Turkey. Total chromosome lengths, relative lengths, long/short arm ratios and centromeric index of mitotic chromosomes were calculated. Somatic chromosome numbers were found to be 2n=2x=18 in A. sipikorensis and 2n=4x=36 in A. sintenisii for the first time. The mean of chromosome length ranged from 6.92±0.08 to 9.23±0.58 µm in A. sipikorensis and from 6.54±0.91 to 9.62±0.79 µm in A. sintenisii. The karyotype consists of six pairs metacentric chromosomes, two submetacentric pairs and a subtelocentric pairs in A. sipikorensis and of fifteen pairs metacentric chromosomes, a submetacentric pairs and two subtelocentric pairs in A. sintenisii. The nuclear DNA C-value was estimated to be 4C= 24.63±0.01 pg of A. sipikorensis and 4C= 35.73±0.05 pg of A. sintenisii. Key words: Achillea chromosomes endemic- karyotype analysis - nuclear DNA content INTRODUCTION Achillea L. includes about 85 taxa, most native to Europe and temperate Asia and with some in North America (Cheers 1999). Several species (44) are recorded in the flora of Turkey (Davis 1975; Davis et al. 1988; Guner et al. 2000). The karyology of the genus has been studied by several authors and reported chromosome numbers include 2n=18, 36, and 54, with basic chromosome number x=9 (Contandriopoulos and Martin 1967; Oswiecimska 1974; Halliday and Beadle 1980; Androschchuk and Kostinenko 1981; Magulaev 1982; Dabrowska, 1989a, 1992; Maffei et al. 1993). The nuclear DNA content varies considerably not only among species, but also among and within populations of species (Bennett and Leitch 1995). The DNA content is one of the genome characteristic, beside the number and morphology of chromosomes, and has been determined for numerous taxa of the family Asteraceae and other plant groups (Rees and Jones 1972; Grime and Mowforth 1982; Dutta 1986). Data on nuclear DNA content of this genus species was given by Dabrowska (1992), firstly. * Corresponding author: turkoglu@cumhuriyet.edu.tr. No cytological works have been published on the Achillea, except for A. multifida, A. setacea, A. spinulifolia, inturkey (Martin-Noguet 1969). A. sipikorensis and A. sintenisii are endemic to Turkey (IUCN 2001). Even chromosome numbers are unknown for most of the approximately 44 Turkey species. The chromosome numbers, karyotypes and DNA C-values estimates of A. sipikorensis Hausskn. & Bornm. and A. sintenisii Hub.-Mor. are presented here for the first time. MATERIALS AND METHODS A. sipikorensis and A. sintenisii plants were collected from the natural populations in the center of Sivas from Turkey. Specimens were deposited at Cumhuriyet University. Seeds of these species were used in the study for karyotype analysis and nuclear DNA content estimation. Karyotype - Mitotic chromosomes in root-tip cells were studied from squash technique. Seeds were germinated in darkness at 25 o C and them put in petri dishes on moist filter paper. Actively growing root tips 1 cm in length were excised from the germinating seeds; them were pretreated with M 8-hydroxyquinoline for 24 hours at 25 o C and fixed in Carnoy (3:1 ethanol absolute: acetic acid) for 24 hours at room temperature. staining was made in 2%orcein after

2 chromosome numbers, karyotypes and 4c dna contents of achillea sipikorensis hausskn. 245 cold hydrolysis in 5NHCl for 7 min. Root tips were squashed in 45 % glacial acetic acid. The slides were examined under a light microscope, and 10 well scattered metaphase plates were selected for karyotype analysis. Some of these metaphases were photographed and ordered in the karyotype on the basis of their length. For each chromosome, total length, short arm and long arm were measured and relative lengths, arm ratios and centromeric index were determined. pairs were identified according nomenclature followed Levan et al.(1964). Nuclear DNA content - For Feulgen cytophotometric estimation of 4C DNA, 10 fixed (Carnoy) root tips from each species were hydrolysed in 5NHCl for 1 hour at room temperature, washed in distilled water and stained in Schiff s reagent for 2 hours at 25 o C; each root tip squash was prepared in 45 % acetic acid on a slide. 4C DNA values were estimated from metaphase chromosomes using reichert-zetopan microspectrophotometer, at 550 nm. On average, 35 4C metaphase nuclei were measured in each of 3 replicates for two species. In situ DNA values were obtained on the basis of optical density, which were converted to picograms (pg) by using Van't Hof's (1965) 4C nuclear DNA value (67,1 pg) for Allium cepa as standard. The standard error and standard deviation of the values were then calculated. The correlation coefficient analysis between total chromosome length and 4C DNA content were done for A. sipikorensis and A. sintenisii. RESULTS Achillea sipikorensis Hausskn. and Bornm. - A. sipikorensis had a chromosome number of 2n=2x=18 (Figs. 1a, 2a and 3). Karyotype formula, chromosome morphology, total and relative chromosome lengths, arm ratio and centromeric index were summarized in Table 1. At mitotic metaphase six pairs of metacentric (m-m), two pairs of submetacentric (sm), and one pair of subtelocentric (st) chromosomes were visible. The chromosome length varied from 6.92±0.08 to 9.23±0.58 µm. The relative chromosome length Fig. 1 Mitotic metaphase chromosomes of Achillea species. a. Achillea sipikorensis (2n=2x=18) b. Achillea sintenisii (2n=4x=36). Scale bar= 10 µm. Table 1 Morphometric characteristics of the chromosomes of A. sipikorensis no Relative Centromeric Arm ratio length (µm) chromosome length index (long/short) Mean ±S. E. (% of 2n) Mean ±S. E. Mean ±S. E. morphology ± ± ± ±0.00 M ± ± ± ±0.14 m ± ± ± ±0.18 m ± ± ± ±0.25 m ± ± ± ±0.32 st ± ± ± ±0.54 sm ± ± ± ±0.76 m ± ± ± ±0.90 sm ± ± ± ±01.11 m T. C. L S. E.= Standard Error; m-m= metacentric; sm= submetacentric; st= subtelocentric; T. C. L.= Total chromosome length (haploid complement).

3 246 turkoglu and askin akpulat Fig. 2 Karyotypes of Achillea species. a. A. sipikorensis,b.a. sintenisii. Scale bar=10 µm. ranged from 10.06±0.60 to 13.41±0.85 %. Total chromosome length was µm. Achillea sintenisii Hub.-Mor. - This species showed 2n=2x=36 chromosomes (Figs. 1b, 2b, 4). The chromosome number, chromosome morphology, total and relative chromosome lengths, arm ratio and centromeric index of A. sintenisii were detailed in Table 2. In the karyotype fifteen chromosome pairs were metacentric (m-m), one pair submetacentric (sm), and 2 pairs subtelocentric (st). The chromosome length ranged from 6.54±0.91 to 9.62±0.79 µm. The relative length varied from 4.41±0.84 to 6.50±0.33 %. Total chromosome length was µm. DNA content - The nuclear DNA C-value of A. sipikorensis (2n=18) was estimated to be 4C=24.63±0.01 pg. The nuclear DNA content was positively correlated with total chromosome length in this species (Table 3). 4C nuclear DNA content of A. sintenisii (2n= 36) was found to be 35.73±0.05 pg. A positive correlation between total chromosome length and DNA content of A. sintenisii was observed (Table 3). Table 2 Morphometric characteristics of the chromosomes of A.sintenisii no Relative Centromeric Arm ratio length (µm) chromosome length index (long/short) Mean ±S. E. (% of 2n) Mean ±S.E. Mean ±S. E. morphology ± ± ± ±0.81 m ± ± ± ±0.00 M ± ± ± ±0.97 m ± ± ± ±0.53 m ± ± ± ±0.64 m ± ± ± ±0.59 m ± ± ± ±0.29 m ± ± ± ±0.39 m ± ± ± ±1.50 st ± ± ± ±0.53 m ± ± ± ±0.00 M ± ± ± ±0.39 st ± ± ± ±0.00 M ± ± ± ±0.00 M ± ± ± ±0.00 M ± ± ± ±0.49 sm ± ± ± ±0.00 M ± ± ± ±0.31 m T. C. L S. E.= Standard Error; m-m= metacentric; sm= submetacentric; st= subtelocentric; T. C. L.= Total chromosome length (haploid complement).

4 chromosome numbers, karyotypes and 4c dna contents of achillea sipikorensis hausskn. 247 Fig. 3 Idiogram of A. sipikorensis. Table 3 4C DNA contents of A. sipikorensis and A. sintenisii and correlation values between total chromosome length and DNA content. Species A. sipikorensis A. sintenisii number r= correlation coefficient Total chromosome length (µm) DISCUSSION 4C DNA content (pg) r r ± ± The genus Achillea is a polyploid complex; with basic chromosome number x=9, widely distributed in the Northern hemisphere. It is interesting because of its diversity, variability of some of its taxa and the existence of problematic populations. The chromosome numbers of diploid A. sipikorensis (2n=2x=18) and tetraploid A. sintenisii (2n=4x=36) were reported for the first time in this study, respectively. Over 48 years have elapsed since Darlington and Wylie (1955) first published the chromosome count for Achillea. numbers of the species have been reported by several investigators. According these reports, A. distans had 2n=6x=54, A. collina had 2n=4x=36; A. ageratum had 2n=2x=18 (Androschchuk and Kostinenko 1981; Maffei et al. 1993); A. setacea had 2n=4x=36 (Magulaev 1982; Maffei et al. 1993); A. ligustica had 2n=2x=18 (Pavone et al. 1981; Maffei et al. 1993); A. aspleniifolia had 2n=2x=18 (Halliday and Beadle 1980; Maffei et al. 1993). In the last, chromosome number was determined in 40 taxa of Achillea L. with different degrees of ploidy (2x, 4x, 6x, 8x) (Dabrowska 1989 a, b, c, 1992). In all species, metacentric chromosomes are the most frequent followed by some submetacentric types. The only report on the chromosome numbers of the Achillea in Turkey, studied in specimens from Bursa and Niğde, was that of Martin-Noguet (1969) that report the chromosome numbers for the following three species, namely A. multifida, A. setacea and A. spinulifolia as 2n=2x=18. Our cytological data for A. sipikorensis and A. sintenisii consistent with those of other investigators (Martin-Noguet 1969; Kuzmanov and Kozuharov 1970; Oswiecimska 1974; Halliday and Beadle 1980; Maffei et al. 1993) for Achillea species in the number of chromosomes (2n=18, 36). Additionally, karyotypes are symmetrical with complements composed of mainly metacentric chromosomes, as the others Achillea species. Polyploidy is widespread in plants and has been a major feature in plant evolution. Estimates of the proportion of species of angiosperms that are polyploid vary from about 20% to 50%, in marked contrast to the rarity of polyploidy in animals. In general, karyotype analysis of Achillea species showed that they were tetraploids and hexaploids (Halliday and Beadle 1980; Androschchuk and Kostinenko 1981; Dabrowska 1992; Maffei et al. 1993). The tetraploid (2n=36) and hexaploid (2n=54) complements found in Achillea are progressive steps in an euploid series, showing that polyploidy is one of the main speciation mechanisms in the perennial species of the genus. Karyotype analysis suggests that all these species have an allopolyploid origin following hybridization process. It is hoped that further evidence on the origin of these polyploids may be obtained by studying meiotic behaviour or using techniques such as genomic in situ hybridization (GISH). On the other hand, the chromosomal homogeneities of these species composed mainly of m and sm type of chromosomes may be responisible for interspecific hybridization (Maffei et al. 1993). Measurable changes in the DNA can result from various sources, in particular, from changes in chromosomal number, deletions or dublications (Price 1976), and aberrant replication of certain chromosomal segments (Keyl 1965). DNA increases and

5 248 turkoglu and askin akpulat Fig. 4 Idiogram of A. sintenisii. decreases have a major role in evolution. These changes and individual nucleotide changes are seen in two of the most important modifications contributing to evolutionary change. Low amounts of DNA are correlated with specialisation in animals and in plants (Stebbins 1966). DNA content was measured cytophotometrically in nuclei of 39 taxa of Achillea A significant diversification was found among the taxa of Achillea between ploidy levels, within ploidy levels (Dabrowska 1992). In present paper, we found that 4C nuclear DNA content of A. sipikorensis was 24.63±0.01 pg, and of A. sintenisii was 35.73±0.05 pg. Correlation coefficient studies revealed significant relationship between karyotype lengths and nuclear genome size of the two species (Table 3). Although the chromosome type of these two species are more similar, DNA contents are different. Differences in DNA content may be due to an increase in DNA by duplication events after polyploid. The results obtained, concerning the number of chromosomes, their structure and nuclear DNA content estimation, may be used for establishing or verifying the taxonomic relationship between species of the genus Achillea. Furthermore, the data given may provide a basis for further population investigations. REFERENCES Androschchuk A. F. and Kostinenko L. D., 1981 numbers of the genus Achillea L. Certain species cultivated in botanical gardens. Ukrajinsk Bot. Zurn. (Kiev), 38: Bennett M. D. and Leitch I. J., 1995 Nuclear DNA amounts in angiosperms. Annals of Botany, 80: Cheers G., 1999 Botanica, Random House Australia Pty Ltd, Australia. Contandriopoulos J. and Martin D., 1967 Contribution a I etude cytotaxonomique des Achillea de Grece. Irregularite de la meiose. Bull. Bot. Soc. France, 114: Dabrowska J., 1989a The chromosome numbers of several taxa of the genus Achillea L. Acta. Soc. Bot. Pol., 58: Dabrowska J., 1989b Problematic octaploid forms and a new chromosome number in the genus Achillea L. 2n= 126. Acta. Soc. Bot. Pol., 58: Dabrowska J., 1989c Number of nucleoli in diploids and polyploids of the genus Achillea L. Acta. Soc. Bot. Pol., 58: Dabrowska J., 1992 number and DNA content in taxa of Achillea L. in relation to the distribution of the genus. Prace Botaniczne, 49: Darlington C. D. and Wylie A. P., 1955 Atlas of Flowering Plants. Allen and Unwin, London. Davis P. H., 1975 Flora of Turkey and the East Aegean Islands, Edinburgh Univ. Press, Edinburgh, 5: Davis P. H., Mill R. R., and Tan K., 1988 Flora of Turkey and the East Aegean Islands Suppl., vo. 10, Edinburgh Univ. Press, Edinburgh. Dutta S. K., 1986 DNA systematics. Vol. II. Plants. CRC pres, Inc. Boca Raton, Florida. Grime J. P. and Mowforth M. A., 1982 Variation in genome size an ecological interpretation. Nature 299:

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