ACTA BOT. CROAT. 77 (1), 2018 93 Acta Bot. Croat. 77 (1), 93–96, 2018 CODEN: ABCRA 25 DOI: 10.2478/botcro-2018-0001 ISSN 0365-0588 eISSN 1847-8476 Short communication Chromosome numbers and karyotype features of Phlomis olivieri Benth. (Lamiaceae) from Iran Hossein Yousefi1, Atefe Amirahmadi2, Reza Naderi2*, Morteza Atri1 1 Department of Biology, Faculty of Sciences, Bu-Ali Sina University, Hamedan, Iran 2 School of Biology and Institute of Biological Science, Damghan University, Damghan, 36716-41167, Iran Abstract – Chromosome numbers were determined in ten accessions of Phlomis olivieri Benth. (Lamiaceae). The seeds were collected from natural habitats in the west of Iran. Chromosome numbers of all accessions were 2n=2x=20. The chromosomes of accessions were metacentric or submetacentric, ranging in length from 2.66 to 8.604 μm. According to the average values of ten accessions, the karyotype of this species consists of 10 pairs of metacentric chromosomes. An ideogram was depicted for the species. This is the first report on the chromo- some number and karyotype analysis of P. olivieri from Iran. Keywords: chromosome count, Iran, karyotype feature, Labiatae, Phlomis * Corresponding author, e-mail: rezanaderia@du.ac.ir Introduction The genus Phlomis L. is one of the largest genera of sub- family Lamioideae (Lamiaceae) with more than 100 rec- ognized species distributed in Asia, southern Europe and northern Africa, which have been divided into two main sec- tions: Phlomoides (Moench) Briq. and Phlomis (Rechinger 1982, Albaladejo et al. 2005). The diagnostic character for separating the sections is corolla shape. Species of the sec- tion Phlomis, which have a corolla with a curved upper lip and trifid lower lip with large median and smaller lateral lobes, differs from species of section Phlomoides that have corolla with straight upper lip and trifid lower lip with sub equal lobes (Azizian and Moore 1982). In Iran, this genus is represented by 19 species (10 species are endemic) including P. olivieri Benth. (sect. Phlomis) which grows wild in north, west and central Iran (Rechinger 1982, Jamzad 2012). Re- cently, the taxonomic value of the indumentum as well as the anatomy and palynology of this species were investigated (Yousefi et al. 2014). There is little cytological information on the genera, though there have been chromosome counts of some spe- cies of Phlomis by workers such as Wagner (1948), Reese (1953), Strid (1965), Zhukova (1967), Chuksanova and Ka- planbekova (1971), Azizian and Cutler (1982), Strid and An- dersson (1985), Aparicio (1997), Aparicio and Albaladejo (2003), Ghaffari (2006) and Özdemir et al. (2014). The chro- mosome number of the species of Phlomis varies from 2n=10 to 2n=46 (Goldblatt and Johnson 1979). Phlomis olivieri is a perennial herb species distributed in Iran and Iraq. This species grows on mountainous regions, adjacent to rocky slopes, steppe vegetation and the over- grazed rangeland soils of the Irano-Turanian region and the Hyrcanian district of Iran (Jamzad 2012) and could be con- sidered one of important destroyed rangeland indicators to- gether with Stachys inflata Benth. (Mozaffarian 2005). The aim of this paper is to determine the chromosome number and karyotype features of P. olivieri for the first time. Materials and methods Plant samples of P. olivieri Benth. were collected in wild populations in the west of Iran. Voucher specimens of all the materials studied were deposited in the herbarium of Isfa- han University, Iran. The locality, collectors and dates of ten accessions are shown in Table 1. Root-tip meristems were obtained from wild collected seeds germinated on wet filter paper in Petri dishes at room temperature in the dark. They YOUSEFI H., AMIRAHMADI A., NADERI R., ATRI M. 94 ACTA BOT. CROAT. 77 (1), 2018 were pretreated using an aqueous solution of colchi (0.05%) at room temperature for 3 h. The material was fixed in ab- solute ethanol and glacial acetic acid (3:1) for 12 h at room temperature and then stored in the fixative at 4 °C. Sam- ples were hydrolyzed in 1 N HCl for 2 min at 60 °C, stained in 1% aqueous aceto-orcein for 2–12 h at room tempera- ture, squashed and mounted in a drop of 45% acetic acid- glycerol (9:1). The best metaphase plates were photographed with a Ziess Axiostar photomicroscope and a Canon dig- ital camera. Karyotype analysis was carried out according to the method described by Levan et al. (1964). Several pa- rameters regarding the karyotypes symmetry/asymmetry including total length of chromosome (C), length of long arm (L), length of short arm (S), arm ratio (R: L/S), centro- meric index (I: (S/C)×100) and centromeric position were calculated for each accession with the use of the DN2 Mi- croscopy Image Processing System. Morphometric data re- garding karyotypes were provided and an ideogram of the species was depicted based on the average of several acces- sions karyotypes which have approximately the same level of chromosome condensation. To assess the existence of pub- lished chromosome counts in the studied species we used the chromosome number database Index to Plant Chromosome Numbers (Goldblatt and Johnson 1979). Results and discussion In the present paper, we determined chromosome num- bers of P. olivieri. The chromosome numbers, karyotype analysis, and ideogram of P. olivieri are shown based on the Tab. 1. Localities and chromosome numbers of the investigated accessions in Phlomis olivieri. No. Location and collection number Geographical character Altitude (m) Chromosome number Date of collection 1 Hamedan Province: Ganjnameh, before Mishan plain, Yousefi 19003. 34°46'03.89"N 48°25'43.67"E 2550 2n = 20 4 June 2011 2 Hamedan province: Asadabad, around Taj abad sofla village, Yousefi 19013. 34°52'49.62"N 48°12'44.58"E 1994 2n = 20 4 June 2011 3 Hamedan province: Malayer, Lashkardar protected area, Yousefi 19011. 34°12'45.14"N 48°58'46.25"E 2172 2n = 20 7 June 2011 4 Hamedan province: Nahavand, Sarabe Giyan, above farmland, Yousefi 19005. 34°08'33.86"N 48°13'03.64"E 1717 2n = 20 5 June 2011 5 Hamedan province: Nahavand, Sarabe Giyan, Yousefi 19006. 34°08'35.16"N 48°13'11.99"E 1698 2n = 20 5 June 2011 6 Hamedan province: Razan, Boghaty Mountains, Yousefi 19012. 35°02'19.5"N 48°50'18.1"E 2350 2n = 20 12 June 2011 7 Hamedan province: Tuyserkan to Malayer, Yousefi 19010. 34°28'25.57"N 48°33'03.63"E 1864 2n = 20 7 June 2011 8 Hamedan province: 5 km Nahavand to Malayer, left side of road, Yousefi 19007. 34°12'45.29"N 48°24'09.62"E 1775 2n = 20 5 June 2011 9 Hamedan province: Nahavand to Malayer, before Kartila- bad village, right side of road, Yousefi 19009. 34°18'44.05"N 48°38'26.69"E 1697 2n = 20 5 June 2011 10 Hamedan province: Kabodarahang, around Gholiabad village, Yousefi 19014. 35°15'03.30"N 48°50'40.01"E 1906 2n = 20 12 June 2011 Tab. 2. Karyomorphological parameters of Phlomis olivieri based on the average values of ten accessions. SD – standard deviation, m – metacentric. Chro- mo- some no. Total length (μm)±SD Long arm length (L) μm Short arm length (S) μm Arm ratio L/S Cen- tro- meric index Chro- mo- some type 1 7.67±0.68 4.09 3.58 1.14 46.67 m 2 7.12±0.82 3.73 3.39 1.10 47.61 m 3 7.11±0.82 3.87 3.24 1.19 45.56 m 4 6.99±0.72 3.81 3.18 1.19 45.49 m 5 6.74±0.73 3.71 3.03 1.22 44.95 m 6 6.64±0.73 3.56 3.08 1.15 46.38 m 7 6.52±0.65 3.65 2.87 1.27 44.01 m 8 6.31±0.72 3.44 2.87 1.19 45.48 m 9 6.20±0.61 3.43 2.77 1.23 44.67 m 10 6.06±0.73 3.39 2.67 1.26 44.05 m 11 6.03±0.69 3.35 2.68 1.25 44.44 m 12 5.86±0.67 3.25 2.61 1.24 44.53 m 13 5.73±0.69 3.07 2.66 1.15 46.42 m 14 5.54±0.48 2.93 2.61 1.12 47.11 m 15 5.48±0.55 3.10 2.38 1.30 43.43 m 16 5.27±0.53 2.97 2.30 1.29 43.64 m 17 5.29±0.52 2.94 2.35 1.25 44.42 m 18 5.10±0.52 2.83 2.27 1.24 44.50 m 19 4.87±0.78 2.83 2.04 1.38 41.88 m 20 4.69±0.74 2.61 2.08 1.25 44.36 m CHROMOSOME NUMBER AND KARYOTYPE OF PHLOMIS OLIVIERI ACTA BOT. CROAT. 77 (1), 2018 95 average values of ten accessions (Tab. 1, Tab. 2, Fig. 1, and Fig. 2). This is the first karyotype determination for this spe- cies. Based on our results the basic chromosome number in the studied accessions was x=10. All accessions have the same chromosome number and represent diploid species with 2n=2x=20. Chromosome sizes vary from 2.66 to 8.60 μm. The longest arm is 4.796 μm (accession 2) and the short- est arm is 1.16 μm (accession 9) (Fig. 1.) Different karyo- types have been found among the accessions of P. olivieri. Generally, the studied karyotypes consist mostly of metacen- tric chromosomes with almost median centromere position (Stace 1989). However, the karyotypes of accessions 1, 4, 6, 7 and 9 beside metacentric chromosomes also possess sub- metacentric chromosomes. Such differences between acces- sion karyotypes could be a result of different chromosome condensation due to the pretreatment procedure. This could be also a reason why secondary constrictions with satellites were not observed in the karyotype of this species. Accord- ing to the average values of ten accessions, the karyotype of this species consists of 10 pairs of metacentric chromo- somes (Tab. 2). Azizian and Cutler (1982) revealed that species of the Phlomis section Phlomis are characterized by 2n=20 large chromosomes, while species of the Phlomis section Phlomoi- des possess 2n=22 and smaller chromosomes. Özdemir et al. (2014) determined 2n=20 chromosomes for P. lunariifo- lia Sm. and P. grandiflora H.S. Thomps., two species of the genus Phlomis sect. Phlomis. The investigated species had 9 pairs of metacentric and 1 pair of submetacentric chromo- somes. Furthermore, the presence of 2n=20 chromosomes within the genus has been confirmed in several species such as P. cypria Post var. cypria (Yildiz and Gücel 2006), P. lych- nitis L., P. purpurea L., P. italica L. and P. herba-venti L. var. tomentosa Boiss. (see Mateu 1986). Although it can be con- cluded that the presence of twenty somatic chromosomes in our study is consistent with the previous studies, karyotypic differences exist within or among species and alteration in chromosome symmetry may arise through translocations, pericentric inversions or fusion (Levin 2002). Our observations as well as comparison of photomi- crographs obtained from previous reports also showed that Phlomis chromosomes are the largest among species of the Lamiaceae genera such as Callicarpa L., Salvia L., Scutellaria L., Sideritis L., Stachys L., Teucrium L. and Thymus L. (Jalas 1948, Boşcaiu et al. 1998, Yildiz and Gücel 2006, Yang et al. 2009, Martin et al. 2011, Contreras and Ruter 2011, Javadi et al. 2011). Acknowledgement The authors would like to thank the graduate depart- ment of Bu-Ali Sina University. A small part of the present study was financially supported by Grant-in-Aids for Scien- tific Research, No. 93035352 from INSF (Iran National Sci- ence Foundation). 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