Chromosome Botany (2008) 3: 53-63 © Copyright 2008 by the International Society of Chromosome Botany Chromosome counts and determination of ploid levels in Iranian species of Trifolium F. Salimpour1, F. Sharifnia, G. Mostafavi, Sh. Hajrasoliha and E. Ukhneh Department of Biology, Faculty of Science, Islamic Azad University, Notrth Tehran Branch, P. O. Box 19585-936, Iran 1Author for correspondence: (dr salimpourgmail.com) Received April 5, 2008; accepted June 10, 2008 ABSTRACT. Chromosome numbers in 140 accessions of 37 annual and perennial species of Trifolium L. in Iran were counted and studied here. The chromosome number of 2n=16 was documented in Trifolium radicasum in Iran. New cytotypes were found in Trifolium ambigum, T. fragiferum, T. montanum, T. nigrescens, T. stellatum and T. tumens. This study deposited more data in chromosome numbers of Trifolium to determine the genus has high diversity of ploid level. Their karotypic formula showed that the most of the chromosomes were median-, submedian- or terminal-centromeric. The smallest chromosomes were found in Sections Versicaria and Mistyllus, while the largest chromosomes were found in Sections Trifolium and Tricocephalum. Thus, Trifolium seemed to be heterogeneous with respect to morphological characters of chromosomes. KEYWORDS: Chromosomes, Iran, Fabaceae Polyploid, Trifolium The Fabaceae is the third largest family of the flowering together with the previous documents in the most standard plants and Trifolium which has 255 species (Gillett and references. Taylor 2001, Zahary and Heller 1984) is the largest genus in the family. The genus name refers after the distinctive MATERIALS AND METHODS leaves usually composed of three leaflets (Trifoliate). Plant materials One hundred and fourty accessions in The natural distribution of Trifolium is from the 37 species of Trifolium were collected in natural habitats temperate to the subtropical regions in the Northern and in Iran, were conserved in Iranian Gene Bank (IGB), and Southern hemispheres. Iran is the second richest center then, they were used in the present study of chromosomes of the species diversity in this genus which has approxi- (Table 1). The plants studied were cultivated in the mately 46 species in six sections such as Lotoidae, Laboratory of Plant Systematics, Islamic Azad University, Versicaria, Mistyllus, Trifolium, Trichocephallum and North Tehran Branch. Chronosemium (Zohary and Heller 1984). Some of these Taxa are important forage and pasture plants. At least Chromosome preparations Root tips of the plants studied 184 species of Trifolium have shown their chromosome were harvested and pretreated in (1-α Bromonaphtalene) numbers (Taylor et al.1979; Zohary and Heller 1984, for 4 h at room temperature, fixed in a solution containing Goldbatt and Johnson 2003). Over 80% of the examined chromic acid and formaldeid (1:1=v/v) for 30 h in the species had the chromosome number of 2n=16 that refrigerator and were, then, macerated in 1N hydrochloric suggested the basic chromosome number of x=8 (Goldblatt acid at 60°C for 12-15 min and stained with hematoxilin 1981). Thirty-one species of the genus examined showed for 18 h. They were squashed. At least ten cells at mitotic some aneuploids such as 2n=10 or 12 or 14 and among metaphase were studied for each species. them 11 species had intraspecific aneuploids of 2n=16 while 24 species (ca 20%) showed intraspecific polyploids RESULTS and of which six were exclusively tetraploid, two were The chromosome numbers were counted in cells at mitotic hexaploid and one was dodecaploid. Most of the ployploid metaphase in apical meristems of root tips in 140 accession species grew naturally in the old world (Zohary and in 37 species of Trifolium in six sections (Table 2). On Heller 1984). Sections Mistyllus or Trichocephallum the basis of the present observation, the basic chromosome seemed not to have polyploidy species according to the number of the species used and placed in the sections of previous documents, however, 11 species of the sections Versicaria, Mistyllus and Lotoidae were x=8. The basic had intraspecific diploid and polyploidy and three had chromosome number found in T. fragiferum and T. tumens intraspecific multi-ployploids at the tetraploid level of the Section Versicaria and in T. ambigum and T. (Ellison et al. 2006). Section Trifolium showed the basic montanum of the Section Lotoidae showed tetraploid for chromosome numbers of x=5, 6, 7 and 8 (El-Khaly 1990, the first time. Four basic chromosome numbers of x=5, Zohary and Heller 1984). 6, 7 and 8 were found in the Section Trifolium. The The present study made chromosome counts in 140 species placed in the Section Chronosomium were diploid accessions, 37 species of Trifolium in Iran and discussed (x=7 and 8). Trifolium physodes, T. repens, T. ambigum, 53 54 SALIMPOUR ET AL. Table 1. The species, the accessions of Trifolium studied and their collecting sites Species Locality and voucher specimen no. T. repens L. Ardebil: Sabalan, Shabil, 2440m, Salimpour 40 T. repens L. Tehran: Plur, 2300m, Salimpour 41 T. repens L. Ardebil: Makohanddar, 1320m, Salimpour 42 T. repens L. Tehran: Siahkoh, 2050m, Salimpour 43 T. repens L. Mazandaran: Siahbishe, Chalous valley, 2300m, Salimpour 44 T. nigrescens Viv. Tehran: Plur, 230m, Salimpour 45 T. nigrescens Viv. Ardebil: Lisar,2540m,Salimpour 46 T. nigrescens Viv. Azarbaijan: Piranshahr, Mirabad, 1300m, Salimpour 47 T. nigrescens Viv. Azarbaijan: Jade oromie, Oshnavie, 1780m, Salimpour 48 T. ambigum M.B. Ardebil: Ghtor su, Kohe Sabalan, 3650m, Salimpour 49 T. ambigum M.B Ardebil: Shabil, Kohe Sabalan, 2440m, Salimpour 53 T. ambigum M.B Azarbaijan: Kohe Sabalan , 2500m, Salimpour 54 T. ambigum M.B Azarbaijane: Marand to Sofian , 1700m, Salimpour 55 T. montanum L. Ardebil: Ghtor su, Kohe Sabalan, 2800m, Salimpour 49 T. montanum L. Ardebil: Shabil, Kohe Sabalan, 2440m, Salimpour 50 T. montanum L. Azarbaijane: Sari chan , Mako, 2600m, Salimpour 51 T. montanum L. Azarbaijane: Northest of Mianeh, 1440m, Salimpour 52 T. hybridum L. Azarbaijane: Northest of Mianeh, 1440m, Salimpour 56 T. hybridum L. Ardebil: Sarein, Salimpour 57 T. hybridum L. Azarbaijaneh: Nari village, Mergor, 1800m, Salimpour 58 T. hybridum L. Azarbaijane: Tergovar to Silvana , 1180m, Salimpour 59 T. hybridum L. Azarbaigan: Arasbaran forest, 2300m , Salimpour 60 T. glomeratum L. Gilan: Anzali, -26m, Salimpour 68 T. glomeratum L. Gilan: Batlagh Beshmen, -21m, Salimpour 69 T. radicosum Boiss et Hohen. Tehran: Tochal, 3750m, Salimpour 61 T. radicosum Boiss et Hohen. Tehran: Tochal, 3850m, Salimpour 62 T. pilular Boiss. Azarbaijaneh: Nari village, Mergor, 1800m, Salimpour 63 T. pilular Boiss. Azarbaijaneh: Sardasht to Mahabad ,1340m, Salimpour 64 T. pilular Boiss. Kermanshah: Biston, 1420m, Salimpour 65 T. pilular Boiss. Lorestan: Reimaleh village, 1500m, Salimpour 66 T. pilular Boiss. Kermanshah: Sarepolezahab, 700m, Salimpour 67 T. subteraneum L. Gilan: Anzali, -26m, Salimpour 70 T. subteraneum L. Gilan: Batlagh Bashm, -21m, Salimpour 71 T. subteraneum L. Golestan: Region toskhstan, 400m, Salimpour 72 T. pretense L. Azarbaijaneh: Nari village, Mergor, 1800m, Salimpour 83 T. pretense L. Tehran: Firozkoh, 1950m, Salimpour 84 T. pretense L. Tehran: Damavand, 2500m, Salimpour 85 T. pretense L. Mazandaran: Dareh Lar, 2420m, Salimpour 86 T. pretense L. Mazandaran: Kelardasht, 1100m , Salimpour 87 T. pretense L. Mazandaran: Mian Kale, 200m, Salimpour 88 T. pretense L. Mazandaran: Ghaemshahr to Keiakola, 80m, Salimpour 89 T. arvense L. Kermanshah: Biston, 1420m, Salimpour 73 T. arvense L. Golestan: Toskastan, 400m, Salimpour 74 T. arvense L. Lorestan: Reimaleh village, 1500m, Salimpour 75 T. arvense L. Mazardaran: Bagh Sarhang, 420m, Salimpour 76 T. arvense L. Mazandaran: Savad koh, 350m, Salimpour 77 T. purpureum Loisel. Lorestan: Reimaleh vilage , 1500m, Salimpour 78 T. purpureum Loisel. Kermanshah: Kerandgharb, 1240m, Salimpour 79 CHROMOSOMES IN IRANIAN TRIFOLIUM 55 Table 1. -- continued T. purpureum Loisel. Azarbaijaneh: Sardasht to Mahabad, 1340m, Salimpour 80 T. purpureum Loisel. Azarbaijaneh: Oshnavie to Piranshahr, 1600m, Salimpour 81 T. purpureum Loisel. Kermanshah: Ghasreshirin, 350m, Salimpour 82 T. angustifolium L. Golestan: Toskastan, 420m, Salimpour 90 T. angustifolium L. Mazandaran: Miankale, 200m, Salimpour 91 T. angustifolium L. Gilan: Asalem to Khalkhal, 1050m, Salimpour 92 T. angustifolium L. Gilan: Gardaneh Heiran to Astara, Salimpour 93 T. scabrum L. Lorestan: kakareza, 1450m, Salimpour 99 T. scabrum L. Mazandaran: Absabad to Kelardasht, 1100m, Salimpour 100 T. scabrum L. Kermanshah: Ghasreshirin, 350m, Salimpour 101 T. echinatum M. B. Kermanshah: Ghasreshirin, 700m, Salimpour 102 T. echinatum M. B. Kermanshah: Ravansar, 1230m, Salimpour 103 T. echinatum M. B. Kermanshah: Sarepolezahab, 700m, Salimpour 104 T. leucantum M. B. Kermanshah: Ghasreshirin, 700m, Salimpour 105 T. leucantum M. B. Kermanshah: Saropolezahab, 700m, Salimpour 106 T. leucantum M. B. Azarbaijane: Arasbaran forest, Salimpour 107 T. leucantum M. B. Kermanshah: Ravansar, 1230m, Salimpour 108 T. cherleri L. Kermanshah: Ghasreshirin, 350m, Salimpour 94 T. cherleri L. Lorestan: Kakareza, 1450m, Salimpour 95 T. cherleri L. Kermanshah: Biston, 1420m, Salimpour 96 T. cherleri L. Kermanshah: Ghareghorighale, 450m, Salimpour 97 T. cherleri L. Azarbaijane: Tergovar to Silvana, 1180m, Salimpour 98 T. hirtum All. Azarbaijane: Sardasht to Mahabad, 350m, Salimpour 109 T. hirtum All. Tehran: Chalos road, 370m, Salimpour 110 T. medium L. Lorestan: