Cytologia 48: 671-677, 1983 Morphological Observations and Chromosome Numbers in Trifolium L. Section Chronosemium Ser. Norman L. Taylor,1 John M. Gillett2 and Nirmala Giri3 Received March 20, 1982 The clover species belonging to Trifolium L. section Chronosemium Ser. are commonlycalled the hop clovers because the heads when dry resemble the inflore scenceof the hop (Humulus lupulus L.). The species are native to Europe, western Asia, and north Africa, and have been introduced by man to other parts of the world. Ofthese, T. dubium, T. campestre, T. aureum and possibly T. micranthum have become naturalizedin North America. Most of the other species have been brought in for experimentalpurposes. Hendrych (1978) was of the opinion that the group (which he considered should be placed in the separate genus Chrysaspis Desv.) had its center of origin in an area extending from southern Italy across the southern part of the Balkan Peninsula to eastern Anatolia in Turkey, and that the species subsequently migrated to cover the present-day ranges. The section includes 16 annual and perennial species, according to Coombe (1968). Hossain (1961) listed 15 species, but his studies covered a limited area. It is evident from these papers that the taxonomic problemsin this group of clovers are far from resolved. Chromosome numbers in the genus Trifolium including section Chronosemium weresurveyed by Britten (1963). The six species of this group had base numbers of 7 and all were diploid except T. dubium Sibth., which was tetraploid (2n=28). Previously,both Wexelsen (1928) and Gadella and Kliphuis (1966) had reported the number 2n=32 for T. dubium. Pritchard (1969) added counts for T. billardieri Spreng.and T. lineare Greene (as T. stenophyllum Boiss.), which were both 2n= 16. Counts for individual species are scattered throughout the literature but have been brought together in the works of Federov (1974) and Love and Love (1974). Giri et al. (1981)found that both T. aureum Poll. and T. strepens Crantz had 16 chromo somesand confirmed the two taxa as one species, T. aureum Poll. The present study seeks to assemble chromosome data for the section and to coordinate them with certain morphological characteristics. Materials and methods Seeds were received from the sources indicated in Table 1. Voucher specimens of all species examined are retained in the Trifolium herbarium of the Department of 1 Department of Agronomy , University of Kentucky, Lexington, Kentucky 40546-0091, U. S. A. 2Botany Division, National Museum of Natural Sciences, Ottawa, Canada K1A OM8. 3 Department of Agronomy, University of Kentucky, Lexington, Kentucky 40546-0091, U. S. A. The investigation reported in this paper (No. 81-3-187) is in connection with a project of the KentuckyAgricultural Experiment Station and is published with approval of the Director. 672 Norman L. Taylor, John M. Gillett and Nirmala Giri Cytologia 48 Agronomy, University of Kentucky, Lexington, Kentucky. Duplicate specimens are in the National Herbarium of Canada, Botany Division, National Museum of Natural Sciences, Ottawa, Ontario, Canada. Seeds of all species were lightly rubbed with sand paper for scarification and placed on a damp filter paper in a petri dish for germination. Plants were grown to the flowering stage in the greenhouse. Vouchers were prepared from them for identification purposes. Root tips were pretreated in .003 M 8-hydroxy-quinoline for 6 to 8 h, fixed in 95% ethyl alcohol: glacial acetic acid (3:1), stained in Feulgen's reagent, placed in 45% glacial acetic acid for 2 h, and squashed in 1% acetocarmine. Somatic chromosome numbers were counted from root tips in all species except T. campestre (S-8-22) where PMC's were also done. Chromosomes for nine species were counted by us. Morpholo gical observations were made from greenhouse plants. Table 1. Origin of Trifolium seed Results and discussion It was evident that identifications of vouchers grown did not always accord with the names of the species supplied by the seed sources. A chromosome number of 2n=16 is reported here for the first time for Trifolium boissieri Guss. ex. Boiss (Table 2). Other species for which chromosome numbers are known and which are not in Table 2, were already reported by others. These are, T. patens Schreb. and T. spadiceum L., reported by Kuzmanov and Stancev (1972), and T. velenovskyi Vandes (in addition to the previous two) reported by Kozuharov et al. (1974 and 1975). The chromosome number of some of the species reported before are con firmed by us (Table 2). A summation of all species for which chromosome numbers Table 2. Characteristics of Trifotium section Chronosemium species 1 Y=yellow; LY=light yellow; P=purple; W=white. 2 S=3mm; M=4-5mm; L=6-10mm. 3 LY=light yellow; YG=yellow green; YB=yellow brown; B=brown. 4 S<1mm; M>1mm; L>1.5mm. 5 S=sessile or subsessile central leaflet attachment; E=central leaflet with elongate petiolule. 6 A=annual; P=perennial. 7 Determined by us. * Reported for the first time. 674 Norman L. Taylor, John M. Gillett and Nirmala Giri Cytologia 48 are presently known is presented in Table 3 and a list of species for which chro mosome numbers are yet required is also given. Only recent reports are given because those earlier than 1969 can be found in Federov (1969, reprinted 1974) and Love and Love (1974). The chromosomes show some variation in size and shape. Chromosomes of T. campestre and T. boissieri are larger than those of other species and chromosomes of T. aureum are the smallest ones. Only one species of the section, T. dubium (Fig. 1), is polyploid with 2n=28 chromosomes. The report of 2n=32 for T. dubium by Wexelsen (1928) was probably based on a mis-identification and, indeed, Wexelsen actually questioned the identity of the plant in the paper. Earlier searches by Mosquin and Gillett (1965) failed to find voucher specimens to verify the identity of any of the species reported. The more recent count of 2n=32 for this speciesby Table 3. Summation of known somatic chromosome numbers of species of Trifolium section Chronosemium * The name T . grandiflorum Schreb. is here employed in lieu of the more familiar name T. speciosum Willd. (1802) according to a study by Hendrych (1976). Gadella and Kliphuis (1966) represents a case of mistaken identity (unpublished communication). We determined the chromosome number of T. dubium as 2n=28, which is in agreement with the previous report of Kliphuis (1962). Chromosomes of all species we have studied are illustrated in Fig. 1. In Table 1, some speciesare listed more than once because seeds were received by us under different names. The names employed in Table 1 are our identifications; the names under which the seed was received are given below in parenthesis. Most of the species of section Chronosemium are annuals (Hossain 1961). The exceptions are T. badium, T. rytidosemium (often considered as a subordinate taxon of T. badium), T. stipitatum and T. spadiceum. The last two species were not examined in the present study. In contrast to the tendency for polyploidy to occur in perennials and diploidy in annuals, the annual species T. dubium is a polyploid. The only other annual Trifolium species ever reported as polyploid, was T. di- 1983 Morphological Observations and Chromosome Numbers in Trifolium L . 675 Figs. IA-O. A, T. campestre (S-67-2), somatic metaphase 2n=14. •~2700. B, T. micranthum (S-15-3), somatic metaphase 2n=16. •~2100. C, T. badium (S-107-2), somatic metaphase 2n=14. •~x2100 . D, T. aureum (S-123-3), somatic metaphase 2n=16. •~2700. E, T. boissieri (S-176-1), somatic metaphase 2n=16. •~2100. F, T. micranthum (S-195-1), somatic metaphase 2n=16. •~2700. G, T. campestre (S-32-1) , somatic metaphase 2n=14, •~2100. H. T. lineare (S-82-3), somatic metaphase 2n=16.•~2100. I, T. billardieri (S-165-1), somatic metaphase 2n=16. •~2700. J , T. grandiflorum (S-90-53), somatic metaphase 2n=16. •~2100. K, T. campestre (S-131-1) , somatic metaphase 2n=14. •~2700. L, T. aureum (5-171-1), somatic meta phase 2n=16. •~2700. M, T. campestre (S-8-22), somatic metaphase 2n=14. •~2700. N, T. campestre (S-8-22) , meiotic metaphase n=7. •~2100. O, T. dubium (S-2-4), somatic metaphase 2n=28. •~2700. 676 Norman L. Taylor, John M. Gillett and Nirmala Giri Cytologia 48 chotomum Hook and Arn. for which Wexelsen (1928) gave a count of 2n=32. No vouchers have been found nor has the report been confirmed. Little relationship of chromosome number to morphology is apparent (Table 2). For example, the presence of pinnate leaves (where the terminal leaflet is stalked) or palmate leaves (where the terminal leaflet is sessile), always employed as a charac teristic to separate species, is not correlated with chromosome number and both types occur in the x=7 and x=8 series. Three species have distinctly different seed colors from the other species grown. Trifolium aureum and T. badium seeds are green along the basal margin and yellow along the terminal margin, while all other species have uniformly yellow, yellow - brown or brown seed. This characteristic, however, is unreliable for classification purposes because the color may change from yellow to brown as seeds age and as a reflection of environmental conditions. The yellow-green seeds of T. aureum and T. badium of the x=8 series are quite distinctive regardless of age. Differences in flower and seed size exist which are also little related to chro mosome number (Table 2). For example, T. micranthum, T. patens, and T. aureum have small flowers and mostly small seeds but have either x=7 or x=8 chromosomes.