The Somatic Chromosomes of Trifolium Cherleri L., T. Hirtum All., T. Ligusticum Balb., and T. Scabrum L.

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: The Somatic Chromosomes of Trifolium Cherleri L., T. Hirtum All., T. Ligusticum Balb., and T. Scabrum L. A. J. Pritchard To cite this article: A. J. Pritchard (1967) The Somatic Chromosomes of Trifolium Cherleri L., T. Hirtum All., T. Ligusticum Balb., and T. Scabrum L., Caryologia, 20:4, , DOI: / To link to this article: Published online: 29 Jan Submit your article to this journal Article views: 51 View related articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 31 December 2016, At: 23:22

2 THE SOMATIC CHROMOSOMES OF TRIFOLIUM CHERLERI L., T. HIRTUM ALL., T. LIGUSTICUM BALB., AND T. SCABRUM L. A. ]. PRITCHARD C.S.I.R.O. Division of Tropical Pastures, Mill Road, St. Lucia, Queensland Received: 20th April 1967 INTRODUCTION The chromosome number of 2n= 10 is unusual in Trifolium. It has been reported only for T. hirtum All. (BRITTEN 1963a) and T. scabrum L. (LARSEN 1960; KLIPHUIS 1962), both of which are included in the subgenus Trifolium by HosSAIN ( 1961 ). A cytological investigation of this subgenus has shown that T. cherleri L. also has a chromosome number of 2n = 10 and T. ligusticum Balb. has 2n= 12. A detailed study was made of the somatic chromosomes of all four of these species in an attempt to determine their relations through chromosome morphology. MATERIAL AND METHODS Chromosome counts were made from root tip preparations after a pretreatment of 24 hr in water at 1 oc. The root-tips were fixed in acetic-alcohol ( 1 : 3 ), hydrolysed in 1N HCl at 60 C for 12 min, stained in Feulgen, and squashed in a drop of 1% acetic-orcein. In each species, eight cells which were flat and had well spread chromosomes were photographed and arm lengths measured on prints enlarged to a total magnification of X The lengths of chromosome arms were converted to microns and also expressed as a percentage of the total length of all chromosome arms in the same cell (per cent length). In the tables and figures the chromosomes of T. hirtum and T. scabrum are numbered in order from the longest to the shortest. The satellited chromosome in T. cherleri is the third longest but is labelled No. 2 in agreement with the number given to the satellited chromosome of the other two species. This avoids confusion when the satellited chromosomes of different species are being compared. Two indices were calculated for each species. The gradient index (GI) measures the size gradient within the genome and the symmetry index (SI) estimates 323] [Caryologia, Vol. 20, n. 4, 1967

3 324 PRITCHARD the overall symmetry of the chromosomes about their centromeres. These indices were calculated as follows: length of shortest chromosome GI = X 100 length of longest chromosome total length of short arms SI = X 100 total length of long arms Chromosome numbers were counted in five accessions of T. hirtum, s1x of T. cherleri, two of T. scabrum, and one of T. ligusticum. The accessions of T. scabrum were collected in Australia, one near Adelaide and the other near Perth. The accessions of T. hirtum were from U.S.A. (2), Bulgaria, Israel, and Cyprus, and those of T. cherleri were from Israel (3 ), Cyprus (2 ), and Algeria. T. ligusticum was collected in Portugal. Voucher specimens of every accession have been retained in the herbarium of the Cunningham Laboratory, Brisbane, Australia. 6 T. hirtum ~ Fig The plot of the mean per cent length of the short arm against that of the long arm of the chromosomes of T. hirtum, T. cherleri and T. scabrum. Each point plotted is marked by the number of the chromosome to which it refers (see Table 1).

4 T. cherleri 4,.., T. scabrum \ 1 1 l ~1/1 2 4~------~5~------=

5 326 PRITCHARD Figs The somatic chromosomes of some species of Trifolium with 2n=l0 and 2n=l2. The chromosomes are numbered in accordance with the text and Table 1. Fig T. hirtum. Fig T. scabrum. Fig T. cherleri. Fig T. ligusticum. OBSERVATIONS In Figure 1 the mean per cent length of the short arm of each of the chromosomes of T. hirtum, T. cherleri and T. scabrum have been plotted

6 SOMATIC CHROMOSOMES OF TRIFOLIUM 327 against the mean per cent length of the long arm. The measurements from each chromosome within a species cluster around a mean value, so that individual chromosomes in any cell can be identified with a high degree of confidence. The major overlap is between the points plotted for Chromosomes No. 2 and No. 3 oft. hirtum, and No. 1 and No. 2 oft. scabrum, but this causes no confusion as in each species No. 2 is the satellited chromosome. T. hirtum T. cherleri T. scabrum IIIII ]It T.ligusticum Fig Idiograms of four Trifolium species. The four shortest chromosomes of T. ligusticum cannot be separated from each other on the basis of size and shape. Details of the other two chromosomes are indicated in Figs. 5 and 6. The longest chromosome is fl. in length and has an arm ratio of The second longest is !J., with an arm ratio of and the mean length of the four short chromosomes is fl.. The total length of the chromosomes is 8.65 fl. and the GI is No measurements of arm ratios were possible in the short chromosomes and no satellites were seen. The karyotype of this species is very different from that of the three species with 2n = 10. Details of the chromosomes of T. hirtum, T. cherleri and T. scabrum

7 328 PRITCHARD are shown in Figs. 2-4 and 6 and the chromosome measurements presented in Table 1. Each karyotype has distinctive features. T. cherleri has a satellited chromosome with the secondary constriction closer to the centromere than it is in the other two species, and the longest chromosome of T. scabrum is the most asymmetrical chromosome of all, with an arm ratio of 2.73 (Table 1 ). The longest chromosome of T. hirtum has an arm ratio of 1.54 and the secondary constriction on the satellited chromosome is approximately halfway between the centromere and the end of the long arm of the chromosome. A comparison of individual chromosomes between species involves several assumptions. Firstly, it is assumed that the differences in absolute chromosome length are due to differences in chromosome coiling, and not due to the accumulated effects of deletions and duplications, secondly that distortions due to pretreatment are at random over the chromosomes of all species, and thirdly that the pretreatment results in a contraction of the chromosomes which is relative to their original lengths. TABLE I Details of somatic chromosomes of T. hirtum, T. cherleri and T. scabrum. Chromosome T. hirtum mean length 11 mean percent length mean arm ratio mean length ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.08 8:71± ± ±0.11 Total Length Gradient Index Symmetry Index Because the data are not independent no analysis of variance is possible for the mean per cent lengths. An analysis of the arm ratio data enables shapes but not lengths to be compared. The least significance difference (p<0.05) for comparing chromosomes within a species is Chromosome No. 2 is significantly different from the other four in T. cherleri, and No. 1 from the others in T. scabrum. For comparing chromosomes between species the least significant difference (p<0.05) is Chromosome No. 3 has a similar shape in all three species and chromosome No. 2 in T. hirtum

8 SOMATIC CHROMOSOMES OF TRIFOLIUM 329 is similar to No. 2 in T. scabrum. Chromosomes No. 5 in T. cheflleri and T. hirtum are similar in shape to chromosome No. 4 in T. scabrum. The other chromosomes cannot be classified into groups and are all different. The three groups of chromosomes with similar shapes can be seen in Figure 1 where chromosomes which are similar in both size and shape occupy similar areas on the graphs. DISCUSSION Basic chromosome numbers of x = 8 and x = 7 are common in Trifolium (BRITTEN 1963b) but the report of x = 6 for T. ligusticum is the first report of this number for the subgenus Trifolium, although x=5 is known, and x=6 has been reported in the subgenus Calycomorphum (Presl.) Hossain (YATES and BRITTAN 1952). Since all of the species with x=5 are, from an evolutionary point of view, advanced and specialized, it is reasonable to T. cherleri T. scabrum mean percent length mean arm ratio mean length 1.1. mean percent lenght mean arm ratio 12.24± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ! assume that this number has been derived by chromosome deletion following chromosome rearrangement through translocations. However it would appear from this study of karyotypes that T. ligusticum has little in common with the three other species and is unlikely to be an ancestral form from which they were derived. A comparison of the karyotypes of the three species with x = 5 shows that several of the chromosomes are similar in shape and relative size. It is possible that these chromosomes are also homologous but this cannot be

9 330 PRITCHARD proved without evidence from chromosomes pairing in interspecific hybrids. Their common and unusual chromosome number emphasises the close relationiship between the species, and in fact, HosSAIN ( 1961) considers that T. hirtum and T. cherleri are easily confused, especially when they occur in mixed stands in their native habitats. However, the distinct differences in the morphology of some chromosomes indicates evolutionary divergence and probable reproduction isolation. STEBBINS ( 1950) considers that an unspecialized karyotype is one which consists of chromosomes which are all the same length and which have median or almost median centromeres. Both GI and SI indicate that, of the species with 2n= 10, T. hirtum has the least specialized karyotype and is probably the most primitive. T. hirtum and T. cherleri have chromosomes which are shorter in absolute length, more variable in size and more asymmetrical than those of T. hirtum. Chromosomal rearrangements have played an important part in the evolution of some species of Trifolium (KATZNELSON and MoRLEY, 1965) but the chromosomes of most species in the genus are very small and only very large translocations or inversions would be readily detectable in somatic chromosomes. BROCK ( 1953) suggested that the large chromosome of T. israelaticum may have resulted from a large translocation in an ancestral species. It is likely that the differences in morphology of some of the chromosomes of the three species of Trifolium with x = 5 resulted from large chromosomal rearrangements. For instance, the distinctive shape of the satellited chromosome of T. cherleri probably resulted from a translocation or inversion which involved the secondary constriction. Acknowledgements. - Thanks are due to R. ]. WILLIAMS, K. P. HAYDOCK and K. F. GouLD of C.S.I.R.O. Cunningham Laboratory, Brisbane, Australia, who provided seed, statistical advice and technical assistance. T. ligusticum was identified by R. D. RoYcE of the Western Australian Herbarium, Perth. REFERENCES BRITTEN E. J., 1963a. - Chromosome numbers in rose clover, Trifolium hirtum. Science N.Y., 142: , 1963b. - Chromosome numbers in the genus Trifolium. Cytologia, 28: BROCK R. D., Species formation in Trifolium subterraneum. Nature Lond., 171: 939. HosSAIN M., A revision of Trifolium in the nearer east. Notes R. Bot. Gdn. Edinb., 23: KATZNELSON J. and MORLEY F. H. W., L. Israel J. Bot., 14: Speciation processes in Trifolium subterraneum KLIPHUIS E., Chromosome numbers of some annual Trifolium species occurring in the Netherlands. Acta Bot. Neerl., 11: LARSEN K., Cytological and experimental studies on the flowering plants of the Canary Islands. Bioi. Skr. Dan. Vid. Selsk., 11: 1-60.

10 SOMATIC CHROMOSOMES OF TRIFOLIUM 331 STEBBINS G. L., Variation and evolution in Plants. Columbia University Press. New York. YATES ]. ]. and BRITTAN N. H., Cytological studies of subterranean clover (Trifolium subterraneum L.). Aust. J. Agric. Res., 3: SUMMARY The chromosome number of T. ligusticum was found to be 2n = 12, and that of T. cherleri 2n=10. A comparison of the chromosomes of these species with those oft. hirtum (2n=10) and T. scabrum (2n=10) showed that the karyotype of each species has distinctive features, but some chromosomes of the species with 2n = 10 are similar in size and shape and may be homologous. It is unlikely that T. ligusticum is ancestral to the other three species.