Chromosome Numbers and Behavior in Some Species of Dioscorea1

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1 96 Cytologia 28 Chromosome Numbers and Behavior in Some Species of Dioscorea1 Franklin _??_. Martin and Sonia Ortiz U. S. Department of Agriculture, Agricultural Research Service, Crops Research Division, Federal Experiment Station, Mayaguez, Puerto Rico Received October 11, 1962 Recent discovery of sapogenins in the tubers of wild Dioscorea species (Correll et al. 1955) has led to the usage of these materials in the synthesis of cortisone and steroidal hormones. For commercial use, the tubers are harvested from the wilds of Mexico and Central America. Such supplies are of variable quality and are subject to depletion. To insure a steady, high quality supply of such plant sapogenins, satisfactory cultivars must be devel oped. As part of a study of the feasibility of breeding Dioscoreas, the chromo somes were observed at mitosis and meiosis in some New-World as well as a few Old-World species. The data are reported herein relation to species distribution and processes of speciation. Previous counts of the chromosomes of species of Dioscorea together with data on probable geographic origin are summarized in Table 1. References are too numerous to be included here, but an annotated list is available to correspondents (Martin and Ortiz 1962). Materials and methods The Dioscorea species studied together with information on geographical source and chromosome counts are listed in Table 2. Sapogenin-bearing species are marked by 3 asterisks. The species were identified with Matuda's key (1954) and through the help of Bernice Schubert. Specimens of all species counted were placed in the herbarium of the Arnold Arboretum, Harvard University, Cambridge, Massachusetts. Meiosis was studied in pollen mother cells, which were fixed in Carnoy's solution for 24 hours, transferred to 70% alcohol, and stained with aceto-carmine containing ferric oxide. Mitosis was studied in root tips collected from germinating seeds, tubers, or bulbils fixed in Carnoy's solution for 24 hours, and transferred to 70% alcohol. The tips were treated with 50% HCl for 15 minutes, washed, stained for 15 minutes in leuco-basic fuchsin, and treated with 5% pectinase for 2 or 3 hours. Finally, the tips were squased in aceto-carmine. 1 In cooperation with New Crops Research Branch.

2 Table 1. Chromosome numbers previously reported and probable geographic origin of Dioscorea species (This list was compiled from the works of numerous authors. An annotated list of pertinent references is available to correspondents.)

3 98 F. W. Martin and S. Ortiz Cytologia 28 Table 2. Dioscorea species studied, their sources and chromosome numbers * P. l. numbers were assigned by the Plant Introduction Branch, USDA, and F. E. S. numbers by the Federal Experiment Station. ** Mi.=mitosis. Me.=Meiosis. *** Sapogenin-bearing species. Results and discussion Meiosis. Because of the small size of the buds of Dioscorea, the pro longed sequence of maturation and flowering, and the absence of discrete external evidences of stage of development, finding appropriate stages for chromosome counts was very difficult. In addition, the shortness of the bivalents (1.0 to 2.5 microns) and the difficulty of isolating PMC's from the anther tissue for proper smears made exact counts difficult. For these reasons, meiosis was studied in only the sapogenin-bearing species. In all cases, except that mentioned below, chromosome number was 36 and meiosis was normal. No irregularities such as univalents, fragments, bridges or lagging bivalents were found.

4 1963 Chromosome Numbers and Behavior in Some Species of Dioscorea 99 In two lines of D. floribunda, the 2n chromosome number was 54, and up to 9 quadrivalents were seen (Fig. 1). In this case, meiosis was characterized by the presence of various abnormalities (Table 3), including univalents, bridges, fragments, and delayed separation of bivalents. It was not possible to determine whether the bridges were due simply to sticky, lagging chromosomes or breakage was involved. The presence of acentic frag ments suggests that the latter process sometimes oc curred. Subsequent stages of meiosis in this line were normal. Cytokinesis occurred after second metaphase. Univalents and fragments probably were usually in cluded in macronuclei, as micronuclei were seldom observed. Mitosis. Obtaining chromosome counts from root tips was much easier and more reliable than obtaining meiotic counts. Nevertheless, at times a preponderance of cells with well-developed prochromosomes of the vesicular type made the actual search confusing and tedious. The mitotic counts of sapogenin-bearing species were in agreement with the meiotic counts, but many more collections and species were counted (Table 2). The chromosome numbers of all Old-World species counted are multiples of 10. These counts are in Fig. 1. Mitotic (above) and meiotic (below) chro mosomes in Dioscorea floribunda. Left, a 36 chromosome line; right, - a 54-chromosome line ( ~1000, from photo micrographs and camera lucida drawings.) agreement with those of previous investigators. The chromosome numbers of the New-World species are multiples of 9. The numbers of these species have not been previously reported, with the exception of an approximate Table 3. Meiotic abnormalities in hexaploid Dioscorea floribunda count of D. spiculiflora. The chromosomes were small and dotlike (0.5 to 2.0 microns). No consistent heterotypic elements were found. The data on chromosome numbers presented here, together with the scanty previous observations, suggest that the progenitors of the New-World 7*

100 F. W. Martin and S. Ortiz Cytologia 28 species had a base chromosome number of 9. Possible exceptions are the North American species complex D. villosa, 2n=60, and the South American D.

5 100 F. W. Martin and S. Ortiz Cytologia 28 species had a base chromosome number of 9. Possible exceptions are the North American species complex D. villosa, 2n=60, and the South American D. discolor, 2n=40 (Smith 1937). It should be noted, however, that D, quaternata, a part of the D. villosa complex, has been found to have a 2n number of 54 (Jensen 1937). A reinvestigation of the chromosome numbers of this species complex seems desirable. Other discrepancies from multiples of 9 occur in the literature (Table 1) and could be errors or approximations. In comparison, Asiatic species have chromosome numbers which are multiples of 10. African species have numbers which are multiples of 9 and 10, and in two species, D. bulbifera, and D. cayennensis, there are both tetraploid and hexaploid races with numbers which are multiples of either 9 or 10. On this basis an African center of origin of the genus can be postulated, with a tendency for 9-chromosome types to spread west and 10-chromosome races to spread east and north. This speculation agrees with Burkill's (1956) belief that most speciation occurred after a continental rift during the late cretacious period. However, from the morphological evidence, he postulates eastern continental Asia as a center of origin. Miege (1954), has postulated a third base number of 12. The chief evidence for this is the occurrence of two 24-chromosome species of Dioscorea in the Pyrenees between France and Spain. Heslot (1953) has questioned, however, whether these should be included in the same genus. Or, these may represent another phylogenetic line from West Africa (Burkill 1956). The existence of two chromosome numbers in D. floribunda suggests a mode of origin of new species in Dioscorea. The anomalous 54-chromosome lines probably represent backcrosses of an allotetraploid to one of its diploid progenitors, followed by chromosomal doubling, as evidenced by the quadri valent pairing of about two-thirds of the chromosomal complement. Struc tural changes in either the diploid or the tetraploid could result in the bridge and fragment abnormalities found. This process has also occurred in other Dioscoreas. A 36-chromosome line of D. bulbifera has a fertile 54-chromo some counterport in Africa (Miege 1954) whereas a 40-chromosome line of the same species has a 60-chromosome counterpart. Similarly, D. cayennensis, which has long been considered a hybrid, has 36-chromosomes in the north of the Ivory Coast, and 54 in the south. D. quaternata, with 54-chromo somes, was found to have irregular meiosis (Jensen 1937). These cases may be similar in origin to the case reported here. Hybridization of races and species followed by chromosomal doubling has been a principal factor in speciation in Dioscorea and has lead to the wide range of chromosome num bers which occur in some species (Table 1). Successful fruit and seed production have already been achieved in crosses of D. composita and D. floribunda. The progeny of these crosses are now being grown for comparison with the parents. As the chromosome numbers of these species are similar, it is highly probable that the hybrids will be

6 1963 Chromosome Numbers and Behavior in Some Species of Dioscorea 101 fertile. On the other hand, the 54-chromosome lines of D. floribunda appear to be already isolated from the 36-chromosome lines of the species by irregular meiosis and fruit set. This isolation is also attested by the fact that the 54 and some 36-chromosome lines came from the same, local geographical areas, - but their intermediates have not been found. Crosses have not been attempted between D. spiculiflora and the related D. friedrichs thalii, but should not be hindered by chromosomal conditions. Summary Chromosomes were counted at meiosis and mitosis in 4 sapogenin-bearing species of Dioscorea and 9 other species. Chromosome numbers of 7 New World species are reported herein for the first time. In these, 2n numbers - were 36 except in two lines of D. floribunda which contained 54-chromosomes and showed irregular meiosis. All New-World species counted had chromo some numbers which are multiples of 9, whereas all Old-World species had numbers which are multiples of 10. The significance of the findings is discussed with reference to origin and distribution of the genus, processes of speciation, and possible barriers to breeding. Literature cited Burkill, I. H The organography and the evolution of Dioscoreaceae, the family of the yams. J. Linn. Soc. (Bot.) 54: Correll, D. S., Schubert, B. G., Gentry, H. S., and Hawley, W. O The search for plant precursors of cortisone. Econ. Bot. 9: Heslot, N Le nombre chromosomique des Dioscoreacees pyreneenes et leur rat tachement au genre Bordorea Miegev. C. R. Acad. Sci. 237: Jensen, H. W Meiosis in several species of dioecious Monocotyledonea: I. The possibility of sex chromosomes. Cytologia, Fujii Jubilee Vol.: Martin, F. W., and Ortiz, S An annotated bibliography of references to the chro mosomes of Dioscorea. Mimeographed circular of the Federal Experiment Station Mayaguez, Puerto Rico. Matuda, E Las Dioscoreas de Mexico. Ann. Instit. de Bot. (Mexico) 24: Miege, J Nombres chromosomiques et repartition geographique quelque plantes tropicales et equatoriales. Rev., Cytol. Paris. 15: Smith, B. W Notes on the cytology and distribution of the Dioscoreaceae. Bull. Torr. Bot. Club. 64:

Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India

Cytologia 44: 739-743, 1979 Received April 10, 1975 Artificial Triploids in Luffa echinato Roxb. P. K. Agarwal,1 R. P. Roy and D. P. Mishra Department of Botany, University of Patna, Patna-5, India Luffa