DISCUSSION. Chromosome numbers in different taxa of Dioscorea investigated

Transcription

1 DISCUSSION Chromosome numbers in different taxa of Dioscorea investigated Dioscorea is one of the most difficult genera for cytotaxonomic and cytogenetic studies (Essad, 1984). Many authors already reported the difficulties encountered in chromosome counting in cultivated species of Dioscorea (Miège,1952.,1954.,Baquar,1980., Zoundjihékpon et al., 1990) and even other Dioscorea species (Suessenguth,1921., Ramachandran, 1968., Baquar, 1980., Araki et al., 1983., Essad, 1984., Gamiette et al., 1999).The presence of extra chromosomes in the cells of some species has been reported by Miège (1954), Baquar (1980), Zoundjihékpon et al., (1990) and Gamiette et al. (1999). The extra chromosomes are reported to be B chromosomes or satellites which in yam are sometimes as large as the chromosomes themselves (Essad, 1984). Chromosome studies, employing classical cytogenetic methods, have proven to be an important source of information for analyzing relationships and evolution of taxa. The presence of more than one ploidy level was observed in the species of Dioscorea investigated in the present study. Almost all the species investigated showed more than one ploidy level. The maximum number of three cytotypes was found in Dioscorea tomentosa and Dioscorea wallichii. Eleven species of Dioscorea showed two cytotypes each while the remaining eight taxa exhibited fixed chromosome counts. Ploidy levels ranging from 2x to 8x chromosomes were observed in the plants investigated (Fig. 130). Of the 36 cytotypes observed in the present study, two were diploids, five were triploids, nine were tetraploids, four were pentaploids, nine were hexaploids and three were heptaploids and four octaploids. Tetraploidy and hexaploidy are the most common ploidy levels shown by the members investigated. The presence of identical chromosome numbers in many species of the genus Dioscorea, irrespective of their morphological differences is noteworthy. Chromosome counts vary from 2n = 16 to 2n = 72 among the members investigated (Table 38). Maximum frequency of chromosome counts was observed in 2n = 36 to 2n = 135

2 54 range. These variations in number of chromosomes are consistent with earlier reports in which different chromosome counts were observed in investigated species by different authors (Table 39). The chromosome counts obtained in the present study are in general confirmation with earlier studies in terms of ploidy in some species. The individual chromosome numbers of species, however, vary from those reported earlier by various authors. Dioscorea hispida,dennst.(dioscorea daemona. Roxb.) (Fig. 58, Fig. 59) Chromosome counts obtained in present investigations are 2n = 16 and 2n = 24 representing two cytotypes of the species. These counts are new and in contrast to the reported count of 2n = 40 (Essad, 1984). Dioscorea rubella, Roxb. (Fig. 60, Fig. 61) The present study reveals two cytotypes for the species with chromosome counts of 2n = 48 and 2n = 56. These observations contradict earlier reports of 2n = 40 (Raghavan, 1958; Ramachandran 1962) and 2n = 30 (Essad, 1984) Dioscorea bulbifera, Linn. var. sativa, Pr. & Burk. (Fig. 62, Fig. 63) Cytotypes with 2n = 54 and 2n = 72 were obtained in present analysis. A review of literature reveals chromosome numbers 2n = 40, 2n = 60 and 2n = 80 for this species. 2n = 80 (Smith 1937); 2n = 36, 40, 54, 60 (Miege, 1954); 2n = 40, 80, , (Raghavan, 1959); 2n = 80, 100 (Ramachandran, 1962), 2n = 40, 60, 70, 80, 100 (Essad, 1984); 2n = 36, 40, 54, 60, 80, (Essad, 1984) in African species. Dioscorea Jacquemontii, Hook. (Fig. 64) Chromosome count of 2n = 56 was found in this species without any somatic variations. Absence of somatic variants may be indicative of the stability of the genome. Chromosome counts pertaining to this species are not available in literature. 136

3 Dioscorea Kalkapershadii, Pr & Burk. (Fig. 65, Fig. 66) Somatic variants with 2n = 45 and 2n = 54 were observed in this species during the present investigations. No previous reports with regard to chromosome counts of this species are available. Dioscorea tomentosa, Heyne.(Dioscorea triphylla, Herb.Russ.)(Fig. 67, Fig. 68, Fig. 69) Intra specific variations in chromosome number are high in this species. Three cytotypes with chromosome counts of 2n = 18, 2n = 27 and 2n = 36 were observed in the species. These counts are not in consistence with the already reported chromosome counts of 2n = 40 and 2n = 60 (Essad, 1984). Dioscorea atropurpurea, Roxb. (Fig. 70, Fig. 71) Present study indicates the existence of two chromosome counts for the species. Somatic variants with 2n = 36 and 2n = 54 were observed. Chromosome counts of this species are not available in literature. Dioscorea bulbifera, var.vera Linnaeus.(Helmia bulbifera, (Linnaeus) Kunth.)(Fig. 72, Fig.73) This species shows a lot of similarities with its edible variety Dioscorea bulbifera var. sativa. Vegetative morphology of these two is so similar rendering their identification almost impossible. Chromosome counts of these two plants are also similar with 2n = 54 and 2n = 72. Previous chromosome counts are not available for this wild species. Dioscorea wallichii, Hook. (D. aculeata, Linn.) (Fig. 74, Fig. 75, Fig. 76) This species is very plastic regarding the chromosome counts. Somatic variations in 137

4 chromosome numbers with 2n = 24, 2n = 40 and 2n = 48 were observed in the present study. Of these, chromosome count of 2n = 40 was reported by Essad (1984). Dioscorea spinosa, Roxburg. (Fig. 77, Fig. 78) Chromosome counts of 2n = 64 and 2n = 72 were found in this species during the present study. The previously reported chromosome count of 2n = 90 (Essad, 1984) is probably a decaploid of the species. Dioscorea esculenta, (Lour.) Burk. (Fig. 79, Fig. 80) Previous reports show chromosome counts of 2n = 80, 2n = 40 (Miege, 1954); 2n = 40, 60, 90, 100, (Martin, 1963); 2n = 90 (Ramachandran, 1963); 2n= 100 (Raghavan, 1958., 1959); 2n = 40, 60, 90, 100 (Essad, 1984).The present study revealed chromosome counts of 2n = 27 and 2n = 54 representing two cytotypes for the species. These counts are in contrast to the previously reported ones,. Dioscorea fasciculata, Roxb. (Dioscorea esculenta, var. esculenta; Dioscorea esculenta, var. fasciculata (Roxburgh) R. Knuth; Oncus esculentus, Loureiro, Fl. Cochinch.) (Fig. 81, Fig. 82) Two cytotypes with 2n = 45 and 2n = 54 chromosomes were found for this species in present investigations. Published chromosome counts are not available probably due to the fact that this plant was considered as Dioscorea esculenta by various authors. Dioscorea Kumaonensis, Kunth. Enum. (Fig. 83, Fig. 84) Chromosome counts for this species are not available in published reports. Present investigation clearly showed two somatic variants with 2n = 24 and 2n =

5 Dioscorea pentaphylla, Linn. (Fig. 85, Fig. 86) Two chromosome counts were observed in this species with 2n =32 and 2n = 64. The reported chromosome number is 2n = 40, 80 (Raghavan, 1958., 1959). 2n = 40, 70, 80, (Essad, 1984) Dioscorea alata, Linn (D. globosa, Roxb.) var. red (Fig. 87) This species is one of the most thoroughly studied one due to the high food value of its tubers. This particular variety is commonly cultivated in our state. Published records show chromosome counts of 2n = 40 (Miege, 1954); 2n = 30, 40, 50, 70 (Sharma, 1956); 2n = 60, 80 (Raghavan, 1958); 2n = 40, 60 (Martin, 1963); 2n =30, 40, 50, 60, 70, 80 (Essad, 1984). Present investigations however show a chromosome count of 2n = 36 without any variation. Dioscorea alata, Linn; (D. globosa, Roxb.) var. white. (Fig. 88) This particular variety is commonly cultivated in our state. Published records show chromosome counts of 2n = 40 (Miege, 1954)., 2n = 30, 40, 50, 70 (Sharma, 1956)., 2n = 60, 80 (Raghavan, 1958)., 2n = 40, 60 (Martin, 1963)., 2n =30, 40, 50, 60, 70, 80 (Essad, 1984). Present investigations however show a chromosome count of 2n = 36 without any variation. Dioscorea oppositifolia, Linn. var.linnaei, Pr & Burk. (Fig. 89) The somatic chromosome count found in present study is 2n = 32. The reported chromosome number is 2n = 40 (Essad, 1984). This count may be a pentaploid species or the plant may be var.dukhunensis, which is morphologically very similar. Dioscorea oppositifolia, Linn. var. dhukunensis, Pr & Burk. (Fig. 90) Present investigations revealed chromosome count of 2n = 40 which is in conformity 139

6 with the previously published chromosome count of 2n = 40 (Essad, 1984). Dioscorea bulbifera, var. A, var. B and var. C (Fig. 91, Fig. 92, Fig. 93) This species which is common in thickets has a chromosome count of 2n = 38 and 2n = 36. Published chromosome counts are not available for these varieties of D. bulbifera. Three variants of this species with visible morphological differences in habit were found in the wild. These variants or cytotypes revealed chromosome counts of 2n = 38 for var. A and 2n = 36 for var.b and var. C. However the chromosome morphology as well as quantitative parameters of these variants differed very much in detail. Basic chromosome numbers in various taxa of Dioscorea Basic chromosome number is regarded as a dependable and stable feature to formulate phylogenetic relationship between various taxa. Basic chromosome numbers varying from 5 to 9 are considered as ancestral forms of Angiosperms. Secondary basic chromosome numbers were evolved from these basic numbers through polyploidy. Basic chromosome number is of importance to determine the systematic position of a taxon at high taxonomic levels (Raven, 1975). Based on available data, the genus Dioscorea has basic chromosome numbers 8, 9, 10, and 12. These variations indicate the advanced nature of the genus. The present investigations were conducted on 21 taxa (including varieties) of Dioscorea. These members showed interesting cytological features, which in many cases contradicts earlier assumptions regarding basic chromosome numbers of species and varieties of Dioscorea Dioscorea hispida, Dennst. (Dioscorea daemona. Roxb.) The cytotypes 2n = 16 and 2n = 24 were found in this species. The presence of these chromosome counts in the cells evidently indicate the base number of this species as X = 8. This is in contrast with earlier reports of X = 10 based on chromosome count of 2n = 40 (Essad, 1984). 140

7 Dioscorea rubella, Roxb. Chromosome counts of 2n = 48 and 2n = 56 were found as cytotypes indicating X = 8 as the basic number. Earlier reports of chromosome counts 2n = 30 and 40 (Raghavan, 1958., Ramachandran, 1962., Essad, 1984) lead to the assumption of X = 10 for this species. Dioscorea bulbifera, Linn. var. sativa, Pr. & Burk. (Dioscorea sativa, Linn.). Chromosome counts of 2n = 54 and 72 obtained in the present investigation shows X = 9 for this species. The new base number contradicts earlier finding based on chromosome counts of 2n =40, 60, 70, 80 and 100 by various authors. The base number X = 9 is supported by the chromosome counts 2n = 36 and 54 reported by Essad (1984) in African species. Dioscorea Jacquemontii, Hook. Only a single chromosome count 2n = 56 was obtained in the analysis of this species. The basic number is X = 8. Published chromosome counts for this species are not available. Dioscorea Kalkapershadii, Pr & Burk. Two cytotypes with 2n = 45 and 2n = 54 were found during analysis of this species. The basic chromosome number is X = 9. No published counts are available for this species. Dioscorea tomentosa. Heyne. (Dioscorea triphylla, Herb. Russ.) Three cytotypes with chromosome counts of 2n = 18, 27 and 36 were found in the species during the present investigation. Basic chromosome number is X = 9. The chromosome number previously recorded is 2n = 40 and 2n = 60 (Essad, 1984), leading to the assumption of base number X= 10 for this species. 141

8 Dioscorea atropurpurea, Roxb. Cytotypes with 2n = 36 and 2n = 54 were observed for the species during present investigation. The base number is X = 9. Published data regarding chromosome counts are not available for this species also. Dioscorea bulbifera. var. vera Linnaeus. (Helmia bulbifera (Linnaeus) Kunth.) Two cytotypes with chromosome counts of 2n = 54 and 2n = 72 were observed indicating a base number of X = 9. Two chromosome counts 2n = 36 and 2n = 54 were reported by Essad (1984) in African species. These chromosome counts corroborate with the base number X = 9 Dioscorea wallichii, Hook. (D. aculeata, Linn.) Cytotypes with 2n = 24, 2n = 40 and 2n = 48 were found during analysis of the species. These counts clearly shows the existence of base number X = 8. Chromosome count of 2n = 40 was reported by Essad (1984). The reported chromosome count reiterate the validity of base number X = 8. Earlier, the base number was assumed to be X = 10. Dioscorea spinosa, Roxburg. Two cytotypes with 2n = 63 and 2n = 72 were obtained for this species during present investigations. The base number for this species is X = 9. Reported chromosome count for this species is 2n = 90 (Essad, 1984). Dioscorea esculenta, (Lour.) Burk. Published chromosome counts of 2n = 40, 60, 90 and 100 were assigned to this species by various authors (Miege,1954., Martin, 1963., Ramachandran, 1963., Raghavan, 1958., 1959., Essad, 1984). Based on these counts, the base number was assumed to be X = 10. The present investigations yielded count of 2n = 27 and 2n = 56 which indicated the base number as X =

9 Dioscorea fasciculata. Roxb. (Dioscorea esculenta var. esculenta; Dioscorea esculenta var. fasciculata (Roxburgh) R. Knuth; Oncus esculentus Loureiro, Fl. Cochinch.) Two cytotypes of this species with 2n = 45 and 2n = 54 were found during the present investigation. These counts indicate the base number as X = 9. Published chromosome counts for this species are not available. Dioscorea Kumaonensis. Kunth Enum. Three cytotypes with 2n = 24, 32 and 40 were observed in this species. These chromosome counts indicate a base number X = 8 for this species. Reported chromosome counts are not available for this species. Dioscorea pentaphylla. Linn. Two chromosome counts were observed in this species with 2n =32 and 2n = 64. These chromosome counts indicate a base number X = 8 for this species. The reported chromosome numbers of 2n = 40, 80 (Raghavan 1958., 1959) and 2n = 40, 70, 80, (Essad, 1984) shows the base number as X = 10 Dioscorea alata, Linn. (D. globosa, Roxb.) var. red Chromosome count of 2n = 36 was found in this species during analysis. The chromosome number indicate a base number X = 9. The published chromosome counts of 2n = 81 (Smith, 1937); 2n = 40 (Miege, 1954); 2n = 30, 40, 50, 70, (Sharma, 1956); 2n = 60, 80, (Raghavan, 1958); 2n = 40, 60 (Martin, 1963); 2n =30, 40, 50, 60, 70, 80, (Essad, 1984) establishes beyond doubt a base number of X= 10. Dioscorea alata, Linn. (D. globosa, Roxb.) var. white. Chromosome counts were similar to Dioscorea alata.var.red. Fixed count of 2n = 36 was observed in all the cells obtained through treatment. Earlier reports regarding the 143

10 chromosome counts of Dioscorea alata may be equally applied to this variety also as varietal differences were not mentioned in published works. Dioscorea oppositifolia, Linn. var.linnaei, Pr & Burk. Only a single cytotype with 2n = 32 was found in this species. The earlier reports indicate a chromosome count of 2n = 40 (Essad, 1984). The basic chromosome count is X = 8 Dioscorea oppositifolia. Linn. var. dukhunensis. Pr & Burk. Single cytotype with 2n = 40 was observed in this species which agrees with published count of 2n = 40, by Essad (1984). The plant is probably a pentaploid with a base number X = 8. Dioscorea bulbifera, var. A, var. B and var. C Three varieties of D. bulbifera differing in morphology were found in the wild. These are small plants which sprout, grow, and flower earlier than others in the wild. They seldom produce fruits. Often these plants reproduce by means of bulbils from the axils of leaves. Of the three varieties, D. bulbifera, var. A has a chromosome count of 2n = 38. D. bulbifera, var.b, and var.c have chromosome counts of 2n = 36.These varieties are considered as tetraploids with a base number X = 9. Polyploidy Polyploidy is the possession of three or more basic chromosome sets in nuclei (Grant, 1981). It is widespread in plant kingdom and forms one of the characteristic features of plants (Stebbins, 1950., Grant, 1973). The Angiosperms in particular have been the subject of considerable speculation regarding the frequency of polyploidy. Muntzing (1936) and Darlington (1973) speculated that about one half of all angiosperms were polyploid, while Stebbins (1950) estimated that between 30 and 35% of Angiosperms were polyploid. Grant (1963) postulated that 47% of all flowering plants were of 144

11 polyploid origin; he proposed that 58% of Monocots and 43% of Dicots were polyploid. Grant hypothesized that flowering plants with numbers of n = 14 or above were of polyploid origin. Using this as a cut-off point, he then estimated the approximate number of chromosome counts in the literature that would be polyploid. Goldblatt, (1980) suggested that Grant s (1973) estimate was too conservative; he thought that numbers above n= 9 and 10 probably had polyploidy in their evolutionary history. Using these lower numbers, he calculated that at least 70% and perhaps 80% of Monocots are of polyploid origin. Lewis, (1980) took an approach similar to that of Goldblatt (1980) with Dicots and estimated that 70 80% of them are polyploids. Masterson, (1994) used a novel approach, comparing stomatal size in fossil and extant taxa. Because stomatal size is often considerably larger in polyploids than in diploids, this provided a reliable method for estimating the incidence of polyploidy. She estimated that 70% of all angiosperms had experienced one or more episodes of polyploidy during evolutional changes. Polyploidy is considered as one of the basic cytogenetic mechanism in evolution and rapid speciation of plant species (Hilu, 1993). It has long been recognized as a prominent force in the evolutionary diversification of both ferns and flowering plants (Winge, 1917., Darlington, 1973., Stebbins, 1940., 1950., 1971., Grant, 1973., Levin, 2002). The most common mechanism for rapid speciation in plants is through polyploidy (Grant, 1981). The appearance of cytotypes - the presence of different chromosome numbers in the somatic cells of the same individual is considered as one of the basic changes in plants leading to polyploidy. This is of much significance in plants reproducing by vegetative structures like bulbils as in the genus Dioscorea. The predominance of one cytotype excludes other cytotypes from reaching high frequencies in a randomly mating population due to the ineffective matings of the rare cytotype (Husband, 2004). This is known as the minority cytotype exclusion principle. A new, and therefore rare, polyploid in a diploid population would be at a major fertility disadvantage, since most pollinations of the polyploids will involve pollen from diploids i.e. rare polyploids will be less fit, since mostly sterile or inviable triploid progeny would be produced (Fowler and Levin, 1984). Levin (1983) argued that although chromosome doubling does have consequences 145

12 at many levels (e.g. cytological, biochemical, physiological, developmental), it can bring about adaptive changes which may cause ecological differentiation between cytotypes. Polyploids can have more than two different alleles at any given locus. The intrinsic advantage of heterozygosity may be a key factor in the growth, performance and adaptability of a polyploid (Grant, 1981., Levin, 1983., Ramsey & Schemske, 1998., 2002., Soltis et al, 2004). Polyploid populations often demonstrate extensive genomic rearrangement including the origin of novel regions of DNA (Song et al., 1995., Arnold, 1997). Increased enzyme activity, novel enzymes and metabolites, and increased metabolic regulation may enable polyploids to invade new habitats that are not occupied by their diploid progenitors. Polyploids have been suggested to be more adaptive in disturbed or novel niches than their diploid progenitors (Ehrendorfer, 1980., Grant, 1981., Stebbins, 1985., Otto & Whitton, 2000., Levin, 2001). Polyploidy seems to be favored in long lived/perennial plants possessing various vegetative means of propagation and in those with frequent occurrences of natural interspecific hybridizations (Hieter and Griffith, 1999).Various possible reasons have been advanced by various workers (Grant, 1981., Lewis, 1980., Stebbins, 1950), to account for the above phenomenon. One of the widely accepted reasons being the enhanced chances of somatic doubling made possible in plants with long lifespan and vegetative means of reproduction (Grant, 1981). Recent studies in several plant groups have revealed that the genesis of allopolyploidy under both synthetic and natural conditions is often accompanied by rapid genetic and sometimes evolutionarily conserved epigenetic changes (Liu et al, 1998., Ozkan et al, 2001., Osborn et al, 2003., Soltis et al, 2004). The capacity of Angiosperms to form new combinations via polyploids may help explain the rapid diversification and high endemism in a given region with high biodiversity. Polyploidization might thus account for the high species diversity and endemism in various plant groups. Several studies pertaining to different genera of Angiosperms suggest evidence of habitat differentiation among cytotypes (Lewis, 1976., Lewis & Suda, 1976., Tyler et al, 1978., 146

13 Hancock & Bringhurst, 1981., Lumaret, 1984., Lumaret et al, 1997., Smith-Huerta, 1984., Felber, 1988., Bayer et al, 1991). In general, greater variability in polyploids for morphological, demographic, and phenotypic traits relative to their diploid progenitors is believed to contribute to habitat differentiation. Polyploidization can be accompanied by the loss of DNA and thus a decrease of chromosome and genome size (Raina et al, 1994). On the contrary, polyploidization and speciation can also be associated with an increase of genome and chromosome size due to, for instance, differential amplification of species-specific repetitive sequences or retrotransposons (Kumar and Bennetzen, 1999., Staginnus et al, 1999). To date, our assumptions about the ploidy level of yams are based on the observations that the basic chromosome number is X = 8, 9, 10 and in rare cases 12 (Essad, 1984., Mustapha et al, 2006). Much of the cytological works in the genus Dioscorea were done in cultivated species due to their importance as a source of food and the steroid diosgenin. As a result, the ploidy levels of these species and their cultivars are widely known and published. Densi et al., (2000) investigated the ploidy levels of 90 cultivars of Dioscorea cayenensis and Dioscorea rotundata of Benin republic. Of the 90 cultivars studied, eighty were found to be tetraploids, three were octoploids, five were hexaploids and two were mixoploid. Majority of the cultivars were tetraploids. This is in agreement with the findings of Essad (1984) which indicated that tetraploid individuals are the most frequent in the Dioscorea species. The same results were obtained in Côte d Ivoire (Zoundjihékpon et al, 1990., Hamon et al, 1992) and Cameroon (Dansi et al, 2000). Based on the published chromosome counts of 72 species of Dioscorea (including wild species) ploidy levels ranging from 2x to 10x was observed by Essad in x forms are the most frequent followed by 2x, 6x, 8x and 10x in more or less equal proportions. 147

14 However, the variation in polyploidy observed within some cultivated species and the lack of obvious relationship between polyploidy and food value suggest that polyploid variation has not actually been involved in speciation in this genus. Dioscorea hispida, Dennst. (Dioscorea daemona. Roxb.) Diploid 2x = 16 and triploid 3x = 24 cytotypes were found in this species. Literature shows, tetraploidy in this species with 2n = 4x = 40. More than 80% of the cells observed in the present study revealed diploid karyotype with 2n = 16. Dioscorea rubella, Roxb Present investigations reveal 6x = 48 and 7x = 56 cytotypes. The already reported counts are 3x = 30 and 4x = 40 (Essad,1984., Raghavan,1958., Ramachandran,1962). The frequency of 7x cytotype was found to be very low in cells observed. The species analyzed appears to be a hexaploid with 2n = 48. Dioscorea bulbifera, Linn. var. sativa, Pr. & Burk. (Dioscorea sativa, Linn.) Ploidy of 6x = 54 and 8x = 72 chromosomes were observed in this species in the present analysis. Polyploids of 4x, 6x, 8x and 10x with a base number of X = 10 were reported by Miege (1954), Raghavan (1959), Ramachandran (1962), Essad (1984). Ploids of 4x = 36 chromosomes and 6x = 54 chromosomes with a base number of X = 9 were reported in African species by the same authors. Present investigations also supports the basic number of X = 9 in this species. Frequency of hexaploid cells with 2n = 54 was found to be lower than that of octaploid cell with a chromosome count of 2n = 72. Dioscorea Jacquemontii, Hook. The chromosome count observed in this species is 2n = 7x = 56 chromosomes with a base number of X = 8. Published chromosome counts are not available for the species. 148

15 Dioscorea Kalkapershadii, Pr & Burk. Two chromosome counts were found in this species with 2n = 5x = 45 chromosomes and 2n = 6x = 54 chromosomes with a base number of X = 9. Chromosome counts for this species are not available in literature. Cells with chromosome count of 2n = 45 occurred in very low frequency among the cells observed. The plant is a hexaploid with a chromosome count of 2n = 54. Dioscorea tomentosa, Heyne. (Dioscorea triphylla, Herb. Russ.) Three chromosome counts of 2n = 2x = 18, 2n = 3x = 27 and 2n = 4x = 36 were observed with a base number of X = 9. Reported chromosome numbers are 2n = 2x = 40 and 2n = 4x = 40 with a base number of X = 10 (Raghavan, 1959., Ramachandran, 1962., Sharma, 1970). Most of the cells analyzed were tetraploids with chromosome count of 2n = 36. Diploid cells with 2n = 18 chromosomes were found to more common than triploid cells with 2n = 27 chromosomes. Dioscorea atropurpurea, Roxb. Two chromosome counts of 2n = 4x = 36 and 2n = 6x = 54 were observed for this species with a base number of X = 9. Reports pertaining to the number of chromosomes are not available for the species. Hexaploid cells with 2n = 54 chromosomes occurred in high frequency than tetraploids with 2n = 36 chromosomes. Dioscorea bulbifera, var. vera Linnaeus. (Helmia bulbifera, (Linnaeus) Kunth.) Two chromosome counts of 2n = 6x = 54 and 2n = 8x = 72, with a base number of X = 9 were observed for the species. The counts obtained for this variety and D. bulbifera. var. sativa are similar. Most of the cells observed were found to be octaploids with chromosome count of 2n =

16 Dioscorea wallichii, Hook. (D. aculeata, Linn.) The reported chromosome count for the species is 2n = 4x = 40 with a base number X = 10 (Raghavan, 1959., Ramachandran, 1962., Sharma, 1970., Essad, 1984). Present investigations reveal chromosome counts of 2n = 3x = 24, 2n = 5x = 40 and 2n = 6x = 48 for this species with a base number of X = 8. Triploid and pentaploid cells were found in low frequency. The species appears to be a hexaploid with 2n = 48 chromosomes. Dioscorea spinosa, Roxburg. Two cytotypes with 2n = 7x = 63 chromosomes and 2n = 8x = 72 chromosomes with a base number of X = 9 were observed in present study. A survey through available counts in published literature shows a chromosome count of 2n = 9x = 90 with a base number of X = 10 (Ramachandran, 1962., Sharma, 1970., Essad, 1984). Frequency of heptaploid cells with chromosome count of 2n = 63 is higher than octaploids with 2n = 72 chromosomes. Dioscorea esculenta, (Lour.) Burk. Chromosome counts of 2n = 3x =27 and 2n = 6x = 54 were obtained for this species. A survey of literature shows chromosome counts of 2n = 40 (Miege, 1952); 2n = 60 (Martin 1978); 2n = 90 (Raghavan, 1958., Ramachandran, 1962., Sharma, 1970) and 2n = 100 (Raghavan, 1958., Sharma, 1970) reported by various authors with a base number of X = 10. Occurrence of triploid cytotypes with chromosome count of 2n = 27 is far less than the hexaploid cytotypes with 2n = 54 chromosomes. Dioscorea fasciculata, Roxb. (Dioscorea esculenta var. esculenta; Dioscorea esculenta var. fasciculata (Roxburgh) R. Knuth; Oncus esculentus Loureiro, Fl. Cochinch.) Chromosome counts of 2n = 5x = 45 and 2n = 6x = 54 were obtained in present investigation. The base number is X = 9. Most of the cells analyzed were found to be hexaploids with chromosome count of 2n =

17 Dioscorea Kumaonensis, Kunth Enum. Three chromosome counts of 2n = 3x = 24 and 2n = 4x = 32 with a base number of X = 8 were found for the species in present investigations. Published reports regarding chromosome counts are not available in literature. Majority of cells observed revealed a chromosome count of 2n = 32. The species is undoubtedly a tetraploid with somatic variation in chromosome numbers. Dioscorea pentaphylla, Linn. Chromosome counts of 2n = 4x = 32 and 2n = 8x = 64 with a base number of X = 8 were found in this species in present investigations. Earlier reports indicated base number X = 10 and chromosome counts 2n = 4x = 40, 2n = 7x = 70 and 2n = 8x = 80 by various authors (Raghavan, 1958., 1959., Essad, 1984). The species appears to be an octaploid as evidenced by the high frequency of cells with chromosome count of 2n = 64 than cells with 2n = 32. Dioscorea alata, Linn. (D. globosa, Roxb.) var. red All the cells observed showed chromosome count of 2n = 4x = 36 with a base number of X = 9. Previous records show chromosome counts of 2n = 30, 40,50,60,70 and 80 corresponding to ploidies of 3x, 4x, 5x, 6x, 7x and 8x respectively. The species is assumed to be a tetraploid with 2n = 36. Dioscorea alata, Linn. (D. globosa, Roxb.) var. white. Variations pertaining to chromosome counts were not observed in this variety. All cells observed revealed a chromosome count of 2n = 36. Previous records show chromosome counts of 2n = 30, 40, 50, 60, 70 and 80 corresponding to polyploids of 3x, 4x, 5x, 6x, 7x and 8x respectively, with a base number X = 10 ( Miege, 1954., Sharma, 1956., 151

18 Raghavan, 1958., Martin,1963., Essad, 1984). The variety examined appears to be a tetraploid with 2n = 36 Dioscorea oppositifolia, Linn. var.linnaei, Pr & Burk. The observed chromosome count is 2n = 4x = 32 with a base number X = 8. The previous documented count is 2n = 4x = 40 (Essad, 1984). Dioscorea oppositifolia, Linn. var.dhukunensis, Pr & Burk. The observed chromosome count of 2n = 5x = 40 with a base number X = 8. The chromosome count of 2n = 40 reported by Essad (1984) is probably a pentaploid, with base number of X = 8. Dioscorea bulbifera, var.a The chromosome count of this variety which resembles D. bulbifera var.vera in general morphology is 2n = 38. Numerical chromosome polymorphism was not observed in this variety. Dioscorea bulbifera, var.b This variety is similar to var.a in morphological features but for the reduced size of leaves and bulbils. Chromosome count of 2n = 36 was observed in this variety without any additional cytotypes. Dioscorea bulbifera, var. C The variety is morphologically the smallest of the three varieties of D. bulbifera. The chromosome count was found to be the same as the D.bulbifera, var.b with 2n = 36. Additional cytotypes were not observed in analysis. 152

19 Karyomorphometrical analysis Different taxa of plants like species, genera, families etc, are related phylogenetically. This relationship is evident in chromosomes since they are the carriers of heredity. Various aspects of chromosomes like their number and molecular as well as morphological structure reflect the evolutionary status of the organism in question. Detailed study of the chromosome characteristics can thus serve as a useful tool to elucidate the evolutionary relationships between different groups of plants and for tracing the path of evolution in these plants. Karyological features like chromosome number, size, nature of centromere, etc, are of much use in working out evolutionary status as well as relationship of species and genera. Phylogenetic relationship of the genus Dioscorea has not been well examined because of the difficulty in species identification due to a high level of polymorphism with respect to morphological characters (Okagami, 1985). Moreover, the chromosomes of different species of Dioscorea are small and numerous, rendering karyotypic analysis all the more difficult. Karyomorphological study using conventional methods is only of limited use in dealing with the chromosomal analysis of the genus Dioscorea. Image analysis system using computer is of much use in analysis of finer details of chromosomes in these plants. Cytological studies on Dioscorea by various authors indicated the presence of large chromosome counts for both wild and cultivated species. Almost all of them observed dot like and rod shaped chromosomes, apparently with out clear constrictions. Image analysis using computer devices is a powerful tool which can generate quantitative chromosome data which is beyond the reach of conventional methods. The magnification of scanned and digitized images in image analysis followed by appropriate adjustment of contrast and colour helps a great deal in identification of primary and secondary constrictions. The constrictions of even very small chromosomes can be clearly demarcated using these techniques. Quantitative data regarding the morphology of chromosomes can be calculated using software Autocad loaded on computer. Data with respect to the length of chromosomes, 153

20 short arm and long arm length, perimeter and area of chromosomes etc, can be accurately measured through analysis of the digitized image. Statistical parameter, forma percentage, or centromeric index, (the ratio of short arm length to total chromosome length) is used to classify chromosomes based on centromere. Uniformity coefficient (perimeter / area), relative chromosome length (RL), and arm difference ratio (AD) are test statics used to ascertain the similarities between chromosomes. These parameters are helpful in tracing homology between metaphase chromosomes. Derived parameters like disparity index (DI) and coefficient of variation (VC) are used to trace the symmetry of the karyotype. The size variations of the chromosomes are reflected in disparity indices. Higher the variation in size of chromosomes, disparity index tends to achieve high values. Variation coefficient reflects the distribution of chromosomes of different size around a mean value. High variation coefficient there fore reflects the heterogeneity of the karyotype. These derived parameters along with other quantitative data are used to trace the evolutionary status of the different species investigated. The genus Dioscorea is characterized by the presence of relatively high chromosome counts. The chromosomes are usually very small often without clear centromeres. Polyploidy within species is very common. The morphometrical features of even cytotypes of the same species differ in details. The quantitative and derived parameters also differ much between the species. These inter and intra specific variations are so complex that an accurate statistical analysis using modern techniques is essential to asses their systematic position and trend of evolution among them. Dioscorea hispida, Dennst. (Dioscorea daemona, Roxb.) (Fig. 94, 95, 131,132) Two cytotypes with chromosome numbers 16 and 24 were observed in this species. One Chromosome with secondary constriction was represented in both types. The ranges of chromosomes for both are more or less same. Chromatin lengths were correspondingly similar. The disparity index and variation coefficient of both cytotypes were not very 154

21 low, indicating asymmetrical karyotypes. The TF values are not so high, which indicate a tendancy towards moderately advanced nature of the karyotype. The mean chromosome sizes are 0.63 and 0.46 in diploid and triploid respectively. Median chromosomes are absent in the cytotypes. In the diploid cytotype, 8 chromosomes have nearly median centromere, six with nearly sub median centromeres and two chromosomes with nearly terminal centromeres which are considered as derived forms. The small chromosome size, high disparity index and comparatively high variation coefficient of the 3x cytotype indicate the advanced nature of triploid cytotype over the diploid one. Dioscorea rubella, Roxb. (Fig. 96, 97, 133, 134) The two cytotypes, 6x and 7x, revealed more or less similar TF values of 41each, which are not so high. This parameter hints at the advanced nature of the species. The variation coefficient of the cytotypes is approximately 36 and 42 respectively. The 6x = 48 cytotype has a better chromosome range than the second cytotype. Greater size differences in chromosome compliments are considered as advanced over narrow range. The disparity index of 6x cytotype is higher than 7x cytotype which shows that 6x cytotype is more advanced of the two. Median chromosomes are absent in both cytotypes. Nearly median chromosomes, nearly sub median chromosomes and nearly sub terminal chromosomes are found in both cytotypes. The number of sub terminal chromosomes in hexaploid cytotype is three times higher than the heptaploid one. Dioscorea bulbifera, Linn. var. sativa, Pr. & Burk. (Dioscorea sativa, Linn.) (Fig. 98, 99, 135, 136) Two cytotypes with 2n = 54 and 2n = 72 were found in the species corresponding to 6x and 8x respectively. Of these the 6x cytotype have high variation coefficient and disparity index of and respectively. These high values indicate the advanced asymmetrical nature of chromosomes. The 8x cytotype in contrast shows relatively low variation coefficient and disparity index of 34.8 and However these values are indicative of asymmetric nature of the chromosomes. Hexaploid cytotype has 17 pairs of nearly median chromosomes and 10 nearly sub median chromosomes. Octaploids have 155

22 one median pair and 17 nearly median pairs and 18 nearly sub median pairs of chromosomes in their cells. The absence or low frequency of median chromosomes is an advanced feature of the species as a whole. Dioscorea Jacquemontii, Hook. (Fig. 100, 137) Only one cytotype with 2n = 56 was found in the species.the karyotype analysis of the species revealed a high disparity index of This indicates the asymmetry of chromosomes which is advanced over symmetry of chromosomes represented by low disparity index. The relatively low TF value of also indicate asymmetrical chromosomes. Nineteen pairs of chromosomes are nearly sub median and nine pairs nearly median. Absence of median chromosomes and the high frequency of nearly sub median ones indicate the derived nature of the karyotype. Dioscorea Kalkapershadii, Pr & Burk. (Fig. 101,102, 138, 139) Cytotypes with 2n = 5x =45 and 2n 6x = 54 having more or less same total chromatin content (TCL of and respectively) were observed. The 5x cytotype showed more asymmetry as evidenced by the high disparity index of and variation coefficient of Variation coefficient of 6x cytotype is indicating a low range in chromosome size. The species as a whole is well advanced in karyomorphology due to high values of disparity index and variation coefficient coupled with low mean centromeric value TF. The chromatin range in 4x cytotype is higher than that of 6x cells. Presence of 5 chromosomes with terminal centromere and the abundance of nearly sub median chromosomes coupled with absence of median chromosomes are derived characters. Dioscorea tomentosa, Heyne. (Dioscorea triphylla, Herb. Russ.) (Fig. 103, 104, 105, 140, 141, 142) The three cytotypes, representing 2x, 3x and 4x sets of chromosomes, recorded high values for disparity index and coefficient of variation. The chromosome complements in 156

23 these karyotypes exhibited great deal of heterogeneity due to disparity indices ranging from 61 to 71. The TF values are comparatively low due to low frequency of metacentrics. Of the three cytotypes, the triploid and tetraploid variants are the most advanced in karyological parameters as they recorded high values for disparity index and coefficient of variation with relatively low values in TF. The chromosomes appear in all forms within the cytotypes. High incidence of near sub terminal and terminal chromosomes in the cytotypes express the flexibility of these species to undergo chromosomal rearrangements leading to speciation. The species as a whole is one of the most advanced in the present investigations. Dioscorea atropurpurea, Roxb. (Fig.106, 107, 143, 144) The two chromosome counts of 36 and 54 representing tetraploidy and hexaploidy have high disparity indices of 61.1 and respectively. Along with a reasonably high value of variation coefficients of and 41.3, the species undoubtedly is advanced with high chromosome heterogeneity and size range. TF values are relatively low and similar in both cytotypes. The chromatin length, in both cytotypes are proportionate indicating a rearrangement of chromatin material in higher ploidy levels. The range of chromosome length in both cytotypes is high indicating the asymmetry of karyotypes. Presence of terminal centromeres and absence of median chromosomes in both cytotypes are derived features. Dioscorea bulbifera, var. vera. Linnaeus. (Helmia bulbifera, (Linnaeus) Kunth.) (Fig. 108, 109, 145, 146) Polyploids with 2n = 6x = 54 and 2n = 8x = 72 were found in the species during the present investigation. These two cytotypes have high disparity indices of 61.1 and respectively indicating chromosome symmetry. However the variation coefficient of 8x cytotype is low indicating the high frequency of median to nearly median chromosomes in karyotype. The TF values are low in both cytotypes indicating the advanced nature of the species. High disparity index values, presence of terminal centromeres in 157

24 chromosomes and relatively high range of chromosome size indicate the advanced nature of karyotypes of both cytotypes. Dioscorea wallichii, Hook. (D. aculeata, Linn.) (Fig. 110, 111, 112, 147, 148, 149) Three cytotypes with 2n = 3x = 24, 2n = 5x = 40 and 2n = 6x = 48 were observed in this species. 6x cytotype is the most common while 3x and 5x cytotypes occurred in low frequencies. Presence of median chromosomes and low variation coefficient among the cytotypes are indicative of the primitiveness of the karyotypes. Relatively high values of disparity indices of 53.72, and 52 in 3x, 5x and 6x cytotypes however indicate deviation from symmetry leading to asymmetry in karyotypes. Dioscorea spinosa, Roxburg. (Fig. 113, 114, 150, 151) Two cytotypes 2n = 7x = 63 and 2n = 8x =72 were found in this species. Of these, the 7x cytotype occurs in high frequency than the octaploid cytotype. The TF values of the two cytotypes are and respectively indicating asymmetry in the two arms of the chromosomes. Variation coefficient of the two cytotypes, are medium with values of and respectively. These values represent moderate size range in chromosomes of the two cytotypes. Disparity indices of the two cytotypes are very high at and respectively. These high values indicate asymmetry and derived nature of the cytotypes. Dioscorea esculenta, (Lour.) Burk. (Fig. 115, 116, 152, 153) Most of the cells in this species have chromosome count of 2n = 6x = 54. Occasional presence of second cytotype with chromosome count of 2n = 3x = 27 was observed in this species. Coefficient of variation and disparity index were found to be very high with 60 and respectively in the 6x cytotype. The 3x cytotype has high TF% of 43.4 and low variation coefficient of Median chromosomes were found in the 3x cytotype indicating its primitive nature. The 6x cytotype is undoubtedly the derived one with low 158

25 TF%, high variation coefficient and disparity index along with high percentage of nearly sub median chromosomes. Dioscorea fasciculata, Roxb. (Dioscorea esculenta, var. esculenta; Dioscorea esculenta, var. fasciculata (Roxburgh) R. Knuth; Oncus esculentus, Loureiro, Fl. Cochinch.) (Fig. 117, 118, 154, 155) Two cytotypes with 2n = 5x = 45 and 2n = 6x = 54 were observed in the species. The 6x cytotype occurred in high frequency than the 5x cytotype. The TF values of 5x and 6x cytotypes are and respectively. Variation coefficient of 5x cytotype is and that of 6x cytotype is The corresponding disparity indices are 67.7 and Relatively high values of variation coefficient and disparity index coupled with low TF values indicate heterogeneity of the karyotypes. The 5x cytotype is more advanced than 6x cytotype due to lower TF values and higher variation coefficient and disparity index. Presence of two pairs of chromosomes with sub terminal constriction substantiates the derived nature of 5x cytotype. Dioscorea Kumaonensis, Kunth Enum. (Fig. 119, 120, 156, 157) Two cytotypes with 2n = 3x = 24 and 2n = 4x = 32 were observed in the species during present investigation. The 4x cytotype occurred in high frequency with occasional 3x cytotypes. TF values obtained for the 3x and 4x cytotypes are and Corresponding variation coefficients are and respectively. Disparity indices obtained are and respectively. Relatively lower values of TF indicate the difference in arm sizes of the chromosomes. Variation coefficient values also indicate tendency towards heterogeneity of the karyotype. Relatively high disparity indices show the asymmetry of chromosome complements of the karyotype. Dioscorea pentaphylla, Linn. (Fig. 121, 122, 158, 159) Cytotypes with 2n = 4x = 32 and 2n = 8x = 64 were present in mitotic cells observed in the species. 8x cells formed the main cytotype. Tetraploid cells were of occasional 159

26 presence in the analysis. In both cytotypes, two median chromosome pairs were represented. The TF values of both cytotypes were more or less equal, in tetraploid cells and in octaploid cytotypes. Variation coefficients were found to be and 44 respectively. The disparity index of tetraploid cytotype was found to be higher at than of the octaploid cytotype. Presence of large numbers of nearly median and nearly sub median chromosomes and high disparity index shows the derived nature of the karyotypes of the species. Dioscorea alata, Linn. (D. globosa, Roxb.) var. red (Fig. 123, 160) All the cells analyzed were found to be similar in their karyotypes with 2n = 4x = 36. Only one pair of metacentric chromosomes was found in the karyotype. Majority of the chromosomes were either nearly median or nearly sub median. The TF value recorded Coefficient of variation was found to be 38 and the disparity index Lower TF value; indicate the advanced nature of the karyotype. Variation coefficient is comparatively low indicating narrow range of chromosome size in the karyotype. Relatively high disparity index shows the asymmetrical nature of the karyotype. Dioscorea alata, Linn. (D. globosa, Roxb.) var. white. (Fig. 124, 161) Only a single cytotype with 2n = 36 was found in this variety during present analysis.two pairs of chromosomes are of median type which is a primitive character. Presence of nine pairs of nearly sub median chromosomes indicates the derived nature of the karyotype. High variation coefficient of and disparity index of indicate the asymmetry and heterogeneity of the karyotype. Dioscorea oppositifolia, Linn. var.linnaei, Pr & Burk. (Fig. 125, 162) One cytotype with a chromosome count of 2n = 40 was found in this variety. Median chromosomes were absent in the karyotype. 14 pairs of chromosomes were with nearly median centromeres and the remaining 6 pairs with nearly sub median centromeres. TF, VC, and DI values were found to be 41.2, 38.46, and respectively. Relatively high 160

27 values of disparity index and low TF values indicate asymmetry and advanced nature of the karyotype. Dioscorea oppositifolia, Linn. var.dhukunensis Pr & Burk. (Fig. 126, 163) Only one cytotype with 2n = 4x = 32 was found in the variety. Median chromosomes are absent in the karyotype. 6 pairs of chromosomes were nearly median and 10 pairs nearly sub median. TF, VC and DI values were found to be 38.1, 40.91, and respectively. High disparity index and low TF value represents the advanced nature and asymmetry of the karyotype. Dioscorea bulbifera var. A (Fig. 127, 164) Chromosome count of 2n = 38 was observed in all the cells of this variety. Median, nearly median and nearly sub median chromosomes were found in the karyotype. TF value of 38.39, variation coefficient of and disparity index of were obtained in analysis. Relatively high disparity index and low TF value represent the advanced nature of the karyotype. Dioscorea bulbifera var. B (Fig. 128, 165) Only a single karyotype with 2n = 36 was found in this variety. Median and nearly median chromosomes were represented in equal proportions within the karyotype. TF value of 36.92, variation coefficient of and disparity index of were obtained through analysis. Lower values of variation coefficient and disparity index indicate the relatively primitive nature of the karyotype. Dioscorea bulbifera. var. C (Fig. 129, 166) Karyotype with 2n = 36 was the only one found in this variety. 14 pairs of chromosomes were nearly median and 4 pairs nearly sub median. Narrow range in chromosomes size represented by relatively high TF value and low variation coefficient indicate the homogeneity of the karyotype. 161

28 Cytological evolution in the genus Dioscorea Investigations of karyological features of the different wild species and varieties of the genus Dioscorea contribute significantly to a better understanding of systematics and evolution of this fascinating group of plants. Distribution of basic chromosome numbers and chromosome morphology confirm the presence of two phylogenetically independent lineages. Group one has a basic chromosome number X = 8 represented by the species Dioscorea hispida, D. sativa, D. jacquemontii, D. wallichii, D. kumaonensis, D. pentaphylla and D. oppositifolia. The second group has a base number of X = 9 represented by Dioscorea bulbifera, var. sativa, D. bulbifera var. vera, three newly found varieties A, B and C of D. bulbifera., D. Kalkapershadi, D. tomentosa, D. atropurpurea, D. spinosa, D. esculenta, and D. fasciculata. Identification of the base numbers X = 8 and X = 9 for the species of Dioscorea investigated in the present study, greatly contradicts the previous assumptions forwarded in studies published by various authors. Investigations by Essad (1984), Dansi et al., (2000), Gamiette et al., (1999), concluded that cultivated Asiatic species of Dioscorea have basic chromosome number X = 10. None of the wild species of Dioscorea investigated in present study showed the existence of this base number. Secondary base numbers are altogether absent in the species investigated. It seems that the genus Dioscorea has not evolved in this direction till date although two species with base number X = 12 were reported from Europe (Essad, 1984). The presence of more than one ploidy level is known in almost all species of Dioscorea. Populations with 2x, 3x,4x, 5x, 6x, 7x and 8x were met with, in present investigations (Fig.130). Polyploidy seems to be favored in these plants which possess vegetative means of propagation by bulbils. Numerical chromosome polymorphism is very rampant in various species of Dioscorea as evidenced by different degrees of polyploidy even in 162