CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX IV. EU- AND ANEUPLOID OFFSPRING FROM ALLO-TRIPLOIDS IN A NATURAL POPULATIONI)

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1 JAPAN. J. GENETICS Vol. 47, No. 2: (1972) CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX IV. EU- AND ANEUPLOID OFFSPRING FROM ALLO-TRIPLOIDS IN A NATURAL POPULATIONI) HIROTO ARAKI Received October 5, 1971 Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812 Scilla scilloides Druce is a perennial complex consisting of two diploids and six polyploids composed of two basic genomes A (x=8) and B (x=9), i. e., 2n=16 (AA), 18 (BB), 26 (ABB), 27 (BBB), 34 (AABB), 35 (ABBB), 36 (BBBB), and 43 (AABBB) (Sato 1942, 1953; Haga and Noda 1971). It has been found that some natural populations are composed of various cytogenetic types (Morinaga 1932; Haga and Noda 1971). For example, the Nakabaru population was found to consist of five types in 1952, BB, BBB, ABB, AABB-1, and ABBB, and their spatial distribution indicated that vegetative propagation predominated (Haga and Noda 1971). Along with euploid types, hypo- and hyperploids, such as 2n=33 (AABB-1), 35 (AABB+ 1), 36 (AABB+2), and 37 (AABB+3), were found occasionally in some natural populations (cf. Haga and Noda 1971). Plant of the constitutions ABB, ABBB, and AABBB showed reduction in seed setting and were sexually unstable, giving various cytogenetic types different from their own in their progeny (Haga and Noda 1971; Araki unpubl.). Thus hypo- and hyper-ploids in natural populations might be the progeny of open-pollinated unstable types (cf. Haga and Noda 1971). The present paper reports the structure of the Nakabaru population of S. scilloides in 1963 at the same location, where eleven years ago Haga and Noda analysed the structure of the population. The structure of the population is revealed to be undergoing change by hypo- and hyper-ploid offspring from sexual reproduction of the unstable cytogenetic type ABB. MATERIALS AND METHODS All the flowering plants of Scilla scilloides collected in 1963 were growing in a natural population at Nakabaru, Saga Prefecture. The sampling area was 12 m x 100 m, covering more than half of the area in which Haga and Noda carried out their sampling in 1952 (cf. Haga and Noda 1971). Bulbs of the flowering plants sampled were transplanted to the experimental garden of the Department of Biology, Kyushu University. In the autumn of 1965, mother plants, bulbs bearing spikes with developed capsules and 1) Contribution from the Department of Biology, Faculty of Science, Kyushu University, No. 186.

2 74 H. ARAKI offspring, and seeds in the capsules were collected in the same area as in Along with the mother plants, offspring from seeds were reared in the experimental garden. For cytological examination, root-tips were pretreated in mol aqueous solution of 8-oxyquinoline for two hours, fixed with La Cour 2BE, and squashed in 45 percent acetic acid after Feulgen-staining. PMCs were fixed in Newcomer's fluid (1953) and squashed using the usual iron-acetocarmine. OBSERVATION (1) Denotation of chromosomal constitution. The chromosome constitutions of genomes A and B are given as follows (cf. Haga and Noda 1971; Araki 1971) : A (x=8) =1V (a1)+5i's (a2-as)+2v's (a7-a8) and B (x=9)=5i's (b1-b5)+4v's (b6-b9). Chromo- Figs Chromosomes of roof-tip cells and PMCs of two cytogenetic types of S. scilloides. la: ABB+3+1f, root-tip cell (2n=29+1f). X1200. ib: ABB+3+lf, PMC ( f). X850. 2a: AABB-3+1f +1F, root-tip cell (2n=31+1f +1F) b: AABB-3+ if +1F, PMC ( f +1F). X 850.

3 CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX VI 75 Table 1. Chromosome constitution of S. scilloides in the Nakabaru population somes a1 and b1 are the markers of the genomes A and B, respectively, and the genome constitution of a given plant is represented on the basis of numbers of the markers and meiotic pairing. For example, a plant with 2n=29+ if showing f at MI is designated as ABB+3+1f (Figs. la-b), a plant with 2n=31+1f+1F showing if +1F as AABB-3+1f +1F (Figs. 2a-b), and so forth. Hereafter, f indicates a telocentric minute supernumerary chromosome, and F a metacentric iso-f. (2) Structure of the population. Plants sampled in 1963 consisted of diploid (BB), polyploids (BBB, ABB, AABB, and ABBB), and hypo- and hyper-ploids at tri- and tetraploid levels (Tables 1 and 2).

4 76 H. ARAKI Table 2. Chromosome constitution and meiotic pairing in hyposcilloides from the and hypo-tetraploids of S. Nakabaru and population hyper-triploids As scored in 1952 by Haga and Noda (1971), ABB and ABBB were predominating in 1963 (Table 1). However, the following differences are noticeable between the samplings: (i) AABB with complete sets of chromosomes were first recorded in 1963, (ii) the structure was more complicated in 1963 than in 1952 due to the occurrence of new types of hypo- and hyper-ploids; as many as 18 types of hypo- and hyper-ploids were found in 1963, in contrast only two types of hypo-ploids in 1952, and (iii) cytogenetic types were more diversified by the addition of supernumerary chromosomes, f and F, in 1963 than in 1952; e. g., eight types of ABB, with the supernumerary chromosomes, in 1963 in contrast to only two types in 1952 (Table 1). As shown in Fig. 3 the plants with the same cytogenetic type were found in clusters of various sizes, indicating each cluster probably represented a clone. (3) Chromosome constitution of hypo- and hyper-ploids. In somatic complements of hypo- and hyper-ploids, the chromosomes al (V), a2, as, b1, b2 (I's), a7, a8, b6, b7, b8, and b9 (v's) are easily identifiable (Figs. la and 2a). The remaining I-shaped chromosomes a3, a4, a5, b3, b4, and b5 are so similar in size and shape that they are difficult to distinguish from one another. At MI in PMCs the chromosomes are classified simply

5 CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX VI 77 Fig. 3. Spatial distribution of various cytogenetic types of S. scilloides.

6 78 H. ARAKI Table 3. Seed settings of five types of S. scilloides Nakabaru population open-pollinated in the into V, I, and v, because of difficulty in identification of individual chromosomes in the classes I and v. As identified so far, lacking or additional chromosomes in the hypoand hyper-ploids were members of the genome A (Table 2). The great majority of PMCs at MI was composed of bivalents and univalents in all the hypo- and hyper-ploids, except for a single plant (Table 2). The configuration and frequency of standard pairing in each cytogenetic type are given in Table 2; 250 PMCs were observed in each plant. An exception was found in one of five plants of the constitution AABB-1+1f, which showed the standard pairing 1m+1511, 82.3 percent. Trivalent in this plant always revealed two free arms, suggesting a reciprocal translocation between a7 and as that was accompanied by loss or elimination of normal a8. Therefore it is convincing that chromosomes of the genome A only are responsible for the formation of the hypo- and hyper-ploids. (4) Chromosome constitutions of progenies and their mother plants. In the sample plants collected in 1965, i. e., mother plants from which seeds were sampled, the frequencies of five euploid types were the same as in 1963 (Table 1). Fertility of the mother plants by natural pollination in the natural population is given in Table 3. ABB set seeds in reduced amount, BBB and ABBB were nearly completely sterile, while BB and AABB yielded many seeds (Table 3). Progenies from the open-pollinated seeds in 1965 in the natural population grew to maturity in The progenies from ABB mother plants showed a wide range of variation in chromosome numbers, including euploid types BB, ABB, AABB, and ABBB, in addition to their hypo- and hyper-ploids (Table 4). Numbers and types of the additional or lacking chromosomes in the hypo- and hyper-ploids differed from plant to plant. Occurrence of ABBB in a progeny from ABB indicates a case of union of an unreduced gamete, n=26 (ABB), from ABB with a reduced normal gamete, n=9 (B), probably from BB. Seeds from BB gave rise to BB, ABB, ABB -1, and those from AABB to ABB, AABB, and AABB minus 1 to 5 (Table 4). ABB -1 from the mother plant BB and AABB minus 1 to 5 from the mother plant AABB indicate that pollen with aneuploid chromosome numbers produced by ABB had certainly functioned in the natural population. The validity of this inference is strengthened by the repeated occurrence of aneuploids in the progenies of AABB ; 11 aneuploids of 10 different types among 64 progenies. The chromosomal variation among the progenies depends on the types of the mother plants (Tables 4 and 5). This indicates that the Nakabaru population of Scilla scilloides

7 CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX VI 79 Table 4. Chromosome constitution of the open-pollinated in the Nakabaru progenies from BB, ABB, and AABB plants population (1965)

8 80 H. ARAKI Table 5. Chromosome constitution of the progenies from hypo- and hyper-ploids openpollinated in the Nakabaru population (1965) has become complicated primarily by sexual reproduction of ABB plants by sexual reproduction of the resulting hypo- and hyper-ploids (Tables and secondarily 4 and 5). DISCUSSION Clusters of plants of the same cytogenetic type in the population of Scilla scilloides are no doubt formed by asexual bulb-multiplication (Fig. 3, cf. Haga and Noda 1971). As mentioned above, a remarkable change in population structure has taken place at the Nakabaru locality during the 11 years from 1952 to 1963, i. e., increase of hypo- and

9 CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX VI 81 hyper-ploid types. All the hypo- and hyper-ploids consisted of two complete sets of genome B and varying numbers of chromosomes from genome A. The same holds true for hypo- and hyper-ploids raised from seeds of the mother plants BB, ABB, and AABB, which were open-pollinated in the natural population. Therefore it is unequivocal that the hypo- and hyper-ploids are produced primarily by (1) selfing of, and/or crossing between ABB, and (2) crossing of ABB with BB and/or AABB. Variations in chromosome numbers in natural populations due to hybridization between plants of different ploidy-levels have been reported in Eleocharis (Saunte 1958; Lewis and John 1961) and in Kalimeris (Shindo 1967). Recently, Shindo (1967), Inoue (1970), and Inoue et al. (1970) have reported inter-generic hybridization between species of Kalimeris, Aster and Heteropappus, which resulted in a wide variation in chromosome numbers in sympatrically mixed natural populations. Different races forming a polyploid series of chromosome numbers have been reported in Cardamine pratensis (Lovkvist 1956). In this case natural hybridizations between the races of different ploidy-levels produced progenies showing a wide range of variation in chromosome numbers (Lovkvist 1956). A similar case of chromosomal variation has been reported in Claytonia virginica (Rothwell 1959; Lewis 1962; Rothwell and Kump 1965). In this species no external morphological distinctions were found among plants with different chromosome numbers, which ranged from 2n=12 to ca. 191 (Rothwell and Kump 1965). This extreme variation in C. virginica is attributable, at least in part, to aneusomaty superimposed upon the aneuploidy (Lewis 1962). In the present case of Scilla scilloides, the variation in chromosome numbers is unequivocally attributed to sexual reproduction of ABB plants. It has been shown that the frequency of good seeds from self ed ABB plants is 1.6 persent, which give rise to ABB, AABB, and hypo- and hyper-ploids (Araki unpubl.). This fact also supports the view that plants of ABB are primarily responsible for making the structure of the Nakabaru population more complicated. The diversity of cytogenetic types of S. scilloides due to supernumeraries has been discussed by Haga and Noda (1971). They suggested that the number of the supernumerary chromosome, f, was subjected to variation in the course of asexual propagation by bulbs, because the number of f varied slightly among the cells within the plant (cf. Haga 1961), and such a mitotic aberration would occur in the primordia of the axillary bud forming bulbs (cf. Sharma 1956). However, the diversity in numbers of the supernumerary chromosome in ABB plants may well come from sexual reproduction of ABB. In 1952, the structure of S. scilloides population at Nakabaru indicated a strong tendency toward asexual propagation by bulb multiplication (Raga and Noda 1971). Then, why in 1963 and 1965 did the Nakabaru population show remarkable effectiveness of sexual reproduction, along with asexual propagation? The change seems to be correlated with mechanization in agricultural cultivation, which replaced cows and horses by cultivators some 15 to 20 years ago. At Nakabaru, the habitat of S. scilloides was an open grazing yard along a tiny river bank. Further, grass had been cut to feed the plowing animals. This makes it difficult for S. scilloides to propagate by seeds. Recently, plowing has been mechanized, cattle no longer being needed. In consequence

10 82 H. ARAKI the habitat has been less disturbed, allowing sexual reproduction to take place there. At any rate, the S. scilloides in the Nakabaru population can propagate by seeds more effectively than before, so that the cytogenetic structure of this population is undergoing remarkable change. SUMMARY 1. Several types of hypo- and hyper-ploids were found along with euploids BB, BBB, ABB, AABB, and ABBB among plants from a natural population of Scilla scilloides Druce at Nakabaru. Here A (x=8) indicates one of two genomes which compose the S. scilloides complex, and B (x=9) another one. Lacking or additional chromosomes in hypo- and hyper-ploids, ABB -1, - 2, ABB + 1, + 2, + 3, + 5, AABB -1, -2, -3, were confirmed to be members of the genome A. 2. The majority of progenies from seeds collected from ABB open-pollinated in the natural population was hypo- and hyper-ploid. Hypo- and hyper-ploids were also found in the progenies from the seeds from the mother plants BB and AABB. These facts indicate frequent occurrence of selfing of, and/or crossing between ABB, as well as crossing of ABB with BB and/or AABB. Therefore, ABB is primarily responsible for the origin of hypo- and hyper-ploids in the natural population. ACKNOWLEDGMENTS The writer wishes to express his appreciation to Prof. T. Haga for his kind guidance and encouragement, to Dr. S. Noda for his kind advice throughout the present study, and to Dr. H. Kayano for his invaluable suggestions in making the manuscript. The writer further wishes to express his gratitude to Dr. W. H. Sharp, Department of Biological Sciences, University of Lethbridge, who kindly polished up the manuscript. The samples from the Nakabaru population in 1963 were collected by Prof. T. Haga, Drs. H. Kayano, T. Watanabe, and T. Egashira, to whom the writer is very grateful. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, No. (A) 0810, to Prof. T. Haga. REFERENCES Araki, H., 1971 Cytogenetics of Scilla scilloides complex. III. Homoeology between genomes A (x=8) and B (x=9). Japan. J. Genetics 46: Haga, T., 1961 Intra-individual variation in number and linear patterning of the chromosomes. I. B-chromosomes in Rumex, Paris, and Scilla. Proc. Jap. Acad. 37: Haga, T., and S. Noda, 1971 Cytogenetics of Scilla scilloides complex. I. Karyotype, genome, and population. (In preparation). Inoue, S., 1970 Cytological studies on the intergeneric natural hybrid between Aster and Kalimeris from Kyushu. I. Chromosome numbers and geographical distribution. Mem. Fac. Gener. Educ. Kumamoto Univ. Ser. Natur. Sci. No. 5: Inoue, S., Shinohara, Y., Tsutsumi, S., and K. Kido, 1970 Cytological studies on the intergeneric natural hybrid between Aster and Kalimeris from Kyushu. II. Natural hybrid between A.

11 CYTOGENETICS OF SCILLA SCILLOIDES COMPLEX VI 83 ageratoides subsp. ovatus and K, yomena. Mem. Fac. Gener. Educ. Kumamoto Univ. Ser. Natur. Sci. No. 5: Lewis, K. R., and B. John, 1961 Hybridization in a wild population of Eleocharis palustries. Chromosoma 12: Lewis, W. H., 1962 Aneusomaty in aneuploid population of Claytonia virginica. Amer. J. Bot. 49: Lovkvist, B., 1956 The Cardamine pratensis complex. Cymb. Bot. Upsal. XIV. 2: Morinaga, T., 1932 A preliminary note of the karyological types of Scilla japonica Bak. Japan. J. Genetics 7: Newcomer, E. H., 1953 A new cytological and histological fixing fluid. Science 118: 161. Rothwell, N. V., 1959 Aneuploidy in Claytonia virginica. Amer. J. Bot. 46: Rothwell, N. V., and J. Kump, 1965 Chromosome numbers in population of Claytonia virginica from the New York metropolitan area. Amer. J. Bot. 52: Sato, D., 1942 Karyotype alteration and phylogeny in Liliaceae and allied families. Jap. J. Bot. 12: Sato, D., 1953 Karyotype analysis and law of homologous series. Sci. Papers of Coll. Gener. Educ. Univ. Tokyo 3: Saunte, L. H., 1958 Chromosome variation in the Heleocharis palustris-uniglumis complex. Nature 181: Sharma, A. K., 1956 A new concept of a means of speciation in plants. Caryologia 9: Shindo, K., 1967 Cytological, morphological and geographical studies on the differentiation of species in section Asteromoea of Kalimeris in Japan. J. Sci. Hiroshima Univ., Ser. B, Div. 2, 11: