C 1998 The Japan Mendel Society Cytologia 63: 191-197, 1998

Chromosome Polymorphism and Bivalent-forming Triploid and Tetraploid pallida ()

Armando Garcia-Velazquez

Especialidad en Genetica Instituto de Recursos Geneticos y Productividad, Colegio de Postgraduados, Montecillo, Mex., Mexico 56230, C.P.

Accepted February 12, 1998

Summary Karyotypes and meiotic chromosome pairing were studied in natural autotriploid and autotetraploid Tradescantia pallida. At both ploidy levels chromosomes appeared as metacentrics,

being larger at the triploid than at the tetraploidy level (9.6-15.3 ƒÊm and 7.5-10.0 ƒÊm, respectively). T pallida exhibits polymorphism with respect to number and position of secondary constrictions. The tetraploid had three large satellites and a small-spheric one. While the triploid had two large satellites and one small-spheric. Both the triploid and tetraploid showed reduced multivalent pairing for triploid, only one trivalent was seen in 32.8% PMC's while 67.2% PMC's had 6 bivalents plus 6 univalents. In the tetraploid, bivalent pairing was very predominant, 87.72% PMC's had 12 bivalents. The presence of a bivalent-forming system in these polyploids, most probably a genetic one, is sug- gested. Key words Chromosome polymorphism, Autopolyploids, Bivalent-forming, Tradescantia.

Tradescantia pallida (Rose) D. R. Hunt Cay. (Syn. Setcreasea purpurea) is a member of the tribe tradescantieae of the family Commelinaceae. The members of the tradescantieae are predomi- nantly Mexican in origin and based on x=6 fairly symmetrical chromosomes (Owens 1981). In the Commelinaceae family there are few bivalent-forming tetraploids including Tradescantia cymbis- patha, T Standleyii (Kenton and Drakeford 1990), T ambigua Mart., T burchii, D. R. Hunt, and T crassifolia group. T ambigua is a South American representative of the genus. There is no data to explain why these self-compatible species are bivalent forming (Owens 1981). It is likely that the diploid-like meiosis in most, if not all, other natural poliploids is genetically regulated, since with- out such a control precise bivalent pairing in these polyploids having several sets of related genomes, would not be achieved (Jauhar 1977). Although allopolyploidy has provided the basis for the evolution of many species including Commelinaceae (Kenton 1981, Jones 1974, 1977), the super-imposition of a precise genetic control on chromosome pairing could be critical for con- ferring meiotic and hence, reproductive stability in sexually reproducing polyploids. Polyploids are usually classified as autopolyploids, segmental allopolyploids or true allopoly- ploids (Stebbins 1971). Such a classification is based on fairly much of information, but most of it deal with chromosome pairing in a hybrid or an individual that is known or presumed to have given rise to a polyploid. The information is generally lacking in natural polyploids. The polyploids consisting of only bivalents are considered allopolyploids, while those of mul- tivalents are treated as autopolyploids or segmental allopolyploids (Jackcson and Casey 1980, 1982). The evolutionary processes of many plant species involve hybridization and polyploidy. An understanding of the genomic relationships between the parental species and the derived polyploids is important, first so that the evolutionary processes may be elucidated and second, for pragmatical reasons (Alonso and Kimber 1981). A complicating factor in polyploidy classification is the presence in some populations and species, of genes affecting homoeologous chromosome pairing. The best known of these is the dominant Ph gene in the hexaploid and tetraploid wheats that prevents homoeologous pairing, and 192 Armando Garcia-Velazquez Cytologia 63 only homologous pairings are observed in normal (Okamoto 1957, Riley and Chapman 1958). Ph-like but not necessarily dominant genes have been reported for diploid wheats and Ph- like effects have been suggested for other plants (Avivi 1976, Jauhar 1977, 1993). The occurrence of diploid (2n= 12), triploid (2n= 18) and tetraploid (2n=24) in Tradescantia pallida could be indicative of autotetraploidy via unreduced gametes. There are several cases in the Commelinaceae in which unreduced gametes are produced. In Rhoeo spathacea Garcia (1991, 1995) observed the production of diplandrogynous autotetraploids, when both male and female ga- metes are unreduced. The similar situation was reported by Jones (1976) in T cymbispatha in which several ploidy levels occurred. Xerophytes of the genus Tradescantia showing their greatest mor- phological diversity are located along the Gulf of Mexico. Tradescantia pallida (Rose) D. R. Hunt is one of these species and plants were located in Ocampo and Ciudad Victoria in the south of

Tamaulipas (23•Ž44'N, 39•Ž08'W), Mexico, at about 400 m altitude. The investigation of triploid and tetraploid cytotypes is the subject of this paper.

Materials and methods

Vegetative material of Tradescantia pallida (Rose) D. R. Hunt was collected from plants grow- ing in their natural habitat. Propagules were potted and cultivated in the greenhouse. All plants pro- duced pink .

Cytological methods The standard cytological techniques used have been described in detail elsewhere (Garcia 1995). tips were pretreated with 2 mM 8-hydroxyquinoline at 18°C for 9 hr before fixing in ethanol : acetic acid (3 : 1, v/v). Karyotypes were directly drawn from metaphase plates at •~3200 magnification by using a drawing attachment. Determination homology in the karyotypes was based on chromosome length, centromere position and presence of satellites.

Meiotic analysis in PMC's For examination of meiosis, anthers were squashed directly into 1.8% propionic orcein and heated gently to spread the chromosomes. Meiosis at MI, AI and TI stages was observed. Pollen fertility was estimated by scoring the percentage of well-filled, stained grains after squashing in propionic orcein. Cells were photographed on Kodalith film using 64•~ and 100 •~ Zeiss planapo chromatic and Neufluar immersion lenses.

Results and discussion Two different chromosome numbers were recorded in the plant material of this study. Out of the six plants, collected in Ocampo, Tamaulipas, one exhibited 2n= 18 and the other five 2n=24. Thus, triploidy and tetraploidy might occur with a basic chromosome number of x=6 as has been reported in the tradescantieae (Hunt 1993). The triploid formation in T pallida even when a rare event, occurs in nature, probably being produced by a cross between 4x and 2x or between 2x and 4x. Several authors have concluded that Tradescantia pallida is a species having diploid, triploid and tetraploid chromosome numbers on the basis of its karyotype (Martinez 1978, Owens 1981, Jones and Kenton 1984).

Karyotype and idiogram Based on Feulgen stained chromosomes in root tips treated with 8HQ the karyotype of T. palli- da consists of 18 or 24 metacentric chromosomes and they showed a graded series of lengths. As expected for an autotriploid and autotetraploid, the chromosomes could be arranged into groups of 1998 Chromosome Polymorphism and Bivalent-forming Triploid and Tetraploid 193

Fig. 1. Drawing of somatic chromosomes of Tradescantia pallida. a) Tetraploid, 2n = 4x = 24,

b) Triploid 2n=3x=18. Scale 10 ƒÊm. three and four on the basis of total length, arm ratio, and position of secondary constrictions (Fig. 1a, b). Two groups can be clearly specialized according to secondary constrictions: they show some polymorphism among putative homologues. In the karyotype of the triploid plant (Fig. 1b) one group includes two metacentrics bearing secondary constrictions in the short arms (Nos. 13, 15) and thereby large satellites. The other group includes a large metacentric chromosome with a small spheric satellite (No. 18). In the tetraploid karyotype (Fig. 1a) there are three chromosomes with large chromosome satellites (Nos. 22, 23, 24), while a small-spheric satellite is located in the short arm of chromosome 7. The standard kary- otype of Tradescantia pallida consists of homologous and perfectly pairable elements (Martinez 1978, Owens 1981, Jones and Kenton 1984, Hunt 1993) at both diploid (2n= 12) and tetraploid (2n=24) levels. Obviously, in the present cytotypes perfect homology is lost for those chromo- somes with satellites. Hunt (1993) has indicated that there is not any cytological diversity as all the Tradescantia species have large metacentric chromosomes even in the north of the trans-Mexican volcanic belt, where the genus shows its greatest morphological diversity. However, the present study on the triploid and tetraploid Tradescantia pallida suggests that a cytogenetic differentiation occurs probably owing to its geographical distribution. In the described karyotypes for triploid and tetraploid cytotypes of Tradescantia pallida, in ad- dition to chromosome homologous groups there is one group of heteromorphics made up by sec- ondary constrictions. Determination of their evolutionary relationship must await further work.

Meiotic behavior Pachytene chromosomes of Tradescantia pallida are not suitable for karyotype analysis. How- ever, at early prophase some multivalent association could be examined. Table 1 presents the chromosome associations in one triploid plant and five tetraploid plants of T pallida. In 1807 PMC's analyzed at MI of triploid plant its high frequencies of bivalents and uni- valents (5.67 per cell in both chromosome associations) contrast with the smaller number of triva- lents per cell (0.32 in average) (Fig. 2d, f). In mathematical meiotic models designed for estimating 194 Armando Garcia-Velazquez Cytologia 63

Table 1. Metaphase I chromosomal association in autotriploid and autotetraploid Tradescantia pallida

A B

C D

E F

Fig. 2. Chromosome behavior in Tradescantia pallida, triploid and tetraploid. a) MI of 2n=24, 12II, b) 10II+ IIV, c) microspore n=12, d) MI of triploid, 5II+1III+5I. Arrow (Triv.), e) MI of tetraploid 10II+ 1IV,f) MI of triploid, 5II+ 8I. 1998 Chromosome Polymorphism and Bivalent-forming Triploid and Tetraploid 195

Table 2. Anaphase I chromosome distribution in autotriploid and autotetraploid Tradescantia pallida

genome affinities in polyploids, 2/3 of chromosome pairs as trivalents in a triploid and the remain- der 1/3 are bivalents accompanied by a univalent (Jackson and Casey 1989). Comparing with the expected values on the models (Sybenga 1996), this natural triploid had significantly few trivalents per cell but it had many bivalents and univalents. However, the presence of only one trivalent in each of 592 PMC's can be an indication of the homology of chromosome groups, and the au- totriploidy origin of this plant may be accepted. Chromosome behavior at AI in triploid and tetraploid plants is shown in Table 2. In all PMC's of the triploid 5 to 6 univalents were observed (Fig. ld, f)..The distribution of such univalents to the poles apparently was not at random, in spite of its high number. In 117 (45.35%) meiocytes a 9 : 9 distribution was observed while a single lagging (9 : 1 : 8) was observed in 48 (18.60%) meiocytes. A 10 : 8 distribution was observed in 93 (36.05%) meiocytes. Upon completion of chromosome separation nuclei began to diffuse and cytokinesis produced dyads. The 9 : 9 chromosome segrega- tion even in the presence of high number of univalents (5 or 6) may indicate that chromosome pair- ing at MI is not always required for the regular distribution of chromosomes at AI. Disjunction of bivalents of the tetraploids is a synchronous and normal event of chromosomes at opposite spindle poles. A 12 : 12 disjunction was observed in 566 (91.88%) meiocytes, while ab- normal disjunction was observed in 50 (8.12%) meiocytes. Only 33 (5.36%) meiocytes presented one lagging chromosome while 17 (2.76%) meiocytes showed unequal (13 : 11) distribution of chromosomes. The normal distribution (12 : 12) was highest (91.88%) than expected on the basis of 86.65% PMC's showing 12 bivalents. It could be an indication that univalents and tetravalents have a regular disjunction as was observed in the triploid and tetraploid PMC's. Five tetraploid plants of Tradescantia pallida were used for cytological analysis, and a total of 1273 PMC's observed at MI primarily formed bivalents with terminal chiasmata (Fig. 2a, b, e). Ex- ceptionally one bivalent with interstitial chiasmata was observed (Fig. 2e). Even when some chro- mosomes do not have homomorphic partners, the structural heterozygosity is not revealed at meio- sis. Chromosome pairing in 1103 PMC's (86.65%) presented 12 bivalents with an overall 11.78 bi- valents/cell (Table 1). Two univalents were observed in 88 (6.91%) PMC's and also 88 PMC's (6.91%) showed one or two quadrivalents or 0.076 quadrivalents per cell (Table 1). These results deviated significantly from the expected values: autotetraploids and autotriploids are required to generate 2/3 multivalents and 1/3 bivalent univalent associations at first meiosis (Sybenga 1975). In both triploid and tetraploid cytotypes univalents at MI behaved normally. In triploid hybrids be- tween Tradescantia ambigua •~ T crassifolia (both bivalent-forming), and in diploid hybrids between

T tepoxtlana (2n= 12) •~ T pallida (2n= 12) Jones (1978) observed univalents that showed the typi- cal fold back appearance when both arms came into contact. In the present study no ring-like univa- lents were observed and the univalents are probably the result of failure of chiasma formation but not of lack of homology. Even though quadrivalents were scarce some degree of homology may be 196 Armando Garcia-Velazquez Cytologia 63

Table 3. Chromosome number at first mitosis in microspores of triploid and tetraploid plants of T pallida and number and percentage of microspores observed

persisted in homologues in T pallida chromosomes. In the triploid one would expect to find high frequency of trivalents; however, both homolo- gous and homoeologus chromosomes paired mostly as bivalents. Evidently there is some mecha- nism controling 2 •~ 2 pairing of the sets of three chromosomes. In tetraploid T pallida (syn. Set- creasea purpurea) Mehra et al. (1971) observed an irregular meiotic behavior with low frequency of qudrivalents (0-4) at metaphase I. The authors reported that multivalents were binomially dis- tributed and the average frequency was low in spite of the fact that all the chromosomes were capa- ble of forming qudrivalents. In the Commelinaceae there are several bivalent-forming species. Kenton and Drakeford (1990) reported that autotetraploid T cymbispatha and T standleyii form only bivalents at meiosis I. In the Tradescantia section there are several tetraploid bivalent-forming species including T acaulis, T ambigua, T burchii, T cirrifere and T crassifolia. Then, several bivalent-forming tetraploids were present in the commelinaceae family suggesting that a genic control of diploidiza- tion or homologous pairing is present in these species.

Pollen stainability By using pollen stainability in 1.8% propionic orcein as index of fertility, 7731 (95.38%) out of 8105 pollen grains observed in the five tetraploid plants were found to be fertile. In the triploid plant the stainability was quite different from the case in the tetraploid; only 267 (15.34%) out of 1740 pollen grains showed stainability and normal shape.

Chromosome numbers in microspore In pollen mitosis of Tradescantia pallida chromosome numbers occurred ranging from to 12 in the triploid plant while in the tetraploid plants occurred from 10 to 14 (Table 3). These chromo- some numbers are apparently result of the behavior of chromosomes at first meiosis. Tetraploid nor- mally segregate their chromosomes and thus almost only haploid number is expected to be ob- served in their pollen grains. As for the triploid, many univalents at MI or achiasmatic homologues tend to be delivered randomly; often giving rise to non-disjunction and thus aneuploid meiotic products (Reyes-Valdes et al. 1996). Apparently the microspores produced by the triploid plant after first mitotic division collapsed. In tetraploids the formation of bivalents could be interpreted as part of an evolved mechanism that insures fertility.

Acknowledgments The author is grateful to Mr. Ricardo Contreras for his technical assistance and to Dr. Carmen Mendoza Castillo for kindly providing the plant material. Also, critical review of the manuscript by Dr. Alfredo Carballo-Quiros is greately appreciated. 1998 Chromosome Polymorphism and Bivalent-forming Triploid and Tetraploid 197

References

Alonso, L. C. and Kimber, G. 1981. The analysis of meiosis in hybrids. II. Triploid hybrids. Can. J. Genet. Cytol. 23 : 221-234. Avivi, L. 1976. The effect of genes controling different degrees of homologous pairing on quadrivalent frequency in induced autotetraploid lines of Triticum longissimum. Can. J. Genet. Cytol. 18: 357-364. Garcia, V. A. 1991. Cytogenetical studies in Rhoeo spathacea. (Commelinaceae). I. A. desynaptic and second division resti- tution mutant. Genome 34: 895-899. - 1995. Cytologenetical studies in Rhoeo spathacea (Commelinaceae). II Characterization of an acrotrisomic plant. Cy- tologia 60: 319-327. Hunt, D. R. 1993. The commelinaceae of mexico. In: Biological Diversity of Mexico. Origins and Distribution. T. P. Ra- manoothy, R. Bye, A. Lot and J. Fa (Eds.). Oxford University Press. 421-437 pp. Jackson, R. C. and Casey, J. 1980. Cytogenetics of polyploids. In: W Lewis (ed.): Polyploidy Biological Relevance 17-44 pp. Pleum Press Corp. N. Y. - and - 1982. Cytologenetic analysis of autopolyploids: models and methods for triploids to octaploids. Am. J. Bot. 69: 487-501. Jauhar, P. P. 1977. Genetic regulation of diploid-like chromosome pairing in Avena. Theor. Appl. Genet. 49: 287-295. - 1993. Cytogenetics of the Festuca-Lolium Complex, Springer-Verlag. Chap. 4: 43-56 pp. Jones, K. 1974. Chromosome evolution by Robertsonian translocation in Gibasis (Commelinaceae). Chromosoma (Berl.) 45: 353-368. - 1976. Multiple Robertsonium functions in the evolution of a plant genes. In: Current Chromosome Research. K. Jones and P. E. Bradham (Eds.). 220-221 pp. - 1977. The role of Robertsonian change in karyotype evolution in higher plants. Chromosomes Today 6: 121-129. - 1978. Aspects of chromosome evolution in plants. Advance Bot. Res. 6: 120-194. - and Kenton, A. 1984. Mechanisms of chromosome change in the evolution of the tribe tradescantieae (Commelinacea). In Sharma, A. and Sharma, K. (Eds.). Chromosomes in Evolution of Eukaryotic Groups C.R.C. Press. Florida 143-168 pp. Kenton, A. 1981. Chromosome evolutions in the Gibasis linearis alliance (Commelinaceae). I. The Robertsonian differentia- tion of G. venustula and G. speciosa. Chromosoma 84: 291-304. - and Drakeford, A. 1990. Genome size and karyotype evolution and Tradescantia section Cybispatha (Commelinacea). Genome 33: 604-610. Martinez, A. J. 1978. Chromosome relationships in the Tradescantia crassifolia alliance. Ph. D. Thesis. University of Read- ing. Mehra, K. L., Sreenath, P. R. and Faruqi, S. A. 1971. Distribution of multivalents and genotypic control of chromosome pairing in Setcreasea species and hybrids. Cytologia 36: 309-320. Okamoto, M. 1957. A synaptic effect of chromosome V. Wheat Inf. Service. Kyoto Univ. 5: 6. Owens, S. J. 1981. Self-incompatibility in the Commelinacea. Ann. Bot. 47: 567-581. Reyes-Valdes, M. H., Ji, Y., Crane, C. E, Taylor, J. F., Islam-Faridi, M. N. H., Price, J. and Stelly, D. M. 1996. ISH Facilitat- ed analysis of meiotic bivalent pairing. Genome 39: 784-792. Riley, R. and Chapman, V. 1958. Genetic control of the cytogenetically diploid behavior of hexaploid wheat. Nature 182: 713-715. Stebbins, G. L. 1971. Chromosomal Evolution in Higher Plants. Edward Arnold, London. Sybenga, J. 1975. Meiotic Configuration. Springer-Verlag, Berlin, Heidelberg. N. Y. - 1996. Chromosome pairing affinity and quadrivalent formation in polyploids: do segmental allopolyploids exist. Genome 39: 1176-1184.