T. cuneiformis (2n=8x=40) and T. oriental is (2n =6x=30) (Turneraceae)

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1 2000 The Japan Mendel Society Cytologia 65: ,2000 Cytogenetic Relationships between Turnera aurelii, T. cuneiformis (2n=8x=40) and T. oriental is (2n =6x=30) (Turneraceae) Aveliano Fernandez 1 * and Maria M. Arbo 2 1 Facultad de Ciencias Exactas y Naturales y Agrimensura (UNNE) 2 Facultad de Ciencias Agrarias (UNNE) Miembros de la Carrera del Investigador Cientifico (CONICET). lnstituto de Bot<inica del Nordeste, C.C. 209, 3400-Corrientes, Argentina Accepted December 6, 1999 Summary We hybridized 2 octoploid species (2n = 8x =40) T. aurelii and T. cuneiformis and one hexaploid species (2n=6x=30), T. orientalis. One octoploid (2n=8x=40) and 2 heptaploid (2n= 7x=35) hybrids were obtained. The hybrids were studied cytologically to determine their genomic relationships. Meiotic behavior in the T. aurelii X T. cuneiform is hybrid was very irregular, with numerous laggard chromosomes and bridges in AI and All. Mean pairing relationships were I 0.10 I, , 0.53 Ill and 0.13 IV. All cells of T. aureliix T. orientalis had 5 I , while T. cuneiformis X T. orientalis presented irregular meiosis, with mean pairing relationships of 4.86 I, II and 0.13 Ill. The genome constitution oft. aurelii is A"AaAo A and oft. orientalis is A o A On the basis of the chromosome associations in the hybrids, we propose the genomic formula A cu A cu A o A for T. cuneiformis. The 3 genomes of T. orientalis are similar to 3 of the 4 genomes oft. aurelii and T. cuneiformis. The 2 latter species show also 3 common genomes and the fourth one is homoeologous. The genus Turnera has 9 series (Urban 1883) with 3 basic chromosome numbers, x=5, x=7 and x = 13. The series Turn era ( = Canaligerae) is the only one with x = 5 and presents species with different ploidy levels, ranging from diploids to octoploids (Fernandez 1987). Since 1982 a controlled crossing program is being carried out and several interspecific hybrids were obtained. Cytogenetic studies of the hybrids between the yellow flowered diploid species, showed that all these species have homoeologous genomes (Fernandez and Arbo 1989). The hybrids between white-blue flowered diploid species, indicated that all the species also have homoeologous genomes. However, the genomes of the yellow flowered are different from the genomes of the white-blue flowered species (Fernandez and Arbo 1996). Hybrids between polyploid and diploid species were also studied, with the result that the polyploid species are segmental allopolyploids (Fernandez 1997, Fernandez and Arbo 1990, 1993a, b). The principal purpose of this work is to determine the genome relationships between T. aurelii, T. cuneiformis, 2n=8x=40 and T. orientalis, 2n=6x=30, through a cytogenetic analysis of their hybrids. Materials and methods The progenitors are indicated with the label used in the greenhouse. Voucher specimens are deposited at the herbaria of the Instituto de Botanica del Nordeste (CTES). The origin of the progenitors is: * Corresponding author, ibone@espacio.com.ar

2 Avcliano Fernandez and Maria M. Arbo Cytologia 65 T aurelii (Gr8). Schinini Paraguay, Dept. Cordillera, rio Salado. T aurelii (Gr9). Arbo Paraguay, Dept. Cordillera, rio Salado. T cuneiformis (04). Krapovickas Brazil, MG, 47 km SW ofpirapora. T cuneiformis (06). Arbo Brazil MG, Santana do Riacho, on the roadside. T orientalis (0). Arbol538. Argentina, Corrientes. T orientalis (02). Cabral 358. Argentina, Misiones, Teyu Cuare. The crossing procedure used is described in Arbo and Fernandez (1987). All crosses were made in greenhouse as follows: flower castration of the maternal plants, pollination with anthers of the plant selected as paternal, and labeling the maternal flowers indicating the pollen donor. The seeds were sowed in individual pots and the hybrids were transplanted and raised in greenhouse after developing few leaves. The preparations for meiotic study were made by fixing the floral buds with 5 parts of absolute ethanol and I part of lactic acid (Fernandez 1973) for 24 h, and stored in 70% ethanol. Pollen mother cells (PMCs) were stained with the Feulgen. For mitotic preparations the same procedure was followed, with a pretreatment of the roots with M 8-hydroxyquinoline during 3 hat room temperature (between C). Pollen stainability, an estimate of viability, was determined for each hybrid using carmin-glycerine (I : I). At least 300 grains per flower were counted. Results and discussion Meiotic behaviour of the progenitors was normal, T aurelii and T cuneiformis always had 20 II and T orientalis 15 II. In the T aurelii (Gr8)X T orientalis (0) hybrid, 2n =7x =35, the only chromosome association at metaphase I was 5 I+ 15 II in 42 PMCs analyzed (Fernandez and Arbo 1993a). Meanwhile the T orientalis (02)XT cuneiformis (04) hybrid, 2n=7x=35, showed II (Fig. la) in 92% of 38 PMCs analysed; cells with I to 3 Ill were also observed. The mean meiotic pairing relationships of this hybrid were: 4.86 univalents, bivalents and 0.13 trivalents (Table 1 ). When the number of trivalents per cell increases, the univalents and bivalents decreases in the same proportion. This fact indicates that the trivalents were formed by autosyndetic and allosyndetic pairing at the same time. In the hybrid T aureliix T orientalis, a complete genome oft aurelii was always without pairing in metaphase I, while in T orientalis X T cuneiform is some chromosomes of the fourth genome oft cuneiformis were paired forming trivalents. This fact would indicate that the fourth genome of T aurelii is more differentiated than that oft cuneiformis with regard to the T orientalis genome. T orientalis has a karyotype composed of 26 metacentric and 4 submetacentric chromosomes. T aurelii and T cuneiformis possess similar karyotypic formula, with 36 metacentrics and 4 submetacentrics, but the chromosomes of T aurelii are larger than those oft cuneiformis (Solis Neffa and Fernandez 1993). The 5 univalents observed in the T aureliix T orientalis hybrid are large, which suggests that they correspond to the largest chromosomes of the T aurelii genome. The hybrid T aurelii (Gr9)XT cuneiformis (06), 2n=8x=40, showed 11 different configurations in the 22 PMCs analyzed. The most frequent ones were 12 I+ 14 II and 10 I+ 15 II (Fig.!b), Table l. Meiotic chromosome pairing, average, ±SE, range and pollen fertility in Turnera hybrids Hybrid 2n 11 Ill IV PMC Fert.% T aureliix T orientalis T orientalis X T cuneifijrmis ± ± ± T aureliix T cuneijijrmis ± ± ± ± (

3 2000 Cytogenetic Relationships between 3 Species of Turnera 99 Fig. l. Meiotic chromosomes in Turnera hybrids. a) Metaphase I with 5 l + 10 I! in T orientalisx T cuneiformis. b) Metaphase I with 10! in T aureliixt cunei/armis. c) Metaphase I with 5!+8 I!+ 1 IV+ 1 XV in T aureliix T cuneiform is. Arrow indicates heteromorphic bivalent and the arrowhead shows XV. d) Anaphase I with laggard chromosomes and bridges in T aureliixt cuneijijrmis. e) Prophase I!, united nuclei, both nuclei are surrounded by one nuclear membrane in T aureliix T cuneiformis. The cell have micronuclei. Scale= l 0 J..lm. which were observed in 31.81% and 18.18% respectively. These configurations show that both species have 3 common genomes and another homoeologous one. The mean meiotic pairing relationships of this hybrid were: univalents, bivalents, 0.19 trivalents and 0.09 quadrivalents (Table 1 ). An heteromorphic bivalent was observed in all the cells at metaphase I. The largest chromosome of this bivalent probably belongs to T aurelii (Fig. 1 c). In another cell an association of 15 chromosomes was observed (XV). This association has been probably formed by multiple translocations among 4 or even more chromosomes (Fig. le). In this hybrid, the main cause oftrivalents and quadrivalents formation would be the autosyndetic and allosyndetic pairing at the same time. Although, trivalent and quadrivalent formation could be also a consequence of translocations. In T aurelii (Gr9)X T cuneiformis (06) a total of 48 PMCs were analyzed in anaphase I, 6.25% of them had only bridges and 81.25% showed laggard chromosomes, 58.33% of which were also accompanied by bridges (Fig. Id). The reciprocal 06XGr9 hybrid showed laggard chromosomes in 78.92% of the 38 PMCs analyzed, 28.94% of which were accompanied by bridges. In Gr9X06 and its reciprocal cross 06XGr9 were analyzed 34 PMCs and 35 PMCs at prophase II respectively. In both hybrids the nuclei were connected by remnant bridges. This phenomenon was observed in 11.76% of the 34 cells in the Gr9X06 cross and 5.7% of the reciprocal cross. In these cells, the nuclei were surrounded by a unique nuclear membrane (Fig. le) and would probably form dyads, as final product of the meiosis. In anaphase II laggard chromosomes were observed in all the studied cells of both hybrids.

4 100 Aveliano Fermindez and Maria M. Arbo Cytologia 65 Fig. 2. Meiotic chromosomes in T aureliix T cune!fhrmis. a) Anaphase 11 with laggard chromosomes and bridges. Two nuclei are joined in one pole. b) Triad which results from the cell observed in a. c) Anaphase 11, with laggard chromosomes and bridges. Two nuclei are joined in both poles. d) Dyad wich results of the cell observed in c. Dyads may also be originated from the cell observed in Fig. le. Scale=lO,um. The laggard chromosomes observed in anaphase I, would be those that were observed as univalents in metaphase I. Apparently most of them were incorporated into the telophase I nuclei, because the number of micronuclei was low compared with the number of laggard chromosomes in metaphase I. The laggard chromosomes observed in anaphase 11, however, would be the univalents that divided equationally in anaphase I. Another interesting fact is the spindle orientation in anaphase 11 and telophase I!. In some cells two telophasic nuclei joined at one pole (Fig. 2a), while in other cells the telophasic nuclei joined at both poles (Fig. 2c), forming triads (Fig. 2b) and dyads (Fig. 2d) respectively. In 496 PMCs observed, there were 1.62% monads ( 4n = 16x = 80), 49.59% dyads, which indicate the formation of unreduced gametes (2n=8x=40). The dyads can or can not be accompanied by micromicrospores, but when they are present the number goes from one to four. Triads were observed in 1% of the cells, which have a non-reduced gamete and two reduced ones. Tetrads appeared in 47.79% of the cells, most of these are tetrahedral and in very low frequency isobilateral (Fig. 3). Probably, the few viable pollen grains produced by the hybrids are non-reduced with 40 chromosomes. The formation of non-reduced gametes would have a fundamental role in the polyploidization (Harlan and de Wet 1975). Asker (1980) considered also that this type of gametes is one of the principal mechanism for the formation of polyploid series. Non-reduced gametes has been observed previously in Turnera hybrids (Fermindez 1997). Besides, some interspecific hybrids were produced by non-reduced gametes (Fermindez and Arbo 1990). This fact indicates that un-reduced gametes have probably played an important role in the origin of poiyploids of Turnera. The pollen fertility was low in all the hybrids. In T. aurelii (Gr9)X T. orientalis (0) it was of

5 2000 Cytogenetic Relationships between 3 Species of Turn era 101 Monads Dyads Triads Tetrads (@00 Cells I % 1.6I I 0.2 I I Fig. 3. Products of a single meiotic cycle oft aureliix T cuneiformis. The small circles represent micromicrospores. 1.4%, in T orientalis (02)X T cuneiformis (04) it was of 4.44%, in T aurelii (Gr9)X T cuneiformis (06) it was of 15.29% and in T cuneiformis (06)XT aurelii (Gr9) it was of 16.41%. These low pollen fertilities result from the irregular meiotic behaviour of these hybrids. T orientalis is probably one of the progenitors of T aurelii (Fernandez and Arbo 1993a) and from the cytogenetic analysis carried out in this work, it raises the possibility that it is also a progenitor of T cuneiformis. The geographical distribution of T orientalis includes the areas oft aurelii and T cuneiformis, fact which supports the hypothesis that T orientalis is the common progenitor oft aurelii and T cuneiformis. The other progenitor oft aurelii and oft cuneiformis would be a diploid species. These diploids should be different for each species, however they should have homoeologous genomes, according to the cytogenetic results found in these hybrids. In conclusion, the cytogenetic analysis of the hybrids indicates that the 3 genomes of T orientalis are similar to 3 of the 4 genomes of T aurelii and T cuneiformis. Between these last two species there are also 3 common genomes and a fourth is homoeologous. We have previously proposed the genomic constitution A"A 0 BBB 0 B 0 for T orientalis and AaAaA 0 A 0 BBB 0 B 0 for T aurelii (Fermindez and Arbo 1993a). According to this formula and the results presented here the genomic constitution AcuAc"A 0 A 0 BBB 0 B 0 is proposed for T cuneiformis. The chromosome associations and configurations found in the hybrids among these species support the genomic formulas proposed above. Acknowledgements This work has been supported by grants of the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) of Argentina and the Secretaria General de Ciencia y Tecnica of the Universidad Nacional del Nordeste. References Arbo, M. M. and Fcrnandez, A Cruzamientos intra e interespecificos en Turnera, serie Canaligerae. Bonplandia 6(1): Asker, S Gametophytic apomixis. Elements and genetic regulation. Hereditas 93: Fermindez, A El acido lactico como fijador cromos6mico. Bol. Soc. Argent. Bot. 15(2-3): Estudios cromos6micos en Tun1era y Piriqueta (Turneraceae ). Bonplandia 6( l ): l Estudio citogenetico en hibridos entre una espccie octoploide, Turnera aurelii y dos diploides, T caerulea y T joelii. Bonplandia 9(3--4): and Arbo, M. M Relaciones gcn6micas entre cuatro espccies diploides de Turnera con flores amarillas (Serie Canaligerae). Bonplandia 6(2):

6 102 Aveliano Fernandez and M aria M. Arbo Cytologia 65 -and Gametas no reducidas y rclaciones genomicas en tres especies de Turnera (Turncraceae). Darwiniana 30(1-4): and a. Relaciones gen6micas entre seis especics de Turnera (Serie Canaligerae) del Paraguay. Candollca 48: and- 1993b. Citogenetica de hibridos entre Turnera grandidentata (4x) y T subulata y T scabra (2x) (Turneraceae). Bonplandia 7(1-4 ): and Relaciones gen6micas entre las especies diploides de ftores blanco-azuladas de Turnera (Scric Canaligerae). Bonplandia 9(1-2): Harlan, J. R. and de Wet, J. M. J On b. Winge and a prayer: the origins of polyploidy. Bot. Rev. 41(4): Solis Neffa, V G. and Fernandez, A Estudios cromos6micos en especies de Turnera (Turncraceae). Bonplandia 7(1-4): Urban, I Monographic dcr familie der Turneraceen. Jahrb. Kiinigl. Bot. Gart. Berlin 2: