Cytologia 68(2): 147–152, 2003

Chromosome Numbers in Brazilian and Argentine Populations of Pfaffia glomerata (Amaranthaceae)

Onildes Maria Taschetto1 and Maria Suely Pagliarini2,*

1 Department of Biology, State University of Western Paraná (UNIOESTE), 85.814-110, Cascavel PR Brazil 2 Department of Cell Biology and Genetics, State University of Maringá, 87.020-900, Maringá PR Brazil

Received February 12, 2003; accepted February 26, 2003

Summary Somatic chromosome numbers were determined in 10 populations of Pfaffia glomerata, also known as “Brazilian ginseng”, collected from different regions of Brazil and Argentina. Nine populations showed 2nϭ34 and one 2nϭ32, 33. Although chromosomes were very small, a pair of satellited chromosomes per karyotype was reported in almost all the populations. Chromosomes were predominantly metacentric and submetacentric. Meristematic interphase nuclei showed areticulate chromatin structure. Prophase chromosomes had deeply stained proximal blocks of condensed chromatin. This is the first cytological report including chromosome counting for the genus Pfaffia, a genus of the Amaranthaceae family with several species of economic importance due to their high content of ecdysteroid glycosides. The latter is a substance similar to that found in the genus Panax, “the Korean ginseng”, widely used as medicine. Key words Pfaffia glomerata, Brazilian ginseng, Chromosome number, Chromatin.

The genus Pfaffia (Amaranthaceae) comprises about 90 species distributed throughout Central and South America. In Brazil, 27 species have been described to date (Taniguchi et al. 1997). Pfaf- fia paniculata (Oliveira 1986, Nishimoto et al. 1986, 1987, Teràn 1990), P. stenophylla (Oliveira 1986, Teràn 1990), P. iresinoides (Nishimoto et al. 1986, 1987, Shiobara et al. 1992, Taniguchi et al. 1997), P. glomerata (Nicoloso et al. 1999), and P. tuberosa (Nishimoto et al. 1986, Siqueira et al. 1993) are widely used in popular medicine. The use of these plants went beyond the regional indigenous populations (Arenas and Azorero 1997, Oliveira 1986) since these transferred their knowledge to the local rural people. Owing to the plant’s properties being concentrated in its roots, it has been commonly employed “for all diseases”. Recently, due to some chemical compounds found in plants of the genus Pfaffia, mainly ecdysteroid glycosides (Nishimoto et al. 1987, 1988), very similar to those in the genus Panax (Korean ginseng), certain species have been popularly called “Brazilian ginseng”. In the folk medicine, the Brazilian ginseng has been used for the treatment of rheumatic diseases, for antidiabetic purposes and inflammatory processes (Taniguchi et al. 1997, Nicoloso et al. 1999). It has also been widely used as a ginseng-like tonic herb against fatigue and stress (Teràn 1990, Nicoloso et al. 1999) and as an aphrodisiac tonic (Teràn 1990, Taniguchi et al. 1997). In the southern region of Brazil, mainly in the area of the floodplain of the Paraná river basin, Pfaffia glomerata is very abundant. However, extensive extraction activities are compromising the wild resources (Agostinho and Zalewski 1996). In spite of their economic and medicinal interest, few studies have been developed on some Pfaffia species. Physicochemical properties (Nishimoto et al. 1984, 1987, 1988, Shiobara et al. 1992, 1993), vegetative propagation methods (Nicoloso et al. 1999), leaf ultrastrutucture characteristics (Estelita-Teixeira and Handro 1984), leaf anatomy (Han- dro, 1964), and medicinal properties (Taniguchi et al. 1997) have been reported. Moreover, there is some confusion in genus Pfaffia systematics. To date, P. iresinoides is being recognized as P. glom-

* Corresponding author. e-mail: [email protected] 148 Onildes Maria Taschetto and Maria Suely Pagliarini Cytologia 68(2) erata (Smith and Downs 1972). Cytological studies, including chromosome number counting and evaluation of the meiotic behavior have never been undertaken. Thus, for the ﬁrst time, we are re- porting the chromosome numbers for 10 populations of P. glomerata collected in different regions of Brazil and Argentina as an initial contribution to the chromosomal knowledge of the genus Pfaf- ﬁa.

Materials and methods Plants of 10 populations of P. glomerata were collected in different sites of Brazil and Argenti- na where they occur wide (Fig. 1). Origin of populations is shown in Table 1. Voucher specimens are deposited in the Herbarium of the Pontifícia Universidade Católica do Rio de Janeiro, Brazil. Fig. 2 illustrates some morphological aspects of P. glomerata, including its characteristic leg-like roots. Plants were kindly identified by Dr Josafá Carlos de Siqueira from the Pontificia Universi- dade Católica do Rio de Janeiro. Plants were maintained at the Medicinal Herb Garden of the State University of Maringá. Shoots from the basis and middle positions were put in water for rooting. Root tips, 1 or 2 cm in length, were pretreated for 3 h in a solution of 8-hidroxiquinoleine 0.002 M at 9°C, fixed in Carnoy’s solution (3: 1, ethanol: glacial acetic acid) and stored in 70% alcohol. Hydrolysis was done in 5 N HCl, for 25 min, at room temperature. Standard chromosome preparations were done according to Feulgen technique (Schiff’s reagent). Photomicrographs were taken with Kodak Im- agelink-HQ, ISO 25, black and white film.

Results and discussion Among the 10 populations analyzed, 9 presented 2nϭ34 chromosomes (Table 1). All

Fig. 1. Geographical distribution of the 10 populations of P. glomerata from Brazil and Argentina. 1. Baitaporã, 2. Itaquiraí, 3. Eldorado, 4. Ilha São Francisco (Guaíra), 5. Ilha do Alemão (Guaíra), 6. Porto Rico, 7. Maringá, 8. Fig. 2. Morphological aspects of P. glomerata (scaleϭ San Ignácio, 9. Posadas, 10. Governador Virasoro. 30 cm). 2003 Chromosome Numbers in Brazilian and Argentine Pfafﬁa glomerata 149

Table 1. Collection sites of P. glomerata populations, voucher number (PUC/RJ), and chromosome numbers (2n)

Country Region Collection site 2n chromosomes

Brazil State of Mato Grosso do Sul Baitaporã 34 Itaquiraí 34 Eldorado 34 State of Paraná Guaíra-Ilha São Francisco 34 Guaíra-Ilha do Alemão 32, 33 Porto Rico 34 Maringá 34 Argentina Province of Missiones San Ignácio 34 Posadas 34 Province of Corrientes Governador Virasoro 34

chromosome counting in the population of Ilha São Francisco (Guaíra municipality) had 2nϭ32 and 33 chromosomes, with a predominance for the latter number. On the other island (Ilha São Francisco), with similar environmental conditions, no changes have been observed in the chromosome number predominantly found in the other populations. Chromosome rearrangements, i.e., inversions, translocations and other events, such as segment transpositions, duplications, and deletions, constitute the raw material for karyotype repatterning during speciation, which finally leads to a homozygous restructured karyotype in the new reproduc- tive community. According to Greilhuber and Ehrendorfer (1988), chromosome number changes in karyotype with monocentric chromosomes, as reported in the Pfaffia glomerata populations (Fig. 3), require changes in centromere number without significant loss, and with only tolerable duplications in coding chromosome parts. This kind of chromosomal rearrangement, known as dysploidy, may occur by centromeric fission or fusion (Robertsonian translocations). In centromeric fission, a 2-armed chromosome breaks in the centromere to give 2 telocentric chromosomes, while in centromeric fusion, a telocentric or acrocentric chromosome is translocated into another chromosome of this type, so that a 2-armed chromosome results. Although karyograms were not undertaken for the present populations of P. glomerata, owing to the quality of karyotypes obtained, reports from many cells of all populations reveal that chromosomes are metacentric and submetacentric. Telo- centric or acrocentric chromosomes that facilitate centromeric fusion were never found (Fig. 3). The karyotype 2nϭ32 and 33 found in the Ilha do Alemão population (Fig. 3e) clearly shows metacentric and submetacentric chromosomes. Robertsonian systems, frequent in animals, have been described in a few plant groups, mainly in Commelinaceae (Jones 1977). The cause of chromosome variation in the Ilha do Alemão population of P. glomerata remains unknown, and more studies are necessary to elucidate it. Despite its economic interest, the Amaranthaceae family is cytologically not well character- ized, while genus Amaranthus is the best studied (Federov 1969, Goldblatt 1981). The predominant base number in the family, including the genus Amaranthus, is xϭ16 and 17, as reported in P. glomerata. Pal et al. (1982) suggested that the base number xϭ17 originated from xϭ16 through primary trisomy. Based on the cytogenetic analysis of interspecific hybrids, Greizerstein and Poggio (1992) support this hypothesis and suggest that the species with 2nϭ32 are polyploid (base number ϭ ϭ ϭ x 8) and that x1 16 is a derived base number. The base number x2 17 would have appeared later by primary trisomy. In Amaranthus hypochondriacus, xϭ16 and xϭ17 have been reported for the same species (Greizerstein and Poggio 1994), and this cytological feature could predict segregation- al sterility observed in some intra- and inter-specific hybrids. One pair of chromosomes with a satellite was found in the karyotype of all populations (Fig. 3). Since chromosomes were very small, it was not easy to identify the chromosome with the sec- 150 Onildes Maria Taschetto and Maria Suely Pagliarini Cytologia 68(2)

Fig. 3. C metaphases of P. glomerata populations. a) Baitaporã, 2nϭ34, b) Itaquiraí, 2nϭ34, c) Eldo- rado, 2nϭ34, d) Ilha São Francisco (Guaíra), 2nϭ34, e) Ilha do Alemão (Guaíra), 2nϭ33, f) Porto Rico, 2nϭ34; g). Maringá, 2nϭ34, h) San Ignácio, 2nϭ34, i) Posadas, 2nϭ34, j) Governador Virasoro, 2nϭ34. Arrows point out sattelites. Barsϭ0.5 µm.

Fig. 4. Meristematic nuclei of P. glomerata. a) Interphase nucleus showing areticulate chromatin structure. b) Prophase nucleus showing deeply stained proximal blocks of condensed chromatin (arrows). ondary constriction in all cells studied. One nucleolus was observed in microsporocytes in prophase I. These data are in agreement with those reported by Greizerstein and Poggio (1994) for some Amaranthus species with 2nϭ32 and 34. 2003 Chromosome Numbers in Brazilian and Argentine Pfafﬁa glomerata 151

In meristematic interphase nuclei the chromatin structure was areticulate in all analyzed populations of P. glomerata (Fig. 4a). The chromatin reticulum was very small and slightly stained. Areticulate nuclei are related mainly to karyotypes with small chromosomes and low DNA content (Barlow 1977), as in the plants under analysis, and are commonly associated with phylogenetically more advanced (Guerra 1987) or more specialized taxa (Barlow 1977). Prophase chromosomes in all populations showed deeply stained proximal blocks of condensed chromatin, allowing an estimate of the chromosome number (Fig. 4b). The presence of high amounts of condensed chromatin as proximal blocks at prophase could be an important feature in the evolution of the genus. Cytogenetic studies on plants have been employed, among other things, to establish genomic relationships in biosystematic research and as an aid in applied crop improvement programs. This is the first report of chromosome counting in the genus Pfaffia, which up to the present has been only poorly studied. Because of its medicinal properties Pfaffia glomerata is highly explored in our region by several commercial groups that extract its active substances for world wide commercializa- tion. Certain populations close to the Paraná river basin are disappearing and are now under govern- mental care. More studies are necessary to better characterize these populations.

Acknowledgements The authors wish to thank Dr Josafá Carlos Siqueira, Pontifícia Universidade Católica do Rio de Janeiro, Brazil, for taxonomical determinations; to Dr. Maude Nancy Joslin Motta, Manager of the Parque Nacional de Ilha Grande (IBAMA) for permitting plant collection in the reserve areas; to Dr. Paulo Renato Taschetto for his collaboration in collection of material and its maintenance.

References

Agostinho, A. A. and Zalewski, M. 1996. A planície alagável do alto rio Paraná; importância e preservação. Eduem, Mar- ingá, PR. Arenas, P. and Azorero, M. R. 1977. Plant used as means of abortion, contraception, sterilization and fecundation by Paraguayan indigenous people. Econ. Bot. 31: 302–306. Barlow, O. 1977. Determinants of nuclear chromatin structure in angiosperms. Ann. Sci. Nat. Bot. (12 Sér) 18: 183–206. Estelita-Teixeira, M. G. and Handro, W. 1984. Leaf ultrastructure in species of Gomphrena and Pfaffia (Amaranthaceae). Can. J. Bot. 62: 812–817. Federov, A. 1969. Chromosome Number of Flowering Plants. V. L. Komarov Botanica Institute. Leningrad: Academy of Sciences of the USSR, pp. 928. Goldblatt, P. 1981. Index to Plant Chromosome Numbers 1975–1978. Missouri Botanical Garden. Monographs in Systemat- ic Botany, Vol. 5, USA, pp. 553. Greilhuber, J. and Ehrendorfer, F. 1988. Karyological approaches to plant taxonomy. ISI Atlas Sci: Plants & Animals 1: 289–297. Greizerstein, E. J. and Poggio, L. 1992. Estudios citogenéticos en seis híbridos del género Amaranthus (Amaranthaceae). Darwiniana 31: 159–165. — and — 1994. Karyological studies in grain Amaranthus. Cytologia 59: 25–30. Guerra, M. 1987. Cytogenetics of Rutaceae. IV. Structure and systematic significance of interphase nuclei. Cytologia 52: 213–222. Handro, W. 1964. Contribuição ao estudo da venação e anatomia foliar das Amarantáceas dos cerrados. Anais Acad. Bras. Cienc. 36: 479–499. Jones, K. 1977. The role of Robertsonian change in karyotype, evolution in higher plants. Chromos. Today 6: 121–129. Nicoloso, F. T., Fortunato, R. P. and Fogaça, M. A. F. 1999. Shoot cutting position effect on rooting of Pfaffia glomerata (Spreng) Pederson in two substrates. Ciência Rural 29: 277–283. Nishimoto, N., Nakai, S. and Takagi, N. 1984. Pfaffosides and nortriterpenoid saponins from Pfaffia paniculata. Phyto- chemistry 23: 139–142. —, Shiobara, Y., Inoue, S. S., Fujino, M., Takemoto, T., Yeoh, C. L. and Hashimoto, G. 1986. Ecdysterone from Pfaffia tuberosa (Spreng) Hicken. Rev. Bras. Farmacog. 1: 188–191. 152 Onildes Maria Taschetto and Maria Suely Pagliarini Cytologia 68(2)

—, —, Fujino, M., Ioune, S. S., Takemoto, T., Oliveira, F., Akiane, G., Akisue, M. K., Hashimoto, G., Tanaka, O., Kasai, R. and Matsura, H. 1987. Ecdysteroids from Pfaffia iresinoides and reassignment of some 13CNMR chemical shifts. Phytochemistry 26: 2505–2507. —, —, and Inoue, S. S. 1988. Three ecdysteroid glycosides from Pfaffia iresinoides. Phytochemistry 27: 1665–1668. Oliveira, F. 1986. Pfaffia paniculata (Martins) Kuntze—The Brazilian ginseng. Rev. Bras. Farmacog. 1: 86–92. Pal, M., Pandley, R. M. and Khoshoo, T. M. 1982. Evolution and improvements of cultivated Amaranthus. IX Cytogenetic relationships between the two basic chromosome numbers. J. Hered. 73: 353–356. Shiobara, Y., Inoue, S. S., Nishiguchi, Y., Kato, K., Takemoto, T. and Nishimoto, G. 1992. Iresinosid, a yellow pigment from Pfaffia iresinoides. Phytochemistry 31: 953–956. —, —, Kato, K., Nishiguchi, Y., Oishi, Y., Nishimoto, N., Oliveira, F., Akisue, G., Akisue, M. K. and Hashimoto, G. 1993. A nortriterpenoid, triterpenoids and ecdysteroids from Pfaffia glomerata. Phytochemistry 32: 1527–1530. Siqueira, V. L., Lopes, A. M. V., Akisue, M. K., Akisue, G. and Paim, A. C. 1993. Anatomic and morphological characteristics of the subterranean system of Pfaffia tuberosa (Spreng) Hicken (Amaranthaceae). LECTA-USF 11: 29–42. Smith, L. B. and Downs. R. J. 1972. Amarantáceas. In: Reitz, P. R. (ed.). Flora Ilustrada Catarinense. Itajaí, Santa Catarina. Taniguchi, S. F., Bersani-Amado, C. A., Sudo, L. S., Assef, S. M. C. and Oga, S. 1997. Effect of Pfaffia iresinoides on the experimental inflammatory process in rats. Phytother. Res. 11: 568–571. Teràn, E. 1990. Plantas de interesse da cosmetologia. Cosmetics & Toiletries (Port.). 2: 18–23.