Chromosome numbers in Bromeliaceae

The present study reports chromosome numbers of 17 species of Bromeliaceae, belonging to the genera Encholirium, Bromelia, Orthophytum, Hohenbergia, Billbergia, Neoglaziovia, Aechmea, Cryptanthus and Ananas. Most species present 2n = 50, however, Bromelia laciniosa, Orthophytum burle-marxii and O. maracasense are polyploids with 2n = 150, 2n = 100 and 2n = 150, respectively, while for Cryptanthus bahianu s, 2n = 34 + 1-4B. B chromosomes were observed in Bromelia plumieri and Hohenbergia aff. utriculosa. The chromosome number of all species was determined for the first time, except for Billbergia chlorosticta and Cryptanthus bahianus. Our data supports the hypothesis of a basic number of x = 25 for the Bromeliaceae family and decreasing aneuploidy in the genus Cryptanthus. Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal da Bahia, Campus Universitário de Ondina, 40170-290 Salvador, BA, Brasil. Send correspondence to A.L.P.C.O. E-mail: ancotias@ufba.br Departamento de Botânica, Instituto de Biologia, Universidade Federal da Bahia, Salvador, BA, Brasil. 174 Cotias-de-Oliveira et al. the Bromelia species did not reach 0.3 μm. Variation in chromosome size appeared within the same karyotype. Bromelia and Billbergia species were more uniform, while Encholirium spectabile, Neoglaziovia variegata and the Aechmea species presented continuous variation from largest to smallest chromosome, but without the clear expression of bimodality, observed by Marchant (1967) in the more advanced Bromelioideae and Tillandsioideae species. Chromosome counts in the subfamily Pitcairnioideae have been published for Brocchinia species (in Goldblat and Johnson, 1993), as well as Deuterocohnia, Dyckia, Fosterella, Hechtia, Pitcairnia, Lindmania and Puya (Marchant, 1967; Sharma and Ghosh, 1971; Brown et al., 1984; Varadarajan and Brown, 1985; Brown and Gilmartin, 1986, 1989). These counts showed n = 25, 2n = 50 in about 60 species analyzed and in only two, 2n = 100 (McWilliams, 1974; Brown et al., 1984; Brown and Gilmartin, 1989; Marchant, 1967; Sharma and Ghosh, 1971). Encholirium spectabile with 2n = 50 represents the first count for the genus. The chromosomes varying from 0.53-1.07 μm are among the largest observed in this study (Figure 1a). Chromosome data are available for the subfamily Bromelioideae for species from the genera Acanthostachys, Aechmea, Ananas, Araeococcus, Billbergia, Bromelia, Canistrum, Cryptanthus, Neoregelia, Nidularium, Portea, Pseudananas, Quesnelia, Streptocalyx nd Wittrockia. The most common number is 2n = 50, although some variations, e.g., 2n = 54, 48, 96, have been found (McWilliams, 1974; Brown et al., 1984; Brown and Gilmartin, 1989; Marchant, 1967; Sharma and Ghosh, 1971). The Aechmea species had 2n = 50 and chromosomes varying from 0.36-0.86 μm in A. aquilega, to 0.46-0.80 μm in A. blanchetiana and 0.46-1.03 μm in A. conifera. The other species in the genus show a certain uniformity in the number n = 25 (Marchant, 1967; Brown and Gilmartin, 1989) and 2n = 50 (Lindschau, 1933, in McWilliams, 1974). The only exception reported is 2n = 54 for A. ornata (Lindschau, 1933, in McWilliams, 1974) and n = 21 for A. tillandsioides (Marchant, 1967). Ananas lucidus with 2n = 50 had more uniform chromosomes, varying from 0.67-0.89 μm (Figure 1e). Ananas also had a certain uniformity of 2n = 50 in the five species already analyzed, but triploids have been previously recorded in A. comosus and tetraploids in A. ananassoides (Lin et al., 1987). The two Billbergia species analyzed showed 2n = 50. B. morelii had small chromosomes, 0.23-0.30 μm, while B. chlorosticta had large ones, 0.86-1.07 μm (Figure 1f, g). The count of 2n = 50 for B. chlorosticta differs from the first report of 2n = 54 by Brown and Gilmartin (1986). Neoglaziovia is a genus with only three species, of which N. variegata had 2n = 100 with chromosome size varying from 0.40-0.93 μm, (Figure 1h). No chromosome data exist for the remaining species of this genus. The two species of Bromelia examined differed in polyploid level and in the presence of B chromosomes: B. plumieri had 2n = 50 + 1-2B and B. laciniosa, 2n = ca. 150, but both had very small chromosomes, in the same range: 0.23-0.26 μm (Figure 1i, j). In B. plumieri B chromosomes had a size similar to others of the same set. Only four species have been analyzed of the Bromelia genus, showing atypical numbers of 2n = 48 for B. alta, 2n = 94 for B. goeldiana (cf. McWilliams, 1974) and 2n = 96 for B. pinguim and B. antiacantha (Lin et al., 1987). Table I Origin, voucher number and chromosome number of species of the Bromeliaceae studied. Subfamily/Species Localities of collection Voucher number 2n


INTRODUCTION
Bromeliaceae is a plant family from tropical and subtropical America, with about 3,000 species in 54 genera (Leme, 1998).It is widely distributed on the American continent, from the States of Virginia and Texas in the southern United States to central Argentina and Chile (Smith, 1934).Pitcairnia feliciana, the only exception, is found in Guine, Africa (Smith and Downs, 1974).Brazil, one of the largest centers of diversity, has approximately 40% of known species, with the more evolved genera and species of the Bromelioideae subfamily and some more advanced taxa of the Pitcairnioideae and Tillandsioideae subfamilies found in the eastern region (Leme and Marigo, 1993).Although numerous botanical, ecological and evolutionary studies exist, cytogenetical analyses are limited to about 9% of the species, most of these cultivated and ornamental (Marchant, 1967;Sharma and Ghosh, 1971;McWilliams, 1974;Brown andGilmartin, 1986, 1989;Lin et al., 1987).There is a predominance of 2n = 50 in these studies and aneuploidy appears to decrease to 2n = 34 in the genus Cryptanthus.However, no cytological data appears for 22 genera, and for the majority of those already studied only a small number of species have been investigated.Besides scarcity of cytological data, recordings of different numbers of chromosomes for the same species justify new counts.This study is the first report of 15 Bromeliaceae species, belonging to nine different genera, and presents new counts for Billbergia chlorosticta and Cryptanthus bahianus.Determination of chromosome number for species of Encholirium, Orthophytum, Hohenbergia and Neoglaziovia represents the first record for these genera.

MATERIAL AND METHODS
Plants were collected from their natural habitat, except for Ananas lucidus, Aechmea blanchetiana and Billbergia morelii which, although native to Bahia, were obtained from specimens in cultivation (Table I).At least three plants per species were analyzed.Voucher specimens were deposited at the ALCB herbarium, Instituto de Biologia, Universidade Federal da Bahia, Brazil.The plants were kept in containers with water up to the point of leaf insertion to encourage rooting.Root tips were pretreated with 0.002 M 8-hydroxyquinoline for 4 h at 18ºC and fixed in 3:1 ethanol-acetic acid for 18-24 h.Root tips were transferred to 70% alcohol and stored at 4ºC, then hydrolyzed in 1 N HCl for 8 min at 60ºC and stained following the Feulgen method (Sharma and Sharma, 1980).Squash preparations were made in a 1% acetic-carmine solution.Coverslips were removed in 45% acetic acid and slides and coverslips mounted in Canada balsam.Chromosome counts and measurements were made in 5-20 metaphases of each species.

RESULTS AND DISCUSSION
The majority of the species analyzed had 2n = 50, but 2n = 34 and 2n = 100 to about 150 were also observed (Table I).The chromosome number of all species was determined for the first time, except for B. chlorosticta and C. bahianus (Figures 1 and 2).Extremely small chromosome size (0.23-1.08 µm) hindered a comparative morphological analysis, but small differences suffice for characterization.While some species such as B. chlorosticta, H. utriculosa and H. stellata had chromosomes varying from 0.41-1.21µm, those of H. littoralis, B. morelii and the Bromelia species did not reach 0.3 µm.Variation in chromosome size appeared within the same karyotype.Bromelia and Billbergia species were more uniform, while Encholirium spectabile, Neoglaziovia variegata and the Aechmea species presented continuous variation from largest to smallest chromosome, but without the clear expression of bimodality, observed by Marchant (1967) in the more advanced Bromelioideae and Tillandsioideae species.
Ananas lucidus with 2n = 50 had more uniform chromosomes, varying from 0.67-0.89µm (Figure 1e).Ananas also had a certain uniformity of 2n = 50 in the five species already analyzed, but triploids have been previously recorded in A. comosus and tetraploids in A. ananassoides (Lin et al., 1987).
Neoglaziovia is a genus with only three species, of which N. variegata had 2n = 100 with chromosome size varying from 0.40-0.93µm, (Figure 1h).No chromosome data exist for the remaining species of this genus.
Generally, a certain uniformity in the somatic number of 2n = 50 prevails in the Bromeliaceae.However, first observations on root tips showed some variation in the number for individual species, and discrepancies exist in numbers found by mitotic and meiotic analyses (cf.Brown and Gilmartin, 1986).The high number and small size of the chromosomes may have contributed to erroneous counts, based on pro-metaphases or metaphases with overlapping chromosomes.However, in some cases variation may be due to the presence of B chromosomes, as observed in B. plumieri, H. aff.utriculosa and C. bahianus (Table I) and 2n = 54 for B. chlorosticta and Aechmea ornata (Brown and Gilmartin, 1986;Lindschau, 1933, cited in McWilliams, 1974).
The karyotype 2n = 50, 100 and 150 observed here supports a hypothesized basic number of x = 25 for the family (Marchant, 1967), while the diploid numbers registered in species of the Cryptanthus and others such as n = 21 in Aechmea tillandsioides, n = 19 in Tillandsia leiboldiana var.leiboldiana, n = 20 and 22 in T. complanata suggest aneuploidy decreasing from this basic number (Marchant, 1967;Brown and Gilmartin, 1989).
Polyploidy as well seems to play an important role in the chromosome evolution of the family, from the original basic number x = 25 (Brown and Gilmartin, 1989) to the diversification of some genera.Till (1984, cited in Brown andGilmartin, 1986) observed a tendency to polyploidy in the subgenus Diaphoranthema (Tillandsia) recording 12 tetraploid species.The Orthophytum species showed a tetraploid karyotype, 2n = 100, and hexaploid, 2n = 150, and the only Neoglaziovia species analyzed had 2n = 100, while four of the six species of Bromelia analyzed were polyploid, with 2n = 100 and 150.

Table I -
Origin, voucher number and chromosome number of species of the Bromeliaceae studied.