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(American Journal of Botany. 2004;91:804-807.)
© 2004 Botanical Society of America, Inc.

Genetics and Molecular Biology

Chromosome numbers, meiotic behavior, and pollen viability of species of Vriesea and Aechmea genera (Bromeliaceae) native to Rio Grande do Sul, Brazil1

Clarisse Palma-Silva, Daniel G. dos Santos, Eliane Kaltchuk-Santos and Maria H. Bodanese-Zanettini2

Departamento de Genética, Universidade Federal do Rio Grande do Sul, CP15053, CEP 91501-970, Porto Alegre, RS, Brazil

Received for publication April 25, 2003. Accepted for publication January 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Chromosome number, meiotic behavior, and pollen viability were analyzed in 15 species of two genera, Vriesea and Aechmea, native to Rio Grande do Sul, Brazil. This study is the first cytogenetic analysis of these taxa. The chromosome numbers are all n = 25, consistent with the proposed base number of x = 25 for Bromeliaceae. All examined taxa displayed regular bivalent pairing and chromosome segregation at meiosis. Observed meiotic abnormalities include univalents in metaphase I; missing or extra chromosomes and precocious division of centromeres in metaphase II; laggards in telophase I and anaphase II/telophase II. The high pollen viability (>88%) reflects a regular meiosis.

Key Words: Aechmea • Bromeliaceae • chromosome numbers • meiotic behavior • pollen viability • Vriesea


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
The family Bromeliaceae has a tropical to subtropical distribution with just one species outside the American continent (McWillams, 1974 ). The nearly 3000 species are divided into three subfamilies: Pitcairnioideae, Bromelioideae, and Tillandsioideae (Smith and Downs, 1974 ).

Vriesea, with approximately 250 species, is the second largest genus in subfamily Tillandsioideae. Vriesea is mostly epiphytic, with species distributed from Mexico to southeast Brazil (Smith and Downs, 1977 ). Twenty species of Vriesea are reported for the Brazilian state of Rio Grande do Sul (Winkler, 1980 , 1982 ; Reitz, 1983 ; Waechter, 1992 ).

The genus Aechmea, comprising approximately 220 species, is common in the Amazon region and the Atlantic Forest of eastern Brazil. Aechmea is a large and diverse genus placed in subfamily Bromelioideae (Smith and Downs, 1979 ), with both epiphytic and terrestrial species. In Rio Grande do Sul state, about 11 species of Aechmea occur (Winkler, 1980 , 1982 ; Reitz, 1983 ; Waechter, 1992 ).

Cytogenetic studies on the family Bromeliaceae are few. And only about 10% of the species have reported chromosome numbers. The chromosome numbers have been recorded for only 15 species of Vriesea and 18 of Aechmea (Lindschau, 1933 ; Gauthé, 1965 ; Weiss, 1965 ; Marchant, 1967 ; Brown and Gilmartin, 1989 ; Cotias-de-Oliveira et al., 2000 ). In Bromeliaceae, the chromosomes are small with sizes varying between 0.23 µm and 1.5 µm (Cotias-de-Oliveira et al., 2000 ). Despite the high homogeneity in its somatic number, 2n = 50, species with 2n = 100 and 150 chromosomes have been reported (Sharma and Ghosh, 1971 ; Cotias-de-Oliveira et al., 2000 ). This chromosome number supports Marchant's (1967) proposal that x = 25 is the base number for the family.

Meiotic analyses are almost nonexistent in Bromeliaceae. Marchant (1967) first used pollen mother cells (PMCs) to access chromosome numbers because of the lack of roots in many epiphytic species, the usual source of somatic chromosomes. Further studies (Brown et al., 1984 , 1997 ; Varadarajan and Brown, 1985 ; Brown and Gilmartin, 1986 , 1989 ) have published chromosome numbers based on PMC analysis. However, to the best of our knowledge, there are few published records on meiotic chromosome behavior of Bromeliaceae. Basic data on meiotic behavior and pollen viability estimations are important for the germplasm characterization, to determine the genetic variability, study biodiversity, and evolution processess of the species analyzed.

This study was done to analyze the meiotic behavior in PMCs and pollen viability of 13 species of Vriesea and two species of Aechmea. The chromosome numbers for these species are reported for the first time, except for Vriesea psittacina (Brown and Gilmartin, 1989 ).


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
The species analyzed in this study are listed in Table 1. The specimens were either collected in the field and from cultivated material at Fundação Zoobotânica (FZB), Porto Alegre, Rio Grande do Sul. Voucher specimens have been deposited at Alarich Schultz herbarium-HAS (FZB). The plants were cultivated in the greenhouse of the Department of Genetics, Universidade Federal do Rio Grande do Sul. Taxonomic nomenclature follows Reitz (1983) .


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  		Table 1. Species of Vriesea and Aechmea analyzed for meiotic chromosome behavior, number of plants examined, chromosome number, voucher, and localities of collection

 
To obtain PMCs undergoing meiosis, young inflorescences were selected when floral buds were 1.7–2.0 mm long for Vriesea guttata, 0.9–1.6 mm long for all other Vriesea species, and 0.3–0.7 mm long for both Aechmea species.

Floral buds were fixed for 24 h in 3 : 1 ethanol : glacial acetic acid with a drop of saturated aqueous ferric chloride (FeCl3·6H2O) at room temperature. After fixation, buds were transferred to 70% alcohol and stored in a freezer at –18°C. Squash preparations were made in a 1% propionic carmine on a microscope slide.

Pollen stainability was used to indicate pollen viability. Flowers at anthesis were collected for this analysis. Floral buds were fixed in 3 : 1 ethanol : glacial acetic acid for 24 h at room temperature and stored in 70% alcohol in a freezer at –18°C. Squash preparations were stained following Alexander's method (1980) . The stainability was determined using samples of 1500 pollen grains per flower. At least three flowers were analyzed per individual. Because the inflorescence of Vriesea species has a small number of floral buds, in some cases it was not possible to analyze both meiosis and pollen viability.

Slides were examined and documented with a Zeiss Axioplan Universal photomicroscope.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
Chromosome number
All species analyzed by mitotic and/or meiotic chromosome counting had a consistent number of n = 25 in PMCs. The chromosome counts presented in this work increased the number of Vriesea species investigated to 24, representing about 11% of the genus. In Aechmea, including the counts presented here, 20 species have available chromosome numbers, representing about 13% of the genera. The data presented here (Table 1) are chromosome numbers published for the first time, except for Vriesea psittacina (Brown and Gilmartin, 1989 ). All species in Vriesea and Aechmea have the number n = 25 (Marchant, 1967 ; Brown and Gilmartin, 1989 ) and 2n = 50 (Lindschau, 1933 ; Cotias-de-Oliveira et al., 2000 ). The only exceptions reported are 2n = 56 for V. hieroglyphica and V. holscheriana (Gauthè, 1965 ), 2n = 48 for V. splendens (Weiss, 1965 ), 2n = 54 for Aechmea ornata (Lindschau, 1933 ) and n = 21 for A. tillansioideae (Marchant, 1967 ).

In all species, the meiotic chromosomes are very small. In Vriesea carinata (Fig. 4), all chromosomes were observed to be metacentric or submetacentric. Satellites were not identified. The lack of information on chromosomal morphology of bromeliads could be imputed to the small chromosome size.



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  		Figs. 1–4. Meiotic behavior in pollen mother cells. 1. Metaphase I in Aechmea gamosepala, n = 25. 2. Metaphase I in Aechmea gamosepala, n = 25, with two univalents (arrows). 3. Anaphase I in Vriesea psittacina, n = 25, with laggards (arrows). 4. Metaphase II in Vriesea carinata, n = 25. Bar = 10 µm

 
Meiotic behavior
Twenty-five plants belonging to nine species of Vriesea and six plants of two species of Aechmea were investigated for meiotic behavior of PMCs. A wide range of meiotic stages were found in anthers within the same flower.

A total of 316 diakinesis/metaphases I (D/MI), 364 anaphase I/telophase I (AI/TI), 327 methaphase II (MII), and 358 anaphase II/telophase II (AII/MII) cells were analyzed and tabulated for Vriesea species (Table 2). The D/MI cells were usually regular with predominant bivalent (II) pairing. There was limited asynapsis/desynapsis with two or four univalents (I). Because of the variation in chromosome size, it was difficult to differentiate univalents and bivalents. However, because the plants had 25II (2n = 50), it could be assumed that the 26 or 27 elements observed in abnormal cells were 24II + 2I or 23II + 4I, respectively. One or two laggard chromosomes were found in AI/TI and AII/TII cells. Abnormal MII cells presented 24, 26, 27, 29, or 31 elements. In this phase, it was also difficult to determine whether the extra elements were chromosomes or chromatids. Missing or extra chromosome(s) could be the result of unequal segregation in anaphase I.


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  		Table 2. Numbers of pollen mother cells (PMCs) analyzed and percentage of PMCs with irregular meiotic behavior

 
Only one plant of Vriesea psittacina presented a highly irregular meiosis II. In this plant, a wide range of numbers of elements in MII and laggards in AII/TII (Fig. 3) was detected. Flowers containing PMCs in meiosis I, as well as flowers presenting pollen grains at an adequate stage for stainability analysis, were not founded in this plant.

The irregularities observed in AI/TI and AII/TII, as well as those recorded in metaphase II, could be explained by the erratic behavior of univalents. The univalents, as a rule, fail to orient properly between the poles, either not segregating, randomly moving to one or the other poles, or dividing, as in mitosis, into their two chromatids (Swanson et al., 1981 ).

In Aechmea species a total of 83 D/MI, 106 AI/TI, 29 MII, and 46 AII/TII cells were analyzed (Table 2). For A. calyculata, the majority of D/MI cells presented the expected 25 bivalent pairing (Fig. 1). Two or four univalents were observed in D/MI abnormal cells. The similarity among chromosome sizes allowed differentiation of univalents and bivalents. A laggard chromosome was found in 1.2% of AI/TI cells of A. calyculata. At meiosis II, a predominance of cells displayed regular behavior. For A. gamosepala, a high frequency (56%) of D/MI cells was observed with irregular pairing (Fig. 2). Unexpectedly, irregularities were not detected in the following phases of meiosis.

Our results are similar to those reported for other bromeliad species. Marchant (1967) analyzed meiosis in PMCs of three species of Vriesea and nine species and a hybrid of Aechmea. All but one species presented regular meiosis. Vriesea splendens had an irregular meiosis presenting univalents at first metaphase. This species was not reported as being of hybrid origin.

Pollen viability
The pollen viability was analyzed in 39 plants from 10 species of Vriesea. The pollen viability of the examined species are described in Table 3 and illustrated in Fig. 5.


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  		Table 3. Percentage of pollen stainability in Vriesea species

 


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  		Fig. 5. Pollen stainability in Vriesea philippo-coburgii. Viable pollen grains are full (dark) and stain purple, and unviable pollen grains are empty (light) and stain green. Bar = 20 µm

 
A high percentage of stained pollen (>88.1%) was registered for all species. These data also reflect the meiotic regularity. The high pollen viability indicates that irregularities observed at meiosis probably are not significant in terms of species fertility.


   FOOTNOTES
 
1 The authors thank FZB (Fundação Zoobotânica do Rio Grande do Sul) for help in collecting samples and Dr. Teresia Strehl for taxonomic assistance. Research was supported by CAPES (Fundação Coordenação de Aperfeiçoamento do Pessoal de Nível Superior) and FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul). Back

2 E-mail: maria.zanettini{at}ufrgs.br Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Alexander M. P. 1980 A versatile stain for pollen fungi, yeast and bacteria. Stain Tecnology 55: 13-18

Brown G. K. A. J. Gilmartin 1986 Chromosomes of the Bromeliaceae. Selbyana 9: 88-93

Brown G. K. A. J. Gilmartin 1989 Chromosome numbers in Bromeliaceae. American Journal of Botany 76: 657-665[CrossRef][ISI]

Brown G. K. C. A. Palací H. E. Luther 1997 Chromosome numbers in Bromeliaceae. Selbyana 18: 85-88

Brown G. K. G. S. Varadarajan A. J. Gilmartin H. E. Luther 1984 Chromosome number reports LXXXV. Bromeliaceae. Taxon 33: 756-760

Cotias-de-Oliveira A. L. P. J. G. A. Assis M. C. Bellintani J. C. Andrade M. L. S. Guedes 2000 Chromosome numbers in Bromeliaceae. Genetics and Molecular Biology 23: 173-177

Gauthé J. 1965 Contribution à l'étude caryologique des Tillandsiées. Mémories du Muséum National d'Histoire Naturelle 16: 39-59

Lindschau M. 1933 Beiträge zur Zytologic der Bromeliaceae. Planta 20: 506-530[CrossRef]

Marchant C. J. 1967 Chromosome evolution in Bromeliaceae. Kew Bulletin 21: 161-170[CrossRef]

McWilliams E. 1974 Chromosome number and evolution. In L. B. Smith and J. Downs [eds.], Pitcairnioideae (Bromeliaceae). Flora Neotropica Monograph 14, vol. 1, 33–39. Hafner Press, New York, New York, USA

Reitz R. 1983 Bromeliáceas e a malária—Bromélia endêmica. Flora Ilustrada Catarinense. Herbário Barbosa Rodrigues. Itajaí, Santa Catarina, Brazil

Sharma A. K. I. Ghosh 1971 Cytotaxonomy of the family Bromeliaceae. Cytologia 36: 237-247[ISI]

Smith L. B. R. J. Downs 1974 Pitcairnioideae (Bromeliaceae). Flora Neotropica Monograph 14, vol. 1. Hafner Press, New York, New York, USA

Smith L. B. R. J. Downs 1977 Tillandsioideae (Bromeliaceae). Flora Neotropica Monograph 14, vol. 2. Hafner Press, New York, New York, USA

Smith L. B. R. J. Downs 1979 Bromeliaceae (Bromelioideae). Flora Neotropica Monograph 14, vol. 3. New York Botanical Garden, Bronx, New York, USA

Swanson C. P. T. Mertz W. J. Young 1981 Cytogenetics, the chromosome in division inheritance and evolution. Prentice Hall, Englewood Cliffs, New Jersey, USA

Varadarajan G. S. G. K. Brown 1985 Chromosome number reports LXXXIX. Taxon 34: 727-730

Waechter J. 1992 O epifitismo vascular na planície costeira do Rio Grande do Sul. Ph.D. dissertation, Universidade de São Carlos, São Carlos, São Paulo, Brazil

Weiss H. E. 1965 Étude caryologique et cyto-taxonomique de quelques Broméliaceés. Mémories du Muséum National d'Histoire Naturelle 16: 9-38

Winkler S. 1980 Ursachen der Verbreitungsmuster einiger Bromeliaceae in Rio Grande do Sul (Südbrasilien). Flora 170: 371-393[ISI]

Winkler S. 1982 Die Bromeliaceae von Rio Grande do Sul (S-Brasilien). Documenta Naturae 3: 90





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