American Journal of Botany 91(6): 804–807. 2004.
CHROMOSOME
NUMBERS, MEIOTIC BEHAVIOR, AND
POLLEN VIABILITY OF SPECIES OF
AECHMEA
ELIANE
VRIESEA
(BROMELIACEAE) NATIVE
GRANDE DO SUL, BRAZIL1
GENERA
AND
TO
RIO
CLARISSE PALMA-SILVA, DANIEL G. DOS SANTOS,
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
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 5
25, consistent with the proposed base number of x 5 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.
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 mm and 1.5 mm (Cotias-de-Oliveira et al., 2000). Despite
the high homogeneity in its somatic number, 2n 5 50, species
with 2n 5 100 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 5 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
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).
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 2188C. 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 2188C. 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.
Manuscript received 25 April 2003; revision accepted 22 January 2004.
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 Nı́vel Superior) and FAPERGS (Fundação de Amparo
à Pesquisa do Estado do Rio Grande do Sul).
2
E-mail: [email protected].
1
804
June 2004]
PALMA-SILVA
ET AL.—CYTOGENETICS OF
BROMELIACEAE
805
TABLE 1. Species of Vriesea and Aechmea analyzed for meiotic chromosome behavior, number of plants examined, chromosome number, voucher,
and localities of collection.
Species
V. carinata Wawra
V. erytrodactylon (E. Morren) E. Morren ex. Mez
V. flammea L.B. Smith
V. friburgensis Mez
V. guttata Linden and André
V. incurvata Gaudchaud
V. platynema Gaudchaud
V. platzmannii E. Morren
V. psittacina (Hooker) Lindley
V. procera (Martius ex. Shultes F.) Wittmack
V. philoppo-coburgii Wawra
V. rodigasiana E. Morren
V. reitzii Leme and A. Costa
Aechmea calyculata (Morren) Baker
A. gamosepala Wittmack
a
Number of
plants
Chromosome
number
7
2
1
4
2
5
3
1
2
4
5
2
2
4
2
50
50
50
50
50
50
50
50
50
—a
—a
—a
—a
50
50
Voucher
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
HAS
—
102424
102427
102321
102411
102414
102438
102406
102404
102415
102418
102306
102421
102310
102467
Localities of
collection
Maquiné
Três Cachoeiras
Morrinhos do Sul
Itapuã
Cambará do Sul
Caraá
Guabiju
Capão da Canoa
Viamão
Maquiné
São Francisco de Paula
Dom Pedro de Alcântara
São Francisco de Paula
Santo Antônio da Patrulha
—
Species analyzed only for pollen stainability.
Figs. 1–4. Meiotic behavior in pollen mother cells. 1. Metaphase I in Aechmea gamosepala, n 5 25. 2. Metaphase I in Aechmea gamosepala, n 5 25, with
two univalents (arrows). 3. Anaphase I in Vriesea psittacina, n 5 25, with laggards (arrows). 4. Metaphase II in Vriesea carinata, n 5 25. Bar 5 10 mm.
806
AMERICAN JOURNAL
TABLE 2.
carinata
erytrodactylon
flammea
friburgensis
guttata
incurvata
platynema
platzmannii
psittacina
calyculata
gamosepala
a
BOTANY
[Vol. 91
Numbers of pollen mother cells (PMCs) analyzed and percentage of PMCs with irregular meiotic behavior.
Species
V.
V.
V.
V.
V.
V.
V.
V.
V.
A.
A.
OF
No. plants
analyzed
No. cells
D/MIa
% Abb
AI/TIc
% Ab
MIId
% Ab
AII/TIIe
% Ab
5
2
1
4
2
5
3
1
2
4
2
329
108
85
141
78
190
237
94
197
190
74
41
39
2
24
0
91
54
32
33
47
36
7.3
7.7
0
0
0
4.4
11.1
15.6
24.2
23.4
56
84
67
30
29
1
56
62
30
5
82
24
0
0
3.3
0
0
1.8
0
0
0
1.2
0
88
2
23
56
44
43
61
2
8
15
14
12.5
0
0
19.6
15.9
6.9
6.6
0
87.5
6.7
0
116
0
30
32
33
0
60
30
57
46
0
1.7
0
0
0
0
0
0
0
73.7
0
0
D/MI 5 diacinese/metaphase I; b Ab 5 abnormal cells; c AI/TI 5 anaphase I/telophase I; d MII 5 Metaphase II; e AII/TII 5 anaphase II/telophase
II.
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
Chromosome number—All species analyzed by mitotic
and/or meiotic chromosome counting had a consistent number
of n 5 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 5 25 (Marchant, 1967; Brown and Gilmartin, 1989) and 2n 5 50 (Lindschau, 1933; Cotias-de-Oliveira et al., 2000). The only exceptions reported are 2n 5 56
for V. hieroglyphica and V. holscheriana (Gauthè, 1965), 2n
5 48 for V. splendens (Weiss, 1965), 2n 5 54 for Aechmea
ornata (Lindschau, 1933) and n 5 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.
TABLE 3.
Percentage of pollen stainability in Vriesea species.
Species
V.
V.
V.
V.
V.
V.
V.
V.
V.
V.
carinata
erytrodactylon
guttata
friburgensis
incurvata
platynema
procera
philoppo-coburgii
rodigasiana
reitzii
a
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 5 50), it could be assumed that the 26 or
27 elements observed in abnormal cells were 24II 1 2I or
23II 1 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.
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
No. plants
analyzed
No. flowers
examined
No. pollen
grains examined
Percentage of
stained grainsa
7
1
1
4
6
7
4
5
2
2
34
4
5
22
28
26
19
25
10
10
50 548
3911
7500
26 990
41 000
3800
26 403
37 101
15 000
15 000
94.3 (84.3–98.7)
92.2
88.1
95.8 (88.9–98.5)
90 (79.9–98.5)
94.1 (87.3–97.7)
93.3 (88.9–98.2)
92.5 (80.5–96.8)
95.4 (94.2–96.6)
98 (97.6–98.4)
Values are means with minimum and maximum ranges.
June 2004]
PALMA-SILVA
ET AL.—CYTOGENETICS OF
BROMELIACEAE
807
tered 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.
LITERATURE CITED
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 5 20 mm.
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.
A high percentage of stained pollen (.88.1%) was regis-
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